Science.gov

Sample records for activate mapks ap-1

  1. Transcriptional suppression of IL-27 production by Mycobacterium tuberculosis-activated p38 MAPK via inhibition of AP-1 binding.

    PubMed

    Zhang, Jidong; Qian, Xuesong; Ning, Huan; Eickhoff, Christopher S; Hoft, Daniel F; Liu, Jianguo

    2011-05-15

    Mycobacterium tuberculosis remains a major global challenge to human health care, and the mechanisms of how M. tuberculosis evades host immune surveillance to favor its survival are still largely unknown. In this study, we found that bacillus Calmette-Guérin (BCG) and viable M. tuberculosis as well as M. tuberculosis lysates could activate IL-27 expression in human and mouse macrophages by induction of p28 subunit transcription. However, in parallel with these effects, BCG and M. tuberculosis lysate stimulation of macrophages induced activation of p38 MAPK signaling molecules MLK3/MKK3/MK2 to prevent maximal IL-27 production. M. tuberculosis lysate-induced p28 transcription was dependent on MyD88 signaling pathway. AP-1/c-Fos was shown to bind directly to the p28 promoter and induce p28 expression after M. tuberculosis lysate stimulation. Overexpression of p38α inhibited the binding of c-Fos to the p28 promoter but had no effect on c-Fos protein expression or phosphorylation in response to M. tuberculosis lysate stimulation. Furthermore, blockade of p38 by SB203580 enhanced M. tuberculosis-induced AP-1 binding to the p28 promoter. Importantly, we show that adding exogenous IL-27 to increase the levels produced by PBMCs stimulated with live mycobacteria enhanced the ability of BCG-expanded T cells to inhibit intracellular mycobacterial growth in human macrophages. Taken together, our data demonstrate that mycobacterial stimulation induces both IL-27 production and p38 MAPK activation. Strategies designed to tip the balance toward positive regulation of p28 induction by mycobacteria could lead to enhanced protective tuberculosis immunity. PMID:21482740

  2. The Role of JNK and p38 MAPK Activities in UVA-Induced Signaling Pathways Leading to AP-1 Activation and c-Fos Expression1

    PubMed Central

    Silvers, Amy L; Bachelor, Michael A; Bowden, G Timothy

    2003-01-01

    Abstract To further delineate ultraviolet A (UVA) signaling pathways in the human keratinocyte cell line HaCaT, we examined the potential role of mitogen-activated protein kinases (MAPKs) in UVA-induced activator protein-1 (AP-1) transactivation and c-Fos expression. UVA-induced phosphorylation of p38 and c-Jun N-terminal kinase (JNK) proteins was detected immediately after irradiation and disappeared after approximately 2 hours. Conversely, phosphorylation of extracellular signal-regulated kinase was significantly inhibited for up to 1 hour post-UVA irradiation. To examine the role of p38 and JNK MAPKs in UVA-induced AP-1 and c-fos transactivations, the selective pharmacologic MAPK inhibitors, SB202190 (p38 inhibitor) and SP600125 (JNK inhibitor), were used to independently treat stably transfected HaCaT cells in luciferase reporter assays. Both SB202190 and SP600125 dose-dependently inhibited UVA-induced AP-1 and c-fos transactivations. SB202190 (0.25–0.5 µM) and SP600125 (62–125 nM) treatments also primarily inhibited UVA-induced c-Fos expression. These results demonstrated that activation of both JNK and p38 play critical role in UVA-mediated AP-1 transactivation and c-Fos expression in these human keratinocyte cells. Targeted inhibition of these MAPKs with their selective pharmacologic inhibitors may be effective chemopreventive strategies for UVA-induced nonmelanoma skin cancer. PMID:14511403

  3. Tiron Inhibits UVB-Induced AP-1 Binding Sites Transcriptional Activation on MMP-1 and MMP-3 Promoters by MAPK Signaling Pathway in Human Dermal Fibroblasts

    PubMed Central

    Zhang, Chao; Zhao, Mei; Zhang, Quan-Wu; Gao, Feng-Hou

    2016-01-01

    Recent research found that Tiron was an effective antioxidant that could act as the intracellular reactive oxygen species (ROS) scavenger or alleviate the acute toxic metal overload in vivo. In this study, we investigated the inhibitory effect of Tiron on matrix metalloproteinase (MMP)-1 and MMP-3 expression in human dermal fibroblast cells. Western blot and ELISA analysis revealed that Tiron inhibited ultraviolet B (UVB)-induced protein expression of MMP-1 and MMP-3. Real-time quantitative PCR confirmed that Tiron could inhibit UVB-induced mRNA expression of MMP-1 and MMP-3. Furthermore, Tiron significantly blocked UVB-induced activation of the MAPK signaling pathway and activator protein (AP)-1 in the downstream of this transduction pathway in fibroblasts. Through the AP-1 binding site mutation, it was found that Tiron could inhibit AP-1-induced upregulation of MMP-1 and MMP-3 expression through blocking AP-1 binding to the AP-1 binding sites in the MMP-1 and MMP-3 promoter region. In conclusion, Tiron may be a novel antioxidant for preventing and treating skin photoaging UV-induced. PMID:27486852

  4. Silibinin suppresses PMA-induced MMP-9 expression by blocking the AP-1 activation via MAPK signaling pathways in MCF-7 human breast carcinoma cells.

    PubMed

    Lee, Syng-Ook; Jeong, Yun-Jeong; Im, Hyo Gwon; Kim, Cheorl-Ho; Chang, Young-Chae; Lee, In-Seon

    2007-03-01

    Matrix metalloproteinase-9 (MMP-9) plays an important role in the invasion and metastasis of cancer cells. In this study, we examined the inhibitory effect of silibinin, a flavonoid antioxidant from milk thistle (Silybum marianum L.) on PMA-induced MMP-9 expression in MCF-7 human breast carcinoma cells. Silibinin significantly and selectively suppressed PMA-induced MMP-9 expression in MCF-7. Silibinin has been found to inhibit PMA-induced MMP-9 gene transcriptional activity by blocking the activation of AP-1 via MAPK signaling pathways. Moreover, the Matrigel invasion assay showed that silibinin reduces PMA-induced invasion of MCF-7 cells. These results suggest that silibinin represents a potential anti-metastatic agent suppressing PMA-induced cancer cell invasion through the specific inhibition of AP-1-dependent MMP-9 gene expression. PMID:17214970

  5. Citrus bergamia Juice Extract Attenuates β-Amyloid-Induced Pro-Inflammatory Activation of THP-1 Cells Through MAPK and AP-1 Pathways

    PubMed Central

    Currò, Monica; Risitano, Roberto; Ferlazzo, Nadia; Cirmi, Santa; Gangemi, Chiara; Caccamo, Daniela; Ientile, Riccardo; Navarra, Michele

    2016-01-01

    Flavonoids have been shown to be effective in protecting against age-related cognitive and motor decline in both in vitro and in vivo models. Recently, a flavonoid-rich extract of Citrus bergamia juice (BJe) has been shown to display anti-oxidant and anti-inflammatory properties against LPS-induced activation of human THP-1 monocytes. In the light of these observations, we wondered whether BJe may be beneficial against neuroinflammatory processes, such as those observed in Alzheimer’s disease. To this aim we used THP-1 monocytes to investigate the mechanisms underlying the beneficial potential of BJe against amyloid-beta1–42 (Aβ1−42) -mediated inflammation. Exposure of THP-1 cells to Aβ1−42 significantly induced the expression and secretion of IL-6 and IL-1β in THP-1 cells and increased the phosphorylation of ERK 1/2 as well as p46 and p54 members of JNK family. Moreover, Aβ1−42 raises AP-1 DNA binding activity in THP-1-treated cells. Interestingly, all these effects were reduced in the presence of BJe. Our data indicate that BJe may effectively counteract the pro-inflammatory activation of monocytes/microglial cells exposed to amyloid fibrils, suggesting a promising role as a natural drug against neuroinflammatory processes. PMID:26853104

  6. Citrus bergamia Juice Extract Attenuates β-Amyloid-Induced Pro-Inflammatory Activation of THP-1 Cells Through MAPK and AP-1 Pathways.

    PubMed

    Currò, Monica; Risitano, Roberto; Ferlazzo, Nadia; Cirmi, Santa; Gangemi, Chiara; Caccamo, Daniela; Ientile, Riccardo; Navarra, Michele

    2016-01-01

    Flavonoids have been shown to be effective in protecting against age-related cognitive and motor decline in both in vitro and in vivo models. Recently, a flavonoid-rich extract of Citrus bergamia juice (BJe) has been shown to display anti-oxidant and anti-inflammatory properties against LPS-induced activation of human THP-1 monocytes. In the light of these observations, we wondered whether BJe may be beneficial against neuroinflammatory processes, such as those observed in Alzheimer's disease. To this aim we used THP-1 monocytes to investigate the mechanisms underlying the beneficial potential of BJe against amyloid-beta1-42 (Aβ1-42) -mediated inflammation. Exposure of THP-1 cells to Aβ1-42 significantly induced the expression and secretion of IL-6 and IL-1β in THP-1 cells and increased the phosphorylation of ERK 1/2 as well as p46 and p54 members of JNK family. Moreover, Aβ1-42 raises AP-1 DNA binding activity in THP-1-treated cells. Interestingly, all these effects were reduced in the presence of BJe. Our data indicate that BJe may effectively counteract the pro-inflammatory activation of monocytes/microglial cells exposed to amyloid fibrils, suggesting a promising role as a natural drug against neuroinflammatory processes. PMID:26853104

  7. Luteolin, a flavonoid, inhibits AP-1 activation by basophils

    SciTech Connect

    Hirano, Toru; Higa, Shinji; Arimitsu, Junsuke; Naka, Tetsuji; Ogata, Atsushi; Shima, Yoshihito; Fujimoto, Minoru; Yamadori, Tomoki; Ohkawara, Tomoharu; Kuwabara, Yusuke; Kawai, Mari; Matsuda, Hisashi; Yoshikawa, Masayuki; Maezaki, Naoyoshi; Tanaka, Tetsuaki; Kawase, Ichiro; Tanaka, Toshio . E-mail: ttanak@imed3.med.osaka-u.ac.jp

    2006-02-03

    Flavonoids including luteolin, apigenin, and fisetin are inhibitors of IL-4 synthesis and CD40 ligand expression by basophils. This study was done to search for compounds with greater inhibitory activity of IL-4 expression and to clarify the molecular mechanisms through which flavonoids inhibit their expression. Of the 37 flavonoids and related compounds examined, ayanin, luteolin, and apigenin were the strongest inhibitors of IL-4 production by purified basophils in response to anti-IgE antibody plus IL-3. Luteolin did not suppress Syk or Lyn phosphorylation in basophils, nor did suppress p54/46 SAPK/JNK, p38 MAPK, and p44/42 MAPK activation by a basophilic cell line, KU812 cells, stimulated with A23187 and PMA. However, luteolin did inhibit phosphorylation of c-Jun and DNA binding activity of AP-1 in nuclear lysates from stimulated KU812 cells. These results provide a fundamental structure of flavonoids for IL-4 inhibition and demonstrate a novel action of flavonoids that suppresses the activation of AP-1.

  8. MAPK/AP-1 signal pathway in tobacco smoke-induced cell proliferation and squamous metaplasia in the lungs of rats.

    PubMed

    Zhong, Cai-Yun; Zhou, Ya-Mei; Douglas, Gordon C; Witschi, Hanspeter; Pinkerton, Kent E

    2005-12-01

    Overwhelming evidence has demonstrated tobacco smoke (TS) is causally associated with various types of cancers, especially lung cancer. Sustained epithelial cell hyperplasia and squamous metaplasia are considered as preneoplastic lesions during the formation of lung cancer. The cellular and molecular mechanisms leading to lung cancer due to TS are not clear. Mitogen-activated protein kinases (MAPK)/activator protein-1 (AP-1) can be activated by various stimuli and play a critical role in the control of cell proliferation and differentiation. To date, information on the response of the MAPK/AP-1 pathway during hyperplasia and squamous metaplasia induced by TS is lacking. We therefore investigated the effects of TS on the development of epithelial hyperplasia and squamous metaplasia, regulation of MAPK/AP-1 activation, and expression of AP-1-regulated cell cycle proteins and differentiation markers in the lungs of rats. Exposure of rats to TS (30 mg/m(3) or 80 mg/m(3), 6 h/day, 3 days/week for 14 weeks) dramatically induced cell proliferation and squamous metaplasia in a dose-dependent manner, effects that paralleled the activation of AP-1-DNA binding activity. Phosphorylated ERK1/2, JNK, p38 and ERK5 were significantly increased by exposure to TS, indicating the activation of these MAPK pathways. Expression of Jun and Fos proteins were differentially regulated by TS. TS upregulated the expression of AP-1-dependent cell cycle proteins including cyclin D1 and proliferating cell nuclear antigen (PCNA). Among the AP-1-dependent cell differentiation markers, keratin 5 and 14 were upregulated, while loricrin, filaggrin and involucrin were downregulated following TS exposure. These findings suggest the important role of MAPK/AP-1 pathway in TS-induced pathogenesis, thus providing new insights into the molecular mechanisms of TS-associated lung diseases including lung cancers. PMID:16051644

  9. Small Molecule Inhibitors Targeting Activator Protein 1 (AP-1)

    PubMed Central

    2015-01-01

    Activator protein 1 (AP-1) is a pivotal transcription factor that regulates a wide range of cellular processes including proliferation, apoptosis, differentiation, survival, cell migration, and transformation. Accumulating evidence supports that AP-1 plays an important role in several severe disorders including cancer, fibrosis, and organ injury, as well as inflammatory disorders such as asthma, psoriasis, and rheumatoid arthritis. AP-1 has emerged as an actively pursued drug discovery target over the past decade. Excitingly, a selective AP-1 inhibitor T-5224 (51) has been investigated in phase II human clinical trials. Nevertheless, no effective AP-1 inhibitors have yet been approved for clinical use. Despite significant advances achieved in understanding AP-1 biology and function, as well as the identification of small molecules modulating AP-1 associated signaling pathways, medicinal chemistry efforts remain an urgent need to yield selective and efficacious AP-1 inhibitors as a viable therapeutic strategy for human diseases. PMID:24831826

  10. Macrophage-derived BAFF induces AID expression through the p38MAPK/CREB and JNK/AP-1 pathways.

    PubMed

    Kim, Hyun-A; Seo, Goo-Young; Kim, Pyeung-Hyeun

    2011-03-01

    BAFF is expressed primarily by macrophages and DCs. BAFF stimulates the differentiation and survival of B cells and induces Ig production. We have demonstrated previously that murine macrophages treated with TGF-β1 or IFN-γ express membrane-bound and soluble forms of BAFF. The ability of these two forms of BAFF to induce expression of AID, which plays a critical role in Ig CSR in B cells, was investigated. Both forms of BAFF, derived from macrophages activated by IFN-γ or TGF-β1, can increase AID expression. Subsequent analysis of BAFF signaling suggested that BAFF induces AID through BCMA, a BAFF-receptor, and p38MAPK and CREB act as intermediates in AID expression. In addition, JNK and AP-1 have similar activities. Our findings suggest that macrophage-derived BAFF stimulates B cells to express AID through BCMA and at least two different pathways, including the p38MAPK/CREB and the JNK/AP-1 pathways. PMID:21169521

  11. Activation of transcription factor AP-1 and mitogen-activated protein kinases in aniline-induced splenic toxicity

    SciTech Connect

    Khan, M. Firoze . E-mail: mfkhan@utmb.edu; Kannan, Subburaj; Wang Jianling

    2006-01-15

    Signaling mechanisms in aniline-induced fibrogenic and/or tumorigenic response in the spleen are not known. Previous studies have shown that aniline exposure leads to iron accumulation and oxidative stress in the spleen, which may cause activation of redox-sensitive transcription factors and regulate the transcription of genes involved in fibrosis and/or tumorigenesis. To test this, male SD rats were treated with 0.5 mmol/kg/day aniline via drinking water for 30 days, and activation of transcription factor AP-1 was determined in the splenocyte nuclear extracts (NEs). AP-1 DNA-binding activity in the NEs of freshly isolated splenocytes from aniline-treated rats increased in comparison to the controls, as determined by electrophoretic mobility shift assay (EMSA). AP-1 binding was also determined in the NEs of cultured splenocytes (2 h and 24 h), which showed even a greater increase in binding activity at 2 h. The specificity of AP-1 binding for relevant DNA motifs was confirmed by competition EMSA and by supershift EMSA using antibodies specific to c-Jun and c-Fos. To further explore the signaling mechanisms in the AP-1 activation, phosphorylation patterns of mitogen-activated protein kinases (MAPKs) were pursued. Aniline exposure induced increases in the phosphorylation of the three classes of MAPKs: extracellular-signal-regulated kinase (ERK 1/2), c-Jun N-terminal kinase (JNK 1/2), and p38 MAPKs. Furthermore, TGF-{beta}1 mRNA expression showed a 3-fold increase in the spleens of aniline-treated rats. These observations suggest a strong association among MAPK phosphorylation, AP-1 activation, and enhanced TGF-{beta}1 gene expression. The observed sequence of events subsequent to aniline exposure could regulate genes that lead to fibrogenic and/or tumorigenic response in the spleen.

  12. ACTIVATION OF AP-1 IN UROTSA CELLS BY METHYLATED ARSENICALS

    EPA Science Inventory

    ACTIVATION OF AP-1 IN UROTSA CELLS BY METHYLATED TRIVALENT ARSENICALS. Z Drobna1, I Jaspers2, D J Thomas3 and M Styblo1. 1Department of Pediatrics; 2Center for Environmental Medicine and Lung Biology, University of North Carolina at Chapel Hill, NC, USA; 3US EPA, RTP, NC, USA.

  13. AP-1-Targeting Anti-Inflammatory Activity of the Methanolic Extract of Persicaria chinensis

    PubMed Central

    Son, Young-Jin; Baek, Kwang-Soo; Yang, Woo Seok; Park, Jae Gwang; Kim, Han Gyung; Chung, Woo-Jae; Yoon, Keejung; Lee, Sang Yeol; Kim, Jong-Hoon

    2015-01-01

    In traditional Chinese medicine, Persicaria chinensis L. has been prescribed to cure numerous inflammatory disorders. We previously analyzed the bioactivity of the methanol extract of this plant (Pc-ME) against LPS-induced NO and PGE2 in RAW264.7 macrophages and found that it prevented HCl/EtOH-induced gastric ulcers in mice. The purpose of the current study was to explore the molecular mechanism by which Pc-ME inhibits activator protein- (AP-) 1 activation pathway and mediates its hepatoprotective activity. To investigate the putative therapeutic properties of Pc-ME against AP-1-mediated inflammation and hepatotoxicity, lipopolysaccharide- (LPS-) stimulated RAW264.7 and U937 cells, a monocyte-like human cell line, and an LPS/D-galactosamine- (D-GalN-) induced acute hepatitis mouse model were employed. The expression of LPS-induced proinflammatory cytokines including interleukin- (IL-) 1β, IL-6, and tumor necrosis factor-α (TNF-α) was significantly diminished by Pc-ME. Moreover, Pc-ME reduced AP-1 activation and mitogen-activated protein kinase (MAPK) phosphorylation in both LPS-stimulated RAW264.7 cells and differentiated U937 cells. Additionally, we highlighted the hepatoprotective and curative effects of Pc-ME pretreated orally in a mouse model of LPS/D-GalN-intoxicated acute liver injury by demonstrating the significant reduction in elevated serum AST and ALT levels and histological damage. Therefore, these results strongly suggest that Pc-ME could function as an antihepatitis remedy suppressing MAPK/AP-1-mediated inflammatory events. PMID:25878717

  14. Thrombin induces endothelial arginase through AP-1 activation.

    PubMed

    Zhu, Weifei; Chandrasekharan, Unni M; Bandyopadhyay, Smarajit; Morris, Sidney M; DiCorleto, Paul E; Kashyap, Vikram S

    2010-04-01

    Arterial thrombosis is a common disease leading to severe ischemia beyond the obstructing thrombus. Additionally, endothelial dysfunction at the site of thrombosis can be rescued by l-arginine supplementation or arginase blockade in several animal models. Exposure of rat aortic endothelial cells (RAECs) to thrombin upregulates arginase I mRNA and protein levels. In this study, we further investigated the molecular mechanism of thrombin-induced arginase changes in endothelial cells. Thrombin strikingly increased arginase I promoter and enzyme activity in primary cultured RAECs. Using different deletion and point mutations of the promoter, we demonstrated that the activating protein-1 (AP-1) consensus site located at -3,157 bp in the arginase I promoter was a thrombin-responsive element. Electrophoretic mobility shift assay and chromatin immunoprecipitation assay further confirmed that upon thrombin stimulation, c-Jun and activating transcription factor-2 (ATF-2) bound to the AP-1 site, which initiated the transactivation. Moreover, loss-of-function studies using small interfering RNA confirmed that recruitment of these two transcription factors to the AP-1 site was required for thrombin-induced arginase upregulation. In the course of defining the signaling pathway leading to the activation of AP-1 by thrombin, we found thrombin-induced phosphorylation of stress-activated protein kinase/c-Jun-NH(2)-terminal kinase (SAPK/JNK or JNK1/2/3) and p38 mitogen-activated protein kinase, which were followed by the phosphorylation of both c-Jun and ATF-2. These findings reveal the basis for thrombin induction of endothelial arginase I and indicate that arginase inhibition may be an attractive therapeutic alternative in the setting of arterial thrombosis and its associated endothelial dysfunction. PMID:20032511

  15. Geniposide suppresses LPS-induced nitric oxide, PGE2 and inflammatory cytokine by downregulating NF-κB, MAPK and AP-1 signaling pathways in macrophages.

    PubMed

    Shi, Qinghai; Cao, Jinjun; Fang, Li; Zhao, Hongyan; Liu, Zhengxiang; Ran, Jihua; Zheng, Xinchuan; Li, Xiaoling; Zhou, Yu; Ge, Di; Zhang, Hongming; Wang, Li; Ran, Ying; Fu, Jianfeng

    2014-06-01

    Inflammatory responses are important to host immune reactions, but uncontrolled inflammatory mediators may aid in the pathogenesis of other inflammatory diseases. Geniposide, an iridoid glycoside found in the herb gardenia, is believed to have broad-spectrum anti-inflammatory effects in murine models but its mechanism of action is unclear. We investigated the action of this compound in murine macrophages stimulated by lipopolysaccharide (LPS), as the stimulation of macrophages by LPS is known to induce inflammatory reactions. We determined the effect of geniposide on LPS-induced production of the inflammatory mediators, nitric oxide (NO) and prostaglandin E2 (PGE2), the mRNA and protein expression of the NO and PGE2 synthases, inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), respectively, and the mRNA and protein expression of the inflammatory cytokine, tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6). Furthermore, nuclear factor (NF)-κB, mitogen-activated protein kinase (MAPK) and activator protein (AP)-1 activity were assayed. To understand the action of geniposide on the NF-κB and MAPK pathways, we studied the effect of NF-κB and MAPK inhibitors on the LPS-induced production of NO, PGE2 and TNF-α. Our findings clearly showed that geniposide mainly exerts its anti-inflammatory effects by inhibiting the LPS-induced NF-κB, MAPK and AP-1 signaling pathways in macrophages, which subsequently reduces overexpression of the inducible enzymes iNOS and COX-2 and suppresses the expression and release of the inflammatory factors, TNF-α, IL-6, NO and PGE2. Thus, geniposide shows promise as a therapeutic agent in inflammatory diseases. PMID:24735815

  16. NF-κB and AP-1 Connection: Mechanism of NF-κB-Dependent Regulation of AP-1 Activity

    PubMed Central

    Fujioka, Shuichi; Niu, Jiangong; Schmidt, Christian; Sclabas, Guido M.; Peng, Bailu; Uwagawa, Tadashi; Li, Zhongkui; Evans, Douglas B.; Abbruzzese, James L.; Chiao, Paul J.

    2004-01-01

    Nuclear factor κB (NF-κB) and activator protein 1 (AP-1) transcription factors regulate many important biological and pathological processes. Activation of NF-κB is regulated by the inducible phosphorylation of NF-κB inhibitor IκB by IκB kinase. In contrast, Fos, a key component of AP-1, is primarily transcriptionally regulated by serum responsive factors (SRFs) and ternary complex factors (TCFs). Despite these different regulatory mechanisms, there is an intriguing possibility that NF-κB and AP-1 may modulate each other, thus expanding the scope of these two rapidly inducible transcription factors. To determine whether NF-κB activity is involved in the regulation of fos expression in response to various stimuli, we analyzed activity of AP-1 and expression of fos, fosB, fra-1, fra-2, jun, junB, and junD, as well as AP-1 downstream target gene VEGF, using MDAPanc-28 and MDAPanc-28/IκBαM pancreatic tumor cells and wild-type, IKK1−/−, and IKK2−/− murine embryonic fibroblast cells. Our results show that elk-1, a member of TCFs, is one of the NF-κB downstream target genes. Inhibition of NF-κB activity greatly decreased expression of elk-1. Consequently, the reduced level of activated Elk-1 protein by extracellular signal-regulated kinase impeded constitutive, serum-, and superoxide-inducible c-fos expression. Thus, our study revealed a distinct and essential role of NF-κB in participating in the regulation of elk-1, c-fos, and VEGF expression. PMID:15314185

  17. Retinoic acid-induced AP-1 transcriptional activity regulates B16 mouse melanoma growth inhibition and differentiation.

    PubMed

    Huang, Ying; Boskovic, Goran; Niles, Richard M

    2003-02-01

    Retinoic acid (RA) inhibits growth and induces differentiation of B16 mouse melanoma cells. These effects are accompanied by a large increase in PKCalpha mRNA and protein levels and surprisingly an increase in activating protein-1 (AP-1) transcriptional activity. To further investigate the RA-induced AP-1 activity we established clones of B16 cells stably expressing an AP-1-luciferase reporter gene. Treatment of these clones with phorbol dibutyrate increased AP-1 activity which peaked at 2-4 h and returned to baseline level by 24 h. In contrast, RA treatment resulted in a slow increase in AP-1 activity that reached a maximum level at 48 h and was maintained for the duration of the treatment. We tested the importance of the RA-induced AP-1 activity by establishing clones which stably express a dominant negative fos gene (A-fos) and have greatly diminished AP-1 activity. Growth rates of untreated A-fos expressing cells were similar to wt B16 and clones not expressing A-fos. However, clones expressing the dominant-negative fos had a markedly decreased sensitivity to RA-induced inhibition of anchorage-dependent and -independent growth. Treatment of wt B16 cells for 48 h with RA increased melanin production by two to fourfold, but this effect was completely lost in the A-fos clones. The ability of RA to induce RARbeta and PKCalpha expression was retained in A-fos clones, suggesting that A-fos was not interfering with RAR transcription activation functions. We tested whether the RA-induced AP-1 activity might be mediated by the ERK1/2 MAPK pathway. Inhibition of ERK1/2 phosphorylation stimulated AP-1 activity, which was not additive to that induced by RA. This finding raises the possibility that this MAPK pathway may be a target of retinoid action. Our observations suggest that AP-1 transcriptional activity induced by RA likely plays an important role in the biological changes mediated by this retinoid in B16 melanoma cells. PMID:12494454

  18. Human TMEM174 that is highly expressed in kidney tissue activates AP-1 and promotes cell proliferation

    SciTech Connect

    Wang, Pingzhang; Sun, Bo; Hao, Dongxia; Zhang, Xiujun; Shi, Taiping; Ma, Dalong

    2010-04-16

    Mitogen-activated protein kinase (MAPK) cascades play an important role in regulation of AP-1 activity through the phosphorylation of distinct substrates. In the present study, we identified a novel protein, TMEM174, whose RNA transcripts are highly expressed in human kidney tissue. TMEM174 is comprised of 243 amino acids, and contains two predicted transmembrane helices which determine its subcellular localization in endoplasmic reticulum and influences its functions. Over-expression of TMME174 enhanced the transcriptional activity of AP-1 and promoted cell proliferation, whereas the truncated mutant TMEM174{Delta}TM without the transmembrane regions did not retain these functions. The possible mechanism of activation of AP-1 by TMEM174 was further examined. Our results suggest the potential role of TMEM174 in renal development and physiological function.

  19. Youngiasides A and C Isolated from Youngia denticulatum Inhibit UVB-Induced MMP Expression and Promote Type I Procollagen Production via Repression of MAPK/AP-1/NF-κB and Activation of AMPK/Nrf2 in HaCaT Cells and Human Dermal Fibroblasts.

    PubMed

    Kim, Myungsuk; Park, Young Gyun; Lee, Hee-Ju; Lim, Sue Ji; Nho, Chu Won

    2015-06-10

    This study investigated the effects of youngiaside A (YA), youngiaside C (YC), and Youngia denticulatum extract (YDE) on extrinsic aging and assessed its molecular mechanisms in UVB-irradiated HaCaT keratinocytes and human dermal fibroblasts (HDFs). The results showed that YA, YC, and YDE decreased matrix metalloproteinase (MMP) expression and production in HaCaT cell and HDFs and increased collagen expression and production in HDFs. In addition, YA, YC, and YDE significantly increased antioxidant enzyme expression, thereby down-regulating UVB-induced reactive oxygen species (ROS) production and ROS-induced mitogen-activated protein kinase (MAPK) and activator protein-1 (AP-1) signaling in HaCaT cells. Furthermore, YA, YC, and YDE reduced phosphorylation of IκBα and IKKα/β, blocked nuclear factor-κB (NF-κB) p65 nuclear translocation, and strongly suppressed pro-inflammatory mediators. Finally, YA, YC, and YDE augmented UVB-induced adenosine monophosphate activated protein kinase (AMPK) phosphorylation and YA and YC did not inhibit MMP-1 production in AMPK inhibitor or nuclear factor-erythroid 2-related factor-2 (Nrf2) siRNA-treated HaCaT cells. The results suggest that these compounds could be potential therapeutic agents for prevention and treatment of skin photoaging. PMID:25994852

  20. AP-1 activity during normal human keratinocyte differentiation: evidence for a cytosolic modulator of AP-1/DNA binding.

    PubMed

    Briata, P; D'Anna, F; Franzi, A T; Gherzi, R

    1993-01-01

    Increased levels of c-fos and c-jun expression have been observed in differentiating epithelial cells. However, no data are available on activator protein 1 (AP-1) activity during keratinocyte differentiation. In this work we investigated c-fos and c-jun gene expression and AP-1-(12-O-tetradecanoylphorbol-13-acetate)-responsive enhancer element (TRE) binding activity during keratinocyte differentiation utilizing both authentic and in culture-reconstituted human epidermis. We demonstrate that: (i) in reconstituted epidermis, non-differentiated and differentiated keratinocytes express equivalent levels of c-Jun, while in reconstituted epidermis permanently grafted onto athymic mice, as well as in authentic epidermis, c-Jun is predominantly expressed in the granular layer of the tissue. Equivalent levels of c-fos expression have been found in all the layers of both reconstituted and authentic epidermis. (ii) Nuclear extracts from cultures enriched in differentiated keratinocytes display an 80-90% reduction of AP-1 activity when compared to extracts from cultures enriched in nondifferentiated cells. (iii) Cytosolic extracts obtained from cultures enriched in differentiated cells reduce, in a concentration-dependent manner, the AP-1 activity present in nuclear extracts of both mammalian and Drosophila cells. (iv) The specific TRE binding activity of a recombinant c-Jun protein is significantly reduced by cytosolic extracts of differentiated keratinocytes, while the specific DNA binding of the purified recombinant human homeoprotein HOX4B is not. (v) The dephosphorylation, by alkaline phosphatase, of cytosolic extracts increases the inhibitory activity already present or makes evident a latent activity. PMID:8416791

  1. The AP-1 transcription factor homolog Pf-AP-1 activates transcription of multiple biomineral proteins and potentially participates in Pinctada fucata biomineralization

    PubMed Central

    Zheng, Xiangnan; Cheng, Minzhang; Xiang, Liang; Liang, Jian; Xie, Liping; Zhang, Rongqing

    2015-01-01

    Activator protein-1 (AP-1) is an important bZIP transcription factor that regulates a series of physiological processes by specifically activating transcription of several genes, and one of its well-chartered functions in mammals is participating in bone mineralization. We isolated and cloned the complete cDNA of a Jun/AP-1 homolog from Pinctada fucata and called it Pf-AP-1. Pf-AP-1 had a highly conserved bZIP region and phosphorylation sites compared with those from mammals. A tissue distribution analysis showed that Pf-AP-1 was ubiquitously expressed in P. fucata and the mRNA level of Pf-AP-1 is extremely high in mantle. Pf-AP-1 expression was positively associated with multiple biomineral proteins in the mantle. The luciferase reporter assay in a mammalian cell line showed that Pf-AP-1 significantly up-regulates the transcriptional activity of the promoters of KRMP, Pearlin, and Prisilkin39. Inhibiting the activity of Pf-AP-1 depressed the expression of multiple matrix proteins. Pf-AP-1 showed a unique expression pattern during shell regeneration and pearl sac development, which was similar to the pattern observed for biomineral proteins. These results suggest that the Pf-AP-1 AP-1 homolog is an important transcription factor that regulates transcription of several biomineral proteins simultaneously and plays a role in P. fucata biomineralization, particularly during pearl and shell formation. PMID:26404494

  2. Japanese encephalitis virus induces matrix metalloproteinase-9 expression via a ROS/c-Src/PDGFR/PI3K/Akt/MAPKs-dependent AP-1 pathway in rat brain astrocytes

    PubMed Central

    2012-01-01

    Background Japanese encephalitis virus (JEV) infection is a major cause of acute encephalopathy in children, which destroys central nervous system (CNS) cells, including astrocytes and neurons. Matrix metalloproteinase (MMP)-9 has been shown to degrade components of the basal lamina, leading to disruption of the blood-brain barrier (BBB) and to contribute to neuroinflammatory responses in many neurological diseases. However, the detailed mechanisms of JEV-induced MMP-9 expression in rat brain astrocytes (RBA-1 cells) are largely unclear. Methods In this study, the effect of JEV on expression of MMP-9 was determined by gelatin zymography, western blot analysis, RT-PCR, and promoter assay. The involvement of AP-1 (c-Jun and c-Fos), c-Src, PDGFR, PI3K/Akt, and MAPKs in these responses were investigated by using the selective pharmacological inhibitors and transfection with siRNAs. Results Here, we demonstrate that JEV induces expression of pro-form MMP-9 via ROS/c-Src/PDGFR/PI3K/Akt/MAPKs-dependent, AP-1 activation in RBA-1 cells. JEV-induced MMP-9 expression and promoter activity were inhibited by pretreatment with inhibitors of AP-1 (tanshinone), c-Src (PP1), PDGFR (AG1296), and PI3K (LY294002), and by transfection with siRNAs of c-Jun, c-Fos, PDGFR, and Akt. Moreover, JEV-stimulated AP-1 activation was inhibited by pretreatment with the inhibitors of c-Src, PDGFR, PI3K, and MAPKs. Conclusion From these results, we conclude that JEV activates the ROS/c-Src/PDGFR/PI3K/Akt/MAPKs pathway, which in turn triggers AP-1 activation and ultimately induces MMP-9 expression in RBA-1 cells. These findings concerning JEV-induced MMP-9 expression in RBA-1 cells imply that JEV might play an important role in CNS inflammation and diseases. PMID:22251375

  3. Guanylyl cyclase/natriuretic peptide receptor-A signaling antagonizes the vascular endothelial growth factor-stimulated MAPKs and downstream effectors AP-1 and CREB in mouse mesangial cells

    PubMed Central

    Tripathi, Satyabha; Pandey, Kailash N.

    2012-01-01

    Along with its natriuretic, diuretic, and vasodilatory properties, atrial natriuretic peptide (ANP) and its guanylyl cyclase/natriuretic peptide receptor-A (GC-A/NPRA) exhibit an inhibitory effect on cell growth and proliferation. However, the signaling pathways mediating this inhibition are not well understood. The objective of this study was to determine the effect of ANP-NPRA system on mitogen-activated protein kinases (MAPKs) and the downstream proliferative transcription factors involving activating protein-1 (AP-1) and cAMP-response element binding protein (CREB) in agonist-stimulated mouse mesangial cells (MMCs). We found that ANP inhibited vascular endothelial growth factor (VEGF)-stimulated phosphorylation of MAPKs (Erk1, Erk2, JNK, and p38), to a greater extent in NPRA-transfected cells (50–60%) relative to vector-transfected cells (25–30%). The analyses of the phosphorylated transcription factors revealed that ANP inhibited VEGF-stimulated activation of CREB, and the AP-1 subunits (c-jun and c-fos). Gel shift assays demonstrated that ANP inhibited VEGF-stimulated AP-1 and CREB DNA-binding ability by 67 % and 62 %, respectively. The addition of the protein kinase G (PKG) inhibitor, KT-5823, restored the VEGF-stimulated activation of MAPKs, AP-1, and CREB, demonstrating the integral role of cGMP/PKG signaling in NPRA-mediated effects. Our results delineate the under lying mechanisms through which ANP-NPRA system exerts an inhibitory effect on MAPKs and down-stream effector molecules, AP-1 and CREB, critical for cell growth and proliferation. PMID:22610792

  4. Hepatopoietin interacts directly with COP9 signalosome and regulates AP-1 activity.

    PubMed

    Wang, Yan; Lu, Chengrong; Wei, Handong; Wang, Na; Chen, Xiaoxiao; Zhang, Lingqiang; Zhai, Yun; Zhu, Yunping; Lu, Yinglin; He, Fuchu

    2004-08-13

    Hepatopoietin (HPO)/augmenter of liver regeneration (ALR) is a specific hepatotrophic growth factor, which plays a key role in liver regeneration. Our previous study indicated that HPO executes its function by an inter-reactive network of the autocrine, paracrine and endocrine pathways. Recently, we have demonstrated that intracellular HPO interacts with Jun activation domain-binding protein 1 (JAB1) and leads to potentiation of activating protein-1 (AP-1) activity in a MAPK independent fashion. JAB1 is the fifth subunit of the COP9 signalosome (CSN), which is first identified as a suppressor of plant morphogenesis. A protein complex kinase activity associated with the CSN has been reported but not identified yet. In this report, we investigated further the association of HPO with the whole CSN. HPO exists in a complex with the eight-component CSN, both when purified from glycerol gradient centrifugation and when reciprocal immunoprecipitated from the lysates of transfected COS-7 cells. Intracellular HPO colocalizes with endogenous CSN in nucleus of hepatic cells. In addition, intracellular function of HPO that increases the phosphorylation of c-Jun leading to potentiate the AP-1 activity is inhibited by curcumin, a potent inhibitor of CSN-associated kinase. Taken together, these results elucidate a novel relationship of intracellular growth factor, HPO with large protein complex, CSN, which suggests a possible linkage between CSN and liver regeneration. PMID:15304329

  5. The E3 ubiquitin ligase Trim7 mediates c-Jun/AP-1 activation by Ras signalling

    PubMed Central

    Chakraborty, Atanu; Diefenbacher, Markus E.; Mylona, Anastasia; Kassel, Olivier; Behrens, Axel

    2015-01-01

    The c-Jun/AP-1 transcription factor controls key cellular behaviours, including proliferation and apoptosis, in response to JNK and Ras/MAPK signalling. While the JNK pathway has been well characterised, the mechanism of activation by Ras was elusive. Here we identify the uncharacterised ubiquitin ligase Trim7 as a critical component of AP-1 activation via Ras. We found that MSK1 directly phosphorylates Trim7 in response to direct activation by the Ras–Raf–MEK–ERK pathway, and this modification stimulates Trim7 E3 ubiquitin ligase activity. Trim7 mediates Lys63-linked ubiquitination of the AP-1 coactivator RACO-1, leading to RACO-1 protein stabilisation. Consequently, Trim7 depletion reduces RACO-1 levels and AP-1-dependent gene expression. Moreover, transgenic overexpression of Trim7 increases lung tumour burden in a Ras-driven cancer model, and knockdown of Trim7 in established xenografts reduces tumour growth. Thus, phosphorylation-ubiquitination crosstalk between MSK1, Trim7 and RACO-1 completes the long sought-after mechanism linking growth factor signalling and AP-1 activation. PMID:25851810

  6. 5-Methoxyl Aesculetin Abrogates Lipopolysaccharide-Induced Inflammation by Suppressing MAPK and AP-1 Pathways in RAW 264.7 Cells.

    PubMed

    Wu, Lei; Li, Xueqin; Wu, Haifeng; Long, Wei; Jiang, Xiaojian; Shen, Ting; Qiang, Qian; Si, Chuanling; Wang, Xinfeng; Jiang, Yunyao; Hu, Weicheng

    2016-01-01

    For the first time, a pale amorphous coumarin derivative, 5-methoxyl aesculetin (MOA), was isolated from the dried bark of Fraxinus rhynchophylla Hance (Oleaceae). MOA modulates cytokine expression in lipopolysaccharide (LPS)-treated RAW 264.7 macrophages, but the precise mechanisms are still not fully understood. We determined the effects of MOA on the production of inflammatory mediators and pro-inflammatory cytokines in the LPS-induced inflammatory responses of RAW 264.7 macrophages. MOA significantly inhibited the LPS-induced production of nitric oxide (NO), prostaglandin E₂ (PGE₂), tumor necrosis factor-α (TNF-α), interleukin-6, and interleukin-1β. It also effectively attenuated inducible nitric oxide (NO) synthase, cyclooxygenase-2, and TNF-α mRNA expression and significantly decreased the levels of intracellular reactive oxygen species. It inhibited phosphorylation of the extracellular signal-regulated kinase (ERK1/2), thus blocking nuclear translocation of activation protein (AP)-1. In a molecular docking study, MOA was shown to target the binding site of ERK via the formation of three hydrogen bonds with two residues of the kinase, which is sufficient for the inhibition of ERK. These results suggest that the anti-inflammatory effects of MOA in RAW 264.7 macrophages derive from its ability to block both the activation of mitogen-activated protein kinases (MAPKs) and one of their downstream transcription factors, activator protein-1 (AP-1). Our observations support the need for further research into MOA as a promising therapeutic agent in inflammatory diseases. PMID:26938526

  7. 5-Methoxyl Aesculetin Abrogates Lipopolysaccharide-Induced Inflammation by Suppressing MAPK and AP-1 Pathways in RAW 264.7 Cells

    PubMed Central

    Wu, Lei; Li, Xueqin; Wu, Haifeng; Long, Wei; Jiang, Xiaojian; Shen, Ting; Qiang, Qian; Si, Chuanling; Wang, Xinfeng; Jiang, Yunyao; Hu, Weicheng

    2016-01-01

    For the first time, a pale amorphous coumarin derivative, 5-methoxyl aesculetin (MOA), was isolated from the dried bark of Fraxinus rhynchophylla Hance (Oleaceae). MOA modulates cytokine expression in lipopolysaccharide (LPS)-treated RAW 264.7 macrophages, but the precise mechanisms are still not fully understood. We determined the effects of MOA on the production of inflammatory mediators and pro-inflammatory cytokines in the LPS-induced inflammatory responses of RAW 264.7 macrophages. MOA significantly inhibited the LPS-induced production of nitric oxide (NO), prostaglandin E2 (PGE2), tumor necrosis factor-α (TNF-α), interleukin-6, and interleukin-1β. It also effectively attenuated inducible nitric oxide (NO) synthase, cyclooxygenase-2, and TNF-α mRNA expression and significantly decreased the levels of intracellular reactive oxygen species. It inhibited phosphorylation of the extracellular signal-regulated kinase (ERK1/2), thus blocking nuclear translocation of activation protein (AP)-1. In a molecular docking study, MOA was shown to target the binding site of ERK via the formation of three hydrogen bonds with two residues of the kinase, which is sufficient for the inhibition of ERK. These results suggest that the anti-inflammatory effects of MOA in RAW 264.7 macrophages derive from its ability to block both the activation of mitogen-activated protein kinases (MAPKs) and one of their downstream transcription factors, activator protein-1 (AP-1). Our observations support the need for further research into MOA as a promising therapeutic agent in inflammatory diseases. PMID:26938526

  8. Anti-inflammatory effects of proanthocyanidin-rich red rice extract via suppression of MAPK, AP-1 and NF-κB pathways in Raw 264.7 macrophages

    PubMed Central

    Yodkeeree, Supachai; Pitchakarn, Pornsiri; Punfa, Wanisa

    2016-01-01

    BACKGROUND/OBJECTIVES Several pharmacological properties of red rice extract have been reported including anti-oxidant, anti-tumor, and reduced cancer cell invasion. This study was conducted to evaluate the anti-inflammatory effects of red rice extract on the production of inflammatory mediators in lipopolysaccharide (LPS)-induced Raw 264.7 macrophages. MATERIALS/METHODS Pro-inflammatory cytokines including tumor necrosis factor-α and interleukin-6 were determined by ELISA and cyclooxygenase-2 and inducible nitric oxide synthase expression was evaluated using western blot analysis. In addition, the signaling pathway controlling the inflammatory cascade such as nuclear factor kappa B (NF-κB), activator proteins-1 (AP-1), and mitogen-activated protein kinase (MAPK) was determined. RESULTS Our results showed that red rice polar extract fraction (RR-P), but not non-polar extract fraction, inhibited interleukin-6, tumor necrosis factor-α, and nitric oxide production in LPS-induced Raw 264.7 cells. RR-P also reduced the expression of inflammatory enzymes, inducible nitric oxide synthase, and cyclooxygenase-2. In addition, activation of AP-1 and NF-κB transcription factor in the nucleus was abrogated by RR-P. RR-P inhibited the phosphorylation of extracellular signaling-regulated kinase 1/2, c-Jun NH2-terminal kinase, and p38 MAPK signaling responsible for the expression of inflammatory mediators in LPS-stimulated Raw 264.7 cells. Based on chemical analysis, high amounts of proanthocyanidin and catechins were detected in the RR-P fraction. However, only proanthocyanidin reduced NF-κB and AP-1 activation in LPS-activated Raw 264.7 cells. CONCLUSION These observations suggest that the anti-inflammatory properties of RR-P may stem from the inhibition of pro-inflammatory mediators via suppression of the AP-1, NF-κB, and MAPKs pathways. PMID:27247720

  9. Curcumin Nanoparticles Ameliorate ICAM-1 Expression in TNF-α-Treated Lung Epithelial Cells through p47 phox and MAPKs/AP-1 Pathways

    PubMed Central

    Yang, Chuen-Mao; Liang, Chan-Jung; Lin, Chun-Ching; Chiang, Yao-Chang; Lee, Hui-Chun; Ko, Horng-Huey; Lee, Chiang-Wen

    2013-01-01

    Upregulation of intercellular adhesion molecule-1 (ICAM-1) involves adhesions between both circulating and resident leukocytes and the human lung epithelial cells during lung inflammatory reactions. We have previously demonstrated that curcumin-loaded polyvinylpyrrolidone nanoparticles (CURN) improve the anti-inflammatory and anti-oxidative properties of curcumin in hepatocytes. In this study, we focused on the effects of CURN on the expression of ICAM-1 in TNF-α-treated lung epithelial cells and compared these to the effects of curcumin water preparation (CURH). TNF-αinduced ICAM-1 expression, ROS production, and cell-cell adhesion were significantly attenuated by the pretreatment with antioxidants (DPI, APO, or NAC) and CURN, but not by CURH, as revealed by western blot analysis, RT-PCR, promoter assay, and ROS detection and adhesion assay. In addition, treatment of TNF-α-treated cells with CURN and antioxidants also resulted in an inhibition of activation of p47 phox and phosphorylation of MAPKs, as compared to that using CURH. Our findings also suggest that phosphorylation of MAPKs may eventually lead to the activation of transcription factors. We also observed that the effects of TNF-α treatment for 30 min, which includes a significant increase in the binding activity of AP-1 and phosphorylation of c-jun and c-fos genes, were reduced by CURN treatment. In vivo studies have revealed that CURN improved the anti-inflammation activities of CURH in the lung epithelial cells of TNF-α-treated mice. Our results indicate that curcumin-loaded polyvinylpyrrolidone nanoparticles may potentially serve as an anti-inflammatory drug for the treatment of respiratory diseases. PMID:23671702

  10. The p38 SAPK pathway regulates the expression of the MMP-9 collagenase via AP-1-dependent promoter activation.

    PubMed

    Simon, C; Simon, M; Vucelic, G; Hicks, M J; Plinkert, P K; Koitschev, A; Zenner, H P

    2001-12-10

    The invasive phenotype of cancers critically depends on the expression of proteases such as the M(R) 92,000 type IV collagenase (MMP-9). Several growth factors and oncogenes were found to increase promoter activity and as a consequence protease expression. This frequently requires the activation of the transcription factor AP-1 by signal transduction cascades such as the ERK and JNK pathways. We have previously demonstrated that the tumor promoter TPA can induce MMP-9 expression via a third signaling cascade, the p38 pathway. Considering that TPA is a potent activator of AP-1, we hypothesized that this transcription factor might also be required for p38 pathway-dependent MMP-9 regulation. While dominant negative p38 and MKK-6 mutants reduced MMP-9 promoter activity in CAT assays, a construct encoding an activating mutation in the MKK-6 protein potently stimulated it. This was mediated via 144 bp of the 5'flanking region of the wild-type promoter, which contains an AP-1 site at -79. Both point mutations in this motif and the expression of a c-jun protein lacking its transactivation domain and therefore acting as a dominant negative AP-1 mutant abrogated MKK-6-dependent promoter stimulation. Finally SB 203580, a specific p38 pathway inhibitor, reduced MMP-9 expression/secretion and in vitro invasion of cancer cells. Thus, our results provide evidence that also the third SAPK/MAPK signaling cascade, the p38 signal transduction pathway, stimulates MMP-9 expression in an AP-1-dependent fashion. PMID:11716547

  11. Suppression of albumin enhancer activity by H-ras and AP-1 in hepatocyte cell lines.

    PubMed Central

    Hu, J; Isom, H C

    1994-01-01

    We demonstrated, using a transient transfection assay, that the albumin enhancer increased the expression of the albumin promoter in a highly differentiated, simian virus 40 (SV40)-immortalized hepatocyte cell line, CWSV1, but was not functional in two ras-transformed cell lines (NR3 and NR4) derived from CWSV1 by stable transfection with the T24ras oncogene. A transient cotransfection assay showed that T24ras and normal c-Ha-ras were each able to inhibit the activity of the albumin enhancer in an immortal hepatocyte cell line. DNase I footprinting and gel mobility shift assays demonstrated that the DNA binding activities specific to the albumin enhancer were not decreased in the ras-transformed cells. ras also did not diminish the expression of HNF1 alpha, C/EBP alpha, HNF3 alpha, HNF3 beta, or HNF3 gamma but did significantly increase AP-1 binding activity. Three AP-1 binding sites were identified within the albumin enhancer, and DNA binding activities specific to these AP-1 sites were induced in the ras-transformed hepatocytes. Subsequent functional assays showed that overexpression of c-jun and c-fos inhibited the activity of the albumin enhancer. Site-directed mutagenesis of the AP-1 binding sites in the albumin enhancer partially abrogated the suppressing effect of ras and c-jun/c-fos on the enhancer. These functional studies therefore supported the results of the structural studies with AP-1. We conclude that the activity of the albumin enhancer is subject to regulation by ras signaling pathways and that the effect of ras on the albumin enhancer activity may be mediated by AP-1. Images PMID:8114691

  12. Modulation of AP-1 activity by the human progesterone receptor in endometrial adenocarcinoma cells.

    PubMed Central

    Bamberger, A M; Bamberger, C M; Gellersen, B; Schulte, H M

    1996-01-01

    The composite transcription factor activating protein 1 (AP-1) integrates various mitogenic signals in a large number of cell types, and is therefore a major regulator of cell proliferation. In the normal human endometrium, proliferation and differentiation alternate in a cyclic fashion, with progesterone being largely implicated in the latter process. However, the effects of progesterone and the progesterone receptor (hPR) on AP-1 activity in the human endometrium are not known. To address this issue, HEC-1-B endometrial adenocarcinoma cells, which are devoid of hPR, were transfected with luciferase reporter constructs driven by two different AP-1-dependent promoters. Unexpectedly, cotransfection of hPR caused a marked induction of luciferase activity in the absence of ligand on both promoters. The magnitude of this induction was similar to that observed in response to the phorbol ester TPA. Addition of ligand reversed the stimulating effect of the unliganded hPR on AM activity in these cells. These effects were specific for hPR, and were not observed with either human estrogen receptor or human glucocorticoid receptor. Furthermore, they strictly depended on the presence of AP-1-responsive sequences within target promoters. Finally, the described effects of hPR on AP-1 activity were shown to be cell-type specific, because they could not be demonstrated in SKUT-1-B, JEG-3, and COS-7 cells. To our knowledge this is the first report of an unliganded steroid receptor stimulating AP-1 activity. This effect and its reversal in the presence of ligand suggest a novel mechanism, through which hPR can act as a key regulator of both proliferation and differentiation in the human endometrium. PMID:8650238

  13. Eugenolol and glyceryl-isoeugenol suppress LPS-induced iNOS expression by down-regulating NF-kappaB AND AP-1 through inhibition of MAPKS and AKT/IkappaBalpha signaling pathways in macrophages.

    PubMed

    Yeh, J L; Hsu, J H; Hong, Y S; Wu, J R; Liang, J C; Wu, B N; Chen, I J; Liou, S F

    2011-01-01

    Eugenol and isoeugenol, two components of clover oil, have been reported to possess several biomedical properties, such as anti-inflammatory, antimicrobial and antioxidant effects. This study aims to examine the anti-inflammatory effects of eugenol, isoeugenol and four of their derivatives on expression of inducible nitric oxide synthase (iNOS) activated by lipopolysaccharide (LPS) in mouse macrophages (RAW 264.7), and to investigate molecular mechanisms underlying these effects. We found that two derivatives, eugenolol and glyceryl-isoeugenol, had potent inhibitory effects on LPS-induced upregulation of nitrite levels, iNOS protein and iNOS mRNA. In addition, they both suppressed the release of tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta) induced by LPS. Moreover, they both attenuated the DNA binding of NF-kB and AP-1, phosphorylation of inhibitory kB-alpha (IkB-alpha), and nuclear translocation of p65 protein induced by LPS. Finally, we demonstrated that glyceryl-isoeugenol suppressed the phosphorylation of ERK1/2, JNK and p38 MAPK, whereas eugenolol suppressed the phosphorylation of ERK1/2 and p38 MAPK. Taken together, these results suggest that that eugenolol and glyceryl-isoeugenol suppress LPS-induced iNOS expression by down-regulating NF-kB and AP-1 through inhibition of MAPKs and Akt/IkB-alpha signaling pathways. Thus, this study implies that eugenolol and glyceryl-isoeugenol may provide therapeutic benefits for inflammatory diseases. PMID:21658309

  14. DIFFERENTIAL ACTIVATION OF AP-1 IN HUMAN BLADDER EPITHELIAL CELLS BY INORGANIC AND METHYLATED ARSENICALS

    EPA Science Inventory

    Differential Activation of AP-1 in Human Bladder Epithelial Cells by Inorganic and Methylated Arsenicals

    Zuzana Drobna, Ilona Jaspers, David J. Thomas, and Miroslav Styblo

    ABSTRACT

    Epidemiological studies have linked chronic ingestion of drinking water contai...

  15. o,p'-DDT induces cyclooxygenase-2 gene expression in murine macrophages: Role of AP-1 and CRE promoter elements and PI3-kinase/Akt/MAPK signaling pathways

    SciTech Connect

    Han, Eun Hee; Kim, Ji Young; Kim, Hyung-Kyun; Hwang, Yong Pil; Jeong, Hye Gwang

    2008-12-01

    Dichlorodiphenyltrichloroethane (DDT) has been used as an insecticide to prevent the devastation of malaria in tropical zones. However, many reports suggest that DDT may act as an endocrine disruptor and may have possible carcinogenic effects. Cyclooxygenase-2 (COX-2) acts as a link between inflammation and carcinogenesis through its involvement in tumor promotion. In the present study, we examined the effect of o,p'-DDT on COX-2 gene expression and analyzed the molecular mechanism of its activity in murine RAW 264.7 macrophages. Exposure to o,p'-DDT markedly enhanced the production of prostaglandin E{sub 2} (PGE{sub 2}), a major COX-2 metabolite, in murine macrophages. Furthermore, o,p'-DDT dose-dependently increased the levels of COX-2 protein and mRNA. Transfection with human COX-2 promoter construct, electrophoretic mobility shift assays and DNA-affinity protein-binding assay experiments revealed that o,p'-DDT activated the activator protein 1 (AP-1) and cyclic AMP response element (CRE) sites, but not the NF-{kappa}B site. Phosphatidylinositol 3 (PI3)-kinase, its downstream signaling molecule, Akt, and mitogen-activated protein kinases (MAPK) were also significantly activated by the o,p'-DDT-induced AP-1 and CRE activation. These results demonstrate that o,p'-DDT induced COX-2 expression via AP-1 and CRE activation through the PI3-K/Akt/ERK, JNK, and p38 MAP kinase pathways. These findings provide further insight into the signal transduction pathways involved in the carcinogenic effects of o,p'-DDT.

  16. YY1 represses human papillomavirus type 16 transcription by quenching AP-1 activity.

    PubMed Central

    O'Connor, M J; Tan, S H; Tan, C H; Bernard, H U

    1996-01-01

    YY1 is a multifunctional transcription factor that has been shown to regulate the expression of a number of cellular and viral genes, including the human papillomavirus (HPV) oncogenes E6 and E7. In this study, we have analyzed the YY1-mediated repression of the HPV type 16 (HPV-16) E6-E7 promoter. A systematic analysis to identify YY1 sites present in the HPV-16 long control region showed that of 30 potential YY1 binding motifs, 24 bound purified recombinant YY1 protein, but only 10 of these were able to bind YY1 when nuclear extracts of HeLa cells were used. Of these, only a cluster of five sites, located in the vicinity of an AP-1 motif, were found to be responsible for repressing the HPV-16 P97 promoter. All five sites were required for repression, the mutation of any one site giving rise to a four- to sixfold increase in transcriptional activity. The target for YY1-mediated repression was identified as being a highly conserved AP-1 site, and we propose that AP-1 may represent a common target for YY1 repression. We also provide data demonstrating that YY1 can bind the transcriptional coactivator CREB-binding protein and propose a potentially novel mechanism by which YY1 represses AP-1 activity as a result of this YY1-CREB-binding protein interaction. PMID:8794287

  17. Apocynin increases glutathione synthesis and activates AP-1 in alveolar epithelial cells.

    PubMed

    Lapperre, T S; Jimenez, L A; Antonicelli, F; Drost, E M; Hiemstra, P S; Stolk, J; MacNee, W; Rahman, I

    1999-01-25

    Apocynin (4-hydroxy-3-methoxy-acetophenone) is a potent intracellular inhibitor of superoxide anion production in neutrophils. In this study, we studied the effect of apocynin on the regulation of the antioxidant glutathione (GSH) and activation of the transcription factor AP-I in human alveolar epithelial cells (A549). Apocynin enhanced intracellular GSH by increasing gamma-glutamylcysteine synthetase activity in A549 cells. Apocynin also increased the expression of gamma-GCS heavy subunit mRNA. This was associated with increased AP-1 DNA binding as measured by the electrophoretic mobility shift assay. These data indicate that apocynin displays antioxidant properties, in part, by increasing glutathione synthesis through activation of AP-1. PMID:9989612

  18. Photodynamic activation as a molecular switch to promote osteoblast cell differentiation via AP-1 activation.

    PubMed

    Kushibiki, Toshihiro; Tu, Yupeng; Abu-Yousif, Adnan O; Hasan, Tayyaba

    2015-01-01

    In photodynamic therapy (PDT), cells are impregnated with a photosensitizing agent that is activated by light irradiation, thereby photochemically generating reactive oxygen species (ROS). The amounts of ROS produced depends on the PDT dose and the nature of the photosensitizer. Although high levels of ROS are cytotoxic, at physiological levels they play a key role as second messengers in cellular signaling pathways, pluripotency, and differentiation of stem cells. To investigate further the use of photochemically triggered manipulation of such pathways, we exposed mouse osteoblast precursor cells and rat primary mesenchymal stromal cells to low-dose PDT. Our results demonstrate that low-dose PDT can promote osteoblast differentiation via the activation of activator protein-1 (AP-1). Although PDT has been used primarily as an anti-cancer therapy, the use of light as a photochemical "molecular switch" to promote differentiation should expand the utility of this method in basic research and clinical applications. PMID:26279470

  19. Photodynamic activation as a molecular switch to promote osteoblast cell differentiation via AP-1 activation

    PubMed Central

    Kushibiki, Toshihiro; Tu, Yupeng; Abu-Yousif, Adnan O.; Hasan, Tayyaba

    2015-01-01

    In photodynamic therapy (PDT), cells are impregnated with a photosensitizing agent that is activated by light irradiation, thereby photochemically generating reactive oxygen species (ROS). The amounts of ROS produced depends on the PDT dose and the nature of the photosensitizer. Although high levels of ROS are cytotoxic, at physiological levels they play a key role as second messengers in cellular signaling pathways, pluripotency, and differentiation of stem cells. To investigate further the use of photochemically triggered manipulation of such pathways, we exposed mouse osteoblast precursor cells and rat primary mesenchymal stromal cells to low-dose PDT. Our results demonstrate that low-dose PDT can promote osteoblast differentiation via the activation of activator protein-1 (AP-1). Although PDT has been used primarily as an anti-cancer therapy, the use of light as a photochemical “molecular switch” to promote differentiation should expand the utility of this method in basic research and clinical applications. PMID:26279470

  20. The expression of the β-defensins hBD-2 and hBD-3 is differentially regulated by NF-κB and MAPK/AP-1 pathways in an in vitro model of Candida esophagitis

    PubMed Central

    Steubesand, Nadine; Kiehne, Karlheinz; Brunke, Gabriele; Pahl, Rene; Reiss, Karina; Herzig, Karl-Heinz; Schubert, Sabine; Schreiber, Stefan; Fölsch, Ulrich R; Rosenstiel, Philip; Arlt, Alexander

    2009-01-01

    Background Candida albicans resides on epithelial surfaces as part of the physiological microflora. However, under certain conditions it may cause life-threatening infections like Candida sepsis. Human β-defensins (hBDs) are critical components of host defense at mucosal surfaces and we have recently shown that hBD-2 and hBD-3 are upregulated in Candida esophagitis. We therefore studied the role of Candidate signalling pathways in order to understand the mechanisms involved in regulation of hBD-expression by C. albicans. We used the esophageal cell line OE21 and analysed the role of paracrine signals from polymorphonuclear leukocytes (PMN) in an in vitro model of esophageal candidiasis. Results Supernatants of C. albicans or indirect coculture with C. albicans induces upregulation of hBD-2 and hBD-3 expression. PMNs strongly amplifies C. albicans-mediated induction of hBDs. By EMSA we demonstrate that C. albicans activates NF-κB and AP-1 in OE21 cells. Inhibition of these pathways revealed that hBD-2 expression is synergistically regulated by both NF-κB and AP-1. In contrast hBD-3 expression is independent of NF-κB and relies solely on an EGFR/MAPK/AP-1-dependent pathway. Conclusion Our analysis of signal transduction events demonstrate a functional interaction of epithelial cells with PMNs in response to Candida infection involving divergent signalling events that differentially govern hBD-2 and hBD-3 expression. PMID:19523197

  1. Temporal coherency between receptor expression, neural activity and AP-1-dependent transcription regulates Drosophila motoneuron dendrite development

    PubMed Central

    Vonhoff, Fernando; Kuehn, Claudia; Blumenstock, Sonja; Sanyal, Subhabrata; Duch, Carsten

    2013-01-01

    Neural activity has profound effects on the development of dendritic structure. Mechanisms that link neural activity to nuclear gene expression include activity-regulated factors, such as CREB, Crest or Mef2, as well as activity-regulated immediate-early genes, such as fos and jun. This study investigates the role of the transcriptional regulator AP-1, a Fos-Jun heterodimer, in activity-dependent dendritic structure development. We combine genetic manipulation, imaging and quantitative dendritic architecture analysis in a Drosophila single neuron model, the individually identified motoneuron MN5. First, Dα7 nicotinic acetylcholine receptors (nAChRs) and AP-1 are required for normal MN5 dendritic growth. Second, AP-1 functions downstream of activity during MN5 dendritic growth. Third, using a newly engineered AP-1 reporter we demonstrate that AP-1 transcriptional activity is downstream of Dα7 nAChRs and Calcium/calmodulin-dependent protein kinase II (CaMKII) signaling. Fourth, AP-1 can have opposite effects on dendritic development, depending on the timing of activation. Enhancing excitability or AP-1 activity after MN5 cholinergic synapses and primary dendrites have formed causes dendritic branching, whereas premature AP-1 expression or induced activity prior to excitatory synapse formation disrupts dendritic growth. Finally, AP-1 transcriptional activity and dendritic growth are affected by MN5 firing only during development but not in the adult. Our results highlight the importance of timing in the growth and plasticity of neuronal dendrites by defining a developmental period of activity-dependent AP-1 induction that is temporally locked to cholinergic synapse formation and dendritic refinement, thus significantly refining prior models derived from chronic expression studies. PMID:23293292

  2. 2,3,5,6-Tetramethylpyrazine (TMP) down-regulated arsenic-induced heme oxygenase-1 and ARS2 expression by inhibiting Nrf2, NF-κB, AP-1 and MAPK pathways in human proximal tubular cells.

    PubMed

    Gong, Xuezhong; Ivanov, Vladimir N; Hei, Tom K

    2016-09-01

    Our recent study demonstrated that sodium arsenite at a clinically relevant dose induced nephrotoxicity in human renal proximal tubular epithelial cell line HK-2, which could be inhibited by natural product 2,3,5,6-tetramethylpyrazine (TMP) with antioxidant activity. The present study demonstrated that arsenic exposure resulted in protein and enzymatic induction of heme oxygenase-1 (HO-1) in dose- and time-dependent manners in HK-2 cells. Blocking HO-1 enzymatic activity by zinc protoporphyrin (ZnPP) augmented arsenic-induced apoptosis, ROS production and mitochondrial dysfunction, suggesting a critical role for HO-1 as a renal protectant in this procession. On the other hand, TMP, upstream of HO-1, inhibited arsenic-induced ROS production and ROS-dependent HO-1 expression. TMP also prevented mitochondria dysfunction and suppressed activation of the intrinsic apoptotic pathway in HK-2 cells. Our results revealed that the regulation of arsenic-induced HO-1 expression was performed through multiple ROS-dependent signal pathways and the corresponding transcription factors, including p38 MAPK and JNK (but not ERK), AP-1, Nrf2 and NF-κB. TMP inhibited arsenic-induced activations of JNK, p38 MAPK, ERK, AP-1 and Nrf2 and block HO-1 protein expression. The present study, furthermore, demonstrated arsenic-induced expression of arsenic response protein 2 (ARS2) that was regulated by p38 MAPK, ERK and NF-κB. To our knowledge, this is the first report showing that ARS2 involved in arsenic-induced nephrotoxicity, while TMP pretreatment prevented such an up-regulation of ARS2 in HK-2 cells. Given ARS2 and HO-1 sharing the similar regulation mechanism, we speculated that ARS2 might also mediate cell survival in this procession. In summary, our study highlighted a role of HO-1 in the protection against arsenic-induced cytotoxicity downstream from the primary targets of TMP and further indicated that TMP may be used as a potential therapeutic agent in the treatment of arsenic

  3. Activation of mitogen-activated protein kinases and AP-1 transcription factor in ovotoxicity induced by 4-vinylcyclohexene diepoxide in rats.

    PubMed

    Hu, Xiaoming; Flaws, Jodi A; Sipes, I Glenn; Hoyer, Patricia B

    2002-09-01

    Previous studies have demonstrated that ovotoxicity induced in small preantral (primordial and primary) ovarian follicles by 4-vinylcyclohexene diepoxide (VCD) in rats is likely via acceleration of the normal process of atresia (apoptosis). This acceleration is associated with increased activities of caspase cascades, changes in subcellular distribution of Bcl-2 family members, and alteration of estrogen receptor-mediated signaling pathways. The present study was designed to investigate possible effects of VCD dosing on the mitogen-activated protein kinases (MAPK)/AP-1 signaling pathways in rat ovarian small follicles. Female F344 rats were given a single dose of VCD (80 mg/kg i.p., 1 day--a time when ovotoxicity has not been initiated) or dosed daily for 10 or 15 days (80 mg/kg i.p.; 10 days--a time when the earliest signs of impending follicular destruction is seen, 15 days--a time when significant ovotoxicity is underway). Four hours following the final dose, ovaries and livers were collected. Ovarian small (25-100 microm) and large (100-250 microm) preantral follicles were isolated, and cytosolic or nuclear extracts were prepared from follicles and livers for analyses. Activities of MAPKs, including extracellular signal-regulated kinase, c-Jun N-terminal protein kinase (JNK), and p38 kinase, were determined in follicular and liver cytosolic extracts, and AP-1 DNA binding activity was determined in follicular and liver nuclear extracts. Compared with control, a single dose of VCD caused a decrease in JNK activity and an increase of AP-1 binding activity in isolated small ovarian follicles. After repeated daily dosing with VCD for 10 or 15 days, JNK and p38 kinase activities in small ovarian follicles were increased (p38 kinase: 1.64 +/- 0.14 for 10 days, 1.48 +/- 0.11 for 15 days, VCD/control, P < 0.01; JNK: 1.44 +/- 0.11 for 10 days, 1.37 +/- 0.06 for 15 days, VCD/control, P < 0.01) and AP-1 binding activity in small ovarian follicles was decreased (10 days, 0

  4. Exogenous avian leukosis virus-induced activation of the ERK/AP1 pathway is required for virus replication and correlates with virus-induced tumorigenesis

    PubMed Central

    Dai, Manman; Feng, Min; Ye, Yu; Wu, Xiaochan; Liu, Di; Liao, Ming; Cao, Weisheng

    2016-01-01

    A proteomics approach was used to reveal the up-regulated proteins involved in the targeted mitogen-activated protein kinase (MAPK) signal transduction pathway in DF-1 cells after ALV subgroup J (ALV-J) infection. Next, we found that ALV-J CHN06 strain infection of DF-1 cells correlated with extracellular signal-regulated kinase 2 (ERK2) activation, which was mainly induced within 15 min, a very early stage of infection, and at a late infection stage, from 108 h to 132 h post-infection. Infection with other ALV subgroup (A/B) strains also triggered ERK/MAPK activation. Moreover, when activating ERK2, ALV subgroups A, B and J simultaneously induced the phosphorylation of c-Jun, an AP1 family member and p38 activation but had no obvious effect on JNK activation at either 15 min or 120 h. Interestingly, only PD98059 inhibited the ALV-induced c-Jun phosphorylation while SP600125 or SB203580 had no influence on c-Jun activation. Furthermore, the viral gp85 and gag proteins were found to contribute to ERK2/AP1 activation. Additionally, the specific ERK inhibitor, PD980509, significantly suppressed ALV replication, as evidenced by extremely low levels of ALV promoter activity and ALV-J protein expression. In vivo analysis of ERK2 activation in tumor cells derived from ALV-J-infected chicken demonstrated a strong correlation between ERK/MAPK activation and virus-associated tumorigenesis. PMID:26754177

  5. Ramalin inhibits VCAM-1 expression and adhesion of monocyte to vascular smooth muscle cells through MAPK and PADI4-dependent NF-kB and AP-1 pathways.

    PubMed

    Park, Bongkyun; Yim, Joung-Han; Lee, Hong-Kum; Kim, Byung-Oh; Pyo, Suhkneung

    2015-01-01

    Cell adhesion molecules play a critical role in inflammatory processes and atherosclerosis. In this study, we investigated the effect of ramalin, a chemical compound from the Antarctic lichen Ramalina terebrata, on vascular cell adhesion molecule-1 (VCAM-1) expression induced by TNF-α in vascular smooth muscular cells (VSMCs). Pretreatment of VSMCs with ramalin (0.1-10 μg/mL) concentration-dependently inhibited TNF-α-induced VCAM-1 expression. Additionally, ramalin inhibited THP-1 (human acute monocytic leukemia cell line) cell adhesion to TNF-α-stimulated VSMCs. Ramalin suppressed TNF-α-induced production of reactive oxygen species (ROS), PADI4 expression, and phosphorylation of p38, ERK, and JNK. Moreover, ramalin inhibited TNF-α-induced translocation of NF-κB and AP-1. Inhibition of PADI4 expression by small interfering RNA or the PADI4-specific inhibitor markedly attenuated TNF-α-induced activation of NF-κB and AP-1 and VCAM-1 expression in VSMCs. Our study provides insight into the mechanisms underlying ramalin activity and suggests that ramalin may be a potential therapeutic agent to modulate inflammation within atherosclerosis. PMID:25494680

  6. Interleukin 1β and tumor necrosis factor α promote hFOB1.19 cell viability via activating AP1

    PubMed Central

    Ying, Hongliang; Li, Qiang; Zhao, Changfu

    2016-01-01

    Bone trauma healing is a complex physiological process, which may involve the function of various inflammatory cytokines. Our study aimed to explore the roles of inflammatory cytokines in bone trauma healing and reveal the potential mechanism. Concentrations of interleukin (IL)-6, IL-1β and tumor necrosis factor alpha (TNF-α) in peripheral blood serum of bone trauma patients after surgery were determined by ELISA. The human osteoblast hFOB1.19 cell line was cultured to determine the effect of these cytokines in cell viability using MTT assay. In addition, luciferase reporter assay was performed to investigate the activator protein 1 (AP1) transcriptional activity, and small interfering RNA was transfected to inhibit FOS, a component of AP1 molecule. IL-6, IL-1β and TNF-α exhibited higher level in patients with more severe bone traumas after surgery. IL-1β and TNF-α, but not IL-6, induced a significant increase of hFOB1.19 viability after three days of treatment (P < 0.05). IL-1β and TNF-α could activate AP1 transcriptional activity in hFOB1.19 cells (P < 0.001), but the activation was inhibited when cells were pretreated with inhibitor of JNKs, SP600125 (P < 0.001). Besides, the effect of IL-1β and TNF-α on promoting viability was significantly inhibited after knockdown of FOS. These findings indicated that IL-1β and TNF-α played an important role in promoting osteoblast viability via the activation of AP1 transcriptional activity, which was likely to involve the JNK/MAPK signaling pathway. Modulating inflammatory cytokines is a potential strategy for improving the outcome of bone trauma healing. PMID:27347349

  7. Apigenin-7-O-β-D-glucuronide inhibits LPS-induced inflammation through the inactivation of AP-1 and MAPK signaling pathways in RAW 264.7 macrophages and protects mice against endotoxin shock.

    PubMed

    Hu, Weicheng; Wang, Xinfeng; Wu, Lei; Shen, Ting; Ji, Lilian; Zhao, Xihong; Si, Chuan-Ling; Jiang, Yunyao; Wang, Gongcheng

    2016-02-01

    Apigenin-7-O-β-D-glucuronide (AG), an active flavonoid derivative isolated from the agricultural residue of Juglans sigillata fruit husks, possesses multiple pharmacological activities, including anti-oxidant, anti-complement, and aldose reductase inhibitory activities. To date, no report has identified the anti-inflammatory mechanisms of AG. This study was therefore designed to characterize the molecular mechanisms of AG on lipopolysaccharide (LPS)-induced inflammatory cytokines in RAW 264.7 cells and on endotoxin-induced shock in mice. AG suppressed the release of nitric oxide (NO), prostaglandin E2 (PGE2), and tumour necrosis factor-α (TNF-α) in LPS-stimulated RAW 264.7 macrophages in a dose-dependent manner without affecting cell viability. Additionally, AG suppressed LPS-induced mRNA expression of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), and TNF-α. AG treatment decreased the translocation of c-Jun into the nucleus, and decreased activator protein-1 (AP-1)-mediated luciferase activity through the inhibition of both p38 mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinase (ERK) phosphorylation. Consistent with the in vitro observations, AG protected mice from LPS-induced endotoxin shock by inhibiting proinflammatory cytokine production. Taken together, these results suggest that AG may be used as a source of anti-inflammatory agents as well as a dietary complement for health promotion. PMID:26750400

  8. Synthetic glucocorticoids that dissociate transactivation and AP-1 transrepression exhibit antiinflammatory activity in vivo.

    PubMed

    Vayssière, B M; Dupont, S; Choquart, A; Petit, F; Garcia, T; Marchandeau, C; Gronemeyer, H; Resche-Rigon, M

    1997-08-01

    Some of the most potent antiinflammatory and immunosuppressive agents are synthetic glucocorticoids. However, major side effects severely limit their therapeutic use. The development of improved glucocorticoid-based drugs will require the separation of beneficial from deleterious effects. One possibility toward this goal is to try to dissociate two main activities of glucocorticoids, i.e. transactivation and transrepression. Screening of a library of compounds using transactivation and AP-1 transrepression models in transiently transfected cells identified dissociated glucocorticoids, which exert strong AP-1 inhibition but little or no transactivation. Importantly, despite high ligand binding affinity, the prototypic dissociated compound, RU24858, acted as a weak agonist and did not efficiently antagonize dexamethasone-induced transcription in transfected cells. Similar results were obtained in hepatic HTC cells for the transactivation of the endogenous tyrosine amino transferase gene (TAT), which encodes one of the enzymes involved in the glucocorticoid-dependent stimulation of neoglucogenesis. To investigate whether dissociated glucocorticoids retained the antiinflammatory and immunosuppressive potential of classic glucocorticoids, several in vitro and in vivo models were used. Indeed, secretion of the proinflammatory lymphokine interleukin-1beta was severely inhibited by dissociated glucocorticoids in human monocytic THP 1 cells. Moreover, in two in vivo models, these compounds exerted an antiinflammatory and immunosuppressive activity as potent as that of the classic glucocorticoid prednisolone. These results may lead to an improvement of antiinflammatory and immunosuppressive therapies and provide a novel concept for drug discovery. PMID:9259316

  9. Sargahydroquinoic acid inhibits TNFα-induced AP-1 and NF-κB signaling in HaCaT cells through PPARα activation

    SciTech Connect

    Jeon, Youngsic; Jung, Yujung; Kim, Min Cheol; Kwon, Hak Cheol; Kang, Ki Sung; Kim, Yong Kee; Kim, Su-Nam

    2014-08-08

    Highlights: • SHQA increases PPARα/γ transactivation and inhibits MMP-2/-9 expression. • SHQA inhibits TNFα-induced AP-1 and MAPK signaling. • SHQA inhibits TNFα-induced p65 translocation and IκBα phosphorylation. • SHQA inhibits TNFα-induced AP-1 and NF-κB signaling via PPARα. - Abstract: Peroxisome proliferator-activated receptors (PPARs) are members of the nuclear hormone receptor superfamily of ligand-activated transcription factors and expressed in various cell types in the skin, including keratinocytes, fibroblasts and infiltrating immune cells. Thus, their ligands are targets for the treatment of various skin disorders, such as photo-aging and chronological aging of skin. Intensive studies have revealed that PPARα/γ functions in photo-aging and age-related inflammation by regulating matrix metalloproteinases (MMPs) via activator protein-1 (AP-1) and nuclear factor kappa B (NF-κB). However, the detailed mechanism of PPARα/γ’s role in skin aging has not yet been elucidated. In this study, we confirmed that sargahydroquinoic acid (SHQA) as a PPARα/γ ligand significantly decreased Tumor Necrosis Factor-alpha (TNFα)-induced MMP-2/-9 expression by downregulating TNFα-induced transcription factors, subsequently reducing IκBα degradation and blocking NF-κB p65 nuclear translocation in HaCaT human epidermal keratinocyte cells. Treatment of cells with SHQA and GW6471 (PPARα antagonist) not bisphenol A diglycidyl ether (PPARγ antagonists), reversed the effect on TNFα-induced inflammatory signaling pathway activation. Taken together, our data suggest that SHQA inhibit TNFα-induced MMP-2/-9 expression and age-related inflammation by suppressing AP-1 and NF-κB pathway via PPARα.

  10. Transcription factor AP-1 in esophageal squamous cell carcinoma: Alterations in activity and expression during Human Papillomavirus infection

    PubMed Central

    2009-01-01

    Background Esophageal squamous cell carcinoma (ESCC) is a leading cause of cancer-related deaths in Jammu and Kashmir (J&K) region of India. A substantial proportion of esophageal carcinoma is associated with infection of high-risk HPV type 16 and HPV18, the oncogenic expression of which is controlled by host cell transcription factor Activator Protein-1 (AP-1). We, therefore, have investigated the role of DNA binding and expression pattern of AP-1 in esophageal cancer with or without HPV infection. Methods Seventy five histopathologically-confirmed esophageal cancer and an equal number of corresponding adjacent normal tissue biopsies from Kashmir were analyzed for HPV infection, DNA binding activity and expression of AP-1 family of proteins by PCR, gel shift assay and immunoblotting respectively. Results A high DNA binding activity and elevated expression of AP-1 proteins were observed in esophageal cancer, which differed between HPV positive (19%) and HPV negative (81%) carcinomas. While JunB, c-Fos and Fra-1 were the major contributors to AP-1 binding activity in HPV negative cases, Fra-1 was completely absent in HPV16 positive cancers. Comparison of AP-1 family proteins demonstrated high expression of JunD and c-Fos in HPV positive tumors, but interestingly, Fra-1 expression was extremely low or nil in these tumor tissues. Conclusion Differential AP-1 binding activity and expression of its specific proteins between HPV - positive and HPV - negative cases indicate that AP-1 may play an important role during HPV-induced esophageal carcinogenesis. PMID:19758438

  11. Saponins from the roots of Platycodon grandiflorum suppress ultraviolet A-induced matrix metalloproteinase-1 expression via MAPKs and NF-κB/AP-1-dependent signaling in HaCaT cells.

    PubMed

    Hwang, Yong Pil; Kim, Hyung Gyun; Choi, Jae Ho; Han, Eun Hee; Kwon, Kwang-Il; Lee, Young Chun; Choi, Jun Min; Chung, Young Chul; Jeong, Tae Cheon; Jeong, Hye Gwang

    2011-12-01

    Saponins from the roots of Platycodon grandiflorum (CKS) have been shown to exhibit many pharmacological activities, including anti-cancer and anti-inflammatory activities and antioxidant effects. However, anti-skin photoaging effects of CKS have not yet been reported. In this study, we investigated the protective effects of CKS against UVA damage on immortalized human keratinocytes (HaCaT). We then explored the inhibitory effects of CKS on UVA-induced MMP-1 and investigated the molecular mechanism underlying those effects. CKS increased the cell viability and inhibited reactive oxygen species (ROS) production in HaCaT cells exposed to UVA irradiation. Pre-treatment of HaCaT cells with CKS inhibited UVA-induced production of MMP-1 and MMP-9. In addition, CKS decreased UVA-induced expression of the inflammatory cytokines IL-1β and IL-6. Western blot analysis further revealed that CKS markedly suppressed the enhancement of collagen degradation in UVA-exposed HaCaT cells. CKS also suppressed UVA-induced activation of NF-κB or c-Jun and c-Fos, and the phosphorylation of MAPKs, which are upstream modulators of NF-κB and AP-1. PMID:22005258

  12. TAK1 regulates NF-{Kappa}B and AP-1 activation in airway epithelial cells following RSV infection

    SciTech Connect

    Dey, Nilay; Liu Tianshuang; Garofalo, Roberto P.; Casola, Antonella

    2011-09-30

    Respiratory syncytial virus (RSV) is the most common cause of epidemic respiratory diseases in infants and young children. RSV infection of airway epithelial cells induces the expression of immune/inflammatory genes through the activation of a subset of transcription factors, including Nuclear Factor-{kappa}B (NF-{kappa}B) and AP-1. In this study, we have investigated the signaling pathway leading to activation of these two transcription factors in response to RSV infection. Our results show that IKK{beta} plays a key role in viral-induced NF-{kappa}B activation, while JNK regulates AP-1-dependent gene transcription, as demonstrated by using kinase inactive proteins and chemical inhibitors of the two kinases. Inhibition of TAK1 activation, by overexpression of kinase inactive TAK1 or using cells lacking TAK1 expression, significantly reduced RSV-induced NF-{kappa}B and AP-1 nuclear translocation and DNA-binding activity, as well as NF-{kappa}B-dependent gene expression, identifying TAK1 as an important upstream signaling molecule regulating RSV-induced NF-{kappa}B and AP-1 activation. - Highlights: > IKK{beta} is a major kinase involved in RSV-induced NF-{kappa}B activation. > JNK regulates AP-1-dependent gene transcription in RSV infection. > TAK1 is a critical upstream signaling molecule for both pathways in infected cells.

  13. Arsenic Directly Binds to and Activates the Yeast AP-1-Like Transcription Factor Yap8

    PubMed Central

    Kumar, Nallani Vijay; Yang, Jianbo; Pillai, Jitesh K.; Rawat, Swati; Solano, Carlos; Kumar, Abhay; Grøtli, Morten; Stemmler, Timothy L.; Rosen, Barry P.

    2015-01-01

    The AP-1-like transcription factor Yap8 is critical for arsenic tolerance in the yeast Saccharomyces cerevisiae. However, the mechanism by which Yap8 senses the presence of arsenic and activates transcription of detoxification genes is unknown. Here we demonstrate that Yap8 directly binds to trivalent arsenite [As(III)] in vitro and in vivo and that approximately one As(III) molecule is bound per molecule of Yap8. As(III) is coordinated by three sulfur atoms in purified Yap8, and our genetic and biochemical data identify the cysteine residues that form the binding site as Cys132, Cys137, and Cys274. As(III) binding by Yap8 does not require an additional yeast protein, and Yap8 is regulated neither at the level of localization nor at the level of DNA binding. Instead, our data are consistent with a model in which a DNA-bound form of Yap8 acts directly as an As(III) sensor. Binding of As(III) to Yap8 triggers a conformational change that in turn brings about a transcriptional response. Thus, As(III) binding to Yap8 acts as a molecular switch that converts inactive Yap8 into an active transcriptional regulator. This is the first report to demonstrate how a eukaryotic protein couples arsenic sensing to transcriptional activation. PMID:26711267

  14. AP-1/IRF-3 Targeted Anti-Inflammatory Activity of Andrographolide Isolated from Andrographis paniculata.

    PubMed

    Shen, Ting; Yang, Woo Seok; Yi, Young-Su; Sung, Gi-Ho; Rhee, Man Hee; Poo, Haryoung; Kim, Mi-Yeon; Kim, Kyung-Woon; Kim, Jong Heon; Cho, Jae Youl

    2013-01-01

    Andrographolide (AG) is an abundant component of plants of the genus Andrographis and has a number of beneficial properties including neuroprotective, anticancer, anti-inflammatory, and antidiabetic effects. Despite numerous pharmacological studies, the precise mechanism of AG is still ambiguous. Thus, in the present study, we investigated the molecular mechanisms of AG and its target proteins as they pertain to anti-inflammatory responses. AG suppressed the production of nitric oxide (NO) and prostaglandin E2 (PGE2), as well as the mRNA abundance of inducible NO synthase (iNOS), tumor necrosis factor-alpha (TNF- α ), cyclooxygenase (COX)-2, and interferon-beta (IFN- β ) in a dose-dependent manner in both lipopolysaccharide- (LPS-) activated RAW264.7 cells and peritoneal macrophages. AG also substantially ameliorated the symptoms of LPS-induced hepatitis and EtOH/HCl-induced gastritis in mice. Based on the results of luciferase reporter gene assays, kinase assays, and measurement of nuclear levels of transcription factors, the anti-inflammatory effects of AG were found to be clearly mediated by inhibition of both (1) extracellular signal-regulated kinase (ERK)/activator protein (AP)-1 and (2) I κ B kinase ε (IKK ε )/interferon regulatory factor (IRF)-3 pathways. In conclusion, we detected a novel molecular signaling pathway by which AG can suppress inflammatory responses. Thus, AG is a promising anti-inflammatory drug with two pharmacological targets. PMID:23840248

  15. Omega 3 but not omega 6 fatty acids inhibit AP-1 activity and cell transformation in JB6 cells

    PubMed Central

    Liu, Guangming; Bibus, Douglas M.; Bode, Ann M.; Ma, Wei-Ya; Holman, Ralph T.; Dong, Zigang

    2001-01-01

    Epidemiological and animal-based investigations have indicated that the development of skin cancer is in part associated with poor dietary practices. Lipid content and subsequently the derived fatty acid composition of the diet are believed to play a major role in the development of tumorigenesis. Omega 3 (ω3) fatty acids, including docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), can effectively reduce the risk of skin cancer whereas omega 6 (ω6) fatty acids such as arachidonic acid (AA) reportedly promote risk. To investigate the effects of fatty acids on tumorigenesis, we performed experiments to examine the effects of the ω3 fatty acids EPA and DHA and of the ω6 fatty acid AA on phorbol 12-tetradecanoate 13-acetate (TPA)-induced or epidermal growth factor (EGF)-induced transcription activator protein 1 (AP-1) transactivation and on the subsequent cellular transformation in a mouse epidermal JB6 cell model. DHA treatment resulted in marked inhibition of TPA- and EGF-induced cell transformation by inhibiting AP-1 transactivation. EPA treatment also inhibited TPA-induced AP-1 transactivation and cell transformation but had no effect on EGF-induced transformation. AA treatment had no effect on either TPA- or EGF-induced AP-1 transactivation or transformation, but did abrogate the inhibitory effects of DHA on TPA- or EGF-induced AP-1 transactivation and cell transformation in a dose-dependent manner. The results of this study demonstrate that the inhibitory effects of ω3 fatty acids on tumorigenesis are more significant for DHA than for EPA and are related to an inhibition of AP-1. Similarly, because AA abrogates the beneficial effects of DHA, the dietary ratio of ω6 to ω3 fatty acids may be a significant factor in mediating tumor development. PMID:11416221

  16. Identification and characterization of Ref-1, a nuclear protein that facilitates AP-1 DNA-binding activity.

    PubMed Central

    Xanthoudakis, S; Curran, T

    1992-01-01

    Fos and Jun form a heterodimeric complex that regulates gene transcription by binding to the activator protein-1 (AP-1) DNA sequence motif. Previously, we demonstrated that the DNA-binding activity of Fos and Jun is regulated in vitro by a novel redox (reduction-oxidation) mechanism. Reduction of a conserved cysteine (cys) residue in the DNA-binding domains of Fos and Jun by chemical reducing agents or by a nuclear redox factor stimulates DNA-binding activity. Here, we describe purification and characterization of a 37 kDa protein (Ref-1) corresponding to the redox factor. Although Ref-1 does not bind to the AP-1 site in association with Fos and Jun, it partially copurifies with a subset of AP-1 proteins. Purified Ref-1 protein stimulates AP-1 DNA-binding activity through the conserved Cys residues in Fos and Jun, but it does not alter the DNA-binding specificity of Fos and Jun. Ref-1 may represent a novel redox component of the signal transduction processes that regulate eukaryotic gene expression. Images PMID:1537340

  17. TNF¿ and GM-CSF-induced activation of the CAEV promoter is independent of AP-1

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Caprine arthritis encephalitis virus transcription is under the control of the viral promoter within the long terminal repeat. Previous studies with the closely related maedi visna lentivirus have indicated that viral transcription is dependent upon the AP-1 transcription factor. Other studies hav...

  18. HIV-1 Nef Induces CCL5 production in astrocytes through p38-MAPK and PI3K/Akt pathway and utilizes NF-kB, CEBP and AP-1 transcription factors

    NASA Astrophysics Data System (ADS)

    Liu, Xun; Shah, Ankit; Gangwani, Mohitkumar R.; Silverstein, Peter S.; Fu, Mingui; Kumar, Anil

    2014-03-01

    The prevalence of HIV-associated neurocognitive disorders (HAND) remains high in patients infected with HIV-1. The production of pro-inflammatory cytokines by astrocytes/microglia exposed to viral proteins is thought to be one of the mechanisms leading to HIV-1- mediated neurotoxicity. In the present study we examined the effects of Nef on CCL5 induction in astrocytes. The results demonstrate that CCL5 is significantly induced in Nef-transfected SVGA astrocytes. To determine the mechanisms responsible for the increased CCL5 caused by Nef, we employed siRNA and chemical antagonists. Antagonists of NF-κB, PI3K, and p38 significantly reduced the expression levels of CCL5 induced by Nef transfection. Furthermore, specific siRNAs demonstrated that the Akt, p38MAPK, NF-κB, CEBP, and AP-1 pathways play a role in Nef-mediated CCL5 expression. The results demonstrated that the PI3K/Akt and p38 MAPK pathways, along with the transcription factors NF-κB, CEBP, and AP-1, are involved in Nef-induced CCL5 production in astrocytes.

  19. HIV-1 Nef Induces CCL5 production in astrocytes through p38-MAPK and PI3K/Akt pathway and utilizes NF-kB, CEBP and AP-1 transcription factors

    PubMed Central

    Liu, Xun; Shah, Ankit; Gangwani, Mohitkumar R.; Silverstein, Peter S.; Fu, Mingui; Kumar, Anil

    2014-01-01

    The prevalence of HIV-associated neurocognitive disorders (HAND) remains high in patients infected with HIV-1. The production of pro-inflammatory cytokines by astrocytes/microglia exposed to viral proteins is thought to be one of the mechanisms leading to HIV-1- mediated neurotoxicity. In the present study we examined the effects of Nef on CCL5 induction in astrocytes. The results demonstrate that CCL5 is significantly induced in Nef-transfected SVGA astrocytes. To determine the mechanisms responsible for the increased CCL5 caused by Nef, we employed siRNA and chemical antagonists. Antagonists of NF-κB, PI3K, and p38 significantly reduced the expression levels of CCL5 induced by Nef transfection. Furthermore, specific siRNAs demonstrated that the Akt, p38MAPK, NF-κB, CEBP, and AP-1 pathways play a role in Nef-mediated CCL5 expression. The results demonstrated that the PI3K/Akt and p38 MAPK pathways, along with the transcription factors NF-κB, CEBP, and AP-1, are involved in Nef-induced CCL5 production in astrocytes. PMID:24658403

  20. Heparin (GAG-hed) inhibits LCR activity of Human Papillomavirus type 18 by decreasing AP1 binding

    PubMed Central

    Villanueva, Rita; Morales-Peza, Néstor; Castelán-Sánchez, Irma; García-Villa, Enrique; Tapia, Rocio; Cid-Arregui, Ángel; García-Carrancá, Alejandro; López-Bayghen, Esther; Gariglio, Patricio

    2006-01-01

    Background High risk HPVs are causative agents of anogenital cancers. Viral E6 and E7 genes are continuously expressed and are largely responsible for the oncogenic activity of these viruses. Transcription of the E6 and E7 genes is controlled by the viral Long Control Region (LCR), plus several cellular transcription factors including AP1 and the viral protein E2. Within the LCR, the binding and activity of the transcription factor AP1 represents a key regulatory event in maintaining E6/E7 gene expression and uncontrolled cell proliferation. Glycosaminoglycans (GAGs), such as heparin, can inhibit tumour growth; they have also shown antiviral effects and inhibition of AP1 transcriptional activity. The purpose of this study was to test the heparinoid GAG-hed, as a possible antiviral and antitumoral agent in an HPV18 positive HeLa cell line. Methods Using in vivo and in vitro approaches we tested GAG-hed effects on HeLa tumour cell growth, cell proliferation and on the expression of HPV18 E6/E7 oncogenes. GAG-hed effects on AP1 binding to HPV18-LCR-DNA were tested by EMSA. Results We were able to record the antitumoral effect of GAG-hed in vivo by using as a model tumours induced by injection of HeLa cells into athymic female mice. The antiviral effect of GAG-hed resulted in the inhibition of LCR activity and, consequently, the inhibition of E6 and E7 transcription. A specific diminishing of cell proliferation rates was observed in HeLa but not in HPV-free colorectal adenocarcinoma cells. Treated HeLa cells did not undergo apoptosis but the percentage of cells in G2/M phase of the cell cycle was increased. We also detected that GAG-hed prevents the binding of the transcription factor AP1 to the LCR. Conclusion Direct interaction of GAG-hed with the components of the AP1 complex and subsequent interference with its ability to correctly bind specific sites within the viral LCR may contribute to the inhibition of E6/E7 transcription and cell proliferation. Our data

  1. Transcriptional activation of the fra-1 gene by AP-1 is mediated by regulatory sequences in the first intron.

    PubMed Central

    Bergers, G; Graninger, P; Braselmann, S; Wrighton, C; Busslinger, M

    1995-01-01

    Constitutive expression of c-Fos, FosB, Fra-1, or c-Jun in rat fibroblasts leads to up-regulation of the immediate-early gene fra-1. Using the posttranslational FosER induction system, we demonstrate that this AP-1-dependent stimulation of fra-1 expression is rapid, depends on a functional DNA-binding domain of FosER, and is a general phenomenon observed in different cell types. In vitro mutagenesis and functional analysis of the rat fra-1 gene in stably transfected Rat-1A-FosER fibroblasts indicated that basal and AP-1-regulated expression of the fra-1 gene depends on regulatory sequences in the first intron which comprise a consensus AP-1 site and two AP-1-like elements. We have also investigated the transactivating and transforming properties of the Fra-1 protein to address the significance of fra-1 up-regulation. The entire Fra-1 protein fused to the DNA-binding domain of Ga14 is shown to lack any transactivation function, and yet it possesses oncogenic potential, as overexpression of Fra-1 in established rat fibroblasts results in anchorage-independent growth in vitro and tumor development in athymic mice, fra-1 is therefore not only induced by members of the Fos family, but its gene product may also contribute to cellular transformation by these proteins. Together, these data identify fra-1 as a unique member of the fos gene family which is under positive control by AP-1 activity. PMID:7791782

  2. S100P/RAGE signaling regulates microRNA-155 expression via AP-1 activation in colon cancer

    PubMed Central

    Onyeagucha, Benjamin Chidi; Mercado-Pimentel, Melania E.; Hutchison, Jennifer; Flemington, Erik K.; Nelson, Mark A.

    2013-01-01

    Accumulating evidence indicates that elevated S100P promotes the pathogenesis of cancers, including colon cancer. S100P exerts its effects by binding to and activating the Receptor for Advance Glycation End-products (RAGE). The effects of up-regulated S100P/RAGE signaling on cell functions are well documented. Despite these observations, little is known about the downstream targets of S100P/RAGE signaling. In the present study, we demonstrated for the first time that activation of RAGE by S100P regulates oncogenic microRNA-155 (miR-155) expression through Activator Protein-1 (AP-1) stimulation in colon cancer cells. Ectopic S100P up-regulated miR-155 levels in human colon cancer cells. Conversely, knockdown of S100P resulted in a decrease in miR-155 levels. Exogenous S100P induced miR-155 expression, but blockage of the RAGE with anti-RAGE antibody suppressed the induction of miR-155 by exogenous S100P. Attenuation of AP-1 activation through pharmacological inhibition of MEK activation or genetic inhibition of c-Jun activation using dominant negative c-Jun (TAM67) suppressed miR-155 induction by exogenous S100P. Also, S100P treatment stimulated the enrichment of c-Fos, an AP-1 family member, at the miR-155 host gene promoter site. Finally, a functional study demonstrated that miR-155 knockdown decreases colon cancer cell growth, motility, and invasion. Altogether, these data demonstrate that the expression of miR-155 is regulated by S100P and is dependent on RAGE activation and stimulation of AP-1. PMID:23693020

  3. Advanced glycation end products upregulate lysyl oxidase and endothelin-1 in human aortic endothelial cells via parallel activation of ERK1/2-NF-κB and JNK-AP-1 signaling pathways.

    PubMed

    Adamopoulos, Christos; Piperi, Christina; Gargalionis, Antonios N; Dalagiorgou, Georgia; Spilioti, Eliana; Korkolopoulou, Penelope; Diamanti-Kandarakis, Evanthia; Papavassiliou, Athanasios G

    2016-04-01

    Endothelial dysfunction involves deregulation of the key extracellular matrix (ECM) enzyme lysyl oxidase (LOX) and the vasoconstrictor protein, endothelin-1 (ET-1), whose gene expression can be modulated by the transcriptional activators nuclear factor kappa B (NF-κB) and activator protein-1 (AP-1). Advanced glycation end products (AGEs) present an aggravating factor of endothelial dysfunction which upon engagement to their receptor RAGE induce upregulation of mitogen-activated protein kinases (MAPKs), leading to NF-κB and AP-1 potentiation. We hypothesized that AGEs could induce NF-κΒ- and AP-1-dependent regulation of LOX and ET-1 expression via the AGE/RAGE/MAPK signaling axis. Western blot, real-time qRT-PCR, FACS analysis and electrophoretic mobility-shift assays were employed in human aortic endothelial cells (HAECs) following treatment with AGE-bovine serum albumin (AGE-BSA) to investigate the signaling pathway towards this hypothesis. Furthermore, immunohistochemical analysis of AGEs, RAGE, LOX and ET-1 expression was conducted in aortic endothelium of a rat experimental model exposed to high- or low-AGE content diet. HAECs exposed to AGE-BSA for various time points exhibited upregulation of LOX and ET-1 mRNA levels in a dose- and time-dependent manner. Exposure of HAECs to AGE-BSA also showed specific elevation of phospho(p)-ERK1/2 and p-JNK levels in a dose- and time-dependent fashion. AGE administration significantly increased NF-κΒ- and AP-1-binding activity to both LOX and ET-1 cognate promoter regions. Moreover, LOX and ET-1 overexpression in rat aortic endothelium upon high-AGE content diet confirmed the functional interrelation of these molecules. Our findings demonstrate that AGEs trigger NF-κΒ- and AP-1-mediated upregulation of LOX and ET-1 via the AGE/RAGE/MAPK signaling cascade in human endothelial cells, thus contributing to distorted endothelial homeostasis by impairing endothelial barrier function, altering ECM biomechanical properties

  4. Trans-repressor activity of nuclear glycosaminoglycans on Fos and Jun/AP-1 oncoprotein-mediated transcription.

    PubMed

    Busch, S J; Martin, G A; Barnhart, R L; Mano, M; Cardin, A D; Jackson, R L

    1992-01-01

    Heparin blocks the phorbol ester-induced progression of nontransformed cells through the G0/G1 phase (Wright, T.C., L.A. Pukac, J.J. Castellot, M.J. Karnovsky, R.A. Levine, H.-Y. Kim-Park, and J. Campisi. 1989. Proc. Natl. Acad. Sci. USA. 86: 3199-3203) or G1 to S phase (Reilly, C. F., M. S. Kindy, K. E. Brown, R. D. Rosenberg, and G. E Sonenshein. 1989. J. Biol. Chem. 264:6990-6995) of the cell cycle. Cell cycle arrest was associated with decreased levels of stage-specific mRNAs suggesting transcriptional regulation of cell growth. In the present report, we show that heparin selectively repressed TPA-inducible AP-1-mediated gene expression. Heparin-induced trans-repression was observed in primary vascular smooth muscle cells, as well as in the transformed HeLa cell line and in nondifferentiated F9 teratocarcinoma cells. Inhibition of AP-1-mediated trans-activation occurred with heparin and pentosan polysulfate but not with chondroitin sulfate A or C. Heparin-binding peptides or heparitinase I addition to nuclear lysates of heparin-treated cells allowed enhanced recovery of endogenous AP-1-specific DNA binding activity. We propose a model in which nuclear glycosaminoglycans play a trans-regulatory role in altering the patterns of inducible gene expression. PMID:1730747

  5. Cdk3-promoted epithelial-mesenchymal transition through activating AP-1 is involved in colorectal cancer metastasis

    PubMed Central

    Tang, Na; Li, Yuejin; Peng, Zhengke; Lu, Chengrong; Dong, Zigang; Tang, Faqing

    2016-01-01

    Cyclin dependent kinase-3 (Cdk3) is a positive regulator of the G1 mammalian cell cycle phase. Cdk3 is involved in cancer progression, but very little is known about its mechanism in cancer development and progression. Herein, we found that Cdk3 increased colorectal cancer metastasis through promoting epithelial-mesenchymal transition (EMT) shift. Cdk3 was found to highly express in metastatic cancer and induce cell motility and invasion. Cdk3 was shown to phosphorylate c-Jun at Ser 63 and Ser 73 in vitro and ex vivo. Cdk3-phosphorylated c-Jun at Ser 63 and Ser 73 resulted in an increased AP-1 activity. Ectopic expression of Cdk3 promoted colorectal cancer from epithelial to mesenchymal transition conjugating AP-1 activation, while AP-1 inhibition dramatically decreased Cdk3-increased EMT shift. These results showed that the Cdk3/c-Jun signaling axis mediating epithelial-mesenchymal transition plays an important role in colorectal cancer metastasis. PMID:26755651

  6. The Synonymous Ala87 Mutation of Estrogen Receptor Alpha Modifies Transcriptional Activation Through Both ERE and AP1 Sites.

    PubMed

    Fernández-Calero, Tamara; Flouriot, Gilles; Marín, Mónica

    2016-01-01

    Estrogen receptor α (ERα) exerts regulatory actions through genomic mechanisms. In the classical pathway, ligand-activated ERα binds directly to DNA through estrogen response elements (ERE) located in the promoter of target genes. ERα can also exert indirect regulation of transcription via protein-protein interaction with other transcription factors such as AP-1.S everal ERα synonymous polymorphisms have been identified and efforts to understand their implications have been made. Nevertheless effects of synonymous polymorphisms are still neglected. This chapter focuses on the experimental procedure employed in order to characterize the transcriptional activity of a synonymous polymorphism of the ERα (rs746432) called Alanine 87 (Ala87). Activity of both WT and Ala87 ERα isoforms on transcriptional pathways can be analyzed in transiently transfected cells using different reporter constructs. ERα efficiency on the classical genomic pathway can be analyzed by determining its transactivation activity on an ERE-driven thymidine kinase (TK) promoter controlling the expression of the luciferase reporter gene. Transcriptional activity through the indirect genomic pathway can be analyzed by employing an AP-1 DNA response element-driven promoter also controlling the expression of luciferase reporter gene. PMID:26585143

  7. Control of Sty1 MAPK activity through stabilisation of the Pyp2 MAPK phosphatase.

    PubMed

    Kowalczyk, Katarzyna M; Hartmuth, Sonya; Perera, David; Stansfield, Peter; Petersen, Janni

    2013-08-01

    In all eukaryotes tight control of mitogen-activated protein kinase (MAPK) activity plays an important role in modulating intracellular signalling in response to changing environments. The fission yeast MAPK Sty1 (also known as Spc1 or Phh1) is highly activated in response to a variety of external stresses. To avoid segregation of damaged organelles or chromosomes, strong Sty1 activation transiently blocks mitosis and cell division until such stresses have been dealt with. MAPK phosphatases dephosphorylate Sty1 to reduce kinase activity. Therefore, tight control of MAPK phosphatases is central for stress adaptation and for cell division to resume. In contrast to Pyp1, the fission yeast Pyp2 MAPK phosphatase is under environmental control. Pyp2 has a unique sequence (the linker region) between the catalytic domain and the N-terminal MAPK-binding site. Here we show that the Pyp2 linker region is a destabilisation domain. Furthermore, the linker region is highly phosphorylated to increase Pyp2 protein stability and this phosphorylation is Sty1 dependent. Our data suggests that Sty1 activation promotes Pyp2 phosphorylation to increase the stability of the phosphatase. This MAPK-dependent Pyp2 stabilisation allows cells to attenuate MAPK signalling and resume cell division, once stresses have been dealt with. PMID:23690545

  8. Activation of NF-κB and AP-1 Mediates Hyperproliferation by Inducing β-Catenin and c-Myc in Helicobacter pylori-Infected Gastric Epithelial Cells

    PubMed Central

    Byun, Eunyoung; Park, Bohye; Lim, Joo Weon

    2016-01-01

    Purpose In the gastric mucosa of Helicobacter pylori (H. pylori)-infected patients with gastritis or adenocarcinoma, proliferation of gastric epithelial cells is increased. Hyperproliferation is related to induction of oncogenes, such as β-catenin and c-myc. Even though transcription factors NF-κB and AP-1 are activated in H. pylori-infected cells, whether NF-κB or AP-1 regulates the expression of β-catenein or c-myc in H. pylori-infected cells has not been clarified. The present study was undertaken to investigate whether H. pylori-induced activation of NF-κB and AP-1 mediates the expression of oncogenes and hyperproliferation of gastric epithelial cells. Materials and Methods Gastric epithelial AGS cells were transiently transfected with mutant genes for IκBα (MAD3) and c-Jun (TAM67) or treated with a specific NF-κB inhibitor caffeic acid phenethyl ester (CAPE) or a selective AP-1 inhibitor SR-11302 to suppress activation of NF-κB or AP-1, respecively. As reference cells, the control vector pcDNA was transfected to the cells. Wild-type cells or transfected cells were cultured with or without H. pylori. Results H. pylori induced activation of NF-κB and AP-1, cell proliferation, and expression of oncogenes (β-catenein, c-myc) in AGS cells, which was inhibited by transfection of MAD3 and TAM67. Wild-type cells and the cells transfected with pcDNA showed similar activities of NF-κB and AP-1, proliferation, and oncogene expression regardless of treatment with H. pylori. Both CAPE and SR-11302 inhibited cell proliferation and expression of oncogenes in H. pylori-infected cells. Conclusion H. pylori-induced activation of NF-κB and AP-1 regulates transcription of oncogenes and mediates hyperproliferation in gastric epithelial cells. PMID:26996564

  9. A cluster region of AP-1 responsive elements is required for transcriptional activity of mouse ODC gene by hepatocyte growth factor.

    PubMed

    Bianchi, Laura; Tacchini, Lorenza; Matteucci, Emanuela; Desiderio, Maria Alfonsina

    2002-05-01

    Ornithine decarboxylase (ODC) activity is regulated by a variety of mechanisms including transcription, translation, and RNA and protein half-life. Since in mouse B16-F1 melanoma cells an early and remarkable (about 6-fold) increase in steady state mRNA levels was observed after hepatocyte growth factor (HGF) treatment, we investigated the transcriptional regulation of mouse ODC promoter. Transient transfection of various ODC-luciferase promoter constructs into the B16-Fl cells in combination with electrophoretic mobility shift assays identified the HGF-responsive element as a cluster of three AP-1 binding sites (-1660 to -1572). Even if each site differs from the canonical TPA responsive element for one nucleotide, only the first two AP-1 consensus sequences seemed to be functional since allowed DNA-binding activity of nuclear proteins after HGF treatment. Comparison of the results of transfection assays with the pOD2.5-luc (2.5 kb gene fragment) and with the construct deprived of the AP-1 cluster pOD-B-luc showed that this 50 bp region was required for ODC transactivating activity in response to HGF. Since in B16-F1 cells HGF increased AP-1 activity and the mRNA expression of various AP-1 subunits, we may conclude that HGF-induced transcription of mouse ODC was largely due to triggering of AP-1 pathway. PMID:12054494

  10. Activin A enhances MMP-7 activity via the transcription factor AP-1 in an esophageal squamous cell carcinoma cell line.

    PubMed

    Yoshinaga, Keiji; Mimori, Koshi; Inoue, Hiroshi; Kamohara, Yukio; Yamashita, Keishi; Tanaka, Fumiaki; Mori, Masaki

    2008-09-01

    Activin A, a member of the transforming growth factor beta (TGF-beta) superfamily, is often overexpressed in solid carcinomas. We have previously reported that the expression of activin A is associated with lymph node metastasis in esophageal cancer. In the current study, our goal was to clarify the molecular mechanisms underlying the aggressive behavior of tumors expressing high levels of activin A. Using cDNA microarrays, the gene expression profile of a human esophageal carcinoma cell line (KYSE170) stably transfected with activin betaA (Act-betaA, a subunit of activin A) was compared with those of control human esophageal carcinoma cell lines. We found that expression of MMP-7 was higher in the Act-betaA transfectants than in the control cells. To reveal the mechanism of expression of MMP-7 mediated by activin A, we evaluated mRNA expression of MMP-7 and Act-betaA with or without activin A neutralizing antibody, using real-time PCR and Northern blot analysis. We also performed promoter analysis of the MMP-7 promoter and assessed c-Jun and Smad2/3 expression. MMP-7 expression in the transfectants was correlated with the level of Act-betaA expression and was reduced by activin A neutralizing antibody. The Act-betaA transfectants had higher MMP-7 promoter activity than control cells. MMP-7 promoter activity was not affected by mutation in the Smad binding site, while mutation of the AP-1 binding site did reduce activity. Moreover, the expression of c-Jun was increased in Act-betaA transfectants. These results indicate that activin A may modulate the expression of MMP-7 via AP-1 and not through Smad2/3. PMID:18695873

  11. Dimerumic Acid Inhibits SW620 Cell Invasion by Attenuating H2O2-Mediated MMP-7 Expression via JNK/C-Jun and ERK/C-Fos Activation in an AP-1-Dependent Manner

    PubMed Central

    Ho, Bing-Ying; Wu, Yao-Ming; Chang, King-Jen; Pan, Tzu-Ming

    2011-01-01

    Reactive oxygen species (ROS) such as hydrogen peroxide (H2O2) in the tumor microenvironment play important roles in tumor invasion and metastasis. Recently, ROS have been reported to cause a significant increase in the production and expression of matrix metalloproteinase (MMP)-7, which is closely correlated with metastatic colorectal cancer. The present study was undertaken to evaluate the scavenging activity of dimerumic acid (DMA) for H2O2 isolated from Monascus-fermented rice to investigate the inhibitory effects of DMA on the invasive potential of SW620 human colon cancer cells, and to explore the mechanisms underlying both these phenomena. Our results showed that increased MMP-7 expression due to H2O2 exposure was mediated by activation of mitogen-activated protein kinases (MAPKs) such as Jun N-terminal kinase (JNK), extracellular-regulated kinase (ERK), and p38 kinase. DMA pretreatment suppressed activation of H2O2-mediated MAPK pathways and cell invasion. Moreover, H2O2-triggered MMP-7 production was demonstrated via JNK/c-Jun and ERK/c-Fos activation in an activating protein 1 (AP-1)-dependent manner. Taken together, these results suggest that DMA suppresses H2O2-induced cell invasion by inhibiting AP-1-mediated MMP-7 gene transcription via the JNK/c-Jun and ERK/c-Fos signaling pathways in SW620 human colon cancer cells. Our data suggest that DMA may be useful in minimizing the development of colorectal metastasis. In the future, DMA supplementation may be a beneficial antioxidant to enhance surgical outcomes. PMID:21814482

  12. Interleukin-18 induces EMMPRIN expression in primary cardiomyocytes via JNK/Sp1 signaling and MMP-9 in part via EMMPRIN and through AP-1 and NF-κB activation

    PubMed Central

    Reddy, Venkatapuram Seenu; Prabhu, Sumanth D.; Mummidi, Srinivas; Valente, Anthony J.; Venkatesan, Balachandar; Shanmugam, Prakashsrinivasan; Delafontaine, Patrice

    2010-01-01

    IL-18 and the extracellular matrix metalloproteinase (MMP) inducer (EMMPRIN) stimulate the expression of proinflammatory cytokines and MMPs and are elevated in myocardial hypertrophy, remodeling, and failure. Here, we report several novel findings in primary cardiomyocytes treated with IL-18. First, IL-18 activated multiple transcription factors, including NF-κB (p50 and p65), activator protein (AP)-1 (cFos, cJun, and JunD), GATA, CCAAT/enhancer-binding protein, myocyte-specific enhancer-binding factor, interferon regulatory factor-1, p53, and specific protein (Sp)-1. Second, IL-18 induced EMMPRIN expression via myeloid differentiation primary response gene 88/IL-1 receptor-associated kinase/TNF receptor-associated factor-6/JNK-dependent Sp1 activation. Third, IL-18 induced a number of MMP genes, particularly MMP-9, at a rapid rate as well as tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-3 at a slower rate. Finally, the IL-18 induction of MMP-9 was mediated in part via EMMPRIN and through JNK- and ERK-dependent AP-1 activation and p38 MAPK-dependent NF-κB activation. These results suggest that the elevated expression of IL-18 during myocardial injury and inflammation may favor EMMPRIN and MMP induction and extracellular matrix degradation. Therefore, targeting IL-18 or its signaling pathways may be of potential therapeutic benefit in adverse remodeling. PMID:20693392

  13. MEKK1 regulates the AP-1 dimer repertoire via control of JunB transcription and Fra-2 protein stability.

    PubMed

    Cuevas, Bruce D; Uhlik, Mark T; Garrington, Timothy P; Johnson, Gary L

    2005-01-27

    Activator protein 1 (AP-1) transcription factor dimers are composed of Jun, Fos, and ATF member proteins, but the mechanisms that determine AP-1 composition are not clearly defined and the function of specific dimers is not well understood. MEKK1 is a mitogen-activated protein kinase (MAPK) kinase kinase and an ubiquitin ligase that regulates both the extracellular signal-regulated kinase 1/2 and the c-Jun amino-terminal kinase. Herein, we demonstrate that MEKK1 regulates the AP-1 protein repertoire. Both FGF-2 and phorbol ester-inducible urokinase-type plasminogen activator (uPA) expression requires AP-1 binding to an enhancer element in the uPA promoter, and we have previously shown that FGF-2 or PMA induction of uPA expression is strongly dependent on MEKK1. JunB mRNA is significantly increased in MEKK1-/- cells, demonstrating that MEKK1 suppresses JunB mRNA expression. Upregulation of JunB expression in MEKK1-/- cells forms an inhibitory AP-1 complex that binds to the uPA promoter and inhibits uPA transcription. MEKK1 also regulates Fra-2 protein stability by inducing Fra-2 ubiquitination and degradation. MEKK1 regulates AP-1-dependent gene expression by regulating the expression, activity and degradation of component members of the AP-1 complex. Controlling the repertoire of a transcription factor complex is a newly defined function for an MAPK kinase kinase. PMID:15558021

  14. Insulin-stimulated expression of c-fos, fra1 and c-jun accompanies the activation of the activator protein-1 (AP-1) transcriptional complex.

    PubMed Central

    Griffiths, M R; Black, E J; Culbert, A A; Dickens, M; Shaw, P E; Gillespie, D A; Tavaré, J M

    1998-01-01

    The activator protein-1 (AP-1) transcriptional complex is made up of members of the Fos (c-Fos, FosB, Fra1, Fra2) and Jun (c-Jun, JunB, JunD) families and is stimulated by insulin in several cell types. The mechanism by which insulin activates this complex is not well understood but it is dependent on the activation of the Erk1 and Erk2 isoforms of mitogen-activated protein kinases. In the current study we show that the AP-1 complex isolated from insulin-stimulated cells contained c-Fos, Fra1, c-Jun and JunB. The activation of the AP-1 complex by insulin was accompanied by (i) a transient increase in c-fos expression, and the transactivation of the ternary complex factors Elk1 and Sap1a, in an Erk1/Erk2-dependent fashion; (ii) a substantial increase in the expression of Fra1 protein and mRNA, which was preceded by a transient decrease in its electrophoretic mobility upon SDS/PAGE, indicative of phosphorylation; and (iii) a sustained increase in c-jun expression without increasing c-Jun phosphorylation on serines 63 and 73 or activation of the stress-activated kinase JNK/SAPK. In conclusion, insulin appears to stimulate the activity of the AP-1 complex primarily through a change in the abundance of the components of this complex, although there may be an additional role for Fra1 phosphorylation. PMID:9742208

  15. Activation of TGF-β1 promoter by hepatitis C virus-induced AP-1 and Sp1: role of TGF-β1 in hepatic stellate cell activation and invasion.

    PubMed

    Presser, Lance D; McRae, Steven; Waris, Gulam

    2013-01-01

    Our previous studies have shown the induction and maturation of transforming growth factor-beta 1 (TGF-β1) in HCV-infected human hepatoma cells. In this study, we have investigated the molecular mechanism of TGF-β1 gene expression in response to HCV infection. We demonstrate that HCV-induced transcription factors AP-1, Sp1, NF-κB and STAT-3 are involved in TGF-β1 gene expression. Using chromatin immunoprecipitation (ChIP) assay, we further show that AP-1 and Sp1 interact with TGF-b1 promoter in vivo in HCV-infected cells. In addition, we demonstrate that HCV-induced TGF-β1 gene expression is mediated by the activation of cellular kinases such as p38 MAPK, Src, JNK, and MEK1/2. Next, we determined the role of secreted bioactive TGF-β1 in human hepatic stellate cells (HSCs) activation and invasion. Using siRNA approach, we show that HCV-induced bioactive TGF-β1 is critical for the induction of alpha smooth muscle actin (α-SMA) and type 1 collagen, the markers of HSCs activation and proliferation. We further demonstrate the potential role of HCV-induced bioactive TGF-β1 in HSCs invasion/cell migration using a transwell Boyden chamber. Our results also suggest the role of HCV-induced TGF-β1 in HCV replication and release. Collectively, these observations provide insight into the mechanism of TGF-β1 promoter activation, as well as HSCs activation and invasion, which likely manifests in liver fibrosis associated with HCV infection. PMID:23437118

  16. p38MAPK activation and DUSP10 expression in meningiomas.

    PubMed

    Johnson, Mahlon D; Reeder, Jay E; O'Connell, Mary

    2016-08-01

    The mitogen activated protein kinase (MAPK) p38MAPK has been implicated in regulation of cell proliferation and apoptosis. However, expression, activation and regulation has not been studied in meningiomas, to our knowledge. p38MAPK is regulated, in part, by dual specificity phosphatases (DUSP) that inactivate signaling by dephosphorylation. DUSP10 is also a likely participant in regulating meningioma proliferation. Five fetal and an adult human leptomeninges and 37 meningioma cultures (MC) were evaluated for DUSP10 as well as phosphorylation of its substrates p38MAPK and p44/42MAPK by western blot and DUSP10 expression by polymerase chain reaction. Platelet derived growth factor-BB (PDGF-BB), transforming growth factor B1 (TGFB1) and cerebrospinal fluid effects on DUSP10 and signaling were also studied in vitro. DUSP10 and phospho-p38MAPK and phospho-p44/42MAPK were detected in all six leptomeninges. DUSP10 was detected in 13 of 17 World Health Organization grade I, 11 of 14 grade II and four of six grade III meningiomas. Phospho-p38MAPK was detected in nine of 17 grade I, two of six grade II, and four of six grade III meningiomas. In the majority of meningiomas DUSP10 expression correlated inversely with phosphorylation of p38MAPK. PDGF-BB increased DUSP10 in MC2 and MC4 and weakly in MC3. TGFB1 increased phosphorylation of p38MAPK and caspase 3 activation. Thus p38MAPK and DUSP10 likely participate in the pathogenesis of meningiomas. PMID:27050915

  17. MAPK-Activated Protein Kinases (MKs): Novel Insights and Challenges

    PubMed Central

    Gaestel, Matthias

    2016-01-01

    Downstream of MAPKs, such as classical/atypical ERKs and p38 MAPKs, but not of JNKs, signaling is often mediated by protein kinases which are phosphorylated and activated by MAPKs and, therefore, designated MAPK-activated protein kinases (MAPKAPKs). Recently, novel insights into the specificity of the assembly of MAPK/MAPKAPK hetero-dimeric protein kinase signaling complexes have been gained. In addition, new functional aspects of MKs have been described and established functions have been challenged. This short review will summarize recent developments including the linear motif (LM) in MKs, the ERK-independent activation of RSK, the RSK-independent effects of some RSK-inhibitors and the challenged role of MK5/PRAK in tumor suppression. PMID:26779481

  18. Mitogen-activated protein kinase (MAPK) in cardiac tissues.

    PubMed

    Page, C; Doubell, A F

    Mitogen-activated protein kinase (MAPK) has recently emerged as a prominent role player in intracellular signalling in the ventricular myocyte with attention being focussed on its possible role in the development of ventricular hypertrophy. It is becoming clear that MAPK is also active in other cells of cardiac origin such as cardiac fibroblasts and possible functions of this signalling pathway in the heart have yet to be explored. In this report the mammalian MAPK pathway is briefly outlined, before reviewing current knowledge of the MAPK pathway in cardiac tissue (ventricular myocytes, vascular smooth muscle cells and cardiac fibroblasts). New data is also presented on the presence and activity of MAPK in two additional cardiac celltypes namely atrial myocytes and vascular endothelial cells from the coronary microcirculation. PMID:8739228

  19. MAPK-Activated Protein Kinases (MKs): Novel Insights and Challenges.

    PubMed

    Gaestel, Matthias

    2015-01-01

    Downstream of MAPKs, such as classical/atypical ERKs and p38 MAPKs, but not of JNKs, signaling is often mediated by protein kinases which are phosphorylated and activated by MAPKs and, therefore, designated MAPK-activated protein kinases (MAPKAPKs). Recently, novel insights into the specificity of the assembly of MAPK/MAPKAPK hetero-dimeric protein kinase signaling complexes have been gained. In addition, new functional aspects of MKs have been described and established functions have been challenged. This short review will summarize recent developments including the linear motif (LM) in MKs, the ERK-independent activation of RSK, the RSK-independent effects of some RSK-inhibitors and the challenged role of MK5/PRAK in tumor suppression. PMID:26779481

  20. CARMA1- and MyD88-dependent activation of Jun/ATF-type AP-1 complexes is a hallmark of ABC diffuse large B-cell lymphomas

    PubMed Central

    Juilland, Mélanie; Gonzalez, Montserrat; Erdmann, Tabea; Banz, Yara; Jevnikar, Zala; Hailfinger, Stephan; Tzankov, Alexandar; Grau, Michael; Lenz, Georg; Novak, Urban

    2016-01-01

    A hallmark of the diffuse large B-cell lymphoma (DLBCL) of the activated B-cell (ABC) type, a molecular subtype characterized by adverse outcome, is constitutive activation of the transcription factor nuclear factor–κB (NF-κB), which controls expression of genes promoting cellular survival and proliferation. Much less, however, is known about the role of the transcription factor activator protein-1 (AP-1) in ABC DLBCL. Here, we show that AP-1, like NF-κB, was controlled by constitutive activation of the B-cell receptor signaling component caspase recruitment domain-containing membrane-associated guanylate kinase 1 (CARMA1) and/or the Toll-like receptor signaling component myeloid differentiation primary response gene 88 (MyD88) in ABC DLBCL cell lines. In contrast to germinal center (GC) B-cell (GCB) DLBCL, ABC DLBCL cell lines expressed high levels of the AP-1 family members c-Jun, JunB, and JunD, which formed heterodimeric complexes with the AP-1 family members activating transcription factor (ATF) 2, ATF3, and ATF7. Inhibition of these complexes by a dominant-negative approach led to impaired growth of a majority of ABC DLBCL cell lines. Individual silencing of c-Jun, ATF2, or ATF3 decreased cellular survival and revealed c-Jun/ATF2-dependent control of ATF3 expression. As a consequence, ATF3 expression was much higher in ABC vs GCB DLBCL cell lines. Samples derived from DLBCL patients showed a clear trend toward high and nuclear ATF3 expression in nodal DLBCL of the non-GC or ABC subtype. These findings identify the activation of AP-1 complexes of the Jun/ATF-type as an important element controlling the growth of ABC DLBCL. PMID:26747248

  1. Anti-Inflammatory Effects of α-Galactosylceramide Analogs in Activated Microglia: Involvement of the p38 MAPK Signaling Pathway

    PubMed Central

    Chung, Young Sun; Park, Seung Bum; Kim, Hee-Sun

    2014-01-01

    Microglial activation plays a pivotal role in the development and progression of neurodegenerative diseases. Thus, anti-inflammatory agents that control microglial activation can serve as potential therapeutic agents for neurodegenerative diseases. Here, we designed and synthesized α-galactosylceramide (α-GalCer) analogs to exert anti-inflammatory effects in activated microglia. We performed biological evaluations of 25 α-GalCer analogs and observed an interesting preliminary structure-activity relationship in their inhibitory influence on NO release and TNF-α production in LPS-stimulated BV2 microglial cells. After identification of 4d and 4e as hit compounds, we further investigated the underlying mechanism of their anti-inflammatory effects using RT-PCR analysis. We confirmed that 4d and 4e regulate the expression of iNOS, COX-2, IL-1β, and IL-6 at the mRNA level and the expression of TNF-α at the post-transcriptional level. In addition, both 4d and 4e inhibited LPS-induced DNA binding activities of NF-κB and AP-1 and phosphorylation of p38 MAPK without affecting other MAP kinases. When we examined the anti-inflammatory effect of a p38 MAPK-specific inhibitor, SB203580, on microglial activation, we observed an identical inhibitory pattern as that of 4d and 4e, not only on NO and TNF-α production but also on the DNA binding activities of NF-κB and AP-1. Taken together, these results suggest that p38 MAPK plays an important role in the anti-inflammatory effects of 4d and 4e via the modulation of NF-κB and AP-1 activities. PMID:24523867

  2. Multiple Protein Kinases via Activation of Transcription Factors NF-κB, AP-1 and C/EBP-δ Regulate the IL-6/IL-8 Production by HIV-1 Vpr in Astrocytes

    PubMed Central

    Gangwani, Mohitkumar R.; Kumar, Anil

    2015-01-01

    Neurocognitive impairments affect a substantial population of HIV-1 infected individuals despite the success of anti-retroviral therapy in controlling viral replication. Astrocytes are emerging as a crucial cell type that might be playing a very important role in the persistence of neuroinflammation seen in patients suffering from HIV-1 associated neurocognitive disorders. HIV-1 viral proteins including Vpr exert neurotoxicity through direct and indirect mechanisms. Induction of IL-8 in microglial cells has been shown as one of the indirect mechanism through which Vpr reduces neuronal survival. We show that HIV-1 Vpr induces IL-6 and IL-8 in astrocytes in a time-dependent manner. Additional experiments utilizing chemical inhibitors and siRNA revealed that HIV-1 Vpr activates transcription factors NF-κB, AP-1 and C/EBP-δ via upstream protein kinases PI3K/Akt, p38-MAPK and Jnk-MAPK leading to the induction of IL-6 and IL-8 in astrocytes. We demonstrate that one of the mechanism for neuroinflammation seen in HIV-1 infected individuals involves induction of IL-6 and IL-8 by Vpr in astrocytes. Understanding the molecular pathways involved in the HIV-1 neuroinflammation would be helpful in the design of adjunct therapy to ameliorate some of the symptoms associated with HIV-1 neuropathogenesis. PMID:26270987

  3. G13-dependent activation of MAPK by thyrotropin.

    PubMed

    Büch, Thomas R H; Biebermann, Heike; Kalwa, Hermann; Pinkenburg, Olaf; Hager, Denise; Barth, Holger; Aktories, Klaus; Breit, Andreas; Gudermann, Thomas

    2008-07-18

    Stimulation of the thyrotropin receptor (TSHR) activates G proteins of all four subfamilies (G(s), G(i/o), G(q/11), and G(12/13)). Whereas G(s)/cAMP-dependent cellular responses upon TSHR stimulation are well established, other signaling pathways are less characterized. We evaluated TSH-elicited cellular responses in human follicular thyroid carcinoma cells stably expressing the TSHR and in primary, nonneoplastic human thyrocytes. In these cellular models, stimulation with TSH caused activation of p44/42 MAPK and subsequent induction of c-Fos. MAPK stimulation occurred independently of G(s), G(i/o), and G(q/11) signaling. Dominant negative constructs of G(12) or G(13) as well as shRNA-mediated suppression of Galpha(12) or Galpha(13) revealed that MAPK activation was dependent on G(13) but not on G(12) signaling. Furthermore, G(13)-dependent transactivation of the epidermal growth factor receptor was necessary for MAPK activation in follicular carcinoma cells, whereas EGFR was not involved in MAPK activation in nonneoplastic primary thyrocytes. The use of bacterial inhibitors of monomeric GTPases revealed that MAPK activation proceeded independently of Rho proteins but was clostridial toxin B-sensitive, suggesting involvement of Cdc42 or Rac. Thus, our data shed new light on cAMP-independent TSHR signaling and identify the first G(13)-dependent TSHR signaling pathway in human thyrocytes. PMID:18445595

  4. Two peptides, TsAP-1 and TsAP-2, from the venom of the Brazilian yellow scorpion, Tityus serrulatus: evaluation of their antimicrobial and anticancer activities.

    PubMed

    Guo, Xiaoxiao; Ma, Chengbang; Du, Qiang; Wei, Ran; Wang, Lei; Zhou, Mei; Chen, Tianbao; Shaw, Chris

    2013-09-01

    Here we report two novel 17-mer amidated linear peptides (TsAP-1 and TsAP-2) whose structures were deduced from cDNAs cloned from a venom-derived cDNA library of the Brazilian yellow scorpion, Tityus serrulatus. Both mature peptides were structurally-characterised following their location in chromatographic fractions of venom and synthetic replicates of each were subjected to a range of biological assays. The peptides were each active against model test micro-organisms but with different potencies. TsAP-1 was of low potency against all three test organisms (MICs 120-160 μM), whereas TsAP-2 was of high potency against the Gram-positive bacterium, Staphylococcus aureus (MIC 5 μM) and the yeast, Candida albicans (10 μM). Haemolytic activity of TsAP-1 was low (4% at 160 μM) and in contrast, that of TsAP-2 was considerably higher (18% at 20 μM). Substitution of four neutral amino acid residues with Lys residues in each peptide had dramatic effects on their antimicrobial potencies and haemolytic activities, particularly those of TsAP-1. The MICs of the enhanced cationic analogue (TsAP-S1) were 2.5 μM for S. aureus/C. albicans and 5 μM for E. coli but with an associated large increase in haemolytic activity (30% at 5 μM). The same Lys residue substitutions in TsAP-2 produced a dramatic effect on its MIC for E. coli lowering this from >320 μM to 5 μM. TsAP-1 was ineffective against three of the five human cancer cell lines tested while TsAP-2 inhibited the growth of all five. Lys residue substitution of both peptides enhanced their potency against all five cell lines with TsAp-S2 being the most potent with IC50 values ranging between 0.83 and 2.0 μM. TsAP-1 and TsAP-2 are novel scorpion venom peptides with broad spectrum antimicrobial and anticancer cell activities the potencies of which can be significantly enhanced by increasing their cationicity. PMID:23770440

  5. The nuclear factor YY1 suppresses the human gamma interferon promoter through two mechanisms: inhibition of AP1 binding and activation of a silencer element.

    PubMed Central

    Ye, J; Cippitelli, M; Dorman, L; Ortaldo, J R; Young, H A

    1996-01-01

    Our group has previously reported that the nuclear factor Yin-Yang 1 (YY1), a ubiquitous DNA-binding protein, is able to interact with a silencer element (BE) in the gamma interferon (IFN-gamma) promoter region. In this study, we demonstrated that YY1 can directly inhibit the activity of the IFN-gamma promoter by interacting with multiple sites in the promoter. In cotransfection assays, a YY1 expression vector significantly inhibited IFN-gamma promoter activity. Mutation of the YY1 binding site in the native IFN-gamma promoter was associated with an increase in the IFN-gamma promoter activity. Analysis of the DNA sequences of the IFN-gamma promoter revealed a second functional YY1 binding site (BED) that overlaps with an AP1 binding site. In this element, AP1 enhancer activity was suppressed by YY1. Since the nuclear level of YY1 does not change upon cell activation, our data support a model that the nuclear factor YY1 acts to suppress basal IFN-gamma transcription by interacting with the promoter at multiple DNA binding sites. This repression can occur through two mechanisms: (i) cooperation with an as-yet-unidentified AP2-like repressor protein and (ii) competition for DNA binding with the transactivating factor AP1. PMID:8756632

  6. Alpha-melanocyte-stimulating hormone modulates activation of NF-kappa B and AP-1 and secretion of interleukin-8 in human dermal fibroblasts.

    PubMed

    Böhm, M; Schulte, U; Kalden, H; Luger, T A

    1999-10-20

    Alpha-melanocyte-stimulating hormone (alpha-MSH) has evolved as a mediator of diverse biological activities in an ever-growing number of non-melanocytic cell types. One mechanism by which alpha-MSH exerts its effects is modulation of AP-1 and NF-kappa B. These two transcription factors also play an important role in fibroblasts, in extracellular matrix composition, and in cytokine expression. By use of electric mobility shift assays, we demonstrate that alpha-MSH (10(-6) to 10(-14) M) activates AP-1 in human dermal fibroblasts, whereas coincubation with interleukin-1 beta (IL-1 beta) results in suppression of its activation. alpha-MSH also induces activation of NF-kappa B but does not modulate DNA binding on costimulation with IL-1 beta. Since AP-1 and NF-kappa B are key elements in controlling interleukin-8 (IL-8) transcription, human fibroblasts were treated with alpha-MSH and IL-1 beta for 24 hours, and cytokine levels in the supernatants were measured by ELISA. alpha-MSH alone had little effect, whereas coincubation with IL-1 beta led to marked downregulation of IL-8 secretion (at most 288 +/- 152 ng/mL) when compared to treatment with IL-1 beta alone (919 +/- 157 ng/mL). Our results indicate that alpha-MSH exerts modulatory effects on the activation of NF-kappa B and AP-1, and that it can regulate chemokine secretion in human dermal fibroblasts. These effects of alpha-MSH may have important regulatory functions in extracellular matrix composition, wound healing, or angiogenesis. PMID:10816661

  7. Cytoskeletal reorganization and TPA differently modify AP-1 to induce the urokinase-type plasminogen activator gene in LLC-PK1 cells.

    PubMed Central

    Lee, J S; von der Ahe, D; Kiefer, B; Nagamine, Y

    1993-01-01

    Urokinase-type plasminogen activator (uPA) is an extracellular protease and expressed in various cells that exhibit dynamic changes in cell morphology, suggesting a link between cytoskeletal reorganization (CSR) and uPA expression. CSR can be induced by pharmacological agents, such as by colchicine for microtubule cytoskeleton and by cytochalasin for microfilament cytoskeleton. Using these agents, we previously showed that CSR induced the uPA gene in LLC-PK1 cells independently of the protein kinase C and cAMP-dependent protein kinase. Here we show that the induction of the uPA gene by CSR is mediated by the activation of c-Jun which interacts with an AP-1-like site located 2 kb upstream of the uPA gene. 12-O-tetradecanoylphorbol 13-acetate (TPA) induces the uPA gene through the same elements, but additionally utilizes an adjacent PEA3 element and induces c-fos. Furthermore, CSR induces a greater accumulation and a more pronounced phosphorylation of c-Jun than TPA induction. AP-1 is a positive regulator of growth and oncogenesis, and CSR is an integral part of these processes. Our results provide a view how CSR and AP-1 could be coupled in these processes. We also show that TPA and CSR act synergistically, suggesting a model where an initial activation signal could be amplified by CSR. Images PMID:8346015

  8. Coenzyme Q0 regulates NFκB/AP-1 activation and enhances Nrf2 stabilization in attenuation of LPS-induced inflammation and redox imbalance: Evidence from in vitro and in vivo studies.

    PubMed

    Yang, Hsin-Ling; Lin, Ming-Wei; Korivi, Mallikarjuna; Wu, Jia-Jiuan; Liao, Chun-Huei; Chang, Chia-Ting; Liao, Jiunn-Wang; Hseu, You-Cheng

    2016-02-01

    Coenzyme Q (CoQ) analogs with variable number of isoprenoid units have been demonstrated as anti-inflammatory and antioxidant/pro-oxidant molecules. In this study we used CoQ0 (2,3-dimethoxy-5-methyl-1,4-benzoquinone, zero isoprenoid side-chains), a novel quinone derivative, and investigated its molecular actions against LPS-induced inflammation and redox imbalance in murine RAW264.7 macrophages and mice. In LPS-stimulated macrophages, non-cytotoxic concentrations of CoQ0 (2.5-10 μM) inhibited iNOS/COX-2 protein expressions with subsequent reductions of NO, PGE2, TNF-α and IL-1β secretions. This inhibition was reasoned by suppression of NFκB (p65) activation, and inhibition of AP-1 (c-Jun., c-Fos, ATF2) translocation. Our findings indicated that IKKα-mediated I-κB degradation and MAPK-signaling are involved in regulation of NFκB/AP-1 activation. Furthermore, CoQ0 triggered HO-1 and NQO-1 genes through increased Nrf2 nuclear translocation and Nrf2/ARE-signaling. This phenomenon was confirmed by diminished CoQ0 protective effects in Nrf2 knockdown cells, where LPS-induced NO, PGE2, TNF-α and IL-1β productions remained high. Molecular evidence revealed that CoQ0 enhanced Nrf2 steady-state level at both transcriptional and translational levels. CoQ0-induced Nrf2 activation appears to be regulated by ROS-JNK-signaling cascades, as evidenced by suppressed Nrf2 activation upon treatment with pharmacological inhibitors of ROS (N-acetylcysteine) and JNK (SP600125). Besides, oral administration of CoQ0 (5 mg/kg) suppressed LPS-induced (1 mg/kg) induction of iNOS/COX-2 and TNF-α/IL-1β through tight regulation of NFκB/Nrf2 signaling in mice liver and spleen. Our findings conclude that pharmacological actions of CoQ0 are mediated via inhibition of NFκB/AP-1 activation and induction of Nrf2/ARE-signaling. Owing to its potent anti-inflammatory and antioxidant properties, CoQ0 could be a promising candidate to treat inflammatory disorders. PMID:26548719

  9. RAF inhibitors that evade paradoxical MAPK pathway activation.

    PubMed

    Zhang, Chao; Spevak, Wayne; Zhang, Ying; Burton, Elizabeth A; Ma, Yan; Habets, Gaston; Zhang, Jiazhong; Lin, Jack; Ewing, Todd; Matusow, Bernice; Tsang, Garson; Marimuthu, Adhirai; Cho, Hanna; Wu, Guoxian; Wang, Weiru; Fong, Daniel; Nguyen, Hoa; Shi, Songyuan; Womack, Patrick; Nespi, Marika; Shellooe, Rafe; Carias, Heidi; Powell, Ben; Light, Emily; Sanftner, Laura; Walters, Jason; Tsai, James; West, Brian L; Visor, Gary; Rezaei, Hamid; Lin, Paul S; Nolop, Keith; Ibrahim, Prabha N; Hirth, Peter; Bollag, Gideon

    2015-10-22

    Oncogenic activation of BRAF fuels cancer growth by constitutively promoting RAS-independent mitogen-activated protein kinase (MAPK) pathway signalling. Accordingly, RAF inhibitors have brought substantially improved personalized treatment of metastatic melanoma. However, these targeted agents have also revealed an unexpected consequence: stimulated growth of certain cancers. Structurally diverse ATP-competitive RAF inhibitors can either inhibit or paradoxically activate the MAPK pathway, depending whether activation is by BRAF mutation or by an upstream event, such as RAS mutation or receptor tyrosine kinase activation. Here we have identified next-generation RAF inhibitors (dubbed 'paradox breakers') that suppress mutant BRAF cells without activating the MAPK pathway in cells bearing upstream activation. In cells that express the same HRAS mutation prevalent in squamous tumours from patients treated with RAF inhibitors, the first-generation RAF inhibitor vemurafenib stimulated in vitro and in vivo growth and induced expression of MAPK pathway response genes; by contrast the paradox breakers PLX7904 and PLX8394 had no effect. Paradox breakers also overcame several known mechanisms of resistance to first-generation RAF inhibitors. Dissociating MAPK pathway inhibition from paradoxical activation might yield both improved safety and more durable efficacy than first-generation RAF inhibitors, a concept currently undergoing human clinical evaluation with PLX8394. PMID:26466569

  10. A novel tribasic Golgi export signal directs cargo protein interaction with activated Rab11 and AP-1–dependent Golgi–plasma membrane trafficking

    PubMed Central

    Parmar, Hirendrasinh B.; Duncan, Roy

    2016-01-01

    The reovirus fusion–associated small transmembrane (FAST) proteins comprise a unique family of viral membrane fusion proteins dedicated to inducing cell–cell fusion. We recently reported that a polybasic motif (PBM) in the cytosolic tail of reptilian reovirus p14 FAST protein functions as a novel tribasic Golgi export signal. Using coimmunoprecipitation and fluorescence resonance energy transfer (FRET) assays, we now show the PBM directs interaction of p14 with GTP-Rab11. Overexpression of dominant-negative Rab11 and RNA interference knockdown of endogenous Rab11 inhibited p14 plasma membrane trafficking and resulted in p14 accumulation in the Golgi complex. This is the first example of Golgi export to the plasma membrane that is dependent on the interaction of membrane protein cargo with activated Rab11. RNA interference and immunofluorescence microscopy further revealed that p14 Golgi export is dependent on AP-1 (but not AP-3 or AP-4) and that Rab11 and AP-1 both colocalize with p14 at the TGN. Together these results imply the PBM mediates interactions of p14 with activated Rab11 at the TGN, resulting in p14 sorting into AP1-coated vesicles for anterograde TGN–plasma membrane transport. PMID:26941330

  11. A novel tribasic Golgi export signal directs cargo protein interaction with activated Rab11 and AP-1-dependent Golgi-plasma membrane trafficking.

    PubMed

    Parmar, Hirendrasinh B; Duncan, Roy

    2016-04-15

    The reovirus fusion-associated small transmembrane (FAST) proteins comprise a unique family of viral membrane fusion proteins dedicated to inducing cell-cell fusion. We recently reported that a polybasic motif (PBM) in the cytosolic tail of reptilian reovirus p14 FAST protein functions as a novel tribasic Golgi export signal. Using coimmunoprecipitation and fluorescence resonance energy transfer (FRET) assays, we now show the PBM directs interaction of p14 with GTP-Rab11. Overexpression of dominant-negative Rab11 and RNA interference knockdown of endogenous Rab11 inhibited p14 plasma membrane trafficking and resulted in p14 accumulation in the Golgi complex. This is the first example of Golgi export to the plasma membrane that is dependent on the interaction of membrane protein cargo with activated Rab11. RNA interference and immunofluorescence microscopy further revealed that p14 Golgi export is dependent on AP-1 (but not AP-3 or AP-4) and that Rab11 and AP-1 both colocalize with p14 at the TGN. Together these results imply the PBM mediates interactions of p14 with activated Rab11 at the TGN, resulting in p14 sorting into AP1-coated vesicles for anterograde TGN-plasma membrane transport. PMID:26941330

  12. Activation of AP-1 and nuclear factor-kappaB transcription factors is involved in hydrogen peroxide-induced apoptotic cell death of oligodendrocytes.

    PubMed

    Vollgraf, U; Wegner, M; Richter-Landsberg, C

    1999-12-01

    H2O2-induced onset and execution of programmed cell death in mature rat brain oligodendrocytes in culture is accompanied by the induction and nuclear translocation of the transcription factors AP-1 and nuclear factor-kappaB (NF-kappaB), both of which have been discussed as regulators of cell death and survival. Supershift analysis of nuclear extracts indicated that the AP-1 complex consists of c-Jun, c-Fos, JunD, and possibly JunB proteins, and that the NF-kappaB complex contains p50, p65, and c-Rel proteins. The first signs of DNA fragmentation were seen already during the first hour after the treatment. DNA fragmentation could be prevented by the antioxidants pyrrolidine dithiocarbamate and vitamin E, by the nuclease inhibitor aurintricarboxylic acid, and by preincubation with the iron chelator deferoxamine (DFO). Additionally, DFO protected oligodendrocytes from H2O2-induced cytotoxic effects as assessed by the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay, and suppressed the formation of free radicals. DFO alone led to a slight increase and in combination with H2O2 synergistically induced DNA-binding activities of AP-1 and NF-kappaB in oligodendrocytes. Our data suggest that although low levels of H2O2 directly activate AP-1 and NF-kappaB and might contribute to signal transduction pathways promoting cell survival, the formation and action of hydroxyl radicals promote cell death mechanisms that can be attenuated by the iron chelator DFO. PMID:10582611

  13. Overexpression of cyclin D1-CDK4 in silica-induced transformed cells is due to activation of ERKs, JNKs/AP-1 pathway.

    PubMed

    Shen, Fuhai; Fan, Xueyun; Liu, Bingci; Jia, Xiaowei; Du, Hongju; You, Baorong; Ye, Meng; Huang, Chuanshu; Shi, Xianglin

    2006-01-25

    Silica has been known to be a factor inducing acute injury and chronic pulmonary fibrosis. Silica has also been listed as a human carcinogen in 1996 by International Agency for Research on Cancer (IARC). However, the molecular mechanisms involved its pathologic effects are not well understood. In these studies, we found that exposure of human embryonic lung fibroblasts (HELF) to crystalline silica could cause increases in activation of extracellular signal-regulated kinases (ERKs), p38K, and c-Jun NH2-terminal amino kinases (JNKs), and HELF transformation. Interestingly, silica-induced transformation of HELF (S-HELF) led to increases in activated levels of ERKs and p46 of JNKs, and decrease in p38K activation, and no effect on activation of p54 of JNKs, as compared with those in parental HELF. Further studies showed that there are differential effects of ERKs, JNKs and p38K, as well as their downstream transcription factor AP-1, in regulation of expression of cyclin D1 and CDK4 and cell cycle alternations induced by silica. Cyclin D1 and CDK4 were increased in S-HELF as compared with those in HELF. Inhibition of ERKs activation by AG126, JNK by SP600125, and AP-1 by curcumin could reduced the induction of cyclin D1 and CDK4. There is no significant difference for cell cycle distribution between groups. These results demonstrate that ERKs and JNKs, but not p38K is responsible for induction of cyclin D1 and CDK4 in S-HELF, suggesting that overexpression of cyclin D1 and CDK4 caused by silica is mediated by ERK, JNK/AP-1signaling pathway. PMID:16125882

  14. A new APE1/Ref-1-dependent pathway leading to reduction of NF-kappaB and AP-1, and activation of their DNA-binding activity.

    PubMed

    Ando, Kozue; Hirao, Satoshi; Kabe, Yasuaki; Ogura, Yuji; Sato, Iwao; Yamaguchi, Yuki; Wada, Tadashi; Handa, Hiroshi

    2008-08-01

    APE1/Ref-1 is thought to be a multifunctional protein involved in reduction-oxidation (redox) regulation and base excision DNA repair, and is required for early embryonic development in mice. APE1/Ref-1 has redox activity and AP endonuclease activity, and is able to enhance DNA-binding activity of several transcription factors, including NF-kappaB, AP-1 and p53, through reduction of their critical cysteine residues. However, it remains elusive exactly how APE1/Ref-1 carries out its essential functions in vivo. Here, we show that APE1/Ref-1 not only reduces target transcription factors directly but also facilitates their reduction by other reducing molecules such as glutathione or thioredoxin. The new activity of APE1/Ref-1, termed redox chaperone activity, is exerted at concentration significantly lower than that required for its redox activity and is neither dependent on its redox activity nor on its AP endonuclease activity. We also show evidence that redox chaperone activity of APE1/Ref-1 is critical to NF-kappaB-mediated gene expression in human cells and is mediated through its physical association with target transcription factors. Thus, APE1/Ref-1 may play multiple roles in an antioxidative stress response pathway through its different biochemical activities. These findings also provide new insight into the mechanism of intracellular redox regulation. PMID:18586825

  15. Two tobacco AP1-like gene promoters with highly specific, tightly regulated and uniquely expressed activity during floral transition, initiation and development

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Biotech engineering of agronomic traits requires an array of highly specific and tightly regulated promoters in flower or other tissues. In this study, we isolated and characterized two tobacco AP1-like promoters (termed NtAP1La and NtAP1Lb1) in transgenic plants using GUS reporter and tissue-speci...

  16. Radical Scavenging Activity-Based and AP-1-Targeted Anti-Inflammatory Effects of Lutein in Macrophage-Like and Skin Keratinocytic Cells

    PubMed Central

    Oh, Jueun; Kim, Ji Hye; Park, Jae Gwang; Yi, Young-Su; Park, Kye Won; Rho, Ho Sik; Lee, Min-Seuk; Yoo, Jae Won; Kang, Seung-Hyun; Hong, Yong Deog; Shin, Song Seok; Cho, Jae Youl

    2013-01-01

    Lutein is a naturally occurring carotenoid with antioxidative, antitumorigenic, antiangiogenic, photoprotective, hepatoprotective, and neuroprotective properties. Although the anti-inflammatory effects of lutein have previously been described, the mechanism of its anti-inflammatory action has not been fully elucidated. Therefore, in the present study, we aimed to investigate the regulatory activity of lutein in the inflammatory responses of skin-derived keratinocytes or macrophages and to elucidate the mechanism of its inhibitory action. Lutein significantly reduced several skin inflammatory responses, including increased expression of interleukin-(IL-) 6 from LPS-treated macrophages, upregulation of cyclooxygenase-(COX-) 2 from interferon-γ/tumor necrosis-factor-(TNF-) α-treated HaCaT cells, and the enhancement of matrix-metallopeptidase-(MMP-) 9 level in UV-irradiated keratinocytes. By evaluating the intracellular signaling pathway and the nuclear transcription factor levels, we determined that lutein inhibited the activation of redox-sensitive AP-1 pathway by suppressing the activation of p38 and c-Jun-N-terminal kinase (JNK). Evaluation of the radical and ROS scavenging activities further revealed that lutein was able to act as a strong anti-oxidant. Taken together, our findings strongly suggest that lutein-mediated AP-1 suppression and anti-inflammatory activity are the result of its strong antioxidative and p38/JNK inhibitory activities. These findings can be applied for the preparation of anti-inflammatory and cosmetic remedies for inflammatory diseases of the skin. PMID:23533312

  17. Characterization of quinolone antibacterial-induced convulsions and increases in nuclear AP-1 DNA- and CRE-binding activities in mouse brain.

    PubMed

    Ito, Y; Ishige, K; Aizawa, M; Fukuda, H

    1999-05-01

    The quinolone antibacterials enoxacin and norfloxacin (2.5 mg/kg, i.v.) provoked clonic convulsions in mice treated concomitantly with biphenylacetic acid (BPAA, 100 mg/kg, i.p.), a major metabolite of the nonsteroidal anti-inflammatory drug fenbufen. Gel-shift assays showed that enoxacin-induced convulsions resulted in increases in nuclear activator protein 1 (AP-1) DNA- and cyclic AMP responsive element (CRE)-binding activities in the cerebral cortex and hippocampus, but not in other regions, such as the cerebellum and thalamus. In contrast, ofloxacin and levofloxacin, at the same doses, in the presence of BPAA did not evoke convulsions or increase these DNA-binding activities. Administration of these quinolones and BPAA alone elicited neither convulsions nor increases in these DNA-binding activities. These results suggest that the increased nuclear AP-1 DNA- and CRE-binding activities in the cerebral cortex and hippocampus induced by quinolones with BPAA correlated with seizure activities and that these brain regions play pivotal roles in quinolone-induced convulsions. PMID:10340309

  18. Terminalia catappa Exerts Antimetastatic Effects on Hepatocellular Carcinoma through Transcriptional Inhibition of Matrix Metalloproteinase-9 by Modulating NF-κB and AP-1 Activity.

    PubMed

    Yeh, Chao-Bin; Hsieh, Ming-Ju; Hsieh, Yih-Shou; Chien, Ming-Hsien; Lin, Pen-Yuan; Chiou, Hui-Ling; Yang, Shun-Fa

    2012-01-01

    High mortality and morbidity rates for hepatocellular carcinoma (HCC) in Taiwan primarily result from uncontrolled tumor metastasis. Previous studies have identified that Terminalia catappa leaf extracts (TCE) exert hepatoprotective, antioxidative, antiinflammatory, anticancer, and antimetastatic activities. However, the effects of TCE on HCC and the underlying molecular mechanisms of its activities have yet to be fully elucidated. The present study's findings demonstrate that TCE concentration dependently inhibits human HCC migration/invasion. Zymographic and western blot analyses revealed that TCE inhibited the activities and expression of matrix metalloproteinase-9 (MMP-9). Assessment of mRNA levels, using reverse transcriptase polymerase chain reaction (PCR) and real-time PCR, and promoter assays confirmed the inhibitory effects of TCE on MMP-9 expression in HCC cells. The inhibitory effects of TCE on MMP-9 proceeded by upregulating tissue inhibitor of metalloproteinase-1 (TIMP-1), as well as suppressing nuclear translocation and DNA binding activity of nuclear factor-kappa B (NF-κB) and activating protein-1 (AP-1) on the MMP-9 promoter in Huh7 cells. In conclusion, TCE inhibits MMP-9 expression and HCC cell metastasis and, thus, has potential use as a chemopreventive agent. Its inhibitory effects are associated with downregulation of the binding activities of the transcription factors NF-κB and AP-1. PMID:23258989

  19. Laa1p, a Conserved AP-1 Accessory Protein Important for AP-1 Localization in Yeast

    PubMed Central

    Fernández, G. Esteban

    2006-01-01

    AP-1 and Gga adaptors participate in clathrin-mediated protein transport between the trans-Golgi network and endosomes. Both adaptors contain homologous domains that act to recruit accessory proteins involved in clathrin-coated vesicle formation, but the spectrum of known adaptor-binding partners is limited. This study describes an evolutionarily conserved protein of Saccharomyces cerevisiae, Laa1p (Yjl207cp), that interacts and functions specifically with AP-1. Deletion of LAA1, when combined with a conditional mutation in clathrin heavy chain or deletion of GGA genes, accentuated growth defects and increased disruption of clathrin-dependent α-factor maturation and transport of carboxypeptidase Y to the vacuole. In contrast, such genetic interactions were not observed between deletions of LAA1 and AP-1 subunit genes. Laa1p preferentially interacted with AP-1 compared with Gga proteins by glutathione S-transferase-fusion affinity binding and coimmunoprecipitations. Localization of AP-1 and Laa1p, but not Gga proteins, was highly sensitive to brefeldin A, an inhibitor of ADP-ribosylation factor (Arf) activation. Importantly, deletion of LAA1 caused mislocalization of AP-1, especially in cells at high density (postdiauxic shift), but it did not affect Gga protein distribution. Our results identify Laa1p as a new determinant of AP-1 localization, suggesting a model in which Laa1p and Arf cooperate to direct stable association of AP-1 with appropriate intracellular membranes. PMID:16687571

  20. Sargahydroquinoic acid inhibits TNFα-induced AP-1 and NF-κB signaling in HaCaT cells through PPARα activation.

    PubMed

    Jeon, Youngsic; Jung, Yujung; Kim, Min Cheol; Kwon, Hak Cheol; Kang, Ki Sung; Kim, Yong Kee; Kim, Su-Nam

    2014-08-01

    Peroxisome proliferator-activated receptors (PPARs) are members of the nuclear hormone receptor superfamily of ligand-activated transcription factors and expressed in various cell types in the skin, including keratinocytes, fibroblasts and infiltrating immune cells. Thus, their ligands are targets for the treatment of various skin disorders, such as photo-aging and chronological aging of skin. Intensive studies have revealed that PPARα/γ functions in photo-aging and age-related inflammation by regulating matrix metalloproteinases (MMPs) via activator protein-1 (AP-1) and nuclear factor kappa B (NF-κB). However, the detailed mechanism of PPARα/γ's role in skin aging has not yet been elucidated. In this study, we confirmed that sargahydroquinoic acid (SHQA) as a PPARα/γ ligand significantly decreased Tumor Necrosis Factor-alpha (TNFα)-induced MMP-2/-9 expression by downregulating TNFα-induced transcription factors, subsequently reducing IκBα degradation and blocking NF-κB p65 nuclear translocation in HaCaT human epidermal keratinocyte cells. Treatment of cells with SHQA and GW6471 (PPARα antagonist) not bisphenol A diglycidyl ether (PPARγ antagonists), reversed the effect on TNFα-induced inflammatory signaling pathway activation. Taken together, our data suggest that SHQA inhibit TNFα-induced MMP-2/-9 expression and age-related inflammation by suppressing AP-1 and NF-κB pathway via PPARα. PMID:25019995

  1. Sp1 binds two sites in the CD11c promoter in vivo specifically in myeloid cells and cooperates with AP1 to activate transcription.

    PubMed Central

    Noti, J D; Reinemann, B C; Petrus, M N

    1996-01-01

    The leukocyte integrin gene, CD11c, is transcriptionally regulated and is expressed predominantly on differentiated cells of the myelomonocytic lineage. In this study we have demonstrated that the regions -72 to -63 and -132 to -104 of the CD11c promoter contain elements responsible for phorbol ester-induced differentiation of the myeloid cell line HL60. DNase I footprinting analysis revealed that these regions can bind purified Sp1, and supershift analysis with Sp1 antibody confirmed that Sp1 in HL60 nuclear extracts could bind these regions. Transfection analysis of CD11c promoter-chloramphenicol acetyltransferase constructs containing deletions of these Sp1-binding sites revealed that these sites are essential for expression of the CD11c gene in HL60 cells but not in the T-cell line Molt4 or the cervical carcinoma cell line HeLa. Moreover, cotransfection of pPacSp1 along with these CD11c promoter-chloramphenicol acetyltransferase constructs into Sp1-deficient Drosophila Schneider 2 cells verified that these sites are essential for Sp1-dependent expression of the CD11c promoter. In vivo genomic footprinting revealed that Sp1 contacts the CD11c promoter within the regions -69 to -63 and -116 to -105 in phorbol 12-myristate 13-acetate-differentiated HL60 cells but not in undifferentiated HL60 cells or in Molt4 or HeLa cells. Cotransfection assays in HL60 cells revealed that Sp1 acts synergistically with Ap1 to activate CD11c. Further, both Sp1 sites are capable of cooperating with AP1. In vitro DNase I footprinting analysis with purified Sp1 and c-jun proteins showed that Sp1 binding could facilitate binding of c-jun. We propose that myeloid-specific expression of the CD11c promoter and is facilitated by cooperative interaction between the Sp1- and Ap1-binding sites. PMID:8649405

  2. Benzyl alcohol derivatives from the mushroom Hericium erinaceum attenuate LPS-stimulated inflammatory response through the regulation of NF-κB and AP-1 activity.

    PubMed

    Noh, Hyung Jun; Yoon, Ju Young; Kim, Geum Sook; Lee, Seung Eun; Lee, Dae Young; Choi, Je Hun; Kim, Seung Yu; Kang, Ki Sung; Cho, Jae Youl; Kim, Ki Hyun

    2014-10-01

    On the search for anti-inflammatory compounds from natural Korean medicinal sources, a bioassay-guided fractionation and chemical investigation of the MeOH extract from the fruiting bodies of Hericium erinaceum resulted in the isolation and identification of five benzyl alcohol derivatives (1-5). In this study, their anti-inflammatory effects on lipopolysaccharide (LPS)-induced production of pro-inflammatory mediators were examined using RAW 264.7 macrophage cells. The structures of isolates were identified by comparing their spectroscopic data with previously reported values. The analysis of their inhibitory activities on LPS-induced nitric oxide (NO) and prostaglandin E2 (PGE2) production in RAW 264.7 macrophage cells showed that erinacerin B (2) and hericenone E (4) decreased the levels of NO and PGE2 production in a concentration-dependent manner. Next, this study was performed to examine their mechanism of action on the regulation of NO and PGE2 production. Compounds 2 and 4 were found to block the LPS-induced phosphorylation of two major inflammatory transcription factors, NF-κB (p65/p50) and AP-1 (c-Jun and c-Fos). Taken together, these results suggest that down-regulation of LPS-induced NO and PGE2 production by compounds 2 and 4 is mediated through the modulation of NF-κB and AP-1 activation in macrophage cells. These results impact the development of potential health products for preventing and treating inflammatory diseases. PMID:25090632

  3. Human lung and bladder carcinoma tumors as compared to their adjacent normal tissue have elevated AP-1 activity associated with the retinoblastoma gene promoter.

    PubMed

    Linardopoulos, S; Papadakis, E; Delakas, D; Theodosiou, V; Cranidis, A; Spandidos, D A

    1993-01-01

    Examination of the nucleotide sequence of the retinoblastoma (Rb) promoter revealed the presence of a DNA region highly homologous to the recognition site for the cellular transcription factor AP-1. A pair of complementary oligonucleotides containing the AP-1 site was synthesized and used in gel retardation assays to determine the role of the AP-1 protein in the regulation of the Rb gene expression. Using nuclear extracts from Hela cells as well as from lung and bladder tumors, we found specific binding of the AP-1 protein to this oligonucleotide. This binding is elevated in Hela cells, in 10/13 lung and 3/8 bladder tumors as compared to adjacent normal tissue. These results suggest that AP-1 could be implicated in Rb gene transcriptional regulation through its interaction with the AP-1 binding site of the Rb gene promoter. PMID:8476221

  4. Cutaneous wound healing through paradoxical MAPK activation by BRAF inhibitors.

    PubMed

    Escuin-Ordinas, Helena; Li, Shuoran; Xie, Michael W; Sun, Lu; Hugo, Willy; Huang, Rong Rong; Jiao, Jing; de-Faria, Felipe Meira; Realegeno, Susan; Krystofinski, Paige; Azhdam, Ariel; Komenan, Sara Marie D; Atefi, Mohammad; Comin-Anduix, Begoña; Pellegrini, Matteo; Cochran, Alistair J; Modlin, Robert L; Herschman, Harvey R; Lo, Roger S; McBride, William H; Segura, Tatiana; Ribas, Antoni

    2016-01-01

    BRAF inhibitors are highly effective therapies for the treatment of BRAF(V600)-mutated melanoma, with the main toxicity being a variety of hyperproliferative skin conditions due to paradoxical activation of the mitogen-activated protein kinase (MAPK) pathway in BRAF wild-type cells. Most of these hyperproliferative skin changes improve when a MEK inhibitor is co-administered, as it blocks paradoxical MAPK activation. Here we show how the BRAF inhibitor vemurafenib accelerates skin wound healing by inducing the proliferation and migration of human keratinocytes through extracellular signal-regulated kinase (ERK) phosphorylation and cell cycle progression. Topical treatment with vemurafenib in two wound-healing mice models accelerates cutaneous wound healing through paradoxical MAPK activation; addition of a mitogen-activated protein kinase kinase (MEK) inhibitor reverses the benefit of vemurafenib-accelerated wound healing. The same dosing regimen of topical BRAF inhibitor does not increase the incidence of cutaneous squamous cell carcinomas in mice. Therefore, topical BRAF inhibitors may have clinical applications in accelerating the healing of skin wounds. PMID:27476449

  5. Cutaneous wound healing through paradoxical MAPK activation by BRAF inhibitors

    PubMed Central

    Escuin-Ordinas, Helena; Li, Shuoran; Xie, Michael W.; Sun, Lu; Hugo, Willy; Huang, Rong Rong; Jiao, Jing; de-Faria, Felipe Meira; Realegeno, Susan; Krystofinski, Paige; Azhdam, Ariel; Komenan, Sara Marie D.; Atefi, Mohammad; Comin-Anduix, Begoña; Pellegrini, Matteo; Cochran, Alistair J.; Modlin, Robert L.; Herschman, Harvey R.; Lo, Roger S.; McBride, William H.; Segura, Tatiana; Ribas, Antoni

    2016-01-01

    BRAF inhibitors are highly effective therapies for the treatment of BRAFV600-mutated melanoma, with the main toxicity being a variety of hyperproliferative skin conditions due to paradoxical activation of the mitogen-activated protein kinase (MAPK) pathway in BRAF wild-type cells. Most of these hyperproliferative skin changes improve when a MEK inhibitor is co-administered, as it blocks paradoxical MAPK activation. Here we show how the BRAF inhibitor vemurafenib accelerates skin wound healing by inducing the proliferation and migration of human keratinocytes through extracellular signal-regulated kinase (ERK) phosphorylation and cell cycle progression. Topical treatment with vemurafenib in two wound-healing mice models accelerates cutaneous wound healing through paradoxical MAPK activation; addition of a mitogen-activated protein kinase kinase (MEK) inhibitor reverses the benefit of vemurafenib-accelerated wound healing. The same dosing regimen of topical BRAF inhibitor does not increase the incidence of cutaneous squamous cell carcinomas in mice. Therefore, topical BRAF inhibitors may have clinical applications in accelerating the healing of skin wounds. PMID:27476449

  6. Bamboo extract reduces interleukin 6 (IL-6) overproduction under lipotoxic conditions through inhibiting the activation of NF-κB and AP-1 pathways.

    PubMed

    Higa, Jason K; Panee, Jun

    2011-07-01

    Interleukin 6 (IL-6) is an inflammatory cytokine overexpressed in obese individuals that contributes to the development of diseases such as insulin resistance, type 2 diabetes, and cardiovascular disease. This study investigated the inhibitory effect of an extract from the bamboo Phyllostachys edulis (BEX) on lipotoxicity-induced over-production of IL-6 in metabolic cell lines. Palmitic acid (PA, 0.4mM) was used to induce lipotoxicity in murine C2C12, 3T3-L1, and Hepa6 cells. Both intra- and extra-cellular protein concentrations of IL-6 were measured in the three cell lines after PA treatment with or without the presence of BEX using cytometric bead assays. IL-6 mRNA levels were quantified using real-time PCR, and nuclear concentrations of c-fos, p50 and p65 proteins were measured using DNA-binding ELISA in 3T3-L1 cells. Lipotoxicity increased IL-6 protein concentration in both cytosol and media collected from myoblast and myotube C2C12, as well as preadipose and adipose 3T3-L1, and the presence of BEX (0.5%, v/v) effectively inhibited this overproduction. IL-6 protein expression in hepatic Hepa6 cells was less affected by lipotoxicity. BEX significantly ameliorated PA-induced upregulation of IL-6 mRNA, which correlated with a reduction in nuclear translocation of p50, p65, and c-fos proteins with the presence of BEX, indicating inhibition of NF-κB and AP-1 activation. In summary, BEX inhibits lipotoxicity-induced IL-6 overproduction in muscle and adipose cell lines through the NF-κB and AP-1 pathways, implicating a potential application of this natural product as a cost-effective anti-inflammation nutraceutical. PMID:21474329

  7. p38γ and p38δ Mitogen Activated Protein Kinases (MAPKs), New Stars in the MAPK Galaxy

    PubMed Central

    Escós, Alejandra; Risco, Ana; Alsina-Beauchamp, Dayanira; Cuenda, Ana

    2016-01-01

    The protein kinases p38γ and p38δ belong to the p38 mitogen-activated protein kinase (MAPK) family. p38MAPK signaling controls many cellular processes and is one of the most conserved mechanisms in eukaryotes for the cellular response to environmental stress and inflammation. Although p38γ and p38δ are widely expressed, it is likely that they perform specific functions in different tissues. Their involvement in human pathologies such as inflammation-related diseases or cancer is starting to be uncovered. In this article we give a general overview and highlight recent advances made in defining the functions of p38γ and p38δ, focusing in innate immunity and inflammation. We consider the potential of the pharmacological targeting of MAPK pathways to treat autoimmune and inflammatory diseases and cancer. PMID:27148533

  8. CR3 and Dectin-1 Collaborate in Macrophage Cytokine Response through Association on Lipid Rafts and Activation of Syk-JNK-AP-1 Pathway.

    PubMed

    Huang, Juin-Hua; Lin, Ching-Yu; Wu, Sheng-Yang; Chen, Wen-Yu; Chu, Ching-Liang; Brown, Gordon D; Chuu, Chih-Pin; Wu-Hsieh, Betty A

    2015-07-01

    Collaboration between heterogeneous pattern recognition receptors (PRRs) leading to synergistic coordination of immune response is important for the host to fight against invading pathogens. Although complement receptor 3 (CR3) and Dectin-1 are major PRRs to detect fungi, crosstalk between these two receptors in antifungal immunity is largely undefined. Here we took advantage of Histoplasma capsulatum which is known to interact with both CR3 and Dectin-1 and specific particulate ligands to study the collaboration of CR3 and Dectin-1 in macrophage cytokine response. By employing Micro-Western Array (MWA), genetic approach, and pharmacological inhibitors, we demonstrated that CR3 and Dectin-1 act collaboratively to trigger macrophage TNF and IL-6 response through signaling integration at Syk kinase, allowing subsequent enhanced activation of Syk-JNK-AP-1 pathway. Upon engagement, CR3 and Dectin-1 colocalize and form clusters on lipid raft microdomains which serve as a platform facilitating their cooperation in signaling activation and cytokine production. Furthermore, in vivo studies showed that CR3 and Dectin-1 cooperatively participate in host defense against disseminated histoplasmosis and instruct adaptive immune response. Taken together, our findings define the mechanism of receptor crosstalk between CR3 and Dectin-1 and demonstrate the importance of their collaboration in host defense against fungal infection. PMID:26132276

  9. Nanofiber diameter-dependent MAPK activity in osteoblasts.

    PubMed

    Jaiswal, Devina; Brown, Justin L

    2012-11-01

    The major challenge for bone tissue engineering lies in the fabrication of scaffolds that can mimic the extracellular matrix and promote osteogenesis. Electrospun fibers are being widely researched for this application due to high porosity, interconnectivity, and mechanical strength of the fibrous scaffolds. Electrospun poly methyl methacrylate (PMMA, 2.416 ± 0.100 μm) fibers were fabricated and etched using a 60% propylene glycol methyl ether acetate (PGMEA)/limonene (vol/vol) solution to obtain fiber diameters ranging from 2.5 to 0.5 μm in a time-dependent manner. The morphology of the fibrous scaffolds was evaluated using scanning electron microscopy and cellular compatibility with etchant-treated scaffold was assessed using immunoflurescence. Mitogen-activated protein kinases (MAPK) activation in response to different fiber diameter was evaluated with western blot as well as quantitative in-cell western. We report that electrospun micro-fibers can be etched to 0.552 ± 0.047 μm diameter without producing beads. Osteoblasts adhered to the fibers and a change in fiber diameter played a major role in modulating the activation of extracellular signal-regulated kinase (ERK) and p38 kinases with 0.882 ± 0.091 μm diameter fibers producing an inverse effect on ERK and p38 phosphorylation. These results indicate that nanofibers produced by wet etching can be effectively utilized to produce diameters that can differentially modulate MAPK activation patterns. PMID:22700490

  10. Staphylococcus aureus induces TGF-β1 and bFGF expression through the activation of AP-1 and NF-κB transcription factors in bovine mammary gland fibroblasts.

    PubMed

    Wu, Jianmei; Ding, Yulin; Bi, Yannan; Wang, Yi; Zhi, Yu; Wang, Jinling; Wang, Fenglong

    2016-06-01

    Staphylococcus aureus is a common Gram-positive pathogen that causes bovine mastitis, a persistent infection of the bovine mammary gland. To better understand the importance of bovine mammary fibroblasts (BMFBs) and the roles of the TLR-NF-κB and TLR-AP-1 signaling pathways in the regulation of S. aureus-associated mastitis and mammary fibosis, BMFBs cultured in vitro were stimulated with different concentrations of heat-inactivated S. aureus to analyze the gene and protein expression of toll-like receptor 2 (TLR2), toll-like receptor 4 (TLR4), transforming growth factor beta 1 (TGF-β1), basic fibroblast growth factor (bFGF) as well as the protein expression of nuclear factor-kappa B (NF-κB) and activation protein-1 (AP-1) by means of quantitative polymerase chain reaction (qPCR) and western blotting, respectively. Specific NF-κB and AP-1 inhibitors were also used to investigate their effects on the regulation of TGF-β1 and bFGF expression. The results indicated that, in addition to increasing mRNA and protein expression of TLR2 and TLR4, S. aureus could also upregulate TGF-β1 and bFGF mRNA expression and secretion through the activation of NF-κB and AP-1. The increase in TGF-β1 and bFGF expression was shown to be inhibited by AP-1- and NF-κB-specific inhibitors. Taken together, S. aureus induces TGF-β1 and bFGF expression through the activation of AP-1 and NF-κB in BMFBs. This information offers new potential targets for the treatment of bovine mammary fibrosis. PMID:26948281

  11. Anti-inflammatory activity of edible oyster mushroom is mediated through the inhibition of NF-κB and AP-1 signaling

    PubMed Central

    2011-01-01

    Background Mushrooms are well recognized for their culinary properties as well as for their potency to enhance immune response. In the present study, we evaluated anti-inflammatory properties of an edible oyster mushroom (Pleurotus ostreatus) in vitro and in vivo. Methods RAW264.7 murine macrophage cell line and murine splenocytes were incubated with the oyster mushroom concentrate (OMC, 0-100 μg/ml) in the absence or presence of lipopolysacharide (LPS) or concanavalin A (ConA), respectively. Cell proliferation was determined by MTT assay. Expression of cytokines and proteins was measured by ELISA assay and Western blot analysis, respectively. DNA-binding activity was assayed by the gel-shift analysis. Inflammation in mice was induced by intraperitoneal injection of LPS. Results OMC suppressed LPS-induced secretion of tumor necrosis factor-α (TNF-α, interleukin-6 (IL-6), and IL-12p40 from RAW264.7 macrophages. OMC inhibited LPS-induced production of prostaglandin E2 (PGE2) and nitric oxide (NO) through the down-regulation of expression of COX-2 and iNOS, respectively. OMC also inhibited LPS-dependent DNA-binding activity of AP-1 and NF-κB in RAW264.7 cells. Oral administration of OMC markedly suppressed secretion of TNF-α and IL-6 in mice challenged with LPS in vivo. Anti-inflammatory activity of OMC was confirmed by the inhibition of proliferation and secretion of interferon-γ (IFN-γ), IL-2, and IL-6 from concanavalin A (ConA)-stimulated mouse splenocytes. Conclusions Our study suggests that oyster mushroom possesses anti-inflammatory activities and could be considered a dietary agent against inflammation. The health benefits of the oyster mushroom warrant further clinical studies. PMID:21575254

  12. The human papillomavirus type 16 E7 gene product interacts with and trans-activates the AP1 family of transcription factors.

    PubMed Central

    Antinore, M J; Birrer, M J; Patel, D; Nader, L; McCance, D J

    1996-01-01

    The E7 gene product of human papillomavirus type 16 (HPV16) binds to the retinoblastoma gene product (pRb) and dissociates pRb-E2F complexes. However, the observation that the ability of E7 to bind pRb is not required for the HPV16-induced immortalization of primary keratinocytes prompted a search for other cellular factors bound by E7. Using a glutathione-S-transferase (GST) fusion protein system, we show that E7 complexes with AP1 transcription factors including c-Jun, JunB, JunD and c-Fos. The ability of E7 to complex with c-Jun in vivo is demonstrated by co-immunoprecipitation and the yeast two-hybrid system. An analysis of E7 point mutants in the GST system indicates that the E7 zinc-finger motif, but not the pRb binding domain, is involved in these interactions. Using c-Jun deletion mutants, E7 binding maps between amino acids 224 and 286 of c-Jun. E7 trans-activates c-Jun-induced transcription from a Jun responsive promoter, and this activity correlates with the ability of E7 mutants to bind Jun proteins. Finally, a transcriptionally inactive c-Jun deletion, which can bind E7, interferes with the E7-induced transformation of rat embryo fibroblasts in cooperation with an activated ras, indicating that the Jun-E7 interaction is physiologically relevant and that Jun factors may be targeted in the E7 transformation pathway. Images PMID:8617242

  13. Evidence for the Involvement of p38 MAPK Activation in Barnacle Larval Settlement

    PubMed Central

    He, Li-Sheng; Xu, Ying; Matsumura, Kiyotaka; Zhang, Yu; Zhang, Gen; Qi, Shu-Hua; Qian, Pei-Yuan

    2012-01-01

    The barnacle Balanus ( = Amphibalanus) amphitrite is a major marine fouling animal. Understanding the molecular mechanism of larval settlement in this species is critical for anti-fouling research. In this study, we cloned one isoform of p38 MAPK (Bar-p38 MAPK) from this species, which shares the significant characteristic of containing a TGY motif with other species such as yeast, Drosophila and humans. The activation of p38 MAPK was detected by an antibody that recognizes the conserved dual phosphorylation sites of TGY. The results showed that phospho-p38 MAPK (pp38 MAPK) was more highly expressed at the cyprid stage, particularly in aged cyprids, in comparison to other stages, including the nauplius and juvenile stages. Immunostaining showed that Bar-p38 MAPK and pp38 MAPK were mainly located at the cyprid antennules, and especially the third and fourth segments, which are responsible for substratum exploration during settlement. The expression and localization patterns of Bar-p38 MAPK suggest its involvement in larval settlement. This postulation was also supported by the larval settlement bioassay with the p38 MAPK inhibitor SB203580. Behavioral analysis by live imaging revealed that the larvae were still capable of exploring the surface of the substratum after SB203580 treatment. This shows that the effect of p38 MAPK on larval settlement might be by regulating the secretion of permanent proteinaceous substances. Furthermore, the level of pp38 MAPK dramatically decreased after full settlement, suggesting that Bar-p38 MAPK maybe plays a role in larval settlement rather than metamorphosis. Finally, we found that Bar-p38 MAPK was highly activated when larvae confronted extracts of adult barnacle containing settlement cues, whereas larvae pre-treated with SB203580 failed to respond to the crude adult extracts. PMID:23115639

  14. A novel synthetic derivative of the natural product berbamine inhibits cell viability and induces apoptosis of human osteosarcoma cells, associated with activation of JNK/AP-1 signaling.

    PubMed

    Yang, Fan; Nam, Sangkil; Zhao, Robin; Tian, Yan; Liu, Lucy; Horne, David A; Jove, Richard

    2013-11-01

    Osteosarcoma is the most common primary bone tumor in children and adolescents. There is a critical need to find more potent drugs for patients with metastatic or recurrent disease. Berbamine (BBM) is a natural compound derived from the Berberis amurensis plants. BBM and its derivatives have been shown to have antitumor effects in several cancers. Here, we report that a novel synthetic berbamine derivative, BBMD3, inhibits cell viability and induces apoptosis of G292, KHOS, and MG-63 human osteosarcoma cells. Induction of apoptosis in these tumor cells depends on activation of caspase-3 and cleavage of poly(ADP-ribose) polymerase (PARP). Since pan-caspase inhibitor (Z-VAD-FMK) and caspase-9 inhibitor (Z-LEHD-FMK) could block the cleavage of PARP, the apoptosis induced by BBMD3 is through intrinsic signaling pathway. BBMD3 increased phosphorylation of c-Jun N-terminal kinase (JNK)/stress-activated protein kinase (SAPK), resulting in increase of phosphorylated c-Jun and total c-Fos, the major components of transcriptional factor AP-1. JNK inhibitor could partially suppress antitumor effect of BBMD3 on osteosarcoma cells. BBMD3 increased the production of reactive oxygen species (ROS) and ROS scavenger, N-acetylcysteine (NAC), could block the phosphorylation of JNK and c-Jun induced by BBMD3. BBMD3 increased the expression of the pro-apototic gene Bad, associated with apoptosis induction. Finally, BBMD3 also decreased the expression of cyclin D1 and D2, the positive cell cycle regulators, which is correlated with growth inhibition in osteosarcoma cells. Collectively, these findings indicate that BBMD3 is a potentially promising drug for the treatment of human osteosarcoma. PMID:24025361

  15. A novel synthetic derivative of the natural product berbamine inhibits cell viability and induces apoptosis of human osteosarcoma cells, associated with activation of JNK/AP-1 signaling

    PubMed Central

    Yang, Fan; Nam, Sangkil; Zhao, Robin; Tian, Yan; Liu, Lucy; Horne, David A; Jove, Richard

    2013-01-01

    Osteosarcoma is the most common primary bone tumor in children and adolescents. There is a critical need to find more potent drugs for patients with metastatic or recurrent disease. Berbamine (BBM) is a natural compound derived from the Berberis amurensis plants. BBM and its derivatives have been shown to have antitumor effects in several cancers. Here, we report that a novel synthetic berbamine derivative, BBMD3, inhibits cell viability and induces apoptosis of G292, KHOS, and MG-63 human osteosarcoma cells. Induction of apoptosis in these tumor cells depends on activation of caspase-3 and cleavage of poly(ADP-ribose) polymerase (PARP). Since pan-caspase inhibitor (Z-VAD-FMK) and caspase-9 inhibitor (Z-LEHD-FMK) could block the cleavage of PARP, the apoptosis induced by BBMD3 is through intrinsic signaling pathway. BBMD3 increased phosphorylation of c-Jun N-terminal kinase (JNK)/stress-activated protein kinase (SAPK), resulting in increase of phosphorylated c-Jun and total c-Fos, the major components of transcriptional factor AP-1. JNK inhibitor could partially suppress antitumor effect of BBMD3 on osteosarcoma cells. BBMD3 increased the production of reactive oxygen species (ROS) and ROS scavenger, N-acetylcysteine (NAC), could block the phosphorylation of JNK and c-Jun induced by BBMD3. BBMD3 increased the expression of the pro-apototic gene Bad, associated with apoptosis induction. Finally, BBMD3 also decreased the expression of cyclin D1 and D2, the positive cell cycle regulators, which is correlated with growth inhibition in osteosarcoma cells. Collectively, these findings indicate that BBMD3 is a potentially promising drug for the treatment of human osteosarcoma. PMID:24025361

  16. Identification of GATA2 and AP-1 activator elements within the enhancer VNTR occurring in intron 5 of the human SIRT3 gene

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Human SIRT3 gene contains an intronic VNTR enhancer. A T > C transition occurring in the second repeat of each VNTR allele implies the presence/absence of a putative GATA binding motif. A partially overlapping AP-1 site, not affected by the transition, was also identified. Aims of the present study ...

  17. Characterization of the human granulocyte-macrophage colony-stimulating factor gene promoter: an AP1 complex and an Sp1-related complex transactivate the promoter activity that is suppressed by a YY1 complex.

    PubMed Central

    Ye, J; Zhang, X; Dong, Z

    1996-01-01

    It is well documented that a repeated CATT element in the human granulocyte-macrophage colony-stimulating factor (GM-CSF) gene promoter is required for promoter activity. However, the transcription factors that are able to transactivate this enhancer element remain unidentified. Recently, we have found that nuclear factor YY1 can interact with the enhancer element. Here, we report that in addition to YY1, two other nuclear factors have been identified in the DNA-protein complexes formed by the CATT oligonucleotide and the Jurkat T-cell nuclear protein. One of these factors is AP1, and the other one is an Sp1-related protein. Results from transient transfection of Jurkat T cells have revealed that formation of both AP1 and the Sp1-related complex is required for the full enhancer activity of the CATT element. This result is supported by cotransfection of a c-jun expression vector and mutational analysis of the AP1 site or the Sp1-related protein binding site. In contrast, formation of the YY1 complex suppresses enhancer activity, since deletion of the YY1 complex induces an augmentation of the enhancer activity and overexpression of YY1 results in an attenuation of the enhancer activity. Results from the mechanism study have revealed that YY1 is able to inhibit transactivation mediated by either AP1 or the Sp1-related protein, and YY1 suppressive activity is DNA binding dependent. Taken together, these data support the ideas that AP1 and the Sp1-related nuclear protein are required for transactivation of the human GM-CSF gene promoter and that YY1 can suppress transactivation of the promoter even under inducible conditions. PMID:8524292

  18. Identification, Nomenclature, and Evolutionary Relationships of Mitogen-Activated Protein Kinase (MAPK) Genes in Soybean

    PubMed Central

    Neupane, Achal; Nepal, Madhav P.; Piya, Sarbottam; Subramanian, Senthil; Rohila, Jai S.; Reese, R. Neil; Benson, Benjamin V.

    2013-01-01

    Mitogen-activated protein kinase (MAPK) genes in eukaryotes regulate various developmental and physiological processes including those associated with biotic and abiotic stresses. Although MAPKs in some plant species including Arabidopsis have been identified, they are yet to be identified in soybean. Major objectives of this study were to identify GmMAPKs, assess their evolutionary relationships, and analyze their functional divergence. We identified a total of 38 MAPKs, eleven MAPKKs, and 150 MAPKKKs in soybean. Within the GmMAPK family, we also identified a new clade of six genes: four genes with TEY and two genes with TQY motifs requiring further investigation into possible legume-specific functions. The results indicated the expansion of the GmMAPK families attributable to the ancestral polyploidy events followed by chromosomal rearrangements. The GmMAPK and GmMAPKKK families were substantially larger than those in other plant species. The duplicated GmMAPK members presented complex evolutionary relationships and functional divergence when compared to their counterparts in Arabidopsis. We also highlighted existing nomenclatural issues, stressing the need for nomenclatural consistency. GmMAPK identification is vital to soybean crop improvement, and novel insights into the evolutionary relationships will enhance our understanding about plant genome evolution. PMID:24137047

  19. Hypertonic saline activation of p38 MAPK primes the PMN respiratory burst.

    PubMed

    Ciesla, D J; Moore, E E; Biffl, W L; Gonzalez, R J; Moore, H B; Silliman, C C

    2001-10-01

    Investigation of hypertonic saline (HTS) modulation of neutrophils (PMN) cytotoxic responses has generated seemingly contradictory results. Clinically relevant levels of HTS attenuate receptor-mediated p38 MAPK signaling, whereas higher levels activate p38 MAPK. Concurrently, HTS exerts a dose-dependent attenuation of the PMN respiratory burst, most notably at concentrations where p38 MAPK is activated. We hypothesized that HTS-mediated p38 MAPK activation augments the PMN respiratory burst on return to normotonicity. We found that although clinically relevant levels of HTS (Na+ > or = 200 mM) did not activate p38 MAPK, higher concentrations (Na+ > or = 300 mM) resulted in activation comparable with that after PAF stimulation. Transient stimulation with high levels of HTS primed the PMN respiratory burst in response to fMLP and PMA. This effect was attenuated by pretreatment with SB 203580, a p38 MAPK specific inhibitor. We conclude that severe osmotic shock primes the respiratory burst via p38 MAPK signaling, further supporting the role of this signaling cascade in PMN priming. PMID:11580111

  20. Ras-GTP dimers activate the Mitogen-Activated Protein Kinase (MAPK) pathway

    PubMed Central

    Nan, Xiaolin; Tamgüney, Tanja M.; Collisson, Eric A.; Lin, Li-Jung; Pitt, Cameron; Galeas, Jacqueline; Lewis, Sophia; Gray, Joe W.; McCormick, Frank; Chu, Steven

    2015-01-01

    Rat sarcoma (Ras) GTPases regulate cell proliferation and survival through effector pathways including Raf-MAPK, and are the most frequently mutated genes in human cancer. Although it is well established that Ras activity requires binding to both GTP and the membrane, details of how Ras operates on the cell membrane to activate its effectors remain elusive. Efforts to target mutant Ras in human cancers to therapeutic benefit have also been largely unsuccessful. Here we show that Ras-GTP forms dimers to activate MAPK. We used quantitative photoactivated localization microscopy (PALM) to analyze the nanoscale spatial organization of PAmCherry1-tagged KRas 4B (hereafter referred to KRas) on the cell membrane under various signaling conditions. We found that at endogenous expression levels KRas forms dimers, and KRasG12D, a mutant that constitutively binds GTP, activates MAPK. Overexpression of KRas leads to formation of higher order Ras nanoclusters. Conversely, at lower expression levels, KRasG12D is monomeric and activates MAPK only when artificially dimerized. Moreover, dimerization and signaling of KRas are both dependent on an intact CAAX (C, cysteine; A, aliphatic; X, any amino acid) motif that is also known to mediate membrane localization. These results reveal a new, dimerization-dependent signaling mechanism of Ras, and suggest Ras dimers as a potential therapeutic target in mutant Ras-driven tumors. PMID:26080442

  1. Ras-GTP dimers activate the mitogen-activated protein kinase (MAPK) pathway

    DOE PAGESBeta

    Nan, Xiaolin; Tamgüney, Tanja M.; Collisson, Eric A.; Lin, Li -Jung; Pitt, Cameron; Galeas, Jacqueline; Lewis, Sophia; Gray, Joe W.; McCormick, Frank; Chu, Steven

    2015-06-16

    Rat sarcoma (Ras) GTPases regulate cell proliferation and survival through effector pathways including Raf-MAPK, and are the most frequently mutated genes in human cancer. Although it is well established that Ras activity requires binding to both GTP and the membrane, details of how Ras operates on the cell membrane to activate its effectors remain elusive. Efforts to target mutant Ras in human cancers to therapeutic benefit have also been largely unsuccessful. Here we show that Ras-GTP forms dimers to activate MAPK. We used quantitative photoactivated localization microscopy (PALM) to analyze the nanoscale spatial organization of PAmCherry1-tagged KRas 4B (hereafter referredmore » to KRas) on the cell membrane under various signaling conditions. We found that at endogenous expression levels KRas forms dimers, and KRasG12D, a mutant that constitutively binds GTP, activates MAPK. Overexpression of KRas leads to formation of higher order Ras nanoclusters. Conversely, at lower expression levels, KRasG12D is monomeric and activates MAPK only when artificially dimerized. Moreover, dimerization and signaling of KRas are both dependent on an intact CAAX (C, cysteine; A, aliphatic; X, any amino acid) motif that is also known to mediate membrane localization. These results reveal a new, dimerization-dependent signaling mechanism of Ras, and suggest Ras dimers as a potential therapeutic target in mutant Ras-driven tumors.« less

  2. Ras-GTP dimers activate the mitogen-activated protein kinase (MAPK) pathway

    SciTech Connect

    Nan, Xiaolin; Tamgüney, Tanja M.; Collisson, Eric A.; Lin, Li -Jung; Pitt, Cameron; Galeas, Jacqueline; Lewis, Sophia; Gray, Joe W.; McCormick, Frank; Chu, Steven

    2015-06-16

    Rat sarcoma (Ras) GTPases regulate cell proliferation and survival through effector pathways including Raf-MAPK, and are the most frequently mutated genes in human cancer. Although it is well established that Ras activity requires binding to both GTP and the membrane, details of how Ras operates on the cell membrane to activate its effectors remain elusive. Efforts to target mutant Ras in human cancers to therapeutic benefit have also been largely unsuccessful. Here we show that Ras-GTP forms dimers to activate MAPK. We used quantitative photoactivated localization microscopy (PALM) to analyze the nanoscale spatial organization of PAmCherry1-tagged KRas 4B (hereafter referred to KRas) on the cell membrane under various signaling conditions. We found that at endogenous expression levels KRas forms dimers, and KRasG12D, a mutant that constitutively binds GTP, activates MAPK. Overexpression of KRas leads to formation of higher order Ras nanoclusters. Conversely, at lower expression levels, KRasG12D is monomeric and activates MAPK only when artificially dimerized. Moreover, dimerization and signaling of KRas are both dependent on an intact CAAX (C, cysteine; A, aliphatic; X, any amino acid) motif that is also known to mediate membrane localization. These results reveal a new, dimerization-dependent signaling mechanism of Ras, and suggest Ras dimers as a potential therapeutic target in mutant Ras-driven tumors.

  3. Pycnogenol Attenuates the Release of Proinflammatory Cytokines and Expression of Perilipin 2 in Lipopolysaccharide-Stimulated Microglia in Part via Inhibition of NF-κB and AP-1 Activation.

    PubMed

    Fan, Bin; Dun, Sai-Hong; Gu, Jian-Qiu; Guo, Yang; Ikuyama, Shoichiro

    2015-01-01

    Over activation of microglia results in the production of proinflammatory agents that have been implicated in various brain diseases. Pycnogenol is a patented extract from French maritime pine bark (Pinus pinaster Aiton) with strong antioxidant and anti-inflammatory potency. The present study investigated whether pycnogenol may be associated with the production of proinflammatory mediators in lipopolysaccharide-stimulated BV2 (mouse-derived) microglia. It was found that pycnogenol treatment was dose-dependently associated with significantly less release of nitricoxide (NO), TNF-α, IL-6 and IL-1β, and lower levels of intercellular adhesion molecule1 (ICAM-1) and perilipin 2 (PLIN2). Furthermore, this effect was replicated in primary brain microglia. Levels of inducible NO synthase mRNA and protein were attenuated, whereas there was no change in the production of the anti-inflammatory cytokine IL-10. Further evidence indicated that pycnogenol treatment led to the suppression of NF-κB activation through inhibition of p65 translocation into the nucleus and inhibited DNA binding of AP-1, suggesting that these proinflammatory factors are associated with NF-κB and AP-1. We conclude that pycnogenol exerts anti-inflammatory effects through inhibition of the NF-κB and AP-1pathway, and may be useful as a therapeutic agent in the prevention of diseases caused by over activation of microglia. PMID:26367267

  4. Pycnogenol Attenuates the Release of Proinflammatory Cytokines and Expression of Perilipin 2 in Lipopolysaccharide-Stimulated Microglia in Part via Inhibition of NF-κB and AP-1 Activation

    PubMed Central

    Fan, Bin; Dun, Sai-Hong; Gu, Jian-Qiu; Guo, Yang; Ikuyama, Shoichiro

    2015-01-01

    Over activation of microglia results in the production of proinflammatory agents that have been implicated in various brain diseases. Pycnogenol is a patented extract from French maritime pine bark (Pinus pinaster Aiton) with strong antioxidant and anti-inflammatory potency. The present study investigated whether pycnogenol may be associated with the production of proinflammatory mediators in lipopolysaccharide-stimulated BV2 (mouse-derived) microglia. It was found that pycnogenol treatment was dose-dependently associated with significantly less release of nitricoxide (NO), TNF-α, IL-6 and IL-1β, and lower levels of intercellular adhesion molecule1 (ICAM-1) and perilipin 2 (PLIN2). Furthermore, this effect was replicated in primary brain microglia. Levels of inducible NO synthase mRNA and protein were attenuated, whereas there was no change in the production of the anti-inflammatory cytokine IL-10. Further evidence indicated that pycnogenol treatment led to the suppression of NF-κB activation through inhibition of p65 translocation into the nucleus and inhibited DNA binding of AP-1, suggesting that these proinflammatory factors are associated with NF-κB and AP-1. We conclude that pycnogenol exerts anti-inflammatory effects through inhibition of the NF-κB and AP-1pathway, and may be useful as a therapeutic agent in the prevention of diseases caused by over activation of microglia. PMID:26367267

  5. An Estrogen Receptor-α/p300 Complex Activates the BRCA-1 Promoter at an AP-1 Site That Binds Jun/Fos Transcription Factors: Repressive Effects of p53 on BRCA-1 Transcription1

    PubMed Central

    Jeffy, Brandon D; Hockings, Jennifer K; Kemp, Michael Q; Morgan, Sherif S; Hager, Jill A; Beliakoff, Jason; Whitesell, Luke J; Bowden, G. Timothy; Romagnolo, Donato F

    2005-01-01

    Abstract One of the puzzles in cancer predisposition is that women carrying BRCA-1 mutations preferentially develop tumors in epithelial tissues of the breast and ovary. Moreover, sporadic breast tumors contain lower levels of BRCA-1 in the absence of mutations in the BRCA-1 gene. The problem of tissue specificity requires analysis of factors that are unique to tissues of the breast. For example, the expression of estrogen receptor-α (ERα) is inversely correlated with breast cancer risk, and 90% of BRCA-1 tumors are negative for ERα. Here, we show that estrogen stimulates BRCA-1 promoter activity in transfected cells and the recruitment of ERα and its cofactor p300 to an AP-1 site that binds Jun/Fos transcription factors. The recruitment of ERα/p300 coincides with accumulation in the S-phase of the cell cycle and is antagonized by the antiestrogen tamoxifen. Conversely, we document that overexpression of wild-type p53 prevents the recruitment of ERα to the AP-1 site and represses BRCA-1 promoter activity. Taken together, our findings support a model in which an ERα/AP-1 complex modulates BRCA-1 transcription under conditions of estrogen stimulation. Conversely, the formation of this transcription complex is abrogated in cells overexpressing p53. PMID:16229810

  6. Nucleolin binds specifically to an AP-1 DNA sequence and represses AP1-dependent transactivation of the matrix metalloproteinase-13 gene.

    PubMed

    Samuel, Shaija; Twizere, Jean-Claude; Beifuss, Katherine K; Bernstein, Lori R

    2008-01-01

    Transcriptional regulation via activator protein-1 (AP-1) protein binding to AP-1 binding sites within gene promoter regions of AP-1 target genes plays a key role in controlling cellular invasion, proliferation, and oncogenesis, and is important to pathogenesis of arthritis and cardiovascular disease. To identify new proteins that interact with the AP-1 DNA binding site, we performed the DNA affinity chromatography-based Nucleotide Affinity Preincubation Specificity TEst of Recognition (NAPSTER) assay, and discovered a 97 kDa protein that binds in vitro to a minimal AP-1 DNA sequence element. Mass spectrometric fragmentation sequencing determined that p97 is nucleolin. Immunoblotting of DNA affinity-purified material with anti-nucleolin antibodies confirmed this identification. Nucleolin also binds the AP-1 site in gel shift assays. Nucleolin interacts in NAPSTER with the AP-1 site within the promoter sequence of the metalloproteinase-13 gene (MMP-13), and binds in vivo in chromatin immunoprecipitation assays in the vicinity of the AP-1 site in the MMP-13 promoter. Overexpression of nucleolin in human HeLa cervical carcinoma cells significantly represses AP-1 dependent gene transactivation of a minimal AP-1 reporter construct and of an MMP-13 promoter reporter sequence. This is the first report of nucleolin binding and transregulation at the AP-1 site. PMID:17626252

  7. Reciprocal effects of varicella-zoster virus (VZV) and AP1: activation of jun, fos and ATF-2 after VZV infection and their importance for the regulation of viral genes.

    PubMed

    Rahaus, Markus; Wolff, Manfred H

    2003-03-01

    Varicella-zoster virus, an alpha-herpesvirus that is pathogenic for man, encodes its own transcription activators, but ubiquitous cellular transcription factors are of great importance, too. Performing quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) we found an increase of transcription of AP1 components jun, fos and of ATF-2 at different times post infection (p.i.). Jun and ATF-2 proteins were detected in infected cells. To study general effects of AP1 in the regulation of VZV encoded genes, oligonucleotide transfections were performed to knockout jun and ATF-2 transcription followed by infection with cell free VZV. RT-PCR analyses of ORFs 4, 9, 21, 29 and 68 belonging to all three kinetic classes of genes and containing different combinations of AP1/TRE and ATF/CREB sites in their respective promoters were carried out. In all cases a reduction of viral transcription was found. Virions produced after this procedure were still infectious, but the amount of newly synthesized virions was clearly reduced. PMID:12606072

  8. Id-1 is induced in MDCK epithelial cells by activated Erk/MAPK pathway in response to expression of the Snail and E47 transcription factors

    SciTech Connect

    Jorda, Mireia; Vinyals, Antonia; Marazuela, Anna; Cubillo, Eva; Olmeda, David; Valero, Eva; Cano, Amparo; Fabra, Angels . E-mail: afabra@idibell.org

    2007-07-01

    Id-1, a member of the helix-loop-helix transcription factor family has been shown to be involved in cell proliferation, angiogenesis and invasion of many types of human cancers. We have previously shown that stable expression of E47 and Snail repressors of the E-cadherin promoter in MDCK epithelial cell line triggers epithelial mesenchymal transition (EMT) concomitantly with changes in gene expression. We show here that both factors activate the Id-1 gene promoter and induce Id-1 mRNA and protein. The upregulation of the Id-1 gene occurs through the transactivation of the promoter by the Erk/MAPK signaling pathway. Moreover, oncogenic Ras is also able to activate Id-1 promoter in MDCK cells in the absence of both E47 and Snail transcription factors. Several transcriptionally active regulatory elements have been identified in the proximal promoter, including AP-1, Sp1 and four putative E-boxes. By EMSA, we only detected an increased binding to Sp1 and AP-1 elements in E47- and Snail-expressing cells. Binding is affected by the treatment of cells with PD 98059 MEK inhibitor, suggesting that MAPK/Erk contributes to the recruitment or assembly of proteins to Id-1 promoter. Small interfering RNA directed against Sp1 reduced Id-1 expression and the upregulation of the promoter, indicating that Sp1 is required for Id-1 induction in E47- and Snail-expressing cells. Our results provide new insights into how some target genes are activated during and/or as a consequence of the EMT triggered by both E47 and Snail transcription factors.

  9. Molecular Changes Involving MEK3-p38 MAPK Activation in Chronic Masticatory Myalgia.

    PubMed

    Meng, H; Gao, Y; Kang, Y F; Zhao, Y P; Yang, G J; Wang, Y; Cao, Y; Gan, Y H; Xie, Q F

    2016-09-01

    The exact mechanism underlying chronic masticatory myalgia (CMM), a conspicuous symptom in temporomandibular disorders, remains unclear. This investigation compared gene expression profiles between CMM patients and healthy subjects. Peripheral blood leukocytes were collected in 8 cases and 8 controls and subjected to whole genome microarray analyses. Data were analyzed with Gene Ontology and interactive pathways analyses. According to Gene Ontology analysis, categories such as ion transport, response to stimuli, and metabolic process were upregulated. The pathway analysis suggested overexpression of the mitogen-activated protein kinase (MAPK) pathway in CMM patients and to a higher degree in a pathway network. Overexpression of representative members of the MAPK pathway-including MAPK kinase 3 (MEK3), calcium voltage-gated channel auxiliary subunit gamma 2 (CACNG2), and growth arrest and DNA damage-inducible gamma (GADD45G)-was validated with real-time polymerase chain reaction. The upregulation of MEK3 was negatively correlated with the age of the CMM group. In the next step, the authors focused on MEK3, the gene that exhibited the greatest degree of differential expression, and its downstream target protein p38 MAPK. The results revealed upregulation of MEK3, as well as phosphorylated MEK3 and phosphorylated p38 MAPK, in CMM patients. These results provide a "fingerprint" for mechanistic studies of CMM in the future and highlight the importance of MEK3-p38 MAPK activation in CMM. PMID:27418173

  10. A functional activating protein 1 (AP-1) site regulates matrix metalloproteinase 2 (MMP-2) transcription by cardiac cells through interactions with JunB-Fra1 and JunB-FosB heterodimers.

    PubMed Central

    Bergman, Marina R; Cheng, Sunfa; Honbo, Norman; Piacentini, Lucia; Karliner, Joel S; Lovett, David H

    2003-01-01

    Enhanced synthesis of a specific matrix metalloproteinase, MMP-2, has been demonstrated in experimental models of ventricular failure and in cardiac extracts from patients with ischaemic cardiomyopathy. Cultured neonatal rat cardiac fibroblasts and myocytes were used to analyse the determinants of MMP-2 synthesis, including the effects of hypoxia. Culture of rat cardiac fibroblasts for 24 h in 1% oxygen enhanced MMP-2 synthesis by more than 5-fold and augmented the MMP-2 synthetic responses of these cells to endothelin-1, angiotensin II and interleukin 1beta. A series of MMP-2 promoter-luciferase constructs were used to map the specific enhancer element(s) that drive MMP-2 transcription in cardiac cells. Deletion studies mapped a region of potent transactivating function within the 91 bp region from -1433 to -1342 bp, the activity of which was increased by hypoxia. Oligonucleotides from this region were cloned in front of a heterologous simian-virus-40 (SV40) promoter and mapped the enhancer activity to a region between -1410 and -1362 bp that included a potential activating protein 1 (AP-1)-binding sequence, C(-1394)CTGACCTCC. Site-specific mutagenesis of the core TGAC sequence (indicated in bold) eliminated the transactivating activity within the -1410 to -1362 bp sequence. Electrophoretic mobility shift assays (EMSAs) using the -1410 to -1362 bp oligonucleotide and rat cardiac fibroblast nuclear extracts demonstrated specific nuclear-protein binding that was eliminated by cold competitor oligonucleotide, but not by the AP-1-mutated oligonucleotide. Antibody-supershift EMSAs of nuclear extracts from normoxic rat cardiac fibroblasts demonstrated Fra1 and JunB binding to the -1410 to -1362 bp oligonucleotide. Nuclear extracts isolated from hypoxic rat cardiac fibroblasts contained Fra1, JunB and also included FosB. Co-transfection of cardiac fibroblasts with Fra1-JunB and FosB-JunB expression plasmids led to significant increases in transcriptional activity. These

  11. Phosphatidylserine enhances IKBKAP transcription by activating the MAPK/ERK signaling pathway.

    PubMed

    Donyo, Maya; Hollander, Dror; Abramovitch, Ziv; Naftelberg, Shiran; Ast, Gil

    2016-04-01

    Familial dysautonomia (FD) is a genetic disorder manifested due to abnormal development and progressive degeneration of the sensory and autonomic nervous system. FD is caused by a point mutation in the IKBKAP gene encoding the IKAP protein, resulting in decreased protein levels. A promising potential treatment for FD is phosphatidylserine (PS); however, the manner by which PS elevates IKAP levels has yet to be identified. Analysis of ChIP-seq results of the IKBKAP promoter region revealed binding of the transcription factors CREB and ELK1, which are regulated by the mitogen-activated protein kinase (MAPK)/extracellular-regulated kinase (ERK) signaling pathway. We show that PS treatment enhanced ERK phosphorylation in cells derived from FD patients. ERK activation resulted in elevated IKBKAP transcription and IKAP protein levels, whereas pretreatment with the MAPK inhibitor U0126 blocked elevation of the IKAP protein level. Overexpression of either ELK1 or CREB activated the IKBKAP promoter, whereas downregulation of these transcription factors resulted in a decrease of the IKAP protein. Additionally, we show that PS improves cell migration, known to be enhanced by MAPK/ERK activation and abrogated in FD cells. In conclusion, our results demonstrate that PS activates the MAPK/ERK signaling pathway, resulting in activation of transcription factors that bind the promoter region of IKBKAP and thus enhancing its transcription. Therefore, compounds that activate the MAPK/ERK signaling pathway could constitute potential treatments for FD. PMID:26769675

  12. Hepatocyte cytoskeleton during ischemia and reperfusion - influence of ANP-mediated p38 MAPK activation

    PubMed Central

    Keller, Melanie; Gerbes, Alexander L; Kulhanek-Heinze, Stefanie; Gerwig, Tobias; Grützner, Uwe; van Rooijen, Nico; Vollmar, Angelika M; Kiemer, Alexandra K

    2005-01-01

    AIM: To determine functional consequences of this activation, whereby we focused on a potential regulation of the hepatocyte cytoskeleton during ischemia and reperfusion. METHODS: For in vivo experiments, animals received ANP (5 μg/kg) intravenously. In a different experimental setting, isolated rat livers were perfused with KH-buffer±ANP (200 nmol/L)±SB203580 (2 μmol/L). Livers were then kept under ischemic conditions for 24 h, and either transplanted or reperfused. Actin, Hsp27, and phosphorylated Hsp27 were determined by Western blotting, p38 MAPK activity by in vitro phosphorylation assay. F-actin distribution was determined by confocal microscopy. RESULTS: We first confirmed that ANP preconditioning leads to an activation of p38 MAPK and observed alterations of the cytoskeleton in hepatocytes of ANP-preconditioned organs. ANP induced an increase of hepatic F-actin after ischemia, which could be prevented by the p38 MAPK inhibitor SB203580 but had no effect on bile flow. After ischemia untreated livers showed a translocation of Hsp27 towards the cytoskeleton and an increase in total Hsp27, whereas ANP preconditioning prohibited translocation but caused an augmentation of Hsp27 phosphorylation. This effect is also mediated via p38 MAPK, since it was abrogated by the p38 MAPK inhibitor SB203580. CONCLUSION: This study reveals that ANP-mediated p38 MAPK activation leads to changes in hepatocyte cytoskeleton involving an elevation of phosphorylated Hsp27 and thereby for the first time shows functional consequences of ANP-induced hepatic p38 MAPK activation. PMID:16437711

  13. AP-1-mediated chromatin looping regulates ZEB2 transcription: new insights into TNFα-induced epithelial-mesenchymal transition in triple-negative breast cancer.

    PubMed

    Qiao, Yichun; Shiue, Chiou-Nan; Zhu, Jian; Zhuang, Ting; Jonsson, Philip; Wright, Anthony P H; Zhao, Chunyan; Dahlman-Wright, Karin

    2015-04-10

    The molecular determinants of malignant cell behaviour in triple-negative breast cancer (TNBC) are poorly understood. Recent studies have shown that regulators of epithelial-mesenchymal transition (EMT) are potential therapeutic targets for TNBC. In this study, we demonstrate that the inflammatory cytokine TNFα induces EMT in TNBC cells via activation of AP-1 signaling and subsequently induces expression of the EMT regulator ZEB2. We also show that TNFα activates both the PI3K/Akt and MAPK/ERK pathways, which act upstream of AP-1. We further investigated in detail AP-1 regulation of ZEB2 expression. We show that two ZEB2 transcripts derived from distinct promoters are both expressed in breast cancer cell lines and breast tumor samples. Using the chromosome conformation capture assay, we demonstrate that AP-1, when activated by TNFα, binds to a site in promoter 1b of the ZEB2 gene where it regulates the expression of both promoter 1b and 1a, the latter via mediating long range chromatin interactions. Overall, this work provides a plausible mechanism for inflammation-induced metastatic potential in TNBC, involving a novel regulatory mechanism governing ZEB2 isoform expression. PMID:25762639

  14. Procyanidin B2 3,3″-di-O-gallate, a biologically active constituent of grape seed extract, induces apoptosis in human prostate cancer cells via targeting NF-κB, Stat3 and AP1 transcription factors

    PubMed Central

    Tyagi, Alpna; Raina, Komal; Shrestha, Suraj Prakash; Miller, Bettina; Thompson, John A.; Wempe, Michael F.; Agarwal, Rajesh; Agarwal, Chapla

    2014-01-01

    Recently, we identified procyanidin B2 3,3″-di-O-gallate (B2G2) as most active constituent of grape seed extract (GSE) for efficacy against prostate cancer (PCa). Isolating large quantities of B2G2 from total GSE is labor intensive and expensive, thereby limiting both efficacy and mechanistic studies with this novel anti-cancer agent. Accordingly, here we synthesized gram-scale quantities of B2G2, compared it with B2G2 isolated from GSE for possible equivalent biological activity, and conducted mechanistic studies. Both B2G2 preparations inhibited cell growth, decreased clonogenicity, and induced cell cycle arrest and apoptotic death, comparable to each other, in various human PCa cell lines. Mechanistic studies focusing on transcription factors involved in apoptotic and survival pathways revealed that B2G2 significantly inhibits NF-κB and AP1 transcriptional activity and nuclear translocation of Stat3 in PCa cell lines, irrespective of their functional androgen receptor status. B2G2 also decreased survivin expression which is regulated by NF-κB, AP1 and Stat3, and increased cleaved PARP level. In summary, we report B2G2 chemical synthesis at gram-quantity with equivalent biological efficacy against human PCa cell lines and same molecular targeting profiles at key transcription factors level. The synthetic B2G2 will stimulate more research on prostate and possibly other malignancies in preclinical models and clinical translation. PMID:24191894

  15. Suppression of B-Raf(V600E) cancers by MAPK hyper-activation

    PubMed Central

    Eldad, Sophia; Smeir, Elia; Bar-Tana, Jacob

    2016-01-01

    B-Raf(V600E) activates MEK/MAPK signalling and acts as oncogenic driver of a variety of cancers, including melanoma, colorectal and papillary thyroid carcinoma. Specific B-Raf(V600E) kinase inhibitors (e.g., Vemurafenib) prove initial efficacy in melanoma followed shortly by acquired resistance, while failing in most other B-Raf(V600E) cancers due to primary resistance. Resistance is due to acquired mutations in the Ras/Raf/MEK/MAPK pathway and/or other oncogenic drivers that bypass B-Raf(V600E). Surprisingly, hyper-activation of MAPK by inhibiting its protein phosphatase 2A by a synthetic long-chain fatty acid analogue (MEDICA), results in oncogene-induced growth arrest and apoptosis of B-Raf(V600E) cancer cells. Growth arrest is accompanied by MAPK-mediated serine/threonine phosphorylation and suppression of a variety of oncogenic drivers that resist treatment by B-Raf(V600E) kinase inhibitors, including ErbB members, c-Met, IGFR, IRS, STAT3 and Akt. The combined activities of mutated B-Raf and MEDICA are required for generating hyper-activated MAPK, growth arrest and apoptosis, implying strict specificity for mutated B-Raf cancer cells. PMID:26959890

  16. Suppression of B-Raf(V600E) cancers by MAPK hyper-activation.

    PubMed

    Atiq, Rawan; Hertz, Rachel; Eldad, Sophia; Smeir, Elia; Bar-Tana, Jacob

    2016-04-01

    B-Raf(V600E) activates MEK/MAPK signalling and acts as oncogenic driver of a variety of cancers, including melanoma, colorectal and papillary thyroid carcinoma. Specific B-Raf(V600E) kinase inhibitors (e.g., Vemurafenib) prove initial efficacy in melanoma followed shortly by acquired resistance, while failing in most other B-Raf(V600E) cancers due to primary resistance. Resistance is due to acquired mutations in the Ras/Raf/MEK/MAPK pathway and/or other oncogenic drivers that bypass B-Raf(V600E). Surprisingly, hyper-activation of MAPK by inhibiting its protein phosphatase 2A by a synthetic long-chain fatty acid analogue (MEDICA), results in oncogene-induced growth arrest and apoptosis of B-Raf(V600E) cancer cells. Growth arrest is accompanied by MAPK-mediated serine/threonine phosphorylation and suppression of a variety of oncogenic drivers that resist treatment by B-Raf(V600E) kinase inhibitors, including ErbB members, c-Met, IGFR, IRS, STAT3 and Akt. The combined activities of mutated B-Raf and MEDICA are required for generating hyper-activated MAPK, growth arrest and apoptosis, implying strict specificity for mutated B-Raf cancer cells. PMID:26959890

  17. Molecular Basis for Enhancement of the Meiotic DMCI Recombinase by RAD51AP1

    SciTech Connect

    Dray, Eloise; Dunlop, Myun Hwa; Kauppi, Liisa; San Filippo, Joseph San; Wiese, Claudia; Tsai, Miaw-Sheue; Begovic, Sead; Schild, David; Jasin, Maria; Keeney, Scott; Sung, Patrick

    2010-11-05

    Homologous recombination is needed for meiotic chromosome segregation, genome maintenance, and tumor suppression. RAD51AP1 (RAD51 Associated Protein 1) has been shown to interact with and enhance the recombinase activity of RAD51. Accordingly, genetic ablation of RAD51AP1 leads to enhanced sensitivity to and also chromosome aberrations upon DNA damage, demonstrating a role for RAD51AP1 in mitotic homologous recombination. Here we show physical association of RAD51AP1 with the meiosis-specific recombinase DMC1 and a stimulatory effect of RAD51AP1 on the DMC1-mediated D-loop reaction. Mechanistic studies have revealed that RAD51AP1 enhances the ability of the DMC1 presynaptic filament to capture the duplex DNA partner and to assemble the synaptic complex, in which the recombining DNA strands are homologously aligned. We also provide evidence that functional co-operation is dependent on complex formation between DMC1 and RAD51AP1, and that distinct epitopes in RAD51AP1 mediate interactions with RAD51 and DMC1. Finally, we show that RAD51AP1 is expressed in mouse testes, and that RAD51AP1 foci co-localize with a subset of DMC1 foci in spermatocytes. These results suggest that RAD51AP1 also serves an important role in meiotic homologous recombination.

  18. Celastrol ameliorates HIV-1 Tat-induced inflammatory responses via NF-kappaB and AP-1 inhibition and heme oxygenase-1 induction in astrocytes

    SciTech Connect

    Youn, Gi Soo; Kwon, Dong-Joo; Ju, Sung Mi; Rhim, Hyangshuk; Bae, Yong Soo; Choi, Soo Young; Park, Jinseu

    2014-10-01

    HIV-1 Tat causes extensive neuroinflammation that may progress to AIDS-related encephalitis and dementia. Celastrol possesses various biological activities such as anti-oxidant, anti-tumor, and anti-inflammatory activities. In this study, we investigated the modulatory effects of celastrol on HIV-1 Tat-induced inflammatory responses and the molecular mechanisms underlying its action in astrocytes. Pre-treatment of CRT-MG human astroglioma cells with celastrol significantly inhibited HIV-1 Tat-induced expression of ICAM-1/VCAM-1 and subsequent monocyte adhesiveness in CRT-MG cells. In addition, celastrol suppressed HIV-1 Tat-induced expression of pro-inflammatory chemokines, such as CXCL10, IL-8, and MCP-1. Celastrol decreased HIV-1 Tat-induced activation of JNK MAPK, AP-1, and NF-κB. Furthermore, celastrol induced mRNA and protein expression of HO-1 as well as Nrf2 activation. Blockage of HO-1 expression using siRNA reversed the inhibitory effect of celastrol on HIV-1 Tat-induced inflammatory responses. These results suggest that celastrol has regulatory effects on HIV-1 Tat-induced inflammatory responses by blocking the JNK MAPK-AP-1/NF-κB signaling pathways and inducing HO-1 expression in astrocytes. - Highlights: • Celastrol suppressed HIV-1 Tat-induced expression of pro-inflammatory genes. • Celastrol inhibited HIV-1 Tat -induced activation of JNK MAPK. • Celastrol inhibited HIV-1 Tat-induced activation of both NF-κB and AP-1. • Celastrol inhibited HIV-1 Tat-induced inflammatory responses via HO-1 induction.

  19. The PHD motif of Map3k1 activates cytokine-dependent MAPK signaling

    PubMed Central

    Gallagher, Ewen; Suddason, Tesha

    2015-01-01

    We generated a mutation in the gene encoding mitogen-activated protein kinase kinase kinase 1 (Map3k1) that results in a protein with an inactive plant homeodomain (PHD). Map3k1mPHD cells are defective in cytokine-mediated MAPK signaling. Protein array identified transforming growth factor (TGF-β)-activated kinase 1 binding protein 1 (Tab1) as a PHD substrate. The Map3k1 PHD transfers Lys63-linked poly-ubiquitin onto Tab1 to activate MAPKs. PMID:27308457

  20. MKK3 was involved in larval settlement of the barnacle Amphibalanus amphitrite through activating the kinase activity of p38MAPK.

    PubMed

    Zhang, Gen; He, Li-Sheng; Wong, Yue Him; Qian, Pei-Yuan

    2013-01-01

    The p38 mitogen-activated protein kinase (p38MAPK) plays a key role in larval settlement of the barnacle Amphibalanus amphitrite. To study the signaling pathway associated with p38MAPK during larval settlement, we sought to identify the upstream kinase of p38MAPK. Three MKKs (MKK3, MKK4 and MKK7) and three MAPKs (p38MAPK, ERK and JNK) in A. amphitrite were cloned and recombinantly expressed in E. coli. Through kinase assays, we found that MKK3, but not MKK4 or MKK7, phosphorylated p38MAPK. Furthermore, MKK3 activity was specific to p38MAPK, as it did not phosphorylate ERK or JNK. To further investigate the functional relationship between MKK3 and p38MAPK in vivo, we studied the localization of phospho-MKK3 (pMKK3) and MKK3 by immunostaining. Consistent with the patterns of p38MAPK and phospho-p38MAPK (pp38MAPK), pMKK3 and MKK3 mainly localized to the antennules of the cyprids. Western blot analysis revealed that pMKK3 levels, like pp38MAPK levels, were elevated at cyprid stage, compared to nauplii and juvenile stages. Moreover, pMKK3 levels increased after treatment with adult barnacle crude extracts, suggesting that MKK3 might mediate the stimulatory effects of adult barnacle extracts on the p38MAPK pathway. PMID:23922727

  1. Tetraspanin CD9 regulates osteoclastogenesis via regulation of p44/42 MAPK activity

    SciTech Connect

    Yi, TacGhee; Kim, Hye-Jin; Cho, Je-Yoel; Woo, Kyung Mi; Ryoo, Hyun-Mo; Kim, Gwan-Shik; Baek, Jeong-Hwa . E-mail: baekjh@snu.ac.kr

    2006-08-18

    Tetraspanin CD9 has been shown to regulate cell-cell fusion in sperm-egg fusion and myotube formation. However, the role of CD9 in osteoclast, another multinucleated cell type, is not still clear. Therefore, we investigated the role of CD9 in osteoclast differentiation. CD9 was expressed in osteoclast lineage cells and its expression level increased during the progression of RANKL-induced osteoclastogenesis. KMC8, a neutralizing antibody specific to CD9, significantly suppressed RANKL-induced multinucleated osteoclast formation and the mRNA expression of osteoclast differentiation marker genes. To define CD9-regulated osteoclastogenic signaling pathway, MAPK pathways were examined. KMC8 induced long-term phosphorylation of p44/42 MAPK, but not of p38 MAPK. Constitutive activation of p44/42 MAPK by overexpressing constitutive-active mutant of MEK1 almost completely blocked osteoclast differentiation. Taken together, these results suggest that CD9 expressed on osteoclast lineage cells might positively regulate osteoclastogenesis via the regulation of p44/42 MAPK activity.

  2. PAK1 is a breast cancer oncogene that coordinately activates MAPK and MET signaling

    PubMed Central

    Shrestha, Yashaswi; Schafer, Eric J.; Boehm, Jesse S.; Thomas, Sapana R.; He, Frank; Du, Jinyan; Wang, Shumei; Barretina, Jordi; Weir, Barbara A.; Zhao, Jean J.; Polyak, Kornelia; Golub, Todd R.; Beroukhim, Rameen; Hahn, William C.

    2011-01-01

    Activating mutations in the RAS family or BRAF frequently occur in many types of human cancers but are rarely detected in breast tumors. However, activation of the RAS-RAF-MEK-ERK Mitogen-Activated Protein Kinase (MAPK) pathway is commonly observed in human breast cancers, suggesting that other genetic alterations lead to activation of this signaling pathway. To identify breast cancer oncogenes that activate the MAPK pathway, we screened a library of human kinases for their ability to induce anchorage-independent growth in a derivative of immortalized human mammary epithelial cells (HMLE). We identified PAK1 as a kinase that permitted HMLE cells to form anchorage-independent colonies. PAK1 is amplified in several human cancer types, including 33% of breast tumor samples and cancer cell lines. The kinase activity of PAK1 is necessary for PAK1-induced transformation. Moreover, we show that PAK1 simultaneously activates MAPK and MET signaling; the latter via inhibition of Merlin. Disruption of these activities inhibits PAK1-driven anchorage-independent growth. These observations establish PAK1 amplification as an alternative mechanism for MAPK activation in human breast cancer and credential PAK1 as a breast cancer oncogene that coordinately regulates multiple signaling pathways, the cooperation of which leads to malignant transformation. PMID:22105362

  3. PDGF-BB-mediated activation of p42(MAPK) is independent of PDGF beta-receptor tyrosine phosphorylation.

    PubMed

    Cartel, N J; Liu, J; Wang, J; Post, M

    2001-10-01

    Herein, we investigated the activity of mitogen-activated protein kinase (MAPK), a key component of downstream signaling events, which is activated subsequent to platelet-derived growth factor (PDGF)-BB stimulation. Specifically, p42(MAPK) activity peaked 60 min after addition of PDGF-BB, declined thereafter, and was determined not to be a direct or necessary component of glycosaminoglycan (GAG) synthesis. PDGF-BB also activated MAPK kinase 2 (MAPKK2) but had no effect on MAPKK1 and Raf-1 activity. Chemical inhibition of Janus kinase, phosphatidylinositol 3-kinase, Src kinase, or tyrosine phosphorylation inhibition of the PDGF beta-receptor (PDGFR-beta) did not abrogate PDGF-BB-induced p42(MAPK) activation or its threonine or tyrosine phosphorylation. A dominant negative cytoplasmic receptor for hyaluronan-mediated motility variant 4 (RHAMMv4), a regulator of MAPKK-MAPK interaction and activation, did not inhibit PDGF-BB-induced p42(MAPK) activation nor did a construct expressing PDGFR-beta with cytoplasmic tyrosines mutated to phenylalanine. However, overexpression of a dominant negative PDGFR-beta lacking the cytoplasmic signaling domain abrogated p42(MAPK) activity. These results suggest that PDGF-BB-mediated activation of p42(MAPK) requires the PDGFR-beta but is independent of its tyrosine phosphorylation. PMID:11557582

  4. Kappa Opioid Receptor-Induced Aversion Requires p38 MAPK Activation in VTA Dopamine Neurons

    PubMed Central

    Ehrich, Jonathan M.; Messinger, Daniel I.; Knakal, Cerise R.; Kuhar, Jamie R.; Schattauer, Selena S.; Bruchas, Michael R.; Zweifel, Larry S.; Kieffer, Brigitte L.; Phillips, Paul E.M.

    2015-01-01

    The endogenous dynorphin-κ opioid receptor (KOR) system encodes the dysphoric component of the stress response and controls the risk of depression-like and addiction behaviors; however, the molecular and neural circuit mechanisms are not understood. In this study, we report that KOR activation of p38α MAPK in ventral tegmental (VTA) dopaminergic neurons was required for conditioned place aversion (CPA) in mice. Conditional genetic deletion of floxed KOR or floxed p38α MAPK by Cre recombinase expression in dopaminergic neurons blocked place aversion to the KOR agonist U50,488. Selective viral rescue by wild-type KOR expression in dopaminergic neurons of KOR−/− mice restored U50,488-CPA, whereas expression of a mutated form of KOR that could not initiate p38α MAPK activation did not. Surprisingly, while p38α MAPK inactivation blocked U50,488-CPA, p38α MAPK was not required for KOR inhibition of evoked dopamine release measured by fast scan cyclic voltammetry in the nucleus accumbens. In contrast, KOR activation acutely inhibited VTA dopaminergic neuron firing, and repeated exposure attenuated the opioid response. This adaptation to repeated exposure was blocked by conditional deletion of p38α MAPK, which also blocked KOR-induced tyrosine phosphorylation of the inwardly rectifying potassium channel (GIRK) subunit Kir3.1 in VTA dopaminergic neurons. Consistent with the reduced response, GIRK phosphorylation at this amino terminal tyrosine residue (Y12) enhances channel deactivation. Thus, contrary to prevailing expectations, these results suggest that κ opioid-induced aversion requires regulation of VTA dopaminergic neuron somatic excitability through a p38α MAPK effect on GIRK deactivation kinetics rather than by presynaptically inhibiting dopamine release. SIGNIFICANCE STATEMENT Kappa opioid receptor (KOR) agonists have the potential to be effective, nonaddictive analgesics, but their therapeutic utility is greatly limited by adverse effects on mood

  5. S100B/RAGE-dependent activation of microglia via NF-kappaB and AP-1 Co-regulation of COX-2 expression by S100B, IL-1beta and TNF-alpha.

    PubMed

    Bianchi, Roberta; Giambanco, Ileana; Donato, Rosario

    2010-04-01

    Extracellular S100B is known to affect astrocytic, neuronal and microglial activities, with different effects depending on its concentration. Whereas at relatively low concentrations S100B exerts trophic effects on neurons and astrocytes, at relatively high concentrations the protein causes neuronal apoptosis and activates astrocytes and microglia, thus potentially representing an endogenous factor implicated in neuroinflammation. We have reported that RAGE ligation by S100B in BV-2 microglia results in the upregulation of expression of the pro-inflammatory cyclo-oxygenase 2 (COX-2) via parallel Ras-Cdc42-Rac1-dependent activation of c-Jun NH(2) terminal protein kinase (JNK) and Ras-Rac1-dependent stimulation of NF-kappaB transcriptional activity. We show here that: (1) S100B also stimulates AP-1 transcriptional activity in microglia via RAGE-dependent activation of JNK; (2) S100B upregulates IL-1beta and TNF-alpha expression in microglia via RAGE engagement; and (3) S100B/RAGE-induced upregulation of COX-2, IL-1beta and TNF-alpha expression requires the concurrent activation of NF-kappaB and AP-1. We also show that S100B synergizes with IL-1beta and TNF-alpha to upregulate on COX-2 expression in microglia. Given the crucial roles of COX-2, IL-1beta and TNF-alpha in the inflammatory response, we propose that, by engaging RAGE, S100B might play an important role in microglia activation in the course of brain damage. PMID:18599158

  6. The Mitogen-Activated Protein Kinase (MAPK) Signaling Pathway as a Discovery Target in Stroke.

    PubMed

    Sun, Jing; Nan, Guangxian

    2016-05-01

    Protein kinases are critical modulators of a variety of intracellular and extracellular signal transduction pathways, and abnormal phosphorylation events can contribute to disease progression in a variety of diseases. As a result, protein kinases have emerged as important new drug targets for small molecule therapeutics. The mitogen-activated protein kinase (MAPK) signaling pathway transmits signals from the cell membrane to the nucleus in response to a variety of different stimuli. Because this pathway controls a broad spectrum of cellular processes, including growth, inflammation, and stress responses, it is accepted as a therapeutic target for cancer and peripheral inflammatory disorders. There is also increasing evidence that MAPK is an important regulator of ischemic and hemorrhagic cerebral vascular disease, raising the possibility that it might be a drug discovery target for stroke. In this review, we discuss the MAPK signaling pathway in association with its activation in stroke-induced brain injury. PMID:26842916

  7. TGF-β2 induces Grb2 to recruit PI3-K to TGF-RII that activates JNK/AP-1-signaling and augments invasiveness of Theileria-transformed macrophages

    PubMed Central

    Haidar, Malak; Whitworth, Jessie; Noé, Gaelle; Liu, Wang Qing; Vidal, Michel; Langsley, Gordon

    2015-01-01

    Theileria-infected macrophages display many features of cancer cells such as heightened invasive capacity; however, the tumor-like phenotype is reversible by killing the parasite. Moreover, virulent macrophages can be attenuated by multiple in vitro passages and so provide a powerful model to elucidate mechanisms related to transformed macrophage virulence. Here, we demonstrate that in two independent Theileria-transformed macrophage cell lines Grb2 expression is down-regulated concomitant with loss of tumor virulence. Using peptidimer-c to ablate SH2 and SH3 interactions of Grb2 we identify TGF-receptor II and the p85 subunit of PI3-K, as Grb2 partners in virulent macrophages. Ablation of Grb2 interactions reduces PI3-K recruitment to TGF-RII and decreases PIP3 production, and dampens JNK phosphorylation and AP-1-driven transcriptional activity down to levels characteristic of attenuated macrophages. Loss of TGF-R>PI3-K>JNK>AP-1 signaling negatively impacts on virulence traits such as reduced JAM-L/ITG4A and Fos-B/MMP9 expression that contribute to virulent macrophage adhesion and invasiveness. PMID:26511382

  8. EPAS1 trans-activation during hypoxia requires p42/p44 MAPK.

    PubMed

    Conrad, P W; Freeman, T L; Beitner-Johnson, D; Millhorn, D E

    1999-11-19

    Hypoxia is a common environmental stress that regulates gene expression and cell function. A number of hypoxia-regulated transcription factors have been identified and have been shown to play critical roles in mediating cellular responses to hypoxia. One of these is the endothelial PAS-domain protein 1 (EPAS1/HIF2-alpha/HLF/HRF). This protein is 48% homologous to hypoxia-inducible factor 1-alpha (HIF1-alpha). To date, virtually nothing is known about the signaling pathways that lead to either EPAS1 or HIF1-alpha activation. Here we show that EPAS1 is phosphorylated when PC12 cells are exposed to hypoxia and that p42/p44 MAPK is a critical mediator of EPAS1 activation. Pretreatment of PC12 cells with the MEK inhibitor, PD98059, completely blocked hypoxia-induced trans-activation of a hypoxia response element (HRE) reporter gene by transfected EPAS1. Likewise, expression of a constitutively active MEK1 mimicked the effects of hypoxia on HRE reporter gene expression. However, pretreatment with PD98059 had no effect on EPAS1 phosphorylation during hypoxia, suggesting that MAPK targets other proteins that are critical for the trans-activation of EPAS1. We further show that hypoxia-induced trans-activation of EPAS1 is independent of Ras. Finally, pretreatment with calmodulin antagonists nearly completely blocked both the hypoxia-induced phosphorylation of MAPK and the EPAS1 trans-activation of HRE-Luc. These results demonstrate that the MAPK pathway is a critical mediator of EPAS1 activation and that activation of MAPK and EPAS1 occurs through a calmodulin-sensitive pathway and not through the GTPase, Ras. These results are the first to identify a specific signaling pathway involved in EPAS1 activation. PMID:10559262

  9. Pheromone-Induced Morphogenesis Improves Osmoadaptation Capacity by Activating the HOG MAPK Pathway**

    PubMed Central

    Baltanás, Rodrigo; Bush, Alan; Couto, Alicia; Durrieu, Lucía; Hohmann, Stefan; Colman-Lerner, Alejandro

    2013-01-01

    Environmental and internal conditions expose cells to a multiplicity of stimuli whose consequences are difficult to predict. Here, we investigate the response to mating pheromone of yeast cells adapted to high osmolarity. Events downstream of pheromone binding involve two mitogen-activated protein kinase (MAPK) cascades: the pheromone response (PR) and the cell-wall integrity response (CWI). Although these MAPK pathways share components with each and a third MAPK pathway, the high osmolarity response (HOG), they are normally only activated by distinct stimuli, a phenomenon called insulation. We found that in cells adapted to high osmolarity, PR activated the HOG pathway in a pheromone- and osmolarity- dependent manner. Activation of HOG by the PR was not due to loss of insulation, but rather a response to a reduction in internal osmolarity, which resulted from an increase in glycerol release caused by the PR. By analyzing single-cell time courses, we found that stimulation of HOG occurred in discrete bursts that coincided with the “shmooing” morphogenetic process. Activation required the polarisome, the cell wall integrity MAPK Slt2, and the aquaglyceroporin Fps1. HOG activation resulted in high glycerol turnover that improved adaptability to rapid changes in osmolarity. Our work shows how a differentiation signal can recruit a second, unrelated sensory pathway to enable responses to yeast to multiple stimuli. PMID:23612707

  10. Pheromone-induced morphogenesis improves osmoadaptation capacity by activating the HOG MAPK pathway.

    PubMed

    Baltanás, Rodrigo; Bush, Alan; Couto, Alicia; Durrieu, Lucía; Hohmann, Stefan; Colman-Lerner, Alejandro

    2013-04-23

    Environmental and internal conditions expose cells to a multiplicity of stimuli whose consequences are difficult to predict. We investigate the response to mating pheromone of yeast cells adapted to high osmolarity. Events downstream of pheromone binding involve two mitogen-activated protein kinase (MAPK) cascades: the pheromone response (PR) and the cell wall integrity (CWI) response. Although the PR MAPK pathway shares components with a third MAPK pathway, the high osmolarity (HOG) response, each one is normally only activated by its cognate stimulus, a phenomenon called insulation. We found that in cells adapted to high osmolarity, PR activated the HOG pathway in a pheromone- and osmolarity-dependent manner. Activation of HOG by the PR was not due to loss of insulation, but rather a response to a reduction in internal osmolarity, which resulted from an increase in glycerol release caused by the PR. By analyzing single-cell time courses, we found that stimulation of HOG occurred in discrete bursts that coincided with the "shmooing" morphogenetic process. Activation required the polarisome, the CWI MAPK Slt2, and the aquaglyceroporin Fps1. HOG activation resulted in high glycerol turnover, which improved adaptability to rapid changes in osmolarity. Our work shows how a differentiation signal can recruit a second, unrelated sensory pathway to fine-tune yeast response in a complex environment. PMID:23612707

  11. Cooperativity between MAPK and PI3K signaling activation is required for glioblastoma pathogenesis

    PubMed Central

    Vitucci, Mark; Karpinich, Natalie O.; Bash, Ryan E.; Werneke, Andrea M.; Schmid, Ralf S.; White, Kristen K.; McNeill, Robert S.; Huff, Byron; Wang, Sophie; Van Dyke, Terry; Miller, C. Ryan

    2013-01-01

    Background Glioblastoma (GBM) genomes feature recurrent genetic alterations that dysregulate core intracellular signaling pathways, including the G1/S cell cycle checkpoint and the MAPK and PI3K effector arms of receptor tyrosine kinase (RTK) signaling. Elucidation of the phenotypic consequences of activated RTK effectors is required for the design of effective therapeutic and diagnostic strategies. Methods Genetically defined, G1/S checkpoint-defective cortical murine astrocytes with constitutively active Kras and/or Pten deletion mutations were used to systematically investigate the individual and combined roles of these 2 RTK signaling effectors in phenotypic hallmarks of glioblastoma pathogenesis, including growth, migration, and invasion in vitro. A novel syngeneic orthotopic allograft model system was used to examine in vivo tumorigenesis. Results Constitutively active Kras and/or Pten deletion mutations activated both MAPK and PI3K signaling. Their combination led to maximal growth, migration, and invasion of G1/S-defective astrocytes in vitro and produced progenitor-like transcriptomal profiles that mimic human proneural GBM. Activation of both RTK effector arms was required for in vivo tumorigenesis and produced highly invasive, proneural-like GBM. Conclusions These results suggest that cortical astrocytes can be transformed into GBM and that combined dysregulation of MAPK and PI3K signaling revert G1/S-defective astrocytes to a primitive gene expression state. This genetically-defined, immunocompetent model of proneural GBM will be useful for preclinical development of MAPK/PI3K-targeted, subtype-specific therapies. PMID:23814263

  12. Activation of MAPK Is Necessary for Long-Term Memory Consolidation Following Food-Reward Conditioning

    ERIC Educational Resources Information Center

    Ribeiro, Maria J.; Schofield, Michael G.; Kemenes, Ildiko; O'Shea, Michael; Kemenes, Gyorgy; Benjamin, Paul R.

    2005-01-01

    Although an important role for the mitogen-activated protein kinase (MAPK) has been established for memory consolidation in a variety of learning paradigms, it is not known if this pathway is also involved in appetitive classical conditioning. We address this question by using a single-trial food-reward conditioning paradigm in the freshwater…

  13. FGFR2c-mediated ERK-MAPK activity regulates coronal suture development.

    PubMed

    Pfaff, Miles J; Xue, Ke; Li, Li; Horowitz, Mark C; Steinbacher, Derek M; Eswarakumar, Jacob V P

    2016-07-15

    Fibroblast growth factor receptor 2 (FGFR2) signaling is critical for proper craniofacial development. A gain-of-function mutation in the 2c splice variant of the receptor's gene is associated with Crouzon syndrome, which is characterized by craniosynostosis, the premature fusion of one or more of the cranial vault sutures, leading to craniofacial maldevelopment. Insight into the molecular mechanism of craniosynostosis has identified the ERK-MAPK signaling cascade as a critical regulator of suture patency. The aim of this study is to investigate the role of FGFR2c-induced ERK-MAPK activation in the regulation of coronal suture development. Loss-of-function and gain-of-function Fgfr2c mutant mice have overlapping phenotypes, including coronal synostosis and craniofacial dysmorphia. In vivo analysis of coronal sutures in loss-of-function and gain-of-function models demonstrated fundamentally different pathogenesis underlying coronal suture synostosis. Calvarial osteoblasts from gain-of-function mice demonstrated enhanced osteoblastic function and maturation with concomitant increase in ERK-MAPK activation. In vitro inhibition with the ERK protein inhibitor U0126 mitigated ERK protein activation levels with a concomitant reduction in alkaline phosphatase activity. This study identifies FGFR2c-mediated ERK-MAPK signaling as a key mediator of craniofacial growth and coronal suture development. Furthermore, our results solve the apparent paradox between loss-of-function and gain-of-function FGFR2c mutants with respect to coronal suture synostosis. PMID:27034231

  14. The Forkhead Transcription Factor FOXK2 Promotes AP-1-Mediated Transcriptional Regulation

    PubMed Central

    Ji, Zongling; Donaldson, Ian J.; Liu, Jingru; Hayes, Andrew; Zeef, Leo A. H.

    2012-01-01

    The transcriptional control circuitry in eukaryotic cells is complex and is orchestrated by combinatorially acting transcription factors. Forkhead transcription factors often function in concert with heterotypic transcription factors to specify distinct transcriptional programs. Here, we demonstrate that FOXK2 participates in combinatorial transcriptional control with the AP-1 transcription factor. FOXK2 binding regions are widespread throughout the genome and are often coassociated with AP-1 binding motifs. FOXK2 acts to promote AP-1-dependent gene expression changes in response to activation of the AP-1 pathway. In this context, FOXK2 is required for the efficient recruitment of AP-1 to chromatin. Thus, we have uncovered an important new molecular mechanism that controls AP-1-dependent gene expression. PMID:22083952

  15. Porcine circovirus type 2 activates PI3K/Akt and p38 MAPK pathways to promote interleukin-10 production in macrophages via Cap interaction of gC1qR.

    PubMed

    Du, Qian; Huang, Yong; Wang, Tongtong; Zhang, Xiujuan; Chen, Yu; Cui, Beibei; Li, Delong; Zhao, Xiaomin; Zhang, Wenlong; Chang, Lingling; Tong, Dewen

    2016-04-01

    Porcine circovirus type 2 (PCV2) infection caused PCV2-associated diseases (PCVAD) is one of the major emerging immunosuppression diseases in pig industry. In this study, we investigated how PCV2 inoculation increases interleukin (IL)-10 expression in porcine alveolar macrophages (PAMs). PCV2 inoculation significantly upregulated IL-10 expression compared with PCV1. Upon initial PCV2 inoculation, PI3K/Akt cooperated with NF-κB pathways to promote IL-10 transcription via p50, CREB and Ap1 transcription factors, whereas inhibition of PI3K/Akt activation blocked Ap1 and CREB binding to the il10 promoter, and decreased the binding level of NF-κB1 p50 with il10 promoter, leading to great reduction in early IL-10 transcription. In the later phase of inoculation, PCV2 further activated p38 MAPK and ERK pathways to enhance IL-10 production by promoting Sp1 binding to the il10 promoter. For PCV2-induced IL-10 production in macrophages, PCV2 capsid protein Cap, but not the replicase Rep or ORF3, was the critical component. Cap activated PI3K/Akt, p38 MAPK, and ERK signaling pathways to enhance IL-10 expression. In the whole process, gC1qR mediated PCV2-induced PI3K/Akt and p38 MAPK activation to enhance IL-10 induction by interaction with Cap. Depletion of gC1qR blocked PI3K/Akt and p38 MAPK activation, resulting in significant decrease in IL-10 production in PCV2-inoculated cells. Thus, gC1qR might be a critical functional receptor for PCV2-induced IL-10 production. Taken together, these data demonstrated that Cap protein binding with host gC1qR induction of PI3K/Akt and p38 MAPK signalings activation is a critical process in enhancing PCV2-induced IL-10 production in porcine alveolar macrophages. PMID:26883107

  16. Porcine circovirus type 2 activates PI3K/Akt and p38 MAPK pathways to promote interleukin-10 production in macrophages via Cap interaction of gC1qR

    PubMed Central

    Wang, Tongtong; Zhang, Xiujuan; Chen, Yu; Cui, Beibei; Li, Delong; Zhao, Xiaomin; Zhang, Wenlong; Chang, Lingling; Tong, Dewen

    2016-01-01

    Porcine circovirus type 2 (PCV2) infection caused PCV2-associated diseases (PCVAD) is one of the major emerging immunosuppression diseases in pig industry. In this study, we investigated how PCV2 inoculation increases interleukin (IL)-10 expression in porcine alveolar macrophages (PAMs). PCV2 inoculation significantly upregulated IL-10 expression compared with PCV1. Upon initial PCV2 inoculation, PI3K/Akt cooperated with NF-κB pathways to promote IL-10 transcription via p50, CREB and Ap1 transcription factors, whereas inhibition of PI3K/Akt activation blocked Ap1 and CREB binding to the il10 promoter, and decreased the binding level of NF-κB1 p50 with il10 promoter, leading to great reduction in early IL-10 transcription. In the later phase of inoculation, PCV2 further activated p38 MAPK and ERK pathways to enhance IL-10 production by promoting Sp1 binding to the il10 promoter. For PCV2-induced IL-10 production in macrophages, PCV2 capsid protein Cap, but not the replicase Rep or ORF3, was the critical component. Cap activated PI3K/Akt, p38 MAPK, and ERK signaling pathways to enhance IL-10 expression. In the whole process, gC1qR mediated PCV2-induced PI3K/Akt and p38 MAPK activation to enhance IL-10 induction by interaction with Cap. Depletion of gC1qR blocked PI3K/Akt and p38 MAPK activation, resulting in significant decrease in IL-10 production in PCV2-inoculated cells. Thus, gC1qR might be a critical functional receptor for PCV2-induced IL-10 production. Taken together, these data demonstrated that Cap protein binding with host gC1qR induction of PI3K/Akt and p38 MAPK signalings activation is a critical process in enhancing PCV2-induced IL-10 production in porcine alveolar macrophages. PMID:26883107

  17. TRAF6 Autoubiquitination-Independent Activation of the NFκB and MAPK Pathways in Response to IL-1 and RANKL

    PubMed Central

    Walsh, Matthew C.; Kim, Gregory K.; Maurizio, Paul L.; Molnar, Elizabeth E.; Choi, Yongwon

    2008-01-01

    The adapter protein TRAF6 is critical for mediating signal transduction from members of the IL-1R/TLR and TNFR superfamilies. The TRAF6 RING finger domain functions as an ubiquitin E3 ligase capable of generating non-degradative K63-linked ubiquitin chains. It is believed that these chains serve as docking sites for formation of signaling complexes, and that K63-linked autoubiquitination of TRAF6 is essential for formation and activation of a complex involving the kinase TAK1 and its adapters, TAB1 and TAB2. In order to assess independently the E3 ligase and ubiquitin substrate functions of TRAF6, we generated, respectively, RING domain and complete lysine-deficient TRAF6 mutants. We found that while the TRAF6 RING domain is required for activation of TAK1, it is dispensable for interaction between TRAF6 and the TAK1-TAB1-TAB2 complex. Likewise, lysine-deficient TRAF6 was found to interact with the TAK1-TAB1-TAB2 complex, but surprisingly was also found to be fully competent to activate TAK1, as well as NFκB and AP-1 reporters. Furthermore, lysine-deficient TRAF6 rescued IL-1-mediated NFκB and MAPK activation, as well as IL-6 elaboration in retrovirally-rescued TRAF6-deficient fibroblasts. Lysine-deficient TRAF6 also rescued RANKL-mediated NFκB and MAPK activation, and osteoclastogenesis in retrovirally-rescued TRAF6-deficient bone marrow macrophages. While incapable of being ubiquitinated itself, we demonstrate that lysine-deficient TRAF6 remains competent to induce ubiquitination of IKKγ/NEMO. Further, this NEMO modification contributes to TRAF6-mediated activation of NFκB. Collectively, our results suggest that while TRAF6 autoubiquitination may serve as a marker of activation, it is unlikely to underpin RING finger-dependent TRAF6 function. PMID:19112497

  18. Regulation of osteosarcoma cell lung metastasis by the c-Fos/AP-1 target FGFR1.

    PubMed

    Weekes, D; Kashima, T G; Zandueta, C; Perurena, N; Thomas, D P; Sunters, A; Vuillier, C; Bozec, A; El-Emir, E; Miletich, I; Patiño-Garcia, A; Lecanda, F; Grigoriadis, A E

    2016-06-01

    Osteosarcoma is the most common primary malignancy of the skeleton and is prevalent in children and adolescents. Survival rates are poor and have remained stagnant owing to chemoresistance and the high propensity to form lung metastases. In this study, we used in vivo transgenic models of c-fos oncogene-induced osteosarcoma and chondrosarcoma in addition to c-Fos-inducible systems in vitro to investigate downstream signalling pathways that regulate osteosarcoma growth and metastasis. Fgfr1 (fibroblast growth factor receptor 1) was identified as a novel c-Fos/activator protein-1(AP-1)-regulated gene. Induction of c-Fos in vitro in osteoblasts and chondroblasts caused an increase in Fgfr1 RNA and FGFR1 protein expression levels that resulted in increased and sustained activation of mitogen-activated protein kinases (MAPKs), morphological transformation and increased anchorage-independent growth in response to FGF2 ligand treatment. High levels of FGFR1 protein and activated pFRS2α signalling were observed in murine and human osteosarcomas. Pharmacological inhibition of FGFR1 signalling blocked MAPK activation and colony growth of osteosarcoma cells in vitro. Orthotopic injection in vivo of FGFR1-silenced osteosarcoma cells caused a marked twofold to fivefold decrease in spontaneous lung metastases. Similarly, inhibition of FGFR signalling in vivo with the small-molecule inhibitor AZD4547 markedly reduced the number and size of metastatic nodules. Thus deregulated FGFR signalling has an important role in osteoblast transformation and osteosarcoma formation and regulates the development of lung metastases. Our findings support the development of anti-FGFR inhibitors as potential antimetastatic therapy. PMID:26387545

  19. Activation of an AP1-Like Transcription Factor of the Maize Pathogen Cochliobolus heterostrophus in Response to Oxidative Stress and Plant Signals

    SciTech Connect

    Lev, Sophie; hadar, Ruthi; Amedeo, Paolo; Baker, Scott E.; Yoder, Olen; Horwitz, Benjamin A.

    2005-02-01

    Redox sensing is a ubiquitous mechanism regulating cellular activity. Fungal pathogens face reactive oxygen species produced by the host plant's oxidative burst in addition to endogenous reactive oxygen species produced during aerobic metabolism. An array of preformed and induced detoxifying enzymes, including superoxide dismutase, catalases, and peroxidases, could allow fungi to infect plants despite the oxidative burst. We isolated a gene (CHAP1) encoding a redox-regulated transcription factor in Cochliobolus heterostrophus, a fungal pathogen of maize. CHAP1 is a bZIP protein that possesses two cysteine-rich domains structurally and functionally related to Saccharomyces cerevisiae YAP1. Deletion of CHAP1 in C. heterostrophus resulted in decreased resistance to oxidative stress caused by hydrogen peroxide and menadione, but the virulence of chap1 mutants was unaffected. Upon activation by oxidizing agents or plant signals, a green fluorescent protein (GFP)-CHAP1 fusion protein became localized in the nucleus. Expression of genes encoding antioxidant proteins was induced in the wild type but not in chap1 mutants. Activation of CHAP1 occurred from the earliest stage of plant infection, in conidial germ tubes on the leaf surface, and persisted during infection. Late in the course of infection, after extensive necrotic lesions were formed, GFP-CHAP1 redistributed to the cytosol in hyphae growing on the leaf surface. Localization of CHAP1 to the nucleus may, through changes in the redox state of the cell, provide a mechanism linking extracellular cues to transcriptional regulation during the plant-pathogen interaction.

  20. Glabridin inhibits migration and invasion by transcriptional inhibition of matrix metalloproteinase 9 through modulation of NF-κB and AP-1 activity in human liver cancer cells

    PubMed Central

    Hsieh, Ming-Ju; Lin, Chiao-Wen; Yang, Shun-Fa; Chen, Mu-Kuan; Chiou, Hui-Ling

    2014-01-01

    BACKGROUND AND PURPOSE High mortality and morbidity rates for hepatocellular carcinoma in Taiwan primarily result from uncontrolled tumour metastasis. Glabridin, a prenylated isoflavonoid of licorice (Glycyrrhiza glabra) roots, is associated with a wide range of biological properties, such as regulation of energy metabolism, oestrogenic, neuroprotective, anti-osteoporotic and skin whitening. However, the effect of glabridin on the metastasis of tumour cells has not been clarified. EXPERIMENTAL APPROACH A wound healing model and Boyden chamber assays in vitro were used to determine the effects of glabridin on the migration and invasion of human hepatocellular carcinoma (HHC) cells. Western blot analysis, gelatin zymography, real-time PCR and promoter assays were used to evaluate the inhibitory effects of glabridin on matrix metalloproteinase 9 (MMP9) expression in these cells. KEY RESULTS Glabridin significantly inhibited migration/invasion capacities of HCC cells, Huh7 and Sk-Hep-1, cell lines that have low cytotoxicity in vitro, even at high concentrations. Western blot analysis and gelatin zymography showed that glabridin inhibited the expression, activities and protein levels of MMP9 and the phosphorylation of ERK1/2 and JNK1/2. These inhibitory effects were associated with an up-regulation of tissue inhibitor of metalloproteinase-1 and a down-regulation of the transcription factors NF-κB and activator protein 1 signalling pathways. Finally, the administration of glabridin effectively suppressed the tumour formation in the hepatoma xenograft model in vivo. CONCLUSION AND IMPLICATIONS Glabridin inhibited the invasion of human HCC cells and may have potential as a chemopreventive agent against liver cancer metastasis. PMID:24641665

  1. Regulation of osteosarcoma cell lung metastasis by the c-Fos/AP-1 target FGFR1

    PubMed Central

    Weekes, Daniel; Zandueta, Carolina; Perurena, Naiara; Thomas, David P; Sunters, Andrew; Vuillier, Céline; Bozec, Aline; El-Emir, Ethaar; Miletich, Isabelle; Patiño-Garcia, Ana; Lecanda, Fernando; Grigoriadis, Agamemnon E

    2015-01-01

    Osteosarcoma is the most common primary malignancy of the skeleton and is prevalent in children and adolescents. Survival rates are poor and have remained stagnant due to chemoresistance and the high propensity to form lung metastases. In this study, we used in vivo transgenic models of c-fos oncogene-induced osteosarcoma and chondrosarcoma in addition to c-Fos-inducible systems in vitro to investigate downstream signaling pathways that regulate osteosarcoma growth and metastasis. Fgfr1 was identified as a novel c-Fos/AP-1 regulated gene. Induction of c-Fos in vitro in osteoblasts and chondroblasts caused an increase in Fgfr1 RNA and FGFR1 protein expression levels that resulted in increased and sustained activation of MAPKs, morphological transformation and increased anchorage-independent growth in response to FGF2 ligand treatment. High levels of FGFR1 protein and activated pFRS2α signalling were observed in murine and human osteosarcomas. Pharmacological inhibition of FGFR1 signalling blocked MAPK activation and colony growth of osteosarcoma cells in vitro. Orthotopic injection in vivo of FGFR1 silenced osteosarcoma cells caused a marked 2- to 5-fold decrease in spontaneous lung metastases. Similarly, inhibition of FGFR signalling in vivo with the small molecule inhibitor AZD4547 markedly reduced the number and size of metastatic nodules. Thus, deregulated FGFR signalling plays an important role in osteoblast transformation and osteosarcoma formation and regulates the development of lung metastases. Our findings support the development of anti-FGFR inhibitors as potential antimetastatic therapy. PMID:26387545

  2. Brominated Flame Retardants, Tetrabromobisphenol A and Hexabromocyclododecane, Activate Mitogen-Activated Protein Kinases (MAPKs) in Human Natural Killer Cells

    PubMed Central

    Cato, Anita; Celada, Lindsay; Kibakaya, Esther Caroline; Simmons, Nadia; Whalen, Margaret M.

    2014-01-01

    NK cells provide a vital surveillance against virally infected cells, tumor cells, and antibody-coated cells through the release of cytolytic mediators and gamma interferon (IFN-γ). Hexabromocyclododecane (HBCD) is a brominated flame retardant used primarily in expanded (EPS) and extruded (XPS) polystyrene foams for thermal insulation in the building and construction industry. Tetrabromobisphenol A (TBBPA) is used both as a reactive and an additive flame retardant in a variety of materials. HBCD and TBBPA contaminate the environment and are found in human blood samples. In previous studies, we have shown that other environmental contaminants, such as the dibutyltin (DBT) and tributyltin (TBT), decrease NK lytic function by activating mitogen-activated protein kinases (MAPKs) in the NK cells. HBCD and TBBPA also interfere with NK cell(s) lytic function. The current study evaluates whether HBCD and/or TBBPA have the capacity to activate MAPKs and MAPK kinases (MAP2Ks). The effects of concentrations of HBCD and TBBPA that inhibited lytic function on the phosphorylation state and total levels of the MAPKs (p44/42, p38, and JNK) and the phosphorylation and total levels of the MAP2Ks (MEK1/2 and MKK3/6) were examined. Results indicate that exposure of human NK cells to 10-0.5 µM HBCD or TBBPA activate MAPKs and MAP2Ks. This HBCD and TBBPA-induced activation of MAPKs may leave them unavailable for activation by virally infected or tumor target cells and thus contributes to the observed decreases in lytic function seen in NK cells exposed to HBCD and TBBPA. PMID:25341744

  3. Nodularin induces tumor necrosis factor-alpha and mitogen-activated protein kinases (MAPK) and leads to induction of endoplasmic reticulum stress.

    PubMed

    Meili, Nicole; Christen, Verena; Fent, Karl

    2016-06-01

    Nodularin is produced by the cyanobacterium Nodularia spumigena. It is of concern due to hepatotoxicity in humans and animals. Here we investigated unexplored molecular mechanisms by transcription analysis in human liver cells, focusing on induction of pro-inflammatory cytokines, the tumor necrosis factor α (TNF-α), endoplasmic reticulum (ER) stress and components of the activator protein-1 complex in human hepatoma cells (Huh7) exposed to non-cytotoxic (0.1 and 1μM) and toxic concentrations (5μM) for 24, 48, and 72h. Transcripts of TNF-α and ER stress marker genes were strongly induced at 1 and 5μM at all time-points. TNF-α led to induction of mitogen-activated protein kinases (MAPK), as demonstrated by induction of CJUN and CFOS, which form the AP-1 complex. Human primary liver cells reacted more sensitive than Huh7 cells. They showed higher cytotoxicity and induction of TNF-α and ER stress at 2.5nM, while HepG2 cells were insensitive up to 10μM due to low expression of organic anion transporting polypeptides. Furthermore, nodularin led to induction of TNF-α protein, and CCAAT/enhancer-binding protein-homologous (CHOP) protein. Our data indicate that nodularin induces inflammation and ER stress and leads to activation of MAPK in liver cells. All of these activated pathways, which were analysed here for the first time in detail, may contribute to the hepatotoxic, and tumorigenic action of nodularin. PMID:27061667

  4. Genkwanin Inhibits Proinflammatory Mediators Mainly through the Regulation of miR-101/MKP-1/MAPK Pathway in LPS-Activated Macrophages

    PubMed Central

    Gao, Yuan; Liu, Fen; Fang, Lei; Cai, Runlan; Zong, Chuanjie; Qi, Yun

    2014-01-01

    Genkwanin is one of the major non-glycosylated flavonoids in many herbs with anti-inflammatory activities. Although its anti-inflammatory activity in vivo has been reported, the potential molecular mechanisms remain obscure. In this study, by pharmacological and genetic approaches, we explore the anti-inflammatory effects of genkwanin in LPS-activated RAW264.7 macrophages. Genkwanin potently decreases the proinflammatory mediators, such as iNOS, TNF-α, IL-1β and IL-6, at the transcriptional and translational levels without cytotoxicity, indicating the excellent anti-inflammatory potency of genkwanin in vitro. Mechanism study shows that genkwanin significantly suppresses the p38- and JNK-mediated AP-1 signaling pathway and increases the mitogen-activated protein kinase (MAPK) phosphatase 1 (MKP-1) expression at the posttranscriptional level. We also confirmed that microRNA-101 (miR-101) is a negative regulator of MKP-1 expression. Moreover, regardless of miR-101-deficient cells or miR-101-abundant cells, the suppression effects of genkwanin on supernatant proinflammatory mediators' levels are far less than that in respective negative control cells, suggesting that genkwanin exerts anti-inflammatory effect mainly through reducing miR-101 production. However, genkwanin can't affect the level of phospho-Akt (p-Akt), indicating that the phosphorylation of Akt may be not responsible for the effect of genkwanin on miR-101 production. We conclude that genkwanin exerts its anti-inflammatory effect mainly through the regulation of the miR-101/MKP-1/MAPK pathway. PMID:24800851

  5. Estrogenic effect of procymidone through activation of MAPK in MCF-7 breast carcinoma cell line.

    PubMed

    Radice, Sonia; Chiesara, Enzo; Frigerio, Silvia; Fumagalli, Roberta; Parolaro, Daniela; Rubino, Tiziana; Marabini, Laura

    2006-05-01

    Procymidone modifies sexual differentiation in vitro and induces estrogenic activity in primary cultured rainbow trout hepatocytes, as shown by an increase in the contents of vitellogenin and heat shock proteins. Since this dicarboximide fungicide is found in human tissues, it was considered of interest to investigate its ability to induce endocrine damage in the MCF-7 human cell line. The mechanism of this estrogenic action was also evaluated. Procymidone 100 microM stimulated cell growth from day 3 up to day 12 and raised the level of pS2 on day 3. Although procymidone does not bind the estrogen receptor (ER), the antiestrogen ICI 182780 inhibited its effect on cell growth and pS2 content, suggesting that the ER is involved indirectly in these effects. In exploring the mechanism of ER indirect activation we found that the antibody against c-Neu receptor (9G6) did not modify procymidone's effects on cell growth and pS2 expression. Thus, procymidone does not bind the c-Neu membrane receptor, excluding this indirect ER activation pathway. We also found that procymidone induced mitogen-activated protein kinase (MAPK) at 15 and 30 min, and that PD 98059, a MAPK (Erk1/2) inhibitor, prevented procymidone's effects on cell growth and pS2, indicating that MAPK activation is responsible for procymidone ER activation. The production of reactive oxygen species (ROS) with these times and elimination of the phenomenon by alpha-tocopherol (alpha-T), a ROS scavenger, is proof that oxygen free-radical production is at the basis of the MAPK activation by procymidone. PMID:16310225

  6. Anti-Inflammatory Effect of Mangostenone F in Lipopolysaccharide-Stimulated RAW264.7 Macrophages by Suppressing NF-κB and MAPK Activation.

    PubMed

    Cho, Byoung Ok; Ryu, Hyung Won; So, Yangkang; Lee, Chang Wook; Jin, Chang Hyun; Yook, Hong Sun; Jeong, Yong Wook; Park, Jong Chun; Jeong, Il Yun

    2014-07-01

    Mangostenone F (MF) is a natural xanthone isolated from Garcinia mangostana. However, little is known about the biological activities of MF. This study was designed to investigate the anti-inflammatory effect and underlying molecular mechanisms of MF in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages. MF dose-dependently inhibited the production of NO, iNOS, and pro-inflammatory cytokines (TNF-α, IL-6, and IL-1β) in LPS-stimulated RAW264.7 macrophages. Moreover, MF decreased the NF-κB luciferase activity and NF-κB DNA binding capacity in LPS-stimulated RAW264.7 macrophages. Furthermore, MF suppressed the NF-κB activation by inhibiting the degradation of IκBα and nuclear translocation of p65 subunit of NF-κB. In addition, MF attenuated the AP-1 luciferase activity and phosphorylation of ERK, JNK, and p38 MAP kinases. Taken together, these results suggest that the anti-inflammatory effect of MF is associated with the suppression of NO production and iNOS expression through the down-regulation of NF-κB activation and MAPK signaling pathway in LPS-stimulated RAW264.7 macrophages. PMID:25143806

  7. N-hydroxycinnamide derivatives of osthole ameliorate hyperglycemia through activation of AMPK and p38 MAPK.

    PubMed

    Lee, Wei-Hwa; Wu, Hsueh-Hsia; Huang, Wei-Jan; Li, Yi-Ning; Lin, Ren-Jye; Lin, Shyr-Yi; Liang, Yu-Chih

    2015-01-01

    Our previous studies found that osthole markedly reduced blood glucose levels in both db/db and ob/ob mice. To improve the antidiabetic activity of osthole, a series of N-hydroxycinnamide derivatives of osthole were synthesized, and their hypoglycemia activities were examined in vitro and in vivo. Both N-hydroxycinnamide derivatives of osthole, OHC-4p and OHC-2m, had the greatest potential for activating AMPK and increasing glucose uptake by L6 skeletal muscle cells. In addition, OHC-4p and OHC-2m time- and dose-dependently increased phosphorylation levels of AMPK and p38 MAPK. The AMPK inhibitor, compound C, and the p38 MAPK inhibitor, SB203580, significantly reversed activation of AMPK and p38 MAPK, respectively, in OHC-4p- and OHC-2m-treated cells. Compound C and SB203580 also inhibited glucose uptake induced by OHC-4p and OHC-2m. Next, we found that OHC-4p and OHC-2m significantly increased glucose transporter 4 (GLUT4) translocation to plasma membranes and counteracted hyperglycemia in mice with streptozotocin-induced diabetes. These results suggest that activation of AMPK and p38 MAPK by OHC-4p and OHC-2m is associated with increased glucose uptake and GLUT4 translocation and subsequently led to amelioration of hyperglycemia. Therefore, OHC-4p and OHC-2m might have potential as antidiabetic agents for treating type 2 diabetes. Our previous studies found that osthole markedly reduced blood glucose levels in both db/db and ob/ob mice. To improve the antidiabetic activity of osthole, a series of N-hydroxycinnamide derivatives of osthole were synthesized, and their hypoglycemia activities were examined in vitro and in vivo. Both N-hydroxycinnamide derivatives of osthole, OHC-4p and OHC-2m, had the greatest potential for activating AMPK and increasing glucose uptake by L6 skeletal muscle cells. In addition, OHC-4p and OHC-2m time- and dose-dependently increased phosphorylation levels of AMPK and p38 MAPK. The AMPK inhibitor, compound C, and the p38 MAPK inhibitor

  8. Premature senescence involving p53 and p16 is activated in response to constitutive MEK/MAPK mitogenic signaling

    PubMed Central

    Lin, Athena W.; Barradas, Marta; Stone, James C.; van Aelst, Linda; Serrano, Manuel; Lowe, Scott W.

    1998-01-01

    Oncogenic Ras transforms immortal rodent cells to a tumorigenic state, in part, by constitutively transmitting mitogenic signals through the mitogen-activated protein kinase (MAPK) cascade. In primary cells, Ras is initially mitogenic but eventually induces premature senescence involving the p53 and p16INK4a tumor suppressors. Constitutive activation of MEK (a component of the MAPK cascade) induces both p53 and p16, and is required for Ras-induced senescence of normal human fibroblasts. Furthermore, activated MEK permanently arrests primary murine fibroblasts but forces uncontrolled mitogenesis and transformation in cells lacking either p53 or INK4a. The precisely opposite response of normal and immortalized cells to constitutive activation of the MAPK cascade implies that premature senescence acts as a fail-safe mechanism to limit the transforming potential of excessive Ras mitogenic signaling. Consequently, constitutive MAPK signaling activates p53 and p16 as tumor suppressors. PMID:9765203

  9. AP-1 family members act with Sox9 to promote chondrocyte hypertrophy.

    PubMed

    He, Xinjun; Ohba, Shinsuke; Hojo, Hironori; McMahon, Andrew P

    2016-08-15

    An analysis of Sox9 binding profiles in developing chondrocytes identified marked enrichment of an AP-1-like motif. Here, we have explored the functional interplay between Sox9 and AP-1 in mammalian chondrocyte development. Among AP-1 family members, Jun and Fosl2 were highly expressed within prehypertrophic and early hypertrophic chondrocytes. Chromatin immunoprecipitation followed by DNA sequencing (ChIP-seq) showed a striking overlap in Jun- and Sox9-bound regions throughout the chondrocyte genome, reflecting direct binding of each factor to the same enhancers and a potential for protein-protein interactions within AP-1- and Sox9-containing complexes. In vitro reporter analysis indicated that direct co-binding of Sox9 and AP-1 at target motifs promoted gene activity. By contrast, where only one factor can engage its DNA target, the presence of the other factor suppresses target activation consistent with protein-protein interactions attenuating transcription. Analysis of prehypertrophic chondrocyte removal of Sox9 confirmed the requirement of Sox9 for hypertrophic chondrocyte development, and in vitro and ex vivo analyses showed that AP-1 promotes chondrocyte hypertrophy. Sox9 and Jun co-bound and co-activated a Col10a1 enhancer in Sox9 and AP-1 motif-dependent manners consistent with their combined action promoting hypertrophic gene expression. Together, the data support a model in which AP-1 family members contribute to Sox9 action in the transition of chondrocytes to the hypertrophic program. PMID:27471255

  10. Atractylodin Inhibits Interleukin-6 by Blocking NPM-ALK Activation and MAPKs in HMC-1.

    PubMed

    Chae, Hee-Sung; Kim, Young-Mi; Chin, Young-Won

    2016-01-01

    Atractylodin is one of the major constituents of the rhizome of Atractylodes lancea, which is widely used in Korean traditional medicine as a remedy for the treatment of gastritis and gastric ulcers. Despite of a major constituent of widely used botanical to treat inflammatory responses little is known about anti-inflammatory effect of atractylodin in the human mast cell (HMC-1). Hence, we evaluated the effect of atractylodin on the release of IL-6, the involvement of nucleophosmin-anaplastic lymphoma kinase (NPM-ALK) and mitogen-activated protein kinases (MAPKs) in phorbol-12-myristate-13-acetate and A23187-induced HMC-1. In addition, Janus kinase 2 (JAK2), signal transducer and activator of transcription 3 (STAT3), phospholipase C (PLC) gamma 1, and AKT phosphorylation relevant to NPM-ALK signal pathway were assessed. IL-6 levels in the HMC-1 stimulated by phorbol-12-myristate-13-acetate and A23187 were apparently decreased by the treatment of atractylodin. Concurrently, atractylodin not only inhibited the phosphorylation of NPM-ALK, but also suppressed the phosphorylation of JAK2, STAT3, PLC gamma 1, and AKT. Furthermore, the activated mitogen-activated protein kinases (MAPKs) by phorbol-12-myristate-13-acetate and A23187 were inhibited by atractylodin. These results suggested that atractylodin might have a potential regulatory effect on inflammatory mediator expression through blockade of both the phosphorylation of MAPKs and the NPM-ALK signaling pathway. PMID:27598116

  11. Dioscorea nipponica Makino inhibits migration and invasion of human oral cancer HSC-3 cells by transcriptional inhibition of matrix metalloproteinase-2 through modulation of CREB and AP-1 activity.

    PubMed

    Chien, Ming-Hsien; Ying, Tsung-Ho; Hsieh, Yih-Shou; Chang, Yu-Chao; Yeh, Chia-Ming; Ko, Jiunn-Liang; Lee, Wen-Sen; Chang, Jer-Hua; Yang, Shun-Fa

    2012-03-01

    Oral cancer mortality has increased during the last decade due to the difficulties in treating related metastasis. Dioscorea nipponica Makino, a popular folk medicine, exerts anti-obesity and anti-inflammation properties. However, the effect of this folk medicine on metastasis of oral cancer has yet to be fully elucidated. The present study demonstrates that D. nipponica extracts (DNE), at a range of concentrations (0-50 μg/mL), concentration-dependently inhibited migration/invasion capacities of human oral cancer cells, HSC-3, without cytotoxic effects. The anti-migration effect of DNE was also observed in two other OSCC cell lines, Ca9-22 and Cal-27. Zymography, real time PCR, and Western blotting analyses revealed that DNE inhibited matrix metalloproteinase-2 (MMP-2) enzyme activity, and RNA and protein expression. The inhibitory effects of DNE on MMP-2 proceeded by up-regulating tissue inhibitor of metalloproteinase-2 (TIMP-2), as well as suppressing nuclear translocation and DNA binding activity of cAMP response element-binding (CREB) and activating protein-1 (AP-1) on the MMP-2 promoter in HSC-3 cells. In conclusion, DNE inhibited the invasion of oral cancer cells and may have potential use as a chemopreventive agent against oral cancer metastasis. PMID:22210353

  12. Ganoderma lucidum polysaccharide exerts anti-tumor activity via MAPK pathways in HL-60 acute leukemia cells.

    PubMed

    Yang, Guohua; Yang, Lei; Zhuang, Yun; Qian, Xifeng; Shen, Yunfeng

    2016-01-01

    In this study, we investigated the anti-tumor activity both in vitro and in vivo of a polysaccharide obtained from Ganoderma lucidum on HL-60 acute myeloid leukemia cells, and focused on its targeting effect on mitogen-activated protein kinase (MAPK) pathways. It was found by the methods such as western blot and flow cytometry (FCM), that G. lucidum polysaccharide (GLP) blocked the extracellular signal-regulated kinase/MAPK signaling pathway, simultaneously activated p38 and JNK MAPK pathways, and therefore regulated their downstream genes and proteins, including p53, c-myc, c-fos, c-jun, Bcl-2, Bax, cleaved caspase-3 and cyclin D1. As a result, cycle arrest and apoptosis of HL-60 cells were induced. Therefore, GLP exerted anti-tumor activity via MAPK pathways in HL-60 acute leukemia cells. PMID:25327706

  13. Basal protein phosphatase 2A activity restrains cytokine expression: role for MAPKs and tristetraprolin.

    PubMed

    Rahman, Md Mostafizur; Rumzhum, Nowshin N; Morris, Jonathan C; Clark, Andrew R; Verrills, Nicole M; Ammit, Alaina J

    2015-01-01

    PP2A is a master controller of multiple inflammatory signaling pathways. It is a target in asthma; however the molecular mechanisms by which PP2A controls inflammation warrant further investigation. In A549 lung epithelial cells in vitro we show that inhibition of basal PP2A activity by okadaic acid (OA) releases restraint on MAPKs and thereby increases MAPK-mediated pro-asthmatic cytokines, including IL-6 and IL-8. Notably, PP2A inhibition also impacts on the anti-inflammatory protein - tristetraprolin (TTP), a destabilizing RNA binding protein regulated at multiple levels by p38 MAPK. Although PP2A inhibition increases TTP mRNA expression, resultant TTP protein builds up in the hyperphosphorylated inactive form. Thus, when PP2A activity is repressed, pro-inflammatory cytokines increase and anti-inflammatory proteins are rendered inactive. Importantly, these effects can be reversed by the PP2A activators FTY720 and AAL(s), or more specifically by overexpression of the PP2A catalytic subunit (PP2A-C). Moreover, PP2A plays an important role in cytokine expression in cells stimulated with TNFα; as inhibition of PP2A with OA or PP2A-C siRNA results in significant increases in cytokine production. Collectively, these data reveal the molecular mechanisms of PP2A regulation and highlight the potential of boosting the power of endogenous phosphatases as novel anti-inflammatory strategies to combat asthmatic inflammation. PMID:25985190

  14. WNT3 Inhibits Cerebellar Granule Neuron Progenitor Proliferation and Medulloblastoma Formation via MAPK Activation

    PubMed Central

    Ayrault, Olivier; Kim, Jee Hae; Zhu, Xiaodong; Murphy, David A.; Van Aelst, Linda; Roussel, Martine F.; Hatten, Mary E.

    2013-01-01

    During normal cerebellar development, the remarkable expansion of granule cell progenitors (GCPs) generates a population of granule neurons that outnumbers the total neuronal population of the cerebral cortex, and provides a model for identifying signaling pathways that may be defective in medulloblastoma. While many studies focus on identifying pathways that promote growth of GCPs, a critical unanswered question concerns the identification of signaling pathways that block mitogenic stimulation and induce early steps in differentiation. Here we identify WNT3 as a novel suppressor of GCP proliferation during cerebellar development and an inhibitor of medulloblastoma growth in mice. WNT3, produced in early postnatal cerebellum, inhibits GCP proliferation by down-regulating pro-proliferative target genes of the mitogen Sonic Hedgehog (SHH) and the bHLH transcription factor Atoh1. WNT3 suppresses GCP growth through a non-canonical Wnt signaling pathway, activating prototypic mitogen-activated protein kinases (MAPKs), the Ras-dependent extracellular-signal-regulated kinases 1/2 (ERK1/2) and ERK5, instead of the classical β-catenin pathway. Inhibition of MAPK activity using a MAPK kinase (MEK) inhibitor reversed the inhibitory effect of WNT3 on GCP proliferation. Importantly, WNT3 inhibits proliferation of medulloblastoma tumor growth in mouse models by a similar mechanism. Thus, the present study suggests a novel role for WNT3 as a regulator of neurogenesis and repressor of neural tumors. PMID:24303070

  15. Concurrent BMP7 and FGF9 signalling governs AP-1 function to promote self-renewal of nephron progenitor cells

    PubMed Central

    Muthukrishnan, Sree Deepthi; Yang, Xuehui; Friesel, Robert; Oxburgh, Leif

    2015-01-01

    Self-renewal of nephron progenitor cells (NPCs) is governed by BMP, FGF and WNT signalling. Mechanisms underlying cross-talk between these pathways at the molecular level are largely unknown. Here we delineate the pathway through which the proliferative BMP7 signal is transduced in NPCs in the mouse. BMP7 activates the MAPKs TAK1 and JNK to phosphorylate the transcription factor JUN, which in turn governs transcription of AP-1-element containing G1-phase cell cycle regulators such as Myc and Ccnd1 to promote NPC proliferation. Conditional inactivation of Tak1 or Jun in cap mesenchyme causes identical phenotypes characterized by premature depletion of NPCs. While JUN is regulated by BMP7, we find that its partner FOS is regulated by FGF9. We demonstrate that BMP7 and FGF9 coordinately regulate AP-1 transcription to promote G1-S cell cycle progression and NPC proliferation. Our findings identify a molecular mechanism explaining the important cooperation between two major NPC self-renewal pathways. PMID:26634297

  16. A novel p38 mitogen activated protein kinase (MAPK) specific inhibitor suppresses respiratory syncytial virus and influenza A virus replication by inhibiting virus-induced p38 MAPK activation.

    PubMed

    Choi, Myung-Soo; Heo, Jinyuk; Yi, Chae-Min; Ban, Junsu; Lee, Noh-Jin; Lee, Na-Rae; Kim, Sang Won; Kim, Nam-Jung; Inn, Kyung-Soo

    2016-08-26

    Respiratory syncytial virus (RSV) and influenza A virus are leading causes of acute lower respiratory infectious disease. Respiratory diseases caused by RSV and influenza A virus result in serious economic burden and life-threatening disease for immunocompromised people. With the revelation that p38 mitogen-activated protein kinase (MAPK) activity in host cells is crucial for infection and replication of RSV and influenza A virus, inhibition of p38 MAPK activity has been suggested as a potential antiviral therapeutic strategy. However, the low selectivity and high toxicity of the p38 MAPK inhibitors necessitate the development of better inhibitors. Herein, we report the synthesis of a novel p38 MAPK inhibitor, NJK14047, with high kinase selectivity. In this work, it was demonstrated that NJK14047 inhibits RSV- and influenza A-mediated p38 MAPK activation in epithelial cells. Subsequently, NJK14047 treatment resulted in decreased viral replication and viral mRNA synthesis. In addition, secretion of interleukin-6 from infected cells was greatly diminished by NJK14047, suggesting that it can ameliorate immunopathological responses to RSV and influenza A. Collectively, the results suggest that NJK14047 has therapeutic potential to treat respiratory viral infection through the suppression of p38 MAPK activation, which is suggested to be an essential step for respiratory virus infection. PMID:27346133

  17. The Mitogen-Activated Protein Kinase (MAPK) Pathway: Role in Immune Evasion by Trypanosomatids

    PubMed Central

    Soares-Silva, Mercedes; Diniz, Flavia F.; Gomes, Gabriela N.; Bahia, Diana

    2016-01-01

    Leishmania spp. and Trypanosoma cruzi are the causative agents of leishmaniasis and Chagas disease, respectively, two neglected tropical diseases that affect about 25 million people worldwide. These parasites belong to the family Trypanosomatidae, and are both obligate intracellular parasites that manipulate host signaling pathways and the innate immune system to establish infection. Mitogen-activated protein kinases (MAPKs) are serine and threonine protein kinases that are highly conserved in eukaryotes, and are involved in signal transduction pathways that modulate physiological and pathophysiological cell responses. This mini-review highlights existing knowledge concerning the mechanisms that Leishmania spp. and T. cruzi have evolved to target the host’s MAPK signaling pathways and highjack the immune response, and, in this manner, promote parasite maintenance in the host. PMID:26941717

  18. The Mitogen-Activated Protein Kinase (MAPK) Pathway: Role in Immune Evasion by Trypanosomatids.

    PubMed

    Soares-Silva, Mercedes; Diniz, Flavia F; Gomes, Gabriela N; Bahia, Diana

    2016-01-01

    Leishmania spp. and Trypanosoma cruzi are the causative agents of leishmaniasis and Chagas disease, respectively, two neglected tropical diseases that affect about 25 million people worldwide. These parasites belong to the family Trypanosomatidae, and are both obligate intracellular parasites that manipulate host signaling pathways and the innate immune system to establish infection. Mitogen-activated protein kinases (MAPKs) are serine and threonine protein kinases that are highly conserved in eukaryotes, and are involved in signal transduction pathways that modulate physiological and pathophysiological cell responses. This mini-review highlights existing knowledge concerning the mechanisms that Leishmania spp. and T. cruzi have evolved to target the host's MAPK signaling pathways and highjack the immune response, and, in this manner, promote parasite maintenance in the host. PMID:26941717

  19. The activation of p38MAPK and JNK pathways in bovine herpesvirus 1 infected MDBK cells.

    PubMed

    Zhu, Liqian; Yuan, Chen; Huang, Liyuan; Ding, Xiuyan; Wang, Jianye; Zhang, Dong; Zhu, Guoqiang

    2016-01-01

    We have shown previously that BHV-1 infection activates Erk1/2 signaling. Here, we show that BHV-1 provoked an early-stage transient and late-stage sustained activation of JNK, p38MAPK and c-Jun signaling in MDBK cells. C-Jun phosphorylation was dependent on JNK. These early events were partially due to the viral entry process. Unexpectedly, reactive oxygen species were not involved in the later activation phase. Interestingly, only activated JNK facilitated the viral multiplication identified through both chemical inhibitor and siRNA. Collectively, this study provides insight into our understanding of early stages of BHV-1 infection. PMID:27590675

  20. Involvement of p38 MAPK in the Anticancer Activity of Cultivated Cordyceps militaris.

    PubMed

    Chou, Shang-Min; Lai, Wan-Jung; Hong, Tzuwen; Tsai, Sheng-Hong; Chen, Yen-Hsun; Kao, Cheng-Hsiang; Chu, Richard; Shen, Tang-Long; Li, Tsai-Kun

    2015-01-01

    Cordyceps militaris is a traditional Chinese medicine frequently used for tonic and therapeutic purposes. Reports from our laboratory and others have demonstrated that extracts of the cultivated fruiting bodies of C. militaris (CM) exhibit a potent cytotoxic effect against many cancer cell lines, especially human leukemia cells. Here, we further investigated the underlying mechanism through which CM is cytotoxic to cancer cells. The CM-mediated induction of PARP cleavage and its related DNA damage signal (γH2AX) was diminished by caspase inhibitor I. In contrast, a ROS scavenger failed to prevent CM-mediated leukemia cell death. Moreover, two signaling molecules, AKT and p38 MAPK, were activated during the course of apoptosis induction. Employing MTT analysis, we found that a p38 MAPK inhibitor but not an AKT inhibitor could rescue cells from CM-mediated cell death, as well as inhibit the cleavage of PARP, formation of apoptotic bodies and up-regulation of the γH2AX signal. These results suggest that CM-mediated leukemia cell death occurs through the activation of the p38 MAPK pathway, indicating its potential therapeutic effects against human leukemia. PMID:26205966

  1. Ribosome Synthesis and MAPK Activity Modulate Ionizing Radiation-Induced Germ Cell Apoptosis in Caenorhabditis elegans

    PubMed Central

    Eberhard, Ralf; Stergiou, Lilli; Hofmann, E. Randal; Hofmann, Jen; Haenni, Simon; Teo, Youjin; Furger, André; Hengartner, Michael O.

    2013-01-01

    Synthesis of ribosomal RNA by RNA polymerase I (RNA pol I) is an elemental biological process and is key for cellular homeostasis. In a forward genetic screen in C. elegans designed to identify DNA damage-response factors, we isolated a point mutation of RNA pol I, rpoa-2(op259), that leads to altered rRNA synthesis and a concomitant resistance to ionizing radiation (IR)-induced germ cell apoptosis. This weak apoptotic IR response could be phenocopied when interfering with other factors of ribosome synthesis. Surprisingly, despite their resistance to DNA damage, rpoa-2(op259) mutants present a normal CEP-1/p53 response to IR and increased basal CEP-1 activity under normal growth conditions. In parallel, rpoa-2(op259) leads to reduced Ras/MAPK pathway activity, which is required for germ cell progression and physiological germ cell death. Ras/MAPK gain-of-function conditions could rescue the IR response defect in rpoa-2(op259), pointing to a function for Ras/MAPK in modulating DNA damage-induced apoptosis downstream of CEP-1. Our data demonstrate that a single point mutation in an RNA pol I subunit can interfere with multiple key signalling pathways. Ribosome synthesis and growth-factor signalling are perturbed in many cancer cells; such an interplay between basic cellular processes and signalling might be critical for how tumours evolve or respond to treatment. PMID:24278030

  2. BMP9-Induced Survival Effect in Liver Tumor Cells Requires p38MAPK Activation

    PubMed Central

    García-Álvaro, María; Addante, Annalisa; Roncero, Cesáreo; Fernández, Margarita; Fabregat, Isabel; Sánchez, Aránzazu; Herrera, Blanca

    2015-01-01

    The study of bone morphogenetic proteins (BMPs) role in tumorigenic processes, and specifically in the liver, has gathered importance in the last few years. Previous studies have shown that BMP9 is overexpressed in about 40% of hepatocellular carcinoma (HCC) patients. In vitro data have also shown evidence that BMP9 has a pro-tumorigenic action, not only by inducing epithelial to mesenchymal transition (EMT) and migration, but also by promoting proliferation and survival in liver cancer cells. However, the precise mechanisms driving these effects have not yet been established. In the present work, we deepened our studies into the intracellular mechanisms implicated in the BMP9 proliferative and pro-survival effect on liver tumor cells. In HepG2 cells, BMP9 induces both Smad and non-Smad signaling cascades, specifically PI3K/AKT and p38MAPK. However, only the p38MAPK pathway contributes to the BMP9 growth-promoting effect on these cells. Using genetic and pharmacological approaches, we demonstrate that p38MAPK activation, although dispensable for the BMP9 proliferative activity, is required for the BMP9 protective effect on serum withdrawal-induced apoptosis. These findings contribute to a better understanding of the signaling pathways involved in the BMP9 pro-tumorigenic role in liver tumor cells. PMID:26343646

  3. Cadmium induces vascular permeability via activation of the p38 MAPK pathway

    SciTech Connect

    Dong, Fengyun; Guo, Fang; Li, Liqun; Guo, Ling; Hou, Yinglong; Hao, Enkui; Yan, Suhua; Allen, Thaddeus D.; Liu, Ju

    2014-07-18

    Highlights: • Low-dose cadmium (Cd) induces vascular hyper-permeability. • p38 MAPK mediates Cd-induced disruption of endothelial cell barrier function. • SB203850 inhibits Cd-induced membrane dissociation of VE-cadherin and β-catenin. • SB203850 reduces Cd-induced expression and secretion of TNF-α. - Abstract: The vasculature of various organs is a targeted by the environmental toxin, cadmium (Cd). However, mechanisms leading to pathological conditions are poorly understood. In the present study, we examined the effect of cadmium chloride (CdCl{sub 2}) on human umbilical vein endothelial cells (HUVECs). At 4 μM, CdCl{sub 2} induced a hyper-permeability defect in HUVECs, but not the inhibition of cell growth up to 24 h. This effect of CdCl{sub 2} was dependent on the activation of the p38 mitogen-activated protein kinase (MAPK) pathway. The p38 MAPK inhibitor SB203850 suppressed the CdCl{sub 2}-induced alteration in trans-endothelial electrical resistance in HUVEC monolayers, a model measurement of vascular endothelial barrier integrity. SB203850 also inhibited the Cd-induced membrane dissociation of vascular endothelial (VE) cadherin and β-catenin, the important components of the adherens junctional complex. In addition, SB203850 reduces the Cd-induced expression and secretion of tumor necrosis factor α (TNF-α). Taken together, our findings suggest that Cd induces vascular hyper-permeability and disruption of endothelial barrier integrity through stimulation of p38 MAPK signaling.

  4. BMP7 retards peripheral myelination by activating p38 MAPK in Schwann cells

    PubMed Central

    Liu, Xiaoyu; Zhao, Yahong; Peng, Su; Zhang, Shuqiang; Wang, Meihong; Chen, Yeyue; Zhang, Shan; Yang, Yumin; Sun, Cheng

    2016-01-01

    Schwann cell (SC) myelination is pivotal for the proper physiological functioning of the nervous system, but the underlying molecular mechanism remains less well understood. Here, we showed that the expression of bone morphogenetic protein 7 (BMP7) inversely correlates with myelin gene expression during peripheral myelination, which suggests that BMP7 is likely a negative regulator for myelin gene expression. Our experiments further showed that the application of BMP7 attenuates the cAMP induced myelin gene expression in SCs. Downstream pathway analysis suggested that both p38 MAPK and SMAD are activated by exogenous BMP7 in SCs. The pharmacological intervention and gene silence studies revealed that p38 MAPK, not SMAD, is responsible for BMP7-mediated suppression of myelin gene expression. In addition, c-Jun, a potential negative regulator for peripheral myelination, was up-regulated by BMP7. In vivo experiments showed that BMP7 treatment greatly impaired peripheral myelination in newborn rats. Together, our results established that BMP7 is a negative regulator for peripheral myelin gene expression and that p38 MAPK/c-Jun axis might be the main downstream target of BMP7 in this process. PMID:27491681

  5. BMP7 retards peripheral myelination by activating p38 MAPK in Schwann cells.

    PubMed

    Liu, Xiaoyu; Zhao, Yahong; Peng, Su; Zhang, Shuqiang; Wang, Meihong; Chen, Yeyue; Zhang, Shan; Yang, Yumin; Sun, Cheng

    2016-01-01

    Schwann cell (SC) myelination is pivotal for the proper physiological functioning of the nervous system, but the underlying molecular mechanism remains less well understood. Here, we showed that the expression of bone morphogenetic protein 7 (BMP7) inversely correlates with myelin gene expression during peripheral myelination, which suggests that BMP7 is likely a negative regulator for myelin gene expression. Our experiments further showed that the application of BMP7 attenuates the cAMP induced myelin gene expression in SCs. Downstream pathway analysis suggested that both p38 MAPK and SMAD are activated by exogenous BMP7 in SCs. The pharmacological intervention and gene silence studies revealed that p38 MAPK, not SMAD, is responsible for BMP7-mediated suppression of myelin gene expression. In addition, c-Jun, a potential negative regulator for peripheral myelination, was up-regulated by BMP7. In vivo experiments showed that BMP7 treatment greatly impaired peripheral myelination in newborn rats. Together, our results established that BMP7 is a negative regulator for peripheral myelin gene expression and that p38 MAPK/c-Jun axis might be the main downstream target of BMP7 in this process. PMID:27491681

  6. OsMAPK6, a mitogen-activated protein kinase, influences rice grain size and biomass production.

    PubMed

    Liu, Shuying; Hua, Lei; Dong, Sujun; Chen, Hongqi; Zhu, Xudong; Jiang, Jun'e; Zhang, Fang; Li, Yunhai; Fang, Xiaohua; Chen, Fan

    2015-11-01

    Grain size is an important agronomic trait in determining grain yield. However, the molecular mechanisms that determine the final grain size are not well understood. Here, we report the functional analysis of a rice (Oryza sativa L.) mutant, dwarf and small grain1 (dsg1), which displays pleiotropic phenotypes, including small grains, dwarfism and erect leaves. Cytological observations revealed that the small grain and dwarfism of dsg1 were mainly caused by the inhibition of cell proliferation. Map-based cloning revealed that DSG1 encoded a mitogen-activated protein kinase (MAPK), OsMAPK6. OsMAPK6 was mainly located in the nucleus and cytoplasm, and was ubiquitously distributed in various organs, predominately in spikelets and spikelet hulls, consistent with its role in grain size and biomass production. As a functional kinase, OsMAPK6 interacts strongly with OsMKK4, indicating that OsMKK4 is likely to be the upstream MAPK kinase of OsMAPK6 in rice. In addition, hormone sensitivity tests indicated that the dsg1 mutant was less sensitive to brassinosteroids (BRs). The endogenous BR levels were reduced in dsg1, and the expression of several BR signaling pathway genes and feedback-inhibited genes was altered in the dsg1 mutant, with or without exogenous BRs, indicating that OsMAPK6 may contribute to influence BR homeostasis and signaling. Thus, OsMAPK6, a MAPK, plays a pivotal role in grain size in rice, via cell proliferation, and BR signaling and homeostasis. PMID:26366992

  7. Voltage-gated Na+ Channel Activity Increases Colon Cancer Transcriptional Activity and Invasion Via Persistent MAPK Signaling

    NASA Astrophysics Data System (ADS)

    House, Carrie D.; Wang, Bi-Dar; Ceniccola, Kristin; Williams, Russell; Simaan, May; Olender, Jacqueline; Patel, Vyomesh; Baptista-Hon, Daniel T.; Annunziata, Christina M.; Silvio Gutkind, J.; Hales, Tim G.; Lee, Norman H.

    2015-06-01

    Functional expression of voltage-gated Na+ channels (VGSCs) has been demonstrated in multiple cancer cell types where channel activity induces invasive activity. The signaling mechanisms by which VGSCs promote oncogenesis remain poorly understood. We explored the signal transduction process critical to VGSC-mediated invasion on the basis of reports linking channel activity to gene expression changes in excitable cells. Coincidentally, many genes transcriptionally regulated by the SCN5A isoform in colon cancer have an over-representation of cis-acting sites for transcription factors phosphorylated by ERK1/2 MAPK. We hypothesized that VGSC activity promotes MAPK activation to induce transcriptional changes in invasion-related genes. Using pharmacological inhibitors/activators and siRNA-mediated gene knockdowns, we correlated channel activity with Rap1-dependent persistent MAPK activation in the SW620 human colon cancer cell line. We further demonstrated that VGSC activity induces downstream changes in invasion-related gene expression via a PKA/ERK/c-JUN/ELK-1/ETS-1 transcriptional pathway. This is the first study illustrating a molecular mechanism linking functional activity of VGSCs to transcriptional activation of invasion-related genes.

  8. Molecular basis for enhancement of the meiotic DMC1 recombinase by RAD51 associated protein 1 (RAD51AP1)

    PubMed Central

    Dray, Eloïse; Dunlop, Myun Hwa; Kauppi, Liisa; Filippo, Joseph San; Wiese, Claudia; Tsai, Miaw-Sheue; Begovic, Sead; Schild, David; Jasin, Maria; Keeney, Scott; Sung, Patrick

    2011-01-01

    Homologous recombination is needed for meiotic chromosome segregation, genome maintenance, and tumor suppression. RAD51AP1 (RAD51 associated protein 1) has been shown to interact with and enhance the recombinase activity of RAD51. Accordingly, genetic ablation of RAD51AP1 leads to enhanced sensitivity to and also chromosome aberrations upon DNA damage, demonstrating a role for RAD51AP1 in mitotic homologous recombination. Here we show physical association of RAD51AP1 with the meiosis-specific recombinase DMC1 and a stimulatory effect of RAD51AP1 on the DMC1-mediated D-loop reaction. Mechanistic studies have revealed that RAD51AP1 enhances the ability of the DMC1 presynaptic filament to capture the duplex-DNA partner and to assemble the synaptic complex, in which the recombining DNA strands are homologously aligned. We also provide evidence that functional cooperation is dependent on complex formation between DMC1 and RAD51AP1 and that distinct epitopes in RAD51AP1 mediate interactions with RAD51 and DMC1. Finally, we show that RAD51AP1 is expressed in mouse testes, and that RAD51AP1 foci colocalize with a subset of DMC1 foci in spermatocytes. These results suggest that RAD51AP1 also serves an important role in meiotic homologous recombination. PMID:21307306

  9. Clathrin interactions with C-terminal regions of the yeast AP-1 beta and gamma subunits are important for AP-1 association with clathrin coats.

    PubMed

    Yeung, B G; Payne, G S

    2001-08-01

    Heterotetrameric adaptor (AP) complexes are thought to coordinate cargo recruitment and clathrin assembly during clathrin-coated vesicle biogenesis. We have identified, and characterized the physiological significance of clathrin-binding activities in the two large subunits of the AP-1 complex in Saccharomyces cerevisiae. Using GST-fusion chromatography, two clathrin-binding sites were defined in the beta1 subunit that match consensus clathrin-binding sequences in other mammalian and yeast clathrin-binding proteins. Clathrin interactions were also identified with the C-terminal region of the gamma subunit. When introduced into chromosomal genes, point mutations in the beta1 clathrin-binding motifs, or deletion of the gamma C-terminal region, reduced association of AP-1 with clathrin in coimmunoprecipitation assays. The beta1 mutations or the gamma truncation individually produced minor effects on AP-1 distribution by subcellular fractionation. However, when beta1 and gamma mutations were combined, severe defects were observed in AP-1 association with membranes and incorporation into clathrin-coated vesicles. The combination of subunit mutations accentuated growth and alpha-factor pheromone maturation defects in chc1-ts cells, though not to the extent caused by complete loss of AP-1 activity. Our results suggest that both the beta1 and gamma subunits contribute interactions with clathrin that are important for stable assembly of AP-1 complexes into clathrin coats in vivo. PMID:11489214

  10. AP-1/σ1A and AP-1/σ1B adaptor-proteins differentially regulate neuronal early endosome maturation via the Rab5/Vps34-pathway

    PubMed Central

    Candiello, Ermes; Kratzke, Manuel; Wenzel, Dirk; Cassel, Dan; Schu, Peter

    2016-01-01

    The σ1 subunit of the AP-1 clathrin-coated-vesicle adaptor-protein complex is expressed as three isoforms. Tissues express σ1A and one of the σ1B and σ1C isoforms. Brain is the tissue with the highest σ1A and σ1B expression. σ1B-deficiency leads to severe mental retardation, accumulation of early endosomes in synapses and fewer synaptic vesicles, whose recycling is slowed down. AP-1/σ1A and AP-1/σ1B regulate maturation of these early endosomes into multivesicular body late endosomes, thereby controlling synaptic vesicle protein transport into a degradative pathway. σ1A binds ArfGAP1, and with higher affinity brain-specific ArfGAP1, which bind Rabex-5. AP-1/σ1A-ArfGAP1-Rabex-5 complex formation leads to more endosomal Rabex-5 and enhanced, Rab5GTP-stimulated Vps34 PI3-kinase activity, which is essential for multivesicular body endosome formation. Formation of AP-1/σ1A-ArfGAP1-Rabex-5 complexes is prevented by σ1B binding of Rabex-5 and the amount of endosomal Rabex-5 is reduced. AP-1 complexes differentially regulate endosome maturation and coordinate protein recycling and degradation, revealing a novel molecular mechanism by which they regulate protein transport besides their established function in clathrin-coated-vesicle formation. PMID:27411398

  11. Curcumin increases gelatinase activity in human neutrophils by a p38 mitogen-activated protein kinase (MAPK)-independent mechanism.

    PubMed

    Antoine, Francis; Girard, Denis

    2015-01-01

    Curcumin has been found to possess anti-inflammatory activities and neutrophils, key players in inflammation, were previously found to be important targets to curcumin in a few studies. For example, curcumin was found to induce apoptosis in neutrophils by a p38 mitogen-activated protein kinase (MAPK)-dependent mechanism. However, the role of curcumin on the biology of neutrophils is still poorly defined. To study the role of curcumin on neutrophil degranulation and to determine the role of p38 MAPK, human neutrophils were freshly isolated from healthy individuals and incubated in vitro with curcumin. Degranulation was studied at three levels: surface expression of granule markers by flow cytometry; release of matrix metallopeptidase-9 (MMP-9 or gelatinase B) enzyme into supernatants by Western blot; and gelatinase B activity by zymography. Activation of p38 MAPK was studied by monitoring its tyrosine phosphorylation levels by western blot and its role by the utilization of a pharmacological inhibitor. The results indicate that curcumin increased the cell surface expression of CD35 (secretory vesicle), CD63 (azurophilic granules), and CD66b (gelatinase granules) in neutrophils. Also, curcumin increased the release and enzymatic activity of gelatinase B in the extracellular milieu and activated p38 MAP kinase in these cells. However, in contrast to fMLP, curcumin-induced enzymatic activity and secretion of gelatinase B were not reversed by use of a p38 inhibitor. Finally, it was found that curcumin was able to enhance phagocytosis. Taken together, the results here demonstrate that curcumin induced degranulation in human neutrophils and that the increased gelatinase activity is not dependent on p38 MAPK activation. Therefore, degranulation is another human neutrophil function that could be modulated by curcumin, as well as phagocytosis. PMID:24926560

  12. Deoxycholic acid mediates non-canonical EGFR-MAPK activation through the induction of calcium signaling in colon cancer cells.

    PubMed

    Centuori, Sara M; Gomes, Cecil J; Trujillo, Jesse; Borg, Jamie; Brownlee, Joshua; Putnam, Charles W; Martinez, Jesse D

    2016-07-01

    Obesity and a western diet have been linked to high levels of bile acids and the development of colon cancer. Specifically, increased levels of the bile acid deoxycholic acid (DCA), an established tumor promoter, has been shown to correlate with increased development of colorectal adenomas and progression to carcinoma. Herein we investigate the mechanism by which DCA leads to EGFR-MAPK activation, a candidate mechanism by which DCA may promote colorectal tumorigenesis. DCA treated colon cancer cells exhibited strong and prolonged activation of ERK1/2 when compared to EGF treatment alone. We also showed that DCA treatment prevents EGFR degradation as opposed to the canonical EGFR recycling observed with EGF treatment. Moreover, the combination of DCA and EGF treatment displayed synergistic activity, suggesting DCA activates MAPK signaling in a non-canonical manner. Further evaluation showed that DCA treatment increased intracellular calcium levels and CAMKII phosphorylation, and that blocking calcium with BAPTA-AM abrogated MAPK activation induced by DCA, but not by EGF. Finally we showed that DCA-induced CAMKII leads to MAPK activation through the recruitment of c-Src. Taken together, we demonstrated that DCA regulates MAPK activation through calcium signaling, an alternative mechanism not previously recognized in human colon cancer cells. Importantly, this mechanism allows for EGFR to escape degradation and thus achieve a constitutively active state, which may explain its tumor promoting effects. PMID:27086143

  13. Extracellular ATP activates MAPK and ROS signaling during injury response in the fungus Trichoderma atroviride

    PubMed Central

    Medina-Castellanos, Elizabeth; Esquivel-Naranjo, Edgardo U.; Heil, Martin; Herrera-Estrella, Alfredo

    2014-01-01

    The response to mechanical damage is crucial for the survival of multicellular organisms, enabling their adaptation to hostile environments. Trichoderma atroviride, a filamentous fungus of great importance in the biological control of plant diseases, responds to mechanical damage by activating regenerative processes and asexual reproduction (conidiation). During this response, reactive oxygen species (ROS) are produced by the NADPH oxidase complex. To understand the underlying early signaling events, we evaluated molecules such as extracellular ATP (eATP) and Ca2+ that are known to trigger wound-induced responses in plants and animals. Concretely, we investigated the activation of mitogen-activated protein kinase (MAPK) pathways by eATP, Ca2+, and ROS. Indeed, application of exogenous ATP and Ca2+ triggered conidiation. Furthermore, eATP promoted the Nox1-dependent production of ROS and activated a MAPK pathway. Mutants in the MAPK-encoding genes tmk1 and tmk3 were affected in wound-induced conidiation, and phosphorylation of both Tmk1 and Tmk3 was triggered by eATP. We conclude that in this fungus, eATP acts as a damage-associated molecular pattern (DAMP). Our data indicate the existence of an eATP receptor and suggest that in fungi, eATP triggers pathways that converge to regulate asexual reproduction genes that are required for injury-induced conidiation. By contrast, Ca2+ is more likely to act as a downstream second messenger. The early steps of mechanical damage response in T. atroviride share conserved elements with those known from plants and animals. PMID:25484887

  14. Activation of ERK induces phosphorylation of MAPK phosphatase-7, a JNK specific phosphatase, at Ser-446.

    PubMed

    Masuda, Kouhei; Shima, Hiroshi; Katagiri, Chiaki; Kikuchi, Kunimi

    2003-08-22

    We previously showed that MKP-7 suppresses MAPK activation in COS-7 cells in the order of selectivity, JNK > p38 > ERK, but interacts with ERK as well as JNK and p38. In this study we found that, when expressed in COS-7 cells with HA-ERK2, the mobility of FLAG-MKP-7 was decreased on SDS-PAGE gels depending on several stimuli, including phorbol 12-myristate 13-acetate, fetal bovine serum, epidermal growth factor, H2O2, and ionomycin. By using U0126, a MEK inhibitor, and introducing several point mutations, we demonstrated that this upward mobility shift is because of phosphorylation and identified Ser-446 of MKP-7 as the phosphorylation site targeted by ERK activation. To determine how MKP-7 interacts with MAPKs, we identified three domains in MKP-7 required for interaction with MAPKs, namely, putative MAP kinase docking domains (D-domain) I and II and a long COOH-terminal stretch unique to MKP-7. The D-domain I is required for interaction with ERK and p38, whereas the D-domain II is required for interaction with JNK and p38, which is likely to be important for MKP-7 to suppress JNK and p38 activations. The COOH-terminal stretch of MKP-7 was shown to determine JNK preference for MKP-7 by masking MKP-7 activity toward p38 and is a domain bound by ERK. These data strongly suggested that Ser-446 of MKP-7 is phosphorylated by ERK. PMID:12794087

  15. Aluminum Nanoparticles Induce ERK and p38MAPK Activation in Rat Brain

    PubMed Central

    Seo, Gyun-Baek; Jo; Lee, Mimi; Shim, Ilseob; Lee, Byung-Woo; Yoon, Byung-Il; Kim, Pilje; Choi, Kyunghee

    2013-01-01

    Aluminum nanoparticles (Al-NPs) are one of the most widely used nanomaterial in cosmetics and medical materials. For this reason, Al-NP exposure is very likely to occur via inhalation in the environment and the workplace. Nevertheless, little is known about the mechanism of Al-NP neurotoxicity via inhalation exposure. In this study, we investigated the effect AL-NPs on the brain. Rats were exposed to Al-NPs by nasal instillation at 1 mg/kg body weight (low exposure group), 20 mg/kg body weight (moderate exposure group), and 40 mg/kg body weight (high exposure group), for a total of 3 times, with a 24-hr interval after each exposure. Inductively coupled plasma mass spectrometry (ICP-MS) analysis indicated that the presence of aluminum was increased in a dose-dependent manner in the olfactory bulb (OFB) and the brain. In microarray analysis, the regulation of mitogen-activated protein kinases (MAPK) activity (GO: 0043405), including Ptprc, P2rx7, Map2k4, Trib3, Trib1, and Fgd4 was significantly over-expressed in the treated mice than in the controls (p = 0.0027). Moreover, Al-NPs induced the activation of ERK1 and p38 MAPK protein expression in the brain, but did not alter the protein expression of JNK, when compared to the control. These data demonstrate that the nasal exposure of Al-NPs can permeate the brain via the olfactory bulb and modulate the gene and protein expression of MAPK and its activity. PMID:24386518

  16. Aluminum Nanoparticles Induce ERK and p38MAPK Activation in Rat Brain.

    PubMed

    Kwon, Jung-Taek; Seo, Gyun-Baek; Jo; Lee, Mimi; Kim, Hyun-Mi; Shim, Ilseob; Lee, Byung-Woo; Yoon, Byung-Il; Kim, Pilje; Choi, Kyunghee

    2013-09-01

    Aluminum nanoparticles (Al-NPs) are one of the most widely used nanomaterial in cosmetics and medical materials. For this reason, Al-NP exposure is very likely to occur via inhalation in the environment and the workplace. Nevertheless, little is known about the mechanism of Al-NP neurotoxicity via inhalation exposure. In this study, we investigated the effect AL-NPs on the brain. Rats were exposed to Al-NPs by nasal instillation at 1 mg/kg body weight (low exposure group), 20 mg/kg body weight (moderate exposure group), and 40 mg/kg body weight (high exposure group), for a total of 3 times, with a 24-hr interval after each exposure. Inductively coupled plasma mass spectrometry (ICP-MS) analysis indicated that the presence of aluminum was increased in a dose-dependent manner in the olfactory bulb (OFB) and the brain. In microarray analysis, the regulation of mitogen-activated protein kinases (MAPK) activity (GO: 0043405), including Ptprc, P2rx7, Map2k4, Trib3, Trib1, and Fgd4 was significantly over-expressed in the treated mice than in the controls (p = 0.0027). Moreover, Al-NPs induced the activation of ERK1 and p38 MAPK protein expression in the brain, but did not alter the protein expression of JNK, when compared to the control. These data demonstrate that the nasal exposure of Al-NPs can permeate the brain via the olfactory bulb and modulate the gene and protein expression of MAPK and its activity. PMID:24386518

  17. Activated p38 MAPK in Peripheral Blood Monocytes of Steroid Resistant Asthmatics

    PubMed Central

    Li, Ling-bo; Leung, Donald Y. M.; Goleva, Elena

    2015-01-01

    Steroid resistance is a significant problem in management of chronic inflammatory diseases, including asthma. Accessible biomarkers are needed to identify steroid resistant patients to optimize their treatment. This study examined corticosteroid resistance in severe asthma. 24 asthmatics with forced expiratory volume in one second of less then 80% predicted were classified as steroid resistant or steroid sensitive based on changes in their lung function following a week of treatment with oral prednisone. Heparinised blood was collected from patients prior to oral prednisone administration. Phosphorylated mitogen activated kinases (MAPK) (extracellular regulated kinase (ERK), p38 and jun kinase (JNK)) were analyzed in whole blood samples using flow cytometry. Activation of phospho-p38 MAPK and phospho-mitogen- and stress-activated protein kinase 1 (MSK1) in asthmatics’ peripheral blood mononuclear cells (PBMC) were confirmed by Western blot. Dexamethasone suppression of the LPS-induced IL-8 mRNA production by steroid resistant asthmatics PBMC in the presence of p38 and ERK inhibitors was evaluated by real time PCR. Flow cytometry analysis identified significantly stronger p38 phosphorylation in CD14+ monocytes from steroid resistant than steroid sensitive asthmatics (p = 0.014), whereas no difference was found in phosphorylation of ERK or JNK in CD14+ cells from these two groups of asthmatics. No difference in phosphorylated p38, ERK, JNK was detected in CD4+, CD8+ T cells, B cells and NK cells from steroid resistant vs. steroid sensitive asthmatics. P38 MAPK pathway activation was confirmed by Western blot, as significantly higher phospho-p38 and phospho-MSK1 levels were detected in the PBMC lysates from steroid resistant asthmatics. P38 inhibitor significantly enhanced DEX suppression of LPS-induced IL-8 mRNA by PBMC of steroid resistant asthmatics. This is the first report demonstrating selective p38 MAPK pathway activation in blood monocytes of steroid

  18. Amarogentin, a Secoiridoid Glycoside, Abrogates Platelet Activation through PLCγ2-PKC and MAPK Pathways

    PubMed Central

    Yen, Ting-Lin; Lu, Wan-Jung; Lien, Li-Ming; Thomas, Philip Aloysius; Lee, Tzu-Yin; Chiu, Hou-Chang; Sheu, Joen-Rong

    2014-01-01

    Amarogentin, an active principle of Gentiana lutea, possess antitumorigenic, antidiabetic, and antioxidative properties. Activation of platelets is associated with intravascular thrombosis and cardiovascular diseases. The present study examined the effects of amarogentin on platelet activation. Amarogentin treatment (15~60 μM) inhibited platelet aggregation induced by collagen, but not thrombin, arachidonic acid, and U46619. Amarogentin inhibited collagen-induced phosphorylation of phospholipase C (PLC)γ2, protein kinase C (PKC), and mitogen-activated protein kinases (MAPKs). It also inhibits in vivo thrombus formation in mice. In addition, neither the guanylate cyclase inhibitor ODQ nor the adenylate cyclase inhibitor SQ22536 affected the amarogentin-mediated inhibition of platelet aggregation, which suggests that amarogentin does not regulate the levels of cyclic AMP and cyclic GMP. In conclusion, amarogentin prevents platelet activation through the inhibition of PLCγ2-PKC cascade and MAPK pathway. Our findings suggest that amarogentin may offer therapeutic potential for preventing or treating thromboembolic disorders. PMID:24868545

  19. PKC theta and p38 MAPK activate the EBV lytic cycle through autophagy induction.

    PubMed

    Gonnella, Roberta; Granato, Marisa; Farina, Antonella; Santarelli, Roberta; Faggioni, Alberto; Cirone, Mara

    2015-07-01

    PKC activation by combining TPA with sodium butyrate (T/B) represents the most effective and widely used strategy to induce the Epstein-Barr virus (EBV) lytic cycle. The results obtained in this study show that novel PKCθ is involved in such process and that it acts through the activation of p38 MAPK and autophagy induction. Autophagy, a mechanism of cellular defense in stressful conditions, is manipulated by EBV to enhance viral replication. Besides promoting the EBV lytic cycle, the activation of p38 and autophagy resulted in a pro-survival effect, as indicated by p38 or ATG5 knocking down experiments. However, this pro-survival role was counteracted by a pro-death activity of PKCθ, due to the dephosphorylation of AKT. In conclusion, this study reports, for the first time, that T/B activates a PKCθ-p38 MAPK axis in EBV infected B cells, that promotes the viral lytic cycle and cell survival and dephosphorylates AKT, balancing cell life and cell death. PMID:25827954

  20. Activation of Egr-1 in Human Lung Epithelial Cells Exposed to Silica through MAPKs Signaling Pathways

    PubMed Central

    Chu, Ling; Wang, Tiansheng; Hu, Yongbin; Gu, Yonghong; Su, Zanshan; Jiang, Haiying

    2013-01-01

    The alveolar type II epithelial cell, regarded historically as a key target cell in initial injury by silica, now appears to be important in both defense from lung damage as well as elaboration of chemokines and cytokines. The molecular basis for silica-induced epithelial cell injury is poorly understood. In this study we explored the activation of nuclear factor Egr-1 and related signal pathway. Human II alveolar epithelial line A549 cells were exposed to silica for indicated time to assay the expression and activation of Egr-1 and upstream MAPKs. Immunofluorescence, western-blot techniques, RT-PCR, Electrophoretic mobility shift assay (EMSA), transient transfection assay, kinase inhibitor experiments were performed. It was found that the expression of Egr-1 at mRNA and protein level was significantly increased in A549 cells after administration with silica and the activity of Egr-1 peaked by silica treatment for 60 minutes. Furthermore, phosphorylated-ERK1/2, P38 MAPKs (the upstream kinase of Egr-1) ballooned during 15-30minutes, 30-60minutes respectively after silica exposure in A549 cells. By administration of ERK1/2, P38 inhibitor, the expression and transcription of Egr-1 were both markedly decreased. But PKC inhibitor did not prevent the increase of Egr-1. These results indicated Egr-1 played a critical role in silica-induced pulmonary fibrosis in an ERK1/2, P38 MAPKs-dependent manner, which suggests Egr-1 is an essential regulator in silicosis, and underlines a new molecular mechanism for fibrosis induced by silica. PMID:23874821

  1. TLR4-Activated MAPK-IL-6 Axis Regulates Vascular Smooth Muscle Cell Function.

    PubMed

    Lee, Guan-Lin; Wu, Jing-Yiing; Tsai, Chien-Sung; Lin, Chih-Yuan; Tsai, Yi-Ting; Lin, Chin-Sheng; Wang, Yi-Fu; Yet, Shaw-Fang; Hsu, Yu-Juei; Kuo, Cheng-Chin

    2016-01-01

    Migration of vascular smooth muscle cells (VSMCs) into the intima is considered to be a vital event in the pathophysiology of atherosclerosis. Despite substantial evidence supporting the pathogenic role of Toll-like receptor 4 (TLR4) in the progression of atherogenesis, its function in the regulation of VSMC migration remains unclear. The goal of the present study was to elucidate the mechanism by which TLR4 regulates VSMC migration. Inhibitor experiments revealed that TLR4-induced IL-6 secretion and VSMC migration were mediated via the concerted actions of MyD88 and TRIF on the activation of p38 MAPK and ERK1/2 signaling. Neutralizing anti-IL-6 antibodies abrogated TLR4-driven VSMC migration and F-actin polymerization. Blockade of p38 MAPK or ERK1/2 signaling cascade inhibited TLR4 agonist-mediated activation of cAMP response element binding protein (CREB). Moreover, siRNA-mediated suppression of CREB production repressed TLR4-induced IL-6 production and VSMC migration. Rac-1 inhibitor suppressed TLR4-driven VSMC migration but not IL-6 production. Importantly, the serum level of IL-6 and TLR4 endogenous ligand HMGB1 was significantly higher in patients with coronary artery diseases (CAD) than in healthy subjects. Serum HMGB1 level was positively correlated with serum IL-6 level in CAD patients. The expression of both HMGB1 and IL-6 was clearly detected in the atherosclerotic tissue of the CAD patients. Additionally, there was a positive association between p-CREB and HMGB1 in mouse atherosclerotic tissue. Based on our findings, we concluded that, upon ligand binding, TLR4 activates p38 MAPK and ERK1/2 signaling through MyD88 and TRIF in VSMCs. These signaling pathways subsequently coordinate an additive augmentation of CREB-driven IL-6 production, which in turn triggers Rac-1-mediated actin cytoskeleton to promote VSMC migration. PMID:27563891

  2. Activation of the MAPK/ERK Cell-Signaling Pathway in Uterine Smooth Muscle Cells of Women With Adenomyosis.

    PubMed

    Streuli, Isabelle; Santulli, Pietro; Chouzenoux, Sandrine; Chapron, Charles; Batteux, Frédéric

    2015-12-01

    We investigated whether the myometrium might be intrinsically different in women with adenomyosis. We studied whether the mitogen-activated protein kinases/extracellular signal-regulated kinases (MAPKs/ERKs) and phosphoinositide 3-kinase/mammalian target of rapamycin/AKT (PI3K/mTOR/AKT) cell-signaling pathways, implicated in the pathogenesis of endometriosis, might also be activated in uterine smooth muscle cells (uSMCs) of women with adenomyosis and measured the production of reactive oxygen species (ROS), proinflammatory mediators that modulate cell proliferation and have been shown to activate the MAPK/ERK pathway in endometriosis. The uSMC cultures were derived from myometrium biopsies obtained during hysterectomy or myomectomy in women with adenomyosis and controls with leiomyoma. Proliferation of uSMCs and in vitro activation of the MAPK/ERK cell-signaling pathway were increased in women with adenomyosis compared to controls. The activation of the PI3K/mTOR/AKT pathway was not significant. The ROS production and ROS detoxification pathways were not different between uSMCs of women with adenomyosis and controls suggesting an ROS-independent activation of the MAPK/ERK pathway. Our results also provide evidence that protein kinase inhibitors and the rapanalogue temsirolimus can control proliferation of uSMCs in vitro suggesting an implication of the MAPK/ERK and the PI3K/mTOR/AKT pathways in proliferation of uSMCs in women with adenomyosis and leiomyomas. PMID:26071388

  3. MEK Inhibition Overcomes Cisplatin Resistance Conferred by SOS/MAPK Pathway Activation in Squamous Cell Carcinoma.

    PubMed

    Kong, Li Ren; Chua, Kian Ngiap; Sim, Wen Jing; Ng, Hsien Chun; Bi, Chonglei; Ho, Jingshan; Nga, Min En; Pang, Yin Huei; Ong, Weijie Richard; Soo, Ross Andrew; Huynh, Hung; Chng, Wee Joo; Thiery, Jean-Paul; Goh, Boon Cher

    2015-07-01

    Genomic analyses of squamous cell carcinoma (SCC) have yet to yield significant strategies against pathway activation to improve treatment. Platinum-based chemotherapy remains the mainstay of treatment for SCC of different histotypes either as a single-agent or alongside other chemotherapeutic drugs or radiotherapy; however, resistance inevitably emerges, which limits the duration of treatment response. To elucidate mechanisms that mediate resistance to cisplatin, we compared drug-induced perturbations to gene and protein expression between cisplatin-sensitive and -resistant SCC cells, and identified MAPK-ERK pathway upregulation and activation in drug-resistant cells. ERK-induced resistance appeared to be activated by Son of Sevenless (SOS) upstream, and mediated through Bim degradation downstream. Clinically, elevated p-ERK expression was associated with shorter disease-free survival in patients with locally advanced head and neck SCC treated with concurrent chemoradiation. Inhibition of MEK/ERK, but not that of EGFR or RAF, augmented cisplatin sensitivity in vitro and demonstrated efficacy and tolerability in vivo. Collectively, these findings suggest that inhibition of the activated SOS-MAPK-ERK pathway may augment patient responses to cisplatin treatment. PMID:25939760

  4. Exocyst Sec10 protects renal tubule cells from injury by EGFR/MAPK activation and effects on endocytosis

    PubMed Central

    Fogelgren, Ben; Zuo, Xiaofeng; Buonato, Janine M.; Vasilyev, Aleksandr; Baek, Jeong-In; Choi, Soo Young; Chacon-Heszele, Maria F.; Palmyre, Aurélien; Polgar, Noemi; Drummond, Iain; Park, Kwon Moo; Lazzara, Matthew J.

    2014-01-01

    Acute kidney injury is common and has a high mortality rate, and no effective treatment exists other than supportive care. Using cell culture models, we previously demonstrated that exocyst Sec10 overexpression reduced damage to renal tubule cells and speeded recovery and that the protective effect was mediated by higher basal levels of mitogen-activated protein kinase (MAPK) signaling. The exocyst, a highly-conserved eight-protein complex, is known for regulating protein trafficking. Here we show that the exocyst biochemically interacts with the epidermal growth factor receptor (EGFR), which is upstream of MAPK, and Sec10-overexpressing cells express greater levels of phosphorylated (active) ERK, the final step in the MAPK pathway, in response to EGF stimulation. EGFR endocytosis, which has been linked to activation of the MAPK pathway, increases in Sec10-overexpressing cells, and gefitinib, a specific EGFR inhibitor, and Dynasore, a dynamin inhibitor, both reduce EGFR endocytosis. In turn, inhibition of the MAPK pathway reduces ligand-mediated EGFR endocytosis, suggesting a potential feedback of elevated ERK activity on EGFR endocytosis. Gefitinib also decreases MAPK signaling in Sec10-overexpressing cells to levels seen in control cells and, demonstrating a causal role for EGFR, reverses the protective effect of Sec10 overexpression following cell injury in vitro. Finally, using an in vivo zebrafish model of acute kidney injury, morpholino-induced knockdown of sec10 increases renal tubule cell susceptibility to injury. Taken together, these results suggest that the exocyst, acting through EGFR, endocytosis, and the MAPK pathway is a candidate therapeutic target for acute kidney injury. PMID:25298525

  5. Measurement of constitutive MAPK and PI3K/AKT signaling activity in human cancer cell lines

    PubMed Central

    Paraiso, Kim H.T.; Van Der Kooi, Kaisa; Messina, Jane L.; Smalley, Keiran S. M.

    2014-01-01

    The growth and survival of cancer cells is often driven by constitutive activity in the mitogen activated protein kinase (MAPK) and phospho-inositide 3-kinase (PI3K)/AKT signaling pathways. Activity in these signal transduction cascades is known to contribute to the uncontrolled growth and resistance to apoptosis that characterizes tumor progression. There is now a great deal of interest in therapeutically targeting these pathways in cancer using small molecule inhibitors. In this chapter we describe methods to measure constitutive MAPK and AKT activity in melanoma cell lines, with a focus upon Western blotting, phospho-flow cytometry and immunofluorescence staining techniques. PMID:21036250

  6. Metabolic Respiration Induces AMPK- and Ire1p-Dependent Activation of the p38-Type HOG MAPK Pathway

    PubMed Central

    Adhikari, Hema; Cullen, Paul J.

    2014-01-01

    Evolutionarily conserved mitogen activated protein kinase (MAPK) pathways regulate the response to stress as well as cell differentiation. In Saccharomyces cerevisiae, growth in non-preferred carbon sources (like galactose) induces differentiation to the filamentous cell type through an extracellular-signal regulated kinase (ERK)-type MAPK pathway. The filamentous growth MAPK pathway shares components with a p38-type High Osmolarity Glycerol response (HOG) pathway, which regulates the response to changes in osmolarity. To determine the extent of functional overlap between the MAPK pathways, comparative RNA sequencing was performed, which uncovered an unexpected role for the HOG pathway in regulating the response to growth in galactose. The HOG pathway was induced during growth in galactose, which required the nutrient regulatory AMP-dependent protein kinase (AMPK) Snf1p, an intact respiratory chain, and a functional tricarboxylic acid (TCA) cycle. The unfolded protein response (UPR) kinase Ire1p was also required for HOG pathway activation in this context. Thus, the filamentous growth and HOG pathways are both active during growth in galactose. The two pathways redundantly promoted growth in galactose, but paradoxically, they also inhibited each other's activities. Such cross-modulation was critical to optimize the differentiation response. The human fungal pathogen Candida albicans showed a similar regulatory circuit. Thus, an evolutionarily conserved regulatory axis links metabolic respiration and AMPK to Ire1p, which regulates a differentiation response involving the modulated activity of ERK and p38 MAPK pathways. PMID:25356552

  7. Manganese modulation of MAPK pathways: effects on upstream mitogen activated protein kinase kinases (MKKs) and mitogen activated kinase phosphatase-1 (MKP-1) in microglial cells

    PubMed Central

    Crittenden, Patrick L.; Filipov, Nikolay M.

    2010-01-01

    Multiple studies demonstrate that manganese (Mn) exposure potentiates inflammatory mediator output from activated glia; this increased output is associated with enhanced mitogen activated protein kinase (MAPK: p38, ERK, and JNK) activity. We hypothesized that Mn activates MAPK by activating the kinases upstream of MAPK, i.e., MKK-3/6, MKK-1/2, and MKK-4 (responsible for activation of p38, ERK, and JNK, respectively), and/or by inhibiting a major phosphatase responsible for MAPK inactivation, MKP-1. Exposure of N9 microglia to Mn (250μM), LPS (100 ng/ml), or Mn+LPS increased MKK-3/6 and MKK-4 activity at 1 h; the effect of Mn+LPS on MKK-4 activation was greater than the rest. At 4 h, Mn, LPS, and Mn+LPS increased MKK-3/6 and MKK-1/2 phosphorylation, whereas MKK-4 was activated only by Mn and Mn+LPS. Besides activating MKK-4 via Ser257/Thr261 phosphorylation, Mn (4 h) prevented MKK-4’s phosphorylation on Ser80, which negatively regulates MKK-4 activity. Exposure to Mn or Mn+LPS (1 h) decreased both mRNA and protein expression of MKP-1, the negative MAPK regulator. In addition, we observed that at 4 h, but not at 1 h, a time point coinciding with increased MAPK activity, Mn+LPS markedly increased TNF-α , IL-6, and Cox-2 mRNA, suggesting a delayed effect. The fact that all three major groups of MKKs, MKK-1/2, MKK-3/6, and MKK-4 are activated by Mn suggests that Mn-induced activation of MAPK occurs via traditional mechanisms, which perhaps involve the MAPKs farthest upstream, MKKKs (MAP3Ks). In addition, for all MKKs, Mn-induced activation was persistent at least for 4 h, indicating a long-term effect. PMID:20589745

  8. Involvement of Ras/Raf/AP-1 in BMP-4 signaling during Xenopus embryonic development.

    PubMed Central

    Xu, R H; Dong, Z; Maeno, M; Kim, J; Suzuki, A; Ueno, N; Sredni, D; Colburn, N H; Kung, H F

    1996-01-01

    Previously, we elucidated the role of bone morphogenetic protein 4 (BMP-4) in the dorsal-ventral patterning of the Xenopus embryo by using a dominant negative mutant of the BMP-4 receptor (DN-BR). The present paper describes the involvement of Ras, Raf, and activator protein 1 (AP-1) in BMP-4 signaling during Xenopus embryonic development. The AP-1 activity was determined by injecting an AP-1-dependent luciferase reporter gene into two-cell-stage Xenopus embryos and measuring the luciferase activity at various developmental stages. We found that injection of BMP-4 mRNA increased AP-1 activity, whereas injection of DN-BR mRNA inhibited AP-1 activity. Similar inhibitory effects were seen with injection of mRNAs encoding dominant negative mutants of c-Ha-Ras, c-Raf, or c-Jun. These results suggest that the endogenous AP-1 activity is regulated by BMP-4/Ras/Raf/Jun signals. We next investigated the effects of Ras/Raf/AP-1 signals on the biological functions of BMP-4. DN-BR-induced dorsalization of the embryo, revealed by the formation of a secondary body axis or dorsalization of the ventral mesoderm explant analyzed by histological and molecular criteria, was significantly reversed by coinjection of [Val12]Ha-Ras, c-Raf, or c-Jun mRNA. Furthermore, the BMP-4-stimulated erythroid differentiation in the ventral mesoderm was substantially inhibited by coinjection with the dominant negative c-Ha-Ras, c-Raf, or c-Jun mutant. Our results suggest the involvement of Ras/Raf/AP-1 in the BMP-4 signaling pathway. Images Fig. 2 Fig. 3 Fig. 4 PMID:8570644

  9. Xenopus H-RasV12 promotes entry into meiotic M phase and cdc2 activation independently of Mos and p42(MAPK).

    PubMed

    Dupré, Aude; Suziedelis, Kestutis; Valuckaite, Ryte; de Gunzburg, Jean; Ozon, René; Jessus, Catherine; Haccard, Olivier

    2002-09-19

    In the Xenopus oocyte, progesterone triggers M phase Promoting Factor (MPF) activation in a protein synthesis dependent manner. Although the synthesis of the p42(MAPK) activator Mos appears to be required for MPF activation, p42(MAPK) activity has been shown to be dispensable. To clarify this paradox, we attempted to activate the p42(MAPK) pathway independently of Mos synthesis by cloning and using Xenopus H-Ras in the oocyte. We demonstrate that the injection of the constitutively active Xe H-RasV12 mutant induces p42(MAPK) and MPF activation through two independent pathways. Xe H-RasV12 induces only a partial activation of p42(MAPK) when protein synthesis and MPF activation are prevented. A full level of p42(MAPK) activation is reached when MPF is activated and Mos is present. In contrast, MPF activation induced by Xe H-RasV12 is achieved independently of Mos synthesis and p42(MAPK) activation but still depends on protein synthesis. Therefore, the amphibian oocyte represents a new model system to analyse an original H-Ras pathway ending to MPF activation and distinct from the p42(MAPK) pathway. The identification of the proteins synthesized in response to Xe H-RasV12 and required for MPF activation, represents an important clue in understanding the mechanism of progesterone action. PMID:12226746

  10. Genome-Wide Survey and Expression Profile Analysis of the Mitogen-Activated Protein Kinase (MAPK) Gene Family in Brassica rapa

    PubMed Central

    Yu, Hao; Qu, Cunmin; Tang, Zhanglin; Li, Jiana; Chai, Yourong; Liang, Ying

    2015-01-01

    Mitogen-activated protein kinase (MAPK) cascades are fundamental signal transduction modules in plants, controlling cell division, development, hormone signaling, and biotic and abiotic stress responses. Although MAPKs have been investigated in several plant species, a comprehensive analysis of the MAPK gene family has hitherto not been performed in Brassica rapa. In this study, we identified 32 MAPKs in the B. rapa genome by conducting BLASTP and syntenic block analyses, and screening for the essential signature motif (TDY or TEY) of plant MAPK proteins. Of the 32 BraMAPK genes retrieved from the Brassica Database, 13 exhibited exon splicing errors, excessive splicing of the 5' sequence, excessive retention of the 5' sequence, and sequencing errors of the 3' end. Phylogenetic trees of the 32 corrected MAPKs from B. rapa and of MAPKs from other plants generated by the neighbor-joining and maximum likelihood methods suggested that BraMAPKs could be divided into four groups (groups A, B, C, and D). Gene number expansion was observed for BraMAPK genes in groups A and D, which may have been caused by the tandem duplication and genome triplication of the ancestral genome of the Brassica progenitor. Except for five members of the BraMAPK10 subfamily, the identified BraMAPKs were expressed in most of the tissues examined, including callus, root, stem, leaf, flower, and silique. Quantitative real-time PCR demonstrated that at least six and five BraMAPKs were induced or repressed by various abiotic stresses and hormone treatments, respectively, suggesting their potential roles in the abiotic stress response and various hormone signal transduction pathways in B. rapa. This study provides valuable insight into the putative physiological and biochemical functions of MAPK genes in B. rapa. PMID:26173020

  11. Genome-Wide Survey and Expression Profile Analysis of the Mitogen-Activated Protein Kinase (MAPK) Gene Family in Brassica rapa.

    PubMed

    Lu, Kun; Guo, Wenjin; Lu, Junxing; Yu, Hao; Qu, Cunmin; Tang, Zhanglin; Li, Jiana; Chai, Yourong; Liang, Ying

    2015-01-01

    Mitogen-activated protein kinase (MAPK) cascades are fundamental signal transduction modules in plants, controlling cell division, development, hormone signaling, and biotic and abiotic stress responses. Although MAPKs have been investigated in several plant species, a comprehensive analysis of the MAPK gene family has hitherto not been performed in Brassica rapa. In this study, we identified 32 MAPKs in the B. rapa genome by conducting BLASTP and syntenic block analyses, and screening for the essential signature motif (TDY or TEY) of plant MAPK proteins. Of the 32 BraMAPK genes retrieved from the Brassica Database, 13 exhibited exon splicing errors, excessive splicing of the 5' sequence, excessive retention of the 5' sequence, and sequencing errors of the 3' end. Phylogenetic trees of the 32 corrected MAPKs from B. rapa and of MAPKs from other plants generated by the neighbor-joining and maximum likelihood methods suggested that BraMAPKs could be divided into four groups (groups A, B, C, and D). Gene number expansion was observed for BraMAPK genes in groups A and D, which may have been caused by the tandem duplication and genome triplication of the ancestral genome of the Brassica progenitor. Except for five members of the BraMAPK10 subfamily, the identified BraMAPKs were expressed in most of the tissues examined, including callus, root, stem, leaf, flower, and silique. Quantitative real-time PCR demonstrated that at least six and five BraMAPKs were induced or repressed by various abiotic stresses and hormone treatments, respectively, suggesting their potential roles in the abiotic stress response and various hormone signal transduction pathways in B. rapa. This study provides valuable insight into the putative physiological and biochemical functions of MAPK genes in B. rapa. PMID:26173020

  12. Endothelium-Derived 5-Methoxytryptophan Protects Endothelial Barrier Function by Blocking p38 MAPK Activation

    PubMed Central

    Chu, Ling-Yun; Wang, Yi-Fu; Cheng, Huei-Hsuan; Kuo, Cheng-Chin; Wu, Kenneth K.

    2016-01-01

    The endothelial junction is tightly controlled to restrict the passage of blood cells and solutes. Disruption of endothelial barrier function by bacterial endotoxins, cytokines or growth factors results in inflammation and vascular damage leading to vascular diseases. We have identified 5-methoxytryptophan (5-MTP) as an anti-inflammatory factor by metabolomic analysis of conditioned medium of human fibroblasts. Here we postulated that endothelial cells release 5-MTP to protect the barrier function. Conditioned medium of human umbilical vein endothelial cells (HUVECs) prevented endothelial hyperpermeability and VE-cadherin downregulation induced by VEGF, LPS and cytokines. We analyzed the metabolomic profile of HUVEC conditioned medium and detected 5-MTP but not melatonin, serotonin or their catabolites, which was confirmed by enzyme-linked immunosorbent assay. Addition of synthetic pure 5-MTP preserved VE-cadherin and maintained barrier function despite challenge with pro-inflammatory mediators. Tryptophan hydroxylase-1, an enzyme required for 5-MTP biosynthesis, was downregulated in HUVECs by pro-inflammatory mediators and it was accompanied by reduction of 5-MTP. 5-MTP protected VE-cadherin and prevented endothelial hyperpermeability by blocking p38 MAPK activation. A chemical inhibitor of p38 MAPK, SB202190, exhibited a similar protective effect as 5-MTP. To determine whether 5-MTP prevents vascular hyperpermeability in vivo, we evaluated the effect of 5-MTP administration on LPS-induced murine microvascular permeability with Evans blue. 5-MTP significantly prevented Evans blue dye leakage. Our findings indicate that 5-MTP is a new class of endothelium-derived molecules which protects endothelial barrier function by blocking p38 MAPK. PMID:27002329

  13. Prenatal choline supplementation advances hippocampal development and enhances MAPK and CREB activation.

    PubMed

    Mellott, Tiffany J; Williams, Christina L; Meck, Warren H; Blusztajn, Jan Krzysztof

    2004-03-01

    Choline is an essential nutrient for animals and humans. Previous studies showed that supplementing the maternal diet with choline during the second half of gestation in rats permanently enhances memory performance of the adult offspring. Here we show that prenatal choline supplementation causes a 3-day advancement in the ability of juvenile rats to use relational cues in a water maze task, indicating that the treatment accelerates hippocampal maturation. Moreover, phosphorylation and therefore activation of hippocampal mitogen-activated protein kinase (MAPK) and cAMP-response element binding protein (CREB) in response to stimulation by glutamate, N-methyl-D-aspartate, or depolarizing concentrations of K+ were increased by prenatal choline supplementation and reduced by prenatal choline deficiency. These data provide the first evidence that developmental plasticity of the hippocampal MAPK and CREB signaling pathways is controlled by the supply of a single essential nutrient, choline, during fetal development and point to these pathways as candidate mechanisms for the developmental and long-term cognitive enhancement induced by prenatal choline supplementation. PMID:14715695

  14. MALT1 Inhibition of Oral Carcinoma Cell Invasion and ERK/MAPK Activation.

    PubMed

    Chiba, T; Soeno, Y; Shirako, Y; Sudo, H; Yagishita, H; Taya, Y; Kawashiri, S; Okada, Y; Imai, K

    2016-04-01

    The expression of mucosa-associated lymphoid tissue 1 (MALT1) that activates nuclear factor (NF)-κB in lymphocyte lineages is rapidly inactivated in oral carcinoma cells at the invasive front and the patients with worst prognosis. However, its mechanism to accelerate carcinoma progression remains unknown, and this study was carried out to examine the role in invasion. HSC2 oral carcinoma cells stably expressing wild-type MALT1 (wtMALT1) reduced the invasion of basement membrane matrices and collagen gels, and the dominant-negative form (∆MALT1)-expressing cells aggressively invaded into collagen gels. MALT1 decelerated proliferation and migration of cells and downregulated expression of matrix metalloproteinase 2 and 9, which were confirmed by short interfering RNA transfections. Reporter assays and immunoblot analysis showed that MALT1 does not affect the NF-κB pathway but inhibits ERK/MAPK activation. This was confirmed by endogenous MALT1 expression in oral carcinoma cell lines. Orthotopic implantation of ∆MALT1-expressing HSC2 cells in mice grew rapid expansive and invasive tongue tumors in contrast to an absence of tumor formation by wtMALT1-expressing cells. These results demonstrate that MALT1 suppresses oral carcinoma invasion by inhibiting proliferation, migration, and extracellular matrix degradation and that the ERK/MAPK pathway is a target of MALT1 and further suggests a role as a suppressor of carcinoma progression. PMID:26701346

  15. IGF-1 protects tubular epithelial cells during injury via activation of ERK/MAPK signaling pathway

    PubMed Central

    Wu, Zengbin; Yu, Yang; Niu, Lei; Fei, Aihua; Pan, Shuming

    2016-01-01

    Injury of renal tubular epithelial cells can induce acute renal failure and obstructive nephropathy. Previous studies have shown that administration of insulin-like growth factor-1 (IGF-1) ameliorates the renal injury in a mouse unilateral ureteral obstruction (UUO) model, whereas the underlying mechanisms are not completely understood. Here, we addressed this question. We found that the administration of IGF-1 significantly reduced the severity of the renal fibrosis in UUO. By analyzing purified renal epithelial cells, we found that IGF-1 significantly reduced the apoptotic cell death of renal epithelial cells, seemingly through upregulation of anti-apoptotic protein Bcl-2, at protein but not mRNA level. Bioinformatics analyses and luciferase-reporter assay showed that miR-429 targeted the 3′-UTR of Bcl-2 mRNA to inhibit its protein translation in renal epithelial cells. Moreover, IGF-1 suppressed miR-429 to increase Bcl-2 in renal epithelial cells to improve survival after UUO. Furthermore, inhibition of ERK/MAPK signaling pathway in renal epithelial cells abolished the suppressive effects of IGF-1 on miR-429 activation, and then the enhanced effects on Bcl-2 in UUO. Thus, our data suggest that IGF-1 may protect renal tubular epithelial cells via activation of ERK/MAPK signaling pathway during renal injury. PMID:27301852

  16. Differential Activation of Mitogen-Activated Protein Kinases, ERK 1/2, p38(MAPK) and JNK p54/p46 During Postnatal Development of Rat Hippocampus.

    PubMed

    Costa, Ana Paula; Lopes, Mark William; Rieger, Débora K; Barbosa, Sabrina Giovana Rocha; Gonçalves, Filipe Marques; Xikota, João Carlos; Walz, Roger; Leal, Rodrigo B

    2016-05-01

    Mitogen-activated protein kinases (MAPKs) are a group of serine-threonine kinases, including p38(MAPK), ERK 1/2 and JNK p54/p46, activated by phosphorylation in response to extracellular stimuli. The early postnatal period is characterized by significant changes in brain structure as well as intracellular signaling. In the hippocampus MAPKs have been involved in the modulation of development and neural plasticity. However, the temporal profile of MAPK activation throughout the early postnatal development is incomplete. An understanding of this profile is important since slight changes in the activity of these enzymes, in response to environmental stress in specific developmental windows, might alter the course of development. The present study was undertaken to investigate the hippocampal differential activation of MAPK during postnatal period. MAPK activation and total content were evaluated by Western blotting of hippocampal tissue obtained from male Wistar rats at postnatal days (P) 1, 4, 7, 10, 14, 21, 30 and 60. The total content and phosphorylation of each MAPK was expressed as mean ± SEM and then calculates as a percentile compared to P1 (set at 100 %). The results showed: (1) phosphorylation peaks of p38(MAPK) at PN4 (p = 0.036) and PN10 to PN60; (2) phosphorylation of ERK1 and ERK2 were increased with age (ERK1 p = 0.0000005 and ERK2 p = 0.003); (3) phosphorylation profile of JNK p54/p46 was not changed during the period analyzed (JNKp56 p = 0.716 and JNKp46 p = 0.192). Therefore, the activity profile of ERK 1/2 and p38(MAPK) during postnatal development of rat hippocampus are differentially regulated. Our results demonstrate that ERK 1/2 and p38(MAPK) are dynamically regulated during postnatal neurodevelopment, suggesting temporal correlation of MAPK activity with critical periods when programmed cell death and synaptogenesis are occurring. This suggests an important role for these MAPKs in postnatal development of rat hippocampus. PMID

  17. p38 MAPK Is Activated but Does Not Play a Key Role during Apoptosis Induction by Saturated Fatty Acid in Human Pancreatic β-Cells

    PubMed Central

    Šrámek, Jan; Němcová-Fürstová, Vlasta; Balušíková, Kamila; Daniel, Petr; Jelínek, Michael; James, Roger F.; Kovář, Jan

    2016-01-01

    Saturated stearic acid (SA) induces apoptosis in the human pancreatic β-cells NES2Y. However, the molecular mechanisms involved are unclear. We showed that apoptosis-inducing concentrations of SA activate the p38 MAPK signaling pathway in these cells. Therefore, we tested the role of p38 MAPK signaling pathway activation in apoptosis induction by SA in NES2Y cells. Crosstalk between p38 MAPK pathway activation and accompanying ERK pathway inhibition after SA application was also tested. The inhibition of p38 MAPK expression by siRNA silencing resulted in a decrease in MAPKAPK-2 activation after SA application, but it had no significant effect on cell viability or the level of phosphorylated ERK pathway members. The inhibition of p38 MAPK activity by the specific inhibitor SB202190 resulted in inhibition of MAPKAPK-2 activation and noticeable activation of ERK pathway members after SA treatment but in no significant effect on cell viability. p38 MAPK overexpression by plasmid transfection produced an increase in MAPKAPK-2 activation after SA exposure but no significant influence on cell viability or ERK pathway activation. The activation of p38 MAPK by the specific activator anisomycin resulted in significant activation of MAPKAPK-2. Concerning the effect on cell viability, application of the activator led to apoptosis induction similar to application of SA (PARP cleavage and caspase-7, -8, and -9 activation) and in inhibition of ERK pathway members. We demonstrated that apoptosis-inducing concentrations of SA activate the p38 MAPK signaling pathway and that this activation could be involved in apoptosis induction by SA in the human pancreatic β-cells NES2Y. However, this involvement does not seem to play a key role. Crosstalk between p38 MAPK pathway activation and ERK pathway inhibition in NES2Y cells seems likely. Thus, the ERK pathway inhibition by p38 MAPK activation does not also seem to be essential for SA-induced apoptosis. PMID:26861294

  18. MEK1-independent activation of MAPK and MEK1-dependent activation of p70 S6 kinase by stem cell factor (SCF) in ovarian cancer cells

    SciTech Connect

    Liu, Lian; Zhang, Xin; Du, Chao; Zhang, Xiaoning; Hou, Nan; Zhao, Di; Sun, Jianzhi; Li, Li; Wang, Xiuwen; Ma, Chunhong

    2009-05-01

    We discovered a stem cell factor (SCF)-triggered, MEK1-independent, and PI3K-dependent MAPK activation pathway in the Kit-expressing ovarian cancer cell line HEY. When we knocked down MEK1 with RNA interference (RNAi) to study the function of MEK1 on the proliferation and survival of ovarian cancer cells, we found that impaired cell growth still occurred after MEK1 expression had been suppressed, although MAPK activation remained intact. This suggests that there is MEK1-independent activation of MAPK in the SCF-induced ovarian cancer cell growth process, and that MEK1 still plays a crucial role in maintaining the malignant properties of ovarian cancer cells even when it fails to activate MAPK as expected.

  19. RAS/MAPK Activation Drives Resistance to Smo Inhibition, Metastasis, and Tumor Evolution in Shh Pathway-Dependent Tumors.

    PubMed

    Zhao, Xuesong; Ponomaryov, Tatyana; Ornell, Kimberly J; Zhou, Pengcheng; Dabral, Sukriti K; Pak, Ekaterina; Li, Wei; Atwood, Scott X; Whitson, Ramon J; Chang, Anne Lynn S; Li, Jiang; Oro, Anthony E; Chan, Jennifer A; Kelleher, Joseph F; Segal, Rosalind A

    2015-09-01

    Aberrant Shh signaling promotes tumor growth in diverse cancers. The importance of Shh signaling is particularly evident in medulloblastoma and basal cell carcinoma (BCC), where inhibitors targeting the Shh pathway component Smoothened (Smo) show great therapeutic promise. However, the emergence of drug resistance limits long-term efficacy, and the mechanisms of resistance remain poorly understood. Using new medulloblastoma models, we identify two distinct paradigms of resistance to Smo inhibition. Sufu mutations lead to maintenance of the Shh pathway in the presence of Smo inhibitors. Alternatively activation of the RAS-MAPK pathway circumvents Shh pathway dependency, drives tumor growth, and enhances metastatic behavior. Strikingly, in BCC patients treated with Smo inhibitor, squamous cell cancers with RAS/MAPK activation emerged from the antecedent BCC tumors. Together, these findings reveal a critical role of the RAS-MAPK pathway in drug resistance and tumor evolution of Shh pathway-dependent tumors. PMID:26130651

  20. RAS/MAPK activation drives resistance to Smo inhibition, metastasis and tumor evolution in Shh pathway-dependent tumors

    PubMed Central

    Zhao, Xuesong; Ponomaryov, Tatyana; Ornell, Kimberly J.; Zhou, Pengcheng; Dabral, Sukriti K.; Pak, Ekaterina; Li, Wei; Atwood, Scott X.; Whitson, Ramon J.; Chang, Anne Lynn S.; Li, Jiang; Oro, Anthony E.; Chan, Jennifer A.; Kelleher, Joseph F.; Segal, Rosalind A.

    2015-01-01

    Aberrant Shh signaling promotes tumor growth in diverse cancers. The importance of Shh signaling is particularly evident in medulloblastoma and basal cell carcinoma (BCC), where inhibitors targeting the Shh pathway component Smoothened (Smo) show great therapeutic promise. However, the emergence of drug resistance limits long-term efficacy and the mechanisms of resistance remain poorly understood. Using new medulloblastoma models, we identify two distinct paradigms of resistance to Smo inhibition. Sufu mutations lead to maintenance of the Shh pathway in the presence of Smo inhibitors. Alternatively activation of the RAS/MAPK pathway circumvents Shh pathway-dependency, drives tumor growth and enhances metastatic behavior. Strikingly, in BCC patients treated with Smo inhibitor, squamous cell cancers with RAS/MAPK activation emerged from the antecedent BCC tumors. Together these findings reveal a critical role of RAS/MAPK pathway in drug resistance and tumor evolution of Shh pathway-dependent tumors. PMID:26130651

  1. Michael addition of dehydroalanine-containing MAPK peptides to catalytic lysine inhibits the activity of phosphothreonine lyase.

    PubMed

    Zhang, Yuan; Yang, Ru; Huang, Juan; Liang, Qiujin; Guo, Yanmin; Bian, Weixiang; Luo, Lingfei; Li, Hongtao

    2015-11-30

    The phosphothreonine lyases OspF and SpvC irreversibly inactivate host dual-phosphorylated mitogen-activated protein kinases (MAPKs) [pThr-X-pTyr motif] through β-elimination. We found that dual-phosphorylated (pSer-X-pTyr) MAPK substrate peptides and their resulting catalytic products cross-link to OspF and SpvC. Mass spectrometry results revealed that these linkages form between lysine, which acts as a general base, and dehydroalanine (Dha) on catalytic products. The nucleophilic addition efficiency is dependent on the K136 residue being in a deprotonated state. Peptide cross-linking inhibits the activity of SpvC and blocks the inactivation of MAPK signaling by SpvC. Small compounds mimicking these sequences may act as phosphothreonine lyase inhibitors. PMID:26519561

  2. REX-1 Expression and p38 MAPK Activation Status Can Determine Proliferation/Differentiation Fates in Human Mesenchymal Stem Cells

    PubMed Central

    Jung, Ji-Won; Kang, Soo-Kyung; Kang, Kyung-Sun

    2010-01-01

    Background REX1/ZFP42 is a well-known embryonic stem cell (ESC) marker. However, the role of REX1, itself, is relatively unknown because the function of REX1 has only been reported in the differentiation of ESCs via STAT signaling pathways. Human mesenchymal stem cells (hMSCs) isolated from young tissues and cancer cells express REX1. Methodology/Principal Finding Human umbilical cord blood-derived MSCs (hUCB-MSCs) and adipose tissue-derived MSCs (hAD-MSCs) strongly express REX1 and have a lower activation status of p38 MAPK, but bone marrow-derived MSCs (hBM-MSCs) have weak REX1 expression and higher activation of p38 MAPK. These results indicated that REX1 expression in hMSCs was positively correlated with proliferation rates but inversely correlated with the phosphorylation of p38 MAPK. In hUCB-MSCs, the roles of REX1 and p38 MAPK were investigated, and a knockdown study was performed using a lentiviral vector-based small hairpin RNA (shRNA). After REX1 knockdown, decreased cell proliferation was observed. In REX1 knocked-down hUCB-MSCs, the osteogenic differentiation ability deteriorated, but the adipogenic potential increased or was similar to that observed in the controls. The phosphorylation of p38 MAPK in hUCB-MSCs significantly increased after REX1 knockdown. After p38 MAPK inhibitor treatment, the cell growth in REX1 knocked-down hUCB-MSCs almost recovered, and the suppressed expression levels of CDK2 and CCND1 were also restored. The expression of MKK3, an upstream regulator of p38 MAPK, significantly increased in REX1 knocked-down hUCB-MSCs. The direct binding of REX1 to the MKK3 gene was confirmed by a chromatin immunoprecipitation (ChIP) assay. Conclusions/Significance These findings showed that REX1 regulates the proliferation/differentiation of hMSCs through the suppression of p38 MAPK signaling via the direct suppression of MKK3. Therefore, p38 MAPK and REX-1 status can determine the cell fate of adult stem cells (ASCs). These results were the

  3. A new water soluble MAPK activator exerts antitumor activity in melanoma cells resistant to the BRAF inhibitor vemurafenib.

    PubMed

    Graziani, Grazia; Artuso, Simona; De Luca, Anastasia; Muzi, Alessia; Rotili, Dante; Scimeca, Manuel; Atzori, Maria Grazia; Ceci, Claudia; Mai, Antonello; Leonetti, Carlo; Levati, Lauretta; Bonanno, Elena; Tentori, Lucio; Caccuri, Anna Maria

    2015-05-01

    Recovery of mitogen activated protein kinase (MAPK) or activation of alternative pathways, such as the PI3K/AKT/mTOR, are involved in acquired resistance to BRAF inhibitors which represent the first-line treatment of BRAF-mutated metastatic melanoma. We recently demonstrated that 6-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)thio)hexan-1-ol (NBDHEX) and its water soluble analog 2-(2-(2-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)thio)ethoxy)ethoxy)ethanol (MC3181) trigger apoptosis in BRAF V600E mutated melanoma cells through activation of the MAPK c-Jun N-terminal kinase (JNK). Herein, we investigated whether NBDHEX and MC3181 might exert antitumor activity against BRAF V600E mutated human melanoma cells rendered resistant to the BRAF inhibitor vemurafenib. To this aim we generated a subline of A375 melanoma resistant in vitro and in vivo to vemurafenib (A375-VR8) and characterized by NRAS G13R mutation, high basal levels of CRAF protein and phospho-activation of AKT. In these cells ERK phosphorylation was not significantly down-modulated by vemurafenib concentrations capable of abrogating ERK phosphorylation in sensitive A375 cells. Both NBDHEX and MC3181 induced marked antiproliferative and apoptotic effects in A375-VR8 cells and, at equitoxic concentrations, caused a strong phosphorylation of JNK, p38, and of the downstream mediators of apoptosis ATF2 and p53. Drug treatment further increased ERK phosphorylation, which was required for the cellular response to the NBD derivatives, as apoptosis was antagonized by the ERK inhibitor FR180204. Finally, in vivo administration of MC3181 provoked JNK activation at the tumor site and markedly reduced A375-VR8 growth. These evidences strongly suggest that the activation of multiple pro-apoptotic MAPK pathways by MC3181 might represent a new strategy for the treatment of melanoma resistant to BRAF inhibitors. PMID:25795251

  4. Variants of the yeast MAPK Mpk1 are fully functional independently of activation loop phosphorylation.

    PubMed

    Goshen-Lago, Tal; Goldberg-Carp, Anat; Melamed, Dganit; Darlyuk-Saadon, Ilona; Bai, Chen; Ahn, Natalie G; Admon, Arie; Engelberg, David

    2016-09-01

    MAP kinases of the ERK family are conserved from yeast to humans. Their catalytic activity is dependent on dual phosphorylation of their activation loop's TEY motif, catalyzed by MAPK kinases (MEKs). Here we studied variants of Mpk1, a yeast orthologue of Erk, which is essential for cell wall integrity. Cells lacking MPK1, or the genes encoding the relevant MEKs, MKK1 and MKK2, do not proliferate under cell wall stress, imposed, for example, by caffeine. Mutants of Mpk1, Mpk1(Y268C) and Mpk1(Y268A), function independently of Mkk1 and Mkk2. We show that these variants are phosphorylated at their activation loop in mkk1∆mkk2∆ and mkk1∆mkk2∆pbs2∆ste7∆ cells, suggesting that they autophosphorylate. However, strikingly, when Y268C/A mutations were combined with the kinase-dead mutation, K54R, or mutations at the TEY motif, T190A+Y192F, the resulting proteins still allowed mkk1∆mkk2∆ cells to proliferate under caffeine stress. Mutating the equivalent residue, Tyr-280/Tyr-261, in Erk1/Erk2 significantly impaired Erk1/2's catalytic activity. This study describes the first case in which a MAPK, Erk/Mpk1, imposes a phenotype via a mechanism that is independent of TEY phosphorylation and an unusual case in which an equivalent mutation in a highly conserved domain of yeast and mammalian Erks causes an opposite effect. PMID:27413009

  5. Small Molecule APY606 Displays Extensive Antitumor Activity in Pancreatic Cancer via Impairing Ras-MAPK Signaling.

    PubMed

    Guo, Na; Liu, Zuojia; Zhao, Wenjing; Wang, Erkang; Wang, Jin

    2016-01-01

    Pancreatic cancer has been found with abnormal expression or mutation in Ras proteins. Oncogenic Ras activation exploits their extensive signaling reach to affect multiple cellular processes, in which the mitogen-activated protein kinase (MAPK) signaling exerts important roles in tumorigenesis. Therapies targeted Ras are thus of major benefit for pancreatic cancer. Although small molecule APY606 has been successfully picked out by virtual drug screening based on Ras target receptor, its in-depth mechanism remains to be elucidated. We herein assessed the antitumor activity of APY606 against human pancreatic cancer Capan-1 and SW1990 cell lines and explored the effect of Ras-MAPK and apoptosis-related signaling pathway on the activity of APY606. APY606 treatment resulted in a dose- and time-dependent inhibition of cancer cell viability. Additionally, APY606 exhibited strong antitumor activity, as evidenced not only by reduction in tumor cell invasion, migration and mitochondrial membrane potential but also by alteration in several apoptotic indexes. Furthermore, APY606 treatment directly inhibited Ras-GTP and the downstream activation of MAPK, which resulted in the down-regulation of anti-apoptotic protein Bcl-2, leading to the up-regulation of mitochondrial apoptosis pathway-related proteins (Bax, cytosolic Cytochrome c and Caspase 3) and of cyclin-dependent kinase 2 and Cyclin A, E. These data suggest that impairing Ras-MAPK signaling is a novel mechanism of action for APY606 during therapeutic intervention in pancreatic cancer. PMID:27223122

  6. R-Ras inhibits VEGF-induced p38MAPK activation and HSP27 phosphorylation in endothelial cells

    PubMed Central

    Sawada, Junko; Li, Fangfei; Komatsu, Masanobu

    2016-01-01

    R-Ras is a Ras family small GTPase highly expressed in mature functional blood vessels in normal tissues. It inhibits pathological angiogenesis and promotes vessel maturation and stabilization. Previous studies suggest that R-Ras affects cellular signaling in endothelial cells, pericytes, and smooth muscle cells to regulate vessel formation and remodeling in adult tissues. R-Ras suppresses VEGF-induced endothelial permeability and vessel sprouting while promoting normalization of pathologically developing vessels in mice. R-Ras attenuates VEGF receptor-2 (VEGFR2) activation by inhibiting internalization of the receptor upon VEGF ligand binding, leading to significant reduction of VEGFR2 autophosphorylation. Here, we show that R-Ras strongly suppresses VEGF-dependent activation of stress-activated protein kinase-2/p38 mitogen-activated protein kinase (SAPK2/p38MAPK) and phosphorylation of downstream heat shock protein 27 (HSP27), a regulator of actin cytoskeleton organization, in endothelial cells. The suppression of p38MAPK activation and HSP27 phosphorylation by R-Ras concurred with altered actin cytoskeleton architecture, reduced membrane protrusion, and inhibition of endothelial cell migration toward VEGF. Silencing of endogenous R-Ras by RNAi increased membrane protrusion and cell migration stimulated by VEGF, and these effects were offset by p38MAPK inhibitor SB203580. These results suggest that R-Ras regulates angiogenic activities of endothelial cells in part via inhibition of the p38MAPK-HSP27 axis of VEGF signaling. PMID:27029009

  7. Small Molecule APY606 Displays Extensive Antitumor Activity in Pancreatic Cancer via Impairing Ras-MAPK Signaling

    PubMed Central

    Guo, Na; Liu, Zuojia; Zhao, Wenjing; Wang, Erkang; Wang, Jin

    2016-01-01

    Pancreatic cancer has been found with abnormal expression or mutation in Ras proteins. Oncogenic Ras activation exploits their extensive signaling reach to affect multiple cellular processes, in which the mitogen-activated protein kinase (MAPK) signaling exerts important roles in tumorigenesis. Therapies targeted Ras are thus of major benefit for pancreatic cancer. Although small molecule APY606 has been successfully picked out by virtual drug screening based on Ras target receptor, its in-depth mechanism remains to be elucidated. We herein assessed the antitumor activity of APY606 against human pancreatic cancer Capan-1 and SW1990 cell lines and explored the effect of Ras-MAPK and apoptosis-related signaling pathway on the activity of APY606. APY606 treatment resulted in a dose- and time-dependent inhibition of cancer cell viability. Additionally, APY606 exhibited strong antitumor activity, as evidenced not only by reduction in tumor cell invasion, migration and mitochondrial membrane potential but also by alteration in several apoptotic indexes. Furthermore, APY606 treatment directly inhibited Ras-GTP and the downstream activation of MAPK, which resulted in the down-regulation of anti-apoptotic protein Bcl-2, leading to the up-regulation of mitochondrial apoptosis pathway-related proteins (Bax, cytosolic Cytochrome c and Caspase 3) and of cyclin-dependent kinase 2 and Cyclin A, E. These data suggest that impairing Ras-MAPK signaling is a novel mechanism of action for APY606 during therapeutic intervention in pancreatic cancer. PMID:27223122

  8. R-Ras Inhibits VEGF-Induced p38MAPK Activation and HSP27 Phosphorylation in Endothelial Cells.

    PubMed

    Sawada, Junko; Li, Fangfei; Komatsu, Masanobu

    2015-01-01

    R-Ras is a Ras family small GTPase that is highly expressed in mature functional blood vessels in normal tissues. It inhibits pathological angiogenesis and promotes vessel maturation and stabilization. Previous studies suggest that R-Ras affects cellular signaling in endothelial cells, pericytes and smooth-muscle cells to regulate vessel formation and remodeling in adult tissues. R-Ras suppresses VEGF-induced endothelial permeability and vessel sprouting while promoting normalization of pathologically developing vessels in mice. It attenuates VEGF receptor-2 (VEGFR2) activation by inhibiting internalization of the receptor upon VEGF ligand binding, leading to significant reduction of VEGFR2 autophosphorylation. Here, we show that R-Ras strongly suppresses the VEGF-dependent activation of stress-activated protein kinase-2/p38 mitogen-activated protein kinase (SAPK2/p38MAPK) and the phosphorylation of downstream heat-shock protein 27 (HSP27), a regulator of actin cytoskeleton organization, in endothelial cells. The suppression of p38MAPK activation and HSP27 phosphorylation by R-Ras concurred with altered actin cytoskeleton architecture, reduced membrane protrusion and inhibition of endothelial cell migration toward VEGF. Silencing of endogenous R-Ras by RNA interference increased membrane protrusion and cell migration stimulated by VEGF, and these effects were offset by p38MAPK inhibitor SB203580. These results suggest that R-Ras regulates angiogenic activities of endothelial cells in part via inhibition of the p38MAPK-HSP27 axis of VEGF signaling. PMID:27029009

  9. AP-1 and clathrin are essential for secretory granule biogenesis in Drosophila

    PubMed Central

    Burgess, Jason; Jauregui, Miluska; Tan, Julie; Rollins, Janet; Lallet, Sylvie; Leventis, Peter A.; Boulianne, Gabrielle L.; Chang, Henry C.; Le Borgne, Roland; Krämer, Helmut; Brill, Julie A.

    2011-01-01

     Regulated secretion of hormones, digestive enzymes, and other biologically active molecules requires the formation of secretory granules. Clathrin and the clathrin adaptor protein complex 1 (AP-1) are necessary for maturation of exocrine, endocrine, and neuroendocrine secretory granules. However, the initial steps of secretory granule biogenesis are only minimally understood. Powerful genetic approaches available in the fruit fly Drosophila melanogaster were used to investigate the molecular pathway for biogenesis of the mucin-containing “glue granules” that form within epithelial cells of the third-instar larval salivary gland. Clathrin and AP-1 colocalize at the trans-Golgi network (TGN) and clathrin recruitment requires AP-1. Furthermore, clathrin and AP-1 colocalize with secretory cargo at the TGN and on immature granules. Finally, loss of clathrin or AP-1 leads to a profound block in secretory granule formation. These findings establish a novel role for AP-1– and clathrin-dependent trafficking in the biogenesis of mucin-containing secretory granules. PMID:21490149

  10. Quantitative cell signalling analysis reveals down-regulation of MAPK pathway activation in colorectal cancer.

    PubMed

    Gulmann, Christian; Sheehan, Katherine M; Conroy, Ronán M; Wulfkuhle, Julia D; Espina, Virginia; Mullarkey, Michelle J; Kay, Elaine W; Liotta, Lance A; Petricoin, Emanuel F

    2009-08-01

    Mitogen-activated protein kinases (MAPK) are considered to play significant roles in colonic carcinogenesis and kinase inhibitor therapy has been proposed as a potential tool in the treatment of this disease. Reverse-phase microarray assays using phospho-specific antibodies can directly measure levels of phosphorylated protein isoforms. In the current study, samples from 35 cases of untreated colorectal cancer colectomies were laser capture-microdissected to isolate epithelium and stroma from cancer as well as normal (i.e. uninvolved) mucosa. Lysates generated from these four tissue types were spotted onto reverse-phase protein microarrays and probed with a panel of antibodies to ERK, p-ERK, p38, p-p38, p-JNK, MEK and p-MEK. Whereas total protein levels were unchanged, or slightly elevated (p38, p = 0.0025) in cancers, activated isoforms, including p-ERK, p-p38 and p-JNK, were decreased two- to four-fold in cancers compared with uninvolved mucosa (p < 0.0023 in all cases except for p-JNK in epithelium, where decrement was non-significant). This was backed up by western blotting. Dukes' stage B and C cancers displayed lower p-ERK and p-p38 expression than Dukes' stage A cancers, although this was not statistically significant. It is concluded that MAPK activity may be down-regulated in colorectal cancer and that further exploration of inhibitory therapy in this system should be carefully evaluated if this finding is confirmed in larger series. PMID:19396842

  11. TNF-α-induced p38MAPK activation regulates TRPA1 and TRPV4 activity in odontoblast-like cells.

    PubMed

    El Karim, Ikhlas; McCrudden, Maeliosa T C; Linden, Gerard J; Abdullah, Hanniah; Curtis, Timothy M; McGahon, Mary; About, Imad; Irwin, Christopher; Lundy, Fionnuala T

    2015-11-01

    The transient receptor potential (TRP) channels are unique cellular sensors that are widely expressed in many neuronal and nonneuronal cells. Among the TRP family members, TRPA1 and TRPV4 are emerging as candidate mechanosensitive channels that play a pivotal role in inflammatory pain and mechanical hyperalgesia. Odontoblasts are nonneuronal cells that possess many of the features of mechanosensitive cells and mediate important defense and sensory functions. However, the effect of inflammation on the activity of the odontoblast's mechanosensitive channels remains unknown. By using immunohistochemistry and calcium microfluorimetry, we showed that odontoblast-like cells express TRPA1 and TRPV4 and that these channels were activated by hypotonicity-induced membrane stretch. Short treatment of odontoblast-like cells with tumor necrosis factor (TNF)-α enhanced TRPA1 and TRPV4 responses to their chemical agonists and membrane stretch. This enhanced channel activity was accompanied by phospho-p38 mitogen-activated protein kinase (MAPK) expression. Treatment of cells with the p38 inhibitor SB202190 reduced TNF-α effects, suggesting modulation of channel activity via p38 MAPK. In addition, TNF-α treatment also resulted in an up-regulation of TRPA1 expression but down-regulation of TRPV4. Unlike TRPV4, enhanced TRPA1 expression was also evident in dental pulp of carious compared with noncarious teeth. SB202190 treatment significantly reduced TNF-α-induced TRPA1 expression, suggesting a role for p38 MAPK signaling in modulating both the transcriptional and non-transcriptional regulation of TRP channels in odontoblasts. PMID:26358221

  12. Bcl-3 Expression Promotes Cell Survival following Interleukin-4 Deprivation and Is Controlled by AP1 and AP1-Like Transcription Factors

    PubMed Central

    Rebollo, Angelita; Dumoutier, Laure; Renauld, Jean-Christophe; Zaballos, Angel; Ayllón, Verónica; Martínez-A., Carlos

    2000-01-01

    We have analyzed the interleukin-4 (IL-4)-triggered mechanisms implicated in cell survival and show here that IL-4 deprivation induces apoptotic cell death but does not modulate Bcl-2 or Bcl-x expression. Since Bcl-x expression is insufficient to ensure cell survival in the absence of IL-4, we speculate that additional molecules replace the antiapoptotic role of Bcl-2 and Bcl-x in an alternative IL-4-triggered pathway. Cell death is associated with Bcl-3 downregulation and Bcl-3 expression blocks IL-4 deprivation-induced apoptosis, suggesting that Bcl-3 acts as a survival factor in the absence of growth factor. To characterize the IL-4-induced regulation of murine Bcl-3 expression, we cloned the promoter of this gene. Sequencing of the promoter showed no TATA box element but did reveal binding sites for AP1, AP1-like, and SP1 transcription factors. Retardation gels showed that IL-4 specifically induces AP1 and AP1-like binding activity and that mutation of these binding sites abolishes the IL-4-induced Bcl-3 promoter activity, suggesting that these transcription factors are important in Bcl-3 promoter transactivation. IL-4 deprivation induces downregulation of Jun expression and upregulation of Fos expression, both of which are proteins involved in the formation of AP1 and AP1-like transcription factors. Overexpression of Jun family proteins transactivates the promoter and restores Bcl-3 expression in the absence of IL-4 stimulation. Taken together, these data describe a new biological role for Bcl-3 and define the regulatory pathway implicated in Bcl-3 expression. PMID:10779330

  13. Hypoxia differentially regulates the mitogen- and stress-activated protein kinases. Role of Ca2+/CaM in the activation of MAPK and p38 gamma.

    PubMed

    Conrad, P W; Millhorn, D E; Beitner-Johnson, D

    2000-01-01

    Hypoxic/ischemic trauma is a primary factor in the pathology of various vascular, pulmonary, and cerebral disease states. Yet, the signaling mechanisms by which cells respond and adapt to changes in oxygen levels are not clearly established. The effects of hypoxia on the stress- and mitogen-activated protein kinase (SAPK and MAPK) signaling pathways were studied in PC12 cells. Exposure to moderate hypoxia (5% O2) was found to progressively stimulate phosphorylation and activation of p38 gamma in particular, and also p38 alpha, two isoforms of the p38 family of stress-activated protein kinases. In contrast, hypoxia had no effect on enzyme activity of p38 beta, p38 beta 2, p38 delta, or on JNK, another stress-activated protein kinase. Prolonged hypoxia also induced phosphorylation and activation of p42/p44 MAPK, although this activation was modest when compared to NGF and UV-induced activation. We further showed that activation of p38 gamma and MAPK during hypoxia requires calcium, as treatment with Ca(2+)-free media or the calmodulin antagonist, W13, blocked the activation of p38 gamma and MAPK, respectively. These studies demonstrate that an extremely typical physiological stress (hypoxia) causes selective activation of specific elements of the SAPKs and MAPKs, and identifies Ca+2/CaM as a critical upstream activator. PMID:10849670

  14. CHIP promotes thyroid cancer proliferation via activation of the MAPK and AKT pathways.

    PubMed

    Zhang, Li; Liu, Lianyong; He, Xiaohua; Shen, Yunling; Liu, Xuerong; Wei, Jing; Yu, Fang; Tian, Jianqing

    2016-08-26

    The carboxyl terminus of Hsp70-interacting protein (CHIP) is a U box-type ubiquitin ligase that plays crucial roles in various biological processes, including tumor progression. To date, the functional mechanism of CHIP in thyroid cancer remains unknown. Here, we obtained evidence of upregulation of CHIP in thyroid cancer tissues and cell lines. CHIP overexpression markedly enhanced thyroid cancer cell viability and colony formation in vitro and accelerated tumor growth in vivo. Conversely, CHIP knockdown impaired cell proliferation and tumor growth. Notably, CHIP promoted cell growth through activation of MAPK and AKT pathways, subsequently decreasing p27 and increasing cyclin D1 and p-FOXO3a expression. Our findings collectively indicate that CHIP functions as an oncogene in thyroid cancer, and is therefore a potential therapeutic target for this disease. PMID:27342662

  15. MAPK Activation Is Essential for Waddlia chondrophila Induced CXCL8 Expression in Human Epithelial Cells

    PubMed Central

    Storrie, Skye; Longbottom, David; Barlow, Peter G.; Wheelhouse, Nick

    2016-01-01

    Background Waddlia chondrophila (W. chondrophila) is an emerging agent of respiratory and reproductive disease in humans and cattle. The organism is a member of the order Chlamydiales, and shares many similarities at the genome level and in growth studies with other well-characterised zoonotic chlamydial agents, such as Chlamydia abortus (C. abortus). The current study investigated the growth characteristics and innate immune responses of human and ruminant epithelial cells in response to infection with W. chondrophila. Methods Human epithelial cells (HEp2) were infected with W. chondrophila for 24h. CXCL8 release was significantly elevated in each of the cell lines by active-infection with live W. chondrophila, but not by exposure to UV-killed organisms. Inhibition of either p38 or p42/44 MAPK significantly inhibited the stimulation of CXCL8 release in each of the cell lines. To determine the pattern recognition receptor through which CXCL8 release was stimulated, wild-type HEK293 cells which express no TLR2, TLR4, NOD2 and only negligible NOD1 were infected with live organisms. A significant increase in CXCL8 was observed. Conclusions/Significance W. chondrophila actively infects and replicates within both human and ruminant epithelial cells stimulating CXCL8 release. Release of CXCL8 is significantly inhibited by inhibition of either p38 or p42/44 MAPK indicating a role for this pathway in the innate immune response to W. chondrophila infection. W. chondrophila stimulation of CXCL8 secretion in HEK293 cells indicates that TLR2, TLR4, NOD2 and NOD1 receptors are not essential to the innate immune response to infection. PMID:27002636

  16. CONTRIBUTION OF INSPIRATORY FLOW TO ACTIVATION OF EGFR, RAS, MAPK, ATF-2 AND C-JUN DURING LUNG STRETCH

    EPA Science Inventory

    Contribution of Inspiratory Flow to Activation of EGFR, Ras, MAPK, ATF-2 and c-Jun during Lung Stretch

    R. Silbajoris 1, Z. Li 2, J. M. Samet 1 and Y. C. Huang 1. 1 NHEERL, ORD, US EPA, RTP, NC and 2 CEMALB, UNC-CH, Chapel Hill, NC .

    Mechanical ventilation with larg...

  17. The p38alpha/beta MAPK functions as a molecular switch to activate the quiescent satellite cell.

    PubMed

    Jones, Nathan C; Tyner, Kristina J; Nibarger, Lisa; Stanley, Heather M; Cornelison, Dawn D W; Fedorov, Yuri V; Olwin, Bradley B

    2005-04-11

    Somatic stem cells cycle slowly or remain quiescent until required for tissue repair and maintenance. Upon muscle injury, stem cells that lie between the muscle fiber and basal lamina (satellite cells) are activated, proliferate, and eventually differentiate to repair the damaged muscle. Satellite cells in healthy muscle are quiescent, do not express MyoD family transcription factors or cell cycle regulatory genes and are insulated from the surrounding environment. Here, we report that the p38alpha/beta family of mitogen-activated protein kinases (MAPKs) reversibly regulates the quiescent state of the skeletal muscle satellite cell. Inhibition of p38alpha/beta MAPKs (a) promotes exit from the cell cycle, (b) prevents differentiation, and (c) insulates the cell from most external stimuli allowing the satellite cell to maintain a quiescent state. Activation of satellite cells and p38alpha/beta MAPKs occurs concomitantly, providing further support that these MAPKs function as a molecular switch for satellite cell activation. PMID:15824134

  18. Increases in cAMP, MAPK Activity and CREB Phosphorylation during REM Sleep: Implications for REM Sleep and Memory Consolidation

    PubMed Central

    Luo, Jie; Phan, Trongha X.; Yang, Yimei; Garelick, Michael G.; Storm, Daniel R.

    2013-01-01

    The cyclic adenosine monophosphate (cAMP), mitogen-activated protein kinase (MAPK) and cAMP response element-binding protein (CREB) transcriptional pathway is required for consolidation of hippocampus-dependent memory. In mice, this pathway undergoes a circadian oscillation required for memory persistence that reaches a peak during the daytime. Since mice exhibit polyphasic sleep patterns during the day, this suggested the interesting possibility that cAMP, MAPK activity and CREB phosphorylation may be elevated during sleep. Here, we report that cAMP, phospho-p44/42 MAPK and phospho-CREB are higher in rapid eye movement (REM) sleep compared to awake mice but are not elevated in non-rapid eye movement (NREM) sleep. This peak of activity during REM sleep does not occur in mice lacking calmodulin-stimulated adenylyl cyclases, a mouse strain that learns but cannot consolidate hippocampus-dependent memory. We conclude that a preferential increase in cAMP, MAPK activity and CREB phosphorylation during REM sleep may contribute to hippocampus-dependent memory consolidation. PMID:23575844

  19. Increases in cAMP, MAPK activity, and CREB phosphorylation during REM sleep: implications for REM sleep and memory consolidation.

    PubMed

    Luo, Jie; Phan, Trongha X; Yang, Yimei; Garelick, Michael G; Storm, Daniel R

    2013-04-10

    The cyclic adenosine monophosphate (cAMP), mitogen-activated protein kinase (MAPK), and cAMP response element-binding protein (CREB) transcriptional pathway is required for consolidation of hippocampus-dependent memory. In mice, this pathway undergoes a circadian oscillation required for memory persistence that reaches a peak during the daytime. Because mice exhibit polyphasic sleep patterns during the day, this suggested the interesting possibility that cAMP, MAPK activity, and CREB phosphorylation may be elevated during sleep. Here, we report that cAMP, phospho-p44/42 MAPK, and phospho-CREB are higher in rapid eye movement (REM) sleep compared with awake mice but are not elevated in non-REM sleep. This peak of activity during REM sleep does not occur in mice lacking calmodulin-stimulated adenylyl cyclases, a mouse strain that learns but cannot consolidate hippocampus-dependent memory. We conclude that a preferential increase in cAMP, MAPK activity, and CREB phosphorylation during REM sleep may contribute to hippocampus-dependent memory consolidation. PMID:23575844

  20. Trimethyltin-Induced Microglial Activation via NADPH Oxidase and MAPKs Pathway in BV-2 Microglial Cells

    PubMed Central

    Kim, Da Jung; Kim, Yong Sik

    2015-01-01

    Trimethyltin (TMT) is known as a potent neurotoxicant that causes neuronal cell death and neuroinflammation, particularly in the hippocampus. Microglial activation is one of the prominent pathological features of TMT neurotoxicity. Nevertheless, it remains unclear how microglial activation occurs in TMT intoxication. In this study, we aimed to investigate the signaling pathways in TMT-induced microglial activation using BV-2 murine microglial cells. Our results revealed that TMT generates reactive oxygen species (ROS) and increases the expression of CD11b and nuclear factor-κB- (NF-κB-) mediated nitric oxide (NO) and tumor necrosis factor- (TNF-) α in BV-2 cells. We also observed that NF-κB activation was controlled by p38 and JNK phosphorylation. Moreover, TMT-induced ROS generation occurred via nicotinamide adenine dinucleotide phosphate (NADPH) oxidase in BV-2 cells. Interestingly, treatment with the NADPH oxidase inhibitor apocynin significantly suppressed p38 and JNK phosphorylation and NF-κB activation and ultimately the production of proinflammatory mediators upon TMT exposure. These findings indicate that NADPH oxidase-dependent ROS generation activated p38 and JNK mitogen-activated protein kinases (MAPKs), which then stimulated NF-κB to release proinflammatory mediators in the TMT-treated BV-2 cells. PMID:26221064

  1. Effect of epidermal growth factor (EGF) on the phosphorylation of mitogen-activated protein kinase (MAPK) in the bovine oviduct in vitro: Alteration by heat stress

    PubMed Central

    WIJAYAGUNAWARDANE, Missaka P. B.; HAMBRUCH, Nina; HAEGER, Jan-Dirk; PFARRER, Christiane

    2015-01-01

    Epidermal growth factor (EGF) has been shown to be involved in control of the oviductal microenvironment. To elucidate the potential mechanisms responsible for the detrimental effect of heat stress and to identify the relation with the endocrine status, the effects of EGF on the level of phosphorylated mitogen-activated-protein kinase (MAPK) and proliferation of bovine oviductal epithelial cells (OECs) exposed to different cyclic ovarian steroidal environments (luteal phase (LP), follicular phase (FP) and postovulatory phase (PO)) and temperatures (mild heat stress (40 C) and severe heat stress (43 C)) were investigated. Western blot was performed to evaluate phosphorylated MAPK, while proliferation was analyzed by MTT assay. Stimulation of OECs with EGF alone or with EGF in the PO and FP environments significantly increased the amount of phosphorylated MAPK, with MAPK 44 phosphorylation being highest during exposure to PO conditions. These effects were not observed in the LP. Heat treatment completely blocked effects of EGF on phosphorylated MAPK. Additionally, severe heat stress led to a significantly lower basal level of phosphorylated MAPK. PD98059 (MAPK inhibitor) completely abolished EGF-stimulated MAPK phosphorylation and OECs proliferation. Overall the results indicate that EGF has the potential to increase the amount of phosphorylated MAPK in OECs and therefore could be involved in regulation of the bovine oviductal microenvironment. However, these regulatory mechanisms may be compromised in the presence of heat stress (high ambient temperature), leading to low fertility rates and impaired embryo survival. PMID:26050642

  2. Importin-7 mediates memory consolidation through regulation of nuclear translocation of training-activated MAPK in Drosophila.

    PubMed

    Li, Qian; Zhang, Xuchen; Hu, Wantong; Liang, Xitong; Zhang, Fang; Wang, Lianzhang; Liu, Zhong-Jian; Zhong, Yi

    2016-03-15

    Translocation of signaling molecules, MAPK in particular, from the cytosol to nucleus represents a universal key element in initiating the gene program that determines memory consolidation. Translocation mechanisms and their behavioral impact, however, remain to be determined. Here, we report that a highly conserved nuclear transporter, Drosophila importin-7 (DIM-7), regulates import of training-activated MAPK for consolidation of long-term memory (LTM). We show that silencing DIM-7 functions results in impaired LTM, whereas overexpression of DIM-7 enhances LTM. This DIM-7-dependent regulation of LTM is confined to a consolidation time window and in mushroom body neurons. Image data show that bidirectional alteration in DIM-7 expression results in proportional changes in the intensity of training-activated MAPK accumulated within the nuclei of mushroom body neurons during LTM consolidation. Such DIM-7-regulated nuclear accumulation of activated MAPK is observed only in the training specified for LTM induction and determines the amplitude, but not the time course, of memory consolidation. PMID:26929354

  3. Naringin inhibits ROS-activated MAPK pathway in high glucose-induced injuries in H9c2 cardiac cells.

    PubMed

    Chen, Jingfu; Guo, Runmin; Yan, Hai; Tian, Lihong; You, Qiong; Li, Shanghai; Huang, Ruina; Wu, Keng

    2014-04-01

    Naringin, an active flavonoid isolated from citrus fruit extracts, exhibits biological and pharmacological properties, such as antioxidant activity and antidiabetic effect. Mitogen-activated protein kinase (MAPK) signalling pathway has been shown to participate in hyperglycaemia-induced injury. The present study tested the hypothesis that naringin protects against high glucose (HG)-induced injuries by inhibiting MAPK pathway in H9c2 cardiac cells. To examine this, the cells were treated with 35 mM glucose (HG) for 24 hr to establish a HG-induced cardiomyocyte injury model. The cells were pre-treated with 80 μM naringin for 2 hr before exposure to HG. The findings of this study showed that exposure of H9c2 cells to HG for 24 hr markedly induced injuries, as evidenced by a decrease in cell viability, increases in apoptotic cells and reactive oxygen species (ROS) production, as well as dissipation of mitochondrial membrance potential (MMP). These injuries were significantly attenuated by the pre-treatment of cells with either naringin or SB203580 (a selective inhibitor of p38 MAPK) or U0126 (a selective inhibitor of extracellular signal regulated kinase 1/2, ERK1/2) or SP600125 (a selective inhibitor of c-jun N-termanal kinase, JNK) before exposure to HG, respectively. Furthermore, exposure of cells to HG increased the phosphorylation of p38 MAPK, ERK1/2 and JNK. The increased activation of MAPK pathway was ameliorated by pre-treatment with either naringin or N-acetyl-L-cysteine (NAC), a ROS scavenger, which also reduced HG-induced cytotoxicity and apoptosis, leading to increase in cell viability and decrease in apoptotic cells. In conclusion, our findings provide new evidence for the first time that naringin protects against HG-induced injuries by inhibiting the activation of MAPK (p38 MAPK, ERK1/2 and JNK) and oxidative stress in H9c2 cells. PMID:24118820

  4. Low concentrations of copper in drinking water increase AP-1 binding in the brain.

    PubMed

    Lung, Shyang; Li, Huihui; Bondy, Stephen C; Campbell, Arezoo

    2015-12-01

    Copper (Cu) in trace amounts is essential for biological organisms. However, dysregulation of the redox-active metal has been implicated in different neurological disorders such as Wilson's, Menkes', Alzheimer's, and Parkinson's diseases. Since many households use Cu tubing in the plumbing system, and corrosion causes the metal to leach into the drinking water, there may be adverse effects on the central nervous system connected with low-level chronic exposure. The present study demonstrates that treatment with a biologically relevant concentration of Cu for 3 months significantly increases activation of the redox-modulated transcription factor AP-1 in mouse brains. This was independent of an upstream kinase indicated in AP-1 activation. Another redox-active transcription factor, NF-κB, was not significantly modified by the Cu exposure. These results indicate that the effect of Cu on AP-1 is unique and may involve direct modulation of DNA binding. PMID:23719850

  5. GSH1, which encodes gamma-glutamylcysteine synthetase, is a target gene for yAP-1 transcriptional regulation.

    PubMed Central

    Wu, A L; Moye-Rowley, W S

    1994-01-01

    Changes in gene dosage of the YAP1 gene, encoding the yAP-1 transcriptional regulatory protein, cause profound alterations in cellular drug and metal resistance. Previous studies on yAP-1 action in yeast cells have used the AP-1 response element (ARE) from simian virus 40 as an artificial site for yAP-1-mediated transcriptional activation. No authentic yeast target sites for control of gene expression by yAP-1 are known. Here we show that the GSH1 gene, encoding gamma-glutamylcysteine synthetase, is transcriptionally responsive to the yAP-1 protein. GSH1 encodes the rate-limiting step in yeast glutathione biosynthesis and contains within its promoter region a DNA element that matches the ARE in 11 of 12 positions. The GSH1 yAP-1 response element (YRE) was recognized by yAP-1 protein in vitro. Northern (RNA) blot analysis showed that GSH1 mRNA levels were responsive to YAP1 gene dosage. A site-directed mutation in the YRE that blocked yAP-1 binding in vitro prevented the mutant GSH1 promoter from responding to elevation in YAP1 gene dosage. A delta gsh1 mutant strain was constructed and unable to grow in the absence of exogenous glutathione. A mutant GSH1 gene lacking the YRE was unable to confer normal cadmium tolerance, although other yAP-1-mediated phenotypes remained normal. Thus, GSH1 is one of several genes that are transcriptionally controlled by yAP-1 and influence drug resistance. Images PMID:7915005

  6. Wnt-11 signaling leads to down-regulation of the Wnt/{beta}-catenin, JNK/AP-1 and NF-{kappa}B pathways and promotes viability in the CHO-K1 cells

    SciTech Connect

    Railo, Antti; Nagy, Irina I.; Kilpelaeinen, Pekka Vainio, Seppo

    2008-08-01

    The Wnt family of glycoprotein growth factors controls a number of central cellular processes such as proliferation, differentiation and ageing. All the Wnt proteins analyzed so far either activate or inhibit the canonical {beta}-catenin signaling pathway that regulates transcription of the target genes. In addition, some of them activate noncanonical signaling pathways that involve components such as the JNK, heterotrimeric G proteins, protein kinase C, and calmodulin-dependent protein kinase II, although the precise signaling mechanisms are only just beginning to be revealed. We demonstrate here that Wnt-11 signaling is sufficient to inhibit not only the canonical {beta}-catenin mediated Wnt signaling but also JNK/AP-1 and NF-{kappa}B signaling in the CHO cells, thus serving as a noncanonical Wnt ligand in this system. Inhibition of the JNK/AP-1 pathway is mediated in part by the MAPK kinase MKK4 and Akt. Moreover, protein kinase C is involved in the regulation of JNK/AP-1 by Wnt-11, but not of the NF-{kappa}B pathway. Consistent with the central role of Akt, JNK and NF-{kappa}B in cell survival and stress responses, Wnt-11 signaling promotes cell viability. Hence Wnt-11 is involved in coordination of key signaling pathways.

  7. Oridonin upregulates PTEN through activating p38 MAPK and inhibits proliferation in human colon cancer cells.

    PubMed

    Wu, Qiu-Xiang; Yuan, Shuang-Xue; Ren, Chun-Mei; Yu, Yu; Sun, Wen-Juan; He, Bai-Cheng; Wu, Ke

    2016-06-01

    Oridonin (ORI) has been reported as an antiproliferation and apoptosis-inducing natural product in various cancer cells. However, the exact molecular mechanism underlying these effects remains unclear. In the present study, we demonstrated the antiproliferation effect of ORI in HCT116 cells, and analyzed the possible molecular mechanism which mediates this effect. We found that ORI inhibits proliferation, induces cell cycle arrest and apoptosis in HCT116 cells, thus also tumor growth. Mechanically, we found that ORI has no substantial effect on mRNA expression of phosphatase and tensin homologue (PTEN), but increases the total protein level of PTEN and markedly reduces the phosphorylation of PTEN; Exogenous expression of PTEN potentiates the anticancer effect of ORI, while knockdown of PTEN attenuates it. ORI also increases the phosphorylation of p38 MAPK, and p38 MAPK-specific inhibitor reduces the antiproliferation effect ORI in HCT116 cells. Moreover, inhibition of p38 MAPK increases the phosphorylation of PTEN, and reverses ORI-induced decrease of PTEN phosphorylation. Our findings suggested that ORI may be a potential anticancer drug for colon cancer, this effect may be mediated by enhancing the function of PTEN through reducing its phosphorylation, which may be resulted from the ORI-induced activation of p38 MAPK. PMID:27108927

  8. Immunosuppressant MPA Modulates Tight Junction through Epigenetic Activation of MLCK/MLC-2 Pathway via p38MAPK

    PubMed Central

    Khan, Niamat; Pantakani, D. V. Krishna; Binder, Lutz; Qasim, Muhammad; Asif, Abdul R.

    2015-01-01

    Background: Mycophenolic acid (MPA) is an important immunosuppressive drug (ISD) prescribed to prevent graft rejection in the organ transplanted patients, however, its use is also associated with adverse side effects like sporadic gastrointestinal (GI) disturbances. Recently, we reported the MPA induced tight junctions (TJs) deregulation which involves MLCK/MLC-2 pathway. Here, we investigated the global histone acetylation as well as gene-specific chromatin signature of several genes associated with TJs regulation in Caco-2 cells after MPA treatment. Results: The epigenetic analysis shows that MPA treatment increases the global histone acetylation levels as well as the enrichment for transcriptional active histone modification mark (H3K4me3) at promoter regions of p38MAPK, ATF-2, MLCK, and MLC-2. In contrast, the promoter region of occludin was enriched for transcriptional repressive histone modification mark (H3K27me3) after MPA treatment. In line with the chromatin status, MPA treatment increased the expression of p38MAPK, ATF-2, MLCK, and MLC-2 both at transcriptional and translational level, while occludin expression was negatively influenced. Interestingly, the MPA induced gene expression changes and functional properties of Caco-2 cells could be blocked by the inhibition of p38MAPK using a chemical inhibitor (SB203580). Conclusions: Collectively, our results highlight that MPA disrupts the structure of TJs via p38MAPK-dependent activation of MLCK/MLC-2 pathway that results in decreased integrity of Caco-2 monolayer. These results led us to suggest that p38MAPK-mediated lose integrity of epithelial monolayer could be the possible cause of GI disturbance (barrier dysfunction) in the intestine, leading to leaky style diarrhea observed in the organ-transplanted patients treated with MPA. PMID:26733876

  9. A Trichoderma atroviride stress-activated MAPK pathway integrates stress and light signals.

    PubMed

    Esquivel-Naranjo, Edgardo Ulises; García-Esquivel, Mónica; Medina-Castellanos, Elizabeth; Correa-Pérez, Víctor Alejandro; Parra-Arriaga, Jorge Luis; Landeros-Jaime, Fidel; Cervantes-Chávez, José Antonio; Herrera-Estrella, Alfredo

    2016-06-01

    Cells possess stress-activated protein kinase (SAPK) signalling pathways, which are activated practically in response to any cellular insult, regulating responses for survival and adaptation to harmful environmental changes. To understand the function of SAPK pathways in T. atroviride, mutants lacking the MAPKK Pbs2 and the MAPK Tmk3 were analysed under several cellular stresses, and in their response to light. All mutants were highly sensitive to cellular insults such as osmotic and oxidative stress, cell wall damage, high temperature, cadmium, and UV irradiation. Under oxidative stress, the Tmk3 pathway showed specific roles during development, which in conidia are essential for tolerance to oxidant agents and appear to play a minor role in mycelia. The function of this pathway was more evident in Δpbs2 and Δtmk3 mutant strains when combining oxidative stress or cell wall damage with light. Light stimulates tolerance to osmotic stress through Tmk3 independently of the photoreceptor Blr1. Strikingly, photoconidiation and expression of blue light regulated genes was severally affected in Δtmk3 and Δpbs2 strains, indicating that this pathway regulates light responses. Furthermore, Tmk3 was rapidly phosphorylated upon light exposure. Thus, our data indicate that Tmk3 signalling cooperates with the Blr photoreceptor complex in the activation of gene expression. PMID:26878111

  10. GNA14 Somatic Mutation Causes Congenital and Sporadic Vascular Tumors by MAPK Activation.

    PubMed

    Lim, Young H; Bacchiocchi, Antonella; Qiu, Jingyao; Straub, Robert; Bruckner, Anna; Bercovitch, Lionel; Narayan, Deepak; McNiff, Jennifer; Ko, Christine; Robinson-Bostom, Leslie; Antaya, Richard; Halaban, Ruth; Choate, Keith A

    2016-08-01

    Vascular tumors are among the most common neoplasms in infants and children; 5%-10% of newborns present with or develop lesions within the first 3 months of life. Most are benign infantile hemangiomas that typically regress by 5 years of age; other vascular tumors include congenital tufted angiomas (TAs), kaposiform hemangioendotheliomas (KHEs), and childhood lobular capillary hemangiomas (LCHs). Some of these lesions can become locally invasive and unresponsive to pharmacologic intervention, leading to significant complications. Recent investigation has revealed that activating mutations in HRAS, KRAS, NRAS, GNAQ, and GNA11 can cause certain types of rare childhood vascular tumors, and we have now identified causal recurrent somatic activating mutations in GNA14 by whole-exome and targeted sequencing. We found somatic activating GNA14 c.614A>T (p.Gln205Leu) mutations in one KHE, one TA, and one LCH and a GNA11 c.547C>T (p.Arg183Cys) mutation in two LCH lesions. We examined mutation pathobiology via expression of mutant GNA14 or GNA11 in primary human endothelial cells and melanocytes. GNA14 and GNA11 mutations induced changes in cellular morphology and rendered cells growth-factor independent by upregulating the MAPK pathway. Our findings identify GNA14 mutations as a cause of childhood vascular tumors, offer insight into mechanisms of oncogenic transformation by mutations affecting Gaq family members, and identify potential targets for therapeutic intervention. PMID:27476652

  11. A Measurable Activation of the bZIP Transcription Factor Atf1 in a Fission Yeast Strain Devoid of Stress-activated and Cell Integrity Mitogen-activated Protein Kinase (MAPK) Activities*

    PubMed Central

    Zhou, Xin; Ma, Yan; Kato, Toshiaki; Kuno, Takayoshi

    2012-01-01

    In Schizosaccharomyces pombe, the stress-activated Sty1 MAPK pathway is essential for cell survival under stress conditions. The Sty1 MAPK regulates Atf1 transcription factor to elicit stress responses in extreme conditions of osmolarity and reactive oxygen species-generating agents such as hydrogen peroxide, heat, low glucose, and heavy metal. Herein, using a newly developed Renilla luciferase reporter assay with enhanced detection sensitivity and accuracy, we show that distinct signaling pathways respond to cadmium and other reactive oxygen species-generating agents for the activation of Atf1. Also, surprisingly, a measurable activation of Atf1 transcription factor was still observed devoid of Sty1 MAPK activity. Further genetic and biological analyses revealed that the residual activation is caused by the activation of the cell wall integrity Pmk1 MAPK pathway and a redox-mediated activation of Atf1. PMID:22661707

  12. Luteolin 8-C-β-fucopyranoside inhibits invasion and suppresses TPA-induced MMP-9 and IL-8 via ERK/AP-1 and ERK/NF-κB signaling in MCF-7 breast cancer cells.

    PubMed

    Park, Su-Ho; Kim, Jung-Hee; Lee, Dong-Hun; Kang, Jeong-Woo; Song, Hyuk-Hwan; Oh, Sei-Ryang; Yoon, Do-Young

    2013-11-01

    Matrix metalloproteinase 9 (MMP-9) and interleukin-8 (IL-8) play major roles in tumor progression and invasion of breast cancer cells. The present study was undertaken to investigate the inhibitory mechanism of cell invasion by luteolin 8-C-β-fucopyranoside (named as LU8C-FP), a C-glycosylflavone, in human breast cancer cells. We investigated whether LU8C-FP would inhibit MMP-9 activation and IL-8 expression in 12-O-tetradecanoylphorbol-13-acetate (TPA)-treated MCF-7 breast cancer cells. LU8C-FP suppressed TPA-induced MMP-9 and IL-8 secretion and mRNA expression via inhibition of the MAPK signaling pathway and down-regulation of nuclear AP-1 and NF-κB. TPA-induced phosphorylation of ERK 1/2 was suppressed by LU8C-FP, whereas JNK and p38 MAPK phosphorylation were unaffected. In addition, LU8C-FP blocked the ERK 1/2 pathways following expression of MMP-9 and IL-8. These results suggest LU8C-FP may function to suppress invasion of breast cancer cells through the ERK/AP-1 and ERK/NF-κB signaling cascades. PMID:23933110

  13. The FGL2/fibroleukin prothrombinase is involved in alveolar macrophage activation in COPD through the MAPK pathway

    SciTech Connect

    Liu, Yanling; Xu, Sanpeng; Xiao, Fei; Xiong, Yan; Wang, Xiaojin; Gao, Sui; Yan, Weiming; Ning, Qin

    2010-05-28

    Fibrinogen-like protein 2 (FGL2)/fibroleukin has been reported to play a vital role in the pathogenesis of some critical inflammatory diseases by possessing immunomodulatory activity through the mediation of 'immune coagulation' and the regulation of maturation and proliferation of immune cells. We observed upregulated FGL2 expression in alveolar macrophages from peripheral lungs of chronic obstructive pulmonary disease (COPD) patients and found a correlation between FGL2 expression and increased macrophage activation markers (CD11b and CD14). The role of FGL2 in the activation of macrophages was confirmed by the detection of significantly decreased macrophage activation marker (CD11b, CD11c, and CD71) expression as well as the inhibition of cell migration and inflammatory cytokine (IL-8 and MMP-9) production in an LPS-induced FGL2 knockdown human monocytic leukemia cell line (THP-1). Increased FGL2 expression co-localized with upregulated phosphorylated p38 mitogen-activated protein kinase (p38-MAPK) in the lung tissues from COPD patients. Moreover, FGL2 knockdown in THP-1 cells significantly downregulated LPS-induced phosphorylation of p38-MAPK while upregulating phosphorylation of c-Jun N-terminal kinase (JNK). Thus, we demonstrate that FGL2 plays an important role in macrophage activation in the lungs of COPD patients through MAPK pathway modulation.

  14. Arachidonic acid stimulates DNA synthesis in brown preadipocytes through the activation of protein kinase C and MAPK.

    PubMed

    Garcia, Bibian; Martinez-de-Mena, Raquel; Obregon, Maria-Jesus

    2012-10-01

    Arachidonic acid (AA) is a polyunsaturated fatty acid that stimulates the proliferation of many cellular types. We studied the mitogenic potential of AA in rat brown preadipocytes in culture and the signaling pathways involved. AA is a potent mitogen which induces 4-fold DNA synthesis in brown preadipocytes. The AA mitogenic effect increases by NE addition. AA also increases the mitogenic action of different growth factor combinations. Other unsaturated and saturated fatty acids do not stimulate DNA synthesis to the same extent as AA. We analyzed the role of PKC and MEK/MAPK signaling pathways. PKC inhibition by bisindolilmaleimide I (BIS) abolishes AA and phorbol ester stimulation of DNA synthesis and reduces the mitogenic activity of different growth factors in brown preadipocytes. Brown preadipocytes in culture express PKC α, δ, ε and ζ isoforms. Pretreatment with high doses of the phorbol ester PDBu, induces downregulation of PKCs ε and δ and reproduces the effect of BIS indicating that AA-dependent induction of DNA synthesis requires PKC activity. AA also activates MEK/MAPK pathway and the inhibition of MEK activity inhibits AA stimulation of DNA synthesis and brown adipocyte proliferation. Inhibition of PKC δ by rottlerin abolishes AA-dependent stimulation of DNA synthesis and MAPK activation, whereas PKC ε inhibition does not produce any effect. In conclusion, our results identify AA as a potent mitogen for brown adipocytes and demonstrate the involvement of the PDBu-sensitive PKC δ isoform and MEK/MAPK pathway in AA-induced proliferation of brown adipocytes. Increased proliferative activity might increase the thermogenic capacity of brown fat. PMID:22766489

  15. atRA-induced apoptosis of mouse embryonic palate mesenchymal cells involves activation of MAPK pathway

    SciTech Connect

    Yu Zengli . E-mail: yuzengli@263.net; Xing Ying . E-mail: xingy@zzu.edu.cn

    2006-08-15

    Our previous studies have shown that atRA treatment resulted in cell-cycle block and growth inhibition in mouse embryonic palatal mesenchymal (MEPM). In the current study, gestation day (GD) 13 MEPM cells were used to test the hypothesis that the growth inhibition by atRA is due to apoptosis. The effects of atRA on apoptosis were assessed by performing MTT assay, Cell Death Detection ELISA and flow cytometry, respectively. Data analysis confirmed that atRA treatment induced apoptosis-like cell death, as shown by decreased cell viability and increased fragmented DNA and sub-G1 fraction. atRA-induced apoptosis was associated with upregulation of bcl-2, translocation of bax protein to the mitochondria from the cytosol, activation of caspase-3 and cytochrome c release into cytosol. atRA-induced apoptosis was abrogated by z-DEVD-fmk, a caspase-3 specific inhibitor, and z-VAD-fmk, a general caspase inhibitor, suggesting that the atRA-induced cell death of MEPM cells occurs through the cytochrome c- and caspase-3-dependent pathways. In addition, atRA treatment caused a strong and sustained activation of c-Jun N-terminal kinase (JNK) and p38 kinase (p38), as well as an early but transient activation of extracellular signal-regulated kinase (ERK). Importantly, atRA-induced DNA fragmentation and capase-3 activation were prevented by pretreatment with the JNK inhibitor (SP600125) and the p38 MAPK inhibitor (SB202190), but not by pretreatment with MEK inhibitor (U0126). From these results, we suggest that mitogen-activated protein kinase-dependent pathways is involved in the atRA-induced apoptosis of MEPM cells.

  16. Interleukin-17A Promotes Aortic Endothelial Cell Activation via Transcriptionally and Post-translationally Activating p38 Mitogen-activated Protein Kinase (MAPK) Pathway.

    PubMed

    Mai, Jietang; Nanayakkara, Gayani; Lopez-Pastrana, Jahaira; Li, Xinyuan; Li, Ya-Feng; Wang, Xin; Song, Ai; Virtue, Anthony; Shao, Ying; Shan, Huimin; Liu, Fang; Autieri, Michael V; Kunapuli, Satya P; Iwakura, Yoichiro; Jiang, Xiaohua; Wang, Hong; Yang, Xiao-Feng

    2016-03-01

    Interleukin-17 (IL-17)-secreting T helper 17 cells were recently identified as a CD4(+) T helper subset and implicated in various inflammatory and autoimmune diseases. The issues of whether and by what mechanism hyperlipidemic stress induces IL-17A to activate aortic endothelial cells (ECs) and enhance monocyte adhesion remained largely unknown. Using biochemical, immunological, microarray, experimental data mining analysis, and pathological approaches focused on primary human and mouse aortic ECs (HAECs and MAECs) and our newly generated apolipoprotein E (ApoE)(-/-)/IL-17A(-/-) mice, we report the following new findings. 1) The hyperlipidemia stimulus oxidized low density lipoprotein up-regulated IL-17 receptor(s) in HAECs and MAECs. 2) IL-17A activated HAECs and increased human monocyte adhesion in vitro. 3) A deficiency of IL-17A reduced leukocyte adhesion to endothelium in vivo. 3) IL-17A activated HAECs and MAECs via up-regulation of proinflammatory cytokines IL-6, granulocyte-macrophage colony-stimulating factor (GM-CSF), chemokine CXC motif ligand 1 (CXCL1), and CXCL2. 4) IL-17A activated ECs specifically via the p38 mitogen-activated protein kinases (MAPK) pathway; the inhibition of p38 MAPK in ECs attenuated IL-17A-mediated activation by ameliorating the expression of the aforementioned proinflammatory cytokines, chemokines, and EC adhesion molecules including intercellular adhesion molecule 1. Taken together, our results demonstrate for the first time that IL-17A activates aortic ECs specifically via p38 MAPK pathway. PMID:26733204

  17. Apoptotic Effects of Cordycepin Through the Extrinsic Pathway and p38 MAPK Activation in Human Glioblastoma U87MG Cells.

    PubMed

    Baik, Ji-Sue; Mun, Seo-Won; Kim, Kyoung-Sook; Park, Shin-Ji; Yoon, Hyun-Kyoung; Kim, Dong-Hyun; Park, Min-Kyu; Kim, Cheorl-Ho; Lee, Young-Choon

    2016-02-28

    We first demonstrated that cordycepin inhibited cell growth and triggered apoptosis in U87MG cells with wild-type p53, but not in T98G cells with mutant-type p53. Western blot data revealed that the levels of procaspase-8, -3, and Bcl-2 were downregulated in cordycepintreated U87MG cells, whereas the levels of Fas, FasL, Bak, cleaved caspase-3, -8, and cleaved PARP were upregulated, indicating that cordycepin induces apoptosis by activating the death receptor-mediated pathway in U87MG cells. Cordycepin-induced apoptosis could be suppressed by only SB203580, a p38 MAPK-specific inhibitor. These results suggest that cordycepin triggered apoptosis in U87MG cells through p38 MAPK activation and inhibition of the Akt survival pathway. PMID:26597532

  18. The endocannabinoid anandamide induces apoptosis of rat decidual cells through a mechanism involving ceramide synthesis and p38 MAPK activation.

    PubMed

    Fonseca, B M; Correia-da-Silva, G; Teixeira, N A

    2013-12-01

    Anandamide (AEA) belongs to an endogenous family of lipid messengers, called endocannabinoids (ECs), which exert pharmacological effects by binding to selective membrane receptors, the CB1 and CB2 receptors. Increasing evidence suggests that AEA is involved in the regulation of a variety of cell signalling pathways both in experimental models and humans. We have previously demonstrated that ECs machinery operates in decidual cells and found that AEA, the principal EC, induced apoptosis in decidual cells through CB1. Here, we investigated in rat primary decidual cells the signal transduction pathways activated upon AEA binding to CB1. We found that AEA induces a significant increase in the level of intracellular ceramide. These effects were reversed by inhibiting CB1 receptor activation with AM251. The ceramide analogue, C6-ceramide, induced a decrease in decidual cell viability and of p38 MAPK phosphorylation. Additionally, the pharmacologic inhibition of de novo ceramide biosynthesis with L-cycloserine and fumonisin B reduced the AEA-effects on cell viability and p38 MAPK phosphorylation. Furthermore, AEA and C6-ceramide induced a drop in ΔΨm, an increase in ROS production and caspase-3/-7 activation, effects partially reverted by inhibitors of ceramide synthesis and of p38 MAPK. Taken together, we showed that AEA induces a reduction in decidual cell viability by a mechanism involving CB1 activation, which results in ceramide synthesis de novo and p38 phosphorylation, followed by mitochondrial stress and ROS production, leading to apoptosis. PMID:24048885

  19. Comparative analysis of MAPK and PI3K/AKT pathway activation and inhibition in human and canine melanoma.

    PubMed

    Fowles, J S; Denton, C L; Gustafson, D L

    2015-09-01

    The lack of advanced animal models of human cancers is considered a barrier to developing effective therapeutics. Canine and human melanomas are histologically disparate but show similar disease progression and response to therapies. The purpose of these studies was to compare human and canine melanoma tumours and cell lines regarding MAPK and PI3K/AKT signalling dysregulation, and response to select molecularly targeted agents. Pathway activation was investigated via microarray and mutational analysis. Growth inhibition and cell cycle effects were assessed for pathway inhibitors AZD6244 (MAPK) and rapamycin (PI3K/AKT) in human and canine melanoma cells. Human and canine melanoma share similar differential gene expression patterns within the MAPK and PI3K/AKT pathways. Constitutive pathway activation and similar sensitivity to AZD6244 and rapamycin was observed in human and canine cells. These results show that human and canine melanoma share activation and sensitivity to inhibition of cancer-related signalling pathways despite differences in activating mutations. PMID:23745794

  20. Inhibition of adipogenic differentiation of bone marrow mesenchymal stem cells by erythropoietin via activating ERK and P38 MAPK.

    PubMed

    Liu, G X; Zhu, J C; Chen, X Y; Zhu, A Z; Liu, C C; Lai, Q; Chen, S T

    2015-01-01

    We examined whether erythropoietin (EPO) can inhibit adipogenic differentiation of mesenchymal stem cells (MSCs) in the mouse bone marrow and its underlying mechanism. We separated and extracted mouse bone marrow MSCs and induced adipogenic differen-tiation using 3-isobutyl-1-methylxanthine, insulin, and dexamethasone. Different concentrations of EPO were added to the cells and observed by Oil Red O staining on the 20th day to quantitatively analyze the degree of cell differentiation. mRNA expression levels of peroxysome proliferator-activated receptor γ (PPARγ), CCAAT enhancer binding protein α, and adiponectin were analyzed by real-time quantitative polymerase chain reaction, and the activity of PPARγ, extracellular sig-nal-regulated kinase (ERK), and p38 mitogen-activated protein kinase (p38 MAPK) were determined by western blotting. EPO significantly inhibited adipogenic differentiation of MSCs after 20 days and reduced absorbance values by Oil Red O staining without affecting proliferation activity. EPO downregulated the mRNA expression of PPARγ, CCAAT enhancer binding protein α, fatty acid binding protein 4, and adiponec-tin during adipogenesis and increased protein phosphorylation of ERK, p38 MAPK, and PPARγ during differentiation. EPO downregulated the mRNA expression of PPARγ, CCAAT enhancer binding protein α, fatty acid binding protein 4, and adiponectin by increasing protein phosphor-ylation of ERK, p38 MAPK, and PPARγ during differentiation, which inhibited adipogenic differentiation of MSCs. PMID:26125905

  1. YES oncogenic activity is specified by its SH4 domain and regulates RAS/MAPK signaling in colon carcinoma cells

    PubMed Central

    Dubois, Fanny; Leroy, Cédric; Simon, Valérie; Benistant, Christine; Roche, Serge

    2015-01-01

    Members of the SRC family of tyrosine kinases (SFK) display important functions in human cancer, but their specific role in tumorigenesis remains unclear. We previously demonstrated that YES regulates a unique oncogenic signaling important for colorectal cancer (CRC) progression that is not shared with SRC. Here, we addressed the underlying mechanism involved in this process. We show that YES oncogenic signaling relies on palmitoylation of its SH4 domain that controls YES localization in cholesterol-enriched membrane micro-domains. Specifically, deletion of the palmitoylation site compromised YES transforming activity, while addition of a palmitoylation site in the SH4 domain of SRC was sufficient for SRC to restore the transforming properties of cells in which YES had been silenced. Subsequently, SILAC phosphoproteomic analysis revealed that micro-domain-associated cell adhesive components and receptor tyrosine kinases are major YES substrates. YES also phosphorylates upstream regulators of RAS/MAPK signaling, including EGFR, SHC and SHP2, which were not targeted by SRC due to the absence of palmitoylation. Accordingly, EGFR-induced MAPK activity was attenuated by YES down-regulation, while increased RAS activity significantly restored cell transformation that was lost upon YES silencing. Collectively, these results uncover a critical role for the SH4 domain in the specification of SFK oncogenic activity and a selective role for YES in the induction of RAS/MAPK signaling in CRC cells. PMID:26269757

  2. 2-Methoxyestradiol induced Bax phosphorylation and apoptosis in human retinoblastoma cells via p38 MAPK activation.

    PubMed

    Min, Hongbo; Ghatnekar, Gautam S; Ghatnekar, Angela V; You, Xiaohong; Bu, Min; Guo, Xinyi; Bu, Shizhong; Shen, Bo; Huang, Qin

    2012-07-01

    Retinoblastoma (Rb) is a common childhood intraocular cancer that affects approximately 300 children each year in the United States alone. 2-Methoxyestradiol (2ME), an endogenous metabolite of 17-β-estradiol that dose not bind to nuclear estrogen receptor, exhibits potent apoptotic activity against rapidly growing tumor cells. Here, we report that 2ME induction of apoptosis was demonstrated by early fragmented DNA after 48 h of incubation with 10 µM 2ME in Rb cell lines. Subsequently, a decrease of proliferation was observed in a time- and dose-dependent manner. Further analysis of the mechanism indicates that p38 kinase plays a critical role in 2ME-induced apoptosis in Y79 cells, even though ERK was also activated by 2ME under the same conditions. Activation of p38 kinase also mediates 2ME induced Bax phosphorylated at Thr(167) after a 6 h treatment of 2ME, which in turn prevents formation of the Bcl-2-Bax heterodimer. Both p38 specific inhibitor, SB 203580, or p38 knockdown by specific siRNA, blocked 2ME induction of Bax phosphorylation. Furthermore, only transiently transfected mutant BaxT167A, but not Bax S163A, inhibited 2ME-induced apoptosis. In summary, our data suggest that 2ME induces apoptosis in human Rb cells by causing phosphorylation of p38 Mitogen-activated protein kinase (MAPK), which appears to be correlated with phosphorlation of Bax. This understanding of 2ME's ability may help develop it as a promising therapeutic candidate by inducing apoptosis in a Rb. PMID:21769948

  3. Chronic inflammation and estradiol interact through MAPK activation to affect TMJ nociceptive processing by trigeminal caudalis neurons.

    PubMed

    Tashiro, A; Okamoto, K; Bereiter, D A

    2009-12-29

    The mitogen-activated protein kinase/extracellular regulated kinase (MAPK/ERK) pathway plays a key role in mediating estrogen actions in the brain and neuronal sensitization during inflammation. Estrogen status is a risk factor in chronic temporomandibular muscle/joint (TMJ) disorders; however, the basis for this relationship is not known. The present study tested the hypothesis that estrogen status acts through the MAPK/ERK signaling pathway to alter TMJ nociceptive processing. Single TMJ-responsive neurons were recorded in laminae I-II at the spinomedullary (Vc/C(1-2)) junction in naïve ovariectomized (OvX) female rats treated for 2 days with high-dose (20 microg/day; HE2) or low-dose estradiol (2 microg/day; LE2) and after chronic inflammation of the TMJ region by complete Freund's adjuvant for 12-14 days. Intra-TMJ injection of ATP (1 mM) was used to activate Vc/C(1-2) neurons. The MAPK/ERK inhibitor (PD98059, 0.01-1 mM) was applied topically to the dorsal Vc/C(1-2) surface at the site of recording 10 min prior to each ATP stimulus. In naïve HE2 rats, low-dose PD98059 caused a maximal inhibition of ATP-evoked activity, whereas even high doses had only minor effects on units in LE2 rats. By contrast, after chronic TMJ inflammation, PD98059 produced a marked and similar dose-related inhibition of ATP-evoked activity in HE2 and LE2 rats. These results suggested that E2 status and chronic inflammation acted, at least in part, through a common MAPK/ERK-dependent signaling pathway to enhance TMJ nociceptive processing by laminae I-II neurons at the spinomedullary junction region. PMID:19786077

  4. Exogenous C2 Ceramide Suppresses Matrix Metalloproteinase Gene Expression by Inhibiting ROS Production and MAPK Signaling Pathways in PMA-Stimulated Human Astroglioma Cells

    PubMed Central

    Jung, Ji-Sun; Ahn, Young-Ho; Moon, Byung-In; Kim, Hee-Sun

    2016-01-01

    Matrix metalloproteinases (MMPs) are a family of zinc-dependent endopeptidases, which play a pivotal role in invasion, migration, and angiogenesis of glioma. Therefore, controlling MMPs is potentially an important therapeutic strategy for glioma. In the present study, we found that exogenous cell-permeable short-chain C2 ceramide inhibits phorbol myristate acetate (PMA)-induced MMP-1, -3, and -9 gene expressions in U87MG and U373MG human astroglioma cells. In addition, C2 ceramide inhibited the protein secretion and enzymatic activities of MMP-1, -3, and -9. The Matrigel invasion assay and wound healing assay showed that C2 ceramide suppresses the in vitro invasion and migration of glioma cells, which appears to be involved in strong inhibition of MMPs by C2 ceramide. Subsequent mechanistic studies revealed that C2 ceramide inhibits PMA-induced mitogen-activated protein kinase (MAPK) phosphorylation and nuclear factor (NF)-κB/activator protein (AP)-1 DNA binding activities. Furthermore, C2 ceramide significantly inhibited PMA-induced reactive oxygen species (ROS) production and NADPH oxidase 4 (NOX4) expression, and inhibition of ROS by diphenylene iodonium (DPI, NADPH oxidase inhibitor) mimicked the effects of C2 ceramide on MMP expression and NF-κB/AP-1 via inhibition of p38 MAPK. The results suggest C2 ceramide inhibits MMP expression and glioma invasion, at least partly, by modulating ROS-p38 MAPK signaling axis and other MAPK signaling pathways. PMID:27043542

  5. Exogenous C2 Ceramide Suppresses Matrix Metalloproteinase Gene Expression by Inhibiting ROS Production and MAPK Signaling Pathways in PMA-Stimulated Human Astroglioma Cells.

    PubMed

    Jung, Ji-Sun; Ahn, Young-Ho; Moon, Byung-In; Kim, Hee-Sun

    2016-01-01

    Matrix metalloproteinases (MMPs) are a family of zinc-dependent endopeptidases, which play a pivotal role in invasion, migration, and angiogenesis of glioma. Therefore, controlling MMPs is potentially an important therapeutic strategy for glioma. In the present study, we found that exogenous cell-permeable short-chain C2 ceramide inhibits phorbol myristate acetate (PMA)-induced MMP-1, -3, and -9 gene expressions in U87MG and U373MG human astroglioma cells. In addition, C2 ceramide inhibited the protein secretion and enzymatic activities of MMP-1, -3, and -9. The Matrigel invasion assay and wound healing assay showed that C2 ceramide suppresses the in vitro invasion and migration of glioma cells, which appears to be involved in strong inhibition of MMPs by C2 ceramide. Subsequent mechanistic studies revealed that C2 ceramide inhibits PMA-induced mitogen-activated protein kinase (MAPK) phosphorylation and nuclear factor (NF)-κB/activator protein (AP)-1 DNA binding activities. Furthermore, C2 ceramide significantly inhibited PMA-induced reactive oxygen species (ROS) production and NADPH oxidase 4 (NOX4) expression, and inhibition of ROS by diphenylene iodonium (DPI, NADPH oxidase inhibitor) mimicked the effects of C2 ceramide on MMP expression and NF-κB/AP-1 via inhibition of p38 MAPK. The results suggest C2 ceramide inhibits MMP expression and glioma invasion, at least partly, by modulating ROS-p38 MAPK signaling axis and other MAPK signaling pathways. PMID:27043542

  6. Secretory pathway retention of mutant prion protein induces p38-MAPK activation and lethal disease in mice

    PubMed Central

    Puig, Berta; Altmeppen, Hermann C.; Ulbrich, Sarah; Linsenmeier, Luise; Krasemann, Susanne; Chakroun, Karima; Acevedo-Morantes, Claudia Y.; Wille, Holger; Tatzelt, Jörg; Glatzel, Markus

    2016-01-01

    Misfolding of proteins in the biosynthetic pathway in neurons may cause disturbed protein homeostasis and neurodegeneration. The prion protein (PrPC) is a GPI-anchored protein that resides at the plasma membrane and may be misfolded to PrPSc leading to prion diseases. We show that a deletion in the C-terminal domain of PrPC (PrPΔ214–229) leads to partial retention in the secretory pathway causing a fatal neurodegenerative disease in mice that is partially rescued by co-expression of PrPC. Transgenic (Tg(PrPΔ214–229)) mice show extensive neuronal loss in hippocampus and cerebellum and activation of p38-MAPK. In cell culture under stress conditions, PrPΔ214–229 accumulates in the Golgi apparatus possibly representing transit to the Rapid ER Stress-induced ExporT (RESET) pathway together with p38-MAPK activation. Here we describe a novel pathway linking retention of a GPI-anchored protein in the early secretory pathway to p38-MAPK activation and a neurodegenerative phenotype in transgenic mice. PMID:27117504

  7. Maternal inflammation activated ROS-p38 MAPK predisposes offspring to heart damages caused by isoproterenol via augmenting ROS generation

    PubMed Central

    Zhang, Qi; Deng, Yafei; Lai, Wenjing; Guan, Xiao; Sun, Xiongshan; Han, Qi; Wang, Fangjie; Pan, Xiaodong; Ji, Yan; Luo, Hongqin; Huang, Pei; Tang, Yuan; Gu, Liangqi; Dan, Guorong; Yu, Jianhua; Namaka, Michael; Zhang, Jianxiang; Deng, Youcai; Li, Xiaohui

    2016-01-01

    Maternal inflammation contributes to the increased incidence of adult cardiovascular disease. The current study investigated the susceptibility of cardiac damage responding to isoproterenol (ISO) in adult offspring that underwent maternal inflammation (modeled by pregnant Sprague-Dawley rats with lipopolysaccharides (LPS) challenge). We found that 2 weeks of ISO treatment in adult offspring of LPS-treated mothers led to augmented heart damage, characterized by left-ventricular systolic dysfunction, cardiac hypertrophy and myocardial fibrosis. Mechanistically, prenatal exposure to LPS led to up-regulated expression of nicotinamide adenine dinucleotide phosphate (NADPH) oxidases, antioxidant enzymes, and p38 MAPK activity in left ventricular of adult offspring at resting state. ISO treatment exaggerated ROS generation, p38 MAPK activation but down-regulated reactive oxygen species (ROS) elimination capacity in the left ventricular of offspring from LPS-treated mothers, while antioxidant N-acetyl-L-cysteine (NAC) reversed these changes together with improved cardiac functions. The p38 inhibitor SB202190 alleviated the heart damage only via inhibiting the expression of NADPH oxidases. Collectively, our data demonstrated that prenatal inflammation programs pre-existed ROS activation in the heart tissue, which switches on the early process of oxidative damages on heart rapidly through a ROS-p38 MAPK-NADPH oxidase-ROS positive feedback loop in response to a myocardial hypertrophic challenge in adulthood. PMID:27443826

  8. Secretory pathway retention of mutant prion protein induces p38-MAPK activation and lethal disease in mice.

    PubMed

    Puig, Berta; Altmeppen, Hermann C; Ulbrich, Sarah; Linsenmeier, Luise; Krasemann, Susanne; Chakroun, Karima; Acevedo-Morantes, Claudia Y; Wille, Holger; Tatzelt, Jörg; Glatzel, Markus

    2016-01-01

    Misfolding of proteins in the biosynthetic pathway in neurons may cause disturbed protein homeostasis and neurodegeneration. The prion protein (PrP(C)) is a GPI-anchored protein that resides at the plasma membrane and may be misfolded to PrP(Sc) leading to prion diseases. We show that a deletion in the C-terminal domain of PrP(C) (PrPΔ214-229) leads to partial retention in the secretory pathway causing a fatal neurodegenerative disease in mice that is partially rescued by co-expression of PrP(C). Transgenic (Tg(PrPΔ214-229)) mice show extensive neuronal loss in hippocampus and cerebellum and activation of p38-MAPK. In cell culture under stress conditions, PrPΔ214-229 accumulates in the Golgi apparatus possibly representing transit to the Rapid ER Stress-induced ExporT (RESET) pathway together with p38-MAPK activation. Here we describe a novel pathway linking retention of a GPI-anchored protein in the early secretory pathway to p38-MAPK activation and a neurodegenerative phenotype in transgenic mice. PMID:27117504

  9. Ursolic Acid Triggers Apoptosis in Human Osteosarcoma Cells via Caspase Activation and the ERK1/2 MAPK Pathway.

    PubMed

    Wu, Chia-Chieh; Cheng, Chun-Hsiang; Lee, Yi-Hui; Chang, Ing-Lin; Chen, Hsin-Yao; Hsieh, Chen-Pu; Chueh, Pin-Ju

    2016-06-01

    Ursolic acid (UA), a naturally occurring pentacyclic triterpene acid found in many medicinal herbs and edible plants, has been shown to trigger apoptosis in several lines of tumor cells in vitro. We found that treatment with UA suppressed the viability of human osteosarcoma MG-63 cells and induced cell cycle arrest at sub-G1 and G2/M phases. Furthermore, exposure to UA induced intracellular oxidative stress and collapse of mitochondrial membrane permeability, resulting in the subsequent activation of apoptotic caspases 8, 9, and 3 as well as PARP cleavage, and ultimately apoptosis in MG-63 cells. Moreover, protein analysis of mitogen-activated protein kinase (MAPK)-related protein expression showed an increase in activated ERK1/2, JNK, and p38 MAPK in UA-treated MG-63 cells. In addition, UA-induced apoptosis was significantly abolished in MG-63 cells that had been pretreated with inhibitors of caspase 3, 8, and 9 and ERK1/2. Furthermore, UA-treated MG-63 cells also exhibited an enhancement in Bax/Bcl-2 ratio, whereas anti-apoptotic XIAP and survivin were down-regulated. Taken together, we provide evidence demonstrating that UA mediates caspase-dependent and ERK1/2 MAPK-associated apoptosis in osteosarcoma MG-63 cells. PMID:27171502

  10. Maternal inflammation activated ROS-p38 MAPK predisposes offspring to heart damages caused by isoproterenol via augmenting ROS generation.

    PubMed

    Zhang, Qi; Deng, Yafei; Lai, Wenjing; Guan, Xiao; Sun, Xiongshan; Han, Qi; Wang, Fangjie; Pan, Xiaodong; Ji, Yan; Luo, Hongqin; Huang, Pei; Tang, Yuan; Gu, Liangqi; Dan, Guorong; Yu, Jianhua; Namaka, Michael; Zhang, Jianxiang; Deng, Youcai; Li, Xiaohui

    2016-01-01

    Maternal inflammation contributes to the increased incidence of adult cardiovascular disease. The current study investigated the susceptibility of cardiac damage responding to isoproterenol (ISO) in adult offspring that underwent maternal inflammation (modeled by pregnant Sprague-Dawley rats with lipopolysaccharides (LPS) challenge). We found that 2 weeks of ISO treatment in adult offspring of LPS-treated mothers led to augmented heart damage, characterized by left-ventricular systolic dysfunction, cardiac hypertrophy and myocardial fibrosis. Mechanistically, prenatal exposure to LPS led to up-regulated expression of nicotinamide adenine dinucleotide phosphate (NADPH) oxidases, antioxidant enzymes, and p38 MAPK activity in left ventricular of adult offspring at resting state. ISO treatment exaggerated ROS generation, p38 MAPK activation but down-regulated reactive oxygen species (ROS) elimination capacity in the left ventricular of offspring from LPS-treated mothers, while antioxidant N-acetyl-L-cysteine (NAC) reversed these changes together with improved cardiac functions. The p38 inhibitor SB202190 alleviated the heart damage only via inhibiting the expression of NADPH oxidases. Collectively, our data demonstrated that prenatal inflammation programs pre-existed ROS activation in the heart tissue, which switches on the early process of oxidative damages on heart rapidly through a ROS-p38 MAPK-NADPH oxidase-ROS positive feedback loop in response to a myocardial hypertrophic challenge in adulthood. PMID:27443826

  11. OXIDATIVE STRESS PARTICIPATES IN ACUTE LUNG INJURY AND ACTIVATION OF MITOGEN ACTIVATED PROTEIN KINASES (MAPK) FOLLOWING AIR POLLUTION PARTICLE EXPOSURE (PM)

    EPA Science Inventory

    OXIDATIVE STRESS PARTICIPATES IN ACUTE LUNG INJURY AND ACTIVATION OF MITOGEN ACTIVATED PROTEIN KINASES (MAPK) FOLLOWING AIR POLLUTION PARTICLE EXPOSURE (PM). E S Roberts1, R Jaskot2, J Richards2, and K L Dreher2. 1College of Veterinary Medicine, NC State University, Raleigh, NC a...

  12. MAPK pathway activation by chronic lead-exposure increases vascular reactivity through oxidative stress/cyclooxygenase-2-dependent pathways

    SciTech Connect

    Simões, Maylla Ronacher; Aguado, Andrea; Fiorim, Jonaína; Silveira, Edna Aparecida; Azevedo, Bruna Fernandes; Toscano, Cindy Medice; Zhenyukh, Olha; Briones, Ana María; Alonso, María Jesús; Vassallo, Dalton Valentim; Salaices, Mercedes

    2015-03-01

    Chronic exposure to low lead concentration produces hypertension; however, the underlying mechanisms remain unclear. We analyzed the role of oxidative stress, cyclooxygenase-2-dependent pathways and MAPK in the vascular alterations induced by chronic lead exposure. Aortas from lead-treated Wistar rats (1st dose: 10 μg/100 g; subsequent doses: 0.125 μg/100 g, intramuscular, 30 days) and cultured aortic vascular smooth muscle cells (VSMCs) from Sprague Dawley rats stimulated with lead (20 μg/dL) were used. Lead blood levels of treated rats attained 21.7 ± 2.38 μg/dL. Lead exposure increased systolic blood pressure and aortic ring contractile response to phenylephrine, reduced acetylcholine-induced relaxation and did not affect sodium nitroprusside relaxation. Endothelium removal and L-NAME left-shifted the response to phenylephrine more in untreated than in lead-treated rats. Apocynin and indomethacin decreased more the response to phenylephrine in treated than in untreated rats. Aortic protein expression of gp91(phox), Cu/Zn-SOD, Mn-SOD and COX-2 increased after lead exposure. In cultured VSMCs lead 1) increased superoxide anion production, NADPH oxidase activity and gene and/or protein levels of NOX-1, NOX-4, Mn-SOD, EC-SOD and COX-2 and 2) activated ERK1/2 and p38 MAPK. Both antioxidants and COX-2 inhibitors normalized superoxide anion production, NADPH oxidase activity and mRNA levels of NOX-1, NOX-4 and COX-2. Blockade of the ERK1/2 and p38 signaling pathways abolished lead-induced NOX-1, NOX-4 and COX-2 expression. Results show that lead activation of the MAPK signaling pathways activates inflammatory proteins such as NADPH oxidase and COX-2, suggesting a reciprocal interplay and contribution to vascular dysfunction as an underlying mechanisms for lead-induced hypertension. - Highlights: • Lead-exposure increases oxidative stress, COX-2 expression and vascular reactivity. • Lead exposure activates MAPK signaling pathway. • ROS and COX-2 activation by

  13. Arsenite suppression of involucrin transcription through AP1 promoter sites in cultured human keratinocytes

    SciTech Connect

    Sinitsyna, Nadezda N.; Reznikova, Tatiana V.; Qin Qin; Song, Hyukhwan; Phillips, Marjorie A.; Rice, Robert H.

    2010-03-15

    While preserving keratinocyte proliferative ability, arsenite suppresses cellular differentiation markers by preventing utilization of AP1 transcriptional response elements. In present experiments, arsenite had a dramatic effect in electrophoretic mobility supershift analysis of proteins binding to an involucrin promoter AP1 response element. Without arsenite treatment, binding of JunB and Fra1 was readily detected in nuclear extracts from preconfluent cultures and was not detected a week after confluence, while c-Fos was detected only after confluence. By contrast, band shift of nuclear extracts from arsenite treated cultures showed only JunB and Fra1 binding in postconfluent as well as preconfluent cultures. Immunoblotting of cell extracts showed that arsenite treatment prevented the loss of Fra1 and the increase in c-Fos proteins that occurred after confluence in untreated cultures. Chromatin immunoprecipitation assays demonstrated substantial reduction of c-Fos and acetylated histone H3 at the proximal and distal AP1 response elements in the involucrin promoter and of coactivator p300 at the proximal element. Alteration of AP1 transcription factors was also examined in response to treatment with four metal containing compounds (chromate, vanadate, hemin, divalent cadmium) that also suppress involucrin transcription. These agents all influenced transcription at AP1 elements in a transcriptional reporter assay, but exhibited less effect than arsenite on binding activity assessed by mobility shift and chromatin immunoprecipitation and displayed variable effects on AP1 protein levels. These findings help trace a mechanism by which transcriptional effects of arsenite become manifest and help rationalize the unique action of arsenite, compared to the other agents, to preserve proliferative ability.

  14. Thrombosis recanalization by paeoniflorin through the upregulation of urokinase-type plasminogen activator via the MAPK signaling pathway

    PubMed Central

    YE, SONGSHAN; MAO, BINGYU; YANG, LEI; FU, WEIYUN; HOU, JUNRAN

    2016-01-01

    Paeoniflorin, the major component of Paeonia lactiflora pall, has previously been reported to prevent thrombosis. Plasminogen activator urokinase (uPA) is a serine protease that markedly facilitates normal thrombosis resolution. Paeoniflorin and uPA have been linked to the mitogen-activated protein kinase (MAPK) signaling pathway. In the current study, the influence of paeoniflorin on the expression of uPA was investigated and the underlying regulatory mechanism was preliminarily determined. The prothrombotic state of the model animals treated with paeoniflorin were assessed by enzyme-linked immunosorbent assay (ELISA). Additionally, the cytotoxicity of paeoniflorin on human umbilical vein endothelial cell (HUVEC) cultures was estimated using a methyl thiazolyl tetrazolium assay and the possible pathways involved in the interaction between paeoniflorin and uPA were evaluated using western blot analysis. The ELISA results demonstrated that the levels of 6-keto prostaglandin F1a, fibronectin and uPA were significantly upregulated by treatment with paeoniflorin compared with control (P<0.05). By contrast, the expression of fibrinogen, D-dimer and thromboxane B2 were inhibited. With an increase in the concentration of paeoniflorin the cell viability of HUVECs decreased gradually. The results of western blot analysis demonstrated that paeoniflorin increased the phosphorylation of MAPK 14 (p38) and MAPK 8 (JNK). The present study demonstrated that paeoniflorin has the potential to improve the prethrombotic state and recanalize thrombosis by increasing the expression of uPA, which may be mediated via regulation of the p38 and JNK MAPK signaling pathways. However, this treatment effect was dependent on the concentration of paeoniflorin used, an unsuitable concentration of the agent would result in a negative effect on the anti-thrombosis pathways. PMID:27082639

  15. Induction of Connective Tissue Growth Factor Expression by Hypoxia in Human Lung Fibroblasts via the MEKK1/MEK1/ERK1/GLI-1/GLI-2 and AP-1 Pathways

    PubMed Central

    Cheng, Yi; Lin, Chien-huang; Chen, Jing-Yun; Li, Chien-Hua; Liu, Yu-Tin; Chen, Bing-Chang

    2016-01-01

    Several reports have indicated that hypoxia, GLI, and connective tissue growth factor (CTGF) contribute to pulmonary fibrosis in idiopathic pulmonary fibrosis. We investigated the participation of mitogen-activated protein kinase kinase (MEK) kinase 1 (MEKK1)/MEK1/ERK1/GLI-1/2 and activator protein-1 (AP-1) signaling in hypoxia-induced CTGF expression in human lung fibroblasts. Hypoxia time-dependently increased CTGF expression, which was attenuated by the small interfering RNA (siRNA) of GLI-1 (GLI-1 siRNA) and GLI-2 (GLI-2 siRNA) in both human lung fibroblast cell line (WI-38) and primary human lung fibroblasts (NHLFs). Moreover, GLI-1 siRNA and GLI-2 siRNA attenuated hypoxia-induced CTGF-luciferase activity, and the treatment of cells with hypoxia induced GLI-1 and GLI-2 translocation. Furthermore, hypoxia-induced CTGF expression was reduced by an MEK inhibitor (PD98059), MEK1 siRNA, ERK inhibitor (U0126), ERK1 siRNA, and MEKK1 siRNA. Both PD98059 and U0126 significantly attenuated hypoxia-induced CTGF-luciferase activity. Hypoxia time-dependently increased MEKK1, ERK, and p38 MAPK phosphorylation. Moreover, SB203580 (a p38 MAPK inhibitor) also apparently inhibited hypoxia-induced CTGF expression. The treatment of cells with hypoxia induced ERK, GLI-1, or GLI-2 complex formation. Hypoxia-induced GLI-1 and GLI-2 translocation into the nucleus was significantly attenuated by U0126. In addition, hypoxia-induced ERK Tyr204 phosphorylation was impeded by MEKK1 siRNA. Moreover, hypoxia-induced CTGF-luciferase activity was attenuated by cells transfected with AP-1 site mutation in a CTGF construct. Exposure to hypoxia caused a time-dependent phosphorylation of c-Jun, but not of c-Fos. Chromatin immunoprecipitation (ChIP) revealed that hypoxia induced the recruitment of c-Jun, GLI-1, and GLI-2 to the AP-1 promoter region of CTGF. Hypoxia-treated cells exhibited an increase in α-smooth muscle actin (α-SMA) and collagen production, which was blocked by GLI-1 siRNA and

  16. Upstream mitogen-activated protein kinase (MAPK) pathway inhibition: MEK inhibitor followed by a BRAF inhibitor in advanced melanoma patients.

    PubMed

    Goldinger, Simone M; Zimmer, Lisa; Schulz, Carsten; Ugurel, Selma; Hoeller, Christoph; Kaehler, Katharina C; Schadendorf, Dirk; Hassel, Jessica C; Becker, Juergen; Hauschild, Axel; Dummer, Reinhard

    2014-01-01

    BRAF-mutant melanoma can be successfully treated by BRAF kinase inhibitors (BRAFi) and MEK kinase inhibitors (MEKi). However, the administration of BRAFi followed by MEKi did not generate promising response rate (RR). The purpose of this investigation was to evaluate the time to progression (TTP) with a mitogen-activated protein kinase (MAPK) pathway upstream inhibition strategy in BRAF mutated melanoma patients. BRAF mutation positive metastatic melanoma patients were identified within the Dermatology Cooperative Oncology Group (DeCOG) network and were treated first with a MEKi and upon progression with a selective BRAFi. A total of 23 melanoma patients (six females, 17 males, aged 47-80 years) were retrospectively analysed for TTP. The total median TTP was 8.9 months. The median TTP for MEKi was 4.8 (1.2-23.2) and subsequent for BRAFi 4.5 (1.2-15.7) months, respectively. A higher RR for MEKi (39%, nine partial responses and 0 complete responses) than previously reported was observed. Our analysis suggests that the reversed inhibition of the MAPK pathway is feasible in BRAF mutated melanoma. The median TTP (8.9 months) is close to the promising BRAF- and MEKi combination therapy (median progression-free survival (PFS) 9.4 months). The total treatment duration of the MAPK inhibition when a MEKi is administered first is similar compared to the reversed sequence, but TTP shifts in favour to the MEKi. This approach is feasible with reasonable tolerability. This clinical investigation encourages further studies in prospective clinical trials to define the optimal treatment schedule for the MAPK pathway inhibition and should be accompanied by molecular monitoring using repeated biopsies. PMID:24183461

  17. Activation of MAPK Is Required for ROS Generation and Exocytosis in HMC-1 Cells Induced by Trichomonas vaginalis-Derived Secretory Products

    PubMed Central

    Narantsogt, Giimaa; Min, Arim; Nam, Young Hee; Lee, Young Ah; Kim, Kyeong Ah; Agvaandaram, Gurbadam; Dorjsuren, Temuulen; El-Benna, Jamel; Shin, Myeong Heon

    2015-01-01

    Trichomonas vaginalis is a flagellated protozoan parasite that causes vaginitis and cervicitis in women and asymptomatic urethritis and prostatitis in men. Mast cells have been reported to be predominant in vaginal smears and vaginal walls of patients infected with T. vaginalis. Mitogen-activated protein kinase (MAPK), activated by various stimuli, have been shown to regulate the transcriptional activity of various cytokine genes in mast cells. In this study, we investigated whether MAPK is involved in ROS generation and exocytotic degranulation in HMC-1 cells induced by T. vaginalis-derived secretory products (TvSP). We found that TvSP induces the activation of MAPK and NADPH oxidase in HMC-1 cells. Stimulation with TvSP induced phosphorylation of MAPK and p47phox in HMC-1 cells. Stimulation with TvSP also induced up-regulation of CD63, a marker for exocytosis, along the surfaces of human mast cells. Pretreatment with MAPK inhibitors strongly inhibited TvSP-induced ROS generation and exocytotic degranulation. Finally, our results suggest that TvSP induces intracellular ROS generation and exocytotic degranulation in HMC-1 via MAPK signaling. PMID:26537039

  18. Hypertonicity-induced p38MAPK activation elicits recovery of corneal epithelial cell volume and layer integrity.

    PubMed

    Bildin, V N; Wang, Z; Iserovich, P; Reinach, P S

    2003-05-01

    In hypertonicity-stressed (i.e., 600 mOsm) SV40-immortalized rabbit and human corneal epithelial cell layers (RCEC and HCEC, respectively), we characterized the relationship between time-dependent changes in translayer resistance, relative cell volume and modulation of MAPK superfamily activities. Sulforhodamine B permeability initially increased by 1.4- and 2-fold in RCEC and HCEC, respectively. Subsequently, recovery to its isotonic level only occurred in RCEC. Light scattering revealed that in RCEC 1) regulatory volume increase (RVI) extent was 20% greater; 2) RVI half-time was 2.5-fold shorter. However, inhibition of Na-K-2Cl cotransporter and Na/K-ATPase activity suppressed the RVI response more in HCEC. MAPK activity changes were as follows: 1) p38 was wave-like and faster as well as larger in RCEC than in HCEC (90- and 18-fold, respectively); 2) increases in SAPK/JNK activity were negligible in comparison to those of p38; 3) Erk1/2 activity declined to 30-40% of their basal values. SB203580, a specific p38 inhibitor, dose dependently suppressed the RVI responses in both cell lines. However, neither U0126, which inhibits MEK, the kinase upstream of Erk, nor SP600125, inhibitor of SAPK/JNK, had any effect on this response. Taken together, sufficient activation of the p38 limb of the MAPK superfamily during a hypertonic challenge is essential for maintaining epithelial cell volume and translayer resistance. On the other hand, Erk1/2 activity restoration seems to be dependent on cell volume recovery. PMID:12879161

  19. Plant MAPK cascades: Just rapid signaling modules?

    PubMed Central

    Boudsocq, Marie; Danquah, Agyemang; de Zélicourt, Axel; Hirt, Heribert; Colcombet, Jean

    2015-01-01

    Abscisic acid (ABA) is a major phytohormone mediating important stress-related processes. We recently unveiled an ABA-activated MAPK signaling module constituted of MAP3K17/18-MKK3-MPK1/2/7/14. Unlike classical rapid MAPK activation, we showed that the activation of the new MAPK module is delayed and relies on the MAP3K protein synthesis. In this addendum, we discuss the role of this original and unexpected activation mechanism of MAPK cascades which suggests that MAPKs can regulate both early and long-term plant stress responses. PMID:26313321

  20. Transcriptional regulation of endothelial nitric oxide synthase expression in uterine artery endothelial cells by c-Jun/AP-1

    PubMed Central

    Qian, Xiao-Xian; Mata-Greenwood, Eugenia; Liao, Wu Xiang; Zhang, Honghai; Zheng, Jing; Chen, Dong-bao

    2007-01-01

    Despite extensive studies have shown that increased endothelial nitric oxide synthase (NOS3) expression in the uterine artery endothelial cells (UAEC) plays a key role in uterine vasodilatation, the molecular mechanism controlling NOS3 expression in UAEC is unknown. According to the sheep NOS3 promoter sequence isolated in our laboratory, we hypothesize that the activator protein-1 (AP-1) site in the proximal sheep NOS3 promoter (TGAGTCA, -682 to -676) is important for NOS3 expression. We developed a c-Jun adenoviral expression system to overexpress c-Jun protein into UAEC to investigate the effects of c-Jun/AP-1 on NOS3 expression. Basal levels of c-Jun protein and mRNA were detected in UAEC. C-Jun protein was overexpressed in a concentration and time-dependent fashion in UAEC infected with sense c-Jun (S-c-Jun), but not sham and antisense c-Jun (A-c-Jun) adenoviruses. Infection with S-c-Jun adenovirus (25 MOI, multiplicity of infection) resulted in efficient c-Jun protein overexpression in UAEC up to 3 days. In S-c-Jun, but not sham and A-c-Jun adenovirus infected UAEC, NOS3 mRNA and protein levels were increased (P<0.05) compared to noninfected controls. Increased NOS3 expression was associated with increased total NOS activity. Transient transfections showed that c-Jun overexpression augmented the transactivation of the sheep NOS3 promoter-driven luciferase/reporter constructs with the AP-1 site but not of deletion constructs without the AP-1 site. When the AP-1 site was mutated, c-Jun failed to trans-activate the sheep NOS3 promoter. AP-1 DNA binding activity also increased in c-Jun overexpressed UAEC. Lastly, the pharmacological AP-1 activator phorbol myristate acetate increased AP-1 binding, trans-activated the wild-type but not the AP-1 mutant NOS3 promoter and dose-dependently stimulated UAEC NOS3 and c-Jun protein expression. Hence, our data show that c-Jun/AP-1 regulates NOS3 transcription involving the proximal AP-1 site in the 5′-regulatory region of

  1. Transcriptomic analysis by RNA-seq reveals AP-1 pathway as key regulator that green tea may rely on to inhibit lung tumorigenesis.

    PubMed

    Pan, Jing; Zhang, Qi; Xiong, Donghai; Vedell, Peter; Yan, Ying; Jiang, Hui; Cui, Peng; Ding, Feng; Tichelaar, Jay W; Wang, Yian; Lubet, Ronald A; You, Ming

    2014-01-01

    Green tea is a promising chemopreventive agent for lung cancer. Multiple signaling events have been reported, however, the relative importance of these mechanisms in mediating the chemopreventive function of green tea is unclear. In the present study, to examine the involvement of AP-1 in green tea polyphenols induced tumor inhibition, human NSCLC cell line H1299 and mouse SPON 10 cells were identified as AP-1 dependent, as these two lines exhibit high constitutive AP-1 activity, and when TAM67 expression was induced with doxycycline, cell growth was inhibited and correlated with suppressed AP-1 activity. RNA-seq was used to determine the global transcriptional effects of AP-1 inhibition and also uncover the possible involvement of AP-1 in tea polyphenols induced chemoprevention. TAM67 mediated changes in gene expression were identified, and within down-regulated genes, AP-1 was identified as a key transcription regulator. RNA-seq analysis revealed that Polyphenon E-treated cells shared 293 commonly down-regulated genes within TAM67 expressing H1299 cells, and by analysis of limited Chip-seq data, over 10% of the down-regulated genes contain a direct AP-1 binding site, indicating that Polyphenon E elicits chemopreventive activity by regulating AP-1 target genes. Conditional TAM67 expressing transgenic mice and NSCLC cell lines were used to further confirm that the chemopreventive activity of green tea is AP-1 dependent. Polyphenon E lost its chempreventive function both in vitro and in vivo when AP-1 was inhibited, indicating that AP-1 inhibition is a major pathway through which green tea exhibits chemopreventive effects. PMID:24343902

  2. Butyrate produced by commensal bacteria potentiates phorbol esters induced AP-1 response in human intestinal epithelial cells.

    PubMed

    Nepelska, Malgorzata; Cultrone, Antonietta; Béguet-Crespel, Fabienne; Le Roux, Karine; Doré, Joël; Arulampalam, Vermulugesan; Blottière, Hervé M

    2012-01-01

    The human intestine is a balanced ecosystem well suited for bacterial survival, colonization and growth, which has evolved to be beneficial both for the host and the commensal bacteria. Here, we investigated the effect of bacterial metabolites produced by commensal bacteria on AP-1 signaling pathway, which has a plethora of effects on host physiology. Using intestinal epithelial cell lines, HT-29 and Caco-2, stably transfected with AP-1-dependent luciferase reporter gene, we tested the effect of culture supernatant from 49 commensal strains. We observed that several bacteria were able to activate the AP-1 pathway and this was correlated to the amount of short chain fatty acids (SCFAs) produced. Besides being a major source of energy for epithelial cells, SCFAs have been shown to regulate several signaling pathways in these cells. We show that propionate and butyrate are potent activators of the AP-1 pathway, butyrate being the more efficient of the two. We also observed a strong synergistic activation of AP-1 pathway when using butyrate with PMA, a PKC activator. Moreover, butyrate enhanced the PMA-induced expression of c-fos and ERK1/2 phosphorylation, but not p38 and JNK. In conclusion, we showed that SCFAs especially butyrate regulate the AP-1 signaling pathway, a feature that may contribute to the physiological impact of the gut microbiota on the host. Our results provide support for the involvement of butyrate in modulating the action of PKC in colon cancer cells. PMID:23300800

  3. Differential Effects of E2 on MAPK Activity in the Brain and Heart of Aged Female Rats

    PubMed Central

    Shults, Cody L.; Rao, Yathindar S.; Pak, Toni R.

    2016-01-01

    Aging and the coincident loss of circulating estrogens at menopause lead to increased risks for neurological and cardiovascular pathologies. Clinical studies show that estrogen therapy (ET) can be beneficial in mitigating these negative effects, in both the brain and heart, when it is initiated shortly after the perimenopausal transition. However, this same therapy is detrimental when initiated >10 years postmenopause. Importantly, the molecular mechanisms underlying this age-related switch in ET efficacy are unknown. Estrogen receptors (ERs) mediate the neuroprotective and cardioprotective functions of estrogens by modulating gene transcription or, non-genomically, by activating second messenger signaling pathways, such as mitogen activated protein kinases (MAPK). These kinases are critical regulators of cell signaling pathways and have widespread downstream effects. Our hypothesis is that age and estrogen deprivation following menopause alters the expression and activation of the MAPK family members p38 and ERK in the brain and heart. To test this hypothesis, we used a surgically induced model of menopause in 18 month old rats through bilateral ovariectomy (OVX) followed by an acute dose of 17β-estradiol (E2) administered at varying time points post-OVX (1 week, 4 weeks, 8 weeks, or 12 weeks). Age and E2 treatment differentially regulated kinase activity in both the brain and heart, and the effects were also brain region specific. MAPK signaling plays an integral role in aging, and the aberrant regulation of those signaling pathways might be involved in age-related disorders. Clinical studies show benefits of ET during early menopause but detrimental effects later, which might be reflective of changes in kinase expression and activation status. PMID:27487271

  4. Kappa Opioid Receptor Activation of p38 MAPK Is GRK3- and Arrestin-dependent in Neurons and Astrocytes*

    PubMed Central

    Bruchas, Michael R.; Macey, Tara A.; Lowe, Janet D.; Chavkin, Charles

    2007-01-01

    AtT-20 cells expressing the wild-type kappa opioid receptor (KOR) increased phospho-p38 MAPK following treatment with the kappa agonist U50,488. The increase was blocked by the kappa antagonist norbinaltorphimine and not evident in untransfected cells. In contrast, U50,488 treatment of AtT-20 cells expressing KOR having alanine substituted for serine-369 (KSA) did not increase phospho-p38. Phosphorylation of serine 369 in the KOR carboxyl terminus by G-protein receptor kinase 3 (GRK3) was previously shown to be required for receptor desensitization, and the results suggest that p38 MAPK activation by KOR may require arrestin recruitment. This hypothesis was tested by transfecting arrestin3-(R170E), a dominant positive form of arrestin that does not require receptor phosphorylation for activation. AtT-20 cells expressing both KSA and arrestin3-(R170E) responded to U50,488 treatment with an increase in phospho-p38 consistent with the hypothesis. Primary cultured astrocytes (glial fibrillary acidic protein-positive) and neurons (γ-aminobutyric acid-positive) isolated from mouse striata also responded to U50,488 by increasing phospho-p38 immunolabeling. p38 activation was not evident in either striatal astrocytes or neurons isolated from KOR knock-out mice or GRK3 knock-out mice. Astrocytes pretreated with small interfering RNA for arrestin3 were also unable to activate p38 in response to U50,488 treatment. Furthermore, in striatal neurons, the kappa-mediated phospho-p38 labeling was colocalized with arrestin3. These findings suggest that KOR may activate p38 MAPK in brain by a GRK3 and arrestin-dependent mechanism. PMID:16648139

  5. Oxidized High-Density Lipoprotein Impairs Endothelial Progenitor Cells' Function by Activation of CD36-MAPK-TSP-1 Pathways

    PubMed Central

    Wu, Jianxiang; He, Zhiqing; Gao, Xiang; Wu, Feng; Ding, Ru; Ren, Yusheng; Jiang, Qijun; Fan, Min

    2015-01-01

    Abstract Aims: High-density lipoprotein (HDL) levels inversely correlate with cardiovascular events due to the protective effects on vascular wall and stem cells, which are susceptible to oxidative modifications and then lead to potential pro-atherosclerotic effects. We proposed that oxidized HDL (ox-HDL) might lead to endothelial progenitor cells (EPCs) dysfunction and investigated underlying mechanisms. Results: ox-HDL was shown to increase apoptosis and intracellular reactive oxygen species levels, but to reduce migration, angiogenesis, and cholesterol efflux of EPCs in a dose-dependent manner. p38 mitogen-activated protein kinase (MAPK) and NF-κB were activated after ox-HDL stimulation, which also upregulated thrombospondin-1 (TSP-1) expression without affecting vascular endothelial growth factor. Effects caused by ox-HDL could be significantly attenuated by pretreatment with short hairpin RNA-mediated CD36 knockdown or probucol. Data of in vivo experiments and the inverse correlation of ox-HDL and circulating EPC numbers among patients with coronary artery diseases (CAD) or CAD and type 2 diabetes also supported it. Meanwhile, HDL separated from such patients could significantly increase cultured EPC's caspase 3 activity, further supporting our proposal. Innovation: This is the most complete study to date of how ox-HDL would impair EPCs function, which was involved with activation of CD36-p38 MAPK-TSP-1 pathways and proved by not only the inverse relationship between ox-HDL and circulating EPCs in clinic but also pro-apoptotic effects of HDL separated from patients' serum. Conclusion: Activation of CD36-p38 MAPK-TSP-1 pathways contributes to the pathological effects of ox-HDL on EPCs' dysfunction, which might be one of the potential etiological factors responsible for the disturbed neovascularization in chronic ischemic disease. Antioxid. Redox Signal. 22, 308–324. PMID:25313537

  6. Modulation of mitogen-activated protein kinases (MAPK) activity in response to different immune stimuli in haemocytes of the common periwinkle Littorina littorea.

    PubMed

    Iakovleva, Nadya V; Gorbushin, Alexander M; Storey, Kenneth B

    2006-09-01

    The modulation of mitogen-activated protein kinase (MAPK) activity in haemocytes of the common periwinkle (Littorina littorea) in response to immune challenges by lipopolysaccharide from Echerichia coli (LPS), mannan from baker's yeast Saccharomyces cerevisiae and secretory-excretory products (SEP) of trematodes Himasthla elongata (Echinostomatidae) or after the treatment with phorbol ester (PMA) has been studied by Western blotting using affinity purified rabbit polyclonal antibodies. Exposure of the cells in suspension to PMA, LPS and mannan triggered an activation of p38 and ERK2. The JNK-mediated cascade was modulated differently by the elicitors examined. PMA treatment caused a transient activation of the JNK54 isoform, LPS exposure resulted in a decrease in activity of JNK46, and mannan had no effect on JNK phosphorylation status. Incubation of periwinkle haemocytes in culture medium containing trematode SEP did not affect the activity of any MAPK. PMID:16533608

  7. Transcription Factor AP1 Potentiates Chromatin Accessibility and Glucocorticoid Receptor Binding

    PubMed Central

    Biddie, Simon C.; John, Sam; Sabo, Pete J.; Thurman, Robert E.; Johnson, Thomas A.; Schiltz, R. Louis; Miranda, Tina B.; Sung, Myong-Hee; Trump, Saskia; Lightman, Stafford L.; Vinson, Charles; Stamatoyannopoulos, John A.; Hager, Gordon L.

    2011-01-01

    Summary Ligand-dependent transcription by the nuclear receptor glucocorticoid receptor (GR) is mediated by interactions with co-regulators. The role of these interactions in determining selective binding of GR to regulatory elements remains unclear. Recent findings indicate a large fraction of genomic GR binding coincides with chromatin that is accessible prior to hormone treatment, suggesting that receptor binding is dictated by proteins that maintain chromatin in an open state. Combining DNaseI accessibility and chromatin immunoprecipitation with high-throughput sequencing, we identify the activator protein 1 (AP1) as a major partner for productive GR-chromatin interactions. AP1 is critical for GR-regulated transcription and recruitment to co-occupied regulatory elements, illustrating an extensive AP1-GR interaction network. Importantly, the maintenance of baseline chromatin accessibility facilitates GR recruitment and is dependent on AP1 binding. We propose a model where the basal occupancy of transcription factors act to prime chromatin and direct inducible transcription factors to select regions in the genome. PMID:21726817

  8. Terminal deoxynucleotidyl transferase is down-regulated by AP-1-like regulatory elements in human lymphoid cells

    PubMed Central

    Peralta-Zaragoza, Oscar; Recillas-Targa, Félix; Madrid-Marina, Vicente

    2004-01-01

    Terminal deoxynucleotidyl transferase (TdT) is a template-independent DNA polymerase that catalyses the incorporation of deoxyribonucleotides into the 3′-hydroxyl end of DNA templates and is thought to increase junctional diversity of antigen receptor genes. TdT is expressed only on immature lymphocytes and acute lymphoblastic leukaemia cells and its transcriptional expression is tightly regulated. We had previously found that protein kinase C (PKC) activation down-regulates TdT expression. PKC-activation induces the synthesis of the Fos and Jun proteins, known as the major components of activation protein 1 (AP-1) transcriptional factor implicated in transcriptional control. Here we report the identification of several DNA–protein interactions within the TdT promoter region in non-TdT expressing human cells. Sequence analysis revealed the presence of a putative AP-1-like DNA-binding site, suggesting that AP-1 may play a relevant role in TdT transcriptional regulation. Using a different source of nuclear extracts and the AP-1–TdT motif as a probe we identified several DNA-protein retarded complexes in electrophoretic mobility shift assays. Super-band shifting analysis using an antibody against c-Jun protein confirmed that the main interaction is produced by a nuclear factor that belongs to the AP-1 family transcription factors. Our findings suggest that the TdT gene expression is down-regulated, at least in part, through AP-1-like transcription factors. PMID:15027905

  9. Terminal deoxynucleotidyl transferase is down-regulated by AP-1-like regulatory elements in human lymphoid cells.

    PubMed

    Peralta-Zaragoza, Oscar; Recillas-Targa, Félix; Madrid-Marina, Vicente

    2004-02-01

    Terminal deoxynucleotidyl transferase (TdT) is a template-independent DNA polymerase that catalyses the incorporation of deoxyribonucleotides into the 3'-hydroxyl end of DNA templates and is thought to increase junctional diversity of antigen receptor genes. TdT is expressed only on immature lymphocytes and acute lymphoblastic leukaemia cells and its transcriptional expression is tightly regulated. We had previously found that protein kinase C (PKC) activation down-regulates TdT expression. PKC-activation induces the synthesis of the Fos and Jun proteins, known as the major components of activation protein 1 (AP-1) transcriptional factor implicated in transcriptional control. Here we report the identification of several DNA-protein interactions within the TdT promoter region in non-TdT expressing human cells. Sequence analysis revealed the presence of a putative AP-1-like DNA-binding site, suggesting that AP-1 may play a relevant role in TdT transcriptional regulation. Using a different source of nuclear extracts and the AP-1-TdT motif as a probe we identified several DNA-protein retarded complexes in electrophoretic mobility shift assays. Super-band shifting analysis using an antibody against c-Jun protein confirmed that the main interaction is produced by a nuclear factor that belongs to the AP-1 family transcription factors. Our findings suggest that the TdT gene expression is down-regulated, at least in part, through AP-1-like transcription factors. PMID:15027905

  10. Opposing Effects of Zac1 and Curcumin on AP-1-Regulated Expressions of S100A7

    PubMed Central

    Chu, Yu-Wen; Liu, Shu-Ting; Cheng, Hsiao-Chun; Huang, Shih-Ming; Chang, Yung-Lung; Chiang, Chien-Ping; Liu, Ying-Chun; Wang, Wei-Ming

    2015-01-01

    ZAC, an encoding gene mapped at chromosome 6q24-q25 within PSORS1, was previously found over-expressed in the lower compartment of the hyperplastic epidermis in psoriatic lesions. Cytokines produced in the inflammatory dermatoses may drive AP-1 transcription factor to induce responsive gene expressions. We demonstrated that mZac1 can enhance AP-1-responsive S100A7 expression of which the encoding gene was located in PSORS4 with HaCaT keratinocytes. However, the mZac1-enhanced AP-1 transcriptional activity was suppressed by curcumin, indicating the anti-inflammatory property of this botanical agent and is exhibited by blocking the AP-1-mediated cross-talk between PSORS1 and PSORS4. Two putative AP-1-binding sites were found and demonstrated to be functionally important in the regulation of S100A7 promoter activity. Moreover, we found curcumin reduced the DNA-binding activity of AP-1 to the recognition element located in the S100A7 promoter. The S100A7 expression was found to be upregulated in the lesioned epidermis of atopic dermatitis and psoriasis, which is where this keratinocyte-derived chemoattractant engaged in the pro-inflammatory feedback loop. Understanding the regulatory mechanism of S100A7 expression will be helpful to develop therapeutic strategies for chronic inflammatory dermatoses via blocking the reciprocal stimuli between the inflammatory cells and keratinocytes. PMID:26633653

  11. Overexpression of members of the AP-1 transcriptional factor family from an early stage of renal carcinogenesis and inhibition of cell growth by AP-1 gene antisense oligonucleotides in the Tsc2 gene mutant (Eker) rat model.

    PubMed

    Urakami, S; Tsuchiya, H; Orimoto, K; Kobayashi, T; Igawa, M; Hino, O

    1997-12-01

    We previously isolated subtracted cDNA clones for genes having increased expression in Tsc2 gene mutant (Eker) rat renal carcinomas (RCs). Among them, fra-1 encoding a transcriptional factor activator protein 1 (AP-1) was identified. We have therefore investigated whether other members of the AP-1 transcription factor family might also be involved in renal carcinogenesis in the Eker rat model. In the present study, overexpression of fra-1, fra-2, c-jun, junB, and junD mRNAs was demonstrated in RCs by Northern blot analysis. Interestingly, AP-1 proteins were highly expressed even in the earliest preneoplastic lesions (e.g., phenotypically altered tubules) as suggested by immunohistochemistry. Moreover, 12-O-tetradecanoylphorbol-13-acetate-responsive element (TRE)-binding activity of AP-1 proteins was observed in RC cell extracts by electrophoretic mobility shift assay. As a next step, we transfected antisense oligonucleotides targeting AP-1 genes into RC cells and demonstrated that their growth was strongly inhibited. Thus, the data suggest that overexpression of AP-1 genes might play a crucial role in renal carcinogenesis in the Eker rat model. PMID:9405228

  12. 17β-estradiol rapidly activates calcium release from intracellular stores via the GPR30 pathway and MAPK phosphorylation in osteocyte-like MLO-Y4 cells.

    PubMed

    Ren, Jian; Wu, Jun Hua

    2012-05-01

    Estrogen regulates critical cellular functions, and its deficiency initiates bone turnover and the development of bone mass loss in menopausal females. Recent studies have demonstrated that 17β-estradiol (E(2)) induces rapid non-genomic responses that activate downstream signaling molecules, thus providing a new perspective to understand the relationship between estrogen and bone metabolism. In this study, we investigated rapid estrogen responses, including calcium release and MAPK phosphorylation, in osteocyte-like MLO-Y4 cells. E(2) elevated [Ca(2+)]( i ) and increased Ca(2+) oscillation frequency in a dose-dependent manner. Immunolabeling confirmed the expression of three estrogen receptors (ERα, ERβ, and G protein-coupled receptor 30 [GPR30]) in MLO-Y4 cells and localized GPR30 predominantly to the plasma membrane. E(2) mobilized calcium from intracellular stores, and the use of selective agonist(s) for each ER showed that this was mediated mainly through the GPR30 pathway. MAPK phosphorylation increased in a biphasic manner, with peaks occurring after 7 and 60 min. GPR30 and classical ERs showed different temporal effects on MAPK phosphorylation and contributed to MAPK phosphorylation sequentially. ICI182,780 inhibited E(2) activation of MAPK at 7 min, while the GPR30 agonist G-1 and antagonist G-15 failed to affect MAPK phosphorylation levels. G-1-mediated MAPK phosphorylation at 60 min was prevented by prior depletion of calcium stores. Our data suggest that E(2) induces the non-genomic responses Ca(2+) release and MAPK phosphorylation to regulate osteocyte function and indicate that multiple receptors mediate rapid E(2) responses. PMID:22392527

  13. Polycyclic aromatic hydrocarbon (PAH)-mediated upregulation of hepatic microRNA-181 family promotes cancer cell migration by targeting MAPK phosphatase-5, regulating the activation of p38 MAPK

    SciTech Connect

    Song, Mi-Kyung; Park, Yong-Keun; Ryu, Jae-Chun

    2013-11-15

    Growing evidence indicates that changes in microRNA (miRNA) expression in cancer induced by chemical carcinogens play an important role in cancer development and progression by regulating related genes. However, the mechanisms underlying miRNA involvement in hepatocarcinogenesis induced by polycyclic aromatic hydrocarbons (PAHs) remain unclear. Thus, the identification of aberrant miRNA expression during PAH-induced cancer cell migration will lead to a better understanding of the substantial role of miRNAs in cancer progression. In the present study, miRNA expression profiling showed significant upregulation of miR-181a, -181b, and -181d in human hepatocellular carcinoma cells (HepG2 line) exposed to benzo[a]anthracene (BA) and benzo[k]fluoranthene (BF). MAPK phosphatase-5 (MKP-5), a validated miR-181 target that deactivates MAPKs, was markedly suppressed while phosphorylation of p38 MAPK was increased after BA and BF exposure. The migration of HepG2 cells, observed using the scratch wound-healing assay, also increased in a dose-dependent manner. Depletion of miR-181 family members by miRNA inhibitors enhanced the expression of MKP-5 and suppressed the phosphorylation of p38 MAPK. Furthermore, the depletion of the miR-181 family inhibited cancer cell migration. Based on these results, we conclude that the miR-181 family plays a critical role in PAH-induced hepatocarcinogenesis by targeting MKP-5, resulting in the regulation of p38 MAPK activation. - Highlights: • We found significant upregulation of miR-181 family in HCC exposed to BA and BF. • We identified the MKP-5 as a putative target of miR-181 family. • MKP-5 was suppressed while p-P38 was increased after BA and BF exposure. • The migration of HepG2 cells increased in a dose-dependent manner.

  14. MAPK Cascades in Guard Cell Signal Transduction

    PubMed Central

    Lee, Yuree; Kim, Yun Ju; Kim, Myung-Hee; Kwak, June M.

    2016-01-01

    Guard cells form stomata on the epidermis and continuously respond to endogenous and environmental stimuli to fine-tune the gas exchange and transpirational water loss, processes which involve mitogen-activated protein kinase (MAPK) cascades. MAPKs form three-tiered kinase cascades with MAPK kinases and MAPK kinase kinases, by which signals are transduced to the target proteins. MAPK cascade genes are highly conserved in all eukaryotes, and they play crucial roles in myriad developmental and physiological processes. MAPK cascades function during biotic and abiotic stress responses by linking extracellular signals received by receptors to cytosolic events and gene expression. In this review, we highlight recent findings and insights into MAPK-mediated guard cell signaling, including the specificity of MAPK cascades and the remaining questions. PMID:26904052

  15. MAPK Cascades in Guard Cell Signal Transduction.

    PubMed

    Lee, Yuree; Kim, Yun Ju; Kim, Myung-Hee; Kwak, June M

    2016-01-01

    Guard cells form stomata on the epidermis and continuously respond to endogenous and environmental stimuli to fine-tune the gas exchange and transpirational water loss, processes which involve mitogen-activated protein kinase (MAPK) cascades. MAPKs form three-tiered kinase cascades with MAPK kinases and MAPK kinase kinases, by which signals are transduced to the target proteins. MAPK cascade genes are highly conserved in all eukaryotes, and they play crucial roles in myriad developmental and physiological processes. MAPK cascades function during biotic and abiotic stress responses by linking extracellular signals received by receptors to cytosolic events and gene expression. In this review, we highlight recent findings and insights into MAPK-mediated guard cell signaling, including the specificity of MAPK cascades and the remaining questions. PMID:26904052

  16. KSR is a scaffold required for activation of the ERK/MAPK module

    PubMed Central

    Roy, François; Laberge, Gino; Douziech, Mélanie; Ferland-McCollough, David; Therrien, Marc

    2002-01-01

    Mechanisms that regulate signal propagation through the ERK/MAPK pathway are still poorly understood. Several proteins are suspected to play critical roles in this process. One of these is Kinase Suppressor of Ras (KSR), a component previously identified in RAS-dependent genetic screens in Drosophila and Caenorhabditis elegans. Here, we show that KSR functions upstream of MEK within the ERK/MAPK module. In agreement with this, we found that KSR facilitates the phosphorylation of MEK by RAF. We further show that KSR associates independently with RAF and MEK, and that these interactions lead to the formation of a RAF/MEK complex, thereby positioning RAF in close proximity to its substrate MEK. These findings suggest that KSR functions as a scaffold that assembles the RAF/MEK functional pair. PMID:11850406

  17. Anti-cancer effect of snake venom toxin through down regulation of AP-1 mediated PRDX6 expression

    PubMed Central

    Son, Dong Ju; Song, Ho Sub; Kim, Jung Hyun; Ko, Seong Cheol; Song, Min Jong; Lee, Won Hyoung; Yoon, Joo Hee; Ham, Young Wan; Han, Sang Bae; Hong, Jin Tae

    2015-01-01

    Snake venom toxin (SVT) from Vipera lebetina turanica contains a mixture of different enzymes and proteins. Peroxiredoxin 6 (PRDX6) is known to be a stimulator of lung cancer cell growth. PRDX6 is a member of peroxidases, and has calcium-independent phospholipase A2 (iPLA2) activities. PRDX6 has an AP-1 binding site in its promoter region of the gene. Since AP-1 is implicated in tumor growth and PRDX6 expression, in the present study, we investigated whether SVT inhibits PRDX6, thereby preventing human lung cancer cell growth (A549 and NCI-H460) through inactivation of AP-1. A docking model study and pull down assay showed that SVT completely fits on the basic leucine zipper (bZIP) region of c-Fos of AP-1. SVT (0–10 μg/ml) inhibited lung cancer cell growth in a concentration dependent manner through induction of apoptotic cell death accompanied by induction of cleaved caspase-3, -8, -9, Bax, p21 and p53, but decreased cIAP and Bcl2 expression via inactivation of AP-1. In an xenograft in vivo model, SVT (0.5 mg/kg and 1 mg/kg) also inhibited tumor growth accompanied with the reduction of PRDX6 expression, but increased expression of proapoptotic proteins. These data indicate that SVT inhibits tumor growth via inhibition of PRDX6 activity through interaction with its transcription factor AP-1. PMID:26061816

  18. Selectivity of docking sites in MAPK kinases.

    PubMed

    Bardwell, A Jane; Frankson, Erlynn; Bardwell, Lee

    2009-05-01

    Protein kinases often recognize their substrates and regulators through docking interactions that occur outside of the active site; these interactions can help us to understand kinase networks, and to target kinases with drugs. During mitogen-activated protein kinase (MAPK) signaling, the ability of MAPK kinases (MKKs, or MEKs) to recognize their cognate MAPKs is facilitated by a short docking motif (the D-site) in the MKK N terminus, which binds to a complementary region on the MAPK. MAPKs then recognize many of their targets using the same strategy, because many MAPK substrates also contain D-sites. The extent to which docking contributes to the specificity of MAPK transactions is incompletely understood. Here we characterize the selectivity of the interaction between MKK-derived D-sites and MAPKs by measuring the ability of D-site peptides to inhibit MAPK-mediated phosphorylation of D-site-containing substrates. We find that all MKK D-sites bind better to their cognate MAPKs than they do to non-cognate MAPKs. For instance, the MKK3 D-site peptide, which is a remarkably potent inhibitor of p38alpha (IC(50) < 10 nm), does not inhibit JNK1 or JNK2. Likewise, MAPKs generally bind as well or better to cognate D-sites than to non-cognate D-sites. For instance, JNK1 and JNK2 do not appreciably bind to any D-sites other than their cognate D-sites from MKK4 and MKK7. In general, cognate, within-pathway interactions are preferred about an order of magnitude over non-cognate interactions. However, the selectivity of MAPKs and their cognate MKK-derived D-sites for each other is limited in some cases; in particular, ERK2 is not very selective. We conclude that MAPK-docking sites in MAPK kinases bind selectively to their cognate MAPKs. PMID:19196711

  19. Requirement of ERα and basal activities of EGFR and Src kinase in Cd-induced activation of MAPK/ERK pathway in human breast cancer MCF-7 cells

    SciTech Connect

    Song, Xiulong Wei, Zhengxi; Shaikh, Zahir A.

    2015-08-15

    Cadmium (Cd) is a common environmental toxicant and an established carcinogen. Epidemiological studies implicate Cd with human breast cancer. Low micromolar concentrations of Cd promote proliferation of human breast cancer cells in vitro. The growth promotion of breast cancer cells is associated with the activation of MAPK/ERK pathway. This study explores the mechanism of Cd-induced activation of MAPK/ERK pathway. Specifically, the role of cell surface receptors ERα, EGFR, and Src kinase was evaluated in human breast cancer MCF-7 cells treated with 1–3 μM Cd. The activation of ERK was studied using a serum response element (SRE) luciferase reporter assay. Receptor phosphorylation was detected by Western blot analyses. Cd treatment increased both the SRE reporter activity and ERK1/2 phosphorylation in a concentration-dependent manner. Cd treatment had no effect on reactive oxygen species (ROS) generation. Also, blocking the entry of Cd into the cells with manganese did not diminish Cd-induced activation of MAPK/ERK. These results suggest that the effect of Cd was likely not caused by intracellular ROS generation, but through interaction with the membrane receptors. While Cd did not appear to activate either EGFR or Src kinase, their inhibition completely blocked the Cd-induced activation of ERK as well as cell proliferation. Similarly, silencing ERα with siRNA or use of ERα antagonist blocked the effects of Cd. Based on these results, it is concluded that not only ERα, but also basal activities of EGFR and Src kinase are essential for Cd-induced signal transduction and activation of MAPK/ERK pathway for breast cancer cell proliferation. - Highlights: • Low micromolar concentrations of Cd rapidly activate ERK1/2 in MCF-7 cells. • Signal transduction and resulting cell proliferation require EGFR, ERα, and Src. • These findings implicate Cd in promotion of breast cancer.

  20. An essential role for the intra-oocyte MAPK activity in the NSN-to-SN transition of germinal vesicle chromatin configuration in porcine oocytes.

    PubMed

    Sun, Ming-Ju; Zhu, Shuai; Li, You-Wei; Lin, Juan; Gong, Shuai; Jiao, Guang-Zhong; Chen, Fei; Tan, Jing-He

    2016-01-01

    The mechanisms for the transition from non-surrounded nucleolus (NSN) to surrounded nucleolus (SN) chromatin configuration during oocyte growth/maturation are unclear. By manipulating enzyme activities and measuring important molecules using small-follicle pig oocytes with a high proportion of NSN configuration and an extended germinal vesicle stage in vitro, this study has the first time up-to-date established the essential role for intra-oocyte mitogen-activated protein kinase (MAPK) in the NSN-to-SN transition. Within the oocyte in 1-2 mm follicles, a cAMP decline activates MAPK, which prevents the NSN-to-SN transition by activating nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) while inhibiting histone deacetylase (HDAC). In cumulus cells of 1-2 mm follicles, a lower level of estradiol and oocyte-derived paracrine factor (ODPF) reduces natriuretic peptide receptor 2 (NPR2) while enhancing FSH and cAMP actions. FSH elevates cAMP levels, which decreases NPR2 while activating MAPK. MAPK closes the gap junctions, which, together with the NPR2 decrease, reduces cyclic guanosine monophosphate (cGMP) delivery leading to the cAMP decline within oocytes. In 3-6 mm follicles, a higher level of estradiol and ODPF and a FSH shortage initiate a reversion of the above events leading to MAPK inactivation and NSN-to-SN transition within oocytes. PMID:27009903

  1. An essential role for the intra-oocyte MAPK activity in the NSN-to-SN transition of germinal vesicle chromatin configuration in porcine oocytes

    PubMed Central

    Sun, Ming-Ju; Zhu, Shuai; Li, You-Wei; Lin, Juan; Gong, Shuai; Jiao, Guang-Zhong; Chen, Fei; Tan, Jing-He

    2016-01-01

    The mechanisms for the transition from non-surrounded nucleolus (NSN) to surrounded nucleolus (SN) chromatin configuration during oocyte growth/maturation are unclear. By manipulating enzyme activities and measuring important molecules using small-follicle pig oocytes with a high proportion of NSN configuration and an extended germinal vesicle stage in vitro, this study has the first time up-to-date established the essential role for intra-oocyte mitogen-activated protein kinase (MAPK) in the NSN-to-SN transition. Within the oocyte in 1–2 mm follicles, a cAMP decline activates MAPK, which prevents the NSN-to-SN transition by activating nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) while inhibiting histone deacetylase (HDAC). In cumulus cells of 1–2 mm follicles, a lower level of estradiol and oocyte-derived paracrine factor (ODPF) reduces natriuretic peptide receptor 2 (NPR2) while enhancing FSH and cAMP actions. FSH elevates cAMP levels, which decreases NPR2 while activating MAPK. MAPK closes the gap junctions, which, together with the NPR2 decrease, reduces cyclic guanosine monophosphate (cGMP) delivery leading to the cAMP decline within oocytes. In 3–6 mm follicles, a higher level of estradiol and ODPF and a FSH shortage initiate a reversion of the above events leading to MAPK inactivation and NSN-to-SN transition within oocytes. PMID:27009903

  2. Ceftiofur impairs pro-inflammatory cytokine secretion through the inhibition of the activation of NF-{kappa}B and MAPK

    SciTech Connect

    Ci Xinxin; Song Yu; Zeng Fanqin; Zhang Xuemei; Li Hongyu; Wang Xinrui; Cui Junqing Deng Xuming

    2008-07-18

    Ceftiofur is a new broad-spectrum, third-generation cephalosporin antibiotic for veterinary use. Immunopharmacological studies can provide new information on the immunomodulatory activities of some drugs, including their effect on cytokine productions. For this reason, we investigated the effect of ceftiofur on cytokine productions in vitro. We found that ceftiofur can downregulate tumor necrosis factor-{alpha} (TNF-{alpha}), interleukin-1{beta} (IL-1{beta}), and interleukin-6 (IL-6), but did not affect interleukin-10 (IL-10) production. We further investigated signal transduction mechanisms to determine how ceftiofur affects. RAW 264.7 cells were pretreated with 1, 5, or 10 mg/L of ceftiofur 1 h prior to treatment with 1 mg/L of LPS. Thirty minutes later, cells were harvested and mitogen activated protein kinases (MAPKs) activation was measured by Western blot. Alternatively, cells were fixed and nuclear factor-{kappa}B (NF-{kappa}B) activation was measured using immunocytochemical analysis. Signal transduction studies showed that ceftiofur significantly inhibited extracellular signal-regulated kinase (ERK), p38, and c-jun NH{sub 2}-terminal kinase (JNK) phosphorylation protein expression. Ceftiofur also inhibited p65-NF-{kappa}B translocation into the nucleus. Therefore, ceftiofur may inhibit LPS-induced production of inflammatory cytokines by blocking NF-{kappa}B and MAPKs signaling in RAW264.7 cells.

  3. GITRL modulates the activities of p38 MAPK and STAT3 to promote Th17 cell differentiation in autoimmune arthritis

    PubMed Central

    Ma, Jie; Wang, Jiemin; Qi, Chen; Rui, Ke; Wang, Yungang; Xu, Huaxi; Lu, Liwei; Wang, Shengjun

    2016-01-01

    The glucocorticoid-induced TNFR family-related protein (GITR) and its ligand play a critical role in the pathogenesis of autoimmune arthritis by enhancing the Th17 cell response, but their molecular mechanisms remain largely unclear. This study aims to define the role of p38 mitogen-activated protein kinases (MAPK) and signal transducer and activator of transcription 3 (STAT3) signaling in GITRL-induced Th17 cells in autoimmune arthritis. We found that the p38 phosphorylation was enhanced by GITRL in activated CD4+T cells, and the p38 inhibitor restrained the GITRL-induced Th17 cell expansion in a dose-dependent manner. Moreover, there was decreased STAT3 activity on Tyr705 and Ser727 with the p38 inhibitor in vitro. Notably, the p38 inhibitor could prevent GITRL-treated arthritis progression and markedly decrease the Th17 cell percentages. The phosphorylation of the Tyr705 site was significantly lower in the GITRL-treated CIA mice administrated with the p38 inhibitor. A significantly higher phosphorylation of p38 was detected in RA patients and had a positive relationship with the serum level of anti-cyclic citrullinated peptide (anti-CCP) antibody. Our findings have indicated that GITRL could promote Th17 cell differentiation by p38 MAPK and STAT3 signaling in autoimmune arthritis. PMID:26657118

  4. REG Iα activates c-Jun through MAPK pathways to enhance the radiosensitivity of squamous esophageal cancer cells.

    PubMed

    Wakita, Akiyuki; Motoyama, Satoru; Sato, Yusuke; Koyota, Souichi; Usami, Shuetsu; Yoshino, Kei; Sasaki, Tomohiko; Imai, Kazuhiro; Saito, Hajime; Minamiya, Yoshihiro

    2015-07-01

    Identification of the key molecules that mediate susceptibility to anticancer treatments would be highly desirable. Based on clinical and cell biological studies, we recently proposed that regenerating gene (REG) Iα may be such a molecule. In the present study, we hypothesized that REG Iα increases radiosensitivity through activation of mitogen-activated protein kinase (MAPK) pathways. To test that idea, we transfected TE-5 and TE-9 squamous esophageal cancer cells with REG Iα and examined its involvement in MAPK signaling and its effect on susceptibility to radiotherapy. We found that REG Iα-expressing cells showed increased expression of c-Jun messenger RNA (mRNA) and phospho-c-Jun protein mediated via the c-Jun N-terminal kinase (JNK) pathway and extracellular signal-regulated kinase (ERK) pathway, as well as increased radiosensitivity. Immunohistochemical analysis confirmed the activation of c-Jun in tumors expressing REG Iα. Collectively, these findings suggest that REG Iα activates c-Jun via the JNK and ERK pathway, thereby enhancing radiosensitivity. PMID:25656613

  5. P4-ATPase Requirement for AP-1/Clathrin Function in Protein Transport from the trans-Golgi Network and Early Endosomes

    PubMed Central

    Liu, Ke; Surendhran, Kavitha; Nothwehr, Steven F.

    2008-01-01

    Drs2p is a resident type 4 P-type ATPase (P4-ATPase) and potential phospholipid translocase of the trans-Golgi network (TGN) where it has been implicated in clathrin function. However, precise protein transport pathways requiring Drs2p and how it contributes to clathrin-coated vesicle budding remain unclear. Here we show a functional codependence between Drs2p and the AP-1 clathrin adaptor in protein sorting at the TGN and early endosomes of Saccharomyces cerevisiae. Genetic criteria indicate that Drs2p and AP-1 operate in the same pathway and that AP-1 requires Drs2p for function. In addition, we show that loss of AP-1 markedly increases Drs2p trafficking to the plasma membrane, but does not perturb retrieval of Drs2p from the early endosome back to the TGN. Thus AP-1 is required at the TGN to sort Drs2p out of the exocytic pathway, presumably for delivery to the early endosome. Moreover, a conditional allele that inactivates Drs2p phospholipid translocase (flippase) activity disrupts its own transport in this AP-1 pathway. Drs2p physically interacts with AP-1; however, AP-1 and clathrin are both recruited normally to the TGN in drs2Δ cells. These results imply that Drs2p acts independently of coat recruitment to facilitate AP-1/clathrin-coated vesicle budding from the TGN. PMID:18508916

  6. The mitogen-activated protein kinase (MAPK) cascade controls phosphatase and tensin homolog (PTEN) expression through multiple mechanisms.

    PubMed

    Ciuffreda, Ludovica; Di Sanza, Cristina; Cesta Incani, Ursula; Eramo, Adriana; Desideri, Marianna; Biagioni, Francesca; Passeri, Daniela; Falcone, Italia; Sette, Giovanni; Bergamo, Paola; Anichini, Andrea; Sabapathy, Kanaga; McCubrey, James A; Ricciardi, Maria Rosaria; Tafuri, Agostino; Blandino, Giovanni; Orlandi, Augusto; De Maria, Ruggero; Cognetti, Francesco; Del Bufalo, Donatella; Milella, Michele

    2012-06-01

    The mitogen-activated protein kinase (MAPK) and PI3K pathways are regulated by extensive crosstalk, occurring at different levels. In tumors, transactivation of the alternate pathway is a frequent "escape" mechanism, suggesting that combined inhibition of both pathways may achieve synergistic antitumor activity. Here we show that, in the M14 melanoma model, simultaneous inhibition of both MEK and mammalian target of rapamycin (mTOR) achieves synergistic effects at suboptimal concentrations, but becomes frankly antagonistic in the presence of relatively high concentrations of MEK inhibitors. This observation led to the identification of a novel crosstalk mechanism, by which either pharmacologic or genetic inhibition of constitutive MEK signaling restores phosphatase and tensin homolog (PTEN) expression, both in vitro and in vivo, and inhibits downstream signaling through AKT and mTOR, thus bypassing the need for double pathway blockade. This appears to be a general regulatory mechanism and is mediated by multiple mechanisms, such as MAPK-dependent c-Jun and miR-25 regulation. Finally, PTEN upregulation appears to be a major effector of MEK inhibitors' antitumor activity, as cancer cells in which PTEN is inactivated are consistently more resistant to the growth inhibitory and anti-angiogenic effects of MEK blockade. PMID:22215152

  7. Tetrahydrocurcumin induces G2/M cell cycle arrest and apoptosis involving p38 MAPK activation in human breast cancer cells.

    PubMed

    Kang, Ning; Wang, Miao-Miao; Wang, Ying-Hui; Zhang, Zhe-Nan; Cao, Hong-Rui; Lv, Yuan-Hao; Yang, Yang; Fan, Peng-Hui; Qiu, Feng; Gao, Xiu-Mei

    2014-05-01

    Curcumin (CUR) is a major naturally-occurring polyphenol of Curcuma species, which is commonly used as a yellow coloring and flavoring agent in foods. In recent years, it has been reported that CUR exhibits significant anti-tumor activity in vivo. However, the pharmacokinetic features of CUR have indicated poor oral bioavailability, which may be related to its extensive metabolism. The CUR metabolites might be responsible for the antitumor pharmacological effects in vivo. Tetrahydrocurcumin (THC) is one of the major metabolites of CUR. In the present study, we examined the efficacy and associated mechanism of action of THC in human breast cancer MCF-7 cells for the first time. Here, THC exhibited significant cell growth inhibition by inducing MCF-7 cells to undergo mitochondrial apoptosis and G2/M arrest. Moreover, co-treatment of MCF-7 cells with THC and p38 MAPK inhibitor, SB203580, effectively reversed the dissipation in mitochondrial membrane potential (Δψm), and blocked THC-mediated Bax up-regulation, Bcl-2 down-regulation, caspase-3 activation as well as p21 up-regulation, suggesting p38 MAPK might mediate THC-induced apoptosis and G2/M arrest. Taken together, these results indicate THC might be an active antitumor form of CUR in vivo, and it might be selected as a potentially effective agent for treatment of human breast cancer. PMID:24593988

  8. Lapatinib increases motility of triple-negative breast cancer cells by decreasing miRNA-7 and inducing Raf-1/MAPK-dependent interleukin-6.

    PubMed

    Hsiao, Yu-Chun; Yeh, Ming-Hsin; Chen, Yun-Ju; Liu, Ju-Fang; Tang, Chih-Hsin; Huang, Wei-Chien

    2015-11-10

    Lapatinib, a dual epidermal growth factor receptor (EGFR) and HER2 tyrosine kinase inhibitor (TKI), has been approved for HER2-positive breast cancer patients. Nevertheless, its inhibitory effect on EGFR did not deliver clinical benefits for triple-negative breast cancer (TNBC) patients even EGFR overexpression was frequently found in this disease. Moreover, lapatinib was unexpectedly found to enhance metastasis of TNBC cells, but the underlying mechanisms are not fully understood. In this study, we explored that the level of interleukin-6 (IL-6) was elevated in lapatinib-treated TNBC cells. Treatment with IL-6 antibody abolished the lapatinib-induced migration. Mechanistically, the signaling axis of Raf-1/mitogen-activated protein kinases (MAPKs), c-Jun N-terminal kinases (JNKs), p38 MAPK, and activator protein 1 (AP-1) was activated in response to lapatinib treatment to induce IL-6 expression. Furthermore, our data showed that microRNA-7 directly binds and inhibits Raf-1 3'UTR activity, and that down-regulation of miR-7 by lapatinib contributes to the activation of Raf-1 signaling pathway and the induction of IL-6 expression. Our results not only revealed IL-6 as a key regulator of lapatinib-induced metastasis, but also explored the requirement of miR7/Raf-1/MAPK/AP-1 axis in lapatinib-induced IL-6 expression. PMID:26513016

  9. Lapatinib increases motility of triple-negative breast cancer cells by decreasing miRNA-7 and inducing Raf-1/MAPK-dependent interleukin-6

    PubMed Central

    Chen, Yun-Ju; Liu, Ju-Fang; Tang, Chih-Hsin; Huang, Wei-Chien

    2015-01-01

    Lapatinib, a dual epidermal growth factor receptor (EGFR) and HER2 tyrosine kinase inhibitor (TKI), has been approved for HER2-positive breast cancer patients. Nevertheless, its inhibitory effect on EGFR did not deliver clinical benefits for triple-negative breast cancer (TNBC) patients even EGFR overexpression was frequently found in this disease. Moreover, lapatinib was unexpectedly found to enhance metastasis of TNBC cells, but the underlying mechanisms are not fully understood. In this study, we explored that the level of interleukin-6 (IL-6) was elevated in lapatinib-treated TNBC cells. Treatment with IL-6 antibody abolished the lapatinib-induced migration. Mechanistically, the signaling axis of Raf-1/mitogen-activated protein kinases (MAPKs), c-Jun N-terminal kinases (JNKs), p38 MAPK, and activator protein 1 (AP-1) was activated in response to lapatinib treatment to induce IL-6 expression. Furthermore, our data showed that microRNA-7 directly binds and inhibits Raf-1 3′UTR activity, and that down-regulation of miR-7 by lapatinib contributes to the activation of Raf-1 signaling pathway and the induction of IL-6 expression. Our results not only revealed IL-6 as a key regulator of lapatinib-induced metastasis, but also explored the requirement of miR7/Raf-1/MAPK/AP-1 axis in lapatinib-induced IL-6 expression. PMID:26513016

  10. Radiolabeled anti-tissue factor antibody (AP-1) for imaging thrombotic disease by PET

    SciTech Connect

    Joshi, V.; Meinken, G.; Srivastava, S.

    1995-05-01

    The objective of this study was to develop and test radioimmunoconjugates of AP-1, an anti-tissue factor (TF) MAb, for PET imaging of vessel wall injury or associated thrombotic disease. Recently, anti rabbit MAb AP-1 was shown to prevent thrombosis following vascular injury in a rabbit model. In the represent study of AP-1 was conjugated with the conventional DTPA dianhydride (DTPA-DA) and with 4-isothiocyanato-cyclohexyl-EDTA (4-ICE) (2 to 2.5 ligands per MAb). Labeling with {sup 57}Co was done by adding {sup 57}CoCl{sub 2} in 0.1 N HCl to 500 {mu}g of conjugate in 0.1 M NaHCO{sub 3} containing 0.12 M acetate. The reaction mixture (pH {approximately} 5.5) was allowed to stand at room temperature for 8 h, and then purified by size exclusion HPLC following EDTA chase (10 {mu}l of 0.1 M EDTA, pH 7.0, 10 min). Labeling efficiencies were >90%. When incubated with mouse serum these conjugates showed similar stability ({approximately}3% activity loss for 4-ICE vs 6% for DTPA-DA at 24 h). The inhibition of tissue factor procoagulant activity was determined for the {sup 67}Co labeled conjugates using a two stage clotting assay. In the first stage, clotting times were determined using serial dilutions of reconstituted TF standards to check linearity. In the second stage, clotting times were determined for {sup 67}Co labeled AP-1 conjugates at various dilutions (1 ng to 1 {mu}g/mL) in presence of 150 ng/ml of TF. Results were compared with those obtained using unlabeled conjugates and the native AP-1. Neither conjugation with chelators nor radiolabelling affected the TF activity of AP-1. These conjugates labeled with {sup 66}Co (t l/1 17.5 h, {beta}{sup +} emission) should prove effective for PET imaging of vessel wall injury or thrombotic disease in our previously established rabbit model. Based on our previous data with other MAbs, the 4-ICE conjugate is expected to provide better biodistribution.

  11. IFN-τ Alleviates Lipopolysaccharide-Induced Inflammation by Suppressing NF-κB and MAPKs Pathway Activation in Mice.

    PubMed

    Wu, Haichong; Zhao, Gan; Jiang, Kangfeng; Chen, Xiuying; Rui, Guangze; Qiu, Changwei; Guo, Mengyao; Deng, Ganzhen

    2016-06-01

    IFN-τ, which is a type I interferon with low cytotoxicity, is defined as a pregnancy recognition signal in ruminants. Type I interferons have been used as anti-inflammatory agents, but their side effects limit their clinical application. The present study aimed to determine the anti-inflammatory effects of IFN-τ in a lipopolysaccharide-stimulated acute lung injury (ALI) model and in RAW264.7 cells and to confirm the mechanism of action involved. The methods used included histopathology, measuring the lung wet/dry ratio, determining the myeloperoxidase activity, ELISA, qPCR, and western blot. The results revealed that IFN-τ greatly ameliorated the infiltration of inflammatory cells and the expression of TNF-α, IL-1β, and IL-6. Further analysis revealed that IFN-τ down-regulated the expression of TLR-2 and TLR-4 mRNA and the activity of the NF-κB and MAPK pathways both in a lipopolysaccharide-induced ALI model and in RAW264.7 cells. The results demonstrated that IFN-τ suppressed the levels of pro-inflammatory cytokines by inhibiting the phosphorylation of the NF-κB and MAPK pathways. Thus, IFN-τ may be an optimal target for the treatment of inflammatory diseases. PMID:27052630

  12. Registered report: RAF inhibitors prime wild-type RAF to activate the MAPK pathway and enhance growth

    PubMed Central

    Bhargava, Ajay; Pelech, Steven; Woodard, Ben; Kerwin, John; Maherali, Nimet

    2016-01-01

    The Reproducibility Project: Cancer Biology seeks to address growing concerns about reproducibility in scientific research by conducting replications of selected experiments from a number of high-profile papers in the field of cancer biology. The papers, which were published between 2010 and 2012, were selected on the basis of citations and Altmetric scores (Errington et al., 2014). This Registered Report describes the proposed replication plan of key experiments from 'RAF inhibitors prime wild-type RAF to activate the MAPK pathway and enhance growth' by Hatzivassiliou and colleagues, published in Nature in 2010 (Hatzivassiliou et al., 2010). Hatzivassiliou and colleagues examined the paradoxical response of RAF-WT tumors to treatment with RAF inhibitors. The key experiments being replicated include Figure 1A, in which the original authors demonstrated that treatment of a subset of BRAFWT tumor cell lines with RAF small molecule inhibitors resulted in an increase in cell viability, Figure 2B, which reported that RAF inhibitor activation of the MAPK pathway was dependent on CRAF but not BRAF, and Figure 4A, where the dimerization of BRAF and CRAF was modulated by the RAF inhibitor PLX4720, but not GDC-0879. The Reproducibility Project: Cancer Biology is a collaboration between the Center for Open Science and Science Exchange, and the results of the replications will be published by eLife. DOI: http://dx.doi.org/10.7554/eLife.09976.001 PMID:26882073

  13. Titanium dioxide nanoparticles stimulate sea urchin immune cell phagocytic activity involving TLR/p38 MAPK-mediated signalling pathway

    PubMed Central

    Pinsino, Annalisa; Russo, Roberta; Bonaventura, Rosa; Brunelli, Andrea; Marcomini, Antonio; Matranga, Valeria

    2015-01-01

    Titanium dioxide nanoparticles (TiO2NPs) are one of the most widespread-engineered particles in use for drug delivery, cosmetics, and electronics. However, TiO2NP safety is still an open issue, even for ethical reasons. In this work, we investigated the sea urchin Paracentrotus lividus immune cell model as a proxy to humans, to elucidate a potential pathway that can be involved in the persistent TiO2NP-immune cell interaction in vivo. Morphology, phagocytic ability, changes in activation/inactivation of a few mitogen-activated protein kinases (p38 MAPK, ERK), variations of other key proteins triggering immune response (Toll-like receptor 4-like, Heat shock protein 70, Interleukin-6) and modifications in the expression of related immune response genes were investigated. Our findings indicate that TiO2NPs influence the signal transduction downstream targets of p38 MAPK without eliciting an inflammatory response or other harmful effects on biological functions. We strongly recommend sea urchin immune cells as a new powerful model for nano-safety/nano-toxicity investigations without the ethical normative issue. PMID:26412401

  14. ANKRD1 acts as a transcriptional repressor of MMP13 via the AP-1 site.

    PubMed

    Almodóvar-García, Karinna; Kwon, Minjae; Samaras, Susan E; Davidson, Jeffrey M

    2014-04-01

    The transcriptional cofactor ANKRD1 is sharply induced during wound repair, and its overexpression enhances healing. We recently found that global deletion of murine Ankrd1 impairs wound contraction and enhances necrosis of ischemic wounds. A quantitative PCR array of Ankrd1(-/-) (KO) fibroblasts indicated that ANKRD1 regulates MMP genes. Yeast two-hybrid and coimmunoprecipitation analyses associated ANKRD1 with nucleolin, which represses AP-1 activation of MMP13. Ankrd1 deletion enhanced both basal and phorbol 12-myristate 13-acetate (PMA)-induced MMP13 promoter activity; conversely, Ankrd1 overexpression in control cells decreased PMA-induced MMP13 promoter activity. Ankrd1 reconstitution in KO fibroblasts decreased MMP13 mRNA, while Ankrd1 knockdown increased these levels. MMP13 mRNA and protein were elevated in intact skin and wounds of KO versus Ankrd1(fl/fl) (FLOX) mice. Electrophoretic mobility shift assay gel shift patterns suggested that additional transcription factors bind to the MMP13 AP-1 site in the absence of Ankrd1, and this concept was reinforced by chromatin immunoprecipitation analysis as greater binding of c-Jun to the AP-1 site in extracts from FLOX versus KO fibroblasts. We propose that ANKRD1, in association with factors such as nucleolin, represses MMP13 transcription. Ankrd1 deletion additionally relieved MMP10 transcriptional repression. Nuclear ANKRD1 appears to modulate extracellular matrix remodeling by MMPs. PMID:24515436

  15. ANKRD1 Acts as a Transcriptional Repressor of MMP13 via the AP-1 Site

    PubMed Central

    Almodóvar-García, Karinna; Kwon, Minjae; Samaras, Susan E.

    2014-01-01

    The transcriptional cofactor ANKRD1 is sharply induced during wound repair, and its overexpression enhances healing. We recently found that global deletion of murine Ankrd1 impairs wound contraction and enhances necrosis of ischemic wounds. A quantitative PCR array of Ankrd1−/− (KO) fibroblasts indicated that ANKRD1 regulates MMP genes. Yeast two-hybrid and coimmunoprecipitation analyses associated ANKRD1 with nucleolin, which represses AP-1 activation of MMP13. Ankrd1 deletion enhanced both basal and phorbol 12-myristate 13-acetate (PMA)-induced MMP13 promoter activity; conversely, Ankrd1 overexpression in control cells decreased PMA-induced MMP13 promoter activity. Ankrd1 reconstitution in KO fibroblasts decreased MMP13 mRNA, while Ankrd1 knockdown increased these levels. MMP13 mRNA and protein were elevated in intact skin and wounds of KO versus Ankrd1fl/fl (FLOX) mice. Electrophoretic mobility shift assay gel shift patterns suggested that additional transcription factors bind to the MMP13 AP-1 site in the absence of Ankrd1, and this concept was reinforced by chromatin immunoprecipitation analysis as greater binding of c-Jun to the AP-1 site in extracts from FLOX versus KO fibroblasts. We propose that ANKRD1, in association with factors such as nucleolin, represses MMP13 transcription. Ankrd1 deletion additionally relieved MMP10 transcriptional repression. Nuclear ANKRD1 appears to modulate extracellular matrix remodeling by MMPs. PMID:24515436

  16. Pterostilbene Is Equally Potent as Resveratrol in Inhibiting 12-O-tetradecanoylphorbol-13-acetate Activated NFkappaB, AP-1, COX-2 and iNOS in Mouse Epidermis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Resveratrol, a phytoalexin present in grapes, has been reported to inhibit multistage mouse skin carcinogenesis. Recent studies showed that topically applied resveratrol significantly inhibited cyclooxygenase-2 (COX-2) expression and activation of nuclear factor-kB (NF-kB) induced by tumor promoter...

  17. The Chromone Alkaloid, Rohitukine, Affords Anti-Cancer Activity via Modulating Apoptosis Pathways in A549 Cell Line and Yeast Mitogen Activated Protein Kinase (MAPK) Pathway

    PubMed Central

    Safia; Kamil, Mohd; Jadiya, Pooja; Sheikh, Saba; Haque, Ejazul; Nazir, Aamir; Lakshmi, Vijai; Mir, Snober S.

    2015-01-01

    The field of cancer research and treatment has made significant progress, yet we are far from having completely safe, efficient and specific therapies that target cancer cells and spare the healthy tissues. Natural compounds may reduce the problems related to cancer treatment. Currently, many plant products are being used to treat cancer. In this study, Rohitukine, a natural occurring chromone alkaloid extracted from Dysoxylum binectariferum, was investigated for cytotoxic properties against budding yeast as well as against lung cancer (A549) cells. We endeavored to specifically study Rohitukine in S. cerevisiae in the context of MAPK pathways as yeast probably represents the experimental model where the organization and regulation of MAPK pathways are best understood. MAPK are evolutionarily conserved protein kinases that transfer extracellular signals to the machinery controlling essential cellular processes like growth, migration, differentiation, cell division and apoptosis. We aimed at carrying out hypothesis driven studies towards targeting the important network of cellular communication, a critical process that gets awry in cancer. Employing mutant strains of genetic model system Saccharomyces cerevisiae. S. cerevisiae encodes five MAPKs involved in control of distinct cellular responses such as growth, differentiation, migration and apoptosis. Our study involves gene knockouts of Slt2 and Hog1 which are functional homologs of human ERK5 and mammalian p38 MAPK, respectively. We performed cytotoxicity assay to evaluate the effect of Rohitukine on cell viability and also determined the effects of drug on generation of reactive oxygen species, induction of apoptosis and expression of Slt2 and Hog1 gene at mRNA level in the presence of drug. The results of this study show a differential effect in the activity of drug between the WT, Slt2 and Hog1 gene deletion strain indicating involvement of MAPK pathway. Further, we investigated Rohitukine induced cytotoxic

  18. Regulation of steatohepatitis and PPARγ signaling by distinct AP-1 dimers

    PubMed Central

    Hasenfuss, Sebastian C.; Bakiri, Latifa; Thomsen, Martin K.; Williams, Evan G.; Auwerx, Johan; Wagner, Erwin F.

    2014-01-01

    Summary Non-alcoholic fatty liver disease (NAFLD) affects up to 30% of the adult population in Western societies, yet the underlying molecular pathways remain poorly understood. Here, we identify the dimeric Activator Protein 1 as a regulator of NAFLD. The Fos-related antigen 1 (Fra-1) and 2 (Fra-2) prevent dietary NAFLD by inhibiting pro-steatotic PPARγ signaling. Moreover, established NAFLD and the associated liver damage can be efficiently reversed by hepatocyte-specific Fra-1 expression. In contrast, c-Fos promotes PPARγ expression, while c-Jun exerts opposing, dimer-dependent functions. Interestingly, JunD was found to be essential for PPARγ signaling and NAFLD development. This unique antagonistic regulation of PPARγ by distinct AP-1 dimers occurs at the transcriptional level and establishes AP-1 as a link between obesity, hepatic lipid metabolism and NAFLD. PMID:24411941

  19. Regulation of steatohepatitis and PPARγ signaling by distinct AP-1 dimers.

    PubMed

    Hasenfuss, Sebastian C; Bakiri, Latifa; Thomsen, Martin K; Williams, Evan G; Auwerx, Johan; Wagner, Erwin F

    2014-01-01

    Nonalcoholic fatty liver disease (NAFLD) affects up to 30% of the adult population in Western societies, yet the underlying molecular pathways remain poorly understood. Here, we identify the dimeric Activator Protein 1 as a regulator of NAFLD. Fos-related antigen 1 (Fra-1) and Fos-related antigen 2 (Fra-2) prevent dietary NAFLD by inhibiting prosteatotic PPARγ signaling. Moreover, established NAFLD and the associated liver damage can be efficiently reversed by hepatocyte-specific Fra-1 expression. In contrast, c-Fos promotes PPARγ expression, while c-Jun exerts opposing, dimer-dependent functions. Interestingly, JunD was found to be essential for PPARγ signaling and NAFLD development. This unique antagonistic regulation of PPARγ by distinct AP-1 dimers occurs at the transcriptional level and establishes AP-1 as a link between obesity, hepatic lipid metabolism, and NAFLD. PMID:24411941

  20. The Conserved MAPK Site in E(spl)-M8, an Effector of Drosophila Notch Signaling, Controls Repressor Activity during Eye Development

    PubMed Central

    Bandyopadhyay, Mohna; Bishop, Clifton P.

    2016-01-01

    The specification of patterned R8 photoreceptors at the onset of eye development depends on timely inhibition of Atonal (Ato) by the Enhancer of split (E(spl) repressors. Repression of Ato by E(spl)-M8 requires the kinase CK2 and is inhibited by the phosphatase PP2A. The region targeted by CK2 harbors additional conserved Ser residues, raising the prospect of regulation via multi-site phosphorylation. Here we investigate one such motif that meets the consensus for modification by MAPK, a well-known effector of Epidermal Growth Factor Receptor (EGFR) signaling. Our studies reveal an important role for the predicted MAPK site of M8 during R8 birth. Ala/Asp mutations reveal that the CK2 and MAPK sites ensure that M8 repression of Ato and the R8 fate occurs in a timely manner and at a specific stage (stage-2/3) of the morphogenetic furrow (MF). M8 repression of Ato is mitigated by halved EGFR dosage, and this effect requires an intact MAPK site. Accordingly, variants with a phosphomimetic Asp at the MAPK site exhibit earlier (inappropriate) activity against Ato even at stage-1 of the MF, where a positive feedback-loop is necessary to raise Ato levels to a threshold sufficient for the R8 fate. Analysis of deletion variants reveals that both kinase sites (CK2 and MAPK) contribute to ‘cis’-inhibition of M8. This key regulation by CK2 and MAPK is bypassed by the E(spl)D mutation encoding the truncated protein M8*, which potently inhibits Ato at stage-1 of R8 birth. We also provide evidence that PP2A likely targets the MAPK site. Thus multi-site phosphorylation controls timely onset of M8 repressor activity in the eye, a regulation that appears to be dispensable in the bristle. The high conservation of the CK2 and MAPK sites in the insect E(spl) proteins M7, M5 and Mγ, and their mammalian homologue HES6, suggest that this mode of regulation may enable E(spl)/HES proteins to orchestrate repression by distinct tissue-specific mechanisms, and is likely to have broader

  1. AP-1-mediated invasion requires increased expression of the hyaluronan receptor CD44.

    PubMed Central

    Lamb, R F; Hennigan, R F; Turnbull, K; Katsanakis, K D; MacKenzie, E D; Birnie, G D; Ozanne, B W

    1997-01-01

    Fibroblasts transformed by Fos oncogenes display increased expression of a number of genes implicated in tumor cell invasion and metastasis. In contrast to normal 208F rat fibroblasts, Fos-transformed 208F fibroblasts are growth factor independent for invasion. We demonstrate that invasion of v-Fos- or epidermal growth factor (EGF)-transformed cells requires AP-1 activity. v-Fos-transformed cell invasion is inhibited by c-jun antisense oligonucleotides and by expression of a c-jun dominant negative mutant, TAM-67. EGF-induced invasion is inhibited by both c-fos and c-jun antisense oligonucleotides. CD44s, the standard form of a transmembrane receptor for hyaluronan, is implicated in tumor cell invasion and metastasis. We demonstrate that increased expression of CD44 in Fos- and EGF-transformed cells is dependent upon AP-1. CD44 antisense oligonucleotides reduce expression of CD44 in v-Fos- or EGF-transformed cells and inhibit invasion but not migration. Expression of a fusion protein between human CD44s and Aequorea victoria green fluorescent protein (GFP) in 208F cells complements the inhibition of invasion by the rat-specific CD44 antisense oligonucleotide. We further show that both v-Fos and EGF transformations result in a concentration of endogenous CD44 or exogenous CD44-GFP at the ends of pseudopodial cell extensions. These results support the hypothesis that one role of AP-1 in transformation is to activate a multigenic invasion program. PMID:9001250

  2. TACE release of TNF-α mediates mechanotransduction-induced activation of p38 MAPK and myogenesis

    PubMed Central

    Zhan, Mei; Jin, Bingwen; Chen, Shuen-Ei; Reecy, James M.; Li, Yi-Ping

    2011-01-01

    Summary Skeletal muscle responds to mechanical stimulation by activating p38 MAPK, a key signal for myogenesis. However, the mechanotransduction mechanism that activates p38 is unknown. Here we show that mechanical stimulation of myoblasts activates p38 and myogenesis through stimulating TNF-α release by TNF-α converting enzyme (TACE). In C2C12 or mouse primary myoblasts cultured in growth medium, static stretch activated p38 along with ERK1/2, JNK and AKT. Disrupting TNF-α signaling by TNF-α-neutralizing antibody or knocking out TNF-α receptors blocked stretch activation of p38, but not ERK1/2, JNK or AKT. Stretch also activated differentiation markers MEF2C, myogenin, p21 and myosin heavy chain in a TNF-α- and p38-dependent manner. Stretch stimulated the cleavage activity of TACE. Conversely, TACE inhibitor TAPI or TACE siRNA abolished stretch activation of p38. In addition, conditioned medium from stretched myoblast cultures activated p38 in unstretched myoblasts, which required TACE activity in the donor myoblasts, and TNF-α receptors in the recipient myoblasts. These results indicate that posttranscriptional activation of TACE mediates the mechanotransduction that activates p38-dependent myogenesis via the release of TNF-α. PMID:17264149

  3. The Pore-Forming α-Toxin from Clostridium septicum Activates the MAPK Pathway in a Ras-c-Raf-Dependent and Independent Manner

    PubMed Central

    Chakravorty, Anjana; Awad, Milena M.; Cheung, Jackie K.; Hiscox, Thomas J.; Lyras, Dena; Rood, Julian I.

    2015-01-01

    Clostridium septicum is the causative agent of atraumatic gas gangrene, with α-toxin, an extracellular pore-forming toxin, essential for disease. How C. septicum modulates the host’s innate immune response is poorly defined, although α-toxin-intoxicated muscle cells undergo cellular oncosis, characterised by mitochondrial dysfunction and release of reactive oxygen species. Nonetheless, the signalling events that occur prior to the initiation of oncosis are poorly characterised. Our aims were to characterise the ability of α-toxin to activate the host mitogen activated protein kinase (MAPK) signalling pathway both in vitro and in vivo. Treatment of Vero cells with purified α-toxin activated the extracellular-signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38 arms of the MAPK pathway and stimulated the release of TNF-α in a dose-dependent manner. Studies using inhibitors of all three MAPK components suggested that activation of ERK occurred in a Ras-c-Raf dependent manner, whereas activation of JNK and p38 occurred by a Ras-independent mechanism. Toxin-mediated activation was dependent on efficient receptor binding and pore formation and on an influx of extracellular calcium ions. In the mouse myonecrosis model we showed that the MAPK pathway was activated in tissues of infected mice, implying that it has an important role in the disease process. PMID:25675415

  4. The pore-forming α-toxin from clostridium septicum activates the MAPK pathway in a Ras-c-Raf-dependent and independent manner.

    PubMed

    Chakravorty, Anjana; Awad, Milena M; Cheung, Jackie K; Hiscox, Thomas J; Lyras, Dena; Rood, Julian I

    2015-02-01

    Clostridium septicum is the causative agent of atraumatic gas gangrene, with α-toxin, an extracellular pore-forming toxin, essential for disease. How C. septicum modulates the host's innate immune response is poorly defined, although α-toxin-intoxicated muscle cells undergo cellular oncosis, characterised by mitochondrial dysfunction and release of reactive oxygen species. Nonetheless, the signalling events that occur prior to the initiation of oncosis are poorly characterised. Our aims were to characterise the ability of α-toxin to activate the host mitogen activated protein kinase (MAPK) signalling pathway both in vitro and in vivo. Treatment of Vero cells with purified α-toxin activated the extracellular-signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38 arms of the MAPK pathway and stimulated the release of TNF-α in a dose-dependent manner. Studies using inhibitors of all three MAPK components suggested that activation of ERK occurred in a Ras-c-Raf dependent manner, whereas activation of JNK and p38 occurred by a Ras-independent mechanism. Toxin-mediated activation was dependent on efficient receptor binding and pore formation and on an influx of extracellular calcium ions. In the mouse myonecrosis model we showed that the MAPK pathway was activated in tissues of infected mice, implying that it has an important role in the disease process. PMID:25675415

  5. Sphingosine kinase inhibitor suppresses IL-18-induced interferon-gamma production through inhibition of p38 MAPK activation in human NK cells

    SciTech Connect

    Cheon, Soyoung; Song, Seok Bean; Jung, Minkyung; Park, Yoorim; Bang, Jung-Wook; Kim, Tae Sung; Park, Hyunjeong; Kim, Cherl-hyun; Yang, Yool-hee; Bang, Sa Ik; Cho, Daeho

    2008-09-12

    Natural killer (NK) cells play an important role in the innate immune response. Interleukin-18 (IL-18) is a well-known interferon-gamma (IFN-{gamma} inducing factor, which stimulates immune response in NK and T cells. Sphingosine kinase (SPHK) catalyzes the formation of sphingosine 1-phosphate (S1P), which acts as a second messenger to function as an anti-apoptotic factor and proliferation stimulator of immune cells. In this study, to elucidate whether SPHK is involved in IL-18-induced IFN-{gamma} production, we measured IL-18-induced IFN-{gamma} production after pre-treatment with SPHK inhibitor (SKI) in NK-92MI cells. We found that IL-18-induced IFN-{gamma} expression was blocked by SKI pre-treatment in both mRNA and protein levels. In addition, the increased IFN-{gamma} production by stimulation with IL-18 is mediated through both SPHK and p38 MAPK. To determine the upstream signals of SKI and p38 MAPK in IL-18-induced IFN-{gamma} production, phosphorylation levels of p38 MAPK was measured after SKI pre-treatment. As a result, inhibition of SPHK by SKI blocked phosphorylation of p38 MAPK, showing that SPHK activation by IL-18 is an upstream signal of p38 MAPK activation. Inhibition of SPHK by SKI also inhibited IL-18-induced IFN-{gamma} production in human primary NK cells. In conclusion, SPHK activation is an essential factor for IL-18-induced IFN-{gamma} production via p38 MAPK.

  6. MAPKs in development: insights from Dictyostelium signaling pathways

    PubMed Central

    Hadwiger, Jeffrey A.; Nguyen, Hoai-Nghia

    2011-01-01

    Mitogen activated protein kinases (MAPKs) play important roles in the development of eukaryotic organisms through the regulation of signal transduction pathways stimulated by external signals. MAPK signaling pathways have been associated with the regulation of cell growth, differentiation, and chemotaxis, indicating MAPKs contribute to a diverse set of developmental processes. In most eukaryotes, the diversity of external signals is likely to far exceed the diversity of MAPKs, suggesting that multiple signaling pathways might share MAPKs. Do different signaling pathways converge before MAPK function or can MAPKs maintain signaling specificity through interactions with specific proteins? The genetic and biochemical analysis of MAPK pathways in simple eukaryotes such as Dictyostelium offers opportunities to investigate functional specificity of MAPKs in G protein-mediated signal transduction pathways. This review considers the regulation and specificity of MAPK function in pathways that control Dictyostelium growth and development. PMID:21666837

  7. Eupafolin inhibits PGE2 production and COX2 expression in LPS-stimulated human dermal fibroblasts by blocking JNK/AP-1 and Nox2/p47{sup phox} pathway

    SciTech Connect

    Tsai, Ming-Horng; Liang, Chan-Jung; Yen, Feng-Lin; Chiang, Yao-Chang; Lee, Chiang-Wen

    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 phox 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 inflammation.

  8. TLR4-NOX4-AP-1 signaling mediates lipopolysaccharide-induced CXCR6 expression in human aortic smooth muscle cells

    SciTech Connect

    Patel, Devang N.; Bailey, Steven R.; Gresham, John K.; Schuchman, David B.; Shelhamer, James H.; Goldstein, Barry J.; Foxwell, Brian M.; Stemerman, Michael B.; Maranchie, Jodi K.; Valente, Anthony J.; Mummidi, Srinivas; Chandrasekar, Bysani . E-mail: chandraseka@uthscsa.edu

    2006-09-08

    CXCL16 is a transmembrane non-ELR CXC chemokine that signals via CXCR6 to induce aortic smooth muscle cell (ASMC) proliferation. While bacterial lipopolysaccharide (LPS) has been shown to stimulate CXCL16 expression in SMC, its effects on CXCR6 are not known. Here, we demonstrate that LPS upregulates CXCR6 mRNA, protein, and surface expression in human ASMC. Inhibition of TLR4 with neutralizing antibodies or specific siRNA interference blocked LPS-mediated CXCR6 expression. LPS stimulated both AP-1 (c-Fos, c-Jun) and NF-{kappa}B (p50 and p65) activation, but only inhibition of AP-1 attenuated LPS-induced CXCR6 expression. Using dominant negative expression vectors and siRNA interference, we demonstrate that LPS induces AP-1 activation via MyD88, TRAF6, ERK1/2, and JNK signaling pathways. Furthermore, the flavoprotein inhibitor diphenyleniodonium chloride significantly attenuated LPS-mediated AP-1-dependent CXCR6 expression, as did inhibition of NOX4 NADPH oxidase by siRNA. Finally, CXCR6 knockdown inhibited CXCL16-induced ASMC proliferation. These results demonstrate that LPS-TLR4-NOX4-AP-1 signaling can induce CXCR6 expression in ASMC, and suggest that the CXCL16-CXCR6 axis may be an important proinflammatory pathway in the pathogenesis of atherosclerosis.

  9. Cinnamon extract induces tumor cell death through inhibition of NFκB and AP1

    PubMed Central

    2010-01-01

    Background Cinnamomum cassia bark is the outer skin of an evergreen tall tree belonging to the family Lauraceae containing several active components such as essential oils (cinnamic aldehyde and cinnamyl aldehyde), tannin, mucus and carbohydrate. They have various biological functions including anti-oxidant, anti-microbial, anti-inflammation, anti-diabetic and anti-tumor activity. Previously, we have reported that anti-cancer effect of cinnamon extracts is associated with modulation of angiogenesis and effector function of CD8+ T cells. In this study, we further identified that anti-tumor effect of cinnamon extracts is also link with enhanced pro-apoptotic activity by inhibiting the activities NFκB and AP1 in mouse melanoma model. Methods Water soluble cinnamon extract was obtained and quality of cinnamon extract was evaluated by HPLC (High Performance Liquid Chromatography) analysis. In this study, we tested anti-tumor activity and elucidated action mechanism of cinnamon extract using various types of tumor cell lines including lymphoma, melanoma, cervix cancer and colorectal cancer in vitro and in vivo mouse melanoma model. Results Cinnamon extract strongly inhibited tumor cell proliferation in vitro and induced active cell death of tumor cells by up-regulating pro-apoptotic molecules while inhibiting NFκB and AP1 activity and their target genes such as Bcl-2, BcL-xL and survivin. Oral administration of cinnamon extract in melanoma transplantation model significantly inhibited tumor growth with the same mechanism of action observed in vitro. Conclusion Our study suggests that anti-tumor effect of cinnamon extracts is directly linked with enhanced pro-apoptotic activity and inhibition of NFκB and AP1 activities and their target genes in vitro and in vivo mouse melanoma model. Hence, further elucidation of active components of cinnamon extract could lead to development of potent anti-tumor agent or complementary and alternative medicine for the treatment of

  10. FGFR2IIIb-MAPK Activity Is Required for Epithelial Cell Fate Decision in the Lower Müllerian Duct.

    PubMed

    Terakawa, Jumpei; Rocchi, Altea; Serna, Vanida A; Bottinger, Erwin P; Graff, Jonathan M; Kurita, Takeshi

    2016-07-01

    Cell fate of lower Müllerian duct epithelium (MDE), to become uterine or vaginal epithelium, is determined by the absence or presence of ΔNp63 expression, respectively. Previously, we showed that SMAD4 and runt-related transcription factor 1 (RUNX1) were independently required for MDE to express ΔNp63. Here, we report that vaginal mesenchyme directs vaginal epithelial cell fate in MDE through paracrine activation of fibroblast growth factor (FGF) receptor-MAPK pathway. In the developing reproductive tract, FGF7 and FGF10 were enriched in vaginal mesenchyme, whereas FGF receptor 2IIIb was expressed in epithelia of both the uterus and vagina. When Fgfr2 was inactivated, vaginal MDE underwent uterine cell fate, and this differentiation defect was corrected by activation of MEK-ERK pathway. In vitro, FGF10 in combination with bone morphogenetic protein 4 and activin A (ActA) was sufficient to induce ΔNp63 in MDE, and ActA was essential for induction of RUNX1 through SMAD-independent pathways. Accordingly, inhibition of type 1 receptors for activin in neonatal mice induced uterine differentiation in vaginal epithelium by down-regulating RUNX1, whereas conditional deletion of Smad2 and Smad3 had no effect on vaginal epithelial differentiation. In conclusion, vaginal epithelial cell fate in MDE is induced by FGF7/10-MAPK, bone morphogenetic protein 4-SMAD, and ActA-RUNX1 pathway activities, and the disruption in any one of these pathways results in conversion from vaginal to uterine epithelial cell fate. PMID:27164167

  11. DNA conformation driven by AP-1 triggers cell-specific expression via a strong epithelial enhancer.

    PubMed

    Virolle, T; Djabari, Z; Ortonne, J P; Aberdam, D

    2000-10-01

    We report here the characterization of the regulatory region of the human LAMA3 gene, coding for the alpha3A chain of laminin-5. A 202 bp fragment is sufficient to confer epithelial-specific expression to a thymidine kinase promoter through the cooperative effect of three AP-1 binding sites. Remarkably, removal of the sequences located between the AP-1 sites does not modify the promoter activity in keratinocytes but allows strong expression in fibroblasts. Replacement of the deleted sequences by non-homologous ones fully restores the restricted enhancement in keratinocytes. Functional analysis and mutagenesis experiments demonstrate that a minimal distance between the AP-1 sites is required for the enhancer DNA fragment to adopt a particular conformation driven by the binding of Jun-Fos heterodimers. In non-permissive cells, this conformation leads to the anchorage of non-DNA-binding fibroblastic cofactors to form an inhibitory ternary complex. Therefore, our results describe for the first time an unusual conformation-dependent epithelial-specific enhancer. PMID:11269498

  12. JUNB/AP-1 controls IFN-γ during inflammatory liver disease

    PubMed Central

    Thomsen, Martin K.; Bakiri, Latifa; Hasenfuss, Sebastian C.; Hamacher, Rainer; Martinez, Lola; Wagner, Erwin F.

    2013-01-01

    Understanding the molecular pathogenesis of inflammatory liver disease is essential to design efficient therapeutic approaches. In hepatocytes, the dimeric transcription factor c-JUN/AP-1 is a major mediator of cell survival during hepatitis, although functions for other JUN proteins in liver disease are less defined. Here, we found that JUNB was specifically expressed in human and murine immune cells during acute liver injury. We analyzed the molecular function of JUNB in experimental models of hepatitis, including administration of concanavalin A (ConA) or α-galactosyl-ceramide, which induce liver inflammation and injury. Mice specifically lacking JUNB in hepatocytes displayed a mild increase in ConA-induced liver damage. However, targeted deletion of Junb in immune cells and hepatocytes protected against hepatitis in experimental models that involved NK/NKT cells. The absence of JUNB in immune cells decreased IFN-γ expression and secretion from NK and NKT cells, leading to reduced STAT1 pathway activation. Systemic IFN-γ treatment or adenovirus-based IRF1 delivery to Junb-deficient mice restored hepatotoxicity, and we demonstrate that Ifng is a direct transcriptional target of JUNB. These findings demonstrate that JUNB/AP-1 promotes cell death during acute hepatitis by regulating IFN-γ production in NK and NKT cells and thus functionally antagonizes the hepatoprotective function of c-JUN/AP-1 in hepatocytes. PMID:24200694

  13. Fra-2/AP-1 controls bone formation by regulating osteoblast differentiation and collagen production.

    PubMed

    Bozec, Aline; Bakiri, Latifa; Jimenez, Maria; Schinke, Thorsten; Amling, Michael; Wagner, Erwin F

    2010-09-20

    The activator protein-1 (AP-1) transcription factor complex, in particular the Fos proteins, is an important regulator of bone homeostasis. Fra-2 (Fosl2), a Fos-related protein of the AP-1 family, is expressed in bone cells, and newborn mice lacking Fra-2 exhibit defects in chondrocytes and osteoclasts. Here we show that Fra-2-deficient osteoblasts display a differentiation defect both in vivo and in vitro. Moreover, Fra-2-overexpressing mice are osteosclerotic because of increased differentiation of osteoblasts, which appears to be cell autonomous. Importantly, the osteoblast-specific osteocalcin (Oc) gene and collagen1α2 (col1α2) are transcriptional targets of Fra-2 in both murine and human bone cells. In addition, Fra-2, Oc, and col1 are expressed in stromal cells of human chondroblastic and osteoblastic osteosarcomas (Os's) as well as during osteoblast differentiation of human Os cell lines. These findings reveal a novel function of Fra-2/AP-1 as a positive regulator of bone and matrix formation in mice and humans. PMID:20837772

  14. Roles for the mitogen-activated protein kinase (MAPK) phosphatase, DUSP1, in feedback control of inflammatory gene expression and repression by dexamethasone.

    PubMed

    Shah, Suharsh; King, Elizabeth M; Chandrasekhar, Ambika; Newton, Robert

    2014-05-01

    Glucocorticoids act on the glucocorticoid receptor (NR3C1) to repress inflammatory gene expression. This is central to their anti-inflammatory effectiveness and rational improvements in therapeutic index depend on understanding the mechanism. Human pulmonary epithelial A549 cells were used to study the role of the mitogen-activated protein kinase (MAPK) phosphatase, dual-specificity phosphatase 1 (DUSP1), in the dexamethasone repression of 11 inflammatory genes induced, in a MAPK-dependent manner, by interleukin-1β (IL1B). Adenoviral over-expression of DUSP1 inactivated MAPK pathways and reduced expression of all 11 inflammatory genes. IL1B rapidly induced DUSP1 expression and RNA silencing revealed a transient role in feedback inhibition of MAPKs and inflammatory gene expression. With dexamethasone, which induced DUSP1 expression, plus IL1B (co-treatment), DUSP1 expression was further enhanced. At 1 h, this was responsible for the dexamethasone inhibition of IL1B-induced MAPK activation and CXCL1 and CXCL2 mRNA expression, with a similar trend for CSF2. Whereas, CCL20 mRNA was not repressed by dexamethasone at 1 h, repression of CCL2, CXCL3, IL6, and IL8 was unaffected, and PTGS2 repression was partially affected by DUSP1 knockdown. At later times, dexamethasone repression of MAPKs was unaffected by DUSP1 silencing. Likewise, 6 h post-IL1B, dexamethasone repression of all 11 mRNAs was essentially unaffected by DUSP1 knockdown. Qualitatively similar data were obtained for CSF2, CXCL1, IL6, and IL8 release. Thus, despite general roles in feedback inhibition, DUSP1 plays a transient, often partial, role in the dexamethasone-dependent repression of certain inflammatory genes. Therefore this also illustrates key roles for DUSP1-independent effectors in mediating glucocorticoid-dependent repression. PMID:24692548

  15. Interleukin 6 Is Required for Pancreatic Cancer Progression by Promoting MAPK Signaling Activation and Oxidative Stress Resistance

    PubMed Central

    Zhang, Yaqing; Yan, Wei; Collins, Meredith A.; Bednar, Filip; Rakshit, Sabita; Zetter, Bruce R.; Stanger, Ben Z.; Chung, Ivy; Rhim, Andrew D.; di Magliano, Marina Pasca

    2013-01-01

    Pancreatic cancer, one of the deadliest human malignancies, is almost invariably associated with the presence of an oncogenic form of Kras. Mice expressing oncogenic Kras in the pancreas recapitulate the step-wise progression of the human disease. The inflammatory cytokine interleukin 6 (IL6) is often expressed by multiple cell types within the tumor microenvironment. Here, we show that IL6 is required for the maintenance and progression of pancreatic cancer precursor lesions. In fact, the lack of IL6 completely ablates cancer progression even in presence of oncogenic Kras. Mechanistically, we show that IL6 synergizes with oncogenic Kras to activate the reactive oxygen species (ROS) detoxification program downstream of the MAPK/ERK signaling cascade. In addition, IL6 regulates the inflammatory microenvironment of pancreatic cancer throughout its progression, providing several signals that are essential for carcinogenesis. Thus, IL6 emerges as a key player at all stages of pancreatic carcinogenesis, and a potential therapeutic target. PMID:24097820

  16. Deficiency of MAPK-activated protein kinase 2 (MK2) prevents adverse remodelling and promotes endothelial healing after arterial injury.

    PubMed

    Kapopara, P R; von Felden, J; Soehnlein, O; Wang, Y; Napp, L C; Sonnenschein, K; Wollert, K C; Schieffer, B; Gaestel, M; Bauersachs, J; Bavendiek, U

    2014-12-01

    Maladaptive remodelling of the arterial wall after mechanical injury (e. g. angioplasty) is characterised by inflammation, neointima formation and media hypertrophy, resulting in narrowing of the affected artery. Moreover, mechanical injury of the arterial wall causes loss of the vessel protecting endothelial cell monolayer. Mitogen-activated protein kinase (MAPK)-activated protein kinase 2 (MK2), a major downstream target of p38 MAPK, regulates inflammation, cell migration and proliferation, essential processes for vascular remodelling and re-endothelialisation. Therefore, we investigated the role of MK2 in remodelling and re-endothelialisation after arterial injury in genetically modified mice in vivo. Hypercholesterolaemic low-density-lipoprotein-receptor-deficient mice (ldlr-/-) were subjected to wire injury of the common carotid artery. MK2-deficiency (ldlr-/-/mk2-/-) nearly completely prevented neointima formation, media hypertrophy, and lumen loss after injury. This was accompanied by reduced proliferation and migration of MK2-deficient smooth muscle cells. In addition, MK2-deficiency severely reduced monocyte adhesion to the arterial wall (day 3 after injury, intravital microscopy), which may be attributed to reduced expression of the chemokine ligands CCL2 and CCL5. In line, MK2-deficiency significantly reduced the content of monocytes, neutrophiles and lymphocytes of the arterial wall (day 7 after injury, flow cytometry). In conclusion, in a model of endothelial injury (electric injury), MK2-deficiency strongly increased proliferation of endothelial cells and improved re-endothelialisation of the arterial wall after injury. Deficiency of MK2 prevents adverse remodelling and promotes endothelial healing of the arterial wall after injury, suggesting that MK2-inhibition is a very attractive intervention to prevent restenosis after percutaneous therapeutic angioplasty. PMID:25120198

  17. Downregulation of Foxc2 enhances apoptosis induced by 5-fluorouracil through activation of MAPK and AKT pathways in colorectal cancer

    PubMed Central

    YANG, CHAO; CUI, XIAOXIAN; DAI, XIAOQIN; LIAO, WENTING

    2016-01-01

    The chemotherapy drug 5-fluorouracil (5-FU) is fundamental for the treatment of colorectal cancer (CRC); however, drug resistance to 5-FU may occasionally occur. Abnormal expression of Forkhead box C2 gene (Foxc2) has been identified in several human cancers, but the role of Foxc2 in the progression of CRC remains unclear. The present study established a stable Foxc2-short hairpin (sh)RNA cell line, which was confirmed by western blot analysis and quantitative polymerase chain reaction. The Foxc2-shRNA cells were treated with 5-FU and the cell viability was determined by an MTT assay. Western blot analysis was performed to investigate the signaling pathway involved in 5-FU treatment. The present study identified that 5-FU increased the percentage of apoptotic CRC cells among the Foxc2/RNA interference-transfected cells compared with cells transfected with an empty vector. Therefore, the downregulation of Foxc2, induced by 5-FU, may enhance apoptosis by the downregulation of apoptotic factors, including B cell lymphoma-2 and pro-caspase-3, in Foxc2-shRNA CRC cells. Furthermore, the mitogen-activated protein kinase (MAPK) and phosphatidylinositide 3-kinases/protein kinase B (PI3K/AKT) pathways were essential for the sensitization effect of Foxc2 to 5-FU treatment. Overall, these findings reveal the mechanisms behind Foxc2 depletion and 5-FU treatment of CRC and suggest that Foxc2 enhances resistance to apoptosis, induced by 5-FU, through the activation of MAPK and P13K/AKT pathways, and may serve as a valuable clinical prognostic marker for CRC. PMID:26893778

  18. Thrombosis recanalization by paeoniflorin through the upregulation of urokinase‑type plasminogen activator via the MAPK signaling pathway.

    PubMed

    Ye, Songshan; Mao, Bingyu; Yang, Lei; Fu, Weiyun; Hou, Junran

    2016-06-01

    Paeoniflorin, the major component of Paeonia lactiflora pall, has previously been reported to prevent thrombosis. Plasminogen activator urokinase (uPA) is a serine protease that markedly facilitates normal thrombosis resolution. Paeoniflorin and uPA have been linked to the mitogen‑activated protein kinase (MAPK) signaling pathway. In the current study, the influence of paeoniflorin on the expression of uPA was investigated and the underlying regulatory mechanism was preliminarily determined. The prothrombotic state of the model animals treated with paeoniflorin were assessed by enzyme‑linked immunosorbent assay (ELISA). Additionally, the cytotoxicity of paeoniflorin on human umbilical vein endothelial cell (HUVEC) cultures was estimated using a methyl thiazolyl tetrazolium assay and the possible pathways involved in the interaction between paeoniflorin and uPA were evaluated using western blot analysis. The ELISA results demonstrated that the levels of 6‑keto prostaglandin F1a, fibronectin and uPA were significantly upregulated by treatment with paeoniflorin compared with control (P<0.05). By contrast, the expression of fibrinogen, D‑dimer and thromboxane B2 were inhibited. With an increase in the concentration of paeoniflorin the cell viability of HUVECs decreased gradually. The results of western blot analysis demonstrated that paeoniflorin increased the phosphorylation of MAPK 14 (p38) and MAPK 8 (JNK). The present study demonstrated that paeoniflorin has the potential to improve the prethrombotic state and recanalize thrombosis by increasing the expression of uPA, which may be mediated via regulation of the p38 and JNK MAPK signaling pathways. However, this treatment effect was dependent on the concentration of paeoniflorin used, an unsuitable concentration of the agent would result in a negative effect on the anti‑thrombosis pathways. PMID:27082639

  19. Role of p38 MAPK activation and mitochondrial cytochrome-c release in allicin-induced apoptosis in SK-N-SH cells.

    PubMed

    Zhuang, Jianhui; Li, Yu; Chi, Yufen

    2016-04-01

    Here, we investigate the apoptotic effect of allicin, the predominant component of freshly crushed garlic, on neuroblastoma cells. In this paper, the authors have first assessed the effect of allicin on human neuroblastoma SK-N-SH cells and then investigated the underlying mechanism. The results indicate that allicin suppresses SK-N-SH cell growth in a dose-dependent and time-dependent manner and that 5 μmol/l of allicin leads to a significant increase in apoptotic rate with annexin-V/PI double staining. Western blot analysis shows that treatment with allicin-induced apoptosis through activation of caspases-3 and 9. Phosphorylation of p38 MAPK contributes to allicin-induced apoptosis upstream of caspase activation. Using p38 MAPK inhibitor, the authors discovered that p38 MAPK activation subsequently induces the release of cytochrome-c from mitochondria into the cytosol. Taken together, the results demonstrate that allicin can activate the p38 MAPK pathway, which leads to mitochondrial release of cytochrome-c, thus inducing SK-N-SH cell apoptosis. Overall, this study suggests that allicin may be used as one of the novel pharmacological treatment strategies in neuroblastoma. PMID:26771864

  20. Antioxidant-induced changes of the AP-1 transcription complex are paralleled by a selective suppression of human papillomavirus transcription.

    PubMed Central

    Rösl, F; Das, B C; Lengert, M; Geletneky, K; zur Hausen, H

    1997-01-01

    Considering the involvement of a redox-regulatory pathway in the expression of human papillomaviruses (HPVs), HPV type 16 (HPV-16)-immortalized human keratinocytes were treated with the antioxidant pyrrolidine-dithiocarbamate (PDTC). PDTC induces elevated binding of the transcription factor AP-1 to its cognate recognition site within the viral regulatory region. Despite of increased AP-1 binding, normally indispensable for efficient HPV-16 transcription, viral gene expression was selectively suppressed at the level of initiation of transcription. Electrophoretic mobility supershift assays showed that the composition of the AP-1 complex, predominantly consisting of Jun homodimers in untreated cells, was altered. Irrespective of enhanced c-fos expression, c-jun was phosphorylated and became primarily heterodimerized with fra-1, which was also induced after PDTC incubation. Additionally, there was also an increased complex formation between c-jun and junB. Because both fra-1 and junB overexpression negatively interferes with c-jun/c-fos trans-activation of AP-1-responsive genes, our results suggest that the observed block in viral transcription is mainly the consequence of an antioxidant-induced reconstitution of the AP-1 transcription complex. Since expression of the c-jun/c-fos gene family is tightly regulated during cellular differentiation, defined reorganization of a central viral transcription factor may represent a novel mechanism controlling the transcription of pathogenic HPVs during keratinocyte differentiation and in the progression to cervical cancer. PMID:8985358

  1. Activation of MAPK and FoxO by Manganese (Mn) in Rat Neonatal Primary Astrocyte Cultures

    PubMed Central

    Exil, Vernat; Ping, Li; Yu, Yingchun; Chakraborty, Sudipta; Caito, Samuel W.; Wells, K. Sam; Karki, Pratap; Lee, Eunsook; Aschner, Michael

    2014-01-01

    Environmental exposure to manganese (Mn) leads to a neurodegenerative disease that has shared clinical characteristics with Parkinson's disease (PD). Mn-induced neurotoxicity is time- and dose-dependent, due in part to oxidative stress. We ascertained the molecular targets involved in Mn-induced neurodegeneration using astrocyte culture as: (1) Astrocytes are vital for information processing within the brain, (2) their redox potential is essential in mitigating reactive oxygen species (ROS) levels, and (3) they are targeted early in the course of Mn toxicity. We first tested protein levels of Mn superoxide dismutase -2 (SOD-2) and glutathione peroxidase (GPx-1) as surrogates of astrocytic oxidative stress response. We assessed levels of the forkhead winged-helix transcription factor O (FoxO) in response to Mn exposure. FoxO is highly regulated by the insulin-signaling pathway. FoxO mediates cellular responses to toxic stress and modulates adaptive responses. We hypothesized that FoxO is fundamental in mediating oxidative stress response upon Mn treatment, and may be a biomarker of Mn-induced neurodegeneration. Our results indicate that 100 or 500 µM of MnCl2 led to increased levels of FoxO (dephosphorylated and phosphorylated) compared with control cells (P<0.01). p-FoxO disappeared from the cytosol upon Mn exposure. Pre-treatment of cultured cells with (R)-(−)-2-oxothiazolidine-4-carboxylic acid (OTC), a cysteine analog rescued the cytosolic FoxO. At these concentrations, MAPK phosphorylation, in particular p38 and ERK, and PPAR gamma coactivator-1 (PGC-1) levels were increased, while AKT phosphorylation remained unchanged. FoxO phosphorylation level was markedly reduced with the use of SB203580 (a p38 MAPK inhibitor) and PD98059 (an ERK inhibitor). We conclude that FoxO phosphorylation after Mn exposure occurs in parallel with, and independent of the insulin-signaling pathway. FoxO levels and its translocation into the nucleus are part of early events

  2. Loss of JUNB/AP-1 promotes invasive prostate cancer

    PubMed Central

    Thomsen, M K; Bakiri, L; Hasenfuss, S C; Wu, H; Morente, M; Wagner, E F

    2015-01-01

    Prostate cancer is a frequent cause of male death in the Western world. Relatively few genetic alterations have been identified, likely owing to disease heterogeneity. Here, we show that the transcription factor JUNB/AP-1 limits prostate cancer progression. JUNB expression is increased in low-grade prostate cancer compared with normal human prostate, but downregulated in high-grade samples and further decreased in all metastatic samples. To model the hypothesis that this downregulation is functionally significant, we genetically inactivated Junb in the prostate epithelium of mice. When combined with Pten (phosphatase and tensin homologue) loss, double-mutant mice were prone to invasive cancer development. Importantly, invasive tumours also developed when Junb and Pten were inactivated in a small cell population of the adult anterior prostate by topical Cre recombinase delivery. The resulting tumours displayed strong histological similarity with human prostate cancer. Loss of JunB expression led to increased proliferation and decreased senescence, likely owing to decreased p16Ink4a and p21CIP1 in epithelial cells. Furthermore, the tumour stroma was altered with increased osteopontin and S100 calcium-binding protein A8/9 expression, which correlated with poor prognoses in patients. These data demonstrate that JUNB/AP-1 cooperates with PTEN signalling as barriers to invasive prostate cancer, whose concomitant genetic or epigenetic suppression induce malignant progression. PMID:25526087

  3. Loss of JUNB/AP-1 promotes invasive prostate cancer.

    PubMed

    Thomsen, M K; Bakiri, L; Hasenfuss, S C; Wu, H; Morente, M; Wagner, E F

    2015-04-01

    Prostate cancer is a frequent cause of male death in the Western world. Relatively few genetic alterations have been identified, likely owing to disease heterogeneity. Here, we show that the transcription factor JUNB/AP-1 limits prostate cancer progression. JUNB expression is increased in low-grade prostate cancer compared with normal human prostate, but downregulated in high-grade samples and further decreased in all metastatic samples. To model the hypothesis that this downregulation is functionally significant, we genetically inactivated Junb in the prostate epithelium of mice. When combined with Pten (phosphatase and tensin homologue) loss, double-mutant mice were prone to invasive cancer development. Importantly, invasive tumours also developed when Junb and Pten were inactivated in a small cell population of the adult anterior prostate by topical Cre recombinase delivery. The resulting tumours displayed strong histological similarity with human prostate cancer. Loss of JunB expression led to increased proliferation and decreased senescence, likely owing to decreased p16(Ink4a) and p21(CIP1) in epithelial cells. Furthermore, the tumour stroma was altered with increased osteopontin and S100 calcium-binding protein A8/9 expression, which correlated with poor prognoses in patients. These data demonstrate that JUNB/AP-1 cooperates with PTEN signalling as barriers to invasive prostate cancer, whose concomitant genetic or epigenetic suppression induce malignant progression. PMID:25526087

  4. A novel mechanism for momordin Ic-induced HepG2 apoptosis: involvement of PI3K- and MAPK-dependent PPARγ activation.

    PubMed

    Wang, Jing; Yuan, Li; Xiao, Haifang; Wang, Chan; Xiao, Chunxia; Wang, Yutang; Liu, Xuebo

    2014-05-01

    Momordin Ic is a natural triterpenoid saponin found in various Chinese and Japanese natural medicines such as the fruit of Kochia scoparia (L.) Schrad. Momordin Ic has been previously demonstrated to induce HepG2 cell apoptosis in a ROS-mediated PI3K and MAPK pathway-dependent manner. In the present study, the underlying mechanisms of PI3K and MAPK pathway-mediated PPARγ, and PGC-1α co-regulator activation, as well as the effects of downstream proteins, COX-2 and FoxO4, on cell apoptosis were investigated. The results demonstrated that momordin Ic activated PPARγ and inhibited COX-2. PGC-1α and FoxO4 expressions were increased by the PI3K or MAPK pathways. Furthermore, PPARγ inhibition decreased p-p38 and FoxO4 expression, and restored COX-2 expression. ROS inhibition exerted little effect on PPARγ, COX-2 and FoxO4 expression but affected PGC-1α expression. These results revealed the involvement of PI3K and MAPK-dependent PPARγ activation in momordin Ic-induced apoptosis, providing more detailed information underlying the pro-apoptotic mechanism of momordin Ic in HepG2 cell apoptosis. PMID:24584198

  5. High Glucose-Induced Oxidative Stress Mediates Apoptosis and Extracellular Matrix Metabolic Imbalances Possibly via p38 MAPK Activation in Rat Nucleus Pulposus Cells

    PubMed Central

    Cheng, Xiaofei; Ni, Bin; Zhang, Feng; Hu, Ying

    2016-01-01

    Objectives. To investigate whether high glucose-induced oxidative stress is implicated in apoptosis of rat nucleus pulposus cells (NPCs) and abnormal expression of critical genes involved in the metabolic balance of extracellular matrix (ECM). Methods. NPCs were cultured with various concentrations of glucose to detect cell viability and apoptosis. Cells cultured with high glucose (25 mM) were untreated or pretreated with N-acetylcysteine or a p38 MAPK inhibitor SB 202190. Reactive oxygen species (ROS) production was evaluated. Activation of p38 MAPK was measured by Western blot. The expression of ECM metabolism-related genes, including type II collagen, aggrecan, SRY-related high-mobility-group box 9 (Sox-9), matrix metalloproteinase 3 (MMP-3), and tissue inhibitor of metalloproteinase 1 (TIMP-1), was analyzed by semiquantitative RT-PCR. Results. High glucose reduced viability of NPCs and induced apoptosis. High glucose resulted in increased ROS generation and p38 MAPK activation. In addition, it negatively regulated the expression of type II collagen, aggrecan, Sox-9, and TIMP-1 and positively regulated MMP-3 expression. These results were changed by pretreatment with N-acetylcysteine or SB 202190. Conclusions. High glucose might promote apoptosis of NPCs, trigger ECM catabolic pathways, and inhibit its anabolic activities, possibly through a p38 MAPK-dependent oxidative stress mechanism.

  6. The combinatorial activation of the PI3K and Ras/MAPK pathways is sufficient for aggressive tumor formation, while individual pathway activation supports cell persistence

    PubMed Central

    Thompson, Keyata N.; Whipple, Rebecca A.; Yoon, Jennifer R.; Lipsky, Michael; Charpentier, Monica S.; Boggs, Amanda E.; Chakrabarti, Kristi R.; Bhandary, Lekhana; Hessler, Lindsay K.; Martin, Stuart S.; Vitolo, Michele I.

    2015-01-01

    A high proportion of human tumors maintain activation of both the PI3K and Ras/MAPK pathways. In basal-like breast cancer (BBC), PTEN expression is decreased/lost in over 50% of cases, leading to aberrant activation of the PI3K pathway. Additionally, BBC cell lines and tumor models have been shown to exhibit an oncogenic Ras-like gene transcriptional signature, indicating activation of the Ras/MAPK pathway. To directly test how the PI3K and Ras/MAPK pathways contribute to tumorigenesis, we deleted PTEN and activated KRas within non-tumorigenic MCF-10A breast cells. Neither individual mutation was sufficient to promote tumorigenesis, but the combination promoted robust tumor growth in mice. However, in vivo bioluminescence reveals that each mutation has the ability to promote a persistent phenotype. Inherent in the concept of tumor cell dormancy, a stage in which residual disease is present but remains asymptomatic, viable cells with each individual mutation can persist in vivo during a period of latency. The persistent cells were excised from the mice and showed increased levels of the cell cycle arrest proteins p21 and p27 compared to the aggressively growing PTEN−/−KRAS(G12V) cells. Additionally, when these persistent cells were placed into growth-promoting conditions, they were able to re-enter the cell cycle and proliferate. These results highlight the potential for either PTEN loss or KRAS activation to promote cell survival in vivo, and the unique ability of the combined mutations to yield rapid tumor growth. This could have important implications in determining recurrence risk and disease progression in tumor subtypes where these mutations are common. PMID:26497685

  7. Inhibition of inflammatory mediators contributes to the anti-inflammatory activity of KYKZL-1 via MAPK and NF-κB pathway

    SciTech Connect

    Xu, Guang-Lin; Du, Yi-Fang; Cheng, Jing; Huan, Lin; Chen, Shi-Cui; Wei, Shao-Hua; Gong, Zhu-Nan; Cai, Jie; Qiu, Ting; Wu, Hao; Sun, Ting; Ao, Gui-Zhen

    2013-10-01

    KYKZL-1, a newly synthesized compound with COX/5-LOX dual inhibition, was subjected to the anti-inflammatory activity test focusing on its modulation of inflammatory mediators as well as intracellular MAPK and NF-κB signaling pathways. In acute ear edema model, pretreatment with KYKZL-1 (p.o.) dose-dependently inhibited the xylene-induced ear edema in mice with a higher inhibition than diclofenac. In a three-day TPA-induced inflammation, KYKZL-1 also showed significant anti-inflammatory activity with inhibition ranging between 20% and 64%. In gastric lesion test, KYKZL-1 elicited markedly fewer stomach lesions with a low index of ulcer as compared to diclofenac in rats. In further studies, KYKZL-1 was found to significantly inhibit the production of NO, PGE{sub 2}, LTB{sub 4} in LPS challenged RAW264.7, which is parallel to its attenuation of the expression of iNOS, COX-2, 5-LOX mRNAs or proteins and inhibition of phosphorylation of p38 and ERK MAPKs and activation of NF-κB. Taken together, our data indicate that KYKZL-1 comprises dual inhibition of COX and 5-LOX and exerts an obvious anti-inflammatory activity with an enhanced gastric safety profile via simultaneous inhibition of phosphorylation of p38 and ERK MAPKs and activation of NF-κB. - Highlights: • KYKZL-1 is designed to exhibit COX/5-LOX dual inhibition. • KYKZL-1 inhibits NO, PGE{sub 2} and LTB{sub 4} and iNOS, COX-2 and 5-LOX mRNAs and MAPKs. • KYKZL-1 inhibits phosphorylation of MAPKs. • KYKZL-1 inactivates NF-κB pathway.

  8. A constitutive active MAPK/ERK pathway due to BRAFV600E positively regulates AHR pathway in PTC.

    PubMed

    Occhi, Gianluca; Barollo, Susi; Regazzo, Daniela; Bertazza, Loris; Galuppini, Francesca; Guzzardo, Vincenza; Jaffrain-Rea, Marie Lise; Vianello, Federica; Ciato, Denis; Ceccato, Filippo; Watutantrige-Fernando, Sara; Bisognin, Andrea; Bortoluzzi, Stefania; Pennelli, Gianmaria; Boscaro, Marco; Scaroni, Carla; Mian, Caterina

    2015-10-13

    The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor mediating the toxicity and tumor-promoting properties of dioxin. AHR has been reported to be overexpressed and constitutively active in a variety of solid tumors, but few data are currently available concerning its role in thyroid cancer. In this study we quantitatively explored a series of 51 paired-normal and papillary thyroid carcinoma (PTC) tissues for AHR-related genes. We identified an increased AHR expression/activity in PTC, independently from its nuclear dimerization partner and repressor but strictly related to a constitutive active MAPK/ERK pathway. The AHR up-regulation followed by an increased expression of AHR target genes was confirmed by a meta-analysis of published microarray data, suggesting a ligand-independent active AHR pathway in PTC. In-vitro studies using a PTC-derived cell line (BCPAP) and HEK293 cells showed that BRAFV600E may directly modulate AHR localization, induce AHR expression and activity in an exogenous ligand-independent manner. The AHR pathway might represent a potential novel therapeutic target for PTC in the clinical practice. PMID:26392334

  9. Carbon monoxide alleviates ethanol-induced oxidative damage and inflammatory stress through activating p38 MAPK pathway

    SciTech Connect

    Li, Yanyan; Gao, Chao; Shi, Yanru; Tang, Yuhan; Liu, Liang; Xiong, Ting; Du, Min; Xing, Mingyou; Liu, Liegang; Yao, Ping

    2013-11-15

    Stress-inducible protein heme oxygenase-1(HO-1) is well-appreciative to counteract oxidative damage and inflammatory stress involving the pathogenesis of alcoholic liver diseases (ALD). The potential role and signaling pathways of HO-1 metabolite carbon monoxide (CO), however, still remained unclear. To explore the precise mechanisms, ethanol-dosed adult male Balb/c mice (5.0 g/kg.bw.) or ethanol-incubated primary rat hepatocytes (100 mmol/L) were pretreated by tricarbonyldichlororuthenium (II) dimmer (CORM-2, 8 mg/kg for mice or 20 μmol/L for hepatocytes), as well as other pharmacological reagents. Our data showed that CO released from HO-1 induction by quercetin prevented ethanol-derived oxidative injury, which was abolished by CO scavenger hemoglobin. The protection was mimicked by CORM-2 with the attenuation of GSH depletion, SOD inactivation, MDA overproduction, and the leakage of AST, ALT or LDH in serum and culture medium induced by ethanol. Moreover, CORM-2 injection or incubation stimulated p38 phosphorylation and suppressed abnormal Tnfa and IL-6, accompanying the alleviation of redox imbalance induced by ethanol and aggravated by inflammatory factors. The protective role of CORM-2 was abolished by SB203580 (p38 inhibitor) but not by PD98059 (ERK inhibitor) or SP600125 (JNK inhibitor). Thus, HO-1 released CO prevented ethanol-elicited hepatic oxidative damage and inflammatory stress through activating p38 MAPK pathway, suggesting a potential therapeutic role of gaseous signal molecule on ALD induced by naturally occurring phytochemicals. - Highlights: • CO alleviated ethanol-derived liver oxidative and inflammatory stress in mice. • CO eased ethanol and inflammatory factor-induced oxidative damage in hepatocytes. • The p38 MAPK is a key signaling mechanism for the protective function of CO in ALD.

  10. CXCL12 induces connective tissue growth factor expression in human lung fibroblasts through the Rac1/ERK, JNK, and AP-1 pathways.

    PubMed

    Lin, Chien-Huang; Shih, Chung-Huang; Tseng, Chih-Chieh; Yu, Chung-Chi; Tsai, Yuan-Jhih; Bien, Mauo-Ying; Chen, Bing-Chang

    2014-01-01

    CXCL12 (stromal cell-derived factor-1, SDF-1) is a potent chemokine for homing of CXCR4+ fibrocytes to injury sites of lung tissue, which contributes to pulmonary fibrosis. Overexpression of connective tissue growth factor (CTGF) plays a critical role in pulmonary fibrosis. In this study, we investigated the roles of Rac1, extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and activator protein-1 (AP-1) in CXCL12-induced CTGF expression in human lung fibroblasts. CXCL12 caused concentration- and time-dependent increases in CTGF expression and CTGF-luciferase activity. CXCL12-induced CTGF expression was inhibited by a CXCR4 antagonist (AMD3100), small interfering RNA of CXCR4 (CXCR4 siRNA), a dominant negative mutant of Rac1 (RacN17), a mitogen-activated protein kinase (MAPK) kinase (MEK) inhibitor (PD98059), a JNK inhibitor (SP600125), a p21-activated kinase inhibitor (PAK18), c-Jun siRNA, and an AP-1 inhibitor (curcumin). Treatment of cells with CXCL12 caused activations of Rac1, Rho, ERK, and c-Jun. The CXCL12-induced increase in ERK phosphorylation was inhibited by RacN17. Treatment of cells with PD98059 and SP600125 both inhibited CXCL12-induced c-Jun phosphorylation. CXCL12 caused the recruitment of c-Jun and c-Fos binding to the CTGF promoter. Furthermore, CXCL12 induced an increase in α-smooth muscle actin (α-SMA) expression, a myofibroblastic phenotype, and actin stress fiber formation. CXCL12-induced actin stress fiber formation and α-SMA expression were respectively inhibited by AMD3100 and CTGF siRNA. Taken together, our results suggest that CXCL12, acting through CXCR4, activates the Rac/ERK and JNK signaling pathways, which in turn initiates c-Jun phosphorylation, and recruits c-Jun and c-Fos to the CTGF promoter and ultimately induces CTGF expression in human lung fibroblasts. Moreover, overexpression of CTGF mediates CXCL12-induced α-SMA expression. PMID:25121739

  11. AP-1-directed human T cell leukemia virus type 1 viral gene expression during monocytic differentiation.

    PubMed

    Grant, Christian; Jain, Pooja; Nonnemacher, Michael; Flaig, Katherine E; Irish, Bryan; Ahuja, Jaya; Alexaki, Aikaterini; Alefantis, Timothy; Wigdahl, Brian

    2006-09-01

    Human T cell leukemia virus type 1 (HTLV-1) has previously been shown to infect antigen-presenting cells and their precursors in vivo. However, the role these important cell populations play in the pathogenesis of HTLV-1-associated myelopathy/tropical spastic paraparesis or adult T cell leukemia remains unresolved. To better understand how HTLV-1 infection of these important cell populations may potentially impact disease progression, the regulation of HTLV-1 viral gene expression in established monocytic cell lines was examined. U-937 promonocytic cells transiently transfected with a HTLV-1 long-terminal repeat (LTR) luciferase construct were treated with phorbol 12-myristate 13-acetate (PMA) to induce cellular differentiation. PMA-induced cellular differentiation resulted in activation of basal and Tax-mediated transactivation of the HTLV-1 LTR. In addition, electrophoretic mobility shift analyses demonstrated that PMA-induced cellular differentiation induced DNA-binding activity of cellular transcription factors to Tax-responsive element 1 (TRE-1) repeat II. Supershift analyses revealed that factors belonging to the activator protein 1 (AP-1) family of basic region/leucine zipper proteins (Fra-1, Fra-2, JunB, and JunD) were induced to bind to TRE-1 repeat II during cellular differentiation. Inhibition of AP-1 DNA-binding activity by overexpression of a dominant-negative c-Fos mutant (A-Fos) in transient expression analyses resulted in severely decreased levels of HTLV-1 LTR activation in PMA-induced U-937 cells. These results have suggested that following infection of peripheral blood monocytes, HTLV-1 viral gene expression may become up-regulated by AP-1 during differentiation into macrophages or dendritic cells. PMID:16829632

  12. A novel synthetic compound MCAP suppresses LPS-induced murine microglial activation in vitro via inhibiting NF-kB and p38 MAPK pathways

    PubMed Central

    Kim, Byung-Wook; More, Sandeep Vasant; Yun, Yo-Sep; Ko, Hyun-Myung; Kwak, Jae-Hwan; Lee, Heesoon; Suk, Kyoungho; Kim, In-Su; Choi, Dong-Kug

    2016-01-01

    Aim: To investigate the anti-neuroinflammatory activity of a novel synthetic compound, 7-methylchroman-2-carboxylic acid N-(2-trifluoromethyl) phenylamide (MCAP) against LPS-induced microglial activation in vitro. Methods: Primary mouse microglia and BV2 microglia cells were exposed to LPS (50 or 100 ng/mL). The expression of iNOS and COX-2, proinflammatory cytokines, NF-κB and p38 MAPK signaling molecules were analyzed by RT-PCR, Western blot and ELISA. The morphological changes of microglia and nuclear translocation of NF-ĸB were visualized using phase contrast and fluorescence microscopy, respectively. Results: Pretreatment with MCAP (0.1, 1, 10 μmol/L) dose-dependently inhibited LPS-induced expression of iNOS and COX-2 in BV2 microglia cells. Similar results were obtained in primary microglia pretreated with MCAP (0.1, 0.5 μmol/L). MCAP dose-dependently abated LPS-induced release of TNF-α, IL-6 and IL-1β, and mitigated LPS-induced activation of NF-κB by reducing the phosphorylation of IκBα in BV2 microglia cells. Moreover, MCAP attenuated LPS-induced phosphorylation of p38 MAPK, whereas SB203580, a p38 MAPK inhibitor, significantly potentiated MCAP-caused inhibition on the expression of MEF-2 (a transcription factor downstream of p38 MAPK). Conclusion: MCAP exerts anti-inflammatory effects in murine microglia in vitro by inhibiting the p38 MAPK and NF-κB signaling pathways and proinflammatory responses. MCAP may be developed as a novel agent for treating diseases involving activated microglial cells. PMID:26838070

  13. Role of P38 MAPK on MMP Activity in Photothrombotic Stroke Mice as Measured using an Ultrafast MMP Activatable Probe

    PubMed Central

    Chang, Di; Wang, Yuan-Cheng; Bai, Ying-Ying; Lu, Chun-Qiang; Xu, Ting-Ting; Zhu, Lei; Ju, Shenghong

    2015-01-01

    Matrix metalloproteinases (MMPs) exert a dual effect in ischemic stroke and thus represent an ideal target for detection and therapy. However, to date, all clinical trials of MMP inhibitors have failed, and alternative drug candidates and therapeutic targets are urgently required. Nonetheless, further investigations are limited by the lack of non-invasive imaging techniques. Here, we report a novel, fast and ultrasensitive MMP activatable optical imaging probe for the dynamic visualization of MMP activity in photothrombotic stroke mice. This probe provides a significant signal enhancement in as little as 15 min, with the highest signal intensity occurring at 1 h post-injection, and shows high sensitivity in measuring MMP activity alterations, which makes it specifically suitable for the real-time visualization of MMP activity and drug discovery in preclinical research. Moreover, using this probe, we successfully demonstrate that the regulation of the p38 mitogen-activated protein kinase (MAPK) signal pathway is capable of modulating MMP activity after stroke, revealing a novel regulatory mechanism of postischemic brain damage and overcoming the limitations of traditional therapeutic strategies associated with MMP inhibitors by using a non-invasive molecular imaging method. PMID:26581247

  14. Hyaluronan Oligosaccharides Induce MMP-1 and -3 via Transcriptional Activation of NF-κB and p38 MAPK in Rheumatoid Synovial Fibroblasts

    PubMed Central

    Hanabayashi, Masahiro; Takahashi, Nobunori; Sobue, Yasumori; Hirabara, Shinya; Ishiguro, Naoki; Kojima, Toshihisa

    2016-01-01

    Objective To explore the effect of hyaluronan oligosaccharides (HAoligos) on interactions between HA and its principal receptor, CD44, in rheumatoid synovial fibroblasts (RSFs) and matrix metalloproteinase (MMP) production. Methods RSFs were isolated from rheumatoid synovial tissue. HA distribution was visualized by immunocytochemistry. MMP-1 and MMP-3 induction was analyzed by real-time RT-PCR and immunoblotting. The interaction between HAoligos and their MMP-producing receptors was tested by blocking with anti-CD44 and anti-Toll-like receptor 4 (TLR-4). Phosphorylation of nuclear factor κB (NF-κB) and mitogen-activated protein kinase (MAPK) was analyzed by immunoblotting. Results Endogenous HA decreased after treatment with HAoligos, while MMP-1 and MMP-3 expression increased in a dose-dependent manner. Pretreatment with anti-CD44 or anti-TLR-4 antibody significantly reduced the effect of HAoligos on MMP-1 and MMP-3 mRNA expression. NF-κB and p38 MAPK phosphorylation was enhanced by HAoligos pretreated with anti-TLR-4, and HAoligo-induced MMP production was blocked with an inhibitor of NF-κB and p38 MAPK pathways. Conclusions Disruptive changes in CD44-HA interactions by HAoligos enhanced MMP-1 and MMP-3 production via activation of NF-κB and p38 MAPK signaling pathways in RSFs. PMID:27564851

  15. Loss of MAPK Pathway Activation in Post-Mitotic Retinal Cells as Mechanism in MEK Inhibition-Related Retinopathy in Cancer Patients

    PubMed Central

    van Dijk, Elon H. C.; Duits, Danique E. M.; Versluis, Mieke; Luyten, Gregrorius P. M.; Bergen, Arthur A. B.; Kapiteijn, Ellen W.; de Lange, Mark J.; Boon, Camiel J. F.; van der Velden, Pieter A.

    2016-01-01

    Abstract Recently, treatment with MEK inhibitors has been shown to be an effective treatment option for metastatic melanoma. Treatment efficacy is dependent on inhibition of MAPK-related melanoma proliferation. However, targeting of MEK can be accompanied by a time-dependent and reversible serous retinopathy of unknown origin. We analyzed the molecular mechanism by which the MEK inhibitor binimetinib may lead to retinopathy, using neuroretina and cell models of retinal pigment epithelium (RPE). Binimetinib inhibited the MAPK pathway while discontinuation of treatment resulted in reactivation. However, cell proliferation was not inhibited correspondingly during binimetinib treatment of ARPE19 cells. Remarkably, post-mitotic neuroretinal tissue displayed a strong MAPK activation that was lost after binimetinib treatment. We propose that binimetinib-associated retinopathy is correlated with inhibition of the MAPK pathway in multiple retinal components. Retinal cells are able to regain the activation after binimetinib treatment, mimicking the reversibility of the retinopathy. As most retinal cells are nonregenerating, other mechanisms than stimulation of proliferation must be involved. PMID:27149444

  16. ESAT-6 induced COX-2 expression involves coordinated interplay between PI3K and MAPK signaling.

    PubMed

    A, Senthil Kumar; Bansal, Kushagra; Holla, Sahana; Verma-Kumar, Shalu; Sharma, Pawan; Balaji, Kithiganahalli Narayanaswamy

    2012-01-01

    Macrophages, as sentinels of robust host immunity, are key regulators of innate immune responses against invading mycobacteria; however, pathogenic mycobacteria survive in the infected host by subverting host innate immunity. Infection dependent expression of early secreted antigenic target protein 6 (ESAT-6) by Mycobacterium tuberculosis is strongly correlated with subversion of innate immune responses against invading mycobacteria. As a part of multifaceted immunity to mycobacterial infection, induced expression of cyclooxygenase-2 (COX-2) may act as an important influencing factor towards effective host immunity. In the current investigation, we demonstrate that ESAT-6 triggers COX-2 expression both in vitro and in vivo in a TLR2 dependent manner. Signaling perturbation data suggest that signaling dynamics of PI3K and p38 and JNK1/2 MAPK assume critical importance in ESAT-6 triggered expression of COX-2 in macrophages. Interestingly, ESAT-6 triggered PI3K-MAPK signaling axis holds the capacity to regulate coordinated activation of NF-κB and AP-1. Overall, current investigation provides mechanistic insights into ESAT-6 induced COX-2 expression and unravels TLR2 mediated interplay of PI3K and MAPK signaling axis as a rate-determining step during intricate host immune responses. These findings would serve as a paradigm to understand pathogenesis of mycobacterial infection and clearly pave a way towards development of novel therapeutics. PMID:22154837

  17. Mitogen-activated Protein Kinase (MAPK) Activated by Prostaglandin E2 Phosphorylates Connexin 43 and Closes Osteocytic Hemichannels in Response to Continuous Flow Shear Stress.

    PubMed

    Riquelme, Manuel A; Burra, Sirisha; Kar, Rekha; Lampe, Paul D; Jiang, Jean X

    2015-11-20

    Cx43 hemichannels serve as a portal for the release of prostaglandins, a critical process in mediating biological responses of mechanical loading on bone formation and remodeling. We have previously observed that fluid flow shear stress (FFSS) opens hemichannels; however, sustained FFSS results in hemichannel closure, as continuous opening of hemichannels is detrimental to cell viability and bone remodeling. However, the mechanism that regulates the closure of the hemichannels is unknown. Here, we show that activation of p44/42 ERK upon continuous FFSS leads to Cx43 phosphorylation at Ser(279)-Ser(282), sites known to be phosphorylated sites by p44/42 MAPK. Incubation of osteocytic MLO-Y4 cells with conditioned media (CM) collected after continuous FFSS increased MAPK-dependent phosphorylation of Cx43. CM treatment inhibited hemichannel opening and this inhibition was reversed when cells were pretreated with the MAPK pathway inhibitor. We found that prostaglandin E2 (PGE2) accumulates in the CM in a time-dependent manner. Treatment with PGE2 increased phospho-p44/42 ERK levels and also Cx43 phosphorylation at Ser(279)-Ser(282) sites. Depletion of PGE2 from CM, and pre-treatment with a p44/42 ERK pathway-specific inhibitor, resulted in a complete inhibition of ERK-dependent Cx43 phosphorylation and attenuated the inhibition of hemichannels by CM and PGE2. Consistently, the opening of hemichannels by FFSS was blocked by PGE2 and CM and this blockage was reversed by U0126 and the CM depleted of PGE2. A similar observation was also obtained in isolated primary osteocytes. Together, results from this study suggest that extracellular PGE2 accumulated after continuous FFSS is responsible for activation of p44/42 ERK signaling and subsequently, direct Cx43 phosphorylation by activated ERK leads to hemichannel closure. PMID:26442583

  18. Selective activation of human dendritic cells by OM-85 through a NF-kB and MAPK dependent pathway.

    PubMed

    Parola, Carmen; Salogni, Laura; Vaira, Xenia; Scutera, Sara; Somma, Paolo; Salvi, Valentina; Musso, Tiziana; Tabbia, Giuseppe; Bardessono, Marco; Pasquali, Christian; Mantovani, Alberto; Sozzani, Silvano; Bosisio, Daniela

    2013-01-01

    OM-85 (Broncho-Vaxom®, Broncho-Munal®, Ommunal®, Paxoral®, Vaxoral®), a product made of the water soluble fractions of 21 inactivated bacterial strain patterns responsible for respiratory tract infections, is used for the prevention of recurrent upper respiratory tract infections and acute exacerbations in chronic obstructive pulmonary disease patients. OM-85 is able to potentiate both innate and adaptive immune responses. However, the molecular mechanisms responsible for OM-85 activation are still largely unknown. Purpose of this study was to investigate the impact of OM-85 stimulation on human dendritic cell functions. We show that OM-85 selectively induced NF-kB and MAPK activation in human DC with no detectable action on the interferon regulatory factor (IRF) pathway. As a consequence, chemokines (i.e. CXCL8, CXCL6, CCL3, CCL20, CCL22) and B-cell activating cytokines (i.e. IL-6, BAFF and IL-10) were strongly upregulated. OM-85 also synergized with the action of classical pro-inflammatory stimuli used at suboptimal concentrations. Peripheral blood mononuclear cells from patients with COPD, a pathological condition often associated with altered PRR expression pattern, fully retained the capability to respond to OM-85. These results provide new insights on the molecular mechanisms of OM-85 activation of the immune response and strengthen the rational for its use in clinical settings. PMID:24386121

  19. Selective Activation of Human Dendritic Cells by OM-85 through a NF-kB and MAPK Dependent Pathway

    PubMed Central

    Scutera, Sara; Somma, Paolo; Salvi, Valentina; Musso, Tiziana; Tabbia, Giuseppe; Bardessono, Marco; Pasquali, Christian; Mantovani, Alberto; Sozzani, Silvano; Bosisio, Daniela

    2013-01-01

    OM-85 (Broncho-Vaxom®, Broncho-Munal®, Ommunal®, Paxoral®, Vaxoral®), a product made of the water soluble fractions of 21 inactivated bacterial strain patterns responsible for respiratory tract infections, is used for the prevention of recurrent upper respiratory tract infections and acute exacerbations in chronic obstructive pulmonary disease patients. OM-85 is able to potentiate both innate and adaptive immune responses. However, the molecular mechanisms responsible for OM-85 activation are still largely unknown. Purpose of this study was to investigate the impact of OM-85 stimulation on human dendritic cell functions. We show that OM-85 selectively induced NF-kB and MAPK activation in human DC with no detectable action on the interferon regulatory factor (IRF) pathway. As a consequence, chemokines (i.e. CXCL8, CXCL6, CCL3, CCL20, CCL22) and B-cell activating cytokines (i.e. IL-6, BAFF and IL-10) were strongly upregulated. OM-85 also synergized with the action of classical pro-inflammatory stimuli used at suboptimal concentrations. Peripheral blood mononuclear cells from patients with COPD, a pathological condition often associated with altered PRR expression pattern, fully retained the capability to respond to OM-85. These results provide new insights on the molecular mechanisms of OM-85 activation of the immune response and strengthen the rational for its use in clinical settings. PMID:24386121

  20. Induction of Apoptosis by PQ1, a Gap Junction Enhancer that Upregulates Connexin 43 and Activates the MAPK Signaling Pathway in Mammary Carcinoma Cells

    PubMed Central

    Shishido, Stephanie N.; Nguyen, Thu A.

    2016-01-01

    The mechanism of gap junction enhancer (PQ1) induced cytotoxicity is thought to be attributed to the change in connexin 43 (Cx43) expression; therefore, the effects of Cx43 modulation in cell survival were investigated in mammary carcinoma cells (FMC2u) derived from a malignant neoplasm of a female FVB/N-Tg(MMTV-PyVT)634Mul/J (PyVT) transgenic mouse. PQ1 was determined to have an IC50 of 6.5 µM in FMC2u cells, while inducing an upregulation in Cx43 expression. The effects of Cx43 modulation in FMC2u cell survival was determined through transfection experiments with Cx43 cDNA, which induced an elevated level of protein expression similar to that seen with PQ1 exposure, or siRNA to silence Cx43 protein expression. Overexpression or silencing of Cx43 led to a reduction or an increase in cell viability, respectively. The mitogen-activated protein kinase (MAPK) family has been implicated in the regulation of cell survival and cell death; therefore, the gap junctional intercellular communication (GJIC)-independent function of PQ1 and Cx43 in the Raf/Mitogen-activated protein kinase/ERK kinase/extracellular-signal-regulated kinase (Raf-MEK-ERK) cascade of cellular survival and p38 MAPK-dependent pathway of apoptosis were explored. PQ1 treatment activated p44/42 MAPK, while the overexpression of Cx43 resulted in a reduced expression. This suggests that PQ1 affects the Raf-MEK-ERK cascade independent of Cx43 upregulation. Both overexpression of Cx43 and PQ1 treatment stimulated an increase in the phosphorylated form of p38-MAPK, reduced levels of the anti-apoptotic protein Bcl-2, and increased the cleavage of pro-caspase-3. Silencing of Cx43 protein expression led to a reduction in the phosphorylation of p38-MAPK and an increase in Bcl-2 expression. The mechanism behind PQ1-induced cytotoxicity in FMC2u mammary carcinoma cells is thought to be attributed to the change in Cx43 expression. Furthermore, PQ1-induced apoptosis through the upregulation of Cx43 may depend on p38

  1. Molecular cloning of the duck mitogen-activated protein kinase 1 (MAPK1) gene and the development of a quantitative real-time PCR assay to detect its expression.

    PubMed

    Cao, Shoulin; Han, Xiangan; Ding, Chan; Wang, Shaohui; Tian, Mingxing; Wang, Xiaolan; Hou, Wanwan; Yue, Jiaping; Wang, Guijun; Yu, Shengqing

    2014-09-01

    Mitogen-activated protein kinase 1 (MAPK1) acts as an integration point for multiple biochemical signals, and is involved in a wide variety of biological processes such as cell proliferation and differentiation, transcription regulation, and development. Mitogen-activated protein kinase 1 plays an important role in inducing cell death in bacterial infections. In this study, the duck MAPK1 gene was cloned for the first time from the Cherry Valley duck. Sequence analysis showed that duck MAPK1 cDNA is 1,557 bp long, with an open reading frame of 1,107 bp. It encodes 368 amino acids, with 85.4, 84.5, and 97.3% homology with the human, mouse, and chicken MAPK1 gene, respectively. Furthermore, a SYBR Green quantitative real-time PCR assay was developed to detect duck MAPK1 expression. Following Riemerella anatipestifer infection by virulent strain Yb2, MAPK1 mRNA level increased more than 200-fold in the duck spleens, suggesting that increased duck MAPK1 expression can be used as an indicator of bacterial infection. Our results provide ground work to warrant further studies of the duck MAPK1 gene in bacterial pathogenesis. PMID:24974389

  2. Baculovirus p35 gene is oppositely regulated by P53 and AP-1 like factors in Spodoptera frugiperda

    SciTech Connect

    Mohareer, Krishnaveni; Sahdev, Sudhir; Hasnain, Seyed E.

    2011-11-04

    Highlights: Black-Right-Pointing-Pointer Baculovirus p35 is regulated by both viral and host factors. Black-Right-Pointing-Pointer Baculovirus p35 is negatively regulated by SfP53-like factor. Black-Right-Pointing-Pointer Baculovirus p35 is positively regulated by SfAP-1-like factor. -- Abstract: Baculovirus p35 belongs to the early class of genes of AcMNPV and requires viral factors like Immediate Early protein-1 for its transcription. To investigate the role of host factors in regulating p35 gene expression, the putative transcription factor binding sites were examined in silico and the role of these factors in influencing the transcription of p35 gene was assessed. We focused our studies on AP-1 and P53-like factors, which are activated under oxidative stress conditions. The AP-1 motif is located at -1401 while P53 motif is at -1912 relative to p35 translation start site. The predicted AP-1 and P53 elements formed specific complexes with Spodoptera frugiperda nuclear extracts. Both AP-1 and P53 motif binding proteins were down regulated as a function of AcMNPV infection in Spodoptera cells. To address the question whether during an oxidative outburst, the p35 transcription is enhanced; we investigated the role of these oxidative stress induced host transcription factors in influencing p35 gene transcription. Reporter assays revealed that AP-1 element enhances the transcription of p35 by a factor of two. Interestingly, P53 element appears to repress the transcription of p35 gene.

  3. Signalling in inflammatory skin disease by AP-1 (Fos/Jun).

    PubMed

    Uluçkan, Özge; Guinea-Viniegra, Juan; Jimenez, Maria; Wagner, Erwin F

    2015-01-01

    Skin inflammation is a physiological reaction to tissue injury, pathogen invasion and irritants. During this process, innate and/or adaptive immune cells are activated and recruited to the site of inflammation to either promote or suppress inflammation. The sequential recruitment and activation of immune cells is modulated by a combination of cytokines and chemokines, which are regulated by transcription factors, such as AP-1 (Fos/Jun), NF-κB, NFATs, and STATs. Here we review the present evidence and the underlying mechanisms of how Jun/AP-1 proteins control skin inflammation. Genetically engineered mouse models (GEMMs) in which AP-1 proteins are deleted in the epidermis have revealed that these proteins control cytokine expression at multiple levels. Constitutive epidermal deletion of JunB in mice leads to a multi-organ disease characterised by increased levels of pro-inflammatory cytokines. These JunB-deficient mutant mice display several phenotypes from skin inflammation to a G-CSF-dependent myeloproliferative disease, as well as kidney atrophy and bone loss, reminiscent of psoriasis and systemic lupus erythematosus. Importantly, epidermal deletion of both JunB and c-Jun in an inducible manner in adult mice leads to a psoriasis-like disease, in which the epidermal proteome expression profile is comparable to the one from psoriasis patient samples. In this GEMM and in psoriasis patient-derived material, S100A8/A9-dependent C3/CFB complement activation, as well as a miR-21-dependent TIMP-3/TACE pathway leading to TNF-α shedding, plays causal roles in disease development. The newly identified therapeutic targets from GEMMs together with investigations in human patient samples open up new avenues for therapeutic interventions for psoriasis and related inflammatory skin diseases. PMID:26458100

  4. PPAR{alpha} agonist fenofibrate protects the kidney from hypertensive injury in spontaneously hypertensive rats via inhibition of oxidative stress and MAPK activity

    SciTech Connect

    Hou, Xiaoyang; Shen, Ying H.; Li, Chuanbao; Wang, Fei; Zhang, Cheng; Bu, Peili; Zhang, Yun

    2010-04-09

    Oxidative stress has been shown to play an important role in the development of hypertensive renal injury. Peroxisome proliferator-activated receptors {alpha} (PPAR{alpha}) has antioxidant effect. In this study, we demonstrated that fenofibrate significantly reduced proteinuria, inflammatory cell recruitment and extracellular matrix (ECM) proteins deposition in the kidney of SHRs without apparent effect on blood pressure. To investigate the mechanisms involved, we found that fenofibrate treatment markedly reduced oxidative stress accompanied by reduced activity of renal NAD(P)H oxidase, increased activity of Cu/Zn SOD, and decreased phosphorylation of p38MAPK and JNK in the kidney of SHRs. Taken together, fenofibrate treatment can protect against hypertensive renal injury without affecting blood pressure by inhibiting inflammation and fibrosis via suppression of oxidative stress and MAPK activity.

  5. Abnormal activity of the MAPK- and cAMP-associated signaling pathways in frontal cortical areas in postmortem brain in schizophrenia.

    PubMed

    Funk, Adam J; McCullumsmith, Robert E; Haroutunian, Vahram; Meador-Woodruff, James H

    2012-03-01

    Recent evidence suggests that schizophrenia may result from alterations of integration of signaling mediated by multiple neurotransmitter systems. Abnormalities of associated intracellular signaling pathways may contribute to the pathophysiology of schizophrenia. Proteins and phospho-proteins comprising mitogen activated protein kinase (MAPK) and 3'-5'-cyclic adenosine monophosphate (cAMP)-associated signaling pathways may be abnormally expressed in the anterior cingulate (ACC) and dorsolateral prefrontal cortex (DLPFC) in schizophrenia. Using western blot analysis we examined proteins of the MAPK- and cAMP-associated pathways in these two brain regions. Postmortem samples were used from a well-characterized collection of elderly patients with schizophrenia (ACC=36, DLPFC=35) and a comparison (ACC=33, DLPFC=31) group. Near-infrared intensity of IR-dye labeled secondary antisera bound to targeted proteins of the MAPK- and cAMP-associated signaling pathways was measured using LiCor Odyssey imaging system. We found decreased expression of Rap2, JNK1, JNK2, PSD-95, and decreased phosphorylation of JNK1/2 at T183/Y185 and PSD-95 at S295 in the ACC in schizophrenia. In the DLPFC, we found increased expression of Rack1, Fyn, Cdk5, and increased phosphorylation of PSD-95 at S295 and NR2B at Y1336. MAPK- and cAMP-associated molecules constitute ubiquitous intracellular signaling pathways that integrate extracellular stimuli, modify receptor expression and function, and regulate cell survival and neuroplasticity. These data suggest abnormal activity of the MAPK- and cAMP-associated pathways in frontal cortical areas in schizophrenia. These alterations may underlie the hypothesized hypoglutamatergic function in this illness. Together with previous findings, these data suggest that abnormalities of intracellular signaling pathways may contribute to the pathophysiology of schizophrenia. PMID:22048463

  6. Melanoma Expressed-CD70 Is Regulated by RhoA and MAPK Pathways without Affecting Vemurafenib Treatment Activity

    PubMed Central

    Sarrabayrouse, Guillaume; Gallardo, Franck; Gence, Rémi; Tilkin-Mariamé, Anne-Françoise

    2016-01-01

    CD70 is a costimulatory molecule member of the Tumor Necrosis Factor family that is expressed on activated immune cells. Its ectopic expression has been described in several types of cancer cells including lymphomas, renal cell carcinomas and glioblastomas. We have recently described its expression in a part of tumor cells from the vast majority of melanoma biopsies and human melanoma cell lines, and found that CD70 expression decreased over time as the disease progressed. Here, we show that RhoA, BRAF and Mitogen Activating Protein Kinase pathways are involved in the positive transcriptional regulation of CD70 expression in melanomas. Interestingly, the clinical inhibitor of the common BRAF V600E/D variants, Vemurafenib (PLX-4032), which is currently used to treat melanoma patients with BRAF V600E/D-mutated metastatic melanomas, decreased CD70 expression in human CD70+ melanoma cell lines. This decrease was seen in melanoma cells both with and without the BRAFV600E/D mutation, although was less efficient in those lacking the mutation. But interestingly, by silencing CD70 in CD70+ melanoma cell lines we show that PLX-4032-induced melanoma cell killing and its inhibitory effect on MAPK pathway activation are unaffected by CD70 expression. Consequently, our work demonstrates that CD70 ectopic expression in melanomas is not a valuable biomarker to predict tumor cells sensitivity to BRAF V600 inhibitors. PMID:26828592

  7. Melanoma Expressed-CD70 Is Regulated by RhoA and MAPK Pathways without Affecting Vemurafenib Treatment Activity.

    PubMed

    Pich, Christine; Teiti, Iotefa; Sarrabayrouse, Guillaume; Gallardo, Franck; Gence, Rémi; Tilkin-Mariamé, Anne-Françoise

    2016-01-01

    CD70 is a costimulatory molecule member of the Tumor Necrosis Factor family that is expressed on activated immune cells. Its ectopic expression has been described in several types of cancer cells including lymphomas, renal cell carcinomas and glioblastomas. We have recently described its expression in a part of tumor cells from the vast majority of melanoma biopsies and human melanoma cell lines, and found that CD70 expression decreased over time as the disease progressed. Here, we show that RhoA, BRAF and Mitogen Activating Protein Kinase pathways are involved in the positive transcriptional regulation of CD70 expression in melanomas. Interestingly, the clinical inhibitor of the common BRAF V600E/D variants, Vemurafenib (PLX-4032), which is currently used to treat melanoma patients with BRAF V600E/D-mutated metastatic melanomas, decreased CD70 expression in human CD70+ melanoma cell lines. This decrease was seen in melanoma cells both with and without the BRAFV600E/D mutation, although was less efficient in those lacking the mutation. But interestingly, by silencing CD70 in CD70+ melanoma cell lines we show that PLX-4032-induced melanoma cell killing and its inhibitory effect on MAPK pathway activation are unaffected by CD70 expression. Consequently, our work demonstrates that CD70 ectopic expression in melanomas is not a valuable biomarker to predict tumor cells sensitivity to BRAF V600 inhibitors. PMID:26828592

  8. ERK/MAPK regulates ERRγ expression, transcriptional activity, and receptor-mediated Tamoxifen resistance in ER+ breast cancer

    PubMed Central

    Heckler, Mary Mazzotta; Thakor, Hemang; Schafer, Cara C.; Riggins, Rebecca B.

    2014-01-01

    Background Selective estrogen receptor modulators (SERMs) such as Tamoxifen (TAM) can significantly improve breast cancer-specific survival for women with ER-positive (ER+) disease. However, resistance to TAM remains a major clinical problem. The resistant phenotype is usually not driven by loss or mutation of ER; instead, changes in multiple proliferative and/or survival pathways override the inhibitory effects of TAM. Estrogen-related receptor gamma (ERRγ) is an orphan member of the nuclear receptor superfamily that promotes TAM resistance in ER+ breast cancer cells. In this study, we sought to clarify the mechanism(s) by which this orphan nuclear receptor is regulated and, in turn, affects TAM resistance. Methods mRNA and protein expression/phosphorylation were monitored by RT-PCR and Western blotting, respectively. Site-directed mutagenesis was used to disrupt consensus ERK target sites. Cell proliferation and cell cycle progression were measured by flow cytometric methods. ERRγ transcriptional activity was assessed by dual-luciferase promoter-reporter assays. Results We show that ERRγ protein levels are affected by the activation state of ERK/MAPK, and mutation of consensus ERK target sites impairs ERRγ-driven transcriptional activity and TAM resistance. Conclusions These findings shed new light on the functional significance of ERRγ in ER+ breast cancer, and are the first to demonstrate a role for kinase regulation of this orphan nuclear receptor. PMID:24684682

  9. Quercetin modulates NF-kappa B and AP-1/JNK pathways to induce cell death in human hepatoma cells.

    PubMed

    Granado-Serrano, Ana Belén; Martín, María Angeles; Bravo, Laura; Goya, Luis; Ramos, Sonia

    2010-01-01

    Quercetin, a dietary flavonoid, has been shown to possess anticarcinogenic properties, but the precise molecular mechanisms of action are not thoroughly elucidated. The aim of this study was to investigate the regulatory effect of quercetin (50 microM) on two main transcription factors (NF-kappa B and AP-1) related to survival/proliferation pathways in a human hepatoma cell line (HepG2) over time. Quercetin induced a significant time-dependent inactivation of the NF-kappa B pathway consistent with a downregulation of the NF-kappa B binding activity (from 15 min onward). These features were in concert with a time-dependent activation (starting at 15 min and maintained up to 18 h) of the AP-1/JNK pathway, which played an important role in the control of the cell death induced by the flavonoid and contributed to the regulation of survival/proliferation (AKT, ERK) and death (caspase-3, p38, unbalance of Bcl-2 proapoptotic and antiapoptotic proteins) signals. These data suggest that NF-kappa B and AP-1 play a main role in the tight regulation of survival/proliferation pathways exerted by quercetin and that the sustained JNK/AP-1 activation and inhibition of NF-kappa B provoked by the flavonoid induced HepG2 death. PMID:20358477

  10. Rho1 GTPase and PKC Ortholog Pck1 Are Upstream Activators of the Cell Integrity MAPK Pathway in Fission Yeast

    PubMed Central

    Sánchez-Mir, Laura; Soto, Teresa; Franco, Alejandro; Madrid, Marisa; Viana, Raúl A.; Vicente, Jero; Gacto, Mariano; Pérez, Pilar; Cansado, José

    2014-01-01

    In the fission yeast Schizosaccharomyces pombe the cell integrity pathway (CIP) orchestrates multiple biological processes like cell wall maintenance and ionic homeostasis by fine tuning activation of MAPK Pmk1 in response to various environmental conditions. The small GTPase Rho2 positively regulates the CIP through protein kinase C ortholog Pck2. However, Pmk1 retains some function in mutants lacking either Rho2 or Pck2, suggesting the existence of additional upstream regulatory elements to modulate its activity depending on the nature of the environmental stimulus. The essential GTPase Rho1 is a candidate to control the activity of the CIP by acting upstream of Pck2, whereas Pck1, a second PKC ortholog, appears to negatively regulate Pmk1 activity. However, the exact regulatory nature of these two proteins within the CIP has remained elusive. By exhaustive characterization of strains expressing a hypomorphic Rho1 allele (rho1-596) in different genetic backgrounds we show that both Rho1 and Pck1 are positive upstream regulatory members of the CIP in addition to Rho2 and Pck2. In this new model Rho1 and Rho2 control Pmk1 basal activity during vegetative growth mainly through Pck2. Notably, whereas Rho2-Pck2 elicit Pmk1 activation in response to most environmental stimuli, Rho1 drives Pmk1 activation through either Pck2 or Pck1 exclusively in response to cell wall damage. Our study reveals the intricate and complex functional architecture of the upstream elements participating in this signaling pathway as compared to similar routes from other simple eukaryotic organisms. PMID:24498240

  11. Transforming growth factor-beta1-induced activation of the Raf-MEK-MAPK signaling pathway in rat lung fibroblasts via a PKC-dependent mechanism.

    PubMed

    Axmann, A; Seidel, D; Reimann, T; Hempel, U; Wenzel, K W

    1998-08-19

    In fibroblasts transforming growth factor-beta1 (TGF-beta1) regulates cell proliferation and turnover of macromolecular components of the extracellular matrix. Here, intracellular signaling events in growth-inhibited embryonic rat lung fibroblasts (RFL-6) upon stimulation with TGF-beta1 were investigated. TGF-beta1 rapidly induced the activation of c-Raf-1, MEK-1, and MAPK p42 and p44. The activation of this pathway by TGF-beta1 did not depend on autocrine platelet-derived growth factor (PDGF) or basic fibroblast growth factor (bFGF). Inhibition of the binding of growth factors to their tyrosine kinase receptors did not affect MAPK activation by TGF-beta1. Ras activation by TGF-beta1 was significantly lower compared to the activation by PDGF or bFGF. The intracellular transduction of the TGF-beta1 signal was completely suppressed by depletion or inhibition of protein kinase C (PKC). It is shown that calcium-dependent isoforms of PKC are required for MAPK activation by TGF-beta1. PMID:9712718

  12. Subanesthetic Isoflurane Reduces Zymosan-Induced Inflammation in Murine Kupffer Cells by Inhibiting ROS-Activated p38 MAPK/NF-κB Signaling

    PubMed Central

    Wang, Hui; Wang, Lei; Li, Nan-lin; Li, Jun-tang; Yu, Feng; Zhao, Ya-li; Wang, Ling; Yi, Jun; Wang, Ling; Bian, Jie-fang; Chen, Jiang-hao; Yuan, Shi-fang; Wang, Ting; Lv, Yong-gang; Liu, Ning-ning; Zhu, Xiao-shan; Ling, Rui; Yun, Jun

    2014-01-01

    Volatile anesthetic isoflurane (ISO) has immunomodulatory effects. The fungal component zymosan (ZY) induces inflammation through toll-like receptor 2 or dectin-1 signaling. We investigated the molecular actions of subanesthetic (0.7%) ISO against ZY-induced inflammatory activation in murine Kupffer cells (KCs), which are known as the resident macrophages within the liver. We observed that ISO reduced ZY-induced cyclooxygenase 2 upregulation and prostaglandin E2 release, as determined by western blot and radioimmunoassay, respectively. ISO also reduced the production of tumor necrosis factor-α, interleukin-1β, IL-6, high-mobility group box-1, macrophage inflammatory protein-1α, macrophage inflammatory protein-2, and monocyte chemoattractant protein-1 as assessed by enzyme-linked immunosorbent assays. ISO blocked the ZY-induced nuclear translocation and DNA-binding activity of nuclear factor- (NF)-κB p65. Moreover, ISO attenuated ZY-induced p38 mitogen-activated protein kinase (MAPK) activation partly by scavenging reactive oxygen species (ROS); the interregulation that ROS activated p38 MAPK followed by NF-κB activation was crucial for the ZY-induced inflammatory responses in KCs. An in vivo study by peritoneal injection of ZY into BALB/C mice confirmed the anti-inflammatory properties of 0.7% ISO against ZY in KCs. These results suggest that ISO ameliorates ZY-induced inflammatory responses in murine KCs by inhibiting the interconnected ROS/p38 MAPK/NF-κB signaling pathways. PMID:25147596

  13. The Mos/mitogen-activated protein kinase (MAPK) pathway regulates the size and degradation of the first polar body in maturing mouse oocytes.

    PubMed Central

    Choi, T; Fukasawa, K; Zhou, R; Tessarollo, L; Borror, K; Resau, J; Vande Woude, G F

    1996-01-01

    Mos is an upstream activator of mitogen-activated protein kinase (MAPK) and, in mouse oocytes, is responsible for metaphase II arrest. This activity has been likened to its function in Xenopus oocytes as a component of cytostatic factor. Thus, Mos-deficient female mice (MOS-/-) are less fertile and oocytes derived from these animals fail to arrest at metaphase II and undergo parthenogenetic activation [Colledge, W. H., Carlton, M. B. L., Udy, C. B. & Evans, M. J. (1994) Nature (London) 370, 65-68 and Hashimoto, N., Watanabe, N., Furuta. Y., Tamemoto, B., Sagata, N., Yokoyama, M., Okazaki, K., Nagayoshi, M., Takeda, N., Ikawa, Y. & Aizawa, S. (1994) Nature (London) 370, 68-71]. Here we show that maturing MOS-/- oocytes fail to activate MAPK throughout meiosis, while p34cdc2 kinase activity is normal until late in metaphase II when it decreases prematurely. Phenotypically, the first meiotic division of MOS-/- oocytes frequently resembles mitotic cleavage or produces an abnormally large polar body. In these oocytes, the spindle shape is altered and the spindle fails to translocate to the cortex, leading to the establishment of an altered cleavage plane. Moreover, the first polar body persists instead of degrading and sometimes undergoes an additional cleavage, thereby providing conditions for parthenogenesis. These studies identify meiotic spindle formation and programmed degradation of the first polar body as new and important roles for the Mos/MAPK pathway. Images Fig. 1 Fig. 2 Fig. 3 PMID:8692939

  14. Neferine, an alkaloid from lotus seed embryo, inhibits human lung cancer cell growth by MAPK activation and cell cycle arrest.

    PubMed

    Poornima, Paramasivan; Weng, Ching Feng; Padma, Viswanadha Vijaya

    2014-01-01

    Neferine is the major bisbenzylisoquinoline alkaloid isolated from the seed embryo of a traditional medicinal plant Nelumbo nucifera (Lotus). Epidemiological studies have revealed the therapeutic potential of lotus seed embryo. Although several mechanisms have been proposed, a clear anticancer action mechanism of neferine on lung cancer cells is still not known. Lung cancer is the most common cause of cancer death in the world, and the patients with advanced stage of nonsmall lung cancer require adjunct chemotherapy after surgical resection for the eradication of cancer cells. In this study, the effects of neferine were evaluated and characterized in A549 cells. Neferine induced apoptosis in a dose-dependent manner with the hypergeneration of reactive oxygen species, activation of MAPKs, lipid peroxidation, depletion of cellular antioxidant pool, loss of mitochondrial membrane potential, and intracellular calcium accumulation. Furthermore, neferine treatment leads to the inhibition of nuclear factor kappaB and Bcl2, upregulation of Bax and Bad, release of cytochrome C, activation of caspase cascade, and DNA fragmentation. In addition, neferine could induce p53 and its effector protein p21 and downregulation of cell cycle regulatory protein cyclin D1 thereby inducing G1 cell cycle arrest. These results suggest a novel function of neferine as an apoptosis inducer in lung cancer cells. PMID:23983146

  15. Activation of transient receptor potential vanilloid 4 induces apoptosis in hippocampus through downregulating PI3K/Akt and upregulating p38 MAPK signaling pathways

    PubMed Central

    Jie, P; Hong, Z; Tian, Y; Li, Y; Lin, L; Zhou, L; Du, Y; Chen, L; Chen, L

    2015-01-01

    Transient receptor potential vanilloid 4 (TRPV4) is a calcium-permeable cation channel that is sensitive to cell swelling, arachidonic acid and its metabolites, epoxyeicosatrienoic acids, which are associated with cerebral ischemia. The activation of TRPV4 induces cytotoxicity in many types of cells, accompanied by an increase in the intracellular free calcium concentration. TRPV4 activation modulates the mitogen-activated protein kinase (MAPK) and phosphatidyl inositol 3 kinase (PI3K)/ protein kinase B (Akt) signaling pathways that regulate cell death and survival. Herein, we examined TRPV4-induced neuronal apoptosis by intracerebroventricular (ICV) injection of a TRPV4 agonist (GSK1016790A) and assessed its involvement in cerebral ischemic injury. ICV injection of GSK1016790A dose-dependently induced apoptosis in the mouse hippocampi (GSK-injected mice). The protein level of phosphorylated p38 MAPK (p-p38 MAPK) was markedly increased and that of phosphorylated c-Jun N-terminal protein kinase (p-JNK) was virtually unchanged. TRPV4 activation also decreased Bcl-2/Bax protein ratio and increased the cleaved caspase-3 protein level, and these effects were blocked by a PI3K agonist and a p38 MAPK antagonist, but were unaffected by a JNK antagonist. ICV injection of the TRPV4 antagonist HC-067047 reduced brain infarction after reperfusion for 48 h in mice with middle cerebral artery occlusion (MCAO). In addition, HC-067047 treatment attenuated the decrease in the phosphorylated Akt protein level and the increase in p-p38 MAPK protein level at 48 h after MCAO, while the increase in p-JNK protein level remained unchanged. Finally, the decreased Bcl-2/Bax protein ratio and the increased cleaved caspase-3 protein level at 48 h after MCAO were markedly attenuated by HC-067047. We conclude that activation of TRPV4 induces apoptosis by downregulating PI3K/Akt and upregulating p38 MAPK signaling pathways, which is involved in cerebral ischemic injury. PMID:26043075

  16. Rhesus macaque θ-defensin RTD-1 inhibits proinflammatory cytokine secretion and gene expression by inhibiting the activation of NF-κB and MAPK pathways.

    PubMed

    Tongaonkar, Prasad; Trinh, Katie K; Schaal, Justin B; Tran, Dat; Gulko, Percio S; Ouellette, André J; Selsted, Michael E

    2015-12-01

    θ-Defensins are pleiotropic, macrocyclic peptides that are expressed uniquely in Old World monkeys. The peptides are potent, broad-spectrum microbicides that also modulate inflammatory responses in vitro and in animal models of viral infection and polymicrobial sepsis. θ-Defensins suppress proinflammatory cytokine secretion by leukocytes stimulated with diverse Toll-like receptor (TLR) ligands. Studies were performed to delineate anti-inflammatory mechanisms of rhesus θ-defensin 1 (RTD-1), the most abundant θ-defensin isoform in macaque granulocytes. RTD-1 reduced the secretion of tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, and IL-8 in lipopolysaccharide (LPS)-stimulated human blood monocytes and THP-1 macrophages, and this was accompanied by inhibition of nuclear factor κB (NF-κB) activation and mitogen-activated protein kinase (MAPK) pathways. Peptide inhibition of NF-κB activation occurred following stimulation of extracellular (TLRs 1/2 and 4) and intracellular (TLR9) receptors. Although RTD-1 did not inhibit MAPK in unstimulated cells, it induced phosphorylation of Akt in otherwise untreated monocytes and THP-1 cells. In the latter, this occurred within 10 min of RTD-1 treatment and produced a sustained elevation of phosphorylated Akt (pAkt) for at least 4 h. pAkt is a negative regulator of MAPK and NF-κB activation. RTD-1 inhibited IκBα degradation and p38 MAPK phosphorylation, and stimulated Akt phosphorylation in LPS-treated human primary monocytes and THP-1 macrophages. Specific inhibition of phosphatidylinositol 3-kinase (PI3K) blocked RTD-1-stimulated Akt phosphorylation and reversed the suppression of NF-κB activation by the peptide. These studies indicate that the anti-inflammatory properties of θ-defensins are mediated by activation of the PI3K/Akt pathway and suppression of proinflammatory signals in immune-stimulated cells. PMID:26269197

  17. Attenuation of the macrophage inflammatory activity by TiO2 nanotubes via inhibition of MAPK and NF-κB pathways

    PubMed Central

    Neacsu, Patricia; Mazare, Anca; Schmuki, Patrik; Cimpean, Anisoara

    2015-01-01

    Biomaterial implantation in a living tissue triggers the activation of macrophages in inflammatory events, promoting the transcription of pro-inflammatory mediator genes. The initiation of macrophage inflammatory processes is mainly regulated by signaling proteins of mitogen-activated protein kinase (MAPK) and by nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathways. We have previously shown that titania nanotubes modified Ti surfaces (Ti/TiO2) mitigate the immune response, compared with flat Ti surfaces; however, little is known regarding the underlying mechanism. Therefore, the aim of this study is to investigate the mechanism(s) by which this nanotopography attenuates the inflammatory activity of macrophages. Thus, we analyzed the effects of TiO2 nanotubes on the activation of MAPK and NF-κB signaling pathways in standard and lipopolysaccharide-evoked conditions. Results showed that the Ti/TiO2 significantly reduce the expression levels of the phosphorylated forms of p38, ERK1/2, c-Jun NH2-terminal kinase (JNK), IKKβ, and IkB-α. Furthermore, a significant reduction in the p65 nuclear accumulation on the nanotubular surface was remarked. Following, by using specific MAPK inhibitors, we observed that lipopolysaccharide-induced production of monocyte chemotactic protein-1 and nitric oxide was significantly inhibited on the Ti/TiO2 surface via p38 and ERK1/2, but not via JNK. However, the selective inhibitor for JNK signaling pathway (SP600125) was effective in reducing tumor necrosis factor alpha release as well as monocyte chemotactic protein-1 and nitric oxide production. Altogether, these data suggest that titania nanotubes can attenuate the macrophage inflammatory response via suppression of MAPK and NF-κB pathways providing a potential mechanism for their anti-inflammatory activity. PMID:26491301

  18. Astragalin inhibits IL-1β-induced inflammatory mediators production in human osteoarthritis chondrocyte by inhibiting NF-κB and MAPK activation.

    PubMed

    Ma, Zhiqiang; Piao, Taikui; Wang, Yanlong; Liu, Jianyu

    2015-03-01

    Astragalin, a bioactive component isolated from Rosa agrestis, has been described to exhibit anti-inflammatory activity. The aim of this study was to investigate the anti-inflammatory effects and the underlying mechanisms of astragalin on IL-1β-stimulated human osteoarthritis chondrocyte. The production of NO and PGE2 was detected by Griess reaction and ELISA. The expression of iNOS and COX-2 was detected by western blotting. The expression of NF-κB and MAPKs was detected by western blot analysis. We found that astragalin dose-dependently inhibited IL-1β-induced NO and PGE2 production, as well as iNOS and COX-2 expression. Meanwhile, western blot analysis showed that astragalin inhibited IL-1β-induced NF-κB and MAPK activation in human osteoarthritis chondrocyte. In addition, astragalin was found to activate PPAR-γ. The inhibition of astragalin on IL-1β-induced NO and PGE2 production can be reversed by PPAR-γ antagonist GW9662. Astragalin suppressed IL-1β-induced inflammatory mediators via activating PPAR-γ, which subsequently inhibited IL-1β-induced NF-κB and MAPK activation. Astragalin may be a potential agent in the treatment of osteoarthritis. PMID:25637445

  19. Vorinostat Enhances Cytotoxicity of SN-38 and Temozolomide in Ewing Sarcoma Cells and Activates STAT3/AKT/MAPK Pathways

    PubMed Central

    Sampson, Valerie B.; Vetter, Nancy S.; Kamara, Davida F.; Collier, Anderson B.; Gresh, Renee C.; Kolb, E. Anders

    2015-01-01

    Histone deacetylase inhibitors (HDACi) have been evaluated in patients with Ewing sarcoma (EWS) but demonstrated limited activity. To better understand the potential for HDACi in EWS, we evaluated the combination of the HDACi vorinostat, with DNA damaging agents SN-38 (the active metabolite of irinotecan and topoisomerase 1 inhibitor) plus the alkylating agent temozolomide (ST). Drugs were evaluated in sequential and simultaneous combinations in two EWS cell lines. Results demonstrate that cell viability, DNA damage and reactive oxygen species (ROS) production are dependent on the sequence of drug administration. Enhanced cytotoxicity is exhibited in vitro in EWS cell lines treated with ST administered before vorinostat, which was modestly higher than concomitant treatment and superior to vorinostat administered before ST. Drug combinations downregulate cyclin D1 to induce G0/G1 arrest and promote apoptosis by cleavage of caspase-3 and PARP. When ST is administered before or concomitantly with vorinostat there is activation of STAT3, MAPK and the p53 pathway. In contrast, when vorinostat is administered before ST, there is DNA repair, increased AKT phosphorylation and reduced H2B acetylation. Inhibition of AKT using the small molecule inhibitor MK-2206 did not restore H2B acetylation. Combining ST with the dual ALK and IGF-1R inhibitor, AZD3463 simultaneously inhibited STAT3 and AKT to enhance the cytotoxic effects of ST and further reduce cell growth suggesting that STAT3 and AKT activation were in part mediated by ALK and IGF-1R signaling. In summary, potent antiproliferative and proapoptotic activity were demonstrated for ST induced DNA damage before or simultaneous with HDAC inhibition and cell death was mediated through the p53 pathway. These observations may aid in designing new protocols for treating pediatric patients with high-risk EWS. PMID:26571493

  20. Resveratrol Inhibits LPS-Induced MAPKs Activation via Activation of the Phosphatidylinositol 3-Kinase Pathway in Murine RAW 264.7 Macrophage Cells

    PubMed Central

    Liu, Bin; Deng, Yi-Shu; Zhan, Dong; Chen, Yuan-Li; He, Ying; Liu, Jing; Zhang, Zong-Ji; Sun, Jun; Lu, Di

    2012-01-01

    Background Resveratrol is a natural polyphenolic compound that has cardioprotective, anticancer and anti-inflammatory properties. We investigated the capacity of resveratrol to protect RAW 264.7 cells from inflammatory insults and explored mechanisms underlying inhibitory effects of resveratrol on RAW 264.7 cells. Methodology/Principal Findings Murine RAW 264.7 cells were treated with resveratrol (1, 5, and 10 µM) and/or LPS (5 µg/ml). Nitric oxide (NO) and prostaglandin E2 (PGE2) were measured by Griess reagent and ELISA. The mRNA and protein levels of proinflammatory proteins and cytokines were analysed by ELISA, RT-PCR and double immunofluorescence labeling, respectively. Phosphorylation levels of Akt, cyclic AMP-responsive element-binding protein (CREB), mitogen-activated protein kinases (MAPKs) cascades, AMP-activated protein kinase (AMPK) and expression of SIRT1(Silent information regulator T1) were measured by western blot. Wortmannin (1 µM), a specific phosphatidylinositol 3-kinase (PI3-K) inhibitor, was used to determine if PI3-K/Akt signaling pathway might be involved in resveratrol’s action on RAW 264.7 cells. Resveratrol significantly attenuated the LPS-induced expression of nitric oxide (NO), prostaglandin E2 (PGE2), inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) in RAW 264.7 cells. Resveratrol increased Akt phosphorylation in a time-dependent manner. Wortmannin, a specific phosphatidylinositol 3-kinase (PI3-K) inhibitor, blocked the effects of resveratrol on LPS-induced RAW 264.7 cells activation. In addition, PI3-K inhibition partially abolished the inhibitory effect of resveratrol on the phosphorylation of cyclic AMP-responsive element-binding protein (CREB) and mitogen-activated protein kinases (MAPKs) cascades. Meanwhile, PI3-K is essential for resveratrol-mediated phosphorylation of AMPK and expression of SIRT1. Conclusion and Implications This investigation

  1. Extracellular Hsp90 serves as a co-factor for MAPK activation and latent viral gene expression during de novo infection by KSHV

    SciTech Connect

    Qin Zhiqiang; DeFee, Michael; Isaacs, Jennifer S.; Parsons, Chris

    2010-07-20

    The Kaposi's sarcoma-associated herpesvirus (KSHV) is the causative agent of Kaposi's sarcoma (KS), an important cause of morbidity and mortality in immunocompromised patients. KSHV interaction with the cell membrane triggers activation of specific intracellular signal transduction pathways to facilitate virus entry, nuclear trafficking, and ultimately viral oncogene expression. Extracellular heat shock protein 90 localizes to the cell surface (csHsp90) and facilitates signal transduction in cancer cell lines, but whether csHsp90 assists in the coordination of KSHV gene expression through these or other mechanisms is unknown. Using a recently characterized non-permeable inhibitor specifically targeting csHsp90 and Hsp90-specific antibodies, we show that csHsp90 inhibition suppresses KSHV gene expression during de novo infection, and that this effect is mediated largely through the inhibition of mitogen-activated protein kinase (MAPK) activation by KSHV. Moreover, we show that targeting csHsp90 reduces constitutive MAPK expression and the release of infectious viral particles by patient-derived, KSHV-infected primary effusion lymphoma cells. These data suggest that csHsp90 serves as an important co-factor for KSHV-initiated MAPK activation and provide proof-of-concept for the potential benefit of targeting csHsp90 for the treatment or prevention of KSHV-associated illnesses.

  2. Apoptosis Induction by the Total Flavonoids from Arachniodes exilis in HepG2 Cells through Reactive Oxygen Species-Mediated Mitochondrial Dysfunction Involving MAPK Activation

    PubMed Central

    Chen, Jing; Xiong, Chaomei; Wei, Han; Yin, Changchang; Ruan, Jinlan

    2014-01-01

    Arachniodes exilis is used as a folk medicine in China and proved to have antibacterial, anti-inflammatory, and sedative activities. In the present study, the antitumor effect of the total flavonoids of A. exilis (TFAE) against HepG2 cells was evaluated. The results showed that TFAE inhibited the growth of HepG2 cells in a dosage- and time-dependent manner. Flow cytometry and Hoechst 33342 fluorescence staining results showed that TFAE could significantly increase the apoptosis ratio of HepG2 cells, which is accompanied with increased intracellular reactive oxygen species (ROS) production and decreased mitochondrial membrane potential (ΔΨm). Western blotting indicated that TFAE downregulated the ratio of Bcl-2/Bax, increased cytochrome c release, and activated the caspases-3 and -9. Further analysis showed that TFAE stimulated the mitogen-activated protein kinase (MAPK). However, treatment with NAC (reactive oxygen species scavenger) and MAPK-specific inhibitors (SP600125 and SB203580) could reverse the changes of these apoptotic-related proteins. These results suggested that TFAE possessed potential anticancer activity in HepG2 cells through ROS-mediated mitochondrial dysfunction involving MAPK pathway. PMID:24976852

  3. Extracellular Hsp90 serves as a co-factor for MAPK activation and latent viral gene expression during de novo infection by KSHV

    PubMed Central

    Qin, Zhiqiang; DeFee, Michael; Isaacs, Jennifer; Parsons, Chris

    2010-01-01

    The Kaposi’s sarcoma-associated herpesvirus (KSHV) is the causative agent of Kaposi’s sarcoma (KS), an important cause of morbidity and mortality in immunocompromised patients. KSHV interaction with the cell membrane triggers activation of specific intracellular signal transduction pathways to facilitate virus entry, nuclear trafficking, and ultimately viral oncogene expression. Extracellular heat shock protein 90 localizes to the cell surface (csHsp90) and facilitates signal transduction in cancer cell lines, but whether csHsp90 assists in the coordination of KSHV gene expression through these or other mechanisms is unknown. Using a recently characterized non-permeable inhibitor specifically targeting csHsp90, we show that csHsp90 inhibition suppresses KSHV gene expression during de novo infection, and that this effect is mediated largely through the inhibition of mitogen-activated protein kinase (MAPK) activation by KSHV. Moreover, we show that targeting csHsp90 reduces constitutive MAPK expression and the release of infectious viral particles by patient-derived, KSHV-infected primary effusion lymphoma cells. These data suggest that csHsp90 serves as an important co-factor for KSHV-initiated MAPK activation and provide proof-of-concept for the potential benefit of targeting csHsp90 for the treatment or prevention of KSHV-associated illnesses. PMID:20451233

  4. Exome sequencing of desmoplastic melanoma identifies recurrent NFKBIE promoter mutations and diverse activating mutations in the MAPK pathway.

    PubMed

    Shain, A Hunter; Garrido, Maria; Botton, Thomas; Talevich, Eric; Yeh, Iwei; Sanborn, J Zachary; Chung, Jongsuk; Wang, Nicholas J; Kakavand, Hojabr; Mann, Graham J; Thompson, John F; Wiesner, Thomas; Roy, Ritu; Olshen, Adam B; Gagnon, Alexander; Gray, Joe W; Huh, Nam; Hur, Joe S; Busam, Klaus J; Scolyer, Richard A; Cho, Raymond J; Murali, Rajmohan; Bastian, Boris C

    2015-10-01

    Desmoplastic melanoma is an uncommon variant of melanoma with sarcomatous histology, distinct clinical behavior and unknown pathogenesis. We performed low-coverage genome and high-coverage exome sequencing of 20 desmoplastic melanomas, followed by targeted sequencing of 293 genes in a validation cohort of 42 cases. A high mutation burden (median of 62 mutations/Mb) ranked desmoplastic melanoma among the most highly mutated cancers. Mutation patterns strongly implicate ultraviolet radiation as the dominant mutagen, indicating a superficially located cell of origin. Newly identified alterations included recurrent promoter mutations of NFKBIE, encoding NF-κB inhibitor ɛ (IκBɛ), in 14.5% of samples. Common oncogenic mutations in melanomas, in particular in BRAF (encoding p.Val600Glu) and NRAS (encoding p.Gln61Lys or p.Gln61Arg), were absent. Instead, other genetic alterations known to activate the MAPK and PI3K signaling cascades were identified in 73% of samples, affecting NF1, CBL, ERBB2, MAP2K1, MAP3K1, BRAF, EGFR, PTPN11, MET, RAC1, SOS2, NRAS and PIK3CA, some of which are candidates for targeted therapies. PMID:26343386

  5. PBX3 promotes migration and invasion of colorectal cancer cells via activation of MAPK/ERK signaling pathway

    PubMed Central

    Han, Hai-Bo; Gu, Jin; Ji, Deng-Bo; Li, Zhao-Wei; Zhang, Yuan; Zhao, Wei; Wang, Li-Min; Zhang, Zhi-Qian

    2014-01-01

    AIM: To investigate the role of pre-B-cell leukemia homeobox (PBX)3 in migration and invasion of colorectal cancer (CRC) cells. METHODS: We detected PBX3 expression in five cell lines and surgical specimens from 111 patients with CRC using real-time reverse transcription-polymerase chain reaction. We forced expression of PBX3 in low metastatic HT-29 and SW480 cells and knocked down expression of PBX3 in highly metastatic LOVO and HCT-8 cells. Wound healing and Boyden chamber assays were used to detect cell migration and invasion after altered expression of PBX3. Western blot was performed to detect the change of signaling molecule ERK1/2 following PBX3 overexpression. RESULTS: High level of PBX3 expression was correlated with the invasive potential of CRC cells, and significantly associated with lymph node invasion (P = 0.02), distant metastasis (P = 0.04), advanced TNM stage (P = 0.03) and poor overall survival of patients (P < 0.05). Ectopic expression of PBX3 in low metastatic cells was shown to promote migration and invasion, while inhibited PBX3 expression in highly metastatic cells suppressed migration and invasion. Furthermore, upregulation of phosphorylated extracellular signal-regulated kinase (ERK)1/2 was found to be one of the targeted molecules responsible for PBX3-induced CRC cell migration and invasion. CONCLUSION: PBX3 induces invasion and metastasis of CRC cells partially through activation of the MAPK/ERK signaling pathway. PMID:25561793

  6. Trim69 regulates zebrafish brain development by ap-1 pathway

    PubMed Central

    Han, Ruiqin; Wang, Renxian; Zhao, Qing; Han, Yongqing; Zong, Shudong; Miao, Shiying; Song, Wei; Wang, Linfang

    2016-01-01

    Proteins belonging to the TRIM family have been implicated in a variety of cellular processes such as apoptosis, differentiation, neurogenesis, muscular physiology and innate immune responses. Trim69, previously identified as a novel gene cloned from a human testis cDNA library, has a homologous gene in zebrafish and this study focused on investigating the function of trim69 in zebrafish neurogenesis. Trim69 was found to be expressed in zebrafish embryo brain at the early stages. Knockdown of trim69 led to deformed brain development, obvious signs of apoptosis present in the head, and decreased expression of neuronal differentiation and stem cell markers. This phenotype was rescued upon co-injection of human mRNA together along with the trim69 knockdown. Results of this study also showed an interaction between TRIM69 and c-Jun in human cells, and upon TRIM69 knock down c-Jun expression subsequently increased, whereas the over-expression of TRIM69 led to the down-regulation of c-Jun. Additionally, knockdown both c-Jun and trim69 can rescue the deformed brain, evident cellular apoptosis in the head and decreased expression of neuronal differentiation and stem cell markers. Overall, our results support a role for trim69 in the development of the zebrafish brain through ap-1 pathway. PMID:27050765

  7. Histamine H4 receptor activation alleviates neuropathic pain through differential regulation of ERK, JNK, and P38 MAPK phosphorylation.

    PubMed

    Sanna, Maria D; Stark, Holger; Lucarini, Laura; Ghelardini, Carla; Masini, Emanuela; Galeotti, Nicoletta

    2015-12-01

    Histamine plays a complex role in pain modulation with opposite roles in nociception for histamine receptor subtypes 1, 2, and 3. The histamine H4 receptor (H4R) is expressed primarily on cells involved in inflammation and immune responses with a proinflammatory activity, but little is known about the role in nociception of neuronal H4R. To investigate the effects of neuronal H4R in pain transmission, the effects produced by the H4R agonist ST-1006 were detected in the spared nerve injury model of neuropathic pain. ST-1006 counteracted mechanical allodynia in neuropathic mice, an effect prevented by the H4R antagonist JNJ 10191584. In spared nerve injury mice, an early over-phosphorylation of ERK1 and ERK2 was observed in the dorsal root ganglia (DRG), spinal cord, and sciatic nerve. A progressive and long-lasting activation of JNK1 was observed in the sciatic nerve and, to a lesser extent, in the spinal cord and DRG. An increased p-P38 content was detected in the spinal cord and DRG, with no modification in the sciatic nerve. Administration of ST-1006 prevented phosphorylation of all 3 MAPK within DRG, and phosphorylation of ERK1, ERK2, and pJNK1 in the sciatic nerve. In the spinal cord, the H4R agonist prevented selectively the pERK2 increase with no effect on pJNK1 and p-P38 levels. Double immunofluorescence experiments showed a neuronal localization and site of action for H4R. These findings suggest a prevalent modulation of ERK activity after H4R stimulation and indicate the DRG as prominent site of action for H4R-mediated antineuropathic activity. Targeting neuronal H4R with selective agonists could have therapeutic potential for neuropathic pain treatment. PMID:26270581

  8. Enhancement of Flow-Induced AP-1 Gene Expression by Cyclosporin A Requires NFAT-Independent Signaling in Bone Cells

    PubMed Central

    WORTON, LEAH E.; KWON, RONALD Y.; GARDINER, EDITH M.; GROSS, TED S.; SRINIVASAN, SUNDAR

    2014-01-01

    Growing evidence suggests that aging compromises the ability of the skeleton to respond to anabolic mechanical stimuli. Recently, we reported that treating senescent mice with Cyclosporin A (CsA) rescued aging-related deficits in loading-induced bone formation. Given that the actions of CsA are often attributed to inhibition of the calcineurin/NFAT axis, we hypothesized that CsA enhances gene expression in bone cells exposed to fluid flow, by inhibiting nuclear NFATc1 accumulation. When exposed to flow, MC3T3-E1 osteoblastic cells exhibited rapid nuclear accumulation of NFATc1 that was abolished by CsA treatment. Under differentiation conditions, intermittent CsA treatment enhanced gene expression of late osteoblastic differentiation markers and activator protein 1 (AP-1) family members. Superimposing flow upon CsA further enhanced expression of the AP-1 members Fra-1 and c-Jun. To delineate the contribution of NFAT in this response, cells were treated with VIVIT, a specific inhibitor of the calcineurin/NFAT interaction. Treatment with VIVIT blocked flow-induced nuclear NFATc1 accumulation but did not recapitulate the CsA-mediated enhancement of flow-induced AP-1 component gene expression. Taken together, our study is the first to demonstrate that CsA enhances mechanically-induced gene expression of AP-1 components in bone cells, and suggests that this response requires calcineurin-dependent mechanisms that are independent of inhibiting NFATc1 nuclear accumulation. PMID:25484988

  9. Selenium inhibits Staphylococcus aureus-induced inflammation by suppressing the activation of the NF-κB and MAPK signalling pathways in RAW264.7 macrophages.

    PubMed

    Bi, Chong-Liang; Wang, Heng; Wang, Yin-Jie; Sun, Jun; Dong, Jun-Sheng; Meng, Xia; Li, Jian-Ji

    2016-06-01

    Inflammation is the hallmark of Staphylococcus aureus (S. aureus)-induced mastitis. Given the interesting relationship between selenium levels and inflammation, this study aimed to demonstrate that selenium modulated the inflammation reaction by suppressing the nuclear factor kappa B (NF-κB) and mitogen activated protein kinase (MAPK) signalling pathways. RAW264.7 macrophages were treated with three different concentrations (1μmol/l, 1.5μmol/l, and 2μmol/l) of Na2SeO3 for 12h before infection with S. aureus for 6h, 8h, and 10h. The results showed that selenium significantly reduced the mRNA expression levels of tumour necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β), and interleukin-6 (IL-6). Furthermore, the release of TNF-α, IL-1β, and IL-6 was decreased significantly with selenium supplementation. In addition, selenium influenced the NF-κB signalling pathway by suppressing the activation of NF-κB p65 and degradation of inhibitory kappa-B (IκB). Selenium also suppressed extracellular regulated protein kinase (Erk), c-Jun N-terminal kinase (Jnk), and p38 phosphorylation through the MAPK signalling pathway. In conclusion, selenium played an anti-inflammation role in RAW264.7 macrophages infected with S. aureus by suppressing the activation of the NF-κB and MAPK signalling pathways. PMID:27036486

  10. Function and Regulation in MAPK Signaling Pathways

    PubMed Central

    Chen, Raymond E.; Thorner, Jeremy

    2007-01-01

    Signaling pathways that activate different mitogen-activated protein kinases (MAPKs) elicit many of the responses that are evoked in cells by changes in certain environmental conditions and upon exposure to a variety of hormonal and other stimuli. These pathways were first elucidated in the unicellular eukaryote Saccharomyces cerevisiae (budding yeast). Studies of MAPK pathways in this organism continue to be especially informative in revealing the molecular mechanisms by which MAPK cascades operate, propagate signals, modulate cellular processes, and are controlled by regulatory factors both internal to and external to the pathways. Here we highlight recent advances and new insights about MAPK-based signaling that have been made through studies in yeast, which provide lessons directly applicable to, and that enhance our understanding of, MAPK-mediated signaling in mammalian cells. PMID:17604854

  11. 4-Hydroxynonenal enhances MMP-9 production in murine macrophages via 5-lipoxygenase-mediated activation of ERK and p38 MAPK

    SciTech Connect

    Lee, Seung J.; Kim, Chae E.; Yun, Mi R.; Seo, Kyo W.; Park, Hye M.; Yun, Jung W.; Shin, Hwa K.; Bae, Sun S.; Kim, Chi D.

    2010-01-15

    Exaggerated levels of 4-hydroxynonenal (HNE) and 5-lipoxygenase (5-LO) co-exist in macrophages in atherosclerotic lesions, and activated macrophages produce MMP-9 that degrades atherosclerotic plaque constituents. This study investigated the effects of HNE on MMP-9 production, and the potential role for 5-LO derivatives in MMP-9 production in murine macrophages. Stimulation of J774A.1 cells with HNE led to activation of 5-LO, as measured by leukotriene B{sub 4} (LTB{sub 4}) production. This was associated with an increased production of MMP-9, which was blunted by inhibition of 5-LO with MK886, a 5-LO inhibitor or with 5-LO siRNA. A cysteinyl-LT{sub 1} (cysLT{sub 1}) receptor antagonist, REV-5901 as well as a BLT{sub 1} receptor antagonist, U-75302, also attenuated MMP-9 production induced by HNE. Furthermore, LTB{sub 4} and cysLT (LTC{sub 4} and LTD{sub 4}) enhanced MMP-9 production in macrophages, suggesting a pivotal role for 5-LO in HNE-mediated production of MMP-9. Among the MAPK pathways, LTB{sub 4} and cysLT enhanced phosphorylation of ERK and p38 MAPK, but not JNK. Linked to these results, a p38 MAPK inhibitor as well as an ERK inhibitor blunted MMP-9 production induced by LT. Collectively, these data suggest that 5-LO-derived LT mediates HNE-induced MMP-9 production via activation of ERK and p38 MAPK pathways, consequently leading to plaque instability in atherosclerosis.

  12. Role of G proteins and modulation of p38 MAPK activation in the protection by nitric oxide against ischemia-reoxygenation injury.

    PubMed

    Rakhit, R D; Kabir, A N; Mockridge, J W; Saurin, A; Marber, M S

    2001-09-01

    Protein kinase C (PKC)-mediated regulation of the mitogen-activated protein kinases (MAPK) may play a role in the protection afforded by ischemic preconditioning (PC). Nitric oxide (NO) can influence MAPK activation via interaction with PKC or farnesylation of low-molecular-weight (LMWT) G proteins. However, we have recently reported the mechanism of NO-induced cardioprotection to be a PKC-independent process. Therefore, we investigated the role of LMWT G proteins and MAPK signaling in NO-induced cardioprotection against simulated ischemia-reoxygenation (SI-R) injury. Neonatal rat cardiomyocytes treated for 90 min with the NO donor S-nitroso-N-acetyl-l,l-penicillamine (SNAP) 1 mM were protected against 6 h of SI (hypoxic conditions at 37 degrees C with 20 mM lactate, 16 mM KCl at pH 6.2) and 24 h reoxygenation under normal culture conditions. NO-induced protection was blocked by the G protein inhibitor alpha-hydroxyfarnesylphosphonic acid (alphaHFP) 10 microM. We studied the time course of p42/44 and p38 MAPK dual-phosphorylation hourly during SI using phospho-specific antibodies. p38 was phosphorylated during SI and the peak phosphorylation was significantly delayed by SNAP pretreatment. The p38 inhibitor SB203580 1 microM, given during SI, protected against injury. Thus the delay in peak p38 activation may contribute to, rather than be the effect of, NO-induced cardioprotection. We have shown that p38beta does not contribute to the total p38 signal in our extracts. Thus there is no detectable beta isoform. We conclude that the main isoform present in these cells and thought to be responsible for the observed phenomenon, is the alpha isoform. PMID:11527399

  13. Activation of peroxisome proliferator-activated receptor-{gamma} (PPAR{gamma}) induces cell death through MAPK-dependent mechanism in osteoblastic cells

    SciTech Connect

    Kim, Sung Hun; Yoo, Chong Il; Kim, Hui Taek; Park, Ji Yeon; Kwon, Chae Hwa; Keun Kim, Yong . E-mail: kim430@pusan.ac.kr

    2006-09-01

    The present study was undertaken to determine the role of the mitogen-activated protein kinase (MAPK) subfamilies in cell death induced by PPAR{gamma} agonists in osteoblastic cells. Ciglitazone and troglitazone, PPAR{gamma} agonists, resulted in a concentration- and time-dependent cell death, which was largely attributed to apoptosis. But a PPAR{alpha} agonist ciprofibrate did not affect the cell death. Ciglitazone caused reactive oxygen species (ROS) generation and ciglitazone-induced cell death was prevented by antioxidants, suggesting an important role of ROS generation in the ciglitazone-induced cell death. ROS generation and cell death induced by ciglitazone were inhibited by the PPAR{gamma} antagonist GW9662. Ciglitazone treatment caused activation of extracellular signal-regulated kinase (ERK) and p38. Activation of ERK was dependent on epidermal growth factor receptor (EGFR) and that of p38 was independent. Ciglitazone-induced cell death was significantly prevented by PD98059, an inhibitor of ERK upstream kinase MEK1/2, and SB203580, a p38 inhibitor. Ciglitazone treatment increased Bax expression and caused a loss of mitochondrial membrane potential, and its effect was prevented by N-acetylcysteine, PD98059, and SB203580. Ciglitazone induced caspase activation, which was prevented by PD98059 and SB203580. The general caspase inhibitor z-DEVD-FMK and the specific inhibitor of caspases-3 DEVD-CHO exerted the protective effect against the ciglitazone-induced cell death. The EGFR inhibitors AG1478 and suramin protected against the ciglitazone-induced cell death. Taken together, these findings suggest that the MAPK signaling pathways play an active role in mediating the ciglitazone-induced cell death of osteoblasts and function upstream of a mitochondria-dependent mechanism. These data may provide a novel insight into potential therapeutic strategies for treatment of osteoporosis.

  14. p12CDK2-AP1 interacts with CD82 to regulate the proliferation and survival of human oral squamous cell carcinoma cells.

    PubMed

    Chai, Juan; Ju, Jun; Zhang, Shao-Wu; Shen, Zhi-Yuan; Liang, Liang; Yang, Xiang-Ming; Ma, Chao; Ni, Qian-Wei; Sun, Mo-Yi

    2016-08-01

    p12 cyclin-dependent kinase 2 (CDK2)-associating protein 1 (p12CDK2-AP1) has been demonstrated to negatively regulate the activity of CDK2. However, the underlying molecular mechanism remains largely unknown. We aimed to determine the potential binding proteins of p12CDK2-AP1 and to elucidate the role of p12CDK2-AP1 in the regulation of the proliferation, invasion, apoptosis, and in vivo growth of human oral squamous cell carcinoma cells. The protein-protein interaction was predicted using computational decision templates. The predicted p12CDK2‑AP1 interacting proteins were overexpressed in human oral squamous cell carcinoma OSCC-15 cells, and the protein binding was examined using co-precipitation (Co-IP). Cell proliferation and invasion were determined via MTT assay and Transwell system, respectively. Cell apoptosis was evaluated using Annexin V-FITC/PI double staining followed by flow cytometric analysis. The in vivo growth of OSCC-15 cells was examined in nude mouse tumor xenografts. We found that overexpression of either p12CDK2-AP1 or CD82 significantly suppressed the proliferation and invasion but promoted the apoptosis of OSCC-15 cells (P<0.05). Importantly, combined overexpression of p12CDK2-AP1 and CD82 showed synergistic antitumor activity compared with the overexpression of a single protein alone (P<0.05). Additionally, the simultaneous overexpression of p12CDK2-AP1 and CD82 significantly suppressed the in vivo tumor growth of OSCC-15 cells in nude mice compared with the negative control (P<0.05). Our findings indicate that p12CDK2-AP1 interacts with CD82 to play a functional role in suppressing the in vitro and in vivo growth of OSCC-15 cells. PMID:27349208

  15. Quantitative proteomic analysis reveals heat stress-induced injury in rat small intestine via activation of the MAPK and NF-κB signaling pathways.

    PubMed

    He, Shasha; Hou, Xiaolin; Xu, Xiaolong; Wan, Changrong; Yin, Peng; Liu, Xiaoxi; Chen, Yuping; Shu, Banchao; Liu, Fenghua; Xu, Jianqin

    2015-03-01

    The intestinal epithelium plays a critical role in absorbing nutrients and maintaining the integrity of the gut barrier. Extreme heat stress induces damage to the intestinal epithelium. However, the protein expression changes and the mechanism behind this damage remain poorly understood. In this study, morphological observation showed that heat stress induced desquamation of intestinal epithelial cells, and destruction of intestinal microvilli and mitochondria. Heat stress-induced changes in the intestinal proteome were quantified using the iTRAQ method followed by mass spectrometry and software analysis. A total of 1689 proteins were identified in rat intestine tissue, of which 41 showed significantly altered expression between the heat stressed and control groups. However, these proteins with significant alterations were involved in biological processes such as cellular assembly and organization, developmental disorder, organismal injury and abnormalities, and inflammation. We found that members of the MAPK and NF-κB signaling pathways act as hub proteins in the network interaction analysis. Furthermore, western blot analysis verified that the MAPK and NF-κB signaling pathways were activated by heat stress as expected. This study suggests that heat stress induces cell cytoskeleton reorganization and an inflammatory response, and the activation of the MAPK and NF-κB signaling pathways, which may ultimately contribute to intestinal injury. PMID:25537883

  16. Fumonisin B1 induces autophagic cell death via activation of ERN1-MAPK8/9/10 pathway in monkey kidney MARC-145 cells.

    PubMed

    Yin, Shutao; Guo, Xiao; Li, Jinghua; Fan, Linghong; Hu, Hongbo

    2016-04-01

    Mycotoxins are secondary fungal metabolites that are capable of inducing a variety of toxic effects in animals and humans resulting from the consumption of the contaminated food. Understanding the mechanisms of the toxicities behind these mycotoxins is required to develop mechanism-based approach to counteract their toxic potential. Fumonisin B1 (FB1) is the most prevalent member of fumonisins that are a group of mycotoxins produced primarily by Fusarium verticillioides and Fusarium proliferatum. Kidney is one of the primary target organs for FB1 action. Using monkey kidney MARC-145 cells as an intro model, we found that FB1 induced caspase-independent programmed cell death accompanied with autophagy induction. Inhibition of autophagy by either chemical inhibitors or RNAi approach led to a significant reduction in cell death by FB1 exposure, indicating possible involvement of autophagy-mediated cell death in nephrotoxicity of FB1. Further mechanistic investigation revealed that activation of ERN1-MAPK8/9/10 axis played a critical role in autophagy induction and autophagy-mediated cell death by FB1 exposure. In addition, we demonstrated that disruption of sphingolipid metabolism was an apical event in FB1-induced ERN1-MAPK8/9/10-mediated autophagic cell death in MARC-145 cells. Lastly, we identified curcumin, a naturally occurring plant phenolic compound, as a possible anti-FB1 agent that can be used to protect kidney cells from FB1-induced cell death through inhibition of MAPK8/9/10 activation. PMID:25925693

  17. Attenuated Leishmania induce pro-inflammatory mediators and influence leishmanicidal activity by p38 MAPK dependent phagosome maturation in Leishmania donovani co-infected macrophages.

    PubMed

    Banerjee, Somenath; Bose, Dipayan; Chatterjee, Nabanita; Das, Subhadip; Chakraborty, Sreeparna; Das, Tanya; Saha, Krishna Das

    2016-01-01

    Promastigote form of Leishmania, an intracellular pathogen, delays phagosome maturation and resides inside macrophages. But till date limited study has been done to manipulate the phagosomal machinery of macrophages to restrict Leishmania growth. Attenuated Leishmania strain exposed RAW 264.7 cells showed a respiratory burst and enhanced production of pro-inflammatory mediators. The augmentation of pro-inflammatory activity is mostly attributed to p38 MAPK and p44/42 MAPK. In our study, these activated macrophages are found to induce phagosome maturation when infected with pathogenic Leishmania donovani. Increased co-localization of carboxyfluorescein succinimidyl ester labeled pathogenic L. donovani with Lysosome was found. Moreover, increased co-localization was observed between pathogenic L. donovani and late phagosomal markers viz. Rab7, Lysosomal Associated Membrane Protein 1, Cathepsin D, Rab9, and V-ATPase which indicate phagosome maturation. It was also observed that inhibition of V-type ATPase caused significant hindrance in attenuated Leishmania induced phagosome maturation. Finally, it was confirmed that p38 MAPK is the key player in acidification and maturation of phagosome in attenuated Leishmania strain pre-exposed macrophages. To our knowledge, this study for the first time reported an approach to induce phagosome maturation in L. donovani infected macrophages which could potentiate short-term prophylactic response in future. PMID:26928472

  18. Attenuated Leishmania induce pro-inflammatory mediators and influence leishmanicidal activity by p38 MAPK dependent phagosome maturation in Leishmania donovani co-infected macrophages

    PubMed Central

    Banerjee, Somenath; Bose, Dipayan; Chatterjee, Nabanita; Das, Subhadip; Chakraborty, Sreeparna; Das, Tanya; Saha, Krishna Das

    2016-01-01

    Promastigote form of Leishmania, an intracellular pathogen, delays phagosome maturation and resides inside macrophages. But till date limited study has been done to manipulate the phagosomal machinery of macrophages to restrict Leishmania growth. Attenuated Leishmania strain exposed RAW 264.7 cells showed a respiratory burst and enhanced production of pro-inflammatory mediators. The augmentation of pro-inflammatory activity is mostly attributed to p38 MAPK and p44/42 MAPK. In our study, these activated macrophages are found to induce phagosome maturation when infected with pathogenic Leishmania donovani. Increased co-localization of carboxyfluorescein succinimidyl ester labeled pathogenic L. donovani with Lysosome was found. Moreover, increased co-localization was observed between pathogenic L. donovani and late phagosomal markers viz. Rab7, Lysosomal Associated Membrane Protein 1, Cathepsin D, Rab9, and V-ATPase which indicate phagosome maturation. It was also observed that inhibition of V-type ATPase caused significant hindrance in attenuated Leishmania induced phagosome maturation. Finally, it was confirmed that p38 MAPK is the key player in acidification and maturation of phagosome in attenuated Leishmania strain pre-exposed macrophages. To our knowledge, this study for the first time reported an approach to induce phagosome maturation in L. donovani infected macrophages which could potentiate short-term prophylactic response in future. PMID:26928472

  19. Glabridin Mediate Caspases Activation and Induces Apoptosis through JNK1/2 and p38 MAPK Pathway in Human Promyelocytic Leukemia Cells

    PubMed Central

    Chien, Ming-Hsien; Chen, Hui-Yu; Yang, Shun-Fa; Hsiao, Pei-Ching

    2014-01-01

    Background Glabridin, a prenylated isoflavonoid of G. glabra L. roots, has been associated with a wide range of biological properties such as regulation of energy metabolism, estrogenic, neuroprotective, anti-osteoporotic, and skin-whitening in previous studies. However, the effect of glabridin on tumor cells metastasis has not been clearly clarified. Here, the molecular mechanism by which glabridin anticancer effects in human promyelocytic leukemia cells was investigated. Methodology and Principal Findings The results showed that glabridin significantly inhibited cell proliferation of four AML cell lines (HL-60, MV4-11, U937, and THP-1). Furthermore, glabridin induced apoptosis of HL-60 cells through caspases-3, -8, and -9 activations and PARP cleavage in dose- and time-dependent manner. Moreover, western blot analysis also showed that glabridin increase phosphorylation of ERK1/2, p38 MAPK and JNK1/2 in dose- and time-dependent manner. Inhibition of p38 MAPK and JNK1/2 by specific inhibitors significantly abolished the glabridin-induced activation of the caspase-3, -8 and -9. Conclusion Taken together, our results suggest that glabridin induced HL-60 cell apoptosis through p38 MAPK and JNK1/2 pathways and could serve as a potential additional chemotherapeutic agent for treating AML. PMID:24901249

  20. Plantamajoside ameliorates lipopolysaccharide-induced acute lung injury via suppressing NF-κB and MAPK activation.

    PubMed

    Wu, Haichong; Zhao, Gan; Jiang, Kangfeng; Chen, Xiuying; Zhu, Zhe; Qiu, Changwei; Li, Chengye; Deng, Ganzhen

    2016-06-01

    Despite developments in the knowledge and therapy of acute lung injury in recent decades, mortality remains high, and there is usually a lack of effective therapy. Plantamajoside, a major ingredient isolated from Plantago asiatica L. (Plantaginaceae), has been reported to have potent anti-inflammatory properties. However, the effect of plantamajoside on lipopolysaccharide (LPS)-induced acute lung injury (ALI) in mice has not been investigated. The present study aimed to reveal the potential mechanism responsible for the anti-inflammatory effects of plantamajoside on LPS-induced acute lung injury in mice and in RAW264.7 cells. The results of histopathological changes as well as the lung wet-to-dry ratio and myeloperoxidase (MPO) activity showed that plantamajoside ameliorated the lung injury that was induced by LPS. qPCR and ELISA assays demonstrated that plantamajoside suppressed the production of IL-1β, IL-6 and TNF-α in a dose-dependent manner. TLR4 is an important sensor in LPS infection. Molecular studies showed that the expression of TLR4 was inhibited by plantamajoside administration. Further study was conducted on nuclear factor (NF)-κB and mitogen-activated protein kinase (MAPK) using pathways using western blots. The results showed that plantamajoside inhibited the phosphorylation of IκBα, p65, p38, JNK and ERK. All results indicated that plantamajoside has protective effect on LPS-induced ALI in mice and in RAW264.7 cells. Thus, plantamajoside may be a potential therapy for the treatment of pulmonary inflammation. PMID:27089391

  1. Oxidized LDL induced extracellular trap formation in human neutrophils via TLR-PKC-IRAK-MAPK and NADPH-oxidase activation.

    PubMed

    Awasthi, Deepika; Nagarkoti, Sheela; Kumar, Amit; Dubey, Megha; Singh, Abhishek Kumar; Pathak, Priya; Chandra, Tulika; Barthwal, Manoj Kumar; Dikshit, Madhu

    2016-04-01

    Neutrophil extracellular traps (NETs) formation was initially linked with host defence and extracellular killing of pathogens. However, recent studies have highlighted their inflammatory potential. Oxidized low density lipoprotein (oxLDL) has been implicated as an independent risk factor in various acute or chronic inflammatory diseases including systemic inflammatory response syndrome (SIRS). In the present study we investigated effect of oxLDL on NETs formation and elucidated the underlying signalling mechanism. Treatment of oxLDL to adhered PMNs led to a time and concentration dependent ROS generation and NETs formation. OxLDL induced free radical formation and NETs release were significantly prevented in presence of NADPH oxidase (NOX) inhibitors suggesting role of NOX activation in oxLDL induced NETs release. Blocking of both toll like receptor (TLR)-2 and 6 significantly reduced oxLDL induced NETs formation indicating requirement of both the receptors. We further identified Protein kinase C (PKC), Interleukin-1 receptor associated kinase (IRAKs), mitogen-activated protein kinase (MAPK) pathway as downstream intracellular signalling mediators involved in oxLDL induced NETs formation. OxLDL components such as oxidized phospholipids (lysophosphatidylcholine (LPC) and oxidized 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphorylcholine (oxPAPC)) were most potent NETs inducers and might be crucial for oxLDL mediating NETs release. Other components like, oxysterols, malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE) were however less potent as compared to oxidized phospholipids. This study thus demonstrates for the first time that treatment of human PMNs with oxLDL or its various oxidized phopholipid component mediated NETs release, implying their role in the pathogenesis of inflammatory diseases such as SIRS. PMID:26774674

  2. Nanosized titanium dioxide resulted in the activation of TGF-β/Smads/p38MAPK pathway in renal inflammation and fibration of mice.

    PubMed

    Hong, F; Wu, N; Ge, Y; Zhou, Y; Shen, T; Qiang, Q; Zhang, Q; Chen, M; Wang, Y; Wang, L; Hong, J

    2016-06-01

    Titanium dioxide nanoparticles (TiO2 NPs) have been demonstrated to damage the kidneys. However, whether chronic nephritis leads to renal fibration or the fibrosis is associated with the activation of TGF-β/Smads/p38MAPK pathway caused by TiO2 NPs exposure is not well understood. Forty male mice were separately exposed to 0, 2.5, 5, or 10 mg/kg body weight TiO2 NPs for 6 months. Renal biochemical functions and levels of TGF-β/Smads/p38MAPK pathway-related markers and extracellular matrix (ECM) expression in the kidneys were investigated. The findings showed that subchronic TiO2 NPs exposure increased levels of urinary creatisix (Cr), N-acetyl-glucosaminidase, and vanin-1, resulted in severe renal inflammation and fibration. Furthermore, TiO2 NP exposure upregulated expression of transforming growth factor-β1 (TGF-β1, 0.07- to 2.72-fold), Smad2 (0.42- to 1.63-fold), Smad3 (0.02- to 1.94-fold), ECM (0.15- to 2.75-fold), α-smooth muscle actin (0.14- to 3.06-fold), p38 mitogen-activated protein kinase (p38MAPK, 0.11- to 3.78-fold), and nuclear factor-κB (0.4- to 2.27-fold), and downregulated Smad7 (0.05- to 0.61-fold) expression in mouse kidney. Subchronic TiO2 NPs exposure induced changes of renal characteristics towards inflammation and fibration may be mediated via TGF-β/Smads/p38MAPK pathway, and the uses of TiO2 NPs should be carried out cautiously, especially in humans. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 1452-1461, 2016. PMID:26850371

  3. Particulate Matter Facilitates C6 Glioma Cells Activation and the Release of Inflammatory Factors Through MAPK and JAK2/STAT3 Pathways.

    PubMed

    Li, Ting; Zhao, Jianya; Ge, Jianbin; Yang, Jianbin; Song, Xinjian; Wang, Cheng; Mao, Jiamin; Zhang, Yan; Zou, Ye; Liu, Yanmei; Chen, Gang

    2016-08-01

    It has been widely accepted that astrocytes, play a role in regulating almost every physiological system. In the present study, we investigated the role of particulate matter (PM) in regulating activation of astrocytes. The glial cell strain C6 was cloned from a rat glioma which was induced by N-nitrosomethylurea. The C6 cells were plated at a density of 5 × 10(6) cells/10 cm diameter dish and incubated with different concentrations (0, 12, 25, 50, 100, 200, and 400 μg/mL) of PM for 24 h and different time (0, 1, 3, 6, 8,12, and 24 h) with 100 μg/mL at 37 °C. The study revealed that PM stimulated the expression of inducible nitric oxide synthase (iNOS) as well as the production of IL-1β in a dose- and time-dependent manner. Furthermore, activation of JAK2/STAT3 and p38/JNK/ERK MAPKs was found in astrocytes following PM treatment. Blockage of JAK and p38/JNK/ERK MAPKs with their specific inhibitors, AG490, SB202190, SP600125 and U0126 significantly reduced PM-induced iNOS expression and IL-1β production. In addition, it was demonstrated that inhibition of p38, JNK and JAK prevented STAT3 tyrosine phosphorylation induced by PM, while blocking ERK did not. MAPKs (p38 and JNK) could regulate tyrosine STAT3 phosphorylation, which suggested that the JAK2/STAT3 pathway might be the downstream of p38/JNK MAPK pathways. PMID:27068033

  4. Khat (Catha edulis) generates reactive oxygen species and promotes hepatic cell apoptosis via MAPK activation.

    PubMed

    Abid, Morad Dirhem Naji; Chen, Juan; Xiang, Min; Zhou, Jie; Chen, Xiaoping; Gong, Feili

    2013-08-01

    A number of studies have suggested an association between khat (Catha edulis) chewing and acute liver lesions or chronic liver disease. However, little is known about the effects of khat on hepatic cells. In the current study, we investigated the mechanism behind khat-induced apoptosis in the L02 human hepatic cell line. We used cell growth inhibition assay, flow cytometry and Hoechst 33258 staining to measure hepatocyte apoptosis induced by khat. Western blot analysis was used to detect the expression levels of caspase-8 and -9, as well as those of Bax and Bcl-2. We also measured reactive oxygen species production. The results indicated that khat induced significant hepatocyte apoptosis in L02 cells. We found that khat activated caspase-8 and -9, upregulated Bax protein expression and downregulated Bcl-2 expression levels, which resulted in the coordination of apoptotic signals. Khat-induced hepatocyte apoptosis is primarily regulated through the sustained activation of the c-Jun NH2-terminal kinase (JNK) pathway and only partially via the extracellular signal-regulated kinase (ERK) cascade. Furthermore, the khat-induced reactive oxygen species (ROS) production and the activation of the ROS scavenger, N-acetyl-L-cysteine (NAC), attenuated the khat-induced activation of JNK and ERK. Our results demonstrate that khat triggers the generation of intracellular ROS and sequentially induces the sustainable activation of JNK, which in turn results in a decrease in cell viability and an increase in cell apoptosis. PMID:23708648

  5. Interplay of Notch and FGF signaling restricts cell fate and MAPK activation in the Drosophila trachea.

    PubMed

    Ikeya, T; Hayashi, S

    1999-10-01

    The patterned branching in the Drosophila tracheal system is triggered by the FGF-like ligand Branchless that activates a receptor tyrosine kinase Breathless and the MAP kinase pathway. A single fusion cell at the tip of each fusion branch expresses the zinc-finger gene escargot, leads branch migration in a stereotypical pattern and contacts with another fusion cell to mediate fusion of the branches. A high level of MAP kinase activation is also limited to the tip of the branches. Restriction of such cell specialization events to the tip is essential for tracheal tubulogenesis. Here we show that Notch signaling plays crucial roles in the singling out process of the fusion cell. We found that Notch is activated in tracheal cells by Branchless signaling through stimulation of &Dgr; expression at the tip of tracheal branches and that activated Notch represses the fate of the fusion cell. In addition, Notch is required to restrict activation of MAP kinase to the tip of the branches, in part through the negative regulation of Branchless expression. Notch-mediated lateral inhibition in sending and receiving cells is thus essential to restrict the inductive influence of Branchless on the tracheal tubulogenesis. PMID:10498681

  6. Role of Mitogen-Activated Protein Kinases in Peptidoglycan-Induced Expression of Inducible Nitric Oxide Synthase and Nitric Oxide in Mouse Peritoneal Macrophages: Extracellular Signal-Related Kinase, a Negative Regulator ▿ †

    PubMed Central

    Bhatt, Kunal H.; Sodhi, Ajit; Chakraborty, Rituparna

    2011-01-01

    The expression of inducible nitric oxide synthase (iNOS) and the production of nitric oxide (NO) are important host defense mechanisms against pathogens in mononuclear phagocytes. The objectives of this study were to examine the roles of mitogen-activated protein kinases (MAPKs) and transcription factors (nuclear factor-κB [NF-κB] and activating protein 1 [AP-1]) in peptidoglycan (PGN)-induced iNOS expression and NO production in macrophages. PGN is a cell wall component of Gram-positive bacteria that stimulates inflammatory responses both ex vivo and in vivo. PGN stimulates the activation of all three classes of MAPKs, extracellular signal-related kinase (ERK), c-Jun N-terminal kinase (JNK), and p38mapk in macrophages, albeit with differential activation kinetics. Using a selective inhibitor of JNK (SP600125) and JNK1/2 small interfering RNA (siRNA) knocked-down macrophages, it was observed that PGN-induced iNOS and NO expression is significantly inhibited. This suggested that JNK MAPK plays an essential role in PGN-induced iNOS expression and NO production. In contrast, inhibition of the ERK pathway using PD98059 dose dependently enhanced PGN-induced iNOS expression and NO production. PGN-induced ERK activation was attenuated in ERK1/2 siRNA knocked-down macrophages; however, NO and iNOS expression were significantly enhanced. An electrophoretic mobility shift assay showed that SP600125 inhibited PGN-induced NF-κB and AP-1 activation, whereas inhibition of the ERK pathway enhanced NF-κB activation, but with no effect on AP-1. These results indicate that the JNK MAPK positively regulate PGN-induced iNOS and NO expression by activating NF-κB and AP-1 transcription factors, whereas the ERK pathway plays a negative regulatory role via affecting NF-κB activity. PMID:21450974

  7. Unconjugated Bilirubin exerts Pro-Apoptotic Effect on Platelets via p38-MAPK activation

    PubMed Central

    NaveenKumar, Somanathapura K.; Thushara, Ram M.; Sundaram, Mahalingam S.; Hemshekhar, Mahadevappa; Paul, Manoj; Thirunavukkarasu, Chinnasamy; Basappa; Nagaraju, Ganesh; Raghavan, Sathees C.; Girish, Kesturu S.; Kemparaju, Kempaiah; Rangappa, Kanchugarakoppal S.

    2015-01-01

    Thrombocytopenia is one of the most frequently observed secondary complications in many pathological conditions including liver diseases, where hyperbilirubinemia is very common. The present study sought to find the cause of thrombocytopenia in unconjugated hyperbilirubinemic conditions. Unconjugated bilirubin (UCB), an end-product of heme catabolism, is known to have pro-oxidative and cytotoxic effects at high serum concentration. We investigated the molecular mechanism underlying the pro-apoptotic effect of UCB on human platelets in vitro, and followed it up with studies in phenylhydrazine-induced hyperbilirubinemic rat model and hyperbilirubinemic human subjects. UCB is indeed found to significantly induce platelet apoptotic events including elevated endogenous reactive oxygen species generation, mitochondrial membrane depolarization, increased intracellular calcium levels, cardiolipin peroxidation and phosphatidylserine externalization (p < 0.001) as evident by FACS analysis. The immunoblots show the elevated levels of cytosolic cytochrome c and caspase activation in UCB-treated platelets. Further, UCB is found to induce mitochondrial ROS generation leading to p38 activation, followed by downstream activation of p53, ultimately resulting in altered expression of Bcl-2 and Bax proteins as evident from immunoblotting. All these parameters conclude that elevated unconjugated bilirubin causes thrombocytopenia by stimulating platelet apoptosis via mitochondrial ROS-induced p38 and p53 activation. PMID:26459859

  8. Electromagnetic pulse activated brain microglia via the p38 MAPK pathway.

    PubMed

    Yang, Long-Long; Zhou, Yan; Tian, Wei-Dong; Li, Hai-Juan; Kang-Chu-Li; Miao, Xia; An, Guang-Zhou; Wang, Xiao-Wu; Guo, Guo-Zhen; Ding, Gui-Rong

    2016-01-01

    Previously, we found that electromagnetic pulses (EMP) induced an increase in blood brain barrier permeability and the leakage of albumin from blood into brain tissue. Albumin is known to activate microglia cells. Thus, we hypothesised that microglia activation could occur in the brain after EMP exposure. To test this hypothesis, the morphology and secretory function of microglia cells, including the expression of OX-42 (a marker of microglia activation), and levels of TNF-α, IL-10, IL-1β, and NO were determined in the rat cerebral cortex after EMP exposure. In addition, to examine the signalling pathway of EMP-induced microglia activation, protein and phosphorylated protein levels of p38, JNK and ERK were determined. It was found that the expression of OX-42increased significantly at 1, 6 and 12h (p<0.05) and recovered to the sham group level at 24h after EMP exposure. Levels of NO, TNF-α and IL-10 also changed significantly in vivo and in vitro after EMP exposure. The protein level of p38 and phosphorylated p38 increased significantly after EMP exposure (p<0.05) and recovered to sham levels at 12 and 24h, respectively. Protein and phosphorylated protein levels of ERK and JNK did not change. SB203580 (p38 inhibitor) partly prevented the change in NO, IL-10, IL-1β, TNF-α levels induced by EMP exposure. Taken together, these results suggested that EMP exposure (200kV/m, 200 pulses) could activate microglia in rat brain and affect its secretory function both in vivo and in vitro, and the p38 pathway is involved in this process. PMID:26688329

  9. Functional Characterization of OsMADS18, a Member of the AP1/SQUA Subfamily of MADS Box Genes1[w

    PubMed Central

    Fornara, Fabio; Pařenicová, Lucie; Falasca, Giuseppina; Pelucchi, Nilla; Masiero, Simona; Ciannamea, Stefano; Lopez-Dee, Zenaida; Altamura, Maria Maddalena; Colombo, Lucia; Kater, Martin M.

    2004-01-01

    MADS box transcription factors controlling flower development have been isolated and studied in a wide variety of organisms. These studies have shown that homologous MADS box genes from different species often have similar functions. OsMADS18 from rice (Oryza sativa) belongs to the phylogenetically defined AP1/SQUA group. The MADS box genes of this group have functions in plant development, like controlling the transition from vegetative to reproductive growth, determination of floral organ identity, and regulation of fruit maturation. In this paper we report the functional analysis of OsMADS18. This rice MADS box gene is widely expressed in rice with its transcripts accumulated to higher levels in meristems. Overexpression of OsMADS18 in rice induced early flowering, and detailed histological analysis revealed that the formation of axillary shoot meristems was accelerated. Silencing of OsMADS18 using an RNA interference approach did not result in any visible phenotypic alteration, indicating that OsMADS18 is probably redundant with other MADS box transcription factors. Surprisingly, overexpression of OsMADS18 in Arabidopsis caused a phenotype closely resembling the ap1 mutant. We show that the ap1 phenotype is not caused by down-regulation of AP1 expression. Yeast two-hybrid experiments showed that some of the natural partners of AP1 interact with OsMADS18, suggesting that the OsMADS18 overexpression phenotype in Arabidopsis is likely to be due to the subtraction of AP1 partners from active transcription complexes. Thus, when compared to AP1, OsMADS18 during evolution seems to have conserved the mechanistic properties of protein-protein interactions, although it cannot complement the AP1 function. PMID:15299121

  10. Neomorphic Mutations in PIK3R1 Confer Sensitivity to MAPK Inhibitors due to Activation of ERK and JNK Pathways | Office of Cancer Genomics

    Cancer.gov

    In a recent publication in Cancer Cell, CTD2 investigators discovered that a known cancer-associated gain-of-function alteration in phosphoinositide-3-kinase regulatory subunit 1 (PIK3R1) results in novel protein activity that confers sensitivity to mitogen-activated protein kinase (MAPK) inhibitors. The PIK3R1 gene encodes the p85α regulatory subunit of PIK3. Under normal conditions, p85α suppresses PIK3 mediated activation of downstream pathways that promote cell growth and survival.

  11. HMGB1 binding to receptor for advanced glycation end products enhances inflammatory responses of human bronchial epithelial cells by activating p38 MAPK and ERK1/2.

    PubMed

    Liang, Yue; Hou, Changchun; Kong, Jinliang; Wen, Hanchun; Zheng, Xiaowen; Wu, Lihong; Huang, Hong; Chen, Yiqiang

    2015-07-01

    The proinflammatory factor high mobility group box protein 1 (HMGB1) has been implicated as an important mediator of many chronic inflammatory diseases, including asthma. Human bronchial epithelial cells (HBECs) play a central role in the pathogenesis of asthma. However, the effects of HMGB1 on HBECs and the underlying mechanisms remain unknown. Here, we investigated receptor expression and proinflammatory cytokine production by primary cultures of HBECs stimulated by HMGB1. We then examined the effects of specific receptor blockade and inhibition of p38 MAPK, ERK1/2, or PI3-K on HMGB1-induced expression of proinflammatory cytokines. HMGB1 increased the expression and secretion of TNF-α, TSLP, MMP-9, and VEGF in a dose- and time-dependent manner. HMGB1 also induced elevated expression of RAGE protein. Secretion of TNF-α, VEGF, MMP-9, and TSLP was significantly decreased by RAGE blockade and p38 MAPK pathway inhibition, while a less pronounced effect was mediated by ERK1/2 inhibition. These observations suggest that HMGB1 binds RAGE and promotes activities of p38 MAPK and ERK1/2 pathways in HBECs. This then enhances the expression of TNF-α, VEGF, MMP-9, and TSLP, which are the important inflammatory factors in asthma. These results demonstrate that HMGB1 enhances the inflammatory responses of HBECs, which are involved in the modulation of inflammatory processes in asthma. PMID:25862459

  12. Characterization of three mitogen-activated protein kinases (MAPK) genes reveals involvement of ERK and JNK, not p38 in defense against bacterial infection in Yesso scallop Patinopecten yessoensis.

    PubMed

    Sun, Yan; Zhang, Lingling; Zhang, Meiwei; Li, Ruojiao; Li, Yangping; Hu, Xiaoli; Wang, Shi; Bao, Zhenmin

    2016-07-01

    Mitogen-activated protein kinases (MAPKs) are protein Ser/Thr kinases that play a vital role in innate immune responses by converting extracellular stimuli into a wide range of cellular responses. Although MAPKs have been extensively studied in various vertebrates and invertebrates, our current understanding of MAPK signaling cascade in scallop is in its infancy. In this study, three MAPK genes (PyERK, PyJNK, and Pyp38) were identified from Yesso scallop Patinopecten yessoensis. The open reading frame of PyERK, PyJNK, and Pyp38 was 1104, 1227, and 1104 bp, encoding 367, 408, and 367 amino acids, respectively. Conservation in some splicing sites was revealed across the three PyMAPKs, suggesting the common descent of MAPKs genes. The expression profiles of PyMAPKs over the course of ten different developmental stages showed that they had different expression patterns. In adult scallops, PyMAPKs were primarily expressed in muscles, hemocytes, gill, and mantle. To gain insights into their role in innate immunity, we investigated their expression profiles after infection with Gram-positive bacteria (Micrococcus luteus) and Gram-negative bacteria (Vibrio anguillarum). Significant difference in gene expression was only found in PyERK and PyJNK, but not Pyp38, suggesting Pyp38 may not participate in immune response to bacterial infection. Besides, PyERK and PyJNK exhibited more drastic change against the invasion of V. anguillarum than M. luteus, suggesting they could be more sensitive to Gram-negative bacteria than Gram-positive bacteria. This study provides valuable resource for elucidating the role of MAPK signal pathway in bivalve innate immune response. PMID:27155450

  13. TRPM7 Activates m-Calpain by Stress-Dependent Stimulation of p38 MAPK and c-Jun N-Terminal Kinase

    PubMed Central

    Su, Li-Ting; Chen, Hsiang-Chin; González-Pagán, Omayra; Overton, Jeffrey D.; Xie, Jia; Yue, Lixia; Runnels, Loren W.

    2010-01-01

    Summary TRPM7 is a Ca2+ and Mg2+ permeant ion channel in possession of its own kinase domain. In a previous study we showed that overexpression of the channel-kinase in HEK-293 cells produced cell rounding and loss of adhesion which was dependent upon the Ca2+-dependent protease m-calpain. The TRPM7-elicited change in cell morphology was channel-dependent and occurred without any significant increase in cytosolic Ca2+. Here we demonstrate that overexpression of TRPM7 increased levels of cellular reactive oxygen species (ROS) and nitric oxide (NO), causing the activation of p38 MAP kinase (p38 MAPK) and c-Jun N-terminal kinase (JNK). Application of inhibitors of p38 MAPK and JNK blocked TRPM7-induced cell rounding and activation of m-calpain, without affecting the phosphorylation state of the protease. Overexpression of TRPM7 increased intracellular Mg2+; however, when the concentrations of either external Ca2+ or Mg2+ was increased to favor permeation of one divalent cation over the other, a similar increase in cell rounding and calpain activity was detected, indicating that TRPM7-mediated activation of m-calpain is not dependent on the nature of the divalent conducted by the channel. Application of inhibitors of nitric oxide synthase and mitochondrial-derived ROS reduced TRPM7-induced increases in nitric oxide and ROS production, blocked the change in cell morphology, and reduced cellular calpain activity. Collectively, our data reveal that excessive TRPM7 channel activity causes oxidative and nitrosative stress, producing cell rounding mediated by p38 MAPK/JNK dependent activation of m-calpain. PMID:20070945

  14. Immunolocalization of the mitogen-activated protein kinases p42MAPK and JNK1, and their regulatory kinases MEK1 and MEK4, in adult rat central nervous system.

    PubMed

    Flood, D G; Finn, J P; Walton, K M; Dionne, C A; Contreras, P C; Miller, M S; Bhat, R V

    1998-08-31

    Cell survival, death, and stress signals are transduced from the cell surface to the cytoplasm and nucleus via a cascade of phosphorylation events involving the mitogen-activated protein kinase (MAPK) family. We compared the distribution of p42 mitogen-activated protein kinase (p42MAPK) and its activator MAPK or ERK kinase (MEK1; involved in transduction of growth and differentiation signals), with c-Jun N-terminal kinase (JNK1) and its activator MEK4 (involved in transduction of stress and death signals) in the adult rat central nervous system. All four kinases were present in the cytoplasm, dendrites, and axons of neurons. The presence of p42MAPK and JNK1 in dendrites and axons, as well as in cell bodies, suggests a role for these kinases in phosphorylation and regulation of cytoplasmic targets. A high degree of correspondence was found between the regional distribution of MEK1 and p42MAPK. Immunostaining for MEK1 and p42MAPK was intense in olfactory structures, neocortex, hippocampus, striatum, midline, and interlaminar thalamic nuclei, hypothalamus, brainstem, Purkinje cells, and spinal cord. In addition to neurons, p42MAPK was also present in oligodendrocytes. Whereas MEK4 was ubiquitously distributed, JNK1 was more selective. Immunostaining for MEK4 and JNK1 was intense in the olfactory bulb, lower cortical layers, the cholinergic basal forebrain, most nuclei of the thalamus, medial habenula, and cranial motor nuclei. The distribution of MEK1 and p42MAPK proteins only partially overlapped with that of MEK4 and JNK1. This suggests that the growth/differentiation and death/stress pathways affected by these kinases may not necessarily act to counterbalance each other in response to extracellular stimuli. The differential distribution of these kinases may control the specificity of neuronal function to extracellular signals. PMID:9714150

  15. Gain-of-Function Mutations in the Toll-Like Receptor Pathway: TPL2-Mediated ERK1/ERK2 MAPK Activation, a Path to Tumorigenesis in Lymphoid Neoplasms?

    PubMed Central

    Rousseau, Simon; Martel, Guy

    2016-01-01

    Lymphoid neoplasms form a family of cancers affecting B-cells, T-cells, and NK cells. The Toll-Like Receptor (TLR) signaling adapter molecule MYD88 is the most frequently mutated gene in these neoplasms. This signaling adaptor relays signals from TLRs to downstream effector pathways such as the Nuclear Factor kappa B (NFκB) and Mitogen Activated Protein Kinase (MAPK) pathways to regulate innate immune responses. Gain-of-function mutations such as MYD88[L265P] activate downstream signaling pathways in absence of cognate ligands for TLRs, resulting in increased cellular proliferation and survival. This article reports an analysis of non-synonymous somatic mutations found in the TLR signaling network in lymphoid neoplasms. In accordance with previous reports, mutations map to MYD88 pro-inflammatory signaling and not TRIF-mediated Type I IFN production. Interestingly, the analysis of somatic mutations found downstream of the core TLR-signaling network uncovered a strong association with the ERK1/2 MAPK cascade. In support of this analysis, heterologous expression of MYD88[L265P] in HEK293 cells led to ERK1/2 MAPK phosphorylation in addition to NFκB activation. Moreover, this activation is dependent on the protein kinase Tumor Promoting Locus 2 (TPL2), activated downstream of the IKK complex. Activation of ERK1/2 would then lead to activation, amongst others, of MYC and hnRNPA1, two proteins previously shown to contribute to tumor formation in lymphoid neoplasms. Taken together, this analysis suggests that TLR-mediated ERK1/2 activation via TPL2 may be a novel path to tumorigenesis. Therefore, the hypothesis proposed is that inhibition of ERK1/2 MAPK activation would prevent tumor growth downstream of MYD88[L265]. It will be interesting to test whether pharmacological inhibitors of this pathway show efficacy in primary tumor cells derived from hematologic malignancies such as Waldenstrom's Macroglobulinemia, where the majority of the cells carry the MYD88[L265P

  16. Down-Regulation of NF-κB Target Genes by the AP-1 and STAT Complex during the Innate Immune Response in Drosophila

    PubMed Central

    Kim, Lark Kyun; Choi, Un Yung; Cho, Hwan Sung; Lee, Jung Seon; Lee, Wook-bin; Kim, Jihyun; Jeong, Kyoungsuk; Shim, Jaewon; Kim-Ha, Jeongsil; Kim, Young-Joon

    2007-01-01

    The activation of several transcription factors is required for the elimination of infectious pathogens via the innate immune response. The transcription factors NF-κB, AP-1, and STAT play major roles in the synthesis of immune effector molecules during innate immune responses. However, the fact that these immune responses can have cytotoxic effects requires their tight regulation to achieve restricted and transient activation, and mis-regulation of the damping process has pathological consequences. Here we show that AP-1 and STAT are themselves the major inhibitors responsible for damping NF-κB–mediated transcriptional activation during the innate immune response in Drosophila. As the levels of dAP-1 and Stat92E increase due to continuous immune signaling, they play a repressive role by forming a repressosome complex with the Drosophila HMG protein, Dsp1. The dAP-1–, Stat92E-, and Dsp1-containing complexes replace Relish at the promoters of diverse immune effector genes by binding to evolutionarily conserved cis-elements, and they recruit histone deacetylase to inhibit transcription. Reduction by mutation of dAP-1, Stat92E, or Dsp1 results in hyperactivation of Relish target genes and reduces the viability of bacterially infected flies despite more efficient pathogen clearance. These defects are rescued by reducing the Relish copy number, thus confirming that mis-regulation of Relish, not inadequate activation of dAP-1, Stat92E, or Dsp1 target genes, is responsible for the reduced survival of the mutants. We conclude that an inhibitory effect of AP-1 and STAT on NF-κB is required for properly balanced immune responses and appears to be evolutionarily conserved. PMID:17803358

  17. Goniothalamin induces apoptosis associated with autophagy activation through MAPK signaling in SK-BR-3 cells.

    PubMed

    Innajak, Sukanda; Mahabusrakum, Wilawan; Watanapokasin, Ramida

    2016-05-01

    Goniothalamin, a plant bioactive styrly-lactone, possesses many biological activities. In the present study, the anticancer effect of goniothalamin on human breast cancer cell line SK-BR-3 was investigated. The results showed that goniothalamin induced nuclear condensation, DNA fragmentation, apoptotic bodies and mitochondrial dysfunction as determined by JC-1 staining. Goniothalamin also increased the Bax/Bcl-2 ratio and expression of cleaved caspase-7, cleaved caspase-9 and cleaved PARP, but decreased Bcl-2 expression. In addition, goniothalamin induced apoptosis via p-JNK1/2 and p-p38 upregulation and inhibited cell survival via p-ERK1/2 and p-Akt downregulation. Notably, goniothalamin induced autophagy through upregulation of Atg7, Atg12-Atg5 conjugation and LC3II. The increased p-p38 and p-JNK1/2 and decreased p-Akt may lead to autophagy induction. Therefore, goniothalamin promoted apoptosis associated with autophagy induction in SK-BR-3 cells through p-p38 and p-JNK1/2 upregulation and p-Akt downregulation. The present study indicated that goniothalamin may be further used as a potential therapeutic candidate or may offer an alternative treatment for breast cancer. PMID:26987063

  18. Nicotine stimulates urokinase-type plasminogen activator receptor expression and cell invasiveness through mitogen-activated protein kinase and reactive oxygen species signaling in ECV304 endothelial cells

    SciTech Connect

    Khoi, Pham Ngoc; Park, Jung Sun; Kim, Nam Ho; Jung, Young Do

    2012-03-01

    Urokinase-type plasminogen activator receptor (uPAR) expression is elevated during inflammation, tissue remodeling and in many human cancers. This study investigated the effect of nicotine, a major alkaloid in tobacco, on uPAR expression and cell invasiveness in ECV304 endothelial cells. Nicotine stimulated uPAR expression in a dose-dependent manner and activated extracellular signal-regulated kinases-1/2 (Erk-1/2), c-Jun amino-terminal kinase (JNK) and p38 mitogen activated protein kinase (MAPK). Specific inhibitors of MEK-1 (PD98059) and JNK (SP600125) inhibited the nicotine-induced uPAR expression, while the p38 MAPK inhibitor SB203580 did not. Expression vectors encoding dominant negative MEK-1 (pMCL-K97M) and JNK (TAM67) also prevented nicotine-induced uPAR promoter activity. The intracellular hydrogen peroxide (H{sub 2}O{sub 2}) content was increased by nicotine treatment. The antioxidant N-acetylcysteine prevented nicotine-activated production of reactive oxygen species (ROS) and uPAR expression. Furthermore, exogenous H{sub 2}O{sub 2} increased uPAR mRNA expression. Deleted and site-directed mutagenesis demonstrated the involvement of the binding sites of transcription factor nuclear factor-kappaB (NF-κB) and activator protein (AP)-1 in the nicotine-induced uPAR expression. Studies with expression vectors encoding mutated NF-κB signaling molecules and AP-1 decoy confirmed that NF-κB and AP-1 were essential for the nicotine-stimulated uPAR expression. MAPK (Erk-1/2 and JNK) and ROS functioned as upstream signaling molecules in the activation of AP-1 and NF-κB, respectively. In addition, ECV304 endothelial cells treated with nicotine displayed markedly enhanced invasiveness, which was partially abrogated by uPAR neutralizing antibodies. The data indicate that nicotine induces uPAR expression via the MAPK/AP-1 and ROS/NF-κB signaling pathways and, in turn, stimulates invasiveness in human ECV304 endothelial cells. -- Highlights: ► Endothelial cells

  19. Apigenin-7-Glycoside Prevents LPS-Induced Acute Lung Injury via Downregulation of Oxidative Enzyme Expression and Protein Activation through Inhibition of MAPK Phosphorylation

    PubMed Central

    Li, Kun-Cheng; Ho, Yu-Ling; Hsieh, Wen-Tsong; Huang, Shyh-Shyun; Chang, Yuan-Shiun; Huang, Guan-Jhong

    2015-01-01

    Apigenin-7-glycoside (AP7Glu) with multiple biological activities is a flavonoid that is currently prescribed to treat inflammatory diseases such as upper respiratory infections. Recently, several studies have shown that its anti-inflammatory activities have been strongly linked to the inhibition of secretion of pro-inflammatory proteins, such as inducible nitric oxide synthase (iNOs) and cyclooxygenase-2 (COX-2) induced through phosphorylation nuclear factor-κB (NF-κB) and mitogen-activated protein kinases (MAPK) pathways. Additionally, inflammation, which can decrease the activities of antioxidative enzymes (AOEs) is also observed in these studies. At the same time, flavonoids are reported to promote the activities of heme oxygenase-1 (HO-1) decreased by LPS. The purpose of this study was to assess these theories in a series of experiments on the suppressive effects of AP7Glu based on LPS-induced nitric oxide production in RAW264.7 macrophages in vitro and acute lung injury in mice in vivo. After six hours of lipopolysaccharide (LPS) stimulation, pulmonary pathological, myeloperoxidase (MPO) activity, total polymorphonuclear leukocytes (PMN) cells, cytokines in bronchoalveolar lavage fluid (BALF) and AOEs, are all affected and changed. Meanwhile, our data revealed that AP7Glu not only did significantly inhibit the LPS-enhanced inflammatory activity in lung, but also exhibited anti-inflammatory effect through the MAPK and inhibitor NF-κB (IκB) pathways. PMID:25590301

  20. The JNK/AP-1 pathway upregulates expression of the recycling endosome rab11a gene in B cells transformed by Theileria.

    PubMed

    Lizundia, Regina; Chaussepied, Marie; Naissant, Bernina; Masse, Guillemette X; Quevillon, Emmanuel; Michel, Fréderique; Monier, Solange; Weitzman, Jonathan B; Langsley, Gordon

    2007-08-01

    Lymphocyte transformation induced by Theileria parasites involves constitutive activation of c-Jun N-terminal kinase (JNK) and the AP-1 transcription factor. We found that JNK/AP-1 activation is associated with elevated levels of Rab11 protein in Theileria-transformed B cells. We show that AP-1 regulates rab11a promoter activity in B cells and that the induction of c-Jun activity in mouse fibroblasts also leads to increased transcription of the endogenous rab11a gene, consistent with it being an AP-1 target. Pharmacological inhibition of the JNK pathway reduced Rab11 protein levels and endosome recycling of transferrin receptor (TfR) and siRNA knockdown of JNK1 and Rab11A levels also reduced TfR surface expression. We propose a model, where activation of the JNK/AP-1 pathway during cell transformation might assure that the regulation of recycling endosomes is co-ordinated with cell-cycle progression. This might be achieved via the simultaneous upregulation of the cell cycle machinery (e.g. cyclin D1) and the recycling endosome regulators (e.g. Rab11A). PMID:17388783

  1. Recombinant human brain natriuretic peptide attenuates LPS-induced cellular injury in human fetal lung fibroblasts via inhibiting MAPK and NF-κB pathway activation.

    PubMed

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

    2016-08-01

    Inflammatory responses are vital in lung injury diseases, particularly acute respiratory distress syndrome (ARDS). Recombinant human brain natriuretic peptide (rhBNP) has been shown to exhibit anti‑inflammatory effects in vivo in our previous studies. The present study aimed to investigate the mechanisms underlying the anti‑inflammatory effects of rhBNP on lipopolysaccharide (LPS)-induced human fetal lung fibroblasts (HFL-1). The results showed that LPS induced a significant increase in the leakage of lactate dehydrogenase and the secretion of interleukin (IL)‑1β. Activation of p38, extracellular-signal regulated kinase (ERK) 1/2, c‑Jun NH2-terminal kinase (JNK) mitogen‑activated protein kinases (MAPK)s, and nuclear factor (NF)‑κB in HFL‑1 cells was also observed following treatment with LPS. Treatment with rhBNP (0.1 µM) reduced the production of IL‑1β at the protein and mRNA levels. Moreover, rhBNP decreased the phosphorylation of p38, ERK1/2 and JNK induced by LPS. However, the JNK inhibitor, SP600125, significantly inhibited LPS‑induced IL‑1β production. These results indicate that the inhibition of IL‑1β by may dependent upon the JNK signaling pathway. The LPS‑induced NF‑κB activation was also suppressed by rhBNP, and IL‑1β production was inhibited by the NF‑κB inhibitor. Furthermore, NF‑κB activation was attenuated by the JNK inhibitor, indicating that NF‑κB activation was dependent on the JNK signaling pathway. The present study suggests that rhBNP exhibits an anti‑inflammatory effect on LPS‑induced HFL‑1 cell injury via the inhibition of MAPK and NF‑κB signaling pathways and may exhibit therapeutic potential for acute lung injury and ARDS. PMID:27314600

  2. Deoxynivalenol induced mouse skin cell proliferation and inflammation via MAPK pathway

    SciTech Connect

    Mishra, Sakshi; Tripathi, Anurag; Chaudhari, Bhushan P.; Dwivedi, Premendra D.; Pandey, Haushila P.; Das, Mukul

    2014-09-01

    Several toxicological manifestations of deoxynivalenol (DON), a mycotoxin, are well documented; however, dermal toxicity is not yet explored. The effect of topical application of DON to mice was studied using markers of skin proliferation, inflammation and tumor promotion. Single topical application of DON (84–672 nmol/mouse) significantly enhanced dermal hyperplasia and skin edema. DON (336 and 672 nmol) caused significant enhancement in [{sup 3}H]-thymidine uptake in DNA along with increased myeloperoxidase and ornithine decarboxylase activities, suggesting tissue inflammation and cell proliferation. Furthermore, DON (168 nmol) caused enhanced expression of RAS, and phosphorylation of PI3K/Akt, ERK, JNK and p38 MAPKs. DON exposure also showed activation of transcription factors, c-fos, c-jun and NF-κB along with phosphorylation of IkBα. Enhanced phosphorylation of NF-κB by DON caused over expression of target proteins, COX-2, cyclin D1 and iNOS in skin. Though a single topical application of DMBA followed by twice weekly application of DON (84 and 168 nmol) showed no tumorigenesis after 24 weeks, however, histopathological studies suggested hyperplasia of the epidermis and hypertrophy of hair follicles. Interestingly, intestine was also found to be affected as enlarged Peyer's patches were observed, suggesting inflammatory effects which were supported by elevation of inflammatory cytokines after 24 weeks of topical application of DON. These results suggest that DON induced cell proliferation in mouse skin is through the activation of MAPK signaling pathway involving transcription factors NFκB and AP-1, further leading to transcriptional activation of downstream target proteins c-fos, c-jun, cyclin D1, iNOS and COX-2 which might be responsible for its inflammatory potential. - Highlights: • Topical application of DON enhanced epidermal inflammation and cell proliferation. • DON follows PI3K/Akt/MAPK signaling cascade, with activation of AP-1 and NF