Hierarchy of protein tyrosine kinases in interleukin-2 (IL-2) signaling: activation of syk depends on Jak3; however, neither Syk nor Lck is required for IL-2-mediated STAT activation.

PubMed

Zhou, Y J; Magnuson, K S; Cheng, T P; Gadina, M; Frucht, D M; Galon, J; Candotti, F; Geahlen, R L; Changelian, P S; O'Shea, J J

2000-06-01

Interleukin-2 (IL-2) activates several different families of tyrosine kinases, but precisely how these kinases interact is not completely understood. We therefore investigated the functional relationships among Jak3, Lck, and Syk in IL-2 signaling. We first observed that in the absence of Jak3, both Lck and Syk had the capacity to phosphorylate Stat3 and Stat5a. However, neither supported IL-2-induced STAT activation, nor did dominant negative alleles of these kinases inhibit. Moreover, pharmacological abrogation of Lck activity did not inhibit IL-2-mediated phosphorylation of Jak3 and Stat5a. Importantly, ligand-dependent Syk activation was dependent on the presence of catalytically active Jak3, whereas Lck activation was not. Interestingly, Syk functioned as a direct substrate of Jak1 but not Jak3. Additionally, Jak3 phosphorylated Jak1, whereas the reverse was not the case. Taken together, our data support a model in which Lck functions in parallel with Jak3, while Syk functions as a downstream element of Jaks in IL-2 signaling. Jak3 may regulate Syk catalytic activity indirectly via Jak1. However, IL-2-mediated Jak3/Stat activation is not dependent on Lck or Syk. While the essential roles of Jak1 and Jak3 in signaling by gammac-utilizing cytokines are clear, it will be important to dissect the exact contributions of Lck and Syk in mediating the effects of IL-2 and related cytokines.

The role of JAK-3 in regulating TLR-mediated inflammatory cytokine production in innate immune cells.

PubMed

Wang, Huizhi; Brown, Jonathan; Gao, Shegan; Liang, Shuang; Jotwani, Ravi; Zhou, Huaxin; Suttles, Jill; Scott, David A; Lamont, Richard J

2013-08-01

The role of JAK-3 in TLR-mediated innate immune responses is poorly understood, although the suppressive function of JAK3 inhibition in adaptive immune response has been well studied. In this study, we found that JAK3 inhibition enhanced TLR-mediated immune responses by differentially regulating pro- and anti- inflammatory cytokine production in innate immune cells. Specifically, JAK3 inhibition by pharmacological inhibitors or specific small interfering RNA or JAK3 gene knockout resulted in an increase in TLR-mediated production of proinflammatory cytokines while concurrently decreasing the production of IL-10. Inhibition of JAK3 suppressed phosphorylation of PI3K downstream effectors including Akt, mammalian target of rapamycin complex 1, glycogen synthase kinase 3β (GSK3β), and CREB. Constitutive activation of Akt or inhibition of GSK3β abrogated the capability of JAK3 inhibition to enhance proinflammatory cytokines and suppress IL-10 production. In contrast, inhibition of PI3K enhanced this regulatory ability of JAK3 in LPS-stimulated monocytes. At the transcriptional level, JAK3 knockout lead to the increased phosphorylation of STATs that could be attenuated by neutralization of de novo inflammatory cytokines. JAK3 inhibition exhibited a GSK3 activity-dependent ability to enhance phosphorylation levels and DNA binding of NF-κB p65. Moreover, JAK3 inhibition correlated with an increased CD4(+) T cell response. Additionally, higher neutrophil infiltration, IL-17 expression, and intestinal epithelium erosion were observed in JAK3 knockout mice. These findings demonstrate the negative regulatory function of JAK3 and elucidate the signaling pathway by which JAK3 differentially regulates TLR-mediated inflammatory cytokine production in innate immune cells.

Activation of JAK3, but not JAK1, is critical to interleukin-4 (IL4) stimulated proliferation and requires a membrane-proximal region of IL4 receptor alpha.

PubMed

Malabarba, M G; Kirken, R A; Rui, H; Koettnitz, K; Kawamura, M; O'Shea, J J; Kalthoff, F S; Farrar, W L

1995-04-21

The tyrosine kinases JAK1 and JAK3 have been shown to undergo tyrosine phosphorylation in response to interleukin-2 (IL), IL4, IL7, and IL9, cytokines which share the common IL2 receptor gamma-chain (IL2R gamma), and evidence has been found for a preferential coupling of JAK3 to IL2R gamma and JAK1 to IL2R beta. Here we show, using human premyeloid TF-1 cells, that IL4 stimulates JAK3 to a larger extent than JAK1, based upon three different evaluation criteria. These include a more vigorous tyrosine phosphorylation of JAK3 as measured by anti-phosphotyrosine immunoblotting, a more marked activation of JAK3 as determined by in vitro tyrosine kinase assays and a more manifest presence of JAK3 in activated IL4-receptor complexes. These observations suggest that IL4 receptor signal transduction does not depend on equimolar heterodimerization of JAK1 and JAK3 following IL4-induced heterodimerization of IL4R alpha and IL2R gamma. Indeed, when human IL4R alpha was stably expressed in mouse BA/F3 cells, robust IL4-induced proliferation and JAK3 activation occurred without detectable involvement of JAK1, JAK2, or TYK2. The present study suggests that JAK1 plays a subordinate role in IL4 receptor signaling, and that in certain cells exclusive JAK3 activation may mediate IL4-induced cell growth. Moreover, mutational analysis of human IL4R alpha showed that a membrane-proximal cytoplasmic region was critical for JAK3 activation, while the I4R motif was not, which is compatible with a role of JAK3 upstream of the recruitment of the insulin receptor substrate-1/4PS signaling proteins by IL4 receptors.

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.

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

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.

JAK inhibitors suppress t(8;21) fusion protein-induced leukemia

PubMed Central

Lo, Miao-Chia; Peterson, Luke F.; Yan, Ming; Cong, Xiuli; Hickman, Justin H.; DeKelver, Russel C.; Niewerth, Denise; Zhang, Dong-Er

2014-01-01

Oncogenic mutations in components of the JAK/STAT pathway, including those in cytokine receptors and JAKs, lead to increased activity of downstream signaling and are frequently found in leukemia and other hematological disorders. Thus, small-molecule inhibitors of this pathway have been the focus of targeted therapy in these hematological diseases. We previously showed that t(8;21) fusion protein AML1-ETO and its alternatively spliced variant AML1-ETO9a (AE9a) enhance the JAK/STAT pathway via down-regulation of CD45, a negative regulator of this pathway. To investigate the therapeutic potential of targeting JAK/STAT in t(8;21) leukemia, we examined the effects of a JAK2-selective inhibitor TG101209 and a JAK1/2-selective inhibitor INCB18424 on t(8;21) leukemia cells. TG101209 and INCB18424 inhibited proliferation and promoted apoptosis of these cells. Furthermore, TG101209 treatment in AE9a leukemia mice reduced tumor burden and significantly prolonged survival. TG101209 also significantly impaired the leukemia-initiating potential of AE9a leukemia cells in secondary recipient mice. These results demonstrate the potential therapeutic efficacy of JAK inhibitors in treating t(8;21) AML. PMID:23812420

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

[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.

Specific Jak3 Downregulation in Lymphocytes Impairs γc Cytokine Signal Transduction and Alleviates Antigen-driven Inflammation In Vivo

PubMed Central

Gómez-Valadés, Alicia G; Llamas, María; Blanch, Sílvia; Perales, José C; Román, Juan; Gómez-Casajús, Lluís; Mascaró, Cristina

2012-01-01

Jak3, one of the four members comprising the Jak family of cytosolic tyrosine kinases, has emerged as a promising target for nontoxic immunotherapies. Although a number of Jak inhibitors has already demonstrated efficacy, they suffer from secondary effects apparently associated to their pan-Jak activity. However, whether selective Jak3 inhibition would afford therapeutic efficacy remains unclear. To address this question we have investigated the immunosuppressive potential of selective Jak3 intervention in lymphocytes using RNA interference (RNAi) technology in vitro and in vivo. Using synthetic small interference RNA (siRNA) sequences we achieved successful transfections into human and mouse primary T lymphocytes. We found that Jak3 knockdown was sufficient to impair not only interleukin-2 (IL-2) and T cell receptor (TCR)-mediated cell activation in vitro, but also antigen-triggereds welling, inflammatory cell infiltration, and proinflammatory cytokine raise in vivo. Furthermore, Jak1 (which mediates γc cytokine signaling in conjunction with Jak3) cosilencing did not provide higher potency to the aforementioned immunosuppressant effects. Our data provides direct evidences indicating that Jak3 protein plays an important role in γc cytokine and antigen-mediated T cell activation and modulates Th1-mediated inflammatory disorders, all in all highlighting its potential as a target in immunosuppressive therapies. PMID:23344234

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

The Janus Kinase (JAK) FERM and SH2 Domains: Bringing Specificity to JAK-Receptor Interactions.

PubMed

Ferrao, Ryan; Lupardus, Patrick J

2017-01-01

The Janus kinases (JAKs) are non-receptor tyrosine kinases essential for signaling in response to cytokines and interferons and thereby control many essential functions in growth, development, and immune regulation. JAKs are unique among tyrosine kinases for their constitutive yet non-covalent association with class I and II cytokine receptors, which upon cytokine binding bring together two JAKs to create an active signaling complex. JAK association with cytokine receptors is facilitated by N-terminal FERM and SH2 domains, both of which are classical mediators of peptide interactions. Together, the JAK FERM and SH2 domains mediate a bipartite interaction with two distinct receptor peptide motifs, the proline-rich "Box1" and hydrophobic "Box2," which are present in the intracellular domain of cytokine receptors. While the general sidechain chemistry of Box1 and Box2 peptides is conserved between receptors, they share very weak primary sequence homology, making it impossible to posit why certain JAKs preferentially interact with and signal through specific subsets of cytokine receptors. Here, we review the structure and function of the JAK FERM and SH2 domains in light of several recent studies that reveal their atomic structure and elucidate interaction mechanisms with both the Box1 and Box2 receptor motifs. These crystal structures demonstrate how evolution has repurposed the JAK FERM and SH2 domains into a receptor-binding module that facilitates interactions with multiple receptors possessing diverse primary sequences.

Glutamine Deprivation Causes Hydrogen Peroxide-induced Interleukin-8 Expression via Jak1/Stat3 Activation in Gastric Epithelial AGS Cells

PubMed Central

Lee, Yun Mi; Kim, Mi Jung; Kim, Youngha; Kim, Hyeyoung

2015-01-01

Background: The Janus kinase (Jak)/Signal transducers of activated transcription (Stat) pathway is an upstream signaling pathway for NF-κB activation in Helicobacter pylori-induced interleukin (IL)-8 production in gastric epithelial AGS cells. H. pylori activates NADPH oxidase and produces hydrogen peroxide, which activates Jak1/Stat3 in AGS cells. Therefore, hydrogen peroxide may be critical for IL-8 production via Jak/Stat activation in gastric epithelial cells. Glutamine is depleted during severe injury and stress and contributes to the formation of glutathione (GSH), which is involved in conversion of hydrogen peroxide into water as a cofactor for GSH peroxidase. Methods: We investigated whether glutamine deprivation induces hydrogen peroxide-mediated IL-8 production and whether hydrogen peroxide activates Jak1/Stat3 to induce IL-8 in AGS cells. Cells were cultured in the presence or absence of glutamine or hydrogen peroxide, with or without GSH or a the Jak/Stat specific inhibitor AG490. Results: Glutamine deprivation decreased GSH levels, but increased levels of hydrogen peroxide and IL-8, an effect that was inhibited by treatment with GSH. Hydrogen peroxide induced the activation of Jak1/Stat3 time-dependently. AG490 suppressed hydrogen peroxide- induced activation of Jak1/Stat3 and IL-8 expression in AGS cells, but did not affect levels of reactive oxygen species in AGS cells. Conclusions: In gastric epithelial AGS cells, glutamine deprivation increases hydrogen peroxide levels and IL-8 expression, which may be mediated by Jak1/Stat3 activation. Glutamine supplementation may be beneficial for preventing gastric inflammation by suppressing hydrogen peroxide-mediated Jak1/Stat3 activation and therefore, reducing IL-8 production. Scavenging hydrogen peroxide or targeting Jak1/Stat3 may also prevent oxidant-mediated gastric inflammation. PMID:26473156

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.

GRAMD1B regulates cell migration in breast cancer cells through JAK/STAT and Akt signaling.

PubMed

Khanna, Puja; Lee, Joan Shuying; Sereemaspun, Amornpun; Lee, Haeryun; Baeg, Gyeong Hun

2018-06-22

Dysregulated JAK/STAT signaling has been implicated in breast cancer metastasis, which is associated with high relapse risks. However, mechanisms underlying JAK/STAT signaling-mediated breast tumorigenesis are poorly understood. Here, we showed that GRAMD1B expression is upregulated on IL-6 but downregulated upon treatment with the JAK2 inhibitor AG490 in the breast cancer MDA-MB-231 cells. Notably, Gramd1b knockdown caused morphological changes of the cells, characterized by the formation of membrane ruffling and protrusions, implicating its role in cell migration. Consistently, GRAMD1B inhibition significantly enhanced cell migration, with an increase in the levels of the Rho family of GTPases. We also found that Gramd1b knockdown-mediated pro-migratory phenotype is associated with JAK2/STAT3 and Akt activation, and that JAK2 or Akt inhibition efficiently suppresses the phenotype. Interestingly, AG490 dose-dependently increased p-Akt levels, and our epistasis analysis suggested that the effect of JAK/STAT inhibition on p-Akt is via the regulation of GRAMD1B expression. Taken together, our results suggest that GRAMD1B is a key signaling molecule that functions to inhibit cell migration in breast cancer by negating both JAK/STAT and Akt signaling, providing the foundation for its development as a novel biomarker in breast cancer.

A role for the JAK-STAT1 pathway in blocking replication of HSV-1 in dendritic cells and macrophages

PubMed Central

Mott, Kevin R; UnderHill, David; Wechsler, Steven L; Town, Terrence; Ghiasi, Homayon

2009-01-01

Background Macrophages and dendritic cells (DCs) play key roles in host defense against HSV-1 infection. Although macrophages and DCs can be infected by herpes simplex virus type 1 (HSV-1), both cell types are resistant to HSV-1 replication. The aim of our study was to determine factor (s) that are involved in the resistance of DCs and macrophages to productive HSV-1 infection. Results We report here that, in contrast to bone marrow-derived DCs and macrophages from wild type mice, DCs and macrophages isolated from signal transducers and activators of transcription-1 deficient (STAT1-/-) mice were susceptible to HSV-1 replication and the production of viral mRNAs and DNA. There were differences in expression of immediate early, early, and late gene transcripts between STAT1+/+ and STAT1-/- infected APCs. Conclusion These results suggest for the first time that the JAK-STAT1 pathway is involved in blocking replication of HSV-1 in DCs and macrophages. PMID:19439086

Role of the JAKs/STATs pathway in the intracellular calcium changes induced by interleukin-6 in hippocampal neurons.

PubMed

Orellana, D I; Quintanilla, R A; Gonzalez-Billault, C; Maccioni, R B

2005-11-01

Recent studies show that inflammation has an active role in the onset of neurodegenerative diseases. It is known that in response to extracellular insults microglia and/or astrocytes produce inflammatory agents. These contribute to the neuropathological events in the aging process and neuronal degeneration. Interleukin-6 (IL-6) has been involved in the pathogenesis of neurodegenerative disorders, such as Alzheimer's and Parkinson's diseases. Here, we show that IL-6 treatment of rat hippocampal neurons increases the calcium influx via NMDA-receptor, an effect that is prevented by the specific NMDA receptor antagonist MK-801 (dizocilpine). We also show that this calcium influx is mediated by the JAKs/STATs pathway, since the inhibitor of JAKs/STATs pathway, JAK 3 inhibitor, blocks calcium influx even in the presence of IL-6. This increase in calcium signal was dependent on external sources, since this signal was not observed in the presence of EGTA. Additional studies indicate that the increase in cytosolic calcium induces tau protein hyperphosphorylation, as revealed by using specific antibodies against Alzheimer phosphoepitopes. This anomalous tau hyperphosphorylation was dependent on both the JAKs/STATs pathway and NMDA receptor. These results suggest that IL-6 would induce a cascade of molecular events that produce a calcium influx through NMDA receptors, mediated by the JAKs/STATs pathway, which subsequently modifies the tau hyperphosphorylation patterns.

Anti-angiogenic and anti-metastatic activity of JAK inhibitor AZD1480

PubMed Central

Xin, Hong; Herrmann, Andreas; Reckamp, Karen; Zhang, Wang; Pal, Sumanta; Hedvat, Michael; Zhang, Chunyan; Liang, Wei; Scuto, Anna; Weng, Shaobu; Morosini, Deborah; Cao, Zhu A.; Zinda, Michael; Figlin, Robert; Huszar, Dennis; Jove, Richard; Yu, Hua

2011-01-01

STAT3 has important functions in both tumor cells and the tumor microenvironment to facilitate cancer progression. The STAT regulatory kinase JAK has been strongly implicated in promoting oncogenesis of various solid tumors, including through the use of JAK kinase inhibitors such as AZD1480. However, direct evidence that JAK drives STAT3 function and cancer pathogenesis at the level of the tumor microenvironment has yet to be established clearly. In this study, we show that AZD1480 inhibits STAT3 in tumor-associated myeloid cells, reducing their number and inhibiting tumor metastasis. Myeloid cell-mediated angiogenesis was also diminished by AZD1480, with additional direct inhibition of endothelial cell function in vitro and in vivo. AZD1480 blocked lung infiltration of myeloid cells and formation of pulmonary metastases in both mouse syngeneic experimental and spontaneous metastatic models. Furthermore, AZD1480 reduced angiogenesis and metastasis in a human xenograft tumor model. Although the effects of AZD1480 on the tumor microenvironment were important for the observed anti-angiogenic activity, constitutive activation of STAT3 in tumor cells themselves could block these anti-angiogenic effects demonstrating the complexity of the JAK/STAT signaling network in tumor progression. Together, our results indicated that AZD1480 can effectively inhibit tumor angiogenesis and metastasis mediated by STAT3 in stromal cells as well as tumor cells. PMID:21920898

JAKs and STATs in Immunoregulation and Immune-Mediated Disease

PubMed Central

O’Shea, John J.; Plenge, Robert

2012-01-01

Summary A landmark in cell biology, the discovery of the JAK-STAT pathway provided a simple mechanism for gene regulation that dramatically advanced our understanding of the action of hormones, interferons, colony stimulating factors, and interleukins. As we learn more about the complexities of immune responses, new insights into the functions of this pathway continue to be revealed, aided by technology that permits genomewide views. As we celebrate the 20th anniversary of the discovery of this paradigm in cell signaling, it is particularly edifying to see how this knowledge has rapidly been translated to human immune disease. Not only have genomewide association studies demonstrated that this pathway is highly relevant to human autoimmunity but targeting JAKs is now a reality in immune-mediated disease. PMID:22520847

Tyrosine phosphorylation of Jak2 in the JH2 domain inhibits cytokine signaling.

PubMed

Feener, Edward P; Rosario, Felicia; Dunn, Sarah L; Stancheva, Zlatina; Myers, Martin G

2004-06-01

Jak family tyrosine kinases mediate signaling by cytokine receptors to regulate diverse biological processes. Although Jak2 and other Jak kinase family members are phosphorylated on numerous sites during cytokine signaling, the identity and function of most of these sites remains unknown. Using tandem mass spectroscopic analysis of activated Jak2 protein from intact cells, we identified Tyr(221) and Tyr(570) as novel sites of Jak2 phosphorylation. Phosphorylation of both sites was stimulated by cytokine treatment of cultured cells, and this stimulation required Jak2 kinase activity. While we observed no gross alteration of signaling upon mutation of Tyr(221), Tyr(570) lies within the inhibitory JH2 domain of Jak2, and mutation of this site (Jak2(Y570F)) results in constitutive Jak2-dependent signaling in the absence of cytokine stimulation and enhances and prolongs Jak2 activation during cytokine stimulation. Mutation of Tyr(570) does not alter the ability of SOCS3 to bind or inhibit Jak2, however. Thus, the phosphorylation of Tyr(570) in vivo inhibits Jak2-dependent signaling independently of SOCS3-mediated inhibition. This Tyr(570)-dependent mechanism of Jak2 inhibition likely represents an important mechanism by which cytokine function is regulated.

JAK1 kinase forms complexes with interleukin-4 receptor and 4PS/insulin receptor substrate-1-like protein and is activated by interleukin-4 and interleukin-9 in T lymphocytes.

PubMed

Yin, T; Tsang, M L; Yang, Y C

1994-10-28

Interleukin (IL)-4 and IL-9 regulate the proliferation of T lymphocytes through interactions with their receptors. Previous studies have shown that unknown tyrosine kinases are involved in the proliferative signaling triggered by IL-4 and IL-9. Here we show that IL-4 and IL-9 induce overlapping (170, 130, and 125 kilodalton (kDa)) and distinct (45 and 88/90 kDa, respectively) protein tyrosine phosphorylation in T lymphocytes. We further identify the 170-kDa tyrosine-phosphorylated protein as 4PS/insulin receptor substrate-1-like (IRS-1L) protein and 130-kDa protein as JAK1 kinase. Furthermore, we demonstrate for the first time that JAK1 forms complexes with the IL-4 receptor and 4PS/IRS-1L protein following ligand-receptor interaction. In addition, we demonstrate that IL-9, but not IL-4, induced tyrosine phosphorylation of Stat 91 transcriptional factor. The overlapping and distinct protein tyrosine phosphorylation and activation of the same JAK1 kinase in T lymphocytes strongly suggests that IL-4 and IL-9 share the common signal transduction pathways and that the specificity for each cytokine could be achieved through the unique tyrosine-phosphorylated proteins triggered by individual cytokines.

Identification of azabenzimidazoles as potent JAK1 selective inhibitors.

PubMed

Vasbinder, Melissa M; Alimzhanov, Marat; Augustin, Martin; Bebernitz, Geraldine; Bell, Kirsten; Chuaqui, Claudio; Deegan, Tracy; Ferguson, Andrew D; Goodwin, Kelly; Huszar, Dennis; Kawatkar, Aarti; Kawatkar, Sameer; Read, Jon; Shi, Jie; Steinbacher, Stefan; Steuber, Holger; Su, Qibin; Toader, Dorin; Wang, Haixia; Woessner, Richard; Wu, Allan; Ye, Minwei; Zinda, Michael

2016-01-01

We have identified a class of azabenzimidazoles as potent and selective JAK1 inhibitors. Investigations into the SAR are presented along with the structural features required to achieve selectivity for JAK1 versus other JAK family members. An example from the series demonstrated highly selective inhibition of JAK1 versus JAK2 and JAK3, along with inhibition of pSTAT3 in vivo, enabling it to serve as a JAK1 selective tool compound to further probe the biology of JAK1 selective inhibitors. Copyright © 2015 Elsevier Ltd. All rights reserved.

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

[Chrysin inhibits lipopolysaccharide-induced inflammatory responses of macrophages via JAK-STATs signaling pathway].

PubMed

Qi, Shi-Mei; Li, Qiang; Jiang, Qi; Qi, Zhi-Lin; Zhang, Yao

2018-03-20

To investigate the mechanism of chrysin in regulating lipopolysaccharide (LPS)-induced inflammation in RAW264.7 cells. RAW264.7 cells were treated with different concentrations (0, 5, 10, 20, 40, 60, 80, 100, 150, and 200 µg/mL) of chrysin for 24 h, and the cell viability was measured using CCK-8. RAW264.7 cells were pre-treated with 10, 30, or 60 µg/mL chrysin for 2 h before stimulation with LPS for different times. The levels of TNF-α, IL-6 and MCP-1 were detected by ELISA, and Western blotting was used to detect the phosphorylation of JAK- 1, JAK-2, STAT-1 and STAT-3. The level of reactive oxygen species in RAW264.7 cells was detected by CM-H2DCFDA fluorescence probe. The effect of ROS on LPS-induced JAK-STATs signal and the inflammatory response of RAW264.7 cells was detected by ROS scavenger NAC. The transcription factors STAT-1 and STAT-3 nuclear translocation were observed by laser confocal microscopy. Chrysin below 60 µg/mL did not significantly affect the viability of RAW264.7 cells. At 10, 30, and 60 µg/mL, chrysin dose-dependently inhibited the expression of iNOS induced by LPS. Chrysin treatment also inhibited LPS-induced phosphorylation of JAK-STATs, nuclear translocation of STAT1 and STAT3, release of TNF-α, IL-6 and MCP-1, and the production of ROS in RAW264.7 cells; ROS acted as an upstream signal to mediate the activation of JAK-STATs signaling pathway. Chrysin blocks the activity of JAK-STATs mediated by ROS to inhibit LPS-induced inflammatory response in RAW264.7 cells.

Structure-Function Correlation of G6, a Novel Small Molecule Inhibitor of Jak2

PubMed Central

Majumder, Anurima; Govindasamy, Lakshmanan; Magis, Andrew; Kiss, Róbert; Polgár, Tímea; Baskin, Rebekah; Allan, Robert W.; Agbandje-McKenna, Mavis; Reuther, Gary W.; Keserű, György M.; Bisht, Kirpal S.; Sayeski, Peter P.

2010-01-01

Somatic mutations in the Jak2 protein, such as V617F, cause aberrant Jak/STAT signaling and can lead to the development of myeloproliferative neoplasms. This discovery has led to the search for small molecule inhibitors that target Jak2. Using structure-based virtual screening, our group recently identified a novel small molecule inhibitor of Jak2 named G6. Here, we identified a structure-function correlation of this compound. Specifically, five derivative compounds of G6 having structural similarity to the original lead compound were obtained and analyzed for their ability to (i) inhibit Jak2-V617F-mediated cell growth, (ii) inhibit the levels of phospho-Jak2, phospho-STAT3, and phospho-STAT5; (iii) induce apoptosis in human erythroleukemia cells; and (iv) suppress pathologic cell growth of Jak2-V617F-expressing human bone marrow cells ex vivo. Additionally, we computationally examined the interactions of these compounds with the ATP-binding pocket of the Jak2 kinase domain. We found that the stilbenoid core-containing derivatives of G6 significantly inhibited Jak2-V617F-mediated cell proliferation in a time- and dose-dependent manner. They also inhibited phosphorylation of Jak2, STAT3, and STAT5 proteins within cells, resulting in higher levels of apoptosis via the intrinsic apoptotic pathway. Finally, the stilbenoid derivatives inhibited the pathologic growth of Jak2-V617F-expressing human bone marrow cells ex vivo. Collectively, our data demonstrate that G6 has a stilbenoid core that is indispensable for maintaining its Jak2 inhibitory potential. PMID:20667821

Heterodimeric JAK-STAT Activation as a Mechanism of Persistence to JAK2 Inhibitor Therapy

PubMed Central

Koppikar, Priya; Bhagwat, Neha; Kilpivaara, Outi; Manshouri, Taghi; Adli, Mazhar; Hricik, Todd; Liu, Fan; Saunders, Lindsay M.; Mullally, Ann; Abdel-Wahab, Omar; Leung, Laura; Weinstein, Abby; Marubayashi, Sachie; Goel, Aviva; Gönen, Mithat; Estrov, Zeev; Ebert, Benjamin L.; Chiosis, Gabriela; Nimer, Stephen D.; Bernstein, Bradley E.; Verstovsek, Srdan; Levine, Ross L.

2012-01-01

The identification of somatic activating mutations in JAK21–4 and in the thrombopoietin receptor (MPL)5 in the majority of myeloproliferative neoplasm (MPN) patients led to the clinical development of JAK2 kinase inhibitors6,7. JAK2 inhibitor therapy improves MPN-associated splenomegaly and systemic symptoms, but does not significantly reduce or eliminate the MPN clone in most MPN patients. We therefore sought to characterize mechanisms by which MPN cells persist despite chronic JAK2 inhibition. Here we show that JAK2 inhibitor persistence is associated with reactivation of JAK-STAT signaling and with heterodimerization between activated JAK2 and JAK1/TYK2, consistent with activation of JAK2 in trans by other JAK kinases. Further, this phenomenon is reversible, such that JAK2 inhibitor withdrawal is associated with resensitization to JAK2 kinase inhibitors and with reversible changes in JAK2 expression. We saw increased JAK2 heterodimerization and sustained JAK2 activation in cell lines, murine models, and patients treated with JAK2 inhibitors. RNA interference and pharmacologic studies demonstrate that JAK2 inhibitor persistent cells remain dependent on JAK2 protein expression. Consequently, therapies that result in JAK2 degradation retain efficacy in persistent cells and may provide additional benefit to patients with JAK2-dependent malignancies treated with JAK2 inhibitors. PMID:22820254

TLR4 and RAGE conversely mediate pro-inflammatory S100A8/9-mediated inhibition of proliferation-linked signaling in myeloproliferative neoplasms.

PubMed

Kovačić, Marijana; Mitrović-Ajtić, Olivera; Beleslin-Čokić, Bojana; Djikić, Dragoslava; Subotički, Tijana; Diklić, Miloš; Leković, Danijela; Gotić, Mirjana; Mossuz, Pascal; Čokić, Vladan P

2018-06-26

Previously, the family of S100A proteins has been found to be associated with inflammation and myelopoiesis and to be able to induce or support myeloproliferation during chronic inflammation. Here, we studied the inflammatory myeloid-related proteins S100A4, S100A8, S100A9 and S100A12 in myeloproliferative neoplasms (MPNs) in order to assess the involvement of chronic inflammation in the pathogenesis of MPN. We analyzed the S100A4, S100A8, S100A9 and S100A12 mRNA and protein levels in the bone marrow and circulation of 140 patients with MPN and 15 healthy controls using Western blotting, microarray-based mRNA expression profiling and ELISA assays, respectively. In addition we performed functional studies on the proliferation-related AKT and ERK1/2 signaling pathways in MPN-derived granulocytes using Western blotting and proteomic analyses. We found that the S100A mRNA levels were increased in MPN patient-derived circulatory CD34 + cells, and that their protein expression levels were also augmented in their granulocytes and bone marrow stroma cells, depending on the JAK2V617F mutation allele burden. We also found that calreticulin (CALR) mutations were related to reduced S100A8 plasma levels in primary myelofibrosis (PMF). The S100A8 plasma levels were found to be increased in MPN, the S100A9 plasma levels in PMF and essential thrombocythemia (ET), and the S100A12 plasma levels in polycythemia vera (PV). These S100A plasma levels showed a positive correlation with the systemic inflammation marker IL-8, as well as with the numbers of leukocytes and thrombocytes, depending on the JAK2V617F mutation status. Additionally, we found that heterodimeric S100A8/9 can inhibit the AKT pathway in MPN-derived granulocytes mediated by the Toll-like receptor 4 (TLR4), depending on the CALR mutation status. Conversely, we found that blocking of the receptor for advanced glycation end products (RAGE) increased the S100A8/9-mediated inhibition of AKT signaling in the MPN

MicroRNA-9 Inhibits NLRP3 Inflammasome Activation in Human Atherosclerosis Inflammation Cell Models through the JAK1/STAT Signaling Pathway.

PubMed

Wang, Yue; Han, Zhihua; Fan, Yuqi; Zhang, Junfeng; Chen, Kan; Gao, Lin; Zeng, Huasu; Cao, Jiatian; Wang, Changqian

2017-01-01

MicroRNA-9 (miR-9) is involved in inflammatory reaction in atherosclerosis; however, its function and regulatory mechanisms remain unclear. We aimed to uncover the exact roles of miR-9 and downstream signaling pathways using in vitro human atherosclerosis models. We used oxidized low-density lipoprotein (oxLDL)-stimulated human THP-1 derived macrophages, oxLDL-stimulated human primary peripheral blood monocytes and lipopolysaccharides (LPS) or Alum-stimulated human THP-1 derived macrophages as in vitro atherosclerosis inflammation models. Transient transfection of over-expression vectors, small interference RNAs (siRNAs) or antisense oligonucleotides was used to regulate intracellular protein or miR-9 levels. Cell responses and signal transduction were detected by multiple assays including Western blotting, enzyme-linked immunosorbent assay (ELISA) and luciferase reporter assay. MiR-9 inhibited while anti-miR-9 antisense oligonucleotides induced interleukin-1 beta (IL-1β) and NLRP3 inflammasome activation in all in vitro models. Janus kinase 1 (JAK1) and matrix metalloproteinase 13 (MMP-13) were identified as the target genes of miR-9. In oxLDL-stimulated human THP-1 derived macrophages, knockdown of JAK1 by siRNA blocked the phosphorylation of signal transducer and activator of transcription 1 (STAT1) and mimicked the effects of miR-9. In the same model, JAK1 knockdown blocked the phosphorylation of NF-κB p65 in the nuclei and the phosphorylation of NF-κB IκBα in the cytoplasm. Our study demonstrated that miR-9 could inhibit activation of the NLRP3 inflammasome and attenuate atherosclerosis-related inflammation, likely through the JAK1/STAT1 signaling pathway. Therefore, miR-9 may serve as a potential therapeutic target for atherosclerosis. © 2017 The Author(s)Published by S. Karger AG, Basel.

miR-204 inhibits angiogenesis and promotes sensitivity to cetuximab in head and neck squamous cell carcinoma cells by blocking JAK2-STAT3 signaling.

PubMed

Wu, Qingwei; Zhao, Yingying; Wang, Peihua

2018-03-01

This study aims to investigate the roles of miR-204 in tumor angiogenesis of head and neck squamous cell carcinoma (HNSCC). Here, we found that miR-204 level was reduced in HNSCC tissues relative to that in normal adjacent tissues. Overexpression of miR-204 promoted tumor angiogenesis in HNSCC cells. Mechanistically, JAK2 was identified as a direct target of miR-204, and miR-204 overexpression blocked JAK2/STAT3 pathway. Moreover, overexpression of JAK2 attenuated the inhibition of miR-204 on tumor angiogenesis of HNSCC. Furthermore, overexpression of miR-204 enhanced sensitivity of cetuximab in HNSCC cells, this effect was attenuated by JAK2 overexpression too. Importantly, JAK2 expression was negatively correlated with miR-204 level in HNSCC tissues. Therefore, miR-204 acts as a tumor suppressor by blocking JAK2/STAT3 pathway in HNSCC cells. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

Proteins mediating DNA loops effectively block transcription.

PubMed

Vörös, Zsuzsanna; Yan, Yan; Kovari, Daniel T; Finzi, Laura; Dunlap, David

2017-07-01

Loops are ubiquitous topological elements formed when proteins simultaneously bind to two noncontiguous DNA sites. While a loop-mediating protein may regulate initiation at a promoter, the presence of the protein at the other site may be an obstacle for RNA polymerases (RNAP) transcribing a different gene. To test whether a DNA loop alters the extent to which a protein blocks transcription, the lac repressor (LacI) was used. The outcome of in vitro transcription along templates containing two LacI operators separated by 400 bp in the presence of LacI concentrations that produced both looped and unlooped molecules was visualized with scanning force microscopy (SFM). An analysis of transcription elongation complexes, moving for 60 s at an average of 10 nt/s on unlooped DNA templates, revealed that they more often surpassed LacI bound to the lower affinity O2 operator than to the highest affinity Os operator. However, this difference was abrogated in looped DNA molecules where LacI became a strong roadblock independently of the affinity of the operator. Recordings of transcription elongation complexes, using magnetic tweezers, confirmed that they halted for several minutes upon encountering a LacI bound to a single operator. The average pause lifetime is compatible with RNAP waiting for LacI dissociation, however, the LacI open conformation visualized in the SFM images also suggests that LacI could straddle RNAP to let it pass. Independently of the mechanism by which RNAP bypasses the LacI roadblock, the data indicate that an obstacle with looped topology more effectively interferes with transcription. © 2017 The Authors Protein Science published by Wiley Periodicals, Inc. on behalf of The Protein Society.

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.

The Small Molecule Inhibitor G6 Significantly Reduces Bone Marrow Fibrosis and the Mutant Burden in a Mouse Model of Jak2-Mediated Myelofibrosis

PubMed Central

Kirabo, Annet; Park, Sung O.; Wamsley, Heather L.; Gali, Meghanath; Baskin, Rebekah; Reinhard, Mary K.; Zhao, Zhizhuang J.; Bisht, Kirpal S.; Keserű, György M.; Cogle, Christopher R.; Sayeski, Peter P.

2013-01-01

Philadelphia chromosome–negative myeloproliferative neoplasms, including polycythemia vera, essential thrombocytosis, and myelofibrosis, are disorders characterized by abnormal hematopoiesis. Among these myeloproliferative neoplasms, myelofibrosis has the most unfavorable prognosis. Furthermore, currently available therapies for myelofibrosis have little to no efficacy in the bone marrow and hence, are palliative. We recently developed a Janus kinase 2 (Jak2) small molecule inhibitor called G6 and found that it exhibits marked efficacy in a xenograft model of Jak2-V617F–mediated hyperplasia and a transgenic mouse model of Jak2-V617F–mediated polycythemia vera/essential thrombocytosis. However, its efficacy in Jak2-mediated myelofibrosis has not previously been examined. Here, we hypothesized that G6 would be efficacious in Jak2-V617F–mediated myelofibrosis. To test this, mice expressing the human Jak2-V617F cDNA under the control of the vav promoter were administered G6 or vehicle control solution, and efficacy was determined by measuring parameters within the peripheral blood, liver, spleen, and bone marrow. We found that G6 significantly reduced extramedullary hematopoiesis in the liver and splenomegaly. In the bone marrow, G6 significantly reduced pathogenic Jak/STAT signaling by 53%, megakaryocytic hyperplasia by 70%, and the Jak2 mutant burden by 68%. Furthermore, G6 significantly improved the myeloid to erythroid ratio and significantly reversed the myelofibrosis. Collectively, these results indicate that G6 is efficacious in Jak2-V617F–mediated myelofibrosis, and given its bone marrow efficacy, it may alter the natural history of this disease. PMID:22796437

The Stilbenoid Tyrosine Kinase Inhibitor, G6, Suppresses Jak2-V617F-mediated Human Pathological Cell Growth in Vitro and in Vivo*

PubMed Central

Kirabo, Annet; Embury, Jennifer; Kiss, Róbert; Polgár, Tímea; Gali, Meghanath; Majumder, Anurima; Bisht, Kirpal S.; Cogle, Christopher R.; Keserű, György M.; Sayeski, Peter P.

2011-01-01

Using structure-based virtual screening, we previously identified a novel stilbenoid inhibitor of Jak2 tyrosine kinase named G6. Here, we hypothesized that G6 suppresses Jak2-V617F-mediated human pathological cell growth in vitro and in vivo. We found that G6 inhibited proliferation of the Jak2-V617F expressing human erythroleukemia (HEL) cell line by promoting marked cell cycle arrest and inducing apoptosis. The G6-dependent increase in apoptosis levels was concomitant with increased caspase 3/7 activity and cleavage of PARP. G6 also selectively inhibited phosphorylation of STAT5, a downstream signaling target of Jak2. Using a mouse model of Jak2-V617F-mediated hyperplasia, we found that G6 significantly decreased the percentage of blast cells in the peripheral blood, reduced splenomegaly, and corrected a pathologically low myeloid to erythroid ratio in the bone marrow by eliminating HEL cell engraftment in this tissue. In addition, drug efficacy correlated with the presence of G6 in the plasma, marrow, and spleen. Collectively, these data demonstrate that the stilbenoid compound, G6, suppresses Jak2-V617F-mediated aberrant cell growth. As such, G6 may be a potential therapeutic lead candidate against Jak2-mediated, human disease. PMID:21127060

Hyperactivation of JAK1 tyrosine kinase induces stepwise, progressive pruritic dermatitis

PubMed Central

Yasuda, Takuwa; Fukada, Toshiyuki; Nishida, Keigo; Nakayama, Manabu; Matsuda, Masashi; Miura, Ikuo; Fukuda, Shinji; Kabashima, Kenji; Nakaoka, Shinji; Bin, Bum-Ho; Kubo, Masato; Hasegawa, Takanori; Ohara, Osamu; Koseki, Haruhiko; Wakana, Shigeharu

2016-01-01

Skin homeostasis is maintained by the continuous proliferation and differentiation of epidermal cells. The skin forms a strong but flexible barrier against microorganisms as well as physical and chemical insults; however, the physiological mechanisms that maintain this barrier are not fully understood. Here, we have described a mutant mouse that spontaneously develops pruritic dermatitis as the result of an initial defect in skin homeostasis that is followed by induction of a Th2-biased immune response. These mice harbor a mutation that results in a single aa substitution in the JAK1 tyrosine kinase that results in hyperactivation, thereby leading to skin serine protease overexpression and disruption of skin barrier function. Accordingly, treatment with an ointment to maintain normal skin barrier function protected mutant mice from dermatitis onset. Pharmacological inhibition of JAK1 also delayed disease onset. Together, these findings indicate that JAK1-mediated signaling cascades in skin regulate the expression of proteases associated with the maintenance of skin barrier function and demonstrate that perturbation of these pathways can lead to the development of spontaneous pruritic dermatitis. PMID:27111231

Hyperactivation of JAK1 tyrosine kinase induces stepwise, progressive pruritic dermatitis.

PubMed

Yasuda, Takuwa; Fukada, Toshiyuki; Nishida, Keigo; Nakayama, Manabu; Matsuda, Masashi; Miura, Ikuo; Dainichi, Teruki; Fukuda, Shinji; Kabashima, Kenji; Nakaoka, Shinji; Bin, Bum-Ho; Kubo, Masato; Ohno, Hiroshi; Hasegawa, Takanori; Ohara, Osamu; Koseki, Haruhiko; Wakana, Shigeharu; Yoshida, Hisahiro

2016-06-01

Skin homeostasis is maintained by the continuous proliferation and differentiation of epidermal cells. The skin forms a strong but flexible barrier against microorganisms as well as physical and chemical insults; however, the physiological mechanisms that maintain this barrier are not fully understood. Here, we have described a mutant mouse that spontaneously develops pruritic dermatitis as the result of an initial defect in skin homeostasis that is followed by induction of a Th2-biased immune response. These mice harbor a mutation that results in a single aa substitution in the JAK1 tyrosine kinase that results in hyperactivation, thereby leading to skin serine protease overexpression and disruption of skin barrier function. Accordingly, treatment with an ointment to maintain normal skin barrier function protected mutant mice from dermatitis onset. Pharmacological inhibition of JAK1 also delayed disease onset. Together, these findings indicate that JAK1-mediated signaling cascades in skin regulate the expression of proteases associated with the maintenance of skin barrier function and demonstrate that perturbation of these pathways can lead to the development of spontaneous pruritic dermatitis.

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.

Calcineurin inhibitors recruit protein kinases JAK2 and JNK, TLR signaling and the UPR to activate NF-κB-mediated inflammatory responses in kidney tubular cells

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

González-Guerrero, Cristian, E-mail: cristian.gonzalez@fjd.es; Ocaña-Salceda, Carlos, E-mail: carlos.ocana@fjd.es; Berzal, Sergio, E-mail: sberzal@fjd.es

The calcineurin inhibitors (CNIs) cyclosporine (CsA) and tacrolimus are key drugs in current immunosuppressive regimes for solid organ transplantation. However, they are nephrotoxic and promote death and profibrotic responses in tubular cells. Moreover, renal inflammation is observed in CNI nephrotoxicity but the mechanisms are poorly understood. We have now studied molecular pathways leading to inflammation elicited by the CNIs in cultured and kidney tubular cells. Both CsA and tacrolimus elicited a proinflammatory response in tubular cells as evidenced by a transcriptomics approach. Transcriptomics also suggested several potential pathways leading to expression of proinflammatory genes. Validation and functional studies disclosed thatmore » in tubular cells, CNIs activated protein kinases such as the JAK2/STAT3 and TAK1/JNK/AP-1 pathways, TLR4/Myd88/IRAK signaling and the Unfolded Protein Response (UPR) to promote NF-κB activation and proinflammatory gene expression. CNIs also activated an Nrf2/HO-1-dependent compensatory response and the Nrf2 activator sulforaphane inhibited JAK2 and JNK activation and inflammation. A murine model of CsA nephrotoxicity corroborated activation of the proinflammatory pathways identified in cell cultures. Human CNIs nephrotoxicity was also associated with NF-κB, STAT3 and IRE1α activation. In conclusion, CNIs recruit several intracellular pathways leading to previously non-described proinflammatory actions in renal tubular cells. Identification of these pathways provides novel clues for therapeutic intervention to limit CNIs nephrotoxicity. - Highlights: • Molecular mechanisms modulating CNI renal inflammation were investigated. • Kinases, immune receptors and ER stress mediate the inflammatory response to CNIs. • Several intracellular pathways activate NF-κB in CNIs-treated tubular cells. • A NF-κB-dependent cytokine profile characterizes CNIs-induced inflammation. • CNI nephrotoxicity was associated to

LOSS OF JAK2 REGULATION VIA VHL-SOCS1 E3 UBIQUITIN HETEROCOMPLEX UNDERLIES CHUVASH POLYCYTHEMIA

PubMed Central

Russell, Ryan C.; Sufan, Roxana I.; Zhou, Bing; Heir, Pardeep; Bunda, Severa; Sybingco, Stephanie S.; Greer, Samantha N.; Roche, Olga; Heathcote, Samuel A.; Chow, Vinca W.K.; Boba, Lukasz M.; Richmond, Terri D.; Hickey, Michele M.; Barber, Dwayne L.; Cheresh, David A.; Simon, M. Celeste; Irwin, Meredith S.; Kim, William Y.; Ohh, Michael

2011-01-01

SUMMARY Chuvash polycythemia (CP) is a rare congenital form of polycythemia caused by homozygous R200W and H191D mutations in the von Hippel-Lindau (VHL) gene whose gene product is the principal negative regulator of hypoxia-inducible factor. However, the molecular mechanisms underlying some of the hallmark features of CP such as hypersensitivity to erythropoietin are unclear. Here, we show that VHL directly binds suppressor of cytokine signalling 1 (SOCS1) to form a heterodimeric E3 ligase that targets phosphorylated (p)JAK2 for ubiquitin-mediated destruction. In contrast, CP-associated VHL mutants have altered affinity for SOCS1 and fail to engage and degrade pJAK2. Systemic administration of a highly selective JAK2 inhibitor, TG101209, reverses the disease phenotype in vhlR200W/R200W knock-in mice, a model that faithfully recapitulates human CP. These results reveal VHL as a SOCS1-cooperative negative regulator of JAK2 and provide compelling biochemical and preclinical evidence for JAK2- targeted therapy in CP patients. PMID:21685897

Antagonistic activities of the immunomodulator and PP2A-activating drug FTY720 (Fingolimod, Gilenya) in Jak2-driven hematologic malignancies

PubMed Central

Oaks, Joshua J.; Santhanam, Ramasamy; Walker, Christopher J.; Roof, Steve; Harb, Jason G.; Ferenchak, Greg; Eisfeld, Ann-Kathrin; Van Brocklyn, James R.; Briesewitz, Roger; Saddoughi, Sahar A.; Nagata, Kyosuke; Bittman, Robert; Caligiuri, Michael A.; Abdel-Wahab, Omar; Levine, Ross; Arlinghaus, Ralph B.; Quintas-Cardama, Alfonso; Goldman, John M.; Apperley, Jane; Reid, Alistair; Milojkovic, Dragana; Ziolo, Mark T.; Marcucci, Guido; Ogretmen, Besim; Neviani, Paolo

2013-01-01

FTY720 (Fingolimod, Gilenya) is a sphingosine analog used as an immunosuppressant in multiple sclerosis patients. FTY720 is also a potent protein phosphatase 2A (PP2A)–activating drug (PAD). PP2A is a tumor suppressor found inactivated in different types of cancer. We show here that PP2A is inactive in polycythemia vera (PV) and other myeloproliferative neoplasms characterized by the expression of the transforming Jak2V617F oncogene. PP2A inactivation occurs in a Jak2V617F dose/kinase-dependent manner through the PI-3Kγ-PKC–induced phosphorylation of the PP2A inhibitor SET. Genetic or PAD-mediated PP2A reactivation induces Jak2V617F inactivation/downregulation and impairs clonogenic potential of Jak2V617F cell lines and PV but not normal CD34+ progenitors. Likewise, FTY720 decreases leukemic allelic burden, reduces splenomegaly, and significantly increases survival of Jak2V617F leukemic mice without adverse effects. Mechanistically, we show that in Jak2V617F cells, FTY720 antileukemic activity requires neither FTY720 phosphorylation (FTY720-P) nor SET dimerization or ceramide induction but depends on interaction with SET K209. Moreover, we show that Jak2V617F also utilizes an alternative sphingosine kinase-1–mediated pathway to inhibit PP2A and that FTY720-P, acting as a sphingosine-1-phosphate-receptor-1 agonist, elicits signals leading to the Jak2-PI-3Kγ-PKC-SET–mediated PP2A inhibition. Thus, PADs (eg, FTY720) represent suitable therapeutic alternatives for Jak2V617F MPNs. PMID:23926298

JAK2 mediates lung fibrosis, pulmonary vascular remodelling and hypertension in idiopathic pulmonary fibrosis: an experimental study.

PubMed

Milara, Javier; Ballester, Beatriz; Morell, Anselm; Ortiz, José L; Escrivá, Juan; Fernández, Estrella; Perez-Vizcaino, Francisco; Cogolludo, Angel; Pastor, Enrique; Artigues, Enrique; Morcillo, Esteban; Cortijo, Julio

2018-06-01

Pulmonary hypertension (PH) is a common disorder in patients with idiopathic pulmonary fibrosis (IPF) and portends a poor prognosis. Recent studies using vasodilators approved for PH have failed in improving IPF mainly due to ventilation ( V )/perfusion ( Q ) mismatching and oxygen desaturation. Janus kinase type 2 (JAK2) is a non-receptor tyrosine kinase activated by a broad spectrum of profibrotic and vasoactive mediators, but its role in PH associated to PH is unknown. The study of JAK2 as potential target to treat PH in IPF. JAK2 expression was increased in pulmonary arteries (PAs) from IPF (n=10; 1.93-fold; P=0.0011) and IPF+PH (n=9; 2.65-fold; P<0.0001) compared with PA from control subjects (n=10). PA remodelling was evaluated in human pulmonary artery endothelial cells (HPAECs) and human pulmonary artery smooth muscle cells (HPASMCs) from patients with IPF in vitro treated with the JAK2 inhibitor JSI-124 or siRNA-JAK2 and stimulated with transforming growth factor beta. Both JSI-124 and siRNA-JAK2 inhibited the HPAEC to mesenchymal transition and the HPASMCs to myofibroblast transition and proliferation. JAK2 inhibition induced small PA relaxation in precision-cut lung slice experiments. PA relaxation was dependent of the large conductance calcium-activated potassium channel (BK Ca ). JAK2 inhibition activated BK Ca channels and reduced intracellular Ca 2+ . JSI-124 1 mg/kg/day, reduced bleomycin-induced lung fibrosis, PA remodelling, right ventricular hypertrophy, PA hypertension and V / Q mismatching in rats. The animal studies followed the ARRIVE guidelines. JAK2 participates in PA remodelling and tension and may be an attractive target to treat IPF associated to PH. © Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2018. All rights reserved. No commercial use is permitted unless otherwise expressly granted.

Roles of germline JAK2 activation mutation JAK2 V625F in the pathology of myeloproliferative neoplasms.

PubMed

Wu, Qing-Yun; Ma, Meng-Meng; Fu, Lin; Zhu, Yuan-Yuan; Liu, Yang; Cao, Jiang; Zhou, Ping; Li, Zhen-Yu; Zeng, Ling-Yu; Li, Feng; Wang, Xiao-Yun; Xu, Kai-Lin

2018-05-18

Janus tyrosine kinase 2 (JAK2) mediates downstream signaling of cytokine receptors in all hematological lineages, constitutively active somatic JAK2 mutations play key roles in the pathology of myeloproliferative neoplasms (MPNs). Recently, germline JAK2 mutations are also associated with triple-negative MPNs. A novel germline mutation JAK2 V625F is reported to be involved in a subset of MPNs patients. However, the pathogenesis of this mutation caused MPN is still unclear. In this study, the homology models of JAK2 V625F showed that the newly formed interaction between F625 and Y613 disrupted the JAK2 JH1-JH2 domain interactions was responsible for its activation, when F625 and Y613 interaction was disrupted, its activity significantly decreased. While, when this interaction was repaired whether by forming hydrogen bond or salt bond, it would cause JAK2 activation. Biochemical studies also demonstrated that JAK2 V625F mutation led to JAK2-STAT5 pathway activation and promoted the proliferation of BaF3 cells. Thus, our results herein provide clues to understand the mechanism JAK2 V625F mutation caused MPNs and give information for the development of JAK2 mutation specific inhibitors. Copyright © 2018 Elsevier B.V. All rights reserved.

Sequential activation of JAKs, STATs and xanthine dehydrogenase/oxidase by hypoxia in lung microvascular endothelial cells.

PubMed

Wang, Guansong; Qian, Pin; Jackson, Fannie R; Qian, Guisheng; Wu, Guangyu

2008-01-01

Xanthine dehydrogenase/oxidase (XDH/XO) is associated with various pathological conditions related to the endothelial injury. However, the molecular mechanism underlying the activation of XDH/XO by hypoxia remains largely unknown. In this report, we determined whether the Janus kinases (JAKs) and signal transducers and activators of transcription (STATs) signaling pathway is involved in hypoxia-induced activation of XDH/XO in primary cultures of lung microvascular endothelial cells (LMVEC). We found that hypoxia significantly increased interleukin 6 (IL6) production in a time-dependent manner in LMVEC. Hypoxia also markedly augmented phosphorylation/activation of JAKs (JAK1, JAK2 and JAK3) and the JAK downstream effectors STATs (STAT3 and STAT5). Hypoxia-induced activation of STAT3 was blocked by IL6 antibodies, the JAK inhibitor AG490 and the suppressor of cytokine signaling 3 (SOCS3), implying that hypoxia-promoted IL6 secretion activates the JAK/STAT pathway in LMVEC. Phosphorylation and DNA-binding activity of STAT3 were also inhibited by the p38 MAPK inhibitor SB203580 and the phosphatidylinositol 3-kinase inhibitor LY294002, suggesting that multiple signaling pathways involved in STAT activation by hypoxia. Importantly, hypoxia promoted XDH/XO activation in LMVEC, which was markedly reversed by inhibiting the JAK-STAT pathway using IL6 antibodies, AG490 and SOCS3. These data demonstrated that JAKs, STATs and XDH/XO were sequentially activated by hypoxia. These data provide the first evidence indicating that the JAK-STAT pathway is involved in hypoxia-mediated XDH/XO activation in LMVEC.

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

Oncogenic activation of JAK3-STAT signaling confers clinical sensitivity to PRN371, a novel selective and potent JAK3 inhibitor, in natural killer/T-cell lymphoma.

PubMed

Nairismägi, M -L; Gerritsen, M E; Li, Z M; Wijaya, G C; Chia, B K H; Laurensia, Y; Lim, J Q; Yeoh, K W; Yao, X S; Pang, W L; Bisconte, A; Hill, R J; Bradshaw, J M; Huang, D; Song, T L L; Ng, C C Y; Rajasegaran, V; Tang, T; Tang, Q Q; Xia, X J; Kang, T B; Teh, B T; Lim, S T; Ong, C K; Tan, J

2018-05-01

Aberrant activation of the JAK3-STAT signaling pathway is a characteristic feature of many hematological malignancies. In particular, hyperactivity of this cascade has been observed in natural killer/T-cell lymphoma (NKTL) cases. Although the first-in-class JAK3 inhibitor tofacitinib blocks JAK3 activity in NKTL both in vitro and in vivo, its clinical utilization in cancer therapy has been limited by the pan-JAK inhibition activity. To improve the therapeutic efficacy of JAK3 inhibition in NKTL, we have developed a highly selective and durable JAK3 inhibitor PRN371 that potently inhibits JAK3 activity over the other JAK family members JAK1, JAK2, and TYK2. PRN371 effectively suppresses NKTL cell proliferation and induces apoptosis through abrogation of the JAK3-STAT signaling. Moreover, the activity of PRN371 has a more durable inhibition on JAK3 compared to tofacitinib in vitro, leading to significant tumor growth inhibition in a NKTL xenograft model harboring JAK3 activating mutation. These findings provide a novel therapeutic approach for the treatment of NKTL.

Secretion pathway of liver IGF-1 via JAK2/STAT3 in chick embryo under the monochromatic light.

PubMed

Wang, Tuanjie; Dong, Yulan; Wang, Zixu; Cao, Jing; Chen, Yaoxing

2016-02-01

This study reveals mechanism of monochromatic light on the IGF-1 secretion of chick embryo liver. The chick embryos were incubated and exposed to continuous red, green, blue light or a dark environment. Compared to other light-treated groups, green light increased IGF-1 and melatonin concentrations both in plasma and liver, and Mel1a, Mel1b and Mel1c receptors expressions in liver but decreased p-JAK2, p-STAT3 and ROS in liver. IGF-1 had a positive correlation with melatonin, but a negative relevance with p-JAK2 and p-STAT3. In vitro, the IGF-1 level in the hepatocyte supernatant was enhanced by melatonin with lower p-JAK2/p-STAT3 and ROS levels, which was suppressed by Mel1c antagonist but not Mel1a/Mel1b or Mel1b antagonists. AG490 (JAK/STAT inhibitor) promoted role of melatonin-Mel1c modulated IGF-1 secretion. These results suggest the antioxidant effect of melatonin mediated the green light-enhanced IGF-1 secretion of chick embryo liver through Mel1c receptor to inhibit the JAK2/STAT3 pathway.

Inhibition of TYK2 and JAK1 Ameliorates Imiquimod-Induced Psoriasis-like Dermatitis by Inhibiting IL-22 and the IL-23/IL-17 axis

PubMed Central

Works, Melissa G.; Yin, Fangfang; Yin, Catherine C.; Yiu, Ying; Shew, Kenneth; Tran, Thanh-Thuy; Dunlap, Nahoko; Lam, Jennifer; Mitchell, Tim; Reader, John; Stein, Paul L.; D’Andrea, Annalisa

2014-01-01

Psoriasis is a chronic autoimmune disease affecting the skin and characterized by aberrant keratinocyte proliferation and function. Immune cells infiltrate the skin and release proinflammatory cytokines that play important roles in psoriasis. The Th17 network, including IL-23 and IL-22, has recently emerged as a critical component in the pathogenesis of psoriasis. IL-22 and IL-23 signaling is dependent on the JAK family of protein tyrosine kinases, making Janus kinase (JAK) inhibition an appealing strategy for the treatment of psoriasis. Here we report the activity of SAR-20347, a small molecule inhibitor with specificity for JAK1 and Tyrosine Kinase 2 (TYK2) over other JAK family members. In cellular assays, SAR-20347 dose-dependently (1 nM-10 μM) inhibited JAK1 and/or TYK2 dependent signaling from the IL-12/IL-23, IL-22, and IFN-α receptors. In vivo, TYK2 mutant mice or treatment of wild type mice with SAR-20347 significantly reduced IL-12 induced IFN-γ production and IL-22-dependent Serum Amyloid A (SAA) to similar extents, indicating that in these models, SAR-20347 is probably acting through inhibition of TYK2. In an imiquimod-induced psoriasis model, the administration of SAR-20347 led to a striking decrease in disease pathology, including reduced activation of keratinocytes, and proinflammatory cytokine levels compared to both TYK2 mutant mice and wild type controls. Taken together, these data indicate that targeting both JAK1 and TYK2-mediated cytokine signaling is more effective than TYK2 inhibition alone in reducing psoriasis pathogenesis. PMID:25156366

Selective targeting of JAK/STAT signaling is potentiated by Bcl-xL blockade in IL-2–dependent adult T-cell leukemia

PubMed Central

Zhang, Meili; Mathews Griner, Lesley A.; Ju, Wei; Duveau, Damien Y.; Guha, Rajarshi; Petrus, Michael N.; Wen, Bernard; Maeda, Michiyuki; Shinn, Paul; Ferrer, Marc; Conlon, Kevin D.; Bamford, Richard N.; O’Shea, John J.; Thomas, Craig J.; Waldmann, Thomas A.

2015-01-01

Adult T-cell leukemia (ATL) develops in individuals infected with human T-cell lymphotropic virus-1 (HTLV-1). Presently there is no curative therapy for ATL. HTLV-1–encoded protein Tax (transactivator from the X-gene region) up-regulates Bcl-xL (B-cell lymphoma-extra large) expression and activates interleukin-2 (IL-2), IL-9, and IL-15 autocrine/paracrine systems, resulting in amplified JAK/STAT signaling. Inhibition of JAK signaling reduces cytokine-dependent ex vivo proliferation of peripheral blood mononuclear cells (PBMCs) from ATL patients in smoldering/chronic stages. Currently, two JAK inhibitors are approved for human use. In this study, we examined activity of multiple JAK inhibitors in ATL cell lines. The selective JAK inhibitor ruxolitinib was examined in a high-throughput matrix screen combined with >450 potential therapeutic agents, and Bcl-2/Bcl-xL inhibitor navitoclax was identified as a strong candidate for multicomponent therapy. The combination was noted to strongly activate BAX (Bcl-2-associated X protein), effect mitochondrial depolarization, and increase caspase 3/7 activities that lead to cleavage of PARP (poly ADP ribose polymerase) and Mcl-1 (myeloid cell leukemia 1). Ruxolitinib and navitoclax independently demonstrated modest antitumor efficacy, whereas the combination dramatically lowered tumor burden and prolonged survival in an ATL murine model. This combination strongly blocked ex vivo proliferation of five ATL patients’ PBMCs. These studies provide support for a therapeutic trial in patients with smoldering/chronic ATL using a drug combination that inhibits JAK signaling and antiapoptotic protein Bcl-xL. PMID:26396258

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

The role of JAK1/2 inhibitors in the treatment of chronic myeloproliferative neoplasms.

PubMed

Keohane, Clodagh; Mesa, Ruben; Harrison, Claire

2013-01-01

In 2005, the description of the JAK2V617F mutation for the first time provided a molecular key to enable more rapid diagnosis and target for novel therapeutics in the myeloproliferative neoplasms. In 2007, the first-in-class agent INC18424, ruxolitinib, JAKafi, or JAKAVI was first tested in patients with intermediate-risk 2 or high-risk myelofibrosis regardless of whether they possessed the JAK2V617F mutation. Patients treated with this agent had major reduction in splenomegaly as well as impressive reduction, and in some cases resolution, of symptoms. This study was followed by the two Controlled Myelofibrosis Study with Oral JAK Inhibitor Therapy (COMFORT) trials (the first-ever phase III trials in myelofibrosis), which confirmed results in these aspects were superior to either placebo or standard care, and updated results show a survival advantage with this therapy. This paper discusses these results and data from other JAK inhibitors while speculating on the future of these therapies. It also reflects on the fact that the true targets and agents' mode of action are uncertain. Unlike targeted therapy for chronic myeloid leukemia (CML), these agents do not deliver molecular remission, and it is not clear whether their predominant benefit is mediated via JAK2, JAK1, or both. Nonetheless, the advent of the JAK inhibitor is a welcome advance and has made a dramatic improvement to the therapeutic landscape of these conditions.

Molecular drug targets in myeloproliferative neoplasms: mutant ABL1, JAK2, MPL, KIT, PDGFRA, PDGFRB and FGFR1

PubMed Central

Tefferi, Ayalew

2009-01-01

Abstract Therapeutically validated oncoproteins in myeloproliferative neoplasms (MPN) include BCR-ABL1 and rearranged PDGFR proteins. The latter are products of intra- (e.g. FIP1L1-PDGFRA) or inter-chromosomal (e.g.ETV6-PDGFRB) gene fusions. BCR-ABL1 is associated with chronic myelogenous leukaemia (CML) and mutant PDGFR with an MPN phenotype characterized by eosinophilia and in addition, in case of FIP1L1-PDGFRA, bone marrow mastocytosis. These genotype-phenotype associations have been effectively exploited in the development of highly accurate diagnostic assays and molecular targeted therapy. It is hoped that the same will happen in other MPN with specific genetic alterations: polycythemia vera (JAK2V617F and other JAK2 mutations), essential thrombocythemia (JAK2V617F and MPL515 mutations), primary myelofibrosis (JAK2V617F and MPL515 mutations), systemic mastocytosis (KITD816V and other KIT mutations) and stem cell leukaemia/lymphoma (ZNF198-FGFR1 and other FGFR1 fusion genes). The current review discusses the above-listed mutant molecules in the context of their value as drug targets. PMID:19175693

IL-15 Activates the Jak3/STAT3 Signaling Pathway to Mediate Glucose Uptake in Skeletal Muscle Cells

PubMed Central

Krolopp, James E.; Thornton, Shantaé M.; Abbott, Marcia J.

2016-01-01

Myokines are specialized cytokines that are secreted from skeletal muscle (SKM) in response to metabolic stimuli, such as exercise. Interleukin-15 (IL-15) is a myokine with potential to reduce obesity and increase lean mass through induction of metabolic processes. It has been previously shown that IL-15 acts to increase glucose uptake in SKM cells. However, the downstream signals orchestrating the link between IL-15 signaling and glucose uptake have not been fully explored. Here we employed the mouse SKM C2C12 cell line to examine potential downstream targets of IL-15-induced alterations in glucose uptake. Following differentiation, C2C12 cells were treated overnight with 100 ng/ml of IL-15. Activation of factors associated with glucose metabolism (Akt and AMPK) and known downstream targets of IL-15 (Jak1, Jak3, STAT3, and STAT5) were assessed with IL-15 stimulation. IL-15 stimulated glucose uptake and GLUT4 translocation to the plasma membrane. IL-15 treatment had no effect on phospho-Akt, phospho-Akt substrates, phospho-AMPK, phospho-Jak1, or phospho-STAT5. However, with IL-15, phospho-Jak3 and phospho-STAT3 levels were increased along with increased interaction of Jak3 and STAT3. Additionally, IL-15 induced a translocation of phospho-STAT3 from the cytoplasm to the nucleus. We have evidence that a mediator of glucose uptake, HIF1α, expression was dependent on IL-15 induced STAT3 activation. Finally, upon inhibition of STAT3 the positive effects of IL-15 on glucose uptake and GLUT4 translocation were abolished. Taken together, we provide evidence for a novel signaling pathway for IL-15 acting through Jak3/STAT3 to regulate glucose metabolism. PMID:28066259

Triazolopyridines as selective JAK1 inhibitors: from hit identification to GLPG0634.

PubMed

Menet, Christel J; Fletcher, Stephen R; Van Lommen, Guy; Geney, Raphael; Blanc, Javier; Smits, Koen; Jouannigot, Nolwenn; Deprez, Pierre; van der Aar, Ellen M; Clement-Lacroix, Philippe; Lepescheux, Liên; Galien, René; Vayssiere, Béatrice; Nelles, Luc; Christophe, Thierry; Brys, Reginald; Uhring, Muriel; Ciesielski, Fabrice; Van Rompaey, Luc

2014-11-26

Janus kinases (JAK1, JAK2, JAK3, and TYK2) are involved in the signaling of multiple cytokines important in cellular function. Blockade of the JAK-STAT pathway with a small molecule has been shown to provide therapeutic immunomodulation. Having identified JAK1 as a possible new target for arthritis at Galapagos, the compound library was screened against JAK1, resulting in the identification of a triazolopyridine-based series of inhibitors represented by 3. Optimization within this chemical series led to identification of GLPG0634 (65, filgotinib), a selective JAK1 inhibitor currently in phase 2B development for RA and phase 2A development for Crohn's disease (CD).

Gα73B is a downstream effector of JAK/STAT signalling and a regulator of Rho1 in Drosophila haematopoiesis.

PubMed

Bausek, Nina; Zeidler, Martin P

2014-01-01

JAK/STAT signalling regulates many essential developmental processes including cell proliferation and haematopoiesis, whereas its inappropriate activation is associated with the majority of myeloproliferative neoplasias and numerous cancers. Furthermore, high levels of JAK/STAT pathway signalling have also been associated with enhanced metastatic invasion by cancerous cells. Strikingly, gain-of-function mutations in the single Drosophila JAK homologue, Hopscotch, result in haemocyte neoplasia, inappropriate differentiation and the formation of melanised haemocyte-derived 'tumour' masses; phenotypes that are partly orthologous to human gain-of-function JAK2-associated pathologies. Here we show that Gα73B, a novel JAK/STAT pathway target gene, is necessary for JAK/STAT-mediated tumour formation in flies. In addition, although Gα73B does not affect haemocyte differentiation, it does regulate haemocyte morphology and motility under non-pathological conditions. We show that Gα73B is required for constitutive, but not injury-induced, activation of Rho1 and for the localisation of Rho1 into filopodia upon haemocyte activation. Consistent with these results, we also show that Rho1 interacts genetically with JAK/STAT signalling, and that wild-type levels of Rho1 are necessary for tumour formation. Our findings link JAK/STAT transcriptional outputs, Gα73B activity and Rho1-dependent cytoskeletal rearrangements and cell motility, therefore connecting a pathway associated with cancer with a marker indicative of invasiveness. As such, we suggest a mechanism by which JAK/STAT pathway signalling may promote metastasis.

JAK2 46/1 haplotype is associated with JAK2 V617F--positive myeloproliferative neoplasms in Brazilian patients.

PubMed

Macedo, L C; Santos, B C; Pagliarini-e-Silva, S; Pagnano, K B B; Rodrigues, C; Quintero, F C; Ferreira, M E; Baraldi, E C; Ambrosio-Albuquerque, E P; Sell, A M; Visentainer, J E L

2015-10-01

This study aimed to verify the association between the JAK2 46/1 haplotype (V617F positive) and some hematological parameters in BCR-ABL-negative chronic myeloproliferative neoplasms (cMPNs) in our population. The blood samples obtained from the patients with cMPN were genotyped for the JAK2 V617F mutation and JAK2 rs10974944 SNP screening using a PCR-RFLP assay. The JAK2 V617F mutation was detected in 80.15% of patients. The G variant of rs10974944 was more frequent in all MPNs, especially those that were JAK2 V617F positive, than in the control population. We also compared the 46/1 haplotype status in each MPN disease entity, polycythemia vera (PV), essential thrombocythemia (ET), primary myelofibrosis (PMF), and MPNu with controls. The G allele frequency relative to controls was significantly enriched in patients with PV and ET, but not in those with PMF and MPNu. PV and ET patients especially, all of whom had the JAK2 V617F mutation, showed significant excess of the G allele. The frequency of JAK2 V617F mutation was associated with elevated hematological parameters, but when we analyze the occurrence of the mutation and the presence of the G allele, just the high hemoglobin was significantly. In agreement with previous reports, JAK2 46/1 haplotype for JAK2 V617F was associated with cMPN positive in Brazilian patients. © 2015 John Wiley & Sons Ltd.

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

SH2-B promotes insulin receptor substrate 1 (IRS1)- and IRS2-mediated activation of the phosphatidylinositol 3-kinase pathway in response to leptin.

PubMed

Duan, Chaojun; Li, Minghua; Rui, Liangyou

2004-10-15

Leptin regulates energy homeostasis primarily by binding and activating its long form receptor (LRb). Deficiency of either leptin or LRb causes morbid obesity. Leptin stimulates LRb-associated JAK2, thus initiating multiple pathways including the Stat3 and phosphatidylinositol (PI) 3-kinase pathways that mediate leptin biological actions. Here we report that SH2-B, a JAK2-interacting protein, promotes activation of the PI 3-kinase pathway by recruiting insulin receptor substrate 1 (IRS1) and IRS2 in response to leptin. SH2-B directly bound, via its PH and SH2 domain, to both IRS1 and IRS2 both in vitro and in intact cells and mediated formation of a JAK2/SH2-B/IRS1 or IRS2 tertiary complex. Consequently, SH2-B dramatically enhanced leptin-stimulated tyrosine phosphorylation of IRS1 and IRS2 in HEK293 cells stably expressing LRb, thus promoting association of IRS1 and IRS2 with the p85 regulatory subunit of PI 3-kinase and phosphorylation and activation of Akt. SH2-B mutants with lower affinity for IRS1 and IRS2 exhibited reduced ability to promote association of JAK2 with IRS1, tyrosine phosphorylation of IRS1, and association of IRS1 with p85 in response to leptin. Moreover, deletion of the SH2-B gene impaired leptin-stimulated tyrosine phosphorylation of endogenous IRS1 in mouse embryonic fibroblasts (MEF), which was reversed by reintroduction of SH2-B. Similarly, SH2-B promoted growth hormone-stimulated tyrosine phosphorylation of IRS1 in both HEK293 and MEF cells. Our data suggest that SH2-B is a novel mediator of the PI 3-kinase pathway in response to leptin or other hormones and cytokines that activate JAK2.

Cell type-specific roles of Jak3 in IL-2-induced proliferative signal transduction

PubMed Central

Fujii, Hodaka

2007-01-01

Binding of IL-2 to its specific receptor induces activation of two members of Jak family protein tyrosine kinases, Jak1 and Jak3. An IL-2R-reconstituted NIH 3T3 fibroblast cell line proliferates in response to IL-2 only when hematopoietic lineage-specific Jak3 is ectopically expressed. However, the mechanism of Jak3-dependent proliferation in the fibroblast cell line is not known. Here, I showed that Jak3 expression is dispensable for IL-2-induced activation of Jak1 and Stat proteins and expression of nuclear proto-oncogenes in the IL-2R-reconstituted fibroblast cell line. However, Jak3 expression markedly enhanced these IL-2-induced signaling events. In contrast, Jak3 expression was essential for induction of cyclin genes involved in the G1-S transition. These data suggest a critical role of Jak3 in IL-2 signaling in the fibroblast cell line and may provide further insight into the cell type-specific mechanism of cytokine signaling. PMID:17266928

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.

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

Cryptochromes regulate IGF-1 production and signaling through control of JAK2-dependent STAT5B phosphorylation

PubMed Central

Chaudhari, Amol; Gupta, Richa; Patel, Sonal; Velingkaar, Nikkhil; Kondratov, Roman

2017-01-01

Insulin-like growth factor (IGF) signaling plays an important role in cell growth and proliferation and is implicated in regulation of cancer, metabolism, and aging. Here we report that IGF-1 level in blood and IGF-1 signaling demonstrates circadian rhythms. Circadian control occurs through cryptochromes (CRYs)—transcriptional repressors and components of the circadian clock. IGF-1 rhythms are disrupted in Cry-deficient mice, and IGF-1 level is reduced by 80% in these mice, which leads to reduced IGF signaling. In agreement, Cry-deficient mice have reduced body (∼30% reduction) and organ size. Down-regulation of IGF-1 upon Cry deficiency correlates with reduced Igf-1 mRNA expression in the liver and skeletal muscles. Igf-1 transcription is regulated through growth hormone–induced, JAK2 kinase–mediated phosphorylation of transcriptional factor STAT5B. The phosphorylation of STAT5B on the JAK2-dependent Y699 site is significantly reduced in the liver and skeletal muscles of Cry-deficient mice. At the same time, phosphorylation of JAK2 kinase was not reduced upon Cry deficiency, which places CRY activity downstream from JAK2. Thus CRYs link the circadian clock and JAK-STAT signaling through control of STAT5B phosphorylation, which provides the mechanism for circadian rhythms in IGF signaling in vivo. PMID:28100634

The JAK inhibitor tofacitinib suppresses synovial JAK1-STAT signalling in rheumatoid arthritis

PubMed Central

Boyle, D L; Soma, K; Hodge, J; Kavanaugh, A; Mandel, D; Mease, P; Shurmur, R; Singhal, A K; Wei, N; Rosengren, S; Kaplan, I; Krishnaswami, S; Luo, Z; Bradley, J; Firestein, G S

2015-01-01

Objective Tofacitinib is an oral Janus kinase (JAK) inhibitor for the treatment of rheumatoid arthritis (RA). The pathways affected by tofacitinib and the effects on gene expression in situ are unknown. Therefore, tofacitinib effects on synovial pathobiology were investigated. Methods A randomised, double-blind, phase II serial synovial biopsy study (A3921073; NCT00976599) in patients with RA with an inadequate methotrexate response. Patients on background methotrexate received tofacitinib 10 mg twice daily or placebo for 28 days. Synovial biopsies were performed on Days -7 and 28 and analysed by immunoassay or quantitative PCR. Clinical response was determined by disease activity score and European League Against Rheumatism (EULAR) response on Day 28 in A3921073, and at Month 3 in a long-term extension study (A3921024; NCT00413699). Results Tofacitinib exposure led to EULAR moderate to good responses (11/14 patients), while placebo was ineffective (1/14 patients) on Day 28. Tofacitinib treatment significantly reduced synovial mRNA expression of matrix metalloproteinase (MMP)-1 and MMP-3 (p<0.05) and chemokines CCL2, CXCL10 and CXCL13 (p<0.05). No overall changes were observed in synovial inflammation score or the presence of T cells, B cells or macrophages. Changes in synovial phosphorylation of signal transducer and activator of transcription 1 (STAT1) and STAT3 strongly correlated with 4-month clinical responses (p<0.002). Tofacitinib significantly decreased plasma CXCL10 (p<0.005) at Day 28 compared with placebo. Conclusions Tofacitinib reduces metalloproteinase and interferon-regulated gene expression in rheumatoid synovium, and clinical improvement correlates with reductions in STAT1 and STAT3 phosphorylation. JAK1-mediated interferon and interleukin-6 signalling likely play a key role in the synovial response. Trial registration number NCT00976599. PMID:25398374

Resolution of bone marrow fibrosis in a patient receiving JAK1/JAK2 inhibitor treatment with ruxolitinib.

PubMed

Wilkins, Bridget S; Radia, Deepti; Woodley, Claire; Farhi, Sarah El; Keohane, Clodagh; Harrison, Claire N

2013-12-01

Ruxolitinib, a JAK1/JAK2 inhibitor, is currently the only pharmacological agent approved for the treatment of myelofibrosis. Approval was based on findings from two phase 3 trials comparing ruxolitinib with placebo (COMFORT-I) and with best available therapy (COMFORT-II) for the treatment of primary or secondary myelofibrosis. In those pivotal trials, ruxolitinib rapidly improved splenomegaly, disease-related symptoms, and quality of life and prolonged survival compared with both placebo and conventional treatments. However, for reasons that are currently unclear, there were only modest histomorphological changes in the bone marrow, and only a subset of patients had significant reductions in JAK2 V617F clonal burden. Here we describe a patient with post-polycythemia vera myelofibrosis who received ruxolitinib at our institution (Guy's and St. Thomas' NHS Foundation Trust, London, United Kingdom) as part of the COMFORT-II study. While on treatment, the patient had dramatic improvements in splenomegaly and symptoms shortly after starting ruxolitinib. With longer treatment, the patient had marked reductions in JAK2 V617F allele burden, and fibrosis of the bone marrow resolved after approximately 3 years of ruxolitinib treatment. To our knowledge, this is the first detailed case report of resolution of fibrosis with a JAK1/JAK2 inhibitor. ClinicalTrials.gov Identifier: NCT00934544.

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

JAK/STAT autocontrol of ligand-producing cell number through apoptosis.

PubMed

Borensztejn, Antoine; Boissoneau, Elisabeth; Fernandez, Guillaume; Agnès, François; Pret, Anne-Marie

2013-01-01

During development, specific cells are eliminated by apoptosis to ensure that the correct number of cells is integrated in a given tissue or structure. How the apoptosis machinery is activated selectively in vivo in the context of a developing tissue is still poorly understood. In the Drosophila ovary, specialised follicle cells [polar cells (PCs)] are produced in excess during early oogenesis and reduced by apoptosis to exactly two cells per follicle extremity. PCs act as an organising centre during follicle maturation as they are the only source of the JAK/STAT pathway ligand Unpaired (Upd), the morphogen activity of which instructs distinct follicle cell fates. Here we show that reduction of Upd levels leads to prolonged survival of supernumerary PCs, downregulation of the pro-apoptotic factor Hid, upregulation of the anti-apoptotic factor Diap1 and inhibition of caspase activity. Upd-mediated activation of the JAK/STAT pathway occurs in PCs themselves, as well as in adjacent terminal follicle and interfollicular stalk cells, and inhibition of JAK/STAT signalling in any one of these cell populations protects PCs from apoptosis. Thus, a Stat-dependent unidentified relay signal is necessary for inducing supernumerary PC death. Finally, blocking apoptosis of PCs leads to specification of excess adjacent border cells via excessive Upd signalling. Our results therefore show that Upd and JAK/STAT signalling induce apoptosis of supernumerary PCs to control the size of the PC organising centre and thereby produce appropriate levels of Upd. This is the first example linking this highly conserved signalling pathway with developmental apoptosis in Drosophila.

Type I/II cytokines, JAKs, and new strategies for treating autoimmune diseases

PubMed Central

Schwartz, Daniella M.; Bonelli, Michael; Gadina, Massimo; O’Shea, John J.

2015-01-01

Cytokines are major drivers of autoimmunity, and biologic agents targeting cytokines have revolutionized the treatment of immune-mediated diseases. Despite the effectiveness of these drugs, they do not induce complete remission in all patients, prompting the development of alternative strategies—including targeting of intracellular signal transduction pathways downstream of cytokines. Many cytokines that bind type I and type II cytokine receptors are critical regulators of immune-mediated diseases and employ the Janus kinase (JAK) and signal transducer and activator of transcription (STAT) pathway to exert their effect. Pharmacological inhibition of JAKs block the actions of type I/II cytokines, and within the past 3 years therapeutic JAK inhibitors, or Jakinibs, have become available to rheumatologists. Jakinibs have proven effective for the treatment of rheumatoid arthritis and other inflammatory diseases. Adverse effects of these agents are largely related to their mode of action and include infections and hyperlipidemia. Jakinibs are currently being investigated for a number of new indications, and second-generation selective Jakinibs are being developed and tested. Targeting STATs could be a future avenue for the treatment of rheumatic diseases, although substantial challenges remain. Nonetheless, the ability to therapeutically target intracellular signalling pathways has already created a new paradigm for the treatment of rheumatologic disease. PMID:26633291

Identification of Tyrosine 972 as a Novel Site of Jak2 Tyrosine Kinase Phosphorylation and its Role in Jak2 Activation

PubMed Central

McDoom, Issam; Ma, Xianyue; Kirabo, Annet; Lee, Kuang-Yung; Ostrov, David A.; Sayeski, Peter P.

2013-01-01

Jak2 is a 130 kDa tyrosine kinase that is important in a number of cellular signaling pathways. Its function is intrinsically regulated by the phosphorylation of a handful of its 49 tyrosines. Here, we report that tyrosine 972 (Y972) is a novel site of Jak2 phosphorylation, and hence auto-regulation. Specifically, we found that Y972 is phosphorylated and confirmed that this residue resides on the surface of the protein. Using expression plasmids that expressed either wild type Jak2 or a full length Jak2 cDNA containing a single Y972F substitution mutation, we investigated the consequences of losing Y972 phosphorylation on Jak2 function. We determined that the loss of Y972 phosphorylation significantly reduced both Jak2 total tyrosine phosphorylation and phosphorylation of Y1007/Y1008. Additionally, Y972 phosphorylation was shown to be important for maximal kinase function. Interestingly, in response to classical cytokine activation, the Jak2-Y972F mutant exhibited a moderately impaired level of activation when compared to wild type protein. However, when Jak2 was activated via a GPCR ligand, the ability of the Y972F mutant to activate was completely lost, therefore suggesting a differential role of Y972 in Jak2 activation. Finally, we found that phosphorylation of Y972 enhances Jak2 kinase function via a mechanism that appears to stabilize the active conformation of the protein. Collectively, our results suggest that Y972 is a novel site of Jak2 phosphorylation and plays an important differential role in ligand-dependent Jak2 activation via a mechanism that involves stabilization of the Jak2 active conformation. PMID:18636744

Regulation of apoptosis by resveratrol through JAK/STAT and mitochondria mediated pathway in human epidermoid carcinoma A431 cells

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

Madan, Esha; Prasad, Sahdeo; Roy, Preeti

2008-12-26

Resveratrol (trans-3,4',5-trihydroxystilbene), a polyphenolic phytoalexin present mainly in grapes, red wine and berries, is known to possess strong chemopreventive and anticancer properties. Here, we demonstrated the anti-proliferative and apoptosis-inducing activities of resveratrol in human epidermoid carcinoma A431 cells. Resveratrol has cytotoxic effects through inhibiting cellular proliferation of A431 cells, which leads to the induction of apoptosis, as evident by an increase in the fraction of cells in the sub-G{sub 1} phase of the cell cycle and Annexin-V binding of externalized phosphatidylserine. Results revealed that inhibition of proliferation is associated with regulation of the JAK/STAT pathway, where resveratrol prevents phosphorylation ofmore » JAK, thereby inhibiting STAT1 phosphorylation. Furthermore, resveratrol treatment actively stimulated reactive oxygen species (ROS) and mitochondrial membrane depolarization. Consequently, an imbalance in the Bax/Bcl-2 ratio triggered the caspase cascade and subsequent cleavage of PARP, thereby shifting the balance in favor of apoptosis. These observations indicate that resveratrol treatment inhibits JAK/STAT-mediated gene transcription and induce the mitochondrial cell death pathway.« less

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.

Pantoprazole blocks the JAK2/STAT3 pathway to alleviate skeletal muscle wasting in cancer cachexia by inhibiting inflammatory response

PubMed Central

Guo, Dunwei; Wang, Chaoyi; Wang, Qiang; Qiao, Zhongpeng; Tang, Hua

2017-01-01

Objective Cancer cachexia is often present in patients with advanced malignant tumors, and the subsequent body weight reduction results in poor quality of life. However, there has been no progress in developing effective clinical therapeutic strategies for skeletal muscle wasting in cancer cachexia. Herein, we explored the functions of pantoprazole on cancer cachexia skeletal muscle wasting. Methods The mouse colon adenocarcinoma cell line C26 was inoculated in the right forelimb of male BALB/C mice to establish a cancer cachexia model. The animals were treated with or without different concentrations of pantoprazole orally, and the body weight, tumor growth, spontaneous activity, and muscle functions were determined at various time points. Two weeks later, the levels of serum IL-6 and TNF-α, the mRNA levels of gastrocnemius JAK2 and STAT3, and the expression levels of p-JAK2, p-STAT3, Fbx32, and MuRF1 were examined with ELISA assay, qRT-PCR assay, and Western blotting, respectively. Further studies were performed to assess the levels of Fbx32 and MuRF1 expression and morphological changes. Results Pantoprazole can alleviate cancer cachexia-induced body weight reduction and inhibit skeletal muscle wasting in a dose-dependent manner. Our results indicated that pantoprazole treatment can decrease the levels of serum IL-6 and TNF-α (56.3% and 67.6%, respectively), and inhibit the activation of the JAK2/STAT3 signaling pathway. Moreover, the expression levels of MuRF1 and Fbx32 were also suppressed after pantoprazole treatment. Conclusion Our findings suggested that pantoprazole can alleviate cancer cachexia skeletal muscle wasting by inhibiting the inflammatory response and blocking the JAK2/STAT3 or ubiquitin proteasome pathway. PMID:28489606

Pantoprazole blocks the JAK2/STAT3 pathway to alleviate skeletal muscle wasting in cancer cachexia by inhibiting inflammatory response.

PubMed

Guo, Dunwei; Wang, Chaoyi; Wang, Qiang; Qiao, Zhongpeng; Tang, Hua

2017-06-13

Cancer cachexia is often present in patients with advanced malignant tumors, and the subsequent body weight reduction results in poor quality of life. However, there has been no progress in developing effective clinical therapeutic strategies for skeletal muscle wasting in cancer cachexia. Herein, we explored the functions of pantoprazole on cancer cachexia skeletal muscle wasting. The mouse colon adenocarcinoma cell line C26 was inoculated in the right forelimb of male BALB/C mice to establish a cancer cachexia model. The animals were treated with or without different concentrations of pantoprazole orally, and the body weight, tumor growth, spontaneous activity, and muscle functions were determined at various time points. Two weeks later, the levels of serum IL-6 and TNF-α, the mRNA levels of gastrocnemius JAK2 and STAT3, and the expression levels of p-JAK2, p-STAT3, Fbx32, and MuRF1 were examined with ELISA assay, qRT-PCR assay, and Western blotting, respectively. Further studies were performed to assess the levels of Fbx32 and MuRF1 expression and morphological changes. Pantoprazole can alleviate cancer cachexia-induced body weight reduction and inhibit skeletal muscle wasting in a dose-dependent manner. Our results indicated that pantoprazole treatment can decrease the levels of serum IL-6 and TNF-α (56.3% and 67.6%, respectively), and inhibit the activation of the JAK2/STAT3 signaling pathway. Moreover, the expression levels of MuRF1 and Fbx32 were also suppressed after pantoprazole treatment. Our findings suggested that pantoprazole can alleviate cancer cachexia skeletal muscle wasting by inhibiting the inflammatory response and blocking the JAK2/STAT3 or ubiquitin proteasome pathway.

Mutant JAK3 phosphoproteomic profiling predicts synergism between JAK3 inhibitors and MEK/BCL2 inhibitors for the treatment of T-cell acute lymphoblastic leukemia

PubMed Central

Degryse, S; de Bock, C E; Demeyer, S; Govaerts, I; Bornschein, S; Verbeke, D; Jacobs, K; Binos, S; Skerrett-Byrne, D A; Murray, H C; Verrills, N M; Van Vlierberghe, P; Cools, J; Dun, M D

2018-01-01

Mutations in the interleukin-7 receptor (IL7R) or the Janus kinase 3 (JAK3) kinase occur frequently in T-cell acute lymphoblastic leukemia (T-ALL) and both are able to drive cellular transformation and the development of T-ALL in mouse models. However, the signal transduction pathways downstream of JAK3 mutations remain poorly characterized. Here we describe the phosphoproteome downstream of the JAK3(L857Q)/(M511I) activating mutations in transformed Ba/F3 lymphocyte cells. Signaling pathways regulated by JAK3 mutants were assessed following acute inhibition of JAK1/JAK3 using the JAK kinase inhibitors ruxolitinib or tofacitinib. Comprehensive network interrogation using the phosphoproteomic signatures identified significant changes in pathways regulating cell cycle, translation initiation, mitogen-activated protein kinase and phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)/AKT signaling, RNA metabolism, as well as epigenetic and apoptotic processes. Key regulatory proteins within pathways that showed altered phosphorylation following JAK inhibition were targeted using selumetinib and trametinib (MEK), buparlisib (PI3K) and ABT-199 (BCL2), and found to be synergistic in combination with JAK kinase inhibitors in primary T-ALL samples harboring JAK3 mutations. These data provide the first detailed molecular characterization of the downstream signaling pathways regulated by JAK3 mutations and provide further understanding into the oncogenic processes regulated by constitutive kinase activation aiding in the development of improved combinatorial treatment regimens. PMID:28852199

Synergistic effect of pacritinib with erlotinib on JAK2-mediated resistance in epidermal gowth factor receptor mutation-positive non-small cell lung Cancer.

PubMed

Ochi, Nobuaki; Isozaki, Hideko; Takeyama, Masami; Singer, Jack W; Yamane, Hiromichi; Honda, Yoshihiro; Kiura, Katsuyuki; Takigawa, Nagio

2016-06-10

The combination effect of pacritinib, a novel JAK2/FLT3 inhibitor, with erlotinib, the epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI), on non-small cell lung cancer cells with EGFR activating mutations was investigated. The combination showed synergistic effects on JAK2-mediated EGFR TKI-resistant PC-9/ER3 cells in some cases. The combination markedly suppressed pAKT and pERK although pSTAT3 expression was similar regardless of treatment with the pacritinib, pacritinib + erlotinib, or control in PC-9/ER3 cells. Receptor tyrosine kinase array profiling demonstrated that pacritinib suppressed MET in the PC-9/ER3 cells. The combined treatment of pacritinib and erlotinib in PC-9/ER3 xenografts showed more tumor shrinkage compared with each drug as monotherapy. Western blotting revealed that pMET in tumor samples was inhibited. These results suggest MET suppression by pacritinib may play a role in overcoming the EGFR-TKI resistance mediated by JAK2 in the PC-9/ER3 cells. In conclusion, pacritinib combined with EGFR-TKI might be a potent strategy against JAK2-mediated EGFR-TKI resistance. Copyright © 2016 Elsevier Inc. All rights reserved.

Selective JAK inhibitors in development for rheumatoid arthritis.

PubMed

Norman, Peter

2014-08-01

The JAK kinases are a family of four tyrosine receptor kinases that play a pivotal role in cytokine receptor signalling pathways via their interaction with signal transducers and activators of transcription proteins. Selective inhibitors of JAK kinases are viewed as of considerable potential as disease-modifying anti-inflammatory drugs for the treatment of rheumatoid arthritis. This article provides a review of the clinical development and available clinical results for those JAK inhibitors currently under investigation. Phase II data for four JAK inhibitors (baricitinib, decernotinib, filgotinib and INCB-039110) are contrasted with that reported for the recently approved JAK inhibitor tofacitinib. The preclinical data on these, in addition to peficitinib, ABT-494, INCB-047986 and AC-410 are also discussed, as are some of the inhibitors in preclinical development. JAK inhibitors are effective in the treatment of rheumatoid arthritis as evidenced by several inhibitors enabling the majority of treated patients to achieve ACR20 responses, with baricitinib and INCB-039110 both effective when administered once daily. JAK inhibitors differ in isoform specificity profiles, with good efficacy achievable by selective inhibition of either JAK1 (filgotinib or INCB-039110) or JAK3 (decernotinib). It remains to be seen what selectivity provides the optimal side-effect profile and to what extent inhibition of JAK2 should be avoided.

Jak2 and Ca2+/calmodulin are key intermediates for bradykinin B2 receptor-mediated activation of Na+/H+ exchange in KNRK and CHO cells.

PubMed

Lefler, David; Mukhin, Yurii V; Pettus, Tobiah; Leeb-Lundberg, L M Fredrik; Garnovskaya, Maria N; Raymond, John R

2003-04-01

Na(+)/H(+) exchangers are ubiquitous in mammalian cells, carrying out key functions, such as cell volume defense, acid-base homeostasis, and regulation of the cytoskeleton. We used two screening technologies (FLIPR and microphysiometry) to characterize the signal transduction pathway used by the bradykinin B(2) receptor to activate Na(+)/H(+) exchange in two cell lines, KNRK and CHO. In both cell types, B(2) receptor activation resulted in rapid increases in the rate of proton extrusion that were sodium-dependent and could be blocked by the Na(+)/H(+) exchange inhibitors EIPA and MIA or by replacing extracellular sodium with TMA. Activation of Na(+)/H(+) exchange by bradykinin was concentration-dependent and could be blocked by the selective B(2) receptor antagonist HOE140, but not by the B(1) receptor antagonist des-Arg10-HOE140. Inhibitors of Jak2 tyrosine kinase (genistein and AG490) and of CAM (W-7 and calmidazolium) attenuated bradykinin-induced activation of Na(+)/H(+) exchange. Bradykinin induced formation of a complex between CAM and Jak2, supporting a regulatory role for Jak2 and CAM in the activation of Na(+)/H(+) exchange in KNRK and CHO cells. We propose that this pathway (B(2) receptor --> Jak2 --> CAM --> Na(+)/H(+) exchanger) is a fundamental regulator of Na(+)/H(+) exchange activity.

Genetic studies reveal an unexpected negative regulatory role for Jak2 in thrombopoiesis

PubMed Central

Meyer, Sara C.; Keller, Matthew D.; Woods, Brittany A.; LaFave, Lindsay M.; Bastian, Lennart; Kleppe, Maria; Bhagwat, Neha; Marubayashi, Sachie

2014-01-01

JAK inhibitor treatment is limited by the variable development of anemia and thrombocytopenia thought to be due to on-target JAK2 inhibition. We evaluated the impact of Jak2 deletion in platelets (PLTs) and megakaryocytes (MKs) on blood counts, stem/progenitor cells, and Jak-Stat signaling. Pf4-Cre–mediated Jak2 deletion in PLTs and MKs did not compromise PLT formation but caused thrombocytosis, and resulted in expansion of MK progenitors and Lin−Sca1+Kit+ cells. Serum thrombopoietin (TPO) was maintained at normal levels in Pf4-Cre–positive Jak2f/f mice, consistent with reduced internalization/turnover by Jak2-deficient PLTs. These data demonstrate that Jak2 in terminal megakaryopoiesis is not required for PLT production, and that Jak2 loss in PLTs and MKs results in non-autonomous expansion of stem/progenitors and of MKs and PLTs via dysregulated TPO turnover. This suggests that the thrombocytopenia frequently seen with JAK inhibitor treatment is not due to JAK2 inhibition in PLTs and MKs, but rather due to JAK2 inhibition in stem/progenitor cells. PMID:25115888

HIP1 functions in clathrin-mediated endocytosis through binding to clathrin and adaptor protein 2.

PubMed

Metzler, M; Legendre-Guillemin, V; Gan, L; Chopra, V; Kwok, A; McPherson, P S; Hayden, M R

2001-10-19

Polyglutamine expansion in huntingtin is the underlying mutation leading to neurodegeneration in Huntington disease. This mutation influences the interaction of huntingtin with different proteins, including huntingtin-interacting protein 1 (HIP1), in which affinity to bind to mutant huntingtin is profoundly reduced. Here we demonstrate that HIP1 colocalizes with markers of clathrin-mediated endocytosis in neuronal cells and is highly enriched on clathrin-coated vesicles (CCVs) purified from brain homogenates. HIP1 binds to the clathrin adaptor protein 2 (AP2) and the terminal domain of the clathrin heavy chain, predominantly through a small fragment encompassing amino acids 276-335. This region, which contains consensus clathrin- and AP2-binding sites, functions in conjunction with the coiled-coil domain to target HIP1 to CCVs. Expression of various HIP1 fragments leads to a potent block of clathrin-mediated endocytosis. Our findings demonstrate that HIP1 is a novel component of the endocytic machinery.

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.

Non-structural protein 1 of influenza viruses inhibits rapid mRNA degradation mediated by double-stranded RNA-binding protein, staufen1.

PubMed

Cho, Hana; Ahn, Sang Ho; Kim, Kyoung Mi; Kim, Yoon Ki

2013-07-11

Although non-structural protein 1 (NS1) of influenza viruses is not essential for virulence, this protein is involved in host-virus interactions, viral replication, and translation. In particular, NS1 is known to interact with the host protein, staufen1 (Stau1). This interaction is important for efficient viral replication. However, the underlying molecular mechanism by which NS1 influences the viral life cycle remains obscure. Here, we show using immunoprecipitation and artificial tethering that the N-terminus of NS1, NS1(1-73), interacts with Stau1, blocks the Stau1-Upf1 interaction, and consequently inhibits the efficiency of Stau1-mediated mRNA decay (SMD), but not nonsense-mediatedmRNA decay (NMD). The regulation of SMD efficiency by NS1 may contribute to building a more favorable cellular environment for viral replication. Copyright © 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

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.

Sympathetic nerve blocks promote anti-inflammatory response by activating the JAK2-STAT3-mediated signaling cascade in rat myocarditis models: A novel mechanism with clinical implications.

PubMed

Park, Hyelim; Park, Hyewon; Mun, Dasom; Kim, Michael; Pak, Hui-Nam; Lee, Moon-Hyoung; Joung, Boyoung

2018-05-01

Left stellectomy has become an important therapeutic option for patients with potentially fatal arrhythmias. However, the antiarrhythmic mechanism of left stellectomy is not well known. The cholinergic anti-inflammatory pathway (CAIP) is a complex immune mechanism that regulates peripheral inflammatory responses. The purpose of this study was to evaluate the effect of left stellectomy on CAIP using rat experimental autoimmune myocarditis (EAM) models. EAM was produced by injecting 2 mg of porcine cardiac myosin into the footpads of rats. Left stellectomy was performed before EAM induction. We evaluated the effect of left stellectomy on arrhythmic events, survival, inflammation, and CAIP in rats without and with EAM. Left stellectomy prevented arrhythmia and improved survival in EAM rats. Left stellectomy decreased the levels of tumor necrosis factor α, interleukin 6, and high mobility group box 1 (P < .05 vs EAM) in serum and heart tissues from EAM rats. In heart rate variability analysis, high-frequency peaks of the power spectrum densities, reflecting parasympathetic cardiovagal tone, were significantly decreased in EAM rats, but increased after left stellectomy. The ratios of phosphorylated STAT3/STAT3 (signal transducer and activator of transcription 3) and phosphorylated JAK2/JAK2 (Janus kinase 2) decreased in cell lysates of the spleen, liver, and heart in EAM rats. However, the same ratios significantly increased after left stellectomy. Nuclear factor κB in cell lysates of the spleen, liver, and heart increased in EAM rats, but decreased after left stellectomy. In EAM models, left stellectomy increased survival of the rats while showing antiarrhythmic effects with reduced inflammation via activation of the JAK2-STAT3-mediated signaling cascade. Our findings suggest an exciting opportunity to develop new and novel therapeutics to attenuate cardiac inflammation. Copyright © 2017 Heart Rhythm Society. Published by Elsevier Inc. All rights reserved.

AMP-activated protein kinase is involved in neural stem cell growth suppression and cell cycle arrest by 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside and glucose deprivation by down-regulating phospho-retinoblastoma protein and cyclin D.

PubMed

Zang, Yi; Yu, Li-Fang; Nan, Fa-Jun; Feng, Lin-Yin; Li, Jia

2009-03-06

The fate of neural stem cells (NSCs), including their proliferation, differentiation, survival, and death, is regulated by multiple intrinsic signals and the extrinsic environment. We had previously reported that 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR) directly induces astroglial differentiation of NSCs by activation of the Janus kinase (JAK)/Signal transducer and activator of transcription 3 (STAT3) pathway independently of AMP-activated protein kinase (AMPK). Here, we reported the observation that AICAR inhibited NSC proliferation and its underlying mechanism. Analysis of caspase activity and cell cycle showed that AICAR induced G1/G0 cell cycle arrest in NSCs, associated with decreased levels of poly(ADP-ribose) polymerase, phospho-retinoblastoma protein (Rb), and cyclin D but did not cause apoptosis. Iodotubericidin and Compound C, inhibitors of adenosine kinase and AMPK, respectively, or overexpression of a dominant-negative mutant of AMPK, but not JAK inhibitor, were able to reverse the anti-proliferative effect of AICAR. Glucose deprivation also activated the AMPK pathway, induced G0/G1 arrest, and suppressed the proliferation of NSCs, an effect associated with decreased levels of phospho-Rb and cyclin D protein. Furthermore, Compound C and overexpression of dominant-negative AMPK in C17.2 NSCs could block the glucose deprivation-mediated down-regulation of cyclin D and partially reverse the suppression of proliferation. These results suggest that AICAR and glucose deprivation might induce G1/G0 cell cycle arrest and suppress proliferation of NSCs via phospho-Rb and cyclin D down-regulation. AMPK, but not JAK/STAT3, activation is key for this inhibitory effect and may play an important role in the responses of NSCs to metabolic stresses such as glucose deprivation.

Leptin-induced ER-α-positive breast cancer cell viability and migration is mediated by suppressing CCN5-signaling via activating JAK/AKT/STAT-pathway.

PubMed

Haque, Inamul; Ghosh, Arnab; Acup, Seth; Banerjee, Snigdha; Dhar, Kakali; Ray, Amitabha; Sarkar, Sandipto; Kambhampati, Suman; Banerjee, Sushanta K

2018-01-25

In menopausal women, one of the critical risk factors for breast cancer is obesity/adiposity. It is evident from various studies that leptin, a 16 kDa protein hormone overproduced in obese people, plays the critical role in neovascularization and tumorigenesis in breast and other organs. However, the mechanisms by which obesity influences the breast carcinogenesis remained unclear. In this study, by analyzing different estrogen receptor-α (ER-α)-positive and ER-α-negative BC cell lines, we defined the role of CCN5 in the leptin-mediated regulation of growth and invasive capacity. We analyzed the effect of leptin on cell viability of ER-α-positive MCF-7 and ZR-75-1 cell lines and ER-α-negative MDA-MB-231 cell line. Additionally, we also determined the effect of leptin on the epithelial-mesenchymal transition (EMT) bio-markers, in vitro invasion and sphere-formation of MCF-7 and ZR-75-1 cell lines. To understand the mechanism, we determined the impact of leptin on CCN5 expression and the functional role of CCN5 in these cells by the treatment of human recombinant CCN5 protein(hrCCN5). Moreover, we also determined the role of JAK-STAT and AKT in the regulation of leptin-induced suppression of CCN5 in BC cells. Present studies demonstrate that leptin can induce cell viability, EMT, sphere-forming ability and migration of MCF-7 and ZR-75-1 cell lines. Furthermore, these studies found that leptin suppresses the expression of CCN5 at the transcriptional level. Although the CCN5 suppression has no impact on the constitutive proliferation of MCF-7 and ZR-75-1 cells, it is critical for leptin-induced viability and necessary for EMT, induction of in vitro migration and sphere formation, as the hrCCN5 treatment significantly inhibits the leptin-induced viability, EMT, migration and sphere-forming ability of these cells. Mechanistically, CCN5-suppression by leptin is mediated via activating JAK/AKT/STAT-signaling pathways. These studies suggest that CCN5 serves as a

Design, synthesis, and evaluation of 4,6-diaminonicotinamide derivatives as novel and potent immunomodulators targeting JAK3.

PubMed

Nakajima, Yutaka; Aoyama, Naohiro; Takahashi, Fumie; Sasaki, Hiroshi; Hatanaka, Keiko; Moritomo, Ayako; Inami, Masamichi; Ito, Misato; Nakamura, Koji; Nakamori, Fumihiro; Inoue, Takayuki; Shirakami, Shohei

2016-10-01

In organ transplantation, T cell-mediated immune responses play a key role in the rejection of allografts. Janus kinase 3 (JAK3) is specifically expressed in hematopoietic cells and associated with regulation of T cell development via interleukin-2 signaling pathway. Here, we designed novel 4,6-diaminonicotinamide derivatives as immunomodulators targeting JAK3 for prevention of transplant rejection. Our optimization of C4- and C6-substituents and docking calculations to JAK3 protein confirmed that the 4,6-diaminonicotinamide scaffold resulted in potent inhibition of JAK3. We also investigated avoidance of human ether-a-go-go related gene (hERG) inhibitory activity. Selected compound 28 in combination with tacrolimus prevented allograft rejection in a rat heterotopic cardiac transplantation model. Copyright © 2016 Elsevier Ltd. All rights reserved.

Methotrexate Is a JAK/STAT Pathway Inhibitor

PubMed Central

Thomas, Sally; Fisher, Katherine H.; Snowden, John A.; Danson, Sarah J.; Brown, Stephen; Zeidler, Martin P.

2015-01-01

Background The JAK/STAT pathway transduces signals from multiple cytokines and controls haematopoiesis, immunity and inflammation. In addition, pathological activation is seen in multiple malignancies including the myeloproliferative neoplasms (MPNs). Given this, drug development efforts have targeted the pathway with JAK inhibitors such as ruxolitinib. Although effective, high costs and side effects have limited its adoption. Thus, a need for effective low cost treatments remains. Methods & Findings We used the low-complexity Drosophila melanogaster pathway to screen for small molecules that modulate JAK/STAT signalling. This screen identified methotrexate and the closely related aminopterin as potent suppressors of STAT activation. We show that methotrexate suppresses human JAK/STAT signalling without affecting other phosphorylation-dependent pathways. Furthermore, methotrexate significantly reduces STAT5 phosphorylation in cells expressing JAK2 V617F, a mutation associated with most human MPNs. Methotrexate acts independently of dihydrofolate reductase (DHFR) and is comparable to the JAK1/2 inhibitor ruxolitinib. However, cells treated with methotrexate still retain their ability to respond to physiological levels of the ligand erythropoietin. Conclusions Aminopterin and methotrexate represent the first chemotherapy agents developed and act as competitive inhibitors of DHFR. Methotrexate is also widely used at low doses to treat inflammatory and immune-mediated conditions including rheumatoid arthritis. In this low-dose regime, folate supplements are given to mitigate side effects by bypassing the biochemical requirement for DHFR. Although independent of DHFR, the mechanism-of-action underlying the low-dose effects of methotrexate is unknown. Given that multiple pro-inflammatory cytokines signal through the pathway, we suggest that suppression of the JAK/STAT pathway is likely to be the principal anti-inflammatory and immunosuppressive mechanism-of-action of low

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

The JAK2 GGCC (46/1) Haplotype in Myeloproliferative Neoplasms: Causal or Random?

PubMed Central

Anelli, Luisa; Zagaria, Antonella; Specchia, Giorgina

2018-01-01

The germline JAK2 haplotype known as “GGCC or 46/1 haplotype” (haplotypeGGCC_46/1) consists of a combination of single nucleotide polymorphisms (SNPs) mapping in a region of about 250 kb, extending from the JAK2 intron 10 to the Insulin-like 4 (INLS4) gene. Four main SNPs (rs3780367, rs10974944, rs12343867, and rs1159782) generating a “GGCC” combination are more frequently indicated to represent the JAK2 haplotype. These SNPs are inherited together and are frequently associated with the onset of myeloproliferative neoplasms (MPN) positive for both JAK2 V617 and exon 12 mutations. The association between the JAK2 haplotypeGGCC_46/1 and mutations in other genes, such as thrombopoietin receptor (MPL) and calreticulin (CALR), or the association with triple negative MPN, is still controversial. This review provides an overview of the frequency and the role of the JAK2 haplotypeGGCC_46/1 in the pathogenesis of different myeloid neoplasms and describes the hypothetical mechanisms at the basis of the association with JAK2 gene mutations. Moreover, possible clinical implications are discussed, as different papers reported contrasting data about the correlation between the JAK2 haplotypeGGCC_46/1 and blood cell count, survival, or disease progression. PMID:29641446

The JAK2 GGCC (46/1) Haplotype in Myeloproliferative Neoplasms: Causal or Random?

PubMed

Anelli, Luisa; Zagaria, Antonella; Specchia, Giorgina; Albano, Francesco

2018-04-11

The germline JAK2 haplotype known as "GGCC or 46/1 haplotype" (haplotype GGCC_46/1 ) consists of a combination of single nucleotide polymorphisms (SNPs) mapping in a region of about 250 kb, extending from the JAK2 intron 10 to the Insulin-like 4 ( INLS4 ) gene. Four main SNPs (rs3780367, rs10974944, rs12343867, and rs1159782) generating a "GGCC" combination are more frequently indicated to represent the JAK2 haplotype. These SNPs are inherited together and are frequently associated with the onset of myeloproliferative neoplasms (MPN) positive for both JAK2 V617 and exon 12 mutations. The association between the JAK2 haplotype GGCC_46/1 and mutations in other genes, such as thrombopoietin receptor ( MPL ) and calreticulin ( CALR ), or the association with triple negative MPN, is still controversial. This review provides an overview of the frequency and the role of the JAK2 haplotype GGCC_46/1 in the pathogenesis of different myeloid neoplasms and describes the hypothetical mechanisms at the basis of the association with JAK2 gene mutations. Moreover, possible clinical implications are discussed, as different papers reported contrasting data about the correlation between the JAK2 haplotype GGCC_46/1 and blood cell count, survival, or disease progression.

Crystal Structure of the FERM-SH2 Module of Human Jak2.

PubMed

McNally, Randall; Toms, Angela V; Eck, Michael J

2016-01-01

Jak-family tyrosine kinases mediate signaling from diverse cytokine receptors. Binding of Jaks to their cognate receptors is mediated by their N-terminal region, which contains FERM and SH2 domains. Here we describe the crystal structure of the FERM-SH2 region of Jak2 at 3.0Å resolution. The structure reveals that these domains and their flanking linker segments interact intimately to form an integrated structural module. The Jak2 FERM-SH2 structure closely resembles that recently described for Tyk2, another member of the Jak family. While the overall architecture and interdomain orientations are preserved between Jak2 and Tyk2, we identify residues in the putative receptor-binding groove that differ between the two and may contribute to the specificity of receptor recognition. Analysis of Jak mutations that are reported to disrupt receptor binding reveals that they lie in the hydrophobic core of the FERM domain, and are thus expected to compromise the structural integrity of the FERM-SH2 unit. Similarly, analysis of mutations in Jak3 that are associated with severe combined immunodeficiency suggests that they compromise Jak3 function by destabilizing the FERM-SH2 structure.

Loss of function JAK1 mutations occur at high frequency in cancers with microsatellite instability and are suggestive of immune evasion.

PubMed

Albacker, Lee A; Wu, Jeremy; Smith, Peter; Warmuth, Markus; Stephens, Philip J; Zhu, Ping; Yu, Lihua; Chmielecki, Juliann

2017-01-01

Immune evasion is a well-recognized hallmark of cancer and recent studies with immunotherapy agents have suggested that tumors with increased numbers of neoantigens elicit greater immune responses. We hypothesized that the immune system presents a common selective pressure on high mutation burden tumors and therefore immune evasion mutations would be enriched in high mutation burden tumors. The JAK family of kinases is required for the signaling of a host of immune modulators in tumor, stromal, and immune cells. Therefore, we analyzed alterations in this family for the hypothesized signature of an immune evasion mutation. Here, we searched a database of 61,704 unique solid tumors for alterations in the JAK family kinases (JAK1/2/3, TYK2). We used The Cancer Genome Atlas and Cancer Cell Line Encyclopedia data to confirm and extend our findings by analyzing gene expression patterns. Recurrent frameshift mutations in JAK1 were associated with high mutation burden and microsatellite instability. These mutations occurred in multiple tumor types including endometrial, colorectal, stomach, and prostate carcinomas. Analyzing gene expression signatures in endometrial and stomach adenocarcinomas revealed that tumors with a JAK1 frameshift exhibited reduced expression of interferon response signatures and multiple anti-tumor immune signatures. Importantly, endometrial cancer cell lines exhibited similar gene expression changes that were expected to be tumor cell intrinsic (e.g. interferon response) but not those expected to be tumor cell extrinsic (e.g. NK cells). From these data, we derive two primary conclusions: 1) JAK1 frameshifts are loss of function alterations that represent a potential pan-cancer adaptation to immune responses against tumors with microsatellite instability; 2) The mechanism by which JAK1 loss of function contributes to tumor immune evasion is likely associated with loss of the JAK1-mediated interferon response.

Platelet-activating factor mediates monocyte chemoattractant protein-1 expression in glomerular immune injury.

PubMed

Jocks, T; Freudenberg, J; Zahner, G; Stahl, R A

1998-01-01

These studies were designed to determine the possible role of platelet-activating factor (PAF) in the production of monocyte chemoattractant protein-1 (MCP-1) in glomerular immune injury. The glomerular lesion was induced in isolated perfused rat kidneys by a rabbit anti-rat-thymocyte serum (ATS) and rat serum (RS) as a complement source. Perfusion of kidneys with ATS and RS results in the selective binding of the antiserum to the glomerular mesangium with consecutive intraglomerular activation of complement. Antibody binding and complement activation induced a significant increase in glomerular MCP-1 mRNA levels when assessed by Northern blotting or RT-PCR. Decomplemented RS or non antibody rabbit IgG had only moderate effects on glomerular MCP-1 mRNA levels. The PAF receptor antagonist WEB 2170 almost completely blocked the ATS and RS induced MCP-1 mRNA levels. Perfusion of control kidneys with PAF increased MCP-1 mRNA expression, an effect which was blocked by WEB 2170. Glomerular MCP-1 protein formation, assessed by Western blotting, was stimulated following ATS and RS and PAF, respectively, was blocked by WEB 2170. These data show that PAF, derived from glomerular resident cells following antibody and complement induced injury, stimulates MCP-1 expression. In addition to the direct effects on leukocyte adhesion and activation PAF may mediate inflammatory cell influx in glomerular injuries due to the release of MCP-1.

Investigation of JAKs/STAT‐3 in lipopolysaccharide‐induced intestinal epithelial cells

PubMed Central

Fu, L.; Wei, L.‐W.; Zhao, M.‐D.; Zhu, J.‐L.; Chen, S.‐Y.; Jia, X.‐B.

2016-01-01

Summary Janus‐activated kinase (JAKs)‐signal transducer and activator of transcription 3 (STAT‐3) signalling play critical roles in immunoregulation and immunopathology, which involve inflammatory responses and enteritis. JAK phosphorylates STAT‐3 in response to stimulation by cytokines or growth factors, and then activates or represses the gene expression. STAT‐3 is activated persistently in cancer cells and contributes to the malignant progression of various types of cancer and inflammation. To elucidate the different roles of JAKs in the activation of STAT‐3, the lipopolysaccharide‐induced primary intestinal epithelial cell (IEC) acute inflammatory model was established. Small interference RNAs (siRNAs) were then employed to attenuate the expression levels of JAKs. Real‐time quantitative reverse transcription–polymerase chain reaction (PCR) (qRT–PCR) revealed that JAK mRNA levels were reduced efficiently by JAK‐specific siRNAs. Under the IEC inflammatory model transfected with si‐JAK, which equates to effective silencing, qRT–PCR and Western blot assays, suggested that knockdowns of JAK attenuated the JAK‐induced down‐regulation of STAT‐3 at the mRNA or protein levels. In particular, JAK1 played a key role, which was consistent with the RNA‐Seq results. Subsequently, the expression levels of proinflammatory cytokines interleukin (IL)‐1β and tumour necrosis factor (TNF)‐α were down‐regulated in the IEC inflammatory model transfected with si‐JAK1. JAK1 appears as a direct activator for STAT‐3, whereas treatments targeting JAK1 repressed STAT‐3 sufficiently pathways in the IEC inflammatory model. Therefore, the control of JAK1 using siRNAs has the potential to be an effective strategy against enteritis. PMID:27357529

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

The SHH/Gli axis regulates CD90-mediated liver cancer stem cell function by activating the IL6/JAK2 pathway.

PubMed

Zhang, Ketao; Che, Siyao; Pan, Chuzhi; Su, Zheng; Zheng, Shangyou; Yang, Shanglin; Zhang, Huayao; Li, Wenda; Wang, Weidong; Liu, Jianping

2018-05-02

The cell surface antigen CD90 has recently been established as a promising marker for liver cancer stem cells. This study aimed to investigate potential implications of SHH/Gli signalling in CD90+ liver cancer stem cells. Correlation of the expression of SHH signalling components and CD90 in liver cancer cells and clinical tissues, as well as in enriched CD90+ liver cancer stem cells and the TCGA database, were analysed by quantitative RT-PCR, Western blotting and flow cytometry. Functional analysis was conducted by siRNA-mediated CD90, Gli1 and Gli3 gene knockdown, SHH treatment and application of the JAK2 inhibitor AZD1480 and IL6 neutralizing antibody in CD90+ liver cancer stem cells, followed by cell proliferation, migration, sphere formation and tumorigenicity assays. CD90 expression exhibited a high positive correlation with Gli1 and Gli3 in multiple liver cancer cell lines and human cancerous liver tissues, both of which showed a significant increase in liver cancer. Analysis of TCGA data revealed an association of CD90, Gli1 and Gli3 with a short overall survival and positive correlation between CD90 expression and Gli3 expression level. The stem cell potentials of CD90+ 97L liver cancer cells were greatly impaired by Gli1/3 knockdown with siRNA but enhanced by SHH treatment. Application of the JAK2 inhibitor AZD1480 and IL6 neutralizing antibody showed the CD90 and SHH/Gli-regulated liver cancer stem cell functions were mediated by the IL6/JAK2/STAT3 pathway. The stem cell properties of CD90+ liver cancer cells are regulated by the downstream SHH/Gli and IL6/JAK2/STAT3 signalling pathways. © 2018 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

Secoisolariciresinol diglucoside prevents the oxidative stress-induced apoptosis of myocardial cells through activation of the JAK2/STAT3 signaling pathway.

PubMed

Huang, Guiqiong; Huang, Xiaofang; Liu, Min; Hua, Yue; Deng, Bo; Jin, Wen; Yan, Wen; Tan, Zhangbin; Wu, Yifen; Liu, Bin; Zhou, Yingchun

2018-06-01

Myocardial cell apoptosis mediated by oxidative stress has previously been identified as a key process in ischemic heart disease. Secoisolariciresinol diglucoside (SDG), a polyphenolic plant lignan primarily found in flaxseed, has been demonstrated to effectively protect myocardial cells from apoptosis. In the present study, the role of the Janus kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) was investigated in mediating the protective effect of SDG. Findings of the present study revealed that treatment with H2O2 reduced cell viability and induced apoptosis in H9C2 rat cardiomyocytes. However, SDG was able to reduce the effect of H2O2 in a dose‑dependent manner. H2O2 reduced the expression level of phosphorylated STAT3 and inhibited the levels of B‑cell lymphoma‑extra‑large and induced myeloid leukemia cell differentiation protein, which are the STAT3 target genes. Conversely, SDG rescued phosphorylation of STAT3 and increased the levels of STAT3 target genes. Treatment with SDG alone led to a dose‑dependent increased phosphorylation of JAK2 and STAT3, without activating Src. Furthermore, the anti‑apoptotic effects of SDG were partially abolished by a JAK2/STAT3 inhibitor. In addition, molecular docking revealed that SDG may bind to the protein kinase domain of JAK2, at a binding energy of ‑8.258 kcal/mol. Molecular dynamics simulations revealed that JAK2‑SDG binding was stable. In conclusion, activation of the JAK2/STAT3 signaling pathway contributed to the anti‑apoptotic activity of SDG, which may be a potential JAK2 activator.

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

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

The JAK2 Inhibitor, AZD1480, Potently Blocks Stat3 Signaling and Oncogenesis in Solid Tumors

PubMed Central

Hedvat, Michael; Huszar, Dennis; Herrmann, Andreas; Gozgit, Joseph M.; Schroeder, Anne; Sheehy, Adam; Buettner, Ralf; Proia, David; Kowolik, Claudia M.; Xin, Hong; Armstrong, Brian; Bebernitz, Geraldine; Weng, Shaobu; Wang, Lin; Ye, Minwei; McEachern, Kristen; Chen, Huawei; Morosini, Deborah; Bell, Kirsten; Alimzhanov, Marat; Ioannidis, Stephanos; McCoon, Patricia; Cao, Zhu A.; Yu, Hua; Jove, Richard; Zinda, Michael

2009-01-01

Summary Persistent activation of Stat3 is oncogenic and is prevalent in a wide variety of human cancers. Chronic cytokine stimulation is associated with Stat3 activation in some tumors, implicating cytokine receptor-associated Jak family kinases. Using Jak2 inhibitors, we demonstrate a central role of Jaks in modulating basal and cytokine-induced Stat3 activation in human solid tumor cell lines. Inhibition of Jak2 activity is associated with abrogation of Stat3 nuclear translocation and tumorigenesis. The Jak2 inhibitor, AZD1480, suppresses the growth of human solid tumor xenografts harboring persistent Stat3 activity. We demonstrate the essential role of Stat3 downstream of Jaks by inhibition of tumor growth using shRNA targeting Stat3. Our data support a key role of Jak kinase activity in Stat3-dependent tumorigenesis. PMID:19962667

Novel mutations in the inhibitory adaptor protein LNK drive JAK-STAT signaling in patients with myeloproliferative neoplasms

PubMed Central

Oh, Stephen T.; Simonds, Erin F.; Jones, Carol; Hale, Matthew B.; Goltsev, Yury; Gibbs, Kenneth D.; Merker, Jason D.; Zehnder, James L.; Nolan, Garry P.

2010-01-01

Dysregulated Janus kinase–signal transducer and activator of transcription (JAK-STAT) signaling due to activation of tyrosine kinases is a common feature of myeloid malignancies. Here we report the first human disease-related mutations in the adaptor protein LNK, a negative regulator of JAK-STAT signaling, in 2 patients with JAK2 V617F–negative myeloproliferative neoplasms (MPNs). One patient exhibited a 5 base-pair deletion and missense mutation leading to a premature stop codon and loss of the pleckstrin homology (PH) and Src homology 2 (SH2) domains. A second patient had a missense mutation (E208Q) in the PH domain. BaF3-MPL cells transduced with these LNK mutants displayed augmented and sustained thrombopoietin-dependent growth and signaling. Primary samples from MPN patients bearing LNK mutations exhibited aberrant JAK-STAT activation, and cytokine-responsive CD34+ early progenitors were abnormally abundant in both patients. These findings indicate that JAK-STAT activation due to loss of LNK negative feedback regulation is a novel mechanism of MPN pathogenesis. PMID:20404132

Autophosphorylation of JAK2 on tyrosines 221 and 570 regulates its activity.

PubMed

Argetsinger, Lawrence S; Kouadio, Jean-Louis K; Steen, Hanno; Stensballe, Allan; Jensen, Ole N; Carter-Su, Christin

2004-06-01

The tyrosine kinase JAK2 is a key signaling protein for at least 20 receptors in the cytokine/hematopoietin receptor superfamily and is a component of signaling by insulin receptor and several G-protein-coupled receptors. However, there is only limited knowledge of the physical structure of JAK2 or which of the 49 tyrosines in JAK2 are autophosphorylated. In this study, mass spectrometry and two-dimensional peptide mapping were used to determine that tyrosines 221, 570, and 1007 in JAK2 are autophosphorylated. Phosphorylation of tyrosine 570 is particularly robust. In response to growth hormone, JAK2 was rapidly and transiently phosphorylated at tyrosines 221 and 570, returning to basal levels by 60 min. Analysis of the sequences surrounding tyrosines 221 and 570 in JAK2 and tyrosines in other proteins that are phosphorylated in response to ligands that activate JAK2 suggests that the YXX[L/I/V] motif is one of the motifs recognized by JAK2. Experiments using JAK2 with tyrosines 221 and 570 mutated to phenylalanine suggest that tyrosines 221 and 570 in JAK2 may serve as regulatory sites in JAK2, with phosphorylation of tyrosine 221 increasing kinase activity and phosphorylation of tyrosine 570 decreasing kinase activity and thereby contributing to rapid termination of ligand activation of JAK2.

Autophosphorylation of JAK2 on Tyrosines 221 and 570 Regulates Its Activity

PubMed Central

Argetsinger, Lawrence S.; Kouadio, Jean-Louis K.; Steen, Hanno; Stensballe, Allan; Jensen, Ole N.; Carter-Su, Christin

2004-01-01

The tyrosine kinase JAK2 is a key signaling protein for at least 20 receptors in the cytokine/hematopoietin receptor superfamily and is a component of signaling by insulin receptor and several G-protein-coupled receptors. However, there is only limited knowledge of the physical structure of JAK2 or which of the 49 tyrosines in JAK2 are autophosphorylated. In this study, mass spectrometry and two-dimensional peptide mapping were used to determine that tyrosines 221, 570, and 1007 in JAK2 are autophosphorylated. Phosphorylation of tyrosine 570 is particularly robust. In response to growth hormone, JAK2 was rapidly and transiently phosphorylated at tyrosines 221 and 570, returning to basal levels by 60 min. Analysis of the sequences surrounding tyrosines 221 and 570 in JAK2 and tyrosines in other proteins that are phosphorylated in response to ligands that activate JAK2 suggests that the YXX[L/I/V] motif is one of the motifs recognized by JAK2. Experiments using JAK2 with tyrosines 221 and 570 mutated to phenylalanine suggest that tyrosines 221 and 570 in JAK2 may serve as regulatory sites in JAK2, with phosphorylation of tyrosine 221 increasing kinase activity and phosphorylation of tyrosine 570 decreasing kinase activity and thereby contributing to rapid termination of ligand activation of JAK2. PMID:15143187

Zap70 functions to maintain stemness of mouse embryonic stem cells by negatively regulating Jak1/Stat3/c-Myc signaling

PubMed Central

Cha, Young; Moon, Bo-Hyun; Lee, Mi-Ok; Ahn, Hee-Jin; Lee, Hye-Jin; Lee, Kyung-Ah; Fornace, Albert J.; Kim, Kwang-Soo; Cha, Hyuk-Jin; Park, Kyung-Soon

2011-01-01

Zeta-chain associated protein kinase-70 (Zap70), a Syk family tyrosine kinase, has been reported to be present exclusively in normal T cells, Natural Killer (NK) cells, and B cells, serving as a pivotal regulator of antigen-mediated receptor signaling and development. In this study, we report that Zap70 is expressed in undifferentiated mouse embryonic stem cells (mESCs) and may critically regulate self-renewal and pluripotency in mESCs. We found that Zap70 knocked-down mESCs (Zap70KD) show sustained self-renewal and defective differentiation. In addition, we present evidence that the sustained self-renewal in Zap70KD is associated with enhanced Jak/Stat3 signaling and c-Myc induction. These altered signaling appears to result from up-regulated LIFR and down-regulated SHP-1 phosphatase activity. Based on these results, we propose that, in undifferentiated mESCs, Zap70 plays important roles in modulating the balance between self-renewal capacity and pluripotent differentiation ability as a key regulator of the Jak/Stat3/c-Myc signaling pathway. PMID:20641039

Controlling nuclear JAKs and STATs for specific gene activation by IFNγ.

PubMed

Noon-Song, Ezra N; Ahmed, Chulbul M; Dabelic, Rea; Canton, Johnathan; Johnson, Howard M

2011-07-08

We previously showed that gamma interferon (IFNγ) and its receptor subunit, IFNGR1, interacted with the promoter region of IFNγ-activated genes along with transcription factor STAT1α. Recent studies have suggested that activated Janus kinases pJAK2 and pJAK1 also played a role in gene activation by phosphorylation of histone H3 on tyrosine 41. This study addresses the question of the role of activated JAKs in specific gene activation by IFNγ. We carried out chromatin immunoprecipitation (ChIP) followed by PCR in IFNγ treated WISH cells and showed association of pJAK1, pJAK2, IFNGR1, and STAT1 on the same DNA sequence of the IRF-1 gene promoter. The β-actin gene, which is not activated by IFNγ, did not show this association. The movement of activated JAK to the nucleus and the IRF-1 promoter was confirmed by the combination of nuclear fractionation, confocal microscopy and DNA precipitation analysis using the biotinylated GAS promoter. Activated JAKs in the nucleus was associated with phosphorylated tyrosine 41 on histone H3 in the region of the GAS promoter. Unphosphorylated JAK2 was found to be constitutively present in the nucleus and was capable of undergoing activation in IFNγ treated cells, most likely via nuclear IFNGR1. Association of pJAK2 and IFNGR1 with histone H3 in IFNγ treated cells was demonstrated by histone H3 immunoprecipitation. Unphosphorylated STAT1 protein was associated with histone H3 of untreated cells. IFNγ treatment resulted in its disassociation and then re-association as pSTAT1. The results suggest a novel role for activated JAKs in epigenetic events for specific gene activation. Copyright © 2011 Elsevier Inc. All rights reserved.

Influenza A virus strains that circulate in humans differ in the ability of their NS1 proteins to block the activation of IRF3 and interferon-β transcription.

PubMed

Kuo, Rei-Lin; Zhao, Chen; Malur, Meghana; Krug, Robert M

2010-12-20

We demonstrate that influenza A virus strains that circulate in humans differ markedly in the ability of their NS1 proteins to block the activation of IRF3 and interferon-β transcription. Strong activation occurs in cells infected with viruses expressing NS1 proteins of seasonal H3N2 and H2N2 viruses, whereas activation is blocked in cells infected with viruses expressing NS1 proteins of some, but not all seasonal H1N1 viruses. The NS1 proteins of the 2009 H1N1 and H5N1 viruses also block these activations. The difference in this NS1 function is mediated largely by the C-terminal region of the effector domain, which contains the only amino acid (K or E at position 196) that covaries with the functional difference. Further, we show that TRIM25 binds the NS1 protein whether or not IRF3 activation is blocked, demonstrating that binding of TRIM25 by the NS1 protein does not necessarily lead to the blocking of IRF3 activation. Copyright © 2010 Elsevier Inc. All rights reserved.

RANKL downregulates cell surface CXCR6 expression through JAK2/STAT3 signaling pathway during osteoclastogenesis

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

Li, Changhong; Zhao, Jinxia; Sun, Lin

Highlights: Black-Right-Pointing-Pointer CXCR6 is down-regulated during RANKL-induced osteoclastogenesis in RAW264.7 cells. Black-Right-Pointing-Pointer CXCR6 reduction was nearly reversed by inhibition of JAK2/STAT3 signaling pathway. Black-Right-Pointing-Pointer CXCL16 alone does not positively regulate osteoclastogenesis. -- Abstract: The receptor activator of nuclear factor-{kappa}B ligand (RANKL), as a member of the tumor necrosis factor (TNF) family, plays an essential role in osteoclast differentiation and function. Chemokines and their receptors have recently been shown to play critical roles in osteoclastogenesis, however, whether CXCL16-CXCR6 plays role in RANKL-mediated osteoclastogenesis is unknown. In this study, we first reported that RANKL decreased CXCR6 in a dose-dependent manner, which maymore » be through deactivation of Akt and STAT3 signaling induced by CXCL16. Interestingly, RANKL-mediated CXCR6 reduction may be associated to the activation of STAT3 by phosphorylation. When STAT3 activation was blocked by JAK2/STAT3 inhibitor AG490, RANKL failed to shut down CXCR6 expression during osteoclastogenesis. However, CXCL16 alone did not augment RANKL-mediated osteoclast differentiation and did not alter RANKL-receptor RANK mRNA expression. These results demonstrate that reduction of CXCL16-CXCR6 is critical in RANKL-mediated osteoclastogenesis, which is mainly through the activation of JAK2/STAT3 signaling. CXCL16-CXCR6 axis may become a novel target for the therapeutic intervention of bone resorbing diseases such as rheumatoid arthritis and osteoporosis.« less

Curcumin blocks RON tyrosine kinase-mediated invasion of breast carcinoma cells.

PubMed

Narasimhan, Madhusudhanan; Ammanamanchi, Sudhakar

2008-07-01

We have recently shown that macrophage-stimulating protein (MSP) promotes the invasion of recepteur d'origine nantais (RON), a tyrosine kinase receptor-positive MDA-MB-231, MDA-MB-468 breast cancer cells, and also identified the regulatory elements required for RON gene expression. In this report, we have analyzed the efficacy of a chemopreventive agent, curcumin, in blocking RON tyrosine kinase-mediated invasion of breast cancer cells. Reverse transcription-PCR and Western analysis indicated the down-regulation of the RON message and protein, respectively, in MDA-MB-231 and MDA-MB-468 cells. Significantly, curcumin-mediated inhibition of RON expression resulted in the blockade of RON ligand, MSP-induced invasion of breast cancer cells. We have identified two putative nuclear factor-kappaB p65 subunit binding sites on the RON promoter. Using chromatin immunoprecipitation analysis and site-directed mutagenesis of the RON promoter, we have confirmed the binding of p65 to the RON promoter. Our data show that curcumin reduces RON expression by affecting p65 protein expression and transcriptional activity. Treatment of MDA-MB-231 cells with pyrrolidine dithiocarbamate, an inhibitor of p65, or small interfering RNA knockdown of p65, blocked RON gene expression and MSP-mediated invasion of MDA-MB-231 cells. This is the first report showing the regulation of human RON gene expression by nuclear factor-kappaB and suggests a potential therapeutic role for curcumin in blocking RON tyrosine kinase-mediated invasion of carcinoma cells.

CYT387, a selective JAK1/JAK2 inhibitor: in vitro assessment of kinase selectivity and preclinical studies using cell lines and primary cells from polycythemia vera patients.

PubMed

Pardanani, A; Lasho, T; Smith, G; Burns, C J; Fantino, E; Tefferi, A

2009-08-01

Somatic mutations in Janus kinase 2 (JAK2), including JAK2V617F, result in dysregulated JAK-signal transducer and activator transcription (STAT) signaling, which is implicated in myeloproliferative neoplasm (MPN) pathogenesis. CYT387 is an ATP-competitive small molecule that potently inhibits JAK1/JAK2 kinases (IC(50)=11 and 18 nM, respectively), with significantly less activity against other kinases, including JAK3 (IC(50)=155 nM). CYT387 inhibits growth of Ba/F3-JAK2V617F and human erythroleukemia (HEL) cells (IC(50) approximately 1500 nM) or Ba/F3-MPLW515L cells (IC(50)=200 nM), but has considerably less activity against BCR-ABL harboring K562 cells (IC=58 000 nM). Cell lines harboring mutated JAK2 alleles (CHRF-288-11 or Ba/F3-TEL-JAK2) were inhibited more potently than the corresponding pair harboring mutated JAK3 alleles (CMK or Ba/F3-TEL-JAK3), and STAT-5 phosphorylation was inhibited in HEL cells with an IC(50)=400 nM. Furthermore, CYT387 selectively suppressed the in vitro growth of erythroid colonies harboring JAK2V617F from polycythemia vera (PV) patients, an effect that was attenuated by exogenous erythropoietin. Overall, our data indicate that the JAK1/JAK2 selective inhibitor CYT387 has potential for efficacious treatment of MPN harboring mutated JAK2 and MPL alleles.

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

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.

The Amelioration of Myelofibrosis with Thrombocytopenia by a JAK1/2 Inhibitor, Ruxolitinib, in a Post-polycythemia Vera Myelofibrosis Patient with a JAK2 Exon 12 Mutation.

PubMed

Ikeda, Kazuhiko; Ueda, Koki; Sano, Takahiro; Ogawa, Kazuei; Ikezoe, Takayuki; Hashimoto, Yuko; Morishita, Soji; Komatsu, Norio; Ohto, Hitoshi; Takeishi, Yasuchika

2017-01-01

Less than 5% of patients with polycythemia vera (PV) show JAK2 exon 12 mutations. Although PV patients with JAK2 exon 12 mutations are known to develop post-PV myelofibrosis (MF) as well as PV with JAK2V617F, the role of JAK inhibitors in post-PV MF patients with JAK2 exon 12 mutations remains unknown. We describe how treatment with a JAK1/2 inhibitor, ruxolitinib, led to the rapid amelioration of marrow fibrosis, erythrocytosis and thrombocytopenia in a 77-year-old man with post-PV MF who carried a JAK2 exon 12 mutation (JAK2H538QK539L). This case suggests that ruxolitinib is a treatment option for post-PV MF in patients with thrombocytopenia or JAK2 exon 12 mutations.

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.

Resveratrol induces cell cycle arrest and apoptosis in malignant NK cells via JAK2/STAT3 pathway inhibition.

PubMed

Quoc Trung, Ly; Espinoza, J Luis; Takami, Akiyoshi; Nakao, Shinji

2013-01-01

Natural killer (NK) cell malignancies, particularly aggressive NK cell leukaemias and lymphomas, have poor prognoses. Although recent regimens with L-asparaginase substantially improved outcomes, novel therapeutic approaches are still needed to enhance clinical response. Resveratrol, a naturally occurring polyphenol, has been extensively studied for its anti-inflammatory, cardioprotective and anti-cancer activities. In this study, we investigated the potential anti-tumour activities of resveratrol against the NK cell lines KHYG-1, NKL, NK-92 and NK-YS. Resveratrol induced robust G0/G1 cell cycle arrest, significantly suppressed cell proliferation and induced apoptosis in a dose- and time-dependent manner for all four cell lines. In addition, resveratrol suppressed constitutively active STAT3 in all the cell lines and inhibited JAK2 phosphorylation but had no effect on other upstream mediators of STAT3 activation, such as PTEN, TYK2, and JAK1. Resveratrol also induced downregulation of the anti-apoptotic proteins MCL1 and survivin, two downstream effectors of the STAT3 pathway. Finally, resveratrol induced synergistic effect on the apoptotic and antiproliferative activities of L-asparaginase against KHYG-1, NKL and NK-92 cells. These results suggest that resveratrol may have therapeutic potential against NK cell malignancies. Furthermore, our finding that resveratrol is a bonafide JAK2 inhibitor extends its potential benefits to other diseases with dysregulated JAK2 signaling.

Resveratrol Induces Cell Cycle Arrest and Apoptosis in Malignant NK Cells via JAK2/STAT3 Pathway Inhibition

PubMed Central

Quoc Trung, Ly; Espinoza, J. Luis; Takami, Akiyoshi; Nakao, Shinji

2013-01-01

Natural killer (NK) cell malignancies, particularly aggressive NK cell leukaemias and lymphomas, have poor prognoses. Although recent regimens with L-asparaginase substantially improved outcomes, novel therapeutic approaches are still needed to enhance clinical response. Resveratrol, a naturally occurring polyphenol, has been extensively studied for its anti-inflammatory, cardioprotective and anti-cancer activities. In this study, we investigated the potential anti-tumour activities of resveratrol against the NK cell lines KHYG-1, NKL, NK-92 and NK-YS. Resveratrol induced robust G0/G1 cell cycle arrest, significantly suppressed cell proliferation and induced apoptosis in a dose- and time-dependent manner for all four cell lines. In addition, resveratrol suppressed constitutively active STAT3 in all the cell lines and inhibited JAK2 phosphorylation but had no effect on other upstream mediators of STAT3 activation, such as PTEN, TYK2, and JAK1. Resveratrol also induced downregulation of the anti-apoptotic proteins MCL1 and survivin, two downstream effectors of the STAT3 pathway. Finally, resveratrol induced synergistic effect on the apoptotic and antiproliferative activities of L-asparaginase against KHYG-1, NKL and NK-92 cells. These results suggest that resveratrol may have therapeutic potential against NK cell malignancies. Furthermore, our finding that resveratrol is a bonafide JAK2 inhibitor extends its potential benefits to other diseases with dysregulated JAK2 signaling. PMID:23372833

JAK2-V617F-induced MAPK activity is regulated by PI3K and acts synergistically with PI3K on the proliferation of JAK2-V617F-positive cells

PubMed Central

Wolf, Alexandra; Eulenfeld, René; Gäbler, Karoline; Rolvering, Catherine; Haan, Serge; Behrmann, Iris; Denecke, Bernd; Haan, Claude; Schaper, Fred

2013-01-01

The identification of a constitutively active JAK2 mutant, namely JAK2-V617F, was a milestone in the understanding of Philadelphia chromosome-negative myeloproliferative neoplasms. The JAK2-V617F mutation confers cytokine hypersensitivity, constitutive activation of the JAK-STAT pathway, and cytokine-independent growth. In this study we investigated the mechanism of JAK2-V617F-dependent signaling with a special focus on the activation of the MAPK pathway. We observed JAK2-V617F-dependent deregulated activation of the multi-site docking protein Gab1 as indicated by constitutive, PI3K-dependent membrane localization and tyrosine phosphorylation of Gab1. Furthermore, we demonstrate that PI3K signaling regulates MAPK activation in JAK2-V617F-positve cells. This cross-regulation of the MAPK pathway by PI3K affects JAK2-V617F-specific target gene induction, erythroid colony formation, and regulates proliferation of JAK2-V617F-positive patient cells in a synergistically manner. PMID:24069558

The nucleocapsid protein of measles virus blocks host interferon response

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

Takayama, Ikuyo; Sato, Hiroki; Watanabe, Akira

2012-03-01

Measles virus (MV) belongs to the genus Morbillivirus of the family Paramyxoviridae. A number of paramyxoviruses inhibit host interferon (IFN) signaling pathways in host immune systems by various mechanisms. Inhibition mechanisms have been described for many paramyxoviruses. Although there are inconsistencies among previous reports concerning MV, it appears that P/V/C proteins interfere with the pathways. In this study, we confirmed the effects of MV P gene products of a wild MV strain on IFN pathways and examined that of other viral proteins on it. Interestingly, we found that N protein acts as an IFN-{alpha}/{beta} and {gamma}-antagonist as strong as Pmore » gene products. We further investigated the mechanisms of MV-N inhibition, and revealed that MV-N blocks the nuclear import of activated STAT without preventing STAT and Jak activation or STAT degradation, and that the nuclear translocation of MV-N is important for the inhibition. The inhibitory effect of the N protein was observed as a common feature of other morbilliviruses. The results presented in this report suggest that N protein of MV as well as P/V/C proteins is involved in the inhibition of host IFN signaling pathways.« less

Discovery and Optimization of a Novel Series of Highly Selective JAK1 Kinase Inhibitors.

PubMed

Grimster, Neil P; Anderson, Erica; Alimzhanov, Marat; Bebernitz, Geraldine; Bell, Kirsten; Chuaqui, Claudio; Deegan, Tracy; Ferguson, Andrew D; Gero, Thomas; Harsch, Andreas; Huszar, Dennis; Kawatkar, Aarti; Kettle, Jason Grant; Lyne, Paul D; Read, Jon A; Rivard Costa, Caroline; Ruston, Linette; Schroeder, Patricia; Shi, Jie; Su, Qibin; Throner, Scott; Toader, Dorin; Vasbinder, Melissa Marie; Woessner, Richard; Wang, Haixia; Wu, Allan; Ye, Minwei; Zheng, Weijia; Zinda, Michael

2018-06-01

Herein, we report the discovery and characterization of a novel series of pyrimidine based JAK1 inhibitors. Optimization of these ATP competitive compounds was guided by X-ray crystallography and a structure-based drug design approach, focusing on selectivity, potency, and pharmaceutical properties. The best compound, 24, displayed remarkable JAK1 selectivity (~1000-fold vs JAK2,3 and TYK2), as well as a good kinase selectivity profile. Moreover, a dose-dependent reduction in pSTAT3, a downstream marker of JAK1 inhibition, was observed when 24 was examined in vivo.

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

Glycosaminoglycans mediate retention of the poxvirus type I interferon binding protein at the cell surface to locally block interferon antiviral responses

PubMed Central

Montanuy, Imma; Alejo, Ali; Alcami, Antonio

2011-01-01

Eradication of smallpox was accomplished 30 yr ago, but poxviral infections still represent a public health concern due to the potential release of variola virus or the emergence of zoonotic poxviruses, such as monkeypox virus. A critical determinant of poxvirus virulence is the inhibition of interferons (IFNs) by the virus-encoded type I IFN-binding protein (IFNα/βBP). This immunomodulatory protein is secreted and has the unique property of interacting with the cell surface in order to prevent IFN-mediated antiviral responses. However, the mechanism of its attachment to the cell surface remains unknown. Using surface plasmon resonance and cell-binding assays, we report that the IFNα/βBP from vaccinia virus, the smallpox vaccine, interacts with cell surface glycosaminoglycans (GAGs). Analysis of the contribution of different regions of the protein to cell surface binding demonstrated that clusters of basic residues in the first immunoglobulin domain mediate GAG interactions. Furthermore, mutation of the GAG-interaction motifs does not affect its IFN-binding and -blocking capacity. Functional conservation of GAG-binding sites is demonstrated for the IFNα/βBP from variola and monkeypox viruses, extending our understanding of immune modulation by the most virulent human poxviruses. These results are relevant for the design of improved vaccines and intervention strategies.—Montanuy, I., Alejo, A., Alcami, A. Glycosaminoglycans mediate retention of the poxvirus type I interferon binding protein at the cell surface to locally block interferon antiviral responses. PMID:21372110

Importin-α-mediated nucleolar localization of potato mop-top virus TRIPLE GENE BLOCK1 (TGB1) protein facilitates virus systemic movement, whereas TGB1 self-interaction is required for cell-to-cell movement in Nicotiana benthamiana.

PubMed

Lukhovitskaya, Nina I; Cowan, Graham H; Vetukuri, Ramesh R; Tilsner, Jens; Torrance, Lesley; Savenkov, Eugene I

2015-03-01

Recently, it has become evident that nucleolar passage of movement proteins occurs commonly in a number of plant RNA viruses that replicate in the cytoplasm. Systemic movement of Potato mop-top virus (PMTV) involves two viral transport forms represented by a complex of viral RNA and TRIPLE GENE BLOCK1 (TGB1) movement protein and by polar virions that contain the minor coat protein and TGB1 attached to one extremity. The integrity of polar virions ensures the efficient movement of RNA-CP, which encodes the virus coat protein. Here, we report the involvement of nuclear transport receptors belonging to the importin-α family in nucleolar accumulation of the PMTV TGB1 protein and, subsequently, in the systemic movement of the virus. Virus-induced gene silencing of two importin-α paralogs in Nicotiana benthamiana resulted in significant reduction of TGB1 accumulation in the nucleus, decreasing the accumulation of the virus progeny in upper leaves and the loss of systemic movement of RNA-CP. PMTV TGB1 interacted with importin-α in N. benthamiana, which was detected by bimolecular fluorescence complementation in the nucleoplasm and nucleolus. The interaction was mediated by two nucleolar localization signals identified by bioinformatics and mutagenesis in the TGB1 amino-terminal domain. Our results showed that while TGB1 self-interaction is needed for cell-to-cell movement, importin-α-mediated nucleolar targeting of TGB1 is an essential step in establishing the efficient systemic infection of the entire plant. These results enabled the identification of two separate domains in TGB1: an internal domain required for TGB1 self-interaction and cell-to-cell movement and the amino-terminal domain required for importin-α interaction in plants, nucleolar targeting, and long-distance movement. © 2015 American Society of Plant Biologists. All Rights Reserved.

RANKL downregulates cell surface CXCR6 expression through JAK2/STAT3 signaling pathway during osteoclastogenesis.

PubMed

Li, Changhong; Zhao, Jinxia; Sun, Lin; Yao, Zhongqiang; Liu, Rui; Huang, Jiansheng; Liu, Xiangyuan

2012-12-14

The receptor activator of nuclear factor-κB ligand (RANKL), as a member of the tumor necrosis factor (TNF) family, plays an essential role in osteoclast differentiation and function. Chemokines and their receptors have recently been shown to play critical roles in osteoclastogenesis, however, whether CXCL16-CXCR6 plays role in RANKL-mediated osteoclastogenesis is unknown. In this study, we first reported that RANKL decreased CXCR6 in a dose-dependent manner, which may be through deactivation of Akt and STAT3 signaling induced by CXCL16. Interestingly, RANKL-mediated CXCR6 reduction may be associated to the activation of STAT3 by phosphorylation. When STAT3 activation was blocked by JAK2/STAT3 inhibitor AG490, RANKL failed to shut down CXCR6 expression during osteoclastogenesis. However, CXCL16 alone did not augment RANKL-mediated osteoclast differentiation and did not alter RANKL-receptor RANK mRNA expression. These results demonstrate that reduction of CXCL16-CXCR6 is critical in RANKL-mediated osteoclastogenesis, which is mainly through the activation of JAK2/STAT3 signaling. CXCL16-CXCR6 axis may become a novel target for the therapeutic intervention of bone resorbing diseases such as rheumatoid arthritis and osteoporosis. Copyright © 2012 Elsevier Inc. All rights reserved.

Complement-mediated bactericidal activity of anti-factor H binding protein monoclonal antibodies against the meningococcus relies upon blocking factor H binding.

PubMed

Giuntini, Serena; Reason, Donald C; Granoff, Dan M

2011-09-01

Binding of the complement-downregulating protein factor H (fH) to the surface of the meningococcus is important for survival of the organism in human serum. The meningococcal vaccine candidate factor H binding protein (fHbp) is an important ligand for human fH. While some fHbp-specific monoclonal antibodies (MAbs) block binding of fH to fHbp, the stoichiometry of blocking in the presence of high serum concentrations of fH and its effect on complement-mediated bactericidal activity are unknown. To investigate this question, we constructed chimeric antibodies in which the human IgG1 constant region was paired with three murine fHbp-specific binding domains designated JAR 3, JAR 5, and MAb502. By surface plasmon resonance, the association rates for binding of all three MAbs to immobilized fHbp were >50-fold higher than that for binding of fH to fHbp, and the MAb dissociation rates were >500-fold lower than that for fH. While all three MAbs elicited similar C1q-dependent C4b deposition on live bacteria (classical complement pathway), only those antibodies that inhibited binding of fH to fHbp (JAR 3 and JAR 5) had bactericidal activity with human complement. MAb502, which did not inhibit fH binding, had complement-mediated bactericidal activity only when tested with fH-depleted human complement. When an IgG1 anti-fHbp MAb binds to sparsely exposed fHbp on the bacterial surface, there appears to be insufficient complement activation for bacteriolysis unless fH binding also is inhibited. The ability of fHbp vaccines to elicit protective antibodies, therefore, is likely to be enhanced if the antibody repertoire is of high avidity and includes fH-blocking activity.

Identification of a novel functional JAK1 S646P mutation in acute lymphoblastic leukemia

PubMed Central

Hu, Liangding; Ning, Hongmei; Jiang, Min; Wang, Danhong; Liu, Tingting; Zhang, Bin; Chen, Hu

2017-01-01

The survival rate of childhood acute lymphoblastic leukemia (ALL) is approaching 90%, while the prognosis of adults remains poor due to the limited therapeutic approaches. In order to identify new targets for ALL, we performed whole-exome sequencing on four adults with B-ALL and discovered a somatic JAK1 S646P mutation. Sanger sequencing of JAK1 was conducted on 53 ALL patients, and two cases exhibited A639G and P960S mutations separately. Functional studies demonstrated that only JAK1 S646P mutation could activate multiple signaling pathways, drive cytokine-independent cell growth, and promote proliferation of malignant cells in nude mice. Moreover, a high sensitivity to the JAK1/2 inhibitor ruxolitinib was observed in S646P mutant model. Exploration in a total of 209 ALL cases showed that JAK1 mutations occur at a frequency of 10.5% in T-ALL (2/19) and 1.6% in B-ALL (3/190). Collectively, our results suggested that JAK1 S646P is an activating mutation in vitro and in vivo. JAK-STAT pathway might represent a promising therapeutic target for ALL. PMID:28410228

Identification of a novel functional JAK1 S646P mutation in acute lymphoblastic leukemia.

PubMed

Li, Qian; Li, Botao; Hu, Liangding; Ning, Hongmei; Jiang, Min; Wang, Danhong; Liu, Tingting; Zhang, Bin; Chen, Hu

2017-05-23

The survival rate of childhood acute lymphoblastic leukemia (ALL) is approaching 90%, while the prognosis of adults remains poor due to the limited therapeutic approaches. In order to identify new targets for ALL, we performed whole-exome sequencing on four adults with B-ALL and discovered a somatic JAK1 S646P mutation. Sanger sequencing of JAK1 was conducted on 53 ALL patients, and two cases exhibited A639G and P960S mutations separately. Functional studies demonstrated that only JAK1 S646P mutation could activate multiple signaling pathways, drive cytokine-independent cell growth, and promote proliferation of malignant cells in nude mice. Moreover, a high sensitivity to the JAK1/2 inhibitor ruxolitinib was observed in S646P mutant model. Exploration in a total of 209 ALL cases showed that JAK1 mutations occur at a frequency of 10.5% in T-ALL (2/19) and 1.6% in B-ALL (3/190). Collectively, our results suggested that JAK1 S646P is an activating mutation in vitro and in vivo. JAK-STAT pathway might represent a promising therapeutic target for ALL.

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.

JAK family members: Molecular cloning, expression profiles and their roles in leptin influencing lipid metabolism in Synechogobius hasta.

PubMed

Wu, Kun; Tan, Xiao-Ying; Xu, Yi-Huan; Shi, Xi; Fan, Yao-Fang; Li, Dan-Dan; Liu, Xu

2017-01-01

Janus kinase (JAK) is a family of non-receptor tyrosine kinases that participate in transducing cytokine signals from the external environment to the nucleus in various biological processes. Currently, information about their genes structure and evolutionary history has been extensively studied in mammals as well as in several fish species. By contrast, limited reports have addressed potential role of diverse JAK in signaling responses to leptin in fish. In this study, we identified and characterized five JAK members of Synechogobius hasta. Compared to mammals, more members of the JAK family were found in S. hasta, which provided evidence that the JAK family members had arisen by the whole genome duplications during vertebrate evolution. For protein structure, all of these members possessed similar domains compared with those of mammals. Their mRNAs were expressed in a wide range of tissues, but at the different levels. Incubation in vitro of freshly isolated hepatocytes of S. hasta with different concentrations of recombinant human leptin decreased the intracellular triglyceride content and lipogenic genes expression, and increased mRNA expression of several JAK and lipolytic genes. AG490, a specific inhibitor of JAK, reversed leptin-induced effects on TG content and JAK2a, JAK2b, hormone-sensitive lipase (HSL2) and acetyl-CoA carboxylase (ACCa), indicating that the JAK2a/b may have mediated the actions of leptin on lipid metabolism at transcriptional level. Copyright © 2016 Elsevier Inc. All rights reserved.

Aggressive B-cell lymphomas in patients with myelofibrosis receiving JAK1/2 inhibitor therapy.

PubMed

Porpaczy, Edit; Tripolt, Sabrina; Hoelbl-Kovacic, Andrea; Gisslinger, Bettina; Bago-Horvath, Zsuzsanna; Casanova-Hevia, Emilio; Clappier, Emmanuelle; Decker, Thomas; Fajmann, Sabine; Fux, Daniela A; Greiner, Georg; Gueltekin, Sinan; Heller, Gerwin; Herkner, Harald; Hoermann, Gregor; Kiladjian, Jean-Jacques; Kolbe, Thomas; Kornauth, Christoph; Krauth, Maria-Theresa; Kralovics, Robert; Muellauer, Leonhard; Mueller, Mathias; Prchal-Murphy, Michaela; Putz, Eva Maria; Raffoux, Emmanuel; Schiefer, Ana-Iris; Schmetterer, Klaus; Schneckenleithner, Christine; Simonitsch-Klupp, Ingrid; Skrabs, Cathrin; Sperr, Wolfgang R; Staber, Philipp Bernhard; Strobl, Birgit; Valent, Peter; Jaeger, Ulrich; Gisslinger, Heinz; Sexl, Veronika

2018-06-14

Inhibition of Janus-kinase 1/2 (JAK1/2) is a mainstay to treat myeloproliferative neoplasms (MPN). Sporadic observations reported the co-incidence of B-cell non-Hodgkin lymphomas during treatment of MPN with JAK1/2 inhibitors. We assessed 626 MPN patients including 69 with myelofibrosis receiving JAK1/2 inhibitors for lymphoma development. B-cell lymphomas evolved in 4/69 patients (5.8%) upon JAK1/2 inhibition compared to 2/557 (0.36%) with conventional treatment (16-fold increased risk). A similar 15-fold increase was observed in an independent cohort of 929 MPN patients. Considering primary myelofibrosis only (N=216), 3 lymphomas were observed in 31 inhibitor-treated patients (9.7%) versus 1/185 controls (0.54%). Lymphomas were of aggressive B-cell type, extra-nodal or leukemic with high MYC expression in the absence of JAK2 V617F or other MPN-associated mutations. Median time from initiation of inhibitor therapy to lymphoma diagnosis was 25 months. Clonal immunoglobulin gene rearrangements were already detected in the bone marrow during myelofibrosis in 16.3% of patients. Lymphomas occurring during JAK1/2 inhibitor treatment were preceded by a pre-existing B-cell clone in all 3 patients tested. Sequencing verified clonal identity in 2 patients. The effects of JAK1/2 inhibition were mirrored in Stat1 -/- mice: 16/24 mice developed a spontaneous myeloid hyperplasia with the concomitant presence of aberrant B-cells. Transplantations of bone marrow from diseased mice unmasked the outgrowth of a malignant B-cell clone evolving into aggressive B-cell leukemia-lymphoma. We conclude that JAK/STAT1 pathway inhibition in myelofibrosis is associated with an elevated frequency of aggressive B-cell lymphomas. Detection of a pre-existing B-cell clone may identify individuals at risk. Copyright © 2018 American Society of Hematology.

Dynamin-related protein 1 mediates mitochondria-dependent apoptosis in chlorpyrifos-treated SH-SY5Y cells.

PubMed

Park, Jae Hyeon; Ko, Juyeon; Hwang, Jungwook; Koh, Hyun Chul

2015-12-01

Recent studies have demonstrated that dynamin-related protein 1 (Drp1), a mitochondrial fission protein, mediates mitochondria-dependent apoptosis through mitochondrial division. However, little is known about the mechanism by which Drp1 modulates apoptosis in response to chlorpyrifos (CPF)-induced toxicity. In this study, we determined that CPF-induced mitochondrial apoptosis is mediated by Drp1 translocation in SH-SY5Y human neuroblastoma cells. Our results showed that CPF treatment induced intrinsic apoptosis by activating caspase-9, caspase-3, and cytochrome c release in SH-SY5Y cells. Cytosolic Drp1 translocated to the mitochondria in CPF-treated cells and was phosphorylated at Ser616. Treating cells with CPF induced the generation of reactive oxygen species (ROS) and activation of mitogen-activated protein kinases (MAPKs). Inhibiting this ROS generation and MAPK activation abolished CPF-induced expression of phospho-Drp1. Furthermore, Drp1 was required for p53 to translocate to the mitochondria under CPF-induced oxidative stress. Treating cells with mitochondrial-division inhibitor-1 (mdivi-1), which blocks Drp1 translocation, increased the viability of CPF-treated cells by abrogating Drp1 translocation and caspase-3 activation. Specifically, pretreating cells with mdivi-1 inhibited Bax translocation to the mitochondria by blocking p53 signaling. Taken together, these data reveal a novel mechanism by which Drp1 activates mitochondrial-dependent apoptosis and indicate that inhibiting Dpr1 function can protect against CPF-induced cytotoxicity. We propose that inhibiting Drp1 is a possible therapeutic approach for pesticide-induced toxicity when hyperactivated Drp1 contributes to pathology. Crown Copyright © 2015. Published by Elsevier B.V. All rights reserved.

Controlling nuclear JAKs and STATs for specific gene activation by IFN{gamma}

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

Noon-Song, Ezra N.; Ahmed, Chulbul M.; Dabelic, Rea

2011-07-08

Highlights: {yields} Gamma interferon (IFN{gamma}) and its receptor subunit, IFNGR1, interact with the promoter region of IFN{gamma}-associated genes along with transcription factor STAT1{alpha}. {yields} We show that activated Janus kinases pJAK2 and pJAK1 also associate with IFNGR1 in the nucleus. {yields} The activated Janus kinases are responsible for phosphorylation of tyrosine 41 on histone H3, an important epigenetic event for specific gene activation. -- Abstract: We previously showed that gamma interferon (IFN{gamma}) and its receptor subunit, IFNGR1, interacted with the promoter region of IFN{gamma}-activated genes along with transcription factor STAT1{alpha}. Recent studies have suggested that activated Janus kinases pJAK2 andmore » pJAK1 also played a role in gene activation by phosphorylation of histone H3 on tyrosine 41. This study addresses the question of the role of activated JAKs in specific gene activation by IFN{gamma}. We carried out chromatin immunoprecipitation (ChIP) followed by PCR in IFN{gamma} treated WISH cells and showed association of pJAK1, pJAK2, IFNGR1, and STAT1 on the same DNA sequence of the IRF-1 gene promoter. The {beta}-actin gene, which is not activated by IFN{gamma}, did not show this association. The movement of activated JAK to the nucleus and the IRF-1 promoter was confirmed by the combination of nuclear fractionation, confocal microscopy and DNA precipitation analysis using the biotinylated GAS promoter. Activated JAKs in the nucleus was associated with phosphorylated tyrosine 41 on histone H3 in the region of the GAS promoter. Unphosphorylated JAK2 was found to be constitutively present in the nucleus and was capable of undergoing activation in IFN{gamma} treated cells, most likely via nuclear IFNGR1. Association of pJAK2 and IFNGR1 with histone H3 in IFN{gamma} treated cells was demonstrated by histone H3 immunoprecipitation. Unphosphorylated STAT1 protein was associated with histone H3 of untreated cells. IFN

Spatiotemporal regulation of cell fusion by JNK and JAK/STAT signaling during Drosophila wound healing.

PubMed

Lee, Ji-Hyun; Lee, Chan-Wool; Park, Si-Hyoung; Choe, Kwang-Min

2017-06-01

Cell-cell fusion is widely observed during development and disease, and imposes a dramatic change on participating cells. Cell fusion should be tightly controlled, but the underlying mechanism is poorly understood. Here, we found that the JAK/STAT pathway suppressed cell fusion during wound healing in the Drosophila larval epidermis, restricting cell fusion to the vicinity of the wound. In the absence of JAK/STAT signaling, a large syncytium containing a 3-fold higher number of nuclei than observed in wild-type tissue formed in wounded epidermis. The JAK/STAT ligand-encoding genes upd2 and upd3 were transcriptionally induced by wounding, and were required for suppressing excess cell fusion. JNK (also known as Basket in flies) was activated in the wound vicinity and activity peaked at ∼8 h after injury, whereas JAK/STAT signaling was activated in an adjoining concentric ring and activity peaked at a later stage. Cell fusion occurred primarily in the wound vicinity, where JAK/STAT activation was suppressed by fusion-inducing JNK signaling. JAK/STAT signaling was both necessary and sufficient for the induction of βPS integrin (also known as Myospheroid) expression, suggesting that the suppression of cell fusion was mediated at least in part by integrin protein. © 2017. Published by The Company of Biologists Ltd.

HIV-1-Specific IgA Monoclonal Antibodies from an HIV-1 Vaccinee Mediate Galactosylceramide Blocking and Phagocytosis

PubMed Central

2018-01-01

ABSTRACT Vaccine-elicited humoral immune responses comprise an array of antibody forms and specificities, with only a fraction contributing to protective host immunity. Elucidation of antibody effector functions responsible for protective immunity against human immunodeficiency virus type 1 (HIV-1) acquisition is a major goal for the HIV-1 vaccine field. Immunoglobulin A (IgA) is an important part of the host defense against pathogens; however, little is known about the role of vaccine-elicited IgA and its capacity to mediate antiviral functions. To identify the antiviral functions of HIV-1-specific IgA elicited by vaccination, we cloned HIV-1 envelope-specific IgA monoclonal antibodies (MAbs) by memory B cell cultures from peripheral blood mononuclear cells from an RV144 vaccinee and produced two IgA clonal cell lines (HG129 and HG130) producing native, nonrecombinant IgA MAbs. The HG129 and HG130 MAbs mediated phagocytosis by monocytes, and HG129 blocked HIV-1 Env glycoprotein binding to galactosylceramide, an alternative HIV-1 receptor. These findings elucidate potential antiviral functions of vaccine-elicited HIV-1 envelope-specific IgA that may act to block HIV-1 acquisition at the portal of entry by preventing HIV-1 binding to galactosylceramide and mediating antibody Fc receptor-mediated virion phagocytosis. Furthermore, these findings highlight the complex and diverse interactions of vaccine-elicited IgA with pathogens that depend on IgA fine specificity and form (e.g., multimeric or monomeric) in the systemic circulation and mucosal compartments. IMPORTANCE Host-pathogen interactions in vivo involve numerous immune mechanisms that can lead to pathogen clearance. Understanding the nature of antiviral immune mechanisms can inform the design of efficacious HIV-1 vaccine strategies. Evidence suggests that both neutralizing and nonneutralizing antibodies can mediate some protection against HIV in animal models. Although numerous studies have characterized the

Recent Progress in JAK Inhibitors for the Treatment of Rheumatoid Arthritis.

PubMed

Nakayamada, Shingo; Kubo, Satoshi; Iwata, Shigeru; Tanaka, Yoshiya

2016-10-01

Rheumatoid arthritis (RA) is a systemic autoimmune disease characterized by synovial inflammation and joint destruction. Considerable advance in the treatment of RA has been made following the advent of biological disease-modifying anti-rheumatic drugs (DMARDs). However, these biologics require intravenous or subcutaneous injection and some patients fail to respond to biological DMARDs or lose their primary response. Various cytokines and cell surface molecules bind to receptors on the cell surface, resulting in the activation of various cell signaling pathways, including phosphorylation of kinase proteins. Among these kinases, the non-receptor tyrosine kinase family Janus kinase (JAK) plays a pivotal role in the pathological processes of RA. Several JAK inhibitors have been developed as new therapies for patients with RA. These are oral synthetic DMARDs that inhibit JAK1, 2, and 3. One JAK inhibitor, tofacitinib, has already been approved in many countries. Results of phase III clinical trials using a JAK1/2 inhibitor, baricitinib, have shown feasible efficacy and tolerable safety. Both drugs are effective in patients who showed inadequate response to biological DMARDs as well as synthetic DMARDs. In addition, clinical phase III trials using filgotinib and ABT-494, specific JAK1 inhibitors, are currently underway. JAK inhibitors are novel therapies for RA, but further studies are needed to determine their risk-benefit ratio and selection of the most appropriate patients for such therapy.

JAK/STAT signaling pathway-mediated immune response in silkworm (Bombyx mori) challenged by Beauveria bassiana.

PubMed

Geng, Tao; Lv, Ding-Ding; Huang, Yu-Xia; Hou, Cheng-Xiang; Qin, Guang-Xing; Guo, Xi-Jie

2016-12-20

Innate immunity was critical in insects defensive system and able to be induced by Janus kinase/signal transducer and activator of transcription cascade transduction (JAK/STAT) signaling pathway. Currently, it had been identified many JAK/STAT signaling pathway-related genes in silkworm, but little function was known on insect innate immunity. To explore the roles of JAK/STAT pathway in antifungal immune response in silkworm (Bombyx mori) against Beauveria bassiana infection, the expression patterns of B. mori C-type lectin 5 (BmCTL5) and genes encoding 6 components of JAK/STAT signaling pathway in silkworm challenged by B. bassiana were analyzed using quantitative real time PCR. Meanwhile the activation of JAK/STAT signaling pathway by various pathogenic micro-organisms and the affect of JAK/STAT signaling pathway inhibitors on antifungal activity in silkworm hemolymph was also detected. Moreover, RNAi assay of BmCTL5 and the affect on expression levels of signaling factors were also analyzed. We found that JAK/STAT pathway could be obviously activated in silkworm challenged with B. bassiana and had no response to bacteria and B. mori cytoplasmic polyhedrosis virus (BmCPV). However, the temporal expression patterns of JAK/STAT signaling pathway related genes were significantly different. B. mori downstream receptor kinase (BmDRK) might be a positive regulator of JAK/STAT signaling pathway in silkworm against B. bassiana infection. Moreover, antifungal activity assay showed that the suppression of JAK/STAT signaling pathway by inhibitors could significantly inhibit the antifungal activity in hemolymph and resulted in increased sensitivity of silkworm to B. bassiana infection, indicating that JAK/STAT signaling pathway might be involved in the synthesis and secretion of antifungal substances. The results of RNAi assays suggested that BmCTL5 might be one pattern recognition receptors for JAK/STAT signaling pathway in silkworm. These findings yield insights for better

The JAK2 46/1 haplotype predisposes to MPL-mutated myeloproliferative neoplasms

PubMed Central

Jones, Amy V.; Campbell, Peter J.; Beer, Philip A.; Schnittger, Susanne; Vannucchi, Alessandro M.; Zoi, Katerina; Percy, Melanie J.; McMullin, Mary Frances; Scott, Linda M.; Tapper, William; Silver, Richard T.; Oscier, David; Harrison, Claire N.; Grallert, Harald; Kisialiou, Aliaksei; Strike, Paul; Chase, Andrew J.; Green, Anthony R.

2010-01-01

The 46/1 JAK2 haplotype predisposes to V617F-positive myeloproliferative neoplasms, but the underlying mechanism is obscure. We analyzed essential thrombocythemia patients entered into the PT-1 studies and, as expected, found that 46/1 was overrepresented in V617F-positive cases (n = 404) versus controls (n = 1492, P = 3.9 × 10−11). The 46/1 haplotype was also overrepresented in cases without V617F (n = 347, P = .009), with an excess seen for both MPL exon 10 mutated and V617F, MPL exon 10 nonmutated cases. Analysis of further MPL-positive, V617F-negative cases confirmed an excess of 46/1 (n = 176, P = .002), but no association between MPL mutations and MPL haplotype was seen. An excess of 46/1 was also seen in JAK2 exon 12 mutated cases (n = 69, P = .002), and these mutations preferentially arose on the 46/1 chromosome (P = .029). No association between 46/1 and clinical or laboratory features was seen in the PT-1 cohort either with or without V617F. The excess of 46/1 in JAK2 exon 12 cases is compatible with both the “hypermutability” and “fertile ground” hypotheses, but the excess in MPL-mutated cases argues against the former. No difference in sequence, splicing, or expression of JAK2 was found on 46/1 compared with other haplotypes, suggesting that any functional difference of JAK2 on 46/1, if it exists, must be relatively subtle. PMID:20304805

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.

JAK and MPL mutations in myeloid malignancies.

PubMed

Tefferi, Ayalew

2008-03-01

The Janus family of non-receptor tyrosine kinases (JAK1, JAK2, JAK3 and tyrosine kinase 2) transduces signals downstream of type I and II cytokine receptors via signal transducers and activators of transcription (STATs). JAK3 is important in lymphoid and JAK2 in myeloid cell proliferation and differentiation. The thrombopoietin receptor MPL is one of several JAK2 cognate receptors and is essential for myelopoiesis in general and megakaryopoiesis in particular. Germline loss-of-function (LOF) JAK3 and MPL mutations cause severe combined immunodeficiency and congenital amegakaryocytic thrombocytopenia, respectively. Germline gain-of-function (GOF) MPL mutation (MPLS505N) causes familial thrombocytosis. Somatic JAK3 (e.g. JAK3A572V, JAK3V722I, JAK3P132T) and fusion JAK2 (e.g. ETV6-JAK2, PCM1-JAK2, BCR-JAK2) mutations have respectively been described in acute megakaryocytic leukemia and acute leukemia/chronic myeloid malignancies. However, current attention is focused on JAK2 (e.g. JAK2V617F, JAK2 exon 12 mutations) and MPL (e.g. MPLW515L/K/S, MPLS505N) mutations associated with myeloproliferative neoplasms (MPNs). A JAK2 mutation, primarily JAK2V617F, is invariably associated with polycythemia vera (PV). The latter mutation also occurs in the majority of patients with essential thrombocythemia (ET) or primary myelofibrosis (PMF). MPL mutational frequency in MPNs is substantially less (<10%). In general, despite a certain degree of genotype - phenotype correlations, the prognostic relevance of harbouring one of these mutations, or their allele burden when present, remains dubious. Regardless, based on the logical assumption that amplified JAK-STAT signalling is central to the pathogenesis of PV, ET and PMF, several anti-JAK2 tyrosine kinase inhibitors have been developed and are currently being tested in humans with these disorders.

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.

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.

A multi-level model accounting for the effects of JAK2-STAT5 signal modulation in erythropoiesis.

PubMed

Lai, Xin; Nikolov, Svetoslav; Wolkenhauer, Olaf; Vera, Julio

2009-08-01

We develop a multi-level model, using ordinary differential equations, based on quantitative experimental data, accounting for murine erythropoiesis. At the sub-cellular level, the model includes a description of the regulation of red blood cell differentiation through Epo-stimulated JAK2-STAT5 signalling activation, while at the cell population level the model describes the dynamics of (STAT5-mediated) red blood cell differentiation from their progenitors. Furthermore, the model includes equations depicting the hypoxia-mediated regulation of hormone erythropoietin blood levels. Take all together, the model constitutes a multi-level, feedback loop-regulated biological system, involving processes in different organs and at different organisational levels. We use our model to investigate the effect of deregulation in the proteins involved in the JAK2-STAT5 signalling pathway in red blood cells. Our analysis results suggest that down-regulation in any of the three signalling system components affects the hematocrit level in an individual considerably. In addition, our analysis predicts that exogenous Epo injection (an already existing treatment for several blood diseases) may compensate the effects of single down-regulation of Epo hormone level, STAT5 or EpoR/JAK2 expression level, and that it may be insufficient to counterpart a combined down-regulation of all the elements in the JAK2-STAT5 signalling cascade.

β-Estradiol-dependent activation of the JAK/STAT pathway requires p/CIP and CARM1.

PubMed

Coughlan, N; Thillainadesan, G; Andrews, J; Isovic, M; Torchia, J

2013-06-01

The steroid receptor coactivator p/CIP, also known as SRC-3, is an oncogene commonly amplified in breast and ovarian cancers. p/CIP is known to associate with coactivator arginine methyltransferase 1 (CARM1) on select estrogen responsive genes. We have shown, using a ChIP-on-chip approach, that in response to stimulation with 17β-estradiol (E2), the p/CIP/CARM1 complex is recruited to 204 proximal promoters in MCF-7 cells. Many of the complex target genes have been previously implicated in signaling pathways related to oncogenesis. Jak2, a member of the Jak/Stat signaling cascade, is one of the direct E2-dependent targets of the p/CIP/CARM1 complex. Following E2-treatment, histone modifications at the Jak2 promoter are reflective of a transcriptionally permissive gene, and modest changes in RNA and protein expression lead us to suggest that an additional factor(s) may be required for a more notable transcriptional and functional response. Bioinformatic examination of the 204 proximal promoter sequences of p/CIP/CARM1 targets supports the idea that transcription factor crosstalk is likely the favored mechanism of E2-dependent p/CIP/CARM1 complex recruitment. This data may have implications towards understanding the oncogenic role of p/CIP in breast cancer and ultimately allow for the identification of new prognostic indicators and/or viable therapeutic targets. Crown Copyright © 2013. Published by Elsevier B.V. All rights reserved.

Protein based Block Copolymers

PubMed Central

Rabotyagova, Olena S.; Cebe, Peggy; Kaplan, David L.

2011-01-01

Advances in genetic engineering have led to the synthesis of protein-based block copolymers with control of chemistry and molecular weight, resulting in unique physical and biological properties. The benefits from incorporating peptide blocks into copolymer designs arise from the fundamental properties of proteins to adopt ordered conformations and to undergo self-assembly, providing control over structure formation at various length scales when compared to conventional block copolymers. This review covers the synthesis, structure, assembly, properties, and applications of protein-based block copolymers. PMID:21235251

Cytokine-mediated inflammation, tumorigenesis, and disease-associated JAK/STAT/SOCS signaling circuits in the CNS.

PubMed

Campbell, Iain L

2005-04-01

Cytokines are plurifunctional mediators of cellular communication. The CNS biology of this family of molecules has been explored by transgenic approaches that targeted the expression of individual cytokine genes to specific cells in the CNS of mice. Such transgenic animals exhibit wide-ranging structural and functional alterations that are linked to the development of distinct neuroinflammatory responses and gene expression profiles specific for each cytokine. The unique actions of individual cytokines result from the activation of specific receptor-coupled cellular signal transduction pathways such as the JAK/STAT tyrosine kinase signaling cascade. The cerebral expression of various STATs, their activation, as well as that of the major physiological inhibitors of this pathway, SOCS1 and SOCS3, is highly regulated in a stimulus- and cell-specific fashion. The role of the key IFN signaling molecules STAT1 or STAT2 was studied in transgenic mice (termed GIFN) with astrocyte-production of IFN-alpha that were null or haploinsufficient for these STAT genes. Surprisingly, these animals developed either more severe and accelerated neurodegeneration with calcification and inflammation (GIFN/STAT1 deficient) or severe immunoinflammation and medulloblastoma (GIFN/STAT2 deficient). STAT dysregulation may result in a signal switch phenomenon in which one cytokine acquires the apparent function of an entirely different cytokine. Therefore, for cytokines such as the IFNs, the receptor-coupled signaling process is complex, involving the coexistence of multiple JAK/STAT as well as alternative pathways. The cellular compartmentalization and balance in the activity of these pathways ultimately determines the repertoire and nature of CNS cytokine actions.

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.

Therapeutic Efficacy of Suppressing the JAK/STAT Pathway in Multiple Models of EAE1

PubMed Central

Liu, Yudong; Holdbrooks, Andrew T.; De Sarno, Patrizia; Rowse, Amber L.; Yanagisawa, Lora L.; McFarland, Braden C.; Harrington, Laurie E.; Raman, Chander; Sabbaj, Steffanie; Benveniste, Etty N.; Qin, Hongwei

2014-01-01

Pathogenic T helper cells and myeloid cells are involved in the pathogenesis of Multiple Sclerosis (MS) and Experimental Autoimmune Encephalomyelitis (EAE), an animal model of MS. The JAK/STAT pathway is utilized by numerous cytokines for signaling, and is critical for development, regulation and termination of immune responses. Dysregulation of the JAK/STAT pathway has pathological implications in autoimmune and neuroinflammatory diseases. Many of the cytokines involved in MS/EAE, including IL-6, IL-12, IL-23, IFN-γ and GM-CSF, use the JAK/STAT pathway to induce biological responses. Thus, targeting JAKs has implications for treating autoimmune inflammation of the brain. We have utilized AZD1480, a JAK1/2 inhibitor, to investigate the therapeutic potential of inhibiting the JAK/STAT pathway in models of EAE. AZD1480 treatment inhibits disease severity in MOG-induced classical and atypical EAE models by preventing entry of immune cells into the brain, suppressing differentiation of Th1 and Th17 cells, deactivating myeloid cells, inhibiting STAT activation in the brain, and reducing expression of pro-inflammatory cytokines and chemokines. Treatment of SJL/J mice with AZD1480 delays disease onset of PLP-induced relapsing-remitting disease, reduces relapses and diminishes clinical severity. AZD1480 treatment was also effective in reducing ongoing paralysis induced by adoptive transfer of either pathogenic Th1 or Th17 cells. In vivo AZD1480 treatment impairs both the priming and expansion of T-cells, and attenuates antigen-presentation functions of myeloid cells. Inhibition of the JAK/STAT pathway has clinical efficacy in multiple pre-clinical models of MS, suggesting the feasibility of the JAK/STAT pathway as a target for neuroinflammatory diseases. PMID:24323580

miR-122-SOCS1-JAK2 axis regulates allergic inflammation and allergic inflammation-promoted cellular interactions

PubMed Central

Kim, Hanearl; Kim, Hyuna; Byun, Jaehwan; Park, Yeongseo; Lee, Hansoo; Lee, Yun Sil; Choe, Jongseon; Kim, Young Myeong; Jeoung, Dooil

2017-01-01

The regulatory role of suppressor of cytokine signaling 1 (SOCS1) in inflammation has been reported. However, its role in allergic inflammation has not been previously reported. SOCS1 mediated in vitro and in vivo allergic inflammation. Histone deacetylase-3 (HDAC3), a mediator of allergic inflammation, interacted with SOCS1, and miR-384 inhibitor, a positive regulator of HDAC3, induced features of allergic inflammation in an SOCS1-dependent manner. miRNA array analysis showed that the expression of miR-122 was decreased by antigen-stimulation. TargetScan analysis predicted the binding of miR-122 to the 3′-UTR of SOCS1. miR-122 inhibitor induced in vitro and in vivo allergic features in SOCS1-dependent manner. SOCS1 was necessary for allergic inflammation-promoted enhanced tumorigenic and metastatic potential of cancer cells. SOCS1 and miR-122 regulated cellular interactions involving cancer cells, mast cells and macrophages during allergic inflammation. SOCS1 mimetic peptide, D-T-H-F-R-T-F-R-S-H-S-D-Y-R-R-I, inhibited in vitro and in vivo allergic inflammation, allergic inflammation-promoted enhanced tumorigenic and metastatic potential of cancer cells, and cellular interactions during allergic inflammation. Janus kinase 2 (JAK2) exhibited binding to SOCS1 mimetic peptide and mediated allergic inflammation. Transforming growth factor- Δ1 (TGF-Δ1) was decreased during allergic inflammation and showed an anti-allergic effect. SOCS1 and JAK2 regulated the production of anti-allergic TGF-Δ1. Taken together, our results show that miR-122-SOCS1 feedback loop can be employed as a target for the development of anti-allergic and anti-cancer drugs. PMID:28968979

Preclinical characterization of GLPG0634, a selective inhibitor of JAK1, for the treatment of inflammatory diseases.

PubMed

Van Rompaey, Luc; Galien, René; van der Aar, Ellen M; Clement-Lacroix, Philippe; Nelles, Luc; Smets, Bart; Lepescheux, Liên; Christophe, Thierry; Conrath, Katja; Vandeghinste, Nick; Vayssiere, Béatrice; De Vos, Steve; Fletcher, Stephen; Brys, Reginald; van 't Klooster, Gerben; Feyen, Jean H M; Menet, Christel

2013-10-01

The JAKs receive continued interest as therapeutic targets for autoimmune, inflammatory, and oncological diseases. JAKs play critical roles in the development and biology of the hematopoietic system, as evidenced by mouse and human genetics. JAK1 is critical for the signal transduction of many type I and type II inflammatory cytokine receptors. In a search for JAK small molecule inhibitors, GLPG0634 was identified as a lead compound belonging to a novel class of JAK inhibitors. It displayed a JAK1/JAK2 inhibitor profile in biochemical assays, but subsequent studies in cellular and whole blood assays revealed a selectivity of ∼30-fold for JAK1- over JAK2-dependent signaling. GLPG0634 dose-dependently inhibited Th1 and Th2 differentiation and to a lesser extent the differentiation of Th17 cells in vitro. GLPG0634 was well exposed in rodents upon oral dosing, and exposure levels correlated with repression of Mx2 expression in leukocytes. Oral dosing of GLPG0634 in a therapeutic set-up in a collagen-induced arthritis model in rodents resulted in a significant dose-dependent reduction of the disease progression. Paw swelling, bone and cartilage degradation, and levels of inflammatory cytokines were reduced by GLPG0634 treatment. Efficacy of GLPG0634 in the collagen-induced arthritis models was comparable to the results obtained with etanercept. In conclusion, the JAK1 selective inhibitor GLPG0634 is a promising novel therapeutic with potential for oral treatment of rheumatoid arthritis and possibly other immune-inflammatory diseases.

Ketamine Corrects Stress-Induced Cognitive Dysfunction through JAK2/STAT3 Signaling in the Orbitofrontal Cortex

PubMed Central

Patton, Michael S; Lodge, Daniel J; Morilak, David A; Girotti, Milena

2017-01-01

Deficits in cognitive flexibility are prominent in stress-related psychiatric disorders, including depression. Ketamine has rapid antidepressant efficacy, but it is unknown if ketamine improves cognitive symptoms. In rats, 2 weeks chronic intermittent cold (CIC) stress impairs reversal learning, a form of cognitive flexibility mediated by the orbitofrontal cortex (OFC) that we have used previously to model cognitive dysfunction in depression. We have shown that activating JAK2/STAT3 signaling in the OFC rescued the CIC stress-induced reversal learning deficit. Thus, in the present study we determined whether ketamine also corrects the stress-induced reversal learning deficit, and if JAK2/STAT3 signaling is involved in this effect. A single injection of ketamine (10 mg/kg, i.p.) 24 h prior to testing rescued the CIC stress-induced reversal learning deficit. CIC stress decreased JAK2 phosphorylation in the OFC, and ketamine restored pJAK2 levels within 2 h post injection. The JAK2 inhibitor AG490 given systemically or into the OFC at the time of ketamine injection prevented its beneficial effect on reversal learning. We then tested the role of JAK2/STAT3 in ketamine-induced plasticity in the OFC. Ketamine depressed local field potentials evoked in the OFC by excitatory thalamic afferent stimulation, and this was prevented by JAK2 inhibition in the OFC. Further, in both the OFC and primary cortical neurons in culture, ketamine increased expression of the neural plasticity-related protein Arc, and this was prevented by JAK2 inhibition. These results suggest that the JAK2/STAT3 signaling pathway is a novel mechanism by which ketamine exerts its therapeutic effects on stress-induced cognitive dysfunction in the OFC. PMID:27748739

JAK2-binding long noncoding RNA promotes breast cancer brain metastasis

PubMed Central

Wang, Shouyu; Liang, Ke; Hu, Qingsong; Li, Ping; Song, Jian; Yang, Yuedong; Yao, Jun; Mangala, Lingegowda Selanere; Li, Chunlai; Park, Peter K.; Hawke, David H.; Zhou, Jianwei; Zhou, Yan; Xia, Weiya; Hung, Mien-Chie; Marks, Jeffrey R.; Gallick, Gary E.; Lopez-Berestein, Gabriel; Flores, Elsa R.; Sood, Anil K.; Huang, Suyun; Yu, Dihua; Yang, Liuqing

2017-01-01

Conventional therapies for breast cancer brain metastases (BCBMs) have been largely ineffective because of chemoresistance and impermeability of the blood-brain barrier. A comprehensive understanding of the underlying mechanism that allows breast cancer cells to infiltrate the brain is necessary to circumvent treatment resistance of BCBMs. Here, we determined that expression of a long noncoding RNA (lncRNA) that we have named lncRNA associated with BCBM (Lnc-BM) is prognostic of the progression of brain metastasis in breast cancer patients. In preclinical murine models, elevated Lnc-BM expression drove BCBM, while depletion of Lnc-BM with nanoparticle-encapsulated siRNAs effectively treated BCBM. Lnc-BM increased JAK2 kinase activity to mediate oncostatin M– and IL-6–triggered STAT3 phosphorylation. In breast cancer cells, Lnc-BM promoted STAT3-dependent expression of ICAM1 and CCL2, which mediated vascular co-option and recruitment of macrophages in the brain, respectively. Recruited macrophages in turn produced oncostatin M and IL-6, thereby further activating the Lnc-BM/JAK2/STAT3 pathway and enhancing BCBM. Collectively, our results show that Lnc-BM and JAK2 promote BCBMs by mediating communication between breast cancer cells and the brain microenvironment. Moreover, these results suggest targeting Lnc-BM as a potential strategy for fighting this difficult disease. PMID:29130936

JAK2 inhibition sensitizes resistant EGFR-mutant lung adenocarcinoma to tyrosine kinase inhibitors

PubMed Central

Gao, Sizhi P.; Chang, Qing; Mao, Ninghui; Daly, Laura A.; Vogel, Robert; Chan, Tyler; Liu, Shu Hui; Bournazou, Eirini; Schori, Erez; Zhang, Haiying; Brewer, Monica Red; Pao, William; Morris, Luc; Ladanyi, Marc; Arcila, Maria; Manova-Todorova, Katia; de Stanchina, Elisa; Norton, Larry; Levine, Ross L.; Altan-Bonnet, Gregoire; Solit, David; Zinda, Michael; Huszar, Dennis; Lyden, David; Bromberg, Jacqueline F.

2016-01-01

Lung adenocarcinomas with mutant epidermal growth factor receptor (EGFR) respond to EGFR-targeted tyrosine kinase inhibitors (TKIs), but resistance invariably occurs. We found that the Janus kinase (JAK)/signal transduction and activator of transcription 3 (STAT3) signaling pathway was aberrantly increased in TKI-resistant EGFR-mutant non–small cell lung cancer (NSCLC) cells. JAK2 inhibition restored sensitivity to the EGFR inhibitor erlotinib in TKI-resistant cell lines and xenograft models of EGFR-mutant TKI-resistant lung cancer. JAK2 inhibition uncoupled EGFR from its negative regulator, suppressor of cytokine signaling 5 (SOCS5), consequently increasing EGFR abundance and restoring the tumor cells’ dependence on EGFR signaling. Furthermore, JAK2 inhibition led to heterodimerization of mutant and wild-type EGFR subunits, the activity of which was then blocked by TKIs. Our results reveal a mechanism whereby JAK2 inhibition overcomes acquired resistance to EGFR inhibitors and support the use of combination therapy with JAK and EGFR inhibitors for the treatment of EGFR-dependent NSCLC. PMID:27025877

Regulation of T cell homeostasis by JAKs and STATs.

PubMed

Ross, Jeremy A; Nagy, Zsuzsanna S; Cheng, Hanyin; Stepkowski, Stanislaw M; Kirken, Robert A

2007-01-01

Regulation of T cell homeostasis is critical for maintaining normal immune function. An imbalance in T cell proliferation can result in disorders ranging from cancer and autoimmunity to immunodeficiencies. Full activation of T cells requires three sequential signals, where signal 3, which is delivered by multiple cytokines, regulates proliferation, differentiation, and survival/death. Signaling from cytokines through their receptors is primarily delivered by two molecular families, namely Janus tyrosine kinases (JAKs) and signal transducers and activators of transcription (STATs). Invaluable knowledge about JAKs and STATs has arisen from studies of mice made genetically deficient in these molecules, analyses of tumor models, and studies of expression patterns by proteomics/genomics, which all have begun to define the role of JAKs and STATs in survival versus apoptosis. These findings also have suggested ways in which JAKs and STATs may be manipulated for therapeutic intervention in lymphoid-derived diseases. This review seeks to focus on the role of JAK tyrosine kinases and STAT transcription factors in mediating the lymphocyte life cycle and how they might be manipulated for therapeutic applications.

Momelotinib inhibits ACVR1/ALK2, decreases hepcidin production, and ameliorates anemia of chronic disease in rodents

PubMed Central

Asshoff, Malte; Petzer, Verena; Warr, Matthew R.; Haschka, David; Tymoszuk, Piotr; Demetz, Egon; Seifert, Markus; Posch, Wilfried; Nairz, Manfred; Maciejewski, Pat; Fowles, Peter; Burns, Christopher J.; Smith, Gregg; Wagner, Kay-Uwe; Weiss, Guenter; Whitney, J. Andrew

2017-01-01

Patients with myelofibrosis (MF) often develop anemia and frequently become dependent on red blood cell transfusions. Results from a phase 2 study for the treatment of MF with the Janus kinase 1/2 (JAK1/2) inhibitor momelotinib (MMB) demonstrated that MMB treatment ameliorated anemia, which was unexpected for a JAK1/2 inhibitor, because erythropoietin-mediated JAK2 signaling is essential for erythropoiesis. Using a rat model of anemia of chronic disease, we demonstrated that MMB treatment can normalize hemoglobin and red blood cell numbers. We found that this positive effect is driven by direct inhibition of the bone morphogenic protein receptor kinase activin A receptor, type I (ACVR1), and the subsequent reduction of hepatocyte hepcidin production. Of note, ruxolitinib, a JAK1/2 inhibitor approved for the treatment of MF, had no inhibitory activity on this pathway. Further, we demonstrated the effect of MMB is not mediated by direct inhibition of JAK2-mediated ferroportin (FPN1) degradation, because neither MMB treatment nor myeloid-specific deletion of JAK2 affected FPN1 expression. Our data support the hypothesis that the improvement of inflammatory anemia by MMB results from inhibition of ACVR1-mediated hepcidin expression in the liver, which leads to increased mobilization of sequestered iron from cellular stores and subsequent stimulation of erythropoiesis. PMID:28188131

Leptin-induced basal Akt phosphorylation and its implication in exercise-mediated improvement of insulin sensitivity.

PubMed

Zheng, Xianjie; Niu, Sen

2018-01-29

Physical exercise is an efficient therapeutical tool in the management of insulin resistance (IR) and related metabolic diseases. Leptin, the well-known obesity hormone and the absence of which leads to IR, showed controversial effects on IR as research continues. Thus, in this study, a detailed investigation of the effect of leptin on exercise-mediated improvement of insulin sensitivity and its underlying mechanism was carried out. Using a rat model of chronic or acute swimming exercise training, we found that serum leptin increased 1 h after either acute exercise or the last session of chronic exercise, when impaired insulin action was observed in previous reports. However, chronic exercise reducd basal serum leptin levels and promoted insulin sensitivity compared with sedentary controls or rats subjected to one bout of aerobic exercise. Our animal results indicated the potential linkage between leptin and insulin sensitivity, which is further investigated in the skeletal muscle L6 cells. Leptin treatment in L6 cells promoted the basal levels of insulin signaling as well as glucose uptake, while blocking JAK2 signaling with either pharmacological intervention (JAK2 inhibitor AG490) or genetic manipulation (siRNA knockdown) decreased the basal levels of insulin signaling. Furthermore, leptin treatment inhibited insulin-stimulated insulin signaling and glucose uptake, while blocking JAK2 signaling restored leptin-attenuated insulin sensitivity. Taken together, our results demonstrated that reduced serum leptin, at least in part, contributes to exercise-mediated improvement of insulin sensitivity, indicating JAK2 as a potent therapeutical target of insulin resistance. Copyright © 2018 Elsevier Inc. All rights reserved.

Strong G-Protein-Mediated Inhibition of Sodium Channels.

PubMed

Mattheisen, Glynis B; Tsintsadze, Timur; Smith, Stephen M

2018-05-29

Voltage-gated sodium channels (VGSCs) are strategically positioned to mediate neuronal plasticity because of their influence on action potential waveform. VGSC function may be strongly inhibited by local anesthetic and antiepileptic drugs and modestly modulated via second messenger pathways. Here, we report that the allosteric modulators of the calcium-sensing receptor (CaSR) cinacalcet, calindol, calhex, and NPS 2143 completely inhibit VGSC current in the vast majority of cultured mouse neocortical neurons. This form of VGSC current block persisted in CaSR-deficient neurons, indicating a CaSR-independent mechanism. Cinacalcet-mediated blockade of VGSCs was prevented by the guanosine diphosphate (GDP) analog GDPβs, indicating that G-proteins mediated this effect. Cinacalcet inhibited VGSCs by increasing channel inactivation, and block was reversed by prolonged hyperpolarization. Strong cinacalcet inhibition of VGSC currents was also present in acutely isolated mouse cortical neurons. These data identify a dynamic signaling pathway by which G-proteins regulate VGSC current to indirectly modulate central neuronal excitability. Published by Elsevier Inc.

CALR, JAK2 and MPL mutation status in Argentinean patients with BCR-ABL1- negative myeloproliferative neoplasms.

PubMed

Ojeda, Mara Jorgelina; Bragós, Irma Margarita; Calvo, Karina Lucrecia; Williams, Gladis Marcela; Carbonell, María Magdalena; Pratti, Arianna Flavia

2018-05-01

To establish the frequency of JAK2, MPL and CALR mutations in Argentinean patients with BCR-ABL1-negative  myeloproliferative neoplasms (MPN) and to compare their clinical and haematological features. Mutations of JAK2V617F, JAK2 exon 12, MPL W515L/K and CALR were analysed in 439 Argentinean patients with BCR-ABL1-negative MPN, including 176 polycythemia vera (PV), 214 essential thrombocythemia (ET) and 49 primary myelofibrosis (PMF). In 94.9% of PV, 85.5% ET and 85.2% PMF, we found mutations in JAK2, MPL or CALR. 74.9% carried JAK2V617F, 12.3% CALR mutations, 2.1% MPL mutations and 10.7% were triple negative. In ET, nine types of CALR mutations were identified, four of which were novel. PMF patients were limited to types 1 and 2, type 2 being more frequent. In ET, patients with CALR mutation were younger and had higher platelet counts than those with JAK2V617F and triple negative. In addition, JAK2V617F patients had high leucocyte and haemoglobin values compared with CALR-mutated and triple-negative patients. In PMF, patients with mutant CALR were associated with higher platelet counts. Our study underscores the importance of JAK2, MPL and CALR genotyping for accurate diagnosis of patients with BCR-ABL1-negative MPN.

Inhibitors of JAK-family kinases: an update on the patent literature 2013-2015, part 1.

PubMed

Kettle, Jason G; Åstrand, Annika; Catley, Matthew; Grimster, Neil P; Nilsson, Magnus; Su, Qibin; Woessner, Richard

2017-02-01

Janus kinases (JAKs) are a family of four enzymes; JAK1, JAK2, JAK3 and tyrosine kinase 2 (TYK2) that are critical in cytokine signalling and are strongly linked to both cancer and inflammatory diseases. There are currently two launched JAK inhibitors for the treatment of human conditions: tofacitinib for Rheumatoid arthritis (RA) and ruxolitinib for myeloproliferative neoplasms including intermediate or high risk myelofibrosis and polycythemia vera. Areas covered: This review covers patents claiming activity against one or more JAK family members in the period 2013-2015 inclusive, and covers 95 patents from 42 applicants, split over two parts. The authors have ordered recent patents according to the primary applicant's name, with part 1 covering A through to I. Expert opinion: Inhibition of JAK-family kinases is an area of growing interest, catalysed by the maturity of data on marketed inhibitors ruxolitinib and tofacitinib in late stage clinical trials. Many applicants are pursuing traditional fast-follower strategies around these inhibitors, with a range of chemical strategies adopted. The challenge will be to show sufficient differentiation to the originator compounds, since dose limiting toxicities with such agents appear to be on target and mechanism-related and also considering that such agents may be available as generic compounds by the time follower agents reach market.

A polyvalent hybrid protein elicits antibodies against the diverse allelic types of block 2 in Plasmodium falciparum merozoite surface protein 1.

PubMed

Tetteh, Kevin K A; Conway, David J

2011-10-13

Merozoite surface protein 1 (MSP1) of Plasmodium falciparum has been implicated as an important target of acquired immunity, and candidate components for a vaccine include polymorphic epitopes in the N-terminal polymorphic block 2 region. We designed a polyvalent hybrid recombinant protein incorporating sequences of the three major allelic types of block 2 together with a composite repeat sequence of one of the types and N-terminal flanking T cell epitopes, and compared this with a series of recombinant proteins containing modular sub-components and similarly expressed in Escherichia coli. Immunogenicity of the full polyvalent hybrid protein was tested in both mice and rabbits, and comparative immunogenicity studies of the sub-component modules were performed in mice. The full hybrid protein induced high titre antibodies against each of the major block 2 allelic types expressed as separate recombinant proteins and against a wide range of allelic types naturally expressed by a panel of diverse P. falciparum isolates, while the sub-component modules had partial antigenic coverage as expected. This encourages further development and evaluation of the full MSP1 block 2 polyvalent hybrid protein as a candidate blood-stage component of a malaria vaccine. Copyright © 2011 Elsevier Ltd. All rights reserved.

Molecular docking, 3D QSAR and dynamics simulation studies of imidazo-pyrrolopyridines as janus kinase 1 (JAK 1) inhibitors.

PubMed

Itteboina, Ramesh; Ballu, Srilata; Sivan, Sree Kanth; Manga, Vijjulatha

2016-10-01

Janus kinase 1 (JAK 1) plays a critical role in initiating responses to cytokines by the JAK-signal transducer and activator of transcription (JAK-STAT). This controls survival, proliferation and differentiation of a variety of cells. Docking, 3D quantitative structure activity relationship (3D-QSAR) and molecular dynamics (MD) studies were performed on a series of Imidazo-pyrrolopyridine derivatives reported as JAK 1 inhibitors. QSAR model was generated using 30 molecules in the training set; developed model showed good statistical reliability, which is evident from r 2 ncv and r 2 loo values. The predictive ability of this model was determined using a test set of 13 molecules that gave acceptable predictive correlation (r 2 Pred ) values. Finally, molecular dynamics simulation was performed to validate docking results and MM/GBSA calculations. This facilitated us to compare binding free energies of cocrystal ligand and newly designed molecule R1. The good concordance between the docking results and CoMFA/CoMSIA contour maps afforded obliging clues for the rational modification of molecules to design more potent JAK 1 inhibitors. Copyright © 2016 Elsevier Ltd. All rights reserved.

High mobility group box 1 induces the activation of the Janus kinase 2 and signal transducer and activator of transcription 3 (JAK2/STAT3) signaling pathway in pancreatic acinar cells in rats, while AG490 and rapamycin inhibit their activation.

PubMed

Wang, Guoliang; Zhang, Jingchao; Dui, Danhua; Ren, Haoyuan; Liu, Jin

2016-11-10

The pathogenesis of severe acute pancreatitis (SAP) remains unclear. The Janus kinase and signal transducer and activator of transcription (JAK/STAT) pathway is important for various cytokines and growth factors. This study investigated the effect of the late inflammatory factor high mobility group box 1 (HMGB1) on the activation of JAK2/STAT3 in pancreatic acinar cells and the inhibitory effects of AG490 (a JAK2 inhibitor) and rapamycin (a STAT3 inhibitor) on this pathway. Rat pancreatic acinar cells were randomly divided into the control, HMGB1, AG490, and rapamycin groups. The mRNA levels of JAK2 and STAT3 at 10, 30, 60, and 120 minutes were detected using reverse transcription polymerase chain reaction (RT-PCR). The protein levels of JAK2 and STAT3 at 60 and 120 minutes were observed using Western blotting. Compared with the control group, the HMGB1 group exhibited significantly increased levels of JAK2 mRNA at each time point; STAT3 mRNA at 30, 60, and 120 minutes; and JAK2 and STAT3 proteins at 60 and 120 minutes (p < 0.01). Compared with the HMGB1 group, the AG490 and rapamycin groups both exhibited significantly decreased levels of JAK2 mRNA at each time point (p < 0.05); STAT3 mRNA at 30, 60, and 120 minutes (p < 0.01); and JAK2 and STAT3 proteins at 60 and 120 minutes (p < 0.01). HMGB1 induces the activation of the JAK2/STAT3 signaling pathway in rat pancreatic acinar cells, and this activation can be inhibited by AG490 and rapamycin. The results of this study may provide new insights for the treatment of SAP.

JAK2 Exon 14 Deletion in Patients with Chronic Myeloproliferative Neoplasms

PubMed Central

Ma, Wanlong; Kantarjian, Hagop; Zhang, Xi; Wang, Xiuqiang; Zhang, Zhong; Yeh, Chen-Hsiung; O'Brien, Susan; Giles, Francis; Bruey, Jean Marie; Albitar, Maher

2010-01-01

Background The JAK2 V617F mutation in exon 14 is the most common mutation in chronic myeloproliferative neoplasms (MPNs); deletion of the entire exon 14 is rarely detected. In our previous study of >10,000 samples from patients with suspected MPNs tested for JAK2 mutations by reverse transcription-PCR (RT-PCR) with direct sequencing, complete deletion of exon 14 (Δexon14) constituted <1% of JAK2 mutations. This appears to be an alternative splicing mutation, not detectable with DNA-based testing. Methodology/Principal Findings We investigated the possibility that MPN patients may express the JAK2 Δexon14 at low levels (<15% of total transcript) not routinely detectable by RT-PCR with direct sequencing. Using a sensitive RT-PCR–based fluorescent fragment analysis method to quantify JAK2 Δexon14 mRNA expression relative to wild-type, we tested 61 patients with confirmed MPNs, 183 with suspected MPNs (93 V617F-positive, 90 V617F-negative), and 46 healthy control subjects. The Δexon14 variant was detected in 9 of the 61 (15%) confirmed MPN patients, accounting for 3.96% to 33.85% (mean  = 12.04%) of total JAK2 transcript. This variant was also detected in 51 of the 183 patients with suspected MPNs (27%), including 20 of the 93 (22%) with V617F (mean [range] expression  = 5.41% [2.13%–26.22%]) and 31 of the 90 (34%) without V617F (mean [range] expression  = 3.88% [2.08%–12.22%]). Immunoprecipitation studies demonstrated that patients expressing Δexon14 mRNA expressed a corresponding truncated JAK2 protein. The Δexon14 variant was not detected in the 46 control subjects. Conclusions/Significance These data suggest that expression of the JAK2 Δexon14 splice variant, leading to a truncated JAK2 protein, is common in patients with MPNs. This alternatively spliced transcript appears to be more frequent in MPN patients without V617F mutation, in whom it might contribute to leukemogenesis. This mutation is missed if DNA rather than RNA is used for

Inborn Errors of Human JAKs and STATs

PubMed Central

Casanova, Jean-Laurent; Holland, Steven M.; Notarangelo, Luigi D.

2012-01-01

Inborn errors of the genes encoding two of the four human JAKs (JAK3 and TYK2) and three of the six human STATs (STAT1, STAT3, and STAT5B) have been described. We review the disorders arising from mutations in these five genes, highlighting the way in which the molecular and cellular pathogenesis of these conditions has been clarified by the discovery of inborn errors of cytokines, hormones, and their receptors, including those interacting with JAKs and STATs. The phenotypic similarities between mice and humans lacking individual JAK-STAT components suggest that the functions of JAKs and STATs are largely conserved in mammals. However, a wide array of phenotypic differences has emerged between mice and humans carrying bi-allelic null alleles of JAK3, TYK2, STAT1, or STAT5B. Moreover, the high level of allelic heterogeneity at the human JAK3, STAT1, and STAT3 loci has revealed highly diverse immunological and clinical phenotypes, which had not been anticipated. PMID:22520845

Inborn errors of human JAKs and STATs.

PubMed

Casanova, Jean-Laurent; Holland, Steven M; Notarangelo, Luigi D

2012-04-20

Inborn errors of the genes encoding two of the four human JAKs (JAK3 and TYK2) and three of the six human STATs (STAT1, STAT3, and STAT5B) have been described. We review the disorders arising from mutations in these five genes, highlighting the way in which the molecular and cellular pathogenesis of these conditions has been clarified by the discovery of inborn errors of cytokines, hormones, and their receptors, including those interacting with JAKs and STATs. The phenotypic similarities between mice and humans lacking individual JAK-STAT components suggest that the functions of JAKs and STATs are largely conserved in mammals. However, a wide array of phenotypic differences has emerged between mice and humans carrying biallelic null alleles of JAK3, TYK2, STAT1, or STAT5B. Moreover, the high degree of allelic heterogeneity at the human JAK3, TYK2, STAT1, and STAT3 loci has revealed highly diverse immunological and clinical phenotypes, which had not been anticipated. Copyright © 2012 Elsevier Inc. All rights reserved.

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.

Preclinical characterization of the selective JAK1/2 inhibitor INCB018424: therapeutic implications for the treatment of myeloproliferative neoplasms

PubMed Central

Quintás-Cardama, Alfonso; Vaddi, Kris; Liu, Phillip; Manshouri, Taghi; Li, Jun; Scherle, Peggy A.; Caulder, Eian; Wen, Xiaoming; Li, Yanlong; Waeltz, Paul; Rupar, Mark; Burn, Timothy; Lo, Yvonne; Kelley, Jennifer; Covington, Maryanne; Shepard, Stacey; Rodgers, James D.; Haley, Patrick; Kantarjian, Hagop

2010-01-01

Constitutive JAK2 activation in hematopoietic cells by the JAK2V617F mutation recapitulates myeloproliferative neoplasm (MPN) phenotypes in mice, establishing JAK2 inhibition as a potential therapeutic strategy. Although most polycythemia vera patients carry the JAK2V617F mutation, half of those with essential thrombocythemia or primary myelofibrosis do not, suggesting alternative mechanisms for constitutive JAK-STAT signaling in MPNs. Most patients with primary myelofibrosis have elevated levels of JAK-dependent proinflammatory cytokines (eg, interleukin-6) consistent with our observation of JAK1 hyperactivation. Accordingly, we evaluated the effectiveness of selective JAK1/2 inhibition in experimental models relevant to MPNs and report on the effects of INCB018424, the first potent, selective, oral JAK1/JAK2 inhibitor to enter the clinic. INCB018424 inhibited interleukin-6 signaling (50% inhibitory concentration [IC50] = 281nM), and proliferation of JAK2V617F+ Ba/F3 cells (IC50 = 127nM). In primary cultures, INCB018424 preferentially suppressed erythroid progenitor colony formation from JAK2V617F+ polycythemia vera patients (IC50 = 67nM) versus healthy donors (IC50 > 400nM). In a mouse model of JAK2V617F+ MPN, oral INCB018424 markedly reduced splenomegaly and circulating levels of inflammatory cytokines, and preferentially eliminated neoplastic cells, resulting in significantly prolonged survival without myelosuppressive or immunosuppressive effects. Preliminary clinical results support these preclinical data and establish INCB018424 as a promising oral agent for the treatment of MPNs. PMID:20130243

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

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

JAK2 inhibitor therapy in myeloproliferative disorders: rationale, preclinical studies and ongoing clinical trials.

PubMed

Pardanani, A

2008-01-01

The recent identification of somatic mutations such as JAK2V617F that deregulate Janus kinase (JAK)-signal transducer and activator of transcription signaling has spurred development of orally bioavailable small-molecule inhibitors that selectively target JAK2 kinase as an approach to pathogenesis-directed therapy of myeloproliferative disorders (MPD). In pre-clinical studies, these compounds inhibit JAK2V617F-mediated cell growth at nanomolar concentrations, and in vivo therapeutic efficacy has been demonstrated in mouse models of JAK2V617F-induced disease. In addition, ex vivo growth of progenitor cells from MPD patients harboring JAK2V617F or MPLW515L/K mutations is also potently inhibited. JAK2 inhibitors currently in clinical trials can be grouped into those designed to primarily target JAK2 kinase (JAK2-selective) and those originally developed for non-MPD indications, but that nevertheless have significant JAK2-inhibitory activity (non-JAK2 selective). This article discusses the rationale for using JAK2 inhibitors for the treatment of MPD, as well as relevant aspects of clinical trial development for these patients. For instance, which group of MPD patients is appropriate for initial Phase I studies? Should JAK2V617F-negative MPD patients be included in the initial studies? What are the likely consequences of 'off-target' JAK3 and wild-type JAK2 inhibition? How should treatment responses be monitored?

Stimulation of the amino acid transporter SLC6A19 by JAK2

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

Bhavsar, Shefalee K.; Hosseinzadeh, Zohreh; Merches, Katja

Highlights: Black-Right-Pointing-Pointer The amino acid transporter SLC6A19 is upregulated by Janus kinase-2 JAK2. Black-Right-Pointing-Pointer The {sup V617F}JAK2 mutant, causing myeloproliferative disease, is more effective. Black-Right-Pointing-Pointer JAK2 inhibitor AG490 reverses stimulation of SLC6A19 by {sup V617F}JAK2. Black-Right-Pointing-Pointer JAK2 enhances SLC6A19 protein insertion into the cell membrane. Black-Right-Pointing-Pointer SLC6A19 may contribute to amino acid uptake into {sup V617F}JAK2 expressing tumor cells. -- Abstract: JAK2 (Janus kinase-2) is expressed in a wide variety of cells including tumor cells and contributes to the proliferation and survival of those cells. The gain of function mutation {sup V617F}JAK2 mutant is found in the majority of myeloproliferativemore » diseases. Cell proliferation depends on the availability of amino acids. Concentrative cellular amino acid uptake is in part accomplished by Na{sup +} coupled amino acid transport through SLC6A19 (B(0)AT). The present study thus explored whether JAK2 activates SLC6A19. To this end, SLC6A19 was expressed in Xenopus oocytes with or without wild type JAK2, {sup V617F}JAK2 or inactive {sup K882E}JAK2 and electrogenic amino acid transport determined by dual electrode voltage clamp. In SLC6A19-expressing oocytes but not in oocytes injected with water or JAK2 alone, the addition of leucine (2 mM) to the bath generated a current (I{sub le}), which was significantly increased following coexpression of JAK2 or {sup V617F}JAK2, but not by coexpression of {sup K882E}JAK2. Coexpression of JAK2 enhanced the maximal transport rate without significantly modifying the affinity of the carrier. Exposure of the oocytes to the JAK2 inhibitor AG490 (40 {mu}M) resulted in a gradual decline of I{sub le}. According to chemiluminescence JAK2 enhanced the carrier protein abundance in the cell membrane. The decline of I{sub le} following inhibition of carrier insertion by brefeldin A (5 {mu}M) was

mTORC1-dependent translation of collapsin response mediator protein-2 drives neuroadaptations underlying excessive alcohol-drinking behaviors

PubMed Central

Liu, F; Laguesse, S; Legastelois, R; Morisot, N; Ben Hamida, S; Ron, D

2017-01-01

Mammalian target of rapamycin complex 1 (mTORC1) has an essential role in dendritic mRNA translation and participates in mechanisms underlying alcohol-drinking and reconsolidation of alcohol-related memories. Here, we report that excessive alcohol consumption increases the translation of downstream targets of mTORC1, including collapsin response mediator protein-2 (CRMP-2), in the nucleus accumbens (NAc) of rodents. We show that alcohol-mediated induction of CRMP-2 translation is mTORC1-dependent, leading to increased CRMP-2 protein levels. Furthermore, we demonstrate that alcohol intake also blocks glycogen synthase kinase-3β (GSK-3β)-phosphorylation of CRMP-2, which results in elevated binding of CRMP-2 to microtubules and a concomitant increase in microtubule content. Finally, we show that systemic administration of the CRMP-2 inhibitor lacosamide, or knockdown of CRMP-2 in the NAc decreases excessive alcohol intake. These results suggest that CRMP-2 in the NAc is a convergent point that receives inputs from two signaling pathways, mTORC1 and GSK-3β, that in turn drives excessive alcohol-drinking behaviors. PMID:26952865

JAK/STAT controls organ size and fate specification by regulating morphogen production and signalling

PubMed Central

Recasens-Alvarez, Carles; Ferreira, Ana; Milán, Marco

2017-01-01

A stable pool of morphogen-producing cells is critical for the development of any organ or tissue. Here we present evidence that JAK/STAT signalling in the Drosophila wing promotes the cycling and survival of Hedgehog-producing cells, thereby allowing the stable localization of the nearby BMP/Dpp-organizing centre in the developing wing appendage. We identify the inhibitor of apoptosis dIAP1 and Cyclin A as two critical genes regulated by JAK/STAT and contributing to the growth of the Hedgehog-expressing cell population. We also unravel an early role of JAK/STAT in guaranteeing Wingless-mediated appendage specification, and a later one in restricting the Dpp-organizing activity to the appendage itself. These results unveil a fundamental role of the conserved JAK/STAT pathway in limb specification and growth by regulating morphogen production and signalling, and a function of pro-survival cues and mitogenic signals in the regulation of the pool of morphogen-producing cells in a developing organ. PMID:28045022

Targeting the Interleukin-6/Jak/Stat Pathway in Human Malignancies

PubMed Central

Sansone, Pasquale; Bromberg, Jacqueline

2012-01-01

The Janus kinase/signal transducer and activator of transcription (Jak/Stat) pathway was discovered 20 years ago as a mediator of cytokine signaling. Since this time, more than 2,500 articles have been published demonstrating the importance of this pathway in virtually all malignancies. Although there are dozens of cytokines and cytokine receptors, four Jaks, and seven Stats, it seems that interleukin-6–mediated activation of Stat3 is a principal pathway implicated in promoting tumorigenesis. This transcription factor regulates the expression of numerous critical mediators of tumor formation and metastatic progression. This review will examine the relative importance and function of this pathway in nonmalignant conditions as well as malignancies (including tumor intrinsic and extrinsic), the influence of other Stats, the development of inhibitors to this pathway, and the potential role of inhibitors in controlling or eradicating cancers. PMID:22355058

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.

An ENU mutagenesis-derived mouse model with a dominant Jak1 mutation resembling phenotypes of systemic autoimmune disease.

PubMed

Sabrautzki, Sibylle; Janas, Eva; Lorenz-Depiereux, Bettina; Calzada-Wack, Julia; Aguilar-Pimentel, Juan A; Rathkolb, Birgit; Adler, Thure; Cohrs, Christian; Hans, Wolfgang; Diener, Susanne; Fuchs, Helmut; Gailus-Durner, Valerie; Busch, Dirk H; Höfler, Heinz; Ollert, Markus; Strom, Tim M; Wolf, Eckhard; Neff, Frauke; Hrabě de Angelis, Martin

2013-08-01

Within the Munich, Germany, N-ethyl-N-nitrosourea mouse mutagenesis program, we isolated a dominant Jak1 mouse model resembling phenotypic characteristics related to autoimmune disease. Chromosomal sequencing revealed a new Jak1 (p.Ser645Pro) point mutation at the conserved serine of the pseudokinase domain, corresponding to a somatic human mutation (p.Ser646Phe) inducing a constitutive activation of the Janus kinase (JAK)/STAT pathway. Morphologically, all Jak1(S645P+/-) mice showed a progressive structural deterioration of ears starting at the age of 4 months, with mononuclear cell infiltration into the dermis. Female mutant mice, in particular, developed severe skin lesions in the neck from 7 months of age. The IHC analysis of these lesions showed an activation of Stat3 downstream to Jak1(S645P) and elevated tissue levels of IL-6. Histopathological analysis of liver revealed a nodular regenerative hyperplasia. In the spleen, the number of Russell bodies was doubled, correlating with significant increased levels of all immunoglobulin isotypes and anti-DNA antibodies in serum. Older mutant mice developed thrombocytopenia and altered microcytic red blood cell counts. Jak1(S645P+/-) mice showed phenotypes related to impaired bone metabolism as increased carboxy-terminal collagen cross-link-1 levels and alkaline phosphatase activities in plasma, hypophosphatemia, and strongly decreased bone morphometric values. Taken together, Jak1(S645P+/-) mice showed an increased activation of the IL-6-JAK-STAT pathway leading to a systemic lupus erythematosus-like phenotype and offering a new valuable tool to study the role of the JAK/STAT pathway in disease development. Copyright © 2013 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

Thymoquinone Augments Cisplatin-Induced Apoptosis on Esophageal Carcinoma Through Mitigating the Activation of JAK2/STAT3 Pathway.

PubMed

Hu, Xue; Ma, Jingjing; Vikash, Vikash; Li, Jiao; Wu, Dandan; Liu, Ya; Zhang, Jixiang; Dong, Weiguo

2018-01-01

Thymoquinone (TQ) is the major constituent of Nigella sativa seed and has shown biological activity in various human carcinomas. However, few studies have reported its effect on esophageal carcinoma (EC). To explore the chemosensitive effect and mechanism of TQ in augmentation of cisplatin (DDP)-induced apoptosis of EC, both in vitro and in vivo. The viability and apoptosis of esophageal carcinoma cells were detected by the Cell Counting Kit-8 assay, flow cytometry, and Hoechst 33258 staining. The expression levels of JAK2, p-JAK2, STAT3, p-STAT3, Bax, Bcl-2, Cyclin D1, Survivin, and caspase-3, 7, 9 were evaluated by western blot analysis. The histological changes were examined by TUNEL technique and immunohistochemical analysis. TQ enhanced the proapoptotic effect of DDP in human esophageal carcinoma cell line Eca-109, while blocking the activation of JAK2/STAT3 signaling pathway. The apoptosis of esophageal carcinoma cells was induced via blocking the activation of JAK2/STAT3 by using a molecular inhibitor (WP1066). Consistent with the in vivo and in vitro results, TQ increased cellular apoptosis and enriched the chemosensitivity of DDP. TQ along with DDP may regulate the progression of EC and has potential to be a chemotherapeutic agent in EC.

Genetic variation at MECOM, TERT, JAK2 and HBS1L-MYB predisposes to myeloproliferative neoplasms

PubMed Central

Tapper, William; Jones, Amy V.; Kralovics, Robert; Harutyunyan, Ashot S.; Zoi, Katerina; Leung, William; Godfrey, Anna L.; Guglielmelli, Paola; Callaway, Alison; Ward, Daniel; Aranaz, Paula; White, Helen E.; Waghorn, Katherine; Lin, Feng; Chase, Andrew; Joanna Baxter, E.; Maclean, Cathy; Nangalia, Jyoti; Chen, Edwin; Evans, Paul; Short, Michael; Jack, Andrew; Wallis, Louise; Oscier, David; Duncombe, Andrew S.; Schuh, Anna; Mead, Adam J.; Griffiths, Michael; Ewing, Joanne; Gale, Rosemary E.; Schnittger, Susanne; Haferlach, Torsten; Stegelmann, Frank; Döhner, Konstanze; Grallert, Harald; Strauch, Konstantin; Tanaka, Toshiko; Bandinelli, Stefania; Giannopoulos, Andreas; Pieri, Lisa; Mannarelli, Carmela; Gisslinger, Heinz; Barosi, Giovanni; Cazzola, Mario; Reiter, Andreas; Harrison, Claire; Campbell, Peter; Green, Anthony R.; Vannucchi, Alessandro; Cross, Nicholas C.P.

2015-01-01

Clonal proliferation in myeloproliferative neoplasms (MPN) is driven by somatic mutations in JAK2, CALR or MPL, but the contribution of inherited factors is poorly characterized. Using a three-stage genome-wide association study of 3,437 MPN cases and 10,083 controls, we identify two SNPs with genome-wide significance in JAK2V617F-negative MPN: rs12339666 (JAK2; meta-analysis P=1.27 × 10−10) and rs2201862 (MECOM; meta-analysis P=1.96 × 10−9). Two additional SNPs, rs2736100 (TERT) and rs9376092 (HBS1L/MYB), achieve genome-wide significance when including JAK2V617F-positive cases. rs9376092 has a stronger effect in JAK2V617F-negative cases with CALR and/or MPL mutations (Breslow–Day P=4.5 × 10−7), whereas in JAK2V617F-positive cases rs9376092 associates with essential thrombocythemia (ET) rather than polycythemia vera (allelic χ2 P=7.3 × 10−7). Reduced MYB expression, previously linked to development of an ET-like disease in model systems, associates with rs9376092 in normal myeloid cells. These findings demonstrate that multiple germline variants predispose to MPN and link constitutional differences in MYB expression to disease phenotype. PMID:25849990

Recent patents in the discovery of small molecule inhibitors of JAK3.

PubMed

Wilson, Lawrence J

2010-05-01

Protein kinase enzymes have become increasingly important as the target of many disease modification drug discovery programs. Disruption of JAK3 function results in quantitative and qualitative deficiencies in both B- and T-cell compartments of the immune system of JAK3 deficient mice and development of severe combined immunodeficiency in humans with the JAK3 genetic aberration. JAK3 plays a specific role in immune function and lymphoid development and it only resides in the hematopoietic system, thus the rationale for selective targeting. Inhibitors of JAK3 have shown utility in many different autoimmune disorders, including allograft rejection during transplantation, acute lymphoblastic leukemia, Type 1 diabetes, rheumatoid arthritis and allergic and asthmatic diseases. These inhibitors are making their way into clinical trials with profound effects, thus, validating the target and strategy. A review that covers around 90 patents and patent applications made in the last 10 years in the area involving JAK3 inhibitors is provided. Specifically, what this content will provide is the genus, highlighted compounds of particular interest, filing organization and some biological measure of these compounds as inhibitors of this protein kinase or none if it is not provided. Some information from original research articles appearing in peer reviewed literature is provided, but this article is not a review of the literature. Furthermore, an overview of the current clinical status and future outcomes of this field is provided as summary. A strong understanding for the current state of the art in patents dealing with inhibitors of JAK3 including genus and species designations, potential commercial interest of this target in the pharmaceutical community, depth of coverage by numbers of examples and selected proof of action against the target. Also, a brief understanding of the biology and pharmacology involved in the processes involving the research, discovery, characterization

Jab1 Mediates Protein Degradation of Rad9/Rad1/Hus1 Checkpoint Complex

PubMed Central

Huang, Jin; Yuan, Honglin; Lu, Chongyuan; Liu, Ximeng; Cao, Xu; Wan, Mei

2009-01-01

Summary The Rad1-Rad9-Hus1 (9-1-1) complex serves a dual role as a DNA-damage sensor in checkpoint signaling and as a mediator in DNA repair pathway. However, the intercellular mechanisms that regulate 9-1-1 complex are poorly understood. Jab1, the fifth component of the COP9 signalosome complex, plays a central role in the degradation of multiple proteins and is emerging as an important regulator in cancer development. Here, we tested the hypothesis that Jab1 controls the protein stability of the 9-1-1 complex via the proteosome pathway. We provide evidence that Jab1 physically associates with the 9-1-1 complex. This association is mediated through direct interaction between Jab1 and Rad1, one of the subunits of 9-1-1 complex. Importantly, Jab1 causes the translocation of the 9-1-1 complex from the nucleus to the cytoplasm, mediating rapid degradation of the 9-1-1 complex via 26S proteasome. Furthermore, Jab1 significantly suppresses checkpoint signaling activation, DNA synthesis recovery from blockage and cell viability after replication stresses such as UV exposure, γ radiation and hydroxyurea treatment. These results suggest that Jab1 is an important regulator for 9-1-1 protein stability control in cells, which may provide novel information on the involvement of Jab1 in checkpoint and DNA repair signaling in response to DNA damage. PMID:17583730

Interaction of Hepatitis C Virus Core Protein with Janus Kinase Is Required for Efficient Production of Infectious Viruses

PubMed Central

Lee, Choongho

2013-01-01

Chronic hepatitis C virus (HCV) infection is responsible for the development of liver cirrhosis and hepatocellular carcinoma. HCV core protein plays not only a structural role in the virion morphogenesis by encapsidating a virus RNA genome but also a non-structural role in HCV-induced pathogenesis by blocking innate immunity. Especially, it has been shown to regulate JAK-STAT signaling pathway through its direct interaction with Janus kinase (JAK) via its proline-rich JAK-binding motif (79PGYPWP84). However, little is known about the physiological significance of this HCV core-JAK association in the context of the virus life cycle. In order to gain an insight, a mutant HCV genome (J6/JFH1-79A82A) was constructed to express the mutant core with a defective JAK-binding motif (79AGYAWP84) using an HCV genotype 2a infectious clone (J6/JFH1). When this mutant HCV genome was introduced into hepatocarcinoma cells, it was found to be severely impaired in its ability to produce infectious viruses in spite of its robust RNA genome replication. Taken together, all these results suggest an essential requirement of HCV core-JAK protein interaction for efficient production of infectious viruses and the potential of using core-JAK blockers as a new anti-HCV therapy. PMID:24009866

Nod-like receptor protein 1 inflammasome mediates neuron injury under high glucose.

PubMed

Meng, Xian-Fang; Wang, Xiao-Lan; Tian, Xiu-Juan; Yang, Zhi-Hua; Chu, Guang-Pin; Zhang, Jing; Li, Man; Shi, Jing; Zhang, Chun

2014-04-01

Diabetic encephalopathy is one of the most common complications of diabetes. Inflammatory events during diabetes may be an important mechanism of diabetic encephalopathy. Inflammasome is a multiprotein complex consisting of Nod-like receptor proteins (NLRPs), apoptosis-associated speck-like protein (ASC), and caspase 1 or 5, which functions to switch on the inflammatory process and the release of inflammatory factors. The present study hypothesized that the formation and activation of NLRP1 inflammasome turns on neuroinflammation and neuron injury during hyperglycemia. The results demonstrated that the levels of interleukin-1 beta (IL-1β) were increased in the cortex of streptozocin (STZ)-induced diabetic rats. The levels of mature IL-1β and IL-18 were also elevated in culture medium of neurons treated with high glucose (50 mM). The expression of three essential components of the NLRP1 inflammasome complex, namely, NLRP1, ASC, and caspase 1, was also upregulated in vivo and in vitro under high glucose. Silencing the ASC gene prevented the caspase-1 activation, and inhibiting caspase 1 activity blocked hyperglycemia-induced release of inflammatory factors and neuron injury. Moreover, we found that pannexin 1 mediated the actvitation of NLRP1 inflammasome under high glucose. These results suggest that hyperglycemia induces neuroinflammation through activation of NLRP1 inflammasome, which represents a novel mechanism of diabetes-associated neuron injury.

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

Inhibition of pyrimidine synthesis reverses viral virulence factor-mediated block of mRNA nuclear export

PubMed Central

Zhang, Liang; Das, Priyabrata; Schmolke, Mirco; Manicassamy, Balaji; Wang, Yaming; Deng, Xiaoyi; Cai, Ling; Tu, Benjamin P.; Forst, Christian V.; Roth, Michael G.; Levy, David E.; García-Sastre, Adolfo; de Brabander, Jef; Phillips, Margaret A.

2012-01-01

The NS1 protein of influenza virus is a major virulence factor essential for virus replication, as it redirects the host cell to promote viral protein expression. NS1 inhibits cellular messenger ribonucleic acid (mRNA) processing and export, down-regulating host gene expression and enhancing viral gene expression. We report in this paper the identification of a nontoxic quinoline carboxylic acid that reverts the inhibition of mRNA nuclear export by NS1, in the absence or presence of the virus. This quinoline carboxylic acid directly inhibited dihydroorotate dehydrogenase (DHODH), a host enzyme required for de novo pyrimidine biosynthesis, and partially reduced pyrimidine levels. This effect induced NXF1 expression, which promoted mRNA nuclear export in the presence of NS1. The release of NS1-mediated mRNA export block by DHODH inhibition also occurred in the presence of vesicular stomatitis virus M (matrix) protein, another viral inhibitor of mRNA export. This reversal of mRNA export block allowed expression of antiviral factors. Thus, pyrimidines play a necessary role in the inhibition of mRNA nuclear export by virulence factors. PMID:22312003

JAK inhibitors for the treatment of myeloproliferative neoplasms and other disorders

PubMed Central

Vainchenker, William; Leroy, Emilie; Gilles, Laure; Marty, Caroline; Plo, Isabelle; Constantinescu, Stefan N.

2018-01-01

JAK inhibitors have been developed following the discovery of the JAK2V617F in 2005 as the driver mutation of the majority of non- BCR-ABL1 myeloproliferative neoplasms (MPNs). Subsequently, the search for JAK2 inhibitors continued with the discovery that the other driver mutations ( CALR and MPL) also exhibited persistent JAK2 activation. Several type I ATP-competitive JAK inhibitors with different specificities were assessed in clinical trials and exhibited minimal hematologic toxicity. Interestingly, these JAK inhibitors display potent anti-inflammatory activity. Thus, JAK inhibitors targeting preferentially JAK1 and JAK3 have been developed to treat inflammation, autoimmune diseases, and graft-versus-host disease. Ten years after the beginning of clinical trials, only two drugs have been approved by the US Food and Drug Administration: one JAK2/JAK1 inhibitor (ruxolitinib) in intermediate-2 and high-risk myelofibrosis and hydroxyurea-resistant or -intolerant polycythemia vera and one JAK1/JAK3 inhibitor (tofacitinib) in methotrexate-resistant rheumatoid arthritis. The non-approved compounds exhibited many off-target effects leading to neurological and gastrointestinal toxicities, as seen in clinical trials for MPNs. Ruxolitinib is a well-tolerated drug with mostly anti-inflammatory properties. Despite a weak effect on the cause of the disease itself in MPNs, it improves the clinical state of patients and increases survival in myelofibrosis. This limited effect is related to the fact that ruxolitinib, like the other type I JAK2 inhibitors, inhibits equally mutated and wild-type JAK2 (JAK2WT) and also the JAK2 oncogenic activation. Thus, other approaches need to be developed and could be based on either (1) the development of new inhibitors specifically targeting JAK2V617F or (2) the combination of the actual JAK2 inhibitors with other therapies, in particular with molecules targeting pathways downstream of JAK2 activation or the stability of JAK2 molecule. In

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

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.

JAK inhibitors in autoinflammation.

PubMed

Hoffman, Hal M; Broderick, Lori

2018-06-11

Interferonopathies are a subset of autoinflammatory disorders with a prominent type I IFN gene signature. Treatment of these patients has been challenging, given the lack of response to common autoinflammatory therapeutics including IL-1 and TNF blockade. JAK inhibitors (Jakinibs) are a family of small-molecule inhibitors that target the JAK/STAT signaling pathway and have shown clinical efficacy, with FDA and European Medicines Agency (EMA) approval for arthritic and myeloproliferative syndromes. Sanchez and colleagues repurposed baricitinib to establish a significant role for JAK inhibition as a novel therapy for patients with interferonopathies, demonstrating the power of translational rare disease research with lifesaving effects.

Crystal Structure of a Complex of the Intracellular Domain of Interferon λ Receptor 1 (IFNLR1) and the FERM/SH2 Domains of Human JAK1.

PubMed

Zhang, Di; Wlodawer, Alexander; Lubkowski, Jacek

2016-11-20

The crystal structure of a construct consisting of the FERM and SH2-like domains of the human Janus kinase 1 (JAK1) bound to a fragment of the intracellular domain of the interferon-λ receptor 1 (IFNLR1) has been determined at the nominal resolution of 2.1Å. In this structure, the receptor peptide forms an 85-Å-long extended chain, in which both the previously identified box1 and box2 regions bind simultaneously to the FERM and SH2-like domains of JAK1. Both domains of JAK1 are generally well ordered, with regions not seen in the crystal structure limited to loops located away from the receptor-binding regions. The structure provides a much more complete and accurate picture of the interactions between JAK1 and IFNLR1 than those given in earlier reports, illuminating the molecular basis of the JAK-cytokine receptor association. A glutamate residue adjacent to the box2 region in IFNLR1 mimics the mode of binding of a phosphotyrosine in classical SH2 domains. It was shown here that a deletion of residues within the box1 region of the receptor abolishes stable interactions with JAK1, although it was previously shown that box2 alone is sufficient to stabilize a similar complex of the interferon-α receptor and TYK2. Published by Elsevier Ltd.

HIV-1 Vpu Antagonizes CD317/Tetherin by Adaptor Protein-1-Mediated Exclusion from Virus Assembly Sites

PubMed Central

Pujol, François M.; Laketa, Vibor; Schmidt, Florian; Mukenhirn, Markus; Müller, Barbara; Boulant, Steeve; Grimm, Dirk; Keppler, Oliver T.

2016-01-01

their release. The viral protein Vpu antagonizes this restriction, and molecular interactions with the restriction factor and adaptor protein complex 1 (AP-1) were suggested to mediate this activity. Vpu modulates intracellular trafficking of CD317/tetherin and excludes the restriction factor from HIV-1 assembly sites at the plasma membrane, but the relative contribution of these effects to antagonism remain elusive. Using a panel of Vpu mutants, as well as interference with AP-1 function and expression, we show here that Vpu antagonizes CD317/tetherin by blocking its recruitment to viral assembly sites in an AP-1-dependent manner. These results refine our understanding of the molecular mechanisms of CD317/tetherin antagonism and suggest complexes of Vpu with the restriction factor and AP-1 as targets for potential therapeutic intervention. PMID:27170757

Role of Per1-interacting protein of the suprachiasmatic nucleus in NGF mediated neuronal survival.

PubMed

Kiyama, Atsuko; Isojima, Yasushi; Nagai, Katsuya

2006-01-13

We previously identified Per1-interacting protein of the suprachiasmatic nucleus (PIPS) in rats. To reveal its role, its tissue distribution was examined by immunoblotting. PIPS-like immunoreactive substance (PIPSLS) was observed in the brain, adrenal gland, and PC12 cells. Since PIPS, which has no nuclear localization signal (NLS), is translocated into nuclei of COS-7 cells in the presence of mPer1, the effect of NGF on nuclear localization of PIPS was examined using PC12 cells. NGF caused nuclear translocation of either PIPSLS or GFP-PIPS. NGF mediated nuclear translocation of PIPSLS was blocked by K252a, a TrkA-inhibitor, or wortmannin, a PI3K-inhibitor. Gab1, which is implicated in TrkA signaling and has NLS, co-immunoprecipitated with PIPSLS from PC12 cells using an anti-PIPS antibody. Inhibition of PIPS expression by RNAi increased levels of apoptosis in PC12 cells. These findings suggest that nuclear translocation of PIPS is involved in NGF mediated neuronal survival via TrkA, PI3K, and Gab1 signaling pathway.

Improved targeting of JAK2 leads to increased therapeutic efficacy in myeloproliferative neoplasms

PubMed Central

Bhagwat, Neha; Koppikar, Priya; Keller, Matthew; Marubayashi, Sachie; Shank, Kaitlyn; Rampal, Raajit; Qi, Jun; Kleppe, Maria; Patel, Hardik J.; Shah, Smit K.; Taldone, Tony; Bradner, James E.; Chiosis, Gabriela

2014-01-01

The discovery of JAK2/MPL mutations in patients with myeloproliferative neoplasms (MPN) led to clinical development of Janus kinase (JAK) inhibitors for treatment of MPN. These inhibitors improve constitutional symptoms and splenomegaly but do not significantly reduce mutant allele burden in patients. We recently showed that chronic exposure to JAK inhibitors results in inhibitor persistence via JAK2 transactivation and persistent JAK–signal transducer and activator of transcription signaling. We performed genetic and pharmacologic studies to determine whether improved JAK2 inhibition would show increased efficacy in MPN models and primary samples. Jak2 deletion in vivo led to profound reduction in disease burden not seen with JAK inhibitors, and deletion of Jak2 following chronic ruxolitinib therapy markedly reduced mutant allele burden. This demonstrates that JAK2 remains an essential target in MPN cells that survive in the setting of chronic JAK inhibition. Combination therapy with the heat shock protein 90 (HSP90) inhibitor PU-H71 and ruxolitinib reduced total and phospho-JAK2 and achieved more potent inhibition of downstream signaling than ruxolitinib monotherapy. Combination treatment improved blood counts, spleen weights, and reduced bone marrow fibrosis compared with ruxolitinib alone. These data suggest alternate approaches that increase JAK2 targeting, including combination JAK/HSP90 inhibitor therapy, are warranted in the clinical setting. PMID:24470592

Jak2 is Necessary for Neuroendocrine Control of Female Reproduction

PubMed Central

Wu, Sheng; Divall, Sara; Hoffman, Gloria E.; Le, Wei Wei; Wagner, Kay-Uwe; Wolfe, Andrew

2011-01-01

GnRH neurons represent the final common output of signals from the brain that regulates reproductive function. A wide range of environmental factors impact GnRH neuron activity including disease, stress, nutrition, and seasonal cues, as well as gonadal steroid hormones. The CNS response is thought to be mediated, at least in part, through intermediate signaling molecules that affect GnRH neuronal activity. In vitro, GnRH neuronal cell lines respond to a variety of ligands which activate the Jak/STAT intracellular signaling pathway. In order to determine its biological function in reproduction, we used Cre/LoxP technology to generate GnRH neuron specific Jak2 conditional knockout (Jak2 G−/−) mice. GnRH mRNA levels were reduced in Jak2 G−/− mice when compared to controls, while the number of GnRH neurons was equivalent, indicating a reduction in GnRH gene expression. Secretion of GnRH is also reduced as basal serum LH levels were significantly lower in female Jak2 G−/− mice while the pituitary responded normally to exogenous GnRH. Preovulatory LH surge levels were blunted in Jak2 G−/− mice, which was correlated with reduced GnRH neuronal activation as assessed by c-Fos. However the activation of GnRH neurons following release from estrogen negative feedback is retained. Female Jak2 G−/− mice exhibited significantly delayed puberty and first estrus, abnormal estrous cyclicity and impaired fertility. These results demonstrate an essential role for Jak2 signaling in GnRH neurons for normal reproductive development and fertility in female mice. PMID:21209203

JAK/STAT3-Regulated Fatty Acid β-Oxidation Is Critical for Breast Cancer Stem Cell Self-Renewal and Chemoresistance.

PubMed

Wang, Tianyi; Fahrmann, Johannes Francois; Lee, Heehyoung; Li, Yi-Jia; Tripathi, Satyendra C; Yue, Chanyu; Zhang, Chunyan; Lifshitz, Veronica; Song, Jieun; Yuan, Yuan; Somlo, George; Jandial, Rahul; Ann, David; Hanash, Samir; Jove, Richard; Yu, Hua

2018-01-09

Cancer stem cells (CSCs) are critical for cancer progression and chemoresistance. How lipid metabolism regulates CSCs and chemoresistance remains elusive. Here, we demonstrate that JAK/STAT3 regulates lipid metabolism, which promotes breast CSCs (BCSCs) and cancer chemoresistance. Inhibiting JAK/STAT3 blocks BCSC self-renewal and expression of diverse lipid metabolic genes, including carnitine palmitoyltransferase 1B (CPT1B), which encodes the critical enzyme for fatty acid β-oxidation (FAO). Moreover, mammary-adipocyte-derived leptin upregulates STAT3-induced CPT1B expression and FAO activity in BCSCs. Human breast-cancer-derived data suggest that the STAT3-CPT1B-FAO pathway promotes cancer cell stemness and chemoresistance. Blocking FAO and/or leptin re-sensitizes them to chemotherapy and inhibits BCSCs in mouse breast tumors in vivo. We identify a critical pathway for BCSC maintenance and breast cancer chemoresistance. Copyright © 2017 Elsevier Inc. All rights reserved.

Pleiotropic AT1 receptor signaling pathways mediating physiological and pathogenic actions of angiotensin II.

PubMed

Hunyady, László; Catt, Kevin J

2006-05-01

Angiotensin II (Ang II) activates a wide spectrum of signaling responses via the AT1 receptor (AT1R) that mediate its physiological control of blood pressure, thirst, and sodium balance and its diverse pathological actions in cardiovascular, renal, and other cell types. Ang II-induced AT1R activation via Gq/11 stimulates phospholipases A2, C, and D, and activates inositol trisphosphate/Ca2+ signaling, protein kinase C isoforms, and MAPKs, as well as several tyrosine kinases (Pyk2, Src, Tyk2, FAK), scaffold proteins (G protein-coupled receptor kinase-interacting protein 1, p130Cas, paxillin, vinculin), receptor tyrosine kinases, and the nuclear factor-kappaB pathway. The AT1R also signals via Gi/o and G11/12 and stimulates G protein-independent signaling pathways, such as beta-arrestin-mediated MAPK activation and the Jak/STAT. Alterations in homo- or heterodimerization of the AT1R may also contribute to its pathophysiological roles. Many of the deleterious actions of AT1R activation are initiated by locally generated, rather than circulating, Ang II and are concomitant with the harmful effects of aldosterone in the cardiovascular system. AT1R-mediated overproduction of reactive oxygen species has potent growth-promoting, proinflammatory, and profibrotic actions by exerting positive feedback effects that amplify its signaling in cardiovascular cells, leukocytes, and monocytes. In addition to its roles in cardiovascular and renal disease, agonist-induced activation of the AT1R also participates in the development of metabolic diseases and promotes tumor progression and metastasis through its growth-promoting and proangiogenic activities. The recognition of Ang II's pathogenic actions is leading to novel clinical applications of angiotensin-converting enzyme inhibitors and AT1R antagonists, in addition to their established therapeutic actions in essential hypertension.

Germline variations at JAK2, TERT, HBS1L-MYB and MECOM and the risk of myeloproliferative neoplasms in Taiwanese population

PubMed Central

Chiang, Yi-Hao; Chang, Yu-Cheng; Lin, Huan-Chau; Huang, Ling; Cheng, Chun-Chia; Wang, Wei-Ting; Cheng, Hung-I; Su, Nai-Wen; Chen, Caleb Gon-Shen; Lin, Johnson; Chang, Yi-Fang; Chang, Ming-Chih; Hsieh, Ruey-Kuen; Chou, Wen-Chien; Lim, Ken-Hong; Kuo, Yuan-Yeh

2017-01-01

Germline variations at JAK2, TERT, HBS1L-MYB and MECOM have been found to associate with myeloproliferative neoplasms (MPNs) in European populations. Whether these germline variations are associated with MPNs in Taiwanese population is obscure. Here we aimed to evaluate the association of five germline variations (JAK2 46/1 haplotype tagged by rs12343867, JAK2 intron 8 rs12339666, TERT rs2736100, HBS1L-MYB rs9376092 and MECOM rs2201862) and the risk of MPNs in Taiwanese population. A total of 178 MPN patients (109 essential thrombocythemia, 54 polycythemia vera and 15 primary myelofibrosis) were enrolled into this study. The information of 17033 control subjects was obtained from Taiwan Biobank database. The JAK2 46/1 haplotype, JAK2 rs12339666 and TERT rs2736100 were significantly associated with Taiwanese MPNs (P = 3.6×10-19, 1.9×10-19 and 3.1×10-6, respectively), and JAK2V617F-positive MPNs (n=121) (P = 5.6×10-21, 4.4×10-21 and 8.6×10-7, respectively). In JAK2V617F-negative cases (n=55), only the JAK2 46/1 haplotype and JAK2 rs12339666 remained statistically significant (P= 0.009 and 0.007, respectively). When stratified by disease subtypes, the JAK2 46/1 haplotype and JAK2 rs12339666 were significantly associated with all three MPN subtypes, but TERT rs2736100 was only associated with essential thrombocythemia and polycythemia vera. We did not find any association of these five SNPs with CALR mutations in our cohort. Furthermore, the risk alleles of MECOM rs2201862 and HBS1L-MYB rs9376092 were demonstrated to be negatively associated with the risk of developing polycythemia vera. In conclusion, germline variations at JAK2 (both the 46/1 haplotype and rs12339666) and TERT rs2736100 were associated with MPNs in Taiwanese population. PMID:29100304

An Auxilin-Like J-Domain Protein, JAC1, Regulates Phototropin-Mediated Chloroplast Movement in Arabidopsis1[w

PubMed Central

Suetsugu, Noriyuki; Kagawa, Takatoshi; Wada, Masamitsu

2005-01-01

The ambient-light conditions mediate chloroplast relocation in plant cells. Under the low-light conditions, chloroplasts accumulate in the light (accumulation response), while under the high-light conditions, they avoid the light (avoidance response). In Arabidopsis (Arabidopsis thaliana), the accumulation response is mediated by two blue-light receptors, termed phototropins (phot1 and phot2) that act redundantly, and the avoidance response is mediated by phot2 alone. A mutant, J-domain protein required for chloroplast accumulation response 1 (jac1), lacks the accumulation response under weak blue light but shows a normal avoidance response under strong blue light. In dark-adapted wild-type cells, chloroplasts accumulate on the bottom of cells. Both the jac1 and phot2 mutants are defective in this chloroplast movement in darkness. Positional cloning of JAC1 reveals that this gene encodes a J-domain protein, resembling clathrin-uncoating factor auxilin at its C terminus. The amounts of JAC1 transcripts and JAC1 proteins are not regulated by light and by phototropins. A green fluorescent protein-JAC1 fusion protein showed a similar localization pattern to green fluorescent protein alone in a transient expression assay using Arabidopsis mesophyll cells and onion (Allium cepa) epidermal cells, suggesting that the JAC1 protein may be a soluble cytosolic protein. Together, these results suggest that JAC1 is an essential component of phototropin-mediated chloroplast movement. PMID:16113208

Model Based Targeting of IL-6-Induced Inflammatory Responses in Cultured Primary Hepatocytes to Improve Application of the JAK Inhibitor Ruxolitinib.

PubMed

Sobotta, Svantje; Raue, Andreas; Huang, Xiaoyun; Vanlier, Joep; Jünger, Anja; Bohl, Sebastian; Albrecht, Ute; Hahnel, Maximilian J; Wolf, Stephanie; Mueller, Nikola S; D'Alessandro, Lorenza A; Mueller-Bohl, Stephanie; Boehm, Martin E; Lucarelli, Philippe; Bonefas, Sandra; Damm, Georg; Seehofer, Daniel; Lehmann, Wolf D; Rose-John, Stefan; van der Hoeven, Frank; Gretz, Norbert; Theis, Fabian J; Ehlting, Christian; Bode, Johannes G; Timmer, Jens; Schilling, Marcel; Klingmüller, Ursula

2017-01-01

IL-6 is a central mediator of the immediate induction of hepatic acute phase proteins (APP) in the liver during infection and after injury, but increased IL-6 activity has been associated with multiple pathological conditions. In hepatocytes, IL-6 activates JAK1-STAT3 signaling that induces the negative feedback regulator SOCS3 and expression of APPs. While different inhibitors of IL-6-induced JAK1-STAT3-signaling have been developed, understanding their precise impact on signaling dynamics requires a systems biology approach. Here we present a mathematical model of IL-6-induced JAK1-STAT3 signaling that quantitatively links physiological IL-6 concentrations to the dynamics of IL-6-induced signal transduction and expression of target genes in hepatocytes. The mathematical model consists of coupled ordinary differential equations (ODE) and the model parameters were estimated by a maximum likelihood approach, whereas identifiability of the dynamic model parameters was ensured by the Profile Likelihood. Using model simulations coupled with experimental validation we could optimize the long-term impact of the JAK-inhibitor Ruxolitinib, a therapeutic compound that is quickly metabolized. Model-predicted doses and timing of treatments helps to improve the reduction of inflammatory APP gene expression in primary mouse hepatocytes close to levels observed during regenerative conditions. The concept of improved efficacy of the inhibitor through multiple treatments at optimized time intervals was confirmed in primary human hepatocytes. Thus, combining quantitative data generation with mathematical modeling suggests that repetitive treatment with Ruxolitinib is required to effectively target excessive inflammatory responses without exceeding doses recommended by the clinical guidelines.

Comprehensive review of JAK inhibitors in myeloproliferative neoplasms

PubMed Central

Sonbol, Mohamad Bassam; Firwana, Belal; Zarzour, Ahmad; Morad, Mohammad; Rana, Vishal

2013-01-01

Myeloproliferative neoplasms (MPNs) are clonal hematopoietic stem-cell disorders, characterized phenotypically by the abnormal accumulation of mature-appearing myeloid cells. Polycythemia vera, essential thrombocythemia, primary myelofibrosis (also known as ‘BCR-ABL1-negative’ MPNs), and chronic myeloid leukemia (CML) are the primary types of MPNs. After the discovery of the BCR-ABL1 fusion protein in CML, several oncogenic tyrosine kinases have been identified in ‘BCR-ABL1-negative’ MPNs, most importantly, JAK2V617F mutation. The similarity in the clinical characteristics of the BCR-ABL1-negative MPN patients along with the prevalence of the Janus kinase mutation in this patient population provided a strong rationale for the development of a new class of pharmacologic inhibitors that target this pathway. The first of its class, ruxolitinib, has now been approved by the food and drug administration (FDA) for the management of patients with intermediate- to high-risk myelofibrosis. Ruxolitinib provides significant and sustained improvements in spleen related and constitutional symptoms secondary to the disease. Although noncurative, ruxolitinib represents a milestone in the treatment of myelofibrosis patients. Other types of JAK2 inhibitors are being tested in various clinical trials at this point and may provide better efficacy data and safety profile than its predecessor. In this article, we comprehensively reviewed and summarized the available preclinical and clinical trials pertaining to JAK inhibitors. PMID:23610611

Cytokine receptor signaling is required for the survival of ALK− anaplastic large cell lymphoma, even in the presence of JAK1/STAT3 mutations

PubMed Central

Chen, Jing; Zhang, Yong; Petrus, Michael N.; Xiao, Wenming; Nicolae, Alina; Raffeld, Mark; Pittaluga, Stefania; Bamford, Richard N.; Nakagawa, Masao; Ouyang, Sunny Tianyi; Epstein, Alan L.; Kadin, Marshall E.; Del Mistro, Annarose; Woessner, Richard; Jaffe, Elaine S.; Waldmann, Thomas A.

2017-01-01

Activating Janus kinase (JAK) and signal transducer and activator of transcription (STAT) mutations have been discovered in many T-cell malignancies, including anaplastic lymphoma kinase (ALK)− anaplastic large cell lymphomas (ALCLs). However, such mutations occur in a minority of patients. To investigate the clinical application of targeting JAK for ALK− ALCL, we treated ALK− cell lines of various histological origins with JAK inhibitors. Interestingly, most exogenous cytokine-independent cell lines responded to JAK inhibition regardless of JAK mutation status. JAK inhibitor sensitivity correlated with the STAT3 phosphorylation status of tumor cells. Using retroviral shRNA knockdown, we have demonstrated that these JAK inhibitor-sensitive cells are dependent on both JAK1 and STAT3 for survival. JAK1 and STAT3 gain-of-function mutations were found in some, but not all, JAK inhibitor-sensitive cells. Moreover, the mutations alone cannot explain the JAK1/STAT3 dependency, given that wild-type JAK1 or STAT3 was sufficient to promote cell survival in the cells that had either JAK1or STAT3 mutations. To investigate whether other mechanisms were involved, we knocked down upstream receptors GP130 or IL-2Rγ. Knockdown of GP130 or IL-2Rγ induced cell death in selected JAK inhibitor-sensitive cells. High expression levels of cytokines, including IL-6, were demonstrated in cell lines as well as in primary ALK− ALCL tumors. Finally, ruxolitinib, a JAK1/2 inhibitor, was effective in vivo in a xenograft ALK− ALCL model. Our data suggest that cytokine receptor signaling is required for tumor cell survival in diverse forms of ALK− ALCL, even in the presence of JAK1/STAT3 mutations. Therefore, JAK inhibitor therapy might benefit patients with ALK− ALCL who are phosphorylated STAT3+. PMID:28356514

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

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.

ATM Is Required for the Prolactin-Induced HSP90-Mediated Increase in Cellular Viability and Clonogenic Growth After DNA Damage.

PubMed

Karayazi Atici, Ödül; Urbanska, Anna; Gopinathan, Sesha Gopal; Boutillon, Florence; Goffin, Vincent; Shemanko, Carrie S

2018-02-01

Prolactin (PRL) acts as a survival factor for breast cancer cells, but the PRL signaling pathway and the mechanism are unknown. Previously, we identified the master chaperone, heat shock protein 90 (HSP90) α, as a prolactin-Janus kinase 2 (JAK2)-signal transducer and activator of transcription 5 (STAT5) target gene involved in survival, and here we investigated the role of HSP90 in the mechanism of PRL-induced viability in response to DNA damage. The ataxia-telangiectasia mutated kinase (ATM) protein plays a critical role in the cellular response to double-strand DNA damage. We observed that PRL increased viability of breast cancer cells treated with doxorubicin or etoposide. The increase in cellular resistance is specific to the PRL receptor, because the PRL receptor antagonist, Δ1-9-G129R-hPRL, prevented the increase in viability. Two different HSP90 inhibitors, 17-allylamino-17-demethoxygeldanamycin and BIIB021, reduced the PRL-mediated increase in cell viability of doxorubicin-treated cells and led to a decrease in JAK2, ATM, and phosphorylated ATM protein levels. Inhibitors of JAK2 (G6) and ATM (KU55933) abolished the PRL-mediated increase in cell viability of DNA-damaged cells, supporting the involvement of each, as well as the crosstalk of ATM with the PRL pathway in the context of DNA damage. Drug synergism was detected between the ATM inhibitor (KU55933) and doxorubicin and between the HSP90 inhibitor (BIIB021) and doxorubicin. Short interfering RNA directed against ATM prevented the PRL-mediated increase in cell survival in two-dimensional cell culture, three-dimensional collagen gel cultures, and clonogenic cell survival, after doxorubicin treatment. Our results indicate that ATM contributes to the PRL-JAK2-STAT5-HSP90 pathway in mediating cellular resistance to DNA-damaging agents. Copyright © 2018 Endocrine Society.

JAK3 as an Emerging Target for Topical Treatment of Inflammatory Skin Diseases.

PubMed

Alves de Medeiros, Ana Karina; Speeckaert, Reinhart; Desmet, Eline; Van Gele, Mireille; De Schepper, Sofie; Lambert, Jo

2016-01-01

The recent interest and elucidation of the JAK/STAT signaling pathway created new targets for the treatment of inflammatory skin diseases (ISDs). JAK inhibitors in oral and topical formulations have shown beneficial results in psoriasis and alopecia areata. Patients suffering from other ISDs might also benefit from JAK inhibition. Given the development of specific JAK inhibitors, the expression patterns of JAKs in different ISDs needs to be clarified. We aimed to analyze the expression of JAK/STAT family members in a set of prevalent ISDs: psoriasis, lichen planus (LP), cutaneous lupus erythematosus (CLE), atopic dermatitis (AD), pyoderma gangrenosum (PG) and alopecia areata (AA) versus healthy controls for (p)JAK1, (p)JAK2, (p)JAK3, (p)TYK2, pSTAT1, pSTAT2 and pSTAT3. The epidermis carried in all ISDs, except for CLE, a strong JAK3 signature. The dermal infiltrate showed a more diverse expression pattern. JAK1, JAK2 and JAK3 were significantly overexpressed in PG and AD suggesting the need for pan-JAK inhibitors. In contrast, psoriasis and LP showed only JAK1 and JAK3 upregulation, while AA and CLE were characterized by a single dermal JAK signal (pJAK3 and pJAK1, respectively). This indicates that the latter diseases may benefit from more targeted JAK inhibitors. Our in vitro keratinocyte psoriasis model displayed reversal of the psoriatic JAK profile following tofacitinib treatment. This direct interaction with keratinocytes may decrease the need for deep skin penetration of topical JAK inhibitors in order to exert its effects on dermal immune cells. In conclusion, these results point to the important contribution of the JAK/STAT pathway in several ISDs. Considering the epidermal JAK3 expression levels, great interest should go to the investigation of topical JAK3 inhibitors as therapeutic option of ISDs.

JAK2 aberrations in childhood B-cell precursor acute lymphoblastic leukemia

PubMed Central

de Goffau-Nobel, Willemieke; Hoogkamer, Alex Q.; Boer, Judith M.; Boeree, Aurélie; van de Ven, Cesca; Koudijs, Marco J.; Besselink, Nicolle J.M.; de Groot-Kruseman, Hester A.; Zwaan, Christian Michel; Horstmann, Martin A.; Pieters, Rob; den Boer, Monique L.

2017-01-01

JAK2 abnormalities may serve as target for precision medicines in pediatric B-cell precursor acute lymphoblastic leukemia (BCP-ALL). In the current study we performed a screening for JAK2 mutations and translocations, analyzed the clinical outcome and studied the efficacy of two JAK inhibitors in primary BCP-ALL cells. Importantly, we identify a number of limitations of JAK inhibitor therapy. JAK2 mutations mainly occurred in the poor prognostic subtypes BCR-ABL1-like and non- BCR-ABL1-like B-other (negative for sentinel cytogenetic lesions). JAK2 translocations were restricted to BCR-ABL1-like cases. Momelotinib and ruxolitinib were cytotoxic in both JAK2 translocated and JAK2 mutated cells, although efficacy in JAK2 mutated cells highly depended on cytokine receptor activation by TSLP. However, our data also suggest that the effect of JAK inhibition may be compromised by mutations in alternative survival pathways and microenvironment-induced resistance. Furthermore, inhibitors induced accumulation of phosphorylated JAK2Y1007, which resulted in a profound re-activation of JAK2 signaling upon release of the inhibitors. This preclinical evidence implies that further optimization and evaluation of JAK inhibitor treatment is necessary prior to its clinical integration in pediatric BCP-ALL. PMID:29163799

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

Low-affinity spermine block mediating outward currents through Kir2.1 and Kir2.2 inward rectifier potassium channels

PubMed Central

Ishihara, Keiko; Yan, Ding-Hong

2007-01-01

The outward component of the strong inward rectifier K+ current (IKir) plays a pivotal role in polarizing the membranes of excitable and non-excitable cells and is regulated by voltage-dependent channel block by internal cations. Using the Kir2.1 channel, we previously showed that a small fraction of the conductance susceptible only to a low-affinity mode of block likely carries a large portion of the outward current. To further examine the relevance of the low-affinity block to outward IKir and to explore its molecular mechanism, we studied the block of the Kir2.1 and Kir2.2 channels by spermine, which is the principal Kir2 channel blocker. Current–voltage relations of outward Kir2.2 currents showed a peak, a plateau and two peaks in the presence of 10, 1 and 0.1 μm spermine, respectively, which was explained by the presence of two conductances that differ in their susceptibility to spermine block. When the current–voltage relations showed one peak, like those of native IKir, outward Kir2.2 currents were mediated mostly by the conductance susceptible to the low-affinity block. They also flowed in a narrower range than the corresponding Kir2.1 currents, because of 3- to 4-fold greater susceptibility to the low-affinity block than in Kir2.1. Reducing external [K+] shifted the voltage dependences of both the high- and low-affinity block of Kir2.1 in parallel with the shift in the reversal potential, confirming the importance of the low-affinity block in mediating outward IKir. When Kir2.1 mutants known to have reduced sensitivity to internal blockers were examined, the D172N mutation in the transmembrane pore region made almost all of the conductance susceptible only to low-affinity block, while the E224G mutation in the cytoplasmic pore region reduced the sensitivity to low-affinity block without markedly altering that to the high-affinity block or the high/low conductance ratio. The effects of these mutations support the hypothesis that Kir2 channels exist in

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

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.

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

The Prevalence of JAK2, MPL, and CALR Mutations in Chinese Patients With BCR-ABL1-Negative Myeloproliferative Neoplasms.

PubMed

Lin, Yani; Liu, Enbin; Sun, Qi; Ma, Jiao; Li, QingHua; Cao, Zeng; Wang, Jun; Jia, Yujiao; Zhang, Hongju; Song, Zhen; Ai, Xiaofei; Shi, Lihui; Feng, Xiaofang; Li, Chenwei; Wang, Jianxiang; Ru, Kun

2015-07-01

To evaluate the mutation frequency of JAK2 V617F, JAK2 exon 12, MPL exon 10, and CALR exon 9 and the value of the combined tests in the diagnosis of BCR-ABL1-negative myeloproliferative neoplasms (MPNs). In the current study, mutations of JAK2 V617F, JAK2 exon 12, MPL exon 10, and CALR exon 9 were analyzed in 929 Chinese patients with BCR-ABL1-negative MPN, including 234 cases of polycythemia vera (PV), 428 ETs, 187 PMFs, and 80 unclassifiable MPNs (MPN-Us). Our result showed that the positive rate of any of four mutations in patients with PV, ET, PMF, and MPN-U was 89.3%, 83.4%, 87.2%, and 77.5%, respectively, which significantly improved the diagnostic rate, especially in ET and PMF. Meanwhile, we also found that the patients without any of four mutations were younger than those with one or more mutations. Unexpectedly, the coexistence of JAK2 V617F and CALR exon 9 was identified in six (0.6%) patients, and JAK2 V617F and MPL exon 10 were present simultaneously in two (0.2%) patients. In addition, we also identified several novel mutation types in CALR exon 9. The combined genetic tests of JAK2 V617F, JAK2 exon 12, MPL exon 10, and CALR exon 9 help improve the diagnostic rate for BCR-ABL1-negative MPN. Copyright© by the American Society for Clinical Pathology.

mom identifies a receptor for the Drosophila JAK/STAT signal transduction pathway and encodes a protein distantly related to the mammalian cytokine receptor family

PubMed Central

Chen, Hua-Wei; Chen, Xiu; Oh, Su-Wan; Marinissen, Maria J.; Gutkind, J. Silvio; Hou, Steven X.

2002-01-01

The JAK/STAT signal transduction pathway controls numerous events in Drosophila melanogaster development. Receptors for the pathway have yet to be identified. Here we have identified a Drosophila gene that shows embryonic mutant phenotypes identical to those in the hopscotch (hop)/JAK kinase and marelle (mrl)/Stat92e mutations. We named this gene master of marelle (mom). Genetic analyses place mom's function between upd (the ligand) and hop. We further show that cultured cells transfected with the mom gene bind UPD and activate the HOP/STAT92E signal transduction pathway. mom encodes a protein distantly related to the mammalian cytokine receptor family. These data show that mom functions as a receptor of the Drosophila JAK/STAT signal transduction pathway. PMID:11825879

LIF-activated Jak signaling determines Esrrb expression during late-stage reprogramming

PubMed Central

Huang, Delun; Wang, Ling; Duan, Jingyue; Huang, Chang; Tian, Xiuchun (Cindy); Zhang, Ming

2018-01-01

ABSTRACT The regulatory process of naïve-state induced pluripotent stem cell (iPSC) generation is not well understood. Leukemia inhibitory factor (LIF)-activated Janus kinase/signal transducer and activator of transcription 3 (Jak/Stat3) is the master regulator for naïve-state pluripotency achievement and maintenance. The estrogen-related receptor beta (Esrrb) serves as a naïve-state marker gene regulating self-renewal of embryonic stem cells (ESCs). However, the interconnection between Esrrb and LIF signaling for pluripotency establishment in reprogramming is unclear. We screened the marker genes critical for complete reprogramming during mouse iPSC generation, and identified genes including Esrrb that are responsive to LIF/Jak pathway signaling. Overexpression of Esrrb resumes the reprogramming halted by inhibition of Jak activity in partially reprogrammed cells (pre-iPSCs), and leads to the generation of pluripotent iPSCs. We further show that neither overexpression of Nanog nor stimulation of Wnt signaling, two upstream regulators of Esrrb in ESCs, stimulates the expression of Esrrb in reprogramming when LIF or Jak activity is blocked. Our study demonstrates that Esrrb is a specific reprogramming factor regulated downstream of the LIF/Jak signaling pathway. These results shed new light on the regulatory role of LIF pathway on complete pluripotency establishment during iPSC generation. PMID:29212799

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.

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

Model Based Targeting of IL-6-Induced Inflammatory Responses in Cultured Primary Hepatocytes to Improve Application of the JAK Inhibitor Ruxolitinib

PubMed Central

Sobotta, Svantje; Raue, Andreas; Huang, Xiaoyun; Vanlier, Joep; Jünger, Anja; Bohl, Sebastian; Albrecht, Ute; Hahnel, Maximilian J.; Wolf, Stephanie; Mueller, Nikola S.; D'Alessandro, Lorenza A.; Mueller-Bohl, Stephanie; Boehm, Martin E.; Lucarelli, Philippe; Bonefas, Sandra; Damm, Georg; Seehofer, Daniel; Lehmann, Wolf D.; Rose-John, Stefan; van der Hoeven, Frank; Gretz, Norbert; Theis, Fabian J.; Ehlting, Christian; Bode, Johannes G.; Timmer, Jens; Schilling, Marcel; Klingmüller, Ursula

2017-01-01

IL-6 is a central mediator of the immediate induction of hepatic acute phase proteins (APP) in the liver during infection and after injury, but increased IL-6 activity has been associated with multiple pathological conditions. In hepatocytes, IL-6 activates JAK1-STAT3 signaling that induces the negative feedback regulator SOCS3 and expression of APPs. While different inhibitors of IL-6-induced JAK1-STAT3-signaling have been developed, understanding their precise impact on signaling dynamics requires a systems biology approach. Here we present a mathematical model of IL-6-induced JAK1-STAT3 signaling that quantitatively links physiological IL-6 concentrations to the dynamics of IL-6-induced signal transduction and expression of target genes in hepatocytes. The mathematical model consists of coupled ordinary differential equations (ODE) and the model parameters were estimated by a maximum likelihood approach, whereas identifiability of the dynamic model parameters was ensured by the Profile Likelihood. Using model simulations coupled with experimental validation we could optimize the long-term impact of the JAK-inhibitor Ruxolitinib, a therapeutic compound that is quickly metabolized. Model-predicted doses and timing of treatments helps to improve the reduction of inflammatory APP gene expression in primary mouse hepatocytes close to levels observed during regenerative conditions. The concept of improved efficacy of the inhibitor through multiple treatments at optimized time intervals was confirmed in primary human hepatocytes. Thus, combining quantitative data generation with mathematical modeling suggests that repetitive treatment with Ruxolitinib is required to effectively target excessive inflammatory responses without exceeding doses recommended by the clinical guidelines. PMID:29062282

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 quinazoline-based sulfonamide derivative (3D) induces apoptosis in colorectal cancer by inhibiting JAK2-STAT3 pathway.

PubMed

Al-Obeed, Omar; Vaali-Mohammed, Mansoor-Ali; Eldehna, Wagdy M; Al-Khayal, Khayal; Mahmood, Amer; Abdel-Aziz, Hatem A; Zubaidi, Ahmed; Alafeefy, Ahmed; Abdulla, Maha; Ahmad, Rehan

2018-01-01

Colorectal cancer (CRC) is a major worldwide health problem owing to its high prevalence and mortality rate. Developments in screening, prevention, biomarker, personalized therapies and chemotherapy have improved detection and treatment. However, despite these advances, many patients with advanced metastatic tumors still succumb to the disease. New anticancer agents are needed for treating advanced stage CRC as most of the deaths occur due to cancer metastasis. A recently developed novel sulfonamide derivative 4-((2-(4-(dimethylamino) phenyl)quinazolin-4-yl)amino)benzenesulfonamide (3D) has shown potent antitumor effect; however, the mechanism underlying the antitumor effect remains unknown. 3D-mediated inhibition on cell viability was evaluated by MTT and real-time cell proliferation was measured by xCelligence RTDP instrument. Western blotting was used to measure pro-apoptotic, anti-apoptotic proteins and JAK2-STAT3 phosphorylation. Flow cytometry was used to measure ROS production and apoptosis. Our study revealed that 3D treatment significantly reduced the viability of human CRC cells HT-29 and SW620. Furthermore, 3D treatment induced the generation of reactive oxygen species (ROS) in human CRC cells. Confirming our observation, N-acetylcysteine significantly inhibited apoptosis. This is further evidenced by the induction of p53 and Bax; release of cytochrome c; activation of caspase-9, caspase-7 and caspase-3; and cleavage of PARP in 3D-treated cells. This compound was found to have a significant effect on the inhibition of antiapoptotic proteins Bcl2 and BclxL. The results further demonstrate that 3D inhibits JAK2-STAT3 pathway by decreasing the constitutive and IL-6-induced phosphorylation of STAT3. 3D also decreases STAT3 target genes such as cyclin D1 and survivin. Furthermore, a combination study of 3D with doxorubicin (Dox) also showed more potent effects than single treatment of Dox in the inhibition of cell viability. Taken together, these findings

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.

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.

DNA triplet repeats mediate heterochromatin-protein-1-sensitive variegated gene silencing.

PubMed

Saveliev, Alexander; Everett, Christopher; Sharpe, Tammy; Webster, Zoë; Festenstein, Richard

2003-04-24

Gene repression is crucial to the maintenance of differentiated cell types in multicellular organisms, whereas aberrant silencing can lead to disease. The organization of DNA into chromatin and heterochromatin is implicated in gene silencing. In chromatin, DNA wraps around histones, creating nucleosomes. Further condensation of chromatin, associated with large blocks of repetitive DNA sequences, is known as heterochromatin. Position effect variegation (PEV) occurs when a gene is located abnormally close to heterochromatin, silencing the affected gene in a proportion of cells. Here we show that the relatively short triplet-repeat expansions found in myotonic dystrophy and Friedreich's ataxia confer variegation of expression on a linked transgene in mice. Silencing was correlated with a decrease in promoter accessibility and was enhanced by the classical PEV modifier heterochromatin protein 1 (HP1). Notably, triplet-repeat-associated variegation was not restricted to classical heterochromatic regions but occurred irrespective of chromosomal location. Because the phenomenon described here shares important features with PEV, the mechanisms underlying heterochromatin-mediated silencing might have a role in gene regulation at many sites throughout the mammalian genome and modulate the extent of gene silencing and hence severity in several triplet-repeat diseases.

Molecular genetic tests for JAK2V617F, Exon12_JAK2 and MPLW515K/L are highly informative in the evaluation of patients suspected to have BCR-ABL1-negative myeloproliferative neoplasms.

PubMed

dos Santos, Marcos Tadeu; Mitne-Neto, Miguel; Miyashiro, Kozue; Chauffaille, Maria de Lourdes L Ferrari; Rizzatti, Edgar Gil

2014-02-01

Polycythaemia vera (PV), essential thrombocythemia (ET) and idiopathic myelofibrosis (MF), are the most common myeloproliferative neoplasms (MPN) in patients without the BCR-ABL1 gene rearrangement. They are caused by clonal expansion of haematopoietic stem cells and share, as a diagnostic criterion, the identification of JAK2V617F mutation. Classically, when other clinical criteria are present, a JAK2V617F negative case requires the analysis of Exon12_JAK2 for the diagnosis of PV, and of MPL515K/L mutations for the diagnosis of ET and MF. Here, we evaluated 78 samples from Brazilian patients suspected to have MPN, without stratification for PV, ET or MF. We found that 28 (35.9%) are JAK2V617F carriers; from the 50 remaining samples, one (2%) showed an Exon12_JAK2 mutation, and another (2%) was positive for MPLW515L mutation. In summary, the investigation of JAK2V617F, Exon12_JAK2 and MPLW515K/L was relevant for the diagnosis of 38.4% of patients suspected to have BCR-ABL1-negative MPN, suggesting that molecular genetic tests are useful for a quick and unequivocal diagnosis of MPN.

Molecular genetic tests for JAK2V617F, Exon12_JAK2 and MPLW515K/L are highly informative in the evaluation of patients suspected to have BCR-ABL1-negative myeloproliferative neoplasms

PubMed Central

dos Santos, Marcos Tadeu; Mitne-Neto, Miguel; Miyashiro, Kozue; Chauffaille, Maria de Lourdes L Ferrari; Rizzatti, Edgar Gil

2014-01-01

Polycythaemia vera (PV), essential thrombocythemia (ET) and idiopathic myelofibrosis (MF), are the most common myeloproliferative neoplasms (MPN) in patients without the BCR-ABL1 gene rearrangement. They are caused by clonal expansion of haematopoietic stem cells and share, as a diagnostic criterion, the identification of JAK2V617F mutation. Classically, when other clinical criteria are present, a JAK2V617F negative case requires the analysis of Exon12_JAK2 for the diagnosis of PV, and of MPL515K/L mutations for the diagnosis of ET and MF. Here, we evaluated 78 samples from Brazilian patients suspected to have MPN, without stratification for PV, ET or MF. We found that 28 (35.9%) are JAK2V617F carriers; from the 50 remaining samples, one (2%) showed an Exon12_JAK2 mutation, and another (2%) was positive for MPLW515L mutation. In summary, the investigation of JAK2V617F, Exon12_JAK2 and MPLW515K/L was relevant for the diagnosis of 38.4% of patients suspected to have BCR-ABL1-negative MPN, suggesting that molecular genetic tests are useful for a quick and unequivocal diagnosis of MPN. PMID:23986553

TIM-family proteins inhibit HIV-1 release

PubMed Central

Li, Minghua; Ablan, Sherimay D.; Miao, Chunhui; Zheng, Yi-Min; Fuller, Matthew S.; Rennert, Paul D.; Maury, Wendy; Johnson, Marc C.; Freed, Eric O.; Liu, Shan-Lu

2014-01-01

Accumulating evidence indicates that T-cell immunoglobulin (Ig) and mucin domain (TIM) proteins play critical roles in viral infections. Herein, we report that the TIM-family proteins strongly inhibit HIV-1 release, resulting in diminished viral production and replication. Expression of TIM-1 causes HIV-1 Gag and mature viral particles to accumulate on the plasma membrane. Mutation of the phosphatidylserine (PS) binding sites of TIM-1 abolishes its ability to block HIV-1 release. TIM-1, but to a much lesser extent PS-binding deficient mutants, induces PS flipping onto the cell surface; TIM-1 is also found to be incorporated into HIV-1 virions. Importantly, TIM-1 inhibits HIV-1 replication in CD4-positive Jurkat cells, despite its capability of up-regulating CD4 and promoting HIV-1 entry. In addition to TIM-1, TIM-3 and TIM-4 also block the release of HIV-1, as well as that of murine leukemia virus (MLV) and Ebola virus (EBOV); knockdown of TIM-3 in differentiated monocyte-derived macrophages (MDMs) enhances HIV-1 production. The inhibitory effects of TIM-family proteins on virus release are extended to other PS receptors, such as Axl and RAGE. Overall, our study uncovers a novel ability of TIM-family proteins to block the release of HIV-1 and other viruses by interaction with virion- and cell-associated PS. Our work provides new insights into a virus-cell interaction that is mediated by TIMs and PS receptors. PMID:25136083

Inhibition of host cell translation elongation by Legionella pneumophila blocks the host cell unfolded protein response.

PubMed

Hempstead, Andrew D; Isberg, Ralph R

2015-12-08

Cells of the innate immune system recognize bacterial pathogens by detecting common microbial patterns as well as pathogen-specific activities. One system that responds to these stimuli is the IRE1 branch of the unfolded protein response (UPR), a sensor of endoplasmic reticulum (ER) stress. Activation of IRE1, in the context of Toll-like receptor (TLR) signaling, induces strong proinflammatory cytokine induction. We show here that Legionella pneumophila, an intravacuolar pathogen that replicates in an ER-associated compartment, blocks activation of the IRE1 pathway despite presenting pathogen products that stimulate this response. L. pneumophila TLR ligands induced the splicing of mRNA encoding XBP1s, the main target of IRE1 activity. L. pneumophila was able to inhibit both chemical and bacterial induction of XBP1 splicing via bacterial translocated proteins that interfere with host protein translation. A strain lacking five translocated translation elongation inhibitors was unable to block XBP1 splicing, but this could be rescued by expression of a single such inhibitor, consistent with limitation of the response by translation elongation inhibitors. Chemical inhibition of translation elongation blocked pattern recognition receptor-mediated XBP1 splicing, mimicking the effects of the bacterial translation inhibitors. In contrast, host cell-promoted inhibition of translation initiation in response to the pathogen was ineffective in blocking XBP1 splicing, demonstrating the need for the elongation inhibitors for protection from the UPR. The inhibition of host translation elongation may be a common strategy used by pathogens to limit the innate immune response by interfering with signaling via the UPR.

Ectodomain shedding of TNF receptor 1 induced by protein synthesis inhibitors regulates TNF-{alpha}-mediated activation of NF-{kappa}B and caspase-8

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

Ogura, Hirotsugu; Tsukumo, Yoshinori; Department of Bioengineering, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama 226-8501

2008-04-01

The transcription factor nuclear factor {kappa}B (NF-{kappa}B) plays a major role in the inducible resistance to death receptor-mediated apoptosis. It has been established that the protein synthesis inhibitor cycloheximide (CHX) sensitizes many types of cells to tumor necrosis factor (TNF)-{alpha}-induced apoptosis, mainly due to its ability to block de novo synthesis of cellular FLICE-inhibitory protein (c-FLIP). Nevertheless, we have surprisingly found that CHX, as well as its structural analogue acetoxycycloheximide (Ac-CHX), prevents TNF-{alpha}-mediated activation of NF-{kappa}B and caspase-8 in human lung carcinoma A549 cells. Both CHX and Ac-CHX reduced the expression of cell surface TNF receptor 1 (TNF-R1) in amore » dose-dependent manner, while Ac-CHX was approximately 100-fold more effective than CHX. Consistent with this observation, Ac-CHX induced the proteolytic cleavage of TNF-R1 and its release into the culture medium. CHX and Ac-CHX profoundly decreased constitutive and inducible expression of c-FLIP, whereas these compounds potentiated TNF-{alpha}-induced caspase-8 activation only when metalloprotease inhibitors were present. Thus, our results indicate that ectodomain shedding of TNF-R1 induced by protein synthesis inhibitors regulates TNF-{alpha}-mediated activation of NF-{kappa}B and caspase-8.« less

Increased frequency of co-existing JAK2 exon-12 or MPL exon-10 mutations in patients with low JAK2(V617F) allelic burden.

PubMed

Nussenzveig, Roberto H; Pham, Ha T; Perkins, Sherrie L; Prchal, Josef T; Agarwal, Archana M; Salama, Mohamed E

2016-01-01

The frequency of co-existing JAK2(V617F)/MPL and JAK2(V617F)/JAK2 exon-12 mutations has not been previously investigated in MPNs. Poor survival was reported in primary myelofibrosis with low JAK2(V617F) allelic burden. However, mutational status of JAK2 exon-12 or MPL were not reported in these patients. This study developed a cost-effective multiplex high resolution melt assay that screens for mutations in JAK2 gene exons-12 and -14 ((V617F)) and MPL gene exon-10. Co-existing mutations with JAK2(V617F) were detected in 2.9% (6/208; two JAK2 exon-12 and four MPL exon-10) patient specimens with known JAK2(V617F) (allelic-burden range: 0.1-96.8%). Co-existing mutations were detected in specimens with < 12% JAK2(V617F) allelic burden. Current WHO guidelines do not recommend further testing once JAK2(V617F) mutation is detected in MPNs. The findings, however, indicate that quantification of JAK2(V617F) allele burden may be clinically relevant in MPNs and in those with low allelic burden additional testing for JAK2 exon-12 and MPL exon-10 mutation should be pursued.

Intracellular Ca2+ homeostasis and JAK1/STAT3 pathway are involved in the protective effect of propofol on BV2 microglia against hypoxia-induced inflammation and apoptosis

PubMed Central

Wang, Jiaqiang; Miao, Changhong

2017-01-01

Background Perioperative hypoxia may induce microglial inflammation and apoptosis, resulting in brain injury. The neuroprotective effect of propofol against hypoxia has been reported, but the underlying mechanisms are far from clear. In this study, we explored whether and how propofol could attenuate microglia BV2 cells from CoCl2-induced hypoxic injury. Methods Mouse microglia BV2 cells were pretreated with propofol, and then stimulated with CoCl2. TNF-α level in the culture medium was measured by ELISA kit. Cell apoptosis and intracellular calcium concentration were measured by flow cytometry analysis. The effect of propofol on CoCl2-modulated expression of Ca2+/Calmodulin (CaM)-dependent protein kinase II (CAMKIIα), phosphorylated CAMKIIα (pCAMKIIα), STAT3, pSTAT3Y705, pSTAT3S727, ERK1/2, pERK1/2, pNFκB(p65), pro-caspase3, cleaved caspase 3, JAK1, pJAK1, JAK2, pJAK2 were detected by Western blot. Results In BV2 cell, CoCl2 treatment time-dependently increased TNF-α release and induced apoptosis, which were alleviated by propofol. CoCl2 (500μmol/L, 8h) treatment increased intracellular Ca2+ level, and caused the phosphorylation of CAMKIIα, ERK1/2 and NFκB (p65), as well as the activation of caspase 3. More importantly, these effects could be modulated by 25μmol/L propofol via maintaining intracellular Ca2+ homeostasis and via up-regulating the phosphorylation of JAK1 and STAT3 at Tyr705. Conclusion Propofol could protect BV2 microglia from hypoxia-induced inflammation and apoptosis. The potential mechanisms may involve the maintaining of intracellular Ca2+ homeostasis and the activation of JAK1/STAT3 pathway. PMID:28542400

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.

Endotoxin-activated microglia injure brain derived endothelial cells via NF-κB, JAK-STAT and JNK stress kinase pathways

PubMed Central

2011-01-01

Background We previously showed that microglia damage blood brain barrier (BBB) components following ischemic brain insults, but the underlying mechanism(s) is/are not well known. Recent work has established the contribution of toll-like receptor 4 (TLR4) activation to several brain pathologies including ischemia, neurodegeneration and sepsis. The present study established the requirement of microglia for lipopolysaccharide (LPS) mediated endothelial cell death, and explored pathways involved in this toxicity. LPS is a classic TLR4 agonist, and is used here to model aspects of brain conditions where TLR4 stimulation occurs. Methods/Results In monocultures, LPS induced death in microglia, but not brain derived endothelial cells (EC). However, LPS increased EC death when cocultured with microglia. LPS led to nitric oxide (NO) and inducible NO synthase (iNOS) induction in microglia, but not in EC. Inhibiting microglial activation by blocking iNOS and other generators of NO or blocking reactive oxygen species (ROS) also prevented injury in these cocultures. To assess the signaling pathway(s) involved, inhibitors of several downstream TLR-4 activated pathways were studied. Inhibitors of NF-κB, JAK-STAT and JNK/SAPK decreased microglial activation and prevented cell death, although the effect of blocking JNK/SAPK was rather modest. Inhibitors of PI3K, ERK, and p38 MAPK had no effect. Conclusions We show that LPS-activated microglia promote BBB disruption through injury to endothelial cells, and the specific blockade of JAK-STAT, NF-κB may prove to be especially useful anti-inflammatory strategies to confer cerebrovascular protection. PMID:21385378

Aurora kinases and protein phosphatase 1 mediate chromosome congression through regulation of CENP-E

PubMed Central

Kim, Yumi; Holland, Andrew J.; Lan, Weijie; Cleveland, Don W.

2010-01-01

Summary Opposing roles of Aurora kinases and protein phosphatase 1 (PP1) during mitosis have long been suggested. Here we demonstrate that Aurora kinases A and B phosphorylate a single residue on the kinetochore motor CENP-E. PP1 binds CENP-E via a motif overlapping this phosphorylation site and binding is disrupted by Aurora phosphorylation. Phosphorylation of CENP-E by the Auroras is enriched at spindle poles, disrupting binding of PP1 and reducing CENP-E’s affinity for individual microtubules. This phosphorylation is required for CENP-E-mediated towing of initially polar chromosomes toward the cell center. Kinetochores on such chromosomes cannot make subsequent stable attachment to spindle microtubules when dephosphorylation of CENP-E or rebinding of PP1 to CENP-E is blocked. Thus, an Aurora/PP1 phosphorylation switch modulates CENP-E motor activity as an essential feature of chromosome congression from poles and localized PP1 delivery by CENP-E to the outer kinetochore is necessary for stable microtubule capture by those chromosomes. PMID:20691903

JIP1-Mediated JNK Activation Negatively Regulates Synaptic Plasticity and Spatial Memory.

PubMed

Morel, Caroline; Sherrin, Tessi; Kennedy, Norman J; Forest, Kelly H; Avcioglu Barutcu, Seda; Robles, Michael; Carpenter-Hyland, Ezekiel; Alfulaij, Naghum; Standen, Claire L; Nichols, Robert A; Benveniste, Morris; Davis, Roger J; Todorovic, Cedomir

2018-04-11

The c-Jun N-terminal kinase (JNK) signal transduction pathway is implicated in learning and memory. Here, we examined the role of JNK activation mediated by the JNK-interacting protein 1 (JIP1) scaffold protein. We compared male wild-type mice with a mouse model harboring a point mutation in the Jip1 gene that selectively blocks JIP1-mediated JNK activation. These male mutant mice exhibited increased NMDAR currents, increased NMDAR-mediated gene expression, and a lower threshold for induction of hippocampal long-term potentiation. The JIP1 mutant mice also displayed improved hippocampus-dependent spatial memory and enhanced associative fear conditioning. These results were confirmed using a second JIP1 mutant mouse model that suppresses JNK activity. Together, these observations establish that JIP1-mediated JNK activation contributes to the regulation of hippocampus-dependent, NMDAR-mediated synaptic plasticity and learning. SIGNIFICANCE STATEMENT The results of this study demonstrate that c-Jun N-terminal kinase (JNK) activation induced by the JNK-interacting protein 1 (JIP1) scaffold protein negatively regulates the threshold for induction of long-term synaptic plasticity through the NMDA-type glutamate receptor. This change in plasticity threshold influences learning. Indeed, mice with defects in JIP1-mediated JNK activation display enhanced memory in hippocampus-dependent tasks, such as contextual fear conditioning and Morris water maze, indicating that JIP1-JNK constrains spatial memory. This study identifies JIP1-mediated JNK activation as a novel molecular pathway that negatively regulates NMDAR-dependent synaptic plasticity and memory. Copyright © 2018 the authors 0270-6474/18/383708-21$15.00/0.

Jak2 FERM Domain Interaction with the Erythropoietin Receptor Regulates Jak2 Kinase Activity▿

PubMed Central

Funakoshi-Tago, Megumi; Pelletier, Stéphane; Moritake, Hiroshi; Parganas, Evan; Ihle, James N.

2008-01-01

Janus kinases are essential for signal transduction by a variety of cytokine receptors and when inappropriately activated can cause hematopoietic disorders and oncogenesis. Consequently, it can be predicted that the interaction of the kinases with receptors and the events required for activation are highly controlled. In a screen to identify phosphorylation events regulating Jak2 activity in EpoR signaling, we identified a mutant (Jak2-Y613E) which has the property of being constitutively activated, as well as an inactivating mutation (Y766E). Although no evidence was obtained to indicate that either site is phosphorylated in signaling, the consequences of the Y613E mutation are similar to those observed with recently described activating mutations in Jak2 (Jak2-V617F and Jak2-L611S). However, unlike the V617F or L611S mutant, the Y613E mutant requires the presence of the receptor but not Epo stimulation for activation and downstream signaling. The properties of the Jak2-Y613E mutant suggest that under normal conditions, Jak2 that is not associated with a receptor is locked into an inactive state and receptor binding through the FERM domain relieves steric constraints, allowing the potential to be activated with receptor engagement. PMID:18160720

Histone deacetylase-related protein inhibits AES-mediated neuronal cell death by direct interaction.

PubMed

Zhang, Xiaoguang; Chen, Hsin-Mei; Jaramillo, Eduardo; Wang, Lulu; D'Mello, Santosh R

2008-08-15

Histone deacetylase-related protein (HDRP), an alternatively spliced and truncated form of histone deacetylase-9 that lacks a C-terminal catalytic domain, protects neurons from death. In an effort to understand the mechanism by which HDRP mediates its neuroprotective effect, we screened for proteins in the brain that interact with HDRP by using a yeast two-hybrid assay. One of the HDRP-interacting proteins identified in this screen was amino enhancer of split (AES), a 197-amino acid protein belonging to the Groucho family. Interaction between HDRP and AES was verified by in vitro binding assays, coimmunoprecipitation, and colocalization studies. To investigate the significance of the HDRP-AES association to the regulation of neuronal survival, we used cultured cerebellar granule neurons, which undergo apoptosis when treated with low potassium (LK) medium. We found that in contrast to HDRP, whose expression is markedly reduced by LK treatment, AES expression was not appreciably altered. Forced expression of AES in healthy neurons results in cell death, an action that is blocked by the coexpression of HDRP. AES is a truncated version of larger Groucho-related proteins, one of which is transducin-like enhancer of split (TLE)-1. We found that the expression of TLE1 is reduced in LK-treated neurons and the forced expression of TLE1 blocks LK-induced neuronal death as well as death induced by AES. Our results show that AES has apoptotic activity in neurons and suggest that neuroprotection by HDRP is mediated by the inhibition of this activity through direct interaction.

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.

JAK-STAT Pathway Activation in Malignant and Non-Malignant Cells Contributes to MPN Pathogenesis and Therapeutic Response

PubMed Central

Kleppe, Maria; Kwak, Minsuk; Koppikar, Priya; Riester, Markus; Keller, Matthew; Bastian, Lennart; Hricik, Todd; Bhagwat, Neha; McKenney, Anna Sophia; Papalexi, Efthymia; Abdel-Wahab, Omar; Rampal, Raajit; Marubayashi, Sachie; Chen, Jonathan J.; Romanet, Vincent; Fridman, Jordan S.; Bromberg, Jacqueline; Teruya-Feldstein, Julie; Murakami, Masato; Radimerski, Thomas; Michor, Franziska; Fan, Rong; Levine, Ross L.

2015-01-01

The identification of JAK2/MPL mutations in patients with myeloproliferative neoplasms (MPN) led to the clinical development of JAK kinase inhibitors, including ruxolitinib. Ruxolitinib reduces splenomegaly and systemic symptoms in myelofibrosis (MF) and improves overall survival; however the mechanism by which JAK inhibitors achieve efficacy has not been delineated. MPN patients present with increased levels of circulating pro-inflammatory cytokines, which are mitigated by JAK inhibitor therapy. We sought to elucidate mechanisms by which JAK inhibitors attenuate cytokine-mediated pathophysiology. Single cell profiling demonstrated that hematopoietic cells from MF models and patient samples aberrantly secrete inflammatory cytokines. Pan-hematopoietic Stat3 deletion reduced disease severity and attenuated cytokine secretion, with similar efficacy as observed with ruxolitinib therapy. By contrast, Stat3 deletion restricted to MPN cells did not reduce disease severity or cytokine production. Consistent with these observations, we found that malignant and non-malignant cells aberrantly secrete cytokines and JAK inhibition reduces cytokine production from both populations. PMID:25572172

Efficacy, Safety, Pharmacokinetics, and Pharmacodynamics of Filgotinib, a Selective JAK-1 Inhibitor, After Short-Term Treatment of Rheumatoid Arthritis: Results of Two Randomized Phase IIa Trials.

PubMed

Vanhoutte, Frédéric; Mazur, Minodora; Voloshyn, Oleksandr; Stanislavchuk, Mykola; Van der Aa, Annegret; Namour, Florence; Galien, René; Meuleners, Luc; van 't Klooster, Gerben

2017-10-01

JAK inhibitors have shown efficacy in rheumatoid arthritis (RA). We undertook this study to test our hypothesis that selective inhibition of JAK-1 would combine good efficacy with a better safety profile compared with less selective JAK inhibitors. In two 4-week exploratory, double-blind, placebo-controlled phase IIa trials, 127 RA patients with an insufficient response to methotrexate (MTX) received filgotinib (GLPG0634, GS-6034) oral capsules (100 mg twice daily or 30, 75, 150, 200, or 300 mg once daily) or placebo, added onto a stable regimen of MTX, to evaluate safety, efficacy, pharmacokinetics (PK), and pharmacodynamics (PD) of filgotinib. The primary efficacy end point was the number and percentage of patients in each treatment group meeting the American College of Rheumatology 20% improvement criteria (achieving an ACR20 response) at week 4. Treatment with filgotinib at 75-300 mg met the primary end point and showed early onset of efficacy. ACR20 response rates progressively increased to week 4, and the Disease Activity Score in 28 joints using the C-reactive protein (CRP) level decreased. Marked and sustained improvements were observed in serum CRP level and other PD markers. The PK of filgotinib and its major metabolite was dose proportional over the 30-300 mg range. Early side effects seen with other less selective JAK inhibitors were not observed (e.g., there was no worsening of anemia [JAK-2 inhibition related], no effects on liver transaminases, and no increase in low-density lipoprotein or total cholesterol). A limited decrease in neutrophils without neutropenia was consistent with immunomodulatory effects through JAK-1 inhibition. There were no infections. Overall, filgotinib was well tolerated. Events related to study drug were mild or moderate and transient during therapy, and the most common such event was nausea. Selective inhibition of JAK-1 with filgotinib shows initial efficacy in RA with an encouraging safety profile in these exploratory

Driver mutations (JAK2V617F, MPLW515L/K or CALR), pentraxin-3 and C-reactive protein in essential thrombocythemia and polycythemia vera.

PubMed

Lussana, Federico; Carobbio, Alessandra; Salmoiraghi, Silvia; Guglielmelli, Paola; Vannucchi, Alessandro Maria; Bottazzi, Barbara; Leone, Roberto; Mantovani, Alberto; Barbui, Tiziano; Rambaldi, Alessandro

2017-02-22

The driver mutations JAK2V617F, MPLW515L/K and CALR influence disease phenotype of myeloproliferative neoplasms (MPNs) and might sustain a condition of chronic inflammation. Pentraxin 3 (PTX3) and high-sensitivity C-reactive protein (hs-CRP) are inflammatory biomarkers potentially useful for refining prognostic classification of MPNs. We evaluated 305 with essential thrombocythemia (ET) and 172 polycythemia vera (PV) patients diagnosed according to the 2016 WHO criteria and with full molecular characterization for driver mutations. PTX3 levels were significantly increased in carriers of homozygous JAK2V617F mutation compared to all the other genotypes and triple negative ET patients, while hs-CRP levels were independent of the mutational profile. The risk of haematological evolution and death from any cause was about 2- and 1.5-fold increased in individuals with high PTX-3 levels, while the thrombosis rate tended to be lower. High hs-CRP levels were associated with risk of haematological evolution, death and also major thrombosis. After sequential adjustment for potential confounders (age, gender, diagnosis and treatments) and the presence of JAK2V617F homozygous status, high hs-CRP levels remained significant for all outcomes, while JAK2V617F homozygous status as well as treatments were the factors independently accounting for adverse outcomes among patients with high PTX3 levels. These results provide evidence that JAK2V617F mutation influences MPN-associated inflammation with a strong correlation between allele burden and PTX3 levels. Plasma levels of hs-CRP and PTX3 might be of prognostic value for patients with ET and PV, but their validation in future prospective studies is needed.

Nuclear degradation of Wilms tumor 1-associating protein and survivin splice variant switching underlie IGF-1-mediated survival.

PubMed

Small, Theodore W; Pickering, J Geoffrey

2009-09-11

WTAP (Wilms tumor 1-associating protein) is a recently identified nuclear protein that is essential for mouse embryo development. The Drosophila homolog of WTAP, Fl(2)d, regulates pre-mRNA splicing; however, the role of WTAP in mammalian cells is uncertain. To elucidate a context for WTAP action, we screened growth and survival factors for their effects on WTAP expression in vascular smooth muscle cells (SMCs), a cell type previously found to express WTAP dynamically. This revealed that insulin-like growth factor-1 (IGF-1) uniquely reduced WTAP abundance. This decline in WTAP proved to be necessary for IGF-1 to confer its antiapoptotic properties, which were blocked by transducing the WTAP gene into SMCs. WTAP down-regulation by IGF-1 was mediated by an IGF-1 receptor-phosphatidylinositol 3-kinase-Akt signaling axis that directed WTAP degradation via a nuclear 26 S proteasome. Moreover, by promoting the degradation of WTAP, IGF-1 shifted the pre-mRNA splicing program for the survival factor, survivin, to reduce expression of survivin-2B, which is proapoptotic, and increase expression of survivin, which is antiapoptotic. Knockdown of survivin-2B rescued the ability of IGF-1 to promote survival when WTAP was overexpressed. These data uncover a novel regulatory cascade for human SMC survival based on adjusting the nuclear abundance of WTAP to define the splice variant balance among survivin isoforms.

Glycogen Synthase Kinase 3 Inactivation Induces Cell Senescence through Sterol Regulatory Element Binding Protein 1-Mediated Lipogenesis in Chang Cells.

PubMed

Kim, You-Mie; Song, Insun; Seo, Yong-Hak; Yoon, Gyesoon

2013-12-01

Enhanced lipogenesis plays a critical role in cell senescence via induction of expression of the mature form of sterol regulatory element binding protein 1 (SREBP1), which contributes to an increase in organellar mass, one of the indicators of senescence. We investigated the molecular mechanisms by which signaling molecules control SREBP1-mediated lipogenesis and senescence. We developed cellular models for stress-induced senescence, by exposing Chang cells, which are immortalized human liver cells, to subcytotoxic concentrations (200 µM) of deferoxamine (DFO) and H2O2. In this model of stress-induced cell senescence using DFO and H2O2, the phosphorylation profile of glycogen synthase kinase 3α (GSK3α) and β corresponded closely to the expression profile of the mature form of SREBP-1 protein. Inhibition of GSK3 with a subcytotoxic concentration of the selective GSK3 inhibitor SB415286 significantly increased mature SREBP1 expression, as well as lipogenesis and organellar mass. In addition, GSK3 inhibition was sufficient to induce senescence in Chang cells. Suppression of GSK3 expression with siRNAs specific to GSK3α and β also increased mature SREBP1 expression and induced senescence. Finally, blocking lipogenesis with fatty acid synthase inhibitors (cerulenin and C75) and siRNA-mediated silencing of SREBP1 and ATP citrate lyase (ACL) significantly attenuated GSK3 inhibition-induced senescence. GSK3 inactivation is an important upstream event that induces SREBP1-mediated lipogenesis and consequent cell senescence.

SH3-binding Protein 5 Mediates the Neuroprotective Effect of the Secreted Bioactive Peptide Humanin by Inhibiting c-Jun NH2-terminal Kinase*

PubMed Central

Takeshita, Yuji; Hashimoto, Yuichi; Nawa, Mikiro; Uchino, Hiroyuki; Matsuoka, Masaaki

2013-01-01

Humanin is a secreted bioactive peptide that suppresses cell toxicity caused by a variety of insults. The neuroprotective effect of Humanin against Alzheimer disease (AD)-related death is mediated by the binding of Humanin to its heterotrimeric Humanin receptor composed of ciliary neurotrophic receptor α, WSX-1, and gp130, as well as the activation of intracellular signaling pathways including a JAK2 and STAT3 signaling axis. Despite the elucidation of the signaling pathways by which Humanin mediates its neuroprotection, the transcriptional targets of Humanin that behaves as effectors of Humanin remains undefined. In the present study, Humanin increased the mRNA and protein expression of SH3 domain-binding protein 5 (SH3BP5), which has been known to be a JNK interactor, in neuronal cells. Similar to Humanin treatment, overexpression of SH3BP5 inhibited AD-related neuronal death, while siRNA-mediated knockdown of endogenous SH3BP5 expression attenuated the neuroprotective effect of Humanin. These results indicate that SH3BP5 is a downstream effector of Humanin. Furthermore, biochemical analysis has revealed that SH3BP5 binds to JNK and directly inhibits JNK through its two putative mitogen-activated protein kinase interaction motifs (KIMs). PMID:23861391

CBL family E3 ubiquitin ligases control JAK2 ubiquitination and stability in hematopoietic stem cells and myeloid malignancies

PubMed Central

Lv, Kaosheng; Jiang, Jing; Donaghy, Ryan; Riling, Christopher R.; Cheng, Ying; Chandra, Vemika; Rozenova, Krasimira; An, Wei; Mohapatra, Bhopal C.; Goetz, Benjamin T.; Pillai, Vinodh; Han, Xu; Todd, Emily A.; Jeschke, Grace R.; Langdon, Wallace Y.; Kumar, Suresh; Hexner, Elizabeth O.

2017-01-01

Janus kinase 2 (JAK2) is a central kinase in hematopoietic stem/progenitor cells (HSPCs), and its uncontrolled activation is a prominent oncogenic driver of hematopoietic neoplasms. However, molecular mechanisms underlying the regulation of JAK2 have remained elusive. Here we report that the Casitas B-cell lymphoma (CBL) family E3 ubiquitin ligases down-regulate JAK2 stability and signaling via the adaptor protein LNK/SH2B3. We demonstrated that depletion of CBL/CBL-B or LNK abrogated JAK2 ubiquitination, extended JAK2 half-life, and enhanced JAK2 signaling and cell growth in human cell lines as well as primary murine HSPCs. Built on these findings, we showed that JAK inhibitor (JAKi) significantly reduced aberrant HSPCs and mitigated leukemia development in a mouse model of aggressive myeloid leukemia driven by loss of Cbl and Cbl-b. Importantly, primary human CBL mutated (CBLmut) leukemias exhibited increased JAK2 protein levels and signaling and were hypersensitive to JAKi. Loss-of-function mutations in CBL E3 ubiquitin ligases are found in a wide range of myeloid malignancies, which are diseases without effective treatment options. Hence, our studies reveal a novel signaling axis that regulates JAK2 in normal and malignant HSPCs and suggest new therapeutic strategies for treating CBLmut myeloid malignancies. PMID:28611190

CD274 (PDL1) and JAK2 genomic amplifications in pulmonary squamous-cell and adenocarcinoma patients.

PubMed

Clavé, Sergi; Pijuan, Lara; Casadevall, David; Taus, Álvaro; Gimeno, Javier; Hernández-Llodrà, Silvia; Rodríguez-Rivera, María; Lorenzo, Marta; Menéndez, Silvia; Albanell, Joan; Espinet, Blanca; Arriola, Edurne; Salido, Marta

2018-01-01

CD274 (PDL1) and JAK2 (9p24.1) gene amplifications have been recently described in pulmonary carcinomas in association with programmed death-ligand 1 (PD-L1) expression. Furthermore, PTEN loss has been explored preclinically in relation to PD-L1 expression. Our aim was to determine whether these genomic alterations affect PD-L1 expression levels in non-small-cell lung cancer. PD-L1 and PTEN expression determined by immunohistochemistry (IHC), and CD274, JAK2 and PTEN copy number alterations (CNAs) determined by fluorescence in-situ hybridisation, were studied in 171 pulmonary carcinoma specimens. PD-L1 expression was positive in 40 cases (23.3%), and CD274 amplification was present in 14 tumours (8.8%). Concordance between both events was found in 12 of 14 amplified cases (P = 0.0001). We found nine JAK2-amplified cases (5.7%), seven with PD-L1 expression (P = 0.0006). Moreover, six of the seven cases had JAK2 and CD274 coamplification (9p24.1 genomic amplification). Remarkably, the average PD-L1 IHC score was higher in these amplified cases (230 versus 80; P = 0.001). Non-statistical associations were observed between PD-L1 expression and PTEN loss and PTEN deletions. We describe a subset of patients (8.2%) who had 9p24.1 amplifications resulting in high expression of PD-L1. Our results provide evidence for genomic up-regulation of PD-L1 expression in non-small-cell lung cancer. © 2017 John Wiley & Sons Ltd.

Responses to Cytokines and Interferons that Depend upon JAKs and STATs.

PubMed

Stark, George R; Cheon, HyeonJoo; Wang, Yuxin

2018-01-02

Many cytokines and all interferons activate members of a small family of kinases (the Janus kinases [JAKs]) and a slightly larger family of transcription factors (the signal transducers and activators of transcription [STATs]), which are essential components of pathways that induce the expression of specific sets of genes in susceptible cells. JAK-STAT pathways are required for many innate and acquired immune responses, and the activities of these pathways must be finely regulated to avoid major immune dysfunctions. Regulation is achieved through mechanisms that include the activation or induction of potent negative regulatory proteins, posttranslational modification of the STATs, and other modulatory effects that are cell-type specific. Mutations of JAKs and STATs can result in gains or losses of function and can predispose affected individuals to autoimmune disease, susceptibility to a variety of infections, or cancer. Here we review recent developments in the biochemistry, genetics, and biology of JAKs and STATs. Copyright © 2018 Cold Spring Harbor Laboratory Press; all rights reserved.

Polypyrimidine tract binding protein 1 protects mRNAs from recognition by the nonsense-mediated mRNA decay pathway

PubMed Central

Ge, Zhiyun; Quek, Bao Lin; Beemon, Karen L; Hogg, J Robert

2016-01-01

The nonsense-mediated mRNA decay (NMD) pathway degrades mRNAs containing long 3'UTRs to perform dual roles in mRNA quality control and gene expression regulation. However, expansion of vertebrate 3'UTR functions has required a physical expansion of 3'UTR lengths, complicating the process of detecting nonsense mutations. We show that the polypyrimidine tract binding protein 1 (PTBP1) shields specific retroviral and cellular transcripts from NMD. When bound near a stop codon, PTBP1 blocks the NMD protein UPF1 from binding 3'UTRs. PTBP1 can thus mark specific stop codons as genuine, preserving both the ability of NMD to accurately detect aberrant mRNAs and the capacity of long 3'UTRs to regulate gene expression. Illustrating the wide scope of this mechanism, we use RNA-seq and transcriptome-wide analysis of PTBP1 binding sites to show that many human mRNAs are protected by PTBP1 and that PTBP1 enrichment near stop codons correlates with 3'UTR length and resistance to NMD. DOI: http://dx.doi.org/10.7554/eLife.11155.001 PMID:26744779

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

Development, Optimization and Structure-Activity Relationships of Covalent-Reversible JAK3 Inhibitors Based on a Tricyclic Imidazo[5,4-d]pyrrolo[2,3-b]pyridine Scaffold.

PubMed

Forster, Michael; Chaikuad, Apirat; Dimitrov, Teodor; Döring, Eva; Holstein, Julia; Berger, Benedict-Tilman; Gehringer, Matthias; Ghoreschi, Kamran; Müller, Susanne; Knapp, Stefan; Laufer, Stefan A

2018-05-31

Janus kinases are major drivers of immune signaling and have been the focus of anti-inflammatory drug discovery for more than a decade. Because of the invariable co-localization of JAK1 and JAK3 at cytokine receptors, the question if selective JAK3 inhibition is sufficient to effectively block downstream signaling has been highly controversial. Recently, we discovered the covalent-reversible JAK3 inhibitor FM-381 (23) featuring high isoform and kinome selectivity. Crystallography revealed that this inhibitor induces an unprecedented binding pocket by interactions of a nitrile substituent with arginine residues in JAK3. Herein we describe detailed structure activity relationships necessary for induction of the arginine pocket and the impact of this structural change on potency, isoform selectivity and efficacy in cellular models. Furthermore, we evaluated the stability of this novel inhibitor class in in vitro metabolic assays and were able to demonstrate an adequate stability of key compound 23 for in vivo use.

MET-oncogenic and JAK2-inactivating alterations are independent factors that affect regulation of PD-L1 expression in lung cancer.

PubMed

Saigi, Maria; Alburquerque-Bejar, Juan J; McLEER-Florin, Anne; Pereira, Carolina; Pros, Eva; Romero, Octavio A; Baixeras, Nuria; Esteve-Codina, Anna; Nadal, Ernest; Brambilla, Elisabeth; Sanchez-Cespedes, Montse

2018-06-13

The blockade of immune checkpoints such as PD-L1 and PD-1 is being exploited therapeutically in several types of malignancies. Here, we aimed to understand the contribution of the genetics of lung cancer (LC) to the ability of tumor cells to escape immunosurveillance checkpoints. Over 150 primary non-small cell lung cancers, including pulmonary sarcomatoid carcinomas, were tested for the levels of HLA-I complex, PD-L1, tumor-infiltrating CD8+ lymphocytes and alterations in main LC genes. Correlations were validated in cancer cell lines using appropriate treatments to activate or inhibit selected pathways. We also performed RNA sequencing to assess changes in gene expression after these treatments Results: MET-oncogenic activation tended to associate with positive PD-L1 immunostaining, whereas STK11 mutations were correlated with negative immunostaining. In MET-altered cancer cells, MET triggered a transcriptional increase of PD-L1 that was independent of the IFNγ-mediated JAK/STAT pathway. The activation of MET also up-regulated other immunosuppressive genes (PDCD1LG2 and SOCS1), and transcripts involved in angiogenesis (VEGFA and NRP1) and in cell proliferation. We also report recurrent inactivating mutations in JAK2 that co-occur with alterations in MET and STK11, which prevented the induction of immunoresponse-related genes following treatment with IFNγ. We show that MET activation promotes the expression of several negative checkpoint regulators of the immunoresponse, including PD-L1. In addition, we report inactivation of JAK2 in LC cells that prevented the response to IFNγ. These alterations are likely to facilitate tumor growth by enabling immune tolerance and may affect the response to immune checkpoint inhibitors. Copyright ©2018, American Association for Cancer Research.

JAK/STAT3 pathway inhibition blocks skeletal muscle wasting downstream of IL-6 and in experimental cancer cachexia.

PubMed

Bonetto, Andrea; Aydogdu, Tufan; Jin, Xiaoling; Zhang, Zongxiu; Zhan, Rui; Puzis, Leopold; Koniaris, Leonidas G; Zimmers, Teresa A

2012-08-01

Cachexia, the metabolic dysregulation leading to sustained loss of muscle and adipose tissue, is a devastating complication of cancer and other chronic diseases. Interleukin-6 and related cytokines are associated with muscle wasting in clinical and experimental cachexia, although the mechanisms by which they might induce muscle wasting are unknown. One pathway activated strongly by IL-6 family ligands is the JAK/STAT3 pathway, the function of which has not been evaluated in regulation of skeletal muscle mass. Recently, we showed that skeletal muscle STAT3 phosphorylation, nuclear localization, and target gene expression are activated in C26 cancer cachexia, a model with high IL-6 family ligands. Here, we report that STAT3 activation is a common feature of muscle wasting, activated in muscle by IL-6 in vivo and in vitro and by different types of cancer and sterile sepsis. Moreover, STAT3 activation proved both necessary and sufficient for muscle wasting. In C(2)C(12) myotubes and in mouse muscle, mutant constitutively activated STAT3-induced muscle fiber atrophy and exacerbated wasting in cachexia. Conversely, inhibiting STAT3 pharmacologically with JAK or STAT3 inhibitors or genetically with dominant negative STAT3 and short hairpin STAT3 reduced muscle atrophy downstream of IL-6 or cancer. These results indicate that STAT3 is a primary mediator of muscle wasting in cancer cachexia and other conditions of high IL-6 family signaling. Thus STAT3 could represent a novel therapeutic target for the preservation of skeletal muscle in cachexia.

JAK/STAT3 pathway inhibition blocks skeletal muscle wasting downstream of IL-6 and in experimental cancer cachexia

PubMed Central

Bonetto, Andrea; Aydogdu, Tufan; Jin, Xiaoling; Zhang, Zongxiu; Zhan, Rui; Puzis, Leopold; Koniaris, Leonidas G.

2012-01-01

Cachexia, the metabolic dysregulation leading to sustained loss of muscle and adipose tissue, is a devastating complication of cancer and other chronic diseases. Interleukin-6 and related cytokines are associated with muscle wasting in clinical and experimental cachexia, although the mechanisms by which they might induce muscle wasting are unknown. One pathway activated strongly by IL-6 family ligands is the JAK/STAT3 pathway, the function of which has not been evaluated in regulation of skeletal muscle mass. Recently, we showed that skeletal muscle STAT3 phosphorylation, nuclear localization, and target gene expression are activated in C26 cancer cachexia, a model with high IL-6 family ligands. Here, we report that STAT3 activation is a common feature of muscle wasting, activated in muscle by IL-6 in vivo and in vitro and by different types of cancer and sterile sepsis. Moreover, STAT3 activation proved both necessary and sufficient for muscle wasting. In C2C12 myotubes and in mouse muscle, mutant constitutively activated STAT3-induced muscle fiber atrophy and exacerbated wasting in cachexia. Conversely, inhibiting STAT3 pharmacologically with JAK or STAT3 inhibitors or genetically with dominant negative STAT3 and short hairpin STAT3 reduced muscle atrophy downstream of IL-6 or cancer. These results indicate that STAT3 is a primary mediator of muscle wasting in cancer cachexia and other conditions of high IL-6 family signaling. Thus STAT3 could represent a novel therapeutic target for the preservation of skeletal muscle in cachexia. PMID:22669242

Tyrosine Kinase 2-mediated Signal Transduction in T Lymphocytes Is Blocked by Pharmacological Stabilization of Its Pseudokinase Domain*

PubMed Central

Tokarski, John S.; Zupa-Fernandez, Adriana; Tredup, Jeffrey A.; Pike, Kristen; Chang, ChiehYing; Xie, Dianlin; Cheng, Lihong; Pedicord, Donna; Muckelbauer, Jodi; Johnson, Stephen R.; Wu, Sophie; Edavettal, Suzanne C.; Hong, Yang; Witmer, Mark R.; Elkin, Lisa L.; Blat, Yuval; Pitts, William J.; Weinstein, David S.; Burke, James R.

2015-01-01

Inhibition of signal transduction downstream of the IL-23 receptor represents an intriguing approach to the treatment of autoimmunity. Using a chemogenomics approach marrying kinome-wide inhibitory profiles of a compound library with the cellular activity against an IL-23-stimulated transcriptional response in T lymphocytes, a class of inhibitors was identified that bind to and stabilize the pseudokinase domain of the Janus kinase tyrosine kinase 2 (Tyk2), resulting in blockade of receptor-mediated activation of the adjacent catalytic domain. These Tyk2 pseudokinase domain stabilizers were also shown to inhibit Tyk2-dependent signaling through the Type I interferon receptor but not Tyk2-independent signaling and transcriptional cellular assays, including stimulation through the receptors for IL-2 (JAK1- and JAK3-dependent) and thrombopoietin (JAK2-dependent), demonstrating the high functional selectivity of this approach. A crystal structure of the pseudokinase domain liganded with a representative example showed the compound bound to a site analogous to the ATP-binding site in catalytic kinases with features consistent with high ligand selectivity. The results support a model where the pseudokinase domain regulates activation of the catalytic domain by forming receptor-regulated inhibitory interactions. Tyk2 pseudokinase stabilizers, therefore, represent a novel approach to the design of potent and selective agents for the treatment of autoimmunity. PMID:25762719

Combination of PIM and JAK2 inhibitors synergistically suppresses cell proliferation and overcomes drug resistance of myeloproliferative neoplasms

PubMed Central

Greco, Rita; Li, Zhifang; Sun, Fangxian; Barberis, Claude; Tabart, Michel; Patel, Vinod; Schio, Laurent; Hurley, Raelene; Chen, Bo; Cheng, Hong; Lengauer, Christoph; Pollard, Jack; Watters, James; Garcia-Echeverria, Carlos; Wiederschain, Dmitri; Adrian, Francisco; Zhang, JingXin

2014-01-01

Inhibitors of JAK2 kinase are emerging as an important treatment modality for myeloproliferative neoplasms (MPN). However, similar to other kinase inhibitors, resistance to JAK2 inhibitors may eventually emerge through a variety of mechanisms. Effective drug combination is one way to enhance therapeutic efficacy and combat resistance against JAK2 inhibitors. To identify potential combination partners for JAK2 compounds in MPN cell lines, we performed pooled shRNA screen targeting 5,000 genes in the presence or absence of JAK2 blockade. One of the top hits identified was MYC, an oncogenic transcription factor that is difficult to inhibit directly, but could be targeted by modulation of upstream regulatory elements such as kinases. We demonstrate herein that PIM kinase inhibitors efficiently suppress MYC protein levels in MPN cell lines. Overexpression of MYC restores the viability of PIM inhibitor-treated cells, revealing causal relationship between MYC down-regulation and cell growth inhibition by PIM compounds. Combination of various PIM inhibitors with a JAK2 inhibitor results in significant synergistic growth inhibition of multiple MPN cancer cell lines and induction of apoptosis. Mechanistic studies revealed strong downregulation of phosphorylated forms of S6 and 4EBP1 by JAK2/PIM inhibitor combination treatment. Finally, such combination was effective in eradicating in vitro JAK2 inhibitor-resistant MPN clones, where MYC is consistently up-regulated. These findings demonstrate that simultaneous suppression of JAK2 and PIM kinase activity by small molecule inhibitors is more effective than either agent alone in suppressing MPN cell growth. Our data suggest that JAK2 and PIM combination might warrant further investigation for the treatment of JAK2-driven hematologic malignancies. PMID:24830942

CHZ868, a Type II JAK2 Inhibitor, Reverses Type I JAK Inhibitor Persistence and Demonstrates Efficacy in Myeloproliferative Neoplasms

PubMed Central

Meyer, Sara C.; Keller, Matthew D.; Chiu, Sophia; Koppikar, Priya; Guryanova, Olga A.; Rapaport, Franck; Xu, Ke; Manova, Katia; Pankov, Dmitry; O’Reilly, Richard J.; Kleppe, Maria; McKenney, Anna Sophia; Shih, Alan H.; Shank, Kaitlyn; Ahn, Jihae; Papalexi, Eftymia; Spitzer, Barbara; Socci, Nick; Viale, Agnes; Mandon, Emeline; Ebel, Nicolas; Andraos, Rita; Rubert, Joëlle; Dammassa, Ernesta; Romanet, Vincent; Dölemeyer, Arno; Zender, Michael; Heinlein, Melanie; Rampal, Rajit; Weinberg, Rona Singer; Hoffman, Ron; Sellers, William R.; Hofmann, Francesco; Murakami, Masato; Baffert, Fabienne; Gaul, Christoph; Radimerski, Thomas; Levine, Ross L.

2015-01-01

Summary Although clinically tested JAK inhibitors reduce splenomegaly and systemic symptoms, molecular responses are not observed in most myeloproliferative neoplasms (MPN) patients. We previously demonstrated that MPN cells become persistent to type I JAK inhibitors that bind the active conformation of JAK2. We investigated if CHZ868, a type II JAK inhibitor, would demonstrate activity in JAK inhibitor persistent cells, murine MPN models, and MPN patient samples. JAK2- and MPL-mutant cell lines were sensitive to CHZ868, including type I JAK inhibitor persistent cells. CHZ868 showed significant activity in murine MPN models and induced reductions in mutant allele burden not observed with type I JAK inhibitors. These data demonstrate that type II JAK inhibition is a viable therapeutic approach for MPN patients. PMID:26175413

G protein-coupled estrogen receptor 1 (GPER 1) mediates estrogen-induced, proliferation of leiomyoma cells.

PubMed

Jiang, Xiuxiu; Ye, Xiaolei; Ma, Junyan; Li, Wen; Wu, Ruijin; Jun, Lin

2015-01-01

G protein-coupled estrogen receptor 1 (GPER-1, formerly known as GPR30) has been proposed as the receptor for estrogen-induced, growth of leiomyomas though its precise mechanisms of action are not clear. We obtained leiomyoma cells (LC) and normal smooth muscle cells from 28 women (n = 28, median age 38 years, median parity 1.0). We incubated them with 17-β estradiol (E(2)), after blocking, or upregulating, expression of GPER-1 with ICI182,780 (a GPER-1 agonist) and siGPR30, respectively. We evaluated the role of GPER-1 in the mitogen-activated protein kinase (MAPK) signaling pathway using Western blot analysis. We studied cell proliferation with 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl tetrazolium bromide, and, mitotic activity with phosphohistone H3 (PPH3) expression in leiomyoma, and, matched, normal, smooth muscle tissues using standard immunohistochemistry. Downregulation of GPER-1 expression with siGPR30 partially attenuated the E(2)-activated MAPK signaling pathway (p < 0.01). Upregulation of GPER-1 with ICI182,780 enhanced the E(2)-activated MAPK signaling pathway (p < 0.01). ICI182,780 enhanced E(2)-induced proliferation of LC (p < 0.01), while knock down of the GPER-1 gene with GPER-1 small interfering RNA partially inhibited E(2)-induced cell proliferation (p < 0.01). There were no significant differences in PPH3 expression between LCs and normal smooth muscle tissues (p > 0.05). Neither ICI182,780 nor siGPR30 increased mitosis in LCs (p > 0.05). Our results indicate that GPER-1 mediates proliferation of estrogen-induced, LC by activating the MAPK pathway, and, not by promoting mitosis.

HSP90 is a therapeutic target in JAK2-dependent myeloproliferative neoplasms in mice and humans

PubMed Central

Marubayashi, Sachie; Koppikar, Priya; Taldone, Tony; Abdel-Wahab, Omar; West, Nathan; Bhagwat, Neha; Caldas-Lopes, Eloisi; Ross, Kenneth N.; Gönen, Mithat; Gozman, Alex; Ahn, James H.; Rodina, Anna; Ouerfelli, Ouathek; Yang, Guangbin; Hedvat, Cyrus; Bradner, James E.; Chiosis, Gabriela; Levine, Ross L.

2010-01-01

JAK2 kinase inhibitors were developed for the treatment of myeloproliferative neoplasms (MPNs), following the discovery of activating JAK2 mutations in the majority of patients with MPN. However, to date JAK2 inhibitor treatment has shown limited efficacy and apparent toxicities in clinical trials. We report here that an HSP90 inhibitor, PU-H71, demonstrated efficacy in cell line and mouse models of the MPN polycythemia vera (PV) and essential thrombocytosis (ET) by disrupting JAK2 protein stability. JAK2 physically associated with both HSP90 and PU-H71 and was degraded by PU-H71 treatment in vitro and in vivo, demonstrating that JAK2 is an HSP90 chaperone client. PU-H71 treatment caused potent, dose-dependent inhibition of cell growth and signaling in JAK2 mutant cell lines and in primary MPN patient samples. PU-H71 treatment of mice resulted in JAK2 degradation, inhibition of JAK-STAT signaling, normalization of peripheral blood counts, and improved survival in MPN models at doses that did not degrade JAK2 in normal tissues or cause substantial toxicity. Importantly, PU-H71 treatment also reduced the mutant allele burden in mice. These data establish what we believe to be a novel therapeutic rationale for HSP90 inhibition in the treatment of JAK2-dependent MPN. PMID:20852385

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.

LIM-domain proteins, LIMD1, Ajuba, and WTIP are required for microRNA-mediated gene silencing

PubMed Central

James, Victoria; Zhang, Yining; Foxler, Daniel E.; de Moor, Cornelia H.; Kong, Yi Wen; Webb, Thomas M.; Self, Tim J.; Feng, Yungfeng; Lagos, Dimitrios; Chu, Chia-Ying; Rana, Tariq M.; Morley, Simon J.; Longmore, Gregory D.; Bushell, Martin; Sharp, Tyson V.

2010-01-01

In recent years there have been major advances with respect to the identification of the protein components and mechanisms of microRNA (miRNA) mediated silencing. However, the complete and precise repertoire of components and mechanism(s) of action remain to be fully elucidated. Herein we reveal the identification of a family of three LIM domain-containing proteins, LIMD1, Ajuba and WTIP (Ajuba LIM proteins) as novel mammalian processing body (P-body) components, which highlight a novel mechanism of miRNA-mediated gene silencing. Furthermore, we reveal that LIMD1, Ajuba, and WTIP bind to Ago1/2, RCK, Dcp2, and eIF4E in vivo, that they are required for miRNA-mediated, but not siRNA-mediated gene silencing and that all three proteins bind to the mRNA 5′ m7GTP cap–protein complex. Mechanistically, we propose the Ajuba LIM proteins interact with the m7GTP cap structure via a specific interaction with eIF4E that prevents 4EBP1 and eIF4G interaction. In addition, these LIM-domain proteins facilitate miRNA-mediated gene silencing by acting as an essential molecular link between the translationally inhibited eIF4E-m7GTP-5′cap and Ago1/2 within the miRISC complex attached to the 3′-UTR of mRNA, creating an inhibitory closed-loop complex. PMID:20616046

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.

Arrestin-dependent angiotensin AT1 receptor signaling regulates Akt and mTor-mediated protein synthesis.

PubMed

Kendall, Ryan T; Lee, Mi-Hye; Pleasant, Dorea L; Robinson, Katherine; Kuppuswamy, Dhandapani; McDermott, Paul J; Luttrell, Louis M

2014-09-19

Control of protein synthesis is critical to both cell growth and proliferation. The mammalian target of rapamycin (mTOR) integrates upstream growth, proliferation, and survival signals, including those transmitted via ERK1/2 and Akt, to regulate the rate of protein translation. The angiotensin AT1 receptor has been shown to activate both ERK1/2 and Akt in arrestin-based signalsomes. Here, we examine the role of arrestin-dependent regulation of ERK1/2 and Akt in the stimulation of mTOR-dependent protein translation by the AT1 receptor using HEK293 and primary vascular smooth muscle cell models. Nascent protein synthesis stimulated by both the canonical AT1 receptor agonist angiotensin II (AngII), and the arrestin pathway-selective agonist [Sar(1)-Ile(4)-Ile(8)]AngII (SII), is blocked by shRNA silencing of βarrestin1/2 or pharmacological inhibition of Akt, ERK1/2, or mTORC1. In HEK293 cells, SII activates a discrete arrestin-bound pool of Akt and promotes Akt-dependent phosphorylation of mTOR and its downstream effector p70/p85 ribosomal S6 kinase (p70/85S6K). In parallel, SII-activated ERK1/2 helps promote mTOR and p70/85S6K phosphorylation, and is required for phosphorylation of the known ERK1/2 substrate p90 ribosomal S6 kinase (p90RSK). Thus, arrestins coordinate AT1 receptor regulation of ERK1/2 and Akt activity and stimulate protein translation via both Akt-mTOR-p70/85S6K and ERK1/2-p90RSK pathways. These results suggest that in vivo, arrestin pathway-selective AT1 receptor agonists may promote cell growth or hypertrophy through arrestin-mediated mechanisms despite their antagonism of G protein signaling. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Somatic CALR Mutations in Myeloproliferative Neoplasms with Nonmutated JAK2

PubMed Central

Baxter, E.J.; Nice, F.L.; Gundem, G.; Wedge, D.C.; Avezov, E.; Li, J.; Kollmann, K.; Kent, D.G.; Aziz, A.; Godfrey, A.L.; Hinton, J.; Martincorena, I.; Van Loo, P.; Jones, A.V.; Guglielmelli, P.; Tarpey, P.; Harding, H.P.; Fitzpatrick, J.D.; Goudie, C.T.; Ortmann, C.A.; Loughran, S.J.; Raine, K.; Jones, D.R.; Butler, A.P.; Teague, J.W.; O’Meara, S.; McLaren, S.; Bianchi, M.; Silber, Y.; Dimitropoulou, D.; Bloxham, D.; Mudie, L.; Maddison, M.; Robinson, B.; Keohane, C.; Maclean, C.; Hill, K.; Orchard, K.; Tauro, S.; Du, M.-Q.; Greaves, M.; Bowen, D.; Huntly, B.J.P.; Harrison, C.N.; Cross, N.C.P.; Ron, D.; Vannucchi, A.M.; Papaemmanuil, E.; Campbell, P.J.; Green, A.R.

2014-01-01

BACKGROUND Somatic mutations in the Janus kinase 2 gene (JAK2) occur in many myeloproliferative neoplasms, but the molecular pathogenesis of myeloproliferative neoplasms with nonmutated JAK2 is obscure, and the diagnosis of these neoplasms remains a challenge. METHODS We performed exome sequencing of samples obtained from 151 patients with myeloproliferative neoplasms. The mutation status of the gene encoding calreticulin (CALR) was assessed in an additional 1345 hematologic cancers, 1517 other cancers, and 550 controls. We established phylogenetic trees using hematopoietic colonies. We assessed calreticulin subcellular localization using immunofluorescence and flow cytometry. RESULTS Exome sequencing identified 1498 mutations in 151 patients, with medians of 6.5, 6.5, and 13.0 mutations per patient in samples of polycythemia vera, essential thrombocythemia, and myelofibrosis, respectively. Somatic CALR mutations were found in 70 to 84% of samples of myeloproliferative neoplasms with nonmutated JAK2, in 8% of myelodysplasia samples, in occasional samples of other myeloid cancers, and in none of the other cancers. A total of 148 CALR mutations were identified with 19 distinct variants. Mutations were located in exon 9 and generated a +1 base-pair frameshift, which would result in a mutant protein with a novel C-terminal. Mutant calreticulin was observed in the endoplasmic reticulum without increased cell-surface or Golgi accumulation. Patients with myeloproliferative neoplasms carrying CALR mutations presented with higher platelet counts and lower hemoglobin levels than patients with mutated JAK2. Mutation of CALR was detected in hematopoietic stem and progenitor cells. Clonal analyses showed CALR mutations in the earliest phylogenetic node, a finding consistent with its role as an initiating mutation in some patients. CONCLUSIONS Somatic mutations in the endoplasmic reticulum chaperone CALR were found in a majority of patients with myeloproliferative neoplasms with

Cdk1, PKCδ and calcineurin-mediated Drp1 pathway contributes to mitochondrial fission-induced cardiomyocyte death

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

Zaja, Ivan; Bai, Xiaowen, E-mail: xibai@mcw.edu; Liu, Yanan

Highlights: • Drp1-mediated increased mitochondrial fission but not fusion is involved the cardiomyocyte death during anoxia-reoxygenation injury. • Reactive oxygen species are upstream initiators of mitochondrial fission. • Increased mitochondrial fission is resulted from Cdk1-, PKCδ-, and calcineurin-mediated Drp1 pathways. - Abstract: Myocardial ischemia–reperfusion (I/R) injury is one of the leading causes of death and disability worldwide. Mitochondrial fission has been shown to be involved in cardiomyocyte death. However, molecular machinery involved in mitochondrial fission during I/R injury has not yet been completely understood. In this study we aimed to investigate molecular mechanisms of controlling activation of dynamin-related protein 1more » (Drp1, a key protein in mitochondrial fission) during anoxia-reoxygenation (A/R) injury of HL1 cardiomyocytes. A/R injury induced cardiomyocyte death accompanied by the increases of mitochondrial fission, reactive oxygen species (ROS) production and activated Drp1 (pSer616 Drp1), and decrease of inactivated Drp1 (pSer637 Drp1) while mitochondrial fusion protein levels were not significantly changed. Blocking Drp1 activity with mitochondrial division inhibitor mdivi1 attenuated cell death, mitochondrial fission, and Drp1 activation after A/R. Trolox, a ROS scavenger, decreased pSer616 Drp1 level and mitochondrial fission after A/R. Immunoprecipitation assay further indicates that cyclin dependent kinase 1 (Cdk1) and protein kinase C isoform delta (PKCδ) bind Drp1, thus increasing mitochondrial fission. Inhibiting Cdk1 and PKCδ attenuated the increases in pSer616 Drp1, mitochondrial fission, and cardiomyocyte death. FK506, a calcineurin inhibitor, blocked the decrease in expression of inactivated pSer637 Drp1 and mitochondrial fission. Our findings reveal the following novel molecular mechanisms controlling mitochondrial fission during A/R injury of cardiomyocytes: (1) ROS are upstream initiators of

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.

Heartland virus NSs protein disrupts host defenses by blocking the TBK1 kinase-IRF3 transcription factor interaction and signaling required for interferon induction.

PubMed

Ning, Yun-Jia; Feng, Kuan; Min, Yuan-Qin; Deng, Fei; Hu, Zhihong; Wang, Hualin

2017-10-06

Heartland virus (HRTV) is a pathogenic phlebovirus related to the severe fever with thrombocytopenia syndrome virus (SFTSV), another phlebovirus causing life-threatening disease in humans. Previous findings have suggested that SFTSV can antagonize the host interferon (IFN) system via viral nonstructural protein (NSs)-mediated sequestration of antiviral signaling proteins into NSs-induced inclusion bodies. However, whether and how HRTV counteracts the host innate immunity is unknown. Here, we report that HRTV NSs (HNSs) also antagonizes IFN and cytokine induction and bolsters viral replication, although no noticeable inclusion body formation was observed in HNSs-expressing cells. Furthermore, HNSs inhibited the virus-triggered activation of IFN-β promoter by specifically targeting the IFN-stimulated response element but not the NF-κB response element. Consistently, HNSs blocked the phosphorylation and nuclear translocation of IFN regulatory factor 3 (IRF3, an IFN-stimulated response element-activating transcription factor). Reporter gene assays next showed that HNSs blockades the antiviral signaling mediated by RIG-I-like receptors likely at the level of TANK-binding kinase 1 (TBK1). Indeed, HNSs strongly interacts with TBK1 as indicated by confocal microscopy and pulldown analyses, and we also noted that the scaffold dimerization domain of TBK1 is required for the TBK1-HNSs interaction. Finally, pulldown assays demonstrated that HNSs expression dose-dependently diminishes a TBK1-IRF3 interaction, further explaining the mechanism for HNSs function. Collectively, these data suggest that HNSs, an antagonist of host innate immunity, interacts with TBK1 and thereby hinders the association of TBK1 with its substrate IRF3, thus blocking IRF3 activation and transcriptional induction of the cellular antiviral responses. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Protein phosphatase 2A mediates resensitization of the neurokinin 1 receptor

PubMed Central

Murphy, Jane E.; Roosterman, Dirk; Cottrell, Graeme S.; Padilla, Benjamin E.; Feld, Micha; Brand, Eva; Cedron, Wendy J.; Steinhoff, Martin

2011-01-01

Activated G protein-coupled receptors (GPCRs) are phosphorylated and interact with β-arrestins, which mediate desensitization and endocytosis. Endothelin-converting enzyme-1 (ECE-1) degrades neuropeptides in endosomes and can promote recycling. Although endocytosis, dephosphorylation, and recycling are accepted mechanisms of receptor resensitization, a large proportion of desensitized receptors can remain at the cell surface. We investigated whether reactivation of noninternalized, desensitized (phosphorylated) receptors mediates resensitization of the substance P (SP) neurokinin 1 receptor (NK1R). Herein, we report a novel mechanism of resensitization by which protein phosphatase 2A (PP2A) is recruited to dephosphorylate noninternalized NK1R. A desensitizing concentration of SP reduced cell-surface SP binding sites by only 25%, and SP-induced Ca2+ signals were fully resensitized before cell-surface binding sites started to recover, suggesting resensitization of cell-surface-retained NK1R. SP induced association of β-arrestin1 and PP2A with noninternalized NK1R. β-Arrestin1 small interfering RNA knockdown prevented SP-induced association of cell-surface NK1R with PP2A, indicating that β-arrestin1 mediates this interaction. ECE-1 inhibition, by trapping β-arrestin1 in endosomes, also impeded SP-induced association of cell-surface NK1R with PP2A. Resensitization of NK1R signaling required both PP2A and ECE-1 activity. Thus, after stimulation with SP, PP2A interacts with noninternalized NK1R and mediates resensitization. PP2A interaction with NK1R requires β-arrestin1. ECE-1 promotes this process by releasing β-arrestin1 from NK1R in endosomes. These findings represent a novel mechanism of PP2A- and ECE-1-dependent resensitization of GPCRs. PMID:21795521

Evaluation of the association between the JAK2 46/1 haplotype and chronic myeloproliferative neoplasms in a Brazilian population

PubMed Central

Silva, Sarah Pagliarini- e-; Santos, Bruna Cunha; de Figueiredo Pereira, Elizangela Mendes; Ferreira, Mari Ellen; Baraldi, Elaine Cristina; Sell, Ana Maria; Visentainer, Jeane Eliete Laguila

2013-01-01

OBJECTIVE: The JAK2 46/1 haplotype has recently been described as a major contributing factor to the development of myeloproliferative neoplasm, whether positive or negative for the JAK2 V617F mutation. The G allele, identified by a single-nucleotide polymorphism known as JAK2 rs10974944, is part of the JAK2 46/1 haplotype. The aim of this study was to verify the association between the presence of the G allele and the development of BCR-ABL-negative chronic myeloproliferative neoplasms in our population. METHODS: Blood and oral mucosa swab samples were obtained from 56 patients of two local Brazilian hospitals who had previously been diagnosed with BCR-ABL-negative chronic myeloproliferative neoplasms. Blood samples from 90 local blood donors were used as controls. The presence of the G allele was assessed using a PCR-RFLP assay after extracting DNA from the samples. RESULTS: The presence of the G allele was strongly associated with the presence of BCR-ABL-negative chronic myeloproliferative neoplasms (p = 0.0001; OR = 2.674; 95% CI = 1.630−4.385) in the studied population. CONCLUSION: In agreement with previous reports, the JAK2 46/1 haplotype, represented in this study by the presence of the G allele, is an important predisposing factor in the oncogenetic development of these neoplasms in our population. PMID:23420150

Regulation of rat heme oxygenase-1 expression by interleukin-6 via the Jak/STAT pathway in hepatocytes.

PubMed

Tron, Kyrylo; Samoylenko, Anatoly; Musikowski, Gernot; Kobe, Fritz; Immenschuh, Stephan; Schaper, Fred; Ramadori, Giuliano; Kietzmann, Thomas

2006-07-01

Heme oxygenase-1 (HO-1) can be induced by various stimuli, one of which is interleukin-6 (IL-6). Therefore, the aim of this study was to elucidate the molecular mechanisms responsible for IL-6-dependent HO-1 induction in the liver. The IL-6-dependent HO-1 regulation in rat primary hepatocytes and HepG2 hepatoma cells was studied by Northern and Western blot analyses, HO-1 promoter reporter gene assays and EMSA. The HO-1 expression was transcriptionally induced by IL-6 in a time- and dose-dependent manner. Activation of signal transducers and activators of transcription (STAT) factors by the IL-6 receptor was crucial for HO-1 induction. By contrast, negative regulation of HO-1 expression appeared to be mediated through the SH2-domain-containing tyrosine phosphatase-2 (SHP2)/ suppressors of cytokine signaling-3 (SOCS3) binding site within the gp130 IL-6 receptor subunit. Among the three putative STAT binding elements (SBE) in the HO-1 promoter, only the distal one was functional and when deleted, the remaining Luc induction was completely obliterated by the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002. The HO-1 SBE3 mediates HO-1 gene induction by IL-6 mainly via activation of the Jak/STAT pathway.

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.

Suppressor of Cytokine Signaling 2 Negatively Regulates NK Cell Differentiation by Inhibiting JAK2 Activity

PubMed Central

Kim, Won Sam; Kim, Mi Jeong; Kim, Dong Oh; Byun, Jae-Eun; Huy, Hangsak; Song, Hae Young; Park, Young-Jun; Kim, Tae-Don; Yoon, Suk Ran; Choi, Eun-Ji; Jung, Haiyoung; Choi, Inpyo

2017-01-01

Suppressor of cytokine signaling (SOCS) proteins are negative regulators of cytokine responses. Although recent reports have shown regulatory roles for SOCS proteins in innate and adaptive immunity, their roles in natural killer (NK) cell development are largely unknown. Here, we show that SOCS2 is involved in NK cell development. SOCS2−/− mice showed a high frequency of NK cells in the bone marrow and spleen. Knockdown of SOCS2 was associated with enhanced differentiation of NK cells in vitro, and the transplantation of hematopoietic stem cells (HSCs) into congenic mice resulted in enhanced differentiation in SOCS2−/− HSCs. We found that SOCS2 could inhibit Janus kinase 2 (JAK2) activity and JAK2-STAT5 signaling pathways via direct interaction with JAK2. Furthermore, SOCS2−/− mice showed a reduction in lung metastases and an increase in survival following melanoma challenge. Overall, our findings suggest that SOCS2 negatively regulates the development of NK cells by inhibiting JAK2 activity via direct interaction. PMID:28383049

Efficacy of the JAK2 inhibitor INCB16562 in a murine model of MPLW515L-induced thrombocytosis and myelofibrosis.

PubMed

Koppikar, Priya; Abdel-Wahab, Omar; Hedvat, Cyrus; Marubayashi, Sachie; Patel, Jay; Goel, Aviva; Kucine, Nicole; Gardner, Jeffrey R; Combs, Andrew P; Vaddi, Kris; Haley, Patrick J; Burn, Timothy C; Rupar, Mark; Bromberg, Jacqueline F; Heaney, Mark L; de Stanchina, Elisa; Fridman, Jordan S; Levine, Ross L

2010-04-08

The discovery of JAK2 and MPL mutations in patients with myeloproliferative neoplasms (MPNs) provided important insight into the genetic basis of these disorders and led to the development of JAK2 kinase inhibitors for MPN therapy. Although recent studies have shown that JAK2 kinase inhibitors demonstrate efficacy in a JAK2V617F murine bone marrow transplantation model, the effects of JAK2 inhibitors on MPLW515L-mediated myeloproliferation have not been investigated. In this report, we describe the in vitro and in vivo effects of INCB16562, a small-molecule JAK2 inhibitor. INCB16562 inhibited proliferation and signaling in cell lines transformed by JAK2 and MPL mutations. Compared with vehicle treatment, INCB16562 treatment improved survival, normalized white blood cell counts and platelet counts, and markedly reduced extramedullary hematopoeisis and bone marrow fibrosis. We observed inhibition of STAT3 and STAT5 phosphorylation in vivo consistent with potent inhibition of JAK-STAT signaling. These data suggest JAK2 inhibitor therapy may be of value in the treatment of JAK2V617F-negative MPNs. However, we did not observe a decrease in the size of the malignant clone in the bone marrow of treated mice at the end of therapy, which suggests that JAK2 inhibitor therapy, by itself, was not curative in this MPN model.

Efficacy of the JAK2 inhibitor INCB16562 in a murine model of MPLW515L-induced thrombocytosis and myelofibrosis

PubMed Central

Koppikar, Priya; Abdel-Wahab, Omar; Hedvat, Cyrus; Marubayashi, Sachie; Patel, Jay; Goel, Aviva; Kucine, Nicole; Gardner, Jeffrey R.; Combs, Andrew P.; Vaddi, Kris; Haley, Patrick J.; Burn, Timothy C.; Rupar, Mark; Bromberg, Jacqueline F.; Heaney, Mark L.; de Stanchina, Elisa; Fridman, Jordan S.

2010-01-01

The discovery of JAK2 and MPL mutations in patients with myeloproliferative neoplasms (MPNs) provided important insight into the genetic basis of these disorders and led to the development of JAK2 kinase inhibitors for MPN therapy. Although recent studies have shown that JAK2 kinase inhibitors demonstrate efficacy in a JAK2V617F murine bone marrow transplantation model, the effects of JAK2 inhibitors on MPLW515L-mediated myeloproliferation have not been investigated. In this report, we describe the in vitro and in vivo effects of INCB16562, a small-molecule JAK2 inhibitor. INCB16562 inhibited proliferation and signaling in cell lines transformed by JAK2 and MPL mutations. Compared with vehicle treatment, INCB16562 treatment improved survival, normalized white blood cell counts and platelet counts, and markedly reduced extramedullary hematopoeisis and bone marrow fibrosis. We observed inhibition of STAT3 and STAT5 phosphorylation in vivo consistent with potent inhibition of JAK-STAT signaling. These data suggest JAK2 inhibitor therapy may be of value in the treatment of JAK2V617F-negative MPNs. However, we did not observe a decrease in the size of the malignant clone in the bone marrow of treated mice at the end of therapy, which suggests that JAK2 inhibitor therapy, by itself, was not curative in this MPN model. PMID:20154217

VEZF1 Elements Mediate Protection from DNA Methylation

PubMed Central

Strogantsev, Ruslan; Gaszner, Miklos; Hair, Alan; Felsenfeld, Gary; West, Adam G.

2010-01-01

There is growing consensus that genome organization and long-range gene regulation involves partitioning of the genome into domains of distinct epigenetic chromatin states. Chromatin insulator or barrier elements are key components of these processes as they can establish boundaries between chromatin states. The ability of elements such as the paradigm β-globin HS4 insulator to block the range of enhancers or the spread of repressive histone modifications is well established. Here we have addressed the hypothesis that a barrier element in vertebrates should be capable of defending a gene from silencing by DNA methylation. Using an established stable reporter gene system, we find that HS4 acts specifically to protect a gene promoter from de novo DNA methylation. Notably, protection from methylation can occur in the absence of histone acetylation or transcription. There is a division of labor at HS4; the sequences that mediate protection from methylation are separable from those that mediate CTCF-dependent enhancer blocking and USF-dependent histone modification recruitment. The zinc finger protein VEZF1 was purified as the factor that specifically interacts with the methylation protection elements. VEZF1 is a candidate CpG island protection factor as the G-rich sequences bound by VEZF1 are frequently found at CpG island promoters. Indeed, we show that VEZF1 elements are sufficient to mediate demethylation and protection of the APRT CpG island promoter from DNA methylation. We propose that many barrier elements in vertebrates will prevent DNA methylation in addition to blocking the propagation of repressive histone modifications, as either process is sufficient to direct the establishment of an epigenetically stable silent chromatin state. PMID:20062523

JAK2 V617F, MPL W515L and JAK2 Exon 12 Mutations in Chinese Patients with Primary Myelofibrosis.

PubMed

Xia, Jun; Lu, Mi-Ze; Jiang, Yuan-Qiang; Yang, Guo-Hua; Zhuang, Yun; Sun, Hong-Li; Shen, Yun-Feng

2012-03-01

JAK2 V617F, MPL W515L and JAK2 exon 12 mutations are novel acquired mutations that induce constitutive cytokine-independent activation of the JAK-STAT pathway in myeloproliferative disorders (MPD). The discovery of these mutations provides novel mechanism for activation of signal transduction in hematopoietic malignancies. This research was to investigate their prevalence in Chinese patients with primary myelofibrosis (PMF). We introduced allele-specific PCR (AS-PCR) combined with sequence analysis to simultaneously screen JAK2 V617F, MPL W515L and JAK2 exon 12 mutations in 30 patients with PMF. Fifteen PMF patients (50.0%) carried JAK2 V617F mutation, and only two JAK2 V617F-negative patients (6.7%) harbored MPL W515L mutation. None had JAK2 exon 12 mutations. Furthermore, these three mutations were not detected in 50 healthy controls. MPL W515L and JAK2 V617F mutations existed in PMF patients but JAK2 exon 12 mutations not. JAK2 V617F and MPL W515L and mutations might contribute to the primary molecular pathogenesis in patients with PMF.

Amino-Terminal β-Amyloid Antibody Blocks β-Amyloid-Mediated Inhibition of the High-Affinity Choline Transporter CHT.

PubMed

Cuddy, Leah K; Seah, Claudia; Pasternak, Stephen H; Rylett, R Jane

2017-01-01

Alzheimer's disease (AD) is a common age-related neurodegenerative disorder that is characterized by progressive cognitive decline. The deficits in cognition and attentional processing that are observed clinically in AD are linked to impaired function of cholinergic neurons that release the neurotransmitter acetylcholine (ACh). The high-affinity choline transporter (CHT) is present at the presynaptic cholinergic nerve terminal and is responsible for the reuptake of choline produced by hydrolysis of ACh following its release. Disruption of CHT function leads to decreased choline uptake and ACh synthesis, leading to impaired cholinergic neurotransmission. We report here that cell-derived β-amyloid peptides (Aβ) decrease choline uptake activity and cell surface CHT protein levels in SH-SY5Y neural cells. Moreover, we make the novel observation that the amount of CHT protein localizing to early endosomes and lysosomes is decreased significantly in cells that have been treated with cell culture medium that contains Aβ peptides released from neural cells. The Aβ-mediated loss of CHT proteins from lysosomes is prevented by blocking lysosomal degradation of CHT with the lysosome inhibitor bafilomycin A1 (BafA 1 ). BafA 1 also attenuated the Aβ-mediated decrease in CHT cell surface expression. Interestingly, however, lysosome inhibition did not block the effect of Aβ on CHT activity. Importantly, neutralizing Aβ using an anti-Aβ antibody directed at the N-terminal amino acids 1-16 of Aβ, but not by an antibody directed at the mid-region amino acids 22-35 of Aβ, attenuates the effect of Aβ on CHT activity and trafficking. This indicates that a specific N-terminal Aβ epitope, or specific conformation of soluble Aβ, may impair CHT activity. Therefore, Aβ immunotherapy may be a more effective therapeutic strategy for slowing the progression of cognitive decline in AD than therapies designed to promote CHT cell surface levels.

Tricyclic Covalent Inhibitors Selectively Target Jak3 through an Active Site Thiol*

PubMed Central

Goedken, Eric R.; Argiriadi, Maria A.; Banach, David L.; Fiamengo, Bryan A.; Foley, Sage E.; Frank, Kristine E.; George, Jonathan S.; Harris, Christopher M.; Hobson, Adrian D.; Ihle, David C.; Marcotte, Douglas; Merta, Philip J.; Michalak, Mark E.; Murdock, Sara E.; Tomlinson, Medha J.; Voss, Jeffrey W.

2015-01-01

The action of Janus kinases (JAKs) is required for multiple cytokine signaling pathways, and as such, JAK inhibitors hold promise for treatment of autoimmune disorders, including rheumatoid arthritis, inflammatory bowel disease, and psoriasis. However, due to high similarity in the active sites of the four members (Jak1, Jak2, Jak3, and Tyk2), developing selective inhibitors within this family is challenging. We have designed and characterized substituted, tricyclic Jak3 inhibitors that selectively avoid inhibition of the other JAKs. This is accomplished through a covalent interaction between an inhibitor containing a terminal electrophile and an active site cysteine (Cys-909). We found that these ATP competitive compounds are irreversible inhibitors of Jak3 enzyme activity in vitro. They possess high selectivity against other kinases and can potently (IC50 < 100 nm) inhibit Jak3 activity in cell-based assays. These results suggest irreversible inhibitors of this class may be useful selective agents, both as tools to probe Jak3 biology and potentially as therapies for autoimmune diseases. PMID:25552479

Tricyclic Covalent Inhibitors Selectively Target Jak3 through an Active Site Thiol

DOE PAGES

Goedken, Eric R.; Argiriadi, Maria A.; Banach, David L.; ...

2014-12-31

The action of Janus kinases (JAKs) is required for multiple cytokine signaling pathways, and as such, JAK inhibitors hold promise for treatment of autoimmune disorders, including rheumatoid arthritis, inflammatory bowel disease, and psoriasis. However, due to high similarity in the active sites of the four members (Jak1, Jak2, Jak3, and Tyk2), developing selective inhibitors within this family is challenging. In this paper, we have designed and characterized substituted, tricyclic Jak3 inhibitors that selectively avoid inhibition of the other JAKs. This is accomplished through a covalent interaction between an inhibitor containing a terminal electrophile and an active site cysteine (Cys-909). Wemore » found that these ATP competitive compounds are irreversible inhibitors of Jak3 enzyme activity in vitro. They possess high selectivity against other kinases and can potently (IC 50 < 100 nM) inhibit Jak3 activity in cell-based assays. Finally, these results suggest irreversible inhibitors of this class may be useful selective agents, both as tools to probe Jak3 biology and potentially as therapies for autoimmune diseases.« less

Tricyclic Covalent Inhibitors Selectively Target Jak3 through an Active Site Thiol

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

Goedken, Eric R.; Argiriadi, Maria A.; Banach, David L.

The action of Janus kinases (JAKs) is required for multiple cytokine signaling pathways, and as such, JAK inhibitors hold promise for treatment of autoimmune disorders, including rheumatoid arthritis, inflammatory bowel disease, and psoriasis. However, due to high similarity in the active sites of the four members (Jak1, Jak2, Jak3, and Tyk2), developing selective inhibitors within this family is challenging. In this paper, we have designed and characterized substituted, tricyclic Jak3 inhibitors that selectively avoid inhibition of the other JAKs. This is accomplished through a covalent interaction between an inhibitor containing a terminal electrophile and an active site cysteine (Cys-909). Wemore » found that these ATP competitive compounds are irreversible inhibitors of Jak3 enzyme activity in vitro. They possess high selectivity against other kinases and can potently (IC 50 < 100 nM) inhibit Jak3 activity in cell-based assays. Finally, these results suggest irreversible inhibitors of this class may be useful selective agents, both as tools to probe Jak3 biology and potentially as therapies for autoimmune diseases.« less

Evolution of JAK-STAT Pathway Components: Mechanisms and Role in Immune System Development

PubMed Central

Liongue, Clifford; O'Sullivan, Lynda A.; Trengove, Monique C.; Ward, Alister C.

2012-01-01

Background Lying downstream of a myriad of cytokine receptors, the Janus kinase (JAK) – Signal transducer and activator of transcription (STAT) pathway is pivotal for the development and function of the immune system, with additional important roles in other biological systems. To gain further insight into immune system evolution, we have performed a comprehensive bioinformatic analysis of the JAK-STAT pathway components, including the key negative regulators of this pathway, the SH2-domain containing tyrosine phosphatase (SHP), Protein inhibitors against Stats (PIAS), and Suppressor of cytokine signaling (SOCS) proteins across a diverse range of organisms. Results Our analysis has demonstrated significant expansion of JAK-STAT pathway components co-incident with the emergence of adaptive immunity, with whole genome duplication being the principal mechanism for generating this additional diversity. In contrast, expansion of upstream cytokine receptors appears to be a pivotal driver for the differential diversification of specific pathway components. Conclusion Diversification of JAK-STAT pathway components during early vertebrate development occurred concurrently with a major expansion of upstream cytokine receptors and two rounds of whole genome duplications. This produced an intricate cell-cell communication system that has made a significant contribution to the evolution of the immune system, particularly the emergence of adaptive immunity. PMID:22412924

Protein extraction from methanol fixed paraffin embedded tissue blocks: A new possibility using cell blocks

PubMed Central

Kokkat, Theresa J.; McGarvey, Diane; Patel, Miral S.; Tieniber, Andrew D.; LiVolsi, Virginia A.; Baloch, Zubair W.

2013-01-01

Background: Methanol fixed and paraffin embedded (MFPE) cellblocks are an essential cytology preparation. However, MFPE cellblocks often contain limited material and their relatively small size has caused them to be overlooked in biomarker discovery. Advances in the field of molecular biotechnology have made it possible to extract proteins from formalin fixed and paraffin embedded (FFPE) tissue blocks. In contrast, there are no established methods for extracting proteins from MFPE cellblocks. We investigated commonly available CHAPS (3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate) buffer, as well as two commercially available Qiagen® kits and compared their effectiveness on MFPE tissue for protein yields. Materials and Methods: MFPE blocks were made by Cellient™ automated system using human tissue specimens from normal and malignant specimens collected in ThinPrep™ Vials. Protein was extracted from Cellient-methanol fixed and paraffin embedded blocks with CHAPS buffer method as well as FFPE and Mammalian Qiagen® kits. Results: Comparison of protein yields demonstrated the effectiveness of various protein extraction methods on MFPE cellblocks. Conclusion: In the current era of minimally invasive techniques to obtain minimal amount of tissue for diagnostic and prognostic purposes, the use of commercial and lab made buffer on low weight MFPE scrapings obtained by Cellient® processor opens new possibilities for protein biomarker research. PMID:24403950

Drosophila glypicans Dally and Dally-like are essential regulators for JAK/STAT signaling and Unpaired distribution in eye development

PubMed Central

Zhang, Yan; You, Jia; Ren, Wenyan; Lin, Xinhua

2013-01-01

The highly conserved janus kinase (JAK)-signal transducer and activator of transcription (STAT) pathway is a well-known signaling system that is involved in many biological processes. In Drosophila, this signaling cascade is activated by ligands of the Unpaired (Upd) family. Therefore, the regulation of Upd distribution is one of the key issues in controlling the JAK/STAT signaling activity and function. Heparan sulfate proteoglycans (HSPGs) are macromolecules that regulate the distribution of many ligand proteins including Wingless, Hedgehog and Decapentaplegic (Dpp). Here we show that during Drosophila eye development, HSPGs are also required in normal Upd distribution and JAK/STAT signaling activity. Loss of HSPG biosynthesis enzyme Brother of tout-velu (Botv), Sulfateless (Sfl), or glypicans Division abnormally delayed (Dally) and Dally-like protein (Dlp) led to reduced levels of extracellular Upd and reduction in JAK/STAT signaling activity. Overexpression of dally resulted in the accumulation of Upd and up-regulation of the signaling activity. Luciferase assay also showed that Dally promotes JAK/STAT signaling activity, and is dependent on its heparin sulfate chains. These data suggest that Dally and Dlp are essential for Upd distribution and JAK/STAT signaling activity. PMID:23313126

JAK2V617F influences epigenomic changes in myeloproliferative neoplasms.

PubMed

Chen, Chih-Cheng; Chiu, Chia-Chen; Lee, Kuan-Der; Hsu, Chia-Chen; Chen, Hong-Chi; Huang, Tim H-M; Hsiao, Shu-Huei; Leu, Yu-Wei

2017-12-16

Negative valine (V) to phenylalanine (F) switch at the Janus kinase (JAK2) 617 codon (V617F) is the dominant driver mutation in patients with myeloproliferative neoplasms (MPNs). JAK2V617F was proved to be sufficient for cell transformation; however, independent mutations might influence the following epigenomic modifications. To assess the JAK2V617F-induced downstream epigenomic changes without interferences, we profiled the epigenomic changes in ectopically expressed JAK2V617F in Ba/F3 cells. Antibodies against phosphorylated signal transducer and activator of transcription 3 (pSTAT3) and enhancer of zeste homolog 2 (EZH2) were used for chromatin-immunoprecipitation sequencing (ChIP-seq) to detect the downstream epigenomic targets in the JAK2-STAT3 signaling pathway. To confirm the JAK2V617F-induced epigenetic changes in vivo, DNA methylation changes in the target loci in patients with MPNs were detected through methylation-specific polymerase chain reaction and were clustered against the changes within controls. We found that ectopically expressed JAK2V617F in Ba/F3 cells reduced the binding specificity; it was associated with cis-regulatory elements and recognized DNA motifs in both pSTAT3-downstream and EZH2-associated targets. Overlapping target loci between the control and JAK2V617F were <3% and 0.4%, respectively, as identified through pSTAT3 and EZH2 ChIP-seq. Furthermore, the methylation changes in the direct target loci (FOXH1, HOXC9, and SRF) were clustered independently from the control locus (L1TD1) and other mutation genes (HMGA2 and Lin28A) in the analyzed MPN samples. Therefore, JAK2V617F influences target binding in both pSTAT3 and EZH2. Without mutations in epigenetic regulators, JAK2V617F can induce downstream epigenomic modifications. Thus, epigenetic changes in JAK2 downstream targets might be trackable in vivo. Copyright © 2017 Elsevier Inc. All rights reserved.

Induction of Suppressor of Cytokine Signaling-3 by Herpes Simplex Virus Type 1 Contributes to Inhibition of the Interferon Signaling Pathway

PubMed Central

Yokota, Shin-ichi; Yokosawa, Noriko; Okabayashi, Tamaki; Suzutani, Tatsuo; Miura, Shunsuke; Jimbow, Kowichi; Fujii, Nobuhiro

2004-01-01

We showed previously that herpes simplex virus type 1 (HSV-1) suppresses the interferon (IFN) signaling pathway during the early infection stage in the human amnion cell line FL. HSV-1 inhibits the IFN-induced phosphorylation of Janus kinases (JAK) in infected FL cells. In the present study, we showed that the suppressor of cytokine signaling-3 (SOCS3), a host negative regulator of the JAK/STAT pathway, is rapidly induced in FL cells after HSV-1 infection. Maximal levels of SOCS3 protein were detected at around 1 to 2 h after infection. This is consistent with the occurrence of HSV-1-mediated inhibition of IFN-induced JAK phosphorylation. The HSV-1 wild-type strain VR3 induced SOCS3 more efficiently than did mutants that are defective in UL41 or UL13 and that are hyperresponsive to IFN. Induction of the IRF-7 protein and transcriptional activation of IFN-α4, which occur in a JAK/STAT pathway-dependent manner, were poorly induced by VR3 but efficiently induced by the mutant viruses. In contrast, phosphorylation of IRF-3 and transcriptional activation of IFN-β, which are JAK/STAT pathway-independent process, were equally well induced by the wild-type strain and the mutants. In conclusion, the SOCS3 protein appears to be mainly responsible for the suppression of IFN signaling and IFN production that occurs during HSV-1 infection. PMID:15163721

Protein degradation in skeletal muscle during experimental hyperthyroidism in rats and the effect of beta-blocking agents.

PubMed

Angerås, U; Hasselgren, P O

1987-04-01

beta-Blocking agents are increasingly used in the management of hyperthyroid patients. The effect of this treatment on increased muscle protein breakdown in the hyperthyroid state is not known. In the present study, experimental hyperthyroidism was induced in rats by daily ip injections of T3 (100 micrograms/100 g BW) during a 10-day period. Control animals received corresponding volumes of solvent. In groups of rats the selective beta-1-blocking agent metoprolol or the nonselective beta-blocker propranolol was infused by miniosmotic pumps implanted sc on the backs of the animals. Protein degradation was measured in incubated intact soleus and extensor digitorum longus muscles by determining tyrosine release into the incubation medium. The protein degradation rate in incubated extensor digitorum longus and soleus muscles was increased by 50-60% during T3 treatment. Metoprolol or propranolol did not influence muscle protein breakdown in either T3-treated or control animals. The results suggest that T3-induced increased muscle proteolysis is not mediated by beta-receptors, and muscle weakness and wasting in hyperthyroidism might not be affected by beta-blockers.

JAK kinases are required for the bacterial RNA and poly I:C induced tyrosine phosphorylation of PKR

PubMed Central

Bleiblo, Farag; Michael, Paul; Brabant, Danielle; Ramana, Chilakamarti V; Tai, TC; Saleh, Mazen; Parrillo, Joseph E; Kumar, Anand; Kumar, Aseem

2013-01-01

Discriminating the molecular patterns associated with RNA is central to innate immunity. The protein kinase PKR is a cytosolic sensor involved in the recognition of viral dsRNA and triggering interferon-induced signaling. Here, we identified bacterial RNA as a novel distinct pattern recognized by PKR. We show that the tyrosine phosphorylation of PKR induced by either bacterial RNA or poly I:C is impaired in mutant cells lacking TYK2, JAK1, or JAK2 kinases. PKR was found to be a direct substrate for the activated JAKs. Our results indicated that the double-stranded structures of bacterial RNA are required to fully activate PKR. These results suggest that bacterial RNA signaling is analogous in some respects to that of viral RNA and interferons and may have implications in bacterial immunity. PMID:23236554

Rho/ROCK-dependent inhibition of 3T3-L1 adipogenesis by G-protein-deamidating dermonecrotic toxins: differential regulation of Notch1, Pref1/Dlk1, and β-catenin signaling

PubMed Central

Bannai, Yuka; Aminova, Leila R.; Faulkner, Melinda J.; Ho, Mengfei; Wilson, Brenda A.

2012-01-01

The dermonecrotic toxins from Pasteurella multocida (PMT), Bordetella (DNT), Escherichia coli (CNF1-3), and Yersinia (CNFY) modulate their G-protein targets through deamidation and/or transglutamination of an active site Gln residue, which results in activation of the G protein and its cognate downstream signaling pathways. Whereas DNT and the CNFs act on small Rho GTPases, PMT acts on the α subunit of heterotrimeric Gq, Gi, and G12/13 proteins. We previously demonstrated that PMT potently blocks adipogenesis and adipocyte differentiation in a calcineurin-independent manner through downregulation of Notch1 and stabilization of β-catenin and Pref1/Dlk1, key proteins in signaling pathways strongly linked to cell fate decisions, including fat and bone development. Here, we report that similar to PMT, DNT, and CNF1 completely block adipogenesis and adipocyte differentiation by preventing upregulation of adipocyte markers, PPARγ and C/EBPα, while stabilizing the expression of Pref1/Dlk1 and β-catenin. We show that the Rho/ROCK inhibitor Y-27632 prevented or reversed these toxin-mediated effects, strongly supporting a role for Rho/ROCK signaling in dermonecrotic toxin-mediated inhibition of adipogenesis and adipocyte differentiation. Toxin treatment was also accompanied by downregulation of Notch1 expression, although this inhibition was independent of Rho/ROCK signaling. We further show that PMT-mediated downregulation of Notch1 expression occurs primarily through G12/13 signaling. Our results reveal new details of the pathways involved in dermonecrotic toxin action on adipocyte differentiation, and the role of Rho/ROCK signaling in mediating toxin effects on Wnt/β-catenin and Notch1 signaling, and in particular the role of Gq and G12/13 in mediating PMT effects on Rho/ROCK and Notch1 signaling. PMID:22919671

A germline JAK2 SNP is associated with predisposition to the development of JAK2V617F-positive myeloproliferative neoplasms

PubMed Central

Kilpivaara, Outi; Mukherjee, Semanti; Schram, Alison M; Wadleigh, Martha; Mullally, Ann; Ebert, Benjamin L; Bass, Adam; Marubayashi, Sachie; Heguy, Adriana; Garcia-Manero, Guillermo; Kantarjian, Hagop; Offit, Kenneth; Stone, Richard M; Gilliland, D Gary; Klein, Robert J; Levine, Ross L

2013-01-01

Polycythemia vera, essential thrombocythemia and primary myelofibrosis are myeloproliferative neoplasms (MPN) characterized by multilineage clonal hematopoiesis1–5. Given that the identical somatic activating mutation in the JAK2 tyrosine kinase gene (JAK2V617F) is observed in most individuals with polycythemia vera, essential thrombocythemia and primary myelofibrosis6–10, there likely are additional genetic events that contribute to the pathogenesis of these phenotypically distinct disorders. Moreover, family members of individuals with MPN are at higher risk for the development of MPN, consistent with the existence of MPN predisposition loci11. We hypothesized that germline variation contributes to MPN predisposition and phenotypic pleiotropy. Genome-wide analysis identified an allele in the JAK2 locus (rs10974944) that predisposes to the development of JAK2V617F-positive MPN, as well as three previously unknown MPN modifier loci. We found that JAK2V617F is preferentially acquired in cis with the predisposition allele. These data suggest that germline variation is an important contributor to MPN phenotype and predisposition. PMID:19287384

Sulforaphane-induced autophagy flux prevents prion protein-mediated neurotoxicity through AMPK pathway.

PubMed

Lee, J-H; Jeong, J-K; Park, S-Y

2014-10-10

Prion diseases are neurodegenerative and infectious disorders that involve accumulation of misfolded scrapie prion protein, and which are characterized by spongiform degeneration. Autophagy, a major homeostatic process responsible for the degradation of cytoplasmic components, has garnered attention as the potential target for neurodegenerative diseases such as prion disease. We focused on protective effects of sulforaphane found in cruciferous vegetables on prion-mediated neurotoxicity and the mechanism of sulforaphane related to autophagy. In human neuroblastoma cells, sulforaphane protected prion protein (PrP) (106-126)-mediated neurotoxicity and increased autophagy flux marker microtubule-associated protein 1 light chain 3-II protein levels, following a decrease of p62 protein level. Pharmacological and genetical inhibition of autophagy by 3MA, wortmannin and knockdown of autophagy-related 5 (ATG5) led to block the effect of sulforaphane against PrP (106-126)-induced neurotoxicity. Furthermore we demonstrated that both sulforaphane-induced autophagy and protective effect of sulforaphane against PrP (106-126)-induced neurotoxicity are dependent on the AMP-activated protein kinase (AMPK) signaling. The present results indicated that sulforaphane of cruciferous vegetables enhanced autophagy flux led to the protection effects against prion-mediated neurotoxicity, which was regulated by AMPK signaling pathways in human neuron cells. Our data also suggest that sulforaphane has a potential value as a therapeutic tool in neurodegenerative disease including prion diseases. Copyright © 2014 IBRO. Published by Elsevier Ltd. All rights reserved.

DOMMINO 2.0: integrating structurally resolved protein-, RNA-, and DNA-mediated macromolecular interactions

PubMed Central

Kuang, Xingyan; Dhroso, Andi; Han, Jing Ginger; Shyu, Chi-Ren; Korkin, Dmitry

2016-01-01

Macromolecular interactions are formed between proteins, DNA and RNA molecules. Being a principle building block in macromolecular assemblies and pathways, the interactions underlie most of cellular functions. Malfunctioning of macromolecular interactions is also linked to a number of diseases. Structural knowledge of the macromolecular interaction allows one to understand the interaction’s mechanism, determine its functional implications and characterize the effects of genetic variations, such as single nucleotide polymorphisms, on the interaction. Unfortunately, until now the interactions mediated by different types of macromolecules, e.g. protein–protein interactions or protein–DNA interactions, are collected into individual and unrelated structural databases. This presents a significant obstacle in the analysis of macromolecular interactions. For instance, the homogeneous structural interaction databases prevent scientists from studying structural interactions of different types but occurring in the same macromolecular complex. Here, we introduce DOMMINO 2.0, a structural Database Of Macro-Molecular INteractiOns. Compared to DOMMINO 1.0, a comprehensive database on protein-protein interactions, DOMMINO 2.0 includes the interactions between all three basic types of macromolecules extracted from PDB files. DOMMINO 2.0 is automatically updated on a weekly basis. It currently includes ∼1 040 000 interactions between two polypeptide subunits (e.g. domains, peptides, termini and interdomain linkers), ∼43 000 RNA-mediated interactions, and ∼12 000 DNA-mediated interactions. All protein structures in the database are annotated using SCOP and SUPERFAMILY family annotation. As a result, protein-mediated interactions involving protein domains, interdomain linkers, C- and N- termini, and peptides are identified. Our database provides an intuitive web interface, allowing one to investigate interactions at three different resolution levels: whole subunit network

Corticosteroids inhibit sphingosine 1-phosphate-induced interleukin-6 secretion from human airway smooth muscle via mitogen-activated protein kinase phosphatase 1-mediated repression of mitogen and stress-activated protein kinase 1.

PubMed

Che, Wenchi; Parmentier, Johannes; Seidel, Petra; Manetsch, Melanie; Ramsay, Emma E; Alkhouri, Hatem; Ge, Qi; Armour, Carol L; Ammit, Alaina J

2014-02-01

Sphingosine 1-phosphate (S1P) is a bioactive sphingolipid that plays an important proinflammatory role in asthmatic airways. Corticosteroids are first-line antiinflammatories in asthma; however, their repressive effects on S1P-induced cytokine secretion have not been investigated. To address this, our in vitro study reveals the molecular mechanisms by which corticosteroids inhibit S1P-induced IL-6 expression in the pivotal immunomodulatory cell type, airway smooth muscle (ASM). We first uncover the cellular signaling pathways responsible: S1P activates a cyclic adenosine monophosphate/cAMP response-element-binding protein (CREB)/CRE-dependent pathway to induce IL-6 transcription, concomitant with stimulation of the mitogen-activated protein kinase (MAPK) superfamily and downstream mitogen and stress-activated protein kinase 1 (MSK1) and histone H3 phosphorylation. In this way, S1P stimulates parallel signaling pathways to induce IL-6 secretion via CRE-driven transcription of the IL-6 gene promoter in a relaxed chromatin environment achieved through histone H3 phosphorylation. Second, we investigated how corticosteroids mediate their repressive effects. The corticosteroid dexamethasone inhibits S1P-induced IL-6 protein secretion and mRNA expression, but CREB/CRE transrepression, inhibition of IL-6 mRNA stability, or subcellular relocation of MSK1 were not responsible for the repressive effects of dexamethasone. Rather, we show that dexamethasone rapidly induces up-regulation of the MAPK deactivator MAPK phosphatase 1 (MKP-1) and that MKP-1 blocks the MAPK-driven activation of MSK1 and phosphorylation of histone H3. This was confirmed by treatment with triptolide, an inhibitor of MKP-1 up-regulation, where repressive effects of corticosteroids were reversed. Our study reveals the molecular mechanism underlying the antiinflammatory capacity of corticosteroids to repress proinflammatory functions induced by the potent bioactive sphingolipid S1P in the lung.

Kinase activation through dimerization by human SH2-B.

PubMed

Nishi, Masahiro; Werner, Eric D; Oh, Byung-Chul; Frantz, J Daniel; Dhe-Paganon, Sirano; Hansen, Lone; Lee, Jongsoon; Shoelson, Steven E

2005-04-01

The isoforms of SH2-B, APS, and Lnk form a family of signaling proteins that have been described as activators, mediators, or inhibitors of cytokine and growth factor signaling. We now show that the three alternatively spliced isoforms of human SH2-B readily homodimerize in yeast two-hybrid and cellular transfections assays, and this is mediated specifically by a unique domain in its amino terminus. Consistent with previous reports, we further show that the SH2 domains of SH2-B and APS bind JAK2 at Tyr813. These findings suggested a model in which two molecules of SH2-B or APS homodimerize with their SH2 domains bound to two JAK2 molecules, creating heterotetrameric JAK2-(SH2-B)2-JAK2 or JAK2-(APS)2-JAK2 complexes. We further show that APS and SH2-B isoforms heterodimerize. At lower levels of SH2-B or APS expression, dimerization approximates two JAK2 molecules to induce transactivation. At higher relative concentrations of SH2-B or APS, kinase activation is blocked. SH2-B or APS homodimerization and SH2-B/APS heterodimerization thus provide direct mechanisms for activating and inhibiting JAK2 and other kinases from the inside of the cell and for potentiating or attenuating cytokine and growth factor receptor signaling when ligands are present.

Membrane-Mediated Cooperativity of Proteins

NASA Astrophysics Data System (ADS)

Weikl, Thomas R.

2018-04-01

Besides direct protein-protein interactions, indirect interactions mediated by membranes play an important role for the assembly and cooperative function of proteins in membrane shaping and adhesion. The intricate shapes of biological membranes are generated by proteins that locally induce membrane curvature. Indirect curvature-mediated interactions between these proteins arise because the proteins jointly affect the bending energy of the membranes. These curvature-mediated interactions are attractive for crescent-shaped proteins and are a driving force in the assembly of the proteins during membrane tubulation. Membrane adhesion results from the binding of receptor and ligand proteins that are anchored in the apposing membranes. The binding of these proteins strongly depends on nanoscale shape fluctuations of the membranes, leading to a fluctuation-mediated binding cooperativity. A length mismatch between receptor-ligand complexes in membrane adhesion zones causes repulsive curvature-mediated interactions that are a driving force for the length-based segregation of proteins during membrane adhesion.

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

Chlorogenic acid induces apoptosis to inhibit inflammatory proliferation of IL-6-induced fibroblast-like synoviocytes through modulating the activation of JAK/STAT and NF-κB signaling pathways

PubMed Central

LOU, LIXIA; ZHOU, JINGWEI; LIU, YUJUN; WEI, YI; ZHAO, JIULI; DENG, JIAGANG; DONG, BIN; ZHU, LINGQUN; WU, AIMING; YANG, YINGXI; CHAI, LIMIN