Sample records for jnk-mediated interleukin-2 mrna

  1. The MAP kinase JNK2 mediates cigarette smoke-induced arterial thrombosis.

    PubMed

    Breitenstein, Alexander; Stämpfli, Simon F; Reiner, Martin F; Shi, Yi; Keller, Stephan; Akhmedov, Alexander; Schaub Clerigué, Ariane; Spescha, Remo D; Beer, Hans-Jürg; Lüscher, Thomas F; Tanner, Felix C; Camici, Giovanni G

    2017-01-05

    Despite public awareness of its deleterious effects, smoking remains a major cause of death. Indeed, it is a risk factor for atherothrombotic complications and in line with this, the introduction of smoking ban in public areas reduced smoking-associated cardiovascular complications. Nonetheless, smoking remains a major concern, and molecular mechanisms by which it causes cardiovascular disease are not known. Peripheral blood monocytes from healthy smokers displayed increased JNK2 and tissue factor (TF) gene expression compared to non-smokers (n=15, p<0.05). Similarly, human aortic endothelial cells exposed to cigarette smoke total particulate matter (CS-TPM) revealed increased TF expression mediated by JNK2 (n=4; p<0.05). Wild-type and JNK2 -/- mice were exposed to cigarette smoke for two weeks after which arterial thrombosis was investigated. Wild-type mice exposed to smoke displayed reduced time to thrombotic arterial occlusion (n=8; p<0.05) and increased tissue factor activity (n=7; p<0.05) as compared to wild-type controls (n=6), while JNK2 -/- mice exposed to smoke maintained an unaltered thrombotic potential (n=8; p=NS) and tissue factor activity (n=8) comparable to that of JNK2 -/- and wild-type controls (n=6; p=NS). Smoking caused an increased production of reactive oxygen species (ROS) in wild-type but not in JNK2 -/- mice (n=7; p<0.05 for wild-type mice and n=5-6; p=NS for JNK2 -/- mice). In conclusion, the MAP kinase JNK2 mediates cigarette smoke-induced TF activation, arterial thrombosis and ROS production. These results underscore a major role of JNK2 in smoke-mediated thrombus formation and may offer an attractive target to prevent smoke-related thrombosis in those subjects which do not manage quitting.

  2. Actinobacillus actinomycetemcomitans Y4 capsular polysaccharide induces IL-1β mRNA expression through the JNK pathway in differentiated THP-1 cells

    PubMed Central

    Iwata, T; Mitani, A; Ishihara, Y; Tanaka, S; Yamamoto, G; Kikuchi, T; Naganawa, T; Matsumura, Y; Suga, T; Koide, M; Sobue, T; Suzuki, T; Noguchi, T

    2005-01-01

    Capsular polysaccharide from Actinobacillus actinomycetemcomitans Y4 (Y4 CP) induces bone resorption in a mouse organ culture system and osteoclast formation in mouse bone marrow cultures, as reported in previous studies. We also found that Y4 CP inhibits the release of interleukin (IL)-6 and IL-8 from human gingival fibroblast (HGF). Thus Y4 CP induces various responses in localized tissue and leads to the secretion of several cytokines. However, the effects of Y4 CP on human monocytes/macrophages are still unclear. In this study, THP-1 cells, which are a human monocytic cell line, were stimulated with Y4 CP, and we measured gene expression in inflammatory cytokine and signal transduction pathways. IL-1β and tumour necrosis factor (TNF)-α mRNA were induced from Y4 CP-treated THP-1 cells. IL-1β mRNA expression was increased according to the dose of Y4 CP, and in a time-dependent manner. IL-1β mRNA expression induced by Y4 CP (100 µg/ml) was approximately 7- to 10-fold greater than that in the control by real-time PCR analysis. Furthermore, neither PD98059, a specific inhibitor of extracellular signal-regulated kinase nor SB203580, a specific inhibitor of p38 kinase prevented the IL-1β expression induced by Y4 CP. However, JNK Inhibitor II, a specific inhibitor of c-Jun N-terminal kinase (JNK) prevented the IL-1β mRNA expression induced by Y4 CP in a concentration-dependent manner. These results indicate that Y4 CP-mediated JNK pathways play an important role in the regulation of IL-1β mRNA. Therefore, Y4 CP-transduced signals for IL-1β induction in the antibacterial action of macrophages may provide a therapeutic strategy for periodontitis. PMID:15996190

  3. Interleukin-1 Acts via the JNK-2 Signaling Pathway to Induce Aggrecan Degradation by Human Chondrocytes.

    PubMed

    Ismail, Heba M; Yamamoto, Kazuhiro; Vincent, Tonia L; Nagase, Hideaki; Troeberg, Linda; Saklatvala, Jeremy

    2015-07-01

    Aggrecan enables articular cartilage to bear load and resist compression. Aggrecan loss occurs early in osteoarthritis and rheumatoid arthritis and can be induced by inflammatory cytokines such as interleukin-1 (IL-1). IL-1 induces cleavage of specific aggrecans characteristic of the ADAMTS proteinases. The aim of this study was to identify the intracellular signaling pathways by which IL-1 causes aggrecan degradation by human chondrocytes and to investigate how aggrecanase activity is controlled by chondrocytes. We developed a cell-based assay combining small interfering RNA (siRNA)-induced knockdown with aggrecan degradation assays. Human articular chondrocytes were overlaid with bovine aggrecan after transfection with siRNAs against molecules of the IL-1 signaling pathway. After IL-1 stimulation, released aggrecan fragments were detected with AGEG and ARGS neoepitope antibodies. Aggrecanase activity and tissue inhibitor of metalloproteinases 3 levels were measured by enzyme-linked immunosorbent assay. Low-density lipoprotein receptor-related protein 1 (LRP-1) shedding was analyzed by Western blotting. ADAMTS-5 is a major aggrecanase in human chondrocytes, regulating aggrecan degradation in response to IL-1. The tumor necrosis factor receptor-associated 6 (TRAF-6)/transforming growth factor β-activated kinase 1 (TAK-1)/MKK-4 signaling axis is essential for IL-1-induced aggrecan degradation, while NF-κB is not. Of the 3 MAPKs (ERK, p38, and JNK), only JNK-2 showed a significant role in aggrecan degradation. Chondrocytes constitutively secreted aggrecanase, which was continuously endocytosed by LRP-1, keeping the extracellular level of aggrecanase low. IL-1 induced aggrecanase activity in the medium in a JNK-2-dependent manner, possibly by reducing aggrecanase endocytosis, because IL-1 caused JNK-2-dependent shedding of LRP-1. The signaling axis TRAF-6/TAK-1/MKK-4/JNK-2 mediates IL-1-induced aggrecanolysis. The level of aggrecanase is controlled by its

  4. Diet-induced obesity mediated by the JNK/DIO2 signal transduction pathway

    PubMed Central

    Vernia, Santiago; Cavanagh-Kyros, Julie; Barrett, Tamera; Jung, Dae Young; Kim, Jason K.; Davis, Roger J.

    2013-01-01

    The cJun N-terminal kinase (JNK) signaling pathway is a key mediator of metabolic stress responses caused by consuming a high-fat diet, including the development of obesity. To test the role of JNK, we examined diet-induced obesity in mice with targeted ablation of Jnk genes in the anterior pituitary gland. These mice exhibited an increase in the pituitary expression of thyroid-stimulating hormone (TSH), an increase in the blood concentration of thyroid hormone (T4), increased energy expenditure, and markedly reduced obesity compared with control mice. The increased amount of pituitary TSH was caused by reduced expression of type 2 iodothyronine deiodinase (Dio2), a gene that is required for T4-mediated negative feedback regulation of TSH expression. These data establish a molecular mechanism that accounts for the regulation of energy expenditure and the development of obesity by the JNK signaling pathway. PMID:24186979

  5. Both internalization and AIP1 association are required for tumor necrosis factor receptor 2-mediated JNK signaling.

    PubMed

    Ji, Weidong; Li, Yonghao; Wan, Ting; Wang, Jing; Zhang, Haifeng; Chen, Hong; Min, Wang

    2012-09-01

    The proinflammtory cytokine tumor necrosis factor (TNF), primarily via TNF receptor 1 (TNFR1), induces nuclear factor-κB (NF-κB)-dependent cell survival, and c-Jun N-terminal kinase (JNK) and caspase-dependent cell death, regulating vascular endothelial cell (EC) activation and apoptosis. However, signaling by the second receptor, TNFR2, is poorly understood. The goal of this study was to dissect how TNFR2 mediates NF-κB and JNK signaling in vascular EC, and its relevance to in vivo EC function. We show that TNFR2 contributes to TNF-induced NF-κB and JNK signaling in EC as TNFR2 deletion or knockdown reduces the TNF responses. To dissect the critical domains of TNFR2 that mediate the TNF responses, we examine the activity of TNFR2 mutant with a specific deletion of the TNFR2 intracellular region, which contains conserved domain I, domain II, domain III, and 2 TNFR-associated factor-2-binding sites. Deletion analyses indicate that different sequences on TNFR2 have distinct roles in NF-κB and JNK activation. Specifically, deletion of the TNFR-associated factor-2-binding sites (TNFR2-59) diminishes the TNFR2-mediated NF-κB, but not JNK activation; whereas, deletion of domain II or domain III blunts TNFR2-mediated JNK but not NF-κB activation. Interestingly, we find that the TNFR-associated factor-2-binding sites ensure TNFR2 on the plasma membrane, but the di-leucine LL motif within the domain II and aa338-355 within the domain III are required for TNFR2 internalization as well as TNFR2-dependent JNK signaling. Moreover, domain III of TNFR2 is responsible for association with ASK1-interacting protein-1, a signaling adaptor critical for TNF-induced JNK signaling. While TNFR2 containing the TNFR-associated factor-2-binding sites prevents EC cell death, a specific activation of JNK without NF-κB activation by TNFR2-59 strongly induces caspase activation and EC apoptosis. Our data reveal that both internalization and ASK1-interacting protein-1 association are

  6. Fucoidan/FGF-2 induces angiogenesis through JNK- and p38-mediated activation of AKT/MMP-2 signalling

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

    Kim, Beom Su; Bonecell Biotech Inc., 77, Dunsan-dong, Seo-gu, Daejeon 302-830; Park, Ji-Yun

    2014-08-08

    Graphical abstract: Schematic diagram of the angiogenic activity mechanism by FGF-2/fucoidan treatment in HUVECs. Fucoidan enhances the FGF-2-induced phosphorylation of p38, JNK, and ERK MAPKs. However, p38 and JNK were involved in AKT phosphorylation and MMP-2 activation and resulted in enhanced angiogenic activity, such as tube formation and migration, in HUVECs. - Highlights: • The angiogenic activity of fucoidan in HUVECs was explored. • Fucoidan enhanced HUVEC proliferation, migration, and tube formation. • Fucoidan enhanced angiogenesis through p38 and JNK but not ERK in HUVECs. • Fucoidan targeted angiogenesis-mediated AKT/MMP-2 signalling in HUVECs. - Abstract: Angiogenesis is an important biologicalmore » process in tissue development and repair. Fucoidan has previously been shown to potentiate in vitro tube formation in the presence of basic fibroblast growth factor (FGF-2). However, the underlying molecular mechanism remains largely unknown. This study was designed to investigate the action of fucoidan in angiogenesis in human umbilical vein endothelial cells (HUVECs) and to explore fucoidan-signalling pathways. First, we evaluated the effect of fucoidan on cell proliferation. Matrigel-based tube formation and wound healing assays were performed to investigate angiogenesis. Matrix metalloproteinase-2 (MMP-2) mRNA expression and activity levels were analysed by reverse transcription polymerase chain reaction (RT-PCR) and zymography, respectively. Additionally, phosphorylation of mitogen-activated protein kinases (MAPKs) and protein kinase B (AKT) was detected by Western blot. The results indicate that fucoidan treatment significantly increased cell proliferation in the presence of FGF-2. Moreover, compared to the effect of FGF-2 alone, fucoidan and FGF-2 had a greater effect on tube formation and cell migration, and this effect was found to be synergistic. Furthermore, fucoidan enhanced the phosphorylation of extracellular signal

  7. Apomorphine prevents LPS-induced IL-23 p19 mRNA expression via inhibition of JNK and ATF4 in HAPI cells.

    PubMed

    Hara, Hirokazu; Kimoto, Dai; Kajita, Miho; Takada, Chisato; Kamiya, Tetsuro; Adachi, Tetsuo

    2017-01-15

    Inflammation has been reported to be closely related to exaggeration of cerebral ischemia and neurodegenerative diseases. Microglia, resident immune cells in the central nervous system, can be activated in response to neuronal injury and produce proinflammatory cytokines, resulting in further aggravation of neuronal injury. Interleukin (IL)-23, which consists of p19 and IL-12 p40 subunits, has been shown to be involved in brain injury associated with neuroinflammation. Apomorphine (Apo), a nonselective dopamine receptor agonist, has been used for clinical therapy of Parkinson's disease. Besides the pharmacological effect, Apo is known to have pleiotropic biological functions. In this study, to elucidate the effect of Apo on lipopolysaccharide (LPS)-induced IL-23 p19 mRNA expression in microglial cell line HAPI cells, we pretreated cells with various concentrations of Apo (10 - 30μM) for 8, 16, and 24h, followed by exposure to LPS (100ng/ml). Pretreatment with Apo dose- and time-dependently suppressed the induction of IL-23 p19 mRNA. However, this effect of Apo was exerted independently of dopamine receptors. JNK and ATF4, an endoplasmic reticulum (ER) stress-inducible transcription factor, were involved in expression of LPS-induced IL-23 p19 mRNA. Pretreatment with Apo (30μM) for 24h inhibited LPS-induced activation of JNK and the nuclear accumulation of ATF4. Thapsigargin (Tg), an ER stress inducer, stimulated IL-23 p19 mRNA expression via an ATF4 dependent mechanism. We also found that Apo inhibited Tg-induced ATF4 accumulation and IL-23 p19 mRNA expression. Taken together, our findings suggest that Apo exerts anti-inflammatory effects through inhibition of JNK and ATF4 signaling pathways. Copyright © 2016 Elsevier B.V. All rights reserved.

  8. Bruton's tyrosine kinase regulates B cell antigen receptor-mediated JNK1 response through Rac1 and phospholipase C-gamma2 activation.

    PubMed

    Inabe, Kazunori; Miyawaki, Toshio; Longnecker, Richard; Matsukura, Hiroyoshi; Tsukada, Satoshi; Kurosaki, Tomohiro

    2002-03-13

    Bruton's tyrosine kinase (Btk) is essential for B cell development and B cell antigen receptor (BCR) function. Recent studies have shown that Btk plays an important role in BCR-mediated c-Jun NH(2)-terminal kinase (JNK) 1 activation; however, the mechanism by which Btk participates in the JNK1 response remains elusive. Here we show that the BCR-mediated Rac1 activation is significantly inhibited by loss of Btk, while this Rac1 activation is not affected by loss of phospholipase C-gamma2 (PLC-gamma2). Since PLC-gamma2 is also required for BCR-mediated JNK1 response, our results suggest that Btk regulates Rac1 pathway as well as PLC-gamma2 pathway, both of which contribute to the BCR-mediated JNK1 response.

  9. p53 mediates bcl-2 phosphorylation and apoptosis via activation of the Cdc42/JNK1 pathway.

    PubMed

    Thomas, A; Giesler, T; White, E

    2000-11-02

    A member of the small G protein family, cdc42, was isolated from a screen undertaken to identify p53-inducible genes during apoptosis in primary baby rat kidney (BRK) cells transformed with E1A and a temperature-sensitive mutant p53 using a PCR-based subtractive hybridization method. Cdc42 is a GTPase that belongs to the Rho/Rac subfamily of Ras-like GTPases. In response to external stimuli, Cdc42 is known to transduce signals to regulate the organization of the actin cytoskeleton, induce DNA synthesis in quiescent fibroblasts, and promote apoptosis in neuronal and immune cells. In this study, we have demonstrated that cdc42 mRNA and protein were up-regulated in the presence of wild-type p53 in BRK cells, followed by cytoplasmic to plasma membrane translocation of Cdc42. Overexpression of Cdc42 in the presence of a dominant-negative mutant p53 induced apoptosis rapidly, indicating that Cdc42 functions downstream of p53. Furthermore, stable expression of a dominant-negative mutant of Cdc42 partially inhibited p53-mediated apoptosis. The Bcl-2 family members Bcl-xL, and the adenovirus protein E1B 19K, inhibited Cdc42-mediated apoptosis, whereas Bcl-2 did not. We provide evidence that PAK1 and JNK1 may play a role downstream of Cdc42 to transduce its apoptotic signal. Cdc42/PAK1 activates JNK1-induced phosphorylation of Bcl-2, thereby inactivating its function, and that a phosphorylation resistant mutant (Bcl-2S70,87A,T56,74A) gains the ability to inhibit Cdc42- and p53-mediated apoptosis. Thus, one mechanism by which p53 promotes apoptosis is through activation of Cdc42 and inactivation of Bcl-2.

  10. 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.

  11. Electroacupuncture attenuates mechanical allodynia by suppressing the spinal JNK1/2 pathway in a rat model of inflammatory pain.

    PubMed

    Du, Jun-Ying; Fang, Jian-Qiao; Liang, Yi; Fang, Jun-Fan

    2014-09-01

    Electroacupuncture (EA) has a substantial analgesic effect on inflammatory pain induced by complete Freund's adjuvant (CFA). The activation of the c-Jun N-terminal kinase 1/2 (JNK1/2) signal transduction pathway in the spinal cord is associated with inflammatory pain. However, the relationship between EA's analgesic effect and the JNK1/2 signal transduction pathway in the inflammatory pain remain unclear. In the present study, we used the established rat model of CFA-induced inflammatory pain to investigate the role of the spinal JNK1/2 pathway in EA-mediated analgesia. We observed a decrease in paw withdrawal thresholds and an increase in paw edema at 1 and 3 days after injecting CFA into the right hindpaw. CFA, 3 days after injection, upregulated expression of phospho-c-Jun N-terminal kinase1/2 (p-JNK1/2) protein and its downstream targets, the transcriptional regulators p-c-Jun and activator protein-1 (AP-1), as well as cyclooxygenase-2 (COX-2) and the transient receptor potential vanilloid 1 (TRPV1). EA significantly alleviated CFA-induced inflammatory pain. In addition, EA reduced p-JNK1/2 protein levels and COX-2 mRNA expressions, a degree of down-regulated p-c-Jun protein level and AP-1 DNA binding activity in the spinal dorsal horn of CFA-administered animals, but it had no effect on TRPV1 mRNA expression. Furthermore, EA and the JNK inhibitor SP600125 synergistically inhibited CFA-induced hyperalgesia and suppressed the COX-2 mRNA expression in the spinal dorsal horn. Our findings indicate that EA alleviates inflammatory pain behavior, at least in part, by reducing COX-2 expression in the spinal cord via the JNK1/2 signaling pathway. Inactivation of the spinal JNK1/2 signal transduction pathway maybe the potential mechanism of EA's antinociception in the inflammatory pain model. Copyright © 2014 Elsevier Inc. All rights reserved.

  12. HCV NS5A Up-Regulates COX-2 Expression via IL-8-Mediated Activation of the ERK/JNK MAPK Pathway

    PubMed Central

    Chen, Wei-Chun; Tseng, Chin-Kai; Chen, Yen-Hsu; Lin, Chun-Kuang; Hsu, Shih-hsien; Wang, Shen-Nien; Lee, Jin-Ching

    2015-01-01

    Chronic hepatitis C virus (HCV) infection leads to intrahepatic inflammation and liver cell injury, which are considered a risk factor for virus-associated hepatitis, cirrhosis, and hepatocellular carcinoma worldwide. Inflammatory cytokines are critical components of the immune system and influence cellular signaling, and genetic imbalances. In this study, we found that cyclooxygenase-2 (COX-2) and interleukin-8 (IL-8) were significantly induced by HCV infection and HCV NS5A expression, and induction of COX-2 correlated with HCV-induced IL-8 production. We also found that the ERK and JNK signaling pathways were involved in the regulation of IL-8-mediated COX-2 induction in response to HCV infection. Using a promoter-linked reporter assay, we identified that the C/EBP regulatory element within the COX-2 promoter was the dominant factor responsible for the induction of COX-2 by HCV. Silencing C/EBP attenuated HCV-induced COX-2 expression. Our results revealed that HCV-induced inflammation promotes viral replication, providing new insights into the involvement of IL-8-mediated COX-2 induction in HCV replication. PMID:26231035

  13. PARP1-mediated necrosis is dependent on parallel JNK and Ca2+/calpain pathways

    PubMed Central

    Douglas, Diana L.; Baines, Christopher P.

    2014-01-01

    ABSTRACT Poly(ADP-ribose) polymerase-1 (PARP1) is a nuclear enzyme that can trigger caspase-independent necrosis. Two main mechanisms for this have been proposed: one involving RIP1 and JNK kinases and mitochondrial permeability transition (MPT), the other involving calpain-mediated activation of Bax and mitochondrial release of apoptosis-inducing factor (AIF). However, whether these two mechanisms represent distinct pathways for PARP1-induced necrosis, or whether they are simply different components of the same pathway has yet to be tested. Mouse embryonic fibroblasts (MEFs) were treated with either N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) or β-Lapachone, resulting in PARP1-dependent necrosis. This was associated with increases in calpain activity, JNK activation and AIF translocation. JNK inhibition significantly reduced MNNG- and β-Lapachone-induced JNK activation, AIF translocation, and necrosis, but not calpain activation. In contrast, inhibition of calpain either by Ca2+ chelation or knockdown attenuated necrosis, but did not affect JNK activation or AIF translocation. To our surprise, genetic and/or pharmacological inhibition of RIP1, AIF, Bax and the MPT pore failed to abrogate MNNG- and β-Lapachone-induced necrosis. In conclusion, although JNK and calpain both contribute to PARP1-induced necrosis, they do so via parallel mechanisms. PMID:25052090

  14. JNK1 Mediates Lipopolysaccharide-Induced CD14 and SR-AI Expression and Macrophage Foam Cell Formation.

    PubMed

    An, Dong; Hao, Feng; Hu, Chen; Kong, Wei; Xu, Xuemin; Cui, Mei-Zhen

    2017-01-01

    Foam cell formation is the key process in the development of atherosclerosis. The uptake of oxidized low-density lipoprotein (oxLDL) converts macrophages into foam cells. We recently reported that lipopolysaccharide (LPS)-induced foam cell formation is regulated by CD14 and scavenger receptor AI (SR-AI). In this study, we employed pharmaceutical and gene knockdown approaches to determine the upstream molecular mediators, which control LPS-induced foam cell formation. Our results demonstrated that the specific c-Jun N-terminal kinase (JNK) pathway inhibitor, SP600125, but neither the specific inhibitor of extracellular signaling-regulated kinase (ERK) kinase MEK1/2, U0126, nor the specific inhibitor of p38 MAPK, SB203580, significantly blocks LPS-induced oxLDL uptake, suggesting that the JNK pathway is the upstream mediator of LPS-induced oxLDL uptake/foam cell formation. To address whether JNK pathway mediates LPS-induced oxLDL uptake is due to JNK pathway-regulated CD14 and SR-AI expression, we assessed whether the pharmaceutical inhibitor of JNK influences LPS-induced expression of CD14 and SR-AI. Our results indicate that JNK pathway mediates LPS-induced CD14 and SR-AI expression. To conclusively address the isoform role of JNK family, we depleted JNK isoforms using the JNK isoform-specific siRNA. Our data showed that the depletion of JNK1, but not JNK2 blocked LPS-induced CD14/SR-AI expression and foam cell formation. Taken together, our results reveal for the first time that JNK1 is the key mediator of LPS-induced CD14 and SR-AI expression in macrophages, leading to LPS-induced oxLDL uptake/foam cell formation. We conclude that the novel JNK1/CD14/SR-AI pathway controls macrophage oxLDL uptake/foam cell formation.

  15. HCV upregulates Bim through the ROS/JNK signalling pathway, leading to Bax-mediated apoptosis.

    PubMed

    Deng, Lin; Chen, Ming; Tanaka, Motofumi; Ku, Yonson; Itoh, Tomoo; Shoji, Ikuo; Hotta, Hak

    2015-09-01

    We previously reported that hepatitis C virus (HCV) infection induces Bax-triggered, mitochondrion-mediated apoptosis by using the HCV J6/JFH1 strain and Huh-7.5 cells. However, it was still unclear how HCV-induced Bax activation. In this study, we showed that the HCV-induced activation and mitochondrial accumulation of Bax were significantly attenuated by treatment with a general antioxidant, N-acetyl cysteine (NAC), or a specific c-Jun N-terminal kinase (JNK) inhibitor, SP600125, with the result suggesting that the reactive oxygen species (ROS)/JNK signalling pathway is upstream of Bax activation in HCV-induced apoptosis. We also demonstrated that HCV infection transcriptionally activated the gene for the pro-apoptotic protein Bim and the protein expression of three major splice variants of Bim (BimEL, BimL and BimS). The HCV-induced increase in the Bim mRNA and protein levels was significantly counteracted by treatment with NAC or SP600125, suggesting that the ROS/JNK signalling pathway is involved in Bim upregulation. Moreover, HCV infection led to a marked accumulation of Bim on the mitochondria to facilitate its interaction with Bax. On the other hand, downregulation of Bim by siRNA (small interfering RNA) significantly prevented HCV-mediated activation of Bax and caspase 3. Taken together, these observations suggest that HCV-induced ROS/JNK signalling transcriptionally activates Bim expression, which leads to Bax activation and apoptosis induction.

  16. Acute ethanol exposure-induced autophagy-mediated cardiac injury via activation of the ROS-JNK-Bcl-2 pathway.

    PubMed

    Zhu, Zhongxin; Huang, Yewei; Lv, Lingchun; Tao, Youli; Shao, Minglong; Zhao, Congcong; Xue, Mei; Sun, Jia; Niu, Chao; Wang, Yang; Kim, Sunam; Cong, Weitao; Mao, Wei; Jin, Litai

    2018-02-01

    Binge drinking is associated with increased cardiac autophagy, and often triggers heart injury. Given the essential role of autophagy in various cardiac diseases, this study was designed to investigate the role of autophagy in ethanol-induced cardiac injury and the underlying mechanism. Our study showed that ethanol exposure enhanced the levels of LC3-II and LC3-II positive puncta and promoted cardiomyocyte apoptosis in vivo and in vitro. In addition, we found that ethanol induced autophagy and cardiac injury largely via the sequential triggering of reactive oxygen species (ROS) accumulation, activation of c-Jun NH2-terminal kinase (JNK), phosphorylation of Bcl-2, and dissociation of the Beclin 1/Bcl-2 complex. By contrast, inhibition of ethanol-induced autophagic flux with pharmacologic agents in the hearts of mice and cultured cells significantly alleviated ethanol-induced cardiomyocyte apoptosis and heart injury. Elimination of ROS with the antioxidant N-acetyl cysteine (NAC) or inhibition of JNK with the JNK inhibitor SP600125 reduced ethanol-induced autophagy and subsequent autophagy-mediated apoptosis. Moreover, metallothionein (MT), which can scavenge reactive oxygen and nitrogen species, also attenuated ethanol-induced autophagy and cell apoptosis in MT-TG mice. In conclusion, our findings suggest that acute ethanol exposure induced autophagy-mediated heart toxicity and injury mainly through the ROS-JNK-Bcl-2 signaling pathway. © 2017 Wiley Periodicals, Inc.

  17. Curcumin suppresses JNK pathway to attenuate BPA-induced insulin resistance in LO2 cells.

    PubMed

    Geng, Shanshan; Wang, Shijia; Zhu, Weiwei; Xie, Chunfeng; Li, Xiaoting; Wu, Jieshu; Zhu, Jianyun; Jiang, Ye; Yang, Xue; Li, Yuan; Chen, Yue; Wang, Xiaoqian; Meng, Yu; Zhong, Caiyun

    2018-01-01

    To examine whether curcumin has protective effect on insulin resistance induced by bisphenol A (BPA) in LO2 cells and whether this effect was mediated by inhibiting the inflammatory mitogen-activated protein kinases (MAPKs) and nuclear factor-κB (NF-κB) pathways. LO2 cells were stimulated with BPA in the presence or absence of curcumin for 5 days. Glucose consumption, activation of insulin signaling, MAPKs and NF-κB pathways, levels of inflammatory cytokines and MDA production were analyzed. Curcumin prevented BPA-induced reduction of glucose consumption and suppression of insulin signaling pathway, indicating curcumin alleviated BPA-triggered insulin resistance in LO2 cells. mRNA and proteins levels of TNF-α and IL-6, as well as MDA level in LO2 cells treated with BPA were decreased by curcumin. Furthermore, curcumin downregulated the activation of p38, JNK, and NF-κB pathways upon stimulation with BPA. Inhibition of JNK pathway, but not p38 nor NF-κB pathway, improved glucose consumption and insulin signaling in BPA-treated LO2 cells. Curcumin inhibits BPA-induced insulin resistance by suppressing JNK pathway. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

  18. Role of Adaptor Protein Toll-Like Interleukin Domain Containing Adaptor Inducing Interferon β in Toll-Like Receptor 3- and 4-Mediated Regulation of Hepatic Drug Metabolizing Enzyme and Transporter Genes.

    PubMed

    Shah, Pranav; Omoluabi, Ozozoma; Moorthy, Bhagavatula; Ghose, Romi

    2016-01-01

    The expressions and activities of hepatic drug-metabolizing enzymes and transporters (DMETs) are altered during infection and inflammation. Inflammatory responses in the liver are mediated primarily by Toll-like receptor (TLR)-signaling, which involves recruitment of Toll/interleukin (IL)-1 receptor (TIR) domain containing adaptor protein (TIRAP) and TIR domain containing adaptor inducing interferon (IFN)-β (TRIF) that eventually leads to induction of proinflammatory cytokines and mitogen-activated protein kinases (MAPKs). Lipopolysaccharide (LPS) activates the Gram-negative bacterial receptor TLR4 and polyinosinic:polycytidylic acid (polyI:C) activates the viral receptor TLR3. TLR4 signaling involves TIRAP and TRIF, whereas TRIF is the only adaptor protein involved in the TLR3 pathway. We have shown previously that LPS-mediated downregulation of DMETs is independent of TIRAP. To determine the role of TRIF, we treated TRIF(+/+) and TRIF(-/-) mice with LPS or polyI:C. LPS downregulated (∼40%-60%) Cyp3a11, Cyp2a4, Ugt1a1, Mrp2 mRNA levels, whereas polyI:C downregulated (∼30%-60%) Cyp3a11, Cyp2a4, Cyp1a2, Cyp2b10, Ugt1a1, Mrp2, and Mrp3 mRNA levels in TRIF(+/+) mice. This downregulation was not attenuated in TRIF(-/-) mice. Induction of cytokines by LPS was observed in both TRIF(+/+) and TRIF(-/-) mice. Cytokine induction was delayed in polyI:C-treated TRIF(-/-) mice, indicating that multiple mechanisms mediating polyI:C signaling exist. To assess the role of MAPKs, primary hepatocytes were pretreated with specific inhibitors before treatment with LPS/polyI:C. We found that only the c-jun-N-terminal kinase (JNK) inhibitor attenuated the down-regulation of DMETs. These results show that TRIF-independent pathways can be involved in the downregulation of DMETs through TLR4 and 3. JNK-dependent mechanisms likely mediate this downregulation. Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.

  19. Increased Expression of Interleukin-18 mRNA is Associated with Carotid Artery Stenosis

    PubMed

    Arapi, Berk; Bayoğlu, Burcu; Cengiz, Müjgan; Dirican, Ahmet; Deser, Serkan Burç; Junusbekov, Yerik; Arslan, Caner

    2018-05-29

    Carotid artery stenosis is the atherosclerotic narrowing of the proximal internal carotid artery and one of the primary causes of stroke. Elevated expression of the pleiotropic proinflammatory cytokine interleukin-18 has been demonstrated in human atherosclerotic plaques. To investigate whether the mRNA expression levels of interleukin-18 and interleukin-18-binding protein and interleukin-18 −137 G/C (rs187238) variants are associated with carotid artery stenosis development. Case-control study. The mRNA expression levels of interleukin-18 and interleukin-18-binding protein and interleukin-18 rs187238 variants were evaluated by quantitative real-time polymerase chain reaction and real-time polymerase chain reaction, respectively, in the peripheral blood mononuclear cells of 70 patients with carotid artery stenosis (36 symptomatic, 34 asymptomatic) and 75 healthy controls. Interleukin-18 mRNA expression was significantly increased in carotid artery stenosis patients compared to that in healthy controls (p=0.01). However, no significant difference was observed between interleukin-18-binding protein mRNA expression levels in patients with carotid artery stenosis and those in controls (p=0.101). Internal carotid artery stenosis severity was significantly higher in symptomatic patients than that in asymptomatic patients (p<0.001). A significant relationship was identified between interleukin-18 expression and internal carotid artery stenosis severity in patients with carotid artery stenosis (p=0.051). Interleukin-18 rs187238 polymorphism genotype frequencies did not significantly differ between patients with carotid artery stenosis and controls (p=0.246). A significant difference was identified between interleukin-18-binding protein gene expression and symptomatic and asymptomatic patients (p=0.026), but there was no difference in interleukin-18 expression between the symptomatic and asymptomatic subgroups (p=0.397). Interleukin-18 mRNA expression may affect carotid

  20. Stathmin Mediates Hepatocyte Resistance to Death from Oxidative Stress by down Regulating JNK

    PubMed Central

    Zhao, Enpeng; Amir, Muhammad; Lin, Yu; Czaja, Mark J.

    2014-01-01

    Stathmin 1 performs a critical function in cell proliferation by regulating microtubule polymerization. This proliferative function is thought to explain the frequent overexpression of stathmin in human cancer and its correlation with a bad prognosis. Whether stathmin also functions in cell death pathways is unclear. Stathmin regulates microtubules in part by binding free tubulin, a process inhibited by stathmin phosphorylation from kinases including c-Jun N-terminal kinase (JNK). The involvement of JNK activation both in stathmin phosphorylation, and in hepatocellular resistance to oxidative stress, led to an examination of the role of stathmin/JNK crosstalk in oxidant-induced hepatocyte death. Oxidative stress from menadione-generated superoxide induced JNK-dependent stathmin phosphorylation at Ser-16, Ser-25 and Ser-38 in hepatocytes. A stathmin knockdown sensitized hepatocytes to both apoptotic and necrotic cell death from menadione without altering levels of oxidant generation. The absence of stathmin during oxidative stress led to JNK overactivation that was the mechanism of cell death as a concomitant knockdown of JNK1 or JNK2 blocked death. Hepatocyte death from JNK overactivation was mediated by the effects of JNK on mitochondria. Mitochondrial outer membrane permeabilization occurred in stathmin knockdown cells at low concentrations of menadione that triggered apoptosis, whereas mitochondrial β-oxidation and ATP homeostasis were compromised at higher, necrotic menadione concentrations. Stathmin therefore mediates hepatocyte resistance to death from oxidative stress by down regulating JNK and maintaining mitochondrial integrity. These findings demonstrate a new mechanism by which stathmin promotes cell survival and potentially tumor growth. PMID:25285524

  1. Endothelial Dysfunction in Human Diabetes is mediated by Wnt5a-JNK Signaling

    PubMed Central

    Bretón-Romero, Rosa; Feng, Bihua; Holbrook, Monika; Farb, Melissa G.; Fetterman, Jessica L.; Linder, Erika A.; Berk, Brittany D.; Masaki, Nobuyuki; Weisbrod, Robert M.; Inagaki, Elica; Gokce, Noyan; Fuster, Jose J.; Walsh, Kenneth; Hamburg, Naomi M.

    2016-01-01

    Objectives Endothelial dysfunction is linked to insulin resistance, inflammatory activation and increased cardiovascular risk in diabetes mellitus; however the mechanisms remain incompletely understood. Recent studies have identified pro-inflammatory signaling of Wnt5a through JNK as a regulator of metabolic dysfunction with potential relevance to vascular function. We sought to gain evidence that increased activation of Wnt5a-JNK signaling contributes to impaired endothelial function in patients with diabetes mellitus. Approach We measured flow-mediated dilation of the brachial artery and characterized freshly isolated endothelial cells by protein expression, eNOS activation, and nitric oxide production in from 85 subjects with Type 2 diabetes mellitus (n=42) and age- and sex-matched non-diabetic controls (n=43) and in human aortic endothelial cells treated with Wnt5a. Results Endothelial cells from patients with diabetes displayed 1.3-fold higher Wnt5a levels (P=0.01) along with 1.4-fold higher JNK activation (P<0.01) without a difference in total JNK levels. Higher JNK activation was associated with lower flow-mediated dilation, consistent with endothelial dysfunction (r=0.53, P=0.02). Inhibition of Wnt5a and JNK signaling restored insulin and A23187-mediated eNOS activation and improved nitric oxide production in endothelial cells from patients with diabetes. In endothelial cells from non-diabetic controls, rWnt5a treatment inhibited eNOS activation replicating the diabetic endothelial phenotype. In HAECs, Wnt5a-induced impairment of eNOS activation and nitric oxide production was reversed by Wnt5a and JNK inhibition. Conclusions Our findings demonstrate that non-canonical Wnt5a signaling and JNK activity contributes to vascular insulin resistance and endothelial dysfunction and may represent a novel therapeutic opportunity to protect the vasculature in patients with diabetes. PMID:26800561

  2. Endothelial Dysfunction in Human Diabetes Is Mediated by Wnt5a-JNK Signaling.

    PubMed

    Bretón-Romero, Rosa; Feng, Bihua; Holbrook, Monika; Farb, Melissa G; Fetterman, Jessica L; Linder, Erika A; Berk, Brittany D; Masaki, Nobuyuki; Weisbrod, Robert M; Inagaki, Elica; Gokce, Noyan; Fuster, Jose J; Walsh, Kenneth; Hamburg, Naomi M

    2016-03-01

    Endothelial dysfunction is linked to insulin resistance, inflammatory activation, and increased cardiovascular risk in diabetes mellitus; however, the mechanisms remain incompletely understood. Recent studies have identified proinflammatory signaling of wingless-type family member (Wnt) 5a through c-jun N-terminal kinase (JNK) as a regulator of metabolic dysfunction with potential relevance to vascular function. We sought to gain evidence that increased activation of Wnt5a-JNK signaling contributes to impaired endothelial function in patients with diabetes mellitus. We measured flow-mediated dilation of the brachial artery and characterized freshly isolated endothelial cells by protein expression, eNOS activation, and nitric oxide production in 85 subjects with type 2 diabetes mellitus (n=42) and age- and sex-matched nondiabetic controls (n=43) and in human aortic endothelial cells treated with Wnt5a. Endothelial cells from patients with diabetes mellitus displayed 1.3-fold higher Wnt5a levels (P=0.01) along with 1.4-fold higher JNK activation (P<0.01) without a difference in total JNK levels. Higher JNK activation was associated with lower flow-mediated dilation, consistent with endothelial dysfunction (r=0.53, P=0.02). Inhibition of Wnt5a and JNK signaling restored insulin and A23187-mediated eNOS activation and improved nitric oxide production in endothelial cells from patients with diabetes mellitus. In endothelial cells from nondiabetic controls, rWnt5a treatment inhibited eNOS activation replicating the diabetic endothelial phenotype. In human aortic endothelial cells, Wnt5a-induced impairment of eNOS activation and nitric oxide production was reversed by Wnt5a and JNK inhibition. Our findings demonstrate that noncanonical Wnt5a signaling and JNK activity contribute to vascular insulin resistance and endothelial dysfunction and may represent a novel therapeutic opportunity to protect the vasculature in patients with diabetes mellitus. © 2016 American Heart

  3. Selection of hammerhead ribozymes for optimum cleavage of interleukin 6 mRNA.

    PubMed Central

    Hendrix, C; Anné, J; Joris, B; Van Aerschot, A; Herdewijn, P

    1996-01-01

    Four GUC triplets in the coding region of the MRNA of interleukin 6 (IL-6) were examined for their suitabilty to serve as a target for hammerhead ribozome-mediated cleavage. This selection procedure was performed with the intention to downregulate IL-6 production as a potential treatment of those diseases in which IL-6 overexpression is involved. Hammerhead ribozymes and their respective short synthetic substrates (19-mers) were synthesized for these four GUC triplets. Notwithstanding the identical catalytic core sequences, the difference in base composition of the helices involved in substrate binding caused substantial variation in cleavage activity. The cleavage reactions on the 1035 nucleotide IL-6 mRNA transcript revealed that two ribozymes were able to cleave this substrate, showing a decrease in catalytic efficiency to 1/30 and 1/300 of the short substrate. This study indicates that the GUC triplet located at nucleotide 510 of the mRNA of IL-6 is the best site for hammerhead ribozyme-mediated cleavage. We suggest that in future targeting of chemically modified hammerhead ribosomes for cleavage of IL-6 RNA should be directed at this location. PMID:8670082

  4. Cot/tpl2-MKK1/2-Erk1/2 controls mTORC1-mediated mRNA translation in Toll-like receptor–activated macrophages

    PubMed Central

    López-Pelaéz, Marta; Fumagalli, Stefano; Sanz, Carlos; Herrero, Clara; Guerra, Susana; Fernandez, Margarita; Alemany, Susana

    2012-01-01

    Cot/tpl2 is the only MAP3K that activates MKK1/2-Erk1/2 in Toll-like receptor–activated macrophages. Here we show that Cot/tpl2 regulates RSK, S6 ribosomal protein, and 4E-BP phosphorylation after stimulation of bone marrow–derived macrophages with lipopolysaccharide (LPS), poly I:C, or zymosan. The dissociation of the 4E-BP–eIF4E complex, a key event in the cap-dependent mRNA translation initiation, is dramatically reduced in LPS-stimulated Cot/tpl2-knockout (KO) macrophages versus LPS-stimulated wild-type (Wt) macrophages. Accordingly, after LPS activation, increased cap-dependent translation is observed in Wt macrophages but not in Cot/tpl2 KO macrophages. In agreement with these data, Cot/tpl2 increases the polysomal recruitment of the 5´ TOP eEF1α and eEF2 mRNAs, as well as of inflammatory mediator gene–encoding mRNAs, such as tumor necrosis factor α (TNFα), interleukin-6 (IL-6), and KC in LPS-stimulated macrophages. In addition, Cot/tpl2 deficiency also reduces total TNFα, IL-6, and KC mRNA expression in LPS-stimulated macrophages, which is concomitant with a decrease in their mRNA half-lives. Macrophages require rapid fine control of translation to provide an accurate and not self-damaging response to host infection, and our data show that Cot/tpl2 controls inflammatory mediator gene–encoding mRNA translation in Toll-like receptor–activated macrophages. PMID:22675026

  5. Cot/tpl2-MKK1/2-Erk1/2 controls mTORC1-mediated mRNA translation in Toll-like receptor-activated macrophages.

    PubMed

    López-Pelaéz, Marta; Fumagalli, Stefano; Sanz, Carlos; Herrero, Clara; Guerra, Susana; Fernandez, Margarita; Alemany, Susana

    2012-08-01

    Cot/tpl2 is the only MAP3K that activates MKK1/2-Erk1/2 in Toll-like receptor-activated macrophages. Here we show that Cot/tpl2 regulates RSK, S6 ribosomal protein, and 4E-BP phosphorylation after stimulation of bone marrow-derived macrophages with lipopolysaccharide (LPS), poly I:C, or zymosan. The dissociation of the 4E-BP-eIF4E complex, a key event in the cap-dependent mRNA translation initiation, is dramatically reduced in LPS-stimulated Cot/tpl2-knockout (KO) macrophages versus LPS-stimulated wild-type (Wt) macrophages. Accordingly, after LPS activation, increased cap-dependent translation is observed in Wt macrophages but not in Cot/tpl2 KO macrophages. In agreement with these data, Cot/tpl2 increases the polysomal recruitment of the 5´ TOP eEF1α and eEF2 mRNAs, as well as of inflammatory mediator gene-encoding mRNAs, such as tumor necrosis factor α (TNFα), interleukin-6 (IL-6), and KC in LPS-stimulated macrophages. In addition, Cot/tpl2 deficiency also reduces total TNFα, IL-6, and KC mRNA expression in LPS-stimulated macrophages, which is concomitant with a decrease in their mRNA half-lives. Macrophages require rapid fine control of translation to provide an accurate and not self-damaging response to host infection, and our data show that Cot/tpl2 controls inflammatory mediator gene-encoding mRNA translation in Toll-like receptor-activated macrophages.

  6. Efavirenz and 8-hydroxyefavirenz induce cell death via a JNK- and BimEL-dependent mechanism in primary human hepatocytes

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

    Bumpus, Namandje N., E-mail: nbumpus1@jhmi.edu

    Chronic use of efavirenz (EFV) has been linked to incidences of hepatotoxicity in patients receiving EFV to treat HIV-1. While recent studies have demonstrated that EFV stimulates hepatic cell death a role for the metabolites of efavirenz in this process has yet to be examined. In the present study, incubation of primary human hepatocytes with synthetic 8-hydroxyEFV (8-OHEFV), which is the primary metabolite of EFV, resulted in cell death, caspase-3 activation and reactive oxygen species formation. The metabolite exerted these effects at earlier time points and using lower concentrations than were required for the parent compound. In addition, pharmacological inhibitionmore » of cytochrome P450-dependent metabolism of EFV using 1-aminobenzotriazole markedly decreased reactive oxygen species formation and cell death. Treatment of primary human hepatocytes with EFV and 8-OHEFV also stimulated phosphorylation of c-Jun N-terminal kinase (JNK) as well as phosphorylation of the JNK substrate c-Jun. Further, the mRNA and protein expression of an isoform of Bim (Bcl-2 interacting mediator of cell death) denoted as BimEL, which is proapoptotic and has been shown to be modulated by JNK, was increased. Inhibition of JNK using SP600125 prevented the EFV- and 8-OHEFV-mediated cell death. Silencing of Bim using siRNA transfected into hepatocytes also prevented cell death resulting from 8-OHEFV-treatment. These data suggest that the oxidative metabolite 8-OHEFV is a more potent inducer of hepatic cell death than the parent compound EFV. Further, activation of the JNK signaling pathway and BimEL mRNA expression appear to be required for EFV- and 8-OHEFV-mediated hepatocyte death. -- Highlights: Black-Right-Pointing-Pointer 8-Hydroxyefavirenz is a more potent stimulator of cell death than efavirenz. Black-Right-Pointing-Pointer Efavirenz and 8-hydroxyefavirenz increase JNK activity and BimEL mRNA expression. Black-Right-Pointing-Pointer JNK and Bim are required for efavirenz- and

  7. A Protein Scaffold Coordinates SRC-Mediated JNK Activation in Response to Metabolic Stress.

    PubMed

    Kant, Shashi; Standen, Claire L; Morel, Caroline; Jung, Dae Young; Kim, Jason K; Swat, Wojciech; Flavell, Richard A; Davis, Roger J

    2017-09-19

    Obesity is a major risk factor for the development of metabolic syndrome and type 2 diabetes. How obesity contributes to metabolic syndrome is unclear. Free fatty acid (FFA) activation of a non-receptor tyrosine kinase (SRC)-dependent cJun NH 2 -terminal kinase (JNK) signaling pathway is implicated in this process. However, the mechanism that mediates SRC-dependent JNK activation is unclear. Here, we identify a role for the scaffold protein JIP1 in SRC-dependent JNK activation. SRC phosphorylation of JIP1 creates phosphotyrosine interaction motifs that bind the SH2 domains of SRC and the guanine nucleotide exchange factor VAV. These interactions are required for SRC-induced activation of VAV and the subsequent engagement of a JIP1-tethered JNK signaling module. The JIP1 scaffold protein, therefore, plays a dual role in FFA signaling by coordinating upstream SRC functions together with downstream effector signaling by the JNK pathway. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

  8. ROS regulation of axonal mitochondrial transport is mediated by Ca2+ and JNK in Drosophila

    PubMed Central

    Liao, Pin-Chao; Tandarich, Lauren C.

    2017-01-01

    Mitochondria perform critical functions including aerobic ATP production and calcium (Ca2+) homeostasis, but are also a major source of reactive oxygen species (ROS) production. To maintain cellular function and survival in neurons, mitochondria are transported along axons, and accumulate in regions with high demand for their functions. Oxidative stress and abnormal mitochondrial axonal transport are associated with neurodegenerative disorders. However, we know little about the connection between these two. Using the Drosophila third instar larval nervous system as the in vivo model, we found that ROS inhibited mitochondrial axonal transport more specifically, primarily due to reduced flux and velocity, but did not affect transport of other organelles. To understand the mechanisms underlying these effects, we examined Ca2+ levels and the JNK (c-Jun N-terminal Kinase) pathway, which have been shown to regulate mitochondrial transport and general fast axonal transport, respectively. We found that elevated ROS increased Ca2+ levels, and that experimental reduction of Ca2+ to physiological levels rescued ROS-induced defects in mitochondrial transport in primary neuron cell cultures. In addition, in vivo activation of the JNK pathway reduced mitochondrial flux and velocities, while JNK knockdown partially rescued ROS-induced defects in the anterograde direction. We conclude that ROS have the capacity to regulate mitochondrial traffic, and that Ca2+ and JNK signaling play roles in mediating these effects. In addition to transport defects, ROS produces imbalances in mitochondrial fission-fusion and metabolic state, indicating that mitochondrial transport, fission-fusion steady state, and metabolic state are closely interrelated in the response to ROS. PMID:28542430

  9. JNK signaling mediates EPHA2-dependent tumor cell proliferation, motility, and cancer stem cell-like properties in non-small cell lung cancer

    PubMed Central

    Song, Wenqiang; Ma, Yufang; Wang, Jialiang; Brantley-Sieders, Dana; Chen, Jin

    2014-01-01

    Recent genome-wide analyses in human lung cancer revealed that EPHA2 receptor tyrosine kinase is overexpressed in non-small cell lung cancer (NSCLC), and high levels of EPHA2 correlate with poor clinical outcome. However, the mechanistic basis for EPHA2-mediated tumor promotion in lung cancer remains poorly understood. Here we show that the JNK/c-JUN signaling mediates EPHA2-dependent tumor cell proliferation and motility. A screen of phospho-kinase arrays revealed a decrease in phospho-c-JUN levels in EPHA2 knockdown cells. Knockdown of EPHA2 inhibited p-JNK and p-c-JUN levels in approximately 50% of NSCLC lines tested. Treatment of parental cells with SP600125, a JNK inhibitor, recapitulated defects in EPHA2-deficient tumor cells; whereas constitutively activated JNK mutants were sufficient to rescue phenotypes. Knockdown of EPHA2 also inhibited tumor formation and progression in xenograft animal models in vivo. Furthermore, we investigated the role of EPHA2 in cancer stem-like cells. RNAi-mediated depletion of EPHA2 in multiple NSCLC lines decreased the ALDH positive cancer stem-like population and tumor spheroid formation in suspension. Depletion of EPHA2 in sorted ALDH positive populations markedly inhibited tumorigenicity in nude mice. Furthermore, analysis of a human lung cancer tissue microarray revealed a significant, positive association between EPHA2 and ALDH expression, indicating an important role for EPHA2 in human lung cancer stem-like cells. Collectively, these studies revealed a critical role of JNK signaling in EPHA2-dependent lung cancer cell proliferation and motility and a role for EPHA2 in cancer stem-like cell function, providing evidence for EPHA2 as a potential therapeutic target in NSCLC. PMID:24607842

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

    PubMed

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

    2007-05-01

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

  11. HER2-induced metastasis is mediated by AKT/JNK/EMT signaling pathway in gastric cancer

    PubMed Central

    Choi, Yiseul; Ko, Young San; Park, Jinju; Choi, Youngsun; Kim, Younghoon; Pyo, Jung-Soo; Jang, Bo Gun; Hwang, Douk Ho; Kim, Woo Ho; Lee, Byung Lan

    2016-01-01

    AIM To investigated the relationships between HER2, c-Jun N-terminal kinase (JNK) and protein kinase B (AKT) with respect to metastatic potential of HER2-positive gastric cancer (GC) cells. METHODS Immunohistochemistry was performed on tissue array slides containing 423 human GC specimens. Using HER2-positve GC cell lines SNU-216 and NCI-N87, HER2 expression was silenced by RNA interference, and the activations of JNK and AKT were suppressed by SP600125 and LY294002, respectively. Transwell assay, Western blot, semi-quantitative reverse transcription-polymerase chain reaction and immunofluorescence staining were used in cell culture experiments. RESULTS In GC specimens, HER2, JNK, and AKT activations were positively correlated with each other. In vitro analysis revealed a positive regulatory feedback loop between HER2 and JNK in GC cell lines and the role of JNK as a downstream effector of AKT in the HER2/AKT signaling pathway. JNK inhibition suppressed migratory capacity through reversing EMT and dual inhibition of JNK and AKT induced a more profound effect on cancer cell motility. CONCLUSION HER2, JNK and AKT in human GC specimens are positively associated with each other. JNK and AKT, downstream effectors of HER2, co-operatively contribute to the metastatic potential of HER2-positive GC cells. Thus, targeting of these two molecules in combination with HER2 downregulation may be a good approach to combat HER2-positive GC. PMID:27895401

  12. Febuxostat, an Inhibitor of Xanthine Oxidase, Suppresses Lipopolysaccharide-Induced MCP-1 Production via MAPK Phosphatase-1-Mediated Inactivation of JNK

    PubMed Central

    Nomura, Johji; Busso, Nathalie; Ives, Annette; Tsujimoto, Syunsuke; Tamura, Mizuho; So, Alexander; Yamanaka, Yoshihiro

    2013-01-01

    Excess reactive oxygen species (ROS) formation can trigger various pathological conditions such as inflammation, in which xanthine oxidase (XO) is one major enzymatic source of ROS. Although XO has been reported to play essential roles in inflammatory conditions, the molecular mechanisms underlying the involvement of XO in inflammatory pathways remain unclear. Febuxostat, a selective and potent inhibitor of XO, effectively inhibits not only the generation of uric acid but also the formation of ROS. In this study, therefore, we examined the effects of febuxostat on lipopolysaccharide (LPS)-mediated inflammatory responses. Here we show that febuxostat suppresses LPS-induced MCP-1 production and mRNA expression via activating MAPK phosphatase-1 (MKP-1) which, in turn, leads to dephosphorylation and inactivation of JNK in macrophages. Moreover, these effects of febuxostat are mediated by inhibiting XO-mediated intracellular ROS production. Taken together, our data suggest that XO mediates LPS-induced phosphorylation of JNK through ROS production and MKP-1 inactivation, leading to MCP-1 production in macrophages. These studies may bring new insights into the novel role of XO in regulating inflammatory process through MAPK phosphatase, and demonstrate the potential use of XO inhibitor in modulating the inflammatory processes. PMID:24086554

  13. Polysaccharides from Smilax glabra inhibit the pro-inflammatory mediators via ERK1/2 and JNK pathways in LPS-induced RAW264.7 cells.

    PubMed

    Lu, Chuan-li; Wei, Zhu; Min, Wang; Hu, Meng-mei; Chen, Wen-long; Xu, Xiao-jie; Lu, Chuan-jian

    2015-05-20

    The rhizomes of Smilax glabra have been used as both food and folk medicine in many countries for a long time. However, little research has been reported on polysaccharides of S. glabra. In the present study, two polysaccharide fractions, SGP-1 and SGP-2, were isolated from the rhizomes of S. glabra with the number average molecular weights of 1.72 × 10(2)kDa and 1.31 × 10(2)kDa, and the weight average molecular weights of 1.31 × 10(5)kDa and 1.18 × 10(5)kDa, respectively, and their mainly monosaccharide compositions were both galactose and rhamnose (2.5:1). Both SGP-1 and SGP-2 significantly suppressed the release of nitric oxide (NO), tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) from LPS-induced RAW 264.7 cells, as well as the mRNA expression of inducible nitric oxide synthase (iNOS), TNF-α and IL-6. Additionally, SGP-1 and SGP-2 repressed the extracellular signal-regulated kinase (ERK) and c-Jun NH2-terminal kinase (JNK). These findings strongly suggested polysaccharides were also the anti-inflammatory active ingredient for S. glabra, and the potential of SGP-1 and SGP-2 as the anti-inflammatory agents. Copyright © 2015. Published by Elsevier Ltd.

  14. Staufen-mediated mRNA decay.

    PubMed

    Park, Eonyoung; Maquat, Lynne E

    2013-01-01

    Staufen1 (STAU1)-mediated mRNA decay (SMD) is an mRNA degradation process in mammalian cells that is mediated by the binding of STAU1 to a STAU1-binding site (SBS) within the 3'-untranslated region (3'-UTR) of target mRNAs. During SMD, STAU1, a double-stranded (ds) RNA-binding protein, recognizes dsRNA structures formed either by intramolecular base pairing of 3'-UTR sequences or by intermolecular base pairing of 3'-UTR sequences with a long-noncoding RNA (lncRNA) via partially complementary Alu elements. Recently, STAU2, a paralog of STAU1, has also been reported to mediate SMD. Both STAU1 and STAU2 interact directly with the ATP-dependent RNA helicase UPF1, a key SMD factor, enhancing its helicase activity to promote effective SMD. Moreover, STAU1 and STAU2 form homodimeric and heterodimeric interactions via domain-swapping. Because both SMD and the mechanistically related nonsense-mediated mRNA decay (NMD) employ UPF1; SMD and NMD are competitive pathways. Competition contributes to cellular differentiation processes, such as myogenesis and adipogenesis, placing SMD at the heart of various physiologically important mechanisms. Copyright © 2013 John Wiley & Sons, Ltd.

  15. Staufen-mediated mRNA decay

    PubMed Central

    Park, Eonyoung; Maquat, Lynne E.

    2013-01-01

    Staufen1 (STAU1)-mediated mRNA decay (SMD) is an mRNA degradation process in mammalian cells that is mediated by the binding of STAU1 to a STAU1-binding site (SBS) within the 3'-untranslated region (3'UTR) of target mRNAs. During SMD, STAU1, a double-stranded (ds) RNA-binding protein, recognizes dsRNA structures formed either by intramolecular base-pairing of 3'UTR sequences or by intermolecular base-pairing of 3'UTR sequences with a long noncoding RNA (lncRNA) via partially complementary Alu elements. Recently, STAU2, a paralog of STAU1, has also been reported to mediate SMD. Both STAU1 and STAU2 interact directly with the ATP-dependent RNA helicase UPF1, a key SMD factor, enhancing its helicase activity to promote effective SMD. Moreover, STAU1 and STAU2 form homodimeric and heterodimeric interactions via domain-swapping. Since both SMD and the mechanistically related nonsense-mediated mRNA decay (NMD) employ UPF1, SMD and NMD are competitive pathways. Competition contributes to cellular differentiation processes, such as myogenesis and adipogenesis, placing SMD at the heart of various physiologically important mechanisms. PMID:23681777

  16. PKCalpha-mediated ERK, JNK and p38 activation regulates the myogenic program in human rhabdomyosarcoma cells.

    PubMed

    Mauro, Annunziata; Ciccarelli, Carmela; De Cesaris, Paola; Scoglio, Arianna; Bouché, Marina; Molinaro, Mario; Aquino, Angelo; Zani, Bianca Maria

    2002-09-15

    We have previously suggested that PKCalpha has a role in 12-O-Tetradecanoylphorbol-13-acetate (TPA)-mediated growth arrest and myogenic differentiation in human embryonal rhabdomyosarcoma cells (RD). Here, by monitoring the signalling pathways triggered by TPA, we demonstrate that PKCalpha mediates these effects by inducing transient activation of c-Jun N-terminal protein kinases (JNKs) and sustained activation of both p38 kinase and extracellular signal-regulated kinases (ERKs) (all referred to as MAPKs). Activation of MAPKs following ectopic expression of constitutively active PKCalpha, but not its dominant-negative form, is also demonstrated. We investigated the selective contribution of MAPKs to growth arrest and myogenic differentiation by monitoring the activation of MAPK pathways, as well as by dissecting MAPK pathways using MEK1/2 inhibitor (UO126), p38 inhibitor (SB203580) and JNK and p38 agonist (anisomycin) treatments. Growth-arresting signals are triggered either by transient and sustained JNK activation (by TPA and anisomycin, respectively) or by preventing both ERK and JNK activation (UO126) and are maintained, rather than induced, by p38. We therefore suggest a key role for JNK in controlling ERK-mediated mitogenic activity. Notably, sarcomeric myosin expression is induced by both TPA and UO126 but is abrogated by the p38 inhibitor. This finding indicates a pivotal role for p38 in controlling the myogenic program. Anisomycin persistently activates p38 and JNKs but prevents myosin expression induced by TPA. In accordance with this negative role, reactivation of JNKs by anisomycin, in UO126-pre-treated cells, also prevents myosin expression. This indicates that, unlike the transient JNK activation that occurs in the TPA-mediated myogenic process, long-lasting JNK activation supports the growth-arrest state but antagonises p38-mediated myosin expression. Lastly, our results with the MEK inhibitor suggest a key role of the ERK pathway in regulating

  17. Calcium/Ask1/MKK7/JNK2/c-Src signalling cascade mediates disruption of intestinal epithelial tight junctions by dextran sulfate sodium.

    PubMed

    Samak, Geetha; Chaudhry, Kamaljit K; Gangwar, Ruchika; Narayanan, Damodaran; Jaggar, Jonathan H; Rao, RadhaKrishna

    2015-02-01

    Disruption of intestinal epithelial tight junctions is an important event in the pathogenesis of ulcerative colitis. Dextran sodium sulfate (DSS) induces colitis in mice with symptoms similar to ulcerative colitis. However, the mechanism of DSS-induced colitis is unknown. We investigated the mechanism of DSS-induced disruption of intestinal epithelial tight junctions and barrier dysfunction in Caco-2 cell monolayers in vitro and mouse colon in vivo. DSS treatment resulted in disruption of tight junctions, adherens junctions and actin cytoskeleton leading to barrier dysfunction in Caco-2 cell monolayers. DSS induced a rapid activation of c-Jun N-terminal kinase (JNK), and the inhibition or knockdown of JNK2 attenuated DSS-induced tight junction disruption and barrier dysfunction. In mice, DSS administration for 4 days caused redistribution of tight junction and adherens junction proteins from the epithelial junctions, which was blocked by JNK inhibitor. In Caco-2 cell monolayers, DSS increased intracellular Ca(2+) concentration, and depletion of intracellular Ca(2+) by 1,2-bis-(o-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid tetrakis(acetoxymethyl ester) (BAPTA/AM) or thapsigargin attenuated DSS-induced JNK activation, tight junction disruption and barrier dysfunction. Knockdown of apoptosis signal-regulated kinase 1 (Ask1) or MKK7 blocked DSS-induced tight junction disruption and barrier dysfunction. DSS activated c-Src by a Ca2+ and JNK-dependent mechanism. Inhibition of Src kinase activity or knockdown of c-Src blocked DSS-induced tight junction disruption and barrier dysfunction. DSS increased tyrosine phosphorylation of occludin, zonula occludens-1 (ZO-1), E-cadherin and β-catenin. SP600125 abrogated DSS-induced tyrosine phosphorylation of junctional proteins. Recombinant JNK2 induced threonine phosphorylation and auto-phosphorylation of c-Src. The present study demonstrates that Ca(2+)/Ask1/MKK7/JNK2/cSrc signalling cascade mediates DSS-induced tight

  18. Peroxiredoxin 2 regulates PGF2α-induced corpus luteum regression in mice by inhibiting ROS-dependent JNK activation.

    PubMed

    Park, Sun-Ji; Kim, Jung-Hak; Kim, Tae-Shin; Lee, Sang-Rae; Park, Jeen-Woo; Lee, Seunghoon; Kim, Jin-Man; Lee, Dong-Seok

    2017-07-01

    Luteal regression is a natural and necessary event to regulate the reproductive process in all mammals. Prostaglandin F2α (PGF2α) is the main factor that causes functional and structural regression of the corpus luteum (CL). It is well known that PGF2α-mediated ROS generation is closely involved in luteal regression. Peroxiredoxin 2 (Prx2) as an antioxidant enzyme plays a protective role against oxidative stress-induced cell death. However, the effect of Prx2 on PGF2α-induced luteal regression has not been reported. Here, we investigated the role of Prx2 in functional and structural CL regression induced by PGF2α-mediated ROS using Prx2-deficient (-/-) mice. We found that PGF2α-induced ROS generation was significantly higher in Prx2-/- MEF cells compared with that in wild-type (WT) cells, which induced apoptosis by activating JNK-mediated apoptotic signaling pathway. Also, PGF2α treatment in the CL derived from Prx2-/- mice promoted the reduction of steroidogenic enzyme expression and the activation of JNK and caspase3. Compared to WT mice, serum progesterone levels and luteal expression of steroidogenic enzymes decreased more rapidly whereas JNK and caspase3 activations were significantly increased in Prx2-/- mice injected with PGF2α. However, the impaired steroidogenesis and PGF2α-induced JNK-dependent apoptosis were rescued by the addition of the antioxidant N-acetyl-L-cysteine (NAC). This is the first study to demonstrate that Prx2 deficiency ultimately accelerated the PGF2α-induced luteal regression through activation of the ROS-dependent JNK pathway. These findings suggest that Prx2 plays a crucial role in preventing accelerated luteal regression via inhibition of the ROS/JNK pathway. Copyright © 2017 Elsevier Inc. All rights reserved.

  19. Interleukin-18 enhances IL-18R/Nox1 binding, and mediates TRAF3IP2-dependent smooth muscle cell migration. Inhibition by simvastatin

    PubMed Central

    Valente, Anthony J.; Yoshida, Tadashi; Izadpanah, Reza; Delafontaine, Patrice; Siebenlist, Ulrich; Chandrasekar, Bysani

    2013-01-01

    We investigated the role of TRAF3 interacting protein 2 (TRAF3IP2), a redox-sensitive adapter protein and an upstream regulator of IKK and JNK in interleukin (IL)-18 induced smooth muscle cell migration, and the mechanism of its inhibition by simvastatin. The pleiotropic cytokine IL-18 induced human coronary artery SMC migration through the induction of TRAF3IP2. IL-18 induced Nox1-dependent ROS generation, TRAF3IP2 expression, and IKK/NF-κB and JNK/AP-1 activation. IL-18 induced its own expression and that of its receptor subunit IL-18Rα. Using co-IP/IB and GST pull-down assays, we show for the first time that the subunits of the IL-18R heterodimer physically associate with Nox1 under basal conditions, and IL-18 appears to enhance their binding. Importantly, the HMG-coA reductase inhibitor simvastatin attenuated IL-18-induced TRAF3IP2 induction. These inhibitory effects were reversed by mevalonate and geranylgeranylpyrophosphate (GGPP), but not by farnesylpyrophosphate (FPP). Interestingly, simvastatin, GGPP, FPP, or Rac1 inhibition did not modulate ectopically expressed TRAF3IP2. The promigratory effects of IL-18 are mediated through TRAF3IP2 in a redox-sensitive manner, and this may involve IL-18R/Nox1 physical association. Further, Simvastatin inhibits inducible, but not ectopically-expressed TRAF3IP2. Targeting TRAF3IP2 may blunt progression of hyperplastic vascular diseases in vivo. PMID:23541442

  20. Regulation of Bovine Leukemia Virus tax and pol mRNA Levels by Interleukin-2 and -10

    PubMed Central

    Pyeon, Dohun; Splitter, Gary A.

    1999-01-01

    Recently, particular cytokines have been identified to affect progression of a variety of diseases and retrovirus infections. Previously, we demonstrated that interleukin-2 (IL-2), IL-12, and gamma interferon increased in peripheral blood mononuclear cells (PBMCs) from animals with early disease and decreased in PBMCs from animals with late disease stages of bovine leukemia virus (BLV) infection. In contrast, IL-10 increased with disease progression. To examine the effects of these cytokines on BLV expression, BLV tax and pol mRNA and p24 protein were quantified by competitive PCR and immunoblotting, respectively. IL-10 inhibited BLV tax and pol mRNA levels in BLV-infected PBMCs; however, the inhibitory effect of IL-10 was prevented in PBMCs depleted of monocytes and/or macrophages (monocyte/macrophages). To determine whether these factors were secreted or monocyte/macrophage associated, monocyte/macrophage-depleted PBMCs were cultured with isolated monocyte/macrophages in transwells where contact between monocyte/macrophages and nonadherent PBMCs was blocked. BLV tax and pol mRNA levels increased in transwell cultures similar to cultures containing nonseparated cells, and IL-10 addition inhibited the increase of BLV tax and pol mRNA. These results suggest that monocyte/macrophages secrete soluble factor(s) that increases BLV mRNA levels and that secretion of these soluble factor(s) could be inhibited by IL-10. In contrast, IL-2 increased BLV tax and pol mRNA and p24 protein production. Thus, IL-10 production by BLV-infected animals with late stage disease may serve to control BLV mRNA levels, while IL-2 may increase BLV mRNA in the early disease stage. To determine a correlation between cell proliferation and BLV expression, the effect of IL-2 and IL-10 on PBMC proliferation was tested. As anticipated, IL-2 stimulated while IL-10 suppressed antigen-specific PBMC proliferation. The present study, combined with our previous findings, suggests that increased IL-10

  1. Nobiletin inhibits human osteosarcoma cells metastasis by blocking ERK and JNK-mediated MMPs expression

    PubMed Central

    Cheng, Hsin-Lin; Hsieh, Ming-Ju; Yang, Jia-Sin; Lin, Chiao-Wen; Lue, Ko-Haung; Lu, Ko-Hsiu; Yang, Shun-Fa

    2016-01-01

    Nobiletin, a polymethoxyflavone, has a few pharmacological activities, including anti-inflammation and anti-cancer effects. However, its effect on human osteosarcoma progression remains uninvestigated. Therefore, we examined the effectiveness of nobiletin against cellular metastasis of human osteosarcoma and the underlying mechanisms. Nobiletin, up to 100 μM without cytotoxicity, significantly decreased motility, migration and invasion as well as enzymatic activities, protein levels and mRNA expressions of matrix metalloproteinase (MMP)-2 and MMP-9 in U2OS and HOS cells. In addition to inhibition of extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK), the inhibitory effect of nobiletin on the DNA-binding activity of the transcription factor nuclear factor-kappa B (NF-κB), cAMP response element-binding protein (CREB), and specificity protein 1 (SP-1) in U2OS and HOS cells. Co-treatment with ERK and JNK inhibitors and nobiletin further reduced U2OS cells migration and invasion. These results indicated that nobiletin inhibits human osteosarcoma U2OS and HOS cells motility, migration and invasion by down-regulating MMP-2 and MMP-9 expressions via ERK and JNK pathways and through the inactivation of downstream NF-κB, CREB, and SP-1. Nobiletin has the potential to serve as an anti-metastatic agent for treating osteosarcoma. PMID:27144433

  2. Fluoride induces oxidative damage and SIRT1/autophagy through ROS-mediated JNK signaling.

    PubMed

    Suzuki, Maiko; Bandoski, Cheryl; Bartlett, John D

    2015-12-01

    Fluoride is an effective caries prophylactic, but at high doses can also be an environmental health hazard. Acute or chronic exposure to high fluoride doses can result in dental enamel and skeletal and soft tissue fluorosis. Dental fluorosis is manifested as mottled, discolored, porous enamel that is susceptible to dental caries. Fluoride induces cell stress, including endoplasmic reticulum stress and oxidative stress, which leads to impairment of ameloblasts responsible for dental enamel formation. Recently we reported that fluoride activates SIRT1 and autophagy as an adaptive response to protect cells from stress. However, it still remains unclear how SIRT1/autophagy is regulated in dental fluorosis. In this study, we demonstrate that fluoride exposure generates reactive oxygen species (ROS) and the resulting oxidative damage is counteracted by SIRT1/autophagy induction through c-Jun N-terminal kinase (JNK) signaling in ameloblasts. In the mouse-ameloblast-derived cell line LS8, fluoride induced ROS, mitochondrial damage including cytochrome-c release, up-regulation of UCP2, attenuation of ATP synthesis, and H2AX phosphorylation (γH2AX), which is a marker of DNA damage. We evaluated the effects of the ROS inhibitor N-acetylcysteine (NAC) and the JNK inhibitor SP600125 on fluoride-induced SIRT1/autophagy activation. NAC decreased fluoride-induced ROS generation and attenuated JNK and c-Jun phosphorylation. NAC decreased SIRT1 phosphorylation and formation of the autophagy marker LC3II, which resulted in an increase in the apoptosis mediators γH2AX and cleaved/activated caspase-3. SP600125 attenuated fluoride-induced SIRT1 phosphorylation, indicating that fluoride activates SIRT1/autophagy via the ROS-mediated JNK pathway. In enamel organs from rats or mice treated with 50, 100, or 125 ppm fluoride for 6 weeks, cytochrome-c release and the DNA damage markers 8-oxoguanine, p-ATM, and γH2AX were increased compared to those in controls (0 ppm fluoride). These

  3. Fluoride induces oxidative damage and SIRT1/autophagy through ROS-mediated JNK signaling

    PubMed Central

    Suzuki, Maiko; Bandoski, Cheryl; Bartlett, John D.

    2015-01-01

    Fluoride is an effective caries prophylactic, but at high doses can also be an environmental health hazard. Acute or chronic exposure to high fluoride doses can result in dental enamel and skeletal and soft tissue fluorosis. Dental fluorosis is manifested as mottled, discolored, porous enamel that is susceptible to dental caries. Fluoride induces cell stress, including endoplasmic reticulum stress and oxidative stress, which leads to impairment of ameloblasts responsible for dental enamel formation. Recently we reported that fluoride activates SIRT1 and autophagy as an adaptive response to protect cells from stress. However, it still remains unclear how SIRT1/autophagy is regulated in dental fluorosis. In this study, we demonstrate that fluoride exposure generates reactive oxygen species (ROS) and the resulting oxidative damage is counteracted by SIRT1/autophagy induction through c-Jun N-terminal kinase (JNK) signaling in ameloblasts. In the mouse-ameloblast-derived cell line LS8, fluoride induced ROS, mitochondrial damage including cytochrome-c release, up-regulation of UCP2, attenuation of ATP synthesis, and H2AX phosphorylation (γH2AX), which is a marker of DNA damage. We evaluated the effects of the ROS inhibitor N-acetylcysteine (NAC) and the JNK inhibitor SP600125 on fluoride-induced SIRT1/autophagy activation. NAC decreased fluoride-induced ROS generation and attenuated JNK and c-Jun phosphorylation. NAC decreased SIRT1 phosphorylation and formation of the autophagy marker LC3II, which resulted in an increase in the apoptosis mediators γH2AX and cleaved/activated caspase-3. SP600125 attenuated fluoride-induced SIRT1 phosphorylation, indicating that fluoride activates SIRT1/autophagy via the ROS-mediated JNK pathway. In enamel organs from rats or mice treated with 50, 100, or 125 ppm fluoride for 6 weeks, cytochrome-c release and the DNA damage markers 8-oxoguanine, p-ATM, and γH2AX were increased compared to those in controls (0 ppm fluoride). These

  4. Leflunomide or A77 1726 protect from acetaminophen-induced cell injury through inhibition of JNK-mediated mitochondrial permeability transition in immortalized human hepatocytes

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

    Latchoumycandane, Calivarathan; Seah, Quee Ming; Tan, Rachel C.H.

    2006-11-15

    Leflunomide, a disease-modifying anti-rheumatic drug, protects against T-cell-mediated liver injury by poorly understood mechanisms. The active metabolite of leflunomide, A77 1726 (teriflunomide) has been shown to inhibit stress-activated protein kinases (JNK pathway), which are key regulators of mitochondria-mediated cell death. Therefore, we hypothesized that leflunomide may protect from drugs that induce the mitochondrial permeability transition (mPT) by blocking the JNK signaling pathway. To this end, we exposed cultured immortalized human hepatocytes (HC-04) to the standard protoxicant drug acetaminophen (APAP), which induces CsA-sensitive mPT-mediated cell death. We determined the effects of leflunomide on the extent of APAP-induced hepatocyte injury and themore » upstream JNK-mediated mitochondrial signaling pathways. We found that leflunomide or A77 1726 concentration-dependently protected hepatocytes from APAP (1 mM)-induced mitochondrial permeabilization and lethal cell injury. This was not due to proximal inhibition of CYP-catalyzed APAP bioactivation to its thiol-reactive metabolite. Instead, we demonstrate that leflunomide (20 {mu}M) inhibited the APAP-induced early (3 h) activation (phosphorylation) of JNK1/2, thus inhibiting phosphorylation of the anti-apoptotic protein Bcl-2 and preventing P-Bcl-2-mediated induction of the mPT. This greatly attenuated mitochondrial cytochrome c release, which we used as a marker for mitochondrial permeabilization. The specific JNK2 inhibitor SP600125 similarly protected from APAP-induced cell death. In conclusion, these findings are consistent with our hypothesis that leflunomide protects from protoxicant-induced hepatocyte injury by inhibiting JNK signaling and preventing mPT induction.« less

  5. Interleukin-22 promotes aerobic glycolysis associated with tumor progression via targeting hexokinase-2 in human colon cancer cells.

    PubMed

    Liu, Yulin; Xiang, Fan; Huang, Yongming; Shi, Liang; Hu, Chaojie; Yang, Yiming; Wang, Di; He, Nan; Tao, Kaixiong; Wu, Ke; Wang, Guobin

    2017-04-11

    Interleukin-22 has been explored extensively in human cancer, but its functions and underlying mechanisms are incompletely understood. Here, we show that aberrant interleukin-22 expression facilitates aerobic glycolysis in colon cancer cells. Elevated interleukin-22 mRNA expression was observed and positively correlated with hexokinase-2 in colon cancer tissues. In vitro, interleukin-22 enhanced glucose consumption and lactate production via targeting hexokinase-2 in colon cancer cells. Moreover, the transcriptional factor c-Myc and signal transducer and activator of transcription 3 were involved in interleukin-22-induced up-regulation of hexokinase-2. We further demonstrated that hexokinase-2 partly accounted for interleukin-22-mediated cellular proliferation in DLD-1 cells. In vivo, our data demonstrated that interleukin-22 significantly promoted tumor growth along with elevated expression of c-Myc and hexokinase-2 in mice. In summary, our findings provide a new perspective on the pro-inflammatory cytokine interleukin-22 in promoting aerobic glycolysis associated with tumor progression in human colon cancer cells.

  6. In vitro effect of short peptides on expression of interleukin-2 gene in splenocytes.

    PubMed

    Kazakova, T B; Barabanova, S V; Khavinson, V Kh; Glushikhina, M S; Parkhomenko, E P; Malinin, V V; Korneva, E A

    2002-06-01

    Synthetic peptides Vilon (Lys-Glu), Epithalon (Ala-Glu-Asp-Gly), and Cortagen (Ala-Glu-Asp-Pro) in vitro activated interleukin-2 mRNA synthesis in splenocytes from CBA mice in the absence of specific inductors. The intensity of interleukin-2 mRNA synthesis in splenocytes depended on the type, concentration, and duration of treatment with the peptides. Vilon and Epithalon were most potent, while Cortagen produced a less pronounced effect on interleukin-2 mRNA synthesis.

  7. Sodium fluoride induces apoptosis in mouse embryonic stem cells through ROS-dependent and caspase- and JNK-mediated pathways

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

    Nguyen Ngoc, Tam Dan; Son, Young-Ok; Lim, Shin-Saeng

    2012-03-15

    Sodium fluoride (NaF) is used as a source of fluoride ions in diverse applications. Fluoride salt is an effective prophylactic for dental caries and is an essential element required for bone health. However, fluoride is known to cause cytotoxicity in a concentration-dependent manner. Further, no information is available on the effects of NaF on mouse embryonic stem cells (mESCs). We investigated the mode of cell death induced by NaF and the mechanisms involved. NaF treatment greater than 1 mM reduced viability and DNA synthesis in mESCs and induced cell cycle arrest in the G{sub 2}/M phase. The addition of NaFmore » induced cell death mainly by apoptosis rather than necrosis. Catalase (CAT) treatment significantly inhibited the NaF-mediated cell death and also suppressed the NaF-mediated increase in phospho-c-Jun N-terminal kinase (p-JNK) levels. Pre-treatment with SP600125 or z-VAD-fmk significantly attenuated the NaF-mediated reduction in cell viability. In contrast, intracellular free calcium chelator, but not of sodium or calcium ion channel blockers, facilitated NaF-induced toxicity in the cells. A JNK specific inhibitor (SP600125) prevented the NaF-induced increase in growth arrest and the DNA damage-inducible protein 45α. Further, NaF-mediated loss of mitochondrial membrane potential was apparently inhibited by pifithrin-α or CAT inhibitor. These findings suggest that NaF affects viability of mESCs in a concentration-dependent manner, where more than 1 mM NaF causes apoptosis through hydroxyl radical-dependent and caspase- and JNK-mediated pathways. -- Highlights: ► The mode of NaF-induced cell death and the mechanisms involved were examined. ► NaF induced mainly apoptotic death of mouse embryonic stem cells (mESCs). ► NaF induced mitochondrial-mediated and caspase-dependent apoptosis. ► JNK- and p53-mediated pathways are involved in NaF-mediated apoptosis in the cells. ► ROS are the up-stream effector in NaF-mediated activation of JNK and p53

  8. Protective Effects of Green Tea Polyphenol Against Renal Injury Through ROS-Mediated JNK-MAPK Pathway in Lead Exposed Rats.

    PubMed

    Wang, Haidong; Li, Deyuan; Hu, Zhongze; Zhao, Siming; Zheng, Zhejun; Li, Wei

    2016-06-30

    To investigate the potential therapeutic effects of polyphenols in treating Pb induced renal dysfunction and intoxication and to explore the detailed underlying mechanisms. Wistar rats were divided into four groups: control groups (CT), Pb exposure groups (Pb), Pb plus Polyphenols groups (Pb+PP) and Polyphenols groups (PP). Animals were kept for 60 days and sacrificed for tests of urea, serum blood urea nitrogen (BUN) and creatinine. Histological evaluations were then performed. In vitro studies were performed using primary kidney mesangial cells to reveal detailed mechanisms. Cell counting kit-8 (CCK-8) was used to evaluate cell viability. Pb induced cell apoptosis was measured by flow cytometry. Reactive oxygen species (ROS) generation and scavenging were tested by DCFH-DA. Expression level of tumor necrosis factor-α (TNF-α), interleukin-1-β (IL-1-β) and IL-6 were assayed by ELISA. Western blot and qPCR were used to measure the expression of ERK1/2, JNK1/2 and p38. Polyphenols have obvious protective effects on Pb induced renal dysfunction and intoxication both in vivo and in vitro. Polyphenols reduced Pb concentration and accumulation in kidney. Polyphenols also protected kidney mesangial cells from Pb induced apoptosis. Polyphenols scavenged Pb induced ROS generation and suppressed ROS-mediated ERK/JNK/p38 pathway. Downstream pro-inflammatory cytokines were inhibited in consistency. Polyphenol is protective in Pb induced renal intoxication and inflammatory responses. The underlying mechanisms lie on the antioxidant activity and ROS scavenging activity of polyphenols.

  9. Protective Effects of Astaxanthin on ConA-Induced Autoimmune Hepatitis by the JNK/p-JNK Pathway-Mediated Inhibition of Autophagy and Apoptosis

    PubMed Central

    Liu, Tong; Wang, Junshan; Dai, Weiqi; Wang, Fan; Zheng, Yuanyuan; Chen, Kan; Li, Sainan; Abudumijiti, Huerxidan; Zhou, Zheng; Wang, Jianrong; Lu, Wenxia; Zhu, Rong; Yang, Jing; Zhang, Huawei; Yin, Qin; Wang, Chengfen; Zhou, Yuqing; Lu, Jie; Zhou, Yingqun; Guo, Chuanyong

    2015-01-01

    Objective Astaxanthin, a potent antioxidant, exhibits a wide range of biological activities, including antioxidant, atherosclerosis and antitumor activities. However, its effect on concanavalin A (ConA)-induced autoimmune hepatitis remains unclear. The aim of this study was to investigate the protective effects of astaxanthin on ConA-induced hepatitis in mice, and to elucidate the mechanisms of regulation. Materials and Methods Autoimmune hepatitis was induced in in Balb/C mice using ConA (25 mg/kg), and astaxanthin was orally administered daily at two doses (20 mg/kg and 40 mg/kg) for 14 days before ConA injection. Levels of serum liver enzymes and the histopathology of inflammatory cytokines and other maker proteins were determined at three time points (2, 8 and 24 h). Primary hepatocytes were pretreated with astaxanthin (80 μM) in vitro 24 h before stimulation with TNF-α (10 ng/ml). The apoptosis rate and related protein expression were determined 24 h after the administration of TNF-α. Results Astaxanthin attenuated serum liver enzymes and pathological damage by reducing the release of inflammatory factors. It performed anti-apoptotic effects via the descending phosphorylation of Bcl-2 through the down-regulation of the JNK/p-JNK pathway. Conclusion This research firstly expounded that astaxanthin reduced immune liver injury in ConA-induced autoimmune hepatitis. The mode of action appears to be downregulation of JNK/p-JNK-mediated apoptosis and autophagy. PMID:25761053

  10. The Anti-Apoptotic and Cardioprotective Effects of Salvianolic Acid A on Rat Cardiomyocytes following Ischemia/Reperfusion by DUSP-Mediated Regulation of the ERK1/2/JNK Pathway

    PubMed Central

    Chen, Qiuping; Zhu, Shasha; Liu, Yang; Pan, Defeng; Chen, Xiaohu; Li, Dongye

    2014-01-01

    /R group. SAA exerts an anti-apoptotic role against myocardial IRI by inhibiting DUSP2-mediated JNK dephosphorylation and activating DUSP4/16-mediated ERK1/2 phosphorylation. PMID:25019380

  11. Syk-mediated tyrosine phosphorylation of mule promotes TNF-induced JNK activation and cell death.

    PubMed

    Lee, C K; Yang, Y; Chen, C; Liu, J

    2016-04-14

    The transcription factor Miz1 negatively regulates TNF-induced JNK activation and cell death by suppressing TRAF2 K63-polyubiquitination; upon TNF stimulation, the suppression is relieved by Mule/ARF-BP1-mediated Miz1 ubiquitination and subsequent degradation. It is not known how Mule is activated by TNF. Here we report that TNF activates Mule by inducing the dissociation of Mule from its inhibitor ARF. ARF binds to and thereby inhibits the E3 ligase activity of Mule in the steady state. TNF induces tyrosine phosphorylation of Mule, which subsequently dissociates from ARF and becomes activated. Inhibition of Mule phosphorylation by silencing of the Spleen Tyrosine Kinase (Syk) prevents its dissociation from ARF, thereby inhibiting Mule E3 ligase activity and TNF-induced JNK activation and cell death. Our data provides a missing link in TNF signaling pathway that leads to JNK activation and cell death.

  12. The fruit juice of Morinda citrifolia (noni) downregulates HIF-1α protein expression through inhibition of PKB, ERK-1/2, JNK-1 and S6 in manganese-stimulated A549 human lung cancer cells.

    PubMed

    Jang, Byeong-Churl

    2012-03-01

    High exposure of manganese is suggested to be a risk factor for many lung diseases. Evidence suggests anticancerous and antiangiogenic effects by products derived from Morinda citrifolia (noni) fruit. In this study, we investigated the effect of noni fruit juice (NFJ) on the expression of HIF-1α, a tumor angiogenic transcription factor in manganese-chloride (manganese)-stimulated A549 human lung carcinoma cells. Treatment with manganese largely induced expression of HIF-1α protein but did not affect HIF-1α mRNA expression in A549 cells, suggesting the metal-mediated co- and/or post-translational HIF-1α upregulation. Manganese treatment also led to increased phosphorylation of extracellular-regulated protein kinase-1/2 (ERK-1/2), c-Jun N-terminal kinase-1 (JNK-1), protein kinase B (PKB), S6 and eukaryotic translation initiation factor-2α (eIF-2α) in A549 cells. Of note, the exposure of NFJ inhibited the manganese-induced HIF-1α protein upregulation in a concentration-dependent manner. Importantly, as assessed by results of pharmacological inhibition and siRNA transfection studies, the effect of NFJ on HIF-1α protein downregulation seemed to be largely associated with the ability of NFJ to interfere with the metal's signaling to activate PKB, ERK-1/2, JNK-1 and S6 in A549 cells. It was further shown that NFJ could repress the induction of HIF-1α protein by desferoxamine or interleukin-1β (IL-1β), another HIF-1α inducer in A549 cells. Thus, the present study provides the first evidence that NFJ has the ability to strongly downregulate manganese-induced HIF-1α protein expression in A549 human lung cancer cells, which may suggest the NFJ-mediated beneficial effects on lung pathologies in which manganese and HIF-1α overexpression play pathogenic roles.

  13. c-Jun N-terminal kinase 3 (JNK3) Mediates Paraquat- and Rotenone-Induced Dopaminergic Neuron Death

    PubMed Central

    Choi, Won Seok; Abel, Glen; Klintworth, Heather; Flavell, Richard A.; Xia, Zhengui

    2011-01-01

    Mechanistic studies underlying dopaminergic neuron death may identify new drug targets for the treatment of Parkinson disease (PD). Epidemiological studies have linked pesticide exposure to increased risk for sporadic PD. Here, we investigated the role of c-Jun N-terminal kinase 3 (JNK3), a neural-specific JNK isoform, in dopaminergic neuron death induced by the pesticides rotenone and paraquat. The role of JNK3 was evaluated using RNA silencing and gene deletion to block JNK3 signaling. Using an antibody that recognizes all isoforms of activated JNKs, we found that paraquat and rotenone stimulate JNK phosphorylation in primary cultured dopaminergic neurons. In cultured neurons transfected with Jnk3-specific siRNA and in neurons from Jnk3−/− mice, JNK phosphorylation was nearly abolished, suggesting that JNK3 is the main JNK isoform activated in dopaminergic neurons by these pesticides. Paraquat- and rotenone-induced death of dopaminergic neurons was also significantly reduced by Jnk3 siRNA or Jnk3 gene deletion and deletion of the Jnk3 gene completely attenuated paraquat-induced dopaminergic neuron death and motor-deficits in vivo. Our data identify JNK3 as a common and critical mediator of dopaminergic neuron death induced by paraquat and rotenone, suggesting that it is a potential drug target for PD treatment. PMID:20418776

  14. JNK-Interacting Protein 3 Mediates the Retrograde Transport of Activated c-Jun N-Terminal Kinase and Lysosomes

    PubMed Central

    Drerup, Catherine M.; Nechiporuk, Alex V.

    2013-01-01

    Retrograde axonal transport requires an intricate interaction between the dynein motor and its cargo. What mediates this interaction is largely unknown. Using forward genetics and a novel in vivo imaging approach, we identified JNK-interacting protein 3 (Jip3) as a direct mediator of dynein-based retrograde transport of activated (phosphorylated) c-Jun N-terminal Kinase (JNK) and lysosomes. Zebrafish jip3 mutants (jip3nl7) displayed large axon terminal swellings that contained high levels of activated JNK and lysosomes, but not other retrograde cargos such as late endosomes and autophagosomes. Using in vivo analysis of axonal transport, we demonstrated that the terminal accumulations of activated JNK and lysosomes were due to a decreased frequency of retrograde movement of these cargos in jip3nl7, whereas anterograde transport was largely unaffected. Through rescue experiments with Jip3 engineered to lack the JNK binding domain and exogenous expression of constitutively active JNK, we further showed that loss of Jip3–JNK interaction underlies deficits in pJNK retrograde transport, which subsequently caused axon terminal swellings but not lysosome accumulation. Lysosome accumulation, rather, resulted from loss of lysosome association with dynein light intermediate chain (dynein accessory protein) in jip3nl7, as demonstrated by our co-transport analyses. Thus, our results demonstrate that Jip3 is necessary for the retrograde transport of two distinct cargos, active JNK and lysosomes. Furthermore, our data provide strong evidence that Jip3 in fact serves as an adapter protein linking these cargos to dynein. PMID:23468645

  15. Ciclopirox induces autophagy through reactive oxygen species-mediated activation of JNK signaling pathway

    PubMed Central

    Zhou, Hongyu; Shen, Tao; Shang, Chaowei; Luo, Yan; Liu, Lei; Yan, Juming; Li, Yan; Huang, Shile

    2014-01-01

    Ciclopirox olamine (CPX), a fungicide, has been demonstrated as a potential anticancer agent. However, the underlying anticancer mechanism is not well understood. Here, we found that CPX induced autophagy in human rhabdomyosarcoma (Rh30 and RD) cells. It appeared that CPX-induced autophagy was attributed to induction of reactive oxygen species (ROS), as N-acetyl-L-cysteine (NAC), a ROS scavenger and antioxidant, prevented this process. Furthermore, we observed that CPX induced activation of mitogen-activated protein kinases (MAPKs), including extracellular signal-regulated kinase 1/2 (ERK1/2), c-Jun N-terminal kinase (JNK) and p38 MAPK, which was also blocked by NAC. However, only inhibition of JNK (with SP600125) or expression of dominant negative c-Jun partially prevented CPX-induced autophagy, indicating that ROS-mediated activation of JNK signaling pathway contributed to CPX-induced autophagy. Of interest, inhibition of autophagy by chloroquine (CQ) enhanced CPX-induced cell death, indicating that CPX-induced autophagy plays a pro-survival role in human rhabdomyosarcoma cells. Our finding suggests that the combination with autophagy inhibitors may be a novel strategy in potentiating the anticancer activity of CPX for treatment of rhabdomyosarcoma. PMID:25294812

  16. In vitro and in vivo expression of interleukin-1alpha and tumor necrosis factor-alpha mRNA in pemphigus vulgaris: interleukin-1alpha and tumor necrosis factor-alpha are involved in acantholysis.

    PubMed

    Feliciani, C; Toto, P; Amerio, P; Pour, S M; Coscione, G; Shivji, G; Wang, B; Sauder, D N

    2000-01-01

    Keratinocyte-derived cytokines have been implicated in the pathogenesis of a number of skin diseases. In this study we examined the possible role of keratinocyte-derived cytokines in the development of acantholysis in pemphigus vulgaris. Nineteen patients with pemphigus vulgaris, demonstrating the characteristic clinical, pathologic, and immunopathologic findings were studied. In situ immunolabeling demonstrated the presence of two cytokines interleukin-1alpha and tumor necrosis factor-alpha, in lesional and perilesional areas. Results were confirmed by reverse transcriptase-polymerase chain reaction, demonstrating overexpression of both cytokines in vivo. To study the role of these cytokines in the pathogenesis of pemphigus vulgaris both in vitro and in vivo studies were performed. The results of the in vitro study demonstrated that pemphigus vulgaris IgG induced interleukin-1alpha and tumor necrosis factor-alpha mRNA in the skin. The potential pathogenic role of these mediators was demonstrated by a blocking study using antibodies against human interleukin-1alpha and tumor necrosis factor-alpha in keratinocytes cultures. A combination of anti-interleukin-1alpha and anti-tumor necrosis factor-alpha antibodies inhibited in vitro pemphigus vulgaris IgG induced acantholysis. To confirm the role of interleukin-1 and tumor necrosis factor-alpha in pemphigus, we utilized passive transfer studies using interleukin-1 deficient mice (ICE-/-, interleukin-1beta-/-) and tumor necrosis factor-alpha receptor deficient mice (TNFR1R2-/-). Both groups demonstrated a decreased susceptibility to the passive transfer of pemphigus. Our data support the role of cytokines interleukin-1 and tumor necrosis factor-alpha in the pathogenesis of pemphigus vulgaris.

  17. Ultra Fine Particles from Diesel Engines Induce Vascular Oxidative Stress via JNK Activation

    PubMed Central

    Li, Rongsong; Ning, Zhi; Cui, Jeffery; Khalsa, Bhavraj; Ai, Lisong; Takabe, Wakako; Beebe, Tyler; Majumdar, Rohit; Sioutas, Constantinos; Hsiai, Tzung

    2011-01-01

    Exposure of particulate air pollution is linked to increased incidences of cardiovascular diseases. Ambient ultra fine particles (UFP) from diesel vehicle engines have been shown to be pro-atherogenic in apoE knockout mice and may constitute a major cardiovascular risk in humans. We posited that circulating nano-sized particles from traffic pollution sources induced vascular oxidative stress via JNK activation in endothelial cells. Diesel UFP were collected from a 1998 Kenworth truck. Intra-cellular superoxide assay revealed that these UFP dose-dependently induced superoxide (O2·-) production in human aortic endothelial cells (HAEC). Flow cytometry (FACS) showed that UFP increased MitoSOX Red intensity specific for mitochondrial superoxide. Protein carbonyl content is increased by UFP as an indication of vascular oxidative stress. UFP also up-regulated hemeoxygenase-1 (HO-1) and tissue factor (TF) mRNA expression, and pre-treatment with antioxidant, N-acetyl cysteine (NAC), significantly decreased their expression. Furthermore, UFP transiently activated JNK in HAEC. Treatment with JNK inhibitor SP600125 and silencing of both JNK1 and JNK2 with siRNA inhibited UFP stimulated O2·- production and mRNA expression of HO-1 and TF. Our findings suggest that JNK activation play an important role in UFP-induced oxidative stress and stress response gene expression. PMID:19154785

  18. Unfolded protein response transducer IRE1-mediated signaling independent of XBP1 mRNA splicing is not required for growth and development of medaka fish

    PubMed Central

    Ishikawa, Tokiro; Kashima, Makoto; Nagano, Atsushi J; Ishikawa-Fujiwara, Tomoko; Kamei, Yasuhiro; Todo, Takeshi

    2017-01-01

    When activated by the accumulation of unfolded proteins in the endoplasmic reticulum, metazoan IRE1, the most evolutionarily conserved unfolded protein response (UPR) transducer, initiates unconventional splicing of XBP1 mRNA. Unspliced and spliced mRNA are translated to produce pXBP1(U) and pXBP1(S), respectively. pXBP1(S) functions as a potent transcription factor, whereas pXBP1(U) targets pXBP1(S) to degradation. In addition, activated IRE1 transmits two signaling outputs independent of XBP1, namely activation of the JNK pathway, which is initiated by binding of the adaptor TRAF2 to phosphorylated IRE1, and regulated IRE1-dependent decay (RIDD) of various mRNAs in a relatively nonspecific manner. Here, we conducted comprehensive and systematic genetic analyses of the IRE1-XBP1 branch of the UPR using medaka fish and found that the defects observed in XBP1-knockout or IRE1-knockout medaka were fully rescued by constitutive expression of pXBP1(S). Thus, the JNK and RIDD pathways are not required for the normal growth and development of medaka. The unfolded protein response sensor/transducer IRE1-mediated splicing of XBP1 mRNA encoding its active downstream transcription factor to maintain the homeostasis of the endoplasmic reticulum is sufficient for growth and development of medaka fish. PMID:28952924

  19. Cypermethrin Induces Macrophages Death through Cell Cycle Arrest and Oxidative Stress-Mediated JNK/ERK Signaling Regulated Apoptosis

    PubMed Central

    Huang, Fang; Liu, Qiaoyun; Xie, Shujun; Xu, Jian; Huang, Bo; Wu, Yihua; Xia, Dajing

    2016-01-01

    Cypermethrin is one of the most highly effective synthetic pyrethroid insecticides. The toxicity of cypermethrin to the reproductive and nervous systems has been well studied. However, little is known about the toxic effect of cypermethrin on immune cells such as macrophages. Here, we investigated the cytotoxicity of cypermethrin on macrophages and the underlying molecular mechanisms. We found that cypermethrin reduced cell viability and induced apoptosis in RAW 264.7 cells. Cypermethrin also increased reactive oxygen species (ROS) production and DNA damage in a dose-dependent manner. Moreover, cypermethrin-induced G1 cell cycle arrest was associated with an enhanced expression of p21, wild-type p53, and down-regulation of cyclin D1, cyclin E and CDK4. In addition, cypermethrin treatment activated MAPK signal pathways by inducing c-Jun N-terminal kinase (JNK) and extracellular regulated protein kinases 1/2 ERK1/2 phosphorylation, and increased the cleaved poly ADP-ribose polymerase (PARP). Further, pretreatment with antioxidant N-acetylcysteine (NAC) effectively abrogated cypermethrin-induced cell cytotoxicity, G1 cell cycle arrest, DNA damage, PARP activity, and JNK and ERK1/2 activation. The specific JNK inhibitor (SP600125) and ERK1/2 inhibitor (PD98059) effectively reversed the phosphorylation level of JNK and ERK1/2, and attenuated the apoptosis. Taken together, these data suggested that cypermethrin caused immune cell death via inducing cell cycle arrest and apoptosis regulated by ROS-mediated JNK/ERK pathway. PMID:27322250

  20. Asthmatic airway smooth muscle CXCL10 production: mitogen-activated protein kinase JNK involvement

    PubMed Central

    Alrashdan, Yazan A.; Alkhouri, Hatem; Chen, Emily; Lalor, Daniel J.; Poniris, Maree; Henness, Sheridan; Brightling, Christopher E.; Burgess, Janette K.; Armour, Carol L.; Ammit, Alaina J.

    2012-01-01

    CXCL10 (IP10) is involved in mast cell migration to airway smooth muscle (ASM) bundles in asthma. We aimed to investigate the role of cytokine-induced MAPK activation in CXCL10 production by ASM cells from people with and without asthma. Confluent growth-arrested ASM cells were treated with inhibitors of the MAPKs ERK, p38, and JNK and transcription factor NF-κB, or vehicle, and stimulated with IL-1β, TNF-α, or IFN-γ, alone or combined (cytomix). CXCL10 mRNA and protein, JNK, NF-κB p65 phosphorylation, and Iκ-Bα protein degradation were assessed using real-time PCR, ELISA, and immunoblotting, respectively. Cytomix, IL-1β, and TNF-α induced CXCL10 mRNA expression more rapidly in asthmatic than nonasthmatic ASM cells. IL-1β and/or TNF-α combined with IFN-γ synergistically increased asthmatic ASM cell CXCL10 release. Inhibitor effects were similar in asthmatic and nonasthmatic cells, but cytomix-induced release was least affected, with only JNK and NF-κB inhibitors halving it. Notably, JNK phosphorylation was markedly less in asthmatic compared with nonasthmatic cells. However, in both, the JNK inhibitor SP600125 reduced JNK phosphorylation and CXCL10 mRNA levels but did not affect CXCL10 mRNA stability or Iκ-Bα degradation. Together, the JNK and NF-κB inhibitors completely inhibited their CXCL10 release. We concluded that, in asthmatic compared with nonasthmatic ASM cells, JNK activation was reduced and CXCL10 gene expression was more rapid following cytomix stimulation. However, in both, JNK activation did not regulate early events leading to NF-κB activation. Thus JNK and NF-κB provide independent therapeutic targets for limiting CXCL10 production and mast cell migration to the ASM in asthma. PMID:22387292

  1. Asthmatic airway smooth muscle CXCL10 production: mitogen-activated protein kinase JNK involvement.

    PubMed

    Alrashdan, Yazan A; Alkhouri, Hatem; Chen, Emily; Lalor, Daniel J; Poniris, Maree; Henness, Sheridan; Brightling, Christopher E; Burgess, Janette K; Armour, Carol L; Ammit, Alaina J; Hughes, J Margaret

    2012-05-15

    CXCL10 (IP10) is involved in mast cell migration to airway smooth muscle (ASM) bundles in asthma. We aimed to investigate the role of cytokine-induced MAPK activation in CXCL10 production by ASM cells from people with and without asthma. Confluent growth-arrested ASM cells were treated with inhibitors of the MAPKs ERK, p38, and JNK and transcription factor NF-κB, or vehicle, and stimulated with IL-1β, TNF-α, or IFN-γ, alone or combined (cytomix). CXCL10 mRNA and protein, JNK, NF-κB p65 phosphorylation, and Iκ-Bα protein degradation were assessed using real-time PCR, ELISA, and immunoblotting, respectively. Cytomix, IL-1β, and TNF-α induced CXCL10 mRNA expression more rapidly in asthmatic than nonasthmatic ASM cells. IL-1β and/or TNF-α combined with IFN-γ synergistically increased asthmatic ASM cell CXCL10 release. Inhibitor effects were similar in asthmatic and nonasthmatic cells, but cytomix-induced release was least affected, with only JNK and NF-κB inhibitors halving it. Notably, JNK phosphorylation was markedly less in asthmatic compared with nonasthmatic cells. However, in both, the JNK inhibitor SP600125 reduced JNK phosphorylation and CXCL10 mRNA levels but did not affect CXCL10 mRNA stability or Iκ-Bα degradation. Together, the JNK and NF-κB inhibitors completely inhibited their CXCL10 release. We concluded that, in asthmatic compared with nonasthmatic ASM cells, JNK activation was reduced and CXCL10 gene expression was more rapid following cytomix stimulation. However, in both, JNK activation did not regulate early events leading to NF-κB activation. Thus JNK and NF-κB provide independent therapeutic targets for limiting CXCL10 production and mast cell migration to the ASM in asthma.

  2. Selenite exacerbates hepatic insulin resistance in mouse model of type 2 diabetes through oxidative stress-mediated JNK pathway

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

    Zhou, Jun, E-mail: hustzhj@hust.edu.cn; Xu, Gang; Bai, Zhaoshuai

    Recent evidence suggests a potential pro-diabetic effect of selenite treatment in type 2 diabetics; however, the underlying mechanisms remain elusive. Here we investigated the effects and the underlying mechanisms of selenite treatment in a nongenetic mouse model of type 2 diabetes. High-fat diet (HFD)/streptozotocin (STZ)-induced diabetic mice were orally gavaged with selenite at 0.5 or 2.0 mg/kg body weight/day or vehicle for 4 weeks. High-dose selenite treatment significantly elevated fasting plasma insulin levels and insulin resistance index, in parallel with impaired glucose tolerance, insulin tolerance and pyruvate tolerance. High-dose selenite treatment also attenuated hepatic IRS1/Akt/FoxO1 signaling and pyruvate kinase genemore » expressions, but elevated the gene expressions of phosphoenolpyruvate carboxyl kinase (PEPCK), glucose 6-phosphatase (G6Pase), peroxisomal proliferator-activated receptor-γ coactivator 1α (PGC-1α) and selenoprotein P (SelP) in the liver. Furthermore, high-dose selenite treatment caused significant increases in MDA contents, protein carbonyl contents, and a decrease in GSH/GSSG ratio in the liver, concurrent with enhanced ASK1/MKK4/JNK signaling. Taken together, these findings suggest that high-dose selenite treatment exacerbates hepatic insulin resistance in mouse model of type 2 diabetes, at least in part through oxidative stress-mediated JNK pathway, providing new mechanistic insights into the pro-diabetic effect of selenite in type 2 diabetes. - Highlights: • Selenite exacerbates hepatic insulin resistance in HFD/STZ-induced diabetic mice. • Selenite elevates hepatic gluconeogenesis and reduces glycolysis in diabetic mice. • Selenite exacerbates hepatic oxidative stress and triggers JNK signaling pathway. • Selenite elevates hepatic selenoprotein P expression in diabetic mice.« less

  3. Granulocyte-macrophage colony-stimulating factor primes interleukin-13 production by macrophages via protease-activated receptor-2.

    PubMed

    Aoki, Manabu; Yamaguchi, Rui; Yamamoto, Takatoshi; Ishimaru, Yasuji; Ono, Tomomichi; Sakamoto, Arisa; Narahara, Shinji; Sugiuchi, Hiroyuki; Hirose, Eiji; Yamaguchi, Yasuo

    2015-04-01

    Chronic inflammation is often linked to the presence of type 2-polarized macrophages, which are induced by the T helper type 2 cytokines interleukin-4 and interleukin-13 (IL-13). IL-13 is a key mediator of tissue fibrosis caused by T helper type 2-based inflammation. Human neutrophil elastase (HNE) plays a pivotal role in the pathogenesis of pulmonary fibrosis. This study investigated the priming effect of granulocyte-macrophage colony-stimulating factor (GM-CSF) on IL-13 expression by macrophages stimulated with HNE. Adherent macrophages were obtained from primary cultures of human mononuclear cells. Expression of IL-13 mRNA and protein by GM-CSF-dependent macrophages was investigated after stimulation with HNE, using the polymerase chain reaction and enzyme-linked immunosorbent assay. GM-CSF had a priming effect on IL-13 mRNA and protein expression by macrophages stimulated with HNE, while this effect was not observed for various other cytokines. GM-CSF-dependent macrophages showed a significant increase in the expression of protease activated receptor-2 (PAR-2) mRNA and protein. The response of IL-13 mRNA to HNE was significantly decreased by pretreatment with alpha1-antitrypsin, a PAR-2 antibody (SAM11), or a PAR-2 antagonist (ENMD-1068). These findings suggest that stimulation with HNE can induce IL-13 production by macrophages, especially GM-CSF-dependent macrophages. Accordingly, neutrophil elastase may have a key role in fibrosis associated with chronic inflammation. Copyright © 2015 Elsevier Inc. All rights reserved.

  4. PPARδ inhibits UVB-induced secretion of MMP-1 through MKP-7-mediated suppression of JNK signaling.

    PubMed

    Ham, Sun A; Kang, Eun S; Lee, Hanna; Hwang, Jung S; Yoo, Taesik; Paek, Kyung S; Park, Chankyu; Kim, Jin-Hoi; Lim, Dae-Seog; Seo, Han G

    2013-11-01

    In the present study, we investigated the role of peroxisome proliferator-activated receptor (PPAR) δ in modulating matrix-degrading metalloproteinases and other mechanisms underlying photoaging processes in the skin. In human dermal fibroblasts (HDFs), activation of PPARδ by its specific ligand GW501516 markedly attenuated UVB-induced secretion of matrix metalloproteinase (MMP)-1, concomitant with decreased generation of reactive oxygen species. These effects were significantly reduced in the presence of PPARδ small interfering RNA and GSK0660. Furthermore, c-Jun N-terminal kinase (JNK), but not p38 or extracellular signal-regulated kinase, mediated PPARδ-dependent inhibition of MMP-1 secretion in HDFs exposed to UVB. PPARδ-mediated messenger RNA stabilization of mitogen-activated protein kinase phosphatase (MKP)-7 was responsible for the GW501516-mediated inhibition of JNK signaling. Inhibition of UVB-induced secretion of MMP-1 by PPARδ was associated with the restoration of types I and III collagen to levels approaching those in cells not exposed to UVB. Finally, in HR-1 hairless mice exposed to UVB, administration of GW501516 significantly reduced wrinkle formation and skin thickness, downregulated MMP-1 and JNK phosphorylation, and restored the levels of MKP-7, types I and III collagen. These results suggest that PPARδ-mediated inhibition of MMP-1 secretion prevents some effects of photoaging and maintains the integrity of skin by inhibiting the degradation of the collagenous extracellular matrix.

  5. Resveratrol Ameliorates Palmitate-Induced Inflammation in Skeletal Muscle Cells by Attenuating Oxidative Stress and JNK/NF-κB Pathway in a SIRT1-Independent Mechanism.

    PubMed

    Sadeghi, Asie; Seyyed Ebrahimi, Shadi Sadat; Golestani, Abolfazl; Meshkani, Reza

    2017-09-01

    Resveratrol has been shown to exert anti-inflammatory and anti-oxidant effects in a variety of cell types, however, its role in prevention of inflammatory responses mediated by palmitate in skeletal muscle cells remains unexplored. In the present study, we investigated the effects of resveratrol on palmitate-induced inflammation and elucidated the underlying mechanisms in skeletal muscle cells. The results showed that palmitate significantly enhanced TNF-α and IL-6 mRNA expression and protein secretion from C2C12 cells at 12, 24, and 36 h treatments. Increased expression of cytokines was accompanied by an enhanced phosphorylation of JNK, P38, ERK1/2, and IKKα/IKKβ. In addition, JNK and P38 inhibitors could significantly attenuate palmitate-induced mRNA expression of TNF-α and IL-6, respectively, whereas NF-κB inhibitor reduced the expression of both cytokines in palmitate-treated cells. Resveratrol pretreatment significantly prevented palmitate-induced TNF-α and IL-6 mRNA expression and protein secretion in C2C12 cells. Importantly, pre-treatment of the cells with resveratrol completely abrogated the phosphorylation of ERK1/2, JNK, and IKKα/IKKβ in palmitate treated cells. The protection from palmitate-induced inflammation by resveratrol was accompanied by a decrease in the generation of reactive oxygen species (ROS). N-acetyl cysteine (NAC), a known scavenger of ROS, could protect palmitate-induced expression of TNF-α and IL-6. Furthermore, inhibition of SIRT1 by shRNA or sirtinol demonstrated that the anti-inflammatory effect of resveratrol in muscle cells is mediated through a SIRT1-independent mechanism. Taken together, these findings suggest that resveratrol may represent a promising therapy for prevention of inflammation in skeletal muscle cells. J. Cell. Biochem. 118: 2654-2663, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  6. MST1 activation by curcumin mediates JNK activation, Foxo3a nuclear translocation and apoptosis in melanoma cells

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

    Yu, Teng, E-mail: tengyu33@yahoo.com; Ji, Jiang; Guo, Yong-li

    2013-11-08

    Highlights: •Curcumin activates MST1 in melanoma cells. •MST1 mediates curcumin-induced apoptosis of melanoma cells. •ROS production is involved in curcumin-induced MST1 activation. •MST1 mediates curcumin-induced JNK activation in melanoma cells. •MST1 mediates curcumin-induced Foxo3a nuclear translocation and Bim expression. -- Abstract: Different groups including ours have shown that curcumin induces melanoma cell apoptosis, here we focused the role of mammalian Sterile 20-like kinase 1 (MST1) in it. We observed that curcumin activated MST1-dependent apoptosis in cultured melanoma cells. MST1 silencing by RNA interference (RNAi) suppressed curcumin-induced cell apoptosis, while MST1 over-expressing increased curcumin sensitivity. Meanwhile, curcumin induced reactive oxygen speciesmore » (ROS) production in melanoma cells, and the ROS scavenger, N-acetyl-cysteine (NAC), almost blocked MST1 activation to suggest that ROS might be required for MST1 activation by curcumin. c-Jun N-terminal protein kinase (JNK) activation by curcumin was dependent on MST1, since MST1 inhibition by RNAi or NAC largely inhibited curcumin-induced JNK activation. Further, curcumin induced Foxo3 nuclear translocation and Bim-1 (Foxo3 target gene) expression in melanoma cells, such an effect by curcumin was inhibited by MST1 RNAi. In conclusion, we suggested that MST1 activation by curcumin mediates JNK activation, Foxo3a nuclear translocation and apoptosis in melanoma cells.« less

  7. Reactive oxygen species mediate nitric oxide production through ERK/JNK MAPK signaling in HAPI microglia after PFOS exposure

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

    Wang, Cheng; Nie, Xiaoke; Zhang, Yan

    2015-10-15

    Perfluorooctane sulfonate (PFOS), an emerging persistent contaminant that is commonly encountered during daily life, has been shown to exert toxic effects on the central nervous system (CNS). However, the molecular mechanisms underlying the neurotoxicity of PFOS remain largely unknown. It has been widely acknowledged that the inflammatory mediators released by hyper-activated microglia play vital roles in the pathogenesis of various neurological diseases. In the present study, we examined the impact of PFOS exposure on microglial activation and the release of proinflammatory mediators, including nitric oxide (NO) and reactive oxidative species (ROS). We found that PFOS exposure led to concentration-dependent NOmore » and ROS production by rat HAPI microglia. We also discovered that there was rapid activation of the ERK/JNK MAPK signaling pathway in the HAPI microglia following PFOS treatment. Moreover, the PFOS-induced iNOS expression and NO production were attenuated after the inhibition of ERK or JNK MAPK by their corresponding inhibitors, PD98059 and SP600125. Interestingly, NAC, a ROS inhibitor, blocked iNOS expression, NO production, and activation of ERK and JNK MAPKs, which suggested that PFOS-mediated microglial NO production occurs via a ROS/ERK/JNK MAPK signaling pathway. Finally, by exposing SH-SY5Y cells to PFOS-treated microglia-conditioned medium, we demonstrated that NO was responsible for PFOS-mediated neuronal apoptosis. - Highlights: • PFOS exposure induced expression of iNOS and production of NO in HAPI microglia. • PFOS induced the production of ROS in HAPI microglia. • ERK/JNK MAPK pathways were activated following PFOS exposure in HAPI microglia. • NO released by HAPI microglia participated in the apoptosis of SH-SY5Y cells.« less

  8. Autophagy blockade sensitizes the anticancer activity of CA-4 via JNK-Bcl-2 pathway

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

    Li, Yangling; Luo, Peihua; Wang, Jincheng

    Combretastatin A-4 (CA-4) has already entered clinical trials of solid tumors over ten years. However, the limited anticancer activity and dose-dependent toxicity restrict its clinical application. Here, we offered convincing evidence that CA-4 induced autophagy in various cancer cells, which was demonstrated by acridine orange staining of intracellular acidic vesicles, the degradation of p62, the conversion of LC3-I to LC3-II and GFP-LC3 punctate fluorescence. Interestingly, CA-4-mediated apoptotic cell death was further potentiated by pretreatment with autophagy inhibitors (3-methyladenine and bafilomycin A1) or small interfering RNAs against the autophagic genes (Atg5 and Beclin 1). The enhanced anticancer activity of CA-4 andmore » 3-MA was further confirmed in the SGC-7901 xenograft tumor model. These findings suggested that CA-4-elicited autophagic response played a protective role that impeded the eventual cell death while autophagy inhibition was expected to improve chemotherapeutic efficacy of CA-4. Meanwhile, CA-4 treatment led to phosphorylation/activation of JNK and JNK-dependent phosphorylation of Bcl-2. Importantly, JNK inhibitor or JNK siRNA inhibited autophagy but promoted CA-4-induced apoptosis, indicating a key requirement of JNK-Bcl-2 pathway in the activation of autophagy by CA-4. We also identified that pretreatment of Bcl-2 inhibitor (ABT-737) could significantly enhance anticancer activity of CA-4 due to inhibition of autophagy. Taken together, our data suggested that the JNK-Bcl-2 pathway was considered as the critical regulator of CA-4-induced protective autophagy and a potential drug target for chemotherapeutic combination. - Highlights: • Autophagy inhibition could be a potential for combretastatin A-4 antitumor efficacy. • The JNK-Bcl-2 pathway plays a critical role in CA-4-induced autophagy. • ABT-737 enhances CA-4 anticancer activity due to inhibition of autophagy.« less

  9. Protective effect of Ginkgo biloba leaves extract, EGb761, on endotoxin-induced acute lung injury via a JNK- and Akt-dependent NFκB pathway.

    PubMed

    Lee, Chien-Ying; Yang, Jiann-Jou; Lee, Shiuan-Shinn; Chen, Chun-Jung; Huang, Yi-Chun; Huang, Kuang-Hua; Kuan, Yu-Hsiang

    2014-07-09

    Acute lung injury (ALI) is a clinical syndrome mainly caused by Gram-negative bacteria which is still in need of an effective therapeutic medicine. EGb761, an extract of Ginkgo biloba leaves, has several bioeffects including anti-inflammation, cardioprotection, neuroprotection, and free radical scavenging. Preadministration of EGb761 inhibited lipopolysaccharide (LPS)-induced histopathological changes and exchange of arterial blood gas. In addition, LPS-induced expression of proinflammatory mediators, such as tumor necrosis factor (TNF)-α, interleukin (IL)-6, macrophage inflammatory protein (MIP)-2, inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2), were suppressed by EGb761. The activation of nuclear factor (NF)κB, a transcription factor of proinflammatory mediators, and phosphorylation of IκB, an inhibitor of NFκB, were also reduced by EGb761. Furthermore, we found the inhibitory concentration of EGb761 on phosphorylation of JNK and Akt was less than those of ERK and p38 MAPK. In conclusion, EGb761 is a potential protective agent for ALI, possibly via downregulating the JNK- and Akt-dependent NFκB activation pathway.

  10. The increased mucosal mRNA expressions of complement C3 and interleukin-17 in inflammatory bowel disease

    PubMed Central

    Sugihara, T; Kobori, A; Imaeda, H; Tsujikawa, T; Amagase, K; Takeuchi, K; Fujiyama, Y; Andoh, A

    2010-01-01

    Recent studies have demonstrated that the complement system participates in the regulation of T cell functions. To address the local biosynthesis of complement components in inflammatory bowel disease (IBD) mucosa, we investigated C3 and interleukin (IL)-17 mRNA expression in mucosal samples obtained from patients with IBD. The molecular mechanisms underlying C3 induction were investigated in human colonic subepithelial myofibroblasts (SEMFs). IL-17 and C3 mRNA expressions in the IBD mucosa were evaluated by real-time polymerase chain reaction. The C3 levels in the supernatant were determined by enzyme-linked immunosorbent assay. IL-17 and C3 mRNA expressions were elevated significantly in the active lesions from ulcerative colitis (UC) and Crohn's disease (CD) patients. There was a significant positive correlation between IL-17 and C3 mRNA expression in the IBD mucosa. IL-17 stimulated a dose- and time-dependent increase in C3 mRNA expression and C3 secretion in colonic SEMFs. The C3 molecules secreted by colonic SEMFs were a 115-kDa α-chain linked to a 70-kDa β-chain by disulphide bonds, which was identical to serum C3. The IL-17-induced C3 mRNA expression was blocked by p42/44 mitogen-activated protein kinase (MAPK) inhibitors (PD98059 and U0216) and a p38 MAPK inhibitor (SB203580). Furthermore, IL-17-induced C3 mRNA expression was inhibited by an adenovirus containing a stable mutant form of IκBα. C3 and IL-17 mRNA expressions are enhanced, with a strong correlation, in the inflamed mucosa of IBD patients. Part of these clinical findings was considered to be mediated by the colonic SEMF response to IL-17. PMID:20089077

  11. Induction of c-fos expression through JNK-mediated TCF/Elk-1 phosphorylation.

    PubMed Central

    Cavigelli, M; Dolfi, F; Claret, F X; Karin, M

    1995-01-01

    Growth factors induce c-fos transcription by stimulating phosphorylation of transcription factor TCF/Elk-1, which binds to the serum response element (SRE). Under such conditions Elk-1 could be phosphorylated by the mitogen-activated protein kinases (MAPKs) ERK1 and ERK2. However, c-fos transcription and SRE activity are also induced by stimuli, such as UV irradiation and activation of the protein kinase MEKK1, that cause only an insignificant increase in ERK1/2 activity. However, both of these stimuli strongly activate two other MAPKs, JNK1 and JNK2, and stimulate Elk-1 transcriptional activity and phosphorylation. We find that the JNKs are the predominant Elk-1 activation domain kinases in extracts of UV-irradiated cells and that immunopurified JNK1/2 phosphorylate Elk-1 on the same major sites recognized by ERK1/2, that potentiate its transcriptional activity. Finally, we show that UV irradiation, but not serum or phorbol esters, stimulate translocation of JNK1 to the nucleus. As Elk-1 is most likely phosphorylated while bound to the c-fos promoter, these results suggest that UV irradiation and MEKK1 activation stimulate TCF/Elk-1 activity through JNK activation, while growth factors induce c-fos through ERK activation. Images PMID:8846788

  12. DISTINCT FUNCTIONS OF JNK AND C-JUN IN OXIDANT-INDUCED HEPATOCYTE DEATH

    PubMed Central

    Amir, Muhammad; Liu, Kun; Zhao, Enpeng; Czaja, Mark J.

    2013-01-01

    Overactivation of c-Jun N-terminal kinase (JNK)/c-Jun signaling is a central mechanism of hepatocyte injury and death including that from oxidative stress. However, the functions of JNK and c-Jun are still unclear, and this pathway also inhibits hepatocyte death. Previous studies of menadione-induced oxidant stress demonstrated that toxicity resulted from sustained JNK/c-Jun activation as death was blocked by the c-Jun dominant negative TAM67. To further delineate the function of JNK/c-Jun signaling in hepatocyte injury from oxidant stress, the effects of direct JNK inhibition on menadione-induced death were examined. In contrast to the inhibitory effect of TAM67, pharmacological JNK inhibition by SP600125 sensitized the rat hepatocyte cell line RALA255-10G to death from menadione. SP600125 similarly sensitized mouse primary hepatocytes to menadione toxicity. Death from SP600125/menadione was c-Jun dependent as it was blocked by TAM67, but independent of c-Jun phosphorylation. Death occurred by apoptosis and necrosis and activation of the mitochondrial death pathway. Short hairpin RNA knockdowns of total JNK or JNK2 sensitized to death from menadione, whereas a jnk1 knockdown was protective. Jnk2 null mouse primary hepatocytes were also sensitized to menadione death. JNK inhibition magnified decreases in cellular ATP content and β-oxidation induced by menadione. This effect mediated cell death as chemical inhibition of β-oxidation also sensitized cells to death from menadione, and supplementation with the β-oxidation substrate oleate blocked death. Components of the JNK/c-Jun signaling pathway have opposing functions in hepatocyte oxidant stress with JNK2 mediating resistance to cell death and c-Jun promoting death. PMID:22644775

  13. A synthetic diosgenin primary amine derivative attenuates LPS-stimulated inflammation via inhibition of NF-κB and JNK MAPK signaling in microglial BV2 cells.

    PubMed

    Cai, Bangrong; Seong, Kyung-Joo; Bae, Sun-Woong; Chun, Changju; Kim, Won-Jae; Jung, Ji-Yeon

    2018-06-08

    Diosgenin, a precursor of steroid hormones in plants, is known to exhibit diverse pharmacological activities including anti-inflammatory properties. In this study, (3β, 25R)‑spirost‑5‑en‑3‑oxyl (2‑((2((2‑aminoethyl)amino)ethyl)amino)ethyl) carbamate (DGP), a new synthetic diosgenin derivative incorporating primary amine was used to investigate its anti-inflammatory effects and underlying mechanisms of action in lipopolysaccharide (LPS)-stimulated microglial BV2 cells. Pretreatment with DGP resulted in significant inhibition of nitric oxide (NO) synthesis, and down-regulation of nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in LPS-stimulated microglial BV2 cells. In addition, DGP decreased the production of reactive oxygen species (ROS) and pro-inflammatory cytokines such as interleukin (IL)-6, IL-1β, and tumor necrosis factor alpha (TNF-α). The inhibitory effects of DGP on these inflammatory mediators in LPS-stimulated microglial BV2 cells were regulated by NF-κB signaling through blocking p65 nuclear translocation and NF-κB p65/DNA binding activity. DGP also blocked the phosphorylation of c-Jun amino-terminal kinase (JNK), but not p38 kinase or extracellular signal-regulated kinases (ERK). The NF-κB inhibitor JSH-23 and JNK-specific inhibitor SP600125 significantly decreased NO production and IL-6 release in LPS-stimulated BV2 cells, respectively. The overall results demonstrate that DGP has anti-inflammatory effects on LPS-stimulated BV2 cells via inhibition of NF-κB and JNK activation, suggesting that DGP is a potential prophylactic agent in various neurodegenerative disorders. Copyright © 2018. Published by Elsevier B.V.

  14. JNK3-Mediated Apoptotic Cell Death in Primary Dopaminergic Neurons

    PubMed Central

    Choi, Won-Seok; Klintworth, Heather M.; Xia, Zhengui

    2012-01-01

    Investigation of mechanisms responsible for dopaminergic neuron death is critical for understanding the pathogenesis of Parkinson’s disease, yet this is often quite challenging technically. Here, we describe detailed methods for culturing primary mesencephalic dopaminergic neurons and examining the activation of c-Jun N-terminal protein Kinase (JNK) in these cultures. We utilized immunocytochemistry and computerized analysis to quantify the number of surviving dopaminergic neurons and JNK activation in dopaminergic neurons. TUNEL staining was used to quantify apoptotic cell death. siRNA was used to specifically inhibit JNK3, the neural specific isoform of JNK. Our data implicate the activation of JNK3 in rotenone-induced dopaminergic neuron apoptosis. PMID:21815073

  15. Effect of angiotensin II type 2 receptor on tyrosine kinase Pyk2 and c-Jun NH2-terminal kinase via SHP-1 tyrosine phosphatase activity: evidence from vascular-targeted transgenic mice of AT2 receptor.

    PubMed

    Matsubara, H; Shibasaki, Y; Okigaki, M; Mori, Y; Masaki, H; Kosaki, A; Tsutsumi, Y; Uchiyama, Y; Fujiyama, S; Nose, A; Iba, O; Tateishi, E; Hasegawa, T; Horiuchi, M; Nahmias, C; Iwasaka, T

    2001-04-20

    Angiotensin II (Ang II) has two major receptor isoforms, AT1 and AT2. AT1 transphosphorylates Ca(2+)-sensitive tyrosine kinase Pyk2 to activate c-Jun NH2-terminal kinase (JNK). Although AT2 inactivates extracellular signal-regulated kinase (ERK) via tyrosine phosphatases (PTP), the action of AT2 on Pyk2 and JNK remains undefined. Using AT2-overexpressing vascular smooth muscle cells (AT2-VSMC) from AT2-transgenic mice, we studied these undefined actions of AT2. AT1-mediated JNK activity was increased 2.2-fold by AT2 inhibition, which was abolished by orthovanadate. AT2 did not affect AT1-mediated Pyk2 phosphorylation, but attenuated c-Jun mRNA accumulation by 32%. The activity of src-homology 2 domain-containing PTP (SHP-1) was significantly upregulated 1 min after AT2 stimulation. Stable overexpression of SHP-1 dominant negative mutant in AT2-VSMC completely abolished AT2-mediated inhibition of JNK activation and c-Jun expression. These findings suggest that AT2 inhibits JNK activity by affecting the downstream signal of Pyk2 in a SHP-1-dependent manner, leading to a decrease in c-Jun expression. Copyright 2001 Academic Press.

  16. Activation of the HMGB1-RAGE axis upregulates TH expression in dopaminergic neurons via JNK phosphorylation.

    PubMed

    Kim, Soo Jeong; Ryu, Min Jeong; Han, Jeongsu; Jang, Yunseon; Kim, Jungim; Lee, Min Joung; Ryu, Ilhwan; Ju, Xianshu; Oh, Eungseok; Chung, Woosuk; Kweon, Gi Ryang; Heo, Jun Young

    2017-11-04

    The derangement of tyrosine hydroxylase (TH) activity reduces dopamine synthesis and is implicated in the pathogenesis of Parkinson's disease. However, the extracellular modulator and intracellular regulatory mechanisms of TH have yet to be identified. Recently, high-mobility group box 1 (HMGB1) was reported to be actively secreted from glial cells and is regarded as a mediator of dopaminergic neuronal loss. However, the mechanism for how HMGB1 affects TH expression, particularly through the receptor for advanced glycation endproducts (RAGE), has not yet been investigated. We found that recombinant HMGB1 (rHMGB1) upregulates TH mRNA expression via simultaneous activation of JNK phosphorylation, and this induction of TH expression is blocked by inhibitors of RAGE and JNK. To investigate how TH expression levels change through the HMGB1-RAGE axis as a result of MPP + toxicity, we co-treated SN4741 dopaminergic cells with MPP + and rHMGB1. rHMGB1 blocked the reduction of TH mRNA following MPP + treatment without altering cell survival rates. Our results suggest that HMGB1 upregulates TH expression to maintain dopaminergic neuronal function via activating RAGE, which is dependent on JNK phosphorylation. Copyright © 2017 Elsevier Inc. All rights reserved.

  17. Perillaldehyde attenuates cerebral ischemia-reperfusion injury-triggered overexpression of inflammatory cytokines via modulating Akt/JNK pathway in the rat brain cortex.

    PubMed

    Xu, Lixing; Li, Yuebi; Fu, Qiang; Ma, Shiping

    2014-11-07

    Perillaldehyde (PAH), one of the major oil components in Perilla frutescens, has anti-inflammatory effects. Few studies have examined the neuroprotective effect of PAH on stroke. So the aim of our study is to investigate the effect of PAH on ischemia-reperfusion-induced injury in the rat brain cortex. Middle cerebral artery occlusion (MCAO) model was selected to make cerebral ischemia-reperfusion injury. Rats were assigned randomly to groups of sham, MCAO, and two treatment groups by PAH at 36.0, 72.0mg/kg. Disease model was set up after intragastrically (i.g.) administering for 7 consecutive days. The neurological deficit, the cerebral infarct size, biochemical parameters and the relative mRNA and protein levels were examined. The results showed that the NO level, the iNOS activity, the neurological deficit scores, the cerebral infarct size and the expression of inflammatory cytokines including interleukin (IL)-1β, interleukin (IL)-6 and tumor necrosis factor (TNF)-α were significantly decreased by PAH treatment. PAH also increased the Phospho-Akt level and decrease the Phospho-JNK level by Western blot analysis. Meanwhile, the PAH groups exhibited a dramatically decrease of apoptosis-related mRNA expression such as Bax and caspase-3. Our findings shown that PAH attenuates cerebral ischemia/reperfusion injury in the rat brain cortex, and suggest its neuroprotective effect is relate to regulating the inflammatory response through Akt /JNK pathway. The activation of this signalling pathway eventually inhibits apoptotic cell death induced by cerebral ischemia-reperfusion. Copyright © 2014 Elsevier Inc. All rights reserved.

  18. Tangeretin suppresses IL-1beta-induced cyclooxygenase (COX)-2 expression through inhibition of p38 MAPK, JNK, and AKT activation in human lung carcinoma cells.

    PubMed

    Chen, Kuan-Hung; Weng, Meng-Shih; Lin, Jen-Kun

    2007-01-15

    Tangeretin (5,6,7,8,4'-pentamethoxyflavone) is a polymethoxylated flavonoid concentrated in the peel of citrus fruits. Recent studies have shown that tangeretin exhibits anti-proliferative, anti-invasive, anti-metastatic, and antioxidant activities. However, the anti-inflammatory properties of tangeretin are unclear. In this study, we examine the effects of tangeretin and its structure-related compound, nobiletin, on the expression of cyclooxygenases-2 (COX-2) in human lung epithelial carcinoma cells, A549, and human non-small cell lung carcinoma cells, H1299. Tangeretin exerts a much better inhibitory activity than nobiletin against IL-1beta-induced production of COX-2 in A549 cells, and it effectively represses the constitutively expressed COX-2 in H1299. RT-PCR was used to investigate the transcriptional inhibition of COX-2 by tangeretin. COX-2 mRNA was rapidly induced by IL-1beta in 3h and markedly suppressed by tangeretin. IL-1beta-induced the activation of ERK, p38 MAPK, JNK, and AKT in A549 cells. COX-2 expression in response to IL-1beta was attenuated by pretreatment with SB203580, SP600125, and LY294002, but not with PD98059, suggesting the involvement of p38 MAPK, JNK, and PI3K in this response. Pretreatment of cells with tangeretin inhibited IL-1beta-induced p38 MAPK, JNK, and AKT phosphorylation and the downstream activation of NF-kappaB. These results may reveal that the tangeretin inhibition of IL-1beta-induced COX-2 expression in A549 cells is, at least in part, mediated through suppression of NF-kappaB transcription factor as well as through suppression of the signaling proteins of p38 MAPK, JNK, and PI3K, but not of ERK.

  19. Cullin-4 regulates Wingless and JNK signaling-mediated cell death in the Drosophila eye

    PubMed Central

    Tare, Meghana; Sarkar, Ankita; Bedi, Shimpi; Kango-Singh, Madhuri; Singh, Amit

    2016-01-01

    In all multicellular organisms, the fundamental processes of cell proliferation and cell death are crucial for growth regulation during organogenesis. Strict regulation of cell death is important to maintain tissue homeostasis by affecting processes like regulation of cell number, and elimination of unwanted/unfit cells. The developing Drosophila eye is a versatile model to study patterning and growth, where complex signaling pathways regulate growth and cell survival. However, the molecular mechanisms underlying regulation of these processes is not fully understood. In a gain-of-function screen, we found that misexpression of cullin-4 (cul-4), an ubiquitin ligase, can rescue reduced eye mutant phenotypes. Previously, cul-4 has been shown to regulate chromatin remodeling, cell cycle and cell division. Genetic characterization of cul-4 in the developing eye revealed that loss-of-function of cul-4 exhibits a reduced eye phenotype. Analysis of twin-spots showed that in comparison with their wild-type counterparts, the cul-4 loss-of-function clones fail to survive. Here we show that cul-4 clones are eliminated by induction of cell death due to activation of caspases. Aberrant activation of signaling pathways is known to trigger cell death in the developing eye. We found that Wingless (Wg) and c-Jun-amino-terminal-(NH2)-Kinase (JNK) signaling are ectopically induced in cul-4 mutant clones, and these signals co-localize with the dying cells. Modulating levels of Wg and JNK signaling by using agonists and antagonists of these pathways demonstrated that activation of Wg and JNK signaling enhances cul-4 mutant phenotype, whereas downregulation of Wg and JNK signaling rescues the cul-4 mutant phenotypes of reduced eye. Here we present evidences to demonstrate that cul-4 is involved in restricting Wg signaling and downregulation of JNK signaling-mediated cell death during early eye development. Overall, our studies provide insights into a novel role of cul-4 in promoting cell

  20. WNT5A-JNK regulation of vascular insulin resistance in human obesity.

    PubMed

    Farb, Melissa G; Karki, Shakun; Park, Song-Young; Saggese, Samantha M; Carmine, Brian; Hess, Donald T; Apovian, Caroline; Fetterman, Jessica L; Bretón-Romero, Rosa; Hamburg, Naomi M; Fuster, José J; Zuriaga, María A; Walsh, Kenneth; Gokce, Noyan

    2016-12-01

    Obesity is associated with the development of vascular insulin resistance; however, pathophysiological mechanisms are poorly understood. We sought to investigate the role of WNT5A-JNK in the regulation of insulin-mediated vasodilator responses in human adipose tissue arterioles prone to endothelial dysfunction. In 43 severely obese (BMI 44±11 kg/m 2 ) and five metabolically normal non-obese (BMI 26±2 kg/m 2 ) subjects, we isolated arterioles from subcutaneous and visceral fat during planned surgeries. Using videomicroscopy, we examined insulin-mediated, endothelium-dependent vasodilator responses and characterized adipose tissue gene and protein expression using real-time polymerase chain reaction and Western blot analyses. Immunofluorescence was used to quantify endothelial nitric oxide synthase (eNOS) phosphorylation. Insulin-mediated vasodilation was markedly impaired in visceral compared to subcutaneous vessels from obese subjects (p<0.001), but preserved in non-obese individuals. Visceral adiposity was associated with increased JNK activation and elevated expression of WNT5A and its non-canonical receptors, which correlated negatively with insulin signaling. Pharmacological JNK antagonism with SP600125 markedly improved insulin-mediated vasodilation by sixfold (p<0.001), while endothelial cells exposed to recombinant WNT5A developed insulin resistance and impaired eNOS phosphorylation (p<0.05). We observed profound vascular insulin resistance in the visceral adipose tissue arterioles of obese subjects that was associated with up-regulated WNT5A-JNK signaling and impaired endothelial eNOS activation. Pharmacological JNK antagonism markedly improved vascular endothelial function, and may represent a potential therapeutic target in obesity-related vascular disease. © The Author(s) 2016.

  1. WNT5A-JNK regulation of vascular insulin resistance in human obesity

    PubMed Central

    Farb, Melissa G; Karki, Shakun; Park, Song-Young; Saggese, Samantha M; Carmine, Brian; Hess, Donald T; Apovian, Caroline; Fetterman, Jessica L; Bretón-Romero, Rosa; Hamburg, Naomi M; Fuster, José J; Zuriaga, María A; Walsh, Kenneth; Gokce, Noyan

    2017-01-01

    Obesity is associated with the development of vascular insulin resistance; however, pathophysiological mechanisms are poorly understood. We sought to investigate the role of WNT5A-JNK in the regulation of insulin-mediated vasodilator responses in human adipose tissue arterioles prone to endothelial dysfunction. In 43 severely obese (BMI 44±11 kg/m2) and five metabolically normal non-obese (BMI 26±2 kg/m2) subjects, we isolated arterioles from subcutaneous and visceral fat during planned surgeries. Using videomicroscopy, we examined insulin-mediated, endothelium-dependent vasodilator responses and characterized adipose tissue gene and protein expression using real-time polymerase chain reaction and Western blot analyses. Immunofluorescence was used to quantify endothelial nitric oxide synthase (eNOS) phosphorylation. Insulin-mediated vasodilation was markedly impaired in visceral compared to subcutaneous vessels from obese subjects (p<0.001), but preserved in non-obese individuals. Visceral adiposity was associated with increased JNK activation and elevated expression of WNT5A and its non-canonical receptors, which correlated negatively with insulin signaling. Pharmacological JNK antagonism with SP600125 markedly improved insulin-mediated vasodilation by sixfold (p<0.001), while endothelial cells exposed to recombinant WNT5A developed insulin resistance and impaired eNOS phosphorylation (p<0.05). We observed profound vascular insulin resistance in the visceral adipose tissue arterioles of obese subjects that was associated with up-regulated WNT5A-JNK signaling and impaired endothelial eNOS activation. Pharmacological JNK antagonism markedly improved vascular endothelial function, and may represent a potential therapeutic target in obesity-related vascular disease. PMID:27688298

  2. Improvement of liver injury and survival by JNK2 and iNOS deficiency in liver transplants from cardiac death mice.

    PubMed

    Liu, Qinlong; Rehman, Hasibur; Krishnasamy, Yasodha; Schnellmann, Rick G; Lemasters, John J; Zhong, Zhi

    2015-07-01

    Inclusion of liver grafts from cardiac death donors (CDD) would increase the availability of donor livers but is hampered by a higher risk of primary non-function. Here, we seek to determine mechanisms that contribute to primary non-function of liver grafts from CDD with the goal to develop strategies for improved function and outcome, focusing on c-Jun-N-terminal kinase (JNK) activation and mitochondrial depolarization, two known mediators of graft failure. Livers explanted from wild-type, inducible nitric oxide synthase knockout (iNOS(-/-)), JNK1(-/-) or JNK2(-/-) mice after 45-min aorta clamping were implanted into wild-type recipients. Mitochondrial depolarization was detected by intravital confocal microscopy in living recipients. After transplantation of wild-type CDD livers, graft iNOS expression and 3-nitrotyrosine adducts increased, but hepatic endothelial NOS expression was unchanged. Graft injury and dysfunction were substantially higher in CDD grafts than in non-CDD grafts. iNOS deficiency and inhibition attenuated injury and improved function and survival of CDD grafts. JNK1/2 and apoptosis signal-regulating kinase-1 activation increased markedly in wild-type CDD grafts, which was blunted by iNOS deficiency. JNK inhibition and JNK2 deficiency, but not JNK1 deficiency, decreased injury and improved function and survival of CDD grafts. Mitochondrial depolarization and binding of phospho-JNK2 to Sab, a mitochondrial protein linked to the mitochondrial permeability transition, were higher in CDD than in non-CDD grafts. iNOS deficiency, JNK inhibition and JNK2 deficiency all decreased mitochondrial depolarization and blunted ATP depletion in CDD grafts. JNK inhibition and deficiency did not decrease 3-nitrotyrosine adducts in CDD grafts. The iNOS-JNK2-Sab pathway promotes CDD graft failure via increased mitochondrial depolarization, and is an attractive target to improve liver function and survival in CDD liver transplantation recipients. Copyright © 2015

  3. Toxoplasma gondii infection inhibits Th17-mediated spontaneous development of arthritis in interleukin-1 receptor antagonist-deficient mice.

    PubMed

    Washino, Takuya; Moroda, Masataka; Iwakura, Yoichiro; Aosai, Fumie

    2012-04-01

    Interleukin 1 receptor antagonist (IL-1Ra)-deficient BALB/c mice develop spontaneous arthritis resembling human rheumatoid arthritis. We herein report that infection with Toxoplasma gondii, an intracellular protozoan, is capable of ameliorating the spontaneous development of arthritis in IL-1Ra-deficient mice. The onset of arthritis development was delayed and the severity score of arthritis was significantly suppressed in T. gondii-infected mice. Expression of IL-12p40 mRNA from CD11c(+) cells of mesenteric lymph nodes (mLN) and spleen markedly increased at 1 week after peroral infection. While CD11c(+) cells also produced IL-10, IL-1β, and IL-6, CD4(+) T cells from T. gondii-infected mice expressed significantly high levels of T-bet and gamma interferon (IFN-γ) mRNA in both mLN and spleen. Levels of GATA-3/IL-4 mRNA or RORγt/IL-17 mRNA decreased in the infected mice, indicating Th1 cell polarization and the reduction of Th2 and Th17 cell polarization. The severity of arthritis was related to Th1 cell polarization accompanied by Th17 cell reduction, demonstrating the protective role of the T. gondii-derived Th1 response against Th17 cell-mediated arthritis in IL-1Ra-deficient mice.

  4. Novel role of TRPV2 in promoting the cytotoxicity of H2O2-mediated oxidative stress in human hepatoma cells.

    PubMed

    Ma, Wenbo; Li, Caiyue; Yin, Shikui; Liu, Jingxin; Gao, Chao; Lin, Zuoxian; Huang, Rongqi; Huang, Jufang; Li, Zhiyuan

    2015-12-01

    Oxidative stress is important for the initiation and progression of cancers, which confers the cells with a survival advantage by inducing oxidative adaption and drug resistance. Therefore, developing strategies to promote oxidative stress-induced cytotoxicity could be important for cancer therapy. Herein, we found that H2O2-mediated oxidative stress increases TRPV2 expression in human hepatoma (HepG2 and Huh-7) cells. This occurred at the mRNA and protein levels in a dose-dependent manner. The significance of TRPV2 in promoting H2O2-induced cell death was demonstrated in gain and loss of function studies with overexpression and knockdown of TRPV2, respectively. Mechanistically, H2O2-induced cell death involves inhibition of pro-survival signaling proteins (Akt, Nrf2) and activation of pro-death signaling proteins (p38, JNK1). Overexpression of TRPV2 in H2O2-treated hepatoma cells aggravates the inhibition of Akt and Nrf2, while it enhances the activation of p38 and JNK1 at the early stage of cell death. Interestingly, increased expression of TRPV2 in HepG2 cells improved the efficacy of stress-associated chemicals to induce cell death. Our findings suggest that TRPV2 acts as an important enhancer for H2O2-induced cytotoxicity. This process occurred by the inhibition of Akt and Nrf2 as well as the early activation of p38 and JNK1. These findings have important implications for inhibition of oxidative adaption and drug resistance. Copyright © 2015 Elsevier Inc. All rights reserved.

  5. 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

  6. IL-17A induces eotaxin-1/CC chemokine ligand 11 expression in human airway smooth muscle cells: role of MAPK (Erk1/2, JNK, and p38) pathways.

    PubMed

    Rahman, Muhammad Shahidur; Yamasaki, Akira; Yang, Jie; Shan, Lianyu; Halayko, Andrew J; Gounni, Abdelilah Soussi

    2006-09-15

    Recently, IL-17A has been shown to be expressed in higher levels in respiratory secretions from asthmatics and correlated with airway hyperresponsiveness. Although these studies raise the possibility that IL-17A may influence allergic disease, the mechanisms remain unknown. In this study, we investigated the molecular mechanisms involved in IL-17A-mediated CC chemokine (eotaxin-1/CCL11) production from human airway smooth muscle (ASM) cells. We found that incubation of human ASM cells with rIL-17A resulted in a significant increase of eotaxin-1/CCL11 release from ASM cells that was reduced by neutralizing anti-IL-17A mAb. Moreover, IL-17A significantly induced eotaxin-1/CCL11 release and mRNA expression, an effect that was abrogated with cycloheximide and actinomycin D treatment. Furthermore, transfection studies using a luciferase-driven reporter construct containing eotaxin-1/CCL11 proximal promoter showed that IL-17A induced eotaxin-1/CCL11 at the transcriptional level. IL-17A also enhanced significantly IL-1beta-mediated eotaxin-1/CCL11 mRNA, protein release, and promoter activity in ASM cells. Primary human ASM cells pretreated with inhibitors of MAPK p38, p42/p44 ERK, JNK, or JAK but not PI3K, showed a significant decrease in eotaxin-1/CCL11 release upon IL-17A treatment. In addition, IL-17A mediated rapid phosphorylation of MAPK (p38, JNK, and p42/44 ERK) and STAT-3 but not STAT-6 or STAT-5 in ASM cells. Taken together, our data provide the first evidence of IL-17A-induced eotaxin-1/CCL11 expression in ASM cells via MAPK (p38, p42/p44 ERK, JNK) signaling pathways. Our results raise the possibility that IL-17A may play a role in allergic asthma by inducing eotaxin-1/CCL11 production.

  7. Interleukin 1 amplifies receptor-mediated activation of phospholipase A2 in 3T3 fibroblasts.

    PubMed Central

    Burch, R M; Connor, J R; Axelrod, J

    1988-01-01

    Human recombinant interleukin 1 alpha (IL-1 alpha) and IL-1 beta stimulated prostaglandin E2 synthesis in 3T3 fibroblasts in a time- and concentration-dependent manner. Enhanced prostaglandin E2 synthesis after IL-1 treatment was apparent by 1 hr and continued to increase for at least 2 days. Half-maximal stimulation occurred at 0.5 pM IL-1 alpha or IL-1 beta, and both interleukins were equally effective, with maximal stimulation occurring in response to 5-10 pM IL-1. In contrast to IL-1, bradykinin stimulation of prostaglandin E2 synthesis is rapid; its effect is maximal by 5 min. In cells that had been pretreated with IL-1 for 24 hr, prostaglandin E2 synthesis in response to bradykinin was amplified more than 10-fold. IL-1 also amplified the receptor-mediated formation of prostaglandin E2 by bombesin and thrombin. The lymphokine did not affect bradykinin receptor number or affinity. IL-1 treatment induced phospholipase A2 and cyclooxygenase but not phospholipase C or prostaglandin E isomerase. It also enhanced bradykinin-stimulated GTPase activity, suggesting possible induction of the GTP-binding regulatory protein coupled to the bradykinin receptor. Thus, IL-1 enhanced receptor-mediated release of prostaglandin E2 in response to bradykinin, bombesin, and thrombin by increasing the cellular levels of phospholipase A2, cyclooxygenase, and GTP-binding regulatory protein(s). PMID:2901097

  8. TMEM16A mediates the hypersecretion of mucus induced by Interleukin-13

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

    Lin, Jiachen; Jiang, Youfan; Li, Li

    2015-06-10

    Previous studies showed that the Ca{sup 2+}-activated Cl{sup −} channel (CaCC) was involved in the pathogenesis of mucus hypersecretion induced by Interleukin-13 (IL-13). However, the mechanisms underlying the process were unknown. Recently, transmembrane protein 16A (TMEM16A) was identified as the channel underlying the CaCC current. The aim of the current study was to investigate whether the TMEM16A channel is part of the mechanism underlying IL-13-induced mucus hypersecretion. We observed that both TMEM16A mRNA and protein expression were significantly up-regulated after treatment with IL-13 in human bronchial epithelial 16 (HBE 16) cells, which correlated with an increase in mucus production. Additionally,more » mucus hypersecretion in rat airways was induced by intratracheal instillation of IL-13 and similar increases were observed in the expression of TMEM16A mRNA and protein in the bronchial epithelium. Niflumic acid (NA), a selective antagonist of CaCC, markedly blocked IL-13-induced mucin (MUC) 5AC mRNA and protein production in vivo and in vitro. Further investigation with HBE16 cells revealed that TMEM16A overexpression clearly promoted mucus production, IκBα phosphorylation, and p65 accumulation in the nucleus. The loss of TMEM16A resulted in inhibition of mucus production, and the TMEM16A-mediated production of MUC5AC was significantly blocked by a nuclear factor-kappa B (NF-κB) inhibitor. Therefore, the TMEM16A channel acts upstream of NF-κB in the regulation of mucus production. This is the first demonstration that the TMEM16A-NF-κB pathway is positively involved in IL-13-induced mucus production, which provides novel insight into the molecular mechanism of mucin overproduction. - Highlights: • TMEM16A acts as downstream events of IL-13 signaling pathway. • Established the link between TMEM16A and mucus hypersecretion. • NF-κB activation might be responsible for TMEM16A mediated mucus secretion.« less

  9. Cleome rutidosperma and Euphorbia thymifolia Suppress Inflammatory Response via Upregulation of Phase II Enzymes and Modulation of NF-κB and JNK Activation in LPS-Stimulated BV2 Microglia

    PubMed Central

    Ding, Hsiou-Yu; Wu, Pei-Shan; Wu, Ming-Jiuan

    2016-01-01

    Cleome rutidosperma DC. and Euphorbia thymifolia L. are herbal medicines used in traditional Indian and Chinese medicine to treat various illnesses. Reports document that they have antioxidant and anti-inflammatory activities; nonetheless, the molecular mechanisms involved in their anti-inflammatory actions have not yet been elucidated. The anti-neuroinflammatory activities and underlying mechanisms of ethanol extracts of Cleome rutidosperma (CR) and Euphorbia thymifolia (ET) were studied using lipopolysaccharide (LPS)-stimulated microglial cell line BV2. The morphology changes and production of pro-inflammatory mediators were assayed. Gene expression of inflammatory genes such as inducible nitric oxide synthase (iNOS), cyclooxygenase (COX)-2, interleukin (IL)-1β, and CC chemokine ligand (CCL)-2, as well as phase II enzymes such as heme oxygenase (HO)-1, the modifier subunit of glutamate cysteine ligase (GCLM) and NAD(P)H quinone dehydrogenase 1 (NQO1), were further investigated using reverse transcription quantitative-PCR (RT-Q-PCR) and Western blotting. The effects of CR and ET on mitogen activated protein kinases (MAPKs) and nuclear factor (NF)-κB signaling pathways were examined using Western blotting and specific inhibitors. CR and ET suppressed BV2 activation, down-regulated iNOS and COX-2 expression and inhibited nitric oxide (NO) overproduction without affecting cell viability. They reduced LPS-mediated tumor necrosis factor (TNF) and IL-6 production, attenuated IL-1β and CCL2 expression, but upregulated HO-1, GCLM and NQO1 expression. They also inhibited p65 NF-κB phosphorylation and modulated Jun-N terminal kinase (JNK) activation in BV2 cells. SP600125, the JNK inhibitor, significantly augmented the anti-IL-6 activity of ET. NF-κB inhibitor, Bay 11-7082, enhanced the anti-IL-6 effects of both CR and ET. Znpp, a competitive inhibitor of HO-1, attenuated the anti-NO effects of CR and ET. Our results show that CR and ET exhibit anti

  10. JNK1 and JNK3 play a significant role in both neuronal apoptosis and necrosis. Evaluation based on in vitro approach using tert-butylhydroperoxide induced oxidative stress in neuro-2A cells and perturbation through 3-aminobenzamide.

    PubMed

    Muthaiah, Vijaya Prakash Krishnan; Michael, Felicia Mary; Palaniappan, Tamilselvi; Rajan, Sridhar Skylab; Chandrasekar, Kirubhanand; Venkatachalam, Sankar

    2017-06-01

    In spinal cord injury (SCI), oxidative stress in the penumbra of the injury site is a characteristic feature. The predominance of necrosis over apoptosis in the ensuing delayed cell death results in progressive waves of necrosis affecting neighboring cells and thus exaggerates the severity of the lesion. Necrosis has been classified into subtypes based on the active molecular players and parthanatos is one among them, which is characterized by the over activation of PARP1 as the pre-mitochondrial event that triggers necrosis. Parthanatos being the necrosis mode reported in SCI, we intended to study the molecular players in the elusive pre-mitochondrial events of PARP1 over activation using an in vitro model. tert-Butylhydroperoxide (tBuOOH) was reported to induce oxidative stress in various cell types including Neuro-2A cells. Using a tailored protocol, a predominantly PARP1 mediated necrotic mode of cell death was obtained in Neuro-2A cells using tBuOOH. By perturbing the progress of necrosis using 3-amniobenzamide, a known PARP1 inhibitor, it was found that JNK1 and JNK3 but not JNK2 were involved in pre-mitochondrial stages of PARP1 mediated cell death. Given that JNK1 and JNK3 play a role in apoptosis also, they may serve as common targets to counter both apoptosis and necrosis. The in vitro model used in the present study may be useful in delineating molecular mechanisms in necrosis. Copyright © 2017 Elsevier B.V. All rights reserved.

  11. Stretch and interleukin 1 beta: pro-labour factors with similar mitogen-activated protein kinase effects but differential patterns of transcription factor activation and gene expression.

    PubMed

    Sooranna, S R; Engineer, N; Liang, Z; Bennett, P R; Johnson, M R

    2007-07-01

    IL-1beta and stretch increase uterine smooth muscle cell (USMC) prostaglandin H synthase 2 (PGHS-2) and interleukin (IL)-8 mRNA expression in a mitogen-activated protein kinase (MAPK) dependent mechanism. We have tested our hypothesis that stretch and IL-1beta activate different components of the MAPK cascade in USMC and investigated the effects of specific MAPK inhibitors on these components. Further, we have used a Jun N-terminal kinase (JNK) and p38 activator, anisomycin, to compare the effect of differential MAPK activation on the expression of PGHS-2, IL-8 and oxytocin receptor (OTR) mRNA with that seen in response to stretch and IL-1beta. Stretch, IL-1beta and anisomycin activated similar components of the MAPK cascade and specific inhibitors of MAPK altered phosphorylation of MAPK and downstream cascade components as expected. Expression of OTR mRNA was increased by stretch and anisomycin in a MAPK-independent manner. All three stimuli increased PGHS-2 and IL-8 mRNA expression in a MAPK-dependent manner, but while the MAPK inhibitors reduced the IL-1beta-induced activation of activating transcription factor (ATF)-2, liver activating protein (LAP) and c-jun, the stretch-induced increase in LAP was unaffected by MAPK-inhibition and only JNK inhibition appeared to reduce c-jun activation. These observations show that stretch, IL-1beta and anisomycin activate the same components of the MAPK cascade, but differentially activate LAP and liver inhibitory protein (LIP) perhaps accounting for the increase in OTR by stretch and anisomycin but not IL-1beta observed in this study.

  12. AMPA receptor-mediated toxicity in oligodendrocyte progenitors involves free radical generation and activation of JNK, calpain and caspase 3.

    PubMed

    Liu, Hsueh-Ning; Giasson, Benoit I; Mushynski, Walter E; Almazan, Guillermina

    2002-07-01

    The molecular mechanisms underlying AMPA (alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate) receptor-mediated excitotoxicity were characterized in rat oligodendrocyte progenitor cultures. Activation of AMPA receptors, in the presence of cyclothiazide to selectively block desensitization, produced a massive Ca(2+) influx and cytotoxicity which were blocked by the antagonists CNQX and GYKI 52466. A role for free radical generation in oligodendrocyte progenitor cell death was deduced from three observations: (i) treatment with AMPA agonists decreased intracellular glutathione; (ii) depletion of intracellular glutathione with buthionine sulfoximine potentiated cell death; and (iii) the antioxidant N -acetylcysteine replenished intracellular glutathione and protected cultures from AMPA receptor-mediated toxicity. Cell death displayed some characteristics of apoptosis, including DNA fragmentation, chromatin condensation and activation of caspase-3 and c-Jun N-terminal kinase (JNK). A substrate of calpain and caspase-3, alpha-spectrin, was cleaved into characteristic products following treatment with AMPA agonists. In contrast, inhibition of either caspase-3 by DEVD-CHO or calpain by PD 150606 protected cells from excitotoxicity. Our results indicate that overactivation of AMPA receptors causes apoptosis in oligodendrocyte progenitors through mechanisms involving Ca(2+) influx, depletion of glutathione, and activation of JNK, calpain, and caspase-3.

  13. Interleukin-2 and subunit alpha of its soluble receptor in autoimmune Addison's disease--an association study and expression analysis.

    PubMed

    Fichna, Marta; Żurawek, Magdalena; Bratland, Eirik; Husebye, Eystein S; Kasperlik-Załuska, Anna; Czarnocka, Barbara; Januszkiewicz-Lewandowska, Danuta; Nowak, Jerzy

    2015-03-01

    Autoimmune Addison's disease (AAD) results from T cell-mediated destruction of the adrenal cortex, commonly accompanied by autoantibodies to 21-hydroxylase (21OH). In order to gain insight into the obscure aetiology of this disease, we investigated the roles of the IL2 and IL2RA genes, encoding interleukin-2 and subunit alpha of its receptor (IL2Ra), respectively. The association of AAD with IL2 and IL2RA polymorphisms (rs6822844, rs2069762, rs3136534, rs11594656, rs3118470 and rs2104286) was tested in 223 patients and 672 healthy controls. Functional studies consisted of gene expression analysis in cultured PBMCs exposed to 21OH and evaluation of serum interleukin by ELISA assays. The frequency of the minor C allele of rs3136534 was significantly decreased in AAD subjects compared to controls (OR 0.71; 95%CI 0.561-0.887; p = 0.003). Only AAD cells responded to 21OH with an elevated IL2 and IL2RA mRNA synthesis (p = 0.004 and p = 0.009 versus controls, respectively), paralleled by increased supernatant levels of both cytokines (p = 0.031 and p = 0.001 versus controls). IL2 mRNA level in 21OH-stimulated AAD PBMCs correlated negatively with age (p = 0.036) and positively with serum antibodies to 21OH (p = 0.006). Carriers of the rs2104286 AA genotype demonstrated higher IL2RA mRNA (p = 0.022) and soluble IL2Ra secretion (p = 0.029) upon 21OH stimulation. Serum interleukin-2 in AAD subjects was significantly higher compared to controls (4.61 ± 4.3 versus 1.71 ± 3.2 pg/mL, p < 0.001), whereas sIL2Ra levels remained similar in both groups (p = 0.885). In conclusion, the study reveals an association between AAD and IL2 locus. It confirms specific 21OH-directed reactivity of the peripheral AAD lymphocytes, which display increased synthesis of interleukin-2 and sIL2Ra.

  14. Prefrontal mRNA expression of long and short isoforms of D2 dopamine receptor: Possible role in delayed learning deficit caused by early life interleukin-1β treatment.

    PubMed

    Schwarz, Alexander P; Trofimov, Alexander N; Zubareva, Olga E; Lioudyno, Victoria I; Kosheverova, Vera V; Ischenko, Alexander M; Klimenko, Victor M

    2017-08-30

    Long (D2L) and short (D2S) isoform of the D2 dopamine receptor are believed to play different roles in behavioral regulation. However, little is known about differential regulation of these isoforms mRNA expression during the process of learning in physiological and pathological states. In this study, we have investigated the combined effect of training in active avoidance (AA) paradigm and chronic early life treatment with pro-inflammatory cytokine interleukin (IL)-1β (1μg/kg i.p., P15-21) on D2S and D2L dopamine receptor mRNA expression in the medial prefrontal cortex (mPFC) of adult rats. We have shown differential regulation of D2 short and long mRNA isoform expression in the mPFC. There was no effect of AA-training on D2S mRNA expression, while D2L mRNA was downregulated in AA-trained control (intact and saline-treated) animals, and this effect was not observed in rats treated with IL-1β. D2S mRNA expression level negatively correlated with learning ability within control (saline-treated and intact) groups but not in IL-1β-treated animals. Thus, prefrontal expression of distinct D2 dopamine receptor splice variants is supposed to be implicated in cognitive decline caused by early life immune challenge. Copyright © 2017 Elsevier B.V. All rights reserved.

  15. ATF3 mediates the inhibitory action of TNF-α on osteoblast differentiation through the JNK signaling pathway.

    PubMed

    Jeong, Byung-Chul

    2018-05-15

    Tumor necrosis factor (TNF)-α, which is a proinflammatory cytokine, inhibits osteoblast differentiation under diverse inflammatory conditions. Activating transcription factor 3 (ATF3), which is a member of the ATF/cAMP response element-binding protein family of transcription factors, has been implicated in the regulation of cell proliferation and differentiation. However, the precise interactions between ATF3 and the TNF-α signaling pathway in the regulation of osteoblast differentiation remain unclear. In this study, we examined the role of ATF3 in the TNF-α-mediated inhibition of osteoblast differentiation and investigated the signaling pathways involved. The treatment of cells with TNF-α downregulated osteogenic markers, but significantly upregulated the expression of Atf3. The inhibition of Atf3 by small interfering RNAs rescued osteogenesis, which was inhibited by TNF-α. Conversely, the enforced expression of Atf3 enhanced the TNF-α-mediated inhibition of osteoblast differentiation, as revealed by the measurement of osteogenic markers and alkaline phosphatase staining. Mechanistically, TNF-α-induced Atf3 expression was significantly suppressed by the inhibition of the c-Jun N-terminal kinase (JNK) pathway. Furthermore, the overexpression of Atf3 did not affect the rescue effect that inhibiting TNF-α expression using a JNK inhibitor had on alkaline phosphatase activity and mineralization. Taken together, these results indicate that ATF3 mediates the inhibitory action of TNF-α on osteoblast differentiation and that the TNF-α-activated JNK pathway is responsible for the induction of Atf3 expression. Copyright © 2018 Elsevier Inc. All rights reserved.

  16. Genetic inhibition of JNK3 ameliorates spinal muscular atrophy.

    PubMed

    Genabai, Naresh K; Ahmad, Saif; Zhang, Zhanying; Jiang, Xiaoting; Gabaldon, Cynthia A; Gangwani, Laxman

    2015-12-15

    Mutation of the Survival Motor Neuron 1 (SMN1) gene causes spinal muscular atrophy (SMA), an autosomal recessive neurodegenerative disorder that occurs in early childhood. Degeneration of spinal motor neurons caused by SMN deficiency results in progressive muscle atrophy and death in SMA. The molecular mechanism underlying neurodegeneration in SMA is unknown. No treatment is available to prevent neurodegeneration and reduce the burden of illness in SMA. We report that the c-Jun NH2-terminal kinase (JNK) signaling pathway mediates neurodegeneration in SMA. The neuron-specific isoform JNK3 is required for neuron degeneration caused by SMN deficiency. JNK3 deficiency reduces degeneration of cultured neurons caused by low levels of SMN. Genetic inhibition of JNK pathway in vivo by Jnk3 knockout results in amelioration of SMA phenotype. JNK3 deficiency prevents the loss of spinal cord motor neurons, reduces muscle degeneration, improves muscle fiber thickness and muscle growth, improves motor function and overall growth and increases lifespan of mice with SMA that shows a systemic rescue of phenotype by a SMN-independent mechanism. JNK3 represents a potential (non-SMN) therapeutic target for the treatment of SMA. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  17. Novel synergistic mechanism for sst2 somatostatin and TNFalpha receptors to induce apoptosis: crosstalk between NF-kappaB and JNK pathways.

    PubMed

    Guillermet-Guibert, J; Saint-Laurent, N; Davenne, L; Rochaix, P; Cuvillier, O; Culler, M D; Pradayrol, L; Buscail, L; Susini, C; Bousquet, C

    2007-02-01

    Somatostatin is a multifunctional hormone that modulates cell proliferation, differentiation and apoptosis. Mechanisms for somatostatin-induced apoptosis are at present mostly unsolved. Therefore, we investigated whether somatostatin receptor subtype 2 (sst2) induces apoptosis in the nontransformed murine fibroblastic NIH3T3 cells. Somatostatin receptor subtype 2 expression induced an executioner caspase-mediated apoptosis through a tyrosine phosphatase SHP-1 (Src homology domain phosphatase-1)-dependent stimulation of nuclear factor kappa B (NF-kappaB) activity and subsequent inhibition of the mitogen-activated protein kinase JNK. Tumor necrosis factor alpha (TNFalpha) stimulated both NF-kappaB and c-Jun NH2-terminal kinase (JNK) activities, which had opposite action on cell survival. Importantly, sst2 sensitized NIH3T3 cells to TNFalpha-induced apoptosis by (1) upregulating TNFalpha receptor protein expression, and sensitizing to TNFalpha-induced caspase-8 activation; (2) enhancing TNFalpha-mediated activation of NF-kappaB, resulting in JNK inhibition and subsequent executioner caspase activation and cell death. We have here unraveled a novel signaling mechanism for a G protein-coupled receptor, which directly triggers apoptosis and crosstalks with a death receptor to enhance death ligand-induced apoptosis.

  18. Absolute Measurements of Macrophage Migration Inhibitory Factor and Interleukin-1-β mRNA Levels Accurately Predict Treatment Response in Depressed Patients.

    PubMed

    Cattaneo, Annamaria; Ferrari, Clarissa; Uher, Rudolf; Bocchio-Chiavetto, Luisella; Riva, Marco Andrea; Pariante, Carmine M

    2016-10-01

    Increased levels of inflammation have been associated with a poorer response to antidepressants in several clinical samples, but these findings have had been limited by low reproducibility of biomarker assays across laboratories, difficulty in predicting response probability on an individual basis, and unclear molecular mechanisms. Here we measured absolute mRNA values (a reliable quantitation of number of molecules) of Macrophage Migration Inhibitory Factor and interleukin-1β in a previously published sample from a randomized controlled trial comparing escitalopram vs nortriptyline (GENDEP) as well as in an independent, naturalistic replication sample. We then used linear discriminant analysis to calculate mRNA values cutoffs that best discriminated between responders and nonresponders after 12 weeks of antidepressants. As Macrophage Migration Inhibitory Factor and interleukin-1β might be involved in different pathways, we constructed a protein-protein interaction network by the Search Tool for the Retrieval of Interacting Genes/Proteins. We identified cutoff values for the absolute mRNA measures that accurately predicted response probability on an individual basis, with positive predictive values and specificity for nonresponders of 100% in both samples (negative predictive value=82% to 85%, sensitivity=52% to 61%). Using network analysis, we identified different clusters of targets for these 2 cytokines, with Macrophage Migration Inhibitory Factor interacting predominantly with pathways involved in neurogenesis, neuroplasticity, and cell proliferation, and interleukin-1β interacting predominantly with pathways involved in the inflammasome complex, oxidative stress, and neurodegeneration. We believe that these data provide a clinically suitable approach to the personalization of antidepressant therapy: patients who have absolute mRNA values above the suggested cutoffs could be directed toward earlier access to more assertive antidepressant strategies

  19. Gefitinib enhances human colon cancer cells to TRAIL-induced apoptosis of via autophagy- and JNK-mediated death receptors upregulation.

    PubMed

    Chen, Lei; Meng, Yue; Guo, Xiaoqing; Sheng, Xiaotong; Tai, Guihua; Zhang, Fenglei; Cheng, Hairong; Zhou, Yifa

    2016-11-01

    Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a potent cancer cell-specific apoptosis-inducing cytokine with little toxicity to most normal cells. Here, we report that gefitinib and TRAIL in combination produce a potent synergistic effect on TRAIL-sensitive human colon cancer HCT116 cells and an additive effect on TRAIL-resistant HT-29 cells. Interestingly, gefitinib increases the expression of cell surface receptors DR4 and DR5, possibly explaining the synergistic effect. Knockdown of DR4 and DR5 by siRNA significantly decreases gefitinib- and TRAIL-mediated cell apoptosis, supporting this idea. Because the inhibition of gefitinib-induced autophagy by 3-MA significantly decreases DR4 and DR5 upregulation, as well as reduces gefitinib- and TRAIL-induced apoptosis, we conclude that death receptor upregulation is autophagy mediated. Furthermore, our results indicate that death receptor expression may also be regulated by JNK activation, because pre-treatment of cells with JNK inhibitor SP600125 significantly decreases gefitinib-induced death receptor upregulation. Interestingly, SP600125 also inhibits the expression CHOP, yet CHOP has no impact on death receptor expressions. We also find here that phosphorylation of Akt and ERK might also be required for TRAIL sensitization. In summary, our results indicate that gefitinib effectively enhances TRAIL-induced apoptosis, likely via autophagy and JNK- mediated death receptor expression and phosphorylation of Akt and ERK.

  20. Protocatechuic Acid from Alpinia oxyphylla Induces Schwann Cell Migration via ERK1/2, JNK and p38 Activation.

    PubMed

    Ju, Da-Tong; Kuo, Wei-Wen; Ho, Tsung-Jung; Paul, Catherine Reena; Kuo, Chia-Hua; Viswanadha, Vijaya Padma; Lin, Chien-Chung; Chen, Yueh-Sheng; Chang, Yung-Ming; Huang, Chih-Yang

    2015-01-01

    Alpinia oxyphylla MIQ (Alpinate Oxyphyllae Fructus, AOF) is an important traditional Chinese medicinal herb whose fruits is widely used to prepare tonics and is used as an aphrodisiac, anti salivary, anti diuretic and nerve-protective agent. Protocatechuic acid (PCA), a simple phenolic compound was isolated from the kernels of AOF. This study investigated the role of PCA in promoting neural regeneration and the underlying molecular mechanisms. Nerve regeneration is a complex physiological response that takes place after injury. Schwann cells play a crucial role in the endogenous repair of peripheral nerves due to their ability to proliferate and migrate. The role of PCA in Schwann cell migration was determined by assessing the induced migration potential of RSC96 Schwann cells. PCA induced changes in the expression of proteins of three MAPK pathways, as determined using Western blot analysis. In order to determine the roles of MAPK (ERK1/2, JNK, and p38) pathways in PCA-induced matrix-degrading proteolytic enzyme (PAs and MMP2/9) production, the expression of several MAPK-associated proteins was analyzed after siRNA-mediated inhibition assays. Treatment with PCA-induced ERK1/2, JNK, and p38 phosphorylation that activated the downstream expression of PAs and MMPs. PCA-stimulated ERK1/2, JNK and p38 phosphorylation was attenuated by individual pretreatment with siRNAs or MAPK inhibitors (U0126, SP600125, and SB203580), resulting in the inhibition of migration and the uPA-related signal pathway. Taken together, our data suggest that PCA extract regulate the MAPK (ERK1/2, JNK, and p38)/PA (uPA, tPA)/MMP (MMP2, MMP9) mediated regeneration and migration signaling pathways in Schwann cells. Therefore, PCA plays a major role in Schwann cell migration and the regeneration of damaged peripheral nerve.

  1. Hyperforin protects against acute cerebral ischemic injury through inhibition of interleukin-17A-mediated microglial activation.

    PubMed

    Ma, Li; Pan, Xia; Zhou, Fang; Liu, Kang; Wang, Long

    2018-01-01

    Hyperforin, a pharmacologically active component of the medicinal plant Hypericum perforatum (St. John's wort), has been shown to be neuroprotective against acute ischemic stroke. However, the underlying mechanisms are still unclear and need to be fully elucidated. C57BL/6 wildtype (WT) mice or interleukin (IL)-17A knock-out mice were subjected to middle cerebral artery occlusion (60min) followed by reperfusion for 72h. Hyperforin (0.5μg) was injected slowly into the right ventricle of WT mice 1, 24 and 48h after middle cerebral artery occlusion (MCAO) onset. Here, we found that hyperforin treatment decreased the mRNA and protein expression of IL-17A at 72h after MCAO onset. Hyperforin reduced infarct volumes and increased neurologic scores accompanied by a decrease in microglial activation and a shift from M1 to M2 phenotypes in the peri-infarct striatum. Furthermore, we revealed that IL-17A was essential to the microglial activation in the acute phase of ischemic stroke. IL-17A knock-out (il-17a -/- ) or anti-IL-17 A monoclonal antibody treatment markedly decreased the microglial activation and induced a shift from M1 to M2 phenotypes of activated microglia. In addition, treatment with recombinant mouse IL-17A abolished the protective effects of hyperforin on acute ischemic brain injury, attenuated the inhibitory effects of hyperforin on the microglial activation, and inhibited the enhanced shift from M1 to M2 phenotypes mediated by hyperforin. In conclusion, our results clearly showed that hyperforin could protect against acute cerebral ischemic injury through inhibition of interleukin-17A-mediated microglial activation and polarization of microglia to M2 phenotype. Copyright © 2017. Published by Elsevier B.V.

  2. Hippo signaling promotes JNK-dependent cell migration.

    PubMed

    Ma, Xianjue; Wang, Hongxiang; Ji, Jiansong; Xu, Wenyan; Sun, Yihao; Li, Wenzhe; Zhang, Xiaoping; Chen, Juxiang; Xue, Lei

    2017-02-21

    Overwhelming studies show that dysregulation of the Hippo pathway is positively correlated with cell proliferation, growth, and tumorigenesis. Paradoxically, the detailed molecular roles of the Hippo pathway in cell invasion remain debatable. Using a Drosophila invasion model in wing epithelium, we show herein that activated Hippo signaling promotes cell invasion and epithelial-mesenchymal transition through JNK, as inhibition of JNK signaling dramatically blocked Hippo pathway activation-induced matrix metalloproteinase 1 expression and cell invasion. Furthermore, we identify bantam -Rox8 modules as essential components downstream of Yorkie in mediating JNK-dependent cell invasion. Finally, we confirm that YAP (Yes-associated protein) expression negatively regulates TIA1 (Rox8 ortholog) expression and cell invasion in human cancer cells. Together, these findings provide molecular insights into Hippo pathway-mediated cell invasion and also raise a noteworthy concern in therapeutic interventions of Hippo-related cancers, as simply inhibiting Yorkie or YAP activity might paradoxically accelerate cell invasion and metastasis.

  3. A liver full of JNK: signaling in regulation of cell function and disease pathogenesis, and clinical approaches.

    PubMed

    Seki, Ekihiro; Brenner, David A; Karin, Michael

    2012-08-01

    c-Jun-N-terminal kinase (JNK) is a mitogen-activated protein kinase family member that is activated by diverse stimuli, including cytokines (such as tumor necrosis factor and interleukin-1), reactive oxygen species (ROS), pathogens, toxins, drugs, endoplasmic reticulum stress, free fatty acids, and metabolic changes. Upon activation, JNK induces multiple biologic events through the transcription factor activator protein-1 and transcription-independent control of effector molecules. JNK isozymes regulate cell death and survival, differentiation, proliferation, ROS accumulation, metabolism, insulin signaling, and carcinogenesis in the liver. The biologic functions of JNK are isoform, cell type, and context dependent. Recent studies using genetically engineered mice showed that loss or hyperactivation of the JNK pathway contributes to the development of inflammation, fibrosis, cancer growth, and metabolic diseases that include obesity, hepatic steatosis, and insulin resistance. We review the functions and pathways of JNK in liver physiology and pathology and discuss findings from preclinical studies with JNK inhibitors. Copyright © 2012 AGA Institute. Published by Elsevier Inc. All rights reserved.

  4. Activation of ERK and JNK signaling pathways by mycotoxin citrinin in human cells

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

    Chang, C.-H.; Yu, F.-Y.; Wang, L.-T.

    2009-06-15

    Mycotoxin citrinin (CTN) is commonly found in foods and feeds that are contaminated/inoculated with Penicillium, Aspergillus and Monascus species. The exposure of human embryonic kidney (HEK293) and HeLa cells to CTN resulted in a dose-dependent increase in the phosphorylation of two major mitogen-activated protein kinases (MAPKs), ERK1/2 and JNK. In HEK293 cultures, the administering of CTN increased both the mRNA and protein levels of egr-1, c-fos and c-jun genes; additionally, the ERK1/2 pathway contributed to the upregulation of Egr-1 and c-Fos protein expression. CTN treatment also induced the transcription activity of Egr-1 and AP-1 proteins, as evidenced by luciferase reportermore » assays. Bioinformatic analyses indicated two genes Gadd45{beta} and MMP3 have Egr-1 and AP-1 response elements in their promoters, respectively. Furthermore, co-exposure of HEK293 cells to CTN and MAPK pathway inhibitors demonstrated that CTN increased the levels of Gadd45{beta} mRNA through ERK1/2 signaling pathway and up-regulated the MMP3 transcripts majorly via JNK pathway. Finally, CTN-triggered caspase 3 activity was significantly reduced in the presence of MAPK inhibitors. Our results suggest that CTN positively regulates ERK1/2 and JNK pathways as well as their downstream effectors in human cells; activated MAPK pathways are also involved in CTN-induced apoptosis.« less

  5. Effect of tocopherol supplementation during last trimester of pregnancy on mRNA abundances of interleukins and angiogenesis in ovine placenta and uterus.

    PubMed

    Kasimanickam, Ramanathan K; Kasimanickam, Vanmathy R; Haldorson, Gary J; Tibary, Ahmed

    2012-01-23

    Interleukins (IL) play an important role in angiogenesis. Tocopherol possesses immunomodulating effect in addition to antioxidant property. The objective of this study was to determine whether gamma tocopherol's (gT) angiogenic activity in placental network is enhanced via promoting interleukins. Pregnant ewes (N=18) were supplemented, orally, with 500 mg of alpha tocopherol (aT; N=6) or 1,000 mg of gT (N=7) or placebo (CON; N=5) once daily from 107 to 137 days post breeding. Uterine and placental tissue samples were obtained at the end of supplementation to evaluate relative mRNA expressions of IL-1b, IL-6, IL-8, Tumor Necrosis Factor (TNF) alpha, Vascular Endothelial Growth Factor (VEGF), kinase insert domain receptor (KDR; VGFR2; a type III receptor tyrosine kinase), and soluble fms-like tyrosine kniase-1 (sFlt1 or sVEGFR1) in uterus, caruncle and cotyledon. Oral supplementation of gT increased IL-6, IL-8, KDR and VEGF mRNA abundances whereas sFlt1 mRNA abundance was suppressed in uterus, caruncle and cotyledon, compared to aT and placebo treated ewes (P<0.05). The TNF alpha and IL-1b mRNA abundances were suppressed in uterus, caruncle and cotyledon but TNF alpha is higher in gT group compared to aT group (P<0.05), whereas IL-1b was similar between treatment groups (P>0.1). Gamma tocopherol supplementation increased IL-6, IL-8, and KDR mRNA abundances and suppressed sFlt1 and TNFalpha mRNA abundances thereby increased VEGF mRNA expression and angiogenesis in placental vascular network during late gestation. It is plausible that the angiogenic effect of gamma tocopherol in placental vascular network is exerted via an alternate path by enhancing IL-6 and IL-8.

  6. Effect of tocopherol supplementation during last trimester of pregnancy on mRNA abundances of interleukins and angiogenesis in ovine placenta and uterus

    PubMed Central

    2012-01-01

    Background Interleukins (IL) play an important role in angiogenesis. Tocopherol possesses immunomodulating effect in addition to antioxidant property. The objective of this study was to determine whether gamma tocopherol's (gT) angiogenic activity in placental network is enhanced via promoting interleukins. Methods Pregnant ewes (N = 18) were supplemented, orally, with 500 mg of alpha tocopherol (aT; N = 6) or 1,000 mg of gT (N = 7) or placebo (CON; N = 5) once daily from 107 to 137 days post breeding. Uterine and placental tissue samples were obtained at the end of supplementation to evaluate relative mRNA expressions of IL-1b, IL-6, IL-8, Tumor Necrosis Factor (TNF) alpha, Vascular Endothelial Growth Factor (VEGF), kinase insert domain receptor (KDR; VGFR2; a type III receptor tyrosine kinase), and soluble fms-like tyrosine kniase-1 (sFlt1 or sVEGFR1) in uterus, caruncle and cotyledon. Results Oral supplementation of gT increased IL-6, IL-8, KDR and VEGF mRNA abundances whereas sFlt1 mRNA abundance was suppressed in uterus, caruncle and cotyledon, compared to aT and placebo treated ewes (P < 0.05). The TNF alpha and IL-1b mRNA abundances were suppressed in uterus, caruncle and cotyledon but TNF alpha is higher in gT group compared to aT group (P < 0.05), whereas IL-1b was similar between treatment groups (P > 0.1). Conclusions Gamma tocopherol supplementation increased IL-6, IL-8, and KDR mRNA abundances and suppressed sFlt1 and TNFalpha mRNA abundances thereby increased VEGF mRNA expression and angiogenesis in placental vascular network during late gestation. It is plausible that the angiogenic effect of gamma tocopherol in placental vascular network is exerted via an alternate path by enhancing IL-6 and IL-8. PMID:22269218

  7. Inflammatory mediator mRNA expression by adenovirus E1A-transfected bronchial epithelial cells.

    PubMed

    Higashimoto, Yuji; Elliott, W Mark; Behzad, Ali R; Sedgwick, Edward G; Takei, Tatsuo; Hogg, James C; Hayashi, Shizu

    2002-07-15

    Lung tissue from patients with emphysema and airway obstruction carries excess adenoviral E1A DNA that is expressed as protein in airway surface epithelium and is associated with an increased inflammatory response. To examine mechanisms by which latent adenoviral infection might amplify the inflammatory process, we transfected primary human bronchial epithelial (HBE) cells from three separate patients undergoing lung resection so that they stably expressed adenovirus E1A. Lipopolysaccharide stimulation of the E1A-transfected HBE cells increased intercellular adhesion molecule-1 and interleukin-8 mRNA and protein expression compared with control cells from the same patient. It also induced greater intercellular adhesion molecule-1 promoter activity and greater nuclear factor-kappa B binding activity of nuclear extracts in E1A transfectants than controls. E1A-positive transfectants constitutively expressed transforming growth factor-beta 1 mRNA and protein, whereas this expression was either very low or not detected in control cells. We conclude that adenoviral E1A transfection transforms primary HBE cells and upregulates their production of mediators that are clinically relevant to the pathogenesis of chronic obstructive pulmonary disease.

  8. Neuroprotective and Anti-Inflammatory Activities of Allyl Isothiocyanate through Attenuation of JNK/NF-κB/TNF-α Signaling.

    PubMed

    Subedi, Lalita; Venkatesan, Ramu; Kim, Sun Yeou

    2017-07-03

    Allyl isothiocyanate (AITC), present in Wasabia japonica (wasabi), is an aliphatic isothiocyanate derived from the precursor sinigrin, which is a glucosinolate present in vegetables of the Brassica family. Traditionally, it has been used to treat rheumatic arthralgia, blood circulation, and pain. This study focuses on its anti-apoptotic activity through the regulation of lipopolysaccharide (LPS)-induced neuroinflammation. Furthermore, we assessed its neuroprotective efficacy, which it achieves through the upregulation of nerve growth factor (NGF) production. Pretreatment with AITC significantly inhibited inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) expression, decreased tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), prostaglandin E2 (PGE2), and nitric oxide (NO) production in activated microglia, and increased the nerve growth factor (NGF) and neurite outgrowth in neuroblastoma cells. AITC inhibited the nuclear factor (NF-κB-mediated transcription by modulating mitogen activated protein kinase (MAPK) signaling, particularly downregulating c-Jun N-terminal kinase (JNK) phosphorylation, which was followed by a reduction in the TNF-α expression in activated microglia. This promising effect of AITC in controlling JNK/NF-κB/TNF-α cross-linking maintains the Bcl-2 gene family and protects neuroblastoma cells from activated microglia-induced toxicity. These findings provide novel insights into the anti-neuroinflammatory effects of AITC on microglial cells, which may have clinical significance in neurodegeneration.

  9. Neuroprotective and Anti-Inflammatory Activities of Allyl Isothiocyanate through Attenuation of JNK/NF-κB/TNF-α Signaling

    PubMed Central

    Subedi, Lalita

    2017-01-01

    Allyl isothiocyanate (AITC), present in Wasabia japonica (wasabi), is an aliphatic isothiocyanate derived from the precursor sinigrin, which is a glucosinolate present in vegetables of the Brassica family. Traditionally, it has been used to treat rheumatic arthralgia, blood circulation, and pain. This study focuses on its anti-apoptotic activity through the regulation of lipopolysaccharide (LPS)-induced neuroinflammation. Furthermore, we assessed its neuroprotective efficacy, which it achieves through the upregulation of nerve growth factor (NGF) production. Pretreatment with AITC significantly inhibited inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) expression, decreased tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), prostaglandin E2 (PGE2), and nitric oxide (NO) production in activated microglia, and increased the nerve growth factor (NGF) and neurite outgrowth in neuroblastoma cells. AITC inhibited the nuclear factor (NF-κB-mediated transcription by modulating mitogen activated protein kinase (MAPK) signaling, particularly downregulating c-Jun N-terminal kinase (JNK) phosphorylation, which was followed by a reduction in the TNF-α expression in activated microglia. This promising effect of AITC in controlling JNK/NF-κB/TNF-α cross-linking maintains the Bcl-2 gene family and protects neuroblastoma cells from activated microglia-induced toxicity. These findings provide novel insights into the anti-neuroinflammatory effects of AITC on microglial cells, which may have clinical significance in neurodegeneration. PMID:28671636

  10. Inhibition of Spinal Interlukin-33/ST2 Signaling and Downstream ERK and JNK Pathways in Electroacupuncture Analgesia in Formalin Mice.

    PubMed

    Han, Ping; Liu, Shenbin; Zhang, Mengting; Zhao, Jing; Wang, Yanqing; Wu, Gencheng; Mi, Wenli

    2015-01-01

    Although acupuncture is widely used to manage pain, it remains highly controversial, largely due to the lack of a clear mechanism for its benefits. Here, we investigated the role of IL-33, a novel interleukin (IL)-1 family member, and its receptor ST2 in the analgesic effects of electroacupuncture (EA) on formalin-induced inflammatory pain. The results showed that 1) EA stimulation of ipsilateral Zusanli (ST 36) and Yanglingquan (GB 34) acupoints for 30 min remarkably suppressed the two phases of formalin-induced spontaneous pain; 2) subcutaneous or intrathecal administration of recombinant IL-33 (rIL-33) significantly inhibited the analgesic effect of EA, whereas the ST2 antibody potentiated EA analgesia in formalin mice; 3) EA treatment decreased the up-regulation of IL-33 and ST2 protein following formalin injection; and 4) the suppression of the formalin-induced expression of spinal phosphorylated ERK and JNK induced by EA treatment was significantly attenuated following subcutaneous rIL-33 delivery, and was further decreased by the ST2 antibody. These data suggest that EA alleviates formalin-induced inflammatory pain, at least partially, by inhibiting of spinal IL-33/ST2 signaling and the downstream ERK and JNK pathways.

  11. Impact of fasting followed by short-term exposure to interleukin-6 on cytochrome P450 mRNA in mice.

    PubMed

    Rasmussen, Martin Krøyer; Bertholdt, Lærke; Gudiksen, Anders; Pilegaard, Henriette; Knudsen, Jakob G

    2018-01-05

    The gene expression of the cytochrome P450 (CYP) enzyme family is regulated by numerous factors. Fasting has been shown to induce increased hepatic CYP mRNA in both humans and animals. However, the coordinated regulation of CYP, CYP-regulating transcription factors, and transcriptional co-factors in the liver linking energy metabolism to detoxification has never been investigated. Interleukin-6 (IL-6) has been suggested to be released during fasting and has been shown to regulate CYP expression. The present study investigated the hepatic mRNA content of selected CYP, AhR, CAR, PXR and PPARα in mice fasted for 18h and subsequently exposed to IL-6. Furthermore, the impact of fasting on PGC-1α, HNF-4α, SIRT1 and SIRT3 mRNA was examined. Fasting induced a marked increase in Cyp2b10, Cyp2e1 and Cyp4a10 mRNA, while CYP1a1, Cyp1a2, Cyp2a4 and Cyp3a11 mRNA levels remained unchanged. In accordance, the mRNA levels of CAR and PPARα were also increased with fasting. The PGC-1α, SIRT1 and SIRT3 mRNA levels were also increased after fasting, while the HNF-4α mRNA levels remained unchanged. In mice subjected to IL-6 injection, the fasting-induced PXR, PPARα and PGC-1α mRNA responses were lower than after saline injection. In conclusion, fasting was demonstrated to be a strong inducer of hepatic CYP mRNA as well as selected transcription factors controlling the expression of the investigated CYP. Moreover, the mRNA levels of transcriptional co-factors acting as energy sensors and co-factors for CYP regulation was also increased in the liver, suggesting crosstalk at the molecular level between regulation of energy metabolism and detoxification. Copyright © 2017 Elsevier B.V. All rights reserved.

  12. Neuroprotection of Scutellarin is mediated by inhibition of microglial inflammatory activation.

    PubMed

    Wang, S; Wang, H; Guo, H; Kang, L; Gao, X; Hu, L

    2011-06-30

    Inhibition of microglial over-reaction and the inflammatory processes may represent a therapeutic target to alleviate the progression of neurological diseases, such as neurodegenerative diseases and stroke. Scutellarin is the major active component of Erigeron breviscapus (Vant.) Hand-Mazz, a herbal medicine in treatment of cerebrovascular diseases for a long time in the Orient. In this study, we explored the mechanisms of neuroprotection by Scutellarin, particularly its anti-inflammatory effects in microglia. We observed that Scutellarin inhibited lipopolysaccharide (LPS)-induced production of proinflammatory mediators such as nitric oxide (NO), tumor necrosis factor α (TNFα), interleukin-1β (IL-1β) and reactive oxygen species (ROS), suppressed LPS-stimulated inducible nitric oxide synthase (iNOS), TNFα, and IL-1β mRNA expression in rat primary microglia or BV-2 mouse microglial cell line. Scutellarin inhibited LPS-induced nuclear translocation and DNA binding activity of nuclear factor κB (NF-κB). It repressed the LPS-induced c-Jun N-terminal kinase (JNK) and p38 phosphorylation without affecting the activity of extracellular signal regulated kinase (ERK) mitogen-activated protein kinase. Moreover, Scutellarin also inhibited interferon-γ (IFN-γ)-induced NO production, iNOS mRNA expression and transcription factor signal transducer and activator of transcription 1α (STAT1α) activation. Concomitantly, conditioned media from Scutellarin pretreated BV-2 cells significantly reduced neurotoxicity compared with conditioned media from LPS treated alone. Together, the present study reported the anti-inflammatory activity of Scutellarin in microglial cells along with their underlying molecular mechanisms, and suggested Scutellarin might have therapeutic potential for various microglia mediated neuroinflammation. Copyright © 2011 IBRO. Published by Elsevier Ltd. All rights reserved.

  13. Inhibiting Src family tyrosine kinase activity blocks glutamate signalling to ERK1/2 and Akt/PKB but not JNK in cultured striatal neurones.

    PubMed

    Crossthwaite, Andrew J; Valli, Haseeb; Williams, Robert J

    2004-03-01

    Glutamate receptor activation of mitogen-activated protein (MAP) kinase signalling cascades has been implicated in diverse neuronal functions such as synaptic plasticity, development and excitotoxicity. We have previously shown that Ca2+-influx through NMDA receptors in cultured striatal neurones mediates the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) and Akt/protein kinase B (PKB) through a phosphatidylinositol 3-kinase (PI 3-kinase)-dependent pathway. Exposing neurones to the Src family tyrosine kinase inhibitor PP2, but not the inactive analogue PP3, inhibited NMDA receptor-induced phosphorylation of ERK1/2 and Akt/PKB in a concentration-dependent manner, and reduced cAMP response element-binding protein (CREB) phosphorylation. To establish a link between Src family tyrosine kinase-mediated phosphorylation and PI 3-kinase signalling, affinity precipitation experiments were performed with the SH2 domains of the PI 3-kinase regulatory subunit p85. This revealed a Src-dependent phosphorylation of a focal adhesion kinase (FAK)-p85 complex on glutamate stimulation. Demonstrating that PI3-kinase is not ubiquitously involved in NMDA receptor signal transduction, the PI 3-kinase inhibitors wortmannin and LY294002 did not prevent NMDA receptor Ca2+-dependent phosphorylation of c-Jun N-terminal kinase 1/2 (JNK1/2). Further, inhibiting Src family kinases increased NMDA receptor-dependent JNK1/2 phosphorylation, suggesting that Src family kinase-dependent cascades may physiologically limit signalling to JNK. These results demonstrate that Src family tyrosine kinases and PI3-kinase are pivotal regulators of NMDA receptor signalling to ERK/Akt and JNK in striatal neurones.

  14. A retained intron in the 3'-UTR of Calm3 mRNA mediates its Staufen2- and activity-dependent localization to neuronal dendrites.

    PubMed

    Sharangdhar, Tejaswini; Sugimoto, Yoichiro; Heraud-Farlow, Jacqueline; Fernández-Moya, Sandra M; Ehses, Janina; Ruiz de Los Mozos, Igor; Ule, Jernej; Kiebler, Michael A

    2017-10-01

    Dendritic localization and hence local mRNA translation contributes to synaptic plasticity in neurons. Staufen2 (Stau2) is a well-known neuronal double-stranded RNA-binding protein (dsRBP) that has been implicated in dendritic mRNA localization. The specificity of Stau2 binding to its target mRNAs remains elusive. Using individual-nucleotide resolution CLIP (iCLIP), we identified significantly enriched Stau2 binding to the 3'-UTRs of 356 transcripts. In 28 (7.9%) of those, binding occurred to a retained intron in their 3'-UTR The strongest bound 3'-UTR intron was present in the longest isoform of Calmodulin 3 ( Calm3 L ) mRNA Calm3 L 3'-UTR contains six Stau2 crosslink clusters, four of which are in this retained 3'-UTR intron. The Calm3 L mRNA localized to neuronal dendrites, while lack of the 3'-UTR intron impaired its dendritic localization. Importantly, Stau2 mediates this dendritic localization via the 3'-UTR intron, without affecting its stability. Also, NMDA-mediated synaptic activity specifically promoted the dendritic mRNA localization of the Calm3 L isoform, while inhibition of synaptic activity reduced it substantially. Together, our results identify the retained intron as a critical element in recruiting Stau2, which then allows for the localization of Calm3 L mRNA to distal dendrites. © 2017 The Authors.

  15. Widespread promoter-mediated coordination of transcription and mRNA degradation

    PubMed Central

    2012-01-01

    Background Previous work showed that mRNA degradation is coordinated with transcription in yeast, and in several genes the control of mRNA degradation was linked to promoter elements through two different mechanisms. Here we show at the genomic scale that the coordination of transcription and mRNA degradation is promoter-dependent in yeast and is also observed in humans. Results We first demonstrate that swapping upstream cis-regulatory sequences between two yeast species affects both transcription and mRNA degradation and suggest that while some cis-regulatory elements control either transcription or degradation, multiple other elements enhance both processes. Second, we show that adjacent yeast genes that share a promoter (through divergent orientation) have increased similarity in their patterns of mRNA degradation, providing independent evidence for the promoter-mediated coupling of transcription to mRNA degradation. Finally, analysis of the differences in mRNA degradation rates between mammalian cell types or mammalian species suggests a similar coordination between transcription and mRNA degradation in humans. Conclusions Our results extend previous studies and suggest a pervasive promoter-mediated coordination between transcription and mRNA degradation in yeast. The diverse genes and regulatory elements associated with this coordination suggest that it is generated by a global mechanism of gene regulation and modulated by gene-specific mechanisms. The observation of a similar coupling in mammals raises the possibility that coupling of transcription and mRNA degradation may reflect an evolutionarily conserved phenomenon in gene regulation. PMID:23237624

  16. Cytosolic calcium mediates RIP1/RIP3 complex-dependent necroptosis through JNK activation and mitochondrial ROS production in human colon cancer cells.

    PubMed

    Sun, Wen; Wu, Xiaxia; Gao, Hongwei; Yu, Jie; Zhao, Wenwen; Lu, Jin-Jian; Wang, Jinhua; Du, Guanhua; Chen, Xiuping

    2017-07-01

    Necroptosis is a form of programmed necrosis mediated by signaling complexes with receptor-interacting protein 1 (RIP1) and RIP3 kinases as the main mediators. However, the underlying execution pathways of this phenomenon have yet to be elucidated in detail. In this study, a RIP1/RIP3 complex was formed in 2-methoxy-6-acetyl-7-methyljuglone (MAM)-treated HCT116 and HT29 colon cancer cells. With this formation, mitochondrial reactive oxygen species (ROS) levels increased, mitochondrial depolarization occurred, and ATP concentrations decreased. This process was identified as necroptosis. This finding was confirmed by experiments showing that MAM-induced cell death was attenuated by the pharmacological or genetic blockage of necroptosis signaling, including RIP1 inhibitor necrostatin-1s (Nec-1s) and siRNA-mediated gene silencing of RIP1 and RIP3, but was unaffected by caspase inhibitor z-vad-fmk or necrosis inhibitor 2-(1H-Indol-3-yl)-3-pentylamino-maleimide (IM54). Transmission electron microscopy (TEM) analysis further revealed the ultrastructural features of MAM-induced necroptosis. MAM-induced RIP1/RIP3 complex triggered necroptosis through cytosolic calcium (Ca 2+ ) accumulation and sustained c-Jun N-terminal kinase (JNK) activation. Both calcium chelator BAPTA-AM and JNK inhibitor SP600125 could attenuate necroptotic features, including mitochondrial ROS elevation, mitochondrial depolarization, and ATP depletion. 2-thenoyltrifluoroacetone (TTFA), which is a mitochondrial complex II inhibitor, was found to effectively reverse both MAM induced mitochondrial ROS generation and cell death, indicating the complex II was the ROS-producing site. The essential role of mitochondrial ROS was confirmed by the protective effect of overexpression of manganese superoxide dismutase (MnSOD). MAM-induced necroptosis was independent of TNFα, p53, MLKL, and lysosomal membrane permeabilization. In summary, our study demonstrated that RIP1/RIP3 complex-triggered cytosolic calcium

  17. PLCγ2 promotes apoptosis while inhibits proliferation in rat hepatocytes through PKCD/JNK MAPK and PKCD/p38 MAPK signalling.

    PubMed

    Chen, Xiaoguang; Lv, Qiongxia; Ma, Jun; Liu, Yumei

    2018-02-11

    The PLCG2 (PLCγ2) gene is a member of PLC gene family encoding transmembrane signalling enzymes involved in various biological processes including cell proliferation and apoptosis. Our earlier study indicated that PLCγ2 may be involved in the termination of regeneration of the liver which is mainly composed of hepatocytes, but its exact biological function and molecular mechanism in liver regeneration termination remains unclear. This study aims to examine the role of PLCγ2 in the growth of hepatocytes. A recombinant adenovirus expressing PLCγ2 was used to infect primary rat hepatocytes. PLCγ2 mRNA and protein levels were detected by qRT-PCR and Western blot. The subcellular location of PLCγ2 protein was tested by an immunofluorescence assay. The proliferation of hepatocytes was measured by MTT assay. The cell cycle and apoptosis were analysed by flow cytometry. Caspase-3, -8 and -9 activities were measured by a spectrophotometry method. Phosphorylation levels of PKCD, JNK and p38 in the infected cells were detected by Western blot. The possible mechanism underlying the role of PLCγ2 in hepatocyte growth was also explored by adding a signalling pathway inhibitor. Hepatocyte proliferation was dramatically reduced, while cell apoptosis was remarkably increased. The results demonstrated that PLCγ2 increased the phosphorylation of PKCD, p38 and JNK in rat hepatocytes. After PKCD activity was inhibited by the inhibitor Go 6983, the levels of both p-p38 and p-JNK MAPKs significantly decreased, and PLCγ2-induced cell proliferation inhibition and cell apoptosis were obviously reversed. This study showed that PLCγ2 regulates hepatocyte growth through PKCD-dependently activating p38 MAPK and JNK MAPK pathways; this result was experimentally based on the further exploration of the effect of PLCγ2 on hepatocyte growth in vivo. © 2018 John Wiley & Sons Ltd.

  18. Long Non-Coding RNA CASC2 Improves Diabetic Nephropathy by Inhibiting JNK Pathway.

    PubMed

    Yang, Huihui; Kan, Quan E; Su, Yong; Man, Hua

    2018-06-11

    It's known that long non-coding RNA CASC2 overexpression inhibit the JNK pathway in some disease models, while JNK pathway activation exacerbates diabetic nephropathy. Therefore we speculate that long non-coding RNA CASC2 can improve diabetic nephropathy by inhibiting JNK pathway. Thus, our study was carried out to investigate the involvement of CASC2 in diabetic nephropathy. We found that serum level of CASC2 was significantly lower in diabetic nephropathy patients than in normal people, and serum level of CASC2 showed no significant correlations with age, gender, alcohol consumption and smoking habits, but was correlated with course of disease. ROC curve analysis showed that serum level of CASC2 could be used to accurately predict diabetic nephropathy. Diabetes mellitus has many complications. This study also included a series of complications of diabetes, such as diabetic retinopathy, diabetic ketoacidosis, diabetic foot infections and diabetic cardiopathy, while serum level of CASC2 was specifically reduced in diabetic nephropathy. CASC2 expression level decreased, while JNK1 phosphorylation level increased in mouse podocyte cells treated with high glucose. CASC2 overexpression inhibited apoptosis of podocyte cells and reduced phosphorylation level of JNK1. We conclude that long non-coding RNA CASC2 may improve diabetic nephropathy by inhibiting JNK pathway. © Georg Thieme Verlag KG Stuttgart · New York.

  19. Interleukin-6 in the injured patient. Marker of injury or mediator of inflammation?

    PubMed Central

    Biffl, W L; Moore, E E; Moore, F A; Peterson, V M

    1996-01-01

    OBJECTIVE: The effects of interleukin (IL)-6 in the injured patient are examined in an attempt to clarify the potential pathophysiologic role of IL-6 in the response to injury. SUMMARY BACKGROUND DATA: Interleukin-6 is an integral cytokine mediator of the acute phase response to injury and infection. However, prolonged and excessive elevations of circulating IL-6 levels in patients after trauma, burns, and elective surgery have been associated with complications and mortality. The mechanistic role of IL-6 in mediating these effects is unclear. METHODS: A review of current literature is performed to summarize the origins, mechanisms of action, and biologic effects of IL-6 and to characterize the IL-6 response to injury. RESULTS: Interleukin-6 is a multifunctional cytokine expressed by a variety of cells after a multitude of stimuli, under complex regulatory control mechanisms. The IL-6 response to injury is uniquely consistent and related to the magnitude of the insult. Moreover, the early postinjury IL-6 response correlates with complications as well as mortality. CONCLUSIONS: Interleukin-6 appears to play an active role in the postinjury immune response, making it an attractive therapeutic target in attempts to control hyperinflammatory provoked organ injury. Images Figure 2. PMID:8916880

  20. Regulation of B7.1 costimulatory molecule is mediated by the IFN regulatory factor-7 through the activation of JNK in lipopolysaccharide-stimulated human monocytic cells.

    PubMed

    Lim, Wilfred; Gee, Katrina; Mishra, Sasmita; Kumar, Ashok

    2005-11-01

    The engagement of CD28 or CTLA-4 with B7.1 provides the essential second costimulatory signal that regulates the development of immune responses, including T cell activation, differentiation, and induction of peripheral tolerance. The signaling molecules and the transcription factors involved in B7.1 regulation are poorly understood. In this study we investigated the role of MAPKs in the regulation of LPS-induced B7.1 expression in human monocytes and the promonocytic THP-1 cells. Our results show that LPS-induced B7.1 expression in monocytic cells did not involve the activation of either p38 or ERKs. Using the JNK-specific inhibitor SP600125, small interfering RNAs specific for JNK1 and JNK2, and agents such as dexamethasone that inhibit JNK activation, we determined that LPS-induced B7.1 expression was regulated by JNK MAPK in both monocytes and THP-1 cells. In addition, we identified a distinct B7.1-responsive element corresponding to the IFN regulatory factor-7 (IRF-7) binding site in the B7.1 promoter responsible for the regulation of LPS-induced B7.1 transcription. Furthermore, SP600125 and dexamethasone inhibited LPS-induced IRF-7 activity. Taken together, these results suggest that LPS-induced B7.1 transcription in human monocytic cells may be regulated by JNK-mediated activation of the IRF-7 transcription factor.

  1. 3-MCPD 1-Palmitate Induced Tubular Cell Apoptosis In Vivo via JNK/p53 Pathways

    PubMed Central

    Liu, Man; Huang, Guoren; Wang, Thomas T.Y.; Sun, Xiangjun; Yu, Liangli (Lucy)

    2016-01-01

    Fatty acid esters of 3-chloro-1, 2-propanediol (3-MCPD esters) are a group of processing induced food contaminants with nephrotoxicity but the molecular mechanism(s) remains unclear. This study investigated whether and how the JNK/p53 pathway may play a role in the nephrotoxic effect of 3-MCPD esters using 3-MCPD 1-palmitate (MPE) as a probe compound in Sprague Dawley rats. Microarray analysis of the kidney from the Sprague Dawley rats treated with MPE, using Gene Ontology categories and KEGG pathways, revealed that MPE altered mRNA expressions of the genes involved in the mitogen-activated protein kinase (JNK and ERK), p53, and apoptotic signal transduction pathways. The changes in the mRNA expressions were confirmed by qRT-PCR and Western blot analyses and were consistent with the induction of tubular cell apoptosis as determined by histopathological, TUNEL, and immunohistochemistry analyses in the kidneys of the Sprague Dawley rats. Additionally, p53 knockout attenuated the apoptosis, and the apoptosis-related protein bax expression and cleaved caspase-3 activation induced by MPE in the p53 knockout C57BL/6 mice, whereas JNK inhibitor SP600125 but not ERK inhibitor U0126 inhibited MPE-induced apoptosis, supporting the conclusion that JNK/p53 might play a critical role in the tubular cell apoptosis induced by MPE and other 3-MCPD fatty acid esters. PMID:27008853

  2. Isorhapontigenin (ISO) inhibited cell transformation by inducing G0/G1 phase arrest via increasing MKP-1 mRNA Stability.

    PubMed

    Gao, Guangxun; Chen, Liang; Li, Jingxia; Zhang, Dongyun; Fang, Yong; Huang, Haishan; Chen, Xiequn; Huang, Chuanshu

    2014-05-15

    The cancer chemopreventive property of Chinese herb new isolate isorhapontigenin (ISO) and mechanisms underlying its activity have never been explored. Here we demonstrated that ISO treatment with various concentrations for 3 weeks could dramatically inhibit TPA/EGF-induced cell transformation of Cl41 cells in Soft Agar assay, whereas co-incubation of cells with ISO at the same concentrations could elicit G0/G1 cell-cycle arrest without redundant cytotoxic effects on non-transformed cells. Further studies showed that ISO treatment resulted in cyclin D1 downregulation in dose- and time-dependent manner. Our results indicated that ISO regulated cyclin D1 at transcription level via targeting JNK/C-Jun/AP-1 activation. Moreover, we found that ISO-inhibited JNK/C-Jun/AP-1 activation was mediated by both upregulation of MKP-1 expression through increasing its mRNA stability and deactivating MKK7. Most importantly, MKP-1 knockdown could attenuate ISO-mediated suppression of JNK/C-Jun activation and cyclin D1 expression, as well as G0/G1 cell cycle arrest and cell transformation inhibition, while ectopic expression of FLAG-cyclin D1 T286A mutant also reversed ISO-induced G0/G1 cell-cycle arrest and inhibition of cell transformation. Our results demonstrated that ISO is a promising chemopreventive agent via upregulating mkp-1 mRNA stability, which is distinct from its cancer therapeutic effect with downregulation of XIAP and cyclin D1 expression.

  3. Hydrogen gas production is associated with reduced interleukin-1β mRNA in peripheral blood after a single dose of acarbose in Japanese type 2 diabetic patients.

    PubMed

    Tamasawa, Atsuko; Mochizuki, Kazuki; Hariya, Natsuyo; Saito, Miyoko; Ishida, Hidenori; Doguchi, Satako; Yanagiya, Syoko; Osonoi, Takeshi

    2015-09-05

    Acarbose, an α-glucosidase inhibitor, leads to the production of hydrogen gas, which reduces oxidative stress. In this study, we examined the effects of a single dose of acarbose immediately before a test meal on postprandial hydrogen gas in breath and peripheral blood interleukin (IL)-1β mRNA expression in Japanese type 2 diabetic patients. Sixteen Japanese patients (14 men, 2 women) participated in this study. The mean±standard deviation age, hemoglobin A1c and body mass index were 52.1±15.4 years, 10.2±2.0%, and 27.7±8.0kg/m(2), respectively. The patients were admitted into our hospital for 2 days and underwent test meals at breakfast without (day 1) or with acarbose (day 2). We performed continuous glucose monitoring and measured hydrogen gas levels in breath, and peripheral blood IL-1β mRNA levels before (0min) and after the test meal (hydrogen gas: 60, 120, 180, and 300min; IL-1β: 180min). The induction of hydrogen gas production and the reduction in peripheral blood IL-1β mRNA after the test meal were not significant between days 1 (without acarbose) and 2 (with acarbose). However, the changes in total hydrogen gas production from day 1 to day 2 were closely and inversely associated with the changes in peripheral blood IL-1β mRNA levels. Our results suggest that an increase in hydrogen gas production is inversely associated with a reduction of the peripheral blood IL-1β mRNA level after a single dose of acarbose in Japanese type 2 diabetic patients. Copyright © 2015 Elsevier B.V. All rights reserved.

  4. Mitochondrial JNK activation triggers autophagy and apoptosis and aggravates myocardial injury following ischemia/reperfusion.

    PubMed

    Xu, Jie; Qin, Xinghua; Cai, Xiaoqing; Yang, Lu; Xing, Yuan; Li, Jun; Zhang, Lihua; Tang, Ying; Liu, Jiankang; Zhang, Xing; Gao, Feng

    2015-02-01

    c-Jun N-terminal kinase (JNK) is a stress-activated mitogen-activated protein kinase that plays a central role in initiating apoptosis in disease conditions. Recent studies have shown that mitochondrial JNK signaling is partly responsible for ischemic myocardial dysfunction; however, the underlying mechanism remains unclear. Here we report for the first time that activation of mitochondrial JNK, rather than JNK localization on mitochondria, induces autophagy and apoptosis and aggravates myocardial ischemia/reperfusion injury. Myocardial ischemia/reperfusion induced a dominant increase of mitochondrial JNK phosphorylation, while JNK mitochondrial localization was reduced. Treatment with Tat-SabKIM1, a retro-inverso peptide which blocks JNK interaction with mitochondria, decreased mitochondrial JNK activation without affecting JNK mitochondrial localization following reperfusion. Tat-SabKIM1 treatment reduced Bcl2-regulated autophagy, cytochrome c-mediated apoptosis and myocardial infarct size. Notably, selective inhibition of mitochondrial JNK activation using Tat-SabKIM1 produced a similar infarct size-reducing effect as inhibiting universal JNK activation with JNK inhibitor SP600125. Moreover, insulin-treated animals exhibited significantly dampened mitochondrial JNK activation accompanied by reduced infarct size and diminished autophagy and apoptosis following reperfusion. Taken together, these findings demonstrate that mitochondrial JNK activation, rather than JNK mitochondrial localization, induces autophagy and apoptosis and exacerbates myocardial ischemia/reperfusion injury. Insulin selectively inhibits mitochondrial JNK activation, contributing to insulin cardioprotection against myocardial ischemic/reperfusion injury. This article is part of a Special Issue entitled: Autophagy and protein quality control in cardiometabolic diseases. Copyright © 2014 Elsevier B.V. All rights reserved.

  5. STAT6: its role in interleukin 4-mediated biological functions.

    PubMed

    Takeda, K; Kishimoto, T; Akira, S

    1997-05-01

    Interleukin (IL) 4 is known to be a cytokine which plays a central role in the regulation of immune response. Studies on cytokine signal transduction have clarified the mechanism by which IL4 exerts its functions. Two cytoplasmic proteins, signal transducer and activator of transcription (STAT) 6 and IL4-induced phosphotyrosine substrate/insulin receptor substrate 2 (4PS/IRS2), are activated in IL4 signal transduction. Recent studies from STAT6-deficient mice have revealed the essential role of STAT6 in IL4-mediated biological actions. In addition, STAT6 has also been demonstrated to be important for the functions mediated by IL13, which is related to IL4. IL4 and IL13 have been shown to induce the production of IgE, which is a major mediator in an allergic response. These findings indicate that STAT6 activation is involved in IL4- and IL13-mediated disorders such as allergy.

  6. Aberrant Axonal Arborization of PDF Neurons Induced by Aβ42-Mediated JNK Activation Underlies Sleep Disturbance in an Alzheimer's Model.

    PubMed

    Song, Qian; Feng, Ge; Huang, Zehua; Chen, Xiaoman; Chen, Zhaohuan; Ping, Yong

    2017-10-01

    Impaired sleep patterns are common symptoms of Alzheimer's disease (AD). Cellular mechanisms underlying sleep disturbance in AD remain largely unknown. Here, using a Drosophila Aβ42 AD model, we show that Aβ42 markedly decreases sleep in a large population, which is accompanied with postdevelopmental axonal arborization of wake-promoting pigment-dispersing factor (PDF) neurons. The arborization is mediated in part via JNK activation and can be reversed by decreasing JNK signaling activity. Axonal arborization and impaired sleep are correlated in Aβ42 and JNK kinase hemipterous mutant flies. Image reconstruction revealed that these aberrant fibers preferentially project to pars intercerebralis (PI), a fly brain region analogous to the mammalian hypothalamus. Moreover, PDF signaling in PI neurons was found to modulate sleep/wake activities, suggesting that excessive release of PDF by these aberrant fibers may lead to the impaired sleep in Aβ42 flies. Finally, inhibition of JNK activation in Aβ42 flies restores nighttime sleep loss, decreases Aβ42 accumulation, and attenuates neurodegeneration. These data provide a new mechanism by which sleep disturbance could be induced by Aβ42 burden, a key initiator of a complex pathogenic cascade in AD.

  7. Involvement of p38 MAPK- and JNK-modulated expression of Bcl-2 and Bax in Naja nigricollis CMS-9-induced apoptosis of human leukemia K562 cells.

    PubMed

    Chen, Ying-Jung; Liu, Wen-Hsin; Kao, Pei-Hsiu; Wang, Jeh-Jeng; Chang, Long-Sen

    2010-06-15

    CMS-9, a phospholipase A(2) (PLA(2)) isolated from Naja nigricollis venom, induced apoptosis of human leukemia K562 cells, characterized by mitochondrial depolarization, modulation of Bcl-2 family members, cytochrome c release and activation of caspases 9 and 3. Moreover, an increase in intracellular Ca2+ concentration and the production of reactive oxygen species (ROS) was noted. Pretreatment with BAPTA-AM (Ca2+ chelator) and N-acetylcysteine (NAC, ROS scavenger) proved that Ca2+ was an upstream event in inducing ROS generation. Upon exposure to CMS-9, activation of p38 MAPK and JNK was observed in K562 cells. BAPTA-AM or NAC abrogated CMS-9-elicited p38 MAPK and JNK activation, and rescued viability of CMS-9-treated K562 cells. SB202190 (p38 MAPK inhibitor) and SP600125 (JNK inhibitor) suppressed CMS-9-induced dissipation of mitochondrial membrane potential, Bcl-2 down-regulation, Bax up-regulation and increased mitochondrial translocation of Bax. Inactivation of PLA(2) activity reduced drastically the cytotoxicity of CMS-9, and a combination of lysophosphatidylcholine and stearic acid mimicked the cytotoxic effects of CMS-9. Taken together, our data suggest that CMS-9-induced apoptosis of K562 cells is catalytic activity-dependent and is mediated through mitochondria-mediated death pathway triggered by Ca2+/ROS-evoked p38 MAPK and JNK activation. 2010 Elsevier Ltd. All rights reserved.

  8. Interleukin-8 mRNA expression in synovial fluid of canine stifle joints with osteoarthritis.

    PubMed

    de Bruin, T; de Rooster, H; van Bree, H; Cox, E

    2005-12-15

    The objective of this study was to examine and compare the presence of interleukin (IL)-8 mRNA in canine stifle osteoarthritis (OA) differing in etiopathogenesis. Synovial fluid (SF) samples were collected from 24 clinically normal stifle joints and 46 diseased stifle joints (32 stifle joints with cranial cruciate ligament rupture (CCLR), 2 joints with CCLR and patella luxation (PL), 7 joints with medial PL and 5 joints with primary OA). The samples were centrifuged to collect synovial fluid cells for RNA extraction. Reverse transcription polymerase chain reaction (RT-PCR) was performed to obtain cDNA from all samples. Canine IL-8 mRNA expression was determined using real time PCR. Synovial fluid glass smears were made of all samples and coloured with H&E for differential cell counts. All stifle joints were radiographed and graded for the severity of OA. Sixty-one percent (28/46) of the samples from canine stifle OA had IL-8 mRNA expression in contrast to 4% (1/24) in the control stifle joints. This difference in prevalence is highly significant. There were no statistically significant pairwise differences among the mean ranks of the various OA groups for the absolute amount of IL-8 mRNA expression. Neither was there a link between the severity of OA (determined by radiographic evaluation) and the presence of IL-8 in the SF nor any significant difference in the absolute amount of IL-8 between the different OA grades. No statistical difference was found in differential cell counts between IL-8-positive and -negative SF samples. IL-8 cannot be used as a specific joint disease marker since IL-8 expression is found in OA differing in etiopathogenesis. It might, however, relate to the ongoing inflammation within the joint.

  9. Inhibition of Spinal Interlukin-33/ST2 Signaling and Downstream ERK and JNK Pathways in Electroacupuncture Analgesia in Formalin Mice

    PubMed Central

    Zhao, Jing; Wang, Yanqing; Wu, Gencheng; Mi, Wenli

    2015-01-01

    Although acupuncture is widely used to manage pain, it remains highly controversial, largely due to the lack of a clear mechanism for its benefits. Here, we investigated the role of IL-33, a novel interleukin (IL)-1 family member, and its receptor ST2 in the analgesic effects of electroacupuncture (EA) on formalin-induced inflammatory pain. The results showed that 1) EA stimulation of ipsilateral Zusanli (ST 36) and Yanglingquan (GB 34) acupoints for 30 min remarkably suppressed the two phases of formalin-induced spontaneous pain; 2) subcutaneous or intrathecal administration of recombinant IL-33 (rIL-33) significantly inhibited the analgesic effect of EA, whereas the ST2 antibody potentiated EA analgesia in formalin mice; 3) EA treatment decreased the up-regulation of IL-33 and ST2 protein following formalin injection; and 4) the suppression of the formalin-induced expression of spinal phosphorylated ERK and JNK induced by EA treatment was significantly attenuated following subcutaneous rIL-33 delivery, and was further decreased by the ST2 antibody. These data suggest that EA alleviates formalin-induced inflammatory pain, at least partially, by inhibiting of spinal IL-33/ST2 signaling and the downstream ERK and JNK pathways. PMID:26067287

  10. Vitamin K2 Induces Mitochondria-Related Apoptosis in Human Bladder Cancer Cells via ROS and JNK/p38 MAPK Signal Pathways

    PubMed Central

    Duan, Fengsen; Yu, Yuejin; Guan, Rijian; Xu, Zhiliang; Liang, Huageng; Hong, Ling

    2016-01-01

    The effects of vitamin K2 on apoptosis in a variety of cancer cells have been well established in previous studies. However, the apoptotic effect of vitamin K2 on bladder cancer cells has not been evaluated. The aim of this study is to examine the apoptotic activity of Vitamin K2 in bladder cancer cells and investigate the underlying mechanism. In this study, Vitamin K2 induced apoptosis in bladder cancer cells through mitochondria pathway including loss of mitochondria membrane potential, cytochrome C release and caspase-3 cascade. Furthermore, the phosphorylation of c-Jun N-terminal kinase (JNK) and p38 MAPK was detected in Vitamin K2-treated cells and both SP600125 (an inhibitor of JNK) and SB203580 (an inhibitor of p38 MAPK) completely abolished the Vitamin K2-induced apoptosis and loss of mitochondria membrane potential. Moreover, the generation of reactive oxygen species (ROS) was detected in bladder cancer cells, upon treatment of vitamin K2 and the anti-oxidant N-acetyl cysteine (NAC) almost blocked the Vitamin K2-triggered apoptosis, loss of mitochondria membrane potential and activation of JNK and p38 MAPK. Taken together, these findings revealed that Vitamin K2 induces apoptosis in bladder cancer cells via ROS-mediated JNK/p38 MAPK and Mitochondrial pathways. PMID:27570977

  11. Vitamin K2 Induces Mitochondria-Related Apoptosis in Human Bladder Cancer Cells via ROS and JNK/p38 MAPK Signal Pathways.

    PubMed

    Duan, Fengsen; Yu, Yuejin; Guan, Rijian; Xu, Zhiliang; Liang, Huageng; Hong, Ling

    2016-01-01

    The effects of vitamin K2 on apoptosis in a variety of cancer cells have been well established in previous studies. However, the apoptotic effect of vitamin K2 on bladder cancer cells has not been evaluated. The aim of this study is to examine the apoptotic activity of Vitamin K2 in bladder cancer cells and investigate the underlying mechanism. In this study, Vitamin K2 induced apoptosis in bladder cancer cells through mitochondria pathway including loss of mitochondria membrane potential, cytochrome C release and caspase-3 cascade. Furthermore, the phosphorylation of c-Jun N-terminal kinase (JNK) and p38 MAPK was detected in Vitamin K2-treated cells and both SP600125 (an inhibitor of JNK) and SB203580 (an inhibitor of p38 MAPK) completely abolished the Vitamin K2-induced apoptosis and loss of mitochondria membrane potential. Moreover, the generation of reactive oxygen species (ROS) was detected in bladder cancer cells, upon treatment of vitamin K2 and the anti-oxidant N-acetyl cysteine (NAC) almost blocked the Vitamin K2-triggered apoptosis, loss of mitochondria membrane potential and activation of JNK and p38 MAPK. Taken together, these findings revealed that Vitamin K2 induces apoptosis in bladder cancer cells via ROS-mediated JNK/p38 MAPK and Mitochondrial pathways.

  12. [The administration of interleukin-1beta during early postnatal develop ment impairs FGF2, but not TIMP1, mRNA expression in brain structures of adult rats].

    PubMed

    Trofimov, A N; Zubareva, O E; Shvarts, A P; Ishchenko, A M; Klimenko, V M

    2014-09-01

    According to the Neurodevelopmental hypothesis, the long-lasting cognitive deficit in schizophrenia and other types of neuropathology may occur by injurious factors, such as hypoxia, traumas, infections that take place during pre- and postnatal development, at least at early stages. These pathological conditions are often associated with the high production of pro-inflammatory cytokine interleukin-1B (IL-1B) by the cells of immune and nervous systems. We investigated the expression of genes involved in the neuroplastic regulation (Fgf2 and Timp2) in medial prefrontal cortex and dorsal and ventral regions of hippocampus of adult rats that were treated with IL-1beta between P15 and P21. The learning impairment in IL-1beta-treated rats is accompanied by lower FGF-2 mRNA levels in medial prefrontal cortex and ventral (not dorsal) hippocampus, but TIMP-1 was not affected. No differences in TIMP-1 and FGF-2 mRNA expressions were observed in untrained IL-1beta-treated when compared to control rats.

  13. TGF-beta and HGF transmit the signals through JNK-dependent Smad2/3 phosphorylation at the linker regions.

    PubMed

    Mori, Shigeo; Matsuzaki, Koichi; Yoshida, Katsunori; Furukawa, Fukiko; Tahashi, Yoshiya; Yamagata, Hideo; Sekimoto, Go; Seki, Toshihito; Matsui, Hirofumi; Nishizawa, Mikio; Fujisawa, Jun-ichi; Okazaki, Kazuichi

    2004-09-23

    Although hepatocyte growth factor (HGF) can act synergistically or antagonistically with transforming growth factor-beta (TGF-beta) signaling, molecular mechanism of their crosstalk remains unknown. Using antibodies which selectively distinguished receptor-regulated Smads (R-Smads) phosphorylated at linker regions from those at C-terminal regions, we herein showed that either HGF or TGF-beta treatment of normal stomach-origin cells activated the JNK pathway, thereafter inducing endogenous R-Smads phosphorylation at linker regions. However, the phosphorylation at their C-terminal regions was not induced by HGF treatment. The activated JNK could directly phosphorylate R-Smads in vitro at the same sites that were phosphorylated in response to TGF-beta or HGF in vivo. Thus, the linker regions of R-Smads were the common phosphorylation sites for HGF and TGF-beta signaling pathways. The phosphorylation induced by simultaneous treatment with HGF and TGF-beta allowed R-Smads to associate with Smad4 and to translocate into the nucleus. JNK pathway involved HGF and TGF-beta-mediated infiltration potency since a JNK inhibitor SP600125 caused the reduction of invasive capacity induced by HGF and TGF-beta signals. Moreover, a combined treatment with HGF and TGF-beta led to a potent increase in plasminogen activator inhibitor type 1 transcriptional activity through Smad3 phosphorylation at the linker region. In contrast, HGF treatment reduced TGF-beta-dependent activation of p15INK4B promoter, in which Smad3 phosphorylation at the C-terminal region was involved. In conclusion, HGF and TGF-beta transmit the signals through JNK-mediated R-Smads phosphorylation at linker regions.

  14. Involvement of MAPKs, NF-{kappa}B and p300 co-activator in IL-1{beta}-induced cytosolic phospholipase A{sub 2} expression in canine tracheal smooth muscle cells

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

    Luo, S.-F.; Lin, C.-C.; Chen, H.-C.

    2008-11-01

    Cytosolic phospholipase A{sub 2} (cPLA{sub 2}) plays a pivotal role in mediating agonist-induced arachidonic acid release for prostaglandin (PG) synthesis during stimulation with interleukin-1{beta} (IL-1{beta}). However, the mechanisms underlying IL-1{beta}-induced cPLA{sub 2} expression and PGE{sub 2} synthesis by canine tracheal smooth muscle cells (CTSMCs) have not been defined. IL-1{beta} induced cPLA{sub 2} protein and mRNA expression, PGE{sub 2} production, and phosphorylation of p42/p44 MAPK, p38 MAPK (ATF{sub 2}), and JNK (c-Jun) in a time- and concentration-dependent manner, determined by Western blotting, RT-PCR, and ELISA, which was attenuated by the inhibitors of MEK1/2 (U0126), p38 MAPK (SB202190), and JNK (SP600125), ormore » transfection with dominant negative mutants of MEK1/2, p38, and JNK, respectively. Furthermore, IL-1{beta}-induced cPLA{sub 2} expression and PGE{sub 2} synthesis was inhibited by a selective NF-{kappa}B inhibitor (helenalin) or transfection with dominant negative mutants of NF-{kappa}B inducing kinase (NIK), I{kappa}B kinase (IKK)-{alpha}, and IKK-{beta}. Consistently, IL-1{beta} stimulated both I{kappa}B-{alpha} degradation and NF-{kappa}B translocation into nucleus in these cells. NF-{kappa}B translocation was blocked by helenalin, but not by U0126, SB202190, and SP600125. MAPKs together with NF-{kappa}B-activated p300 recruited to cPLA{sub 2} promoter thus facilitating the binding of NF-{kappa}B to cPLA{sub 2} promoter region and expression of cPLA{sub 2} mRNA. IL-1{beta}-induced cPLA{sub 2} expression and PGE{sub 2} production was inhibited by actinomycin D and cycloheximide, indicating the involvement of transcriptional and translational events in these responses. These results suggest that in CTSMCs, IL-1{beta}-induced cPLA{sub 2} expression and PGE{sub 2} synthesis was independently mediated through activation of MAPKs and NF-{kappa}B pathways and was connected to p300 recruitment and activation.« less

  15. Determining if an mRNA is a Substrate of Nonsense-Mediated mRNA Decay in Saccharomyces cerevisiae.

    PubMed

    Johansson, Marcus J O

    2017-01-01

    Nonsense-mediated mRNA decay (NMD) is a conserved eukaryotic quality control mechanism which triggers decay of mRNAs harboring premature translation termination codons. In this chapter, I describe methods for monitoring the influence of NMD on mRNA abundance and decay rates in Saccharomyces cerevisiae. The descriptions include detailed methods for growing yeast cells, total RNA isolation, and Northern blotting. Although the chapter focuses on NMD, the methods can be easily adapted to assess the effect of other mRNA decay pathways.

  16. Cafeteria feeding induces interleukin-1beta mRNA expression in rat liver and brain.

    PubMed

    Hansen, M K; Taishi, P; Chen, Z; Krueger, J M

    1998-06-01

    intake affects gut-immune function and can provide a strong intestinal antigen challenge resulting in activation of host defense mechanisms in the digestive system. Previously, we showed that feeding rats a cafeteria diet increases non-rapid eye movement sleep by a subdiaphragmatic mechanism. Food intake and sleep regulation and the immune system share the regulatory molecule interleukin-1beta (IL-1beta). Thus this study examined the effects of a cafeteria diet on IL-1beta mRNA and IL-1 receptor accessory protein (IL-1RAP) mRNA expression in rat liver and brain. Rats were fed normal rat chow or a palatable diet consisting of bread, chocolate, and shortbread cookies (cafeteria diet). After 3 days, midway between the light period of the light-dark cycle, rats were killed by decapitation. Feeding rats a cafeteria diet resulted in increased IL-1beta mRNA expression in the liver and hypothalamus compared with rats fed only the normal rat chow. In addition, cafeteria feeding decreased IL-1RAP mRNA levels in the liver and brain stem. These results indicate that feeding has direct effects on cytokine production and together with other data suggest that the increased sleep that accompanies increased feeding may be the result of increased brain IL-1beta. These results further suggest that cytokine-to-brain communication may be important in normal physiological conditions, such as feeding, as well as being important during inflammatory responses.

  17. Growth Cone MKK7 mRNA Targeting Regulates MAP1b-Dependent Microtubule Bundling to Control Neurite Elongation

    PubMed Central

    Feltrin, Daniel; Fusco, Ludovico; Witte, Harald; Moretti, Francesca; Martin, Katrin; Letzelter, Michel; Fluri, Erika; Scheiffele, Peter; Pertz, Olivier

    2012-01-01

    Local mRNA translation in neurons has been mostly studied during axon guidance and synapse formation but not during initial neurite outgrowth. We performed a genome-wide screen for neurite-enriched mRNAs and identified an mRNA that encodes mitogen-activated protein kinase kinase 7 (MKK7), a MAP kinase kinase (MAPKK) for Jun kinase (JNK). We show that MKK7 mRNA localizes to the growth cone where it has the potential to be translated. MKK7 is then specifically phosphorylated in the neurite shaft, where it is part of a MAP kinase signaling module consisting of dual leucine zipper kinase (DLK), MKK7, and JNK1. This triggers Map1b phosphorylation to regulate microtubule bundling leading to neurite elongation. We propose a model in which MKK7 mRNA localization and translation in the growth cone allows for a mechanism to position JNK signaling in the neurite shaft and to specifically link it to regulation of microtubule bundling. At the same time, this uncouples activated JNK from its functions relevant to nuclear translocation and transcriptional activation. PMID:23226105

  18. CYP3A5 mRNA degradation by nonsense-mediated mRNA decay.

    PubMed

    Busi, Florent; Cresteil, Thierry

    2005-09-01

    The total CYP3A5 mRNA level is significantly greater in carriers of the CYP3A5*1 allele than in CYP3A5*3 homozygotes. Most of the CYP3A5*3 mRNA includes an intronic sequence (exon 3B) containing premature termination codons (PTCs) between exons 3 and 4. Two models were used to investigate the degradation of CYP3A5 mRNA: a CYP3A5 minigene consisting of CYP3A5 exons and introns 3 to 6 transfected into MCF7 cells, and the endogenous CYP3A5 gene expressed in HepG2 cells. The 3'-untranslated region g.31611C>T mutation has no effect on CYP3A5 mRNA decay. Splice variants containing exon 3B were more unstable than wild-type (wt) CYP3A5 mRNA. Cycloheximide prevents the recognition of PTCs by ribosomes: in transfected MCF7 and HepG2 cells, cycloheximide slowed down the degradation of exon 3B-containing splice variants, suggesting the participation of nonsense-mediated decay (NMD). When PTCs were removed from pseudoexon 3B or when UPF1 small interfering RNA was used to impair the NMD mechanism, the decay of the splice variant was reduced, confirming the involvement of NMD in the degradation of CYP3A5 splice variants. Induction could represent a source of variability for CYP3A5 expression and could modify the proportion of splice variants. The extent of CYP3A5 induction was investigated after exposure to barbiturates or steroids: CYP3A4 was markedly induced in a pediatric population compared with untreated neonates. However, no effect could be detected in either the total CYP3A5 RNA, the proportion of splice variant RNA, or the protein level. Therefore, in these carriers, induction is unlikely to switch on the phenotypic CYP3A5 expression in carriers of CYP3A5*3/*3.

  19. Blockade of the spinal BDNF-activated JNK pathway prevents the development of antiretroviral-induced neuropathic pain.

    PubMed

    Sanna, Maria Domenica; Ghelardini, Carla; Galeotti, Nicoletta

    2016-06-01

    Although antiretroviral agents have been used successfully in suppressing viral production, they have also been associated with a number of side effects. The antiretroviral toxic neuropathy induces debilitating and extremely difficult to treat pain syndromes that often lead to discontinuation of antiretroviral therapy. Due to the critical need for the identification of novel therapeutic targets to improve antiretroviral neuropathic pain management, we investigated the role of the JNK signalling pathway in the mechanism of antiretroviral painful neuropathy. Mice were exposed to zalcitabine (2',3'-dideoxycytidine, ddC) and stavudine (2',3'-didehydro-3'-deoxythymidine, d4T) that induced a persistent mechanical allodynia and a transient cold allodynia. Treatment with the JNK blocker SP600125 before antiretroviral administration abolished mechanical hypersensitivity with no effect on thermal response. A robust spinal JNK overphosphorylation was observed on post-injection day 1 and 3, along with a JNK-dependent increase in p-c-Jun and ATF3 protein levels. Co-immunoprecipitation experiments showed the presence of a heterodimeric complex between ATF3 and c-Jun indicating that these transcription factors can act together to regulate transcription through heterodimerization. A rise in BDNF and caspase-3 protein levels was detected on day 1 and BDNF sequestration prevented both caspase-3 and p-JNK increase. These data suggest that BDNF plays a role in the early stages of ddC-induced allodynia by promoting apoptotic events and the activation of a hypernociceptive JNK-mediated pathway. We illustrated the activation of a BDNF-mediated JNK pathway involved in the early events responsible for the promotion of neuropathic pain, leading to a better knowledge of the mechanisms involved in the antiretroviral neuropathy. JNK blockade prevents antiretroviral-induced pain hypersensitivity. This may represent a potential prophylactic treatment of neuropathic pain to improve antiretroviral

  20. Phospholipase C-{delta}{sub 1} regulates interleukin-1{beta} and tumor necrosis factor-{alpha} mRNA expression

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

    Chung, Eric; Jakinovich, Paul; Bae, Aekyung

    Phospholipase C-{delta}{sub 1} (PLC{delta}{sub 1}) is a widely expressed highly active PLC isoform, modulated by Ca{sup 2+} that appears to operate downstream from receptor signaling and has been linked to regulation of cytokine production. Here we investigated whether PLC{delta}{sub 1} modulated expression of the pro-inflammatory cytokines interleukin-1{beta} (IL-1{beta}), tumor necrosis factor-{alpha} (TNF-{alpha}) and interleukin-6 (IL-6) in rat C6 glioma cells. Expression of PLC{delta}{sub 1} was specifically suppressed by small interfering RNA (siRNA) and the effects on cytokine mRNA expression, stimulated by the Toll-like receptor (TLR) agonist, lipopolysaccharide (LPS), were examined. Real-time polymerase chain reaction (RT-PCR) results showed that PLC{delta}{sub 1}more » knockdown enhanced expression IL-1{beta} and tumor necrosis factor-{alpha} (TNF-{alpha}) mRNA by at least 100 fold after 4 h of LPS stimulation compared to control siRNA treatment. PLC{delta}{sub 1} knock down caused persistently high Nf{kappa}b levels at 4 h of LPS stimulation compared to control siRNA-treated cells. PLC{delta}{sub 1} knockdown was also associated with elevated nuclear levels of c-Jun after 30 min of LPS stimulation, but did not affect LPS-stimulated p38 or p42/44 MAPK phosphorylation, normally associated with TLR activation of cytokine gene expression; rather, enhanced protein kinase C (PKC) phosphorylation of cellular proteins was observed in the absence of LPS stimulation. An inhibitor of PKC, bisindolylmaleimide II (BIM), reversed phosphorylation, prevented elevation of nuclear c-Jun levels, and inhibited LPS-induced increases of IL-1{beta} and TNF-{alpha} mRNA's induced by PLC{delta}{sub 1} knockdown. Our results show that loss of PLC{delta}{sub 1} enhances PKC/c-Jun signaling and up-modulates pro-inflammatory cytokine gene transcription in concert with the TLR-stimulated p38MAPK/Nf{kappa}b pathway. Our findings are consistent with the idea that PLC{delta}{sub 1} is

  1. Dual p38/JNK Mitogen Activated Protein Kinase Inhibitors Prevent Ozone-Induced Airway Hyperreactivity in Guinea Pigs

    PubMed Central

    Verhein, Kirsten C.; Salituro, Francesco G.; Ledeboer, Mark W.; Fryer, Allison D.; Jacoby, David B.

    2013-01-01

    Ozone exposure causes airway hyperreactivity and increases hospitalizations resulting from pulmonary complications. Ozone reacts with the epithelial lining fluid and airway epithelium to produce reactive oxygen species and lipid peroxidation products, which then activate cell signaling pathways, including the mitogen activated protein kinase (MAPK) pathway. Both p38 and c-Jun NH2 terminal kinase (JNK) are MAPK family members that are activated by cellular stress and inflammation. To test the contribution of both p38 and JNK MAPK to ozone-induced airway hyperreactivity, guinea pigs were pretreated with dual p38 and JNK MAPK inhibitors (30 mg/kg, ip) 60 minutes before exposure to 2 ppm ozone or filtered air for 4 hours. One day later airway reactivity was measured in anesthetized animals. Ozone caused airway hyperreactivity one day post-exposure, and blocking p38 and JNK MAPK completely prevented ozone-induced airway hyperreactivity. Blocking p38 and JNK MAPK also suppressed parasympathetic nerve activity in air exposed animals, suggesting p38 and JNK MAPK contribute to acetylcholine release by airway parasympathetic nerves. Ozone inhibited neuronal M2 muscarinic receptors and blocking both p38 and JNK prevented M2 receptor dysfunction. Neutrophil influx into bronchoalveolar lavage was not affected by MAPK inhibitors. Thus p38 and JNK MAPK mediate ozone-induced airway hyperreactivity through multiple mechanisms including prevention of neuronal M2 receptor dysfunction. PMID:24058677

  2. JNK Signaling: Regulation and Functions Based on Complex Protein-Protein Partnerships

    PubMed Central

    Zeke, András; Misheva, Mariya

    2016-01-01

    SUMMARY The c-Jun N-terminal kinases (JNKs), as members of the mitogen-activated protein kinase (MAPK) family, mediate eukaryotic cell responses to a wide range of abiotic and biotic stress insults. JNKs also regulate important physiological processes, including neuronal functions, immunological actions, and embryonic development, via their impact on gene expression, cytoskeletal protein dynamics, and cell death/survival pathways. Although the JNK pathway has been under study for >20 years, its complexity is still perplexing, with multiple protein partners of JNKs underlying the diversity of actions. Here we review the current knowledge of JNK structure and isoforms as well as the partnerships of JNKs with a range of intracellular proteins. Many of these proteins are direct substrates of the JNKs. We analyzed almost 100 of these target proteins in detail within a framework of their classification based on their regulation by JNKs. Examples of these JNK substrates include a diverse assortment of nuclear transcription factors (Jun, ATF2, Myc, Elk1), cytoplasmic proteins involved in cytoskeleton regulation (DCX, Tau, WDR62) or vesicular transport (JIP1, JIP3), cell membrane receptors (BMPR2), and mitochondrial proteins (Mcl1, Bim). In addition, because upstream signaling components impact JNK activity, we critically assessed the involvement of signaling scaffolds and the roles of feedback mechanisms in the JNK pathway. Despite a clarification of many regulatory events in JNK-dependent signaling during the past decade, many other structural and mechanistic insights are just beginning to be revealed. These advances open new opportunities to understand the role of JNK signaling in diverse physiological and pathophysiological states. PMID:27466283

  3. JNK Signaling: Regulation and Functions Based on Complex Protein-Protein Partnerships.

    PubMed

    Zeke, András; Misheva, Mariya; Reményi, Attila; Bogoyevitch, Marie A

    2016-09-01

    The c-Jun N-terminal kinases (JNKs), as members of the mitogen-activated protein kinase (MAPK) family, mediate eukaryotic cell responses to a wide range of abiotic and biotic stress insults. JNKs also regulate important physiological processes, including neuronal functions, immunological actions, and embryonic development, via their impact on gene expression, cytoskeletal protein dynamics, and cell death/survival pathways. Although the JNK pathway has been under study for >20 years, its complexity is still perplexing, with multiple protein partners of JNKs underlying the diversity of actions. Here we review the current knowledge of JNK structure and isoforms as well as the partnerships of JNKs with a range of intracellular proteins. Many of these proteins are direct substrates of the JNKs. We analyzed almost 100 of these target proteins in detail within a framework of their classification based on their regulation by JNKs. Examples of these JNK substrates include a diverse assortment of nuclear transcription factors (Jun, ATF2, Myc, Elk1), cytoplasmic proteins involved in cytoskeleton regulation (DCX, Tau, WDR62) or vesicular transport (JIP1, JIP3), cell membrane receptors (BMPR2), and mitochondrial proteins (Mcl1, Bim). In addition, because upstream signaling components impact JNK activity, we critically assessed the involvement of signaling scaffolds and the roles of feedback mechanisms in the JNK pathway. Despite a clarification of many regulatory events in JNK-dependent signaling during the past decade, many other structural and mechanistic insights are just beginning to be revealed. These advances open new opportunities to understand the role of JNK signaling in diverse physiological and pathophysiological states. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  4. Sulforaphane attenuates microglia-mediated neuronal necroptosis through down-regulation of MAPK/NF-κB signaling pathways in LPS-activated BV-2 microglia.

    PubMed

    Qin, Sisi; Yang, Canhong; Huang, Weihua; Du, Shuhua; Mai, Hantao; Xiao, Jijie; Lü, Tianming

    2018-01-31

    Sulforaphane (SFN), a natural dietary isothiocyanate in cruciferous vegetables such as broccoli and cabbage, has very strong anti-inflammatory activity. Activation of microglia leads to overexpression of a series of pro-inflammatory mediators, which play a vital role in neuronal damage. SFN may have neuroprotective effects in different neurodegenerative diseases related to inflammation. However, the mechanisms underlying SFN's protection of neurons against microglia-mediated neuronal damage are not fully understood. Here, we investigated how SFN attenuated microglia-mediated neuronal damage. Our results showed that SFN could not directly protect the viability of neurons following pro-inflammatory mediators, but increased the viability of BV-2 microglia and down-regulated the mRNA and protein levels of pro-inflammatory mediators including TNF-α, IL-1β, IL-6 and iNOS in a concentration-dependent manner in BV-2 cells. SFN also significantly blocked the phosphorylation of MAPKs (p38, JNK, and ERK1/2) and NF-κB p65, both by itself and with MAPK inhibitors (SB203580, SP 600125, and U0126) or an NF-κB inhibitor (PDTC). The expression of pro-inflammatory proteins was also blocked by SFN with or without inhibitors. Further, SFN indirectly increased the viability and maintained the morphology of neurons, and the protein expression of RIPK3 and MLKL was significantly suppressed by SFN in neuronal necroptosis through p38, JNK, and NF-κB p65 but not ERK1/2 signaling pathways. Together, our results demonstrate that SFN attenuates LPS-induced pro-inflammatory responses through down-regulation of MAPK/NF-κB signaling pathway in BV-2 microglia and thus indirectly suppresses microglia-mediated neuronal damage. Copyright © 2018 Elsevier Ltd. All rights reserved.

  5. l-Theanine prevents ETEC-induced liver damage by reducing intrinsic apoptotic response and inhibiting ERK1/2 and JNK1/2 signaling pathways.

    PubMed

    Gong, Zhihua; Liu, Qiuling; Lin, Ling; Deng, Yanli; Cai, Shuxian; Liu, Zunying; Zhang, Sheng; Xiao, Wenjun; Xiong, Shuo; Chen, Dong

    2018-01-05

    l-Theanine (LTA; γ-glutamylethylamide), a peculiar non-protein-derived amino acid isolated from tea, is widely used as a functional ingredient and dietary supplement. l-Theanine has been confirmed to have hepatoprotective effects, but the underlying mechanism remains unknown. This study investigated the protective effect of l-Theanine-in vivo, using an enterotoxigenic Escherichia coli (ETEC)-infected mouse model. l-Theanine significantly decreased the elevated serum activities of both aspartate aminotransferase (AST) and alanine aminotransferase (ALT), two biomarkers of hepatic impairment. This was consistent with histopathological images from the microscopic observation of liver tissue. In addition, l-theanine significantly increased the mRNA and protein expression of Bcl-2 and decreased the expression of Bax, anti- and pro-apoptotic molecules, respectively, compared with levels in the ETEC control group. The expression of cleaved caspase-3 protein in the group pre-treated with l-theanine was significantly lower than that in the ETEC group. Additionally, decreases in extracellular signal-regulated kinase (ERK1/2) and c-Jun NH 2 -terminal kinase(JNK1/2) MAPK phosphorylation were observed in the l-theanine pre-treated group. Our study demonstrates that l-theanine possesses anti-apoptotic activity, which can be attributed to suppression of the intrinsic mitochondria-mediated apoptosis and MAPK phosphorylation signaling pathways. Copyright © 2017 Elsevier B.V. All rights reserved.

  6. Akt mediates 17beta-estradiol and/or estrogen receptor-alpha inhibition of LPS-induced tumor necresis factor-alpha expression and myocardial cell apoptosis by suppressing the JNK1/2-NFkappaB pathway.

    PubMed

    Liu, Chung-Jung; Lo, Jeng-Fan; Kuo, Chia-Hua; Chu, Chun-Hsien; Chen, Li-Ming; Tsai, Fuu-Jen; Tsai, Chang-Hai; Tzang, Bor-Show; Kuo, Wei-Wen; Huang, Chih-Yang

    2009-09-01

    Evidence shows that women have lower tumour necrosis factor-alpha (TNF-alpha) levels and lower incidences of heart dysfunction and sepsis-related morbidity and mortality. To identify the cardioprotective effects and precise cellular/molecular mechanisms behind estrogen and estrogen receptors (ERs), we investigated the effects of 17beta-estradiol (E(2)) and estrogen receptor alpha (ERalpha) on LPS-induced apoptosis by analyzing the activation of survival and death signalling pathways in doxycycline (Dox)-inducible Tet-On/ERalpha H9c2 myocardial cells and ERalpha-transfected primary cardiomyocytes overexpressing ERalpha. We found that LPS challenge activated JNK1/2, and then induced IkappaB degradation, NFkappaB activation, TNF-alpha up-regulation and subsequent myocardial apoptotic responses. In addition, treatments involving E(2), membrane-impermeable BSA-E(2) and/or Dox, which induces ERalpha overexpression, significantly inhibited LPS-induced apoptosis by suppressing LPS-up-regulated JNK1/2 activity, IkappaB degradation, NFkappaB activation and pro-apoptotic proteins (e.g. TNF-alpha, active caspases-8, t-Bid, Bax, released cytochrome c, active caspase-9, active caspase-3) in myocardial cells. However, the cardioprotective properties of E(2), BSA-E(2) and ERalpha overexpression to inhibit LPS-induced apoptosis and promote cell survival were attenuated by applying LY294002 (PI3K inhibitor) and PI3K siRNA. These findings suggest that E(2), BSA-E(2) and ERalpha expression exert their cardioprotective effects by inhibiting JNK1/2-mediated LPS-induced TNF-alpha expression and cardiomyocyte apoptosis through activation of Akt.

  7. Tristetraprolin regulation of interleukin 23 mRNA stability prevents a spontaneous inflammatory disease

    PubMed Central

    Molle, Céline; Zhang, Tong; Ysebrant de Lendonck, Laure; Gueydan, Cyril; Andrianne, Mathieu; Sherer, Félicie; Van Simaeys, Gaetan; Blackshear, Perry J.; Leo, Oberdan

    2013-01-01

    Interleukin (IL) 12 and IL23 are two related heterodimeric cytokines produced by antigen-presenting cells. The balance between these two cytokines plays a crucial role in the control of Th1/Th17 responses and autoimmune inflammation. Most studies focused on their transcriptional regulation. Herein, we explored the role of the adenine and uridine–rich element (ARE)–binding protein tristetraprolin (TTP) in influencing mRNA stability of IL12p35, IL12/23p40, and IL23p19 subunits. LPS-stimulated bone marrow–derived dendritic cells (BMDCs) from TTP−/− mice produced normal levels of IL12/23p40. Production of IL12p70 was modestly increased in these conditions. In contrast, we observed a strong impact of TTP on IL23 production and IL23p19 mRNA stability through several AREs in the 3′ untranslated region. TTP−/− mice spontaneously develop an inflammatory syndrome characterized by cachexia, myeloid hyperplasia, dermatitis, and erosive arthritis. We observed IL23p19 expression within skin lesions associated with exacerbated IL17A and IL22 production by infiltrating γδ T cells and draining lymph node CD4 T cells. We demonstrate that the clinical and immunological parameters associated with TTP deficiency were completely dependent on the IL23–IL17A axis. We conclude that tight control of IL23 mRNA stability by TTP is critical to avoid severe inflammation. PMID:23940256

  8. Tristetraprolin regulation of interleukin 23 mRNA stability prevents a spontaneous inflammatory disease.

    PubMed

    Molle, Céline; Zhang, Tong; Ysebrant de Lendonck, Laure; Gueydan, Cyril; Andrianne, Mathieu; Sherer, Félicie; Van Simaeys, Gaetan; Blackshear, Perry J; Leo, Oberdan; Goriely, Stanislas

    2013-08-26

    Interleukin (IL) 12 and IL23 are two related heterodimeric cytokines produced by antigen-presenting cells. The balance between these two cytokines plays a crucial role in the control of Th1/Th17 responses and autoimmune inflammation. Most studies focused on their transcriptional regulation. Herein, we explored the role of the adenine and uridine-rich element (ARE)-binding protein tristetraprolin (TTP) in influencing mRNA stability of IL12p35, IL12/23p40, and IL23p19 subunits. LPS-stimulated bone marrow-derived dendritic cells (BMDCs) from TTP(-/-) mice produced normal levels of IL12/23p40. Production of IL12p70 was modestly increased in these conditions. In contrast, we observed a strong impact of TTP on IL23 production and IL23p19 mRNA stability through several AREs in the 3' untranslated region. TTP(-/-) mice spontaneously develop an inflammatory syndrome characterized by cachexia, myeloid hyperplasia, dermatitis, and erosive arthritis. We observed IL23p19 expression within skin lesions associated with exacerbated IL17A and IL22 production by infiltrating γδ T cells and draining lymph node CD4 T cells. We demonstrate that the clinical and immunological parameters associated with TTP deficiency were completely dependent on the IL23-IL17A axis. We conclude that tight control of IL23 mRNA stability by TTP is critical to avoid severe inflammation.

  9. The Role of Interleukin-1 and Interleukin-18 in Pro-Inflammatory and Anti-Viral Responses to Rhinovirus in Primary Bronchial Epithelial Cells

    PubMed Central

    Kay, Linda; Parker, Lisa C.; Sabroe, Ian; Sleeman, Matthew A.; Briend, Emmanuel; Finch, Donna K.

    2013-01-01

    Human Rhinovirus (HRV) is associated with acute exacerbations of chronic respiratory disease. In healthy individuals, innate viral recognition pathways trigger release of molecules with direct anti-viral activities and pro-inflammatory mediators which recruit immune cells to support viral clearance. Interleukin-1alpha (IL-1α), interleukin-1beta (IL-1β) and interleukin-18 (IL-18) have critical roles in the establishment of neutrophilic inflammation, which is commonly seen in airways viral infection and thought to be detrimental in respiratory disease. We therefore investigated the roles of these molecules in HRV infection of primary human epithelial cells. We found that all three cytokines were released from infected epithelia. Release of these cytokines was not dependent on cell death, and only IL-1β and IL-18 release was dependent on caspase-1 catalytic activity. Blockade of IL-1 but not IL-18 signaling inhibited up-regulation of pro-inflammatory mediators and neutrophil chemoattractants but had no effect on virus induced production of interferons and interferon-inducible genes, measured at both mRNA and protein level. Similar level of virus mRNA was detected with and without IL-1RI blockade. Hence IL-1 signaling, potentially involving both IL-1β and IL-1α, downstream of viral recognition plays a key role in induction of pro-inflammatory signals and potentially in recruitment and activation of immune cells in response to viral infection instigated by the epithelial cells, whilst not participating in direct anti-viral responses. PMID:23723976

  10. Prostaglandin E2 mediates growth arrest in NFS-60 cells by down-regulating interleukin-6 receptor expression.

    PubMed

    de Silva, Kumudika I; Daud, Asif N; Deng, JiangPing; Jones, Stephen B; Gamelli, Richard L; Shankar, Ravi

    2003-02-15

    Interleukin-6 (IL-6), a potent myeloid mitogen, and the immunosuppressive prostanoid prostaglandin E2 (PGE2) are elevated following thermal injury and sepsis. We have previously demonstrated that bone marrow myeloid commitment shifts toward monocytopoiesis and away from granulocytopoiesis during thermal injury and sepsis and that PGE2 plays a central role in this alteration. Here we investigated whether PGE2 can modulate IL-6-stimulated growth in the promyelocytic cell line, NFS-60, by down-regulating IL-6 receptor (IL-6r) expression. Exposure of NFS-60 cells to PGE2 suppressed IL-6-stimulated proliferation as well as IL-6r expression. Receptor down-regulation is functionally significant since IL-6-induced signal transduction through activators of transcription (STAT)-3 is also decreased. Down-regulation of IL-6r correlated with the ability of PGE2 to arrest cells in the G0/G1 phase of the cell cycle. PGE2 appears to signal through EP2 receptors. Butaprost (EP2 agonist) but not sulprostone (EP3 agonist) inhibited IL-6-stimulated proliferation. In addition, an EP2 antagonist (AH6809) alleviated the anti-proliferative effects of PGE2. NFS-60 cells express predominantly EP2 and EP4 receptors. While PGE2 down-regulated both the IL-6r protein and mRNA expression, it had no influence on EP2 or EP4 mRNA expression. The present study demonstrates that PGE2 is a potent down-regulator of IL-6r expression and thus may provide a mechanistic explanation for the granulocytopenia seen in thermal injury and sepsis.

  11. Bornyl caffeate induces apoptosis in human breast cancer MCF-7 cells via the ROS- and JNK-mediated pathways

    PubMed Central

    Yang, Chuan-bin; Pei, Wei-jing; Zhao, Jia; Cheng, Yuan-yuan; Zheng, Xiao-hui; Rong, Jian-hui

    2014-01-01

    Aim: To investigate the effects of bornyl caffeate discovered in several species of plant on human breast cancer cells in vitro and the underlying mechanisms. Methods: Human breast cancer cell line MCF-7 and other tumor cell lines (T47D, HepG2, HeLa, and PC12) were tested. Cell viability was determined using MTT assay, and apoptosis was defined by monitoring the morphology of the nuclei and staining with Annexin V-FITC. Mitochondrial membrane potential (MMP) was measured using JC-1 under fluorescence microscopy. Intracellular reactive oxygen species (ROS) were assessed by flow cytometry. The expression of apoptosis-associated proteins was determined by Western blotting analysis. Results: Bornyl caffeate (10, 25, and 50 μmol/L) suppressed the viability of MCF-7 cells in dose- and time-dependent manners, but neither caffeic acid nor borneol showed cytotoxicity at a concentration of 50 μmol/L. Bornyl caffeate also exerted cytotoxicity to HepG2, Hela, T47D, and PC12 cells. Bornyl caffeate dose-dependently induced apoptosis of MCF-7 cells, increased the expression of Bax and decreased the expression of Bcl-xl, resulting in the disruption of MMP and subsequent activation of caspase-3. Moreover, bornyl caffeate triggered the formation of ROS and activated p38 and c-Jun JNK. In MCF-7 cells, the cytotoxicity of bornyl caffeate was significantly attenuated by SB203580 (p38 inhibitor), SP600125 (JNK inhibitor), z-VAD (pan-caspase inhibitor) or the thiol antioxidant L-NAC. Conclusion: Bornyl caffeate exerts non-selective cytotoxicity against cancer cells of different origin in vitro. The compound induces apoptosis in human breast cancer MCF-7 cells via the ROS- and JNK-mediated pathways. PMID:24335836

  12. Neuroprotective Effects of the Absence of JNK1 or JNK3 Isoforms on Kainic Acid-Induced Temporal Lobe Epilepsy-Like Symptoms.

    PubMed

    de Lemos, Luisa; Junyent, Felix; Camins, Antoni; Castro-Torres, Rubén Darío; Folch, Jaume; Olloquequi, Jordi; Beas-Zarate, Carlos; Verdaguer, Ester; Auladell, Carme

    2018-05-01

    The activation of c-Jun-N-terminal kinases (JNK) pathway has been largely associated with the pathogenesis and the neuronal death that occur in neurodegenerative diseases. Altogether, this justifies why JNKs have become a focus of screens for new therapeutic strategies. The aim of the present study was to identify the role of the different JNK isoforms (JNK1, JNK2, and JNK3) in apoptosis and inflammation after induction of brain damage. To address this aim, we induced excitotoxicity in wild-type and JNK knockout mice (jnk1 -/- , jnk2 -/- , and jnk3 -/- ) via an intraperitoneal injection of kainic acid, an agonist of glutamic-kainate-receptors, that induce status epilepticus.Each group of animals was divided into two treatments: a single intraperitoneal dose of saline solution, used as a control, and a single intraperitoneal dose (30 mg/kg) of kainic acid. Our results reported a significant decrease in neuronal degeneration in the hippocampus of jnk1 -/- and jnk3 -/- mice after kainic acid treatment, together with reduced or unaltered expression of several apoptotic genes compared to WT treated mice. In addition, both jnk1 -/- and jnk3 -/- mice exhibited a reduction in glial reactivity, as shown by the lower expression of inflammatory genes and a reduction of JNK phosphorylation. In addition, in jnk3 -/- mice, the c-Jun phosphorylation was also diminished.Collectively, these findings provide compelling evidence that the absence of JNK1 or JNK3 isoforms confers neuroprotection against neuronal damage induced by KA and evidence, for the first time, the implication of JNK1 in excitotoxicity. Accordingly, JNK1 and/or JNK3 are promising targets for the prevention of cell death and inflammation during epileptogenesis.

  13. 3'UTR AU-Rich Elements (AREs) and the RNA-Binding Protein Tristetraprolin (TTP) Are Not Required for the LPS-Mediated Destabilization of Phospholipase-Cβ-2 mRNA in Murine Macrophages.

    PubMed

    Shukla, Smita; Elson, Genie; Blackshear, Perry J; Lutz, Carol S; Leibovich, S Joseph

    2017-04-01

    We have shown previously that bacterial lipopolysaccharide (LPS)-mediated suppression of phospholipase-Cβ-2 (PLCβ-2) expression is involved in M1 (inflammatory) to M2-like (wound healing) phenotypic switching of macrophages triggered by adenosine. This suppression is mediated post-transcriptionally by destabilization of PLCβ-2 mRNA (messenger ribonucleic acid). To investigate the mechanism of this LPS-mediated destabilization, we examined the roles of RNA-binding agents including microRNAs and RNA-binding proteins that are involved in regulating stability of mRNAs encoding growth factors, inflammatory mediators, and proto-oncogenes. Adenylate and uridylate (AU)-rich elements (AREs) in 3'UTRs are specific recognition sites for RNA-binding proteins including tristetraprolin (TTP), HuR, and AUF1 and for microRNAs that are involved in regulating mRNA stability. In this study, we investigated the role of TTP and AREs in regulating PLCβ-2 mRNA stability. The 3'UTR of the PLCβ-2 gene was inserted into the pLightswitch luciferase reporter plasmid and transfected into RAW264.7 cells. LPS suppressed luciferase expression from this reporter. Luciferase expression from mutant 3'UTR constructs lacking AREs was similarly downregulated, suggesting that these regions are not required for LPS-mediated suppression of PLCβ-2. TTP was rapidly upregulated in both primary murine macrophages and RAW264.7 cells in response to LPS. Suppression of PLCβ-2 by LPS was examined using macrophages from mice lacking TTP (TTP -/- ). LPS suppressed PLCβ-2 expression to the same extent in wild type (WT) and TTP -/- macrophages. Also, the rate of decay of PLCβ-2 mRNA in LPS-treated macrophages following transcriptional blockade was similar in WT and TTP -/- macrophages, clearly indicating that TTP is not involved in LPS-mediated destabilization of PLCβ-2 mRNA in macrophages.

  14. WIG1 is crucial for AGO2-mediated ACOT7 mRNA silencing via miRNA-dependent and -independent mechanisms.

    PubMed

    Lee, Hyung Chul; Jung, Seung Hee; Hwang, Hyun Jung; Kang, Donghee; De, Supriyo; Dudekula, Dawood B; Martindale, Jennifer L; Park, Byungkyu; Park, Seung Kuk; Lee, Eun Kyung; Lee, Jeong-Hwa; Jeong, Sunjoo; Han, Kyungsook; Park, Heon Joo; Ko, Young-Gyu; Gorospe, Myriam; Lee, Jae-Seon

    2017-06-20

    RNA-binding proteins (RBPs) are involved in mRNA splicing, maturation, transport, translation, storage and turnover. Here, we identified ACOT7 mRNA as a novel target of human WIG1. ACOT7 mRNA decay was triggered by the microRNA miR-9 in a WIG1-dependent manner via classic recruitment of Argonaute 2 (AGO2). Interestingly, AGO2 was also recruited to ACOT7 mRNA in a WIG1-dependent manner in the absence of miR-9, which indicates an alternative model whereby WIG1 controls AGO2-mediated gene silencing. The WIG1-AGO2 complex attenuated translation initiation via an interaction with translation initiation factor 5B (eIF5B). These results were confirmed using a WIG1 tethering system based on the MS2 bacteriophage coat protein and a reporter construct containing an MS2-binding site, and by immunoprecipitation of WIG1 and detection of WIG1-associated proteins using liquid chromatography-tandem mass spectrometry. We also identified WIG1-binding motifs using photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation analyses. Altogether, our data indicate that WIG1 governs the miRNA-dependent and the miRNA-independent recruitment of AGO2 to lower the stability of and suppress the translation of ACOT7 mRNA. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  15. The protective effect of lycopene on hypoxia/reoxygenation-induced endoplasmic reticulum stress in H9C2 cardiomyocytes.

    PubMed

    Gao, Yang; Jia, Pengyu; Shu, WenQi; Jia, Dalin

    2016-03-05

    Nowadays, drugs protecting ischemia/reperfusion (I/R) myocardium become more suitable for clinic. It has been confirmed lycopene has various protections, but lacking the observation of its effect on endoplasmic reticulum stress (ERS)-mediated apoptosis caused by hypoxia/reoxygenation (H/R). This study aims to clarify the protective effect of lycopene on ERS induced by H/R in H9C2 cardiomyocytes. Detect the survival rate, lactic dehydrogenase (LDH) activity, apoptosis ratio, glucose-regulated proteins 78 (GRP78), C/EBP homologous protein (CHOP), c-Jun-N-terminal protein Kinase (JNK) and Caspase-12 mRNA and protein expression and phosphorylation of JNK (p-JNK) protein expression. LDH activity, apoptosis ratio and GRP78 protein expression increase in the H/R group, reduced by lycopene. The survival rate reduces in the H/R and thapsigargin (TG) groups; lycopene and 4-phenyl butyric acid (4-PBA) can improve it caused by H/R, lycopene also can improve it caused by TG. The apoptosis ratio, the expression of GRP78, CHOP and Caspase-12 mRNA and protein and p-JNK protein increase in the H/R and TG groups, weaken in the lycopene+H/R, 4-PBA+H/R and lycopene+TG groups. There is no obvious change in the expression of JNK mRNA or protein. Hence, our results provide the evidence that 10 μM lycopene plays an obviously protective effect on H/R H9C2 cardiomyocytes, realized through reducing ERS and apoptosis. The possible mechanism may be related to CHOP, p-JNK and Caspase-12 pathways. Copyright © 2016. Published by Elsevier B.V.

  16. 3′UTR AU-rich elements (AREs) and the RNA-binding protein Tristetraprolin (TTP) are not required for the LPS-mediated destabilization of phospholipase-Cβ-2 mRNA in murine macrophages

    PubMed Central

    Shukla, Smita; Elson, Genie; Blackshear, Perry J.; Lutz, Carol S.; Leibovich, S. Joseph

    2017-01-01

    We have shown previously that bacterial lipopolysaccharide (LPS)-mediated suppression of Phospholipase-Cβ-2 (PLCβ-2) expression is involved in M1 (inflammatory) to M2-like (wound healing) phenotypic switching of macrophages triggered by adenosine. This suppression is mediated post-transcriptionally by destabilization of PLCβ-2 mRNA. To investigate the mechanism of this LPS-mediated destabilization, we examined the roles of RNA-binding agents including microRNAs and RNA-binding proteins that are involved in regulating stability of mRNAs encoding growth factors, inflammatory mediators and proto-oncogenes. Adenylate and Uridylate (AU)-rich elements (AREs) in 3′UTRs are specific recognition sites for RNA-binding proteins including Tristetraprolin (TTP), HuR and AUF1, and for microRNAs that are involved in regulating mRNA stability. In this study, we investigated the role of TTP and AREs in regulating PLCβ-2 mRNA stability. The 3′UTR of the PLCβ-2 gene was inserted into the pLightswitch luciferase reporter plasmid and transfected into RAW264.7 cells. LPS suppressed Luciferase expression from this reporter. Luciferase expression from mutant 3′UTR constructs lacking AREs was similarly down-regulated, suggesting that these regions are not required for LPS-mediated suppression of PLCβ-2. TTP was rapidly upregulated in both primary murine macrophages and RAW264.7 cells in response to LPS. Suppression of PLCβ-2 by LPS was examined using macrophages from mice lacking TTP. LPS suppressed PLCβ-2 expression to the same extent in wild type and TTP−/− macrophages. Also, the rate of decay of PLCβ-2 mRNA in LPS-treated macrophages following transcriptional blockade was similar in wild type and TTP−/− macrophages, clearly indicating that TTP is not involved in LPS-mediated destabilization of PLCβ-2 mRNA in macrophages. PMID:28124257

  17. TRX-ASK1-JNK signaling regulation of cell density-dependent cytotoxicity in cigarette smoke-exposed human bronchial epithelial cells.

    PubMed

    Lee, Yong Chan; Chuang, Chun-Yu; Lee, Pak-Kei; Lee, Jin-Soo; Harper, Richart W; Buckpitt, Alan B; Wu, Reen; Oslund, Karen

    2008-05-01

    Cigarette smoke is a major environmental air pollutant that injures airway epithelium and incites subsequent diseases including chronic obstructive pulmonary disease. The lesion that smoke induces in airway epithelium is still incompletely understood. Using a LIVE/DEAD cytotoxicity assay, we observed that subconfluent cultures of bronchial epithelial cells derived from both human and monkey airway tissues and an immortalized normal human bronchial epithelial cell line (HBE1) were more susceptible to injury by cigarette smoke extract (CSE) and by direct cigarette smoke exposure than cells in confluent cultures. Scraping confluent cultures also caused an enhanced cell injury predominately in the leading edge of the scraped confluent cultures by CSE. Cellular ATP levels in both subconfluent and confluent cultures were drastically reduced after CSE exposure. In contrast, GSH levels were significantly reduced only in subconfluent cultures exposed to smoke and not in confluent cultures. Western blot analysis demonstrated ERK activation in both confluent and subconfluent cultures after CSE. However, activation of apoptosis signal-regulating kinase 1 (ASK1), JNK, and p38 were demonstrated only in subconfluent cultures and not in confluent cultures after CSE. Using short interfering RNA (siRNA) to JNK1 and JNK2 and a JNK inhibitor, we attenuated CSE-mediated cell death in subconfluent cultures but not with an inhibitor of the p38 pathway. Using the tetracycline (Tet)-on inducible approach, overexpression of thioredoxin (TRX) attenuated CSE-mediated cell death and JNK activation in subconfluent cultures. These results suggest that the TRX-ASK1-JNK pathway may play a critical role in mediating cell density-dependent CSE cytotoxicity.

  18. 5, 8, 11, 14-eicosatetraynoic acid suppresses CCL2/MCP-1 expression in IFN-γ-stimulated astrocytes by increasing MAPK phosphatase-1 mRNA stability.

    PubMed

    Lee, Jee Hoon; Kim, Hyunmi; Woo, Joo Hong; Joe, Eun-hye; Jou, Ilo

    2012-02-18

    The peroxisome proliferator-activated receptor (PPAR)-α activator, 5,8,11,14-eicosatetraynoic acid (ETYA), is an arachidonic acid analog. It is reported to inhibit up-regulation of pro-inflammatory genes; however, its underlying mechanism of action is largely unknown. In the present study, we focused on the inhibitory action of ETYA on the expression of the chemokine, CCL2/MCP-1, which plays a key role in the initiation and progression of inflammation. To determine the effect of ETYA, primary cultured rat astrocytes and microglia were stimulated with IFN-γ in the presence of ETYA and then, expression of CCL2/MCP-1 and MAPK phosphatase (MKP-1) were determined using RT-PCR and ELISA. MKP-1 mRNA stability was evaluated by treating actinomycin D. The effect of MKP-1 and human antigen R (HuR) was analyzed by using specific siRNA transfection system. The localization of HuR was analyzed by immunocytochemistry and subcellular fractionation experiment. We found that ETYA suppressed CCL2/MCP-1 transcription and secretion of CCL2/MCP-1 protein through up-regulation of MKP-1mRNA levels, resulting in suppression of c-Jun N-terminal kinase (JNK) phosphorylation and activator protein 1 (AP1) activity in IFN-γ-stimulated brain glial cells. Moreover, these effects of ETYA were independent of PPAR-α. Experiments using actinomycin D revealed that the ETYA-induced increase in MKP-1 mRNA levels reflected an increase in transcript stability. Knockdown experiments using small interfering RNA demonstrated that this increase in MKP-1 mRNA stability depended on HuR, an RNA-binding protein known to promote enhanced mRNA stability. Furthermore, ETYA-induced, HuR-mediated mRNA stabilization resulted from HuR-MKP-1 nucleocytoplasmic translocation, which served to protect MKP-1 mRNA from the mRNA degradation machinery. ETYA induces MKP-1 through HuR at the post-transcriptional level in a receptor-independent manner. The mechanism revealed here suggests eicosanoids as potential therapeutic

  19. Glycyrrhetinic acid inhibits ICAM-1 expression via blocking JNK and NF-κB pathways in TNF-α-activated endothelial cells

    PubMed Central

    Chang, Ying-ling; Chen, Chien-lin; Kuo, Chao-Lin; Chen, Bor-chyuan; You, Jyh-sheng

    2010-01-01

    Aim: To investigate the effects of glycyrrhetinic acid (GA), an active component extracted from the root of Glycyrrhizae glabra, on the expression of intercellular adhesion molecule-1 (ICAM-1) in tumor necrosis factor-α (TNF-α)-activated human umbilical vein endothelial cells (HUVEC). Methods: ICAM-1 mRNA and protein levels were detected using RT-PCR and cell enzyme-linked immunosorbent assays. The adherence of human monocytic THP-1 cells labeled with [3H]thymidine to HUVEC was determined by counting radioactivity with a scintillation counter. The activation of mitogen-activated protein kinases as well as the degradation of IκB and nuclear factor-κB (NF-κB) or phospho-c-Jun in the nucleus were detected by western blots. NF-κB binding activity was detected using electrophoretic mobility shift assay. Results: GA (50 and 100 μmol/L) significantly inhibits TNF-α-induced ICAM-1 mRNA and protein expressions, as well as THP-1 cell adhesiveness in HUVEC. GA selectively inhibited TNF-α-activated signal pathway of c-Jun N-terminal kinase (JNK), without affecting extracellular signal-regulated kinase 1/2 and p38. Furthermore, GA apparently inhibited IκB/NF-κB signaling system by preventing IκB degradation, NF-κB translocation, and NF-κB/DNA binding activity. Finally, pretreatment with GA or the inhibitors of NF-κB, JNK, and p38 reduced the ICAM-1 protein expression induced by TNF-α. Conclusion: GA inhibits TNF-α-stimulated ICAM-1 expression, leading to a decrease in adherent monocytes to HUVEC. This inhibition is attributed to GA interruption of both JNK/c-Jun and IκB/NF-κB signaling pathways, which decrease activator protein-1 (AP-1) and NF-κB mediated ICAM-1 expressions. The results suggest that GA may provide a beneficial effect in treating vascular diseases associated with inflammation, such as atherosclerosis. PMID:20418897

  20. Elevated interleukin 6 is induced by prostaglandin E2 in a murine model of inflammation: possible role of cyclooxygenase-2.

    PubMed Central

    Hinson, R M; Williams, J A; Shacter, E

    1996-01-01

    Injection of mineral oils such as pristane into the peritoneal cavities of BALB/c mice results in a chronic peritonitis associated with high tissue levels of interleukin 6 (IL-6). Here we show that increased prostaglandin E2 (PGE2) synthesis causes induction of IL-6 and that expression of an inducible cyclooxygenase, Cox-2, may mediate this process. Levels of both PGE2 and IL-6 are elevated in inflammatory exudates from pristane-treated mice compared with lavage samples from untreated mice. The Cox-2 gene is induced in the peritoneal macrophage fraction isolated from the mice. A cause and effect relationship between increased macrophage PGE2 and IL-6 production is shown in vitro. When peritoneal macrophages are activated with an inflammatory stimulus (polymerized albumin), the Cox-2 gene is induced and secretion of PGE2 and IL-6 increases, with elevated PGE2 appearing before IL-6. Cotreatment with 1 microM indomethacin inhibits PGE2 production by the cells and reduces the induction of IL-6 mRNA but has no effect on Cox-2 mRNA, consistent with the fact that the drug inhibits catalytic activity of the cyclooxygenase but does not affect expression of the gene. Addition of exogenous PGE2 to macrophages induces IL-6 protein and mRNA synthesis, indicating that the eicosanoid stimulates IL-6 production at the level of gene expression. PGE2-stimulated IL-6 production is unaffected by addition of indomethacin. Taken together with the earlier finding that indomethacin diminishes the elevation of IL-6 in pristane-treated mice, the results show that PGE2 can induce IL-6 production in vivo and implicate expression of the Cox-2 gene in the regulation of this cytokine. Images Fig. 2 Fig. 4 Fig. 5 Fig. 6 Fig. 8 PMID:8643498

  1. In Vitro Interleukin-1 and 2 Production and Interleukin 2 Receptor Expression in the Rhesus Monkey

    NASA Technical Reports Server (NTRS)

    Schmitt, Didier A.; Sonnenfeld, Gerald; Husson, David; Tkaczuk, Jean; Andre, Eric; Schaffar, Laurance

    1996-01-01

    Anti-human monoclonal antibodies were used to detect and quantify interleukins-1 and 2 and interleukin-2 receptor expression in peripheral blood mononuclear cells from a rhesus monkey. Interleukin-1 production could be induced by phorbol esters (PMA) and was potentiated by phytohemagglutinin (PHA). Interleukin-2 secretion could also be induced by the combination of PHA and PMA, but only weakly with PHA alone. Interleukin-2 receptor expression was present in a subpopulation of unstimulated lymphocytes and could be enhanced by PHA or PMA. These data show once again that the rhesus monkey immune system is cross-reactive with the human one and that rhesus macaque could be a good model to study interleukin therapy.

  2. Anthrapyrazolone analogues intercept inflammatory JNK signals to moderate endotoxin induced septic shock

    NASA Astrophysics Data System (ADS)

    Prasad, Karothu Durga; Trinath, Jamma; Biswas, Ansuman; Sekar, Kanagaraj; Balaji, Kithiganahalli N.; Guru Row, Tayur N.

    2014-11-01

    Severe sepsis or septic shock is one of the rising causes for mortality worldwide representing nearly 10% of intensive care unit admissions. Susceptibility to sepsis is identified to be mediated by innate pattern recognition receptors and responsive signaling pathways of the host. The c-Jun N-terminal Kinase (JNK)-mediated signaling events play critical role in bacterial infection triggered multi-organ failure, cardiac dysfunction and mortality. In the context of kinase specificities, an extensive library of anthrapyrazolone analogues has been investigated for the selective inhibition of c-JNK and thereby to gain control over the inflammation associated risks. In our comprehensive biochemical characterization, it is observed that alkyl and halogen substitution on the periphery of anthrapyrazolone increases the binding potency of the inhibitors specifically towards JNK. Further, it is demonstrated that hydrophobic and hydrophilic interactions generated by these small molecules effectively block endotoxin-induced inflammatory genes expression in in vitro and septic shock in vivo, in a mouse model, with remarkable efficacies. Altogether, the obtained results rationalize the significance of the diversity oriented synthesis of small molecules for selective inhibition of JNK and their potential in the treatment of severe sepsis.

  3. Organizing Principles of Mammalian Nonsense-Mediated mRNA Decay

    PubMed Central

    Popp, Maximilian Wei-Lin; Maquat, Lynne E.

    2014-01-01

    Cells use messenger RNAs (mRNAs) to ensure the accurate dissemination of genetic information encoded by DNA. Given that mRNAs largely direct the synthesis of a critical effector of cellular phenotype, i.e., proteins, tight regulation of both the quality and quantity of mRNA is a prerequisite for effective cellular homeostasis. Here, we review nonsense-mediated mRNA decay (NMD), which is the best-characterized posttranscriptional quality control mechanism that cells have evolved in their cytoplasm to ensure transcriptome fidelity. We use protein quality control as a conceptual framework to organize what is known about NMD, highlighting overarching similarities between these two polymer quality control pathways, where the protein quality control and NMD pathways intersect, and how protein quality control can suggest new avenues for research into mRNA quality control. PMID:24274751

  4. Ras promotes cell survival by antagonizing both JNK and Hid signals in the Drosophila eye.

    PubMed

    Wu, Yue; Zhuang, Yuan; Han, Min; Xu, Tian; Deng, Kejing

    2009-10-20

    Programmed cell death, or apoptosis, is a fundamental physiological process during normal development or in pathological conditions. The activation of apoptosis can be elicited by numerous signalling pathways. Ras is known to mediate anti-apoptotic signals by inhibiting Hid activity in the Drosophila eye. Here we report the isolation of a new loss-of-function ras allele, rasKP, which causes excessive apoptosis in the Drosophila eye. This new function is likely to be mediated through the JNK pathway since the inhibition of JNK signalling can significantly suppress rasKP-induced apoptosis, whereas the removal of hid only weakly suppresses the phenotype. Furthermore, the reduction of JNK signalling together with the expression of the baculovirus caspase inhibitor p35, which blocks Hid activity, strongly suppresses the rasKP cell death. In addition, we find a strong correlation between rasKP-induced apoptosis in the eye disc and the activation of JNK signalling. In the Drosophila eye, Ras may protect cells from apoptosis by inhibiting both JNK and Hid activities. Surprisingly, reducing Ras activity in the wing, however, does not cause apoptosis but rather affects cell and organ size. Thus, in addition to its requirement for cell viability, Ras appears to mediate different biological roles depending on the developmental context and on the level of its expression.

  5. Iodine-131 treatment of thyroid cancer cells leads to suppression of cell proliferation followed by induction of cell apoptosis and cell cycle arrest by regulation of B-cell translocation gene 2-mediated JNK/NF-κB pathways.

    PubMed

    Zhao, L M; Pang, A X

    2017-01-16

    Iodine-131 (131I) is widely used for the treatment of thyroid-related diseases. This study aimed to investigate the expression of p53 and BTG2 genes following 131I therapy in thyroid cancer cell line SW579 and the possible underlying mechanism. SW579 human thyroid squamous carcinoma cells were cultured and treated with 131I. They were then assessed for 131I uptake, cell viability, apoptosis, cell cycle arrest, p53 expression, and BTG2 gene expression. SW579 cells were transfected with BTG2 siRNA, p53 siRNA and siNC and were then examined for the same aforementioned parameters. When treated with a JNK inhibitor of SP600125 and 131I or with a NF-κB inhibitor of BMS-345541 and 131I, non-transfected SW579 cells were assessed in JNK/NFκB pathways. It was observed that 131I significantly inhibited cell proliferation, promoted cell apoptosis and cell cycle arrest. Both BTG2 and p53 expression were enhanced in a dose-dependent manner. An increase in cell viability by up-regulation in Bcl2 gene, a decrease in apoptosis by enhanced CDK2 gene expression and a decrease in cell cycle arrest at G0/G1 phase were also observed in SW579 cell lines transfected with silenced BTG2 gene. When treated with SP600125 and 131I, the non-transfected SW579 cell lines significantly inhibited JNK pathway, NF-κB pathway and the expression of BTG2. However, when treated with BMS-345541 and 131I, only the NF-κB pathway was suppressed. 131I suppressed cell proliferation, induced cell apoptosis, and promoted cell cycle arrest of thyroid cancer cells by up-regulating B-cell translocation gene 2-mediated activation of JNK/NF-κB pathways.

  6. Caffeic Acid Phenethyl Ester (Propolis Extract) Ameliorates Insulin Resistance by Inhibiting JNK and NF-κB Inflammatory Pathways in Diabetic Mice and HepG2 Cell Models.

    PubMed

    Nie, Jiarui; Chang, Yaning; Li, Yujia; Zhou, Yingjun; Qin, Jiawen; Sun, Zhen; Li, Haibin

    2017-10-18

    Caffeic acid phenethyl ester (CAPE), extracted from propolis, was evaluated for the ameliorative effects on insulin resistance and the mechanisms were identified, using non-insulin-dependent diabetes mellitus (NIDDM) model mice and insulin resistance (IR) model cells. After 5 weeks of CAPE supplementation, insulin sensitivity, hyperlipidemia, and peroxisome proliferator-activated receptor-α (PPAR-α) levels were improved in mice. Proinflammatory cytokines in serum and the expressions of tumor necrosis factor-alpha (TNF-α) mRNA in tissues were markedly downregulated from CAPE-treated mice. In vitro, CAPE supplement significantly improved glucose consumption, glucose uptake, glycogen content, and oxidative stress and decreased expression of glucose-6-phosphatase (G6Pase) mRNA in cells. Both in vivo and in vitro, CAPE enhanced p-Akt (Ser473) and p-insulin receptor substrate (IRS)-1 (Tyr612), but inhibited p-JNK (Thr183/Tyr185), p-NF-κB p65 (Ser536), and nuclear translocation of p-NF-κB p65 (Ser536). In summary, CAPE can ameliorate insulin resistance through modulation of JNK and NF-κB signaling pathway in mice and HepG2 cells.

  7. cAMP-Mediated Stimulation of Tyrosine Hydroxylase mRNA Translation Is Mediated by Polypyrimidine-Rich Sequences within Its 3′-Untranslated Region and Poly(C)-Binding Protein 2

    PubMed Central

    Xu, Lu; Sterling, Carol R.

    2009-01-01

    Tyrosine hydroxylase (TH) plays a critical role in maintaining the appropriate concentrations of catecholamine neurotransmitters in brain and periphery, particularly during long-term stress, long-term drug treatment, or neurodegenerative diseases. Its expression is controlled by both transcriptional and post-transcriptional mechanisms. In a previous report, we showed that treatment of rat midbrain slice explant cultures or mouse MN9D cells with cAMP analog or forskolin leads to induction of TH protein without concomitant induction of TH mRNA. We further showed that cAMP activates mechanisms that regulate TH mRNA translation via cis-acting sequences within its 3′-untranslated region (UTR). In the present report, we extend these studies to show that MN9D cytoplasmic proteins bind to the same TH mRNA 3′-UTR domain that is required for the cAMP response. RNase T1 mapping demonstrates binding of proteins to a 27-nucleotide polypyrimidine-rich sequence within this domain. A specific mutation within the polypyrimidine-rich sequence inhibits protein binding and cAMP-mediated translational activation. UV-cross-linking studies identify a ∼44-kDa protein as a major TH mRNA 3′-UTR binding factor, and cAMP induces the 40- to 42-kDa poly(C)-binding protein-2 (PCBP2) in MN9D cells. We show that PCBP2 binds to the TH mRNA 3′-UTR domain that participates in the cAMP response. Overexpression of PCBP2 induces TH protein without concomitant induction of TH mRNA. These results support a model in which cAMP induces PCBP2, leading to increased interaction with its cognate polypyrimidine binding site in the TH mRNA 3′-UTR. This increased interaction presumably plays a role in the activation of TH mRNA translation by cAMP in dopaminergic neurons. PMID:19620256

  8. Role of interleukin-1beta and tumor necrosis factor-alpha-dependent expression of cyclooxygenase-2 mRNA in thermal hyperalgesia induced by chronic inflammation in mice.

    PubMed

    Narita, M; Shimamura, M; Imai, S; Kubota, C; Yajima, Y; Takagi, T; Shiokawa, M; Inoue, T; Suzuki, M; Suzuki, T

    2008-03-18

    The present study investigated whether the endogenous pro-inflammatory cytokines [interleukin (IL)-1beta and tumor necrosis factor-alpha (TNF-alpha)]-dependent expression of cyclooxygenase-2 (COX-2) mRNA within the spinal cord could be involved in the development of chronic inflammatory pain-like behaviors in mice. We demonstrated that the expression of COX-2 mRNA on the ipsilateral side of the spinal cord was significantly increased 6 h and 3 days after intraplantar injection of complete Freund's adjuvant (CFA), compared with the expression in saline-treated mice. In addition, the chronic pain-like behaviors following CFA injection were markedly suppressed by repeated intrathecal (i.t.) pre-treatment with the COX-2 inhibitor etodolac, but not with the COX-1 inhibitor mofezolac. The cytosolic level of the activated form of nuclear factor-kappa B (NF-kappaB), which is a major contributor to the induction of COX-2, on the ipsilateral side of the mouse spinal cord was also increased compared with that in the saline-treated mice. The key finding in the present study was that a single i.t. injection with either IL-1beta or TNF-alpha induced a marked increase in spinal COX-2 mRNA and persistent thermal hyperalgesia in mice. Furthermore, CFA-induced hypersensitivity to inflammatory pain was significantly reduced by repeated i.t. pre-injection of the recombinant Fc chimera of IL-1 receptor I or soluble TNF receptor I, which sequesters endogenous IL-1beta or TNF-alpha, respectively. In contrast, the expression of spinal COX-2 mRNA in CFA-treated mice was similar to that in saline-treated mice at 7 days after CFA injection. The present findings strongly indicate the early intrathecal use of the COX-2 inhibitor for the relief of chronic inflammatory pain. Furthermore, together with the result in a previous study that pro-inflammatory cytokines lead to stimulation of a NF-kappaB-dependent transcriptional pathway, these findings suggest that a spinal cytokine/NF-kappaB/COX-2

  9. Hit-to-lead optimization and kinase selectivity of imidazo[1,2-a]quinoxalin-4-amine derived JNK1 inhibitors.

    PubMed

    Li, Bei; Cociorva, Oana M; Nomanbhoy, Tyzoon; Weissig, Helge; Li, Qiang; Nakamura, Kai; Liyanage, Marek; Zhang, Melissa C; Shih, Ann Y; Aban, Arwin; Hu, Yi; Cajica, Julia; Pham, Lan; Kozarich, John W; Shreder, Kevin R

    2013-09-15

    As the result of a rhJNK1 HTS, the imidazo[1,2-a]quinoxaline 1 was identified as a 1.6 μM rhJNK1 inhibitor. Optimization of this compound lead to AX13587 (rhJNK1 IC50=160 nM) which was co-crystallized with JNK1 to identify key molecular interactions. Kinase profiling against 125+ kinases revealed AX13587 was an inhibitor of JNK, MAST3, and MAST4 whereas its methylene homolog AX14373 (native JNK1 IC50=47 nM) was a highly specific JNK inhibitor. Copyright © 2013 Elsevier Ltd. All rights reserved.

  10. Up-regulation of IL-23 expression in human dental pulp fibroblasts by IL-17 via activation of the NF-κB and MAPK pathways.

    PubMed

    Wei, L; Liu, M; Xiong, H; Peng, B

    2017-11-06

    To investigate the effects of the pro-inflammatory and Th17-polarizing mediator IL-17 on HDPFs-mediated IL-23 production and the molecular mechanism involved. Interleukin (IL)-17R expression was determined by semi-quantitative reverse transcriptase-polymerase chain reaction and Western blot in cultured human dental pulp fibroblasts (HDPFs). Quantitative real-time polymerase chain reaction and enzyme-linked immunosorbent assay were used to determine IL-23 mRNA and protein levels in IL-17-stimulated HDPFs, respectively. The nuclear factor-kappa B (NF-κB) and mitogen-activated protein kinases (MAPKs) signalling pathways that mediate the IL-17-stimulated production of IL-23 was investigated using Western blot and specific signalling inhibitor analyses. Statistical analyses were performed using Kruskal-Wallis tests followed by the Mann-Whitney U-test. Statistical significance was considered when the P value < 0.05. Primary HDPFs steadily expressed IL-17R mRNA and surface-bound protein. IL-17 stimulated the expression of IL-23 mRNA and protein in cultured human dental pulp fibroblasts, which was attenuated by IL-17 or IL-17R neutralizing antibodies. In accordance with the enhanced expression of IL-23, IL-17 stimulation resulted in rapid activation of p38 MAPK, extracellular signal-regulated kinase (ERK) 1/2, c-Jun-N-terminal kinase (JNK) and NF-κB in HDPFs. Inhibitors of p38 MAPK, ERK 1/2 or NF-κB significantly suppressed, whereas blocking JNK substantially augmented IL-23 production from IL-17-stimulated HDPFs. HDPFs expressed IL-17R and responded to IL-17 to produce IL-23 via the activation of the NF-κB and MAPK signalling pathways. The findings provide insights into the cellular mechanisms of the participation of IL-17 in the activation of HDPFs in inflamed pulp tissue. © 2017 International Endodontic Journal. Published by John Wiley & Sons Ltd.

  11. ROCK activity affects IL-1-induced signaling possibly through MKK4 and p38 MAPK in Caco-2 cells.

    PubMed

    Banerjee, Sayantan; McGee, Dennis W

    2016-09-01

    Elevated levels of interleukin-1 (IL-1) accompany inflammatory bowel disease. IL-1-stimulated intestinal epithelial cells can secrete potent chemokines like CXCL8 to exacerbate inflammation. Previously, we found that inhibiting the Rho-associated kinase (ROCK) could inhibit IL-1- or TNF-α-induced CXCL8 secretion by the Caco-2 colonic epithelial cell line. This ROCK inhibition did not affect IκBα phosphorylation and degradation, but suppressed the phosphorylation of c-Jun N-terminal kinase (JNK). Therefore, ROCK must play an important role in epithelial cell CXCL8 responses through an effect on the JNK signaling pathway. Here, we extend these studies by showing that inhibiting ROCK suppressed the IL-1-induced phosphorylation of MKK4, a known activator of JNK, but not MKK7. Yet, ROCK inhibition had no significant effect on the IL-1-induced phosphorylation of extracellular-signal-regulated kinase (ERK) 1/2. Inhibiting ROCK also suppressed the phosphorylation of p38 MAPK after IL-1 stimulation, but this inhibition had no significant effect on the stability of CXCL8 messenger RNA (mRNA) after IL-1 stimulation. These results suggest that ROCK may be important in IL-1-induced signaling through MKK4 to JNK and the activation of p38 MAPK. Finally, inhibiting ROCK in IL-1 and TNF-α co-stimulated Caco-2 cells also resulted in a significant suppression of CXCL8 secretion and mRNA levels suggesting that inhibiting ROCK may be a mechanism to inhibit the overall response of epithelial cells to both cytokines. These studies indicate a novel signaling event, which could provide a target for suppressing intestinal epithelial cells (IEC) chemokine responses involved in mucosal inflammation.

  12. Cannabinoid Receptor 2 Suppresses Leukocyte Inflammatory Migration by Modulating the JNK/c-Jun/Alox5 Pathway*

    PubMed Central

    Liu, Yi-Jie; Fan, Hong-Bo; Jin, Yi; Ren, Chun-Guang; Jia, Xiao-E; Wang, Lei; Chen, Yi; Dong, Mei; Zhu, Kang-Yong; Dong, Zhi-Wei; Ye, Bai-Xin; Zhong, Zhong; Deng, Min; Liu, Ting Xi; Ren, Ruibao

    2013-01-01

    Inflammatory migration of immune cells is involved in many human diseases. Identification of molecular pathways and modulators controlling inflammatory migration could lead to therapeutic strategies for treating human inflammation-associated diseases. The role of cannabinoid receptor type 2 (Cnr2) in regulating immune function had been widely investigated, but the mechanism is not fully understood. Through a chemical genetic screen using a zebrafish model for leukocyte migration, we found that both an agonist of the Cnr2 and inhibitor of the 5-lipoxygenase (Alox5, encoded by alox5) inhibit leukocyte migration in response to acute injury. These agents have a similar effect on migration of human myeloid cells. Consistent with these results, we found that inactivation of Cnr2 by zinc finger nuclease-mediated mutagenesis enhances leukocyte migration, while inactivation of Alox5 blocks leukocyte migration. Further investigation indicates that there is a signaling link between Cnr2 and Alox5 and that alox5 is a target of c-Jun. Cnr2 activation down-regulates alox5 expression by suppressing the JNK/c-Jun activation. These studies demonstrate that Cnr2, JNK, and Alox5 constitute a pathway regulating leukocyte migration. The cooperative effect between the Cnr2 agonist and Alox5 inhibitor also provides a potential therapeutic strategy for treating human inflammation-associated diseases. PMID:23539630

  13. Inhibition of interleukin-2-mediated lymphocyte activation in patients with Cushing's syndrome: a comparison with hypocortisolemic patients.

    PubMed

    Sauer, J; Stalla, G K; Müller, O A; Arzt, E

    1994-02-01

    We investigated the lymphocyte interleukin-2 (IL-2) system, which is critically involved in lymphocyte activation, in patients with disorders or the hypothalamic-pituitary-adrenal (HPA) axis. Patients with Cushing's syndrome (n = 9) showed a significant (p < 0.05) inhibition of phytohemagglutinin (PHA)-stimulated IL-2 secretion by peripheral lymphocytes and a decrease of sensitivity to cortisol inhibition in vitro compared to normal subjects (n = 9). Circulating soluble interleukin-2 receptor (sIL-2R) levels were significantly decreased (p < 0.05), whereas no significant difference was observed in PHA-induced sIL-2R release in vitro. In patients with hypocortisolism (n = 12), in vitro IL-2 synthesis was increased compared to normal subjects and to patients with Cushing's syndrome (p < 0.01). In vitro sIL-2R release was significantly higher (p < 0.01) compared to patients with Cushing's syndrome. In contrast to patients with secondary adrenal insufficiency (n = 7), patients with an adrenal origin of hypocortisolism (Addison's disease, bilateral adrenalectomy; n = 5) showed significantly elevated circulating sIL-2R levels compared to normal subjects and patients with Cushing's syndrome (p < 0.005). There was no significant difference between the study groups in mitogen-induced DNA synthesis. This is the first description of alterations of cytokine secretion in patients with HPA axis disorders. The contrary effects of long-term hypercortisolism and insufficient or absent adrenal glucocorticoid secretion on IL-2-mediated lymphocyte activation could account for the immune states previously observed in these patients.

  14. Cone arrestin binding to JNK3 and Mdm2: conformational preference and localization of interaction sites

    PubMed Central

    Song, Xiufeng; Gurevich, Eugenia V.; Gurevich, Vsevolod V.

    2008-01-01

    Arrestins are multi-functional regulators of G protein-coupled receptors. Receptor-bound arrestins interact with >30 remarkably diverse proteins and redirect the signaling to G protein-independent pathways. The functions of free arrestins are poorly understood, and the interaction sites of the non-receptor arrestin partners are largely unknown. In this study, we show that cone arrestin, the least studied member of the family, binds c-Jun N-terminal kinase (JNK3) and Mdm2 and regulates their subcellular distribution. Using arrestin mutants with increased or reduced structural flexibility, we demonstrate that arrestin in all conformations binds JNK3 comparably, whereas Mdm2 preferentially binds cone arrestin ‘frozen’ in the basal state. To localize the interaction sites, we expressed separate N- and C-domains of cone and rod arrestins and found that individual domains bind JNK3 and remove it from the nucleus as efficiently as full-length proteins. Thus, the arrestin binding site for JNK3 includes elements in both domains with the affinity of partial sites on individual domains sufficient for JNK3 relocalization. N-domain of rod arrestin binds Mdm2, which localizes its main interaction site to this region. Comparable binding of JNK3 and Mdm2 to four arrestin subtypes allowed us to identify conserved residues likely involved in these interactions. PMID:17680991

  15. TRPM2 contributes to LPS/IFNγ-induced production of nitric oxide via the p38/JNK pathway in microglia.

    PubMed

    Miyake, Takahito; Shirakawa, Hisashi; Kusano, Ayaka; Sakimoto, Shinya; Konno, Masakazu; Nakagawa, Takayuki; Mori, Yasuo; Kaneko, Shuji

    2014-02-07

    Microglia are immune cells that maintain brain homeostasis at a resting state by surveying the environment and engulfing debris. However, in some pathological conditions, microglia can produce neurotoxic factors such as pro-inflammatory cytokines and nitric oxide (NO) that lead to neuronal degeneration. Inflammation-induced calcium (Ca(2+)) signaling is thought to underlie this abnormal activation of microglia, but the mechanisms are still obscure. We previously showed that combined application of lipopolysaccharide and interferon γ (LPS/IFNγ) induced-production of NO in microglia from wild-type (WT) mice is significantly reduced in microglia from transient receptor potential melastatin 2 (TRPM2)-knockout (KO) mice. Here, we found that LPS/IFNγ produced a late-onset Ca(2+) signaling in WT microglia, which was abolished by application of the NADPH oxidase inhibitor diphenylene iodonium (DPI) and ML-171. In addition, pharmacological blockade or gene deletion of TRPM2 channel in microglia did not show this Ca(2+) signaling. Furthermore, pharmacological manipulation and Western blotting revealed that Ca(2+) mobilization, the proline-rich tyrosine kinase 2 (Pyk2), p38 mitogen-activated protein kinase (p38 MAPK) and c-Jun NH2-terminal kinase (JNK) contributed to TRPM2-mediated LPS/IFNγ-induced activation, while the extracellular signal-regulated protein kinase (ERK) did not. These results suggest that LPS/IFNγ activates TRPM2-mediated Ca(2+) signaling, which in turn increases downstream p38 MAPK and JNK signaling and results in increased NO production in microglia. Copyright © 2014 Elsevier Inc. All rights reserved.

  16. A macrophage NBR1-MEKK3 complex triggers JNK-mediated adipose-tissue inflammation in obesity

    PubMed Central

    Hernandez, Eloy D.; Lee, Sang Jun; Kim, Ji Young; Duran, Angeles; Linares, Juan F.; Yajima, Tomoko; Müller, Timo D.; Tschöp, Matthias H.; Smith, Steven R.; Diaz-Meco, Maria T.; Moscat, Jorge

    2014-01-01

    SUMMARY The c-Jun NH(2)-terminal kinase (JNK) is a critical determinant of obesity-associated inflammation and glucose intolerance. The upstream mechanisms controlling this pathway are still unknown. Here we report that the levels of the PB1 domain-containing adapter NBR1 correlated with the expression of pro-inflammatory molecules in adipose tissue from human patients with metabolic syndrome, suggesting that NBR1 plays a key role in adipose-tissue inflammation. We also show that NBR1 inactivation in the myeloid compartment impairs the function, M1 polarization and chemotactic activity of macrophages, prevents inflammation of adipose tissue, and improves glucose tolerance in obese mice. Furthermore, we demonstrate that an interaction between the PB1 domains of NBR1 and the mitogen-activated kinase kinase 3 (MEKK3) enables the formation of a signaling complex required for the activation of JNK. Together these discoveries identify an NBR1-MEKK3 complex as a key regulator of JNK signaling and adipose-tissue inflammation in obesity. PMID:25043814

  17. The Core Molecular Machinery Used for Engulfment of Apoptotic Cells Regulates the JNK Pathway Mediating Axon Regeneration in Caenorhabditis elegans.

    PubMed

    Pastuhov, Strahil Iv; Fujiki, Kota; Tsuge, Anna; Asai, Kazuma; Ishikawa, Sho; Hirose, Kazuya; Matsumoto, Kunihiro; Hisamoto, Naoki

    2016-09-14

    The mechanisms that govern the ability of specific neurons to regenerate their axons after injury are not well understood. In Caenorhabditis elegans, the initiation of axon regeneration is positively regulated by the JNK-MAPK pathway. In this study, we identify two components functioning upstream of the JNK pathway: the Ste20-related protein kinase MAX-2 and the Rac-type GTPase CED-10. CED-10, when bound by GTP, interacts with MAX-2 and functions as its upstream regulator in axon regeneration. CED-10, in turn, is activated by axon injury via signals initiated from the integrin α-subunit INA-1 and the nonreceptor tyrosine kinase SRC-1 and transmitted via the signaling module CED-2/CrkII-CED-5/Dock180-CED-12/ELMO. This module is also known to regulate the engulfment of apoptotic cells during development. Our findings thus reveal that the molecular machinery used for engulfment of apoptotic cells also promotes axon regeneration through activation of the JNK pathway. The molecular mechanisms of axon regeneration after injury remain poorly understood. In Caenorhabditis elegans, the initiation of axon regeneration is positively regulated by the JNK-MAPK pathway. In this study, we show that integrin, Rac-GTPase, and several other molecules, all of which are known to regulate engulfment of apoptotic cells during development, also regulate axon regeneration. This signaling module activates the JNK-MAPK cascade via MAX-2, a PAK-like protein kinase that binds Rac. Our findings thus reveal that the molecular machinery used for engulfment of apoptotic cells also promotes axon regeneration through activation of the JNK pathway. Copyright © 2016 the authors 0270-6474/16/369710-12$15.00/0.

  18. MnO2 nanosheet mediated "DD-A" FRET binary probes for sensitive detection of intracellular mRNA.

    PubMed

    Ou, Min; Huang, Jin; Yang, Xiaohai; Quan, Ke; Yang, Yanjing; Xie, Nuli; Wang, Kemin

    2017-01-01

    The donor donor-acceptor (DD-A) FRET model has proven to have a higher FRET efficiency than donor-acceptor acceptor (D-AA), donor-acceptor (D-A), and donor donor-acceptor acceptor (DD-AA) FRET models. The in-tube and in-cell experiments clearly demonstrate that the "DD-A" FRET binary probes can indeed increase the FRET efficiency and provide higher imaging contrast, which is about one order of magnitude higher than the ordinary "D-A" model. Furthermore, MnO 2 nanosheets were employed to deliver these probes into living cells for intracellular TK1 mRNA detection because they can adsorb ssDNA probes, penetrate across the cell membrane and be reduced to Mn 2+ ions by intracellular GSH. The results indicated that the MnO 2 nanosheet mediated "DD-A" FRET binary probes are capable of sensitive and selective sensing gene expression and chemical-stimuli changes in gene expression levels in cancer cells. We believe that the MnO 2 nanosheet mediated "DD-A" FRET binary probes have the potential as a simple but powerful tool for basic research and clinical diagnosis.

  19. Dexmedetomidine-induced Contraction Involves Phosphorylation of Caldesmon by JNK in Endothelium-denuded Rat Aortas

    PubMed Central

    Baik, Jiseok; Ok, Seong-Ho; Cho, Hyunhoo; Yu, Jongsun; Kim, Woochan; Nam, In-Koo; Choi, Mun-Jeoung; Lee, Heon-Keun; Sohn, Ju-Tae

    2014-01-01

    Caldesmon, an inhibitory actin binding protein, binds to actin and inhibits actin-myosin interactions, whereas caldesmon phosphorylation reverses the inhibitory effect of caldesmon on actin-myosin interactions, potentially leading to enhanced contraction. The goal of this study was to investigate the cellular signaling pathway responsible for caldesmon phosphorylation, which is involved in the regulation of the contraction induced by dexmedetomidine (DMT), an alpha-2 adrenoceptor agonist, in endothelium-denuded rat aortas. SP600125 (a c-Jun NH2-terminal kinase [JNK] inhibitor) dose-response curves were generated in aortas that were pre-contracted with DMT or phorbol 12,13-dibutyrate (PDBu), a protein kinase C (PKC) activator. Dose-response curves to the PKC inhibitor chelerythrine were generated in rat aortas pre-contracted with DMT. The effects of SP600125 and rauwolscine (an alpha-2 adrenoceptor inhibitor) on DMT-induced caldesmon phosphorylation in rat aortic vascular smooth muscle cells (VSMCs) were investigated by western blot analysis. PDBu-induced caldesmon and DMT-induced PKC phosphorylation in rat aortic VSMCs was investigated by western blot analysis. The effects of GF109203X (a PKC inhibitor) on DMT- or PDBu-induced JNK phosphorylation in VSMCs were assessed. SP600125 resulted in the relaxation of aortas that were pre-contracted with DMT or PDBu, whereas rauwolscine attenuated DMT-induced contraction. Chelerythrine resulted in the vasodilation of aortas pre-contracted with DMT. SP600125 and rauwolscine inhibited DMT-induced caldesmon phosphorylation. Additionally, PDBu induced caldesmon phosphorylation, and GF109203X attenuated the JNK phosphorylation induced by DMT or PDBu. DMT induced PKC phosphorylation in rat aortic VSMCs. These results suggest that alpha-2 adrenoceptor-mediated, DMT-induced contraction involves caldesmon phosphorylation that is mediated by JNK phosphorylation by PKC. PMID:25332685

  20. Arsenic trioxide mediates HAPI microglia inflammatory response and subsequent neuron apoptosis through p38/JNK MAPK/STAT3 pathway

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

    Mao, Jiamin

    Arsenic is a widely distributed toxic metalloid all over the world. Inorganic arsenic species are supposed to affect astrocytic functions and to cause neuron apoptosis in CNS. Microglias are the key cell type involved in innate immune responses in CNS, and microglia activation has been linked to inflammation and neurotoxicity. In this study, using ELISA, we showed that Arsenic trioxide up-regulated the expression and secretion of IL-1β in a dose-dependent manner and a time-dependent manner in cultured HAPI microglia cells. The secretion of IL-1β caused the apoptosis of SH-SY5Y. These pro-inflammatory responses were inhibited by the STAT3 blocker, AG490 andmore » P38/JNK MAPK blockers SB202190, SP600125. Further, Arsenic trioxide exposure could induce phosphorylation and activation of STAT3, and the translocation of STAT3 from the cytosol to the nucleus in this HAPI microglia cell line. Thus, the STAT3 signaling pathway can be activated after Arsenic trioxide treatment. However, P38/JNK MAPK blockers SB202190, SP600125 also obviously attenuated STAT3 activation and transnuclear transport induced by Arsenic trioxide. In concert with these results, we highlighted that the secretion of IL-1β and STAT3 activation induced by Arsenic trioxide can be mediated by elevation of P38/JNK MAPK in HAPI microglia cells and then induced the toxicity of neurons. - Highlights: • Arsenic trioxide exposure induced expression of IL-β in HAPI microglia. • Arsenic trioxide exposure induced activation of MAPK pathways in HAPI microglia. • Arsenic trioxide exposure induced activation of STAT3 pathways in HAPI microglia. • The expression of IL-β though P38/JNK MAPK/STAT3 pathways in HAPI microglia.« less

  1. Identification of Relationships Between Interleukin 15 mRNA and Brain-Derived Neurotrophic Factor II mRNA Levels With Formal Components of Temperament in Asthmatic Patients.

    PubMed

    Panek, Michał; Jonakowski, Mateusz; Zioło, Jan; Pietras, Tadeusz; Wieteska, Łukasz; Małachowska, Beata; Mokros, Łukasz; Szemraj, Janusz; Kuna, Piotr

    2017-04-01

    Asthma is a chronic inflammatory and heterogeneous disease developing mostly through allergic inflammation, which modifies the expression of various cytokines and neurotrophins. Previous studies suggest the involvement of interleukin (IL)-15 in the regulation of immune response in asthma. Brain-derived neurotrophic factor (BDNF) II plays an important role as a regulator of development and survival of neurons as well as maintenance of their physiological activity. Chronic stress associated with asthma and elevated IL-15 mRNA and BDNFII mRNA levels may affect the mood and a subjective sensation of dyspnoea-inducing anxiety. Psychopathological variables and numerous cytokine/neurotrophin interactions influence the formation of temperament and strategies of coping with stress. The aim of the study was to identify the role of IL-15 mRNA and BDNFII mRNA expressions and their effect on components of temperament and strategies of coping with stress in asthmatics. A total of 352 subjects (176 healthy volunteers and 176 asthmatic patients) participated in the study. The Formal Characteristic of Behaviour-Temperament Inventory (FCB-TI), Coping Inventory for Stressful Situations (CISS), Beck Depression Inventory, State-Trait Anxiety Inventory, and Borg Rating of Perceived Exertion (RPE) Scale were applied in all the subjects. The expression of IL-15 and BDNFII gene was measured using quantitative real-time polymerase chain reaction (qRT-PCR). Different levels of IL-15 and BDNFII expressions between healthy volunteers and patients were revealed in the study. IL-15 enhanced the BDNFII mRNA expression among patients with bronchial asthma. The depression level negatively correlated with the BDNFII mRNA expression. This neurotrophin modified the temperament variable. BDNFII significantly affected (proportional relationship) the level of briskness in asthmatic patients. BDNFII might influence the level and style of coping with stress (emotion-oriented style). This hypothesis

  2. Taurine zinc solid dispersions enhance bile-incubated L02 cell viability and improve liver function by inhibiting ERK2 and JNK phosphorylation during cholestasis.

    PubMed

    Wang, Yu; Mei, Xueting; Yuan, Jingquan; Lai, Xiaofang; Xu, Donghui

    2016-07-29

    Dietary intakes of taurine and zinc are associated with decreased risk of liver disease. In this study, solid dispersions (SDs) of a taurine zinc complex on hepatic injury were examined in vitro using the immortalized human hepatocyte cell line L02 and in a rat model of bile duct ligation. Sham-operated and bile duct ligated Sprague-Dawley rats were treated with the vehicle alone or taurine zinc (40, 80, 160mg/kg) for 17days. Bile duct ligation significantly increased blood lipid levels, and promoted hepatocyte apoptosis, inflammation and compensatory biliary proliferation. In vitro, incubation with bile significantly reduced L02 cell viability; this effect was significantly attenuated by pretreatment with SP600125 (a JNK inhibitor) and enhanced when co-incubated with taurine zinc SDs. In vivo, administration of taurine zinc SDs decreased serum alanine aminotransferase and aspartate aminotransferase activities in a dose-dependent manner and attenuated the increases in serum total bilirubin, total cholesterol and low density lipoprotein cholesterol levels after bile duct ligation. Additionally, taurine zinc SDs downregulated the expression of interleukin-1β and inhibited the phosphorylation of Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase2 (ERK2) in the liver after bile duct ligation. Moreover, taurine zinc SDs had more potent blood lipid regulatory and anti-apoptotic effects than the physical mixture of taurine and zinc acetate. Therefore, we speculate that taurine zinc SDs protect liver function at least in part via a mechanism linked to reduce phosphorylation of JNK and ERK2, which suppresses inflammation, apoptosis and cholangiocyte proliferation during cholestasis. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  3. Interleukins 1alpha and 1beta secreted by some melanoma cell lines strongly reduce expression of MITF-M and melanocyte differentiation antigens.

    PubMed

    Kholmanskikh, Olga; van Baren, Nicolas; Brasseur, Francis; Ottaviani, Sabrina; Vanacker, Julie; Arts, Nathalie; van der Bruggen, Pierre; Coulie, Pierre; De Plaen, Etienne

    2010-10-01

    We report that melanoma cell lines expressing the interleukin-1 receptor exhibit 4- to 10-fold lower levels of mRNA of microphthalmia-associated transcription factor (MITF-M) when treated with interleukin-1beta. This effect is NF-kappaB and JNK-dependent. MITF-M regulates the expression of melanocyte differentiation genes such as MLANA, tyrosinase and gp100, which encode antigens recognized on melanoma cells by autologous cytolytic T lymphocytes. Accordingly, treating some melanoma cells with IL-1beta reduced by 40-100% their ability to activate such antimelanoma cytolytic T lymphocytes. Finally, we observed large amounts of biologically active IL-1alpha or IL-1beta secreted by two melanoma cell lines that did not express MITF-M, suggesting an autocrine MITF-M downregulation. We estimate that approximately 13% of melanoma cell lines are MITF-M-negative and secrete IL-1 cytokines. These results indicate that the repression of melanocyte-differentiation genes by IL-1 produced by stromal cells or by tumor cells themselves may represent an additional mechanism of melanoma immune escape.

  4. Vitex rotundifolia Fruit Extract Induces Apoptosis Through the Downregulation of ATF3-Mediated Bcl-2 Expression in Human Colorectal Cancer Cells.

    PubMed

    Song, Hun Min; Park, Gwang Hun; Koo, Jin Suk; Jeong, Hyung Jin; Jeong, Jin Boo

    2017-01-01

    Fruit from Vitex rotundifolia L. (VF) has been reported to initiate apoptosis in human colorectal cancer cells through the accumulation of reactive oxygen species. Since various regulatory factors are involved in the apoptotic pathway, further study of the potential mechanisms of VF associated with the induction of apoptosis may be important despite the fact that the molecular target of VF for apoptosis has already been elucidated. In this study, we showed a new potential mechanism for the relationship between VF-mediated ATF3 expression and apoptosis to better understand the apoptotic mechanism of VF in human colorectal cancer cells. VF reduced the cell viability and induced apoptosis in human colorectal cancer cells. VF treatment increased both the protein and mRNA level of ATF3 and upregulated ATF3 promoter activity. The cis-element responsible for ATF3 transcriptional activation by VF was CREB which is located between [Formula: see text]147 to [Formula: see text]85 of ATF3 promoter. Inhibitions of ERK1/2, p38, JNK and GSK3[Formula: see text] blocked VF-mediated ATF3 expression. ATF3 knockdown by ATF3 siRNA attenuated the cleavage of PARP by VF, while ATF3 overexpression increased VF-mediated cleaved PARP. ATF3 knockdown also attenuated VF-mediated cell viability and cell death. In addition, VF downregulated Bcl-2 expression at both protein and mRNA level. ATF3 knockdown by ATF3 siRNA blocked VF-mediated downregulation of Bcl-2. In conclusion, VF may activate ATF3 expression through transcriptional regulation and subsequently suppress Bcl-2 expression as an anti-apoptotic protein, which may result in the induction of apoptosis in human colorectal cancer cells.

  5. Intrinsic catch-up growth of hypoplastic fetal lungs is mediated by interleukin-6.

    PubMed

    Nogueira-Silva, Cristina; Moura, Rute S; Esteves, Nuno; Gonzaga, Sílvia; Correia-Pinto, Jorge

    2008-07-01

    Fetal lung hypoplasia is a common finding in several fetal conditions such as congenital diaphragmatic hernia (CDH). Interestingly, previous studies have demonstrated that hypoplastic lungs have the ability to recover to normal size, when relieved from mechanical factors. However, the underlying mechanisms remain largely unknown. Recently, interleukin-6 (IL-6) has been involved in catch-up growth phenomenon in children. Thus, we hypothesized that IL-6 could mediate fetal growth recover from hypoplastic lungs. Control and nitrofen-induced hypoplastic lung explants were cultured either in normal conditions or with IL-6 neutralizing antibodies. The total number of peripheral airway buds, epithelial perimeter, and total explant area were analyzed and daily branching rates were calculated. Additionally, IL-6 mRNA and protein expression was assessed both in qualitative (by in situ hybridization and immunohistochemistry) and in quantitative (by real-time PCR and Western blot) approaches, in control and hypoplastic lungs (nitrofen and CDH groups). Nitrofen-induced hypoplastic lungs showed in vitro, out of systemic environment, the ability to recover from hypoplasia and presented daily branching rates significantly higher than controls. Blocking IL-6 activity significantly diminished the intrinsic capacity of hypoplastic fetal lungs to recover from hypoplasia and attenuated their daily branching rates. Although more exacerbated in CDH, both nitrofen-exposed lungs presented significant IL-6 mRNA and protein over-expression throughout all studied gestational ages. The present study suggests, for the first time, that fetal lung is able to recover from growth retardation through a way that resembles the catch-up growth phenomenon, and it seems to be, at least partially, orchestrated by intrinsic mechanisms implicating IL-6.

  6. 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

  7. mRNA–mRNA duplexes that auto-elicit Staufen1-mediated mRNA decay

    PubMed Central

    Gong, Chenguang; Tang, Yalan; Maquat, Lynne E.

    2013-01-01

    We report a new mechanism by which human mRNAs crosstalk: an Alu element in the 3'-untranslated region (3' UTR) of one mRNA can base-pair with a partially complementary Alu element in the 3' UTR of a different mRNA thereby creating a Staufen1 (STAU1)-binding site (SBS). STAU1 binding to a 3' UTR SBS was previously shown to trigger STAU1-mediated mRNA decay (SMD) by directly recruiting the ATP-dependent RNA helicase UPF1, which is also a key factor in the mechanistically related nonsense-mediated mRNA decay (NMD) pathway. In the case of a 3' UTR SBS created via mRNA–mRNA base-pairing, we show that SMD targets both mRNAs in the duplex provided that both mRNAs are translated. If only one mRNA is translated, then it alone is targeted for SMD. We demonstrate the importance of mRNA–mRNA-triggered SMD to the processes of cell migration and invasion. PMID:24056942

  8. Eicosapentaenoic acid (EPA) induced apoptosis in HepG2 cells through ROS–Ca{sup 2+}–JNK mitochondrial pathways

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

    Zhang, Yuanyuan; Han, Lirong; Qi, Wentao

    Highlights: • EPA evoked ROS formation, [Ca{sup 2+}]{sub c} accumulation, the opening of MPTP and the phosphorylation of JNK. • EPA-induced [Ca{sup 2+}]{sub c} elevation was depended on production of ROS. • EPA-induced ROS generation, [Ca{sup 2+}]{sub c} increase, and JNK activated caused MPTP opening. • The apoptosis induced by EPA was related to release of cytochrome C through the MPTP. • EPA induced HepG2 cells apoptosis through ROS–Ca{sup 2+}–JNK mitochondrial pathways. - Abstract: Eicosapentaenoic acid (EPA), a well-known dietary n−3 PUFAS, has been considered to inhibit proliferation of tumor cells. However, the molecular mechanism related to EPA-induced liver cancermore » cells apoptosis has not been reported. In this study, we investigated the effect of EPA on HepG2 cells proliferation and apoptosis mechanism through mitochondrial pathways. EPA inhibited proliferation of HepG2 cells in a dose-dependent manner and had no significant effect on the cell viability of humor normal liver L-02 cells. It was found that EPA initially evoked ROS formation, leading to [Ca{sup 2+}]{sub c} accumulation and the mitochondrial permeability transition pore (MPTP) opening; EPA-induced HepG2 cells apoptosis was inhibited by N-acetylcysteine (NAC, an inhibitor of ROS), 1,2-bis (2-aminophenoxy) ethane-N,N,N′,N′-tetraacetic acid (BAPTA-AM, a chelator of calcium) and CsA (inhibitor of MPTP). The relationship between ROS production, the increase of cytoplasmic Ca and MPTP opening was detected. It seems that ROS may act as an upstream regulator of EPA-induced [Ca{sup 2+}]{sub c} generation, moreover, generation of ROS, overload of mitochondrial [Ca{sup 2+}]{sub c}, and JNK activated cause the opening of MPTP. Western blotting results showed that EPA elevated the phosphorylation status of JNK, processes associated with the ROS generation. Simultaneously, the apoptosis induced by EPA was related to release of cytochrome C from mitochondria to cytoplasm through

  9. Silibinin strongly inhibits the growth kinetics of colon cancer stem cell-enriched spheroids by modulating interleukin 4/6-mediated survival signals

    PubMed Central

    Agarwal, Chapla; Agarwal, Rajesh

    2014-01-01

    Involvement of cancer stem cells (CSC) in initiation, progression, relapse, and therapy-resistance of colorectal cancer (CRC) warrants search for small molecules as ‘adjunct-therapy’ to target both colon CSC and bulk tumor population. Herein, we assessed the potential of silibinin to eradicate colon CSC together with associated molecular mechanisms. In studies examining how silibinin modulates dynamics of CSC spheroids in terms of its effect on kinetics of CSC spheroids generated in presence of mitogenic and interleukin (IL)-mediated signaling which provides an autocrine/paracrine amplification loop in CRC, silibinin strongly decreased colon CSC pool together with cell survival of bulk tumor cells. Silibinin effect on colon CSC was mediated via blocking of pro-tumorigenic signaling, notably IL-4/-6 signaling that affects CSC population. These silibinin effects were associated with decreased mRNA and protein levels of various CSC-associated transcription factors, signaling molecules and markers. Furthermore, 2D and 3D differentiation assays indicated formation of more differentiated clones by silibinin. These results highlight silibinin potential to interfere with kinetics of CSC pool by shifting CSC cell division to asymmetric type via targeting various signals associated with the survival and multiplication of colon CSC pool. Together, our findings further support clinical usefulness of silibinin in CRC intervention and therapy. PMID:24970802

  10. Glucocorticoid receptor interacts with PNRC2 in a ligand-dependent manner to recruit UPF1 for rapid mRNA degradation.

    PubMed

    Cho, Hana; Park, Ok Hyun; Park, Joori; Ryu, Incheol; Kim, Jeonghan; Ko, Jesang; Kim, Yoon Ki

    2015-03-31

    Glucocorticoid receptor (GR), which was originally known to function as a nuclear receptor, plays a role in rapid mRNA degradation by acting as an RNA-binding protein. The mechanism by which this process occurs remains unknown. Here, we demonstrate that GR, preloaded onto the 5'UTR of a target mRNA, recruits UPF1 through proline-rich nuclear receptor coregulatory protein 2 (PNRC2) in a ligand-dependent manner, so as to elicit rapid mRNA degradation. We call this process GR-mediated mRNA decay (GMD). Although GMD, nonsense-mediated mRNA decay (NMD), and staufen-mediated mRNA decay (SMD) share upstream frameshift 1 (UPF1) and PNRC2, we find that GMD is mechanistically distinct from NMD and SMD. We also identify de novo cellular GMD substrates using microarray analysis. Intriguingly, GMD functions in the chemotaxis of human monocytes by targeting chemokine (C-C motif) ligand 2 (CCL2) mRNA. Thus, our data provide molecular evidence of a posttranscriptional role of the well-studied nuclear hormone receptor, GR, which is traditionally considered a transcription factor.

  11. A role for protein phosphatase-2A in p38 mitogen-activated protein kinase-mediated regulation of the c-Jun NH(2)-terminal kinase pathway in human neutrophils.

    PubMed

    Avdi, Natalie J; Malcolm, Kenneth C; Nick, Jerry A; Worthen, G Scott

    2002-10-25

    Human neutrophil accumulation in inflammatory foci is essential for the effective control of microbial infections. Although exposure of neutrophils to cytokines such as tumor necrosis factor-alpha (TNFalpha), generated at sites of inflammation, leads to activation of MAPK pathways, mechanisms responsible for the fine regulation of specific MAPK modules remain unknown. We have previously demonstrated activation of a TNFalpha-mediated JNK pathway module, leading to apoptosis in adherent human neutrophils (Avdi, N. J., Nick, J. A., Whitlock, B. B., Billstrom, M. A., Henson, P. M., Johnson, G. L., and Worthen, G. S. (2001) J. Biol. Chem. 276, 2189-2199). Herein, evidence is presented linking regulation of the JNK pathway to p38 MAPK and the Ser/Thr protein phosphatase-2A (PP2A). Inhibition of p38 MAPK by SB 203580 and M 39 resulted in significant augmentation of TNFalpha-induced JNK and MKK4 (but not MKK7 or MEKK1) activation, whereas prior exposure to a p38-activating agent (platelet-activating factor) diminished the TNFalpha-induced JNK response. TNFalpha-induced apoptosis was also greatly enhanced upon p38 inhibition. Studies with a reconstituted cell-free system indicated the absence of a direct inhibitory effect of p38 MAPK on the JNK module. Neutrophil exposure to the Ser/Thr phosphatase inhibitors okadaic acid and calyculin A induced JNK activation. Increased phosphatase activity following TNFalpha stimulation was shown to be PP2A-associated and p38-dependent. Furthermore, PP2A-induced dephosphorylation of MKK4 resulted in its inactivation. Thus, in neutrophils, p38 MAPK, through a PP2A-mediated mechanism, regulates the JNK pathway, thus determining the extent and nature of subsequent responses such as apoptosis.

  12. JNK and NADPH Oxidase Involved in Fluoride-Induced Oxidative Stress in BV-2 Microglia Cells

    PubMed Central

    Yan, Ling; Liu, Shengnan; Wang, Chen; Wang, Fei; Song, Yingli; Yan, Nan; Xi, Shuhua; Liu, Ziyou; Sun, Guifan

    2013-01-01

    Excessive fluoride may cause central nervous system (CNS) dysfunction, and oxidative stress is a recognized mode of action of fluoride toxicity. In CNS, activated microglial cells can release more reactive oxygen species (ROS), and NADPH oxidase (NOX) is the major enzyme for the production of extracellular superoxide in microglia. ROS have been characterized as an important secondary messenger and modulator for various mammalian intracellular signaling pathways, including the MAPK pathways. In this study we examined ROS production and TNF-α, IL-1β inflammatory cytokines releasing, and the expression of MAPKs in BV-2 microglia cells treated with fluoride. We found that fluoride increased JNK phosphorylation level of BV-2 cells and pretreatment with JNK inhibitor SP600125 markedly reduced the levels of intracellular O2 ·− and NO. NOX inhibitor apocynin and iNOS inhibitor SMT dramatically decreased NaF-induced ROS and NO generations, respectively. Antioxidant melatonin (MEL) resulted in a reduction in JNK phosphorylation in fluoride-stimulated BV-2 microglia. The results confirmed that NOX and iNOS played an important role in fluoride inducing oxidative stress and NO production and JNK took part in the oxidative stress induced by fluoride and meanwhile also could be activated by ROS in fluoride-treated BV-2 cells. PMID:24072958

  13. Curcumol suppresses RANKL-induced osteoclast formation by attenuating the JNK signaling pathway

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

    Yu, Mingxiang, E-mail: yu.mingxiang@zs-hospital.sh.cn; Chen, Xianying; Lv, Chaoyang

    Highlights: • Curcumol suppresses osteoclasts differentiation in vitro. • Curcumol impairs JNK/AP-1 signaling pathway. • Curcumol may be used for treating osteoclast related diseases. - Abstract: Osteoclasts, derived from hemopoietic progenitors of the monocyte/macrophage lineage, have a unique role in bone resorption, and are considered a potential therapeutic target in the treatment of such pathologic bone diseases as osteoporosis, rheumatoid arthritis, and periodontitis. In the present study, we demonstrate that curcumol, one of the major components of the essential oil of Rhizoma Curcumae, exhibits an inhibitory effect on receptor activator of nuclear factor kappaB ligand (RANKL)-induced osteoclast differentiation with bothmore » bone marrow-derived macrophages and RAW264.7 cells in a dose-dependent manner. In addition, RANKL-induced mRNA expression of osteoclast-specific genes, such as tartrate-resistant acid phosphatase, calcitonin receptor, and cathepsin K, is prominently reduced in the presence of curcumol. Furthermore, the molecular mechanism of action was investigated, and curcumol inhibited osteoclastogenesis by specifically impairing RANKL-induced c-Jun N-terminal kinase (JNK)/activator protein-1 (AP-1) signaling, which was further identified in rescue studies by means of anisomycin, a JNK signaling-specific activator. Taken together, these findings suggest that curcumol suppresses RANKL-induced osteoclast differentiation through the JNK/AP-1 signaling pathway, and may be useful as a therapeutic treatment for bone resorption-associated diseases.« less

  14. Ectopic expression of H2AX protein promotes TrkA-induced cell death via modulation of TrkA tyrosine-490 phosphorylation and JNK activity upon DNA damage

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

    Jung, Eun Joo; Kim, Deok Ryong, E-mail: drkim@gnu.ac.kr

    2011-01-21

    Research highlights: {yields} We established TrkA-inducible U2OS cells stably expressing GFP-H2AX proteins. {yields} GFP-H2AX was colocalized with TrkA in the cytoplasm. {yields} {gamma}H2AX production was significantly increased upon activation of TrkA and suppressed by TrkA inhibitor or JNK inhibitor. {yields} Ectopic expression of H2AX promoted TrkA-mediated cell death through the modulation of TrkA tyrosine-490 phosphorylation and JNK activity upon DNA damage. -- Abstract: We previously reported that TrkA overexpression causes accumulation of {gamma}H2AX proteins in the cytoplasm, subsequently leading to massive cell death in U2OS cells. To further investigate how cytoplasmic H2AX is associated with TrkA-induced cell death, we establishedmore » TrkA-inducible cells stably expressing GFP-tagged H2AX. We found that TrkA co-localizes with ectopically expressed GFP-H2AX proteins in the cytoplasm, especially at the juxta-nuclear membranes, which supports our previous results about a functional connection between TrkA and {gamma}H2AX in TrkA-induced cell death. {gamma}H2AX production from GFP-H2AX proteins was significantly increased when TrkA was overexpressed. Moreover, ectopic expression of H2AX activated TrkA-mediated signal pathways via up-regulation of TrkA tyrosine-490 phosphorylation. In addition, suppression of TrkA tyrosine-490 phosphorylation under a certain condition was removed by ectopic expression of H2AX, indicating a functional role of H2AX in the maintenance of TrkA activity. Indeed, TrkA-induced cell death was highly elevated by ectopic H2AX expression, and it was further accelerated by DNA damage via JNK activation. These all results suggest that cytoplasmic H2AX could play an important role in TrkA-mediated cell death by modulating TrkA upon DNA damage.« less

  15. Exon 2-mediated c-myc mRNA decay in vivo is independent of its translation.

    PubMed Central

    Pistoi, S; Roland, J; Babinet, C; Morello, D

    1996-01-01

    We have previously shown that the steady-state level of c-myc mRNA in vivo is primarily controlled by posttranscriptional regulatory mechanisms. To identify the sequences involved in this process, we constructed a series of H-2/myc transgenic lines in which various regions of the human c-MYC gene were placed under the control of the quasi-ubiquitous H-2K class I regulatory sequences. We demonstrated that the presence of one of the two coding exons, exon 2 or exon 3, is sufficient to confer a level of expression of transgene mRNA similar to that of endogenous c-myc in various adult tissues as well as after partial hepatectomy or after protein synthesis inhibition. We now focus on the molecular mechanisms involved in modulation of expression of mRNAs containing c-myc exon 2 sequences, with special emphasis on the coupling between translation and c-myc mRNA turnover. We have undertaken an analysis of expression, both at the mRNA level and at the protein level, of new transgenic constructs in which the translation is impaired either by disruption of the initiation codon or by addition of stop codons upstream of exon 2. Our results show that the translation of c-myc exon 2 is not required for regulated expression of the transgene in the different situations analyzed, and therefore they indicate that the mRNA destabilizing function of exon 2 is independent of translation by ribosomes. Our investigations also reveal that, in the thymus, some H-2/myc transgenes express high levels of mRNA but low levels of protein. Besides the fact that these results suggest the existence of tissue-specific mechanisms that control c-myc translatability in vivo, they also bring another indication of the uncoupling of c-myc mRNA translation and degradation. PMID:8756668

  16. Induction of an angiogenic phenotype in endometriotic stromal cell cultures by interleukin-1beta.

    PubMed

    Lebovic, D I; Bentzien, F; Chao, V A; Garrett, E N; Meng, Y G; Taylor, R N

    2000-03-01

    Activated peritoneal macrophages are associated with endometriosis and may play a central role in its aetiology by releasing interleukin-1beta (IL-1beta) in response to refluxed endometrium. Pari passu with the establishment of endometriotic implants is the development of a vascular supply. In this study we investigated the angiogenic properties of two endometrial proteins, vascular endothelial growth factor (VEGF) and interleukin-6 (IL-6), and assessed their production in response to IL-1beta stimulation in human stromal cells isolated from normal endometrium (NE) and endometriotic lesions (EI). Proliferation of bovine brain capillary endothelial cells (BBCE) with a [(3)H]-thymidine incorporation assay was observed when VEGF (2.1 +/- 0.2-fold; P < 0.05) or VEGF and IL-6 (1.8 +/- 0.1-fold; P < 0.05) were added in vitro, relative to saline-treated control cultures. Northern blot analysis showed induction of VEGF mRNA (2.6-fold; P < 0.05) and IL-6 mRNA (6.3-fold; P < 0.05) transcripts in EI cells, but not NE cells, exposed to IL-1beta. A similar induction was seen with VEGF and IL-6 protein secretion in the responsive EI cells. Reverse transcription-polymerase chain reaction (RT-PCR) for the IL-1 receptor type I (IL-1 RI) indicated that the differential effects of IL-1beta on NE and EI cells was associated with 2.4 +/- 0.1-fold more receptor mRNA in EI versus NE cells. We propose that the ability of IL-1beta to activate an angiogenic phenotype in EI stromal cells but not in NE cells, is mediated by the IL-1 RI.

  17. JNK1 Inhibition Attenuates Hypoxia-Induced Autophagy and Sensitizes to Chemotherapy.

    PubMed

    Vasilevskaya, Irina A; Selvakumaran, Muthu; Roberts, David; O'Dwyer, Peter J

    2016-08-01

    Inhibition of hypoxia-induced stress signaling through JNK potentiates the effects of oxaliplatin. The JNK pathway plays a role in both autophagy and apoptosis; therefore, it was determined how much of the effect of JNK inhibition on oxaliplatin sensitivity is dependent on its effect on autophagy. We studied the impact of JNK isoform downregulation in the HT29 colon adenocarcinoma cell line on hypoxia- and oxaliplatin-induced responses. Electron microscopic analyses demonstrated that both oxaliplatin- and hypoxia-induced formations of autophagosomes were reduced significantly in HT29 cells treated with the JNK inhibitor SP600125. The role of specific JNK isoforms was defined using HT29-derived cell lines stably expressing dominant-negative constructs for JNK1 and JNK2 (HTJ1.3 and HTJ2.2, respectively). These cell lines demonstrated that functional JNK1 is required for hypoxia-induced autophagy and that JNK2 does not substitute for it. Inhibition of autophagy in HTJ1.3 cells also coincided with enhancement of intrinsic apoptosis. Analysis of Bcl2-family proteins revealed hyperphosphorylation of Bcl-XL in the HTJ1.3 cell line, but this did not lead to the expected dissociation from Beclin 1. Consistent with this, knockdown of Bcl-XL in HT29 cells did not significantly affect the induction of autophagy, but abrogated hypoxic resistance to oxaliplatin due to the faster and more robust activation of apoptosis. These data suggest that balance between autophagy and apoptosis is shifted toward apoptosis by downregulation of JNK1, contributing to oxaliplatin sensitization. These findings further support the investigation of JNK inhibition in colorectal cancer treatment. Mol Cancer Res; 14(8); 753-63. ©2016 AACR. ©2016 American Association for Cancer Research.

  18. Friedelane-type triterpenoids as selective anti-inflammatory agents by regulation of differential signaling pathways in LPS-stimulated macrophages

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

    Villar-Lorenzo, Andrea, E-mail: avillar@iib.uam.es

    A series of 31 pentacyclic triterpenoids isolated from the root barks of Celastrus vulcanicola and Maytenus jelskii were tested for cytotoxicity and inhibitory activity against lipopolysaccharide (LPS)-induced nitric oxide (NO) production in RAW 264.7 macrophages. Compounds 18 (C18) and 25 (C25) exhibited significant inhibition of LPS-induced NO release at 50 and 25 μM concentrations, respectively, and decreased mRNAs of pro-inflammatory cytokines. At the molecular level, C18 neither inhibited LPS-mediated phosphorylation of mitogen activated protein kinases (MAPKs) nor nuclear translocation of nuclear factor kappa beta (NFκB). Instead, C18 enhanced and prolonged nuclear translocation of nuclear factor-erythroid 2-related factor 2 (Nrf2) andmore » increased the expression of its target genes including hemeoxigenase 1 (HO1). C25 efficiently inhibited LPS-mediated phosphorylation of JNK, p38 and ERK, without affecting NFκB or Nrf2 signaling pathways. Both compounds reduced LPS-mediated processing of caspase-1 and the cleavage of interleukin 1β (IL1β) proform, reflecting their ability to target the inflammasome. C25 also counteracted LPS effects on iNOS expression and pro-inflammatory cytokines mRNA levels in Bv-2 microglial cells. The anti-inflammatory effect of both compounds was also assessed in human macrophages. Our results suggest that triterpenoids C18 and C25 possess anti-inflammatory effects, which may be therapeutically relevant for diseases linked to inflammation. - Highlights: • Compounds 18 (C18) and 25 (C25) exert anti-inflammatory effects in macrophages. • C18 enhanced nuclear translocation of Nrf2 and increased HO1 expression. • C25 inhibited the phosphorylation of JNK, p38 and ERK, members of the MAPKs family. • C25 reduced LPS-mediated processing of caspase-1 and the cleavage of interleukin 1β. • C18 and C25 may be therapeutic agents for diseases linked to inflammation.« less

  19. MicroRNA-155 attenuates late sepsis-induced cardiac dysfunction through JNK and β-arrestin 2.

    PubMed

    Zhou, Yu; Song, Yan; Shaikh, Zahir; Li, Hui; Zhang, Haiju; Caudle, Yi; Zheng, Shouhua; Yan, Hui; Hu, Dan; Stuart, Charles; Yin, Deling

    2017-07-18

    Cardiac dysfunction is correlated with detrimental prognosis of sepsis and contributes to a high risk of mortality. After an initial hyperinflammatory reaction, most patients enter a protracted state of immunosuppression (late sepsis) that alters both innate and adaptive immunity. The changes of cardiac function in late sepsis are not yet known. MicroRNA-155 (miR-155) is previously found to play important roles in both regulations of immune activation and cardiac function. In this study, C57BL/6 mice were operated to develop into early and late sepsis phases, and miR-155 mimic was injected through the tail vein 48 h after cecal ligation and puncture (CLP). The effect of miR-155 on CLP-induced cardiac dysfunction was explored in late sepsis. We found that increased expression of miR-155 in the myocardium protected against cardiac dysfunction in late sepsis evidenced by attenuating sepsis-reduced cardiac output and enhancing left ventricular systolic function. We also observed that miR-155 markedly reduced the infiltration of macrophages and neutrophils into the myocardium and attenuated the inflammatory response via suppression of JNK signaling pathway. Moreover, overexpression of β-arrestin 2 (Arrb2) exacerbated the mice mortality and immunosuppression in late sepsis. Furthermore, transfection of miR-155 mimic reduced Arrb2 expression, and then restored immunocompetence and improved survival in late septic mice. We conclude that increased miR-155 expression through systemic administration of miR-155 mimic attenuates cardiac dysfunction and improves late sepsis survival by targeting JNK associated inflammatory signaling and Arrb2 mediated immunosuppression.

  20. Phosphorylation of Glutathione S-Transferase P1 (GSTP1) by Epidermal Growth Factor Receptor (EGFR) Promotes Formation of the GSTP1-c-Jun N-terminal kinase (JNK) Complex and Suppresses JNK Downstream Signaling and Apoptosis in Brain Tumor Cells*

    PubMed Central

    Okamura, Tatsunori; Antoun, Gamil; Keir, Stephen T.; Friedman, Henry; Bigner, Darell D.; Ali-Osman, Francis

    2015-01-01

    Under normal physiologic conditions, the glutathione S-transferase P1 (GSTP1) protein exists intracellularly as a dimer in reversible equilibrium with its monomeric subunits. In the latter form, GSTP1 binds to the mitogen-activated protein kinase, JNK, and inhibits JNK downstream signaling. In tumor cells, which frequently are characterized by constitutively high GSTP1 expression, GSTP1 undergoes phosphorylation by epidermal growth factor receptor (EGFR) at tyrosine residues 3, 7, and 198. Here we report on the effect of this EGFR-dependent GSTP1 tyrosine phosphorylation on the interaction of GSTP1 with JNK, on the regulation of JNK downstream signaling by GSTP1, and on tumor cell survival. Using in vitro and in vivo growing human brain tumors, we show that tyrosine phosphorylation shifts the GSTP1 dimer-monomer equilibrium to the monomeric state and facilitates the formation of the GSTP1-JNK complex, in which JNK is functionally inhibited. Targeted mutagenesis and functional analysis demonstrated that the increased GSTP1 binding to JNK results from phosphorylation of the GSTP1 C-terminal Tyr-198 by EGFR and is associated with a >2.5-fold decrease in JNK downstream signaling and a significant suppression of both spontaneous and drug-induced apoptosis in the tumor cells. The findings define a novel mechanism of regulatory control of JNK signaling that is mediated by the EGFR/GSTP1 cross-talk and provides a survival advantage for tumors with activated EGFR and high GSTP1 expression. The results lay the foundation for a novel strategy of dual EGFR/GSTP1 for treating EGFR+ve, GSTP1 expressing GBMs. PMID:26429914

  1. Phosphorylation of Glutathione S-Transferase P1 (GSTP1) by Epidermal Growth Factor Receptor (EGFR) Promotes Formation of the GSTP1-c-Jun N-terminal kinase (JNK) Complex and Suppresses JNK Downstream Signaling and Apoptosis in Brain Tumor Cells.

    PubMed

    Okamura, Tatsunori; Antoun, Gamil; Keir, Stephen T; Friedman, Henry; Bigner, Darell D; Ali-Osman, Francis

    2015-12-25

    Under normal physiologic conditions, the glutathione S-transferase P1 (GSTP1) protein exists intracellularly as a dimer in reversible equilibrium with its monomeric subunits. In the latter form, GSTP1 binds to the mitogen-activated protein kinase, JNK, and inhibits JNK downstream signaling. In tumor cells, which frequently are characterized by constitutively high GSTP1 expression, GSTP1 undergoes phosphorylation by epidermal growth factor receptor (EGFR) at tyrosine residues 3, 7, and 198. Here we report on the effect of this EGFR-dependent GSTP1 tyrosine phosphorylation on the interaction of GSTP1 with JNK, on the regulation of JNK downstream signaling by GSTP1, and on tumor cell survival. Using in vitro and in vivo growing human brain tumors, we show that tyrosine phosphorylation shifts the GSTP1 dimer-monomer equilibrium to the monomeric state and facilitates the formation of the GSTP1-JNK complex, in which JNK is functionally inhibited. Targeted mutagenesis and functional analysis demonstrated that the increased GSTP1 binding to JNK results from phosphorylation of the GSTP1 C-terminal Tyr-198 by EGFR and is associated with a >2.5-fold decrease in JNK downstream signaling and a significant suppression of both spontaneous and drug-induced apoptosis in the tumor cells. The findings define a novel mechanism of regulatory control of JNK signaling that is mediated by the EGFR/GSTP1 cross-talk and provides a survival advantage for tumors with activated EGFR and high GSTP1 expression. The results lay the foundation for a novel strategy of dual EGFR/GSTP1 for treating EGFR+ve, GSTP1 expressing GBMs. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  2. Protective Role of Taurine against Arsenic-Induced Mitochondria-Dependent Hepatic Apoptosis via the Inhibition of PKCδ-JNK Pathway

    PubMed Central

    Das, Joydeep; Ghosh, Jyotirmoy; Manna, Prasenjit; Sil, Parames C.

    2010-01-01

    Background Oxidative stress-mediated hepatotoxic effect of arsenic (As) is mainly due to the depletion of glutathione (GSH) in liver. Taurine, on the other hand, enhances intracellular production of GSH. Little is known about the mechanism of the beneficial role of taurine in As-induced hepatic pathophysiology. Therefore, in the present study we investigated its beneficial role in As-induced hepatic cell death via mitochondria-mediated pathway. Methodology/Principal Findings Rats were exposed to NaAsO2 (2 mg/kg body weight for 6 months) and the hepatic tissue was used for oxidative stress measurements. In addition, the pathophysiologic effect of NaAsO2 (10 µM) on hepatocytes was evaluated by determining cell viability, mitochondrial membrane potential and ROS generation. As caused mitochondrial injury by increased oxidative stress and reciprocal regulation of Bcl-2, Bcl-xL/Bad, Bax, Bim in association with increased level of Apaf-1, activation of caspase 9/3, cleavage of PARP protein and ultimately led to apoptotic cell death. In addition, As markedly increased JNK and p38 phosphorylation with minimal disturbance of ERK. Pre-exposure of hepatocytes to a JNK inhibitor SP600125 prevented As-induced caspase-3 activation, ROS production and loss in cell viability. Pre-exposure of hepatocytes to a p38 inhibitor SB2035, on the other hand, had practically no effect on these events. Besides, As activated PKCδ and pre-treatment of hepatocytes with its inhibitor, rottlerin, suppressed the activation of JNK indicating that PKCδ is involved in As-induced JNK activation and mitochondrial dependent apoptosis. Oral administration of taurine (50 mg/kg body weight for 2 weeks) both pre and post to NaAsO2 exposure or incubation of the hepatocytes with taurine (25 mM) were found to be effective in counteracting As-induced oxidative stress and apoptosis. Conclusions/Significance Results indicate that taurine treatment improved As-induced hepatic damages by inhibiting PKCδ-JNK

  3. Joining the dots - protein-RNA interactions mediating local mRNA translation in neurons.

    PubMed

    Gallagher, Christopher; Ramos, Andres

    2018-06-01

    Establishing and maintaining the complex network of connections required for neuronal communication requires the transport and in situ translation of large groups of mRNAs to create local proteomes. In this Review, we discuss the regulation of local mRNA translation in neurons and the RNA-binding proteins that recognise RNA zipcode elements and connect the mRNAs to the cellular transport networks, as well as regulate their translation control. However, mRNA recognition by the regulatory proteins is mediated by the combinatorial action of multiple RNA-binding domains. This increases the specificity and affinity of the interaction, while allowing the protein to recognise a diverse set of targets and mediate a range of mechanisms for translational regulation. The structural and molecular understanding of the interactions can be used together with novel microscopy and transcriptome-wide data to build a mechanistic framework for the regulation of local mRNA translation. © 2018 Federation of European Biochemical Societies.

  4. Anti-inflammatory effects of ursodeoxycholic acid by lipopolysaccharide-stimulated inflammatory responses in RAW 264.7 macrophages

    PubMed Central

    Ko, Wan-Kyu; Lee, Soo-Hong; Kim, Sung Jun; Jo, Min-Jae; Kumar, Hemant; Han, In-Bo; Sohn, Seil

    2017-01-01

    Purpose The aim of this study was to investigate the anti-inflammatory effects of Ursodeoxycholic acid (UDCA) in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. Methods We induced an inflammatory process in RAW 264.7 macrophages using LPS. The anti-inflammatory effects of UDCA on LPS-stimulated RAW 264.7 macrophages were analyzed using nitric oxide (NO). Pro-inflammatory and anti-inflammatory cytokines were analyzed by quantitative real time polymerase chain reaction (qRT-PCR) and enzyme-linked immunosorbent assay (ELISA). The phosphorylations of extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 in mitogen-activated protein kinase (MAPK) signaling pathways and nuclear factor kappa-light polypeptide gene enhancer in B-cells inhibitor, alpha (IκBα) signaling pathways were evaluated by western blot assays. Results UDCA decreased the LPS-stimulated release of the inflammatory mediator NO. UDCA also decreased the pro-inflammatory cytokines tumor necrosis factor-α (TNF-α), interleukin 1-α (IL-1α), interleukin 1-β (IL-1β), and interleukin 6 (IL-6) in mRNA and protein levels. In addition, UDCA increased an anti-inflammatory cytokine interleukin 10 (IL-10) in the LPS-stimulated RAW 264.7 macrophages. UDCA inhibited the expression of inflammatory transcription factor nuclear factor kappa B (NF-κB) in LPS-stimulated RAW 264.7 macrophages. Furthermore, UDCA suppressed the phosphorylation of ERK, JNK, and p38 signals related to inflammatory pathways. In addition, the phosphorylation of IκBα, the inhibitor of NF-κB, also inhibited by UDCA. Conclusion UDCA inhibits the pro-inflammatory responses by LPS in RAW 264.7 macrophages. UDCA also suppresses the phosphorylation by LPS on ERK, JNK, and p38 in MAPKs and NF-κB pathway. These results suggest that UDCA can serve as a useful anti-inflammatory drug. PMID:28665991

  5. Oxidized Low-Density Lipoprotein-Activated c-Jun NH2-Terminal Kinase Regulates Manganese Superoxide Dismutase Ubiquitination

    PubMed Central

    Takabe, Wakako; Li, Rongsong; Ai, Lisong; Yu, Fei; Berliner, Judith A.; Hsiai, Tzung K.

    2012-01-01

    Objective Oxidized low-density lipoprotein (oxLDL) modulates intracellular redox status and induces apoptosis in endothelial cells. However, the signal pathways and molecular mechanism remain unknown. In this study, we investigated the role of manganese superoxide dismutase (Mn-SOD) on oxLDL-induced apoptosis via c-Jun NH2-terminal kinase (JNK)-mediated ubiquitin/proteasome pathway. Methods and Results OxLDL induced JNK phosphorylation that peaked at 30 minutes in human aortic endothelial cells. Fluorescence-activated cell sorting analysis revealed that oxLDL increased mitochondrial superoxide production by 1.88±0.19-fold and mitochondrial membrane potential by 18%. JNK small interference RNA (siJNK) reduced oxLDL-induced mitochondrial superoxide production by 88.4% and mitochondrial membrane potential by 61.7%. OxLDL did not affect Mn-SOD mRNA expression, but it significantly reduced Mn-SOD protein level, which was restored by siJNK. Immunoprecipitation by ubiquitin antibody revealed that oxLDL increased ubiquitination of Mn-SOD, which was inhibited by siJNK. OxLDL-induced caspase-3 activities were also attenuated by siJNK but were enhanced by Mn-SOD small interfering RNA. Furthermore, overexpression of Mn-SOD abrogated oxLDL-induced caspase-3 activities. Conclusion OxLDL-induced JNK activation regulates mitochondrial redox status and Mn-SOD protein degradation via JNK-dependent ubiquitination, leading to endothelial cell apoptosis. PMID:20139358

  6. Secreted phospholipase A2 of Clonorchis sinensis activates hepatic stellate cells through a pathway involving JNK signalling.

    PubMed

    Wu, Yinjuan; Li, Ye; Shang, Mei; Jian, Yu; Wang, Caiqin; Bardeesi, Adham Sameer A; Li, Zhaolei; Chen, Tingjin; Zhao, Lu; Zhou, Lina; He, Ai; Huang, Yan; Lv, Zhiyue; Yu, Xinbing; Li, Xuerong

    2017-03-16

    Secreted phospholipase A2 (sPLA2) is a protein secreted by Clonorchis sinensis and is a component of excretory and secretory products (CsESPs). Phospholipase A2 is well known for its role in liver fibrosis and inhibition of tumour cells. The JNK signalling pathway is involved in hepatic stellate cells (HSCs) activation. Blocking JNK activity with SP600125 inhibits HSCs activation. In a previous study, the protein CssPLA2 was expressed in insoluble inclusion bodies. Therefore, it's necessary to express CssPLA2 in water-soluble form and determine whether the enzymatic activity of CssPLA2 or cell signalling pathways is involved in liver fibrosis caused by clonorchiasis. Balb/C mice were given an abdominal injection of MBP-CssPLA2. Liver sections with HE and Masson staining were observed to detect accumulation of collagen. Western blot of mouse liver was done to detect the activation of JNK signalling pathway. In vitro, HSCs were incubated with MBP-CssPLA2 to detect the activation of HSCs as well as the activation of JNK signalling pathway. The mutant of MBP-CssPLA2 without enzymatic activity was constructed and was also incubated with HSCs to check whether activation of the HSCs was related to the enzymatic activity of MBP-CssPLA2. The recombinant protein MBP-CssPLA2 was expressed soluble and of good enzymatic activity. A mutant of CssPLA2, without enzymatic activity, was also constructed. In vivo liver sections of Balb/C mice that were given an abdominal injection of 50 μg/ml MBP-CssPLA2 showed an obvious accumulation of collagen and a clear band of P-JNK1 could be seen by western blot of the liver tissue. In vitro, MBP-CssPLA2, as well as the mutant, was incubated with HSCs and it was proved that activation of HSCs was related to activation of the JNK signalling pathway instead of the enzymatic activity of MBP-CssPLA2. Activation of HSCs by CssPLA2 is related to the activation of the JNK signalling pathway instead of the enzymatic activity of CssPLA2. This finding

  7. Activation of colo-rectal high-threshold afferent nerves by Interleukin-2 is tetrodotoxin-sensitive and upregulated in a mouse model of chronic visceral hypersensitivity.

    PubMed

    Campaniello, M A; Harrington, A M; Martin, C M; Ashley Blackshaw, L; Brierley, S M; Hughes, P A

    2016-01-01

    Chronic visceral pain is a defining feature of irritable bowel syndrome (IBS). IBS patients often show alterations in innate and adaptive immune function which may contribute to symptoms. Immune mediators are known to modulate the activity of viscero-sensory afferent nerves, but the focus has been on the innate immune system. Interleukin-2 (IL-2) is primarily associated with adaptive immune responses but its effects on colo-rectal afferent function in health or disease are unknown. Myeloperoxidase (MPO) activity determined the extent of inflammation in health, acute trinitrobenzene-sulfonic acid (TNBS) colitis, and in our post-TNBS colitis model of chronic visceral hypersensitivity (CVH). The functional effects of IL-2 on high-threshold colo-rectal afferents and the expression of IL-2R and NaV 1.7 mRNA in colo-rectal dorsal root ganglia (DRG) neurons were compared between healthy and CVH mice. MPO activity was increased during acute colitis, but subsided to levels comparable to health in CVH mice. IL-2 caused direct excitation of colo-rectal afferents that was blocked by tetrodotoxin. IL-2 did not affect afferent mechanosensitivity in health or CVH. However, an increased proportion of afferents responded directly to IL-2 in CVH mice compared with controls (73% vs 33%; p < 0.05), and the abundance of IL-2R and NaV 1.7 mRNA was increased 3.5- and 2-fold (p < 0.001 for both) in colo-rectal DRG neurons. IL-2, an immune mediator from the adaptive arm of the immune response, affects colo-rectal afferent function, indicating these effects are not restricted to innate immune mediators. Colo-rectal afferent sensitivity to IL-2 is increased long after healing from inflammation. © 2015 John Wiley & Sons Ltd.

  8. Kinetics of lipid-nanoparticle-mediated intracellular mRNA delivery and function

    NASA Astrophysics Data System (ADS)

    Zhdanov, Vladimir P.

    2017-10-01

    mRNA delivery into cells forms the basis for one of the new and promising ways to treat various diseases. Among suitable carriers, lipid nanoparticles (LNPs) with a size of about 100 nm are now often employed. Despite high current interest in this area, the understanding of the basic details of LNP-mediated mRNA delivery and function is limited. To clarify the kinetics of mRNA release from LNPs, the author uses three generic models implying (i) exponential, (ii) diffusion-controlled, and (iii) detachment-controlled kinetic regimes, respectively. Despite the distinct differences in these kinetics, the associated transient kinetics of mRNA translation to the corresponding protein and its degradation are shown to be not too sensitive to the details of the mRNA delivery by LNPs (or other nanocarriers). In addition, the author illustrates how this protein may temporarily influence the expression of one gene or a few equivalent genes. The analysis includes positive or negative regulation of the gene transcription via the attachment of the protein without or with positive or negative feedback in the gene expression. Stable, bistable, and oscillatory schemes have been scrutinized in this context.

  9. Angiotensin II initiates tyrosine kinase Pyk2-dependent signalings leading to activation of Rac1-mediated c-Jun NH2-terminal kinase.

    PubMed

    Murasawa, S; Matsubara, H; Mori, Y; Masaki, H; Tsutsumi, Y; Shibasaki, Y; Kitabayashi, I; Tanaka, Y; Fujiyama, S; Koyama, Y; Fujiyama, A; Iba, S; Iwasaka, T

    2000-09-01

    Ca(2+)-sensitive tyrosine kinase Pyk2 was shown to be involved in angiotensin (Ang) II-mediated activation of extracellular signal-regulated kinase (ERK) via transactivation of epidermal growth factor receptor (EGF-R). In this study, we tested the involvement of Pyk2 and EGF-R in Ang II-induced activation of JNK and c-Jun in cardiac fibroblasts. Ang II markedly stimulated JNK activities, which were abolished by genistein and intracellular Ca(2+) chelators but partially by protein kinase C depletion. Inhibition of EGF-R did not affect Pyk2 and JNK activation by Ang II. Stable transfection with a dominant negative (DN) mutant for Pyk2 (PKM) completely blocked JNK activation by Ang II. DN mutants of Rac1 (DN-Rac1) and MEK kinase (DN-MEKK1) also abolished it, whereas those of Cdc42, RhoA, and Ha-Ras had no effect. Induction of c-Jun gene transcription by Ang II was abolished in PKM, DN-Rac1, and DN-MEKK1, in which Ang II-induced binding of ATF2/c-Jun heterodimer to the activator protein-1 sequence at -190 played a key role. These results suggest that 1) in cardiac fibroblasts activation of JNK and c-Jun by Ang II is initiated by Pyk2-dependent signalings but not by downstream signals of EGF-R or Ras, 2) Rac1 but not Cdc42 is required for JNK activation by Ang II upstream of MEKK1, and 3) ATF-2/c-Jun binding to the activator protein-1 sequence at -190 plays a key role for induction of c-Jun gene by Ang II.

  10. ZNF9 Activation of IRES-Mediated Translation of the Human ODC mRNA Is Decreased in Myotonic Dystrophy Type 2

    PubMed Central

    Sammons, Morgan A.; Antons, Amanda K.; Bendjennat, Mourad; Udd, Bjarne; Krahe, Ralf; Link, Andrew J.

    2010-01-01

    Myotonic dystrophy types 1 and 2 (DM1 and DM2) are forms of muscular dystrophy that share similar clinical and molecular manifestations, such as myotonia, muscle weakness, cardiac anomalies, cataracts, and the presence of defined RNA-containing foci in muscle nuclei. DM2 is caused by an expansion of the tetranucleotide CCTG repeat within the first intron of ZNF9, although the mechanism by which the expanded nucleotide repeat causes the debilitating symptoms of DM2 is unclear. Conflicting studies have led to two models for the mechanisms leading to the problems associated with DM2. First, a gain-of-function disease model hypothesizes that the repeat expansions in the transcribed RNA do not directly affect ZNF9 function. Instead repeat-containing RNAs are thought to sequester proteins in the nucleus, causing misregulation of normal cellular processes. In the alternative model, the repeat expansions impair ZNF9 function and lead to a decrease in the level of translation. Here we examine the normal in vivo function of ZNF9. We report that ZNF9 associates with actively translating ribosomes and functions as an activator of cap-independent translation of the human ODC mRNA. This activity is mediated by direct binding of ZNF9 to the internal ribosome entry site sequence (IRES) within the 5′UTR of ODC mRNA. ZNF9 can activate IRES-mediated translation of ODC within primary human myoblasts, and this activity is reduced in myoblasts derived from a DM2 patient. These data identify ZNF9 as a regulator of cap-independent translation and indicate that ZNF9 activity may contribute mechanistically to the myotonic dystrophy type 2 phenotype. PMID:20174632

  11. Expression and purification of functional JNK2beta2: perspectives on high-level production of recombinant MAP kinases.

    PubMed

    Savopoulos, John W; Dowd, Stephen; Armour, Carolyn; Carter, Paul S; Greenwood, Catherine J; Mills, David; Powell, David; Pettman, Gary R; Jenkins, Owen; Walsh, Frank S; Philpott, Karen L

    2002-02-01

    The mitogen-activated protein (MAP) kinases are a group of serine/threonine kinases that mediate intracellular signal transduction in response to environmental stimuli including stress, growth factors, and various cytokines. Of this family, the c-Jun N-terminal kinases (JNKs) are members which, depending on cell type, have been shown to activate the transcription of genes involved in the inflammatory response, apoptosis, and hypertrophy. Here we report the use Baculovirus/Sf9 cells to produce milligram quantities of recombinant JNK2beta2 substrate which could be purified to >90% as judged by SDS-PAGE. In addition, we report a novel method for the site-specific biotinylation for this enzyme and demonstrate that the biotinylated product is an authentic substrate of the upstream kinases MKK4 and 7 and can phosphorylate a downstream target, ATF-2. We also show that the phosphorylated product can be captured efficiently on streptavidin-coated beads for use in scintillation proximity assays. Copyright 2002 Elsevier Science (USA).

  12. Adenosine triphosphate induces P2Y2 activation and interleukin-8 release in human esophageal epithelial cells.

    PubMed

    Wu, Liping; Oshima, Tadayuki; Fukui, Hirokazu; Watari, Jiro; Miwa, Hiroto

    2017-07-01

    Immune-mediated mucosal inflammation characterized by the release of interleukin (IL)-8 is associated with gastroesophageal reflux disease. ATP released by human esophageal epithelial cells (HEECs) mediates the release of cytokines through P2 nucleotide receptors that are present on various cells, including HEECs. This study characterized and identified human esophageal epithelial P2 receptors that are responsible for ATP-mediated release of IL-8 by using a human esophageal stratified squamous epithelial model. Primary HEECs were cultured with the use of an air-liquid interface (ALI) system. The ATP analogue adenosine 5'-O-3-thiotriphosphate (ATP-γ-S) was added to the basolateral compartment, and IL-8 release was measured. Involvement of the P2Y2 receptor was assessed with the use of selective and non-selective receptor antagonists and a P2Y2 receptor agonist. Expression of the P2Y2 receptor was assessed using western blotting and immunohistochemistry. Adenosine triphosphate-γ-S induced IL-8 release through the P2Y2 receptor. A P2Y2 receptor antagonist but not a P2X3 receptor antagonist or a P2Y1 receptor antagonist blocked ATP-γ-S-mediated IL-8 release. Conversely, a P2Y2 receptor agonist induced IL-8 release. Western blotting and immunohistochemistry of the P2Y2 receptor showed strong expression of the P2Y2 receptor on ALI-cultured HEECs and in human esophagus. Inhibition of extracellular signal-regulated kinase but not of protein kinase C blocked the ATP-mediated release of IL-8. ATP-γ-S induced phosphorylation of extracellular signal-regulated kinase, and a P2Y2 receptor antagonist blocked this phosphorylation. Interleukin-8 release after purinergic stimulation in ALI-cultured HEECs is mediated through P2Y2 receptor activation. ATP-induced IL-8 release maybe involved in the pathogenesis of refractory gastroesophageal reflux disease. © 2016 Journal of Gastroenterology and Hepatology Foundation and John Wiley & Sons Australia, Ltd.

  13. Complete inhibition of anisomycin and UV radiation but not cytokine induced JNK and p38 activation by an aryl-substituted dihydropyrrolopyrazole quinoline and mixed lineage kinase 7 small interfering RNA.

    PubMed

    Wang, Xushan; Mader, Mary M; Toth, John E; Yu, Xiaohong; Jin, Najia; Campbell, Robert M; Smallwood, Jeffrey K; Christe, Michael E; Chatterjee, Arindam; Goodson, Theodore; Vlahos, Chris J; Matter, William F; Bloem, Laura J

    2005-05-13

    Mixed lineage kinase 7 (MLK7) is a mitogen-activated protein kinase kinase kinase (MAPKKK) that activates the pro-apoptotic signaling pathways p38 and JNK. A library of potential kinase inhibitors was screened, and a series of dihydropyrrolopyrazole quinolines was identified as highly potent inhibitors of MLK7 in vitro catalytic activity. Of this series, an aryl-substituted dihydropyrrolopyrazole quinoline (DHP-2) demonstrated an IC50 of 70 nM for inhibition of pJNK formation in COS-7 cell MLK7/JNK co-transfection assays. In stimulated cells, DHP-2 at 200 nM or MLK7 small interfering RNA completely blocked anisomycin and UV induced but had no effect on interleukin-1beta or tumor necrosis factor-alpha-induced p38 and JNK activation. Additionally, the compound blocked anisomycin and UV-induced apoptosis in COS-7 cells. Heart tissue homogenates from MLK7 transgenic mice treated with DHP-2 at 30 mg/kg had reduced JNK and p38 activation with no apparent effect on ERK activation, demonstrating that this compound can be used to block MLK7-driven MAPK pathway activation in vivo. Taken together, these data demonstrate that MLK7 is the MAPKKK required for modulation of the stress-activated MAPKs downstream of anisomycin and UV stimulation and that DHP-2 can be used to block MLK7 pathway activation in cells as well as in vivo.

  14. Apigenin potentiates the antitumor activity of 5-FU on solid Ehrlich carcinoma: Crosstalk between apoptotic and JNK-mediated autophagic cell death platforms

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

    Gaballah, Hanaa H., E-mail: hanaahibishy@hotmail.c

    Background: Although 5- Fluorouracil (5-FU) has exhibited effectiveness against cancer, novel therapeutic strategies are needed to enhance its antitumor efficiency and modulate its cytotoxity. Apigenin, a flavonoid present in fruits and vegetables, is a potent dietary phytochemical effective in cancer chemoprevention. Aim: This study was undertaken to investigate the potential synergistic antitumor activity of apigenin and 5-FU on Solid Ehrlich carcinoma (SEC). Methods: Eighty Swiss albino male mice were divided into four equal groups: vehicle treated control SEC, SEC + 5-FU, SEC + apigenin, SEC + 5-FU + apigenin. Beclin-1 and caspases 3, 9 and JNK activities were estimated bymore » ELISA; mRNA expression levels of the antiapoptotic gene Mcl-1 were estimated using quantitative real-time RT-PCR, while tissue malondialdehyde (MDA), glutathione peroxidase and total antioxidant capacity were evaluated spectrophotometrically. A part of the tumor was examined for histopathological and Ki-67 immunohistochemistry analysis. Results: 5-FU and/or apigenin caused significant increase in tissue levels of Beclin-1, caspases 3, 9 and JNK activities, MDA with significant decrease in tumor volume, Mcl-1expression, tissue glutathione peroxidase and total antioxidant capacity and alleviated the histopathological changes with significant decrease of Ki-67 proliferation index compared to vehicle treated SEC control group. In conclusion: The combination of 5-FU and apigenin had a greater effect than each of 5-FU or apigenin alone against solid Ehrlich carcinoma in mice. - Highlights: • Apigenin potentiated 5-FU cytotoxicity in EAC solid tumor models in vivo. • It acted via autophagy stimulation, downregulating MCL-1 and Ki-67 expression. • It caused JNK activation and ROS accumulation; resulted in tumor growth inhibition. • Apigenin can be used as a co-adjuvant agent in cancer therapy.« less

  15. Mechanism of interleukin-13 production by granulocyte-macrophage colony-stimulating factor-dependent macrophages via protease-activated receptor-2.

    PubMed

    Yamaguchi, Rui; Yamamoto, Takatoshi; Sakamoto, Arisa; Ishimaru, Yasuji; Narahara, Shinji; Sugiuchi, Hiroyuki; Hirose, Eiji; Yamaguchi, Yasuo

    2015-06-01

    Granulocyte-macrophage colony-stimulating factor (GM-CSF) promotes classically activated M1 macrophages. GM-CSF upregulates protease-activated receptor-2 (PAR-2) protein expression and activation of PAR-2 by human neutrophil elastase (HNE) regulates cytokine production. This study investigated the mechanism of PAR-2-mediated interleukin (IL)-13 production by GM-CSF-dependent macrophages stimulated with HNE. Adherent macrophages were obtained from primary cultures of human mononuclear cells. After stimulation with HNE to activate the mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) signaling pathway, IL-13 mRNA and protein levels were assessed by the reverse transcriptase-polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. PAR-2 protein was detected in GM-CSF-dependent macrophages by Western blotting. Unexpectedly, PD98059 (an ERK1 inhibitor) increased IL-13 production, even at higher concentrations. Interestingly, U0126 (an ERK1/2 inhibitor) reduced IL-13 production in a concentration-dependent manner. Neither SB203580 (a p38alpha/p38beta inhibitor) nor BIRB796 (a p38gamma/p38delta inhibitor) affected IL-13 production, while TMB-8 (a calcium chelator) diminished IL-13 production. Stimulation with HNE promoted the production of IL-13 (a Th2 cytokine) by GM-CSF-dependent M1 macrophages. PAR-2-mediated IL-13 production may be dependent on the Ca(2+)/ERK2 signaling pathway. Copyright © 2015 Elsevier Inc. All rights reserved.

  16. Activation of the transcription factor nuclear factor-kappa B in uterine luminal epithelial cells by interleukin 1 Beta 2: a novel interleukin 1 expressed by the elongating pig conceptus.

    PubMed

    Mathew, Daniel J; Newsom, Emily M; Guyton, Jennifer M; Tuggle, Christopher K; Geisert, Rodney D; Lucy, Matthew C

    2015-04-01

    Conceptus mortality is greatest in mammals during the peri-implantation period, a time when conceptuses appose and attach to the uterine surface epithelium while releasing proinflammatory molecules. Interleukin 1 beta (IL1B), a master proinflammatory cytokine, is released by the primate, rodent, and pig blastocyst during the peri-implantation period and is believed to be essential for establishment of pregnancy. The gene encoding IL1B has duplicated in the pig, resulting in a novel gene. Preliminary observations indicate that the novel IL1B is specifically expressed by pig conceptuses during the peri-implantation period. To verify this, IL1B was cloned from mRNA isolated from Day 12 pig conceptuses and compared with IL1B cloned from mRNA isolated from pig peripheral blood leukocytes (PBLs). The pig conceptuses, but not the PBLs, expressed a novel IL1B, referred to here as interleukin 1 beta 2 (IL1B2). Porcine endometrium was treated with recombinant porcine interleukin 1 beta 1 (IL1B1), the prototypical cytokine, and IL1B2 proteins. Immunohistochemistry and real-time RT-PCR were used to measure activation of nuclear factor-kappa B (NFKB) and NFKB-regulated transcripts, respectively, within the endometrium. Both IL1B1 and IL1B2 activated NFKB in the uterine luminal epithelium within 4 h. The NFKB activation and related gene expression, however, were lower in endometrium treated with IL1B2, suggesting that the conceptus-derived cytokine may have reduced activity within the uterus. In conclusion, the peri-implantation pig conceptus expresses a novel IL1B that can activate NFKB within the uterine surface epithelium, likely creating a proinflammatory microenvironment during establishment of pregnancy in the pig. © 2015 by the Society for the Study of Reproduction, Inc.

  17. Dual regulation of HMGB1 by combined JNK1/2-ATF2 axis with miR-200 family in nonalcoholic steatohepatitis in mice.

    PubMed

    Chen, Xin; Ling, Yan; Wei, Yanping; Tang, Jing; Ren, Yibing; Zhang, Baohua; Jiang, Feng; Li, Hengyu; Wang, Ruoyu; Wen, Wen; Lv, Guishuai; Wu, Mengchao; Chen, Lei; Li, Liang; Wang, Hongyang

    2018-05-01

    In the context of diabetes, obesity, and metabolic syndrome, the inflammatory signaling has critical roles in the pathogenesis of nonalcoholic fatty liver disease (NAFLD), but the underlying mechanisms remain poorly delineated. Herein, early and persistently elevated, proinflammatory cytokine HMGB1 expression was detected in a high-fat diet (HFD)-induced NAFLD model in C57BL/6 mice. The expression and extracellular release of HMGB1 was rapidly and dramatically induced by saturated palmitic acid in vitro. HFD-induced inflammatory response and liver function impairment were both mitigated after the inhibition of endogenous HMGB1 by neutralizing antibody in vivo. The up-regulation of HMGB1 was thought to be modified by dual channels: in the transcriptional level, it was regulated by JNK1/JNK2-ATF2 axis; post-transcriptionally, it was regulated by the microRNA (miR)-200 family, especially miR-429. miR-429 liver conditional knockout mice (miR-429 Δhep ), fed either a normal diet or an HFD, showed severe liver inflammation and dysfunction, accompanied by greater expression of HMGB1. Intriguingly, the up-regulation and release of HMGB1 could in turn self-activate TLR4-JNK1/JNK2-ATF2 signaling, thus forming a positive feedback. Our findings reveal a novel mechanism by which HMGB1 expression was regulated by both the JNK1/2-ATF2 axis and the miR-200 family, which provides a potential new approach for the treatment of NAFLD.-Chen, X., Ling, Y., Wei, Y., Tang, J., Ren, Y., Zhang, B., Jiang, F., Li, H., Wang, R., Wen, W., Lv, G., Wu, M., Chen, L., Li, L., Wang, H. Dual regulation of HMGB1 by combined JNK1/2-ATF2 axis with miR-200 family in nonalcoholic steatohepatitis in mice.

  18. Cannabinoid inhibition of adenylate cyclase-mediated signal transduction and interleukin 2 (IL-2) expression in the murine T-cell line, EL4.IL-2.

    PubMed

    Condie, R; Herring, A; Koh, W S; Lee, M; Kaminski, N E

    1996-05-31

    Cannabinoid receptors negatively regulate adenylate cyclase through a pertussis toxin-sensitive GTP-binding protein. In the present studies, signaling via the adenylate cyclase/cAMP pathway was investigated in the murine thymoma-derived T-cell line, EL4.IL-2. Northern analysis of EL4.IL-2 cells identified the presence of 4-kilobase CB2 but not CB1 receptor-subtype mRNA transcripts. Southern analysis of genomic DNA digests for the CB2 receptor demonstrated identical banding patterns for EL4.IL-2 cells and mouse-derived DNA, both of which were dissimilar to DNA isolated from rat. Treatment of EL4.IL-2 cells with either cannabinol or Delta9-THC disrupted the adenylate cyclase signaling cascade by inhibiting forskolin-stimulated cAMP accumulation which consequently led to a decrease in protein kinase A activity and the binding of transcription factors to a CRE consensus sequence. Likewise, an inhibition of phorbol 12-myristate 13-acetate (PMA)/ionomycin-induced interleukin 2 (IL-2) protein secretion, which correlated to decreased IL-2 gene transcription, was induced by both cannabinol and Delta9-THC. Further, cannabinoid treatment also decreased PMA/ionomycin-induced nuclear factor binding to the AP-1 proximal site of the IL-2 promoter. Conversely, forskolin enhanced PMA/ionomycin-induced AP-1 binding. These findings suggest that inhibition of signal transduction via the adenylate cyclase/cAMP pathway induces T-cell dysfunction which leads to a diminution in IL-2 gene transcription.

  19. [The mRNA expression of mitogen-activated protein kinase signal pathway related genes in the blood of arseniasis patients caused by burning coal].

    PubMed

    Luo, Peng; Zhang, Ai-hua; Xiao, Yun; Pan, Xue-li; Dong, Xue-xin; Huang, Xiao-xin

    2013-09-01

    To detect the mRNA expression of ERK1, ERK2, JNK1 and P38 gene in mitogen-activated protein kinase(MAPK) path way in the arseniasis patients caused by burning coal. 70 arseniasis patients caused by burning coal at Jiaole village XingRen county in December 2006 were selected as case group, and another 30 villagers with similar living habits, matched gender and age, healthy physical condition without history of burning high arsenic coal were selected as control group from 12 km nearby the same village.Silver diethyl dithiocarbamate method (Ag-DDC) was taken to detect the arsenic contents in the environmental media, food, and arsenic level in the urine and hair of arseniasis patients.On the principle of informed consent, the peripheral blood was collected from the patients. The total RNA was extracted with Trizol method and cDNA was reversed from it. The mRNA expression of ERK1, ERK2, JNK1 and P38 gene in MAPK path way were tested by real-time fluorescent quantitative PCR (QT-PCR). A total of 70 cases of arseniasis patients (31 cases of mild, 25 cases of moderate and 14 cases of severe) and 30 cases of control were chosen. The median (quartile) of arsenic contents in the indoor air, outdoor air, coal, chili and corn were 0.079 (0.053-0.117) mg/m(3) ,0.007 (0.002-0.015) mg/m(3) , 93.010 (39.460-211.740) mg/kg, 3.460(0.550-16.760) mg/kg and 1.500(0.300-4.140) mg/kg respectively. They were above the national health standards. The median (quartile) of arsenic contents in the soil, rice and drinking water were separately 12.130(4.230-24.820) mg/kg, 0.650(0.300-0.980) mg/kg and 0.043(0.012-0.089)mg/kg, which were within the national health standards. Compared with the control group ((26.97 ± 9.71)µg/g Cr), arsenic level in the patients' urine ((71.48 ± 22.74)µg/g Cr) increased significantly, the differences were significant (F = 90.38, P < 0.01). Compared with the control group ((1.58 ± 1.07)µg/g), arsenic level in the patients' hair ((4.45 ± 2.78) µg/g) increased

  20. Prolonged duration of isoflurane anesthesia impairs spatial recognition memory through the activation of JNK1/2 in the hippocampus of mice.

    PubMed

    Jiang, Shan; Miao, Bei; Chen, Ying

    2017-05-03

    Postoperative cognitive dysfunction is a frequent complication with surgery and anesthesia, and the underlying mechanism is unclear. Our aim was to investigate the effect of different durations of isoflurane anesthesia on spatial recognition memory and activation of JNK1/2 in the hippocampus of mice. In the present study, adult male mice were anesthetized with isoflurane for different durations (1.5% isoflurane for 1, 2, and 4 h). Spatial recognition memory was determined using spontaneous alternation and two-trial recognition memory in Y-maze at 24 h after anesthesia. The activation of JNK1/2 in the hippocampus was tested using western blot. Mice treated with isoflurane for 4 h showed significantly decreased spontaneous alternations and decreased exploration parameters compared with the no anesthesia group, but this was not observed in mice treated with isoflurane for 1 or 2 h. The protein levels of p-JNK1/2 in the hippocampus were significantly increased at 10 min after isoflurane anesthesia for 1, 2, and 4 h compared with no anesthesia. However, only isoflurane anesthesia for 4 h still increased JNK1/2 and p-JNK1/2 levels at 24 h after anesthesia. We concluded that prolonged duration of isoflurane anesthesia maintained the activation of JNK1/2, which led to memory impairment at 24 h after anesthesia.

  1. N-Acetylcysteine Attenuates Ischemia-Reperfusion-Induced Apoptosis and Autophagy in Mouse Liver via Regulation of the ROS/JNK/Bcl-2 Pathway

    PubMed Central

    Xia, Yujing; Dai, Weiqi; Wang, Fan; Shen, Miao; Cheng, Ping; Wang, Junshan; Lu, Jie; Zhang, Yan; Yang, Jing; Zhu, Rong; Zhang, Huawei; Li, Jingjing; Zheng, Yuanyuan; Zhou, Yingqun; Guo, Chuanyong

    2014-01-01

    Background Hepatic ischemia–reperfusion injury (HIRI) remains a pivotal clinical problem after hemorrhagic shock, transplantation, and some types of toxic hepatic injury. Apoptosis and autophagy play important roles in cell death during HIRI. It is also known that N-acetylcysteine (NAC) has significant pharmacologic effects on HIRI including elimination of reactive oxygen species (ROS) and attenuation of hepatic apoptosis. However, the effects of NAC on HIRI-induced autophagy have not been reported. In this study, we evaluated the effects of NAC on autophagy and apoptosis in HIRI, and explored the possible mechanism involved. Methods A mouse model of segmental (70%) hepatic warm ischemia was adopted to determine hepatic injury. NAC (150 mg/kg), a hepatoprotection agent, was administered before surgery. We hypothesized that the mechanism of NAC may involve the ROS/JNK/Bcl-2 pathway. We evaluated the expression of JNK, P-JNK, Bcl-2, Beclin 1 and LC3 by western blotting and immunohistochemical staining. Autophagosomes were evaluated by transmission electron microscopy (TEM). Results We found that ALT, AST and pathological changes were significantly improved in the NAC group. Western blotting analysis showed that the expression levels of Beclin 1 and LC3 were significantly decreased in NAC-treated mice. In addition, JNK, p-JNK, Bax, TNF-α, NF-κB, IL2, IL6 and levels were also decreased in NAC-treated mice. Conclusion NAC can prevent HIRI-induced autophagy and apoptosis by influencing the JNK signal pathway. The mechanism is likely to involve attenuation of JNK and p-JNK via scavenged ROS, an indirect increase in Bcl-2 level, and finally an alteration in the balance of Beclin 1 and Bcl-2. PMID:25264893

  2. [Houttuynia Cordata induces expression of human beta-defensin-2 mRNA in pulmonary epithelial cells in vitro].

    PubMed

    Luo, Li; Dong, Bi-rong; Teng, Li-hua

    2008-07-01

    To explore the effects of Houttuynia Cordata on expression of human beta-defensin-2 (HBD-2) in pulmonary epithelial cells (SPC-A-1) in vitro; and to observe the correlationship between the level of HBD-2 mRNA and the concentrations or treatment times of Houttuynia Cordata. The SPC-A-1 cells were cultured with different concentrations of Houttuynia Cordata in vitro, including 0, 12.5, 25, 50, 100 and 200 microg/ml. And then, the SPC-A-1 cells were cultured with the optimal concentration of Houttuynia Cordata in different lengths of time, including 1, 2, 4, 8, 16 and 24 hours. After the treatment, the mRNA level of HBD-2 in pulmonary epithelial cells was detected by means of semi-quantitative reverse transcription polymerase chain reaction (RT-PCR). After being cultured with Houttuynia Cordata, the expression of HBD-2 mRNA had positive correlation with the stimulus concentrations (rs=0.829, P=0.042) and stimulus time (rs=0.914, P=0.003). The highest expression of HBD-2 mRNA was induced by 100 microg/ml Houttuynia Cordata after 8-hour treatment. In comparison with the normal control group and the interleukin-1beta group, 100 microg/ml Houttuynia Cordata could significantly up-regulate the expression of HBD-2 mRNA in SPC-A-1 cells after 8-hour treatment (P<0.01). Houttuynia Cordata can up-regulate expression of HBD-2 mRNA in SPC-A-1 cells, and the highest expression level of HBD-2 mRNA can be obtained by culture with 100 microg/ml Houttuynia Cordata for 8 hours.

  3. Vitamin E and Lycopene Reduce Coal Burning Fluorosis-induced Spermatogenic Cell Apoptosis via Oxidative Stress-mediated JNK and ERK Signaling Pathways.

    PubMed

    Tian, Yuan; Xiao, Yuehai; Wang, Bolin; Sun, Chao; Tang, Kaifa; Sun, Fa

    2017-12-22

    Although fluoride has been widely used in toothpaste, mouthwash, and drinking water to prevent dental caries, the excessive intake of fluoride can cause fluorosis which is associated with dental, skeletal, and soft tissue fluorosis. Recent evidences have drawn the attention to its adverse effects on male reproductive system that include spermatogenesis defect, sperm count loss, and sperm maturation impairment. Fluoride induces oxidative stress through the activation of mitogen activated protein kinase (MAPK) cascade which can lead to cell apoptosis. Vitamin E (VE) and lycopene are two common anti-oxidants, being protective to reactive oxygen species (ROS)-induced toxic effects. However, whether and how these two anti-oxidants prevent fluoride-induced spermatogenic cell apoptosis are largely unknown. In the present study, a male rat model for coal burning fluorosis was established and the histological lesions and spermatogenic cell apoptosis in rat testes were observed. The decreased expression of clusterin, a heterodimeric glycoprotein reported to regulate spermatogenic cell apoptosis, is detected in fluoride-treated rat testes. Interestingly, the co-administration with VE or lycopene reduced fluorosis-mediated testicular toxicity and rescued clusterin expression. Further, fluoride caused the enhanced Jun N-terminal kinase (JNK) and extracellular signal-regulated protein kinase (ERK) phosphorylation, which was reduced by VE or lycopene. Thus, VE and lycopene prevent coal burning fluorosis-induced spermatogenic cell apoptosis through the suppression of oxidative stress-mediated JNK and ERK signaling pathway, which could be an alternative therapeutic strategy for the treatment of fluorosis. ©2017 The Author(s).

  4. Characterization of interleukin-4-stimulated nasal polyp fibroblasts.

    PubMed

    Steinke, John W; Crouse, Charles D; Bradley, Dewayne; Hise, Kathleen; Lynch, Kevin; Kountakis, Stilianos E; Borish, Larry

    2004-02-01

    Chronic hyperplastic eosinophilic sinusitis is an inflammatory disease that results in the accumulation of eosinophils, fibroblasts, mast cells, and goblet cells at the site of injury. A common feature of this disease is the presence of nasal polyposis (NP). The current studies were designed to assess the contribution of interleukin (IL)-4 to fibroblast-mediated inflammation in chronic hyperplastic eosinophilic sinusitis/NP. In addition, we hypothesized that cysteinyl leukotrienes (CysLT) may directly influence fibroblast-mediated fibrotic and remodeling pathways in this disorder. Fibroblasts were isolated from NP tissue. All fibroblast lines expressed the IL-4 receptor. IL-4 induced changes in mRNA and protein expression of fibrotic (transforming growth factor-beta1 and -beta2) and inflammatory cytokines and chemokines (IL-6 and CCL11) by fibroblasts as measured by semiquantitative and quantitative polymerase chain reaction, RNase protection assay, and enzyme-linked immunosorbent assay. The expression of CysLT and other proinflammatory lipid receptors on fibroblasts was evaluated. CysLT1 and CysLT2 receptors were not expressed on fibroblasts; however, LPA(1) receptor was constitutively expressed and LPA(2) receptor expression was upregulated by IL-4. The metabolic cascade involved in CysLT synthesis was not expressed in fibroblasts and could not be induced by IL-4 treatment.

  5. Crosstalk between Fas and JNK determines lymphocyte apoptosis after ionizing radiation.

    PubMed

    Praveen, Koganti; Saxena, Nandita

    2013-06-01

    Radiation simultaneously activate Fas and JNK pathway in lymphocytes but their precise interaction is not clearly understood. Activation of Fas pathway is required for radiation induced apoptosis, however induction of JNK pathway may or may not contribute in apoptosis. Here we report that Fas, Fas associated death domain and total JNK are activated in a dose- and time-dependent radiation exposure. A biphasic pattern of phospho-JNK was found at lower doses (1 and 2 Gy), however at higher doses of radiation phospho-JNK was continuously activated. Interestingly, Fas ligand expression remained biphasic at all the doses of radiation. Our results suggest that the Fas pathway is the major player in radiation-induced apoptosis, with JNK playing a contributory role. We also observed that Fas ligand expression by radiation is dependent on JNK activation. We also propose that radiation activates JNK pathway, but sustained activation is required for maximal induction of apoptosis at later times. Our findings define a mechanism for crosstalk between JNK and Fas pathway in radiation-induced apoptosis, which may lead to the development of new therapeutic strategies.

  6. Midazolam suppresses interleukin-1β-induced interleukin-6 release from rat glial cells

    PubMed Central

    2011-01-01

    Background Peripheral-type benzodiazepine receptor (PBR) expression levels are low in normal human brain, but their levels increase in inflammation, brain injury, neurodegenerative states and gliomas. It has been reported that PBR functions as an immunomodulator. The mechanisms of action of midazolam, a benzodiazepine, in the immune system in the CNS remain to be fully elucidated. We previously reported that interleukin (IL)-1β stimulates IL-6 synthesis from rat C6 glioma cells and that IL-1β induces phosphorylation of inhibitory kappa B (IκB), p38 mitogen-activated protein (MAP) kinase, stress-activated protein kinase (SAPK)/c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase 1/2, and signal transducer and activator of transcription (STAT)3. It has been shown that p38 MAP kinase is involved in IL-1β-induced IL-6 release from these cells. In the present study, we investigated the effect of midazolam on IL-1β-induced IL-6 release from C6 cells, and the mechanisms of this effect. Methods Cultured C6 cells were stimulated by IL-1β. IL-6 release from C6 cells was measured using an enzyme-linked immunosorbent assay, and phosphorylation of IκB, the MAP kinase superfamily, and STAT3 was analyzed by Western blotting. Results Midazolam, but not propofol, inhibited IL-1β-stimulated IL-6 release from C6 cells. The IL-1β-stimulated levels of IL-6 were suppressed by wedelolactone (an inhibitor of IκB kinase), SP600125 (an inhibitor of SAPK/JNK), and JAK inhibitor I (an inhibitor of JAK 1, 2 and 3). However, IL-6 levels were not affected by PD98059 (an inhibitor of MEK1/2). Midazolam markedly suppressed IL-1β-stimulated STAT3 phosphorylation without affecting the phosphorylation of p38 MAP kinase, SAPK/JNK or IκB. Conclusion These results strongly suggest that midazolam inhibits IL-1β-induced IL-6 release in rat C6 glioma cells via suppression of STAT3 activation. Midazolam may affect immune system function in the CNS. PMID:21682888

  7. Adhesion- and stress-related adaptation of glioma radiochemoresistance is circumvented by β1 integrin/JNK co-targeting.

    PubMed

    Vehlow, Anne; Klapproth, Erik; Storch, Katja; Dickreuter, Ellen; Seifert, Michael; Dietrich, Antje; Bütof, Rebecca; Temme, Achim; Cordes, Nils

    2017-07-25

    Resistance of cancer stem-like and cancer tumor bulk cells to radiochemotherapy and destructive infiltration of the brain fundamentally influence the treatment efficiency to cure of patients suffering from Glioblastoma (GBM). The interplay of adhesion and stress-related signaling and activation of bypass cascades that counteract therapeutic approaches remain to be identified in GBM cells. We here show that combined inhibition of the adhesion receptor β1 integrin and the stress-mediator c-Jun N-terminal kinase (JNK) induces radiosensitization and blocks invasion in stem-like and patient-derived GBM cultures as well as in GBM cell lines. In vivo, this treatment approach not only significantly delays tumor growth but also increases median survival of orthotopic, radiochemotherapy-treated GBM mice. Both, in vitro and in vivo, effects seen with β1 integrin/JNK co-inhibition are superior to the monotherapy. Mechanistically, the in vitro radiosensitization provoked by β1 integrin/JNK targeting is caused by defective DNA repair associated with chromatin changes, enhanced ATM phosphorylation and prolonged G2/M cell cycle arrest. Our findings identify a β1 integrin/JNK co-dependent bypass signaling for GBM therapy resistance, which might be therapeutically exploitable.

  8. Investigation of Dracocephalum kostchyi plant extract on the effective inflammatory transcription factors and mediators in activated macrophages.

    PubMed

    Kalantar, Kurosh; Gholijani, Nasser; Mousaei, Nashmin; Yazdani, Malihe; Amirghofran, Zahra

    2018-06-07

    Dracocephalum kotschyi is traditionally used for its anti-inflammatory effects. We aimed to investigate the effects of ethyl acetate extract of D. kotschyi on the expression of key inflammatory mediators and main signaling molecules involved in regulation of inflammation. Lipopolysaccharide (LPS)-stimulated J774.1 mouse macrophages were cultured in the presence of the plant extract and examined by the real time-PCR for gene expressions of interleukin (IL)-1β, tumor necrosis factor (TNF)-α, inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2. Cytokine levels and phosphorylated forms of stress-activated protein kinases/c-Jun N-terminal kinase (SAPK/JNK), signal transducer and activator of transcription (STAT)-3, p38, IκB-α and nuclear factor (NF)-κB p65 were determined using ELISA. The extract significantly reduced the expression of key mediators of inflammation. iNOS expression level decreased from 138±8.5 fold in LPS-only treated cells to 6.5±2.6 fold after treatment with 25 ug/ml of the extract (p<0.001). Similarly, COX-2 expression decreased from 632 ±98.8 fold in control to 124 ±24.6 fold (p<0.01). Treatment of cells with the extract significantly reduced IL-1β and TNF-α cytokines at both gene and protein expression levels. The extract at 25 µg/ml caused significant decreases in phospho-SAPK/JNK and phospho-STAT3 levels in macrophages (p<0.01). Proteins of phospho-p38, NFκB-p65 and phospho-NF-κB p65 had a reduced levels in treated cells (p<0.05). No significant change in phospho-IĸB level was observed. These findings suggested that D. kotschyi with inhibition of NF-κB, SAPK/JNK, STAT-3 and p-38 might have reduced the expression levels of key inflammatory mediators and thus possibly have potential beneficial impact on inflammatory diseases. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  9. The contribution of interleukin-2 to effective wound healing.

    PubMed

    Doersch, Karen M; DelloStritto, Daniel J; Newell-Rogers, M Karen

    2017-02-01

    Ineffective skin wound healing is a significant source of morbidity and mortality. Roughly 6.5 million Americans experience chronically open wounds and the cost of treating these wounds numbers in the billions of dollars annually. In contrast, robust wound healing can lead to the development of either hypertrophic scarring or keloidosis, both of which can cause discomfort and can be cosmetically undesirable. Appropriate wound healing requires the interplay of a variety of factors, including the skin, the local microenvironment, the immune system, and the external environment. When these interactions are perturbed, wounds can be a nidus for infection, which can cause them to remain open an extended period of time, or can scar excessively. Interleukin-2, a cytokine that directs T-cell expansion and phenotypic development, appears to play an important role in wound healing. The best-studied role for Interleukin-2 is in influencing T-cell development. However, other cell types, including fibroblasts, the skin cells responsible for closing wounds, express the Interleukin-2 receptor, and therefore may respond to Interleukin-2. Studies have shown that treatment with Interleukin-2 can improve the strength of healed skin, which implicates Interleukin-2 in the wound healing process. Furthermore, diseases that involve impaired wound healing, such as diabetes and systemic lupus erythematosus, have been linked to deficiencies in Interleukin-2 or defects Interleukin-2-receptor signaling. The focus of this review is to summarize the current understanding of the role of Interleukin-2 in wound healing, to highlight diseases in which Interleukin-2 and its receptor may contribute to impaired wound healing, and to assess Interleukin-2-modulating approaches as potential therapies to improve wound healing.

  10. The contribution of interleukin-2 to effective wound healing

    PubMed Central

    DelloStritto, Daniel J; Newell-Rogers, M Karen

    2016-01-01

    Ineffective skin wound healing is a significant source of morbidity and mortality. Roughly 6.5 million Americans experience chronically open wounds and the cost of treating these wounds numbers in the billions of dollars annually. In contrast, robust wound healing can lead to the development of either hypertrophic scarring or keloidosis, both of which can cause discomfort and can be cosmetically undesirable. Appropriate wound healing requires the interplay of a variety of factors, including the skin, the local microenvironment, the immune system, and the external environment. When these interactions are perturbed, wounds can be a nidus for infection, which can cause them to remain open an extended period of time, or can scar excessively. Interleukin-2, a cytokine that directs T-cell expansion and phenotypic development, appears to play an important role in wound healing. The best-studied role for Interleukin-2 is in influencing T-cell development. However, other cell types, including fibroblasts, the skin cells responsible for closing wounds, express the Interleukin-2 receptor, and therefore may respond to Interleukin-2. Studies have shown that treatment with Interleukin-2 can improve the strength of healed skin, which implicates Interleukin-2 in the wound healing process. Furthermore, diseases that involve impaired wound healing, such as diabetes and systemic lupus erythematosus, have been linked to deficiencies in Interleukin-2 or defects Interleukin-2-receptor signaling. The focus of this review is to summarize the current understanding of the role of Interleukin-2 in wound healing, to highlight diseases in which Interleukin-2 and its receptor may contribute to impaired wound healing, and to assess Interleukin-2-modulating approaches as potential therapies to improve wound healing. PMID:27798123

  11. Structural and functional characterisation of ferret interleukin-2.

    PubMed

    Ren, Bin; McKinstry, William J; Pham, Tam; Newman, Janet; Layton, Daniel S; Bean, Andrew G; Chen, Zhenjun; Laurie, Karen L; Borg, Kathryn; Barr, Ian G; Adams, Timothy E

    2016-02-01

    While the ferret is a valuable animal model for a number of human viral infections, such as influenza, Hendra and Nipah, evaluating the cellular immune response following infection has been hampered by the lack of a number of species-specific immunological reagents. Interleukin 2 (IL-2) is one such key cytokine. Ferret recombinant IL-2 incorporating a C-terminal histidine tag was expressed and purified and the three-dimensional structure solved and refined at 1.89 Å by X-ray crystallography, which represents the highest resolution and first non-human IL-2 structure. While ferret IL-2 displays the classic cytokine fold of the four-helix bundle structure, conformational flexibility was observed at the second helix and its neighbouring region in the bundle, which may result in the disruption of the spatial arrangement of residues involved in receptor binding interactions, implicating subtle differences between ferret and human IL-2 when initiating biological functions. Ferret recombinant IL-2 stimulated the proliferation of ferret lymph node cells and induced the expression of mRNA for IFN-γ and Granzyme A. Crown Copyright © 2015. Published by Elsevier Ltd. All rights reserved.

  12. Sulforaphane inhibits IL-1β-induced proliferation of rheumatoid arthritis synovial fibroblasts and the production of MMPs, COX-2, and PGE2.

    PubMed

    Choi, Yun Jung; Lee, Won-Seok; Lee, Eun-Gyeong; Sung, Myung-Soon; Yoo, Wan-Hee

    2014-10-01

    This study was performed to define the effects of sulforaphane on interleukin-1β (IL-1β)-induced proliferation of rheumatoid arthritis synovial fibroblasts (RASFs), the expression of matrix metalloproteinases (MMPs) and cyclooxygenase (COX), and the production of prostaglandin E2 (PGE2) by RASFs. The proliferation of RASFs was evaluated with CCK-8 reagent in the presence of IL-1β with/without sulforaphane. The expression of MMPs, tissue inhibitor of metalloproteinase-1, COXs, intracellular mitogen-activated protein kinase signalings, including p-ERK, p-p38, p-JNK, and nuclear factor-kappaB (NF-kB), and the production of PGE2 were examined by Western blotting or semi-quantitative RT-PCR and ELISA. Sulforaphane inhibits unstimulated and IL-1β-induced proliferation of RASFs; the expression of MMP-1, MMP-3, and COX-2 mRNA and protein; and the PGE2 production induced by IL-1β. Sulforaphane also inhibits the phosphorylation of ERK-1/2, p-38, and JNK and activation of NF-kB by IL-1β. These results indicate that sulforaphane inhibits the proliferation of synovial fibroblasts, the expression of MMPs and COX-2, and the production of PGE2, which are involved in synovitis and destruction of RA, and suggest that sulforaphane might be a new therapeutic agent for RA.

  13. Expression of Tac antigen component of bovine interleukin-2 receptor in different leukocyte populations infected with Theileria parva or Theileria annulata.

    PubMed Central

    Dobbelaere, D A; Prospero, T D; Roditi, I J; Kelke, C; Baumann, I; Eichhorn, M; Williams, R O; Ahmed, J S; Baldwin, C L; Clevers, H

    1990-01-01

    The Tac antigen component of the bovine interleukin-2 receptor was expressed as a Cro-beta-galactosidase fusion protein in Escherichia coli and used to raise antibodies in rabbits. These antibodies were used for flow cytofluorimetric analysis to investigate the expression of Tac antigen in a variety of Theileria parva-infected cell lines and also in three Theileria annulata-infected cell lines. Cells expressing Tac antigen on their surface were found in all T. parva-infected cell lines tested whether these were of T- or B-cell origin. T cells expressing Tac antigen could be CD4- CD8-, CD4+ CD8-, CD4- CD8+, or CD4+ CD8+. Tac antigen expression was observed both in cultures which had been maintained in the laboratory for several years and in transformed cell lines which had recently been established by infection of lymphocytes in vitro with T. parva. Northern (RNA) blot analysis demonstrated Tac antigen transcripts in RNA isolated from all T. parva-infected cell lines. Three T. annulata-infected cell lines which were not of T-cell origin were also tested. Two of them expressed Tac antigen on their surface. Abundant Tac antigen mRNA was detected in these T. annulata-infected cell lines, but only trace amounts were demonstrated in the third cell line, which contained very few Tac antigen-expressing cells. In all cell lines tested, whether cloned or uncloned, a proportion of the cells did not express detectable levels of Tac antigen on their surface. This was also the case for a number of other leukocyte surface markers. In addition, we showed that the interleukin-2 receptors were biologically functional, because addition of recombinant interleukin-2 to cultures stimulated cell proliferation. Recombinant interleukin-2 treatment also resulted in increased amounts of steady-state Tac antigen mRNA. The relevance of interleukin-2 receptor expression on Theileria-infected cells is discussed. Images PMID:1979317

  14. Role of nuclear factor of activated T-cells and activator protein-1 in the inhibition of interleukin-2 gene transcription by cannabinol in EL4 T-cells.

    PubMed

    Yea, S S; Yang, K H; Kaminski, N E

    2000-02-01

    We previously reported that immunosuppressive cannabinoids inhibited interleukin (IL)-2 steady-state mRNA expression and secretion by phorbol-12-myristate-13-acetate plus ionomycin-activated mouse splenocytes and EL4 murine T-cells. Here we show that inhibition of IL-2 production by cannabinol, a modest central nervous system-active cannabinoid, is mediated through the inhibition of IL-2 gene transcription. Moreover, electrophoretic mobility shift assays demonstrated that cannabinol markedly inhibited the DNA binding activity of nuclear factor of activated T-cells (NF-AT) and activator protein-1 (AP-1) in a time- and concentration-dependent manner in activated EL4 cells. The inhibitory effects produced by cannabinol on AP-1 DNA binding were quite transient, showing partial recovery by 240 min after cell activation and no effect on the activity of a reporter gene under the control of AP-1. Conversely, cannabinol-mediated inhibition of NF-AT was robust and sustained as demonstrated by an NF-AT-regulated reporter gene. Collectively, these results suggest that decreased IL-2 production by cannabinol in EL4 cells is due to the inhibition of transcriptional activation of the IL-2 gene and is mediated, at least in part, through a transient inhibition of AP-1 and a sustained inhibition of NF-AT.

  15. Interleukin-6 overexpression induces pulmonary hypertension.

    PubMed

    Steiner, M Kathryn; Syrkina, Olga L; Kolliputi, Narasaish; Mark, Eugene J; Hales, Charles A; Waxman, Aaron B

    2009-01-30

    Inflammatory cytokine interleukin (IL)-6 is elevated in the serum and lungs of patients with pulmonary artery hypertension (PAH). Several animal models of PAH cite the potential role of inflammatory mediators. We investigated role of IL-6 in the pathogenesis of pulmonary vascular disease. Indices of pulmonary vascular remodeling were measured in lung-specific IL-6-overexpressing transgenic mice (Tg(+)) and compared to wild-type (Tg(-)) controls in both normoxic and chronic hypoxic conditions. The Tg(+) mice exhibited elevated right ventricular systolic pressures and right ventricular hypertrophy with corresponding pulmonary vasculopathic changes, all of which were exacerbated by chronic hypoxia. IL-6 overexpression increased muscularization of the proximal arterial tree, and hypoxia enhanced this effect. It also reproduced the muscularization and proliferative arteriopathy seen in the distal arteriolar vessels of PAH patients. The latter was characterized by the formation of occlusive neointimal angioproliferative lesions that worsened with hypoxia and were composed of endothelial cells and T-lymphocytes. IL-6-induced arteriopathic changes were accompanied by activation of proangiogenic factor, vascular endothelial growth factor, the proproliferative kinase extracellular signal-regulated kinase, proproliferative transcription factors c-MYC and MAX, and the antiapoptotic proteins survivin and Bcl-2 and downregulation of the growth inhibitor transforming growth factor-beta and proapoptotic kinases JNK and p38. These findings suggest that IL-6 promotes the development and progression of pulmonary vascular remodeling and PAH through proproliferative antiapoptotic mechanisms.

  16. JNK1 induces hedgehog signaling from stellate cells to accelerate liver regeneration in mice.

    PubMed

    Langiewicz, Magda; Graf, Rolf; Humar, Bostjan; Clavien, Pierre A

    2018-04-28

    To improve outcomes of two-staged hepatectomies for large/multiple liver tumors, portal vein ligation (PVL) has been combined with parenchymal transection (associating liver partition and portal vein ligation for staged hepatectomy [coined ALPPS]) to greatly accelerate liver regeneration. In a novel ALPPS mouse model, we have reported paracrine Indian hedgehog (IHH) signaling from stellate cells as an early contributor to augmented regeneration. Here, we sought to identify upstream regulators of IHH. ALPPS in mice was compared against PVL and additional control surgeries. Potential IHH regulators were identified through in silico mining of transcriptomic data. c-Jun N-terminal kinase (JNK1 [Mapk8]) activity was reduced through SP600125 to evaluate its effects on IHH signaling. Recombinant IHH was injected after JNK1 diminution to substantiate their relationship during accelerated liver regeneration. Transcriptomic analysis linked Ihh to Mapk8. JNK1 upregulation after ALPPS was validated and preceded the IHH peak. On immunofluorescence, JNK1 and IHH co-localized in alpha-smooth muscle actin-positive non-parenchymal cells. Inhibition of JNK1 prior to ALPPS surgery reduced liver weight gain to PVL levels and was accompanied by downregulation of hepatocellular proliferation and the IHH-GLI1-CCND1 axis. In JNK1-inhibited mice, recombinant IHH restored ALPPS-like acceleration of regeneration and re-elevated JNK1 activity, suggesting the presence of a positive IHH-JNK1 feedback loop. JNK1-mediated induction of IHH paracrine signaling from hepatic stellate cells is essential for accelerated regeneration of parenchymal mass. The JNK1-IHH axis is a mechanism unique to ALPPS surgery and may point to therapeutic alternatives for patients with insufficient regenerative capacity. Associating liver partition and portal vein ligation for staged hepatectomy (so called ALPPS), is a new two-staged approach to hepatectomy, which induces an unprecedented acceleration of liver

  17. Interleukin-34 inhibits hepatitis B virus replication in vitro and in vivo.

    PubMed

    Cheng, Sheng-Tao; Tang, Hua; Ren, Ji-Hua; Chen, Xiang; Huang, Ai-Long; Chen, Juan

    2017-01-01

    The hepatitis B virus (HBV) infection could activate the immune system and induce extensive inflammatory response. As the most important inflammatory factor, interleukins are critical for anti-viral immunity. Here we investigated whether interleukin-34 (IL-34) play a role in HBV infection. In this study, we first found that both serum IL-34 and IL-34 mRNA in PBMCs in chronic HBV patients was significantly decreased compared to the healthy controls. Furthermore, both IL-34 protein and mRNA levels were declined hepatoma cells expressing HBV. In addition, the clinical parameters analysis found that serum IL-34 was significantly associated with HBV DNA (P = 0.0066), ALT (P = 0.0327), AST (P = 0.0435), TB (P = 0.0406), DB (P = 0.0368) and AFP (P = 0.0225). Correlation analysis also found that serum IL-34 negatively correlated with HBV DNA copies, ALT and AST. In vitro studies found that IL-34 treatment in HepAD38 and HepG2.2.15 cells markedly inhibited HBV DNA, total RNA, 3.5kb mRNA and HBc protein. In vivo studies further demonstrated IL-34 treatment in HBV transgenic mice exhibited greater inhibition on HBV DNA, total RNA, 3.5kb mRNA and HBc protein, suggesting the effect to IL-34 on HBV is likely due to host innate or adaptive immune response. Our study identified a novel interleukin, IL-34, which has anti-viral activity in HBV replication in hepatocytes in vitro and in vivo. These data suggest a rationale for the use of IL-34 in the HBV treatment.

  18. Poly-adenine-mediated fluorescent spherical nucleic acid probes for live-cell imaging of endogenous tumor-related mRNA.

    PubMed

    Zhu, Dan; Zhao, Dongxia; Huang, Jiaxuan; Zhu, Yu; Chao, Jie; Su, Shao; Li, Jiang; Wang, Lihua; Shi, Jiye; Zuo, Xiaolei; Weng, Lixing; Li, Qian; Wang, Lianhui

    2018-05-16

    Identification of tumor-related mRNA in living cells hold great promise for early cancer diagnosis and pathological research. Herein, we present poly-adenine (polyA)-mediated fluorescent spherical nucleic acid (FSNA) probes for intracellular mRNA detection with regulable sensitivities by programmably adjusting the loading density of DNA on gold nano-interface. Gold nanoparticles (AuNPs) functionalized with polyA-tailed recognition sequences were hybridized to fluorescent "reporter" strands to fabricate fluorescence-quenched FSNA probes. While exposed to target gene, the "reporter" strands were released from FSNA through strand displacement and fluorescence was recovered. With polyA20 tail as the attaching block, the detection limit of FSNA probes was calculated to be 0.31 nM, which is ~55 fold lower than that of thiolated probes without surface density regulation. Quantitative intracellular mRNA detection and imaging could be achieved with polyA-mediated FSNA probes within 2 hours, indicating their application potential in rapid and sensitive intracellular target imaging. Copyright © 2018. Published by Elsevier Inc.

  19. dMyc is required in retinal progenitors to prevent JNK-mediated retinal glial activation

    PubMed Central

    Correia, Andreia; Santos, Marília A.; Relvas, João B.; Pereira, Paulo S.

    2017-01-01

    In the nervous system, glial cells provide crucial insulation and trophic support to neurons and are important for neuronal survival. In reaction to a wide variety of insults, glial cells respond with changes in cell morphology and metabolism to allow repair. Additionally, these cells can acquire migratory and proliferative potential. In particular, after axonal damage or pruning the clearance of axonal debris by glial cells is key for a healthy nervous system. Thus, bidirectional neuron-glial interactions are crucial in development, but little is known about the cellular sensors and signalling pathways involved. In here, we show that decreased cellular fitness in retinal progenitors caused by reduced Drosophila Myc expression triggers non cell-autonomous activation of retinal glia proliferation and overmigration. Glia migration occurs beyond its normal limit near the boundary between differentiated photoreceptors and precursor cells, extending into the progenitor domain. This overmigration is stimulated by JNK activation (and the function of its target Mmp1), while proliferative responses are mediated by Dpp/TGF-β signalling activation. PMID:28267791

  20. Differential utilization of decapping enzymes in mammalian mRNA decay pathways

    PubMed Central

    Li, You; Song, Mangen; Kiledjian, Megerditch

    2011-01-01

    mRNA decapping is a crucial step in the regulation of mRNA stability and gene expression. Dcp2 is an mRNA decapping enzyme that has been widely studied. We recently reported the presence of a second mammalian cytoplasmic decapping enzyme, Nudt16. Here we address the differential utilization of the two decapping enzymes in specified mRNA decay processes. Using mouse embryonic fibroblast (MEF) cell lines derived from a hypomorphic knockout of the Dcp2 gene with undetectable levels of Dcp2 or MEF cell lines harboring a Nudt16-directed shRNA to generate reduced levels of Nudt16, we demonstrate the distinct roles for Dcp2 and Nudt16 in nonsense-mediated mRNA decay (NMD), decay of ARE-containing mRNA and miRNA-mediated silencing. Our results indicated that NMD preferentially utilizes Dcp2 rather than Nudt16; Dcp2 and Nudt16 are redundant in miRNA-mediated silencing; and Dcp2 and Nudt16 are differentially utilized for ARE-mRNA decay. These data demonstrate that the two distinct decapping enzymes can uniquely function in specific mRNA decay processes in mammalian cells. PMID:21224379

  1. Induction of an interleukin-1 receptor (IL-1R) on monocytic cells. Evidence that the receptor is not encoded by a T cell-type IL-1R mRNA.

    PubMed

    Spriggs, M K; Lioubin, P J; Slack, J; Dower, S K; Jonas, U; Cosman, D; Sims, J E; Bauer, J

    1990-12-25

    Primary human monocytes and the human monocytic cell line THP-1 were induced to express receptors for interleukin-1 alpha (IL-1 alpha) and IL-1 beta. Treatment of primary monocytes with dexamethasone resulted in a 10-fold increase in receptor number over untreated cells, to approximately 2,000 receptors/cell. Treatment of THP-1 cells with phorbol ester followed by prostaglandin E2 and dexamethasone resulted in the expression of approximately 30,000 receptors/cell. Competitive binding assays on THP-1 cells showed that both IL-1 alpha and IL-1 beta bind to the same receptor. The monocyte IL-1R is significantly smaller (63 kDa) than the T cell IL-1R (80 kDa) and is immunologically distinct. However, induction of monocytes and monocytic cell lines leads to the appearance of an abundant mRNA of approximately 5,000 bases which hybridizes to a cDNA probe from the T cell-type IL-1R. Sequence data obtained from a cDNA clone of this mRNA indicate that the message is identical to the T cell IL-1R mRNA throughout the coding region. A smaller mRNA, also homologous to the T cell IL-1R mRNA, accumulated in induced THP-1 cells and has a shorter 3'-untranslated region than the larger. Data are presented which suggest that neither form of this message encodes the 63-kDa IL-1R, but rather that this protein is the product of a separate nonhomologous mRNA.

  2. Activation of the EGFR/p38/JNK Pathway by Mitochondrial-Derived Hydrogen Peroxide Contributes To Oxygen-induced Contraction Of Ductus Arteriosus

    PubMed Central

    Hong, Zhigang; Cabrera, Jésus A; Mahapatra, Saswati; Kutty, Shelby; Weir, E. Kenneth; Archer, Stephen L.

    2014-01-01

    Oxygen-induced contraction of the ductus arteriosus (DA) involves a mitochondrial oxygen-sensor, which signals pO2 in the DA smooth muscle cell (DASMC) by increasing production of diffusible hydrogen peroxide (H2O2). H2O2 stimulates vasoconstriction by regulating ion channels and rho kinase, leading to calcium influx and calcium sensitization. Because epidermal growth factor receptor (EGFR) signaling is also redox regulated and participates in oxygen sensing and vasoconstriction in other systems, we explored the role of the EGFR and its signaling cascade (p38 and JNK) in DA contraction. Experiments were performed in DA rings isolated from full-term New Zealand White rabbits and human DASMC. In human DASMCs increasing pO2 from hypoxia to normoxia (40 to 100 mmHg) significantly increased cytosolic calcium, p<0.01. This normoxic rise in intracellular calcium was mimicked by EGF and inhibited by EGFR siRNA. In DA rings, EGF caused contraction whilst the specific EGFR inhibitor (AG1478) and the tyrosine kinase inhibitors (genistein or tyrphostin A23) selectively attenuated oxygen-induced contraction (p <0.01). Conversely, orthovanadate, a tyrosine phosphatase inhibitor known to activate EGFR signaling, caused dose-dependent contraction of hypoxic DA and superimposed increases in oxygen caused minimal additional contraction. Ansomycin, an activator of EGFR’s downstream kinases, p38 and JNK, caused DA contraction; conversely, oxygen-induced DA contraction was blocked by inhibitors of p38 MAPK (SB203580) or JNK (JNK inhibitor II). O2-induced phosphorylation of EGFR occurred within 5-minutes of increasing pO2 and was inhibited by mitochondrial-targeted overexpression of catalase. AG1478 prevented the oxygen-induced p38 and JNK phosphorylation. In conclusion, O2-induced EGFR transactivation initiates p38/JNK-mediated increases in cytosolic calcium and contributes to DA contraction. The EGFR/p38/JNK pathway is regulated by mitochondrial redox signaling and is a promising

  3. Fluid Shear Stress-Induced JNK Activity Leads to Actin Remodeling for Cell Alignment

    PubMed Central

    Mengistu, Meron; Brotzman, Hannah; Ghadiali, Samir; Lowe-Krentz, Linda

    2012-01-01

    Fluid shear stress (FSS) exerted on endothelial cell surfaces induces actin cytoskeleton remodeling through mechanotransduction. This study was designed to determine whether FSS activates Jun N-terminal kinase (JNK), to examine the spatial and temporal distribution of active JNK relative to the actin cytoskeleton in endothelial cells exposed to different FSS conditions, and to evaluate the effects of active JNK on actin realignment. Exposure to 15 and 20 dyn/cm2 FSS induced higher activity levels of JNK than the lower 2 and 4 dyn/cm2 flow conditions. At the higher FSS treatments, JNK activity increased with increasing exposure time, peaking 30 minutes after flow onset with an 8-fold activity increase compared to cells in static culture. FSS-induced phospho-JNK co-localized with actin filaments at cell peripheries, as well as with stress fibers. Pharmacologically blocking JNK activity altered FSS-induced actin structure and distribution as a response to FSS. Our results indicate that FSS-induced actin remodeling occurs in three phases, and that JNK plays a role in at least one, suggesting that this kinase activity is involved in mechanotransduction from the apical surface to the actin cytoskeleton in endothelial cells. PMID:20626006

  4. Interleukin-6 mediates low-threshold mechanical allodynia induced by intrathecal HIV-1 envelope glycoprotein gp120

    PubMed Central

    Schoeniger-Skinner, Diana K.; Ledeboer, Annemarie; Frank, Matthew G.; Milligan, Erin D.; Poole, Stephen; Martin, David; Maier, Steven F.; Watkins, Linda R.

    2007-01-01

    Spinal cord glia (microglia and astrocytes) contribute to enhanced pain states. One model that has been used to study this phenomenon is intrathecal (i.t.) administration of gp120, an envelope glycoprotein of HIV-1 known to activate spinal cord glia and thereby induce low-threshold mechanical allodynia, a pain symptom where normally innocuous (non-painful) stimuli are perceived as painful. Previous studies have shown that i.t. gp120-induced allodynia is mediated via the release of the glial pro-inflammatory cytokines, tumor necrosis factor-α (TNF), and interleukin-1β (IL-1). As we have recently reported that i.t. gp120 induces the release of interleukin-6 (IL-6), in addition to IL-1 and TNF, the present study tested whether this IL-6 release in spinal cord contributes to gp120-induced mechanical allodynia and/or to gp120-induced increases in TNF and IL-1. An i.t. anti-rat IL-6 neutralizing antibody was used to block IL-6 actions upon its release by i.t. gp120. This IL-6 blockade abolished gp120-induced mechanical allodynia. While the literature predominantly documents the cascade of pro-inflammatory cytokines as beginning with TNF, followed by the stimulation of IL-1, and finally TNF plus IL-1 stimulating the release of IL-6, the present findings indicate that a blockade of IL-6 inhibits the gp120-induced elevations of TNF, IL-1, and IL-6 mRNA in dorsal spinal cord, elevation of IL-1 protein in lumbar dorsal spinal cord, and TNF and IL-1 protein release into the surrounding lumbosacral cerebrospinal fluid. These results would suggest that IL-6 induces pain facilitation, and may do so in part by stimulating the production and release of other proinflammatory cytokines. PMID:17204394

  5. Anti-inflammatory activity of 4-methoxyhonokiol is a function of the inhibition of iNOS and COX-2 expression in RAW 264.7 macrophages via NF-kappaB, JNK and p38 MAPK inactivation.

    PubMed

    Zhou, Hong Yu; Shin, Eun Myoung; Guo, Lian Yu; Youn, Ui Joung; Bae, KiHwan; Kang, Sam Sik; Zou, Li Bo; Kim, Yeong Shik

    2008-05-31

    The extracts or constituents from the bark of Magnolia (M.) obovata are known to have many pharmacological activities. 4-Methoxyhonokiol, a neolignan compound isolated from the stem bark of M. obovata, was found to exhibit a potent anti-inflammatory effect in different experimental models. Pretreatment with 4-methoxyhonokiol (i.p.) dose-dependently inhibited the dye leakage and paw swelling in an acetic-acid-induced vascular permeability assay and a carrageenan-induced paw edema assay in mice, respectively. In the lipopolysaccharide (LPS)-induced systemic inflammation model, 4-methoxyhonokiol significantly inhibited plasma nitric oxide (NO) release in mice. To identify the mechanisms underlying this anti-inflammatory action, we investigated the effect of 4-methoxyhonokiol on LPS-induced responses in a murine macrophage cell line, RAW 264.7. The results demonstrated that 4-methoxyhonokiol significantly inhibited LPS-induced NO production as well as the protein and mRNA expressions of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). Furthermore, 4-methoxyhonokiol inhibited LPS-mediated nuclear factor-kappaB (NF-kappaB) activation via the prevention of inhibitor kappaB (IkappaB) phosphorylation and degradation. 4-Methoxyhonokiol had no effect on the LPS-induced phosphorylation of extracellular signal-regulated kinase (ERK), whereas it attenuated the phosphorylation of p38 mitogen-activated protein kinase (p38 MAPK) and c-Jun NH2-terminal kinase (JNK) in a concentration-dependent manner. Taken together, our data suggest that 4-methoxyhonokiol is an active anti-inflammatory constituent of the bark of M. obovata, and that its anti-inflammatory property might be a function of the inhibition of iNOS and COX-2 expression via down-regulation of the JNK and p38 MAP kinase signal pathways and inhibition of NF-kappaB activation in RAW 264.7 macrophages.

  6. Stem cell factor and interleukin-2/15 combine to enhance MAPK-mediated proliferation of human natural killer cells

    PubMed Central

    Benson, Don M.; Yu, Jianhua; Becknell, Brian; Wei, Min; Freud, Aharon G.; Ferketich, Amy K.; Trotta, Rossana; Perrotti, Danilo; Briesewitz, Roger

    2009-01-01

    Stem cell factor (SCF) promotes synergistic cellular proliferation in combination with several growth factors, and appears important for normal natural killer (NK)–cell development. CD34+ hematopoietic precursor cells (HPCs) require interleukin-15 (IL-15) for differentiation into human NK cells, and this effect can be mimicked by IL-2. Culture of CD34+ HPCs or some primary human NK cells in IL-2/15 and SCF results in enhanced growth compared with either cytokine alone. The molecular mechanisms responsible for this are unknown and were investigated in the present work. Activation of NK cells by IL-2/15 increases expression of c-kit whose kinase activity is required for synergy with IL-2/15 signaling. Mitogen-activated protein kinase (MAPK) signaling intermediaries that are activated both by SCF and IL-2/15 are enhanced in combination to facilitate earlier cell-cycle entry. The effect results at least in part via enhanced MAPK-mediated modulation of p27 and CDK4. Collectively the data reveal a novel mechanism by which SCF enhances cellular proliferation in combination with IL-2/15 in primary human NK cells. PMID:19060242

  7. Eicosapentaenoic acid (EPA) induced apoptosis in HepG2 cells through ROS-Ca(2+)-JNK mitochondrial pathways.

    PubMed

    Zhang, Yuanyuan; Han, Lirong; Qi, Wentao; Cheng, Dai; Ma, Xiaolei; Hou, Lihua; Cao, Xiaohong; Wang, Chunling

    2015-01-24

    Eicosapentaenoic acid (EPA), a well-known dietary n-3 PUFAS, has been considered to inhibit proliferation of tumor cells. However, the molecular mechanism related to EPA-induced liver cancer cells apoptosis has not been reported. In this study, we investigated the effect of EPA on HepG2 cells proliferation and apoptosis mechanism through mitochondrial pathways. EPA inhibited proliferation of HepG2 cells in a dose-dependent manner and had no significant effect on the cell viability of humor normal liver L-02 cells. It was found that EPA initially evoked ROS formation, leading to [Ca(2+)]c accumulation and the mitochondrial permeability transition pore (MPTP) opening; EPA-induced HepG2 cells apoptosis was inhibited by N-acetylcysteine (NAC, an inhibitor of ROS), 1,2-bis (2-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid (BAPTA-AM, a chelator of calcium) and CsA (inhibitor of MPTP). The relationship between ROS production, the increase of cytoplasmic Ca and MPTP opening was detected. It seems that ROS may act as an upstream regulator of EPA-induced [Ca(2+)]c generation, moreover, generation of ROS, overload of mitochondrial [Ca(2+)]c, and JNK activated cause the opening of MPTP. Western blotting results showed that EPA elevated the phosphorylation status of JNK, processes associated with the ROS generation. Simultaneously, the apoptosis induced by EPA was related to release of cytochrome C from mitochondria to cytoplasm through the MPTP and activation of caspase-9 and caspase-3. These results suggest that EPA induces apoptosis through ROS-Ca(2+)-JNK mitochondrial pathways. Copyright © 2014 Elsevier Inc. All rights reserved.

  8. Signaling pathways of interleukin-1 actions in the brain: anatomical distribution of phospho-ERK1/2 in the brain of rat treated systemically with interleukin-1beta.

    PubMed

    Nadjar, A; Combe, C; Busquet, P; Dantzer, R; Parnet, P

    2005-01-01

    Interleukin-1beta is released at the periphery during infection and acts on the nervous system to induce fever, neuroendocrine activation, and behavioral changes. These effects are mediated by brain type I IL-1 receptors. In vitro studies have shown the ability of interleukin-1beta to activate mitogen-activated protein kinase signaling pathways including p38, c-Jun N-terminal kinase and extracellular signal-regulated protein kinase 1 and 2 (ERK1/2). In contrast to other mitogen-activated protein kinases, little is known about ERK1/2 activation in the rat brain in response to interleukin-1beta. The aim of the present study was therefore to investigate spatial and temporal activation of ERK1/2 in the rat brain after peripheral administration of interleukin-1beta using immunohistochemistry to detect the phosphorylated form of the kinase. In non-stimulated conditions, phosphorylated ERK1/2 immunoreactivity was observed in neurons throughout the brain. Administration of interleukin-1beta (60 microg/kg, i.p.) induced the phosphorylation of ERK1/2 in areas at the interface between brain and blood or cerebrospinal fluid: meninges, circumventricular organs, endothelial like cells of the blood vessels, and in brain nuclei involved in behavioral depression, fever and neuroendocrine activation: paraventricular nucleus of the hypothalamus, supraoptic nucleus, central amygdala and arcuate nucleus. Double labeling of phosphorylated ERK1/2 and cell markers revealed the expression of phosphorylated ERK1/2 in neurons, astrocytes and microglia. Since phosphorylated ERK1/2 was found in structures in which type I IL-1 receptor has already been identified as well as in structures lacking this receptor, activation of ERK1/2 is likely to occur in response to both direct and indirect action of interleukin-1beta on its target cells.

  9. Icariin attenuates angiotensin II-induced hypertrophy and apoptosis in H9c2 cardiomyocytes by inhibiting reactive oxygen species-dependent JNK and p38 pathways

    PubMed Central

    ZHOU, HENG; YUAN, YUAN; LIU, YUAN; DENG, WEI; ZONG, JING; BIAN, ZHOU-YAN; DAI, JIA; TANG, QI-ZHU

    2014-01-01

    Icariin, the major active component isolated from plants of the Epimedium family, has been reported to have potential protective effects on the cardiovascular system. However, it is not known whether icariin has a direct effect on angiotensin II (Ang II)-induced cardiomyocyte enlargement and apoptosis. In the present study, embryonic rat heart-derived H9c2 cells were stimulated by Ang II, with or without icariin administration. Icariin treatment was found to attenuate the Ang II-induced increase in mRNA expression levels of hypertrophic markers, including atrial natriuretic peptide and B-type natriuretic peptide, in a concentration-dependent manner. The cell surface area of Ang II-treated H9c2 cells also decreased with icariin administration. Furthermore, icariin repressed Ang II-induced cell apoptosis and protein expression levels of Bax and cleaved-caspase 3, while the expression of Bcl-2 was increased by icariin. In addition, 2′,7′-dichlorofluorescein diacetate incubation revealed that icariin inhibited the production of intracellular reactive oxygen species (ROS), which were stimulated by Ang II. Phosphorylation of c-Jun N-terminal kinase (JNK) and p38 in Ang II-treated H9c2 cells was blocked by icariin. Therefore, the results of the present study indicated that icariin protected H9c2 cardiomyocytes from Ang II-induced hypertrophy and apoptosis by inhibiting the ROS-dependent JNK and p38 pathways. PMID:24940396

  10. Apigenin potentiates the antitumor activity of 5-FU on solid Ehrlich carcinoma: Crosstalk between apoptotic and JNK-mediated autophagic cell death platforms.

    PubMed

    Gaballah, Hanaa H; Gaber, Rasha A; Mohamed, Darin A

    2017-02-01

    Although 5- Fluorouracil (5-FU) has exhibited effectiveness against cancer, novel therapeutic strategies are needed to enhance its antitumor efficiency and modulate its cytotoxity. Apigenin, a flavonoid present in fruits and vegetables, is a potent dietary phytochemical effective in cancer chemoprevention. This study was undertaken to investigate the potential synergistic antitumor activity of apigenin and 5-FU on Solid Ehrlich carcinoma (SEC). Eighty Swiss albino male mice were divided into four equal groups: vehicle treated control SEC, SEC+5-FU, SEC+apigenin, SEC+ 5-FU+apigenin. Beclin-1 and caspases 3, 9 and JNK activities were estimated by ELISA; mRNA expression levels of the antiapoptotic gene Mcl-1 were estimated using quantitative real-time RT-PCR, while tissue malondialdehyde (MDA), glutathione peroxidase and total antioxidant capacity were evaluated spectrophotometrically. A part of the tumor was examined for histopathological and Ki-67 immunohistochemistry analysis. 5-FU and/or apigenin caused significant increase in tissue levels of Beclin-1, caspases 3, 9 and JNK activities, MDA with significant decrease in tumor volume, Mcl-1expression, tissue glutathione peroxidase and total antioxidant capacity and alleviated the histopathological changes with significant decrease of Ki-67 proliferation index compared to vehicle treated SEC control group. The combination of 5-FU and apigenin had a greater effect than each of 5-FU or apigenin alone against solid Ehrlich carcinoma in mice. Copyright © 2016 Elsevier Inc. All rights reserved.

  11. Influenza A viruses suppress cyclooxygenase-2 expression by affecting its mRNA stability.

    PubMed

    Dudek, Sabine Eva; Nitzsche, Katja; Ludwig, Stephan; Ehrhardt, Christina

    2016-06-06

    Infection with influenza A viruses (IAV) provokes activation of cellular defence mechanisms contributing to the innate immune and inflammatory response. In this process the cyclooxygenase-2 (COX-2) plays an important role in the induction of prostaglandin-dependent inflammation. While it has been reported that COX-2 is induced upon IAV infection, in the present study we observed a down-regulation at later stages of infection suggesting a tight regulation of COX-2 by IAV. Our data indicate the pattern-recognition receptor RIG-I as mediator of the initial IAV-induced COX-2 synthesis. Nonetheless, during on-going IAV replication substantial suppression of COX-2 mRNA and protein synthesis could be detected, accompanied by a decrease in mRNA half-life. Interestingly, COX-2 mRNA stability was not only imbalanced by IAV replication but also by stimulation of cells with viral RNA. Our results reveal tristetraprolin (TTP), which is known to bind COX-2 mRNA and promote its rapid degradation, as regulator of COX-2 expression in IAV infection. During IAV replication and viral RNA accumulation TTP mRNA synthesis was induced, resulting in reduced COX-2 levels. Accordingly, the down-regulation of TTP resulted in increased COX-2 protein expression after IAV infection. These findings indicate a novel IAV-regulated cellular mechanism, contributing to the repression of host defence and therefore facilitating viral replication.

  12. Staufen2 functions in Staufen1-mediated mRNA decay by binding to itself and its paralog and promoting UPF1 helicase but not ATPase activity

    PubMed Central

    Park, Eonyoung; Gleghorn, Michael L.; Maquat, Lynne E.

    2013-01-01

    Staufen (STAU)1-mediated mRNA decay (SMD) is a posttranscriptional regulatory mechanism in mammals that degrades mRNAs harboring a STAU1-binding site (SBS) in their 3′-untranslated regions (3′ UTRs). We show that SMD involves not only STAU1 but also its paralog STAU2. STAU2, like STAU1, is a double-stranded RNA-binding protein that interacts directly with the ATP-dependent RNA helicase up-frameshift 1 (UPF1) to reduce the half-life of SMD targets that form an SBS by either intramolecular or intermolecular base-pairing. Compared with STAU1, STAU2 binds ∼10-fold more UPF1 and ∼two- to fivefold more of those SBS-containing mRNAs that were tested, and it comparably promotes UPF1 helicase activity, which is critical for SMD. STAU1- or STAU2-mediated augmentation of UPF1 helicase activity is not accompanied by enhanced ATP hydrolysis but does depend on ATP binding and a basal level of UPF1 ATPase activity. Studies of STAU2 demonstrate it changes the conformation of RNA-bound UPF1. These findings, and evidence for STAU1−STAU1, STAU2−STAU2, and STAU1−STAU2 formation in vitro and in cells, are consistent with results from tethering assays: the decrease in mRNA abundance brought about by tethering siRNA-resistant STAU2 or STAU1 to an mRNA 3′ UTR is inhibited by downregulating the abundance of cellular STAU2, STAU1, or UPF1. It follows that the efficiency of SMD in different cell types reflects the cumulative abundance of STAU1 and STAU2. We propose that STAU paralogs contribute to SMD by “greasing the wheels” of RNA-bound UPF1 so as to enhance its unwinding capacity per molecule of ATP hydrolyzed. PMID:23263869

  13. Staufen2 functions in Staufen1-mediated mRNA decay by binding to itself and its paralog and promoting UPF1 helicase but not ATPase activity.

    PubMed

    Park, Eonyoung; Gleghorn, Michael L; Maquat, Lynne E

    2013-01-08

    Staufen (STAU)1-mediated mRNA decay (SMD) is a posttranscriptional regulatory mechanism in mammals that degrades mRNAs harboring a STAU1-binding site (SBS) in their 3'-untranslated regions (3' UTRs). We show that SMD involves not only STAU1 but also its paralog STAU2. STAU2, like STAU1, is a double-stranded RNA-binding protein that interacts directly with the ATP-dependent RNA helicase up-frameshift 1 (UPF1) to reduce the half-life of SMD targets that form an SBS by either intramolecular or intermolecular base-pairing. Compared with STAU1, STAU2 binds ~10-fold more UPF1 and ~two- to fivefold more of those SBS-containing mRNAs that were tested, and it comparably promotes UPF1 helicase activity, which is critical for SMD. STAU1- or STAU2-mediated augmentation of UPF1 helicase activity is not accompanied by enhanced ATP hydrolysis but does depend on ATP binding and a basal level of UPF1 ATPase activity. Studies of STAU2 demonstrate it changes the conformation of RNA-bound UPF1. These findings, and evidence for STAU1-STAU1, STAU2-STAU2, and STAU1-STAU2 formation in vitro and in cells, are consistent with results from tethering assays: the decrease in mRNA abundance brought about by tethering siRNA-resistant STAU2 or STAU1 to an mRNA 3' UTR is inhibited by downregulating the abundance of cellular STAU2, STAU1, or UPF1. It follows that the efficiency of SMD in different cell types reflects the cumulative abundance of STAU1 and STAU2. We propose that STAU paralogs contribute to SMD by "greasing the wheels" of RNA-bound UPF1 so as to enhance its unwinding capacity per molecule of ATP hydrolyzed.

  14. Differential mRNA expression of neuroinflammatory modulators in the spinal cord and thalamus of type 2 diabetic monkeys.

    PubMed

    Ding, Huiping; Kiguchi, Norikazu; Kishioka, Shiroh; Ma, Tao; Peters, Christopher M; Ko, Mei-Chuan

    2018-05-11

    Given that diabetes-associated complications are closely associated with neuroinflammation, it is imperative to study potential changes in neuroinflammatory modulators in the central nervous system of diabetic primates. The mRNA levels of pro- and anti-inflammatory cytokines, toll-like receptors (TLRs), growth factors, and cannabinoid receptors were compared in the spinal dorsal horn (SDH) and thalamus of naturally occurring type 2 diabetic monkeys and an age-matched control group using reverse transcription and quantitative real-time polymerase chain reaction. In the SDH of diabetic monkeys, mRNA levels of proinflammatory cytokines (i.e. interleukin [IL]-1β and tumor necrosis factor [TNF] α), TLR1, and TLR2 were increased, whereas mRNA levels of IL-10, an anti-inflammatory cytokine, were decreased. No changes were observed in the mRNA levels of growth factors and cannabinoid receptors. In line with the mRNA data, TNFα immunoreactivity was significantly increased in diabetic monkeys. Moreover, mRNA expression levels of IL-1β, TNFα, TLR1, and TLR2 in the SDH were positively correlated with plasma glucose concentrations in all monkeys. Several ligands and receptors involved in neuroinflammation are simultaneously dysregulated in the spinal cord of diabetic monkeys. This primate disease model will facilitate the design of novel treatment approaches to ameliorate neuroinflammation-driven adverse effects in diabetic patients. © 2018 Ruijin Hospital, Shanghai Jiaotong University School of Medicine and John Wiley & Sons Australia, Ltd.

  15. Interleukin-17B Antagonizes Interleukin-25-Mediated Mucosal Inflammation.

    PubMed

    Reynolds, Joseph M; Lee, Young-Hee; Shi, Yun; Wang, Xiaohu; Angkasekwinai, Pornpimon; Nallaparaju, Kalyan C; Flaherty, Stephanie; Chang, Seon Hee; Watarai, Hiroshi; Dong, Chen

    2015-04-21

    The interleukin-17 (IL-17) family of cytokines has emerged as a critical player in inflammatory diseases. Among them, IL-25 has been shown to be important in allergic inflammation and protection against parasitic infection. Here we have demonstrated that IL-17B, a poorly understood cytokine, functions to inhibit IL-25-driven inflammation. IL-17B and IL-25, both binding to the interleukin-17 receptor B (IL-17RB), were upregulated in their expression after acute colonic inflammation. Individual inhibition of these cytokines revealed opposing functions in colon inflammation: IL-25 was pathogenic but IL-17B was protective. Similarly opposing phenotypes were observed in Citrobacter rodentium infection and allergic asthma. Moreover, IL-25 was found to promote IL-6 production from colon epithelial cells, which was inhibited by IL-17B. Therefore, our data demonstrate that IL-17B is an anti-inflammatory cytokine in the IL-17 family. Copyright © 2015 Elsevier Inc. All rights reserved.

  16. Activation of the Liver X Receptor by Agonist TO901317 Improves Hepatic Insulin Resistance via Suppressing Reactive Oxygen Species and JNK Pathway.

    PubMed

    Dong, Ying; Gao, Guirong; Fan, Hongyan; Li, Shengxian; Li, Xuhang; Liu, Wei

    2015-01-01

    Activation of Liver X receptors (LXRs), key transcriptional regulators of glucose metabolism, normalizes glycemia and improves insulin sensitivity in rodent models with insulin resistance. However, the molecular mechanism is unclear. This study is aimed to elucidate the mechanism of LXRs-mediated liver glucose metabolic regulation in vitro and in vivo. Db/db mice were used as an in vivo model of diabetes; palmitate (PA)-stimulated HepG2 cells were used as an in vitro cell model with impairment of insulin signaling. TO901317 (TO) was chosen as the LXRs agonist. We demonstrated that TO treatment for 14 days potently improved the hepatic glucose metabolism in db/db mice, including fasting blood glucose, fasting insulin level, and HOMA-IR. TO had no effect on the glucose metabolism in normal WT mice. TO-mediated activation of hepatic LXRs led to strong inhibition of ROS production accompanied by inactivation of JNK pathway and re-activation of Akt pathway. TO also suppressed the expression of gluconeogenic genes such as PEPCK and G-6-pase in db/db mice, but not in WT mice. In HepG2 cells, TO almost completely restored PA-induced Akt inactivation, and suppressed PA-stimulated ROS production and JNK activation. Interestingly, basal level of ROS was also inhibited by TO in HepG2 cells. TO significantly inhibited PA-stimulated expressions of gluconeogenic genes. Finally, we found that anti-oxidative genes, such as Nrf2, were up-regulated after LXRs activation by TO. These results strongly support the notion that activation of LXRs is critical in suppression of liver gluconeogenesis and improvement of insulin sensitivity in diabetic individuals. At molecular levels, the mode of action appears to be as fellows: under diabetic condition, ROS production is increased, JNK is activated, and Akt activity is inhibited; TO-mediated LXR activation potently inhibits ROS production, increases anti-oxidative gene expressions, suppresses JNK activation, and restores Akt activity. Our

  17. JNK1 ablation in mice confers long-term metabolic protection from diet-induced obesity at the cost of moderate skin oxidative damage.

    PubMed

    Becattini, Barbara; Zani, Fabio; Breasson, Ludovic; Sardi, Claudia; D'Agostino, Vito Giuseppe; Choo, Min-Kyung; Provenzani, Alessandro; Park, Jin Mo; Solinas, Giovanni

    2016-09-01

    Obesity and insulin resistance are associated with oxidative stress, which may be implicated in the progression of obesity-related diseases. The kinase JNK1 has emerged as a promising drug target for the treatment of obesity and type 2 diabetes. JNK1 is also a key mediator of the oxidative stress response, which can promote cell death or survival, depending on the magnitude and context of its activation. In this article, we describe a study in which the long-term effects of JNK1 inactivation on glucose homeostasis and oxidative stress in obese mice were investigated for the first time. Mice lacking JNK1 (JNK1(-/-)) were fed an obesogenic high-fat diet (HFD) for a long period. JNK1(-/-) mice fed an HFD for the long term had reduced expression of antioxidant genes in their skin, more skin oxidative damage, and increased epidermal thickness and inflammation compared with the effects in control wild-type mice. However, we also observed that the protection from obesity, adipose tissue inflammation, steatosis, and insulin resistance, conferred by JNK1 ablation, was sustained over a long period and was paralleled by decreased oxidative damage in fat and liver. We conclude that compounds targeting JNK1 activity in brain and adipose tissue, which do not accumulate in the skin, may be safer and most effective.-Becattini, B., Zani, F., Breasson, L., Sardi, C., D'Agostino, V. G., Choo, M.-K., Provenzani, A., Park, J. M., Solinas, G. JNK1 ablation in mice confers long-term metabolic protection from diet-induced obesity at the cost of moderate skin oxidative damage. © FASEB.

  18. Modification of tissue-factor mRNA and protein response to thrombin and interleukin 1 by high glucose in cultured human endothelial cells.

    PubMed

    Boeri, D; Almus, F E; Maiello, M; Cagliero, E; Rao, L V; Lorenzi, M

    1989-02-01

    Because diabetic vascular disease is accompanied by a state of hypercoagulability, manifested by increased thrombin activity and foci of intravascular coagulation, we investigated whether a specific procoagulant property of the endothelium--production and surface expression of tissue factor--is modified by elevated glucose concentrations. In unperturbed human vascular endothelial cells, tissue factor mRNA and expression of the functional protein were undetectable and were not induced by 10-12 days of exposure to 30 mM glucose. In thrombin-stimulated cultures, tissue-factor expression was related inversely to cellular density, with confluent cultures producing (per 10(5) cells) half the amount of tissue factor measured in sparse cultures. Cells exposed to high glucose and studied when cell number and thymidine incorporation were identical to control cells manifested increased tissue-factor mRNA level and functional protein production in response to thrombin (P = .002). This effect was not attributable to hypertonicity and was not observed after short exposure to high glucose. In contrast, the tissue-factor response to interleukin 1, a modulator of endothelial function in the context of host defense, was decreased in cells cultured in high glucose (P = .04). These findings indicate that exposure to high glucose can alter tissue-factor gene expression in perturbed vascular endothelium. The reciprocal effects of high glucose on the tissue-factor response to thrombin and interleukin 1 points to different pathways of tissue-factor stimulation by the two agents and suggests functional consequences pertinent to the increased thrombin activity and compromised host-defense mechanisms observed in diabetes.

  19. Mu opioid receptor stimulation activates c-Jun N-terminal kinase 2 by distinct arrestin-dependent and independent mechanisms.

    PubMed

    Kuhar, Jamie Rose; Bedini, Andrea; Melief, Erica J; Chiu, Yen-Chen; Striegel, Heather N; Chavkin, Charles

    2015-09-01

    G protein-coupled receptor desensitization is typically mediated by receptor phosphorylation by G protein-coupled receptor kinase (GRK) and subsequent arrestin binding; morphine, however, was previously found to activate a c-Jun N-terminal kinase (JNK)-dependent, GRK/arrestin-independent pathway to produce mu opioid receptor (MOR) inactivation in spinally-mediated, acute anti-nociceptive responses [Melief et al.] [1]. In the current study, we determined that JNK2 was also required for centrally-mediated analgesic tolerance to morphine using the hotplate assay. We compared JNK activation by morphine and fentanyl in JNK1(-/-), JNK2(-/-), JNK3(-/-), and GRK3(-/-) mice and found that both compounds specifically activate JNK2 in vivo; however, fentanyl activation of JNK2 was GRK3-dependent, whereas morphine activation of JNK2 was GRK3-independent. In MOR-GFP expressing HEK293 cells, treatment with either arrestin siRNA, the Src family kinase inhibitor PP2, or the protein kinase C (PKC) inhibitor Gö6976 indicated that morphine activated JNK2 through an arrestin-independent Src- and PKC-dependent mechanism, whereas fentanyl activated JNK2 through a Src-GRK3/arrestin-2-dependent and PKC-independent mechanism. This study resolves distinct ligand-directed mechanisms of JNK activation by mu opioid agonists and understanding ligand-directed signaling at MOR may improve opioid therapeutics. Copyright © 2015 Elsevier Inc. All rights reserved.

  20. Berberine attenuates CCN2-induced IL-1β expression and prevents cartilage degradation in a rat model of osteoarthritis

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

    Liu, Shan-Chi; Lee, Hsiang-Ping; Department of Chinese Medicine, China Medical University Hospital, Taichung, Taiwan

    Connective tissue growth factor (CTGF; also known as CCN2) is an inflammatory mediator that is abundantly expressed in osteoarthritis (OA). Interleukin-1β (IL-1β) plays a pivotal role in OA pathogenesis. Berberine exhibits an anti-inflammatory effect, but the mechanisms by which it modulates CCN2-induced IL-1β expression in OA synovial fibroblasts (OASFs) remain unknown. We showed that CCN2-induced IL-1β expression is mediated by the activation of α{sub v}β{sub 3}/α{sub v}β{sub 5} integrin-dependent reactive oxygen species (ROS) generation, and subsequent activation of apoptosis signal-regulating kinase 1 (ASK1), p38/JNK, and nuclear factor-κB (NF-κB) signaling pathways. This IL-1β expression in OASFs is attenuated by N-acetylcysteine (NAC),more » inhibitors of ASK1, p38, or JNK, or treatment with berberine. Furthermore, berberine also reverses cartilage damage in an experimental model of collagenase-induced OA (CIOA). We observed that CCN2 increased IL-1β expression via α{sub v}β{sub 3}/α{sub v}β{sub 5} integrins, ROS, and ASK1, p38/JNK, and NF-κB signaling pathways. Berberine was found to inhibit these signaling components in OASFs in vitro and prevent cartilage degradation in vivo. We suggest a novel therapeutic strategy of using berberine for managing OA. - Highlights: • CCN2 induce IL-1β production via αvβ3/αvβ5 integrin, ROS, ASK1, p38/JNK, and NF-κB. • Berberine attenuates CCN2-induced IL-1β expression in vitro and in OA rat model. • Berberine as natural drug of choice for anti-inflammatory effect to ameliorates OA.« less

  1. β1 integrin- and JNK-dependent tumor growth upon hypofractionated radiation.

    PubMed

    Sayeed, Aejaz; Lu, Huimin; Liu, Qin; Deming, David; Duffy, Alexander; McCue, Peter; Dicker, Adam P; Davis, Roger J; Gabrilovich, Dmitry; Rodeck, Ulrich; Altieri, Dario C; Languino, Lucia R

    2016-08-16

    Radiation therapy is an effective cancer treatment modality although tumors invariably become resistant. Using the transgenic adenocarcinoma of mouse prostate (TRAMP) model system, we report that a hypofractionated radiation schedule (10 Gy/day for 5 consecutive days) effectively blocks prostate tumor growth in wild type (β1wt /TRAMP) mice as well as in mice carrying a conditional ablation of β1 integrins in the prostatic epithelium (β1pc-/- /TRAMP). Since JNK is known to be suppressed by β1 integrins and mediates radiation-induced apoptosis, we tested the effect of SP600125, an inhibitor of c-Jun amino-terminal kinase (JNK) in the TRAMP model system. Our results show that SP600125 negates the effect of radiation on tumor growth in β1pc-/- /TRAMP mice and leads to invasive adenocarcinoma. These effects are associated with increased focal adhesion kinase (FAK) expression and phosphorylation in prostate tumors in β1pc-/- /TRAMP mice. In marked contrast, radiation-induced tumor growth suppression, FAK expression and phosphorylation are not altered by SP600125 treatment of β1wt /TRAMP mice. Furthermore, we have reported earlier that abrogation of insulin-like growth factor receptor (IGF-IR) in prostate cancer cells enhances the sensitivity to radiation. Here we further explore the β1/IGF-IR crosstalk and report that β1 integrins promote cell proliferation partly by enhancing the expression of IGF-IR. In conclusion, we demonstrate that β1 integrin-mediated inhibition of JNK signaling modulates tumor growth rate upon hypofractionated radiation.

  2. Optimization of a nonviral transfection system to evaluate Cox-2 controlled interleukin-4 expression for osteoarthritis gene therapy in vitro.

    PubMed

    Lang, Annemarie; Neuhaus, Johannes; Pfeiffenberger, Moritz; Schröder, Erik; Ponomarev, Igor; Weber, Yvonne; Gaber, Timo; Schmidt, Michael F G

    2014-01-01

    Gene therapy appears to have the potential for achieving a long-term remedy for osteoarthritis (OA). However, there is a risk of adverse reactions, especially when using cytomegalovirus-controlled expression. To provide a safe application, we focused on the expression of therapeutic cytokines [e.g. interleukin (IL)-4] in a disease-responsive manner by use of the previously cloned Cox-2 promoter as 'genetic switch'. In the present study, we report the functionality of a controlled gene therapeutic system in an equine osteoarthritic cell model. Different nonviral transfection reagents were tested for their efficiency on equine chondrocytes stimulated with equine IL-1β or lipopolysaccharide to create an inflammatory environment. To optimize the transfection, we successfully redesigned the vector by excluding the internal ribosomal entry site (IRES). The functionality of our Cox-2 promoter construct with respect to expressing IL-4 was proven at the mRNA and protein levels and the anti-inflammatory potential of IL-4 was confirmed by analyzing the expression of IL-1β, IL-6, IL-8, matrix metalloproteinase (MMP)-1, MMP-3 and tumor necrosis factor (TNF)-α using a quantitative polymerase chain reaction. Nonviral transfection reagents yielded transfection rates from 21% to 44% with control vectors with and without IRES, respectively. Stimulation of equine chondrocytes resulted in a 20-fold increase of mRNA expression of IL-1β. Such exogenous stimulation of chondrocytes transfected with pNCox2-IL4 led to an increase of IL-4 mRNA expression, whereas expression of inflammatory mediators decreased. The timely link between these events confirms the anti-inflammatory potential of synthesized IL-4. We consider that this approach has significant potential for translation into a useful anti-inflammation therapy. Molecular tools such as the described therapeutic plasmid pave the way for a local-controlled, self-limiting gene therapy. Copyright © 2014 John Wiley & Sons, Ltd.

  3. N-acetyl cysteine inhibits lipopolysaccharide-mediated induction of interleukin-6 synthesis in MC3T3-E1 cells through the NF-kB signaling pathway.

    PubMed

    Guo, Ling; Zhang, Hui; Li, Wangyang; Zhan, Danting; Wang, Min

    2018-06-06

    Interleukin-6 (IL-6) is a potent stimulator of osteoclastic activity. Lipopolysaccharide (LPS) has been shown to regulate the expression of potent inflammatory factors, including TNF-α and IL-6. Currently, effective therapeutic treatments for bacteria-caused bone destruction are limited. N-acetyl cysteine (NAC) is an antioxidant small molecule that possibly modulates osteoblastic differentiation. However, whether NAC can affect the LPS-mediated reduction of IL-6 synthesis in MC3T3-E1 cells is still unknown. The aim of this study was to investigate the role of NAC in the LPS -mediated reduction of IL-6 synthesis by MC3T3-E1 cells and to explore the underlying molecular mechanisms. In addition, we aimed to determine the involvement of the NF-kB pathway in any changes in IL-6 expression observed in response to LPS and NAC. MC3T3-E1 cells (ATCC, CRL-2593) were cultured in α-minimum essential medium. Cells were stimulated using NAC or LPS at various concentrations. Cell proliferation was observed at multiple time points using a cell counting kit 8 (CCK-8). IL-6 mRNA expression and protein synthesis were determined using quantitative polymerase chain reaction (qPCR) and enzyme-linked immunosorbent assay analyses. NF-kB mRNA expression and protein synthesis was determined using qPCR and Western blots analyses. The results demonstrate that LPS induced IL-6 and NF-kB mRNA expression and protein synthesis in the cultured MC3T3-E1 cells. However, these effects were abolished following pre-treatment with NAC. Pretreatment with NAC (1 mmol/l) or BAY11-7082 (10 μmol/l) both significantly inhibited the NF-kB activity induced by LPS. NAC inhibits the LPS-mediated induction of IL-6 synthesis in MC3T3-E1 cells through the NF-kB pathway. Copyright © 2018 Elsevier Ltd. All rights reserved.

  4. Interleukin-34 inhibits hepatitis B virus replication in vitro and in vivo

    PubMed Central

    Cheng, Sheng-Tao; Tang, Hua; Ren, Ji-Hua; Chen, Xiang; Huang, Ai-Long

    2017-01-01

    Background The hepatitis B virus (HBV) infection could activate the immune system and induce extensive inflammatory response. As the most important inflammatory factor, interleukins are critical for anti-viral immunity. Here we investigated whether interleukin-34 (IL-34) play a role in HBV infection. Methodology/Principal findings In this study, we first found that both serum IL-34 and IL-34 mRNA in PBMCs in chronic HBV patients was significantly decreased compared to the healthy controls. Furthermore, both IL-34 protein and mRNA levels were declined hepatoma cells expressing HBV. In addition, the clinical parameters analysis found that serum IL-34 was significantly associated with HBV DNA (P = 0.0066), ALT (P = 0.0327), AST (P = 0.0435), TB (P = 0.0406), DB (P = 0.0368) and AFP (P = 0.0225). Correlation analysis also found that serum IL-34 negatively correlated with HBV DNA copies, ALT and AST. In vitro studies found that IL-34 treatment in HepAD38 and HepG2.2.15 cells markedly inhibited HBV DNA, total RNA, 3.5kb mRNA and HBc protein. In vivo studies further demonstrated IL-34 treatment in HBV transgenic mice exhibited greater inhibition on HBV DNA, total RNA, 3.5kb mRNA and HBc protein, suggesting the effect to IL-34 on HBV is likely due to host innate or adaptive immune response. Conclusions/Significance Our study identified a novel interleukin, IL-34, which has anti-viral activity in HBV replication in hepatocytes in vitro and in vivo. These data suggest a rationale for the use of IL-34 in the HBV treatment. PMID:28614380

  5. Adoptive Cell Therapy for Patients with Melanoma, Using Tumor-Infiltrating Lymphocytes Genetically Engineered to Secrete Interleukin-2

    PubMed Central

    HEEMSKERK, BIANCA; LIU, KE; DUDLEY, MARK. E.; JOHNSON, LAURA A.; KAISER, ANDREW; DOWNEY, STEPHANIE; ZHENG, ZHILI; SHELTON, THOMAS E.; MATSUDA, KANT; ROBBINS, PAUL F.; MORGAN, RICHARD A.; ROSENBERG, STEVEN A.

    2008-01-01

    Adoptive cell transfer of tumor-infiltrating lymphocytes (TILs) after lymphodepletion mediates regression in 50% of patients with metastatic melanoma. In vivo persistence and telomere length of the transferred cells correlate with antitumor response. In an attempt to prolong the in vivo survival of the transferred cells, TILs were genetically engineered to produce interleukin (IL)-2. In vitro, these transduced TILs secreted IL-2 while retaining tumor specificity and exhibited prolonged survival after IL-2 withdrawal. In a phase I/II clinical trial, seven evaluable patients received transduced TILs and one patient experienced a partial response associated with in vivo persistence of IL-2-transduced TILs in circulating lymphocytes. An additional five patients received transduced TILs in conjunction with IL-2 administration. Persistence of IL-2-transduced TILs was observed in three patients, including one partial responder. The transgene DNA as well as vector-derived IL-2 mRNA could be detected for 4 months in responding patients. The low response rate in this trial was possibly due to a reduction in telomere length in cells as a result of prolonged in vitro culture. In this study, insertion of the IL-2 gene into antitumor TILs increased their ability to survive after IL-2 withdrawal in vitro but did not increase their in vivo persistence or clinical effectiveness. PMID:18444786

  6. The coffee diterpene kahweol suppresses the cell proliferation by inducing cyclin D1 proteasomal degradation via ERK1/2, JNK and GKS3β-dependent threonine-286 phosphorylation in human colorectal cancer cells.

    PubMed

    Park, Gwang Hun; Song, Hun Min; Jeong, Jin Boo

    2016-09-01

    Kahweol as a coffee-specific diterpene has been reported to exert anti-cancer properties. However, the mechanism responsible for the anti-cancer effects of kahweol is not fully understood. The main aim of this investigation was to determine the effect of kahweol on cell proliferation and the possible mechanisms in human colorectal cancer cells. Kahweol inhibited markedly the proliferation of human colorectal cancer cell lines such as HCT116, SW480. Kahweol decreased cyclin D1 protein level in HCT116 and SW480 cells. Contrast to protein levels, cyclin D1 mRNA level and promoter activity did not be changed by kahweol treatment. MG132 treatment attenuated kahweol-mediated cyclin D1 downregulation and the half-life of cyclin D1 was decreased in kahweol-treated cells. Kahweol increased phosphorylation of cyclin D1 at threonine-286 and a point mutation of threonine-286 to alanine attenuated cyclin D1 degradation by kahweol. Inhibition of ERK1/2 by PD98059, JNK by SP600125 or GSK3β by LiCl suppressed cyclin D1 phosphorylation and downregulation by kahweol. Furthermore, the inhibition of nuclear export by LMB attenuated cyclin D1 degradation by kahweol. In conclusion, kahweol-mediated cyclin D1 degradation may contribute to the inhibition of the proliferation in human colorectal cancer cells. Copyright © 2016 Elsevier Ltd. All rights reserved.

  7. Reduced beta 2-microglobulin mRNA levels in transgenic mice expressing a designed hammerhead ribozyme.

    PubMed Central

    Larsson, S; Hotchkiss, G; Andäng, M; Nyholm, T; Inzunza, J; Jansson, I; Ahrlund-Richter, L

    1994-01-01

    We have generated three artificial hammerhead ribozymes, denoted 'Rz-b', 'Rz-c' and 'Rz-d', with different specificities for exon II of the mouse beta-2-microglobulin (beta 2M) mRNA. In this study we tested for ribozyme mediated reduction of beta 2M mRNA in a cell line and in transgenic mice. Transfections of either of the Rz-b, Rz-c or Rz-d plasmids into a mouse cell-line (NIH/3T3) revealed reductions of beta 2M mRNA substrate in each case. Ribozyme expression in individual transfected clones was accompanied with an up to 80% reduction of beta 2M mRNA levels. Rz-c was selected for a transgenic study. Seven Rz-c transgenic founder animals were identified from which three ribozyme expressing families were established and analysed. Expression of the ribozyme transgene was tested for and detected in lung, kidney and spleen. Expression was accompanied with reduction of the beta 2M mRNA levels of heterozygous (Rz+/-) animals compared to non-transgenic litter mates. The effect was most pronounced in lung with more than 90% beta 2M mRNA reduction in individual mice. In summary, expression of our ribozymes in a cell free system, in a cell-line and in transgenic mice were all accompanied with reductions of beta 2M mRNA levels. Images PMID:8036151

  8. The adaptor protein Cindr regulates JNK activity to maintain epithelial sheet integrity.

    PubMed

    Yasin, Hannah W R; van Rensburg, Samuel H; Feiler, Christina E; Johnson, Ruth I

    2016-02-15

    Epithelia are essential barrier tissues that must be appropriately maintained for their correct function. To achieve this a plethora of protein interactions regulate epithelial cell number, structure and adhesion, and differentiation. Here we show that Cindr (the Drosophila Cin85 and Cd2ap ortholog) is required to maintain epithelial integrity. Reducing Cindr triggered cell delamination and movement. Most delaminating cells died. These behaviors were consistent with JNK activation previously associated with loss of epithelial integrity in response to ectopic oncogene activity. We confirmed a novel interaction between Cindr and Drosophila JNK (dJNK), which when perturbed caused inappropriate JNK signaling. Genetically reducing JNK signaling activity suppressed the effects of reducing Cindr. Furthermore, ectopic JNK signaling phenocopied loss of Cindr and was partially rescued by concomitant cindr over-expression. Thus, correct Cindr-dJNK stoichiometry is essential to maintain epithelial integrity and disturbing this balance may contribute to the pathogenesis of disease states, including cancer. Copyright © 2016 Elsevier Inc. All rights reserved.

  9. Chromatoid Body Protein TDRD6 Supports Long 3’ UTR Triggered Nonsense Mediated mRNA Decay

    PubMed Central

    Fanourgakis, Grigorios; Akpinar, Müge; Dahl, Andreas; Jessberger, Rolf

    2016-01-01

    Chromatoid bodies (CBs) are spermiogenesis-specific organelles of largely unknown function. CBs harbor various RNA species, RNA-associated proteins and proteins of the tudor domain family like TDRD6, which is required for a proper CB architecture. Proteome analysis of purified CBs revealed components of the nonsense-mediated mRNA decay (NMD) machinery including UPF1. TDRD6 is essential for UPF1 localization to CBs, for UPF1-UPF2 and UPF1-MVH interactions. Upon removal of TDRD6, the association of several mRNAs with UPF1 and UPF2 is disturbed, and the long 3’ UTR-stimulated but not the downstream exon-exon junction triggered pathway of NMD is impaired. Reduced association of the long 3’ UTR mRNAs with UPF1 and UPF2 correlates with increased stability and enhanced translational activity. Thus, we identified TDRD6 within CBs as required for mRNA degradation, specifically the extended 3’ UTR-triggered NMD pathway, and provide evidence for the requirement of NMD in spermiogenesis. This function depends on TDRD6-promoted assembly of mRNA and decay enzymes in CBs. PMID:27149095

  10. Cytochrome P450-2C11 mRNA is not expressed in endothelial cells dissected from rat renal arterioles.

    PubMed

    Heil, Sandra G; De Vriese, An S; Kluijtmans, Leo A J; Dijkman, Henry; van Strien, Denise; Akkers, Robert; Blom, Henk J

    2005-01-01

    Cytochrome P450 (CYP) isoenzymes (CYP2C and CYP2J) are involved in the production of epoxyeicosatrienoic acids, which are postulated as endothelium-derived hyperpolarizing factors (EDHFs). We hypothesized that if CYP2C11 is involved in the EDHF-mediated responses, its mRNA should be expressed in endothelial cells. We, therefore, examined the mRNA expression of CYP2C11 in endothelial cells of renal arterioles. Laser microdissection was applied to isolate endothelial cells from the renal arterioles of 4 male and 4 female Wistar rats. As a positive control of CYP2C11 expression, hepatocytes were also dissected from these rats. RNA was isolated and real-time quantitative polymerase chain reaction (Q-PCR) analysis was applied. Q-PCR analysis showed that CYP2C11 mRNA was not expressed in laser microdissected endothelial cells of renal arterioles of male and female rats. CYP2C11 mRNA expression was highly abundant in hepatocytes dissected from male livers, but in female livers hardly any CYP2C11 mRNA was detected. We have shown that endothelial cells can be dissected from small renal arterioles by laser microdissection to study the mRNA expression of specific genes by Q-PCR. Using this novel tool, we demonstrated that the CYP2C11 mRNA was not expressed in the endothelial cells of renal arterioles. Therefore, we speculate that CYP2C11 does not contribute to the EDHF-mediated responses in renal arterioles. Copyright (c) 2005 S. Karger AG, Basel.

  11. Induction of the SHARP-2 mRNA level by insulin is mediated by multiple signaling pathways.

    PubMed

    Kanai, Yukiko; Asano, Kosuke; Komatsu, Yoshiko; Takagi, Katsuhiro; Ono, Moe; Tanaka, Takashi; Tomita, Koji; Haneishi, Ayumi; Tsukada, Akiko; Yamada, Kazuya

    2017-02-01

    The rat enhancer of split- and hairy-related protein-2 (SHARP-2) is an insulin-inducible transcription factor which represses transcription of the rat phosphoenolpyruvate carboxykinase gene. In this study, a regulatory mechanism of the SHARP-2 mRNA level by insulin was analyzed. Insulin rapidly induced the level of SHARP-2 mRNA. This induction was blocked by inhibitors for phosphoinositide 3-kinase (PI 3-K), protein kinase C (PKC), and mammalian target of rapamycin (mTOR), actinomycin D, and cycloheximide. Whereas an adenovirus infection expressing a dominant negative form of atypical PKC lambda (aPKCλ) blocked the insulin-induction of the SHARP-2 mRNA level, insulin rapidly activated the mTOR. Insulin did not enhance transcriptional activity from a 3.7 kb upstream region of the rat SHARP-2 gene. Thus, we conclude that insulin induces the expression of the rat SHARP-2 gene at the transcription level via both a PI 3-K/aPKCλ- and a PI 3-K/mTOR- pathways and that protein synthesis is required for this induction.

  12. TNF-α potentiates uric acid-induced interleukin-1β (IL-1β) secretion in human neutrophils.

    PubMed

    Yokose, Kohei; Sato, Shuzo; Asano, Tomoyuki; Yashiro, Makiko; Kobayashi, Hiroko; Watanabe, Hiroshi; Suzuki, Eiji; Sato, Chikako; Kozuru, Hideko; Yatsuhashi, Hiroshi; Migita, Kiyoshi

    2018-05-01

    Monosodium urate (MSU) has been shown to promote interleukin-1β (IL-1β) secretion in human monocytes, but the priming signals for NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome pathway remains elusive. In this study, we investigated the role of Tumor necrosis factor-alpha (TNF-α) on MSU-mediated IL-1β induction in human neutrophils. Human neutrophils were stimulated with MSU, in the presence or absence of TNF-α priming. The cellular supernatants were analyzed for IL-1β, IL-18, and caspase-1 by enzyme-linked immunosorbent assay (ELISA) methods. Pro-IL-1β mRNA expressions in human neutrophils were analyzed by real-time PCR method. TNF-α stimulation induced pro-IL-1β mRNA expression; however, MSU stimulation did not induce pro-IL-1β mRNA expression in human neutrophils. TNF-α alone or MSU stimulation did not result in efficient IL-1β secretion in human neutrophils, whereas in TNF-α-primed neutrophils, MSU stimulation resulted in a marked IL-1β and IL-18 secretion. TNF-α-primed neutrophils secreted cleaved caspase-1 (p20), in response to MSU stimulation. Our data demonstrate that priming of human neutrophils with TNF-α promotes uric acid-mediated IL-1β secretion in the absence of microbial stimulation. These findings provide insights into the neutrophils-mediated inflammatory processes in gouty arthritis.

  13. SMAD4 Loss Is Associated with Cetuximab Resistance and Induction of MAPK/JNK Activation in Head and Neck Cancer Cells.

    PubMed

    Ozawa, Hiroyuki; Ranaweera, Ruchira S; Izumchenko, Evgeny; Makarev, Eugene; Zhavoronkov, Alex; Fertig, Elana J; Howard, Jason D; Markovic, Ana; Bedi, Atul; Ravi, Rajani; Perez, Jimena; Le, Quynh-Thu; Kong, Christina S; Jordan, Richard C; Wang, Hao; Kang, Hyunseok; Quon, Harry; Sidransky, David; Chung, Christine H

    2017-09-01

    Purpose: We previously demonstrated an association between decreased SMAD4 expression and cetuximab resistance in head and neck squamous cell carcinoma (HNSCC). The purpose of this study was to further elucidate the clinical relevance of SMAD4 loss in HNSCC. Experimental Design: SMAD4 expression was assessed by IHC in 130 newly diagnosed and 43 patients with recurrent HNSCC. Correlative statistical analysis with clinicopathologic data was also performed. OncoFinder, a bioinformatics tool, was used to analyze molecular signaling in TCGA tumors with low or high SMAD4 mRNA levels. The role of SMAD4 was investigated by shRNA knockdown and gene reconstitution of HPV-negative HNSCC cell lines in vitro and in vivo Results: Our analysis revealed that SMAD4 loss was associated with an aggressive, HPV-negative, cetuximab-resistant phenotype. We found a signature of prosurvival and antiapoptotic pathways that were commonly dysregulated in SMAD4 -low cases derived from TCGA-HNSCC dataset and an independent oral cavity squamous cell carcinoma (OSCC) cohort obtained from GEO. We show that SMAD4 depletion in an HNSCC cell line induces cetuximab resistance and results in worse survival in an orthotopic mouse model in vivo We implicate JNK and MAPK activation as mediators of cetuximab resistance and provide the foundation for the concomitant EGFR and JNK/MAPK inhibition as a potential strategy for overcoming cetuximab resistance in HNSCCs with SMAD4 loss. Conclusions: Our study demonstrates that loss of SMAD4 expression is a signature characterizing the cetuximab-resistant phenotype and suggests that SMAD4 expression may be a determinant of sensitivity/resistance to EGFR/MAPK or EGFR/JNK inhibition in HPV-negative HNSCC tumors. Clin Cancer Res; 23(17); 5162-75. ©2017 AACR . ©2017 American Association for Cancer Research.

  14. TIPE attenuates the apoptotic effect of radiation and cisplatin and promotes tumor growth via JNK and p38 activation in Raw264.7 and EL4 cells.

    PubMed

    Liu, Yao; Ni, Xiao Yan; Chen, Rui Ling; Li, Juan; Gao, Feng Guang

    2018-06-01

    Tumor necrosis factor α‑induced protein 8 (TIPE) is highly expressed in many types of malignancies. Apoptosis is the process of programmed cell death which maintains the balance of cell survival and death. TIPE is involved in the carcinogenesis of many tumor types, yet the exact role of TIPE in defective apoptosis‑associated carcinogenesis remains uncertain. In the present study, TIPE‑overexpressing Raw264.7 and EL4 cells and vector control cells were treated with 4 mJ/cm2 ultraviolet radiation or 2 µg/ml cisplatin. Following ultraviolet irradiation, TIPE overexpression decreased the percentage of apoptotic cells as detected by flow cytometric and reversed the cisplatin‑mediated decrease in mitochondrial membrane potential by JC‑1 assay. Western blot analyses also revealed that TIPE overexpression inhibited cisplatin‑induced activation of caspase‑3 and ‑9 and PARP. Secondly, TIPE overexpression increased the levels of phosphorylated JNK, MEK and p38. Moreover, inhibition of JNK and p38, but not MEK, efficiently abolished the cell pro‑survival effect of TIPE. Most importantly, an in vivo tumor implantation model revealed that TIPE overexpression augmented the volume and weight of the implanted tumors, indicating that TIPE facilitated tumor formation. We found that TIPE exhibited an anti‑apoptotic effect via JNK and p38 activation, which ultimately promoted tumor. Hence, the present study revealed that activation of JNK and p38 kinases contribute to the TIPE‑mediated anti‑apoptotic effect, indicating that JNK and p38 may be potential therapeutic molecules for TIPE overexpression‑associated diseases.

  15. 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

  16. Fluoxetine a novel anti-hepatitis C virus agent via ROS-, JNK-, and PPARβ/γ-dependent pathways.

    PubMed

    Young, Kung-Chia; Bai, Chyi-Huey; Su, Hui-Chen; Tsai, Pei-Ju; Pu, Chien-Yu; Liao, Chao-Sheng; Lin, Yu-Min; Lai, Hsin-Wen; Chong, Lee-Won; Tsai, Yau-Sheng; Tsao, Chiung-Wen

    2014-10-01

    More than 20% of chronic hepatitis C (CHC) patients receiving interferon-alpha (IFN-α)-based anti-hepatitis C virus (HCV) therapy experienced significant depression, which was relieved by treatment with fluoxetine. However, whether and how fluoxetine affected directly the anti-HCV therapy remained unclear. Here, we demonstrated that fluoxetine inhibited HCV infection and blocked the production of reactive oxygen species (ROS) and lipid accumulation in Huh7.5 cells. Fluoxetine facilitated the IFN-α-mediated antiviral actions via activations of signal transducer and activator of transcription (STAT)-1 and c-Jun amino-terminal kinases (JNK). Alternatively, fluoxetine elevated peroxisome proliferator-activated receptor (PPAR) response element activity under HCV infection. The inhibitory effects of fluoxetine on HCV infection and lipid accumulation, but not production of ROS, were partially reversed by the PPAR-β, -γ, and JNK antagonists. Furthermore, fluoxetine intervention to the IFN-α-2b regimen facilitated to reduce HCV titer and alanine transaminase level for CHC patients. Therefore, fluoxetine intervention to the IFN-α-2b regimen improved the efficacy of anti-HCV treatment, which might be related to blockades of ROS generation and lipid accumulation and activation of host antiviral JNK/STAT-1 and PPARβ/γ signals. Copyright © 2014 Elsevier B.V. All rights reserved.

  17. An Interleukin-33-Mast Cell-Interleukin-2 Axis Suppresses Papain-Induced Allergic Inflammation by Promoting Regulatory T Cell Numbers.

    PubMed

    Morita, Hideaki; Arae, Ken; Unno, Hirotoshi; Miyauchi, Kousuke; Toyama, Sumika; Nambu, Aya; Oboki, Keisuke; Ohno, Tatsukuni; Motomura, Kenichiro; Matsuda, Akira; Yamaguchi, Sachiko; Narushima, Seiko; Kajiwara, Naoki; Iikura, Motoyasu; Suto, Hajime; McKenzie, Andrew N J; Takahashi, Takao; Karasuyama, Hajime; Okumura, Ko; Azuma, Miyuki; Moro, Kazuyo; Akdis, Cezmi A; Galli, Stephen J; Koyasu, Shigeo; Kubo, Masato; Sudo, Katsuko; Saito, Hirohisa; Matsumoto, Kenji; Nakae, Susumu

    2015-07-21

    House dust mite-derived proteases contribute to allergic disorders in part by disrupting epithelial barrier function. Interleukin-33 (IL-33), produced by lung cells after exposure to protease allergens, can induce innate-type airway eosinophilia by activating natural helper (NH) cells, a member of group 2 innate lymphoid cells (ILC2), to secrete Th2 type-cytokines. Because IL-33 also can induce mast cells (MCs) to secrete Th2 type-cytokines, MCs are thought to cooperate with NH cells in enhancing protease or IL-33-mediated innate-type airway eosinophilia. However, we found that MC-deficient Kit(W-sh/W-sh) mice exhibited exacerbated protease-induced lung inflammation associated with reduced numbers of regulatory T (Treg) cells. Moreover, IL-2 produced by IL-33-stimulated MCs promoted expansion of numbers of Treg cells, thereby suppressing development of papain- or IL-33-induced airway eosinophilia. We have thus identified a unique anti-inflammatory pathway that can limit induction of innate-type allergic airway inflammation mediated by NH cells. Copyright © 2015 Elsevier Inc. All rights reserved.

  18. Interleukin 31 mediates MAP kinase and STAT1/3 activation in intestinal epithelial cells and its expression is upregulated in inflammatory bowel disease

    PubMed Central

    Dambacher, Julia; Beigel, Florian; Seiderer, Julia; Haller, Dirk; Göke, Burkhard; Auernhammer, Christoph J; Brand, Stephan

    2007-01-01

    Background/aim Interleukin 31 (IL31), primarily expressed in activated lymphocytes, signals through a heterodimeric receptor complex consisting of the IL31 receptor alpha (IL31Rα) and the oncostatin M receptor (OSMR). The aim of this study was to analyse IL31 receptor expression, signal transduction, and specific biological functions of this cytokine system in intestinal inflammation. Methods Expression studies were performed by RT‐PCR, quantitative PCR, western blotting, and immunohistochemistry. Signal transduction was analysed by western blotting. Cell proliferation was measured by MTS assays, cell migration by restitution assays. Results Colorectal cancer derived intestinal epithelial cell (IEC) lines express both IL31 receptor subunits, while their expression in unstimulated primary murine IEC was low. LPS and the proinflammatory cytokines TNF‐α, IL1β, IFN‐γ, and sodium butyrate stimulation increased IL31, IL31Rα, and OSMR mRNA expression, while IL31 itself enhanced IL8 expression in IEC. IL31 mediates ERK‐1/2, Akt, STAT1, and STAT3 activation in IEC resulting in enhanced IEC migration. However, at low cell density, IL31 had significant antiproliferative capacities (p<0.005). IL31 mRNA expression was not increased in the TNFΔARE mouse model of ileitis but in inflamed colonic lesions compared to non‐inflamed tissue in patients with Crohn's disease (CD; average 2.4‐fold increase) and in patients with ulcerative colitis (UC; average 2.6‐fold increase) and correlated with the IL‐8 expression in these lesions (r = 0.564 for CD; r = 0.650 for UC; total number of biopsies analysed: n = 88). Conclusion IEC express the functional IL31 receptor complex. IL31 modulates cell proliferation and migration suggesting a role in the regulation of intestinal barrier function particularly in intestinal inflammation. PMID:17449633

  19. 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.

  20. Molecular cloning and mRNA expression analysis of interleukin-8 gene in Japanese sea perch (Lateolabrax japonicus).

    PubMed

    Qiu, Lihua; Zhang, Hanhua; Yang, Keng; Jiang, Shigui

    2009-05-01

    Interleukin-8 (IL-8), the first known chemokine, is a CXC chemokine, which is cable of attracting neutrophils and inducing them to release lysozomal enzymes, triggering the respiratory burst. In the present study, the cDNA of an IL-8 was cloned from Japanese sea perch Lateolabrax japonicus (designated LjIL-8) by homology cloning and rapid amplification of cDNA ends (RACE) approaches. The full-length cDNA of LjIL-8 consisted of 803 nucleotides with a canonical polyadenylation signal sequence AATAAA and a poly(A) tail, and an open reading frame (ORF) of 300 bp encoding a polypeptide of 99 amino acid residues with a predicted molecular weight of 6.6 kDa. The high identity of LjIL-8 with IL-8 in other organisms indicated that LjIL-8 should be a new member of the IL-8 family. By fluorescent quantitative real-time PCR, mRNA transcript of LjIL-8 was detectable in all the examined tissues with higher level in spleen and head-kidney. The temporal expression of LjIL-8 mRNA in the spleen was up-regulated by lipopolyssacharide (LPS) stimulation and reached the maximum level at 6 h post-stimulation, and then dropped back to the original level gradually. These results indicated that LjIL-8 was a constitutive and inducible acute-phase protein that perhaps involved in the immune defense of L. japonicus.

  1. Sex-lethal promotes nuclear retention of msl2 mRNA via interactions with the STAR protein HOW

    PubMed Central

    Graindorge, Antoine; Carré, Clément; Gebauer, Fátima

    2013-01-01

    Female-specific repression of male-specific-lethal-2 (msl2) mRNA in Drosophila melanogaster provides a paradigm for coordinated control of gene expression by RNA-binding complexes. Repression is orchestrated by Sex-lethal (SXL), which binds to the 5′ and 3′ untranslated regions (UTRs) of the mRNA and inhibits splicing in the nucleus and subsequent translation in the cytoplasm. Here we show that SXL ensures msl2 silencing by yet a third mechanism that involves inhibition of nucleocytoplasmic transport of msl2 mRNA. To identify SXL cofactors in msl2 regulation, we devised a two-step purification method termed GRAB (GST pull-down and RNA affinity binding) and identified Held-Out-Wings (HOW) as a component of the msl2 5′ UTR-associated complex. HOW directly interacts with SXL and binds to two sequence elements in the msl2 5′ UTR. Depletion of HOW reduces the capacity of SXL to repress the expression of msl2 reporters without affecting SXL-mediated regulation of splicing or translation. Instead, HOW is required for SXL to retain msl2 transcripts in the nucleus. Cooperation with SXL confers a sex-specific role to HOW. Our results uncover a novel function of SXL in nuclear mRNA retention and identify HOW as a mediator of this function. PMID:23788626

  2. Interleukin-8 is associated with adhesion, migration and invasion in human gastric cancer SCG-7901 cells.

    PubMed

    Ju, Dawei; Sun, Dazhi; Xiu, Lijuan; Meng, Xianze; Zhang, Cian; Wei, Pinkang

    2012-03-01

    Interleukin-8 is known as an important chemokine involved in tumor angiogenesis and progression. Overexpression of interleukin-8 has been detected in a variety of human tumors, including gastric cancer, and is negatively correlated with prognosis. The aim of our study is to determine the effects of interleukin-8 on proliferation, adhesion, migration and invasion abilities and correlated molecular mechanisms in gastric cancer. We made recombinant interleukin-8 ranged from 0 ng/ml to 100 ng/ml interferes in human gastric cancer SCG-7901 cells in vitro. The results shown that interleukin-8 did not change cell proliferation, but promoted cell adhesion to endothelial cell and extracellular matrix components (collagen, laminin and fibronectin) as detected by Cell Counting Kit-8. And it induced migration and invasion ability based on scratch and transwell-chamber assays. Also, interleukin-8 regulated the protein and mRNA expression of matrix metalloproteinase-9, intercellular adhesion molecule-1 and E-cad and there was obviously a dose-dependent relationship, but the protein or mRNA expression of matrix metalloproteinase-2 was not obviously changed under the tested conditions. Our findings indicate that interleukin-8 is associated with adhesion, migration and invasion in gastric cancer and the regulation of matrix metalloproteinase-9, intercellular adhesion molecule-1 and E-cad expression is one of the potential molecule mechanisms. The studies imply interleukin-8 may be an alternative treatment strategy against gastric cancer.

  3. Tumor necrosis factor-{alpha} induces MMP-9 expression via p42/p44 MAPK, JNK, and nuclear factor-{kappa}B in A549 cells

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

    Lin, C.-C.; Tseng, Hsiao-Wei; Hsieh, Hsi-Lung

    2008-06-15

    Matrix metalloproteinases (MMPs), in particular MMP-9, have been shown to be induced by cytokines including tumor necrosis factor-{alpha} (TNF-{alpha}) and contributes to airway inflammation. However, the mechanisms underlying MMP-9 expression induced by TNF-{alpha} in human A549 cells remain unclear. Here, we showed that TNF-{alpha} induced production of MMP-9 protein and mRNA is determined by zymographic, Western blotting, RT-PCR and ELISA assay, which were attenuated by inhibitors of MEK1/2 (U0126), JNK (SP600125), and NF-{kappa}B (helenalin), and transfection with dominant negative mutants of ERK2 ({delta}ERK) and JNK ({delta}JNK), and siRNAs for MEK1, p42 and JNK2. TNF-{alpha}-stimulated phosphorylation of p42/p44 MAPK and JNKmore » were attenuated by pretreatment with the inhibitors U0126 and SP600125 or transfection with dominant negative mutants of {delta}ERK and {delta}JNK. Furthermore, the involvement of NF-{kappa}B in TNF-{alpha}-induced MMP-9 production was consistent with that TNF-{alpha}-stimulated degradation of I{kappa}B-{alpha} and translocation of NF-{kappa}B into the nucleus which were blocked by helenalin, but not by U0126 and SP600125, revealed by immunofluorescence staining. The regulation of MMP-9 gene transcription by MAPKs and NF-{kappa}B was further confirmed by gene luciferase activity assay. MMP-9 promoter activity was enhanced by TNF-{alpha} in A549 cells transfected with wild-type MMP-9-Luc, which was inhibited by helenalin, U0126, or SP600125. In contrast, TNF-{alpha}-stimulated MMP-9 luciferase activity was totally lost in cells transfected with mutant-NF-{kappa}B MMP-9-luc. Moreover, pretreatment with actinomycin D and cycloheximide attenuated TNF-{alpha}-induced MMP-9 expression. These results suggest that in A549 cells, phosphorylation of p42/p44 MAPK, JNK, and transactivation of NF-{kappa}B are essential for TNF-{alpha}-induced MMP-9 gene expression.« less

  4. Inhibitory effect of phloretin and biochanin A on IgE-mediated allergic responses in rat basophilic leukemia RBL-2H3 cells.

    PubMed

    Chung, Mi Ja; Sohng, Jae Kyung; Choi, Doo Jin; Park, Yong Il

    2013-09-17

    Anti-allergic effects and action mechanism of phloretin (Phl) and biochanin A (BioA) on the IgE-antigen complex-mediated allergic responses in rat basophilic leukemia RBL-2H3 cells were investigated. Cell viability, formation of reactive oxygen species (ROS), DPPH radical-scavenging activity, β-hexosaminidase release, production of interleukin (IL)-4, IL-13, and tumor necrosis factor-alpha (TNF-α) and phosphorylation of Akt and mitogen-activated protein kinase (MAPK) were determined by MTT assay, 2,7-dichlorofluorescein diacetate (DCF-DA) assay, DPPH radical-scavenging assay, reverse transcriptase polymerase chain reaction (RT-PCR), enzyme-linked immunosorbent assay (ELISA) and western blot analysis, respectively. Ph1 and BioA dose-dependently inhibited the formation of ROS and the release of β-hexosaminidase from the RBL-2H3 cells and also showed DPPH radical-scavenging activity. Ph1 and BioA suppressed the antigen-induced phosphorylation of the downstream signaling intermediates, including MAPK and Akt, which are critical for the production of pro-inflammatory cytokines, and also significantly attenuated the production of IgE-mediated pro-inflammatory cytokines, such as IL-4, IL-13, and TNF-α. Phloretin and biochanin A attenuate the degranulation and allergic cytokine production through inhibition of intracellular ROS production and the phosphorylation of Akt and the MAPKs, such as ERK1/2, p38, and JNK. The results of this study suggested that these two plant flavonoids may have potent anti-allergic activity in vitro. © 2013.

  5. Induction of nitric oxide synthase in rat intestine by interleukin-1alpha may explain diarrhea associated with zinc deficiency.

    PubMed

    Cui, L; Takagi, Y; Wasa, M; Iiboshi, Y; Khan, J; Nezu, R; Okada, A

    1997-09-01

    Synthesis of inducible nitric oxide synthase (iNOS) in the intestine may result in local tissue damage. We investigated whether a challenge with interleukin-1alpha could give rise to intestinal iNOS expression and diarrhea in rats of differing zinc status. Weaning male rats were fed a zinc-deficient (ZD) diet (2 mg zinc/kg) for 4 wk to induce zinc deficiency or a zinc-supplemented diet [50.8 mg zinc/kg; controls, including pair-fed (PF ) and ad libitum (AL) consumption groups], and then subcutaneously injected with interleukin-1alpha (2 x 10(7) units/kg body wt). Without the interleukin-1alpha challenge, ZD rats had significantly lower plasma zinc concentration than the other groups. Intestinal metallothionein-1 mRNA abundance was lower in ZD rats than in AL rats. iNOS was expressed in the intestine of ZD rats but not in the others. None of the rats experienced diarrhea during the feeding period. Interleukin-1alpha led to a reduction in plasma zinc concentration, enhancement in intestinal metallothionein-1 mRNA levels, and expression of the intestinal iNOS gene in all groups. However, the abundance of iNOS mRNA was significantly higher in ZD rats than in the other groups. The presence of iNOS protein was demonstrated by immunohistochemical staining in the intestine of ZD rats that had been treated with interleukin-1alpha 12 h earlier. In addition, diarrhea occurred in most of the ZD rats and some of the PF rats but not in AL rats after interleukin-1alpha treatment. We conclude that ZD rats respond to interleukin-1alpha challenge more severely than controls, reflected by a more marked and prolonged iNOS expression and a greater incidence of diarrhea.

  6. Nucleolin Mediates MicroRNA-directed CSF-1 mRNA Deadenylation but Increases Translation of CSF-1 mRNA*

    PubMed Central

    Woo, Ho-Hyung; Baker, Terri; Laszlo, Csaba; Chambers, Setsuko K.

    2013-01-01

    CSF-1 mRNA 3′UTR contains multiple unique motifs, including a common microRNA (miRNA) target in close proximity to a noncanonical G-quadruplex and AU-rich elements (AREs). Using a luciferase reporter system fused to CSF-1 mRNA 3′UTR, disruption of the miRNA target region, G-quadruplex, and AREs together dramatically increased reporter RNA levels, suggesting important roles for these cis-acting regulatory elements in the down-regulation of CSF-1 mRNA. We find that nucleolin, which binds both G-quadruplex and AREs, enhances deadenylation of CSF-1 mRNA, promoting CSF-1 mRNA decay, while having the capacity to increase translation of CSF-1 mRNA. Through interaction with the CSF-1 3′UTR miRNA common target, we find that miR-130a and miR-301a inhibit CSF-1 expression by enhancing mRNA decay. Silencing of nucleolin prevents the miRNA-directed mRNA decay, indicating a requirement for nucleolin in miRNA activity on CSF-1 mRNA. Downstream effects followed by miR-130a and miR-301a inhibition of directed cellular motility of ovarian cancer cells were found to be dependent on nucleolin. The paradoxical effects of nucleolin on miRNA-directed CSF-1 mRNA deadenylation and on translational activation were explored further. The nucleolin protein contains four acidic stretches, four RNA recognition motifs (RRMs), and nine RGG repeats. All three domains in nucleolin regulate CSF-1 mRNA and protein levels. RRMs increase CSF-1 mRNA, whereas the acidic and RGG domains decrease CSF-1 protein levels. This suggests that nucleolin has the capacity to differentially regulate both CSF-1 RNA and protein levels. Our finding that nucleolin interacts with Ago2 indirectly via RNA and with poly(A)-binding protein C (PABPC) directly suggests a nucleolin-Ago2-PABPC complex formation on mRNA. This complex is in keeping with our suggestion that nucleolin may work with PABPC as a double-edged sword on both mRNA deadenylation and translational activation. Our findings underscore the complexity of

  7. Multiple lipopolysaccharide (LPS) injections alter interleukin 6 (IL-6), IL-7, IL-10 and IL-6 and IL-7 receptor mRNA in CNS and spleen.

    PubMed

    Szot, Patricia; Franklin, Allyn; Figlewicz, Dianne P; Beuca, Timothy Petru; Bullock, Kristin; Hansen, Kim; Banks, William A; Raskind, Murray A; Peskind, Elaine R

    2017-07-04

    Neuroinflammation is proposed to be an important component in the development of several central nervous system (CNS) disorders including depression, Alzheimer's disease, Parkinson's disease, and traumatic brain injury. However, exactly how neuroinflammation leads to, or contributes to, these central disorders is unclear. The objective of the study was to examine and compare the expression of mRNAs for interleukin-6 (IL-6), IL-7, IL-10 and the receptors for IL-6 (IL-6R) and IL-7 (IL-7R) using in situ hybridization in discrete brain regions and in the spleen after multiple injections of 3mg/kg lipopolysaccharide (LPS), a model of neuroinflammation. In the spleen, LPS significantly elevated IL-6 mRNA expression, then IL-10 mRNA, with no effect on IL-7 or IL-7R mRNA, while significantly decreasing IL-6R mRNA expression. In the CNS, LPS administration had the greatest effect on IL-6 and IL-6R mRNA. LPS increased IL-6 mRNA expression only in non-neuronal cells throughout the brain, but significantly elevated IL-6R mRNA in neuronal populations, where observed, except the cerebellum. LPS resulted in variable effects on IL-10 mRNA, and had no effect on IL-7 or IL-7R mRNA expression. These studies indicate that LPS-induced neuroinflammation has substantial but variable effects on the regional and cellular patterns of CNS IL-6, IL-7 and IL-10, and for IL-6R and IL-7R mRNA expression. It is apparent that administration of LPS can affect non-neuronal and neuronal cells in the brain. Further research is required to determine how CNS inflammatory changes associated with IL-6, IL-10 and IL-6R could in turn contribute to the development of CNS neurological disorders. Published by Elsevier Ltd.

  8. Protective effect of resveratrol against nigrostriatal pathway injury in striatum via JNK pathway.

    PubMed

    Li, Dan; Liu, Nan; Zhao, Liang; Tong, Lei; Kawano, Hitoshi; Yan, Hong-Jing; Li, Hong-Peng

    2017-01-01

    Nigrostriatal pathway injury is one of the traumatic brain injury models that usually lead to neurological dysfunction or neuron necrosis. Resveratrol-induced benefits have recently been demonstrated in several models of neuronal degeneration diseases. However, the protective properties of resveratrol against neurodegeneration have not been explored definitely. Thus, we employ the nigrostriatal pathway injury model to mimic the insults on the brain. Resveratrol decreased the p-ERK expression and increased the p-JNK expression compared to the DMSO group, but not alter the p38 MAPK proteins around the lesion site by Western blot. Prior to the injury, mice were infused with resveratrol intracerebroventricularly with or without JNK-IN-8, a specific c-JNK pathway inhibitor for JNK1, JNK2 and JNK4. The study assessed modified improved neurological function score (mNSS) and beam/walking test, the level of inflammatory cytokines IL-1β, IL-6 and TNF-α, and striatal expression of Bax and Bcl-2 proteins associated with neuronal apoptosis. The results revealed that resveratrol exerted a neuroprotective effect as shown by the improved mNSS and beam latency, anti-inflammatory effects as indicated by the decreased level of IL-1β, TNF-α and IL-6. Furthermore, resveratrol up-regulated the protein expression of p-JNK and Bcl-2, down-regulated the expression of Bax and the number of Fluoro-Jade C (FJC) positive neurons. However, these advantages of resveratrol were abolished by JNK-IN-8 treatment. Overall, we demonstrated that resveratrol treatment attenuates the nigrostriatal pathway injury-induced neuronal apoptosis and inflammation via activation of c-JNK signaling. Copyright © 2016 Elsevier B.V. All rights reserved.

  9. The Nuclear Pore-Associated TREX-2 Complex Employs Mediator to Regulate Gene Expression

    PubMed Central

    Schneider, Maren; Hellerschmied, Doris; Schubert, Tobias; Amlacher, Stefan; Vinayachandran, Vinesh; Reja, Rohit; Pugh, B. Franklin; Clausen, Tim; Köhler, Alwin

    2015-01-01

    Summary Nuclear pore complexes (NPCs) influence gene expression besides their established function in nuclear transport. The TREX-2 complex localizes to the NPC basket and affects gene-NPC interactions, transcription, and mRNA export. How TREX-2 regulates the gene expression machinery is unknown. Here, we show that TREX-2 interacts with the Mediator complex, an essential regulator of RNA Polymerase (Pol) II. Structural and biochemical studies identify a conserved region on TREX-2, which directly binds the Mediator Med31/Med7N submodule. TREX-2 regulates assembly of Mediator with the Cdk8 kinase and is required for recruitment and site-specific phosphorylation of Pol II. Transcriptome and phenotypic profiling confirm that TREX-2 and Med31 are functionally interdependent at specific genes. TREX-2 additionally uses its Mediator-interacting surface to regulate mRNA export suggesting a mechanism for coupling transcription initiation and early steps of mRNA processing. Our data provide mechanistic insight into how an NPC-associated adaptor complex accesses the core transcription machinery. PMID:26317468

  10. G protein-coupled estrogen receptor inhibits the P2Y receptor-mediated Ca(2+) signaling pathway in human airway epithelia.

    PubMed

    Hao, Yuan; Chow, Alison W; Yip, Wallace C; Li, Chi H; Wan, Tai F; Tong, Benjamin C; Cheung, King H; Chan, Wood Y; Chen, Yangchao; Cheng, Christopher H; Ko, Wing H

    2016-08-01

    P2Y receptor activation causes the release of inflammatory cytokines in the bronchial epithelium, whereas G protein-coupled estrogen receptor (GPER), a novel estrogen (E2) receptor, may play an anti-inflammatory role in this process. We investigated the cellular mechanisms underlying the inhibitory effect of GPER activation on the P2Y receptor-mediated Ca(2+) signaling pathway and cytokine production in airway epithelia. Expression of GPER in primary human bronchial epithelial (HBE) or 16HBE14o- cells was confirmed on both the mRNA and protein levels. Stimulation of HBE or 16HBE14o- cells with E2 or G1, a specific agonist of GPER, attenuated the nucleotide-evoked increases in [Ca(2+)]i, whereas this effect was reversed by G15, a GPER-specific antagonist. G1 inhibited the secretion of two proinflammatory cytokines, interleukin (IL)-6 and IL-8, in cells stimulated by adenosine 5'-(γ-thio)triphosphate (ATPγS). G1 stimulated a real-time increase in cAMP levels in 16HBE14o- cells, which could be inhibited by adenylyl cyclase inhibitors. The inhibitory effects of E2 or G1 on P2Y receptor-induced increases in Ca(2+) were reversed by treating the cells with a protein kinase A (PKA) inhibitor. These results demonstrated that the inhibitory effects of G1 or E2 on P2Y receptor-mediated Ca(2+) mobilization and cytokine secretion were due to GPER-mediated activation of a cAMP-dependent PKA pathway. This study has reported, for the first time, the expression and function of GPER as an anti-inflammatory component in human bronchial epithelia, which may mediate through its opposing effects on the pro-inflammatory pathway activated by the P2Y receptors in inflamed airway epithelia.

  11. RhoA/ROCK signaling regulates smooth muscle phenotypic modulation and vascular remodeling via the JNK pathway and vimentin cytoskeleton.

    PubMed

    Tang, Lian; Dai, Fan; Liu, Yan; Yu, Xiaoqiang; Huang, Chao; Wang, Yuqin; Yao, Wenjuan

    2018-05-20

    The RhoA/ROCK signaling pathway regulates cell morphology, adhesion, proliferation, and migration. In this study, we investigated the regulatory role of RhoA/ROCK signaling on PDGF-BB-mediated smooth muscle phenotypic modulation and vascular remodeling and clarified the molecular mechanisms behind these effects. PDGF-BB treatment induced the activation of RhoA, ROCK, PDGF-Rβ, and the expression of PDGF-Rβ in HA-VSMCs (human aortic vascular smooth muscle cells). PDGF-Rβ inhibition and RhoA suppression blocked PDGF-BB-induced RhoA activation and ROCK induction. In addition, PDGF-BB-mediated cell proliferation and migration were suppressed by PDGF-Rβ inhibition, RhoA suppression, and ROCK inhibition, suggesting that PDGF-BB promotes phenotypic modulation of HA-VSMCs by activating the RhoA/ROCK pathway via the PDGF receptor. Moreover, suppressing both ROCK1 and ROCK2 blocked cell cycle progression from G0/G1 to S phase by decreasing the transcription and protein expression of cyclin D1, CDK2, and CDK4 via JNK/c-Jun pathway, thus reducing cell proliferation in PDGF-BB-treated HA-VSMCs. ROCK1 deletion, rather than ROCK2 suppression, significantly inhibited PDGF-BB-induced migration by reducing the expression of vimentin and preventing the remodeling of vimentin and phospho-vimentin. Furthermore, ROCK1 deletion suppressed vimentin by inhibiting the phosphorylation of Smad2/3 and the nuclear translocation of Smad4. These findings suggested that ROCK1 and ROCK2 might play different roles in PDGF-BB-mediated cell proliferation and migration in HA-VSMCs. In addition, PDGF-BB and its receptor participated in neointima formation and vascular remodeling by promoting cell cycle protein expression via the JNK pathway and enhancing vimentin expression in a rat balloon injury model; effects that were inhibited by treatment with fasudil. Together, the results of this study reveal a novel mechanism through which RhoA/ROCK signaling regulates smooth muscle phenotypic modulation and

  12. The nucleolus as a stress sensor: JNK2 inactivates the transcription factor TIF-IA and down-regulates rRNA synthesis.

    PubMed

    Mayer, Christine; Bierhoff, Holger; Grummt, Ingrid

    2005-04-15

    Cells respond to a variety of extracellular and intracellular forms of stress by down-regulating rRNA synthesis. We have investigated the mechanism underlying stress-dependent inhibition of RNA polymerase I (Pol I) transcription and show that the Pol I-specific transcription factor TIF-IA is inactivated upon stress. Inactivation is due to phosphorylation of TIF-IA by c-Jun N-terminal kinase (JNK) at a single threonine residue (Thr 200). Phosphorylation at Thr 200 impairs the interaction of TIF-IA with Pol I and the TBP-containing factor TIF-IB/SL1, thereby abrogating initiation complex formation. Moreover, TIF-IA is translocated from the nucleolus into the nucleoplasm. Substitution of Thr 200 by valine as well as knock-out of Jnk2 prevent inactivation and translocation of TIF-IA, leading to stress-resistance of Pol I transcription. Our data identify TIF-IA as a downstream target of the JNK pathway and suggest a critical role of JNK2 to protect rRNA synthesis against the harmful consequences of cellular stress.

  13. Nicotine impairs cyclooxygenase-2-dependent kinin-receptor-mediated murine airway relaxations

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

    Xu, Yuan, E-mail: yuan.xu@ki.se; Cardell, Lars-Olaf

    Introduction: Cigarette smoke induces local inflammation and airway hyperreactivity. In asthmatics, it worsens the symptoms and increases the risk for exacerbation. The present study investigates the effects of nicotine on airway relaxations in isolated murine tracheal segments. Methods: Segments were cultured for 24 h in the presence of vehicle, nicotine (10 μM) and/or dexamethasone (1 μM). Airway relaxations were assessed in myographs after pre-contraction with carbachol (1 μM). Kinin receptors, cyclooxygenase (COX) and inflammatory mediator expressions were assessed by real-time PCR and confocal-microscopy-based immunohistochemistry. Results: The organ culture procedure markedly increased bradykinin- (selective B{sub 2} receptor agonist) and des-Arg{sup 9}-bradykinin-more » (selective B{sub 1} receptor agonist) induced relaxations, and slightly increased relaxation induced by isoprenaline, but not that induced by PGE{sub 2}. The kinin receptor mediated relaxations were epithelium-, COX-2- and EP2-receptor-dependent and accompanied by drastically enhanced mRNA levels of kinin receptors, as well as inflammatory mediators MCP-1 and iNOS. Increase in COX-2 and mPGES-1 was verified both at mRNA and protein levels. Nicotine selectively suppressed the organ-culture-enhanced relaxations induced by des-Arg{sup 9}-bradykinin and bradykinin, at the same time reducing mPGES-1 mRNA and protein expressions. α7-nicotinic acetylcholine receptor inhibitors α-bungarotoxin and MG624 both blocked the nicotine effects on kinin B{sub 2} receptors, but not those on B{sub 1}. Dexamethasone completely abolished kinin-induced relaxations. Conclusion: It is tempting to conclude that a local inflammatory process per se could have a bronchoprotective component by increasing COX-2 mediated airway relaxations and that nicotine could impede this safety mechanism. Dexamethasone further reduced airway inflammation together with relaxations. This might contribute to the steroid resistance

  14. The effect of menadione on glutathione S-transferase A1 (GSTA1): c-Jun N-terminal kinase (JNK) complex dissociation in human colonic adenocarcinoma Caco-2 cells.

    PubMed

    Adnan, Humaira; Antenos, Monica; Kirby, Gordon M

    2012-10-02

    Glutathione S-transferases (GSTs) act as modulators of mitogen-activated protein kinase signal transduction pathways via a mechanism involving protein-protein interactions. We have demonstrated that GSTA1 forms complexes with JNK and modifies JNK activation during cellular stress, but the factors that influence complex association and dissociation are unknown. We hypothesized that menadione causes dissociation of GSTA1-JNK complexes, activates JNK, and the consequences of menadione exposure depend on GSTA1 expression. We demonstrate that menadione causes GSTA1-JNK dissociation and JNK activation in preconfluent Caco-2 cells, whereas postconfluent cells are resistant to this effect. Moreover, preconfluent cells are more sensitive than postconfluent cells to menadione-induced cytotoxicity. Activation of JNK is transient since removal of menadione causes GSTA1 to re-associate with JNK reducing cytotoxicity. Over-expression and knockdown of GSTA1 did not alter JNK activation by menadione or sensitivity to menadione-induced cytotoxicity. These results indicate that GSTA1-JNK complex integrity does not affect the ability of menadione to activate JNK. N-acetyl cysteine prevents GSH depletion and blocks menadione-induced complex dissociation, JNK activation and inhibits menadione-induced cytotoxicity. JNK activation and inhibits menadione-induced cytotoxicity. The data suggest that the mechanism of menadione-induced JNK activation involves the production of reactive oxygen species, likely superoxide anion, and intracellular GSH levels play an important role in preventing GSTA1-JNK complex dissociation, subsequent JNK activation and induction of cytotoxicity. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

  15. Autoantibodies to BP180 associated with bullous pemphigoid release interleukin-6 and interleukin-8 from cultured human keratinocytes.

    PubMed

    Schmidt, E; Reimer, S; Kruse, N; Jainta, S; Bröcker, E B; Marinkovich, M P; Giudice, G J; Zillikens, D

    2000-11-01

    Bullous pemphigoid is an inflammatory subepidermal blistering disease that is associated with auto- antibodies to the keratinocyte surface protein, BP180. In addition to the binding of autoantibodies, the infiltration of inflammatory cells is necessary for blister formation. Cytokines, including interleukin-6 and interleukin-8, have been implicated in the disease process of both human and experimental murine bullous pemphigoid. This study was aimed at testing the hypothesis that the binding of anti-BP180 antibodies to their target antigen triggers a signal transduction event that results in the secretion of these pro-inflammatory cytokines. Consistent with this hypothesis, treatment of cultured normal human epidermal keratinocytes with bullous pemphigoid IgG, but not control IgG, led to increased levels of interleukin-6 and interleukin-8, but not interleukin-1alpha, interleukin-1beta, tumor necrosis factor-alpha, interleukin-10, or monocyte chemoattractant protein-1, in the culture medium. This effect was concentration- and time-dependent and was abolished by depleting the bullous pemphigoid IgG of reactivity to two distinct epitopes on the BP180 NC16A domain. Upregulation of interleukin-6 and interleukin-8 was found at both protein and mRNA levels. In addition, bullous pemphigoid IgG did not induce the release of interleukin-6 and interleukin-8 from BP180-deficient keratinocytes obtained from a patient with generalized atrophic benign epidermolysis bullosa. These data indicate that bullous pemphigoid-associated autoantibodies to the human BP180 ectodomain trigger a signal transducing event that leads to expression and secretion of interleukin-6 and interleukin-8 from human keratinocytes.

  16. Role of JNK isoforms in the kainic acid experimental model of epilepsy and neurodegeneration.

    PubMed

    Auladell, Carme; de Lemos, Luisa; Verdaguer, Ester; Ettcheto, Miren; Busquets, Oriol; Lazarowski, Alberto; Beas-Zarate, Carlos; Olloquequi, Jordi; Folch, Jaume; Camins, Antoni

    2017-01-01

    Chemoconvulsants that induce status epilepticus in rodents have been widely used over the past decades due to their capacity to reproduce with high similarity neuropathological and electroencephalographic features observed in patients with temporal lobe epilepsy (TLE). Kainic acid  is one of the most used chemoconvulsants in experimental models. KA administration mainly induces neuronal loss in the hippocampus. We focused the present review inthe c-Jun N-terminal kinase-signaling pathway (JNK), since it has been shown to play a key role in the process of neuronal death following KA activation. Among the three isoforms of JNK (JNK1, JNK2, JNK3), JNK3 is widely localized in the majority of areas of the hippocampus, whereas JNK1 levels are located exclusively in the CA3 and CA4 areas and in dentate gyrus. Disruption of the gene encoding JNK3 in mice renders neuroprotection to KA, since these animals showed a reduction in seizure activity and a diminution in hippocampal neuronal apoptosis. In light of this, JNK3 could be a promising subcellular target for future therapeutic interventions in epilepsy.

  17. Identification of interleukin-1 beta as a key mediator in the upregulation of Cav3.2–USP5 interactions in the pain pathway

    PubMed Central

    Stemkowski, Patrick L; Garcia-Caballero, Agustin; Gadotti, Vinicius M; M’Dahoma, Said; Chen, Lina; Souza, Ivana A

    2017-01-01

    We recently reported that nerve injury or peripheral inflammation triggers an upregulation of the deubiquitinase, USP5 in mouse dorsal root ganglion and spinal dorsal horn. This leads to dysregulated ubiquitination of Cav3.2 T-type calcium channels, thus increasing Cav3.2 channel plasma membrane expression and nociceptive signaling in the primary afferent pain pathway. This phenomenon could be recapitulated by noninvasive, optogenetic activation of transient receptor potential vanilloid-1–expressing nociceptors, indicating that neuronal activity is a key player in this process. Given the relevance of the pro-inflammatory cytokine interleukin-1 beta in many forms of pathological pain, we hypothesized that interleukin-1 beta may be a critical cofactor required to drive upregulation of interactions between USP5 and Cav3.2 channels. Here, we report that gene expression, as well as protein levels for interleukin-1 beta and the endogenous interleukin-1 receptor-I antagonist, IL-1Ra are unaltered following conditioning stimulation of optogenetically targeted cutaneous nociceptors, indicating that neuronal activity is not a driver of interleukin-1 beta signaling. In contrast, co-immunoprecipitation experiments revealed that intrathecal administration of interleukin-1 beta in wild-type mice led to an increase in the interaction between USP5 and Cav3.2 in the spinal dorsal horn. Moreover, disruption of the interaction between USP5 and Cav3.2 with TAT peptides suppressed acute nocifensive responses produced by interleukin-1 beta, which was similar to that achieved by elimination of T-type channel activity with the channel blockers, mibefradil, or TTA-A2. Finally, this upregulation could be maintained in dorsal root ganglion neuron cultures exposed overnight to interleukin-1 beta, while the copresence of interleukin-1 receptor antagonist or the dampening of neuronal cell activity with tetrodotoxin attenuated this response. Altogether, our findings identify interleukin-1 beta

  18. JNK: bridging the insulin signaling and inflammatory pathway.

    PubMed

    Liu, Gang; Rondinone, Cristina M

    2005-10-01

    Obesity and insulin resistance are strongly associated with systemic markers of inflammation and endoplasmic reticulum stress. c-Jun N-terminal kinases (JNK) are activated by inflammatory cytokines and have a key role in beta-cell apoptosis and in negative regulation of insulin signaling. JNK1-deficient mice are protected from diet-induced obesity and insulin resistance, while genetically obese mice with targeted mutations in JNK1 are leaner and have reduced insulin and blood glucose levels. These studies validate JNK as a link between inflammation and metabolic diseases and as a promising drug target. This review highlights recent advances in small-molecule inhibitors of JNK that have also been targeted for other diseases with an inflammatory component such as stroke, rheumatoid arthritis, and Alzheimer's and Parkinson's diseases.

  19. WISP-2 in human gastric cancer and its potential metastatic suppressor role in gastric cancer cells mediated by JNK and PLC-γ pathways.

    PubMed

    Ji, Jiafu; Jia, Shuqin; Jia, Yongning; Ji, Ke; Hargest, Rachel; Jiang, Wen G

    2015-09-15

    It has recently been shown that WISP proteins (Wnt-inducted secreted proteins), a group of intra- and extra-cellular regulatory proteins, have been implicated in the initiation and progression of a variety of tumour types including colorectal and breast cancer. However, the role of WISP proteins in gastric cancer (GC) cells and their clinical implications have not yet been elucidated. The expression of WISP molecules in a cohort of GC patients was analysed using real-time quantitative PCR and immunohistochemistry. The expression of a panel of recognised epithelial-mesenchymal transition (EMT) markers was quantified using Q-PCR in paired tumour and normal tissues. WISP-2 knockdown (kd) sublines using ribozyme transgenes were created in the GC cell lines AGS and HGC27. Subsequently, several biological functions, including cell growth, adhesion, migration and invasion, were studied. Potential pathways for the interaction of EMT, extracellular matrix and MMP were evaluated. Overexpression of WISP-2 was detected in GC and significantly correlated with early tumour node-metastasis staging, differentiation status and positively correlated with overall survival and disease-free survival of the patients. WISP-2 expression was inversely correlated with that of Twist and Slug in paired samples. Kd of WISP-2 expression promoted the proliferation, migration and invasion of GC cells. WISP-2 suppressed GC cell metastasis through reversing EMT and suppressing the expression and activity of MMP9 and MMP2 via JNK and ERK. Cell motility analysis indicated that WISP-2 kd contributed to GC cells' motility and can be attenuated by PLC-γ and JNK small inhibitors. Increased expression of WISP-2 in GC is positively correlated with favourable clinical features and the survival of patients with GC and is a negative regulator of growth, migration and invasion in GC cells. These findings suggest that WISP-2 is a potential tumour suppressor in GC.

  20. [Over-expressed Bax inhibitor 1 (BI-1) inhibits apoptosis of hippocampal neurons via endoplasmic reticulum IRE1-JNK pathway in rats with subarachnoid hemorrhage].

    PubMed

    Liu, Jiaxin; Zhou, Shuai; Qian, Xiying; Zhang, Yueting; Zhao, Jianhua

    2017-10-01

    Objective To investigate the protective effect of lentivirus-mediated BI-1 overexpression on hippocampal neurons in rats with subarachnoid hemorrhage (SAH) and the relationship with endoplasmic reticulum IRE1-JNK signaling pathway. Methods The lentivirus solution of BI-1 over-expression was injected into the brain of rats 24 hours before SAH rat model was established by intravascular puncture method. At 24 hours after modeling, the brain water content and neurological score of the rats were measured. The apoptosis of hippocampal neurons was detected by TUNEL assay. Western blotting was used to detect the expressions of BI-1 protein and endoplasmic reticulum stress (ERS) marker proteins GRP78 and IRE1. ERS in hippocampal neurons of the rats with SAH was intervened by IRE1α-specific inhibitor KIRA6, and then the protein expressions of p-IRE1, p-JNK, Bax, Bcl2 and caspase-3 were detected by Western blotting. Results BI-1 over-expression improved neurobehavioral score, decreased brain water content and hippocampal neuron apoptosis rate, and also down-regulated GRP78 and IRE1 protein levels in the rats with SAH. Both the interference of KIRA6 and the over-expression of BI-1 inhibited the expressions of p-IRE1, p-JNK, Bax and caspase-3, and promoted the expression of anti-apoptotic protein Bcl2. Conclusion Over-expression of BI-1 can inhibit the apoptosis of hippocampal neurons in rats with SAH by inhibiting the activation of ERS-mediated IRE1-JNK signaling pathway, thus ultimately attenuating the early brain injury following SAH.

  1. Endophilin-1 regulates blood-brain barrier permeability via EGFR-JNK signaling pathway.

    PubMed

    Chen, Lin; Liu, Wenjing; Wang, Ping; Xue, Yixue; Su, Qingjie; Zeng, Chaosheng; Shang, Xiuli

    2015-05-05

    Endophilin-1 (Endo1), a multifunctional protein, is essential for synaptic vesicle endocytosis. However, the role and mechanism of endophilin-1 in blood-brain barrier (BBB) function are still unclear. This study was performed to determine whether endophilin-1 regulated BBB permeability via the EGFR-JNK signaling pathway. In the present study, we found that endophilin-1 over-expression in human cerebral microvascular endothelial cell (hCMEC/D3) increased BBB permeability and meanwhile reduced the expression levels of epidermal growth factor receptor (EGFR), phosphorylated c-Jun N-terminal kinase (p-JNK). While endophilin-1 knockdown led to the contrary results. After JNK inhibitor SP600125 was administered to the endophilin-1 silenced hCMEC/D3 cells, the transendothelial electrical resistance (TEER) value was decreased and the permeability coefficient values to 4kDa and 40kDa FITC-dextran were increased. Results observed by Transmission electron microscopy (TEM) showed that tight junctions (TJs) were opened. Moreover, immunofluorescence and Western blot assays revealed the discontinuous distribution of TJ-associated proteins ZO-1, occludin on cell-cell boundaries and a significant decrease in protein expressing levels. Therefore, these results indicated that endophilin-1 positively regulated BBB permeability via the EGFR-JNK signaling pathway in hCMEC/D3 cells, which would provide an experimental basis for further research on endophilin-1 mediated the opening of BBB. Copyright © 2015 Elsevier B.V. All rights reserved.

  2. Circulating interleukin-6 induces fever through a STAT3-linked activation of COX-2 in the brain.

    PubMed

    Rummel, Christoph; Sachot, Christelle; Poole, Stephen; Luheshi, Giamal N

    2006-11-01

    Interleukin (IL)-6 is an important humoral mediator of fever following infection and inflammation and satisfies a number of criteria for a circulating pyrogen. However, evidence supporting such a role is diminished by the moderate or even absent ability of the recombinant protein to induce fever and activate the cyclooxygenase-2 (COX-2) pathway in the brain, a prerequisite step in the initiation and maintenance of fever. In the present study, we investigated the role of endogenous circulating IL-6 in a rodent model of localized inflammation, by neutralizing its action using a specific antiserum (IL-6AS). Rats were injected with LPS (100 microg/kg) or saline into a preformed air pouch in combination with an intraperitoneal injection of either normal sheep serum or IL-6AS (1.8 ml/rat). LPS induced a febrile response, which was accompanied by a significant rise in plasma IL-6 and nuclear STAT3 translocation in endothelial cells throughout the brain 2 h after treatment, including areas surrounding the sensory circumventricular organs and the median preoptic area (MnPO), important regions in mediating fever. These responses were abolished in the presence of the IL-6AS, which also significantly inhibited the LPS-induced upregulation of mRNA expression or immunoreactivity (IR) of the inducible form of COX, the rate-limiting enzyme for PGE2-synthesis. Interestingly, nuclear signal transducer and activator of transcription (STAT)3-positive cells colocalized with COX-2-IR, signifying that IL-6-activated cells are directly involved in PGE2 production. These observations suggest that IL-6 is an important circulating pyrogen that activates the COX-2-pathway in cerebral microvasculature, most likely through a STAT3-dependent pathway.

  3. A Fat-Facets-Dscam1-JNK Pathway Enhances Axonal Growth in Development and after Injury

    PubMed Central

    Koch, Marta; Nicolas, Maya; Zschaetzsch, Marlen; de Geest, Natalie; Claeys, Annelies; Yan, Jiekun; Morgan, Matthew J.; Erfurth, Maria-Luise; Holt, Matthew; Schmucker, Dietmar; Hassan, Bassem A.

    2018-01-01

    Injury to the adult central nervous systems (CNS) can result in severe long-term disability because damaged CNS connections fail to regenerate after trauma. Identification of regulators that enhance the intrinsic growth capacity of severed axons is a first step to restore function. Here, we conducted a gain-of-function genetic screen in Drosophila to identify strong inducers of axonal growth after injury. We focus on a novel axis the Down Syndrome Cell Adhesion Molecule (Dscam1), the de-ubiquitinating enzyme Fat Facets (Faf)/Usp9x and the Jun N-Terminal Kinase (JNK) pathway transcription factor Kayak (Kay)/Fos. Genetic and biochemical analyses link these genes in a common signaling pathway whereby Faf stabilizes Dscam1 protein levels, by acting on the 3′-UTR of its mRNA, and Dscam1 acts upstream of the growth-promoting JNK signal. The mammalian homolog of Faf, Usp9x/FAM, shares both the regenerative and Dscam1 stabilizing activities, suggesting a conserved mechanism. PMID:29472843

  4. Lymphotoxin β receptor activation promotes mRNA expression of RelA and pro-inflammatory cytokines TNFα and IL-1β in bladder cancer cells.

    PubMed

    Shen, Mo; Zhou, Lianlian; Zhou, Ping; Zhou, Wu; Lin, Xiangyang

    2017-07-01

    The role of inflammation in tumorigenesis and development is currently well established. Lymphotoxin β receptor (LTβR) activation induces canonical and noncanonical nuclear factor (NF)‑κB signaling pathways, which are linked to inflammation‑induced carcinogenesis. In the present study, 5,637 bladder cancer cells were cultured and the activation of LTβR was induced by functional ligand, lymphotoxin (LT) α1β2, and silencing with shRNA. Reverse transcription‑quantitative polymerase chain reaction was utilized to detect the mRNA expression levels of NF‑κB family members RelA and RelB, cytokines including LTα, LTβ, tumor necrosis factor (TNF)α, TNF superfamily member 14, interleukin (IL)‑6 and IL‑1β, and proliferation‑related genes including CyclinD1 and Survivin. The expression of phospho‑p65 was determined by western blotting. Activation of LTβR on bladder cancer 5,637 cells was demonstrated to upregulate the mRNA expression levels of the RELA proto‑oncogene, RelA, by 2.5‑fold compared with unstimulated cells, while no significant change was observed in the RELB proto‑oncogene NF‑κB member mRNA levels. Expression of pro‑inflammatory cytokines tumor necrosis factor (TNF)α and interleukin (IL)‑1β mRNA levels were significantly increased nearly 5‑fold and 1.5‑fold, respectively, following LTβR activation compared with unstimulated cells. The LTβR‑induced upregulation of RelA, TNFα and IL‑1β was decreased by ~33, 27, and 26% respectively when LTβR was silenced via short hairpin RNA. Activation of LTβR had no effect on 5,637 cell growth, despite CyclinD1 and Survivin mRNA levels increasing by ~2.7 and 1.3‑fold, respectively, compared with unstimulated cells. In conclusion, activation of LTβR induced the expression of RelA mRNA levels. LTβR activation might be an important mediator in promoting an inflammatory microenvironment in bladder cancer, via the upregulation of TNFα and IL‑1β mRNA levels. LTβR may

  5. Inhibition of JNK Sensitizes Hypoxic Colon Cancer Cells to DNA Damaging Agents

    PubMed Central

    Vasilevskaya, Irina A.; Selvakumaran, Muthu; Hierro, Lucia Cabal; Goldstein, Sara R.; Winkler, Jeffrey D.; O'Dwyer, Peter J.

    2015-01-01

    Purpose We showed previously that in HT29 colon cancer cells, modulation of hypoxia-induced stress signaling affects oxaliplatin cytotoxicity. To further study the significance of hypoxia-induced signaling through JNK, we set out to investigate how modulation of kinase activities influences cellular responses of hypoxic colon cancer cells to cytotoxic drugs. Experimental design In a panel of cell lines we investigated effects of pharmacological and molecular inhibition of JNK on sensitivity to oxaliplatin, SN-38 and 5-FU. Combination studies for the drugs and JNK inhibitor CC-401 were carried out in vitro and in vivo. Results Hypoxia-induced JNK activation was associated with resistance to oxaliplatin. CC-401 in combination with chemotherapy demonstrates synergism in colon cancer cell lines, though synergy is not always hypoxia-specific. A more detailed analysis focused on HT29 and SW620 (responsive), and HCT116 (non-responsive) lines. In HT29 and SW620 cells CC-401 treatment results in greater DNA damage in the sensitive cells. In vivo, potentiation of bevacizumab, oxaliplatin, and the combination by JNK inhibition was confirmed in HT29-derived mouse xenografts, where tumor growth delay was greater in the presence of CC-401. Finally, stable introduction of a dominant negative JNK1, but not JNK2, construct into HT29 cells rendered them more sensitive to oxaliplatin under hypoxia, suggesting differing input of JNK isoforms in cellular responses to chemotherapy. Conclusions These findings demonstrate that signaling through JNK is a determinant of response to therapy in colon cancer models, and support the testing of JNK inhibition to sensitize colon tumors in the clinic. PMID:26023085

  6. Growth Suppression and Therapy Sensitization of Breast Cancer

    DTIC Science & Technology

    1999-07-01

    northern analysis, the same membrane was hybridized successively with JNKI probe, JNK2 probe and the G3PDH probe. Figure 10. A549 cells were treated with the...90 0 00 z SO E 40 04. z 20 to JNK2 Oligonucleotide n* 47 JNK1AS TREATED 2 JNlK2AS TREATED. 0) c a 0 mRNA mRNA JNK1 J&,I ~R JNK1 JNK2 JNK2 G3PDH SI... G3PDH 48 iil vý 0 z z Z O! zz C13~ A.LOR 496 Reference #28 PREFERENTIAL PLATINATION OF AN ACTIVATED CELLULAR PROMOTER BY CIS-DIAMMINEDICHLOROPLATINUM

  7. AIP1 recruits phosphatase PP2A to ASK1 in tumor necrosis factor-induced ASK1-JNK activation.

    PubMed

    Min, Wang; Lin, Yan; Tang, Shibo; Yu, Luyang; Zhang, Haifeng; Wan, Ting; Luhn, Tricia; Fu, Haian; Chen, Hong

    2008-04-11

    Previously we have shown that AIP1 (apoptosis signal-regulating kinase [ASK]1-interacting protein 1), a novel member of the Ras-GAP protein family, facilitates dephosphorylation of ASK1 at pSer967 and subsequently 14-3-3 release from ASK1, leading to enhanced ASK1-JNK signaling. However, the phosphatase(s) responsible for ASK1 dephosphorylation at pSer967 has not been identified. In the present study, we identified protein phosphatase (PP)2A as a potential phosphatase in vascular endothelial cells (ECs). Tumor necrosis factor (TNF)-induced dephosphorylation of ASK1 pSer967 in ECs was blocked by PP2A inhibitor okadaic acid. Overexpression of PP2A catalytic subunit induced dephosphorylation of ASK1 pSer967 and activation of c-Jun N-terminal kinase (JNK). In contrast, a catalytic inactive form of PP2A or PP2A small interfering RNA blunted TNF-induced dephosphorylation of ASK1 pSer967 and activation of JNK without effects on NF-kappaB activation. Whereas AIP1, via its C2 domain, binds to ASK1, PP2A binds to the GAP domain of AIP1. Endogenous AIP1-PP2A complex can be detected in the resting state, and TNF induces a complex formation of AIP1-PP2A with ASK1. Furthermore, TNF-induced association of PP2A with ASK1 was diminished in AIP1-knockdown ECs, suggesting a critical role of AIP1 in recruiting PP2A to ASK1. TNF-signaling molecules TRAF2 and RIP1, known to be in complex with AIP1 and activate AIP1 by phosphorylating AIP1 at Ser604, are critical for TNF-induced ASK1 dephosphorylation. Finally, PP2A and AIP1 cooperatively induce activation of ASK1-JNK signaling and EC apoptosis, as demonstrated by both overexpression and small interfering RNA knockdown approaches. Taken together, our data support a critical role of PP2A-AIP1 complex in TNF-induced activation of ASK1-JNK apoptotic signaling.

  8. A ginseng metabolite, compound K, induces autophagy and apoptosis via generation of reactive oxygen species and activation of JNK in human colon cancer cells

    PubMed Central

    Kim, A D; Kang, K A; Kim, H S; Kim, D H; Choi, Y H; Lee, S J; Kim, H S; Hyun, J W

    2013-01-01

    Compound K (20-O-(β-D-glucopyranosyl)-20(S)-protopanaxadiol) is an active metabolite of ginsenosides and induces apoptosis in various types of cancer cells. This study investigated the role of autophagy in compound K-induced cell death of human HCT-116 colon cancer cells. Compound K activated an autophagy pathway characterized by the accumulation of vesicles, the increased positive acridine orange-stained cells, the accumulation of LC3-II, and the elevation of autophagic flux. Whereas blockade of compound K-induced autophagy by 3-methyladenein and bafilomycin A1 significantly increased cell viability. In addition, compound K augmented the time-dependent expression of the autophagy-related proteins Atg5, Atg6, and Atg7. However, knockdown of Atg5, Atg6, and Atg7 markedly inhibited the detrimental impact of compound K on LC3-II accumulation and cell vitality. Compound K-provoked autophagy was also linked to the generation of intracellular reactive oxygen species (ROS); both of these processes were mitigated by the pre-treatment of cells with the antioxidant N-acetylcysteine. Moreover, compound K activated the c-Jun NH2-terminal kinase (JNK) signaling pathway, whereas downregulation of JNK by its specific inhibitor SP600125 or by small interfering RNA against JNK attenuated autophagy-mediated cell death in response to compound K. Compound K also provoked apoptosis, as evidenced by an increased number of apoptotic bodies and sub-G1 hypodiploid cells, enhanced activation of caspase-3 and caspase-9, and modulation of Bcl-2 and Bcl-2-associated X protein expression. Notably, compound K-stimulated autophagy as well as apoptosis was induced by disrupting the interaction between Atg6 and Bcl-2. Taken together, these results indicate that the induction of autophagy and apoptosis by compound K is mediated through ROS generation and JNK activation in human colon cancer cells. PMID:23907464

  9. LC3-mediated fibronectin mRNA translation induces fibrosarcoma growth by increasing connective tissue growth factor

    PubMed Central

    Ying, Lihua; Lau, Agatha; Alvira, Cristina M.; West, Robert; Cann, Gordon M.; Zhou, Bin; Kinnear, Caroline; Jan, Eric; Sarnow, Peter; Van de Rijn, Matt; Rabinovitch, Marlene

    2009-01-01

    Summary Previously, we related fibronectin (Fn1) mRNA translation to an interaction between an AU-rich element in the Fn1 3′ UTR and light chain 3 (LC3) of microtubule-associated proteins 1A and 1B. Since human fibrosarcoma (HT1080) cells produce little fibronectin and LC3, we used these cells to investigate how LC3-mediated Fn1 mRNA translation might alter tumor growth. Transfection of HT1080 cells with LC3 enhanced fibronectin mRNA translation. Using polysome analysis and RNA-binding assays, we show that elevated levels of translation depend on an interaction between a triple arginine motif in LC3 and the AU-rich element in Fn1 mRNA. Wild-type but not mutant LC3 accelerated HT1080 cell growth in culture and when implanted in SCID mice. Comparison of WT LC3 with vector-transfected HT1080 cells revealed increased fibronectin-dependent proliferation, adhesion and invasion. Microarray analysis of genes differentially expressed in WT and vector-transfected control cells indicated enhanced expression of connective tissue growth factor (CTGF). Using siRNA, we show that enhanced expression of CTGF is fibronectin dependent and that LC3-mediated adhesion, invasion and proliferation are CTGF dependent. Expression profiling of soft tissue tumors revealed increased expression of both LC3 and CTGF in some locally invasive tumor types. PMID:19366727

  10. Identification of a second murine interleukin-11 receptor alpha-chain gene (IL11Ra2) with a restricted pattern of expression.

    PubMed

    Robb, L; Hilton, D J; Brook-Carter, P T; Begley, C G

    1997-03-15

    The interleukin-11 receptor alpha-chain, a member of the hematopoietin receptor superfamily, forms, together with gp130, a functional high-affinity receptor complex for interleukin 11. We, and others, reported the cloning of the murine interleukin 11 receptor alpha-chain cDNA (IL11Ra) and recently described the structure of the IL11Ra locus. We also described the presence of a second IL11Ra-like locus in some mouse strains. In this study we report that the second locus, designated IL11Ra2, encodes an mRNA species. The transcript was 99% identical to the IL11Ra transcript in the coding and 3'-untranslated region, but had a different 5'-untranslated region. The complete genomic organization of the IL11Ra2 locus is presented, and the two loci are shown to be located on a 200-kb NaeI genomic fragment. Comparison of the expression pattern of the IL11Ra and IL11Ra2 genes using an RT-PCR restriction fragment length polymorphism strategy revealed that while the expression of IL11Ra was widespread, expression of IL11Ra2 was restricted to testis, lymph node, and thymus.

  11. Alternative Polyadenylation and Nonsense-Mediated Decay Coordinately Regulate the Human HFE mRNA Levels

    PubMed Central

    Martins, Rute; Proença, Daniela; Silva, Bruno; Barbosa, Cristina; Silva, Ana Luísa; Faustino, Paula; Romão, Luísa

    2012-01-01

    Nonsense-mediated decay (NMD) is an mRNA surveillance pathway that selectively recognizes and degrades defective mRNAs carrying premature translation-termination codons. However, several studies have shown that NMD also targets physiological transcripts that encode full-length proteins, modulating their expression. Indeed, some features of physiological mRNAs can render them NMD-sensitive. Human HFE is a MHC class I protein mainly expressed in the liver that, when mutated, can cause hereditary hemochromatosis, a common genetic disorder of iron metabolism. The HFE gene structure comprises seven exons; although the sixth exon is 1056 base pairs (bp) long, only the first 41 bp encode for amino acids. Thus, the remaining downstream 1015 bp sequence corresponds to the HFE 3′ untranslated region (UTR), along with exon seven. Therefore, this 3′ UTR encompasses an exon/exon junction, a feature that can make the corresponding physiological transcript NMD-sensitive. Here, we demonstrate that in UPF1-depleted or in cycloheximide-treated HeLa and HepG2 cells the HFE transcripts are clearly upregulated, meaning that the physiological HFE mRNA is in fact an NMD-target. This role of NMD in controlling the HFE expression levels was further confirmed in HeLa cells transiently expressing the HFE human gene. Besides, we show, by 3′-RACE analysis in several human tissues that HFE mRNA expression results from alternative cleavage and polyadenylation at four different sites – two were previously described and two are novel polyadenylation sites: one located at exon six, which confers NMD-resistance to the corresponding transcripts, and another located at exon seven. In addition, we show that the amount of HFE mRNA isoforms resulting from cleavage and polyadenylation at exon seven, although present in both cell lines, is higher in HepG2 cells. These results reveal that NMD and alternative polyadenylation may act coordinately to control HFE mRNA levels, possibly varying its

  12. Targeting p53 via JNK pathway: a novel role of RITA for apoptotic signaling in multiple myeloma.

    PubMed

    Saha, Manujendra N; Jiang, Hua; Yang, Yijun; Zhu, Xiaoyun; Wang, Xiaoming; Schimmer, Aaron D; Qiu, Lugui; Chang, Hong

    2012-01-01

    The low frequency of p53 alterations e.g., mutations/deletions (∼10%) in multiple myeloma (MM) makes this tumor type an ideal candidate for p53-targeted therapies. RITA is a small molecule which can induce apoptosis in tumor cells by activating the p53 pathway. We previously showed that RITA strongly activates p53 while selectively inhibiting growth of MM cells without inducing genotoxicity, indicating its potential as a drug lead for p53-targeted therapy in MM. However, the molecular mechanisms underlying the pro-apoptotic effect of RITA are largely undefined. Gene expression analysis by microarray identified a significant number of differentially expressed genes associated with stress response including c-Jun N-terminal kinase (JNK) signaling pathway. By Western blot analysis we further confirmed that RITA induced activation of p53 in conjunction with up-regulation of phosphorylated ASK-1, MKK-4 and c-Jun. These results suggest that RITA induced the activation of JNK signaling. Chromatin immunoprecipitation (ChIP) analysis showed that activated c-Jun binds to the activator protein-1 (AP-1) binding site of the p53 promoter region. Disruption of the JNK signal pathway by small interfering RNA (siRNA) against JNK or JNK specific inhibitor, SP-600125 inhibited the activation of p53 and attenuated apoptosis induced by RITA in myeloma cells carrying wild type p53. On the other hand, p53 transcriptional inhibitor, PFT-α or p53 siRNA not only inhibited the activation of p53 transcriptional targets but also blocked the activation of c-Jun suggesting the presence of a positive feedback loop between p53 and JNK. In addition, RITA in combination with dexamethasone, known as a JNK activator, displays synergistic cytotoxic responses in MM cell lines and patient samples. Our study unveils a previously undescribed mechanism of RITA-induced p53-mediated apoptosis through JNK signaling pathway and provides the rationale for combination of p53 activating drugs with JNK

  13. Targeting p53 via JNK Pathway: A Novel Role of RITA for Apoptotic Signaling in Multiple Myeloma

    PubMed Central

    Saha, Manujendra N.; Jiang, Hua; Yang, Yijun; Zhu, Xiaoyun; Wang, Xiaoming; Schimmer, Aaron D.; Qiu, Lugui; Chang, Hong

    2012-01-01

    The low frequency of p53 alterations e.g., mutations/deletions (∼10%) in multiple myeloma (MM) makes this tumor type an ideal candidate for p53-targeted therapies. RITA is a small molecule which can induce apoptosis in tumor cells by activating the p53 pathway. We previously showed that RITA strongly activates p53 while selectively inhibiting growth of MM cells without inducing genotoxicity, indicating its potential as a drug lead for p53-targeted therapy in MM. However, the molecular mechanisms underlying the pro-apoptotic effect of RITA are largely undefined. Gene expression analysis by microarray identified a significant number of differentially expressed genes associated with stress response including c-Jun N-terminal kinase (JNK) signaling pathway. By Western blot analysis we further confirmed that RITA induced activation of p53 in conjunction with up-regulation of phosphorylated ASK-1, MKK-4 and c-Jun. These results suggest that RITA induced the activation of JNK signaling. Chromatin immunoprecipitation (ChIP) analysis showed that activated c-Jun binds to the activator protein-1 (AP-1) binding site of the p53 promoter region. Disruption of the JNK signal pathway by small interfering RNA (siRNA) against JNK or JNK specific inhibitor, SP-600125 inhibited the activation of p53 and attenuated apoptosis induced by RITA in myeloma cells carrying wild type p53. On the other hand, p53 transcriptional inhibitor, PFT-α or p53 siRNA not only inhibited the activation of p53 transcriptional targets but also blocked the activation of c-Jun suggesting the presence of a positive feedback loop between p53 and JNK. In addition, RITA in combination with dexamethasone, known as a JNK activator, displays synergistic cytotoxic responses in MM cell lines and patient samples. Our study unveils a previously undescribed mechanism of RITA-induced p53-mediated apoptosis through JNK signaling pathway and provides the rationale for combination of p53 activating drugs with JNK

  14. 1,25-Dihydroxyvitamin D3 attenuates endotoxin-induced production of inflammatory mediators by inhibiting MAPK activation in primary cortical neuron-glia cultures.

    PubMed

    Huang, Ya-Ni; Ho, Yi-Jung; Lai, Chien-Cheng; Chiu, Chien-Tsai; Wang, Jia-Yi

    2015-08-12

    Neuroinflammation occurs in insulted regions of the brain and may be due to reactive oxygen species (ROS), nitric oxide (NO), cytokines, and chemokines produced by activated glia. Excessive production of neurotoxic molecules causes further neuronal damage. Low levels of vitamin D3 are a risk factor for various brain diseases. Using the bacterial endotoxin, lipopolysaccharide (LPS), to induce neuroinflammation in primary cortical neuron-glia cultures, we investigated how 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) affected neuroinflammation. LPS (100 ng/ml) induced the accumulation of nitrite and the production of ROS, interleukin (IL)-6, and macrophage inflammatory protein (MIP)-2 in time-dependent manners. Inhibition of p38 and extracellular signal-regulated kinase (ERK) but not c-Jun N-terminal kinase (JNK) mitogen-activated protein kinase (MAPK) by 20 μM of SB203580, PD98059, and SP600125, significantly reduced LPS-induced ROS production, NO accumulation, and inducible NO synthase (iNOS) expression, respectively. LPS-induced IL-6 and MIP-2 were significantly attenuated by inhibition of p38, ERK, and JNK MAPK. Cotreatment with 1,25(OH)2D3 attenuated LPS-induced ROS production, NO accumulation, and iNOS expression in concentration-dependent manners. 1,25(OH)2D3 also reduced LPS-induced production of IL-6 and MIP-2. Similarly, iNOS, IL-6, and MIP-2 mRNA expression in cells treated with LPS significantly increased, whereas this effect was attenuated by 1,25(OH)2D3. Moreover, LPS-induced phosphorylation of p38, ERK, and JNK MAPK was significantly inhibited by 1,25(OH)2D3. Our findings indicate that 1,25(OH)2D3 reduced the LPS-stimulated production of inflammatory molecules in neuron-glia cultures by inhibiting MAPK pathways and the production of downstream inflammatory molecules. We suggest that 1,25(OH)2D3 can be used to alleviate neuroinflammation in various brain injuries.

  15. Coal-induced interleukin-6 gene expression is mediated through ERKs and p38 MAPK pathways.

    PubMed

    Huang, X; Zhang, Q

    2003-08-15

    In the present study, we have tested the ability of coal dust to stimulate kinase phosphorylation of activator protein-1 (AP-1) signal transduction pathways and production of interleukin-6 (IL-6) in both mouse epidermal JB6 and human lung epithelial A549 cells. Seven coal samples from three coalmine regions of Pennsylvania (PA), West Virginia (WV), and Utah (UT) with high, medium, and low prevalence of coal workers' pneumoconiosis (CWP), respectively, were investigated. Results from the present study indicate that three PA coals stimulated the mitogen-activated protein kinase (MAPK) family of extracellular signal-regulated kinases (ERKs) and p38 MAPK, but not c-Jun-NH2-terminal kinases (JNKs) in human lung A549 cells. The effects of three UT coals on the kinase phosphorylation were less as compared to those of the PA coals. Coal dusts from three coalmine regions induced IL-6 in a dose-dependent manner in both JB6 and A549 cells. Interestingly, levels of IL-6 in both cells treated with coals from three coalmine regions correlated well with CWP prevalence from that region. To assess the role of AP-1 pathways in coal-mediated transcriptional activation of IL-6, various inhibitors were used in cells treated with one PA coal, which induced a maximal response. It was found that the increase in IL-6 protein and mRNA by the PA coal was completely eliminated by the pretreatment of both cell types with PD98059, a specific MEK1 inhibitor, and SB202190, a p38 kinase inhibitor. Our results indicate that coal dust can stimulate IL-6 release from mouse epidermal JB6 cells and human lung epithelial A549 cells, and the coal-induced IL-6 increase may involve ERKs and p38 MAPK pathways.

  16. Nonsense-mediated mRNA decay factor Upf2 exists in both the nucleoplasm and the cytoplasm

    PubMed Central

    TATSUNO, TAKANORI; NAKAMURA, YUKA; MA, SHAOFU; TOMOSUGI, NAOHISA; ISHIGAKI, YASUHITO

    2016-01-01

    Upf2 protein predominantly localizes to the cytoplasmic fraction, and binds to the exon junction complex (EJC) on spliced mRNA. The present study aimed to determine the cellular site where the interaction between Upf2 and EJC occurs. First, the cell lysate was fractionated into the cytoplasm and nucleoplasm, and western blotting to detect levels of Upf2 protein was performed. Upf2 was clearly detected in the cytoplasm and in the nucleoplasm. Secondly, immunostaining was performed, and the majority of Upf2 was detected in the cytoplasmic perinuclear region; a small quantity of Upf2 was detected in the intranuclear region. RNase treatment of the cells reduced the Upf2 immunostained signal. The immune-purified fractions containing nuclear and cytoplasmic Upf2 also contained one of the EJC core factors, RBM8A. These results implied the existence of Upf2 in the nucleoplasm and the cytoplasm, and it appeared to be involved in the construction of the mRNA complex. In order to verify the construction of Upf2-binding EJC in the nucleoplasm, an in situ proximity ligation assay was performed with anti-Upf2 and anti-RBM8A antibodies. These results demonstrated that their interaction occurred not only in the cytoplasmic region, but also in the intranuclear region. Taken together, these results suggested that Upf2 combines with EJC in both the cytoplasmic and the intranuclear fractions, and that it is involved in mRNA metabolism in human cells. PMID:27221324

  17. Antitumor effects of the flavone chalcone: inhibition of invasion and migration through the FAK/JNK signaling pathway in human gastric adenocarcinoma AGS cells.

    PubMed

    Lin, Su-Hsuan; Shih, Yuan-Wei

    2014-06-01

    Chalcones (benzylideneacetophenone) are cancer-preventive food components found in a human diet rich in fruits and vegetables. In this study, we first report the chemopreventive effect of chalcone in human gastric adenocarcinoma cell lines: AGS. The results showed that chalcone could inhibit the abilities of the adhesion, invasion, and migration by cell-matrix adhesion assay, Boyden chamber invasion/migration assay, and wound-healing assay. Molecular data showed that the effect of chalcone in AGS cells might be mediated via sustained inactivation of the phosphorylation of focal adhesion kinase (FAK) and c-Jun N-terminal kinase 1 and 2 (JNK1/2) signal involved in the downregulation of the expressions of matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9). Next, chalcone-treated AGS cells showed tremendous decrease in the phosphorylation and degradation of inhibitor of kappaBα (IκBα), the nuclear level of NF-κB, and the binding ability of NF-κB to NF-κB response element. Furthermore, treating FAK small interfering RNA (FAK siRNA) and specific inhibitor for JNK (SP600125) to AGS cells could reduce the phosphorylation of JNK1/2 and the activity of MMP-2 and MMP-9. Our results revealed that chalcone significantly inhibited the metastatic ability of AGS cells by reducing MMP-2 and MMP-9 expressions concomitantly with a marked reduction on cell invasion and migration through suppressing and JNK signaling pathways. We suggest that chalcone may offer the application in clinical medicine.

  18. Cell death caused by the synergistic effects of zinc and dopamine is mediated by a stress sensor gene Gadd45b - implication in the pathogenesis of Parkinson's disease.

    PubMed

    Yang, Tien-Chun; Wu, Pei-Chun; Chung, I-Fang; Jiang, Jhih-Hang; Fann, Ming-Ji; Kao, Lung-Sen

    2016-10-01

    The pathogenesis of Parkinson's disease (PD) is not completely understood, Zinc (Zn(2+) ) and dopamine (DA) have been shown to involve in the degeneration of dopaminergic cells. By microarray analysis, we identified Gadd45b as a candidate molecule that mediates Zn(2+) and DA-induced cell death; the mRNA and protein levels of Gadd45b are increased by Zn(2+) treatment and raised to an even higher level by Zn(2+) plus DA treatment. Zn(2+) plus DA treatment-induced PC12 cell death was enhanced when there was over-expression of Gadd45b and was decreased by knock down of Gadd45b. MAPK p38 and JNK signaling was able to cross-talk with Gadd45b during Zn(2+) and DA treatment. The synergistic effects of Zn(2+) and DA on PC12 cell death can be accounted for by an activation of the Gadd45b-induced cell death pathway and an inhibition of p38/JNK survival pathway. Furthermore, the in vivo results show that the levels of Gadd45b protein expression and phosphorylation of p38 were increased in the substantia nigra by the infusion of Zn(2+) /DA in the mouse brain and the level of Gadd45b mRNA is significantly higher in the substantia nigra of male PD patients than normal controls. The novel role of Gadd45b and its interactions with JNK and p38 will help our understanding of the pathogenesis of PD and help the development of future treatments for PD. Zinc and dopamine are implicated in the degeneration of dopaminergic neurons. We previously demonstrated that zinc and dopamine induced synergistic effects on PC12 cell death. Results from this study show that these synergistic effects can be accounted for by activation of the Gadd45b-induced cell death pathway and inhibition of the p38/JNK survival pathway. We provide in vitro and in vivo evidence to support a novel role for Gadd45b in the pathogenesis of Parkinson's disease. © 2016 International Society for Neurochemistry.

  19. Methotrexate increases expression of cell cycle checkpoint genes via Jun-N-terminal kinase activation

    PubMed Central

    Spurlock, Charles F.; Tossberg, John T.; Fuchs, Howard A.; Olsen, Nancy J.; Aune, Thomas M.

    2011-01-01

    Objective To assess defects in expression of critical cell cycle checkpoint genes and proteins in subjects with rheumatoid arthritis relative to presence or absence of methotrexate medication and assess the role of Jun N-terminal kinase in methotrexate induction of these genes. Methods Flow cytometry analysis was used to quantify changes in intracellular proteins, measure reactive oxygen species (ROS), and determine apoptosis in different lymphoid populations. Quantitative reverse transcriptase polymerase chain reaction (Q-RT-PCR) was employed to determine changes in cell cycle checkpoint target genes. Results RA subjects express lower baseline levels of MAPK9, TP53, CDKN1A, CDKN1B, CHEK2, and RANGAP1 messenger RNA (mRNA) and total JNK protein. MAPK9, TP53, CDKN1A, and CDKN1B mRNA expression, but not CHEK2, and RANGAP1, is higher in patients on low-dose MTX therapy. Further, JNK levels inversely correlate with CRP levels in RA patients. In tissue culture, MTX induces expression of both p53 and p21 by JNK2 and JNK1-dependent mechanisms, respectively, while CHEK2 and RANGAP1 are not induced by MTX. MTX also induces ROS production, JNK activation, and sensitivity to apoptosis in activated T cells. Supplementation with tetrahydrobiopterin blocks these MTX-mediated effects. Conclusions Our findings support the notion that MTX restores some, but not all of the proteins contributing to cell cycle checkpoint deficiencies in RA T cells by a JNK dependent pathway. PMID:22183962

  20. SCIL-STROKE (Subcutaneous Interleukin-1 Receptor Antagonist in Ischemic Stroke): A Randomized Controlled Phase 2 Trial.

    PubMed

    Smith, Craig J; Hulme, Sharon; Vail, Andy; Heal, Calvin; Parry-Jones, Adrian R; Scarth, Sylvia; Hopkins, Karen; Hoadley, Margaret; Allan, Stuart M; Rothwell, Nancy J; Hopkins, Stephen J; Tyrrell, Pippa J

    2018-05-01

    The proinflammatory cytokine IL-1 (interleukin-1) has a deleterious role in cerebral ischemia, which is attenuated by IL-1 receptor antagonist (IL-1Ra). IL-1 induces peripheral inflammatory mediators, such as interleukin-6, which are associated with worse prognosis after ischemic stroke. We investigated whether subcutaneous IL-1Ra reduces the peripheral inflammatory response in acute ischemic stroke. SCIL-STROKE (Subcutaneous Interleukin-1 Receptor Antagonist in Ischemic Stroke) was a single-center, double-blind, randomized, placebo-controlled phase 2 trial of subcutaneous IL-1Ra (100 mg administered twice daily for 3 days) in patients presenting within 5 hours of ischemic stroke onset. Randomization was stratified for baseline National Institutes of Health Stroke Scale score and thrombolysis. Measurement of plasma interleukin-6 and other peripheral inflammatory markers was undertaken at 5 time points. The primary outcome was difference in concentration of log(interleukin-6) as area under the curve to day 3. Secondary outcomes included exploratory effect of IL-1Ra on 3-month outcome with the modified Rankin Scale. We recruited 80 patients (mean age, 72 years; median National Institutes of Health Stroke Scale, 12) of whom 73% received intravenous thrombolysis with alteplase. IL-1Ra significantly reduced plasma interleukin-6 ( P <0.001) and plasma C-reactive protein ( P <0.001). IL-1Ra was well tolerated with no safety concerns. Allocation to IL-1Ra was not associated with a favorable outcome on modified Rankin Scale: odds ratio (95% confidence interval)=0.67 (0.29-1.52), P =0.34. Exploratory mediation analysis suggested that IL-1Ra improved clinical outcome by reducing inflammation, but there was a statistically significant, alternative mechanism countering this benefit. IL-1Ra reduced plasma inflammatory markers which are known to be associated with worse clinical outcome in ischemic stroke. Subcutaneous IL-1Ra is safe and well tolerated. Further experimental

  1. Catalpol ameliorates high-fat diet-induced insulin resistance and adipose tissue inflammation by suppressing the JNK and NF-κB pathways

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

    Zhou, Jun, E-mail: hustzhj@hust.edu.cn; Xu, Gang; Ma, Shuai

    Catalpol, a bioactive component from the root of Rehmannia glutinosa, has been shown to possess hypoglycemic effects in type 2 diabetic animal models, however, the underlying mechanisms remain poorly understood. Here we investigated the effect of catalpol on high-fat diet (HFD)-induced insulin resistance and adipose tissue inflammation in mice. Oral administration of catalpol at 100 mg/kg for 4 weeks had no effect on body weight of HFD-induced obese mice, but it significantly improved fasting glucose and insulin levels, glucose tolerance and insulin tolerance. Moreover, macrophage infiltration into adipose tissue was markedly reduced by catalpol. Intriguingly, catalpol also significantly reduced mRNA expressionsmore » of M1 pro-inflammatory cytokines, but increased M2 anti-inflammatory gene expressions in adipose tissue. Concurrently, catalpol significantly suppressed the c-Jun NH2-terminal kinase (JNK) and nuclear factor-kappa B (NF-κB) signaling pathways in adipose tissue. Collectively, these results suggest that catalpol may ameliorate HFD-induced insulin resistance in mice by attenuating adipose tissue inflammation and suppressing the JNK and NF-κB pathways, and thus provide important new insights into the underlying mechanisms of the antidiabetic effect of catalpol. - Highlights: • Catalpol ameliorates high-fat diet (HFD)-induced insulin resistance in mice. • Catalpol reduces adipose tissue macrophage infiltration in HFD-fed mice. • Catalpol regulates M1 and M2 inflammatory gene expression in obese adipose tissue. • Catalpol suppresses the JNK and NF-κB signaling pathways in obese adipose tissue.« less

  2. Effect of azithromycin on Prevotella intermedia lipopolysaccharide-induced production of interleukin-6 in murine macrophages.

    PubMed

    Choi, Eun-Young; Jin, Ji-Young; Choi, Jeom-Il; Choi, In Soon; Kim, Sung-Jo

    2014-04-15

    Interleukin-6 (IL-6) is a key proinflammatory cytokine which plays a central role in the pathogenesis of periodontal disease. Host modulatory agents targeting at inhibiting IL-6, therefore, appear to be beneficial in slowing the progression of periodontal disease and potentially reducing destructive aspects of the host response. The present study was designed to investigate the effect of the macrolide antibiotic azithromycin on IL-6 generation in murine macrophages treated with lipopolysaccharide (LPS) from Prevotella intermedia, a pathogen implicated in inflammatory periodontal disease, and its mechanisms of action. Azithromycin significantly suppressed IL-6 production as well as its mRNA expression in P. intermedia LPS-activated RAW264.7 cells. LPS-induced activation of JNK and p38 was not affected by azithromycin treatment. Azithromycin failed to prevent P. intermedia LPS from degrading IκB-α. Instead, azithromycin significantly diminished nuclear translocation and DNA binding activity of NF-κB p50 subunit induced with LPS. Azithromycin inhibited P. intermedia LPS-induced STAT1 and STAT3 phosphorylation. In addition, azithromycin up-regulated the mRNA level of SOCS1 in cells treated with LPS. In conclusion, azithromycin significantly attenuated P. intermedia LPS-induced production of IL-6 in murine macrophages via inhibition of NF-κB, STAT1 and STAT3 activation, which is possibly related to the activation of SOCS1 signaling. Further in vivo studies are required to better evaluate the potential of azithromycin in the treatment of periodontal disease. Copyright © 2014 Elsevier B.V. All rights reserved.

  3. Linking JNK Activity to the DNA Damage Response

    PubMed Central

    Picco, Vincent

    2013-01-01

    The activity of c-Jun N-terminal kinase (JNK) was initially described as ultraviolet- and oncogene-induced kinase activity on c-Jun. Shortly after this initial discovery, JNK activation was reported for a wider variety of DNA-damaging agents, including γ-irradiation and chemotherapeutic compounds. As the DNA damage response mechanisms were progressively uncovered, the mechanisms governing the activation of JNK upon genotoxic stresses became better understood. In particular, a recent set of papers links the physical breakage in DNA, the activation of the transcription factor NF-κB, the secretion of TNF-α, and an autocrine activation of the JNK pathway. In this review, we will focus on the pathway that is initiated by a physical break in the DNA helix, leading to JNK activation and the resultant cellular consequences. The implications of these findings will be discussed in the context of cancer therapy with DNA-damaging agents. PMID:24349633

  4. Reactive oxygen species activate differentiation gene transcription of acute myeloid leukemia cells via the JNK/c-JUN signaling pathway.

    PubMed

    Lam, Chung Fan; Yeung, Hoi Ting; Lam, Yuk Man; Ng, Ray Kit

    2018-05-01

    Reactive oxygen species (ROS) and altered cellular redox status are associated with many malignancies. Acute myeloid leukemia (AML) cells are maintained at immature state by differentiation blockade, which involves deregulation of transcription factors in myeloid differentiation. AML cells can be induced to differentiate by phorbol-12-myristate-13-acetate (PMA), which possesses pro-oxidative activity. However, the signaling events mediated by ROS in the activation of transcriptional program during AML differentiation has not been fully elucidated. Here, we investigated AML cell differentiation by treatment with PMA and ROS scavenger N-acetyl-l-cysteine (NAC). We observed elevation of intracellular ROS level in the PMA-treated AML cells, which correlated with differentiated cell morphology and increased CD11b + mature cell population. The effect of PMA can be abolished by NAC co-treatment, supporting the involvement of ROS in the process. Moreover, we demonstrated that short ROS elevation mediated cell cycle arrest, but failed to activate myeloid gene transcription; whereas prolonged ROS elevation activated JNK/c-JUN signaling pathway. Inhibition of JNK suppressed the expression of key myeloid transcriptional regulators c-JUN, SPI-1 and MAFB, and prevented AML cells from undergoing terminal differentiation. These findings provide new insights into the crucial role of JNK/c-Jun signaling pathway in the activation of transcriptional program during ROS-mediated AML differentiation. Copyright © 2018 Elsevier Ltd. All rights reserved.

  5. Glabridin induces apoptosis and cell cycle arrest in oral cancer cells through the JNK1/2 signaling pathway.

    PubMed

    Chen, Chang-Tai; Chen, Yi-Tzu; Hsieh, Yi-Hsien; Weng, Chia-Jui; Yeh, Jung-Chun; Yang, Shun-Fa; Lin, Chiao-Wen; Yang, Jia-Sin

    2018-06-01

    Glabridin, a flavonoid extracted from licorice (Glycyrrhiza glabra), possesses various biological properties, including anticancer activities. However, the effect of glabridin on oral cancer cell apoptosis and the underlying molecular mechanisms has not been elucidated. In this study, we demonstrated that glabridin treatment significantly inhibits cell proliferation in human oral cancer SCC-9 and SAS cell lines. Flow cytometric assays demonstrated that glabridin induced several features of apoptosis, such as sub-G1 phase cell increase and phosphatidylserine externalization. Furthermore, glabridin induced apoptosis dose-dependently in SCC-9 cells through caspase-3, -8, and -9 activation and poly (ADP-ribose) polymerase cleavage. Moreover, glabridin increased the phosphorylation of the extracellular signal-regulated kinase, p38, and c-Jun N-terminal kinase (JNK) pathways in a dose-dependent manner. Moreover, the inhibition of the JNK1/2 inhibitor significantly reversed the glabridin-induced activation of the caspase pathway. In conclusion, our findings suggest that glabridin induces oral cancer cell apoptosis through the JNK1/2 pathway and is a potential therapeutic agent for oral cancer. © 2018 Wiley Periodicals, Inc.

  6. Salmonella enteritidis Effector AvrA Stabilizes Intestinal Tight Junctions via the JNK Pathway.

    PubMed

    Lin, Zhijie; Zhang, Yong-Guo; Xia, Yinglin; Xu, Xiulong; Jiao, Xinan; Sun, Jun

    2016-12-23

    Salmonella pathogenesis studies to date have focused on Salmonella typhimurium, and the pathogenesis of a second major serotype, Salmonella enteritidis, is poorly understood. Salmonella spp. possess effector proteins that display biochemical activities and modulate host functions. Here, we generated a deletion mutant of the effector AvrA, S.E-AvrA - , and a plasmid-mediated complementary strain, S.E-AvrA - /pAvrA + (S.E-AvrA + ), in S. Enteritidis. Using in vitro and in vivo infection models, we showed that AvrA stabilizes epithelial tight junction (TJ) proteins, such as ZO-1, in human intestinal epithelial cells. Transepithelial electrical resistance was significantly higher in cells infected with S.E-AvrA + than in cells infected with S.E-AvrA - Inhibition of the JNK pathway suppresses the disassembly of TJ proteins; we found that enteritidis AvrA inhibited JNK activity in cells infected with wild type or S.E-AvrA + strains. Therefore, Enteritidis AvrA-induced ZO-1 stability is achieved via suppression of the JNK pathway. Furthermore, the S.E-AvrA - strain led to enhanced bacterial invasion, both in vitro and in vivo Taken together, our data reveal a novel role for AvrA in S. Enteritidis: Enteritidis AvrA stabilizes intestinal TJs and attenuates bacterial invasion. The manipulation of JNK activity and TJs in microbial-epithelial interactions may be a novel therapeutic approach for the treatment of infectious diseases. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  7. Salmonella enteritidis Effector AvrA Stabilizes Intestinal Tight Junctions via the JNK Pathway*

    PubMed Central

    Lin, Zhijie; Zhang, Yong-Guo; Xia, Yinglin; Xu, Xiulong; Jiao, Xinan

    2016-01-01

    Salmonella pathogenesis studies to date have focused on Salmonella typhimurium, and the pathogenesis of a second major serotype, Salmonella enteritidis, is poorly understood. Salmonella spp. possess effector proteins that display biochemical activities and modulate host functions. Here, we generated a deletion mutant of the effector AvrA, S.E-AvrA−, and a plasmid-mediated complementary strain, S.E-AvrA−/pAvrA+ (S.E-AvrA+), in S. Enteritidis. Using in vitro and in vivo infection models, we showed that AvrA stabilizes epithelial tight junction (TJ) proteins, such as ZO-1, in human intestinal epithelial cells. Transepithelial electrical resistance was significantly higher in cells infected with S.E-AvrA+ than in cells infected with S.E-AvrA−. Inhibition of the JNK pathway suppresses the disassembly of TJ proteins; we found that enteritidis AvrA inhibited JNK activity in cells infected with wild type or S.E-AvrA+ strains. Therefore, Enteritidis AvrA-induced ZO-1 stability is achieved via suppression of the JNK pathway. Furthermore, the S.E-AvrA− strain led to enhanced bacterial invasion, both in vitro and in vivo. Taken together, our data reveal a novel role for AvrA in S. Enteritidis: Enteritidis AvrA stabilizes intestinal TJs and attenuates bacterial invasion. The manipulation of JNK activity and TJs in microbial-epithelial interactions may be a novel therapeutic approach for the treatment of infectious diseases. PMID:27875307

  8. UVC-induced apoptosis in Dubca cells is independent of JNK activation and p53{sup Ser-15} phosphorylation

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

    Chathoth, Shahanas; Thayyullathil, Faisal; Hago, Abdulkader

    2009-06-12

    Ultraviolet C (UVC) irradiation in mammalian cell lines activates a complex signaling network that leads to apoptosis. By using Dubca cells as a model system, we report the presence of a UVC-induced apoptotic pathway that is independent of c-Jun N-terminal kinases (JNKs) activation and p53 phosphorylation at Ser{sup 15}. Irradiation of Dubca cells with UVC results in a rapid JNK activation and phosphorylation of its downstream target c-Jun, as well as, phosphorylation of activating transcription factor 2 (ATF2). Pre-treatment with JNK inhibitor, SP600125, inhibited UVC-induced c-Jun phosphorylation without preventing UVC-induced apoptosis. Similarly, inhibition of UVC-induced p53 phosphorylation did not preventmore » Dubca cell apoptosis, suggesting that p53{sup Ser-15} phosphorylation is not associated with UVC-induced apoptosis signaling. The pan-caspase inhibitor z-VAD-fmk inhibited UVC-induced PARP cleavage, DNA fragmentation, and ultimately apoptosis of Dubca cells. Altogether, our study clearly indicates that UVC-induced apoptosis is independent of JNK and p53 activation in Dubca cells, rather, it is mediated through a caspase dependent pathway. Our findings are not in line with the ascribed critical role for JNKs activation, and downstream phosphorylation of targets such as c-Jun and ATF2 in UVC-induced apoptosis.« less

  9. The insulin receptor substrate-1-related 4PS substrate but not the interleukin-2R gamma chain is involved in interleukin-13-mediated signal transduction.

    PubMed

    Wang, L M; Michieli, P; Lie, W R; Liu, F; Lee, C C; Minty, A; Sun, X J; Levine, A; White, M F; Pierce, J H

    1995-12-01

    Interleukin-13 (IL-13) induced a potent mitogenic response in IL-3-dependent TF-1 cells and DNA synthesis to a lesser extent in MO7E and FDC-P1 cells. IL-13 stimulation of these lines, like IL-4 and insulin-like growth factor-1 (IGF-1), resulted in tyrosine phosphorylation of a 170-kD substrate. The tyrosine-phosphorylated 170-kD substrate strongly associated with the 85-kD subunit of phosphoinositol-3 (PI-3) kinase and with Grb-2. Anti-4PS serum readily detected the 170-kD substrate in lysates from both TF-1 and FDC-P1 cells stimulated with IL-13 or IL-4. These data provide evidence that IL-13 induces tyrosine phosphorylation of the 4PS substrate, providing an essential interface between the IL-13 receptor and signaling molecules containing SH2 domains. IL-13 and IL-4 stimulation of murine L cell fibroblasts, which endogenously express the IL-4 receptor (IL-4R alpha) and lack expression of the IL-2 receptor gamma subunit (IL-2R gamma), resulted in tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1)/4PS. Enhanced tyrosine phosphorylation of IRS-1/4PS was observed in response to IL-4, but not IL-13 treatment of L cells transfected with the IL-2R gamma chain. These results indicate that IL-13 does not use the IL-2R gamma subunit in its receptor complex and that expression of IL-2R gamma enhances, but is not absolutely required for mediating IL-4-induced tyrosine phosphorylation of IRS-1/4PS.

  10. Phosphorylation of interleukin (IL)-24 is required for mediating its anti-cancer activity.

    PubMed

    Panneerselvam, Janani; Shanker, Manish; Jin, Jiankang; Branch, Cynthia D; Muralidharan, Ranganayaki; Zhao, Yan D; Chada, Sunil; Munshi, Anupama; Ramesh, Rajagopal

    2015-06-30

    Interleukin (IL)-24 is a tumor suppressor/cytokine gene that undergoes post-translational modifications (PTMs). Glycosylation and ubiquitination are important for IL-24 protein stabilization and degradation respectively. Little is known about IL-24 protein phosphorylation and its role in IL-24-mediated anti-tumor activities. In this study we conducted molecular studies to determine whether IL-24 phosphorylation is important for IL-24-mediated anti-cancer activity.Human H1299 lung tumor cell line that was stably transfected with a doxycycline (DOX)-inducible (Tet-on) plasmid vector carrying the cDNA of IL-24-wild-type (IL-24wt) or IL-24 with all five phosphorylation sites replaced (IL-24mt) was used in the present study. Inhibition of tumor cell proliferation, cell migration and invasion, and induction of G2/M cell cycle arrest was observed in DOX-induced IL-24wt-expressing cells but not in IL-24mt-expressing cells. Secretion of IL-24mt protein was greatly reduced compared to IL-24wt protein. Further, IL-24wt and IL-24mt proteins markedly differed in their subcellular organelle localization. IL-24wt but not IL-24mt inhibited the AKT/mTOR signaling pathway. SiRNA-mediated AKT knockdown and overexpression of myristolyated AKT protein confirmed that IL-24wt but not IL-24mt mediated its anti-cancer activity by inhibiting the AKT signaling pathway.Our results demonstrate that IL-24 phosphorylation is required for inhibiting the AKT/mTOR signaling pathway and exerting its anti-cancer activities.

  11. Interleukin-1 homologues IL-1F7b and IL-18 contain functional mRNA instability elements within the coding region responsive to lipopolysaccharide

    PubMed Central

    2004-01-01

    IL-1F7b, a novel homologue of the IL-1 (interleukin 1) family, was discovered by computational cloning. We demonstrated that IL-1F7b shares critical amino acid residues with IL-18 and binds to the IL-18-binding protein enhancing its ability to inhibit IL-18-induced interferon-γ. We also showed that low levels of IL-1F7b are constitutively present intracellularly in human blood monocytes. In this study, we demonstrate that similar to IL-18, both mRNA and intracellular protein expression of IL-1F7b are up-regulated by LPS (lipopolysaccharide) in human monocytes. In stable transfectants of murine RAW264.7 macrophage cells, there was no IL-1F7b protein expression despite a highly active CMV promoter. We found that IL-1F7b-specific mRNA was rapidly degraded in transfected cells, via a 3′-UTR (untranslated region)-independent control of IL-1F7b transcript stability. After LPS stimulation, there was a rapid transient increase in IL-1F7b-specific mRNA and concomitant protein levels. Using sequence alignment, we found a conserved ten-nucleotide homology box within the open reading frame of IL-F7b, which is flanking the coding region instability elements of some selective genes. In-frame deletion of downstream exon 5 from the full-length IL-1F7b cDNA markedly increased the levels of IL-1F7b mRNA. A similar coding region element is located in IL-18. When transfected into RAW264.7 macrophages, IL-18 mRNA was also unstable unless treated with LPS. These results indicate that both IL-1F7b and IL-18 mRNA contain functional instability determinants within their coding region, which influence mRNA decay as a novel mechanism to regulate the expression of IL-1 family members. PMID:15046617

  12. Smad, but not MAPK, pathway mediates the expression of type I collagen in radiation induced fibrosis

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

    Yano, Hiroyuki; Division of Radioisotope Research, Department of Research Support, Research Promotion Project, Oita University, 1-1 Idaigaoka Hasama-machi, Yufu, Oita 879-5593; Hamanaka, Ryoji

    Highlights: Black-Right-Pointing-Pointer We examine how radiation affects the expression level and signal pathway of collagen. Black-Right-Pointing-Pointer TGF-{beta}1 mRNA is elevated earlier than those of collagen genes after irradiation. Black-Right-Pointing-Pointer Smad pathway mediates the expression of collagen in radiation induced fibrosis. Black-Right-Pointing-Pointer MAPK pathways are not affected in the expression of collagen after irradiation. -- Abstract: Radiation induced fibrosis occurs following a therapeutic or accidental radiation exposure in normal tissues. Tissue fibrosis is the excessive accumulation of collagen and other extracellular matrix components. This study investigated how ionizing radiation affects the expression level and signal pathway of type I collagen. Realmore » time RT-RCR showed that both {alpha}1and {alpha}2 chain of type I collagen mRNA were elevated from 48 h after irradiation with 10 Gy in NIH3T3 cells. The relative luciferase activities of both genes and type I collagen marker were elevated at 72 h. TGF-{beta}1 mRNA was elevated earlier than those of type I collagen genes. A Western blot analysis showed the elevation of Smad phosphorylation at 72 h. Conversely, treatment with TGF-{beta} receptor inhibitor inhibited the mRNA and relative luciferase activity of type I collagen. The phosphorylation of Smad was repressed with the inhibitor, and the luciferase activity was cancelled using a mutant construct of Smad binding site of {alpha}2(I) collagen gene. However, the MAPK pathways, p38, ERK1/2 and JNK, were not affected with specific inhibitors or siRNA. The data showed that the Smad pathway mediated the expression of type I collagen in radiation induced fibrosis.« less

  13. Anti-neuroinflammatory effects of grossamide from hemp seed via suppression of TLR-4-mediated NF-κB signaling pathways in lipopolysaccharide-stimulated BV2 microglia cells.

    PubMed

    Luo, Qian; Yan, Xiaoli; Bobrovskaya, Larisa; Ji, Mei; Yuan, Huiqing; Lou, Hongxiang; Fan, Peihong

    2017-04-01

    Grossamide, a representative lignanamide in hemp seed, has been reported to possess potential anti-inflammatory effects. However, the potential anti-neuroinflammatory effects and underlying mechanisms of action of grossamide are still unclear. Therefore, the present study investigated the possible effects and underlying mechanisms of grossamide against lipopolysaccharide (LPS)-induced inflammatory response in BV2 microglia cells. BV2 microglia cells were pre-treated with various concentrations of grossamide before being stimulated with LPS to induce inflammation. The levels of pro-inflammatory cytokines were determined using the enzyme-linked immunoassay (ELISA) and mRNA expression levels were measured by real-time PCR. The translocation of nuclear factor-kappa B (NF-κB) and contribution of TLR4-mediated NF-κB activation on inflammatory effects were evaluated by immunostaining and Western blot analysis. This study demonstrated that grossamide significantly inhibited the secretion of pro-inflammatory mediators such as interleukin 6 (IL-6) and tumor necrosis factor α (TNF-α), and decreased the level of LPS-mediated IL-6 and TNF-α mRNA. In addition, it significantly reduced the phosphorylation levels of NF-κB subunit p65 in a concentration-dependent manner and suppressed translocation of NF-κB p65 into the nucleus. Furthermore, grossamide markedly attenuated the LPS-induced expression of Toll-like receptor 4 (TLR4) and myeloid differentiation factor 88 (MyD88). Taken together, these data suggest that grossamide could be a potential therapeutic candidate for inhibiting neuroinflammation in neurodegenerative diseases.

  14. Theiler's virus infection induces the expression of cyclooxygenase-2 in murine astrocytes: inhibition by the anti-inflammatory cytokines interleukin-4 and interleukin-10.

    PubMed

    Molina-Holgado, Eduardo; Arévalo-Martín, Angel; Ortiz, Sergio; Vela, José M; Guaza, Carmen

    2002-05-24

    Theiler's murine encephalomyelitis virus (TMEV) causes an acute encephalomyelitis followed by a persistent infection of the central nervous system (CNS) resulting in a chronic inflammation and axonal demyelination in susceptible strains of mice. The pathogenesis of TMEV-induced demyelinating disease remains unknown, but infection of brain glial cells is a critical factor for virus persistence in the CNS. In the present study we investigated the effects of the anti-inflammatory cytokines interleukin-4 (IL-4) and interleukin-10 (IL-10) on the production of inflammatory mediators, such as prostaglandins, after infection of primary astroglial SJL/J murine cultures with TMEV. This infection resulted in a time-dependent transcription of the gene encoding cyclooxygenase-2 (COX-2) and an increased production of prostaglandin E2 (PGE(2)). Both, IL-4 but mainly, IL-10 (1 and 10 ng/ml) decreased the TMEV-induced expression of COX-2 as well as the synthesis of PGE(2). Interestingly, treatment with IL-10 completely abrogated COX-2 induction. The molecular mechanisms involved in the regulation of COX-2 expression by TMEV are unknown, but the effects of anti-inflammatory cytokines may involve the inhibition of the transcription factor nuclear factor B activity and lead to strategies capable of interrupting the inflammatory cascade triggered by TMEV in brain glial cells.

  15. Spatiotemporal regulation of ERK2 by dual specificity phosphatases.

    PubMed

    Caunt, Christopher J; Armstrong, Stephen P; Rivers, Caroline A; Norman, Michael R; McArdle, Craig A

    2008-09-26

    Although many stimuli activate extracellular signal-regulated kinases 1 and 2 (ERK1/2), the kinetics and compartmentalization of ERK1/2 signals are stimulus-dependent and dictate physiological consequences. ERKs can be inactivated by dual specificity phosphatases (DUSPs), notably the MAPK phosphatases (MKPs) and atypical DUSPs, that can both dephosphorylate and scaffold ERK1/2. Using a cell imaging model (based on knockdown of endogenous ERKs and add-back of wild-type or mutated ERK2-GFP reporters), we explored possible effects of DUSPs on responses to transient or sustained ERK2 activators (epidermal growth factor and phorbol 12,13-dibutyrate, respectively). For both stimuli, a D319N mutation (which impairs DUSP binding) increased ERK2 activity and reduced nuclear accumulation. These stimuli also increased mRNA levels for eight DUSPs. In a short inhibitory RNA screen, 12 of 16 DUSPs influenced ERK2 responses. These effects were evident among nuclear inducible MKP, cytoplasmic ERK MKP, JNK/p38 MKP, and atypical DUSP subtypes and, with the exception of the nuclear inducible MKPs, were paralleled by corresponding changes in Egr-1 luciferase activation. Simultaneous removal of all JNK/p38 MKPs or nuclear inducible MKPs revealed them as positive and negative regulators of ERK2 signaling, respectively. The effects of JNK/p38 MKP short inhibitory RNAs were not dependent on protein neosynthesis but were reversed in the presence of JNK and p38 kinase inhibitors, indicating DUSP-mediated cross-talk between MAPK pathways. Overall, our data reveal that a large number of DUSPs influence ERK2 signaling. Together with the known tissue-specific expression of DUSPs and the importance of ERK1/2 in cell regulation, our data support the potential value of DUSPs as targets for drug therapy.

  16. Sirtuin 3 enhanced drug sensitivity of human hepatoma cells through glutathione S-transferase pi 1/JNK signaling pathway

    PubMed Central

    Cai, Xue-Fei; Zhang, Wen-Lu; Ren, Ji-Hua; Zhou, Li; Chen, Xiang; Chen, Ke; Li, Wan-Yu; Liu, Bo; Yang, Qiu-Xia; Cheng, Sheng-Tao; Huang, Li-Xia; Huang, Ai-Long; Chen, Juan

    2016-01-01

    SIRT3, a class III histone deacetylase, has been implicated in various cancers as a novel therapeutic target. In hepatocellular carcinoma (HCC), we previously reported that SIRT3 induced cell apoptosis by regulating GSK-3β/Bax signaling pathway. Downregulation of SIRT3 in HCC cells facilitates tumor cell survival. In this study, we found that chemotherapeutic agents (doxorubicin, cisplatin and epirubicin) and sorafenib treatment downregulated SIRT3 mRNA and protein levels in three HCC cell lines. MTS assay found that SIRT3 overexpression sensitized liver cancer cells to chemotherapeutic agents and sorafenib in SMMC-7721, Huh-7 and PLC/PRF/5 cell lines. Moreover, SIRT3 overexpression promoted chemotherapeutic agents-induced or sorafenib-induced apoptosis as evidenced by flow cytometry, enhanced PARP cleavage and enhanced Caspase-9 cleavage in three HCC cells. In contrast, SIRT3 silencing increased drug resistance of HCC cells to chemotherapeutic agents. Mechanistic study found that SIRT3 downregulated the mRNA and protein levels of glutathione S-transferase pi 1 (GSTP1), which is a member of phase II detoxification enzymes families involved in metabolizing for chemotherapeutic agents. Moreover, SIRT3 decreased the amount of GSTP1 that was associated with JNK, which finally contributed the activation of JNK activity and activation of downstream target c-Jun and Bim. Importantly, GSTP1 overexpression or JNK inhibitor abolished SIRT3-induced apoptosis in HCC cells exposed to chemotherapeutic agents. Finally, there was a negative correlation between SIRT3 expression and GSTP1 expression in human HCC tissues. Together, our findings revealed SIRT3 could enhance the drug sensitivity of HCC cells to an array of chemotherapeutic agents. SIRT3 may serve as a potential target for improving the chemosensitivity of HCC patients. PMID:27367026

  17. Sirtuin 3 enhanced drug sensitivity of human hepatoma cells through glutathione S-transferase pi 1/JNK signaling pathway.

    PubMed

    Tao, Na-Na; Zhou, Hong-Zhong; Tang, Hua; Cai, Xue-Fei; Zhang, Wen-Lu; Ren, Ji-Hua; Zhou, Li; Chen, Xiang; Chen, Ke; Li, Wan-Yu; Liu, Bo; Yang, Qiu-Xia; Cheng, Sheng-Tao; Huang, Li-Xia; Huang, Ai-Long; Chen, Juan

    2016-08-02

    SIRT3, a class III histone deacetylase, has been implicated in various cancers as a novel therapeutic target. In hepatocellular carcinoma (HCC), we previously reported that SIRT3 induced cell apoptosis by regulating GSK-3β/Bax signaling pathway. Downregulation of SIRT3 in HCC cells facilitates tumor cell survival. In this study, we found that chemotherapeutic agents (doxorubicin, cisplatin and epirubicin) and sorafenib treatment downregulated SIRT3 mRNA and protein levels in three HCC cell lines. MTS assay found that SIRT3 overexpression sensitized liver cancer cells to chemotherapeutic agents and sorafenib in SMMC-7721, Huh-7 and PLC/PRF/5 cell lines. Moreover, SIRT3 overexpression promoted chemotherapeutic agents-induced or sorafenib-induced apoptosis as evidenced by flow cytometry, enhanced PARP cleavage and enhanced Caspase-9 cleavage in three HCC cells. In contrast, SIRT3 silencing increased drug resistance of HCC cells to chemotherapeutic agents. Mechanistic study found that SIRT3 downregulated the mRNA and protein levels of glutathione S-transferase pi 1 (GSTP1), which is a member of phase II detoxification enzymes families involved in metabolizing for chemotherapeutic agents. Moreover, SIRT3 decreased the amount of GSTP1 that was associated with JNK, which finally contributed the activation of JNK activity and activation of downstream target c-Jun and Bim. Importantly, GSTP1 overexpression or JNK inhibitor abolished SIRT3-induced apoptosis in HCC cells exposed to chemotherapeutic agents. Finally, there was a negative correlation between SIRT3 expression and GSTP1 expression in human HCC tissues. Together, our findings revealed SIRT3 could enhance the drug sensitivity of HCC cells to an array of chemotherapeutic agents. SIRT3 may serve as a potential target for improving the chemosensitivity of HCC patients.

  18. [Effect of lipopolysaccharides from Porphyromonas endodontalis on the expression of interleukin-34 in mouse osteoblasts].

    PubMed

    Yu, Ya-Qiong; Guo, Jia-Jie; Qiu, Li-Hong; Li, Xiao-Lin; Yang, Di; Guo, Yan

    2017-02-01

    To investigate the effects of lipopolysaccharides (LPS) extracted from Porphyromonas endodontalis (P.e) on the expression of interleukin-34 (IL-34) mRNA in MC3T3-E1 cells and the role of p38MAPK, ERK1/2, NF-κB and SIRT1 in the process. MC3T3-E1 cells were treated with different concentrations of P.e-LPS(0-50 mg/L) and 20 mg/L P.e-LPS for different time (0-24 h). The expression of IL-34 mRNA was detected by real-time reverse transcription-polymerase chain reaction (real time RT-PCR). MC3T3-E1 cells were pretreated with inhibitor of NF-κB(BAY 11-7082),inhibitor of p38MAPK (SB203580), inhibitor of ERK1/2 (PD98059), agonist of sirtuin1 (SIRT1) [resveratrol (RES)] and inhibitor of SIRT1 (EX-527) for 1 h, and then were treated with 20 mg/L P.e-LPS. The expression of IL-34 mRNA was detected by real time RT-PCR. Statistical analysis was performed using one-way ANOVA and Dunnett t test with SPSS 13.0 software package. The level of IL-34 mRNA increased significantly after treatment with different concentrations of P.e-LPS(0-50 mg/L),which indicated that P.e-LPS induced osteoblasts to express IL-34 mRNA in a dose-dependent manner. Maximal induction of IL-34 mRNA expression was observed in MC3T3-E1 cells treated with 20 mg/L P.e-LPS for 24 h.At 48 h, the expression of IL-34 mRNA decreased gradually. The mRNA of IL-34 decreased significantly after pretreatment with 10 μmol/L BAY-117082, SB203580 and PD98059 for 1 h. P.e-LPS-induced IL-34 upregulation was attenuated by pretreatment with RES, but increased by EX-527. These results suggest that P.e-LPS may mediate IL-34 mRNA expression in MC3T3-E1 cells. This process is dependent, at least in part, on p38MAPK, ERK1/2, NF-κB and SIRT1 signaling pathways.

  19. Involvement of hypothalamic cyclooxygenase-2, interleukin-1β and melanocortin in the development of docetaxel-induced anorexia in rats.

    PubMed

    Yamamoto, Kouichi; Asano, Keiko; Ito, Yui; Matsukawa, Naoki; Kim, Seikou; Yamatodani, Atsushi

    2012-12-16

    Docetaxel, a taxane derivative, is frequently used for the treatment of advanced breast cancer, non-small cell lung cancer, and metastatic prostate cancer. Clinical reports demonstrated that docetaxel-based chemotherapy often induces anorexia, but the etiology is not completely understood. To elucidate possible mechanisms, we investigated the involvement of central interleukin (IL)-1β, cyclooxygenase (COX)-2, and pro-opiomelanocortin (POMC) in the development of docetaxel-induced anorexia in rats. Rats received docetaxel (10mg/kg, i.p.) with or without pretreatment with selective COX-2 inhibitors, NS-398 (10 and 30 mg/kg, i.g.) or celecoxib (10 and 30 mg/kg, i.g.), and a non-selective COX inhibitor, indomethacin (10mg/kg, i.g.), then food intake was monitored for 24h after administration. We also examined expression of IL-1β, COX-2, and POMC mRNA in hypothalamus of docetaxel-treated rats and the effect of a COX-2 inhibitor on docetaxel-induced POMC mRNA expression. Food consumption in rats was significantly decreased 24h after administration of docetaxel and anorexia was partially reversed by all COX inhibitors. Administration of docetaxel increased IL-1β, COX-2, and POMC mRNA expression in the hypothalamus of rats. The time required to increase these gene expressions was comparable to the latency period of docetaxel-induced anorexia in rats. In addition, pretreatment with COX-2 inhibitors suppressed docetaxel-induced expression of POMC mRNA. These results suggest that IL-1β and COX-2 mRNA expression and subsequent activation of POMC in the hypothalamus may contribute to the development of docetaxel-induced anorexia in rats. Copyright © 2012. Published by Elsevier Ireland Ltd.

  20. NF-κB and JNK mediated apoptosis and G0/G1 arrest of HeLa cells induced by rubiarbonol G, an arborinane-type triterpenoid from Rubia yunnanensis.

    PubMed

    Zeng, Guang-Zhi; Wang, Zhe; Zhao, Li-Mei; Fan, Jun-Ting; Tan, Ning-Hua

    2018-06-28

    Rubia yunnanensis is a medicinal plant mainly grown in Yunnan province in Southwest China, and its root named "Xiaohongshen" has been used as a herb in Yunnan for the treatment of cancers. Three major types of chemical components, Rubiaceae-type cyclopeptides, quinones, and triterpenoids, were identified from R. yunnanensis, in which some of compounds including rubiarbonol G (RG), a unique arboriane-type triterpenoid, showed cytotoxicity on cancer cells. But the cytotoxic mechanism of RG has not been reported. To investigate the cytotoxic mechanism of RG on cancer cells. RG was evaluated its cytotoxicity on 7 cancer cell lines by the SRB assay, and detected the effect on apoptosis and cell cycle arrest by Annexin V-FITC/PI apoptosis assay and DNA contents analysis. The expression and activity of apoptosis and cell cycle related proteins were also investigated by western blot and caspase activity assay. Furthermore, the effect of RG on NF-κB signaling was also tested by luciferase assay, western blot, and immunofluorescence staining. RG showed potent cytotoxicity on 7 human cancer cell lines, whose activity was attributed to apoptosis induction and G 0 /G 1 arrest in HeLa cells. Results from the mechanism study showed that RG promoted the activation of ERK1/2 and JNK pathway in MAPK family, which in turn increased the expression of p53, thereby triggering the G 0 /G 1 arrest through p53/p21/cyclin D1 signaling. Moreover, RG-mediated JNK activation down-regulated the expression of the anti-apoptotic protein Bcl-2, which caused the release of cytochrome c to the cytosol and activated the cleavage of caspase cascade and poly(ADP-ribose) polymerase, thereby inducing apoptosis in HeLa cells. In addition, RG was also found to inhibit the activation of NF-κB signaling by down-regulating the expression and attenuating the translocation to nucleus of NF-κB p65, by which the down-stream p53, cyclin D1, Bcl-2, and caspases were regulated, thereby triggering apoptosis and G

  1. Epiregulin (EREG) is upregulated through an IL-1β autocrine loop in Caco-2 epithelial cells with reduced CFTR function.

    PubMed

    Massip-Copiz, Macarena; Clauzure, Mariángeles; Valdivieso, Ángel G; Santa-Coloma, Tomás A

    2018-03-01

    CFTR is a cAMP-regulated chloride channel, whose mutations produce cystic fibrosis. The impairment of CFTR activity increases the intracellular Cl - concentration, which in turn produces an increased interleukin-1β (IL-1β) secretion. The secreted IL-1β then induces an autocrine positive feedback loop, further stimulating IL-1β priming and secretion. Since IL-1β can transactivate the epidermal growth factor receptor (EGFR), we study here the levels of expression for different EGFR ligands in Caco-2/pRS26 cells (expressing shRNA against CFTR resulting in a reduced CFTR expression and activity). The epiregulin (EREG), amphiregulin (AREG), and heparin binding EGF like growth factor (HBEGF) mRNAs, were found overexpressed in Caco-2/pRS26 cells. The EREG mRNA had the highest differential expression and was further characterized. In agreement with its mRNA levels, Western blots (WB) showed increased EREG levels in CFTR-impaired cells. In addition, EREG mRNA and protein levels were stimulated by incubation with exogenous IL-1β and inhibited by the Interleukin 1 receptor type I (IL1R1) antagonist IL1RN, suggesting that the overexpression of EREG is a consequence of the autocrine IL-1β loop previously described for these cells. In addition, the JNK inhibitor SP600125, and the EGFR inhibitors AG1478 and PD168393, also had an inhibitory effect on EREG expression, suggesting that EGFR, activated in Caco-2/pRS26 cells, is involved in the observed EREG upregulation. In conclusion, in Caco-2 CFTR-shRNA cells, the EGFR ligand EREG is overexpressed due to an active IL-1β autocrine loop that indirectly activates EGFR, constituting new signaling effectors for the CFTR signaling pathway, downstream of CFTR, Cl - , and IL-1β. © 2017 Wiley Periodicals, Inc.

  2. Novel mechanism of JNK pathway activation by adenoviral E1A

    PubMed Central

    Morrison, Helen; Pospelova, Tatiana V.; Pospelov, Valery A.; Herrlich, Peter

    2014-01-01

    The adenoviral oncoprotein E1A influences cellular regulation by interacting with a number of cellular proteins. In collaboration with complementary oncogenes, E1A fully transforms primary cells. As part of this action, E1A inhibits transcription of c-Jun:Fos target genes while promoting that of c-Jun:ATF2-dependent genes including jun. Both c-Jun and ATF2 are hyperphosphorylated in response to E1A. In the current study, E1A was fused with the ligand binding domain of the estrogen receptor (E1A-ER) to monitor the immediate effect of E1A activation. With this approach we now show that E1A activates c-Jun N-terminal kinase (JNK), the upstream kinases MKK4 and MKK7, as well as the small GTPase Rac1. Activation of the JNK pathway requires the N-terminal domain of E1A, and, importantly, is independent of transcription. In addition, it requires the presence of ERM proteins. Downregulation of signaling components upstream of JNK inhibits E1A-dependent JNK/c-Jun activation. Taking these findings together, we show that E1A activates the JNK/c-Jun signaling pathway upstream of Rac1 in a transcription-independent manner, demonstrating a novel mechanism of E1A action. PMID:24742962

  3. Hypothalamic AMPK-ER Stress-JNK1 Axis Mediates the Central Actions of Thyroid Hormones on Energy Balance.

    PubMed

    Martínez-Sánchez, Noelia; Seoane-Collazo, Patricia; Contreras, Cristina; Varela, Luis; Villarroya, Joan; Rial-Pensado, Eva; Buqué, Xabier; Aurrekoetxea, Igor; Delgado, Teresa C; Vázquez-Martínez, Rafael; González-García, Ismael; Roa, Juan; Whittle, Andrew J; Gomez-Santos, Beatriz; Velagapudi, Vidya; Tung, Y C Loraine; Morgan, Donald A; Voshol, Peter J; Martínez de Morentin, Pablo B; López-González, Tania; Liñares-Pose, Laura; Gonzalez, Francisco; Chatterjee, Krishna; Sobrino, Tomás; Medina-Gómez, Gema; Davis, Roger J; Casals, Núria; Orešič, Matej; Coll, Anthony P; Vidal-Puig, Antonio; Mittag, Jens; Tena-Sempere, Manuel; Malagón, María M; Diéguez, Carlos; Martínez-Chantar, María Luz; Aspichueta, Patricia; Rahmouni, Kamal; Nogueiras, Rubén; Sabio, Guadalupe; Villarroya, Francesc; López, Miguel

    2017-07-05

    Thyroid hormones (THs) act in the brain to modulate energy balance. We show that central triiodothyronine (T3) regulates de novo lipogenesis in liver and lipid oxidation in brown adipose tissue (BAT) through the parasympathetic (PSNS) and sympathetic nervous system (SNS), respectively. Central T3 promotes hepatic lipogenesis with parallel stimulation of the thermogenic program in BAT. The action of T3 depends on AMP-activated protein kinase (AMPK)-induced regulation of two signaling pathways in the ventromedial nucleus of the hypothalamus (VMH): decreased ceramide-induced endoplasmic reticulum (ER) stress, which promotes BAT thermogenesis, and increased c-Jun N-terminal kinase (JNK) activation, which controls hepatic lipid metabolism. Of note, ablation of AMPKα1 in steroidogenic factor 1 (SF1) neurons of the VMH fully recapitulated the effect of central T3, pointing to this population in mediating the effect of central THs on metabolism. Overall, these findings uncover the underlying pathways through which central T3 modulates peripheral metabolism. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  4. Nicotinamide mononucleotide inhibits JNK activation to reverse Alzheimer disease.

    PubMed

    Yao, Zhiwen; Yang, Wenhao; Gao, Zhiqiang; Jia, Peng

    2017-04-24

    Amyloid-β (Aβ) oligomers have been accepted as major neurotoxic agents in the therapy of Alzheimer's disease (AD). It has been shown that the activity of nicotinamide adenine dinucleotide (NAD+) is related with the decline of Aβ toxicity in AD. Nicotinamide mononucleotide (NMN), the important precursor of NAD+, is produced during the reaction of nicotinamide phosphoribosyl transferase (Nampt). This study aimed to figure out the potential therapeutic effects of NMN and its underlying mechanisms in APPswe/PS1dE9 (AD-Tg) mice. We found that NMN gave rise to a substantial improvement in behavioral measures of cognitive impairments compared to control AD-Tg mice. In addition, NMN treatment significantly decreased β-amyloid production, amyloid plaque burden, synaptic loss, and inflammatory responses in transgenic animals. Mechanistically, NMN effectively controlled JNK activation. Furthermore, NMN potently progressed nonamyloidogenic amyloid precursor protein (APP) and suppressed amyloidogenic APP by mediating the expression of APP cleavage secretase in AD-Tg mice. Based on our findings, it was suggested that NMN substantially decreases multiple AD-associated pathological characteristically at least partially by the inhibition of JNK activation. Copyright © 2017 Elsevier B.V. All rights reserved.

  5. Roles of p38 and JNK protein kinase pathways activated by compound cantharidin capsules containing serum on proliferation inhibition and apoptosis of human gastric cancer cell line

    PubMed Central

    Sun, Yonghao; Zhang, Dejuan; Mao, Mao; Lu, Yangping; Jiao, Ning

    2017-01-01

    The aim of the present study was to investigate the inhibitory effect of compound cantharides capsules (CCCs) on the viability and apoptosis of human gastric cancer cell lines, BGC-823 and SGC-7901, and to detect its regulation of gene expression levels, as well as its inhibition mechanisms. Each cell line was grouped into a control group, CCC serum group, 5-fluorouracil (5-FU) group, combination therapy group (CCC serum + 5-FU) and serum control group. Growth curves were measured and flow cytometry was used to detect cell apoptosis and cell viability. The mRNA expression level of proliferation-related C-MYC and p53 genes were assayed by reverse transcription-quantitative polymerase chain reaction. Protein phosphorylation levels of proliferating cell nuclear antigen, p38 mitogen-activated protein kinase, extracellular signal-related kinase 1/2, c-Jun N-terminal kinase (JNK) and IκB were assayed by western blotting. The combined CCC serum and 5-FU group exhibited a higher inhibition rate in both cell lines and CCC serum therapy demonstrated a similar effect to 5-FU treatment, as demonstrated in the MTT and cell growth assay. Combined therapy significantly decreased the C-MYC mRNA expression levels and increased p53 mRNA expression levels (P<0.05). Combined therapy of 5-FU and CCC was more significant compared with CCC serum or 5-FU only (P<0.05). P38 and JNK-related protein phosphorylation are involved in apoptosis initiated by CCC combined 5-FU therapy. Combined therapy was able to significantly inhibit human gastric cancer cell growth (P<0.05), and advance cell apoptosis compared with CCC serum only. CCC serum resulted in downregulation of the c-Myc gene and upregulation of the p53 gene. p38 and JNK-related protein phosphorylation is involved in the inhibition of cell viability and apoptosis of human gastric cancer cell lines. PMID:28810654

  6. Roles of p38 and JNK protein kinase pathways activated by compound cantharidin capsules containing serum on proliferation inhibition and apoptosis of human gastric cancer cell line.

    PubMed

    Sun, Yonghao; Zhang, Dejuan; Mao, Mao; Lu, Yangping; Jiao, Ning

    2017-08-01

    The aim of the present study was to investigate the inhibitory effect of compound cantharides capsules (CCCs) on the viability and apoptosis of human gastric cancer cell lines, BGC-823 and SGC-7901, and to detect its regulation of gene expression levels, as well as its inhibition mechanisms. Each cell line was grouped into a control group, CCC serum group, 5-fluorouracil (5-FU) group, combination therapy group (CCC serum + 5-FU) and serum control group. Growth curves were measured and flow cytometry was used to detect cell apoptosis and cell viability. The mRNA expression level of proliferation-related C-MYC and p53 genes were assayed by reverse transcription-quantitative polymerase chain reaction. Protein phosphorylation levels of proliferating cell nuclear antigen, p38 mitogen-activated protein kinase, extracellular signal-related kinase 1/2, c-Jun N-terminal kinase (JNK) and IκB were assayed by western blotting. The combined CCC serum and 5-FU group exhibited a higher inhibition rate in both cell lines and CCC serum therapy demonstrated a similar effect to 5-FU treatment, as demonstrated in the MTT and cell growth assay. Combined therapy significantly decreased the C-MYC mRNA expression levels and increased p53 mRNA expression levels (P<0.05). Combined therapy of 5-FU and CCC was more significant compared with CCC serum or 5-FU only (P<0.05). P38 and JNK-related protein phosphorylation are involved in apoptosis initiated by CCC combined 5-FU therapy. Combined therapy was able to significantly inhibit human gastric cancer cell growth (P<0.05), and advance cell apoptosis compared with CCC serum only. CCC serum resulted in downregulation of the c-Myc gene and upregulation of the p53 gene. p38 and JNK-related protein phosphorylation is involved in the inhibition of cell viability and apoptosis of human gastric cancer cell lines.

  7. 2',5'-Dihydroxychalcone-induced glutathione is mediated by oxidative stress and kinase signaling pathways.

    PubMed

    Kachadourian, Remy; Pugazhenthi, Subbiah; Velmurugan, Kalpana; Backos, Donald S; Franklin, Christopher C; McCord, Joe M; Day, Brian J

    2011-09-15

    Hydroxychalcones are naturally occurring compounds that continue to attract considerable interest because of their anti-inflammatory and antiangiogenic properties. They have been reported to inhibit the synthesis of the inducible nitric oxide synthase and to induce the expression of heme oxygenase-1. This study examines the mechanisms by which 2',5'-dihydroxychalcone (2',5'-DHC) induces an increase in cellular glutathione (GSH) levels using a cell line stably expressing a luciferase reporter gene driven by antioxidant-response elements (MCF-7/AREc32). The 2',5'-DHC-induced increase in cellular GSH levels was partially inhibited by the catalytic antioxidant MnTDE-1,3-IP(5+), suggesting that reactive oxygen species (ROS) mediate the antioxidant adaptive response. 2',5'-DHC treatment induced phosphorylation of the c-Jun N-terminal kinase (JNK) pathway, which was also inhibited by MnTDE-1,3-IP(5+). These findings suggest a ROS-dependent activation of the AP-1 transcriptional response. However, whereas 2',5'-DHC triggered the NF-E2-related factor 2 (Nrf2) transcriptional response, cotreatment with MnTDE-1,3-IP(5+) did not decrease 2',5'-DHC-induced Nrf2/ARE activity, showing that this pathway is not dependent on ROS. Moreover, pharmacological inhibitors of mitogen-activated protein kinase (MAPK) pathways showed a role for JNK and p38MAPK in mediating the 2',5'-DHC-induced Nrf2 response. These findings suggest that the 2',5'-DHC-induced increase in GSH levels results from a combination of ROS-dependent and ROS-independent pathways. Copyright © 2011 Elsevier Inc. All rights reserved.

  8. Single-Factor SOX2 Mediates Direct Neural Reprogramming of Human Mesenchymal Stem Cells via Transfection of In Vitro Transcribed mRNA.

    PubMed

    Kim, Bo-Eun; Choi, Soon Won; Shin, Ji-Hee; Kim, Jae-Jun; Kang, Insung; Lee, Byung-Chul; Lee, Jin Young; Kook, Myoung Geun; Kang, Kyung-Sun

    2018-01-01

    Neural stem cells (NSCs) are a prominent cell source for understanding neural pathogenesis and for developing therapeutic applications to treat neurodegenerative disease because of their regenerative capacity and multipotency. Recently, a variety of cellular reprogramming technologies have been developed to facilitate in vitro generation of NSCs, called induced NSCs (iNSCs). However, the genetic safety aspects of established virus-based reprogramming methods have been considered, and non-integrating reprogramming methods have been developed. Reprogramming with in vitro transcribed (IVT) mRNA is one of the genetically safe reprogramming methods because exogenous mRNA temporally exists in the cell and is not integrated into the chromosome. Here, we successfully generated expandable iNSCs from human umbilical cord blood-derived mesenchymal stem cells (UCB-MSCs) via transfection with IVT mRNA encoding SOX2 (SOX2 mRNA) with properly optimized conditions. We confirmed that generated human UCB-MSC-derived iNSCs (UM-iNSCs) possess characteristics of NSCs, including multipotency and self-renewal capacity. Additionally, we transfected human dermal fibroblasts (HDFs) with SOX2 mRNA. Compared with human embryonic stem cell-derived NSCs, HDFs transfected with SOX2 mRNA exhibited neural reprogramming with similar morphologies and NSC-enriched mRNA levels, but they showed limited proliferation ability. Our results demonstrated that human UCB-MSCs can be used for direct reprogramming into NSCs through transfection with IVT mRNA encoding a single factor, which provides an integration-free reprogramming tool for future therapeutic application.

  9. The mechanism by which MEK/ERK regulates JNK and p38 activity in polyamine depleted IEC-6 cells during apoptosis

    PubMed Central

    Bavaria, Mitul N.; Jin, Shi; Ray, Ramesh M.; Johnson, Leonard R.

    2014-01-01

    Polyamine-depletion inhibited apoptosis by activating ERK1/2, while, preventing JNK1/2 activation. MKP-1 knockdown by SiRNA increased ERK1/2, JNK1/2, and p38 phosphorylation and apoptosis. Therefore, we predicted that polyamines might regulate MKP1 via MEK/ERK and thereby apoptosis. We examined the role of MEK/ERK in the regulation of MKP1 and JNK, and p38 activities and apoptosis. Inhibition of MKP-1 activity with a pharmacological inhibitor, sanguinarine (SA), increased JNK1/2, p38, and ERK1/2 activities without causing apoptosis. However, pre-activation of these kinases by SA significantly increased camptothecin (CPT)-induced apoptosis suggesting different roles for MAPKs during survival and apoptosis. Inhibition of MEK1 activity prevented the expression of MKP-1 protein and augmented CPT-induced apoptosis, which correlated with increased activities of JNK1/2, caspases, and DNA fragmentation. Polyamine depleted cells had higher levels of MKP-1 protein and decreased JNK1/2 activity and apoptosis. Inhibition of MEK1 prevented MKP-1 expression and increased JNK1/2 and apoptosis. Phospho-JNK1/2, phospho-ERK2, MKP-1, and the catalytic subunit of protein phosphatase 2A (PP2Ac) formed a complex in response to TNF/CPT. Inactivation of PP2Ac had no effect on the association of MKP-1 and JNK1. However, inhibition of MKP-1 activity decreased the formation of the MKP-1, PP2Ac and JNK complex. Following inhibition by SA, MKP-1 localized in the cytoplasm, while basal and CPT-induced MKP-1 remained in the nuclear fraction. These results suggest that nuclear MKP-1 translocates to the cytoplasm, binds phosphorylated JNK and p38 resulting in dephosphorylation and decreased activity. Thus, MEK/ERK activity controls the levels of MKP-1 and, thereby, regulates JNK activity in polyamine-depleted cells. PMID:24253595

  10. PI3-K/Akt/JNK/NF-κB is essential for MMP-9 expression and outgrowth in human limbal epithelial cells on intact amniotic membrane.

    PubMed

    Cheng, Ching-Yi; Hsieh, Hsi-Lung; Hsiao, Li-Der; Yang, Chuen-Mao

    2012-07-01

    Matrix metalloproteinase-9 (MMP-9) plays an important role in the outgrowth of expanded human limbal epithelial cells on intact amniotic membranes (AM). The mechanisms of MMP-9 expression and cell outgrowth remain unknown. Here, we demonstrated that MMP-9 is preferentially expressed at the leading edge of limbal epithelial outgrowth. Treatment with the inhibitors of PI3-K (LY294002), Akt (SH-5), MEK1/2 (U0126), and JNK1/2 (SP600125) attenuated the outgrowth area, indicating that PI3-K/Akt, p42/p44 MAPK, and JNK1/2 are involved in the outgrowth of intact AM-expanded limbal epithelial cells. However, MMP-9 expression at both transcriptional and translational levels was attenuated by treatment with SP600125, LY294002, or SH-5, not by U0126 and SB202190, suggesting that JNK1/2 and PI3-K/Akt participate in MMP-9 expression. Moreover, NF-κB phosphorylation and nuclear translocation was especially noted at the leading edge, which was attenuated by treatment with SP600125 or LY294002. Helenalin, a selective NF-κB inhibitor, reduced both the limbal epithelial outgrowth and MMP-9 expression. Finally, the data reveal that PI3-K/Akt is an upstream component of the JNK1/2 pathway in MMP-9 expression. Thus, both MAPKs and PI3-K/Akt are required for limbal epithelial outgrowth on intact AM, only the PI3-K/Akt/JNK is essential for MMP-9 expression mediated through activation of transcriptional factor NF-κB in this model. Copyright © 2012 Elsevier B.V. All rights reserved.

  11. Histamine Promotes the Release of Interleukin-6 via the H1R/p38 and NF-κB Pathways in Nasal Fibroblasts.

    PubMed

    Park, Il-Ho; Um, Ji-Young; Cho, Jung-Sun; Lee, Seung Hoon; Lee, Sang Hag; Lee, Heung-Man

    2014-11-01

    Based on the close relationship between histamine and interleukin 6 (IL-6), we hypothesized that histamine may regulate the production of cytokines, such as IL-6, during allergic inflammation. Here, we examined the role of histamine in IL-6 production and histamine receptor activity in nasal fibroblasts, along with the mechanisms underlying these effects. Experiments were performed using nasal fibroblasts from 8 normal patients. RT-PCR was used to identify the major histamine receptors expressed in nasal fibroblasts. Fibroblasts were then treated with histamine with or without histamine-receptor antagonists, and monitored for IL-6 production using an ELISA. Four potential downstream signaling molecules, p38, extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and NF-κB, were evaluated by Western blot, and a luciferase reporter assay. Elevated expression was seen for all histamine receptors, with IL-6 protein levels increasing significantly following histamine stimulation. Among the histamine-receptor specific antagonists, only the H1R antagonist significantly decreased IL-6 production in histamine-stimulated nasal fibroblasts. Histamine increased the expression level of phosphorylated p38 (pp38), pERK, and pJNK, as well as NF-κB induction. The H1R antagonist actively suppressed pp38 and NF-κB expression in histamine-induced nasal fibroblasts, but not pERK and pJNK. The p38 inhibitor strongly attenuated IL-6 production in histamine-stimulated nasal fibroblasts. The data presented here suggest that antihistamines may be involved in the regulation of cytokines, such as IL-6, due to the role of histamine as an inflammatory mediator in nasal fibroblasts.

  12. Airborne fine particulate matter causes murine bronchial hyperreactivity via MAPK pathway-mediated M3 muscarinic receptor upregulation.

    PubMed

    Wang, Rong; Xiao, Xue; Shen, Zhenxing; Cao, Lei; Cao, Yongxiao

    2017-02-01

    Regarding the human health effects, airborne fine particulate matter 2.5 (PM 2.5 ) is an important environmental risk factor. However, the underlying molecular mechanisms are largely unknown. The present study examined the hypothesis that PM 2.5 causes bronchial hyperreactivity by upregulated muscarinic receptors via the mitogen-activated protein kinase (MAPK) pathway. The isolated rat bronchi segments were cultured with different concentration of PM 2.5 for different time. The contractile response of the bronchi segments were recorded by a sensitive myograph. The mRNA and protein expression levels of M 3 muscarinic receptors were studied by quantitative real-time PCR and immunohistochemistry, respectively. The muscarinic receptors agonist, carbachol induced a remarkable contractile response on fresh and DMSO cultured bronchial segments. Compared with the fresh or DMSO culture groups, 1.0 µg/mL of PM 2.5 cultured for 24 h significantly enhanced muscarinic receptor-mediated contractile responses in bronchi with a markedly increased maximal contraction. In addition, the expression levels of mRNA and protein for M 3 muscarinic receptors in bronchi of PM 2.5 group were higher than that of fresh or DMSO culture groups. SB203580 (p38 inhibitor) and U0126 (MEK1/2 inhibitor) significantly inhibited the PM 2.5 -induced enhanced contraction and increased mRNA and protein expression of muscarinic receptors. However, JNK inhibitor SP600125 had no effect on PM 2.5 -induced muscarinic receptor upregulation and bronchial hyperreactivity. In conclusion, airborne PM 2.5 upregulates muscarinic receptors, which causes subsequently bronchial hyperreactivity shown as enhanced contractility in bronchi. This process may be mediated by p38 and MEK1/2 MAPK pathways. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 371-381, 2017. © 2016 Wiley Periodicals, Inc.

  13. β3-adrenergic receptor activation induces TGFβ1 expression in cardiomyocytes via the PKG/JNK/c-Jun pathway.

    PubMed

    Xu, Zhongcheng; Wu, Jimin; Xin, Junzhou; Feng, Yenan; Hu, Guomin; Shen, Jing; Li, Mingzhe; Zhang, Youyi; Xiao, Han; Wang, Li

    2018-06-05

    In heart failure, the expression of cardiac β 3 -adrenergic receptors (β 3 -ARs) increases. However, the precise role of β 3 -AR signaling within cardiomyocytes remains unclear. Transforming growth factor β1 (TGFβ1) is a crucial cytokine mediating the cardiac remodeling that plays a causal role in the progression of heart failure. Here, we set out to determine the effect of β 3 -AR activation on TGFβ1 expression in rat cardiomyocytes and examine the underlying mechanism. The selective β 3 -AR agonist BRL37344 induced an increase in TGFβ1 expression and the phosphorylation of c-Jun N-terminal kinase (JNK) and c-Jun in β 3 -AR-overexpressing cardiomyocytes. Those effects of BRL37344 were suppressed by a β 3 -AR antagonist. Moreover, the inhibition of JNK and c-Jun activity by a JNK inhibitor and c-Jun siRNA blocked the increase in TGFβ1 expression upon β 3 -AR activation. A protein kinase G (PKG) inhibitor also attenuated β 3 -AR-agonist-induced TGFβ1 expression and the phosphorylation of JNK and c-Jun. In conclusion, the β 3 -AR activation in cardiomyocytes increases the expression of TGFβ1 via the PKG/JNK/c-Jun pathway. These results help us further understand the role of β 3 -AR signaling in heart failure. Copyright © 2018 Elsevier Inc. All rights reserved.

  14. Recruitment of Staufen2 Enhances Dendritic Localization of an Intron-Containing CaMKIIα mRNA.

    PubMed

    Ortiz, Raúl; Georgieva, Maya V; Gutiérrez, Sara; Pedraza, Neus; Fernández-Moya, Sandra M; Gallego, Carme

    2017-07-05

    Regulation of mRNA localization is a conserved cellular process observed in many types of cells and organisms. Asymmetrical mRNA distribution plays a particularly important role in the nervous system, where local translation of localized mRNA represents a key mechanism in synaptic plasticity. CaMKIIα is a very abundant mRNA detected in neurites, consistent with its crucial role at glutamatergic synapses. Here, we report the presence of CaMKIIα mRNA isoforms that contain intron i16 in dendrites, RNA granules, and synaptoneurosomes from primary neurons and brain. This subpopulation of unspliced mRNA preferentially localizes to distal dendrites in a synaptic-activity-dependent manner. Staufen2, a well-established marker of RNA transport in dendrites, interacts with intron i16 sequences and enhances its distal dendritic localization, pointing to the existence of intron-mediated mechanisms in the molecular pathways that modulate dendritic transport and localization of synaptic mRNAs. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

  15. Arctic ground squirrel (Spermophilus parryii) hippocampal neurons tolerate prolonged oxygen– glucose deprivation and maintain baseline ERK1/2 and JNK activation despite drastic ATP loss

    PubMed Central

    Christian, Sherri L; Ross, Austin P; Zhao, Huiwen W; Kristenson, Heidi J; Zhan, Xinhua; Rasley, Brian T; Bickler, Philip E; Drew, Kelly L

    2009-01-01

    Oxygen–glucose deprivation (OGD) initiates a cascade of intracellular responses that culminates in cell death in sensitive species. Neurons from Arctic ground squirrels (AGS), a hibernating species, tolerate OGD in vitro and global ischemia in vivo independent of temperature or torpor. Regulation of energy stores and activation of mitogen-activated protein kinase (MAPK) signaling pathways can regulate neuronal survival. We used acute hippocampal slices to investigate the role of ATP stores and extracellular signal-regulated kinase (ERK)1/2 and Jun NH2-terminal kinase (JNK) MAPKs in promoting survival. Acute hippocampal slices from AGS tolerated 30 mins of OGD and showed a small but significant increase in cell death with 2 h OGD at 37°C. This tolerance is independent of hibernation state or season. Neurons from AGS survive OGD despite rapid ATP depletion by 3 mins in interbout euthermic AGS and 10 mins in hibernating AGS. Oxygen–glucose deprivation does not induce JNK activation in AGS and baseline ERK1/2 and JNK activation is maintained even after drastic depletion of ATP. Surprisingly, inhibition of ERK1/2 or JNK during OGD had no effect on survival, whereas inhibition of JNK increased cell death during normoxia. Thus, protective mechanisms promoting tolerance to OGD by AGS are downstream from ATP loss and are independent of hibernation state or season. PMID:18398417

  16. JNK-1 Inhibition Leads to Antitumor Activity in Ovarian Cancer

    PubMed Central

    Vivas-Mejia, Pablo; Benito, Juliana Maria; Fernandez, Ariel; Han, Hee-Dong; Mangala, Lingegowda; Rodriguez-Aguayo, Cristian; Chavez-Reyes, Arturo; Lin, Yvonne G.; Nick, Alpa M.; Stone, Rebecca L.; Kim, Hye Sun; Claret, Francois-Xavier; Bornmann, William; Hennessy, Bryan TJ.; Sanguino, Angela; Peng, Zhengong; Sood, Anil K.; Lopez-Berestein, Gabriel

    2011-01-01

    Purpose To demonstrate the functional, clinical and biological significance of JNK-1 in ovarian carcinoma. Experimental Design Analysis of the impact of JNK on 116 epithelial ovarian cancers was conducted. The role of JNK in vitro and in experimental models of ovarian cancer was assessed. We studied the role of WBZ_4, a novel JNK inhibitor redesigned from imatinib based on targeting wrapping defects, in cell lines and in experimental models of ovarian cancer. Results We found a significant association of pJNK with progression free survival in the 116 epithelial ovarian cancers obtained at primary debulking therapy. WBZ_4 led to cell growth inhibition and increased apoptosis in a dose dependent fashion in four ovarian cancer cell lines. In vivo, while imatinib had no effect on tumor growth, WBZ_4 inhibited tumor growth in orthotopic murine models of ovarian cancer. The anti-tumor effect was further increased in combination with docetaxel. Silencing of JNK-1 with systemically administered siRNA led to significantly reduced tumor weights as compared to non-silencing siRNA controls, indicating that indeed the antitumor effects observed were due to JNK-1 inhibition. Conclusions These studies identify JNK-1 as an attractive therapeutic target in ovarian carcinoma and that the re-designed WBZ_4 compound should be considered for further clinical development. PMID:20028751

  17. (E)-3-(3,4-Dimethoxyphenyl)-1-(5-hydroxy-2,2-dimethyl-2H-chromen-6-yl)prop-2-en-1-one ameliorates the collagen-arthritis via blocking ERK/JNK and NF-κB signaling pathway.

    PubMed

    Li, Xiuxia; Peng, Fei; Xie, Caifeng; Wu, Wenshuang; Han, Xiaolei; Chen, Lijuan

    2013-12-01

    Our previous report has shown a natural pyranochalcones-derived compound, (E)-3-(3,4-Dimethoxyphenyl)-1-(5-hydroxy-2,2-dimethyl-2H-chromen-6-yl)prop-2-en-1-one (5b), that exerted protection against carrageenan-induced hind paw edema and adjuvant-induced arthritis. In this study, collagen-induced arthritis (CIA) model was used to further examine the anti-arthritic effects of 5b in vivo; the underlying molecular mechanisms of action were also investigated using a murine monocytic cell line, RAW264.7 cells. Here we showed that oral administration of 5b (20mg/kg) significantly suppressed the progression of arthritis. Improvement in disease severity was accompanied by inhibition of CD68-positive cells in knee joint and reduced pro-inflammatory cytokines TNF-α, IL-1β and IL-6 in serum. In vitro, 5b suppressed expressions of iNOS, cyclooxygenase-2 (COX-2), TNF-α, IL-6 and IL-1β as well as productions of nitric oxide (NO) and prostaglandin E2 (PGE2) in lipopolysaccharide (LPS)-treated macrophages. This compound also significantly suppressed LPS-induced NF-κB activation, including phosphorylation of I-κB, degradation of I-κB, and nuclear translocation of p65 and p50. Treatment with 5b also blocked LPS-induced expression of TLR4 remarkably, suppressed degradation of IRAKs and phosphorylations of JNK and ERK, but had little effect to p38 kinase activation. These findings indicated that 5b might be a therapeutic agent for rheumatoid arthritis, and exerted an anti-inflammatory effect mainly through mediating TLR4, NF-κB and ERK/JNK signaling pathways in monocytes. © 2013.

  18. Enteroaggregative Escherichia coli flagellin-induced interleukin-8 secretion requires Toll-like receptor 5-dependent p38 MAP kinase activation

    PubMed Central

    Khan, Mohammed A S; Kang, Jian; Steiner, Theodore S

    2004-01-01

    Enteroaggregative Escherichia coli (EAEC) is an emerging enteric pathogen that causes acute and chronic diarrhoea in a number of clinical settings. EAEC diarrhoea involves bacterial aggregation, adherence to intestinal epithelial cells and elaboration of several toxigenic bacterial mediators. Flagellin (FliC-EAEC), a major bacterial surface protein of EAEC, causes interleukin (IL)-8 release from several epithelial cell lines. The host response to flagellins from E. coli and several other bacteria is mediated by Toll-like receptor 5 (TLR5), which signals through nuclear factor kappa B (NF-κB) to induce transcription of pro-inflammatory cytokines. p38 mitogen-activating protein (MAP) kinase (MAPK) is a member of a family of stress-related kinases that influences a diverse range of cellular functions including host inflammatory responses to microbial products. We studied the role of p38 MAPK in FliC-EAEC-induced IL-8 secretion from Caco-2 human intestinal epithelial cells and THP-1 human monocytic cells. We found that IL-8 secretion from both cell types is dependent on p38 MAPK, which is phospho-activated in response to FliC-EAEC. The role of TLR5 in p38 MAPK-dependent IL-8 secretion was verified in HEp-2 cells transiently transfected with a TLR5 expression construct. Activation of interleukin-1 receptor-associated kinase (IRAK) was also observed in Caco-2 and TLR5-transfected HEp-2 cells after exposure to FliC-EAEC. Finally, we demonstrated that pharmacological inhibition of p38 MAPK reduced IL-8 transcription and mRNA levels, but did not affect NF-κB activation. Collectively, our results suggest that TLR5 mediates p38 MAPK-dependent IL-8 secretion from epithelial and monocytic cells incubated with FliC-EAEC, and that this effect requires IL-8 promoter activation independent of NF-κB nuclear migration. PMID:15270737

  19. Activation of the Jnk signaling pathway by a dual-specificity phosphatase, JSP-1

    PubMed Central

    Shen, Yu; Luche, Ralf; Wei, Bo; Gordon, Marcia L.; Diltz, Curtis D.; Tonks, Nicholas K.

    2001-01-01

    The mitogen-activated protein kinases (MAPKs) are integral to the mechanisms by which cells respond to physiological stimuli, such as growth factors, hormones, and cytokines, and to a wide variety of environmental stresses. The MAPKs, which are stimulated by phosphorylation of a TXY motif in their activation loop, are components of signal transduction cascades in which sequential activation of protein kinases culminates in their activation and their subsequent phosphorylation of various effector proteins that mediate the physiological response. MAPKs are also subject to dephosphorylation and inactivation, both by enzymes that recognize the residues of the TXY motif independently and by dual specificity phosphatases, which dephosphroylate both Tyr and Ser/Thr residues. We report the identification and characterization of a novel dual specificity phosphatase. Contrary to expectation, this broadly expressed enzyme did not inactivate MAPKs in transient cotransfection assays but instead displayed the capacity to function as a selective activator of the MAPK Jnk, hence the name, Jnk Stimulatory Phosphatase-1 (JSP-1). This study illustrates a new aspect of the regulation of MAPK-dependent signal transduction and raises the possibility that JSP-1 may offer a different perspective to the study of various inflammatory and proliferative disorders associated with dysfunctional Jnk signaling. PMID:11717427

  20. Activation of the Jnk signaling pathway by a dual-specificity phosphatase, JSP-1.

    PubMed

    Shen, Y; Luche, R; Wei, B; Gordon, M L; Diltz, C D; Tonks, N K

    2001-11-20

    The mitogen-activated protein kinases (MAPKs) are integral to the mechanisms by which cells respond to physiological stimuli, such as growth factors, hormones, and cytokines, and to a wide variety of environmental stresses. The MAPKs, which are stimulated by phosphorylation of a TXY motif in their activation loop, are components of signal transduction cascades in which sequential activation of protein kinases culminates in their activation and their subsequent phosphorylation of various effector proteins that mediate the physiological response. MAPKs are also subject to dephosphorylation and inactivation, both by enzymes that recognize the residues of the TXY motif independently and by dual specificity phosphatases, which dephosphroylate both Tyr and Ser/Thr residues. We report the identification and characterization of a novel dual specificity phosphatase. Contrary to expectation, this broadly expressed enzyme did not inactivate MAPKs in transient cotransfection assays but instead displayed the capacity to function as a selective activator of the MAPK Jnk, hence the name, Jnk Stimulatory Phosphatase-1 (JSP-1). This study illustrates a new aspect of the regulation of MAPK-dependent signal transduction and raises the possibility that JSP-1 may offer a different perspective to the study of various inflammatory and proliferative disorders associated with dysfunctional Jnk signaling.

  1. Dihydroartemisinin induces endothelial cell anoikis through the activation of the JNK signaling pathway

    PubMed Central

    Zhang, Jiao; Guo, Ling; Zhou, Xia; Dong, Fengyun; Li, Liqun; Cheng, Zuowang; Xu, Yinghua; Liang, Jiyong; Xie, Qi; Liu, Ju

    2016-01-01

    Angiogenesis is required for the growth and metastasis of solid tumors. The anti-malarial agent dihydroartemisinin (DHA) demonstrates potent anti-angiogenic activity, but the underlying molecular mechanisms are not yet fully understood. During the process of angiogenesis, endothelial cells migrating from existing capillaries may undergo programmed cell death after detaching from the extracellular matrix, a process that is defined as anchorage-dependent apoptosis or anoikis. In the present study, DHA-induced cell death was compared in human umbilical vein endothelial cells (HUVECs) cultured in suspension and attached to culture plates. In suspended HUVECs, the cell viability was decreased and apoptosis was increased with the treatment of 50 µM DHA for 5 h, while the same treatment did not affect the attached HUVECs. In addition, 50 µM DHA increased the phosphorylation of c-Jun N-terminal kinase (JNK) in suspended HUVECs, but not in attached HUVECs, for up to 5 h of treatment. The JNK inhibitor, SP600125, reversed DHA-induced cell death in suspended HUVECs, suggesting that the JNK pathway may mediate DHA-induced endothelial cell anoikis. The data from the present study indicates a novel mechanism for understanding the anti-angiogenic effects of DHA, which may be used as a component for chemotherapy. PMID:27602117

  2. Insulin Signaling Augments eIF4E-Dependent Nonsense-Mediated mRNA Decay in Mammalian Cells.

    PubMed

    Park, Jungyun; Ahn, Seyoung; Jayabalan, Aravinth K; Ohn, Takbum; Koh, Hyun Chul; Hwang, Jungwook

    2016-07-01

    Nonsense-mediated mRNA decay (NMD) modulates the level of mRNA harboring a premature termination codon (PTC) in a translation-dependent manner. Inhibition of translation is known to impair NMD; however, few studies have investigated the correlation between enhanced translation and increased NMD. Here, we demonstrate that insulin signaling events increase translation, leading to an increase in NMD of eIF4E-bound transcripts. We provide evidence that (i) insulin-mediated enhancement of translation augments NMD and rapamycin abrogates this enhancement; (ii) an increase in AKT phosphorylation due to inhibition of PTEN facilitates NMD; (iii) insulin stimulation increases the binding of up-frameshift factor 1 (UPF1), most likely to eIF4E-bound PTC-containing transcripts; and (iv) insulin stimulation induces the colocalization of UPF1 and eIF4E in processing bodies. These results illustrate how extracellular signaling promotes the removal of eIF4E-bound NMD targets. Copyright © 2015 Elsevier B.V. All rights reserved.

  3. Low humidity environmental challenge causes barrier disruption and cornification of the mouse corneal epithelium via a c-jun N-terminal kinase 2 (JNK2) pathway.

    PubMed

    Pelegrino, F S A; Pflugfelder, S C; De Paiva, C S

    2012-01-01

    Patients with tear dysfunction often experience increased irritation symptoms when subjected to drafty and/or low humidity environmental conditions. The purpose of this study was to investigate the effects of low humidity stress (LHS) on corneal barrier function and expression of cornified envelope (CE) precursor proteins in the epithelium of C57BL/6 and c-jun N-terminal kinase 2 (JNK2) knockout (KO) mice. LHS was induced in both strains by exposure to an air draft for 15 (LHS15D) or 30 days (LHS30D) at a relative humidity <30%RH. Nonstressed (NS) mice were used as controls. Oregon-green-dextran uptake was used to measure corneal barrier function. Levels of small proline-rich protein (SPRR)-2, involucrin, occludin, and MMP-9 were evaluated by immunofluorescent staining in cornea sections. Wholemount corneas immunostained for occludin were used to measure mean apical cell area. Gelatinase activity was evaluated by in situ zymography. Expression of MMP, CE and inflammatory cytokine genes was evaluated by qPCR. C57BL/6 mice exposed to LHS15D showed corneal barrier dysfunction, decreased apical corneal epithelial cell area, higher MMP-9 expression and gelatinase activity and increased involucrin and SPRR-2 immunoreactivity in the corneal epithelium compared to NS mice. JNK2KO mice were resistant to LHS-induced corneal barrier disruption. MMP-3,-9,-13, IL-1α, IL-1β, involucrin and SPRR-2a RNA transcripts were significantly increased in C57BL/6 mice at LHS15D, while no change was noted in JNK2KO mice. LHS is capable of altering corneal barrier function, promoting pathologic alteration of the TJ complex and stimulating production of CE proteins by the corneal epithelium. Activation of the JNK2 signaling pathway contributes to corneal epithelial barrier disruption in LHS. Copyright © 2011 Elsevier Ltd. All rights reserved.

  4. Interleukin-10 Overexpression Promotes Fas-Ligand-Dependent Chronic Macrophage-Mediated Demyelinating Polyneuropathy

    PubMed Central

    Dace, Dru S.; Khan, Aslam A.; Stark, Jennifer L.; Kelly, Jennifer; Cross, Anne H.; Apte, Rajendra S.

    2009-01-01

    Background Demyelinating polyneuropathy is a debilitating, poorly understood disease that can exist in acute (Guillain-Barré syndrome) or chronic forms. Interleukin-10 (IL-10), although traditionally considered an anti-inflammatory cytokine, has also been implicated in promoting abnormal angiogenesis in the eye and in the pathobiology of autoimmune diseases such as lupus and encephalomyelitis. Principal Findings Overexpression of IL-10 in a transgenic mouse model leads to macrophage-mediated demyelinating polyneuropathy. IL-10 upregulates ICAM-1 within neural tissues, promoting massive macrophage influx, inflammation-induced demyelination, and subsequent loss of neural tissue resulting in muscle weakness and paralysis. The primary insult is to perineural myelin followed by secondary axonal loss. Infiltrating macrophages within the peripheral nerves demonstrate a highly pro-inflammatory signature. Macrophages are central players in the pathophysiology, as in vivo depletion of macrophages using clodronate liposomes reverses the phenotype, including progressive nerve loss and paralysis. Macrophage-mediate demyelination is dependent on Fas-ligand (FasL)-mediated Schwann cell death. Significance These findings mimic the human disease chronic idiopathic demyelinating polyneuropathy (CIDP) and may also promote further understanding of the pathobiology of related conditions such as acute idiopathic demyelinating polyneuropathy (AIDP) or Guillain-Barré syndrome. PMID:19771172

  5. Glutamine deprivation stimulates mTOR-JNK-dependent chemokine secretion

    PubMed Central

    Shanware, Naval P.; Bray, Kevin; Eng, Christina H.; Wang, Fang; Follettie, Maximillian; Myers, Jeremy; Fantin, Valeria R.; Abraham, Robert T.

    2014-01-01

    The non-essential amino acid, glutamine, exerts pleiotropic effects on cell metabolism, signalling and stress resistance. Here we demonstrate that short-term glutamine restriction triggers an endoplasmic reticulum (ER) stress response that leads to production of the pro-inflammatory chemokine, interleukin-8 (IL-8). Glutamine deprivation-induced ER stress triggers colocalization of autophagosomes, lysosomes and the Golgi into a subcellular structure whose integrity is essential for IL-8 secretion. The stimulatory effect of glutamine restriction on IL-8 production is attributable to depletion of tricarboxylic acid cycle intermediates. The protein kinase, mTOR, is also colocalized with the lysosomal membrane clusters induced by glutamine deprivation, and inhibition of mTORC1 activity abolishes both endomembrane reorganization and IL-8 secretion. Activated mTORC1 elicits IL8 gene expression via the activation of an IRE1-JNK signalling cascade. Treatment of cells with a glutaminase inhibitor phenocopies glutamine restriction, suggesting that these results will be relevant to the clinical development of glutamine metabolism inhibitors as anticancer agents. PMID:25254627

  6. Inactivation of JNK1 enhances innate IL-10 production and dampens autoimmune inflammation in the brain.

    PubMed

    Tran, Elise H; Azuma, Yasu-Taka; Chen, Manchuan; Weston, Claire; Davis, Roger J; Flavell, Richard A

    2006-09-05

    Environmental insults such as microbial pathogens can contribute to the activation of autoreactive T cells, leading to inflammation of target organs and, ultimately, autoimmune disease. Various infections have been linked to multiple sclerosis and its animal counterpart, autoimmune encephalomyelitis. The molecular process by which innate immunity triggers autoreactivity is not currently understood. By using a mouse model of multiple sclerosis, we found that the genetic loss of the MAPK, c-Jun N-terminal kinase 1 (JNK1), enhances IL-10 production, rendering innate myeloid cells unresponsive to certain microbes and less capable of generating IL-17-producing, encephalitogenic T cells. Moreover, JNK1-deficient central nervous system myeloid cells are unable to respond to effector T cell inflammatory cytokines, preventing further progression to neuroinflammation. Thus, we have identified the JNK1 signal transduction pathway in myeloid cells to be a critical component of a regulatory circuit mediating inflammatory responses in autoimmune disease. Our findings provide further insights into the pivotal MAPK-regulated network of innate and adaptive cytokines in the progression to autoimmunity.

  7. The induction of cytochrome P450 2E1 by ethanol leads to the loss of synaptic proteins via PPARα down-regulation.

    PubMed

    Na, Shufang; Li, Jie; Zhang, Huibo; Li, Yueran; Yang, Zheqiong; Zhong, Yanjun; Dong, Guicheng; Yang, Jing; Yue, Jiang

    2017-06-15

    Ethanol, one of the most commonly abused substances throughout history, is a substrate and potent inducer of cytochrome P450 2E1 (CYP2E1). Our previous study showed that brain CYP2E1 was induced by chronic ethanol treatment and was associated with ethanol-induced neurotoxicity in rats. We therefore investigated the possible mechanism of brain CYP2E1 involvement in ethanol-induced neurodegeneration. Compared with the controls, chronic ethanol treatment (3.0g/kg, i.g., 160days) significantly increased CYP2E1 mRNA levels in the rat cortex, but the mRNA levels of peroxisome proliferator-activated receptor α (PPARα) and the pre- and post-synaptic proteins (synaptophysin, SYP, and drebrin1, DBN1) were decreased. Ethanol treatment dose-dependently induced CYP2E1 mRNA expression, and CYP2E1 overexpression exacerbated the ethanol-induced neurotoxicity. Pretreatment with p38 inhibitor (SB202190) and ERK1/2 inhibitor (U0126) attenuated the induction of CYP2E1 mRNA and protein levels by ethanol; however, no change was observed with JNK inhibitor pretreatment. Ethanol exposure or CYP2E1 overexpression significantly decreased PPARα, SYP, and DBN1 expression as indicated by the data from real-time RT-PCR, Western blotting and immunocytochemistry. The activation of PPARα by WY14643 increased the activity of the SYP and DBN1 promoters and attenuated the inhibition of these genes by ethanol. The specific siRNA for CYP2E1 significantly attenuated the ethanol-induced inhibition of PPARα, SYP and DBN1 mRNA levels. These results suggest that the induction of CYP2E1 by ethanol may be mediated via the p38 and ERK1/2 signaling pathways in neurons but not via the JNK pathway. The CYP2E1-PPARα axis may play a role in ethanol-induced neurotoxicity via the alteration of the genes related with synaptic function. Copyright © 2017. Published by Elsevier B.V.

  8. A case of cervical cancer expressed three mRNA variant of Hyaluronan-mediated motility receptor

    PubMed Central

    Villegas-Ruíz, Vanessa; Salcedo, Mauricio; Zentella-Dehesa, Alejandro; de Oca, Edén V Montes; Román-Basaure, Edgar; Mantilla-Morales, Alejandra; Dávila-Borja, Víctor M; Juárez-Méndez, Sergio

    2014-01-01

    Cervical cancer is the second malignancy in Mexico, little is known about the prognostic factors associated with this disease. Several cellular components are important in their transformation and progression. Alternative mRNA splice is an important mechanism for generating protein diversity, nevertheless, in cancer unknown mRNA diversity is expressed. Hyaluronan-mediated motility receptor (HMMR, RHAMM, CD168) is a family member of proteins, hyaluronan acid dependent, and has been associated with different malignant processes such as: angiogenesis, cell invasiveness, proliferation, metastasis and poor outcome in some tumors. In the present study we identified expression of HMMR in cervical cancer by means of RT-PCR and sequencing. Our results indicate co-expression of two HMMR variants in all samples, and one case expressed three alternative HMMR splice transcripts. These results showed the heterogeneity of mRNA transcripts of HMMR that could express in cancer and the expression of HMMR could be marker of malignancy in CC. PMID:24966934

  9. Albumin-induced podocyte injury and protection are associated with regulation of COX-2.

    PubMed Central

    Agrawal, Shipra; Guess, Adam J.; Chanley, Melinda A.; Smoyer, and William E.

    2014-01-01

    Albuminuria is both a hallmark and a risk factor for progressive glomerular disease, and results in increased exposure of podocytes to serum albumin with its associated factors. Here in vivo and in vitro models of serum albumin overload were used to test the hypothesis that albumin-induced proteinuria and podocyte injury directly correlate with COX-2 induction. Albumin induced COX-2, MCP-1, CXCL1 and the stress protein HSP25 in both rat glomeruli and cultured podocytes, while B7-1 and HSP70i were also induced in podocytes. Podocyte exposure to albumin induced both mRNA and protein and enhanced the mRNA stability of COX-2, a key regulator of renal hemodynamics and inflammation, which renders podocytes susceptible to injury. Podocyte exposure to albumin also stimulated several kinases (p38 MAPK, MK2, JNK/SAPK and ERK1/2), inhibitors of which (except JNK/SAPK) down-regulated albumin-induced COX-2. Inhibition of AMPK, PKC and NFκB also down-regulated albumin-induced COX-2. Critically, albumin-induced COX-2 was also inhibited by glucocorticoids and thiazolidinediones, both of which directly protect podocytes against injury. Furthermore, specific albumin-associated fatty acids were identified as important contributors to COX-2 induction, podocyte injury and proteinuria. Thus, COX-2 is associated with podocyte injury during albuminuria, as well as with the known podocyte protection imparted by glucocorticoids and thiazolidinediones. Moreover, COX-2 induction, podocyte damage and albuminuria appear mediated largely by serum albumin-associated fatty acids. PMID:24918154

  10. Regulation of 1-alpha, 25-dihydroxyvitamin D3 on interleukin-6 and interleukin-8 induced by sulfur mustard (HD) on human skin cells.

    PubMed

    Arroyo, Carmen M; Kan, Robert K; Burman, Damon L; Kahler, David W; Nelson, Marian R; Corun, Charlene M; Guzman, Juanita J; Broomfield, Clarence A

    2003-05-01

    The regulatory effects of the active form of vitamin D, 1-alpha, 25-dihydroxyvitamin D3 (1-alpha, 25 (OH)2D3) were assessed on the cytokine and chemokine secretion induced by sulfur mustard on human skin fibroblasts and human epidermal keratinocytes. Stimulation of human skin fibroblasts with sulfur mustard (10(-4) M for 24 hr at 37 degrees ) resulted in approximately a 5 times increase in the secretion of interleukin-6 and over a 10 times increase for interleukin-8, which was inhibited by 1-alpha, 25 (OH)2D3, at 2D3 also suppressed interleukin-8 secretion by 5 times and interleukin-6 by 4 times on sulfur mustard-stimulated human epidermal keratinocytes at concentrations 2D3 was dose-dependent for the suppression of interleukin-6 and interleukin-8 induced by sulfur mustard on human skin fibroblasts/human epidermal keratinocytes, apparent at nanomolar concentrations. Our results indicate that the suppression of these inflammatory mediators by 1-alpha, 25 (OH)2D3 is dependent on the source of the primary cultures, cell densities, and kinetics of pretreatments. In contrast to the inhibition of cytokine/chemokine production, cell proliferation was enhanced by almost 1.7 times on treated human epidermal keratinocytes with 1-alpha, 25 (OH)2D3 (1 x 10(-9) M) after sulfur mustard-stimulation (10(-4) M for 24 hr at 37 degrees C). The observed enhancement diversified based on cell density, and kinetics of pretreatment with a maximal synergism (s) observed at 1 x 10(-9) M. Photomicrographs show typical signs of cellular degeneration caused by sulfur mustard such as chromatin condensation. The observed cellular degeneration was lessened when human epidermal keratinocytes were treated with 1-alpha, 25 (OH)2D3 (2 x 10(-9) M). 1-alpha, 25(OH)2D3 could be an alternative treatment for cutaneous inflammation disorders caused by sulfur mustard because we have demonstrated its ability to suppress inflammatory

  11. Hypoxia induces IGFBP3 in esophageal squamous cancer cells through HIF-1α-mediated mRNA transcription and continuous protein synthesis

    PubMed Central

    Natsuizaka, Mitsuteru; Naganuma, Seiji; Kagawa, Shingo; Ohashi, Shinya; Ahmadi, Azal; Subramanian, Harry; Chang, Sanders; Nakagawa, Kei J.; Ji, Xinjun; Liebhaber, Stephen A.; Klein-Szanto, Andres J.; Nakagawa, Hiroshi

    2012-01-01

    Insulin-like growth factor binding protein (IGFBP)-3 regulates cell proliferation and apoptosis in esophageal squamous cell carcinoma (ESCC) cells. We have investigated how the hypoxic tumor microenvironment in ESCC fosters the induction of IGFBP3. RNA interference experiments revealed that hypoxia-inducible factor (HIF)-1α, but not HIF-2α, regulates IGFBP3 mRNA induction. By chromatin immunoprecipitation and transfection assays, HIF-1α was found to transactivate IGFBP3 through a novel hypoxia responsive element (HRE) located at 57 kb upstream from the transcription start site. Metabolic labeling experiments demonstrated hypoxia-mediated inhibition of global protein synthesis. 7-Methyl GTP-cap binding assays suggested that hypoxia suppresses cap-dependent translation. Experiments using pharmacological inhibitors for mammalian target of rapamycin (mTOR) suggested that a relatively weak mTOR activity may be sufficient for cap-dependent translation of IGFBP3 under hypoxic conditions. Bicistronic RNA reporter transfection assays did not validate the possibility of an internal ribosome entry site as a potential mechanism for cap-independent translation for IGFBP3 mRNA. Finally, IGFBP3 mRNA was found enriched to the polysomes. In aggregate, our study establishes IGFBP3 as a direct HIF-1α target gene and that polysome enrichment of IGFBP3 mRNA may permit continuous translation under hypoxic conditions.—Natsuizaka, M., Naganuma, S., Kagawa, S., Ohashi, S., Ahmadi, A., Subramanian, H., Chang, S., Nakagawa, K. J., Ji, X., Liebhaber, S. A., Klein-Szanto, A. J., Nakagawa, H. Hypoxia induces IGFBP3 in esophageal squamous cancer cells through HIF-1α-mediated mRNA transcription and continuous protein synthesis. PMID:22415309

  12. Tethering of human Ago proteins to mRNA mimics the miRNA-mediated repression of protein synthesis.

    PubMed

    Pillai, Ramesh S; Artus, Caroline G; Filipowicz, Witold

    2004-10-01

    MicroRNAs (miRNAs) are approximately 21-nt-long RNAs involved in regulating development, differentiation, and other processes in eukaryotes. In metazoa, nearly all miRNAs control gene expression by imperfectly base-pairing with the 3'-untranslated region (3'-UTR) of target mRNAs and repressing protein synthesis by an unknown mechanism. It is also unknown whether miRNA-mRNA duplexes containing mismatches and bulges provide specific features that are recognized by factors mediating the repression. miRNAs form part of ribonucleoprotein complexes, miRNPs, that contain Argonaute (Ago) and other proteins. Here we demonstrate that effects of miRNAs on translation can be mimicked in human HeLa cells by the miRNA-independent tethering of Ago proteins to the 3'-UTR of a reporter mRNA. Inhibition of protein synthesis occurred without a change in the reporter mRNA level and was dependent on the number, but not the position, of the hairpins tethering hAgo2 to the 3'-UTR. These findings indicate that a primary function of miRNAs is to guide their associated proteins to the mRNA. Copyright 2004 RNA Society

  13. Effects of RNAi-Mediated Knockdown of Histone Methyltransferases on the Sex-Specific mRNA Expression of Imp in the Silkworm Bombyx mori

    PubMed Central

    Suzuki, Masataka G.; Ito, Haruka; Aoki, Fugaku

    2014-01-01

    Sexual differentiation in Bombyx mori is controlled by sex-specific splicing of Bmdsx, which results in the omission of exons 3 and 4 in a male-specific manner. In B. mori, insulin-like growth factor II mRNA-binding protein (Imp) is a male-specific factor involved in male-specific splicing of Bmdsx. Male-specific Imp mRNA results from the male-specific inclusion of exon 8. To verify the link between histone methylation and alternative RNA processing in Imp, we examined the effects of RNAi-mediated knockdown of several histone methyltransferases on the sex-specific mRNA expression of Imp. As a result, male-specific expression of Imp mRNA was completely abolished when expression of the H3K79 methyltransferase DOT1L was repressed to <10% of that in control males. Chromatin immunoprecipitation-quantitative PCR analysis revealed a higher distribution of H3K79me2 in normal males than in normal females across Imp. RNA polymerase II (RNAP II) processivity assays indicated that RNAi knockdown of DOT1L in males caused a twofold decrease in RNAP II processivity compared to that in control males, with almost equivalent levels to those observed in normal females. Inhibition of RNAP II-mediated elongation in male cells repressed the male-specific splicing of Imp. Our data suggest the possibility that H3K79me2 accumulation along Imp is associated with the male-specific alternative processing of Imp mRNA that results from increased RNAP II processivity. PMID:24758924

  14. Protein kinase-A-dependent osteoprotegerin production on interleukin-1 stimulation in human gingival fibroblasts is distinct from periodontal ligament fibroblasts

    PubMed Central

    Hormdee, D; Nagasawa, T; Kiji, M; Yashiro, R; Kobayashi, H; Koshy, G; Noguchi, K; Nitta, H; Ishikawa, I

    2005-01-01

    Periodontitis, a chronic inflammatory disease, is characterized by increased expression of interleukin (IL)-1 and other inflammatory mediators resulting in extensive osteoclast formation and bone loss. Expression of receptor activator of nuclear factor kappa B ligand (RANKL) and its decoy receptor, osteoprotegerin (OPG), by osteoblasts is important to regulate osteoclast differentiation. The aim of the present study was to investigate the regulatory effects of IL-1 on RANKL and OPG production by mesenchymal fibroblasts in periodontal tissue. Human gingival fibroblasts (HGF) and periodontal ligament fibroblasts (PDL) were stimulated with IL-1α with or without protein synthesis inhibitor cycloheximide (CHX), protein kinase A (PKA) inhibitors, protein kinase C (PKC) inhibitors and prostaglandin E2 (PGE2) inhibitor. In some experiments, the cultured cells were directly stimulated with either PKA or PKC activators. In HGF, IL-1α-stimulated OPG mRNA expression was high and could be reduced by CHX. PKA inhibitor completely abrogated IL-1α-induced OPG mRNA expression and OPG production. Endogenous PGE2 further enhanced IL-1α-induced OPG production in HGF. In PDL, RANKL mRNA expression was greatly augmented by IL-1α. IL-1α induced OPG mRNA expression and protein production. PKC inhibitor partially reduced IL-1α-induced OPG production and PKC activator enhanced OPG production in PDL. The IL-1α-stimulated OPG mRNA expression in HGF was greater than PDL. These results provide new evidence for the possible osteoclastogenesis-inhibitory function of HGF through PKA activity pathway. PDL utilized PKC for OPG production. Thus, we emphasize that HGF and PDL have different characteristics of host defence mechanism against inflammatory process. PMID:16297161

  15. Propofol protects hippocampal neurons from apoptosis in ischemic brain injury by increasing GLT-1 expression and inhibiting the activation of NMDAR via the JNK/Akt signaling pathway.

    PubMed

    Gong, Hong-Yan; Zheng, Fang; Zhang, Chao; Chen, Xi-Yan; Liu, Jing-Jing; Yue, Xiu-Qin

    2016-09-01

    Ischemic brain injury (IBI) can cause nerve injury and is a leading cause of morbidity and mortality worldwide. The neuroprotective effects of propofol against IBI have been previously demonstrated. However, the neuroprotective effects of propofol on hippocampal neurons are not yet entirely clear. In the present study, models of IBI were established in hypoxia-exposed hippocampal neuronal cells. Cell viability assay and apoptosis assay were performed to examine the neuroprotective effects of propofol on hippocampal neurons in IBI. A significant decrease in cell viability and a significant increase in cell apoptosis were observed in the IBI group compared with the control group, accompanied by a decrease in glial glutamate transporter-1 (GLT‑1) expression as determined by RT-qPCR and western blot analysis. The effects of IBI were reversed by propofol treatment. The siRNA-mediated knockdown of GLT‑1 in the hypoxia-exposed hippocampal neuronal cells led to an increase in cell apoptosis, Jun N-terminal kinase (JNK) activation and N-methyl-D‑aspartate (NMDA) receptor (NR1 and NR2B) activation, as well as to a decrease in cell viability and a decrease in Akt activation. The effects of RNA interference-mediated GLT‑1 gene silencing on cell viability, JNK activation, NMDAR activation, cell apoptosis and Akt activation in the hippocampal neuronal cells were slightly reversed by propofol treatment. The JNK agonist, anisomycin, and the Akt inhibitor, LY294002, both significantly blocked the effects of propofol on hippocampal neuronal cell viability and apoptosis in IBI. The decrease in JNK activation and the increase in Akt activation caused by GLT‑1 overexpression were reversed by NMDA. Collectively, our findings suggest that propofol treatment protects hippocampal neurons against IBI by enhancing GLT‑1 expression and inhibiting the activation of NMDAR via the JNK/Akt signaling pathway.

  16. Induced expression of mRNA for IL-5, IL-6, TNF-alpha, MIP-2 and IFN-gamma in immunologically activated rat peritoneal mast cells: inhibition by dexamethasone and cyclosporin A.

    PubMed Central

    Williams, C M; Coleman, J W

    1995-01-01

    We examined the capacity of purified rat peritoneal connective tissue-type mast cells (PMC) to express mRNA for several cytokines. Stimulation of PMC with anti-IgE for 4 hr induced the expression of mRNA encoding interleukin-5 (IL-5), IL-6, tumour necrosis factor-alpha (TNF-alpha), macrophage inflammatory protein-2 (MIP-2) and interferon-gamma (IFN-gamma). Unstimulated PMC expressed detectable mRNA for TNF-alpha but not for the other four cytokines. Incubation of PMC with cyclosporin A (CsA) or dexamethasone (DEX), each at 10(-6) M for 24 hr, significantly inhibited the induced expression of mRNA for each of the five cytokines, and also inhibited release of biologically active TNF-alpha. Throughout these experiments mRNA levels of the housekeeping gene G3PDH were not altered by stimulation with anti-IgE or incubation with CsA or DEX. We conclude that immunological activation of rat PMC induces gene expression of several cytokines and that expression of these genes can be inhibited by immunosuppressive drugs. Images Figure 1 Figure 2 Figure 3 PMID:7490125

  17. ROCK mediates phorbol ester-induced apoptosis in prostate cancer cells via p21Cip1 up-regulation and JNK.

    PubMed

    Xiao, Liqing; Eto, Masumi; Kazanietz, Marcelo G

    2009-10-23

    It is established that androgen-dependent prostate cancer cells undergo apoptosis upon treatment with phorbol esters and related analogs, an effect primarily mediated by PKCdelta. Treatment of LNCaP prostate cancer cells with phorbol 12-myristate 13-acetate (PMA) causes a strong and sustained activation of RhoA and its downstream effector ROCK (Rho kinase) as well as the formation of stress fibers. These effects are impaired in cells subjected to PKCdelta RNA interference depletion. Functional studies revealed that expression of a dominant negative RhoA mutant or treatment with the ROCK inhibitor Y-27632 inhibits the apoptotic effect of PMA in LNCaP cells. Remarkably, the cytoskeleton inhibitors cytochalasin B and blebbistatin blocked not only PMA-induced apoptosis but also the activation of JNK, a mediator of the cell death effect by the phorbol ester. In addition, we found that up-regulation of the cell cycle inhibitor p21(Cip1) is required for PMA-induced apoptosis and that inhibitors of ROCK or the cytoskeleton organization prevent p21(Cip1) induction. Real time PCR analysis and reporter gene assay revealed that PMA induces p21(Cip1) transcriptionally in a ROCK- and cytoskeleton-dependent manner. p21(Cip1) promoter analysis revealed that PMA induction is dependent on Sp1 elements in the p21(Cip1) promoter but independent of p53. Taken together, our studies implicate ROCK-mediated up-regulation of p21(Cip1) and the cytoskeleton in PKCdelta-dependent apoptosis in prostate cancer cells.

  18. Power Frequency Magnetic Fields Affect the p38 MAPK-Mediated Regulation of NB69 Cell Proliferation Implication of Free Radicals.

    PubMed

    Martínez, María Antonia; Úbeda, Alejandro; Moreno, Jorge; Trillo, María Ángeles

    2016-04-06

    The proliferative response of the neuroblastoma line NB69 to a 100 µT, 50 Hz magnetic field (MF) has been shown mediated by activation of the MAPK-ERK1/2 pathway. This work investigates the MF effect on the cell cycle of NB69, the participation of p38 and c-Jun N-terminal (JNK) kinases in the field-induced proliferative response and the potential involvement of reactive oxygen species (ROS) in the activation of the MAPK-ERK1/2 and -p38 signaling pathways. NB69 cultures were exposed to the 100 µT MF, either intermittently for 24, 42 or 63 h, or continuously for periods of 15 to 120 min, in the presence or absence of p38 or JNK inhibitors: SB203580 and SP600125, respectively. Antioxidant N-acetylcysteine (NAC) was used as ROS scavenger. Field exposure induced transient activation of p38, JNK and ERK1/2. The MF proliferative effect, which was mediated by changes in the cell cycle, was blocked by the p38 inhibitor, but not by the JNK inhibitor. NAC blocked the field effects on cell proliferation and p38 activation, but not those on ERK1/2 activation. The MF-induced proliferative effects are exerted through sequential upregulation of MAPK-p38 and -ERK1/2 activation, and they are likely mediated by a ROS-dependent activation of p38.

  19. Antiallergic Activity of Ethanol Extracts of Arctium lappa L. Undried Roots and Its Active Compound, Oleamide, in Regulating FcεRI-Mediated and MAPK Signaling in RBL-2H3 Cells.

    PubMed

    Yang, Woong-Suk; Lee, Sung Ryul; Jeong, Yong Joon; Park, Dae Won; Cho, Young Mi; Joo, Hae Mi; Kim, Inhye; Seu, Young-Bae; Sohn, Eun-Hwa; Kang, Se Chan

    2016-05-11

    The antiallergic potential of Arctium lappa L. was investigated in Sprague-Dawley rats, ICR mice, and RBL-2H3 cells. Ethanol extract (90%) of A. lappa (ALE, 100 μg/mL) inhibited the degranulation rate by 52.9%, determined by the level of β-hexosaminidase. ALE suppressed passive cutaneous anaphylaxis (PCA) in rats and attenuated anaphylaxis and histamine release in mice. To identify the active compound of ALE, we subsequently fractionated and determined the level of β-hexosaminidase in all subfractions. Oleamide was identified as an active compound of ALE, which attenuated the secretion of histamine and the production of tumor necrosis factor (TNF)-α and interleukin-4 (IL-4) in cells treated with compound 48/80 or A23187/phorbol myristate acetate (PMA). Oleamide suppressed FcεRI-tyrosine kinase Lyn-mediated pathway, c-Jun N-terminal kinases (JNK/SAPK), and p38 mitogen-activated protein kinases (p38-MAPKs). These results showed that ALE and oleamide attenuated allergic reactions and should serve as a platform to search for compounds with antiallergic activity.

  20. Inhibitory effect of Styrax Japonica Siebold et al. Zuccarini glycoprotein (38 kDa) on interleukin-1β and induction proteins in chromium(VI)-treated BNL CL.2 cells.

    PubMed

    Lee, Jin; Lim, Kye-Taek

    2012-08-01

    Chromium(VI) [Cr(VI)] induces chronic inflammation in hepatocytes. Inflammation has been shown to play an important role in tumorigenesis, tumor progression, and metastasis. To examine the effects of the Styrax Japonica Siebold et al. Zuccarini (SJSZ) glycoprotein on inflammation in BNL CL.2 cells, we evaluated the activities of c-Jun NH(2)-terminal kinase (JNK), extracellular signal-regulated kinase (ERK), nuclear factor (NF)-κB (p50 and p65), and inflammation-related factors [cyclooxygenase (COX)-2, inducible nitric oxide syntheses (iNOS) and interleukin (IL)-1β] in Cr-induced BNL CL.2 cells using immunoblot analysis and RT-PCR. We also used two-dimensional gel electrophoresis (2-DE) to compare between treatments. To determine which proteins are induced by Cr(VI), we evaluated total protein lysates using 2-DE. After Cr(VI) treatment, total protein lysates were prepared and resolved by 2-DE. The results obtained from this study demonstrated that the SJSZ glycoprotein (50 μg/ml) inhibits expression of JNK, ERK, NF-κB, and the expression of COX-2, iNOS, and IL-1β. Moreover, the results obtained from 2DE showed that four proteins out of nine proteins were relatively expressed strongly, while the rest of them were relatively appeared weakly on the gel. Taken together, these data indicate that the SJSZ glycoprotein prevents expression of COX-2, iNOS, and IL-1β by blocking NF-κB and MAPKs in Cr(VI)-induced BNL CL.2 cells.

  1. Interleukin Expression after Injury and the Effects of Interleukin-1 Receptor Antagonist

    PubMed Central

    Chamberlain, Connie S.; Leiferman, Ellen M.; Frisch, Kayt E.; Brickson, Stacey L.; Murphy, William L.; Baer, Geoffrey S.; Vanderby, Ray

    2013-01-01

    Ligament healing follows a series of complex coordinated events involving various cell types, cytokines, as well as other factors, producing a mechanically inferior tissue more scar-like than native tissue. Macrophages provide an ongoing source of cytokines to modulate inflammatory cell adhesion and migration as well as fibroblast proliferation. Studying interleukins inherent to ligament healing during peak macrophage activation and angiogenesis may elucidate inflammatory mediators involved in subsequent scar formation. Herein, we used a rat healing model assayed after surgical transection of their medial collateral ligaments (MCLs). On days 3 and 7 post-injury, ligaments were collected and used for microarray analysis. Of the 12 significantly modified interleukins, components of the interleukin-1 family were significantly up-regulated. We therefore examined the influence of interleukin-1 receptor antagonist (IL-1Ra) on MCL healing. Transected rat MCLs received PBS or IL-1Ra at the time of surgery. Inhibition of IL-1 activation decreased pro-inflammatory cytokines (IL-1α, IL-1β, IL-12, IL-2, and IFN-γ), myofibroblasts, and proliferating cells, as well as increased anti-inflammatory cytokines (IL-10), endothelial cells/blood vessel lumen, M2 macrophages, and granulation tissue size without compromising the mechanical properties. These results support the concept that IL-1Ra modulates MCL-localized granulation tissue components and cytokine production to create a transient environment that is less inflammatory. Overall, IL-1Ra may have therapeutic potential early in the healing cascade by stimulating the M2 macrophages and altering the granulation tissue components. However, the single dose of IL-1Ra used in this study was insufficient to maintain the more regenerative early response. Due to the transient influence on most of the healing components tested, IL-1Ra may have greater therapeutic potential with sustained delivery. PMID:23936523

  2. Kaposi's Sarcoma-Associated Herpesvirus G-Protein-Coupled Receptor Prevents AU-Rich-Element-Mediated mRNA Decay

    PubMed Central

    Corcoran, Jennifer A.; Khaperskyy, Denys A.; Johnston, Benjamin P.; King, Christine A.; Cyr, David P.; Olsthoorn, Alisha V.

    2012-01-01

    During lytic Kaposi's sarcoma-associated herpesvirus (KSHV) infection, host gene expression is severely restricted by a process of global mRNA degradation known as host shutoff, which rededicates translational machinery to the expression of viral proteins. A subset of host mRNAs is spared from shutoff, and a number of these contain cis-acting AU-rich elements (AREs) in their 3′ untranslated regions. AREs are found in labile mRNAs encoding cytokines, growth factors, and proto-oncogenes. Activation of the p38/MK2 signal transduction pathway reverses constitutive decay of ARE-mRNAs, resulting in increased protein production. The viral G-protein-coupled receptor (vGPCR) is thought to play an important role in promoting the secretion of angiogenic molecules from KSHV-infected cells during lytic replication, but to date it has not been clear how vGPCR circumvents host shutoff. Here, we demonstrate that vGPCR activates the p38/MK2 pathway and stabilizes ARE-mRNAs, augmenting the levels of their protein products. Using MK2-deficient cells, we demonstrate that MK2 is essential for maximal vGPCR-mediated ARE-mRNA stabilization. ARE-mRNAs are normally delivered to cytoplasmic ribonucleoprotein granules known as processing bodies (PBs) for translational silencing and decay. We demonstrate that PB formation is prevented during KSHV lytic replication or in response to vGPCR-mediated activation of RhoA subfamily GTPases. Together, these data show for the first time that vGPCR impacts gene expression at the posttranscriptional level, coordinating an attack on the host mRNA degradation machinery. By suppressing ARE-mRNA turnover, vGPCR may facilitate escape of certain target mRNAs from host shutoff and allow secretion of angiogenic factors from lytically infected cells. PMID:22696654

  3. Inhibition of Vascular c-Jun N-Terminal Kinase 2 Improves Obesity-Induced Endothelial Dysfunction After Roux-en-Y Gastric Bypass.

    PubMed

    Doytcheva, Petia; Bächler, Thomas; Tarasco, Erika; Marzolla, Vincenzo; Engeli, Michael; Pellegrini, Giovanni; Stivala, Simona; Rohrer, Lucia; Tona, Francesco; Camici, Giovanni G; Vanhoutte, Paul M; Matter, Christian M; Lutz, Thomas A; Lüscher, Thomas F; Osto, Elena

    2017-11-14

    Roux-en-Y gastric bypass (RYGB) reduces obesity-associated comorbidities and cardiovascular mortality. RYGB improves endothelial dysfunction, reducing c-Jun N-terminal kinase (JNK) vascular phosphorylation. JNK activation links obesity with insulin resistance and endothelial dysfunction. Herein, we examined whether JNK1 or JNK2 mediates obesity-induced endothelial dysfunction and if pharmacological JNK inhibition can mimic RYGB vascular benefits. After 7 weeks of a high-fat high-cholesterol diet, obese rats underwent RYGB or sham surgery; sham-operated ad libitum-fed rats received, for 8 days, either the control peptide D-TAT or the JNK peptide inhibitor D-JNKi-1 (20 mg/kg per day subcutaneous). JNK peptide inhibitor D-JNKi-1 treatment improved endothelial vasorelaxation in response to insulin and glucagon-like peptide-1, as observed after RYGB. Obesity increased aortic phosphorylation of JNK2, but not of JNK1. RYGB and JNK peptide inhibitor D-JNKi-1 treatment blunted aortic JNK2 phosphorylation via activation of glucagon-like peptide-1-mediated signaling. The inhibitory phosphorylation of insulin receptor substrate-1 was reduced, whereas the protein kinase B/endothelial NO synthase pathway was increased and oxidative stress was decreased, resulting in improved vascular NO bioavailability. Decreased aortic JNK2 phosphorylation after RYGB rapidly improves obesity-induced endothelial dysfunction. Pharmacological JNK inhibition mimics the endothelial protective effects of RYGB. These findings highlight the therapeutic potential of novel strategies targeting vascular JNK2 against the severe cardiovascular disease associated with obesity. © 2017 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley.

  4. Downregulation of Ras C-terminal processing by JNK inhibition

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

    Mouri, Wataru; Department of Neurosurgery, Yamagata University School of Medicine, Yamagata 990-9585; Biology Division, National Cancer Center Research Institute, Tokyo 104-0045

    2008-06-27

    After translation, Ras proteins undergo a series of modifications at their C-termini. This post-translational C-terminal processing is essential for Ras to become functional, but it remains unknown whether and how Ras C-terminal processing is regulated. Here we show that the C-terminal processing and subsequent plasma membrane localization of H-Ras as well as the activation of the downstream signaling pathways by H-Ras are prevented by JNK inhibition. Conversely, JNK activation by ultraviolet irradiation resulted in promotion of C-terminal processing of H-Ras. Furthermore, increased cell density promoted C-terminal processing of H-Ras most likely through an autocrine/paracrine mechanism, which was also blocked undermore » JNK-inhibited condition. Ras C-terminal processing was sensitive to JNK inhibition in the case of H- and N-Ras but not K-Ras, and in a variety of cell types. Thus, our results suggest for the first time that Ras C-terminal processing is a regulated mechanism in which JNK is involved.« less

  5. Smad3 phosphoisoform-mediated signaling during sporadic human colorectal carcinogenesis.

    PubMed

    Matsuzaki, K

    2006-06-01

    Transforming growth factor-beta (TGF-beta) signaling occurring during human colorectal carcinogenesis involves a shift in TGF-beta function, reducing the cytokine's antiproliferative effect, while increasing actions that promote invasion and metastasis. TGF-beta signaling involves phosphorylation of Smad3 at serine residues 208 and 213 in the linker region and serine residues 423 and 425 in the C-terminal region. Exogenous TGF-beta activates not only TGF-beta type I receptor (TbetaRI) but also c-Jun N-terminal kinase (JNK), changing unphosphorylated Smad3 to its phosphoisoforms: C-terminally phosphorylated Smad3 (pSmad3C) and linker phosphorylated Smad3 (pSmad3L). Either pSmad3C or pSmad3L oligomerizes with Smad4, and translocates into nuclei. While the TbetaRI/pSmad3C pathway inhibits growth of normal epithelial cells in vivo, JNK/pSmad3L-mediated signaling promotes tumor cell invasion and extracellular matrix synthesis by activated mesenchymal cells. Furthermore, hepatocyte growth factor signaling interacts with TGF-beta to activate the JNK/pSmad3L pathway, accelerating nuclear transport of cytoplasmic pSmad3L. This reduces accessibility of unphosphorylated Smad3 to membrane-anchored TbetaRI, preventing Smad3C phosphorylation, pSmad3C-mediated transcription, and antiproliferative effects of TGF-beta on epithelial cells. As neoplasia progresses from normal colorectal epithelium through adenoma to invasive adenocarcinoma with distant metastasis, nuclear pSmad3L gradually increases while pSmad3C decreases. The shift from TbetaRI/pSmad3C-mediated to JNK/pSmad3L-mediated signaling is a major mechanism orchestrating a complex transition of TGF-beta signaling during sporadic human colorectal carcinogenesis. This review summarizes the recent understanding of Smad3 phosphoisoform-mediated signaling, particularly 'cross-talk' between Smad3 and JNK pathways that cooperatively promote oncogenic activities. Understanding of these actions should help to develop more effective

  6. The Role of High Dose Interleukin-2 in the Era of Targeted Therapy.

    PubMed

    Gills, Jessie; Parker, William P; Pate, Scott; Niu, Sida; Van Veldhuizen, Peter; Mirza, Moben; Holzbeierlein, Jeffery M; Lee, Eugene K

    2017-09-01

    We assessed survival outcomes following high dose interleukin-2 in a contemporary cohort of patients during the era of targeted agents. We retrospectively reviewed the records of patients with metastatic renal cell carcinoma treated with high dose interleukin-2 between July 2007 and September 2014. Clinicopathological data were abstracted and patient response to therapy was based on RECIST (Response Evaluation Criteria In Solid Tumors), version 1.1 criteria. The Kaplan-Meier method was used to estimate progression-free and overall survival in the entire cohort, the response to high dose interleukin-2 in regard to previous targeted agent therapy and the response to the targeted agent in relation to the response to high dose interleukin-2. We identified 92 patients, of whom 87 had documentation of a response to high dose interleukin-2. Median overall survival was 34.4 months from the initiation of high dose interleukin-2 therapy in the entire cohort. Patients who received targeted therapy before high dose interleukin-2 had overall survival (median 34.4 and 30.0 months, p = 0.88) and progression-free survival (median 1.5 and 1.7 months, p = 0.8) similar to those in patients who received no prior therapy, respectively. Additionally, patients with a complete or partial response to high dose interleukin-2 had similar outcomes for subsequent targeted agents compared to patients whose best response was stable or progressive disease (median overall survival 30.1 vs 25.4 months, p = 0.4). Our data demonstrate that patient responses to high dose interleukin-2 and to targeted agents before and after receiving high dose interleukin-2 are independent. As such, carefully selected patients should be offered high dose interleukin-2 for the possibility of a complete and durable response without the fear of limiting the treatment benefit of targeted agents. Copyright © 2017 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.

  7. Interleukin-17-induced protein lipocalin 2 is dispensable for immunity to oral candidiasis.

    PubMed

    Ferreira, Maria Carolina; Whibley, Natasha; Mamo, Anna J; Siebenlist, Ulrich; Chan, Yvonne R; Gaffen, Sarah L

    2014-03-01

    Oropharyngeal candidiasis (OPC; thrush) is an opportunistic fungal infection caused by the commensal microbe Candida albicans. Immunity to OPC is strongly dependent on CD4+ T cells, particularly those of the Th17 subset. Interleukin-17 (IL-17) deficiency in mice or humans leads to chronic mucocutaneous candidiasis, but the specific downstream mechanisms of IL-17-mediated host defense remain unclear. Lipocalin 2 (Lcn2; 24p3; neutrophil gelatinase-associated lipocalin [NGAL]) is an antimicrobial host defense factor produced in response to inflammatory cytokines, particularly IL-17. Lcn2 plays a key role in preventing iron acquisition by bacteria that use catecholate-type siderophores, and lipocalin 2(-/-) mice are highly susceptible to infection by Escherichia coli and Klebsiella pneumoniae. The role of Lcn2 in mediating immunity to fungi is poorly defined. Accordingly, in this study, we evaluated the role of Lcn2 in immunity to oral infection with C. albicans. Lcn2 is strongly upregulated following oral infection with C. albicans, and its expression is almost entirely abrogated in mice with defective IL-17 signaling (IL-17RA(-/-) or Act1(-/-) mice). However, Lcn2(-/-) mice were completely resistant to OPC, comparably to wild-type (WT) mice. Moreover, Lcn2 deficiency mediated protection from OPC induced by steroid immunosuppression. Therefore, despite its potent regulation during C. albicans infection, Lcn2 is not required for immunity to mucosal candidiasis.

  8. Bacteriolytic Activity Of Human Interleukin-2, Chicken Egg Lysozyme In The Presence Of Potential Effectors

    PubMed Central

    Levashov, P. A.; Matolygina, D. A.; Ovchinnikova, E. D.; Atroshenko, D. L.; Savin, S. S.; Belogurova, N. G.; Smirnov, S. A.; Tishkov, V. I.; Levashov, A. V.

    2017-01-01

    The bacteriolytic activity of interleukin-2 and chicken egg lysozyme in the presence of various substances has been studied. Glycine and lysine do not affect the activity of interleukin-2 but increase that of lysozyme, showing a bell-shape concentration dependence peaking at 1.5 mM glycine and 18 mM lysine. Arginine and glutamate activate both interleukin-2 and lysozyme with a concentration dependence of the saturation type. Aromatic amino acids have almost no effect on the activity of both interleukin-2 and lysozyme. Aromatic amines, tryptamine, and tyramine activate interleukin-2 but inhibit lysozyme. Peptide antibiotics affect interleukin and lysozyme similarly and exhibit maximum activity in the micromolar range of antibiotics. Taurine has no effect on the activity of interleukin-2 and lysozyme. Mildronate showed no influence on lysozyme, but it activated interleukin-2 with the activity maximum at 3 mM. EDTA activates both interleukin-2 and lysozyme at concentrations above 0.15 mM. PMID:28740730

  9. mRNA export: threading the needle

    PubMed Central

    Gaouar, Ouassila; Germain, Hugo

    2013-01-01

    After mRNA biogenesis, several proteins interact with the messenger to ensure its proper export to the cytoplasm. Some of these proteins will bind RNA early on, at the onset of transcription by RNA polymerase II holoenzyme, while others will join later for downstream processing steps, such as poly-adenylation or splicing, or may direct mRNA ribonucleoprotein particle migration to the nucleopore. We recently discovered that Arabidopsis plant knockout for the protein MOS11 (MODIFIER OF SNC1, 11) partially suppresses autoimmune responses observed in the TNL-type [TIR/NBS/LRR (Toll-interleukin-like receptor/nucleotide-binding site/C-terminal leucine-rich repeat)] R gene gain-of-function variant snc1 (suppressor of npr1-1, constitutive 1). This suppression of resistance to pathogens appears to be caused by a decrease in nuclear mRNA export in mos11-1 snc1 plants. In humans, the putative ortholog of MOS11, CIP29 (29-kDa cytokine-induced protein), interacts with three proteins that are also involved in mRNA export: DDX39 (DEAD-box RNA helicase), TAF15 of the FUS family (FUSED IN SARCOMA), and ALY (ALWAYS EARLY), a protein implicated in mRNA export in mammalian systems. These proteins have received very little attention in plants. Here, we will discuss their particularities and role in mRNA export and biotic stress. PMID:23526740

  10. Amyloid-β Reduces Exosome Release from Astrocytes by Enhancing JNK Phosphorylation.

    PubMed

    Abdullah, Mohammad; Takase, Hiroshi; Nunome, Mari; Enomoto, Hiroyuki; Ito, Jin-Ichi; Gong, Jian-Sheng; Michikawa, Makoto

    2016-07-02

    Exosomes are small extracellular vesicles secreted by variety of cell types such as neurons, astrocytes, and oligodendrocytes. It is suggested that exosomes play essential role in the maintenance of the neuronal functions and also in the clearance of amyloid-β (Aβ) from the brain. Aβ is well known to cause neuronal cell death, whereas little is known about its effect on astrocytes. In this study, we examined the effect of Aβ on release of exosomes from astrocytes in culture. We analyzed release of exosomes and apoE, both of which are known to remove/clear Aβ from the brain, in the culture medium of astrocytes. We found that exosome and apoE-HDL were successfully separated by density gradient ultracentrifugation demonstrated by distribution of their specific markers, flotillin and HSP90, and cholesterol, and morphological analysis using electron microscopy. Exosome release was significantly reduced by Aβ1-42 treatment in cultured astrocytes accompanied by an increased JNK phosphorylation. Whereas, apoE-HDL release remained unchanged. A JNK inhibitor restored the decreased levels of exosome release induced by Aβ treatment to levels similar to those of control, suggesting that Aβ1-42 inhibits exosome release via stimulation of JNK signal pathway. Because exosomes are shown to remove Aβ in the brain, our findings suggest that increased Aβ levels in the brain may impair the exosome-mediated Aβ clearance pathway.

  11. Drosophila MOF regulates DIAP1 and induces apoptosis in a JNK dependent pathway.

    PubMed

    Pushpavalli, Sreerangam N C V L; Sarkar, Arpita; Ramaiah, M Janaki; Koteswara Rao, G; Bag, Indira; Bhadra, Utpal; Pal-Bhadra, Manika

    2016-03-01

    Histone modulations have been implicated in various cellular and developmental processes where in Drosophila Mof is involved in acetylation of H4K16. Reduction in the size of larval imaginal discs is observed in the null mutants of mof with increased apoptosis. Deficiency involving Hid, Reaper and Grim [H99] alleviated mof (RNAi) induced apoptosis in the eye discs. mof (RNAi) induced apoptosis leads to activation of caspases which is suppressed by over expression of caspase inhibitors like P35 and Diap1 clearly depicting the role of caspases in programmed cell death. Also apoptosis induced by knockdown of mof is rescued by JNK mutants of bsk and tak1 indicating the role of JNK in mof (RNAi) induced apoptosis. The adult eye ablation phenotype produced by ectopic expression of Hid, Rpr and Grim, was restored by over expression of Mof. Accumulation of Mof at the Diap1 promoter 800 bp upstream of the transcription start site in wild type larvae is significantly higher (up to twofolds) compared to mof (1) mutants. This enrichment coincides with modification of histone H4K16Ac indicating an induction of direct transcriptional up regulation of Diap1 by Mof. Based on these results we propose that apoptosis triggered by mof (RNAi) proceeds through a caspase-dependent and JNK mediated pathway.

  12. l-Arginine modulates neonatal lymphocyte proliferation through an interleukin-2 independent pathway

    PubMed Central

    Yu, Hong-Ren; Kuo, Ho-Chang; Huang, Li-Tung; Chen, Chih-Cheng; Tain, You-Lin; Sheen, Jiunn-Ming; Tiao, Mao-Meng; Huang, Hsin-Chun; Yang, Kuender D; Ou, Chia-Yo; Hsu, Te-Yao

    2014-01-01

    In cases of arginine depletion, lymphocyte proliferation, cytokine production and CD3ζ chain expression are all diminished. In addition to myeloid suppressor cells, polymorphonuclear cells (PMN) also exert T-cell immune suppressive effects through arginase-induced l-arginine depletion, especially during pregnancy. In this study, we investigated how arginase/l-arginine modulates neonatal lymphocyte proliferation. Results showed that the neonatal plasma l-arginine level was lower than in adults (48·1 ± 11·3 versus 86·5 ± 14·6 μm; P = 0·003). Neonatal PMN had a greater abundance of arginase I protein than adult PMN. Both transcriptional regulation and post-transcriptional regulation were responsible for the higher arginase I expression of neonatal PMN. Exogenous l-arginine enhanced neonate lymphocyte proliferation but not that of adult cells. The RNA-binding protein HuR was important but was not the only modulation factor in l-arginine-regulated neonatal T-cell proliferation. l-Arginine-mediated neonatal lymphocyte proliferation could not be blocked by interleukin-2 receptor blocking antibodies. These results suggest that the altered arginase/l-arginine cascade may be one of the mechanisms that contribute to altered neonatal immune responses. Exogenous l-arginine could enhance neonate lymphocyte proliferation through an interleukin-2-independent pathway. PMID:24697328

  13. Inorganic arsenic represses interleukin-17A expression in human activated Th17 lymphocytes

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

    Morzadec, Claudie; Macoch, Mélinda; Robineau, Marc

    2012-08-01

    Trivalent inorganic arsenic [As(III)] is an efficient anticancer agent used to treat patients suffering from acute promyelocytic leukemia. Recently, experimental studies have clearly demonstrated that this metalloid can also cure lymphoproliferative and/or pro-inflammatory syndromes in different murine models of chronic immune-mediated diseases. T helper (Th) 1 and Th17 lymphocytes play a central role in development of these diseases, in mice and humans, especially by secreting the potent pro-inflammatory cytokine interferon-γ and IL-17A, respectively. As(III) impairs basic functions of human T cells but its ability to modulate secretion of pro-inflammatory cytokines by differentiated Th lymphocytes is unknown. In the present study,more » we demonstrate that As(III), used at concentrations clinically achievable in plasma of patients, has no effect on the secretion of interferon-γ from Th1 cells but almost totally blocks the expression and the release of IL-17A from human Th17 lymphocytes co-stimulated for five days with anti-CD3 and anti-CD28 antibodies, in the presence of differentiating cytokines. In addition, As(III) specifically reduces mRNA levels of the retinoic-related orphan receptor (ROR)C gene which encodes RORγt, a key transcription factor controlling optimal IL-17 expression in fully differentiated Th17 cells. The metalloid also blocks initial expression of IL-17 gene induced by the co-stimulation, probably in part by impairing activation of the JNK/c-Jun pathway. In conclusion, our results demonstrate that As(III) represses expression of the major pro-inflammatory cytokine IL-17A produced by human Th17 lymphocytes, thus strengthening the idea that As(III) may be useful to treat inflammatory immune-mediated diseases in humans. -- Highlights: ► Arsenic inhibits secretion of IL-17A from human naïve and memory Th17 lymphocytes. ► Arsenic represses early expression of IL-17A gene in human activated T lymphocytes. ► Arsenic interferes with

  14. [Biology and immunotherapy advance of interleukin 2 and interleukin 15-review].

    PubMed

    Chen, Guang-Hua; Wu, De-Pei

    2009-08-01

    IL-2 and IL-15 play an important roles in regulating the lymphocyte function and homeostasis. Advances in understanding of the cellular and molecular biology of IL-2 and IL-15 and their receptor complex have provided rationale to better utilize them to expand and activate immune effectors in patients with cancer. These two cytokines stimulate similar responses from lymphocytes in vitro, but play markedly distinct roles in lymphoid biology in vivo. Their distinct physiological functions can be ascribed to distinct signaling pathways initiated by distinct cytokine receptor subunits, differential expression patterns of their receptors. Recently, the discovery of a novel mechanism of IL-15 cytokine signaling, trans-presentation, has provided insights into the divergent ways of these cytokine function. Although their heterotrimeric receptors have two receptor subunits in common, these two cytokines have contrasting roles in adaptive immune responses. The unique role of interleukin 2 is in the elimination of self-reactive T cells to prevent autoimmunity. By contrast, interleukin 15 is dedicated to the prolonged maintenance of memory T-cell responses to pathogens. As discussed in this article, the biology of IL-2 and IL-15 two cytokines will affect the development of novel treatment for malignancies and autoimmune diseases.

  15. Luteolin and Apigenin Attenuate 4-Hydroxy-2-Nonenal-Mediated Cell Death through Modulation of UPR, Nrf2-ARE and MAPK Pathways in PC12 Cells

    PubMed Central

    Wu, Pei-Shan; Yen, Jui-Hung; Kou, Mei-Chun; Wu, Ming-Jiuan

    2015-01-01

    Luteolin and apigenin are dietary flavones and exhibit a broad spectrum of biological activities including antioxidant, anti-inflammatory, anti-cancer and neuroprotective effects. The lipid peroxidation product 4-hydroxy-2-nonenal (4-HNE) has been implicated as a causative agent in the development of neurodegenerative disorders. This study investigates the cytoprotective effects of luteolin and apigenin against 4-HNE-mediated cytotoxicity in neuronal-like catecholaminergic PC12 cells. Both flavones restored cell viability and repressed caspase-3 and PARP-1 activation in 4-HNE-treated cells. Luteolin also mitigated 4-HNE-mediated LC3 conversion and reactive oxygen species (ROS) production. Luteolin and apigenin up-regulated 4-HNE-mediated unfolded protein response (UPR), leading to an increase in endoplasmic reticulum chaperone GRP78 and decrease in the expression of UPR-targeted pro-apoptotic genes. They also induced the expression of Nrf2-targeted HO-1 and xCT in the absence of 4-HNE, but counteracted their expression in the presence of 4-HNE. Moreover, we found that JNK and p38 MAPK inhibitors significantly antagonized the increase in cell viability induced by luteolin and apigenin. Consistently, enhanced phosphorylation of JNK and p38 MAPK was observed in luteolin- and apigenin-treated cells. In conclusion, this result shows that luteolin and apigenin activate MAPK and Nrf2 signaling, which elicit adaptive cellular stress response pathways, restore 4-HNE-induced ER homeostasis and inhibit cytotoxicity. Luteolin exerts a stronger cytoprotective effect than apigenin possibly due to its higher MAPK, Nrf2 and UPR activation, and ROS scavenging activity. PMID:26087007

  16. Cleavage of HPV-16 E6/E7 mRNA mediated by modified 10-23 deoxyribozymes.

    PubMed

    Reyes-Gutiérrez, Pablo; Alvarez-Salas, Luis M

    2009-09-01

    Deoxyribozymes (DXZs) are small oligodeoxynucleotides capable of mediating phosphodiester bond cleavage of a target RNA in a sequence-specific manner. These molecules are a new generation of artificial catalytic nucleic acids currently used to silence many disease-related genes. The present study describes a DXZ (Dz1023-434) directed against the polycistronic mRNA from the E6 and E7 genes of human papillomavirus type 16 (HPV-16), the main etiological agent of cervical cancer. Dz1023-434 showed efficient cleavage against a bona fide antisense window at nt 410-445 within HPV-16 E6/E7 mRNA even in low [Mg(2+)] conditions. Using a genetic analysis as guidance, we introduced diverse chemical modifications within Dz1023-434 catalytic core to produce a stable locked nucleic acid (LNA)-modified DXZ (Dz434-LNA) with significant cleavage activity of full E6/E7 transcripts. Cell culture testing of Dz434-LNA produced a sharp decrement of E6/E7 mRNA levels in HPV-16-positive cells resulting in decreased proliferation and considerable cell death in a specific and dose-dependent manner. No significant effects were observed with inactive or scrambled control DXZs nor from using HPV-negative cells, suggesting catalysis-dependent effect and high specificity. The biological effects of Dz434-LNA suggest a potential use for the treatment of cervical cancer.

  17. Induced expression of mRNA for IL-5, IL-6, TNF-alpha, MIP-2 and IFN-gamma in immunologically activated rat peritoneal mast cells: inhibition by dexamethasone and cyclosporin A.

    PubMed

    Williams, C M; Coleman, J W

    1995-10-01

    We examined the capacity of purified rat peritoneal connective tissue-type mast cells (PMC) to express mRNA for several cytokines. Stimulation of PMC with anti-IgE for 4 hr induced the expression of mRNA encoding interleukin-5 (IL-5), IL-6, tumour necrosis factor-alpha (TNF-alpha), macrophage inflammatory protein-2 (MIP-2) and interferon-gamma (IFN-gamma). Unstimulated PMC expressed detectable mRNA for TNF-alpha but not for the other four cytokines. Incubation of PMC with cyclosporin A (CsA) or dexamethasone (DEX), each at 10(-6) M for 24 hr, significantly inhibited the induced expression of mRNA for each of the five cytokines, and also inhibited release of biologically active TNF-alpha. Throughout these experiments mRNA levels of the housekeeping gene G3PDH were not altered by stimulation with anti-IgE or incubation with CsA or DEX. We conclude that immunological activation of rat PMC induces gene expression of several cytokines and that expression of these genes can be inhibited by immunosuppressive drugs.

  18. Curcumin attenuates BPA-induced insulin resistance in HepG2 cells through suppression of JNK/p38 pathways.

    PubMed

    Geng, Shanshan; Wang, Shijia; Zhu, Weiwei; Xie, Chunfeng; Li, Xiaoting; Wu, Jieshu; Zhu, Jianyun; Jiang, Ye; Yang, Xue; Li, Yuan; Chen, Yue; Wang, Xiaoqian; Meng, Yu; Zhu, Mingming; Wu, Rui; Huang, Cong; Zhong, Caiyun

    2017-04-15

    Bisphenol A (BPA) is an artificial environmental endocrine disrupting chemicals. Accumulating evidence indicates that exposure to BPA contributes to insulin resistance through diverse mechanism including inflammation and oxidative stress. Previous studies have suggested curcumin as a safe phytochemical which can improve obesity-related insulin resistance, inflammation and oxidative stress. The present study aimed to investigate the ability of curcumin to prevent BPA-induced insulin resistance in vitro and the underlying mechanism. Following the establishmet of in vitro insulin resistance via BPA treatment in human liver HepG2 cells, the protective effects of curcumin were determiend. We showed that treatment of HepG2 cells with 100nM BPA for 5days induced significantly decreased glucose consumption, impaired insulin signaling, elevation of pro-inflammatory cytokines and oxidative stress, and activation of signaling pathways; inhibition of JNK and p38 pathways, but not ERK nor NF-κB pathways, improved glucose consumption and insulin signaling in BPA-treated HepG2 cells. Moreover, we revealed that curcumin effectively attenuated the spectrum of effects of BPA-triggered insulin resistance, whereas pretreatment with JNK and p38 agonist anisomycin could significantly compensate the effects caused by curcumin. These data illustrated the role of JNK/p38 activation in BPA-induced insulin resistance and suggested curcumin as a promising candidate for the intervention of BPA-induced insulin resistance. Copyright © 2017 Elsevier B.V. All rights reserved.

  19. JNK-induced MCP-1 production in spinal cord astrocytes contributes to central sensitization and neuropathic pain.

    PubMed

    Gao, Yong-Jing; Zhang, Ling; Samad, Omar Abdel; Suter, Marc R; Yasuhiko, Kawasaki; Xu, Zhen-Zhong; Park, Jong-Yeon; Lind, Anne-Li; Ma, Qiufu; Ji, Ru-Rong

    2009-04-01

    Our previous study showed that activation of c-jun-N-terminal kinase (JNK) in spinal astrocytes plays an important role in neuropathic pain sensitization. We further investigated how JNK regulates neuropathic pain. In cultured astrocytes, tumor necrosis factor alpha (TNF-alpha) transiently activated JNK via TNF receptor-1. Cytokine array indicated that the chemokine CCL2/MCP-1 (monocyte chemoattractant protein-1) was strongly induced by the TNF-alpha/JNK pathway. MCP-1 upregulation by TNF-alpha was dose dependently inhibited by the JNK inhibitors SP600125 (anthra[1,9-cd]pyrazol-6(2H)-one) and D-JNKI-1. Spinal injection of TNF-alpha produced JNK-dependent pain hypersensitivity and MCP-1 upregulation in the spinal cord. Furthermore, spinal nerve ligation (SNL) induced persistent neuropathic pain and MCP-1 upregulation in the spinal cord, and both were suppressed by D-JNKI-1. Remarkably, MCP-1 was primarily induced in spinal cord astrocytes after SNL. Spinal administration of MCP-1 neutralizing antibody attenuated neuropathic pain. Conversely, spinal application of MCP-1 induced heat hyperalgesia and phosphorylation of extracellular signal-regulated kinase in superficial spinal cord dorsal horn neurons, indicative of central sensitization (hyperactivity of dorsal horn neurons). Patch-clamp recordings in lamina II neurons of isolated spinal cord slices showed that MCP-1 not only enhanced spontaneous EPSCs but also potentiated NMDA- and AMPA-induced currents. Finally, the MCP-1 receptor CCR2 was expressed in neurons and some non-neuronal cells in the spinal cord. Together, we have revealed a previously unknown mechanism of MCP-1 induction and action. MCP-1 induction in astrocytes after JNK activation contributes to central sensitization and neuropathic pain facilitation by enhancing excitatory synaptic transmission. Inhibition of the JNK/MCP-1 pathway may provide a new therapy for neuropathic pain management.

  20. Oolong tea prevents cardiomyocyte loss against hypoxia by attenuating p-JNK mediated hypertrophy and enhancing P-IGF1R, p-akt, and p-Badser136 activity and by fortifying NRF2 antioxidation system.

    PubMed

    Shibu, Marthandam Asokan; Kuo, Chia-Hua; Chen, Bih-Cheng; Ju, Da-Tong; Chen, Ray-Jade; Lai, Chao-Hung; Huang, Pei-Jane; Viswanadha, Vijaya Padma; Kuo, Wei-Wen; Huang, Chih-Yang

    2018-02-01

    Tea, the most widely consumed natural beverage has been associated with reduced mortality risk from cardiovascular disease. Oolong tea is a partially fermented tea containing high levels of catechins, their degree of oxidation varies between 20%-80% causing differences in their active metabolites. In this study we examined the effect of oolong tea extract (OTE) obtained by oxidation at low-temperature for short-time against hypoxic injury and found that oolong tea provides cyto-protective effects by suppressing the JNK mediated hypertrophic effects and by enhancing the innate antioxidant mechanisms in neonatal cardiomyocytes and in H9c2 cells. OTE effectively attenuates 24 h hypoxia-triggered cardiomyocyte loss by suppressing caspase-3-cleavage and apoptosis in a dose-dependent manner. OTE also enhances the IGFIR/p-Akt associated survival-mechanism involving the elevation of p-Bad ser136 in a dose-dependent manner to aid cellular adaptations against hypoxic challenge. The results show the effects and mechanism of Oolong tea to provide cardio-protective benefits during hypoxic conditions. © 2017 Wiley Periodicals, Inc.

  1. Salmon trypsin stimulates the expression of interleukin-8 via protease-activated receptor-2

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

    Larsen, Anett K.; Department of Pharmacology, Institute of Medical Biology, University of Tromso, Tromso; Seternes, Ole-Morten

    2008-08-01

    In this study, we focus on salmon trypsin as an activator of inflammatory responses in airway cells in vitro. The rationale behind the investigation is that salmon industry workers are exposed to aerosols containing enzymes, which are generated during industrial processing of the fish. Knowing that serine proteases such as trypsin are highly active mediators with diverse biological activities, the stimulation of nuclear factor-kappa B (NF-{kappa}B) and interleukin (IL)-8 and the role of protease-activated receptors (PAR) in inflammatory signal mediation were investigated. Protease-activated receptors are considered important under pathological situations in the human airways, and a thorough understanding of PAR-inducedmore » cellular events and their consequences in airway inflammation is necessary. Human airway epithelial cells (A549) were exposed to trypsin isolated from fish (Salmo salar), and we observed that purified salmon trypsin could generate secretion of IL-8 in a concentration-dependent manner. Furthermore, we demonstrate that PAR-2 activation by salmon trypsin is coupled to an induction of NF-{kappa}B-mediated transcription using a PAR-2 transfected HeLa cell model. Finally, we show that the release of IL-8 from A549 following stimulation with purified salmon trypsin is mediated through activation of PAR-2 using specific small interfering RNAs (siRNAs). The results presented suggest that salmon trypsin, via activation of PAR-2, might influence inflammation processes in the airways if inhaled in sufficient amounts.« less

  2. DUSP5 functions as a feedback regulator of TNFα-induced ERK1/2 dephosphorylation and inflammatory gene expression in adipocytes.

    PubMed

    Habibian, Justine S; Jefic, Mitra; Bagchi, Rushita A; Lane, Robert H; McKnight, Robert A; McKinsey, Timothy A; Morrison, Ron F; Ferguson, Bradley S

    2017-10-10

    Adipose tissue inflammation is a central pathological element that regulates obesity-mediated insulin resistance and type II diabetes. Evidence demonstrates that extracellular signal-regulated kinase (ERK 1/2) activation (i.e. phosphorylation) links tumor necrosis factor α (TNFα) to pro-inflammatory gene expression in the nucleus. Dual specificity phosphatases (DUSPs) inactivate ERK 1/2 through dephosphorylation and can thus inhibit inflammatory gene expression. We report that DUSP5, an ERK1/2 phosphatase, was induced in epididymal white adipose tissue (WAT) in response to diet-induced obesity. Moreover, DUSP5 mRNA expression increased during obesity development concomitant to increases in TNFα expression. Consistent with in vivo findings, DUSP5 mRNA expression increased in adipocytes in response to TNFα, parallel with ERK1/2 dephosphorylation. Genetic loss of DUSP5 exacerbated TNFα-mediated ERK 1/2 signaling in 3T3-L1 adipocytes and in adipose tissue of mice. Furthermore, inhibition of ERK 1/2 and c-Jun N terminal kinase (JNK) signaling attenuated TNFα-induced DUSP5 expression. These data suggest that DUSP5 functions in the feedback inhibition of ERK1/2 signaling in response to TNFα, which resulted in increased inflammatory gene expression. Thus, DUSP5 potentially acts as an endogenous regulator of adipose tissue inflammation; although its role in obesity-mediated inflammation and insulin signaling remains unclear.

  3. Interconnections between mRNA degradation and RDR-dependent siRNA production in mRNA turnover in plants.

    PubMed

    Tsuzuki, Masayuki; Motomura, Kazuki; Kumakura, Naoyoshi; Takeda, Atsushi

    2017-03-01

    Accumulation of an mRNA species is determined by the balance between the synthesis and the degradation of the mRNA. Individual mRNA molecules are selectively and actively degraded through RNA degradation pathways, which include 5'-3' mRNA degradation pathway, 3'-5' mRNA degradation pathway, and RNA-dependent RNA polymerase-mediated mRNA degradation pathway. Recent studies have revealed that these RNA degradation pathways compete with each other in mRNA turnover in plants and that plants have a hidden layer of non-coding small-interfering RNA production from a set of mRNAs. In this review, we summarize the current information about plant mRNA degradation pathways in mRNA turnover and discuss the potential roles of a novel class of the endogenous siRNAs derived from plant mRNAs.

  4. In vivo reduction or blockade of interleukin-1β in primary osteoarthritis influences expression of mediators implicated in pathogenesis

    PubMed Central

    Santangelo, K. S.; Nuovo, G. J.; Bertone, A. L.

    2012-01-01

    Summary Objective Diminish interleukin-1β (IL-1β) signaling in a model of primary osteoarthritis by RNA interference-based transcript reduction or receptor blockade, and quantify changes incurred on transcript expression of additional mediators. Methods Knees of Hartley guinea pigs were collected at 120 and 180 days of age following injection with viral vectors (N=4/treatment group/date) at 60 days. Two groups received either adeno-associated viral serotype 5 vector containing a knockdown sequence (TV), or adenoviral vector encoding for IL-1 receptor antagonist protein (Ad-IRAP); treatments were contrasted with opposite knees administered corresponding vector controls. A third group evaluated TV relative to saline-only injected knees. Chondropathy and immunohistochemistry findings were compared to untreated guinea pigs. Transcript expression levels in cartilage were calculated using the comparative CT (2−ΔΔCT) method and analyzed by one-way ANOVA with pairwise comparisons using Tukey 95% confidence intervals. Results Vector transduction was confirmed at both harvest dates. TV and Ad-IRAP, relative to vector controls, significantly decreased IL-1β. Inflammatory mediators [tumor necrosis factor-α (TNF-α), interleukin-8 (IL-8), interferon-γ (IFN-γ)], and catabolic matrix metalloproteinase 13 (MMP13) were also decreased, while anabolic transforming growth factor-β1 (TGF-β1) was increased. IL-1β was also decreased by TV versus saline, with a decrease in MMP13 and increase TGF-β1; TNF-α, IL-8, and IFN-γ were transiently increased. Conclusions This work confirmed that a reduction in IL-1β signaling was accomplished by either method, resulting in decreased expression of three inflammatory mediators and one catabolic agent, and increased expression of an anabolic molecule. Thus, evidence is provided that IL-1β serves a role in vivo in spontaneous osteoarthritis and that these translational tools may provide beneficial disease modification. PMID:22935786

  5. Power Frequency Magnetic Fields Affect the p38 MAPK-Mediated Regulation of NB69 Cell Proliferation Implication of Free Radicals

    PubMed Central

    Martínez, María Antonia; Úbeda, Alejandro; Moreno, Jorge; Trillo, María Ángeles

    2016-01-01

    The proliferative response of the neuroblastoma line NB69 to a 100 µT, 50 Hz magnetic field (MF) has been shown mediated by activation of the MAPK-ERK1/2 pathway. This work investigates the MF effect on the cell cycle of NB69, the participation of p38 and c-Jun N-terminal (JNK) kinases in the field-induced proliferative response and the potential involvement of reactive oxygen species (ROS) in the activation of the MAPK-ERK1/2 and -p38 signaling pathways. NB69 cultures were exposed to the 100 µT MF, either intermittently for 24, 42 or 63 h, or continuously for periods of 15 to 120 min, in the presence or absence of p38 or JNK inhibitors: SB203580 and SP600125, respectively. Antioxidant N-acetylcysteine (NAC) was used as ROS scavenger. Field exposure induced transient activation of p38, JNK and ERK1/2. The MF proliferative effect, which was mediated by changes in the cell cycle, was blocked by the p38 inhibitor, but not by the JNK inhibitor. NAC blocked the field effects on cell proliferation and p38 activation, but not those on ERK1/2 activation. The MF-induced proliferative effects are exerted through sequential upregulation of MAPK-p38 and -ERK1/2 activation, and they are likely mediated by a ROS-dependent activation of p38. PMID:27058530

  6. RNase MRP cleaves the CLB2 mRNA to promote cell cycle progression: novel method of mRNA degradation.

    PubMed

    Gill, Tina; Cai, Ti; Aulds, Jason; Wierzbicki, Sara; Schmitt, Mark E

    2004-02-01

    RNase mitochondrial RNA processing (RNase MRP) mutants have been shown to have an exit-from-mitosis defect that is caused by an increase in CLB2 mRNA levels, leading to increased Clb2p (B-cyclin) levels and a resulting late anaphase delay. Here we describe the molecular defect behind this delay. CLB2 mRNA normally disappears rapidly as cells complete mitosis, but the level remains high in RNase MRP mutants. This is in direct contrast to other exit-from-mitosis mutants and is the result of an increase in CLB2 mRNA stability. We found that highly purified RNase MRP cleaved the 5' untranslated region (UTR) of the CLB2 mRNA in several places in an in vitro assay. In vivo, we identified RNase MRP-dependent cleavage products on the CLB2 mRNA that closely matched in vitro products. Disposal of these products was dependent on the 5'-->3' exoribonuclease Xrn1 and not the exosome. Our results demonstrate that the endoribonuclease RNase MRP specifically cleaves the CLB2 mRNA in its 5'-UTR to allow rapid 5' to 3' degradation by the Xrn1 nuclease. Degradation of the CLB2 mRNA by the RNase MRP endonuclease provides a novel way to regulate the cell cycle that complements the protein degradation machinery. In addition, these results denote a new mechanism of mRNA degradation not seen before in the yeast Saccharomyces cerevisiae.

  7. NADPH oxidase/ROS-dependent PYK2 activation is involved in TNF-α-induced matrix metalloproteinase-9 expression in rat heart-derived H9c2 cells

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

    Yang, Chuen-Mao, E-mail: chuenmao@mail.cgu.edu.tw; Heart Failure Center, Division of Cardiology, Department of Internal Medicine, Chang Gung Memorial Hospital at Keelung, Keelung, Taiwan; Lee, I-Ta

    TNF-α plays a mediator role in the pathogenesis of chronic heart failure contributing to cardiac remodeling and peripheral vascular disturbances. The implication of TNF-α in inflammatory responses has been shown to be mediated through up-regulation of matrix metalloproteinase-9 (MMP-9). However, the detailed mechanisms of TNF-α-induced MMP-9 expression in rat embryonic-heart derived H9c2 cells are largely not defined. We demonstrated that in H9c2 cells, TNF-α induced MMP-9 mRNA and protein expression associated with an increase in the secretion of pro-MMP-9. TNF-α-mediated responses were attenuated by pretreatment with the inhibitor of ROS (N-acetyl-L-cysteine, NAC), NADPH oxidase [apocynin (APO) or diphenyleneiodonium chloride (DPI)],more » MEK1/2 (U0126), p38 MAPK (SB202190), JNK1/2 (SP600125), NF-κB (Bay11-7082), or PYK2 (PF-431396) and transfection with siRNA of TNFR1, p47{sup phox}, p42, p38, JNK1, p65, or PYK2. Moreover, TNF-α markedly induced NADPH oxidase-derived ROS generation in these cells. TNF-α-enhanced p42/p44 MAPK, p38 MAPK, JNK1/2, and NF-κB (p65) phosphorylation and in vivo binding of p65 to the MMP-9 promoter were inhibited by U0126, SB202190, SP600125, NAC, DPI, or APO. In addition, TNF-α-mediated PYK2 phosphorylation was inhibited by NAC, DPI, or APO. PYK2 inhibition could reduce TNF-α-stimulated MAPKs and NF-κB activation. Thus, in H9c2 cells, we are the first to show that TNF-α-induced MMP-9 expression is mediated through a TNFR1/NADPH oxidase/ROS/PYK2/MAPKs/NF-κB cascade. We demonstrated that NADPH oxidase-derived ROS generation is involved in TNF-α-induced PYK2 activation in these cells. Understanding the regulation of MMP-9 expression and NADPH oxidase activation by TNF-α on H9c2 cells may provide potential therapeutic targets of chronic heart failure. - Highlights: • TNF-α induces MMP-9 secretion and expression via a TNFR1-dependent pathway. • TNF-α induces ROS/PYK2-dependent MMP-9 expression in H9c2 cells. • TNF

  8. Gallic acid prevents isoproterenol-induced cardiac hypertrophy and fibrosis through regulation of JNK2 signaling and Smad3 binding activity

    PubMed Central

    Ryu, Yuhee; Jin, Li; Kee, Hae Jin; Piao, Zhe Hao; Cho, Jae Yeong; Kim, Gwi Ran; Choi, Sin Young; Lin, Ming Quan; Jeong, Myung Ho

    2016-01-01

    Gallic acid, a type of phenolic acid, has been shown to have beneficial effects in inflammation, vascular calcification, and metabolic diseases. The present study was aimed at determining the effect and regulatory mechanism of gallic acid in cardiac hypertrophy and fibrosis. Cardiac hypertrophy was induced by isoproterenol (ISP) in mice and primary neonatal cardiomyocytes. Gallic acid pretreatment attenuated concentric cardiac hypertrophy. It downregulated the expression of atrial natriuretic peptide, brain natriuretic peptide, and beta-myosin heavy chain in vivo and in vitro. Moreover, it prevented interstitial collagen deposition and expression of fibrosis-associated genes. Upregulation of collagen type I by Smad3 overexpression was observed in cardiac myoblast H9c2 cells but not in cardiac fibroblasts. Gallic acid reduced the DNA binding activity of phosphorylated Smad3 in Smad binding sites of collagen type I promoter in rat cardiac fibroblasts. Furthermore, it decreased the ISP-induced phosphorylation of c-Jun N-terminal kinase (JNK) and extracellular signal regulated kinase (ERK) protein in mice. JNK2 overexpression reduced collagen type I and Smad3 expression as well as GATA4 expression in H9c2 cells and cardiac fibroblasts. Gallic acid might be a novel therapeutic agent for the prevention of cardiac hypertrophy and fibrosis by regulating the JNK2 and Smad3 signaling pathway. PMID:27703224

  9. Lipopolysaccharide-regulated production of bone sialoprotein and interleukin-8 in human periodontal ligament fibroblasts: the role of toll-like receptors 2 and 4 and the MAPK pathway.

    PubMed

    Zhang, Y; Li, X

    2015-04-01

    Lipopolysaccharide (LPS) on the cell wall of periodontal pathogens is a major mediator of the inflammatory response and can enhance alveolar bone resorption in periodontitis. Bone sialoprotein is an early marker of osteoblast differentiation. The proinflammatory cytokine, interleukin-8 (IL-8), induces osteoclast differentiation, maturation and maintenance of bone resorption activity. However, the effects of LPS from periodontal pathogens on the expression of bone sialoprotein and IL-8 in human osteoblasts and the mechanism of periodontal bone metabolism regulation are rather unclear. The objectives of this study were to determine the effects of Porphyromonas gingivalis LPS on the production of bone sialoprotein and IL-8 in human periodontal ligament fibroblasts (hPDLFs), and to investigate whether toll-like receptor (TLR) 2, TLR4 and MAPKs pathways are involved in the regulation of production of bone sialoprotein and IL-8 by P. gingivalis LPS. The third-generation of hPDLFs were cultured with mineralization-inducing culture medium. After hPDLFs were treated with P. gingivalis LPS, bone sialoprotein and IL-8 mRNA expression were detected using Real time PCR. Then hPDLFs were transiently transfected with siTLR2 or siTLR4 (20 nm) or inhibited by MAPK signaling pathways inhibitors, and then bone sialoprotein and IL-8 mRNA and protein expression were also detected using Real time PCR and western blotting. Treatments with 0.01 and 0.1 mg/L of P. gingivalis LPS for 8 h up-regulated bone sialoprotein mRNA expression, whereas 10 and 100 mg/L of P. gingivalis LPS induced a significant decrease in the expression of bone sialoprotein mRNA. In contrast, IL8 mRNA levels were increased significantly by 10 mg/L of P. gingivalis LPS. Interestingly, small interfering RNA (siRNA) knock down of the TLR2 and ERK1/2 inhibitor, PD98059, abolished the effects of P. gingivalis LPS on the bone sialoprotein mRNA level, whereas siRNA knock down of the TLR2 and p38 MAPK inhibitor

  10. Interleukin-17-Induced Protein Lipocalin 2 Is Dispensable for Immunity to Oral Candidiasis

    PubMed Central

    Ferreira, Maria Carolina; Whibley, Natasha; Mamo, Anna J.; Siebenlist, Ulrich; Chan, Yvonne R.

    2014-01-01

    Oropharyngeal candidiasis (OPC; thrush) is an opportunistic fungal infection caused by the commensal microbe Candida albicans. Immunity to OPC is strongly dependent on CD4+ T cells, particularly those of the Th17 subset. Interleukin-17 (IL-17) deficiency in mice or humans leads to chronic mucocutaneous candidiasis, but the specific downstream mechanisms of IL-17-mediated host defense remain unclear. Lipocalin 2 (Lcn2; 24p3; neutrophil gelatinase-associated lipocalin [NGAL]) is an antimicrobial host defense factor produced in response to inflammatory cytokines, particularly IL-17. Lcn2 plays a key role in preventing iron acquisition by bacteria that use catecholate-type siderophores, and lipocalin 2−/− mice are highly susceptible to infection by Escherichia coli and Klebsiella pneumoniae. The role of Lcn2 in mediating immunity to fungi is poorly defined. Accordingly, in this study, we evaluated the role of Lcn2 in immunity to oral infection with C. albicans. Lcn2 is strongly upregulated following oral infection with C. albicans, and its expression is almost entirely abrogated in mice with defective IL-17 signaling (IL-17RA−/− or Act1−/− mice). However, Lcn2−/− mice were completely resistant to OPC, comparably to wild-type (WT) mice. Moreover, Lcn2 deficiency mediated protection from OPC induced by steroid immunosuppression. Therefore, despite its potent regulation during C. albicans infection, Lcn2 is not required for immunity to mucosal candidiasis. PMID:24343647

  11. Interleukin-4 and RANTES expression in maturing eosinophils derived from human cord blood CD34+ progenitors

    PubMed Central

    Velazquez, J R; Lacy, P; Mahmudi-Azer, S; Bablitz, B; Milne, C D; Denburg, J A; Moqbel, R

    2000-01-01

    Eosinophils elaborate a number of proinflammatory mediators, including immunoregulatory cytokines and chemokines. Interleukin (IL)-4 and RANTES are important cytokines that have previously been shown to be expressed by mature eosinophils. We hypothesized that de novo synthesis of IL-4 and RANTES occurs in nascent eosinophils, leading to storage of newly produced proteins in crystalloid granule-like structures. Cytokine mRNA and protein expression were examined in cultured eosinophil colonies, which were derived from purified cord blood CD34+ cells and generated in semisolid media (methylcellulose) in the presence of recombinant human (rh)IL-3 and rhIL-5. Cytokine mRNA profiles were analysed by the reverse transcription–polymerase chain reaction (RT–PCR) to determine transcription of IL-4 and RANTES in cells on days 0, 7, 14, 21 and 28 of culture. The expression of translated cytokine products and granule major basic protein (MBP) was confirmed, from day 23 onwards, for colonies cultured in semisolid media, by immunofluorescent labelling and confocal laser-scanning microscopy (CLSM). We found that mRNA sequences encoding IL-4 and RANTES were expressed in freshly prepared, non-differentiated CD34+ cells. Furthermore, RANTES mRNA localized to carbol chromotrope 2R-positive colony cells, as assessed using in situ RT–PCR on day 21 of culture in semisolid media, and was found to gradually decrease (relative to β2-microglobulin) in rhIL-3- and rhIL-5-treated colony cells (comprising > 90% eosinophil-like cells) up to day 28. Immunoreactivity for IL-4 and RANTES co-localized with MBP in maturing colony eosinophils on day 23 of culture in semisolid media, as judged by CLSM. These results suggest that synthesis and storage of immunoregulatory cytokines, essential for processes associated with adaptive immunity, occurs in nascent eosinophils during their growth and differentiation. PMID:11106947

  12. c-Jun localizes to the nucleus independent of its phosphorylation by and interaction with JNK and vice versa promotes nuclear accumulation of JNK

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

    Schreck, Ilona; Al-Rawi, Marco; Mingot, Jose-Manuel

    2011-04-22

    Highlights: {yields} HSP70, Ku70 and 80 as well as importin 8 are novel interactors of c-Jun. {yields} Nuclear accumulation of c-Jun does not require its functions as a transcription factor. {yields} Nuclear accumulation of c-Jun does not require the interaction with its kinase JNK. {yields} Nuclear accumulation of JNK is regulated by interaction with c-Jun. -- Abstract: In order to activate gene expression, transcription factors such as c-Jun have to reside in the nucleus. The abundance of c-Jun in the nucleus correlates with the activity of its target genes. As a consequence of excessive c-Jun activation, cells undergo apoptosis ormore » changes in differentiation whereas decreased c-Jun function can reduce proliferation. In the present study we addressed how nuclear accumulation of the transcription factor c-Jun is regulated. First, we analyzed which functions of c-Jun are required for efficient nuclear accumulation. Mutants of c-Jun deficient in dimerization or DNA-binding show no defect in nuclear transport. Furthermore, c-Jun import into the nucleus of living cells occurred when the c-Jun phosphorylation sites were mutated as well in cells that lack the major c-Jun kinase, JNK, suggesting that c-Jun transport into the nucleus does not require JNK signaling. Conversely, however, binding of c-Jun seemed to enhance nuclear accumulation of JNK. In order to identify proteins that might be relevant for the nuclear translocation of c-Jun we searched for novel binding partners by a proteomic approach. In addition to the heat shock protein HSP70 and the DNA damage repair factors Ku70 and 80, we isolated human importin 8 as a novel interactor of c-Jun. Interaction of Imp 8 with c-Jun in human cells was confirmed by co-immunoprecipitation experiments. Nuclear accumulation of c-Jun does not require its functions as a transcription factor or the interaction with its kinase JNK. Interestingly, nuclear accumulation of JNK is regulated by interaction with c-Jun. Unraveling

  13. Post-Transcriptional Regulation of BCL2 mRNA by the RNA-Binding Protein ZFP36L1 in Malignant B Cells

    PubMed Central

    Zekavati, Anna; Nasir, Asghar; Alcaraz, Amor; Aldrovandi, Maceler; Marsh, Phil; Norton, John D.; Murphy, John J.

    2014-01-01

    The human ZFP36 zinc finger protein family consists of ZFP36, ZFP36L1, and ZFP36L2. These proteins regulate various cellular processes, including cell apoptosis, by binding to adenine uridine rich elements in the 3′ untranslated regions of sets of target mRNAs to promote their degradation. The pro-apoptotic and other functions of ZFP36 family members have been implicated in the pathogenesis of lymphoid malignancies. To identify candidate mRNAs that are targeted in the pro-apoptotic response by ZFP36L1, we reverse-engineered a gene regulatory network for all three ZFP36 family members using the ‘maximum information coefficient’ (MIC) for target gene inference on a large microarray gene expression dataset representing cells of diverse histological origin. Of the three inferred ZFP36L1 mRNA targets that were identified, we focussed on experimental validation of mRNA for the pro-survival protein, BCL2, as a target for ZFP36L1. RNA electrophoretic mobility shift assay experiments revealed that ZFP36L1 interacted with the BCL2 adenine uridine rich element. In murine BCL1 leukemia cells stably transduced with a ZFP36L1 ShRNA lentiviral construct, BCL2 mRNA degradation was significantly delayed compared to control lentiviral expressing cells and ZFP36L1 knockdown in different cell types (BCL1, ACHN, Ramos), resulted in increased levels of BCL2 mRNA levels compared to control cells. 3′ untranslated region luciferase reporter assays in HEK293T cells showed that wild type but not zinc finger mutant ZFP36L1 protein was able to downregulate a BCL2 construct containing the BCL2 adenine uridine rich element and removal of the adenine uridine rich core from the BCL2 3′ untranslated region in the reporter construct significantly reduced the ability of ZFP36L1 to mediate this effect. Taken together, our data are consistent with ZFP36L1 interacting with and mediating degradation of BCL2 mRNA as an important target through which ZFP36L1 mediates its pro-apoptotic effects in

  14. Murine knockin model for progranulin-deficient frontotemporal dementia with nonsense-mediated mRNA decay

    PubMed Central

    Nguyen, Thi A.; Zhang, Jiasheng; Devireddy, Swathi; Zhou, Ping; Karydas, Anna M.; Xu, Xialian; Miller, Bruce L.; Rigo, Frank; Ferguson, Shawn M.; Walther, Tobias C.; Farese, Robert V.

    2018-01-01

    Frontotemporal dementia (FTD) is the most common neurodegenerative disorder in individuals under age 60 and has no treatment or cure. Because many cases of FTD result from GRN nonsense mutations, an animal model for this type of mutation is highly desirable for understanding pathogenesis and testing therapies. Here, we generated and characterized GrnR493X knockin mice, which model the most common human GRN mutation, a premature stop codon at arginine 493 (R493X). Homozygous GrnR493X mice have markedly reduced Grn mRNA levels, lack detectable progranulin protein, and phenocopy Grn knockout mice, with CNS microgliosis, cytoplasmic TDP-43 accumulation, reduced synaptic density, lipofuscinosis, hyperinflammatory macrophages, excessive grooming behavior, and reduced survival. Inhibition of nonsense-mediated mRNA decay (NMD) by genetic, pharmacological, or antisense oligonucleotide-based approaches showed that NMD contributes to the reduced mRNA levels in GrnR493X mice and cell lines and in fibroblasts from patients containing the GRNR493X mutation. Moreover, the expressed truncated R493X mutant protein was functional in several assays in progranulin-deficient cells. Together, these findings establish a murine model for in vivo testing of NMD inhibition or other therapies as potential approaches for treating progranulin deficiency caused by the R493X mutation. PMID:29511098

  15. Mitotic inheritance of mRNA facilitates translational activation of the osteogenic-lineage commitment factor Runx2 in progeny of osteoblastic cells

    PubMed Central

    Varela, Nelson; Aranguiz, Alejandra; Lizama, Carlos; Sepulveda, Hugo; Antonelli, Marcelo; Thaler, Roman; Moreno, Ricardo D.; Montecino, Martin; Stein, Gary S.; van Wijnen, Andre J.; Galindo, Mario

    2017-01-01

    Epigenetic mechanisms mediate the acquisition of specialized cellular phenotypes during tissue development, maintenance and repair. When phenotype-committed cells transit through mitosis, chromosomal condensation counteracts epigenetic activation of gene expression. Subsequent post-mitotic re-activation of transcription depends on epigenetic DNA and histone modifications, as well as other architecturally bound proteins that ‘bookmark’ the genome. Osteogenic lineage commitment, differentiation and progenitor proliferation require the bone-related runt-related transcription factor Runx2. Here, we characterized a non-genomic mRNA mediated mechanism by which osteoblast precursors retain their phenotype during self-renewal. We show that osteoblasts produce maximal levels of Runx2 mRNA, but not protein, prior to mitotic cell division. Runx2 mRNA partitions symmetrically between daughter cells in a non-chromosomal tubulin-containing compartment. Subsequently, transcription-independent de novo synthesis of Runx2 protein in early G1 phase results in increased functional interactions of Runx2 with a representative osteoblast-specific target gene (osteocalcin/BGLAP2) in chromatin. Somatic transmission of Runx2 mRNAs in osteoblasts and osteosarcoma cells represents a versatile mechanism for translational rather than transcriptional induction of this principal gene regulator to maintain osteoblast phenotype identity after mitosis. PMID:26381402

  16. [Impacts of the formula of Suoquanwan(SQW) on expression of AQP-2 mRNA and AVPR-V2 mRNA in the kidney of rat polyuria model of Yang-deficiency].

    PubMed

    Cao, Hong-Ying; Wu, Qing-He; Huang, Ping; He, Jin-Yang

    2009-06-01

    To observe the impacts of the formula of Suoquanwan (SQW) on the expression of AQP-2 mRNA and AVPR-V2 mRNA in the kidney of rat polyuria model of Yang-deficiency. The model rats were induced by adenine (250 mg/kg) for 4 weeks, then treated respectively with SQW or dDAVP. The expression of AQP-2 mRNA and AVPR-V2 mRNA in kidney of Yang-deficiency model by realtime fluorescence quantitative PCR method were investigated. In model rats, the expression of AQP-2 mRNA and AVPR-V2 mRNA in the kidney decreased, dDAVP and SQW high dose could increased the expression of AQP-2 mRNA and AVPR-V2 mRNA in the kidney. The others had no influence on the expression of AQP-2 mRNA and AVPR-V2 mRNA in the kidney. SQW can increase the expression of AQP-2 mRNA and AVPR-V2 mRNA in the kidney of rat polyuria model of Yang-deficiency.

  17. Rutin hydrate ameliorates cadmium chloride-induced spatial memory loss and neural apoptosis in rats by enhancing levels of acetylcholine, inhibiting JNK and ERK1/2 activation and activating mTOR signalling.

    PubMed

    Abdel-Aleem, Ghada A; Khaleel, Eman F

    2017-12-07

    This study aimed at studying the potential neuroprotective effect of Rutin hydrate (RH) alone or in conjugation with α-tocopherol against cadmium chloride (CdCl 2 )-induced neurotoxicity and cognitive impairment in rats and to investigate the mechanisms of action. Rats intoxicated with CdCl 2 were treated with the vehicle, RH, α-tocopherol or combined treatment were examined, and compared to control rats received vehicle or individual doses of either drug. Data confirmed that RH improves spatial memory function by increasing acetylcholine availability, boosting endogenous antioxidant potential, activating cell survival and inhibiting apoptotic pathways, an effect that is more effective when RH was conjugated with α-tocopherol. Mechanism of RH action includes activation of PP2A mediated inhibiting of ERK1/2 and JNK apoptotic pathways and inhibition of PTEN mediated activation of mTOR survival pathway. In conclusion, RH affords a potent neuroprotection against CdCl 2 -induced brain damage and memory dysfunction and co-administration of α-tocopherol enhances its activity.

  18. A Novel c-Jun N-terminal Kinase (JNK) Signaling Complex Involved in Neuronal Migration during Brain Development.

    PubMed

    Zhang, Feng; Yu, Jingwen; Yang, Tao; Xu, Dan; Chi, Zhixia; Xia, Yanheng; Xu, Zhiheng

    2016-05-27

    Disturbance of neuronal migration may cause various neurological disorders. Both the transforming growth factor-β (TGF-β) signaling and microcephaly-associated protein WDR62 are important for neuronal migration during brain development; however, the underlying molecular mechanisms involved remain unclear. We show here that knock-out or knockdown of Tak1 (TGFβ-activated kinase 1) and Jnk2 (c-Jun N-terminal kinase 2) perturbs neuronal migration during cortical development and that the migration defects incurred by knock-out and/or knockdown of Tβr2 (type II TGF-β receptor) or Tak1 can be partially rescued by expression of TAK1 and JNK2, respectively. Furthermore, TAK1 forms a protein complex with RAC1 and two scaffold proteins of the JNK pathway, the microcephaly-associated protein WDR62 and the RAC1-interacting protein POSH (plenty of Src homology). Components of the complex coordinate with each other in the regulation of TAK1 as well as JNK activities. We suggest that unique JNK protein complexes are involved in the diversified biological and pathological functions during brain development and pathogenesis of diseases. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  19. 2′,5′-Dihydroxychalcone-induced glutathione is mediated by oxidative stress and kinase signaling pathways

    PubMed Central

    Kachadourian, Remy; Pugazhenthi, Subbiah; Velmurugan, Kalpana; Backos, Donald S.; Franklin, Christopher C.; McCord, Joe M.; Day, Brian J.

    2011-01-01

    Hydroxychalcones are naturally occurring compounds that continue to attract considerable interest due to their anti-inflammatory and anti-angiogenic properties. They have been reported to inhibit the synthesis of the inducible nitric oxide (NO) synthase and to induce the expression of heme oxygenase-1 (HO-1). This study examines the mechanisms by which 2′,5′-dihydroxychalcone (2′,5′-DHC) induces an increase in cellular glutathione (GSH) levels using a cell line stably expressing a luciferase reporter gene driven by antioxidant response elements (MCF-7/AREc32). 2′,5′-DHC-induced increase in cellular GSH levels was partially inhibited by the catalytic antioxidant MnTDE-1,3-IP5+, suggesting that reactive oxygen species (ROS) mediate the antioxidant adaptive response. 2′,5′-DHC treatment induced the phosphorylation of c-Jun N-terminal kinase (JNK) pathway that was also inhibited by MnTDE-1,3-IP5+. These findings suggest a ROS-dependent activation of the AP-1 transcriptional response. However, while 2′,5′-DHC triggered the NF-E2-related factor 2 (Nrf2) transcriptional response, co-treatment with MnTDE-1,3-IP5+ did not decrease 2′,5′-DHC-induced Nrf2/ARE activity, showing that this pathway is not dependent on ROS. Moreover, pharmacological inhibitors of mitogen-activated protein (MAP) kinase pathways showed a role for JNK and p38MAPK in mediating the 2′,5′-DHC-induced Nrf2 response. These findings suggest that the 2′,5′-DHC-induced increase in GSH levels results from a combination of ROS-dependent and ROS-independent pathways. PMID:21712085

  20. MYC Mediates mRNA Cap Methylation of Canonical Wnt/β-catenin Signaling Transcripts by Recruiting CDK7 and RNA Methyltransferase

    PubMed Central

    Posternak, Valeriya; Ung, Matthew H.; Cheng, Chao; Cole, Michael D.

    2016-01-01

    MYC is a pleiotropic transcription factor that activates and represses a wide range of target genes and is frequently deregulated in human tumors. While much is known about the role of MYC in transcriptional activation and repression, MYC can also regulate mRNA cap methylation through a mechanism that has remained poorly understood. Here it is reported that MYC enhances mRNA cap methylation of transcripts globally, specifically increasing mRNA cap methylation of genes involved in Wnt/β-catenin signaling. Elevated mRNA cap methylation of Wnt signaling transcripts in response to MYC leads to augmented translational capacity, elevated protein levels, and enhanced Wnt signaling activity. Mechanistic evidence indicates that MYC promotes recruitment of RNA methyltransferase (RNMT) to Wnt signaling gene promoters by enhancing phosphorylation of serine 5 on the RNA Polymerase II Carboxy-Terminal Domain, mediated in part through an interaction between the TIP60 acetyltransferase complex and TFIIH. Implications MYC enhances mRNA cap methylation above and beyond transcriptional induction. PMID:27899423

  1. Molecular cloning, characterization and mRNA expression of duck interleukin-17F

    USDA-ARS?s Scientific Manuscript database

    Interleukin-17F (IL-17F) is a proinflammatory cytokine that plays an important role in gut homeostasis. A full-length duck IL-17F (duIL-17F) cDNA with a 501-bp coding region was identified in ConA-activated splenic lymphocytes. duIL-17F is predicted to encode 166 amino acids, including a 26-amino ...

  2. The JNK inhibitor XG-102 protects against TNBS-induced colitis.

    PubMed

    Reinecke, Kirstin; Eminel, Sevgi; Dierck, Franziska; Roessner, Wibke; Kersting, Sabine; Chromik, Ansgar Michael; Gavrilova, Olga; Laukevicience, Ale; Leuschner, Ivo; Waetzig, Vicki; Rosenstiel, Philip; Herdegen, Thomas; Sina, Christian

    2012-01-01

    The c-Jun N-terminal kinase (JNK)-inhibiting peptide D-JNKI-1, syn. XG-102 was tested for its therapeutic potential in acute inflammatory bowel disease (IBD) in mice. Rectal instillation of the chemical irritant trinitrobenzene sulfonic acid (TNBS) provoked a dramatic acute inflammation in the colon of 7-9 weeks old mice. Coincident subcutaneous application of 100 µg/kg XG-102 significantly reduced the loss of body weight, rectal bleeding and diarrhoea. After 72 h, the end of the study, the colon was removed and immuno-histochemically analysed. XG-102 significantly reduced (i) pathological changes such as ulceration or crypt deformation, (ii) immune cell pathology such as infiltration and presence of CD3- and CD68-positive cells, (iii) the production of tumor necrosis factor (TNF)-α in colon tissue cultures from TNBS-treated mice, (iv) expression of Bim, Bax, FasL, p53, and activation of caspase 3, (v) complexation of JNK2 and Bim, and (vi) expression and activation of the JNK substrate and transcription factor c-Jun. A single application of subcutaneous XG-102 was at least as effective or even better depending on the outcome parameter as the daily oral application of sulfasalazine used for treatment of IBD.The successful and substantial reduction of the severe, TNBS-evoked intestinal damages and clinical symptoms render the JNK-inhibiting peptide XG-102 a powerful therapeutic principle of IBD.

  3. Stimulation of EphB2/ephrin-B1 signalling by tumour necrosis factor alpha in human dental pulp stem cells.

    PubMed

    Zhu, Lifang; Dissanayaka, Waruna Lakmal; Green, David William; Zhang, Chengfei

    2015-04-01

    The aim of this study was to investigate whether in vitro stimulation of dental pulp stem cells (DPSCs) by tumour necrosis factor alpha (TNF-α) would induce secretion of EphB2/ephrin-B1 signalling. Dental pulp stem cells isolated from human dental pulp were treated with TNF-α (5-100 ng/ml) over 2-48 h. EphB2/ephrin-B1 mRNA and protein levels were measured by real-time polymerase chain reaction (RT-PCR) and western blot analysis respectively. Additionally, DPSCs were pre-incubated with TNF-α receptor neutralizing antibodies or infected with nuclear factor-kappa B (NF-ĸB) inhibitor, p38 MAPK inhibitor, Jun N-terminal kinase (JNK) inhibitor and MEK inhibitor before TNF-α treatment. Results were analysed by one-way ANOVA. Tumour necrosis factor alpha increased EphB2 mRNA expression in DPSCs at concentrations up to 20 ng/ml and ephrin-B1 at concentrations up to 40 ng/ml (P < 0.05). Its mRNA expression reached maximum at 24 h when treated with TNF-α at 20 ng/ml (P < 0.05). EphB2/ephrin-B1 protein expression levels were high at 16 and 24 h as shown by western blotting. Neutralizing antibodies for TNFR1/2 receptors down-regulated EphB2/ephrin-B1 mRNA expression (P < 0.05) and ephrin-B1 protein expression, but not EphB2 protein expression. JNK-inhibitor inhibited EphB2 mRNA expression only (P < 0.05). EphB2/ephrin-B1 were invoked in DPSCs with TNF-α treatment via the JNK-dependent pathway, but not NF-ĸB, p38 MAPK or MEK signalling. © 2015 John Wiley & Sons Ltd.

  4. AAV serotype 2/1-mediated gene delivery of anti-inflammatory interleukin-10 enhances neurogenesis and cognitive function in APP+PS1 mice.

    PubMed

    Kiyota, T; Ingraham, K L; Swan, R J; Jacobsen, M T; Andrews, S J; Ikezu, T

    2012-07-01

    Brain inflammation is a double-edged sword. It is required for brain repair in acute damage, whereas chronic inflammation and autoimmune disorders are neuropathogenic. Certain proinflammatory cytokines and chemokines are closely related to cognitive dysfunction and neurodegeneration. Representative anti-inflammatory cytokines, such as interleukin (IL)-10, can suppress neuroinflammation and have significant therapeutic potentials in ameliorating neurodegenerative disorders such as Alzheimer's disease (AD). Here, we show that adeno-associated virus (AAV) serotype 2/1 hybrid-mediated neuronal expression of the mouse IL-10 gene ameliorates cognitive dysfunction in amyloid precursor protein+ presenilin-1 bigenic mice. AAV2/1 infection of hippocampal neurons resulted in sustained expression of IL-10 without its leakage into the blood, reduced astro/microgliosis, enhanced plasma amyloid-β peptide (Aβ) levels and enhanced neurogenesis. Moreover, increased levels of IL-10 improved spatial learning, as determined by the radial arm water maze. Finally, IL-10-stimulated microglia enhanced proliferation but not differentiation of primary neural stem cells in the co-culture system, whereas IL-10 itself had no effect. Our data suggest that IL-10 gene delivery has a therapeutic potential for a non-Aβ-targeted treatment of AD.

  5. 5-methylcytosine promotes mRNA export — NSUN2 as the methyltransferase and ALYREF as an m5C reader

    PubMed Central

    Yang, Xin; Yang, Ying; Sun, Bao-Fa; Chen, Yu-Sheng; Xu, Jia-Wei; Lai, Wei-Yi; Li, Ang; Wang, Xing; Bhattarai, Devi Prasad; Xiao, Wen; Sun, Hui-Ying; Zhu, Qin; Ma, Hai-Li; Adhikari, Samir; Sun, Min; Hao, Ya-Juan; Zhang, Bing; Huang, Chun-Min; Huang, Niu; Jiang, Gui-Bin; Zhao, Yong-Liang; Wang, Hai-Lin; Sun, Ying-Pu; Yang, Yun-Gui

    2017-01-01

    5-methylcytosine (m5C) is a post-transcriptional RNA modification identified in both stable and highly abundant tRNAs and rRNAs, and in mRNAs. However, its regulatory role in mRNA metabolism is still largely unknown. Here, we reveal that m5C modification is enriched in CG-rich regions and in regions immediately downstream of translation initiation sites and has conserved, tissue-specific and dynamic features across mammalian transcriptomes. Moreover, m5C formation in mRNAs is mainly catalyzed by the RNA methyltransferase NSUN2, and m5C is specifically recognized by the mRNA export adaptor ALYREF as shown by in vitro and in vivo studies. NSUN2 modulates ALYREF's nuclear-cytoplasmic shuttling, RNA-binding affinity and associated mRNA export. Dysregulation of ALYREF-mediated mRNA export upon NSUN2 depletion could be restored by reconstitution of wild-type but not methyltransferase-defective NSUN2. Our study provides comprehensive m5C profiles of mammalian transcriptomes and suggests an essential role for m5C modification in mRNA export and post-transcriptional regulation. PMID:28418038

  6. JNK1 regulates histone acetylation in trigeminal neurons following chemical stimulation

    PubMed Central

    Wu, Jing; Zhang, Xuan; Nauta, Haring J; Lin, Qing; Li, Junfa; Fang, Li

    2008-01-01

    Trigeminal nerve fibers in nasal and oral cavities are sensitive to various environmental hazardous stimuli, which trigger many neurotoxic problems such as chronic migraine headache and trigeminal irritated disorders. However, the role of JNK kinase cascade and its epigenetic modulation of histone remodeling in trigeminal ganglion (TG) neurons activated by environmental neurotoxins remains unknown. Here we investigated the role of JNK/c-Jun cascade in the regulation of acetylation of H3 histone in TG neurons following in vitro stimulation by a neuro-inflammatory agent, mustard oil (MO). We found that MO stimulation elicited JNK/c-Jun pathway significantly by enhancing phospho-JNK1, phospho-c-Jun expression, and c-Jun activity, which were correlated with an elevated acetylated H3 histone in TG neurons. However, increases in phospho-c-Jun and c-Jun activity were significantly blocked by a JNK inhibitor, SP600125. We also found that altered H3 histone remodeling, assessed by H3 acetylation in triggered TG neurons, was reduced by SP600125. The study suggests that the activated JNK signaling in regulation of histone remodeling may contribute to neuro-epigentic changes in peripheral sensory neurons following environmental neurotoxic exposure. PMID:18822271

  7. JNK-induced MCP-1 production in spinal cord astrocytes contributes to central sensitization and neuropathic pain

    PubMed Central

    Gao, Yong-Jing; Zhang, Ling; Samad, Omar Abdel; Suter, Marc R.; Yasuhiko, Kawasaki; Xu, Zhen-Zhong; Park, Jong-Yeon; Lind, Anne-Li; Ma, Qiufu; Ji, Ru-Rong

    2009-01-01

    Our previous study showed that activation of c-jun-N-terminal kinase (JNK) in spinal astrocytes plays an important role in neuropathic pain sensitization. We further investigated how JNK regulates neuropathic pain. In cultured astrocytes, TNF-α transiently activated JNK via TNF receptor-1. Cytokine array indicated that the chemokine CCL2/MCP-1 (monocyte chemoattractant protein-1) was strongly induced by the TNF-α/JNK pathway. MCP-1 upregulation by TNF-α was dose-dependently inhibited by the JNK inhibitors SP600125 and D-JNKI-1. Spinal injection of TNF-α produced JNK-dependent pain hypersensitivity and MCP-1 upregulation in the spinal cord. Further, spinal nerve ligation (SNL) induced persistent neuropathic pain and MCP-1 upregulation in the spinal cord, and both were suppressed by D-JNKI-1. Remarkably, MCP-1 was primarily induced in spinal cord astrocytes after SNL. Spinal administration of MCP-1 neutralizing antibody attenuated neuropathic pain. Conversely, spinal application of MCP-1 induced heat hyperalgesia and phosphorylation of extracellular signal-regulated kinase (ERK) in superficial spinal cord dorsal horn neurons, indicative of central sensitization (hyperactivity of dorsal horn neurons). Patch clamp recordings in lamina II neurons of isolated spinal cord slices showed that MCP-1 not only enhanced spontaneous excitatory synaptic currents (sEPSCs) but also potentiated NMDA- and AMPA-induced currents. Finally, the MCP-1 receptor CCR2 was expressed in neurons and some non-neuronal cells in the spinal cord. Taken together, we have revealed a previously unknown mechanism of MCP-1 induction and action. MCP-1 induction in astrocytes following JNK activation contributes to central sensitization and neuropathic pain facilitation by enhancing excitatory synaptic transmission. Inhibition of the JNK/MCP-1 pathway may provide a new therapy for neuropathic pain management. PMID:19339605

  8. Nonsense-mediated mRNA decay: inter-individual variability and human disease

    PubMed Central

    Nguyen, Lam Son; Wilkinson, Miles; Gecz, Jozef

    2013-01-01

    Nonsense-Mediated mRNA Decay (NMD) is a regulatory pathway that functions to degrade transcripts containing premature termination codons (PTCs) and to maintain normal transcriptome homeostasis. Nonsense and frameshift mutations that generate PTCs cause approximately one-third of all known human genetic diseases and thus NMD has a potentially important role in human disease. In genetic disorders in which the affected genes carry PTC-generating mutations, NMD acts as a double-edge sword. While it can benefit the patient by degrading PTC-containing mRNAs that encode detrimental, dominant-negative truncated proteins, it can also make the disease worse when a PTC-containing mRNA is degraded that encodes a mutant but still functional protein. There is evidence that the magnitude of NMD varies between individuals, which, in turn, has been shown to correlate with both clinical presentations and the patients’ responses to drugs that promote read-through of PTCs. In this review, we examine the evidence supporting the existence of inter-individual variability in NMD efficiency and discuss the genetic factors that underlie this variability. We propose that inter-individual variability in NMD efficiency is a common phenomenon in human populations and that an individual’s NMD efficiency should be taken into consideration when testing, developing, and making therapeutic decisions for diseases caused by genes harboring PTCs. PMID:24239855

  9. Cyclooxygenase-2 mediates the febrile response of mice to interleukin-1beta.

    PubMed

    Li, S; Ballou, L R; Morham, S G; Blatteis, C M

    2001-08-10

    Various lines of evidence have implicated cyclooxygenase (COX)-2 as a modulator of the fever induced by the exogenous pyrogen lipopolysaccharide (LPS). Thus, treatment with specific inhibitors of COX-2 suppresses the febrile response without affecting basal body (core) temperature (T(c)). Furthermore, COX-2 gene-ablated mice are unable to develop a febrile response to intraperitoneal (i.p.) LPS, whereas their COX-1-deficient counterparts produce fevers not different from their wild-type (WT) controls. To extend the apparently critical role of COX-2 for LPS-induced fevers to fevers produced by endogenous pyrogens, we studied the thermal responses of COX-1- and COX-2 congenitally deficient mice to i.p. and intracerebroventricular (i.c.v.) injections of recombinant murine (rm) interleukin (IL)-1beta. We also assessed the effects of one selective COX-1 inhibitor, SC-560, and two selective COX-2 inhibitors, nimesulide (NIM) and dimethylfuranone (DFU), on the febrile responses of WT and COX-1(-/-) mice to LPS and rmIL-1beta, i.p. Finally, we verified the integrity of the animals' responses to PGE2, i.c.v. I.p. and i.c.v. rmIL-1beta induced similar fevers in WT and COX-1 knockout mice, but provoked no rise in the T(c)s of COX-2 null mutants. The fever produced in WT mice by i.p. LPS was not affected by SC-560, but it was attenuated and abolished by NIM and DFU, respectively, while that caused by i.p. rmIL-1beta was converted into a T(c) fall by DFU. There were no differences in the responses to i.c.v. PGE2 among the WT and COX knockout mice. These results, therefore, further support the notion that the production of PGE2 in response to pyrogens is critically dependent on COX-2 expression.

  10. Phloretin induces apoptosis of non-small cell lung carcinoma A549 cells via JNK1/2 and p38 MAPK pathways.

    PubMed

    Min, Jie; Huang, Kenan; Tang, Hua; Ding, Xinyu; Qi, Chen; Qin, Xiong; Xu, Zhifei

    2015-12-01

    Phloretin (Ph) existing in apples, pears and various vegetables is known to have antitumor activities in several cancer cell lines. However, little is known about its effect on human lung cancer cells. The aim of the present study was to see whether Ph could induce apoptosis of non-small cell lung cancer (NSCLC) cells, and explore the possible underlying mechanism of action. We found that Ph markedly induced cell apoptosis of NSCLC cell line A549, and inhibited the migration of A549 cells in a dose-dependent manner. The expression level of BAX, cleaved caspase-3 and -9, and degraded form of PARP was increased and Bcl-2 was decreased after Ph treatment. In addition, the phosphorylation of P38 MAPK, ERK1/2 and JNK1/2 was increased in a dose‑dependent manner in parallel with Ph treatment. Inhibition of P38 MAPK and JNK1/2 by specific inhibitors significantly abolished the Ph-induced activation of the caspase-3 and -9. In vivo tumor-suppression assay further indicated that Ph (20 mg/kg) displayed a more significant inhibitory effect on A549 xenografts in tumor growth. All these findings indicate that Ph is able to inhibit NSCLC A549 cell growth by inducing apoptosis through P38 MAPK and JNK1/2 pathways, and therefore may prove to be an adjuvant to the treatment of NSCLC.

  11. Gasdermin C is induced by ultraviolet light and contributes to MMP-1 expression via activation of ERK and JNK pathways.

    PubMed

    Kusumaningrum, Novi; Lee, Dong Hun; Yoon, Hyun-Sun; Kim, Yeon Kyung; Park, Chi-Hyun; Chung, Jin Ho

    2018-05-01

    Ultraviolet (UV) radiation plays important roles in various skin diseases including premature aging and cancer. UV has been shown to regulate the expressions of many genes including matrix metalloproteinases (MMPs). Gasdermin C (GSDMC) belongs to Gasdermin family and is known to be expressed in the epithelial cells of many tissues including the skin. However, the functions of GSDMC remain poorly understood. We aimed to investigate the role of GSDMC in UV-induced MMP-1, MMP-3, and MMP-9 expressions in human skin keratinocytes. Primary human skin keratinocytes and an immortalized human skin keratinocyte cell line (HaCaT cells) were irradiated with UV. Knockdown and overexpression of GSDMC were performed to study the effect of GSDMC. The mRNA and protein levels were analyzed by quantitative real-time polymerase chain reaction (qRT-PCR) and western blotting, respectively. We found that GSDMC expression is increased by UV irradiation in human skin keratinocytes. Further studies showed that GSDMC expression is increased at relatively late time points after UV irradiation and that this GSDMC induction plays important roles in the expressions of MMP-1, but not of MMP-3 and MMP-9, and the activations of ERK and JNK induced by UV. In addition, we found that overexpression of GSDMC increases the MMP-1 expression and the activities of ERK and JNK and that GSDMC-induced MMP-1 expression is suppressed by inhibition of ERK or JNK activities. Our results suggest that GSDMC is increased by UV radiation and contributes to UV-induced MMP-1 expression through the activation of ERK and JNK pathways. Copyright © 2018 Japanese Society for Investigative Dermatology. Published by Elsevier B.V. All rights reserved.

  12. Interleukin-1 receptor (IL-1R) mediates epilepsy-induced sleep disruption.

    PubMed

    Huang, Tzu-Rung; Jou, Shuo-Bin; Chou, Yu-Ju; Yi, Pei-Lu; Chen, Chun-Jen; Chang, Fang-Chia

    2016-11-22

    Sleep disruptions are common in epilepsy patients. Our previous study demonstrates that homeostatic factors and circadian rhythm may mediate epilepsy-induced sleep disturbances when epilepsy occurs at different zeitgeber hours. The proinflammatory cytokine, interleukin-1 (IL-1), is a somnogenic cytokine and may also be involved in epileptogenesis; however, few studies emphasize the effect of IL-1 in epilepsy-induced sleep disruption. We herein hypothesized that IL-1 receptor type 1 (IL-1R1) mediates the pathogenesis of epilepsy and epilepsy-induced sleep disturbances. We determined the role of IL-1R1 by using IL-1R1 knockout (IL-1R1 -/- KO) mice. Our results elucidated the decrease of non-rapid eye movement (NREM) sleep during the light period in IL-1R -/- mice and confirmed the somnogenic role of IL-1R1. Rapid electrical amygdala kindling was performed to induce epilepsy at the particular zeitgeber time (ZT) point, ZT13. Our results demonstrated that seizure thresholds induced by kindling stimuli, such as the after-discharge threshold and successful kindling rates, were not altered in IL-1R -/- mice when compared to those obtained from the wildtype mice (IL-1R +/+ mice). This result suggests that IL-1R1 is not involved in kindling-induced epileptogenesis. During sleep, ZT13 kindling stimulation significantly enhanced NREM sleep during the subsequent 6 h (ZT13-18) in wildtype mice, and sleep returned to the baseline the following day. However, the kindling-induced sleep alteration was absent in the IL-1R -/- KO mice. These results indicate that the IL-1 signal mediates epilepsy-induced sleep disturbance, but dose not participate in kindling-induced epileptogenesis.

  13. JNK Activation Contributes to Oxidative Stress-Induced Parthanatos in Glioma Cells via Increase of Intracellular ROS Production.

    PubMed

    Zheng, Linjie; Wang, Chen; Luo, Tianfei; Lu, Bin; Ma, Hongxi; Zhou, Zijian; Zhu, Dong; Chi, Guangfan; Ge, Pengfei; Luo, Yinan

    2017-07-01

    Parthanatos is a form of PARP-1-dependent programmed cell death. The induction of parthanatos is emerging as a new strategy to kill gliomas which are the most common type of primary malignant brain tumor. Oxidative stress is thought to be a critical factor triggering parthanatos, but its underlying mechanism is poorly understood. In this study, we used glioma cell lines and H 2 O 2 to investigate the role of JNK in glioma cell parthanatos induced by oxidative stress. We found that exposure to H 2 O 2 not only induced intracellular accumulation of ROS but also resulted in glioma cell death in a concentration- and incubation time-dependent manner, which was accompanied with cytoplasmic formation of PAR polymer, expressional upregulation of PARP-1, mitochondrial depolarization, and AIF translocation to nucleus. Pharmacological inhibition of PARP-1 with 3AB or genetic knockdown of its level with siRNA rescued glioma cell death, as well as suppressed cytoplasmic accumulation of PAR polymer and nuclear translocation of AIF, which were consistent with the definition of parthanatos. Moreover, the phosphorylated level of JNK increased markedly with the extension of H 2 O 2 exposure time. Either attenuation of intracellular ROS with antioxidant NAC or inhibition of JNK phosphorylation with SP600125 or JNK siRNA could significantly prevent H 2 O 2 -induced parthanatos in glioma cells. Additionally, inhibition of JNK with SP600125 alleviated intracellular accumulation of ROS and attenuated mitochondrial generation of superoxide. Thus, we demonstrated that JNK activation contributes to glioma cell parthanatos caused by oxidative stress via increase of intracellular ROS generation.

  14. Effect of JNK inhibitor SP600125 on hair cell regeneration in zebrafish (Danio rerio) larvae

    PubMed Central

    Sun, Shaoyang; Wang, Xu; Li, Wenyan; Li, Huawei

    2016-01-01

    The c-Jun amino-terminal kinase (JNK) proteins are a subgroup of the mitogen-activated protein kinase family. They play a complex role in cell proliferation, survival, and apoptosis. Here, we report a novel role of JNK signalling in hair cell regeneration. We eliminated hair cells of 5-day post-fertilization zebrafish larvae using neomycin followed by JNK inhibition with SP600125. JNK inhibition strongly decreased the number of regenerated hair cells in response to neomycin damage. These changes were associated with reduced proliferation. JNK inhibition also increased cleaved caspase-3 activity and induced apoptosis in regenerating neuromasts. Finally, JNK inhibition with SP600125 decreased the expression of genes related to Wnt. Over-activation of the Wnt signalling pathway partly rescued the hair cell regeneration defects induced by JNK inhibition. Together, our findings provide novel insights into the function of JNK and show that JNK inhibition blocks hair cell regeneration by controlling the Wnt signalling pathway. PMID:27438150

  15. High-Dose Fluoride Impairs the Properties of Human Embryonic Stem Cells via JNK Signaling.

    PubMed

    Fu, Xin; Xie, Fang-Nan; Dong, Ping; Li, Qiu-Chen; Yu, Guang-Yan; Xiao, Ran

    2016-01-01

    Fluoride is a ubiquitous natural substance that is often used in dental products to prevent dental caries. The biphasic actions of fluoride imply that excessive systemic exposure to fluoride can cause harmful effects on embryonic development in both animal models and humans. However, insufficient information is available on the effects of fluoride on human embryonic stem cells (hESCs), which is a novel in vitro humanized model for analyzing the embryotoxicities of chemical compounds. Therefore, we investigated the effects of sodium fluoride (NaF) on the proliferation, differentiation and viability of H9 hESCs. For the first time, we showed that 1 mM NaF did not significantly affect the proliferation of hESCs but did disturb the gene expression patterns of hESCs during embryoid body (EB) differentiation. Higher doses of NaF (2 mM and above) markedly decreased the viability and proliferation of hESCs. The mode and underlying mechanism of high-dose NaF-induced cell death were further investigated by assessing the sub-cellular morphology, mitochondrial membrane potential (MMP), caspase activities, cellular reactive oxygen species (ROS) levels and activation of mitogen-activated protein kinases (MAPKs). High-dose NaF caused the death of hESCs via apoptosis in a caspase-mediated but ROS-independent pathway, coupled with an increase in the phospho-c-Jun N-terminal kinase (p-JNK) levels. Pretreatment with a p-JNK-specific inhibitor (SP600125) could effectively protect hESCs from NaF-induced cell death in a concentration- and time-dependent manner. These findings suggest that NaF might interfere with early human embryogenesis by disturbing the specification of the three germ layers as well as osteogenic lineage commitment and that high-dose NaF could cause apoptosis through a JNK-dependent pathway in hESCs.

  16. Tilmicosin does not inhibit interleukin-8 gene expression in the bovine lung experimentally infected with Mannheimia (Pasteurella) haemolytica.

    PubMed Central

    Goubau, S; Morck, D W; Buret, A

    2000-01-01

    The expression of the interleukin-8 (IL-8) gene was examined by in situ hybridization in lung tissues from calves experimentally infected with Mannheimia (Pasteurella) haemolytica and treated with tilmicosin. Interleukin-8 mRNA expression was detected in alveolar areas, particularly along interlobular septa, in the lumen, and in the epithelial cells of some bronchioles. In lesional lung tissues from animals that had received tilmicosin, we found large areas with limited inflammation. There was no staining for IL-8 mRNA in these areas. In contrast, in strongly inflamed areas, the same patterns and intensities of staining for IL-8 mRNA were detected in tilmicosin- and sham-treated animals. We conclude that tilmicosin does not affect the expression of IL-8 mRNA in tissue showing microscopic signs of inflammation. Together with previous reports, this supports the view that the pro-apoptotic properties of tilmicosin on neutrophils do not compromise the host defense mechanisms required to control the infection. Images Figure 1. PMID:11041503

  17. A Quantitative RNAi Screen for JNK Modifiers Identifies Pvr as a Novel Regulator of Drosophila Immune Signaling

    PubMed Central

    Bond, David; Foley, Edan

    2009-01-01

    Drosophila melanogaster responds to gram-negative bacterial challenges through the IMD pathway, a signal transduction cassette that is driven by the coordinated activities of JNK, NF-κB and caspase modules. While many modifiers of NF-κB activity were identified in cell culture and in vivo assays, the regulatory apparatus that determines JNK inputs into the IMD pathway is relatively unexplored. In this manuscript, we present the first quantitative screen of the entire genome of Drosophila for novel regulators of JNK activity in the IMD pathway. We identified a large number of gene products that negatively or positively impact on JNK activation in the IMD pathway. In particular, we identified the Pvr receptor tyrosine kinase as a potent inhibitor of JNK activation. In a series of in vivo and cell culture assays, we demonstrated that activation of the IMD pathway drives JNK-dependent expression of the Pvr ligands, Pvf2 and Pvf3, which in turn act through the Pvr/ERK MAP kinase pathway to attenuate the JNK and NF-κB arms of the IMD pathway. Our data illuminate a poorly understood arm of a critical and evolutionarily conserved innate immune response. Furthermore, given the pleiotropic involvement of JNK in eukaryotic cell biology, we believe that many of the novel regulators identified in this screen are of interest beyond immune signaling. PMID:19893628

  18. Interleukin-9 enhances interleukin-5 receptor expression, differentiation, and survival of human eosinophils.

    PubMed

    Gounni, A S; Gregory, B; Nutku, E; Aris, F; Latifa, K; Minshall, E; North, J; Tavernier, J; Levit, R; Nicolaides, N; Robinson, D; Hamid, Q

    2000-09-15

    Interleukin-9 (IL-9) has been implicated in the pathogenesis of allergic disorders. To examine the interaction between IL-9 and eosinophils, we evaluated mature peripheral blood eosinophils for their expression of the specific alpha-subunit of the IL-9 receptor (IL-9R-alpha). The expression of IL-9R-alpha by human eosinophils was detected at the messenger RNA (mRNA) and protein levels by reverse transcriptase-polymerase chain reaction (RT-PCR), flow cytometry, and immunocytochemical analysis, respectively. Functional analyses demonstrated that recombinant human (rh)IL-9 inhibited in vitro peripheral blood human eosinophil apoptosis in a concentration-dependent manner. We then examined the role of IL-9 in eosinophil differentiation using the human cord blood CD34(+) cells and human promyelocytic leukemia cells (HL-60). The addition of IL-9 to CD34(+) cells cultured in IL-3 and IL-5 enhanced eosinophil development, and IL-9 alone induced the expression of IL-5R-alpha. IL-9 also up-regulated the IL-5R-alpha chain cell surface expression during terminal eosinophil differentiation of the HL-60 cell line. Our findings suggest that IL-9 may potentiate in vivo eosinophil function by increasing their survival and IL-5-mediated differentiation and maturation. Taken together, these results suggest a mechanism by which IL-9 potentiates airway and tissue eosinophilia.

  19. JNK pathway activation is controlled by Tao/TAOK3 to modulate ethanol sensitivity.

    PubMed

    Kapfhamer, David; King, Ian; Zou, Mimi E; Lim, Jana P; Heberlein, Ulrike; Wolf, Fred W

    2012-01-01

    Neuronal signal transduction by the JNK MAP kinase pathway is altered by a broad array of stimuli including exposure to the widely abused drug ethanol, but the behavioral relevance and the regulation of JNK signaling is unclear. Here we demonstrate that JNK signaling functions downstream of the Sterile20 kinase family gene tao/Taok3 to regulate the behavioral effects of acute ethanol exposure in both the fruit fly Drosophila and mice. In flies tao is required in neurons to promote sensitivity to the locomotor stimulant effects of acute ethanol exposure and to establish specific brain structures. Reduced expression of key JNK pathway genes substantially rescued the structural and behavioral phenotypes of tao mutants. Decreasing and increasing JNK pathway activity resulted in increased and decreased sensitivity to the locomotor stimulant properties of acute ethanol exposure, respectively. Further, JNK expression in a limited pattern of neurons that included brain regions implicated in ethanol responses was sufficient to restore normal behavior. Mice heterozygous for a disrupted allele of the homologous Taok3 gene (Taok3Gt) were resistant to the acute sedative effects of ethanol. JNK activity was constitutively increased in brains of Taok3Gt/+ mice, and acute induction of phospho-JNK in brain tissue by ethanol was occluded in Taok3Gt/+ mice. Finally, acute administration of a JNK inhibitor conferred resistance to the sedative effects of ethanol in wild-type but not Taok3Gt/+ mice. Taken together, these data support a role of a TAO/TAOK3-JNK neuronal signaling pathway in regulating sensitivity to acute ethanol exposure in flies and in mice.

  20. Effect of electroacupuncture on the expression of interlukin-1beta mRNA after transient focal cerebral ischemia.

    PubMed

    Xu, Zhen-Feng; Wu, Gen-Cheng; Cao, Xiao-Ding

    2002-01-01

    It has been reported that interleukin-1beta (IL-1beta ) play a key role in the pathogenesis of cerebral ischemia. Acupuncture is an effective traditional medical therapy in China. The aim of present study was to evaluate the effect of electroacupuncture (EA) on IL-1beta mRNA expression after middle cerebral artery occlusion (MCAO) in rats. Using in situ hybridization technique, it was found that in the MCAO group the expression of IL-1beta mRNA was significantly increased at 2h, 6h, 12h after reperfusion in cerebral ischemic cortex compared with normal group. In EA+ MCAO group the expression of IL-1beta mRNA was significantly decreased at 2h, 6h and 12h in ischemic cortex compared with MCAO group. The results indicated that EA might decrease the IL-1beta protein expression by reducing the IL-beta mRNA expression in ischemic cortex.

  1. O-GlcNAc-mediated interaction between VER2 and TaGRP2 elicits TaVRN1 mRNA accumulation during vernalization in winter wheat

    PubMed Central

    Xiao, Jun; Xu, Shujuan; Li, Chunhua; Xu, Yunyuan; Xing, Lijing; Niu, Yuda; Huan, Qing; Tang, Yimiao; Zhao, Changping; Wagner, Doris; Gao, Caixia; Chong, Kang

    2014-01-01

    Vernalization, sensing of prolonged cold, is important for seasonal flowering in eudicots and monocots. While vernalization silences a repressor (FLC, MADS-box transcription factor) in eudicots, it induces an activator (TaVRN1, an AP1 clade MADS-box transcription factor) in monocots. The mechanism for TaVRN1 induction during vernalization is not well understood. Here we reveal a novel mechanism for controlling TaVRN1 mRNA accumulation in response to prolonged cold sensing in wheat. The carbohydrate-binding protein VER2, a jacalin lectin, promotes TaVRN1 upregulation by physically interacting with the RNA-binding protein TaGRP2. TaGRP2 binds to TaVRN1 pre-mRNA and inhibits TaVRN1 mRNA accumulation. The physical interaction between VER2 and TaGRP2 is controlled by TaGRP2 O-GlcNAc modification, which gradually increases during vernalization. The interaction between VER2 and O-GlcNAc-TaGRP2 reduces TaGRP2 protein accumulation in the nucleus and/or promotes TaGRP2 dissociation from TaVRN1, leading to TaVRN1 mRNA accumulation. Our data reveal a new mechanism for sensing prolonged cold in temperate cereals. PMID:25091017

  2. Hydrogen-rich saline attenuates skin ischemia/reperfusion induced apoptosis via regulating Bax/Bcl-2 ratio and ASK-1/JNK pathway.

    PubMed

    Liu, Yun-Qi; Liu, Yi-Fang; Ma, Xue-Mei; Xiao, Yi-Ding; Wang, You-Bin; Zhang, Ming-Zi; Cheng, Ai-Xin; Wang, Ting-Ting; Li, Jia-La; Zhao, Peng-Xiang; Xie, Fei; Zhang, Xin

    2015-07-01

    Many pathways have been reported involving the effect of hydrogen-rich saline on protecting skin flap partial necrosis induced by the inflammation of ischemia/reperfusion injury. This study focused on the influence of hydrogen-rich saline treatment on apoptosis pathway of ASK-1/JNK and Bcl-2/Bax radio in I/R injury of skin flaps. Adult male Sprague-Dawley rats were divided into three groups. Group 1 was sham surgery group, Group 2 and 3 were ischemia/reperfusion surgery treated with physiological saline and hydrogen-rich saline respectively. Blood perfusion of flap was measured by Laser doppler flowmeters. Hematoxylin and eosin staining was used to observe morphological changes. Early apoptosis in skin flap was observed through TUNEL staining and presented as the percentage of TUNEL-positive cells of total cells. pASK-1, pJNK, Bcl-2 and Bax were examined by immunodetection. In addition Bcl-2, Bax and caspase-3 were detected by qPCR. Caspase-3 activity was also measured. Compared to the Group 2, tissues from the group 3 were observed with a high expression of Bcl-2 and a low expression of pASK-1, pJNK, and Bax, a larger survival area and a high level of blood perfusion. Hydrogen-rich saline ameliorated inflammatory infiltration and decreased cell apoptosis. The results indicate that hydrogen-rich saline could ameliorate ischemia/reperfusion injury and improve flap survival rate by inhibiting the apoptosis factor and, at the same time, promoting the expression of anti-apoptosis factor. Copyright © 2015. Published by Elsevier Ltd.

  3. TNF Receptor 1/2 Predict Heart Failure Risk in Type 2 Diabetes Mellitus Patients.

    PubMed

    Ping, Zhang; Aiqun, Ma; Jiwu, Li; Liang, Shao

    2017-04-06

    Inflammation plays an important role in heart failure and diabetes mellitus. Traditional serum markers have limited predictive value in heart failure and diabetes. TNFR1 and TNFR2 (TNFR1/2) have been proven to be strongly associated with heart failure and diabetes complications. This study aimed to assess the association of sTNFR1 and sTNFR2 levels and incidental HF risk in diabetes patients.We detected the mRNA, protein, and serum expression of TNFR1/2, their downstream signaling pathway protein NF-kB, and JNK expression and some traditional serum inflammatory markers in a heart failure group without diabetes mellitus or abnormal glucose tolerance (n = 84), a diabetes mellitus group without heart failure (n = 86), and a heart failure with diabetes mellitus group (n = 86).TNFR1/2 were significantly higher in patients with heart failure and diabetes mellitus based on mRNA expression to protein expression and serum expression. However, there were no differences in mRNA, protein, and serum levels of TNFR1/2 between the HF group and DM group. Furthermore, there were no differences between the groups in some traditional serum inflammatory markers.This study demonstrated higher expressions of TNFR, NF-kB, and JNK in patients with heart failure and diabetes mellitus. Compared with traditional serum markers, TNFR1 and TNFR2 are associated with heart failure risk in type 2 diabetes mellitus patients.

  4. The effects of a cytokine suppressive anti-inflammatory drug on the output of prostaglandin E(2) and interleukin-1 beta from human fetal membranes.

    PubMed

    Sullivan, M H F; Alvi, S A; Brown, N L; Elder, M G; Bennett, P R

    2002-03-01

    Fetal membranes are a primary source of prostaglandins and pro-inflammatory cytokines implicated in human parturition, so the inhibition of inflammatory pathways may be of benefit in pregnancies complicated by premature labour. We have therefore investigated the effects of a cytokine-suppressant anti-inflammatory drug (CSAID) on the output of prostaglandin E(2) (PGE(2)) and interleukin (IL)-1 beta from human fetal membranes in vitro. Bacterial endotoxin increased the expression of mRNA for IL-1 beta and type-2 cyclo-oxygenase (COX-2), and there were corresponding increases in the output of IL-1 beta protein and PGE(2). The CSAID decreased IL-1 beta protein, COX-2 expression and PGE(2) output, but not mRNA for IL-1 beta, indicating a post-translational effect on the production of IL-1 beta and a transcriptional affect on COX-2, with an overall reduction in PGE(2). These findings are consistent with the effects of CSAIDs in other systems, and indicate that they are of possible use in premature labour.

  5. IL-4 mRNA Is Downregulated in the Liver of Pancreatic Cancer Patients Suffering from Cachexia.

    PubMed

    Prokopchuk, Olga; Steinacker, Jürgen M; Nitsche, Ulrich; Otto, Stephanie; Bachmann, Jeannine; Schubert, Elaine C; Friess, Helmut; Martignoni, Marc E

    2017-01-01

    Interleukin-4 (IL-4) together with interleukin-13 (IL-13) play an important role in inflammation and wound repair, and are known to be upregulated in human skeletal muscle after strenuous physical exercise. Additionally, these cytokines may act as autocrine growth factors in pancreatic cancer cells. We hypothesize that IL-4, IL-13, and their corresponding receptors are involved in mechanism of cancer cachexia. Tissue samples from human skeletal muscle, white fat, liver, healthy pancreas, and pancreatic ductal adenocarcinoma were analyzed by quantitative real-time polymerase chain reaction for mRNA expression levels of IL-4, IL-13, IL-4 receptor α, and IL-13 receptor α1. We demonstrate for the first time that liver IL-4 mRNA is downregulated in vivo in patients with pancreatic cancer and cachexia. Additionally, IL-4 mRNA in the liver inversely correlated with musculus psoas thickness. We speculate that suppression of IL-4 is involved in cancer cachexia, although the exact mechanisms have to be further elucidated.

  6. Thunbergia alata inhibits inflammatory responses through the inactivation of ERK and STAT3 in macrophages.

    PubMed

    Cho, Young-Chang; Kim, Ye Rang; Kim, Ba Reum; Bach, Tran The; Cho, Sayeon

    2016-11-01

    Thunbergia alata (Acanthaceae) has been used traditionally to treat various inflammatory diseases such as fever, cough and diarrhea in East African countries including Uganda and Kenya. However, systemic studies elucidating the anti-inflammatory effects and precise mechanisms of action of T. alata have not been conducted, to the best of our knowledge. To address these concerns, we explored the anti-inflammatory effects of a methanol extract of T. alata (MTA) in macrophages. Non-cytotoxic concentrations of MTA (≤300 µg/ml) inhibited nitric oxide (NO) production in lipopolysaccharide (LPS)‑stimulated RAW 264.7 macrophages by transcriptional regulation of inducible NO synthase in a dose-dependent manner. The expression of cyclooxygenase-2, the enzyme responsible for the production of prostaglandin E2, was unchanged by MTA at the mRNA and protein levels. MTA treatment inhibited interleukin (IL)-6 production and decreased the mRNA expression of pro‑inflammatory cytokines, including IL-6 and IL-1β. Tumor necrosis factor-α production and mRNA expression were not regulated by MTA treatment. The decreased production of inflammatory mediators by MTA was followed by the reduced phosphorylation of extracellular signal‑regulated kinase (ERK) and signal transducer and activator of transcription 3 (STAT3). MTA treatment had no effect on activity of other mitogen‑activated protein kinases (MAPKs), p38, c-Jun N-terminal kinase (JNK), and nuclear factor-κB (NF-κB). These results indicate that MTA selectively inhibits the excessive production of inflammatory mediators in LPS-stimulated murine macrophages by reducing the activity of ERK and STAT3, suggesting that MTA plays an important inhibitory role in the modulation of severe inflammation.

  7. Hypo-responsiveness of interleukin-8 production in human embryonic epithelial intestine 407 cells independent of NF-{kappa}B pathway: New lessons from endotoxin and ribotoxic deoxynivalenol

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

    Moon, Yuseok; Yang, Hyun; Park, Seung-Hwan

    Mucosal epithelium senses external toxic insults and transmits the danger signals into the epithelial cells in order to activate a broad range of inflammatory responses. However, pre-exposure to the commensal endotoxins can induce inflammatory tolerance and maintain the homeostasis without excessive immune responses. We recently reported that ribotoxin deoxynivalenol (DON) and its derivatives elicited the pro-inflammatory response as the mucosal insults in human epithelial cells. Taking the knowledge into consideration, we tested the hypothesis that endotoxin pre-exposure can attenuate ribotoxin-induced epithelial interleukin-8 (IL-8) production via a tolerance mechanism. Pre-exposure to endotoxin repressed IL-8 release and its gene expression. However, inflammatorymore » tolerance was not mediated by the attenuated NF-{kappa}B activation which has been generally recognized as the major mediator of LPS-mediated toll-like receptor (TLR) signaling pathway. Instead, pre-exposure to endotoxin was observed to trigger the delayed induction of peroxisome proliferator-activated receptor gamma (PPAR-{gamma}) which contributed to the diminished IL-8 production in the human epithelial cells. Moreover, endogenous PPAR-{gamma} agonist suppressed toxicant-mediated interleukin-8 production and IL-8 mRNA stability. Taken together, endotoxin induced hypo-production of pro-inflammatory cytokine IL-8 in the human epithelial cells, which was associated with the delayed activation of PPAR-{gamma} expression by pre-existing endotoxin.« less

  8. TRAIL Enhances Shikonin Induced Apoptosis through ROS/JNK Signaling in Cholangiocarcinoma Cells.

    PubMed

    Zhou, Guangyao; Yang, Zuqin; Wang, Xiaodong; Tao, Ran; Zhou, Yuanping

    2017-01-01

    Cholangiocarcinoma (CCA), arising from varying locations within the biliary tree, is the second most common primary liver malignancy worldwide. Shikonin, an active compound extracted from the Chinese herb Zicao, holds anti-bacterial, anti-inflammatory, and anti-tumor activities. However, the effect of shikonin on human cholangiocarcinoma and detailed mechanisms of TRAIL enhancement remains to be elucidated. The purpose of the study was to investigate the protective functions of TRAIL enhancement for shikonin induced apoptosis in cholangiocarcinoma cells. We use MTT assay, apoptosis assay, caspase activity assay, flow cytometry assay, real time PCR and Western blot to observe the effects of TRAIL on shikonin induced cholangiocarcinoma cells apoptosis and its mechanism. Shikonin inhibited cell viability and induced apoptosis of CCA cells, effects enhanced by TRAIL treatment via activation of caspase-3, -8, -9. Furhermore, TRAIL enhanced anti-proliferation of shikonin and shikonin induced apoptosis through induction of ROS mediated JNK activation, while AKT activation had an effect on shikonin anti-proliferation activity, but not in the TRAIL enhanced counterparts. Finally, shikonin upregulated DR5 expression, an effect essential for TRAIL-enhanced activities of shikonin in RBE cells. Our results revealed that shikonin could inhibit cells viability and induce apoptosis of CCA cells, effects enhanced by TRAIL treatment via ROS mediated JNK signalling pathways, involving up-regulation of DR5 expression. Our results provide further insight into the mechanism underlying the anti-tumor effects of shikonin by TRAIL enhanced in CCA and a new therapeutic strategy to CCA treatment. © 2017 The Author(s). Published by S. Karger AG, Basel.

  9. Murine knockin model for progranulin-deficient frontotemporal dementia with nonsense-mediated mRNA decay.

    PubMed

    Nguyen, Andrew D; Nguyen, Thi A; Zhang, Jiasheng; Devireddy, Swathi; Zhou, Ping; Karydas, Anna M; Xu, Xialian; Miller, Bruce L; Rigo, Frank; Ferguson, Shawn M; Huang, Eric J; Walther, Tobias C; Farese, Robert V

    2018-03-20

    Frontotemporal dementia (FTD) is the most common neurodegenerative disorder in individuals under age 60 and has no treatment or cure. Because many cases of FTD result from GRN nonsense mutations, an animal model for this type of mutation is highly desirable for understanding pathogenesis and testing therapies. Here, we generated and characterized Grn R493X knockin mice, which model the most common human GRN mutation, a premature stop codon at arginine 493 (R493X). Homozygous Grn R493X mice have markedly reduced Grn mRNA levels, lack detectable progranulin protein, and phenocopy Grn knockout mice, with CNS microgliosis, cytoplasmic TDP-43 accumulation, reduced synaptic density, lipofuscinosis, hyperinflammatory macrophages, excessive grooming behavior, and reduced survival. Inhibition of nonsense-mediated mRNA decay (NMD) by genetic, pharmacological, or antisense oligonucleotide-based approaches showed that NMD contributes to the reduced mRNA levels in Grn R493X mice and cell lines and in fibroblasts from patients containing the GRN R493X mutation. Moreover, the expressed truncated R493X mutant protein was functional in several assays in progranulin-deficient cells. Together, these findings establish a murine model for in vivo testing of NMD inhibition or other therapies as potential approaches for treating progranulin deficiency caused by the R493X mutation. Copyright © 2018 the Author(s). Published by PNAS.

  10. Down-regulation of ATF2 in the inhibition of T-2-toxin-induced chondrocyte apoptosis by selenium chondroitin sulfate nanoparticles

    NASA Astrophysics Data System (ADS)

    Han, Jing; Guo, Xiong

    2013-12-01

    Selenium chondroitin sulfate nanoparticles (SeCS) with a size range of 30-200 nm were obtained in our previous study. Meanwhile, the up-regulated expression of ATF2 mRNA and protein levels could be observed in the cartilage from Kashin-Beck disease (KBD) patients. In this paper, we investigated the inhibition effect of SeCS on T-2-toxin-induced apoptosis of chondrocyte from KBD patients. Here, we found that when the chondrocytes were treated with T-2 toxin, the chondrocyte apoptosis performed in a concentration-dependent manner. The apoptosis of chondrocyte induced by T-2 toxin involved the increased levels of ATF2, JNK and p38 mRNAs and related protein expression. SeCS could partly block the T-2-toxin-induced chondrocyte apoptosis by decreasing the expression of ATF2, JNK and p38 mRNAs and p-JNK, p-38, ATF2 and p-ATF2 proteins. JNK and p38 pathways involved in the apoptosis of chondrocyte induced by T-2 toxin, and SeCS was efficient in the inhibition of chondrocyte apoptosis by T-2 toxin. These results suggested that SeCS had a potential for further prevention and treatment for KBD as well as other selenium deficiency disease.

  11. Modulation of Interleukins in Sepsis-Associated Clotting Disorders: Interplay With Hemostatic Derangement.

    PubMed

    Low, Christopher; Syed, Daneyal; Khan, Daniel; Tetik, Sermin; Walborn, Amanda; Hoppensteadt, Debra; Mosier, Michael; Fareed, Jawed

    2017-01-01

    Interleukins play a central role in the immune system and are involved in a variety of immunological, inflammatory, and infectious disease states including sepsis syndrome. Levels of interleukins may correlate with overall survival and may directly or indirectly affect some of the regulators of coagulation and fibrinolysis, thereby disrupting hemostasis and thrombosis. Our hypothesis is that in sepsis-associated coagulopathies (SACs), interleukins may be upregulated, leading to hemostatic imbalance by generating thrombogenic mediators. We profiled the levels of interleukins IL-1α, IL-1β, IL-2, IL-4, IL-6, IL-8, and IL-10 in addition to d-dimer (DD) in patients with SAC and in normal donors. We observed the highest increase in interleukins IL-6 (322-fold), IL-8 (48-fold), IL-10 (72-fold), and DD (18-fold). This suggests that interleukins such as IL-6 and IL-10 have a close association with coagulopathy and fibrinolytic dysregulation in sepsis and can be considered as candidates for potential therapeutic targets in SAC. © The Author(s) 2016.

  12. Tumor necrosis factor and interleukin 1 as mediators of endotoxin-induced beneficial effects

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

    Urbaschek, R.; Urbaschek, B.

    Bacterial lipopolysaccharides or endotoxins are known to induce tumor necrosis; enhanced nonspecific resistance to bacterial, viral, and parasitic infections and to radiation sickness; and tolerance to lethal doses of endotoxin. These beneficial effects are achieved by pretreatment with minute amounts of endotoxin. Recombinant tumor necrosis factor (TNF) and interleukin 1 (IL-1) are among the mediators capable of invoking radioprotection or resistance to the consequences of cecal ligation and puncture. Both cytokines are potent inducers of serum colony-stimulating factor (CSF) in C3H/HeJ mice (low responders to endotoxin). The number of splenic granulocyte-macrophage precursors was found to increase 5 days after injectionmore » of TNF in these mice. Although with IL-1 no increase in the number of granulocyte-macrophage colonies occurred in culture in the presence of serum CSF, a marked stimulation was observed when TNF was added. This stimulation of myelopoiesis observed in vivo and in vitro may be related to the radioprotective effect of TNF. The data presented suggest that TNF and IL-1 released after injection of endotoxin participate in the mediation of endotoxin-induced enhancement of nonspecific resistance and stimulation of hematopoiesis. 76 references.« less

  13. Resveratrol Inhibits the Epidermal Growth Factor-Induced Migration of Osteoblasts: the Suppression of SAPK/JNK and Akt.

    PubMed

    Kawabata, Tetsu; Tokuda, Haruhiko; Fujita, Kazuhiko; Kainuma, Shingo; Sakai, Go; Matsushima-Nishiwaki, Rie; Kozawa, Osamu; Otsuka, Takanobu

    2017-01-01

    Resveratrol is a polyphenol enriched in the skins of grapes and berries, that shows various beneficial effects for human health. In the present study, we investigated the mechanism behind the epidermal growth factor (EGF)-induced migration of osteoblast-like MC3T3-E1 cells, and the effect of resveratrol on this cell migration. The cell migration was examined using Boyden chamber, and phosphorylation of each kinase was analyzed by Western blotting. The EGF-induced migration was suppressed by PD98059, an inhibitor of MEK1/2, as well as SB203580, an inhibitor of p38 MAP kinase, SP600125, an inhibitor of SAPK/JNK, and deguelin, an inhibitor of Akt. In contrast, rapamycin, an inhibitor of upstream kinase of p70 S6 kinase, and fasudil, an inhibitor of Rho-kinase, hardly affected the migration. Resveratrol significantly reduced the EGF-induced migration in a dose-dependent manner. SRT1720, an SIRT1 activator, suppressed the migration by EGF. In addition, resveratrol markedly attenuated the EGF-induced phosphorylation of SAPK/JNK and Akt without affecting the phosphorylation of p44/p42 MAP kinase or p38 MAP kinase. The phosphorylation of SAPK/JNK and Akt induced by EGF was down-regulated by SRT1720. Our results strongly suggest that resveratrol reduces the EGF-stimulated migration of osteoblasts via suppression of SAPK and Akt, and that the inhibitory effect of resveratrol is mediated in part via SIRT1. © 2017 The Author(s). Published by S. Karger AG, Basel.

  14. TSG-6 secreted by human umbilical cord-MSCs attenuates severe burn-induced excessive inflammation via inhibiting activations of P38 and JNK signaling.

    PubMed

    Liu, Lingying; Song, Huifeng; Duan, Hongjie; Chai, Jiake; Yang, Jing; Li, Xiao; Yu, Yonghui; Zhang, Xulong; Hu, Xiaohong; Xiao, Mengjing; Feng, Rui; Yin, Huinan; Hu, Quan; Yang, Longlong; Du, Jundong; Li, Tianran

    2016-07-22

    The hMSCs have become a promising approach for inflammation treatment in acute phase. Our previous study has demonstrated that human umbilical cord-MSCs could alleviate the inflammatory reaction of severely burned wound. In this study, we further investigated the potential role and mechanism of the MSCs on severe burn-induced excessive inflammation. Wistar rats were randomly divided into following groups: Sham, Burn, Burn+MSCs, Burn+MAPKs inhibitors, and Burn, Burn+MSCs, Burn+Vehicle, Burn+siTSG-6, Burn+rhTSG-6 in the both experiments. It was found that MSCs could only down-regulate P38 and JNK signaling, but had no effect on ERK in peritoneal macrophages of severe burn rats. Furthermore, suppression of P38 and JNK activations significantly reduced the excessive inflammation induced by severe burn. TSG-6 was secreted by MSCs using different inflammatory mediators. TSG-6 from MSCs and recombinant human (rh)TSG-6 all significantly reduced activations of P38 and JNK signaling induced by severe burn and then attenuated excessive inflammations. On the contrary, knockdown TSG-6 in the cells significantly increased phosphorylation of P38 and JNK signaling and reduced therapeutic effect of the MSCs on excessive inflammation. Taken together, this study suggested TSG-6 from MSCs attenuated severe burn-induced excessive inflammation via inhibiting activation of P38 and JNK signaling.

  15. Effects of retinoids and thiazolidinediones on proliferation, insulin release, insulin mRNA, GLUT 2 transporter protein and mRNA of INS-1 cells.

    PubMed

    Blumentrath, J; Neye, H; Verspohl, E J

    2001-09-01

    Both 9-cis-retinoic acid (9cRA) and all-trans-retinoic acid (ATRA) are active metabolites of vitamin A (retinol). There exists an interaction between retinoid receptors and peroxisome proliferator-activated receptors (PPARgamma). To define their functions in an insulin secreting system the effects of ATRA, 9cRA and the PPARgamma agonist rosiglitazone on cell proliferation, insulin release and glucose transporter (GLUT) 2 of INS-1 cells were tested. Retinoic acid receptor (RAR-alpha and -gamma) and retinoid X receptor (RXR-alpha and -beta) proteins are present (immunoblots). Both 9cRA and ATRA inhibit INS-1 cell proliferation ([3H]-thymidine assay) in a concentration dependent manner. Both 9cRA and ATRA increased insulin release, but only ATRA ralsed the GLUT 2 mRNA in a bell-shaped concentration response curve after 48 h. The insulinotropic effect of one compound is not significantly superimposed by the other indicating that the same binding sites are used by 9cRA and ATRA. The acute and chronic effects of the PPARgamma agonist rosiglitazone on insulin release were additionally determined since glitazones act as transcription factors together with RXR agonists. At high concentrations (100 microM) rosiglitazone inhibited glucose (8.3 mM) stimulated insulin secretion (acute experiment over 60 min). Insulin secretion, however, was increased during a 24 h treatment at a concentration of 10 microM and again inhibited at 100 microM. Changes in preproinsulin mRNA expression were not observed. Rosiglitazone (100 microM) increased GLUT 2 mRNA paralleled by an increase of GLUT 2 protein, but only after 24 h of treatment. This data indicate that RAR and RXR mediate insulin release. The changes in GLUT 2 have no direct impact on insulin release; the inhibition seen at high concentrations of either compound is possibly the result of the observed inhibition of cell proliferation. Effects of rosiglitazone on preproinsulin mRNA and GLUT 2 (mRNA and protein) do not play a role in

  16. Galectin-9 exhibits anti-myeloma activity through JNK and p38 MAP kinase pathways.

    PubMed

    Kobayashi, T; Kuroda, J; Ashihara, E; Oomizu, S; Terui, Y; Taniyama, A; Adachi, S; Takagi, T; Yamamoto, M; Sasaki, N; Horiike, S; Hatake, K; Yamauchi, A; Hirashima, M; Taniwaki, M

    2010-04-01

    Galectins constitute a family of lectins that specifically exhibit the affinity for beta-galactosides and modulate various biological events. Galectin-9 is a tandem-repeat type galectin with two carbohydrate recognition domains and has recently been shown to have an anti-proliferative effect on cancer cells. We investigated the effect of recombinant protease-resistant galectin-9 (hGal9) on multiple myeloma (MM). In vitro, hGal9 inhibited the cell proliferation of five myeloma cell lines examined, including a bortezomib-resistant subcell line, with IC(50) between 75.1 and 280.0 nM, and this effect was mediated by the induction of apoptosis with the activation of caspase-8, -9, and -3. hGal9-activated Jun NH(2)-terminal kinase (JNK) and p38 MAPK signaling pathways followed by H2AX phosphorylation. Importantly, the inhibition of either JNK or p38 MAPK partly inhibited the anti-proliferative effect of hGal9, indicating the crucial role of these pathways in the anti-MM effect of hGal9. hGal9 also induced cell death in patient-derived myeloma cells, some with poor-risk factors, such as chromosomal deletion of 13q or translocation t(4;14)(p16;q32). Finally, hGal9 potently inhibited the growth of human myeloma cells xenografted in nude mice. These suggest that hGal9 is a new therapeutic target for MM that may overcome resistance to conventional chemotherapy.

  17. An antigen receptor-driven, interleukin 2-independent pathway for proliferation of murine cytolytic T lymphocyte clones

    PubMed Central

    1986-01-01

    Proliferation of T lymphocytes can be induced by IL-2, either through an autocrine pathway in which the responding cell produces its own IL-2 or through an exocrine pathway in which IL-2 secreted by Th stimulates proliferation of IL-2-dependent CTL. However, proliferation of at least some CTL clones, such as CTL L3 and CTL dB45, also can be induced by stimulation of the antigen receptor in the absence of IL-2. Stimulation of these cloned CTL with T cell-depleted allogeneic spleen cells, allogeneic tumor cells, or immobilized mAb reactive with the T cell antigen receptor (TCR) induced thymidine incorporation, entry into cell cycle, and secretion of macrophage activating factor, but these stimuli did not induce the secretion of IL-2. Several observations indicated that such proliferation of cloned CTL induced by stimulation of the TCR was independent of IL-2; IL-2 could not be detected in supernatants from stimulated CTL cells. mAbs reactive with the murine IL-2-R efficiently blocked IL-2-mediated thymidine incorporation in cloned CTL and Th, but had no inhibitory effect on TCR-driven thymidine incorporation in the CTL clones. TCR-driven thymidine incorporation in cloned Th L2 cells was profoundly inhibited by these antibodies, indicating the operation of an IL-2-mediated autocrine pathway for proliferation in this cloned Th. When antibodies to the TCR were used to stimulate cloned CTL and Th, IFN-gamma mRNA was easily shown in the cloned CTL and Th. Although IL-2 mRNA could be detected in the cloned Th, it was never observed in the cloned CTL. These findings provide evidence for the existence of a TCR-mediated, IL-2-independent pathway for induction of cellular proliferation in cloned murine CTL. PMID:3486939

  18. Influenza polymerase encoding mRNAs utilize atypical mRNA nuclear export.

    PubMed

    Larsen, Sean; Bui, Steven; Perez, Veronica; Mohammad, Adeba; Medina-Ramirez, Hilario; Newcomb, Laura L

    2014-08-28

    Influenza is a segmented negative strand RNA virus. Each RNA segment is encapsulated by influenza nucleoprotein and bound by the viral RNA dependent RNA polymerase (RdRP) to form viral ribonucleoproteins responsible for RNA synthesis in the nucleus of the host cell. Influenza transcription results in spliced mRNAs (M2 and NS2), intron-containing mRNAs (M1 and NS1), and intron-less mRNAs (HA, NA, NP, PB1, PB2, and PA), all of which undergo nuclear export into the cytoplasm for translation. Most cellular mRNA nuclear export is Nxf1-mediated, while select mRNAs utilize Crm1. Here we inhibited Nxf1 and Crm1 nuclear export prior to infection with influenza A/Udorn/307/1972(H3N2) virus and analyzed influenza intron-less mRNAs using cellular fractionation and reverse transcription-quantitative polymerase chain reaction (RT-qPCR). We examined direct interaction between Nxf1 and influenza intron-less mRNAs using immuno purification of Nxf1 and RT-PCR of associated RNA. Inhibition of Nxf1 resulted in less influenza intron-less mRNA export into the cytoplasm for HA and NA influenza mRNAs in both human embryonic kidney cell line (293 T) and human lung adenocarcinoma epithelial cell line (A549). However, in 293 T cells no change was observed for mRNAs encoding the components of the viral ribonucleoproteins; NP, PA, PB1, and PB2, while in A549 cells, only PA, PB1, and PB2 mRNAs, encoding the RdRP, remained unaffected; NP mRNA was reduced in the cytoplasm. In A549 cells NP, NA, HA, mRNAs were found associated with Nxf1 but PA, PB1, and PB2 mRNAs were not. Crm1 inhibition also resulted in no significant difference in PA, PB1, and PB2 mRNA nuclear export. These results further confirm Nxf1-mediated nuclear export is functional during the influenza life cycle and hijacked for select influenza mRNA nuclear export. We reveal a cell type difference for Nxf1-mediated nuclear export of influenza NP mRNA, a reminder that cell type can influence molecular mechanisms. Importantly, we

  19. Enhancing Interleukin-6 and Interleukin-11 receptor cleavage.

    PubMed

    Lokau, Juliane; Wandel, Marieke; Garbers, Christoph

    2017-04-01

    Proteolytic cleavage of the membrane-bound Interleukin-6 receptor (IL-6R) by the metalloprotease ADAM17 releases an agonistic soluble form of the IL-6R (sIL-6R), which is responsible for the pro-inflammatory trans-signaling branch of the cytokine's activities. This proteolytic step, which is also called ectodomain shedding, is critically regulated by the cleavage site within the IL-6R stalk, because mutations or small deletions within this region are known to render the IL-6R irresponsive towards proteolysis. In the present study, we employed cleavage site profiling data of ADAM17 to generate an IL-6R with increased cleavage susceptibility. Using site-directed mutagenesis, we showed that the non-prime sites P3 and P2 and the prime site P1' were critical for this increase in proteolysis, whereas other positions within the cleavage site were of minor importance. Insertion of this optimized cleavage site into the stalk of the Interleukin-11 receptor (IL-11R) was not sufficient to enable ADAM17-mediated proteolysis, but transfer of different parts of the IL-6R stalk enabled shedding by ADAM17. These findings shed light on the cleavage site specificities of ADAM17 using a native substrate and reveal further differences in the proteolysis of IL-6R and IL-11R. Copyright © 2017 Elsevier Ltd. All rights reserved.

  20. Phloretin induces apoptosis of non-small cell lung carcinoma A549 cells via JNK1/2 and p38 MAPK pathways

    PubMed Central

    MIN, JIE; LI, XU; HUANG, KENAN; TANG, HUA; DING, XINYU; QI, CHEN; QIN, XIONG; XU, ZHIFEI

    2015-01-01

    Phloretin (Ph) existing in apples, pears and various vegetables is known to have antitumor activities in several cancer cell lines. However, little is known about its effect on human lung cancer cells. The aim of the present study was to see whether Ph could induce apoptosis of non-small cell lung cancer (NSCLC) cells, and explore the possible underlying mechanism of action. We found that Ph markedly induced cell apoptosis of NSCLC cell line A549, and inhibited the migration of A549 cells in a dose-dependent manner. The expression level of BAX, cleaved caspase-3 and -9, and degraded form of PARP was increased and Bcl-2 was decreased after Ph treatment. In addition, the phosphorylation of P38 MAPK, ERK1/2 and JNK1/2 was increased in a dose-dependent manner in parallel with Ph treatment. Inhibition of P38 MAPK and JNK1/2 by specific inhibitors significantly abolished the Ph-induced activation of the caspase-3 and -9. In vivo tumor-suppression assay further indicated that Ph (20 mg/kg) displayed a more significant inhibitory effect on A549 xenografts in tumor growth. All these findings indicate that Ph is able to inhibit NSCLC A549 cell growth by inducing apoptosis through P38 MAPK and JNK1/2 pathways, and therefore may prove to be an adjuvant to the treatment of NSCLC. PMID:26503828

  1. Mild Electrical Stimulation and Heat Shock Ameliorates Progressive Proteinuria and Renal Inflammation in Mouse Model of Alport Syndrome

    PubMed Central

    Fukuda, Ryosuke; Morino-Koga, Saori; Suico, Mary Ann; Koyama, Kosuke; Sato, Takashi; Shuto, Tsuyoshi; Kai, Hirofumi

    2012-01-01

    Alport syndrome is a hereditary glomerulopathy with proteinuria and nephritis caused by defects in genes encoding type IV collagen in the glomerular basement membrane. All male and most female patients develop end-stage renal disease. Effective treatment to stop or decelerate the progression of proteinuria and nephritis is still under investigation. Here we showed that combination treatment of mild electrical stress (MES) and heat stress (HS) ameliorated progressive proteinuria and renal injury in mouse model of Alport syndrome. The expressions of kidney injury marker neutrophil gelatinase-associated lipocalin and pro-inflammatory cytokines interleukin-6, tumor necrosis factor-α and interleukin-1β were suppressed by MES+HS treatment. The anti-proteinuric effect of MES+HS treatment is mediated by podocytic activation of phosphatidylinositol 3-OH kinase (PI3K)-Akt and heat shock protein 72 (Hsp72)-dependent pathways in vitro and in vivo. The anti-inflammatory effect of MES+HS was mediated by glomerular activation of c-jun NH2-terminal kinase 1/2 (JNK1/2) and p38-dependent pathways ex vivo. Collectively, our studies show that combination treatment of MES and HS confers anti-proteinuric and anti-inflammatory effects on Alport mice likely through the activation of multiple signaling pathways including PI3K-Akt, Hsp72, JNK1/2, and p38 pathways, providing a novel candidate therapeutic strategy to decelerate the progression of patho-phenotypes in Alport syndrome. PMID:22937108

  2. The interleukins-1 alpha, -1 beta, and -2 do not acutely disrupt the murine blood-brain barrier.

    PubMed

    Banks, W A; Kastin, A J

    1992-05-01

    Previous studies have suggested that some of the central nervous system (CNS) effects of interleukin-2 (IL-2) and perhaps other cytokines might be mediated through disruption of the blood-brain barrier (BBB). We investigated the ability of human IL-2 and, in selected studies, human IL-1 alpha and human IL-1 beta to disrupt the BBB to radioiodinated bovine serum albumin (RISA) after intravenous (i.v.) and intracerebroventricular (i.c.v.) injection. No disruption of the BBB occurred for up to 2 h after the i.v. injection of 2 micrograms/mouse of IL-2 (10(5) U/kg of body weight), 2 micrograms of IL-1 alpha (10(7) U/kg), or 2 micrograms of IL-1 beta (10(7) U/kg). This dose of i.v. IL-2 also did not affect BBB permeability to RISA in the brain to blood direction. Damage to the BBB induced by hypertension elicited by i.v. epinephrine was not enhanced or prolonged by IL-2. When given directly into the CNS by the i.c.v. route, 100 ng of IL-2 (2.2 x 10(5) U/kg of brain), 100 ng of IL-1 alpha (2.2 x 10(7) U/kg of brain), or 100 ng of IL-1 beta (2.2 x 10(7) U/kg of brain) had no effect on BBB integrity in either the blood to brain or the brain to blood direction. We conclude that the effects of IL-1 alpha, IL-1 beta, and IL-2 on the CNS, as studied under these conditions, are not due to disruption of the BBB but are mediated by other mechanisms including the ability of some interleukins to cross the BBB by a saturable transport system described previously.

  3. Induction apoptosis of luteolin in human hepatoma HepG2 cells involving mitochondria translocation of Bax/Bak and activation of JNK

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

    Lee, H.-J.; Wang, C.-J.; Kuo, H.-C.

    2005-03-01

    Since hepatocellular carcinoma remains a major challenging clinical problem in many parts of the world including Eastern Asia and Southern Africa, it is imperative to develop more effective chemopreventive and chemotherapy agents. Herein, we present an investigation regarding the anticancer potential of luteolin, a natural flavonoid, and the mechanism of its action in human hepatoma HepG2 cells. Using DNA fragmentation assay and nuclear staining assay, it showed that luteolin induced apoptosis of HepG2 cells. Luteolin induced the cytosolic release of cytochrome c and activated CPP32. We found that Bax and Bak translocated to mitochondria apparently, whereas Fas ligand (FasL) wasmore » unchanged after a treatment with luteolin for 3 h. In addition, it showed that c-Jun NH{sub 2}-terminal kinase (JNK) was activated after the treatment of luteolin for 3-12 h. Further investigation showed that a specific JNK inhibitor, SP600125, reduced the activation of CPP 32, the mitochondrial translocation of Bax, as well as the cytosolic release of cytochrome c that induced by luteolin. Finally, the apoptosis induced by luteolin was suppressed by a pretreatment with SP600125 via evaluating annexin V-FITC binding assay. These data suggest that luteolin induced apoptosis via mechanisms involving mitochondria translocation of Bax/Bak and activation of JNK.« less

  4. Visfatin Is Regulated by Rosiglitazone in Type 2 Diabetes Mellitus and Influenced by NFκB and JNK in Human Abdominal Subcutaneous Adipocytes

    PubMed Central

    da Silva, Nancy F.; Al-Daghri, Nasser; Creely, Steven J.; Kusminski, Christine M.; Tripathi, Gyanendra; Levick, Paul L.; Khanolkar, Manish; Evans, Marc; Chittari, Madhu V.; Patel, Vinod; Kumar, Sudhesh; McTernan, Philip G.

    2011-01-01

    Visfatin has been proposed as an insulin-mimicking adipocytokine, predominantly secreted from adipose tissue and correlated with obesity. However, recent studies suggest visfatin may act as a proinflammatory cytokine. Our studies sought to determine the significance of this adipocytokine and its potential role in the pathogenesis of T2DM. Firstly, we examined the effects of diabetic status on circulating visfatin levels, and several other adipocytokines, demonstrating that diabetic status increased visfatin*, TNF-α*** and IL-6*** compared with non-diabetic subjects (*p<0.05, **p<0.01, ***p<0.001, respectively). We then assessed the effects of an insulin sensitizer, rosiglitazone (RSG), in treatment naïve T2DM subjects, on circulating visfatin levels. Our findings showed that visfatin was reduced post-RSG treatment [vs. pre-treatment (*p<0.05)] accompanied by a reduction in HOMA-IR**, thus implicating a role for insulin in visfatin regulation. Further studies addressed the intracellular mechanisms by which visfatin may be regulated, and may exert pro-inflammatory effects, in human abdominal subcutaneous (Abd Sc) adipocytes. Following insulin (Ins) and RSG treatment, our in vitro findings highlighted that insulin (100 nM), alone, upregulated visfatin protein expression whereas, in combination with RSG (10 nM), it reduced visfatin*, IKKβ** and p-JNK1/2*. Furthermore, inhibition of JNK protein exacted a significant reduction in visfatin expression (**p<0.01), whilst NF-κB blockade increased visfatin (*p<0.05), thus identifying JNK as the more influential factor in visfatin regulation. Additional in vitro analysis on adipokines regulating visfatin showed that only Abd Sc adipocytes treated with recombinant human (rh)IL-6 increased visfatin protein (*p<0.05), whilst rh visfatin treatment, itself, had no influence on TNF-α, IL-6 or resistin secretion from Sc adipocytes. These data highlight visfatin's regulation by insulin and RSG, potentially acting through NF

  5. MarvelD3 couples tight junctions to the MEKK1–JNK pathway to regulate cell behavior and survival

    PubMed Central

    Steed, Emily; Elbediwy, Ahmed; Vacca, Barbara; Dupasquier, Sébastien; Hemkemeyer, Sandra A.; Suddason, Tesha; Costa, Ana C.; Beaudry, Jean-Bernard; Zihni, Ceniz; Gallagher, Ewen; Pierreux, Christophe E.

    2014-01-01

    MarvelD3 is a transmembrane component of tight junctions, but there is little evidence for a direct involvement in the junctional permeability barrier. Tight junctions also regulate signaling mechanisms that guide cell proliferation; however, the transmembrane components that link the junction to such signaling pathways are not well understood. In this paper, we show that MarvelD3 is a dynamic junctional regulator of the MEKK1–c-Jun NH2-terminal kinase (JNK) pathway. Loss of MarvelD3 expression in differentiating Caco-2 cells resulted in increased cell migration and proliferation, whereas reexpression in a metastatic tumor cell line inhibited migration, proliferation, and in vivo tumor formation. Expression levels of MarvelD3 inversely correlated with JNK activity, as MarvelD3 recruited MEKK1 to junctions, leading to down-regulation of JNK phosphorylation and inhibition of JNK-regulated transcriptional mechanisms. Interplay between MarvelD3 internalization and JNK activation tuned activation of MEKK1 during osmotic stress, leading to junction dissociation and cell death in MarvelD3-depleted cells. MarvelD3 thus couples tight junctions to the MEKK1–JNK pathway to regulate cell behavior and survival. PMID:24567356

  6. Hypothermia increases interleukin-6 and interleukin-10 in juvenile endotoxemic mice.

    PubMed

    Stewart, Corrine R; Landseadel, Jessica P; Gurka, Matthew J; Fairchild, Karen D

    2010-01-01

    To develop a juvenile mouse model to establish effects of in vivo hypothermia on expression of the inflammation-modulating cytokines tumor necrosis factor-alpha, interleukin-1beta, interleukin-6, and interleukin-10. Although induced hypothermia is neuroprotective in some patients, the mechanisms of protection are not well understood and concerns remain over potential detrimental effects, particularly in the setting of infection. We previously showed that in vitro hypothermia increases production of tumor necrosis factor-alpha and interleukin-1beta in lipopolysaccharide-treated monocytes. : Laboratory investigation. Research laboratory. Juvenile (4-wk) male C57BL/6 mice. : Mice were given chlorpromazine to suspend thermoregulation and lipopolysaccharide to stimulate cytokine production. Core temperature was maintained at 32 degrees C or 37 degrees C for 6 hrs by adjusting environmental temperature. In separate experiments, lipopolysaccharide-treated mice were kept in a cooling chamber without chlorpromazine treatment. Plasma and organs were collected for cytokine quantitation. Chlorpromazine-treated hypothermic mice had 2.3-fold and 1.8-fold higher plasma interleukin-6 and interleukin-10 levels at 6 hrs compared with identically treated normothermic mice (p < .05), whereas plasma tumor necrosis factor-alpha and interleukin-1beta were not significantly different at 2 hrs or 6 hrs. Liver tumor necrosis factor-alpha and interleukin-6 were significantly higher in hypothermic vs. normothermic mice, but lung and brain cytokines were not different. Lipopolysaccharide-treated mice kept in a cooling chamber without chlorpromazine treatment developed varying degrees of hypothermia with associated increases in plasma interleukin-6 and interleukin-10. A nonspecific marker of stress (plasma corticosterone) was not affected by hypothermia in lipopolysaccharide-treated mice. Further studies are necessary to determine the mechanism and physiologic consequences of augmented systemic

  7. Convergent regulation of the lysosomal two-pore channel-2 by Mg2+, NAADP, PI(3,5)P2 and multiple protein kinases

    PubMed Central

    Jha, Archana; Ahuja, Malini; Patel, Sandip; Brailoiu, Eugen; Muallem, Shmuel

    2014-01-01

    Lysosomal Ca2+ homeostasis is implicated in disease and controls many lysosomal functions. A key in understanding lysosomal Ca2+ signaling was the discovery of the two-pore channels (TPCs) and their potential activation by NAADP. Recent work concluded that the TPCs function as a PI(3,5)P2 activated channels regulated by mTORC1, but not by NAADP. Here, we identified Mg2+ and the MAPKs, JNK and P38 as novel regulators of TPC2. Cytoplasmic Mg2+ specifically inhibited TPC2 outward current, whereas lysosomal Mg2+ partially inhibited both outward and inward currents in a lysosomal lumen pH-dependent manner. Under controlled Mg2+, TPC2 is readily activated by NAADP with channel properties identical to those in response to PI(3,5)P2. Moreover, TPC2 is robustly regulated by P38 and JNK. Notably, NAADP-mediated Ca2+ release in intact cells is regulated by Mg2+, PI(3,5)P2, and P38/JNK kinases, thus paralleling regulation of TPC2 currents. Our data affirm a key role for TPC2 in NAADP-mediated Ca2+ signaling and link this pathway to Mg2+ homeostasis and MAP kinases, pointing to roles for lysosomal Ca2+ in cell growth, inflammation and cancer. PMID:24502975

  8. Low-level lasers on microRNA and uncoupling protein 2 mRNA levels in human breast cancer cells

    NASA Astrophysics Data System (ADS)

    Canuto, K. S.; Teixeira, A. F.; Rodrigues, J. A.; Paoli, F.; Nogueira, E. M.; Mencalha, A. L.; Fonseca, A. S.

    2017-06-01

    MicroRNA is short non-coding RNA and is a mediator of post-transcriptional regulation of gene expression. In addition, uncoupling proteins (UCPs) regulate thermogenesis, metabolic and energy balance, and decrease reactive oxygen species production. Both microRNA and UCP2 expression can be altered in cancer cells. At low power, laser wavelength, frequency, fluence and emission mode deternube photobiological responses, which are the basis of low-level laser therapy. There are few studies on miRNA and UCP mRNA levels after low-level laser exposure on cancer cells. In this work, we evaluate the micrRNA (mir-106b and mir-15a) and UCP2 mRNA levels in human breast cancer cells exposed to low-level lasers. MDA-MB-231 human breast cancer cells were exposed to low-level red and infrared lasers, total RNA was extracted for cDNA synthesis and mRNA levels by real time quantitative polymerase chain reaction were evaluated. Data show that mir-106b and mir-15a relative levels are not altered, but UCP2 mRNA relative levels are increased in MDA-MB-231 human breast cancer cells exposed to low-level red and infrared lasers at fluences used in therapeutic protocols.

  9. The tumor promoter arsenite stimulates AP-1 activity by inhibiting a JNK phosphatase.

    PubMed Central

    Cavigelli, M; Li, W W; Lin, A; Su, B; Yoshioka, K; Karin, M

    1996-01-01

    Trivalent arsenic (As3+) is highly carcinogenic, but devoid of known mutagenic activity. Therefore, it is likely to act as a tumor promoter. To understand the molecular basis for the tumor-promoting activity of As3+, we examined its effect on transcription factor AP-1, whose activity is stimulated by several other tumor promoters. We found that As3+, but not As5+, which is toxic but not carcinogenic, is a potent stimulator of AP-1 transcriptional activity and an efficient inducer of c-fos and c-jun gene expression. Induction of c-jun and c-fos transcription by As3+ correlates with activation of Jun kinases (JNKs) and p38/Mpk2, which phosphorylate transcription factors that activate these immediate early genes. No effect on ERK activity was observed. As5+, on the other hand, had a negligible effect on JNK or p38/Mpk2 activity. Biochemical analysis and co-transfection experiments strongly suggest that the primary mechanism by which As3+ stimulates JNK activity involves the inhibition of a constitutive dual-specificity JNK phosphatase. This phosphatase activity appears to be responsible for maintaining low basal JNK activity in non-stimulated cells and its inhibition may lead to tumor promotion through induction of proto-oncogenes such as c-jun and c-fos, and stimulation of AP-1 activity. The same phosphatase may also regulate p38/Mpk2 activity. Images PMID:8947050

  10. Interleukin-33/ST2 system attenuates aldosterone-induced adipogenesis and inflammation.

    PubMed

    Martínez-Martínez, Ernesto; Cachofeiro, Victoria; Rousseau, Elodie; Álvarez, Virginia; Calvier, Laurent; Fernández-Celis, Amaya; Leroy, Céline; Miana, María; Jurado-López, Raquel; Briones, Ana M; Jaisser, Frederic; Zannad, Faiez; Rossignol, Patrick; López-Andrés, Natalia

    2015-08-15

    Interleukin-33 (IL-33) but not soluble ST2 (sST2) exerts anti-inflammatory and protective effects in several tissues. Aldosterone, a proinflammatory mediator which promotes adipogenesis, is elevated in obese patients. The aim of this study was to investigate the interactions between IL-33/ST2 system and Aldosterone in adipose tissue. Rats fed a high fat diet presented increased sST2 expression, diminished IL-33/sST2 ratio and enhanced levels of differentiation and inflammation in adipose tissue as compared to controls. A similar pattern was observed in adipose tissue from C57BL/6 Aldosterone-treated mice. In both animal models, Aldosterone was correlated with sST2. Treatment of 3T3-L1 adipocytes with IL-33 delayed adipocyte differentiation diminished lipid accumulation and decreased inflammation. Aldosterone decreased IL-33 and increased sST2 expressions in differentiated adipocytes. Aldosterone-induced adipocyte differentiation and inflammation were blocked by IL-33 treatment, but sST2 did not exert any effects. The crosstalk between IL-33/ST2 and Aldosterone could be relevant in the metabolic consequences of obesity. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

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

    Gandhi, Adarsh, E-mail: adarsh.gandhi@nih.gov; Guo, Tao, E-mail: tguo4@jhu.edu; Shah, Pranav

    Inflammation is a major component of idiosyncratic adverse drug reactions (IADRs). To understand the molecular mechanism of inflammation-mediated IADRs, we determined the role of the Toll-like receptor (TLR) signaling pathway in idiosyncratic hepatotoxicity of the anti-psychotic drug, chlorpromazine (CPZ). Activation of TLRs recruits the first adaptor protein, Toll-interleukin 1 receptor domain containing adaptor protein (TIRAP) to the TIR domain of TLRs leading to the activation of the downstream kinase, c-Jun-N-terminal kinase (JNK). Prolonged activation of JNK leads to cell-death. We hypothesized that activation of TLR2 by lipoteichoic acid (LTA) or TLR4 by lipopolysaccharide (LPS) will augment the hepatotoxicity of CPZmore » by TIRAP-dependent mechanism involving prolonged activation of JNK. Adult male C57BL/6, TIRAP{sup +/+} and TIRAP{sup −/−} mice were pretreated with saline, LPS (2 mg/kg) or LTA (6 mg/kg) for 30 min or 16 h followed by CPZ (5 mg/kg) or saline (vehicle) up to 24 h. We found that treatment of mice with CPZ in presence of LPS or LTA leads to ∼ 3–4 fold increase in serum ALT levels, a marked reduction in hepatic glycogen content, significant induction of serum tumor necrosis factor (TNF) α and prolonged JNK activation, compared to LPS or LTA alone. Similar results were observed in TIRAP{sup +/+} mice, whereas the effects of LPS or LTA on CPZ-induced hepatotoxicity were attenuated in TIRAP{sup −/−} mice. For the first time, we show that inflammation-mediated hepatotoxicity of CPZ is dependent on TIRAP, and involves prolonged JNK activation in vivo. Thus, TIRAP-dependent pathways may be targeted to predict and prevent inflammation-mediated IADRs. -- Highlights: ► Inflammation augments the toxicity of an idiosyncratic hepatotoxin chlorpromazine. ► Activation of Toll-like receptors by LPS or LTA induces chlorpromazine toxicity. ► Sustained stress kinase (JNK) activation is associated with chlorpromazine toxicity. ► These

  12. Evidence for convergent evolution of SINE-directed Staufen-mediated mRNA decay.

    PubMed

    Lucas, Bronwyn A; Lavi, Eitan; Shiue, Lily; Cho, Hana; Katzman, Sol; Miyoshi, Keita; Siomi, Mikiko C; Carmel, Liran; Ares, Manuel; Maquat, Lynne E

    2018-01-30

    Primate-specific Alu short interspersed elements (SINEs) as well as rodent-specific B and ID (B/ID) SINEs can promote Staufen-mediated decay (SMD) when present in mRNA 3'-untranslated regions (3'-UTRs). The transposable nature of SINEs, their presence in long noncoding RNAs, their interactions with Staufen, and their rapid divergence in different evolutionary lineages suggest they could have generated substantial modification of posttranscriptional gene-control networks during mammalian evolution. Some of the variation in SMD regulation produced by SINE insertion might have had a similar regulatory effect in separate mammalian lineages, leading to parallel evolution of the Staufen network by independent expansion of lineage-specific SINEs. To explore this possibility, we searched for orthologous gene pairs, each carrying a species-specific 3'-UTR SINE and each regulated by SMD, by measuring changes in mRNA abundance after individual depletion of two SMD factors, Staufen1 (STAU1) and UPF1, in both human and mouse myoblasts. We identified and confirmed orthologous gene pairs with 3'-UTR SINEs that independently function in SMD control of myoblast metabolism. Expanding to other species, we demonstrated that SINE-directed SMD likely emerged in both primate and rodent lineages >20-25 million years ago. Our work reveals a mechanism for the convergent evolution of posttranscriptional gene regulatory networks in mammals by species-specific SINE transposition and SMD.

  13. Dusuqing granules (DSQ) suppress inflammation in Klebsiella pneumonia rat via NF-κB/MAPK signaling.

    PubMed

    Mei, Xue; Wang, Hao-Xun; Li, Jian-Sheng; Liu, Xiao-Hui; Lu, Xiao-Fan; Li, Ya; Zhang, Wei-Yu; Tian, Yan-Ge

    2017-04-17

    Dusuqing granules (DSQ) have been used in the treatment of bacterial pneumonia clinically, with remarkable benefits. This study was initiated to explore the effects of DSQ on pulmonary inflammation by regulating nuclear factor (NF)-κB/mitogen-activated protein kinase (MAPK) signaling in bacterial pneumonia rats. Rat model was duplicated with Klebsiella pneumonia by a one-time intratracheal injection. Rats were randomized into control, model, DSQ and levofloxacin (LVX) groups. After administrated with appropriate medicines for 7 days, lung tissues were harvested and prepared for pathological analysis, and interleukin (IL)-1, IL-6, monocyte chemotactic protein (MCP)-1and macrophage inflammatory protein (MIP)-2 detections. NF-κB mRNA was measured by real-time qPCR, and the phosphorylation and total proteins of P38MAPK, JNK46/54, ERK42/44 were determined by Western blotting. Marked pathological impairments were observed in model rats, whereas were improved in DSQ group. The cytokines levels, NF-κB mRNA expression and the phosphorylation of P38MAPK, JNK46/54 and ERK42/44 proteins were significantly higher in model group, and were significantly depressed in DSQ group. The protective effects of DSQ on Klebsiella pneumonia might be attributed to its inactivative effects of NF-κB/ MAPK pathway.

  14. Downregulation of Programmed Cell Death 4 by Inflammatory Conditions Contributes to the Generation of the Tumor Promoting Microenvironment

    PubMed Central

    Yasuda, Michiko; Schmid, Tobias; Rübsamen, Daniela; Colburn, Nancy H.; Irie, Kazuhiro; Murakami, Akira

    2012-01-01

    Ample evidence has shown key roles of inflammation in tumor promotion and carcinogenesis, and tumor-associated macrophages are known to promote tumor growth and dissemination. Programmed cell death 4 (Pdcd4) is a novel tumor suppressor, and although various studies have revealed that the functions and expression mechanisms of Pdcd4 in tumor promotion, those in regard to inflammation remain unclear. In the present study, we examined whether inflammatory stimuli regulate Pdcd4 expression. 12-O-tetradecanoylphorbol 13-acetate (TPA) suppressed expression of pdcd4 mRNA in human monocytic cell lines (U937, THP-1). Similarly, the bacterial endotoxin lipopolysaccharide (LPS) downregulated pdcd4 level in mouse RAW264.7 and peritoneal macrophages. Furthermore, conditioned medium from LPS-stimulated RAW264.7 macrophages suppressed pdcd4 mRNA in RAW264.7 macrophages, and findings obtained with recombinant tumor necrosis factor-α (TNF-α) and TNF-α-specific siRNA suggested that TNF-α partly mediates LPS-triggered Pdcd4 downregulation via an autocrine mechanism. Specific inhibitors of phosphoinositide-3-kinase (PI3K) and c-jun N-terminus kinase (JNK) restored LPS-abolished pdcd4 mRNA. Consistently, in MCF7 mammary carcinoma cells, conditioned medium from TPA-differentiated/activated U937 cells suppressed pdcd4 mRNA. Additionally, knockdown of pdcd4 in RAW264.7 macrophages using siRNA significantly enhanced LPS-induced TNF-α protein production, and interferon-γ, CC chemokine ligand (Ccl) 1, Ccl20, and interleukin-10 mRNA expression. These results suggest that Pdcd4 suppresses the induction of these inflammatory mediators. Taken together, loss of Pdcd4 in macrophages may be a critical step in establishing the inflammatory environment while that in tumor cells contributes to tumor progression. PMID:20607724

  15. Eupafolin inhibits PGE2 production and COX2 expression in LPS-stimulated human dermal fibroblasts by blocking JNK/AP-1 and Nox2/p47{sup phox} pathway

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

    Tsai, Ming-Horng; Lin, Zih-Chan; Liang, Chan-Jung

    2014-09-01

    Eupafolin, a major active component found in the methanol extracts of Phyla nodiflora, has been used to treat inflammation of skin. We examined its effects on cyclooxygenase-2 (COX-2) expression in LPS-treated human dermal fibroblasts. Lipopolysaccharide (LPS) significantly increased prostaglandin-E2 (PGE2) production associated with increased COX-2 expression in Hs68 cells. This effect was blocked by eupafolin, TLR-4 antibody, antioxidants (APO and NAC), as well as inhibitors, including U0126 (ERK1/2), SB202190 (p38), SP600125 (JNK1/2), and Tanshinone IIA (AP-1). In gene regulation level, qPCR and promoter assays revealed that COX-2 expression was attenuated by eupafolin. In addition, eupafolin also ameliorated LPS-induced p47 phoxmore » activation and decreased reactive oxygen species (ROS) generation and NADPH oxidase (Nox) activity. Moreover, pretreatment with eupafolin and APO led to reduced LPS-induced phosphorylation of ERK1/2, JNK, and p38. Further, eupafolin attenuated LPS-induced increase in AP-1 transcription factor binding activity as well as the increase in the phosphorylation of c-Jun and c-Fos. In vivo studies have shown that in dermal fibroblasts of LPS treated mice, eupafolin exerted anti-inflammation effects by decreasing COX-2 protein levels. Our results reveal a novel mechanism for anti-inflammatory and anti-oxidative effects of eupafolin that involved inhibition of LPS-induced ROS generation, suppression of MAPK phosphorylation, diminished DNA binding activity of AP-1 and attenuated COX-2 expression leading to reduced production of prostaglandin E2 (PGE2). Our results demonstrate that eupafolin may be used to treat inflammatory responses associated with dermatologic diseases. - Highlights: • LPS activates the Nox2/p47{sup phox}/JNK/AP-1 and induces COX2 expression in Hs68 cells. • Eupafolin inhibits LPS-induced COX-2 expression via Nox2/p47{sup phox} inhibition. • Eupafolin may be used in the treatment of skin diseases involving

  16. Antiallergic effect of fisetin on IgE-mediated mast cell activation in vitro and on passive cutaneous anaphylaxis (PCA).

    PubMed

    Jo, Woo-Ri; Park, Hye-Jin

    2017-10-01

    Fisetin (3,7,3',4'-tetrahydroxyflavone), a naturally occurring bioactive flavonoid, has been shown to inhibit inflammation. However, little is known about the effect of fisetin on immunoglobulin E (IgE)-mediated allergic responses. In this study, the effect of fisetin on rat basophilic leukemia (RBL-2H3) cell-mediated allergic reactions was investigated. Fisetin inhibited β-hexosaminidase release and decreased the level of interleukin-4 and tumor necrosis factor-α mRNA in IgE/antigen (IgE/Ag)-stimulated RBL-2H3 cells. To elucidate the antiallergic mechanism, we examined the levels of signaling molecules responsible for degranulation and release of inflammatory cytokines. Fisetin decreased the levels of activated spleen tyrosine kinase, Gab2 proteins, linker of activated T cells, extracellular signal-related kinase 1/2 in the IgE/Ag-stimulated RBL2H3 cells, and NFκB and STAT3 proteins activated in the ear tissue of mice with passive cutaneous anaphylaxis (PCA). In addition, fisetin significantly lowered of FcɛRI α-subunit mRNA expression. Consistent with the cellular data, fisetin markedly suppressed RBL-2H3 cell-dependent PCA in IgE/Ag-sensitized mice. These results suggest that fisetin may have potential as a therapeutic agent for the treatment of allergic diseases. Copyright © 2017 Elsevier Inc. All rights reserved.

  17. Expression of Cytokines Interleukin-2, Interleukin-4, Interleukin-10 and Transforming Growth Factor β in Gastric Adenocarcinoma Biopsies Obtained from Mexican Patients

    PubMed Central

    Orea, Maria Alicia Diaz; Perez, Veronica Muñoz; Conde, Eduardo Gómez; Sánchez, Victor Omar Castellanos; Lopez, Rogelio Gonzalez; Alonso, J Carlos Flores; Cárdenas, M Elena; Galicia, A Luisa; Mendoza, Aurelio

    2017-01-01

    Objective: In this study, expression of Interleukin-2, Interleukin-4, Interleukin-10 and transforming growth factor beta in diffuse and intestinal type gastric cancers from Mexican patients was assessed for use as markers of malignancy. Methods: A total of 30 biopsies from gastric adenocarcinomas, 60% diffuse, 20% intestinal and 20% mixed in type, were studied by immunohistochemistry. Results: Regarding expression of cytokines, 23% were positive for IL-2, 26.7% for IL-4, 16.6% for IL-10 and none for TGF-β. There were found Significant statistically stage differences were noted. For example, for stages I-II 100% were IL-2 positive (p = 0.009), 87.5% were IL-4 positive (p = 0.005) and 100.0% IL-10 positive (p = 0.009). Young women were more likely to suffer gastric adenocarcinoma. In biopsies of male patients with gastric cancer, there was an increased expression of IL-2 and in biopsies from female patients in IL4. There was significantly greater detection of IL-4 and IL-10 expression in stages I and II than in stages III and IV. It was also found that IL-4, IL-10 had a higher positive expression in patients biopsies with low-level differentiations than patients with well differentiated gastric cancer in which cases were undetected. Conclusions: These results suggest that positive expression of IL-4 and IL-10 may be useful as a molecular marker to distinguish stage I and II diffuse gastric cancers which can be more readily controlled. PMID:28350427

  18. Barium inhibits arsenic-mediated apoptotic cell death in human squamous cell carcinoma cells.

    PubMed

    Yajima, Ichiro; Uemura, Noriyuki; Nizam, Saika; Khalequzzaman, Md; Thang, Nguyen D; Kumasaka, Mayuko Y; Akhand, Anwarul A; Shekhar, Hossain U; Nakajima, Tamie; Kato, Masashi

    2012-06-01

    Our fieldwork showed more than 1 μM (145.1 μg/L) barium in about 3 μM (210.7 μg/L) arsenic-polluted drinking well water (n = 72) in cancer-prone areas in Bangladesh, while the mean concentrations of nine other elements in the water were less than 3 μg/L. The types of cancer include squamous cell carcinomas (SCC). We hypothesized that barium modulates arsenic-mediated biological effects, and we examined the effect of barium (1 μM) on arsenic (3 μM)-mediated apoptotic cell death of human HSC-5 and A431 SCC cells in vitro. Arsenic promoted SCC apoptosis with increased reactive oxygen species (ROS) production and JNK1/2 and caspase-3 activation (apoptotic pathway). In contrast, arsenic also inhibited SCC apoptosis with increased NF-κB activity and X-linked inhibitor of apoptosis protein (XIAP) expression level and decreased JNK activity (antiapoptotic pathway). These results suggest that arsenic bidirectionally promotes apoptotic and antiapoptotic pathways in SCC cells. Interestingly, barium in the presence of arsenic increased NF-κB activity and XIAP expression and decreased JNK activity without affecting ROS production, resulting in the inhibition of the arsenic-mediated apoptotic pathway. Since the anticancer effect of arsenic is mainly dependent on cancer apoptosis, barium-mediated inhibition of arsenic-induced apoptosis may promote progression of SCC in patients in Bangladesh who keep drinking barium and arsenic-polluted water after the development of cancer. Thus, we newly showed that barium in the presence of arsenic might inhibit arsenic-mediated cancer apoptosis with the modulation of the balance between arsenic-mediated promotive and suppressive apoptotic pathways.

  19. The interleukin-1, interleukin-2, interleukin-6 and tumour necrosis factor alpha serological levels in localised and systemic sclerosis.

    PubMed

    Alecu, M; Geleriu, L; Coman, G; Gălăţescu, L

    1998-01-01

    Serological level of interleukin-1 (IL-1), Interleukin-2 (IL-2), Interleukin-6 (IL-6) and tumour necrosis factor (TNF) alpha was investigated in 26 patients with scleroderma, divided into three lots, by the extension and the progress of the disease. Determinations were performed by ELISA in attack and in remission (after treatment with prednison). Normal values: IL-1 (0-5 pg/ml), IL-2 (0-5 pg/ml), IL-6 (5-15 pg/ml), TNF (0-16 pg/ml). Lot A. Results obtained at the first determination showed that IL-1 is elevated in 4 cases (10-15 pg/ml), IL-2 in 5 cases (10-32 pg/ml), IL-6 in 5 cases (15-42 pg/ml) and TNF in 4 cases (18-34 pg/ml). In the second determination IL-1 was increased in 1 case (8 pg/ml), IL-2 in 1 case (9 pg/ml), IL-6 in 2 cases (12 pg/ml) and TNF was normal. Lot B. In the first determination IL-1 was elevated in 5 cases (8-12 pg/ml), IL-2 in 5 cases (10-15 pg/ml), IL-6 in 7 cases (16-20 pg/ml) and TNF was raised in 3 cases (18-25 pg/ml). At the second determination IL-1 showed normal values in all the cases, IL-2 was raised in 2 cases (10 pg/ml), IL-6 in 2 cases (12.15 pg/ml), TNF in 1 case (20 pg/ml). Lot C. In the first determination there were raised values in 4 cases for IL-1 (6-8 pg/ml), 3 cases for IL-2 (10-18 pg/ml), 5 cases for IL-6 (18-20 pg/ml), 2 cases for TNF (20 pg/ml). At the second determination IL-2 was elevated in 1 case (10 pg/ml), IL-6 in 1 case (15 pg/ml). We consider that in scleroderma there is a disturbance of the investigated cytokines due to the activation and involvement of the secretory cells into the pathogenesis of the disease. The increase of the serological levels of IL-1, IL-2, IL-6 and TNF depends on the extension of the lesions and the clinical and biological activity periods of the disease. The absence of the increase of the serological levels does not exclude their activity at the lesional site.

  20. Cell type-specific regulation of beta2-adrenoceptor mRNA by agonists.

    PubMed

    Danner, S; Lohse, M J

    1997-07-16

    Prolonged agonist stimulation of beta2-adrenoceptors results in receptor down-regulation which is often paralleled by a reduction of the corresponding mRNA. In this study, we investigated the agonist-dependent regulation of beta2-adrenoceptor mRNA in DDT1-MF2 smooth muscle cells and C6 glioma cells. In DDT1-MF2 cells the half-life of the mRNA was 12 h in monolayer compared to 2 h in suspension cultures. Under both conditions, the agonist isoproterenol reduced this half-life by a factor of 2. In contrast, in C6 glioma cells isoproterenol had no effect on the mRNA stability, even though it reduced mRNA levels by approximately 50%. Isoproterenol-induced downregulation of beta2-adrenoceptor mRNA was completely blocked in C6 cells by the presence of a protein synthesis inhibitor, while this was not so in DDT1-MF2-cells. These data show that beta2-adrenoceptor downregulation occurs via cell-type specific mechanisms.

  1. [Study of the effect of JNK signal transduction pathway in intense noise-induced apoptosis in cochlea of guinea pig].

    PubMed

    Xue, Qiuhong; Chen, Jia; Gong, Shusheng; Xie, Jing; He, Jian; Chen, Xiaolin

    2009-12-01

    To investigate the mechanism of intense noise-induced cochlea cells death in guinea pig, and the effect of JNK signal transduction pathway in the procedure of cochlea cells apoptosis by intense noise-induced. Thirty-two guinea pigs were randomly divided into 4 groups. The guinea pigs in the experiment groups were exposed to 4 kHz narrow band noise at 120 dB SPL for 4 h. After the noise expose for 1, 4, 14 days of the experiment guinea pigs, ABR of the guinea pigs on experiment and control groups were tested before put them to death. Four guinea pig's cochleas of every group were taken to paraffin section, and the rest was extracted the total cochlear's protein. Apoptosis was tested by terminal deoxynucleotidyl Transferase (TdT)-mediated deoxyuridine triphosphate (d-UTP) nick and labeling method (TUNEL). The phosphorylation of JNK and c-Jun were tested by immunohistochemistry and western blot methods. Tunel-Positive cells in the Corti's, SGC and SV of experiment groups, and there have significant differences compared with the control group (P<0.01) and Tunel-Positive cells are most in 1 d experiment group. The positive cells of P-JNK and P-c-Jun could be detected in guinea pig's cochleas after noise exposed, but no positive cells were found in the control. Protein levels of P-JNK and P-c-Jun were risen up and activated quickly after noise exposed, and achieved peak in 1 d, 4 d and then fallen-offs, but still maintained higher levels within 14 d. Intense noise causes cochlea cell lesion by inducing apoptosis to result in and JNK signal transduction pathway plays an important role in the procedure of apoptosis.

  2. Rac1b enhances cell survival through activation of the JNK2/c-JUN/Cyclin-D1 and AKT2/MCL1 pathways

    PubMed Central

    Wang, Hong; Wei, Si-Si; Chen, Jie; Chen, Yi-He; Xu, Wei-Ping; Jie, Qi-Qiang; Zhou, Qing; Li, Yi-Gang; Wei, Yi-Dong; Wang, Yue-Peng

    2016-01-01

    Rac1b is a constitutively activated, alternatively spliced form of the small GTPase Rac1. Previous studies showed that Rac1b promotes cell proliferation and inhibits apoptosis. In the present study, we used microarray analysis to detect genes differentially expressed in HEK293T cells and SW480 human colon cancer cells stably overexpressing Rac1b. We found that the pro-proliferation genes JNK2, c-JUN and cyclin-D1 as well as anti-apoptotic AKT2 and MCL1 were all upregulated in both lines. Rac1b promoted cell proliferation and inhibited apoptosis by activating the JNK2/c-JUN/cyclin-D1 and AKT2/MCL1 pathways, respectively. Very low Rac1b levels were detected in the colonic epithelium of wild-type Sprague-Dawley rats. Knockout of the rat Rac1 gene exon-3b or knockdown of endogenous Rac1b in HT29 human colon cancer cells downregulated only the AKT2/MCL1 pathway. Our study revealed that very low levels of endogenous Rac1b inhibit apoptosis, while Rac1b upregulation both promotes cell proliferation and inhibits apoptosis. It is likely the AKT2/MCL1 pathway is more sensitive to Rac1b regulation. PMID:26918455

  3. Schwann Cell Migration Induced by Earthworm Extract via Activation of PAs and MMP2/9 Mediated through ERK1/2 and p38

    PubMed Central

    Chang, Yung-Ming; Shih, Ying-Ting; Chen, Yueh-Sheng; Liu, Chien-Liang; Fang, Wen-Kuei; Tsai, Chang-Hai; Tsai, Fuu-Jen; Kuo, Wei-Wen; Lai, Tung-Yuan; Huang, Chih-Yang

    2011-01-01

    The earthworm, which has stasis removal and wound-healing functions, is a widely used Chinese herbal medicine in China. Schwann cell migration is critical for the regeneration of injured nerves. Schwann cells provide an essentially supportive activity for neuron regeneration. However, the molecular migration mechanisms induced by earthworms in Schwann cells remain unclear. Here, we investigate the roles of MAPK (ERK1/2, JNK and p38) pathways for earthworm-induced matrix-degrading proteolytic enzyme (PAs and MMP2/9) production in Schwann cells. Moreover, earthworm induced phosphorylation of ERK1/2 and p38, but not JNK, activate the downstream signaling expression of PAs and MMPs in a time-dependent manner. Earthworm-stimulated ERK1/2 and p38 phosphorylation was attenuated by pretreatment with U0126 and SB203580, resulting in migration and uPA-related signal pathway inhibition. The results were confirmed using small interfering ERK1/2 and p38 RNA. These results demonstrated that earthworms can stimulate Schwann cell migration and up-regulate PAs and MMP2/9 expression mediated through the MAPK pathways, ERK1/2 and p38. Taken together, our data suggests the MAPKs (ERK1/2, p38)-, PAs (uPA, tPA)-, MMP (MMP2, MMP9) signaling pathway of Schwann cells regulated by earthworms might play a major role in Schwann cell migration and nerve regeneration. PMID:19808845

  4. Hawthorn (Crataegus oxyacantha L.) bark extract regulates antioxidant response element (ARE)-mediated enzyme expression via Nrf2 pathway activation in normal hepatocyte cell line.

    PubMed

    Krajka-Kuźniak, Violetta; Paluszczak, Jarosław; Oszmiański, Jan; Baer-Dubowska, Wanda

    2014-04-01

    Hawthorn (Crataegus oxyacantha L.), a plant used in traditional medicine, is a rich source of procyanidins which have been reported to exhibit antioxidant and anti-carcinogenic activity. In this study, we assessed the effect of hawthorn bark extract (HBE) on Nrf2 pathway activation in THLE-2 and HepG2 cells. Treatment with 1.1 µg/mL, 5.5 µg/mL and 11 µg/mL of HBE resulted in the translocation of Nrf2 from the cytosol to the nucleus in both cell lines; however, the accumulation of phosphorylated Nrf2 was observed only in THLE-2. Accordingly, treatment of cells with HBE was associated with an increase in the mRNA and protein level of such Nrf2-dependent genes as glutathione S-transferases (GSTA, GSTP, GSTM, GSTT), NAD(P)H:quinone oxidoreductase 1 (NQO1) and heme oxygenase-1 (HO-1) (0.2-1.1-fold change, p < 0.05), however, only in normal THLE-2 hepatocytes. The induction of NQO1 correlated with an increased level of p53 (0.21-0.42-fold change, p < 0.05). These effects may be related to induction of phosphorylation of upstream ERK and JNK kinases. Collectively, the results suggest that the Nrf2/ARE pathway may play an important role in the regulation of procyanidin-mediated antioxidant/detoxifying effects in hepatocytes, and this may explain the hepatoprotective and chemopreventive properties of these phytochemicals. Copyright © 2013 John Wiley & Sons, Ltd.

  5. Silencing Glycogen Synthase Kinase-3β Inhibits Acetaminophen Hepatotoxicity and Attenuates JNK Activation and Loss of Glutamate Cysteine Ligase and Myeloid Cell Leukemia Sequence 1*

    PubMed Central

    Shinohara, Mie; Ybanez, Maria D.; Win, Sanda; Than, Tin Aung; Jain, Shilpa; Gaarde, William A.; Han, Derick; Kaplowitz, Neil

    2010-01-01

    Previously we demonstrated that c-Jun N-terminal kinase (JNK) plays a central role in acetaminophen (APAP)-induced liver injury. In the current work, we examined other possible signaling pathways that may also contribute to APAP hepatotoxicity. APAP treatment to mice caused glycogen synthase kinase-3β (GSK-3β) activation and translocation to mitochondria during the initial phase of APAP-induced liver injury (∼1 h). The silencing of GSK-3β, but not Akt-2 (protein kinase B) or glycogen synthase kinase-3α (GSK-3α), using antisense significantly protected mice from APAP-induced liver injury. The silencing of GSK-3β affected several key pathways important in conferring protection against APAP-induced liver injury. APAP treatment was observed to promote the loss of glutamate cysteine ligase (GCL, rate-limiting enzyme in GSH synthesis) in liver. The silencing of GSK-3β decreased the loss of hepatic GCL, and promoted greater GSH recovery in liver following APAP treatment. Silencing JNK1 and -2 also prevented the loss of GCL. APAP treatment also resulted in GSK-3β translocation to mitochondria and the degradation of myeloid cell leukemia sequence 1 (Mcl-1) in mitochondrial membranes in liver. The silencing of GSK-3β reduced Mcl-1 degradation caused by APAP treatment. The silencing of GSK-3β also resulted in an inhibition of the early phase (0–2 h), and blunted the late phase (after 4 h) of JNK activation and translocation to mitochondria in liver following APAP treatment. Taken together our results suggest that activation of GSK-3β is a key mediator of the initial phase of APAP-induced liver injury through modulating GCL and Mcl-1 degradation, as well as JNK activation in liver. PMID:20061376

  6. Stress- and Rho-activated ZO-1–associated nucleic acid binding protein binding to p21 mRNA mediates stabilization, translation, and cell survival

    PubMed Central

    Nie, Mei; Balda, Maria S.; Matter, Karl

    2012-01-01

    A central component of the cellular stress response is p21WAF1/CIP1, which regulates cell proliferation, survival, and differentiation. Inflammation and cell stress often up-regulate p21 posttranscriptionally by regulatory mechanisms that are poorly understood. ZO-1–associated nucleic acid binding protein (ZONAB)/DbpA is a Y-box transcription factor that is regulated by components of intercellular junctions that are affected by cytokines and tissue damage. We therefore asked whether ZONAB activation is part of the cellular stress response. Here, we demonstrate that ZONAB promotes cell survival in response to proinflammatory, hyperosmotic, and cytotoxic stress and that stress-induced ZONAB activation involves the Rho regulator GEF-H1. Unexpectedly, stress-induced ZONAB activation does not stimulate ZONAB’s activity as a transcription factor but leads to the posttranscriptional up-regulation of p21 protein and mRNA. Up-regulation is mediated by ZONAB binding to specific sites in the 3′-untranslated region of the p21 mRNA, resulting in mRNA stabilization and enhanced translation. Binding of ZONAB to mRNA is activated by GEF-H1 via Rho stimulation and also mediates Ras-induced p21 expression. We thus identify a unique type of stress and Rho signaling activated pathway that drives mRNA stabilization and translation and links the cellular stress response to p21 expression and cell survival. PMID:22711822

  7. miR-328 Functions as an RNA Decoy to Modulate hnRNP E2 Regulation of mRNA Translation in Leukemic Blasts

    PubMed Central

    Eiring, Anna M.; Harb, Jason G.; Neviani, Paolo; Garton, Christopher; Oaks, Joshua J.; Spizzo, Riccardo; Liu, Shujun; Schwind, Sebastian; Santhanam, Ramasamy; Hickey, Christopher J.; Becker, Heiko; Chandler, Jason C.; Andino, Raul; Cortes, Jorge; Hokland, Peter; Huettner, Claudia S.; Bhatia, Ravi; Roy, Denis C.; Liebhaber, Stephen A.; Caligiuri, Michael A.; Marcucci, Guido; Garzon, Ramiro; Croce, Carlo M.; Calin, George A.; Perrotti, Danilo

    2010-01-01

    SUMMARY MicroRNAs and heterogeneous ribonucleoproteins (hnRNPs) are posttranscriptional gene regulators that bind mRNA in a sequence-specific manner. Here, we report that loss of miR-328 occurs in blast crisis chronic myelogenous leukemia (CML-BC) in a BCR/ABL dose- and kinase-dependent manner through the MAPK-hnRNP E2 pathway. Restoration of miR-328 expression rescues differentiation and impairs survival of leukemic blasts by simultaneously interacting with the translational regulator poly(rC)-binding protein hnRNP E2 and with the mRNA encoding the survival factor PIM1, respectively. The interaction with hnRNP E2 is independent of the microRNA’s seed sequence and it leads to release of CEBPA mRNA from hnRNP E2-mediated translational inhibition. Altogether, these data reveal the dual ability of a microRNA to control cell fate both through base pairing with mRNA targets and through a decoy activity that interferes with the function of regulatory proteins. PMID:20211135

  8. Boehmeria nivea attenuates LPS-induced inflammatory markers by inhibiting p38 and JNK phosphorylations in RAW264.7 macrophages.

    PubMed

    Sung, Mi Jeong; Davaatseren, Munkhtugs; Kim, Sung Hee; Kim, Min Jung; Hwang, Jin-Taek

    2013-09-01

    Boehmeria nivea (Linn.) Gaudich (Urticaceae), a natural herb, has a long history of treating several diseases including wound healing. However, the anti-inflammatory effect of B. nivea has not been investigated. We investigated whether the 70% ethanol extract of B. nivea (Ebn) can exert anti-inflammatory activity. Several phenolic compounds of extracts were determined to provide further information on the correlation between anti-inflammatory effects and phenolic compounds. We prepared a 70% ethanol extract of B. nivea leaves and evaluated its anti-inflammatory activity (200, 400, 800, 1200 µg/mL) by measuring the secretions of nitric oxide (NO), tumor necrosis factor alpha (TNF-α) and interleukin 6 (IL-6), which were stimulated by lipopolysaccharide (LPS) in RAW264.7 macrophages. The total phenolic compounds were determined by the Folin-Ciocalteu method and major compounds were determined by HPLC. Ebn was able to abolish the LPS-induced secretions of NO, TNF-α and IL-6. It also decreased the protein levels (IC₅₀ = 186 µg/mL) of LPS-induced inducible nitric oxide synthase (iNOS). The LPS stimulated p38, JNK and ERK phosphorylations significantly more than the controls. Surprisingly, although Ebn reduced p38 and JNK phosphorylations, it did not influence ERK phosphorylation. We found that Ebn revealed several major compounds such as chlorogenic acid (1.96 mg/100 g), rutin (46.48 mg/100 g), luteolin-7-glucoside (11.29 mg/100 g), naringin (1.13 mg/100 g), hesperidin (23.69 mg/100 g) and tangeretin (1.59 mg/100 g). Boehmeria nivea exerts an anti-inflammatory effect on macrophages by inhibiting p38 and JNK, suggesting that it may be used as a functional ingredient against inflammation.

  9. Detection of siRNA Mediated Target mRNA Cleavage Activities in Human Cells by a Novel Stem-Loop Array RT-PCR Analysis

    DTIC Science & Technology

    2016-09-07

    sequences of the target mRNA, and a double stranded stem at the 5′ end that forms a stem -loop to function as a forceps to stabilize the secondary...E-mjournal homepage: www.elsevier.com/locate/bbrepDetection of siRNA-mediated target mRNA cleavage activities in human cells by a novel stem -loop...challenges for the accurate and efficient detection and verification of cleavage sites on target mRNAs. Here we used a sensitive stem -loop array reverse

  10. Proteolytic Inhibition of Salmonella enterica Serovar Typhimurium-Induced Activation of the Mitogen-Activated Protein Kinases ERK and JNK in Cultured Human Intestinal Cells

    PubMed Central

    Mynott, Tracey L.; Crossett, Ben; Prathalingam, S. Radhika

    2002-01-01

    Bromelain, a mixture of cysteine proteases from pineapple stems, blocks signaling by the mitogen-activated protein (MAP) kinases extracellular regulated kinase 1 (ERK-1) and ERK-2, inhibits inflammation, and protects against enterotoxigenic Escherichia coli infection. In this study, we examined the effect of bromelain on Salmonella enterica serovar Typhimurium infection, since an important feature of its pathogenesis is its ability to induce activation of ERK-1 and ERK-2, which leads to internalization of bacteria and induction of inflammatory responses. Our results show that bromelain dose dependently blocks serovar Typhimurium-induced ERK-1, ERK-2, and c-Jun NH2-terminal kinase (JNK) activation in Caco-2 cells. Bromelain also blocked signaling induced by carbachol and anisomycin, pharmacological MAP kinase agonists. Despite bromelain inhibition of serovar Typhimurium-induced MAP kinase signaling, it did not prevent subsequent invasion of the Caco-2 cells by serovar Typhimurium or alter serovar Typhimurium -induced decreases in resistance across Caco-2 monolayers. Surprisingly, bromelain also did not block serovar Typhimurium-induced interleukin-8 (IL-8) secretion but synergized with serovar Typhimurium to enhance IL-8 production. We also found that serovar Typhimurium does not induce ERK phosphorylation in Caco-2 cells in the absence of serum but that serovar Typhimurium-induced invasion and decreases in monolayer resistance are unaffected. Collectively, these data indicate that serovar Typhimurium-induced invasion of Caco-2 cells, changes in the resistance of epithelial cell monolayers, and IL-8 production can occur independently of the ERK and JNK signaling pathways. Data also confirm that bromelain is a novel inhibitor of MAP kinase signaling pathways and suggest a novel role for proteases as inhibitors of signal transduction pathways in intestinal epithelial cells. PMID:11748167

  11. 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.

  12. LncRNA uc.48+ is involved in the diabetic immune and inflammatory responses mediated by P2X7 receptor in RAW264.7 macrophages.

    PubMed

    Wu, Hong; Wen, Fang; Jiang, Mei; Liu, Qiang; Nie, Yijun

    2018-08-01

    High glucose combined with high FFAs can contribute to the unfavorable development of type 2 diabetes mellitus (T2DM) and monocytes/macrophages are important in the occurrence and development of T2DM, which is regarded as a type of low‑grade inflammation. Although our previous study demonstrated that increased expression of P2X7 receptor (P2X7R) in peripheral blood monocytes may alter the innate immune system and that long non‑coding (lnc)RNA uc.48+ was involved in diabetic neuropathic pain, the involvement of uc.48+ mediated by the P2X7R in monocyte/macrophages during T2DM has not been reported. In the present study, the effectsof uc.48+ small interference RNA (siRNA) on factors, including the mRNA and protein expression of P2X7R, apoptosis and proliferation, levels of reactive oxygen species (ROS), cytokine levels, and expression of phosphorylated (p‑) extracellular signal‑regulated kinase (ERK)1/2, were examined in RAW264.7 macrophages following exposure to high glucose and high plasma free fatty acids (FFAs). After RAW264.7 cells were transfected with uc.48+ siRNA under high glucose conditions and FFAs treatment, the mRNA expression levels of uc.48+ and P2X7 receptor were detected by reverse transcription‑polymerase chain reaction. The protein mass of P2X7 receptor and ERK signaling pathway were assessed by western blotting. ROS and calcium concentrations, and culture supernatant cytokine content [tumor necrosis factor‑α, interleukin (IL)‑10, IL‑1β] were detected by fluorescent probes and ELISA respectively. Cell viability and apoptosis were determined by MTS test and flow cytometry, respectively. It was found that treatment of RAW264.7 cells with high glucose and FFAs, which exhibited increased expression of uc.48+, evoked P2X7R‑mediated immune and inflammatory responses through several means, including cytokine secretion, ROS formation, and activation of the ERK signaling pathway. The uc.48+ siRNA regulated these factors and thus

  13. Protective role for ovarian glutathione S-transferase isoform pi during 7,12-dimethylbenz[a]anthracene-induced ovotoxicity

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

    Bhattacharya, Poulomi, E-mail: poulomib@iastate.edu; Keating, Aileen F., E-mail: akeating@iastate.edu

    2012-04-15

    7,12-Dimethylbenz[a]anthracene (DMBA) destroys ovarian follicles at all developmental stages. This study investigated a role for the glutathione S-transferase (Gst) isoforms alpha (a), mu (m) and pi (p) and the transcription factors, Ahr and Nrf2, during DMBA-induced ovotoxicity, and their regulation by phosphatidylinositol-3 kinase (PI3K) signaling. Negative regulation of JNK by GSTP during DMBA exposure was also studied. Post-natal day (PND) 4 Fischer 344 rat ovaries were exposed to vehicle control (1% DMSO) ± DMBA (1 μM) or vehicle control (1% DMSO) ± LY294002 (PI3K inhibitor; 20 μM) for 1, 2, 4, or 6 days. Total RNA or protein was isolated,more » followed by RT-PCR or Western blotting to determine mRNA or protein level, respectively. Immunoprecipitation using an anti-GSTP antibody was performed to determine interaction between GSTP and JNK, followed by Western blotting to determine JNK and p-c-Jun protein level. DMBA had no impact on Gsta, Gstm or Nrf2 mRNA level, but increased Gstp mRNA and protein after 2 days. Ahr mRNA and protein increased after 2 and 4 days of DMBA exposure, respectively and DMBA increased NRF2 protein level after 4 days. JNK bound to GSTP was increased during DMBA exposure, with a concomitant decrease in unbound JNK and p-c-Jun. Ahr and Gstp mRNA were decreased (2 days) and increased (4 days) by PI3K inhibition, while Gstm mRNA increased (P < 0.05) after both time points, and there was no effect on Nrf2 mRNA. PI3K inhibition increased AHR, NRF2 and GSTP protein level. These findings support involvement of ovarian GSTP during DMBA exposure, and indicate a regulatory role for the PI3K signaling pathway on ovarian xenobiotic metabolism gene expression. -- Highlights: ► Ovarian GSTP is activated in response to DMBA exposure. ► AhR and Nrf2 transcription factors are up-regulated by DMBA. ► PI3K signaling regulates Ahr, Nrf2 and Gstp expression. ► GSTP negatively regulates ovarian JNK in response to DMBA exposure.« less

  14. Divergent responses to thermogenic stimuli in BAT and subcutaneous adipose tissue from interleukin 18 and interleukin 18 receptor 1-deficient mice.

    PubMed

    Pazos, Patricia; Lima, Luis; Tovar, Sulay; González-Touceda, David; Diéguez, Carlos; García, María C

    2015-12-10

    Brown and beige adipocytes recruitment in brown (BAT) or white adipose tissue, mainly in the inguinal fat pad (iWAT), meet the need for temperature adaptation in cold-exposure conditions and protect against obesity in face of hypercaloric diets. Using interleukin18 (Il18) and Il18 receptor 1- knockout (Il18r1-KO) mice, this study aimed to investigate the role of IL18 signaling in BAT and iWAT activation and thermogenesis under both stimuli. Il18-KO, extremely dietary obesity-prone as previously described, failed to develop diet-induced thermogenesis as assessed by BAT and iWAT Ucp1 mRNA levels. Overweight when fed standard chow but not HFD, HFD-fed Il18r1-KO mice exhibited increased iWAT Ucp1 gene expression. Energy expenditure was reduced in pre-obese Il18r1-KO mice and restored upon HFD-challenge. Cold exposure lead to similar results; Il18r1-KO mice were protected against acute body temperature drop, displaying a more brown-like structure, alternative macrophage activation and thermogenic gene expression in iWAT than WT controls. Opposite effects were observed in Il18-KO mice. Thus, Il18 and Il18r1 genetic ablation disparate effects on energy homeostasis are likely mediated by divergent BAT responses to thermogenic stimuli as well as iWAT browning. These results suggest that a more complex receptor-signaling system mediates the IL18 adipose-tissue specific effects in energy expenditure.

  15. Blockage of JNK pathway enhances arsenic trioxide-induced apoptosis in human keratinocytes

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

    Huang, H.-S., E-mail: huanghs@mail.ncku.edu.t; Liu, Z.-M.; Hong, D.-Y.

    2010-04-15

    Arsenic is well known as a carcinogen predisposing humans to some severe diseases and also as an effective medicine for treating acute promyelocytic leukemia, syphilis, and psoriasis. Multiple active mechanisms, including cell cycle arrest and apoptosis, have been proposed in therapy; however, the opposing effects of arsenic remain controversial. Our previous study found that arsenic trioxide (ATO)-induced activation of p21{sup WAF1/CIP1} (p21) led to A431 cell death through the antagonistic effects of the signaling of ERK1/2 and JNK1. In the current study, the inhibitory effects of JNK1 on ATO-induced p21 expression were explored. Over-expression of JNK1 in A431 cells couldmore » inhibit p21 expression, which was associated with HDAC1 and TGIF. Using the GST pull-down assay and fluorescence resonance energy transfer analysis, N-terminal domain (amino acids 1-108) of TGIF, critical to its binding with c-Jun, was found. Using reporter assays, requirement of the C-terminal domain (amino acids 138-272) of TGIF to suppress ATO-induced p21 expression was observed. Thus, the domains of TGIF that carried out its inhibitory effects on p21 were identified. Finally, treatment with JNK inhibitor SP600125 could enhance ATO-induced apoptosis of HaCaT keratinocytes by using flow cytometry.« less

  16. Unusual Water-mediated Antigenic Recognition of the Proinflammatory Cytokine Interleukin-18

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

    Argiriadi, Maria A.; Xiang, Tao; Wu, Chengbin

    2009-10-21

    The unique cytokine interleukin-18 (IL-18) acts synergistically with IL-12 to regulate T-helper 1 and 2 lymphocytes and, as such, seems to underlie the pathogenesis of various autoimmune and allergic diseases. Several anti-IL-18 agents are in clinical development, including the recombinant human antibody ABT-325, which is entering trials for autoimmune diseases. Given competing cytokine/receptor and cytokine/receptor decoy interactions, understanding the structural basis for recognition is critical for effective development of anti-cytokine therapies. Here we report three crystal structures: the murine antibody 125-2H Fab fragment bound to human IL-18, at 1.5 {angstrom} resolution; the 125-2H Fab (2.3 {angstrom}); and the ABT-325 Fabmore » (1.5 {angstrom}). These structures, along with human/mouse IL-18 chimera binding data, allow us to make three key observations relevant to the biology and antigenic recognition of IL-18 and related cytokines. First, several IL-18 residues shift dramatically (>10 {angstrom}) upon binding 125-2H, compared with unbound IL-18 (Kato, Z., Jee, J., Shikano, H., Mishima, M., Ohki, I., Ohnishi, H., Li, A., Hashimoto, K., Matsukuma, E., Omoya, K., Yamamoto, Y., Yoneda, T., Hara, T., Kondo, N., and Shirakawa, M. (2003) Nat. Struct. Biol. 10, 966-971). IL-18 thus exhibits plasticity that may be common to its interactions with other receptors. Related cytokines may exhibit similar plasticity. Second, ABT-325 and 125-2H differ significantly in combining site character and architecture, thus explaining their ability to bind IL-18 simultaneously at distinct epitopes. These data allow us to define the likely ABT-325 epitope and thereby explain the distinct neutralizing mechanisms of both antibodies. Third, given the high 125-2H potency, 10 well ordered water molecules are trapped upon complex formation in a cavity between two IL-18 loops and all six 125-2H complementarity-determining regions. Thus, counterintuitively, tight and specific

  17. Effects of interleukins on connective tissue type mast cells co-cultured with fibroblasts.

    PubMed Central

    Levi-Schaffer, F; Segal, V; Shalit, M

    1991-01-01

    We investigated the effects of interleukin-2 (IL-2), interleukin-3 (IL-3) and interleukin-4 (IL-4) on mouse and rat peritoneal mast cells (MC) co-cultured with 3T3 fibroblasts (MC/3T3). The continuous presence of these cytokines for 7-9 days in the culture media was neither toxic nor caused proliferation of MC, as determined by the stability of MC numbers in culture. Long-term incubation of mouse MC/3T3 with IL-2 (100 U/ml), IL-3 (50 U/ml), IL-4 (50 U/ml) or a mixture of IL-3 and IL-4 (25 U/ml) induced an increase in basal histamine release of 79.3 +/- 19.0%, 41.0 +/- 17.3%, 25.2 +/- 10.4% and 30.2 +/- 3.2%, respectively, over control cells incubated with medium alone. When rat MC/3T3 were incubated for 7 days with the various interleukins an enhancement in histamine release similar to that observed with mouse MC/3T3 was found. Preincubation (1 hr) of rat MC/3T3 with interleukins prior to immunological activation with anti-IgE antibodies enhanced histamine release. The highest effect was observed with IL-3 + IL-4 (60.4 +/- 10.8% increase) followed by IL-2 (51.5 +/- 4.5%), IL-4 (28.6 +/- 10.3%) and IL-3 (13.2 +/- 4.2%). This study demonstrates that when mouse and rat peritoneal MC are cultured with fibroblasts in the presence of interleukins they do not proliferate, suggesting that they preserve their connective tissue type MC phenotype. Moreover, interleukins display a pro-inflammatory effect on these cells by enhancing both basal and anti-IgE-mediated histamine release. PMID:2016117

  18. JNK1/2-dependent phosphorylation of angulin-1/LSR is required for the exclusive localization of angulin-1/LSR and tricellulin at tricellular contacts in EpH4 epithelial sheet.

    PubMed

    Nakatsu, Daiki; Kano, Fumi; Taguchi, Yuki; Sugawara, Taichi; Nishizono, Takashi; Nishikawa, Kiyotaka; Oda, Yukako; Furuse, Mikio; Murata, Masayuki

    2014-07-01

    Tricellular tight junctions (tTJs) are specialized structural variants of tight junctions within tricellular contacts of an epithelial sheet and comprise several transmembrane proteins including lipolysis-stimulated lipoprotein receptor (angulin-1/LSR) and tricellulin. To elucidate the mechanism of its formation, we carried out stepwise screening of kinase inhibitors followed by RNAi screening to identify kinases that regulate intracellular localization of angulin-1/LSR to the tTJs using a fluorescence image-based screen. We found that the activity of JNK1 and JNK2, but not JNK3, was required for the exclusive localization of angulin-1/LSR at the tTJs. Based on a bioinformatics approach, we estimated the potential phosphorylation site of angulin-1/LSR by JNK1 to be serine 288 and experimentally confirmed that JNK1 directly phosphorylates angulin-1/LSR at this site. We found that JNK2 was also involved in the phosphorylation of angulin-1/LSR. Furthermore, GFP-tagged angulin-1/LSR(S288A), in which serine 288 was substituted by alanine, was observed to be dispersed to bicellular junctions, indicating that phosphorylation of Ser288 is crucial for the exclusive localization of angulin-1/LSR and tricellulin at tTJs. Our fluorescence image-based screening for kinases inhibitor or siRNAs combined with the phosphorylation site prediction could become a versatile and useful tool to elucidate the mechanisms underlying the maintenance of tTJs regulated by kinase networks. © 2014 The Authors Genes to Cells © 2014 by the Molecular Biology Society of Japan and Wiley Publishing Asia Pty Ltd.

  19. TAB2 Is Essential for Prevention of Apoptosis in Fetal Liver but Not for Interleukin-1 Signaling

    PubMed Central

    Sanjo, Hideki; Takeda, Kiyoshi; Tsujimura, Tohru; Ninomiya-Tsuji, Jun; Matsumoto, Kunihiro; Akira, Shizuo

    2003-01-01

    The proinflammatory cytokine interleukin-1 (IL-1) transmits a signal via several critical cytoplasmic proteins such as MyD88, IRAKs and TRAF6. Recently, serine/threonine kinase TAK1 and TAK1 binding protein 1 and 2 (TAB1/2) have been identified as molecules involved in IL-1-induced TRAF6-mediated activation of AP-1 and NF-κB via mitogen-activated protein (MAP) kinases and IκB kinases, respectively. However, their physiological functions remain to be clarified. To elucidate their roles in vivo, we generated TAB2-deficient mice. The TAB2 deficiency was embryonic lethal due to liver degeneration and apoptosis. This phenotype was similar to that of NF-κB p65-, IKKβ-, and NEMO/IKKγ-deficient mice. However, the IL-1-induced activation of NF-κB and MAP kinases was not impaired in TAB2-deficient embryonic fibroblasts. These findings demonstrate that TAB2 is essential for embryonic development through prevention of liver apoptosis but not for the IL-1 receptor-mediated signaling pathway. PMID:12556483

  20. MKP-7, a JNK phosphatase, blocks ERK-dependent gene activation by anchoring phosphorylated ERK in the cytoplasm

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

    Masuda, Kouhei; Katagiri, Chiaki; Division of Biochemical Oncology and Immunology, Institute for Genetic Medicine, Hokkaido University, Sapporo

    2010-03-05

    MAPK phosphatase-7 (MKP-7) was identified as a JNK-specific phosphatase. However, despite its high specificity for JNK, MKP-7 interacts also with ERK. We previously showed that as a physiological consequence of their interaction, activated ERK phosphorylates MKP-7 at Ser-446, and stabilizing MKP-7. In the present study, we analyzed MKP-7 function in activation of ERK. A time-course experiment showed that both MKP-7 and its phosphatase-dead mutant prolonged mitogen-induced ERK phosphorylation, suggesting that MKP-7 functions as a scaffold for ERK. An important immunohistological finding was that nuclear translocation of phospho-ERK following PMA stimulation was blocked by co-expressed MKP-7 and, moreover, that phospho-ERK co-localizedmore » with MKP-7 in the cytoplasm. Reporter gene analysis indicated that MKP-7 blocks ERK-mediated transcription. Overall, our data indicate that MKP-7 down-regulates ERK-dependent gene expression by blocking nuclear accumulation of phospho-ERK.« less