Expression of Iron-Related Proteins Differentiate Non-Cancerous and Cancerous Breast Tumors

PubMed Central

Pizzamiglio, Sara; De Bortoli, Maida; Taverna, Elena; Signore, Michele; Veneroni, Silvia; Chi-shing Cho, William; Orlandi, Rosaria; Verderio, Paolo; Bongarzone, Italia

2017-01-01

We have previously reported hepcidin and ferritin increases in the plasma of breast cancer patients, but not in patients with benign breast disease. We hypothesized that these differences in systemic iron homeostasis may reflect alterations in different iron-related proteins also play a key biochemical and regulatory role in breast cancer. Thus, here we explored the expression of a bundle of molecules involved in both iron homeostasis and tumorigenesis in tissue samples. Enzyme-linked immunosorbent assay (ELISA) or reverse-phase protein array (RPPA), were used to measure the expression of 20 proteins linked to iron processes in 24 non-cancerous, and 56 cancerous, breast tumors. We found that cancerous tissues had higher level of hepcidin than benign lesions (p = 0.012). The univariate analysis of RPPA data highlighted the following seven proteins differentially expressed between non-cancerous and cancerous breast tissue: signal transducer and transcriptional activator 5 (STAT5), signal transducer and activator of transcription 3 (STAT3), bone morphogenetic protein 6 (BMP6), cluster of differentiation 74 (CD74), transferrin receptor (TFRC), inhibin alpha (INHA), and STAT5_pY694. These findings were confirmed for STAT5, STAT3, BMP6, CD74 and INHA when adjusting for age. The multivariate statistical analysis indicated an iron-related 10-protein panel effective in separating non-cancerous from cancerous lesions including STAT5, STAT5_pY694, myeloid differentiation factor 88 (MYD88), CD74, iron exporter ferroportin (FPN), high mobility group box 1 (HMGB1), STAT3_pS727, TFRC, ferritin heavy chain (FTH), and ferritin light chain (FTL). Our results showed an association between some iron-related proteins and the type of tumor tissue, which may provide insight in strategies for using iron chelators to treat breast cancer. PMID:28216608

Obatoclax analog SC-2001 inhibits STAT3 phosphorylation through enhancing SHP-1 expression and induces apoptosis in human breast cancer cells.

PubMed

Liu, Chun-Yu; Su, Jung-Chen; Ni, Mei-Huei; Tseng, Ling-Ming; Chu, Pei-Yi; Wang, Duen-Shian; Tai, Wei-Tien; Kao, Yuan-Ping; Hung, Man-Hsin; Shiau, Chung-Wai; Chen, Kuen-Feng

2014-07-01

Interfering oncogenic STAT3 signaling is a promising anti-cancer strategy. We examined the efficacy and drug mechanism of an obatoclax analog SC-2001, a novel STAT3 inhibitor, in human breast cancer cells. Human breast cancer cell lines were used for in vitro studies. Apoptosis was examined by both flow cytometry and western blot. Signaling pathways were assessed by western blot. In vivo efficacy of SC-2001 was tested in xenograft nude mice. SC-2001 inhibited cell growth and induced apoptosis in association with downregulation of p-STAT3 (Tyr 705) in breast cancer cells. STAT3-regulated proteins, including Mcl-1, survivin, and cyclin D1, were repressed by SC-2001. Over-expression of STAT3 in MDA-MB-468 cells protected cells from SC-2001-induced apoptosis. Moreover, SC-2001 enhanced the expression of protein tyrosine phosphatase SHP-1, a negative regulator of STAT3. Furthermore, the enhanced SHP-1 expression, in conjunction with increased SHP-1 phosphatase activity, was mediated by upregulated transcription by RFX-1. Chromatin immunoprecipitation assay revealed that SC-2001 increased the binding capacity of RFX-1 to the SHP-1 promoter. Knockdown of either RFX-1 or SHP-1 reduced SC-2001-induced apoptosis, whereas ectopic expression of RFX-1 increased SHP-1 expression and enhanced the apoptotic effect of SC-2001. Importantly, SC-2001 suppressed tumor growth in association with enhanced RFX-1 and SHP-1 expression and p-STAT3 downregulation in MDA-MB-468 xenograft tumors. SC-2001 induced apoptosis in breast cancer cells, an effect that was mediated by RFX-1 upregulated SHP-1 expression and SHP-1-dependent STAT3 inactivation. Our study indicates targeting STAT3 signaling pathway may be a useful approach for the development of targeted agents for anti-breast cancer.

Inhibition of Stat3 activation suppresses caspase-3 and the ubiquitin-proteasome system, leading to preservation of muscle mass in cancer cachexia.

PubMed

Silva, Kleiton Augusto Santos; Dong, Jiangling; Dong, Yanjun; Dong, Yanlan; Schor, Nestor; Tweardy, David J; Zhang, Liping; Mitch, William E

2015-04-24

Cachexia occurs in patients with advanced cancers. Despite the adverse clinical impact of cancer-induced muscle wasting, pathways causing cachexia are controversial, and clinically reliable therapies are not available. A trigger of muscle protein loss is the Jak/Stat pathway, and indeed, we found that conditioned medium from C26 colon carcinoma (C26) or Lewis lung carcinoma cells activates Stat3 (p-Stat3) in C2C12 myotubes. We identified two proteolytic pathways that are activated in muscle by p-Stat3; one is activation of caspase-3, and the other is p-Stat3 to myostatin, MAFbx/Atrogin-1, and MuRF-1 via CAAT/enhancer-binding protein δ (C/EBPδ). Using sequential deletions of the caspase-3 promoter and CHIP assays, we determined that Stat3 activation increases caspase-3 expression in C2C12 cells. Caspase-3 expression and proteolytic activity were stimulated by p-Stat3 in muscles of tumor-bearing mice. In mice with cachexia caused by Lewis lung carcinoma or C26 tumors, knock-out of p-Stat3 in muscle or with a small chemical inhibitor of p-Stat3 suppressed muscle mass losses, improved protein synthesis and degradation in muscle, and increased body weight and grip strength. Activation of p-Stat3 stimulates a pathway from C/EBPδ to myostatin and expression of MAFbx/Atrogin-1 and increases the ubiquitin-proteasome system. Indeed, C/EBPδ KO decreases the expression of MAFbx/Atrogin-1 and myostatin, while increasing muscle mass and grip strength. In conclusion, cancer stimulates p-Stat3 in muscle, activating protein loss by stimulating caspase-3, myostatin, and the ubiquitin-proteasome system. These results could lead to novel strategies for preventing cancer-induced muscle wasting. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Inhibition of Stat3 Activation Suppresses Caspase-3 and the Ubiquitin-Proteasome System, Leading to Preservation of Muscle Mass in Cancer Cachexia*

PubMed Central

Silva, Kleiton Augusto Santos; Dong, Jiangling; Dong, Yanjun; Dong, Yanlan; Schor, Nestor; Tweardy, David J.; Zhang, Liping; Mitch, William E.

2015-01-01

Cachexia occurs in patients with advanced cancers. Despite the adverse clinical impact of cancer-induced muscle wasting, pathways causing cachexia are controversial, and clinically reliable therapies are not available. A trigger of muscle protein loss is the Jak/Stat pathway, and indeed, we found that conditioned medium from C26 colon carcinoma (C26) or Lewis lung carcinoma cells activates Stat3 (p-Stat3) in C2C12 myotubes. We identified two proteolytic pathways that are activated in muscle by p-Stat3; one is activation of caspase-3, and the other is p-Stat3 to myostatin, MAFbx/Atrogin-1, and MuRF-1 via CAAT/enhancer-binding protein δ (C/EBPδ). Using sequential deletions of the caspase-3 promoter and CHIP assays, we determined that Stat3 activation increases caspase-3 expression in C2C12 cells. Caspase-3 expression and proteolytic activity were stimulated by p-Stat3 in muscles of tumor-bearing mice. In mice with cachexia caused by Lewis lung carcinoma or C26 tumors, knock-out of p-Stat3 in muscle or with a small chemical inhibitor of p-Stat3 suppressed muscle mass losses, improved protein synthesis and degradation in muscle, and increased body weight and grip strength. Activation of p-Stat3 stimulates a pathway from C/EBPδ to myostatin and expression of MAFbx/Atrogin-1 and increases the ubiquitin-proteasome system. Indeed, C/EBPδ KO decreases the expression of MAFbx/Atrogin-1 and myostatin, while increasing muscle mass and grip strength. In conclusion, cancer stimulates p-Stat3 in muscle, activating protein loss by stimulating caspase-3, myostatin, and the ubiquitin-proteasome system. These results could lead to novel strategies for preventing cancer-induced muscle wasting. PMID:25787076

GLI1 inhibitor GANT61 exhibits antitumor efficacy in T-cell lymphoma cells through down-regulation of p-STAT3 and SOCS3

PubMed Central

Geng, Lingyun; Lu, Kang; Li, Peipei; Li, Xinyu; Zhou, Xiangxiang; Li, Ying; Wang, Xin

2017-01-01

T-cell lymphomas are lymphoid malignancies with aggressive clinical course and poor prognosis. Increasing evidences suggest that deregulation of signal transducer and activator of transcription-3 (STAT3) and suppressor of cytokine signaling 3 (SOCS3) is associated with the pathogenesis of T-cell lymphomas. The hedgehog (Hh)/glioma-associated oncogene-1 (GLI1) pathway, aberrantly activated in a number of tumors, has also been extensively studied. We found that protein expressions of GL11, p-STAT3, STAT3, and SOCS3 were up-regulated in T-cell lymphoma tissues and cell lines. Moreover, the protein expressions of p-STAT3 and SOCS3 were positively correlated with GLI1 in T-cell lymphomas. GLI1 inhibitor GANT61 and lentivirus-mediated siGLI1 exhibited inhibitory effects in the three T-cell lines (Jurkat, Karpass299 and Myla3676 cells). The protein expressions of p-STAT3 and SOCS3 were decreased accompanied with the inhibition of GLI1. These findings indicated that GANT61 is a promising agent against T-cell lymphoma and the antitumor activity might be partly mediated by down-regulating p-STAT3 and SOCS3. PMID:27275540

Signal Transducer and Activator of Transcription 1 (STAT1) Knock-down Induces Apoptosis in Malignant Pleural Mesothelioma.

PubMed

Arzt, Lisa; Halbwedl, Iris; Gogg-Kamerer, Margit; Popper, Helmut H

2017-07-01

Malignant pleural mesothelioma (MPM) is the most common primary tumor of the pleura. Its incidence is still increasing in Europe and the prognosis remains poor. We investigated the oncogenic function of signal transducer and activator of transcription 1 (STAT1) in MPM in more detail. A miRNA profiling was performed on 52 MPM tissue samples. Upregulated miRNAs (targeting SOCS1/3) were knocked-down using miRNA inhibitors. mRNA expression levels of STAT1/3, SOCS1/3 were detected in MPM cell lines. STAT1 has been knocked-down using siRNA and qPCR was used to detect mRNA expression levels of all JAK/STAT family members and genes that regulate them. An immunohistochemical staining was performed to detect the expression of caspases. STAT1 was upregulated and STAT3 was downregulated, SOCS1/3 protein was not detected but it was possible to detect SOCS1/3 mRNA in MPM cell lines. The upregulated miRNAs were successfully knocked-down, however the expected effect on SOCS1 expression was not detected. STAT1 knock-down had different effects on STAT3/5 expression. Caspase 3a and 8 expression was found to be increased after STAT1 knock-down. The physiologic regulation of STAT1 via SOCS1 is completely lost in MPM and it does not seem that the miRNAs identified by now, do inhibit the expression of SOCS1. MPM cell lines compensate STAT1 knock-down by increasing the expression of STAT3 or STAT5a, two genes which are generally considered to be oncogenes. And much more important, STAT1 knock-down induces apoptosis in MPM cell lines and STAT1 might therefore be a target for therapeutic intervention.

Stat3-induced S1PR1 expression is critical for persistent Stat3 activation in tumors

PubMed Central

Lee, Heehyoung; Deng, Jiehui; Kujawski, Maciej; Yang, Chunmei; Liu, Yong; Herrmann, Andreas; Kortylewski, Marcin; Horne, David; Somlo, George; Forman, Stephen; Jove, Richard; Yu, Hua

2011-01-01

IL-6/Jak2 signaling is viewed critical for persistent Stat3 activation in cancer. However, IL-6-induced Stat3 activity is transient in normal physiology. Here we identify a mechanism important for persistent Stat3 activation in tumor cells and the tumor microenvironment. We show that sphingosine-1-phosphate receptor 1 (S1PR1), a G-protein-coupled receptor for lysophospholipid sphingosine-1-phosphate (S1P), is elevated in Stat3-positive tumors. Stat3 is a transcription factor for the S1pr1 gene. Enhanced S1pr1 expression activates Stat3 and upregulates Il6 gene expression, thereby accelerating tumor growth and metastasis. Conversely, silencing S1pr1 in tumor cells or immune cells inhibits tumor Stat3 activity, tumor growth and metastasis. S1P/S1PR1-induced Stat3 activation is persistent, in contrast to transient Stat3 activation by IL-6. S1PR1 activates Stat3 in part by upregulating Jak2 tyrosine kinase activity. We demonstrate that Stat3-induced S1pr1 expression, as well as S1P/S1PR1 pathway, is important for persistent Stat3 activation in cancer cells and the tumor microenvironment and for malignant progression. PMID:21102457

Nipah Virus V Protein Evades Alpha and Gamma Interferons by Preventing STAT1 and STAT2 Activation and Nuclear Accumulation

PubMed Central

Rodriguez, Jason J.; Parisien, Jean-Patrick; Horvath, Curt M.

2002-01-01

Characterization of recent outbreaks of fatal encephalitis in southeast Asia identified the causative agent to be a previously unrecognized enveloped negative-strand RNA virus of the Paramyxoviridae family, Nipah virus. One feature linking Nipah virus to this family is a conserved cysteine-rich domain that is the hallmark of paramyxovirus V proteins. The V proteins of other paramyxovirus species have been linked with evasion of host cell interferon (IFN) signal transduction and subsequent antiviral responses by inducing proteasomal degradation of the IFN-responsive transcription factors, STAT1 or STAT2. Here we demonstrate that Nipah virus V protein escapes IFN by a distinct mechanism involving direct inhibition of STAT protein function. Nipah virus V protein differs from other paramyxovirus V proteins in its subcellular distribution but not in its ability to inhibit cellular IFN responses. Nipah virus V protein does not induce STAT degradation but instead inhibits IFN responses by forming high-molecular-weight complexes with both STAT1 and STAT2. We demonstrate that Nipah virus V protein accumulates in the cytoplasm by a Crm1-dependent mechanism, alters the STAT protein subcellular distribution in the steady state, and prevents IFN-stimulated STAT redistribution. Consistent with the formation of complexes, STAT protein tyrosine phosphorylation is inhibited in cells expressing the Nipah virus V protein. As a result, Nipah virus V protein efficiently prevents STAT1 and STAT2 nuclear translocation in response to IFN, inhibiting cellular responses to both IFN-α and IFN-γ. PMID:12388709

The dipeptide Pro-Asp promotes IGF-1 secretion and expression in hepatocytes by enhancing JAK2/STAT5 signaling pathway.

PubMed

Wang, Songbo; Wang, Guoqing; Zhang, Mengyuan; Zhuang, Lu; Wan, Xiaojuan; Xu, Jingren; Wang, Lina; Zhu, Xiaotong; Gao, Ping; Xi, Qianyun; Zhang, Yongliang; Shu, Gang; Jiang, Qingyan

2016-11-15

It has been implicated that IGF-1 secretion can be regulated by dietary protein. However, whether the dipeptides, one of digested products of dietary protein, have influence on IGF-1 secretion remain largely unknown. Our study aimed to investigate the effects of the dipeptide Pro-Asp on IGF-1 secretion and expression in hepatocytes and to explore the possible underlying mechanisms. Our findings demonstrated that Pro-Asp promoted the secretion and gene expression of IGF-1 in HepG2 cells and primary porcine hepatocytes. Meanwhile, Pro-Asp activated the ERK and Akt signaling pathways, downstream of IGF-1. In addition, Pro-Asp enhanced GH-mediated JAK2/STAT5 signaling pathway, while inhibition of JAK2/STAT5 blocked the promotive effect of Pro-Asp on IGF-1 secretion and expression. Moreover, acute injection of Pro-Asp stimulated IGF-1 expression and activated JAK2/STAT5 signaling pathway in mice liver. Together, these results suggested that the dipeptide Pro-Asp promoted IGF-1 secretion and expression in hepatocytes by enhancing GH-mediated JAK2/STAT5 signaling pathway. Copyright © 2016. Published by Elsevier Ireland Ltd.

[Expressions of VEGF/VEGFRs and activation of STATs in ovarian carcinoma].

PubMed

Chen, Bing-Ya; Ye, Da-Feng; Xie, Xing; Chen, Huai-Zeng; Lü, Wei-Guo

2005-01-01

To study the expressions of VEGF/VEGFRs and activation of STATs in ovarian epithelial carcinoma, and to elucidate direct effect of VEGF on ovarian carcinoma cells. Tissue samples from 42 women with primary ovarian epithelial carcinoma (OVCA), 29 with begnin ovarian tumor (OVBT) and 11 with normal ovarian tissue (NOV) were collected. LSAB immunohistochemical staining was used to determine the expression of VEGF, VEGFR1, VEGFR2 and activated STATS (P-STAT1, P-STAT3, P-STAT5, P-STAT6) proteins. (1) Semi-quantitative scoring showed that VEGF expression in OVCA was significantly higher than that in OVBT and NOV (P < 0.01). Expressions of VEGFR1 and VEGFR2 were significantly elevated in OVCA, including tumor cells and stromal vascular endothelial cells (P < 0.01, compared with OVBT and NOV). There was no difference in VEGFRs expressions between OVBT and NOV. (2) In OVCA, tumor cells and endothelial cells expressed P-STAT3 and P-STAT5 at significantly higher levels than those in OVBT and NOV (P = 0.000). The staining of P-STAT1 and P-STAT6 was weak with no significant differences among OVCA, OVBT and NOV. (3) Expressions of VEGFR1 and VEGFR2 in endothelial cells were significantly correlated with P-STAT5 and P-STAT3, respectively (P = 0.006 and 0.001). In cancer cells, VEGF, VEGFR1 and VEGFR2 were all significantly correlated with P-STAT3 and P-STAT5 (P = 0.000), but not with P-STAT1 or P-STAT6. VEGF affects ovarian carcinoma cells via VEGFRs, and STATs probably participate in intracellular signaling of VEGF.

Transcriptional deregulation of oncogenic myocyte enhancer factor 2C in T-cell acute lymphoblastic leukemia.

PubMed

Nagel, Stefan; Venturini, Letizia; Meyer, Corinna; Kaufmann, Maren; Scherr, Michaela; Drexler, Hans G; Macleod, Roderick A F

2011-02-01

Myocyte enhancer factor 2C (MEF2C) encodes a transcription factor which is ectopically expressed in T-cell acute lymphoblastic leukemia (T-ALL) cell lines, deregulated directly by ectopically expressed homeodomain protein NKX2-5 or by loss of promoter regions via del(5)(q14). Here, we analyzed the MEF2C 5'-region, thus identifying potential regulatory binding sites for GFI1B, basic helix-loop-helix proteins, STAT5, and HOXA9/HOXA10. Chromatin immunoprecipitation and overexpression analyses demonstrated direct activation by GFI1B and LYL1 and inhibition by STAT5. HOXA9/HOXA10 activated expression of NMYC which in turn mediated MEF2C repression, indicating an indirect mode of regulation via NMYC interactor (NMI) and STAT5. Lacking comma: Chromosomal deletion of the STAT5 binding site in LOUCY cells reduced protein levels of STAT5 in some MEF2C-positve T-ALL cell lines, and the presence of inhibitory IL7-JAK-STAT5 signaling highlighted the repressive impact of this factor in MEF2C regulation. Taken together, our results indicate that the expression of MEF2C in T-ALL cells is principally deregulated via activating leukemic transcription factors GFI1B or NKX2-5 and by escaping inhibitory developmental STAT5 signaling.

Expression and Purification of Soluble STAT5b/STAT3 Proteins for SH2 Domain Binding Assay.

PubMed

Asai, Akira; Takakuma, Kazuyuki

2017-01-01

When a large hydrophobic full-length protein is expressed in bacteria, it is often challenging to obtain recombinant proteins in the soluble fraction. One way to overcome this challenge is expression of deletion mutants that have improved solubility while maintaining biological activity. In this chapter, we describe a protocol for expression of truncated forms of STAT5b and STAT3 proteins that are soluble and retain SH2-mediated activity for phospho-Tyr peptide recognition.

PTP1B is a negative regulator of interleukin 4–induced STAT6 signaling

PubMed Central

Lu, Xiaoqing; Malumbres, Raquel; Shields, Benjamin; Jiang, Xiaoyu; Sarosiek, Kristopher A.; Natkunam, Yasodha

2008-01-01

Protein tyrosine phosphatase 1B (PTP1B) is a ubiquitously expressed enzyme shown to negatively regulate multiple tyrosine phosphorylation-dependent signaling pathways. PTP1B can modulate cytokine signaling pathways by dephosphorylating JAK2, TYK2, and STAT5a/b. Herein, we report that phosphorylated STAT6 may serve as a cytoplasmic substrate for PTP1B. Overexpression of PTP1B led to STAT6 dephosphorylation and the suppression of STAT6 transcriptional activity, whereas PTP1B knockdown or deficiency augmented IL-4–induced STAT6 signaling. Pretreatment of these cells with the PTK inhibitor staurosporine led to sustained STAT6 phosphorylation consistent with STAT6 serving as a direct substrate of PTP1B. Furthermore, PTP1B-D181A “substrate-trapping” mutants formed stable complexes with phosphorylated STAT6 in a cellular context and endogenous PTP1B and STAT6 interacted in an interleukin 4 (IL-4)–inducible manner. We delineate a new negative regulatory loop of IL-4–JAK-STAT6 signaling. We demonstrate that IL-4 induces PTP1B mRNA expression in a phosphatidylinositol 3-kinase–dependent manner and enhances PTP1B protein stability to suppress IL-4–induced STAT6 signaling. Finally, we show that PTP1B expression may be preferentially elevated in activated B cell–like diffuse large B-cell lymphomas. These observations identify a novel regulatory loop for the regulation of IL-4–induced STAT6 signaling that may have important implications in both neoplastic and inflammatory processes. PMID:18716132

Measles virus C protein suppresses gamma-activated factor formation and virus-induced cell growth arrest

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yokota, Shin-ichi; Okabayashi, Tamaki; Fujii, Nobuhiro, E-mail: fujii@sapmed.ac.j

2011-05-25

Measles virus (MeV) produces two accessory proteins, V and C, from the P gene. These accessory proteins have been reported to contribute to efficient virus proliferation through the modulation of host cell events. Our previous paper described that Vero cell-adapted strains of MeV led host cells to growth arrest through the upregulation of interferon regulatory factor 1 (IRF-1), and wild strains did not. In the present study, we found that C protein expression levels varied among MeV strains in infected SiHa cells. C protein levels were inversely correlated with IRF-1 expression levels and with cell growth arrest. Forced expression ofmore » C protein released cells from growth arrest. C-deficient recombinant virus efficiently upregulated IRF-1 and caused growth arrest more efficiently than the wild-type virus. C protein preferentially bound to phosphorylated STAT1 and suppressed STAT1 dimer formation. We conclude that MeV C protein suppresses IFN-{gamma} signaling pathway via inhibition of phosphorylated STAT1 dimerization.« less

Expression of JAKs/STATs pathway molecules in rat model of rapid focal segmental glomerulosclerosis.

PubMed

Liang, Yaojun; Jin, Yu; Li, Yuning

2009-09-01

The objective of this study was to investigate the role of the Janus kinase-signal transducers and activators of transcription (JAKs/STATs) pathway in focal segmental glomerulosclerosis. Sixty specific pathogen-free male Wistar rats were randomly divided into two groups: a model group (MG) and a control group (CG). In the MG group, nephropathy was induced by unilateral nephrectomy and a single tail vein injection of adriamycin (5 mg/kg). Ten rats were sacrificed every 2 weeks in each group. The expressions of smooth muscle alpha actin (alpha-SMA), collagen (COL)-IV, STAT1, and STAT3 were examined using histochemical techniques, and Western blotting was used to examine the protein levels of STAT1, STAT3, phosphorylated (P)-STAT1, P-STAT3, and transforming growth factor beta1 (TGFbeta(1)). The expressions of JAK1, JAK2, STAT1, STAT3, suppressors of cytokine signaling (SOCS)1, SOCS3, protein inhibitors of activated STAT (PIAS)1, and PIAS3 were also measured by real-time quantitative reverse transcriptase-PCR. A steady and significant increase in the expressions of alpha-SMA, COL-IV and TGFbeta(1) were observed in MG rats over the whole experimental course. Increased STAT1 and P-STAT1 levels in MG rats were observed by week 6, whereas increased levels of STAT3 and P-STAT3 were noted by week 2. At the mRNA levels, JAK1, STAT1, and PIAS1 were significantly increased in MG rats in week 2, whereas JAK2 mRNA showed a significant decrease by weeks 2 and 4, followed by an significant increase in week 6. Significantly increased STAT3 levels were noted in week 2, followed by a steady and significant decrease in weeks 4 and 6. Significantly reduced levels of SOCS1, SOCS3, and PIAS3 mRNA were noted at all time points. We conclude that the JAKs/STATs signaling pathway may play an important role in the pathological process of rapid focal segmental glomerulosclerosis in the rat model.

STAT6 is amplified in a subset of dedifferentiated liposarcoma.

PubMed

Doyle, Leona A; Tao, Derrick; Mariño-Enríquez, Adrián

2014-09-01

A recurrent intrachromosomal rearrangement on chromosome 12q in solitary fibrous tumor leads to the formation of a NAB2-STAT6 fusion oncogene. As a result, nuclear expression of the cytoplasmic transcription factor STAT6 is found in solitary fibrous tumor and serves as a useful diagnostic marker. STAT6 is located in 12q13, a region containing well-characterized oncogenes that are commonly amplified in dedifferentiated liposarcoma; we have previously reported that STAT6 is expressed in a subset of dedifferentiated liposarcoma. The aim of this study was to determine the frequency of STAT6 expression in dedifferentiated liposarcoma and the underlying genetic mechanism. STAT6 protein expression was analyzed by immunohistochemistry in a well-characterized series of 35 previously unpublished cases of dedifferentiated liposarcoma, all with nuclear MDM2 and/or CDK4 expression by immunohistochemistry and/or cytogenetic features of dedifferentiated liposarcoma. FISH for STAT6 was performed in all cases with STAT6 expression, and a subset of control cases without STAT6 expression. In total 4/35 cases (11%) showed STAT6 expression (three with multifocal staining of moderate to strong intensity and one with weak focal staining). FISH demonstrated amplification of STAT6 in all cases positive for STAT6 by immunohistochemistry; in contrast, FISH performed on four STAT6-negative dedifferentiated liposarcomas demonstrated no STAT6 amplification (P=0.0286). Of the four STAT6 amplified cases, three patients were male and one was female, ranging in age from 51 to 76 years. Tumors were located in the mediastinum (n=2), paratesticular soft tissue (n=1), and perirenal soft tissue (n=1). Three patients received pre-operative chemotherapy +/- radiation therapy. In conclusion, STAT6 is amplified in a subset of dedifferentiated liposarcoma, resulting in STAT6 protein expression that can be detected by immunohistochemistry and may be a potential pitfall in the differential diagnosis of dedifferentiated liposarcoma and solitary fibrous tumor. These findings suggest a role for STAT6-mediated transcriptional activity in some cases of dedifferentiated liposarcoma and highlight the genomic complexity and heterogeneity of dedifferentiated liposarcoma.

Deficiency of activated STAT1 in head and neck cancer cells mediates TAP1-dependent escape from cytotoxic T lymphocytes

PubMed Central

Leibowitz, Michael S.; Filho, Pedro A. Andrade; Ferrone, Soldano; Ferris, Robert L.

2012-01-01

Squamous cell carcinoma of the head and neck (SCCHN) cells can escape recognition by tumor antigen (TA)-specific cytotoxic T lymphocytes (CTL) by downregulation of antigen processing machinery (APM) components, such as the transporter associated with antigen processing (TAP)-1/2 heterodimer. APM component upregulation by interferon gamma (IFN-γ) restores SCCHN cell recognition and susceptibility to lysis by CTL, but the mechanism underlying TAP1/2 downregulation in SCCHN cells is not known. Because IFN-γ activates signal transducer and activator of transcription (STAT)-1, we investigated phosphorylated (p)-STAT1 as a mediator of low basal TAP1/2 expression in SCCHN cells. SCCHN cells were found to express basal total STAT1 but low to undetectable levels of activated STAT1. The association of increased pSTAT1 levels and APM components likely reflects a cause–effect relationship, since STAT1 knockdown significantly reduced both IFN-γ-mediated APM component expression and TA-specific CTL recognition of IFN-γ-treated SCCHN cells. On the other hand, since oncogenic pSTAT3 is overexpressed in SCCHN cells and was found to heterodimerize with pSTAT1, we also tested whether pSTAT3 and pSTAT1:pSTAT3 heterodimers inhibited IFN-γ-induced STAT1 activation and APM component expression. First, STAT3 activation or depletion did not affect basal or IFN-γ-induced expression of pSTAT1 and APM components or recognition of SCCHN cells by TA-specific CTL. Second, pSTAT1:pSTAT3 heterodimers did not interfere with IFN-γ-induced STAT1 binding to the TAP1 promoter or APM protein expression. These findings demonstrate that APM component downregulation is regulated primarily by an IFN-γ-pSTAT1-mediated signaling pathway, independent of oncogenic STAT3 overexpression in SCCHN cells. PMID:21207025

Mumps Virus V Protein Antagonizes Interferon without the Complete Degradation of STAT1

PubMed Central

Kubota, Toru; Yokosawa, Noriko; Yokota, Shin-ichi; Fujii, Nobuhiro; Tashiro, Masato; Kato, Atsushi

2005-01-01

Mumps virus (MuV) has been shown to antagonize the antiviral effects of interferon (IFN) through proteasome-mediated complete degradation of STAT1 by using the viral V protein (T. Kubota et al., Biochem. Biophys. Res. Commun. 283:255-259, 2001). However, we found that MuV could inhibit IFN signaling and the generation of a subsequent antiviral state long before the complete degradation of cellular STAT1 in infected cells. In MuV-infected cells, nuclear translocation and phosphorylation of STAT1 and STAT2 tyrosine residue (Y) at 701 and 689, respectively, by IFN-β were significantly inhibited but the phosphorylation of Jak1 and Tyk2 was not inhibited. The transiently expressed MuV V protein also inhibited IFN-β-induced Y701-STAT1 and Y689-STAT2 phosphorylation, suggesting that the V protein could block IFN-β-induced signal transduction without the aid of other viral components. Finally, a substitution of an alanine residue in place of a cysteine residue in the C-terminal V-unique region known to be required for STAT1 degradation and inhibition of anti-IFN signaling resulted in the loss of V protein function to inhibit the Y701-STAT1 and Y689-STAT2 phosphorylation. PMID:15767445

A Bioinformatics Approach Identifies Signal Transducer and Activator of Transcription-3 and Checkpoint Kinase 1 as Upstream Regulators of Kidney Injury Molecule-1 after Kidney Injury

PubMed Central

Ajay, Amrendra Kumar; Kim, Tae-Min; Ramirez-Gonzalez, Victoria; Park, Peter J.; Frank, David A.

2014-01-01

Kidney injury molecule-1 (KIM-1)/T cell Ig and mucin domain-containing protein-1 (TIM-1) is upregulated more than other proteins after AKI, and it is highly expressed in renal damage of various etiologies. In this capacity, KIM-1/TIM-1 acts as a phosphatidylserine receptor on the surface of injured proximal tubular epithelial cells, mediating phagocytosis of apoptotic cells, and it may also act as a costimulatory molecule for immune cells. Despite recognition of KIM-1 as an important therapeutic target for kidney disease, the regulators of KIM-1 transcription in the kidney remain unknown. Using a bioinformatics approach, we identified upstream regulators of KIM-1 after AKI. In response to tubular injury in rat and human kidneys or oxidant stress in human proximal tubular epithelial cells (HPTECs), KIM-1 expression increased significantly in a manner that corresponded temporally and regionally with increased phosphorylation of checkpoint kinase 1 (Chk1) and STAT3. Both ischemic and oxidant stress resulted in a dramatic increase in reactive oxygen species that phosphorylated and activated Chk1, which subsequently bound to STAT3, phosphorylating it at S727. Furthermore, STAT3 bound to the KIM-1 promoter after ischemic and oxidant stress, and pharmacological or genetic induction of STAT3 in HPTECs increased KIM-1 mRNA and protein levels. Conversely, inhibition of STAT3 using siRNAs or dominant negative mutants reduced KIM-1 expression in a kidney cancer cell line (769-P) that expresses high basal levels of KIM-1. These observations highlight Chk1 and STAT3 as critical upstream regulators of KIM-1 expression after AKI and may suggest novel approaches for therapeutic intervention. PMID:24158981

APPL1-mediated activation of STAT3 contributes to inhibitory effect of adiponectin on hepatic gluconeogenesis.

PubMed

Ding, Youming; Zhang, Deling; Wang, Bin; Zhang, Yemin; Wang, Lei; Chen, Xiaoyan; Li, Mingxin; Tang, Zhao; Wang, Changhua

2016-09-15

Adiponectin has been shown to suppress hepatic gluconeogenesis. However, the signaling pathways underlying its action remain ill-defined. The purpose of this study was to examine the potential role of APPL1 in mediating anti-gluconeogenic ability of adiponectin. Primary hepatocytes were isolated from male C57BL/6 mice. Western blot and RT-PCR were performed to detect protein expression and mRNA level, respectively. The protein-protein association was determined by immunoprecipitation and GST pull-down assay. We found that APPL1 protein levels were negatively associated with expressions of proteins and mRNAs of gluconeogenesis enzymes under stimulation with adiponectin. In addition, adiponectin-stimulated STAT3 phosphorylation and acetylation were positively regulated by APPL1 and negative regulated by SirT1. Pharmacological and genetic inhibition of STAT3 mitigated impact of adiponectin on hepatic gluconeogenesis. Furthermore, adiponectin administration facilitated the binding of APPL1 to SirT1 and suppressed the association of SirT1 with STAT3. Taken together, our study showed that APPL1-SirT1-STAT3 pathway mediated adiponectin signaling in primary hepatocytes. This new finding provides a novel mechanism by which adiponectin suppresses hepatic gluconeogenesis. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

A soluble receptor for advanced glycation end-products inhibits myocardial apoptosis induced by ischemia/reperfusion via the JAK2/STAT3 pathway.

PubMed

Jiang, Xue; Guo, Cai-xia; Zeng, Xiang-jun; Li, Hui-hua; Chen, Bu-xing; Du, Feng-he

2015-08-01

sRAGE can protect cardiomyocytes from apoptosis induced by ischemia/reperfusion (I/R). However, the signaling mechanisms in cardioprotection by sRAGE are currently unknown. We investigated the cardioprotective effect and potential molecular mechanisms of sRAGE inhibition on apoptosis in the mouse myocardial I/R as an in vivo model and neonatal rat cardiomyocyte subjected to ischemic buffer as an in vitro model. Cardiac function and myocardial infarct size following by I/R were evaluated with echocardiography and Evans blue/2,3,5-triphenyltetrazolium chloride. Apoptosis was detected by TUNEL staining and caspase-3 activity. Expression of the apoptosis-related proteins p53, Bax, Bcl-2, JAK2/p-JAK2, STAT3/p-STAT3, AKT/p-AKT, ERK/p-ERK, STAT5A/p-STAT5A and STAT6/p-STAT6 were detected by western blot analysis in the presence and absence of the JAK2 inhibitor AG 490. sRAGE (100 µg/day) improved the heart function in mice with I/R: the left ventricular ejection fraction and fractional shortening were increased by 42 and 57%, respectively; the infarct size was decreased by 52%, the TUNEL-positive myocytes by 66%, and activity of caspase-3 by 24%, the protein expression of p53 and ratio of Bax to Bcl-2 by 29 and 88%, respectively; protein expression of the p-JAK2, p-STAT3 and p-AKT were increased by 92, 280 and 31%, respectively. sRAGE have no effect on protein expression of p-ERK1/2, p-STAT5A and p-STAT6 following by I/R. sRAGE (900 nmol/L) exhibited anti-apoptotic effects in cardiomyocytes by decreasing TUNEL-positive myocytes by 67% and caspase-3 activity by 20%, p53 protein level and the Bax/Bcl-2 ratio by 58 and 86%, respectively; increasing protein expression of the p-JAK2 and p-STAT3 by 26 and 156%, respectively, p-AKT protein level by 33%. The anti-apoptotic effects of sRAGE following I/R were blocked by JAK2 inhibitor AG 490. The effect of sRAGE reduction on TUNEL-positive myocytes and caspase-3 activity were abolished by PI3K inhibitor LY294002, but not ERK 1/2 inhibitor PD98059. These results suggest that sRAGE protects cardiomyocytes from apoptosis induced by I/R in vitro and in vivo by activating the JAK2/STAT3 signaling pathway.

Vaccinia Virus Blocks Stat1-Dependent and Stat1-Independent Gene Expression Induced by Type I and Type II Interferons

PubMed Central

Mann, Brandon A.; Huang, Julia He; Li, Ping; Chang, Hua-Chen; Slee, Roger B.; O'Sullivan, Audrey; Mathur, Anita; Yeh, Norman; Klemsz, Michael J.; Brutkiewicz, Randy R.; Blum, Janice S.

2008-01-01

Blocking the function of Stat (signal transducer and activator of transcription) proteins, which are critical for antiviral responses, has evolved as a common mechanism for pathogen immune evasion. The poxvirus-encoded phosphatase H1 is critical for viral replication, and may play an additional role in the evasion of host defense by dephosphorylating Stat1 and blocking interferon (IFN)-stimulated innate immune responses. Vaccinia virus (VACV) H1 can inhibit the phosphorylation of the transcription factor Stat1 after IFN-γ stimulation of epithelial cells, greatly attenuating IFN-induced biological functions. In this study, we demonstrate that VACV infection is capable of inhibiting the phosphorylation of Stat1 and Stat2 after stimulation of fibroblasts or bone marrow-derived macrophages with either type I or type II IFNs, but did not inhibit the activation of Stat3 or Stat5 in either cell type. By using recombinant proteins for in vitro assays, we observe that variola virus H1 is more active than VACV H1, although it has similar selectivity for Stat targets. Differential effects of VACV infection were observed on the induction of IFN-stimulated genes, with complete inhibition of some genes by VACV infection, while others were less affected. Despite the IFN-γ-induced expression of some genes in VACV-infected cells, IFN-γ was unable to rescue the VACV-mediated inhibition of MHC class II antigen presentation. Moreover, VACV infection can affect the IFN-induced expression of Stat1-dependent and Stat1-independent genes, suggesting that the virus may target additional IFN-activated pathways. Thus, VACV targets multiple signaling pathways in the evasion of antiviral immune responses. PMID:18593332

Suppression of STAT3 NH2 -terminal domain chemosensitizes medulloblastoma cells by activation of protein inhibitor of activated STAT3 via de-repression by microRNA-21.

PubMed

Ray, Sutapa; Coulter, Don W; Gray, Shawn D; Sughroue, Jason A; Roychoudhury, Shrabasti; McIntyre, Erin M; Chaturvedi, Nagendra K; Bhakat, Kishor K; Joshi, Shantaram S; McGuire, Timothy R; Sharp, John G

2018-04-01

Medulloblastoma (MB) is a malignant pediatric brain tumor with poor prognosis. Signal transducers and activators of transcription-3 (STAT3) is constitutively activated in MB where it functions as an oncoprotein, mediating cancer progression and metastasis. Here, we have delineated the functional role of activated STAT3 in MB, by using a cell permeable STAT3-NH 2 terminal domain inhibitor (S3-NTDi) that specifically perturbs the structure/function of STAT3. We have implemented several biochemical experiments using human MB tumor microarray (TMA) and pediatric MB cell lines, derived from high-risk SHH-TP53-mutated and MYC-amplified Non-WNT/SHH tumors. Treatment of MB cells with S3-NTDi leads to growth inhibition, cell cycle arrest, and apoptosis. S3-NTDi downregulated expression of STAT3 target genes, delayed migration of MB cells, attenuated epithelial-mesenchymal transition (EMT) marker expressions and reduced cancer stem-cell associated protein expressions in MB-spheres. To elucidate mechanisms, we showed that S3-NTDi induce expression of pro-apoptotic gene, C/EBP-homologous protein (CHOP), and decrease association of STAT3 to the proximal promoter of CCND1 and BCL2. Of note, S3-NTDi downregulated microRNA-21, which in turn, de-repressed Protein Inhibitor of Activated STAT3 (PIAS3), a negative regulator of STAT3 signaling pathway. Furthermore, combination therapy with S3-NTDi and cisplatin significantly decreased highly aggressive MYC-amplified MB cell growth and induced apoptosis by downregulating STAT3 regulated proliferation and anti-apoptotic gene expression. Together, our results revealed an important role of STAT3 in regulating MB pathogenesis. Disruption of this pathway with S3-NTDi, therefore, may serves as a promising candidate for targeted MB therapy by enhancing chemosensitivity of MB cells and potentially improving outcomes in high-risk patients. © 2017 Wiley Periodicals, Inc.

Stat1-independent regulation of gene expression in response to IFN-γ

PubMed Central

Ramana, Chilakamarti V.; Gil, M. Pilar; Han, Yulong; Ransohoff, Richard M.; Schreiber, Robert D.; Stark, George R.

2001-01-01

Although Stat1 is essential for cells to respond fully to IFN-γ, there is substantial evidence that, in the absence of Stat1, IFN-γ can still regulate the expression of some genes, induce an antiviral state and affect cell growth. We have now identified many genes that are regulated by IFN-γ in serum-starved Stat1-null mouse fibroblasts. The proteins induced by IFN-γ in Stat1-null cells can account for the substantial biological responses that remain. Some genes are induced in both wild-type and Stat1-null cells and thus are truly Stat1-independent. Others are subject to more complex regulation in response to IFN-γ, repressed by Stat1 in wild-type cells and activated in Stat1-null cells. Many genes induced by IFN-γ in Stat1-null fibroblasts also are induced by platelet-derived growth factor in wild-type cells and thus are likely to be involved in cell proliferation. In mouse cells expressing the docking site mutant Y440F of human IFN-γ receptor subunit 1, the mouse Stat1 is not phosphorylated in response to human IFN-γ, but c-myc and c-jun are still induced, showing that the Stat1 docking site is not required for Stat1-independent signaling. PMID:11390994

Natural and synthetic STAT3 inhibitors reduce hepcidin expression in differentiated mouse hepatocytes expressing the active phosphorylated STAT3 form.

PubMed

Fatih, Nadia; Camberlein, Emilie; Island, Marie Laure; Corlu, Anne; Abgueguen, Emmanuelle; Détivaud, Lénaïck; Leroyer, Patricia; Brissot, Pierre; Loréal, Olivier

2010-05-01

During the inflammatory process, hepcidin overexpression favours the development of anaemia of chronic diseases which represents the second most common form of anaemia worldwide. The identification of therapeutic agents decreasing hepcidin expression is therefore an important goal. The aim of this study was to target the STAT3 signalling involved in the development of increased hepcidin expression related to chronic inflammation. In a co-culture model associating mouse hepatocytes and rat liver epithelial cells, the mRNA levels of hepcidin1, albumin, aldolase B, Cyp3a4, Stat3, Smad4 and iron regulatory genes were measured by real-time PCR. STAT3 and phosphorylated SMAD1/5/8 proteins were analysed by Western blot. At variance of hepatocyte pure culture, co-culture provided high levels of hepcidin1 mRNA, reaching 400% of the freshly isolated hepatocyte values after 6 days of culture. Hepcidin expression was associated with the maintenance of hepatocyte phenotype, STAT3 phosphorylation and functional BMP/SMAD pathway. Stat3 siRNAs inhibited the hepcidin1 mRNA expression. STAT3 inhibitors, including curcumin, AG490 and a peptide (PpYLKTK), reduced hepcidin1 mRNA expression even when cells were additionally exposed to IL-6. Hepcidin1 mRNA was expressed at high levels by hepatocytes in the co-culture model, and STAT3 pathway activation was controlled through STAT3 inhibitors. Such inhibitors could be useful to prevent anaemia related to hepcidin overexpression during chronic inflammation.

Enhancement of ICAM-1 via the JAK2/STAT3 signaling pathway in a rat model of severe acute pancreatitis-associated lung injury

PubMed Central

HAN, XIAO; WANG, YUXI; CHEN, HAILONG; ZHANG, JINGWEN; XU, CAIMING; LI, JIAN; LI, MINGYUE

2016-01-01

Acute lung injury (ALI), which is associated with severe acute pancreatitis (SAP), results from damage to the pulmonary microvascular endothelial cells (PMVECs), which in turn leads to high levels of inflammatory cytokines that destroy PMVECs. However, the molecular mechanisms underlying SAP-associated ALI (SAP-ALI) are currently not well understood. Intercellular adhesion molecule-1 (ICAM-1) has been implicated in the persistent migration and accumulation of neutrophils and macrophages, which in turn has been associated with the increased permeability of microvascular endothelial cells. Signal transduction via the Janus kinase-2 (JAK2)/signal transducer and activator of transcription-3 (STAT3) transcription factors has been shown to be involved in inflammation. The present study aimed to investigate the expression levels of ICAM-1 and JAK2/STAT3 signaling components in a rat model of SAP-ALI. SAP was induced in the rat model, and dexamethasone (DEX) was administered to the treatment group. Subsequently, ICAM-1, interleukin (IL)-6, IL-8, tumor necrosis factor (TNF)-α, JAK2, STAT3 and nuclear factor (NF)-κB mRNA expression levels were determined using reverse transcription-polymerase chain reaction; ICAM-1 protein expression levels were determined using western blotting; and IL-6, IL-8 and TNF-α levels were measured via an enzyme-linked immunosorbent assay. In addition, an immunohistochemical analysis of ICAM-1, NF-κB, JAK2 and STAT3 was conducted, and the protein expression and cell morphology of the lungs in all rats was analyzed. ICAM-1 mRNA and protein expression levels were significantly increased following induction of SAP, and were significantly decreased in the DEX-treated group. Furthermore, treatment with DEX significantly reduced serum expression levels of IL-6, IL-8 and TNF-α and decreased expression levels of NF-κB, JAK2 and STAT3 in the lung tissue, as compared with the untreated SAP group. The present study demonstrated that DEX treatment was able to suppress ICAM-1 mRNA and protein expression in a rat model of SAP-ALI via the inhibition of IL-6 and TNF-α-induced JAK2/STAT3 activation; thus suggesting that DEX treatment may be considered a potential strategy in the treatment of patients with SAP-ALI. PMID:26997994

Characterization of STAT5B phosphorylation correlating with expression of cytokine-inducible SH2-containing protein (CIS).

PubMed

Cooper, John C; Boustead, Jared N; Yu, Chao-Lan

2006-06-01

Cytokine-inducible SH2-containing protein (CIS) is the first identified member of genes encoding for the suppressor of cytokine signaling (SOCS). CIS is also a well-known target gene of signal transducer and activator of transcription 5 (STAT5) pathways, providing normal negative feedback control of signaling by cytokines and growth factors. Three other SOCS genes, SOCS1, SOCS2, and SOCS3, can be silenced by DNA hypermethylation in human cancers, suggesting a potential mechanism for constitutive STAT activation. However, it is not known whether CIS expression is similarly perturbed in tumor cells. We report here the absence of CIS expression in T lymphoma LSTRA that overexpresses the Lck protein tyrosine kinase and exhibits elevated STAT5 activity. Pervanadate-induced CIS expression and STAT5 binding to the CIS promoter in vivo over a short time course implies that mechanisms other than DNA hypermethylation may contribute to defective CIS expression in LSTRA cells. Comparison with cytokine-dependent BaF3 cells stimulated with interleukin-3 (IL-3) further reveals that CIS induction correlates with specific STAT5b post-translational modifications. It exhibits as the slowest migrating form through SDS-polyacrylamide gel electrophoresis (SDS-PAGE) analysis. This distinctly modified STAT5b is the predominant form that binds to the consensus STAT5 sites in the CIS promoter and accumulates in the nucleus. In vitro phosphatase assays and phosphoamino acid analysis suggest the involvement of phosphorylation on residues other than the highly conserved tyrosine and serine sites in this distinct STAT5b mobility shift. All together, our results provide a novel link between incomplete STAT5b phosphorylation and defective SOCS gene expression in cancer cells.

IL-15 regulates Bcl-2 family members Bim and Mcl-1 through JAK/STAT and PI3K/AKT pathways in T cells.

PubMed

Shenoy, Aparna R; Kirschnek, Susanne; Häcker, Georg

2014-08-01

Maintenance of T cells is determined by their survival capacity, which is regulated by Bcl-2 proteins. Cytokines signalling through the common gamma chains such as IL-2, IL-7 and IL-15 are important for T-cell survival but how these cytokines determine the expression of Bcl-2-family proteins is not clear. We report signalling events of cytokines that regulate expression of two key Bcl-2 proteins, pro-apoptotic Bim and anti-apoptotic Mcl-1, in resting C57BL/6 mouse T cells. IL-2, IL-7 and IL-15 inhibited apoptosis but paradoxically induced the expression of Bim, countered by concomitant induction of Mcl-1. Bim induction by IL-15 was found at the mRNA and protein levels and depended on both JAK/STAT and PI3K signals. A new STAT5-binding site was identified in the Bim promoter, which was occupied by STAT5 upon IL-15 stimulation. Although it also depended on JAK/STAT- and PI3K signalling, Mcl-1 regulation was independent of Mcl-1 mRNA levels and of regulation of protein stability, suggesting translational regulation. Concurrent CD3 signals inhibited some of the IL-7 effect but not the IL-15 effect on Bcl-2 proteins. The data suggest that cytokines induce Bim and prime T cells for apoptosis, but also inhibit apoptosis by stabilising Mcl-1. Later downregulation of short-lived Mcl-1 may induce efficient, Bim-dependent apoptosis. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Expression of STATs and their inhibitors SOCS and PIAS in brain tumors. In vitro and in vivo study.

PubMed

Ehrmann, J; Strakova, N; Vrzalikova, K; Hezova, R; Kolar, Z

2008-01-01

Proteins of STAT family belongs to the transcription factors. Through their binding to the DNA specific sites and consequent regulation of transcription of various genes, these signaling proteins play an important role in many cell functions. Recent studies demonstrated persistent activation of STATs and loss of their natural inhibitors SOCS and PIAS in various human cancers. There is also evidence that experimental pharmacologic or genetic modulation of their function mignt by a new approach in anticancer treatment. The aim of this study was in vitro assesment and analysis of expression of STATs, SOCS and PIAS in glioblastoma cell lines undergoing treatment by PPARgamma agonists/antagonists because PPARgamma and STATs are tightly regulated by an overlapping set of nuclear regulatory proteins. We further analysed immunohistochemical expression of these proteins in vivo, with its correlation to grading in various brain tumors. The results of in vitro study showed decreased expression of phosphorylated form of STAT3 and increase of its inhibitors SOCS3 and PIAS3 in glioblastoma cell lines after treatment with IC50 of PPARgamma agonist ciglitazone. In vivo study failed to reveal changes in STAT3 and SOCS3 expression in either low and high grade astrocytomas, however we detect lower expression of STAT2 in low grade astrocytomas when comparing with high grade astrocytomas and lower expression of STAT3 in ependymomas when comparing with anaplastic ones. The results showed existing relationship between STAT and PPARgamma signaling in glial tumors and further suppport expected important role of STATs in regulation of growth and differentiation in these tumors.

Suppression of microRNA-135b-5p protects against myocardial ischemia/reperfusion injury by activating JAK2/STAT3 signaling pathway in mice during sevoflurane anesthesia.

PubMed

Xie, Xiao-Juan; Fan, Dong-Mei; Xi, Kai; Chen, Ya-Wei; Qi, Peng-Wei; Li, Qian-Hui; Fang, Liang; Ma, Li-Gang

2017-06-30

The study aims to explore the effects of miR-135b-5p on myocardial ischemia/reperfusion (I/R) injuries by regulating Janus protein tyrosine kinase 2 (JAK2)/signal transducer and activator of transcription (STAT) signaling pathway by mediating inhalation anesthesia with sevoflurane. A sum of 120 healthy Wistar male mice was assigned into six groups. Left ventricular ejection fraction (LVEF) and left ventricular shortening fraction (LVSF) were detected. Cardiomyocyte apoptosis was determined by terminal dexynucleotidyl transferase mediated dUTP-biotin nick end labeling (TUNEL) assay. MiR-135b-5p expression, mRNA and protein expression of p-STAT3, p-JAK2, STAT3, JAK2, B-cell lymphoma-2 (Bcl-2) and Bcl-2 associated X protein B (Bax) were detected by quantitative real-time PCR (qRT-PCR) and Western blotting. Target relationship between miR-135b-5p and JAK2 was confirmed by dual-luciferase reporter assay. The other five groups exhibited increased cardiomyocyte necrosis, apoptosis, miR-135b-5p and Bax expression, mRNA expression of JAK2 and STAT3, and protein expression of p-STAT3 and p-JAK2 compared with the sham group, but showed decreased LVEF, LVFS, and Bcl-2 expression. Compared with the model and AG490 + Sevo groups, the Sevo, inhibitor + Sevo and inhibitor + AG490 + Sevo groups displayed reduced cardiomyocyte necrosis, apoptosis, miR-135b-5p and Bax expression, but displayed elevated mRNA expression of JAK2 and STAT3, protein expression of p-STAT3 and p-JAK2, LVEF, LVFS and Bcl-2 expression. Compared with the Sevo and inhibitor + AG490 + Sevo groups, the AG490 + Sevo group showed decreased LVEF, LVFS, Bcl-2 expression, mRNA expressions of JAK2 and STAT3, and protein expressions of p-STAT3 and p-JAK2, but increased cardiomyocyte necrosis, apoptosis, and Bax expressions. MiR-135b-5p negatively targetted JAK2. Inhibition of miR-135b-5p can protect against myocardial I/R injury by activating JAK2/STAT3 signaling pathway through mediation of inhalation anesthesia with sevoflurane. © 2017 The Author(s).

Negative regulation of STAT3 protein-mediated cellular respiration by SIRT1 protein.

PubMed

Bernier, Michel; Paul, Rajib K; Martin-Montalvo, Alejandro; Scheibye-Knudsen, Morten; Song, Shaoming; He, Hua-Jun; Armour, Sean M; Hubbard, Basil P; Bohr, Vilhelm A; Wang, Lili; Zong, Yaping; Sinclair, David A; de Cabo, Rafael

2011-06-03

In mammals, the transcriptional activity of signal transducer and activator of transcription 3 (STAT3) is regulated by the deacetylase SIRT1. However, whether the newly described nongenomic actions of STAT3 toward mitochondrial oxidative phosphorylation are dependent on SIRT1 is unclear. In this study, Sirt1 gene knock-out murine embryonic fibroblast (MEF) cells were used to delineate the role of SIRT1 in the regulation of STAT3 mitochondrial function. Here, we show that STAT3 mRNA and protein levels and the accumulation of serine-phosphorylated STAT3 in mitochondria were increased significantly in Sirt1-KO cells as compared with wild-type MEFs. Various mitochondrial bioenergetic parameters, such as the oxygen consumption rate in cell cultures, enzyme activities of the electron transport chain complexes in isolated mitochondria, and production of ATP and lactate, indicated that Sirt1-KO cells exhibited higher mitochondrial respiration as compared with wild-type MEFs. Two independent approaches, including ectopic expression of SIRT1 and siRNA-mediated knockdown of STAT3, led to reduction in intracellular ATP levels and increased lactate production in Sirt1-KO cells that were approaching those of wild-type controls. Comparison of profiles of phospho-antibody array data indicated that the deletion of SirT1 was accompanied by constitutive activation of the pro-inflammatory NF-κB pathway, which is key for STAT3 induction and increased cellular respiration in Sirt1-KO cells. Thus, SIRT1 appears to be a functional regulator of NF-κB-dependent STAT3 expression that induces mitochondrial biogenesis. These results have implications for understanding the interplay between STAT3 and SIRT1 in pro-inflammatory conditions.

Negative Regulation of STAT3 Protein-mediated Cellular Respiration by SIRT1 Protein*

PubMed Central

Bernier, Michel; Paul, Rajib K.; Martin-Montalvo, Alejandro; Scheibye-Knudsen, Morten; Song, Shaoming; He, Hua-Jun; Armour, Sean M.; Hubbard, Basil P.; Bohr, Vilhelm A.; Wang, Lili; Zong, Yaping; Sinclair, David A.; de Cabo, Rafael

2011-01-01

In mammals, the transcriptional activity of signal transducer and activator of transcription 3 (STAT3) is regulated by the deacetylase SIRT1. However, whether the newly described nongenomic actions of STAT3 toward mitochondrial oxidative phosphorylation are dependent on SIRT1 is unclear. In this study, Sirt1 gene knock-out murine embryonic fibroblast (MEF) cells were used to delineate the role of SIRT1 in the regulation of STAT3 mitochondrial function. Here, we show that STAT3 mRNA and protein levels and the accumulation of serine-phosphorylated STAT3 in mitochondria were increased significantly in Sirt1-KO cells as compared with wild-type MEFs. Various mitochondrial bioenergetic parameters, such as the oxygen consumption rate in cell cultures, enzyme activities of the electron transport chain complexes in isolated mitochondria, and production of ATP and lactate, indicated that Sirt1-KO cells exhibited higher mitochondrial respiration as compared with wild-type MEFs. Two independent approaches, including ectopic expression of SIRT1 and siRNA-mediated knockdown of STAT3, led to reduction in intracellular ATP levels and increased lactate production in Sirt1-KO cells that were approaching those of wild-type controls. Comparison of profiles of phospho-antibody array data indicated that the deletion of SirT1 was accompanied by constitutive activation of the pro-inflammatory NF-κB pathway, which is key for STAT3 induction and increased cellular respiration in Sirt1-KO cells. Thus, SIRT1 appears to be a functional regulator of NF-κB-dependent STAT3 expression that induces mitochondrial biogenesis. These results have implications for understanding the interplay between STAT3 and SIRT1 in pro-inflammatory conditions. PMID:21467030

Macrophages of multiple sclerosis patients display deficient SHP-1 expression and enhanced inflammatory phenotype.

PubMed

Christophi, George P; Panos, Michael; Hudson, Chad A; Christophi, Rebecca L; Gruber, Ross C; Mersich, Akos T; Blystone, Scott D; Jubelt, Burk; Massa, Paul T

2009-07-01

Recent studies in mice have demonstrated that the protein tyrosine phosphatase SHP-1 is a crucial negative regulator of proinflammatory cytokine signaling, TLR signaling, and inflammatory gene expression. Furthermore, mice genetically lacking SHP-1 (me/me) display a profound susceptibility to inflammatory CNS demyelination relative to wild-type mice. In particular, SHP-1 deficiency may act predominantly in inflammatory macrophages to increase CNS demyelination as SHP-1-deficient macrophages display coexpression of inflammatory effector molecules and increased demyelinating activity in me/me mice. Recently, we reported that PBMCs of multiple sclerosis (MS) patients have a deficiency in SHP-1 expression relative to normal control subjects indicating that SHP-1 deficiency may play a similar role in MS as to that seen in mice. Therefore, it became essential to examine the specific expression and function of SHP-1 in macrophages from MS patients. Herein, we document that macrophages of MS patients have deficient SHP-1 protein and mRNA expression relative to those of normal control subjects. To examine functional consequences of the lower SHP-1, the activation of STAT6, STAT1, and NF-kappaB was quantified and macrophages of MS patients showed increased activation of these transcription factors. In accordance with this observation, several STAT6-, STAT1-, and NF-kappaB-responsive genes that mediate inflammatory demyelination were increased in macrophages of MS patients following cytokine and TLR agonist stimulation. Supporting a direct role of SHP-1 deficiency in altered macrophage function, experimental depletion of SHP-1 in normal subject macrophages resulted in an increased STAT/NF-kappaB activation and increased inflammatory gene expression to levels seen in macrophages of MS patients. In conclusion, macrophages of MS patients display a deficiency of SHP-1 expression, heightened activation of STAT6, STAT1, and NF-kappaB and a corresponding inflammatory profile that may be important in controlling macrophage-mediated demyelination in MS.

[Study on the correlation between EGFR-STAT3 signal pathway and laryngeal papilloma].

PubMed

Wang, Xinhua; Sun, Jingwu

2009-09-01

To explore the relationship between the expression of EGFR and STAT3 in human laryngeal papilloma and its biological behavior. Reverse transcription polymerase chain reaction(RT-PCR), immunohistochemical staining and Western blot were used to evaluate the mRNA and protein expression of EGFR and STAT3 (p-STAT3) in 42 laryngeal papilloma tissues and 15 samples of normal laryngeal tissue, and the relationship between the protein expression of them and clinic pathological parameters was also analyzed. The mRNA expression levels of EGFR and STAT3 in laryngeal papilloma tissue were significantly higher than that in normal laryngeal tissue (P < 0.05, P < 0.01). Protein positive expression of EGFR and p-STAT3 were also detected in a significantly greater proportion of laryngeal papilloma than normal laryngeal tissue by immunohistochemistry and western blot (P < 0.01, P < 0.05). There was relationship between EGFR and p-STAT3 overexpression in laryngeal papilloma (P < 0.05). The expression p-STAT3 was correlated with the recurrence and canceration of laryngeal papilloma (P < 0.05). The EGFR-STAT3 signal transduction pathway may be involved in the pathogenesis of laryngeal papilloma,, and the persistent activation of STAT3 gene plays an important role in the recurrence and canceration of laryngeal papilloma.

S100A6 regulates endothelial cell cycle progression by attenuating antiproliferative STAT1 signaling

PubMed Central

Lerchenmüller, Carolin; Heißenberg, Julian; Damilano, Federico; Bezzeridis, Vassilios J.; Krämer, Isabel; Bochaton-Piallat, Marie-Luce; Hirschberg, Kristóf; Busch, Martin; Katus, Hugo A.; Peppel, Karsten; Rosenzweig, Anthony; Busch, Hauke; Boerries, Melanie; Most, Patrick

2016-01-01

Objective S100A6, a member of the S100-protein family, has been described as relevant for cell cycle entry and progression in endothelial cells (ECs). The molecular mechanism conferring S100A6’s proliferative actions, however, remained elusive. Approach and Results Originating from the clinically relevant observation of enhanced S100A6 protein expression in proliferating ECs in remodeling coronary and carotid arteries, our study unveiled S100A6 as a suppressor of antiproliferative signal transducers and activators of transcription 1 (STAT1) signaling. Discovery of the molecular liaison was enabled by combining gene expression time series analysis with bioinformatic pathway modeling in S100A6 silenced human ECs stimulated with vascular endothelial growth factor A (VEGF-A). This unbiased approach led to successful identification and experimental validation of interferon-inducible transmembrane protein 1 (IFITM1) and protein inhibitors of activated STAT (PIAS) as key components of the link between S100A6 and STAT1. Conclusions Given the important role of coordinated EC cell cycle activity for integrity and reconstitution of the inner lining of arterial blood vessels in health and disease, STAT1 suppression by S100A6 may represent a promising therapeutic target to facilitate reendothelialization in damaged vessels. PMID:27386938

Increased EGFR expression induced by a novel oncogene, CUG2, confers resistance to doxorubicin through Stat1-HDAC4 signaling.

PubMed

Kaowinn, Sirichat; Jun, Seung Won; Kim, Chang Seok; Shin, Dong-Myeong; Hwang, Yoon-Hwae; Kim, Kyujung; Shin, Bosung; Kaewpiboon, Chutima; Jeong, Hyeon Hee; Koh, Sang Seok; Krämer, Oliver H; Johnston, Randal N; Chung, Young-Hwa

2017-12-01

Previously, it has been found that the cancer upregulated gene 2 (CUG2) and the epidermal growth factor receptor (EGFR) both contribute to drug resistance of cancer cells. Here, we explored whether CUG2 may exert its anticancer drug resistance by increasing the expression of EGFR. EGFR expression was assessed using Western blotting, immunofluorescence and capacitance assays in A549 lung cancer and immortalized bronchial BEAS-2B cells, respectively, stably transfected with a CUG2 expression vector (A549-CUG2; BEAS-CUG2) or an empty control vector (A549-Vec; BEAS-Vec). After siRNA-mediated EGFR, Stat1 and HDAC4 silencing, antioxidant and multidrug resistance protein and mRNA levels were assessed using Western blotting and RT-PCR. In addition, the respective cells were treated with doxorubicin after which apoptosis and reactive oxygen species (ROS) levels were measured. Stat1 acetylation was assessed by immunoprecipitation. We found that exogenous CUG2 overexpression induced EGFR upregulation in A549 and BEAS-2B cells, whereas EGFR silencing sensitized these cells to doxorubicin-induced apoptosis. In addition, we found that exogenous CUG2 overexpression reduced the formation of ROS during doxorubicin treatment by enhancing the expression of antioxidant and multidrug resistant proteins such as MnSOD, Foxo1, Foxo4, MRP2 and BCRP, whereas EGFR silencing congruently increased the levels of ROS by decreasing the expression of these proteins. We also found that EGFR silencing and its concomitant Akt, ERK, JNK and p38 MAPK inhibition resulted in a decreased Stat1 phosphorylation and, thus, a decreased activation. Since also acetylation can affect Stat1 activation via a phospho-acetyl switch, HDAC inhibition may sensitize cells to doxorubicin-induced apoptosis. Interestingly, we found that exogenous CUG2 overexpression upregulated HDAC4, but not HDAC2 or HDAC3. Conversely, we found that HDAC4 silencing sensitized the cells to doxorubicin resistance by decreasing Stat1 phosphorylation and EGFR expression, thus indicating an interplay between HDAC4, Stat1 and EGFR. Taken together, we conclude that CUG2-induced EGFR upregulation confers doxorubicin resistance to lung (cancer) cells through Stat1-HDAC4 signaling.

Enhanced phosphorylation of STAT1 is dependent on PKR signaling in HLA-B27 expressing U937 monocytic cells

PubMed Central

Ruuska, Marja; Sahlberg, Anna S.; Colbert, Robert A.; Granfors, Kaisa; Penttinen, Markus A.

2011-01-01

Objective To study the phosphorylation of STAT1 in HLA-B27-transfected human monocytic cells and the role of signaling molecules PKR and p38 in STAT1 phosphorylation. Methods U937 human monocytic cell transfectants stably expressing wild type HLA-B27 or mutated HLA-B27 heavy chains (HC) with amino acid substitutions in the B pocket were prepared. Mock transfected cells were prepared using the antibiotic resistance vectors (pSV2neo or RSV5neo) alone. PMA differentiated cells were stimulated with LPS or infected with S. enteritidis. Western blotting and flow cytometry were used to detect the phosphorylation and expression levels of STAT1 protein. Specific inhibitors were added in cell culture to study the role of PKR and p38 on STAT1 phosphorylation. Results STAT1 is constitutively highly phosphorylated on tyrosine 701 residue in HLA-B27 positive monocytic cells when compared to control cells, even prior to stimulation with LPS or bacteria. This phenotype is associated with the expression of HLA-B27 HCs that misfold. In addition, phosphorylation of STAT1 is dependent on PKR. Conclusion Our results show that STAT1 tyrosine 701 is constitutively highly phosphorylated in HLA-B27 expressing monocyte-macrophage cell line. Since phosphorylation of tyrosine 701 on STAT1 is sufficient to induce interferon-dependent genes, constitutive activity of this phosphorylation site may lead to overexpression of interferon-dependent genes, as well as other STAT1-dependent genes, in HLA-B27 monocyte-macrophages. Our results offer a mechanism by which B27 expression alone, without any external trigger, is potentially capable of inducing activation of STAT1, a critical regulator of the inflammatory response. PMID:21968657

Identification of Novel STAT6-Regulated Proteins in Mouse B Cells by Comparative Transcriptome and Proteome Analysis.

PubMed

Mokada-Gopal, Lavanya; Boeser, Alexander; Lehmann, Christian H K; Drepper, Friedel; Dudziak, Diana; Warscheid, Bettina; Voehringer, David

2017-05-01

The transcription factor STAT6 plays a key role in mediating signaling downstream of the receptors for IL-4 and IL-13. In B cells, STAT6 is required for class switch recombination to IgE and for germinal center formation during type 2 immune responses directed against allergens or helminths. In this study, we compared the transcriptomes and proteomes of primary mouse B cells from wild-type and STAT6-deficient mice cultured for 4 d in the presence or absence of IL-4. Microarray analysis revealed that 214 mRNAs were upregulated and 149 were downregulated >3-fold by IL-4 in a STAT6-dependent manner. Across all samples, ∼5000 proteins were identified by label-free quantitative liquid chromatography/mass spectrometry. A total of 149 proteins was found to be differentially expressed >3-fold between IL-4-stimulated wild-type and STAT6 -/- B cells (75 upregulated and 74 downregulated). Comparative analysis of the proteome and transcriptome revealed that expression of these proteins was mainly regulated at the transcriptional level, which argues against a major role for posttranscriptional mechanisms that modulate the STAT6-dependent proteome. Nine proteins were selected for confirmation by flow cytometry or Western blot. We show that CD30, CD79b, SLP-76, DEC205, IL-5Rα, STAT5, and Thy1 are induced by IL-4 in a STAT6-dependent manner. In contrast, Syk and Fc receptor-like 1 were downregulated. This dataset provides a framework for further functional analysis of newly identified IL-4-regulated proteins in B cells that may contribute to germinal center formation and IgE switching in type 2 immunity. Copyright © 2017 by The American Association of Immunologists, Inc.

Opposite nuclear level and binding activity of STAT5B and STAT3 proteins with rat haptoglobin gene under normal and turpentine induced acute phase conditions.

PubMed

Grigorov, I; Lazić, T; Cvetković, I; Milosavljević, T; Petrović, M

2001-01-01

Transcription of the rat gene encoding haptoglobin (Hp) is highly induced during acute phase (AP) response which has been previously shown to be mediated by inducible STAT3 member of the Signal Transducer and Activators of Transcription (STATs) family proteins. In this study, we observed that under normal but not in the turpentine induced AP conditions, another member of the STAT family proteins, STAT5b is expressed and binds to the hormone regulatory element (HRE) of the rat Hp gene. We found that the nuclear amounts of constitutively active STAT5b in rat liver decreased significantly with time of turpentine treatment as opposed to that of cytosol STAT5b, suggesting possible export of constitutive STAT5b from the nucleus. Nuclear accumulation and binding of inducible STAT3 proteins to the rat Hp gene HRE following turpentine treatment implicated that STAT5b negatively regulates Hp gene expression during normal conditions.

Stat6 activity-related Th2 cytokine profile and tumor growth advantage of human colorectal cancer cells in vitro and in vivo.

PubMed

Li, Ben Hui; Xu, Shuang Bing; Li, Feng; Zou, Xiao Guang; Saimaiti, Abudukeyoumu; Simayi, Dilixia; Wang, Ying Hong; Zhang, Yan; Yuan, Jia; Zhang, Wen Jie

2012-03-01

Signal transducer and activator of transcription 6 (Stat6) is critical in Th2 polarization of immune cells and active Stat6 activity has been suggested in anti-tumor immunity in animal models. The present study aims at investigating the impact of natural Stat6 activity on tumor microenvironment in human colorectal cancer cells in vitro and in vivo. Using colorectal cancer cell lines HT-29 and Caco-2 whose IL-4/Stat6 activities were known and nude mice as a model, we examined correlative relationships between Stat6 activities and gene expression profiles together with cellular behaviors in vitro and in vivo. HT-29 cells carrying active Stat6 signaling displayed spontaneous expression profiles favoring Th2 cytokines, cell cycle promotion, anti-apoptosis and pro-metastasis with increased mRNA levels of IL-4, IL-13, GATA-3, CDK4, CD44v6 and S100A4 using RT-PCR. In contrast, Caco-2 cells carrying defective Stat6 signaling exhibited spontaneous expression profiles favoring Th1 and Th17 cytokines, cell cycle inhibition, pro-apoptosis and anti-metastasis with elevated mRNA expression of IFNγ, TNFα, IL-12A, IL-17, IL-23, T-bet, CDKN1A, CDKNIB, CDKN2A and NM23-H1. Xenograft tumors of Stat6-active HT-29 cells showed a growth advantage over those of Stat6-defective Caco-2 cells. Furthermore, mice bearing HT-29 tumors expressed increased levels of Th2 cytokines IL-4 and IL-5 in the blood and pro-growth and/or pro-metastasis proteins CDK4 and CD44v6 in the tumor. To the contrary, mice bearing Caco-2 tumors expressed heightened levels of Th1 cytokines IFNγ and TNF in the blood and pro-apoptosis and anti-metastatic proteins p53 and p27(kip1) in the tumor. Colorectal cancer cells carrying active Stat6 signaling may create a microenvironment favoring Th2 cytokines and promoting expression of genes related to pro-growth, pro-metastasis and anti-apoptosis, which leads to a tumor growth advantage in vivo. These findings may imply why Stat6 pathway is constitutively activated in a number of human malignancies. Copyright © 2011 Elsevier Inc. All rights reserved.

p38 Mitogen-Activated Protein Kinase/Signal Transducer and Activator of Transcription-3 Pathway Signaling Regulates Expression of Inhibitory Molecules in T Cells Activated by HIV-1–Exposed Dendritic Cells

PubMed Central

Che, Karlhans Fru; Shankar, Esaki Muthu; Muthu, Sundaram; Zandi, Sasan; Sigvardsson, Mikael; Hinkula, Jorma; Messmer, Davorka; Larsson, Marie

2012-01-01

Human immunodeficiency virus type 1 (HIV-1) infection enhances the expression of inhibitory molecules on T cells, leading to T-cell impairment. The signaling pathways underlying the regulation of inhibitory molecules and subsequent onset of T-cell impairment remain elusive. We showed that both autologous and allogeneic T cells exposed to HIV-pulsed dendritic cells (DCs) upregulated cytotoxic T-lymphocyte antigen (CTLA-4), tumor-necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), lymphocyte-activation gene-3 (LAG3), T-cell immunoglobulin mucin-3 (TIM-3), CD160 and certain suppression-associated transcription factors, such as B-lymphocyte induced maturation protein-1 (BLIMP-1), deltex homolog 1 protein (DTX1) and forkhead box P3 (FOXP3), leading to T-cell suppression. This induction was regulated by p38 mitogen-activated protein kinase/signal transducer and activator of transcription-3 (P38MAPK/STAT3) pathways, because their blockade significantly abrogated expression of all the inhibitory molecules studied and a subsequent recovery in T-cell proliferation. Neither interleukin-6 (IL-6) nor IL-10 nor growth factors known to activate STAT3 signaling events were responsible for STAT3 activation. Involvement of the P38MAPK/STAT3 pathways was evident because these proteins had a higher level of phosphorylation in the HIV-1–primed cells. Furthermore, blockade of viral CD4 binding and fusion significantly reduced the negative effects DCs imposed on primed T cells. In conclusion, HIV-1 interaction with DCs modulated their functionality, causing them to trigger the activation of the P38MAPK/STAT3 pathway in T cells, which was responsible for the upregulation of inhibitory molecules. PMID:22777388

Requirement for STAT1 in LPS-induced gene expression in macrophages.

PubMed

Ohmori, Y; Hamilton, T A

2001-04-01

This study examines the role of the signal transducer and activator of transcription 1 (STAT1) in induction of lipopolysaccharide (LPS)-stimulated gene expression both in vitro and in vivo. LPS-induced expression of an interferon (IFN)-inducible 10-kDa protein (IP-10), IFN regulatory factor-1 (IRF-1), and inducible nitric oxide synthase (iNOS) mRNAs was severely impaired in macrophages prepared from Stat1-/- mice, whereas levels of tumor necrosis factor alpha and KC (a C-X-C chemokine) mRNA in LPS-treated cell cultures were unaffected. A similar deficiency in LPS-induced gene expression was observed in livers and spleens from Stat1-/- mice. The reduced LPS-stimulated gene expression seen in Stat1-/- macrophages was not the result of reduced activation of nuclear factor kappaB. LPS stimulated the delayed activation of both IFN-stimulated response element and IFN-gamma-activated sequence binding activity in macrophages from wild-type mice. Activation of these STAT1-containing transcription factors was mediated by the intermediate induction of type I IFNs, since the LPS-induced IP-10, IRF-1, and iNOS mRNA expression was markedly reduced in macrophages from IFN-alpha/betaR-/- mice and blocked by cotreatment with antibodies against type I IFN. These results indicate that indirect activation of STAT1 by LPS-induced type I IFN participates in promoting optimal expression of LPS-inducible genes, and they suggest that STAT1 may play a critical role in innate immunity against gram-negative bacterial infection.

Nuclear envelope proteins Nesprin2 and LaminA regulate proliferation and apoptosis of vascular endothelial cells in response to shear stress.

PubMed

Han, Yue; Wang, Lu; Yao, Qing-Ping; Zhang, Ping; Liu, Bo; Wang, Guo-Liang; Shen, Bao-Rong; Cheng, Binbin; Wang, Yingxiao; Jiang, Zong-Lai; Qi, Ying-Xin

2015-05-01

The dysfunction of vascular endothelial cells (ECs) influenced by flow shear stress is crucial for vascular remodeling. However, the roles of nuclear envelope (NE) proteins in shear stress-induced EC dysfunction are still unknown. Our results indicated that, compared with normal shear stress (NSS), low shear stress (LowSS) suppressed the expression of two types of NE proteins, Nesprin2 and LaminA, and increased the proliferation and apoptosis of ECs. Targeted small interfering RNA (siRNA) and gene overexpression plasmid transfection revealed that Nesprin2 and LaminA participate in the regulation of EC proliferation and apoptosis. A protein/DNA array was further used to detect the activation of transcription factors in ECs following transfection with target siRNAs and overexpression plasmids. The regulation of AP-2 and TFIID mediated by Nesprin2 and the activation of Stat-1, Stat-3, Stat-5 and Stat-6 by LaminA were verified under shear stress. Furthermore, using Ingenuity Pathway Analysis software and real-time RT-PCR, the effects of Nesprin2 or LaminA on the downstream target genes of AP-2, TFIID, and Stat-1, Stat-3, Stat-5 and Stat-6, respectively, were investigated under LowSS. Our study has revealed that NE proteins are novel mechano-sensitive molecules in ECs. LowSS suppresses the expression of Nesprin2 and LaminA, which may subsequently modulate the activation of important transcription factors and eventually lead to EC dysfunction. Copyright © 2015 Elsevier B.V. All rights reserved.

The Circular RNA Interacts with STAT3, Increasing Its Nuclear Translocation and Wound Repair by Modulating Dnmt3a and miR-17 Function.

PubMed

Yang, Zhen-Guo; Awan, Faryal Mehwish; Du, William W; Zeng, Yan; Lyu, Juanjuan; Wu, De; Gupta, Shaan; Yang, Weining; Yang, Burton B

2017-09-06

Delayed or impaired wound healing is a major health issue worldwide, especially in patients with diabetes and atherosclerosis. Here we show that expression of the circular RNA circ-Amotl1 accelerated healing process in a mouse excisional wound model. Further studies showed that ectopic circ-Amotl1 increased protein levels of Stat3 and Dnmt3a. The increased Dnmt3a then methylated the promoter of microRNA miR-17, decreasing miR-17-5p levels but increasing fibronectin expression. We found that Stat3, similar to Dnmt3a and fibronectin, was a target of miR-17-5p. Decreased miR-17-5p levels would increase expression of fibronectin, Dnmt3a, and Stat3. All of these led to increased cell adhesion, migration, proliferation, survival, and wound repair. Furthermore, we found that circ-Amotl1 not only increased Stat3 expression but also facilitated Stat3 nuclear translocation. Thus, the ectopic expressed circ-Amotl1 and Stat3 were mainly translocated to nucleus. In the presence of circ-Amotl1, Stat3 interacted with Dnmt3a promoter with increased affinity, facilitating Dnmt3a transcription. Ectopic application of circ-Amotl1 accelerating wound repair may shed light on skin wound healing clinically. Copyright © 2017. Published by Elsevier Inc.

Analysis of gene expression profile microarray data in complex regional pain syndrome.

PubMed

Tan, Wulin; Song, Yiyan; Mo, Chengqiang; Jiang, Shuangjian; Wang, Zhongxing

2017-09-01

The aim of the present study was to predict key genes and proteins associated with complex regional pain syndrome (CRPS) using bioinformatics analysis. The gene expression profiling microarray data, GSE47603, which included peripheral blood samples from 4 patients with CRPS and 5 healthy controls, was obtained from the Gene Expression Omnibus (GEO) database. The differentially expressed genes (DEGs) in CRPS patients compared with healthy controls were identified using the GEO2R online tool. Functional enrichment analysis was then performed using The Database for Annotation Visualization and Integrated Discovery online tool. Protein‑protein interaction (PPI) network analysis was subsequently performed using Search Tool for the Retrieval of Interaction Genes database and analyzed with Cytoscape software. A total of 257 DEGs were identified, including 243 upregulated genes and 14 downregulated ones. Genes in the human leukocyte antigen (HLA) family were most significantly differentially expressed. Enrichment analysis demonstrated that signaling pathways, including immune response, cell motion, adhesion and angiogenesis were associated with CRPS. PPI network analysis revealed that key genes, including early region 1A binding protein p300 (EP300), CREB‑binding protein (CREBBP), signal transducer and activator of transcription (STAT)3, STAT5A and integrin α M were associated with CRPS. The results suggest that the immune response may therefore serve an important role in CRPS development. In addition, genes in the HLA family, such as HLA‑DQB1 and HLA‑DRB1, may present potential biomarkers for the diagnosis of CRPS. Furthermore, EP300, its paralog CREBBP, and the STAT family genes, STAT3 and STAT5 may be important in the development of CRPS.

5-hydroxy-2-methyl-1,4-naphthoquinone, a vitamin K3 analogue, suppresses STAT3 activation pathway through induction of protein tyrosine phosphatase, SHP-1: potential role in chemosensitization.

PubMed

Sandur, Santosh K; Pandey, Manoj K; Sung, Bokyung; Aggarwal, Bharat B

2010-01-01

The activation of signal transducers and activators of transcription 3 (STAT3) has been linked with carcinogenesis through survival, proliferation, and angiogenesis of tumor cells. Agents that can suppress STAT3 activation have potential not only for prevention but also for treatment of cancer. In the present report, we investigated whether 5-hydroxy-2-methyl-1,4-naphthoquinone (plumbagin), an analogue of vitamin K, and isolated from chitrak (Plumbago zeylanica), an Ayurvedic medicinal plant, can modulate the STAT3 pathway. We found that plumbagin inhibited both constitutive and interleukin 6-inducible STAT3 phosphorylation in multiple myeloma (MM) cells and this correlated with the inhibition of c-Src, Janus-activated kinase (JAK)1, and JAK2 activation. Vanadate, however, reversed the plumbagin-induced downregulation of STAT3 activation, suggesting the involvement of a protein tyrosine phosphatase. Indeed, we found that plumbagin induced the expression of the protein tyrosine phosphatase, SHP-1, and silencing of the SHP-1 abolished the effect of plumbagin. This agent also downregulated the expression of STAT3-regulated cyclin D1, Bcl-xL, and vascular endothelial growth factor; activated caspase-3; induced poly (ADP ribose) polymerase cleavage; and increased the sub-G(1) population of MM cells. Consistent with these results, overexpression of constitutive active STAT3 significantly reduced the plumbagin-induced apoptosis. When compared with AG490, a rationally designed STAT3/JAK2 inhibitor, plumbagin was found more potent in suppressing the proliferation of cells. Plumbagin also significantly potentiated the apoptotic effects of thalidomide and bortezomib in MM cells. Overall, these results suggest that the plumbagin inhibits STAT3 activation pathway through the induction of SHP-1 and this may mediate the sensitization of STAT3 overexpressing cancers to chemotherapeutic agents.

Maternal gestational betaine supplementation-mediated suppression of hepatic cyclin D2 and presenilin1 gene in newborn piglets is associated with epigenetic regulation of the STAT3-dependent pathway.

PubMed

Cai, Demin; Yuan, Mengjie; Jia, Yimin; Liu, Haoyu; Hu, Yun; Zhao, Ruqian

2015-12-01

Betaine, which donates methyl groups through methionine metabolism for DNA and protein methylation, is critical for epigenetic gene regulation, especially during fetal development. Here we fed gestational sows with control or betaine supplemented diets (3 g/kg) throughout the pregnancy to explore the effects of maternal betaine on hepatic cell proliferation in neonatal piglets. Neonatal piglets born to betaine-supplemented sows demonstrated a reduction of cell number and DNA content in the liver, which was associated with significantly down-regulated hepatic expression of cell cycle regulatory genes, cyclin D2 (CCND2) and presenilin1 (PSEN1). Moreover, STAT3 binding to the promoter of CCND2 and PSEN1 was also lower in betaine-exposed piglets, accompanied by strong reduction of STAT3 mRNA and protein expression, along with its phosphorylation at Tyr705 and Ser727 residues. Also, prenatal betaine exposure significantly attenuated upstream kinases of STAT3 signaling pathway (phospho-ERK1/2, phospho-SRC and phospho-JAK2) in the livers of neonates. Furthermore, the repressed STAT3 expression in the liver of betaine-exposed piglets was associated with DNA hypermethylation and more enriched repression histone mark H3K27me3 on its promoter, together with significantly up-regulated expression of H3K27me3 and enhancer of zeste homolog 2 (EZH2) proteins, as well as miR-124a, which targets STAT3. Taken together, our results suggest that maternal dietary betaine supplementation during gestation inhibits hepatic cell proliferation in neonatal piglets, at least partly, through epigenetic regulation of hepatic CCND2 and PSEN1 genes via a STAT3-dependent pathway. These neonatal changes in cell cycle and proliferation regulation may lead to lower liver weight and hepatic DNA content at weaning. Copyright © 2015 Elsevier Inc. All rights reserved.

JAK-STAT signalling and the atrial fibrillation promoting fibrotic substrate

PubMed Central

Chen, Yu; Surinkaew, Sirirat; Naud, Patrice; Qi, Xiao-Yan; Gillis, Marc-Antoine; Shi, Yan-Fen; Tardif, Jean-Claude; Dobrev, Dobromir; Nattel, Stanley

2017-01-01

Aims Left-atrial (LA) fibrosis is an important feature of many atrial fibrillation (AF) substrates. The JAK-STAT system contributes to cardiac remodelling, but its role in AF is unknown. Here we investigated JAK-STAT changes in an AF-model and their potential contributions to LA-fibrosis. Methods and results LA-remodelling was studied in dogs with heart failure (HF) induced by ventricular tachypacing (VTP, 240 bpm), and in mice with left-ventricular (LV) dysfunction due to myocardial infarction (MI). The selective STAT-3 inhibitor S3I-201 was administered to fibroblasts in vitro or mice in vivo (10 mg/kg/d, osmotic mini-pump). HF-dogs developed LA-selective fibrosis and AF-susceptibility at 1-week VTP. The mRNA-expression of platelet-derived growth factor (PDGF, a JAK-STAT activator) isoforms A, C and D, as well as JAK2, increased in LA fibroblasts from 1-week VTP. HF upregulated protein-expression of PDGF-receptor-β and phosphorylated (activated) signal transducer and activator of transcription 3 (STAT3) in LA. PDGF-AB stimulation of LA fibroblasts increased PDGFR-α, STAT3 and phosphorylated-STAT3 expression, as well as collagen-1 and fibronectin-1 protein secretion (by 1.6- to 20-fold), with smaller changes in LV fibroblasts. Phosphorylated-STAT3 and collagen upregulation were suppressed by the JAK2 inhibitor AG-490, PDGF receptor inhibitor AG1296 and STAT3-inhibitor SI3-201. In vivo S3I-201 treatment of MI-mice attenuated LA-fibrosis, LA-dilation and P-wave duration changes versus vehicle-control. Conclusions HF activates the LA JAK-STAT system and enhances PDGF-signalling. JAK-STAT inhibition reduces the profibrotic effects of PDGF stimulation on canine fibroblasts in vitro while attenuating in vivo LA-fibrosis and remodelling in post-MI mice, suggesting that the JAK/STAT pathway contributes to LA-fibrogenesis and might be a potential target for LA-fibrosis prevention. PMID:28158495

SARS coronavirus papain-like protease up-regulates the collagen expression through non-Samd TGF-β1 signaling.

PubMed

Wang, Ching-Ying; Lu, Chien-Yi; Li, Shih-Wen; Lai, Chien-Chen; Hua, Chun-Hung; Huang, Su-Hua; Lin, Ying-Ju; Hour, Mann-Jen; Lin, Cheng-Wen

2017-05-02

SARS coronavirus (CoV) papain-like protease (PLpro) reportedly induced the production of TGF-β1 through p38 MAPK/STAT3-meidated Egr-1-dependent activation (Sci. Rep. 6, 25754). This study investigated the correlation of PLpro-induced TGF-β1 with the expression of Type I collagen in human lung epithelial cells and mouse pulmonary tissues. Specific inhibitors for TGF-βRI, p38 MAPK, MEK, and STAT3 proved that SARS-CoV PLpro induced TGF-β1-dependent up-regulation of Type I collagen in vitro and in vivo. Subcellular localization analysis of SMAD3 and SMAD7 indicated that non-SMAD pathways in TGF-β1 signaling involved in the production of Type I collagen in transfected cells with pSARS-PLpro. Comprehensive analysis of ubiquitin-conjugated proteins using immunoprecipitation and nanoLC-MS/MS indicated that SARS-CoV PLpro caused the change in the ubiquitination profile of Rho GTPase family proteins, in which linked with the increase of Rho-like GTPase family proteins. Moreover, selective inhibitors TGF-βRI and STAT6 (AS1517499) ascertained that STAT6 activation was required for PLpro-induced TGF-β1-dependent up-regulation of Type I collagen in human lung epithelial cells. The results showed that SARS-CoV PLpro stimulated TGF-β1-dependent expression of Type I collagen via activating STAT6 pathway. Copyright © 2017 Elsevier B.V. All rights reserved.

Role of STAT1 in Chlamydia-Induced Type-1 Interferon Production in Oviduct Epithelial Cells

PubMed Central

Hosey, Kristen Lynette; Hu, Sishun

2015-01-01

We previously reported that Chlamydia muridarum-infected murine oviduct epithelial cells (OE cells) secrete interferon β (IFN-β) in a mostly TLR3-dependent manner. However, C. muridarum-infected TLR3-deficient OE cells were still able to secrete detectable levels of IFN-β into the supernatants, suggesting that other signaling pathways contribute to Chlamydia-induced IFN-β synthesis in these cells. We investigated the role of STAT1 as a possible contributor in the Chlamydia-induced type-1 IFN production in wild-type (WT) and TLR3-deficient OE cells to ascertain its putative role at early- and late-times during Chlamydia infection. Our data show that C. muridarum infection significantly increased STAT1 gene expression and protein activation in WT OE cells; however, TLR3-deficient OE cells showed diminished STAT1 protein activation and gene expression. There was significantly less IFN-β detected in the supernatants of C. muridarum-infected OE cells derived from mice deficient in STAT1 when compared with WT OE cells, which suggest that STAT1 is required for the optimal synthesis of IFN-β during infection. Real-time quantitative polymerase chain reaction analyses of signaling components of the type-1 IFN signaling pathway demonstrated equal upregulation in the expression of STAT2 and IRF7 genes in the WT and TLR3-deficient OE cells, but no upregulation in these genes in the STAT1-deficient OE cells. Finally, experiments in which INFAR1 was blocked with neutralizing antibody revealed that IFNAR1-mediated signaling was critical to the Chlamydia-induced upregulation in IFN-α gene transcription, but had no role in the Chlamydia-induced upregulation in IFN-β gene transcription. PMID:26262558

Granulin, a novel STAT3-interacting protein, enhances STAT3 transcriptional function and correlates with poorer prognosis in breast cancer

PubMed Central

Yeh, Jennifer E.; Kreimer, Simion; Walker, Sarah R.; Emori, Megan M.; Krystal, Hannah; Richardson, Andrea; Ivanov, Alexander R.; Frank, David A.

2015-01-01

Since the neoplastic phenotype of a cell is largely driven by aberrant gene expression patterns, increasing attention has been focused on transcription factors that regulate critical mediators of tumorigenesis such as signal transducer and activator of transcription 3 (STAT3). As proteins that interact with STAT3 may be key in addressing how STAT3 contributes to cancer pathogenesis, we took a proteomics approach to identify novel STAT3-interacting proteins. We performed mass spectrometry-based profiling of STAT3-containing complexes from breast cancer cells that have constitutively active STAT3 and are dependent on STAT3 function for survival. We identified granulin (GRN) as a novel STAT3-interacting protein that was necessary for both constitutive and maximal leukemia inhibitory factor (LIF)induced STAT3 transcriptional activity. GRN enhanced STAT3 DNA binding and also increased the time-integrated amount of LIF-induced STAT3 activation in breast cancer cells. Furthermore, silencing GRN neutralized STAT3-mediated tumorigenic phenotypes including viability, clonogenesis, and migratory capacity. In primary breast cancer samples, GRN mRNA levels were positively correlated with STAT3 gene expression signatures and with reduced patient survival. These studies identify GRN as a functionally important STAT3-interacting protein that may serve as an important prognostic biomarker and potential therapeutic target in breast cancer. PMID:26000098

Direct Interaction of Jak1 and v-Abl Is Required for v-Abl-Induced Activation of STATs and Proliferation

PubMed Central

Danial, Nika N.; Losman, Julie A.; Lu, Tianhong; Yip, Natalie; Krishnan, Kartik; Krolewski, John; Goff, Stephen P.; Wang, Jean Y. J.; Rothman, Paul B.

1998-01-01

In Abelson murine leukemia virus (A-MuLV)-transformed cells, members of the Janus kinase (Jak) family of non-receptor tyrosine kinases and the signal transducers and activators of transcription (STAT) family of signaling proteins are constitutively activated. In these cells, the v-Abl oncoprotein and the Jak proteins physically associate. To define the molecular mechanism of constitutive Jak-STAT signaling in these cells, the functional significance of the v-Abl–Jak association was examined. Mapping the Jak1 interaction domain in v-Abl demonstrates that amino acids 858 to 1080 within the carboxyl-terminal region of v-Abl bind Jak1 through a direct interaction. A mutant of v-Abl lacking this region exhibits a significant defect in Jak1 binding in vivo, fails to activate Jak1 and STAT proteins, and does not support either the proliferation or the survival of BAF/3 cells in the absence of cytokine. Cells expressing this v-Abl mutant show extended latency and decreased frequency in generating tumors in nude mice. In addition, inducible expression of a kinase-inactive mutant of Jak1 protein inhibits the ability of v-Abl to activate STATs and to induce cytokine-independent proliferation, indicating that an active Jak1 is required for these v-Abl-induced signaling pathways in vivo. We propose that Jak1 is a mediator of v-Abl-induced STAT activation and v-Abl induced proliferation in BAF/3 cells, and may be important for efficient transformation of immature B cells by the v-abl oncogene. PMID:9774693

Regulation of PSMB5 Protein and β Subunits of Mammalian Proteasome by Constitutively Activated Signal Transducer and Activator of Transcription 3 (STAT3)

PubMed Central

Vangala, Janakiram Reddy; Dudem, Srikanth; Jain, Nishant; Kalivendi, Shasi V.

2014-01-01

The ubiquitin-proteasome system facilitates the degradation of ubiquitin-tagged proteins and performs a regulatory role in cells. Elevated proteasome activity and subunit expression are found in several cancers. However, the inherent molecular mechanisms responsible for increased proteasome function in cancers remain unclear despite the well investigated and defined role of the mammalian proteasome. This study was initiated to elucidate the mechanisms involved in the regulation of β subunits of the mammalian proteasome. Suppression of STAT3 tyrosine phosphorylation coordinately decreased the mRNA and protein levels of the β subunits of the 20 S core complex in DU145 cells. Notably, PSMB5, a molecular target of bortezomib, was shown to be a target of STAT3. Knockdown of STAT3 decreased PSMB5 protein. Inhibition of phospho-STAT3 substantially reduced PSMB5 protein levels in cells expressing constitutively active-STAT3. Accumulation of activated STAT3 resulted in the induction of PSMB5 promoter and protein levels. In addition, a direct correlation was observed between the endogenous levels of PSMB5 and constitutively active STAT3. PSMB5 and STAT3 protein levels remained unaltered following the inhibition of proteasome activity. The EGF-induced concerted increase of β subunits was blocked by inhibition of the EGF receptor or STAT3 but not by the PI3K/AKT or MEK/ERK pathways. Decreased proteasome activities were due to reduced protein levels of catalytic subunits of the proteasome in STAT3-inhibited cells. Combined treatments with bortezomib and inhibitor of STAT3 abrogated proteasome activity and enhanced cellular apoptosis. Overall, we demonstrate that aberrant activation of STAT3 regulates the expression of β subunits, in particular PSMB5, and the catalytic activity of the proteasome. PMID:24627483

STAT proteins: from normal control of cellular events to tumorigenesis.

PubMed

Calò, Valentina; Migliavacca, Manuela; Bazan, Viviana; Macaluso, Marcella; Buscemi, Maria; Gebbia, Nicola; Russo, Antonio

2003-11-01

Signal transducers and activators of transcription (STAT) proteins comprise a family of transcription factors latent in the cytoplasm that participate in normal cellular events, such as differentiation, proliferation, cell survival, apoptosis, and angiogenesis following cytokine, growth factor, and hormone signaling. STATs are activated by tyrosine phosphorylation, which is normally a transient and tightly regulates process. Nevertheless, several constitutively activated STATs have been observed in a wide number of human cancer cell lines and primary tumors, including blood malignancies and solid neoplasias. STATs can be divided into two groups according to their specific functions. One is made up of STAT2, STAT4, and STAT6, which are activated by a small number of cytokines and play a distinct role in the development of T-cells and in IFNgamma signaling. The other group includes STAT1, STAT3, and STAT5, activated in different tissues by means of a series of ligands and involved in IFN signaling, development of the mammary gland, response to GH, and embriogenesis. This latter group of STATS plays an important role in controlling cell-cycle progression and apoptosis and thus contributes to oncogenesis. Although an increased expression of STAT1 has been observed in many human neoplasias, this molecule can be considered a potential tumor suppressor, since it plays an important role in growth arrest and in promoting apoptosis. On the other hand, STAT3 and 5 are considered as oncogenes, since they bring about the activation of cyclin D1, c-Myc, and bcl-xl expression, and are involved in promoting cell-cycle progression, cellular transformation, and in preventing apoptosis.

Role for herpes simplex virus 1 ICP27 in the inhibition of type I interferon signaling

DOE Office of Scientific and Technical Information (OSTI.GOV)

Johnson, Karen E.; Song, Byeongwoon; Knipe, David M.

2008-05-10

Host cells respond to viral infection by many mechanisms, including the production of type I interferons which act in a paracrine and autocrine manner to induce the expression of antiviral interferon-stimulated genes (ISGs). Viruses have evolved means to inhibit interferon signaling to avoid induction of the innate immune response. Herpes simplex virus 1 (HSV-1) has several mechanisms to inhibit type I interferon production, the activities of ISGs, and the interferon signaling pathway itself. We report that the inhibition of the Jak/STAT pathway by HSV-1 requires viral gene expression and that viral immediate-early protein ICP27 plays a role in downregulating STAT-1more » phosphorylation and in preventing the accumulation of STAT-1 in the nucleus. We also show that expression of ICP27 by transfection causes an inhibition of IFN-induced STAT-1 nuclear accumulation. Therefore, ICP27 is necessary and sufficient for at least some of the effects of HSV infection on STAT-1.« less

Bortezomib inhibits STAT5-dependent degradation of LEF-1, inducing granulocytic differentiation in congenital neutropenia CD34+ cells

PubMed Central

Gupta, Kshama; Kuznetsova, Inna; Klimenkova, Olga; Klimiankou, Maksim; Meyer, Johann; Moore, Malcolm A. S.; Zeidler, Cornelia; Welte, Karl

2014-01-01

The transcription factor lymphoid enhancer–binding factor 1 (LEF-1), which plays a definitive role in granulocyte colony-stimulating factor (G-CSF) receptor-triggered granulopoiesis, is downregulated in granulocytic progenitors of severe congenital neutropenia (CN) patients. However, the exact mechanism of LEF-1 downregulation is unclear. CN patients are responsive to therapeutically high doses of G-CSF and are at increased risk of developing acute myeloid leukemia. The normal expression of LEF-1 in monocytes and lymphocytes, whose differentiation is unaffected in CN, suggests the presence of a granulopoiesis-specific mechanism downstream of G-CSF receptor signaling that leads to LEF-1 downregulation. Signal transducer and activator of transcription 5 (STAT5) is activated by G-CSF and is hyperactivated in acute myeloid leukemia. Here, we investigated the effects of activated STAT5 on LEF-1 expression and functions in hematopoietic progenitor cells. We demonstrated that constitutively active STAT5a (caSTAT5a) inhibited LEF-1–dependent autoregulation of the LEF-1 gene promoter by binding to the LEF-1 protein, recruiting Nemo-like kinase and the E3 ubiquitin-ligase NARF to LEF-1, leading to LEF-1 ubiquitination and a reduction in LEF-1 protein levels. The proteasome inhibitor bortezomib reversed the defective G-CSF–triggered granulocytic differentiation of CD34+ cells from CN patients in vitro, an effect that was accompanied by restoration of LEF-1 protein levels and LEF-1 messenger RNA autoregulation. Taken together, our data define a novel mechanism of LEF-1 downregulation in CN patients via enhanced ubiquitination and degradation of LEF-1 protein by hyperactivated STAT5. PMID:24394665

Target research on tumor biology characteristics of mir-155-5p regulation on gastric cancer cell.

PubMed

Feng, Jun-an

2016-03-01

After the mir-155-5p over expressed in gastric cancer cells, the expression profile chip was adopted to screen its target genes. Some of the intersection of target genes were selected based on the bioinformatics prediction, in order to study the mechanism of its function and role of research. Affymetrix eukaryotic gene expression spectrum was conducted to screen mir-155-5p regulated genetic experiment. Western blot technique was employed to detect and screen the protein expression of target genes. Mimics was transfected in BGC-823 of gastric cancer cells. Compared with mimics-nc group and mock group, the mRNA expression quantities of SMAD1, STAT1, CAB39, CXCR4 and CA9 were significantly lower. After the gastric cancer cells BGC-823 and MKN-45 had been transfected by mimics, compared with mimics-nc (MNC) group and mock (MOCK) group, it was decreased for the protein expression of SMAD1, STAT1 and CAB39 in mimics (MIMICS) group. The verification of qRT-PCR demonstrated that SMAD1, STAT1, CAB39, CXCR4 and CA9 were the predicted target genes and target proteins of mir-155-5p, the over expression of mir-155-5p could enable the decreasing of its expression level in gastric cancer cells MKN-45 and BGC-823.

Transforming properties of the Huntingtin interacting protein 1/ platelet-derived growth factor beta receptor fusion protein.

PubMed

Ross, T S; Gilliland, D G

1999-08-06

We have previously reported that the Huntingtin interacting protein 1 (HIP1) gene is fused to the platelet-derived growth factor beta receptor (PDGFbetaR) gene in a patient with chronic myelomonocytic leukemia. We now show that HIP1/PDGFbetaR oligomerizes, is constitutively tyrosine-phosphorylated, and transforms the murine hematopoietic cell line, Ba/F3, to interleukin-3-independent growth. A kinase-inactive mutant is neither tyrosine-phosphorylated nor able to transform Ba/F3 cells. Oligomerization and kinase activation required the 55-amino acid carboxyl-terminal TALIN homology region but not the leucine zipper domain. Tyrosine phosphorylation of a 130-kDa protein and STAT5 correlates with transformation in cells expressing HIP1/PDGFbetaR and related mutants. A deletion mutant fusion protein that contains only the TALIN homology region of HIP1 fused to PDGFbetaR is incapable of transforming Ba/F3 cells and does not tyrosine-phosphorylate p130 or STAT5, although it is itself constitutively tyrosine-phosphorylated. We have also analyzed cells expressing Tyr --> Phe mutants of HIP1/PDGFbetaR in the known PDGFbetaR SH2 docking sites and report that none of these sites are necessary for STAT5 activation, p130 phosphorylation, or Ba/F3 transformation. The correlation of factor-independent growth of hematopoietic cells with p130 and STAT5 phosphorylation/activation in both the HIP1/PDGFbetaR Tyr --> Phe and deletion mutational variants suggests that both STAT5 and p130 are important for transformation mediated by HIP1/PDGFbetaR.

Vaccinia Virus Protein C6 Inhibits Type I IFN Signalling in the Nucleus and Binds to the Transactivation Domain of STAT2.

PubMed

Stuart, Jennifer H; Sumner, Rebecca P; Lu, Yongxu; Snowden, Joseph S; Smith, Geoffrey L

2016-12-01

The type I interferon (IFN) response is a crucial innate immune signalling pathway required for defense against viral infection. Accordingly, the great majority of mammalian viruses possess means to inhibit this important host immune response. Here we show that vaccinia virus (VACV) strain Western Reserve protein C6, is a dual function protein that inhibits the cellular response to type I IFNs in addition to its published function as an inhibitor of IRF-3 activation, thereby restricting type I IFN production from infected cells. Ectopic expression of C6 inhibits the induction of interferon stimulated genes (ISGs) in response to IFNα treatment at both the mRNA and protein level. C6 inhibits the IFNα-induced Janus kinase/signal transducer and activator of transcription (JAK/STAT) signalling pathway at a late stage, downstream of STAT1 and STAT2 phosphorylation, nuclear translocation and binding of the interferon stimulated gene factor 3 (ISGF3) complex to the interferon stimulated response element (ISRE). Mechanistically, C6 associates with the transactivation domain of STAT2 and this might explain how C6 inhibits the type I IFN signalling very late in the pathway. During virus infection C6 reduces ISRE-dependent gene expression despite the presence of the viral protein phosphatase VH1 that dephosphorylates STAT1 and STAT2. The ability of a cytoplasmic replicating virus to dampen the immune response within the nucleus, and the ability of viral immunomodulators such as C6 to inhibit multiple stages of the innate immune response by distinct mechanisms, emphasizes the intricacies of host-pathogen interactions and viral immune evasion.

Impact of STAT/SOCS mRNA Expression Levels after Major Injury

PubMed Central

Brumann, M.; Matz, M.; Kusmenkov, T.; Stegmaier, J.; Biberthaler, P.; Kanz, K.-G.; Mutschler, W.; Bogner, V.

2014-01-01

Background. Fulminant changes in cytokine receptor signalling might provoke severe pathological alterations after multiple trauma. The aim of this study was to evaluate the posttraumatic imbalance of the innate immune system with a special focus on the STAT/SOCS family. Methods. 20 polytraumatized patients were included. Blood samples were drawn 0 h–72 h after trauma; mRNA expression profiles of IL-10, STAT 3, SOCS 1, and SOCS 3 were quantified by qPCR. Results. IL-10 mRNA expression increased significantly in the early posttraumatic period. STAT 3 mRNA expressions showed a significant maximum at 6 h after trauma. SOCS 1 levels significantly decreased 6 h–72 h after trauma. SOCS 3 levels were significantly higher in nonsurvivors 6 h after trauma. Conclusion. We present a serial, sequential investigation in human neutrophil granulocytes of major trauma patients evaluating mRNA expression profiles of IL-10, STAT 3, SOCS 1, and SOCS 3. Posttraumatically, immune disorder was accompanied by a significant increase of IL-10 and STAT 3 mRNA expression, whereas SOCS 1 mRNA levels decreased after injury. We could demonstrate that death after trauma was associated with higher SOCS 3 mRNA levels already at 6 h after trauma. To support our results, further investigations have to evaluate protein levels of STAT/SOCS family in terms of posttraumatic immune imbalance. PMID:24648661

CoCl2 , a mimic of hypoxia, enhances bone marrow mesenchymal stem cells migration and osteogenic differentiation via STAT3 signaling pathway.

PubMed

Yu, Xin; Wan, Qilong; Cheng, Gu; Cheng, Xin; Zhang, Jing; Pathak, Janak L; Li, Zubing

2018-06-16

Mesenchymal stem cells homing and migration is a crucial step during bone fracture healing. Hypoxic environment in fracture site induces bone marrow mesenchymal stem cells (BMSCs) migration, but its mechanism remains unclear. Our previous study and studies by other groups have reported the involvement of signal transducer and activator of transcription 3 (STAT3) pathway in cell migration. However, the role of STAT3 pathway in hypoxia-induced cell migration is still unknown. In this study, we investigated the role of STAT3 signaling in hypoxia-induced BMSCs migration and osteogenic differentiation. BMSCs isolated from C57BL/6 male mice were cultured in the presence of cobalt chloride (CoCl 2 ) to simulate intracellular hypoxia. Hypoxia enhanced BMSCs migration, and upregulated cell migration related gene expression i.e., metal-loproteinase (MMP) 7, MMP9 and C-X-C motif chemokine receptor 4. Hypoxia enhanced the phosphorylation of STAT3, and cell migration related proteins: c-jun n-terminal kinase (JNK), focal of adhesion kinase (FAK), extracellular regulated protein kinases and protein kinase B 1/2 (ERK1/2). Moreover, hypoxia enhanced expression of osteogenic differentiation marker. Inhibition of STAT3 suppressed the hy-poxia-induced BMSCs migration, cell migration related signaling molecules phos-phorylation, and osteogenic differentiation related gene expression. In conclusion, our result indicates that hypoxia-induced BMSCs migration and osteogenic differentiation is via STAT3 phosphorylation and involves the cooperative activity of the JNK, FAK and MMP9 signaling pathways. This article is protected by copyright. All rights reserved.

Adenovirus-mediated tissue factor pathway inhibitor gene transfer induces apoptosis by blocking the phosphorylation of JAK-2/STAT-3 pathway in vascular smooth muscle cells.

PubMed

Fu, Yu; Zhao, Yong; Liu, Yue; Zhu, Yejing; Chi, Jinyu; Hu, Jing; Zhang, Xiaohui; Yin, Xinhua

2012-10-01

In our previous study, we have demonstrated that tissue factor pathway inhibitor (TFPI) gene could induce vascular smooth muscle cell (VSMC) apoptosis. This study was conducted to investigate whether the overexpression of the TFPI gene can induce VSMC apoptosis by inhibiting JAK-2/STAT-3 pathway phosphorylation and thereby inhibiting the expression of such downstream targets as the apoptotic protein Bcl-2 and cell cycle protein cyclin D1. The effect of TFPI on the expression of survivin, a central molecule in cell survival, was also investigated. Rat VSMCs were infected with recombinant adenovirus containing either the TFPI (Ad-TFPI) or LacZ (Ad-LacZ) gene or DMEM in vitro. TFPI expression was detected by ELISA. TUNEL staining and electron microscope were carried out to determine the apoptosis of VSMCs. The expression levels of JAK-2, p-JAK-2, STAT-3, p-STAT-3, cyclin D1, Bcl-2 and survivin were examined by western blot analysis. TFPI protein was detected in the TFPI group after gene transfer and the peak expression was at the 3rd day. At the 3rd, 5th and 7th days after gene transfer, the apoptotic rates by TUNEL assay in the TFPI group were 10.91 ± 1.66%, 13.46 ± 1.28% and 17.04 ± 1.95%, respectively, whereas those in the LacZ group were 3.28 ± 0.89%, 4.01 ± 0.72% and 4.89 ± 1.17%, respectively. We observed cell contraction, slight mitochondrial swelling, nuclear pyknosis and apoptotic body formation in TFPI-treated VSMCs using electron microscopy. JAK-2, p-JAK-2, STAT-3, p-STAT-3, cyclin D1 and Bcl-2, which are all involved in the JAK-2/STAT-3 pathway, were detected in the VSMCs on the 3rd, 5th and 7th days after gene transfer, which is consistent with previously demonstrated time points when VSMCs apoptosis occurred. The expression levels of p-JAK-2, p-STAT-3, cyclin D1 and Bcl-2 were significantly decreased over time in the TFPI group (each P<0.05) but not in the Ad-LacZ and DMEM groups. However, this attenuation of expression was not observed for JAK-2 and STAT-3 in any of the groups at any time points after gene transfer (each P>0.05). The expression level of survivin in the TFPI group also weakened significantly over time compared with the levels in the Ad-LacZ and DMEM groups (each P<0.05) at the 3rd, 5th and 7th days after gene transfer. The results demonstrated that TFPI played an apoptosis-inducing role in VSMCs in a manner that involves both the suppression of JAK-2/STAT-3 pathway phosphorylation and the down-regulation of survivin. Our data show for the first time that targeting the JAK-2/STAT-3 pathway and survivin by overexpressing TFPI may be a new avenue for the treatment of restenosis. Copyright © 2012 Elsevier Inc. All rights reserved.

Inhibition of DNA methyltransferase induces G2 cell cycle arrest and apoptosis in human colorectal cancer cells via inhibition of JAK2/STAT3/STAT5 signalling.

PubMed

Xiong, Hua; Chen, Zhao-Fei; Liang, Qin-Chuan; Du, Wan; Chen, Hui-Min; Su, Wen-Yu; Chen, Guo-Qiang; Han, Ze-Guang; Fang, Jing-Yuan

2009-09-01

DNA methyltransferase inhibitors (MTIs) have recently emerged as promising chemotherapeutic or preventive agents for cancer, despite their poorly characterized mechanisms of action. The present study shows that DNA methylation is integral to the regulation of SH2-containing protein tyrosine phosphatase 1 (SHP1) expression, but not for regulation of suppressors of cytokine signalling (SOCS)1 or SOCS3 in colorectal cancer (CRC) cells. SHP1 expression correlates with down-regulation of Janus kinase/signal transducers and activators of transcription (JAK2/STAT3/STAT5) signalling, which is mediated in part by tyrosine dephosphorylation events and modulation of the proteasome pathway. Up-regulation of SHP1 expression was achieved using a DNA MTI, 5-aza-2'-deoxycytidine (5-aza-dc), which also generated significant down-regulation of JAK2/STAT3/STAT5 signalling. We demonstrate that 5-aza-dc suppresses growth of CRC cells, and induces G2 cell cycle arrest and apoptosis through regulation of downstream targets of JAK2/STAT3/STAT5 signalling including Bcl-2, p16(ink4a), p21(waf1/cip1) and p27(kip1). Although 5-aza-dc did not significantly inhibit cell invasion, 5-aza-dc did down-regulate expression of focal adhesion kinase and vascular endothelial growth factor in CRC cells. Our results demonstrate that 5-aza-dc can induce SHP1 expression and inhibit JAK2/STAT3/STAT5 signalling. This study represents the first evidence towards establishing a mechanistic link between inhibition of JAK2/STAT3/STAT5 signalling and the anticancer action of 5-aza-dc in CRC cells that may lead to the use of MTIs as a therapeutic intervention for human colorectal cancer.

Influence of Maternal Undernutrition and Overfeeding on Cardiac Ciliary Neurotrophic Factor Receptor and Ventricular Size in Fetal Sheep

PubMed Central

Dong, Feng; Ford, Stephen P.; Nijland, Mark J.; Nathanielsz, Peter W.; Ren, Jun

2008-01-01

Intrauterine nutrition status is reported to correlate with risk of cardiovascular diseases in adulthood. Either under- or over-nutrition during early to mid gestation contributes to altered fetal growth and ventricular geometry. This study was designed to examine myocardial expression of ciliary neurotrophic factor receptor α (CTNFRα) and its down-stream mediator signal transducer and activator of transcription 3 (STAT3) on maternal under- or over-nutrition-induced changes in fetal heart weight. Multiparous ewes were fed with 50% (nutrient-restricted, NR), 100% (control) or 150% (overfed, OF) of NRC requirements from 28 to 78 days of gestation (dG; Term 148 dG). Ewes were euthanized on day 78, and the gravid uteri and fetuses recovered. Ventricular protein expression of CTNFRα, STAT3, phosphorylated STAT3, insulin-like growth factor I receptor (IGF-1R) and IGF binding protein 3 (IGFBP3) were quantitated using western blot. Plasma cortisol levels were higher in both NR and OF fetuses whereas plasma IGF-1 levels were lower and higher, in NR and OF fetuses. Fetal weights were reduced by 29.9% in NR ewes and were increased by 22.2% in fetuses from OF ewes compared to control group. Nutrient restriction did not affect fetal heart or ventricular weights whereas overfeeding increased heart and ventricular weights. Protein expression of CTNFRα in fetal ventricular tissue was reduced in OF group whereas STAT3 and pSTAT3 levels were reduced in both NR and OF groups. Expression of IGF-1R and IGFBP3 was unaffected in either NR or OF group. These data suggested that compared with maternal undernutrition, intrauterine overfeeding during early to mid gestation is associated with increases fetal blood concentrations of cortisol and IGF-1 in association with ventricular hypertrophy where reduced expression of CNTFRα and STAT3 may play a role. PMID:17869083

La Piedad Michoacán Mexico Virus V protein antagonizes type I interferon response by binding STAT2 protein and preventing STATs nuclear translocation.

PubMed

Pisanelli, Giuseppe; Laurent-Rolle, Maudry; Manicassamy, Balaji; Belicha-Villanueva, Alan; Morrison, Juliet; Lozano-Dubernard, Bernardo; Castro-Peralta, Felipa; Iovane, Giuseppe; García-Sastre, Adolfo

2016-02-02

La Piedad Michoacán Mexico Virus (LPMV) is a member of the Rubulavirus genus within the Paramyxoviridae family. LPMV is the etiologic agent of "blue eye disease", causing a significant disease burden in swine in Mexico with long-term implications for the agricultural industry. This virus mainly affects piglets and is characterized by meningoencephalitis and respiratory distress. It also affects adult pigs, causing reduced fertility and abortions in females, and orchitis and epididymitis in males. Viruses of the Paramyxoviridae family evade the innate immune response by targeting components of the interferon (IFN) signaling pathway. The V protein, expressed by most paramyxoviruses, is a well-characterized IFN signaling antagonist. Until now, there were no reports on the role of the LPMV-V protein in inhibiting the IFN response. In this study we demonstrate that LPMV-V protein antagonizes type I but not type II IFN signaling by binding STAT2, a component of the type I IFN cascade. Our results indicate that the last 18 amino acids of LPMV-V protein are required for binding to STAT2 in human and swine cells. While LPMV-V protein does not affect the protein levels of STAT1 or STAT2, it does prevent the IFN-induced phosphorylation and nuclear translocation of STAT1 and STAT2 thereby inhibiting cellular responses to IFN α/β. Copyright © 2015 Elsevier B.V. All rights reserved.

La Piedad Michoacán Mexico Virus V protein antagonizes type I interferon response by binding STAT2 protein and preventing STATs nuclear translocation

PubMed Central

Pisanelli, Giuseppe; Laurent-Rolle, Maudry; Manicassamy, Balaji; Belicha-Villanueva, Alan; Morrison, Juliet; Lozano-Dubernard, Bernardo; Castro-Peralta, Felipa; Iovane, Giuseppe; García-Sastre, Adolfo

2017-01-01

La Piedad Michoacán Mexico Virus (LPMV) is a member of the Rubulavirus genus within the Paramyxoviridae family. LPMV is the etiologic agent of “blue eye disease”, causing a significant disease burden in swine in Mexico with long-term implications for the agricultural industry. This virus mainly affects piglets and is characterized by meningoencephalitis and respiratory distress. It also affects adult pigs, causing reduced fertility and abortions in females, and orchitis and epididymitis in males. Viruses of the Paramyxoviridae family evade the innate immune response by targeting components of the interferon (IFN) signaling pathway. The V protein, expressed by most paramyxoviruses, is a well-characterized IFN signaling antagonist. Until now, there were no reports on the role of the LPMV-V protein in inhibiting the IFN response. In this study we demonstrate that LPMV-V protein antagonizes type I but not type II IFN signaling by binding STAT2, a component of the type I IFN cascade. Our results indicate that the last 18 amino acids of LPMV-V protein are required for binding to STAT2 in human and swine cells. While LPMV-V protein does not affect the protein levels of STAT1 or STAT2, it does prevent the IFN-induced phosphorylation and nuclear translocation of STAT1 and STAT2 thereby inhibiting cellular responses to IFN α/β PMID:26546155

Phosphorylation of STAT-1 Serine 727 Is Prolonged in HLA-B27-Expressing Human Monocytic Cells

PubMed Central

Ruuska, Marja; Sahlberg, Anna S.; Granfors, Kaisa; Penttinen, Markus A.

2013-01-01

A tissue antigen, HLA-B27, is strongly associated with a group of rheumatic diseases called spondyloarthritides. Despite the intensive research, the exact role of HLA-B27 in the pathogenesis of these diseases is still unclear. Here we studied whether HLA-B27 modulates the phosphorylation of signal transducer and activator of transcription 1 (STAT-1) serine 727 residue and the localization of STAT-1 in Salmonella-infected human monocytic cells. In addition, we studied the role of signaling molecule double-stranded RNA activated protein kinase (PKR) in these modulatory effects. U937 human monocytic cell transfectants stably expressing wild type HLA-B27 or mutated HLA-B27 heavy chains with amino acid substitutions in the B pocket were prepared. The PMA-differentiated cells were infected with S. enteritidis. Western blotting was used to detect the phosphorylation of STAT-1, and to visualize the localization of STAT-1 in the cells confocal microscopy was used. Specific inhibitors were employed to study the role of PKR in STAT-1 phosphorylation. We discovered that the phosphorylation of STAT-1 serine 727 is prolonged in cells expressing misfolding forms of HLA-B27 after S. enteritidis infection, whereas in mock cells and in cells expressing mutated, non-misfolding HLA-B27 the phosphorylation of serine 727 is transient. Interestingly, STAT-1 serine 727 phosphorylation is partly dependent on PKR. In addition, more STAT-1 is localized in the nucleus of HLA-B27-expressing cells, even before an external trigger, when compared to mock cells. In conclusion, our results show that the phosphorylation of STAT-1 serine 727 residue is prolonged in HLA-B27-expressing monocyte-macrophage U937 cells after bacterial infection. This is of interest since the phosphorylation of serine 727 on STAT-1 is suggested to contribute to macrophage activation and promote inflammatory responses. Therefore, our results provide a mechanism which explains how the expression of an HLA-B27 molecule can impact the course of Salmonella infection and reactive arthritis. PMID:23349666

Toxoplasma gondii induces FAK-Src-STAT3 signaling during infection of host cells that prevents parasite targeting by autophagy.

PubMed

Portillo, Jose-Andres C; Muniz-Feliciano, Luis; Lopez Corcino, Yalitza; Lee, So Jung; Van Grol, Jennifer; Parsons, Sarah J; Schiemman, William P; Subauste, Carlos S

2017-10-01

Targeting of Toxoplasma gondii by autophagy is an effective mechanism by which host cells kill the protozoan. Thus, the parasite must avoid autophagic targeting to survive. Here we show that the mammalian cytoplasmic molecule Focal Adhesion Kinase (FAK) becomes activated during invasion of host cells. Activated FAK appears to accompany the formation of the moving junction (as assessed by expression the parasite protein RON4). FAK activation was inhibited by approaches that impaired β1 and β3 integrin signaling. FAK caused activation of Src that in turn mediated Epidermal Growth Factor Receptor (EGFR) phosphorylation at the unique Y845 residue. Expression of Src-resistant Y845F EGFR mutant markedly inhibited ROP16-independent activation of STAT3 in host cells. Activation of FAK, Y845 EGFR or STAT3 prevented activation of PKR and eIF2α, key stimulators of autophagy. Genetic or pharmacologic inhibition of FAK, Src, EGFR phosphorylation at Y845, or STAT3 caused accumulation of the autophagy protein LC3 and LAMP-1 around the parasite and parasite killing dependent on autophagy proteins (ULK1 and Beclin 1) and lysosomal enzymes. Parasite killing was inhibited by expression of dominant negative PKR. Thus, T. gondii activates a FAK→Src→Y845-EGFR→STAT3 signaling axis within mammalian cells, thereby enabling the parasite to survive by avoiding autophagic targeting through a mechanism likely dependent on preventing activation of PKR and eIF2α.

Expression of the JAK/STAT Signaling Pathway in Bullous Pemphigoid and Dermatitis Herpetiformis

PubMed Central

Wozniacka, A.; Waszczykowska, E.; Zebrowska, A.

2017-01-01

A family of eleven proteins comprises the Janus kinases (JAK) and signal transducers and activators of transcription (STAT) signaling pathway, which enables transduction of signal from cytokine receptor to the nucleus and activation of transcription of target genes. Irregular functioning of the cascade may contribute to pathogenesis of autoimmune diseases; however, there are no reports concerning autoimmune bullous diseases yet to be published. The aim of this study was to evaluate the expression of proteins constituting the JAK/STAT signaling pathway in skin lesions and perilesional area in dermatitis herpetiformis (DH) and bullous pemphigoid (BP), as well as in the control group. Skin biopsies were collected from 21 DH patients, from 20 BP patients, and from 10 healthy volunteers. The localization and expression of selected STAT and JAK proteins were examined by immunohistochemistry and immunoblotting. We found significantly higher expression of JAK/STAT proteins in skin lesions in patients with BP and DH, in comparison to perilesional skin and the control group, which may be related to proinflammatory cytokine network and induction of inflammatory infiltrate in tissues. Our findings suggest that differences in the JAK and STAT expression may be related to distinct cytokines activating them and mediating neutrophilic and/or eosinophilic infiltrate. PMID:29203970

Hepatic expression of the GH/JAK/STAT/IGF pathway, acute-phase response signalling and complement system are affected in mouse offspring by prenatal and early postnatal exposure to maternal high-protein diet.

PubMed

Vanselow, Jens; Kucia, Marzena; Langhammer, Martina; Koczan, Dirk; Rehfeldt, Charlotte; Metges, Cornelia C

2011-12-01

Effects of pre- and early postnatal exposure to maternal high-protein diets are not well understood. Transcription profiling was performed in male mouse offspring exposed to maternal high-protein diet during pregnancy and/or lactation to identify affected hepatic molecular pathways. Dams were fed isoenergetic diets with control (20% w/w) or high protein levels (40%). The hepatic expression profiles were evaluated by differential microarray analysis 3 days (d3) and 3 weeks (d21) after birth. Offspring from three different high-protein dietary groups, HP (d3, high-protein diet during pregnancy), HPHP (d21, high-protein diet during pregnancy and lactation) and CHP (d21, control diet during pregnancy and high-protein diet during lactation), were compared with age-matched offspring from dams fed control diet. Offspring body and liver mass of all high-protein groups were decreased. Prenatal high-protein diet affected hepatic expression of genes mapping to the acute response/complement system and the GH/JAK/STAT/IGF signalling pathways. Maternal exposure to high-protein diet during lactation affected hepatic gene expression of the same pathways but additionally affected genes mapping to protein, fatty acid, hexose and pyruvate metabolism. (1) Genes of the acute response/complement system and GH/JAK/STAT/IGF pathways were down-regulated in offspring of dams exposed to high-protein diets during pregnancy and/or lactation. (2) Genes related to nutrient and energy metabolism, however, were only affected when high-protein diet was administered during lactation. (3) Modulation of the GH/JAK/STAT/IGF pathway might be responsible for reduced body and liver masses by maternal high-protein diet.

The role of JAK/STAT3 signaling pathway on apoptosis of lung adenocarcinoma cell line PC-9 induced by icotinib.

PubMed

Zhang, Yuping; Meng, Xia; Shi, Hongyang; Li, Wei; Ming, Zongjuan; Zhong, Yujie; Deng, Wenjing; Zhang, Qiuhong; Fan, Na; Niu, Zequn; Chen, Guo'an; Yang, Shuanying

2016-01-01

The aim of this study is to estimate the role of JAK/STAT3 signaling pathway on apoptosis of lung adenocarcinoma induced by icotinib. EGFR mutation was detected in lung adenocarcinoma cell line PC-9 by ARMS assay; The inhibitory rates of cell proliferation of PC-9 cells which were exposed to different concentrations of icotinib (0~100 μMol/L) for different time (24~72 h) respectively were evaluated by MTT assay; Apoptosis of PC-9 cells exposed to different concentrations of icotinib (0, 0.1, 1 and 10 μMol/L) for 48 h were evaluated by TUNEL assay; JAK2, STAT3, Bcl-2, Bax mRNA expressions were evaluated by Real-time PCR assay; The protein levels of P-STAT3 and IL-6 were evaluated by Western-blot assay. Human lung adenocarcinoma cell line PC-9 had an exon 19 deletion mutation in EGFR gene; Followed by treatment of icotinib, the proliferation of PC-9 cells were all inhibited significantly, especially in 48 and 72 h (P<0.01) in all concentrations; The inhibitory rates of cell proliferation in different treating time had statistical significance (P<0.01); Cell apoptosis in different concentrations were increased significantly (P<0.05); Along with the increasing concentrations, gene expression levels of JAK2, STAT3 and Bcl-2 decreased significantly (P<0.05), Bax increased significantly (P<0.05), JAK2/STAT3 ratios increased significantly (P<0.01), and Bcl-2/bax ratios decreased significantly (P<0.01); P-STAT3 and IL-6 protein levels were inhibited significantly in higher concentration. JAK/STAT3 signaling pathway participates in apoptosis of PC-9 cells induced by icotinib. The most likely mechanism is icotinib inhibited the gene expression levels of JAK2, STAT3 and Bcl-2, so with the P-STAT3 and IL-6 protein levels, and mediated gene Bax overexpression.

The role of JAK/STAT3 signaling pathway on apoptosis of lung adenocarcinoma cell line PC-9 induced by icotinib

PubMed Central

Zhang, Yuping; Meng, Xia; Shi, Hongyang; Li, Wei; Ming, Zongjuan; Zhong, Yujie; Deng, Wenjing; Zhang, Qiuhong; Fan, Na; Niu, Zequn; Chen, Guo’an; Yang, Shuanying

2016-01-01

Objective: The aim of this study is to estimate the role of JAK/STAT3 signaling pathway on apoptosis of lung adenocarcinoma induced by icotinib. Methods: EGFR mutation was detected in lung adenocarcinoma cell line PC-9 by ARMS assay; The inhibitory rates of cell proliferation of PC-9 cells which were exposed to different concentrations of icotinib (0~100 μMol/L) for different time (24~72 h) respectively were evaluated by MTT assay; Apoptosis of PC-9 cells exposed to different concentrations of icotinib (0, 0.1, 1 and 10 μMol/L) for 48 h were evaluated by TUNEL assay; JAK2, STAT3, Bcl-2, Bax mRNA expressions were evaluated by Real-time PCR assay; The protein levels of P-STAT3 and IL-6 were evaluated by Western-blot assay. Results: Human lung adenocarcinoma cell line PC-9 had an exon 19 deletion mutation in EGFR gene; Followed by treatment of icotinib, the proliferation of PC-9 cells were all inhibited significantly, especially in 48 and 72 h (P<0.01) in all concentrations; The inhibitory rates of cell proliferation in different treating time had statistical significance (P<0.01); Cell apoptosis in different concentrations were increased significantly (P<0.05); Along with the increasing concentrations, gene expression levels of JAK2, STAT3 and Bcl-2 decreased significantly (P<0.05), Bax increased significantly (P<0.05), JAK2/STAT3 ratios increased significantly (P<0.01), and Bcl-2/bax ratios decreased significantly (P<0.01); P-STAT3 and IL-6 protein levels were inhibited significantly in higher concentration. Conclusions: JAK/STAT3 signaling pathway participates in apoptosis of PC-9 cells induced by icotinib. The most likely mechanism is icotinib inhibited the gene expression levels of JAK2, STAT3 and Bcl-2, so with the P-STAT3 and IL-6 protein levels, and mediated gene Bax overexpression. PMID:27186296

Altered expression and activation of signal transducers and activators of transcription (STATs) in hepatitis C virus infection: in vivo and in vitro studies.

PubMed

Larrea, E; Aldabe, R; Molano, E; Fernandez-Rodriguez, C M; Ametzazurra, A; Civeira, M P; Prieto, J

2006-08-01

Signal transducers and activators of transcription (STATs) play a critical role in antiviral defence. STAT3 is also important in cell protection against inflammatory damage. STAT proteins are activated by interferons and by hepatoprotective cytokines of the interleukin 6 superfamily, including cardiotrophin 1. We analysed the status of STATs in hepatitis C virus (HCV) infected livers and the relationship between expression and activation of STATs and HCV replication in Huh7 cells transfected with HCV genomic replicon. STAT3alpha expression was reduced in HCV infected livers showing an inverse correlation with serum alanine aminotransferase. In patients with HCV infection, nuclear staining for phosphorylated STAT3 was faint in parenchymal cells (although conspicuous in infiltrating leucocytes), in contrast with strong nuclear staining in hepatocytes from control livers. Expression and activation of STAT1 (a factor activated by both interferon (IFN)-alpha and IFN-gamma) were increased in HCV infected livers, particularly in those with high inflammatory activity. Conversely, phosphorylated STAT2 (a factor selectively activated by IFN-alpha) was undetectable in livers with HCV infection, a finding that was associated with marked downregulation of the two functional subunits of the IFN-alpha receptor. HCV replication in Huh7 cells caused STAT3alpha downregulation and blocked STAT3 phosphorylation by either IFN-alpha or cardiotrophin 1. HCV replication in Huh7 cells also inhibited STAT1 and STAT2 activation by IFN-alpha while there was no impairment of STAT1 phosphorylation by the proinflammatory cytokine IFN-gamma. STAT3 is downregulated in HCV infected livers and in Huh7 cells bearing the full length HCV replicon. HCV replication is associated with impaired Jak-STAT signalling by antiviral and cytoprotective cytokines. These effects may favour viral replication while facilitating the progression of liver disease.

Expression and activation of STAT3 in ischemia-induced retinopathy.

PubMed

Mechoulam, Hadas; Pierce, Eric A

2005-12-01

Signal transducer and activator of transcription protein-3 (STAT3) is a transcription factor that participates in many biological processes, including tumor angiogenesis. The expression and activation of Stat3 in the mouse model of ischemia-induced retinal neovascularization was investigated to evaluate the possible role of STAT3 in retinal vascular disease. Retinal neovascularization was induced in mice pups by exposure to hyperoxia. Gene microarrays were used to identify genes whose expression in the retina is altered at postnatal day (P)12 and P18. The relative levels of Stat3 mRNA were determined by semiquantitative RT-PCR. Stat3 protein levels and the levels of the activated form of Stat3 (pStat3) at P12, P15, P18, and P22 were determined by immunoblot analysis. Stat3 and pStat3 were demonstrated by immunofluorescence in retinal sections at P12, P15, and P18. In a series of microarray experiments, increased Stat3 mRNA levels in the retina were detected at P18. This result was validated by RT-PCR and demonstrated that Stat3 and pStat3 protein levels also increase during the development of neovascularization. Stat3 partially colocalized with blood vessels at the peak of neovascularization. pStat3 colocalized completely with blood vessels in both experimental samples and age-matched controls. pStat3 staining increased notably in the neovascular vessels at P15 and P18 and was more strongly associated with the epiretinal vessels than with inner retinal vessels. It was not detected in larger blood vessels, such as those of the optic nerve. The level of Stat3 expression increased, and pStat3 was observed in association with retinal neovascularization. Activated Stat3 was preferentially localized to neovascular retinal vessels. These data suggest that STAT3 may have a role in proliferative retinopathy.

Meningeal hemangiopericytoma and solitary fibrous tumors carry the NAB2-STAT6 fusion and can be diagnosed by nuclear expression of STAT6 protein.

PubMed

Schweizer, Leonille; Koelsche, Christian; Sahm, Felix; Piro, Rosario M; Capper, David; Reuss, David E; Pusch, Stefan; Habel, Antje; Meyer, Jochen; Göck, Tanja; Jones, David T W; Mawrin, Christian; Schittenhelm, Jens; Becker, Albert; Heim, Stephanie; Simon, Matthias; Herold-Mende, Christel; Mechtersheimer, Gunhild; Paulus, Werner; König, Rainer; Wiestler, Otmar D; Pfister, Stefan M; von Deimling, Andreas

2013-05-01

Non-central nervous system hemangiopericytoma (HPC) and solitary fibrous tumor (SFT) are considered by pathologists as two variants of a single tumor entity now subsumed under the entity SFT. Recent detection of frequent NAB2-STAT6 fusions in both, HPC and SFT, provided additional support for this view. On the other hand, current neuropathological practice still distinguishes between HPC and SFT. The present study set out to identify genes involved in the formation of meningeal HPC. We performed exome sequencing and detected the NAB2-STAT6 fusion in DNA of 8/10 meningeal HPC thereby providing evidence of close relationship of these tumors with peripheral SFT. Due to the considerable effort required for exome sequencing, we sought to explore surrogate markers for the NAB2-STAT6 fusion protein. We adopted the Duolink proximity ligation assay and demonstrated the presence of NAB2-STAT6 fusion protein in 17/17 HPC and the absence in 15/15 meningiomas. More practical, presence of the NAB2-STAT6 fusion protein resulted in a strong nuclear signal in STAT6 immunohistochemistry. The nuclear reallocation of STAT6 was detected in 35/37 meningeal HPC and 25/25 meningeal SFT but not in 87 meningiomas representing the most important differential diagnosis. Tissues not harboring the NAB2-STAT6 fusion protein presented with nuclear expression of NAB2 and cytoplasmic expression of STAT6 proteins. In conclusion, we provide strong evidence for meningeal HPC and SFT to constitute variants of a single entity which is defined by NAB2-STAT6 fusion. In addition, we demonstrate that this fusion can be rapidly detected by STAT6 immunohistochemistry which shows a consistent nuclear reallocation. This immunohistochemical assay may prove valuable for the differentiation of HPC and SFT from other mesenchymal neoplasms.

STATs shape the active enhancer landscape of T cell populations.

PubMed

Vahedi, Golnaz; Takahashi, Hayato; Nakayamada, Shingo; Sun, Hong-Wei; Sartorelli, Vittorio; Kanno, Yuka; O'Shea, John J

2012-11-21

Signaling pathways are intimately involved in cellular differentiation, allowing cells to respond to their environment by regulating gene expression. Although enhancers are recognized as key elements that regulate selective gene expression, the interplay between signaling pathways and actively used enhancer elements is not clear. Here, we use CD4(+) T cells as a model of differentiation, mapping the activity of cell-type-specific enhancer elements in T helper 1 (Th1) and Th2 cells. Our data establish that STAT proteins have a major impact on the activation of lineage-specific enhancers and the suppression of enhancers associated with alternative cell fates. Transcriptome analysis further supports a functional role for enhancers regulated by STATs. Importantly, expression of lineage-defining master regulators in STAT-deficient cells fails to fully recover the chromatin signature of STAT-dependent enhancers. Thus, these findings point to a critical role of STATs as environmental sensors in dynamically molding the specialized enhancer architecture of differentiating cells. Copyright © 2012 Elsevier Inc. All rights reserved.

STATs Shape the Active Enhancer Landscape of T Cell Populations

PubMed Central

Vahedi, Golnaz; Takahashi, Hayato; Nakayamada, Shingo; Sun, Hong-wei; Sartorelli, Vittorio; Kanno, Yuka; O’Shea, John J.

2012-01-01

SUMMARY Signaling pathways are intimately involved in cellular differentiation, allowing cells to respond to their environment by regulating gene expression. While enhancers are recognized as key elements that regulate selective gene expression, the interplay between signaling pathways and actively used enhancer elements is not clear. Here, we use CD4+ T cells as a model of differentiation, mapping the acquisition of cell-type-specific enhancer elements in T-helper 1 (Th1) and Th2 cells. Our data establish that STAT proteins have a major impact on the acquisition of lineage-specific enhancers and the suppression of enhancers associated with alternative cell fates. Transcriptome analysis further supports a functional role for enhancers regulated by STATs. Importantly, expression of lineage-defining master regulators in STAT-deficient cells fails to fully recover the chromatin signature of STAT-dependent enhancers. Thus, these findings point to a critical role of STATs as environmental sensors in dynamically molding the specialized enhancer architecture of differentiating cells. PMID:23178119

An oncogenic axis of STAT-mediated BATF3 upregulation causing MYC activity in classical Hodgkin lymphoma and anaplastic large cell lymphoma.

PubMed

Lollies, A; Hartmann, S; Schneider, M; Bracht, T; Weiß, A L; Arnolds, J; Klein-Hitpass, L; Sitek, B; Hansmann, M-L; Küppers, R; Weniger, M A

2018-01-01

Classical Hodgkin lymphoma (cHL) and anaplastic large cell lymphoma (ALCL) feature high expression of activator protein-1 (AP-1) transcription factors, which regulate various physiological processes but also promote lymphomagenesis. The AP-1 factor basic leucine zipper transcription factor, ATF-like 3 (BATF3), is highly transcribed in cHL and ALCL; however, its functional importance in lymphomagenesis is unknown. Here we show that proto-typical CD30 + lymphomas, namely cHL (21/30) and primary mediastinal B-cell lymphoma (8/9), but also CD30 + diffuse large B-cell lymphoma (15/20) frequently express BATF3 protein. Mass spectrometry and co-immunoprecipitation established interactions of BATF3 with JUN and JUNB in cHL and ALCL lines. BATF3 knockdown using short hairpin RNAs was toxic for cHL and ALCL lines, reducing their proliferation and survival. We identified MYC as a critical BATF3 target and confirmed binding of BATF3 to the MYC promoter. JAK/STAT signaling regulated BATF3 expression, as chemical JAK2 inhibition reduced and interleukin 13 stimulation induced BATF3 expression in cHL lines. Chromatin immunoprecipitation substantiated a direct regulation of BATF3 by STAT proteins in cHL and ALCL lines. In conclusion, we identified STAT-mediated BATF3 expression that is essential for lymphoma cell survival and promoted MYC activity in cHL and ALCL, hence we recognized a new oncogenic axis in these lymphomas.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Park, Yu-Kyoung; Lee, Tae-Yoon; Choi, Jong-Soon

Highlights: • Compound 7b, a meridianin C derivative, inhibits adipogenesis. • Compound 7b inhibits C/EBP-α, PPAR-γ, FAS, STAT-3, and STAT-5 in 3T3-L1 adipocytes. • Compound 7b inhibits leptin, but not adiponectin, expression in 3T3-L1 adipocytes. • Compound 7b thus may have therapeutic potential against obesity. - Abstract: Meridianin C, a marine alkaloid, is a potent protein kinase inhibitor and has anti-cancer activity. We have recently developed a series of meridianin C derivatives (compound 7a–7j) and reported their proviral integration Moloney Murine Leukemia Virus (pim) kinases’ inhibitory and anti-proliferative effects on human leukemia cells. Here we investigated the effect of thesemore » meridianin C derivatives on adipogenesis. Strikingly, among the derivatives tested, compound 7b most strongly inhibited lipid accumulation during the differentiation of 3T3-L1 preadipocytes into adipocytes. However, meridianin C treatment was largely cytotoxic to 3T3-L1 adipocytes. On mechanistic levels, compound 7b reduced not only the expressions of CCAAT/enhancer-binding protein-α (C/EBP-α), peroxisome proliferator-activated receptor-γ (PPAR-γ), and fatty acid synthase (FAS) but also the phosphorylation levels of signal transducer and activator of transcription-3 (STAT-3) and STAT-5 during adipocyte differentiation. Moreover, compound 7b repressed leptin, but not adiponectin, expression during adipocyte differentiation. Collectively, these findings demonstrate that a meridianin C derivative inhibits adipogenesis by down-regulating expressions and/or phosphorylations of C/EBP-α, PPAR-γ, FAS, STAT-3 and STAT-5.« less

[Inhibition of suppressor of cytokine signaling 1 on lipopolysaccharide-induced mucin5AC hypersecretion and the mechanism in human bronchial epithelial cells].

PubMed

Liu, C Y; Li, Q; Zhou, X D

2016-03-29

To explore the inhibitory role of suppressor of cytokine signaling 1 (SOCS1) in lipopolysaccharide (LPS)-induced mucin5AC (MUC5AC) hypersecretion and the potential mechanism involved in this process. The human bronchial epithelial cells 16HBE were divided into 0, 0.5, 1, 6, 12 and 24 h groups according to the time of LPS challenge. In gain- and loss- of functions experiments, wild-type SOCS1 and SOCS1-targeted siRNA (SOCS1-siRNA) were synthesized to identify the function of SOCS1 in LPS-mediated MUC5AC hypersecretion, and named wild-type SOCS1 group and SOCS1-siRNA group, respectively, and the non-transfected group and non-targeted siRNA group were used as controls. In Filgotinib group, the specific inhibitor of Janus kinase 1 (JAK1), Filgotinib, was used to detect the role of JAK1/signal transducer and activator of transcription 1 (STAT1) signaling pathway in LPS challenge, and the aqueous physiological buffer group was used as the control. The production of MUC5AC protein was measured by enzyme linked immunosorbent assay (ELISA), and the amount of MUC5AC protein was normalized to the total protein in cell lysates and was expressed as μg/mg cell lysates. The proteins expressions of SOCS1, phosphorylation of JAK1 and STAT1 were measured by Western blot, and the total expression of its protein (for JAK1 and STAT1) or β-actin (for SOCS1) was used as the loading control. Compared to 0 h group, LPS induced a robust induction in MUC5AC expression, the expression levels of MUC5AC in 0, 0.5, 1, 6, 12 and 24 h groups were (2.86±0.20), (3.42±0.29), (3.43±0.12), (10.22±0.96), (14.56±1.12), (14.15±1.34) μg/mg, in association with a decrease of SOCS1 expression. And in 6 h group, the expressions of MUC5AC and SOCS1 were both medium up-regulated (all P<0.05). Consequently, the application of LPS for 6 h was selected as the optimal responses period in the ensuing experiments. Compared to the expression of MUC5AC protein in non-transfected group, high level of SOCS1 in wild-type SOCS1 group led to a reduced phosphorylation of JAK1/STAT1, as well as Filgotinib did, thereby suppressing excessive MUC5AC production in wild-type SOCS1 group and Filgotinib group [(4.04±0.65), (7.02±0.83) vs (10.37±1.00) μg/mg] (all P<0.05). Conversely, compared to the expression of MUC5AC in non-targeted siRNA group, down-regulation of SOCS1 in SOCS1-siRNA group promoted the phosphorylation of JAK1/STAT1, and then further increased MUC5AC production [(13.69±1.32) vs (11.01±1.41) μg/mg] (all P<0.05). These results show that SOCS1 suppresses LPS-promoted MUC5AC hypersecretion through the inhibition of JAK1/STAT1 signaling pathway.

IL-8 induces miR-424-5p expression and modulates SOCS2/STAT5 signaling pathway in oral squamous cell carcinoma.

PubMed

Peng, Hsuan-Yu; Jiang, Shih-Sheng; Hsiao, Jenn-Ren; Hsiao, Michael; Hsu, Yuan-Ming; Wu, Guan-Hsun; Chang, Wei-Min; Chang, Jang-Yang; Jin, Shiow-Lian Catherine; Shiah, Shine-Gwo

2016-06-01

Suppressor of cytokine signaling (SOCS) proteins are negative feedback regulators of the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway. Dysregulation of SOCS protein expression in cancers can be one of the mechanisms that maintain STAT activation, but this mechanism is still poorly understood in oral squamous cell carcinoma (OSCC). Here, we report that SOCS2 protein is significantly downregulated in OSCC patients and its levels are inversely correlated with miR-424-5p expression. We identified the SOCS2 protein, which modulates STAT5 activity, as a direct target of miR-424-5p. The miR-424-5p-induced STAT5 phosphorylation, matrix metalloproteinases (MMPs) expression, and cell migration and invasion were blocked by SOCS2 restoration, suggesting that miR-424-5p exhibits its oncogenic activity through negatively regulating SOCS2 levels. Furthermore, miR-424-5p expression could be induced by the cytokine IL-8 primarily through enhancing STAT5 transcriptional activity rather than NF-κB signaling. Antagomir-mediated inactivation of miR-424-5p prevented the IL-8-induced cell migration and invasion, indicating that miR-424-5p is required for IL-8-induced cellular invasiveness. Taken together, these data indicate that STAT5-dependent expression of miR-424-5p plays an important role in mediating IL-8/STAT5/SOCS2 feedback loop, and scavenging miR-424-5p function using antagomir may have therapeutic potential for the treatment of OSCC. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

STAT4 Deficiency Fails To Induce Lung Th2 or Th17 Immunity following Primary or Secondary Respiratory Syncytial Virus (RSV) Challenge but Enhances the Lung RSV-Specific CD8+ T Cell Immune Response to Secondary Challenge

PubMed Central

Dulek, Daniel E.; Newcomb, Dawn C.; Toki, Shinji; Goliniewska, Kasia; Cephus, Jacqueline; Reiss, Sara; Bates, John T.; Crowe, James E.; Boyd, Kelli L.; Moore, Martin L.; Zhou, Weisong

2014-01-01

ABSTRACT Immune-mediated lung injury is a hallmark of lower respiratory tract illness caused by respiratory syncytial virus (RSV). STAT4 plays a critical role in CD4+ Th1 lineage differentiation and gamma interferon (IFN-γ) protein expression by CD4+ T cells. As CD4+ Th1 differentiation is associated with negative regulation of CD4+ Th2 and Th17 differentiation, we hypothesized that RSV infection of STAT4−/− mice would result in enhanced lung Th2 and Th17 inflammation and impaired lung Th1 inflammation compared to wild-type (WT) mice. We performed primary and secondary RSV challenges in WT and STAT4−/− mice and used STAT1−/− mice as a positive control for the development of RSV-specific lung Th2 and Th17 inflammation during primary challenge. Primary RSV challenge of STAT4−/− mice resulted in decreased T-bet and IFN-γ expression levels in CD4+ T cells compared to those of WT mice. Lung Th2 and Th17 inflammation did not develop in primary RSV-challenged STAT4−/− mice. Decreased IFN-γ expression by NK cells, CD4+ T cells, and CD8+ T cells was associated with attenuated weight loss and enhanced viral clearance with primary challenge in STAT4−/− mice compared to WT mice. Following secondary challenge, WT and STAT4−/− mice also did not develop lung Th2 or Th17 inflammation. In contrast to primary challenge, secondary RSV challenge of STAT4−/− mice resulted in enhanced weight loss, an increased lung IFN-γ expression level, and an increased lung RSV-specific CD8+ T cell response compared to those of WT mice. These data demonstrate that STAT4 regulates the RSV-specific CD8+ T cell response to secondary infection but does not independently regulate lung Th2 or Th17 immune responses to RSV challenge. IMPORTANCE STAT4 is a protein critical for both innate and adaptive immune responses to viral infection. Our results show that STAT4 regulates the immune response to primary and secondary challenge with RSV but does not restrain RSV-induced lung Th2 or Th17 immune responses. These findings suggest that STAT4 expression may influence lung immunity and severity of illness following primary and secondary RSV infections. PMID:24920804

Two host microRNAs influence WSSV replication via STAT gene regulation.

PubMed

Huang, Ying; Wang, Wen; Ren, Qian

2016-03-31

MicroRNAs (miRNAs) have important roles in post-transcriptional regulation of gene expression. During viral infection, viruses utilize hosts to enhance their replication by altering cellular miRNAs. The Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway plays crucial roles in the antiviral responses. In this study, two miRNAs (miR-9041 and miR-9850) from Macrobrachium rosenbergii were found to promote white spot syndrome virus (WSSV) replication. The up-regulation of miR-9041 or miR-9850 suppresses STAT expression in the gills of M. rosenbergii, which subsequently down-regulates the expression of its downstream dynamin (Dnm) genes: Dnm1, Dnm2, and Dnm3. Knockdown of miR-9041 and miR-9850 restricts WSSV replication by up-regulating STAT and Dnm gene expression. The silencing of STAT, Dnm1, Dnm2, or Dnm3 led to an increase of the number of WSSV copies in shrimp. The injection of recombinant Dnm1, Dnm2, or Dnm3 proteins could inhibit WSSV replication in vivo. Overall, our research indicates the roles of host miRNAs in the enhancement of WSSV replication by regulating the host JAK/STAT pathway.

The Inhibition of Stat5 by a Peptide Aptamer Ligand Specific for the DNA Binding Domain Prevents Target Gene Transactivation and the Growth of Breast and Prostate Tumor Cells

PubMed Central

Weber, Axel; Borghouts, Corina; Brendel, Christian; Moriggl, Richard; Delis, Natalia; Brill, Boris; Vafaizadeh, Vida; Groner, Bernd

2013-01-01

The signal transducer and activator of transcription Stat5 is transiently activated by growth factor and cytokine signals in normal cells, but its persistent activation has been observed in a wide range of human tumors. Aberrant Stat5 activity was initially observed in leukemias, but subsequently also found in carcinomas. We investigated the importance of Stat5 in human tumor cell lines. shRNA mediated downregulation of Stat5 revealed the dependence of prostate and breast cancer cells on the expression of this transcription factor. We extended these inhibition studies and derived a peptide aptamer (PA) ligand, which directly interacts with the DNA-binding domain of Stat5 in a yeast-two-hybrid screen. The Stat5 specific PA sequence is embedded in a thioredoxin (hTRX) scaffold protein. The resulting recombinant protein S5-DBD-PA was expressed in bacteria, purified and introduced into tumor cells by protein transduction. Alternatively, S5-DBD-PA was expressed in the tumor cells after infection with a S5-DBD-PA encoding gene transfer vector. Both strategies impaired the DNA-binding ability of Stat5, suppressed Stat5 dependent transactivation and caused its intracellular degradation. Our experiments describe a peptide based inhibitor of Stat5 protein activity which can serve as a lead for the development of a clinically useful compound for cancer treatment. PMID:24276378

Nipah and Hendra Virus Nucleoproteins Inhibit Nuclear Accumulation of Signal Transducer and Activator of Transcription 1 (STAT1) and STAT2 by Interfering with Their Complex Formation

PubMed Central

Sugai, Akihiro; Sato, Hiroki; Takayama, Ikuyo; Yoneda, Misako

2017-01-01

ABSTRACT Henipaviruses, such as Nipah (NiV) and Hendra (HeV) viruses, are highly pathogenic zoonotic agents within the Paramyxoviridae family. The phosphoprotein (P) gene products of the paramyxoviruses have been well characterized for their interferon (IFN) antagonist activity and their contribution to viral pathogenicity. In this study, we demonstrated that the nucleoprotein (N) of henipaviruses also prevents the host IFN signaling response. Reporter assays demonstrated that the NiV and HeV N proteins (NiV-N and HeV-N, respectively) dose-dependently suppressed both type I and type II IFN responses and that the inhibitory effect was mediated by their core domains. Additionally, NiV-N prevented the nuclear transport of signal transducer and activator of transcription 1 (STAT1) and STAT2. However, NiV-N did not associate with Impα5, Impβ1, or Ran, which are members of the nuclear transport system for STATs. Although P protein is known as a binding partner of N protein and actively retains N protein in the cytoplasm, the IFN antagonist activity of N protein was not abolished by the coexpression of P protein. This suggests that the IFN inhibition by N protein occurs in the cytoplasm. Furthermore, we demonstrated that the complex formation of STATs was hampered in the N protein-expressing cells. As a result, STAT nuclear accumulation was reduced, causing a subsequent downregulation of interferon-stimulated genes (ISGs) due to low promoter occupancy by STAT complexes. This novel route for preventing host IFN responses by henipavirus N proteins provides new insight into the pathogenesis of these viruses. IMPORTANCE Paramyxoviruses are well known for suppressing interferon (IFN)-mediated innate immunity with their phosphoprotein (P) gene products, and the henipaviruses also possess P, V, W, and C proteins for evading host antiviral responses. There are numerous studies providing evidence for the relationship between viral pathogenicity and antagonistic activities against IFN responses by P gene products. Meanwhile, little attention has been paid to the influence of nucleoprotein (N) on host innate immune responses. In this study, we demonstrated that both the NiV and HeV N proteins have antagonistic activity against the JAK/STAT signaling pathway by preventing the nucleocytoplasmic trafficking of STAT1 and STAT2. This inhibitory effect is due to an impairment of the ability of STATs to form complexes. These results provide new insight into the involvement of N protein in viral pathogenicity via its IFN antagonism. PMID:28835499

Nipah and Hendra Virus Nucleoproteins Inhibit Nuclear Accumulation of Signal Transducer and Activator of Transcription 1 (STAT1) and STAT2 by Interfering with Their Complex Formation.

PubMed

Sugai, Akihiro; Sato, Hiroki; Takayama, Ikuyo; Yoneda, Misako; Kai, Chieko

2017-11-01

Henipaviruses, such as Nipah (NiV) and Hendra (HeV) viruses, are highly pathogenic zoonotic agents within the Paramyxoviridae family. The phosphoprotein (P) gene products of the paramyxoviruses have been well characterized for their interferon (IFN) antagonist activity and their contribution to viral pathogenicity. In this study, we demonstrated that the nucleoprotein (N) of henipaviruses also prevents the host IFN signaling response. Reporter assays demonstrated that the NiV and HeV N proteins (NiV-N and HeV-N, respectively) dose-dependently suppressed both type I and type II IFN responses and that the inhibitory effect was mediated by their core domains. Additionally, NiV-N prevented the nuclear transport of signal transducer and activator of transcription 1 (STAT1) and STAT2. However, NiV-N did not associate with Impα5, Impβ1, or Ran, which are members of the nuclear transport system for STATs. Although P protein is known as a binding partner of N protein and actively retains N protein in the cytoplasm, the IFN antagonist activity of N protein was not abolished by the coexpression of P protein. This suggests that the IFN inhibition by N protein occurs in the cytoplasm. Furthermore, we demonstrated that the complex formation of STATs was hampered in the N protein-expressing cells. As a result, STAT nuclear accumulation was reduced, causing a subsequent downregulation of interferon-stimulated genes (ISGs) due to low promoter occupancy by STAT complexes. This novel route for preventing host IFN responses by henipavirus N proteins provides new insight into the pathogenesis of these viruses. IMPORTANCE Paramyxoviruses are well known for suppressing interferon (IFN)-mediated innate immunity with their phosphoprotein (P) gene products, and the henipaviruses also possess P, V, W, and C proteins for evading host antiviral responses. There are numerous studies providing evidence for the relationship between viral pathogenicity and antagonistic activities against IFN responses by P gene products. Meanwhile, little attention has been paid to the influence of nucleoprotein (N) on host innate immune responses. In this study, we demonstrated that both the NiV and HeV N proteins have antagonistic activity against the JAK/STAT signaling pathway by preventing the nucleocytoplasmic trafficking of STAT1 and STAT2. This inhibitory effect is due to an impairment of the ability of STATs to form complexes. These results provide new insight into the involvement of N protein in viral pathogenicity via its IFN antagonism. Copyright © 2017 American Society for Microbiology.

Herbal remedy magnolol suppresses IL-6-induced STAT3 activation and gene expression in endothelial cells

PubMed Central

Chen, Shih-Chung; Chang, Ying-Ling; Wang, Danny Ling; Cheng, Jing-Jy

2006-01-01

Magnolol (Mag), an active constituent isolated from the Chinese herb Hou p'u (Magnolia officinalis) has long been used to suppress inflammatory processes. Chronic inflammation is well known to be involved in vascular injuries such as atherosclerosis in which interleukin (IL)-6 may participate. Signal transducer and activator of transcription protein 3 (STAT3), a transcription factor involved in inflammation and the cell cycle, is activated by IL-6. In this study, we evaluated whether Mag can serve as an anti-inflammatory agent during endothelial injuries. The effects of Mag on IL-6-induced STAT3 activation and downstream target gene induction in endothelial cells (ECs) were examined. Pretreatment of ECs with Mag dose dependently inhibited IL-6-induced Tyr705 and Ser727 phosphorylation in STAT3 without affecting the phosphorylation of JAK1, JAK2, and ERK1/2. Mag pretreatment of these ECs dose dependently suppressed IL-6-induced promoter activity of intracellular cell adhesion molecule (ICAM)-1 that contains functional IL-6 response elements (IREs). An electrophoretic mobility shift assay (EMSA) revealed that Mag treatment significantly reduced STAT3 binding to the IRE region. Consistently, Mag treatment markedly inhibited ICAM-1 expression on the endothelial surface. As a result, reduced monocyte adhesion to IL-6-activated ECs was observed. Furthermore, Mag suppressed IL-6-induced promoter activity of cyclin D1 and monocyte chemotactic protein (MCP)-1 for which STAT3 activation plays a role. In conclusion, our results indicate that Mag inhibits IL-6-induced STAT3 activation and subsequently results in the suppression of downstream target gene expression in ECs. These results provide a therapeutic basis for the development of Mag as an anti-inflammatory agent for vascular disorders including atherosclerosis. PMID:16520748

Herpes simplex virus-1 infection causes the secretion of a type I interferon-antagonizing protein and inhibits signaling at or before Jak-1 activation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Johnson, Karen E.; Knipe, David M., E-mail: david_knipe@hms.harvard.ed

2010-01-05

Host cells respond to viral infection by the production of type I interferons (IFNs), which induce the expression of antiviral genes. Herpes simplex virus I (HSV-1) encodes many mechanisms that inhibit the type I IFN response, including the ICP27-dependent inhibition of type I IFN signaling. Here we show inhibition of Stat-1 nuclear accumulation in cells that express ICP27. ICP27 expression also induces the secretion of a small, heat-stable type I IFN antagonizing protein that inhibits Stat-1 nuclear accumulation. We show that the inhibition of IFN-induced Stat-1 phosphorylation occurs at or upstream of Jak-1 phosphorylation. Finally, we show that ISG15 expressionmore » is induced after IFNalpha treatment in mock-infected cells, but not cells infected with WT HSV-1 or ICP27{sup -} HSV-1. These data suggest that HSV-1 has evolved multiple mechanisms to inhibit IFN signaling not only in infected cells, but also in neighboring cells, thereby allowing for increased viral replication and spread.« less

MicroRNA-194 promotes osteoblast differentiation via downregulating STAT1

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li, Jun; He, Xijing; Wei, Wenzhi

Osteoblast differentiation is a vital process in maintaining bone homeostasis in which various transcriptional factors, signaling molecules, and microRNAs (miRNAs) are involved. Recently, signal transducer and activator of transcription 1 (STAT1) has been found to play an important role in regulating osteoblast differentiation. Here, we identified that STAT1 expression was regulated by miR-194. Using mouse bone mesenchymal stem cells (BMSCs), we found that miR-194 expression was significantly increased following osteoblast differentiation induction. Overexpression of miR-194 by lentivirus-mediated gene transfer markedly increased osteoblast differentiation, whereas inhibition of miR-194 significantly suppressed osteoblast differentiation of BMSCs. Using a dual-luciferase reporter assay, a directmore » interaction between miR-194 and the 3′-untranslated region (UTR) of STAT1 was confirmed. Additionally, miR-194 regulated mRNA and protein expression of STAT1 in BMSCs. Further analysis showed that miR-194 overexpression promoted the nuclear translocation of runt-related transcription factor 2 (Runx2), which is critical for osteoblast differentiation. In contrast, inhibition of miR-194 blocked the nuclear translocation of Runx2. Moreover, overexpression of STAT1 significantly blocked Runx2 nuclear translocation and osteoblast differentiation mediated by miR-194 overexpression. Taken together, our data suggest that miR-194 regulates osteoblast differentiation through modulating STAT1-mediated Runx2 nuclear translocation. - Highlights: • Overexpression of miR-194 significantly increased osteoblast differentiation. • miR-194 directly targeted the 3′- UTR of STAT1. • miR-194 regulated the expression of STAT1. • Overexpression of miR-194 promoted the nuclear translocation of Runx2.« less

Expression of interferon-induced antiviral genes is delayed in a STAT1 knockout mouse model of Crimean-Congo hemorrhagic fever.

PubMed

Bowick, Gavin C; Airo, Adriana M; Bente, Dennis A

2012-06-19

Crimean Congo hemorrhagic fever (CCHF) is a tick-borne hemorrhagic zoonosis associated with high mortality. Pathogenesis studies and the development of vaccines and antivirals against CCHF have been severely hampered by the lack of suitable animal model. We recently developed and characterized a mature mouse model for CCHF using mice carrying STAT1 knockout (KO). Given the importance of interferons in controlling viral infections, we investigated the expression of interferon pathway-associated genes in KO and wild-type (WT) mice challenged with CCHF virus. We expected that the absence of the STAT1 protein would result in minimal expression of IFN-related genes. Surprisingly, the KO mice showed high levels of IFN-stimulated gene expression, beginning on day 2 post-infection, while in WT mice challenged with virus the same genes were expressed at similar levels on day 1. We conclude that CCHF virus induces similar type I IFN responses in STAT1 KO and WT mice, but the delayed response in the KO mice permits rapid viral dissemination and fatal illness.

Berberine-induced Inactivation of Signal Transducer and Activator of Transcription 5 Signaling Promotes Male-specific Expression of a Bile Acid Uptake Transporter*

PubMed Central

Bu, Pengli; Le, Yuan; Zhang, Yue; Zhang, Youcai; Cheng, Xingguo

2017-01-01

Sodium-taurocholate co-transporting polypeptide (Ntcp/NTCP) is the major uptake transporter of bile salts in mouse and human livers. In certain diseases, including endotoxemia, cholestasis, diabetes, and hepatocarcinoma, Ntcp/NTCP expression is markedly reduced, which interferes with enterohepatic circulation of bile salts, impairing the absorption of lipophilic compounds. Therefore, normal Ntcp/NTCP expression in the liver is physiologically important. Berberine is an herbal medicine used historically to improve liver function and has recently been shown to repress STAT signaling. However, berberine effects on Ntcp/NTCP expression are unknown, prompting use to investigate this possible connection. Our results showed that berberine dose-dependently increased Ntcp expression in male mouse liver and decreased taurocholic acid levels in serum but increased them in the liver. In mouse and human hepatoma cells, berberine induced Ntcp/NTCP mRNA and protein expression and increased cellular uptake of [3H] taurocholate. Mechanistically, berberine decreased nuclear protein levels of phospho-JAK2 and phospho-STAT5, thus disrupting the JAK2-STAT5 signaling. Moreover, berberine stimulated luciferase reporter expression from the mouse Ntcp promoter when one putative STAT5 response element (RE) (−1137 bp) was deleted and from the human NTCP promoter when three putative STAT5REs (−2898, −2164, and −691 bp) were deleted. Chromatin immunoprecipitation demonstrated that berberine decreased binding of phospho-STAT5 protein to the−2164 and −691 bp STAT5REs in the human NTCP promoter. In summary, berberine-disrupted STAT5 signaling promoted mouse and human Ntcp/NTCP expression, resulting in enhanced bile acid uptake. Therefore, berberine may be a therapeutic candidate compound for maintaining bile acid homeostasis. PMID:28154180

Berberine-induced Inactivation of Signal Transducer and Activator of Transcription 5 Signaling Promotes Male-specific Expression of a Bile Acid Uptake Transporter.

PubMed

Bu, Pengli; Le, Yuan; Zhang, Yue; Zhang, Youcai; Cheng, Xingguo

2017-03-17

Sodium-taurocholate co-transporting polypeptide (Ntcp/NTCP) is the major uptake transporter of bile salts in mouse and human livers. In certain diseases, including endotoxemia, cholestasis, diabetes, and hepatocarcinoma, Ntcp/NTCP expression is markedly reduced, which interferes with enterohepatic circulation of bile salts, impairing the absorption of lipophilic compounds. Therefore, normal Ntcp/NTCP expression in the liver is physiologically important. Berberine is an herbal medicine used historically to improve liver function and has recently been shown to repress STAT signaling. However, berberine effects on Ntcp/NTCP expression are unknown, prompting use to investigate this possible connection. Our results showed that berberine dose-dependently increased Ntcp expression in male mouse liver and decreased taurocholic acid levels in serum but increased them in the liver. In mouse and human hepatoma cells, berberine induced Ntcp/NTCP mRNA and protein expression and increased cellular uptake of [3H] taurocholate. Mechanistically, berberine decreased nuclear protein levels of phospho-JAK2 and phospho-STAT5, thus disrupting the JAK2-STAT5 signaling. Moreover, berberine stimulated luciferase reporter expression from the mouse Ntcp promoter when one putative STAT5 response element (RE) (-1137 bp) was deleted and from the human NTCP promoter when three putative STAT5REs (-2898, -2164, and -691 bp) were deleted. Chromatin immunoprecipitation demonstrated that berberine decreased binding of phospho-STAT5 protein to the-2164 and -691 bp STAT5REs in the human NTCP promoter. In summary, berberine-disrupted STAT5 signaling promoted mouse and human Ntcp/NTCP expression, resulting in enhanced bile acid uptake. Therefore, berberine may be a therapeutic candidate compound for maintaining bile acid homeostasis. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Unphosphorylated STATs go nuclear.

PubMed

Brown, Stephen; Zeidler, Martin P

2008-10-01

The JAK/STAT signal transduction pathway has traditionally been viewed as a cytokine-stimulated activator of gene expression consisting of a straightforward receptor/JAK kinase/STAT transcription factor cascade. Recent studies in Drosophila, have, however consistently identified a range of chromatin-remodelling factors as regulators of in vivo JAK/STAT signalling. Now, the detailed analysis of one of these, heterochromatin protein 1 (HP1), has provided an insight into an unexpected non-canonical in vivo role for STAT. In this model, unphosphorylated STATs associate with and maintain the stability of transcriptionally repressed heterochromatin--an effect countered by the recruitment of STAT to the canonical pathway. We examine the background of this new model and its implications for JAK/STAT pathway requirements in stem cell maintenance and cancer.

15-deoxy-Delta12,14-prostaglandin J2 inhibits INF-gamma-induced JAK/STAT1 signalling pathway activation and IP-10/CXCL10 expression in mesangial cells.

PubMed

Panzer, Ulf; Zahner, Gunther; Wienberg, Ulrike; Steinmetz, Oliver M; Peters, Anett; Turner, Jan-Eric; Paust, Hans-Joachim; Wolf, Gunter; Stahl, Rolf A K; Schneider, André

2008-12-01

Activators of the peroxisome proliferator-activated receptor gamma (PPARgamma), originally found to be implicated in lipid metabolism and glucose homeostasis, have been shown to modulate inflammatory responses through interference with cytokine and chemokine production. Given the central role of mesangial cell-derived chemokines in glomerular leukocyte recruitment in human and experimental glomerulonephritis, we studied the influence of natural and synthetic PPARgamma activators on INF-gamma-induced expression of the T cell-attracting chemokines IP-10/CXCL10, Mig/CXCL9 and I-TAC/CXCL11 in mouse mesangial cells. INF-gamma-treated mesangial cells were cultured in the presence or absence of either the naturally occurring PPARgamma ligand 15-deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)) or synthetic PPARgamma activators of the glitazone group. Chemokine mRNA and protein expression and activation of the JAK/STAT signalling pathway were analysed. The 15d-PGJ(2), but not synthetic PPARgamma ligands, dose-dependently inhibited INF-gamma-induced chemokine gene (mRNA and protein) expression. Combined results from EMSA and western blot analysis revealed the inhibitory ability of 15d-PGJ(2), but not of synthetic PPARgamma ligands, on IFN-gamma-induced tyrosine phosphorylation of JAK1, JAK2, STAT1 and nuclear STAT1 translocation and DNA binding. Our results demonstrate that 15d-PGJ(2) inhibits INF-gamma-induced chemokine expression in mesangial cells by targeting the JAK/STAT signalling pathway. This effect is independent of an interference with PPARgamma.

C/EBPβ Promotes STAT3 Expression and Affects Cell Apoptosis and Proliferation in Porcine Ovarian Granulosa Cells.

PubMed

Yuan, Xiaolong; Zhou, Xiaofeng; He, Yingting; Zhong, Yuyi; Zhang, Ailing; Zhang, Zhe; Zhang, Hao; Li, Jiaqi

2018-06-13

Previous studies suggest that signal transducer and activator of transcription 3 (STAT3) and CCAAT/enhancer binding protein beta (C/EBPβ) play an essential role in ovarian granulosa cells (GCs) for mammalian follicular development. Several C/EBPβ putative binding sites were previously predicted on the STAT3 promoter in mammals. However, the molecular regulation of C/EBPβ on STAT3 and their effects on cell proliferation and apoptosis remain virtually unexplored in GCs. Using porcine GCs as a model, the 5′-deletion, luciferase report assay, mutation, chromatin immunoprecipitation, Annexin-V/PI staining and EdU assays were applied to investigate the molecular mechanism for C/EBPβ regulating the expression of STAT3 and their effects on the cell proliferation and apoptosis ability. We found that over and interfering with the expression of C/EBPβ significantly increased and decreased the messenger RNA (mRNA) and protein levels of STAT3 , respectively. The dual luciferase reporter assay showed that C/EBPβ directly bound at −1397/−1387 of STAT3 to positively regulate the mRNA and protein expressions of STAT3 . Both C/EBPβ and STAT3 were observed to inhibit cell apoptosis and promote cell proliferation. Furthermore, C/EBPβ might enhance the antiapoptotic and pro-proliferative effects of STAT3 . These results would be of great insight in further exploring the molecular mechanism of C/EBPβ and STAT3 on the function of GCs and the development of ovarian follicles in mammals.

Sophoraflavanone G induces apoptosis of human cancer cells by targeting upstream signals of STATs.

PubMed

Kim, Byung-Hak; Won, Cheolhee; Lee, Yun-Han; Choi, Jung Sook; Noh, Kum Hee; Han, Songhee; Lee, Haeri; Lee, Chang Seok; Lee, Dong-Sup; Ye, Sang-Kyu; Kim, Myoung-Hwan

2013-10-01

Aberrantly activated signal transducer and activator of transcription (STAT) proteins are implicated with human cancers and represent essential roles for cancer cell survival and proliferation. Therefore, the development of small-molecule inhibitors of STAT signaling bearing pharmacological activity has therapeutic potential for the treatment of human cancers. In this study, we identified sophoraflavanone G as a novel small-molecule inhibitor of STAT signaling in human cancer cells. Sophoraflavanone G inhibited tyrosine phosphorylation of STAT proteins in Hodgkin's lymphoma and tyrosine phosphorylation of STAT3 in solid cancer cells by inhibiting phosphorylation of the Janus kinase (JAK) proteins, Src family tyrosine kinases, such as Lyn and Src, Akt, and ERK1/2. In addition, sophoraflavanone G inhibited STAT5 phosphorylation in murine-bone-marrow-derived pro-B cells transfected with translocated Ets Leukemia (TEL)-JAKs and cytokine-induced rat pre-T lymphoma cells, as well as STAT5b reporter activity in TEL-JAKs and STAT5b reporter systems. Sophoraflavanone G also inhibited nuclear factor-κB (NF-κB) signaling in multiple myeloma cells. Furthermore, sophoraflavanone G inhibited cancer cell proliferation and induced apoptosis by regulating the expression of apoptotic and anti-apoptotic proteins. Our data suggest that sophoraflavanone G is a novel small-molecule inhibitor of STAT signaling by targeting upstream signals of STATs that may have therapeutic potential for cancers caused by persistently activated STAT proteins. Copyright © 2013 Elsevier Inc. All rights reserved.

First-in-human trial of a STAT3 decoy oligonucleotide in head and neck tumors: implications for cancer therapy

PubMed Central

Sen, Malabika; Thomas, Sufi. M.; Kim, Seungwon; Yeh, Joanne I.; Ferris, Robert L.; Johnson, Jonas T.; Duvvuri, Umamaheswar; Lee, Jessica; Sahu, Nivedita; Joyce, Sonali; Freilino, Maria L.; Shi, Haibin; Li, Changyou; Ly, Danith; Rapireddy, Srinivas; Etter, Jonathan P.; Li, Pui-Kai; Wang, Lin; Chiosea, Simion; Seethala, Raja R.; Gooding, William. E.; Chen, Xiaomin; Kaminski, Naftali; Pandit, Kusum; Johnson, Daniel. E.; Grandis, Jennifer R.

2013-01-01

Despite evidence implicating transcription factors, including STAT3, in oncogenesis, these proteins have been regarded as “undruggable”. We developed a decoy targeting STAT3 and performed a phase 0 trial. Expression levels of STAT3 target genes were decreased in the head and neck cancers following injection with the STAT3 decoy compared with tumors receiving saline control. Decoys have not been amenable to systemic administration due to instability. To overcome this barrier, we linked the oligonucleotide strands using hexa-ethyleneglycol spacers. This cyclic STAT3 decoy bound with high affinity to STAT3 protein, reduced cellular viability, and suppressed STAT3 target gene expression in cancer cells. Intravenous injection of the cyclic STAT3 decoy inhibited xenograft growth and downregulated STAT3 target genes in the tumors. These results provide the first demonstration of a successful strategy to inhibit tumor STAT3 signaling via systemic administration of a selective STAT3 inhibitor, thereby paving the way for broad clinical development. PMID:22719020

Phosphoinositide 3-kinase/protein kinase B (PI3K/AKT) and Janus kinase/signal transducer and activator of transcription (JAK/STAT) follicular signalling is conserved in the mare ovary.

PubMed

Hall, Sally E; Upton, Rose M O; McLaughlin, Eileen A; Sutherland, Jessie M

2017-09-26

The mare ovary is unique in its anatomical structure; however, the signalling pathways responsible for physiological processes, such as follicular activation, remain uncharacterised. This provided us with the impetus to explore whether signalling molecules from important folliculogenesis pathways, phosphoinositide 3-kinase/protein kinase B (PI3K/AKT) and Janus kinase/signal transducer and activator of transcription (JAK/STAT), are conserved in the mare ovary. Messenger RNA expression of six genes important in follicle development was measured using quantitative polymerase chain reaction and protein localisation of key pathway members (PI3K, AKT1, phosphatase and tensin homologue (PTEN), JAK1, STAT3 and suppressor of cytokine signalling 4 (SOCS4)) was compared in tissue from fetal and adult mare ovaries. Tissue from adult ovaries exhibited significantly increased levels of mRNA expression of PI3K, AKT1, PTEN, JAK1, STAT3 and SOCS4 compared with tissue from fetal ovaries. PI3K, AKT1, JAK1 and STAT3 demonstrated redistributed localisation, from pregranulosa cells in fetal development, to both the oocyte and granulosa cells of follicles in the adult ovary, whilst negative feedback molecules PTEN and SOCS4 were only localised to the granulosa cells in the adult ovary. These findings suggest that the PI3K/AKT and JAK/STAT signalling pathways are utilised during folliculogenesis in the mare, similarly to previously studied mammalian species, and may serve as useful biomarkers for assessment of ovary development in the horse.

Interleukin-6 inhibits hepatic growth hormone signaling via upregulation of Cis and Socs-3.

PubMed

Denson, Lee A; Held, Matthew A; Menon, Ram K; Frank, Stuart J; Parlow, Albert F; Arnold, Dodie L

2003-04-01

Cytokines may cause an acquired growth hormone (GH) resistance in patients with inflammatory diseases. Anabolic effects of GH are mediated through activation of STAT5 transcription factors. We have reported that TNF-alpha suppresses hepatic GH receptor (GHR) gene expression, whereas the cytokine-inducible SH2-containing protein 1 (Cis)/suppressors of cytokine signaling (Socs) genes are upregulated by TNF-alpha and IL-6 and inhibit GH activation of STAT5. However, the relative importance of these mechanisms in inflammatory GH resistance was not known. We hypothesized that IL-6 would prevent GH activation of STAT5 and that this would involve Cis/Socs protein upregulation. GH +/- LPS was administered to TNF receptor 1 (TNFR1) or IL-6 null mice and wild-type (WT) controls. STAT5, STAT3, GHR, Socs 1-3, and Cis phosphorylation and abundance were assessed by using immunoblots, EMSA, and/or real time RT-PCR. TNF-alpha and IL-6 abundance were assessed by using ELISA. GH activated STAT5 in WT and TNFR1 or IL-6 null mice. LPS pretreatment prevented STAT5 activation in WT and TNFR1 null mice; however, STAT5 activation was preserved in IL-6 null mice. GHR abundance did not change with LPS administration. Inhibition of STAT5 activation by LPS was temporally associated with phosphorylation of STAT3 and upregulation of Cis and Socs-3 protein in WT and TNFR1 null mice; STAT3, Cis, and Socs-3 were not induced in IL-6 null mice. IL-6 inhibits hepatic GH signaling by upregulating Cis and Socs-3, which may involve activation of STAT3. Therapies that block IL-6 may enhance GH signaling in inflammatory diseases.

The JAK-STAT signaling pathway: input and output integration.

PubMed

Murray, Peter J

2007-03-01

Universal and essential to cytokine receptor signaling, the JAK-STAT pathway is one of the best understood signal transduction cascades. Almost 40 cytokine receptors signal through combinations of four JAK and seven STAT family members, suggesting commonality across the JAK-STAT signaling system. Despite intense study, there remain substantial gaps in understanding how the cascades are activated and regulated. Using the examples of the IL-6 and IL-10 receptors, I will discuss how diverse outcomes in gene expression result from regulatory events that effect the JAK1-STAT3 pathway, common to both receptors. I also consider receptor preferences by different STATs and interpretive problems in the use of STAT-deficient cells and mice. Finally, I consider how the suppressor of cytokine signaling (SOCS) proteins regulate the quality and quantity of STAT signals from cytokine receptors. New data suggests that SOCS proteins introduce additional diversity into the JAK-STAT pathway by adjusting the output of activated STATs that alters downstream gene activation.

Caffeic Acid Inhibits Chronic UVB-Induced Cellular Proliferation Through JAK-STAT3 Signaling in Mouse Skin.

PubMed

Agilan, Balupillai; Rajendra Prasad, N; Kanimozhi, Govindasamy; Karthikeyan, Ramasamy; Ganesan, Muthusamy; Mohana, Shanmugam; Velmurugan, Devadasan; Ananthakrishnan, Dhanapalan

2016-05-01

Signal transducers and activators of transcription 3 (STAT3) play a critical role in inflammation, proliferation and carcinogenesis. Inhibition of JAK-STAT3 signaling is proved to be a novel target for prevention of UVB-induced skin carcinogenesis. In this study, chronic UVB irradiation (180 mJ cm(-2) ; weekly thrice for 30 weeks) induces the expression of IL-10 and JAK1 that eventually activates the STAT3 which leads to the transcription of proliferative and antiapoptotic markers such as PCNA, Cyclin-D1, Bcl2 and Bcl-xl, respectively. Caffeic acid (CA) inhibits JAK-STAT3 signaling, thereby induces apoptotic cell death by upregulating Bax, Cytochrome-C, Caspase-9 and Caspase-3 expression in mouse skin. Furthermore, TSP-1 is an antiangiogeneic protein, which is involved in the inhibition of angiogenesis and proliferation. Chronic UVB exposure decreased the expression of TSP-1 and pretreatment with CA prevented the UVB-induced loss of TSP-1 in UVB-irradiated mouse skin. Thus, CA offers protection against UVB-induced photocarcinogenesis probably through modulating the JAK-STAT3 in the mouse skin. © 2016 The American Society of Photobiology.

Inhibitory effect of fluvoxamine on β-casein expression via a serotonin-independent mechanism in human mammary epithelial cells.

PubMed

Chiba, Takeshi; Maeda, Tomoji; Kimura, Soichiro; Morimoto, Yasunori; Sanbe, Atsushi; Ueda, Hideo; Kudo, Kenzo

2015-11-05

Selective serotonin reuptake inhibitors (SSRIs) are widely used as a first-line therapy in postpartum depression. The objective of this study was to determine the mechanism underlying the inhibitory effects of the SSRI, fluvoxamine, on β-casein expression, an indicator of lactation, in MCF-12A human mammary epithelial cells. Expression levels of serotonin (5-hydroxytryptamine; 5-HT) transporter, an SSRI target protein, and tryptophan hydroxylase 1, a rate-limiting enzyme in 5-HT biosynthesis, were increased in MCF-12A cells by prolactin treatment. Treatment with 1 μM fluvoxamine for 72 h significantly decreased protein levels of β-casein and phosphorylated signal transducer and activator transcription 5 (pSTAT5). Extracellular 5-HT levels were significantly increased after exposure to 1 μM fluvoxamine, in comparison with those of untreated and vehicle-treated cells; however, extracellular 5-HT had little effect on the decrease in β-casein expression. Expression of glucose-related protein 78/binding immunoglobulin protein, a regulator of endoplasmic reticulum (ER) stress, was significantly increased after treatment with 1 μM fluvoxamine for 48 h. Exposure to tunicamycin, an inducer of ER stress, also decreased expression of β-casein and pSTAT5 in a manner similar to fluvoxamine. Our results indicate that fluvoxamine suppresses β-casein expression in MCF-12A cells via inhibition of STAT5 phosphorylation caused by induction of ER stress. Further studies are required to confirm the effect of fluvoxamine on the function of mammary epithelial cells. Copyright © 2015 Elsevier B.V. All rights reserved.

MRTF-A mediated FN and ICAM-1 expression in AGEs-induced rat glomerular mesangial cells via activating STAT5.

PubMed

Chen, Qiuhong; Huang, Junying; Gong, Wenyan; Chen, Zhiquan; Huang, Jiani; Liu, Peiqing; Huang, Heqing

2018-01-15

Advanced glycation end products (AGEs), formed at an accelerated rate under diabetes, play a role in inflammation and fibrosis in mesangial areas in diabetic nephropathy (DN). However, the transcriptional modulator that mediates the cellular response to AGEs remains largely obscure. Our goal was to determine whether myocardin-related transcription factor (MRTF)-A, a key protein involved in the transcriptional regulation of smooth muscle cell phenotype, was responsible for the glomerular mesangial cells (GMCs) injury by AGEs, and, if so, how MRTF-A promoted mesangial dysfunction initiated by AGEs. In this study, MRTF-A was activated by AGEs in terms of protein expression and nuclear translocation in rat GMCs. MRTF-A overexpression synergistically enhanced the induction of FN and ICAM-1 by AGEs. In contract, depletion of MRTF-A abrogated the pathogenic program triggered by AGEs. Then, by interfering with MRTF-A, STAT1, STAT3 and STAT5 nuclear translocation were observed and we screened out STAT5, which was decreased obviously when MRTF-A depleted. Further investigation showed that MRTF-A interacted with STAT5 and promoted its nuclear accumulation and transcriptional activity. Therefore, our present findings suggested a role of MRTF-A in AGEs-induced GMCs injury, and further revealed that the underlying molecular mechanism was related to activating the nuclear factor STAT5. Copyright © 2017 Elsevier B.V. All rights reserved.

Constitutive IDO expression in human cancer is sustained by an autocrine signaling loop involving IL-6, STAT3 and the AHR

PubMed Central

Sahm, Felix; Rauschenbach, Katharina J.; Trump, Saskia; Winter, Marcus; Ott, Martina; Ochs, Katharina; Lutz, Christian; Liu, Xiangdong; Anastasov, Natasa; Lehmann, Irina; Höfer, Thomas; von Deimling, Andreas; Wick, Wolfgang; Platten, Michael

2014-01-01

Indoleamine-2,3-dioxygenase (IDO) inhibitors have entered clinical trials based on their ability to restore anti-tumor immunity in preclinical studies. However, the mechanisms leading to constitutive expression of IDO in human tumors are largely unknown. Here we analyzed the pathways mediating constitutive IDO expression in human cancer. IDO-positive tumor cells and tissues showed basal phosphorylation and acetylation of STAT3 as evidenced by western blotting and immunoprecipitation. Inhibition of IL-6 or STAT3 using siRNA and/or pharmacological inhibitors reduced IDO mRNA and protein expression as well as kynurenine formation. In turn, IDO enzymatic activity activated the AHR as shown by the induction of AHR target genes. IDO-mediated AHR activation induced IL-6 expression, while inhibition or knockdown of the AHR reduced IL-6 expression. IDO activity thus sustains its own expression via an autocrine AHR–IL-6–STAT3 signaling loop. Inhibition of the AHR–IL-6–STAT3 signaling loop restored T-cell proliferation in mixed leukocyte reactions performed in the presence of IDO-expressing human cancer cells. Identification of the IDO-AHR-IL-6-STAT3 signaling loop maintaining IDO expression in human cancers reveals novel therapeutic targets for the inhibition of this core pathway promoting immunosuppression of human cancers. The relevance of the IDO-AHR-IL-6-STAT3 transcriptional circuit is underscored by the finding that high expression of its members IDO, STAT3 and the AHR target gene CYP1B1 is associated with reduced relapse-free survival in lung cancer patients. PMID:24657910

The effect of leptin and resveratrol on JAK/STAT pathways and Sirt-1 gene expression in the renal tissue of ischemia/reperfusion induced rats.

PubMed

Erkasap, S; Erkasap, N; Bradford, B; Mamedova, L; Uysal, O; Ozkurt, M; Ozyurt, R; Kutlay, O; Bayram, B

2017-01-01

Our study aimed to investigate the possible modifying effects of leptin and combined use of resveratrol on rat renal I/R injury and their relationship on signal pathways and apoptosis-related mechanisms. Renal ischemia-reperfusion (I/R) injury is an important cause of acute renal failure. Male Sprague Dawley rats were divided into 5 groups: Control, I/R, I/R+leptin, I/R+resveratrol and I/R+leptin+resveratrol. Leptin (10 μg/kg BW) was administered (i.p.) 30 min prior to I/R. Resveratrol was administered by gavage at 20 mg/kg BW per d for 12 d prior to I/R. The left renal artery was exposed to 1 h of ischemia and 1 h of reperfusion. Resveratrol treatment alone increased TNF-α, TNF-α R1, NF-κB, SIRT-1, STAT1 and STAT3 mRNA levels and decreased caspase 3 protein levels. Leptin treatment alone significantly decreased the caspase 3 protein levels. The combined use of resveratrol and leptin significantly increased STAT3, and caspase 3 mRNA levels, and decreased the caspase 3 protein levels. Apoptosis was significantly decreased especially in the leptin and leptin+resveratrol groups. The present study suggest that a combined use of resveratrol and leptin has preventive and regulatory effects on renal I/R injury; the mechanism involves decreasing apoptosis, likely by altering the JAK/STAT pathway and SIRT1 expression (Fig. 8, Ref. 24).

Antimycotics suppress the Malassezia extract-induced production of CXC chemokine ligand 10 in human keratinocytes.

PubMed

Hau, Carren S; Kanda, Naoko; Makimura, Koichi; Watanabe, Shinichi

2014-02-01

Malassezia, a lipophilic yeast, exacerbates atopic dermatitis. Malassezia products can penetrate the disintegrated stratum corneum and encounter subcorneal keratinocytes in the skin of atopic dermatitis patients. Type 1 helper T (Th1) cells infiltrate chronic lesions with atopic dermatitis, and antimycotic agents improve its symptoms. We aimed to identify Malassezia-induced chemokines in keratinocytes and examine whether antimycotics suppressed this induction. Normal human keratinocytes were incubated with a Malassezia restricta extract and antimycotics. Chemokine expression was analyzed by enzyme-linked immunosorbent assays and real-time polymerase chain reaction. Signal transducer and activator of transcription (STAT)1 activity was examined by luciferase assays. The tyrosine-phosphorylation of STAT1 was analyzed by western blotting. The M. restricta extract increased the mRNA and protein expression of Th1-attracting CXC chemokine ligand (CXCL)10 and STAT1 activity and phosphorylation in keratinocytes, which was suppressed by a Janus kinase inhibitor. The antimycotics itraconazole, ketoconazole, luliconazole, terbinafine, butenafine and amorolfine suppressed M. restricta extract-induced CXCL10 mRNA and protein expression and STAT1 activity and phosphorylation. These effects were similarly induced by 15-deoxy-Δ-(12,14) -prostaglandin J2 (15d-PGJ2 ), a prostaglandin D2 metabolite. Antimycotics increased the release of 15d-PGJ2 from keratinocytes. The antimycotic-induced suppression of CXCL10 production and STAT1 activity was counteracted by a lipocalin-type prostaglandin D synthase inhibitor. The antimycotics itraconazole, ketoconazole, luliconazole, terbinafine, butenafine and amorolfine may suppress the M. restricta-induced production of CXCL10 by inhibiting STAT1 through an increase in 15d-PGJ2 production in keratinocytes. These antimycotics may block the Th1-mediated inflammation triggered by Malassezia in the chronic phase of atopic dermatitis. © 2014 Japanese Dermatological Association.

Trichostatin A, a histone deacetylase inhibitor, suppresses JAK2/STAT3 signaling via inducing the promoter-associated histone acetylation of SOCS1 and SOCS3 in human colorectal cancer cells.

PubMed

Xiong, Hua; Du, Wan; Zhang, Yan-Jie; Hong, Jie; Su, Wen-Yu; Tang, Jie-Ting; Wang, Ying-Chao; Lu, Rong; Fang, Jing-Yuan

2012-02-01

Aberrant janus kinase/signal transducers and activators of transcription (JAK/STAT) signaling is involved in the oncogenesis of several cancers. Suppressors of cytokine signaling (SOCS) genes and SH2-containing protein tyrosine phosphatase 1 (SHP1) proteins, which are negative regulators of JAK/STAT signaling, have been reported to have tumor suppressor functions. However, in colorectal cancer (CRC) cells, the mechanisms that regulate SOCS and SHP1 genes, and the cause of abnormalities in the JAK/STAT signaling pathway, remain largely unknown. The present study shows that trichostatin A (TSA), a histone deacetylase (HDAC) inhibitor, leads to the hyperacetylation of histones associated with the SOCS1 and SOCS3 promoters, but not the SHP1 promoter in CRC cells. This indicates that histone modifications are involved in the regulation of SOCS1 and SOCS3. Moreover, upregulation of SOCS1 and SOCS3 expression was achieved using TSA, which also significantly downregulated JAK2/STAT3 signaling in CRC cells. We also demonstrate that TSA suppresses the growth of CRC cells, and induces G1 cell cycle arrest and apoptosis through the regulation of downstream targets of JAK2/STAT3 signaling, including Bcl-2, survivin and p16(ink4a) . Therefore, our data demonstrate that TSA may induce SOCS1 and SOCS3 expression by inducing histone modifications and consequently inhibits JAK2/STAT3 signaling in CRC cells. These results also establish a mechanistic link between the inhibition of JAK2/STAT3 signaling and the anticancer action of TSA in CRC cells. Copyright © 2011 Wiley Periodicals, Inc.

Withaferin A down-regulates lipopolysaccharide-induced cyclooxygenase-2 expression and PGE2 production through the inhibition of STAT1/3 activation in microglial cells.

PubMed

Min, Kyoung-Jin; Choi, Kyounghwa; Kwon, Taeg Kyu

2011-08-01

Microglia are the major immune effector cells in the brain, and microglia activated by injury and infection can produce inflammatory mediators. A number of studies have reported that withaferin A has anti-inflammatory functions. However, the effects of withaferin A on the microglial inflammatory response have not been investigated. Our results show that withaferin A inhibited lipopolysaccharide (LPS)-induced cyclooxygenase (COX)-2 mRNA and protein expression and prostaglandin E2 (PGE(2)) production in BV2 murine microglial cells. Withaferin A had no effect on LPS-induced Akt and ERK phosphorylation, but phosphorylation of p38 and JNK was slightly decreased by withaferin A. Withaferin A significantly inhibited LPS-induced STAT1 and STAT3 phosphorylation in a dose-dependent manner. Furthermore, withaferin A inhibited nuclear translocation of STAT1 and interferon-gamma activated sequence (GAS)-promoter activity. Taken together, these results suggest that withaferin A inhibits LPS-induced PGE(2) production and COX-2 expression, at least in part, by blocking STAT1 and STAT3 activation. Copyright © 2011 Elsevier B.V. All rights reserved.

Follistatin-like protein 1 induction of matrix metalloproteinase 1, 3 and 13 gene expression in rheumatoid arthritis synoviocytes requires MAPK, JAK/STAT3 and NF-κB pathways.

PubMed

Ni, Su; Li, Chenkai; Xu, Nanwei; Liu, Xi; Wang, Wei; Chen, Wenyang; Wang, Yuji; van Wijnen, Andre J

2018-06-22

Elevated levels of follistatin-like protein 1 (FSTL1) have been found both in mouse models for human rheumatoid arthritis (RA) and collagen-induced arthritis (CIA). In this study, we elucidated the potential mechanisms by which FSTL1 contributes to the pathogenesis of RA. Fibroblast-like synoviocytes (FLSs) were established from synovial tissues of RA patients and stimulated with human recombinant FSTL1. Protein and mRNA expression levels of select matrix metalloproteinases (i.e., MMP1, MMP3, MMP13) in FLS were measured by, respectively, real-time RT-qPCR and ELISA. Activation of MAPK and other pathways that affect MMPs were evaluated by Western blotting. We also compared concentrations of MMPs in plasma in RA patients versus healthy controls (HC). Expression levels of MMP1, MMP3, and MMP13 were clearly stimulated by FSTL1 in vitro. FSTL1 activated the inflammation-related NF-κB signaling pathway, as well as all three mitogen-activated protein kinase (MAPK) pathways and the JAK/STAT3 pathway. Moreover, select chemical inhibitors that target p38 (SB203580), Erk1/2 (SP600125), JNK (SCH772984), STAT3 (AG490), and NF-κB (BAY 11-7082) significantly attenuated MMP expression. Inhibition of Toll-like receptor 4 by compound TAK-242 significantly abolished those effects of FSTL1. Importantly, elevated plasma concentrations of MMP3 were found to correlate with plasma FSTL1 levels in RA patients. These findings suggest that FSTL1 accelerates RA progression by activating MAPK, JAK/STAT3, and NF-κB pathways to enhance secretion of different MMPs and this enhancement is via TLR4. Targeting FSTL1 may provide a promising pharmacological drug therapy to ameliorate RA symptoms and perhaps reverse disease progression. © 2018 Wiley Periodicals, Inc.

Tyrosine 110 in the measles virus phosphoprotein is required to block STAT1 phosphorylation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Devaux, Patricia; Messling, Veronika von; Songsungthong, Warangkhana

2007-03-30

The measles virus (MV) P gene encodes three proteins: P, an essential polymerase cofactor, and C and V, which have multiple functions including immune evasion. We show here that the MV P protein also contributes to immune evasion, and that tyrosine 110 is required to block nuclear translocation of the signal transducer and activator of transcription factors (STAT) after interferon type I treatment. In particular, MV P inhibits STAT1 phosphorylation. This is shown not only by transient expression but also by reverse genetic analyses based on a new functional infectious cDNA derived from a MV vaccine vial (Moraten strain). Ourmore » study also identifies a conserved sequence around P protein tyrosine 110 as a candidate interaction site with a cellular protein.« less

STAT6 is a cargo of exportin 1: Biological relevance in primary mediastinal B-cell lymphoma.

PubMed

Miloudi, Hadjer; Leroy, Karen; Jardin, Fabrice; Sola, Brigitte

2018-06-01

Primary mediastinal B-cell lymphoma (PMBL) is a distinct B-cell lymphoma subtype with unique clinicopathological and molecular features. PMBL cells are characterised by several genetic abnormalities that conduct to the constitutive activation of the Janus kinase 2/signal transducer and activator of transcription 6 (JAK2/STAT6) signalling pathway. Among recurrent genetic changes in PMBL, we previously reported that the XPO1 gene encoding exportin 1 that controls the nuclear export of cargo proteins and RNAs, is mutated (p.E571K) in about 25% of PMBL cases. We therefore hypothesized that STAT6 could be a cargo of XPO1 and that STAT6 cytoplasm/nucleus shuttle could be altered in a subset of PMBL cells. Using immunocytochemistry techniques as well as the proximity ligation assay, we showed that STAT6 bound XPO1 in PBML cell lines and in HEK-293 cells genetically engineered to produce STAT6. Moreover, XPO1-mediated export of STAT6 occurs in cells expressing either a wild-type or the E571K mutated XPO1 protein. Copyright © 2018 Elsevier Inc. All rights reserved.

Deficiency of PTP1B Attenuates Hypothalamic Inflammation via Activation of the JAK2-STAT3 Pathway in Microglia.

PubMed

Tsunekawa, Taku; Banno, Ryoichi; Mizoguchi, Akira; Sugiyama, Mariko; Tominaga, Takashi; Onoue, Takeshi; Hagiwara, Daisuke; Ito, Yoshihiro; Iwama, Shintaro; Goto, Motomitsu; Suga, Hidetaka; Sugimura, Yoshihisa; Arima, Hiroshi

2017-02-01

Protein tyrosine phosphatase 1B (PTP1B) regulates leptin signaling in hypothalamic neurons via the JAK2-STAT3 pathway. PTP1B has also been implicated in the regulation of inflammation in the periphery. However, the role of PTP1B in hypothalamic inflammation, which is induced by a high-fat diet (HFD), remains to be elucidated. Here, we showed that STAT3 phosphorylation (p-STAT3) was increased in microglia in the hypothalamic arcuate nucleus of PTP1B knock-out mice (KO) on a HFD, accompanied by decreased Tnf and increased Il10 mRNA expression in the hypothalamus compared to wild-type mice (WT). In hypothalamic organotypic cultures, incubation with TNFα led to increased p-STAT3, accompanied by decreased Tnf and increased Il10 mRNA expression, in KO compared to WT. Incubation with p-STAT3 inhibitors or microglial depletion eliminated the differences in inflammation between genotypes. These data indicate an important role of JAK2-STAT3 signaling negatively regulated by PTP1B in microglia, which attenuates hypothalamic inflammation under HFD conditions. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

Recombinant human brain natriuretic peptide ameliorates trauma-induced acute lung injury via inhibiting JAK/STAT signaling pathway in rats.

PubMed

Song, Zhi; Zhao, Xiu; Gao, Yan; Liu, Martin; Hou, Mingxiao; Jin, Hongxu; Cui, Yan

2015-05-01

JAK/STAT signal pathway plays an important role in the inflammation process of acute lung injury (ALI). This study aimed to investigate the correlation between recombinant human brain natriuretic peptide (rhBNP) and the JAK/STAT signaling pathway and to explore the protective mechanism of rhBNP against trauma-induced ALI. The arterial partial pressure in oxygen, lung wet-dry weight ratios, protein content in bronchoalveolar lavage fluid, the histopathologic of the lung, as well as the protein expressions of STAT1, JAK2, and STAT3 were detected. Sprague-Dawley rats were randomly divided into five groups: a control group, a sham-operated group, an ALI group, an ALI + rhBNP group, and an ALI + AG490 group. At 4 hours, 12 hours, 1 day, 3 days, and 7 days after injury, injured lung specimens were harvested. rhBNP pretreatment significantly ameliorated hypoxemia and histopathologic changes and alleviated pulmonary edema in trauma-induced ALI rats. rhBNP pretreatment reduced the phosphorylated protein and total protein level of STAT1. Similarly to JAK-specific inhibitor AG490, rhBNP was shown to significantly inhibit the phosphorylation of JAK2 and STAT3 in rats with trauma-induced ALI. Our experimental findings indicated that rhBNP can protect rats against trauma-induced ALI and that its underlying mechanism may be related to the inhibition of JAK/STAT signaling pathway activation.

Identification of Fat Mass and Obesity Associated (FTO) Protein Expression in Cardiomyocytes: Regulation by Leptin and Its Contribution to Leptin-Induced Hypertrophy

PubMed Central

Gan, Xiaohong Tracey; Zhao, Ganjian; Huang, Cathy X.; Rowe, Adrianna C.; Purdham, Daniel M.; Karmazyn, Morris

2013-01-01

The recently-identified fat mass and obesity-associated (FTO) protein is associated with various physiological functions including energy and body weight regulation. Ubiquitously expressed, FTO was identified in heart homogenates although its function is unknown. We studied whether FTO is specifically expressed within the cardiac myocyte and its potential role pertaining to the hypertrophic effect of the adipokine leptin. Most experiments were performed using cultured neonatal rat cardiomyocytes which showed nuclei-specific FTO expression. Leptin significantly increased FTO expression which was associated with myocyte hypertrophy although both events were abrogated by FTO knockdown with siRNA. Administration of a leptin receptor antibody to either normal or obese rats significant reduced myocardial FTO protein expression. Responses in cardiomyocytes were accompanied by JAK2/STAT3 activation whereas JAK2/STAT3 inhibition abolished these effects. Expression of the cut-like homeobox 1(CUX1) transcriptional factor was significantly increased by leptin although this was restricted to the cathepsin L-dependent, proteolytically-derived shorter p110CUX1 isoform whereas the longer p200CUX1 protein was not significantly affected. Cathepsin L expression and activity were both significantly increased by leptin whereas a cathepsin L peptide inhibitor or siRNA specific for CUX1 completely prevented the leptin-induced increase in FTO expression. The cathepsin L peptide inhibitor or siRNA-induced knockdown of either CUX1 or FTO abrogated the hypertrophic response to leptin. Two other pro-hypertrophic factors, endothelin-1 or angiotensin II had no effect on FTO expression and FTO knockdown did not alter the hypertrophic response to either agent. This study demonstrates leptin-induced FTO upregulation in cardiomyocytes via JAK2/STAT3- dependent CUX1 upregulation and suggests an FTO regulatory function of leptin. It also demonstrates for the first time a functional role of FTO in the cardiomyocyte. PMID:24019958

Identification of fat mass and obesity associated (FTO) protein expression in cardiomyocytes: regulation by leptin and its contribution to leptin-induced hypertrophy.

PubMed

Gan, Xiaohong Tracey; Zhao, Ganjian; Huang, Cathy X; Rowe, Adrianna C; Purdham, Daniel M; Karmazyn, Morris

2013-01-01

The recently-identified fat mass and obesity-associated (FTO) protein is associated with various physiological functions including energy and body weight regulation. Ubiquitously expressed, FTO was identified in heart homogenates although its function is unknown. We studied whether FTO is specifically expressed within the cardiac myocyte and its potential role pertaining to the hypertrophic effect of the adipokine leptin. Most experiments were performed using cultured neonatal rat cardiomyocytes which showed nuclei-specific FTO expression. Leptin significantly increased FTO expression which was associated with myocyte hypertrophy although both events were abrogated by FTO knockdown with siRNA. Administration of a leptin receptor antibody to either normal or obese rats significant reduced myocardial FTO protein expression. Responses in cardiomyocytes were accompanied by JAK2/STAT3 activation whereas JAK2/STAT3 inhibition abolished these effects. Expression of the cut-like homeobox 1(CUX1) transcriptional factor was significantly increased by leptin although this was restricted to the cathepsin L-dependent, proteolytically-derived shorter p110CUX1 isoform whereas the longer p200CUX1 protein was not significantly affected. Cathepsin L expression and activity were both significantly increased by leptin whereas a cathepsin L peptide inhibitor or siRNA specific for CUX1 completely prevented the leptin-induced increase in FTO expression. The cathepsin L peptide inhibitor or siRNA-induced knockdown of either CUX1 or FTO abrogated the hypertrophic response to leptin. Two other pro-hypertrophic factors, endothelin-1 or angiotensin II had no effect on FTO expression and FTO knockdown did not alter the hypertrophic response to either agent. This study demonstrates leptin-induced FTO upregulation in cardiomyocytes via JAK2/STAT3- dependent CUX1 upregulation and suggests an FTO regulatory function of leptin. It also demonstrates for the first time a functional role of FTO in the cardiomyocyte.

Interferon-gamma regulates nucleoside transport systems in macrophages through signal transduction and activator of transduction factor 1 (STAT1)-dependent and -independent signalling pathways.

PubMed Central

Soler, Concepció; Felipe, Antonio; García-Manteiga, José; Serra, Maria; Guillén-Gómez, Elena; Casado, F Javier; MacLeod, Carol; Modolell, Manuel; Pastor-Anglada, Marçal; Celada, Antonio

2003-01-01

The expressions of CNT and ENT (concentrative and equilibrative nucleoside transporters) in macrophages are differentially regulated by IFN-gamma (interferon-gamma). This cytokine controls gene expression through STAT1-dependent and/or -independent pathways (where STAT1 stands for signal transduction and activator of transcription 1). In the present study, the role of STAT1 in the response of nucleoside transporters to IFN-gamma was studied using macrophages from STAT1 knockout mice. IFN-gamma triggered an inhibition of ENT1-related nucleoside transport activity through STAT1-dependent mechanisms. Such inhibition of macrophage growth and ENT1 activity by IFN-gamma is required for DNA synthesis. Interestingly, IFN-gamma led to an induction of the CNT1- and CNT2-related nucleoside transport activities independent of STAT1, thus ensuring the supply of extracellular nucleosides for the STAT1-independent RNA synthesis. IFN-gamma up-regulated CNT2 mRNA and CNT1 protein levels and down-regulated ENT1 mRNA in both wild-type and STAT1 knockout macrophages. This is consistent with a STAT1-independent, long-term-mediated, probably transcription-dependent, regulation of nucleoside transporter genes. Moreover, STAT1-dependent post-transcriptional mechanisms are implicated in the regulation of ENT1 activity. Although nitric oxide is involved in the regulation of ENT1 activity in B-cells at a post-transcriptional level, our results show that STAT1-dependent induction of nitric oxide by IFN-gamma is not implicated in the regulation of ENT1 activity in macrophages. Our results indicate that both STAT1-dependent and -independent pathways are involved in the regulation of nucleoside transporters by IFN-gamma in macrophages. PMID:12868960

SOCS3 promotes apoptosis of mammary differentiated cells.

PubMed

Le Provost, Fabienne; Miyoshi, Keiko; Vilotte, Jean-Luc; Bierie, Brian; Robinson, Gertraud W; Hennighausen, Lothar

2005-12-30

Growth and function of the mammary gland is regulated by cytokines and modulated by suppressor of cytokine signalling (SOCS) proteins. In vitro experiments demonstrated that SOCS3 can inhibit PRL induction of milk protein gene expression and STAT5 activation. We explored the SOCS3 expression pattern during mouse mammary development and its regulation by PRL and GH in wild-type and STAT5a-null mammary tissue. Our results suggest that, in vivo, PRL stimulates SOCS3 expression in stromal adipocytes, independently of STAT5a stimulation. In mammary epithelial cells, SOCS3 expression appears to be related to STAT3 activation. Together, our results are consistent with a role of SOCS3 in the mammary gland by promoting apoptosis of differentiated cells (adipocytes during gestation and epithelial cells during involution).

STAT3 Knockdown Reduces Pancreatic Cancer Cell Invasiveness and Matrix Metalloproteinase-7 Expression in Nude Mice

PubMed Central

Huang, Ke jian; Wu, Wei dong; Jiang, Tao; Cao, Jun; Feng, Zhen zhong; Qiu, Zheng jun

2011-01-01

Aims Transducer and activator of transcription-3 (STAT3) plays an important role in tumor cell invasion and metastasis. The aim of the present study was to investigate the effects of STAT3 knockdown in nude mouse xenografts of pancreatic cancer cells and underlying gene expression. Methods A STAT3 shRNA lentiviral vector was constructed and infected into SW1990 cells. qRT-PCR and western immunoblot were performed to detect gene expression. Nude mouse xenograft assays were used to assess changes in phenotypes of these stable cells in vivo. HE staining was utilized to evaluate tumor cell invasion and immunohistochemistry was performed to analyze gene expression. Results STAT3 shRNA successfully silenced expression of STAT3 mRNA and protein in SW1990 cells compared to control cells. Growth rate of the STAT3-silenced tumor cells in nude mice was significantly reduced compared to in the control vector tumors and parental cells-generated tumors. Tumor invasion into the vessel and muscle were also suppressed in the STAT3-silenced tumors compared to controls. Collagen IV expression was complete and continuous surrounding the tumors of STAT3-silenced SW1990 cells, whereas collagen IV expression was incomplete and discontinuous surrounding the control tumors. Moreover, microvessel density was significantly lower in STAT3-silenced tumors than parental or control tumors of SW1990 cells. In addition, MMP-7 expression was reduced in STAT3-silenced tumors compared to parental SW1990 xenografts and controls. In contrast, expression of IL-1β and IgT7α was not altered. Conclusion These data clearly demonstrate that STAT3 plays an important role in regulation of tumor growth, invasion, and angiogenesis, which could be act by reducing MMP-7 expression in pancreatic cancer cells. PMID:21991388

Retinoic acid induces signal transducer and activator of transcription (STAT) 1, STAT2, and p48 expression in myeloid leukemia cells and enhances their responsiveness to interferons.

PubMed

Matikainen, S; Ronni, T; Lehtonen, A; Sareneva, T; Melén, K; Nordling, S; Levy, D E; Julkunen, I

1997-06-01

IFNs are antiproliferative cytokines that have growth-inhibitory effects on various normal and malignant cells. Therefore, they have been used in the treatment of certain forms of cancer, such as chronic myelogenous leukemia and hairy cell leukemia. However, there is little evidence that IFNs would be effective in the treatment of acute myelogenous leukemia, and molecular mechanisms underlying IFN unresponsiveness have not been clarified. Here we have studied the activation and induction of IFN-specific transcription factors signal transducer and activator of transcription (STAT) 1, STAT2, and p48 in all-trans-retinoic acid (ATRA)-differentiated myeloid leukemia cells using promyelocytic NB4, myeloblastic HL-60, and monoblastic U937 cells as model systems. These cells respond to ATRA by growth inhibition and differentiation. We show that in undifferentiated NB4 cells, 2',5'-oligoadenylate synthetase and MxB gene expression is not activated by IFN-alpha, possibly due to a relative lack of signaling molecules, especially p48 protein. However, during ATRA-induced differentiation, steady-state STAT1, STAT2, and especially p48 mRNA and corresponding protein levels were elevated both in NB4 and U937 cells, apparently correlating to an enhanced responsiveness of these cells to IFNs. ATRA treatment of NB4 cells sensitized them to IFN action as seen by increased IFN-gamma activation site DNA-binding activity or by efficient formation of IFN-alpha-specific ISGF3 complex and subsequent oligoadenylate synthetase and MxB gene expression. Lack of p48 expression could be one of the mechanisms of promyelocytic leukemia cell escape from growth-inhibitory effects of IFN-alpha.

T-Cell Protein Tyrosine Phosphatase Attenuates STAT3 and Insulin Signaling in the Liver to Regulate Gluconeogenesis

PubMed Central

Fukushima, Atsushi; Loh, Kim; Galic, Sandra; Fam, Barbara; Shields, Ben; Wiede, Florian; Tremblay, Michel L.; Watt, Matthew J.; Andrikopoulos, Sofianos; Tiganis, Tony

2010-01-01

OBJECTIVE Insulin-induced phosphatidylinositol 3-kinase (PI3K)/Akt signaling and interleukin-6 (IL-6)-instigated JAK/STAT3-signaling pathways in the liver inhibit the expression of gluconeogenic genes to decrease hepatic glucose output. The insulin receptor (IR) and JAK1 tyrosine kinases and STAT3 can serve as direct substrates for the T-cell protein tyrosine phosphatase (TCPTP). Homozygous TCPTP-deficiency results in perinatal lethality prohibiting any informative assessment of TCPTP's role in glucose homeostasis. Here we have used Ptpn2+/− mice to investigate TCPTP's function in glucose homeostasis. RESEARCH DESIGN AND METHODS We analyzed insulin sensitivity and gluconeogenesis in chow versus high-fat–fed (HFF) Ptpn2+/− and Ptpn2+/+ mice and insulin and IL-6 signaling and gluconeogenic gene expression in Ptpn2+/− and Ptpn2+/+ hepatocytes. RESULTS HFF Ptpn2+/− mice exhibited lower fasted blood glucose and decreased hepatic glucose output as determined in hyperinsulinemic euglycemic clamps and by the decreased blood glucose levels in pyruvate tolerance tests. The reduced hepatic glucose output coincided with decreased expression of the gluconeogenic genes G6pc and Pck1 and enhanced hepatic STAT3 phosphorylation and PI3K/Akt signaling in the fasted state. Insulin-induced IR-β–subunit Y1162/Y1163 phosphorylation and PI3K/Akt signaling and IL-6–induced STAT3 phosphorylation were also enhanced in isolated Ptpn2+/− hepatocytes. The increased insulin and IL-6 signaling resulted in enhanced suppression of G6pc and Pck1 mRNA. CONCLUSIONS Liver TCPTP antagonises both insulin and STAT3 signaling pathways to regulate gluconeogenic gene expression and hepatic glucose output. PMID:20484139

Validation of nuclear STAT6 immunostaining as a diagnostic marker of meningeal solitary fibrous tumor (SFT)/hemangiopericytoma.

PubMed

Berghoff, Anna S; Kresl, Philip; Bienkowski, Michal; Koelsche, Christian; Rajky, Ursula; Hainfellner, Johannes A; Preusser, Matthias

NAB2-STAT6 gene fusion is a molecular characteristic of solitary fibrous tumors (SFT) and hemangiopericytoma, underscoring their definition as one diagnostic entity. NAB2-STAT6 fusion is associated with nuclear relocation of STAT6 protein that can be detected by immunohistochemistry. We evaluated the diagnostic value of STAT6 expression in meningeal tumors. 77 meningeal tumors (17/77 (22.0%) SFT/hemangiopericytoma, 11/77 meningothelial meningioma, 10/77 atypical meningioma 8/77 chordoid meningioma, 9/77 fibroblastic meningioma, 10/77 transitional meningioma, 3/77 rhabdoid meningioma and 9/77 anaplastic meningioma) were included. STAT6 immunohistochemistry was performed on FFPE specimens using a fully automated slide-staining system and anti-STAT6 antibody SC-20:sc621. Two independent observers analyzed all specimens blinded to histological diagnoses, and a third observer was consulted in case of discordancy. STAT6 immunohistochemistry yielded an exclusively nuclear immunostaining signal. 16/17 (94%) SFT/hemangiopericytoma specimens presented with clear-cut, wide-spread, and moderate to strong staining in tumor cell nuclei and were rated as STAT6-positive. In only 1 SFT case with weak and focal nuclear STAT6 immunostaining signal, STAT6 expression was rated discordant (observer 1: STAT6-negative, observers 2 and 3: STAT6-positive). All non-SFT/hemangiopericytoma cases were unanimously rated as STAT6-negative. In 76/77 (98.7%) cases the evaluation of STAT6 immunostaining results was in agreement among observers. STAT6 immunohistochemistry is a robust method to verify diagnosis of SFT/hemangiopericytoma and should therefore be included in the diagnostic work-up of meningeal tumors. In singular cases, weak and focal STAT6 expression may lead to false-negative evaluation and may prompt further molecular work-up.
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Apoptosis in response to heat stress is positively associated with heat-shock protein 90 expression in chicken myocardial cells in vitro.

PubMed

Zhang, Xiao-Hui; Wu, Hong; Tang, Shu; Li, Qiao-Ning; Xu, Jiao; Zhang, Miao; Su, Ya-Nan; Yin, Bin; Zhao, Qi-Ling; Kemper, Nicole; Hartung, Joerg; Bao, En-Dong

2017-06-30

To determine heat-shock protein (Hsp)90 expression is connected with cellular apoptotic response to heat stress and its mechanism, chicken ( Gallus gallus ) primary myocardial cells were treated with the Hsp90 promoter, aspirin, and its inhibitor, geldanamycin (GA), before heat stress. Cellular viability, heat-stressed apoptosis and reactive oxygen species level under different treatments were measured, and the expression of key proteins of the signaling pathway related to Hsp90 and their colocalization with Hsp90 were detected. The results showed that aspirin treatment increased the expression of protein kinase B (Akt), the signal transducer and activator of transcription (STAT)-3 and p-IKKα/β and the colocalization of Akt and STAT-3 with Hsp90 during heat stress, which was accompanied by improved viability and low apoptosis. GA significantly inhibited Akt expression and p-IKKα/β level, but not STAT-3 quantity, while the colocalization of Akt and STAT-3 with Hsp90 was weakened, followed by lower cell viability and higher apoptosis. Aspirin after GA treatment partially improved the stress response and apoptosis rate of tested cells caused by the recovery of Akt expression and colocalization, rather than the level of STAT-3 (including its co-localization with Hsp90) and p-IKKα/β. Therefore, Hsp90 expression has a positive effect on cellular capacity to resist heat-stressed injury and apoptosis. Moreover, inhibition of Hsp90 before stress partially attenuated its positive effects.

Janus kinase (JAK) inhibitors in the treatment of inflammatory and neoplastic diseases.

PubMed

Roskoski, Robert

2016-09-01

The Janus kinase (JAK) family of non-receptor protein-tyrosine kinases consists of JAK1, JAK2, JAK3, and TYK2 (tyrosine kinase-2). Each of these proteins contains a JAK homology pseudokinase (JH2) domain that regulates the adjacent protein kinase domain (JH1). JAK1/2 and TYK2 are ubiquitously expressed whereas JAK3 is found predominantly in hematopoietic cells. The Janus kinase family is regulated by numerous cytokines including interleukins, interferons, and hormones such as erythropoietin, thrombopoietin, and growth hormone. Ligand binding to cytokine and hormone receptors leads to the activation of associated Janus kinases, which then mediate the phosphorylation of the receptors. The SH2 domain of STATs (signal transducers and activators of transcription) binds to the receptor phosphotyrosines thereby promoting STAT phosphorylation by the Janus kinases and consequent activation. STAT dimers are translocated to the nucleus where they participate in the regulation of the expression of thousands of proteins. JAK-STAT dysregulation results in autoimmune disorders such as rheumatoid arthritis, ulcerative colitis, and Crohn disease. JAK-STAT dysregulation also plays a role in the pathogenesis of myelofibrosis, polycythemia vera, and other myeloproliferative illnesses. An activating JAK2 V617F mutation occurs in 95% of people with polycythemia vera and in a lower percentage of people with other neoplasms. JAK1/3 signaling participates in the pathogenesis of inflammatory afflictions while JAK1/2 signaling participates in the development of several malignancies including leukemias and lymphomas as well as myeloproliferative neoplasms. Tofacitinib is a pan-JAK inhibitor that is approved by the FDA for the treatment of rheumatoid arthritis and ruxolitinib is a JAK1/2 inhibitor that is approved for the treatment of polycythemia vera and myelofibrosis. Copyright © 2016 Elsevier Ltd. All rights reserved.

Aging impairs transcriptional regulation of vascular endothelial growth factor in human microvascular endothelial cells: implications for angiogenesis and cell survival.

PubMed

Ahluwalia, A; Jones, M K; Szabo, S; Tarnawski, A S

2014-04-01

In some tissues, aging impairs angiogenesis and reduces expression of vascular endothelial growth factor A (VEGF), a fundamental regulator of angiogenesis. We previously examined angiogenesis in aging and young gastric mucosa in vivo and in vitro and showed that an imbalance between expressions of VEGF (pro-angiogenic factor) and endostatin (anti-angiogenic protein) results in an aging-related impairment of angiogenesis in rats. However, the human relevance of these findings, and whether these mechanisms apply to endothelial cells derived from other tissues, is not clear. Since P-STAT3 and P-CREB are transcription factors that, in association with HIF-1α, can activate VEGF gene expression in some cells (e.g., liver cancer cells, vascular smooth muscle cells), we examined the expression of these two proteins in human dermal microvascular endothelial cells (HMVECs) derived from aging and neonatal individuals. We examined and quantified in vitro angiogenesis, expression of VEGF, P-STAT3, P-CREB and importin-α in HMVECs isolated from neonates (neonatal) and a 66 year old subject (aging). We also examined the effects of treatment with exogenous VEGF and endostatin on in vitro angiogenesis in these cells. Endothelial cells isolated from aging individuals had impaired angiogenesis (vs. neonatal endothelial cells) and reduced expression of VEGF mRNA and protein. Aged HMVECs also had reduced importin-α expression, and reduced expression and nuclear translocation of P-STAT3 and P-CREB. Reduced VEGF gene expression in aged HMVECs strongly correlated with the decreased levels of P-STAT3, P-CREB and importin-α in these cells. Our study clearly demonstrates that endothelial cells from aging individuals have impaired angiogenesis and reduced expression of VEGF likely due to impaired nuclear transport of P-STAT3 and P-CREB transcription factors in these cells.

Screening for MPL mutations in essential thrombocythemia and primary myelofibrosis: normal Mpl expression and absence of constitutive STAT3 and STAT5 activation in MPLW515L-positive platelets.

PubMed

Glembotsky, Ana C; Korin, Laura; Lev, Paola R; Chazarreta, Carlos D; Marta, Rosana F; Molinas, Felisa C; Heller, Paula G

2010-05-01

To evaluate the frequency of MPL W515L, W515K and S505N mutations in essential thrombocythemia (ET) and primary myelofibrosis (PMF) and to determine whether MPLW515L leads to impaired Mpl expression, constitutive STAT3 and STAT5 activation and enhanced response to thrombopoietin (TPO). Mutation detection was performed by allele-specific PCR and sequencing. Platelet Mpl expression was evaluated by flow cytometry, immunoblotting and real-time RT-PCR. Activation of STAT3 and STAT5 before and after stimulation with increasing concentrations of TPO was studied by immunoblotting. Plasma TPO was measured by ELISA. MPLW515L was detected in 1 of 100 patients with ET and 1 of 11 with PMF. Platelets from the PMF patient showed 100% mutant allele, which was <50% in platelets from the ET patient, who also showed the mutation in granulocytes, monocytes and B cells. Mpl surface and total protein expression were normal, and TPO levels were mildly increased in the MPLW515L-positive ET patient, while MPL transcripts did not differ from controls in both MPLW515L-positive patients. Constitutive STAT3 and STAT5 phosphorylation was absent and dose response to TPO-induced phosphorylation was not enhanced. The low frequency of MPL mutations in this cohort is in agreement with previous studies. The finding of normal Mpl levels in MPLW515L-positive platelets indicates this mutation does not lead to dysregulated Mpl expression, as frequently shown for myeloproliferative neoplasms. The lack of spontaneous STAT3 and STAT5 activation and the normal response to TPO is unexpected as MPLW515L leads to constitutive receptor activation and hypersensitivity to TPO in experimental models.

Erythroblast Transformation by the Friend Spleen Focus-Forming Virus Is Associated with a Block in Erythropoietin-Induced STAT1 Phosphorylation and DNA Binding and Correlates with High Expression of the Hematopoietic Phosphatase SHP-1

PubMed Central

Nishigaki, Kazuo; Hanson, Charlotte; Ohashi, Takashi; Spadaccini, Angelo; Ruscetti, Sandra

2006-01-01

Infection of mice with Friend spleen focus-forming virus (SFFV) results in a multistage erythroleukemia. In the first stage, the SFFV envelope glycoprotein interacts with the erythropoietin receptor and a short form of the receptor tyrosine kinase sf-Stk, resulting in constitutive activation of signal transducing molecules and the development of erythropoietin (Epo)-independent erythroid hyperplasia and polycythemia. The second stage results from the outgrowth of a rare virus-infected erythroid cell that expresses nonphysiological levels of the myeloid transcription factor PU.1. These cells exhibit a differentiation block and can be grown as murine erythroleukemia (MEL) cell lines. In this study, we examined SFFV MEL cells to determine whether their transformed phenotype was associated with a block in the activation of any Epo signal-transducing molecules. Our studies indicate that Epo- or SFFV-induced activation of STAT1/3 DNA binding activity is blocked in SFFV MEL cells. The block is at the level of tyrosine phosphorylation of STAT1, although Jak2 phosphorylation is not blocked in these cells. In contrast to Epo, alpha interferon can induce STAT1 phosphorylation and DNA binding in SFFV MEL cells. The SFFV-transformed cells were shown to express elevated levels of the hematopoietic phosphatase SHP-1, and treatment of the cells with a phosphatase inhibitor restored STAT1 tyrosine phosphorylation. MEL cells derived from Friend murine leukemia virus (MuLV) or ME26 MuLV-infected mice, which do not express PU.1, express lower levels of SHP-1 and are not blocked in STAT1/3 DNA-binding activity. Our studies suggest that SFFV-infected erythroid cells become transformed when differentiation signals activated by STAT1/3 are blocked due to high SHP-1 levels induced by inappropriate expression of the PU.1 protein. PMID:16731906

The G-protein-coupled bile acid receptor Gpbar1 (TGR5) protects against renal inflammation and renal cancer cell proliferation and migration through antagonizing NF-κB and STAT3 signaling pathways.

PubMed

Su, Jia; Zhang, Qiqi; Qi, Hui; Wu, Linlin; Li, Yuanqiang; Yu, Donna; Huang, Wendong; Chen, Wei-Dong; Wang, Yan-Dong

2017-08-15

Gpbar1 (TGR5), a G-protein-coupled bile acid membrane receptor, is well known for its roles in regulation of glucose metabolism and energy homeostasis. In the current work, we found that TGR5 activation by its ligand suppressed lipopolysaccharide (LPS)-induced proinflammatory gene expression in wild-type (WT) but not TGR5 -/- mouse kidney. Furthermore, we found that TGR5 is a suppressor of kidney cancer cell proliferation and migration. We show that TGR5 activation antagonized NF-κB and STAT3 signaling pathways through suppressing the phosphorylation of IκBα, the translocation of p65 and the phosphorylation of STAT3. TGR5 overexpression with ligand treatment inhibited gene expression mediated by NF-κB and STAT3. These results suggest that TGR5 antagonizes kidney inflammation and kidney cancer cell proliferation and migration at least in part by inhibiting NF-κB and STAT3 signaling. These findings identify TGR5 may serve as an attractive therapeutic tool for human renal inflammation related diseases and cancer.

Tofacitinib regulates synovial inflammation in psoriatic arthritis, inhibiting STAT activation and induction of negative feedback inhibitors

PubMed Central

Gao, W; McGarry, T; Orr, C; McCormick, J; Veale, D J; Fearon, U

2016-01-01

Background Psoriatic arthritis (PsA) is a chronic inflammatory disease, characterised by synovitis and destruction of articular cartilage/bone. Janus-kinase and signal transducer and activator of transcription (JAK-STAT) signalling pathway is implicated in the pathogenesis of PsA. Objectives To examine the effect of tofacitinib (JAK inhibitor) on proinflammatory mechanisms in PsA. Methods Primary PsA synovial fibroblasts (PsAFLS) and ex vivo PsA synovial explants were cultured with tofacitinib (1 µM). PhosphoSTAT3 (pSTAT3), phosphoSTAT1 (pSTAT1), suppressor of cytokine signaling-3 (SOCS3), protein inhibitor of activated Stat3 (PIAS3) and nuclear factor kappa B cells (NFκBp65) were quantified by western blot. The effect of tofacitinib on PsAFLS migration, invasion, Matrigel network formation and matrix metallopeptidase (MMP)2/9 was quantified by invasion/migration assays and zymography. Interleukin (IL)-6, IL-8, IFN-gamma-inducible protein 10 (IP-10) monocyte chemoattractant protein (MCP)-1, IL-17, IL-10, MMP3 and tissue inhibitor of metalloproteinases 3 (TIMP3) were assessed by ELISA. Results Tofacitinib significantly decreased pSTAT3, pSTAT1, NFκBp65 and induced SOCS3 and PIAS3 expression in PsAFLS and synovial explant cultures (p<0.05). Functionally, PsAFLS invasion, network formation and migration were inhibited by tofacitinib (all p<0.05). In PsA explant, tofacitinib significantly decreased spontaneous secretion of IL-6, IL-8, MCP-1, MMP9/MMP2, MMP3 (all p<0.05) and decreased the MMP3/TIMP3 ratio (p<0.05), with no effect observed for IP-10 or IL-10. Conclusions This study further supports JAK-STAT inhibition as a therapeutic target for the treatment of PsA. PMID:26353790

Targeting constitutively-activated STAT3 in hypoxic ovarian cancer, using a novel STAT3 inhibitor

PubMed Central

McCann, Georgia A.; Naidu, Shan; Rath, Kellie S.; Bid, Hemant K.; Tierney, Brent J.; Suarez, Adrian; Varadharaj, Saradhadevi; Zhang, Jianying; Hideg, Kálmán; Houghton, Peter; Kuppusamy, Periannan; Cohn, David E.; Selvendiran, Karuppaiyah

2014-01-01

Tumor hypoxia, a feature of many solid tumors including ovarian cancer, is associated with resistance to therapies. We previously demonstrated that hypoxic exposure results in increased expression of phosphorylated signal transducer and activator of transcription 3 (pSTAT3). We hypothesized the activation of STAT3 could lead to chemotherapeutic resistance in ovarian cancer cells in hypoxic conditions. In this study, we demonstrate the level of pSTAT3 Tyr705 is increased in the hypoxic regions of human epithelial ovarian cancer (EOC) specimens, as determined by HIF-1α and CD-31 staining. In vitro mutagenesis studies proved that pSTAT3 Tyr705 is necessary for cell survival and proliferation under hypoxic conditions. In addition, we show that S1PR1, a regulator of STAT3 transcription via the JAK/STAT pathway, is highly expressed in hypoxic ovarian cancer cells (HOCCs). Knock down of S1PR1 in HOCCs reduced pSTAT3 Tyr705 levels and was associated with decreased cell survival. Treatment of HOCCs with the STAT3 inhibitor HO-3867 resulted in a rapid and dramatic decrease in pSTAT3 Tyr705 levels as a result of ubiquitin proteasome degradation. STAT3-target proteins Bcl-xL, cyclin D2 and VEGF showed similar decreases in HO-3867 treated cells. Taken together, these findings suggest that activation of STAT3 Tyr705 promotes cell survival and proliferation in HOCCs, and that S1PR1 is involved in the initiation of STAT3 activation. Targeting hypoxia-mediated STAT3 activation represents a therapeutic option for ovarian cancer and other solid tumors. PMID:25594014

Combination Treatment with Apricoxib and IL-27 Enhances Inhibition of Epithelial-Mesenchymal Transition in Human Lung Cancer Cells through a STAT1 Dominant Pathway

PubMed Central

Lee, Mi-Heon; Kachroo, Puja; Pagano, Paul C; Yanagawa, Jane; Wang, Gerald; Walser, Tonya C; Krysan, Kostyantyn; Sharma, Sherven; John, Maie St.; Dubinett, Steven M; Lee, Jay M

2015-01-01

Background The cyclooxygenase 2 (COX-2) pathway has been implicated in the molecular pathogenesis of many malignancies, including lung cancer. Apricoxib, a selective COX-2 inhibitor, has been described to inhibit epithelial-mesenchymal transition (EMT) in human malignancies. The mechanism by which apricoxib may alter the tumor microenvironment by affecting EMT through other important signaling pathways is poorly defined. IL-27 has been shown to have anti-tumor activity and our recent study showed that IL-27 inhibited EMT through a STAT1 dominant pathway. Objective The purpose of this study is to investigate the role of apricoxib combined with IL-27 in inhibiting lung carcinogenesis by modulation of EMT through STAT signaling. Methods and Results Western blot analysis revealed that IL-27 stimulation of human non-small cell lung cancer (NSCLC) cell lines results in STAT1 and STAT3 activation, decreased Snail protein and mesenchymal markers (N-cadherin and vimentin) and a concomitant increase in expression of epithelial markers (E-cadherin, β-and γ-catenins), and inhibition of cell migration. The combination of apricoxib and IL-27 resulted in augmentation of STAT1 activation. However, IL-27 mediated STAT3 activation was decreased by the addition of apricoxib. STAT1 siRNA was used to determine the involvement of STAT1 pathway in the enhanced inhibition of EMT and cell migration by the combined IL-27 and apricoxib treatment. Pretreatment of cells with STAT1 siRNA inhibited the effect of combined IL-27 and apricoxib in the activation of STAT1 and STAT3. In addition, the augmented expression of epithelial markers, decreased expression mesenchymal markers, and inhibited cell migration by the combination treatment were also inhibited by STAT1 siRNA, suggesting that the STAT1 pathway is important in the enhanced effect from the combination treatment. Conclusion Combined apricoxib and IL-27 has an enhanced effect in inhibition of epithelial-mesenchymal transition and cell migration in human lung cancer cells through a STAT1 dominant pathway. PMID:26523208

Immune function of a Rab-related protein by modulating the JAK-STAT signaling pathway in the silkworm, Bombyx mori.

PubMed

Chen, Chen; Eldein, Salah; Zhou, Xiaosan; Sun, Yu; Gao, Jin; Sun, Yuxuan; Liu, Chaoliang; Wang, Lei

2018-01-01

The Rab-family GTPases mainly regulate intracellular vesicle transport, and play important roles in the innate immune response in invertebrates. However, the function and signal transduction of Rab proteins in immune reactions remain unclear in silkworms. In this study, we analyzed a Rab-related protein of silkworm Bombyx mori (BmRABRP) by raising antibodies against its bacterially expressed recombinant form. Tissue distribution analysis showed that BmRABRP mRNA and protein were high expressed in the Malpighian tubule and fat body, respectively. However, among the different stages, only the fourth instar larvae and pupae showed significant BmRABRP levels. After challenge with four pathogenic microorganisms (Escherichia coli, BmNPV, Beauveria bassiana, Micrococcus luteus), the expression of BmRABRP mRNA in the fat body was significantly upregulated. In contrast, the BmRABRP protein was significantly upregulated after infection with BmNPV, while it was downregulated by E. coli, B. bassiana, and M. luteus. A specific dsRNA was used to explore the immune function and relationship between BmRABRP and the JAK-STAT signaling pathway. After BmRABRP gene interference, significant reduction in the number of nodules and increased mortality suggested that BmRABRP plays an important role in silkworm's response to bacterial challenge. In addition, four key genes (BmHOP, BmSTAT, BmSOCS2, and BmSOCS6) of the JAK-STAT signaling pathway showed significantly altered expressions after BmRABRP silencing. BmHOP and BmSOCS6 expressions were significantly decreased, while BmSTAT and BmSOCS2 were significantly upregulated. Our results suggested that BmRABRP is involved in the innate immune response against pathogenic microorganisms through the JAK-STAT signaling pathway in silkworm. © 2017 Wiley Periodicals, Inc.

Severe energy deficit upregulates leptin receptors, leptin signaling, and PTP1B in human skeletal muscle.

PubMed

Perez-Suarez, Ismael; Ponce-González, Jesús Gustavo; de La Calle-Herrero, Jaime; Losa-Reyna, Jose; Martin-Rincon, Marcos; Morales-Alamo, David; Santana, Alfredo; Holmberg, Hans-Christer; Calbet, Jose A L

2017-11-01

In obesity, leptin receptors (OBR) and leptin signaling in skeletal muscle are downregulated. To determine whether OBR and leptin signaling are upregulated with a severe energy deficit, 15 overweight men were assessed before the intervention (PRE), after 4 days of caloric restriction (3.2 kcal·kg body wt -1 ·day -1 ) in combination with prolonged exercise (CRE; 8 h walking + 45 min single-arm cranking/day) to induce an energy deficit of ~5,500 kcal/day, and following 3 days of control diet (isoenergetic) and reduced exercise (CD). During CRE, the diet consisted solely of whey protein ( n = 8) or sucrose ( n = 7; 0.8 g·kg body wt -1 ·day -1 ). Muscle biopsies were obtained from the exercised and the nonexercised deltoid muscles and from the vastus lateralis. From PRE to CRE, serum glucose, insulin, and leptin were reduced. OBR expression was augmented in all examined muscles associated with increased maximal fat oxidation. Compared with PRE, after CD, phospho-Tyr 1141 OBR, phospho-Tyr 985 OBR, JAK2, and phospho-Tyr 1007/1008 JAK2 protein expression were increased in all muscles, whereas STAT3 and phospho-Tyr 705 STAT3 were increased only in the arms. The expression of protein tyrosine phosphatase 1B (PTP1B) in skeletal muscle was increased by 18 and 45% after CRE and CD, respectively ( P < 0.05). Suppressor of cytokine signaling 3 (SOCS3) tended to increase in the legs and decrease in the arm muscles (ANOVA interaction: P < 0.05). Myosin heavy chain I isoform was associated with OBR protein expression ( r  = -0.75), phospho-Tyr 985 OBR ( r  = 0.88), and phospho-Tyr 705 STAT3/STAT3 ( r = 0.74). In summary, despite increased PTP1B expression, skeletal muscle OBR and signaling are upregulated by a severe energy deficit with greater response in the arm than in the legs likely due to SOCS3 upregulation in the leg muscles. NEW & NOTEWORTHY This study shows that the skeletal muscle leptin receptors and their corresponding signaling cascade are upregulated in response to a severe energy deficit, contributing to increase maximal fat oxidation. The responses are more prominent in the arm muscles than in the legs but partly blunted by whey protein ingestion and high volume of exercise. This occurs despite an increase of protein tyrosine phosphatase 1B protein expression, a known inhibitor of insulin and leptin signaling. Copyright © 2017 the American Physiological Society.

Inhibition of Interferon-beta Responses in Multiple Sclerosis Immune Cells Associated With High-Dose Statins

PubMed Central

Feng, Xuan; Han, Diana; Kilaru, Bharat K.; Franek, Beverly S.; Niewold, Timothy B.; Reder, Anthony T.

2014-01-01

Objective To determine whether statins affect type 1 interferon responses in relapsing-remitting multiple sclerosis (RRMS). Design Study effects of atorvastatin on type 1 interferon responses in Jurkat cells, mononuclear cells (MNCs) from therapy-naive patients with RRMS in vitro, and MNCs from interferon-treated RRMS patients in vivo in 4 conditions: no drug, statin only, interferon-beta only, and statin added on to interferon-beta therapy. Patients The study examined clinically stable patients with RRMS: 21 therapy-naive patients and 14 patients receiving interferon-beta with a statin. Interventions Statin effects on in vitro and in vivo interferon-beta–induced STAT1 transcription factor activation, expression of interferon-stimulated proteins in MNCs, and serum type 1 interferon activity. Results In vitro, atorvastatin dose dependently inhibited expression of interferon-stimulated P-Y-STAT1 by 44% (P< .001), interferon regulatory factor 1 protein by 30% (P= .006), and myxovirus resistance 1 protein by 32% (P=.004) compared with no-statin control in MNCs from therapy-naive RRMS patients. In vivo, 9 of 10 patients who received high-dose statins (80 mg) had a significant reduction in interferon-beta therapy–induced serum interferon-α/β activity, whereas only 2 of 4 patients who received medium-dose statins (40 mg) had reductions. High-dose add-on statin therapy significantly blocked interferon-beta function, with less P-Y-STAT1 transcription factor activation, and reduced myxovirus resistance 1 protein and viperin protein production. Medium doses of statins did not change STAT1 activation. Conclusions High-dose add-on statin therapy significantly reduces interferon-beta function and type 1 interferon responses in RRMS patients. These data provide a putative mechanism for how statins could counteract the beneficial effects of interferon-beta and worsen disease. PMID:22801747

Toxicological effect of TiO2 nanoparticle-induced myocarditis in mice

NASA Astrophysics Data System (ADS)

Hong, Fashui; Wang, Ling; Yu, Xiaohong; Zhou, Yingjun; Hong, Jie; Sheng, Lei

2015-08-01

Currently, impacts of exposure to TiO2 nanoparticles (NPs) on the cardiovascular system are not well understood. The aim of this study was to investigate whether TiO2 NPs induce myocarditis and its underlying molecular mechanism in the cardiac inflammation in mice. Mice were exposed to TiO2 NPs for 6 months; biochemical parameters of serum and expression of Th1-related and Th2-related cytokines in the heart were investigated. The results showed that TiO2 NP exposure resulted in cardiac lesions coupling with pulmonary inflammation; increases of aspartate aminotransferase (AST), creatine kinase (CK), C-reaction protein (CRP), lactate dehydrogenase (LDH), alpha-hydroxybutyrate dehydrogenase (HBDH), adhesion molecule-1 (ICAM-1), and monocyte chemoattractant protein-1 (MCP-1) levels; and a reduction of nitric oxide (NOx) level in the serum. These were associated with increases of nuclear factor-κB (NF-κB), tumor necrosis factor-α (TNF-α), interleukin (IL)-4, IL-6, transforming growth factor-β (TGF-β), creatine kinase, CRP, adhesion molecule-1, and monocyte chemoattractant protein-1, interferon-γ (IFN-γ), signal transducers and activators of transcription (STAT)1, STAT3, or STAT6, GATA-binding domain-3, GATA-binding domain-4, endothelin-1 expression levels, and T-box expressed in T cells expression level that is the master regulator of pro-inflammatory cytokines and transcription factors in the heart. These findings imply that TiO2 NP exposure may increase the occurrence and development of cardiovascular diseases.

GPR30 mediates anorectic estrogen-induced STAT3 signaling in the hypothalamus.

PubMed

Kwon, Obin; Kang, Eun Seok; Kim, Insook; Shin, Sora; Kim, Mijung; Kwon, Somin; Oh, So Ra; Ahn, Young Soo; Kim, Chul Hoon

2014-11-01

Estrogen plays an important role in the control of energy balance in the hypothalamus. Leptin-independent STAT3 activation (i.e., tyrosine(705)-phosphorylation of STAT3, pSTAT3) in the hypothalamus is hypothesized as the primary mechanism of the estrogen-induced anorexic response. However, the type of estrogen receptor that mediates this regulation is unknown. We investigated the role of the G protein-coupled receptor 30 (GPR30) in estradiol (E2)-induced STAT3 activation in the hypothalamus. Regulation of STAT3 activation by E2, G-1, a specific agonist of GPR30 and G-15, a specific antagonist of GPR30 was analyzed in vitro and in vivo. Effect of GPR30 activation on eating behavior was analyzed in vivo. E2 stimulated pSTAT3 in cells expressing GPR30, but not expressing estrogen receptor ERα and ERβ. G-1 induced pSTAT3, and G-15 inhibited E2-induced pSTAT3 in primary cultures of hypothalamic neurons. A cerebroventricular injection of G-1 increased pSTAT3 in the arcuate nucleus of mice, which was associated with a decrease in food intake and body weight gain. These results suggest that GPR30 is the estrogen receptor that mediates the anorectic effect of estrogen through the STAT3 pathway in the hypothalamus. Copyright © 2014 Elsevier Inc. All rights reserved.

Interleukin-6 and intercellular cell adhesion molecule-1 expression remains elevated in revived live endothelial cells following spaceflight.

PubMed

Muid, S; Froemming, G R A; Ali, A M; Nawawi, H

2013-12-01

The effects of spaceflight on cardiovascular health are not necessarily seen immediately after astronauts have returned but can be delayed. It is important to investigate the long term effects of spaceflight on protein and gene expression of inflammation and endothelial activation as a predictor for the development of atherosclerosis and potential cardiovascular problems. The objectives of this study were to investigate the (a) protein and gene expression of inflammation and endothelial activation, (b) expression of nuclear factor kappa B (NFκB), signal transducer and activator of transcription-3 (STAT-3) and endothelial nitric oxide synthase (eNOS) in human umbilical vein endothelial cells (HUVEC) 3 months post-space flight travel compared to ground controls. HUVEC cultured on microcarriers in fluid processing apparatus were flown to the International Space Station (ISS) by the Soyuz TMA-11 rocket. After landing, the cells were detached from microcarriers and recultured in T-25 cm(2) culture flasks (Revived HUVEC). Soluble protein expression of IL-6, TNF-α, ICAM-1, VCAM-1 and e-selectin were measured by ELISA. Gene expression of these markers and in addition NFκB, STAT-3 and eNOS were measured. Spaceflight induced IL-6 and ICAM-1 remain elevated even after 3 months post spaceflight travel and this is mediated via STAT-3 pathway. The downregulation of eNOS expression in revived HUVEC cells suggests a reduced protection of the cells and the surrounding vessels against future insults that may lead to atherosclerosis. It would be crucial to explore preventive measures, in relation to atherosclerosis and its related complications.

Novel compounds TAD-1822-7-F2 and F5 inhibited HeLa cells growth through the JAK/Stat signaling pathway.

PubMed

Yang, Tianfeng; Shi, Xianpeng; Kang, Yuan; Zhu, Man; Fan, Mengying; Zhang, Dongdong; Zhang, Yanmin

2018-07-01

Cervical carcinoma remains the second most common malignancy with a high mortality rate among women worldwide. TAD-1822-7-F2 (F2) and TAD-1822-7-F5 (F5) are novel compounds synthesized on the chemical structure of taspine derivatives, and show an effective suppression for HeLa cells. Our study aims to confirm the potential targets of F2 and F5, and investigate the underlying mechanism of the inhibitory effect on HeLa cells. In this study, Real Time Cell Analysis and crystal violet staining assay were conducted to investigate the effect of F2 and F5 on HeLa cells proliferation. And the analytical methods of surface plasmon resonance and quartz crystal microbalance were established and employed to study the interaction between F2 and F5 and potential target protein JAK2, suggesting that both compounds have strong interaction with the JAK2 protein. Western blot analysis, immunofluorescence staining study and PCR was conducted to investigate the molecules of JAK/Stat signaling pathway. Interestingly, F2 and F5 showed diverse regulation for signaling molecules because of their different chemical structure. F2 increased the expression of JAK2 and downregulated the level of P-JAK1 and P-JAK2, and decreased P-Stat3 (Ser727). While F5 could increase the expression of JAK2 and naturally decrease the phosphorylation of JAK1 and Tyk2, and decreased the expression of P-Stat6. Moreover, F2 and F5 showed the same downregulation on the P-Stat3 (Tyr705). Therefore, F2 and F5 could target the JAK2 protein and prevent the phosphorylation of JAKs to suppress the phosphorylation of the downstream effector Stats, which suggested that F2 and F5 have great potential to be the inhibitors of the JAK/Stat signaling pathway. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

The synthetic α-bromo-2',3,4,4'-tetramethoxychalcone (α-Br-TMC) inhibits the JAK/STAT signaling pathway.

PubMed

Pinz, Sophia; Unser, Samy; Brueggemann, Susanne; Besl, Elisabeth; Al-Rifai, Nafisah; Petkes, Hermina; Amslinger, Sabine; Rascle, Anne

2014-01-01

Signal transducer and activator of transcription STAT5 and its upstream activating kinase JAK2 are essential mediators of cytokine signaling. Their activity is normally tightly regulated and transient. However, constitutive activation of STAT5 is found in numerous cancers and a driving force for malignant transformation. We describe here the identification of the synthetic chalcone α-Br-2',3,4,4'-tetramethoxychalcone (α-Br-TMC) as a novel JAK/STAT inhibitor. Using the non-transformed IL-3-dependent B cell line Ba/F3 and its oncogenic derivative Ba/F3-1*6 expressing constitutively activated STAT5, we show that α-Br-TMC targets the JAK/STAT pathway at multiple levels, inhibiting both JAK2 and STAT5 phosphorylation. Moreover, α-Br-TMC alters the mobility of STAT5A/B proteins in SDS-PAGE, indicating a change in their post-translational modification state. These alterations correlate with a decreased association of STAT5 and RNA polymerase II with STAT5 target genes in chromatin immunoprecipitation assays. Interestingly, expression of STAT5 target genes such as Cis and c-Myc was differentially regulated by α-Br-TMC in normal and cancer cells. While both genes were inhibited in IL-3-stimulated Ba/F3 cells, expression of the oncogene c-Myc was down-regulated and that of the tumor suppressor gene Cis was up-regulated in transformed Ba/F3-1*6 cells. The synthetic chalcone α-Br-TMC might therefore represent a promising novel anticancer agent for therapeutic intervention in STAT5-associated malignancies.

RNA editing is induced by type I interferon in esophageal squamous cell carcinoma.

PubMed

Zhang, Jinyao; Chen, Zhaoli; Tang, Zefang; Huang, Jianbing; Hu, Xueda; He, Jie

2017-07-01

In recent years, abnormal RNA editing has been shown to play an important role in the development of esophageal squamous cell carcinoma, as such abnormal editing is catalyzed by ADAR (adenosine deaminases acting on RNA). However, the regulatory mechanism of ADAR1 in esophageal squamous cell carcinomas remains largely unknown. In this study, we investigated ADAR1 expression and its association with RNA editing in esophageal squamous cell carcinomas. RNA sequencing applied to esophageal squamous cell carcinoma clinical samples showed that ADAR1 expression was correlated with the expression of STAT1, STAT2, and IRF9. In vitro experiments showed that the abundance of ADAR1 protein was associated with the induced activation of the JAK/STAT pathway by type I interferon. RNA sequencing results showed that treatment with type I interferon caused an increase in the number and degree of RNA editing in esophageal squamous cell carcinoma cell lines. In conclusion, the activation of the JAK/STAT pathway is a regulatory mechanism of ADAR1 expression and causes abnormal RNA editing profile in esophageal squamous cell carcinoma. This mechanism may serve as a new target for esophageal squamous cell carcinoma therapy.

Endothelial Cell Proteomic Response to Rickettsia conorii Infection Reveals Activation of the Janus Kinase (JAK)-Signal Transducer and Activator of Transcription (STAT)-Inferferon Stimulated Gene (ISG)15 Pathway and Reprogramming Plasma Membrane Integrin/Cadherin Signaling.

PubMed

Zhao, Yingxin; Valbuena, Gustavo; Walker, David H; Gazi, Michal; Hidalgo, Marylin; DeSousa, Rita; Oteo, Jose Antonio; Goez, Yenny; Brasier, Allan R

2016-01-01

Rickettsia conorii is the etiologic agent of Mediterranean spotted fever, a re-emerging infectious disease with significant mortality. This Gram-negative, obligately intracellular pathogen is transmitted via tick bites, resulting in disseminated vascular endothelial cell infection with vascular leakage. In the infected human, Rickettsia conorii infects endothelial cells, stimulating expression of cytokines and pro-coagulant factors. However, the integrated proteomic response of human endothelial cells to R. conorii infection is not known. In this study, we performed quantitative proteomic profiling of primary human umbilical vein endothelial cells (HUVECs) with established R conorii infection versus those stimulated with endotoxin (LPS) alone. We observed differential expression of 55 proteins in HUVEC whole cell lysates. Of these, we observed induction of signal transducer and activator of transcription (STAT)1, MX dynamin-like GTPase (MX1), and ISG15 ubiquitin-like modifier, indicating activation of the JAK-STAT signaling pathway occurs in R. conorii-infected HUVECs. The down-regulated proteins included those involved in the pyrimidine and arginine biosynthetic pathways. A highly specific biotinylated cross-linking enrichment protocol was performed to identify dysregulation of 11 integral plasma membrane proteins that included up-regulated expression of a sodium/potassium transporter and down-regulation of α-actin 1. Analysis of Golgi and soluble Golgi fractions identified up-regulated proteins involved in platelet-endothelial adhesion, phospholipase activity, and IFN activity. Thirty four rickettsial proteins were identified with high confidence in the Golgi, plasma membrane, or secreted protein fractions. The host proteins associated with rickettsial infections indicate activation of interferon-STAT signaling pathways; the disruption of cellular adhesion and alteration of antigen presentation pathways in response to rickettsial infections are distinct from those produced by nonspecific LPS stimulation. These patterns of differentially expressed proteins suggest mechanisms of pathogenesis as well as methods for diagnosis and monitoring Rickettsia infections. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

MMP-2 participates in the sclera of guinea pig with form-deprivation myopia via IGF-1/STAT3 pathway.

PubMed

Liu, Y-X; Sun, Y

2018-05-01

To investigate the expression changes of MMP-2 (matrix metalloproteinases-2) mediated by IGF-1 (insulin-like growth factors-1) STAT3 (signal transducer and activator of transcription 3) pathway in the sclera of the form-deprivation myopia guinea pigs. Twenty-four three-week-old guinea pigs were randomly divided into 4 groups: group A (Control), B, C and D. Guinea pigs in group A were sacrificed after 21 days without any special treatment. Guinea pigs in group B were sacrificed 7 days after receiving stitch in the right eye. Guinea pigs in group C were sacrificed 14 days after receiving stitch in the right eye. Guinea pigs in group D were sacrificed 21 days after receiving stitch in the right eye. Eyeball refraction and axial length of guinea pigs were measured before sacrifice. Eyeballs of guinea pigs were enucleated after sacrifice. The expressions of IGF-1, STAT3 and MMP-2 in scleral tissue were detected by Western blot. Axial length extension and myopia appeared in the right eye of guinea pigs in group B. The expressions of IGF-1, STAT3 and MMP-2 in the sclera significantly increased after 7 days of occlusion compared with that in control group A (p<0.05). In the right eye of group C, the axial prolongation and myopia formation appeared after 14-day occlusion. The expressions of IGF-1, STAT3 and MMP-2 in sclera significantly increased compared with that in group A (p<0.05). In the right eye of group D, the axial extension and myopia formation occurred. IGF-1, STAT3 and MMP-2 in scleral significantly upregulated 21 days after occlusion (p<0.05). Furthermore, at different stages of deprivation, protein expressions of MMP-2 and IGF-1 in sclera were positively correlated (r = 0.962, p<0.01). Form-deprivation of guinea pigs lead to increased expressions of IGF-1, STAT3 and MMP-2 in the sclera and myopia of guinea pigs. The expressions of IGF-1, STAT3 and MMP-2 increased progressively over the time of deprivation. Additionally, overexpression of MMP-2 mediated by IGF-1/STAT3 pathway in sclera might promote the formation of myopia.

Coffee Polyphenols Change the Expression of STAT5B and ATF-2 Modifying Cyclin D1 Levels in Cancer Cells

PubMed Central

Oleaga, Carlota; Ciudad, Carlos J.; Noé, Véronique; Izquierdo-Pulido, Maria

2012-01-01

Background. Epidemiological studies suggest that coffee consumption reduces the risk of cancer, but the molecular mechanisms of its chemopreventive effects remain unknown. Objective. To identify differentially expressed genes upon incubation of HT29 colon cancer cells with instant caffeinated coffee (ICC) or caffeic acid (CA) using whole-genome microarrays. Results. ICC incubation of HT29 cells caused the overexpression of 57 genes and the underexpression of 161, while CA incubation induced the overexpression of 12 genes and the underexpression of 32. Using Venn-Diagrams, we built a list of five overexpressed genes and twelve underexpressed genes in common between the two experimental conditions. This list was used to generate a biological association network in which STAT5B and ATF-2 appeared as highly interconnected nodes. STAT5B overexpression was confirmed at the mRNA and protein levels. For ATF-2, the changes in mRNA levels were confirmed for both ICC and CA, whereas the decrease in protein levels was only observed in CA-treated cells. The levels of cyclin D1, a target gene for both STAT5B and ATF-2, were downregulated by CA in colon cancer cells and by ICC and CA in breast cancer cells. Conclusions. Coffee polyphenols are able to affect cyclin D1 expression in cancer cells through the modulation of STAT5B and ATF-2. PMID:22919439

Protein inhibitor of activated STAT3 inhibits adipogenic gene expression

DOE Office of Scientific and Technical Information (OSTI.GOV)

Deng Jianbei; Hua Kunjie; Caveney, Erica J.

2006-01-20

Protein inhibitor of activated STAT3 (PIAS3), a cytokine-induced repressor of signal transducer and activator of transcription 3 (STAT3) and a modulator of a broad array of nuclear proteins, is expressed in white adipose tissue, but its role in adipogenesis is not known. Here, we determined that PIAS3 was constitutively expressed in 3T3-L1 cells at all stages of adipogenesis. However, it translocated from the nucleus to the cytoplasm 4 days after induction of differentiation by isobutylmethylxanthine, dexamethasone, and insulin (MDI). In ob/ob mice, PIAS3 expression was increased in white adipose tissue depots compared to lean mice and was found in themore » cytoplasm of adipocytes. Overexpression of PIAS3 in differentiating preadipocytes, which localized primarily to the nucleus, inhibited mRNA level gene expression of adipogenic transcription factors C/EBP{alpha} and PPAR{gamma}, as well as their downstream target genes aP2 and adiponectin. PIAS3 also inhibited C/EBP{alpha} promoter activation mediated specifically by insulin, but not dexamethasone or isobutylmethylxanthine. Taken together, these data suggest that PIAS3 may play an inhibitory role in adipogenesis by modulating insulin-activated transcriptional activation events. Increased PIAS3 expression in adipose tissue may play a role in the metabolic disturbances of obesity.« less

Signal transducer and activator of transcription 3 is the dominant mediator of the anti-inflammatory effects of IL-10 in human macrophages.

PubMed

Williams, Lynn; Bradley, Laura; Smith, Alexandra; Foxwell, Brian

2004-01-01

The signaling mechanism by which the anti-inflammatory cytokine IL-10 mediates suppression of proinflammatory cytokine synthesis remains largely unknown. Macrophage-specific STAT3-null mice have demonstrated that STAT3 plays a critical role in the suppression of LPS-induced TNF-alpha release, although the mechanism by which STAT3 mediates this inhibition is still not clear. Using an adenoviral system, we have expressed a dominant negative (DN) STAT3 in human macrophages to broaden the investigation to determine the role of STAT3 in IL-10-mediated anti-inflammatory signaling and gene expression. Overexpression of STAT3 DN completely inhibited IL-10-induced suppressor of cytokine signaling 3, tissue inhibitor of MMP-1, TNF receptor expression, and the recently identified IL-10-inducible genes, T cell protein tyrosine phosphatase and signaling lymphocyte activation molecule. STAT3 DN also blocked IL-10-mediated inhibition of MHC class II and COX2 expression. In agreement with the studies in STAT3-null mice, overexpression of the STAT3 DN completely reversed the ability of IL-10 to inhibit LPS-mediated TNF-alpha and IL-6 production. However, real-time PCR analysis showed that STAT3 DN expression did not affect immediate suppression of TNF-alpha mRNA, but did reverse the suppression observed at later time points, suggesting a biphasic regulation of TNF-alpha mRNA levels by IL-10. In conclusion, although STAT3 does appear to be the dominant mediator of the majority of IL-10 functions, there are elements of its anti-inflammatory activity that are STAT3 independent.

Erythropoietin activates telomerase through transcriptional and posttranscriptional regulation in human erythroleukemic JAS-REN-A cells.

PubMed

Akiyama, Masaharu; Kawano, Takeshi; Mikami-Terao, Yoko; Agawa-Ohta, Miyuki; Yamada, Osamu; Ida, Hiroyuki; Yamada, Hisashi

2011-03-01

We evaluated the molecular mechanism of telomerase activation by erythropoietin (EPO) in human erythroleukemic JAS-REN-A cells. Telomerase activity increased 3-4 fold after 3-24h of culture with EPO and was associated with increases in c-myc mRNA after 1-3h, of c-Myc protein after 3-6h, and of human telomerase reverse transcriptase (hTERT) mRNA and hTERT protein after 6-24h. Simultaneously EPO induced phosphorylation of signal transducer activator of transcription 5 (STAT5), AKT, and extracellular signal-regulated kinase (ERK). Telomerase activity induced by EPO was significantly inhibited by AG490, PD98059, and LY294002. AG490 downregulated c-myc and hTERT mRNA expression with inhibited STAT5 and AKT phosphorylation. PD98059 also reduced c-myc and hTERT expression and inhibited ERK phosphorylation. However, LY294002 did not inhibit c-myc or hTERT mRNA expression despite inhibiting STAT5 and AKT phosphorylation. These results suggest that EPO activates telomerase in JAS-REN-A cells through dual regulation: hTERT gene transcription by Janus tyrosine kinase 2/STAT5/c-Myc and hTERT protein phosphorylation by phosphatidylinositol 3'-kinase/AKT. Copyright © 2010 Elsevier Ltd. All rights reserved.

The PPARdelta agonist GW501516 suppresses interleukin-6-mediated hepatocyte acute phase reaction via STAT3 inhibition.

PubMed

Kino, T; Rice, K C; Chrousos, G P

2007-05-01

Interleukin-6 and downstream liver effectors acute phase reactants are implicated in the systemic inflammatory reaction. Peroxisome proliferator-activated receptor delta (PPARdelta), which binds to and is activated by a variety of fatty acids, was recently shown to have anti-inflammatory actions. We examined the ability of the synthetic PPARdelta agonist GW501516 to suppress interleukin-6-induced expression of acute phase proteins in human hepatoma HepG2 cells and rat primary hepatocytes. Results GW501516 dose-dependently suppressed interleukin-6-induced mRNA expression of the acute phase protein alpha1-antichymotrypsin in HepG2 cells. The compound also suppressed interleukin-6-induced mRNA expression of alpha2-acid glycoprotein, beta-fibrinogen and alpha2-macroglobulin in and the secretion of C-reactive protein by rat primary hepatocytes. Depletion of the PPARdelta receptor, but not of PPARalpha or gamma, attenuated the suppressive effect of GW501516 on interleukin-6-induced alpha1-antichymotrypsin mRNA expression, indicating that PPARdelta specifically mediated this effect. Since interleukin-6 stimulates the transcriptional activity of the alpha1-antichymotrypsin promoter by activating the signal transducer and activator of transcription (STAT) 3, we examined functional interaction of this transcription factor and PPARdelta on this promoter. Overexpression of PPARdelta enhanced the suppressive effect of GW501516 on STAT3-activated transcriptional activity of the alpha1-antichymotrypsin promoter, while GW501516 suppressed interleukin-6-induced binding of this transcription factor to this promoter. These findings indicate that agonist-activated PPARdelta interferes with interleukin-6-induced acute phase reaction in the liver by inhibiting the transcriptional activity of STAT3. PPARdelta agonists might be useful for the suppression of systemic inflammatory reactions in which IL-6 plays a central role.

Developmental expression of STATs, nuclear factor-κB and inflammatory genes in the jejunum of piglets during weaning.

PubMed

Yi, Hongbo; Jiang, Denghu; Zhang, Lin; Xiong, Haitao; Han, Feifei; Wang, Yizhen

2016-07-01

The signal transducer and activator of transcription (STAT) proteins play essential roles in apoptosis, proliferation and survival. However, the role of STATs in intestinal inflammation during weaning is unclear. This study aimed to investigate developmental expression of STATs, nuclear factor-κB (NF-κB) and inflammatory genes in the jejunum of piglets during weaning. Thirty-two piglets were weaned at 21d and sacrificed at 0, 1, 7, or 14d (n=8) after weaning. Villus height and the villus height/crypt depth ratio were decreased, whereas crypt depth was increased in the jejunum at 7 and 14d after weaning. In addition, the mRNA levels of interferon-γ (IFN-γ), inducible nitric oxide synthase (iNOS), IL-6, IL-8, IL-12 and IL-22 were increased in the jejunum at 7 and 14d after weaning, whereas transforming growth factor-β (TGF-β), suppressor of cytokine signaling 3 (SCOS3) and arginase-1 was decreased. Neutrophil infiltration was increased in the mucosa of the jejunum after weaning. Moreover, phosphorylation of IκB-α, NF-κB, AKT and STAT-3 was increased. However, the phosphorylation of STAT-1 (at 7 and 14d) and STAT-6 (at 1 and 7d) was suppressed in the jejunum after weaning. Treatment of porcine jejunal epithelial (IPEC-J2) cells with the STAT inhibitors fludarabine, niclosamide and teriflunomide, which inhibit the phosphorylation of STAT-1, STAT-3 and STAT-6, respectively, weakened the defense capacity of these cells against bacterial infection. In conclusion, weaning caused severe inflammation associated with activation of the NF-κB and STAT-3 pathways and suppression of STAT-1 and STAT-6 in the jejunum of piglets. Copyright © 2016 Elsevier B.V. All rights reserved.

Alantolactone suppresses APOC3 expression and alters lipid homeostasis in L02 liver cells.

PubMed

Yang, Meiting; Zhao, Hanhan; Ai, Huihan; Zhu, Hongbin; Wang, Shuyue; Bao, Yongli; Li, Yuxin

2018-06-05

A high level of APOC3 expression is an independent risk factor for some lipid metabolism-related diseases, such as cardiovascular disease (CVD), nonalcoholic fatty liver disease (NAFLD) and atherosclerosis (AS). This suggests that down-regulating APOC3 expression is a potential way of regulating lipid levels. In this study, we used luciferase reporter screening to identify a natural compound, alantolactone (ALA), that can inhibit the promoter activity of APOC3. ALA decreased APOC3 expression at both mRNA and protein levels. Then we pretreated L02 liver cells with oxLDL to investigate the function of ALA in lipid homeostasis. Intriguingly, ALA attenuated oxLDL-induced foam cell formation by reducing total cholesterol (TC) and triglyceride (TG) contents. Furthermore, these results could be reversed by overexpressing APOC3 protein. ALA inhibited tyrosine phosphorylation (Tyr705pho) of STAT3 to down-regulate APOC3 expression. Intriguingly, overexpression of a wild-type STAT3 or a constitutively active form of STAT3 (STAT3-C) up-regulated APOC3 expression and partly reversed the effect of ALA in oxLDL-induced L02 cells. Overexpression of wild-type STAT3 also increased TC but not TG contents in L02 cells. However, overexpression of STAT3-C significantly increased TC and TG contents, and the effect of ALA was partly attenuated by STAT3-C, although this was not statistically significant. These results suggest that ALA attenuates lipid accumulation through down-regulation of APOC3 expression, at least in part by inhibiting STAT3 signaling. Copyright © 2018 Elsevier B.V. All rights reserved.

FRNK negatively regulates IL-4-mediated inflammation.

PubMed

Sharma, Ritu; Colarusso, Pina; Zhang, Hong; Stevens, Katarzyna M; Patel, Kamala D

2015-02-15

Focal adhesion kinase (FAK)-related nonkinase (PTK2 isoform 6 in humans, hereafter referred to as FRNK) is a cytoskeletal regulatory protein that has recently been shown to dampen lung fibrosis, yet its role in inflammation is unknown. Here, we show for the first time that expression of FRNK negatively regulates IL-4-mediated inflammation in a human model of eosinophil recruitment. Mechanistically, FRNK blocks eosinophil accumulation, firm adhesion and transmigration by preventing transcription and protein expression of VCAM-1 and CCL26. IL-4 activates STAT6 to induce VCAM-1 and CCL26 transcription. We now show that IL-4 also increases GATA6 to induce VCAM-1 expression. FRNK blocks IL-4-induced GATA6 transcription but has little effect on GATA6 protein expression and no effect on STAT6 activation. FRNK can block FAK or Pyk2 signaling and we, thus, downregulated these proteins using siRNA to determine whether signaling from either protein is involved in the regulation of VCAM-1 and CCL26. Knockdown of FAK, Pyk2 or both had no effect on VCAM-1 or CCL26 expression, which suggests that FRNK acts independently of FAK and Pyk2 signaling. Finally, we found that IL-4 induces the late expression of endogenous FRNK. In summary, FRNK represents a novel mechanism to negatively regulate IL-4-mediated inflammation. © 2015. Published by The Company of Biologists Ltd.

Hes1 promotes the IL-22-mediated antimicrobial response by enhancing STAT3-dependent transcription in human intestinal epithelial cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Murano, Tatsuro; Okamoto, Ryuichi, E-mail: rokamoto.gast@tmd.ac.jp; Department of Advanced GI Therapeutics, Graduate School, Tokyo Medical and Dental University, Tokyo

Highlights: •Hes1 enhances IL-22-STAT3 signaling in human intestinal epithelial cells. •Hes1 enhances REG family gene induction by IL-22-STAT3 signaling. •Protein level of Hes1 restricts the response to IL-22. •Present regulation of a cytokine signal represents a new mode of Hes1 function. -- Abstract: Notch signaling plays an essential role in the proliferation and differentiation of intestinal epithelial cells (IECs). We have previously shown that Notch signaling is up-regulated in the inflamed mucosa of ulcerative colitis (UC) and thereby plays an indispensable role in tissue regeneration. Here we show that in addition to Notch signaling, STAT3 signaling is highly activated inmore » the inflamed mucosa of UC. Forced expression of the Notch target gene Hes1 dramatically enhanced the IL-22-mediated STAT3-dependent transcription in human IECs. This enhancement of STAT3-dependent transcription was achieved by the extended phosphorylation of STAT3 by Hes1. Microarray analysis revealed that Hes1-mediated enhancement of IL-22-STAT3 signaling significantly increased the induction of genes encoding antimicrobial peptides, such as REG1A, REG3A and REG3G, in human IECs. Conversely, the reduction of Hes1 protein levels with a γ-secretase inhibitor significantly down-regulated the induction of those genes in IECs, resulting in a markedly poor response to IL-22. Our present findings identify a new role for the molecular function of Hes1 in which the protein can interact with cytokine signals and regulate the immune response of IECs.« less

Heat shock protein-27 (HSP27) regulates STAT3 and eIF4G levels in first trimester human placenta.

PubMed

Shochet, Gali Epstein; Komemi, Oded; Sadeh-Mestechkin, Dana; Pomeranz, Meir; Fishman, Ami; Drucker, Liat; Lishner, Michael; Matalon, Shelly Tartakover

2016-12-01

During placental implantation, cytotrophoblast cells differentiate to extravillous trophoblast (EVT) cells that invade from the placenta into the maternal uterine blood vessels. The heat shock protein-27 (HSP27), the signal transducer and activator of transcription-3 (STAT3) and the eukaryotic translation initiation factor 4E (EIF4E) are involved in regulating EVT cell differentiation/migration. EIF4E and EIF4G compose the translation initiation complex, which is a major control point in protein translation. The molecular chaperone distinctiveness of HSP27 implies that it directly interferes with many target proteins. STAT3, EIF4E, and EIF4G were found to be HSP27 client proteins in tumor cells. We aimed to analyze if HSP27 regulate STAT3 and EIF4G levels in first trimester human placenta. We found that like STAT3, EIF4G is highly expressed in the EVT cells (immunohistochemistry). Silencing HSP27 in HTR-8/SVneo cells (siRNA, EVT cell line) and in placental explants reduced STAT3 level (47 and 33 %, respectively, p < 0.05). HSP27 silencing reduced the levels of STAT3 phosphorylation (33 % reduction, p < 0.05) and targets (IRF1, MUC1, MMP2/9 and EIF4E, 30-49 % reduction, p < 0.05) in the HTR-8/SVneo cells. Moreover, HSP27 silencing significantly reduced EIF4G level and elevated the level of its fragments in HTR-8/SVneo cells and in the placental explants (p < 0.05). In conclusion, Placental implantation and development are accompanied by trophoblast cell proliferation and differentiation, which necessitates intense protein translation and STAT3 activation. HSP27 was found to be regulator of translation initiation and STAT3 level. Therefore, it suggests that HSP27 is a key protein during placental development and trophoblast cell differentiation.

PDGF-BB induces PRMT1 expression through ERK1/2 dependent STAT1 activation and regulates remodeling in primary human lung fibroblasts.

PubMed

Sun, Qingzhu; Liu, Li; Mandal, Jyotshna; Molino, Antonio; Stolz, Daiana; Tamm, Michael; Lu, Shemin; Roth, Michael

2016-04-01

Tissue remodeling of sub-epithelial mesenchymal cells is a major pathology occurring in chronic obstructive pulmonary disease (COPD) and asthma. Fibroblasts, as a major source of interstitial connective tissue extracellular matrix, contribute to the fibrotic and inflammatory changes in these airways diseases. Previously, we described that protein arginine methyltransferase-1 (PRMT1) participates in airway remodeling in a rat model of pulmonary inflammation. In this study we investigated the mechanism by which PDGF-BB regulates PRMT1 in primary lung fibroblasts, isolated from human lung biopsies. Fibroblasts were stimulated with PDGF-BB for up-to 48h and the regulatory and activation of signaling pathways controlling PRMT1 expression were determined. PRMT1 was localized by immuno-histochemistry in human lung tissue sections and by immunofluorescence in isolated fibroblasts. PRMT1 activity was suppressed by the pan-PRMT inhibitor AMI1. ERK1/2 mitogen activated protein kinase (MAPK) was blocked by PD98059, p38 MAPK by SB203580, and STAT1 by small interference (si) RNA treatment. The results showed that PDGF-BB significantly increased PRMT1 expression after 1h lasting over 48h, through ERK1/2 MAPK and STAT1 signaling. The inhibition of ERK1/2 MAPK or of PRMT1 activity decreased PDGF-BB induced fibroblast proliferation, COX2 production, collagen-1A1 secretion, and fibronectin production. These findings suggest that PRMT1 is a central regulator of tissue remodeling and that the signaling sequence controlling its expression in primary human lung fibroblast is PDGF-ERK-STAT1. Therefore, PRMT1 presents a novel therapeutic and diagnostic target for the control of airway wall remodeling in chronic lung diseases. Copyright © 2016 Elsevier Inc. All rights reserved.

Identification of STAT3 and STAT5 proteins in the rat suprachiasmatic nucleus and the Day/Night difference in astrocytic STAT3 phosphorylation in response to lipopolysaccharide.

PubMed

Moravcová, Simona; Červená, Kateřina; Pačesová, Dominika; Bendová, Zdeňka

2016-01-01

Signal transducers and activators of transcription (STAT) proteins regulate many aspects of cellular physiology from growth and differentiations to immune responses. Using immunohistochemistry, we show the daily rhythm of STAT3 protein in the rat suprachiasmatic nucleus (SCN), with low but significant amplitude peaking in the morning. We also reveal the strong expression of STAT5A in astrocytes of the SCN and the STAT5B signal in nonastrocytic cells. Administration of lipopolysaccharide (LPS) acutely induced phosphorylation of STAT3 on Tyr705 during both the day and the night and induced phosphorylation on Ser727 but only after the daytime application. The LPS-induced phospho-STAT3 (Tyr705) remained elevated for 24 hr after the daytime application but declined within 8 hr when LPS was applied at night. © 2015 Wiley Periodicals, Inc.

Effects of obesity and exercise on testicular leptin signal transduction and testosterone biosynthesis in male mice.

PubMed

Yi, Xuejie; Gao, Haining; Chen, Dequan; Tang, Donghui; Huang, Wanting; Li, Tao; Ma, Tie; Chang, Bo

2017-04-01

To explore the role of the testicular leptin and JAK-STAT[leptin (LEP)-JAK-STAT] pathway in testosterone biosynthesis during juvenile stages and exercise for weight loss, male C57BL/6J mice were randomly divided into normal-diet and high-fat diet groups. After 10 wk, mice in the high-fat diet-fed group were further divided randomly into obese control, obese moderate-volume exercise, and obese high-volume exercise groups. Mice in the obese moderate-volume exercise group were provided with 2 h/day, 6 days/wk swimming exercise for 8 wk, and mice in the obese high-volume exercise group underwent twice the amount of daily exercise intervention as the obese moderate-volume exercise group. The results showed that a high-fat diet causes obesity, leptin resistance, inhibition of the testicular LEP-JAK-STAT pathway, decreased mRNA and protein expression of steroidogenic factor-1, steroidogenic acute regulatory protein, and the P -450 side-chain cleavage enzyme, a decrease in the serum testosterone-to-estradiol ratio, and declines in sperm quality parameters. Both moderate and high-volume exercise were able to reduce body fat and increase the mRNA and protein expression of LEP-JAK-STAT, but only moderate exercise significantly increased the mRNA and protein expression of steroidogenic factor-1, steroidogenic acute regulatory protein, and P -450 side-chain cleavage enzyme and significantly reversed the serum testosterone-to-estradiol ratio and sperm quality parameters. These findings suggest that by impairing the testicular LEP-JAK-STAT pathway, early-stage obesity inhibits the biosynthesis of testosterone and sexual development and reduces male reproductive potential. Long-term moderate and high-volume exercise can effectively reduce body fat and improve obesity-induced abnormalities in testicular leptin signal transduction, whereas only moderate-volume exercise can reverse the negative impacts of obesity on male reproductive function. Copyright © 2017 the American Physiological Society.

Reelin promotes the adhesion and drug resistance of multiple myeloma cells via integrin β1 signaling and STAT3

PubMed Central

Lv, Meng; Liang, Xiaodong; Dai, Hui; Qin, Xiaodan; Zhang, Yan; Hao, Jie; Sun, Xiuyuan; Yin, Yanhui; Huang, Xiaojun; Zhang, Jun; Lu, Jin; Ge, Qing

2016-01-01

Reelin is an extracellular matrix (ECM) protein that is essential for neuron migration and positioning. The expression of reelin in multiple myeloma (MM) cells and its association with cell adhesion and survival were investigated. Overexpression, siRNA knockdown, and the addition of recombinant protein of reelin were used to examine the function of reelin in MM cells. Clinically, high expression of reelin was negatively associated with progression-free survival and overall survival. Functionally, reelin promoted the adhesion of MM cells to fibronectin via activation of α5β1 integrin. The resulting phosphorylation of Focal Adhesion Kinase (FAK) led to the activation of Src/Syk/STAT3 and Akt, crucial signaling molecules involved in enhancing cell adhesion and protecting cells from drug-induced cell apoptosis. These findings indicate reelin's important role in the activation of integrin-β1 and STAT3/Akt pathways in multiple myeloma and highlight the therapeutic potential of targeting reelin/integrin/FAK axis. PMID:26848618

Reelin promotes the adhesion and drug resistance of multiple myeloma cells via integrin β1 signaling and STAT3.

PubMed

Lin, Liang; Yan, Fan; Zhao, Dandan; Lv, Meng; Liang, Xiaodong; Dai, Hui; Qin, Xiaodan; Zhang, Yan; Hao, Jie; Sun, Xiuyuan; Yin, Yanhui; Huang, Xiaojun; Zhang, Jun; Lu, Jin; Ge, Qing

2016-03-01

Reelin is an extracellular matrix (ECM) protein that is essential for neuron migration and positioning. The expression of reelin in multiple myeloma (MM) cells and its association with cell adhesion and survival were investigated. Overexpression, siRNA knockdown, and the addition of recombinant protein of reelin were used to examine the function of reelin in MM cells. Clinically, high expression of reelin was negatively associated with progression-free survival and overall survival. Functionally, reelin promoted the adhesion of MM cells to fibronectin via activation of α5β1 integrin. The resulting phosphorylation of Focal Adhesion Kinase (FAK) led to the activation of Src/Syk/STAT3 and Akt, crucial signaling molecules involved in enhancing cell adhesion and protecting cells from drug-induced cell apoptosis. These findings indicate reelin's important role in the activation of integrin-β1 and STAT3/Akt pathways in multiple myeloma and highlight the therapeutic potential of targeting reelin/integrin/FAK axis.

Regulatory effect of calcineurin inhibitor, tacrolimus, on IL-6/sIL-6R-mediated RANKL expression through JAK2-STAT3-SOCS3 signaling pathway in fibroblast-like synoviocytes

PubMed Central

2013-01-01

Introduction This study investigated whether the calcineurin inhibitor, tacrolimus, suppresses receptor activator of NF-κB ligand (RANKL) expression in fibroblast-like synoviocytes (FLS) through regulation of IL-6/Janus activated kinase (JAK2)/signal transducer and activator of transcription-3 (STAT3) and suppressor of cytokine signaling (SOCS3) signaling. Methods The expression of RANKL, JAK2, STAT3, and SOCS3 proteins was assessed by western blot analysis, real-time PCR and ELISA in IL-6 combined with soluble IL-6 receptor (sIL-6R)-stimulated rheumatoid arthritis (RA)-FLS with or without tacrolimus treatment. The effects of tacrolimus on synovial inflammation and bone erosion were assessed using mice with arthritis induced by K/BxN serum. Immunofluorescent staining was performed to identify the effect of tacrolimus on RANKL and SOCS3. The tartrate-resistant acid phosphatase staining assay was performed to assess the effect of tacrolimus on osteoclast differentiation. Results We found that RANKL expression in RA FLS is regulated by the IL-6/sIL-6R/JAK2/STAT3/SOCS3 pathway. Inhibitory effects of tacrolimus on RANKL expression in a serum-induced arthritis mice model were identified. Tacrolimus inhibits RANKL expression in IL-6/sIL-6R-stimulated FLS by suppressing STAT3. Among negative regulators of the JAK/STAT pathway, such as CIS1, SOCS1, and SOCS3, only SOCS3 is significantly induced by tacrolimus. As compared to dexamethasone and methotrexate, tacrolimus more potently suppresses RANKL expression in FLS. By up-regulating SOCS3, tacrolimus down-regulates activation of the JAK-STAT pathway by IL-6/sIL-6R trans-signaling, thus decreasing RANKL expression in FLS. Conclusions These data suggest that tacrolimus might affect the RANKL expression in IL-6 stimulated FLS through STAT3 suppression, together with up-regulation of SOCS3. PMID:23406906

Activated Rac1 requires gp130 for Stat3 activation, cell proliferation and migration

DOE Office of Scientific and Technical Information (OSTI.GOV)

Arulanandam, Rozanne; Geletu, Mulu; Feracci, Helene

2010-03-10

Rac1 (Rac) is a member of the Rho family of small GTPases which controls cell migration by regulating the organization of actin filaments. Previous results suggested that mutationally activated forms of the Rho GTPases can activate the Signal Transducer and Activator of Transcription-3 (Stat3), but the exact mechanism is a matter of controversy. We recently demonstrated that Stat3 activity of cultured cells increases dramatically following E-cadherin engagement. To better understand this pathway, we now compared Stat3 activity levels in mouse HC11 cells before and after expression of the mutationally activated Rac1 (Rac{sup V12}), at different cell densities. The results revealedmore » for the first time a dramatic increase in protein levels and activity of both the endogenous Rac and Rac{sup V12} with cell density, which was due to inhibition of proteasomal degradation. In addition, Rac{sup V12}-expressing cells had higher Stat3, tyrosine-705 phosphorylation and activity levels at all densities, indicating that Rac{sup V12} is able to activate Stat3. Further examination of the mechanism of Stat3 activation showed that Rac{sup V12} expression caused a surge in mRNA of Interleukin-6 (IL6) family cytokines, known potent Stat3 activators. Knockdown of gp130, the common subunit of this family reduced Stat3 activity, indicating that these cytokines may be responsible for the Stat3 activation by Rac{sup V12}. The upregulation of IL6 family cytokines was required for cell migration and proliferation induced by Rac{sup V12}, as shown by gp130 knockdown experiments, thus demonstrating that the gp130/Stat3 axis represents an essential effector of activated Rac for the regulation of key cellular functions.« less

Topical N-Acetylcysteine Accelerates Wound Healing in Vitro and in Vivo via the PKC/Stat3 Pathway

PubMed Central

Tsai, Min-Ling; Huang, Hui-Pei; Hsu, Jeng-Dong; Lai, Yung-Rung; Hsiao, Yu-Ping; Lu, Fung-Jou; Chang, Horng-Rong

2014-01-01

N-Acetylcysteine (Nac) is an antioxidant administered in both oral and injectable forms. In this study, we used Nac topically to treat burn wounds in vitro and in vivo to investigate mechanisms of action. In vitro, we monitored glutathione levels, cell proliferation, migration, scratch-wound healing activities and the epithelialization-related proteins, matrixmetalloproteinase-1 (MMP-1) and proteins involved in regulating the expression of MMP-1 in CCD-966SK cells treated with Nac. Various Nac concentrations (0.1, 0.5, and 1.0 mM) increased glutathione levels, cell viability, scratch-wound healing activities and migration abilities of CCD-966SK cells in a dose-dependent manner. The MMP-1 expression of CCD-966SK cells treated with 1.0 mM Nac for 24 h was significantly increased. Levels of phosphatidylinositol 3-kinase (PI3K), protein kinase C (PKC), janus kinase 1 (Jak1), signal transducer and activator of transcription 3 (Stat3), c-Fos and Jun, but not extracellular signal-regulated protein kinases 1 and 2 (Erk1/2), were also significantly increased in a dose-dependent manner compared to the controls. In addition, Nac induced collagenous expression of MMP-1 via the PKC/Stat3 signaling pathway. In vivo, a burn wound healing rat model was applied to assess the stimulation activity and histopathological effects of Nac, with 3.0% Nac-treated wounds being found to show better characteristics on re-epithelialization. Our results demonstrated that Nac can potentially promote wound healing activity, and may be a promising drug to accelerate burn wound healing. PMID:24798751

Activation of the protein tyrosine phosphatase SHP2 via the interleukin-6 signal transducing receptor protein gp130 requires tyrosine kinase Jak1 and limits acute-phase protein expression.

PubMed

Schaper, F; Gendo, C; Eck, M; Schmitz, J; Grimm, C; Anhuf, D; Kerr, I M; Heinrich, P C

1998-11-01

Stimulation of the interleukin-6 (IL-6) signalling pathway occurs via the IL-6 receptor-glycoprotein 130 (IL-6R-gp130) receptor complex and results in the regulation of acute-phase protein genes in liver cells. Ligand binding to the receptor complex leads to tyrosine phosphorylation and activation of Janus kinases (Jak), phosphorylation of the signal transducing subunit gp130, followed by recruitment and phosphorylation of the signal transducer and activator of transcription factors STAT3 and STAT1 and the src homology domain (SH2)-containing protein tyrosine phosphatase (SHP2). The tyrosine phosphorylated STAT factors dissociate from the receptor, dimerize and translocate to the nucleus where they bind to enhancer sequences of IL-6 target genes. Phosphorylated SHP2 is able to bind growth factor receptor bound protein (grb2) and thus might link the Jak/STAT pathway to the ras/raf/mitogen-activated protein kinase pathway. Here we present data on the dose-dependence, kinetics and kinase requirements for SHP2 phosphorylation after the activation of the signal transducer, gp130, of the IL-6-type family receptor complex. When human fibrosarcoma cell lines deficient in Jak1, Jak2 or tyrosine kinase 2 (Tyk2) were stimulated with IL-6-soluble IL-6R complexes it was found that only in Jak1-, but not in Jak 2- or Tyk2-deficient cells, SHP2 activation was greatly impaired. It is concluded that Jak1 is required for the tyrosine phosphorylation of SHP2. This phosphorylation depends on Tyr-759 in the cytoplasmatic domain of gp130, since a Tyr-759-->Phe exchange abrogates SHP2 activation and in turn leads to elevated and prolonged STAT3 and STAT1 activation as well as enhanced acute-phase protein gene induction. Therefore, SHP2 plays an important role in acute-phase gene regulation.

STAT3-Activated GM-CSFRα Translocates to the Nucleus and Protects CLL Cells from Apoptosis

PubMed Central

Li, Ping; Harris, David; Liu, Zhiming; Rozovski, Uri; Ferrajoli, Alessandra; Wang, Yongtao; Bueso-Ramos, Carlos; Hazan-Halevy, Inbal; Grgurevic, Srdana; Wierda, William; Burger, Jan; O'Brien, Susan; Faderl, Stefan; Keating, Michael; Estrov, Zeev

2014-01-01

Here it was determined that Chronic Lymphocytic Leukemia (CLL) cells express the α-subunit but not the β-subunit of the granulocyte-macrophage colony-stimulating factor receptor (GM-CSFR/CSF3R). GM-CSFRα was detected on the surface, in the cytosol, and the nucleus of CLL cells via confocal microscopy, cell fractionation, and GM-CSFRα antibody epitope mapping. Because STAT3 is frequently activated in CLL and the GM-CSFRα promoter harbors putative STAT3 consensus binding sites, MM1 cells were transfected with truncated forms of the GM-CSFRα promoter, then stimulated with IL-6 to activate STAT3 to identify STAT3 binding sites. Chromatin immunoprecipitation (ChIP) and an electoromobility shift assay (EMSA) confirmed STAT3 occupancy to those promoter regions in both IL-6 stimulated MM1 and CLL cells. Transfection of MM1 cells with STAT3 siRNA or CLL cells with STAT3 shRNA significantly down-regulated GM-CSFRα mRNA and protein levels. RNA transcripts, involved in regulating cell-survival pathways, and the proteins KAP1 (TRIM28) and ISG15 co-immunoprecipitated with GM-CSFRα. GM-CSFRα-bound KAP1 enhanced the transcriptional activity of STAT3, whereas ISG15 inhibited the NF-κB pathway. Nevertheless, overexpression of GM-CSFRα protected MM1 cells from dexamethasone-induced apoptosis, and GM-CSFRα knockdown induced apoptosis in CLL cells, suggesting that GM-CSFRα provides a ligand-independent survival advantage. PMID:24836891

A20 Regulates Atherogenic Interferon (IFN)-γ Signaling in Vascular Cells by Modulating Basal IFNβ Levels*

PubMed Central

Moll, Herwig P.; Lee, Andy; Minussi, Darlan C.; da Silva, Cleide G.; Csizmadia, Eva; Bhasin, Manoj; Ferran, Christiane

2014-01-01

IFNγ signaling in endothelial (EC) and smooth muscle cells (SMC) is a key culprit of pathologic vascular remodeling. The impact of NF-κB inhibitory protein A20 on IFNγ signaling in vascular cells remains unknown. In gain- and loss-of-function studies, A20 inversely regulated expression of IFNγ-induced atherogenic genes in human EC and SMC by modulating STAT1 transcription. In vivo, inadequate A20 expression in A20 heterozygote mice aggravated intimal hyperplasia following partial carotid artery ligation. This outcome uniquely associated with increased levels of Stat1 and super-induction of Ifnγ-dependent genes. Transcriptome analysis of the aortic media from A20 heterozygote versus wild-type mice revealed increased basal Ifnβ signaling as the likely cause for higher Stat1 transcription. We confirmed higher basal IFNβ levels in A20-silenced human SMC and showed that neutralization or knockdown of IFNβ abrogates heightened STAT1 levels in these cells. Upstream of IFNβ, A20-silenced EC and SMC demonstrated higher levels of phosphorylated/activated TANK-binding kinase-1 (TBK1), a regulator of IFNβ transcription. This suggested that A20 knockdown increased STAT1 transcription by enhancing TBK1 activation and subsequently basal IFNβ levels. Altogether, these results uncover A20 as a key physiologic regulator of atherogenic IFNγ/STAT1 signaling. This novel function of A20 added to its ability to inhibit nuclear factor-κB (NF-κB) activation solidifies its promise as an ideal therapeutic candidate for treatment and prevention of vascular diseases. In light of recently discovered A20/TNFAIP3 (TNFα-induced protein 3) single nucleotide polymorphisms that impart lower A20 expression or function, these results also qualify A20 as a reliable clinical biomarker for vascular risk assessment. PMID:25217635

Emodin inhibits growth and induces apoptosis in an orthotopic hepatocellular carcinoma model by blocking activation of STAT3

PubMed Central

Subramaniam, Aruljothi; Shanmugam, Muthu K; Ong, Tina H; Li, Feng; Perumal, Ekambaram; Chen, Luxi; Vali, Shireen; Abbasi, Taher; Kapoor, Shweta; Ahn, Kwang Seok; Kumar, Alan Prem; Hui, Kam M; Sethi, Gautam

2013-01-01

BACKGROUND AND PURPOSE Aberrant activation of STAT3 is frequently encountered and promotes proliferation, survival, metastasis and angiogenesis in hepatocellular carcinoma (HCC). Here, we have investigated whether emodin mediates its effect through interference with the STAT3 activation pathway in HCC. EXPERIMENTAL APPROACH The effect of emodin on STAT3 activation, associated protein kinases and apoptosis was investigated using various HCC cell lines. Additionally, we also used a predictive tumour technology to analyse the effects of emodin. The in vivo effects of emodin were assessed in an orthotopic mouse model of HCC. KEY RESULTS Emodin suppressed STAT3 activation in a dose- and time-dependent manner in HCC cells, mediated by the modulation of activation of upstream kinases c-Src, JAK1 and JAK2. Vanadate treatment reversed emodin-induced down-regulation of STAT3, suggesting the involvement of a tyrosine phosphatase and emodin induced the expression of the tyrosine phosphatase SHP-1 that correlated with the down-regulation of constitutive STAT3 activation. Interestingly, silencing of the SHP-1 gene by siRNA abolished the ability of emodin to inhibit STAT3 activation. Finally, when administered i.p., emodin inhibited the growth of human HCC orthotopic tumours in male athymic nu/nu mice and STAT3 activation in tumour tissues. CONCLUSIONS AND IMPLICATIONS Emodin mediated its effects predominantly through inhibition of the STAT3 signalling cascade and thus has a particular potential for the treatment of cancers expressing constitutively activated STAT3. PMID:23848338

The Synthetic α-Bromo-2′,3,4,4′-Tetramethoxychalcone (α-Br-TMC) Inhibits the JAK/STAT Signaling Pathway

PubMed Central

Brueggemann, Susanne; Besl, Elisabeth; Al-Rifai, Nafisah; Petkes, Hermina; Amslinger, Sabine; Rascle, Anne

2014-01-01

Signal transducer and activator of transcription STAT5 and its upstream activating kinase JAK2 are essential mediators of cytokine signaling. Their activity is normally tightly regulated and transient. However, constitutive activation of STAT5 is found in numerous cancers and a driving force for malignant transformation. We describe here the identification of the synthetic chalcone α-Br-2′,3,4,4′-tetramethoxychalcone (α-Br-TMC) as a novel JAK/STAT inhibitor. Using the non-transformed IL-3-dependent B cell line Ba/F3 and its oncogenic derivative Ba/F3-1*6 expressing constitutively activated STAT5, we show that α-Br-TMC targets the JAK/STAT pathway at multiple levels, inhibiting both JAK2 and STAT5 phosphorylation. Moreover, α-Br-TMC alters the mobility of STAT5A/B proteins in SDS-PAGE, indicating a change in their post-translational modification state. These alterations correlate with a decreased association of STAT5 and RNA polymerase II with STAT5 target genes in chromatin immunoprecipitation assays. Interestingly, expression of STAT5 target genes such as Cis and c-Myc was differentially regulated by α-Br-TMC in normal and cancer cells. While both genes were inhibited in IL-3-stimulated Ba/F3 cells, expression of the oncogene c-Myc was down-regulated and that of the tumor suppressor gene Cis was up-regulated in transformed Ba/F3-1*6 cells. The synthetic chalcone α-Br-TMC might therefore represent a promising novel anticancer agent for therapeutic intervention in STAT5-associated malignancies. PMID:24595334

Inactivation of JAK2/STAT3 Signaling Axis and Downregulation of M1 mAChR Cause Cognitive Impairment in klotho Mutant Mice, a Genetic Model of Aging

PubMed Central

Park, Seok-Joo; Shin, Eun-Joo; Min, Sun Seek; An, Jihua; Li, Zhengyi; Hee Chung, Yoon; Hoon Jeong, Ji; Bach, Jae-Hyung; Nah, Seung-Yeol; Kim, Won-Ki; Jang, Choon-Gon; Kim, Yong-Sun; Nabeshima, Yo-ichi; Nabeshima, Toshitaka; Kim, Hyoung-Chun

2013-01-01

We previously reported cognitive dysfunction in klotho mutant mice. In the present study, we further examined novel mechanisms involved in cognitive impairment in these mice. Significantly decreased janus kinase 2 (JAK2) and signal transducer and activator of transcription3 (STAT3) phosphorylation were observed in the hippocampus of klotho mutant mice. A selective decrease in protein expression and binding density of the M1 muscarinic cholinergic receptor (M1 mAChR) was observed in these mice. Cholinergic parameters (ie, acetylcholine (ACh), choline acetyltransferase (ChAT), and acetylcholinesterase (AChE)) and NMDAR-dependent long-term potentiation (LTP) were significantly impaired in klotho mutant mice. McN-A-343 (McN), an M1 mAChR agonist, significantly attenuated these impairments. AG490 (AG), a JAK2 inhibitor, counteracted the attenuating effects of McN, although AG did not significantly alter the McN-induced effect on AChE. Furthermore, AG significantly inhibited the attenuating effects of McN on decreased NMDAR-dependent LTP, protein kinase C βII, p-ERK, p-CREB, BDNF, and p-JAK2/p-STAT3-expression in klotho mutant mice. In addition, k252a, a BDNF receptor tyrosine kinase B (TrkB) inhibitor, significantly counteracted McN effects on decreased ChAT, ACh, and M1 mAChR and p-JAK2/p-STAT3 expression. McN-induced effects on cognitive impairment in klotho mutant mice were consistently counteracted by either AG or k252a. Our results suggest that inactivation of the JAK2/STAT3 signaling axis and M1 mAChR downregulation play a critical role in cognitive impairment observed in klotho mutant mice. PMID:23389690

Autophagy Facilitates IFN-γ-induced Jak2-STAT1 Activation and Cellular Inflammation*

PubMed Central

Chang, Yu-Ping; Tsai, Cheng-Chieh; Huang, Wei-Ching; Wang, Chi-Yun; Chen, Chia-Ling; Lin, Yee-Shin; Kai, Jui-In; Hsieh, Chia-Yuan; Cheng, Yi-Lin; Choi, Pui-Ching; Chen, Shun-Hua; Chang, Shih-Ping; Liu, Hsiao-Sheng; Lin, Chiou-Feng

2010-01-01

Autophagy is regulated for IFN-γ-mediated antimicrobial efficacy; however, its molecular effects for IFN-γ signaling are largely unknown. Here, we show that autophagy facilitates IFN-γ-activated Jak2-STAT1. IFN-γ induces autophagy in wild-type but not in autophagy protein 5 (Atg5−/−)-deficient mouse embryonic fibroblasts (MEFs), and, autophagy-dependently, IFN-γ induces IFN regulatory factor 1 and cellular inflammatory responses. Pharmacologically inhibiting autophagy using 3-methyladenine, a known inhibitor of class III phosphatidylinositol 3-kinase, confirms these effects. Either Atg5−/− or Atg7−/− MEFs are, independent of changes in IFN-γ receptor expression, resistant to IFN-γ-activated Jak2-STAT1, which suggests that autophagy is important for IFN-γ signal transduction. Lentivirus-based short hairpin RNA for Atg5 knockdown confirmed the importance of autophagy for IFN-γ-activated STAT1. Without autophagy, reactive oxygen species increase and cause SHP2 (Src homology-2 domain-containing phosphatase 2)-regulated STAT1 inactivation. Inhibiting SHP2 reversed both cellular inflammation and the IFN-γ-induced activation of STAT1 in Atg5−/− MEFs. Our study provides evidence that there is a link between autophagy and both IFN-γ signaling and cellular inflammation and that autophagy, because it inhibits the expression of reactive oxygen species and SHP2, is pivotal for Jak2-STAT1 activation. PMID:20592027

AAG8 promotes carcinogenesis by activating STAT3.

PubMed

Sun, Bing; Kawahara, Masahiro; Ehata, Shogo; Nagamune, Teruyuki

2014-09-01

Dysregulation of signalling pathways by changes of gene expression contributes to hallmarks of cancer. The ubiquitously expressed chaperone protein AAG8 (aging-associated gene 8 protein, encoded by the SIGMAR1 gene) is often found to be overexpressed in various cancers. AAG8 is involved in ER (endoplasmic reticulum)-associated degradation and has been intensively elaborated in neuroscience. However, its rationale in carcinogenesis has rarely been noticed. In this study, we explored the intrinsic oncogenetic roles of AAG8 in cancer cells and found that AAG8 promoted carcinogenesis both in vitro and in vivo. We further characterized AAG8, for the first time to our knowledge, as a STAT3 activator and elucidated that it alternatively activated STAT3 in addition to IL6/JAK pathway. Based on these findings and a drug screening study, we demonstrated that combined inhibition of AAG8 and IL6/JAK signalling synergistically limits cancer cell growth. Taken together, our findings shed light on the fundamental evidences for identification of AAG8 as an oncoprotein and potential target for cancer prevention, as well as highlight the importance of ER proteins in contributing to JAK/STAT signaling and carcinogenesis. Copyright © 2014 Elsevier Inc. All rights reserved.

Nuclear relocation of STAT6 reliably predicts NAB2-STAT6 fusion for the diagnosis of solitary fibrous tumour.

PubMed

Koelsche, Christian; Schweizer, Leonille; Renner, Marcus; Warth, Arne; Jones, David T W; Sahm, Felix; Reuss, David E; Capper, David; Knösel, Thomas; Schulz, Birte; Petersen, Iver; Ulrich, Alexis; Renker, Eva Kristin; Lehner, Burkhard; Pfister, Stefan M; Schirmacher, Peter; von Deimling, Andreas; Mechtersheimer, Gunhild

2014-11-01

Nuclear relocation of STAT6 has been shown in tumours with NAB2-STAT6 fusion, and has been proposed as an ancillary marker for the diagnosis of solitary fibrous tumours (SFTs). The aim of this study was to verify the utility of STAT6 immunohistology in diagnosing SFT. A total of 689 formalin-fixed paraffin-embedded tumours comprising 35 pleural SFTs and 654 other mesenchymal tumours were investigated for STAT6 expression using immunohistochemistry. Nine dedifferentiated liposarcomas (DDLSs) and five SFTs were also examined for the presence of NAB2-STAT6 fusion at the protein level using the proximity ligation assay (PLA), and for copy number variants (CNVs) with the Illumina Infinium Human Methylation450 array. Thirty-four of 35 SFTs showed strong nuclear STAT6 expression. Furthermore, five of 68 DDLSs, two of 130 undifferentiated pleomorphic sarcomas and one of 63 cases of nodular fasciitis showed moderate to strong nuclear STAT6 expression. The PLA indicated the presence of NAB2-STAT6 fusion protein in SFTs, but signal was also detected in some DDLSs. Copy number analysis showed an overall low frequency of chromosomal imbalances in SFTs, but complex karyotypes in DDLSs, including amplification of STAT6 and MDM2 loci. The detection of nuclear relocation of STAT6 with immunohistochemistry is a characteristic of SFTs, and may serve as a diagnostic marker that indicates NAB2-STAT6 fusion and helps to discriminate SFTs from histological mimics. © 2014 John Wiley & Sons Ltd.

PDGFRα depletion attenuates glioblastoma stem cells features by modulation of STAT3, RB1 and multiple oncogenic signals.

PubMed

Cenciarelli, Carlo; Marei, Hany E; Felsani, Armando; Casalbore, Patrizia; Sica, Gigliola; Puglisi, Maria Ausiliatrice; Cameron, Angus J M; Olivi, Alessandro; Mangiola, Annunziato

2016-08-16

Platelet derived growth factor receptors (PDGFRs) play an important role in tumor pathogenesis, and they are frequently overexpressed in glioblastoma (GBM). Earlier we have shown a higher protein expression of PDGFR isoforms (α and β) in peritumoral-tissue derived cancer stem cells (p-CSC) than in tumor core (c-CSC) of several GBM affected patients. In the current study, in order to assess the activity of PDGFRα/PDGF-AA signaling axis, we performed time course experiments to monitor the effects of exogenous PDGF-AA on the expression of downstream target genes in c-CSC vs p-CSC. Interestingly, in p-CSC we detected the upregulation of Y705-phosphorylated Stat3, concurrent with a decrement of Rb1 protein in its active state, within minutes of PDGF-AA addition. This finding prompted us to elucidate the role of PDGFRα in self-renewal, invasion and differentiation in p-CSC by using short hairpin RNA depletion of PDGFRα expression. Notably, in PDGFRα-depleted cells, protein analysis revealed attenuation of stemness-related and glial markers expression, alongside early activation of the neuronal marker MAP2a/b that correlated with the induction of tumor suppressor Rb1. The in vitro reduction of the invasive capacity of PDGFRα-depleted CSC as compared to parental cells correlated with the downmodulation of markers of epithelial-mesenchymal transition phenotype and angiogenesis. Surprisingly, we observed the induction of anti-apoptotic proteins and compensatory oncogenic signals such as EDN1, EDNRB, PRKCB1, PDGF-C and PDGF-D. To conclude, we hypothesize that the newly discovered PDGFRα/Stat3/Rb1 regulatory axis might represent a potential therapeutic target for GBM treatment.

Upregulation of survivin by leptin/STAT3 signaling in MCF-7 cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jiang Haiping; Tianjin Medical University Cancer Hospital, Tianjin; Yu Jinming

2008-03-28

Leptin and its receptors are overexpressed in breast cancer tissues and correlate with poor prognosis. Survivin, a member of the inhibitor of apoptosis protein (IAP) gene family, is generally upregulated in tumor tissues and prevents tumor cells from apoptosis. Here we showed that leptin upregulated survivin mRNA and protein expression in MCF-7 breast cancer cells. Meanwhile, leptin suppressed docetaxel-induced apoptosis by inhibiting caspase activity. Knockdown of signal transducer and activator transcription 3 (STAT3) expression by small interfering RNA (siRNA) blocked leptin-induced upregulation of survivin. TransAM ELISA showed that leptin increased nuclear translocation of active STAT3. In addition, chromatin immunoprecipitation (ChIP)more » assay detected an enhanced binding of STAT3 to survivin promoter in MCF-7 cells after treatment by leptin. Further studies showed that leptin enhanced the transcriptional activity of survivin promoter. Collectively, our findings identify leptin/STAT3 signaling as a novel pathway for survivin expression in breast cancer cells.« less

Severe fever with thrombocytopenia syndrome virus inhibits exogenous Type I IFN signaling pathway through its NSs invitro.

PubMed

Chen, Xu; Ye, Haiyan; Li, Shilin; Jiao, Baihai; Wu, Jianqin; Zeng, Peibin; Chen, Limin

2017-01-01

Severe fever with thrombocytopenia syndrome (SFTS) is an emerging infectious disease caused by a novel bunyavirus (SFTS virus, SFTSV). At present there is still no specific antiviral treatment for SFTSV; To understand which cells support SFTSV life cycle and whether SFTSV infection activates host innate immunity, four different cell lines (Vero, Hela, Huh7.5.1, and Huh7.0) were infected with SFTSV. Intracellular/extracellular viral RNA and expression of IFNα, and IFNß were detected by real-time RT- PCR following infection. To confirm the role of non-structural protein (NSs) of SFTSV in exogenous IFNα-induced Jak/STAT signaling, p-STAT1 (Western Blot), ISRE activity (Luciferase assay) and ISG expression (real-time PCR) were examined following IFNα stimulation in the presence or absence of over-expression of NSs in Hela cells. Our study showed that all the four cell lines supported SFTSV life cycle and SFTSV activated host innate immunity to produce type I IFNs in Hela cells but not in Huh7.0, Huh7.5.1 or Vero cells. NSs inhibited exogenous IFNα-induced Jak/STAT signaling as shown by decreased p-STAT1 level, suppressed ISRE activity and down-regulated ISG expression. Suppression of the exogenous Type I IFN-induced Jak/STAT signaling by NSs might be one of the mechanisms of SFTSV to evade host immune surveillance.

IL-10/STAT3 is reduced in childhood obesity with hypertriglyceridemia and is related to triglyceride level in diet-induced obese rats.

PubMed

Liu, Yuesheng; Xu, Dong; Yin, Chunyan; Wang, Sisi; Wang, Min; Xiao, Yanfeng

2018-06-13

The prevalence of childhood obesity and obesity-related metabolic disorder such as dyslipidemia has sharply increased in the past few decades. Chronic low-grade inflammation is associated with the development of comorbidities and poor prognosis in obesity. This study aims to evaluate interleukin-10 (IL-10) in childhood obesity with hypertriglyceridemia. We evaluated IL-10 and signal transducer and activator of transcription 3 (STAT3) mRNA expression in adipose tissue (AT) as well as serum IL-10 in 62 children of 3 groups and in high-fat diet (HFD) induced obese rat. Expression of IL-10 and STAT3 protein in AT of diet-induced obese rats were examined over feed period. Adipose IL-10 and STAT3 mRNA expression and serum IL-10 reduced in obese children with hypertriglyceridemia and in HFD obese rats. The protein expression of IL-10 and STAT3 decreased in AT of obese rats compared with the control rats at end time. Expression of IL-10 mRNA was negatively correlated to TG and LDL-C levels, and positively correlated to HDL-C, adiponectin and serum IL-10 levels. IL-10 expression and its downstream JAK-STAT pathway are down-regulated in obese children with hypertriglyceridemia and in HFD obese rats.

Downregulation of PTP1B and TC-PTP phosphatases potentiate dendritic cell-based immunotherapy through IL-12/IFNγ signaling.

PubMed

Penafuerte, Claudia; Feldhammer, Matthew; Mills, John R; Vinette, Valerie; Pike, Kelly A; Hall, Anita; Migon, Eva; Karsenty, Gerard; Pelletier, Jerry; Zogopoulos, George; Tremblay, Michel L

2017-01-01

PTP1B and TC-PTP are highly related protein-tyrosine phosphatases (PTPs) that regulate the JAK/STAT signaling cascade essential for cytokine-receptor activation in immune cells. Here, we describe a novel immunotherapy approach whereby monocyte-derived dendritic cell (moDC) function is enhanced by modulating the enzymatic activities of PTP1B and TC-PTP. To downregulate or delete the activity/expression of these PTPs, we generated mice with PTP-specific deletions in the dendritic cell compartment or used PTP1B and TC-PTP specific inhibitor. While total ablation of PTP1B or TC-PTP expression leads to tolerogenic DCs via STAT3 hyperactivation, downregulation of either phosphatase remarkably shifts the balance toward an immunogenic DC phenotype due to hyperactivation of STAT4, STAT1 and Src kinase. The resulting increase in IL-12 and IFNγ production subsequently amplifies the IL-12/STAT4/IFNγ/STAT1/IL-12 positive autocrine loop and enhances the therapeutic potential of mature moDCs in tumor-bearing mice. Furthermore, pharmacological inhibition of both PTPs improves the maturation of defective moDCs derived from pancreatic cancer (PaC) patients. Our study provides a new advance in the use of DC-based cancer immunotherapy that is complementary to current cancer therapeutics.

17β-Estradiol and ICI182,780 Differentially Regulate STAT5 Isoforms in Female Mammary Epithelium, With Distinct Outcomes

PubMed Central

Jallow, Fatou; Brockman, Jennifer L; Helzer, Kyle T; Rugowski, Debra E; Goffin, Vincent; Alarid, Elaine T; Schuler, Linda A

2018-01-01

Abstract Prolactin (PRL) and estrogen cooperate in lobuloalveolar development of the mammary gland and jointly regulate gene expression in breast cancer cells in vitro. Canonical PRL signaling activates STAT5A/B, homologous proteins that have different target genes and functions. Although STAT5A/B are important for physiological mammary function and tumor pathophysiology, little is known about regulation of their expression, particularly of STAT5B, and the consequences for hormone action. In this study, we examined the effect of two estrogenic ligands, 17β-estradiol (E2) and the clinical antiestrogen, ICI182,780 (ICI, fulvestrant) on expression of STAT5 isoforms and resulting crosstalk with PRL in normal and tumor murine mammary epithelial cell lines. In all cell lines, E2 and ICI significantly increased protein and corresponding nascent and mature transcripts for STAT5A and STAT5B, respectively. Transcriptional regulation of STAT5A and STAT5B by E2 and ICI, respectively, is associated with recruitment of estrogen receptor alpha and increased H3K27Ac at a common intronic enhancer 10 kb downstream of the Stat5a transcription start site. Further, E2 and ICI induced different transcripts associated with differentiation and tumor behavior. In tumor cells, E2 also significantly increased proliferation, invasion, and stem cell-like activity, whereas ICI had no effect. To evaluate the role of STAT5B in these responses, we reduced STAT5B expression using short hairpin (sh) RNA. shSTAT5B blocked ICI-induced transcripts associated with metastasis and the epithelial mesenchymal transition in both cell types. shSTAT5B also blocked E2-induced invasion of tumor epithelium without altering E2-induced transcripts. Together, these studies indicate that STAT5B mediates a subset of protumorigenic responses to both E2 and ICI, underscoring the need to understand regulation of its expression and suggesting exploration as a possible therapeutic target in breast cancer. PMID:29594259

JAK/Stat signaling regulates heart precursor diversification in Drosophila

PubMed Central

Johnson, Aaron N.; Mokalled, Mayssa H.; Haden, Tom N.; Olson, Eric N.

2011-01-01

Intercellular signal transduction pathways regulate the NK-2 family of transcription factors in a conserved gene regulatory network that directs cardiogenesis in both flies and mammals. The Drosophila NK-2 protein Tinman (Tin) was recently shown to regulate Stat92E, the Janus kinase (JAK) and Signal transducer and activator of transcription (Stat) pathway effector, in the developing mesoderm. To understand whether the JAK/Stat pathway also regulates cardiogenesis, we performed a systematic characterization of JAK/Stat signaling during mesoderm development. Drosophila embryos with mutations in the JAK/Stat ligand upd or in Stat92E have non-functional hearts with luminal defects and inappropriate cell aggregations. Using strong Stat92E loss-of-function alleles, we show that the JAK/Stat pathway regulates tin expression prior to heart precursor cell diversification. tin expression can be subdivided into four phases and, in Stat92E mutant embryos, the broad phase 2 expression pattern in the dorsal mesoderm does not restrict to the constrained phase 3 pattern. These embryos also have an expanded pericardial cell domain. We show the E(spl)-C gene HLHm5 is expressed in a pattern complementary to tin during phase 3 and that this expression is JAK/Stat dependent. In addition, E(spl)-C mutant embryos phenocopy the cardiac defects of Stat92E embryos. Mechanistically, JAK/Stat signals activate E(spl)-C genes to restrict Tin expression and the subsequent expression of the T-box transcription factor H15 to direct heart precursor diversification. This study is the first to characterize a role for the JAK/Stat pathway during cardiogenesis and identifies an autoregulatory circuit in which tin limits its own expression domain. PMID:21965617

Nipah virus sequesters inactive STAT1 in the nucleus via a P gene-encoded mechanism.

PubMed

Ciancanelli, Michael J; Volchkova, Valentina A; Shaw, Megan L; Volchkov, Viktor E; Basler, Christopher F

2009-08-01

The Nipah virus (NiV) phosphoprotein (P) gene encodes the C, P, V, and W proteins. P, V, and W, have in common an amino-terminal domain sufficient to bind STAT1, inhibiting its interferon (IFN)-induced tyrosine phosphorylation. P is also essential for RNA-dependent RNA polymerase function. C is encoded by an alternate open reading frame (ORF) within the common amino-terminal domain. Mutations within residues 81 to 113 of P impaired its polymerase cofactor function, as assessed by a minireplicon assay, but these mutants retained STAT1 inhibitory function. Mutations within the residue 114 to 140 region were identified that abrogated interaction with and inhibition of STAT1 by P, V, and W without disrupting P polymerase cofactor function. Recombinant NiVs were then generated. A G121E mutation, which abrogated inhibition of STAT1, was introduced into a C protein knockout background (C(ko)) because the mutation would otherwise also alter the overlapping C ORF. In cell culture, relative to the wild-type virus, the C(ko) mutation proved attenuating but the G121E mutant virus replicated identically to the C(ko) virus. In cells infected with the wild-type and C(ko) viruses, STAT1 was nuclear despite the absence of tyrosine phosphorylation. This latter observation mirrors what has been seen in cells expressing NiV W. In the G121E mutant virus-infected cells, STAT1 was not phosphorylated and was cytoplasmic in the absence of IFN stimulation but became tyrosine phosphorylated and nuclear following IFN addition. These data demonstrate that the gene for NiV P encodes functions that sequester inactive STAT1 in the nucleus, preventing its activation and suggest that the W protein is the dominant inhibitor of STAT1 in NiV-infected cells.

Novel signal transducer and activator of transcription 1 mutation disrupts small ubiquitin-related modifier conjugation causing gain of function.

PubMed

Sampaio, Elizabeth P; Ding, Li; Rose, Stacey R; Cruz, Phillip; Hsu, Amy P; Kashyap, Anuj; Rosen, Lindsey B; Smelkinson, Margery; Tavella, Tatyana A; Ferre, Elise M N; Wierman, Meredith K; Zerbe, Christa S; Lionakis, Michail S; Holland, Steven M

2018-05-01

Sumoylation is a posttranslational reversible modification of cellular proteins through the conjugation of small ubiquitin-related modifier (SUMO) and comprises an important regulator of protein function. We sought to characterize the molecular mechanism of a novel mutation at the SUMO motif on signal transducer and activator of transcription 1 (STAT1). STAT1 sequencing and functional characterization were performed in transfection experiments by using immunoblotting and immunoprecipitation in STAT1-deficient cell lines. Transcriptional response and target gene activation were also investigated in PBMCs. We identified a novel STAT1 mutation (c.2114A>T, p.E705V) within the SUMO motif ( 702 IKTE 705 ) in a patient with disseminated Rhodococcus species infection, Norwegian scabies, chronic mucocutaneous candidiasis, hypothyroidism, and esophageal squamous cell carcinoma. The mutation is located in the tail segment and is predicted to disrupt STAT1 sumoylation. Immunoprecipitation experiments performed in transfected cells confirmed absent STAT1 sumoylation for E705V, whereas it was present in wild-type (WT) STAT1 cells, as well as the loss-of-function mutants L706S and Y701C. Furthermore, stimulation with IFN-γ led to enhanced STAT1 phosphorylation, enhanced transcriptional activity, and target gene expression in the E705V-transfected compared with WT-transfected cells. Computer modeling of WT and mutant STAT1 molecules showed variations in the accessibility of the phosphorylation site Y701, which corresponded to the loss-of-function and gain-of-function variants. This is the first report of a mutation in the STAT1 sumoylation motif associated with clinical disease. These data reinforce sumoylation as a key posttranslational regulatory modification of STAT1 and identify a novel mechanism for gain-of-function STAT1 disease in human subjects. Copyright © 2017 American Academy of Allergy, Asthma & Immunology. All rights reserved.

[Astaxanthin inhibits proliferation and promotes apoptosis of A549 lung cancer cells via blocking JAK1/STAT3 pathway].

PubMed

Wu, Chuntao; Zhang, Jinji; Liu, Tienan; Jiao, Guimei; Li, Changzai; Hu, Baoshan

2016-06-01

Objective To investigate the anti-tumor effects of astaxanthin on A549 lung cancer cells and the related mechanisms. Methods A549 cells were cultured with various concentrations of astaxanthin (20, 40, 60, 80, 100 μmol/L), and DMSO at the same concentrations served as vehicle controls. The viability of A549 cells was detected by CCK-8 assay; cell cycle and apoptosis were observed by flow cytometry; and the expressions of B-cell lymphoma-2 (Bcl-2), Bcl-2 associated X protein (Bax), signal transducers and activators of transcription 3 (STAT3), and Janus kinase 1 (JAK1) were evaluated by Western blotting. Results CCK-8 assay showed that astaxanthin decreased the proliferation of A549 cells in a dose-dependent manner. Flow cytometry showed that astaxanthin increased the number of cells in the G0/G1 phase and induced apoptosis in A549 cells. Western blotting showed that astaxanthin up-regulated the expression of Bax and down-regulated the expressions of Bcl-2, STAT3 and JAK1. Conclusion Astaxanthin functions as a potent inhibitor of A549 lung cancer cell growth by targeting JAK1/STAT3 signaling pathway.

Surfactant protein C dampens inflammation by decreasing JAK/STAT activation during lung repair.

PubMed

Jin, Huiyan; Ciechanowicz, Andrzej K; Kaplan, Alanna R; Wang, Lin; Zhang, Ping-Xia; Lu, Yi-Chien; Tobin, Rachel E; Tobin, Brooke A; Cohn, Lauren; Zeiss, Caroline J; Lee, Patty J; Bruscia, Emanuela M; Krause, Diane S

2018-05-01

Surfactant protein C (SPC), a key component of pulmonary surfactant, also plays a role in regulating inflammation. SPC deficiency in patients and mouse models is associated with increased inflammation and delayed repair, but the key drivers of SPC-regulated inflammation in response to injury are largely unknown. This study focuses on a new mechanism of SPC as an anti-inflammatory molecule using SPC-TK/SPC-KO (surfactant protein C-thymidine kinase/surfactant protein C knockout) mice, which represent a novel sterile injury model that mimics clinical acute respiratory distress syndrome (ARDS). SPC-TK mice express the inducible suicide gene thymidine kinase from by the SPC promoter, which targets alveolar type 2 (AT2) cells for depletion in response to ganciclovir (GCV). We compared GCV-induced injury and repair in SPC-TK mice that have normal endogenous SPC expression with SPC-TK/SPC-KO mice lacking SPC expression. In contrast to SPC-TK mice, SPC-TK/SPC-KO mice treated with GCV exhibited more severe inflammation, resulting in over 90% mortality; there was only 8% mortality of SPC-TK animals. SPC-TK/SPC-KO mice had highly elevated inflammatory cytokines and granulocyte infiltration in the bronchoalveolar lavage (BAL) fluid. Consistent with a proinflammatory phenotype, immunofluorescence revealed increased phosphorylated signal transduction and activation of transcription 3 (pSTAT3), suggesting enhanced Janus kinase (JAK)/STAT activation in inflammatory and AT2 cells of SPC-TK/SPC-KO mice. The level of suppressor of cytokine signaling 3, an anti-inflammatory mediator that decreases pSTAT3 signaling, was significantly decreased in the BAL fluid of SPC-TK/SPC-KO mice. Hyperactivation of pSTAT3 and inflammation were rescued by AZD1480, a JAK1/2 inhibitor. Our findings showing a novel role for SPC in regulating inflammation via JAK/STAT may have clinical applications.

The JAK2 pathway is activated in idiopathic pulmonary fibrosis.

PubMed

Milara, Javier; Hernandez, Gracia; Ballester, Beatriz; Morell, Anselm; Roger, Inés; Montero, P; Escrivá, Juan; Lloris, José M; Molina-Molina, Maria; Morcillo, Esteban; Cortijo, Julio

2018-02-06

Idiopathic pulmonary fibrosis (IPF) is the most rapidly progressive and fatal fibrotic disorder, with no curative therapies. The signal transducer and activator of transcription 3 (STAT3) protein is activated in lung fibroblasts and alveolar type II cells (ATII), thereby contributing to lung fibrosis in IPF. Although activation of Janus kinase 2 (JAK2) has been implicated in proliferative disorders, its role in IPF is unknown. The aim of this study was to analyze JAK2 activation in IPF, and to determine whether JAK2/STAT3 inhibition is a potential therapeutic strategy for this disease. JAK2/p-JAK2 and STAT3/pSTAT3 expression was evaluated using quantitative real time-PCR, western blotting, and immunohistochemistry. Compared to human healthy lung tissue (n = 10) both proteins were upregulated in the lung tissue of IPF patients (n = 12). Stimulating primary ATII and lung fibroblasts with transforming growth factor beta 1 or interleukin (IL)-6/IL-13 activated JAK2 and STAT3, inducing epithelial to mesenchymal and fibroblast to myofibroblast transitions. Dual p-JAK2/p-STAT3 inhibition with JSI-124 or silencing of JAK2 and STAT3 genes suppressed ATII and the fibroblast to myofibroblast transition, with greater effects than the sum of those obtained using JAK2 or STAT3 inhibitors individually. Dual rather than single inhibition was also more effective for inhibiting fibroblast migration, preventing increases in fibroblast senescence and Bcl-2 expression, and ameliorating impaired autophagy. In rats administered JSI-124, a dual inhibitor of p-JAK2/p-STAT3, at a dose of 1 mg/kg/day, bleomycin-induced lung fibrosis was reduced and collagen deposition in the lung was inhibited, as were JAK2 and STAT3 activation and several markers of fibrosis, autophagy, senescence, and anti-apoptosis. JAK2 and STAT3 are activated in IPF, and their dual inhibition may be an attractive strategy for treating this disease.

α-Lipoic Acid Inhibits Expression of IL-8 by Suppressing Activation of MAPK, Jak/Stat, and NF-κB in H. pylori-Infected Gastric Epithelial AGS Cells.

PubMed

Choi, Ji Hyun; Cho, Soon Ok; Kim, Hyeyoung

2016-01-01

The epithelial cytokine response, associated with reactive oxygen species (ROS), is important in Helicobacter pylori (H. pylori)-induced inflammation. H. pylori induces the production of ROS, which may be involved in the activation of mitogen-activated protein kinases (MAPK), janus kinase/signal transducers and activators of transcription (Jak/Stat), and oxidant-sensitive transcription factor, nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), and thus, expression of interleukin-8 (IL-8) in gastric epithelial cells. α-lipoic acid, a naturally occurring thiol compound, is a potential antioxidant. It shows beneficial effects in treatment of oxidant-associated diseases including diabetes. The present study is purposed to investigate whether α-lipoic acid inhibits expression of inflammatory cytokine IL-8 by suppressing activation of MAPK, Jak/Stat, and NF-κB in H. pylori-infected gastric epithelial cells. Gastric epithelial AGS cells were pretreated with or without α-lipoic acid for 2 h and infected with H. pylori in a Korean isolate (HP99) at a ratio of 300:1. IL-8 mRNA expression was analyzed by RT-PCR analysis. IL-8 levels in the medium were determined by enzyme-linked immunosorbent assay. NF-κB-DNA binding activity was determined by electrophoretic mobility shift assay. Phospho-specific and total forms of MAPK and Jak/Stat were assessed by Western blot analysis. ROS levels were determined using dichlorofluorescein fluorescence. As a result, H. pylori induced increases in ROS levels, mRNA, and protein levels of IL-8, as well as the activation of MAPK [extracellular signal-regulated kinase 1/2 (ERK1/2), c-Jun NH2-terminal kinase 1/2 (JNK1/2), p38], Jak/Stat (Jak1/2, Stat3), and NF-κB in AGS cells, which was inhibited by α-lipoic acid. In conclusion, α-lipoic acid may be beneficial for prevention and/or treatment of H. pylori infection-associated gastric inflammation.

The platelet-derived growth factor receptor/STAT3 signaling pathway regulates the phenotypic transition of corpus cavernosum smooth muscle in rats.

PubMed

Yan, Jun-Feng; Huang, Wen-Jie; Zhao, Jian-Feng; Fu, Hui-Ying; Zhang, Gao-Yue; Huang, Xiao-Jun; Lv, Bo-Dong

2017-01-01

Erectile dysfunction (ED) is a common clinical disease that is difficult to treat. We previously found that hypoxia modulates the phenotype of primary corpus cavernosum smooth muscle cells (CCSMCs) in rats, but the underlying molecular mechanism is still unknown. Platelet-derived growth factor receptor (PDGFR)-related signaling pathways are correlated with cell phenotypic transition, but research has been focused more on vascular smooth muscle and tracheal smooth muscle and less on CCSMCs. Here, we investigated the role of PDGFR-related signaling pathways in penile CCSMCs, which were successfully isolated from rats and cultured in vitro. PDGF-BB at 5, 10, or 20 ng/ml altered CCSMC morphology from the original elongated, spindle shape to a broader shape and promoted the synthetic phenotype and expression of the related proteins vimentin and collagen-I, while inhibiting the contractile phenotype and expression of the related proteins smooth muscle (SM) α-actin (α-SMA) and desmin. Inhibition of PDGFR activity via siRNA or the PDGFR inhibitor crenolanib inhibited vimentin and collagen-I expression, increased α-SMA and desmin expression, and considerably inhibited serine-threonine protein kinase (AKT) and signal transducer and activator of transcription 3 (STAT3) phosphorylation. STAT3 knockdown promoted the contractile phenotype, inhibited vimentin and collagen-I expression, and increased α-SMA and desmin expression, whereas AKT knockdown did not affect phenotype-associated proteins. STAT3 overexpression in CCSMC cells weakened the suppressive effect of PDGFR inhibition on the morphology and phenotypic transformation induced by PDGF-BB. Through activation of the PDGFR/STAT3 signaling pathway, PDGF promoted the synthetic phenotype transition; thus, regulation of this pathway might contribute to ED therapy.

Molecular cloning, subcelluar location and expression profile of signal transducer and activator of transcription 2 (STAT2) from turbot, Scophthalmus maximus.

PubMed

Wang, Na; Wang, Xian-Li; Yang, Chang-Geng; Chen, Song-Lin

2013-10-01

Signal transducer and activator of transcription 2 (STAT2) is an important molecule involved in the type I interferon signalling pathway. To date, little STAT2 homologue is available in fish except Atlantic salmon and goldfish. In this paper, STAT2 was firstly cloned and characterized from turbot, a marine flatfish with high economic value. Briefly, turbot STAT2 cDNA is 3206 bp in length encoding a predicted protein of 793 amino acids. The phylogenetic tree shows that turbot STAT2 protein shared the closest relationship with Atlantic salmon. Analysis of subcellular distribution indicates that STAT2 is mainly present in the cytoplasm of TK cells. Stat2 mRNA is constitutively expressed in widespread tissues and induced by several folds in turbot tissues and TK cells after stimulation with Vibrio anguillarum and lymphocystis disease virus (LCDV). Unlike the higher vertebrate STAT2, turbot STAT2 nuclear export signal (NES) exists not in the C-terminal 79 amino acids but in N-terminal 137-312 amino acids (STAT_alpha domain). The nuclear translocation of turbot STAT2 after Poly(I:C) treatment proved its transcription activity in TK cells. All these results suggested that STAT2 may be involved in the immune response in turbot as a transcription factor. Copyright © 2013 Elsevier Ltd. All rights reserved.

[The effect of leptin and its mechanisms on the migration and invasion of human breast cancer MCF-7 cells].

PubMed

Wang, Lin; Cao, Hong; Pang, Xueli; Li, Kuangfa; Dang, Weiqi; Tang, Hao; Chen, Tingmei

2013-12-01

To investigate the effect and the relevant molecular mechanisms of leptin on the migration and invasion of human breast cancer MCF-7 cells. The expression of OB-R in MCF-7 cells was measured by RT-PCR and Western blotting. The effects of leptin (100 ng/mL) on the the phosphorylation of a few key cell signaling proteins, p-ERK1/2, p-STAT3, p-AKT in MCF-7 cells were examined by Western blotting. Cell scratch assay and Transwell(TM); assay were utilized to measure the effects of leptin on the migration and invasion capability of MCF-7 cells, respectively. The effects of leptin on the mRNA and protein expression of matrix metalloproteinas 9 (MMP-9) and transforming growth factor β (TGF-β) were measured by RT-PCR and Western blotting. Both OB-Rb and OB-Rt were expressed in MCF-7 cells. This indicated that leptin may have significant activities in MCF7 cells. Indeed, leptin increased the phosphorylation of p-ERK1/2, p-STAT3, and p-AKT in MCF-7 cells (P < 0.05). Further, leptin promoted migration and invasion of MCF-7 cells, which were attenuated by the JAK/STAT inhibitor AG490 (50 μmol/L), and the PI3K/AKT inhibitor LY294002 (10 μmol/L) (P < 0.05). Similarly, leptin also increased the mRNA and protein expression of MMP-9 and TGF-β, and these effects were blocked by AG490 and LY294002 as well (P < 0.05). Leptin promoted the migration and invasion capabilities of MCF-7 cells. These activities may be achieved by the upregulation of MMP-9 and TGF-β through JAK/STAT and PI3K/AKT signaling pathways.

STAT3, p-STAT3 and HIF-1α are associated with vasculogenic mimicry and impact on survival in gastric adenocarcinoma

PubMed Central

SONG, YAN-YAN; SUN, LI-DAN; LIU, MIN-LI; LIU, ZHONG-LIANG; CHEN, FEI; ZHANG, YING-ZHE; ZHENG, YAN; ZHANG, JIAN-PING

2014-01-01

Vasculogenic mimicry (VM) formation is important for invasion and metastasis of tumor cells in gastric adenocarcinoma (GAC). The present study aimed to investigate the association between signal transducer and activator of transcription-3 (STAT3), phosphor-STAT3 (p-STAT3), hypoxia-inducible factor-1α (HIF-1α) and VM formation in GAC, and discuss their clinical significance and correlation with the prognosis of patients with GAC. The expression levels of STAT3, p-STAT3, HIF-1α and VM were assessed in 60 cases of patients with GAC and 20 cases of patients with gastritis on tissue microarrays by immunohistochemical methods. The expression levels of STAT3, p-STAT3, HIF-1α and VM were higher in patients with GAC (particularly in poorly differentiated GAC) than in those with gastritis (P<0.05). The expression levels of STAT3, p-STAT3 and HIF-1α were higher in VM tissues compared with non-VM tissues (P<0.05). Positive correlations existed between STAT3, p-STAT3, HIF-1α and VM expression (P<0.05). The expression levels of STAT3, p-STAT3 and HIF-1α, VM, status of lymph node metastasis and tumor differentiation degree were associated with the overall survival time of patients with GAC (P<0.05). However, only p-STAT3 and VM expression were identified as the independent risk factors of GAC OS when analyzed with multivariate analysis. p-STAT3 and VM play a significant role in indicating the prognosis of patients with GAC. STAT3 activation may play a positive role in VM formation of GAC by the STAT3-p-STAT3-HIF-1α-VM effect axis. PMID:24959290

Effects of total flavonoids of sea buckthorn ( Hippophae rhamnoides L.) on cytotoxicity of NK92-MI cells.

PubMed

Hou, Diandong; Wang, Decheng; Ma, Xiande; Chen, Wenna; Guo, Shengnan; Guan, Hongquan

2017-12-01

Sea buckthorn ( Hippophae rhamnoides L.) has multifarious medicinal properties including immunoregulatory effect. The total flavonoids of Hippophae rhamnoides L. (TFH) are the main active components isolated from berries of sea buckthorn. The aim of this study was to evaluate the effects of TFH on the cytotoxicity of NK92-MI cells and its possible mechanisms. NK92-MI cells were treated with TFH (2.5 or 5.0 mg/L) or phosphate-buffered saline (PBS) for 24 h, the cytotoxicity against K562 was detected by measuring the release of lactate dehydrogenase (LDH), expression levels of NCRs (NKp30, NKp44, NKp46) and NKG2D were detected by flow cytometry, and expression levels of perforin and granzyme B were detected by western blot. Cytokine Antibody Arrays with 80 cytokine proteins were used to profile the effect of TFH on cytokines. Western blot was adopted to detect the effects of TFH on STAT1, STAT4, and STAT5 signal pathway. Compared with the normal control group, TFH could significantly enhance NK92-MI cell cytotoxicity against K562 cells, upregulate expressions of NKp44, NKp46, perforin, and granzyme B. TFH could upregulate expressions of IL-1α, IL-2, IL-7, IL-15, CSF-2, CSF-3, MCP-1, MIG, IFN-γ, TNF-α, and TNF-β and downregulate expressions of IL-16, MIP-1β, CX3CL-1, and MIF. TFH could increase expressions of phospho-STAT1 and phospho-STAT5. The results suggest that TFH stimulated NK92-MI cells to activate and enhance cytotoxicity of NK92-MI cells.

The transcriptional co-activator p/CIP (NCoA-3) is up-regulated by STAT6 and serves as a positive regulator of transcriptional activation by STAT6.

PubMed

Arimura, Akinori; vn Peer, Maartje; Schröder, Andreas J; Rothman, Paul B

2004-07-23

Transcriptional activation by signal transducer and activator of transcription 6 (STAT6) has been shown to require the direct interaction not only with co-activators such as p300 and cAMP-responsive element-binding protein-binding protein (CBP) but also with nuclear co-activator 1, a member of the p160/steroid receptor co-activator family. Among the p160/steroid receptor co-activators, only p/CIP (nuclear co-activator 3) has been shown to be up-regulated by interleukin (IL)-4 in B cells through a STAT-6-dependent mechanism using Gene-Chip analysis. In this study, we have investigated the function of p/CIP in the transcriptional activation by STAT6. We found that p/CIP indirectly interacted with STAT6 via p300, and overexpression of the CBP-interacting domain of p/CIP (p/CIP(947-1084)) prevented the interaction of p/CIP with STAT6 by blocking the binding of p/CIP to p300. Whereas expression of p/CIP(947-1084) resulted in a marked reduction of STAT6-mediated transactivation, overexpression of wild type p/CIP resulted in significant enhancement of it. In addition, p/CIP(947-1084) markedly reduced CD23 expression on B cells stimulated with IL-4, whereas overexpression of wild type p/CIP enhanced it. Chromatin immunoprecipitations demonstrate that IL-4 increases the interaction of p/CIP with the murine immunoglobulin heavy chain germ line epsilon promoter in B cells. These results suggest that p/CIP positively regulates STAT6 transcriptional activation through formation of a STAT6, p300/CBP, and p/CIP complex.

Proteomic and functional analyses reveal MAPK1 regulates milk protein synthesis.

PubMed

Lu, Li-Min; Li, Qing-Zhang; Huang, Jian-Guo; Gao, Xue-Jun

2012-12-27

L-Lysine (L-Lys) is an essential amino acid that plays fundamental roles in protein synthesis. Many nuclear phosphorylated proteins such as Stat5 and mTOR regulate milk protein synthesis. However, the details of milk protein synthesis control at the transcript and translational levels are not well known. In this current study, a two-dimensional gel electrophoresis (2-DE)/MS-based proteomic technology was used to identify phosphoproteins responsible for milk protein synthesis in dairy cow mammary epithelial cells (DCMECs). The effect of L-Lys on DCMECs was analyzed by CASY technology and reversed phase high performance liquid chromatography (RP-HPLC). The results showed that cell proliferation ability and β-casein expression were enhanced in DCMECs treated with L-Lys. By phosphoproteomics analysis, six proteins, including MAPK1, were identified up-expressed in DCMECs treated with 1.2 mM L-Lys for 24 h, and were verified by quantitative real-time PCR (qRT-PCR) and western blot. Overexpression and siRNA inhibition of MAPK1 experiments showed that MAPK1 upregulated milk protein synthesis through Stat5 and mTOR pathway. These findings that MAPK1 involves in regulation of milk synthesis shed new insights for understanding the mechanisms of milk protein synthesis.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Park, Sun Young; Kim, Ji-Hee; Lee, Sang Joon

Surfactin, one of the most powerful biosurfactants, is a bacterial cyclic lipopeptide. Here, we investigated the anti-neuroinflammatory properties of surfactin in lipoteichoic acid (LTA)-stimulated BV-2 microglial cells. Surfactin significantly inhibited excessive production of the pro-inflammatory mediators TNF-α, IL-1β, IL-6, monocyte chemoattractant protein-1 (MCP-1), prostaglandin E{sub 2} (PGE{sub 2}), nitric oxide (NO) and reactive oxygen species (ROS), and suppressed the expression of matrix metalloproteinase-9 (MMP-9), inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2). Subsequent mechanistic studies revealed that surfactin inhibited LTA-induced nuclear factor-kappaB (NF-κB) and signal transducer and activator of transcription-1 (STAT-1) activation. However, surfactin increases the phosphorylation of the STAT-3, amore » component of the homeostatic mechanism causing anti-inflammatory events. We also demonstrated that surfactin induces heme oxygenase-1 (HO-1) expression and nuclear factor-regulated factor-2 (Nrf-2) activation, and that the anti-inflammatory effects of surfactin are abrogated by small interfering RNA-mediated knock-down of HO-1 or Nrf-2. Interestingly, we found that surfactin increased the level of cAMP and induced phosphorylation of cAMP responsive element binding protein (CREB) in microglial cells. Furthermore, treatment with the protein kinase A (PKA) inhibitor, H-89, blocked HO-1 induction by surfactin and abolished surfactin's suppressive effects on ROS and NO production. These results indicate that HO-1 and its upstream effector, PKA, play a pivotal role in the anti-neuroinflammatory response of surfactin in LTA-stimulated microglia. Therefore, surfactin might have therapeutic potential for neuroprotective agents to treat inflammatory and neurodegenerative diseases. - Highlights: ► Surfactin inhibits proinflammatory mediator synthesis in LTA-activated BV-2 cells. ► Surfactin suppresses NF-κB and STAT-1, but potentiates phosphorylation of STAT-3. ► Surfactin induces HO-1 expression and Nrf-2 activation. ► Surfactin induces cAMP and CREB phosphorylation. ► PKA inhibitor blocks HO-1 induction and surfactin’s antiinflammatory effects.« less

Interferon-γ biphasically regulates angiotensinogen expression via a JAK-STAT pathway and suppressor of cytokine signaling 1 (SOCS1) in renal proximal tubular cells

PubMed Central

Satou, Ryousuke; Miyata, Kayoko; Gonzalez-Villalobos, Romer A.; Ingelfinger, Julie R.; Navar, L. Gabriel; Kobori, Hiroyuki

2012-01-01

Renal inflammation modulates angiotensinogen (AGT) production in renal proximal tubular cells (RPTCs) via inflammatory cytokines, including interleukin-6, tumor necrosis factor α, and interferon-γ (IFN-γ). Among these, the effects of IFN-γ on AGT regulation in RPTCs are incompletely delineated. This study aimed to elucidate mechanisms by which IFN-γ regulates AGT expression in RPTCs. RPTCs were incubated with or without IFN-γ up to 48 h. AGT expression, STAT1 and STAT3 activities, and SOCS1 expression were evaluated. RNA interference studies against STAT1, SOCS1, and STAT3 were performed to elucidate a signaling cascade. IFN-γ decreased AGT expression at 6 h (0.61±0.05, ratio to control) and 12 h (0.47±0.03). In contrast, longer exposure for 24 and 48 h increased AGT expression (1.76±0.18, EC50=3.4 ng/ml, and 1.45±0.08, respectively). IFN-γ treatment for 6 h strongly induced STAT1 phosphorylation and SOCS1 augmentation, and decreased STAT3 activity. However, STAT1 phosphorylation and SOCS1 augmentation waned at 24 h, while STAT3 activity increased. RNA interference studies revealed that activation of STAT1-SOCS1 axis decreased STAT3 activity. Thus, IFN-γ biphasically regulates AGT expression in RPTCs via STAT3 activity modulated by STAT1-SOCS1 axis, suggesting the STAT1-SOCS1 axis is important in IFN-γ-induced activation of the intrarenal renin-angiotensin system.—Satou, R., Miyata, K., Gonzalez-Villalobos, R. A., Ingelfinger, J. R., Navar, L. G., Kobori, H. Interferon-γ biphasically regulates angiotensinogen expression via a JAK-STAT pathway and suppressor of cytokine signaling 1 (SOCS1) in renal proximal tubular cells. PMID:22302831

JaK/STAT Inhibition to Prevent Post-Traumatic Epileptogenesis

DTIC Science & Technology

2013-07-31

months 22-28) 2i. Assess mossy fiber sprouting, cell loss and glial proliferation 10 weeks post injury using Timm and Nissl staining (40 mice...1e. Assess protein levels and regional/cellular expression of JaK1 and 2, pSTAT1-5 using fluorescent immunohistochemistry with co- staining for cell...treated with CCI, 10 of which were treated with WP1066. Early post-injury experiments are underway; Timm staining has not revealed mossy fiber

Histidine augments the suppression of hepatic glucose production by central insulin action.

PubMed

Kimura, Kumi; Nakamura, Yusuke; Inaba, Yuka; Matsumoto, Michihiro; Kido, Yoshiaki; Asahara, Shun-Ichiro; Matsuda, Tomokazu; Watanabe, Hiroshi; Maeda, Akifumi; Inagaki, Fuyuhiko; Mukai, Chisato; Takeda, Kiyoshi; Akira, Shizuo; Ota, Tsuguhito; Nakabayashi, Hajime; Kaneko, Shuichi; Kasuga, Masato; Inoue, Hiroshi

2013-07-01

Glucose intolerance in type 2 diabetes is related to enhanced hepatic glucose production (HGP) due to the increased expression of hepatic gluconeogenic enzymes. Previously, we revealed that hepatic STAT3 decreases the expression of hepatic gluconeogenic enzymes and suppresses HGP. Here, we show that increased plasma histidine results in hepatic STAT3 activation. Intravenous and intracerebroventricular (ICV) administration of histidine-activated hepatic STAT3 reduced G6Pase protein and mRNA levels and augmented HGP suppression by insulin. This suppression of hepatic gluconeogenesis by histidine was abolished by hepatic STAT3 deficiency or hepatic Kupffer cell depletion. Inhibition of HGP by histidine was also blocked by ICV administration of a histamine H1 receptor antagonist. Therefore, histidine activates hepatic STAT3 and suppresses HGP via central histamine action. Hepatic STAT3 phosphorylation after histidine ICV administration was attenuated in histamine H1 receptor knockout (Hrh1KO) mice but not in neuron-specific insulin receptor knockout (NIRKO) mice. Conversely, hepatic STAT3 phosphorylation after insulin ICV administration was attenuated in NIRKO but not in Hrh1KO mice. These findings suggest that central histidine action is independent of central insulin action, while both have additive effects on HGP suppression. Our results indicate that central histidine/histamine-mediated suppression of HGP is a potential target for the treatment of type 2 diabetes.

Histidine Augments the Suppression of Hepatic Glucose Production by Central Insulin Action

PubMed Central

Kimura, Kumi; Nakamura, Yusuke; Inaba, Yuka; Matsumoto, Michihiro; Kido, Yoshiaki; Asahara, Shun-ichiro; Matsuda, Tomokazu; Watanabe, Hiroshi; Maeda, Akifumi; Inagaki, Fuyuhiko; Mukai, Chisato; Takeda, Kiyoshi; Akira, Shizuo; Ota, Tsuguhito; Nakabayashi, Hajime; Kaneko, Shuichi; Kasuga, Masato; Inoue, Hiroshi

2013-01-01

Glucose intolerance in type 2 diabetes is related to enhanced hepatic glucose production (HGP) due to the increased expression of hepatic gluconeogenic enzymes. Previously, we revealed that hepatic STAT3 decreases the expression of hepatic gluconeogenic enzymes and suppresses HGP. Here, we show that increased plasma histidine results in hepatic STAT3 activation. Intravenous and intracerebroventricular (ICV) administration of histidine-activated hepatic STAT3 reduced G6Pase protein and mRNA levels and augmented HGP suppression by insulin. This suppression of hepatic gluconeogenesis by histidine was abolished by hepatic STAT3 deficiency or hepatic Kupffer cell depletion. Inhibition of HGP by histidine was also blocked by ICV administration of a histamine H1 receptor antagonist. Therefore, histidine activates hepatic STAT3 and suppresses HGP via central histamine action. Hepatic STAT3 phosphorylation after histidine ICV administration was attenuated in histamine H1 receptor knockout (Hrh1KO) mice but not in neuron-specific insulin receptor knockout (NIRKO) mice. Conversely, hepatic STAT3 phosphorylation after insulin ICV administration was attenuated in NIRKO but not in Hrh1KO mice. These findings suggest that central histidine action is independent of central insulin action, while both have additive effects on HGP suppression. Our results indicate that central histidine/histamine-mediated suppression of HGP is a potential target for the treatment of type 2 diabetes. PMID:23474485

IL-7–dependent STAT1 activation limits homeostatic CD4+ T cell expansion

PubMed Central

Le Saout, Cecile; Luckey, Megan A.; Villarino, Alejandro V.; Smith, Mindy; Hasley, Rebecca B.; Myers, Timothy G.; Imamichi, Hiromi; Park, Jung-Hyun; O’Shea, John J.; Lane, H. Clifford

2017-01-01

IL-7 regulates homeostatic mechanisms that maintain the overall size of the T cell pool throughout life. We show that, under steady-state conditions, IL-7 signaling is principally mediated by activation of signal transducers and activators of transcription 5 (STAT5). In contrast, under lymphopenic conditions, there is a modulation of STAT1 expression resulting in an IL-7–dependent STAT1 and STAT5 activation. Consequently, the IL-7–induced transcriptome is altered with enrichment of IFN-stimulated genes (ISGs). Moreover, STAT1 overexpression was associated with reduced survival in CD4+ T cells undergoing lymphopenia-induced proliferation (LIP). We propose a model in which T cells undergoing LIP upregulate STAT1 protein, “switching on” an alternate IL-7–dependent program. This mechanism could be a physiological process to regulate the expansion and size of the CD4+ T cell pool. During HIV infection, the virus could exploit this pathway, leading to the homeostatic dysregulation of the T cell pools observed in these patients. PMID:29202461

STAT3 inhibition attenuates the progressive phenotypes of Alport syndrome mouse model.

PubMed

Yokota, Tsubasa; Omachi, Kohei; Suico, Mary Ann; Kamura, Misato; Kojima, Haruka; Fukuda, Ryosuke; Motomura, Keishi; Teramoto, Keisuke; Kaseda, Shota; Kuwazuru, Jun; Takeo, Toru; Nakagata, Naomi; Shuto, Tsuyoshi; Kai, Hirofumi

2018-02-01

Alport syndrome (AS) is a hereditary, progressive nephritis caused by mutation of type IV collagen. Previous studies have shown that activation of signal transducer and activator of transcription 3 (STAT3) exacerbates other renal diseases, but whether STAT3 activation exacerbates AS pathology is still unknown. Here we aim to investigate the involvement of STAT3 in the progression of AS. Phosphorylated STAT3 expression was assessed by immunoblotting analysis of kidneys and glomeruli of an AS mouse model (Col4a5 G5X mutant). To determine the effect of blocking STAT3 signaling, we treated AS mice with the STAT3 inhibitor stattic (10 mg/kg i.p., three times per week for 10 weeks; n = 10). We assessed the renal function [proteinuria, blood urea nitrogen (BUN), serum creatinine] and analyzed the glomerular injury score, fibrosis and inflammatory cell invasion by histological staining. Moreover, we analyzed the gene expression of nephritis-associated molecules. Phosphorylated STAT3 was upregulated in AS kidneys and glomeruli. Treatment with stattic ameliorated the progressive renal dysfunction, such as increased levels of proteinuria, BUN and serum creatinine. Stattic also significantly suppressed the gene expression levels of renal injury markers (Lcn2, Kim-1), pro-inflammatory cytokines (Il-6, KC), pro-fibrotic genes (Tgf-β, Col1a1, α-Sma) and Mmp9. Stattic treatment decreased the renal fibrosis congruently with the decrease of transforming growth factor beta (TGF-β) protein and increase of antifibrosis-associated markers p-Smad1, 5 and 8, which are negative regulators of TGF-β signaling. STAT3 inhibition significantly ameliorated the renal dysfunction in AS mice. Our finding identifies STAT3 as an important regulator in AS progression and provides a promising therapeutic target for AS. © The Author 2017. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved.

Hypothalamic S1P/S1PR1 axis controls energy homeostasis.

PubMed

Silva, Vagner R R; Micheletti, Thayana O; Pimentel, Gustavo D; Katashima, Carlos K; Lenhare, Luciene; Morari, Joseane; Mendes, Maria Carolina S; Razolli, Daniela S; Rocha, Guilherme Z; de Souza, Claudio T; Ryu, Dongryeol; Prada, Patrícia O; Velloso, Lício A; Carvalheira, José B C; Pauli, José Rodrigo; Cintra, Dennys E; Ropelle, Eduardo R

2014-09-25

Sphingosine 1-phosphate receptor 1 (S1PR1) is a G-protein-coupled receptor for sphingosine-1-phosphate (S1P) that has a role in many physiological and pathophysiological processes. Here we show that the S1P/S1PR1 signalling pathway in hypothalamic neurons regulates energy homeostasis in rodents. We demonstrate that S1PR1 protein is highly enriched in hypothalamic POMC neurons of rats. Intracerebroventricular injections of the bioactive lipid, S1P, reduce food consumption and increase rat energy expenditure through persistent activation of STAT3 and the melanocortin system. Similarly, the selective disruption of hypothalamic S1PR1 increases food intake and reduces the respiratory exchange ratio. We further show that STAT3 controls S1PR1 expression in neurons via a positive feedback mechanism. Interestingly, several models of obesity and cancer anorexia display an imbalance of hypothalamic S1P/S1PR1/STAT3 axis, whereas pharmacological intervention ameliorates these phenotypes. Taken together, our data demonstrate that the neuronal S1P/S1PR1/STAT3 signalling axis plays a critical role in the control of energy homeostasis in rats.

Inhibition of STAT-3 Results in Radiosensitization of Human Squamous Cell Carcinoma

PubMed Central

Bonner, James A.; Trummell, Hoa Q.; Willey, Christopher D.; Plants, Brian A.; Raisch, Kevin P.

2009-01-01

Background Signal Transducer and Activator of Transcription – 3 (STAT-3) is a downstream component of the Epidermal Growth Factor Receptor (EGFr) signaling process that may facilitate the resistance of tumor cells to conventional cancer treatments. Studies were performed to determine if inhibition of this downstream protein may produce radiosensitization. Methods/Results A431 cells (human squamous cell carcinoma cells with EGFr overexpression) were found to be sensitized to radiation after treatment with STAT-3 small interfering RNA (siRNA). Therefore, a short hairpin RNA (shRNA) against STAT-3 was designed and cloned into a pBABE vector system modified for shRNA expression. Following transfection, clone 2.1 was selected for further study as it showed a dramatic reduction of STAT-3 protein (and mRNA) when compared to A431 parental cells or a negative control shRNA cell line (transfected with STAT-3 shRNA with 2 base pairs mutated). A431 2.1 showed doubling times of 25-31 h as compared to 18-24 h for the parental cell line. The A431 shRNA knockdown STAT-3 cells A431 were more sensitive to radiation than A431 parental or negative STAT-3 control cells. Conclusion A431 cells stably transfected with shRNA against STAT-3 resulted in enhanced radiosensitivity. Further work will be necessary to determine whether inhibition of STAT-3 phosphorylation is a necessary step for the radiosensitization that is induced by inhibition of EGFr. PMID:19616333

Possible involvement of the JAK/STAT signaling pathway in N-acetylcysteine-mediated antidepressant-like effects.

PubMed

Al-Samhari, Marwa M; Al-Rasheed, Nouf M; Al-Rejaie, Salim; Al-Rasheed, Nawal M; Hasan, Iman H; Mahmoud, Ayman M; Dzimiri, Nduna

2016-03-01

Advances in depression research have targeted inflammation and oxidative stress to develop novel types of treatment. The JAK/STAT signaling pathway plays pivotal roles in immune and inflammatory responses. The present study was designed to investigate the effects of N-acetylcysteine, a putative precursor of the antioxidant glutathione, in an animal model of depression, with an emphasis on the JAK/STAT signaling pathway. Fluoxetine, a classical antidepressant drug was also under investigation. Male Wistar rats were subjected to forced swimming test and given N-acetylcysteine and fluoxetine immediately after the pre-test session, 5 h later and 1 h before the test session of the forced swimming test. N-acetylcysteine decreased immobility time (P < 0.05), serum corticosterone (P < 0.001), and hydrogen peroxide (P < 0.001), while restored glutathione concentration. Treatment of the rats with N-acetylcysteine produced significant (P < 0.001) down-regulation of STAT3 mRNA expression and protein phosphorylation. On the other hand, N-acetylcysteine significantly (P < 0.001) increased SOCS3 gene expression; however, SOCS3 protein was not changed. In conclusion, our study suggests that modulation of the JAK/STAT pathway might mediate the antidepressant-like effects of N-acetylcysteine. Therefore, depression research may target the JAK/STAT signaling pathway to provide a novel effective therapy. © 2015 by the Society for Experimental Biology and Medicine.

Possible involvement of the JAK/STAT signaling pathway in N-acetylcysteine-mediated antidepressant-like effects

PubMed Central

Al-Samhari, Marwa M; Al-Rasheed, Nouf M; Al-Rejaie, Salim; Al-Rasheed, Nawal M; Hasan, Iman H; Dzimiri, Nduna

2015-01-01

Advances in depression research have targeted inflammation and oxidative stress to develop novel types of treatment. The JAK/STAT signaling pathway plays pivotal roles in immune and inflammatory responses. The present study was designed to investigate the effects of N-acetylcysteine, a putative precursor of the antioxidant glutathione, in an animal model of depression, with an emphasis on the JAK/STAT signaling pathway. Fluoxetine, a classical antidepressant drug was also under investigation. Male Wistar rats were subjected to forced swimming test and given N-acetylcysteine and fluoxetine immediately after the pre-test session, 5 h later and 1 h before the test session of the forced swimming test. N-acetylcysteine decreased immobility time (P < 0.05), serum corticosterone (P < 0.001), and hydrogen peroxide (P < 0.001), while restored glutathione concentration. Treatment of the rats with N-acetylcysteine produced significant (P < 0.001) down-regulation of STAT3 mRNA expression and protein phosphorylation. On the other hand, N-acetylcysteine significantly (P < 0.001) increased SOCS3 gene expression; however, SOCS3 protein was not changed. In conclusion, our study suggests that modulation of the JAK/STAT pathway might mediate the antidepressant-like effects of N-acetylcysteine. Therefore, depression research may target the JAK/STAT signaling pathway to provide a novel effective therapy. PMID:26643864

Anticancer effect of cucurbitacin B on MKN-45 cells via inhibition of the JAK2/STAT3 signaling pathway

PubMed Central

Xie, You-Li; Tao, Wen-Hui; Yang, Ti-Xiong; Qiao, Jian-Guo

2016-01-01

The aim of the present study was to investigate the effect of cucurbitacin B on MKN-45 gastric carcinoma cells. Cell proliferation was determined using a cell counting kit-8 assay, and commercial cell cycle and apoptosis analysis kits were used to determine the cell cycle by flow cytometry. The mRNA expression of genes which mediate cell cycle checkpoints and apoptosis was detected using reverse transcription-quantitative polymerase chain reaction, and a terminal deoxynucleotidyl transferase dUTP nick end labeling assay was used to determine apoptosis rate. Western blot analysis was used to detect the protein expression levels of JAK2/STAT3 signaling pathway-associated proteins. The presented data show that cucurbitacin B significantly inhibited the proliferation of MKN-45 cells in a dose- and time-dependent manner. In accordance with these findings, cucurbitacin B blocked the progression of the cell cycle from G0/G1 to S phase, which was confirmed by the mRNA expression analysis. Cucurbitacin B treatment significantly suppressed the expression of cyclin D1, cyclin E, cyclin-dependent kinase 4 (CDK4) and CDK2, while increasing the expression of p27. Cucurbitacin B also promoted cell apoptosis, as was determined by TUNEL assay and evaluation of mRNA expression. Further experiments suggested that the beneficial effect of cucurbitacin B on blocking the proliferation and inducing the apoptosis of MKN-45 cells may have been associated with suppression of the JAK2/STAT3 signaling pathway. Thus, the present results indicate that cucurbitacin B suppresses proliferation and promoted apoptosis of MKN-45 cells, which may be mediated by inhibition of the JAK2/STAT3 signaling pathway. Cucurbitacin B therefore may warrant further investigation as a feasible therapy for gastric carcinoma. PMID:27698776

Non-genomic STAT5-dependent effects at the endoplasmic reticulum and Golgi apparatus and STAT6-GFP in mitochondria

PubMed Central

Sehgal, Pravin B

2013-01-01

STAT protein species are well-known as transcription factors that regulate nuclear gene expression. Recent novel lines of research suggest new non-genomic functions of STAT5A/B and STAT6. It was discovered in human pulmonary arterial endothelial cells that STAT5A, including STAT5A-GFP, constitutively associated with the Golgi apparatus, and both STAT5A and B with the endoplasmic reticulum. Acute siRNA-mediated knockdown of STAT5A/B led to the rapid development of a dramatic cystic change in the endoplasmic reticulum (ER) characterized by deposition of the ER structural protein reticulon-4 (RTN4; also called Nogo-B) and the ER-resident GTPase atlastin-3 (ATL3) along cyst membranes and cyst-zone boundaries, accompanied by Golgi fragmentation. Functional consequences included reduced anterograde trafficking, an ER stress response (increased GRP78/BiP) and eventual mitochondrial fragmentation. This phenotype was “non-genomic” in that it was elicited in enucleated cytoplasts. In cross-immunopanning assays STAT5A and B species associated with ATL3, and the ER-lumen spacer CLIMP63 (also called cytoskeleton-associated protein 4, CKAP4) but not RTN4. From a disease significance perspective we posit that STAT5, which is known to be affected by estradiol-17β and prolactin, represents the gender-sensitive determinant in the pathogenesis of idiopathic pulmonary hypertension (IPAH), a disease which includes ER/Golgi dysfunctions but with a 2- to 4-fold higher prevalence in postpubertal women. A separate line of recent research produced evidence for the association of STAT6-GFP, but not STAT3-GFP, STAT3-DsRed, or STAT3-Flag, with mitochondria in live-cell, immunofluorescence, and immunoelectron microscopy. An N-terminal truncation of STAT6-GFP (1–459), which lacked the SH2 domain and Tyr-phosphorylation site, constitutively associated with mitochondria. Thus, the emergent new of biology STAT proteins includes non-genomic roles—structurally and functionally—in the three closely related membrane organelles consisting of the endoplasmic reticulum, Golgi apparatus, and mitochondria. PMID:24470974

Evaluation of STAT5A Gene Expression in Aflatoxin B1 Treated Bovine Mammary Epithelial Cells

PubMed Central

Forouharmehr, Ali; Harkinezhad, Taher; Qasemi-Panahi, Babak

2013-01-01

Purpose: Aflatoxin B1 (AFB1) is a potent mycotoxin which has been produced by fungi such as Aspergillus flavus and Aspergillus parasiticus as secondary metabolites due to their growth on food stuffs and induces hepatocellular carcinoma in many animal species, including humans. In the present study, the effect of AFB1 on STAT5A gene expression was investigated in bovine mammary epithelial cells using real time RT-PCR. Methods: Bovine mammary epithelial cells were seeded in a 24-well culture plate for three-dimensional (3D) culture in Matrigel matrix. After 21 days of 3D culture and reaching the required number of cells, cells were treated with AFB1 and incubated for 8 h. For real time PCR reaction, total RNA from the cultured and treated cells was extracted and used for complementary DNA synthesis. Results: The expression of STAT5A gene was significantly down regulated by AFB1 in dose- dependent manner and led to the reduction of proliferation and differentiation of epithelial cells, which has direct effect in milk protein quantity and quality. Conclusion: According to the results, it seems that down regulation of STAT5A gene in AFB1-treated cells maybe due to DNA damage induced by AFB1 in bovine mammary epithelial cells. PMID:24312879

DOE Office of Scientific and Technical Information (OSTI.GOV)

Unterberger, Claudia; Hanson, Steven; Department of Infection, Immunity and Inflammation, University of Leicester, University Road, Leicester LE1 9HN

Little is known about determinants regulating expression of Mannan-binding lectin associated serine protease-2 (MASP-2), the effector component of the lectin pathway of complement activation. Comparative bioinformatic analysis of the MASP2 promoter regions in human, mouse, and rat, revealed conservation of two putative Stat binding sites, termed StatA and StatB. Site directed mutagenesis specific for these sites was performed. Transcription activity was decreased 5-fold when StatB site was mutated in the wildtype reporter gene construct. Gel retardation and competition assays demonstrated that proteins contained in the nuclear extract prepared from HepG2 specifically bound double-stranded StatB oligonucleotides. Supershift analysis revealed Stat3 tomore » be the major specific binding protein. We conclude that Stat3 binding is important for MASP2 promoter activity.« less

p-STAT3 in luminal breast cancer: Integrated RNA-protein pooled analysis and results from the BIG 2-98 phase III trial.

PubMed

Sonnenblick, Amir; Salgado, Roberto; Brohée, Sylvain; Zahavi, Tamar; Peretz, Tamar; Van den Eynden, Gert; Rouas, Ghizlane; Salmon, Asher; Francis, Prudence A; Di Leo, Angelo; Crown, John P A; Viale, Giuseppe; Daly, Laura; Javdan, Bahar; Fujisawa, Sho; De Azambuja, Evandro; Lieveke, Ameye; Piccart, Martine J; Bromberg, Jacqueline F; Sotiriou, Christos

2018-02-01

In the present study, in order to investigate the role of signal transducer and activator of transcription 3 (STAT3) in estrogen receptor (ER)-positive breast cancer prognosis, we evaluated the phosphorylated STAT3 (p-STAT3) status and investigated its effect on the outcome in a pooled analysis and in a large prospective adjuvant trial. By using the TCGA repository, we developed gene signatures that reflected the level of p-STAT3. Using pooled analysis of the expression data from luminal breast cancer patients, we assessed the effects of the p-STAT3 expression signature on prognosis. We further validated the p-STAT3 prognostic effect using immunohistochemistry (IHC) and immunofluorescence staining of p-STAT3 tissue microarrays from a large randomised prospective trial. Our analysis demonstrated that p-STAT3 expression was elevated in luminal A-type breast cancer (Kruskal-Wallis test, P<10e-10) and was significantly associated with a good prognosis (log-rank, P<10e-10). Notably, the p-STAT3 expression signature identified patients with a good prognosis irrespective of the luminal subtype (log-rank: luminal A, P=0.026; luminal B, P=0.006). p-STAT3 staining by IHC in the stroma or tumour was detected in 174 out of 610 ER-positive samples (28.5%) from the BIG 2-98 randomised trial. With a median follow-up of 10.1 years, p-STAT3 was associated with a reduced risk of recurrence in ER-positive/HER2-negative breast cancer (Cox univariate HR, 0.66; 95% CI, 0.44-0.98; P=0.04). On the whole, our data indicate that p-STAT3 is associated with an improved outcome in ER-positive breast cancer.

Different competitive capacities of Stat4- and Stat6-deficient CD4+ T cells during lymphophenia-driven proliferation.

PubMed

Sanchez-Guajardo, Vanesa; Borghans, José A M; Marquez, Maria-Elena; Garcia, Sylvie; Freitas, Antonio A

2005-02-01

The outcome of an immune response relies on the competitive capacities acquired through differentiation of CD4(+) T cells into Th1 or Th2 effector cells. Because Stat4 and Stat6 proteins are implicated in the Th1 vs Th2 generation and maintenance, respectively, we compare in this study the kinetics of Stat4(-/-) and Stat6(-/-) CD4(+) T cells during competitive bone marrow reconstitution and lymphopenia-driven proliferation. After bone marrow transplantation, both populations reconstitute the peripheral T cell pools equally well. After transfer into lymphopenic hosts, wild-type and Stat6(-/-) CD4(+) T cells show a proliferation advantage, which is early associated with the expression of an active phospho-Stat4 and the down-regulation of Stat6. Despite these differences, Stat4- and Stat6-deficient T cells reach similar steady state numbers. However, when both Stat4(-/-) and Stat6(-/-) CD4(+) T cells are coinjected into the same hosts, the Stat6(-/-) cells become dominant and out-compete Stat4(-/-) cells. These findings suggest that cell activation, through the Stat4 pathway and the down-regulation of Stat6, confers to pro-Th1 T cells a slight proliferation advantage that in a competitive situation has major late repercussions, because it modifies the final homeostatic equilibrium of the populations and favors the establishment of Th1 CD4(+) T cell dominance.

Angiotensin II up-regulates PAX2 oncogene expression and activity in prostate cancer via the angiotensin II type I receptor.

PubMed

Bose, Sudeep K; Gibson, Willietta; Giri, Shailendra; Nath, Narender; Donald, Carlton D

2009-09-01

Paired homeobox 2 gene (PAX2) is a transcriptional regulator, aberrantly expressed in prostate cancer cells and its down-regulation promotes cell death in these cells. The molecular mechanisms of tumor progression by PAX2 over-expression are still unclear. However, it has been reported that angiotensin-II (A-II) induces cell growth in prostate cancer via A-II type 1 receptor (AT1R) and is mediated by the phosphorylation of mitogen activated protein kinase (MAPK) as well as signal transducer and activator of transcription 3 (STAT3). Here we have demonstrated that A-II up-regulates PAX2 expression in prostate epithelial cells and prostate cancer cell lines resulting in increased cell growth. Furthermore, AT1R receptor antagonist losartan was shown to inhibit A-II induced PAX2 expression in prostate cancer. Moreover, analysis using pharmacological inhibitors against MEK1/2, ERK1/2, JAK-II, and phospho-STAT3 demonstrated that AT1R-mediated stimulatory effect of A-II on PAX2 expression was regulated in part by the phosphorylation of ERK1/2, JAK II, and STAT3 pathways. In addition, we have showed that down-regulation of PAX2 by an AT1R antagonist as well as JAK-II and STAT3 inhibitors suppress prostate cancer cell growth. Collectively, these findings show for the first time that the renin-angiotensin system (RAS) may promote prostate tumorigenesis via up-regulation of PAX2 expression. Therefore, PAX2 may be a novel therapeutic target for the treatment of carcinomas such as prostate cancer via the down-regulation of its expression by targeting the AT1R signaling pathways.

Inhibition of STAT3 and ErbB2 Suppresses Tumor Growth, Enhances Radiosensitivity, and Induces Mitochondria-Dependent Apoptosis in Glioma Cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gao Ling; Li Fengsheng; Dong Bo

2010-07-15

Purpose: Constitutively activated signal transducer and activator of transcription 3 (STAT3) and ErbB2 are involved in the pathogenesis of many tumors, including astrocytoma. Inactivation of these molecules is reported to result in radiosensitization. The purpose of this study was to investigate whether inhibition of STAT3, ErbB2, or both could enhance radiotherapy in the human glioma model (U251 and U87 cell lines). Methods and Materials: The RNAi plasmids targeting STAT3 or ErbB2 were constructed, and their downregulatory effects on target proteins were examined by immunoblotting. After combination treatment of RNAi with or without irradiation, the cell viability was determined using 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazoliummore » bromide (MTT) and clonogenic assays. The in vivo effect of combined treatment was determined using the U251 xenograft model. The apoptosis caused by the inhibition of STAT3 and ErbB2 was detected, and the mechanism involved in the apoptosis was investigated, including increases in caspase proteins, mitochondrial damage, and the expression of key modulating protein of different apoptosis pathways. Results: Transfection of U251 cells with STAT3 or ErbB2 siRNA plasmids specifically reduced their target gene expressions. Inhibition of STAT3 or ErbB2 greatly decreased glioma cell survival after 2, 4, or 6 Gy irradiation. Inhibition of STAT3 and ErbB2 also enhanced radiation-induced tumor growth inhibition in the U251 xenograft model. Furthermore, the suppression of either STAT3 or ErbB2 could induce U251 cell apoptosis, which was related primarily to the mitochondrial apoptotic pathway. Conclusions: These results indicated that simultaneous inhibition of STAT3 and ErbB2 expression can promote potent antitumor activity and radiosensitizing activity in human glioma.« less

APC loss in breast cancer leads to doxorubicin resistance via STAT3 activation.

PubMed

VanKlompenberg, Monica K; Leyden, Emily; Arnason, Anne H; Zhang, Jian-Ting; Stefanski, Casey D; Prosperi, Jenifer R

2017-11-28

Resistance to chemotherapy is one of the leading causes of death from breast cancer. We recently established that loss of Adenomatous Polyposis Coli (APC) in the Mouse Mammary Tumor Virus - Polyoma middle T (MMTV-PyMT) transgenic mouse model results in resistance to cisplatin or doxorubicin-induced apoptosis. Herein, we aim to establish the mechanism that is responsible for APC-mediated chemotherapeutic resistance. Our data demonstrate that MMTV-PyMT; Apc Min/+ cells have increased signal transducer and activator of transcription 3 (STAT3) activation. STAT3 can be constitutively activated in breast cancer, maintains the tumor initiating cell (TIC) population, and upregulates multidrug resistance protein 1 (MDR1). The activation of STAT3 in the MMTV-PyMT; Apc Min/+ model is independent of interleukin 6 (IL-6); however, enhanced EGFR expression in the MMTV-PyMT; Apc Min/+ cells may be responsible for the increased STAT3 activation. Inhibiting STAT3 with a small molecule inhibitor A69 in combination with doxorubicin, but not cisplatin, restores drug sensitivity. A69 also decreases doxorubicin enhanced MDR1 gene expression and the TIC population enhanced by loss of APC. In summary, these results have revealed the molecular mechanisms of APC loss in breast cancer that can guide future treatment plans to counteract chemotherapeutic resistance.

STAT3/NF-κB interactions determine the level of haptoglobin expression in male rats exposed to dietary restriction and/or acute phase stimuli.

PubMed

Uskoković, Aleksandra; Dinić, Svetlana; Mihailović, Mirjana; Grdović, Nevena; Arambašić, Jelena; Vidaković, Melita; Bogojević, Desanka; Ivanović-Matić, Svetlana; Martinović, Vesna; Petrović, Miodrag; Poznanović, Goran; Grigorov, Ilijana

2012-01-01

Haptoglobin is a constitutively expressed protein which is predominantly synthesized in the liver. During the acute-phase (AP) response haptoglobin is upregulated along with other AP proteins. Its upregulation during the AP response is mediated by cis-trans interactions between the hormone-responsive element (HRE) residing in the haptoglobin gene and inducible transcription factors STAT3 and C/EBP β. In male rats that have been subjected to chronic 50% dietary restriction (DR), the basal haptoglobin serum level is decreased. The aim of this study was to characterize the trans-acting factor(s) responsible for the reduction of haptoglobin expression in male rats subjected to 50% DR for 6 weeks. Protein-DNA interactions between C/EBP and STAT families of transcription factors and the HRE region of the haptoglobin gene were examined in livers of male rats subjected to DR, as well as during the AP response that was induced by turpentine administration. In DR rats, we observed associations between the HRE and C/EBPα/β, STAT5b and NF-κB p50, and the absence of interactions between STAT3 and NF-kB p65. Subsequent induction of the AP response in DR rats by turpentine administration elicited a normal, almost 2-fold increase in the serum haptoglobin level that was accompanied by HRE-binding of C/EBPβ, STAT3/5b and NF-kB p65/p50, and the establishment of interaction between STAT3 and NF-κB p65. These results suggest that STAT3 and NF-κB p65 crosstalk plays a central role while C/EBPβ acquires an accessory role in establishing the level of haptoglobin gene expression in male rats exposed to DR and AP stimuli.

The Role of Epithelial Stat3 in Amelogenesis during Mouse Incisor Renewal.

PubMed

Zhang, Bin; Meng, Bo; Viloria, Edward; Naveau, Adrien; Ganss, Bernhard; Jheon, Andrew H

2018-03-16

The aim of this study was to evaluate the role of epithelial signal transducer and activator of transcription 3 (STAT3) in mouse incisor amelogenesis. Since Stat3 is expressed in the epithelial component of developing and adult mouse teeth, we generated and analyzed Krt14Cre/+;Stat3fl/fl mutant mice in which Stat3 was inactivated in epithelia including ameloblast progenitors and ameloblasts, the cells responsible for enamel formation. Histological analysis showed little enamel matrix in mutant incisors compared to controls. Delayed incisor enamel mineralization was demonstrated using micro-computed X-ray tomography analysis and was supported by an increase in the pre-expression distance of enamel-enriched proteins such as amelogenin, ameloblastin, and kallikrein-4. Lastly, scanning electron microscopy analysis showed little enamel mineralization in mutant incisors underneath the mesial root of the 1st molar; however, the micro-architecture of enamel mineralization was similar in the erupted portion of control and mutant incisors. Taken together, our findings demonstrate for the first time that the absence of epithelial Stat3 in mice leads to delayed incisor amelogenesis. © 2018 S. Karger AG, Basel.

Long non-coding RNA PICART1 suppresses proliferation and promotes apoptosis in lung cancer cells by inhibiting JAK2/STAT3 signaling.

PubMed

Zhao, J M; Cheng, W; He, X G; Liu, Y L; Wang, F F; Gao, Y F

2018-06-26

Lung cancer remains the most common cause of tumor-related death worldwide. Recent studies have revealed that long non-coding RNAs (lncRNAs) are involved in the development of various cancers, including lung cancer. This study aimed to investigate the effect and the molecular basis of lncRNA PICART1 on lung cancer. We first assessed the PICART1 expression in lung cancer in vitro and vivo by qRT-PCR. Then the expression of PICART1 in SPC-A-1 and NCI-H1975 cell lines was inhibited and overexpressed by transient transfections. Thereafter, cell viability, cell cycle, migration and apoptosis were respectively measured by MTT, Transwell and flow cytometry assay. Furthermore, qRT-PCR and western blot analysis were mainly performed to assess the expression levels of apoptosis- and migration-related proteins and JAK2/STAT3 pathway proteins. Tumor formation was measured by xenograft tumor model assay in vivo. PICART1 expression was down-regulated in human lung cancer tissues and cell lines. Knockdown of PICART1 increased cell viability of lung cancer cell lines. However, PICART1 overexpression inhibited cell cycle progression and promoted apoptosis in SPC-A-1 and NCI-H1975 cell lines. PICART1 overexpression also inhibited migration, as evidenced by up-regulation of E-cadherin, and down-regulation of Twist1, MMP2 and MMP9. Furthermore, we found PICART1 inhibition may regulate cell apoptosis and migration through activating JAK2/STAT3 pathway. In vivo experiments revealed that PICART1 knockdown significantly promoted tumor formation.This study demonstrates that PICART1 overexpression represents an anti-growth and anti-metastasis role in lung cancer cells. Additionally, PICART1 acts as a tumor suppressor may be via regulation of JAK2/STAT3 pathway.

Novel function of STAT1beta in B cells: induction of cell death by a mechanism different from that of STAT1alpha.

PubMed

Najjar, Imen; Schischmanoff, Pierre Olivier; Baran-Marszak, Fanny; Deglesne, Pierre-Antoine; Youlyouz-Marfak, Ibtissam; Pampin, Mathieu; Feuillard, Jean; Bornkamm, Georg W; Chelbi-Alix, Mounira K; Fagard, Remi

2008-12-01

Alternate splicing of STAT1 produces two isoforms: alpha, known as the active form, and beta, previously shown to act as a dominant-negative factor. Most studies have dealt with STAT1alpha, showing its involvement in cell growth control and cell death. To examine the specific function of either isoform in cell death, a naturally STAT1-deficient human B cell line was transfected to express STAT1alpha or STAT1beta. STAT1alpha, expressed alone, enhanced cell death, potentiated the fludarabine-induced apoptosis, and enhanced the nuclear location, the phosphorylation, and the transcriptional activity of p53. Unexpectedly, STAT1beta, expressed alone, induced cell death through a mechanism that was independent of the nuclear function of p53. Indeed, in STAT1beta-expressing B cells, p53 was strictly cytoplasmic where it formed clusters, and there was no induction of the transcriptional activity of p53. These data reveal a novel role of STAT1beta in programmed cell death, which is independent of p53.

Radiosensitization by inhibiting STAT1 in renal cell carcinoma.

PubMed

Hui, Zhouguang; Tretiakova, Maria; Zhang, Zhongfa; Li, Yan; Wang, Xiaozhen; Zhu, Julie Xiaohong; Gao, Yuanhong; Mai, Weiyuan; Furge, Kyle; Qian, Chao-Nan; Amato, Robert; Butler, E Brian; Teh, Bin Tean; Teh, Bin S

2009-01-01

Renal cell carcinoma (RCC) has been historically regarded as a radioresistant malignancy, but the molecular mechanism underlying its radioresistance is not understood. This study investigated the role of signal transducer and activator of transcription 1 (STAT1), a transcription factor downstream of the interferon-signaling pathway, in radioresistant RCC. The expressions of STAT1 and STAT3 in 164 human clear cell RCC samples, 47 papillary RCC samples, and 15 normal kidney tissue samples were examined by microarray expression profiling and immunohistochemistry. Western blotting was performed to evaluate the total and phosphorylated STAT1 expression in CRL-1932 (786-O) (human clear cell RCC), SKRC-39 (human papillary RCC), CCL-116 (human fibroblast), and CRL-1441 (G-401) (human Wilms tumor). STAT1 was reduced or inhibited by fludarabine and siRNA, respectively, and the effects on radiation-induced cell death were investigated using clonogenic assays. STAT1 expression, but not STAT3 expression, was significantly greater in human RCC samples (p = 1.5 x 10(-8) for clear cell; and p = 3.6 x 10(-4) for papillary). Similarly, the expression of STAT1 was relatively greater in the two RCC cell lines. STAT1 expression was reduced by both fludarabine and siRNA, significantly increasing the radiosensitivity in both RCC cell lines. This is the first study reporting the overexpression of STAT1 in human clear cell and papillary RCC tissues. Radiosensitization in RCC cell lines was observed by a reduction or inhibition of STAT1 signaling, using fludarabine or siRNA. Our data suggest that STAT1 may play a key role in RCC radioresistance and manipulation of this pathway may enhance the efficacy of radiotherapy.

Nuclear localization of activated STAT6 and STAT3 in epidermis of prurigo nodularis.

PubMed

Fukushi, S; Yamasaki, K; Aiba, S

2011-11-01

Prurigo nodularis (PN) is a chronic dermatitis characterized by discrete, raised, and firm papulonodules with intense pruritus. The pathogenesis still remains to be elucidated. To clarify the role of Th1 and Th2 cytokines in the pathogenesis of PN. We examined the cytokine signatures, such as phosphorylation of STAT1, STAT3 and STAT6, HLA-DR and hyaluronan accumulation, to reveal the Th1 and Th2 cytokine influence on the lesional epidermis of PN. We first optimized antigen retrieval methods to detect these signatures with antibodies for phospho-STAT1 (pSTAT1), phospho-STAT3 (pSTAT3), phospho-STAT6 (pSTAT6), HLA-DR and hyaluronic acid binding protein (HABP) on the formalin-fixed paraffin-embedded sections of psoriasis, lichen planus and atopic dermatitis biopsy samples. Activation of STAT1 and STAT6 in epidermis by Th1 and Th2 cytokines was further confirmed in a cultured skin equivalent model treated with interferon-γ or interleukin (IL)-4/IL-13. With the relevant immunostaining methods, we examined the cytokine signatures in 22 cases of PN. The results revealed that (i) the entire epidermis of 19 cases was stained with anti-pSTAT6 antibody, (ii) 21 cases demonstrated nuclear staining with anti-pSTAT3 antibody, (iii) the entire epidermis of 21 cases was stained with HABP, (iv) the epidermis of eight cases showed scattered staining with anti-pSTAT1 antibody, and (v) six cases were positive for HLA-DR membrane expression. These data indicated that Th2 cytokines related to STAT6 activation together with some unknown stimuli that activate STAT3 play a principal role in the pathogenesis of PN. © 2011 The Authors. BJD © 2011 British Association of Dermatologists.

Intracellular staining for analysis of the expression and phosphorylation of signal transducers and activators of transcription (STATs) in NK cells.

PubMed

Miyagi, Takuya; Lee, Seung-Hwan; Biron, Christine A

2010-01-01

Cytokines stimulate biological responses by activating intracellular signaling pathways. We have been adapting flow cytometric techniques to measure the levels of expression and activation of signaling molecules within mixed populations containing NK cells and to characterize their differences within NK cell subpopulations. Approaches for evaluating the total levels of the signal transducers and activators of transcription STAT1 and STAT4, of STAT1 in cells expressing IFNgamma, and of the type 1 interferon (type 1 IFN) activation by phosphorylation, i.e., induction of pSTAT1 and pSTAT4, have been developed. The results of experiments using these techniques have demonstrated that an unusual feature of NK cells is high basal expression of STAT4 but reduced STAT1 levels. The condition predisposes for pSTAT4 activation by type 1 IFNs. The work has also shown, however, that total STAT1 levels are induced during viral infections as a result of IFN exposure, and that this change acts to promote the activation of STAT1 but limit both the activation of STAT4 and IFNgamma expression. The intracellular staining approaches used for the studies described here have utility in characterizing other mechanisms regulating cytokine-mediated signaling, and defining additional pathways shaping cellular responses to cytokines.

Myeloproliferative disease induced by TEL-PDGFRB displays dynamic range sensitivity to Stat5 gene dosage

PubMed Central

Cain, Jennifer A.; Xiang, Zhifu; O'Neal, Julie; Kreisel, Friederike; Colson, AnnaLynn; Luo, Hui; Hennighausen, Lothar

2007-01-01

Expression of the constitutively activated TEL/PDGFβR fusion protein is associated with the t(5;12)(q33;p13) chromosomal translocation found in a subset of patients with chronic myelomonocytic leukemia. TEL/PDGFβR activates multiple signal transduction pathways in cell-culture systems, and expression of the TEL-PDGFRB fusion gene induces myeloproliferative disease (MPD) in mice. We used gene-targeted mice to characterize the contribution of signal transducer and activator of transcription (Stat) and Src family genes to TEL-PDGFRB–mediated transformation in methylcellulose colony and murine bone marrow transduction/transplantation assays. Fetal liver hematopoietic stem and progenitor cells harboring targeted deletion of both Stat5a and Stat5b (Stat5abnull/null) genes were refractory to transformation by TEL-PDGFRB in methylcellulose colony assays. Notably, these cell populations were maintained in Stat5abnull/null fetal livers and succumbed to transformation by c-Myc. Surprisingly, targeted disruption of either Stat5a or Stat5b alone also impaired TEL-PDGFRB–mediated transformation. Survival of TPiGFP→Stat5a−/− and TPiGFP→Stat5a+/− mice was significantly prolonged, demonstrating significant sensitivity of TEL-PDGFRB–induced MPD to the dosage of Stat5a. TEL-PDGFRB–mediated MPD was incompletely penetrant in TPiGFP→Stat5b−/− mice. In contrast, Src family kinases Lyn, Hck, and Fgr and the Stat family member Stat1 were dispensable for TEL-PDGFRB disease. Together, these data demonstrate that Stat5a and Stat5b are dose-limiting mediators of TEL-PDGFRB–induced myeloproliferation. PMID:17218386

Porcine bocavirus NP1 negatively regulates interferon signaling pathway by targeting the DNA-binding domain of IRF9

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, Ruoxi; The Cooperative Innovation Center for Sustainable Pig Production, Wuhan 430070; Fang, Liurong, E-mail: fanglr@mail.hzau.edu.cn

2015-11-15

To subvert host antiviral immune responses, many viruses have evolved countermeasures to inhibit IFN signaling pathway. Porcine bocavirus (PBoV), a newly identified porcine parvovirus, has received attention because it shows clinically high co-infection prevalence with other pathogens in post-weaning multisystemic wasting syndrome (PWMS) and diarrheic piglets. In this study, we screened the structural and non-structural proteins encoded by PBoV and found that the non-structural protein NP1 significantly suppressed IFN-stimulated response element (ISRE) activity and subsequent IFN-stimulated gene (ISG) expression. However, NP1 affected neither the activation and translocation of STAT1/STAT2, nor the formation of the heterotrimeric transcription factor complex ISGF3 (STAT1/STAT2/IRF9).more » Detailed analysis demonstrated that PBoV NP1 blocked the ISGF3 DNA-binding activity by combining with the DNA-binding domain (DBD) of IRF9. In summary, these results indicate that PBoV NP1 interferes with type I IFN signaling pathway by blocking DNA binding of ISGF3 to attenuate innate immune responses. - Highlights: • Porcine bocavirus (PBoV) NP1 interferes with the IFN α/β signaling pathway. • PBoV NP1 does not prevent STAT1/STAT2 phosphorylation and nuclear translocation. • PBoV NP1 inhibits the DNA-binding activity of ISGF3. • PBoV NP1 interacts with the DNA-binding domain of IRF9.« less

[IL-12 induces autophagy via AKT/mTOR/STAT3 signaling pathway in human hepatoma cells].

PubMed

Liu, Cuiying; Xie, Changli; Lin, Yan; Wu, Bitao; Wang, Qin; Li, Ziwei; Tu, Zhiguang

2016-07-01

Objective To investigate the effect of IL-12 on autophagy and the relative possible mechanism in HepG2 and SMMC-7721 human hepatoma cells. Methods The hepatoma cells were treated with IL-12 (10 ng/mL) for 6 hours. Western blotting was applied to detect the expressions of microtubule-associated protein 1 light chain 3 (LC-3), Beclin 1 and the phosphorylated levels of protein kinase B (AKT), mammalian target of rapamycin (mTOR), signal transducer and activator of transcription 3 (STAT3); immunofluorescence assay (IFA) and transmission electron microscopy (TEM) were used to observe the formation of autophagosome. After STAT3 was inhibited by STATTIC or siSTAT3 and AKT was activated by insulin-like growth factor (IGF-1), Western blotting and IFA were performed again to analyze the change of IL-12-induced autophagy. After the cells were treated with IL-12 (10 ng/mL) for 1, 2, 3, 4, 5 days, CCK-8 assay was used to determine the growth ability. After the hepatoma cells were treated with IL-12 (10 ng/mL) for 48 hours, trypan blue staining was used to detect the death rate of the cells. After cell autophagy was inhibit by siBeclin 1, CCK-8 assay and trypan blue staining were performed again to study the effect of IL-12 on the proliferation and death of human hepatoma cells. Results IL-12 induced autophagy and inhibited cell growth in the hepatoma cells. Silencing Beclin 1 gene enhanced IL-12-mediated growth inhibition and cell death. Furthermore, IL-12 treatment also decreased the expressions of p-AKT, p-mTOR and p-STAT3. The pretreatment of siSTAT3 or STATTIC inhibited STAT3-enhanced IL-12-induced autophagy. Accordingly, activation of AKT with IGF-1 decreased IL-12-induced autophagy. Conclusion IL-12 could induce autophagy through AKT/mTOR/STAT3 signaling pathways and the induction of autophagy attenuates the growth-inhibitory effect of IL-12 on hepatoma cells.

Pyruvate dehydrogenase complex (PDC) subunits moonlight as interaction partners of phosphorylated STAT5 in adipocytes and adipose tissue.

PubMed

Richard, Allison J; Hang, Hardy; Stephens, Jacqueline M

2017-12-01

STAT5 proteins play a role in adipocyte development and function, but their specific functions are largely unknown. To this end, we used an unbiased MS-based approach to identify novel STAT5-interacting proteins. We observed that STAT5A bound the E1β and E2 subunits of the pyruvate dehydrogenase complex (PDC). Whereas STAT5A typically localizes to the cytosol or nucleus, PDC normally resides within the mitochondrial matrix where it converts pyruvate to acetyl-CoA. We employed affinity purification and immunoblotting to validate the interaction between STAT5A and PDC subunits in murine and human cultured adipocytes, as well as in adipose tissue. We found that multiple PDC subunits interact with hormone-activated STAT5A in a dose- and time-dependent manner that coincides with tyrosine phosphorylation of STAT5. Using subcellular fractionation and immunofluorescence microscopy, we observed that PDC-E2 is present within the adipocyte nucleus where it associates with STAT5A. Because STAT5A is a transcription factor, we used chromatin immunoprecipitation (ChIP) to assess PDC's ability to interact with STAT5 DNA-binding sites. These analyses revealed that PDC-E2 is bound to a STAT5-binding site in the promoter of the STAT5 target gene c ytokine- i nducible SH 2-containing protein ( cish ). We have demonstrated a compelling interaction between STAT5A and PDC subunits in adipocytes under physiological conditions. There is previous evidence that PDC localizes to cancer cell nuclei where it plays a role in histone acetylation. On the basis of our ChIP data and these previous findings, we hypothesize that PDC may modulate STAT5's ability to regulate gene expression by controlling histone or STAT5 acetylation. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

INTERFERON α ACTIVATES NF-κ B IN JAK1-DEFICIENT CELLS THROUGH A TYK2-DEPENDENT PATHWAY

PubMed Central

Yang, Chuan He; Murti, Aruna; Valentine, William J.; Du, Ziyun; Pfeffer, Lawrence M.

2005-01-01

In addition to activating members of the STAT transcription factor family, IFN α/β activates the NF-κ B transcription factor. To determine the role of the JAK-STAT pathway in NF-κ B activation by IFN, we examined NF-κ B activation in JAK1-deficient mutant human fibrosarcoma cells. In wild-type fibrosarcoma cells (2fTGH) IFN activates STAT1, STAT2 and STAT3, as well as NF-κB complexes comprised of p50 and p65. In contrast, in JAK1-deficient cells IFN induces NF-κB activation and NF-κB dependent gene transcription, but does not activate these STAT proteins and has no effect on STAT-dependent gene transcription. Expression of a catalytically-inactive TYK2 tyrosine kinase in JAK1-deficient cells, as well as in the highly IFN-sensitive Daudi lymphoblastoid cell line, abrogates NF-κB activation by IFN. Moreover, IFN does not promote NF-κB activation in TYK2-deficient mutant fibrosarcoma cells. Our results demonstrate a dichotomy between the classical JAK-STAT pathway and the NF-κB signaling pathway. In the IFN signaling pathway leading to STAT activation both JAK1 and TYK2 are essential, while NF-κB activation requires only TYK2. PMID:15883164

TLR4 Signaling via NANOG Cooperates With STAT3 to Activate Twist1 and Promote Formation of Tumor-initiating Stem-like Cells in Livers of Mice

PubMed Central

Kumar, Dinesh Babu Uthaya; Chen, Chia-Lin; Liu, Jian-Chang; Feldman, Douglas E.; Sher, Linda S.; French, Samuel; DiNorcia, Joseph; French, Samuel W.; Naini, Bita V.; Junrungsee, Sunhawit; Agopian, Vatche Garen; Zarrinpar, Ali; Machida, Keigo

2015-01-01

BACKGROUND & AIMS Obesity and alcohol consumption contribute to steatohepatitis, which increases risk for hepatitis C virus (HCV)-associated hepatocellular carcinomas (HCCs). Mice Hepatocytes that express HCV-NS5A in liver upregulate expression of Toll-like receptor-4 (TLR4), and develop liver tumors containing tumor-initiating stem-like cells (TICs) that express NANOG. We investigated whether the TLR4 signals to NANOG to promote development of TICs and tumorigenesis in mice placed on Western diet high in cholesterol and saturated fat (HCFD). METHODS We expressed HCV-NS5A from a transgene (NS5A Tg) in Tlr4−/− (C57Bl6/10ScN), and wild type control mice. Mice were fed a HCFD for 12 months. TICs were identified and isolated based on being CD133+, CD49f+, and CD45-. We obtained 142 paraffin-embedded sections of different stage HCCs and adjacent non-tumor areas from the same patients, and performed gene expression, immunofluorescence, and immunohistochemical analyses. RESULTS A higher proportion of NS5A Tg mice developed liver tumors (39%) than mice that did not express HCV NS5A following the HCFD (6%); only 9% of Tlr4−/− NS5A Tg mice fed HCFD developed liver tumors. Livers from NS5A Tg mice fed the HCFD had increased levels of TLR4, NANOG, pSTAT3, and TWIST1 proteins, and increases in Tlr4, Nanog, Stat3, and Twist1 mRNAs. In TICs from NS5A Tg mice. NANOG and pSTAT3 directly interacts to activate expression of Twist1. Levels of TLR4, NANOG, pSTAT3, and TWIST were increased in HCC compared with non-tumor tissues from patients. CONCLUSIONS HCFD and HCV-NS5A together stimulated TLR4-NANOG and the OB-R-pSTAT3 signaling pathways resulting in liver tumorigenesis through an exaggerated mesenchymal phenotype with prominent Twist1-expressing TICs. PMID:26582088

STAT3 activation in skeletal muscle links muscle wasting and the acute phase response in cancer cachexia.

PubMed

Bonetto, Andrea; Aydogdu, Tufan; Kunzevitzky, Noelia; Guttridge, Denis C; Khuri, Sawsan; Koniaris, Leonidas G; Zimmers, Teresa A

2011-01-01

Cachexia, or weight loss despite adequate nutrition, significantly impairs quality of life and response to therapy in cancer patients. In cancer patients, skeletal muscle wasting, weight loss and mortality are all positively associated with increased serum cytokines, particularly Interleukin-6 (IL-6), and the presence of the acute phase response. Acute phase proteins, including fibrinogen and serum amyloid A (SAA) are synthesized by hepatocytes in response to IL-6 as part of the innate immune response. To gain insight into the relationships among these observations, we studied mice with moderate and severe Colon-26 (C26)-carcinoma cachexia. Moderate and severe C26 cachexia was associated with high serum IL-6 and IL-6 family cytokines and highly similar patterns of skeletal muscle gene expression. The top canonical pathways up-regulated in both were the complement/coagulation cascade, proteasome, MAPK signaling, and the IL-6 and STAT3 pathways. Cachexia was associated with increased muscle pY705-STAT3 and increased STAT3 localization in myonuclei. STAT3 target genes, including SOCS3 mRNA and acute phase response proteins, were highly induced in cachectic muscle. IL-6 treatment and STAT3 activation both also induced fibrinogen in cultured C2C12 myotubes. Quantitation of muscle versus liver fibrinogen and SAA protein levels indicates that muscle contributes a large fraction of serum acute phase proteins in cancer. These results suggest that the STAT3 transcriptome is a major mechanism for wasting in cancer. Through IL-6/STAT3 activation, skeletal muscle is induced to synthesize acute phase proteins, thus establishing a molecular link between the observations of high IL-6, increased acute phase response proteins and muscle wasting in cancer. These results suggest a mechanism by which STAT3 might causally influence muscle wasting by altering the profile of genes expressed and translated in muscle such that amino acids liberated by increased proteolysis in cachexia are synthesized into acute phase proteins and exported into the blood.

STAT3 Activation in Skeletal Muscle Links Muscle Wasting and the Acute Phase Response in Cancer Cachexia

PubMed Central

Kunzevitzky, Noelia; Guttridge, Denis C.; Khuri, Sawsan; Koniaris, Leonidas G.; Zimmers, Teresa A.

2011-01-01

Background Cachexia, or weight loss despite adequate nutrition, significantly impairs quality of life and response to therapy in cancer patients. In cancer patients, skeletal muscle wasting, weight loss and mortality are all positively associated with increased serum cytokines, particularly Interleukin-6 (IL-6), and the presence of the acute phase response. Acute phase proteins, including fibrinogen and serum amyloid A (SAA) are synthesized by hepatocytes in response to IL-6 as part of the innate immune response. To gain insight into the relationships among these observations, we studied mice with moderate and severe Colon-26 (C26)-carcinoma cachexia. Methodology/Principal Findings Moderate and severe C26 cachexia was associated with high serum IL-6 and IL-6 family cytokines and highly similar patterns of skeletal muscle gene expression. The top canonical pathways up-regulated in both were the complement/coagulation cascade, proteasome, MAPK signaling, and the IL-6 and STAT3 pathways. Cachexia was associated with increased muscle pY705-STAT3 and increased STAT3 localization in myonuclei. STAT3 target genes, including SOCS3 mRNA and acute phase response proteins, were highly induced in cachectic muscle. IL-6 treatment and STAT3 activation both also induced fibrinogen in cultured C2C12 myotubes. Quantitation of muscle versus liver fibrinogen and SAA protein levels indicates that muscle contributes a large fraction of serum acute phase proteins in cancer. Conclusions/Significance These results suggest that the STAT3 transcriptome is a major mechanism for wasting in cancer. Through IL-6/STAT3 activation, skeletal muscle is induced to synthesize acute phase proteins, thus establishing a molecular link between the observations of high IL-6, increased acute phase response proteins and muscle wasting in cancer. These results suggest a mechanism by which STAT3 might causally influence muscle wasting by altering the profile of genes expressed and translated in muscle such that amino acids liberated by increased proteolysis in cachexia are synthesized into acute phase proteins and exported into the blood. PMID:21799891

The role of signal transducer and activator of transcription 3 in Rift Valley fever virus infection

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pinkham, Chelsea; An, Soyeon; Lundberg, Lindsay

Rift Valley fever (RVF) is a zoonotic disease that can cause severe illness in humans and livestock, triggering spontaneous abortion in almost 100% of pregnant ruminants. In this study, we demonstrate that signal transducer and activator of transcription 3 (STAT3) is phosphorylated on its conserved tyrosine residue (Y705) following RVFV infection. This phosphorylation was dependent on a major virulence factor, the viral nonstructural protein NSs. Loss of STAT3 had little effect on viral replication, but rather resulted in cells being more susceptible to RVFV-induced cell death. Phosphorylated STAT3 translocated to the nucleus, coinciding with inhibition of fos, jun, and nr4a2more » gene expression, and the presence of STAT3 and NSs at the nr4a2 promoter. NSs was found predominantly in the cytoplasm of STAT3 null cells, indicating that STAT3 influences NSs nuclear localization. Collectively, these data demonstrate that STAT3 functions in a pro-survival capacity through modulation of NSs localization. - Highlights: • STAT3 is phosphorylated on tyrosine residue 705 following RVFV infection. • Phosphorylation of STAT3 was dependent on the viral protein NSs. • STAT3 -/- MEFs were more susceptible to RVFV-induced cell death. • Loss of STAT3 led to an increase in pro-apoptotic gene expression. • STAT3 functions in a pro-survival capacity by modulation of NSs localization.« less

Increasing Maternal Body Mass Index Is Associated with Systemic Inflammation in the Mother and the Activation of Distinct Placental Inflammatory Pathways1

PubMed Central

Aye, Irving L.M.H.; Lager, Susanne; Ramirez, Vanessa I.; Gaccioli, Francesca; Dudley, Donald J.; Jansson, Thomas; Powell, Theresa L.

2014-01-01

ABSTRACT Obese pregnant women have increased levels of proinflammatory cytokines in maternal circulation and placental tissues. However, the pathways contributing to placental inflammation in obesity are largely unknown. We tested the hypothesis that maternal body mass index (BMI) was associated with elevated proinflammatory cytokines in maternal and fetal circulations and increased activation of placental inflammatory pathways. A total of 60 women of varying pre-/early pregnancy BMI, undergoing delivery by Cesarean section at term, were studied. Maternal and fetal (cord) plasma were collected for analysis of insulin, leptin, IL-1beta, IL-6, IL-8, monocyte chemoattractant protein (MCP) 1, and TNFalpha by multiplex ELISA. Activation of the inflammatory pathways in the placenta was investigated by measuring the phosphorylated and total protein expression of p38-mitogen-activated protein kinase (MAPK), c-Jun-N-terminal kinase (JNK)-MAPK, signal transducer-activated transcription factor (STAT) 3, caspase-1, IL-1beta, IkappaB-alpha protein, and p65 DNA-binding activity. To determine the link between activated placental inflammatory pathways and elevated maternal cytokines, cultured primary human trophoblast (PHT) cells were treated with physiological concentrations of insulin, MCP-1, and TNFalpha, and inflammatory signaling analyzed by Western blot. Maternal BMI was positively correlated with maternal insulin, leptin, MCP-1, and TNFalpha, whereas only fetal leptin was increased with BMI. Placental phosphorylation of p38-MAPK and STAT3, and the expression of IL-1beta protein, were increased with maternal BMI; phosphorylation of p38-MAPK was also correlated with birth weight. In contrast, placental NFkappaB, JNK and caspase-1 signaling, and fetal cytokine levels were unaffected by maternal BMI. In PHT cells, p38-MAPK was activated by MCP-1 and TNFalpha, whereas STAT3 phosphorylation was increased following TNFalpha treatment. Maternal BMI is associated with elevated maternal cytokines and activation of placental p38-MAPK and STAT3 inflammatory pathways, without changes in fetal systemic inflammatory profile. Activation of p38-MAPK by MCP-1 and TNFalpha, and STAT3 by TNFalpha, suggests a link between elevated proinflammatory cytokines in maternal plasma and activation of placental inflammatory pathways. We suggest that inflammatory processes associated with elevated maternal BMI may influence fetal growth by altering placental function. PMID:24759787

Acute myotube protein synthesis regulation by IL-6-related cytokines.

PubMed

Gao, Song; Durstine, J Larry; Koh, Ho-Jin; Carver, Wayne E; Frizzell, Norma; Carson, James A

2017-11-01

IL-6 and leukemia inhibitory factor (LIF), members of the IL-6 family of cytokines, play recognized paradoxical roles in skeletal muscle mass regulation, being associated with both growth and atrophy. Overload or muscle contractions can induce a transient increase in muscle IL-6 and LIF expression, which has a regulatory role in muscle hypertrophy. However, the cellular mechanisms involved in this regulation have not been completely identified. The induction of mammalian target of rapamycin complex 1 (mTORC1)-dependent myofiber protein synthesis is an established regulator of muscle hypertrophy, but the involvement of the IL-6 family of cytokines in this process is poorly understood. Therefore, we investigated the acute effects of IL-6 and LIF administration on mTORC1 signaling and protein synthesis in C2C12 myotubes. The role of glycoprotein 130 (gp130) receptor and downstream signaling pathways, including phosphoinositide 3-kinase (PI3K)-Akt-mTORC1 and signal transducer and activator of transcription 3 (STAT3)-suppressor of cytokine signaling 3 (SOCS3), was investigated by administration of specific siRNA or pharmaceutical inhibitors. Acute administration of IL-6 and LIF induced protein synthesis, which was accompanied by STAT3 activation, Akt-mTORC1 activation, and increased SOCS3 expression. This induction of protein synthesis was blocked by both gp130 siRNA knockdown and Akt inhibition. Interestingly, STAT3 inhibition or Akt downstream mTORC1 signaling inhibition did not fully block the IL-6 or LIF induction of protein synthesis. SOCS3 siRNA knockdown increased basal protein synthesis and extended the duration of the protein synthesis induction by IL-6 and LIF. These results demonstrate that either IL-6 or LIF can activate gp130-Akt signaling axis, which induces protein synthesis via mTORC1-independent mechanisms in cultured myotubes. However, IL-6- or LIF-induced SOCS3 negatively regulates the activation of myotube protein synthesis. Copyright © 2017 the American Physiological Society.

RTVP-1 promotes mesenchymal transformation of glioma via a STAT-3/IL-6-dependent positive feedback loop

PubMed Central

Giladi, Nis David; Ziv-Av, Amotz; Lee, Hae Kyung; Finniss, Susan; Cazacu, Simona; Xiang, Cunli; Ben-Asher, Hiba Waldman; deCarvalho, Ana; Mikkelsen, Tom; Poisson, Laila; Brodie, Chaya

2015-01-01

Glioblastomas (GBMs), the most aggressive primary brain tumors, exhibit increased invasiveness and resistance to anti-tumor treatments. We explored the role of RTVP-1, a glioma-associated protein that promotes glioma cell migration, in the mesenchymal transformation of GBM. Analysis of The Cancer Genome Atlas (TCGA) demonstrated that RTVP-1 expression was higher in mesenchymal GBM and predicted tumor recurrence and poor clinical outcome. ChiP analysis revealed that the RTVP-1 promoter binds STAT3 and C/EBPβ, two master transcription factors that regulate mesenchymal transformation of GBM. In addition, IL-6 induced RTVP-1 expression in a STAT3-dependent manner. RTVP-1 increased the migration and mesenchymal transformation of glioma cells. Similarly, overexpression of RTVP-1 in human neural stem cells induced mesenchymal differentiation, whereas silencing of RTVP-1 in glioma stem cells (GSCs) decreased the mesenchymal transformation and stemness of these cells. Silencing of RTVP-1 also increased the survival of mice bearing GSC-derived xenografts. Using gene array analysis of RTVP-1 silenced glioma cells we identified IL-6 as a mediator of RTVP-1 effects on the mesenchymal transformation and migration of GSCs, therefore acting in a positive feedback loop by upregulating RTVP-1 expression via the STAT3 pathway. Collectively, these results implicate RTVP-1 as a novel prognostic marker and therapeutic target in GBM. PMID:26267319

Propofol enhances the cisplatin-induced apoptosis on cervical cancer cells via EGFR/JAK2/STAT3 pathway.

PubMed

Li, Haoran; Lu, Yan; Pang, Yangyang; Li, Mengjiao; Cheng, Xi; Chen, Jiawei

2017-02-01

The main purpose of this study was to evaluate propofol and its combined effect with cisplatin on apoptosis of cervical cancer cells and molecular mechanisms of this phenomenon. The effects of propofol and cisplatin on cell viability and apoptosis were detected by cell counting kit-8 (CCK-8) assay, colony formation assay and flow cytometry assay. Besides, protein expression of EGFR/JAK2/STAT3 pathway was determined by western blot. STAT3 was over-expressed in cervical cancer cells by STAT3 cDNA. Expression of EGFR and STAT3 protein of human tissues was evaluated by immunohistochemistry (IHC) assay. In this study, we found that not only propofol alone could inhibit cervical cancer cells viability but also could increase the inhibitory effect of cisplatin on cervical cancer cells growth. Meanwhile, propofol sensitized cervical cancer cells to cisplatin-induced apoptosis but not affected normal cervical cells. In genetic level, propofol could enhance the anti-tumor effect of cisplatin through EGFR/JAK2/STAT3 pathway. Further studies indicated that overexpression of EGFR and STAT3 is related to poor prognoses in cervical cancer patients, which contributed to confirm the clinical role of combined application of propofol and cisplatin. Propofol enhances the cisplatin-induced cell apoptosis cervical cancer cells via EGFR/JAK2/STAT3 pathway and may be developed as a potential therapeutic agent to treat cervical cancer. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

Visual detection of STAT5B gene expression in living cell using the hairpin DNA modified gold nanoparticle beacon.

PubMed

Xue, Jianpeng; Shan, Lingling; Chen, Haiyan; Li, Yang; Zhu, Hongyan; Deng, Dawei; Qian, Zhiyu; Achilefu, Samuel; Gu, Yueqing

2013-03-15

Signal transducer and activator of transcription 5B (STAT5B) is an important protein in JAK-STAT signaling pathway that is responsible for the metastasis and proliferation of tumor cells. Determination of the STAT5B messenger Ribonucleic Acid (mRNA) relating to the STAT5B expression provides insight into the mechanism of tumor progression. In this study, we designed and used a special hairpin deoxyribonucleic acid (DNA) for human STAT5B mRNA to functionalize gold nanoparticles, which served as a beacon for detecting human STAT5B expression. Up to 90% quenching efficiency was achieved. Upon hybridizing with the target mRNA, the hairpin DNA modified gold nanoparticle beacons (hDAuNP beacons) release the fluorophores attached at 5' end of the oligonucleotide sequence. The fluorescence properties of the beacon before and after the hybridization with the complementary DNA were confirmed in vitro. The stability of hDAuNP beacons against degradation by DNase I and GSH indicated that the prepared beacon is stable inside cells. The detected fluorescence in MCF-7 cancer cells correlates with the specific STAT5B mRNA expression, which is consistent with the result from PCR measurement. Fluorescence microscopy showed that the hDAuNP beacons internalized in cells without using transfection agents, with intracellular distribution in the cytoplasm rather than the nucleus. The results demonstrated that this beacon could directly provide quantitative measurement of the intracellular STAT5B mRNA in living cells. Compared to the previous approaches, this beacon has advantages of higher target to background ratio of detection and an increased resistance to nuclease degradation. The strategy reported in this study is a promising approach for the intracellular measurement of RNA or protein expression in living cells, and has great potential in the study of drug screening and discovery. Copyright © 2012 Elsevier B.V. All rights reserved.

Increasing maternal body mass index is associated with systemic inflammation in the mother and the activation of distinct placental inflammatory pathways.

PubMed

Aye, Irving L M H; Lager, Susanne; Ramirez, Vanessa I; Gaccioli, Francesca; Dudley, Donald J; Jansson, Thomas; Powell, Theresa L

2014-06-01

Obese pregnant women have increased levels of proinflammatory cytokines in maternal circulation and placental tissues. However, the pathways contributing to placental inflammation in obesity are largely unknown. We tested the hypothesis that maternal body mass index (BMI) was associated with elevated proinflammatory cytokines in maternal and fetal circulations and increased activation of placental inflammatory pathways. A total of 60 women of varying pre-/early pregnancy BMI, undergoing delivery by Cesarean section at term, were studied. Maternal and fetal (cord) plasma were collected for analysis of insulin, leptin, IL-1beta, IL-6, IL-8, monocyte chemoattractant protein (MCP) 1, and TNFalpha by multiplex ELISA. Activation of the inflammatory pathways in the placenta was investigated by measuring the phosphorylated and total protein expression of p38-mitogen-activated protein kinase (MAPK), c-Jun-N-terminal kinase (JNK)-MAPK, signal transducer-activated transcription factor (STAT) 3, caspase-1, IL-1beta, IkappaB-alpha protein, and p65 DNA-binding activity. To determine the link between activated placental inflammatory pathways and elevated maternal cytokines, cultured primary human trophoblast (PHT) cells were treated with physiological concentrations of insulin, MCP-1, and TNFalpha, and inflammatory signaling analyzed by Western blot. Maternal BMI was positively correlated with maternal insulin, leptin, MCP-1, and TNFalpha, whereas only fetal leptin was increased with BMI. Placental phosphorylation of p38-MAPK and STAT3, and the expression of IL-1beta protein, were increased with maternal BMI; phosphorylation of p38-MAPK was also correlated with birth weight. In contrast, placental NFkappaB, JNK and caspase-1 signaling, and fetal cytokine levels were unaffected by maternal BMI. In PHT cells, p38-MAPK was activated by MCP-1 and TNFalpha, whereas STAT3 phosphorylation was increased following TNFalpha treatment. Maternal BMI is associated with elevated maternal cytokines and activation of placental p38-MAPK and STAT3 inflammatory pathways, without changes in fetal systemic inflammatory profile. Activation of p38-MAPK by MCP-1 and TNFalpha, and STAT3 by TNFalpha, suggests a link between elevated proinflammatory cytokines in maternal plasma and activation of placental inflammatory pathways. We suggest that inflammatory processes associated with elevated maternal BMI may influence fetal growth by altering placental function. © 2014 by the Society for the Study of Reproduction, Inc.

Activation of Stat3 Transcription Factor by Herpesvirus Saimiri STP-A Oncoprotein

PubMed Central

Chung, Young-Hwa; Cho, Nam-hyuk; Garcia, Maria Ines; Lee, Sun-Hwa; Feng, Pinghui; Jung, Jae U.

2004-01-01

The saimiri transforming protein (STP) oncogene of Herpesvirus saimiri subgroup A strain 11 (STP-A11) is not required for viral replication but is required for lymphoid cell immortalization in culture and lymphoma induction in primates. We previously showed that STP-A11 interacts with cellular Src kinase through its SH2 binding motif and that this interaction elicits Src signal transduction. Here we demonstrate that STP-A11 interacts with signal transducer and activator of transcription 3 (Stat3) independently of Src association and that the amino-terminal short proline-rich motif of STP-A11 and the central linker region of Stat3 are necessary for their interaction. STP-A11 formed a triple complex with Src kinase and Stat3 where Src kinase phosphorylated Stat3, resulting in the nuclear localization and transcriptional activation of Stat3. Consequently, the constitutively active Stat3 induced by STP-A11 elicited cellular signal transduction, which ultimately induced cell survival and proliferation upon serum deprivation. Furthermore, this activity was strongly correlated with the induction of Fos, cyclin D1, and Bcl-XL expression. These results demonstrate that STP-A11 independently targets two important cellular signaling molecules, Src and Stat3, and that these proteins cooperate efficiently to induce STP-A11-mediated transformation. PMID:15163742

Activation of peroxisome proliferator-activated receptor-β/-δ (PPAR-β/-δ) ameliorates insulin signaling and reduces SOCS3 levels by inhibiting STAT3 in interleukin-6-stimulated adipocytes.

PubMed

Serrano-Marco, Lucía; Rodríguez-Calvo, Ricardo; El Kochairi, Ilhem; Palomer, Xavier; Michalik, Liliane; Wahli, Walter; Vázquez-Carrera, Manuel

2011-07-01

It has been suggested that interleukin (IL)-6 is one of the mediators linking obesity-derived chronic inflammation with insulin resistance through activation of STAT3, with subsequent upregulation of suppressor of cytokine signaling 3 (SOCS3). We evaluated whether peroxisome proliferator-activated receptor (PPAR)-β/-δ prevented activation of the IL-6-STAT3-SOCS3 pathway and insulin resistance in adipocytes. Adipocytes and white adipose tissue from wild-type and PPAR-β/-δ-null mice were used to evaluate the effect of PPAR-β/-δ on the IL-6-STAT3-SOCS3 pathway. First, we observed that the PPAR-β/-δ agonist GW501516 prevented both IL-6-dependent reduction in insulin-stimulated Akt phosphorylation and glucose uptake in adipocytes. In addition, this drug treatment abolished IL-6-induced SOCS3 expression in differentiated 3T3-L1 adipocytes. This effect was associated with the capacity of the drug to prevent IL-6-induced STAT3 phosphorylation on Tyr(705) and Ser(727) residues in vitro and in vivo. Moreover, GW501516 prevented IL-6-dependent induction of extracellular signal-related kinase (ERK)1/2, a serine-threonine-protein kinase involved in serine STAT3 phosphorylation. Furthermore, in white adipose tissue from PPAR-β/-δ-null mice, STAT3 phosphorylation (Tyr(705) and Ser(727)), STAT3 DNA-binding activity, and SOCS3 protein levels were higher than in wild-type mice. Several steps in STAT3 activation require its association with heat shock protein 90 (Hsp90), which was prevented by GW501516 as revealed in immunoprecipitation studies. Consistent with this finding, the STAT3-Hsp90 association was enhanced in white adipose tissue from PPAR-β/-δ-null mice compared with wild-type mice. Collectively, our findings indicate that PPAR-β/-δ activation prevents IL-6-induced STAT3 activation by inhibiting ERK1/2 and preventing the STAT3-Hsp90 association, an effect that may contribute to the prevention of cytokine-induced insulin resistance in adipocytes. © 2011 by the American Diabetes Association.

Overexpression of Human Papillomavirus Type 16 Oncoproteins Enhances Epithelial-Mesenchymal Transition via STAT3 Signaling Pathway in Non-Small Cell Lung Cancer Cells.

PubMed

Zhang, Wenzhang; Wu, Xin; Hu, Liang; Ma, Yuefan; Xiu, Zihan; Huang, Bingyu; Feng, Yun; Tang, Xudong

2017-05-24

The human papillomavirus (HPV) infection may be associated with the development and progression of non-small cell lung cancer (NSCLC). However, the role of HPV-16 oncoproteins in the development and progression of NSCLC is not completely clear. Epithelial-mesenchymal transition (EMT), a crucial step for invasion and metastasis, plays a key role in the development and progression of NSCLC. Here we explored the effect of HPV-16 oncoproteins on EMT and the underlying mechanisms. NSCLC cell lines, A549 and NCI-H460, were transiently transfected with the EGFP-N1-HPV-16 E6 or E7 plasmid. Real-time PCR and Western blot analysis were performed to analyze the expression of EMT markers. A protein microarray was used to screen the involved signaling pathway. Our results showed that overexpression of HPV-16 E6 and E7 oncoproteins in NSCLC cells significantly promoted EMT-like morphologic changes, downregulated the mRNA and protein levels of EMT epithelial markers (E-cadherin and ZO-1), and upregulated the mRNA and protein levels of EMT mesenchymal markers (N-cadherin and vimentin) and transcription factors (ZEB-1 and Snail-1). Furthermore, the HPV-16 E6 oncoprotein promoted STAT3 activation. Moreover, WP1066, a specific signal transducer and activator of transcription 3 (STAT3) inhibitor, reversed the effect of HPV-16 E6 on the expression of ZO-1, vimentin, and ZEB-1 in transfected NSCLC cells. Taken together, our results suggest that overexpression of HPV-16 E6 and E7 oncoproteins enhances EMT, and the STAT3 signaling pathway may be involved in HPV-16 E6-induced EMT in NSCLC cells.

Intrinsic and Extrinsic Regulation of PD-L2 Expression in Oncogene-Driven Non-Small Cell Lung Cancer.

PubMed

Shibahara, Daisuke; Tanaka, Kentaro; Iwama, Eiji; Kubo, Naoki; Ota, Keiichi; Azuma, Koichi; Harada, Taishi; Fujita, Jiro; Nakanishi, Yoichi; Okamoto, Isamu

2018-03-27

The interaction of programmed cell death ligand 2 (PD-L2) with programmed cell death 1 is implicated in tumor immune escape. The regulation of PD-L2 expression in tumor cells has remained unclear, however. We here examined intrinsic and extrinsic regulation of PD-L2 expression in NSCLC. PD-L2 expression was evaluated by reverse transcription and real-time polymerase chain reaction analysis and by flow cytometry. BEAS-2B cells stably expressing an activated mutant form of EGFR or the echinoderm microtubule associated protein like 4 (EML4)-ALK receptor tyrosine kinase fusion oncoprotein manifested increased expression of PD-L2 at both the mRNA and protein levels. Furthermore, treatment of NSCLC cell lines that harbor such driver oncogenes with corresponding EGFR or ALK tyrosine kinase inhibitors or depletion of EGFR or ALK by small interfering RNA transfection suppressed expression of PD-L2, demonstrating that activating EGFR mutations or echinoderm microtubule associated protein like 4 gene (EML4)-ALK receptor tyrosine kinase gene (ALK) fusion intrinsically induce PD-L2 expression. We also found that interferon gamma (IFN-γ) extrinsically induced expression of PD-L2 through signal transducer and activator of transcription 1 signaling in NSCLC cells. Oncogene-driven expression of PD-L2 in NSCLC cells was inhibited by knockdown of the transcription factors signal transducer and activator of transcription 3 (STAT3) or c-FOS. IFN-γ also activated STAT3 and c-FOS, suggesting that these proteins may also contribute to the extrinsic induction of PD-L2 expression. Expression of PD-L2 is induced intrinsically by activating EGFR mutations or EML4-ALK fusion and extrinsically by IFN-γ, with STAT3 and c-FOS possibly contributing to both intrinsic and extrinsic pathways. Our results thus provide insight into the complexity of tumor immune escape in NSCLC. Copyright © 2018 International Association for the Study of Lung Cancer. Published by Elsevier Inc. All rights reserved.

Malignant transformation of CD4+ T lymphocytes mediated by oncogenic kinase NPM/ALK recapitulates IL-2-induced cell signaling and gene expression reprogramming

PubMed Central

Marzec, Michal; Halasa, Krzysztof; Liu, Xiaobin; Wang, Hong Y.; Cheng, Mangeng; Baldwin, Donald; Tobias, John W.; Schuster, Stephen J.; Woetmann, Anders; Zhang, Qian; Turner, Suzanne D.; Odum, Niels; Wasik, Mariusz A.

2013-01-01

Anaplastic lymphoma kinase (ALK) physiologically expressed only by nervous system cells displays remarkable capacity to transform CD4+ T lymphocytes and other types of non-neural cells. Here we report that activity of nucleophosphmin (NPM)/ALK chimeric protein, the dominant form of ALK expressed in T-cell lymphomas (ALK+TCL), closely resembles cell activation induced by interleukin 2 (IL-2), the key cytokine supporting growth and survival of normal CD4+ T lymphocytes. Direct comparison of gene expression by ALK+TCL cells treated with an ALK inhibitor and IL-2-dependent ALK-TCL cells stimulated with the cytokine revealed a very similar, albeit inverse, gene regulation pattern. Depending on the analysis method, up to 67% of the modulated genes could be defined as modulated in common by NPM/ALK and IL-2. Based on the gene expression patterns, Jak/STAT and IL-2 signaling pathways topped the list of pathways identified as affected by both IL-2 and NPM/ALK. The expression dependence on NPM/ALK and IL-2 of the five selected genes: CD25 (IL-2Rα), Egr-1, Fosl-1, SOCS3, and Irf-4 was confirmed at the protein level. In both ALK+TCL and IL-2-stimulated ALK-TCL cells, CD25, SOCS3, and Irf-4 genes were activated predominantly by the STAT5 and STAT3 transcription factors, while transcription of Egr-1 and Fosl-1 was induced by the MEK-ERK pathway. Finally, we found that Egr-1, a protein not associated previously with either IL-2 or ALK, contributes to the cell proliferation. These findings indicate that NPM/ALK transforms the target CD4+ T lymphocytes, at least in part, by utilizing the pre-existing, IL-2-dependent signaling pathways. PMID:24218456

Combined targeting of STAT3 and STAT5: a novel approach to overcome drug resistance in chronic myeloid leukemia.

PubMed

Gleixner, Karoline V; Schneeweiss, Mathias; Eisenwort, Gregor; Berger, Daniela; Herrmann, Harald; Blatt, Katharina; Greiner, Georg; Byrgazov, Konstantin; Hoermann, Gregor; Konopleva, Marina; Waliul, Islam; Cumaraswamy, Abbarna A; Gunning, Patrick T; Maeda, Hiroshi; Moriggl, Richard; Deininger, Michael; Lion, Thomas; Andreeff, Michael; Valent, Peter

2017-09-01

In chronic myeloid leukemia, resistance against BCR-ABL1 tyrosine kinase inhibitors can develop because of BCR-ABL1 mutations, activation of additional pro-oncogenic pathways, and stem cell resistance. Drug combinations covering a broad range of targets may overcome resistance. CDDO-Me (bardoxolone methyl) is a drug that inhibits the survival of leukemic cells by targeting different pro-survival molecules, including STAT3. We found that CDDO-Me inhibits proliferation and survival of tyrosine kinase inhibitor-resistant BCR-ABL1 + cell lines and primary leukemic cells, including cells harboring BCR-ABL1 T315I or T315I + compound mutations. Furthermore, CDDO-Me was found to block growth and survival of CD34 + /CD38 - leukemic stem cells (LSC). Moreover, CDDO-Me was found to produce synergistic growth-inhibitory effects when combined with BCR-ABL1 tyrosine kinase inhibitors. These drug-combinations were found to block multiple signaling cascades and molecules, including STAT3 and STAT5. Furthermore, combined targeting of STAT3 and STAT5 by shRNA and STAT5-targeting drugs also resulted in synergistic growth-inhibition, pointing to a new efficient concept of combinatorial STAT3 and STAT5 inhibition. However, CDDO-Me was also found to increase the expression of heme-oxygenase-1, a heat-shock-protein that triggers drug resistance and cell survival. We therefore combined CDDO-Me with the heme-oxygenase-1 inhibitor SMA-ZnPP, which also resulted in synergistic growth-inhibitory effects. Moreover, SMA-ZnPP was found to sensitize BCR-ABL1 + cells against the combination 'CDDO-Me+ tyrosine kinase inhibitor'. Together, combined targeting of STAT3, STAT5, and heme-oxygenase-1 overcomes resistance in BCR-ABL1 + cells, including stem cells and highly resistant sub-clones expressing BCR-ABL1 T315I or T315I-compound mutations. Whether such drug-combinations are effective in tyrosine kinase inhibitor-resistant patients with chronic myeloid leukemia remains to be elucidated. Copyright© 2017 Ferrata Storti Foundation.

STATs profiling reveals predominantly-activated STAT3 in cholangiocarcinoma genesis and progression.

PubMed

Dokduang, Hasaya; Techasen, Anchalee; Namwat, Nisana; Khuntikeo, Narong; Pairojkul, Chawalit; Murakami, Yoshinori; Loilome, Watcharin; Yongvanit, Puangrat

2014-10-01

We investigated the aberrant expression of the STAT family in humans and liver fluke (Opisthorchis viverrini, Ov)-induced hamster cholangiocarcinoma (CCA) tissues. The expression and phosphorylation of STAT1, STAT2, STAT3, STAT4, STAT5a, STAT5b and STAT6 in human hamster CCA tissues were immunohistochemistry-profiled. Localizations of STAT5 in macrophages and lipopolysaccharide (LPS)-induced macrophage-conditioned media mediated STAT3 activation in CCA cells were demonstrated. The expressions of STAT 1-4 and 6 were detected in the cytoplasm of hyperplastic bile ducts and tumor cells, whereas STAT5a and STAT5b were observed in macrophages and connective tissues surrounding tumor, respectively. The expressions of STAT3 and STAT5b were significantly observed in tumors with a poorer histological differentiation. STAT3 expression was significantly associated with shorter survival of CCA patients and was predominately activated in CCA cell lines. In the CCA-hamsters, STATs expression was gradually increased along the carcinogenesis, especially at 30 days post-infection in which the inflammatory response was markedly observed, showing the correlation between the inflammation and STATs activation. Moreover, LPS-induced macrophage-conditioned media could mediate STAT3 activation in CCA cells. STAT3 is the major STAT, which plays roles in the inflammation that contributes to CCA carcinogenesis and progression and may serve as a marker for a poor prognosis of CCA. © 2014 Japanese Society of Hepato-Biliary-Pancreatic Surgery.

Testicular Lumicrine Factors Regulate ERK, STAT, and NFKB Pathways in the Initial Segment of the Rat Epididymis to Prevent Apoptosis1

PubMed Central

Xu, Bingfang; Abdel-Fattah, Rana; Yang, Ling; Crenshaw, Sallie A.; Black, Michael B.; Hinton, Barry T.

2011-01-01

The initial segment of the epididymis is vital for male fertility; therefore, it is important to understand the mechanisms that regulate this important region. Deprival of testicular luminal fluid factors/lumicrine factors from the epididymis results in a wave of apoptosis in the initial segment. In this study, a combination of protein array and microarray analyses was used to examine the early changes in downstream signal transduction pathways following loss of lumicrine factors. We discovered the following cascade of events leading to the loss of protection and eventual apoptosis: in the first 6 h after loss of lumicrine factors, down-regulation of the ERK pathway components was observed at the mRNA expression and protein activity levels. Microarray analysis revealed that mRNA levels of several key components of the ERK pathway, Dusp6, Dusp5, and Etv5, decreased sharply, while the analysis from the protein array revealed a decline in the activities of MAP2K1/2 and MAPK1. Immunostaining of phospho-MAPK3/1 indicated that down-regulation of the ERK pathway was specific to the epithelial cells of the initial segment. Subsequently, after 12 h of loss of lumicrine factors, levels of mRNA expression of STAT and NFKB pathway components increased, mRNA levels of several genes encoding cell cycle inhibitors increased, and levels of protein expression of several proapoptotic phosphatases increased. Finally, after 18 h of loss of protection from lumicrine factors, apoptosis was observed. In conclusion, testicular lumicrine factors protect the cells of the initial segment by activating the ERK pathway, repressing STAT and NFKB pathways, and thereby preventing apoptosis. PMID:21311037

Regulation of c–myc expression by IFN–γ through Stat1-dependent and -independent pathways

PubMed Central

Ramana, Chilakamarti V.; Grammatikakis, Nicholas; Chernov, Mikhail; Nguyen, Hannah; Goh, Kee Chuan; Williams, Bryan R.G.; Stark, George R.

2000-01-01

Interferons (IFNs) inhibit cell growth in a Stat1-dependent fashion that involves regulation of c–myc expression. IFN–γ suppresses c–myc in wild-type mouse embryo fibroblasts, but not in Stat1-null cells, where IFNs induce c–myc mRNA rapidly and transiently, thus revealing a novel signaling pathway. Both tyrosine and serine phosphorylation of Stat1 are required for suppression. Induced expression of c–myc is likely to contribute to the proliferation of Stat1-null cells in response to IFNs. IFNs also suppress platelet-derived growth factor (PDGF)-induced c–myc expression in wild-type but not in Stat1-null cells. A gamma-activated sequence element in the promoter is necessary but not sufficient to suppress c–myc expression in wild-type cells. In PKR-null cells, the phosphorylation of Stat1 on Ser727 and transactivation are both defective, and c–myc mRNA is induced, not suppressed, in response to IFN–γ. A role for Raf–1 in the Stat1-independent pathway is revealed by studies with geldanamycin, an HSP90-specific inhibitor, and by expression of a mutant of p50cdc37 that is unable to recruit HSP90 to the Raf–1 complex. Both agents abrogated the IFN–γ-dependent induction of c–myc expression in Stat1-null cells. PMID:10637230

Three STATs are involved in the regulation of the expression of antimicrobial peptides in the triangle sail mussel, Hyriopsis cumingii.

PubMed

Dai, Yun-Jia; Hui, Kai-Min; Zhang, Ying-Hao; Liu, Yan; Wang, Yu-Qing; Zhao, Li-Juan; Lin, Li; Chai, Lian-Qin; Wei, Shun; Lan, Jiang-Feng

2017-04-01

Janus kinase (Jak) and signal transducers and activators of transcription (STAT) signaling pathway is associated in antiviral and antibacterial immune response. Previous studies primarily investigated the function of STATs in mammals. For most invertebrates, only one STAT was found in each species, such as STAT92E was found in Drosophila melanogaster. The studies, which focus on the functional difference between various STATs in the same species of invertebrate, are limited. In the present study, three STATs (HcSTAT1, HcSTAT2 and HcSTAT3) were identified in triangle shell pearl mussel, Hyriopsis cumingii. Phylogenetic analysis showed that HcSTAT1 and HcSTAT3 were clustered with Homo sapiens STAT5, and HcSTAT2 was clustered with Pinctada fucata STAT and Crassostea gigas STAT6. All three STATs could be detected in all tested tissues (hemocytes, hepatopancreas, gill, mantle and foot), and were induced expression when challenged with Staphylococcus aureus or Aeromonas hydrophilia in hemocytes and hepatopancreas. HcSTAT1 regulated the expression of HcDef, HcWAP, HcThe and HcTNF. The expression of HcWAP and HcTNF was down-regulated in HcSTAT2-RNAi mussel. And HcSTAT3 affected the expression of HcTNF. The study is the first report of different functions in antibacterial immune responses between STATs in mollusks. Copyright © 2017 Elsevier Ltd. All rights reserved.

STAT1-Induced HLA class I Upregulation Enhances Immunogenicity and Clinical Response to anti-EGFR mAb Cetuximab Therapy in HNC Patients

PubMed Central

Srivastava, Raghvendra M.; Trivedi, Sumita; Concha-Benavente, Fernando; Hyun-bae, Jie; Wang, Lin; Seethala, Raja R.; Branstetter, Barton F.; Ferrone, Soldano; Ferris, Robert L.

2015-01-01

The goal of this study was to characterize the molecular mechanisms underlying cetuximab-mediated upregulation of HLA class I antigen-processing machinery components in head and neck cancer (HNC) cells and to determine the clinical significance of these changes in cetuximab-treated HNC patients. Flow cytometry, signaling studies and chromatin immunoprecipitation (ChIP) assays were performed using HNC cells treated with cetuximab alone or with Fcγ receptor (FcγR)-bearing lymphocytes to establish the mechanism of EGFR-dependent regulation of HLA APM expression. A prospective phase II clinical trial of neoadjuvant cetuximab was utilized to correlate HLA class I expression with clinical response in HNC patients. EGFR blockade triggered STAT1 activation and HLA upregulation, in a src homology-containing protein (SHP)-2-dependent fashion, more prominently in HLA-B/C than in HLA-A alleles. EGFR signaling blockade also enhanced IFNγ receptor 1 (IFNAR) expression, augmenting induction of HLA class I and TAP1/2 expression by IFNγ, which was abrogated in STAT1−/− cells. Cetuximab enhanced HNC cell recognition by EGFR853–861-specific CTLs, and notably enhanced surface presentation of a non-EGFR peptide (MAGE-3271–279). HLA class I upregulation was significantly associated with clinical response in cetuximab-treated HNC patients. EGFR induces HLA downregulation through SHP-2/STAT1 suppression. Reversal of HLA class I downregulation was more prominent in clinical responders to cetuximab therapy, supporting an important role for adaptive immunity in cetuximab antitumor activity. Abrogating EGFR-induced immune escape mechanisms and restoring STAT1 signaling to reverse HLA downregulation using cetuximab should be combined with strategies to enhance adaptive cellular immunity. PMID:25972070

Nuclear matrix protein SMAR1 control regulatory T-cell fate during inflammatory bowel disease (IBD)

PubMed Central

Mirlekar, B; Ghorai, S; Khetmalas, M; Bopanna, R; Chattopadhyay, S

2015-01-01

Regulatory T (Treg) cells are essential for self-tolerance and immune homeostasis. Transcription factor Foxp3, a positive regulator of Treg cell differentiation, has been studied to some extent. Signal transducer and activator of transcription factor 3 (STAT3) is known to negatively regulate Foxp3. It is not clear how STAT3 is regulated during Treg differentiation. We show that SMAR1, a known transcription factor and tumor suppressor, is directly involved in maintaining Treg cell fate decision. T-cell-specific conditional knockdown of SMAR1 exhibits increased susceptibility towards inflammatory disorders, such as colitis. The suppressive function of Treg cells is compromised in the absence of SMAR1 leading to increased T helper type 17 (Th17) differentiation and inflammation. Compared with wild-type, the SMAR1−/− Treg cells showed increased susceptibility of inflammatory bowel disease in Rag1−/− mice, indicating the role of SMAR1 in compromising Treg cell differentiation resulting in severe colitis. We show that SMAR1 negatively regulate STAT3 expression favoring Foxp3 expression and Treg cell differentiation. SMAR1 binds to the MAR element of STAT3 promoter, present adjacent to interleukin-6 response elements. Thus Foxp3, a major driver of Treg cell differentiation, is regulated by SMAR1 via STAT3 and a fine-tune balance between Treg and Th17 phenotype is maintained. PMID:25993445

Evidence for a functional link between Dd-STATa and Dd-PIAS, a Dictyostelium PIAS homologue.

PubMed

Kawata, Takefumi; Hirano, Tatsunori; Ogasawara, Shun; Aoshima, Ryota; Yachi, Ayako

2011-09-01

Several mammalian protein families inhibit the activity of signal transducer and activator of transcription (STAT) proteins. The protein inhibitor of activated STAT (PIAS) was initially identified through its ability to interact with human STAT proteins. We isolated a gene (pisA) encoding a Dictyostelium orthologue of PIAS, Dd-PIAS, which possesses almost all the representative motifs and domains of mammalian PIAS proteins. A Dd-PIAS null mutant strain displays a normal terminal morphology but with accelerated development once cells are aggregated. In contrast, Dd-PIAS overexpressor strains demonstrate delayed aggregation, almost no slug phototaxis, impaired slug motility, and a prolonged slug migration period. This strain is a near phenocopy of the Dd-STATa null mutant, although it eventually forms a fruiting body, albeit inefficiently. The expression of several Dd-STATa-activated genes is upregulated in the Dd-PIAS null mutant and there is ectopic expression of pstAB makers. The concentration of a PIAS-green fluorescent protein (GFP) fusion protein, expressed under the PIAS promoter, is greatest in the pstO cells and gradually decreases with proximity to the tip of the slug and culminant: a pattern diametrically opposite to that of Dd-STATa. Our results suggest a functional interrelationship between Dd-PIAS and Dd-STATa that influences gene expression and development. © 2011 The Authors. Development, Growth & Differentiation © 2011 Japanese Society of Developmental Biologists.

STAT3 activation is associated with cerebrospinal fluid interleukin-10 (IL-10) in primary central nervous system diffuse large B cell lymphoma.

PubMed

Mizowaki, Takashi; Sasayama, Takashi; Tanaka, Kazuhiro; Mizukawa, Katsu; Takata, Kumi; Nakamizo, Satoshi; Tanaka, Hirotomo; Nagashima, Hiroaki; Nishihara, Masamitsu; Hirose, Takanori; Itoh, Tomoo; Kohmura, Eiji

2015-09-01

Signal transducers and activators of transcription 3 (STAT3) are activated by various cytokines and oncogenes; however, the activity and pathogenesis of STAT3 in diffuse large B cell lymphoma of the central nervous system have not been thoroughly elucidated. We investigated the phosphorylation levels of STAT3 in 40 specimens of primary central nervous system diffuse large B-cell lymphoma (PCNS DLBCL) and analyzed the association between phsopho-STAT3 (pSTAT3) expression and cerebrospinal fluid (CSF) concentration of interleukin-10 (IL-10) or IL-6. Immunohistochemistry and Western blot analysis revealed that most of the specimens in PCNS DLBCL expressed pSTST3 protein, and a strong phosphorylation levels of STAT3 was statistically associated with high CSF IL-10 levels, but not with CSF IL-6 levels. Next, we demonstrated that recombinant IL-10 and CSF containing IL-10 induced the phosphorylation of STAT3 in PCNS DLBCL cells. Furthermore, molecular subtype classified by Hans' algorithm was correlated with pSTAT3 expression levels and CSF IL-10 levels. These results suggest that the STAT3 activity is correlated with CSF IL-10 level, which is a useful marker for STAT3 activity in PCNS DLBCLs.

MAPK regulation of IL-4/IL-13 receptors contributes to the synergistic increase in CCL11/eotaxin-1 in response to TGF-β1 and IL-13 in human airway fibroblasts.

PubMed

Zhou, Xiuxia; Hu, Haizhen; Balzar, Silvana; Trudeau, John B; Wenzel, Sally E

2012-06-15

CCL11/eotaxin-1 is a potent eosinophilic CC chemokine expressed by primary human fibroblasts. The combination of TGF-β1 and IL-13 synergistically increases CCL11 expression, but the mechanisms behind the synergy are unclear. To address this, human airway fibroblast cultures from normal and asthmatic subjects were exposed to IL-13 alone or TGF-β1 plus IL-13. Transcriptional (nuclear run-on) and posttranscriptional (mRNA stability) assays confirmed that transcriptional regulation is critical for synergistic expression of CCL11. TGF-β1 plus IL-13 synergistically increased STAT-6 phosphorylation, nuclear translocation, and binding to the CCL11 promoter as compared with IL-13 alone. STAT-6 small interfering RNA significantly knocked down both STAT-6 mRNA expression and phosphorylation and inhibited CCL11 mRNA and protein expression. Regulation of the IL-4Rα complex by TGF-β1 augmented IL-13 signaling by dampening IL-13Rα2 expression, overcoming IL-13's autoregulation of its pathway and enhancing the expression of CCL11. Our data suggest that TGF-β1 induced activation of the MEK/ERK pathway reduces IL-13Rα2 expression induced by IL-13. Thus, TGF-β1, a pleiotropic cytokine upregulated in asthmatic airways, can augment eosinophilic inflammation by interfering with IL-13's negative feedback autoregulatory loop under MEK/ERK-dependent conditions.

[Diagnostic value of STAT6 immunohistochemistry in solitary fibrous tumor/meningeal hemangiopericytoma].

PubMed

Zhang, Xialing; Cheng, Haixia; Bao, Yun; Tang, Feng; Wang, Yin

2016-02-01

To investigate the diagnostic role of STAT6 immunohistochemistry in solitary fibrous tumors (SFT)/meningeal hemangiopericytomas (HPC). Evaluated the expression of STAT6, vimentin, CD34, EMA, PR, S-100, CD56, GFAP and Ki-67 in a cohort of 37 SFT/meningeal HPC, 30 meningiomas and 30 schwannomas by immunohistochemistry staining. All SFT/meningeal HPC demonstrated nuclear positivity for STAT6, and the proportion of positive tumor cells ranged from 60% to 95%, with no significant difference cases.Vimentin was strongly positive in all cases. CD34, EMA and PR positivity was found in 32 cases, 1 case and 4 cases, respectively.S-100 protein, CD56 and GFAP were negative; Ki-67 labeling index was 1%-8%. However, the meningiomas and schwannomas were negative for STAT6. STAT6 is a relatively specific biomarker for SFT/meningeal HPC, and may be used in the diagnosis and differential diagnosis of SFT/meningeal HPC, especially for the atypical cases, and allows the precise pathologic diagnosis of SFT/meningeal HPC.

Liver Inflammation and Metabolic Signaling in ApcMin/+ Mice: The Role of Cachexia Progression

PubMed Central

Narsale, Aditi A.; Enos, Reilly T.; Puppa, Melissa J.; Chatterjee, Saurabh; Murphy, E. Angela; Fayad, Raja; Pena, Majorette O’; Durstine, J. Larry; Carson, James A.

2015-01-01

The ApcMin/+ mouse exhibits an intestinal tumor associated loss of muscle and fat that is accompanied by chronic inflammation, insulin resistance and hyperlipidemia. Since the liver governs systemic energy demands through regulation of glucose and lipid metabolism, it is likely that the liver is a pathological target of cachexia progression in the ApcMin/+ mouse. The purpose of this study was to determine if cancer and the progression of cachexia affected liver endoplasmic reticulum (ER)-stress, inflammation, metabolism, and protein synthesis signaling. The effect of cancer (without cachexia) was examined in wild-type and weight-stable ApcMin/+ mice. Cachexia progression was examined in weight-stable, pre-cachectic, and severely-cachectic ApcMin/+ mice. Livers were analyzed for morphology, glycogen content, ER-stress, inflammation, and metabolic changes. Cancer induced hepatic expression of ER-stress markers BiP (binding immunoglobulin protein), IRE-1α (endoplasmic reticulum to nucleus signaling 1), and inflammatory intermediate STAT-3 (signal transducer and activator of transcription 3). While gluconeogenic enzyme phosphoenolpyruvate carboxykinase (PEPCK) mRNA expression was suppressed by cancer, glycogen content or protein synthesis signaling remained unaffected. Cachexia progression depleted liver glycogen content and increased mRNA expression of glycolytic enzyme PFK (phosphofrucktokinase) and gluconeogenic enzyme PEPCK. Cachexia progression further increased pSTAT-3 but suppressed p-65 and JNK (c-Jun NH2-terminal kinase) activation. Interestingly, progression of cachexia suppressed upstream ER-stress markers BiP and IRE-1α, while inducing its downstream target CHOP (DNA-damage inducible transcript 3). Cachectic mice exhibited a dysregulation of protein synthesis signaling, with an induction of p-mTOR (mechanistic target of rapamycin), despite a suppression of Akt (thymoma viral proto-oncogene 1) and S6 (ribosomal protein S6) phosphorylation. Thus, cancer induced ER-stress markers in the liver, however cachexia progression further deteriorated liver ER-stress, disrupted protein synthesis regulation and caused a differential inflammatory response related to STAT-3 and NF-κB (Nuclear factor—κB) signaling. PMID:25789991

Traumatically injured astrocytes release a proteomic signature modulated by STAT3 dependent cell survival

PubMed Central

Levine, Jaclynn; Kwon, Eunice; Paez, Pablo; Yan, Weihong; Czerwieniec, Gregg; Loo, Joseph A.; Sofroniew, Michael V.; Wanner, Ina-Beate

2015-01-01

Molecular markers associated with CNS injury are of diagnostic interest. Mechanical trauma generates cellular deformation associated with membrane permeability with unknown molecular consequences. We used an in vitro model of stretch-injury and proteomic analyses to determine protein changes in murine astrocytes and their surrounding fluids. Abrupt pressure-pulse stretching resulted in the rapid release of 59 astrocytic proteins with profiles reflecting cell injury and cell death, i.e. mechanoporation and cell lysis. This acute trauma-release proteome was overrepresented with metabolic proteins compared to the uninjured cellular proteome, bearing relevance for post-traumatic metabolic depression. Astrocyte-specific deletion of signal transducer and activator of transcription 3 (STAT3-CKO) resulted in reduced stretch-injury tolerance, elevated necrosis and increased protein release. Consistent with more lysed cells, more protein complexes, nuclear and transport proteins were released from STAT3-CKO versus non-transgenic astrocytes. STAT3-CKO astrocytes had reduced basal expression of GFAP, lactate dehydrogenase B (LDHB), aldolase C (ALDOC) and astrocytic phosphoprotein 15 (PEA15), and elevated levels of tropomyosin (TPM4) and α actinin 4 (ACTN4). Stretching caused STAT3 dependent cellular depletion of PEA15 and GFAP, and its filament disassembly in subpopulations of injured astrocytes. PEA15 and ALDOC signals were low in injured astrocytes acutely after mouse spinal cord crush injury and robustly expressed in reactive astrocytes one day post-injury. In contrast, α crystallin (CRYAB) was present in acutely injured astrocytes, and absent from uninjured and reactive astrocytes, demonstrating novel marker differences among post-injury astrocytes. These findings reveal a proteomic signature of traumatically-injured astrocytes reflecting STAT3-dependent cellular survival with potential diagnostic value. PMID:26683444

The antihypertension drug doxazosin suppresses JAK/STATs phosphorylation and enhances the effects of IFN-α/γ-induced apoptosis.

PubMed

Park, Mi Sun; Kim, Boh-Ram; Kang, Sokbom; Kim, Dae-Yong; Rho, Seung Bae

2014-11-01

Doxazosin, a commonly prescribed treatment for patients with benign prostatic hyperplasia, serves as an α1-blocker of the adrenergic receptors. In this study, we calculated its effect on the ovarian carcinoma cells. Doxazosin induces dose-dependent growth suppression and is additively activated through IFN-α or IFN-γ stimulation. They both enhanced G1 phase arrest, as well as the activity of caspase-3, and the reduction of cyclin D1 and CDK4 protein levels. Doxazosin growth suppression was abolished either by the Janus family of tyrosine kinase (JAK) or the signal transducer and activator of transcription (STAT) inhibitor treatment. The activity of JAK/STAT was dependent on the level of doxazosin, suggesting a requirement of doxazosin for the activation of JAK/STAT. Furthermore, doxazosin plus IFN-α or doxazosin plus IFN-γ additively suppressed the activation of the JAK/STAT signals through phosphorylation of JAK and STAT, thus affecting the activation of subsequent downstream signaling components PI3K, mTOR, 70S6K, and PKCδ. In vivo study demonstrated that doxazosin significantly suppressed tumor growth in an ovarian cancer cell xenograft mouse model, inducing apoptotic cell death by up-regulating the expression of p53, whereas c-Myc expression was markedly reduced. Our data indicate that doxazosin can modulate the apoptotic effects of IFN-α- and IFN-γ through the JAK/STAT signaling pathways. Collectively, we indicate that this action may be a potent chemotherapeutic property against ovarian carcinoma.

Sphingosylphosphorylcholine promotes the differentiation of resident Sca-1 positive cardiac stem cells to cardiomyocytes through lipid raft/JNK/STAT3 and β-catenin signaling pathways.

PubMed

Li, Wenjing; Liu, Honghong; Liu, Pingping; Yin, Deling; Zhang, Shangli; Zhao, Jing

2016-07-01

Resident cardiac Sca-1-positive (+) stem cells may differentiate into cardiomyocytes to improve the function of damaged hearts. However, little is known about the inducers and molecular mechanisms underlying the myogenic conversion of Sca-1(+) stem cells. Here we report that sphingosylphosphorylcholine (SPC), a naturally occurring bioactive lipid, induces the myogenic conversion of Sca-1(+) stem cells, as evidenced by the increased expression of cardiac transcription factors (Nkx2.5 and GATA4), structural proteins (cardiac Troponin T), transcriptional enhancer (Mef2c) and GATA4 nucleus translocation. First, SPC activated JNK and STAT3, and the JNK inhibitor SP600125 or STAT3 inhibitor stattic impaired the SPC-induced expression of cardiac transcription factors and GATA4 nucleus translocation, which suggests that JNK and STAT3 participated in SPC-promoted cardiac differentiation. Moreover, STAT3 activation was inhibited by SP600125, whereas JNK was inhibited by β-cyclodextrin as a lipid raft breaker, which indicates a lipid raft/JNK/STAT3 pathway involved in SPC-induced myogenic transition. β-Catenin, degraded by activated GSK3β, was inhibited by SPC. Furthermore, GSK3β inhibitors weakened but the β-catenin inhibitor promoted SPC-induced differentiation. We found no crosstalk between the lipid raft/JNK/STAT3 and β-catenin pathway. Our study describes a lipid, SPC, as an endogenic inducer of myogenic conversion in Sca-1(+) stem cells with low toxicity and high efficiency for uptake. Copyright © 2016 Elsevier B.V. All rights reserved.

The role of STATs in lung carcinogenesis: an emerging target for novel therapeutics.

PubMed

Karamouzis, Michalis V; Konstantinopoulos, Panagiotis A; Papavassiliou, Athanasios G

2007-05-01

The signal transducer and activator of transcription (STAT) proteins are a family of latent cytoplasmic transcription factors, which form dimers when activated by cytokine receptors, tyrosine kinase growth factor receptors as well as non-receptor tyrosine kinases. Dimeric STATs translocate to the nucleus, where they bind to specific DNA-response elements in the promoters of target genes, thereby inducing unique gene expression programs often in association with other transcription regulatory proteins. The functional consequence of different STAT proteins activation varies, as their target genes play diverse roles in normal cellular/tissue functions, including growth, apoptosis, differentiation and angiogenesis. Certain activated STATs have been implicated in human carcinogenesis, albeit only few studies have focused into their role in lung tumours. Converging evidence unravels their molecular interplays and complex multipartite regulation, rendering some of them appealing targets for lung cancer treatment with new developing strategies.

Proliferating cell nuclear antigen promotes cell proliferation and tumorigenesis by up-regulating STAT3 in non-small cell lung cancer.

PubMed

Wang, Liuxin; Kong, Weixiang; Liu, Bing; Zhang, Xueqing

2018-08-01

Proliferating cell nuclear antigen (PCNA) functions as a bridging molecule, which targets proteins that have distinct roles in cell growth. The expression of PCNA is dysregulated in some tumors and takes part in the progression of oncogenesis. However, the roles of PCNA in the progression of non-small cell lung cancer (NSCLC) remain unknown. The present study aimed to investigate the function of PCNA in the occurrence and development of NSCLC and its underlying molecular mechanisms. Western blotting, RT-PCR, and immunohistochemistry assays were used to detect the expression pattern of PCNA in NSCLC tissues and cells. A log rank test was performed to compare the overall survival (OS) of patients with high/low expression of PCNA. Besides, the relationship between PCNA and signal transducer and activator of transcription-3 (STAT3) proteins were evaluated. Then, MTT, flow cytometry, clonal formation, and in vivo xenograft assays were conducted to investigate the effects of PCNA/STAT3 on cell growth, clonal formation, apoptosis, and tumorigenesis. Results showed that PCNA expression was elevated in NSCLC tissues and cells and it could combine with STAT3 and increased its expression and phosphorylation. Moreover, the expression of PCNA showed a positive correlation with the TNM grade and occurrence rate of the lymphatic metastasis and poor prognosis of NSCLC patients. Overexpression of PCNA promoted cell proliferation, clonal formation, and tumorigenesis in lung cancer cells and inhibited cell apoptosis. In contrast, these effects were inhibited when knockdown of STAT3. In conclusion, this study demonstrates that PCNA functions as an oncogene in the progression of NSCLC through up-regulation of STAT3. These findings point to a potentially new therapeutic strategy for NSCLC. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

The milk protein α-casein functions as a tumor suppressor via activation of STAT1 signaling, effectively preventing breast cancer tumor growth and metastasis

PubMed Central

Bonuccelli, Gloria; Castello-Cros, Remedios; Capozza, Franco; Martinez-Outschoorn, Ubaldo E.; Lin, Zhao; Tsirigos, Aristotelis; Xuanmao, Jiao; Whitaker-Menezes, Diana; Howell, Anthony; Lisanti, Michael P.; Sotgia, Federica

2012-01-01

Here, we identified the milk protein α-casein as a novel suppressor of tumor growth and metastasis. Briefly, Met-1 mammary tumor cells expressing α-casein showed a ~5-fold reduction in tumor growth and a near 10-fold decrease in experimental metastasis. To identify the molecular mechanism(s), we performed genome-wide transcriptional profiling. Interestingly, our results show that α-casein upregulates gene transcripts associated with interferon/STAT1 signaling and downregulates genes associated with “stemness.” These findings were validated by immunoblot and FACS analysis, which showed the upregulation and hyperactivation of STAT1 and a decrease in the number of CD44(+) “cancer stem cells.” These gene signatures were also able to predict clinical outcome in human breast cancer patients. Thus, we conclude that a lactation-based therapeutic strategy using recombinant α-casein would provide a more natural and non-toxic approach to the development of novel anticancer therapies. PMID:23047602

Roles of unphosphorylated STATs in signaling.

PubMed

Yang, Jinbo; Stark, George R

2008-04-01

The seven members of the signal transducer and activator of transcription (STAT) family of transcription factors are activated in response to many different cytokines and growth factors by phosphorylation of specific tyrosine residues. The STAT1 and STAT3 genes are specific targets of activated STATs 1 and 3, respectively, resulting in large increases in the levels of these unphosphorylated STATs (U-STATs) in response to the interferons (STAT1) or ligands that active gp130, such as IL-6 (STAT3). U-STATs drive gene expression by novel mechanisms distinct from those used by phosphorylated STAT (P-STAT) dimers. In this review, we discuss the roles of U-STATs in transcription and regulation of gene expression.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ma, Xusheng, E-mail: maxushengtt@163.com; Yang, Xing; Nian, Xiaofeng

Peste des petits ruminants virus (PPRV) causes a fatal disease in small ruminants. V protein of PPRV plays a pivotal role in interfering with host innate immunity by blocking IFNs signaling through interacting with STAT1 and STAT2. In the present study, the results demonstrated that PPRV V protein blocks IFN actions in a dose dependent manner and restrains the translocation of STAT1/2 proteins. We speculate that the translocation inhibition might be caused by the interfering of the downstream of STAT protein. Mutagenesis defines that Cys cluster and Trp motif of PPRV V protein are essential for STAT-mediated IFN signaling. Thesemore » findings give a new sight for the further studies to understand the delicate mechanism of PPRV to escape the IFN signaling. - Highlights: • PPRV V protein inhibits type I IFN production and blocks its activation. • PPRV V protein negatively regulates activation of ISRE and GAS promoter. • PPRV V protein inhibits nuclear translocation of STAT protein by non-degradation. • PNT and VCT domain of PPRV V protein inhibit IFN transduction. • PPRV V protein binds with STAT protein via some conserved motifs.« less

Inhibition of STAT3 phosphorylation by sulforaphane reduces adhesion molecule expression in vascular endothelial cell.

PubMed

Cho, Young S; Kim, Chan H; Ha, Tae S; Ahn, Hee Y

2015-11-18

Intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1) play key roles in the initiation of vascular inflammation. In this study, we explored whether sulforaphane, a dietary phytochemical, can inhibit the expression of ICAM-1 and VCAM-1 in human umbilical vein endothelial cells (HUVEC) stimulated with lipopolysaccharide (LPS), and the mechanisms involved. Sulforaphane prevented the LPS-mediated increase in ICAM-1 and VCAM-1 expression, (P < 0.01) in HUVEC. Sulforaphane also prevented the LPS-mediated increase in the phosphorylation of signal transducer and activator of transcription 3 (STAT3) (P < 0.01). Stattic, a STAT3 inhibitor, reduced the LPS-induced expression of ICAM-1 and VCAM-1, and STAT3 phosphorylation (P < 0.01). STAT3 small interfering RNA treatment reduced the LPS-induced expression of ICAM-1, VCAM-1, and STAT3 (P < 0.01). Sulforaphane reduced LPS-mediated THP-1 monocyte adhesion to HUVEC (P < 0.01). In C57BL/6 mice, injection of LPS increased aortic ICAM-1 and VCAM-1 expression, and this effect was prevented by sulforaphane. These data provide insight into the mechanism through which sulforaphane partly reduces the expression of ICAM-1 and VCAM-1 on the vascular wall by inhibiting STAT3 phosphorylation.

Essential role for Stat5a/b in myeloproliferative neoplasms induced by BCR-ABL1 and JAK2V617F in mice

PubMed Central

Walz, Christoph; Ahmed, Wesam; Lazarides, Katherine; Betancur, Monica; Patel, Nihal; Hennighausen, Lothar; Zaleskas, Virginia M.

2012-01-01

STAT5 proteins are constitutively activated in malignant cells from many patients with leukemia, including the myeloproliferative neoplasms (MPNs) chronic myeloid leukemia (CML) and polycythemia vera (PV), but whether STAT5 is essential for the pathogenesis of these diseases is not known. In the present study, we used mice with a conditional null mutation in the Stat5a/b gene locus to determine the requirement for STAT5 in MPNs induced by BCR-ABL1 and JAK2V617F in retroviral transplantation models of CML and PV. Loss of one Stat5a/b allele resulted in a decrease in BCR-ABL1–induced CML-like MPN and the appearance of B-cell acute lymphoblastic leukemia, whereas complete deletion of Stat5a/b prevented the development of leukemia in primary recipients. However, BCR-ABL1 was expressed and active in Stat5-null leukemic stem cells, and Stat5 deletion did not prevent progression to lymphoid blast crisis or abolish established B-cell acute lymphoblastic leukemia. JAK2V617F failed to induce polycythemia in recipients after deletion of Stat5a/b, although the loss of STAT5 did not prevent the development of myelofibrosis. These results demonstrate that STAT5a/b is essential for the induction of CML-like leukemia by BCR-ABL1 and of polycythemia by JAK2V617F, and validate STAT5a/b and the genes they regulate as targets for therapy in these MPNs. PMID:22234689

Isolation and Expression Analysis of STAT Members from Synechogobius hasta and Their Roles in Leptin Affecting Lipid Metabolism

PubMed Central

Wu, Kun; Tan, Xiao-Ying; Wei, Chuan-Chuan; You, Wen-Jing; Zhuo, Mei-Qin; Song, Yu-Feng

2016-01-01

Signal transducers and activators of transcription proteins (STATs) act as important mediators in multiple biological processes induced by a large number of cytokines. In the present study, full-length cDNA sequences of seven STAT members, including some splicing variants different from those in mammals, were obtained from Synechogobius hasta. The phylogenetic analysis revealed that the seven STAT members were derived from paralogous genes that might have arisen by whole genome duplication (WGD) events during vertebrate evolution. All of these members share similar domain structure compared with those of mammals, and were widely expressed across the tested tissues (brain, gill, heart, intestine, liver, muscle and spleen), but at variable levels. Incubation in vitro of recombinant human leptin changed the intracellular triglyceride (TG) content and mRNA levels of several STATs members, as well as expressions and activities of genes involved in lipid metabolism. Furthermore, Tyrphostin B42 (AG490), a specific inhibitor of the Janus Kinase 2(JAK2)-STAT pathway, partially reversed leptin-induced change on STAT3 and its two spliced isoforms expression, as well as expressions and activities of genes involved in lipid metabolism. As a consequence, the decrease of TG content was also reversed. Thus, our study suggests that STAT3 is the requisite for the leptin signal and the activation of the STAT3 member may account for the leptin-induced changes in lipid metabolism in S. hasta. PMID:27011172

Isolation and Expression Analysis of STAT Members from Synechogobius hasta and Their Roles in Leptin Affecting Lipid Metabolism.

PubMed

Wu, Kun; Tan, Xiao-Ying; Wei, Chuan-Chuan; You, Wen-Jing; Zhuo, Mei-Qin; Song, Yu-Feng

2016-03-22

Signal transducers and activators of transcription proteins (STATs) act as important mediators in multiple biological processes induced by a large number of cytokines. In the present study, full-length cDNA sequences of seven STAT members, including some splicing variants different from those in mammals, were obtained from Synechogobius hasta. The phylogenetic analysis revealed that the seven STAT members were derived from paralogous genes that might have arisen by whole genome duplication (WGD) events during vertebrate evolution. All of these members share similar domain structure compared with those of mammals, and were widely expressed across the tested tissues (brain, gill, heart, intestine, liver, muscle and spleen), but at variable levels. Incubation in vitro of recombinant human leptin changed the intracellular triglyceride (TG) content and mRNA levels of several STATs members, as well as expressions and activities of genes involved in lipid metabolism. Furthermore, Tyrphostin B42 (AG490), a specific inhibitor of the Janus Kinase 2(JAK2)-STAT pathway, partially reversed leptin-induced change on STAT3 and its two spliced isoforms expression, as well as expressions and activities of genes involved in lipid metabolism. As a consequence, the decrease of TG content was also reversed. Thus, our study suggests that STAT3 is the requisite for the leptin signal and the activation of the STAT3 member may account for the leptin-induced changes in lipid metabolism in S. hasta.

A chimeric cyclic interferon-α2b peptide induces apoptosis by sequential activation of phosphatidylinositol 3-kinase, protein kinase Cδ and p38 MAP kinase.

PubMed

Blank, V C; Bertucci, L; Furmento, V A; Peña, C; Marino, V J; Roguin, L P

2013-06-10

We have previously demonstrated that tyrosine phosphorylation of STAT1/3 and p38 mitogen-activated protein kinase (p38 MAPK) activation are involved in the apoptotic response triggered by a chimeric cyclic peptide of the interferon-α2b (IFN-α2b) in WISH cells. Since the peptide also induced serine phosphorylation of STAT proteins, in the present study we examined the kinase involved in serine STAT1 phosphorylation and the signaling effectors acting upstream such activation. We first found that p38 MAPK is involved in serine STAT1 phosphorylation, since a reduction of phophoserine-STAT1 levels was evident after incubating WISH cells with cyclic peptide in the presence of a p38 pharmacological inhibitor or a dominant-negative p38 mutant. Next, we demonstrated that the peptide induced activation of protein kinase Cδ (PKCδ). Based on this finding, the role of this kinase was then evaluated. After incubating WISH cells with a PKCδ inhibitor or after decreasing PKCδ expression levels by RNA interference, both peptide-induced serine STAT1 and p38 phosphorylation levels were significantly decreased, indicating that PKCδ functions as an upstream regulator of p38. We also showed that PKCδ and p38 activation stimulated by the peptide was inhibited by a specific pharmacological inhibitor of phosphatidylinositol 3-kinase (PI3K) or by a dominant-negative p85 PI3K-regulatory subunit, suggesting that PI3K is upstream in the signaling cascade. In addition, the role of PI3K and PKCδ in cyclic peptide-induced apoptosis was examined. Both signaling effectors were found to regulate the antiproliferative activity and the apoptotic response triggered by the cyclic peptide in WISH cells. In conclusion, we herein demonstrated that STAT1 serine phosphorylation is mediated by the sequential activation of PI3K, PKCδ and p38 MAPK. This signaling cascade contributes to the antitumor effect induced by the chimeric IFN-α2b cyclic peptide in WISH cells. Copyright © 2013 Elsevier Inc. All rights reserved.

Sustained phosphorylation of mutated FGFR3 is a crucial feature of genetic dwarfism and induces apoptosis in the ATDC5 chondrogenic cell line via PLCgamma-activated STAT1.

PubMed

Harada, Daisuke; Yamanaka, Yoshitaka; Ueda, Koso; Nishimura, Riko; Morishima, Tsuneo; Seino, Yoshiki; Tanaka, Hiroyuki

2007-08-01

The most frequent type of rhizomelic dwarfism, achondroplasia (ACH), is caused by mutations in the fibroblast growth factor receptor 3 (FGFR3) gene. Mutations in FGFR3 result in skeletal dysplasias of variable severity, including mild phenotypic effects in hypochondroplasia (HCH), severe phenotypic effects in thanatophoric dysplasia types I (TDI) and II (TDII), and severe but survivable phenotypic effects in severe achondroplasia with developmental delay and acanthosis nigricans (SADDAN). To explore the molecular mechanisms that result in the different phenotypes, we investigated the kinetics of mutated versions of FGFR3. First, we assayed the phosphorylation states of the mutated FGFR3s and found that the level of phosphorylation in TDI-FGFR3 was lower than in ACH-FGFR3, although the other mutants were phosphorylated according to phenotypic severity. Second, we analyzed the duration of the phosphorylation. TDI-FGFR3 was not highly phosphorylated under ligand-free conditions, but the peak phosphorylation levels of TDI-FGFR3 and ACH-FGFR3 were maintained for 30 min after stimulation with FGF-1. Moreover, ligand-dependent phosphorylation of TDI-FGFR3, but not ACH-FGFR3, lasted for more than 8 h after FGF-1 administration. The other mutant proteins showed sustained phosphorylation independent of ligand presence. Third, we investigated the intracellular localization of the mutant proteins. Immunofluorescence analysis showed accumulations of TDII-FGFR3, SADDAN-FGFR3, and a portion of TDI-FGFR3 in the endoplasmic reticulum (ER). Based on these data, we concluded that sustained phosphorylation of FGFR3 causes chondrodysplasia, and the phenotypic severity depends on the proportion of ER-localized mutant FGFR3. In FGFR3 signaling, the transcription factor, signal transducer and activator of transcription 1 (STAT1) inhibit proliferation and induce apoptosis of chondrocytes. Here we reveal that phospholipase C gamma (PLCgamma) mediates FGFR3-induced STAT1 activation. Both PLCgamma and STAT1 were activated by FGFR3 signaling, but a dominant-negative form of PLCgamma (DN-PLCgamma) remarkably reduced STAT1 phosphorylation. Apoptosis assays revealed that the constitutively active forms of FGFR3 (TDII-FGFR3) and STAT1 (STAT1-C) induce apoptosis of chondrogenic ATDC5 cells via caspase activity. DN-PLCgamma reduced the apoptosis of ATDC5 cells expressing TDII-FGFR3, but over-expression of both DN-PLCgamma and STAT1-C induced apoptosis. Therefore, we conclude that a PLCgamma-STAT1 pathway mediates apoptotic signaling by FGFR3.

Mesenchymal stem cells protect against obstruction-induced renal fibrosis by decreasing STAT3 activation and STAT3-dependent MMP-9 production

PubMed Central

Matsui, Futoshi; Babitz, Stephen A.; Rhee, Audrey; Hile, Karen L.; Zhang, Hongji

2017-01-01

STAT3 is a transcription factor implicated in renal fibrotic injury, but the role of STAT3 in mesenchymal stem cell (MSC)-induced renoprotection during renal fibrosis remains unknown. We hypothesized that MSCs protect against obstruction-induced renal fibrosis by downregulating STAT3 activation and STAT3-induced matrix metalloproteinase-9 (MMP-9) expression. Male Sprague-Dawley rats underwent renal arterial injection of vehicle or MSCs (1 × 106/rat) immediately before sham operation or induction of unilateral ureteral obstruction (UUO). The kidneys were harvested after 4 wk and analyzed for collagen I and III gene expression, collagen deposition (Masson’s trichrome), fibronectin, α-smooth muscle actin, active STAT3 (p-STAT3), MMP-9, and tissue inhibitor of matrix metalloproteinases 1 (TIMP-1) expression. In a separate arm, the STAT3 inhibitor S3I-201 (10 mg/kg) vs. vehicle was administered to rats intraperitoneally just after induction of UUO and daily for 14 days thereafter. The kidneys were harvested after 2 wk and analyzed for p-STAT3 and MMP-9 expression, and collagen and fibronectin deposition. Renal obstruction induced a significant increase in collagen, fibronectin, α-SMA, p-STAT3, MMP-9, and TIMP-1 expression while exogenously administered MSCs significantly reduced these indicators of obstruction-induced renal fibrosis. STAT3 inhibition with S3I-201 significantly reduced obstruction-induced MMP-9 expression and tubulointerstitial fibrosis. These results demonstrate that MSCs protect against obstruction-induced renal fibrosis, in part, by decreasing STAT3 activation and STAT3-dependent MMP-9 production. PMID:27760767

Alternative Splicing of STAT3 Is Affected by RNA Editing.

PubMed

Goldberg, Lior; Abutbul-Amitai, Mor; Paret, Gideon; Nevo-Caspi, Yael

2017-05-01

A-to-I RNA editing, carried out by adenosine deaminase acting on RNA (ADAR) enzymes, is an epigenetic phenomenon of posttranscriptional modifications on pre-mRNA. RNA editing in intronic sequences may influence alternative splicing of flanking exons. We have previously shown that conditions that induce editing result in elevated expression of signal transducer and activator of transcription 3 (STAT3), preferentially the alternatively-spliced STAT3β isoform. Mechanisms regulating alternative splicing of STAT3 have not been elucidated. STAT3 undergoes A-to-I RNA editing in an intron residing in proximity to the alternatively spliced exon. We hypothesized that RNA editing plays a role in regulating alternative splicing toward STAT3β. In this study we extend our observation connecting RNA editing to the preferential induction of STAT3β expression. We study the involvement of ADAR1 in STAT3 editing and reveal the connection between editing and alternative splicing of STAT3. Deferoaxamine treatment caused the induction in STAT3 RNA editing and STAT3β expression. Silencing ADAR1 caused a decrease in STAT3 editing and expression with a preferential decrease in STAT3β. Cells transfected with a mutated minigene showed preferential splicing toward the STAT3β transcript. Editing in the STAT3 intron is performed by ADAR1 and affects STAT3 alternative splicing. These results suggest that RNA editing is one of the molecular mechanisms regulating the expression of STAT3β.

STAT1:DNA sequence-dependent binding modulation by phosphorylation, protein:protein interactions and small-molecule inhibition

PubMed Central

Bonham, Andrew J.; Wenta, Nikola; Osslund, Leah M.; Prussin, Aaron J.; Vinkemeier, Uwe; Reich, Norbert O.

2013-01-01

The DNA-binding specificity and affinity of the dimeric human transcription factor (TF) STAT1, were assessed by total internal reflectance fluorescence protein-binding microarrays (TIRF-PBM) to evaluate the effects of protein phosphorylation, higher-order polymerization and small-molecule inhibition. Active, phosphorylated STAT1 showed binding preferences consistent with prior characterization, whereas unphosphorylated STAT1 showed a weak-binding preference for one-half of the GAS consensus site, consistent with recent models of STAT1 structure and function in response to phosphorylation. This altered-binding preference was further tested by use of the inhibitor LLL3, which we show to disrupt STAT1 binding in a sequence-dependent fashion. To determine if this sequence-dependence is specific to STAT1 and not a general feature of human TF biology, the TF Myc/Max was analysed and tested with the inhibitor Mycro3. Myc/Max inhibition by Mycro3 is sequence independent, suggesting that the sequence-dependent inhibition of STAT1 may be specific to this system and a useful target for future inhibitor design. PMID:23180800

Multifaceted Intervention by the Hsp90 Inhibitor Ganetespib (STA-9090) in Cancer Cells with Activated JAK/STAT Signaling

PubMed Central

Proia, David A.; Foley, Kevin P.; Korbut, Tim; Sang, Jim; Smith, Don; Bates, Richard C.; Liu, Yuan; Rosenberg, Alex F.; Zhou, Dan; Koya, Keizo; Barsoum, James; Blackman, Ronald K.

2011-01-01

There is accumulating evidence that dysregulated JAK signaling occurs in a wide variety of cancer types. In particular, mutations in JAK2 can result in the constitutive activation of STAT transcription factors and lead to oncogenic growth. JAK kinases are established Hsp90 client proteins and here we show that the novel small molecule Hsp90 inhibitor ganetespib (formerly STA-9090) exhibits potent in vitro and in vivo activity in a range of solid and hematological tumor cells that are dependent on JAK2 activity for growth and survival. Of note, ganetespib treatment results in sustained depletion of JAK2, including the constitutively active JAK2V617F mutant, with subsequent loss of STAT activity and reduced STAT-target gene expression. In contrast, treatment with the pan-JAK inhibitor P6 results in only transient effects on these processes. Further differentiating these modes of intervention, RNA and protein expression studies show that ganetespib additionally modulates cell cycle regulatory proteins, while P6 does not. The concomitant impact of ganetespib on both cell growth and cell division signaling translates to potent antitumor efficacy in mouse models of xenografts and disseminated JAK/STAT-driven leukemia. Overall, our findings support Hsp90 inhibition as a novel therapeutic approach for combating diseases dependent on JAK/STAT signaling, with the multimodal action of ganetespib demonstrating advantages over JAK-specific inhibitors. PMID:21533169

The fruits of Gleditsia sinensis Lam. inhibits adipogenesis through modulation of mitotic clonal expansion and STAT3 activation in 3T3-L1 cells.

PubMed

Lee, Ji-Hye; Go, Younghoon; Lee, Bonggi; Hwang, Youn-Hwan; Park, Kwang Il; Cho, Won-Kyung; Ma, Jin Yeul

2018-08-10

Gleditsia sinensis Lam. (G. sinensis) has been used in Oriental medicine for tumor, thrombosis, inflammation-related disease, and obesity. The pharmacological inhibitory effects of fruits of G. sinensis (GFE) on hyperlipidemia have been reported, but its inhibitory effects on adipogenesis and underlying mechanisms have not been elucidated. Herein we evaluated the anti-adipogenic effects of GFE and described the underlying mechanisms. The effects of ethanol extracts of GFE on adipocyte differentiation were examined in 3T3-L1 cells using biochemical and molecular analyses. During the differentiation of 3T3-L1 cells, GFE significantly reduced lipid accumulation and downregulated master adipogenic transcription factors, including CCAAT/enhancer-binding protein-α and peroxisome proliferator-activated receptor-γ, at mRNA and protein levels. These changes led to the suppression of several adipogenic-specific genes and proteins, including fatty acid synthase, sterol regulatory element-binding protein 1, stearoyl-CoA desaturase-1, and acetyl CoA carboxylase. However, the inhibitory effects of GFE on lipogenesis were only shown when GFE is treated in the early stage of adipogenesis within the first two days of differentiation. As a potential mechanism, during the early stages of differentiation, GFE inhibited cell proliferation by a decrease in the expression of DNA synthesis-related proteins and increased p27 expression and suppressed signal transducer and activator of transcription 3 (STAT3) activation induced in a differentiation medium. GFE inhibits lipogenesis by negative regulation of adipogenic transcription factors, which is associated with GFE-mediated cell cycle arrest and STAT3 inhibition. Copyright © 2018 Elsevier B.V. All rights reserved.

Expression of unique chimeric human papilloma virus type 16 (HPV-16) L1-L2 proteins in Pichia pastoris and Hansenula polymorpha.

PubMed

Bredell, Helba; Smith, Jacques J; Görgens, Johann F; van Zyl, Willem H

2018-04-30

Cervical cancer is ranked the fourth most common cancer in women worldwide. Despite two commercially available prophylactic vaccines, it is unaffordable for most women in developing countries. We compared the optimized expression of monomers of the unique HPV type 16 L1-L2 chimeric protein (SAF) in two yeast strains of Pichia pastoris, KM71 (Mut s ) and GS115 (Mut + ), with Hansenula polymorpha NCYC 495 to determine the preferred host in bioreactors. SAF was uniquely created by replacing the h4 helix of the HPV-16 capsid L1 protein with a L2 peptide. Two different feeding strategies in fed-batch cultures of P. pastoris Mut s were evaluated: a predetermined feed rate versus feeding based on the oxygen consumption by maintaining constant dissolved oxygen levels (DO stat). All cultures showed a significant increase in biomass when methanol was fed using the DO stat method. In P. pastoris the SAF concentrations were higher in the Mut s strains than in the Mut + strains. However, H. polymorpha produced the highest level of SAF at 132.10 mg.L -1 culture while P. pastoris Mut s only produced 23.61 mg.L -1 . H. polymorpha showed greater potential for the expression of HPV-16 L1/L2 chimeric proteins despite the track record of P. pastoris as a high level producer of heterologous proteins. This article is protected by copyright. All rights reserved.

Biologic consequences of Stat1-independent IFN signaling

PubMed Central

Gil, M. Pilar; Bohn, Erwin; O'Guin, Andrew K.; Ramana, Chilakamarti V.; Levine, Beth; Stark, George R.; Virgin, Herbert W.; Schreiber, Robert D.

2001-01-01

Although Stat1 is required for many IFN-dependent responses, recent work has shown that IFNγ functions independently of Stat1 to affect the growth of tumor cells or immortalized fibroblasts. We now demonstrate that both IFNγ and IFNα/β regulate proliferative responses in cells of the mononuclear phagocyte lineage derived from Stat1-null mice. Using both representational difference analysis and gene arrays, we show that IFNγ exerts its Stat1-independent actions on mononuclear phagocytes by regulating the expression of many genes. This result was confirmed by monitoring changes in expression and function of the corresponding gene products. Regulation of the expression of these genes requires the IFNγ receptor and Jak1. The physiologic relevance of IFN-dependent, Stat1-independent signaling was demonstrated by monitoring antiviral responses in Stat1-null mice. Thus, the IFN receptors engage alternative Stat1-independent signaling pathways that have important physiological consequences. PMID:11390995

Myeloid-cell protein tyrosine phosphatase-1B deficiency in mice protects against high-fat diet and lipopolysaccharide-induced inflammation, hyperinsulinemia, and endotoxemia through an IL-10 STAT3-dependent mechanism.

PubMed

Grant, Louise; Shearer, Kirsty D; Czopek, Alicja; Lees, Emma K; Owen, Carl; Agouni, Abdelali; Workman, James; Martin-Granados, Cristina; Forrester, John V; Wilson, Heather M; Mody, Nimesh; Delibegovic, Mirela

2014-02-01

Protein tyrosine phosphatase-1B (PTP1B) negatively regulates insulin and leptin signaling, rendering it an attractive drug target for treatment of obesity-induced insulin resistance. However, some studies suggest caution when targeting macrophage PTP1B, due to its potential anti-inflammatory role. We assessed the role of macrophage PTP1B in inflammation and whole-body metabolism using myeloid-cell (LysM) PTP1B knockout mice (LysM PTP1B). LysM PTP1B mice were protected against lipopolysaccharide (LPS)-induced endotoxemia and hepatic damage associated with decreased proinflammatory cytokine secretion in vivo. In vitro, LPS-treated LysM PTP1B bone marrow-derived macrophages (BMDMs) displayed increased interleukin (IL)-10 mRNA expression, with a concomitant decrease in TNF-α mRNA levels. These anti-inflammatory effects were associated with increased LPS- and IL-10-induced STAT3 phosphorylation in LysM PTP1B BMDMs. Chronic inflammation induced by high-fat (HF) feeding led to equally beneficial effects of macrophage PTP1B deficiency; LysM PTP1B mice exhibited improved glucose and insulin tolerance, protection against LPS-induced hyperinsulinemia, decreased macrophage infiltration into adipose tissue, and decreased liver damage. HF-fed LysM PTP1B mice had increased basal and LPS-induced IL-10 levels, associated with elevated STAT3 phosphorylation in splenic cells, IL-10 mRNA expression, and expansion of cells expressing myeloid markers. These increased IL-10 levels negatively correlated with circulating insulin and alanine transferase levels. Our studies implicate myeloid PTP1B in negative regulation of STAT3/IL-10-mediated signaling, highlighting its inhibition as a potential anti-inflammatory and antidiabetic target in obesity.

Zinc improves learning and memory abilities of fetal growth restriction rats and promotes trophoblast cell invasion and migration via enhancing STAT3-MMP-2/9 axis activity.

PubMed

Zong, Lu; Wei, Xiaohua; Gou, Wenli; Huang, Pu; Lv, Ye

2017-12-29

Fetal growth restriction (FGR) is a well-known risk factor for cognitive dysfunction, especially for learning and memory abilities. However, knowledge about prevention and treatment methods of learning and memory abilities of fetal are limit. Here, Morris water maze and passive avoidance tests showed zinc supplementation could protect the impairment of the learning and memory abilities caused by FGR. As accumulating evidence suggested that insufficiency of placental trophoblast cell invasion was closely related to FGR fetal neurodevelopmental dysplasia, we further explored the relationship between zinc supplementation during pregnancy and placental trophoblast. Microarray identified 346 differently expressed genes in placental tissues with and without zinc supplementation, and GO and KEGG analyses showed these differently expressed genes were highly enriched in cell invasion and migration and STAT3 pathway. Protein-protein interaction(PPI) analysis found that STAT3 interacted with matrix metalloproteinase-2/9 (MMP-2/9). In vivo , western blot results authenticated that the expression levels of phospho-STAT3, STAT3, MMP-2 and MMP-9 were up-regulated in placental tissues after zinc treatment. To validate whether zinc could promotes trophoblast cell invasion and migration via enhancing STAT3-MMP-2/9 activity. In vitro , Transwell assay was performed, and we observed that abilities of invasion and migration were obviously increased in zinc treated trophoblast cells. And phospho-STAT3, STAT3, MMP-2 and MMP-9 expression levels were correspondingly increased in zinc treated trophoblast cells, which were dose-dependent. Moreover, gain-of-function and loss-of-function of STAT3 confirmed that zinc promotes cell invasion and migration via regulating STAT3 mediated up-regulation of MMP-2/9 activity. We propose that activation of MMP-2/9 mediated by STAT3 may contribute to invasion and migration of trophoblast cells, which improved neurodevelopmental impairment of FGR rats probably via contributing to placental development. Our findings are the first to show a possible mechanism of reversing neurodevelopmental impairment of FGR rats by zinc supplementation, holding promise for the development of novel therapeutic modalities for learning and memory abilities impairment caused by FGR.

Rosmanol potently inhibits lipopolysaccharide-induced iNOS and COX-2 expression through downregulating MAPK, NF-kappaB, STAT3 and C/EBP signaling pathways.

PubMed

Lai, Ching-Shu; Lee, Jong Hun; Ho, Chi-Tang; Liu, Cheng Bin; Wang, Ju-Ming; Wang, Ying-Jan; Pan, Min-Hsiung

2009-11-25

Rosmanol is a natural polyphenol from the herb rosemary (Rosmarinus officinalis L.) with high antioxidant activity. In this study, we investigated the inhibitory effects of rosmanol on the induction of NO synthase (NOS) and COX-2 in RAW 264.7 cells induced by lipopolysaccharide (LPS). Rosmanol markedly inhibited LPS-stimulated iNOS and COX-2 protein and gene expression, as well as the downstream products, NO and PGE2. Treatment with rosmanol also reduced translocation of the nuclear factor-kappaB (NF-kappaB) subunits by prevention of the degradation and phosphorylation of inhibitor kappaB (IkappaB). Western blot analysis showed that rosmanol significantly inhibited translocation and phosphorylation of NF-kappaB, signal transducer and activator of transcription-3 (STAT3), and the protein expression of C/EBPbeta and C/EBPdelta. We also found that rosmanol suppressed LPS-induced phosphorylation of ERK1/2, p38 mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K)/Akt signaling. Our results demonstrate that rosmanol downregulates inflammatory iNOS and COX-2 gene expression by inhibiting the activation of NF-kappaB and STAT3 through interfering with the activation of PI3K/Akt and MAPK signaling. Taken together, rosmanol might contribute to the potent anti-inflammatory effect of rosemary and may have potential to be developed into an effective anti-inflammatory agent.

Morbillivirus Control of the Interferon Response: Relevance of STAT2 and mda5 but Not STAT1 for Canine Distemper Virus Virulence in Ferrets

PubMed Central

Svitek, Nicholas; Gerhauser, Ingo; Goncalves, Christophe; Grabski, Elena; Döring, Marius; Kalinke, Ulrich; Anderson, Danielle E.; Cattaneo, Roberto

2014-01-01

ABSTRACT The V proteins of paramyxoviruses control the innate immune response. In particular, the V protein of the genus Morbillivirus interferes with the signal transducer and activator of transcription 1 (STAT1), STAT2, and melanoma differentiation-associated protein 5 (mda5) signaling pathways. To characterize the contributions of these pathways to canine distemper virus (CDV) pathogenesis, we took advantage of the knowledge about the mechanisms of interaction between the measles virus V protein with these key regulators of innate immunity. We generated recombinant CDVs with V proteins unable to properly interact with STAT1, STAT2, or mda5. A virus with combined STAT2 and mda5 deficiencies was also generated, and available wild-type and V-protein-knockout viruses were used as controls. Ferrets infected with wild-type and STAT1-blind viruses developed severe leukopenia and loss of lymphocyte proliferation activity and succumbed to the disease within 14 days. In contrast, animals infected with viruses with STAT2 or mda5 defect or both STAT2 and mda5 defects developed a mild self-limiting disease similar to that associated with the V-knockout virus. This study demonstrates the importance of interference with STAT2 and mda5 signaling for CDV immune evasion and provides a starting point for the development of morbillivirus vectors with reduced immunosuppressive properties. IMPORTANCE The V proteins of paramyxoviruses interfere with the recognition of the virus by the immune system of the host. For morbilliviruses, the V protein is known to interact with the signal transducer and activator of transcription 1 (STAT1) and STAT2 and the melanoma differentiation-associated protein 5 (mda5), which are involved in interferon signaling. Here, we examined the contribution of each of these signaling pathways to the pathogenesis of the carnivore morbillivirus canine distemper virus. Using viruses selectively unable to interfere with the respective signaling pathway to infect ferrets, we found that inhibition of STAT2 and mda5 signaling was critical for lethal disease. Our findings provide new insights in the mechanisms of morbillivirus immune evasion and may lead to the development of new vaccines and oncolytic vectors. PMID:24371065

Morbillivirus control of the interferon response: relevance of STAT2 and mda5 but not STAT1 for canine distemper virus virulence in ferrets.

PubMed

Svitek, Nicholas; Gerhauser, Ingo; Goncalves, Christophe; Grabski, Elena; Döring, Marius; Kalinke, Ulrich; Anderson, Danielle E; Cattaneo, Roberto; von Messling, Veronika

2014-03-01

The V proteins of paramyxoviruses control the innate immune response. In particular, the V protein of the genus Morbillivirus interferes with the signal transducer and activator of transcription 1 (STAT1), STAT2, and melanoma differentiation-associated protein 5 (mda5) signaling pathways. To characterize the contributions of these pathways to canine distemper virus (CDV) pathogenesis, we took advantage of the knowledge about the mechanisms of interaction between the measles virus V protein with these key regulators of innate immunity. We generated recombinant CDVs with V proteins unable to properly interact with STAT1, STAT2, or mda5. A virus with combined STAT2 and mda5 deficiencies was also generated, and available wild-type and V-protein-knockout viruses were used as controls. Ferrets infected with wild-type and STAT1-blind viruses developed severe leukopenia and loss of lymphocyte proliferation activity and succumbed to the disease within 14 days. In contrast, animals infected with viruses with STAT2 or mda5 defect or both STAT2 and mda5 defects developed a mild self-limiting disease similar to that associated with the V-knockout virus. This study demonstrates the importance of interference with STAT2 and mda5 signaling for CDV immune evasion and provides a starting point for the development of morbillivirus vectors with reduced immunosuppressive properties. The V proteins of paramyxoviruses interfere with the recognition of the virus by the immune system of the host. For morbilliviruses, the V protein is known to interact with the signal transducer and activator of transcription 1 (STAT1) and STAT2 and the melanoma differentiation-associated protein 5 (mda5), which are involved in interferon signaling. Here, we examined the contribution of each of these signaling pathways to the pathogenesis of the carnivore morbillivirus canine distemper virus. Using viruses selectively unable to interfere with the respective signaling pathway to infect ferrets, we found that inhibition of STAT2 and mda5 signaling was critical for lethal disease. Our findings provide new insights in the mechanisms of morbillivirus immune evasion and may lead to the development of new vaccines and oncolytic vectors.

Defective interleukin-4/Stat6 activity correlates with increased constitutive expression of negative regulators SOCS-3, SOCS-7, and CISH in colon cancer cells.

PubMed

Liu, Xiao Hong; Xu, Shuang Bing; Yuan, Jia; Li, Ben Hui; Zhang, Yan; Yuan, Qin; Li, Pin Dong; Li, Feng; Zhang, Wen Jie

2009-12-01

Interleukin-4 (IL-4)-induced Stat6 activities (phenotypes) vary among human cancer cells, of which the HT-29 cell line carries an active Stat6(high) phenotype, while Caco-2 carries a defective Stat6(null) phenotype, respectively. Cancer cells with Stat6(high) show resistance to apoptosis and exaggerated metastasis, suggesting the clinical significance of Stat6 phenotypes. We previously showed that Stat6(high) HT-29 cells exhibited low constitutive expression of Stat6-negative regulators SOCS-1 and SHP-1 because of gene hypermethylation. This study further examined the constitutive expression of other closely related SOCS family numbers including SOCS-3, SOCS-5, SOCS-7, and CISH using RT-PCR. Similar to SOCS-1 and SHP-1, Stat6(high) HT-29 cells expressed low constitutive mRNA of SOCS-3, SOCS-7, and CISH than Stat6(null) Caco-2 cells. Interestingly, DNA demethylation using 5-aza-2'-deoxycytidine in HT-29 cells up-regulated mRNA expression of the above genes, indicating a hypermethylation status, which was confirmed by methylation-specific sequencing in selected SOCS-3 gene. Furthermore, defective Stat6(null) Caco-2 exhibited impaired phosphorylation of Stat6 after IL-4 stimulation by flow cytometry, in keeping with the notion of an over-performed negative regulation. The findings that IL-4/Stat6 phenotypes show differential expression of multiple negative regulators suggest a model that a collective force of powerful negative regulators, directly and indirectly, acts on Stat6 activation, which may result in differential Stat6 phenotypes.

High expression of survivin and down-regulation of Stat-3 characterize the feto-maternal interface in failing murine pregnancies during the implantation period.

PubMed

Garcia, Mariana G; Tirado-Gonzalez, Irene; Handjiski, Bori; Tometten, Mareike; Orsal, Arif S; Hajos, Silvia E; Fernández, Nelson; Arck, Petra C; Blois, Sandra M

2007-07-01

The materno-fetal interface has for long been considered as an immune privileged biological site and thus understanding the mechanisms underlying fetal survival have been the focus of intense research. In adults, survivin and Stat-3 proteins are involved in tolerance as well as the induction of apoptosis. However, the role of these molecules in pregnancy and development has not been addressed. We have evaluated the expression of survivin and Stat-3 in allogeneic mouse models of low abortions (CBA/J x Balb/c), abortion prone (CBA/J x DBA/2J) and stress-triggered abortions from DBA/2J-mated CBA/J mice. We show that survivin is over-expressed in abortion-prone mating on gestation day 7.5. This effect was also found in stress-exposed mice, whereas expression was low in normal pregnancy mice. The phosphorylated Stat-3 (p-Stat-3) was down regulated in high abortion mating compared with low abortion mating, CBA/J x Balb/c. The level of apoptosis was similar in the three groups studied. Our results suggest that high expression of survivin and low expression of p-Stat-3 are involved in pregnancy loss in mice.

Titrated extract of Centella asiatica increases hair inductive property through inhibition of STAT signaling pathway in three-dimensional spheroid cultured human dermal papilla cells.

PubMed

Choi, Yeong Min; An, Sungkwan; Lee, Junwoo; Lee, Jae Ho; Lee, Jae Nam; Kim, Young Sam; Ahn, Kyu Joong; An, In-Sook; Bae, Seunghee

2017-12-01

Dermal papilla (DP) is a pivotal part of hair follicle, and the smaller size of the DP is related with the hair loss. In this study, we investigated the effect of titrated extract of Centella asiatica (TECA) on hair growth inductive property on 3D spheroid cultured human DP cells (HDP cells). Significantly increased effect of TECA on cell viability was only shown in 3D sphered HPD cells, not in 2D cultured HDP cells. Also, TECA treatment increased the sphere size of HDP cells. The luciferase activity of STAT reporter genes and the expression of STAT-targeted genes, SOCS1 and SOCS3, were significantly decreased. Also, TECA treatment increased the expression of the hair growth-related signature genes in 3D sphered HDP cells. Furthermore, TECA led to downregulation of the level of phosphorylated STAT proteins in 3D sphered HDP cells. Overall, TECA activates the potential of hair inductive capacity in HDP cells.

MIR506 induces autophagy-related cell death in pancreatic cancer cells by targeting the STAT3 pathway.

PubMed

Sun, Longhao; Hu, Limei; Cogdell, David; Lu, Li; Gao, Chao; Tian, Weijun; Zhang, Zhixiang; Kang, Ya'an; Fleming, Jason B; Zhang, Wei

2017-04-03

Pancreatic ductal adenocarcinoma (PDAC) is the most aggressive and lethal cancer. The role of autophagy in the pathobiology of PDAC is intricate, with opposing functions manifested in different cellular contexts. MIR506 functions as a tumor suppressor in many cancer types through the regulation of multiple pathways. In this study, we hypothesized that MIR506 exerted a tumor suppression function in PDAC by inducing autophagy-related cell death. Our results provided evidence that downregulation of MIR506 expression was associated with disease progression in human PDAC. MIR506 triggered autophagic flux in PDAC cells, which led to autophagy-related cell death through direct targeting of the STAT3 (signal transducer and activator of transcription 3)-BCL2-BECN1 axis. Silencing and inhibiting STAT3 recapitulated the effects of MIR506, whereas forced expression of STAT3 abrogated the effects of MIR506. We propose that the apoptosis-inhibitory protein BCL2, which also inhibits induction of autophagy by blocking BECN1, was inhibited by MIR506 through targeting STAT3, thus augmenting BECN1 and promoting autophagy-related cell death. Silencing BECN1 and overexpression of BCL2 abrogated the effects of MIR506. These findings expand the known mechanisms of MIR506-mediated tumor suppression to activation of autophagy-related cell death and suggest a strategy for using MIR506 as an anti-STAT3 approach to PDAC treatment.

Sevoflurane postconditioning protects the myocardium against ischemia/reperfusion injury via activation of the JAK2–STAT3 pathway

PubMed Central

Wu, Jianjiang; Yu, Jin; Xie, Peng; Maimaitili, Yiliyaer; Wang, Jiang; Yang, Long; Ma, Haiping; Zhang, Xing; Yang, Yining

2017-01-01

Background Sevoflurane postconditioning (S-post) has similar cardioprotective effects as ischemic preconditioning. However, the underlying mechanism of S-post has not been fully elucidated. Janus kinase signaling transduction/transcription activator (JAK2–STAT3) plays an important role in cardioprotection. The purpose of this study was to determine whether the cardioprotective effects of S-post are associated with activation of the JAK2–STAT3 signal pathway. Methods An adult male Sprague–Dawley (SD) rat model of myocardial ischemia/reperfusion (I/R) injury was established using the Langendorff isolated heart perfusion apparatus. At the beginning of reperfusion, 2.4% sevoflurane alone or in combination with AG490 (a JAK2 selective inhibitor) was used as a postconditioning treatment. The cardiac function indicators, myocardial infarct size, lactic dehydrogenase (LDH) release, mitochondrial ultrastructure, mitochondrial reactive oxygen species (ROS) generation rates, ATP content, protein expression of p-JAK, p-STAT3, Bcl-2 and Bax were measured. Results Compared with the I/R group, S-post significantly increased the expression of p-JAK, p-STAT3 and Bcl-2 and reduced the protein expression of Bax, which markedly decreased the myocardial infarction areas, improved the cardiac function indicators and the mitochondrial ultrastructure, decreased the mitochondrial ROS and increased the ATP content. However, the cardioprotective effects of S-post were abolished by treatment with a JAK2 selective inhibitor (p < 0.05). Conclusion This study demonstrates that the cardioprotective effects of S-post are associated with the activation of JAK2–STAT3. The mechanism may be related to an increased expression of p-JAK2 and p-STAT3 after S-post, which reduced mitochondrial ROS generation and increased mitochondrial ATP content, thereby reducing apoptosis and myocardial infarct size. PMID:28392989

Protein kinase C epsilon, which sensitizes skin to sun's UV radiation-induced cutaneous damage and development of squamous cell carcinomas, associates with Stat3.

PubMed

Aziz, Moammir H; Manoharan, Herbert T; Verma, Ajit K

2007-02-01

Chronic exposure to UV radiation (UVR) is the major etiologic factor in the development of human skin cancers including squamous cell carcinoma (SCC). We have shown that protein kinase C(epsilon) (PKC(epsilon)), a Ca(2+)-independent, phospholipid-dependent serine/threonine kinase, is an endogenous photosensitizer. PKC(epsilon) is among the six isoforms (alpha, delta, epsilon, eta, mu, and zeta) expressed in both mouse and human skin. PKC(epsilon) transgenic mice, which overexpress PKC(epsilon) in the basal epidermal cells and cells of the hair follicle, are highly sensitive to UVR-induced cutaneous damage and development of SCC. We now present that PKC(epsilon)-overexpressing, but not PKC(delta)-overexpressing, transgenic mice, when exposed to a single (4 kJ/m(2)) or repeated (four doses, 2 kJ/m(2)/dose, thrice weekly) UVR, emitted by Kodacel-filtered FS-40 sun lamps, elicit constitutive phosphorylation of signal transducers and activators of transcription 3 (Stat3) at both Tyr705 and Ser727 residues. UVR-induced phosphorylation of Stat3 accompanied increased expression of Stat3-regulated genes (c-myc, cyclin D1, cdc25A, and COX-2). In reciprocal immunoprecipitation/blotting experiments, phosphorylated Stat3 co-immunoprecipitated with PKC(epsilon). As observed in vivo using PKC(epsilon) knockout mice and in vitro in an immunocomplex kinase assay, PKC(epsilon) phosphorylated Stat3 at Ser727 residue. These results indicate for the first time that (a) PKC(epsilon) is a Stat3Ser727 kinase; (b) PKC(epsilon)-mediated phosphorylation of StatSer727 may be essential for transcriptional activity of Stat3; and (c) UVR-induced phosphorylation of Ser727 may be a key component of the mechanism by which PKC(epsilon) imparts sensitivity to UVR-induced development of SCC.

Tetrandrine has anti-adipogenic effect on 3T3-L1 preadipocytes through the reduced expression and/or phosphorylation levels of C/EBP-α, PPAR-γ, FAS, perilipin A, and STAT-3

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jang, Byeong-Churl, E-mail: jangbc123@gw.kmu.ac.kr

Tetrandrine is a bisbenzylisoquinoline alkaloid isolated from the roots of Stephania tetrandra S. Moore and has been shown to possess anti-inflammatory and anti-cancerous activities. In this study, the effect of tetrandrine on adipogenesis in 3T3-L1 preadipocytes was investigated. Tetrandrine at 10 μM concentration strongly inhibited lipid accumulation and triglyceride (TG) synthesis during the differentiation of 3T3-L1 preadipocytes into adipocytes. On mechanistic levels, tetrandrine reduced not only the expressions of CCAAT/enhancer-binding protein-α (C/EBP-α), peroxisome proliferator-activated receptor-γ (PPAR-γ), fatty acid synthase (FAS), and perilipin A but also the phosphorylation levels of signal transducer and activator of transcription-3 (STAT-3) during 3T3-L1 adipocyte differentiation. Tetrandrinemore » also reduced the mRNA expression of leptin, but not adiponectin, during 3T3-L1 adipocyte differentiation. Collectively, these findings show that tetrandrine has strong anti-adipogenic effect on 3T3-L1 preadipocytes and the effect is largely attributable to the reduced expression and/or phosphorylation levels of C/EBP-α, PPAR-γ, FAS, perilipin A, and STAT-3. - Highlights: • Tetrandrine, a bisbenzylisoquinoline alkaloid, inhibits adipogenesis. • Tetrandrine inhibits C/EBP-α, PPAR-γ, FAS, perilipin A, and STAT-3 in 3T3-L1 adipocytes. • Tetrandrine reduces leptin, but not adiponectin, expression in 3T3-L1 adipocytes. • Tetrandrine may thus have therapeutic potential against obesity.« less

Interleukin-4 upregulates RhoA protein via an activation of STAT6 in cultured human bronchial smooth muscle cells.

PubMed

Chiba, Yoshihiko; Todoroki, Michiko; Misawa, Miwa

2010-02-01

Interleukin-4 (IL-4) is believed to play a role in allergic bronchial asthma, and has been suggested to cause hyperresponsiveness of airway smooth muscle. In the present study, the effects of IL-4 on the expression of RhoA protein, a monomeric GTP-binding protein that contributes to the contraction of smooth muscle, were determined in cultured human bronchial smooth muscle cells (hBSMCs). Incubation of hBSMCs with IL-4 (100ng/mL) caused a distinct phosphorylation of signal transducer and activator of transcription 6 (STAT6), a major signal transducer activated by IL-4, indicating that IL-4 is capable of activating signal transduction in the hBSMCs directly. IL-4 also caused a significant increase in the expression level of RhoA protein: the peak of the upregulation of RhoA protein was observed at 12-24h after the IL-4 treatment. Both the phosphorylation of STAT6 and the upregulation of RhoA protein induced by IL-4 were inhibited by the co-incubation with AS1517499, a selective inhibitor of STAT6, in a concentration-dependent fashion. These findings suggest that IL-4 is capable of inducing an upregulation of RhoA via an activation of STAT6 in cultured hBSMCs. The RhoA upregulation induced by IL-4, one of the Th2 cytokines upregulated in the airways of allergic bronchial asthmatics, might result in an augmentation of bronchial smooth muscle contractility, that is one of the causes of airway hyperresponsiveness. Copyright 2009 Elsevier Ltd. All rights reserved.

Essential oil of Artemisia argyi suppresses inflammatory responses by inhibiting JAK/STATs activation.

PubMed

Chen, Lin-Lin; Zhang, Hao-Jun; Chao, Jung; Liu, Jun-Feng

2017-05-23

Artemisia argyi is a herbal medicine traditionally used in Asia for the treatment of bronchitis, dermatitis and arthritis. Recent studies revealed the anti-inflammatory effect of essential oil in this plant. However, the mechanisms underlying the therapeutic potential have not been well elucidated. The present study is aimed to verify its anti-inflammatory effect and investigate the probable mechanisms. The essential oil from Artemisia argyi (AAEO) was initially tested against LPS-induced production of inflammatory mediators and cytokines in RAW264.7 macrophages. Protein and mRNA expressions of iNOS and COX-2 were determined by Western blotting and RT-PCR analysis, respectively. The effects on the activation of MAPK/NF-κB/AP-1 and JAK/STATs pathway were also investigated by western blot. Meanwhile, in vivo anti-inflammatory effect was examined by histologic and immunohistochemical analysis in TPA-induced mouse ear edema model. The results of in vitro experiments showed that AAEO dose-dependently suppressed the release of pro-inflammatory mediators (NO, PGE 2 and ROS) and cytokines (TNF-α, IL-6, IFN-β and MCP-1) in LPS-induced RAW264.7 macrophages. It down-regulated iNOS and COX-2 protein and mRNA expression but did not affect the activity of these two enzymes. AAEO significantly inhibited the phosphorylation of JAK2 and STAT1/3, but not the activation of MAPK and NF-κB cascades. In animal model, oral administration of AAEO significantly attenuated TPA-induced mouse ear edema and decreased the protein level of COX-2. AAEO suppresses inflammatory responses via down-regulation of the JAK/STATs signaling and ROS scavenging, which could contribute, at least in part, to the anti-inflammatory effect of AAEO. Copyright © 2017 Elsevier Ireland Ltd. All rights reserved.

Differential Inflammatory-Response Kinetics of Human Keratinocytes upon Cytosolic RNA- and DNA-Fragment Induction.

PubMed

Danis, Judit; Janovák, Luca; Gubán, Barbara; Göblös, Anikó; Szabó, Kornélia; Kemény, Lajos; Bata-Csörgő, Zsuzsanna; Széll, Márta

2018-03-08

Keratinocytes are non-professional immune cells contributing actively to innate immune responses partially by reacting to a wide range of molecular patterns by activating pattern recognition receptors. Cytosolic nucleotide fragments as pathogen- or self-derived trigger factors are activating inflammasomes and inducing anti-viral signal transduction pathways as well as inducing expression of inflammatory cytokines. We aimed to compare the induced inflammatory reactions in three keratinocyte cell types-normal human epidermal keratinocytes, the HaCaT cell line and the HPV-KER cell line-upon exposure to the synthetic RNA and DNA analogues poly(I:C) and poly(dA:dT) to reveal the underlying signaling events. Both agents induced the expression of interleukin-6 and tumor necrosis factor α in all cell types; however, notable kinetic and expression level differences were found. Western blot analysis revealed rapid activation of the nuclear factor κB (NF-κB), mitogen activated protein kinase and signal transducers of activator of transcription (STAT) signal transduction pathways in keratinocytes upon poly(I:C) treatment, while poly(dA:dT) induced slower activation. Inhibition of NF-κB, p38, STAT-1 and STAT-3 signaling resulted in decreased cytokine expression, whereas inhibition of mitogen-activated protein kinase kinase 1/2 (MEK1/2) signaling showed a negative feedback role in both poly(I:C)- and poly(dA:dT)-induced cytokine expression. Based on our in vitro results nucleotide fragments are able to induce inflammatory reactions in keratinocytes, but with different rate and kinetics of cytokine expression, explained by faster activation of signaling routes by poly(I:C) than poly(dA:dT).

Differential Inflammatory-Response Kinetics of Human Keratinocytes upon Cytosolic RNA- and DNA-Fragment Induction

PubMed Central

Danis, Judit; Janovák, Luca; Gubán, Barbara; Göblös, Anikó; Szabó, Kornélia; Bata-Csörgő, Zsuzsanna; Széll, Márta

2018-01-01

Keratinocytes are non-professional immune cells contributing actively to innate immune responses partially by reacting to a wide range of molecular patterns by activating pattern recognition receptors. Cytosolic nucleotide fragments as pathogen- or self-derived trigger factors are activating inflammasomes and inducing anti-viral signal transduction pathways as well as inducing expression of inflammatory cytokines. We aimed to compare the induced inflammatory reactions in three keratinocyte cell types—normal human epidermal keratinocytes, the HaCaT cell line and the HPV-KER cell line—upon exposure to the synthetic RNA and DNA analogues poly(I:C) and poly(dA:dT) to reveal the underlying signaling events. Both agents induced the expression of interleukin-6 and tumor necrosis factor α in all cell types; however, notable kinetic and expression level differences were found. Western blot analysis revealed rapid activation of the nuclear factor κB (NF-κB), mitogen activated protein kinase and signal transducers of activator of transcription (STAT) signal transduction pathways in keratinocytes upon poly(I:C) treatment, while poly(dA:dT) induced slower activation. Inhibition of NF-κB, p38, STAT-1 and STAT-3 signaling resulted in decreased cytokine expression, whereas inhibition of mitogen-activated protein kinase kinase 1/2 (MEK1/2) signaling showed a negative feedback role in both poly(I:C)- and poly(dA:dT)-induced cytokine expression. Based on our in vitro results nucleotide fragments are able to induce inflammatory reactions in keratinocytes, but with different rate and kinetics of cytokine expression, explained by faster activation of signaling routes by poly(I:C) than poly(dA:dT). PMID:29518010

Mesenchymal stem cells protect against obstruction-induced renal fibrosis by decreasing STAT3 activation and STAT3-dependent MMP-9 production.

PubMed

Matsui, Futoshi; Babitz, Stephen A; Rhee, Audrey; Hile, Karen L; Zhang, Hongji; Meldrum, Kirstan K

2017-01-01

STAT3 is a transcription factor implicated in renal fibrotic injury, but the role of STAT3 in mesenchymal stem cell (MSC)-induced renoprotection during renal fibrosis remains unknown. We hypothesized that MSCs protect against obstruction-induced renal fibrosis by downregulating STAT3 activation and STAT3-induced matrix metalloproteinase-9 (MMP-9) expression. Male Sprague-Dawley rats underwent renal arterial injection of vehicle or MSCs (1 × 10 6 /rat) immediately before sham operation or induction of unilateral ureteral obstruction (UUO). The kidneys were harvested after 4 wk and analyzed for collagen I and III gene expression, collagen deposition (Masson's trichrome), fibronectin, α-smooth muscle actin, active STAT3 (p-STAT3), MMP-9, and tissue inhibitor of matrix metalloproteinases 1 (TIMP-1) expression. In a separate arm, the STAT3 inhibitor S3I-201 (10 mg/kg) vs. vehicle was administered to rats intraperitoneally just after induction of UUO and daily for 14 days thereafter. The kidneys were harvested after 2 wk and analyzed for p-STAT3 and MMP-9 expression, and collagen and fibronectin deposition. Renal obstruction induced a significant increase in collagen, fibronectin, α-SMA, p-STAT3, MMP-9, and TIMP-1 expression while exogenously administered MSCs significantly reduced these indicators of obstruction-induced renal fibrosis. STAT3 inhibition with S3I-201 significantly reduced obstruction-induced MMP-9 expression and tubulointerstitial fibrosis. These results demonstrate that MSCs protect against obstruction-induced renal fibrosis, in part, by decreasing STAT3 activation and STAT3-dependent MMP-9 production. Copyright © 2017 the American Physiological Society.

Feed-Forward Reciprocal Activation of PAFR and STAT3 Regulates Epithelial-Mesenchymal Transition in Non-Small Cell Lung Cancer.

PubMed

Chen, Jie; Lan, Tian; Zhang, Weimin; Dong, Lijia; Kang, Nan; Zhang, Shumin; Fu, Ming; Liu, Bing; Liu, Kangtai; Zhan, Qimin

2015-10-01

Platelet-activating factor receptor (PAFR), a G-protein-coupled receptor, has been implicated in tumorigenesis, but its contributions to metastatic progression have not been investigated. Here, we show that PAFR is overexpressed in non-small cell lung cancer (NSCLC) as well as in breast, colorectal, and gastric carcinomas. Expression of PAFR correlates closely with clinical stages, survival time, and distant metastasis. In human NSCLC cells, activation of the PAF/PAFR signaling axis accentuated malignant character, including by stimulating epithelial-mesenchymal transition (EMT). In contrast, silencing PAFR in aggressive NSCLC cells inhibited these effects. Mechanistic investigations showed that PAFR stimulated EMT by activating STAT3 via upregulation of G-protein-dependent SRC or JAK2 kinase activity. Notably, STAT3 transcriptionally elevated PAFR expression. Thus, activation of PAFR in NSCLC cells initiated a forward feedback loop responsible for mediating the aggressive malignant character of NSCLC cells in vitro and in vivo. Reinforcing this reciprocal activation loop, PAF/PAFR signaling also upregulated IL6 expression and thereby STAT3 activation. Overall, our results elucidated an important role for PAFR dysregulation in the pathogenicity of NSCLC and unraveled a forward feedback loop between PAFR and STAT3 that acts to drive the malignant progression of NSCLC. ©2015 American Association for Cancer Research.

Activation of Peroxisome Proliferator–Activated Receptor-β/-δ (PPAR-β/-δ) Ameliorates Insulin Signaling and Reduces SOCS3 Levels by Inhibiting STAT3 in Interleukin-6–Stimulated Adipocytes

PubMed Central

Serrano-Marco, Lucía; Rodríguez-Calvo, Ricardo; El Kochairi, Ilhem; Palomer, Xavier; Michalik, Liliane; Wahli, Walter; Vázquez-Carrera, Manuel

2011-01-01

OBJECTIVE It has been suggested that interleukin (IL)-6 is one of the mediators linking obesity-derived chronic inflammation with insulin resistance through activation of STAT3, with subsequent upregulation of suppressor of cytokine signaling 3 (SOCS3). We evaluated whether peroxisome proliferator–activated receptor (PPAR)-β/-δ prevented activation of the IL-6-STAT3-SOCS3 pathway and insulin resistance in adipocytes. RESEARCH DESIGN AND METHODS Adipocytes and white adipose tissue from wild-type and PPAR-β/-δ-null mice were used to evaluate the effect of PPAR-β/-δ on the IL-6-STAT3-SOCS3 pathway. RESULTS First, we observed that the PPAR-β/-δ agonist GW501516 prevented both IL-6–dependent reduction in insulin-stimulated Akt phosphorylation and glucose uptake in adipocytes. In addition, this drug treatment abolished IL-6–induced SOCS3 expression in differentiated 3T3-L1 adipocytes. This effect was associated with the capacity of the drug to prevent IL-6–induced STAT3 phosphorylation on Tyr705 and Ser727 residues in vitro and in vivo. Moreover, GW501516 prevented IL-6–dependent induction of extracellular signal–related kinase (ERK)1/2, a serine-threonine-protein kinase involved in serine STAT3 phosphorylation. Furthermore, in white adipose tissue from PPAR-β/-δ–null mice, STAT3 phosphorylation (Tyr705 and Ser727), STAT3 DNA-binding activity, and SOCS3 protein levels were higher than in wild-type mice. Several steps in STAT3 activation require its association with heat shock protein 90 (Hsp90), which was prevented by GW501516 as revealed in immunoprecipitation studies. Consistent with this finding, the STAT3-Hsp90 association was enhanced in white adipose tissue from PPAR-β/-δ–null mice compared with wild-type mice. CONCLUSIONS Collectively, our findings indicate that PPAR-β/-δ activation prevents IL-6–induced STAT3 activation by inhibiting ERK1/2 and preventing the STAT3-Hsp90 association, an effect that may contribute to the prevention of cytokine-induced insulin resistance in adipocytes. PMID:21617181

MAPK Regulation of IL-4/-13 Receptors Contributes to the Synergistic Increase in CCL11/Eotaxin-1 in Response to TGF-β1 and IL-13 in Human Airway Fibroblasts

PubMed Central

Zhou, Xiuxia; Hu, Haizhen; Balzar, Silvana; Trudeau, John B.; Wenzel, Sally E.

2012-01-01

CCL11/eotaxin-1 is a potent eosinophilic CC chemokine expressed by primary human fibroblasts. The combination of TGF-β1 and IL-13 synergistically increases CCL11 expression, but the mechanisms behind the synergy are unclear. To address this, human airway fibroblast cultures from normal and asthmatic subjects were exposed to IL-13 alone or TGF-β1 plus IL-13. Transcriptional (nuclear run-on) and post-transcriptional (mRNA stability) assays confirmed that transcriptional regulation is critical for synergistic expression of CCL11. TGF-β1 plus IL-13 synergistically increased STAT-6 phosphorylation, nuclear translocation and binding to the CCL11 promoter as compared to IL-13 alone. STAT-6 siRNA significantly knocked down both STAT-6 mRNA expression and phosphorylation, and inhibited CCL11 mRNA and protein expression. Regulation of the IL-4 receptor α (IL-4Rα) complex by TGF-β1 augmented IL-13 signaling by dampening IL-13 receptor α2 (IL-13Rα2) expression, overcoming IL-13's autoregulation of its pathway and enhancing the expression of CCL11. Our data suggest that TGF-β1 induced activation of the MEK-ERK pathway reduces IL-13Rα2 expression induced by IL-13. Thus, TGF-β1, a pleiotropic cytokine upregulated in asthmatic airways, can augment eosinophilic inflammation by interfering with IL-13's negative feedback autoregulatory loop under MEK/ERK dependent conditions. PMID:22573806

Effects of the Combination of the Main Active Components of Astragalus and Panax notoginseng on Inflammation and Apoptosis of Nerve Cell after Cerebral Ischemia-Reperfusion.

PubMed

Huang, Xiao-Ping; Ding, Huang; Lu, Jin-Dong; Tang, Ying-Hong; Deng, Bing-Xiang; Deng, Chang-Qing

2015-01-01

Astragalus and Panax notoginseng are commonly used to treat cardio-cerebrovascular diseases in China and are often combined together to promote curative effect. We speculate that the enhancement of the combination on anticerebral ischemia injury may come from the main active components. The purpose of this work was to probe the effects and mechanisms of Astragaloside IV (the active component of Astragalus) combined with Ginsenoside Rg1, Ginsenoside Rb1, and Notoginsenoside R1 (the active components of P. notoginseng) to antagonize ischemia/reperfusion (I/R) injury via inflammation and apoptosis. C57BL/6 mice were randomly divided into sham, model, Astragaloside IV, Ginsenoside Rg1, Ginsenoside Rb1, Notoginsenoside R1, four active components combination, and Edaravone groups. After administration for 3 days, bilateral common carotid arteries (CCA) were occluded with artery clip for 20[Formula: see text]min followed by reperfusion for 24[Formula: see text]h. Our results showed that the survival rate of nerve cell in hippocampal CA1 decreased while the apoptotic rate increased, and the level of caspase-3 protein in brain tissues was elevated, the expressions of TNF-a, IL-1, and ICAM-1 mRNA as well as phosphorylated nuclear factor kappa B (NF-κB) inhibitor protein α (p-IκBa) in brain tissues were up-regulated, and the nuclear translocation rate of NF-κB was raised. Additionally, the protein expressions of phosphorylated tyrosine kinase 1 (p-JAK1), phosphorylated signal transducer and activator of transcription-1 (p-STAT1), glucose regulated protein 78 (GRP78), caspase-12, and phosphorylated c-Jun N-terminal kinases 1/2 (p-JNK1/2) in brain tissues were also significantly strengthened after I/R for 24 h. All drugs could increase neurocyte survival rate in hippocampal CA1, decrease the apoptotic rate, and inhibit caspase-3 protein expression, in contrast, the effects of four active components combination were better than those of active components alone. In addition, Astragaloside IV and Ginsenoside Rg1 could down-regulate the level of TNF-α, and ICAM-1 mRNA, respectively, Notoginsenoside R1 reduced both TNF-α and ICAM-1 mRNA, and the combination of the 4 effective components had inhibitory effects on the expressions of TNF-α, IL-1β, and ICAM-1 mRNA. Astragaloside IV, Ginsenoside Rg1, Notoginsenoside R1, and 4 effective components combination were able to restrain the phosphorylation of IκBα, and relieve the nuclear translocation rate of NF-κB. Moreover, the effects of the combination are greater than those of active components alone. All drugs could suppress the phosphorylation of JAK1 induced by I/R; meanwhile the expression of p-STAT1 exhibited a decrease in Ginsenoside Rg1 and four active components combination groups. The decreases of p-JAK1 and p-STAT1 in the four active components combination group were more obvious than those in active components alone groups. Astragaloside IV, Ginsenoside Rg1, and Notoginsenoside R1 further augmented GRP78 expression caused by I/R, Notoginsenoside R1 attenuated caspase-12 protein expression, Astragaloside IV and Ginsenoside Rg1 lessened the phosphorylation of JNK1/2, and the four active components combination was capable of up-regulating GRP78 protein while down-regulating the expressions of caspase-12 and p-JNK1/2. Similarly, the effects of the four active components combination were greater than those of effective components alone. These suggested that the combination of the main active components of Astragalus and Panax notoginseng could strengthen protective effects on cerebral ischemia injury via anti-apoptosis and anti-inflammation, and the mechanisms might be associated with restraining the activation of NF-κB and JAK1/STAT1 signal pathways and regulating endoplasmic reticulum stress (ERS) after cerebral ischemia.

The JAK/STAT pathway is involved in the upregulation of PD-L1 expression in pancreatic cancer cell lines.

PubMed

Doi, Toshifumi; Ishikawa, Takeshi; Okayama, Tetsuya; Oka, Kaname; Mizushima, Katsura; Yasuda, Tomoyo; Sakamoto, Naoyuki; Katada, Kazuhiro; Kamada, Kazuhiro; Uchiyama, Kazuhiko; Handa, Osamu; Takagi, Tomohisa; Naito, Yuji; Itoh, Yoshito

2017-03-01

Although improvements in the chemotherapy modalities for pancreatic cancer have been realized, pancreatic cancer remains one of the most lethal malignancies. New-generation cancer immunotherapy methods, such as blocking of the PD-1/PD-L1 pathway, are consistently being investigated to improve the survival of pancreatic cancer patients. In the present study, we evaluated the influence of anticancer agents 5-fluorouracil, gemcitabine and paclitaxel on PD-L1 expression in human pancreatic cancer cell lines MIA PaCa-2 and AsPC-1 and in murine pancreatic cancer cell line Pan02. Additionally, we analyzed the molecular mechanisms that facilitated the regulation of PD-L1 expression in these cell lines. We observed that when AsPC-1, MIA PaCa-2 and Pan02 cells were stimulated by 5-fluorouracil, gemcitabine or paclitaxel, PD-L1 surface protein expression was enhanced. Similarly, the mRNA level of PD-L1 was upregulated in the AsPC-1 and Pan02 cells when stimulated by each of the three anticancer agents. The phosphorylation of STAT1 and an increase in total STAT1 were also observed in the AsPC-1 cells when stimulated by each anticancer agent. In response to JAK2 inhibitor treatment, PD-L1 upregulation induced by the anticancer agents was reduced in a dose-dependent manner. These results suggest that i) the JAK2/STAT1 pathway is involved in the anticancer agent-mediated PD-L1 transcription; and ii) the anticancer agents altered the tumor immune response which may induce tumor immune escape. These findings can have an influence on the design of treatments that combine chemotherapy and immunotherapy.

Long non-coding RNA AK093407 promotes proliferation and inhibits apoptosis of human osteosarcoma cells via STAT3 activation

PubMed Central

Wang, Yongkun; Liang, Tingting; Wang, Yao; Huang, Yan; Li, Ye

2017-01-01

Osteosarcoma is a malignant tumor of the skeletal system. Long non-coding RNAs (lncRNAs) have been shown to play significant role in osteosarcoma. The present study evaluated the effects and mechanism of lncRNA AK093407 in osteosarcoma. The study included human osteosarcoma cell line, U-2OS. Cell proliferation, viability, and apoptosis were measured using Ki-67 proliferation assay, MTT assay, and Annexin V/PI staining assay, respectively. Relative mRNA and protein expressions were measured using qRT-PCR and western blot, respectively. Interaction between AK093407 and STAT3 was identified using mass spectrometry and RNA pull-down assay. Results revealed that AK093407 was highly expressed in osteosarcoma cells and tissues. Then we demonstrated that overexpression of AK093407 promoted cell proliferation and viability and inhibited apoptosis, whereas suppression of AK093407 showed opposite effects. In addition, AK093407 regulated the expression of genes and proteins (Bcl-2, TGF-β, NF-κB, and PCNA) involved in the cell proliferation, viability, and apoptosis. Furthermore, we showed that AK093407 interacted with STAT3, and promoted its phosphorylation. Lastly, we showed that STAT3 activation was essential for the effects of AK093407 on cell proliferation and apoptosis as the overexpression of AK093407 in the presence of STAT3 inhibitor did not promote cell proliferation and inhibit cell apoptosis. AK093407 is highly expressed in osteosarcoma cells and tissues, and promotes cell proliferation and viability and inhibits apoptosis of osteosarcoma cell line U-2OS via STAT3 activation. PMID:28469961

TC-PTP and PTP1B: Regulating JAK-STAT signaling, controlling lymphoid malignancies.

PubMed

Pike, Kelly A; Tremblay, Michel L

2016-06-01

Lymphoid malignancies are characterized by an accumulation of genetic lesions that act co-operatively to perturb signaling pathways and alter gene expression programs. The Janus kinases (JAK)-signal transducers and activators of transcription (STATs) pathway is one such pathway that is frequently mutated in leukemia and lymphoma. In response to cytokines and growth factors, a cascade of reversible tyrosine phosphorylation events propagates the JAK-STAT pathway from the cell surface to the nucleus. Activated STAT family members then play a fundamental role in establishing the transcriptional landscape of the cell. In leukemia and lymphoma, somatic mutations have been identified in JAK and STAT family members, as well as, negative regulators of the pathway. Most recently, inactivating mutations in the protein tyrosine phosphatase (PTP) genes PTPN1 (PTP1B) and PTPN2 (TC-PTP) were sequenced in B cell lymphoma and T cell acute lymphoblastic leukemia (T-ALL) respectively. The loss of PTP1B and TC-PTP phosphatase activity is associated with an increase in cytokine sensitivity, elevated JAK-STAT signaling, and changes in gene expression. As inactivation mutations in PTPN1 and PTPN2 are restricted to distinct subsets of leukemia and lymphoma, a future challenge will be to identify in which cellular contexts do they contributing to the initiation or maintenance of leukemogenesis or lymphomagenesis. As well, the molecular mechanisms by which PTP1B and TC-PTP loss co-operates with other genetic aberrations will need to be elucidated to design more effective therapeutic strategies. Copyright © 2016 Elsevier Ltd. All rights reserved.

AKT-induced PKM2 phosphorylation signals for IGF-1-stimulated cancer cell growth

PubMed Central

Park, Young Soo; Kim, Dong Joon; Koo, Han; Jang, Se Hwan; You, Yeon-Mi; Cho, Jung Hee; Yang, Suk-Jin; Yu, Eun Sil; Jung, Yuri; Lee, Dong Chul; Kim, Jung-Ae; Park, Zee-Yong; Park, Kyung Chan; Yeom, Young Il

2016-01-01

Pyruvate kinase muscle type 2 (PKM2) exhibits post-translational modifications in response to various signals from the tumor microenvironment. Insulin-like growth factor 1 (IGF-1) is a crucial signal in the tumor microenvironment that promotes cell growth and survival in many human cancers. Herein, we report that AKT directly interacts with PKM2 and phosphorylates it at Ser-202, which is essential for the nuclear translocation of PKM2 protein under stimulation of IGF-1. In the nucleus, PKM2 binds to STAT5A and induces IGF-1-stimulated cyclin D1 expression, suggesting that PKM2 acts as an important factor inducing STAT5A activation under IGF-1 signaling. Concordantly, overexpression of STAT5A in cells deficient in PKM2 expression failed to restore IGF-induced growth, whereas reconstitution of PKM2 in PKM2 knockdown cells restored the IGF-induced growth capacity. Our findings suggest a novel role of PKM2 in promoting the growth of cancers with dysregulated IGF/phosphoinositide 3-kinase/AKT signaling. PMID:27340866

STAT inhibitors for cancer therapy

PubMed Central

2013-01-01

Signal Transducer and Activator of Transcription (STAT) proteins are a family of cytoplasmic transcription factors consisting of 7 members, STAT1 to STAT6, including STAT5a and STAT5b. STAT proteins are thought to be ideal targets for anti-cancer therapy since cancer cells are more dependent on the STAT activity than their normal counterparts. Inhibitors targeting STAT3 and STAT5 have been developed. These included peptidomimetics, small molecule inhibitors and oligonucleotides. This review summarized advances in preclinical and clinical development of these compounds. PMID:24308725

Transcriptional network profile on synovial fluid T cells in psoriatic arthritis.

PubMed

Fiocco, Ugo; Martini, Veronica; Accordi, Benedetta; Caso, Francesco; Costa, Luisa; Oliviero, Francesca; Scanu, Anna; Facco, Monica; Boso, Daniele; Gatto, Mariele; Felicetti, Mara; Frallonardo, Paola; Ramonda, Roberta; Piva, Lucia; Zambello, Renato; Agostini, Carlo; Scarpa, Raffaele; Basso, Giuseppe; Semenzato, Gianpietro; Dayer, Jean-Michel; Punzi, Leonardo; Doria, Andrea

2015-09-01

The objective of the study was to quantify the transcriptional profile, as the main T cell lineage-transcription factors on synovial fluid (SF) T cells, in relation to SF cytokines and T cell frequencies (%) of psoriatic arthritis (PsA) patients. Reverse phase protein array was employed to identify interleukin (IL)-23Rp19-, FOXP3- and related orphan receptor gamma T (RORγt)- protein and Janus associated tyrosine kinases 1 (JAK1), signal transducer and activator and transcription 1 (STAT1), STAT3 and STAT5 phosphoproteins in total T cell lysates from SF of PsA patients. IL-1β, IL-2, IL-6, IL-21 and interferon (INF)-γ were measured using a multiplex bead immunoassay in SF from PsA patients and peripheral blood (PB) from healthy controls (HC). Frequencies of CD4(+)CD25(-), CD4(+)CD25(high) FOXP3(+) and CD4(+)CD25(high) CD127(low) Treg, and either mean fluorescence intensity (MFI) of FOXP3(+) on CD4(+) Treg or MFI of classic IL-6 receptor (IL-6R) α expression on CD4(+)CD25(-) helper/effector T cells (Th/eff) and Treg cells, were quantified in SF of PsA patients and in PB from HC by flow cytometry (FC). In PsA SF samples, IL-2, IL-21 and IFN-γ were not detectable, whereas IL-6 and IL-1β levels were higher than in SF of non-inflammatory osteoarthritis patients. Higher levels of IL-23R-, FOXP3- and RORγt proteins and JAK1, STAT1, STAT3 and STAT5 were found in total T cells from SF of PsA patients compared with PB from HC. Direct correlations between JAK1 Y1022/Y1023 and STAT5 Y694, and STAT3 Y705 and IL6, were found in SF of PsA patients. Increased proportion of CD4(+)CD25(high) FOXP3(+) and CD4(+)CD25(high) CD127(low) Treg cells and brighter MFI of IL-6Rα were observed both on CD4(+)CD25(high)- and CD4(+)CD25(-) T cells in PsA SF. The study showed a distinctive JAK1/STAT3/STAT5 transcriptional network on T cells in the joint microenvironment, outlining the interplay of IL-6, IL-23, IL-1β and γC cytokines in the polarization and plasticity of Th17 and Treg cells, which might participate in the perpetuation of joint inflammation in PsA patients.

Pirfenidone exerts a suppressive effect on CCL18 expression in U937-derived macrophages partly by inhibiting STAT6 phosphorylation.

PubMed

Saito, Yoshinobu; Azuma, Arata; Matsuda, Kuniko; Kamio, Koichiro; Abe, Shinji; Gemma, Akihiko

2016-10-27

CC chemokine ligand 18 (CCL18) is suggested to play a role in the development of pulmonary fibrosis. Macrophages are thought to be the main source of CCL18, and the effect of pirfenidone, an anti-fibrotic agent for idiopathic pulmonary fibrosis, on the expression of CCL18 in macrophages warrants investigation. The purpose of this study was to investigate the effect of pirfenidone on the expression of CCL18 in macrophages. U937 cells were differentiated into macrophages by phorbol myristate acetate and then stimulated with recombinant IL-4 to induce the production of CCL18. The cells were treated with pirfenidone, and the mRNA and protein levels for CCL18 were measured by a reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. The effects of pirfenidone on the IL-4 receptor (IL-4R) expression and STAT6 activation were investigated and on the JAK kinase activity were measured using the Z'-LYTE™ kinase assay. Pirfenidone significantly suppressed the expression of CCL18 when the cells were treated with concentrations of 50-250 μg/mL. Pirfenidone did not affect the expression of the IL-4R components. The selective STAT6 inhibitor AS1517499 suppressed CCL18 expression. Both AS1517499 and pirfenidone suppressed STAT6 phosphorylation (p < .05), although the effect of pirfenidone was less marked than that of AS1517499. The Z'-LYTE™ kinase assay showed a reduction in the activities of JAK1, JAK3 and TYK2 by pirfenidone. Pirfenidone suppresses CCL18 expression in macrophages and this effect is thought to be attributed partly to the inhibition of STAT6 phosphorylation.

Biological responses to PDGF-BB versus PDGF-DD in human mesangial cells.

PubMed

van Roeyen, C R C; Ostendorf, T; Denecke, B; Bokemeyer, D; Behrmann, I; Strutz, F; Lichenstein, H S; LaRochelle, W J; Pena, C E; Chaudhuri, A; Floege, J

2006-04-01

Platelet-derived growth factor (PDGF)-BB and PDGF-DD mediate mesangial cell proliferation in vitro and in vivo. While PDGF-BB is a ligand for the PDGF alpha- and beta-receptor chains, PDGF-DD binds more selectively to the beta-chain, suggesting potential differences in the biological activities. Signal transduction and regulation of gene expression induced by PDGF-BB and -DD were compared in primary human mesangial cells (HMCs), which expressed PDGF alpha- and beta-receptor subunits. The growth factor concentrations used were chosen based on their equipotency in inducing HMCs proliferation and binding to the betabeta-receptor. Both growth factors, albeit at different concentrations induced phosphorylation and activation of extracellular signal-regulated kinase 1 (ERK1) and ERK2. In addition, PDGFs led to the phosphorylation and activation of signal transducers and activators of transcription 1 (STAT1) and STAT3. HMCs proliferation induced by either PDGF-BB or -DD could be blocked by signal transduction inhibitors of the mitogen-activated protein kinase-, Janus kinase (JAK)/STAT-, or phosphatidyl-inositol 3-kinase pathways. Using a gene chip array and subsequent verification by real-time reverse transcriptase (RT)-polymerase chain reaction, we found that in HMC genes for matrix metalloproteinase 13 (MMP-13) and MMP-14 and, to a low extent, cytochrome B5 and cathepsin L were exclusively regulated by PDGF-BB, whereas no exclusive gene regulation was detected by PDGF-DD. However, at the protein level, both MMP-13 and -14 were equally induced by PDGF-BB and -DD. PDGF-BB and -DD effect similar biological responses in HMCs albeit at different potencies. Rare apparently differential gene regulation did not result in different protein expression, suggesting that in HMCs both PDGFs exert their biological activity almost exclusively via the PDGF beta-receptor.

Artesunate inhibits adipogeneis in 3T3-L1 preadipocytes by reducing the expression and/or phosphorylation levels of C/EBP-α, PPAR-γ, FAS, perilipin A, and STAT-3

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jang, Byeong-Churl, E-mail: jangbc123@gw.kmu.ac.kr

Differentiation of preadipocyte, also called adipogenesis, leads to the phenotype of mature adipocyte. However, excessive adipogenesis is closely linked to the development of obesity. Artesunate, one of artemisinin-type sesquiterpene lactones from Artemisia annua L., is known for anti-malarial and anti-cancerous activities. In this study, we investigated the effect of artesunate on adipogenesis in 3T3-L1 preadipocytes. Artesunate strongly inhibited lipid accumulation and triglyceride (TG) synthesis during the differentiation of 3T3-L1 preadipocytes into adipocytes at 5 μM concentration. Artesunate at 5 μM also reduced not only the expressions of CCAAT/enhancer-binding protein-α (C/EBP-α), peroxisome proliferator-activated receptor-γ (PPAR-γ), fatty acid synthase (FAS), and perilipin A butmore » also the phosphorylation levels of signal transducer and activator of transcription-3 (STAT-3) during adipocyte differentiation. Moreover, artesunate at 5 μM reduced leptin, but not adiponectin, mRNA expression during adipocyte differentiation. Taken together, these findings demonstrate that artesunate inhibits adipogenesis in 3T3-L1 preadipoytes through the reduced expression and/or phosphorylation levels of C/EBP-α, PPAR-γ, FAS, perilipin A, and STAT-3. -- Highlights: •Artesunate, an artemisinin derivative, inhibits adipogenesis. •Artesunate inhibits C/EBP-α, PPAR-γ, FAS, perilipin A, and STAT-3 in 3T3-L1 adipocytes. •Artesunate reduces leptin, but not adiponectin, expression in 3T3-L1 adipocytes. •Artesunate thus may have therapeutic potential against obesity.« less

Sodium Benzoate, a Food Additive and a Metabolite of Cinnamon, Enriches Regulatory T Cells via STAT6-Mediated Upregulation of TGF-β.

PubMed

Kundu, Madhuchhanda; Mondal, Susanta; Roy, Avik; Martinson, Jeffrey L; Pahan, Kalipada

2016-10-15

Upregulation and/or maintenance of regulatory T cells (Tregs) during autoimmune insults may have therapeutic efficacy in autoimmune diseases. Earlier we have reported that sodium benzoate (NaB), a metabolite of cinnamon and a Food and Drug Administration-approved drug against urea cycle disorders, upregulates Tregs and protects mice from experimental allergic encephalomyelitis, an animal model of multiple sclerosis. However, mechanisms by which NaB increases Tregs are poorly understood. Because TGF-β is an important inducer of Tregs, we examined the effect of NaB on the status of TGF-β. In this study, we demonstrated that NaB induced the expression of TGF-β mRNA and protein in normal as well as proteolipid protein-primed splenocytes. The presence of a consensus STAT6 binding site in the promoter of the TGF-β gene, activation of STAT6 in splenocytes by NaB, recruitment of STAT6 to the TGF-β promoter by NaB, and abrogation of NaB-induced expression of TGF-β in splenocytes by small interfering RNA knockdown of STAT6 suggest that NaB induces the expression of TGF-β via activation of STAT6. Furthermore, we demonstrated that blocking of TGF-β by neutralizing Abs abrogated NaB-mediated protection of Tregs and experimental allergic encephalomyelitis. These studies identify a new function of NaB in upregulating TGF-β via activation of STAT6, which may be beneficial in MS patients. Copyright © 2016 by The American Association of Immunologists, Inc.

Silibinin Inhibits ICAM-1 Expression via Regulation of N-Linked and O-Linked Glycosylation in ARPE-19 Cells

PubMed Central

Chen, Yi-Hao; Chen, Ching-Long; Liang, Chang-Min; Liang, Jy-Been; Tai, Ming-Cheng; Chang, Yun-Hsiang; Lu, Da-Wen; Chen, Jiann-Torng

2014-01-01

To evaluate the effects of silibinin on intercellular adhesion molecule-1 (ICAM-1) expression, we used ARPE-19 cells as a model in which tumor necrosis factor (TNF-α) and interferon (IFN-γ) enhanced ICAM-1 expression. This upregulation was inhibited by silibinin. In an adherence assay using ARPE-19 and THP-1 cells, silibinin inhibited the cell adhesion function of ICAM-1. The inhibitory effects of silibinin on ICAM-1 expression were mediated via the blockage of nuclear translocation of p65 proteins in TNF-α and phosphorylation of STAT1 in IFN-γ-stimulated cells. In addition, silibinin altered the degree of N-linked glycosylation posttranslationally in ARPE-19 cells by significantly enhancing MGAT3 gene expression. Silibinin can increase the O-GlcNAc levels of glycoproteins in ARPE-19 cells. In a reporter gene assay, PUGNAc, which can also increase O-GlcNAc levels, inhibited NF-κB reporter activity in TNF-α-induced ARPE-19 cells and this process was augmented by silibinin treatment. Overexpression of OGT gene was associated with reduced TNF-α-induced ICAM-1 levels, which is consistent with that induced by silibinin treatment. Taken together, silibinin inhibits ICAM-1 expression and its function through altered O-linked glycosylation in NF-κB and STAT1 signaling pathways and decreases the N-linked glycosylation of ICAM-1 transmembrane protein in proinflammatory cytokine-stimulated ARPE-19 cells. PMID:25032222

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lu, Yanxin; Biomedical Research Institute, Shenzhen-PKU-HKUST Medical Center, Guangdong Province, Shenzhen 518036; Yue, Xupeng

Highlights: •miR-124 is down-regulated in hepatocellular carcinoma HepG2 cells. •Over-expression of miR-124 suppresses proliferation and induces apoptosis in HepG2 cells. •miR-124 inhibits xenograft tumor growth in nude mice implanted with HepG2 cells by reducing STAT3 expression. •STATs function as a novel target of miR-124 in HCC HepG2 cells. -- Abstract: The aberrant expression of microRNAs is associated with development and progression of cancers. Down-regulation of miR-124 has been demonstrated in the hepatocellular carcinoma (HCC), but the underlying mechanism by which miR-124 suppresses tumorigenesis in HCC remains elusive. In this study, we found that miR-124 suppresses the tumor growth of HCCmore » through targeting the signal transducers and activators of transcription 3 (STAT3). Overexpression of miR-124 suppressed proliferation and induced apoptosis in HepG-2 cells. Luciferase assay confirmed that miR-124 binding to the 3′-UTR region of STAT3 inhibited the expression of STAT3 and phosphorylated STAT3 proteins in HepG-2 cells. Knockdown of STAT3 by siRNA in HepG-2 cells mimicked the effect induced by miR-124. Overexpression of STAT3 in miR-124-transfected HepG-2 cells effectively rescued the inhibition of cell proliferation caused by miR-124. Furthermore, miR-124 suppressed xenograft tumor growth in nude mice implanted with HepG-2 cells by reducing STAT3 expression. Taken together, our findings show that miR-124 functions as tumor suppressor in HCC by targeting STAT3, and miR-124 may therefore serve as a biomarker for diagnosis and therapeutics in HCC.« less

Regulation of immunophenotype modulation of monocytes-macrophages from M1 into M2 by prostate cancer cell-culture supernatant via transcription factor STAT3.

PubMed

Solís-Martínez, R; Cancino-Marentes, M; Hernández-Flores, G; Ortiz-Lazareno, P; Mandujano-Álvarez, G; Cruz-Gálvez, C; Sierra-Díaz, E; Rodríguez-Padilla, C; Jave-Suárez, L F; Aguilar-Lemarroy, A; Bravo-Cuellar, A

2018-04-01

Transcription factor STAT3 has a prominent innate immunity effect on cancer progression. We determined the regulation of STAT3 in the immunophenotype modulation of macrophages from M1 into M2 induced by the cell-culture supernatant of the Prostate-Cancer line PC3. Monocytes-macrophages from healthy donors were cultured in the supernatant of PC3 cells, membrane proteins, and intracytoplasmic and phosphorylated STAT3 were measured using flow cytometry, while cytokines and growth factors were studied using luminescence. Cytotoxicity and nitric oxide were evaluated via colorimetric assays. The supernatant of PC3 prostate-tumor cells effectively induced macrophages toward an M2 profile, and the expression of phosphorylated STAT3 in the monocytes-macrophages notably increased, and mainly related to IL-10. In the group of monocytes-macrophages treated with a STAT3 inhibitor, the macrophages were induced toward an M1 phenotype. In this study, we showed that the secretion profile of PC3 prostate-cancer cells induces a change in macrophage phenotype from M1 into M2, and that the phenomenon is related to phosphorylation of transcription factor STAT3 and IL-10. Copyright © 2018. Published by Elsevier B.V.

Pleiotrophin (PTN) is expressed in vascularized human atherosclerotic plaques: IFN-γ/JAK/STAT1 signaling is critical for the expression of PTN in macrophages

PubMed Central

Li, Fuqiang; Tian, Fang; Wang, Lai; Williamson, Ian K.; Sharifi, Behrooz G.; Shah, Prediman K.

2010-01-01

Neovascularization is critical to destabilization of atheroma. We previously reported that the angiogenic growth factor pleiotrophin (PTN) coaxes monocytes to assume the phenotype of functional endothelial cells in vitro and in vivo. In this study we show that PTN expression is colocalized with capillaries of human atherosclerotic plaques. Among the various reagents that are critical to the pathogenesis of atherosclerosis, interferon (IFN)-γ was found to markedly induce PTN mRNA expression in a dose-dependent manner in macrophages. Mechanistic studies revealed that the Janus kinase inhibitors, WHI-P154 and ATA, efficiently blocked STAT1 phosphorylation in a concentration- and time-dependent manner. Notably, the level of phosphorylated STAT1 was found to correlate directly with the PTN mRNA levels. In addition, STAT1/STAT3/p44/42 signaling molecules were found to be phosphorylated by IFN-γ in macrophages, and they were translocated into the nucleus. Further, PTN promoter analysis showed that a gamma-activated sequence (GAS) located at −2086 to −2078 bp is essential for IFN-γ-regulated promoter activity. Moreover, electrophoretic mobility shift, supershift, and chromatin immunoprecipitation analyses revealed that both STAT1 and STAT3 bind to the GAS at the chromatin level in the IFN-γ stimulated cells. Finally, to test whether the combined effect of STAT1/STAT3/p44/42 signaling is required for the expression of PTN in macrophages, gene knockdowns of these transcription factors were performed using siRNA. Cells lacking STAT1, but not STAT3 or p42, have markedly reduced PTN mRNA levels. These data suggest that PTN expression in the human plaques may be in part regulated by IFN-γ and that PTN is involved in the adaptive immunity.—Li, F., Tian, F., Wang, L., Williamson, I. K., Sharifi, B. G., Shah, P. K. Pleiotrophin (PTN) is expressed in vascularized human atherosclerotic plaques: IFN-γ/JAK/STAT1 signaling is critical for the expression of PTN in macrophages PMID:19917672

Anti-cancer effect of bee venom toxin and melittin in ovarian cancer cells through induction of death receptors and inhibition of JAK2/STAT3 pathway

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jo, Miran; Park, Mi Hee; Kollipara, Pushpa Saranya

We investigated whether bee venom and melittin, a major component of bee venom, inhibit cell growth through enhancement of death receptor expressions in the human ovarian cancer cells, SKOV3 and PA-1. Bee venom (1–5 μg/ml) and melittin (0.5–2 μg/ml) inhibited the growth of SKOV3 and PA-1 ovarian cancer cells by the induction of apoptotic cell death in a dose dependent manner. Consistent with apoptotic cell death, expression of death receptor (DR) 3 and DR6 was increased in both cancer cells, but expression of DR4 was increased only in PA-1 cells. Expression of DR downstream pro-apoptotic proteins including caspase-3, 8, andmore » Bax was concomitantly increased, but the phosphorylation of JAK2 and STAT3 and the expression of Bcl-2 were inhibited by treatment with bee venom and melittin in SKOV3 and PA-1 cells. Expression of cleaved caspase-3 was increased in SKOV3, but cleaved caspase-8 was increased in PA-1 cells. Moreover, deletion of DR3, DR4, and DR6 by small interfering RNA significantly reversed bee venom and melittin-induced cell growth inhibitory effect as well as down regulation of STAT3 by bee venom and melittin in SKOV3 and PA-1 ovarian cancer cell. These results suggest that bee venom and melittin induce apoptotic cell death in ovarian cancer cells through enhancement of DR3, DR4, and DR6 expression and inhibition of STAT3 pathway. -- Highlights: ► Some studies have showed that bee venom and/or melittin have anti-cancer effects. ► We found that bee venom and melittin inhibited cell growth in ovarian cancer cells. ► Bee venom and melittin induce apoptosis in SKOV3 and PA-1.« less

Binding of galectin-1 to integrin β1 potentiates drug resistance by promoting survivin expression in breast cancer cells.

PubMed

Nam, KeeSoo; Son, Seog-Ho; Oh, Sunhwa; Jeon, Donghwan; Kim, Hyungjoo; Noh, Dong-Young; Kim, Sangmin; Shin, Incheol

2017-05-30

Galectin-1 is a β-galactoside binding protein secreted by many types of aggressive cancer cells. Although many studies have focused on the role of galectin-1 in cancer progression, relatively little attention has been paid to galectin-1 as an extracellular therapeutic target. To elucidate the molecular mechanisms underlying galectin-1-mediated cancer progression, we established galectin-1 knock-down cells via retroviral delivery of short hairpin RNA (shRNA) against galectin-1 in two triple-negative breast cancer (TNBC) cell lines, MDA-MB-231 and Hs578T. Ablation of galectin-1 expression decreased cell proliferation, migration, invasion, and doxorubicin resistance. We found that these effects were caused by decreased galectin-1-integrin β1 interactions and suppression of the downstream focal adhesion kinase (FAK)/c-Src pathway. We also found that silencing of galectin-1 inhibited extracellular signal-regulated kinase (ERK)/signal transducer and activator of transcription 3 (STAT3) signaling, thereby down-regulating survivin expression. This finding implicates STAT3 as a transcription factor for survivin. Finally, rescue of endogenous galectin-1 knock-down and recombinant galectin-1 treatment both recovered signaling through the FAK/c-Src/ERK/STAT3/survivin pathway. Taken together, these results suggest that extracellular galectin-1 contributes to cancer progression and doxorubicin resistance in TNBC cells. These effects appear to be mediated by galectin-1-induced up-regulation of the integrin β1/FAK/c-Src/ERK/STAT3/survivin pathway. Our results imply that extracellular galectin-1 has potential as a therapeutic target for triple-negative breast cancer.

Epigenetic silencing of the NR4A3 tumor suppressor, by aberrant JAK/STAT signaling, predicts prognosis in gastric cancer

NASA Astrophysics Data System (ADS)

Yeh, Chung-Min; Chang, Liang-Yu; Lin, Shu-Hui; Chou, Jian-Liang; Hsieh, Hsiao-Yen; Zeng, Li-Han; Chuang, Sheng-Yu; Wang, Hsiao-Wen; Dittner, Claudia; Lin, Cheng-Yu; Lin, Jora M. J.; Huang, Yao-Ting; Ng, Enders K. W.; Cheng, Alfred S. L.; Wu, Shu-Fen; Lin, Jiayuh; Yeh, Kun-Tu; Chan, Michael W. Y.

2016-08-01

While aberrant JAK/STAT signaling is crucial to the development of gastric cancer (GC), its effects on epigenetic alterations of its transcriptional targets remains unclear. In this study, by expression microarrays coupled with bioinformatic analyses, we identified a putative STAT3 target gene, NR4A3 that was downregulated in MKN28 GC daughter cells overexpressing a constitutively activated STAT3 mutant (S16), as compared to an empty vector control (C9). Bisulphite pyrosequencing and demethylation treatment showed that NR4A3 was epigenetically silenced by promoter DNA methylation in S16 and other GC cell lines including AGS cells, showing constitutive activation of STAT3. Subsequent experiments revealed that NR4A3 promoter binding by STAT3 might repress its transcription. Long-term depletion of STAT3 derepressed NR4A3 expression, by promoter demethylation, in AGS GC cells. NR4A3 re-expression in GC cell lines sensitized the cells to cisplatin, and inhibited tumor growth in vitro and in vivo, in an animal model. Clinically, GC patients with high NR4A3 methylation, or lower NR4A3 protein expression, had significantly shorter overall survival. Intriguingly, STAT3 activation significantly associated only with NR4A3 methylation in low-stage patient samples. Taken together, aberrant JAK/STAT3 signaling epigenetically silences a potential tumor suppressor, NR4A3, in gastric cancer, plausibly representing a reliable biomarker for gastric cancer prognosis.

Oncostatin M Mediates STAT3-Dependent Intestinal Epithelial Restitution via Increased Cell Proliferation, Decreased Apoptosis and Upregulation of SERPIN Family Members

PubMed Central

Beigel, Florian; Friedrich, Matthias; Probst, Corina; Sotlar, Karl; Göke, Burkhard; Diegelmann, Julia; Brand, Stephan

2014-01-01

Objective Oncostatin M (OSM) is produced by activated T cells, monocytes, and dendritic cells and signals through two distinct receptor complexes consisting of gp130 and LIFR (I) or OSMR-β and gp130 (II), respectively. Aim of this study was to analyze the role of OSM in intestinal epithelial cells (IEC) and intestinal inflammation. Methods OSM expression and OSM receptor distribution was analyzed by PCR and immunohistochemistry experiments, signal transduction by immunoblotting. Gene expression studies were performed by microarray analysis and RT-PCR. Apoptosis was measured by caspases-3/7 activity. IEC migration and proliferation was studied in wounding and water soluble tetrazolium assays. Results The IEC lines Caco-2, DLD-1, SW480, HCT116 and HT-29 express mRNA for the OSM receptor subunits gp130 and OSMR-β, while only HCT116, HT-29 and DLD-1 cells express LIFR mRNA. OSM binding to its receptor complex activates STAT1, STAT3, ERK-1/2, SAPK/JNK-1/2, and Akt. Microarray analysis revealed 79 genes that were significantly up-regulated (adj.-p≤0.05) by OSM in IEC. Most up-regulated genes belong to the functional categories “immunity and defense” (p = 2.1×10−7), “apoptosis” (p = 3.7×10−4) and “JAK/STAT cascade” (p = 3.4×10−6). Members of the SERPIN gene family were among the most strongly up-regulated genes. OSM significantly increased STAT3- and MEK1-dependent IEC cell proliferation (p<0.05) and wound healing (p = 3.9×10−5). OSM protein expression was increased in colonic biopsies of patients with active inflammatory bowel disease (IBD). Conclusions OSM promotes STAT3-dependent intestinal epithelial cell proliferation and wound healing in vitro. Considering the increased OSM expression in colonic biopsy specimens of patients with active IBD, OSM upregulation may modulate a barrier-protective host response in intestinal inflammation. Further in vivo studies are warranted to elucidate the exact role of OSM in intestinal inflammation and the potential of OSM as a drug target in IBD. PMID:24710357

Yes-Associated Protein Promotes Angiogenesis via Signal Transducer and Activator of Transcription 3 in Endothelial Cells.

PubMed

He, Jinlong; Bao, Qiankun; Zhang, Yan; Liu, Mingming; Lv, Huizhen; Liu, Yajin; Yao, Liu; Li, Bochuan; Zhang, Chenghu; He, Shuang; Zhai, Guijin; Zhu, Yan; Liu, Xin; Zhang, Kai; Wang, Xiu-Jie; Zou, Ming-Hui; Zhu, Yi; Ai, Ding

2018-02-16

Angiogenesis is a complex process regulating endothelial cell (EC) functions. Emerging lines of evidence support that YAP (Yes-associated protein) plays an important role in regulating the angiogenic activity of ECs. The objective of this study was to specify the effect of EC YAP on angiogenesis and its underlying mechanisms. In ECs, vascular endothelial growth factor reduced YAP phosphorylation time and dose dependently and increased its nuclear accumulation. Using Tie2Cre-mediated YAP transgenic mice, we found that YAP promoted angiogenesis in the postnatal retina and tumor tissues. Mass spectrometry revealed signal transducer and activator of transcription 3 (STAT3) as a potential binding partner of YAP in ECs. Western blot and immunoprecipitation assays indicated that binding with YAP prolonged interleukin 6-induced STAT3 nuclear accumulation by blocking chromosomal maintenance 1-mediated STAT3 nuclear export without affecting its phosphorylation. Moreover, angiopoietin-2 expression induced by STAT3 was enhanced by YAP overexpression in ECs. Finally, a selective STAT3 inhibitor or angiopoietin-2 blockage partly attenuated retinal angiogenesis in Tie2Cre-mediated YAP transgenic mice. YAP binding sustained STAT3 in the nucleus to enhance the latter's transcriptional activity and promote angiogenesis via regulation of angiopoietin-2. © 2018 American Heart Association, Inc.

Curcumin induces growth-arrest and apoptosis in association with the inhibition of constitutively active JAK-STAT pathway in T cell leukemia

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rajasingh, Johnson; Raikwar, Himanshu P.; Muthian, Gladson

2006-02-10

Adult T cell leukemia is an aggressive and frequently fatal malignancy that expressess constitutively activated growth-signaling pathways in association with deregulated growth and resistance to apoptosis. Curcumin (diferuloylmethane) is a naturally occurring yellow pigment, isolated from the rhizomes of the plant Curcuma longa that has traditionally been used in the treatment of injury and inflammation. But the effect and mechanism of action of curcumin on T cell leukemia is not known. To investigate the antitumor activity of curcumin in T cell leukemia, we examined its effect on constitutive phosphorylation of JAK and STAT proteins, proliferation, and apoptosis in HTLV-I-transformed Tmore » cell lines. HTLV-I-transformed T cell leukemia lines, MT-2, HuT-102, and SLB-1, express constitutively phosphorylated JAK3, TYK2, STAT3, and STAT5 signaling proteins. In vitro treatment with curcumin induced a dose-dependent decrease in JAK and STAT phosphorylation resulting in the induction of growth-arrest and apoptosis in T cell leukemia. The induction of growth-arrest and apoptosis in association with the blockade of constitutively active JAK-STAT pathway suggests this be a mechanism by which curcumin induces antitumor activity in T cell leukemia.« less

OPB-31121, a novel small molecular inhibitor, disrupts the JAK2/STAT3 pathway and exhibits an antitumor activity in gastric cancer cells.

PubMed

Kim, Mi-Jung; Nam, Hyun-Jin; Kim, Hwang-Phill; Han, Sae-Won; Im, Seock-Ah; Kim, Tae-You; Oh, Do-Youn; Bang, Yung-Jue

2013-07-10

We investigated the mechanisms of action and antitumor effects of OPB-31121, a novel STAT3 inhibitor, in gastric cancer cells. OPB-31121 downregulated JAK2 and gp130 expression and inhibited JAK2 phosphorylation which leads to inhibition of STAT3 phosphorylation. OPB-31121 inhibited constitutively activated and IL-6-induced JAK/STAT signaling pathway. OPB-31121 decreased cell proliferation in both gastric cancer cells and in a xenograft model, induced the apoptosis of gastric cancer cells, inhibited the expression of antiapoptotic proteins, and showed synergism with 5-fluorouracil and cisplatin. Taken together, our study suggests that STAT3 inhibition with OPB-31121 can be tested in patients with gastric cancer. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

Hydrogen ameliorates pulmonary hypertension in rats by anti-inflammatory and antioxidant effects.

PubMed

Kishimoto, Yasuaki; Kato, Taichi; Ito, Mikako; Azuma, Yoshiteru; Fukasawa, Yoshie; Ohno, Kinji; Kojima, Seiji

2015-09-01

The pathogenesis of pulmonary arterial hypertension (PAH) involves reactive oxygen species and inflammation. Beneficial effects of molecular hydrogen, which exerts both anti-inflammatory and antioxidative effects, have been reported for various pathologic conditions. We therefore hypothesized that molecular hydrogen would improve monocrotaline (MCT)-induced PAH in rats. Nineteen male Sprague-Dawley rats (body weight: 200-300 g) were divided into groups, receiving: (1) MCT + hydrogen-saturated water (group H); (2) MCT + dehydrogenized water (group M); or (3) saline + dehydrogenized water (group C). Sixteen days after substance administration, we evaluated hemodynamics, harvested the lungs and heart, and performed morphometric analysis of the pulmonary vasculature. Macrophage infiltration, antiproliferating cell nuclear antigen-positive cells, 8-hydroxy-deoxyguanosine (8-OHdG)-positive cells, and expressions of phosphorylated signal transducers and activators of transcription-3 (STAT3) and nuclear factor of activated T-cells (NFAT) were evaluated immunohistochemically. Stromal cell-derived factor-1 and monocyte chemoattractant protein-1 expressions were evaluated by quantitative reverse-transcription polymerase chain reaction. Pulmonary arterial hypertension was significantly exacerbated in group M compared to group C, but was significantly improved in group H. Vascular density was significantly reduced in group M, but not in group H. Adventitial macrophages, antiproliferating cell nuclear antigen - and 8-OHdG-positive cells, and stromal cell-derived factor-1 and monocyte chemoattractant protein-1 expressions were significantly increased in group M, but improved in group H. Expressions of phosphorylated STAT3 and NFAT were up-regulated in group M, but improved in group H. Molecular hydrogen ameliorates MCT-induced PAH in rats by suppressing macrophage accumulation, reducing oxidative stress and modulating the STAT3/NFAT axis. Copyright © 2015 The American Association for Thoracic Surgery. Published by Elsevier Inc. All rights reserved.

Cardioprotective Effects of Quercetin in Cardiomyocyte under Ischemia/Reperfusion Injury

PubMed Central

Chen, Yi-Wen; Chou, Hsiu-Chuan; Lin, Szu-Ting; Chen, You-Hsuan; Chang, Yu-Jung; Chen, Linyi; Chan, Hong-Lin

2013-01-01

Quercetin, a polyphenolic compound existing in many vegetables, fruits, has antiinflammatory, antiproliferation, and antioxidant effect on mammalian cells. Quercetin was evaluated for protecting cardiomyocytes from ischemia/reperfusion injury, but its protective mechanism remains unclear in the current study. The cardioprotective effects of quercetin are achieved by reducing the activity of Src kinase, signal transducer and activator of transcription 3 (STAT3), caspase 9, Bax, intracellular reactive oxygen species production, and inflammatory factor and inducible MnSOD expression. Fluorescence two-dimensional differential gel electrophoresis (2D-DIGE) and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) can reveal the differentially expressed proteins of H9C2 cells treated with H2O2 or quercetin. Although 17 identified proteins were altered in H2O2-induced cells, these proteins such as alpha-soluble NSF attachment protein (α-SNAP), Ena/VASP-like protein (Evl), and isopentenyl-diphosphate delta-isomerase 1 (Idi-1) were reverted by pretreatment with quercetin, which correlates with kinase activation, DNA repair, lipid, and protein metabolism. Quercetin dephosphorylates Src kinase in H2O2-induced H9C2 cells and likely blocks the H2O2-induced inflammatory response through STAT3 kinase modulation. This probably contributes to prevent ischemia/reperfusion injury in cardiomyocytes. PMID:23573126

The potency of STAT (signal transducers and activators of transcription) 3 protein as growth promoter for chicken

NASA Astrophysics Data System (ADS)

Ma'ruf, Anwar; Iswati, Sri; Hidajati, Nove; Damayanti, Ratna

2017-09-01

The long-term objective of this study was to produce STAT synthetic protein in chicken during growth period resulting from the increase of growth hormone (GH) as growth promoter. This study used ten male chicken Lohman from PT. Multibreeder Indonesia. The chicken were kept within batteried cage, with a capacity of one chicken in each cage. The chickens were fed twice a day, at 6 a.m. and 6 p.m. with the amount of feed 10% less than standard. On day 21 the chicken were slaughtered to obtain the samples, i.e., adipose, liver and muscles for the following examinations (1) isolation of STAT-3 signaling protein from adipose, liver and muscles of the chicken, (2) analysis of STAT-3 signaling protein using SDS-PAGE method, and (3) identification of STAT-3 signaling protein using Western blot method by means of protein detection using electrophoresis with polyacrylamide gels. Results of examination on protein in hepatic, muscle and adipose of chickens in growth period revealed that STAT protein was positively present in those tissues. This finding was followed-up with SDS-PAGE examination, from which we found the presence of protein band between the markers of 116 kDa and 14.4 kDa. The protein band was supposedly the STAT-3 protein. To prove that protein band formed was the STAT-3, Western blot examination was conducted using rabbit polyclonal antibody STAT-3. The result showed the formation of the protein band, indicating the presence of reaction between antigen (STAT-3 protein) and STAT-3 protein antibody. In conclusion, STAT-3 protein is present in hepatic, muscular, and adipose tissues, with molecular weight of 59.4 kDa.

Epstein-Barr Virus Latent Membrane Protein 2A (LMP2A) enhances IL-10 production through the activation of Bruton's tyrosine kinase and STAT3.

PubMed

Incrocci, Ryan; Barse, Levi; Stone, Amanda; Vagvala, Sai; Montesano, Michael; Subramaniam, Vijay; Swanson-Mungerson, Michelle

2017-01-01

Previous data demonstrate that Epstein-Barr Virus Latent Membrane Protein 2A (LMP2A) enhances IL-10 to promote the survival of LMP2A-expressing B cell lymphomas. Since STAT3 is an important regulator of IL-10 production, we hypothesized that LMP2A activates a signal transduction cascade that increases STAT3 phosphorylation to enhance IL-10. Using LMP2A-negative and -positive B cell lines, the data indicate that LMP2A requires the early signaling molecules of the Syk/RAS/PI3K pathway to increase IL-10. Additional studies indicate that the PI3K-regulated kinase, BTK, is responsible for phosphorylating STAT3, which ultimately mediates the LMP2A-dependent increase in IL-10. These data are the first to show that LMP2A signaling results in STAT3 phosphorylation in B cells through a PI3K/BTK-dependent pathway. With the use of BTK and STAT3 inhibitors to treat B cell lymphomas in clinical trials, these findings highlight the possibility of using new pharmaceutical approaches to treat EBV-associated lymphomas that express LMP2A. Copyright © 2016 Elsevier Inc. All rights reserved.

Oncostatin M suppresses metastasis of lung adenocarcinoma by inhibiting SLUG expression through coordination of STATs and PIASs signalings.

PubMed

Pan, Chih-Ming; Wang, Mong-Lien; Chiou, Shih-Hwa; Chen, Hsiao-Yun; Wu, Cheng-Wen

2016-09-13

Oncostatin M (OSM) is linked with multiple biological responses including growth and differentiation. Previous reports showed inhibitory effects of OSM in tumor progression while others showed promoting effects. The dual role of OSM in the development of various cancers is still unclear. We previously described OSM-mediated SLUG suppression, leading to repressed metastasis of lung adenocarcinoma (LAC) cells. However, the underlying mechanism remains elusive. Here, we showed that OSM suppresses SLUG express in LAC cells through a STAT1-dependent transcriptional inhibition. Knockdown of STAT1 reversed the OSM-suppressed SLUG expression and rescued the OSM-mediated inhibition of cell proliferation, migration, and invasion in vitro, as well as pulmonary metastasis in vivo. STAT1 suppressed SLUG transcription through binding to its promoter region in response to OSM. Furthermore, PIAS4, a co-repressor of STAT, and HDAC1 were able to bind to STAT1 on SLUG promoter region, resulting in reduced H3K9 acetylation and suppressed SLUG expression upon OSM treatment. In contrast, PIAS3 bound to activated STAT3, another effector of OSM, in response to OSM and blocked the binding of STAT3 to SLUG promoter region, preventing STAT3-dependent activation of SLUG transcription. Our findings suggested that OSM suppresses SLUG expression and tumor metastasis of LAC through inducing the inhibitory effect of the STAT1-dependent pathway and suppressing the activating effect of STAT3-dependent signaling. These results can serve as a scientific basis for the potential therapeutic intervention of OSM in cancer cells.

Inhibition of interleukin-3- and interferon- α-induced JAK/STAT signaling by the synthetic α-X-2',3,4,4'-tetramethoxychalcones α-Br-TMC and α-CF3-TMC.

PubMed

Jobst, Belinda; Weigl, Julia; Michl, Carina; Vivarelli, Fabio; Pinz, Sophia; Amslinger, Sabine; Rascle, Anne

2016-11-01

The JAK/STAT pathway is an essential mediator of cytokine signaling, often upregulated in human diseases and therefore recognized as a relevant therapeutic target. We previously identified the synthetic chalcone α-bromo-2',3,4,4'-tetramethoxychalcone (α-Br-TMC) as a novel JAK2/STAT5 inhibitor. We also found that treatment with α-Br-TMC resulted in a downward shift of STAT5 proteins in SDS-PAGE, suggesting a post-translational modification that might affect STAT5 function. In the present study, we show that a single cysteine within STAT5 is responsible for the α-Br-TMC-induced protein shift, and that this modification does not alter STAT5 transcriptional activity. We also compared the inhibitory activity of α-Br-TMC to that of another synthetic chalcone, α-trifluoromethyl-2',3,4,4'-tetramethoxychalcone (α-CF3-TMC). We found that, like α-Br-TMC, α-CF3-TMC inhibits JAK2 and STAT5 phosphorylation in response to interleukin-3, however without altering STAT5 mobility in SDS-PAGE. Moreover, we demonstrate that both α-Br-TMC and α-CF3-TMC inhibit interferon-α-induced activation of STAT1 and STAT2, by inhibiting their phosphorylation and the expression of downstream interferon-stimulated genes. Together with the previous finding that α-Br-TMC and α-CF3-TMC inhibit the response to inflammation by inducing Nrf2 and blocking NF-κB activities, our data suggest that synthetic chalcones might be useful as anti-inflammatory, anti-cancer and immunomodulatory agents in the treatment of human diseases.

Arctigenin inhibits lipopolysaccharide-induced iNOS expression in RAW264.7 cells through suppressing JAK-STAT signal pathway.

PubMed

Kou, Xianjuan; Qi, Shimei; Dai, Wuxing; Luo, Lan; Yin, Zhimin

2011-08-01

Arctigenin has been demonstrated to have an anti-inflammatory function, but the precise mechanisms of its action remain to be fully defined. In the present study, we determined the effects of arctigenin on lipopolysaccharide (LPS)-induced production of proinflammatory mediators and the underlying mechanisms involved in RAW264.7 cells. Our results indicated that arctigenin exerted its anti-inflammatory effect by inhibiting ROS-dependent STAT signaling through its antioxidant activity. Arctigenin also significantly reduced the phosphorylation of STAT1 and STAT 3 as well as JAK2 in LPS-stimulated RAW264.7 cells. The inhibitions of STAT1 and STAT 3 by arctigenin prevented their translocation to the nucleus and consequently inhibited expression of iNOS, thereby suppressing the expression of inflammation-associated genes, such as IL-1β, IL-6 and MCP-1, whose promoters contain STAT-binding elements. However, COX-2 expression was slightly inhibited at higher drug concentrations (50 μM). Our data demonstrate that arctigenin inhibits iNOS expression via suppressing JAK-STAT signaling pathway in macrophages. Crown Copyright © 2011. Published by Elsevier B.V. All rights reserved.

Targeting mitochondrial STAT3 with the novel phospho-valproic acid (MDC-1112) inhibits pancreatic cancer growth in mice.

PubMed

Mackenzie, Gerardo G; Huang, Liqun; Alston, Ninche; Ouyang, Nengtai; Vrankova, Kvetoslava; Mattheolabakis, George; Constantinides, Panayiotis P; Rigas, Basil

2013-01-01

New agents are needed to treat pancreatic cancer, one of the most lethal human malignancies. We synthesized phospho-valproic acid, a novel valproic acid derivative, (P-V; MDC-1112) and evaluated its efficacy in the control of pancreatic cancer. P-V inhibited the growth of human pancreatic cancer xenografts in mice by 60%-97%, and 100% when combined with cimetidine. The dominant molecular target of P-V was STAT3. P-V inhibited the phosphorylation of JAK2 and Src, and the Hsp90-STAT3 association, suppressing the activating phosphorylation of STAT3, which in turn reduced the expression of STAT3-dependent proteins Bcl-xL, Mcl-1 and survivin. P-V also reduced STAT3 levels in the mitochondria by preventing its translocation from the cytosol, and enhanced the mitochondrial levels of reactive oxygen species, which triggered apoptosis. Inhibition of mitochondrial STAT3 by P-V was required for its anticancer effect; mitochondrial STAT3 overexpression rescued animals from the tumor growth inhibition by P-V. Our results indicate that P-V is a promising candidate drug against pancreatic cancer and establish mitochondrial STAT3 as its key molecular target.

Effects and mechanisms of vitamin A and vitamin E on the levels of serum leptin and other related cytokines in rats with rheumatoid arthritis

PubMed Central

XIONG, RI-BO; LI, QING; WAN, WEI-REN; GUO, JIN-QIANG; LUO, BING-DE; GAN, LU

2014-01-01

Leptin has been identified as an important cytokine in the inflammatory networks of rheumatoid arthritis (RA). Higher serum leptin levels may accelerate the development of RA. This study aimed to examine the effects of vitamin A (VitA) and vitamin E (VitE) on the levels of leptin and other related experimental and clinical indices, and to explore the mechanisms of these effects through the Janus kinase/signal transducer and activator of transcription (STAT) signal transduction pathway in rats with collagen-induced arthritis (CIA). CIA model rats were established by the intradermal injection of bovine type II collagen emulsified in incomplete Freund’s adjuvant, followed by a booster intradermal injection. Four weeks later, the CIA model rats were treated with 42.86 μg retinol equivalents/kg body weight (b.w.) VitA or 200 mg/kg b.w. VitE for four weeks. The levels of leptin, tumor necrosis factor-α (TNF-α), interleukin (IL)-6, IL-10, IL-4, C-reactive protein (CRP) and rheumatic factor were measured by ELISA using commercial kits, and the erythrocyte sedimentation rate (ESR) was determined. In addition, the expression levels of phosphorylated (p)-STAT1, p-STAT3 and leptin in the synovium were evaluated by western blot analysis. The results indicated that VitA and VitE significantly reduced the levels of leptin, TNF-α, IL-6 and CRP and the ESR and significantly increased the levels of IL-10 compared with those of the model group. Furthermore, significantly reduced p-STAT3 protein expression levels were observed in the VitA and VitE groups. In conclusion, VitA and VitE reduced the levels of serum leptin protein and other cytokines. Furthermore, VitA and VitE also reduced the p-STAT3 protein levels. The present study may provide a novel approach for the treatment of RA. PMID:25009608

Adenylyl cyclase A expression is tip-specific in Dictyostelium slugs and directs StatA nuclear translocation and CudA gene expression.

PubMed

Verkerke-van Wijk, I; Fukuzawa, M; Devreotes, P N; Schaap, P

2001-06-01

cAMP oscillations, generated by adenylyl cyclase A (ACA), coordinate cell aggregation in Dictyostelium and have also been implicated in organizer function during multicellular development. We used a gene fusion of the ACA promoter with a labile lacZ derivative to study the expression pattern of ACA. During aggregation, most cells expressed ACA, but thereafter expression was lost in all cells except those of the anterior tip. Before aggregation, ACA transcription was strongly upregulated by nanomolar cAMP pulses. Postaggregative transcription was sustained by nanomolar cAMP pulses, but downregulated by a continuous micromolar cAMP stimulus and by the stalk-cell-inducing factor DIF. Earlier work showed that the transcription factor StatA displays tip-specific nuclear translocation and directs tip-specific expression of the nuclear protein CudA, which is essential for culmination. Both StatA and CudA were present in nuclei throughout the entire slug in an aca null mutant that expresses ACA from the constitutive actin15 promoter. This suggests that the tip-specific expression of ACA directs tip-specific nuclear translocation of StatA and tip-specific expression of CudA. Copyright 2001 Academic Press.

Role of PELP1 in EGFR-ER Signaling Crosstalk in Ovarian Cancer Cells

DTIC Science & Technology

2009-04-01

expression of genes involved in metastasis using a focused microarray approach. We have used Human Tumor Metastasis Microarray (Oligo GE array from...ovarian cancer progression. Analysis of human genome databases and SAGE data suggested deregulation of PELP1 expression in ovarian cancer cells...PI3K, and STAT3 in the cytosol. PELP1/MNAR regulates meiosis via its interactions with heterotimeric Gbc protein, androgen receptor (AR), and by

Mechanism for the differentiation of EoL-1 cells into eosinophils by histone deacetylase inhibitors.

PubMed

Kaneko, Motoko; Ishihara, Kenji; Takahashi, Aki; Hong, Jangja; Hirasawa, Noriyasu; Zee, Okpyo; Ohuchi, Kazuo

2007-01-01

EoL-1 cells have a FIP1L1-PDGFRA fusion gene which causes the transformation of eosinophilic precursor cells into leukemia cells. Recently, we suggested that the induction of differentiation of EoL-1 cells into eosinophils by the HDAC inhibitors apicidin and n-butyrate is due to the continuous inhibition of HDACs. However, neither apicidin nor n-butyrate inhibited the expression of FIP1L1-PDGFRA mRNA, although both these inhibitors suppressed cell proliferation. Therefore, in this study, we analyzed whether the levels of FIP1L1-PDGFRalpha protein and phosphorylated-Stat5 involved in the signaling for the proliferation of EoL-1 cells are attenuated by HDAC inhibitors. EoL-1 cells were incubated in the presence of apicidin, TSA or n-butyrate. FIP1L1-PDGFRalpha and phosphorylated-Stat5 were detected by Western blotting. Treatment of EoL-1 cells with apicidin at 100 nM or n-butyrate at 500 microM decreased the levels of FIP1L1-PDGFRalpha protein and phosphorylated-Stat5, while that with trichostatin A at 30 nM did not. The decrease in the level of FIP1L1-PDGFRalpha protein caused by apicidin and n-butyrate might be one of the mechanisms by which EoL-1 cells are induced to differentiate into eosinophils by these HDAC inhibitors.

DIESEL EXHAUST PARTICULATE (DEP)-INDUCED ACTIV ATION OF STAT3 REQUIRES ACTIVITIES OF EGFR AND SRC IN AIRWAY EPITHELIAL CELLS

EPA Science Inventory

In vivo exposure to diesel exhaust particles (DEP) elicits acute inflammatory responses in the lung characterized by inflammatory cell influx and elevated expression of mediators such as cytokines, and chemokines. Signal transducers and activators of transcription (STAT) protein...

Investigation of Dracocephalum kostchyi plant extract on the effective inflammatory transcription factors and mediators in activated macrophages.

PubMed

Kalantar, Kurosh; Gholijani, Nasser; Mousaei, Nashmin; Yazdani, Malihe; Amirghofran, Zahra

2018-06-07

Dracocephalum kotschyi is traditionally used for its anti-inflammatory effects. We aimed to investigate the effects of ethyl acetate extract of D. kotschyi on the expression of key inflammatory mediators and main signaling molecules involved in regulation of inflammation. Lipopolysaccharide (LPS)-stimulated J774.1 mouse macrophages were cultured in the presence of the plant extract and examined by the real time-PCR for gene expressions of interleukin (IL)-1β, tumor necrosis factor (TNF)-α, inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2. Cytokine levels and phosphorylated forms of stress-activated protein kinases/c-Jun N-terminal kinase (SAPK/JNK), signal transducer and activator of transcription (STAT)-3, p38, IκB-α and nuclear factor (NF)-κB p65 were determined using ELISA. The extract significantly reduced the expression of key mediators of inflammation. iNOS expression level decreased from 138±8.5 fold in LPS-only treated cells to 6.5±2.6 fold after treatment with 25 ug/ml of the extract (p<0.001). Similarly, COX-2 expression decreased from 632 ±98.8 fold in control to 124 ±24.6 fold (p<0.01). Treatment of cells with the extract significantly reduced IL-1β and TNF-α cytokines at both gene and protein expression levels. The extract at 25 µg/ml caused significant decreases in phospho-SAPK/JNK and phospho-STAT3 levels in macrophages (p<0.01). Proteins of phospho-p38, NFκB-p65 and phospho-NF-κB p65 had a reduced levels in treated cells (p<0.05). No significant change in phospho-IĸB level was observed. These findings suggested that D. kotschyi with inhibition of NF-κB, SAPK/JNK, STAT-3 and p-38 might have reduced the expression levels of key inflammatory mediators and thus possibly have potential beneficial impact on inflammatory diseases. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Cell signaling and transcription factor genes expressed during whole body regeneration in a colonial chordate.

PubMed

Rinkevich, Yuval; Rinkevich, Baruch; Reshef, Ram

2008-10-12

The restoration of adults from fragments of blood vessels in botryllid ascidians (termed whole body regeneration [WBR]) represents an inimitable event in the chordates, which is poorly understood on the mechanistic level. To elucidate mechanisms underlying this phenomenon, a subtracted EST library for early WBR stages was previously assembled, revealing 76 putative genes belonging to major signaling pathways, including Notch/Delta, JAK/STAT, protein kinases, nuclear receptors, Ras oncogene family members, G-Protein coupled receptor (GPCR) and transforming growth factor beta (TGF-beta) signaling. RT-PCR on selected transcripts documented specific up-regulation in only regenerating fragments, pointing to a broad activation of these signaling pathways at onset of WBR. The followed-up expression pattern of seven representative transcripts from JAK/STAT signaling (Bl-STAT), the Ras oncogene family (Bl-Rap1A, Bl-Rab-33), the protein kinase family (Bl-Mnk), Bl-Cnot, Bl-Slit and Bl-Bax inhibitor, revealed systemic and site specific activations during WBR in a sub-population of circulatory cells. WBR in the non-vertebrate chordate Botrylloides leachi is a multifaceted phenomenon, presided by a complex array of cell signaling and transcription factors. Above results, provide a first insight into the whole genome molecular machinery of this unique regeneration process, and reveal the broad participation of cell signaling and transcription factors in the process. While regeneration involves the participation of specific cell populations, WBR signals are systemically expressed at the organism level.

Loss of TIMP3 underlies diabetic nephropathy via FoxO1/STAT1 interplay.

PubMed

Fiorentino, Loredana; Cavalera, Michele; Menini, Stefano; Marchetti, Valentina; Mavilio, Maria; Fabrizi, Marta; Conserva, Francesca; Casagrande, Viviana; Menghini, Rossella; Pontrelli, Paola; Arisi, Ivan; D'Onofrio, Mara; Lauro, Davide; Khokha, Rama; Accili, Domenico; Pugliese, Giuseppe; Gesualdo, Loreto; Lauro, Renato; Federici, Massimo

2013-03-01

ADAM17 and its inhibitor TIMP3 are involved in nephropathy, but their role in diabetic kidney disease (DKD) is unclear. Diabetic Timp3(-/-) mice showed increased albuminuria, increased membrane thickness and mesangial expansion. Microarray profiling uncovered a significant reduction of Foxo1 expression in diabetic Timp3(-/-) mice compared to WT, along with FoxO1 target genes involved in autophagy, while STAT1, a repressor of FoxO1 transcription, was increased. Re-expression of Timp3 in Timp3(-/-) mesangial cells rescued the expression of Foxo1 and its targets, and decreased STAT1 expression to control levels; abolishing STAT1 expression led to a rescue of FoxO1, evoking a role of STAT1 in linking Timp3 deficiency to FoxO1. Studies on kidney biopsies from patients with diabetic nephropathy confirmed a significant reduction in TIMP3, FoxO1 and FoxO1 target genes involved in autophagy compared to controls, while STAT1 expression was strongly increased. Our study suggests that loss of TIMP3 is a hallmark of DKD in human and mouse models and designates TIMP3 as a new possible therapeutic target for diabetic nephropathy. Copyright © 2013 The Authors. Published by John Wiley and Sons, Ltd on behalf of EMBO.

STAT3 and STAT1 mediate IL-11–dependent and inflammation-associated gastric tumorigenesis in gp130 receptor mutant mice

PubMed Central

Ernst, Matthias; Najdovska, Meri; Grail, Dianne; Lundgren-May, Therese; Buchert, Michael; Tye, Hazel; Matthews, Vance B.; Armes, Jane; Bhathal, Prithi S.; Hughes, Norman R.; Marcusson, Eric G.; Karras, James G.; Na, Songqing; Sedgwick, Jonathon D.; Hertzog, Paul J.; Jenkins, Brendan J.

2008-01-01

Deregulated activation of STAT3 is frequently associated with many human hematological and epithelial malignancies, including gastric cancer. While exaggerated STAT3 signaling facilitates an antiapoptotic, proangiogenic, and proproliferative environment for neoplastic cells, the molecular mechanisms leading to STAT3 hyperactivation remain poorly understood. Using the gp130Y757F/Y757F mouse model of gastric cancer, which carries a mutated gp130 cytokine receptor signaling subunit that cannot bind the negative regulator of cytokine signaling SOCS3 and is characterized by hyperactivation of the signaling molecules STAT1 and STAT3, we have provided genetic evidence that IL-11 promotes chronic gastric inflammation and associated tumorigenesis. Expression of IL-11 was increased in gastric tumors in gp130Y757F/Y757F mice, when compared with unaffected gastric tissue in wild-type mice, while gp130Y757F/Y757F mice lacking the IL-11 ligand–binding receptor subunit (IL-11Rα) showed normal gastric STAT3 activation and IL-11 expression and failed to develop gastric tumors. Furthermore, reducing STAT3 activity in gp130Y757F/Y757F mice, either genetically or by therapeutic administration of STAT3 antisense oligonucleotides, normalized gastric IL-11 expression and alleviated gastric tumor burden. Surprisingly, the genetic reduction of STAT1 expression also reduced gastric tumorigenesis in gp130Y757F/Y757F mice and coincided with reduced gastric inflammation and IL-11 expression. Collectively, our data have identified IL-11 as a crucial cytokine promoting chronic gastric inflammation and associated tumorigenesis mediated by excessive activation of STAT3 and STAT1. PMID:18431520

IGF-1 induces the epithelial-mesenchymal transition via Stat5 in hepatocellular carcinoma.

PubMed

Zhao, Chuanzong; Wang, Qian; Wang, Ben; Sun, Qi; He, Zhaobin; Hong, Jianguo; Kuehn, Florian; Liu, Enyu; Zhang, Zongli

2017-12-19

It has been reported that the epithelial-mesenchymal transition (EMT) plays an important role in hepatocellular carcinoma (HCC). However, the relationship between the insulin-like growth factor-1 (IGF-1) and EMT of HCC was not fully elucidated. In the present work, we found that the expression of N-cadherin, Vimentin, Snail1, Snail2, and Twist1 was positively associated with IGF-1R expression, while E-cadherin expression was negatively associated with IGF-1 expression in human HCC samples. Furthermore, we observed that IGF-1 up-regulated the expression of N-cadherin, Vimentin, Snail1, Snail2 and Twist1, and down-regulated the expression of E-cadherin. In addition, Stat5 was induced in IGF-1-treated HepG2 and Hep3B cells, and Stat5 inhibition or siRNA significantly affected IGF-1-induced EMT in HepG2 and Hep3B cells. In conclusion, IGF-1 induces EMT of HCC via Stat5 signaling pathway. Thus, IGF-1/Stat5 can be recommended as a potential and novel therapeutic strategy for HCC patients.

Identification of JAK2 as a Mediator of FIP1L1-PDGFRA-Induced Eosinophil Growth and Function in CEL

PubMed Central

Li, Bin; Zhang, Guangsen; Li, Cui; He, Dan; Li, Xinying; Zhang, Chunfang; Tang, Faqing; Deng, Xiyun; Lu, Jingchen; Tang, Youhong; Li, Ruijuan; Chen, Zhuchu; Duan, Chaojun

2012-01-01

The Fip1-like1 (FIP1L1)-platelet-derived growth factor receptor alpha fusion gene (F/P) arising in the pluripotent hematopoietic stem cell (HSC),causes 14% to 60% of patients with hypereosinophilia syndrome (HES). These patients, classified as having F/P (+) chronic eosinophilic leukemia (CEL), present with clonal eosinophilia and display a more aggressive disease phenotype than patients with F/P (–) HES patients. The mechanisms underlying predominant eosinophil lineage targeting and the cytotoxicity of eosinophils in this leukemia remain unclear. Given that the Janus tyrosine kinase (JAK)/signal transducers and activators of transcription (Stat) signaling pathway is key to cytokine receptor-mediated eosinophil development and activated Stat3 and Stat5 regulate the expression of genes involved in F/P malignant transformation, we investigated whether and how JAK proteins were involved in the pathogenesis of F/P-induced CEL. F/P activation of JAK2, Stat3 and Stat5, were confirmed in all the 11 F/P (+) CEL patients examined. In vitro inhibition of JAK2 in EOL-1, primary F/P(+) CEL cells (PC) and T674I F/P Imatinib resistant cells(IR) by either JAK2-specific short interfering RNA (siRNA) or the tryphostin derivative AG490(AG490), significantly reduced cellular proliferation and induced cellular apoptosis. The F/P can enhance the IL-5-induced JAK2 activation, and further results indicated that JAK2 inhibition blocked IL-5-induced cellular migration and activation of the EOL-1 and PC cells in vitro. F/P-stimulation of the JAK2 suppressed cells led to a significantly reduction in Stat3 activation, but relatively normal induction of Stat5 activation. Interestingly, JAK2 inhibition also reduced PI3K, Akt and NF-κB activity in a dose-dependent manner, and suppressed expression levels of c-Myc and Survivin. These results strongly suggest that JAK2 is activated by F/P and is required for F/P stimulation of cellular proliferation and infiltration, possibly through induction of c-Myc and Survivin expression via activation of multiple signaling pathways, including NF-κB, Stat3, and PI3K/Akt. PMID:22523564

Over-expression of Stat5b confers protection against diabetes in the non-obese diabetic (NOD) mice via up-regulation of CD4{sup +}CD25{sup +} regulatory T cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jin, Yulan; Purohit, Sharad; Department of Pathology, Medical College of Georgia, Georgia Health Sciences University, GA

Highlights: Black-Right-Pointing-Pointer This is the first study to provide direct evidence of the role of Stat5b in NOD mice. Black-Right-Pointing-Pointer Over-expression of wild type Stat5b transgene protects NOD mice against diabetes. Black-Right-Pointing-Pointer This protection may be mediated by the up-regulation of CD4{sup +}CD25{sup +} Tregs. -- Abstract: The signal transducers and activators of transcription (STAT) family of proteins play a critical role in cytokine signaling required for fine tuning of immune regulation. Previous reports showed that a mutation (L327M) in the Stat5b protein leads to aberrant cytokine signaling in the NOD mice. To further elaborate the role of Stat5b inmore » diabetes, we established a NOD transgenic mouse that over-expresses the wild type Stat5b gene. The incidences of spontaneous diabetes as well as cyclophosphamide-induced diabetes were significantly reduced and delayed in the Stat5b transgenic NOD mice compared to their littermate controls. The total cell numbers of CD4{sup +} T cells and especially CD8{sup +} T cells in the spleen and pancreatic lymph node were increased in the Stat5b transgenic NOD mice. Consistent with these findings, CD4{sup +} and CD8{sup +} T cells from the Stat5b transgenic NOD mice showed a higher proliferation capacity and up-regulation of multiple cytokines including IL-2, IFN-{gamma}, TNF-{alpha} and IL-10 as well as anti-apoptotic gene Bcl-xl. Furthermore, the number and proportion of CD4{sup +}CD25{sup +} regulatory T cells were significantly increased in transgenic mice although in vitro suppression ability of the regulatory T-cells was not affected by the transgene. Our results suggest that Stat5b confers protection against diabetes in the NOD mice by regulating the numbers and function of multiple immune cell types, especially by up-regulating CD4{sup +}CD25{sup +} regulatory T cells.« less

COX-2 contributes to LPS-induced Stat3 activation and IL-6 production in microglial cells

PubMed Central

Zhu, Jie; Li, Shuzhen; Zhang, Yue; Ding, Guixia; Zhu, Chunhua; Huang, Songming; Zhang, Aihua; Jia, Zhanjun; Li, Mei

2018-01-01

Many stimuli including lipopolysaccharide (LPS) could activate microglial cells to subsequently cause inflammatory nerve injury. However, the mechanism of LPS-induced neuroinflammation in microglial cells is still elusive. Thus, the present study was undertaken to examine the role of COX-2 in mediating the activation of Stat3 and the production of IL-6 in BV2 cells challenged with LPS. After 24 h treatment, LPS dose-dependently enhanced COX-2 expression at both mRNA and protein levels. Meanwhile, IL-6 with other inflammatory cytokines including IL-1β, TNF-α, and MCP-1 were similarly enhanced by LPS. Then a specific COX-2 inhibitor (NS-398) was administered to BV2 before LPS treatment. Significantly, COX-2 inhibition suppressed the upregulation of IL-6 at both mRNA and protein levels in line with the trend blockade on IL-1β, TNF-α, and MCP-1. Stat3 drives proinflammatory signaling pathways and contributes to IL-6 production via a transcriptional mechanism in many diseases. Here we found that inhibition of COX-2 entirely blocked LPS-induced Stat3 phosphorylation, which might contribute to the blockade of IL-6 production to some extent. Meanwhile, COX-2 siRNA approach largely reproduced the phenotypes shown by specific COX-2 inhibitor in LPS-treated BV2 cells. Together, these findings suggested that COX-2 might contribute to LPS-induced IL-6 production possibly through activating Stat3 signaling pathway in microglial cells. PMID:29636886

IL-6 mediates differentiation disorder during spermatogenesis in obesity-associated inflammation by affecting the expression of Zfp637 through the SOCS3/STAT3 pathway.

PubMed

Huang, Guizhen; Yuan, Miao; Zhang, Jie; Li, Jun; Gong, Di; Li, Yanyan; Zhang, Jie; Lin, Ping; Huang, Lugang

2016-06-22

Zfp637 is a recently identified zinc finger protein, and its functions remain largely unknown. Here, we innovatively demonstrate the effects of Zfp637 on the differentiation of mouse spermatogonia and on its downstream target gene SOX2 in vitro. Obesity has been recognized as a chronic inflammatory disease that leads to decreased sexual function and sexual development disorders. We observed higher levels of IL-6 in serum and testis homogenates from obese mice compared with control mice. We also demonstrated that high levels of IL-6 inhibited Zfp637 expression, and we elucidated the underlying mechanisms. SOCS3 overexpression and STAT3 phosphorylation inhibitor (AG490) were used to investigate the function of the SOCS3/STAT3 pathway during this process. Our results showed that exposure of mouse spermatogonial cells to high levels of IL-6 inhibited Zfp637 expression by increasing SOCS3 expression and inhibiting the phosphorylation of STAT3, further reducing cellular differentiation. Consistent with the in vitro results, we observed increasing expression levels of SOCS3 and SOX2, but a reduction of Zfp637 expression, in obese mouse testes. In conclusion, Zfp637 plays a crucial role in spermatogenesis by downregulating SOX2 expression, and IL-6 can decrease the expression of Zfp637 through the SOCS3/STAT3 signaling pathway.

Multiple Functional Domains and Complexes of the Two Nonstructural Proteins of Human Respiratory Syncytial Virus Contribute to Interferon Suppression and Cellular Location▿

PubMed Central

Swedan, Samer; Andrews, Joel; Majumdar, Tanmay; Musiyenko, Alla; Barik, Sailen

2011-01-01

Human respiratory syncytial virus (RSV), a major cause of severe respiratory diseases, efficiently suppresses cellular innate immunity, represented by type I interferon (IFN), using its two unique nonstructural proteins, NS1 and NS2. In a search for their mechanism, NS1 was previously shown to decrease levels of TRAF3 and IKKε, whereas NS2 interacted with RIG-I and decreased TRAF3 and STAT2. Here, we report on the interaction, cellular localization, and functional domains of these two proteins. We show that recombinant NS1 and NS2, expressed in lung epithelial A549 cells, can form homo- as well as heteromers. Interestingly, when expressed alone, substantial amounts of NS1 and NS2 localized to the nuclei and to the mitochondria, respectively. However, when coexpressed with NS2, as in RSV infection, NS1 could be detected in the mitochondria as well, suggesting that the NS1-NS2 heteromer localizes to the mitochondria. The C-terminal tetrapeptide sequence, DLNP, common to both NS1 and NS2, was required for some functions, but not all, whereas only the NS1 N-terminal region was important for IKKε reduction. Finally, NS1 and NS2 both interacted specifically with host microtubule-associated protein 1B (MAP1B). The contribution of MAP1B in NS1 function was not tested, but in NS2 it was essential for STAT2 destruction, suggesting a role of the novel DLNP motif in protein-protein interaction and IFN suppression. PMID:21795342

Multiple functional domains and complexes of the two nonstructural proteins of human respiratory syncytial virus contribute to interferon suppression and cellular location.

PubMed

Swedan, Samer; Andrews, Joel; Majumdar, Tanmay; Musiyenko, Alla; Barik, Sailen

2011-10-01

Human respiratory syncytial virus (RSV), a major cause of severe respiratory diseases, efficiently suppresses cellular innate immunity, represented by type I interferon (IFN), using its two unique nonstructural proteins, NS1 and NS2. In a search for their mechanism, NS1 was previously shown to decrease levels of TRAF3 and IKKε, whereas NS2 interacted with RIG-I and decreased TRAF3 and STAT2. Here, we report on the interaction, cellular localization, and functional domains of these two proteins. We show that recombinant NS1 and NS2, expressed in lung epithelial A549 cells, can form homo- as well as heteromers. Interestingly, when expressed alone, substantial amounts of NS1 and NS2 localized to the nuclei and to the mitochondria, respectively. However, when coexpressed with NS2, as in RSV infection, NS1 could be detected in the mitochondria as well, suggesting that the NS1-NS2 heteromer localizes to the mitochondria. The C-terminal tetrapeptide sequence, DLNP, common to both NS1 and NS2, was required for some functions, but not all, whereas only the NS1 N-terminal region was important for IKKε reduction. Finally, NS1 and NS2 both interacted specifically with host microtubule-associated protein 1B (MAP1B). The contribution of MAP1B in NS1 function was not tested, but in NS2 it was essential for STAT2 destruction, suggesting a role of the novel DLNP motif in protein-protein interaction and IFN suppression.

A Review: Phytochemicals Targeting JAK/STAT Signaling and IDO Expression in Cancer.

PubMed

Arumuggam, Niroshaathevi; Bhowmick, Neil A; Rupasinghe, H P Vasantha

2015-06-01

Cancer remains a major health problem worldwide. Among many other factors, two regulatory defects that are present in most cancer cells are constitutive activation of Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling pathway and the induction of indoleamine 2, 3-dioxygenase (IDO), an enzyme that catalyzes tryptophan degradation, through JAK/STAT signaling. Cytokine signaling activates STAT proteins in regulating cell proliferation, differentiation, and survival through modulation of target genes. Many phytochemicals can inhibit both JAK/STAT signaling and IDO expression in antigen-presenting cells by targeting different pathways. Some of the promising phytochemicals that are discussed in this review include resveratrol, cucurbitacin, curcumin, (-)-epigallocatechin gallate, and others. It is now evident that phytochemicals play key roles in inhibition of tumor proliferation and development and provide novel means for therapeutic targeting of cancer. Copyright © 2015 John Wiley & Sons, Ltd.

Endosomal accumulation of Toll-like receptor 4 causes constitutive secretion of cytokines and activation of signal transducers and activators of transcription in Niemann-Pick disease type C (NPC) fibroblasts: a potential basis for glial cell activation in the NPC brain.

PubMed

Suzuki, Michitaka; Sugimoto, Yuko; Ohsaki, Yuki; Ueno, Makoto; Kato, Shinsuke; Kitamura, Yukisato; Hosokawa, Hiroshi; Davies, Joanna P; Ioannou, Yiannis A; Vanier, Marie T; Ohno, Kousaku; Ninomiya, Haruaki

2007-02-21

Niemann-Pick disease type C (NPC) is an inherited lipid storage disorder caused by mutations in NPC1 or NPC2 genes. Loss of function of either protein results in the endosomal accumulation of cholesterol and other lipids, progressive neurodegeneration, and robust glial cell activation. Here, we report that cultured human NPC fibroblasts secrete interferon-beta, interleukin-6 (IL-6), and IL-8, and contain increased levels of signal transducers and activators of transcription (STATs). These cells also contained increased levels of Toll-like receptor 4 (TLR4) that accumulated in cholesterol-enriched endosomes/lysosomes, and small interfering RNA knockdown of this receptor reduced cytokine secretion. In the NPC1-/- mouse brain, glial cells expressed TLR4 and IL-6, whereas both glial and neuronal cells expressed STATs. Genetic deletion of TLR4 in NPC1-/- mice reduced IL-6 secretion by cultured fibroblasts but failed to alter STAT levels or glial cell activation in the brain. In contrast, genetic deletion of IL-6 normalized STAT levels and suppressed glial cell activation. These findings indicate that constitutive cytokine secretion leads to activation of STATs in NPC fibroblasts and that this secretion is partly caused by an endosomal accumulation of TLR4. These results also suggest that similar signaling events may underlie glial cell activation in the NPC1-/- mouse brain.

Knockdown of NogoA prevents MPP+‑induced neurotoxicity in PC12 cells via the mTOR/STAT3 signaling pathway.

PubMed

Zhong, Jianbin; Li, Xie; Wan, Limei; Chen, Zhibang; Zhong, Simin; Xiao, Songhua; Yan, Zhengwen

2016-02-01

NogoA is a myelin‑associated protein, which is important in the inhibition of axonal fiber growth and in regeneration following injury of the mammalian central nervous system. A previous study suggested that NogoA may be key in the process of Parkinson's disease (PD), which is the second most common chronic neurodegenerative disorder worldwide. The regulatory mechanism underlying the effect of NogoA on the process of PD remains to be fully elucidated. The present study aimed to investigate the effect and underlying mechanism of NogoA on cellular viability, apoptosis and autophagy induced by 1-methyl-4-phenylpyridinium (MPP+) in PC12 cells, a commonly used in vitro PD model. PC12 cells were treated with 1 mM MPP+ for 24 h and the cells were harvested for western blotting. The results demonstrated that the protien expression levels of NogoA were increased in the PC12 cells treated with MPP+. Subsequently, NogoA small interfering RNA was synthesized and transfected into PC12 cells to silence the expression of NogoA. NogoA knockdown significantly reduced the MPP+‑induced decrease in cell viability and apoptosis, detected using a cell counting kit‑8 and flow cytometric analysis, respectively. Interference in the expression of NogoA increased the MPP+‑induced decrease in mitochondrial membrane potential, determined quantitatively by flow cytometry using JC-1 dye, and the protein levels of Beclin‑1. In addition, MPP+ treatment activated the mammalian target of rapamycin (mTOR)/signal transducer and activator of transcription 3 (STAT3) signaling pathway. Knockdown of NogoA significantly inhibited the expression levels of mTOR and STAT3. Furthermore, overexpression of NogoA had similar neurotoxic effects on the PC12 cells as MPP+ treatment. Treatment with rapamycin, an inhibitor of the mTOR/STAT3 signaling pathway had a similar effect to that of NogoA knockdown in the MPP+‑treated PC12 cells. Taken together, the results from the present study demonstrated that NogoA may regulate MPP+‑induced neurotoxicity in PC12 cells via the mTOR/STAT3 signaling pathway and provided an explanation regarding the regulatory mechanism of NogoA on the process of PD.

The Human Respiratory Syncytial Virus Nonstructural Protein 1 Regulates Type I and Type II Interferon Pathways*

PubMed Central

Hastie, Marcus L.; Headlam, Madeleine J.; Patel, Nirav B.; Bukreyev, Alexander A.; Buchholz, Ursula J.; Dave, Keyur A.; Norris, Emma L.; Wright, Cassandra L.; Spann, Kirsten M.; Collins, Peter L.; Gorman, Jeffrey J.

2012-01-01

Respiratory syncytial viruses encode a nonstructural protein (NS1) that interferes with type I and III interferon and other antiviral responses. Proteomic studies were conducted on human A549 type II alveolar epithelial cells and type I interferon-deficient Vero cells (African green monkey kidney cells) infected with wild-type and NS1-deficient clones of human respiratory syncytial virus to identify other potential pathway and molecular targets of NS1 interference. These analyses included two-dimensional differential gel electrophoresis and quantitative Western blotting. Surprisingly, NS1 was found to suppress the induction of manganese superoxide dismutase (SOD2) expression in A549 cells and to a much lesser degree Vero cells in response to infection. Because SOD2 is not directly inducible by type I interferons, it served as a marker to probe the impact of NS1 on signaling of other cytokines known to induce SOD2 expression and/or indirect effects of type I interferon signaling. Deductive analysis of results obtained from cell infection and cytokine stimulation studies indicated that interferon-γ signaling was a potential target of NS1, possibly as a result of modulation of STAT1 levels. However, this was not sufficient to explain the magnitude of the impact of NS1 on SOD2 induction in A549 cells. Vero cell infection experiments indicated that NS1 targeted a component of the type I interferon response that does not directly induce SOD2 expression but is required to induce another initiator of SOD2 expression. STAT2 was ruled out as a target of NS1 interference using quantitative Western blot analysis of infected A549 cells, but data were obtained to indicate that STAT1 was one of a number of potential targets of NS1. A label-free mass spectrometry-based quantitative approach is proposed as a means of more definitive identification of NS1 targets. PMID:22322095

Reciprocal activation of α5-nAChR and STAT3 in nicotine-induced human lung cancer cell proliferation.

PubMed

Zhang, Yao; Jia, Yanfei; Li, Ping; Li, Huanjie; Xiao, Dongjie; Wang, Yunshan; Ma, Xiaoli

2017-07-20

Cigarette smoking is the top environmental risk factor for lung cancer. Nicotine, the addictive component of cigarettes, induces lung cancer cell proliferation, invasion and migration via the activation of nicotinic acetylcholine receptors (nAChRs). Genome-wide association studies (GWAS) show that CHRNA5 gene encoding α5-nAChR is especially relevant to lung cancer. However, the mechanism of this subunit in lung cancer is not clear. In the present study, we demonstrate that the expression of α5-nAChR is correlated with phosphorylated STAT3 (pSTAT3) expression, smoking history and lower survival of non-small cell lung cancer (NSCLC) samples. Nicotine increased the levels of α5-nAChR mRNA and protein in NSCLC cell lines and activated the JAK2/STAT3 signaling cascade. Nicotine-induced activation of JAK2/STAT3 signaling was inhibited by the silencing of α5-nAChR. Characterization of the CHRNA5 promoter revealed four STAT3-response elements. ChIP assays confirmed that the CHRNA5 promoter contains STAT3 binding sites. By silencing STAT3 expression, nicotine-induced upregulation of α5-nAChR was suppressed. Downregulation of α5-nAChR and/or STAT3 expression inhibited nicotine-induced lung cancer cell proliferation. These results suggest that there is a feedback loop between α5-nAChR and STAT3 that contributes to the nicotine-induced tumor cell proliferation, which indicates that α5-nAChR is an important therapeutic target involved in tobacco-associated lung carcinogenesis. Copyright © 2017 Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, and Genetics Society of China. Published by Elsevier Ltd. All rights reserved.

STAT3 and importins are novel mediators of early molecular and cellular responses in experimental duodenal ulceration.

PubMed

Khomenko, Tetyana; Deng, Xiaoming; Ahluwalia, Amrita; Tarnawski, Andrzej; Patel, Khushin N; Sandor, Zsuzsanna; Szabo, Sandor

2014-02-01

Signal transducer and activator of transcription 3 (STAT3) is a transcription factor that directly upregulates VEGF, Ref-1, p21, and anti-apoptotic genes such as Bcl-xL. In this study, we hypothesized that STAT3 signaling is activated and provides a critical protective role that is required for enterocyte survival during the early phases of cysteamine-induced duodenal ulcers. We studied the effect of inhibition of STAT3 activity on cysteamine-induced duodenal ulcers in rats and egr-1 knockout mice using STAT3/DNA binding assay, immunohistochemistry, immunoblot, and quantitative reverse transcriptase PCR analyses. We found that G-quartet oligodeoxynucleotides T40214, a specific inhibitor of STAT3/DNA binding, aggravated cysteamine-induced duodenal ulcers in rats 2.8-fold (p < 0.05). In the pre-ulcerogenic stage, cysteamine induced STAT3 tyrosine phosphorylation, its translocation to nuclei, an increased expression and nuclear translocation of importin α and β in the rat duodenal mucosa. Cysteamine enhanced the binding of STAT3 to its DNA consensus sequences at 6, 12, and 24 h after cysteamine by 1.5-, 1.8-, and 3.5-fold, respectively, and activated the expression of STAT3 target genes such as VEGF, Bcl-xL, Ref-1, and STAT3-induced feedback inhibitor, a suppressor of cytokine signaling 3. We also demonstrated that egr-1 knockout mice, which are more susceptible to cysteamine-induced duodenal ulcers, had lower levels of STAT3 expression, its phosphorylation, expression of importin α or β, and STAT3/DNA binding than wild-type mice in response to cysteamine. Thus, STAT3 represents an important new molecular mechanism in experimental duodenal ulceration.

Stat5 Signaling Specifies Basal versus Stress Erythropoietic Responses through Distinct Binary and Graded Dynamic Modalities

PubMed Central

Porpiglia, Ermelinda; Hidalgo, Daniel; Koulnis, Miroslav; Tzafriri, Abraham R.; Socolovsky, Merav

2012-01-01

Erythropoietin (Epo)-induced Stat5 phosphorylation (p-Stat5) is essential for both basal erythropoiesis and for its acceleration during hypoxic stress. A key challenge lies in understanding how Stat5 signaling elicits distinct functions during basal and stress erythropoiesis. Here we asked whether these distinct functions might be specified by the dynamic behavior of the Stat5 signal. We used flow cytometry to analyze Stat5 phosphorylation dynamics in primary erythropoietic tissue in vivo and in vitro, identifying two signaling modalities. In later (basophilic) erythroblasts, Epo stimulation triggers a low intensity but decisive, binary (digital) p-Stat5 signal. In early erythroblasts the binary signal is superseded by a high-intensity graded (analog) p-Stat5 response. We elucidated the biological functions of binary and graded Stat5 signaling using the EpoR-HM mice, which express a “knocked-in” EpoR mutant lacking cytoplasmic phosphotyrosines. Strikingly, EpoR-HM mice are restricted to the binary signaling mode, which rescues these mice from fatal perinatal anemia by promoting binary survival decisions in erythroblasts. However, the absence of the graded p-Stat5 response in the EpoR-HM mice prevents them from accelerating red cell production in response to stress, including a failure to upregulate the transferrin receptor, which we show is a novel stress target. We found that Stat5 protein levels decline with erythroblast differentiation, governing the transition from high-intensity graded signaling in early erythroblasts to low-intensity binary signaling in later erythroblasts. Thus, using exogenous Stat5, we converted later erythroblasts into high-intensity graded signal transducers capable of eliciting a downstream stress response. Unlike the Stat5 protein, EpoR expression in erythroblasts does not limit the Stat5 signaling response, a non-Michaelian paradigm with therapeutic implications in myeloproliferative disease. Our findings show how the binary and graded modalities combine to generate high-fidelity Stat5 signaling over the entire basal and stress Epo range. They suggest that dynamic behavior may encode information during STAT signal transduction. PMID:22969412

The Janus kinase inhibitor tofacitinib inhibits TNF-α-induced gliostatin expression in rheumatoid fibroblast-like synoviocytes.

PubMed

Kawaguchi, Yohei; Waguri-Nagaya, Yuko; Tatematsu, Naoe; Oguri, Yusuke; Kobayashi, Masaaki; Nozaki, Masahiro; Asai, Kiyofumi; Aoyama, Mineyoshi; Otsuka, Takanobu

2018-01-15

Gliostatin (GLS) is known to have angiogenic and arthritogenic activity, and GLS expression levels in serum from patients with rheumatoid arthritis (RA) are significantly correlated with the disease activity. Tofacitinib is a novel oral Janus kinase (JAK) inhibitor and is effective in treating RA. However, the mechanism of action of tofacitinib in fibroblast-like synoviocytes (FLSs) has not been elucidated. The purpose of this study was to investigate the modulatory effects of tofacitinib on serum GLS levels in patients with RA and GLS production in FLSs derived from patients with RA. Six patients with RA who had failed therapy with at least one TNF inhibitor and were receiving tofacitinib therapy were included in the study. Serum samples were collected to measure CRP, MMP-3 and GLS expression. FLSs derived from patients with RA were cultured and stimulated by TNFα with or without tofacitinib. GLS expression levels were determined using reverse transcription-polymerase chain reaction (RT-PCR), EIA and immunocytochemistry, and signal transducer and activator of transcription (STAT) protein phosphorylation levels were determined by western blotting. Treatment with tofacitinib decreased serum GLS levels in all patients. GLS mRNA and protein expression levels were significantly increased by treatment with TNF-α alone, and these increases were suppressed by treatment with tofacitinib, which also inhibited TNF-α-induced STAT1 phosphorylation. JAK/STAT activation plays a pivotal role in TNF-α-mediated GLS up-regulation in RA. Suppression of GLS expression in FLSs has been suggested to be one of the mechanisms through which tofacitinib exerts its anti-inflammatory effects.

Notch1-STAT3-ETBR signaling axis controls reactive astrocyte proliferation after brain injury.

PubMed

LeComte, Matthew D; Shimada, Issei S; Sherwin, Casey; Spees, Jeffrey L

2015-07-14

Defining the signaling network that controls reactive astrogliosis may provide novel treatment targets for patients with diverse CNS injuries and pathologies. We report that the radial glial cell antigen RC2 identifies the majority of proliferating glial fibrillary acidic protein-positive (GFAP(+)) reactive astrocytes after stroke. These cells highly expressed endothelin receptor type B (ETB(R)) and Jagged1, a Notch1 receptor ligand. To study signaling in adult reactive astrocytes, we developed a model based on reactive astrocyte-derived neural stem cells isolated from GFAP-CreER-Notch1 conditional knockout (cKO) mice. By loss- and gain-of-function studies and promoter activity assays, we found that Jagged1/Notch1 signaling increased ETB(R) expression indirectly by raising the level of phosphorylated signal transducer and activator of transcription 3 (STAT3), a previously unidentified EDNRB transcriptional activator. Similar to inducible transgenic GFAP-CreER-Notch1-cKO mice, GFAP-CreER-ETB(R)-cKO mice exhibited a defect in reactive astrocyte proliferation after cerebral ischemia. Our results indicate that the Notch1-STAT3-ETB(R) axis connects a signaling network that promotes reactive astrocyte proliferation after brain injury.

Notch1–STAT3–ETBR signaling axis controls reactive astrocyte proliferation after brain injury

PubMed Central

LeComte, Matthew D.; Shimada, Issei S.; Sherwin, Casey; Spees, Jeffrey L.

2015-01-01

Defining the signaling network that controls reactive astrogliosis may provide novel treatment targets for patients with diverse CNS injuries and pathologies. We report that the radial glial cell antigen RC2 identifies the majority of proliferating glial fibrillary acidic protein-positive (GFAP+) reactive astrocytes after stroke. These cells highly expressed endothelin receptor type B (ETBR) and Jagged1, a Notch1 receptor ligand. To study signaling in adult reactive astrocytes, we developed a model based on reactive astrocyte-derived neural stem cells isolated from GFAP-CreER-Notch1 conditional knockout (cKO) mice. By loss- and gain-of-function studies and promoter activity assays, we found that Jagged1/Notch1 signaling increased ETBR expression indirectly by raising the level of phosphorylated signal transducer and activator of transcription 3 (STAT3), a previously unidentified EDNRB transcriptional activator. Similar to inducible transgenic GFAP-CreER-Notch1-cKO mice, GFAP-CreER-ETBR-cKO mice exhibited a defect in reactive astrocyte proliferation after cerebral ischemia. Our results indicate that the Notch1–STAT3–ETBR axis connects a signaling network that promotes reactive astrocyte proliferation after brain injury. PMID:26124113

The IGF-I/JAK2-STAT3/miR-21 signaling pathway may be associated with human renal cell carcinoma cell growth.

PubMed

Su, Ying; Zhao, An; Cheng, Guoping; Xu, Jingjing; Ji, Enming; Sun, Wenyong

2017-07-04

Renal cell carcinoma (RCC) is the highest mortality rate of the genitourinary cancers, and the treatment options are very limited. Thus, identification of molecular mechanisms underlying RCC tumorigenesis, is critical for identifying biomarkers for RCC diagnosis and prognosis. To validate whether the IGF-I/JAK2-STAT3/miR-21 signaling pathway is associated with human RCC cell growth. qRT-PCR and Western blotting were used to detect the mRNA and protein expression levels, respectively. The MTT assay was performed to determine cell survival rate. The Annexin V-FITC/PI apoptosis detection kit was used to detect cell apoptosis. We employed RCC tissues and cell lines (A498; ACHN; Caki-1; Caki-2 and 786-O) in the study. IGF-I, and its inhibitor (NT-157) were administrated to detect the effects of IGF-I on the expression of miR-21 and p-JAK2. JAK2 inhibitor (AG490), and si-STAT3 were used to detect the effects of JAK2/STAT3 signaling pathway on the expression of miR-21. In our study, we firstly showed that the expression levels of IGF-I and miR-21 were up-regulated in RCC tissues and cell lines. After exogenous IGF-I treatment, the expression levels of miR-21, p-IGF-IR and p-JAK2 were significantly increased, whereas NT-157 treatment showed the reversed results. Further study indicated that JAK2 inhibitor or si-STAT3 significantly reversed the IGF-I-induced miR-21 expression level. Finally, we found that IGF-I treatment significantly prompted human RCC cell survival and inhibited cell apoptosis, and NT-157 treatment showed the reversed results. The IGF-I/JAK2-STAT3/miR-21 signaling pathway may be associated with human RCC cell growth.

Overexpression of B7-H3 augments anti-apoptosis of colorectal cancer cells by Jak2-STAT3.

PubMed

Zhang, Ting; Jiang, Bo; Zou, Shi-Tao; Liu, Fen; Hua, Dong

2015-02-14

To investigate the role of the overexpression of B7-H3 in apoptosis in colorectal cancer cell lines and the underlying molecular mechanisms. SW620 cells that highly overexpressed B7-H3 (SW620-B7-H3-EGFP) and HCT8 cells stably transfected with B7-H3 shRNA (HCT8-shB7-H3) were previously constructed in our laboratory. Cells transfected with pIRES2-EGFP were used as negative controls (SW620-NC and HCT8-NC). Real-time PCR and western blotting analysis were used to detect the mRNA and protein expressions of the apoptosis regulator proteins Bcl-2, Bcl-xl and Bax. A cell proliferation assay was used to evaluate the survival rate and drug sensitivity of the cells. The effect of drug resistance was detected by a cell cycle assay. Active caspase-3 western blotting was used to reflect the anti-apoptotic ability of cells. Western blotting was also performed to determine the expression of proteins associated with the Jak2-STAT3 signaling pathway and the apoptosis regulator proteins after the treatment with AG490, a Jak2 specific inhibitor, in B7-H3 overexpressing cells. The data were analyzed by GraphPad Prism 6 using a non-paired t-test. Whether by overexpression in SW620 cells or downregulation in HCT8, B7-H3 significantly affected the expression of anti- and pro-apoptotic proteins, at both the transcriptional and translational levels, compared with the negative control (P < 0.05). A cell proliferation assay revealed that B7-H3 overexpression increased the drug resistance of cells and resulted in a higher survival rate (P < 0.05). In addition, the results of cell cycle and active caspase-3 western blotting proved that B7-H3 overexpression inhibited apoptosis in colorectal cancer cell lines (P < 0.05). B7-H3 overexpression improved Jak2 and STAT3 phosphorylation and, in turn, increased the expression of the downstream anti-apoptotic proteins B-cell CLL/lymphoma 2 (Bcl-2) and Bcl-xl, based on western blotting (P < 0.05). After treating B7-H3 overexpressing cells with the Jak2-specific inhibitor AG490, the phosphorylation of Jak2 and STAT3, and the expression of Bcl-2 and Bcl-xl, decreased accordingly (P < 0.05). This finding suggested that the Jak2-STAT3 pathway is involved in the mechanism mediating the anti-apoptotic ability of B7-H3. The overexpression of B7-H3 induces resistance to apoptosis in colorectal cancer cell lines by upregulating the Jak2-STAT3 signaling pathway, potentially providing new approaches to the treatment of colorectal cancer.

Interferon Alpha Signalling and Its Relevance for the Upregulatory Effect of Transporter Proteins Associated with Antigen Processing (TAP) in Patients with Malignant Melanoma

PubMed Central

Ensslen, Silke; Marquardt, Yvonne; Czaja, Katharina; Joussen, Sylvia; Beer, Daniel; Abele, Rupert; Plewnia, Gabriele; Tampé, Robert; Merk, Hans F.; Hermanns, Heike M.; Baron, Jens M.

2016-01-01

Introduction Interferon alpha (IFNα) is routinely used in the clinical practice for adjuvant systemic melanoma therapy. Understanding the molecular mechanism of IFNα effects and prediction of response in the IFNα therapy regime allows initiation and continuation of IFNα treatment for responder and exclusion of non-responder to avoid therapy inefficacy and side-effects. The transporter protein associated with antigen processing-1 (TAP1) is part of the MHC class I peptide-loading complex, and important for antigen presentation in tumor and antigen presenting cells. In the context of personalized medicine, we address this potential biomarker TAP1 as a target of IFNα signalling. Results We could show that IFNα upregulates TAP1 expression in peripheral blood mononuclear cells (PBMCs) of patients with malignant melanoma receiving adjuvant high-dose immunotherapy. IFNα also induced expression of TAP1 in mouse blood and tumor tissue and suppressed the formation of melanoma metastasis in an in vivo B16 tumor model. Besides its expression, TAP binding affinity and transport activity is induced by IFNα in human monocytic THP1 cells. Furthermore, our data revealed that IFNα clearly activates phosphorylation of STAT1 and STAT3 in THP1 and A375 melanoma cells. Inhibition of Janus kinases abrogates the IFNα-induced TAP1 expression. These results suggest that the JAK/STAT pathway is a crucial mediator for TAP1 expression elicited by IFNα treatment. Conclusion We suppose that silencing of TAP1 expression provides tumor cells with a mechanism to escape cytotoxic T-lymphocyte recognition. The observed benefit of IFNα treatment could be mediated by the shown dual effect of TAP1 upregulation in antigen presenting cells on the one hand, and of TAP1 upregulation in ‘silent’ metastatic melanoma cells on the other hand. In conclusion, this work contributes to a better understanding of the mode of action of IFNα which is essential to identify markers to predict, assess and monitor therapeutic response of IFNα treatment in the future. PMID:26735690

Suppressive effects of formoterol and salmeterol on eotaxin-1 in bronchial epithelial cells.

PubMed

Chu, Yu-Te; Chang, Tai-Tsung; Jong, Yuh-Jyh; Kuo, Po-Lin; Lee, Hsi-Ming; Lee, Min-Sheng; Chang, Hui-Wen; Hung, Chih-Hsing

2010-03-01

Eotaxin-1 (CCL11), an eosinophil-specific C-C chemokine, is a potent chemoattractant for mobilization of eosinophils into airways after allergic stimulation. Eotaxin-1 recruits eosinophils into inflammatory sites, and may play a role in the pathogenesis of asthma. Formoterol and salmeterol are two inhaled long acting beta(2) adrenoceptor agonists (LABAs), widely used for the local treatment of asthma. However, little is known about their effects on the eotaxin-1 expression of bronchial epithelial cells. BEAS-2B cells were stimulated by adding IL-4 with or without 2 h pre-treatment of formoterol or salmeterol. The protein and mRNA expression of eotaxin-1 were measured by ELISA assay and real-time PCR, respectively. Effects of formoterol and salmeterol on nuclear and cytosolic pSTAT-6 expression were evaluated by Western blot and immunofluorescence study. Formoterol and salmeterol (10(-7)-10(-10) m) significantly down-regulated IL-4- induced eotaxin-1 expression in BEAS-2B cells. A specific beta(2) adrenoceptor antagonist (ICI 118,551) reversed their suppression of eotaxin-1 production. Forskolin, an cAMP activator, could also suppress the expression of eotaxin-1 by IL-4 in a dose dependent manner (10(-7)-10(-10 )m). The western blot and immunofluorescence studies demonstrated that formoterol 10(-7 )m suppressed the nuclear expression of pSTAT-6. Formoterol and salmeterol, two inhaled long-acting beta(2) agonists, down-regulated IL-4- induced eotaxin-1 expression in BEAS-2B cells. The effect was mediated via the beta(2) adrenoceptor, and cAMP. Formoterol significantly down-regulated pSTAT6 at higher concentration, and further turned off the IL-4 signaling pathway.

Spatio-temporal kinetics of growth hormone receptor signaling in single cells using FRET microscopy.

PubMed

Biener-Ramanujan, Eva; Ramanujan, V Krishnan; Herman, Brian; Gertler, Arieh

2006-08-01

The growth hormone (GH) receptor (R)-mediated JAK2 (Janus kinase-2)-STAT5 (signaling transducer and activator of transcription-5) pathway involves a cascade of protein-protein interactions and tyrosine phosphorylations that occur in a spatially and temporally sensitive manner in cells. To study GHR dimerization or GH-induced conformational change of predimerized GHRs and STAT5 activation kinetics in intact cells, fluorescence resonance energy transfer (FRET) and live-cell imaging methods were employed. FRET measurements at the membrane of HEK-293T cells co-expressing GHRs tagged at the C-terminus with cyan (C) and yellow (Y) fluorescent proteins (FPs) revealed transient GHR dimerization lasting 2-3 min, with a maximum at 3 min after GH stimulation, which was sufficient to induce STAT5 activation. The transient nature of the dimerization or GH-induced conformational change of predimerized GHRs kinetics was not a result of GHR internalization, as neither potassium- nor cholesterol-depletion treatments prolonged the FRET signal. YFP-tagged STAT5 recruitment to the membrane, binding to GHR-CFP, and phosphorylation, occurred within minutes of GH stimulation. Activated STAT5a-YFP did not show nuclear accumulation, despite nuclear pSTAT5 increase, suggesting high turnover of STAT5 nuclear shuttling. Although GHR dimerization and STAT5 activation have been reported previously, this is the first spatially resolved demonstration of GHR-signaling kinetics in intact cells.

The novel anti-adipogenic effect and mechanisms of action of SGI-1776, a Pim-specific inhibitor, in 3T3-L1 adipocytes.

PubMed

Park, Yu-Kyoung; Hong, Victor Sukbong; Lee, Tae-Yoon; Lee, Jinho; Choi, Jong-Soon; Park, Dong-Soon; Park, Gi-Young; Jang, Byeong-Churl

2016-01-01

The proviral integration site for moloney murine leukemia virus (Pim) kinases, consisting of Pim-1, Pim-2 and Pim-3, belongs to a family of serine/threonine kinases that are involved in controlling cell growth and differentiation. Pim kinases are emerging as important mediators of adipocyte differentiation. SGI-1776, an inhibitor of Pim kinases, is widely used to assess the physiological roles of Pim kinases, particularly cell functions. In the present study, we examined the effects of SGI-1776 on adipogenesis. The anti‑adipogenic effect of SGI‑1776 was measured by Oil Red O staining and AdipoRed assays. The effect of SGI‑1776 on the growth of 3T3‑L1 adipocytes was determined by cell count analysis. The effects of SGI‑1776 on the protein and mRNA expression of adipogenesis-related proteins and adipokines in 3T3‑L1 adipocytes were also evaluated by western blot analysis and RT‑PCR, respectively. Notably, SGI-1776 markedly inhibited lipid accumulation during the differentiation of 3T3-L1 preadipocytes into adipocytes. On a mechanistic level, SGI-1776 inhibited not only the expression of CCAAT/enhancer-binding protein-α (C/EBP-α), peroxisome proliferator-activated receptor-γ (PPAR-γ) and fatty acid synthase (FAS), but also the phosphorylation of signal transducer and activator of transcription-3 (STAT-3). Moreover, SGI-1776 decreased the expression of adipokines, including the expression of leptin and regulated on activation, normal T cell expressed and secreted (RANTES) during adipocyte differentiation. These findings demonstrate that SGI-1776 inhibits adipogenesis by downregulating the expression and/or phosphorylation levels of C/EBP-α, PPAR-γ, FAS and STAT-3.

Preventing intimal thickening of vein grafts in vein artery bypass using STAT-3 siRNA

PubMed Central

2012-01-01

Background Proliferation and migration of vascular smooth muscle cells (VSMCs) play a key role in neointimal formation which leads to restenosis of vein graft in venous bypass. STAT-3 is a transcription factor associated with cell proliferation. We hypothesized that silencing of STAT-3 by siRNA will inhibit proliferation of VSMCs and attenuate intimal thickening. Methods Rat VSMCs were isolated and cultured in vitro by applying tissue piece inoculation methods. VSMCs were transfected with STAT 3 siRNA using lipofectamine 2000. In vitro proliferation of VSMC was quantified by the MTT assay, while in vivo assessment was performed in a venous transplantation model. In vivo delivery of STAT-3 siRNA plasmid or scramble plasmid was performed by admixing with liposomes 2000 and transfected into the vein graft by bioprotein gel applied onto the adventitia. Rat jugular vein-carotid artery bypass was performed. On day 3 and7 after grafting, the vein grafts were extracted, and analyzed morphologically by haematoxylin eosin (H&E), and assessed by immunohistochemistry for expression of Ki-67 and proliferating cell nuclear antigen (PCNA). Western-blot and reverse transcriptase polymerase chain reaction (RT-PCR) were used to detect the protein and mRNA expression in vivo and in vitro. Cell apoptosis in vein grafts was detected by TUNEL assay. Results MTT assay shows that the proliferation of VSMCs in the STAT-3 siRNA treated group was inhibited. On day 7 after operation, a reduced number of Ki-67 and PCNA positive cells were observed in the neointima of the vein graft in the STAT-3 siRNA treated group as compared to the scramble control. The PCNA index in the control group (31.3 ± 4.7) was higher than that in the STAT-3 siRNA treated group (23.3 ± 2.8) (P < 0.05) on 7d. The neointima in the experimental group(0.45 ± 0.04 μm) was thinner than that in the control group(0.86 ± 0.05 μm) (P < 0.05).Compared with the control group, the protein and mRNA levels in the experimental group in vivo and in vitro decreased significantly. Down regulation of STAT-3 with siRNA resulted in a reduced expression of Bcl-2 and cyclin D1. However, apoptotic cells were not obviously found in all grafts on day 3 and 7 post surgery. Conclusions The STAT-3 siRNA can inhibit the proliferation of VSMCs in vivo and in vitro and attenuate neointimal formation. PMID:22216901

IL-22/STAT3-Induced Increases in SLURP1 Expression within Psoriatic Lesions Exerts Antimicrobial Effects against Staphylococcus aureus.

PubMed

Moriwaki, Yasuhiro; Takada, Kiyoko; Nagasaki, Toshinori; Kubo, Natsuki; Ishii, Tomohiro; Kose, Kazuaki; Kageyama, Taihei; Tsuji, Shoutaro; Kawashima, Koichiro; Misawa, Hidemi

2015-01-01

SLURP1 is the causal gene for Mal de Meleda (MDM), an autosomal recessive skin disorder characterized by diffuse palmoplantar keratoderma and transgressive keratosis. Moreover, although SLURP1 likely serves as an important proliferation/differentiation factor in keratinocytes, the possible relation between SLURP1 and other skin diseases, such as psoriasis and atopic dermatitis, has not been studied, and the pathophysiological control of SLURP1 expression in keratinocytes is largely unknown. Our aim was to examine the involvement of SLURP1 in the pathophysiology of psoriasis using an imiquimod (IMQ)-induced psoriasis model mice and normal human epidermal keratinocytes (NHEKs). SLURP1 expression was up-regulated in the skin of IMQ-induced psoriasis model mice. In NHEKs stimulated with the inflammatory cytokines IL-17, IL-22 and TNF-α, which are reportedly expressed in psoriatic lesions, SLURP1 mRNA expression was significantly up-regulated by IL-22 but not the other two cytokines. The stimulatory effect of IL-22 was completely suppressed in NHEKs treated with a STAT3 inhibitor or transfected with siRNA targeting STAT3. Because IL-22 induces production of antimicrobial proteins in epithelial cells, the antibacterial activity of SLURP1 was assessed against Staphylococcus aureus (S. aureus), which is known to be associated with disease severity in psoriasis. SLURP1 significantly suppressed the growth of S. aureus. These results indicate SLURP1 participates in pathophysiology of psoriasis by regulating keratinocyte proliferation and differentiation, and by suppressing the growth of S. aureus.

Inhibition of the EGFR/STAT3/CEBPD Axis Reverses Cisplatin Cross-resistance with Paclitaxel in the Urothelial Carcinoma of the Urinary Bladder.

PubMed

Wang, Wei-Jan; Li, Chien-Feng; Chu, Yu-Yi; Wang, Yu-Hui; Hour, Tzyh-Chyuan; Yen, Chia-Jui; Chang, Wen-Chang; Wang, Ju-Ming

2017-01-15

Cisplatin (CDDP) is frequently used in combination chemotherapy with paclitaxel for treating urothelial carcinoma of the urinary bladder (UCUB). CDDP cross-resistance has been suggested to develop with paclitaxel, thus hindering successful UCUB treatment. Therefore, elucidating the mechanisms underlying CDDP-induced anticancer drug resistance is imperative and may provide an insight in developing novel therapeutic strategy. Loss-of-function assays were performed to elucidate the role of the EGFR and STAT3 in CDDP-induced CCAAT/enhancer-binding protein delta (CEBPD) expression in UCUB cells. Reporter and in vivo DNA-binding assays were employed to determine whether CEBPD directly regulates ATP binding cassette subfamily B member 1 (ABCB1) and ATP binding cassette subfamily C member 2 (ABCC2) activation. Finally, a xenograft animal assay was used to examine the abilities of gefitinib and S3I-201 (a STAT3 inhibitor) to reverse CDDP and paclitaxel sensitivity. CEBPD expression was maintained in postoperative chemotherapy patients, and this expression was induced by CDDP even in CDDP-resistant UCUB cells. Upon CDDP treatment, CEBPD activated ABCB1 and ABCC2. Furthermore, the EGFR/STAT3 pathway contributed to CDDP-induced CEBPD expression in UCUB cells. Gefitinib and S3I-201 treatment significantly reduced the expression of CEBPD and enhanced the sensitivity of CDDP-resistant UCUB cells to CDDP and paclitaxel. Our results revealed the risk of CEBPD activation in CDDP-resistant UCUB cells and suggested a therapeutic strategy for patients with UCUB or UCUB resisted to CDDP and paclitaxel by combination with either gefitinib or S3I-201. Clin Cancer Res; 23(2); 503-13. ©2016 AACR. ©2016 American Association for Cancer Research.

JAK-STAT and G-protein-coupled receptor signaling pathways are frequently altered in epitheliotropic intestinal T-cell lymphoma

PubMed Central

Nairismägi, M-L; Tan, J; Lim, J Q; Nagarajan, S; Ng, C C Y; Rajasegaran, V; Huang, D; Lim, W K; Laurensia, Y; Wijaya, G C; Li, Z M; Cutcutache, I; Pang, W L; Thangaraju, S; Ha, J; Khoo, L P; Chin, S T; Dey, S; Poore, G; Tan, L H C; Koh, H K M; Sabai, K; Rao, H-L; Chuah, K L; Ho, Y-H; Ng, S-B; Chuang, S-S; Zhang, F; Liu, Y-H; Pongpruttipan, T; Ko, Y H; Cheah, P-L; Karim, N; Chng, W-J; Tang, T; Tao, M; Tay, K; Farid, M; Quek, R; Rozen, S G; Tan, P; Teh, B T; Lim, S T; Tan, S-Y; Ong, C K

2016-01-01

Epitheliotropic intestinal T-cell lymphoma (EITL, also known as type II enteropathy-associated T-cell lymphoma) is an aggressive intestinal disease with poor prognosis and its molecular alterations have not been comprehensively characterized. We aimed to identify actionable easy-to-screen alterations that would allow better diagnostics and/or treatment of this deadly disease. By performing whole-exome sequencing of four EITL tumor-normal pairs, followed by amplicon deep sequencing of 42 tumor samples, frequent alterations of the JAK-STAT and G-protein-coupled receptor (GPCR) signaling pathways were discovered in a large portion of samples. Specifically, STAT5B was mutated in a remarkable 63% of cases, JAK3 in 35% and GNAI2 in 24%, with the majority occurring at known activating hotspots in key functional domains. Moreover, STAT5B locus carried copy-neutral loss of heterozygosity resulting in the duplication of the mutant copy, suggesting the importance of mutant STAT5B dosage for the development of EITL. Dysregulation of the JAK-STAT and GPCR pathways was also supported by gene expression profiling and further verified in patient tumor samples. In vitro overexpression of GNAI2 mutants led to the upregulation of pERK1/2, a member of MEK-ERK pathway. Notably, inhibitors of both JAK-STAT and MEK-ERK pathways effectively reduced viability of patient-derived primary EITL cells, indicating potential therapeutic strategies for this neoplasm with no effective treatment currently available. PMID:26854024

JAK-STAT and G-protein-coupled receptor signaling pathways are frequently altered in epitheliotropic intestinal T-cell lymphoma.

PubMed

Nairismägi, M-L; Tan, J; Lim, J Q; Nagarajan, S; Ng, C C Y; Rajasegaran, V; Huang, D; Lim, W K; Laurensia, Y; Wijaya, G C; Li, Z M; Cutcutache, I; Pang, W L; Thangaraju, S; Ha, J; Khoo, L P; Chin, S T; Dey, S; Poore, G; Tan, L H C; Koh, H K M; Sabai, K; Rao, H-L; Chuah, K L; Ho, Y-H; Ng, S-B; Chuang, S-S; Zhang, F; Liu, Y-H; Pongpruttipan, T; Ko, Y H; Cheah, P-L; Karim, N; Chng, W-J; Tang, T; Tao, M; Tay, K; Farid, M; Quek, R; Rozen, S G; Tan, P; Teh, B T; Lim, S T; Tan, S-Y; Ong, C K

2016-06-01

Epitheliotropic intestinal T-cell lymphoma (EITL, also known as type II enteropathy-associated T-cell lymphoma) is an aggressive intestinal disease with poor prognosis and its molecular alterations have not been comprehensively characterized. We aimed to identify actionable easy-to-screen alterations that would allow better diagnostics and/or treatment of this deadly disease. By performing whole-exome sequencing of four EITL tumor-normal pairs, followed by amplicon deep sequencing of 42 tumor samples, frequent alterations of the JAK-STAT and G-protein-coupled receptor (GPCR) signaling pathways were discovered in a large portion of samples. Specifically, STAT5B was mutated in a remarkable 63% of cases, JAK3 in 35% and GNAI2 in 24%, with the majority occurring at known activating hotspots in key functional domains. Moreover, STAT5B locus carried copy-neutral loss of heterozygosity resulting in the duplication of the mutant copy, suggesting the importance of mutant STAT5B dosage for the development of EITL. Dysregulation of the JAK-STAT and GPCR pathways was also supported by gene expression profiling and further verified in patient tumor samples. In vitro overexpression of GNAI2 mutants led to the upregulation of pERK1/2, a member of MEK-ERK pathway. Notably, inhibitors of both JAK-STAT and MEK-ERK pathways effectively reduced viability of patient-derived primary EITL cells, indicating potential therapeutic strategies for this neoplasm with no effective treatment currently available.

STAT5A and STAT5B have opposite correlations with drug response gene expression

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lamba, V., E-mail: vlamba@ufl.edu; Jia, B.; Liang, F.

Introduction: STAT5A and STAT5B are important transcription factors that play a key role in regulation of several important physiological processes including proliferation, survival, mediation of responses to cytokines and in regulating gender differences in drug response genes such as the hepatic cytochrome P450s (CYPs) that are responsible for a large majority of drug metabolism reactions in the human body. STAT5A and STAT5b have a high degree of sequence homology and have been reported to have largely similar functions. Recent studies have, however, indicated that they can also often have distinct and unique roles in regulating gene expression. Objective: In thismore » study, we evaluated the association of STAT5A and STAT5B mRNA expression levels with those of several key hepatic cytochrome P450s (CYPs) and hepatic transcription factors (TFs) and evaluated the potential roles of STAT5A and 5b in mediating gender differences in these CYPs and TFs. Methods: Expression profiling for major hepatic CYP isoforms and transcription factors was performed using RNA sequencing (RNA-seq) in 102 human liver samples (57 female, 45 male). Real time PCR gene expression data for selected CYPs and TFs was available on a subset of 50 human liver samples (25 female, 25 male) and was used to validate the RNA-seq findings. Results: While STAT5A demonstrated significant negative correlation with expression levels of multiple hepatic transcription factors (including NR1I2 and HNF4A) and DMEs such as CYP3A4 and CYP2C19, STAT5B expression was observed to demonstrate positive associations with several CYPs and TFs analyzed. As STAT5A and STAT5B have been shown to be important in regulation of gender differences in CYPs, we also analyzed STAT5A and 5b associations with CYPs and TFs separately in males and females and observed gender dependent differential associations of STATs with several CYPs and TFs. Results from the real time PCR validation largely supported our RNA-seq findings. Conclusions: Using both RNA sequencing and real time PCR, we examined the association of STAT5A and STAT5B mRNA expression with CYP and TF gene expression. While STAT5A demonstrated significant negative correlations with expression levels of multiple hepatic TFs (including NR1I2 and HNF4α) and CYPs (eg. CYP3A4, CYP2C19), STAT5B expression was observed to demonstrate positive association with most of the CYPs/TFs analyzed suggesting that STAT5A and STAT5b have potentially different and distinct roles in regulating expression of hepatic drug response genes. Further studies are needed to elucidate the potential roles of STAT5A and 5b in regulation of CYPs/TFs and the potential implications of these findings.« less

Naringin attenuates the development of carrageenan-induced acute lung inflammation through inhibition of NF-κb, STAT3 and pro-inflammatory mediators and enhancement of IκBα and anti-inflammatory cytokines.

PubMed

Ahmad, Sheikh Fayaz; Attia, Sabry M; Bakheet, Saleh A; Zoheir, Khairy M A; Ansari, Mushtaq Ahmad; Korashy, Hesham M; Abdel-Hamied, Hala E; Ashour, Abdelkader E; Abd-Allah, Adel R A

2015-04-01

Naringin has been reported to possess diverse pharmacological properties, including anti-arthritic and anti-inflammatory activities. The aim of the present study was to determine the potential anti-inflammatory effect of naringin in a mouse model of carrageenan-induced pleurisy. A single dose of naringin (40 and 80 mg/kg) was administered per oral (p.o.) 1 h before carrageenan (Cg) administration. Pro- and anti-inflammatory cytokines were analysed in pleural fluid. We also assessed the effects of naringin on the expression levels of iNOS, inducible cyclooxygenase isoform (COX-2), ICAM-1, MIP-2, PGE2, STAT3, TGF-β1, nuclear factor kappa B (NF-κB) and inhibitor of kappa B (IκBα) in lung tissue. The histological examinations revealed anti-inflammatory effect of naringin while Cg group deteriorated. Naringin downregulated Th1 and upregulated Th2 cytokines. Western blot analyses revealed increased protein expression of NF-κB, STAT3 and COX-2 and decreased IκBα in response to Cg treatment, which were reversed by the treatment with naringin. In the Cg group, mRNA expression levels of pro-inflammatory mediators upregulated and anti-inflammatory mediators downregulated. Naringin reversed these actions.

Long non-coding RNA HOTAIR and STAT3 synergistically regulate the cervical cancer cell migration and invasion.

PubMed

Zhang, Yan; Cheng, Xiaoling; Liang, Hua; Jin, Zhenzhen

2018-04-25

Homeobox (HOX) transcript antisense RNA (HOTAIR) is a long intergenic non-coding RNA (lncRNA) that has been reported to be highly upregulated in several types of cancers. However, the role of HOTAIR in human cervical cancer is still unclear. We therefore investigated the expression and probable function of HOTAIR in cervical cancer cells. The expression of HOTAIR was examined in (HeLa, CaSki, ME-180, HT-3) and Human Cervical Epithelial Cells (HCerEpiC) by qRT-PCR. Transfection of si-NC, si-HOTAIR or si-STAT3 was carried out with the help of Lipofectamine 2000. The cell viability was assessed by CCK-8 assay. The cell migration and invasion was examined by wound healing and Boyden chamber assays. Protein expression was determined by western blotting. Our results showed that expression of HOTAIR was significantly upregulated in cervical cancer cells and inhibition of the expression of HOTAIR in HeLa cervical cancer cells resulted in suppression of cell proliferation, migration and invasion. Further, analysis of the promoter of HOTAIR, revealed that STAT3 could potentially regulate the activity of the HOTAIR in cervical cancer cells and inhibition of STAT3 had similar effects on the proliferation, migration and invasion of the cervical cancer cells as that of HOTAIR. Further, the suppression of STAT3 expression was associated with concomitant downregulation of IncRNA HOTAIR as indicated by the qRT-PCR. To unveil if STAT3 and HOTAIR have synergistic effects on the cell migration and invasion, si-STAT3 and si-HOTAIR were co-transformed into cervical HeLa cancer cells and it was observed that STAT3 and HOTAIR could synergistically inhibit the proliferation, migration and invasion of the cervical cancer cells. Taken together we conclude that HOTAIR and STAT3 synergistically regulate the proliferation, migration and invasion of cervical cancer cells. Copyright © 2018. Published by Elsevier B.V.

Truncated p110 ERBB2 induces mammary epithelial cell migration, invasion and orthotopic xenograft formation, and is associated with loss of phosphorylated STAT5.

PubMed

Ward, T M; Iorns, E; Liu, X; Hoe, N; Kim, P; Singh, S; Dean, S; Jegg, A-M; Gallas, M; Rodriguez, C; Lippman, M; Landgraf, R; Pegram, M D

2013-05-09

Truncated-ERBB2 isoforms (t-ERBB2s), resulting from receptor proteolysis or alternative translation of the ERBB2 mRNA, exist in a subset of human breast tumors. t-ERBB2s lack the receptor extracellular domain targeted by therapeutic anti-ERBB2 antibodies and antibody-drug conjugates, including trastuzumab, trastuzumab-DM1 and pertuzumab. In clinical studies, expression of t-ERBB2 in breast tumors correlates with metastasis as well as trastuzumab resistance. By using a novel immuno-microarray method, we detect a significant t-ERBB2 fraction in 18 of 31 (58%) of immunohistochemistry (IHC)3+ ERBB2+ human tumor specimens, and further show that t-ERBB2 isoforms are phosphorylated in a subset of IHC3+ samples (10 of 31, 32%). We investigated t-ERBB2 biological activity via engineered expression of full-length and truncated ERBB2 isoforms in human mammary epithelial cells (HMECs), including HMEC and MCF10A cells. Expression of p110 t-ERBB2, but not p95m (m=membrane, also 648CTF) or intracellular ERBB2s, significantly enhanced cell migration and invasion in multiple cell types. In addition, only expression of the p110 isoform led to human breast epithelial cell (HMLE) xenograft formation in vivo. Expression of t-ERBB2s did not result in hyperactivation of the phosphoinositide kinase-3/AKT or mitogen-activated protein kinase signaling pathways in these cells; rather, phosphoproteomic array profiling revealed attenuation of phosphorylated signal transducer and activator of transcription 5 (STAT5) in p110-t-ERBB2-expressing cells compared to controls. Short hairpin-mediated silencing of STAT5 phenocopied p110-t-ERBB2-driven cell migration and invasion, while expression of constitutively active STAT5 reversed these effects. Thus, we provide novel evidence that (1) expression of p110 t-ERBB2 is sufficient for full transformation of HMEC, yielding in vivo xenograft formation, and (2) truncated p110 t-ERBB2 expression is associated with decreased phosphorylation of STAT5.

Truncated p110 ERBB2 induces mammary epithelial cell migration, invasion and orthotopic xenograft formation, and is associated with loss of phosphorylated STAT5

PubMed Central

Ward, T M; Iorns, E; Liu, X; Hoe, N; Kim, P; Singh, S; Dean, S; Jegg, A-M; Gallas, M; Rodriguez, C; Lippman, M; Landgraf, R; Pegram, M D

2013-01-01

Truncated-ERBB2 isoforms (t-ERBB2s), resulting from receptor proteolysis or alternative translation of the ERBB2 mRNA, exist in a subset of human breast tumors. t-ERBB2s lack the receptor extracellular domain targeted by therapeutic anti-ERBB2 antibodies and antibody–drug conjugates, including trastuzumab, trastuzumab-DM1 and pertuzumab. In clinical studies, expression of t-ERBB2 in breast tumors correlates with metastasis as well as trastuzumab resistance. By using a novel immuno-microarray method, we detect a significant t-ERBB2 fraction in 18 of 31 (58%) of immunohistochemistry (IHC)3+ ERBB2+ human tumor specimens, and further show that t-ERBB2 isoforms are phosphorylated in a subset of IHC3+ samples (10 of 31, 32%). We investigated t-ERBB2 biological activity via engineered expression of full-length and truncated ERBB2 isoforms in human mammary epithelial cells (HMECs), including HMEC and MCF10A cells. Expression of p110 t-ERBB2, but not p95m (m=membrane, also 648CTF) or intracellular ERBB2s, significantly enhanced cell migration and invasion in multiple cell types. In addition, only expression of the p110 isoform led to human breast epithelial cell (HMLE) xenograft formation in vivo. Expression of t-ERBB2s did not result in hyperactivation of the phosphoinositide kinase-3/AKT or mitogen-activated protein kinase signaling pathways in these cells; rather, phosphoproteomic array profiling revealed attenuation of phosphorylated signal transducer and activator of transcription 5 (STAT5) in p110-t-ERBB2-expressing cells compared to controls. Short hairpin-mediated silencing of STAT5 phenocopied p110-t-ERBB2-driven cell migration and invasion, while expression of constitutively active STAT5 reversed these effects. Thus, we provide novel evidence that (1) expression of p110 t-ERBB2 is sufficient for full transformation of HMEC, yielding in vivo xenograft formation, and (2) truncated p110 t-ERBB2 expression is associated with decreased phosphorylation of STAT5. PMID:22751112

High expression of N-myc (and STAT) interactor predicts poor prognosis and promotes tumor growth in human glioblastoma

PubMed Central

Yun, Dapeng; Zhao, Yingjie; Wang, Jingkun; Xu, Tao; Li, Xiaoying; Wang, Yuqi; Yuan, Li; Sun, Ruochuan; Song, Xiao; Huai, Cong; Hu, Lingna; Yang, Song; Min, Taishan; Chen, Juxiang; Chen, Hongyan; Lu, Daru

2015-01-01

Glioma is the most malignant brain tumor and glioblastoma (GBM) is the most aggressive type. The involvement of N-myc (and STAT) interactor (NMI) in tumorigenesis was sporadically reported but far from elucidation. This study aims to investigate roles of NMI in human glioma. Three independent cohorts, the Chinese tissue microarray (TMA) cohort (N = 209), the Repository for Molecular Brain Neoplasia Data (Rembrandt) cohort (N = 371) and The Cancer Genome Atlas (TCGA) cohort (N = 528 or 396) were employed. Transcriptional or protein levels of NMI expression were significantly increased according to tumor grade in all three cohorts. High expression of NMI predicted significantly unfavorable clinical outcome for GBM patients, which was further determined as an independent prognostic factor. Additionally, expression and prognostic value of NMI were associated with molecular features of GBM including PTEN deletion and EGFR amplification in TCGA cohort. Furthermore, overexpression or depletion of NMI revealed its regulation on G1/S progression and cell proliferation (both in vitro and in vivo), and this effect was partially dependent on STAT1, which interacted with and was regulated by NMI. These data demonstrate that NMI may serve as a novel prognostic biomarker and a potential therapeutic target for glioblastoma. PMID:25669971

High expression of N-myc (and STAT) interactor predicts poor prognosis and promotes tumor growth in human glioblastoma.

PubMed

Meng, Delong; Chen, Yuanyuan; Yun, Dapeng; Zhao, Yingjie; Wang, Jingkun; Xu, Tao; Li, Xiaoying; Wang, Yuqi; Yuan, Li; Sun, Ruochuan; Song, Xiao; Huai, Cong; Hu, Lingna; Yang, Song; Min, Taishan; Chen, Juxiang; Chen, Hongyan; Lu, Daru

2015-03-10

Glioma is the most malignant brain tumor and glioblastoma (GBM) is the most aggressive type. The involvement of N-myc (and STAT) interactor (NMI) in tumorigenesis was sporadically reported but far from elucidation. This study aims to investigate roles of NMI in human glioma. Three independent cohorts, the Chinese tissue microarray (TMA) cohort (N = 209), the Repository for Molecular Brain Neoplasia Data (Rembrandt) cohort (N = 371) and The Cancer Genome Atlas (TCGA) cohort (N = 528 or 396) were employed. Transcriptional or protein levels of NMI expression were significantly increased according to tumor grade in all three cohorts. High expression of NMI predicted significantly unfavorable clinical outcome for GBM patients, which was further determined as an independent prognostic factor. Additionally, expression and prognostic value of NMI were associated with molecular features of GBM including PTEN deletion and EGFR amplification in TCGA cohort. Furthermore, overexpression or depletion of NMI revealed its regulation on G1/S progression and cell proliferation (both in vitro and in vivo), and this effect was partially dependent on STAT1, which interacted with and was regulated by NMI. These data demonstrate that NMI may serve as a novel prognostic biomarker and a potential therapeutic target for glioblastoma.

The Role of p-STAT3 as a Prognostic and Clinicopathological Marker in Colorectal Cancer: A Systematic Review and Meta-Analysis

PubMed Central

Chu, Qi; Gan, Yong; Ren, Hui; Zhang, Liyan; Wang, Liwei; Li, Xiaoxiu; Wang, Wei

2016-01-01

Objective High expression of phosphorylated signal transducer and activator of transcription 3 (p-STAT3) has been detected in a variety of human tumors. However, the association of positive p-STAT3 expression with clinicopathological parameters and the prognosis of colorectal cancer patients remain controversial. To identify the relationship between p-STAT3 expression and clinicopathological parameters and prognosis in patients with colorectal cancer, a systematic review and meta-analysis were performed. Methods We performed a comprehensive literature search from PubMed, EMBASE, and SinoMed through 27 March, 2016. Hazard ratios (HRs) with 95% confidence intervals (CI) were combined to evaluate the association between p-STAT3 expression and overall survival of colorectal cancer patients. Odds ratios (ORs) with 95% CI were combined to evaluate the association between p-STAT3 expression and clinicopathological parameters in patients with colorectal cancer. Results Seventeen studies including a total of 2,346 colorectal cancer patients were included in this meta-analysis. The combined HR was 1.43 (95% CI: 1.23–1.67, P < 0.001), which suggested a positive relationship between p-STAT3 overexpression and poorer overall survival of colorectal cancer patients. In addition, the results indicated that positive p-STAT3 expression was significantly associated with the presence of lymph node metastasis (OR: 2.43, 95% CI: 1.18–5.01, P = 0.02) but was not associated with TNM stage, tumor differentiation or gender. Conclusion The meta-analysis results suggest that p-STAT3 overexpression is unfavorable for the prognosis of colorectal cancer patients, and p-STAT3 overexpression is associated with the presence of lymph node metastasis among colorectal cancer patients. PMID:27504822

Knocking off the suppressors of cytokine signaling (SOCS): their roles in mammalian pregnancy.

PubMed

Fitzgerald, Justine S; Toth, Bettina; Jeschke, Udo; Schleussner, Ekkehard; Markert, Udo R

2009-12-01

This review discusses the possible role of the suppressor of cytokine signaling (SOCS) proteins in mammalian reproduction. SOCS are regulatory proteins that are rapidly transcribed in response to intracellular Janus kinase-signal transducer and activator of transcription (JAK-STAT) signaling, a cascade governing biological functions including cytokine-induced immunological responses and reproductive processes. For instance STAT3 appears to mediate trophoblast invasion induced by LIF. The SOCS family includes 8 members (cytokine-inducible SH2 protein [CIS] and SOCS1-7) that orchestrate distinct reactions by antagonizing STAT activation. Emerging evidence points to a role of some family members in synchronizing Th1/Th2 cell profiles, the balance in which is considered vital to pregnancy maintenance. The reproductive phenotypes of mutant mice harboring targeted disruption of SOCS gene isoforms offer insights for reproductive immunology, trophoblast function and human pregnancy. CIS transgenic mice display impaired responses to IL-2 and resemble STAT5 deficient mice, except they are fertile. SOCS1 deficiency leads to an overabundance of IFNgamma signaling, yet SOCS1 null mutant mice are able to reproduce. Lack of SOCS3 is embryonically lethal due to placental insufficiency, while SOCS3 over-expression leads to elevated Th2 responses. SOCS3 seems to be vital for reproduction by regulating LIF-driven trophoblast differentiation. SOCS5 inhibits IL-4 signaling, yet the SOCS5 transgenic mouse has no conspicuous reproductive phenotype. SOCS-6 and SOCS-7 null mutant mice display growth retardation. In summary, SOCS proteins are avidly involved in fine regulation of immunological and other vital cellular responses. Many of the above phenotypes present contradictions to accepted reproductive immunological paradigms.

Molecular cloning, transcriptional profiling, and subcellular localization of signal transducer and activator of transcription 2 (STAT2) ortholog from rock bream, Oplegnathus fasciatus.

PubMed

Bathige, S D N K; Umasuthan, Navaneethaiyer; Priyathilaka, Thanthrige Thiunuwan; Thulasitha, William Shanthakumar; Jayasinghe, J D H E; Wan, Qiang; Nam, Bo-Hye; Lee, Jehee

2017-08-30

Signal transducer and activator of transcription 2 (STAT2) is a key element that transduces signals from the cell membrane to the nucleus via the type I interferon-signaling pathway. Although the structural and functional aspects of STAT proteins are well studied in mammals, information on teleostean STATs is very limited. In this study, a STAT paralog, which is highly homologous to the STAT2 members, was identified from a commercially important fish species called rock bream and designated as RbSTAT2. The RbSTAT2 gene was characterized at complementary DNA (cDNA) and genomic sequence levels, and was found to possess structural features common with its mammalian counterparts. The complete cDNA sequence was distributed into 24 exons in the genomic sequence. The promoter proximal region was analyzed and found to contain potential transcription factor binding sites to regulate the transcription of RbSTAT2. Phylogenetic studies and comparative genomic structure organization revealed the distinguishable evolution for fish and other vertebrate STAT2 orthologs. Transcriptional quantification was performed by SYBR Green quantitative real-time PCR (qPCR) and the ubiquitous expression of RbSTAT2 transcripts was observed in all tissues analyzed from healthy fish, with a remarkably high expression in blood cells. Significantly (P<0.05) altered transcription of RbSTAT2 was detected after immune challenge experiments with viral (rock bream irido virus; RBIV), bacterial (Edwardsiella tarda and Streptococcus iniae), and immune stimulants (poly I:C and LPS). Antiviral potential was further confirmed by WST-1 assay, by measuring the viability of rock bream heart cells treated with RBIV. In addition, results of an in vitro challenge experiment signified the influence of rock bream interleukin-10 (RbIL-10) on transcription of RbSTAT2. Subcellular localization studies by transfection of pEGFP-N1/RbSTAT2 into rock bream heart cells revealed that the RbSTAT2 was usually located in the cytoplasm and translocated near to the nucleus upon poly I:C administration. Altogether, these findings suggest that RbSTAT2 is involved in various biologically crucial mechanisms, and provides immune protection to the rock bream. Copyright © 2017 Elsevier B.V. All rights reserved.

Parathyroid hormone inhibition of Na{sup +}/H{sup +} exchanger 3 transcription: Intracellular signaling pathways and transcription factor expression

DOE Office of Scientific and Technical Information (OSTI.GOV)

Neri, Elida Adalgisa; Bezerra, Camila Nogueira Alves, E-mail: camilab@icb.usp.br; Queiroz-Leite, Gabriella Duarte

2015-06-12

The main transport mechanism of reabsorption of sodium bicarbonate and fluid in the renal proximal tubules involves Na{sup +}/H{sup +} exchanger 3 (NHE3), which is acutely and chronically downregulated by parathyroid hormone (PTH). Although PTH is known to exert an inhibitory effect on NHE3 expression and transcription, the molecular mechanisms involved remain unclear. Here, we demonstrated that, in opossum kidney proximal tubule (OKP) cells, PTH-induced inhibition of Nhe3 gene promoter occurs even in the core promoter that controls expression of the reporter gene. We found that inhibition of the protein kinase A (PKA) and Janus kinase/signal transducer and activator ofmore » transcription (JAK/STAT) pathways transformed PTH from an inhibitor of promoter activity into an activator of that same activity, as did point mutations in the EGR1, Sp1, and Sp3 binding consensus elements in the promoter. In nuclear extracts of PTH-treated OKP cells, we also observed increased expression of EGR1 mRNA and of some Sp3 isoforms. Electrophoretic mobility shift assay showed a supershift of the −61 to −42-bp probe with an anti-EGR1 antibody in PTH-treated cells, suggesting that EGR1 binding is relevant for the inhibitory activity of PTH. We conclude that PTH-induced inhibition of NHE3 transcription is related to higher EGR1 expression; to EGR1 binding to the proximal and core promoters; and to PKA and JAK/STAT pathway activation. This mechanism might be responsible, at least in part, for lower NHE3 expression and sodium reabsorption in renal proximal tubules in the presence of high PTH levels. - Highlights: • PTH regulation of Nhe3 promoter depends on EGR1 binding. • EGR1, PKA and JAK/STAT are involved in PTH inhibition of the Nhe3 promoter. • PTH alters expression of EGR1 and Sp3. • PTH inhibits the Nhe3 promoter by regulating PKA and JAK/STAT signaling.« less

The inhibitory effects of capillarisin on cell proliferation and invasion of prostate carcinoma cells.

PubMed

Tsui, Ke-Hung; Chang, Ying-Ling; Yang, Pei-Shan; Hou, Chen-Pang; Lin, Yu-Hsiang; Lin, Bing-Wei; Feng, Tsui-Hsia; Juang, Horng-Heng

2018-04-01

Capillarisin (Cap), an active component of Artemisia capillaris root extracts, is characterized by its anti-inflammatory, anti-oxidant and anti-cancer properties. Nevertheless, the functions of Cap in prostate cancer have not been fully explored. We evaluated the potential actions of Cap on the cell proliferation, migration and invasion of prostate carcinoma cells. Cell proliferation and cell cycle distribution were measured by water-soluble tetrazolium-1 and flow cytometry assays. The expression of cyclins, p21, p27, survivin, matrix metallopeptidase (MMP2 and MMP9) were assessed by immunoblotting assays. Effects of Cap on invasion and migration were determined by wound closure and matrigel transmigration assays. The constitutive and interlukin-6 (IL-6)-inducible STAT3 activation of prostate carcinoma cells were determined by immunoblotting and reporter assays. Capillarisin inhibited androgen-independent DU145 and androgen-dependent LNCaP cell growth through the induction of cell cycle arrest at the G0/G1 phase by upregulating p21 and p27 while downregulating expression of cyclin D1, cyclin A and cyclin B. Cap decreased protein expression of survivin, MMP-2, and MMP-9 and therefore blocked the migration and invasion of DU145 cells. Cap suppressed constitutive and IL-6-inducible STAT3 activation in DU145 and LNCaP cells. Our data indicate that Cap blocked cell growth by modulation of p21, p27 and cyclins. The inhibitory effects of Cap on survivin, MMP-2, MMP-9 and STAT3 activation may account for the suppression of invasion in prostate carcinoma cells. Our data suggest that Cap might be a therapeutic agent in treating advanced prostate cancer with constitutive STAT3 or IL-6-inducible STAT3 activation. © 2017 John Wiley & Sons Ltd.

Kyungheechunggan-Tang-01, a New Herbal Medication, Suppresses LPS-Induced Inflammatory Responses through JAK/STAT Signaling Pathway in RAW 264.7 Macrophages

PubMed Central

Han, Hee-Soo; Shin, Ji-Sun; Inn, Kyung-Soo; Lee, Jang-Hoon; Park, Geonha

2017-01-01

Medicinal plants have been used as alternative therapeutic tools to alleviate inflammatory diseases. The objective of this study was to evaluate anti-inflammatory properties of Kyungheechunggan-tang- (KCT-) 01, KCT-02, and Injinchunggan-tang (IJCGT) as newly developed decoctions containing 3–11 herbs in LPS-induced macrophages. KCT-01 showed the most potent inhibitory effects on LPS-induced NO, PGE2, TNF-α, and IL-6 production among those three herbal formulas. In addition, KCT-01 significantly inhibited LPS-induced iNOS and COX-2 at protein levels and expression of iNOS, COX-2, TNF-α, and IL-6 at mRNA levels. Molecular data revealed that KCT-01 attenuated the activation of JAK/STAT signaling cascade without affecting NF-κB or AP-1 activation. In ear inflammation induced by croton oil, KCT-01 significantly reduced edema, MPO activity, expression levels of iNOS and COX-2, and STAT3 phosphorylation in ear tissues. Taken together, our findings suggest that KCT-01 can downregulate the expression of proinflammatory genes by inhibiting JAK/STAT signaling pathway under inflammatory conditions. This study provides useful data for further exploration and application of KCT-01 as a potential anti-inflammatory medicine. PMID:29348772

Desensitization of the growth hormone-induced Janus kinase 2 (Jak 2)/signal transducer and activator of transcription 5 (Stat5)-signaling pathway requires protein synthesis and phospholipase C.

PubMed

Fernández, L; Flores-Morales, A; Lahuna, O; Sliva, D; Norstedt, G; Haldosén, L A; Mode, A; Gustafsson, J A

1998-04-01

Signal transducers and activators of transcription (Stat) proteins are latent cytoplasmic transcription factors that are tyrosine phosphorylated by Janus kinases (Jak) in response to GH and other cytokines. GH activates Stat5 by a mechanism that involves tyrosine phosphorylation and nuclear translocation. However, the mechanisms that turn off the GH-activated Jak2/Stat5 pathway are unknown. Continuous exposure to GH of BRL-4 cells, a rat hepatoma cell line stably transfected with rat GH receptor, induces a rapid but transient activation of Jak2 and Stat5. GH-induced Stat5 DNA-binding activity was detected after 2 min and reached a maximum at 10 min. Continued exposure to GH resulted in a desensitization characterized by 1) a rapid decrease in Stat5 DNA-binding activity. The rate of decrease of activity was rapid up to 1 h of GH treatment, and the remaining activity declined slowly thereafter. The activity of Stat5 present after 5 h is still higher than the control levels and almost 10-20% with respect to maximal activity at 10 min; and 2) the inability of further GH treatment to reinduce activation of Stat5. In contrast, with transient exposures of BRL-4 cells to GH, Stat5 DNA-binding activity could repeatedly be induced. GH-induced Jak2 and Stat5 activities were independent of ongoing protein synthesis. However, Jak2 tyrosine phosphorylation and Stat5 DNA-binding activity were prolonged for at least 4 h in the presence of cycloheximide, which suggests that the maintenance of desensitization requires ongoing protein synthesis. Furthermore, inhibition of protein synthesis potentiated GH-induced transcriptional activity in BRL-4 cells transiently transfected with SPIGLE1CAT, a reporter plasmid activated by Stat5. GH-induced Jak2 and Stat5 activation were not affected by D609 or mepacrine, both inhibitors of phospholipase C. However, in the presence of D609 and mepacrine, GH maintained prolonged Jak2 and Stat5 activation. Transactivation of SPIGLE1 by GH was potentiated by mepacrine and D609 but not by the phospholipase A2 inhibitor AACOCF3. Thus, a regulatory circuit of GH-induced transcription through the Jak2/Stat5-signaling pathway includes a prompt GH-induced activation of Jak2/Stat5 followed by a negative regulatory response; ongoing protein synthesis and intracellular signaling pathways, where phospholipase C activity is involved, play a critical role to desensitize the GH-activated Jak2/Stat5-signaling pathway.

IFNγ producing CD8+ T cells modified to resist major immune checkpoints induce regression of MHC class I-deficient melanomas

PubMed Central

Buferne, Michel; Chasson, Lionel; Grange, Magali; Mas, Amandine; Arnoux, Fanny; Bertuzzi, Mélanie; Naquet, Philippe; Leserman, Lee; Schmitt-Verhulst, Anne-Marie; Auphan-Anezin, Nathalie

2015-01-01

Tumors with reduced expression of MHC class I (MHC-I) molecules may be unrecognized by tumor antigen-specific CD8+ T cells and thus constitute a challenge for cancer immunotherapy. Here we monitored development of autochthonous melanomas in TiRP mice that develop tumors expressing a known tumor antigen as well as a red fluorescent protein (RFP) reporter knock in gene. The latter permits non-invasive monitoring of tumor growth by biofluorescence. One developing melanoma was deficient in cell surface expression of MHC-I, but MHC-I expression could be rescued by exposure of these cells to IFNγ. We show that CD8+ T cells specific for tumor antigen/MHC-I were efficient at inducing regression of the MHC-I-deficient melanoma, provided that the T cells were endowed with properties permitting their migration into the tumor and their efficient production of IFNγ. This was the case for CD8+ T cells transfected to express an active form of STAT5 (STAT5CA). The amount of IFNγ produced ex vivo from T cells present in tumors after adoptive transfer of the CD8+ T cells was correlated with an increase in surface expression of MHC-I molecules by the tumor cells. We also show that these CD8+ T cells expressed PD-1 and upregulated its ligand PDL-1 on melanoma cells within the tumor. Despite upregulation of this immunosuppressive pathway, efficient IFNγ production in the melanoma microenvironment was found associated with resistance of STAT5CA-expressing CD8+ T cells to inhibition both by PD-1/PDL-1 engagement and by TGFβ1, two main immune regulatory mechanisms hampering the efficiency of immunotherapy in patients. PMID:25949872

E-cadherin and, in its absence, N-cadherin promotes Nanog expression in mouse embryonic stem cells via STAT3 phosphorylation.

PubMed

Hawkins, Kate; Mohamet, Lisa; Ritson, Sarah; Merry, Catherine L R; Ward, Christopher M

2012-09-01

We have recently shown that loss of E-cadherin in mouse embryonic stem cells (mESCs) results in significant alterations to both the transcriptome and hierarchy of pluripotency-associated signaling pathways. Here, we show that E-cadherin promotes kruppel-like factor 4 (Klf4) and Nanog transcript and protein expression in mESCs via STAT3 phosphorylation and that β-catenin, and its binding region in E-cadherin, is required for this function. To further investigate the role of E-cadherin in leukemia inhibitory factor (LIF)-dependent pluripotency, E-cadherin null (Ecad(-/-)) mESCs were cultured in LIF/bone morphogenetic protein supplemented medium. Under these conditions, Ecad(-/-) mESCs exhibited partial restoration of cell-cell contact and STAT3 phosphorylation and upregulated Klf4, Nanog, and N-cadherin transcripts and protein. Abrogation of N-cadherin using an inhibitory peptide caused loss of phospho STAT3, Klf4, and Nanog in these cells, demonstrating that N-cadherin supports LIF-dependent pluripotency in this context. We therefore identify a novel molecular mechanism linking E- and N-cadherin to the core circuitry of pluripotency in mESCs. This mechanism may explain the recently documented role of E-cadherin in efficient induced pluripotent stem cell reprogramming. Copyright © 2012 AlphaMed Press.

Polychlorinated biphenyls (PCB 101, PCB 153 and PCB 180) alter leptin signaling and lipid metabolism in differentiated 3T3-L1 adipocytes.

PubMed

Ferrante, Maria C; Amero, Paola; Santoro, Anna; Monnolo, Anna; Simeoli, Raffaele; Di Guida, Francesca; Mattace Raso, Giuseppina; Meli, Rosaria

2014-09-15

Non-dioxin-like polychlorinated biphenyls (NDL-PCBs) are highly lipophilic environmental contaminants that accumulate in lipid-rich tissues, such as adipose tissue. Here, we reported the effects induced by PCBs 101, 153 and 180, three of the six NDL-PCBs defined as indicators, on mature 3T3-L1 adipocytes. We observed an increase in lipid content, in leptin gene expression and a reduction of leptin receptor expression and signaling, when cells were exposed to PCBs, alone or in combination. These modifications were consistent with the occurrence of "leptin-resistance" in adipose tissue, a typical metabolic alteration related to obesity. Therefore, we investigated how PCBs affect the expression of pivotal proteins involved in the signaling of leptin receptor. We evaluated the PCB effect on the intracellular pathway JAK/STAT, determining the phosphorylation of STAT3, a downstream activator of the transcription of leptin gene targets, and the expression of SOCS3 and PTP1B, two important regulators of leptin resistance. In particular, PCBs 153 and 180 or all PCB combinations induced a significant reduction in pSTAT3/STAT3 ratio and an increase in PTP1B and SOCS3, evidencing an additive effect. The impairment of leptin signaling was associated with the reduction of AMPK/ACC pathway activation, leading to the increase in lipid content. These pollutants were also able to increase the transcription of inflammatory cytokines (IL-6 and TNFα). It is worthy to note that the PCB concentrations used are comparable to levels detectable in human adipose tissue. Our data strongly support the hypothesis that NDL-PCBs may interfere with the lipid metabolism contributing to the development of obesity and related diseases. Copyright © 2014 Elsevier Inc. All rights reserved.

Astaxanthin Inhibits JAK/STAT-3 Signaling to Abrogate Cell Proliferation, Invasion and Angiogenesis in a Hamster Model of Oral Cancer

PubMed Central

Kowshik, J.; Baba, Abdul Basit; Giri, Hemant; Deepak Reddy, G.; Dixit, Madhulika; Nagini, Siddavaram

2014-01-01

Identifying agents that inhibit STAT-3, a cytosolic transcription factor involved in the activation of various genes implicated in tumour progression is a promising strategy for cancer chemoprevention. In the present study, we investigated the effect of dietary astaxanthin on JAK-2/STAT-3 signaling in the 7,12-dimethylbenz[a]anthracene (DMBA)-induced hamster buccal pouch (HBP) carcinogenesis model by examining the mRNA and protein expression of JAK/STAT-3 and its target genes. Quantitative RT-PCR, immunoblotting and immunohistochemical analyses revealed that astaxanthin supplementation inhibits key events in JAK/STAT signaling especially STAT-3 phosphorylation and subsequent nuclear translocation of STAT-3. Furthermore, astaxanthin downregulated the expression of STAT-3 target genes involved in cell proliferation, invasion and angiogenesis, and reduced microvascular density, thereby preventing tumour progression. Molecular docking analysis confirmed inhibitory effects of astaxanthin on STAT signaling and angiogenesis. Cell culture experiments with the endothelial cell line ECV304 substantiated the role of astaxanthin in suppressing angiogenesis. Taken together, our data provide substantial evidence that dietary astaxanthin prevents the development and progression of HBP carcinomas through the inhibition of JAK-2/STAT-3 signaling and its downstream events. Thus, astaxanthin that functions as a potent inhibitor of tumour development and progression by targeting JAK/STAT signaling may be an ideal candidate for cancer chemoprevention. PMID:25296162

The role of STATs in transcriptional control and their impact on cellular function.

PubMed

Bromberg, J; Darnell, J E

2000-05-15

The STAT proteins (Signal Transducers and Activators of Transcription), were identified in the last decade as transcription factors which were critical in mediating virtually all cytokine driven signaling. These proteins are latent in the cytoplasm and become activated through tyrosine phosphorylation which typically occurs through cytokine receptor associated kinases (JAKs) or growth factor receptor tyrosine kinases. Recently a number of non-receptor tyrosine kinases (for example src and abl) have been found to cause STAT phosphorylation. Phosphorylated STATs form homo- or hetero-dimers, enter the nucleus and working coordinately with other transcriptional co-activators or transcription factors lead to increased transcriptional initiation. In normal cells and in animals, ligand dependent activation of the STATs is a transient process, lasting for several minutes to several hours. In contrast, in many cancerous cell lines and tumors, where growth factor dysregulation is frequently at the heart of cellular transformation, the STAT proteins (in particular Stats 1, 3 and 5) are persistently tyrosine phosphorylated or activated. The importance of STAT activation to growth control in experiments using anti-sense molecules or dominant negative STAT protein encoding constructs performed in cell lines or studies in animals lacking specific STATs strongly indicate that STATs play an important role in controlling cell cycle progression and apoptosis. Stat1 plays an important role in growth arrest, in promoting apoptosis and is implicated as a tumor suppressor; while Stats 3 and 5 are involved in promoting cell cycle progression and cellular transformation and preventing apoptosis. Many questions remain including: (1) a better understanding of how the STAT proteins through association with other factors increase transcription initiation; (2) a more complete definition of the sets of genes which are activated by different STATs and (3) how these sets of activated genes differ as a function of cell type. Finally, in the context of many cancers, where STATs are frequently persistently activated, an understanding of the mechanisms leading to their constitutive activation and defining the potential importance of persistent STAT activation in human tumorigenesis remains. Oncogene (2000).

Caveolin-1-deficient mice show accelerated mammary gland development during pregnancy, premature lactation, and hyperactivation of the Jak-2/STAT5a signaling cascade.

PubMed

Park, David S; Lee, Hyangkyu; Frank, Philippe G; Razani, Babak; Nguyen, Andrew V; Parlow, Albert F; Russell, Robert G; Hulit, James; Pestell, Richard G; Lisanti, Michael P

2002-10-01

It is well established that mammary gland development and lactation are tightly controlled by prolactin signaling. Binding of prolactin to its cognate receptor (Prl-R) leads to activation of the Jak-2 tyrosine kinase and the recruitment/tyrosine phosphorylation of STAT5a. However, the mechanisms for attenuating the Prl-R/Jak-2/STAT5a signaling cascade are just now being elucidated. Here, we present evidence that caveolin-1 functions as a novel suppressor of cytokine signaling in the mammary gland, akin to the SOCS family of proteins. Specifically, we show that caveolin-1 expression blocks prolactin-induced activation of a STAT5a-responsive luciferase reporter in mammary epithelial cells. Furthermore, caveolin-1 expression inhibited prolactin-induced STAT5a tyrosine phosphorylation and DNA binding activity, suggesting that caveolin-1 may negatively regulate the Jak-2 tyrosine kinase. Because the caveolin-scaffolding domain bears a striking resemblance to the SOCS pseudosubstrate domain, we examined whether Jak-2 associates with caveolin-1. In accordance with this homology, we demonstrate that Jak-2 cofractionates and coimmunoprecipitates with caveolin-1. We next tested the in vivo relevance of these findings using female Cav-1 (-/-) null mice. If caveolin-1 normally functions as a suppressor of cytokine signaling in the mammary gland, then Cav-1 null mice should show premature development of the lobuloalveolar compartment because of hyperactivation of the prolactin signaling cascade via disinhibition of Jak-2. In accordance with this prediction, Cav-1 null mice show accelerated development of the lobuloalveolar compartment, premature milk production, and hyperphosphorylation of STAT5a (pY694) at its Jak-2 phosphorylation site. In addition, the Ras-p42/44 MAPK cascade is hyper-activated. Because a similar premature lactation phenotype is observed in SOCS1 (-/-) null mice, we conclude that caveolin-1 is a novel suppressor of cytokine signaling.

CS-09RTVP-1 PROMOTES THE MESENCHYMAL TRANSFORMATION OF GLIOMA STEM CELLS VIA THE CXCR4 AND IL-6 PATHWAYS

PubMed Central

Giladi, Nis; Lee, Hae Kyung; Finniss, Susan; Cazacu, Simona; Xiang, Cunli; Poisson, Laila; Mikkelsen, Tom; Ziv-Av, Amotz; Brodie, Chaya

2014-01-01

Glioblastoma (GBM), the most aggressive primary brain tumors, are categorized into the major subgroups: proneural, neural, classical and mesenchymal, the latter being characterized by increased invasion and poor prognosis. We recently identified RTVP-1 as a glioma-associated protein that regulates glioma cell migration and invasion. Using ChiP analyses, we found that the RTVP-1 promoter binds STAT3 and C/EBPbeta, the two master transcription factors that regulate mesenchymal transformation of GBM. Analysis of TCGA tumor specimens demonstrated that the expression of RTVP-1 was higher in the mesenchymal GBM and was inversely correlated with patient survival. We further found that RTVP-1 was expressed in glioma stem cells (GSCs) but not in human neural stem cells (NSCs). Overexpression of RTVP-1 in NSCs induced their mesenchymal transformation, whereas silencing of RTVP-1 in GSCs decreased their mesenchymal signature, increased their neural phenotypes and inhibited the self renewal and stemness of these cells. Silencing of RTVP-1 also decreased tumor volume of GSC-derived xenografts and increased animal survival. Using gene array analysis of RTVP-1 silenced cells we identified IL-6 and CXCR4 as major mediators of RTVP-1 effects on the mesenchymal transformation and self-renewal of GSCs. Using a pull down assay with His-tagged RTVP-1 and FRET analysis, we identified HSP27, N-WASP and hnRNPK as novel interacting proteins of RTVP-1, that mediate its effects on GSC migration and invadopodia formation. In summary, RTVP-1 expression is regulated by STAT3 and CEBPbeta and is promoting the mesenchymal transformation of GSCs. RTVP-1 induces self-renewal and migration of GSCs by the increased expression of IL-6 and CXCR4 and via its interaction with N-WASP, hnRNPK and HSP27. The upregulation of IL-6 by RTVP-1 acts in a positive feedback loop to further increase RTVP-1 expression by activating the STAT3 pathway. Collectively, these results implicate RTVP-1 as a novel prognostic marker and therapeutic target in GBM.

IL-27 inhibits epithelial-mesenchymal transition and angiogenic factor production in a STAT1-dominant pathway in human non-small cell lung cancer

PubMed Central

2013-01-01

Background Interleukin-27 signaling is mediated by the JAK-STAT pathway via activation of STAT1 and STAT3, which have tumor suppressive and oncogenic activities, respectively. Epithelial–mesenchymal transition (EMT) and angiogenesis are key processes in carcinogenesis. Although IL-27 has been shown to have potent anti-tumor activity in various cancer models, the role of IL-27 in EMT and angiogenesis is poorly understood. In this study, we investigated the role of IL-27 in regulating EMT and angiogenesis through modulation of the STAT pathways in human non-small cell lung carcinoma (NSCLC) cells. Methods STAT activation following IL-27 exposure was measured in human NSCLC cell lines. Expression of epithelial (E-cadherin, γ-catenin) and mesenchymal (N-cadherin, vimentin) markers were assessed by Western blot analysis. Production of pro-angiogenic factors (VEGF, IL-8/CXCL8, CXCL5) were examined by ELISA. Cell motility was examined by an in vitro scratch and transwell migration assays. Selective inhibitors of STAT1 (STAT1 siRNAs) and STAT3 (Stattic) were used to determine whether both STAT1 and STAT3 are required for IL-27 mediated inhibition of EMT and secretion of angiogenic factors. Results Our results demonstrate that IL-27 stimulation in NSCLC resulted in 1) STAT1 and STAT3 activation in a JAK-dependent manner, 2) development of epithelial phenotypes, including a decrease in the expression of a transcriptional repressor for E-cadherin (SNAIL), and mesenchymal marker (vimentin) with a reciprocal increase in the expression of epithelial markers, 3) inhibition of cell migration, and 4) reduced production of pro-angiogenic factors. STAT1 inhibition in IL-27–treated cells reversed the IL-27 effect with resultant increased expression of Snail, vimentin and the pro-angiogenic factors. The inhibition of STAT3 activation had no effect on the development of the epithelial phenotype. Conclusion IL-27 induces mesenchymal to epithelial transition and inhibits the production of pro-angiogenic factors in a STAT1–dominant pathway. These findings highlight the importance of STAT1 in repressing lung carcinogenesis and describe a new anti-tumor mechanism of IL-27. PMID:24274066

Effects of MicroRNA-23a on Differentiation and Gene Expression Profiles in 3T3-L1 Adipocytes

PubMed Central

Huang, Yong; Huang, Jinxiu; Qi, Renli; Wang, Qi; Wu, Yongjiang; Wang, Jing

2016-01-01

MicroRNAs (miRNAs) are small non-coding RNA molecules that regulate growth, development, and programmed death of cells. A newly-published study has shown that miRNA-23a could regulate 3T3-L1 adipocyte differentiation. Here, we identified miRNA-23a as a negative regulator of 3T3-L1 adipocyte differentiation again. Over-expression of miRNA-23a inhibited differentiation and decreased lipogenesis as well as down-regulated mRNA and protein expression of both peroxisome proliferator-activated receptor (PPAR) γ and fatty acid binding protein (FABP) 4, whereas knock down of miRNA-23a showed the opposite effects on differentiation as well as increasing the number of apoptotic cells. Additionally, digital gene expression profiling sequencing (DGE-Seq) was used to assay changes in gene expression profiles following alterations in the level of miR-23a. In total, over-expression or knock down of miRNA-23a significantly changed the expression of 313 and 425 genes, respectively. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses indicated that these genes were mainly involved in the stress response, immune system, metabolism, cell cycle, among other pathways. Additionally, the signal transducer and activator of transcription 1 (Stat1) was shown to be a target of miRNA-23a by computational and dual-luciferase reporter assays that indicated Janus Kinase (Jak)-Stat signal pathway was implicated in regulating adipogenesis mediated by miRNA-23a in adipocytes. PMID:27783036

Activation of Stat1 by mutant fibroblast growth-factor receptor in thanatophoric dysplasia type II dwarfism.

PubMed

Su, W C; Kitagawa, M; Xue, N; Xie, B; Garofalo, S; Cho, J; Deng, C; Horton, W A; Fu, X Y

1997-03-20

The achondroplasia class of chondrodysplasias comprises the most common genetic forms of dwarfism in humans and includes achondroplasia, hypochondroplasia and thanatophoric dysplasia types I and II (TDI and TDII), which are caused by different mutations in a fibroblast growth-factor receptor FGFR3 (ref. 1). The molecular mechanism and the mediators of these FGFR3-related growth abnormalities are not known. Here we show that mutant TDII FGFR3 has a constitutive tyrosine kinase activity which can specifically activate the transcription factor Stat1 (for signal transducer and activator of transcription). Furthermore, expression of TDII FGFR3 induced nuclear translocation of Stat1, expression of the cell-cycle inhibitor p21(WAF1/CIP1), and growth arrest of the cell. Thus, TDII FGFR3 may use Stat1 as a mediator of growth retardation in bone development. Consistent with this, Stat1 activation and increased p21(WAF1/CIP1) expression was found in the cartilage cells from the TDII fetus, but not in those from the normal fetus. Thus, abnormal STAT activation and p21(WAF1/CIP1) expression by the TDII mutant receptor may be responsible for this FGFR3-related bone disease.

MACC1 regulates Fas mediated apoptosis through STAT1/3 - Mcl-1 signaling in solid cancers.

PubMed

Radhakrishnan, Harikrishnan; Ilm, Katharina; Walther, Wolfgang; Shirasawa, Senji; Sasazuki, Takehiko; Daniel, Peter T; Gillissen, Bernhard; Stein, Ulrike

2017-09-10

MACC1 was identified as a novel player in cancer progression and metastasis, but its role in death receptor-mediated apoptosis is still unexplored. We show that MACC1 knockdown sensitizes cancer cells to death receptor-mediated apoptosis. For the first time, we provide evidence for STAT signaling as a MACC1 target. MACC1 knockdown drastically reduced STAT1/3 activating phosphorylation, thereby regulating the expression of its apoptosis targets Mcl-1 and Fas. STAT signaling inhibition by the JAK1/2 inhibitor ruxolitinib mimicked MACC1 knockdown-mediated molecular signatures and apoptosis sensitization to Fas activation. Despite the increased Fas expression, the reduced Mcl-1 expression was instrumental in apoptosis sensitization. This reduced Mcl-1-mediated apoptosis sensitization was Bax and Bak dependent. MACC1 knockdown also increased TRAIL-induced apoptosis. MACC1 overexpression enhanced STAT1/3 phosphorylation and increased Mcl-1 expression, which was abrogated by ruxolitinib. The central role of Mcl-1 was strengthened by the resistance of Mcl-1 overexpressing cells to apoptosis induction. The clinical relevance of Mcl-1 regulation by MACC1 was supported by their positive expression correlation in patient-derived tumors. Altogether, we reveal a novel death receptor-mediated apoptosis regulatory mechanism by MACC1 in solid cancers through modulation of the STAT1/3-Mcl-1 axis. Copyright © 2017 Elsevier B.V. All rights reserved.

IGF-1-induced MMP-11 expression promotes the proliferation and invasion of gastric cancer cells through the JAK1/STAT3 signaling pathway.

PubMed

Su, Chao; Wang, Wenchang; Wang, Cunchuan

2018-05-01

The present study aimed to investigate the association between insulin-like growth factor-1 (IGF-1) and matrix metalloproteinase-11 (MMP-11) expression in gastric cancer (GC) and the underlying mechanisms in SGC-7901 cells. Reverse transcription-quantitative polymerase chain reaction analysis revealed that the expression of IGF-1 and MMP-11 was significantly upregulated in GC tissues compared with normal gastric tissue. Furthermore, IGF-1 significantly and dose-dependently promoted MMP-11. Western blotting revealed that the addition of IGF-1 to SGC-7901 cells led to an evident enhancement in signal transducer and activator of transcription 3 (STAT3), IGF-1R and Janus kinase 1 (JAK1) phosphorylation at 20 and 40 min. A decrease in the extent of the elevated expression of MMP-11 and the enhanced phosphorylation of STAT3, JAK1 and IGF-1 receptor (IGF-1R) induced by IGF-1 in SGC-7901 cells were observed following treatment with NT157 (an IGF-1R inhibitor). Furthermore, piceatannol (a JAK1 inhibitor) or small interfering RNA against STAT3 reduced the extent of the increased expression of MMP-11 induced by IGF-1 in SGC-7901 cells. Piceatannol treatment induced the dose-dependent decline in the enhancement of STAT3 phosphorylation induced by IGF-1, indicating that the JAK1/STAT3 pathway may be implicated in the elevated expression of MMP-11 induced by IGF-1 in SGC-7901 cells. Finally, IGF-1 treatment significantly promoted the proliferation and invasion of SGC-7901 cells, which was inhibited following NT157, piceatannol or si-STAT3 treatment. The present study therefore demonstrated that IGF-1-induced MMP-11 may have facilitated the proliferation and invasion of SGC-7901 cells via the JAK1/STAT3 pathway.

The measles virus phosphoprotein interacts with the linker domain of STAT1

DOE Office of Scientific and Technical Information (OSTI.GOV)

Devaux, Patricia, E-mail: devaux.patricia@mayo.edu; Priniski, Lauren; Cattaneo, Roberto

2013-09-15

The measles virus (MV) phosphoprotein (P) and V proteins block the interferon (IFN) response by impeding phosphorylation of the signal transducer and activator of transcription 1 (STAT1) by the Janus kinase 1 (JAK1). We characterized how STAT1 mutants interact with P and JAK1 phosphorylation. Certain mutants of the linker, the Src-homology 2 domain (SH2), or the transactivation domain had reduced or abolished phosphorylation through JAK1 after IFN treatment. Other mutants, mainly localized in the linker, failed to interact with P as documented by the lack of interference with nuclear translocation. Thus the functional footprint of P on STAT1 localizes mainlymore » to the linker domain; there is also some overlap with the STAT1 phosphorylation functional footprint on the SH2 domain. Based on these observations, we discuss how the MV-P might operate to inhibit the JAK/STAT pathway. - Highlights: • Residue in the linker and SH2 domains of STAT1 are important for MV-P interaction. • Residue in the linker and SH2 domains of STAT1 are important for STAT1 phosphorylation. • Residues interferring with both functions have similar location on STAT1. • The viral P and V proteins may operate in concert to inhibit the JAK/STAT pathway.« less

Viral-induced Modulation of Multiple Checkpoint Proteins in Cancers.

PubMed

Nuovo, Gerard J; Folcik, Virginia A; Magro, Cynthia

2017-07-01

Therapy with checkpoint inhibitors represents a major advance in cancer treatment. The purpose of this study was to examine the expression patterns of the checkpoint proteins programmed death ligand 1 (PD L1), PD L2, indoleamine 2,3-dioxygenase 1 (IDO1), and cytotoxic T-lymphocyte antigen 4 (CTLA4) in cancers including those associated with viral infections. Normal, noninflamed tissues rarely express checkpoint proteins with exceptions including the placenta and stomach. Expression of PD L1 was noted in 30%, PD L2 in 18%, IDO1 in 13%, and CTLA4 in 14% of 333 nonviral malignancies including endometrial, ovarian, lung, and breast cancers. The expression of each checkpoint protein was significantly higher among 166 cases of viral-related (mostly human papillomavirus) cancers where expression of PD L1 was noted in 84%, PD L2 in 67%, IDO1 in 61%, and CTLA4 in 37% (each P value <0.001); 97% of the viral-related cancers showed expression of at least 1 checkpoint protein. In addition, over 90% of the CD8 cells in the viral-associated cancers were quiescent based on low coexpression of Ki-67 as well as pSTAT1. It is concluded that viral infection in cancers is associated with the increased expression of key checkpoint proteins. This indicates that cancers with productive viral infection may be better targets for checkpoint inhibitor therapy.

The use of dissolved oxygen-controlled, fed-batch aerobic cultivation for recombinant protein subunit vaccine manufacturing.

PubMed

Farrell, Patrick; Sun, Jacob; Champagne, Paul-Philippe; Lau, Heron; Gao, Meg; Sun, Hong; Zeiser, Arno; D'Amore, Tony

2015-11-27

A simple "off-the-shelf" fed-batch approach to aerobic bacterial cultivation for recombinant protein subunit vaccine manufacturing is presented. In this approach, changes in the dissolved oxygen levels are used to adjust the nutrient feed rate (DO-stat), so that the desired dissolved oxygen level is maintained throughout cultivation. This enables high Escherichia coli cell densities and recombinant protein titers. When coupled to a kLa-matched scale-down model, process performance is shown to be consistent at the 2L, 20L, and 200L scales for two recombinant E. coli strains expressing different protein subunit vaccine candidates. Additionally, by mining historical DO-stat nutrient feeding data, a method to transition from DO-stat to a pre-determined feeding profile suitable for larger manufacturing scales without using feedback control is demonstrated at the 2L, 20L, and 200L scales. Copyright © 2015 Elsevier Ltd. All rights reserved.

Human parvovirus B19 VP1u Protein as inflammatory mediators induces liver injury in naïve mice.

PubMed

Hsu, Tsai-Ching; Chiu, Chun-Ching; Chang, Shun-Chih; Chan, Hsu-Chin; Shi, Ya-Fang; Chen, Tzy-Yen; Tzang, Bor-Show

2016-01-01

Human parvovirus B19 (B19V) is a human pathogen known to be associated with many non-erythroid diseases, including hepatitis. Although B19V VP1-unique region (B19-VP1u) has crucial roles in the pathogenesis of B19V infection, the influence of B19-VP1u proteins on hepatic injury is still obscure. This study investigated the effect and possible inflammatory signaling of B19-VP1u in livers from BALB/c mice that were subcutaneously inoculated with VP1u-expressing COS-7 cells. The in vivo effects of B19-VP1u were analyzed by using live animal imaging system (IVIS), Haematoxylin-Eosin staining, gel zymography, and immunoblotting after inoculation. Markedly hepatocyte disarray and lymphocyte infiltration, enhanced matrix metalloproteinase (MMP)-9 activity and increased phosphorylation of p38, ERK, IKK-α, IκB and NF-κB (p-p65) proteins were observed in livers from BALB/c mice receiving COS-7 cells expressing B19-VP1u as well as the significantly increased CRP, IL-1β and IL-6. Notably, IFN-γ and phosphorylated STAT1, but not STAT3, were also significantly increased in the livers of BALB/c mice that were subcutaneously inoculated with VP1u-expressing COS-7 cells. These findings revealed the effects of B19-VP1u on liver injury and suggested that B19-VP1u may have a role as mediators of inflammation in B19V infection.

Peretinoin, an acyclic retinoid, suppresses steatohepatitis and tumorigenesis by activating autophagy in mice fed an atherogenic high-fat diet

PubMed Central

Honda, Masao; Takegoshi, Kai; Yamashita, Taro; Nakamura, Mikiko; Shirasaki, Takayoshi; Sakai, Yoshio; Shimakami, Tetsuro; Nagata, Naoto; Takamura, Toshinari; Tanaka, Takuji; Kaneko, Shuichi

2017-01-01

The pathogenesis of non-alcoholic steatohepatitis (NASH) is still unclear and the prevention of the development of hepatocellular carcinoma (HCC) has not been established. We established an atherogenic and high-fat diet mouse model that develops hepatic steatosis, inflammation, fibrosis, and liver tumors at a high frequency. Using two NASH-HCC mouse models, we showed that peretinoin, an acyclic retinoid, significantly improved liver histology and reduced the incidence of liver tumors. Interestingly, we found that peretinoin induced autophagy in the liver of mice, which was characterized by the increased co-localized expression of microtubule-associated protein light chain 3B-II and lysosome-associated membrane protein 2, and increased autophagosome formation and autophagy flux in the liver. These findings were confirmed using primary mouse hepatocytes. Among representative autophagy pathways, the autophagy related (Atg) 5-Atg12-Atg16L1 pathway was impaired; especially, Atg16L1 was repressed at both the mRNA and protein level. Decreased Atg16L1 mRNA expression was also found in the liver of patients with NASH according to disease progression. Promoter analysis revealed that peretinoin activated the promoter of Atg16L1 by increasing the expression of CCAAT/enhancer-binding-protein-alpha. Interestingly, Atg16L1 overexpression in HepG2 cells inhibited palmitate-induced NF-kB activation and interleukin-6-induced STAT3 activation. We showed that Atg16L1 induced the de-phosphorylation of Gp130, a receptor subunit of interleukin-6 family cytokines, which subsequently repressed phosphorylated-STAT3 (Tyr705) levels, and this process might be independent of autophagy function. Thus, peretinoin prevents the progression of NASH and the development of HCC through activating the autophagy pathway by increased Atg16L1 expression, which is an essential regulator of autophagy and anti-inflammatory proteins. PMID:28591717

Prognostic Value and Clinicopathological Significance of p-stat3 Among Gastric Carcinoma Patients: A Systematic Review and Meta-Analysis.

PubMed

Ji, Kun; Zhang, Liyan; Zhang, Mingxuan; Chu, Qi; Li, Xin; Wang, Wei

2016-02-01

The overexpression of phosphorylated signal transducer and activator of transcription 3 (p-stat3) was detected in a variety of human tumors. The published studies on p-stat3 expression among gastric carcinoma patients remain controversial.In order to clarify the prognosis value of p-stat3 with overall survival and its association with clinicopathological characteristics in gastric carcinoma, we performed a systematic review and meta-analysis.Eligible studies were retrieved by searching PubMed, Embase, Cochrane library, and Chinese biomedical literature service system databases.Studies described the association between p-stat3 expression and clinicopathological characteristics and overall survival in gastric carcinoma patients; p-stat3 expression was detected by immunohistochemistry (IHC).Odds ratio (OR) and hazard ratio (HR) were considered as a measure of evaluating the association in meta-analysis; I was used to assess the heterogeneity across studies; publication bias was assessed with funnel plot, Egger test, and Begg test.Twenty-three studies including 2872 patients which evaluated the p-stat3 expression by IHC in gastric carcinoma were included. The pooled HR (HR = 2.02, 95% CI: 1.49-2.73, P < 0.00001) indicated that the increased p-stat3 expression was significantly associated with poor overall survival. In addition, when we investigated the association between p-stat3 overexpression and clinicopathological characteristics of gastric carcinoma, we found that the increased p-stat3 expression was significantly associated with tumor differentiation (poorly vs well-moderately: OR = 3.70, 95% CI: 1.98-6.93, P < 0.0001) and lymph node metastasis (present vs absent: OR = 2.40, 95% CI: 1.28-4.50, P = 0.007).The different type of primary antibody was used; the assessment methods of p-stat3 positive expression were defined differently; the locations of p-stat3 expression were different; the method of extrapolating HR from Kaplan-Meier survival curves did seem to be less reliable than when HR was extracted directly from literatures; sample sizes, the age of patients, and the follow-up durations are different.In conclusion, our meta-analysis indicates that the increased p-stat3 expression may be not only predict poor prognosis, but also be associated with worse tumor differentiation and lymph node metastasis in patients with gastric carcinoma.

Upregulated STAT3 and RhoA signaling in colorectal cancer (CRC) regulate the invasion and migration of CRC cells.

PubMed

Zhang, G-Y; Yang, W-H; Chen, Z

2016-05-01

We aimed to reveal the expression and activation of signal transducers and activators of transcription 3 (STAT3) and RhoA/Rho-associated coiled-coil forming kinase 1 (ROCK1) signaling in CRC tissues, and to investigate the regulatory role of STAT3 and RhoA signaling in the invasion and migration of colorectal cancer cells. We examined the expression of STAT3, RhoA and ROCK1 in CRC tissues with real-time PCR and Western blotting methods. And then we examined the interaction between STAT3 and RhoA/ROCK1 signaling in CRC HT-29 cells with gain-of-function and loss-of-function strategies. In addition, we determined the regulation by STAT3 and RhoA/ROCK1 on the invasion and migration of CRC HT-29 cells. Our study demonstrated a significant upregulation of RhoA and ROCK1 expression and STAT3-Y705 phosphorylation in 32 CRC specimens, compared to the 17 normal CRC tissues. Further study demonstrated there was a coordination between STAT3 and RhoA/Rock signaling in the HT-29 cells. Moreover, STAT3 knockdown or RhoA knockdown significantly repressed the migration and invasion in HT-29 cells and vice versa. STAT3 and RhoA signaling regulate the invasion and migration of CRC cells, implying the orchestrated and oncogenic roles of STAT3 and RhoA/ROCK1 signaling in CRC.

STATs in Lung Development: Distinct Early and Late Expression, Growth Modulation and Signaling Dysregulation in Congenital Diaphragmatic Hernia.

PubMed

Piairo, Paulina; Moura, Rute S; Baptista, Maria João; Correia-Pinto, Jorge; Nogueira-Silva, Cristina

2018-01-01

Congenital diaphragmatic hernia (CDH) is a life-threatening developmental anomaly, intrinsically combining severe pulmonary hypoplasia and hypertension. During development, signal transducers and activators of transcription (STAT) are utilized to elicit cell growth, differentiation, and survival. We used the nitrofen-induced CDH rat model. At selected gestational time points, lungs were divided into two experimental groups, i.e., control or CDH. We performed immunohistochemistry and western blotting analysis to investigate the developmental expression profile of the complete family of STATs (STAT1-6), plus specific STATs activation (p-STAT3, p-STAT6) and regulation by SOCS (SOCS3) in normal lungs against those of diseased lungs. The normal fetal lung explants were treated with piceatannol (STAT3 inhibitor) in vitro followed by morphometrical analysis. Molecular profiling of STATs during the lung development revealed distinct early and late expression signatures. Experimental CDH altered the STATs expression, activation, and regulation in the fetal lungs. In particular, STAT3 and STAT6 were persistently over-expressed and early over-activated. Piceatannol treatment dose-dependently stimulated the fetal lung growth. These findings suggest that STATs play an important role during normal fetal lung development and CDH pathogenesis. Moreover, functionally targeting STAT signaling modulates fetal lung growth, which highlights that STAT3 and STAT6 signaling might be promising therapeutic targets in reducing or preventing pulmonary hypoplasia in CDH. © 2018 The Author(s). Published by S. Karger AG, Basel.

Fatty acid binding protein 5 promotes tumor angiogenesis and activates the IL6/STAT3/VEGFA pathway in hepatocellular carcinoma.

PubMed

Pan, Long; Xiao, Heng; Liao, Rui; Chen, Qingsong; Peng, Chong; Zhang, Yuchi; Mu, Tong; Wu, Zhongjun

2018-06-25

Tumor angiogenesis is an essential process for facilitating tumor growth and metastasis. Fatty acid binding protein 5(FABP5)is highly expressed in hepatocellular carcinoma (HCC). Thus, we investigated the role of FABP5 in tumor angiogenesis during HCC development. In this study, the protein and mRNA levels of FABP5 in matched HCC and adjacent noncancerous liver tissues from 43 patients were determined using immunohistochemistry and real-time quantitative PCR, respectively. Two HCC cell lines (Huh7 and SMMC-7721) and human umbilical vein endothelial cells (HUVECS) were used to investigate the pro-angiogenic effect of FABP5 by tube formation, CCK8 and Transwell migration assays. The expression levels of interleukin 6 (IL6) and vascular endothelial growth factor A (VEGFA) secreted from HCC cells were detected by enzyme-linked immunosorbent assay (ELISA). In 43 HCC patients, the expression of FABP5 mRNA was positively correlated with intratumoral VEGFA mRNA expression. FABP5 mRNA expression was also associated with adverse HCC characteristics. In vitro, cell viability, cell migration and tube formation in HUVECs were enhanced with increasing expression of FABP5 in HCC cells. Downregulation of FABP5 expression inhibited the IL6/STAT3/VEGFA pathway in HCC cells and inhibited tumor angiogenesis. FABP5 was shown to promote angiogenesis and activate the IL6/STAT3/VEGFA pathway in HCC. FABP5 may be a potential antiangiogenic target in the treatment of HCC. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

HO-1 inhibits IL-13-induced goblet cell hyperplasia associated with CLCA1 suppression in normal human bronchial epithelial cells.

PubMed

Mishina, Kei; Shinkai, Masaharu; Shimokawaji, Tadasuke; Nagashima, Akimichi; Hashimoto, Yusuke; Inoue, Yoriko; Inayama, Yoshiaki; Rubin, Bruce K; Ishigatsubo, Yoshiaki; Kaneko, Takeshi

2015-12-01

Mucus hypersecretion and goblet cell hyperplasia are common features that characterize asthma. IL-13 increases mucin (MUC) 5AC, the major component of airway mucus, in airway epithelial cells. According to the literature, IL-13 receptor activation leads to STAT6 activation and consequent induction of chloride channel accessory 1 (CLCA1) gene expression, associated with the induction of MUC5AC. Heme oxygenase-1 (HO-1) is an enzyme that catalyzes oxidation of heme to biliverdin, and has anti-inflammatory and anti-oxidant properties. We examined the effects of HO-1 on mucin production and goblet cell hyperplasia induced by IL-13. Moreover, we assessed the cell signaling intermediates that appear to be responsible for mucin production. Normal human bronchial epithelial (NHBE) cells were grown at air liquid interface (ALI) in the presence or absence of IL-13 and hemin, a HO-1 inducer, for 14 days. Protein concentration was analyzed using ELISA, and mRNA expression was examined by real-time PCR. Histochemical analysis was performed using HE staining, andWestern blotting was performed to evaluate signaling transduction pathway. Hemin (4 μM) significantly increased HO-1 protein expression (p b 0.01) and HO-1 mRNA expression (p b 0.001). IL-13 significantly increased goblet cells, MUC5AC protein secretion (p b 0.01) and MUC5AC mRNA (p b 0.001), and these were decreased by hemin by way of HO-1. Tin protoporphyrin (SnPP)-IX, a HO-1 inhibitor, blocked the effect of hemin restoring MUC5AC protein secretion (p b 0.05) and goblet cell hyperplasia. Hemin decreased the expression of CLCA1 mRNA (p b 0.05) and it was reversed by SnPP-IX, but could not suppress IL-13-induced phosphorylation of STAT6 or SAM pointed domain-containing ETS transcription factor (SPDEF) and Forkhead box A2 (FOXA2) mRNA expression. In summary, HO-1 overexpression suppressed IL-13-induced goblet cell hyperplasia and MUC5AC production, and involvement of CLCA1 in the mechanism was suggested.

SIRT1 counteracted the activation of STAT3 and NF-κB to repress the gastric cancer growth.

PubMed

Lu, Juanjuan; Zhang, Liping; Chen, Xiang; Lu, Qiming; Yang, Yuxia; Liu, Jingping; Ma, Xin

2014-01-01

Sirtuin-1 (SIRT1) possesses apparently dual roles in regulation of tumor. Previous reports have documented the crosstalk between SIRT1 with signal transducer and activator of transcription 3 (STAT3) and nuclear factor kappa-B (NF-κB) signaling in leukemia, lymphoma and myeloma. In this study, the purpose was to survey the regulatory effects of SIRT1 on gastric cancer (GC) cells (AGS and MKN-45) and the relationships between SIRT1 and activation of STAT3 and NF-κB in GC cells. We found the SIRT1 activator (resveratrol RSV) contributed to the repression of viability and increase of senescence, which were rescued by SIRT1 inhibitor (nicotinamide NA) and SIRT1 depletion by CCK-8 assay and SA-β-gal assay respectively. Further study found SIRT1 activation (RSV supplement) not only inhibited the activation of STAT3 including STAT3 mRNA level, c-myc mRNA level phosphorylated STAT3 (pSTAT3) proteins and acetylizad STAT3 (acSTAT3) proteins, but also repression of pNF-κB p65 and acNF-κB p65. NA reversed the effects of RSV. In addition, either RSV or NA application could not change the cellular viability and senescence in MKN-45 cells with STAT3 knockdown or NF-κB knockdown. Overall, our findings suggested SIRT1 activation could induced the loss of viability and increases of senescence in GC in vitro. Moreover, our observations revealed SIRT1 displayed growth inhibitory activity in GC cells highly associated with causing repression of activation of STAT3 and NF-κB proteins via deacetylation.

The role of the BMP signaling cascade in regulation of stem cell activity following massive small bowel resection in a rat.

PubMed

Sukhotnik, I; Berkowitz, D; Dorfman, T; Halabi, Salim; Pollak, Y; Bejar, J; Bitterman, A; Coran, A G

2016-02-01

Bone morphogenetic proteins (BMPs) are a group of growth factors that are implicated in intestinal growth, morphogenesis, differentiation, and homeostasis. The role of the BMP signaling cascade in stimulation of cell proliferation after massive small bowel resection is unknown. The purpose of this study was to evaluate the role of BMP signaling during intestinal adaptation in a rat model of short bowel syndrome (SBS). Male rats were divided into two groups: Sham rats underwent bowel transection and SBS rats underwent a 75 % bowel resection. Parameters of intestinal adaptation, enterocyte proliferation and apoptosis were determined 2 weeks after operation. Illumina's Digital Gene Expression analysis was used to determine the BMP signaling gene expression profiling. BMP-related genes and protein expression were determined using real-time PCR, Western blotting and immunohistochemistry. From the total number of 20,000 probes, 8 genes related to BMP signaling were investigated. From these genes, five genes were found to be up-regulated in jejunum (BMP1-10 %, BMP2-twofold increase, BMP3-10 %, BMP2R-12 % and STAT3-28 %) and four genes to be up-regulated in ileum (BMP1-16 %, BMP2-27 %, BMP3-10 %, and STAT3-20 %) in SBS vs sham animals with a relative change in gene expression level of 10 % or more. SBS rats also demonstrated a significant increase in BMP2 and STAT3 mRNA and protein levels (determined by real-time PCR and Western blot) compared to control animals. Two weeks following massive bowel resection in rats, the BMP signaling pathway is stimulated. BMP signaling may serve as an important mediator of reciprocal interactions between the epithelium and the underlying mesenchymal stroma during intestinal adaptation following massive bowel resection in a rat.

Single administration of recombinant IL-6 restores the gene expression of lipogenic enzymes in liver of fasting IL-6-deficient mice.

PubMed

Gavito, A L; Cabello, R; Suarez, J; Serrano, A; Pavón, F J; Vida, M; Romero, M; Pardo, V; Bautista, D; Arrabal, S; Decara, J; Cuesta, A L; Valverde, A M; Rodríguez de Fonseca, F; Baixeras, E

2016-03-01

Lipogenesis is intimately controlled by hormones and cytokines as well as nutritional conditions. IL-6 participates in the regulation of fatty acid metabolism in the liver. We investigated the role of IL-6 in mediating fasting/re-feeding changes in the expression of hepatic lipogenic enzymes. Gene and protein expression of lipogenic enzymes were examined in livers of wild-type (WT) and IL-6-deficient (IL-6(-/-) ) mice during fasting and re-feeding conditions. Effects of exogenous IL-6 administration on gene expression of these enzymes were evaluated in vivo. The involvement of STAT3 in mediating these IL-6 responses was investigated by using siRNA in human HepG2 cells. During feeding, the up-regulation in the hepatic expression of lipogenic genes presented similar time kinetics in WT and IL-6(-/-) mice. During fasting, expression of lipogenic genes decreased gradually over time in both strains, although the initial drop was more marked in IL-6(-/-) mice. Protein levels of hepatic lipogenic enzymes were lower in IL-6(-/-) than in WT mice at the end of the fasting period. In WT, circulating IL-6 levels paralleled gene expression of hepatic lipogenic enzymes. IL-6 administration in vivo and in vitro showed that IL-6-mediated signalling was associated with the up-regulation of hepatic lipogenic enzyme genes. Moreover, silencing STAT3 in HepG2 cells attenuated IL-6 mediated up-regulation of lipogenic gene transcription levels. IL-6 sustains levels of hepatic lipogenic enzymes during fasting through activation of STAT3. Our findings indicate that clinical use of STAT3-associated signalling cytokines, particularly against steatosis, should be undertaken with caution. © 2016 The British Pharmacological Society.

Association of Mumps Virus V Protein with RACK1 Results in Dissociation of STAT-1 from the Alpha Interferon Receptor Complex

PubMed Central

Kubota, Toru; Yokosawa, Noriko; Yokota, Shin-ichi; Fujii, Nobuhiro

2002-01-01

It has been reported that mumps virus protein V or the C-terminal Cys-rich region of protein V (Vsp) is associated with blocking of the interferon (IFN) signal transduction pathway through a decrease in STAT-1 production. The intracellular target of the V protein was investigated by using a two-hybrid screening system with Vsp as bait. Full-length V protein and Vsp were able to bind to RACK1, and the interaction did not require two WD domains, WD1 and WD2, in RACK1. A significant interaction between V protein and RACK1 was also demonstrated in cells persistently infected with mumps virus (FLMT cells), and the formation of the complex was not affected by treatment with IFN. On the other hand, in uninfected cells, STAT-1 was associated with the long form of the β subunit of the alpha IFN receptor, and this association was mediated by the function of RACK1 as an adaptor protein. Immunoprecipitation and glutathione S-transferase pull-down experiments revealed that the association of RACK1 or mumps virus V protein with the IFN receptor was undetectable in mumps virus-infected cells. Furthermore, RACK1 interacted with mumps virus V protein with a higher affinity than STAT-1 did. Therefore, it is suggested that mumps virus V protein has the ability to interact strongly with RACK1 and consequently to bring about the disruption of the complex formed from STAT-1, RACK1, and the IFN receptor. PMID:12438593

The novel curcumin analog FLLL32 decreases STAT3 DNA binding activity and expression, and induces apoptosis in osteosarcoma cell lines

PubMed Central

2011-01-01

Background Curcumin is a naturally occurring phenolic compound shown to have a wide variety of antitumor activities; however, it does not attain sufficient blood levels to do so when ingested. Using structure-based design, a novel compound, FLLL32, was generated from curcumin. FLLL32 possesses superior biochemical properties and more specifically targets STAT3, a transcription factor important in tumor cell survival, proliferation, metastasis, and chemotherapy resistance. In our previous work, we found that several canine and human osteosarcoma (OSA) cell lines, but not normal osteoblasts, exhibit constitutive phosphorylation of STAT3. Compared to curcumin, we hypothesized that FLLL32 would be more efficient at inhibiting STAT3 function in OSA cells and that this would result in enhanced downregulation of STAT3 transcriptional targets and subsequent death of OSA cells. Methods Human and canine OSA cells were treated with vehicle, curcumin, or FLLL32 and the effects on proliferation (CyQUANT®), apoptosis (SensoLyte® Homogeneous AMC Caspase- 3/7 Assay kit, western blotting), STAT3 DNA binding (EMSA), and vascular endothelial growth factor (VEGF), survivin, and matrix metalloproteinase-2 (MMP2) expression (RT-PCR, western blotting) were measured. STAT3 expression was measured by RT-PCR, qRT- PCR, and western blotting. Results Our data showed that FLLL32 decreased STAT3 DNA binding by EMSA. FLLL32 promoted loss of cell proliferation at lower concentrations than curcumin leading to caspase-3- dependent apoptosis, as evidenced by PARP cleavage and increased caspase 3/7 activity; this could be inhibited by treatment with the pan-caspase inhibitor Z-VAD-FMK. Treatment of OSA cells with FLLL32 decreased expression of survivin, VEGF, and MMP2 at both mRNA and protein levels with concurrent decreases in phosphorylated and total STAT3; this loss of total STAT3 occurred, in part, via the ubiquitin-proteasome pathway. Conclusions These data demonstrate that the novel curcumin analog FLLL32 has biologic activity against OSA cell lines through inhibition of STAT3 function and expression. Future work with FLLL32 will define the therapeutic potential of this compound in vivo. PMID:21443800

Gene expression profiling combined with bioinformatics analysis identify biomarkers for Parkinson disease.

PubMed

Diao, Hongyu; Li, Xinxing; Hu, Sheng; Liu, Yunhui

2012-01-01

Parkinson disease (PD) progresses relentlessly and affects approximately 4% of the population aged over 80 years old. It is difficult to diagnose in its early stages. The purpose of our study is to identify molecular biomarkers for PD initiation using a computational bioinformatics analysis of gene expression. We downloaded the gene expression profile of PD from Gene Expression Omnibus and identified differentially coexpressed genes (DCGs) and dysfunctional pathways in PD patients compared to controls. Besides, we built a regulatory network by mapping the DCGs to known regulatory data between transcription factors (TFs) and target genes and calculated the regulatory impact factor of each transcription factor. As the results, a total of 1004 genes associated with PD initiation were identified. Pathway enrichment of these genes suggests that biological processes of protein turnover were impaired in PD. In the regulatory network, HLF, E2F1 and STAT4 were found have altered expression levels in PD patients. The expression levels of other transcription factors, NKX3-1, TAL1, RFX1 and EGR3, were not found altered. However, they regulated differentially expressed genes. In conclusion, we suggest that HLF, E2F1 and STAT4 may be used as molecular biomarkers for PD; however, more work is needed to validate our result.

Gene Expression Profiling Combined with Bioinformatics Analysis Identify Biomarkers for Parkinson Disease

PubMed Central

Diao, Hongyu; Li, Xinxing; Hu, Sheng; Liu, Yunhui

2012-01-01

Parkinson disease (PD) progresses relentlessly and affects approximately 4% of the population aged over 80 years old. It is difficult to diagnose in its early stages. The purpose of our study is to identify molecular biomarkers for PD initiation using a computational bioinformatics analysis of gene expression. We downloaded the gene expression profile of PD from Gene Expression Omnibus and identified differentially coexpressed genes (DCGs) and dysfunctional pathways in PD patients compared to controls. Besides, we built a regulatory network by mapping the DCGs to known regulatory data between transcription factors (TFs) and target genes and calculated the regulatory impact factor of each transcription factor. As the results, a total of 1004 genes associated with PD initiation were identified. Pathway enrichment of these genes suggests that biological processes of protein turnover were impaired in PD. In the regulatory network, HLF, E2F1 and STAT4 were found have altered expression levels in PD patients. The expression levels of other transcription factors, NKX3-1, TAL1, RFX1 and EGR3, were not found altered. However, they regulated differentially expressed genes. In conclusion, we suggest that HLF, E2F1 and STAT4 may be used as molecular biomarkers for PD; however, more work is needed to validate our result. PMID:23284986

Effect of hyperthyroidism on circulating prolactin and hypothalamic expression of tyrosine hydroxylase, prolactin signaling cascade members and estrogen and progesterone receptors during late pregnancy and lactation in the rat.

PubMed

Pennacchio, Gisela E; Neira, Flavia J; Soaje, Marta; Jahn, Graciela A; Valdez, Susana R

2017-02-15

Hyperthyroidism (HyperT) compromises pregnancy and lactation, hindering suckling-induced PRL release. We studied the effect of HyperT on hypothalamic mRNA (RT-qPCR) and protein (Western blot) expression of tyrosine hydroxylase (TH), PRL receptor (PRLR) and signaling pathway members, estrogen-α (ERα) and progesterone (PR) receptors on late pregnancy (days G19, 20 and 21) and early lactation (L2) in rats. HyperT advanced pre-partum PRL release, reduced circulating PRL on L2 and increased TH mRNA (G21 and L2), p-TH, PRLR mRNA, STAT5 protein (G19 and L2), PRLR protein (G21) and CIS protein (G19). PRs mRNAs and protein decreased on G19 but afterwards PRA mRNA (G20), PRB mRNA (G21) and PRA mRNA and protein (L2) increased. ERα protein increased on G19 and decreased on G20. Thus, the altered hypothalamic PRLR, STAT5, PR and ERα expression in hyperthyroid rats may induce elevated TH expression and activation, that consequently, elevate dopaminergic tone during lactation, blunting suckling-induced PRL release and litter growth. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Prognostic Role of Phospho-STAT3 in Patients with Cancers of the Digestive System: A Systematic Review and Meta-Analysis.

PubMed

Li, Mu-xing; Bi, Xin-yu; Huang, Zhen; Zhao, Jian-jun; Han, Yue; Li, Zhi-Yu; Zhang, Ye-fan; Li, Yuan; Chen, Xiao; Hu, Xu-hui; Zhao, Hong; Cai, Jian-qiang

2015-01-01

The definite prognostic role of p-STAT3 has not been well defined. We performed a meta-analysis evaluating the prognostic role of p-STAT3 expression in patients with digestive system cancers. We searched the available articles reporting the prognostic value of p-STAT3 in patients with cancers of the digestive system, mainly including colorectal cancer, gastric cancer, hepatocellular carcinoma, esophagus cancer and pancreatic cancer. The pooled hazard ratios (HRs) with 95 % confidence intervals (95 % CIs) of overall survival (OS) and disease-free survival (DFS) were used to assess the prognostic role of p-STAT3 expression level in cancer tissues. And the association between p-STAT3 expression and clinicopathological characteristics was evaluated. A total of 22 studies with 3585 patients were finally enrolled in the meta-analysis. The results showed that elevated p-STAT3 expression level predicted inferior OS (HR = 1.809, 95% CI: 1.442-2.270, P < 0.001) and DFS (HR = 1.481, 95% CI: 1.028-2.133, P = 0.035) in patients with malignant cancers of the digestive system. Increased expression of p-STAT3 is significantly related with tumor cell differentiation (Odds ratio (OR) = 1.895, 95% CI: 1.364-2.632, P < 0.001) and lymph node metastases (OR = 2.108, 95% CI: 1.104-4.024, P = 0.024). Sensitivity analysis suggested that the pooled HR was stable and omitting a single study did not change the significance of the pooled HR. Funnel plots and Egger's tests revealed there was no significant publication bias in the meta-analysis. Phospho-STAT3 might be a prognostic factor of patients with digestive system cancers. More well designed studies with adequate follow-up are needed to gain a thorough understanding of the prognostic role of p-STAT3.

Ononitol monohydrate enhances PRDM16 & UCP-1 expression, mitochondrial biogenesis and insulin sensitivity via STAT6 and LTB4R in maturing adipocytes.

PubMed

Subash-Babu, P; Alshatwi, Ali A

2018-03-01

Ononitol monohydrate (OMH), a glycoside was originally isolated from Cassia tora (Linn.). Glycosides regulate lipid metabolism but scientific validation desired. Hence, we aimed to evaluate the effect of OMH on enhancing mitochondrial potential, mitochondrial biogenesis, upregulate the expression of brown adipogenesis specific genes in maturing adipocytes. In addition, we observed the inter-relation between adipocyte and T-lymphocyte; whether, OMH treated adipocyte-condition medium stimulate T-cell chemokine linked with insulin resistance. In a dose dependent manner OMH treated to preadipocyte significantly inhibited maturation and enhanced mitochondrial biogenesis, it was confirmed by Oil red 'O and Nile red stain without inducing cytotoxicity. The mRNA levels of adipocyte browning related genes such as, PR domain containing 16 (PRDM16), peroxisome proliferator activated receptor gamma coactivator 1 alpha (PPARγC1α) and uncoupling protein-1 (UCP-1) have been significantly upregulated. In addition, adipogenic transcription factors [such as proliferator activated receptor γ (PPARγ), CCAAT/enhancer binding protein (C/EBPα) and sterol regulatory element binding protein-1c (SREBP-1c)] and adipogenic genes were significantly down-regulated by treatment with OMH when compared to control cells. Protein expression levels of adiponectin have been increased; leptin, C/EBPα and leukotriene B4 receptor (LTB4R) were down regulated by OMH in mature adipocytes. In addition, adipocyte condition medium and OMH treated T-lymphocyte, significantly increased insulin signaling pathway related mRNAs, such as interlukin-4 (IL-4), signal transducer and activator of transcription 6 (STAT 6 ) and decreased leukotriene B4 (LTB 4 ). The present findings suggest that OMH increased browning factors in differentiating and maturing preadipocyte also decreased adipose tissue inflammation as well as the enhanced insulin signaling. Copyright © 2018. Published by Elsevier Masson SAS.

Constitutively Activated STAT3 Frequently Coexpresses with Epidermal Growth Factor Receptor in High-Grade Gliomas and Targeting STAT3 Sensitizes Them to Iressa and Alkylators

PubMed Central

Lo, Hui-Wen; Cao, Xinyu; Zhu, Hu; Ali-Osman, Francis

2009-01-01

Purpose The goals of this study are to elucidate the relationship of the oncogenic transcription factor signal transducer and activator of transcription 3(STAT3) with glioma aggressiveness and to understand the role of high STAT3 activity in the resistance of malignant gliomas and medulloblastomas to chemotherapy. Experimental Design Immunohistochemical staining and biochemical methods were used to examine the extent of STAT3 activation and EGFR expression in primary specimens and cell lines, respectively. Cellular response to drug treatments was determined using cell cytotoxicity and clonogenic growth assays. Results We found STAT3 to be constitutively activated in 60% of primary high-grade/malignant gliomas and the extent of activation correlated positively with glioma grade. High levels of activated/phosphorylated STAT3 were also present in cultured human malignant glioma and medulloblastoma cells. Three STAT3-activating kinases, Janus-activated kinase 2 (JAK2), EGFR, and EGFRvIII, contributed to STAT3 activation. An inhibitor toJAK2/STAT3, JSI-124, significantly reduced expression of STAT3 target genes, suppressed cancer cell growth, and induced apoptosis. Furthermore, we found that STAT3 constitutive activation coexisted with EGFR expression in 27.2% of primary high-grade/malignant gliomas and such coexpression correlated positively with glioma grade. Combination of an anti-EGFR agent Iressa and a JAK2/STAT3 inhibitor synergistically suppressed STAT3 activation and potently killed glioblastoma cell lines that expressed EGFR or EGFRvIII. JSI-124 also sensitized malignant glioma and medulloblastoma cells to temozolomide, 1,3-bis(2-chloroethyl)-1-nitrosourea, and cisplatin in which a synergism existed between JSI-124 and cisplatin. Conclusion STAT3 constitutive activation, alone and in concurrence with EGFR expression, plays an important role in high-grade/malignant gliomas and targeting STAT3/JAK2 sensitizes these tumors to anti-EGFR and alkylating agents. PMID:18829483

Adiponectin inhibits leptin signalling via multiple mechanisms to exert protective effects against hepatic fibrosis.

PubMed

Handy, Jeffrey A; Fu, Ping P; Kumar, Pradeep; Mells, Jamie E; Sharma, Shvetank; Saxena, Neeraj K; Anania, Frank A

2011-12-15

Adiponectin is protective against hepatic fibrosis, whereas leptin promotes fibrosis. In HSCs (hepatic stellate cells), leptin signals via a JAK2 (Janus kinase 2)/STAT3 (signal transducer and activator of transcription 3) pathway, producing effects that enhance ECM (extracellular matrix) deposition. SOCS-3 (suppressor of cytokine signalling-3) and PTP1B (protein tyrosine phosphatase 1B) are both negative regulators of JAK/STAT signalling, and recent studies have demonstrated a role for adiponectin in regulating SOCS-3 expression. In the present study we investigate mechanisms whereby adiponectin dampens leptin signalling and prevents excess ECM production. We treated culture-activated rat HSCs with recombinant adiponectin, leptin, both or neither, and also treated adiponectin knockout (Ad-/-) and wild-type mice with leptin and/or carbon tetrachloride (CCl4) or saline. We analyse JAK2 and Ob-Rb (long form of the leptin receptor) phosphorylation, and PTP1B expression and activity. We also explore potential mechanisms through which adiponectin regulates SOCS-3-Ob-Rb association. Adiponectin inhibits leptin-stimulated JAK2 activation and Ob-Rb phosphorylation in HSCs, whereas both were increased in Ad-/- mice. Adiponectin stimulates PTP1B expression and activity in vitro, whereas PTP1B expression was lower in Ad-/-mice than in wild-type mice. Adiponectin also promotes SOCS-3-Ob-R association and blocks leptin-stimulated formation of extracellular TIMP-1 (tissue inhibitor of metalloproteinases-1)-MMP-1 (matrix metalloproteinase-1) complexes in vitro. These results suggest two novel mechanisms whereby adiponectin inhibits hepatic fibrosis: (i) by promoting binding of SOCS-3 to Ob-Rb, and (ii) by stimulating PTP1B expression and activity, thus inhibiting JAK2/STAT3 signalling at multiple points.

Revealing the cellular localization of STAT1 during the cell cycle by super-resolution imaging

PubMed Central

Gao, Jing; Wang, Feng; Liu, Yanhou; Cai, Mingjun; Xu, Haijiao; Jiang, Junguang; Wang, Hongda

2015-01-01

Signal transducers and activators of transcription (STATs) can transduce cytokine signals and regulate gene expression. The cellular localization and nuclear trafficking of STAT1, a representative of the STAT family with multiple transcriptional functions, is tightly related with transcription process, which usually happens in the interphase of the cell cycle. However, these priority questions regarding STAT1 distribution and localization at the different cell-cycle stages remain unclear. By using direct stochastic optical reconstruction microscopy (dSTORM), we found that the nuclear expression level of STAT1 increased gradually as the cell cycle carried out, especially after EGF stimulation. Furthermore, STAT1 formed clusters in the whole cell during the cell cycle, with the size and the number of clusters also increasing significantly from G1 to G2 phase, suggesting that transcription and other cell-cycle related activities can promote STAT1 to form more and larger clusters for fast response to signals. Our work reveals that the cellular localization and clustering distribution of STAT1 are associated with the cell cycle, and further provides an insight into the mechanism of cell-cycle regulated STAT1 signal transduction. PMID:25762114

Mitochondrial translocation of signal transducer and activator of transcription 5 (STAT5) in leukemic T cells and cytokine-stimulated cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chueh, Fu-Yu; Leong, King-Fu; Yu, Chao-Lan, E-mail: chaolan.yu@rosalindfranklin.edu

2010-11-26

Research highlights: {yields} STAT5 interacts with a mitochondrial protein PDC-E2 in a leukemic T cell line LSTRA. {yields} Tyrosine-phosphorylated STAT5, but not STAT3, is present in LSTRA mitochondria. {yields} Cytokines induce mitochondrial translocation of STAT5, but not STAT1 or STAT3. {yields} Cytokine-induced mitochondrial translocation of tyrosine-phosphorylated STAT5 is transient. {yields} Mitochondrial STAT5 binds to a putative STAT5 site in the mitochondrial DNA in vitro. -- Abstract: Signal transducers and activators of transcription (STATs) were first identified as key signaling molecules in response to cytokines. Constitutive STAT activation also has been widely implicated in oncogenesis. We analyzed STAT5-associated proteins in amore » leukemic T cell line LSTRA, which exhibits constitutive tyrosine phosphorylation and activation of STAT5. A cellular protein was found to specifically interact with STAT5 in LSTRA cells by co-immunoprecipitation. Sequencing analysis and subsequent immunoblotting confirmed the identity of this STAT5-associated protein as the E2 component of mitochondrial pyruvate dehydrogenase complex (PDC-E2). Consistent with this interaction, both subcellular fractionation and immunofluorescence microscopy revealed mitochondrial localization of STAT5 in LSTRA cells. Mitochondrial localization of tyrosine-phosphorylated STAT5 also occurred in cytokine-stimulated cells. A time course experiment further demonstrated the transient kinetics of STAT5 mitochondrial translocation after cytokine stimulation. In contrast, cytokine-induced STAT1 and STAT3 activation did not result in their translocation into mitochondria. Furthermore, we showed that mitochondrial STAT5 bound to the D-loop regulatory region of mitochondrial DNA in vitro. It suggests a potential role of STAT5 in regulating the mitochondrial genome. Proliferative metabolism toward aerobic glycolysis is well known in cancer cells as the Warburg effect and is also observed in cytokine-stimulated cells. Our novel findings of cytokine-induced STAT5 translocation into mitochondria and its link to oncogenesis provide important insights into the underlying mechanisms of this characteristic metabolic shift.« less

A plausibly causal functional lupus-associated risk variant in the STAT1-STAT4 locus.

PubMed

Patel, Zubin; Lu, Xiaoming; Miller, Daniel; Forney, Carmy R; Lee, Joshua; Lynch, Arthur; Schroeder, Connor; Parks, Lois; Magnusen, Albert F; Chen, Xiaoting; Pujato, Mario; Maddox, Avery; Zoller, Erin E; Namjou, Bahram; Brunner, Hermine I; Henrickson, Michael; Huggins, Jennifer L; Williams, Adrienne H; Ziegler, Julie T; Comeau, Mary E; Marion, Miranda C; Glenn, Stuart B; Adler, Adam; Shen, Nan; Nath, Swapan K; Stevens, Anne M; Freedman, Barry I; Pons-Estel, Bernardo A; Tsao, Betty P; Jacob, Chaim O; Kamen, Diane L; Brown, Elizabeth E; Gilkeson, Gary S; Alarcón, Graciela S; Martin, Javier; Reveille, John D; Anaya, Juan-Manuel; James, Judith A; Sivils, Kathy L; Criswell, Lindsey A; Vilá, Luis M; Petri, Michelle; Scofield, R Hal; Kimberly, Robert P; Edberg, Jeffrey C; Ramsey-Goldman, Rosalind; Bang, So-Young; Lee, Hye-Soon; Bae, Sang-Cheol; Boackle, Susan A; Cunninghame Graham, Deborah; Vyse, Timothy J; Merrill, Joan T; Niewold, Timothy B; Ainsworth, Hannah C; Silverman, Earl D; Weisman, Michael H; Wallace, Daniel J; Raj, Prithvi; Guthridge, Joel M; Gaffney, Patrick M; Kelly, Jennifer A; Alarcón-Riquelme, Marta E; Langefeld, Carl D; Wakeland, Edward K; Kaufman, Kenneth M; Weirauch, Matthew T; Harley, John B; Kottyan, Leah C

2018-04-18

Systemic Lupus Erythematosus (SLE or lupus) (OMIM: 152700) is a chronic autoimmune disease with debilitating inflammation that affects multiple organ systems. The STAT1-STAT4 locus is one of the first and most highly-replicated genetic loci associated with lupus risk. We performed a fine-mapping study to identify plausible causal variants within the STAT1-STAT4 locus associated with increased lupus disease risk. Using complementary frequentist and Bayesian approaches in trans-ancestral Discovery and Replication cohorts, we found one variant whose association with lupus risk is supported across ancestries in both the Discovery and Replication cohorts: rs11889341. In B cell lines from patients with lupus and healthy controls, the lupus risk allele of rs11889341 was associated with increased STAT1 expression. We demonstrated that the transcription factor HMGA1, a member of the HMG transcription factor family with an AT-hook DNA-binding domain, has enriched binding to the risk allele compared to the non-risk allele of rs11889341. We identified a genotype-dependent repressive element in the DNA within the intron of STAT4 surrounding rs11889341. Consistent with expression quantitative trait locus (eQTL) analysis, the lupus risk allele of rs11889341 decreased the activity of this putative repressor. Altogether, we present a plausible molecular mechanism for increased lupus risk at the STAT1-STAT4 locus in which the risk allele of rs11889341, the most probable causal variant, leads to elevated STAT1 expression in B cells due to decreased repressor activity mediated by increased binding of HMGA1.

Integrity of the LXXLL motif in Stat6 is required for the inhibition of breast cancer cell growth and enhancement of differentiation in the context of progesterone

PubMed Central

2014-01-01

Background Progesterone is essential for the proliferation and differentiation of mammary gland epithelium. Studies of breast cancer cells have demonstrated a biphasic progesterone response consisting of an initial proliferative burst followed by sustained growth arrest. However, the transcriptional factors acting with the progesterone receptor (PR) to mediate the effects of progesterone on mammary cell growth and differentiation remain to be determined. Recently, it was demonstrated that signal transducer and activator of transcription 6 (Stat6) is a cell growth suppressor. Similar to progesterone-bound PR, Stat6 acts by inducing the expression of the G1 cyclin-dependent kinase inhibitors p21 and p27. The possible interaction between Stat6 and progesterone pathways in mammary cells was therefore investigated in the present study. Methods ChIP and luciferase were assayed to determine whether Stat6 induces p21 and p27 expression by recruitment at the proximal Sp1-binding sites of the gene promoters. Immunoprecipitation and Western blotting were performed to investigate the interaction between Stat6 and PR-B. The cellular DNA content and cell cycle distribution in breast cancer cells were analyzed by FACS. Results We found that Stat6 interacts with progesterone-activated PR in T47D cells. Stat6 synergizes with progesterone-bound PR to transactivate the p21 and p27 gene promoters at the proximal Sp1-binding sites. Moreover, Stat6 overexpression and knockdown, respectively, increased or prevented the induction of p21 and p27 gene expression by progesterone. Stat6 knockdown also abolished the inhibitory effects of progesterone on pRB phosphorylation, G1/S cell cycle progression, and cell proliferation. In addition, knockdown of Stat6 expression prevented the induction of breast cell differentiation markers, previously identified as progesterone target genes. Finally, Stat6 gene expression levels increased following progesterone treatment, indicating a positive auto-regulatory loop between PR and Stat6. Conclusions Taken together, these data identify Stat6 as a coactivator of PR mediating the growth-inhibitory and differentiation effects of progesterone on breast cancer cells. PMID:24401087

Electrical stimulation induces IL-6 in skeletal muscle through extracellular ATP by activating Ca(2+) signals and an IL-6 autocrine loop.

PubMed

Bustamante, Mario; Fernández-Verdejo, Rodrigo; Jaimovich, Enrique; Buvinic, Sonja

2014-04-15

Interleukin-6 (IL-6) is an important myokine that is highly expressed in skeletal muscle cells upon exercise. We assessed IL-6 expression in response to electrical stimulation (ES) or extracellular ATP as a known mediator of the excitation-transcription mechanism in skeletal muscle. We examined whether the canonical signaling cascade downstream of IL-6 (IL-6/JAK2/STAT3) also responds to muscle cell excitation, concluding that IL-6 influences its own expression through a positive loop. Either ES or exogenous ATP (100 μM) increased both IL-6 expression and p-STAT3 levels in rat myotubes, a process inhibited by 100 μM suramin and 2 U/ml apyrase. ATP also evoked IL-6 expression in both isolated skeletal fibers and extracts derived from whole FDB muscles. ATP increased IL-6 release up to 10-fold. STAT3 activation evoked by ATP was abolished by the JAK2 inhibitor HBC. Blockade of secreted IL-6 with a neutralizing antibody or preincubation with the STAT3 inhibitor VIII reduced STAT3 activation evoked by extracellular ATP by 70%. Inhibitor VIII also reduced by 70% IL-6 expression evoked by ATP, suggesting a positive IL-6 loop. In addition, ATP increased up to 60% the protein levels of SOCS3, a negative regulator of the IL-6 signaling pathway. On the other hand, intracellular calcium chelation or blockade of IP3-dependent calcium signals abolished STAT3 phosphorylation evoked by either extracellular ATP or ES. These results suggest that expression of IL-6 in stimulated skeletal muscle cells is mediated by extracellular ATP and nucleotide receptors, involving IP3-dependent calcium signals as an early step that triggers a positive IL-6 autocrine loop.

Chlorogenic acid suppresses lipopolysaccharide‑induced nitric oxide and interleukin‑1β expression by inhibiting JAK2/STAT3 activation in RAW264.7 cells.

PubMed

Kim, Sang-Hun; Park, Sun-Young; Park, Young-Lan; Myung, Dae-Seong; Rew, Jong-Sun; Joo, Young-Eun

2017-12-01

Chlorogenic acid (CA) is a phenolic compound purified from coffee, fruits and their associated beverages, which possess various biological properties, such as antioxidant and anticarcinogenic activities. The present study evaluated the effects of CA on lipopolysaccharide (LPS)‑induced inflammation in RAW264.7 cells and the associated intracellular signaling pathways using reverse transcription‑quantitative polymerase chain reaction, western blotting and enzyme‑linked immunosorbent assays. CA pretreatment inhibited LPS‑induced expression of inducible nitric oxide synthase (iNOS), nitric oxide (NO) and pro‑inflammatory mediators including interleukin (IL)‑6, tumor necrosis factor‑α (TNF‑α), macrophage inflammatory protein‑2 (MIP‑2) and IL‑1β in RAW264.7 cells. In addition, phosphorylation of Janus kinase 2/signal transducer and activator of transcription 3 (JAK2/STAT3) with LPS was inhibited by CA pretreatment. CA and STAT3 inhibitor (STAT3i) pretreatment inhibited LPS‑induced nuclear translocation of phosphorylated STAT3. In addition, STAT3i inhibited the LPS‑induced expression of iNOS, NO and IL‑1β similar to the results of CA pretreatment. By contrast, STAT3i did not inhibit the LPS‑induced increase in IL‑6, TNF‑α and MIP‑2 expression. These results indicate that CA may suppress LPS‑induced NO and IL‑1β expression by inhibiting JAK2/STAT3 activation in RAW264.7 cells.

Regulation of Spi 2.1 and 2.2 gene expression after turpentine inflammation: discordant responses to IL-6.

PubMed

Berry, S A; Bergad, P L; Stolz, A M; Towle, H C; Schwarzenberg, S J

1999-06-01

The rat serine protease inhibitor (Spi) 2 gene family includes both positive (Spi 2.2) and negative (Spi 2.1) acute phase reactants, facilitating modeling of regulation of hepatic acute phase response (APR). To examine the role of signal transducer and activation of transcription (STAT) proteins in the divergent regulation of these model genes after induction of APR, we evaluated the proximal promoters of the genes, focusing on STAT binding sites contained in these promoter elements. Induction of APR by turpentine injection includes activation of a STAT3 complex that can bind to a gamma-activated sequence (GAS) in the Spi 2.2 gene promoter, although the Spi 2.2 GAS site can bind STAT1 or STAT5 as well. To create an in vitro model of APR, primary hepatocytes were treated with combinations of cytokines and hormones to mimic the hormonal milieu of the whole animal after APR induction. Incubation of primary rat hepatocytes with interleukin (IL)-6, a critical APR cytokine, leads to activation of STAT3 and a 28-fold induction of a chloramphenicol acetyltransferase reporter construct containing the -319 to +85 region of the Spi 2.2 promoter. This suggests the turpentine-induced increase of Spi 2.2 is mediated primarily by IL-6. In contrast, although turpentine treatment reduces Spi 2.1 mRNA in vivo and IL-6 does not increase Spi 2.1 mRNA in primary rat hepatocytes, treatment of hepatocytes with IL-6 results in a 5. 4-fold induction of Spi 2.1 promoter activity mediated through the paired GAS elements in this promoter. Differential regulation of Spi 2.1 and 2.2 genes is due in part to differences in the promoters of these genes at the GAS sites. IL-6 alone fails to reproduce the pattern of rat Spi 2 gene expression that results from turpentine-induced inflammation.

Remote Control of Intestinal Stem Cell Activity by Haemocytes in Drosophila

PubMed Central

Chakrabarti, Sveta; Li, Xiaoxue; Collas, Esther Jeanne; Boquete, Jean-Phillipe; Lemaitre, Bruno

2016-01-01

The JAK/STAT pathway is a key signaling pathway in the regulation of development and immunity in metazoans. In contrast to the multiple combinatorial JAK/STAT pathways in mammals, only one canonical JAK/STAT pathway exists in Drosophila. It is activated by three secreted proteins of the Unpaired family (Upd): Upd1, Upd2 and Upd3. Although many studies have established a link between JAK/STAT activation and tissue damage, the mode of activation and the precise function of this pathway in the Drosophila systemic immune response remain unclear. In this study, we used mutations in upd2 and upd3 to investigate the role of the JAK/STAT pathway in the systemic immune response. Our study shows that haemocytes express the three upd genes and that injury markedly induces the expression of upd3 by the JNK pathway in haemocytes, which in turn activates the JAK/STAT pathway in the fat body and the gut. Surprisingly, release of Upd3 from haemocytes upon injury can remotely stimulate stem cell proliferation and the expression of Drosomycin-like genes in the intestine. Our results also suggest that a certain level of intestinal epithelium renewal is required for optimal survival to septic injury. While haemocyte-derived Upd promotes intestinal stem cell activation and survival upon septic injury, haemocytes are dispensable for epithelium renewal upon oral bacterial infection. Our study also indicates that intestinal epithelium renewal is sensitive to insults from both the lumen and the haemocoel. It also reveals that release of Upds by haemocytes coordinates the wound-healing program in multiple tissues, including the gut, an organ whose integrity is critical to fly survival. PMID:27231872

Conjugated bilirubin affects cytokine profiles in hepatitis A virus infection by modulating function of signal transducer and activator of transcription factors

PubMed Central

Castro-García, Flor P; Corral-Jara, Karla F; Escobedo-Melendez, Griselda; Sandoval-Hernandez, Monserrat A; Rosenstein, Yvonne; Roman, Sonia; Panduro, Arturo; Fierro, Nora A

2014-01-01

Hepatitis A virus (HAV) infection is the major cause of acute liver failure in paediatric patients. The clinical spectrum of infection is variable, and liver injury is determined by altered hepatic enzyme function and bilirubin concentration. We recently reported differences in cytokine profiles between distinct HAV-induced clinical courses, and bilirubin has been recognized as a potential immune-modulator. However, how bilirubin may affect cytokine profiles underlying the variability in the course of infection has not been determined. Herein, we used a transcription factor (TF) binding site identification approach to retrospectively analyse cytokine expression in HAV-infected children and to predict the entire set of TFs associated with the expression of specific cytokine profiles. The results suggested that modulation of the activity of signal transducers and activators of transcription proteins (STATs) may play a central role during HAV infection. This led us to compare the degree of STAT phosphorylation in peripheral blood lymphoid cells (PBLCs) from paediatric patients with distinct levels of conjugated bilirubin (CB). Low CB levels in sera were associated with increased STAT-1 and STAT-5 phosphorylation. A positive correlation was observed between the serum interleukin-6 (IL-6) content and CB values, whereas higher levels of CB correlated with reduced serum IL-8 values and with a reduction in the proportion of PBLCs positive for STAT-5 phosphorylation. When CB was used to stimulate patients’ PBLCs in vitro, the levels of IL-6 and tumour necrosis factor-α were increased. The data showed that bilirubin plays a role in STAT function and affects cytokine profile expression during HAV infection. PMID:24943111

Remote Control of Intestinal Stem Cell Activity by Haemocytes in Drosophila.

PubMed

Chakrabarti, Sveta; Dudzic, Jan Paul; Li, Xiaoxue; Collas, Esther Jeanne; Boquete, Jean-Phillipe; Lemaitre, Bruno

2016-05-01

The JAK/STAT pathway is a key signaling pathway in the regulation of development and immunity in metazoans. In contrast to the multiple combinatorial JAK/STAT pathways in mammals, only one canonical JAK/STAT pathway exists in Drosophila. It is activated by three secreted proteins of the Unpaired family (Upd): Upd1, Upd2 and Upd3. Although many studies have established a link between JAK/STAT activation and tissue damage, the mode of activation and the precise function of this pathway in the Drosophila systemic immune response remain unclear. In this study, we used mutations in upd2 and upd3 to investigate the role of the JAK/STAT pathway in the systemic immune response. Our study shows that haemocytes express the three upd genes and that injury markedly induces the expression of upd3 by the JNK pathway in haemocytes, which in turn activates the JAK/STAT pathway in the fat body and the gut. Surprisingly, release of Upd3 from haemocytes upon injury can remotely stimulate stem cell proliferation and the expression of Drosomycin-like genes in the intestine. Our results also suggest that a certain level of intestinal epithelium renewal is required for optimal survival to septic injury. While haemocyte-derived Upd promotes intestinal stem cell activation and survival upon septic injury, haemocytes are dispensable for epithelium renewal upon oral bacterial infection. Our study also indicates that intestinal epithelium renewal is sensitive to insults from both the lumen and the haemocoel. It also reveals that release of Upds by haemocytes coordinates the wound-healing program in multiple tissues, including the gut, an organ whose integrity is critical to fly survival.

Conjugated bilirubin affects cytokine profiles in hepatitis A virus infection by modulating function of signal transducer and activator of transcription factors.

PubMed

Castro-García, Flor P; Corral-Jara, Karla F; Escobedo-Melendez, Griselda; Sandoval-Hernandez, Monserrat A; Rosenstein, Yvonne; Roman, Sonia; Panduro, Arturo; Fierro, Nora A

2014-12-01

Hepatitis A virus (HAV) infection is the major cause of acute liver failure in paediatric patients. The clinical spectrum of infection is variable, and liver injury is determined by altered hepatic enzyme function and bilirubin concentration. We recently reported differences in cytokine profiles between distinct HAV-induced clinical courses, and bilirubin has been recognized as a potential immune-modulator. However, how bilirubin may affect cytokine profiles underlying the variability in the course of infection has not been determined. Herein, we used a transcription factor (TF) binding site identification approach to retrospectively analyse cytokine expression in HAV-infected children and to predict the entire set of TFs associated with the expression of specific cytokine profiles. The results suggested that modulation of the activity of signal transducers and activators of transcription proteins (STATs) may play a central role during HAV infection. This led us to compare the degree of STAT phosphorylation in peripheral blood lymphoid cells (PBLCs) from paediatric patients with distinct levels of conjugated bilirubin (CB). Low CB levels in sera were associated with increased STAT-1 and STAT-5 phosphorylation. A positive correlation was observed between the serum interleukin-6 (IL-6) content and CB values, whereas higher levels of CB correlated with reduced serum IL-8 values and with a reduction in the proportion of PBLCs positive for STAT-5 phosphorylation. When CB was used to stimulate patients' PBLCs in vitro, the levels of IL-6 and tumour necrosis factor-α were increased. The data showed that bilirubin plays a role in STAT function and affects cytokine profile expression during HAV infection. © 2014 John Wiley & Sons Ltd.

Activation of the NF-κB pathway by the STAT3 inhibitor JSI-124 in human glioblastoma cells

PubMed Central

McFarland, Braden C.; Gray, G. Kenneth; Nozell, Susan E.; Hong, Suk W.; Benveniste, Etty N.

2013-01-01

Glioblastoma tumors are characterized by their invasiveness and resistance to therapies. The transcription factor STAT3 was recently identified as a master transcriptional regulator in the mesenchymal subtype of GBM, which has generated an increased interest in targeting STAT3. We have evaluated more closely the mechanism of action of one particular STAT3 inhibitor, JSI-124 (cucurbitacin I). In this study, we confirmed that JSI-124 inhibits both constitutive and stimulus-induced JAK2 and STAT3 phosphorylation, and decreases cell proliferation while inducing apoptosis in cultured GBM cells. However, we discovered that prior to the inhibition of STAT3, JSI-124 activates the NF-κB pathway, via NF-κB p65 phosphorylation and nuclear translocation. In addition, JSI-124 treatment induces the expression of IL-6, IL-8 and SOCS3 mRNA, which leads to a corresponding increase in IL-6, IL-8 and SOCS3 protein expression. Moreover, the NF-κB driven SOCS3 expression acts as a negative regulator of STAT3, abrogating any subsequent STAT3 activation and provides a mechanism of STAT3 inhibition following JSI-124 treatment. Chromatin immunoprecipitation analysis confirms that NF-κB p65 in addition to other activating co-factors are found at the promoters of IL-6, IL-8 and SOCS3, following JSI-124 treatment. Using pharmacological inhibition of NF-κB and inducible knockdown of NF-κB p65, we found that JSI-124-induced expression of IL-6, IL-8 and SOCS3 was significantly inhibited, demonstrating an NF-κB dependent mechanism. Our data indicate that although JSI-124 may demonstrate potential anti-tumor effects through inhibition of STAT3, other off-target pro-inflammatory pathways are activated, emphasizing that more careful and thorough pre-clinical investigations must be implemented to prevent potential harmful effects. PMID:23386688

REGULATION OF INFLAMMATORY TRANSCRIPTION FACTORS BY HEAT SHOCK PROTEIN 70 IN PRIMARY CULTURED ASTROCYTES EXPOSED TO OXYGEN–GLUCOSE DEPRIVATION

PubMed Central

KIM, J. Y.; YENARI, M. A.; LEE, J. E.

2018-01-01

Inflammation is an important event in ischemic injury. These immune responses begin with the expression of pro-inflammatory genes modulating transcription factors, such as nuclear factor-κB (NF-κB), activator protein-1 (AP-1), and signal transducers and activator of transcription-1 (STAT-1). The 70-kDa heat shock protein (Hsp70) can both induce and arrest inflammatory reactions and lead to improved neurological outcome in experimental brain injury and ischemia. Since Hsp70 are induced under heat stress, we investigated the link between Hsp70 neuroprotection and phosphorylation of inhibitor of κB (IκB), c-Jun N-terminal kinases (JNK) and p38 through co-immunoprecipitation and enzyme-linked immunosorbent assay (ELISA) assay. Transcription factors and pro-inflammatory genes were quantified by immunoblotting, electrophoretic-mobility shift assay and reverse transcription-polymerase chain reaction assays. The results showed that heat stress led to Hsp70 overexpression which rendered neuroprotection after ischemia-like injury. Overexpression Hsp70 also interrupts the phosphorylation of IκB, JNK and p38 and bluntsDNA binding of their transcription factors (NF-κB, AP-1 and STAT-1), effectively downregulating the expression of pro-inflammatory genes inheat-pretreatedastrocytes. Takentogether, these results suggest that overexpression of Hsp70 may protect against brain ischemia via an anti-inflammatory mechanism by interrupting the phosphorylation of upstream of transcription factors. PMID:25485480

STAT3/IRF1 Pathway Activation Sensitizes Cervical Cancer Cells to Chemotherapeutic Drugs.

PubMed

Walch-Rückheim, Barbara; Pahne-Zeppenfeld, Jennifer; Fischbach, Jil; Wickenhauser, Claudia; Horn, Lars Christian; Tharun, Lars; Büttner, Reinhard; Mallmann, Peter; Stern, Peter; Kim, Yoo-Jin; Bohle, Rainer Maria; Rübe, Christian; Ströder, Russalina; Juhasz-Böss, Ingolf; Solomayer, Erich-Franz; Smola, Sigrun

2016-07-01

Neoadjuvant radio/chemotherapy regimens can markedly improve cervical cancer outcome in a subset of patients, while other patients show poor responses, but may encounter severe adverse effects. Thus, there is a strong need for predictive biomarkers to improve clinical management of cervical cancer patients. STAT3 is considered as a critical antiapoptotic factor in various malignancies. We therefore investigated STAT3 activation during cervical carcinogenesis and its impact on the response of cervical cancer cells to chemotherapeutic drugs. Tyr705-phosphorylated STAT3 increased from low-grade cervical intraepithelial neoplasia (CIN1) to precancerous CIN3 lesions. Notably, pTyr705-STAT3 activation significantly declined from CIN3 to invasive cancer, also when compared in the same clinical biopsy. pTyr705-STAT3 was also low or absent in cultured human cervical cancer cell lines, consistent with the in vivo expression data. Unexpectedly, IL6-type cytokine signaling inducing STAT3 activation rendered cervical cancer cells significantly more susceptible to chemotherapeutic drugs, that is, cisplatin or etoposide. This chemosensitization was STAT3-dependent and we identified IFN regulatory factor-1 (IRF1) as the STAT3-inducible mediator required for cell death enhancement. In line with these data, pTyr705-STAT3 significantly correlated with nuclear IRF1 expression in cervical cancer in vivo Importantly, high IRF1 expression in pretreatment cervical cancer biopsy cells was associated with a significantly better response to neoadjuvant radio/chemotherapy of the patients. In summary, our study has identified a key role of the STAT3/IRF1 pathway for chemosensitization in cervical cancer. Our results suggest that pretherapeutic IRF1 expression should be evaluated as a novel predictive biomarker for neoadjuvant radio/chemotherapy responses. Cancer Res; 76(13); 3872-83. ©2016 AACR. ©2016 American Association for Cancer Research.

Sustained activation of STAT5 is essential for chromatin remodeling and maintenance of mammary-specific function

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xu, Ren; Nelson, Celeste M.; Muschler, John L.

2009-06-03

Epithelial cells, once dissociated and placed in two-dimensional (2D) cultures, rapidly lose tissue-specific functions. We showed previously that in addition to prolactin, signaling by laminin-111 was necessary to restore functional differentiation of mammary epithelia. Here, we elucidate two additional aspects of laminin-111 action. We show that in 2D cultures, the prolactin receptor is basolaterally localized and physically segregated from its apically placed ligand. Detachment of the cells exposes the receptor to ligation by prolactin leading to signal transducers and activators of transcription protein 5 (STAT5) activation, but only transiently and not sufficiently for induction of milk protein expression. We showmore » that laminin-111 reorganizes mammary cells into polarized acini, allowing both the exposure of the prolactin receptor and sustained activation of STAT5. The use of constitutively active STAT5 constructs showed that the latter is necessary and sufficient for chromatin reorganization and {beta}-casein transcription. These results underscore the crucial role of continuous laminin signaling and polarized tissue architecture in maintenance of transcription factor activation, chromatin organization, and tissue-specific gene expression.« less

Silymarin suppresses the PGE2 -induced cell migration through inhibition of EP2 activation; G protein-dependent PKA-CREB and G protein-independent Src-STAT3 signal pathways.

PubMed

Woo, Seon Min; Min, Kyoung-Jin; Chae, In Gyeong; Chun, Kyung-Soo; Kwon, Taeg Kyu

2015-03-01

Silymarin has been known as a chemopreventive agent, and possesses multiple anti-cancer activities including induction of apoptosis, inhibition of proliferation and growth, and blockade of migration and invasion. However, whether silymarin could inhibit prostaglandin (PG) E2 -induced renal cell carcinoma (RCC) migration and what are the underlying mechanisms are not well elucidated. Here, we found that silymarin markedly inhibited PGE2 -stimulated migration. PGE2 induced G protein-dependent CREB phosphorylation via protein kinase A (PKA) signaling, and PKA inhibitor (H89) inhibited PGE2 -mediated migration. Silymarin reduced PGE2 -induced CREB phosphorylation and CRE-promoter activity. PGE2 also activated G protien-independent signaling pathways (Src and STAT3) and silymarin reduced PGE2 -induced phosphorylation of Src and STAT3. Inhibitor of Src (Saracatinib) markedly reduced PGE2 -mediated migration. We found that EP2, a PGE2 receptor, is involved in PGE2 -mediated cell migration. Down regulation of EP2 by EP2 siRNA and EP2 antagonist (AH6809) reduced PGE2 -inudced migration. In contrast, EP2 agonist (Butaprost) increased cell migration and silymarin effectively reduced butaprost-mediated cell migration. Moreover, PGE2 increased EP2 expression through activation of positive feedback mechanism, and PGE2 -induced EP2 expression, as well as basal EP2 levels, were reduced in silymarin-treated cells. Taken together, our study demonstrates that silymarin inhibited PGE2 -induced cell migration through inhibition of EP2 signaling pathways (G protein dependent PKA-CREB and G protein-independent Src-STAT3). © 2013 Wiley Periodicals, Inc.

Survival response of hippocampal neurons under low oxygen conditions induced by Hippophae rhamnoides is associated with JAK/STAT signaling.

PubMed

Manickam, Manimaran; Tulsawani, Rajkumar

2014-01-01

Janus activated kinase/signal transducers and activators of transcription (JAK/STATs) pathway are associated with various neuronal functions including cell survival and inflammation. In the present study, it is hypothesized that protective action of aqueous extract of Hippophae rhamnoides in hippocampal neurons against hypoxia is mediated via JAK/STATs. Neuronal cells exposed to hypoxia (0.5% O2) display higher reactive oxygen species with compromised antioxidant status compared to unexposed control cells. Further, these cells had elevated levels of pro-inflammatory cytokines; tumor necrosis factor α and interleukin 6 and nuclear factor κappa B. Moreover, the expression of JAK1 was found to be highly expressed with phosphorylation of STAT3 and STAT5. Cells treated with JAK1, STAT3 and STAT5 specific inhibitors resulted in more cell death compared to hypoxic cells. Treatment of cells with extract prevented oxidative stress and inflammatory response associated with hypoxia. The extract treated cells had more cell survival than hypoxic cells with induction of JAK1 and STAT5b. Cells treated with extract having suppressed JAK1 or STAT3 or STAT5 expression showed reduced cell viability than the cell treated with extract alone. Overall, the findings from these studies indicate that the aqueous extract of Hippophae rhamnoides treatment inhibited hypoxia induced oxidative stress by altering cellular JAK1, STAT3 and STAT5 levels thereby enhancing cellular survival response to hypoxia and provide a basis for possible use of aqueous extract of Hippophae rhamnoides in facilitating tolerance to hypoxia.

Survival Response of Hippocampal Neurons under Low Oxygen Conditions Induced by Hippophae rhamnoides is Associated with JAK/STAT Signaling

PubMed Central

Manickam, Manimaran; Tulsawani, Rajkumar

2014-01-01

Janus activated kinase/signal transducers and activators of transcription (JAK/STATs) pathway are associated with various neuronal functions including cell survival and inflammation. In the present study, it is hypothesized that protective action of aqueous extract of Hippophae rhamnoides in hippocampal neurons against hypoxia is mediated via JAK/STATs. Neuronal cells exposed to hypoxia (0.5% O2) display higher reactive oxygen species with compromised antioxidant status compared to unexposed control cells. Further, these cells had elevated levels of pro-inflammatory cytokines; tumor necrosis factor α and interleukin 6 and nuclear factor κappa B. Moreover, the expression of JAK1 was found to be highly expressed with phosphorylation of STAT3 and STAT5. Cells treated with JAK1, STAT3 and STAT5 specific inhibitors resulted in more cell death compared to hypoxic cells. Treatment of cells with extract prevented oxidative stress and inflammatory response associated with hypoxia. The extract treated cells had more cell survival than hypoxic cells with induction of JAK1 and STAT5b. Cells treated with extract having suppressed JAK1 or STAT3 or STAT5 expression showed reduced cell viability than the cell treated with extract alone. Overall, the findings from these studies indicate that the aqueous extract of Hippophae rhamnoides treatment inhibited hypoxia induced oxidative stress by altering cellular JAK1, STAT3 and STAT5 levels thereby enhancing cellular survival response to hypoxia and provide a basis for possible use of aqueous extract of Hippophae rhamnoides in facilitating tolerance to hypoxia. PMID:24516559

Adiponectin inhibits leptin signaling via multiple mechanisms to exert protective effects against hepatic fibrosis

PubMed Central

HANDY, Jeffrey A.; FU, Ping P.; KUMAR, Pradeep; MELLS, Jamie E.; SHARMA, Shvetank; SAXENA, Neeraj K.; ANANIA, Frank A.

2011-01-01

SYNOPSIS Adiponectin is protective against hepatic fibrosis, while leptin promotes fibrosis. In hepatic stellate cells (HSCs), leptin signals via a Janus Kinase 2/Signal Transducers and Activators of Transcription 3 (Jak2/Stat3) pathway, producing effects that enhance extracellular matrix deposition. Suppressors of Cytokine Signaling-3 (SOCS-3) and Protein Tyrosine Phosphatase-1B (PTP1B) are both negative regulators of Jak/Stat signaling, and recent studies demonstrated a role for adiponectin in regulating SOCS-3 expression. In this study we investigated mechanisms whereby adiponectin dampens leptin signaling and prevents excess ECM production. We treated culture-activated rat HSCs with recombinant adiponectin, leptin, both or neither, and also treated adiponectin knockout (Ad−/−) and wild-type mice with leptin and/or carbon tetrachloride (CCl4), or saline. We analyzed Jak2 and Ob-Rb phosphorylation, and PTP1B expression and activity. We also explored potential mechanisms through which adiponectin regulates SOCS-3/Ob-Rb association. Adiponectin inhibited leptin-stimulated Jak2 activation and Ob-Rb phosphorylation in HSCs, while both were increased in Ad−/− mice. Adiponectin stimulated PTP1B expression and activity, in vitro, while PTP1B expression was lower in Ad−/−mice than in wild-type mice. Adiponectin also promoted SOCS-3/Ob-R association, and blocked leptin-stimulated formation of extracellular TIMP-1/MMP-1 complexes, in vitro. These data suggest two novel mechanisms whereby adiponectin inhibits hepatic fibrosis: by promoting binding of SOCS-3 to Ob-Rb, and stimulating PTP1B expression and activity, thus inhibiting Jak2-Stat3 signaling at multiple points. PMID:21846328

Uterine Deletion of Gp130 or Stat3 Shows Implantation Failure with Increased Estrogenic Responses

PubMed Central

Sun, Xiaofei; Bartos, Amanda; Whitsett, Jeffrey A.

2013-01-01

Leukemia inhibitory factor (LIF), a downstream target of estrogen, is essential for implantation in mice. LIF function is thought to be mediated by its binding to LIF receptor (LIFR) and recruitment of coreceptor GP130 (glycoprotein 130), and this receptor complex then activates signal transducer and activator of transcription (STAT)1/3. However, the importance of LIFR and GP130 acting via STAT3 in implantation remains uncertain, because constitutive inactivation of Lifr, Gp130, or Stat3 shows embryonic lethality in mice. To address this issue, we generated mice with conditional deletion of uterine Gp130 or Stat3 and show that both GP130 and STAT3 are critical for uterine receptivity and implantation. Implantation failure in these deleted mice is associated with higher uterine estrogenic responses prior to the time of implantation. These heightened estrogenic responses are not due to changes in ovarian hormone levels or expression of their nuclear receptors. In the deleted mice, estrogen-responsive gene, Lactoferrin (Ltf), and Mucin 1 protein, were up-regulated in the uterus. In addition, progesterone-responsive genes, Hoxa10 and Indian hedgehog (Ihh), were markedly down-regulated in STAT3-inactivated uteri. These changes in uteri of deleted mice were reflected by the failure of differentiation of the luminal epithelium, which is essential for blastocyst attachment. PMID:23885093

Inhibition of cerebral ischemia/reperfusion injury-induced apoptosis: nicotiflorin and JAK2/STAT3 pathway.

PubMed

Hu, Guang-Qiang; Du, Xi; Li, Yong-Jie; Gao, Xiao-Qing; Chen, Bi-Qiong; Yu, Lu

2017-01-01

Nicotiflorin is a flavonoid extracted from Carthamus tinctorius. Previous studies have shown its cerebral protective effect, but the mechanism is undefined. In this study, we aimed to determine whether nicotiflorin protects against cerebral ischemia/reperfusion injury-induced apoptosis through the JAK2/STAT3 pathway. The cerebral ischemia/reperfusion injury model was established by middle cerebral artery occlusion/reperfusion. Nicotiflorin (10 mg/kg) was administered by tail vein injection. Cell apoptosis in the ischemic cerebral cortex was examined by hematoxylin-eosin staining and terminal deoxynucleotidyl transferase dUTP nick end labeling assay. Bcl-2 and Bax expression levels in ischemic cerebral cortex were examined by immunohistochemial staining. Additionally, p-JAK2, p-STAT3, Bcl-2, Bax, and caspase-3 levels in ischemic cerebral cortex were examined by western blot assay. Nicotiflorin altered the shape and structure of injured neurons, decreased the number of apoptotic cells, down-regulates expression of p-JAK2, p-STAT3, caspase-3, and Bax, decreased Bax immunoredactivity, and increased Bcl-2 protein expression and immunoreactivity. These results suggest that nicotiflorin protects against cerebral ischemia/reperfusion injury-induced apoptosis via the JAK2/STAT3 pathway.

Cardiotrophin-1 Induces Matrix Metalloproteinase-1 in Human Aortic Endothelial Cells

PubMed Central

Tokito, Akinori; Jougasaki, Michihisa; Ichiki, Tomoko; Hamasaki, Shuichi

2013-01-01

Rupture of an atherosclerotic plaque is a key event in the development of cardiovascular disorders, in which matrix metalloproteinase-1 (MMP-1) plays a crucial role by degradation of extracellular matrix resulting in plaque instability. Cardiotrophin-1 (CT-1), a member of interleukin-6-type proinflammatory cytokines, has potent cardiovascular actions and is highly expressed in vascular endothelium, however its role in atherosclerosis has not been fully elucidated to date. The present study was designed to investigate whether CT-1 induces MMP-1 in human aortic endothelial cells (HAECs). Ribonuclease protection assay demonstrated that MMP-1 gene level in HAECs was enhanced by the treatment of CT-1 in a dose- and time-dependent manner. Immunocytochemical staining, Western immunoblot analysis and enzyme-linked immunosorbent assay revealed that CT-1 augmented MMP-1 protein synthesis and secretion. MMP-1 activity assay revealed that MMP-1 present in the supernatant of HAECs was exclusively precursor form. Casein zymography disclosed proteolytic activity in the supernatant of HAECs, which was enhanced by CT-1 treatment. Furthermore, pharmacological inhibitor study indicated the important roles of extracellular signal-regulated kinase (ERK) 1/2, p38 mitogen-activated protein (MAP) kinase, c-Jun N-terminal kinase (JNK) and Janus kinase/signal transducers and activators of transcription (JAK/STAT) signaling pathways in mediating CT-1-induced MMP-1 gene and protein expression. These data reveal for the first time that CT-1 induces the proteolytic potential in HAECs by upregulating MMP-1 expression through ERK1/2, p38 MAP kinase, JNK and JAK/STAT pathways, and suggest that CT-1 may play an important role in the pathophysiology of atherosclerosis and plaque instability. PMID:23935888

Apigenin overcomes drug resistance by blocking the signal transducer and activator of transcription 3 signaling in breast cancer cells

PubMed Central

Seo, Hye-Sook; Ku, Jin Mo; Choi, Hyeong Sim; Woo, Jong-Kyu; Lee, Byung Hoon; Kim, Doh Sun; Song, Hyun Jong; Jang, Bo-Hyoung; Shin, Yong Cheol; Ko, Seong-Gyu

2017-01-01

Drug resistance in chemotherapy is a serious obstacle for the successful treatment of cancer. Drug resistance is caused by various factors, including the overexpression of P-glycoprotein (P-gp, MDR1). The development of new, useful compounds that overcome drug resistance is urgent. Apigenin, a dietary flavonoid, has been reported as an anticancer drug in vivo and in vitro. In the present study, we investigated whether apigenin is able to reverse drug resistance using adriamycin-resistant breast cancer cells (MCF-7/ADR). In our experiments, apigenin significantly decreased cell growth and colony formation in MCF-7/ADR cells and parental MCF-7 cells. This growth inhibition was related to the accumulation of cells in the sub-G0/G1 apoptotic population and an increase in the number of apoptotic cells. Apigenin reduced the mRNA expression of multidrug resistance 1 (MDR1) and multidrug resistance-associated proteins (MRPs) in MCF-7/ADR cells. Apigenin also downregulated the expression of P-gp. Apigenin reversed drug efflux from MCF-7/ADR cells, resulting in rhodamine 123 (Rho123) accumulation. Inhibition of drug resistance by apigenin is related to the suppression of the signal transducer and activator of transcription 3 (STAT3) signaling pathway. Apigenin decreased STAT3 activation (p-STAT3) and its nuclear translocation and inhibited the secretion of VEGF and MMP-9, which are STAT3 target genes. A STAT3 inhibitor, JAK inhibitor I and an HIF-1α inhibitor decreased cell growth in MCF-7 and MCF-7/ADR cells. Taken together, these results demonstrate that apigenin can overcome drug resistance. PMID:28656316

Apigenin overcomes drug resistance by blocking the signal transducer and activator of transcription 3 signaling in breast cancer cells.

PubMed

Seo, Hye-Sook; Ku, Jin Mo; Choi, Hyeong Sim; Woo, Jong-Kyu; Lee, Byung Hoon; Kim, Doh Sun; Song, Hyun Jong; Jang, Bo-Hyoung; Shin, Yong Cheol; Ko, Seong-Gyu

2017-08-01

Drug resistance in chemotherapy is a serious obstacle for the successful treatment of cancer. Drug resistance is caused by various factors, including the overexpression of P‑glycoprotein (P‑gp, MDR1). The development of new, useful compounds that overcome drug resistance is urgent. Apigenin, a dietary flavonoid, has been reported as an anticancer drug in vivo and in vitro. In the present study, we investigated whether apigenin is able to reverse drug resistance using adriamycin‑resistant breast cancer cells (MCF‑7/ADR). In our experiments, apigenin significantly decreased cell growth and colony formation in MCF‑7/ADR cells and parental MCF‑7 cells. This growth inhibition was related to the accumulation of cells in the sub‑G0/G1 apoptotic population and an increase in the number of apoptotic cells. Apigenin reduced the mRNA expression of multidrug resistance 1 (MDR1) and multidrug resistance‑associated proteins (MRPs) in MCF‑7/ADR cells. Apigenin also downregulated the expression of P‑gp. Apigenin reversed drug efflux from MCF‑7/ADR cells, resulting in rhodamine 123 (Rho123) accumulation. Inhibition of drug resistance by apigenin is related to the suppression of the signal transducer and activator of transcription 3 (STAT3) signaling pathway. Apigenin decreased STAT3 activation (p‑STAT3) and its nuclear translocation and inhibited the secretion of VEGF and MMP‑9, which are STAT3 target genes. A STAT3 inhibitor, JAK inhibitor I and an HIF‑1α inhibitor decreased cell growth in MCF‑7 and MCF‑7/ADR cells. Taken together, these results demonstrate that apigenin can overcome drug resistance.

Identification and expression analysis of leptin-regulated immediate early response and late target genes.

PubMed

Waelput, W; Verhee, A; Broekaert, D; Eyckerman, S; Vandekerckhove, J; Beattie, J H; Tavernier, J

2000-05-15

Using PC12 cells as an in vitro model system, we have identified a series of transcripts induced through activation of the leptin receptor. On the basis of kinetic studies, two distinct gene sets could be discerned: signal transducer and activator of transciption-3 (STAT-3), suppressor of cytokine signalling-3 (SOCS-3), MT-II (metallothionein-II), the serine/threonine kinase fibroblast-growth-factor-inducible kinase (Fnk) and modulator recognition factor (MRF-1), which are immediate early response genes, and pancreatitis-associated protein I (PAP I), squalene epoxidase, uridine diphosphate glucuronosyltransferase and annexin VIII, which are late induced target genes. At late time points a strong co-stimulation with beta-nerve growth factor or with the adenylate cyclase activator forskolin was observed. To assess the validity of the PC12-cell model system, we examined the effect of leptin administration on the gene transcription of STAT-3, MT-II, Fnk and PAP I in vivo. Leptin treatment of leptin-deficient ob/ob mice increased the STAT-3, SOCS-3, MT-II and Fnk mRNA, and MT-I protein levels in liver, whereas, in jejunum, expression of PAP I mRNA was down-regulated. Furthermore, administration of leptin to starved wild-type mice enhanced the expression of MT-II and Fnk mRNA in liver, but decreased MT-II and PAP I mRNA expression in jejunum. These findings may help to explain the obese phenotype observed in some colonies of MT-I- and MT-II-null mice and/or the observation that leptin protects against tumour-necrosis-factor toxicity in vivo.

Acanthamoeba castellanii STAT protein.

PubMed

Kicinska, Anna; Leluk, Jacek; Jarmuszkiewicz, Wieslawa

2014-01-01

STAT (signal transducers and activators of transcription) proteins are one of the important mediators of phosphotyrosine-regulated signaling in metazoan cells. We described the presence of STAT protein in a unicellular, free-living amoebae with a simple life cycle, Acanthamoeba castellanii. A. castellanii is the only, studied to date, Amoebozoan that does not belong to Mycetozoa but possesses STATs. A sequence of the A. castellanii STAT protein includes domains similar to those of the Dictyostelium STAT proteins: a coiled coil (characteristic for Dictyostelium STAT coiled coil), a STAT DNA-binding domain and a Src-homology domain. The search for protein sequences homologous to A. castellanii STAT revealed 17 additional sequences from lower eukaryotes. Interestingly, all of these sequences come from Amoebozoa organisms that belong to either Mycetozoa (slime molds) or Centramoebida. We showed that there are four separated clades within the slime mold STAT proteins. The A. castellanii STAT protein branches next to a group of STATc proteins from Mycetozoa. We also demonstrate that Amoebozoa form a distinct monophyletic lineage within the STAT protein world that is well separated from the other groups.

Hepatitis B Virus X Protein Impairs Hepatic Insulin Signaling Through Degradation of IRS1 and Induction of SOCS3

PubMed Central

Kim, KyeongJin; Kim, Kook Hwan; Cheong, JaeHun

2010-01-01

Background Hepatitis B virus (HBV) is a major cause of chronic liver diseases, and frequently results in hepatitis, cirrhosis, and ultimately hepatocellular carcinoma. The role of HCV in associations with insulin signaling has been elucidated. However, the pathogenesis of HBV-associated insulin signaling remains to be clearly characterized. Therefore, we have attempted to determine the mechanisms underlying the HBV-associated impairment of insulin signaling. Methodology The expressions of insulin signaling components were investigated in HBx-transgenic mice, HBx-constitutive expressing cells, and transiently HBx-transfected cells. Protein and gene expression was examined by Western blot, immunohistochemistry, RT-PCR, and promoter assay. Protein-protein interaction was detected by coimmunoprecipitation. Principal Findings HBx induced a reduction in the expression of IRS1, and a potent proteasomal inhibitor blocked the downregulation of IRS1. Additionally, HBx enhanced the expression of SOCS3 and induced IRS1 ubiquitination. Also, C/EBPα and STAT3 were involved in the HBx-induced expression of SOCS3. HBx interfered with insulin signaling activation and recovered the insulin-mediated downregulation of gluconeogenic genes. Conclusions/Significance These results provide direct experimental evidences for the contribution of HBx in the impairment of insulin signaling. PMID:20351777

Expression of p53/HGF/c-met/STAT3 signal in fetuses with neural tube defects.

PubMed

Trovato, Maria; D'Armiento, Maria; Lavra, Luca; Ulivieri, Alessandra; Dominici, Roberto; Vitarelli, Enrica; Grosso, Maddalena; Vecchione, Raffaella; Barresi, Gaetano; Sciacchitano, Salvatore

2007-02-01

Neural tube defects (NTD) are morphogenetic alterations due to a defective closure of neural tube. Hepatocyte growth factor (HGF)/c-met system plays a role in morphogenesis of nervous system, lung, and kidney. HGF/c-met morphogenetic effects are mediated by signal transducers and activators of transcription (STAT)3 and both HGF and c-met genes are regulated from p53. The aim of our study was to analyze mRNA and protein expressions of p53, HGF, c-met, and STAT3 in fetuses with NTD. By reverse transcriptase-polymerase chain reaction and immunohistochemistry, we analyzed neural tissues from four NTD fetuses and the corresponding non-malformed lungs, kidneys and placentas. We found a reduced mRNA expression of HGF/c-met/STAT3 pathway, in the malformed nervous systems and placentas. The reduced expression of this pathway correlated with the absence of p53 in all these samples. On the contrary, detectable expression levels of p53, HGF, c-met, and STAT3 were observed in non-malformed lungs and kidneys obtained from the same fetuses. Comparable results were obtained by immunohistochemistry, with the exception of p53, which was undetected in all fetal tissues. In conclusion, in NTD fetuses, both the defective neural tube tissue and the placenta have a reduction in all components of the p53/HGF/c-met/STAT3 cascade. This raises the possibility of using the suppression of these genes for early diagnosis of NTD especially on chorionic villus sampling.

Constitutive STAT5 Activation Correlates With Better Survival in Cervical Cancer Patients Treated With Radiation Therapy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chen, Helen H.W.; Institute of Clinical Medicine, College of Medicine, National Cheng Kung University, Tainan, Taiwan; Chou, Cheng-Yang

2012-02-01

Purpose: Constitutively activated signal transducers and activators of transcription (STAT) factors, in particular STAT1, STAT3, and STAT5, have been detected in a wide variety of human primary tumors and have been demonstrated to directly contribute to oncogenesis. However, the expression pattern of these STATs in cervical carcinoma is still unknown, as is whether or not they have prognostic significance. This study investigated the expression patterns of STAT1, STAT3, and STAT5 in cervical cancer and their associations with clinical outcomes in patients treated with radical radiation therapy. Methods and Materials: A total of 165 consecutive patients with International Federation of Gynecologymore » and Obstetrics (FIGO) Stages IB to IVA cervical cancer underwent radical radiation therapy, including external beam and/or high-dose-rate brachytherapy between 1989 and 2002. Immunohistochemical studies of their formalin-fixed, paraffin-embedded tissues were performed. Univariate and multivariate analyses were performed to identify and to evaluate the effects of these factors affecting patient survival. Results: Constitutive activations of STAT1, STAT3, and STAT5 were observed in 11%, 22%, and 61% of the participants, respectively. While STAT5 activation was associated with significantly better metastasis-free survival (p < 0.01) and overall survival (p = 0.04), STAT1 and STAT3 activation were not. Multivariate analyses showed that STAT5 activation, bulky tumor ({>=}4 cm), advanced stage (FIGO Stages III and IV), and brachytherapy (yes vs. no) were independent prognostic factors for cause-specific overall survival. None of the STATs was associated with local relapse. STAT5 activation (odds ratio = 0.29, 95% confidence interval = 0.13-0.63) and advanced stage (odds ratio = 2.54; 95% confidence interval = 1.03-6.26) were independent predictors of distant metastasis. Conclusions: This is the first report to provide the overall expression patterns and prognostic significance of specific STATs in cervical carcinoma. Our results indicate that constitutive STAT5 activation correlates with better metastasis-free survival and overall survival in cervical cancer patients who have received radiation therapy.« less

Leptin upregulates telomerase activity and transcription of human telomerase reverse transcriptase in MCF-7 breast cancer cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ren, He, E-mail: herenrh@yahoo.com.cn; Zhao, Tiansuo; Wang, Xiuchao

2010-03-26

The aim was to analyze the mechanism of leptin-induced activity of telomerase in MCF-7 breast cancer cells. We found that leptin activated telomerase in a dose-dependent manner; leptin upregulated the expression of Human Telomerase Reverse Transcriptase (hTERT) at mRNA and protein levels; blockade of signal transducer and activator of transcription 3 (STAT3) phosphorylation significantly counteracted leptin-induced hTERT transcription and protein expression; chromatin immunoprecipitation analysis showed that leptin enhanced the binding of STAT3 to the hTERT promoter. This study uncovers a new mechanism of the proliferative effect of leptin on breast cancer cells and provides a new explanation of obesity-related breastmore » cancer.« less

The role of the JAK/STAT signal pathway in rheumatoid arthritis

PubMed Central

Malemud, Charles J.

2018-01-01

Proinflammatory cytokine activation of the Janus kinase/signal transducers and activators of transcription (JAK/STAT) signal transduction pathway is a critical event in the pathogenesis and progression of rheumatoid arthritis. Under normal conditions, JAK/STAT signaling reflects the influence of negative regulators of JAK/STAT, exemplified by the suppressor of cytokine signaling and protein inhibitor of activated STAT. However, in rheumatoid arthritis (RA) both of these regulators are dysfunctional. Thus, continuous activation of JAK/STAT signaling in RA synovial joints results in the elevated level of matrix metalloproteinase gene expression, increased frequency of apoptotic chondrocytes and most prominently ‘apoptosis resistance’ in the inflamed synovial tissue. Tofacitinib, a JAK small molecule inhibitor, with selectivity for JAK2/JAK3 was approved by the United States Food and Drug Administration (US FDA) for the therapy of RA. Importantly, tofacitinib has demonstrated significant clinical efficacy for RA in the post-US FDA-approval surveillance period. Of note, the success of tofacitinib has spurred the development of JAK1, JAK2 and other JAK3-selective small molecule inhibitors, some of which have also entered the clinical setting, whereas other JAK inhibitors are currently being evaluated in RA clinical trials. PMID:29942363

IFN-λ Inhibits MiR-122 Transcription through a Stat3-HNF4α Inflammatory Feedback Loop in an IFN-α Resistant HCV Cell Culture System

PubMed Central

Aboulnasr, Fatma; Hazari, Sidhartha; Nayak, Satyam; Chandra, Partha K.; Panigrahi, Rajesh; Ferraris, Pauline; Chava, Srinivas; Kurt, Ramazan; Song, Kyongsub; Dash, Asha; Balart, Luis A.; Garry, Robert F.; Wu, Tong; Dash, Srikanta

2015-01-01

Background HCV replication in persistently infected cell culture remains resistant to IFN-α/RBV combination treatment, whereas IFN-λ1 induces viral clearance. The antiviral mechanisms by which IFN-λ1 induces sustained HCV clearance have not been determined. Aim To investigate the mechanisms by which IFN-λ clears HCV replication in an HCV cell culture model. Methods IFN-α sensitive (S3-GFP) and resistant (R4-GFP) cells were treated with equivalent concentrations of either IFN-α or IFN-λ. The relative antiviral effects of IFN-α and IFN-λ1 were compared by measuring the HCV replication, quantification of HCV-GFP expression by flow cytometry, and viral RNA levels by real time RT-PCR. Activation of Jak-Stat signaling, interferon stimulated gene (ISG) expression, and miRNA-122 transcription in S3-GFP and R4-GFP cells were examined. Results We have shown that IFN-λ1 induces HCV clearance in IFN-α resistant and sensitive replicon cell lines in a dose dependent manner through Jak-Stat signaling, and induces STAT 1 and STAT 2 activation, ISRE-luciferase promoter activation and ISG expression. Stat 3 activation is also involved in IFN-λ1 induced antiviral activity in HCV cell culture. IFN-λ1 induced Stat 3 phosphorylation reduces the expression of hepatocyte nuclear factor 4 alpha (HNF4α) through miR-24 in R4-GFP cells. Reduced expression of HNF4α is associated with decreased expression of miR-122 resulting in an anti-HCV effect. Northern blot analysis confirms that IFN-λ1 reduces miR-122 levels in R4-GFP cells. Our results indicate that IFN-λ1 activates the Stat 3-HNF4α feedback inflammatory loop to inhibit miR-122 transcription in HCV cell culture. Conclusions In addition to the classical Jak–Stat antiviral signaling pathway, IFN-λ1 inhibits HCV replication through the suppression of miRNA-122 transcription via an inflammatory Stat 3–HNF4α feedback loop. Inflammatory feedback circuits activated by IFNs during chronic inflammation expose non-responders to the risk of hepatocellular carcinoma. PMID:26657215

Essential role of Stat6 in IL-4 signalling.

PubMed

Takeda, K; Tanaka, T; Shi, W; Matsumoto, M; Minami, M; Kashiwamura, S; Nakanishi, K; Yoshida, N; Kishimoto, T; Akira, S

1996-04-18

Interleukin-4 (IL-4) is a pleiotropic lymphokine which plays an important role in the immune system. IL-4 activates two distinct signalling pathways through tyrosine phosphorylation of Stat6, a signal transducer and activator of transcription, and of a 170K protein called 4PS. To investigate the functional role of Stat6 in IL-4 signalling, we generated mice deficient in Stat6 by gene targeting. We report here that in the mutant mice, expression of CD23 and major histocompatibility complex (MHC) class II in resting B cells was not enhanced in response to IL-4. IL-4 induced B-cell proliferation costimulated by anti-IgM antibody was abolished. The T-cell proliferative response was also notably reduced. Furthermore, production of Th2 cytokines from T cells as well as IgE and IgG1 responses after nematode infection were profoundly reduced. These findings agreed with those obtained in IL-4 deficient mice or using antibodies to IL-4 and the IL-4 receptor. We conclude that Stat6 plays a central role in exerting IL-4 mediated biological responses.

Peracetylated hydroxytyrosol, a new hydroxytyrosol derivate, attenuates LPS-induced inflammatory response in murine peritoneal macrophages via regulation of non-canonical inflammasome, Nrf2/HO1 and JAK/STAT signaling pathways.

PubMed

Montoya, Tatiana; Aparicio-Soto, Marina; Castejón, María Luisa; Rosillo, María Ángeles; Sánchez-Hidalgo, Marina; Begines, Paloma; Fernández-Bolaños, José G; Alarcón-de-la-Lastra, Catalina

2018-03-18

The present study was designed to investigate the anti-inflammatory effects of a new derivative of hydroxytyrosol (HTy), peracetylated hydroxytyrosol (Per-HTy), compared with its parent, HTy, on lipopolysaccharide (LPS)-stimulated murine macrophages as well as potential signaling pathways involved. In particular, we attempted to characterize the role of the inflammasome underlying Per-HTy possible anti-inflammatory effects. Isolated murine peritoneal macrophages were treated with HTy or its derivative in the presence or absence of LPS (5 μg/ml) for 18 h. Cell viability was determined using sulforhodamine B (SRB) assay. Nitric oxide (NO) production was analyzed by Griess method. Production of pro-inflammatory cytokines was evaluated by enzyme-linked immunosorbent assay (ELISA) and inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2, janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway (STAT3), haem oxigenase 1 (HO1), nuclear factor (erythroid-derived 2)-like 2 (Nrf2) expression and mitogen-activated protein kinases (MAPKs) activation was determined by Western blot. Per-HTy significantly reduced the levels of NO and pro-inflammatory cytokines as well as both COX-2 and iNOS expressions. Furthermore, Per-HTy treatment inhibited STAT3 and increased Nrf2 and HO1 protein levels in murine macrophages exposed to LPS. In addition, Per-HTy anti-inflammatory activity was related with an inhibition of non-canonical nucleotide binding domain (NOD)-like receptor (NLRP3) inflammasome pathways by decreasing pro-inflammatory interleukin (IL)-1β and IL-18 cytokine levels as consequence of regulation of cleaved caspase-11 enzyme. These results support that this new HTy derivative may offer a new promising nutraceutical therapeutic strategy in the management of inflammatory-related pathologies. Copyright © 2018. Published by Elsevier Inc.

Lloviu virus VP24 and VP35 proteins function as innate immune antagonists in human and bat cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Feagins, Alicia R.; Basler, Christopher F., E-mail: chris.basler@mssm.edu

Lloviu virus (LLOV) is a new member of the filovirus family that also includes Ebola virus (EBOV) and Marburg virus (MARV). LLOV has not been cultured; however, its genomic RNA sequence indicates the coding capacity to produce homologs of the EBOV and MARV VP24, VP35, and VP40 proteins. EBOV and MARV VP35 proteins inhibit interferon (IFN)-alpha/beta production and EBOV VP35 blocks activation of the antiviral kinase PKR. The EBOV VP24 and MARV VP40 proteins inhibit IFN signaling, albeit by different mechanisms. Here we demonstrate that LLOV VP35 suppresses Sendai virus induced IFN regulatory factor 3 (IRF3) phosphorylation, IFN-α/β production, andmore » PKR phosphorylation. Additionally, LLOV VP24 blocks tyrosine phosphorylated STAT1 binding to karyopherin alpha 5 (KPNA5), STAT1 nuclear accumulation, and IFN-induced gene expression. LLOV VP40 lacks detectable IFN antagonist function. These activities parallel EBOV IFN inhibitory functions. EBOV and LLOV VP35 and VP24 proteins also inhibit IFN responses in bat cells. These data suggest that LLOV infection will block innate immune responses in a manner similar to EBOV. - Highlights: • Lloviu virus (LLOV) is a new member of the filovirus family. • LLOV VP35 blocks IRF3 phosphorylation, IFN-α/β production and PKR phosphorylation. • LLOV VP24 inhibits IFN responses by targeting phospho-STAT1 KPNA interaction. • Infection by LLOV may block innate immune responses in a manner similar to EBOV.« less

STAT5A-mediated NOX5-L expression promotes the proliferation and metastasis of breast cancer cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dho, So Hee; Radioisotope Research Division, Department of Research Reactor Utilization, Korea Atomic Energy Research Institute, Daejeon 305-353; Kim, Ji Young

NADPH oxidase (NOX) generates reactive oxygen species (ROS) and has been suggested to mediate cell proliferation in some cancers. Here, we show that an increase in the expression of NOX5 long form (NOX5-L) is critical for tumor progression in breast tumor tissues. Immunostaining of clinical samples indicated that NOX5 was overexpressed in 41.1% of breast ductal carcinoma samples. NOX5-L depletion consistently suppressed cell proliferation, invasion, and migration in vitro. Antibody-mediated neutralization of NOX5-L attenuated tumor progression in a mouse xenograft model. Promoter analysis revealed that NOX5-L expression is regulated by STAT5A in breast cancer cells. Based on our novel findings,more » we suggest that inhibition of NOX5-L may be a promising therapeutic strategy that exerts anti-cancer effects via the modulation of ROS-mediated cell signaling. - Highlights: • The ROS-generating protein, NOX5-L, determines cellular proliferation and metastasis in subset of breast tumor. • Tumor growth was attenuated by the treatment of anti-NOX5-L antibody in a xenograft model. • NOX5-L expression is transcriptionally regulated by STAT5A in breast cancer cells.« less

Sulforaphane inhibits the interferon-γ-induced expression of MIG, IP-10 and I-TAC in INS‑1 pancreatic β-cells through the downregulation of IRF-1, STAT-1 and PKB.

PubMed

Park, Yu-Kyoung; Ramalingam, Mahesh; Kim, Shin; Jang, Byeong-Churl; Park, Jong Wook

2017-09-01

Sulforaphane (SFN) is a dietary isothiocyanate abundantly available in cruciferous vegetables and has been shown to possess anti-inflammatory and immunomodulatory activities. Chemokines are important mediators of inflammation and immune responses due to their ability to recruit and activate macrophages and leukocytes. To date, little is known about the SFN-mediated regulation of chemokine expression in pancreatic β-cells. In this study, we investigated the inhibitory effects and mechanisms of SFN on the interferon-γ (IFN-γ)-induced expression of a subset of chemokines, including monokine induced by IFN-γ (MIG), IFN-inducible protein of 10 kDa (IP-10) and IFN-inducible T‑cell alpha chemoattractant (I-TAC), in INS‑1 cells, a rat pancreatic β-cell line. Notably, IFN-γ treatment led to an increase in the mRNA expression levels of MIG, IP-10 and I-TAC in the INS‑1 cells. However, SFN strongly blocked the mRNA expressions of MIG, IP-10 and I-TAC induced by IFN-γ in INS‑1 cells. On the mechanistic level, SFN significanlty decreased not only the mRNA expression levels of interferon regulatory factor-1 (IRF-1), but also the phosphorylation levels of signal transducer and activator of transcription-1 (STAT-1) and protein kinase B (PKB) which were induced by IFN-γ in the INS‑1 cells. Pharmacological inhibition experiments further revealed that treatment with JAK inhibitor I weakly inhibited the IFN-γ-induced expression of IP-10, whereas it strongly suppressed the IFN-γ-induced expression of MIG and I-TAC in the INS‑1 cells. Moreover, treatment with LY294002, a PI3K/PKB inhibitor, was able to slightly repress IFN‑γ‑induced expressions of MIG and I-TAC, but not IP-10, in INS‑1 cells. Importantly, the IFN-γ-induced increase in the expression levels of MIG, IP-10 and I-TAC in the INS-1 cells was strongly inhibited by SFN, but not by other natural substances, such as curcumin, sanguinarine, resveratrol, triptolide and epigallocatechin gallate (EGCG), suggesting the specificity of SFN in downregulating the levels of these chemokines. To the best of our knowledge, these results collectively demonstrate for the first time that SFN strongly inhibits the IFN-γ-induced expression of MIG, IP-10 and I-TAC in INS‑1 cells and this inhibition is, at least in part, mediated through the reduced expression and phosphorylation levels of IRF-1, STAT-1 and PKB.

JAK and STAT members in channel catfish: Identification, phylogenetic analysis and expression profiling after Edwardsiella ictaluri infection.

PubMed

Jin, Yulin; Zhou, Tao; Li, Ning; Liu, Shikai; Xu, Xiaoyan; Pan, Ying; Tan, Suxu; Shi, Huitong; Yang, Yujia; Yuan, Zihao; Wang, Wenwen; Luo, Jian; Gao, Dongya; Dunham, Rex; Liu, Zhanjiang

2018-04-01

The Janus kinase/signal transducers and activators of transcription (JAK/STAT) signaling pathway is one of the main pleiotropic cascades used to transmit information from extracellular receptors to the nucleus, which results in DNA transcription and expression of genes involved in immunity, proliferation, differentiation, migration, apoptosis, and cell survival. Members of JAK family and STAT family have been extensively studied in different mammalian species because of their important roles in innate and adaptive immune responses. However, they have not been systematically studied among teleost fish species. In this study, five JAK family members and eight STAT family members were identified and characterized from channel catfish. Phylogenetic analysis was conducted to properly annotate these genes. Syntenic analysis was also conducted to establish orthology, and confirm the results from phylogenetic analysis. Compared to mammals, more members of the JAK and STAT family were identified in channel catfish genome. Expression of JAK and STAT family members was detected in healthy catfish tissues, but was induced in gill, liver, and intestine after bacterial challenge. Notably, the significant upregulation of STAT1b gene in catfish liver, gill and intestine after Edwardsiella ictaluri infection supported the notion that high STAT1 expression are involved in defense against pathogens. Collectively, the increased expression of JAK and STAT members in tested tissues suggested their crucial function in defending the host against pathogen invasion. Copyright © 2017 Elsevier Ltd. All rights reserved.

Inflammation and Cancer: Two Pieces of the Same Puzzle? | Center for Cancer Research

Cancer.gov

Chronic inflammation, in Crohn’s disease for example, is a known risk factor for malignant transformation, however the role inflammation plays in cancer initiation is poorly understood. STAT2, an important protein that regulates gene activation, is known to be stimulated by immune factors that inhibit cell growth. STAT2 also has reduced expression in the immune cells of

Emodin alleviates jejunum injury in rats with sepsis by inhibiting inflammation response.

PubMed

Chen, Yi-Kun; Xu, Ying-Kun; Zhang, Hao; Yin, Jiang-Tao; Fan, Xin; Liu, Da-Dong; Fu, Hai-Yan; Wan, Bing

2016-12-01

Emodin is an anthraquinone derived from Chinese herb that exerts anti-inflammation effects. This study aimed to investigate whether emodin provides the protection for jejunum injury by inhibiting inflammation. We established a model of sepsis caused by cecal ligation and puncture. Forty-eight male Wistar rats were divided into four groups (n=12). Jejunum injury was assessed by pathological examination. The activity of pJAK1/pSTAT3 and protein levels of Bcl-2 and Bax were detected by Western blot analysis. Inflammatory factors IL-6, TNF-α and procalcitonin were detected by ELISA. Apoptosis was detected by TUNEL. We found that emodin alleviated jejunum damage and apoptosis induced by sepsis and decreased the levels of IL-6, TNF-α and procalcitonin in septic rats. Furthermore, we observed that emodin increased the levels of pJAK1 and of pSTAT3, which were decreased in rats with sepsis. In addition, emodin enhanced the expression of Bcl-2 which was downregulated by sepsis and decreased the expression of Bax which was upregulated by sepsis. In conclusion, these results indicate that emodin suppresses inflammatory response induced by sepsis. Emodin activates JAK1/STAT3 signaling pathway and regulates Bcl-2 and Bax expression to protect the jejunum in rats with sepsis. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

Cancer-associated fibroblasts promote endometrial cancer growth via activation of interleukin-6/STAT-3/c-Myc pathway.

PubMed

Subramaniam, Kavita S; Omar, Intan Sofia; Kwong, Soke Chee; Mohamed, Zahurin; Woo, Yin Ling; Mat Adenan, Noor Azmi; Chung, Ivy

2016-01-01

Cancer-associated fibroblasts (CAFs) secrete various pro-tumorigenic cytokines, yet the role of these cytokines in the progression of endometrial cancer remains unclear. We found that CAFs isolated from human endometrial cancer (EC) tissues secreted high levels of interleukin-6 (IL-6), which promotes EC cell proliferation in vitro. Neutralizing IL-6 in CAF-conditioned media reduced (47% inhibition) while IL-6 recombinant protein increased cell proliferation (~2.4 fold) of both EC cell lines and primary cultures. IL-6 receptors (IL-6R and gp130) were expressed only in EC epithelial cells but not in CAF, indicating a one-way paracrine signaling. In the presence of CAF-conditioned media, Janus kinase/signal transducers and activators of transcription (JAK/STAT3) pathway was activated in EC cells. Treatment with JAK and STAT3 specific inhibitors, AD412 and STATTIC, respectively, significantly abrogated CAF-mediated cell proliferation, indicating the role of IL-6 activation in EC cell proliferation. We further showed that one of STAT-3 target genes, c-Myc, was highly induced in EC cells after exposure to CAF-conditioned medium at both mRNA (>105-fold vs. control) and protein level (>2-fold vs. control). EC cell proliferation was dependent on c-Myc expression, as RNAi-mediated c-Myc down-regulation led to a significant 46% reduction in cell viability when compared with scrambled control. Interestingly, CAF-conditioned media failed to promote proliferation in EC cells with reduced c-Myc expression, suggesting that CAF-mediated cell proliferation was also dependent on c-Myc expression. Subcutaneous tumor xenograft model showed that EC cells grew at least 1.4 times larger when co-injected with CAF, when compared to those injected with EC cells alone. Mice injected with EC cells with down-regulated c-Myc expression, however, showed at least 2.5 times smaller tumor compared to those in control group. Notably, there was no increase of tumor size when co-injected with CAFs. Further immunohistochemical staining on human tissues showed positive expression of IL-6 receptors, phosphorylated-STAT3 and c-Myc in human EC tissues with less signals in benign endometrium. Taken together, our data suggests that IL-6 secreted by CAF induces c-Myc expression to promote EC proliferation in vitro and in vivo. IL-6 pathway can be a potential target to disrupt tumor-stroma interaction in endometrial cancer progression.

STAT3 or USF2 Contributes to HIF Target Gene Specificity

PubMed Central

Pawlus, Matthew R.; Wang, Liyi; Murakami, Aya; Dai, Guanhai; Hu, Cheng-Jun

2013-01-01

The HIF1- and HIF2-mediated transcriptional responses play critical roles in solid tumor progression. Despite significant similarities, including their binding to promoters of both HIF1 and HIF2 target genes, HIF1 and HIF2 proteins activate unique subsets of target genes under hypoxia. The mechanism for HIF target gene specificity has remained unclear. Using siRNA or inhibitor, we previously reported that STAT3 or USF2 is specifically required for activation of endogenous HIF1 or HIF2 target genes. In this study, using reporter gene assays and chromatin immuno-precipitation, we find that STAT3 or USF2 exhibits specific binding to the promoters of HIF1 or HIF2 target genes respectively even when over-expressed. Functionally, HIF1α interacts with STAT3 to activate HIF1 target gene promoters in a HIF1α HLH/PAS and N-TAD dependent manner while HIF2α interacts with USF2 to activate HIF2 target gene promoters in a HIF2α N-TAD dependent manner. Physically, HIF1α HLH and PAS domains are required for its interaction with STAT3 while both N- and C-TADs of HIF2α are involved in physical interaction with USF2. Importantly, addition of functional USF2 binding sites into a HIF1 target gene promoter increases the basal activity of the promoter as well as its response to HIF2+USF2 activation while replacing HIF binding site with HBS from a HIF2 target gene does not change the specificity of the reporter gene. Importantly, RNA Pol II on HIF1 or HIF2 target genes is primarily associated with HIF1α or HIF2α in a STAT3 or USF2 dependent manner. Thus, we demonstrate here for the first time that HIF target gene specificity is achieved by HIF transcription partners that are required for HIF target gene activation, exhibit specific binding to the promoters of HIF1 or HIF2 target genes and selectively interact with HIF1α or HIF2α protein. PMID:23991099

Response of the JAK-STAT signaling pathway to oxygen deprivation in the red eared slider turtle, Trachemys scripta elegans.

PubMed

Bansal, Saumya; Biggar, Kyle K; Krivoruchko, Anastasia; Storey, Kenneth B

2016-11-15

The red-eared slider turtle, Trachemys scripta elegans, is a model organism commonly used to study the environmental stress of anoxia. It exhibits multiple biochemical adaptations to ensure its survival during the winter months where quantities of oxygen are largely depleted. We proposed that JAK-STAT signaling would display stress responsive regulation to mediate the survival of the red-eared slider turtle, Trachemys scripta elegans, during anoxic stress. Importantly, the JAK-STAT signaling pathway is involved in transmitting extracellular signals to the nucleus resulting in the expression of select genes that aid cell survival and growth. Immunoblotting was used to compare the relative phosphorylation levels of JAK proteins, STAT proteins, and two of its inhibitors, SOCS and PIAS, in response to anoxia. A clear activation of the JAK-STAT pathway was observed in the liver tissue while no significant changes were found in the skeletal muscle. To further support our findings we also found an increase in mRNA transcripts of downstream targets of STATs, namely bcl-xL and bcl-2, using PCR analysis in the liver tissues. These findings suggest an important role for the JAK-STAT pathway in exhibiting natural anoxia tolerance by the red-eared slider turtle. Copyright © 2016 Elsevier B.V. All rights reserved.

Genome-wide identification of novel expression signatures reveal distinct patterns and prevalence of binding motifs for p53, nuclear factor-κB and other signal transcription factors in head and neck squamous cell carcinoma

PubMed Central

Yan, Bin; Yang, Xinping; Lee, Tin-Lap; Friedman, Jay; Tang, Jun; Van Waes, Carter; Chen, Zhong

2007-01-01

Background Differentially expressed gene profiles have previously been observed among pathologically defined cancers by microarray technologies, including head and neck squamous cell carcinomas (HNSCCs). However, the molecular expression signatures and transcriptional regulatory controls that underlie the heterogeneity in HNSCCs are not well defined. Results Genome-wide cDNA microarray profiling of ten HNSCC cell lines revealed novel gene expression signatures that distinguished cancer cell subsets associated with p53 status. Three major clusters of over-expressed genes (A to C) were defined through hierarchical clustering, Gene Ontology, and statistical modeling. The promoters of genes in these clusters exhibited different patterns and prevalence of transcription factor binding sites for p53, nuclear factor-κB (NF-κB), activator protein (AP)-1, signal transducer and activator of transcription (STAT)3 and early growth response (EGR)1, as compared with the frequency in vertebrate promoters. Cluster A genes involved in chromatin structure and function exhibited enrichment for p53 and decreased AP-1 binding sites, whereas clusters B and C, containing cytokine and antiapoptotic genes, exhibited a significant increase in prevalence of NF-κB binding sites. An increase in STAT3 and EGR1 binding sites was distributed among the over-expressed clusters. Novel regulatory modules containing p53 or NF-κB concomitant with other transcription factor binding motifs were identified, and experimental data supported the predicted transcriptional regulation and binding activity. Conclusion The transcription factors p53, NF-κB, and AP-1 may be important determinants of the heterogeneous pattern of gene expression, whereas STAT3 and EGR1 may broadly enhance gene expression in HNSCCs. Defining these novel gene signatures and regulatory mechanisms will be important for establishing new molecular classifications and subtyping, which in turn will promote development of targeted therapeutics for HNSCC. PMID:17498291

The Jak-STAT pathway stimulated by interferon alpha or interferon beta.

PubMed

Horvath, Curt M

2004-11-23

Type I interferons, such as interferon alpha and interferon beta (IFN-alpha and beta), signal through a Janus kinase (Jak) to signal transduction and activator of transcription (STAT) pathway to stimulate gene expression. In response to ligand binding, the receptors dimerize, Jaks phosphorylate STAT1 and STAT2, which then dimerize and interact with a third transcriptional regulator IFN regulatory factor 9 (IRF9) to stimulate gene expression. IFN-alpha is the main innate antiviral cytokine and is essential for effective immune response to viral infection. The animation shows activation of STAT-responsive gene expression in response to type I IFNs.

Inhibitory Effects of Bangladeshi Medicinal Plant Extracts on Interactions between Transcription Factors and Target DNA Sequences

PubMed Central

Lampronti, Ilaria; Khan, Mahmud T.H.; Borgatti, Monica; Bianchi, Nicoletta

2008-01-01

Several transcription factors (TFs) play crucial roles in governing the expression of different genes involved in the immune response, embryo or cell lineage development, cell apoptosis, cell cycle progression, oncogenesis, repair and fibrosis processes and inflammation. As far as inflammation, TFs playing pivotal roles are nuclear factor kappa B (NF-kB), activator protein (AP-1), signal transducer and activator of transcription (STATs), cAMP response element binding protein (CREB) and GATA-1 factors. All these TFs regulate the expression of pro-inflammatory cytokines and are involved in the pathogenesis of a number of human disorders, particularly those with an inflammatory component. Since several medicinal plants can be employed to produce extracts exhibiting biological effects and because alteration of gene transcription represents a very interesting approach to control the expression of selected genes, this study sought to verify the ability of several extracts derived from Bangladeshi medicinal plants in interfering with molecular interactions between different TFs and specific DNA sequences. We first analyzed the antiproliferative activity of 19 medicinal plants on different human cell lines, including erythroleukemia K562, B lymphoid Raji and T lymphoid Jurkat cell lines. Secondly, we employed the electrophoretic mobility shift assay as a suitable technique for a fast screening of plant extracts altering the binding between NF-kB, AP-1, GATA-1, STAT-3, CREB and the relative target DNA elements. PMID:18830455

[Knockdown of STAT3 inhibits proliferation and migration of HepG2 hepatoma cells induced by IFN1].

PubMed

Li, Xiaofang; Wang, Yuqi; Yan, Ben; Fang, Peipei; Ma, Chao; Xu, Ning; Fu, Xiaoyan; Liang, Shujuan

2018-02-01

Objective To prepare lentiviruses expressing shRNA sequences targeting human signal transducer and activator of transcription 3 (STAT3) and detect the effect of STAT3 knockdown on type I interferon (IFN1)-induced proliferation and migration in HepG2 cells. Methods Four STAT3-targeting shRNA sequences (shRNA1-shRNA4) and one control sequence (Ctrl shRNA) were selected and cloned respectively into pLKO.1-sp6-pgk-GFP to construct shRNA-expressing vectors. Along with backbone psPAX2 and pMD2.G vectors, they were separately transfected into HEK293T cells to prepare lentiviruses. HepG2 cells were infected with the lentiviruses. Cytoplastic STAT3 level was detected by Western blotting to screen effective shRNA sequence(s) targeting STAT3. Proliferation and migration of HepG2 cells were analyzed by CCK-8 assay and Transwell TM migration and scratching assay, respectively. To detect the effect of IFN1 on cell proliferation and migration of HepG2 cells, the cells were treated with 2000 U/mL IFNα2b for indicated time and the activation of IFN-triggered STAT1 signal transduction was assayed by Western blotting. Results Two most effective STAT3-targeting shRNA sequences shRNA1 and shRNA2 were selected, and the expression of both STAT3 shRNA significantly decreased proliferation and migration of HepG2 cells. When treated with IFNα2b, 2000 U/mL of IFN1 showed more competent in attenuating growth and migration of HepG2 cells. Our data further proved that knockdown of STAT3 increased the phosphorylation of STAT1, and IFNα2b further enhanced the activation of STAT1 signaling in HepG2 cells. Conclusion Knockdown of STAT3 inhibits cell migration and growth, and rescues IFN response through up-regulating STAT1 signal transduction in HepG2 hepatoma cells.

Interferon gamma, an important marker of response to immune checkpoint blockade in non-small cell lung cancer and melanoma patients

PubMed Central

Karachaliou, Niki; Gonzalez-Cao, Maria; Crespo, Guillermo; Drozdowskyj, Ana; Aldeguer, Erika; Gimenez-Capitan, Ana; Teixido, Cristina; Molina-Vila, Miguel Angel; Viteri, Santiago; De Los Llanos Gil, Maria; Algarra, Salvador Martin; Perez-Ruiz, Elisabeth; Marquez-Rodas, Ivan; Rodriguez-Abreu, Delvys; Blanco, Remedios; Puertolas, Teresa; Royo, Maria Angeles; Rosell, Rafael

2018-01-01

Background: Programmed death-ligand 1 (PD-L1) may be induced by oncogenic signals or can be upregulated via interferon gamma (IFN-γ). We have explored whether the expression of IFNG, the gene encoding IFN-γ, is associated with clinical response to the immune checkpoint blockade in non-small cell lung cancer (NSCLC) and melanoma patients. The role of inflammation-associated transcription factors STAT3, IKBKE, STAT1 and other associated genes has also been examined. Methods: Total RNA from 17 NSCLC and 21 melanoma patients was analyzed by quantitative reverse transcription PCR. STAT3 and Rantes, YAP1 and CXCL5, DNMT1, RIG1 and TET1, EOMES, IFNG, PD-L1 and CTLA4, IKBKE and NFATC1 mRNA were examined. PD-L1 protein expression in tumor and immune cells and stromal infiltration of CD8+ T-cells were also evaluated. Progression-free survival and overall survival were estimated. Results: A total of 17 NSCLC patients received nivolumab and 21 melanoma patients received pembrolizumab. Progression-free survival with nivolumab was significantly longer in NSCLC patients with high versus low IFNG expression (5.1 months versus 2 months, p = 0.0124). Progression-free survival with pembrolizumab was significantly longer in melanoma patients with high versus low IFNG expression (5.0 months versus 1.9 months, p = 0.0099). Significantly longer overall survival was observed for melanoma patients with high versus low IFNG expression (not reached versus 10.2 months p = 0.0183). There was a trend for longer overall survival for NSCLC patients with high versus low IFNG expression. Conclusions: IFN-γ is an important marker for prediction of response to immune checkpoint blockade. Further research is warranted in order to validate whether IFNG is more accurate than PD-L1. PMID:29383037

Analysis of JAK-STAT signaling pathway genes and their microRNAs in the intestinal mucosa of genetically disparate chicken lines induced with necrotic enteritis.

PubMed

Truong, Anh Duc; Rengaraj, Deivendran; Hong, Yeojin; Hoang, Cong Thanh; Hong, Yeong Ho; Lillehoj, Hyun S

2017-05-01

The JAK-STAT signaling pathway plays a key role in cytokine and growth factor activation and is involved in several cellular functions and diseases. The main objective of this study was to investigate the expression of candidate JAK-STAT pathway genes and their regulators and interactors in the intestinal mucosal layer of two genetically disparate chicken lines [Marek's disease (MD)-resistant line 6.3 and MD-susceptible line 7.2] induced with necrotic enteritis (NE). Through RNA-sequencing, we investigated 116 JAK-STAT signaling pathway-related genes that were significant and differentially expressed between the intestinal mucosa of the two lines compared with respective uninfected controls. About 15 JAK-STAT pathway genes were further verified by qRT-PCR, and the results were in agreement with our sequencing data. All the identified 116 genes were annotated through Gene Ontology and mapped to the KEGG chicken JAK-STAT signaling pathway. To the best of our knowledge, this is the first study to represent the transcriptional analysis of a large number of candidate genes, regulators, and potential interactors in the JAK-STAT pathway of the two chicken lines induced with NE. Several key genes of the interactome, namely, STAT1/3/4, STAT5B, JAK1-3, TYK2, AKT1/3, SOCS1-5, PIAS1/2/4, PTPN6/11, and PIK3, were determined to be differentially expressed in the two lines. Moreover, we detected 68 known miRNAs variably targeting JAK-STAT pathway genes and differentially expressed in the two lines induced with NE. The RNA-sequencing and bioinformatics analyses in this study provided an abundance of data that will be useful for future studies on JAK-STAT pathways associated with the functions of two genetically disparate chicken lines induced with NE. Copyright © 2017 Elsevier B.V. All rights reserved.

Prolonged activation of innate antiviral gene signature after childbirth is determined by IFNL3 genotype.

PubMed

Price, Aryn A; Tedesco, Dana; Prasad, Mona R; Workowski, Kimberly A; Walker, Christopher M; Suthar, Mehul S; Honegger, Jonathan R; Grakoui, Arash

2016-09-20

Maternal innate and adaptive immune responses are modulated during pregnancy to concurrently defend against infection and tolerate the semiallogeneic fetus. The restoration of these systems after childbirth is poorly understood. We reasoned that enhanced innate immune activation may extend beyond gestation while adaptive immunity recovers. To test this hypothesis, the transcriptional profiles of total peripheral blood mononuclear cells following delivery in healthy women were compared with those of nonpregnant control subjects. Interestingly, interferon-stimulated genes (ISGs) encoding proteins such as IFIT1, IFIT2, and IFIT3, as well as signaling proteins such as STAT1, STAT2, and MAVS, were enriched postpartum. Antiviral genes were primarily expressed in CD14(+) cells and could be stratified according to genetic variation at the interferon-λ3 gene (IFNL3, also named IL28B) SNP rs12979860. Antiviral gene expression was sustained beyond 6 mo following delivery in mothers with a CT or TT genotype, but resembled baseline nonpregnant control levels following delivery in mothers with a CC genotype. CT and TT IFNL3 genotypes have been associated with persistent elevated ISG expression in individuals chronically infected with hepatitis C virus. Together, these data suggest that postpartum, the normalization of the physiological rheostat controlling IFN signaling depends on IFNL3 genotype.

Lipopolysaccharides upregulate hepcidin in neuron via microglia and the IL-6/STAT3 signaling pathway.

PubMed

Qian, Zhong-Ming; He, Xuan; Liang, Tuo; Wu, Ka-Chun; Yan, Yik-Chun; Lu, Li-Na; Yang, Guang; Luo, Qian Qian; Yung, Wing-Ho; Ke, Ya

2014-12-01

Neuroinflammation is closely related to brain iron homeostasis. Our previous study demonstrated that lipopolysaccharides (LPS) can regulate expression of iron-regulatory peptide hepcidin; however, the mechanism is undefined. Here, we demonstrated that intracerebroventricular injection of LPS in rat brain upregulated hepcidin and downregulated ferroportin 1 in the cortex and substantia nigra. LPS increased hepcidin expression in neurons only when they were co-cultured with BV-2 microglia, and the upregulation was suppressed by IL-6 neutralizing antibody in vitro. In addition, IL-6 but not IL-1α, IL-1β, or tumor necrosis factor-alpha increased hepcidin expression and signal transducer and activator of transcription 3 (STAT3) phosphorylation in cortical neurons and MES23.5 dopaminergic neurons. These effects were blocked by the STAT3 inhibitor, stattic. Our results show that neurons are the major source of increased hepcidin expression in response to LPS challenge but microglia play a key mediator role by releasing IL-6 and recruiting the STAT3 pathway. We conclude that LPS upregulates hepcidin expression in neurons via microglia and the IL-6/STAT3 signaling pathway.

Under-expression of CK2β subunit in ccRCC represents a complementary biomarker of p-STAT3 Ser727 that correlates with patient survival

PubMed Central

Vilardell, Jordi; Alcaraz, Estefania; Sarró, Eduard; Trilla, Enric; Cuadros, Thaïs; de Torres, Inés; Plana, Maria; Ramón y Cajal, Santiago; Pinna, Lorenzo A.; Ruzzene, Maria; Morote, Juan; Meseguer, Anna; Itarte, Emilio

2018-01-01

Clear cell renal cell carcinoma (ccRCC) is the most common and aggressive subtype of renal cancer. STAT3 pathway is altered in these tumors and p-STAT3 Ser727 is an independent prognostic factor for ccRCC. Protein kinase CK2 is altered in different types of tumors and overexpression of CK2α is considered predictive of bad prognosis and metastatic risk. CK2 subunits analyses in ccRCC samples showed increased CK2α/α’ nuclear content in all cases, but decreased cytosolic CK2β (CK2βcyt) levels in the more advanced tumors. Stable downregulation of CK2β in renal proximal tubular (HK-2) and clear cell adenocarcinoma (786-O) cells triggered changes in E-cadherin, vimentin and Snail1 protein levels indicative of epithelial-to-mesenchymal transition (EMT), and increased HIF-α. Moreover, CK2β was required in order to observe STAT3 Ser727 phosphorylation in HK-2 but not in 786-O cells. We also observed that CK2β improved the prognostic value of p-STAT3 Ser727, as CK2βcyt>41 (median value) discriminates patients free of disease for a period of 10 years upon surgery, from those with CK2βcyt<41, when p-STAT3 Ser727levels are low. We conclude that CK2β down-regulation might represent a mechanism to support EMT and angiogenesis and that CK2βcyt levels are instrumental to refine prognosis of ccRCC patients with low p-STAT3 Ser727 levels. PMID:29464030

Under-expression of CK2β subunit in ccRCC represents a complementary biomarker of p-STAT3 Ser727 that correlates with patient survival.

PubMed

Vilardell, Jordi; Alcaraz, Estefania; Sarró, Eduard; Trilla, Enric; Cuadros, Thaïs; de Torres, Inés; Plana, Maria; Ramón Y Cajal, Santiago; Pinna, Lorenzo A; Ruzzene, Maria; Morote, Juan; Meseguer, Anna; Itarte, Emilio

2018-01-19

Clear cell renal cell carcinoma (ccRCC) is the most common and aggressive subtype of renal cancer. STAT3 pathway is altered in these tumors and p-STAT3 Ser727 is an independent prognostic factor for ccRCC. Protein kinase CK2 is altered in different types of tumors and overexpression of CK2α is considered predictive of bad prognosis and metastatic risk. CK2 subunits analyses in ccRCC samples showed increased CK2α/α' nuclear content in all cases, but decreased cytosolic CK2β (CK2βcyt) levels in the more advanced tumors. Stable downregulation of CK2β in renal proximal tubular (HK-2) and clear cell adenocarcinoma (786-O) cells triggered changes in E-cadherin, vimentin and Snail1 protein levels indicative of epithelial-to-mesenchymal transition (EMT), and increased HIF-α. Moreover, CK2β was required in order to observe STAT3 Ser727 phosphorylation in HK-2 but not in 786-O cells. We also observed that CK2β improved the prognostic value of p-STAT3 Ser727, as CK2βcyt>41 (median value) discriminates patients free of disease for a period of 10 years upon surgery, from those with CK2βcyt<41, when p-STAT3 Ser727levels are low. We conclude that CK2β down-regulation might represent a mechanism to support EMT and angiogenesis and that CK2βcyt levels are instrumental to refine prognosis of ccRCC patients with low p-STAT3 Ser727 levels.

Combination therapy of chitosan, gynostemma, and motherwort alleviates the progression of experimental rat chronic renal failure by inhibiting STAT1 activation

PubMed Central

Bai, Wenxia; Wang, Shudong; An, Shanshan; Guo, Mengjie; Gong, Guangming; Liu, Wenya; Ma, Shaoxin; Li, Xin; Fu, Jihua; Yao, Wenbing

2018-01-01

This study aimed to investigate the effect of single and combination therapy using chitosan (K), gynostemma (J), and motherwort (Y) on an experimental rat model of chronic renal failure (CRF) induced by adenine and the underlying mechanisms. CRF rats were treated with individual or combinational therapy with two or three of these agents. Biochemical indicators showed that the levels of blood urea nitrogen, creatinine and uric acid decreased and the levels of albumin and hemoglobin increased by single or combination therapy of these drugs. Drug treatment also decreased oxidative stress damage of renal tissues in CRF rats. Histopathological lesions were attenuated in each drug treatment group by various degrees. Additionally, drug treatment affected the expression of extracellular matrix (ECM) proteins including plasminogen activator inhibitor 1, collagen I, matrix metalloprotease-1, and tissue inhibitor of metalloproteinases 1. In particular, the combination therapy of K, J, and Y was superior to the respective monotherapy, which supported the prescription of KJY combination. We further studied the inhibitory effect of KJY on LPS-induced inflammation in RAW264.7 macrophages. The results showed that KJY inhibited LPS-induced secretion of inflammatory cytokines (Interferon-gamma, Interleukin-1 Beta, chemokine (C-X-C motif) ligand 10, cyclooxygenase-2 and Tumor necrosis factor-α in RAW264.7 macrophages. Combination therapy of KJY suppressed the protein expression of Cyclooxygenase-2 and inducible nitric oxide synthase in vivo and in vitro. Further study indicated that KJY inhibited STAT1 activation by down regulating p-STAT1 to exert anti-inflammatory effect and improve renal function in rats with chronic renal failure. PMID:29643988

IL-10 plays a pivotal role in anti-inflammatory effects of resveratrol in activated microglia cells.

PubMed

Cianciulli, Antonia; Dragone, Teresa; Calvello, Rosa; Porro, Chiara; Trotta, Teresa; Lofrumento, Dario Domenico; Panaro, Maria Antonietta

2015-02-01

The development of agents that can modulate microglial activation has been suggested as one potential strategy for the treatment or prevention of neurodegenerative diseases. Among these agents, resveratrol, with its anti-inflammatory action, has been described to have neuroprotective effects. In this paper we demonstrate that in LPS-stimulated microglia resveratrol pretreatment reduced, in a dose-dependent manner, pro-inflammatory cytokines IL-1β, TNF-α and IL-6 mRNA expression and increased the release of anti-inflammatory interleukin (IL)-10. Moreover, resveratrol pretreatment up-regulated the phosphorylated forms of JAK1 and STAT3, as well as suppressor of cytokine signaling (SOCS)3 protein expression in LPS activated cells, demonstrating that the JAK-STAT signaling pathway is involved in the anti-inflammatory effect exerted by resveratrol. By supplementing the cultures with an IL-10 neutralizing antibody (IL-10NA) we obtained the opposite effect. Taken together, these data allow us to conclude that the LPS-induced pro-inflammatory response in microglial cells can be markedly reduced by resveratrol, through IL-10 dependent up-regulation of SOCS3, requiring the JAK-STAT signaling pathway. Copyright © 2015 Elsevier B.V. All rights reserved.

An SH2 domain model of STAT5 in complex with phospho-peptides define ``STAT5 Binding Signatures''

NASA Astrophysics Data System (ADS)

Gianti, Eleonora; Zauhar, Randy J.

2015-05-01

The signal transducer and activator of transcription 5 (STAT5) is a member of the STAT family of proteins, implicated in cell growth and differentiation. STAT activation is regulated by phosphorylation of protein monomers at conserved tyrosine residues, followed by binding to phospho-peptide pockets and subsequent dimerization. STAT5 is implicated in the development of severe pathological conditions, including many cancer forms. However, nowadays a few STAT5 inhibitors are known, and only one crystal structure of the inactive STAT5 dimer is publicly available. With a view to enabling structure-based drug design, we have: (1) analyzed phospho-peptide binding pockets on SH2 domains of STAT5, STAT1 and STAT3; (2) generated a model of STAT5 bound to phospho-peptides; (3) assessed our model by docking against a class of known STAT5 inhibitors (Müller et al. in ChemBioChem 9:723-727, 2008); (4) used molecular dynamics simulations to optimize the molecular determinants responsible for binding and (5) proposed unique "Binding Signatures" of STAT5. Our results put in place the foundations to address STAT5 as a target for rational drug design, from sequence, structural and functional perspectives.

Epigallocatechin-3-gallate Ameliorates Seawater Aspiration-Induced Acute Lung Injury via Regulating Inflammatory Cytokines and Inhibiting JAK/STAT1 Pathway in Rats

PubMed Central

Liu, Wei; Dong, Mingqing; Bo, Liyan; Li, Congcong; Liu, Qingqing; Li, Yanyan; Ma, Lijie; Xie, Yonghong; Fu, Enqing; Mu, Deguang; Pan, Lei; Jin, Faguang; Li, Zhichao

2014-01-01

Signal transducers and activators of transcriptions 1 (STAT1) play an important role in the inflammation process of acute lung injury (ALI). Epigallocatechin-3-gallate (EGCG) exhibits a specific and strong anti-STAT1 activity. Therefore, our study is to explore whether EGCG pretreatment can ameliorate seawater aspiration-induced ALI and its possible mechanisms. We detected the arterial partial pressure of oxygen, lung wet/dry weight ratios, protein content in bronchoalveolar lavage fluid, and the histopathologic and ultrastructure staining of the lung. The levels of IL-1, TNF-α, and IL-10 and the total and the phosphorylated protein level of STAT1, JAK1, and JAK2 were assessed in vitro and in vivo. The results showed that EGCG pretreatment significantly improved hypoxemia and histopathologic changes, alleviated pulmonary edema and lung vascular leak, reduced the production of TNF-α and IL-1, and increased the production of IL-10 in seawater aspiration-induced ALI rats. EGCG also prevented the seawater aspiration-induced increase of TNF-α and IL-1 and decrease of IL-10 in NR8383 cell line. Moreover, EGCG pretreatment reduced the total and the phosphorylated protein level of STAT1 in vivo and in vitro and reduced the phosphorylated protein level of JAK1 and JAK2. The present study demonstrates that EGCG ameliorates seawater aspiration-induced ALI via regulating inflammatory cytokines and inhibiting JAK/STAT1 pathway in rats. PMID:24692852

Adenosine A1 Receptor Protects Against Cisplatin Ototoxicity by Suppressing the NOX3/STAT1 Inflammatory Pathway in the Cochlea

PubMed Central

Kaur, Tejbeer; Borse, Vikrant; Sheth, Sandeep; Sheehan, Kelly; Ghosh, Sumana; Tupal, Srinivasan; Jajoo, Sarvesh; Mukherjea, Debashree; Rybak, Leonard P.

2016-01-01

Cisplatin is a commonly used antineoplastic agent that produces ototoxicity that is mediated in part by increasing levels of reactive oxygen species (ROS) via the NOX3 NADPH oxidase pathway in the cochlea. Recent studies implicate ROS generation in mediating inflammatory and apoptotic processes and hearing loss by activating signal transducer and activator of transcription (STAT1). In this study, we show that the adenosine A1 receptor (A1AR) protects against cisplatin ototoxicity by suppressing an inflammatory response initiated by ROS generation via NOX3 NADPH oxidase, leading to inhibition of STAT1. Trans-tympanic administration of the A1AR agonist R-phenylisopropyladenosine (R-PIA) inhibited cisplatin-induced ototoxicity, as measured by auditory brainstem responses and scanning electron microscopy in male Wistar rats. This was associated with reduced NOX3 expression, STAT1 activation, tumor necrosis factor-α (TNF-α) levels, and apoptosis in the cochlea. In vitro studies in UB/OC-1 cells, an organ of Corti immortalized cell line, showed that R-PIA reduced cisplatin-induced phosphorylation of STAT1 Ser727 (but not Tyr701) and STAT1 luciferase activity by suppressing the ERK1/2, p38, and JNK mitogen-activated protein kinase (MAPK) pathways. R-PIA also decreased the expression of STAT1 target genes, such as TNF-α, inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) and reduced cisplatin-mediated apoptosis. These data suggest that the A1AR provides otoprotection by suppressing NOX3 and inflammation in the cochlea and could serve as an ideal target for otoprotective drug therapy. SIGNIFICANCE STATEMENT Cisplatin is a widely used chemotherapeutic agent for the treatment of solid tumors. Its use results in significant and permanent hearing loss, for which no US Food and Drug Administration-approved treatment is currently available. In this study, we targeted the cochlear adenosine A1 receptor (A1AR) by trans-tympanic injections of the agonist R-phenylisopropyladenosine (R-PIA) and showed that it reduced cisplatin-induced inflammation and apoptosis in the rat cochlea and preserved hearing. The mechanism of protection involves suppression of the NOX3 NADPH oxidase enzyme, a major target of cisplatin-induced reactive oxygen species (ROS) generation in the cochlea. ROS initiates an inflammatory and apoptotic cascade in the cochlea by activating STAT1 transcription factor, which is attenuated by R-PIA. Therefore, trans-tympanic delivery of A1AR agonists could effectively treat cisplatin ototoxicity. PMID:27053204

Adenosine A1 Receptor Protects Against Cisplatin Ototoxicity by Suppressing the NOX3/STAT1 Inflammatory Pathway in the Cochlea.

PubMed

Kaur, Tejbeer; Borse, Vikrant; Sheth, Sandeep; Sheehan, Kelly; Ghosh, Sumana; Tupal, Srinivasan; Jajoo, Sarvesh; Mukherjea, Debashree; Rybak, Leonard P; Ramkumar, Vickram

2016-04-06

Cisplatin is a commonly used antineoplastic agent that produces ototoxicity that is mediated in part by increasing levels of reactive oxygen species (ROS) via the NOX3 NADPH oxidase pathway in the cochlea. Recent studies implicate ROS generation in mediating inflammatory and apoptotic processes and hearing loss by activating signal transducer and activator of transcription (STAT1). In this study, we show that the adenosine A1 receptor (A1AR) protects against cisplatin ototoxicity by suppressing an inflammatory response initiated by ROS generation via NOX3 NADPH oxidase, leading to inhibition of STAT1. Trans-tympanic administration of the A1AR agonist R-phenylisopropyladenosine (R-PIA) inhibited cisplatin-induced ototoxicity, as measured by auditory brainstem responses and scanning electron microscopy in male Wistar rats. This was associated with reduced NOX3 expression, STAT1 activation, tumor necrosis factor-α (TNF-α) levels, and apoptosis in the cochlea. In vitro studies in UB/OC-1 cells, an organ of Corti immortalized cell line, showed that R-PIA reduced cisplatin-induced phosphorylation of STAT1 Ser(727) (but not Tyr(701)) and STAT1 luciferase activity by suppressing the ERK1/2, p38, and JNK mitogen-activated protein kinase (MAPK) pathways.R-PIA also decreased the expression of STAT1 target genes, such as TNF-α, inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) and reduced cisplatin-mediated apoptosis. These data suggest that the A1AR provides otoprotection by suppressing NOX3 and inflammation in the cochlea and could serve as an ideal target for otoprotective drug therapy. Cisplatin is a widely used chemotherapeutic agent for the treatment of solid tumors. Its use results in significant and permanent hearing loss, for which no US Food and Drug Administration-approved treatment is currently available. In this study, we targeted the cochlear adenosine A1 receptor (A1AR) by trans-tympanic injections of the agonist R-phenylisopropyladenosine (R-PIA) and showed that it reduced cisplatin-induced inflammation and apoptosis in the rat cochlea and preserved hearing. The mechanism of protection involves suppression of the NOX3 NADPH oxidase enzyme, a major target of cisplatin-induced reactive oxygen species (ROS) generation in the cochlea. ROS initiates an inflammatory and apoptotic cascade in the cochlea by activating STAT1 transcription factor, which is attenuated byR-PIA. Therefore, trans-tympanic delivery of A1AR agonists could effectively treat cisplatin ototoxicity. Copyright © 2016 the authors 0270-6474/16/363962-16$15.00/0.

Expression of Par3 polarity protein correlates with poor prognosis in ovarian cancer.

PubMed

Nakamura, Hiroe; Nagasaka, Kazunori; Kawana, Kei; Taguchi, Ayumi; Uehara, Yuriko; Yoshida, Mitsuyo; Sato, Masakazu; Nishida, Haruka; Fujimoto, Asaha; Inoue, Tomoko; Adachi, Katsuyuki; Nagamatsu, Takeshi; Arimoto, Takahide; Oda, Katsutoshi; Osuga, Yutaka; Fujii, Tomoyuki

2016-11-17

Previous studies have shown that the cell polarity protein partitioning defective 3 (Par3) plays an essential role in the formation of tight junctions and definition of apical-basal polarity. Aberrant function of this protein has been reported to be involved in epithelial-mesenchymal transition (EMT) and cancer invasion. The aim of this study was to examine the functional mechanism of Par3 in ovarian cancer. First, we investigated the association between Par3 expression level and survival of 50 ovarian cancer patients. Next, we conducted an in vitro analysis of ovarian cancer cell lines, focusing on the cell line JHOC5, to investigate Par3 function. To investigate the function of Par3 in invasion, the IL-6/STAT3 pathway was analyzed upon Par3 knockdown with siRNA. The effect of siRNA treatment was assessed by qPCR, ELISA, and western blotting. Invasiveness and cell proliferation following treatment with siRNA against Par3 were investigated using Matrigel chamber, wound healing, and cell proliferation assays. Expression array data for ovarian cancer patient samples revealed low Par3 expression was significantly associated with good prognosis. Univariate analysis of clinicopathological factors revealed significant association between high Par3 levels and peritoneal dissemination at the time of diagnosis. Knockdown of Par3 in JHOC5 cells suppressed cell invasiveness, migration, and cell proliferation with deregulation of IL-6/STAT3 activity. Taken together, these results suggest that Par3 expression is likely involved in ovarian cancer progression, especially in peritoneal metastasis. The underlying mechanism may be that Par3 modulates IL-6 /STAT3 signaling. Here, we propose that the expression of Par3 in ovarian cancer may control disease outcome.

Induction of caspase-dependent extrinsic apoptosis by apigenin through inhibition of signal transducer and activator of transcription 3 (STAT3) signalling in HER2-overexpressing BT-474 breast cancer cells.

PubMed

Seo, Hye-Sook; Jo, Jae Kyung; Ku, Jin Mo; Choi, Han-Seok; Choi, Youn Kyung; Woo, Jong-Kyu; Kim, Hyo In; Kang, Soo-Yeon; Lee, Kang Min; Nam, Koong Won; Park, Namkyu; Jang, Bo-Hyoung; Shin, Yong Cheol; Ko, Seong-Gyu

2015-10-23

Phytoestrogen intake is known to be beneficial to decrease breast cancer incidence and progression. But its molecular mechanisms of action are still unknown. The present study aimed to examine the effect of apigenin on proliferation and apoptosis in HER2-expressing breast cancer cells. In our experiments, apigenin inhibited the proliferation of BT-474 cells in a dose- and time-dependent manner. Apigenin also inhibited clonogenic survival (anchorage-dependent and -independent) of BT-474 cells in a dose-dependent manner. These growth inhibitions were accompanied with an increase in sub-G0/G1 apoptotic populations. Apigenin-induced extrinsic a caspase-dependent apoptosis up-regulating the levels of cleaved caspase-8 and cleaved caspase-3, and inducing the cleavage of poly (ADP-ribose) polymerase (PARP). Whereas, apigenin did not induce apoptosis via intrinsic mitochondrial apoptosis pathway since this compound did not decrease mitochondrial membrane potential without affecting the levels of B-cell lymphoma 2 (Bcl-2) and Bcl-2-associated X protein (BAX). Apigenin reduced the expression of phospho-JAK1, phospho-JAK2 and phospho-STAT3 and decreased signal transducer and activator of transcription 3 (STAT3) dependent luciferase reporter gene activity in BT-474 cells. Apigenin inhibited CoCl2-induced VEGF secretion and decreased the nuclear translocation of STAT3. Our study indicates that apigenin induces apoptosis through inhibition of STAT3 signalling and could serve as a useful compound to prevent or treat HER2-overexpressing breast cancer. © 2015 Authors.

Induction of caspase-dependent extrinsic apoptosis by apigenin through inhibition of signal transducer and activator of transcription 3 (STAT3) signalling in HER2-overexpressing BT-474 breast cancer cells

PubMed Central

Seo, Hye-Sook; Jo, Jae Kyung; Ku, Jin Mo; Choi, Han-Seok; Choi, Youn Kyung; Woo, Jong-Kyu; in Kim, Hyo; Kang, Soo-yeon; Lee, Kang min; Nam, Koong Won; Park, Namkyu; Jang, Bo-Hyoung; Shin, Yong Cheol; Ko, Seong-Gyu

2015-01-01

Phytoestrogen intake is known to be beneficial to decrease breast cancer incidence and progression. But its molecular mechanisms of action are still unknown. The present study aimed to examine the effect of apigenin on proliferation and apoptosis in HER2-expressing breast cancer cells. In our experiments, apigenin inhibited the proliferation of BT-474 cells in a dose- and time-dependent manner. Apigenin also inhibited clonogenic survival (anchorage-dependent and -independent) of BT-474 cells in a dose-dependent manner. These growth inhibitions were accompanied with an increase in sub-G0/G1 apoptotic populations. Apigenin-induced extrinsic a caspase-dependent apoptosis up-regulating the levels of cleaved caspase-8 and cleaved caspase-3, and inducing the cleavage of poly (ADP-ribose) polymerase (PARP). Whereas, apigenin did not induce apoptosis via intrinsic mitochondrial apoptosis pathway since this compound did not decrease mitochondrial membrane potential without affecting the levels of B-cell lymphoma 2 (Bcl-2) and Bcl-2-associated X protein (BAX). Apigenin reduced the expression of phospho-JAK1, phospho-JAK2 and phospho-STAT3 and decreased signal transducer and activator of transcription 3 (STAT3) dependent luciferase reporter gene activity in BT-474 cells. Apigenin inhibited CoCl2-induced VEGF secretion and decreased the nuclear translocation of STAT3. Our study indicates that apigenin induces apoptosis through inhibition of STAT3 signalling and could serve as a useful compound to prevent or treat HER2-overexpressing breast cancer. PMID:26500281

IFNβ/TNFα synergism induces a non-canonical STAT2/IRF9-dependent pathway triggering a novel DUOX2 NADPH Oxidase-mediated airway antiviral response

PubMed Central

Fink, Karin; Martin, Lydie; Mukawera, Esperance; Chartier, Stéfany; De Deken, Xavier; Brochiero, Emmanuelle; Miot, Françoise; Grandvaux, Nathalie

2013-01-01

Airway epithelial cells are key initial innate immune responders in the fight against respiratory viruses, primarily via the secretion of antiviral and proinflammatory cytokines that act in an autocrine/paracrine fashion to trigger the establishment of an antiviral state. It is currently thought that the early antiviral state in airway epithelial cells primarily relies on IFNβ secretion and the subsequent activation of the interferon-stimulated gene factor 3 (ISGF3) transcription factor complex, composed of STAT1, STAT2 and IRF9, which regulates the expression of a panoply of interferon-stimulated genes encoding proteins with antiviral activities. However, the specific pathways engaged by the synergistic action of different cytokines during viral infections, and the resulting physiological outcomes are still ill-defined. Here, we unveil a novel delayed antiviral response in the airways, which is initiated by the synergistic autocrine/paracrine action of IFNβ and TNFα, and signals through a non-canonical STAT2- and IRF9-dependent, but STAT1-independent cascade. This pathway ultimately leads to the late induction of the DUOX2 NADPH oxidase expression. Importantly, our study uncovers that the development of the antiviral state relies on DUOX2-dependent H2O2 production. Key antiviral pathways are often targeted by evasion strategies evolved by various pathogenic viruses. In this regard, the importance of the novel DUOX2-dependent antiviral pathway is further underlined by the observation that the human respiratory syncytial virus is able to subvert DUOX2 induction. PMID:23545780

Plant HDAC inhibitor chrysin arrest cell growth and induce p21WAF1 by altering chromatin of STAT response element in A375 cells

PubMed Central

2012-01-01

Background Chrysin and its analogues, belongs to flavonoid family and possess potential anti-tumour activity. The aim of this study is to determine the molecular mechanism by which chrysin controls cell growth and induce apoptosis in A375 cells. Methods Effect of chrysin and its analogues on cell viability and cell cycle analysis was determined by MTT assay and flowcytometry. A series of Western blots was performed to determine the effect of chrysin on important cell cycle regulatory proteins (Cdk2, cyclin D1, p53, p21, p27). The fluorimetry and calorimetry based assays was conducted for characterization of chrysin as HDAC inhibitor. The changes in histone tail modification such as acetylation and methylation was studied after chrysin treatment was estimated by immuno-fluorescence and western blot analysis. The expression of Bcl-xL, survivin and caspase-3 was estimated in chrysin treated cells. The effect of chrysin on p21 promoter activity was studied by luciferase and ChIP assays. Results Chrysin cause G1 cell cycle arrest and found to inhibit HDAC-2 and HDAC-8. Chrysin treated cells have shown increase in the levels of H3acK14, H4acK12, H4acK16 and decrease in H3me2K9 methylation. The p21 induction by chrysin treatment was found to be independent of p53 status. The chromatin remodelling at p21WAF1 promoter induces p21 activity, increased STAT-1 expression and epigenetic modifications that are responsible for ultimate cell cycle arrest and apoptosis. Conclusion Chrysin shows in vitro anti-cancer activity that is correlated with induction of histone hyperacetylation and possible recruitment of STAT-1, 3, 5 proteins at STAT (−692 to −684) region of p21 promoter. Our results also support an unexpected action of chrysin on the chromatin organization of p21WAF1 promoter through histone methylation and hyper-acetylation. It proposes previously unknown sequence specific chromatin modulations in the STAT responsive elements for regulating cell cycle progression negatively via the induction of the CDK inhibitor p21WAF1. PMID:22591439

Metformin combined with aspirin significantly inhibit pancreatic cancer cell growth in vitro and in vivo by suppressing anti-apoptotic proteins Mcl-1 and Bcl-2

PubMed Central

Yue, Wen; Zheng, Xi; Lin, Yong; Yang, Chung S.; Xu, Qing; Carpizo, Darren; Huang, Huarong; DiPaola, Robert S.; Tan, Xiang-Lin

2015-01-01

Metformin and aspirin have been studied extensively as cancer preventive or therapeutic agents. However, the effects of their combination on pancreatic cancer cells have not been investigated. Herein, we evaluated the effects of metformin and aspirin, alone or in combination, on cell viability, migration, and apoptosis as well as the molecular changes in mTOR, STAT3 and apoptotic signaling pathways in PANC-1 and BxPC3 cells. Metformin and aspirin, at relatively low concentrations, demonstrated synergistically inhibitory effects on cell viability. Compared to the untreated control or individual drug, the combination of metformin and aspirin significantly inhibited cell migration and colony formation of both PANC-1 and BxPC-3 cells. Metformin combined with aspirin significantly inhibited the phosphorylation of mTOR and STAT3, and induced apoptosis as measured by caspase-3 and PARP cleavage. Remarkably, metformin combined with aspirin significantly downregulated the anti-apoptotic proteins Mcl-1 and Bcl-2, and upregulated the pro-apoptotic proteins Bim and Puma, as well as interrupted their interactions. The downregulation of Mcl-1 and Bcl-2 was independent of AMPK or STAT3 pathway but partially through mTOR signaling and proteasome degradation. In a PANC-1 xenograft mouse model, we demonstrated that the combination of metformin and aspirin significantly inhibited tumor growth and downregulated the protein expression of Mcl-1 and Bcl-2 in tumors. Taken together, the combination of metformin and aspirin significantly inhibited pancreatic cancer cell growth in vitro and in vivo by regulating the pro- and anti-apoptotic Bcl-2 family members, supporting the continued investigation of this two drug combination as chemopreventive or chemotherapeutic agents for pancreatic cancer. PMID:26056043

Characterization of two mosquito STATs, AaSTAT and CtSTAT. Differential regulation of tyrosine phosphorylation and DNA binding activity by lipopolysaccharide treatment and by Japanese encephalitis virus infection.

PubMed

Lin, Chang-Chi; Chou, Chih-Ming; Hsu, Ya-Li; Lien, Jih-Ching; Wang, Yu-Ming; Chen, Shui-Tsung; Tsai, Shu-Chuan; Hsiao, Pei-Wen; Huang, Chang-Jen

2004-01-30

Two mosquito STATs, AaSTAT and CtSTAT, have been cloned from Aedes albopictus and Culex tritaeniorhynchus mosquitoes, respectively. These two STATs are more similar to those of Drosophila, Anopheles, and mammalian STAT5 in the DNA binding and Src homology 2 domains. The mRNA transcripts are expressed at all developmental stages, and the proteins are present predominantly at the pupal and adult stages in both mosquitoes. Stimulation with lipopolysaccharide resulted in an increase of tyrosine phosphorylation and DNA binding activity of AaSTAT and CtSTAT as well as an increase of luciferase activity of a reporter gene containing Drosophila STAT binding motif in mosquito C6/36 cells. After being infected with Japanese encephalitis virus, nuclear extracts of C6/36 cells revealed a decrease of tyrosine phosphorylation and DNA binding activity of AaSTAT which could be restored by sodium orthovanadate treatment. Taking all of the data together, this is the first report to clone and characterize two mosquito STATs with 81% identity and to demonstrate a different response of tyrosine phosphorylation and DNA binding of these two STATs by lipopolysaccharide treatment and by Japanese encephalitis virus infection.

Two Domains of the V Protein of Virulent Canine Distemper Virus Selectively Inhibit STAT1 and STAT2 Nuclear Import▿

PubMed Central

Röthlisberger, Anne; Wiener, Dominique; Schweizer, Matthias; Peterhans, Ernst; Zurbriggen, Andreas; Plattet, Philippe

2010-01-01

Canine distemper virus (CDV) causes in dogs a severe systemic infection, with a high frequency of demyelinating encephalitis. Among the six genes transcribed by CDV, the P gene encodes the polymerase cofactor protein (P) as well as two additional nonstructural proteins, C and V; of these V was shown to act as a virulence factor. We investigated the molecular mechanisms by which the P gene products of the neurovirulent CDV A75/17 strain disrupt type I interferon (IFN-α/β)-induced signaling that results in the establishment of the antiviral state. Using recombinant knockout A75/17 viruses, the V protein was identified as the main antagonist of IFN-α/β-mediated signaling. Importantly, immunofluorescence analysis illustrated that the inhibition of IFN-α/β-mediated signaling correlated with impaired STAT1/STAT2 nuclear import, whereas the phosphorylation state of these proteins was not affected. Coimmunoprecipitation assays identified the N-terminal region of V (VNT) responsible for STAT1 targeting, which correlated with its ability to inhibit the activity of the IFN-α/β-mediated antiviral state. Conversely, while the C-terminal domain of V (VCT) could not function autonomously, when fused to VNT it optimally interacted with STAT2 and subsequently efficiently suppressed the IFN-α/β-mediated signaling pathway. The latter result was further supported by a single mutation at position 110 within the VNT domain of CDV V protein, resulting in a mutant that lost STAT1 binding while retaining a partial STAT2 association. Taken together, our results identified the CDV VNT and VCT as two essential modules that complement each other to interfere with the antiviral state induced by IFN-α/β-mediated signaling. Hence, our experiments reveal a novel mechanism of IFN-α/β evasion among the morbilliviruses. PMID:20427537

Integrated analysis reveals that STAT3 is central to the crosstalk between HER/ErbB receptor signaling pathways in human mammary epithelial cells

DOE PAGES

Gong, Chunhong; Zhang, Yi; Shankaran, Harish; ...

2014-10-02

Human epidermal growth factor receptors (HER, also known as ErbB) drive cellular proliferation, pro-survival and stress responses by activating several downstream kinases, in particular ERK, p38, JNK (SAPK), the PI3K/AKT, as well as various transcriptional regulators such as STAT3. When co-expressed, first three members of HER family (HER1-3) can form homo- and hetero-dimers. Based on the considerable evidence which suggest that every receptor dimer activates intracellular signaling pathways differentially, we hypothesized that the HER dimerization pattern is a better predictor of downstream signaling than the total receptor activation levels. We validated our hypothesis using a combination of model-based analysis tomore » quantify the HER dimerization patterns and multi-factorial experiments where HER dimerization patterns and signaling crosstalk were rationally perturbed. We have measured the activation of HER1-3 receptors and of the sentinel signaling proteins ERK, AKT, p38, JNK, STAT3 as a function of time in a panel of human mammary epithelial (HME) cells expressing different levels of HER1-3 stimulated with various ligand combinations. Our analysis using multiple ways of clustering the activation data has confirmed that the HER receptor dimer is a better predictor of the signaling through p38, ERK and AKT pathways than the total HER receptor expression and activation levels. Targeted inhibition studies to identify the causal effects allowed us to obtain a consensus regulatory interaction model, which revealed that STAT3 occupies a central role in the crosstalk between the studied pathways.« less

Liraglutide, leptin, and their combined effects on feeding: additive intake reduction through common intracellular signaling mechanisms

PubMed Central

Kanoski, Scott E.; Ong, Zhi Yi; Fortin, Samantha M.; Schlessinger, Elizabeth S.; Grill, Harvey J.

2014-01-01

Aims Glucagon like peptide-1 receptor (GLP-1R) agonists and leptin each exert anorexigenic effects. In combination, the intake inhibitory and weight loss effects are greater than either treatment alone, however the mechanisms unclear. Materials and methods Effects of liraglutide (a long-acting GLP-1 analogue) and leptin co-treatment, delivered in low or moderate doses subcutaneously (SC) or to the 3rd ventricle respectively, on cumulative intake, meal patterns, and hypothalamic expression of intracellular signaling proteins [phosphorylated signal transducer and activator of transcription-3 (pSTAT3) and protein tyrosine phosphatase-1B (PTP1B)] were examined in lean rats. Results A low-dose combination of liraglutide (25μg/kg) and leptin (0.75μg) additively reduced cumulative food intake and body weight, a result mediated predominantly through a significant reduction in meal frequency that was not present with either drug alone. Liraglutide treatment alone also reduced meal size; an effect not enhanced with leptin co-administration. Moderate doses of liraglutide (75μg/kg) and leptin (4μg) examined separately each reduced meal frequency, cumulative food intake, and body weight; only liraglutide reduced meal size. In combination these doses did not further enhance the anorexigenic effects of either treatment alone. Ex vivo immunoblot showed elevated pSTAT3 in hypothalamic tissue following liraglutide-leptin co-treatment, an effect greater than leptin treatment alone. In addition, SC liraglutide reduced expression of PTP1B (a negative regulator of leptin receptor signaling), revealing a potential mechanism for the enhanced pSTAT3 response following liraglutide-leptin co-administration. Conclusions Collectively, these results provide novel behavioral and molecular mechanisms underlying the additive reduction in food intake and body weight following liraglutide-leptin combination treatment. PMID:25475828

Feedback activation of STAT3 mediates trastuzumab resistance via upregulation of MUC1 and MUC4 expression

PubMed Central

Li, Wei; Fan, Kexing; Qian, Weizhu; Hou, Sheng; Wang, Hao; Dai, Jianxin; Wei, Huafeng; Guo, Yajun

2014-01-01

Although HER2-targeting antibody trastuzumab confers a substantial benefit for patients with HER2-overexpressing breast and gastric cancer, overcoming trastuzumab resistance remains a large unmet need. In this study, we revealed a STAT3-centered positive feedback loop that mediates the resistance of trastuzumab. Mechanistically, chronic exposure of trastuzumab causes the upregulation of fibronection (FN), EGF and IL-6 in parental trastuzumab-sensitive breast and gastric cells and convergently leads to STAT3 hyperactivation. Activated STAT3 enhances the expression of FN, EGF and IL-6, thus constituting a positive feedback loop which amplifies and maintains the STAT3 signal; furthermore, hyperactivated STAT3 signal promotes the expression of MUC1 and MUC4, consequently mediating trastuzumab resistance via maintenance of persistent HER2 activation and masking of trastuzumab binding to HER2 respectively. Genetic or pharmacological inhibition of STAT3 disrupted STAT3-dependent positive feedback loop and recovered the trastuzumab sensitivity partially due to increased apoptosis induction. Combined trastuzumab with STAT3 inhibition synergistically suppressed the growth of the trastuzumab-resistant tumor xenografts in vivo. Taken together, our results suggest that feedback activation of STAT3 constitutes a key node mediating trastuzumab resistance. Combinatorial targeting on both HER2 and STAT3 may enhance the efficacy of trastuzumab or other HER2-targeting agents in HER2-positive breast and gastric cancer. PMID:25327561

Feedback activation of STAT3 mediates trastuzumab resistance via upregulation of MUC1 and MUC4 expression.

PubMed

Li, Guangchao; Zhao, Likun; Li, Wei; Fan, Kexing; Qian, Weizhu; Hou, Sheng; Wang, Hao; Dai, Jianxin; Wei, Huafeng; Guo, Yajun

2014-09-30

Although HER2-targeting antibody trastuzumab confers a substantial benefit for patients with HER2-overexpressing breast and gastric cancer, overcoming trastuzumab resistance remains a large unmet need. In this study, we revealed a STAT3-centered positive feedback loop that mediates the resistance of trastuzumab. Mechanistically, chronic exposure of trastuzumab causes the upregulation of fibronection (FN), EGF and IL-6 in parental trastuzumab-sensitive breast and gastric cells and convergently leads to STAT3 hyperactivation. Activated STAT3 enhances the expression of FN, EGF and IL-6, thus constituting a positive feedback loop which amplifies and maintains the STAT3 signal; furthermore, hyperactivated STAT3 signal promotes the expression of MUC1 and MUC4, consequently mediating trastuzumab resistance via maintenance of persistent HER2 activation and masking of trastuzumab binding to HER2 respectively. Genetic or pharmacological inhibition of STAT3 disrupted STAT3-dependent positive feedback loop and recovered the trastuzumab sensitivity partially due to increased apoptosis induction. Combined trastuzumab with STAT3 inhibition synergistically suppressed the growth of the trastuzumab-resistant tumor xenografts in vivo. Taken together, our results suggest that feedback activation of STAT3 constitutes a key node mediating trastuzumab resistance. Combinatorial targeting on both HER2 and STAT3 may enhance the efficacy of trastuzumab or other HER2-targeting agents in HER2-positive breast and gastric cancer.

PVT1-derived miR-1207-5p promotes breast cancer cell growth by targeting STAT6.

PubMed

Yan, Chen; Chen, Yaqing; Kong, Weiwei; Fu, Liya; Liu, Yunde; Yao, Qingjuan; Yuan, Yuhua

2017-05-01

Accumulating evidence indicates that ectopic expression of non-coding RNAs are responsible for breast cancer progression. Increased non-coding RNA PVT1, the host gene of microRNA-1207-5p (miR-1207-5p), has been associated with breast cancer proliferation. However, how PVT1 functions in breast cancer is still not clear. In this study, we show a PVT1-derived microRNA, miR-1207-5p, that promotes the proliferation of breast cancer cells by directly regulating STAT6. We first confirm the positive correlated expression pattern between PVT1 and miR-1207-5p by observing consistent induced expression by estrogen, and overexpression in breast cancer cell lines and breast cancer patient specimens. Moreover, silence of PVT1 also decreased miR-1207-5p expression. Furthermore, increased miR-1207-5p expression promoted, while decreased miR-1207-5p expression suppressed, cell proliferation, colony formation, and cell cycle progression in breast cancer cell lines. Mechanistically, a novel target of miR-1207-5p, STAT6, was identified by a luciferase reporter assay. Overexpression of miR-1207-5p decreased the levels of STAT6, which activated CDKN1A and CDKN1B to regulate the cell cycle. We also confirmed the reverse correlation of miR-1207-5p and STAT6 expression levels in breast cancer samples. Therefore, our findings reveal that PVT1-derived miR-1207-5p promotes the proliferation of breast cancer cells by targeting STAT6, which in turn controls CDKN1A and CDKN1B expression. These findings suggest miR-1207-5p might be a potential target for breast cancer therapy. © 2017 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.

Transcriptional regulation of the novel monoamine oxidase renalase: Crucial roles of transcription factors Sp1, STAT3, and ZBP89.

PubMed

Sonawane, Parshuram J; Gupta, Vinayak; Sasi, Binu K; Kalyani, Ananthamohan; Natarajan, Bhargavi; Khan, Abrar A; Sahu, Bhavani S; Mahapatra, Nitish R

2014-11-11

Renalase, a novel monoamine oxidase, is emerging as an important regulator of cardiovascular, metabolic, and renal diseases. However, the mechanism of transcriptional regulation of this enzyme remains largely unknown. We undertook a systematic analysis of the renalase gene to identify regulatory promoter elements and transcription factors. Computational analysis coupled with transfection of human renalase promoter/luciferase reporter plasmids (5'-promoter-deletion constructs) into various cell types (HEK-293, IMR32, and HepG2) identified two crucial promoter domains at base pairs -485 to -399 and -252 to -150. Electrophoretic mobility shift assays using renalase promoter oligonucleotides with and without potential binding sites for transcription factors Sp1, STAT3, and ZBP89 displayed formation of specific complexes with HEK-293 nuclear proteins. Consistently, overexpression of Sp1, STAT3, and ZBP89 augmented renalase promoter activity; additionally, siRNA-mediated downregulation of Sp1, STAT3, and ZBP89 reduced the level of endogenous renalase transcription as well as the transfected renalase promoter activity. In addition, chromatin immunoprecipitation assays showed in vivo interactions of these transcription factors with renalase promoter. Interestingly, renalase promoter activity was augmented by nicotine and catecholamines; while Sp1 and STAT3 synergistically activated the nicotine-induced effect, Sp1 appeared to enhance epinephrine-evoked renalase transcription. Moreover, renalase transcript levels in mouse models of human essential hypertension were concomitantly associated with endogenous STAT3 and ZBP89 levels, suggesting crucial roles for these transcription factors in regulating renalase gene expression in cardiovascular pathological conditions.

Ethanol metabolism by alcohol dehydrogenase or cytochrome P450 2E1 differentially impairs hepatic protein trafficking and growth hormone signaling.

PubMed

Doody, Erin E; Groebner, Jennifer L; Walker, Jetta R; Frizol, Brittnee M; Tuma, Dean J; Fernandez, David J; Tuma, Pamela L

2017-12-01

The liver metabolizes alcohol using alcohol dehydrogenase (ADH) and cytochrome P 450 2E1 (CYP2E1). Both enzymes metabolize ethanol into acetaldehyde, but CYP2E1 activity also results in the production of reactive oxygen species (ROS) that promote oxidative stress. We have previously shown that microtubules are hyperacetylated in ethanol-treated polarized, hepatic WIF-B cells and livers from ethanol-fed rats. We have also shown that enhanced protein acetylation correlates with impaired clathrin-mediated endocytosis, constitutive secretion, and nuclear translocation and that the defects are likely mediated by acetaldehyde. However, the roles of CYP2E1-generated metabolites and ROS in microtubule acetylation and these alcohol-induced impairments have not been examined. To determine if CYP2E1-mediated alcohol metabolism is required for enhanced acetylation and the trafficking defects, we coincubated cells with ethanol and diallyl sulfide (DAS; a CYP2E1 inhibitor) or N -acetyl cysteine (NAC; an antioxidant). Both agents failed to prevent microtubule hyperacetylation in ethanol-treated cells and also failed to prevent impaired secretion or clathrin-mediated endocytosis. Somewhat surprisingly, both DAS and NAC prevented impaired STAT5B nuclear translocation. Further examination of microtubule-independent steps of the pathway revealed that Jak2/STAT5B activation by growth hormone was prevented by DAS and NAC. These results were confirmed in ethanol-exposed HepG2 cells expressing only ADH or CYP2E1. Using quantitative RT-PCR, we further determined that ethanol exposure led to blunted growth hormone-mediated gene expression. In conclusion, we determined that alcohol-induced microtubule acetylation and associated defects in microtubule-dependent trafficking are mediated by ADH metabolism whereas impaired microtubule-independent Jak2/STAT5B activation is mediated by CYP2E1 activity. NEW & NOTEWORTHY Impaired growth hormone-mediated signaling is observed in ethanol-exposed hepatocytes and is explained by differential effects of alcohol dehydrogenase (ADH)- and cytochrome P 450 2E1 (CYP2E1)-mediated ethanol metabolism on the Jak2/STAT5B pathway. Copyright © 2017 the American Physiological Society.

Molecular mechanisms of mucocutaneous immunity against Candida and Staphylococci

PubMed Central

Maródi, László; Cypowyj, Sophie; Tóth, Beáta; Chernyshova, Liudmyla; Puel, Anne; Casanova, Jean-Laurent

2013-01-01

Signal transducer and activator of transcription (STAT) proteins are key components of the innate and adaptive immune responses to pathogenic microorganisms. Recent research on primary immunodeficiency disorders and the identification of patients carrying germline mutations in STAT1, STAT3, and STAT5B have highlighted the role of human STATs in host defense against various viruses, bacteria, and fungi. Mutations in STAT1 and STAT3 may disrupt various cytokine pathways that control mucocutaneous immunity against Candida species, especially Candida albicans, and Staphylococci, especially Staphylococcus aureus. Here, we consider inborn errors of immunity arising from mutations in either STAT1 or STAT3 that affect mucocutaneous immunity to Candida and Staphylococci. PMID:23040277

Transcriptional activation of the suppressor of cytokine signaling-3 (SOCS-3) gene via STAT3 is increased in F9 REX1 (ZFP-42) knockout teratocarcinoma stem cells relative to wild-type cells.

PubMed

Xu, Juliana; Sylvester, Renia; Tighe, Ann P; Chen, Siming; Gudas, Lorraine J

2008-03-14

Rex1 (Zfp42), first identified as a gene that is transcriptionally repressed by retinoic acid (RA), encodes a zinc finger transcription factor expressed at high levels in F9 teratocarcinoma stem cells, embryonic stem cells, and other stem cells. Loss of both alleles of Rex1 by homologous recombination alters the RA-induced differentiation of F9 cells, a model of pluripotent embryonic stem cells. We identified Suppressor of Cytokine Signaling-3 (SOCS-3) as a gene that exhibits greatly increased transcriptional activation in RA, cAMP, and theophylline (RACT)-treated F9 Rex1(-/-) cells (approximately 25-fold) as compared to wild-type (WT) cells ( approximately 2.5-fold). By promoter deletion, mutation, and transient transfection analyses, we have shown that this transcriptional increase is mediated by the STAT3 DNA-binding elements located between -99 to -60 in the SOCS-3 promoter. Overexpression of STAT3 dominant-negative mutants greatly diminishes this SOCS-3 transcriptional increase in F9 Rex1(-/-) cells. This increase in SOCS-3 transcription is associated with a four- to fivefold higher level of tyrosine-phosphorylated STAT3 in the RACT-treated F9 Rex1(-/-) cells as compared to WT. Dominant-negative Src tyrosine kinase, Jak2, and protein kinase A partially reduce the transcriptional activation of the SOCS 3 gene in RACT-treated F9 Rex1 null cells. In contrast, parathyroid hormone peptide enhances the effect of RA in F9 Rex1(-/-) cells, but not in F9 WT. Thus, Rex1, which is highly expressed in stem cells, inhibits signaling via the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway, thereby modulating the differentiation of F9 cells.

FAP Promotes Immunosuppression by Cancer-Associated Fibroblasts in the Tumor Microenvironment via STAT3-CCL2 Signaling.

PubMed

Yang, Xuguang; Lin, Yuli; Shi, Yinghong; Li, Bingji; Liu, Weiren; Yin, Wei; Dang, Yongjun; Chu, Yiwei; Fan, Jia; He, Rui

2016-07-15

Cancer-associated fibroblasts (CAF) are components of the tumor microenvironment whose contributions to malignant progression are not fully understood. Here, we show that the fibroblast activation protein (FAP) triggers induction of a CAF subset with an inflammatory phenotype directed by STAT3 activation and inflammation-associated expression signature marked by CCL2 upregulation. Enforcing FAP expression in normal fibroblasts was sufficient to endow them with an inflammatory phenotype similar to FAP(+)CAFs. We identified FAP as a persistent activator of fibroblastic STAT3 through a uPAR-dependent FAK-Src-JAK2 signaling pathway. In a murine liver tumor model, we found that FAP(+)CAFs were a major source of CCL2 and that fibroblastic STAT3-CCL2 signaling in this setting promoted tumor growth by enhancing recruitment of myeloid-derived suppressor cells (MDSC). The CCL2 receptor CCR2 was expressed on circulating MDSCs in tumor-bearing subjects and FAP(+)CAF-mediated tumor promotion and MDSC recruitment was abrogated in Ccr2-deficient mice. Clinically, we observed a positive correlation between stromal expression of FAP, p-STAT3, and CCL2 in human intrahepatic cholangiocarcinoma, a highly aggressive liver cancer with dense desmoplastic stroma, where elevated levels of stromal FAP predicted a poor survival outcome. Taken together, our results showed how FAP-STAT3-CCL2 signaling in CAFs was sufficient to program an inflammatory component of the tumor microenvironment, which may have particular significance in desmoplasia-associated cancers. Cancer Res; 76(14); 4124-35. ©2016 AACR. ©2016 American Association for Cancer Research.