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Sample records for activating p38 mitogen-activated

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

    PubMed

    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

  2. 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

  3. The case for inhibiting p38 mitogen-activated protein kinase in heart failure.

    PubMed

    Arabacilar, Pelin; Marber, Michael

    2015-01-01

    This minireview discusses the evidence that the inhibition of p38 mitogen-activated protein kinases (p38 MAPKs) maybe of therapeutic value in heart failure. Most previous experimental studies, as well as past and ongoing clinical trials, have focussed on the role of p38 MAPKs in myocardial infarction and acute coronary syndromes. There is now growing evidence that these kinases are activated within the myocardium of the failing human heart and in the heart and blood vessels of animal models of heart failure. Furthermore, from a philosophical viewpoint the chronic activation of the adaptive stress pathways that lead to the activation of p38 MAPKs in heart failure is analogous to the chronic activation of the sympathetic, renin-aldosterone-angiotensin and neprilysin systems. These have provided some of the most effective therapies for heart failure. This minireview questions whether similar and synergistic advantages would follow the inhibition of p38 MAPKs.

  4. Activation of p38 Mitogen-Activated Protein Kinase Promotes Epidermal Growth Factor Receptor Internalization

    PubMed Central

    Vergarajauregui, Silvia; Miguel, Anitza San; Puertollano, Rosa

    2006-01-01

    Endocytic trafficking plays an important role in the regulation of the epidermal growth factor receptor (EGFR). To address if cellular kinases regulate EGFR internalization, we used anisomycin, a potent activator of kinase cascades in mammalian cells, especially the stress-activated mitogen-activated protein (MAP) kinase subtypes. Here, we report that activation of p38 MAP kinase by anisomycin is sufficient to induce internalization of EGFR. Anisomycin and EGF employ different mechanisms to promote EGFR endocytosis as anisomycin-induced internalization does not require tyrosine kinase activity or ubiquitination of the receptor. In addition, anisomycin treatment did not result in delivery and degradation of EGFR at lysosomes. Incubation with a specific inhibitor of p38, or depletion of endogenous p38 by small interfering RNAs, abolished anisomycin-induced internalization of EGFR while having no effect on transferrin endocytosis, indicating that the effect of p38 activation on EGFR endocytosis is specific. Interestingly, inhibition of p38 activation also abolished endocytosis of EGFR induced by UV radiation. Our results reveal a novel role for p38 in the regulation of EGFR endocytosis and suggest that stimulation of EGFR internalization by p38 might represent a general mechanism to prevent generation of proliferative or anti-apoptotic signals under stress conditions. PMID:16683917

  5. p38 Mitogen-Activated Protein Kinase Pathway Regulates Genes during Proliferation and Differentiation in Oligodendrocytes

    PubMed Central

    Haines, Jeffery D.; Fulton, Debra L.; Richard, Stephane; Almazan, Guillermina

    2015-01-01

    We have previously shown that p38 mitogen-activated protein kinase (p38 MAPK) is important for oligodendrocyte (OLG) differentiation and myelination. However, the precise cellular mechanisms by which p38 regulates OLG differentiation remain largely unknown. To determine whether p38 functions in part through transcriptional events in regulating OLG identity, we performed microarray analysis on differentiating oligodendrocyte progenitors (OLPs) treated with a p38 inhibitor. Consistent with a role in OLG differentiation, pharmacological inhibition of p38 down-regulated the transcription of genes that are involved in myelin biogenesis, transcriptional control and cell cycle. Proliferation assays showed that OLPs treated with the p38 inhibitor retained a proliferative capacity which could be induced upon application of mitogens demonstrating that after two days of p38-inhibition OLGs remained poised to continue mitosis. Together, our results suggest that the p38 pathway regulates gene transcription which can coordinate OLG differentiation. Our microarray dataset will provide a useful resource for future studies investigating the molecular mechanisms by which p38 regulates oligodendrocyte differentiation and myelination. PMID:26714323

  6. p38 mitogen-activated protein kinase plays a key role in regulating MAPKAPK2 expression

    SciTech Connect

    Sudo, Tatsuhiko . E-mail: sudo@riken.jp; Kawai, Kayoko; Matsuzaki, Hiroshi; Osada, Hiroyuki

    2005-11-18

    One of three major families of the mitogen-activated kinases (MAPK), p38 as well as JNK, has been shown to transduce extracellular stress stimuli into cellular responses by phospho-relay cascades. Among p38 families, p38{alpha} is a widely characterized isoform and the biological phenomena are explained by its kinase activity regulating functions of its downstream substrates. However, its specific contributions to each phenomenon are yet not fully elucidated. For better understanding of the role of MAPKs, especially p38{alpha}, we utilized newly established mouse fibroblast cell lines originated from a p38{alpha} null mouse, namely, a parental cell line without p38{alpha} gene locus, knockout of p38{alpha} (KOP), Zeosin-resistant (ZKOP), revertant of p38{alpha} (RKOP), and Exip revertant (EKOP). EKOP is smaller in size but grows faster than the others. Although comparable amounts of ERK and JNK are expressed in each cell line, ERK is highly phosphorylated in EKOP even in normal culture conditions. Serum stimulation after serum starvation led to ERK phosphorylation in RKOP and ZKOP, but not in EKOP as much. On the contrary, relative phosphorylation level of JNK to total JNK in response to UV was low in RKOP. And its phosphorylation as well as total JNK is slightly lower in EKOP. RKOP is less sensitive to UV irradiation as judged by the survival rate. Stress response upon UV or sorbitol stimuli, leading to mitogen activate protein kinase activated kinase 2 (MAPKAPK2) phosphorylation, was only observed in RKOP. Further experiments reveal that MAPKAPK2 expression is largely suppressed in ZKOP and EKOP. Its expression was recovered by re-introduction of p38{alpha}. The loss of MAPKAPK2 expression accompanied by the defect of p38{alpha} is confirmed in an embryonic extract prepared from p38{alpha} null mice. These data demonstrate that p38 signal pathway is regulated not only by phosphorylation but also by modulation of the expression of its component. Together, we have

  7. Heat Shock Factor 1 Is a Substrate for p38 Mitogen-Activated Protein Kinases

    PubMed Central

    Dayalan Naidu, Sharadha; Sutherland, Calum; Zhang, Ying; Risco, Ana; de la Vega, Laureano; Caunt, Christopher J.; Hastie, C. James; Lamont, Douglas J.; Torrente, Laura; Chowdhry, Sudhir; Benjamin, Ivor J.; Keyse, Stephen M.; Cuenda, Ana

    2016-01-01

    Heat shock factor 1 (HSF1) monitors the structural integrity of the proteome. Phosphorylation at S326 is a hallmark for HSF1 activation, but the identity of the kinase(s) phosphorylating this site has remained elusive. We show here that the dietary agent phenethyl isothiocyanate (PEITC) inhibits heat shock protein 90 (Hsp90), the main negative regulator of HSF1; activates p38 mitogen-activated protein kinase (MAPK); and increases S326 phosphorylation, trimerization, and nuclear translocation of HSF1, and the transcription of a luciferase reporter, as well as the endogenous prototypic HSF1 target Hsp70. In vitro, all members of the p38 MAPK family rapidly and stoichiometrically catalyze the S326 phosphorylation. The use of stable knockdown cell lines and inhibitors indicated that among the p38 MAPKs, p38γ is the principal isoform responsible for the phosphorylation of HSF1 at S326 in cells. A protease-mass spectrometry approach confirmed S326 phosphorylation and unexpectedly revealed that p38 MAPK also catalyzes the phosphorylation of HSF1 at S303/307, previously known repressive posttranslational modifications. Thus, we have identified p38 MAPKs as highly efficient catalysts for the phosphorylation of HSF1. Furthermore, our findings suggest that the magnitude and persistence of activation of p38 MAPK are important determinants of the extent and duration of the heat shock response. PMID:27354066

  8. p38 Mitogen-activated protein kinase inhibitors: a review on pharmacophore mapping and QSAR studies.

    PubMed

    Gangwal, Rahul P; Bhadauriya, Anuseema; Damre, Mangesh V; Dhoke, Gaurao V; Sangamwar, Abhay T

    2013-01-01

    p38 mitogen-activated protein (MAP) kinases are the serine/threonine protein kinases, which play a vital role in cellular responses to external stress signals. p38 MAP kinase inhibitors have shown anti-inflammatory effects in the preclinical disease models, primarily through inhibition of the expression of inflammatory mediators. A number of structurally diverse p38 MAP kinase inhibitors have been developed as potential anti-inflammatory agents. Most of the inhibitors have failed in the clinical trials either due to poor pharmacokinetic profile or selectivity issue, which makes p38 MAP kinase a promising target for molecular modelling studies. Several quantitative structure activity relationships (QSAR) and pharmacophore models have been developed to identify the structural requirements essential for p38 MAP kinase inhibitory activity. In this review, we provide an overview of the presently known p38 MAP kinase inhibitors and how QSAR analyses among series of compounds have led to the development of molecular models and pharmacophores, allowing the design of novel inhibitors.

  9. Glucose deprivation increases tau phosphorylation via P38 mitogen-activated protein kinase.

    PubMed

    Lauretti, Elisabetta; Praticò, Domenico

    2015-12-01

    Alterations of glucose metabolism have been observed in Alzheimer's disease (AD) brain. Previous studies showed that glucose deprivation increases amyloidogenesis via a BACE-1-dependent mechanism. However, no data are available on the effect that this condition may have on tau phosphorylation. In this study, we exposed neuronal cells to a glucose-free medium and investigated the effect on tau phosphorylation. Compared with controls, cells incubated in the absence of glucose had a significant increase in tau phosphorylation at epitopes Ser202/Thr205 and Ser404, which was associated with a selective activation of the P38 mitogen-activated protein kinase. Pharmacological inhibition of this kinase prevented the increase in tau phosphorylation, while fluorescence studies revealed its co-localization with phosphorylated tau. The activation of P38 was secondary to the action of the apoptosis signal-regulating kinase 1, as its down-regulation prevented it. Finally, glucose deprivation induced cell apoptosis, which was associated with a significant increase in both caspase 3 and caspase 12 active forms. Taken together, our studies reveal a new mechanism whereby glucose deprivation can modulate AD pathogenesis by influencing tau phosphorylation and suggest that this pathway may be a new therapeutic target for AD.

  10. A role for p38 mitogen-activated protein kinase in early post-embryonic development of Schistosoma mansoni.

    PubMed

    Ressurreição, Margarida; Rollinson, David; Emery, Aidan M; Walker, Anthony J

    2011-11-01

    The importance of p38 mitogen-activated protein kinase (p38 MAPK) to Schistosoma mansoni miracidium to mother-sporocyst development was investigated. Western blotting revealed that phosphorylation (activation) of p38 MAPK was low in larvae after 4h development in vitro but increased markedly during transformation, with ∼2.7- and ∼3.7-fold increases after 19h and 28h culture, respectively. Immunohistochemistry of larvae undergoing transformation revealed activated p38 MAPK associated with regions including the tegument, neural mass and germinal cells. Inhibition of larval p38 MAPK with SB203580 reduced significantly the rate of development of miracidia to mother sporocysts, whereas activation of p38 MAPK with anisomycin had the opposite effect. These results provide insight into p38 MAPK signalling in schistosomes and support a role for p38 MAPK in the early post-embryonic development of S. mansoni. PMID:21787807

  11. Anti-inflammatory effects of a p38 mitogen-activated protein kinase inhibitor during human endotoxemia.

    PubMed

    Branger, Judith; van den Blink, Bernt; Weijer, Sebastiaan; Madwed, Jeffrey; Bos, Carina L; Gupta, Abhya; Yong, Chan-Loi; Polmar, Stephen H; Olszyna, Dariusz P; Hack, C Erik; van Deventer, Sander J H; Peppelenbosch, Maikel P; van der Poll, Tom

    2002-04-15

    The p38 mitogen-activated protein kinase (MAPK) participates in intracellular signaling cascades resulting in inflammatory responses. Therefore, inhibition of the p38 MAPK pathway may form the basis of a new strategy for treatment of inflammatory diseases. However, p38 MAPK activation during systemic inflammation in humans has not yet been shown, and its functional significance in vivo remains unclear. Hence, we exposed 24 healthy male subjects to an i.v. dose of LPS (4 ng/kg), preceded 3 h earlier by orally administered 600 or 50 mg BIRB 796 BS (an in vitro p38 MAPK inhibitor) or placebo. Both doses of BIRB 796 BS significantly inhibited LPS-induced p38 MAPK activation in the leukocyte fraction of the volunteers. Cytokine production (TNF-alpha, IL-6, IL-10, and IL-1R antagonist) was strongly inhibited by both low and high dose p38 MAPK inhibitor. In addition, p38 MAPK inhibition diminished leukocyte responses, including neutrophilia, release of elastase-alpha(1)-antitrypsin complexes, and up-regulation of CD11b with down-regulation of L-selectin. Finally, blocking p38 MAPK decreased C-reactive protein release. These data identify p38 MAPK as a principal mediator of the inflammatory response to LPS in humans. Furthermore, the anti-inflammatory potential of an oral p38 MAPK inhibitor in humans in vivo suggests that p38 MAPK inhibitors may provide a new therapeutic option in the treatment of inflammatory diseases.

  12. Mitogen-activated protein kinase p38b interaction with delta class glutathione transferases from the fruit fly, Drosophila melanogaster.

    PubMed

    Wongtrakul, Jeerang; Sukittikul, Suchada; Saisawang, Chonticha; Ketterman, Albert J

    2012-01-01

    Glutathione transferases (GSTs) are a family of multifunctional enzymes involved in xenobiotic biotransformation, drug metabolism, and protection against oxidative damage. The p38b mitogen-activated protein kinase is involved in cellular stress response. This study screened interactions between Drosophila melanogaster Meigen (Diptera: Drosophilidae) Delta class glutathione transferases (DmGSTs) and the D. melanogaster p38b MAPK. Therefore, 12 DmGSTs and p38b kinase were obtained as recombinant proteins. The study showed that DmGSTD8 and DmGSTD11b significantly increased p38b activity toward ATF2 and jun, which are transcription factor substrates. DmGSTD3 and DmGSTD5 moderately increased p38b activity for jun. In addition, GST activity in the presence of p38b was also measured. It was found that p38b affected substrate specificity toward CDNB (1-chloro-2,4-dinitrobenzene) and DCNB (1,2-dichloro-4-nitrobenzene) of several GST isoforms, i.e., DmGSTD2, DmGSTD5, DmGSTD8, and DmGSTD11b. The interaction of a GST and p38b can affect the substrate specificity of either enzyme, which suggests induced conformational changes affecting catalysis. Similar interactions do not occur for all the Delta enzymes and p38b, which suggests that these interactions could be specific. PMID:23438069

  13. The p38α mitogen-activated protein kinase is a key regulator of myelination and remyelination in the CNS.

    PubMed

    Chung, S-H; Biswas, S; Selvaraj, V; Liu, X-B; Sohn, J; Jiang, P; Chen, C; Chmilewsky, F; Marzban, H; Horiuchi, M; Pleasure, D E; Deng, W

    2015-05-07

    The p38α mitogen-activated protein kinase (MAPK) is one of the serine/threonine kinases regulating a variety of biological processes, including cell-type specification, differentiation and migration. Previous in vitro studies using pharmacological inhibitors suggested that p38 MAPK is essential for oligodendrocyte (OL) differentiation and myelination. To investigate the specific roles of p38α MAPK in OL development and myelination in vivo, we generated p38α conditional knockout (CKO) mice under the PLP and nerve/glial antigen 2 (NG2) gene promoters, as these genes are specifically expressed in OL progenitor cells (OPCs). Our data revealed that myelin synthesis was completely inhibited in OLs differentiated from primary OPC cultures derived from the NG2 Cre-p38α CKO mouse brains. Although an in vivo myelination defect was not obvious after gross examination of these mice, electron microscopic analysis showed that the ultrastructure of myelin bundles was severely impaired. Moreover, the onset of myelination in the corpus callosum was delayed in the knockout mice compared with p38α fl/fl control mice. A delay in OL differentiation in the central nervous system was observed with concomitant downregulation in the expression of OPC- and OL-specific genes such as Olig1 and Zfp488 during early postnatal development. OPC proliferation was not affected during this time. These data indicate that p38α is a positive regulator of OL differentiation and myelination. Unexpectedly, we observed an opposite effect of p38α on remyelination in the cuprizone-induced demyelination model. The p38α CKO mice exhibited better remyelination capability compared with p38α fl/fl mice following demyelination. The opposing roles of p38α in myelination and remyelination could be due to a strong anti-inflammatory effect of p38α or a dual reciprocal regulatory action of p38α on myelin formation during development and on remyelination after demyelination.

  14. Early activation of p38 mitogen activated protein kinase is associated with interferon-alpha-induced depression and fatigue

    PubMed Central

    Felger, Jennifer C.; Alagbe, Oyetunde; Pace, Thaddeus W. W.; Woolwine, Bobbi J.; Hu, Fang; Raison, Charles L.; Miller, Andrew H.

    2011-01-01

    Cytokine-induced stimulation of p38 mitogen activated protein kinase (MAPK) has been shown to influence behaviorally-relevant pathophysiologic pathways including monoamine neurotransmission and neuroendocrine function and thus may contribute to behavioral changes that occur during chronic administration of the innate immune cytokine, interferon (IFN)-alpha. Accordingly, in the current study, phosphorylation (activation) of intracellular p38 MAPK in peripheral blood lymphocytes was analyzed by flow cytometry every 2 hours for 12 hours following the initial injection of IFN-alpha in eleven patients with chronic hepatitis C. Hourly assessments of plasma concentrations of adrenocorticotropic hormone, cortisol and interleukin-6 were also obtained. Symptoms of depression and fatigue were measured at baseline and after 4 and 12 weeks of IFN-alpha treatment. Acute administration of IFN-alpha significantly increased the percentage of lymphocytes staining positive for intracellular phosphorylated p38 (p-p38). IFN-alpha-induced increases in p-p38 were significantly greater in patients that developed clinically significant depressive symptoms [Montgomery Asberg Depression Rating Scale (MADRS) score ≥15] during the first 12 weeks of IFN-alpha treatment. Increases in the percentage of p-p38-positive lymphocytes following the first IFN-alpha injection also highly correlated with depression severity at weeks 4 (r=0.85, p=0.001) and 12 (r=0.70, p=0.018). Similar relationships were observed for fatigue. Examination of relationships between p-p38 induction and factors previously reported to predict IFN-alpha-induced depressive symptoms revealed strong associations of p-p38 with baseline MADRS (r=0.82, p=0.002) and cortisol responses to the initial injection of IFN-alpha (r=0.91, p=0.000). Taken together, these findings indicate that sensitivity of p38 MAPK signaling pathways to immune stimulation is associated with depressive symptoms during chronic IFN-alpha treatment. PMID

  15. The Mitogen-Activated Protein Kinase p38α Regulates Tubular Damage in Murine Anti-Glomerular Basement Membrane Nephritis

    PubMed Central

    Müller, Ralf; Daniel, Christoph; Hugo, Christian; Amann, Kerstin; Mielenz, Dirk; Endlich, Karlhans; Braun, Tobias; van der Veen, Betty; Heeringa, Peter; Schett, Georg; Zwerina, Jochen

    2013-01-01

    p38 mitogen-activated protein kinase (MAPK) is thought to play a central role in acute and chronic inflammatory responses. Whether p38MAPK plays a pathogenic role in crescentic GN (GN) and which of its four isoforms is preferentially involved in kidney inflammation is not definitely known. We thus examined expression and activation of p38MAPK isoforms during anti-glomerular basement membrane (GBM) nephritis. Therefore, p38α conditional knockout mice (MxCre-p38αΔ/Δ) were used to examine the role of p38α in anti-GBM induced nephritis. Both wild type and MxCre-p38αΔ/Δ mice developed acute renal failure over time. Histological examinations revealed a reduced monocyte influx and less tubular damage in MxCre-p38αΔ/Δ mice, whereas glomerular crescent formation and renal fibrosis was similar. Likewise, the levels of pro- and anti-inflammatory cytokines such as TNF, IL-1 and IL-10 were similar, but IL-8 was even up-regulated in MxCre-p38αΔ/Δ mice. In contrast, we could detect strong down-regulation of chemotactic cytokines such as CCL-2, -5 and -7, in the kidneys of MxCre-p38αΔ/Δ mice. In conclusion, p38α is the primary p38MAPK isoform expressed in anti-GBM nephritis and selectively affects inflammatory cell influx and tubular damage. Full protection from nephritis is however not achieved as renal failure and structural damage still occurs. PMID:23441175

  16. p38 Mitogen-Activated Protein Kinase in beryllium-induced dendritic cell activation.

    PubMed

    Li, L; Huang, Z; Gillespie, M; Mroz, P M; Maier, L A

    2014-12-01

    Dendritic cells (DC) play a role in the regulation of immune responses to haptens, which in turn impact DC maturation. Whether beryllium (Be) is able to induce DC maturation and if this occurs via the MAPK pathway is not known. Primary monocyte-derived DCs (moDCs) models were generated from Be non-exposed healthy volunteers as a non-sensitized cell model, while PBMCs from BeS (Be sensitized) and CBD (chronic beryllium disease) were used as disease models. The response of these cells to Be was evaluated. The expression of CD40 was increased significantly (p<0.05) on HLA-DP Glu69+ moDCs after 100 μM BeSO₄-stimulation. BeSO₄ induced p38MAPK phosphorylation, while IκB-α was degraded in Be-stimulated moDCs. The p38 MAPK inhibitor SB203580 blocked Be-induced NF-κB activation in moDCs, suggesting that p38MAPK and NF-κB are dependently activated by BeSO₄. Furthermore, in BeS and CBD subjects, SB203580 downregulated Be-stimulated proliferation in a dose-dependent manner, and decreased Be-stimulated TNF-α and IFNγ cytokine production. Taken together, this study suggests that Be-induces non-sensitized Glu69+ DCs maturation, and that p38MAPK signaling is important in the Be-stimulated DCs activation as well as subsequent T cell proliferation and cytokine production in BeS and CBD. In total, the MAPK pathway may serve as a potential therapeutic target for human granulomatous lung diseases.

  17. p38 Mitogen-Activated Protein Kinase in beryllium-induced dendritic cell activation

    PubMed Central

    Li, L.; Huang, Z.; Gillespie, M.; Mroz, P.M.; Maier, L.A.

    2014-01-01

    Dendritic cells (DC) play a role in the regulation of immune responses to haptens, which in turn impact DC maturation. Whether beryllium (Be) is able to induce DC maturation and if this occurs via the MAPK pathway is not known. Primary monocyte-derived DCs (moDCs) models were generated from Be non-exposed healthy volunteers as a non-sensitized cell model, while PBMCs from BeS (Be sensitized) and CBD (chronic beryllium disease) were used as disease models. The response of these cells to Be was evaluated. The expression of CD40 was increased significantly (p<0.05) on HLA-DP Glu69+ moDCs after 100 μM BeSO4-stimulation. BeSO4 induced p38MAPK phosphorylation, while IκB-α was degraded in Be-stimulated moDCs. The p38 MAPK inhibitor SB203580 blocked Be-induced NF-κB activation in moDCs, suggesting that p38MAPK and NF-κB are dependently activated by BeSO4. Furthermore, in BeS and CBD subjects, SB203580 downregulated Be-stimulated proliferation in a dose-dependent manner, and decreased Be-stimulated TNF-α and IFNγ cytokine production. Taken together, this study suggests that Be-induces non-sensitized Glu69+ DCs maturation, and that p38MAPK signaling is important in the Be-stimulated DCs activation as well as subsequent T cell proliferation and cytokine production in BeS and CBD. In total, the MAPK pathway may serve as a potential therapeutic target for human granulomatous lung diseases. PMID:25454621

  18. Role of p38 Mitogen-Activated Protein Kinase in Middle Ear Mucosa Hyperplasia during Bacterial Otitis Media

    PubMed Central

    Palacios, Sean D.; Pak, Kwang; Rivkin, Alexander Z.; Kayali, Ayse G.; Austen, Darrell; Aletsee, Christoph; Melhus, Åsa; Webster, Nicholas J. G.; Ryan, Allen F.

    2004-01-01

    Hyperplasia of the middle ear mucosa contributes to the sequelae of acute otitis media. Understanding the signal transduction pathways that mediate hyperplasia could lead to the development of new therapeutic interventions for this disease and its sequelae. Endotoxin derived from bacteria involved in middle ear infection can contribute to the hyperplastic response. The p38 mitogen-activated protein kinase (MAPK) is known to be activated by endotoxin as well as cytokines and other inflammatory mediators that have been documented in otitis media. We assessed the activation of p38 in the middle ear mucosa of an in vivo rat bacterial otitis media model. Strong activity of p38 was observed 1 to 6 h after bacterial inoculation. Activity continued at a lower level for at least 7 days. The effects of p38 activation were assessed using an in vitro model of rat middle ear mucosal hyperplasia in which mucosal growth is stimulated by nontypeable Haemophilus influenzae during acute otitis media. Hyperplastic mucosal explants treated with the p38α and p38β inhibitor SB203580 demonstrated significant inhibition of otitis media-stimulated mucosal growth. The results of this study suggest that intracellular signaling via p38 MAPK influences the hyperplastic response of the middle ear mucosa during bacterial otitis media. PMID:15271927

  19. p38 mitogen-activated protein kinase activation during platelet storage: consequences for platelet recovery and hemostatic function in vivo

    PubMed Central

    Canault, Matthias; Duerschmied, Daniel; Brill, Alexander; Stefanini, Lucia; Schatzberg, Daphne; Cifuni, Stephen M.

    2010-01-01

    Platelets undergo several modifications during storage that reduce their posttransfusion survival and functionality. One important feature of these changes, which are known as platelet storage lesion, is the shedding of the surface glycoproteins GPIb-α and GPV. We recently demonstrated that tumor necrosis factor-α converting enzyme (TACE/ADAM17) mediates mitochondrial injury-induced shedding of adhesion receptors and that TACE activity correlates with reduced posttransfusion survival of these cells. We now confirm that TACE mediates receptor shedding and clearance of platelets stored for 16 hours at 37°C or 22°C. We further demonstrate that both storage and mitochondrial injury lead to the phosphorylation of p38 mitogen-activated kinase (MAPK) in platelets and that TACE-mediated receptor shedding from mouse and human platelets requires p38 MAP kinase signaling. Protein kinase C, extracellular regulated-signal kinase MAPK, and caspases were not involved in TACE activation. Both inhibition of p38 MAPK and inactivation of TACE during platelet storage led to a markedly improved posttransfusion recovery and hemostatic function of platelets in mice. p38 MAPK inhibitors had only minor effects on the aggregation of fresh platelets under static or flow conditions in vitro. In summary, our data suggest that inhibition of p38 MAPK or TACE during storage may significantly improve the quality of stored platelets. PMID:19965619

  20. p38 mitogen-activated protein kinase activation during platelet storage: consequences for platelet recovery and hemostatic function in vivo.

    PubMed

    Canault, Matthias; Duerschmied, Daniel; Brill, Alexander; Stefanini, Lucia; Schatzberg, Daphne; Cifuni, Stephen M; Bergmeier, Wolfgang; Wagner, Denisa D

    2010-03-01

    Platelets undergo several modifications during storage that reduce their posttransfusion survival and functionality. One important feature of these changes, which are known as platelet storage lesion, is the shedding of the surface glycoproteins GPIb-alpha and GPV. We recently demonstrated that tumor necrosis factor-alpha converting enzyme (TACE/ADAM17) mediates mitochondrial injury-induced shedding of adhesion receptors and that TACE activity correlates with reduced posttransfusion survival of these cells. We now confirm that TACE mediates receptor shedding and clearance of platelets stored for 16 hours at 37 degrees C or 22 degrees C. We further demonstrate that both storage and mitochondrial injury lead to the phosphorylation of p38 mitogen-activated kinase (MAPK) in platelets and that TACE-mediated receptor shedding from mouse and human platelets requires p38 MAP kinase signaling. Protein kinase C, extracellular regulated-signal kinase MAPK, and caspases were not involved in TACE activation. Both inhibition of p38 MAPK and inactivation of TACE during platelet storage led to a markedly improved posttransfusion recovery and hemostatic function of platelets in mice. p38 MAPK inhibitors had only minor effects on the aggregation of fresh platelets under static or flow conditions in vitro. In summary, our data suggest that inhibition of p38 MAPK or TACE during storage may significantly improve the quality of stored platelets.

  1. p38 mitogen-activated protein kinase mediates IL-8 induction by the ribotoxin deoxynivalenol in human monocytes

    SciTech Connect

    Islam, Zahidul; Gray, Jennifer S.; Pestka, James J. . E-mail: pestka@msu.edu

    2006-06-15

    The effects of the ribotoxic trichothecene deoxynivalenol (DON) on mitogen-activated protein kinase (MAPK)-mediated IL-8 expression were investigated in cloned human monocytes and peripheral blood mononuclear cells (PBMC). DON (250 to 1000 ng/ml) induced both IL-8 mRNA and IL-8 heteronuclear RNA (hnRNA), an indicator of IL-8 transcription, in the human U937 monocytic cell line in a concentration-dependent manner. Expression of IL-8 hnRNA, mRNA and protein correlated with p38 phosphorylation and was completely abrogated by the p38 MAPK inhibitor SB203580. DON at 500 ng/ml similarly induced p38-dependent IL-8 protein and mRNA expression in PBMC cultures from healthy volunteers. Significantly increased IL-6 and IL-1{beta} intracellular protein and mRNA expression was also observed in PBMC treated with DON (500 ng/ml) which were also partially p38-dependent. Flow cytometry of PBMC revealed that DON-induced p38 phosphorylation varied among individuals relative to both threshold toxin concentrations (25-100 ng/ml) and relative increases in percentages of phospho-p38{sup +} cells. DON-induced p38 activation occurred exclusively in the CD14{sup +} monocyte population. DON was devoid of agonist activity for human Toll-like receptors 2, 3, 4, 5, 7, 8 and 9. However, two other ribotoxins, emetine and anisomycin, induced p38 phosphorylation in PBMC similarly to DON. Taken together, these data suggest that (1) p38 activation was required for induction of IL-8 and proinflammatory gene expression in the monocyte and (2) DON induced p38 activation in human monocytes via the ribotoxic stress response.

  2. p38 mitogen-activated protein kinase (p38 MAPK)-mediated autoimmunity: lessons to learn from ANCA vasculitis and pemphigus vulgaris.

    PubMed

    Mavropoulos, Athanasios; Orfanidou, Timoklia; Liaskos, Christos; Smyk, Daniel S; Billinis, Charalambos; Blank, Miri; Rigopoulou, Eirini I; Bogdanos, Dimitrios P

    2013-03-01

    Evidence is beginning to accumulate that p38 mitogen activated protein kinase (p38 MAPK) signaling pathway plays an important role in the regulation of cellular and humoral autoimmune responses. The exact mechanisms and the degree by which the p38 MAPK pathway participates in the immune-mediated induction of diseases have started to emerge. This review discusses the recent advances in the molecular dissection of the p38 MAPK pathway and the findings generated by reports investigating its role in the pathogenesis of autoimmune diseases such as rheumatoid arthritis, systemic lupus erythematosus, and autoimmune hepatitis. Application of newly-developed protocols based on sensitive flow cytometric detection has proven to be a useful tool in the investigation of the phosphorylation of p38 MAPK within different peripheral blood mononuclear cell populations and may help us to better understand the enigmatic role of this signaling cascade in the induction of autoimmunity as well as its role in immunosuppressive-induced remission. Special attention is paid to reported data proposing a specific role for autoantibody-induced activation of p38 MAPK-mediated immunopathology in the pathogenesis of autoimmune blistering diseases and anti-neutrophilic antibody-mediated vasculitides.

  3. The p38/RK mitogen-activated protein kinase pathway regulates interleukin-6 synthesis response to tumor necrosis factor.

    PubMed Central

    Beyaert, R; Cuenda, A; Vanden Berghe, W; Plaisance, S; Lee, J C; Haegeman, G; Cohen, P; Fiers, W

    1996-01-01

    Tumour necrosis factor (TNF) is a pleiotropic cytokine, the activities of which include effects on gene expression, cell growth and cell death. The biological signalling mechanisms which are responsible for these TNF effects remain largely unknown. Here we demonstrate that the stress-responsive p38 mitogen-activated protein (MAP) kinase is involved in TNF-induced cytokine expression. TNF Treatment of cell activated the p38 MAP kinase pathway, as revealed by increased phosphorylation of p38 MAP kinase itself, activation of the substrate protein MAPKAP kinase-2, and culminating in the phosphorylation of the heat shock protein 27 (hsp27). Pretreatment of cells with the highly specific p38 MAP kinase inhibitor SB203580 completely blocked this TNF-induced activation of MAPKAP kinase-2 and hsp27 phosphorylation. Under the same conditions, SB203580 also completely inhibited TNF-induced synthesis of interleukin (IL)-6 and expression of a reporter gene that was driven by a minimal promoter containing two NF-Kappa B elements. However, neither TNF-induced DNA binding of TNF-Kappa B nor TNF-induced phosphorylation of its subunits was modulated by SB203580, suggesting that NF-Kappa B is not a direct target for the p38 MAP kinase pathway. Interestingly, TNF-induced cytotoxicity was not affected by SB203580, indicating that p38 MAP kinase might be an interesting target to interfere selectively with TNF-induced gene activation. Images PMID:8617238

  4. p38 mitogen-activated protein kinase interacts with vinculin at focal adhesions during fatty acid-stimulated cell adhesion

    PubMed Central

    George, Margaret D.; Wine, Robert N.; Lackford, Brad; Kissling, Grace E.; Akiyama, Steven K.; Olden, Kenneth; Roberts, John D.

    2014-01-01

    Arachidonic acid stimulates cell adhesion by activating α2β1 integrins in a process that depends on protein kinases, including p38 mitogen activated protein kinase. Here, we describe the interaction of cytoskeletal components with key signaling molecules that contribute to spreading of, and morphological changes in, arachidonic acid-treated MDA-MB-435 human breast carcinoma cells. Arachidonic acid-treated cells showed increased attachment and spreading on collagen type IV as measured by electric cell-substrate impedance sensing. Fatty acid-treated cells displayed short cortical actin filaments associated with an increased number of β1 integrin-containing pseudopodia whereas untreated cells displayed elongated stress fibers and fewer clusters of β1 integrins. Confocal microscopy of arachidonic acid-treated cells showed that vinculin and phospho-p38 both appeared enriched in pseudopodia and at the tips of actin filaments, and fluorescence ratio imaging indicated the increase was specific for the phospho-(active) form of p38. Immunoprecipitates of phospho-p38 from extracts of arachidonic acid-treated cells contained vinculin, and GST-vinculin fusion proteins carrying the central region of vinculin bound phospho-p38, whereas fusion proteins expressing the terminal portions of vinculin did not. These data suggest that phospho-p38 associates with particular domains on critical focal adhesion proteins that are involved in tumor cell adhesion and spreading and that this association can be regulated by factors in the tumor microenvironment. PMID:24219282

  5. Role of p38 mitogen-activated protein kinase in Kupffer cell secretion of the proinflammatory cytokines after burn trauma.

    PubMed

    Chen, Xu-Lin; Xia, Zhao-Fan; Wei, Duo; Han, Sheng; Ben, Dao-Feng; Wang, Guang-Qing

    2003-09-01

    This study was designed to investigate the role of p38 mitogen-activated protein (MAP) kinase on Kupffer cells (KCs) secretion of proinflammatory cytokines such as tumor necrosis factor (TNF)-alpha and interleukin (IL)-1beta and hepatic injury following burn trauma. Sprague-Dawley rats were randomized into four groups: (1) sham burn rats given vehicle, (2) sham burn rats given the p38 MAP kinase inhibitor SB203580 (10mg/kg i.v., 15min and 12h after sham burn), (3) rats given a 30% total body surface area (TBSA) full-thickness burn and fluid resuscitation plus vehicle, and (4) burn rats given injury and fluid resuscitation plus SB203580. Rats from each group were killed at 24h post-burn to examine plasma aspartate transaminase (AST) and alanine transaminase (ALT) and KCs were isolated. The KCs secretion of TNF-alpha and IL-1beta and p38 MAP kinase activity (by Western blot analysis) were also examined. These studies showed by more significant activation of p38 MAP kinase in KCs harvested from burn rats than from shams. Burn trauma resulted in hepatic dysfunction and promoted KCs secretion of TNF-alpha and IL-1beta. SB203580 inhibited p38 MAP kinase activity, reduced KCs secretion of proinflammatory cytokines, and alleviated burn-mediated hepatic dysfunction. These data suggest p38 MAP kinase activation is one important aspect of the signaling event that may mediate the KCs secretion of proinflammatory cytokines TNF-alpha and IL-1beta following burn trauma.

  6. Prp19 facilitates invasion of hepatocellular carcinoma via p38 mitogen-activated protein kinase/Twist1 pathway

    PubMed Central

    Zhu, Ji-Min; Yu, Qian; Xue, Ru-Yi; Fang, Ying; Zhang, Yi-An; Chen, Yan-Jie; Liu, Tao-Tao; Dong, Ling; Shen, Xi-Zhong

    2016-01-01

    Pre-mRNA processing factor 19 (Prp19) is involved in many cellular events including pre-mRNA processing and DNA damage response. However, the pathological role of Prp19 in hepatocellular carcinoma (HCC) is still elusive. Here, we reported that Prp19 was increased in most HCC tissues and HCC cell lines, and its overexpression in HCC tissues was positively correlated with vascular invasion, tumor capsule breakthrough and poor prognosis. Prp19 potentiated migratory and invasive abilities of HCC cells in vitro and in vivo. Furthermore Prp19 facilitated Twist1-induced epithelial-mesenchymal transition. Mechanistic insights revealed that Prp19 directly binded with TGF-β-activated kinase1 (TAK1) and promoted the activation of p38 mitogen-activated protein kinase (MAPK), preventing Twist1 from degradation. Finally Prp19/p38 MAPK/Twist1 axis was attested in nude mice xenografts and HCC patient specimens. This work implies that the gain of Prp19 is a critical event during the progression of HCC, making it a promising target for malignancies with aberrant Prp19 expression. PMID:26959880

  7. On the participation of hippocampal p38 mitogen-activated protein kinase in extinction and reacquisition of inhibitory avoidance memory.

    PubMed

    Rossato, J I; Bevilaqua, L R M; Lima, R H; Medina, J H; Izquierdo, I; Cammarota, M

    2006-11-17

    Inhibitory avoidance (IA) learning relies on the formation of an association between stepping down from a platform present in a certain context (conditioned stimulus; CS) with an aversive unconditioned stimulus (US; i.e. a footshock). A single CS-US pairing establishes a robust long-term memory expressed as an increase in step-down latency at testing. However, repeated retrieval of the avoidance response in the absence of the US induces extinction of IA memory. That is, recurring presentation of the CS alone results in a new learning indicating that the CS no longer predicts the US. Although the signaling pathways involved in the consolidation of IA and other fear-motivated memories have been profusely studied, little is known about the molecular requirements of fear memory extinction. Here we report that, as happens with its consolidation, extinction of IA long-term memory requires activity of the p38 subfamily of mitogen-activated protein kinases (MAPK) in the CA1 region of the dorsal hippocampus. Moreover, we found that inhibition of hippocampal p38MAPK blocked memory reacquisition after extinction without affecting either the increase in IA memory retention induced by a second training session or animal's locomotor/exploratory activity and anxiety state.

  8. Increased expression of p38 mitogen-activated protein kinase is related to the acute renal lesions induced by gentamicin.

    PubMed

    Volpini, R A; Balbi, A P C; Costa, R S; Coimbra, T M

    2006-06-01

    Mitogen-activated protein kinases (MAPK) may be involved in the pathogenesis of acute renal failure. This study investigated the expression of p-p38 MAPK and nuclear factor kappa B (NF-kappaB) in the renal cortex of rats treated with gentamicin. Twenty rats were injected with gentamicin, 40 mg/kg, i.m., twice a day for 9 days, 20 with gentamicin + pyrrolidine dithiocarbamate (PDTC, an NF-kappaB inhibitor), 14 with 0.15 M NaCl, i.m., twice a day for 9 days, and 14 with 0.15 M NaCl , i.m., twice a day for 9 days and PDTC, 50 mg kg(-1) day(-1), i.p., twice a day for 15 days. The animals were killed 5 and 30 days after the last of the injections and the kidneys were removed for histological, immunohistochemical and Western blot analysis and for nitrate determination. The results of the immunohistochemical study were evaluated by counting the p-p38 MAPK-positive cells per area of renal cortex measuring 0.05 mm2. Creatinine was measured by the Jaffé method in blood samples collected 5 and 30 days after the end of the treatments. Gentamicin-treated rats presented a transitory increase in plasma creatinine levels. In addition, animals killed 5 days after the end of gentamicin treatment presented acute tubular necrosis and increased nitrate levels in the renal cortex. Increased expression of p-p38 MAPK and NF-kappaB was also observed in the kidneys from these animals. The animals killed 30 days after gentamicin treatment showed residual areas of interstitial fibrosis in the renal cortex, although the expression of p-p38 MAPK in their kidneys did not differ from control. Treatment with PDTC reduced the functional and structural changes induced by gentamicin as well as the expression of p-p38 MAPK and NF-kappaB. The increased expression of p-p38 MAPK and NF-kappaB observed in these rats suggests that these signaling molecules may be involved in the pathogenesis of tubulointerstitial nephritis induced by gentamicin.

  9. MKK3- and MKK6-regulated gene expression is mediated by the p38 mitogen-activated protein kinase signal transduction pathway.

    PubMed Central

    Raingeaud, J; Whitmarsh, A J; Barrett, T; Dérijard, B; Davis, R J

    1996-01-01

    The p38 mitogen-activated protein (MAP) kinase signal transduction pathway is activated by proinflammatory cytokines and environmental stress. The detection of p38 MAP kinase in the nucleus of activated cells suggests that p38 MAP kinase can mediate signaling to the nucleus. To test this hypothesis, we constructed expression vectors for activated MKK3 and MKK6, two MAP kinase kinases that phosphorylate and activate p38 MAP kinase. Expression of activated MKK3 and MKK6 in cultured cells caused a selective increase in p38 MAP kinase activity. Cotransfection experiments demonstrated that p38 MAP kinase activation causes increased reporter gene expression mediated by the transcription factors ATF2 and Elk-1. These data demonstrate that the nucleus is one target of the p38 MAP kinase signal transduction pathway. PMID:8622669

  10. Phosphorylation of sodium channel Na(v)1.8 by p38 mitogen-activated protein kinase increases current density in dorsal root ganglion neurons.

    PubMed

    Hudmon, Andy; Choi, Jin-Sung; Tyrrell, Lynda; Black, Joel A; Rush, Anthony M; Waxman, Stephen G; Dib-Hajj, Sulayman D

    2008-03-19

    The sensory neuron-specific sodium channel Na(v)1.8 and p38 mitogen-activated protein kinase are potential therapeutic targets within nociceptive dorsal root ganglion (DRG) neurons in inflammatory, and possibly neuropathic, pain. Na(v)1.8 channels within nociceptive DRG neurons contribute most of the inward current underlying the depolarizing phase of action potentials. Nerve injury and inflammation of peripheral tissues cause p38 activation in DRG neurons, a process that may contribute to nociceptive neuron hyperexcitability, which is associated with pain. However, how substrates of activated p38 contribute to DRG neuron hyperexcitability is currently not well understood. We report here, for the first time, that Na(v)1.8 and p38 are colocalized in DRG neurons, that Na(v)1.8 within DRG neurons is a substrate for p38, and that direct phosphorylation of the Na(v)1.8 channel by p38 regulates its function in these neurons. We show that direct phosphorylation of Na(v)1.8 at two p38 phospho-acceptor serine residues on the L1 loop (S551 and S556) causes an increase in Na(v)1.8 current density that is not accompanied by changes in gating properties of the channel. Our study suggests a mechanism by which activated p38 contributes to inflammatory, and possibly neuropathic, pain through a p38-mediated increase of Na(v)1.8 current density. PMID:18354022

  11. Molecular Basis of the Interaction of the Human Protein Tyrosine Phosphatase Non-receptor Type 4 (PTPN4) with the Mitogen-activated Protein Kinase p38γ.

    PubMed

    Maisonneuve, Pierre; Caillet-Saguy, Célia; Vaney, Marie-Christine; Bibi-Zainab, Edoo; Sawyer, Kristi; Raynal, Bertrand; Haouz, Ahmed; Delepierre, Muriel; Lafon, Monique; Cordier, Florence; Wolff, Nicolas

    2016-08-01

    The human protein tyrosine phosphatase non-receptor type 4 (PTPN4) prevents cell death induction in neuroblastoma and glioblastoma cell lines in a PDZ·PDZ binding motifs-dependent manner, but the cellular partners of PTPN4 involved in cell protection are unknown. Here, we described the mitogen-activated protein kinase p38γ as a cellular partner of PTPN4. The main contribution to the p38γ·PTPN4 complex formation is the tight interaction between the C terminus of p38γ and the PDZ domain of PTPN4. We solved the crystal structure of the PDZ domain of PTPN4 bound to the p38γ C terminus. We identified the molecular basis of recognition of the C-terminal sequence of p38γ that displays the highest affinity among all endogenous partners of PTPN4. We showed that the p38γ C terminus is also an efficient inducer of cell death after its intracellular delivery. In addition to recruiting the kinase, the binding of the C-terminal sequence of p38γ to PTPN4 abolishes the catalytic autoinhibition of PTPN4 and thus activates the phosphatase, which can efficiently dephosphorylate the activation loop of p38γ. We presume that the p38γ·PTPN4 interaction promotes cellular signaling, preventing cell death induction.

  12. Inhibition of p38 Mitogen-Activated Protein Kinase Enhances the Apoptosis Induced by Oxidized Low-Density Lipoprotein in Endothelial Progenitor Cells.

    PubMed

    Tie, Guodong; Yan, Jinglian; Messina, Julia A; Raffai, Robert L; Messina, Louis M

    2015-01-01

    Oxidized low-density lipoprotein (oxLDL) is an important risk factor in the development of atherosclerosis. oxLDL has been shown to decrease endothelial progenitor cell (EPC) number by inducing apoptosis. p38 mitogen-activated protein kinase (MAPK) was shown to be activated by oxLDL and participated in the regulation of EPC number and function. However, the role of p38 remains unknown. Here, we show that oxLDL-induced p38 phosphorylation in EPCs is time and dose dependent. Treatment with antioxidant N-acetyl cysteine restored oxLDL-induced p38 phosphorylation to basal levels. LOX-1-blocking antibody also significantly decreased oxLDL-induced p38 phosphorylation. Interestingly, TUNEL staining showed that pretreatment with the p38 inhibitor SB203580 further increased oxLDL-induced apoptosis in EPCs. In accordance with these findings, pretreatment with SB203580 further attenuated Akt phosphorylation in EPCs challenged with oxLDL, indicating an interaction between Akt and p38 MAPK pathways. In agreement, inhibition of p38 MAPK further attenuated Akt phosphorylation and increased apoptosis in EPCs isolated from hypercholesterolemic ApoE-/- mice. In conclusion, p38 MAPK serves as an anti-apoptotic pathway by supporting Akt activity when EPCs are challenged with oxLDL. PMID:27031525

  13. Activation of p38 mitogen-activated protein kinase in the early and peak phases of autoimmune neuritis in rat sciatic nerves.

    PubMed

    Moon, Changjong; Ahn, Meejung; Kim, Heechul; Lee, Yongduk; Koh, Chang Sung; Matsumoto, Yoh; Shin, Taekyun

    2005-04-01

    To examine the involvement of p38 mitogen-activated protein kinase (MAPK) in autoimmune disorders of the peripheral nerve system, we analyzed the phosphorylation of p38 MAPK protein in the sciatic nerves of Lewis rats with experimental autoimmune neuritis (EAN). Western blot analysis showed that phosphorylated p38 (p-p38) MAPK protein was significantly increased in the sciatic nerves of rats in the early and peak phases of EAN, and declined gradually thereafter. Immunohistochemistry showed that p-p38 MAPK levels were increased in the infiltrating inflammatory cells, including T cells and macrophages, as well as in blood vessels and some Schwann cells in EAN-affected sciatic nerves, as compared to the sciatic nerves of controls. Some inflammatory cells and a few Schwann cells were also positive for TUNEL reaction at the peak and recovery phases of EAN. In conclusion, we postulate that the phosphorylation of p38 MAPK is involved in the elimination of infiltrating inflammatory cells during the course of EAN and may possibly modulate recovery in autoimmune disorders of the peripheral nervous system.

  14. Stearoyl lysophosphatidylcholine enhances the phagocytic ability of macrophages through the AMP-activated protein kinase/p38 mitogen activated protein kinase pathway.

    PubMed

    Quan, Hui; Hur, Young-Hoe; Xin, Chun; Kim, Joung-Min; Choi, Jeong-Il; Kim, Man-Young; Bae, Hong-Beom

    2016-10-01

    A previous study showed that stearoyl lysophosphatidylcholine (sLPC) suppressed extracellular high mobility group box 1 translocation in macrophages stimulated with lipopolysaccharide through AMP-activated protein kinase (AMPK) activation. In the present study, we investigated whether sLPC-induced AMPK activation could enhance macrophages phagocytosis of bacteria. We found that sLPC increased phosphorylation of AMPK and acetyl-CoA carboxylase, a downstream target of AMPK, in a time- and dose-dependent manner in macrophages. Furthermore, sLPC increased the uptake of FITC-conjugated Escherichia coli by macrophages in a dose-dependent manner, and treatment with an AMPK inhibitor (compound C) or siRNA to AMPKα1 reversed this uptake. sLPC increased the phosphorylation of p38 mitogen-activated protein kinase (MAPK), but inhibition of AMPK activity with compound C or siRNA to AMPKα1 prevented the sLPC-induced increase in p38 MAPK phosphorylation. SB203580, a p38 MAPK inhibitor, decreased sLPC-induced phagocytosis. In vivo, systemic administration of sLPC to mice led to increased AMPK and p38 MAPK activity in the lung and to increased phagocytosis of fluorescent E. coli in bronchoalveolar lavage cells. These results suggest that sLPC increases macrophages phagocytosis through activation of the AMPK/p38 MAPK pathway. Therefore, sLPC is a candidate pharmacological agent for the treatment of bacterial infections in clinically relevant conditions. PMID:27517519

  15. The chemokine CCL2 activates p38 mitogen-activated protein kinase pathway in cultured rat hippocampal cells

    PubMed Central

    Cho, Jungsook; Gruol, Donna L.

    2008-01-01

    Emerging evidence indicates that chemokines can regulate both the physiology and biochemistry of CNS neurons and glia. In the current study, Western blot analysis showed that in rat hippocampal neuronal/glial cultures the signal transduction pathway activated by CCL2, a chemokine expressed in the normal brain and at elevated levels during neuroinflammation, involves a G-protein coupled receptor, p38 MAPK as well as its immediate upstream kinase MKK3/6, and the downstream transcription factor CREB. ERK 1/2 and the transcription factors STAT1 and STAT3 do not play a prominent role. CCL2 also altered Ca2+ influx and synaptic network activity in the hippocampal neurons. These results suggest an important role for p38 MAPK and CREB in hippocampal actions of CCL2. PMID:18584881

  16. Isoflurane attenuates mouse microglial engulfment induced by lipopolysaccharide and interferon-γ possibly by inhibition of p38 mitogen-activated protein kinase.

    PubMed

    Ryu, Jung-Hee; Wang, Zhi; Fan, Dan; Han, Sung-Hee; Do, Sang-Hwan; Zuo, Zhiyi

    2016-09-28

    Microglial engulfment is a basic function to clean up dead and injured cells and invaders, such as bacteria. This study was designed to assess the effects of isoflurane on the microglial engulfment induced by lipopolysaccharide (LPS) plus interferon-γ (IFN-γ) and the involvement of p38 mitogen-activated protein kinase (MAPK) in these effects. C8-B4 microglial cells were exposed to 1, 2, and 3% isoflurane at 2 h after the initiation of LPS (100 ng/ml) and IFN-γ (1 ng/ml) stimulation. Fluorescent immunostaining was performed to assess the percentage of cells with engulfment of fluorescent microspheres after stimulation for 24 h. P38 and phosphorylated p38 were determined by Western blotting. Isoflurane concentration dependently decreased microglial engulfment stimulated by LPS and IFN-γ. LPS and IFN-γ increased the phosphorylated p38 in microglial cells. This upregulation was decreased by isoflurane. SB203580, a p38 MAPK inhibitor, abolished the LPS-induced and IFN-γ-induced increase of engulfment activity, whereas anisomycin, a p38 MAPK activator, partly reversed the isoflurane-decreased microglial engulfment activity. These results suggest that isoflurane reduces LPS-induced and IFN-γ-induced microglial engulfment and that these effects may be mediated by inhibiting p38 MAPK. PMID:27513199

  17. Stress-activated mitogen-activated protein kinases c-Jun NH2-terminal kinase and p38 target Cdc25B for degradation.

    PubMed

    Uchida, Sanae; Yoshioka, Katsuji; Kizu, Ryoichi; Nakagama, Hitoshi; Matsunaga, Tsukasa; Ishizaka, Yukihito; Poon, Randy Y C; Yamashita, Katsumi

    2009-08-15

    Cdc25 dual specificity phosphatases positively regulate the cell cycle by activating cyclin-dependent kinase/cyclin complexes. Of the three mammalian Cdc25 isoforms, Cdc25A is phosphorylated by genotoxic stress-activated Chk1 or Chk2, which triggers its SCFbeta-TrCP-mediated degradation. However, the roles of Cdc25B and Cdc25C in cell stress checkpoints remain inconclusive. We herein report that c-Jun NH2-terminal kinase (JNK) induces the degradation of Cdc25B. Nongenotoxic stress induced by anisomycin caused rapid degradation of Cdc25B as well as Cdc25A. Cdc25B degradation was dependent mainly on JNK and partially on p38 mitogen-activated protein kinase (p38). Accordingly, cotransfection with JNK1, JNK2, or p38 destabilized Cdc25B. In vitro kinase assays and site-directed mutagenesis experiments revealed that the critical JNK and p38 phosphorylation site in Cdc25B was Ser101. Cdc25B with Ser101 mutated to alanine was refractory to anisomycin-induced degradation, and cells expressing such mutant Cdc25B proteins were able to override the anisomycin-induced G2 arrest. These results highlight the importance of a novel JNK/p38-Cdc25B axis for a nongenotoxic stress-induced cell cycle checkpoint.

  18. OSU-DY7, a novel D-tyrosinol derivative, mediates cytotoxicity in chronic lymphocytic leukaemia and Burkitt lymphoma through p38 mitogen-activated protein kinase pathway

    PubMed Central

    Bai, Li-Yuan; Ma, Yihui; Kulp, Samuel K.; Wang, Shu-Huei; Chiu, Chang-Fang; Frissora, Frank; Mani, Rajeswaran; Mo, Xiaokui; Jarjoura, David; Byrd, John C.; Chen, Ching-Shih; Muthusamy, Natarajan

    2013-01-01

    Summary Drug resistance and associated immune deregulation limit use of current therapies in chronic lymphocytic leukaemia (CLL), thus warranting alternative therapy development. Herein we demonstrate that OSU-DY7, a novel D-tyrosinol derivative targeting p38 mitogen-activated protein kinase (MAPK), mediates cytotoxicity in lymphocytic cell lines representing CLL (MEC-1), acute lymphoblastic leukaemia (697 cells), Burkitt lymphoma (Raji and Ramos) and primary B cells from CLL patients in a dose- and time-dependent manner. The OSU-DY7-induced cytotoxicity is dependent on caspase activation, as evidenced by induction of caspase-3 activation and poly (ADP-ribose) polymerase (PARP) cleavage and rescue of cytotoxicity by Z-VAD-FMK. Interestingly, OSU-DY7-induced cytotoxicity is mediated through activation of p38 MAPK, as evidenced by increased phosphorylation of p38 MAPK and downstream target protein MAPKAPK2. Pretreatment of B-CLL cells with SB202190, a specific p38 MAPK inhibitor, results in decreased MAPKAPK2 protein level with concomitant rescue of the cells from OSU-DY7-mediated cytotoxicity. Furthermore, OSU-DY7-induced cytotoxicity is associated with down regulation of p38 MAPK target BIRC5, that is rescued at protein and mRNA levels by SB202190. This study provides evidence for a role of OSU-DY7 in p38 MAPK activation and BIRC5 down regulation associated with apoptosis in B lymphocytic cells, thus warranting development of this alternative therapy for lymphoid malignancies. PMID:21470196

  19. 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

  20. Inhibition of coagulation, fibrinolysis, and endothelial cell activation by a p38 mitogen-activated protein kinase inhibitor during human endotoxemia.

    PubMed

    Branger, Judith; van den Blink, Bernt; Weijer, Sebastiaan; Gupta, Abhya; van Deventer, Sander J H; Hack, C Erik; Peppelenbosch, Maikel P; van der Poll, Tom

    2003-06-01

    P38 mitogen-activated protein kinase (MAPK) is an important component of intracellular signaling cascades that initiate various inflammatory cellular responses. To determine the role of p38 MAPK in the procoagulant response to lipopolysaccharide (LPS), 24 healthy subjects were exposed to an intravenous dose of LPS (4 ng/kg), preceded 3 hours earlier by orally administered 600 or 50 mg BIRB 796 BS (a specific p38 MAPK inhibitor), or placebo. The 600-mg dose of BIRB 796 BS strongly inhibited LPS-induced coagulation activation, as measured by plasma concentrations of the prothrombin fragment F1 + 2. BIRB 796 BS also dose dependently attenuated the activation and subsequent inhibition of the fibrinolytic system (plasma tissue-type plasminogen activator, plasmin-alpha2-antiplasmin complexes, and plasminogen activator inhibitor type 1) and endothelial cell activation (plasma soluble E-selectin and von Willebrand factor). Activation of p38 MAPK plays an important role in the procoagulant and endothelial cell response after in vivo exposure to LPS.

  1. Reactive oxygen species and p38 mitogen-activated protein kinase activate Bax to induce mitochondrial cytochrome c release and apoptosis in response to malonate.

    PubMed

    Gomez-Lazaro, M; Galindo, M F; Melero-Fernandez de Mera, R M; Fernandez-Gómez, F J; Concannon, C G; Segura, M F; Comella, J X; Prehn, J H M; Jordan, J

    2007-03-01

    Malonate, an inhibitor of mitochondrial complex II, is a widely used toxin to study neurodegeneration in Huntington's disease and ischemic stroke. We have shown previously that malonate increased reactive oxygen species (ROS) production in human SH-SY5Y neuroblastoma cells, leading to oxidative stress, cytochrome c release, and apoptotic cell death. Expression of a green fluorescent protein-Bax fusion protein in SH-SY5Y neuroblastoma cells demonstrated a Bax redistribution from the cytosol to mitochondria after 12 to 24 h of malonate treatment that coincided with mitochondrial potential collapse and chromatin condensation. Inhibition of Bax translocation using furosemide, as well as Bax gene deletion, afforded significant protection against malonate-induced apoptosis. Further experiments revealed that malonate induced a prominent increase in the level of activated p38 mitogen-activated protein (MAP) kinase and that treatment with the p38 MAP kinase inhibitor SKF86002 potently blocked malonate-induced Bax translocation and apoptosis. Treatment with vitamin E diminished ROS production, reduced the activation status of p38 MAP kinase, inhibited Bax translocation, and protected against malonate-induced apoptosis. Our data suggest that malonate-induced ROS production and subsequent p38 MAP kinase activation mediates the activation of the pro-apoptotic Bax protein to induce mitochondrial membrane permeabilization and neuronal apoptosis.

  2. Novel Antiplatelet Activity of Minocycline Involves Inhibition of MLK3-p38 Mitogen Activated Protein Kinase Axis

    PubMed Central

    Jackson, Joseph W.; Singh, Meera V.; Singh, Vir B.; Jones, Letitia D.; Davidson, Gregory A.; Ture, Sara; Morrell, Craig N.; Schifitto, Giovanni; Maggirwar, Sanjay B.

    2016-01-01

    Platelets play an essential role in hemostasis and wound healing by facilitating thrombus formation at sites of injury. Platelets also mediate inflammation and contain several pro-inflammatory molecules including cytokines and chemokines that mediate leukocyte recruitment and activation. Not surprisingly, platelet dysfunction is known to contribute to several inflammatory disorders. Antiplatelet therapies, such as aspirin, adenosine diphosphate (ADP) antagonists, glycoprotein IIb/IIIa (GPIIb/IIIa) inhibitors, and anticoagulants such as warfarin, dampen platelet activity at the risk of unwarranted bleeding. Thus, the development of drugs that reduce platelet-mediated inflammation without interfering with thrombus formation is of importance to combat platelet-associated disorders. We have shown here for the first time that the tetracycline antibiotic, minocycline, administered to HIV-infected individuals reduces plasma levels of soluble CD40L and platelet factor 4 levels, host molecules predominately released by platelets. Minocycline reduced the activation of isolated platelets in the presence of the potent platelet activator, thrombin, as measured by ELISA and flow cytometry. Platelet degranulation was reduced upon exposure to minocycline as shown by mepacrine retention and flow cytometry. However, minocycline had no effect on spreading, aggregation, GPIIb/IIIa activation, or in vivo thrombus formation. Lastly, immunoblot analysis suggests that the antiplatelet activity of minocycline is likely mediated by inhibition of mixed lineage kinase 3 (MLK3)-p38 MAPK signaling axis and loss of p38 activity. Our findings provide a better understanding of platelet biology and a novel repurposing of an established antibiotic, minocycline, to specifically reduce platelet granule release without affecting thrombosis, which may yield insights in generating novel, specific antiplatelet therapies. PMID:27270236

  3. LY2228820 dimesylate, a selective inhibitor of p38 mitogen-activated protein kinase, reduces angiogenic endothelial cord formation in vitro and in vivo.

    PubMed

    Tate, Courtney M; Blosser, Wayne; Wyss, Lisa; Evans, Glenn; Xue, Qi; Pan, Yong; Stancato, Louis

    2013-03-01

    LY2228820 dimesylate is a highly selective small molecule inhibitor of p38α and p38β mitogen-activated protein kinases (MAPKs) that is currently under clinical investigation for human malignancies. p38 MAPK is implicated in a wide range of biological processes, in particular those that support tumorigenesis. One such process, angiogenesis, is required for tumor growth and metastasis, and many new cancer therapies are therefore directed against the tumor vasculature. Using an in vitro co-culture endothelial cord formation assay, a surrogate of angiogenesis, we investigated the role of p38 MAPK in growth factor- and tumor-driven angiogenesis using LY2228820 dimesylate treatment and by shRNA gene knockdown. p38 MAPK was activated in endothelial cells upon growth factor stimulation, with inhibition by LY2228820 dimesylate treatment causing a significant decrease in VEGF-, bFGF-, EGF-, and IL-6-induced endothelial cord formation and an even more dramatic decrease in tumor-driven cord formation. In addition to involvement in downstream cytokine signaling, p38 MAPK was important for VEGF, bFGF, EGF, IL-6, and other proangiogenic cytokine secretion in stromal and tumor cells. LY2228820 dimesylate results were substantiated using p38α MAPK-specific shRNA and shRNA against the downstream p38 MAPK effectors MAPKAPK-2 and HSP27. Using in vivo models of functional neoangiogenesis, LY2228820 dimesylate treatment reduced hemoglobin content in a plug assay and decreased VEGF-A-stimulated vascularization in a mouse ear model. Thus, p38α MAPK is implicated in tumor angiogenesis through direct tumoral effects and through reduction of proangiogenic cytokine secretion via the microenvironment.

  4. LY2228820 dimesylate, a selective inhibitor of p38 mitogen-activated protein kinase, reduces angiogenic endothelial cord formation in vitro and in vivo.

    PubMed

    Tate, Courtney M; Blosser, Wayne; Wyss, Lisa; Evans, Glenn; Xue, Qi; Pan, Yong; Stancato, Louis

    2013-03-01

    LY2228820 dimesylate is a highly selective small molecule inhibitor of p38α and p38β mitogen-activated protein kinases (MAPKs) that is currently under clinical investigation for human malignancies. p38 MAPK is implicated in a wide range of biological processes, in particular those that support tumorigenesis. One such process, angiogenesis, is required for tumor growth and metastasis, and many new cancer therapies are therefore directed against the tumor vasculature. Using an in vitro co-culture endothelial cord formation assay, a surrogate of angiogenesis, we investigated the role of p38 MAPK in growth factor- and tumor-driven angiogenesis using LY2228820 dimesylate treatment and by shRNA gene knockdown. p38 MAPK was activated in endothelial cells upon growth factor stimulation, with inhibition by LY2228820 dimesylate treatment causing a significant decrease in VEGF-, bFGF-, EGF-, and IL-6-induced endothelial cord formation and an even more dramatic decrease in tumor-driven cord formation. In addition to involvement in downstream cytokine signaling, p38 MAPK was important for VEGF, bFGF, EGF, IL-6, and other proangiogenic cytokine secretion in stromal and tumor cells. LY2228820 dimesylate results were substantiated using p38α MAPK-specific shRNA and shRNA against the downstream p38 MAPK effectors MAPKAPK-2 and HSP27. Using in vivo models of functional neoangiogenesis, LY2228820 dimesylate treatment reduced hemoglobin content in a plug assay and decreased VEGF-A-stimulated vascularization in a mouse ear model. Thus, p38α MAPK is implicated in tumor angiogenesis through direct tumoral effects and through reduction of proangiogenic cytokine secretion via the microenvironment. PMID:23335506

  5. Phosphorylation by p38 Mitogen-Activated Protein Kinase Promotes Estrogen Receptor α Turnover and Functional Activity via the SCFSkp2 Proteasomal Complex

    PubMed Central

    Bhatt, Shweta; Xiao, Zhen; Meng, Zhaojing

    2012-01-01

    The nuclear hormone receptor estrogen receptor α (ERα) mediates the actions of estrogens in target cells and is a master regulator of the gene expression and proliferative programs of breast cancer cells. The presence of ERα in breast cancer cells is crucial for the effectiveness of endocrine therapies, and its loss is a hallmark of endocrine-insensitive breast tumors. However, the molecular mechanisms underlying the regulation of the cellular levels of ERα are not fully understood. Our findings reveal a unique cellular pathway involving the p38 mitogen-activated protein kinase (p38MAPK)-mediated phosphorylation of ERα at Ser-294 that specifies its turnover by the SCFSkp2 proteasome complex. Consistently, we observed an inverse relationship between ERα and Skp2 or active p38MAPK in breast cancer cell lines and human tumors. ERα regulation by Skp2 was cell cycle stage dependent and critical for promoting the mitogenic effects of estradiol via ERα. Interestingly, by the knockdown of Skp2 or the inhibition of p38MAPK, we restored functional ERα protein levels and the control of gene expression and proliferation by estrogen and antiestrogen in ERα-negative breast cancer cells. Our findings highlight a novel pathway with therapeutic potential for restoring ERα and the responsiveness to endocrine therapy in some endocrine-insensitive ERα-negative breast cancers. PMID:22431515

  6. Sensitization of apoptotically-resistant breast carcinoma cells to TNF and TRAIL by inhibition of p38 mitogen-activated protein kinase signaling.

    PubMed

    Weldon, Christopher B; Parker, Amanda P; Patten, Daniel; Elliott, Steven; Tang, Yan; Frigo, Daniel E; Dugan, Christine M; Coakley, Erin L; Butler, Nancy N; Clayton, John L; Alam, Jawed; Curiel, Tyler J; Beckman, Barbara S; Jaffe, Bernard M; Burow, Matthew E

    2004-06-01

    The mitogen-activated protein kinase (MAPK) cascade is a critical component in the regulation of cell survival and proliferation decisions. In breast carcinoma cells, activation of the p38-MAPK member of this family occurs in response to pro-inflammatory cytokines and cellular stress. The involvement of p38-MAPK in the activation of the transcription factor, NF-kappaB, suggests a potential role and mechanism for regulation of cell survival and drug resistance. Generation of the resistant MCF-7 variant (MCF-7TN-R) was achieved by prolonged exposure of MCF-7N cells to increasing concentrations of TNF. Differences in MAPK activation and function in the MCF-7 cell variants were determined. The role of the p38-MAPK pathway in regulation of resistance was determined using pharmacological (SB 203580) or molecular [Dominant Inhibitory (DI)-p38] inhibition. The effect of p38 inhibition on NF-kappaB transcriptional activation was analyzed. As compared to the sensitive MCF-7N parent cell line, the MCF-7TN-R cell line displayed significant resistance to TNF- and TRAIL-induced cell death. Analysis of the expression and phosphorylation of members of the MAPK family revealed an increased basal activation of p38 in the MCF-7TN-R variant. The p38-mediated phosphorylation and transcriptional activity were suppressed by pharmacologic inhibition with SB 230580. Treatment of MCF-7TN-R cells with SB partially restored sensitivity to TNF-induced cell death. In addition, use of a DI-p38 construct with or without the addition to TNF induced cell death, thus restoring TNF-sensitivity to these cells. The ability of p38 inhibition to restore apoptotic sensitivity was correlated with suppression of the TNF-induced cell survival pathway, NF-kappaB. The increased activation of p38-MAPK in MCF-7TN-R cells demonstrates that this signaling pathway through activation of NF-kappaB is an important route for control of resistance to cell death in breast carcinoma. Molecular and pharmacological

  7. Toll-Like Receptor 4-Mediated Activation of p38 Mitogen-Activated Protein Kinase Is a Determinant of Respiratory Virus Entry and Tropism▿

    PubMed Central

    Marchant, David; Singhera, Gurpreet K.; Utokaparch, Soraya; Hackett, Tillie L.; Boyd, John H.; Luo, Zongshu; Si, Xiaoning; Dorscheid, Delbert R.; McManus, Bruce M.; Hegele, Richard G.

    2010-01-01

    Respiratory viruses exert a heavy toll of morbidity and mortality worldwide. Despite this burden there are few specific treatments available for respiratory virus infections. Since many viruses utilize host cell enzymatic machinery such as protein kinases for replication, we determined whether pharmacological inhibition of kinases could, in principle, be used as a broad antiviral strategy for common human respiratory virus infections. A panel of green fluorescent protein (GFP)-expressing recombinant respiratory viruses, including an isolate of H1N1 influenza virus (H1N1/Weiss/43), was used to represent a broad range of virus families responsible for common respiratory infections (Adenoviridae, Paramyxoviridae, Picornaviridae, and Orthomyxoviridae). Kinase inhibitors were screened in a high-throughput assay that detected virus infection in human airway epithelial cells (1HAEo-) using a fluorescent plate reader. Inhibition of p38 mitogen-activated protein kinase (MAPK) signaling was able to significantly inhibit replication by all viruses tested. Therefore, the pathways involved in virus-mediated p38 and extracellular signal-regulated kinase (ERK) MAPK activation were investigated using bronchial epithelial cells and primary fibroblasts derived from MyD88 knockout mouse lungs. Influenza virus, which activated p38 MAPK to approximately 10-fold-greater levels than did respiratory syncytial virus (RSV) in 1HAEo- cells, was internalized about 8-fold faster and more completely than RSV. We show for the first time that p38 MAPK is a determinant of virus infection that is dependent upon MyD88 expression and Toll-like receptor 4 (TLR4) ligation. Imaging of virus-TLR4 interactions showed significant clustering of TLR4 at the site of virus-cell interaction, triggering phosphorylation of downstream targets of p38 MAPK, suggesting the need for a signaling receptor to activate virus internalization. PMID:20702616

  8. Protein-bound polysaccharide-K induces apoptosis via mitochondria and p38 mitogen-activated protein kinase-dependent pathways in HL-60 promyelomonocytic leukemia cells.

    PubMed

    Hirahara, Noriyuki; Edamatsu, Takeo; Fujieda, Ayako; Fujioka, Masaki; Wada, Tsutomu; Tajima, Yoshitsugu

    2013-07-01

    Protein-bound polysaccharide-K (PSK) is extracted from Coriolus versicolor (CM101). PSK is a biological response modifier (BRM), and its mechanism of action is partly mediated by modulating host immune systems; however, recent studies showed antiproliferative activity of PSK. Therefore, we examined the mechanism underlying the antiproliferative activity of PSK using seven different human malignant cell lines (WiDr, HT29, SW480, KATOIII, AGS, HL-60 and U937), and PSK was found to inhibit the proliferation of HL-60 cells most profoundly. Therefore, HL-60 cells were used to elucidate the mechanism of the antiproliferative activity. Western blotting was performed to detect phosphorylated p38 mitogen-activated protein kinase (MAPK). A p38 MAPK inhibitor, SB203580, was used to examine the roles in PSK-induced apoptosis and growth inhibition. Flow cytometry was performed for mitochondrial membrane potential detection. PSK activated caspase-3 and induced p38 MAPK phosphorylation. Co-treatment with SB203580 blocked PSK-induced apoptosis, caspase-3 activation and growth inhibition. PSK induced apoptosis via the mitochondrial pathway. The depolarization of mitochondria induced by PSK was reversed by co-treatment with SB203580. The present study revealed that PSK induced apoptosis in HL-60 cells via a mitochondrial and p38 MAPK-dependent pathway. PMID:23604455

  9. p38 Mitogen-Activated Protein Kinase (MAPK) Increases Arginase Activity and Contributes to Endothelial Dysfunction in Corpora Cavernosa from Angiotensin-II Treated Mice

    PubMed Central

    Toque, Haroldo A.; Romero, Maritza J.; Tostes, Rita C.; Shatanawi, Alia; Chandra, Surabhi; Carneiro, Zidonia N.; Inscho, Edward W.; Webb, R. Clinton; Caldwell, Ruth B.; Caldwell, R. William

    2010-01-01

    Introduction Angiotensin II (AngII) activates p38 mitogen-activated protein kinase (MAPK) and elevates arginase activity in endothelial cells. Upregulation of arginase activity has been implicated in endothelial dysfunction by reducing NO bioavailability. However, signaling pathways activated by AngII in the penis are largely unknown. Aim We hypothesized that activation of p38 MAPK increases arginase activity and thus impairs penile vascular function in AngII-treated mice. Methods Male C57BL/6 mice were implanted with osmotic minipumps containing saline or AngII (42 μg/kg/h) for 14 days and co-treated with p38 MAPK inhibitor, SB 203580 (5 μg/kg/day), beginning 2 days before minipump implantation. Systolic blood pressure (SBP) was measured. Corpus cavernosum (CC) tissue was used for vascular functional studies and protein expression levels of p38 MAPK, arginase and constitutive NOS, and arginase activity. Main Outcome Measures Arginase expression and activity; expression of phospho-p38 MAPK, -eNOS and nNOS proteins; endothelium-dependent and nitrergic nerve-mediated relaxations were determined in CC from control and AngII-infused mice. Results AngII increased SBP (22%) and increased CC arginase activity and expression (~2-fold), and phosphorylated P38 MAPK levels (30%) over control. Treatment with SB 203580 prevented these effects. Endothelium-dependent NO-mediated relaxation to acetylcholine was significantly reduced by AngII and this effect was prevented by SB 203580 (P<0.01). AngII (2-week) did not alter nitrergic function. However, SB 203580 significantly increased nitrergic relaxation in both control and AngII tissue at lower frequencies. Maximum contractile responses for phenylephrine and electrical field stimulation were increased by AngII (56% and 171%, respectively), and attenuated by SB 203580 treated. AngII treatment also decreased eNOS phosphorylation at Ser-1177 compared to control. Treatment with SB 203580 prevented all these changes. Conclusion p38

  10. Src Family Kinases and p38 Mitogen-Activated Protein Kinases Regulate Pluripotent Cell Differentiation in Culture

    PubMed Central

    Tan, Boon Siang Nicholas; Kwek, Joly; Wong, Chong Kum Edwin; Saner, Nicholas J.; Yap, Charlotte; Felquer, Fernando; Morris, Michael B.; Gardner, David K.; Rathjen, Peter D.; Rathjen, Joy

    2016-01-01

    Multiple pluripotent cell populations, which together comprise the pluripotent cell lineage, have been identified. The mechanisms that control the progression between these populations are still poorly understood. The formation of early primitive ectoderm-like (EPL) cells from mouse embryonic stem (mES) cells provides a model to understand how one such transition is regulated. EPL cells form from mES cells in response to l-proline uptake through the transporter Slc38a2. Using inhibitors of cell signaling we have shown that Src family kinases, p38 MAPK, ERK1/2 and GSK3β are required for the transition between mES and EPL cells. ERK1/2, c-Src and GSK3β are likely to be enforcing a receptive, primed state in mES cells, while Src family kinases and p38 MAPK are involved in the establishment of EPL cells. Inhibition of these pathways prevented the acquisition of most, but not all, features of EPL cells, suggesting that other pathways are required. L-proline activation of differentiation is mediated through metabolism and changes to intracellular metabolite levels, specifically reactive oxygen species. The implication of multiple signaling pathways in the process suggests a model in which the context of Src family kinase activation determines the outcomes of pluripotent cell differentiation. PMID:27723793

  11. Decreased hepatic phosphorylated p38 mitogen-activated protein kinase contributes to attenuation of thioacetamide-induced hepatic necrosis in diet-induced obese mice.

    PubMed

    Shirai, Makoto; Arakawa, Shingo; Teranishi, Munehiro; Kai, Kiyonori

    2016-04-01

    We previously reported that thioacetamide (TA)-induced hepatocellular necrosis was attenuated in mice fed a high-fat diet (HFD mice) compared with mice fed a normal rodent diet (ND mice). In this study, we investigated whether p38 mitogen-activated protein kinase (p38 MAPK) was involved in this attenuation. Western blot analysis revealed that hepatic phosphorylated p38 MAPK protein decreased at 8 and 24 hours (hr) after TA dosing in the HFD mice, while it decreased only at 24 hr in the ND mice in comparison to the time- and diet-matched, vehicle-treated mice. p38 MAPK regulates various biological functions including inflammation, therefore, hepatic metabolomics analysis focusing on pro-inflammatory lipid mediators was performed. At 24 hr after TA dosing, only one pro-inflammatory mediator, 12-hydroxyeicosatetraenoic acid (HETE), was higher in the HFD mice. On the other hand, in addition to 12-HETE, 15-HETE and 12-hydroxyeicosapentaenoic acid (HEPE) were higher and omega-3/omega-6 polyunsaturated fatty acids ratios were lower in the ND mice at 24 hr. These results of metabolomics indicated that less pro-inflammatory state was seen in HFD mice than in ND mice at 24 hr. Finally, to confirm whether the observed decrease in phosphorylated p38 MAPK could attenuate TA-induced hepatocellular necrosis, we showed that SB203580 hydrochloride, an inhibitor of p38 MAPK, partially attenuated TA-induced hepatic necrosis in ND mice. Collectively, these results suggest that a prompt decrease in phosphorylation of p38 MAPK after TA administration is one of the factors that attenuate TA-induced hepatic necrosis in HFD mice. PMID:26961609

  12. Activation of p38 Mitogen-Activated Protein Kinase by Clotrimazole Induces Multidrug Resistance-Associated Protein 3 Activation through a Novel Transcriptional Element.

    PubMed

    Sasaki, Takamitsu; Inami, Keita; Numata, Yoshihiro; Funakoshi, Kodai; Yoshida, Midori; Kumagai, Takeshi; Kanno, Shuichi; Matsui, Satomi; Toriyabe, Takayoshi; Yamazoe, Yasushi; Yoshinari, Kouichi; Nagata, Kiyoshi

    2016-10-01

    Multidrug resistance-associated protein 3 (MRP3) is a basolaterally localized transporter in the liver and contributes to the transport of various metabolites such as conjugates of endogenous compounds and drugs from hepatocytes. MRP3 expression in the human liver is low under normal physiologic conditions but is induced by drug treatment. Although several studies have identified a region necessary for the basal transcription of MRP3, no region that responds to drugs has been reported. To identify the xenobiotic-responsive elements of MRP3, we constructed a luciferase reporter plasmid containing the MRP3 5'-flanking region up to -10 kb upstream from the transcription start site. Among typical nuclear receptor ligands, clotrimazole dramatically enhanced MRP3 reporter activity in HepG2 cells, whereas rifampicin had no effect. We then conducted MRP3 reporter assays with deletion or mutation constructs to identify a clotrimazole-responsive element. The element was located approximately -6.8 kb upstream from the MRP3 transcription start site. Overexpression of the pregnane X receptor did not enhance clotrimazole-mediated transcription. We found that clotrimazole was toxic to HepG2 cells and we therefore investigated whether mitogen-activated protein kinase (MAPK) activation is involved in the transactivation of MRP3 by clotrimazole. p38 MAPK inhibitor SB203580 [4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole] suppressed MRP3 mRNA expression induced by clotrimazole, whereas c-Jun N-terminal kinase inhibitor SP600125 (1,9-pyrazoloanthrone) and extracellular signal-regulated kinase inhibitor PD98059 [2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one] did not. Phosphorylated p38 MAPK was detected in HepG2 cells treated with clotrimazole. These results suggest that activation of the p38 MAPK pathway induces the transcriptional activation of MRP3. PMID:27507784

  13. Interaction of Omega, Sigma, and Theta glutathione transferases with p38b mitogen-activated protein kinase from the fruit fly, Drosophila melanogaster.

    PubMed

    Wongtrakul, J; Janphen, K; Saisawang, C; Ketterman, A J

    2014-05-01

    Glutathione S-transferases (GSTs) are a diverse family of phase II detoxification enzymes found in almost all organisms. Besides playing a major role in the detoxification of xenobiotic and toxic compounds, GSTs are also involved in the regulation of mitogen activated protein (MAP) kinase signal transduction by interaction with proteins in the pathway. An in vitro study was performed for Theta, Omega, Sigma GSTs and their interaction with MAP kinase p38b protein from the fruit fly Drosophila melanogaster Meigen (Diptera: Drosophilidae). The study included the effects of all five Omega class GSTs (DmGSTO1, DmGSTO2a, DmGSTO2b, DmGSTO3, DmGSTO4), all five Theta class GSTs (DmGSTT1, DmGSTT2, DmGSTT3a, DmGSTT3b, DmGSTT4), and one Sigma class glutathione transferase on the activity of Drosophila p38b, including the reciprocal effect of this kinase protein on glutathione transferase activity. It was found that DmGSTT2, DmGSTT3b, DmGSTO1, and DmGSTO3 activated p38b significantly. Substrate specificities of GSTs were also altered after co-incubation with p38b. Although p38b activated DmGSTO1, DmGSTO2a, and DmGSTT2, it inhibited DmGSTT3b and DmGSTO3 activity toward xenobiotic and physiological substrates tested. These results suggest a novel link between Omega and Theta GSTs with the p38b MAP kinase pathway.

  14. Interaction of Omega, Sigma, and Theta Glutathione Transferases with p38b Mitogen-Activated Protein Kinase from the Fruit Fly, Drosophila melanogaster

    PubMed Central

    Wongtrakul, J.; Janphen, K.; Saisawang, C.; Ketterman, A.J.

    2014-01-01

    Glutathione S-transferases (GSTs) are a diverse family of phase II detoxification enzymes found in almost all organisms. Besides playing a major role in the detoxification of xenobiotic and toxic compounds, GSTs are also involved in the regulation of mitogen activated protein (MAP) kinase signal transduction by interaction with proteins in the pathway. An in vitro study was performed for Theta, Omega, Sigma GSTs and their interaction with MAP kinase p38b protein from the fruit fly Drosophila melanogaster Meigen (Diptera: Drosophilidae). The study included the effects of all five Omega class GSTs (DmGSTO1, DmGSTO2a, DmGSTO2b, DmGSTO3, DmGSTO4), all five Theta class GSTs (DmGSTT1, DmGSTT2, DmGSTT3a, DmGSTT3b, DmGSTT4), and one Sigma class glutathione transferase on the activity of Drosophila p38b, including the reciprocal effect of this kinase protein on glutathione transferase activity. It was found that DmGSTT2, DmGSTT3b, DmGSTO1, and DmGSTO3 activated p38b significantly. Substrate specificities of GSTs were also altered after co-incubation with p38b. Although p38b activated DmGSTO1, DmGSTO2a, and DmGSTT2, it inhibited DmGSTT3b and DmGSTO3 activity toward xenobiotic and physiological substrates tested. These results suggest a novel link between Omega and Theta GSTs with the p38b MAP kinase pathway. PMID:25373207

  15. Activation of p38 mitogen-activated protein kinase by celecoxib oppositely regulates survivin and gamma-H2AX in human colorectal cancer cells

    SciTech Connect

    Hsiao, P.-W.; Chang, C.-C.; Liu, H.-F.; Tsai, C.-M.; Chiu, Ted H.; Chao, J.-I . E-mail: chaoji@mail.tcu.edu.tw

    2007-07-01

    Cancer cells express survivin that facilitates tumorigenesis. Celecoxib has been shown to reduce human colorectal cancers. However, the role and regulation of survivin by celecoxib in colorectal carcinoma cells remain unclear. Treatment with 40-80 {mu}M celecoxib for 24 h induced cytotoxicity and proliferation inhibition via a concentration-dependent manner in RKO colorectal carcinoma cells. Celecoxib blocked the survivin protein expression and increased the phosphorylation of H2AX at serine-193 ({gamma}-H2AX). The survivin gene knockdown by transfection with a survivin siRNA revealed that the loss of survivin correlated with the expression of {gamma}-H2AX. Meanwhile, celecoxib increased caspase-3 activation and apoptosis. Celecoxib activated the phosphorylation of p38 mitogen-activated protein (MAP) kinase. The phosphorylated proteins of p38 MAP kinase and {gamma}-H2AX were observed in the apoptotic cells. SB203580, a specific p38 MAP kinase inhibitor, protected the survivin protein expression and decreased the levels of {gamma}-H2AX and apoptosis in the celecoxib-exposed cells. The blockade of survivin expression increased the celecoxib-induced cytotoxicity; conversely, overexpression of survivin by transfection with a survivin-expressing vector raised the cancer cell proliferation and resisted the celecoxib-induced cell death. Our results provide for the first time that p38 MAP kinase participates in the down-regulation of survivin and subsequently induces the activation of {gamma}-H2AX for mediating apoptosis following treatment with celecoxib in human colorectal cancer cells.

  16. Cross-interactions of two p38 mitogen-activated protein (MAP) kinase inhibitors and two cholecystokinin (CCK) receptor antagonists with the CCK1 receptor and p38 MAP kinase.

    PubMed

    Morel, Caroline; Ibarz, Géraldine; Oiry, Catherine; Carnazzi, Eric; Bergé, Gilbert; Gagne, Didier; Galleyrand, Jean-Claude; Martinez, Jean

    2005-06-01

    Although SB202190 and SB203580 are described as specific p38 MAP kinase inhibitors, several reports have indicated that other enzymes are also sensitive to SB203580. Using a pharmacological approach, we report for the first time that compounds SB202190 and SB203580 were able to directly and selectively interact with a G-protein-coupled receptor, namely the cholecystokinin receptor subtype CCK1, but not with the CCK2 receptor. We demonstrated that these compounds were non-competitive antagonists of the CCK1 receptor at concentrations typically used to inhibit protein kinases. By chimeric construction of the CCK2 receptor, we determined the involvement of two CCK1 receptor intracellular loops in the binding of SB202190 and SB203580. We also showed that two CCK antagonists, L364,718 and L365,260, were able to regulate p38 mitogen-activated protein (MAP) kinase activity. Using a reporter gene strategy and immunoblotting experiments, we demonstrated that both CCK antagonists inhibited selectively the enzymatic activity of p38 MAP kinase. Kinase assays suggested that this inhibition resulted from a direct interaction with both CCK antagonists. Molecular modeling simulations suggested that this interaction occurs in the ATP binding pocket of p38 MAP kinase. These results suggest that SB202190 and SB203580 bind to the CCK1 receptor and, as such, these compounds should be used with caution in models that express this receptor. We also found that L364,718 and L365,260, two CCK receptor antagonists, directly interacted with p38 MAP kinase and inhibited its activity. These findings suggest that the CCK1 receptor shares structural analogies with the p38 MAP kinase ATP binding site. They open the way to potential design of either a new family of MAP kinase inhibitors from CCK1 receptor ligand structures or new CCK1 receptor ligands based on p38 MAP kinase inhibitor structures.

  17. A stress-activated, p38 mitogen-activated protein kinase-ATF/CREB pathway regulates posttranscriptional, sequence-dependent decay of target RNAs.

    PubMed

    Gao, Jun; Wagnon, Jacy L; Protacio, Reine M; Glazko, Galina V; Beggs, Marjorie; Raj, Vinay; Davidson, Mari K; Wahls, Wayne P

    2013-08-01

    Broadly conserved, mitogen-activated/stress-activated protein kinases (MAPK/SAPK) of the p38 family regulate multiple cellular processes. They transduce signals via dimeric, basic leucine zipper (bZIP) transcription factors of the ATF/CREB family (such as Atf2, Fos, and Jun) to regulate the transcription of target genes. We report additional mechanisms for gene regulation by such pathways exerted through RNA stability controls. The Spc1 (Sty1/Phh1) kinase-regulated Atf1-Pcr1 (Mts1-Mts2) heterodimer of the fission yeast Schizosaccharomyces pombe controls the stress-induced, posttranscriptional stability and decay of sets of target RNAs. Whole transcriptome RNA sequencing data revealed that decay is associated nonrandomly with transcripts that contain an M26 sequence motif. Moreover, the ablation of an M26 sequence motif in a target mRNA is sufficient to block its stress-induced loss. Conversely, engineered M26 motifs can render a stable mRNA into one that is targeted for decay. This stress-activated RNA decay (SARD) provides a mechanism for reducing the expression of target genes without shutting off transcription itself. Thus, a single p38-ATF/CREB signal transduction pathway can coordinately induce (promote transcription and RNA stability) and repress (promote RNA decay) transcript levels for distinct sets of genes, as is required for developmental decisions in response to stress and other stimuli. PMID:23732911

  18. A Stress-Activated, p38 Mitogen-Activated Protein Kinase–ATF/CREB Pathway Regulates Posttranscriptional, Sequence-Dependent Decay of Target RNAs

    PubMed Central

    Gao, Jun; Wagnon, Jacy L.; Protacio, Reine M.; Glazko, Galina V.; Beggs, Marjorie; Raj, Vinay

    2013-01-01

    Broadly conserved, mitogen-activated/stress-activated protein kinases (MAPK/SAPK) of the p38 family regulate multiple cellular processes. They transduce signals via dimeric, basic leucine zipper (bZIP) transcription factors of the ATF/CREB family (such as Atf2, Fos, and Jun) to regulate the transcription of target genes. We report additional mechanisms for gene regulation by such pathways exerted through RNA stability controls. The Spc1 (Sty1/Phh1) kinase-regulated Atf1-Pcr1 (Mts1-Mts2) heterodimer of the fission yeast Schizosaccharomyces pombe controls the stress-induced, posttranscriptional stability and decay of sets of target RNAs. Whole transcriptome RNA sequencing data revealed that decay is associated nonrandomly with transcripts that contain an M26 sequence motif. Moreover, the ablation of an M26 sequence motif in a target mRNA is sufficient to block its stress-induced loss. Conversely, engineered M26 motifs can render a stable mRNA into one that is targeted for decay. This stress-activated RNA decay (SARD) provides a mechanism for reducing the expression of target genes without shutting off transcription itself. Thus, a single p38-ATF/CREB signal transduction pathway can coordinately induce (promote transcription and RNA stability) and repress (promote RNA decay) transcript levels for distinct sets of genes, as is required for developmental decisions in response to stress and other stimuli. PMID:23732911

  19. Bone morphogenetic protein 2-induced human dental pulp cell differentiation involves p38 mitogen-activated protein kinase-activated canonical WNT pathway

    PubMed Central

    Yang, Jing; Ye, Ling; Hui, Tian-Qian; Yang, Dong-Mei; Huang, Ding-Ming; Zhou, Xue-Dong; Mao, Jeremy J; Wang, Cheng-Lin

    2015-01-01

    Both bone morphogenetic protein 2 (BMP2) and the wingless-type MMTV integration site (WNT)/β-catenin signalling pathway play important roles in odontoblast differentiation and dentinogenesis. Cross-talk between BMP2 and WNT/β-catenin in osteoblast differentiation and bone formation has been identified. However, the roles and mechanisms of the canonical WNT pathway in the regulation of BMP2 in dental pulp injury and repair remain largely unknown. Here, we demonstrate that BMP2 promotes the differentiation of human dental pulp cells (HDPCs) by activating WNT/β-catenin signalling, which is further mediated by p38 mitogen-activated protein kinase (MAPK) in vitro. BMP2 stimulation upregulated the expression of β-catenin in HDPCs, which was abolished by SB203580 but not by Noggin or LDN193189. Furthermore, BMP2 enhanced cell differentiation, which was not fully inhibited by Noggin or LDN193189. Instead, SB203580 partially blocked BMP2-induced β-catenin expression and cell differentiation. Taken together, these data suggest a possible mechanism by which the elevation of β-catenin resulting from BMP2 stimulation is mediated by the p38 MAPK pathway, which sheds light on the molecular mechanisms of BMP2-mediated pulp reparative dentin formation. PMID:26047580

  20. Cry1Ac toxin induces macrophage activation via ERK1/2, JNK and p38 mitogen-activated protein kinases.

    PubMed

    Torres-Martínez, Marilu; Rubio-Infante, Néstor; García-Hernández, Ana Lilia; Nava-Acosta, Raúl; Ilhuicatzi-Alvarado, Damaris; Moreno-Fierros, Leticia

    2016-09-01

    The Cry1Ac toxin from Bacillus thuringiensis is used commercially as a bio-insecticide and is expressed in transgenic plants that are used for human and animal consumption. Although it was originally considered innocuous for mammals, the Cry1Ac toxin is not inert and has the ability to induce mucosal and systemic immunogenicity. Herein, we examined whether the Cry1Ac toxin promotes macrophage activation and explored the signalling pathways that may mediate this effect. Treatment of primary and RAW264.7 macrophages with the Cry1Ac toxin resulted in upregulation of the costimulatory molecules CD80, CD86 and ICOS-L and enhanced production of nitric oxide, the chemokine MCP-1 and the proinflammatory cytokines TNF-α and IL-6. Remarkably, the Cry1Ac toxin induced phosphorylation of the mitogen-activated protein kinases (MAPKs) ERK1/2, JNK and p38 and promoted nuclear translocation of nuclear factor-kappa B (NF-κB) p50 and p65. p38 and ERK1/2 MAPKs were involved in this effect, as indicated by the Cry1Ac-induced upregulation of CD80 and IL-6 and TNF-α abrogation by the p38 MAPK inhibitor SB203580. Furthermore, treatment the MEK1/2 kinase inhibitor PD98059 blocked increases in MCP-1 secretion and augmented Cry1Ac-induced ICOS-L upregulation. These data demonstrate the capacity of the Cry1Ac toxin to induce macrophage activation via the MAPK and NF-κB pathways. PMID:27394658

  1. A Role for p38 Mitogen-activated Protein Kinase-mediated Threonine 30-dependent Norepinephrine Transporter Regulation in Cocaine Sensitization and Conditioned Place Preference*

    PubMed Central

    Mannangatti, Padmanabhan; NarasimhaNaidu, Kamalakkannan; Damaj, Mohamad Imad; Ramamoorthy, Sammanda; Jayanthi, Lankupalle Damodara

    2015-01-01

    The noradrenergic and p38 mitogen-activated protein kinase (p38 MAPK) systems are implicated in cocaine-elicited behaviors. Previously, we demonstrated a role for p38 MAPK-mediated norepinephrine transporter (NET) Thr30 phosphorylation in cocaine-induced NET up-regulation (Mannangatti, P., Arapulisamy, O., Shippenberg, T. S., Ramamoorthy, S., and Jayanthi, L. D. (2011) J. Biol. Chem. 286, 20239–20250). The present study explored the functional interaction between p38 MAPK-mediated NET regulation and cocaine-induced behaviors. In vitro cocaine treatment of mouse prefrontal cortex synaptosomes resulted in enhanced NET function, surface expression, and phosphorylation. Pretreatment with PD169316, a p38 MAPK inhibitor, completely blocked cocaine-mediated NET up-regulation and phosphorylation. In mice, in vivo administration of p38 MAPK inhibitor SB203580 completely blocked cocaine-induced NET up-regulation and p38 MAPK activation in the prefrontal cortex and nucleus accumbens. When tested for cocaine-induced locomotor sensitization and conditioned place preference (CPP), mice receiving SB203580 on cocaine challenge day or on postconditioning test day exhibited significantly reduced cocaine sensitization and CPP. A transactivator of transcription (TAT) peptide strategy was utilized to test the involvement of the NET-Thr30 motif. In vitro treatment of synaptosomes with TAT-NET-Thr30 (wild-type peptide) completely blocked cocaine-mediated NET up-regulation and phosphorylation. In vivo administration of TAT-NET-Thr30 peptide but not TAT-NET-T30A (mutant peptide) completely blocked cocaine-mediated NET up-regulation and phosphorylation. In the cocaine CPP paradigm, mice receiving TAT-NET-Thr30 but not TAT-NET-T30A on postconditioning test day exhibited significantly reduced cocaine CPP. Following extinction, TAT-NET-Thr30 when given prior to cocaine challenge significantly reduced reinstatement of cocaine CPP. These results demonstrate that the direct inhibition of p38

  2. Synergistic effects of p38 mitogen-activated protein kinase inhibition with a corticosteroid in alveolar macrophages from patients with chronic obstructive pulmonary disease.

    PubMed

    Armstrong, J; Harbron, C; Lea, S; Booth, G; Cadden, P; Wreggett, K A; Singh, D

    2011-09-01

    Corticosteroids partially suppress cytokine production by chronic obstructive pulmonary disease (COPD) alveolar macrophages. p38 mitogen-activated protein kinase (MAPK) inhibitors are a novel class of anti-inflammatory drug. We have studied the effects of combined treatment with a corticosteroid and a p38 MAPK inhibitor on cytokine production by COPD alveolar macrophages, with the aim of investigating dose-sparing and efficacy-enhancing effects. Alveolar macrophages from 10 patients with COPD, six smokers, and six nonsmokers were stimulated with lipopolysaccharide (LPS) after preincubation with five concentrations of dexamethasone alone, five concentrations of the p38 MAPK inhibitor 1-(5-tert-butyl-2-p-tolyl-2H-pyrazol-3-yl)-3(4-(2-morpholin-4-yl-ethoxy)naphthalen-1-yl)urea (BIRB-796) alone, and all combinations of these concentrations. After 24 h, the supernatants were analyzed for interleukin (IL)-8, IL-6, tumor necrosis factor α (TNFα), granulocyte macrophage-colony-stimulating factor (GM-CSF), IL-1α, IL-1β, IL-1ra, IL-10, monocyte chemoattractant protein 3, macrophage-derived chemokine (MDC), and regulated on activation normal T cell expressed and secreted (RANTES). The effect of dexamethasone on p38 MAPK activation was analyzed by Western blotting. Dexamethasone and BIRB-796 both reduced LPS-induced cytokine production in a dose-dependent manner in all subject groups, with no differences between groups. Increasing the concentration of BIRB-796 in combination with dexamethasone produced progressively greater inhibition of cytokine production than dexamethasone alone. There were significant efficacy-enhancing benefits and synergistic dose-sparing effects (p < 0.05) for the combination treatment for IL-8, IL-6, TNFα, GM-CSF, IL-1ra, IL-10, MDC, and RANTES in one or more subject groups. Dexamethasone had no effect on LPS-induced p38 MAPK activation. We conclude that p38 MAPK activation in alveolar macrophages is corticosteroid-insensitive. Combining a p38

  3. 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.

  4. 2,2',4,4'-Tetrachlorobiphenyl upregulates cyclooxygenase-2 in HL-60 cells via p38 mitogen-activated protein kinase and NF-{kappa}B

    SciTech Connect

    Bezdecny, Steven A.; Karmaus, Peer; Roth, Robert A.; Ganey, Patricia E. . E-mail: ganey@msu.edu

    2007-06-15

    Polychlorinated biphenyls (PCBs) are ubiquitous, persistent environmental contaminants that affect a number of cellular systems, including neutrophils. Among the effects caused by the noncoplanar PCB 2,2',4,4'-tetrachlorobiphenyl (2244-TCB) in granulocytic HL-60 cells are increases in superoxide anion production, activation of phospholipase A{sub 2} with subsequent release of arachidonic acid (AA) and upregulation of the inflammatory gene cyclooxygenase-2 (COX-2). The objective of this study was to determine the signal transduction pathways involved in the upregulation of COX-2 by 2244-TCB. Treatment of HL-60 cells with 2244-TCB led to increased expression of COX-2 mRNA. This increase was prevented by the transcriptional inhibitor actinomycin D in cells pretreated with 2244-TCB for 10 min. The increase in COX-2 mRNA was associated with release of {sup 3}H-AA, phosphorylation of p38 and extracellular signal-regulated kinase (ERK) mitogen-activated protein (MAP) kinases, increased levels of nuclear NF-{kappa}B and increased superoxide anion production. Bromoenol lactone, an inhibitor of the calcium-independent phospholipase A{sub 2}, reduced {sup 3}H-AA release but had no effect on COX-2 mRNA, protein or activity. Pretreatment with SB-202190 or SB-203580, inhibitors of the p38 MAP kinase pathway, prevented the 2244-TCB-mediated induction of COX-2 and phosphorylation of p38 and ERK MAP kinases. These inhibitors did not alter {sup 3}H-AA release. Treatment with PD 98059 or U 0126, inhibitors of the MAP/ERK (MEK) pathway, prevented the 2244-TCB-mediated activation of ERK but had no effect on COX-2 induction or p38 phosphorylation. 2244-TCB treatment did not affect c-Jun N-terminal kinase (JNK) phosphorylation. 2244-TCB exposure increased the amount of nuclear NF-{kappa}B. This increase was prevented by pretreatment with p38 MAP kinase inhibitors, but not by pretreatment with MEK inhibitors. Pretreatment with inhibitors of NF-{kappa}B prevented the 2244-TCB

  5. Octacalcium phosphate crystals directly stimulate expression of inducible nitric oxide synthase through p38 and JNK mitogen-activated protein kinases in articular chondrocytes

    PubMed Central

    Ea, Hang-Korng; Uzan, Benjamin; Rey, Christian; Lioté, Frédéric

    2005-01-01

    Basic calcium phosphate (BCP) crystals, including hydroxyapatite, octacalcium phosphate (OCP) and carbonate-apatite, have been associated with severe osteoarthritis and several degenerative arthropathies. Most studies have considered the chondrocyte to be a bystander in the pathogenesis of calcium crystal deposition disease, assuming that synovial cell cytokines were the only triggers of chondrocyte activation. In the present study we identified direct activation of articular chondrocytes by OCP crystals, which are the BCP crystals with the greatest potential for inducing inflammation. OCP crystals induced nitric oxide (NO) production and inducible nitric oxide synthase (NOS) mRNA expression by isolated articular chondrocytes and cartilage fragments, in a dose-dependent manner and with variations over time. OCP crystals also induced IL-1β mRNA expression. Using pharmacological and cytokine inhibitors, we observed that OCP crystals induced NO production and inducible NOS mRNA activation were regulated at both the transcriptional and the translational levels; were independent from IL-1β gene activation; and involved p38 and c-Jun amino-terminal kinase (JNK) mitogen-activated protein kinase (MAPK) pathways, as further confirmed by OCP crystal-induced p38 and JNK MAPK phosphorylation. Taken together, our data suggest that the transcriptional inducible NOS response to OCP crystals involved both the p38 and the JNK MAPK pathways, probably under the control of activator protein-1. NO, a major mediator of cartilage degradation, can be directly produced by BCP crystals in chondrocytes. Together with synovial activation, this direct mechanism may be important in the pathogenesis of destructive arthropathies triggered by microcrystals. PMID:16207333

  6. p38 mitogen-activated protein kinase inhibitor LY2228820 enhances bortezomib-induced cytotoxicity and inhibits osteoclastogenesis in multiple myeloma; therapeutic implications.

    PubMed

    Ishitsuka, Kenji; Hideshima, Teru; Neri, Paola; Vallet, Sonia; Shiraishi, Norihiko; Okawa, Yutaka; Shen, Zhenxin; Raje, Noopur; Kiziltepe, Tanyel; Ocio, Enrique M; Chauhan, Dharminder; Tassone, Pierfrancesco; Munshi, Nikhil; Campbell, Robert M; Dios, Alfonso De; Shih, Chuan; Starling, James J; Tamura, Kazuo; Anderson, Kenneth C

    2008-05-01

    The interaction between multiple myeloma (MM) cells and the bone marrow (BM) microenvironment induces proliferation and survival of MM cells, as well as osteoclastogenesis. This study investigated the therapeutic potential of novel p38 mitogen-activated protein kinase (p38MAPK) inhibitor LY2228820 (LY) in MM. Although cytotoxicity against MM cell lines was modest, LY significantly enhanced the toxicity of bortezomib by down-regulating bortezomib-induced heat shock protein 27 phosphorylation. LY inhibited interleukin-6 secretion from long term cultured-BM stromal cells and BM mononuclear cells (BMMNCs) derived from MM patients in remission. LY also inhibited macrophage inflammatory protein-1alpha secretion from patient MM cells and BMMNCs as well as normal CD14 positive osteoclast precursor cells. Moreover, LY significantly inhibited in vitro osteoclastogenesis from CD14 positive cells induced by macrophage-colony stimulating factor and soluble receptor activator of nuclear factor-kappaB ligand. Finally, LY also inhibited in vivo osteoclatogenesis in a severe combined immunodeficiency mouse model of human MM. These results suggest that LY represents a promising novel targeted approach to improve MM patient outcome both by enhancing the effect of bortezomib and by reducing osteoskeletal events.

  7. Perivascular adipose tissue-derived leptin promotes vascular smooth muscle cell phenotypic switching via p38 mitogen-activated protein kinase in metabolic syndrome rats.

    PubMed

    Li, Hao; Wang, Ya-Ping; Zhang, Li-Na; Tian, Gang

    2014-04-01

    Perivascular adipose tissue (PVAT)-derived leptin is a detrimental adipocytokine and plays a critical role in the development of cardiovascular diseases in metabolic syndrome (MetS). During vascular remodeling, vascular smooth muscle cells (VSMCs) undergo phenotypic switching into a synthetic phenotype characterized by decreased expression of differentiation markers (smooth muscle myosin heavy chain, α-smooth muscle actin, and calponin) and increased proliferation. We aimed to determine whether PVAT-derived leptin influences VSMC phenotypic switching and to explore the underlying mechanisms in MetS rats. In vivo, 32 Wistar rats were divided into two groups that received either a normal diet (control rat) or a high-fat diet (MetS rats). After 16 weeks, rat aortas were stained using hematoxylin-eosin and imaged. VSMC differentiation markers and proliferating cell nuclear antigen (PCNA), PVAT-derived leptin, aortic leptin receptor (ObR), and p38 mitogen-activated protein kinase (MAPK) expression were detected. In vitro, aortic VSMCs were incubated with MetS rat PVAT conditioned medium (PVAT-CM) to mimic in vivo conditions and were pretreated with a p38 MAPK inhibitor (SB 203580) or leptin antagonist. Differentiation marker expression, including PCNA and p38 MAPK, was detected. MetS rats exhibited pronounced insulin resistance, hyperglycemia, hyperlipidemia, hypertension, obesity, and an associated increase in PVAT weight. VSMCs underwent phenotypic switching in MetS rat aorta and contributed to vascular remodeling. PVAT-derived leptin expression was higher in MetS rats than in control rats (P < 0.01). ObRa expression and p38 MAPK phosphorylation were upregulated in MetS rat aorta. In vitro, VSMCs incubated with MetS rat PVAT-CM underwent phenotypic switching, associated with increased p38 MAPK phosphorylation. This VSMC phenotypic switching was inhibited by pretreatment with SB 203580 or a leptin antagonist. These results suggest that in MetS rats, PVAT

  8. Mitogen-activated protein kinases (p38 and c-Jun NH2-terminal kinase) are differentially regulated during cardiac volume and pressure overload hypertrophy.

    PubMed

    Sopontammarak, Somkiat; Aliharoob, Assad; Ocampo, Catherina; Arcilla, Rene A; Gupta, Mahesh P; Gupta, Madhu

    2005-01-01

    Chronic pressure overload (PO) and volume overload (VO) result in morphologically and functionally distinct forms of myocardial hypertrophy. However, the molecular mechanism initiating these two types of hypertrophy is not yet understood. Data obtained from different cell types have indicated that the mitogen-activated protein kinases (MAPKs) comprising c-Jun NH2-terminal kinase (JNK), extracellular signal-regulated kinase (ERK), and p38 play an important role in transmitting signals of stress stimuli to elicit the cellular response. We tested the hypothesis that early induction of MAPKs differs in two types of overload on the heart and associates with distinct expression of hypertrophic marker genes, namely ANF, alpha-myosin heavy chain (alpha-MHC), and beta-MHC. In rats, VO was induced by aortocaval shunt and PO by constriction of the abdominal aorta. The PO animals were further divided into two groups depending on the severity of the constriction, mild (MPO) and severe pressure overload (SPO), having 35 and 85% aortic constriction, respectively. Early changes in MAPK activity (2-120 min and 1 to 2 d) were analyzed by the in vitro kinase assay using kinase-specific antibodies for p38, JNK, and ERK2. The change in expression of hypertrophy marker genes was examined by Northern blot analysis. In VO hypertrophy, the activity of p38 was markedly increased (10-fold), without changing the activity of ERK and JNK. However, during PO hypertrophy, the activity of JNK was significantly increased (two- to sixfold) and depended on the severity of the load. The activity of p38 was not changed in MPO hypertrophy, whereas it was slightly elevated (50%) in hearts with SPO. Similarly, ERK activity was not changed in hearts with MPO, but a transient rise in activity was observed in hearts with SPO. The expression of ANF and beta-MHC genes was elevated in both PO and VO hypertrophy; however, this change was much greater in hearts subjected to PO than VO hypertrophy. Alpha

  9. Pro-oncogenic role of alternative p38 mitogen-activated protein kinases p38γ and p38δ, linking inflammation and cancer in colitis-associated colon cancer.

    PubMed

    Del Reino, Paloma; Alsina-Beauchamp, Dayanira; Escós, Alejandra; Cerezo-Guisado, Ma Isabel; Risco, Ana; Aparicio, Noelia; Zur, Rafal; Fernandez-Estévez, Marian; Collantes, Elena; Montans, Jose; Cuenda, Ana

    2014-11-01

    p38 MAPK signaling has been implicated in the regulation of processes leading to cancer development and progression. Chronic inflammation is a known risk factor for tumorigenesis, yet the precise mechanism of this association remains largely unknown. The related p38αMAPK (MAPK14) proteins p38γ (MAPK12) and p38δ (MAPK13) were recently shown to modulate the immune response, although their role in tumorigenesis remains controversial and their function in inflammation-associated cancer has not been studied. We analyzed the role of p38γ and p38δ in colon cancer associated to colitis using the azoxymethane/dextran sodium sulphate (AOM/DSS) colitis-associated colon cancer model in wild-type (WT), p38γ-, p38δ-, and p38γ/δ-deficient (p38γ/δ(-/-)) mice. We found that p38γ/δ deficiency significantly decreased tumor formation, in parallel with a decrease in proinflammatory cytokine and chemokine production. Analysis of leukocyte populations in p38γ/δ(-/-) mouse colon showed less macrophage and neutrophil recruitment than in WT mice. Furthermore, WT chimeric mice with transplanted p38γ/δ(-/-) bone marrow had less tumors than WT mice transplanted with WT bone marrow, whereas tumor number was significantly increased in p38γ/δ(-/-) chimeric mice with WT bone marrow compared with p38γ/δ(-/-) mice transplanted with p38γ/δ(-/-) bone marrow. Together, our results establish that p38γ and p38δ are central to colitis-associated colon cancer formation through regulation of hematopoietic cell response to injury, and validate p38γ and p38δ as potential targets for cancer therapy.

  10. Artocarpol A stimulation of superoxide anion generation in neutrophils involved the activation of PLC, PKC and p38 mitogen-activated PK signaling pathways.

    PubMed

    Kuan, Yu-Hsiang; Lin, Ruey-Hseng; Tsao, Lo-Ti; Lin, Chun-Nan; Wang, Jih-Pyang

    2005-06-01

    1 Artocarpol A (ART), a natural phenolic compound isolated from Artocarpus rigida, stimulated a slow onset and long-lasting superoxide anion generation in rat neutrophils, whereas only slightly activated the NADPH oxidase in a cell-free system. 2 Pretreatment of neutrophils with pertussis toxin (1 microg ml(-1)), 50 microM 2'-amino-3'-methoxyflavone (PD 98059), or 1 microM 1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio)butadiene (U0126) had no effect on ART-stimulated superoxide anion generation. ART (30 microM) did not induce extracellular signal-regulated kinase (ERK) phosphorylation. 3 4-(4-Fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)-1H-imidazole (SB 203580) markedly attenuated the ART-stimulated superoxide anion generation (IC50 value of 4.3+/-0.3 microM). Moreover, ART induced p38 mitogen-activated PK (MAPK) phosphorylation and activation. 4 The superoxide anion generation in response to ART was also substantially inhibited in a Ca2+-free medium, and by pretreatment with 1 microM 1-[6-((17beta-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl]-1H-pyrrole-2,5-dione (U-73122) and 100 microM 2-aminoethyldiphenyl borate (2-APB). ART (30 microM) stimulated the [Ca2+]i elevation in the presence or absence of external Ca2+, and also increased the D-myo-inositol 1,4,5-trisphosphate formation. 5 2-[1-(3-Dimethylaminopropyl)-1H-indol-3-yl]-3-(1H-indol-3-yl)-maleimide (GF 109203X) greatly inhibited the ART-stimulated superoxide anion generation (IC50 value of 7.8+/-1.0 nM). ART increased the recruitment of PKC-alpha, -betaI, and -betaII to the plasma membrane of neutrophils, and stimulated Ca2+-dependent PKC activation in the cytosol preparation. 6 ART induced the phosphorylation of p47phox, which was attenuated by GF 109203X. Moreover, ART evoked the membrane association of p47(phox), which was inhibited by GF 109203X and SB 203580. 7 These results indicate that the ART stimulation of superoxide anion generation involved the activation of p38 MAPK, PLC/Ca2

  11. p38β Mitogen-Activated Protein Kinase Modulates Its Own Basal Activity by Autophosphorylation of the Activating Residue Thr180 and the Inhibitory Residues Thr241 and Ser261

    PubMed Central

    Beenstock, Jonah; Melamed, Dganit; Mooshayef, Navit; Mordechay, Dafna; Garfinkel, Benjamin P.; Ahn, Natalie G.; Admon, Arie

    2016-01-01

    Many enzymes are self-regulated and can either inhibit or enhance their own catalytic activity. Enzymes that do both are extremely rare. Many protein kinases autoactivate by autophosphorylating specific sites at their activation loop and are inactivated by phosphatases. Although mitogen-activated protein kinases (MAPKs) are usually activated by dual phosphorylation catalyzed by MAPK kinases (MAPKKs), the MAPK p38β is exceptional and is capable of self-activation by cis autophosphorylation of its activation loop residue T180. We discovered that p38β also autophosphorylates in trans two previously unknown sites residing within a MAPK-specific structural element known as the MAPK insert: T241 and S261. Whereas phosphorylation of T180 evokes catalytic activity, phosphorylation of S261 reduces the activity of T180-phosphorylated p38β, and phosphorylation of T241 reduces its autophosphorylation in trans. Both phosphorylations do not affect the activity of dually phosphorylated p38β. T241 of p38β is found phosphorylated in vivo in bone and muscle tissues. In myogenic cell lines, phosphorylation of p38β residue T241 is correlated with differentiation to myotubes. T241 and S261 are also autophosphorylated in intrinsically active variants of p38α, but in this protein, they probably play a different role. We conclude that p38β is an unusual enzyme that automodulates its basal, MAPKK-independent activity by several autophosphorylation events, which enhance and suppress its catalytic activity. PMID:26976637

  12. Thermodynamic characterization of pyrazole and azaindole derivatives binding to p38 mitogen-activated protein kinase using Biacore T100 technology and van't Hoff analysis.

    PubMed

    Papalia, Giuseppe A; Giannetti, Anthony M; Arora, Nidhi; Myszka, David G

    2008-12-15

    Biacore T100 technology was used in conjunction with a van't Hoff analysis to characterize the thermodynamic binding parameters of 85 small-molecule inhibitors of adenosine triphosphate (ATP) binding to p38 mitogen-activated protein (MAP) kinase. The compounds were selected from a large panel of azaindole and pyrazole derivatives for which IC(50) data exist. We showed a strong relationship between the K(D) and IC(50) of a compound, but only a modest relationship between k(off) and IC(50) was detected and an apparent relationship between a compound's k(on) and its IC(50) could not be discerned. Similarly, a correlation between a compound's IC(50) and its thermodynamic parameters DeltaH degrees and DeltaS degrees could not be established. The lack of a predominant kinetic or thermodynamic signature associated with the inhibitory potential of these compounds demonstrates that there exists, even within a single well-defined system, a library of kinetic routes or, alternatively, a library of initial and final enthalpic and entropic states from which to effect inhibition. As a complement to these studies, selected double mutant thermodynamic cycles were performed to probe the energetic coupling, if any, between common sites of fluorination in both the azaindole and pyrazole classes and two different substituents. Although both cycles indicated negligible coupling free energies, both revealed significant coupling enthalpies, an observation made in other similarly dissected systems. The possible significance and caveats associated with these findings along with the advantages of using Biacore technology to derive thermodynamic parameters in drug discovery efforts are discussed.

  13. A requirement of serotonergic p38α mitogen-activated protein kinase for peripheral immune system activation of CNS serotonin uptake and serotonin-linked behaviors

    PubMed Central

    Baganz, N L; Lindler, K M; Zhu, C B; Smith, J T; Robson, M J; Iwamoto, H; Deneris, E S; Hewlett, W A; Blakely, R D

    2015-01-01

    Alterations in central serotonin (5-hydroxytryptamine, 5-HT) neurotransmission and peripheral immune activation have been linked to multiple neuropsychiatric disorders, including depression, schizophrenia and autism. The antidepressant-sensitive 5-HT transporter (SERT, SLC6A4), a critical determinant of synaptic 5-HT inactivation, can be regulated by pro-inflammatory cytokine signaling. Systemic innate immune system activation via intraperitoneal lipopolysaccharide (LPS) injection rapidly elevates brain SERT activity and 5-HT clearance. Moreover, the pro-inflammatory cytokine interleukin (IL)-1β rapidly stimulates SERT activity in raphe nerve terminal preparations ex vivo, effects that are attenuated by pharmacological p38 MAPK inhibition. To establish a role of serotonergic p38α MAPK signaling in LPS/IL-1β-induced SERT regulation and attendant behavioral responses, we pursued studies in mice that afford conditional elimination of p38α MAPK in 5-HT neurons (p38α5HT−). We found p38α5HT− and control (p38α5HT+) littermates to be indistinguishable in viability and growth and to express equivalent levels of SERT protein and synaptosomal 5-HT transport activity. Consistent with pharmacological studies, however, IL-1β fails to increase SERT activity in midbrain synaptosomes prepared from p38α5HT− animals. Moreover, although LPS elevated plasma corticosterone and central/peripheral pro-inflammatory cytokines in p38α5HT− animals, elevations in midbrain SERT activity were absent nor were changes in depressive and anxiety-like behaviors observed. Our studies support an obligate role of p38α MAPK signaling in 5-HT neurons for the translation of immune activation to SERT regulation and 5-HT-modulated behaviors. PMID:26529424

  14. 5-Aminolevulinic Acid-Mediated Sonodynamic Therapy Promotes Phenotypic Switching from Dedifferentiated to Differentiated Phenotype via Reactive Oxygen Species and p38 Mitogen-Activated Protein Kinase in Vascular Smooth Muscle Cells.

    PubMed

    Dan, Juhua; Sun, Xin; Li, Wanlu; Zhang, Yun; Li, Xuesong; Xu, Haobo; Li, Zhitao; Tian, Zhen; Guo, Shuyuan; Yao, Jianting; Gao, Weidong; Tian, Ye

    2015-06-01

    Sonodynamic therapy (SDT) has been found to inhibit in-stent restenosis in animal models. However, the mechanism is not fully elucidated. Here, we investigated the effects of 5-aminolevulinic acid (ALA)-mediated SDT (ALA-SDT) on vascular smooth muscle cells (VSMCs), a cause of restenosis, with a focus on SDT-induced phenotypic switching. Serum-induced dedifferentiated VSMCs were cultured with ALA (1 mm, 24 h) and exposed to ultrasound (0.8 W/cm(2)) for 5 min. Results indicated that ALA-SDT inhibited the migration and proliferation of VSMCs and enhanced the expression of differentiated phenotypic markers in VSMCs. Additionally, ALA-SDT increased intracellular reactive oxygen species accumulation and phosphorylated p38 mitogen-activated protein kinase in VSMCs. Inhibition of reactive oxygen species elevation or p38 mitogen-activated protein kinase activity abolished the expression of smooth muscle 22α (SM22α) in VSMCs induced by ALA-SDT. Taken together, these results suggest that ALA-SDT promotes transformation of the VSMC phenotype from the dedifferentiated to differentiated status via reactive oxygen species and activated p38 mitogen-activated protein kinase.

  15. Inhibition of p38 mitogen-activated protein kinase ameliorates radiation-induced ototoxicity in zebrafish and cochlea-derived cell lines.

    PubMed

    Shin, Yoo Seob; Hwang, Hye Sook; Kang, Sung Un; Chang, Jae Won; Oh, Young-Taek; Kim, Chul-Ho

    2014-01-01

    Radiation is a widely used treatment for head and neck cancers, and one of its most severe side effects is ototoxicity. Radiation-induced ototoxicity has been demonstrated to be linked to the increased production of ROS and MAPK. We intended to investigate the effect of p38 inhibition on radiation-induced ototoxicity in cochlea-derived HEI-OC1 cells and in a zebrafish model. The otoprotective effect of p38 inhibition against radiation was tested in vitro in the organ of Corti-derived cell line, HEI-OC1, and in vivo in a zebrafish model. Radiation-induced apoptosis, mitochondrial dysfunction, and an increase of intracellular NO generation were demonstrated in HEI-OC1 cells. The p38-specific inhibitor, SB203580, ameliorated radiation-induced apoptosis and mitochondrial injury in HEI-OC1 cells. p38 inhibition reduced radiation-induced activation of JNK, p38, cytochrome c, and cleavage of caspase-3 and PARP in HEI-OC1 cells. Scanning electron micrography showed that SB203580 prevented radiation-induced destruction of kinocilium and stereocilia in zebrafish neuromasts. The results of this study suggest that p38 plays an important role in mediating radiation-induced ototoxicity and inhibition of p38 could be a plausible option for preventing radiation ototoxicity. PMID:24374476

  16. Sphingosine kinase 1 regulates tumor necrosis factor-mediated RANTES induction through p38 mitogen-activated protein kinase but independently of nuclear factor κB activation.

    PubMed

    Adada, Mohamad M; Orr-Gandy, K Alexa; Snider, Ashley J; Canals, Daniel; Hannun, Yusuf A; Obeid, Lina M; Clarke, Christopher J

    2013-09-20

    Sphingosine kinase 1 (SK1) produces the pro-survival sphingolipid sphingosine 1-phosphate and has been implicated in inflammation, proliferation, and angiogenesis. Recent studies identified TRAF2 as a sphingosine 1-phosphate target, implicating SK1 in activation of the NF-κB pathway, but the functional consequences of this connection on gene expression are unknown. Here, we find that loss of SK1 potentiates induction of the chemokine RANTES (regulated on activation, normal T cell expressed and secreted; also known as CCL5) in HeLa cells stimulated with TNF-α despite RANTES induction being highly dependent on the NF-κB pathway. Additionally, we find that SK1 is not required for TNF-induced IKK phosphorylation, IκB degradation, nuclear translocation of NF-κB subunits, and transcriptional NF-κB activity. In contrast, loss of SK1 prevented TNF-induced phosphorylation of p38 MAPK, and inhibition of p38 MAPK, like SK1 knockdown, also potentiates RANTES induction. Finally, in addition to RANTES, loss of SK1 also potentiated the induction of multiple chemokines and cytokines in the TNF response. Taken together, these data identify a potential and novel anti-inflammatory function of SK1 in which chemokine levels are suppressed through SK1-mediated activation of p38 MAPK. Furthermore, in this system, activation of NF-κB is dissociated from SK1, suggesting that the interaction between these pathways may be more complex than currently thought.

  17. Insulin-like growth factor 1 receptor and p38 mitogen-activated protein kinase signals inversely regulate signal transducer and activator of transcription 3 activity to control human dental pulp stem cell quiescence, propagation, and differentiation.

    PubMed

    Vandomme, Jerome; Touil, Yasmine; Ostyn, Pauline; Olejnik, Cecile; Flamenco, Pilar; El Machhour, Raja; Segard, Pascaline; Masselot, Bernadette; Bailliez, Yves; Formstecher, Pierre; Polakowska, Renata

    2014-04-15

    Dental pulp stem cells (DPSCs) remain quiescent until activated in response to severe dental pulp damage. Once activated, they exit quiescence and enter regenerative odontogenesis, producing reparative dentin. The factors and signaling molecules that control the quiescence/activation and commitment to differentiation of human DPSCs are not known. In this study, we determined that the inhibition of insulin-like growth factor 1 receptor (IGF-1R) and p38 mitogen-activated protein kinase (p38 MAPK) signaling commonly activates DPSCs and promotes their exit from the G0 phase of the cell cycle as well as from the pyronin Y(low) stem cell compartment. The inhibition of these two pathways, however, inversely determines DPSC fate. In contrast to p38 MAPK inhibitors, IGF-1R inhibitors enhance dental pulp cell sphere-forming capacity and reduce the cells' colony-forming capacity without inducing cell death. The inverse cellular changes initiated by IGF-1R and p38 MAPK inhibitors were accompanied by inverse changes in the levels of active signal transducer and activator of transcription 3 (STAT3) factor, inactive glycogen synthase kinase 3, and matrix extracellular phosphoglycoprotein, a marker of early odontoblast differentiation. Our data suggest that there is cross talk between the IGF-1R and p38 MAPK signaling pathways in DPSCs and that the signals provided by these pathways converge at STAT3 and inversely regulate its activity to maintain quiescence or to promote self-renewal and differentiation of the cells. We propose a working model that explains the possible interactions between IGF-1R and p38 MAPK at the molecular level and describes the cellular consequences of these interactions. This model may inspire further fundamental study and stimulate research on the clinical applications of DPSC in cellular therapy and tissue regeneration. PMID:24266654

  18. Elevation of cortical serotonin transporter activity upon peripheral immune challenge is regulated independently of p38 mitogen-activated protein kinase activation and transporter phosphorylation.

    PubMed

    Schwamborn, Robert; Brown, Eric; Haase, Jana

    2016-05-01

    The serotonin transporter (SERT) is responsible for high-affinity serotonin (5-HT) uptake from extracellular fluid and is a prominent pharmacological target in the treatment of depression. In recent years, depression has also been linked to immune system activation. Inflammatory conditions can cause sickness behaviour and depression-like symptoms in both animals and humans. Since SERT has been proposed as one of the molecular targets in inflammation-induced depression, we applied the widely used lipopolysaccharides (LPS) model to study the effects of peripheral inflammation on SERT activity in the brain. We show that 24 h after intraperitoneal LPS administration, SERT-mediated 5-HT uptake is significantly enhanced in the frontal cortex. Analysis of uptake kinetics revealed that the transport capacity (Vmax ) of cortical SERT was increased in LPS-injected animals, while the Km value remained unchanged. The increase in Vmax was neither due to increased SERT protein expression nor increased synaptic surface exposure. The suppression of SERT activity upon inhibition of p38 MAPK was not selective for LPS-induced enhancement of SERT function. In addition, SERT activity changes in LPS-treated rats are unaffected by nitric oxide synthase and protein kinase G inhibitors. Using the Phos-Tag method, we identified five SERT-specific protein bands representing distinct phosphorylation states of SERT. However, the enhancement of SERT activity in LPS-treated rats was not correlated with altered transporter phosphorylation. Together with previous studies by others, our results suggest that SERT is regulated by multiple mechanisms in response to peripheral immune system activation. Peripheral injection of lipopolysaccharide (LPS) induces characteristic sickness and depression-like behaviour in rats over a period of at least 24 h. We show here that the activity of the serotonin transporter (SERT), a prominent antidepressant target, is up-regulated 24 h following LPS

  19. Elevation of cortical serotonin transporter activity upon peripheral immune challenge is regulated independently of p38 mitogen-activated protein kinase activation and transporter phosphorylation.

    PubMed

    Schwamborn, Robert; Brown, Eric; Haase, Jana

    2016-05-01

    The serotonin transporter (SERT) is responsible for high-affinity serotonin (5-HT) uptake from extracellular fluid and is a prominent pharmacological target in the treatment of depression. In recent years, depression has also been linked to immune system activation. Inflammatory conditions can cause sickness behaviour and depression-like symptoms in both animals and humans. Since SERT has been proposed as one of the molecular targets in inflammation-induced depression, we applied the widely used lipopolysaccharides (LPS) model to study the effects of peripheral inflammation on SERT activity in the brain. We show that 24 h after intraperitoneal LPS administration, SERT-mediated 5-HT uptake is significantly enhanced in the frontal cortex. Analysis of uptake kinetics revealed that the transport capacity (Vmax ) of cortical SERT was increased in LPS-injected animals, while the Km value remained unchanged. The increase in Vmax was neither due to increased SERT protein expression nor increased synaptic surface exposure. The suppression of SERT activity upon inhibition of p38 MAPK was not selective for LPS-induced enhancement of SERT function. In addition, SERT activity changes in LPS-treated rats are unaffected by nitric oxide synthase and protein kinase G inhibitors. Using the Phos-Tag method, we identified five SERT-specific protein bands representing distinct phosphorylation states of SERT. However, the enhancement of SERT activity in LPS-treated rats was not correlated with altered transporter phosphorylation. Together with previous studies by others, our results suggest that SERT is regulated by multiple mechanisms in response to peripheral immune system activation. Peripheral injection of lipopolysaccharide (LPS) induces characteristic sickness and depression-like behaviour in rats over a period of at least 24 h. We show here that the activity of the serotonin transporter (SERT), a prominent antidepressant target, is up-regulated 24 h following LPS

  20. Non-mutagenic Suppression of Enterocyte Ferroportin 1 by Chemical Ribosomal Inactivation via p38 Mitogen-activated Protein Kinase (MAPK)-mediated Regulation: EVIDENCE FOR ENVIRONMENTAL HEMOCHROMATOSIS.

    PubMed

    Oh, Chang-Kyu; Park, Seong-Hwan; Kim, Juil; Moon, Yuseok

    2016-09-16

    Iron transfer across the basolateral membrane of an enterocyte into the circulation is the rate-limiting step in iron absorption and is regulated by various pathophysiological factors. Ferroportin (FPN), the only known mammalian iron exporter, transports iron from the basolateral surface of enterocytes, macrophages, and hepatocytes into the blood. Patients with genetic mutations in FPN or repeated blood transfusion develop hemochromatosis. In this study, non-mutagenic ribosomal inactivation was assessed as an etiological factor of FPN-associated hemochromatosis in enterocytes. Non-mutagenic chemical ribosomal inactivation disrupted iron homeostasis by regulating expression of the iron exporter FPN-1, leading to intracellular accumulation in enterocytes. Mechanistically, a xenobiotic insult stimulated the intracellular sentinel p38 MAPK signaling pathway, which was positively involved in FPN-1 suppression by ribosomal dysfunction. Moreover, ribosomal inactivation-induced iron accumulation in Caenorhabditis elegans as a simplified in vivo model for gut nutrition uptake was dependent on SEK-1, a p38 kinase activator, leading to suppression of FPN-1.1 expression and iron accumulation. In terms of gene regulation, ribosomal stress-activated p38 signaling down-regulated NRF2 and NF-κB, both of which were positive transcriptional regulators of FPN-1 transcription. This study provides molecular evidence for the modulation of iron bioavailability by ribosomal dysfunction as a potent etiological factor of non-mutagenic environmental hemochromatosis in the gut-to-blood axis.

  1. Pro-life role for c-Jun N-terminal kinase and p38 mitogen-activated protein kinase at rostral ventrolateral medulla in experimental brain stem death

    PubMed Central

    2012-01-01

    Background Based on an experimental brain stem death model, we demonstrated previously that activation of the mitogen-activated protein kinase kinase 1/2 (MEK1/2)/extracellular signal-regulated kinase 1/2 (ERK1/2)/ mitogen-activated protein kinase signal-interacting kinase 1/2 (MNK1/2) cascade plays a pro-life role in the rostral ventrolateral medulla (RVLM), the origin of a life-and-death signal detected from systemic arterial pressure, which sequentially increases (pro-life) and decreases (pro-death) to reflect progressive dysfunction of central cardiovascular regulation during the advancement towards brain stem death in critically ill patients. The present study assessed the hypothesis that, in addition to ERK1/2, c-Jun NH2-terminal kinase (JNK) and p38 mitogen-activated protein kinase (p38MAPK), the other two mammalian members of MAPKs that are originally identified as stress-activated protein kinases, are activated specifically by MAPK kinase 4 (MAP2K4) or MAP2K6 and play a pro-life role in RVLM during experimental brain stem death. We further delineated the participation of phosphorylating activating transcriptional factor-2 (ATF-2) and c-Jun, the classical transcription factor activated by JNK or p38MAPK, in this process. Results An experimental model of brain stem death that employed microinjection of the organophosphate insecticide mevinphos (Mev; 10 nmol) bilaterally into RVLM of Sprague–Dawley rats was used, alongside cardiovascular, pharmacological and biochemical evaluations. Results from ELISA showed that whereas the total JNK, p38MAPK, MAP2K4 and MAP2K6 were not affected, augmented phosphorylation of JNK at Thr183 and Tyr185 and p38MAPK at Thr180 and Tyr182, accompanied by phosphorylation of their upstream activators MAP2K4 at Ser257 and Thr261 and MAP2K6 at Ser207 and Thr211 in RVLM occurred preferentially during the pro-life phase of experimental brain stem death. Moreover, the activity of transcription factors ATF-2 at Thr71 and c-Jun at Ser73

  2. Methoxychlor-induced inducible nitric oxide synthase and proinflammatory cytokines expression in macrophages via NF-kappaB, ERK, and p38 mitogen-activated protein kinases.

    PubMed

    Kim, Ji Young; Oh, Kyo Nyeo; Han, Eun Hee; Kim, Dong Hee; Jeong, Tae Cheon; Lee, Eung Seok; Jeong, Hye Gwang

    2005-08-12

    Methoxychlor (MXC) is a pesticide that was developed as a replacement for dichlorodiphenyltrichloroethane. The influence of MXC on cytokine production or the functions of macrophages is unclear. This study examined the effects of MXC on the production of nitric oxide (NO) and the proinflammatory cytokines (IL-1beta, IL-6, and TNF-alpha), and analyzed the molecular mechanism in mouse macrophages. The addition of MXC to macrophages induced the production of NO and proinflammatory cytokines and expression levels of these genes in a dose-dependent manner. The NF-kappaB sites were identified in the promoter of the iNOS and proinflammatory cytokines genes. The transient expression and electrophoretic mobility shift assays revealed that the NF-kappaB transcription factor mediated the MXC-induced increase in the iNOS and proinflammatory cytokines expression levels. In addition, MXC induced the rapid phosphorylation of the ERK1/2 and p38 MAPK. This demonstrates that MXC stimulates the production of NO and proinflammatory cytokines and can up-regulate the expression levels of these genes via NF-kappaB transactivation and ERK1/2 and p38 MAPK phosphorylation. Overall, this study provides evidence showing that MXC has inflammatory potential that is previously unrecognized immunomodulating activity.

  3. Attenuation of acute lung injury in mice by oxymatrine is associated with inhibition of phosphorylated p38 mitogen-activated protein kinase.

    PubMed

    Xu, G L; Yao, L; Rao, S Y; Gong, Z N; Zhang, S Q; Yu, S Q

    2005-04-01

    Oxymatrine is one of the alkaloids extracted from Chinese herb Sophora japonica (Sophora flavescens Ait.) with activities of anti-inflammation, inhibiting immune reaction, antivirus, protecting hepatocytes and antihepatic fibrosis. However, the effect of oxymatrine on acute lung injury (ALI) has not been known yet. In this study, the effect of oxymatrine on ALI was investigated using an oleic acid-induced ALI mouse model. Morphological findings showed that the oleic acid group demonstrated a marked lung injury represented by prominent atelectasis, intraalveolar and interstitial patchy hemorrhage, edema, thickened alveolar septum, formation of hyaline membranes and the existence of inflammatory cells in alveolar spaces. While in the oxymatrine/dexamethasone group, these changes were less severe and in the vicinity of the control group. Furthermore, pretreatment with oxymatrine significantly alleviated oleic acid-induced lung injury accompanied by reduction of lung index and wet-to-dry weight ratio, decreases in serum TNF-alpha level and inhibition of phosphorylated p38 MAPK. These findings suggest that oxymatrine has a beneficial effect on acute lung injury induced by oleic acid in mice and may inhibit the production of proinflammatory cytokine, TNF-alpha, by means of the inhibition of p38 MAPK. PMID:15763380

  4. Delayed Cell Cycle Progression in Selenoprotein W-depleted Cells Is Regulated by a Mitogen-activated Protein Kinase Kinase 4-p38/c-Jun NH2-terminal Kinase-p53 Pathway*

    PubMed Central

    Hawkes, Wayne Chris; Alkan, Zeynep

    2012-01-01

    Selenoprotein W (SEPW1) is a ubiquitous, highly conserved thioredoxin-like protein whose depletion causes a transient p53- and p21Cip1-dependent G1-phase cell cycle arrest in breast and prostate epithelial cells. SEPW1 depletion increases phosphorylation of Ser-33 in p53, which is associated with decreased p53 ubiquitination and stabilization of p53. We report here that delayed cell cycle progression, Ser-33 phosphorylation, and p53 nuclear accumulation from SEPW1 depletion require mitogen-activated protein kinase kinase 4 (MKK4). Silencing MKK4 rescued G1 arrest, Ser-33 phosphorylation, and nuclear accumulation of p53 induced by SEPW1 depletion, but silencing MKK3, MKK6, or MKK7 did not. SEPW1 silencing did not change the phosphorylation state of MKK4 but increased total MKK4 protein. Silencing p38γ, p38δ, or JNK2 partially rescued G1 arrest from SEPW1 silencing, suggesting they signal downstream from MKK4. These results imply that SEPW1 silencing increases MKK4, which activates p38γ, p38δ, and JNK2 to phosphorylate p53 on Ser-33 and cause a transient G1 arrest. PMID:22730327

  5. Novel human neutrophil agonistic properties of arsenic trioxide: involvement of p38 mitogen-activated protein kinase and/or c-jun NH2-terminal MAPK but not extracellular signal-regulated kinases-1/2.

    PubMed

    Binet, François; Girard, Denis

    2008-12-01

    Arsenic trioxide (ATO) is known for treating acute promyelocytic leukemia and for inducing apoptosis and mitogen-activated protein kinases (MAPKs) in promyelocytes and cancer cells. We recently reported that ATO induces neutrophil apoptosis. The aim of this study was to establish whether or not ATO recruits MAPKs in neutrophils, as well as to further investigate its agonistic properties. We found that ATO activates p38 and that, unlike H2O2, this response was not inhibited by exogenous catalase. Also, we demonstrated that ATO-induced p38 activation occurs before H2O2 generation and without a calcium burst. We next established that ATO recruits c-jun NH2-terminal (JNK) but not extracellular signal-regulated kinase 1 and 2 (Erk-1/2). Using pharmacological inhibitors, we found that the proapoptotic activity of ATO occurs by a MAPK-independent mechanism. In contrast, the ability of ATO to enhance adhesion, migration, phagocytosis, release, and activity of gelatinase and degranulation of secretory, specific, and gelatinase, but not azurophilic granules, is dependent upon activation of p38 and/or JNK. This is the first study establishing that ATO possesses important agonistic properties in human neutrophils. Given the central role of neutrophils in various inflammatory disorders, we propose that ATO might have broader therapeutic implications in clinics, especially for regulating inflammation.

  6. Luteolin prevents solar radiation-induced matrix metalloproteinase-1 activation in human fibroblasts: a role for p38 mitogen-activated protein kinase and interleukin-20 released from keratinocytes.

    PubMed

    Wölfle, Ute; Heinemann, Anja; Esser, Philipp R; Haarhaus, Birgit; Martin, Stefan F; Schempp, Christoph M

    2012-10-01

    Human skin is continuously exposed to solar radiation, which can result in photoaging, a process involving both dermal and, to a lesser extent, epidermal structures. Previously, we have shown that the flavonoid luteolin protects the epidermis from ultraviolet (UV)-induced damage by a combination of UV-absorbing, antioxidant, and antiinflammatory properties. The aim of the present study was to determine direct and indirect effects of luteolin on dermal fibroblasts as major targets of photoaging. Stimulation of fibroblasts with UVA light or the proinflammatory cytokine interleukin-20 (IL-20) is associated with wrinkled skin, increased IL-6 secretion, matrix metalloproteinase (MMP-1) expression, and hyaluronidase activity. All of these targets were inhibited by luteolin via interference with the p38 mitogen-activated protein kinase (MAPK) pathway. Next, we assessed the role of conditioned supernatants from keratinocytes irradiated with solar-simulated radiation (SSR) on nonirradiated dermal fibroblasts. In keratinocytes, luteolin inhibited SSR-induced production of IL-20, also via interference with the p38 MAPK pathway. Similarly, keratinocyte supernatant-induced IL-6 and MMP-1 expression in fibroblasts was reduced by pretreatment of keratinocytes with luteolin. Finally, these results were confirmed ex vivo on skin explants treated with luteolin before UV irradiation. Our results suggest that SSR-mediated production of soluble factors in keratinocytes is modulated by luteolin and may attenuate photoaging in dermal fibroblasts. PMID:23004935

  7. Design, Synthesis, and Anti-inflammatory Properties of Orally Active 4-(Phenylamino)-pyrrolo[2,1-f][1,2,4]triazine p38[alpha] Mitogen-Activated Protein Kinase Inhibitors

    SciTech Connect

    Hynes, Jr., John; Dyckman, Alaric J.; Lin, Shuqun; Wrobleski, Stephen T.; Wu, Hong; Gillooly, Kathleen M.; Kanner, Steven B.; Lonial, Herinder; Loo, Derek; McIntyre, Kim W.; Pitt, Sidney; Shen, Ding Ren; Shuster, David J.; Yang, XiaoXia; Zhang, Rosemary; Behnia, Kamelia; Zhang, Hongjian; Marathe, Punit H.; Doweyko, Arthur M.; Tokarski, John S.; Sack, John S.; Pokross, Matthew; Kiefer, Susan E.; Newitt, John A.; Barrish, Joel C.; Dodd, John; Schieven, Gary L.; Leftheris, Katerina

    2008-06-30

    A novel structural class of p38 mitogen-activated protein (MAP) kinase inhibitors consisting of substituted 4-(phenylamino)-pyrrolo[2,1- f][1,2,4]triazines has been discovered. An initial subdeck screen revealed that the oxindole-pyrrolo[2,1- f][1,2,4]triazine lead 2a displayed potent enzyme inhibition (IC 50 60 nM) and was active in a cell-based TNFalpha biosynthesis inhibition assay (IC 50 210 nM). Replacement of the C4 oxindole with 2-methyl-5- N-methoxybenzamide aniline 9 gave a compound with superior p38 kinase inhibition (IC 50 10 nM) and moderately improved functional inhibition in THP-1 cells. Further replacement of the C6 ester of the pyrrolo[2,1- f][1,2,4]triazine with amides afforded compounds with increased potency, excellent oral bioavailability, and robust efficacy in a murine model of acute inflammation (murine LPS-TNFalpha). In rodent disease models of chronic inflammation, multiple compounds demonstrated significant inhibition of disease progression leading to the advancement of 2 compounds 11b and 11j into further preclinical and toxicological studies.

  8. A Network of Mitogen-Activated Protein Kinases Links G Protein-Coupled Receptors to the c-jun Promoter: a Role for c-Jun NH2-Terminal Kinase, p38s, and Extracellular Signal-Regulated Kinase 5

    PubMed Central

    Marinissen, Maria Julia; Chiariello, Mario; Pallante, Michael; Gutkind, J. Silvio

    1999-01-01

    The expression of the c-jun proto-oncogene is rapidly induced in response to mitogens acting on a large variety of cell surface receptors. The resulting functional activity of c-Jun proteins appears to be critical for cell proliferation. Recently, we have shown that a large family of G protein-coupled receptors (GPCRs), represented by the m1 muscarinic receptor, can initiate intracellular signaling cascades that result in the activation of mitogen-activated protein kinases (MAPK) and c-Jun NH2-terminal kinases (JNK) and that the activation of JNK but not of MAPK correlated with a remarkable increase in the expression of c-jun mRNA. Subsequently, however, we obtained evidence that GPCRs can potently stimulate the activity of the c-jun promoter through MEF2 transcription factors, which do not act downstream from JNK. In view of these observations, we set out to investigate further the nature of the signaling pathway linking GPCRs to the c-jun promoter. Utilizing NIH 3T3 cells, we found that GPCRs can activate the c-jun promoter in a JNK-independent manner. Additionally, we demonstrated that these GPCRs can elevate the activity of novel members of the MAPK family, including ERK5, p38α, p38γ, and p38δ, and that the activation of certain kinases acting downstream from MEK5 (ERK5) and MKK6 (p38α and p38γ) is necessary to fully activate the c-jun promoter. Moreover, in addition to JNK, ERK5, p38α, and p38γ were found to stimulate the c-jun promoter by acting on distinct responsive elements. Taken together, these results suggest that the pathway linking GPCRs to the c-jun promoter involves the integration of numerous signals transduced by a highly complex network of MAPK, rather than resulting from the stimulation of a single linear protein kinase cascade. Furthermore, our findings suggest that each signaling pathway affects one or more regulatory elements on the c-jun promoter and that the transcriptional response most likely results from the temporal integration

  9. Interleukin-25-induced chemokines and interleukin-6 release from eosinophils is mediated by p38 mitogen-activated protein kinase, c-Jun N-terminal kinase, and nuclear factor-kappaB.

    PubMed

    Wong, Chun K; Cheung, Phyllis F Y; Ip, Wai K; Lam, Christopher W K

    2005-08-01

    Interleukin (IL)-25, a novel Th2 cytokine, is capable of amplifying allergic inflammation. We investigated the modulation of nuclear factor (NF)-kappaB and mitogen-activated protein kinases (MAPK) pathways in IL-25-activated eosinophils, the principal effector cells of allergic inflammation, for the in vitro release of chemokines including monocyte chemoattractant protein-1 (MCP-1), IL-8, and macrophage inflammatory protein (MIP)-1alpha, and inflammatory cytokine IL-6. Gene expression of chemokines and IL-6 was evaluated by RT-PCR, and concentrations of chemokines and cytokine were measured by cytokine protein array, cytometric bead array, and enzyme-linked immunosorbent assay. NF-kappaB, c-Jun amino-terminal kinase (JNK), and p38 MAPK activities in eosinophils were assessed by electrophoretic mobility shift assay and Western blot. IL-25 was found to upregulate the gene expression of chemokines MCP-1, MIP-1alpha, and IL-8, and cytokine IL-6, in eosinophils, and to significantly increase the release of the above chemokines and IL-6 from eosinophils. IL-25 could also activate the JNK, p38 MAPK, and NF-kappaB activities of eosinophils, while inhibitor of IkappaB-alpha phosphorylation (BAY11-7082), JNK (SP600125), and p38 MAPK (SB203580) could suppress the release of IL-8, MIP-1alpha, MCP-1, and IL-6. Together, the above results showed that the induction of MCP-1, MIP-1alpha, IL-8, and IL-6 in IL-25-activated eosinophils are regulated by JNK, p38 MAPK, and NF-kappaB pathways.

  10. Ghrelin protects against depleted uranium-induced apoptosis of MC3T3-E1 cells through oxidative stress-mediated p38-mitogen-activated protein kinase pathway.

    PubMed

    Hao, Yuhui; Liu, Cong; Huang, Jiawei; Gu, Ying; Li, Hong; Yang, Zhangyou; Liu, Jing; Wang, Weidong; Li, Rong

    2016-01-01

    Depleted uranium (DU) mainly accumulates in the bone over the long term. Osteoblast cells are responsible for the formation of bone, and they are sensitive to DU damage. However, studies investigating methods of reducing DU damage in osteoblasts are rarely reported. Ghrelin is a stomach hormone that stimulates growth hormones released from the hypothalamic-pituitary axis, and it is believed to play an important physiological role in bone metabolism. This study evaluates the impact of ghrelin on DU-induced apoptosis of the osteoblast MC3T3-E1 and investigates its underlying mechanisms. The results show that ghrelin relieved the intracellular oxidative stress induced by DU, eliminated reactive oxygen species (ROS) and reduced lipid peroxidation by increasing intracellular GSH levels; in addition, ghrelin effectively suppressed apoptosis, enhanced mitochondrial membrane potential, and inhibited cytochrome c release and caspase-3 activation after DU exposure. Moreover, ghrelin significantly reduced the expression of DU-induced phosphorylated p38-mitogen-activated protein kinase (MAPK). A specific inhibitor (SB203580) or specific siRNA of p38-MAPK could significantly suppress DU-induced apoptosis and related signals, whereas ROS production was not affected. In addition, ghrelin receptor inhibition could reduce the anti-apoptosis effect of ghrelin on DU and reverse the effect of ghrelin on intracellular ROS and p38-MAPK after DU exposure. These results suggest that ghrelin can suppress DU-induced apoptosis of MC3T3-E1 cells, reduce DU-induced oxidative stress by interacting with its receptor, and inhibit downstream p38-MAPK activation, thereby suppressing the mitochondrial-dependent apoptosis pathway.

  11. Allicin induces apoptosis of the MGC-803 human gastric carcinoma cell line through the p38 mitogen-activated protein kinase/caspase-3 signaling pathway.

    PubMed

    Zhang, Xuecheng; Zhu, Yong; Duan, Wei; Feng, Chen; He, Xuan

    2015-04-01

    Gastric cancer is one of the most common forms of malignant tumor, and the development of anti‑gastric cancer drugs with minimal toxicity is of clinical importance. Allicin is extracted from Allium sativum (garlic). Recent research, including clinical experiments, has shown that garlic has anticancer and tumor suppressive effects. The present study aimed to investigate the effects of allicin on the MGC‑803 human gastric carcinoma cell line, and to further explore the possible mechanisms of its tumor suppressor effects. The effects of allicin on the MGC‑803 cells were initially examined using an 3‑(4,5‑dimethylthiazol‑2‑yl)‑2,5‑diphenyltetrazolium bromide assay. Hoechst staining was also used, in order to demonstrate the impact of allicin on MGC‑803 cell apoptosis. In addition, western blot analysis was performed to determine the abnormal expression levels of apoptosis‑associated proteins, following the treatment of MGC‑803 cells with allicin. Western blotting was also used to investigate the specific mechanisms underlying allicin‑induced apoptosis of MGC‑803 cells. The rate of MGC‑803 apoptosis was significantly increased, when the concentration and treatment time of allicin were increased. Hoechst staining detected an enhanced rate of apoptosis, and enhanced expression levels of cleaved caspase 3 were determined by western blotting. Notably, the protein expression levels of p38 were increased when the MGC‑803 cells were treated with allicin. The results of the present study suggest that allicin may inhibit the proliferation and induce the apoptosis of MGC‑803 human gastric carcinoma cells, and this may partially be achieved through the enhanced expression of p38 and cleaved caspase 3. PMID:25523417

  12. Global Analysis of Serine/Threonine and Tyrosine Protein Phosphatase Catalytic Subunit Genes in Neurospora crassa Reveals Interplay Between Phosphatases and the p38 Mitogen-Activated Protein Kinase

    PubMed Central

    Ghosh, Arit; Servin, Jacqueline A.; Park, Gyungsoon; Borkovich, Katherine A.

    2013-01-01

    Protein phosphatases are integral components of the cellular signaling machinery in eukaryotes, regulating diverse aspects of growth and development. The genome of the filamentous fungus and model organism Neurospora crassa encodes catalytic subunits for 30 protein phosphatase genes. In this study, we have characterized 24 viable N. crassa phosphatase catalytic subunit knockout mutants for phenotypes during growth, asexual development, and sexual development. We found that 91% of the mutants had defects in at least one of these traits, whereas 29% possessed phenotypes in all three. Chemical sensitivity screens were conducted to reveal additional phenotypes for the mutants. This resulted in the identification of at least one chemical sensitivity phenotype for 17 phosphatase knockout mutants, including novel chemical sensitivities for two phosphatase mutants lacking a growth or developmental phenotype. Hence, chemical sensitivity or growth/developmental phenotype was observed for all 24 viable mutants. We investigated p38 mitogen-activated protein kinase (MAPK) phosphorylation profiles in the phosphatase mutants and identified nine potential candidates for regulators of the p38 MAPK. We demonstrated that the PP2C class phosphatase pph-8 (NCU04600) is an important regulator of female sexual development in N. crassa. In addition, we showed that the Δcsp-6 (ΔNCU08380) mutant exhibits a phenotype similar to the previously identified conidial separation mutants, Δcsp-1 and Δcsp-2, that lack transcription factors important for regulation of conidiation and the circadian clock. PMID:24347630

  13. A supercritical CO₂ extract from seabuckthorn leaves inhibits pro-inflammatory mediators via inhibition of mitogen activated protein kinase p38 and transcription factor nuclear factor-κB.

    PubMed

    Jayashankar, Bindhya; Mishra, K P; Kumar, M S Y; Udayasankar, K; Misra, K; Ganju, L; Singh, S B

    2012-08-01

    In the present study, we have demonstrated the anti-inflammatory properties of supercritical CO₂ extract of seabuckthorn leaves (SCE) on mouse alveolar macrophage cell line (MH-S), human peripheral blood mononuclear cells (hPBMCs) in-vitro and in-vivo. Treatment of MH-S cells with SCE (0.5-100 μg/ml) significantly inhibited lipopolysaccharide (LPS)-stimulated nitric oxide (NO) production. It also inhibited the release of LPS-induced pro-inflammatory cytokines IL-6 and TNF-α, which was further confirmed by suppression of LPS induced TNF-α in hPBMCs by ELISPOT assay. In addition, western blot analysis demonstrated that SCE decreased LPS-induced inducible nitric-oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) protein expression in MH-S cells. Furthermore, SCE treatment also reduced nuclear factor-κB (NF-κB) translocation in nucleus induced by LPS in MH-S cells. To elucidate the molecular mechanism for inhibition of pro-inflammatory mediators by SCE (100 μg/ml), we further studied the effect of SCE on LPS-induced p38 mitogen-activated protein kinase (MAPK). It was observed that the phosphorylation of p38 MAPK in LPS-stimulated MH-S cells was significantly inhibited by SCE, which was further proven by suppression of LPS induced CD40 expression. The in-vivo model of AIA mice also showed a significant reduction in the inflammation of paw edema. These data collectively suggest that SCE suppressed the LPS-induced production of NO, IL-6, and TNF-α and expression of CD40, iNOS and COX-2 proteins by inhibiting NF-κB activation and phosphorylation of p38 MAPK. Hence, the SCE has potent anti-inflammatory activity and might be useful in chronic inflammatory diseases. PMID:22664145

  14. Enterohaemorrhagic Escherichia coli O157:H7 Shiga-like toxin 1 is required for full pathogenicity and activation of the p38 mitogen-activated protein kinase pathway in Caenorhabditis elegans.

    PubMed

    Chou, T-C; Chiu, H-C; Kuo, C-J; Wu, C-M; Syu, W-J; Chiu, W-T; Chen, C-S

    2013-01-01

    Enterohaemorrhagic Escherichia coli (EHEC) causes life-threatening infections in humans as a consequence of the production of Shiga-like toxins. Lack of a good animal model system currently hinders in vivo study of EHEC virulence by systematic genetic methods. Here we applied the genetically tractable animal, Caenorhabditis elegans, as a surrogate host to study the virulence of EHEC as well as the host immunity to this human pathogen. Our results show that E. coli O157:H7, a serotype of EHEC, infects and kills C. elegans. Bacterial colonization and induction of the characteristic attaching and effacing (A/E) lesions in the intact intestinal epithelium of C. elegans by E. coli O157:H7 were concomitantly demonstrated in vivo. Genetic analysis indicated that the Shiga-like toxin 1 (Stx1) of E. coli O157:H7 is a virulence factor in C. elegans and is required for full toxicity. Moreover, the C. elegans p38 mitogen-activated protein kinase (MAPK) pathway, an evolutionarily conserved innate immune and stress response signalling pathway, is activated in the regulation of host susceptibility to EHEC infection in a Stx1-dependent manner. Our results validate the EHEC-C. elegans interaction as suitable for future comprehensive genetic screens for both novel bacterial and host factors involved in the pathogenesis of EHEC infection.

  15. Tumor necrosis factor alpha promotes the proliferation of human nucleus pulposus cells via nuclear factor-κB, c-Jun N-terminal kinase, and p38 mitogen-activated protein kinase

    PubMed Central

    Wang, Xiao-Hu; Hong, Xin; Zhu, Lei; Wang, Yun-Tao; Bao, Jun-Ping; Liu, Lei; Wang, Feng

    2015-01-01

    Although tumor necrosis factor alpha (TNF-α) is known to play a critical role in intervertebral disc (IVD) degeneration, the effect of TNF-α on nucleus pulposus (NP) cells has not yet been elucidated. The aim of this study was to explore the effect of TNF-α on proliferation of human NP cells. NP cells were treated with different concentrations of TNF-α. Cell proliferation was determined by cell counting kit-8 (CCK-8) analysis and Ki67 immunofluorescence staining, and expression of cyclin B1 was studied by quantitative real-time RT-PCR. Cell cycle was measured by flow cytometry and cell apoptosis was analyzed using an Annexin V–fluorescein isothiocyanate (FITC) & propidium iodide (PI) apoptosis detection kit. To identify the mechanism by which TNF-α induced proliferation of NP cells, selective inhibitors of major signaling pathways were used and Western blotting was carried out. Treatment with TNF-α increased cell viability (as determined by CCK-8 analysis) and expression of cyclin B1 and the number of Ki67-positive and S-phase NP cells, indicating enhancement of proliferation. Consistent with this, NP cell apoptosis was suppressed by TNF-α treatment. Moreover, inhibition of NF-κB, c-Jun N-terminal kinase (JNK), and p38 mitogen-activated protein kinase (MAPK) blocked TNF-α-stimulated proliferation of NP cells. In conclusion, the current findings suggest that the effect of TNF-α on IVD degeneration involves promotion of the proliferation of human NP cells via the NF-κB, JNK, and p38 MAPK pathways. PMID:25304312

  16. Tumor necrosis factor alpha promotes the proliferation of human nucleus pulposus cells via nuclear factor-κB, c-Jun N-terminal kinase, and p38 mitogen-activated protein kinase.

    PubMed

    Wang, Xiao-Hu; Hong, Xin; Zhu, Lei; Wang, Yun-Tao; Bao, Jun-Ping; Liu, Lei; Wang, Feng; Wu, Xiao-Tao

    2015-04-01

    Although tumor necrosis factor alpha (TNF-α) is known to play a critical role in intervertebral disc (IVD) degeneration, the effect of TNF-α on nucleus pulposus (NP) cells has not yet been elucidated. The aim of this study was to explore the effect of TNF-α on proliferation of human NP cells. NP cells were treated with different concentrations of TNF-α. Cell proliferation was determined by cell counting kit-8 (CCK-8) analysis and Ki67 immunofluorescence staining, and expression of cyclin B1 was studied by quantitative real-time RT-PCR. Cell cycle was measured by flow cytometry and cell apoptosis was analyzed using an Annexin V-fluorescein isothiocyanate (FITC) & propidium iodide (PI) apoptosis detection kit. To identify the mechanism by which TNF-α induced proliferation of NP cells, selective inhibitors of major signaling pathways were used and Western blotting was carried out. Treatment with TNF-α increased cell viability (as determined by CCK-8 analysis) and expression of cyclin B1 and the number of Ki67-positive and S-phase NP cells, indicating enhancement of proliferation. Consistent with this, NP cell apoptosis was suppressed by TNF-α treatment. Moreover, inhibition of NF-κB, c-Jun N-terminal kinase (JNK), and p38 mitogen-activated protein kinase (MAPK) blocked TNF-α-stimulated proliferation of NP cells. In conclusion, the current findings suggest that the effect of TNF-α on IVD degeneration involves promotion of the proliferation of human NP cells via the NF-κB, JNK, and p38 MAPK pathways.

  17. Combined glutamine and arginine decrease proinflammatory cytokine production by biopsies from Crohn's patients in association with changes in nuclear factor-kappaB and p38 mitogen-activated protein kinase pathways.

    PubMed

    Lecleire, Stéphane; Hassan, Aktham; Marion-Letellier, Rachel; Antonietti, Michel; Savoye, Guillaume; Bôle-Feysot, Christine; Lerebours, Eric; Ducrotté, Philippe; Déchelotte, Pierre; Coëffier, Moïse

    2008-12-01

    Glutamine (Gln) and arginine (Arg) are conditionally essential amino acids with immunomodulatory properties. The aim of the study was to assess the effects of Gln and Arg alone or in combination on cytokine release by cultured colonic biopsies from patients with active Crohn's disease (CD). Ten consecutive patients [mean (range) age 26 (18-39) y] with active colonic CD (mean CD activity index: 383.7 +/- 129.8) were prospectively included in the study. Eight colonic biopsies were obtained via a colonoscopy and incubated during 18 h with low (physiological) or high (pharmacological) doses of Arg (0.1 or 2 mmol/L designated as Arg(low) or Arg(high), respectively) and Gln (0.6 or 10 mmol/L designated as Gln(low) or Gln(high), respectively). The concentrations of cytokines [interleukin (IL)-4, IL-10, IL-8, IL-6, tumor necrosis factor-alpha (TNFalpha), IL-1beta, interferon-gamma) were assessed by ELISA, and nitric oxide (NO) production was evaluated by Griess assay. Nuclear factor (NF)-kappaB p65 subunit, inhibitor of NFkappaB-alpha, and p38 mitogen-activated protein kinase (MAPK) were assessed by immunoblotting. Arg(high)/Gln(high) decreased the production of TNFalpha, IL-1beta, IL-8, and IL-6 (each P < 0.01). Arg(low)/Gln(high) decreased IL-6 and IL-8 production (both P < 0.01), whereas Arg(high)/Gln(low) did not affect cytokine and NO production. Arg(low)/Gln(high) and Arg(high)/Gln(high) decreased NF-kappaB p65 subunit expression, whereas p38 MAPK was decreased only by Arg(high)/Gln(high). Combined pharmacological doses of Arg and Gln decreased TNFalpha and the main proinflammatory cytokines release in active colonic CD biopsies via NF-kappaB and p38 MAPK pathways. These results could be the basis of prospective studies evaluating the effects of enteral supply of combined Arg and Gln during active CD.

  18. Liver X receptor agonist GW3965 dose-dependently regulates lps-mediated liver injury and modulates posttranscriptional TNF-alpha production and p38 mitogen-activated protein kinase activation in liver macrophages.

    PubMed

    Wang, Yun Yong; Dahle, Maria K; Steffensen, Knut R; Reinholt, Finn P; Collins, Jon L; Thiemermann, Christoph; Aasen, Ansgar O; Gustafsson, Jan-Ake; Wang, Jacob E

    2009-11-01

    Modulation of the host inflammatory response to infection may be a key approach to improve the outcome of patients with sepsis and organ injury. We previously reported that pretreatment of rats with the liver X receptor (LXR) agonist GW3965 reduced the liver injury associated with endotoxemia and attenuated the production of TNF-alpha by rat Kupffer cells. Here, we examine the dose-dependent effect of GW3965 on liver injury and cytokine production in a rat model of endotoxemia and explore the mechanisms underlying TNF-alpha attenuation in Kupffer cells. Low doses of GW3965 (0.1 or 0.3 mg/kg) administered 30 min before infusion of LPS and peptidoglycan significantly attenuated the increase in plasma levels of the liver injury markers alanine aminotransferase and bilirubin (6 h) as well as the inflammatory mediators TNF-alpha (1 h) and prostaglandin E2 (6 h) associated with endotoxemia. In contrast, pretreatment with a higher dose of GW3965 (1.0 mg/kg) had no such effect. Studies in primary cultures of rat Kupffer cells demonstrated that LXR agonist treatment attenuated both the secreted and cell-associated levels of TNF-alpha, whereas TNF-alpha mRNA levels were not altered. Phosphorylated p38 mitogen-activated protein kinase, which plays a major role in production of TNF-alpha at the posttranscriptional level, was attenuated by GW3965 treatment in Kupffer cells. Experiments in murine LXR-deficient Kupffer cells demonstrated enhanced production of TNF-alpha in Kupffer cells from LXR-alpha(-/-) mice when challenged with LPS compared with LXR-beta(-/-) and wild-type Kupffer cells. Taken together, these results argue in favor of a novel mechanism for LXR-mediated attenuation of liver injury by interfering with posttranscriptional regulation of TNF-alpha in Kupffer cells. PMID:19295476

  19. Anti-hepatitis B virus effect of matrine-type alkaloid and involvement of p38 mitogen-activated protein kinase and tumor necrosis factor receptor-associated factor 6.

    PubMed

    Chen, Jia-Xin; Shen, Hong-Hui; Niu, Ming; Guo, Yu-Ming; Liu, Xiao-Qiong; Han, Yan-Zhong; Zhang, Ya-Ming; Zhao, Yan-Ling; Bai, Bing-Ke; Zhou, Wen-Jun; Xiao, Xiao-He

    2016-04-01

    The matrine-type alkaloid, oxymatrine inhibits hepatitis B virus (HBV) replication but very little is known about these effects in other matrine-type alkaloids, including sophoridine and sophocarpine. Therefore, we compared the in vitro anti-HBV effects of matrine, oxymatrine, sophocarpine, and sophoridine by treating an HBV-transfected cell line (HepG2.2.15) with 0.4-1.6mM of the compounds for 24 or 72h. The levels of the HBV surface antigen (HBsAg) and e antigen (HBeAg) in the culture medium, as well as the intracellular and extracellular HBV DNA levels, were determined. Metabolomic analysis and detection of the mRNA level of p38 mitogen-activated protein kinase (MAPK), tumor necrosis factor receptor-associated factor (TRAF) 6, extracellular signal-regulated kinase (ERK) 1, NOD-like receptor family pyrin domain containing 10 (NLRP10), and caspase-1 were conducted in sophoridine-treated HepG2.2.15 cells. HepG2.2.15 cell exposure to 0.4-1.6mM sophocarpine or sophoridine for 24h reduced the HBsAg level of the medium more effectively than exposure to matrine and oxymatrine did, and reduced the HBeAg levels more effectively than these compounds did at 1.6mM. Sophoridine (0.4-1.6mM) reduced the cell medium HBV DNA levels more than the same concentrations of matrine, oxymatrine, or sophocarpine did. After 72h, 0.4 and 0.8mM sophoridine reduced HBsAg and intracellular HBV DNA levels more potently than matrine, oxymatrine, or sophocarpine did. Furthermore, sophoridine (0.8mM) potently reduced the cell medium HBeAg levels while the metabolomic analyses revealed that HepG2.2.15 cells exposed to 0.8mM sophoridine for 72h exhibited reduced cycloleucine and phytosphingosine levels. In addition, the mRNA expression analyses revealed that HepG2.2.15 cells exposed to 0.8mM sophoridine showed reduced levels of p38 MAPK, TRAF6, ERK1, NLRP10, and caspase-1. Sophoridine produced more potent anti-HBV effects than matrine, oxymatrine, and sophocarpine did. These effects may be related

  20. PDTC, metal chelating compound, induces G1 phase cell cycle arrest in vascular smooth muscle cells through inducing p21Cip1 expression: involvement of p38 mitogen activated protein kinase.

    PubMed

    Moon, Sung-Kwon; Jung, Sun-Young; Choi, Yung-Hyun; Lee, Young-Choon; Patterson, Cam; Kim, Cheorl-Ho

    2004-02-01

    Pyrrolidine dithiocarbamate (PDTC), a metal chelating compound, is known to induce cell death in vascular smooth muscle cells (VSMC). However, the molecular mechanism for PDTC-induced VSMC death is not well understood. Addition of PDTC reduced cell growth and DNA synthesis on VSMC in low density conditions. However, in serum depleted medium, PDTC did not affect the cell viability, suggesting that certain factors in serum may mediate the cytotoxic effect of PDTC. Several metal chelators prevented the cell death induced by PDTC. In a serum-deprived condition, addition of exogenous metals, copper, iron, and zinc, restored the cytotoxic effect of PDTC. These data indicate that metals such as copper, iron, and zinc in serum may mediate the cytotoxic effect of PDTC. At low VSMC density in 10% FBS, treatment of PDTC, which induced a cell-cycle block in G1-phase, induced down-regulation of cyclins and CDKs and up-regulation of the CDK inhibitor p21 expression, whereas up-regulation of p27 or p53 by PDTC was not observed. Finally, we determined PDTC-mediated signaling pathway involved in VSMC death. Among relevant pathways, PDTC induced marked activation of p38MAPK and JNK. Expression of dominant negative p38MAPK and SB203580, a p38MAPK specific inhibitor, blocked PDTC-dependent p38MAPK, growth inhibition, and p21 expression. These data demonstrate that the p38MAPK pathway participates in p21 induction, which consequently leads to decrease of cyclin D1/cdk4 and cyclin E/cdk2 complexes and PDTC-dependent VSMC growth inhibition. In conclusion, an understanding of the molecular mechanisms of PDTC in VSMC provides a theoretical basis for clinical approaches using antioxidant therapies in atherosclerosis. PMID:14603533

  1. Early treatment with UR13870, a novel inhibitor of p38α mitogenous activated protein kinase, prevents hyperreflexia and anxiety behaviors, in the spared nerve injury model of neuropathic pain.

    PubMed

    Galan-Arriero, Iriana; Avila-Martin, Gerardo; Ferrer-Donato, Agueda; Gomez-Soriano, Julio; Piazza, Stefano; Taylor, Julian

    2015-09-14

    Microglia cell activation plays a role in the development of neuropathic pain partly due to the activation of the p38α MAPK signaling pathway after nerve injury. In this study we assessed the effect of UR13870, a p38α MAPK inhibitor, in the "spared nerve injury" (SNI) model, to study its effects on modulation of spinal microglial activation and to test behavioral hyperreflexia responses and cerebral-mediated pain behavior. The effect of daily administration of UR13870 (10mg/kg p.o.) and Pregabalin (50mg/kg p.o.) on reflex hypersensitivity to mechanical and cold test stimuli and on affective related pain responses measured with the place escape avoidance paradigm and the open field-induced anxiety test, were evaluated after SNI in Sprague Dawley rats. Microglial reactivity in the ipsilateral lumbar laminae I/II dorsal horn was evaluated with OX-42 immunohistochemistry. UR13870 treatment significantly decreased hindlimb hyperreflexia to both mechanical and cold stimuli after SNI without loss of general motor function, in addition to a reduction in pain-related anxiety behavior at day 21 after SNI, accompanied by normalization of OX-42 immunoreactivity within the ipsilateral lumbar dorsal horn. Pregabalin treatment only reduced mechanical hyperreflexia and affected general motor function. Oral administration of the p38α MAPK inhibitor, UR13870, mediates antinociception to both mechanical and cold stimuli, and significantly restored inner-zone exploration in the open field test, accompanied by normalization in dorsal horn microglial activation in the SNI model. PMID:26240995

  2. Early treatment with UR13870, a novel inhibitor of p38α mitogenous activated protein kinase, prevents hyperreflexia and anxiety behaviors, in the spared nerve injury model of neuropathic pain.

    PubMed

    Galan-Arriero, Iriana; Avila-Martin, Gerardo; Ferrer-Donato, Agueda; Gomez-Soriano, Julio; Piazza, Stefano; Taylor, Julian

    2015-09-14

    Microglia cell activation plays a role in the development of neuropathic pain partly due to the activation of the p38α MAPK signaling pathway after nerve injury. In this study we assessed the effect of UR13870, a p38α MAPK inhibitor, in the "spared nerve injury" (SNI) model, to study its effects on modulation of spinal microglial activation and to test behavioral hyperreflexia responses and cerebral-mediated pain behavior. The effect of daily administration of UR13870 (10mg/kg p.o.) and Pregabalin (50mg/kg p.o.) on reflex hypersensitivity to mechanical and cold test stimuli and on affective related pain responses measured with the place escape avoidance paradigm and the open field-induced anxiety test, were evaluated after SNI in Sprague Dawley rats. Microglial reactivity in the ipsilateral lumbar laminae I/II dorsal horn was evaluated with OX-42 immunohistochemistry. UR13870 treatment significantly decreased hindlimb hyperreflexia to both mechanical and cold stimuli after SNI without loss of general motor function, in addition to a reduction in pain-related anxiety behavior at day 21 after SNI, accompanied by normalization of OX-42 immunoreactivity within the ipsilateral lumbar dorsal horn. Pregabalin treatment only reduced mechanical hyperreflexia and affected general motor function. Oral administration of the p38α MAPK inhibitor, UR13870, mediates antinociception to both mechanical and cold stimuli, and significantly restored inner-zone exploration in the open field test, accompanied by normalization in dorsal horn microglial activation in the SNI model.

  3. Acquisition of contextual discrimination involves the appearance of a RAS-GRF1/p38 mitogen-activated protein (MAP) kinase-mediated signaling pathway that promotes long term potentiation (LTP).

    PubMed

    Jin, Shan-Xue; Arai, Junko; Tian, Xuejun; Kumar-Singh, Rajendra; Feig, Larry A

    2013-07-26

    RAS-GRF1 is a guanine nucleotide exchange factor with the ability to activate RAS and RAC GTPases in response to elevated calcium levels. We previously showed that beginning at 1 month of age, RAS-GRF1 mediates NMDA-type glutamate receptor (NMDAR)-induction of long term depression in the CA1 region of the hippocampus of mice. Here we show that beginning at 2 months of age, when mice first acquire the ability to discriminate between closely related contexts, RAS-GRF1 begins to contribute to the induction of long term potentiation (LTP) in the CA1 hippocampus by mediating the action of calcium-permeable, AMPA-type glutamate receptors (CP-AMPARs). Surprisingly, LTP induction by CP-AMPARs through RAS-GRF1 occurs via activation of p38 MAP kinase rather than ERK MAP kinase, which has more frequently been linked to LTP. Moreover, contextual discrimination is blocked by knockdown of Ras-Grf1 expression specifically in the CA1 hippocampus, infusion of a p38 MAP kinase inhibitor into the CA1 hippocampus, or the injection of an inhibitor of CP-AMPARs. These findings implicate the CA1 hippocampus in the developmentally dependent capacity to distinguish closely related contexts through the appearance of a novel LTP-supporting signaling pathway.

  4. 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

  5. Serotonin via 5-HT7 receptors activates p38 mitogen-activated protein kinase and protein kinase C epsilon resulting in interleukin-6 synthesis in human U373 MG astrocytoma cells.

    PubMed

    Lieb, Klaus; Biersack, Lisa; Waschbisch, Anne; Orlikowski, Sonja; Akundi, Ravi Shankar; Candelario-Jalil, Eduardo; Hüll, Michael; Fiebich, Bernd L

    2005-05-01

    Serotonin [5-hydroxytryptamine (5-HT)] is a widely distributed neurotransmitter which is involved in neuroimmunomodulatory processes. Previously, it has been demonstrated that 5-HT may induce interleukin (IL)-6 expression in primary rat hippocampal astrocytes. The present study was undertaken to investigate the molecular pathways underlying this induction of IL-6 synthesis. As a model system, we used the human astrocytoma cell line U373 MG, which synthesizes IL-6 upon stimulation with various inducers. 5-HT dose- and time-dependently induced IL-6 protein synthesis. We identified several 5-HT receptors to be expressed on U373 MG cells, including the 5-HT1D, 5-HT2A, 5-HT3 and 5-HT7 receptors. In this report, we show that the 5-HT-induced IL-6 release is mediated by the 5-HT7 receptor based on several agonist/antagonists that were used. 5-HT-induced IL-6 synthesis is inhibited by the partially selective 5-HT7 receptor antagonist, pimozide, and the selective antagonist SB269970. Furthermore, IL-6 synthesis was induced by the 5-HT7 receptor agonist carboxamidotryptamin. In addition, we found p38 MAPKs and protein kinase C (PKC) epsilon to be involved in 5-HT-induced IL-6 synthesis as specific inhibitors of these enzymes (SB202190 and RO-31-8425, respectively) blocked 5-HT-induced IL-6 synthesis. Furthermore, 5-HT mediated the phosphorylation of both p38 MAPK as well as the PKC epsilon isoform. The p42/44 MAPKs, however, were not involved in 5-HT-induced IL-6 synthesis. This study shows, for the first time, a central role of 5-HT7 receptor linked to p38 MAPK and PKC epsilon for the induction of cytokine synthesis in astrocytic cells. PMID:15836614

  6. Lysophosphatidic acid-mediated signal-transduction pathways involved in the induction of the early-response genes prostaglandin G/H synthase-2 and Egr-1: a critical role for the mitogen-activated protein kinase p38 and for Rho proteins.

    PubMed Central

    Reiser, C O; Lanz, T; Hofmann, F; Hofer, G; Rupprecht, H D; Goppelt-Struebe, M

    1998-01-01

    During inflammatory processes of the kidney, lesions of the glomerulus lead to aggregation of thrombocytes and infiltration of macrophages, which can release bioactive mediators. One of these important signalling molecules is lysophosphatidic acid (LPA). Incubation of rat mesangial cells with LPA induced mRNA and protein expression of the early-response genes pghs-2 (for prostaglandin G/H synthase-2/cyclo-oxygenase-2) and egr-1. As shown by antisense experiments, induction of egr-1 was related to the strong mitogenic effect of LPA. LPA-mediated gene expression was inhibited by pertussis toxin, indicating coupling to G-proteins of the Gi family. Specific inhibition of proteins of the small G-protein subfamily Rho with toxin B from Clostridium difficile led to changes in mesangial cell morphology without induction of apoptosis. LPA-mediated expression of pghs-2 and egr-1 was reduced to base-line levels by toxin B, indicating a role for Rho proteins in LPA-mediated gene induction. Of the two mitogen-activated protein kinase (MAPK) pathways investigated, the MAPK kinase-extracellular signal-regulated kinase pathway was involved in the induction of both pghs-2 and egr-1 mRNA expression, as shown by the inhibitory effect of PD98059. Activation of the MAPK p38, however, was only related to pghs-2 expression, whereas egr-1 expression was not affected by treatment of mesangial cells with the specific inhibitor SB203580. Taken together our data provide evidence that LPA-mediated activation of MAPK kinase and Rho proteins leads to the induction of the functionally distinct early-response genes pghs-2 and egr-1, whereas activation of MAPK p38 revealed considerable differences between the regulation of these two genes. PMID:9494074

  7. Inducement of mitogen-activated protein kinases in frozen shoulders

    PubMed Central

    Kanbe, Katsuaki; Inoue, Kazuhiko; Inoue, Yasuo; Chen, Qian

    2010-01-01

    Background Mitogen-activated protein (MAP) kinases are well-known molecules that play key roles in mechanical stress signals during skeletal development. To test our hypothesis that the synovium in frozen shoulders is induced by MAP kinases, immunohistochemical analyses for detecting expression and signal transduction of MAP kinases were performed in synovial tissue obtained from the rotator interval (RI) in frozen shoulders. Methods Synovial tissues were examined from 10 frozen shoulder patients with a mean age of 55.4 years (46–62 years). Synovial tissues between the long head of the biceps tendon (LHB) and the RI in frozen shoulders were stained with hematoxylin and eosin (H&E) and then examined with immunohistochemical staining. Extracellular signal-regulated (ERK), the Jun N-terminal (JNK), and p38 mitogen-activated protein (MAP) kinases, nuclear factor κB (NF-κB), p50, CD29 (β1-integrin), matrix metalloproteinase (MMP)-3, interleukin-6 (IL-6), CD56, CD68, S-100, and vascular endothelial growth factor (VEGF) were analyzed to detect expression patterns. Results H&E showed vascular proliferation with fibrin and fibrous tissue in the synovium of frozen shoulders. ERK was expressed in the epithelial cells of vascular tissue, and JNK was expressed strongly in the interstitial cells around vascular tissue; p38 MAPK was not expressed. NF-κB was expressed in vascular tissue, and IL-6 was expressed around vascular tissue. CD29 (β1-integrin) was expressed in vascular tissue and in superficial cells of synovial tissue. MMP-3 and VEGF were expressed on the surface layer of synovial tissue and vascular tissue, and CD68 was expressed on the surface layer. Nerve-related proteins, CD56 and S-100, were expressed weakly. Conclusions Mechanical stress on the LHB and RI in the shoulder may induce ERK and JNK to express NF-κB by CD29 to develop capsule contracture, producing MMP-3, IL-6, and VEGF. PMID:19214689

  8. Mitogen-Activated Protein Kinases and Hypoxic/Ischemic Nephropathy.

    PubMed

    Luo, Fengbao; Shi, Jian; Shi, Qianqian; Xu, Xianlin; Xia, Ying; He, Xiaozhou

    2016-01-01

    Tissue hypoxia/ischemia is a pathological feature of many human disorders including stroke, myocardial infarction, hypoxic/ischemic nephropathy, as well as cancer. In the kidney, the combination of limited oxygen supply to the tissues and high oxygen demand is considered the main reason for the susceptibility of the kidney to hypoxic/ischemic injury. In recent years, increasing evidence has indicated that a reduction in renal oxygen tension/blood supply plays an important role in acute kidney injury, chronic kidney disease, and renal tumorigenesis. However, the underlying signaling mechanisms, whereby hypoxia alters cellular behaviors, remain poorly understood. Mitogen-activated protein kinases (MAPKs) are key signal-transducing enzymes activated by a wide range of extracellular stimuli, including hypoxia/ischemia. There are four major family members of MAPKs: the extracellular signal-regulated kinases-1 and -2 (ERK1/2), the c-Jun N-terminal kinases (JNK), p38 MAPKs, and extracellular signal-regulated kinase-5 (ERK5/BMK1). Recent studies, including ours, suggest that these MAPKs are differentially involved in renal responses to hypoxic/ischemic stress. This review will discuss their changes in hypoxic/ischemic pathophysiology with acute kidney injury, chronic kidney diseases and renal carcinoma. PMID:27544204

  9. Acidic pH stimulates the production of the angiogenic CXC chemokine, CXCL8 (interleukin-8), in human adult mesenchymal stem cells via the extracellular signal-regulated kinase, p38 mitogen-activated protein kinase, and NF-kappaB pathways.

    PubMed

    Bischoff, David S; Zhu, Jian-Hua; Makhijani, Nalini S; Yamaguchi, Dean T

    2008-07-01

    Blood vessel injury results in limited oxygen tension and diffusion leading to hypoxia, increased anaerobic metabolism, and elevated production of acidic metabolites that cannot be easily removed due to the reduced blood flow. Therefore, an acidic extracellular pH occurs in the local microenvironment of disrupted bone. The potential role of acidic pH and glu-leu-arg (ELR(+)) CXC chemokines in early events in bone repair was studied in human mesenchymal stem cells (hMSCs) treated with medium of decreasing pH (7.4, 7.0, 6.7, and 6.4). The cells showed a reciprocal increase in CXCL8 (interleukin-8, IL-8) mRNA levels as extracellular pH decreased. At pH 6.4, CXCL8 mRNA was induced >60x in comparison to levels at pH 7.4. hMSCs treated with osteogenic medium (OGM) also showed an increase in CXCL8 mRNA with decreasing pH; although, at a lower level than that seen in cells grown in non-OGM. CXCL8 protein was secreted into the medium at all pHs with maximal induction at pH 6.7. Inhibition of the G-protein-coupled receptor alpha, G(alphai), suppressed CXCL8 levels in response to acidic pH; whereas phospholipase C inhibition had no effect on CXCL8. The use of specific mitogen-activated protein kinase (MAPK) signal transduction inhibitors indicated that the pH-dependent increase in CXCL8 mRNA is due to activation of ERK and p38 pathways. The JNK pathway was not involved. NF-kappaB inhibition resulted in a decrease in CXCL8 levels in hMSCs grown in non-OGM. However, OGM-differentiated hMSCs showed an increase in CXCL8 levels when treated with the NF-kappaB inhibitor PDTC, a pyrrolidine derivative of dithiocarbamate.

  10. Activity of mitogen-activated protein kinases in the esophageal epithelium of patients with Barrett's esophagus.

    PubMed

    Chwiesko, A; Baniukiewicz, A; Semeniuk, J; Kaczmarski, M; Wasielica-Berger, J; Milewski, R; Dabrowski, A

    2015-01-01

    Barrett's esophagus (BE), a complication of gastroesophageal reflux disease, is associated with an increased risk of esophageal cancer. Mitogen-activated protein kinases may play an important role in the pathogenesis of this process. We aimed to evaluate mitogen-activated protein kinases activity in esophageal mucosa of patients with BE and find possible relationship between reflux type and BE. Twenty-four patients (mean age: 59 years) with gastroesophageal reflux disease symptoms and endoscopically suspected esophageal metaplasia (ESEM) were prospectively enrolled for testing by a multichannel intraluminal impedance monitoring along with a Bilitec 2000. Endoscopic biopsies were taken from methylene blue-positive pit patterns (sites suggesting specialized intestinal metaplasia [SIM]), from 2 cm above the Z-line and from cardial parts of the stomach. The biopsies were analyzed for extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), p38 activity by Western blot. Seventeen ESEMs had histologically proven metaplasia: eight patients had SIM and nine had gastric-type epithelia (GE). Biliary reflux was more evident in SIM (P = 0.019) but not in GE (P = 0.019); non-biliary reflux was typical for GE (P = 0.005) but not for SIM (P = 0.04). Strong activations of ERK and p38 were found predominantly in SIM, but not in normal esophageal mucosa (NE) (P = 0.01 and P < 0.001 respectively). Strong signals for active JNK and p38 were detected in GE, but not in NE (P = 0.006 and P = 0.02 respectively). ERK activity was significantly higher than p38 activity in ESEM patients only with GE (P = 0.02). The strong activation of ERK, but not JNK is indicative of SIM. The presence of bile in gastroesophageal refluxate is predisposing to SIM, but not to GE in esophageal mucosa.

  11. Dehydrocorydaline promotes myogenic differentiation via p38 MAPK activation

    PubMed Central

    Yoo, Miran; Lee, Sang-Jin; Kim, Yong Kee; Seo, Dong-Wan; Baek, Nam-In; Ryu, Jae-Ha; Kang, Jong-Sun; Bae, Gyu-Un

    2016-01-01

    Muscle regeneration is a coordinated process that involves proliferation and differentiation of muscle progenitor cells. Activation of MyoD is a key event in myogenic differentiation, which is regulated by p38 mitogen-activated protein kinases (MAPK). In a screen of natural compounds for the enhancement of MyoD activity, dehydrocorydaline (DHC) from the Corydalis tuber was identified. Treatment of C2C12 myoblasts with DHC increased the expression levels of muscle-specific proteins, including MyoD, myogenin and myosin heavy chain. In addition, C2C12 myoblasts exhibited enhanced multinucleated myotube formation without any cytotoxicity. Treatment with DHC elevated p38 MAPK activation and the interaction of MyoD with an E protein, which is likely to result in activation of MyoD and promotion of myoblast differentiation. Furthermore, defects in differentiation-induced p38 MAPK activation and myoblast differentiation induced by depletion of the promyogenic receptor protein Cdo in C2C12 myoblasts were restored by DHC treatment. In conclusion, these results indicated that DHC stimulates p38 MAPK activation, which can enhance heterodimerization of MyoD and E proteins, thus resulting in MyoD activation and myoblast differentiation. These findings suggested that DHC may be considered a potential therapeutic compound for the improvement of muscle stem cell regenerative capacity in injured muscle. PMID:27573543

  12. Selective activation of p38alpha and p38gamma by hypoxia. Role in regulation of cyclin D1 by hypoxia in PC12 cells.

    PubMed

    Conrad, P W; Rust, R T; Han, J; Millhorn, D E; Beitner-Johnson, D

    1999-08-13

    Hypoxic/ischemic trauma is a primary factor in the pathology of a multitude of disease states. The effects of hypoxia on the stress- and mitogen-activated protein kinase signaling pathways were studied in PC12 cells. Exposure to moderate hypoxia (5% O(2)) progressively stimulated phosphorylation and activation of p38gamma in particular, and also p38alpha, two stress-activated protein kinases. In contrast, hypoxia had no effect on enzyme activity of p38beta, p38beta(2), p38delta, or on c-Jun N-terminal kinase, another stress-activated protein kinase. Prolonged hypoxia also induced phosphorylation and activation of p42/p44 mitogen-activated protein kinase, although this activation was modest compared with nerve growth factor- and ultraviolet light-induced activation. Hypoxia also dramatically down-regulated immunoreactivity of cyclin D1, a gene that is known to be regulated negatively by p38 at the level of gene expression (Lavoie, J. N., L'Allemain, G., Brunet, A., Muller, R., and Pouyssegur, J. (1996) J. Biol. Chem. 271, 20608-20616). This effect was partially blocked by SB203580, an inhibitor of p38alpha but not p38gamma. Overexpression of a kinase-inactive form of p38gamma was also able to reverse in part the effect of hypoxia on cyclin D1 levels, suggesting that p38alpha and p38gamma converge to regulate cyclin D1 during hypoxia. These studies demonstrate that an extremely typical physiological stress (hypoxia) causes selective activation of specific p38 signaling elements; and they also identify a downstream target of these pathways. PMID:10438538

  13. Crosstalk and Signaling Switches in Mitogen-Activated Protein Kinase Cascades

    PubMed Central

    Fey, Dirk; Croucher, David R.; Kolch, Walter; Kholodenko, Boris N.

    2012-01-01

    Mitogen-activated protein kinase (MAPK) cascades control cell fate decisions, such as proliferation, differentiation, and apoptosis by integrating and processing intra- and extracellular cues. However, similar MAPK kinetic profiles can be associated with opposing cellular decisions depending on cell type, signal strength, and dynamics. This implies that signaling by each individual MAPK cascade has to be considered in the context of the entire MAPK network. Here, we develop a dynamic model of feedback and crosstalk for the three major MAPK cascades; extracellular signal-regulated kinase (ERK), p38 mitogen-activated protein kinase (p38), c-Jun N-terminal kinase (JNK), and also include input from protein kinase B (AKT) signaling. Focusing on the bistable activation characteristics of the JNK pathway, this model explains how pathway crosstalk harmonizes different MAPK responses resulting in pivotal cell fate decisions. We show that JNK can switch from a transient to sustained activity due to multiple positive feedback loops. Once activated, positive feedback locks JNK in a highly active state and promotes cell death. The switch is modulated by the ERK, p38, and AKT pathways. ERK activation enhances the dual specificity phosphatase (DUSP) mediated dephosphorylation of JNK and shifts the threshold of the apoptotic switch to higher inputs. Activation of p38 restores the threshold by inhibiting ERK activity via the PP1 or PP2A phosphatases. Finally, AKT activation inhibits the JNK positive feedback, thus abrogating the apoptotic switch and allowing only proliferative signaling. Our model facilitates understanding of how cancerous deregulations disturb MAPK signal processing and provides explanations for certain drug resistances. We highlight a critical role of DUSP1 and DUSP2 expression patterns in facilitating the switching of JNK activity and show how oncogene induced ERK hyperactivity prevents the normal apoptotic switch explaining the failure of certain drugs to

  14. Analysis of mitogen-activated protein kinase activity in yeast.

    PubMed

    Elion, Elaine A; Sahoo, Rupam

    2010-01-01

    Mitogen-activated protein (MAP) kinases play central roles in transmitting extracellular and intracellular information in a wide variety of situations in eukaryotic cells. Their activities are perturbed in a large number of diseases, and their activating kinases are currently therapeutic targets in cancer. MAPKs are highly conserved among all eukaryotes. MAPKs were first cloned from the yeast Saccharomyces cerevisiae. Yeast has five MAPKs and one MAPK-like kinase. The mating MAPK Fus3 is the best characterized yeast MAPK. Members of all subfamilies of human MAPKs can functionally substitute S. cerevisiae MAPKs, providing systems to use genetic approaches to study the functions of either yeast or human MAPKs and to identify functionally relevant amino acid residues that enhance or reduce the effects of therapeutically relevant inhibitors and regulatory proteins. Here, we describe an assay to measure Fus3 activity in immune complexes prepared from S. cerevisiae extracts. The assay conditions are applicable to other MAPKs, as well. PMID:20811996

  15. Pyrrolopyridine inhibitors of mitogen-activated protein kinase-activated protein kinase 2 (MK-2).

    PubMed

    Anderson, David R; Meyers, Marvin J; Vernier, William F; Mahoney, Matthew W; Kurumbail, Ravi G; Caspers, Nicole; Poda, Gennadiy I; Schindler, John F; Reitz, David B; Mourey, Robert J

    2007-05-31

    A new class of potent kinase inhibitors selective for mitogen-activated protein kinase-activated protein kinase 2 (MAPKAP-K2 or MK-2) for the treatment of rheumatoid arthritis has been prepared and evaluated. These inhibitors have IC50 values as low as 10 nM against the target and have good selectivity profiles against a number of kinases including CDK2, ERK, JNK, and p38. These MK-2 inhibitors have been shown to suppress TNFalpha production in U397 cells and to be efficacious in an acute inflammation model. The structure-activity relationships of this series, the selectivity for MK-2 and their activity in both in vitro and in vivo models are discussed. The observed selectivity is discussed with the aid of an MK-2/inhibitor crystal structure.

  16. Activation of multiple mitogen-activated protein kinases by recombinant calcitonin gene-related peptide receptor.

    PubMed

    Parameswaran, N; Disa, J; Spielman, W S; Brooks, D P; Nambi, P; Aiyar, N

    2000-02-18

    Calcitonin gene-related peptide is a 37-amino-acid neuropeptide and a potent vasodilator. Although calcitonin gene-related peptide has been shown to have a number of effects in a variety of systems, the mechanisms of action and the intracellular signaling pathways, especially the regulation of mitogen-activated protien kinase (MAPK) pathway, is not known. In the present study we investigated the role of calcitonin gene-related peptide in the regulation of MAPKs in human embryonic kidney (HEK) 293 cells stably transfected with a recombinant porcine calcitonin gene-related peptide-1 receptor. Calcitonin gene-related peptide caused a significant dose-dependent increase in cAMP response and the effect was inhibited by calcitonin gene-related peptide(8-37), the calcitonin gene-related peptide-receptor antagonist. Calcitonin gene-related peptide also caused a time- and concentration-dependent increase in extracellular signal-regulated kinase (ERK) and P38 mitogen-activated protein kinase (P38 MAPK) activities, with apparently no significant change in cjun-N-terminal kinase (JNK) activity. Forskolin, a direct activator of adenylyl cyclase also stimulated ERK and P38 activities in these cells suggesting the invovement of cAMP in this process. Calcitonin gene-related peptide-stimulated ERK and P38 MAPK activities were inhibited significantly by calcitonin gene-related peptide receptor antagonist, calcitonin gene-related peptide-(8-37) suggesting the involvement of calcitonin gene-related peptide-1 receptor. Preincubation of the cells with the cAMP-dependent protein kinase inhibitor, H89 [¿N-[2-((p-bromocinnamyl)amino)ethyl]-5-isoquinolinesulfonamide, hydrochloride¿] inhibited calcitonin gene-related peptide-mediated activation of ERK and p38 kinases. On the other hand, preincubation of the cells with wortmannin ¿[1S-(1alpha,6balpha,9abeta,11alpha, 11bbeta)]-11-(acetyloxy)-1,6b,7,8,9a,10,11, 11b-octahydro-1-(methoxymethyl)-9a,11b-dimethyl-3H-furo[4,3, 2-de]indeno[4,5-h]-2

  17. Pivotal Role of Mitogen-Activated Protein Kinase-Activated Protein Kinase 2 in Inflammatory Pulmonary Diseases

    PubMed Central

    Qian, Feng; Deng, Jing; Wang, Gang; Ye, Richard D.; Christman, John W.

    2016-01-01

    Mitogen-activated protein kinase (MAPK)-activated protein kinase (MK2) is exclusively regulated by p38 MAPK in vivo. Upon activation of p38 MAPK, MK2 binds with p38 MAPK, leading to phosphorylation of TTP, Hsp27, Akt and Cdc25 that are involved in regulation of various essential cellular functions. In this review, we discuss current knowledge about molecular mechanisms of MK2 in regulation of TNF-α production, NADPH oxidase activation, neutrophil migration, and DNA-damage-induced cell cycle arrest which are involved in the molecular pathogenesis of acute lung injury, pulmonary fibrosis, and non-small-cell lung cancer. Collectively current and emerging new information indicate that developing MK2 inhibitors and blocking MK2-mediated signal pathways is a potential therapeutic strategy for treatment of inflammatory and fibrotic lung diseases and lung cancer. PMID:26119506

  18. Cellular reprogramming through mitogen-activated protein kinases

    PubMed Central

    Lee, Justin; Eschen-Lippold, Lennart; Lassowskat, Ines; Böttcher, Christoph; Scheel, Dierk

    2015-01-01

    Mitogen-activated protein kinase (MAPK) cascades are conserved eukaryote signaling modules where MAPKs, as the final kinases in the cascade, phosphorylate protein substrates to regulate cellular processes. While some progress in the identification of MAPK substrates has been made in plants, the knowledge on the spectrum of substrates and their mechanistic action is still fragmentary. In this focused review, we discuss the biological implications of the data in our original paper (Sustained mitogen-activated protein kinase activation reprograms defense metabolism and phosphoprotein profile in Arabidopsis thaliana; Frontiers in Plant Science 5: 554) in the context of related research. In our work, we mimicked in vivo activation of two stress-activated MAPKs, MPK3 and MPK6, through transgenic manipulation of Arabidopsis thaliana and used phosphoproteomics analysis to identify potential novel MAPK substrates. Here, we plotted the identified putative MAPK substrates (and downstream phosphoproteins) as a global protein clustering network. Based on a highly stringent selection confidence level, the core networks highlighted a MAPK-induced cellular reprogramming at multiple levels of gene and protein expression—including transcriptional, post-transcriptional, translational, post-translational (such as protein modification, folding, and degradation) steps, and also protein re-compartmentalization. Additionally, the increase in putative substrates/phosphoproteins of energy metabolism and various secondary metabolite biosynthesis pathways coincides with the observed accumulation of defense antimicrobial substances as detected by metabolome analysis. Furthermore, detection of protein networks in phospholipid or redox elements suggests activation of downstream signaling events. Taken in context with other studies, MAPKs are key regulators that reprogram cellular events to orchestrate defense signaling in eukaryotes. PMID:26579181

  19. Parallel regulation of mitogen-activated protein kinase kinase 3 (MKK3) and MKK6 in Gq-signaling cascade.

    PubMed

    Yamauchi, J; Tsujimoto, G; Kaziro, Y; Itoh, H

    2001-06-29

    Heterotrimeric G protein G(q) stimulates the activity of p38 mitogen-activated protein kinase (MAPK) in mammalian cells. To investigate the signaling mechanism whereby alpha and betagamma subunits of G(q) activate p38 MAPK, we introduced kinase-deficient mutants of mitogen-activated protein kinase kinase 3 (MKK3), MKK4, and MKK6 into human embryonal kidney 293 cells. The activation of p38 MAPK by Galpha(q) and Gbetagamma was blocked by kinase-deficient MKK3 and MKK6 but not by kinase-deficient MKK4. In addition, Galpha(q) and Gbetagamma stimulated MKK3 and MKK6 activities. The MKK3 and MKK6 activations by Galpha(q), but not by Gbetagamma, were dependent on phospholipase C and c-Src. Galpha(q) stimulated MKK3 in a Rac- and Cdc42-dependent manner and MKK6 in a Rho-dependent manner. On the other hand, Gbetagamma activated MKK3 in a Rac- and Cdc42-dependent manner and MKK6 in a Rho-, Rac-, and Cdc42-dependent manner. Gbetagamma-induced MKK3 and MKK6 activations were dependent on a tyrosine kinase other than c-Src. These results suggest that Galpha(q) and Gbetagamma stimulate the activity of p38 MAPK by regulating MKK3 and MKK6 through parallel signaling pathways.

  20. The Role of Endosomal Escape and Mitogen-Activated Protein Kinases in Adenoviral Activation of the Innate Immune Response

    PubMed Central

    Smith, Jeffrey S.; Xu, Zhili; Tian, Jie; Palmer, Donna J.; Ng, Philip; Byrnes, Andrew P.

    2011-01-01

    Adenoviral vectors (AdV) activate multiple signaling pathways associated with innate immune responses, including mitogen-activated protein kinases (MAPKs). In this study, we investigated how systemically-injected AdVs activate two MAPK pathways (p38 and ERK) and the contribution of these kinases to AdV-induced cytokine and chemokine responses in mice. Mice were injected intravenously either with a helper-dependent Ad2 vector that does not express viral genes or transgenes, or with the Ad2 mutant ts1, which is defective in endosomal escape. We found that AdV induced rapid phosphorylation of p38 and ERK as well as a significant cytokine response, but ts1 failed to activate p38 or ERK and induced only a limited cytokine response. These results demonstrate that endosomal escape of virions is a critical step in the induction of these innate pathways and responses. We then examined the roles of p38 and ERK pathways in the innate cytokine response by administering specific kinase inhibitors to mice prior to AdV. The cytokine and chemokine response to AdV was only modestly suppressed by a p38 inhibitor, while an ERK inhibitor has mixed effects, lowering some cytokines and elevating others. Thus, even though p38 and ERK are rapidly activated after i.v. injection of AdV, cytokine and chemokine responses are mostly independent of these kinases. PMID:22046344

  1. Thymic Stromal Lymphopoietin Promotes Fibrosis and Activates Mitogen-Activated Protein Kinases in MRC-5 Cells

    PubMed Central

    Li, Li; Tang, Su; Tang, Xiaodong

    2016-01-01

    Background Acute lung injury (ALI) is a life-threatening hypoxemic respiratory disorder with high incidence and mortality. ALI usually manifests as widespread inflammation and lung fibrosis with the accumulation of pro-inflammatory and pro-fibrotic factors and collagen. Thymic stromal lymphopoietin (TSLP) has a significant role in regulation of inflammation but little is known about its roles in lung fibrosis or ALI. This study aimed to define the role and possible regulatory mechanism of TSLP in lung fibrosis. Material/Methods We cultured human lung fibroblast MRC-5 cells and overexpressed or inhibited TSLP by the vector or small interfering RNA transfection. Then, the pro-fibrotic factors skeletal muscle actin alpha (α-SMA) and collagen I, and the 4 mitogen-activated protein kinases (MAPKs) – MAPK7, p38, extracellular signal-regulated kinase 1 (ERK1), and c-Jun N-terminal kinase 1 (JNK1) – were detected by Western blot. Results Results showed that TSLP promoted the production of α-SMA and collagen I (P<0.001), suggesting that it can accelerate MRC-5 cell fibrosis. It also activated the expression of MAPK7, p-p38, p-ERK1, and p-JNK1, but the total MAPK7, p-38, ERK1, and JNK1 protein levels were mostly unchanged, indicating the activated MAPK pathways that might contribute to the promotion of cell fibrosis. Conclusions This study shows the pro-fibrotic role of TSLP in MRC-5 cells, suggesting TSLP is a potential therapeutic target for treating lung fibrosis in ALI. It possibly functions via activating MAPKs. These findings add to our understanding of the mechanism of fibrosis. PMID:27385084

  2. A hemagglutinin with mitogenic activity from dark red kidney beans.

    PubMed

    Xia, Lixin; Ng, T B

    2006-12-01

    A 67-kDa hemagglutinin composed of two identical subunits was purified from Phaseolus vulgaris cv. 'Dark Red Kidney Bean'. It was unadsorbed on DEAE-cellulose but adsorbed on Affi-gel blue gel. The hemagglutinin was highly purified after the two aforementioned chromatographic steps as revealed by a single peak in gel filtration on Superdex 75 and a single band in SDS-PAGE. The hemagglutinating activity was stable between 25 degrees C and 70 degrees C, and between pH 4 and pH 11, and in the presence of a variety of divalent metal chlorides at 500 mM concentration. The activity was reduced by 50% at 80 degrees C, and also when the pH was lowered to 3 or elevated to 12. The activity was reduced by 75% in the presence of 250 mM KCl or NaCl. A variety of sugars tested failed to inhibit the hemagglutinating activity of the hemagglutinin. Although the hemagglutinin exhibited mitogenic activity toward murine splenocytes, it had no effect on the activity of HIV-1 reverse transcriptase or mycelial growth in the fungi Botrytis cinerea, Fusarium oxysporum and Mycosphaerella arachidicola. It exerted an antiproliferative activity on leukemia L1210 cells.

  3. A hemagglutinin with mitogenic activity from dark red kidney beans.

    PubMed

    Xia, Lixin; Ng, T B

    2006-12-01

    A 67-kDa hemagglutinin composed of two identical subunits was purified from Phaseolus vulgaris cv. 'Dark Red Kidney Bean'. It was unadsorbed on DEAE-cellulose but adsorbed on Affi-gel blue gel. The hemagglutinin was highly purified after the two aforementioned chromatographic steps as revealed by a single peak in gel filtration on Superdex 75 and a single band in SDS-PAGE. The hemagglutinating activity was stable between 25 degrees C and 70 degrees C, and between pH 4 and pH 11, and in the presence of a variety of divalent metal chlorides at 500 mM concentration. The activity was reduced by 50% at 80 degrees C, and also when the pH was lowered to 3 or elevated to 12. The activity was reduced by 75% in the presence of 250 mM KCl or NaCl. A variety of sugars tested failed to inhibit the hemagglutinating activity of the hemagglutinin. Although the hemagglutinin exhibited mitogenic activity toward murine splenocytes, it had no effect on the activity of HIV-1 reverse transcriptase or mycelial growth in the fungi Botrytis cinerea, Fusarium oxysporum and Mycosphaerella arachidicola. It exerted an antiproliferative activity on leukemia L1210 cells. PMID:16945595

  4. Ubiquitin plays an atypical role in GPCR-induced p38 MAP kinase activation on endosomes.

    PubMed

    Grimsey, Neil J; Aguilar, Berenice; Smith, Thomas H; Le, Phillip; Soohoo, Amanda L; Puthenveedu, Manojkumar A; Nizet, Victor; Trejo, JoAnn

    2015-09-28

    Protease-activated receptor 1 (PAR1) is a G protein-coupled receptor (GPCR) for thrombin and promotes inflammatory responses through multiple pathways including p38 mitogen-activated protein kinase signaling. The mechanisms that govern PAR1-induced p38 activation remain unclear. Here, we define an atypical ubiquitin-dependent pathway for p38 activation used by PAR1 that regulates endothelial barrier permeability. Activated PAR1 K63-linked ubiquitination is mediated by the NEDD4-2 E3 ubiquitin ligase and initiated recruitment of transforming growth factor-β-activated protein kinase-1 binding protein-2 (TAB2). The ubiquitin-binding domain of TAB2 was essential for recruitment to PAR1-containing endosomes. TAB2 associated with TAB1, which induced p38 activation independent of MKK3 and MKK6. The P2Y1 purinergic GPCR also stimulated p38 activation via NEDD4-2-mediated ubiquitination and TAB1-TAB2. TAB1-TAB2-dependent p38 activation was critical for PAR1-promoted endothelial barrier permeability in vitro, and p38 signaling was required for PAR1-induced vascular leakage in vivo. These studies define an atypical ubiquitin-mediated signaling pathway used by a subset of GPCRs that regulates endosomal p38 signaling and endothelial barrier disruption.

  5. Ubiquitin plays an atypical role in GPCR-induced p38 MAP kinase activation on endosomes

    PubMed Central

    Grimsey, Neil J.; Aguilar, Berenice; Smith, Thomas H.; Le, Phillip; Soohoo, Amanda L.; Puthenveedu, Manojkumar A.; Nizet, Victor

    2015-01-01

    Protease-activated receptor 1 (PAR1) is a G protein–coupled receptor (GPCR) for thrombin and promotes inflammatory responses through multiple pathways including p38 mitogen-activated protein kinase signaling. The mechanisms that govern PAR1-induced p38 activation remain unclear. Here, we define an atypical ubiquitin-dependent pathway for p38 activation used by PAR1 that regulates endothelial barrier permeability. Activated PAR1 K63-linked ubiquitination is mediated by the NEDD4-2 E3 ubiquitin ligase and initiated recruitment of transforming growth factor-β–activated protein kinase-1 binding protein-2 (TAB2). The ubiquitin-binding domain of TAB2 was essential for recruitment to PAR1-containing endosomes. TAB2 associated with TAB1, which induced p38 activation independent of MKK3 and MKK6. The P2Y1 purinergic GPCR also stimulated p38 activation via NEDD4-2–mediated ubiquitination and TAB1–TAB2. TAB1–TAB2-dependent p38 activation was critical for PAR1-promoted endothelial barrier permeability in vitro, and p38 signaling was required for PAR1-induced vascular leakage in vivo. These studies define an atypical ubiquitin-mediated signaling pathway used by a subset of GPCRs that regulates endosomal p38 signaling and endothelial barrier disruption. PMID:26391660

  6. Mitogen-Activated Protein Kinase Phosphatase 2 Regulates the Inflammatory Response in Sepsis▿

    PubMed Central

    Cornell, Timothy T.; Rodenhouse, Paul; Cai, Qing; Sun, Lei; Shanley, Thomas P.

    2010-01-01

    Sepsis results from a dysregulation of the regulatory mechanisms of the pro- and anti-inflammatory response to invading pathogens. The mitogen-activated protein (MAP) kinase cascades are key signal transduction pathways involved in the cellular production of cytokines. The dual-specific phosphatase 1 (DUSP 1), mitogen-activated protein kinase phosphatase-1 (MKP-1), has been shown to be an important negative regulator of the inflammatory response by regulating the p38 and Jun N-terminal protein kinase (JNK) MAP kinase pathways to influence pro- and anti-inflammatory cytokine production. MKP-2, also a dual-specific phosphatase (DUSP 4), is a phosphatase highly homologous with MKP-1 and is known to regulate MAP kinase signaling; however, its role in regulating the inflammatory response is not known. We hypothesized a regulatory role for MKP-2 in the setting of sepsis. Mice lacking the MKP-2 gene had a survival advantage over wild-type mice when challenged with intraperitoneal lipopolysaccharide (LPS) or a polymicrobial infection via cecal ligation and puncture. The MKP-2−/− mice also exhibited decreased serum levels of both pro-inflammatory cytokines (tumor necrosis factor alpha [TNF-α], interleukin-1β [IL-1β], IL-6) and anti-inflammatory cytokines (IL-10) following endotoxin challenge. Isolated bone marrow-derived macrophages (BMDMs) from MKP-2−/− mice showed increased phosphorylation of the extracellular signal-regulated kinase (ERK), decreased phosphorylation of JNK and p38, and increased induction of MKP-1 following LPS stimulation. The capacity for cytokine production increased in MKP-2−/− BMDMs following MKP-1 knockdown. These data support a mechanism by which MKP-2 targets ERK deactivation, thereby decreasing MKP-1 and thus removing the negative inhibition of MKP-1 on cytokine production. PMID:20351138

  7. Mitogen-activated protein kinase cascades in Vitis vinifera

    PubMed Central

    Çakır, Birsen; Kılıçkaya, Ozan

    2015-01-01

    Protein phosphorylation is one of the most important mechanisms to control cellular functions in response to external and endogenous signals. Mitogen-activated protein kinases (MAPK) are universal signaling molecules in eukaryotes that mediate the intracellular transmission of extracellular signals resulting in the induction of appropriate cellular responses. MAPK cascades are composed of four protein kinase modules: MAPKKK kinases (MAPKKKKs), MAPKK kinases (MAPKKKs), MAPK kinases (MAPKKs), and MAPKs. In plants, MAPKs are activated in response to abiotic stresses, wounding, and hormones, and during plant pathogen interactions and cell division. In this report, we performed a complete inventory of MAPK cascades genes in Vitis vinifera, the whole genome of which has been sequenced. By comparison with MAPK, MAPK kinases, MAPK kinase kinases and MAPK kinase kinase kinase kinase members of Arabidopsis thaliana, we revealed the existence of 14 MAPKs, 5 MAPKKs, 62 MAPKKKs, and 7 MAPKKKKs in Vitis vinifera. We identified orthologs of V. vinifera putative MAPKs in different species, and ESTs corresponding to members of MAPK cascades in various tissues. This work represents the first complete inventory of MAPK cascades in V. vinifera and could help elucidate the biological and physiological functions of these proteins in V. vinifera. PMID:26257761

  8. Regulation of mitogen-activated protein kinases by a calcium/calmodulin-dependent protein kinase cascade.

    PubMed Central

    Enslen, H; Tokumitsu, H; Stork, P J; Davis, R J; Soderling, T R

    1996-01-01

    Membrane depolarization of NG108 cells gives rapid (< 5 min) activation of Ca2+/calmodulin-dependent protein kinase IV (CaM-KIV), as well as activation of c-Jun N-terminal kinase (JNK). To investigate whether the Ca2+-dependent activation of mitogen-activated protein kinases (ERK, JNK, and p38) might be mediated by the CaM kinase cascade, we have transfected PC12 cells, which lack CaM-KIV, with constitutively active mutants of CaM kinase kinase and/or CaM-KIV (CaM-KKc and CaM-KIVc, respectively). In the absence of depolarization, CaM-KKc transfection had no effect on Elk-dependent transcription of a luciferase reporter gene, whereas CaM-KIVc alone or in combination with CaM-KKc gave 7- to 10-fold and 60- to 80-fold stimulations, respectively, which were blocked by mitogen-activated protein (MAP) kinase phosphatase cotransfection. When epitope-tagged constructs of MAP kinases were co-transfected with CaM-KKc plus CaM-KIVc, the immunoprecipitated MAP kinases were activated 2-fold (ERK-2) and 7- to 10-fold (JNK-1 and p38). The JNK and p38 pathways were further investigated using specific c-Jun or ATF2-dependent transcriptional assays. We found that c-Jun/ATF2-dependent transcriptions were enhanced 7- to 10-fold by CaM-KIVc and 20- to 30-fold by CaM-KKc plus CaM-KIVc. In the case of the Jun-dependent transcription, this effect was not due to direct phosphorylation of c-Jun by activated CaM-KIV, since transcription was blocked by a dominant-negative JNK and by two MAP kinase phosphatases. Mutation of the phosphorylation site (Thr196) in CaM-KIV, which mediates its activation by CaM-KIV kinase, prevented activation of Elk-1, c-Jun, and ATF2 by the CaM kinase cascade. These results establish a new Ca2+-dependent mechanism for regulating MAP kinase pathways and resultant transcription. Images Fig. 1 Fig. 3 Fig. 4 PMID:8855261

  9. Tryptase Activation of Immortalized Human Urothelial Cell Mitogen-Activated Protein Kinase

    PubMed Central

    Marentette, John O.; Hauser, Paul J.; Hurst, Robert E.; Klumpp, David J.; Rickard, Alice; McHowat, Jane

    2013-01-01

    The pathogenesis of interstitial cystitis/painful bladder syndrome (IC/PBS) is multifactorial, but likely involves urothelial cell dysfunction and mast cell accumulation in the bladder wall. Activated mast cells in the bladder wall release several inflammatory mediators, including histamine and tryptase. We determined whether mitogen-activated protein (MAP) kinases are activated in response to tryptase stimulation of urothelial cells derived from human normal and IC/PBS bladders. Tryptase stimulation of normal urothelial cells resulted in a 2.5-fold increase in extracellular signal regulated kinase 1/2 (ERK 1/2). A 5.5-fold increase in ERK 1/2 activity was observed in urothelial cells isolated from IC/PBS bladders. No significant change in p38 MAP kinase was observed in tryptase-stimulated normal urothelial cells but a 2.5-fold increase was observed in cells isolated from IC/PBS bladders. Inhibition of ERK 1/2 with PD98059 or inhibition of p38 MAP kinase with SB203580 did not block tryptase-stimulated iPLA2 activation. Incubation with the membrane phospholipid-derived PLA2 hydrolysis product lysoplasmenylcholine increased ERK 1/2 activity, suggesting the iPLA2 activation is upstream of ERK 1/2. Real time measurements of impedance to evaluate wound healing of cell cultures indicated increased healing rates in normal and IC/PBS urothelial cells in the presence of tryptase, with inhibition of ERK 1/2 significantly decreasing the wound healing rate of IC/PBS urothelium. We conclude that activation of ERK 1/2 in response to tryptase stimulation may facilitate wound healing or cell motility in areas of inflammation in the bladder associated with IC/PBS. PMID:23922867

  10. Protein Kinase Cδ Mediates Neurogenic but Not Mitogenic Activation of Mitogen-Activated Protein Kinase in Neuronal Cells

    PubMed Central

    Corbit, Kevin C.; Foster, David A.; Rosner, Marsha Rich

    1999-01-01

    In several neuronal cell systems, fibroblast-derived growth factor (FGF) and nerve growth factor (NGF) act as neurogenic agents, whereas epidermal growth factor (EGF) acts as a mitogen. The mechanisms responsible for these different cellular fates are unclear. We report here that although FGF, NGF, and EGF all activate mitogen-activated protein (MAP) kinase (extracellular signal-related kinase [ERK]) in rat hippocampal (H19-7) and pheochromocytoma (PC12) cells, the activation of ERK by the neurogenic agents FGF and NGF is dependent upon protein kinase Cδ (PKCδ), whereas ERK activation in response to the mitogenic EGF is independent of PKCδ. Antisense PKCδ oligonucleotides or the PKCδ-specific inhibitor rottlerin inhibited FGF- and NGF-induced, but not EGF-induced, ERK activation. In contrast, EGF-induced ERK activation was inhibited by the phosphatidylinositol-3-kinase inhibitor wortmannin, which had no effect upon FGF-induced ERK activation. Rottlerin also inhibited the activation of MAP kinase kinase (MEK) in response to activated Raf, but had no effect upon c-Raf activity or ERK activation by activated MEK. These results indicate that PKCδ functions either downstream from or in parallel with c-Raf, but upstream of MEK. Inhibition of PKCδ also blocked neurite outgrowth induced by FGF and NGF in PC12 cells and by activated Raf in H19-7 cells, indicating a role for PKCδ in the neurogenic effects of FGF, NGF, and Raf. Interestingly, the PKCδ requirement is apparently cell type specific, since FGF-induced ERK activation was independent of PKCδ in NIH 3T3 murine fibroblasts, in which FGF is a mitogen. These data demonstrate that PKCδ contributes to growth factor specificity and response in neuronal cells and may also promote cell-type-specific differences in growth factor signaling. PMID:10330161

  11. Rapamycin induces mitogen-activated protein (MAP) kinase phosphatase-1 (MKP-1) expression through activation of protein kinase B and mitogen-activated protein kinase kinase pathways.

    PubMed

    Rastogi, Ruchi; Jiang, Zhongliang; Ahmad, Nisar; Rosati, Rita; Liu, Yusen; Beuret, Laurent; Monks, Robert; Charron, Jean; Birnbaum, Morris J; Samavati, Lobelia

    2013-11-22

    Mitogen-activated protein kinase phosphatase-1 (MKP-1), also known as dual specificity phosphatase-1 (DUSP-1), plays a crucial role in the deactivation of MAPKs. Several drugs with immune-suppressive properties modulate MKP-1 expression as part of their mechanism of action. We investigated the effect of mTOR inhibition through rapamycin and a dual mTOR inhibitor (AZD2014) on MKP-1 expression. Low dose rapamycin led to a rapid activation of both AKT and ERK pathways with a subsequent increase in MKP-1 expression. Rapamycin treatment led to phosphorylation of CREB, transcription factor 1 (ATF1), and ATF2, three transcription factors that bind to the cyclic AMP-responsive elements on the Mkp-1 promoter. Inhibition of either the MEK/ERK or the AKT pathway attenuated rapamycin-mediated MKP-1 induction. AZD2014 did not activate AKT but activated the ERK pathway, leading to a moderate MKP-1 induction. Using bone marrow-derived macrophages (BMDMs) derived from wild-type (WT) mice or mice deficient in AKT1 and AKT2 isoforms or BMDM from targeted deficiency in MEK1 and MEK2, we show that rapamycin treatment led to an increased MKP1 expression in BMDM from WT but failed to do so in BMDMs lacking the AKT1 isoform or MEK1 and MEK2. Importantly, rapamycin pretreatment inhibited LPS-mediated p38 activation and decreased nitric oxide and IL-6 production. Our work provides a conceptual framework for the observed immune modulatory effect of mTOR inhibition.

  12. Hydrogen peroxide activates activator protein-1 and mitogen-activated protein kinases in pancreatic stellate cells.

    PubMed

    Kikuta, Kazuhiro; Masamune, Atsushi; Satoh, Masahiro; Suzuki, Noriaki; Satoh, Kennichi; Shimosegawa, Tooru

    2006-10-01

    Activated pancreatic stellate cells (PSCs) are implicated in the pathogenesis of pancreatic inflammation and fibrosis, where oxidative stress is thought to play a key role. Reactive oxygen species such as hydrogen peroxide (H(2)O(2)) may act as a second messenger to mediate the actions of growth factors and cytokines. But the role of reactive oxygen species in the activation and regulation of cell functions in PSCs remains largely unknown. We here examined the effects of H(2)O(2) on the activation of signal transduction pathways and cell functions in PSCs. PSCs were isolated from the pancreas of male Wistar rats, and used in their culture-activated, myofibroblast-like phenotype unless otherwise stated. Activation of transcription factors was examined by electrophoretic mobility shift assay and luciferase assay. Activation of mitogen-activated protein (MAP) kinases was assessed by Western blotting using anti-phosphospecific antibodies. The effects of H(2)O(2) on proliferation, alpha(1)(I)procollagen gene expression, and monocyte chemoattractant protein-1 production were evaluated. The effect of H(2)O(2) on the transformation of freshly isolated PSCs in culture was also assessed. H(2)O(2) at non-cytotoxic concentrations (up to 100 microM) induced oxidative stress in PSCs. H(2)O(2) activated activator protein-1, but not nuclear factor kappaB. In addition, H(2)O(2) activated three classes of MAP kinases: extracellular signal-regulated kinase, c-Jun N-terminal kinase, and p38 MAP kinase. H(2)O(2) induced alpha(1)(I)procollagen gene expression but did not induce proliferation or monocyte chemoattractant protein-1 production. H(2)O(2) did not initiate the transformation of freshly isolated PSCs to myofibroblast-like phenotype. Specific activation of these signal transduction pathways and collagen gene expression by H(2)O(2) may play a role in the pathogenesis of pancreatic fibrosis.

  13. Context Specificity of Stress-activated Mitogen-activated Protein (MAP) Kinase Signaling: The Story as Told by Caenorhabditis elegans.

    PubMed

    Andrusiak, Matthew G; Jin, Yishi

    2016-04-01

    Stress-associated p38 and JNK mitogen-activated protein (MAP) kinase signaling cascades trigger specific cellular responses and are involved in multiple disease states. At the root of MAP kinase signaling complexity is the differential use of common components on a context-specific basis. The roundwormCaenorhabditis eleganswas developed as a system to study genes required for development and nervous system function. The powerful genetics ofC. elegansin combination with molecular and cellular dissections has led to a greater understanding of how p38 and JNK signaling affects many biological processes under normal and stress conditions. This review focuses on the studies revealing context specificity of different stress-activated MAPK components inC. elegans.

  14. Context Specificity of Stress-activated Mitogen-activated Protein (MAP) Kinase Signaling: The Story as Told by Caenorhabditis elegans.

    PubMed

    Andrusiak, Matthew G; Jin, Yishi

    2016-04-01

    Stress-associated p38 and JNK mitogen-activated protein (MAP) kinase signaling cascades trigger specific cellular responses and are involved in multiple disease states. At the root of MAP kinase signaling complexity is the differential use of common components on a context-specific basis. The roundwormCaenorhabditis eleganswas developed as a system to study genes required for development and nervous system function. The powerful genetics ofC. elegansin combination with molecular and cellular dissections has led to a greater understanding of how p38 and JNK signaling affects many biological processes under normal and stress conditions. This review focuses on the studies revealing context specificity of different stress-activated MAPK components inC. elegans. PMID:26907690

  15. Baicalin attenuates acute myocardial infarction of rats via mediating the mitogen-activated protein kinase pathway.

    PubMed

    Liu, Xiaobing; Gu, Jianmin; Fan, Yuqi; Shi, Huihua; Jiang, Mier

    2013-01-01

    Baicalin is a bioactive ingredient from the herb and has possessed various pharmacological actions. The present study was performed to evaluate the cardioprotective potential of baicalin against myocardial infarction and explore the potential mechanism. Baicalin was intraperitoneally injected into the rats by the doses of 50, 100 and 200 mg/kg, respectively, once a day for 7 d and, 30 min after the last administration, the left coronary artery was ligated. Infarct size was measured to analyze the myocardial damage. Myocardial specific enzymes, including creatine kinase (CK), the MB isoenzyme of creatine kinase (CK-MB), lactate dehydrogenase (LDH) and cardiac troponin T (cTnT) were determined with the colorimetric method. Evidence for myocardial apoptosis was detected by caspase-3 activity measurement and Western blot analysis. We also examined the protein levels of three major subgroups of mitogen-activated protein kinases (MAPKs), namely, extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38 by immuoblotting. Our results indicated that baicalin significantly reduced the infarct size and myocardial enzymes (CK, CK-MB, LDH and cTnT). Administration of baicalin also suppressed the activity and protein expression of caspase-3. Moreover, the protein level of phosphorylated ERK (p-ERK) was found to be evidently augmented while the phosphorylated JNK (p-JNK) and phosphorylated p38 (p-p38) were strikingly diminished in infarcted rats with baicalin treatment. These findings suggest that the baicalin's cardioprotection associates with mediation of MAPK cascades in acute myocardial infarction of rats.

  16. Wounding activates p38 map kinase and activation transcription factor 3 in leading keratinocytes.

    PubMed

    Harper, Erin G; Alvares, Stacy M; Carter, William G

    2005-08-01

    Quiescent epidermis anchors to laminin 5 in the basement membrane via integrin alpha6beta4. Wounding elevates expression of laminin 5, generating leading keratinocytes (LKs) that migrate via beta1 integrins. Laminin 5 was evaluated as a regulator of cell signaling, and mRNA and protein expression in LKs. An in vitro wound model was developed based on suspension and re-adhesion of quiescent human keratinocytes (HKs). DNA microarrays identified multiple mRNAs elevated 1.5 hours after suspension and re-adhesion including activation transcription factor 3 (ATF3). In vitro and in vivo, levels of ATF3 protein elevate in nuclei of LKs, but not in nuclei of the following cells, 2 hours after suspension or wounding but decline by 12-18 hours post injury. Significantly, null defects in laminin 5 or integrin beta4 that inhibit anchorage chronically elevate ATF3 in vivo. This suggests that adhesion to laminin 5, but not other ligands, suppresses activation. On suspension, ATF3 and other transcripts in the microarrays are elevated by phosphorylated p38 mitogen-activated protein kinase (P-p38), a stress kinase that regulates mRNA and cell motility. Inhibition of P-p38 with SB203580 prevents phosphorylation of ATF2, a transcription factor for ATF3 in LKs. Re-adhesion to laminin 5 via alpha6beta4 dephosphorylates P-p38 and suppresses ATF3 protein relative to cells in suspension. Thus, wounding of quiescent HKs disrupts laminin 5 adhesion to activate p38, generating mRNA transcripts that define LKs. Adhesion to deposits of laminin 5 via alpha6beta4 suppresses P-p38 and activation mRNAs including ATF3. Defects in laminin 5 and alpha6beta4 sustain P-p38 with probable pathological effects on transcription and migration.

  17. Inhibition of respiratory syncytial virus replication and virus-induced p38 kinase activity by berberine.

    PubMed

    Shin, Han-Bo; Choi, Myung-Soo; Yi, Chae-Min; Lee, Jun; Kim, Nam-Jung; Inn, Kyung-Soo

    2015-07-01

    Respiratory syncytial virus (RSV) causes severe lower respiratory tract infection and poses a major public health threat worldwide. No effective vaccines or therapeutics are currently available; berberine, an isoquinoline alkaloid from various medicinal plants, has been shown to exert antiviral and several other biological effects. Recent studies have shown that p38 mitogen-activated protein kinase (MAPK) activity is implicated in infection by and replication of viruses such as RSV and the influenza virus. Because berberine has previously been implicated in modulating the activity of p38 MAPK, its effects on RSV infection and RSV-mediated p38 MAPK activation were examined. Replication of RSV in epithelial cells was significantly reduced by treatment with berberine. Berberine treatment caused decrease in viral protein and mRNA syntheses. Similar to previously reported findings, RSV infection caused phosphorylation of p38 MAPK at a very early time point of infection, and phosphorylation was dramatically reduced by berberine treatment. In addition, production of interleukin-6 mRNA upon RSV infection was significantly suppressed by treatment with berberine, suggesting the anti-inflammatory role of berberine during RSV infection. Taken together, we showed that berberine, a natural compound already proven to be safe for human consumption, suppresses the replication of RSV. In addition, the current study suggests that inhibition of RSV-mediated early p38 MAPK activation, which has been implicated as an early step in viral infection, as a potential molecular mechanism.

  18. Crosstalk between mitogen-activated protein kinases and mitochondria in cardiac diseases: therapeutic perspectives

    PubMed Central

    Javadov, Sabzali; Jang, Sehwan; Agostini, Bryan

    2014-01-01

    Cardiovascular diseases cause more mortality and morbidity worldwide than any other diseases. Although many intracellular signaling pathways influence cardiac physiology and pathology, the mitogen-activated protein kinase (MAPK) family has garnered significant attention because of its vast implications in signaling and cross-talk with other signaling networks. The extensively studied MAPKs ERK1/2, p38, JNK, and ERK5, demonstrate unique intracellular signaling mechanisms, responding to a myriad of mitogens and stressors and influencing the signaling of cardiac development, metabolism, performance, and pathogenesis. Definitive relationships between MAPK signaling and cardiac dysfunction remain elusive, despite 30 years of extensive clinical studies and basic research of various animal/cell models, severities of stress, and types of stimuli. Still, several studies have proven the importance of MAPK cross-talk with mitochondria, powerhouses of the cell that provide over 80% of ATP for normal cardiomyocyte function and play a crucial role in cell death. Although many questions remain unanswered, there exists enough evidence to consider the possibility of targeting MAPK-mitochondria interactions in the prevention and treatment of heart disease. The goal of this review is to integrate previous studies into a discussion of MAPKs and MAPK-mitochondria signaling in cardiac diseases, such as myocardial infarction (ischemia), hypertrophy and heart failure. A comprehensive understanding of relevant molecular mechanisms, as well as challenges for studies in this area, will facilitate the development of new pharmacological agents and genetic manipulations for therapy of cardiovascular diseases. PMID:24924700

  19. Crosstalk between mitogen-activated protein kinases and mitochondria in cardiac diseases: therapeutic perspectives.

    PubMed

    Javadov, Sabzali; Jang, Sehwan; Agostini, Bryan

    2014-11-01

    Cardiovascular diseases cause more mortality and morbidity worldwide than any other diseases. Although many intracellular signaling pathways influence cardiac physiology and pathology, the mitogen-activated protein kinase (MAPK) family has garnered significant attention because of its vast implications in signaling and crosstalk with other signaling networks. The extensively studied MAPKs ERK1/2, p38, JNK, and ERK5, demonstrate unique intracellular signaling mechanisms, responding to a myriad of mitogens and stressors and influencing the signaling of cardiac development, metabolism, performance, and pathogenesis. Definitive relationships between MAPK signaling and cardiac dysfunction remain elusive, despite 30 years of extensive clinical studies and basic research of various animal/cell models, severities of stress, and types of stimuli. Still, several studies have proven the importance of MAPK crosstalk with mitochondria, powerhouses of the cell that provide over 80% of ATP for normal cardiomyocyte function and play a crucial role in cell death. Although many questions remain unanswered, there exists enough evidence to consider the possibility of targeting MAPK-mitochondria interactions in the prevention and treatment of heart disease. The goal of this review is to integrate previous studies into a discussion of MAPKs and MAPK-mitochondria signaling in cardiac diseases, such as myocardial infarction (ischemia), hypertrophy and heart failure. A comprehensive understanding of relevant molecular mechanisms, as well as challenges for studies in this area, will facilitate the development of new pharmacological agents and genetic manipulations for therapy of cardiovascular diseases.

  20. Chemerin Stimulates Vascular Smooth Muscle Cell Proliferation and Carotid Neointimal Hyperplasia by Activating Mitogen-Activated Protein Kinase Signaling

    PubMed Central

    Xiong, Wei; Luo, Yu; Wu, Lin; Liu, Feng; Liu, Huadong; Li, Jianghua; Liao, Bihong; Dong, Shaohong

    2016-01-01

    Vascular neointimal hyperplasia and remodeling arising from local inflammation are characteristic pathogeneses of proliferative cardiovascular diseases, such as atherosclerosis and post angioplasty restenosis. The molecular mechanisms behind these pathological processes have not been fully determined. The adipokine chemerin is associated with obesity, metabolism, and control of inflammation. Recently, chemerin has gained increased attention as it was found to play a critical role in the development of cardiovascular diseases. In this study, we investigated the effects of chemerin on the regulation of vascular smooth muscle cells and carotid neointimal formation after angioplasty. We found that circulating chemerin levels increased after carotid balloon injury, and that knockdown of chemerin significantly inhibited the proliferative aspects of vascular smooth muscle cells induced by platelet-derived growth factor-BB and pro-inflammatory chemokines in vitro as well as prohibited carotid neointimal hyperplasia and pro-inflammatory chemokines in vivo after angioplasty. Additionally, inhibition of chemerin down-regulated the expression of several proteins, including phosphorylated p38 mitogen-activated protein kinase, phosphorylated extracellular signal regulated kinase 1/2, nuclear factor-kappa B p65, and proliferation cell nuclear antigen. The novel finding of this study is that chemerin stimulated vascular smooth muscle cells proliferation and carotid intimal hyperplasia through activation of the mitogen-activated protein kinase signaling pathway, which may lead to vascular inflammation and remodeling, and is relevant to proliferative cardiovascular diseases. PMID:27792753

  1. Roles of mitogen activated protein kinases and EGF receptor in arsenite-stimulated matrix metalloproteinase-9 production

    SciTech Connect

    Cooper, Karen L.; Myers, Terrance Alix; Rosenberg, Martina; Chavez, Miquella; Hudson, Laurie G. . E-mail: lghudson@unm.edu

    2004-11-01

    The dermatotoxicity of arsenic is well established and epidemiological studies identify an increased incidence of keratinocytic tumors (basal cell and squamous cell carcinoma) associated with arsenic exposure. Little is known about the underlying mechanisms of arsenic-mediated skin carcinogenesis, but activation of mitogen-activated protein (MAP) kinases and subsequent regulation of downstream target genes may contribute to tumor promotion and progression. In this study, we investigated activation of the extracellular signal regulated kinase (ERK) and the stress-associated kinase p38 by arsenite in HaCat cells, a spontaneously immortalized human keratinocyte cell line. Arsenite concentrations {>=}100 {mu}M stimulate rapid activation of p38 and ERK MAP kinases. However, upon extended exposure (24 h), persistent stimulation of p38 and ERK MAP kinases was detected at low micromolar concentrations of arsenite. Although ERK and p38 were activated with similar time and concentration dependence, the mechanism of activation differed for these two MAP kinases. ERK activation by arsenite was fully dependent on the catalytic activity of the epidermal growth factor (EGF) receptor and partially dependent on Src-family kinase activity. In contrast, p38 activation was independent of EGF receptor or Src-family kinase activity. Arsenite-stimulated MAP kinase signal transduction resulted in increased production of matrix metalloproteinase (MMP)-9, an AP-1 regulated gene product. MMP-9 induction by arsenite was prevented when EGF receptor or MAP kinase signaling was inhibited. These studies indicate that EGF receptor activation is a component of arsenite-mediated signal transduction and gene expression in keratinocytes and that low micromolar concentrations of arsenite stimulate key signaling pathways upon extended exposure. Stimulation of MAP kinase cascades by arsenic and subsequent regulation of genes including c-fos, c-jun, and the matrix degrading proteases may play an important

  2. p38 MAP Kinase Links CAR Activation and Inactivation in the Nucleus via Phosphorylation at Threonine 38

    PubMed Central

    Hori, Takeshi; Moore, Rick

    2016-01-01

    Nuclear receptor constitutive androstane receptor (CAR, NR1I3), which regulates hepatic drug and energy metabolisms as well as cell growth and death, is sequestered in the cytoplasm as its inactive form phosphorylated at threonine 38. CAR activators elicit dephosphorylation, and nonphosphorylated CAR translocates into the nucleus to activate its target genes. CAR was previously found to require p38 mitogen-activated protein kinase (MAPK) to transactivate the cytochrome P450 2B (CYP2B) genes. Here we have demonstrated that p38 MAPK forms a complex with CAR, enables it to bind to the response sequence, phenobarbital-responsive enhancer module (PBREM), within the CYP2B promoter, and thus recruits RNA polymerase II to activate transcription. Subsequently, p38 MAPK elicited rephosphorylation of threonine 38 to inactivate CAR and exclude it from the nucleus. Thus, nuclear p38 MAPK exerted dual regulation by sequentially activating and inactivating CAR-mediated transcription through phosphorylation of threonine 38. PMID:27074912

  3. Mitogen-activated protein kinases: a new therapeutic target in cardiac pathology.

    PubMed

    Ravingerová, Tána; Barancík, Miroslav; Strnisková, Monika

    2003-05-01

    Eukaryotic cells respond to different external stimuli by activation of mechanisms of cell signaling. One of the major systems participating in the transduction of signal from the cell membrane to nuclear and other intracellular targets is the highly conserved mitogen-activated protein kinase (MAPK) superfamily. The members of MAPK family are involved in the regulation of a large variety of cellular processes such as cell growth, differentiation, development, cell cycle, death and survival. Several MAPK subfamilies, each with apparently unique signaling pathway, have been identified in the mammalian myocardium. These cascades differ in their upstream activation sequence and in downstream substrate specifity. Each pathway follows the same conserved three-kinase module consisting of MAPK, MAPK kinase (MAPKK, MKK or MEK), and MAPK kinase kinase (MAPKKK, MEKK). The major groups of MAPKs found in cardiac tissue include the extracellular signal-regulated kinases (ERKs), the stress-activated/c-Jun NH2-terminal kinases (SAPK/JNKs), p38-MAPK, and ERK5/big MAPK 1 (BMK1). The ERKs are strongly activated by mitogenic and growth factors and by physical stress, whereas SAPK/JNKs and p38-MAPK can be activated by various cell stresses, such as hyperosmotic shock, metabolic stress or protein synthesis inhibitors, UV radiation, heat shock, cytokines, and ischemia. Activation of MAPKs family plays a key role in the pathogenesis of various processes in the heart, e.g. myocardial hypertrophy and its transition to heart failure, in ischemic and reperfusion injury, as well in the cardioprotection conferred by ischemia- or pharmacologically-induced preconditioning. The following approaches are currently utilized to elucidate the role of MAPKs in the myocardium: (i) studies of the effects of myocardial processes on the activity of these kinases; (ii) pharmacological modulations of MAPKs activity and evaluation of their impact on the (patho)physiological processes in the heart; (iii) gene

  4. Centrifugal force induces human ligamentum flavum fibroblasts inflammation through activation of JNK and p38 pathways.

    PubMed

    Chao, Yuan-Hung; Tsuang, Yang-Hwei; Sun, Jui-Sheng; Sun, Man-Ger; Chen, Ming-Hong

    2012-01-01

    Inflammation has been proposed to be an important causative factor in ligamentum flavum hypertrophy. However, the mechanisms of mechanical load on inflammation of ligamentum flavum remain unclear. In this study, we used an in vitro model of human ligamentum flavum fibroblasts subjected to centrifugal force to elucidate the effects of mechanical load on cultured human ligamentum flavum fibroblasts; we further studied its molecular and biochemical mechanisms. Human ligamentum flavum fibroblasts were obtained from six patients undergoing lumbar spine surgery. Monolayer cultures of human ligamentum flavum fibroblasts were subjected to different magnitudes of centrifugal forces. Cell viability, cell death, biochemical response, and molecular response to centrifugal forces were analyzed. It was found that centrifugal stress significantly suppressed cell viability without inducing cell death. Centrifugal force at 67.1 g/cm(2) for 60 min significantly increases the production of prostaglandin E2 and nitric oxide as well as gene expression of proinflammatory cytokines, including interleukin (IL)-1α, IL-1β and IL-6, showed that centrifugal force-dependent induction of cyclooxygense-2 and inducible NO synthase required JNK and p38 mitogen-activated protein kinase, but not ERK 1/2 activities. This study suggested that centrifugal force does induce inflammatory responses in human ligamentum flavum fibroblasts. The activation of both JNK and p38 mitogen-activated protein kinase mechanotransduction cascades is a crucial intracellular mechanism that mediates cyclooxygense-2/prostaglandin E2 and inducible NO synthase/nitric oxide production.

  5. Phosphorylation of Mitogen-Activated Protein Kinases Contributes to Interferon γ Production in Response to Mycobacterium tuberculosis

    PubMed Central

    Pasquinelli, Virginia; Rovetta, Ana I.; Alvarez, Ivana B.; Jurado, Javier O.; Musella, Rosa M.; Palmero, Domingo J.; Malbrán, Alejandro; Samten, Buka; Barnes, Peter F.; García, Verónica E.

    2013-01-01

    Immune control of Mycobacterium tuberculosis depends on interferon γ (IFN-γ)–producing CD4+ lymphocytes. Previous studies have shown that T cells from patients with tuberculosis produce less IFN-γ, compared with healthy donors, in response to mycobacterial antigens, although IFN-γ responses to mitogens are preserved. In this work, we found that M. tuberculosis–induced IFN-γ production by human T cells correlated with phosphorylation of the mitogen-activated protein kinases (MAPKs), extracellular signal-regulated kinase (ERK), and p38. Moreover, the majority of IFN-γ–producing T cells expressed signaling lymphocyte activation molecule (SLAM), and SLAM activation further increased ERK phosphorylation. Interestingly, patients with tuberculosis had delayed activation of ERK and p38, and this was most marked in patients with the poorest IFN-γ responses (ie, low responders). Besides, SLAM signaling failed to phosphorylate ERK in low responders. Our findings suggest that activation of p38 and ERK, in part through SLAM, mediates T-cell IFN-γ production in response to M. tuberculosis, a pathway that is defective in patients with tuberculosis. PMID:23125442

  6. Involvement of the mitogen-activated protein kinase pathway in soft-shelled turtle iridovirus-induced apoptosis.

    PubMed

    Huang, Youhua; Huang, Xiaohong; Cai, Jia; Ye, Fuzhou; Qin, Qiwei

    2011-06-01

    Iridoviruses are large DNA viruses that infect invertebrates and poikilothermic vertebrates, and result in significant economic losses in aquaculture production, and drastic declines in amphibian populations. Soft-shelled turtle iridovirus (STIV) is the causative agent of severe systemic diseases in farm-raised soft-shelled turtles (Trionyx sinensis). In the present study, the mechanisms of STIV-induced cell death and the roles of the mitogen-activated protein kinase (MAPK) signaling pathway were investigated. STIV infection evoked typical apoptosis in fish cells, as demonstrated by the formation of apoptotic bodies, positive terminal deoxynucleotidyl transferase-mediated nicked-end labeling, and caspase-3 activation. The translocation of cytochrome c from mitochondria to cytoplasm, and caspase-9 activation suggested that a mitochondria-mediated pathway was involved in STIV-induced apoptosis. Moreover, MAPK pathways, including extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38 MAPK signaling were activated during STIV infection. Using specific inhibitors, we found that MAPK signaling molecules, including ERK, JNK and p38 MAPK, were important for virus release, whereas, only ERK and p38 MAPK were involved in STIV-induced apoptosis by modulating caspase-3 activity. Taken together, our findings shed light on the roles of the MAPK signaling pathway in iridovirus-induced apoptosis and virus replication, which provides new insights into understanding iridovirus-host interaction.

  7. Conformation-selective ATP-competitive inhibitors control regulatory interactions and noncatalytic functions of mitogen-activated protein kinases.

    PubMed

    Hari, Sanjay B; Merritt, Ethan A; Maly, Dustin J

    2014-05-22

    Most potent protein kinase inhibitors act by competing with ATP to block the phosphotransferase activity of their targets. However, emerging evidence demonstrates that ATP-competitive inhibitors can affect kinase interactions and functions in ways beyond blocking catalytic activity. Here, we show that stabilizing alternative ATP-binding site conformations of the mitogen-activated protein kinases (MAPKs) p38α and Erk2 with ATP-competitive inhibitors differentially, and in some cases divergently, modulates the abilities of these kinases to interact with upstream activators and deactivating phosphatases. Conformation-selective ligands are also able to modulate Erk2's ability to allosterically activate the MAPK phosphatase DUSP6, highlighting how ATP-competitive ligands can control noncatalytic kinase functions. Overall, these studies underscore the relationship between the ATP-binding and regulatory sites of MAPKs and provide insight into how ATP-competitive ligands can be designed to confer graded control over protein kinase function.

  8. Constitutive activation of p38 MAPK in tumor cells contributes to osteolytic bone lesions in multiple myeloma.

    PubMed

    Yang, J; He, J; Wang, J; Cao, Y; Ling, J; Qian, J; Lu, Y; Li, H; Zheng, Y; Lan, Y; Hong, S; Matthews, J; Starbuck, M W; Navone, N M; Orlowski, R Z; Lin, P; Kwak, L W; Yi, Q

    2012-09-01

    Bone destruction is a hallmark of multiple myeloma and affects more than 80% of patients. However, current therapy is unable to completely cure and/or prevent bone lesions. Although it is accepted that myeloma cells mediate bone destruction by inhibition of osteoblasts and activation of osteoclasts, the underlying mechanism is still poorly understood. This study demonstrates that constitutive activation of p38 mitogen-activated protein kinase in myeloma cells is responsible for myeloma-induced osteolysis. Our results show that p38 is constitutively activated in most myeloma cell lines and primary myeloma cells from patients. Myeloma cells with high/detectable p38 activity, but not those with low/undetectable p38 activity, injected into severe combined immunodeficient (SCID) or SCID-hu mice caused bone destruction. Inhibition or knockdown of p38 in human myeloma reduced or prevented myeloma-induced osteolytic bone lesions without affecting tumor growth, survival, or homing to bone. Mechanistic studies showed that myeloma cell p38 activity inhibited osteoblastogenesis and bone formation and activated osteoclastogenesis and bone resorption in myeloma-bearing SCID mice. This study elucidates a novel molecular mechanism-activation of p38 signaling in myeloma cells-by which myeloma cells induce osteolytic bone lesions, and indicates that targeting myeloma cell p38 may be a viable approach to treating or preventing myeloma bone disease. PMID:22425892

  9. Inhibitory effect of panduratin A on UV-induced activation of mitogen-activated protein kinases (MAPKs) in dermal fibroblast cells.

    PubMed

    Shim, Jae-Seok; Kwon, Yi-Young; Han, Young-Sun; Hwang, Jae-Kwan

    2008-10-01

    Exposure of the skin to ultraviolet (UV) induces photoaging associated with up-regulated matrix metalloproteinases (MMPs) activities and decreased collagen synthesis. We previously reported that panduratin A, a chalcone compound isolated from KAEMPFERIA PANDURATA Roxb ., decreased MMP-1 expression in UV-irradiated human skin fibroblasts. Here, we have investigated the effect of panduratin A on UV-induced activation of mitogen-activated protein kinases (MAPKs) signaling modules such as extracellular-regulated protein kinase (ERK), Jun-N-terminal kinase (JNK) and p38 kinase. Treatment with panduratin A in the range of 0.001 - 0.1 microM significantly inhibited UV-induced ERK, JNK and p38 activation. Moreover, inhibition of ERK, JNK and p38 by panduratin A resulted in decreased c-Fos expression and c-Jun phosphorylation induced by UV, which led to inhibition of activator protein-1 (AP-1) DNA binding activity. Panduratin A showed stronger activity than epigallocatechin 3- O-gallate (EGCG) known as a natural anti-aging agent. The results suggest that panduratin A can down-regulate UV-induced MMP-1 expression by inhibiting the MAPKs pathways and AP-1 activation. AP-1:activator protein-1 EGCG:epigallocatechin 3- O-gallate ERK:extracellular-regulated protein kinase JNK:c-Jun N-terminal kinase MAPK:mitogen-activated protein kinase MMP:matrix metalloproteinase UV:ultraviolet.

  10. Constitutive activation of p38 MAPK in tumor cells contributes to osteolytic bone lesions in multiple myeloma

    PubMed Central

    Yang, Jing; He, Jin; Wang, Ji; Cao, Yabing; Ling, Jianhua; Qian, Jianfei; Lu, Yong; Li, Haiyan; Zheng, Yuhuan; Lan, Yongsheng; Hong, Sungyoul; Matthews, Jairo; Starbuck, Michael W; Navone, Nora M; Orlowski, Robert Z.; Lin, Pei; Kwak, Larry W.; Yi, Qing

    2012-01-01

    Bone destruction is a hallmark of multiple myeloma and affects more than 80% of patients. However, current therapy is unable to completely cure and/or prevent bone lesions. Although it is accepted that myeloma cells mediate bone destruction by inhibition of osteoblasts and activation of osteoclasts, the underlying mechanism is still poorly understood. This study demonstrates that constitutive activation of p38 mitogen-activated protein kinase in myeloma cells is responsible for myeloma-induced osteolysis. Our results show that p38 is constitutively activated in most myeloma cell lines and primary myeloma cells from patients. Myeloma cells with high/detectable p38 activity, but not those with low/undetectable p38 activity, injected into SCID or SCID-hu mice caused bone destruction. Inhibition or knockdown of p38 in human myeloma reduced or prevented myeloma-induced osteolytic bone lesions without affecting tumor growth, survival, or homing to bone. Mechanistic studies showed that myeloma cell p38 activity inhibited osteoblastogenesis and bone formation and activated osteoclastogenesis and bone resorption in myeloma-bearing SCID mice. This study elucidates a novel molecular mechanism—sactivation of p38 signaling in myeloma cells—by which myeloma cells induce osteolytic bone lesions and indicates that targeting myeloma cell p38 may be a viable approach to treating or preventing myeloma bone disease. PMID:22425892

  11. Differential activation by cytokines of mitogen-activated protein kinases in bovine temporomandibular-joint disc cells.

    PubMed

    Landesberg, R; Takeuchi, E; Puzas, J E

    1999-01-01

    Temporomandibular disorders affect a significant proportion of the population. While their aetiology is not well defined, recent histological studies suggest that the majority are similar to the osteoarthritis seen in other joints. Inflammatory cytokines such as interleukin-1 and tumour necrosis factor-alpha appear to be important in the cascade of events leading to joint destruction in osteoarthritis. Here, cells from the disc of bovine temporomandibular joint were used to examine the response to various cytokines in vitro. Disc cells were stimulated with interleukin-1alpha, tumour necrosis factor-alpha, transforming growth factor-beta, platelet-derived growth factor, and basic fibroblast growth factor. Their effects were monitored by assessing the phosphorylation of selected signal-transduction intermediates using western blot. Mitogen-activated protein kinases (Erk 1, Erk 2) were rapidly phosphorylated by exposure to basic fibroblast growth factor, platelet-derived growth factor, and tumour necrosis factor-alpha, while interleukin-1alpha showed a weak response. Transforming growth factor-beta failed to activate these kinases. Examination of the effect of these cytokines on p38 (an intermediate in the stress-activated protein-kinase pathway) showed an increase in phosphorylated p38 when stimulated with tumour necrosis factor-alpha and interleukin-1alpha. The amounts of phosphorylated signal transducer and activator of transcription-3 did not significantly increase when the cells were exposed to any of the cytokines. PMID:10075149

  12. Adult-onset hyperthyroidism impairs spatial learning: possible involvement of mitogen-activated protein kinase signaling pathways.

    PubMed

    Bitiktaş, Soner; Kandemir, Başak; Tan, Burak; Kavraal, Şehrazat; Liman, Narin; Dursun, Nurcan; Dönmez-Altuntaş, Hamiyet; Aksan-Kurnaz, Işil; Suer, Cem

    2016-08-01

    Given evidence that mitogen-activated protein kinase (MAPK) activation is part of the nongenomic actions of thyroid hormones, we investigated the possible consequences of hyperthyroidism for the cognitive functioning of adult rats. Young adult rats were treated with L-thyroxine or saline. Twenty rats in each group were exposed to Morris water maze testing, measuring their performance in a hidden-platform spatial task. In a separate set of rats not exposed to Morris water maze testing (untrained rats), the expression and phosphorylated levels of p38-MAPK and of its two downstream effectors, Elk-1 and cAMP response element-binding protein, were evaluated using quantitative reverse transcriptase-PCR and western blotting. Rats with hyperthyroidism showed delayed acquisition of learning compared with their wild-type counterparts, as shown by increased escape latencies and distance moved on the last two trials of daily training in the water maze. The hyperthyroid rats, however, showed no difference during probe trials. Western blot analyses of the hippocampus showed that hyperthyroidism increased phosphorylated p38-MAPK levels in untrained rats. Although our study is correlative in nature and does not exclude the contribution of other molecular targets, our findings suggest that the observed impairments in acquisition during actual learning in rats with hyperthyroidism may result from the increased phosphorylation of p38-MAPK. PMID:27258653

  13. Transcriptional activation of MMP-13 by periodontal pathogenic LPS requires p38 MAP kinase.

    PubMed

    Rossa, Carlos; Liu, Min; Bronson, Paul; Kirkwood, Keith L

    2007-01-01

    Matrix metalloprotease-13 (MMP-13) is induced by pro-inflammatory cytokines and increased expression is associated with a number of pathological conditions such as tumor metastasis, osteoarthritis, rheumatoid arthritis and periodontal diseases. MMP-13 gene regulation and the signal transduction pathways activated in response to bacterial LPS are largely unknown. In these studies, the role of the mitogen-activated protein kinase (MAPK) pathways in the regulation of MMP-13 induced by lipopolysaccharide was investigated. Lipopolysaccharide from Escherichia coli and Actinobacillus actinomycetemcomitans significantly (P < 0.05) increased MMP-13 steady-state mRNA (average of 27% and 46% increase, respectively) in murine periodontal ligament fibroblasts. MMP-13 mRNA induction was significantly reduced by inhibition of p38 MAP kinase. Immunoblot analysis indicated that p38 signaling was required for LPS-induced MMP-13 expression. Lipopolysaccharide induced proximal promoter reporter (-660/+32 mMMP-13) gene activity required p38 signaling. Collectively, these results indicate that lipopolysaccharide-induced murine MMP-13 is regulated by p38 signaling through a transcriptional mechanism.

  14. Hesperetin exerts apoptotic effect on A431 skin carcinoma cells by regulating mitogen activated protein kinases and cyclins.

    PubMed

    Smina, T P; Mohan, A; Ayyappa, K A; Sethuraman, S; Krishnan, U M

    2015-10-30

    Dietary agents and phytochemicals have been utilised for the management of cancer for many years. Hesperetin, a dietary flavonoid found abundantly in citrus fruits, was evaluated for its cytotoxic and pro-apoptotic activities in A431 human skin carcinoma cells. Effect of hesperetin in regulating MAPK (Mitogen-Activated Protein Kinase) signalling pathway and levels of various cyclins and other downstream apoptotic proteins were investigated. Its critical role in regulating other apoptotic proteins especially p21, Bcl-2 and Bax were also assessed. Hesperetin stimulated alterations in MAPK (Mitogen-Activated Protein Kinase) signalling pathway by modulating the expression levels of ERK (Extracellular signal Regulated Kinase), JNK (c-Jun NH2-terminal Kinase) and p38; thereby induced apoptosis in A431 cells. Hesperetin regulated the levels of cyclin A2, B1, D1, D3 and E1. It also modulated the levels of various proteins involved in apoptotic pathway especially p21, Bcl-2 and Bax. The study revealed the efficiency of hesperetin against human skin carcinoma cells and proposed its mechanism of action; there by opens up new avenues for the use of this dietary flavonoid against skin malignancies.

  15. Social interaction reward decreases p38 activation in the nucleus accumbens shell of rats.

    PubMed

    Salti, Ahmad; Kummer, Kai K; Sadangi, Chinmaya; Dechant, Georg; Saria, Alois; El Rawas, Rana

    2015-12-01

    We have previously shown that animals acquired robust conditioned place preference (CPP) to either social interaction alone or cocaine alone. Recently it has been reported that drugs of abuse abnormally activated p38, a member of mitogen-activated protein kinase family, in the nucleus accumbens. In this study, we aimed to investigate the expression of the activated form of p38 (pp38) in the nucleus accumbens shell and core of rats expressing either cocaine CPP or social interaction CPP 1 h, 2 h and 24 h after the CPP test. We hypothesized that cocaine CPP will increase pp38 in the nucleus accumbens shell/core as compared to social interaction CPP. Surprisingly, we found that 24 h after social interaction CPP, pp38 neuronal levels were decreased in the nucleus accumbens shell to the level of naïve rats. Control saline rats that received saline in both compartments of the CPP apparatus and cocaine CPP rats showed similar enhanced p38 activation as compared to naïve and social interaction CPP rats. We also found that the percentage of neurons expressing dopaminergic receptor D2R and pp38 was also decreased in the shell of the nucleus accumbens of social interaction CPP rats as compared to controls. Given the emerging role of p38 in stress/anxiety behaviors, these results suggest that (1) social interaction reward has anti-stress effects; (2) cocaine conditioning per se does not affect p38 activation and that (3) marginal stress is sufficient to induce p38 activation in the shell of the nucleus accumbens.

  16. Social interaction reward decreases p38 activation in the nucleus accumbens shell of rats

    PubMed Central

    Salti, Ahmad; Kummer, Kai K.; Sadangi, Chinmaya; Dechant, Georg; Saria, Alois; El Rawas, Rana

    2016-01-01

    We have previously shown that animals acquired robust conditioned place preference (CPP) to either social interaction alone or cocaine alone. Recently it has been reported that drugs of abuse abnormally activated p38, a member of mitogen-activated protein kinase family, in the nucleus accumbens. In this study, we aimed to investigate the expression of the activated form of p38 (pp38) in the nucleus accumbens shell and core of rats expressing either cocaine CPP or social interaction CPP 1 h, 2 h and 24 h after the CPP test. We hypothesized that cocaine CPP will increase pp38 in the nucleus accumbens shell/core as compared to social interaction CPP. Surprisingly, we found that 24 h after social interaction CPP, pp38 neuronal levels were decreased in the nucleus accumbens shell to the level of naïve rats. Control saline rats that received saline in both compartments of the CPP apparatus and cocaine CPP rats showed similar enhanced p38 activation as compared to naïve and social interaction CPP rats. We also found that the percentage of neurons expressing dopaminergic receptor D2R and pp38 was also decreased in the shell of the nucleus accumbens of social interaction CPP rats as compared to controls. Given the emerging role of p38 in stress/anxiety behaviors, these results suggest that (1) social interaction reward has anti-stress effects; (2) cocaine conditioning per se does not affect p38 activation and that (3) marginal stress is sufficient to induce p38 activation in the shell of the nucleus accumbens. PMID:26300300

  17. Social interaction reward decreases p38 activation in the nucleus accumbens shell of rats.

    PubMed

    Salti, Ahmad; Kummer, Kai K; Sadangi, Chinmaya; Dechant, Georg; Saria, Alois; El Rawas, Rana

    2015-12-01

    We have previously shown that animals acquired robust conditioned place preference (CPP) to either social interaction alone or cocaine alone. Recently it has been reported that drugs of abuse abnormally activated p38, a member of mitogen-activated protein kinase family, in the nucleus accumbens. In this study, we aimed to investigate the expression of the activated form of p38 (pp38) in the nucleus accumbens shell and core of rats expressing either cocaine CPP or social interaction CPP 1 h, 2 h and 24 h after the CPP test. We hypothesized that cocaine CPP will increase pp38 in the nucleus accumbens shell/core as compared to social interaction CPP. Surprisingly, we found that 24 h after social interaction CPP, pp38 neuronal levels were decreased in the nucleus accumbens shell to the level of naïve rats. Control saline rats that received saline in both compartments of the CPP apparatus and cocaine CPP rats showed similar enhanced p38 activation as compared to naïve and social interaction CPP rats. We also found that the percentage of neurons expressing dopaminergic receptor D2R and pp38 was also decreased in the shell of the nucleus accumbens of social interaction CPP rats as compared to controls. Given the emerging role of p38 in stress/anxiety behaviors, these results suggest that (1) social interaction reward has anti-stress effects; (2) cocaine conditioning per se does not affect p38 activation and that (3) marginal stress is sufficient to induce p38 activation in the shell of the nucleus accumbens. PMID:26300300

  18. 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

  19. Mitogen-activated protein kinase pathway mediates DBP-maf-induced apoptosis in RAW 264.7 macrophages.

    PubMed

    Gumireddy, Kiranmai; Reddy, C Damodar; Swamy, Narasimha

    2003-09-01

    Vitamin D-binding protein-macrophage-activating factor (DBP-maf) is derived from serum vitamin D binding protein (DBP) by selective deglycosylation during inflammation. In the present study, we investigated the effect of DBP-maf on RAW 264.7 macrophages and the underlying intracellular signal transduction pathways. DBP-maf increased proapoptotic caspase-3, -8, and -9 activities and induced apoptosis in RAW 264.7 cells. However, DBP, the precursor to DBP-maf did not induce apoptosis in these cells. Cell cycle analysis of DBP-maf-treated RAW 264.7 cells revealed growth arrest with accumulation of cells in sub-G(0)/G(1) phase. We also investigated the role of mitogen-activated protein kinase (MAPK) pathways in the DBP-maf-induced apoptosis of RAW264.7 cells. DBP-maf increased the phosphorylation of p38 and JNK1/2, while it decreased the ERK1/2 phosphorylation. Treatment with the p38 MAPK inhibitor, SB202190, attenuated DBP-maf-induced apoptosis. PD98059, a MEK specific inhibitor, did not show a significant inhibition of apoptosis induced by DBP-maf. Taken together, these results suggest that the p38 MAPK pathway plays a crucial role in DBP-maf-mediated apoptosis of macrophages. Our studies indicate that, during inflammation DBP-maf may function positively by causing death of the macrophages when activated macrophages are no longer needed at the site of inflammation. In summary, we report for the first time that DBP-maf induces apoptosis in macrophages via p38 and JNK1/2 pathway. PMID:12938159

  20. Liquiritigenin Induces Tumor Cell Death through Mitogen-Activated Protein Kinase- (MPAKs-) Mediated Pathway in Hepatocellular Carcinoma Cells

    PubMed Central

    Lu, Jiahui; Liu, Yan; Meng, Qingfan; Xie, Jing; Wang, Zhenzuo

    2014-01-01

    Liquiritigenin (LQ), separated from Glycyrrhiza radix, possesses anti-inflammatory, antihyperlipidemic, and antiallergic effects. Our present study aims to investigate the antihepatocellular carcinoma effects of LQ both in cell and animal models. LQ strikingly reduced cell viability, enhanced apoptotic rate, induced lactate dehydrogenase over-release, and increased intracellular reactive oxygen species (ROS) level and caspase 3 activity in both PLC/PRL/5 and HepG2 cells. The expression of cleaved PARP, the hall-marker of apoptosis, was enhanced by LQ. LQ treatment resulted in a reduction of the expressions of B-cell lymphoma 2 (Bcl-2) and B-cell lymphoma-extra large (Bcl-xL), and an increase of the phosphorylation of c-Jun N-terminal kinases (JNK) and P38. LQ-mediated cell viability reduction, mitochondrial dysfunction, apoptosis related protein abnormal expressions, and JNK and P38 activation were partially abolished by N-Acetyl-L-cysteine (a ROS inhibitor) pretreatment. Moreover, LQ suppressed the activation of extracellular signaling-regulated kinase (ERKs) and reduced the translocation of phosphor-ERKs from cytoplasm to nucleus. This antitumor activity was further confirmed in PLC/PRL/5-xenografted mice model. All these data indicate that the antihepatocellular carcinoma effects of LQ are related to its modulation of the activations of mitogen-activated protein kinase (MAPKs). The study provides experimental evidence supporting LQ as a potential therapeutic agent for hepatocellular carcinoma treatment. PMID:24738081

  1. HIV-1 Nef-induced FasL induction and bystander killing requires p38 MAPK activation

    PubMed Central

    Muthumani, Karuppiah; Choo, Andrew Y.; Hwang, Daniel S.; Premkumar, Arumugam; Dayes, Nathanael S.; Harris, Crafford; Green, Douglas R.; Wadsworth, Scott A.; Siekierka, John J.; Weiner, David B.

    2005-01-01

    The human immunodeficiency virus (HIV) has been reported to target noninfected CD4 and CD8 cells for destruction. This effect is manifested in part through up-regulation of the death receptor Fas ligand (FasL) by HIV-1 negative factor (Nef), leading to bystander damage. However, the signal transduction and transcriptional regulation of this process remains elusive. Here, we provide evidence that p38 mitogen-activated protein kinase (MAPK) is required for this process. Loss-of-function experiments through dominant-negative p38 isoform, p38 siRNA, and chemical inhibitors of p38 activation suggest that p38 is necessary for Nef-induced activator protein-1 (AP-1) activation, as inhibition leads to an attenuation of AP-1-dependent transcription. Furthermore, mutagenesis of the FasL promoter reveals that its AP-1 enhancer element is required for Nef-mediated transcriptional activation. Therefore, a linear pathway for Nef-induced FasL expression that encompasses p38 and AP-1 has been elucidated. Furthermore, chemical inhibition of the p38 pathway attenuates HIV-1-mediated bystander killing of CD8 cells in vitro. (Blood. 2005;106:2059-2068) PMID:15928037

  2. Mitogen-activated protein kinase and abscisic acid signal transduction.

    PubMed

    Heimovaara-Dijkstra, S; Testerink, C; Wang, M

    2000-01-01

    The phytohormone abscisic acid (ABA) is a classical plant hormone, responsible for regulation of abscission, diverse aspects of plant and seed development, stress responses and germination. It was found that ABA signal transduction in plants can involve the activity of type 2C-phosphatases (PP2C), calcium, potassium, pH and a transient activation of MAP kinase. The ABA signal transduction cascades have been shown to be tissue-specific, the transient activation of MAP kinase has until now only been found in barley aleurone cells. However, type 2C phosphatases are involved in the induction of most ABA responses, as shown by the PP2C-deficient abi-mutants. These phosphatases show high homology with phosphatases that regulate MAP kinase activity in yeast. In addition, the role of farnesyl transferase as a negative regulator of ABA responses also indicates towards involvement of MAP kinase in ABA signal transduction. Farnesyl transferase is known to regulate Ras proteins, Ras proteins in turn are known to regulate MAP kinase activation. Interestingly, Ras-like proteins were detected in barley aleurone cells. Further establishment of the involvement of MAP kinase in ABA signal transduction and its role therein, still awaits more study.

  3. Localised mitogenic activity in horses following infection with Streptococcus equi.

    PubMed

    McLean, R; Rash, N L; Robinson, C; Waller, A S; Paillot, R

    2015-06-01

    Streptococcus equi subspecies equi (S. equi) is the causative agent of strangles, a highly contagious upper respiratory disease of equids. Streptococcus equi produces superantigens (sAgs), which are thought to contribute to strangles pathogenicity through non-specific T-cell activation and pro-inflammatory response. Streptococcus equi infection induces abscesses in the lymph nodes of the head and neck. In some individuals, some abscess material remains into the guttural pouch and inspissates over time to form chondroids which can harbour live S. equi. The aim of this study was to determine the sites of sAg production during infection and therefore improve our understanding of their role. Abscess material, chondroids and serum collected from Equidae with signs of strangles were tested in mitogenic assays. Mitogenic sAg activity was only detected in abscess material and chondroids. Our data support the localised in vivo activity of sAg during both acute and carrier phases of S. equi infection.

  4. 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

  5. Monoclonal antibodies that inhibit mitogenic activity of Mycoplasma pulmonis.

    PubMed Central

    Lapidot, Z; Siman-Tov, R; Naot, Y

    1995-01-01

    Previous studies have suggested a correlation between mitogenic, polyclonal activation of host lymphocytes and the respiratory tract inflammatory diseases induced by Mycoplasma pulmonis. This study describes the generation of monoclonal antibodies (MAbs) to M. pulmonis membrane antigens with different capacities to inhibit stimulation of cultured rat lymphocytes by mycoplasmal membranes and with variable effects on M. pulmonis growth. We show that the inhibitory effects exerted on mitogenesis by purified MAbs are inversely related to the effects of MAbs on M. pulmonis growth. Immunoblotting of electrophoretically separated membrane proteins, with both growth- and mitogenesis-inhibiting antibodies, revealed significant changes in the reactions obtained with both types of MAb following short exposure of membranes to heat. Growth-inhibiting MAbs strongly react with heat-labile antigenic complexes with molecular weights of 65,000 to 75,000. Inhibition of mitogenesis is mainly associated with recognition of membrane complexes of 84 to 113 kDa that exhibit disperse smears and variable heat sensitivities. Following brief heating of membranes, more distinct bands of 103, 90, and 84 kDa are obtained with MAbs that inhibit mitogenesis. Experiments with other mitogenic mycoplasma species and MAb 3.3.10.2, a potent inhibitor of mitogenesis reveal that whereas the antigenic epitope recognized by this antibody is present on unheated membranes from different mycoplasmas, with heated membranes the MAb yields reactions only with M. pulmonis and M. arthritidis. Our studies suggest that M. pulmonis mitogens are unique membrane complexes of variable molecular weights, highly susceptible to heat and less sensitive to reducing agents. PMID:7806349

  6. The role of p38 MAPK activation in auranofin-induced apoptosis of human promyelocytic leukaemia HL-60 cells

    PubMed Central

    Park, Seon-Joo; Kim, In-Sook

    2005-01-01

    In a previous study, we reported an antileukaemic activity of auranofin (AF), demonstrating its dual effects: on the induction of apoptotic cell death and its synergistic action with retinoic acid on cell differentiation. In this study, we investigated the downstream signalling events of AF-induced apoptosis to determine the molecular mechanisms of AF activity. Treatment of HL-60 cells with AF induced apoptosis in a concentration- and time-dependent manner. Western blot analysis showed that AF-induced apoptosis was accompanied by the activation of caspase-8, caspase-9, and caspase-3, and the release of cytochrome c from the mitochondria. The phosphorylation and kinase activities of p38 mitogen-activated protein kinase (p38 MAPK) increased gradually until 12 h after AF (2 μM) treatment, and p38 MAPK was also activated concentration-dependently. Pretreatment with SB203580, a specific inhibitor of p38 MAPK, significantly blocked DNA fragmentation and the cleavage of procaspase-8, procaspase-3, and poly-ADP-ribose polymerase (PARP), whereas SB203580 alone had no effect. Reactive oxygen species (ROS) were also detected within 1 h after AF treatment, and the antioxidant N-acetyl-L-cysteine (NAC) effectively protected the cells from apoptosis by inhibiting the phosphorylation of p38 MAPK and the activation of caspases. These results suggest that ROS generation and the subsequent activation of p38 MAPK are essential for the proapoptotic effects of AF in human promyelocytic leukaemia HL-60 cells. PMID:16086031

  7. 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

  8. Mitochondrial DNA is released by shock and activates neutrophils via p38 map kinase.

    PubMed

    Zhang, Qin; Itagaki, Kiyoshi; Hauser, Carl J

    2010-07-01

    Bacterial DNA (bDNA) can activate an innate-immune stimulatory "danger" response via toll-like receptor 9 (TLR9). Mitochondrial DNA (mtDNA) is unique among endogenous molecules in that mitochondria evolved from prokaryotic ancestors. Thus, mtDNA retains molecular motifs similar to bDNA. It is unknown, however, whether mtDNA is released by shock or is capable of eliciting immune responses like bDNA. We hypothesized shock-injured tissues might release mtDNA and that mtDNA might act as a danger-associated molecular pattern (or "alarmin") that can activate neutrophils (PMNs) and contribute to systemic inflammatory response syndrome. Standardized trauma/hemorrhagic shock caused circulation of mtDNA as well as nuclear DNA. Human PMNs were incubated in vitro with purified mtDNA or nuclear DNA, with or without pretreatment by chloroquine (an inhibitor of endosomal receptors like TLR9). Neutrophil activation was assessed as matrix metalloproteinase (MMP) 8 and MMP-9 release as well as p38 and p44/42 mitogen-activated protein kinase (MAPK) phosphorylation. Mitochondrial DNA induced PMN MMP-8/MMP-9 release and p38 phosphorylation but did not activate p44/42. Responses were inhibited by chloroquine. Nuclear DNA did not induce PMN activation. Intravenous injection of disrupted mitochondria (mitochondrial debris) into rats induced p38 MAPK activation and IL-6 and TNF-alpha accumulation in the liver. In summary, mtDNA is released into the circulation by shock. Mitochondrial DNA activates PMN p38 MAPK, probably via TLR9, inducing an inflammatory phenotype. Mitochondrial DNA may act as a danger-associated molecular pattern or alarmin after shock, contributing to the initiation of systemic inflammatory response syndrome.

  9. Functional analysis of oxidative stress-activated mitogen-activated protein kinase cascade in plants

    PubMed Central

    Kovtun, Yelena; Chiu, Wan-Ling; Tena, Guillaume; Sheen, Jen

    2000-01-01

    Despite the recognition of H2O2 as a central signaling molecule in stress and wounding responses, pathogen defense, and regulation of cell cycle and cell death, little is known about how the H2O2 signal is perceived and transduced in plant cells. We report here that H2O2 is a potent activator of mitogen-activated protein kinases (MAPKs) in Arabidopsis leaf cells. Using epitope tagging and a protoplast transient expression assay, we show that H2O2 can activate a specific Arabidopsis mitogen-activated protein kinase kinase kinase, ANP1, which initiates a phosphorylation cascade involving two stress MAPKs, AtMPK3 and AtMPK6. Constitutively active ANP1 mimics the H2O2 effect and initiates the MAPK cascade that induces specific stress-responsive genes, but it blocks the action of auxin, a plant mitogen and growth hormone. The latter observation provides a molecular link between oxidative stress and auxin signal transduction. Finally, we show that transgenic tobacco plants that express a constitutively active tobacco ANP1 orthologue, NPK1, display enhanced tolerance to multiple environmental stress conditions without activating previously described drought, cold, and abscisic acid signaling pathways. Thus, manipulation of key regulators of an oxidative stress signaling pathway, such as ANP1/NPK1, provides a strategy for engineering multiple stress tolerance that may greatly benefit agriculture. PMID:10717008

  10. Metabolic and cytoskeletal modulation of transferrin receptor mobility in mitogen-activated human lymphocytes.

    PubMed Central

    Galbraith, G M; Galbraith, R M

    1980-01-01

    The transferrin receptors which appear on mitogen-activated human peripheral blood lymphocytes were found by the use of immunofluorescence techniques to display temperature-dependent patching and capping reactions upon binding of transferrin. Lateral mobility of ligand-occupied membrane sites was accompanied by both shedding and endocytosis of receptor-transferrin complexes. In the presence of sodium azide or the microfilament inhibitor cytochalasin B, cap formation and shedding were markedly inhibited. In contrast, endocytosis of patched receptor-ligand complexes was inhibited by azide and microtubule inhibitors, including colchicine, vinblastine and vincristine. Co-capping experiments performed to elucidate further the alterations in membrane configuration involved in these reactions failed to reveal any topographical relationship between transferrin receptors and lectin-binding sites in these cells. These studied indicate that temperature-dependent mobility of transferrin receptors upon mitogen-activated peripheral blood lymphocytes is dependent upon the integrity of the cytoskeletal system and metabolic function of the cell. PMID:6258830

  11. Mitogenic activity of new lectins from seeds of wild Artocarpus species from Vietnam.

    PubMed

    Blasco, E; Ngoc, L D; Aucouturier, P; Preud'Homme, J L; Barra, A

    1996-05-01

    Proliferative response of human peripheral blood mononuclear cells (PBMC) stimulated by new lectins purified from seeds of differents Artocarpus species from Vietnam (A. asperulus, A. heterophyllus, A. masticata, A. melinoxylus, A. parva and A. petelotii) was studied and compared to those of the lectin jacalin purified from jackfruit (A. heterophyllus) seeds collected in the island La Réunion. All lectins stimulated human PBMC to proliferate, with a variable efficiency of the mitogenic activity. Phenotypic analysis of cells recovered after 7 day-cultures showed that these lectins mostly stimulated CD4+ T lymphocytes. These results suggest that these lectins from different Artocarpus species are similar in terms of their mitogenic activity although their structural features are not identical.

  12. Effects of pentylenetetrazole kindling on mitogen-activated protein kinases levels in neocortex and hippocampus of mice.

    PubMed

    Ben, Juliana; de Oliveira, Paulo Alexandre; Gonçalves, Filipe Marques; Peres, Tanara Vieira; Matheus, Filipe Carvalho; Hoeller, Alexandre Ademar; Leal, Rodrigo Bainy; Walz, Roger; Prediger, Rui Daniel

    2014-12-01

    The epileptogenesis process involves cell signaling events associated with neuroplasticity. The mitogen-activated protein kinases (MAPKs) integrate signals originating from a variety of extracellular stimuli and may regulate cell differentiation, survival, cell death and synaptic plasticity. Here we compared the total and phosphorylated MAPKs (ERK1/2, JNK1/2 and p38(MAPK)) levels in the neocortex and hippocampus of adult Swiss male mice quantified by western blotting analysis 48 h after the last injection of pentylenetetrazole (PTZ), according to the kindling protocol (35 mg/kg, i.p., on alternated days, with a total of eight injections). The total levels of the investigated MAPKs and the phospho-p38(MAPK) in the neocortex and hippocampus were not affected by the PTZ injections. The MAPKs phosphorylation levels remain unaltered in PTZ-treated animals without convulsive seizures. The phospho-JNK2 phosphorylation, but not the phospho-JNK1, was increased in the hippocampus of PTZ-treated animals showing 1-3 days with convulsive seizures, whereas no significant changes were observed in those animals with more than 3 days with convulsive seizures. The phospho-ERK1/2 phosphorylation decreased in the neocortex and increased in the hippocampus of animals with 1-4 days with convulsive seizures and became unaltered in mice that showed convulsive seizures for more than 4 days. These findings indicate that resistance to PTZ kindling is associated with unaltered ERK1/2, JNK1/2 and p38(MAPK) phosphorylation levels in the neocortex and hippocampus. Moreover, when the PTZ kindling-induced epileptogenesis manifests behaviorally, the activation of the different MAPKs sub-families shows a variable and non-linear pattern in the neocortex and hippocampus.

  13. Neospora caninum Activates p38 MAPK as an Evasion Mechanism against Innate Immunity

    PubMed Central

    Mota, Caroline M.; Oliveira, Ana C. M.; Davoli-Ferreira, Marcela; Silva, Murilo V.; Santiago, Fernanda M.; Nadipuram, Santhosh M.; Vashisht, Ajay A.; Wohlschlegel, James A.; Bradley, Peter J.; Silva, João S.; Mineo, José R.; Mineo, Tiago W. P.

    2016-01-01

    Due to the high prevalence and economic impact of neosporosis, the development of safe and effective vaccines and therapies against this parasite has been a priority in the field and is crucial to limit horizontal and vertical transmission in natural hosts. Limited data is available regarding factors that regulate the immune response against this parasite and such knowledge is essential in order to understand Neospora caninum induced pathogenesis. Mitogen-activated protein kinases (MAPKs) govern diverse cellular processes, including growth, differentiation, apoptosis, and immune-mediated responses. In that sense, our goal was to understand the role of MAPKs during the infection by N. caninum. We found that p38 phosphorylation was quickly triggered in macrophages stimulated by live tachyzoites and antigen extracts, while its chemical inhibition resulted in upregulation of IL-12p40 production and augmented B7/MHC expression. In vivo blockade of p38 resulted in an amplified production of cytokines, which preceded a reduction in latent parasite burden and enhanced survival against the infection. Additionally, the experiments indicate that the p38 activation is induced by a mechanism that depends on GPCR, PI3K and AKT signaling pathways, and that the phenomena here observed is distinct that those induced by Toxoplasma gondii’s GRA24 protein. Altogether, these results showed that N. caninum manipulates p38 phosphorylation in its favor, in order to downregulate the host’s innate immune responses. Additionally, those results infer that active interference in this signaling pathway may be useful for the development of a new therapeutic strategy against neosporosis. PMID:27679624

  14. Neospora caninum Activates p38 MAPK as an Evasion Mechanism against Innate Immunity

    PubMed Central

    Mota, Caroline M.; Oliveira, Ana C. M.; Davoli-Ferreira, Marcela; Silva, Murilo V.; Santiago, Fernanda M.; Nadipuram, Santhosh M.; Vashisht, Ajay A.; Wohlschlegel, James A.; Bradley, Peter J.; Silva, João S.; Mineo, José R.; Mineo, Tiago W. P.

    2016-01-01

    Due to the high prevalence and economic impact of neosporosis, the development of safe and effective vaccines and therapies against this parasite has been a priority in the field and is crucial to limit horizontal and vertical transmission in natural hosts. Limited data is available regarding factors that regulate the immune response against this parasite and such knowledge is essential in order to understand Neospora caninum induced pathogenesis. Mitogen-activated protein kinases (MAPKs) govern diverse cellular processes, including growth, differentiation, apoptosis, and immune-mediated responses. In that sense, our goal was to understand the role of MAPKs during the infection by N. caninum. We found that p38 phosphorylation was quickly triggered in macrophages stimulated by live tachyzoites and antigen extracts, while its chemical inhibition resulted in upregulation of IL-12p40 production and augmented B7/MHC expression. In vivo blockade of p38 resulted in an amplified production of cytokines, which preceded a reduction in latent parasite burden and enhanced survival against the infection. Additionally, the experiments indicate that the p38 activation is induced by a mechanism that depends on GPCR, PI3K and AKT signaling pathways, and that the phenomena here observed is distinct that those induced by Toxoplasma gondii’s GRA24 protein. Altogether, these results showed that N. caninum manipulates p38 phosphorylation in its favor, in order to downregulate the host’s innate immune responses. Additionally, those results infer that active interference in this signaling pathway may be useful for the development of a new therapeutic strategy against neosporosis.

  15. Neospora caninum Activates p38 MAPK as an Evasion Mechanism against Innate Immunity.

    PubMed

    Mota, Caroline M; Oliveira, Ana C M; Davoli-Ferreira, Marcela; Silva, Murilo V; Santiago, Fernanda M; Nadipuram, Santhosh M; Vashisht, Ajay A; Wohlschlegel, James A; Bradley, Peter J; Silva, João S; Mineo, José R; Mineo, Tiago W P

    2016-01-01

    Due to the high prevalence and economic impact of neosporosis, the development of safe and effective vaccines and therapies against this parasite has been a priority in the field and is crucial to limit horizontal and vertical transmission in natural hosts. Limited data is available regarding factors that regulate the immune response against this parasite and such knowledge is essential in order to understand Neospora caninum induced pathogenesis. Mitogen-activated protein kinases (MAPKs) govern diverse cellular processes, including growth, differentiation, apoptosis, and immune-mediated responses. In that sense, our goal was to understand the role of MAPKs during the infection by N. caninum. We found that p38 phosphorylation was quickly triggered in macrophages stimulated by live tachyzoites and antigen extracts, while its chemical inhibition resulted in upregulation of IL-12p40 production and augmented B7/MHC expression. In vivo blockade of p38 resulted in an amplified production of cytokines, which preceded a reduction in latent parasite burden and enhanced survival against the infection. Additionally, the experiments indicate that the p38 activation is induced by a mechanism that depends on GPCR, PI3K and AKT signaling pathways, and that the phenomena here observed is distinct that those induced by Toxoplasma gondii's GRA24 protein. Altogether, these results showed that N. caninum manipulates p38 phosphorylation in its favor, in order to downregulate the host's innate immune responses. Additionally, those results infer that active interference in this signaling pathway may be useful for the development of a new therapeutic strategy against neosporosis. PMID:27679624

  16. 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

  17. 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.

  18. Nobiletin suppresses MMP-9 expression through modulation of p38 MAPK activity in human dermal fibrobalsts.

    PubMed

    Kim, Jin-Ju; Korm, Sovannarith; Kim, Won-Seok; Kim, Ok-Seon; Lee, Ji-Seon; Min, Hyung-Geun; Chin, Young-Won; Cha, Hyuk-Jin

    2014-01-01

    We aimed to identify a novel flavonoid from the in-house natural products to suppress matrix metalloproteases (MMPs), which is responsible for degradation of collagen and other extracellular matrix proteins. Total eight natural products were screened for identification of a novel MMP-9 suppressor using MMP-9 reporter system, where the prompt initial screening with multiple samples is readily examined. Among the extracts used in the present study, one extract (Citrus unshiu) was found active in this assay system. Furthermore, three representative flavonoids in this active extract of Citrus unshiu peel were tested in MMP-9 reporter system. Nobiletin (NB) of the tested flavonoids suppressed MMP-9 expression without cytotoxicity, which was validated by both real-time polymerase chain reaction (PCR) and zymography analyses. Sustained p38 mitogen activated protein kinase (MAPK) activity, closely associated with induction of MMP-9 under stress condition, was markedly reduced by NB treatment, which implies that modulation of p38MAPK by nobiletin is responsible for reduction of MMP9 expression. Hence, nobiletin, identified from MMP-9 reporter system based screening, may be further applied for the purpose of delaying collagen degradation in skin fibroblasts.

  19. Halofuginone inhibits NF-kappaB and p38 MAPK in activated T cells.

    PubMed

    Leiba, M; Cahalon, L; Shimoni, A; Lider, O; Zanin-Zhorov, A; Hecht, I; Sela, U; Vlodavsky, I; Nagler, A

    2006-08-01

    Halofuginone, a low molecular weight plant alkaloid, inhibits collagen alpha1 (I) gene expression in several animal models and in patients with fibrotic disease, including scleroderma and graft-versus-host disease. In addition, halofuginone has been shown to inhibit angiogenesis and tumor progression. It was demonstrated recently that halofuginone inhibits transforming growth factor-beta (TGF-beta), an important immunomodulator. The present study was undertaken to explore the effects of halofuginone on activated T cells. Peripheral blood T cells were activated by anti-CD3 monoclonal antibodies in the absence and presence of halofuginone and assessed for nuclear factor (NF)-kappaB activity, production of tumor necrosis factor alpha (TNF-alpha) and interferon-gamma (IFN-gamma), T cell apoptosis, chemotaxis, and phosphorylation of p38 mitogen-activated protein kinase (MAPK). A delayed-type hypersensitivity (DTH) model was applied to investigate the effect of halofuginone on T cells in vivo. Preincubation of activated peripheral blood T cells with 10-40 ng/ml halofuginone resulted in a significant dose-dependent decrease in NF-kappaB activity (80% inhibition following incubation with 40 ng halofuginone, P = 0.002). In addition, 40 ng/ml halofuginone inhibited secretion of TNF-alpha, IFN-gamma, interleukin (IL)-4, IL-13, and TGF-beta (P < 0.005). Similarly, halofuginone inhibited the phosphorylation of p38 MAPK and apoptosis in activated T cells (P = 0.0001 and 0.005, respectively). In contrast, T cell chemotaxis was not affected. Halofuginone inhibited DTH response in mice, indicating suppression of T cell-mediated inflammation in vivo. Halofuginone inhibits activated peripheral blood T cell functions and proinflammatory cytokine production through inhibition of NF-kappaB activation and p38 MAPK phosphorylation. It also inhibited DTH response in vivo, making it an attractive immunomodulator and anti-inflammatory agent. PMID:16769768

  20. Contributions of reactive oxygen species and mitogen-activated protein kinase signaling in arsenite-stimulated hemeoxygenase-1 production

    SciTech Connect

    Cooper, Karen L.; Liu, Ke Jian; Hudson, Laurie G. . E-mail: lhudson@salud.unm.edu

    2007-01-15

    Hemeoxygenase-1 (HO-1) is an oxidative stress responsive gene upregulated by various physiological and exogenous stimuli. HO-1 has cytoprotective activities and arsenite is a potent inducer of HO-1 in many cell types and tissues, including epidermal keratinocytes. We investigated the potential contributions of reactive oxygen species (ROS) generation and mitogen-activated protein kinase (MAPK) activation to arsenite-dependent regulation of HO-1 in HaCaT cells, an immortalized human keratinocyte line. Both epidermal growth factor (EGF) and arsenite stimulated ROS production was detected by dihydroethidium (DHE) staining and fluorescence microscopy. Arsenite induced HO-1 in a time- and concentration-dependent manner, while HO-1 expression in response to EGF was modest and evident at extended time points (48-72 h). Inhibition of EGF receptor, MEK I/II or Src decreased arsenite-stimulated HO-1 expression by 20-30%. In contrast, addition of a superoxide scavenger or inhibition of p38 activity decreased the arsenite-dependent response by 80-90% suggesting that ROS and p38 are required for HO-1 induction. However, ROS generation alone was insufficient for the observed arsenite-dependent response as use of a xanthine/xanthine oxidase system to generate ROS did not produce an equivalent upregulation of HO-1. Cooperation between ERK signaling and ROS generation was demonstrated by synergistic induction of HO-1 in cells co-treated with EGF and xanthine/xanthine oxidase resulting in a response nearly equivalent to that observed with arsenite. These findings suggest that the ERK/MAPK activation is necessary but not sufficient for optimal arsenite-stimulated HO-1 induction. The robust and persistent upregulation of HO-1 may have a role in cellular adaptation to chronic arsenic exposure.

  1. 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.

  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. 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

  4. Modeling anhydrobiosis: activation of the mitogen-activated protein kinase ERK by dehydration in both human cells and nematodes.

    PubMed

    Huang, Zebo; Banton, Matthew C; Tunnacliffe, Alan

    2010-12-01

    Anhydrobiosis ("life without water") is the state of suspended animation that certain organisms, including some nematodes, tardigrades, and bdelloid rotifers, enter during desiccation. Extreme water loss imposes considerable stress on biomolecules, cells, and tissues, and must require specific sensing and response mechanisms for survival. However, these mechanisms are poorly understood, in part owing to the lack of amenable model systems. We have, therefore, begun to develop mammalian cell lines as tools for investigating the eukaryotic response to desiccation, and have an additional long-term goal of generating a desiccation-tolerant mammalian cell. Here, we investigate the role of the mitogen-activated protein kinases (MAPKs) in controlling gene expression in response to evaporative water loss. We report that the ERK MAPK pathway inhibitor U0126 can almost completely block induction of desiccation early response genes in a human cell line, suggesting a role for the ERK signal transduction pathway in the stress response. Accordingly, ERK is activated by phosphorylation during desiccation of human cells. Importantly, nematodes also activate ERK on drying, showing that the mammalian cell model behaves similarly to invertebrates experiencing similar stress conditions. We further reveal that, in response to desiccation, human cells can rapidly initiate complex stress signaling networks involving all three MAPK pathways, with transient activation of ERK and sustained activation of JNK and p38. These results are consistent with a role for MAPK pathways in anhydrobiotic adaptation and suggest that non-anhydrobiotes are able to sense and, at least to some extent, respond appropriately to evaporative water loss.

  5. Parasite Mitogen-Activated Protein Kinases as Drug Discovery Targets to Treat Human Protozoan Pathogens

    PubMed Central

    Brumlik, Michael J.; Pandeswara, Srilakshmi; Ludwig, Sara M.; Murthy, Kruthi; Curiel, Tyler J.

    2011-01-01

    Protozoan pathogens are a highly diverse group of unicellular organisms, several of which are significant human pathogens. One group of protozoan pathogens includes obligate intracellular parasites such as agents of malaria, leishmaniasis, babesiosis, and toxoplasmosis. The other group includes extracellular pathogens such as agents of giardiasis and amebiasis. An unfortunate unifying theme for most human protozoan pathogens is that highly effective treatments for them are generally lacking. We will review targeting protozoan mitogen-activated protein kinases (MAPKs) as a novel drug discovery approach towards developing better therapies, focusing on Plasmodia, Leishmania, and Toxoplasma, about which the most is known. PMID:21637385

  6. Targeting the RAS pathway by mitogen-activated protein kinase inhibitors.

    PubMed

    Kiessling, Michael K; Rogler, Gerhard

    2015-01-01

    Targeting of oncogenic driver mutations with small-molecule inhibitors resulted in powerful treatment options for cancer patients in recent years. The RAS (rat sarcoma) pathway is among the most frequently mutated pathways in human cancer. Whereas targeting mutant Kirsten RAS (KRAS) remains difficult, mutant B rapidly accelerated fibrosarcoma (BRAF) kinase is an established drug target in cancer. Now data show that neuroblastoma RAS (NRAS) and even Harvey RAS (HRAS) mutations could be predictive markers for treatment with mitogen-activated protein kinase (MEK) inhibitors. This review discusses recent preclinical and clinical studies of MEK inhibitors in BRAF and RAS mutant cancer. PMID:26691679

  7. The dual specificity phosphatases M3/6 and MKP-3 are highly selective for inactivation of distinct mitogen-activated protein kinases.

    PubMed

    Muda, M; Theodosiou, A; Rodrigues, N; Boschert, U; Camps, M; Gillieron, C; Davies, K; Ashworth, A; Arkinstall, S

    1996-11-01

    The mitogen-activated protein (MAP) kinase family includes extracellular signal-regulated kinase (ERK), c-Jun NH2-terminal kinase/stress-activated protein kinase (JNK/SAPK) and p38/RK/CSBP (p38) as structurally and functionally distinct enzyme classes. Here we describe two new dual specificity phosphatases of the CL100/MKP-1 family that are selective for inactivating ERK or JNK/SAPK and p38 MAP kinases when expressed in COS-7 cells. M3/6 is the first phosphatase of this family to display highly specific inactivation of JNK/SAPK and p38 MAP kinases. Although stress-induced activation of p54 SAPKbeta, p46 SAPKgamma (JNK1) or p38 MAP kinases is abolished upon co-transfection with increasing amounts of M3/6 plasmid, epidermal growth factor-stimulated ERK1 is remarkably insensitive even to the highest levels of M3/6 expression obtained. In contrast to M3/6, the dual specificity phosphatase MKP-3 is selective for inactivation of ERK family MAP kinases. Low level expression of MKP-3 blocks totally epidermal growth factor-stimulated ERK1, whereas stress-induced activation of p54 SAPKbeta and p38 MAP kinases is inhibited only partially under identical conditions. Selective regulation by M3/6 and MKP-3 was also observed upon chronic MAP kinase activation by constitutive p21(ras) GTPases. Hence, although M3/6 expression effectively blocked p54 SAPKbeta activation by p21(rac) (G12V), ERK1 activated by p21(ras) (G12V) was insensitive to this phosphatase. ERK1 activation by oncogenic p21(ras) was, however, blocked totally by co-expression of MKP-3. This is the first report demonstrating reciprocally selective inhibition of different MAP kinases by two distinct dual specificity phosphatases.

  8. Cadmium-induced apoptosis and necrosis in human osteoblasts: role of caspases and mitogen-activated protein kinases pathways.

    PubMed

    Brama, M; Politi, L; Santini, P; Migliaccio, S; Scandurra, R

    2012-02-01

    Cadmium is a widespread environmental pollutant which induces severe toxic alterations, including osteomalacia and osteoporosis, likely by estrogen receptor-dependent mechanisms. Indeed, cadmium has been described to act as an endocrine disruptor and its toxicity is exerted both in vivo and in vitro through induction of apoptosis and/or necrosis by not fully clarified intracellular mechanism(s) of action. Aim of the present study was to further investigate the molecular mechanism by which cadmium might alter homeostasis of estrogen target cells, such as osteoblast homeostasis, inducing cell apoptosis and/or necrosis. Human osteoblastic cells (hFOB 1.19) in culture were used as an in vitro model to characterize the intracellular mechanisms induced by this heavy metal. Cells were incubated in the presence/ absence of 10-50 μM cadmium chloride at different times and DNA fragmentation and activation of procaspases- 8 and -3 were induced upon CdCl(2) treatment triggering apoptotic and necrotic pathways. Addition of caspase-8 and -3 inhibitors (Z-IETD-FMK and Z-DQMD-FMK) partially blocked these effects. No activation of procaspase-9 was observed. To determine the role of mitogen-activated protein kinases (MAPK) in these events, we investigated c-jun N-terminal kinase (JNK), p38 and extracellular signal-regulated protein kinase (ERK1/2) phosphorylation which were activated by 10 μM CdCl(2). Chemical inhibitors of JNK, p38, and ERK1/2, SP600125, SB202190, and PD98059, significantly reduced the phosphorylation of the kinases and blunted apoptosis. In contrast, caspase inhibitors did not reduce the cadmium-induced MAPK phosphorylation, suggesting an independent activation of these pathways. In conclusion, at least 2 pathways appear activated by cadmium in osteoblasts: a direct induction of caspase-8 followed by activation of caspase-3 and an indirect induction by phosphorylation of ERK1/2, p38, and JNK MAPK triggering activation of caspase-8 and -3.

  9. Mitogen-Activated Protein Kinase Signaling in the Heart: Angels Versus Demons in a Heart-Breaking Tale

    PubMed Central

    ROSE, BETH A.; FORCE, THOMAS; WANG, YIBIN

    2013-01-01

    Among the myriad of intra-cellular signaling networks that govern the cardiac development and pathogenesis, mitogen-activated protein kinases (MAPKs) are prominent players that have been the focus of extensive investigations in the past decades. The four best characterized MAPK subfamilies, ERK1/2, JNK, p38, and ERK5, are the targets of pharmacological and genetic manipulations to uncover their roles in cardiac development, function, and diseases. However, information reported in the literature from these efforts has not yet resulted in a clear view about the roles of specific MAPK pathways in heart. Rather, controversies from contradictive results have led to a perception that MAPKs are ambiguous characters in heart with both protective and detrimental effects. The primary object of this review is to provide a comprehensive overview of the current progress, in an effort to highlight the areas where consensus is established verses the ones where controversy remains. MAPKs in cardiac development, cardiac hypertrophy, ischemia/reperfusion injury, and pathological remodeling are the main focuses of this review as these represent the most critical issues for evaluating MAPKs as viable targets of therapeutic development. The studies presented in this review will help to reveal the major challenges in the field and the limitations of current approaches and point to a critical need in future studies to gain better understanding of the fundamental mechanisms of MAPK function and regulation in the heart. PMID:20959622

  10. 4-Anilino-6-phenyl-quinoline inhibitors of mitogen activated protein kinase-activated protein kinase 2 (MK2).

    PubMed

    Olsson, Henric; Sjö, Peter; Ersoy, Oguz; Kristoffersson, Anna; Larsson, Joakim; Nordén, Bo

    2010-08-15

    A class of inhibitors of mitogen activated protein kinase-activated kinase 2 (MK2) was discovered via high-throughput screening. This compound class demonstrates activity against the enzyme with sub-microM IC(50) values, and suppresses LPS-induced TNFalpha levels in THP-1 cells. MK2 inhibition kinetic measurements indicated mixed binding approaching non-ATP competitive inhibition.

  11. Decreased expression of mitochondrial aldehyde dehydrogenase-2 induces liver injury via activation of the mitogen-activated protein kinase pathway.

    PubMed

    Zhong, Zibiao; Ye, Shaojun; Xiong, Yan; Wu, Lianxi; Zhang, Meng; Fan, Xiaoli; Li, Ling; Fu, Zhen; Wang, Huanglei; Chen, Mingyun; Yan, Xiaomin; Huang, Wei; Ko, Dicken Shiu-Chung; Wang, Yanfeng; Ye, Qifa

    2016-01-01

    The aim of this study was to determine the role of ALDH2 in the injury of liver from brain-dead donors. Using brain-dead rabbit model and hypoxia model, levels of ALDH2 and apoptosis in tissues and cell lines were determined by Western blot, flow cytometry (FCM), and transferase (TdT)-mediated biotin-16-dUTP nick-end labeling (TUNEL) assays. After the expression of ALDH2 during hypoxia had been inhibited or activated, the accumulations of 4-hydroxynonenal (4-HNE) and molecules involved in mitogen-activated protein kinase (MAPK) signaling pathway were analyzed using ELISA kit and Western blot. The low expression of phosphorylated ALDH2 in liver was time-dependent in the brain-dead rabbit model. Immunohistochemistry showed ALDH2 was primarily located in endothelial, and the rates of cell apoptosis in the donation after brain-death (DBD) rabbit groups significantly increased with time. Following the treatment of inhibitor of ALDH2, daidzein, in combination with hypoxia for 8 h, the apoptosis rate and the levels of 4-HNE, P-JNK, and cleaved caspase-3 significantly increased in contrast to that in hypoxic HUVECs; however, they all decreased after treatment with Alda-1 and hypoxia compared with that in hypoxic HUVECs (P < 0.05). Instead, the levels of P-P38, P-ERK, P-JNK, and cleaved caspase-3 decreased and the ratio of bcl-2/bax increased with ad-ALDH2 (10(6) pfu/ml) in combination with hypoxia for 8 h, which significantly alleviated in contrast to that in hypoxic HUVECs. We found low expression of ALDH2 and high rates of apoptosis in the livers of brain-dead donor rabbits. Furthermore, decreased ALDH2 led to apoptosis in HUVECs through MAPK pathway.

  12. Inhibition of allograft inflammatory factor-1 expression reduces development of neointimal hyperplasia and p38 kinase activity

    PubMed Central

    Sommerville, Laura J.; Xing, Chen; Kelemen, Sheri E.; Eguchi, Satoru; Autieri, Michael V.

    2009-01-01

    Aims Allograft inflammatory factor-1 (AIF-1) is a calcium-binding, scaffold-signalling protein expressed in vascular smooth muscle cells (VSMCs) in response to injury. The effects of AIF-1 attenuation on development of intimal hyperplasia are unknown, and the molecular mechanisms of these effects remain uncharacterized. The goals of the present study were to determine whether AIF-1 knockdown reduced VSMC proliferation, migration, and intimal hyperplasia, and determine AIF-1 effects on signal transduction in VSMCs. Methods and results Balloon angioplasty-injured rat carotid arteries transduced with adenovirus to overexpress AIF-1 (AdAIF-1) significantly increased, and adenovirus to knock down AIF-1 (AdsiRNA) expression significantly decreased neointimal formation compared with green fluorescent protein (AdGFP) and Adscrambled controls (P < 0.05 and P < 0.01, n = 6). Primary rat VSMCs transduced with AdAIF-1 displayed a significant increase in proliferation, and AdsiRNA-transduced VSMCs proliferated significantly more slowly than controls (P < 0.05). VSMCs transduced with AdAIF-1 show increased migration when compared with control VSMCs (P < 0.01). Rat VSMCs transduced with AdAIF-1 showed constitutive and prolonged activation of the mitogen-activated protein kinase p38, whereas AdsiRNA-treated VSMCs showed decreased p38 activation compared with AdGFP (P < 0.05). Immunohistochemical analysis of AdAIF-1-transduced carotid arteries showed increased staining with a phospho-specific p38 antibody compared with AdGFP-transduced arteries. A specific p38 inhibitor abrogated AIF-1-induced VSMC proliferation, but not AIF-1-induced migration. Conclusion Taken together, AIF-1 expression plays a key role in the development of neointimal hyperplasia. AIF-1 expression enhances the activation of p38 MAP kinase. AIF-1-enhanced proliferation is p38 kinase dependent, but AIF-1-enhanced VSMC migration is p38 independent. PMID:18779232

  13. Abscisic Acid Induces Mitogen-Activated Protein Kinase Activation in Barley Aleurone Protoplasts.

    PubMed

    Knetsch, MLW.; Wang, M.; Snaar-Jagalska, B. E.; Heimovaara-Dijkstra, S.

    1996-06-01

    Abscisic acid (ABA) induces a rapid and transient mitogen-activated protein (MAP) kinase activation in barley aleurone protoplasts. MAP kinase activity, measured as myelin basic protein phosphorylation by MAP kinase immunoprecipitates, increased after 1 min, peaked after 3 min, and decreased to basal levels after ~5 min of ABA treatment in vivo. Antibodies recognizing phosphorylated tyrosine residues precipitate with myelin basic protein kinase activity that has identical ABA activation characteristics and demonstrate that tyrosine phosphorylation of MAP kinase occurs during activation. The half-maximal concentration of ABA required for MAP kinase activation, 3 x 10-7 M, is very similar to that required for ABA-induced rab16 gene expression. The tyrosine phosphatase inhibitor phenylarsine oxide can completely block ABA-induced MAP kinase activation and rab16 gene expression. These results lead us to conclude that ABA activates MAP kinase via a tyrosine phosphatase and that these steps are a prerequisite for ABA induction of rab16 gene expression.

  14. Abscisic Acid Induces Mitogen-Activated Protein Kinase Activation in Barley Aleurone Protoplasts.

    PubMed Central

    Knetsch, MLW.; Wang, M.; Snaar-Jagalska, B. E.; Heimovaara-Dijkstra, S.

    1996-01-01

    Abscisic acid (ABA) induces a rapid and transient mitogen-activated protein (MAP) kinase activation in barley aleurone protoplasts. MAP kinase activity, measured as myelin basic protein phosphorylation by MAP kinase immunoprecipitates, increased after 1 min, peaked after 3 min, and decreased to basal levels after ~5 min of ABA treatment in vivo. Antibodies recognizing phosphorylated tyrosine residues precipitate with myelin basic protein kinase activity that has identical ABA activation characteristics and demonstrate that tyrosine phosphorylation of MAP kinase occurs during activation. The half-maximal concentration of ABA required for MAP kinase activation, 3 x 10-7 M, is very similar to that required for ABA-induced rab16 gene expression. The tyrosine phosphatase inhibitor phenylarsine oxide can completely block ABA-induced MAP kinase activation and rab16 gene expression. These results lead us to conclude that ABA activates MAP kinase via a tyrosine phosphatase and that these steps are a prerequisite for ABA induction of rab16 gene expression. PMID:12239411

  15. A vaccine carrier derived from Neisseria meningitidis with mitogenic activity for lymphocytes.

    PubMed Central

    Liu, M A; Friedman, A; Oliff, A I; Tai, J; Martinez, D; Deck, R R; Shieh, J T; Jenkins, T D; Donnelly, J J; Hawe, L A

    1992-01-01

    Protein carriers vary in their ability to increase the immunogenicity of poorly immunogenic or T-lymphocyte-independent antigens. We examined one such carrier, the outer membrane protein complex derived from Neisseria meningitidis serogroup B strain B11, in an attempt to determine why this outer membrane protein complex was more immunogenic in young infants and in relevant animal models than two other carriers used in conjugates made with Haemophilus influenzae type b polysaccharide, a T-cell-independent antigen. A single protein of the outer membrane protein complex, the class 2 porin protein, was purified and shown to function as a T-helper lymphocyte carrier protein. Unexpectedly, it was also found to have mitogenic activity for lymphocytes that was not due to lipopolysaccharide. This mitogenic activity appears to date to be unique to this carrier protein of the carrier proteins tested and may contribute to the ability of the H. influenzae type b conjugate vaccine made with the outer membrane protein complex to generate IgG anti-polysaccharide antibody responses in mice and infant monkeys and protective immune responses in infants less than 6 months of age. Images PMID:1533934

  16. Brucella abortus induces TNF-α-dependent astroglial MMP-9 secretion through mitogen-activated protein kinases

    PubMed Central

    2013-01-01

    Background Central nervous system (CNS) invasion by bacteria of the genus Brucella results in an inflammatory disorder called neurobrucellosis. We have recently demonstrated that B. abortus infects microglia and astrocytes, eliciting the production of a variety of pro-inflammatory cytokines which contribute to CNS damage. Matrix metalloproteinases (MMP) have been implicated in inflammatory tissue destruction in a range of pathological situations in the CNS. Increased MMP secretion is induced by pro-inflammatory cytokines in a variety of CNS diseases characterized by tissue-destructive pathology. Methods In this study, the molecular mechanisms that regulate MMP secretion from Brucella-infected astrocytes in vitro were investigated. MMP-9 was evaluated in culture supernatants by ELISA, zymography and gelatinolytic activity. Involvement of mitogen-activated protein kinases (MAPK) signaling pathways was evaluated by Western blot and using specific inhibitors. The role of TNF-α was evaluated by ELISA and by assays with neutralizing antibodies. Results B. abortus infection induced the secretion of MMP-9 from murine astrocytes in a dose-dependent fashion. The phenomenon was independent of bacterial viability and was recapitulated by L-Omp19, a B. abortus lipoprotein model, but not its LPS. B. abortus and L-Omp19 readily activated p38 and Erk1/2 MAPK, thus enlisting these pathways among the kinase pathways that the bacteria may address as they invade astrocytes. Inhibition of p38 or Erk1/2 significantly diminished MMP-9 secretion, and totally abrogated production of this MMP when both MAPK pathways were inhibited simultaneously. A concomitant abrogation of B. abortus- and L-Omp19-induced TNF-α production was observed when p38 and Erk1/2 pathways were inhibited, indicating that TNF-α could be implicated in MMP-9 secretion. MMP-9 secretion induced by B. abortus or L-Omp19 was completely abrogated when experiments were conducted in the presence of a TNF-α neutralizing

  17. Sustained mitogen-activated protein kinase activation reprograms defense metabolism and phosphoprotein profile in Arabidopsis thaliana

    PubMed Central

    Lassowskat, Ines; Böttcher, Christoph; Eschen-Lippold, Lennart; Scheel, Dierk; Lee, Justin

    2014-01-01

    Mitogen-activated protein kinases (MAPKs) target a variety of protein substrates to regulate cellular signaling processes in eukaryotes. In plants, the number of identified MAPK substrates that control plant defense responses is still limited. Here, we generated transgenic Arabidopsis thaliana plants with an inducible system to simulate in vivo activation of two stress-activated MAPKs, MPK3, and MPK6. Metabolome analysis revealed that this artificial MPK3/6 activation (without any exposure to pathogens or other stresses) is sufficient to drive the production of major defense-related metabolites, including various camalexin, indole glucosinolate and agmatine derivatives. An accompanying (phospho)proteome analysis led to detection of hundreds of potential phosphoproteins downstream of MPK3/6 activation. Besides known MAPK substrates, many candidates on this list possess typical MAPK-targeted phosphosites and in many cases, the corresponding phosphopeptides were detected by mass spectrometry. Notably, several of these putative phosphoproteins have been reported to be associated with the biosynthesis of antimicrobial defense substances (e.g., WRKY transcription factors and proteins encoded by the genes from the “PEN” pathway required for penetration resistance to filamentous pathogens). Thus, this work provides an inventory of candidate phosphoproteins, including putative direct MAPK substrates, for future analysis of MAPK-mediated defense control. (Proteomics data are available with the identifier PXD001252 via ProteomeXchange, http://proteomecentral.proteomexchange.org). PMID:25368622

  18. Regulation of mitogen-activated protein kinase 3/1 activity during meiosis resumption in mammals

    PubMed Central

    PROCHAZKA, Radek; BLAHA, Milan

    2015-01-01

    In vivo, resumption of oocyte meiosis occurs in large ovarian follicles after the preovulatory surge of luteinizing hormone (LH). The LH surge leads to the activation of a broad signaling network in mural granulosa cells equipped with LH receptors. The signals generated in the mural granulosa cells are further augmented by locally produced peptides or steroids and transferred to the cumulus cell compartment and the oocyte itself. Over the last decade, essential progress has been made in the identification of molecular events associated with the final maturation and ovulation of mammalian oocytes. All new evidence argues for a multiple roles of mitogen-activated protein kinase 3/1 (MAPK3/1) in the gonadotropin-induced ovulation processes. However, the knowledge of gonadotropin-induced signaling pathways leading to MAPK3/1 activation in follicular cells seems limited. To date, only the LH-induced transactivation of the epidermal growth factor receptor/MAPK3/1 pathway has been described in granulosa/cumulus cells even though other mechanisms of MAPK3/1 activation have been detected in other types of cells. In this review, we aimed to summarize recent advances in the elucidation of gonadotropin-induced mechanisms leading to the activation of MAPK3/1 in preovulatory follicles and cultured cumulus-oocyte complexes and to point out a specific role of this kinase in the processes accompanying final maturation of the mammalian oocyte. PMID:26688146

  19. Evidence for a role of mitogen-activated protein kinase 3/mitogen-activated protein kinase in the development of testicular ischemia-reperfusion injury.

    PubMed

    Minutoli, Letteria; Antonuccio, Pietro; Romeo, Carmelo; Nicòtina, Piero Antonio; Bitto, Alessandra; Arena, Salvatore; Polito, Francesca; Altavilla, Domenica; Turiaco, Nunzio; Cutrupi, Antonio; Zuccarello, Biagio; Squadrito, Francesco

    2005-10-01

    Mitogen-activated protein kinase (MAPK) 3/MAPK1 (also known as ERK1/ERK2) plays an important role in the signal transduction pathways. To our knowledge, however, its role in the development of testicular ischemia-reperfusion injury has not yet been investigated. Therefore, we studied the pattern of MAPK3/MAPK1 activation in a experimental model of testicular ischemia-reperfusion injury. We also investigated MAPK8 to understand whether an association exists between these two MAPKs. Adult male Sprague-Dawley rats were subjected to 1 h of testicular ischemia followed by 24 h of reperfusion or to a sham testicular ischemia-reperfusion. Animals were randomized to receive PD98059, which is an inhibitor of MAPK3/MAPK1 (10 mg/kg i.p. administered immediately after detorsion), or its vehicle. The time course of MAPK3/MAPK1, MAPK8, and tumor necrosis factor (TNF; also known as TNF alpha) expression and a histological examination in both the ischemic-reperfused testis and the contralateral one were performed. In both testes, MAPK3/MAPK1 and MAPK8 expression appeared following 10 min of reperfusion and reached their highest activation after 30 min. The MAPK levels slowly decreased, and no significant expression of either kinase was observed following 2 h of reperfusion. Expression of TNF was evident after 1 h of reperfusion and reached its maximum increase after 3 h. PD98059 blunted MAPK3/MAPK1 and MAPK8, reduced TNF expression, and improved the testicular damage caused by ischemia-reperfusion injury in both testes. These data emphasize that MAPK3/MAPK1 has a role in testicular damage and that its blockade might have a future therapeutic role for the management of patients with unilateral testicular torsion.

  20. Actions of Rho family small G proteins and p21-activated protein kinases on mitogen-activated protein kinase family members.

    PubMed Central

    Frost, J A; Xu, S; Hutchison, M R; Marcus, S; Cobb, M H

    1996-01-01

    The mitogen-activated protein (MAP) kinases are a family of serine/threonine kinases that are regulated by distinct extracellular stimuli. The currently known members include extracellular signal-regulated protein kinase 1 (ERK1), ERK2, the c-Jun N-terminal kinase/stress-activated protein kinases (JNK/SAPKs), and p38 MAP kinases. We find that overexpression of the Ste20-related enzymes p21-activated kinase 1 (PAK1) and PAK2 in 293 cells is sufficient to activate JNK/SAPK and to a lesser extent p38 MAP kinase but not ERK2. Rat MAP/ERK kinase kinase 1 can stimulate the activity of each of these MAP kinases. Although neither activated Rac nor the PAKs stimulate ERK2 activity, overexpression of either dominant negative Rac2 or the N-terminal regulatory domain of PAK1 inhibits Ras-mediated activation of ERK2, suggesting a permissive role for Rac in the control of the ERK pathway. Furthermore, constitutively active Rac2, Cdc42hs, and RhoA synergize with an activated form of Raf to increase ERK2 activity. These findings reveal a previously unrecognized connection between Rho family small G proteins and the ERK pathway. PMID:8668187

  1. Mitogen-Activated Protein Kinase Kinase 3 Is Required for Regulation during Dark-Light Transition.

    PubMed

    Lee, Horim

    2015-07-01

    Plant growth and development are coordinately orchestrated by environmental cues and phytohormones. Light acts as a key environmental factor for fundamental plant growth and physiology through photosensory phytochromes and underlying molecular mechanisms. Although phytochromes are known to possess serine/threonine protein kinase activities, whether they trigger a signal transduction pathway via an intracellular protein kinase network remains unknown. In analyses of mitogen-activated protein kinase kinase (MAPKK, also called MKK) mutants, the mkk3 mutant has shown both a hypersensitive response in plant hormone gibberellin (GA) and a less sensitive response in red light signaling. Surprisingly, light-induced MAPK activation in wild-type (WT) seedlings and constitutive MAPK phosphorylation in dark-grown mkk3 mutant seedlings have also been found, respectively. Therefore, this study suggests that MKK3 acts in negative regulation in darkness and in light-induced MAPK activation during dark-light transition. PMID:26082029

  2. Complexity of the primary genetic response to mitogenic activation of human T cells

    SciTech Connect

    Zipfel, P.F.; Siebenlist, U. ); Irving, S.G.; Kelly, K. )

    1989-03-01

    The authors describe the isolation and characterization of more than 60 novel cDNA clones that constitute part of the immediate genetic response to resting human peripheral blood T cells after mitogen activation. This primary response was highly complex, both in the absolute number of inducible genes and in the diversity of regulation. Although most of the genes expressed in activated T cells were shared with the activation response of normal human fibroblasts, a significant number were more restricted in tissue specificity and thus likely encode or effect the differentiated functions of activated T cells. The activatable genes could be further differentiated on the basis of kinetics of induction, response to cycloheximide, and sensitivity to the immunosuppressive drug cylcosporin A. It is of note that cyclosporin A inhibited the expression of more than 10 inducible genes, which suggests that this drug has a broad genetic mechanism of action.

  3. Genome-wide identification and transcriptional expression analysis of mitogen-activated protein kinase and mitogen-activated protein kinase kinase genes in Capsicum annuum.

    PubMed

    Liu, Zhiqin; Shi, Lanping; Liu, Yanyan; Tang, Qian; Shen, Lei; Yang, Sheng; Cai, Jinsen; Yu, Huanxin; Wang, Rongzhang; Wen, Jiayu; Lin, Youquan; Hu, Jiong; Liu, Cailing; Zhang, Yangwen; Mou, Shaoliang; He, Shuilin

    2015-01-01

    The tripartite mitogen-activated protein kinase (MAPK) signaling cascades have been implicated in plant growth, development, and environment adaptation, but a comprehensive understanding of MAPK signaling at genome-wide level is limited in Capsicum annuum. Herein, genome-wide identification and transcriptional expression analysis of MAPK and MAPK kinase (MAPKK) were performed in pepper. A total of 19 pepper MAPK (CaMAPKs) genes and five MAPKK (CaMAPKKs) genes were identified. Phylogenetic analysis indicated that CaMAPKs and CaMAPKKs could be classified into four groups and each group contains similar exon-intron structures. However, significant divergences were also found. Notably, five members of the pepper MAPKK family were much less conserved than those found in Arabidopsis, and 9 Arabidopsis MAPKs did not have orthologs in pepper. Additionally, 7 MAPKs in Arabidopsis had either two or three orthologs in the pepper genome, and six pepper MAPKs and one MAPKK differing in sequence were found in three pepper varieties. Quantitative real-time RT-PCR analysis showed that the majority of MAPK and MAPKK genes were ubiquitously expressed and transcriptionally modified in pepper leaves after treatments with heat, salt, and Ralstonia solanacearum inoculation as well as exogenously applied salicylic acid, methyl jasmonate, ethephon, and abscisic acid. The MAPKK-MAPK interactome was tested by yeast two-hybrid assay, the results showed that one MAPKK might interact with multiple MAPKs, one MAPK might also interact with more than one MAPKKs, constituting MAPK signaling networks which may collaborate in transmitting upstream signals into appropriate downstream cellular responses and processes. These results will facilitate future functional characterization of MAPK cascades in pepper. PMID:26442088

  4. Genome-wide identification and transcriptional expression analysis of mitogen-activated protein kinase and mitogen-activated protein kinase kinase genes in Capsicum annuum

    PubMed Central

    Liu, Zhiqin; Shi, Lanping; Liu, Yanyan; Tang, Qian; Shen, Lei; Yang, Sheng; Cai, Jinsen; Yu, Huanxin; Wang, Rongzhang; Wen, Jiayu; Lin, Youquan; Hu, Jiong; Liu, Cailing; Zhang, Yangwen; Mou, Shaoliang; He, Shuilin

    2015-01-01

    The tripartite mitogen-activated protein kinase (MAPK) signaling cascades have been implicated in plant growth, development, and environment adaptation, but a comprehensive understanding of MAPK signaling at genome-wide level is limited in Capsicum annuum. Herein, genome-wide identification and transcriptional expression analysis of MAPK and MAPK kinase (MAPKK) were performed in pepper. A total of 19 pepper MAPK (CaMAPKs) genes and five MAPKK (CaMAPKKs) genes were identified. Phylogenetic analysis indicated that CaMAPKs and CaMAPKKs could be classified into four groups and each group contains similar exon-intron structures. However, significant divergences were also found. Notably, five members of the pepper MAPKK family were much less conserved than those found in Arabidopsis, and 9 Arabidopsis MAPKs did not have orthologs in pepper. Additionally, 7 MAPKs in Arabidopsis had either two or three orthologs in the pepper genome, and six pepper MAPKs and one MAPKK differing in sequence were found in three pepper varieties. Quantitative real-time RT-PCR analysis showed that the majority of MAPK and MAPKK genes were ubiquitously expressed and transcriptionally modified in pepper leaves after treatments with heat, salt, and Ralstonia solanacearum inoculation as well as exogenously applied salicylic acid, methyl jasmonate, ethephon, and abscisic acid. The MAPKK-MAPK interactome was tested by yeast two-hybrid assay, the results showed that one MAPKK might interact with multiple MAPKs, one MAPK might also interact with more than one MAPKKs, constituting MAPK signaling networks which may collaborate in transmitting upstream signals into appropriate downstream cellular responses and processes. These results will facilitate future functional characterization of MAPK cascades in pepper. PMID:26442088

  5. Mitogen-Activated Protein Kinases and Mitogen Kinase Phosphatase 1: A Critical Interplay in Macrophage Biology

    PubMed Central

    Lloberas, Jorge; Valverde-Estrella, Lorena; Tur, Juan; Vico, Tania; Celada, Antonio

    2016-01-01

    Macrophages are necessary in multiple processes during the immune response or inflammation. This review emphasizes the critical role of the mitogen-activated protein kinases (MAPKs) and mitogen kinase phosphatase-1 (MKP-1) in the functional activities of macrophages. While the phosphorylation of MAPKs is required for macrophage activation or proliferation, MKP-1 dephosphorylates these kinases, thus playing a balancing role in the control of macrophage behavior. MKP-1 is a nuclear-localized dual-specificity phosphatase whose expression is regulated at multiple levels, including at the transcriptional and post-transcriptional level. The regulatory role of MKP-1 in the interplay between MAPK phosphorylation/dephosphorylation makes this molecule a critical regulator of macrophage biology and inflammation. PMID:27446931

  6. Association of mitogen-activated protein kinase with the microtubule cytoskeleton.

    PubMed Central

    Reszka, A A; Seger, R; Diltz, C D; Krebs, E G; Fischer, E H

    1995-01-01

    Using indirect immunofluorescence microscopy and biochemical techniques, we have determined that approximately one-third of the total mitogen-activated protein kinase (MAPK) is associated with the microtubule cytoskeleton in NIH 3T3 mouse fibroblasts. This population of enzyme can be separated from the soluble form that is found distributed throughout the cytosol and is also present in the nucleus after mitogen stimulation. The microtubule-associated enzyme pool constitutes half of all detectable MAPK activity after mitogenic stimulation. These findings extend the known in vivo associations of MAPK with microtubules to include the entire microtubule cytoskeleton of proliferating cells, and they suggest that a direct association of MAPK with microtubules may be in part responsible for the observed correlations between MAPK activities and cytoskeletal alteration. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 6 PMID:7568036

  7. Mitogen-Activated Protein Kinases and Mitogen Kinase Phosphatase 1: A Critical Interplay in Macrophage Biology.

    PubMed

    Lloberas, Jorge; Valverde-Estrella, Lorena; Tur, Juan; Vico, Tania; Celada, Antonio

    2016-01-01

    Macrophages are necessary in multiple processes during the immune response or inflammation. This review emphasizes the critical role of the mitogen-activated protein kinases (MAPKs) and mitogen kinase phosphatase-1 (MKP-1) in the functional activities of macrophages. While the phosphorylation of MAPKs is required for macrophage activation or proliferation, MKP-1 dephosphorylates these kinases, thus playing a balancing role in the control of macrophage behavior. MKP-1 is a nuclear-localized dual-specificity phosphatase whose expression is regulated at multiple levels, including at the transcriptional and post-transcriptional level. The regulatory role of MKP-1 in the interplay between MAPK phosphorylation/dephosphorylation makes this molecule a critical regulator of macrophage biology and inflammation. PMID:27446931

  8. Mitogen-activated protein kinase activation in UV-induced signal transduction.

    PubMed

    Bode, Ann M; Dong, Zigang

    2003-01-28

    Experimental evidence supported by epidemiological findings suggests that solar ultraviolet (UV) irradiation is the most important environmental carcinogen leading to the development of skin cancers. Because the ozone layer blocks UVC (wavelength, 180 to 280 nm) exposure, UVA (UVA I, 340 to 400 nm; UVA II, 320 to 340 nm) and UVB (280 to 320 nm) are probably the chief carcinogenic components of sunlight with relevance for human skin cancer. Substantial contributions to the elucidation of the specific signal transduction pathways involved in UV-induced skin carcinogenesis have been made over the past few years, and most evidence suggests that the cellular signaling response is UV wavelength-dependent. The mitogen-activated protein kinase (MAPK) signaling cascades are targets for UV and are important in the regulation of the multitude of UV-induced cellular responses. Experimental studies have used a range of UVA, UVB, UVC, and various combinations in multiple doses, and the observed effects on activation and phosphorylation of MAPKs are varied. This review focuses on the mechanistic data supporting a role for MAPKs in UV-induced skin carcinogenesis. Progress in understanding the mechanisms of UV-induced signal transduction could lead to the use of these protein kinases as specific targets for the prevention and control of skin cancer.

  9. Mitogen-activated protein kinase phosphatase 1 negatively regulates MAPK signaling in mouse hypothalamus.

    PubMed

    Adachi, Koichi; Goto, Motomitsu; Onoue, Takeshi; Tsunekawa, Taku; Shibata, Miyuki; Hagimoto, Shigeru; Ito, Yoshihiro; Banno, Ryoichi; Suga, Hidetaka; Sugimura, Yoshihisa; Oiso, Yutaka; Arima, Hiroshi

    2014-05-21

    Mitogen-activated protein kinase phosphatase 1 (MKP-1) is shown to negatively regulate MAPK signaling in various peripheral tissues as well as the central nervous system such as cortex, striatum and hippocampus. In this study, we examined whether MKP-1 regulates MAPK signaling in the mouse hypothalamus. Intraperitoneal injection of TNFα significantly increased MKP-1 mRNA expression in paraventricular and arcuate nuclei in the hypothalamus. TNFα treatment induced increases in MKP-1 expression at both mRNA and protein levels, accompanied by the inactivation of MAPK signaling in mouse hypothalamic explants. Inhibition of MKP-1 by its inhibitor or siRNA increased MAPK activity in the explants. Our data indicate that MKP-1 negatively regulates MAPK signaling in the mouse hypothalamus.

  10. Sphingosine induces phospholipase D and mitogen activated protein kinase in vascular smooth muscle cells.

    PubMed

    Taher, M M; Abd-Elfattah, A S; Sholley, M M

    1998-12-01

    The enzymes phospholipase D and diacylglycerol kinase generate phosphatidic acid which is considered to be a mitogen. Here we report that sphingosine produced a significant amount of phosphatidic acid in vascular smooth muscle cells from the rat aorta. The diacylglycerol kinase inhibitor R59 949 partially depressed sphingosine induced phosphatidic acid formation, suggesting that activation of phospholipase C and diacylglycerol kinase can not account for the bulk of phosphatidic acid produced and that additional pathways such as phospholipase D may contribute to this. Further, we have shown that phosphatidylethanol was produced by sphingosine when vascular smooth muscle cells were stimulated in the presence of ethanol. Finally, as previously shown for other cell types, sphingosine stimulated mitogen-activated protein kinase in vascular smooth muscle cells.

  11. 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

  12. Mitogen-activated protein kinase cascades in signaling plant growth and development.

    PubMed

    Xu, Juan; Zhang, Shuqun

    2015-01-01

    Mitogen-activated protein kinase (MAPK) cascades are ubiquitous signaling modules in eukaryotes. Early research of plant MAPKs has been focused on their functions in immunity and stress responses. Recent studies reveal that they also play essential roles in plant growth and development downstream of receptor-like protein kinases (RLKs). With only a limited number of MAPK components, multiple functional pathways initiated from different receptors often share the same MAPK components or even a complete MAPK cascade. In this review, we discuss how MAPK cascades function as molecular switches in response to spatiotemporal-specific ligand-receptor interactions and the availability of downstream substrates. In addition, we discuss other possible mechanisms governing the functional specificity of plant MAPK cascades, a question central to our understanding of MAPK functions.

  13. 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

  14. Homology modeling studies of yeast Mitogen-Activated Protein Kinases (MAPKS): structural motifs as a basis for specificity.

    PubMed

    Smith, D L; Nilar, S H

    2010-06-01

    Mitogen-activated protein kinases (MAPKs) are key components of cellular signal transduction. It is the objective of this communication to demonstrate that insight into protein-protein interactions in the Common Docking motif of yeast mitogen-activated protein kinases can be obtained based on homology models. Homology models for four yeast MAPKs, FUS3, KSS1, HOG1 and MPK1 were built based on the X-ray structures of active and inactive rat ERK2. The structural motifs required for the basis of specificity were rationalized based on these structures. PMID:19995338

  15. Tighter αC-helix–αL16-helix interactions seem to make p38α less prone to activation by autophosphorylation than Hog1

    PubMed Central

    Tesker, Masha; Selamat, Sadiduddin Edbe; Beenstock, Jonah; Hayouka, Ruchama; Livnah, Oded; Engelberg, David

    2016-01-01

    Many eukaryotic protein kinases (EPKs) are autoactivated through autophosphorylation of their activation loop. Mitogen-activated protein (MAP) kinases do not autophosphorylate spontaneously; relying instead upon mitogen-activated protein kinase (MAPK) kinases (MKKs) for their activation loop phosphorylation. Yet, in previous studies we identified mutations in the yeast MAPK high osmolarity glycerol (Hog1) that render it capable of spontaneous autophosphorylation and consequently intrinsically active (MKK-independent). Four of the mutations occurred in hydrophobic residues, residing in the αC-helix, which is conserved in all EPKs, and in the αL16-helix that is unique to MAPKs. These four residues interact together forming a structural element termed ‘hydrophobic core’. A similar element exists in the Hog1’s mammalian orthologues p38s. Here we show that the ‘hydrophobic core’ is a loose suppressor of Hog1’s autophosphorylation. We inserted 18 point mutations into this core, 17 of which were able to render Hog1 MKK-independent. In p38s, however, only a very few mutations in the equivalent residues rendered these proteins intrinsically active. Structural analysis revealed that a salt bridge between the αC-helix and the αL16-helix that exists in p38α may not exist in Hog1. This bond further stabilizes the ‘hydrophobic core’ of p38, making p38 less prone to de-repressing its concealed autophosphorylation. Mutating equivalent hydrophobic residues in Jnk1 and Erk2 has no effect on their autophosphorylation. We propose that specific structural elements developed in the course of evolution to suppress spontaneous autophosphorylation of Hog1/p38. The suppressors were kept wobbly, probably to allow activation by induced autophosphorylation, but became stricter in mammalian p38s than in the yeast Hog1. PMID:26987986

  16. A sensitive flow cytometry-based nucleotide excision repair assay unexpectedly reveals that mitogen-activated protein kinase signaling does not regulate the removal of UV-induced DNA damage in human cells.

    PubMed

    Rouget, Raphael; Auclair, Yannick; Loignon, Martin; Affar, El Bachir; Drobetsky, Elliot A

    2008-02-29

    In response to diverse genotoxic stimuli (e.g. UV and cisplatin), the mitogen-activated protein kinases ERK1/2, JNK1/2, and p38alpha/beta become rapidly phosphorylated and in turn activate multiple downstream effectors that modulate apoptosis and/or growth arrest. Furthermore, previous lines of evidence have strongly suggested that ERK1/2 and JNK1/2 participate in global-genomic nucleotide excision repair, a critical antineoplastic pathway that removes helix-distorting DNA adducts induced by a variety of mutagenic agents, including UV. To rigorously evaluate the potential role of mitogen-activated protein kinases in global-genomic nucleotide excision repair, various human cell strains (primary skin fibroblasts, primary lung fibroblasts, and HCT116 colon carcinoma cells) were treated with highly specific chemical inhibitors, which, following UV exposure, (i) abrogated the capacities of ERK1/2, JNK1/2, or p38alpha/beta to phosphorylate specific downstream effectors and (ii) characteristically modulated cellular proliferation, clonogenic survival, and/or apoptosis. A highly sensitive flow cytometry-based nucleotide excision repair assay recently optimized and validated in our laboratory was then employed to directly demonstrate that the kinetics of UV DNA photoadduct repair are highly similar in mock-treated versus mitogen-activated protein kinase inhibitor-treated cells. These data on primary and tumor cells treated with pharmacological inhibitors were fully corroborated by repair studies using (i) short hairpin RNA-mediated knockdown of ERK1/2 or JNK1/2 in human U2OS osteosarcoma cells and (ii) expression of a dominant negative p38alpha mutant in human primary lung fibroblasts. Our results provide solid evidence for the first time, in disaccord with a burgeoning perception, that mitogen-activated protein kinase signaling does not influence the efficiency of human global-genomic nucleotide excision repair.

  17. Noise exposure immediately activates cochlear mitogen-activated protein kinase signaling.

    PubMed

    Alagramam, Kumar N; Stepanyan, Ruben; Jamesdaniel, Samson; Chen, Daniel H-C; Davis, Rickie R

    2014-01-01

    Noise-induced hearing loss (NIHL) is a major public health issue worldwide. Uncovering the early molecular events associated with NIHL would reveal mechanisms leading to the hearing loss. Our aim is to investigate the immediate molecular responses after different levels of noise exposure and identify the common and distinct pathways that mediate NIHL. Previous work showed mice exposed to 116 decibels sound pressure level (dB SPL) broadband noise for 1 h had greater threshold shifts than the mice exposed to 110 dB SPL broadband noise, hence we used these two noise levels in this study. Groups of 4-8-week-old CBA/CaJ mice were exposed to no noise (control) or to broadband noise for 1 h, followed by transcriptome analysis of total cochlear RNA isolated immediately after noise exposure. Previously identified and novel genes were found in all data sets. Following exposure to noise at 116 dB SPL, the earliest responses included up-regulation of 243 genes and down-regulation of 61 genes, while a similar exposure at 110 dB SPL up-regulated 155 genes and down-regulated 221 genes. Bioinformatics analysis indicated that mitogen-activated protein kinase (MAPK) signaling was the major pathway in both levels of noise exposure. Nevertheless, both qualitative and quantitative differences were noticed in some MAPK signaling genes, after exposure to different noise levels. Cacna1b , Cacna1g , and Pla2g6 , related to calcium signaling were down-regulated after 110 dB SPL exposure, while the fold increase in the expression of Fos was relatively lower than what was observed after 116 dB SPL exposure. These subtle variations provide insight on the factors that may contribute to the differences in NIHL despite the activation of a common pathway.

  18. Cigarette smoke extract-induced BEAS-2B cell apoptosis and anti-oxidative Nrf-2 up-regulation are mediated by ROS-stimulated p38 activation.

    PubMed

    Lin, Xi-Xi; Yang, Xin-Fu; Jiang, Jun-Xia; Zhang, Shui-Juan; Guan, Yan; Liu, Ya-Nan; Sun, Yan-Hong; Xie, Qiang-Min

    2014-12-01

    Cigarette smoke contains reactive oxygen (ROS) that can cause oxidative stress. It increases the number of apoptotic and necrotic lung cells and further induces the development of chronic airway disease. In this study, we investigated the effects of cigarette smoke extract (CSE) on apoptosis in human bronchial epithelial cells (BEAS-2B). CSE exposure induced ROS generation and p38 mitogen-activated protein kinase (MAPK) activation that are associated with the activation of apoptosis-regulating signal kinase 1 (ASK-1). N-acetylcysteine (a general antioxidant) attenuated the CSE-induced ASK-1 and p38 MAPK activation and cell apoptosis, suggesting a triggering role of ROS in ASK-1/p38 MAPK activation during apoptotic progression. In contrast, the inhibition and knockdown of p38 attenuated the expression of anti-oxidant master NF-E2-related factor 2 (Nrf-2) and CSE-induced apoptosis, suggesting that p38 MAPK modulates Nrf-2 expression and presumably prevents cell apoptosis. Taken together, the data presented in this manuscript demonstrate that the ROS-dependent ASK-1/p38 signaling cascade regulates CSE-induced BEAS-2B cell apoptosis. In addition, anti-oxidative Nrf-2 is also up-regulated by the ROS/p38 signaling cascade in this progression. PMID:25134437

  19. The food contaminant deoxynivalenol activates the mitogen activated protein kinases in the intestine: interest of ex vivo models as an alternative to in vivo experiments.

    PubMed

    Lucioli, Joelma; Pinton, Philippe; Callu, Patrick; Laffitte, Joëlle; Grosjean, François; Kolf-Clauw, Martine; Oswald, Isabelle P; Bracarense, Ana Paula F R L

    2013-05-01

    Trichothecenes induce changes in the intestinal barrier function through decreased expression of cell junction proteins and apoptosis of enterocytes. The mitogen activated protein kinases (MAPK) play an important role in the signaling pathways of cell turnover and differentiation. Using ex vivo and in vivo approaches, the purpose of this study was to investigate the ability of low doses of DON to induce histological changes in the intestine and to activate the MAPK ERK 1/2, p38 and JNK. Twelve weaning piglets received during four weeks a control diet or a DON-contaminated diet (2.3 mg DON/kg feed). Six weaning piglets were used to prepare jejunal explants (ex vivo model). Explants were exposed during 4 h to vehicle, 5 or 10 μM DON. Intestinal changes were graded using a histological score. Pigs fed a DON-diet and explants exposed to DON showed a significant decrease in the jejunal score. In both models, the toxin significantly enhanced phosphorylation of ERK 1/2 and p38, whereas the increased phosphorylation of JNK was non significant. Taken together these results indicate that in vivo or ex vivo exposure of intestinal tissue to DON lead to similar intestinal lesions and activation of MAPK. These effects could impair the homeostasis of intestinal tissue in the aspects of barrier function and immune protection. The similarity of the in vivo and ex vivo results provides also strong evidence that the jejunal explant model is a good alternative for toxicological studies in intestinal tissue.

  20. Induction of prostaglandin D2 through the p38 MAPK pathway is responsible for the antipruritic activity of sertaconazole nitrate.

    PubMed

    Kaur, Simarna; Sur, Runa; Liebel, Frank T; Southall, Michael D

    2010-10-01

    Prostaglandin D(2) (PGD(2)) is known to have antipruritic activity by suppressing histamine release. However, agents that can topically induce PGD(2) for itch relief are not well established. The antimycotic sertaconazole nitrate (STZ) has been shown to exhibit anti-itch properties; however, the mechanism for this activity has not been elucidated. STZ mitigated degranulation of RBL-2H3 (rat basophilic leukemia) mast cells induced by compound 48/80, a pruritogenic agent known to promote the release of histamine, and augmented PGD(2) production in mast cells and macrophages. Addition of exogenous PGD(2) abrogated compound 48/80-induced degranulation by acting through the prostanoid D receptor 1 (DP1). STZ induced p38 mitogen-activated protein kinase (MAPK) phosphorylation in mast cells and a pharmacological inhibitor of p38 MAPK, SB203580, resulted in the attenuation of PGD(2) levels. Finally, in a murine model of pruritus, the scratching behavior induced by compound 48/80 was mitigated by topical application of STZ. This effect was reversed by the addition of the cyclooxygenase inhibitor, ibuprofen, or a DP1 receptor antagonist (MK0524). Collectively, these results suggest that STZ mediates its anti-itch effects by boosting the antipruritic agent, PGD(2), by the activation of the p38-MAPK pathway. This is the first report to demonstrate a promising approach to topically induce PGD(2) for improving pruritus. PMID:20505747

  1. Cell cycle arrest mediated by the MEK/mitogen-activated protein kinase pathway

    PubMed Central

    Pumiglia, Kevin M.; Decker, Stuart J.

    1997-01-01

    The mitogen-activated protein kinase (MAPK) cascade plays a crucial role in the transduction of extracellular signals into responses governing growth and differentiation. The effects of a specific inhibitor of the MAPK kinase (MEK)/MAPK pathway (PD98059) on nerve growth factor (NGF)-induced growth arrest and inhibition of cell cycle-dependent kinases (CDKs) have been examined. Treatment of NIH 3T3 cells expressing TRKA with PD98059 dramatically reversed the complete inhibition of growth of these cells caused by NGF. PD98059 also blocked the ability of NGF to inhibit the activities of CDK4 and CDK2, while partially preventing NGF induction of p21Cip1/WAF1. To independently evaluate the involvement of the MEK/MAPK pathway in growth arrest, an inducible activated form of the Raf-1 protooncogene (ΔRAF-1:ER) was expressed in these cells. Activation of ΔRAF-1:ER resulted in a prolonged increase in MAPK activity and growth arrest of these cells, with concomitant induction of p21Cip1/WAF1 and inhibition of CDK2 activity. These effects of ΔRAF-1:ER activation were all reversed by treatment of cells with PD98059. These data indicate that in addition to functioning as a positive effector of growth, stimulation of the MEK/MAPK pathway can result in an inhibition of CDK activity and cell cycle arrest. PMID:9012803

  2. Molecular Profiles of Mitogen Activated Protein Kinase Signaling Pathways in Orofacial Development

    PubMed Central

    Singh, Saurabh; Yin, Xiaolong; Pisano, M. Michele; Greene, Robert M.

    2010-01-01

    Background Formation of the mammalian orofacial region involves multiple signaling pathways regulating sequential expression of and interaction between molecular signals during embryogenesis. The present study examined the expression patterns of members of the MAPK family in developing murine orofacial tissue. Methods Total RNA was extracted from developing embryonic orofacial tissue during gestational days (GDs) 12–14 and used to prepare biotinylated cDNA probes, which were then denatured and hybridized to murine MAP kinase signaling pathways gene arrays. Results Expression of a number of genes involved in the (ERK1/2) cascade transiently increased in the embryonic orofacial tissue over the developmental period examined. Numerous members of the SAPK/JNK cascade were constitutively expressed in the tissue. Genes known to play a role in p38 MAPK signaling exhibited constitutive expression during orofacial development. Western blot analysis demonstrated that ERK2/1, p38 and SAPK/JNK kinases are present in embryonic orofacial tissue on each of GD 12, 13 and 14. By using phospho-specific antibodies, active ERK was shown to be temporally regulated during orofacial development. Minimal amounts of active p38 and active SAPK/JNK were detected in orofacial tissue during GDs 12–14. Conclusions Our study documents specific expression patterns of genes coding for proteins belonging to the ERK1/2, p38 and SAPK/JNK MAP kinase families in embryonic orofacial tissue. We also demonstrate that active, phosphorylated forms of ERK1/2 only, were detected in the embryonic tissue investigated, suggesting a more central role for members of this family in embryonic orofacial development. PMID:17177285

  3. Association of mitogen-activated protein kinases with microtubules in mouse macrophages

    PubMed Central

    1996-01-01

    Taxol, a microtubule-binding diterpene, mimics many effects of lipopolysaccharide (LPS) on mouse macrophages. The LPS-mimetic effects of taxol appear to be under the same genetic control as responses to LPS itself. Thus we have postulated a role for microtubule-associated proteins (MAP) in the response of macrophages to LPS. Stimulation of macrophages by LPS quickly induces the activation of mitogen-activated protein kinases (MAPK). MAPK are generally considered cytosolic enzymes. Herein we report that much of the LPS-activatable pool of MAPK in primary mouse peritoneal macrophages is microtubule associated. By immunofluorescence, MAPK were localized to colchicine- and nocodazole- disruptible filaments. From both mouse brain and RAW 264.7 macrophages, MAPK could be coisolated with polymerized tubulin. Fractionation of primary macrophages into cytosol-, microfilament-, microtubule-, and intermediated filament-rich extracts revealed that approximately 10% of MAPK but none of MAPK kinase (MEK1A and MEK2) was microtubule bound. Exposure of macrophages to LPS did not change the proportion of MAPK bound to microtubules, but preferentially activated the microtubule- associated pool. These findings confirm the prediction that LPS activates a kinase bound to microtubules. Together with LPS-mimetic actions of taxol and the shared genetic control of responses to LPS and taxol, these results support the hypothesis that a major LPS-signaling pathway in mouse macrophages may involve activation of one or more microtubule-associated kinases. PMID:8666946

  4. Biological significance of nuclear localization of mitogen-activated protein kinase Pmk1 in fission yeast.

    PubMed

    Sánchez-Mir, Laura; Franco, Alejandro; Madrid, Marisa; Vicente-Soler, Jero; Villar-Tajadura, M Antonia; Soto, Teresa; Pérez, Pilar; Gacto, Mariano; Cansado, José

    2012-07-27

    Mitogen-activated protein kinase (MAPK) signaling pathways play a fundamental role in the response of eukaryotic cells to environmental changes. Also, much evidence shows that the stimulus-dependent nuclear targeting of this class of regulatory kinases is crucial for adequate regulation of distinct cellular events. In the fission yeast Schizosaccharomyces pombe, the cell integrity MAPK pathway, whose central element is the MAPK Pmk1, regulates multiple processes such as cell wall integrity, vacuole fusion, cytokinesis, and ionic homeostasis. In non-stressed cells Pmk1 is constitutively localized in both cytoplasm and nucleus, and its localization pattern appears unaffected by its activation status or in response to stress, thus questioning the biological significance of the presence of this MAPK into the nucleus. We have addressed this issue by characterizing mutants expressing Pmk1 versions excluded from the cell nucleus and anchored to the plasma membrane in different genetic backgrounds. Although nuclear Pmk1 partially regulates cell wall integrity at a transcriptional level, membrane-tethered Pmk1 performs many of the biological functions assigned to wild type MAPK like regulation of chloride homeostasis, vacuole fusion, and cellular separation. However, we found that down-regulation of nuclear Pmk1 by MAPK phosphatases induced by the stress activated protein kinase pathway is important for the fine modulation of extranuclear Pmk1 activity. These results highlight the importance of the control of MAPK activity at subcellular level.

  5. Mitogen-activated Protein Kinase Kinase Kinase 1 Protects against Nickel-induced Acute Lung Injury

    PubMed Central

    Mongan, Maureen; Tan, Zongqing; Chen, Liang; Peng, Zhimin; Dietsch, Maggie; Su, Bing; Leikauf, George; Xia, Ying

    2008-01-01

    Nickel compounds are environmental and occupational hazards that pose serious health problems and are causative factors of acute lung injury. The c-jun N-terminal kinases (JNKs) are regulated through a mitogen-activated protein (MAP) 3 kinase-MAP2 kinase cascade and have been implicated in nickel toxicity. In this study, we used genetically modified cells and mice to investigate the involvement of two upstream MAP3Ks, MAP3K1 and 2, in nickel-induced JNK activation and acute lung injury. In mouse embryonic fibroblasts, levels of JNK activation and cytotoxicity induced by nickel were similar in the Map3k2-null and wild-type cells but were much lower in the Map3k1/Map3k2 double-null cells. Conversely, the levels of JNK activation and cytotoxicity were unexpectedly much higher in the Map3k1-null cells. In adult mouse tissue, MAP3K1 was widely distributed but was abundantly expressed in the bronchiole epithelium of the lung. Accordingly, MAP3K1 ablation in mice resulted in severe nickel-induced acute lung injury and reduced survival. Based on these findings, we propose a role for MAP3K1 in reducing JNK activation and protecting the mice from nickel-induced acute lung injury. PMID:18467339

  6. Activation of a mitogen-activated protein kinase pathway in Arabidopsis by chitin.

    PubMed

    Wan, Jinrong; Zhang, Shuqun; Stacey, Gary

    2004-03-01

    SUMMARY Chitin, a polysaccharide composed of beta-1-->4-linked N-acetyl-d-glucosamine, has been shown or implicated as a signal in plant defence and development. However, the key components of chitin perception and downstream signalling in non-leguminous plants are largely unknown. In recent years, mitogen-activated protein kinases (MAPKs) and their cascades were shown to transduce various extracellular stimuli into internal cellular responses. To investigate the possible involvement of MAPKs in chitin signalling in plants, the model plant Arabidopsis thaliana was treated with crab-shell chitin and also with the purified chitin oligomers (degree of polymerization, d.p. = 2-8). Both mRNA levels and kinase activity of two MAPK genes, AtMPK6 and AtMPK3, were monitored after treatment. The mRNA of AtMPK3 was strongly up-regulated by both chitin and its larger oligomers (d.p. = 6-8), but the mRNA of AtMPK6 did not appear to be regulated by these treatments. However, the kinase activity of both MAPKs was induced by chitin and the larger oligomers (d.p. = 6-8), with AtMPK6 much more strongly induced. In addition, WRKY22, WRKY29, WRKY33 and WRKY53, which encode four WRKY transcription factors that recognize TTGAC(C/T) W-box elements in promoters of numerous plant defence-related genes, were up-regulated by these treatments. WRKY33 and WRKY53 expression was induced by the transgenic expression of the tobacco MAPKK NtMEK2 active mutant NtMEK2(DD), suggesting a potential role for these WRKY transcription factors in relaying the signal generated from the MAPK cascade to downstream genes. These data suggest that AtMPK6/AtMPK3 and WRKY transcription factors (such as WRKY33 and WRKY53) may be important components of a pathway involved in chitin signalling in Arabidopsis plants.

  7. Context-dependent transcriptional interpretation of mitogen activated protein kinase signaling in the Drosophila embryo

    NASA Astrophysics Data System (ADS)

    Kim, Yoosik; Iagovitina, Antonina; Ishihara, Keisuke; Fitzgerald, Kate M.; Deplancke, Bart; Papatsenko, Dmitri; Shvartsman, Stanislav Y.

    2013-06-01

    Terminal regions of the Drosophila embryo are patterned by the localized activation of Mitogen Activated Protein Kinase (MAPK), which induces zygotic genes through relief of their repression by transcriptional repressor Capicua. The levels of MAPK activation at the anterior and posterior termini are close to each other, but the expression patterns of MAPK-target genes, such as zerknüllt (zen) and tailless (tll), display strong anterior-posterior (AP) asymmetry. This region-specific response to MAPK activation provides a clear example of context-dependent interpretation of inductive signaling, a common developmental effect that remains poorly understood. In the past, the AP asymmetry of zen expression was attributed to a mechanism that depends on MAPK substrate competition. We present data suggesting that the asymmetric expression of tll is generated by a different mechanism, based on feedforward control and multiple enhancers of the tll gene. A simple mathematical model of this mechanism correctly predicts how the wild-type expression pattern of tll changes in mutants affecting the anterior, dorsoventral, and terminal patterning systems and some of their direct targets.

  8. Regulation of WRKY46 Transcription Factor Function by Mitogen-Activated Protein Kinases in Arabidopsis thaliana

    PubMed Central

    Sheikh, Arsheed H.; Eschen-Lippold, Lennart; Pecher, Pascal; Hoehenwarter, Wolfgang; Sinha, Alok K.; Scheel, Dierk; Lee, Justin

    2016-01-01

    Mitogen-activated protein kinase (MAPK) cascades are central signaling pathways activated in plants after sensing internal developmental and external stress cues. Knowledge about the downstream substrate proteins of MAPKs is still limited in plants. We screened Arabidopsis WRKY transcription factors as potential targets downstream of MAPKs, and concentrated on characterizing WRKY46 as a substrate of the MAPK, MPK3. Mass spectrometry revealed in vitro phosphorylation of WRKY46 at amino acid position S168 by MPK3. However, mutagenesis studies showed that a second phosphosite, S250, can also be phosphorylated. Elicitation with pathogen-associated molecular patterns (PAMPs), such as the bacterial flagellin-derived flg22 peptide led to in vivo destabilization of WRKY46 in Arabidopsis protoplasts. Mutation of either phosphorylation site reduced the PAMP-induced degradation of WRKY46. Furthermore, the protein for the double phosphosite mutant is expressed at higher levels compared to wild-type proteins or single phosphosite mutants. In line with its nuclear localization and predicted function as a transcriptional activator, overexpression of WRKY46 in protoplasts raised basal plant defense as reflected by the increase in promoter activity of the PAMP-responsive gene, NHL10, in a MAPK-dependent manner. Thus, MAPK-mediated regulation of WRKY46 is a mechanism to control plant defense. PMID:26870073

  9. Context-dependent transcriptional interpretation of mitogen activated protein kinase signaling in the Drosophila embryo

    PubMed Central

    Kim, Yoosik; Iagovitina, Antonina; Ishihara, Keisuke; Fitzgerald, Kate M.; Deplancke, Bart; Papatsenko, Dmitri; Shvartsman, Stanislav Y.

    2013-01-01

    Terminal regions of the Drosophila embryo are patterned by the localized activation of Mitogen Activated Protein Kinase (MAPK), which induces zygotic genes through relief of their repression by transcriptional repressor Capicua. The levels of MAPK activation at the anterior and posterior termini are close to each other, but the expression patterns of MAPK-target genes, such as zerknüllt (zen) and tailless (tll), display strong anterior-posterior (AP) asymmetry. This region-specific response to MAPK activation provides a clear example of context-dependent interpretation of inductive signaling, a common developmental effect that remains poorly understood. In the past, the AP asymmetry of zen expression was attributed to a mechanism that depends on MAPK substrate competition. We present data suggesting that the asymmetric expression of tll is generated by a different mechanism, based on feedforward control and multiple enhancers of the tll gene. A simple mathematical model of this mechanism correctly predicts how the wild-type expression pattern of tll changes in mutants affecting the anterior, dorsoventral, and terminal patterning systems and some of their direct targets. PMID:23822503

  10. VIP1 response elements mediate mitogen-activated protein kinase 3-induced stress gene expression.

    PubMed

    Pitzschke, Andrea; Djamei, Armin; Teige, Markus; Hirt, Heribert

    2009-10-27

    The plant pathogen Agrobacterium tumefaciens transforms plant cells by delivering its T-DNA into the plant cell nucleus where it integrates into the plant genome and causes tumor formation. A key role of VirE2-interacting protein 1 (VIP1) in the nuclear import of T-DNA during Agrobacterium-mediated plant transformation has been unravelled and VIP1 was shown to undergo nuclear localization upon phosphorylation by the mitogen-activated protein kinase MPK3. Here, we provide evidence that VIP1 encodes a functional bZIP transcription factor that stimulates stress-dependent gene expression by binding to VIP1 response elements (VREs), a DNA hexamer motif. VREs are overrepresented in promoters responding to activation of the MPK3 pathway such as Trxh8 and MYB44. Accordingly, plants overexpressing VIP1 accumulate high levels of Trxh8 and MYB44 transcripts, whereas stress-induced expression of these genes is impaired in mpk3 mutants. Trxh8 and MYB44 promoters are activated by VIP1 in a VRE-dependent manner. VIP1 strongly enhances expression from a synthetic promoter harboring multiple VRE copies and directly interacts with VREs in vitro and in vivo. Chromatin immunoprecipitation assays of the MYB44 promoter confirm that VIP1 binding to VREs is enhanced under conditions of MPK3 pathway stimulation. These results provide molecular insight into the cellular mechanism of target gene regulation by the MPK3 pathway. PMID:19820165

  11. Nedd4-induced monoubiquitination of IRS-2 enhances IGF signalling and mitogenic activity.

    PubMed

    Fukushima, Toshiaki; Yoshihara, Hidehito; Furuta, Haruka; Kamei, Hiroyasu; Hakuno, Fumihiko; Luan, Jing; Duan, Cunming; Saeki, Yasushi; Tanaka, Keiji; Iemura, Shun-Ichiro; Natsume, Tohru; Chida, Kazuhiro; Nakatsu, Yusuke; Kamata, Hideaki; Asano, Tomoichiro; Takahashi, Shin-Ichiro

    2015-04-16

    Insulin-like growth factors (IGFs) induce proliferation of various cell types and play important roles in somatic growth and cancer development. Phosphorylation of insulin receptor substrate (IRS)-1/2 by IGF-I receptor tyrosine kinase is essential for IGF action. Here we identify Nedd4 as an IRS-2 ubiquitin ligase. Nedd4 monoubiquitinates IRS-2, which promotes its association with Epsin1, a ubiquitin-binding protein. Nedd4 recruits IRS-2 to the membrane, probably through promoting Epsin1 binding, and enhances IGF-I receptor-induced IRS-2 tyrosine phosphorylation. In thyroid FRTL-5 cells, activation of the cyclic AMP pathway increases the association of Nedd4 with IRS-2, thereby enhancing IRS-2-mediated signalling and cell proliferation induced by IGF-I. The Nedd4 and IRS-2 association is also required for maximal activation of IGF-I signalling and cell proliferation in prostate cancer PC-3 cells. Nedd4 overexpression accelerates zebrafish embryonic growth through IRS-2 in vivo. We conclude that Nedd4-induced monoubiquitination of IRS-2 enhances IGF signalling and mitogenic activity.

  12. The Time Course of JNK and P38 Activation in Cerebellar Granule Neurons Following Glucose Deprivation and BDNF Treatment

    PubMed Central

    Vakili Zahir, Niki; Abkhezr, Mousa; Khaje Piri, Zahra; Ostad, Seyyed Nasser; Kebriaezade, Abbass; Ghahremani, Mohammad Hossein

    2012-01-01

    Low glucose condition induces neuronal cell-death via intracellular mechanisms including mitogen-activated protein kinases (MAPK) signaling pathways. It has been shown that low glucose medium decreases neuronal survival in cerebellar granule neurons (CGNs). In this study, we have examined the activation of JNK, p38kinase and ERK1/2 pathways in low glucose medium in CGNs. The CGNs were prepared from new-born (P-2 and P-5) rats and cultured in Dulbecco′s Modified Eagle′s Medium high (DMEM-HIGH) glucose supplemented with Fetal Bovine Serum (FBS) 10% for 7 days. The glucose deprivation was induced through replacing the culture medium with the low glucose (5 mM) medium. The MAPK pathways activation was evaluated through phospho specific antibodies using western blot. The viability of cells was measuring using MTT assay. The results indicated that low glucose reduces the cell survival and brain-derived neurotrophic factor (BDNF) elevates the cell viability in CGNs. The basal c-Jun N-terminal kinase (JNK) activity was high in CGNs and glucose deprivation for 24 h had increased phospho-JNK level to 2-fold compared to basal. BDNF treatment reduced the basal JNK activity within 30 min but had no effect in longer incubations. BDNF also blocked the low glucose-induced JNK activation. In addition, CGNs exhibited high p38 phosphorylation in low glucose medium in 48 h. These results demonstrated that in sustained low glucose conditions, CGNs had high activity of stress-activated MAPK which could induce cellular damage. Moreover, BDNF can prevent JNK and p38 activation in stress conditions and increase cell viability. Our results suggest that in sustained stress conditions, inhibition of JNK and/or p38 pathways might protect neurons from damage in low glucose conditions. PMID:24250454

  13. Interferon-β counter-regulates its own pro-apoptotic action by activating p38 MAPK signalling in human SH-SY5Y neuroblastoma cells.

    PubMed

    Dedoni, Simona; Olianas, Maria C; Onali, Pierluigi

    2014-10-01

    Type I interferons (IFNs) induce apoptosis of neuroblastoma cells, but the molecular mechanisms regulating this event have not been completely elucidated. Here, we investigated the role of p38 mitogen activated protein kinase (MAPK) activity, a key regulator of apoptosis and a known modulator of IFN-induced responses in non-neuronal cells. We show that in SH-SY5Y human neuroblastoma cells IFN-β induced a delayed and sustained increase of p38 MAPK activity through a novel mechanism involving the sequential activation of Janus kinase-signal transducer and activator of transcription-1 signalling, enhanced expression of the NADPH oxidase catalytic subunit gp91(phox), increased reactive oxygen species production and stimulation of the MAPK kinase kinase transforming growth factor-β-activated kinase 1. Either blockade of p38 MAPK by the second generation inhibitors BIRB0796 and VX745 or siRNA knockdown of p38α MAPK enhanced IFN-β-induced apoptosis of neuroblastoma cells. Exposure to IFN-β increased the phosphorylation of the small heat shock protein HSP27 at Ser15, Ser78 and Ser82 with a time course similar to p38 MAPK activation and this response was suppressed by either p38α MAPK depletion or pharmacological inhibition of p38 MAPK and MAPK-activated protein kinase 2 (MK2). Either silencing of HSP27 expression by siRNA or MK2 inhibition potentiated IFN-β-induced apoptotic death. These results indicate that IFN-β-induced apoptosis of human SH-SY5Y neuroblastoma cells is associated with a long-lasting up-regulation of p38 MAPK activity, stimulation of MK2 and phosphorylation of the pro-survival protein HSP27. Moreover, the data show that inhibition of p38 MAPK signalling potentiates the anti-neuroblastoma activity of the cytokine, indicating that this pathway mediates a counter-regulatory response.

  14. Ras-dependent and -independent pathways target the mitogen-activated protein kinase network in macrophages.

    PubMed Central

    Büscher, D; Hipskind, R A; Krautwald, S; Reimann, T; Baccarini, M

    1995-01-01

    Mitogen-activated protein kinases (MAPKs) are activated upon a variety of extracellular stimuli in different cells. In macrophages, colony-stimulating factor 1 (CSF-1) stimulates proliferation, while bacterial lipopolysaccharide (LPS) inhibits cell growth and causes differentiation and activation. Both CSF-1 and LPS rapidly activate the MAPK network and induce the phosphorylation of two distinct ternary complex factors (TCFs), TCF/Elk and TCF/SAP. CSF-1, but not LPS, stimulated the formation of p21ras. GTP complexes. Expression of a dominant negative ras mutant reduced, but did not abolish, CSF-1-mediated stimulation of MEK and MAPK. In contrast, activation of the MEK kinase Raf-1 was Ras independent. Treatment with the phosphatidylcholine-specific phospholipase C inhibitor D609 suppressed LPS-mediated, but not CSF-1-mediated, activation of Raf-1, MEK, and MAPK. Similarly, down-regulation or inhibition of protein kinase C blocked MEK and MAPK induction by LPS but not that by CSF-1. Phorbol 12-myristate 13-acetate pretreatment led to the sustained activation of the Raf-1 kinase but not that of MEK and MAPK. Thus, activated Raf-1 alone does not support MEK/MAPK activation in macrophages. Phosphorylation of TCF/Elk but not that of TCF/SAP was blocked by all treatments that interfered with MAPK activation, implying that TCF/SAP was targeted by a MAPK-independent pathway. Therefore, CSF-1 and LPS target the MAPK network by two alternative pathways, both of which induce Raf-1 activation. The mitogenic pathway depends on Ras activity, while the differentiation signal relies on protein kinase C and phosphatidylcholine-specific phospholipase C activation. PMID:7799956

  15. Ozone induces a proinflammatory response in primary human bronchial epithelial cells through mitogen-activated protein kinase activation without nuclear factor-κB activation.

    PubMed

    McCullough, Shaun D; Duncan, Kelly E; Swanton, Samantha M; Dailey, Lisa A; Diaz-Sanchez, David; Devlin, Robert B

    2014-09-01

    Ground-level ozone (O3) is a ubiquitous environmental air pollutant that is a potent inducer of airway inflammation and has been linked with respiratory and cardiovascular morbidity and mortality. Some studies using transformed or immortalized cells have attributed O3-mediated expression of inflammatory cytokines with activation of the canonical NF-κB pathway. In this study, we sought to characterize the O3-mediated activation of cellular signaling pathways using primary human bronchial epithelial cells obtained from a panel of donors. We demonstrate that the O3-induced expression of proinflammatory cytokines requires the activation of the epidermal growth factor receptor/MEK/ERK and MKK4/p38 mitogen-activated signaling pathways but does not appear to involve activation of canonical NF-κB signaling. In addition to providing a novel mechanistic model for the O3-mediated induction of proinflammatory cytokines, these findings highlight the importance of using primary cells over cell lines in mechanistic studies.

  16. Trichoderma mitogen-activated protein kinase signaling is involved in induction of plant systemic resistance.

    PubMed

    Viterbo, Ada; Harel, Michal; Horwitz, Benjamin A; Chet, Ilan; Mukherjee, Prasun K

    2005-10-01

    The role of a mitogen-activated protein kinase (MAPK) TmkA in inducing systemic resistance in cucumber against the bacterial pathogen Pseudomonas syringae pv. lacrymans was investigated by using tmkA loss-of-function mutants of Trichoderma virens. In an assay where Trichoderma spores were germinated in proximity to cucumber roots, the mutants were able to colonize the plant roots as effectively as the wild-type strain but failed to induce full systemic resistance against the leaf pathogen. Interactions with the plant roots enhanced the level of tmkA transcript in T. virens and its homologue in Trichoderma asperellum. At the protein level, we could detect the activation of two forms reacting to the phospho-p44/42 MAPK antibody. Biocontrol experiments demonstrated that the tmkA mutants retain their biocontrol potential against Rhizoctonia solani in soil but are not effective against Sclerotium rolfsii in reducing disease incidence. Our results show that, unlike in many plant-pathogen interactions, Trichoderma TmkA MAPK is not involved in limited root colonization. Trichoderma, however, needs MAPK signaling in order to induce full systemic resistance in the plant.

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

    SciTech Connect

    Kim, Beom Su; Park, Ji-Yun; Kang, Hyo-Jin; Kim, Hyung-Jin; Lee, Jun

    2014-08-08

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

  18. Inhibition of endocytosis exacerbates TNF-α-induced endothelial dysfunction via enhanced JNK and p38 activation.

    PubMed

    Choi, Hyehun; Nguyen, Hong N; Lamb, Fred S

    2014-04-15

    Tumor necrosis factor-α (TNF-α) is a pro-inflammatory cytokine that causes endothelial dysfunction. Endocytosis of TNF-α receptors (TNFR) precedes endosomal reactive oxygen species (ROS) production, which is required for NF-κB activation in vascular smooth muscle cells. It is unknown how endocytosis of TNFRs impacts signaling in endothelial cells. We hypothesized that TNF-α-induced endothelial dysfunction is induced by both endosomal and cell surface events, including NF-κB and mitogen-activated protein kinases (MAPKs) activation, and endocytosis of the TNFR modifies signaling. Mesenteric artery segments from C57BL/6 mice were treated with TNF-α (10 ng/ml) for 22 h in tissue culture, with or without signaling inhibitors (dynasore for endocytosis, SP600125 for JNK, SB203580 for p38, U0126 for ERK), and vascular function was assessed. Endothelium-dependent relaxation to acetylcholine (ACh) was impaired by TNF-α, and dynasore exacerbated this, whereas JNK or p38 inhibition prevented these effects. In cultured endothelial cells from murine mesenteric arteries, dynasore potentiated JNK and p38 but not ERK phosphorylation and promoted cell death. NF-κB activation by TNF-α was decreased by dynasore. JNK inhibition dramatically increased both the magnitude and duration of TNF-α-induced NF-κB activation and potentiated intercellular adhesion molecule-1 (ICAM-1) activation. Dynasore still inhibited NF-κB activation in the presence of SP600125. Thus TNF-α-induced endothelial dysfunction is both JNK and p38 dependent. Endocytosis modulates the balance of NF-κB and MAPK signaling, and inhibition of NF-κB activation by JNK limits this pro-proliferative signal, which may contribute to endothelial cell death in response to TNF-α.

  19. Control of thrombopoietin-induced megakaryocytic differentiation by the mitogen-activated protein kinase pathway.

    PubMed Central

    Rouyez, M C; Boucheron, C; Gisselbrecht, S; Dusanter-Fourt, I; Porteu, F

    1997-01-01

    Thrombopoietin (TPO) is the major regulator of both growth and differentiation of megakaryocytes. We previously showed that both functions can be generated by TPO in the megakaryoblastic cell line UT7, in which murine Mpl was introduced, and are independently controlled by distinct regions of the cytoplasmic domain of Mpl. Particularly, residues 71 to 94 of this domain (deleted in the mutant mpl delta3) were found to be required for megakaryocytic maturation but dispensable for proliferation. We show here that TPO-induced differentiation in UT7 cells is tightly dependent on a strong, long-lasting activation of the mitogen-activated protein kinase (MAPK) pathway. Indeed, (i) in UT7-mpl cells, TPO induced a strong activation of extracellular signal-regulated kinases (ERK) which was persistent until at least 4 days in TPO-containing medium; (ii) a specific MAPK kinase (MEK) inhibitor inhibited TPO-induced megakaryocytic gene expression; (iii) the Mpl mutant mpl delta3, which displayed no maturation activity, transduced only a weak and transient ERK activation in UT7 cells; and (iv) TPO-induced megakaryocytic differentiation in UT7-mpl delta3 cells was partially restored by expression of a constitutively activated mutant of MEK. The capacity of TPO to trigger a strong and prolonged MAPK signal depended on the cell in which Mpl was introduced. In BAF3-mpl cells, TPO triggered a weak and transient ERK activation, similar to that induced in UT7-mpl delta3 cells. In these cells, no difference in MAPK activation was found between normal Mpl and mpl delta3. Thus, depending on the cellular context, several distinct regions of the cytoplasmic domain of Mpl and signaling pathways may contribute to generate quantitative variations in MAPK activation. PMID:9271377

  20. Inhibition of the mitogen-activated protein kinase pathway triggers B16 melanoma cell differentiation.

    PubMed

    Englaro, W; Bertolotto, C; Buscà, R; Brunet, A; Pagès, G; Ortonne, J P; Ballotti, R

    1998-04-17

    In B16 melanoma cells, mitogen-activated protein (MAP) kinases are activated during cAMP-induced melanogenesis (Englaro, W., Rezzonico, R., Durand-Clément, M., Lallemand, D., Ortonne, J. P., and Ballotti, R. (1995) J. Biol. Chem. 270, 24315-24320). To establish the role of the MAP kinases in melanogenesis, we studied the effects of a specific MAP kinase kinase (MEK) inhibitor PD 98059 on different melanogenic parameters. We showed that PD 98059 inhibits the activation of MAP kinase extracellular signal-regulated kinase 1 by cAMP, but does not impair the effects of cAMP either on the morphological differentiation, characterized by an increase in dendrite outgrowth, or on the up-regulation of tyrosinase that is the key enzyme in melanogenesis. On the contrary, PD 98059 promotes by itself cell dendricity and increases the tyrosinase amount and activity. Moreover, down-regulation of the MAP kinase pathway by PD 98059, or with dominant negative mutants of p21(ras) and MEK, triggers a stimulation of the tyrosinase promoter activity and enhances the effect of cAMP on this parameter. Conversely, activation of the MAP kinase pathway, using constitutive active mutants of p21(ras) and MEK, leads to an inhibition of basal and cAMP-induced tyrosinase gene transcription. These results demonstrate that the MAP kinase pathway activation is not required for cAMP-induced melanogenesis. Furthermore, the inhibition of this pathway induces B16 melanoma cell differentiation, while a sustained activation impairs the melanogenic effect of cAMP-elevating agents. PMID:9545341

  1. Isolation and characterization of a novel lectin with mitogenic activity from Pleurotus ferulae.

    PubMed

    Xu, Cheng-Jian; Wang, Yue-Xiang; Niu, Bo-Nan; Liu, Bing; Li, Ying-Biao; Wang, Xue-Ming; Lu, Shi-Ling

    2014-07-01

    Lectins are the tools for the determination of sugar chain structure. Recently, lectin arrays have become a popular new technology; therefore, lectins with specific sugar-binding properties are required. The objective of the study was to isolate a novel lectin from Pleurotus ferulae mushrooms and characterize its various biological activities. A novel lectin was extracted with deionized water, precipitated from the aqueous extract using 75% saturated (NH4)2SO4, and subjected on DEAE-cellulose followed by affinity chromatography on sepharose-6B. The activity was tested using hemagglutination assays, and carbohydrate-binding specificity was determined by glycan microarray analysis. Its effects on the mitogenic activity of mouse splenocytes were determined by MTT assay. The novel lectin was adsorbed on ion-exchange chromatography DEAE-cellulose and shown as a band with the molecular mass of 17.5 kDa on a SDS-PAGE and as a single 35.0-kDa peak in gel filtration on Superdex G-75. The hemagglutinating activity of the lectin was inhibited by D-glucose, lactose, D-galactose, and galactosamine. The lectin was stable on 60°C. The hemagglutinating activity of lectin was reduced by 50% at 70°C. At 80°C, it was further reduced to 6.25% of its original activity. The hemagglutinating activity was the highest at pH 6-9. Moreover, its hemagglutinating activity was inhibited by Mg2+ and Ca2+ ions. The lectin isolated from P. ferulae in the current study possessed highly potent hemagglutinating and proliferative activities toward mouse splenocytes. PMID:25016256

  2. 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

  3. A conserved mitogen-activated protein kinase pathway is required for mating in Candida albicans.

    PubMed

    Chen, Jiangye; Chen, Jing; Lane, Shelley; Liu, Haoping

    2002-12-01

    Candida albicans had been thought to lack a mating process until the recent discovery of a mating type-like locus and mating between MTLa and MTL(alpha) strains. To elucidate the molecular mechanisms that regulate mating in C. albicans, we examined the function of Cph1 and its upstream mitogen-activated protein (MAP) kinase pathway in mating, as they are homologues of the pheromone-responsive MAP kinase pathway in Saccharomyces cerevisiae. We found that overexpressing CPH1 in MTLa, but not in MTLa/alpha strains, induced the transcription of orthologues of S. cerevisiae pheromone-induced genes and also increased mating efficiency. Furthermore, cph1 and hst7 mutants were completely defective in mating, and cst20 and cek1 mutants showed reduced mating efficiency, as in S. cerevisiae. The partial mating defect in cek1 results from the presence of a functionally redundant MAP kinase, Cek2. CEK2 complemented the mating defect of a fus3 kss1 mutant of S. cerevisiae and was expressed only in MTLa or MTL(alpha), but not in MTLa/alpha cell types. Moreover, a cek1 cek2 double mutant was completely defective in mating. Our data suggest that the conserved MAP kinase pathway regulates mating in C. albicans. We also observed that C. albicans mating efficiency was greatly affected by medium composition, indicating the potential involvement of nutrient-sensing pathways in mating in addition to the MAP kinase pathway. PMID:12453219

  4. How Mitogen-Activated Protein Kinases Recognize and Phosphorylate Their Targets: A QM/MM Study

    PubMed Central

    Turjanski, Adrian Gustavo; Hummer, Gerhard; Gutkind, J. Silvio

    2009-01-01

    Mitogen-activated protein kinase (MAPK) signaling pathways play an essential role in the transduction of environmental stimuli to the nucleus, thereby regulating a variety of cellular processes, including cell proliferation, differentiation and programmed cell death. The components of the MAPK extracellular activated protein kinase (ERK) cascade represent attractive targets for cancer therapy as their aberrant activation is a frequent event among highly prevalent human cancers. To understand how MAPKs recognize and phosphorylate their targets is key to unravel their function. However, these events are still poorly understood due to the lack of complex structures of MAPKs with their bound targets in the active site. Here, we have modeled the interaction of ERK with a target peptide and analyzed the specificity towards Ser/Thr-Pro motifs. By using a Quantum Mechanics/Molecular Mechanics (QM/MM) approach we propose a mechanism for the phosphoryl transfer catalyzed by ERK that offers new insights into MAPK function. Our results suggest that 1) the proline residue has a role both in specificity and phospho transfer efficiency; 2) the reaction occurs in one step with ERK2 Asp147 acting as the catalytic base; 3) a conserved Lys in the kinase superfamily usually mutated to check kinase activity strongly stabilizes the transition state; and 4) the reaction mechanism is similar with either one or two Mg2+ ions in the active site. Taken together, our results provide a detailed description of the molecular events involved in the phosphorylation reaction catalyzed by MAPK and contributes to the general understanding of kinase activity. PMID:19361221

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

    PubMed

    Kim, Beom Su; Park, Ji-Yun; Kang, Hyo-Jin; Kim, Hyung-Jin; Lee, Jun

    2014-08-01

    Angiogenesis is an important biological process in tissue development and repair. Fucoidan has previously been shown to potentiate in vitro tube formation in the presence of basic fibroblast growth factor (FGF-2). However, the underlying molecular mechanism remains largely unknown. This study was designed to investigate the action of fucoidan in angiogenesis in human umbilical vein endothelial cells (HUVECs) and to explore fucoidan-signalling pathways. First, we evaluated the effect of fucoidan on cell proliferation. Matrigel-based tube formation and wound healing assays were performed to investigate angiogenesis. Matrix metalloproteinase-2 (MMP-2) mRNA expression and activity levels were analysed by reverse transcription polymerase chain reaction (RT-PCR) and zymography, respectively. Additionally, phosphorylation of mitogen-activated protein kinases (MAPKs) and protein kinase B (AKT) was detected by Western blot. The results indicate that fucoidan treatment significantly increased cell proliferation in the presence of FGF-2. Moreover, compared to the effect of FGF-2 alone, fucoidan and FGF-2 had a greater effect on tube formation and cell migration, and this effect was found to be synergistic. Furthermore, fucoidan enhanced the phosphorylation of extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), p38, and AKT. MMP-2 activation was also significantly increased. Specific inhibitors of p38 (SB203580) and JNK (SP600125) inhibited tube formation and wound healing, while an ERK inhibitor (PD98059) did not. MMP-2 activation and AKT phosphorylation were also attenuated and associated with the suppression of p38 and JNK phosphorylation, but not with that of ERK. These results indicate that fucoidan, in the presence of FGF-2, induces angiogenesis through AKT/MMP-2 signalling by activating p38 and JNK. These findings provide basic molecular information on the effect of fucoidan on angiogenesis in the presence of FGF-2.

  6. 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

  7. Porins from Salmonella enterica Serovar Typhimurium Activate the Transcription Factors Activating Protein 1 and NF-κB through the Raf-1-Mitogen-Activated Protein Kinase Cascade

    PubMed Central

    Galdiero, Massimiliano; Vitiello, Mariateresa; Sanzari, Emma; D’Isanto, Marina; Tortora, Annalisa; Longanella, Anna; Galdiero, Stefania

    2002-01-01

    In this study we examined the ability of Salmonella enterica serovar Typhimurium porins to activate activating protein 1 (AP-1) and nuclear factor κB (NF-κB) through the mitogen-activated protein kinase (MAPK) cascade, and we identified the AP-1-induced protein subunits. Our results demonstrate that these enzymes may participate in cell signaling pathways leading to AP-1 and NF-κB activation following porin stimulation of cells. Raf-1 was phosphorylated in response to the treatment of U937 cells with porins; moreover, the porin-mediated increase in Raf-1 phosphorylation is accompanied by the phosphorylation of MAPK kinase 1/2 (MEK1/2), p38, extracellular-signal-regulated kinase 1/2, and c-Jun N-terminal kinase. We used three different inhibitors of phosphorylation pathways: 2′-amino-3′-methoxyflavone (PD-098059), a selective inhibitor of MEK1 activator and the MAPK cascade; 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole (SB203580), a specific inhibitor of the p38 pathway; and 7β-acetoxy-1α,6β,9α-trihydroxy-8,13-epoxy-labd-14-en-11-one (forskolin), an inhibitor at the level of Raf-1 kinase. PD-098059 pretreatment of cells decreases AP-1 and NF-κB activation by lipopolysaccharide (LPS) but not by porins, and SB203580 pretreatment of cells decreases mainly AP-1 and NF-κB activation by porins; in contrast, forskolin pretreatment of cells does not affect AP-1 and NF-κB activation following either porin or LPS stimulation. Our data suggest that the p38 signaling pathway mainly regulates AP-1 and NF-κB activation in cells treated with S. enterica serovar Typhimurium porins. Antibody electrophoretic mobility shift assays showed that JunD and c-Fos binding is found in cells treated with porins, in cells treated with LPS, and in unstimulated cells. However, by 30 to 60 min of stimulation, a different complex including c-Jun appears in cells treated with porins or LPS, while the Fra-2 subunit is present only after porin stimulation

  8. Key signalling nodes in mammary gland development and cancer. Mitogen-activated protein kinase signalling in experimental models of breast cancer progression and in mammary gland development.

    PubMed

    Whyte, Jacqueline; Bergin, Orla; Bianchi, Alessandro; McNally, Sara; Martin, Finian

    2009-01-01

    Seven classes of mitogen-activated protein kinase (MAPK) intracellular signalling cascades exist, four of which are implicated in breast disease and function in mammary epithelial cells. These are the extracellular regulated kinase (ERK)1/2 pathway, the ERK5 pathway, the p38 pathway and the c-Jun N-terminal kinase (JNK) pathway. In some forms of human breast cancer and in many experimental models of breast cancer progression, signalling through the ERK1/2 pathway, in particular, has been implicated as being important. We review the influence of ERK1/2 activity on the organised three-dimensional association of mammary epithelial cells, and in models of breast cancer cell invasion. We assess the importance of epidermal growth factor receptor family signalling through ERK1/2 in models of breast cancer progression and the influence of ERK1/2 on its substrate, the oestrogen receptor, in this context. In parallel, we consider the importance of these MAPK-centred signalling cascades during the cycle of mammary gland development. Although less extensively studied, we highlight the instances of signalling through the p38, JNK and ERK5 pathways involved in breast cancer progression and mammary gland development.

  9. A role for mitogen-activated protein kinase in the spindle assembly checkpoint in XTC cells.

    PubMed

    Wang, X M; Zhai, Y; Ferrell, J E

    1997-04-21

    The spindle assembly checkpoint prevents cells whose spindles are defective or chromosomes are misaligned from initiating anaphase and leaving mitosis. Studies of Xenopus egg extracts have implicated the Erk2 mitogen-activated protein kinase (MAP kinase) in this checkpoint. Other studies have suggested that MAP kinases might be important for normal mitotic progression. Here we have investigated whether MAP kinase function is required for mitotic progression or the spindle assembly checkpoint in vivo in Xenopus tadpole cells (XTC). We determined that Erk1 and/or Erk2 are present in the mitotic spindle during prometaphase and metaphase, consistent with the idea that MAP kinase might regulate or monitor the status of the spindle. Next, we microinjected purified recombinant XCL100, a Xenopus MAP kinase phosphatase, into XTC cells in various stages of mitosis to interfere with MAP kinase activation. We found that mitotic progression was unaffected by the phosphatase. However, XCL100 rendered the cells unable to remain arrested in mitosis after treatment with nocodazole. Cells injected with phosphatase at prometaphase or metaphase exited mitosis in the presence of nocodazole-the chromosomes decondensed and the nuclear envelope re-formed-whereas cells injected with buffer or a catalytically inactive XCL100 mutant protein remained arrested in mitosis. Coinjection of constitutively active MAP kinase kinase-1, which opposes XCL100's effects on MAP kinase, antagonized the effects of XCL100. Since the only known targets of MAP kinase kinase-1 are Erk1 and Erk2, these findings argue that MAP kinase function is required for the spindle assembly checkpoint in XTC cells.

  10. Molecular cloning and characterization of mitogen-activated protein kinase 2 in Toxoplasma gondii

    PubMed Central

    Huang, Huan; Ma, Yan Fen; Bao, Yi; Lee, Hattie; Lisanti, Michael P; Tanowitz, Herbert B

    2011-01-01

    Mitogen-activated protein kinase (MAPK) pathways are major signal transduction systems by which eukaryotic cells convert environmental cues to intracellular events, such as cell proliferation and differentiation. Toxoplasma gondii is an obligate intracellular protozoan that is both a human and animal pathogen. This Apicomplexan causes significant morbidity and mortality in immune-competent and immune-compromised hosts. In humans, the most common manifestations of T. gondii infections are chorioretinitis in congenital infection and encephalitis in immune-compromised patients, such as patients with advanced AIDS. We have identified a T. gondii homolog of the MAPK family that we have called TgMAPK2. Sequence analyses demonstrated that TgMAPK2 has homology with lower eukaryotic ERK2 but has significant differences from mammalian ERK2. TgMAPK2 has an open reading frame of 2,037 bp, 678 amino acids, and its molecular weight is 73.1 kDa. It contains the typical 12 subdomains of a MAPK and has a TDY motif in the dual phosphorylation and activation subdomains. This suggests that TgMAPK2 may play an important role in stress response. Recombinant TgMAPK2 was catalytically active and was not inhibited by a human ERK2 inhibitor, FR180204. A partial TgMAPK2 lacking the ATP-binding motifs GxGxxGxV was successfully regulated by a ligand-controlled destabilization domain (ddFKBP) expression vector system in T. gondii. Since TgMAPK2 is significantly different from its mammalian counterpart, it may be useful as a drug target. This work establishes a foundation for further study for this unique kinase. PMID:22030559

  11. Mercuric ions inhibit mitogen-activated protein kinase dephosphorylation by inducing reactive oxygen species

    SciTech Connect

    Haase, Hajo; Engelhardt, Gabriela; Hebel, Silke; Rink, Lothar

    2011-01-01

    Mercury intoxication profoundly affects the immune system, in particular, signal transduction of immune cells. However, the mechanism of the interaction of mercury with cellular signaling pathways, such as mitogen activated protein kinases (MAPK), remains elusive. Therefore, the objective of this study is to investigate three potential ways in which Hg{sup 2+} ions could inhibit MAPK dephosphorylation in the human T-cell line Jurkat: (1) by direct binding to phosphatases; (2) by releasing cellular zinc (Zn{sup 2+}); and (3) by inducing reactive oxygen species (ROS). Hg{sup 2+} causes production of ROS, measured by dihydrorhodamine 123, and triggers ROS-mediated Zn{sup 2+} release, detected with FluoZin-3. Yet, phosphatase-inhibition is not mediated by binding of Zn{sup 2+} or Hg{sup 2+}. Rather, phosphatases are inactivated by at least two forms of thiol oxidation; initial inhibition is reversible with reducing agents such as Tris(2-carboxyethyl)phosphine. Prolonged inhibition leads to non-reversible phosphatase oxidation, presumably oxidizing the cysteine thiol to sulfinic- or sulfonic acid. Notably, phosphatases are a particularly sensitive target for Hg{sup 2+}-induced oxidation, because phosphatase activity is inhibited at concentrations of Hg{sup 2+} that have only minor impact on over all thiol oxidation. This phosphatase inhibition results in augmented, ROS-dependent MAPK phosphorylation. MAPK are important regulators of T-cell function, and MAPK-activation by inhibition of phosphatases seems to be one of the molecular mechanisms by which mercury affects the immune system.

  12. Mitogen-Activated Protein Kinase Kinase 3 Regulates Seed Dormancy in Barley.

    PubMed

    Nakamura, Shingo; Pourkheirandish, Mohammad; Morishige, Hiromi; Kubo, Yuta; Nakamura, Masako; Ichimura, Kazuya; Seo, Shigemi; Kanamori, Hiroyuki; Wu, Jianzhong; Ando, Tsuyu; Hensel, Goetz; Sameri, Mohammad; Stein, Nils; Sato, Kazuhiro; Matsumoto, Takashi; Yano, Masahiro; Komatsuda, Takao

    2016-03-21

    Seed dormancy has fundamental importance in plant survival and crop production; however, the mechanisms regulating dormancy remain unclear [1-3]. Seed dormancy levels generally decrease during domestication to ensure that crops successfully germinate in the field. However, reduction of seed dormancy can cause devastating losses in cereals like wheat (Triticum aestivum L.) and barley (Hordeum vulgare L.) due to pre-harvest sprouting, the germination of mature seed (grain) on the mother plant when rain occurs before harvest. Understanding the mechanisms of dormancy can facilitate breeding of crop varieties with the appropriate levels of seed dormancy [4-8]. Barley is a model crop [9, 10] and has two major seed dormancy quantitative trait loci (QTLs), SD1 and SD2, on chromosome 5H [11-19]. We detected a QTL designated Qsd2-AK at SD2 as the single major determinant explaining the difference in seed dormancy between the dormant cultivar "Azumamugi" (Az) and the non-dormant cultivar "Kanto Nakate Gold" (KNG). Using map-based cloning, we identified the causal gene for Qsd2-AK as Mitogen-activated Protein Kinase Kinase 3 (MKK3). The dormant Az allele of MKK3 is recessive; the N260T substitution in this allele decreases MKK3 kinase activity and appears to be causal for Qsd2-AK. The N260T substitution occurred in the immediate ancestor allele of the dormant allele, and the established dormant allele became prevalent in barley cultivars grown in East Asia, where the rainy season and harvest season often overlap. Our findings show fine-tuning of seed dormancy during domestication and provide key information for improving pre-harvest sprouting tolerance in barley and wheat. PMID:26948880

  13. Evolutionary history of mitogen-activated protein kinase (MAPK) genes in Lotus, Medicago, and Phaseolus

    PubMed Central

    Neupane, Achal; Nepal, Madhav P; Benson, Benjamin V; MacArthur, Kenton J; Piya, Sarbottam

    2013-01-01

    Mitogen-Activated Protein Kinase (MAPK) genes encode proteins that mediate various signaling pathways associated with biotic and abiotic stress responses in eukaryotes. The MAPK genes form a 3-tier signal transduction cascade between cellular stimuli and physiological responses. Recent identification of soybean MAPKs and availability of genome sequences from other legume species allowed us to identify their MAPK genes. The main objectives of this study were to identify MAPKs in 3 legume species, Lotus japonicus, Medicago truncatula, and Phaseolus vulgaris, and to assess their phylogenetic relationships. We used approaches in comparative genomics for MAPK gene identification and named the newly identified genes following Arabidopsis MAPK nomenclature model. We identified 19, 18, and 15 MAPKs and 7, 4, and 9 MAPKKs in the genome of Lotus japonicus, Medicago truncatula, and Phaseolus vulgaris, respectively. Within clade placement of MAPKs and MAPKKs in the 3 legume species were consistent with those in soybean and Arabidopsis. Among 5 clades of MAPKs, 4 founder clades were consistent to MAPKs of other plant species and orthologs of MAPK genes in the fifth clade-"Clade E" were consistent with those in soybean. Our results also indicated that some gene duplication events might have occurred prior to eudicot-monocot divergence. Highly diversified MAPKs in soybean relative to those in 3 other legume species are attributable to the polyploidization events in soybean. The identification of the MAPK genes in the legume species is important for the legume crop improvement; and evolutionary relationships and functional divergence of these gene members provide insights into plant genome evolution. PMID:24317362

  14. Hyperpigmentation mechanism of methyl 3,5-di-caffeoylquinate through activation of p38 and MITF induction of tyrosinase.

    PubMed

    Kim, Hyo Jung; Kim, Jin Sook; Woo, Je-Tae; Lee, Ik-Soo; Cha, Byung-Yoon

    2015-07-01

    Methyl 3,5-di-caffeoylquinate (3,5-diCQM) has been used for the treatment of various diseases in oriental medicine, but its effect on melanogenesis has not been reported yet. In this study, the molecular mechanism of 3,5-diCQM-induced melanogenesis was investigated. It was found that 3,5-diCQM induced synthesis of melanin pigments in murine B16F10 melanoma cells in a concentration-dependent manner. Treatment of cells with 3,5-diCQM for 48 h increased extracellular and intracellular melanin production and tyrosinase activity. The expressions of tyrosinase, tyrosinase-related protein 1 (TRP1), and TRP2 were up-regulated in a dose-dependent manner 48 h after 3,5-diCQM treatment. Western blot analysis showed that 3,5-diCQM increased the phosphorylation of p38 mitogen-activated protein kinase and cAMP responsive element binding as well as the expression of microphthalmia-associated transcription factor. In addition, 3,5-diCQM-stimulated cAMP production, and 3,5-diCQM-induced tyrosinase activity and melanin synthesis were attenuated by H89, a protein kinase A inhibitor. These results suggested that 3,5-diCQM-mediated activation of the p38 pathway may represent a novel approach for an effective therapy for vitiligo and hair graying. PMID:26018825

  15. Infectious bursal disease virus infection induces macrophage activation via p38 MAPK and NF-kappaB pathways.

    PubMed

    Khatri, Mahesh; Sharma, Jagdev M

    2006-06-01

    In the present study, we show that infection with infectious bursal disease virus (IBDV) causes activation of macrophages, the key cells involved in inflammatory and immune-regulatory functions. Exposure of cultured spleen macrophages (SM) from SPF chickens to IBDV resulted in the production of nitric oxide (NO). In addition, there was upregulation of mRNA expression of inducible nitric oxide synthase (iNOS), IL-8 and cyclooxygenase-2 (COX-2). The signal transduction pathways involved in macrophage activation were examined. The role of mitogen-activated protein kinases (MAPKs) and nuclear factor-kappaB (NF-kappaB) was tested by using specific pharmacological inhibitors. Addition of p38 MAPK inhibitor, SB-203580 and NF-kappaB inhibitor Bay 11-7082, suppressed IBDV-induced NO production and mRNA expression of iNOS, IL-8 and COX-2. The results suggest that IBDV uses cellular signal transduction machinery, in particular the p38 MAPK and NF-kappaB pathways, to elicit macrophage activation. The increased production of NO, IL-8 and COX-2 by macrophages may contribute to bursa inflammatory responses commonly seen during the acute IBDV infection.

  16. Structural characterization and antitumor and mitogenic activity of a lectin from the gill of bighead carp (Aristichthys nobilis).

    PubMed

    Yao, Dongrui; Pan, Saikun; Zhou, Mingqian

    2012-12-01

    In this study, we investigated the gross structure, secondary structure, and antitumor and mitogenic activity of GANL, a lectin from the gill of bighead carp (Aristichthys nobilis). We used infrared spectroscopy, β-elimination, and circular dichroism spectroscopy to determine the structure of GANL. We measured antiproliferation activity against six human tumor cell lines and mitogenic activity against murine splenocytes using the MTT assay. Based on infrared spectroscopy and β-elimination, we conclude that GANL is a glycoprotein. The protein and carbohydrate moieties are joined by O-glycosidic linkage. A circular dichroism spectroscopic analysis revealed that the secondary structure of GANL consists of α-helices (34.8 %), β-sheets (12.1 %), β-turns (24.5 %), and unordered structures (33.0 %). GANL exerted potent antitumor activity against the HeLa cell line (IC(50) = 11.86 μg/mL) and a mitogenic effect on murine splenocytes in the MTT assay. GANL, a lectin that is isolated from the gills of bighead carp, is a glycoprotein with potent antitumor and mitogenic activity. PMID:22714932

  17. Pyroglutamic acid stimulates DNA synthesis in rat primary hepatocytes through the mitogen-activated protein kinase pathway.

    PubMed

    Inoue, Shinjiro; Okita, Yoichi; de Toledo, Andreia; Miyazaki, Hiroyuki; Hirano, Eiichi; Morinaga, Tetsuo

    2015-01-01

    We purified pyroglutamic acid from human placental extract and identified it as a potent stimulator of rat primary hepatocyte DNA synthesis. Pyroglutamic acid dose-dependently stimulated DNA synthesis, and this effect was inhibited by PD98059, a dual specificity mitogen-activated protein kinase kinase 1 (MAP2K1) inhibitor. Therefore, pyroglutamic acid stimulated DNA synthesis in rat primary hepatocytes via MAPK signaling.

  18. Characterization of p38 MAPKs from orange-spotted grouper, Epinephelus coioides involved in SGIV infection.

    PubMed

    Cai, Jia; Huang, Youhua; Wei, Shina; Huang, Xiaohong; Ye, Fuzhou; Fu, Jing; Qin, Qiwei

    2011-12-01

    p38 mitogen-activated protein kinases (MAPKs) are broadly expressed signaling molecules that involves in the regulation of cellular responsible for various extracellular stimuli. In this study, three p38 MAPK genes (Ec-p38a, p38b and p38β) were cloned from grouper, Epinephelus coioides and their characteristics were investigated in vitro. Although Ec-p38a, p38b and p38β showed high homologies to other fish p38a MPAK, p38b MAPK and p38β MAPK, respectively, they all contained the conserved structures of Thr-Gly-Tyr (TGY) motif and substrate binding site Ala-Thr-Arg-Trp (ATRW). Phylogenetic analysis indicated that Ec-p38a, p38b and p38β are more closely related to those from fish than mammals. The tissue distribution patterns of Ec-p38a, p38b and p38β were different, and Ec-p38β was up-regulated most obviously in head kidney after Singapore grouper iridovirus (SGIV) infection. Overexpression of Ec-p38β in FHM cells delayed the occurrence of CPE induced by SGIV infection. Further analysis indicated that overexpression of Ec-p38β inhibited viral gene transcription and protein synthesis, as well as SGIV induced typical apoptosis in fish cells. Taken together, our data indicated that Ec-p38β played a crucial role in regulating apoptosis and virus replication during iridovirus infection.

  19. Excessive L-cysteine induces vacuole-like cell death by activating endoplasmic reticulum stress and mitogen-activated protein kinase signaling in intestinal porcine epithelial cells.

    PubMed

    Ji, Yun; Wu, Zhenlong; Dai, Zhaolai; Sun, Kaiji; Zhang, Qing; Wu, Guoyao

    2016-01-01

    High intake of dietary cysteine is extremely toxic to animals and the underlying mechanism remains largely unknown. This study was conducted to test the hypothesis that excessive L-cysteine induces cell death by activating endoplasmic reticulum (ER) stress and mitogen-activated protein kinase (MAPK) signaling in intestinal porcine epithelial cells. Jejunal enterocytes were cultured in the presence of 0-10 mmol/L L-cysteine. Cell viability, morphologic alterations, mRNA levels for genes involved in ER stress, protein abundances for glucose-regulated protein 78, C/EBP homologous protein (CHOP), alpha subunit of eukaryotic initiation factor-2 (eIF2α), extracellular signal-regulated kinase (ERK1/2), p38 MAPK, and c-Jun N-terminal protein kinase (JNK1/2) were determined. The results showed that L-cysteine (5-10 mmol/L) reduced cell viability (P < 0.05) and led to vacuole-like cell death in intestinal porcine epithelial cells. These adverse effects of L-cysteine were not affected by the autophagy inhibitor 3-methyladenine. The protein abundances for CHOP, phosphorylated (p)-eIF2α, p-JNK1/2, p-p38 MAPK, and the spliced form of XBP-1 mRNA were enhanced (P < 0.05), whereas those for p-ERK1/2 were reduced (P < 0.05). Collectively, excessive L-cysteine induces vacuole-like cell death via the activation of ER stress and MAPK signaling in small intestinal epithelial cells. These signaling pathways may be potential targets for developing effective strategies to prevent the toxicity of dietary cysteine.

  20. The food contaminant deoxynivalenol activates the mitogen activated protein kinases in the intestine: interest of ex vivo models as an alternative to in vivo experiments.

    PubMed

    Lucioli, Joelma; Pinton, Philippe; Callu, Patrick; Laffitte, Joëlle; Grosjean, François; Kolf-Clauw, Martine; Oswald, Isabelle P; Bracarense, Ana Paula F R L

    2013-05-01

    Trichothecenes induce changes in the intestinal barrier function through decreased expression of cell junction proteins and apoptosis of enterocytes. The mitogen activated protein kinases (MAPK) play an important role in the signaling pathways of cell turnover and differentiation. Using ex vivo and in vivo approaches, the purpose of this study was to investigate the ability of low doses of DON to induce histological changes in the intestine and to activate the MAPK ERK 1/2, p38 and JNK. Twelve weaning piglets received during four weeks a control diet or a DON-contaminated diet (2.3 mg DON/kg feed). Six weaning piglets were used to prepare jejunal explants (ex vivo model). Explants were exposed during 4 h to vehicle, 5 or 10 μM DON. Intestinal changes were graded using a histological score. Pigs fed a DON-diet and explants exposed to DON showed a significant decrease in the jejunal score. In both models, the toxin significantly enhanced phosphorylation of ERK 1/2 and p38, whereas the increased phosphorylation of JNK was non significant. Taken together these results indicate that in vivo or ex vivo exposure of intestinal tissue to DON lead to similar intestinal lesions and activation of MAPK. These effects could impair the homeostasis of intestinal tissue in the aspects of barrier function and immune protection. The similarity of the in vivo and ex vivo results provides also strong evidence that the jejunal explant model is a good alternative for toxicological studies in intestinal tissue. PMID:23403092

  1. Low concentrations of trichosanthin induce apoptosis and cell cycle arrest via c-Jun N-terminal protein kinase/mitogen-activated protein kinase activation.

    PubMed

    Zhang, Duo; Chen, Bin; Zhou, Jian; Zhou, Lin; Li, Qing; Liu, Fei; Chou, Kuang-Yen; Tao, Lei; Lu, Li-Ming

    2015-01-01

    Trichosanthin (TCS) is a type I ribosome--inactivating protein, which inhibits cell viability in human epithelial type 2 (HEp-2) and AMC-HN-8 human laryngeal epidermoid carcinoma cells. Although TCS is a potential chemotherapeutic agent, its mechanism of action remains to be elucidated. In the present study, HEp-2 and AMC-HN-8 cells were treated with different concentrations of TCS combined with or without cisplatin. After 5 days of successive treatment, different experimental groups were detected using a cell counting kit-8 and the collected supernatants were analyzed using a lactate dehydrogenase kit. Flow cytometric assays were performed to detect apoptosis and cell cycle arrest in the HEp-2 and AMC-HN-8 cells, reverse transcription quantitative polymerase chain reaction was performed to detect the levels of p27, p21WAF and western blot analysis was performed to detect changes in c-Jun N-terminal protein kinase (JNK)/phosphorylated (phospho)-JNK, p38/phospho-p38, extracellular signal-regulated kinase (ERK)/phospho-ERK, caspase-3 and caspase-9 in the HEp-2 and AMC-HN-8 cancer cells. TCS significantly inhibited the cell viability of the HEp-2 and AMC-HN-8 cells, independently of necrosis. TCS induced apoptosis and increased the percentage of HEp-2 and AMC-HN-8 cells in the S-phase of the cell cycle. In addition, the JNK/mitogen-activated protein kinase (MAPK) pathway was activated by TCS in the HEp-2 and AMC-HN-8 cells. Low concentrations of TCS also induced apoptosis and S-phase cell cycle arrest in the HEp-2 and AMC-HN-8 cells. The antitumor effects of TCS may be associated with JNK/MAPK activation.

  2. Titanium dioxide nanoparticles stimulate sea urchin immune cell phagocytic activity involving TLR/p38 MAPK-mediated signalling pathway.

    PubMed

    Pinsino, Annalisa; Russo, Roberta; Bonaventura, Rosa; Brunelli, Andrea; Marcomini, Antonio; Matranga, Valeria

    2015-09-28

    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.

  3. 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

  4. Protein tyrosine kinase and mitogen-activated protein kinase signalling pathways contribute to differences in heterophil-mediated innate immune responsiveness between two lines of broilers

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Protein tyrosine phosphorylation mediates signal transduction of cellular processes, with protein tyrosine kinases (PTKs) regulating virtually all signaling events. The mitogen-activated protein kinase (MAPK) super-family consists of three conserved pathways that convert receptor activation into ce...

  5. Prostaglandins from Cytosolic Phospholipase A2α/Cyclooxygenase-1 Pathway and Mitogen-activated Protein Kinases Regulate Gene Expression in Candida albicans-infected Macrophages.

    PubMed

    Yun, Bogeon; Lee, HeeJung; Jayaraja, Sabarirajan; Suram, Saritha; Murphy, Robert C; Leslie, Christina C

    2016-03-25

    In Candida albicans-infected resident peritoneal macrophages, activation of group IVA cytosolic phospholipase A2(cPLA2α) by calcium- and mitogen-activated protein kinases triggers the rapid production of prostaglandins I2 and E2 through cyclooxygenase (COX)-1 and regulates gene expression by increasing cAMP. InC. albicans-infected cPLA2α(-/-)or COX-1(-/-)macrophages, expression ofI l10,Nr4a2, and Ptgs2 was lower, and expression ofTnfα was higher, than in wild type macrophages. Expression was reconstituted with 8-bromo-cAMP, the PKA activator 6-benzoyl-cAMP, and agonists for prostaglandin receptors IP, EP2, and EP4 in infected but not uninfected cPLA2α(-/-)or COX-1(-/-)macrophages. InC. albicans-infected cPLA2α(+/+)macrophages, COX-2 expression was blocked by IP, EP2, and EP4 receptor antagonists, indicating a role for both prostaglandin I2 and E2 Activation of ERKs and p38, but not JNKs, by C. albicansacted synergistically with prostaglandins to induce expression of Il10,Nr4a2, and Ptgs2. Tnfα expression required activation of ERKs and p38 but was suppressed by cAMP. Results using cAMP analogues that activate PKA or Epacs suggested that cAMP regulates gene expression through PKA. However, phosphorylation of cAMP-response element-binding protein (CREB), the cAMP-regulated transcription factor involved inIl10,Nr4a2,Ptgs2, andTnfα expression, was not mediated by cAMP/PKA because it was similar inC. albicans-infected wild type and cPLA2α(-/-)or COX-1(-/-)macrophages. CREB phosphorylation was blocked by p38 inhibitors and induced by the p38 activator anisomycin but not by the PKA activator 6-benzoyl-cAMP. Therefore, MAPK activation inC. albicans-infected macrophages plays a dual role by promoting the cPLA2α/prostaglandin/cAMP/PKA pathway and CREB phosphorylation that coordinately regulate immediate early gene expression.

  6. Low concentration of arsenic could induce caspase-3 mediated head kidney macrophage apoptosis with JNK-p38 activation in Clarias batrachus

    SciTech Connect

    Datta, Soma; Mazumder, Shibnath; Ghosh, Debabrata; Dey, Saibal; Bhattacharya, Shelley

    2009-12-15

    We had earlier demonstrated that chronic exposure (30 days) to micro-molar concentration (0.50 muM) of arsenic induced head kidney macrophage (HKM) death in Clarias batrachus. The purpose of the present study is to characterize the nature of HKM death induced by arsenic and elucidate the signal transduction pathways involved in the process. Arsenic-induced HKM death was apoptotic in nature as evident from DNA gel, Annexin V-propidium iodide, Hoechst 33342 staining and TdT-mediated dUTP nick end labeling (TUNEL) assays. Inhibitor studies and immunoblot analyses further demonstrated that arsenic-induced HKM apoptosis involved activation of caspase-3 and cleavage of poly(ADP-ribose) polymerase, a well-characterized caspase-3 substrate. Preincubation with antioxidants N-acetyl-cysteine or dimethyl sulfoxide significantly lowered reactive oxygen species (ROS) levels in arsenic-treated HKM and prevented caspase activation, malondialdehyde formation and HKM apoptosis. Arsenic induced membrane translocation of the NADPH oxidase subunit p47{sup phox}. Preincubation with apocynin and diphenyleneiodonium chloride, both selective inhibitors of NADPH oxidases, prevented p47{sup phox} translocation, ROS production and HKM death. Exposure of HKM to arsenic induced the activation of mitogen-activated protein kinase family (MAPK) proteins including c-Jun NH{sub 2}-terminal protein kinase (JNK) and p38 mitogen-activated protein kinase (p38). Preincubation of HKM with p38 inhibitor SB203580 and JNK inhibitor SP600125 protected the HKM against arsenic-induced apoptosis. We conclude that exposure to micro-molar concentration of arsenic induces ROS generation through the activation of NADPH oxidases, which in turn causes caspase-3 mediated HKM apoptosis. In addition, the study also indicates a role of p38-JNK pathway in arsenic-induced HKM apoptosis in C. batrachus.

  7. Diphenylarsinic Acid Induced Activation of Cultured Rat Cerebellar Astrocytes: Phosphorylation of Mitogen-Activated Protein Kinases, Upregulation of Transcription Factors, and Release of Brain-Active Cytokines.

    PubMed

    Negishi, Takayuki; Matsumoto, Mami; Kojima, Mikiya; Asai, Ryota; Kanehira, Tomoko; Sakaguchi, Fumika; Takahata, Kazuaki; Arakaki, Rina; Aoyama, Yohei; Yoshida, Hikari; Yoshida, Kenji; Yukawa, Kazunori; Tashiro, Tomoko; Hirano, Seishiro

    2016-03-01

    Diphenylarsinic acid (DPAA) was detected as the primary compound responsible for the arsenic poisoning that occurred in Kamisu, Ibaraki, Japan, where people using water from a well that was contaminated with a high level of arsenic developed neurological (mostly cerebellar) symptoms and dysregulation of regional cerebral blood flow. To understand the underlying molecular mechanism of DPAA-induced cerebellar symptoms, we focused on astrocytes, which have a brain-protective function. Incubation with 10 µM DPAA for 96 h promoted cell proliferation, increased the expression of antioxidative stress proteins (heme oxygenase-1 and heat shock protein 70), and induced the release of cytokines (MCP-1, adrenomedullin, FGF2, CXCL1, and IL-6). Furthermore, DPAA overpoweringly increased the phosphorylation of three major mitogen-activated protein kinases (MAPKs) (ERK1/2, p38MAPK, and SAPK/JNK), which indicated MAPK activation, and subsequently induced expression and/or phosphorylation of transcription factors (Nrf2, CREB, c-Jun, and c-Fos) in cultured rat cerebellar astrocytes. Structure-activity relationship analyses of DPAA and other related pentavalent organic arsenicals revealed that DPAA at 10 µM activated astrocytes most effective among organic arsenicals tested at the same dose. These results suggest that in a cerebellum exposed to DPAA, abnormal activation of the MAPK-transcription factor pathway and irregular secretion of these neuroactive, glioactive, and/or vasoactive cytokines in astrocytes can be the direct/indirect cause of functional abnormalities in surrounding neurons, glial cells, and vascular cells: This in turn might lead to the onset of cerebellar symptoms and disruption of cerebral blood flow. PMID:26645585

  8. Oncogenic Ras activation of Raf/mitogen-activated protein kinase-independent pathways is sufficient to cause tumorigenic transformation.

    PubMed Central

    Khosravi-Far, R; White, M A; Westwick, J K; Solski, P A; Chrzanowska-Wodnicka, M; Van Aelst, L; Wigler, M H; Der, C J

    1996-01-01

    Substantial evidence supports a critical role for the activation of the Raf-1/MEK/mitogen-activated protein kinase pathway in oncogenic Ras-mediated transformation. For example, dominant negative mutants of Raf-1, MEK, and mitogen-activated protein kinase all inhibit Ras transformation. Furthermore, the observation that plasma membrane-localized Raf-1 exhibits the same transforming potency as oncogenic Ras suggests that Raf-1 activation alone is sufficient to mediate full Ras transforming activity. However, the recent identification of other candidate Ras effectors (e.g., RalGDS and phosphatidylinositol-3 kinase) suggests that activation of other downstream effector-mediated signaling pathways may also mediate Ras transforming activity. In support of this, two H-Ras effector domain mutants, H-Ras(12V, 37G) and H-Ras(12V, 40C), which are defective for Raf binding and activation, induced potent tumorigenic transformation of some strains of NIH 3T3 fibroblasts. These Raf-binding defective mutants of H-Ras induced a transformed morphology that was indistinguishable from that induced by activated members of Rho family proteins. Furthermore, the transforming activities of both of these mutants were synergistically enhanced by activated Raf-1 and inhibited by the dominant negative RhoA(19N) mutant, indicating that Ras may cause transformation that occurs via coordinate activation of Raf-dependent and -independent pathways that involves Rho family proteins. Finally, cotransfection of H-Ras(12V, 37G) and H-Ras(12V, 40C) resulted in synergistic cooperation of their focus-forming activities, indicating that Ras activates at least two Raf-independent, Ras effector-mediated signaling events. PMID:8668210

  9. Discovery and Characterization of Non-ATP Site Inhibitors of the Mitogen Activated Protein (MAP) Kinases

    SciTech Connect

    Comess, Kenneth M.; Sun, Chaohong; Abad-Zapatero, Cele; Goedken, Eric R.; Gum, Rebecca J.; Borhani, David W.; Argiriadi, Maria; Groebe, Duncan R.; Jia, Yong; Clampit, Jill E.; Haasch, Deanna L.; Smith, Harriet T.; Wang, Sanyi; Song, Danying; Coen, Michael L.; Cloutier, Timothy E.; Tang, Hua; Cheng, Xueheng; Quinn, Christopher; Liu, Bo; Xin, Zhili; Liu, Gang; Fry, Elizabeth H.; Stoll, Vincent; Ng, Teresa I.; Banach, David; Marcotte, Doug; Burns, David J.; Calderwood, David J.; Hajduk, Philip J.

    2012-03-02

    Inhibition of protein kinases has validated therapeutic utility for cancer, with at least seven kinase inhibitor drugs on the market. Protein kinase inhibition also has significant potential for a variety of other diseases, including diabetes, pain, cognition, and chronic inflammatory and immunologic diseases. However, as the vast majority of current approaches to kinase inhibition target the highly conserved ATP-binding site, the use of kinase inhibitors in treating nononcology diseases may require great selectivity for the target kinase. As protein kinases are signal transducers that are involved in binding to a variety of other proteins, targeting alternative, less conserved sites on the protein may provide an avenue for greater selectivity. Here we report an affinity-based, high-throughput screening technique that allows nonbiased interrogation of small molecule libraries for binding to all exposed sites on a protein surface. This approach was used to screen both the c-Jun N-terminal protein kinase Jnk-1 (involved in insulin signaling) and p38{alpha} (involved in the formation of TNF{alpha} and other cytokines). In addition to canonical ATP-site ligands, compounds were identified that bind to novel allosteric sites. The nature, biological relevance, and mode of binding of these ligands were extensively characterized using two-dimensional {sup 1}H/{sup 13}C NMR spectroscopy, protein X-ray crystallography, surface plasmon resonance, and direct enzymatic activity and activation cascade assays. Jnk-1 and p38{alpha} both belong to the MAP kinase family, and the allosteric ligands for both targets bind similarly on a ledge of the protein surface exposed by the MAP insertion present in the CMGC family of protein kinases and distant from the active site. Medicinal chemistry studies resulted in an improved Jnk-1 ligand able to increase adiponectin secretion in human adipocytes and increase insulin-induced protein kinase PKB phosphorylation in human hepatocytes, in

  10. Natural feed contaminant zearalenone decreases the expressions of important pro- and anti-inflammatory mediators and mitogen-activated protein kinase/NF-κB signalling molecules in pigs.

    PubMed

    Pistol, Gina Cecilia; Gras, Mihail Alexandru; Marin, Daniela Eliza; Israel-Roming, Florentina; Stancu, Mariana; Taranu, Ionelia

    2014-02-01

    Zearalenone (ZEA) is an oestrogenic mycotoxin produced by Fusarium species, considered to be a risk factor from both public health and agricultural perspectives. In the present in vivo study, a feeding trial was conducted to evaluate the in vivo effect of a ZEA-contaminated diet on immune response in young pigs. The effect of ZEA on pro-inflammatory (TNF-α, IL-8, IL-6, IL-1β and interferon-γ) and anti-inflammatory (IL-10 and IL-4) cytokines and other molecules involved in inflammatory processes (matrix metalloproteinases (MMP)/tissue inhibitors of matrix metalloproteinases (TIMP), nuclear receptors: PPARγ and NF-κB1, mitogen-activated protein kinases (MAPK): mitogen-activated protein kinase kinase kinase 7 (TAK1)/mitogen-activated protein kinase 14 (p38α)/mitogen-activated protein kinase 8 (JNK1)/ mitogen-activated protein kinase 9 (JNK2)) in the liver of piglets was investigated. The present results showed that a concentration of 316 parts per billion ZEA leads to a significant decrease in the levels of pro- and anti-inflammatory cytokines at both gene expression and protein levels, correlated with a decrease in the levels of other inflammatory mediators, MMP and TIMP. The results also showed that dietary ZEA induces a dramatic reduction in the expressions of NF-κB1 and TAK1/p38α MAPK genes in the liver of the experimentally intoxicated piglets, and has no effect on the expression of PPARγ mRNA. The present results suggest that the toxic action of ZEA begins in the upstream of the MAPK signalling pathway by the inhibition of TAK1, a MAPK/NF-κB activator. In conclusion, the present study shows that ZEA alters several important parameters of the hepatic cellular immune response. From an economic point of view, these data suggest that, in pigs, ZEA is not only a powerful oestrogenic mycotoxin but also a potential hepatotoxin when administered through the oral route. Therefore, the present results represent additional data from cellular and molecular levels

  11. Natural feed contaminant zearalenone decreases the expressions of important pro- and anti-inflammatory mediators and mitogen-activated protein kinase/NF-κB signalling molecules in pigs.

    PubMed

    Pistol, Gina Cecilia; Gras, Mihail Alexandru; Marin, Daniela Eliza; Israel-Roming, Florentina; Stancu, Mariana; Taranu, Ionelia

    2014-02-01

    Zearalenone (ZEA) is an oestrogenic mycotoxin produced by Fusarium species, considered to be a risk factor from both public health and agricultural perspectives. In the present in vivo study, a feeding trial was conducted to evaluate the in vivo effect of a ZEA-contaminated diet on immune response in young pigs. The effect of ZEA on pro-inflammatory (TNF-α, IL-8, IL-6, IL-1β and interferon-γ) and anti-inflammatory (IL-10 and IL-4) cytokines and other molecules involved in inflammatory processes (matrix metalloproteinases (MMP)/tissue inhibitors of matrix metalloproteinases (TIMP), nuclear receptors: PPARγ and NF-κB1, mitogen-activated protein kinases (MAPK): mitogen-activated protein kinase kinase kinase 7 (TAK1)/mitogen-activated protein kinase 14 (p38α)/mitogen-activated protein kinase 8 (JNK1)/ mitogen-activated protein kinase 9 (JNK2)) in the liver of piglets was investigated. The present results showed that a concentration of 316 parts per billion ZEA leads to a significant decrease in the levels of pro- and anti-inflammatory cytokines at both gene expression and protein levels, correlated with a decrease in the levels of other inflammatory mediators, MMP and TIMP. The results also showed that dietary ZEA induces a dramatic reduction in the expressions of NF-κB1 and TAK1/p38α MAPK genes in the liver of the experimentally intoxicated piglets, and has no effect on the expression of PPARγ mRNA. The present results suggest that the toxic action of ZEA begins in the upstream of the MAPK signalling pathway by the inhibition of TAK1, a MAPK/NF-κB activator. In conclusion, the present study shows that ZEA alters several important parameters of the hepatic cellular immune response. From an economic point of view, these data suggest that, in pigs, ZEA is not only a powerful oestrogenic mycotoxin but also a potential hepatotoxin when administered through the oral route. Therefore, the present results represent additional data from cellular and molecular levels

  12. Interferon-Gamma Increases Endothelial Permeability by Causing Activation of p38 MAP Kinase and Actin Cytoskeleton Alteration.

    PubMed

    Ng, Chin Theng; Fong, Lai Yen; Sulaiman, Mohd Roslan; Moklas, Mohamad Aris Mohd; Yong, Yoke Keong; Hakim, Muhammad Nazrul; Ahmad, Zuraini

    2015-07-01

    Interferon-gamma (IFN-γ) is known to potentiate the progression of inflammatory diseases, such as inflammatory bowel disease and atherosclerosis. IFN-γ has been found to disrupt the barrier integrity of epithelial and endothelial cell both in vivo and in vitro. However, the mechanisms of IFN-γ underlying increased endothelial cell permeability have not been extensively elucidated. We reported that IFN-γ exhibits a biphasic nature in increasing endothelial permeability. The changes observed in the first phase (4-8 h) involve cell retraction and rounding in addition to condensed peripheral F-actin without a significant change in the F-/G-actin ratio. However, cell elongation, stress fiber formation, and an increased F-/G-actin ratio were noticed in the second phase (16-24 h). Consistent with our finding from the permeability assay, IFN-γ induced the formation of intercellular gaps in both phases. A delayed phase of increased permeability was observed at 12 h, which paralleled the onset of cell elongation, stress fiber formation, and increased F-/G-actin ratio. In addition, IFN-γ stimulated p38 mitogen-activated protein (MAP) kinase phosphorylation over a 24 h period. Inhibition of p38 MAP kinase by SB203580 prevented increases in paracellular permeability, actin rearrangement, and increases in the F-/G-actin ratio caused by IFN-γ. Our results suggest that p38 MAP kinase is activated in response to IFN-γ and causes actin rearrangement and altered cell morphology, which in turn mediates endothelial cell hyperpermeability. The F-/G-actin ratio might be involved in the regulation of actin distribution and cell morphology rather than the increased permeability induced by IFN-γ.

  13. Insulin stimulates mitogen-activated protein kinase by a Ras-independent pathway in 3T3-L1 adipocytes.

    PubMed

    Carel, K; Kummer, J L; Schubert, C; Leitner, W; Heidenreich, K A; Draznin, B

    1996-11-29

    To characterize tissue-specific differences in insulin signaling, we compared the mechanisms of mitogen-activated protein (MAP) kinase activation by insulin in the mitogenically active 3T3-L1 fibroblasts with the metabolically active 3T3-L1 adipocytes. In both cell lines, insulin significantly increased p21(ras).GTP loading (1.5-2-fold) and MAP kinase activity (5-8-fold). Inhibition of Ras farnesylation with lovastatin blocked activation of p21(ras) and Raf-1 kinase in both 3T3-L1 fibroblasts and 3T3-L1 adipocytes. In 3T3-L1 fibroblasts, this was accompanied by an inhibition of the stimulatory effect of insulin on MAP kinase. In contrast, in 3T3-L1 adipocytes, despite an inhibition of activation of p21(ras) and Raf-1 by lovastatin, insulin continued to stimulate MAP kinase activity. Fractionation of the cell lysates on the FPLC Mono-Q column revealed that lovastatin inhibited insulin stimulation of ERK2 (and, to a lesser extent, ERK1) in 3T3-L1 fibroblasts and had no effect on the insulin-stimulated ERK2 in 3T3-L1 adipocytes. These results demonstrate an important distinction between the mechanism of insulin signaling in the metabolically and mitogenically active cells. Insulin activates MAP kinase by the Ras-dependent pathway in the 3T3-L1 fibroblasts and by the Ras-independent pathway in the 3T3-L1 adipocytes.

  14. Additive effect of heat on the UVB-induced tyrosinase activation and melanogenesis via ERK/p38/MITF pathway in human epidermal melanocytes.

    PubMed

    Gu, Wei-Jie; Ma, Hui-Jun; Zhao, Guang; Yuan, Xiao-Ying; Zhang, Ping; Liu, Wen; Ma, Li-Juan; Lei, Xiao-Bing

    2014-08-01

    Heat is known as an environmental factor that causes significant skin pigmentation, but its effects on melanogenesis have been poorly studied. It has been shown that mitogen-activated protein kinase (MAPK) is involved in ultraviolet B (UVB) and stress-induced melanogenesis in melanocytes. In this study, we investigated the effects of heat and UVB, on melanocyte melanogenesis, differentiation, and MAPK phosphorylation. The results showed that heat (1 h at 40 °C for 5 days) increased cell dendrites, enlarged cell bodies, and induced extracellular signal-regulated kinases (ERK)/p38/MITF activation but did not influence melanogenesis of human epidermal melanocytes from skin phototype III. UVB irradiation (20 mJ/cm(2) for 5 days) induced melanogenesis and c-jun N-terminal kinases (JNK)/p38/MITF/tyrosinase activation in melanocytes from skin phototype III. UVB combined with heat resulted in much more significant tyrosinase activation and melanogenesis as compared with UVB alone in melanocytes from skin phototype III. Furthermore, heat treatment and UVB irradiation induced JNK, ERK, and p38 activation but not melanogenic and morphological changes in melanocytes from skin phototype I. These findings suggested that heat promoted melanocyte differentiation, probably via heat-induced ERK/p38/MITF/activation. Furthermore, heat had an additive effect on the UVB-induced tyrosinase activation and melanogenesis. These results provide a new clue for dermatologists for the treatment of hypopigmented skin disease with heat combined with UVB irradiation.

  15. Role of mitogen activated protein kinases in skin tumorigenicity of Patulin

    SciTech Connect

    Saxena, Neha; Ansari, Kausar M.; Kumar, Rahul; Chaudhari, Bhushan P.; Dwivedi, Premendra D.; Das, Mukul

    2011-12-15

    WHO has highlighted the need to evaluate dermal toxicity of mycotoxins including Patulin (PAT), detected in several fruits. In this study the skin carcinogenic potential of topically applied PAT was investigated. Single topical application of PAT (400 nmol) showed enhanced cell proliferation ({approx} 2 fold), along with increased generation of ROS and activation of ERK, p38 and JNK MAPKs, in mouse skin. PAT exposure also showed activation of downstream target proteins, c-fos, c-Jun and NF-{kappa}B transcription factors. Further, single topical application of PAT (400 nmol) followed by twice weekly application of TPA resulted in tumor formation after 14 weeks, indicating the tumor initiating activity of PAT. However no tumors were observed when PAT was used either as a complete carcinogen (80 nmol) or as a tumor promoter (20 nmol and 40 nmol) for 25 weeks. Histopathological findings of tumors found in PAT/TPA treated mice showed that these tumors were of squamous cell carcinoma type and similar to those found in the positive control group (DMBA/TPA) along with significant increase of lipid peroxidation and decrease in free sulfydryls, catalase, superoxide dismutase and glutathione reductase activities. The results suggest the possible role of free radicals in PAT mediated dermal tumorigenicity involving MAPKs. -- Highlights: Black-Right-Pointing-Pointer Single topical application of Patulin showed enhanced cell proliferation. Black-Right-Pointing-Pointer Patulin activate MAPKs, c-fos, c-Jun and NF-{kappa}B transcription factors. Black-Right-Pointing-Pointer Patulin showed skin tumor initiating potential. Black-Right-Pointing-Pointer We could not detect skin tumor promoting potential of Patulin at the tested dose. Black-Right-Pointing-Pointer However prolonged exposure of Patulin at a higher dose may promote tumor.

  16. Synthesis and p38 Inhibitory Activity of Some Novel Substituted N,N'-Diarylurea Derivatives.

    PubMed

    Zhu, Dianxi; Xing, Qifeng; Cao, Ruiyuan; Zhao, Dongmei; Zhong, Wu

    2016-01-01

    We have identified a novel series of substituted N,N'-diarylurea p38α inhibitors. The inhibitory activity of the target compounds against the enzyme p38α, MAPKAPK2 in BHK cells, TNF-α release in LPS-stimulated THP-1 cells and p38α binding experiments were tested. Among these compounds, 25a inhibited the p38α enzyme with an IC50 value of 0.47 nM and a KD value of 1.54 × 10(-8) and appears to be the most promising one in the series. PMID:27223276

  17. Paroxetine-induced apoptosis in human osteosarcoma cells: Activation of p38 MAP kinase and caspase-3 pathways without involvement of [Ca{sup 2+}]{sub i} elevation

    SciTech Connect

    Chou, C.-T.; He Shiping; Jan, C.-R. . E-mail: crjan@isca.vghks.gov.tw

    2007-02-01

    Selective serotonin reuptake inhibitors (SSRIs), a group of antidepressants, are generally used for treatment of various mood and anxiety disorders. There has been much research showing the anti-tumor and cytotoxic activities of some antidepressants; but the detailed mechanisms were unclear. In cultured human osteosarcoma cells (MG63), paroxetine reduced cell viability in a concentration- and time-dependent manner. Paroxetine caused apoptosis as assessed by propidium iodide-stained cells and increased caspase-3 activation. Although immunoblotting data revealed that paroxetine could activate the phosphorylation of extracellular signal-regulated kinase (ERK), c-Jun NH{sub 2}-terminal kinase (JNK) and p38 mitogen-activated protein kinase (p38 MAPK), only SB203580 (a p38 MAPK inhibitor) partially prevented cells from apoptosis. Paroxetine also induced [Ca{sup 2+}]{sub i} increases which involved the mobilization of intracellular Ca{sup 2+} stored in the endoplasmic reticulum and Ca{sup 2+} influx from extracellular medium. However, pretreatment with BAPTA/AM, a Ca{sup 2+} chelator, to prevent paroxetine-induced [Ca{sup 2+}]{sub i} increases did not protect cells from death. The results suggest that in MG63 cells, paroxetine caused Ca{sup 2+}-independent apoptosis via inducing p38 MAPK-associated caspase-3 activation.

  18. Mitogen-Activated Protein Kinase Phosphorylation of Splicing Factor 45 (SPF45) Regulates SPF45 Alternative Splicing Site Utilization, Proliferation, and Cell Adhesion

    PubMed Central

    Al-Ayoubi, Adnan M.; Zheng, Hui; Liu, Yuying; Bai, Tao

    2012-01-01

    The regulation of alternative mRNA splicing factors by extracellular cues and signal transduction cascades is poorly understood. Using an engineered extracellular signal-regulated kinase 2 (ERK2) that can utilize ATP analogs, we have identified the alternative mRNA splicing factor 45 (SPF45), which is overexpressed in cancer, as a novel coimmunoprecipitating ERK2 substrate. ERK2 phosphorylated SPF45 on Thr71 and Ser222 in vitro and in cells in response to H-RasV12, B-RAF-V600E, and activated MEK1. Jun N-terminal kinase 1 (JNK1) and p38α also phosphorylated SPF45 in vitro and associated with SPF45 in cells. SPF45 was differentially phosphorylated in cells by all three mitogen-activated protein (MAP) kinases in response to phorbol myristate acid (PMA), H2O2, UV, and anisomycin stimulation. ERK and p38 activation decreased SPF45-dependent exon 6 exclusion from fas mRNA in a minigene assay in cells. Stable overexpression of SPF45 in SKOV-3 cells dramatically inhibited cell proliferation in a phosphorylation-dependent manner through inhibition of ErbB2 expression. SPF45 overexpression also induced EDA inclusion into fibronectin transcripts and fibronectin expression in a phosphorylation-dependent and -independent manner, respectively, specifically affecting cellular adhesion to a fibronectin matrix. These data identify SPF45 as the first splicing factor regulated by multiple MAP kinase pathways and show effects of both SPF45 overexpression and phosphorylation. PMID:22615491

  19. Signaling of chloroquine-induced stress in the yeast Saccharomyces cerevisiae requires the Hog1 and Slt2 mitogen-activated protein kinase pathways.

    PubMed

    Baranwal, Shivani; Azad, Gajendra Kumar; Singh, Vikash; Tomar, Raghuvir S

    2014-09-01

    Chloroquine (CQ) has been under clinical use for several decades, and yet little is known about CQ sensing and signaling mechanisms or about their impact on various biological pathways. We employed the budding yeast Saccharomyces cerevisiae as a model organism to study the pathways targeted by CQ. Our screening with yeast mutants revealed that it targets histone proteins and histone deacetylases (HDACs). Here, we also describe the novel role of mitogen-activated protein kinases Hog1 and Slt2, which aid in survival in the presence of CQ. Cells deficient in Hog1 or Slt2 are found to be CQ hypersensitive, and both proteins were phosphorylated in response to CQ exposure. CQ-activated Hog1p is translocated to the nucleus and facilitates the expression of GPD1 (glycerol-3-phosphate dehydrogenase), which is required for the synthesis of glycerol (one of the major osmolytes). Moreover, cells treated with CQ exhibited an increase in intracellular reactive oxygen species (ROS) levels and the effects were rescued by addition of reduced glutathione to the medium. The deletion of SOD1, the superoxide dismutase in yeast, resulted in hypersensitivity to CQ. We have also observed P38 as well as P42/44 phosphorylation in HEK293T human cells upon exposure to CQ, indicating that the kinds of responses generated in yeast and human cells are similar. In summary, our findings define the multiple biological pathways targeted by CQ that might be useful for understanding the toxicity modulated by this pharmacologically important molecule.

  20. Fisetin Ameliorated Photodamage by Suppressing the Mitogen-Activated Protein Kinase/Matrix Metalloproteinase Pathway and Nuclear Factor-κB Pathways.

    PubMed

    Chiang, Hsiu-Mei; Chan, Shih-Yun; Chu, Yin; Wen, Kuo-Ching

    2015-05-13

    Ultraviolet (UV) irradiation is one of the most important extrinsic factors contributing to skin photodamage. After UV irradiation, a series of signal transductions in the skin will be activated, leading to inflammatory response and photoaged skin. In this study, fisetin, a flavonol that exists in fruits and vegetables, was investigated for its photoprotective effects. The results revealed that 5-25 μM fisetin inhibits cyclooxygenase-2 (COX-2) and matrix metalloproteinase (MMP)-1, MMP-3, MMP-9 expression induced by ultraviolet B (UVB) irradiation in human skin fibroblasts. In addition, fisetin suppressed UVB-induced collagen degradation. With regard to its effect on upper-stream signal transduction, we found that fisetin reduced the expression of ultraviolet (UV)-induced ERK, JNK, and p38 phosphorylation in the mitogen-activated protein kinase (MAP kinase) pathway. Furthermore, fisetin reduced inhibitor κB (IκB) degradation and increased the amount of p65, which is a major subunit of nuclear factor-κB (NF-κB), in cytoplasm. It also suppressed NF-κB translocated to the nucleus and inhibited cAMP response element-binding protein (CREB) Ser-133 phosphorylation level in the phosphoinositide 3-kinase/protein kinase B/CREB (PI3K/AKT/CREB) pathway. Finally, fisetin inhibited UV-induced intracellular reactive oxygen species (ROS), prostaglandin E2 (PGE2), and nitric oxide (NO) generation. The mentioned effects and mechanisms suggest that fisetin can be used in the development of photoprotective agents.

  1. Myocilin Stimulates Osteogenic Differentiation of Mesenchymal Stem Cells through Mitogen-activated Protein Kinase Signaling*

    PubMed Central

    Kwon, Heung Sun; Johnson, Thomas V.; Tomarev, Stanislav I.

    2013-01-01

    Myocilin is a secreted glycoprotein that is expressed in ocular and non-ocular tissues. Mutations in the MYOCILIN gene may lead to juvenile- and adult-onset primary open-angle glaucoma. Here we report that myocilin is expressed in bone marrow-derived mesenchymal stem cells (MSCs) and plays a role in their differentiation into osteoblasts in vitro and in osteogenesis in vivo. Expression of myocilin was detected in MSCs derived from mouse, rat, and human bone marrow, with human MSCs exhibiting the highest level of myocilin expression. Expression of myocilin rose during the course of human MSC differentiation into osteoblasts but not into adipocytes, and treatment with exogenous myocilin further enhanced osteogenesis. MSCs derived from Myoc-null mice had a reduced ability to differentiate into the osteoblastic lineage, which was partially rescued by exogenous extracellular myocilin treatment. Myocilin also stimulated osteogenic differentiation of wild-type MSCs, which was associated with activation of the p38, Erk1/2, and JNK MAP kinase signaling pathways as well as up-regulated expression of the osteogenic transcription factors Runx2 and Dlx5. Finally, cortical bone thickness and trabecular volume, as well as the expression level of osteopontin, a known factor of bone remodeling and osteoblast differentiation, were reduced dramatically in the femurs of Myoc-null mice compared with wild-type mice. These data suggest that myocilin should be considered as a target for improving the bone regenerative potential of MSCs and may identify a new role for myocilin in bone formation and/or maintenance in vivo. PMID:23629661

  2. Breast tumor kinase (protein tyrosine kinase 6) regulates heregulin-induced activation of ERK5 and p38 MAP kinases in breast cancer cells.

    PubMed

    Ostrander, Julie Hanson; Daniel, Andrea R; Lofgren, Kristopher; Kleer, Celina G; Lange, Carol A

    2007-05-01

    Total tyrosine kinase activity is often elevated in both cytosolic and membrane fractions of malignant breast tissue and correlates with a decrease in disease-free survival. Breast tumor kinase (Brk; protein tyrosine kinase 6) is a soluble tyrosine kinase that was cloned from a metastatic breast tumor and found to be overexpressed in a majority of breast tumors. Herein, we show that Brk is overexpressed in 86% of invasive ductal breast tumors and coexpressed with ErbB family members in breast cancer cell lines. Additionally, the ErbB ligand, heregulin, activates Brk kinase activity. Knockdown of Brk by stable expression of short hairpin RNA (shRNA) in T47D breast cancer cells decreases proliferation and blocks epidermal growth factor (EGF)- and heregulin-induced activation of Rac GTPase, extracellular signal-regulated kinase (ERK) 5, and p38 mitogen-activated protein kinase (MAPK) but not Akt, ERK1/2, or c-Jun NH(2)-terminal kinase. Furthermore, EGF- and heregulin-induced cyclin D1 expression is dependent on p38 signaling and inhibited by Brk shRNA knockdown. The myocyte enhancer factor 2 transcription factor target of p38 MAPK and ERK5 signaling is also sensitive to altered Brk expression. Finally, heregulin-induced migration of T47D cells requires p38 MAPK activity and is blocked by Brk knockdown. These results place Brk in a novel signaling pathway downstream of ErbB receptors and upstream of Rac, p38 MAPK, and ERK5 and establish the ErbB-Brk-Rac-p38 MAPK pathway as a critical mediator of breast cancer cell migration.

  3. Protein-tyrosine-phosphatase 2C is phosphorylated and inhibited by 44-kDa mitogen-activated protein kinase.

    PubMed Central

    Peraldi, P; Zhao, Z; Filloux, C; Fischer, E H; Van Obberghen, E

    1994-01-01

    Protein-tyrosine-phosphatase 2C (PTP2C, also named SHPTP2, SHPTP3, or PTP1D) is a cytosolic enzyme with two Src homology 2 domains. We have investigated its regulation by phosphorylation in PC12 rat pheochromocytoma cells. In untreated cells, PTP2C was phosphorylated predominantly on serine residues. A 5-min treatment with epidermal growth factor (EGF) induced an increase in phosphorylation on threonine and, to a lesser degree, on serine. After 45 min of exposure to EGF, PTP2C phosphorylation returned to basal levels. Using an in vitro kinase assay, we found that the 44-kDa mitogen-activated protein kinase, p44mapk, phosphorylated PTP2C on serine and threonine residues. This phosphorylation resulted in a pronounced inhibition of PTP2C enzyme activity measured with phosphorylated EGF receptors as substrate. Moreover, in intact PC12 cells, PTP2C was also inhibited following a short EGF treatment, but its activity returned to normal when the exposure to EGF was maintained for 45 min. The profile of this response to EGF can be inversely correlated to that of the stimulatory action of EGF on p44mapk. These data suggest that the EGF-induced regulation of PTP2C activity is mediated by p44mapk. These findings provide evidence for an additional role of the mitogen-activated protein kinase cascade--namely, the regulation of a PTP. Images PMID:8197172

  4. 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

  5. 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

  6. Signalling pathway leading to an activation of mitogen-activated protein kinase by stimulating M3 muscarinic receptor.

    PubMed

    Kim, J Y; Yang, M S; Oh, C D; Kim, K T; Ha, M J; Kang, S S; Chun, J S

    1999-01-15

    The signalling pathway leading to an activation of mitogen-activated protein (MAP) kinase subtypes Erk-1 and -2 upon stimulation of muscarinic receptor with carbachol in human neuroblastoma SK-N-BE2(C) cells was investigated. Carbachol activated Erk-1/-2 by stimulating M3 muscarinic receptor, as determined by specific antagonists for individual muscarinic receptors. The activation of Erk-1/-2 by carbachol was blocked by the inhibition or down-regulation of protein kinase C (PKC). Among the multiple PKC isoforms expressed in SK-N-BE2(C) cells, only PKCepsilon was activated by the treatment of carbachol, and selective down-regulation of PKCepsilon was sufficient to block Erk-1/-2 activation. Carbachol treatment induced activation of the serine/threonine protein kinase Raf, and an inhibition of Raf blocked Erk-1/-2 activation. Ectopic expression of inhibitory small GTPase Ras, RasN17, blocked the carbachol-induced Raf activation without affecting the activation of PKCepsilon, while the inhibition of PKC blocked the Raf activation. Thus, these results suggest that carbachol-induced activation of PKCepsilon mediates Erk-1/-2 activation by a sequential activation of Ras, Raf and MAP kinase kinase.

  7. Dynamic ubiquitination of the mitogen-activated protein kinase kinase (MAPKK) Ste7 determines mitogen-activated protein kinase (MAPK) specificity.

    PubMed

    Hurst, Jillian H; Dohlman, Henrik G

    2013-06-28

    Ubiquitination is a post-translational modification that tags proteins for proteasomal degradation. In addition, there is a growing appreciation that ubiquitination can influence protein activity and localization. Ste7 is a prototype MAPKK in yeast that participates in both the pheromone signaling and nutrient deprivation/invasive growth pathways. We have shown previously that Ste7 is ubiquitinated upon pheromone stimulation. Here, we show that the Skp1/Cullin/F-box ubiquitin ligase SCF(Cdc4) and the ubiquitin protease Ubp3 regulate Ste7 ubiquitination and signal specificity. Using purified components, we demonstrate that SCF(Cdc4) ubiquitinates Ste7 directly. Using gene deletion mutants, we show that SCF(Cdc4) and Ubp3 have opposing effects on Ste7 ubiquitination. Although SCF(Cdc4) is necessary for proper activation of the pheromone MAPK Fus3, Ubp3 is needed to limit activation of the invasive growth MAPK Kss1. Finally, we show that Fus3 phosphorylates Ubp3 directly and that phosphorylation of Ubp3 is necessary to limit Kss1 activation. These results reveal a feedback loop wherein one MAPK limits the ubiquitination of an upstream MAPKK and thereby prevents spurious activation of a second competing MAPK. PMID:23645675

  8. Involvement of mitogen-activated protein kinase activation in the signal-transduction pathways of the soya bean oxidative burst.

    PubMed Central

    Taylor, A T; Kim, J; Low, P S

    2001-01-01

    The oxidative burst constitutes one of the most rapid defence responses characterized in the Plant Kingdom. We have observed that four distinct elicitors of the soya bean oxidative burst activate kinases of masses approximately 44 kDa and approximately 47 kDa. Evidence that these kinases regulate production of reactive oxygen species include: (i) their rapid activation by oxidative burst elicitors, (ii) their tight temporal correlation between activation/deactivation of the kinases and activation/deactivation of the oxidative burst, (iii) the identical pharmacological profile of kinase activation and oxidant production for 13 commonly used inhibitors, and (iv) the autologous activation of both kinases and oxidant production by calyculin A and cantharidin, two phosphatase inhibitors. Immunological and biochemical studies reveal that the activated 44 kDa and 47 kDa kinases are mitogen-activated protein (MAP) kinase family members. The kinases prefer myelin basic protein as a substrate, and they phosphorylate primarily on threonine residues. The kinases are themselves phosphorylated on tyrosine residues, and this phosphorylation is required for activity. Finally, both kinases are recognized by an antibody against activated MAP kinase immediately after (but not before) cell stimulation by elicitors. Based on these and other observations, a preliminary sequence of signalling steps linking elicitor stimulation, kinase activation and Ca(2+) entry, to initiation of oxidant production, is proposed. PMID:11311144

  9. Isolation of a Glucosamine Binding Leguminous Lectin with Mitogenic Activity towards Splenocytes and Anti-Proliferative Activity towards Tumor Cells

    PubMed Central

    Chan, Yau Sang; Wong, Jack Ho; Fang, Evandro Fei; Pan, Wenliang; Ng, Tzi Bun

    2012-01-01

    A dimeric 64-kDa glucosamine-specific lectin was purified from seeds of Phaseolus vulgaris cv. “brown kidney bean.” The simple 2-step purification protocol involved affinity chromatography on Affi-gel blue gel and gel filtration by FPLC on Superdex 75. The lectin was absorbed on Affi-gel blue gel and desorbed using 1M NaCl in the starting buffer. Gel filtration on Superdex 75 yielded a major absorbance peak that gave a single 32-kDa band in SDS-PAGE. Hemagglutinating activity was completely preserved when the ambient temperature was in the range of 20°C–60°C. However, drastic reduction of the activity occurred at temperatures above 65°C. Full hemagglutinating activity of the lectin was observed at an ambient pH of 3 to 12. About 50% activity remained at pH 0–2, and only residual activity was observed at pH 13–14. Hemagglutinating activity of the lectin was inhibited by glucosamine. The brown kidney bean lectin elicited maximum mitogenic activity toward murine splenocytes at 2.5 µM. The mitogenic activity was nearly completely eliminated in the presence of 250 mM glucosamine. The lectin also increased mRNA expression of the cytokines IL-2, TNF-α and IFN-γ. The lectin exhibited antiproliferative activity toward human breast cancer (MCF7) cells, hepatoma (HepG2) cells and nasopharyngeal carcinoma (CNE1 and CNE2) cells with IC50 of 5.12 µM, 32.85 µM, 3.12 µM and 40.12 µM respectively after treatment for 24 hours. Flow cytometry with Annexin V and propidum iodide staining indicated apoptosis of MCF7 cells. Hoechst 33342 staining also indicated formation of apoptotic bodies in MCF7 cells after exposure to brown kidney bean lectin. Western blotting revealed that the lectin-induced apoptosis involved ER stress and unfolded protein response. PMID:22720002

  10. Isolation of a glucosamine binding leguminous lectin with mitogenic activity towards splenocytes and anti-proliferative activity towards tumor cells.

    PubMed

    Chan, Yau Sang; Wong, Jack Ho; Fang, Evandro Fei; Pan, Wenliang; Ng, Tzi Bun

    2012-01-01

    A dimeric 64-kDa glucosamine-specific lectin was purified from seeds of Phaseolus vulgaris cv. "brown kidney bean." The simple 2-step purification protocol involved affinity chromatography on Affi-gel blue gel and gel filtration by FPLC on Superdex 75. The lectin was absorbed on Affi-gel blue gel and desorbed using 1M NaCl in the starting buffer. Gel filtration on Superdex 75 yielded a major absorbance peak that gave a single 32-kDa band in SDS-PAGE. Hemagglutinating activity was completely preserved when the ambient temperature was in the range of 20 °C-60 °C. However, drastic reduction of the activity occurred at temperatures above 65 °C. Full hemagglutinating activity of the lectin was observed at an ambient pH of 3 to 12. About 50% activity remained at pH 0-2, and only residual activity was observed at pH 13-14. Hemagglutinating activity of the lectin was inhibited by glucosamine. The brown kidney bean lectin elicited maximum mitogenic activity toward murine splenocytes at 2.5 µM. The mitogenic activity was nearly completely eliminated in the presence of 250 mM glucosamine. The lectin also increased mRNA expression of the cytokines IL-2, TNF-α and IFN-γ. The lectin exhibited antiproliferative activity toward human breast cancer (MCF7) cells, hepatoma (HepG2) cells and nasopharyngeal carcinoma (CNE1 and CNE2) cells with IC(50) of 5.12 µM, 32.85 µM, 3.12 µM and 40.12 µM respectively after treatment for 24 hours. Flow cytometry with Annexin V and propidum iodide staining indicated apoptosis of MCF7 cells. Hoechst 33342 staining also indicated formation of apoptotic bodies in MCF7 cells after exposure to brown kidney bean lectin. Western blotting revealed that the lectin-induced apoptosis involved ER stress and unfolded protein response. PMID:22720002

  11. Fibroblast growth factor, but not activin, is a potent activator of mitogen-activated protein kinase in Xenopus explants.

    PubMed Central

    Graves, L M; Northrop, J L; Potts, B C; Krebs, E G; Kimelman, D

    1994-01-01

    Isolated explants from the animal hemisphere of Xenopus embryos were incubated with Xenopus basic fibroblast growth factor (XbFGF) or human activin A. XbFGF incubation resulted in the rapid activation of mitogen-activated protein kinase (MAPK) and ribosomal S6 protein kinase (pp90rsk) in a dose-dependent manner with the highest levels of activation occurring at 50 ng/ml. Maximal activation occurred within 6-10 min after the addition of growth factor, and the activity of both kinases declined to unstimulated levels after 30 min. Activin was unable to activate either MAPK or pp90rsk in the Xenopus explants to a substantial level, although it induced dorsal mesoderm better than XbFGF under the same experimental conditions. The regulatory protein Xwnt-8 did not activate MAPK, nor did it enhance the activation of MAPK by XbFGF. XbFGF was able to activate MAPK through at least the midgastrula stage, suggesting that this family of growth factors may have a role in gastrula-stage events. Images PMID:7510404

  12. p38γ and p38δ reprogram liver metabolism by modulating neutrophil infiltration.

    PubMed

    González-Terán, Bárbara; Matesanz, Nuria; Nikolic, Ivana; Verdugo, María Angeles; Sreeramkumar, Vinatha; Hernández-Cosido, Lourdes; Mora, Alfonso; Crainiciuc, Georgiana; Sáiz, María Laura; Bernardo, Edgar; Leiva-Vega, Luis; Rodríguez, Elena; Bondía, Victor; Torres, Jorge L; Perez-Sieira, Sonia; Ortega, Luis; Cuenda, Ana; Sanchez-Madrid, Francisco; Nogueiras, Rubén; Hidalgo, Andrés; Marcos, Miguel; Sabio, Guadalupe

    2016-03-01

    Non-alcoholic fatty liver disease (NAFLD) is a major health problem and the main cause of liver disease in Western countries. Although NAFLD is strongly associated with obesity and insulin resistance, its pathogenesis remains poorly understood. The disease begins with an excessive accumulation of triglycerides in the liver, which stimulates an inflammatory response. Alternative p38 mitogen-activated kinases (p38γ and p38δ) have been shown to contribute to inflammation in different diseases. Here we demonstrate that p38δ is elevated in livers of obese patients with NAFLD and that mice lacking p38γ/δ in myeloid cells are resistant to diet-induced fatty liver, hepatic triglyceride accumulation and glucose intolerance. This protective effect is due to defective migration of p38γ/δ-deficient neutrophils to the damaged liver. We further show that neutrophil infiltration in wild-type mice contributes to steatosis development by means of inflammation and liver metabolic changes. Therefore, p38γ and p38δ in myeloid cells provide a potential target for NAFLD therapy. PMID:26843485

  13. 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...

  14. Piperine ameliorates the severity of cerulein-induced acute pancreatitis by inhibiting the activation of mitogen activated protein kinases.

    PubMed

    Bae, Gi-Sang; Kim, Min-Sun; Jeong, Jinsu; Lee, Hye-Youn; Park, Kyoung-Chel; Koo, Bon Soon; Kim, Byung-Jin; Kim, Tae-Hyeon; Lee, Seung Ho; Hwang, Sung-Yeon; Shin, Yong Kook; Song, Ho-Joon; Park, Sung-Joo

    2011-07-01

    Piperine is a phenolic component of black pepper (Piper nigrum) and long pepper (Piper longum), fruits used in traditional Asian medicine. Our previous study showed that piperine inhibits lipopolysaccharide-induced inflammatory responses. In this study, we investigated whether piperine reduces the severity of cerulein-induced acute pancreatitis (AP). Administration of piperine reduced histologic damage and myeloperoxidase (MPO) activity in the pancreas and ameliorated many of the examined laboratory parameters, including the pancreatic weight (PW) to body weight (BW) ratio, as well as serum levels of amylase and lipase and trypsin activity. Furthermore, piperine pretreatment reduced the production of tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6 during cerulein-induced AP. In accordance with in vivo results, piperine reduced cell death, amylase and lipase activity, and cytokine production in isolated cerulein-treated pancreatic acinar cells. In addition, piperine inhibited the activation of mitogen-activated protein kinases (MAPKs). These findings suggest that the anti-inflammatory effect of piperine in cerulein-induced AP is mediated by inhibiting the activation of MAPKs. Thus, piperine may have a protective effect against AP.

  15. Involvement of mitogen-activated protein kinase pathway in T-2 toxin-induced cell cycle alteration and apoptosis in human neuroblastoma cells.

    PubMed

    Agrawal, Mona; Bhaskar, A S B; Lakshmana Rao, P V

    2015-01-01

    T-2 toxin is the most toxic trichothecene and a frequent contaminant in many agriculture products. Dietary ingestion represents the most common route of T-2 toxin exposure in humans. T-2 toxin exposure leads to many pathological conditions like nervous disorders, cardiovascular alterations, immune depression and dermal inflammation. However, the neuronal toxicity of T-2 toxin in vitro remains unclear. In the present study, we investigated the mechanism of T-2 toxin-induced apoptosis in human neuroblastoma cells (IMR-32). T-2 toxin was cytotoxic at a low concentration of 10 ng/ml. The 50% inhibitory concentration (IC50) of T-2 toxin was found to be 40 ng/ml as assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, crystal violet dye exclusion test and lactate dehydrogenase (LDH) leakage. T-2 toxin increased intracellular reactive oxygen species generation as early as 15 min and peaked at 60 min as analyzed by flow cytometry. Annexin V + propidium iodide staining showed time-dependent increase in percent apoptotic cells. DNA gel electrophoresis showed oligonucleosomal DNA fragmentation typical of apoptotic cells. Additionally, casapse-3 activation and PARP cleavage indicated involvement of mitochondrial mediated caspase-dependent pathway of apoptosis. Cell cycle analysis revealed time-dependent increase in sub-G1 population of cells and significant up-regulation of CDK2, CDK6, cyclin A and p21 messenger RNA (mRNA) levels. Exposure to T-2 toxin induced the phosphorylation of extracellular signal-regulated kinase (ERK), p38-mitogen-activated protein kinase and c-jun N-terminal kinases (JNK). Analysis of human phospho-mitogen-activated protein kinase (MAPK) antibody array revealed time-dependent increase in phosphorylation. Upstream of ERK pathway Grb2, Ras and Raf and downstream transcription factors c-fos and c-jun were significantly up-regulated. Z-VAD-FMK and MAPK inhibitors (PD 98059, SB 203580 and ZM 336372) exposure prior to T-2

  16. Mammary gland specific expression of Brk/PTK6 promotes delayed involution and tumor formation associated with activation of p38 MAPK

    PubMed Central

    2011-01-01

    Introduction Protein tyrosine kinases (PTKs) are frequently overexpressed and/or activated in human malignancies, and regulate cancer cell proliferation, cellular survival, and migration. As such, they have become promising molecular targets for new therapies. The non-receptor PTK termed breast tumor kinase (Brk/PTK6) is overexpressed in approximately 86% of human breast tumors. The role of Brk in breast pathology is unclear. Methods We expressed a WAP-driven Brk/PTK6 transgene in FVB/n mice, and analyzed mammary glands from wild-type (wt) and transgenic mice after forced weaning. Western blotting and immunohistochemistry (IHC) studies were conducted to visualize markers of mammary gland involution, cell proliferation and apoptosis, as well as Brk, STAT3, and activated p38 mitogen-activated protein kinase (MAPK) in mammary tissues and tumors from WAP-Brk mice. Human (HMEC) or mouse (HC11) mammary epithelial cells were stably or transiently transfected with Brk cDNA to assay p38 MAPK signaling and cell survival in suspension or in response to chemotherapeutic agents. Results Brk-transgenic dams exhibited delayed mammary gland involution and aged mice developed infrequent tumors with reduced latency relative to wt mice. Consistent with delayed involution, mammary glands of transgenic animals displayed decreased STAT3 phosphorylation, a marker of early-stage involution. Notably, p38 MAPK, a pro-survival signaling mediator downstream of Brk, was activated in mammary glands of Brk transgenic relative to wt mice. Brk-dependent signaling to p38 MAPK was recapitulated by Brk overexpression in the HC11 murine mammary epithelial cell (MEC) line and human MEC, while Brk knock-down in breast cancer cells blocked EGF-stimulated p38 signaling. Additionally, human or mouse MECs expressing Brk exhibited increased anchorage-independent survival and resistance to doxorubicin. Finally, breast tumor biopsies were subjected to IHC analysis for co-expression of Brk and phospho-p38 MAPK

  17. Whey protein concentrate enhances intestinal integrity and influences transforming growth factor-β1 and mitogen-activated protein kinase signalling pathways in piglets after lipopolysaccharide challenge.

    PubMed

    Xiao, Kan; Jiao, Lefei; Cao, Shuting; Song, Zehe; Hu, Caihong; Han, Xinyan

    2016-03-28

    Whey protein concentrate (WPC) has been reported to have protective effects on the intestinal barrier. However, the molecular mechanisms involved are not fully elucidated. Transforming growth factor-β1 (TGF-β1) is an important component in the WPC, but whether TGF-β1 plays a role in these processes is not clear. The aim of this study was to investigate the protective effects of WPC on the intestinal epithelial barrier as well as whether TGF-β1 is involved in these protection processes in a piglet model after lipopolysaccharide (LPS) challenge. In total, eighteen weanling pigs were randomly allocated to one of the following three treatment groups: (1) non-challenged control and control diet; (2) LPS-challenged control and control diet; (3) LPS+5 %WPC diet. After 19 d of feeding with control or 5 %WPC diets, pigs were injected with LPS or saline. At 4 h after injection, pigs were killed to harvest jejunal samples. The results showed that WPC improved (P<0·05) intestinal morphology, as indicated by greater villus height and villus height:crypt depth ratio, and intestinal barrier function, which was reflected by increased transepithelial electrical resistance and decreased mucosal-to-serosal paracellular flux of dextran (4 kDa), compared with the LPS group. Moreover, WPC prevented the LPS-induced decrease (P<0·05) in claudin-1, occludin and zonula occludens-1 expressions in the jejunal mucosae. WPC also attenuated intestinal inflammation, indicated by decreased (P<0·05) mRNA expressions of TNF-α, IL-6, IL-8 and IL-1β. Supplementation with WPC also increased (P<0·05) TGF-β1 protein, phosphorylated-Smad2 expression and Smad4 and Smad7 mRNA expressions and decreased (P<0·05) the ratios of the phosphorylated to total c-jun N-terminal kinase (JNK) and p38 (phospho-JNK:JNK and p-p38:p38), whereas it increased (P<0·05) the ratio of extracellular signal-regulated kinase (ERK) (phospho-ERK:ERK). Collectively, these results suggest that dietary inclusion of WPC

  18. A mitogen-activated protein kinase of the corn leaf pathogen Cochliobolus heterostrophus is involved in conidiation, appressorium formation, and pathogenicity: diverse roles for mitogen-activated protein kinase homologs in foliar pathogens.

    PubMed

    Lev, S; Sharon, A; Hadar, R; Ma, H; Horwitz, B A

    1999-11-01

    Fungal pathogens perceive and respond to molecules from the plant, triggering pathogenic development. Transduction of these signals may use heterotrimeric G proteins, and it is thought that protein phosphorylation cascades are also important. We have isolated a mitogen-activated protein kinase homolog from the corn pathogen Cochliobolus heterostrophus to test its role as a component of the transduction pathways. The new gene, CHK1, has a deduced amino acid sequence 90% identical to Pmk1 of the rice blast fungus Magnaporthe grisea and 59% identical to Fus3 of Saccharomyces cerevisiae. A series of chk1 deletion mutants has poorly developed aerial hyphae, autolysis, and no conidia. No pseudothecia are formed when a cross between two Deltachk1 mutants is attempted. The ability of Deltachk1 mutants to infect corn plants is reduced severely. The growth pattern of hyphae on a glass surface is strikingly altered from that of the wild type, forming coils or loops, but no appressoria. This set of phenotypes overlaps only partially with that of pmk1 mutants, the homologous gene of the rice blast fungus. In particular, sexual and asexual sporulation both require Chk1 function in Cochliobolus heterostrophus, in contrast to Pmk1, but perhaps more similar to yeast, where Fus3 transmits the mating signal. Chk1 is required for efficient colonization of leaf tissue, which can be compared with filamentous invasive growth of yeast, modulated through another closely related mitogen-activated protein kinase, Kss1. Ubiquitous signaling elements thus are used in diverse ways in different plant pathogens, perhaps the result of coevolution of the transducers and their targets. PMID:10557357

  19. Combined deletion of p38γ and p38δ reduces skin inflammation and protects from carcinogenesis

    PubMed Central

    Zur, Rafal; Garcia-Ibanez, Laura; Nunez-Buiza, Angel; Aparicio, Noelia; Liappas, Georgios; Escós, Alejandra; Risco, Ana; Page, Angustias; Saiz-Ladera, Cristina; Alsina-Beauchamp, Dayanira; Montans, José; Paramio, Jesús M.; Cuenda, Ana

    2015-01-01

    The contribution of chronic skin inflammation to the development of squamous cell carcinoma (SCC) is poorly understood. While the mitogen-activated protein kinase p38α regulates inflammatory responses and tumour development, little is known about the role of p38γ and p38δ in these processes. Here we show that combined p38γ and p38δ (p38γ/δ) deletion blocked skin tumour development in a chemically induced carcinogenesis model. p38γ/δ deletion reduced TPA-induced epidermal hyperproliferation and inflammation; it inhibited expression of proinflammatory cytokines and chemokines in keratinocytes in vitro and in whole skin in vivo, resulting in decreased neutrophil recruitment to skin. Our data indicate that p38γ/δ in keratinocytes promote carcinogenesis by enabling formation of a proinflammatory microenvironment that fosters epidermal hyperproliferation and tumourigenesis. These findings provide genetic evidence that p38γ and p38δ have essential roles in skin tumour development, and suggest that targeting inflammation through p38γ/δ offers a therapeutic strategy for SCC treatment and prevention. PMID:26079427

  20. 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.

  1. 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

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

    DOE PAGES

    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

  3. Comparative chemical array screening for p38γ/δ MAPK inhibitors using a single gatekeeper residue difference between p38α/β and p38γ/δ

    PubMed Central

    Kondoh, Yasumitsu; Honda, Kaori; Hiranuma, Sayoko; Hayashi, Teruo; Shimizu, Takeshi; Watanabe, Nobumoto; Osada, Hiroyuki

    2016-01-01

    Mammalian p38 mitogen activated protein kinases (MAPKs) are responsive to a variety of cellular stresses. The development of specific pyridinyl imidazole inhibitors has permitted the characterization of the p38 MAPK isoform p38α, which is expressed in most cell types, whereas the physiological roles of p38γ and p38δ are poorly understood. In this study, we report an approach for identifying selective inhibitors against p38γ and p38δ by focusing on the difference in gatekeeper residues between p38α/β and p38γ/δ. Using GST-fused p38α wild type and T106M mutant constructs, wherein the p38α gatekeeper residue (Thr-106) was substituted by the p38γ/δ-type (Met), we performed comparative chemical array screening to identify specific binders of the mutant and identified SU-002 bound to p38αT106M specifically. SU-002 was found to inhibit p38αT106M but not p38α kinase activity in in vitro kinase assays. SU-005, the analog of SU-002, had inhibitory effects against the kinase activity of p38γ and p38δ in vitro but not p38α. In addition, SU-005 inhibited both p38γ and p38δ auto-phosphorylation in HeLa and HEK293T cells. These results demonstrate that the comparative chemical array screening approach is a powerful technique to explore specific inhibitors for mutant proteins with even single amino-acid substitutions in a high-throughput manner. PMID:27431267

  4. Differential activation of mitogen-activated protein kinase in response to basic fibroblast growth factor in skeletal muscle cells.

    PubMed Central

    Campbell, J S; Wenderoth, M P; Hauschka, S D; Krebs, E G

    1995-01-01

    In the MM14 mouse myoblast cell line, fibroblast growth factor (FGF) stimulates proliferation and represses differentiation. However, the intracellular signaling pathways used by FGF to affect these cellular processes are unknown. The predominant FGF receptor present on MM14 cells, FGFR1, is a receptor tyrosine kinase capable of activating the mitogen-activated protein kinase (MAPK) cascade in fibroblast and neuronal cell lines. To determine whether the FGF signal is mediated via the MAPK cascade in myoblasts, MM14 cells were stimulated with basic FGF (bFGF) and activities of the various kinases were measured. After withdrawal from serum and bFGF for 3 hr, bFGF stimulated MAPK kinase (MAPKK) activity, but MAPK and S6 peptide kinase activities were not detected. In contrast, when serum and bFGF were withdrawn for 10 hr, the activities of MAPKK, MAPK, and S6 peptide kinase were all stimulated by bFGF treatment. The inability of bFGF to stimulate MAPK after 3 hr of withdrawal may be due, in part, to the presence of a MAPK phosphatase activity that was detected in MM14 cell extracts. This dephosphorylating activity diminishes during commitment to terminal differentiation and is inhibited by sodium orthovanadate. Thus, the ability of bFGF to stimulate MAPK in MM14 cells is correlated with the loss of a MAPK phosphatase activity. These results show that although bFGF activates MAPKK in proliferating myoblasts, the mitogenic signal does not progress to the downstream kinases, providing a physiological example of an uncoupling of the MAPK cascade. Images Fig. 4 Fig. 5 PMID:7846069

  5. Baicalin Attenuates Hypoxia-Induced Pulmonary Arterial Hypertension to Improve Hypoxic Cor Pulmonale by Reducing the Activity of the p38 MAPK Signaling Pathway and MMP-9.

    PubMed

    Yan, Shuangquan; Wang, Yiran; Liu, Panpan; Chen, Ali; Chen, Mayun; Yao, Dan; Xu, Xiaomei; Wang, Liangxing; Huang, Xiaoying

    2016-01-01

    Baicalin has a protective effect on hypoxia-induced pulmonary hypertension in rats, but the mechanism of this effect remains unclear. Thus, investigating the potential mechanism of this effect was the aim of the present study. Model rats that display hypoxic pulmonary hypertension and cor pulmonale under control conditions were successfully generated. We measured a series of indicators to observe the levels of pulmonary arterial hypertension, pulmonary arteriole remodeling, and right ventricular remodeling. We assessed the activation of p38 mitogen-activated protein kinase (MAPK) in the pulmonary arteriole walls and pulmonary tissue homogenates using immunohistochemistry and western blot analyses, respectively. The matrix metalloproteinase- (MMP-) 9 protein and mRNA levels in the pulmonary arteriole walls were measured using immunohistochemistry and in situ hybridization. Our results demonstrated that baicalin not only reduced p38 MAPK activation in both the pulmonary arteriole walls and tissue homogenates but also downregulated the protein and mRNA expression levels of MMP-9 in the pulmonary arteriole walls. This downregulation was accompanied by the attenuation of pulmonary hypertension, arteriole remodeling, and right ventricular remodeling. These results suggest that baicalin may attenuate pulmonary hypertension and cor pulmonale, which are induced by chronic hypoxia, by downregulating the p38 MAPK/MMP-9 pathway. PMID:27688788

  6. Baicalin Attenuates Hypoxia-Induced Pulmonary Arterial Hypertension to Improve Hypoxic Cor Pulmonale by Reducing the Activity of the p38 MAPK Signaling Pathway and MMP-9

    PubMed Central

    Wang, Yiran; Chen, Ali; Chen, Mayun; Yao, Dan; Xu, Xiaomei; Wang, Liangxing

    2016-01-01

    Baicalin has a protective effect on hypoxia-induced pulmonary hypertension in rats, but the mechanism of this effect remains unclear. Thus, investigating the potential mechanism of this effect was the aim of the present study. Model rats that display hypoxic pulmonary hypertension and cor pulmonale under control conditions were successfully generated. We measured a series of indicators to observe the levels of pulmonary arterial hypertension, pulmonary arteriole remodeling, and right ventricular remodeling. We assessed the activation of p38 mitogen-activated protein kinase (MAPK) in the pulmonary arteriole walls and pulmonary tissue homogenates using immunohistochemistry and western blot analyses, respectively. The matrix metalloproteinase- (MMP-) 9 protein and mRNA levels in the pulmonary arteriole walls were measured using immunohistochemistry and in situ hybridization. Our results demonstrated that baicalin not only reduced p38 MAPK activation in both the pulmonary arteriole walls and tissue homogenates but also downregulated the protein and mRNA expression levels of MMP-9 in the pulmonary arteriole walls. This downregulation was accompanied by the attenuation of pulmonary hypertension, arteriole remodeling, and right ventricular remodeling. These results suggest that baicalin may attenuate pulmonary hypertension and cor pulmonale, which are induced by chronic hypoxia, by downregulating the p38 MAPK/MMP-9 pathway. PMID:27688788

  7. Baicalin Attenuates Hypoxia-Induced Pulmonary Arterial Hypertension to Improve Hypoxic Cor Pulmonale by Reducing the Activity of the p38 MAPK Signaling Pathway and MMP-9

    PubMed Central

    Wang, Yiran; Chen, Ali; Chen, Mayun; Yao, Dan; Xu, Xiaomei; Wang, Liangxing

    2016-01-01

    Baicalin has a protective effect on hypoxia-induced pulmonary hypertension in rats, but the mechanism of this effect remains unclear. Thus, investigating the potential mechanism of this effect was the aim of the present study. Model rats that display hypoxic pulmonary hypertension and cor pulmonale under control conditions were successfully generated. We measured a series of indicators to observe the levels of pulmonary arterial hypertension, pulmonary arteriole remodeling, and right ventricular remodeling. We assessed the activation of p38 mitogen-activated protein kinase (MAPK) in the pulmonary arteriole walls and pulmonary tissue homogenates using immunohistochemistry and western blot analyses, respectively. The matrix metalloproteinase- (MMP-) 9 protein and mRNA levels in the pulmonary arteriole walls were measured using immunohistochemistry and in situ hybridization. Our results demonstrated that baicalin not only reduced p38 MAPK activation in both the pulmonary arteriole walls and tissue homogenates but also downregulated the protein and mRNA expression levels of MMP-9 in the pulmonary arteriole walls. This downregulation was accompanied by the attenuation of pulmonary hypertension, arteriole remodeling, and right ventricular remodeling. These results suggest that baicalin may attenuate pulmonary hypertension and cor pulmonale, which are induced by chronic hypoxia, by downregulating the p38 MAPK/MMP-9 pathway.

  8. Epiderstatin, a new inhibitor of the mitogenic activity induced by epidermal growth factor. I. Taxonomy, fermentation, isolation and characterization.

    PubMed

    Osada, H; Sonoda, T; Kusakabe, H; Isono, K

    1989-11-01

    Inhibitors of mitogenic activity induced by epidermal growth factor (EGF) were screened from culture broths of soil microorganisms. A strain of actinomycetes has been found to produce a new glutarimide antibiotic named epiderstatin which inhibits the incorporation of [3H]thymidine into quiescent animal cells stimulated by EGF. Taxonomic studies have revealed that the producing strain belongs to a subspecies of Streptomyces pulveraceus, thus the name, Streptomyces pulveraceus subsp. epiderstagenes was given to this strain. The molecular formula (C15H20N2O4) and UV profile (lambda max 295 nm) of the antibiotic are distinct from other known antibiotics. It inhibited the incorporation of [3H]thymidine into quiescent cells stronger than into growing cells. PMID:2584144

  9. Genome-wide identification and analysis of expression profiles of maize mitogen-activated protein kinase kinase kinase.

    PubMed

    Kong, Xiangpei; Lv, Wei; Zhang, Dan; Jiang, Shanshan; Zhang, Shizhong; Li, Dequan

    2013-01-01

    Mitogen-activated protein kinase (MAPK) cascades are highly conserved signal transduction model in animals, yeast and plants. Plant MAPK cascades have been implicated in development and stress responses. Although MAPKKKs have been investigated in several plant species including Arabidopsis and rice, no systematic analysis has been conducted in maize. In this study, we performed a bioinformatics analysis of the entire maize genome and identified 74 MAPKKK genes. Phylogenetic analyses of MAPKKKs from maize, rice and Arabidopsis have classified them into three subgroups, which included Raf, ZIK and MEKK. Evolutionary relationships within subfamilies were also supported by exon-intron organizations and the conserved protein motifs. Further expression analysis of the MAPKKKs in microarray databases revealed that MAPKKKs were involved in important signaling pathways in maize different organs and developmental stages. Our genomics analysis of maize MAPKKK genes provides important information for evolutionary and functional characterization of this family in maize.

  10. Asymmetric p38 activation in zebrafish: its possible role in symmetric and synchronous cleavage.

    PubMed

    Fujii, R; Yamashita, S; Hibi, M; Hirano, T

    2000-09-18

    Cleavage is one of the initial steps of embryogenesis, and is characterized by a series of symmetric and synchronous cell divisions. We showed that p38 MAP kinase (p38) is asymmetrically activated on one side of the blastodisc during the early cleavage period in zebrafish (Danio rerio) embryos. When a dominant negative (DN) form of p38 was uniformly expressed, blastomere cleavage was impaired on one side of the blastodisc, resulting in the formation of blastomeres with a large mass of cytoplasm and an enlarged nucleus on the affected side. The area affected by the DN-p38 expression did not correlate with the initial cleavage plane, but coincided with the side where dharma/bozozok, a dorsal-specific zygotic gene, was expressed (Yamanaka et al. 1998). Furthermore, UV irradiation and removal of the vegetal yolk mass before the first cleavage, both of which inhibit the initiation of the dorsalizing signals, abolished the asymmetric p38 activation. Our findings suggest that asymmetric p38 activation is required for symmetric and synchronous cleavage, and may be regulated by the same machinery that controls the initiation of dorsalizing signals. PMID:10995439

  11. Inhibitory effect of caffeic acid on ADP-induced thrombus formation and platelet activation involves mitogen-activated protein kinases

    PubMed Central

    Lu, Yu; Li, Quan; Liu, Yu-Ying; Sun, Kai; Fan, Jing-Yu; Wang, Chuan-She; Han, Jing-Yan

    2015-01-01

    Caffeic acid (CA), one of the active constituents of Radix Salvia miltiorrhizae, exhibits antioxidant and anti-inflammatory activities. However, few studies have assessed the ability of CA to inhibit platelet mediated thrombus generation in vivo. In this study, we investigated the antithrombotic effect of CA in mouse cerebral arterioles and venules using intravital microscopy. The antiplatelet activity of CA in ADP stimulated mouse platelets in vitro was also examined in attempt to explore the underlying mechanism. Our results demonstrated that CA (1.25–5 mg/kg) significantly inhibited thrombus formation in vivo. In vitro, CA (25–100 μM) inhibited ADP-induced platelet aggregation, P-selectin expression, ATP release, Ca2+ mobilization, and integrin αIIbβ3 activation. Additionally, CA attenuated p38, ERK, and JNK activation, and enhanced cAMP levels. Taken together, these data provide evidence for the inhibition of CA on platelet-mediated thrombosis in vivo, which is, at least partly, mediated by interference in phosphorylation of ERK, p38, and JNK leading to elevation of cAMP and down-regulation of P-selectin expression and αIIbβ3 activation. These results suggest that CA may have potential for the treatment of aberrant platelet activation-related diseases. PMID:26345207

  12. Suppression of UVB-induced phosphorylation of mitogen-activated protein kinases and nuclear factor kappa B by green tea polyphenol in SKH-1 hairless mice.

    PubMed

    Afaq, Farrukh; Ahmad, Nihal; Mukhtar, Hasan

    2003-12-18

    Studies from our laboratory have shown that epigallocatechin-3-gallate, the major polyphenol present in green tea, inhibits ultraviolet (UV)B-exposure-mediated phosphorylation of mitogen-activated protein kinases (MAPKs) (Toxicol. Appl. Pharmacol. 176: 110-117, 2001) and activation of nuclear factor kappa B (NF-kappaB) (Oncogene 22: 1035-1044, 2003) pathways in normal human epidermal keratinocytes. This study was designed to investigate the relevance of these findings to the in vivo situations in SKH-1 hairless mouse model, which is regarded to have relevance to human situations. SKH-1 hairless mice were topically treated with GTP (5 mg/0.2 ml acetone/mouse) and were exposed to UVB 30 min later (180 mJ/cm2). These treatments were repeated every alternate day for 2 weeks, for a total of seven treatments. The animals were killed 24 h after the last UVB exposure. Topical application of GTP resulted in significant decrease in UVB-induced bifold-skin thickness, skin edema and infiltration of leukocytes. Employing Western blot analysis and immunohistochemical studies, we found that GTP resulted in inhibition of UVB-induced: (i) phosphorylation of extracellular-signal-regulated kinases (ERK1/2), (ii) c-Jun N-terminal kinases, and (iii) p38 protein expression. Since NF-kappaB plays a major role in inflammation and cell proliferation, we assessed the effect of GTP on UVB-mediated modulations in the NF-kappaB pathway. Our data demonstrated that GTP inhibited UVB-induced: (i) activation of NF-kappaB, (ii) activation of IKKalpha, and (iii) phosphorylation and degradation of IkappaBalpha. Our data suggest that GTP protects against the adverse effects of UV radiation via modulations in MAPK and NF-kappaB signaling pathways, and provides molecular basis for the photochemopreventive effect of GTP in an in vivo animal model system.

  13. Enhanced IL-1{beta}-induced IL-8 production in cystic fibrosis lung epithelial cells is dependent of both mitogen-activated protein kinases and NF-{kappa}B signaling

    SciTech Connect

    Muselet-Charlier, Celine; Roque, Telma; Boncoeur, Emilie; Chadelat, Katarina; Clement, Annick; Jacquot, Jacky; Tabary, Olivier . E-mail: olivier.tabary@st-antoine.inserm.fr

    2007-06-01

    Transcription nuclear factor-{kappa}B (NF-{kappa}B) is hyperactivated in cystic fibrosis (CF) lung epithelial cells, and participates in exaggerated IL-8 production in the CF lung. We recently found that rapid activation of NF-{kappa}B occurred in a CF lung epithelial IB3-1 cell line (CF cells) upon IL-1{beta} stimulation, which was not observed in its CFTR-corrected lung epithelial S9 cell line (corrected cells). To test whether other signaling pathways such as that of mitogen-activated protein kinases (MAPKs) could be involved in IL-1{beta}-induced IL-8 production of CF cells, we investigated ERK1/2, JNK, and p38MAP signaling compared to NF-{kappa}B. Within 30 min, exposure to IL-1{beta} caused high activation of NF-{kappa}B, ERK1/2, p38MAP but not JNK in CF cells compared to corrected cells. Treatment of IL-1{beta}-stimulated CF cells with a series of chemical inhibitors of NF-{kappa}B, ERK1/2, and p38MAP, when used separately, reduced slightly IL-8 production. However, when used together, these inhibitors caused a blockade in IL-1{beta}-induced IL-8 production in CF cells. Understanding of the cross-talk between NF-{kappa}B and MAPKs signaling in CF lung epithelial cells may help in developing new therapeutics to reduce lung inflammation in patients with CF.

  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. Chlamydial plasmid-encoded protein pORF5 induces production of IL-1β and IL-18 via NALP3 inflammasome activation and p38 MAPK pathway

    PubMed Central

    Cao, Wenjuan; Zou, Yan; Su, Shengmei; He, Zhansheng; Liu, Yan; Huang, Qiulin; Li, Zhongyu

    2015-01-01

    The pathogenesis of Chlamydia-induced inflammation is poorly understood. pORF5 is the only secreted protein encoded by Chlamydial plasmid. This study aims to investigate the effects of pORF5 on the production of interleukin-1β (IL-1β) and interleukin-18 (IL-18) and the underlying mechanisms of these effects. THP-1 (a human acute monocytic leukemia cell line) cells were stimulated by pORF5 with or without pretreatment with Natch domain, Leucine-rich repeat and PYD-containing protein 3 (NALP3) siRNA, apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) siRNA, cysteine aspartate-specific protease-1 (caspase-1) specific inhibitor and p38 mitogen-activated protein kinase (p38 MAPK) inhibitor. IL-1β, IL-18 and caspase-1 expression was detected through both ELISA and qRT-PCR. NALP3 and ASC expression was detected by qRT-PCR. The expression of caspase-1 and phosphorylated-p38 MAPK was detected by western blot analysis. pORF5 induced IL-1β, IL-18, caspase-1 and NALP3 inflammasome expression in THP-1 cells. Caspase-1 inhibitor significantly reduced pORF5-induced IL-1β and IL-18 expression. The siRNAs for NALP3 inflammasome significantly reduced pORF5-induced IL-1β, IL-18 and caspase-1 expression. Furthermore, p38 MAPK inhibitor significantly reduced pORF5-induced IL-1β, IL-18, caspase-1 and NALP3 inflammasome expression. pORF5 could induce production of IL-1β and IL-18 via NALP3 inflammasome activation and p38MAPK pathway. pORF5 protein might play an important role in Chlamydia pathogenesis. This study provides a new insight into the molecular pathogenesis of Chlamydial diseases. PMID:26884953

  16. 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.

  17. Docosahexaenoic acid attenuates mycotoxin-induced immunoglobulin a nephropathy, interleukin-6 transcription, and mitogen-activated protein kinase phosphorylation in mice.

    PubMed

    Jia, Qunshan; Zhou, Hui-Ren; Bennink, Maurice; Pestka, James J

    2004-12-01

    The purpose of this investigation was to evaluate the dose-dependent effects of docosahexaenoic acid (DHA) on deoxynivalenol (DON)-induced IgA nephropathy in mice and their relation to proinflammatory gene expression and mitogen-activated protein kinase (MAPK) activation. Consumption of a modified AIN-93G diet containing 1, 5, and 30 g/kg DHA resulted in dose-dependent increases of DHA in liver phospholipids with concomitant decreases in arachidonic acid compared with control diets. DHA dose dependently inhibited increases in serum IgA and IgA immune complexes (IC) as well as IgA deposition in the kidney in DON-fed mice; the 30 g/kg DHA diet had the earliest detectable effects and maximal efficacy. Both splenic interleukin-6 (IL-6) mRNA and heterogeneous nuclear RNA (hnRNA), an indicator of IL-6 transcription, were significantly reduced in DON-fed mice that consumed 5 and 30 g/kg DHA; a similar reduction was observed for cyclooxygenase (COX-2) mRNA. In a subsequent study, acute DON exposure (25 mg/kg body weight) induced splenic IL-6 mRNA and hnRNA as well as COX-2 mRNA in mice fed the control diet, whereas induction of both RNA species was significantly inhibited in mice fed 30 g/kg DHA. These latter inhibitory effects corresponded to a reduction in DON-induced phosphorylation of p38, extracellular-signal related kinase 1/2, and c-Jun N-terminal kinase 1/2 MAPKs in the spleen. Taken together, the results indicate that DHA dose-dependently inhibited DON-induced IgA dysregulation and nephropathy, and that impairment of MAPK activation and expression of COX-2 and IL-6 are potential critical upstream mechanisms.

  18. Ethanol Impairs Intestinal Barrier Function in Humans through Mitogen Activated Protein Kinase Signaling: A Combined In Vivo and In Vitro Approach

    PubMed Central

    Elamin, Elhaseen; Masclee, Ad; Troost, Freddy; Pieters, Harm-Jan; Keszthelyi, Daniel; Aleksa, Katarina; Dekker, Jan; Jonkers, Daisy

    2014-01-01

    Background Ethanol-induced gut barrier disruption is associated with several gastrointestinal and liver disorders. Aim Since human data on effects of moderate ethanol consumption on intestinal barrier integrity and involved mechanisms are limited, the objectives of this study were to investigate effects of a single moderate ethanol dose on small and large intestinal permeability and to explore the role of mitogen activated protein kinase (MAPK) pathway as a primary signaling mechanism. Methods Intestinal permeability was assessed in 12 healthy volunteers after intraduodenal administration of either placebo or 20 g ethanol in a randomised cross-over trial. Localization of the tight junction (TJ) and gene expression, phosphorylation of the MAPK isoforms p38, ERK and JNK as indicative of activation were analyzed in duodenal biopsies. The role of MAPK was further examined in vitro using Caco-2 monolayers. Results Ethanol increased small and large intestinal permeability, paralleled by redistribution of ZO-1 and occludin, down-regulation of ZO-1 and up-regulation of myosin light chain kinase (MLCK) mRNA expression, and increased MAPK isoforms phosphorylation. In Caco-2 monolayers, ethanol increased permeability, induced redistribution of the junctional proteins and F-actin, and MAPK and MLCK activation, as indicated by phosphorylation of MAPK isoforms and myosin light chain (MLC), respectively, which could be reversed by pretreatment with either MAPK inhibitors or the anti-oxidant L-cysteine. Conclusions Administration of moderate ethanol dosage can increase both small and colon permeability. Furthermore, the data indicate a pivotal role for MAPK and its crosstalk with MLCK in ethanol-induced intestinal barrier disruption. Trial Registration ClinicalTrials.gov NCT00928733 PMID:25226407

  19. Mitogen-activated protein kinase activation by oxidative and bacterial stress in an amphibian cell culture model.

    PubMed

    Carter, Lisa A; Tabor, Maija B; Bonner, James C; Bonner, Lisa A

    2002-07-01

    The decline of many amphibian species could be caused by their susceptibility to environmental pollutants that cause cellular stress and cell death. A variety of intracellular signal transduction pathways are activated by environmental stress factors, which result in cell death. Mitogen-activated protein kinases are intracellular signaling molecules that include the extracellular signal-regulated kinases (ERK-1 and ERK-2). We used cultured (italic)Xenopus(/italic) tadpole cells (XTC-2 cells) to investigate the activation of ERK by oxidative or bacterial stress, two environmental factors that could contribute to pollution in aquatic systems. We exposed XTC-2 cell monolayers to hydrogen peroxide or bacterial lipopolysaccharide and measured ERK activation by Western blotting using antibodies raised against phosphorylated ERK-1 and ERK-2. Only ERK-2 was detected in XTC-2 cells. Both hydrogen peroxide and lipopolysaccharide caused ERK-2 phosphorylation in a time- and concentration-dependent manner. Hydrogen peroxide caused a 20- to 30-fold increase in ERK-2 activation that peaked 30 min after treatment, and lipopolysaccharide induced a 5- to 10-fold increase in ERK-2 activation that peaked 60 min after treatment. PD98059, an inhibitor of the ERK pathway, reduced the cytotoxic response of XTC-2 cells to hydrogen peroxide or lipopolysaccharide. These data suggest that ERK-2 is an intracellular target of oxidative and bacterial stress in amphibians that mediates, at least in part, the cytotoxic response to hydrogen peroxide or lipopolysaccharide. Moreover, the (italic)Xenopus(/italic) (XTC-2) cell culture system could serve as a useful model to identify agents that might threaten amphibian populations and human health.

  20. Two hydrophobic residues can determine the specificity of mitogen-activated protein kinase docking interactions.

    PubMed

    Bardwell, A Jane; Bardwell, Lee

    2015-10-30

    MAPKs bind to many of their upstream regulators and downstream substrates via a short docking motif (the D-site) on their binding partner. MAPKs that are in different families (e.g. ERK, JNK, and p38) can bind selectively to D-sites in their authentic substrates and regulators while discriminating against D-sites in other pathways. Here we demonstrate that the short hydrophobic region at the distal end of the D-site plays a critical role in determining the high selectivity of JNK MAPKs for docking sites in their cognate MAPK kinases. Changing just 1 or 2 key hydrophobic residues in this submotif is sufficient to turn a weak JNK-binding D-site into a strong one, or vice versa. These specificity-determining differences are also found in the D-sites of the ETS family transcription factors Elk-1 and Net. Moreover, swapping two hydrophobic residues between these D-sites switches the relative efficiency of Elk-1 and Net as substrates for ERK versus JNK, as predicted. These results provide new insights into docking specificity and suggest that this specificity can evolve rapidly by changes to just 1 or 2 amino acids.

  1. Inhibitory effects of [6]-gingerol on PMA-induced COX-2 expression and activation of NF-kappaB and p38 MAPK in mouse skin.

    PubMed

    Kim, Sue Ok; Chun, Kyung-Soo; Kundu, Joydeb Kumar; Surh, Young-Joon

    2004-01-01

    [6]-Gingerol, a major pungent ingredient of ginger (Zingiber officinale Roscoe, Zingiberaceae), has a wide array of pharmacologic effects. Previous studies have demonstrated that [6]-gingerol inhibits mouse skin tumor promotion and anchorage-independent growth of cultured mouse epidermal cells stimulated with epidermal growth factor. Cyclooxygenase-2 (COX-2), a key enzyme in the prostaglandin biosynthesis, has been recognized as a molecular target for many anti-inflammatory as well as chemopreventive agents. Topical application of [6]-gingerol inhibited phorbol 12-myristate 13-acetate -induced COX-2 expression. One of the essential transcription factors responsible for COX-2 induction is NF-kappaB. [6]-Gingerol suppressed NF-kappaB DNA binding activity in mouse skin. In addition, [6]-gingerol inhibited the phoshorylation of p38 mitogen-activated protein kinase which may account for its inactivation of NF-kappaB and suppression of COX-2 expression. PMID:15630166

  2. Overexpression of human insulin receptor substrate 1 induces cellular transformation with activation of mitogen-activated protein kinases.

    PubMed Central

    Ito, T; Sasaki, Y; Wands, J R

    1996-01-01

    The receptor insulin substrate 1 protein (IRS-1) is a specific substrate for insulin receptor tyrosine kinase. Expression and tyrosyl phosphorylation of IRS-1 play an important role during normal hepatocyte growth, and the gene is overexpressed in hepatocellular carcinoma tissue. We determined if IRS-1 overexpression directly contributes to cellular transformation. The human IRS-1 gene was subcloned into a mammalian expression vector driven by the cytomegalovirus early promoter. NIH 3T3 cells transiently transfected with this vector subsequently developed transformed foci. Several stably transfected cell lines were established, and they grew efficiently under low-serum conditions and formed colonies when plated in soft agar. Cell lines overexpressing IRS-1 displayed increased tyrosyl phosphorylation of IRS-1 and association with Grb2 but not with the p85 subunit of phosphatidylinositol 3' kinase. Since Grb2 is a component of the son-of-sevenless-Ras pathway and upstream in the mitogen-activated protein kinase (MAPK) cascade, enzymatic activities of the major components of this cascade, such as MAPK kinase and MAPK were evaluated and found to be substantially increased in three independent cell lines with IRS-1 protein overexpression. Such cells, when injected into nude mice, were highly tumorigenic, and there may be a correlation between the degree of MAPK activation and tumor growth rate. This report describes the generation of a transformed phenotype by overexpression of a molecule without a catalytic domain far upstream in the signal transduction cascade and suggests that prolonged activation of MAPKs by this mechanism may be one of the molecular events related to hepatocellular transformation. PMID:8622697

  3. Stimulation of Periodontal Ligament Stem Cells by Dentin Matrix Protein 1 Activates Mitogen-Activated Protein Kinase and Osteoblast Differentiation

    PubMed Central

    Chandrasekaran, Sangeetha; Ramachandran, Amsaveni; Eapen, Asha; George, Anne

    2013-01-01

    Background Periodontitis can ultimately result in tooth loss. Many natural and synthetic materials have been tried to achieve periodontal regeneration, but the results remain variable and unpredictable. We hypothesized that exogenous treatment with dentin matrix protein 1 (DMP1) activates specific genes and results in phenotypic and functional changes in human periodontal ligament stem cells (hPDLSCs). Methods hPDLSCs were isolated from extracted teeth and cultured in the presence or absence of DMP1. Quantitative polymerase chain reactions were performed to analyze the expression of several genes involved in periodontal regeneration. hPDLSCs were also processed for immunocytochemical and Western blot analysis using phosphorylated extracellular signal-regulated kinase (pERK) and ERK antibodies. Alkaline phosphatase and von Kossa staining were performed to characterize the differentiation of hPDLSCs into osteoblasts. Field emission scanning electron microscopic analysis of the treated and control cell cultures were also performed. Results Treatment with DMP1 resulted in the upregulation of genes, such as matrix metalloproteinase-2, alkaline phosphatase, and transforming growth factor β1. Activation of ERK mitogen-activated protein kinase signaling pathway and translocation of pERK from the cytoplasm to the nucleus was observed. Overall, DMP1-treated cells showed increased expression of alkaline phosphatase, increased matrix, and mineralized nodule formation when compared with untreated controls. Conclusion DMP1 can orchestrate a coordinated expression of genes and phenotypic changes in hPDLSCs by activation of the ERK signaling pathway, which may provide a valuable strategy for tissue engineering approaches in periodontal regeneration. PMID:22612367

  4. Luteolin is a bioflavonoid that attenuates adipocyte-derived inflammatory responses via suppression of nuclear factor-κB/mitogen-activated protein kinases pathway

    PubMed Central

    Nepali, Sarmila; Son, Ji-Seon; Poudel, Barun; Lee, Ji-Hyun; Lee, Young-Mi; Kim, Dae-Ki

    2015-01-01

    Background: Inflammation of adipocytes has been a therapeutic target for treatment of obesity and metabolic disorders which cause insulin resistance and hence lead to type II diabetes. Luteolin is a bioflavonoid with many beneficial properties such as antioxidant, antiproliferative, and anti-cancer. Objectives: To elucidate the potential anti-inflammatory response and the underlying mechanism of luteolin in 3T3-L1 adipocytes. Materials and Methods: We stimulated 3T3-L1 adipocytes with the mixture of tumor necrosis factor-α, lipopolysaccharide, and interferon-γ (TLI) in the presence or absence of luteolin. We performed Griess’ method for nitric oxide (NO) production and measure mRNA and protein expressions by real-time polymerase chain reaction and western blotting, respectively. Results: Luteolin opposed the stimulation of inducible nitric oxide synthase and NO production by simultaneous treatment of adipocytes with TLI. Furthermore, it reduced the pro-inflammatory genes such as cyclooxygenase-2, interleukin-6, resistin, and monocyte chemoattractant protein-1. Furthermore, luteolin improved the insulin sensitivity by enhancing the expression of insulin receptor substrates (IRS1/2) and glucose transporter-4 via phosphatidylinositol-3K signaling pathway. This inhibition was associated with suppression of Iκ-B-α degradation and subsequent inhibition of nuclear factor-κB (NF-κB) p65 translocation to the nucleus. In addition, luteolin blocked the phosphorylation of ERK1/2, c-Jun N-terminal Kinases and also p38 mitogen-activated protein kinases (MAPKs). Conclusions: These results illustrate that luteolin attenuates inflammatory responses in the adipocytes through suppression of NF-κB and MAPKs activation, and also improves insulin sensitivity in 3T3-L1 cells, suggesting that luteolin may represent a therapeutic agent to prevent obesity-associated inflammation and insulin resistance. PMID:26246742

  5. Kainate receptor-mediated apoptosis in primary cultures of cerebellar granule cells is attenuated by mitogen-activated protein and cyclin-dependent kinase inhibitors

    PubMed Central

    Giardina, Sarah F; Beart, Philip M

    2002-01-01

    Previous studies have suggested that neuronal apoptosis is the result of an abortive attempt to re-enter the cell cycle, and more recently the cyclin-dependent (CDKs) and the mitogen-activated protein (MAP) kinases, two superfamilies of kinases that influence and control cell cycle progression, have been implicated in neuronal apoptosis. Here, to examine whether CDK/MAPK related pathways are involved in excitotoxicity, we studied the actions of various kinase inhibitors on apoptosis induced by the ionotropic glutamate (Glu) receptor agonist, kainate (KA), in primary cultures of murine cerebellar granule cells (CGCs). KA-mediated neurotoxicity was concentration-dependent, as determined by a cell viability assay monitoring the reduction of 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and largely apoptotic in nature, as shown by morphological examination and labelling of DNA fragmentation in situ using terminal deoxynucleotidyl transferase (TdT)-mediated dUTP digoxigenin nick-end labelling (TUNEL). KA-mediated neurotoxicity and apoptosis was completely attenuated by the mixed CDK and MAP kinase inhibitor, olomoucine, in a concentration-dependent manner (50 – 600 μM), and partially by roscovitine (1 – 100 μM), a more selective CDK inihibitor. The p38 MAP kinase inhibitor, SB203580 (1 – 100 μM), partially attenuated KA receptor-mediated apoptosis, as did the MAP kinase kinase inhibitors PD98509 (1 – 100 μM) and U0126 (1 – 100 μM). These findings provide new evidence for a complex network of interacting pathways involving CDK/MAPK that control apoptosis downstream of KA receptor activation in excitotoxic neuronal cell death. PMID:11934814

  6. Activation of Raf-1 and mitogen-activated protein kinase in murine macrophages partially mimics lipopolysaccharide-induced signaling events

    PubMed Central

    1995-01-01

    Lipopolysaccharide (LPS), a highly conserved component of the outer membrane of gram-negative bacteria, stimulates macrophages to release various cytokine and eicosanoid mediators of the immune response. The mechanism by which LPS stimulates these cells is poorly characterized. One of the most rapid LPS-stimulated events is the phosphorylation and activation of the p42 and p44 isoforms of mitogen-activated protein (MAP) kinase. We wished to examine the role of MAP kinase in LPS- induced signaling in murine macrophages by activating MAP kinase independently of LPS. An expression vector encoding a Raf-1:estrogen receptor (ER) chimeric protein was transfected into the murine macrophage cell line RAW 264.7. Activation of this chimeric protein (delta Raf-1:ER) by estradiol resulted in rapid and prolonged activation of MAP kinase, as expected from previous results implicating Raf-1 as an upstream activator of this signaling cascade. LPS stimulation induced accumulation of MAP kinase phosphatase 1 messenger RNA, whereas delta Raf-1:ER activation did not, perhaps accounting for the more prolonged activation of MAP kinase seen in response to delta Raf-1:ER activation. Similarly, activation of DNA binding by the transcription factor, nuclear factor (NF) kappa B, as assessed by electrophoretic mobility shift assay, occurred in response to LPS stimulation but not in response to delta Raf-1:ER activation or phorbol myristate acetate (PMA) stimulation. Using an enzyme-linked immunosorbent assay for murine tumor necrosis factor alpha (TNF-alpha), we found that LPS and PMA stimulation and delta Raf-1:ER activation induced secretion of TNF-alpha, although the amount of TNF-alpha secreted in response to delta Raf-1:ER activation and PMA stimulation was approximately 20-fold less than that secreted in response to LPS. Correspondingly, accumulation of TNF-alpha messenger RNA was weakly induced by delta Raf-1:ER activation or PMA stimulation, whereas strong induction was noted in

  7. Activation of Raf-1 and mitogen-activated protein kinase in murine macrophages partially mimics lipopolysaccharide-induced signaling events.

    PubMed

    Hambleton, J; McMahon, M; DeFranco, A L

    1995-07-01

    Lipopolysaccharide (LPS), a highly conserved component of the outer membrane of gram-negative bacteria, stimulates macrophages to release various cytokine and eicosanoid mediators of the immune response. The mechanism by which LPS stimulates these cells is poorly characterized. One of the most rapid LPS-stimulated events is the phosphorylation and activation of the p42 and p44 isoforms of mitogen-activated protein (MAP) kinase. We wished to examine the role of MAP kinase in LPS-induced signaling in murine macrophages by activating MAP kinase independently of LPS. An expression vector encoding a Raf-1:estrogen receptor (ER) chimeric protein was transfected into the murine macrophage cell line RAW 264.7. Activation of this chimeric protein (delta Raf-1:ER) by estradiol resulted in rapid and prolonged activation of MAP kinase, as expected from previous results implicating Raf-1 as an upstream activator of this signaling cascade. LPS stimulation induced accumulation of MAP kinase phosphatase 1 messenger RNA, whereas delta Raf-1:ER activation did not, perhaps accounting for the more prolonged activation of MAP kinase seen in response to delta Raf-1:ER activation. Similarly, activation of DNA binding by the transcription factor, nuclear factor (NF) kappa B, as assessed by electrophoretic mobility shift assay, occurred in response to LPS stimulation but not in response to delta Raf-1:ER activation or phorbol myristate acetate (PMA) stimulation. Using an enzyme-linked immunosorbent assay for murine tumor necrosis factor alpha (TNF-alpha), we found that LPS and PMA stimulation and delta Raf-1:ER activation induced secretion of TNF-alpha, although the amount of TNF-alpha secreted in response to delta Raf-1:ER activation and PMA stimulation was approximately 20-fold less than that secreted in response to LPS. Correspondingly, accumulation of TNF-alpha messenger RNA was weakly induced by delta Raf-1:ER activation or PMA stimulation, whereas strong induction was noted in

  8. Human immunodeficiency virus type 1 Nef binds directly to Lck and mitogen-activated protein kinase, inhibiting kinase activity.

    PubMed Central

    Greenway, A; Azad, A; Mills, J; McPhee, D

    1996-01-01

    It is now well established that human immunodeficiency virus type I (HIV-1) Nef contributes substantially to disease pathogenesis by augmenting virus replication and markedly perturbing T-cell function. The effect of Nef on host cell activation could be explained in part by its interaction with specific cellular proteins involved in signal transduction, including at least a member of the src family kinase, Lck, and the serine/threonine kinase, mitogen-activated protein kinase (MAPK). Recombinant Nef directly interacted with purified Lck and MAPK in coprecipitation experiments and binding assays. A proline-rich repeat sequence [(Pxx)4] in Nef occurring between amino acid residues 69 to 78 is highly conserved and bears strong resemblance to a defined consensus sequence identified as an SH3 binding domain present in several proteins which can interact with the SH3 domain of various signalling and cytoskeletal proteins. Binding and coprecipitation assays with short synthetic peptides corresponding to the proline-rich repeat sequence [(Pxx)4] of Nef and the SH2, SH3, or SH2 and SH3 domains of Lck revealed that the interaction between these two proteins is at least in part mediated by the proline repeat sequence of Nef and the SH3 domain of Lck. In addition to direct binding to full-length Nef, MAPK was also shown to bind the same proline repeat motif. Nef protein significantly decreased the in vitro kinase activity of Lck and MAPK. Inhibition of key members of signalling cascades, including those emanating from the T-cell receptor, by the HIV-1 Nef protein undoubtedly alters the ability of the infected T cell to respond to antigens or cytokines, facilitating HIV-1 replication and contributing to HIV-1-induced disease pathogenesis. PMID:8794306

  9. Simian virus 40 small t antigen cooperates with mitogen-activated kinases to stimulate AP-1 activity.

    PubMed Central

    Frost, J A; Alberts, A S; Sontag, E; Guan, K; Mumby, M C; Feramisco, J R

    1994-01-01

    The simian virus 40 small tumor antigen (small t) specifically interacts with protein phosphatase type 2A (PP2A) in vivo and alters its catalytic activity in vitro. Among the substrates for PP2A in vitro are the activated forms of MEK and ERK kinases. Dephosphorylation of the activating phosphorylation sites on MEK and ERKs by PP2A in vitro results in a decrease in their respective kinase activities. Recently, it has been shown that overexpression of small t in CV-1 cells results in an inhibition of PP2A activity toward MEK and ERK2 and a constitutive upregulation of MEK and ERK2 activity. Previously, we have observed that overexpression of either ERK1, MEK1, or a constitutively active truncated form of c-Raf-1 (BXB) is insufficient to activate AP-1 in REF52 fibroblasts. We therefore examined whether overexpression of small t either alone or in conjunction with ERK1, MEK1, or BXB could activate AP-1. We found that coexpression of small t and either ERK1, MEK1, or BXB resulted in an increase in AP-1 activity, whereas expression of either small t or any of the kinases alone did not have any effect. Similarly, coexpression of small t and ERK1 activated serum response element-regulated promoters. Coexpression of kinase-deficient mutants of ERK1 and ERK2 inhibited the activation of AP-1 caused by expression of small t and either MEK1 or BXB. Coexpression of an interfering MEK, which inhibited AP-1 activation by small t and BXB, did not inhibit the activation of AP-1 caused by small t and ERK1. In contrast to REF52 cells, we observed that overexpression of either small or ERK1 alone in CV-1 cells was sufficient to stimulate AP-1 activity and that this stimulation was not enhanced by expression of small t and ERK1 together. These results show that the effects of small t on immediate-early gene expression depend on the cell type examined and suggest that the mitogen-activated protein kinase activation pathway is distinctly regulated in different cell types. Images PMID

  10. EP2 Induces p38 Phosphorylation via the Activation of Src in HEK 293 Cells

    PubMed Central

    Chun, Kyung-Soo; Shim, Minsub

    2015-01-01

    Prostaglandin E2 (PGE2), a major product of cyclooxygenase, binds to four different prostaglandin E2 receptors (EP1, EP2, EP3, and EP4) which are G-protein coupled transmembrane receptors (GPCRs). Although GPCRs including EP receptors have been shown to be associated with their specific G proteins, recent evidences suggest that GPCRs can regulate MAPK signaling via non-G protein coupled pathways including Src. EP2 is differentially expressed in various tissues and the expression of EP2 is induced by extracellular stimuli. We hypothesized that an increased level of EP2 expression may affect MAPK signaling. The overexpression of EP2 in HEK 293 cells resulted in significant increase in intracellular cAMP levels response to treatment with butaprost, a specific EP2 agonist, while overexpression of EP2 alone did not increase intracellular cAMP levels. However, EP2 overexpression in the absence of PGE2 induced an increase in the level of p38 phosphorylation as well as the kinase activity of p38, suggesting that up-regulation of EP2 may promote p38 activation via non-G protein coupled pathway. Inhibition of Src completely blocked EP2-induced p38 phosphorylation and overexpression of Src increased the level of p38 phosphorylation, indicating that Src is upstream kinase for EP2-induced p38 phosphorylation. EP2 overexpression also increased the Src activity and EP2 protein was co-immunoprecipitated with Src. Furthermore, sequential co-immunoprecipitation studies showed that EP2, Src, and β-arrestin can form a complex. Our study found a novel pathway in which EP2 is associated with Src, regulating p38 pathway. PMID:26535079

  11. N-Farnesyloxy-norcantharimide inhibits progression of human leukemic Jurkat T cells through regulation of mitogen-activated protein kinase and interleukin-2 production

    PubMed Central

    Chang, Ming-Che; Wu, Jin-Yi; Liao, Hui-Fen; Chen, Yu-Jen

    2015-01-01

    This study investigated the anticancer effects of N-farnesyloxy-norcantharimide (NOC15), a newly synthesized norcantharidin (NCTD) analogue, on human leukemic Jurkat T cells and the signaling pathway underlying its effects. We found that the half maximal inhibitory concentration (IC50) of NOC15 on Jurkat T cells is 1.4 μmol/l, which is 11.14-fold (=15.6÷1.4) smaller than the 15.6 μmol/l of NCTD on Jurkat T cells, whereas the IC50 of NOC15 on human normal lymphoblast (HNL) is 207.9 μmol/l, which is 8.17-fold (=1698.0÷207.8) smaller than the 1698.0 μmol/l of NCTD on HNL cells. These results indicated that NOC15 exerts a higher anticancer effect on Jurkat T cells and has higher toxicity toward HNL cells than NCTD. Thus, NOC15 is 1.36-fold (=11.14÷8.17) beneficial as an anticancer agent toward Jurkat T cells compared with NCTD. Moreover, NOC15 can increase the percentage of cells in the sub-G1 phase and reduce the cell viability of Jurkat T cells, stimulate p38 and extracellular signal-regulated protein kinase 1/2 (ERK1/2) of mitogen-activated protein kinases (MAPKs) signaling pathway, and inhibit calcineurin expression and interleukin-2 (IL-2) production. However, NOC15 exerted no effects on the Jun-N-terminal kinase 1/2 (JNK1/2) signaling pathway, the production of IL-8, and tumor necrosis factor-α. We conclude that the anticancer activity of the newly synthesized NOC15 is 1.36-fold beneficial than NCTD as an anticancer agent and that NOC15 can increase the percentage of cells in the sub-G1 phase through the stimulation of p38 and ERK1/2 of the MAPK signaling pathway and the inhibition of calcineurin expression and IL-2 production. The NOC15 may have the potential of being developed into an anticancer agent in the future. PMID:26288134

  12. Apoptosis during spontaneous and prostaglandin F(2alpha)-induced luteal regression in the buffalo cow (Bubalus bubalis): involvement of mitogen-activated protein kinases.

    PubMed

    Yadav, Vijay K; Sudhagar, Ranga R; Medhamurthy, R

    2002-09-01

    The present study was conducted to evaluate whether the corpus luteum (CL) of the water buffalo (Bubalus bubalis) cow undergoes luteal regression by the process of apoptosis and to examine the involvement of mitogen-activated protein (MAP) kinases during prostaglandin (PG) F(2alpha)-induced luteolysis. Sections of CL from late in the estrous cycle, i.e., during spontaneous luteolysis, stained for 4',6'-diamidino-2-phenylindole revealed increased numbers of condensed nuclei, indicating cell death by apoptosis, which was confirmed further by the occurrence of pronounced oligonucleosome formation. For morphological and biochemical characterization during PGF(2alpha)-induced apoptosis, CL were collected at 0, 4, 12, and 18 h after injection of 750 micro g of Tiaprost, a synthetic analogue of PGF(2alpha), to midestrous buffalo cows. Serum progesterone concentrations fell within 4 h and decreased (P < 0.05) maximally by 18 h. Concomitant decreases (P < 0.05) in the levels of steroidogenic acute regulatory mRNA and protein were observed in CL during 12-18 h, with the more profound effect on mRNA levels. Quantitative analysis of the genomic DNA showed a >5-fold increase (P < 0.05) in the low molecular weight DNA fragments by 18 h postinjection. Immunoblot analysis of CL tissue lysates showed increased (P < 0.05) levels of phospho-Jun N-terminal kinase (JNK) 1 (4- to 14-fold during 4-18 h) and phospho-p38 (2- to 4-fold at 18 h). Immunohistochemical evaluation of CL sections revealed an increased nuclear localization of phospho-JNK after treatment. These findings demonstrate that the CL of the buffalo cow undergoes cell death by the process of apoptosis both during spontaneous and PGF(2alpha)-induced luteolysis and that MAP kinases are involved during PGF(2alpha)-mediated apoptosis in the CL.

  13. EhMAPK, the mitogen-activated protein kinase from Entamoeba histolytica is associated with cell survival.

    PubMed

    Ghosh, Anupama Sardar; Ray, Doel; Dutta, Suman; Raha, Sanghamitra

    2010-10-08

    Mitogen Activated Protein Kinases (MAPKs) are a class of serine/threonine kinases that regulate a number of different cellular activities including cell proliferation, differentiation, survival and even death. The pathogen Entamoeba histolytica possess a single homologue of a typical MAPK gene (EhMAPK) whose identification was previously reported by us but its functional implications remained unexplored. EhMAPK, the only mitogen-activated protein kinase from the parasitic protist Entamoeba histolytica with Threonine-X-Tyrosine (TXY) phosphorylation motif was cloned, expressed in E. coli and functionally characterized under different stress conditions. The expression profile of EhMAPK at the protein and mRNA level remained similar among untreated, heat shocked and hydrogen peroxide-treated samples in all cases of dose and time. But a significant difference was obtained in the phosphorylation status of the protein in response to different stresses. Heat shock at 43°C or 0.5 mM H(2)O(2) treatment enhanced the phosphorylation status of EhMAPK and augmented the kinase activity of the protein whereas 2.0 mM H(2)O(2) treatment induced dephosphorylation of EhMAPK and loss of kinase activity. 2.0 mM H(2)O(2) treatment reduced parasite viability significantly but heat shock and 0.5 mM H(2)O(2) treatment failed to adversely affect E. histolytica viability. Therefore, a distinct possibility that activation of EhMAPK is associated with stress survival in E. histolytica is seen. Our study also gives a glimpse of the regulatory mechanism of the protein under in vivo conditions. Since the parasite genome lacks any typical homologue of mammalian MEK, the dual specificity kinases which are the upstream activators of MAPK, indications of the existence of some alternate regulatory mechanisms of the EhMAPK activity is perceived. These may include the autophosphorylation activity of the protein itself in combination with some upstream phosphatases which are not yet identified.

  14. Postextinction Infusion of a Mitogen-Activated Protein Kinase Inhibitor into the Medial Prefrontal Cortex Impairs Memory of the Extinction of Conditioned Fear

    ERIC Educational Resources Information Center

    Hugues, Sandrine; Deschaux, Olivier; Garcia, Rene

    2004-01-01

    We investigated whether postextinction training infusion of PD098059, a selective inhibitor of mitogen-activated protein kinase (MAPK) activation, into the medial prefrontal cortex, would impair retention of extinction learning in rats. We found that immediate, but not late (2 or 4 h), postextinction infusion of PD098059 provoked a full return of…

  15. Beta interferon and oncostatin M activate Raf-1 and mitogen-activated protein kinase through a JAK1-dependent pathway.

    PubMed Central

    Stancato, L F; Sakatsume, M; David, M; Dent, P; Dong, F; Petricoin, E F; Krolewski, J J; Silvennoinen, O; Saharinen, P; Pierce, J; Marshall, C J; Sturgill, T; Finbloom, D S; Larner, A C

    1997-01-01

    Activation of early response genes by interferons (IFNs) and other cytokines requires tyrosine phosphorylation of a family of transcription factors termed signal transducers and activators of transcription (Stats). The Janus family of tyrosine kinases (Jak1, Jak2, Jak3, and Tyk2) is required for cytokine-induced tyrosine phosphorylation and dimerization of the Stat proteins. In order for IFNs to stimulate maximal expression of Stat1alpha-regulated genes, phosphorylation of a serine residue in the carboxy terminus by mitogen-activated protein kinase (MAPK) is also required. In HeLa cells, both IFN-beta and oncostatin M (OSM) stimulated MAPK and Raf-1 enzyme activity, in addition to Stat1 and Stat3 tyrosine phosphorylation. OSM stimulation of Raf-1 correlated with GTP loading of Ras, whereas IFN-beta activation of Raf-1 was Ras independent. IFN-beta- and OSM-induced Raf-1 activity could be coimmunoprecipitated with either Jak1 or Tyk2. Furthermore, HeLa cells lacking Jak1 displayed no activation of STAT1alpha, STAT3, and Raf-1 by IFN-beta or OSM and also demonstrated no increase in the relative level of GTP-bound p21ras in response to OSM. The requirement for Jak1 for IFN-beta- and OSM-induced activation of Raf-1 was also seen in Jak1-deficient U4A fibrosarcoma cells. Interestingly, basal MAPK, but not Raf-1, activity was constitutively enhanced in Jak1-deficient HeLa cells. Transient expression of Jak1 in both Jak-deficient HeLa cells and U4A cells reconstituted the ability of IFN-beta and OSM to activate Raf-1 and decreased the basal activity of MAPK, while expression of a kinase-inactive form of the protein showed no effect. Moreover, U4A cells selected for stable expression of Jak1, or COS cells transiently expressing Jak1 or Tyk2 but not Jak3, exhibited enhanced Raf-1 activity. Therefore, it appears that Jak1 is required for Raf-1 activation by both IFN-beta and OSM. These results provide evidence for a link between the Jaks and the Raf/MAPK signaling pathways

  16. Mitogen-activated protein kinase 3/mitogen-activated protein kinase 1 activates apoptosis during testicular ischemia-reperfusion injury in a nuclear factor-kappaB-independent manner.

    PubMed

    Minutoli, Letteria; Antonuccio, Pietro; Polito, Francesca; Bitto, Alessandra; Squadrito, Francesco; Di Stefano, Vincenzo; Nicotina, Piero Antonio; Fazzari, Carmine; Maisano, Daniele; Romeo, Carmelo; Altavilla, Domenica

    2009-02-14

    Nuclear factor kappa-B (NF-kappaB), mitogen-activated protein kinase3/MAPK1 and MAPK8 are involved in testicular ischemia reperfusion injury (testicular-I/R). NF-kappaB knock-out mice (KO) subjected to testicular-I/R have a reduced testicular damage, blunted MAPK8 activation and enhanced MAPK3/MAPK1 activity. To better understand the role of MAPK3/MAPK1 up-regulation during testicular-I/R, we investigated the effects of PD98059, an inhibitor of MAPK3/MAPK1, in KO mice during testicular-I/R. KO and wild-type (WT) animals underwent 1 h testicular ischemia followed by 24 h reperfusion or a sham testicular-I/R. Animals received either PD98059 (5 mg/kg/ip) or its vehicle. MAPK3/MAPK1, BAX, caspase-3 and -9 and TNF-alpha expression were assessed along with histological examination and an immunostaining for protein of apoptosis. Testicular-I/R caused a greater increase in MAPK3/MAPK1 in KO than in WT animals in both testes. KO mice had a lower expression of the apoptotic proteins and TNF-alpha as well as reduced histological damage compared to WT. Immunostaining confirmed the lower expression of BAX in the Leydig cells of KO mice. Administration of PD98059, abrogated MAPK3/MAPK1 expression and slightly reduced TNF-alpha but did not improve or reverse the histological damage in KO. PD98059 significantly reduced the histological damage in WT mice and markedly reduced the apoptotic proteins in KO and WT mice. These results suggest that testicular-I/R triggers also a pathway of organ damage involving MAPK3/MAPK1, TNF-alpha, BAX, caspase-3 and -9 that activates an apoptotic machinery in an NF-kappaB independent manner. These findings should contribute to better understand testicular torsion-induced damage.

  17. Inhibition of spinal constitutive NOS-2 by 1400W attenuates tissue injury and inflammation-induced hyperalgesia and spinal p38 activation.

    PubMed

    Tang, Qingbo; Svensson, Camilla I; Fitzsimmons, Bethany; Webb, Michael; Yaksh, Tony L; Hua, Xiao-Ying

    2007-05-01

    Nitric oxide (NO) and its synthesizing enzymes, including NO synthase-2 (NOS-2, also called inducible NOS, iNOS), have been implicated in spinal nociception. 1400W is a highly selective NOS-2 inhibitor, as compared with either NOS-1 (neuronal NOS, nNOS) or NOS-3 (endothelial NOS). Here we examined the anti-nociceptive effects of intrathecal (IT) administration of 1400W in two experimental models of hyperalgesia (formalin and carrageenan models), in addition to the effect of 1400W on stimulation-induced activation of spinal p38 mitogen-activated protein kinase (p38). IT treatment of rats with 1400W produced a dose-dependent inhibition of paw formalin-induced phase II flinches, and attenuated carrageenan-induced thermal hyperalgesia. In contrast, IT injection of a selective inhibitor of NOS-1, nNOS inhibitor-I, had no effect in either model. Furthermore, 1400W at a dose that suppressed formalin-induced flinching behavior also blocked formalin-evoked p38 phosphorylation (activation) in the spinal cord, while nNOS inhibitor-I displayed no activity. The prompt effects of IT 1400W suggest involvement of constitutively expressed NOS-2 in spinal nociception. The NOS-2 protein in rat spinal cords was undetectable by Western blotting. However, when the protein was immunoprecipitated prior to Western blotting, NOS-2-immunoreactive bands were detected in the tissues, including naïve spinal cords. The presence of constitutive spinal NOS-2 was further confirmed by reverse transcriptase-polymerase chain reaction. Taken together, the present studies suggest that constitutively expressed spinal NOS-2 mediates tissue injury and inflammation-induced hyperalgesia, and that activation of p38 is one of the downstream factors in NO-mediated signaling in the initial processing of spinal nociception.

  18. L1 cell adhesion molecule induces melanoma cell motility by activation of mitogen-activated protein kinase pathways.

    PubMed

    Yi, Young-Su; Baek, Kwang-Soo; Cho, Jae Youl

    2014-06-01

    L1 cell adhesion molecule (L1CAM) is highly expressed in various types of cancer cells and has been implicated in the control of cell proliferation and motility. Recently, L1CAM was reported to induce the motility of melanoma cells, but the mechanism of this induction remains poorly understood. In this study, we investigated the molecular mechanisms by which L1CAM induces the motility of melanoma cells. Unlike other types of cancer cells, B16F10 melanoma cells highly expressed L1CAM at both the RNA and protein levels, and the expression of L1CAM induced AP-1 activity. In accordance to AP-1 activation, MAPK signaling pathways were activated by L1CAM. Inhibition of L1CAM expression by L1CAM-specific siRNA suppressed the activation of MAPKs such as ERK and p38. However, no significant change was observed in JNK activation. As expected, upstream MAP2K, MKK3/6, MAP3K, and TAK1 were also deactivated by the inhibition of L1CAM expression. L1CAM induced the motility of B16F10 cells. Inhibition of L1CAM expression suppressed migration and invasion of B16F10 cells, but no suppressive effect was observed on their proliferation and anti-apoptotic resistance. Treatment of B16F10 cells with U0126, an ERK inhibitor, or SB203580, a p38 inhibitor, suppressed the migration and invasion abilities of B16F10 cells. Taken together, our results suggest that L1CAM induces the motility of B16F10 melanoma cells via the activation of MAPK pathways. This finding provides a more detailed molecular mechanism of L1CAM-mediated induction of melanoma cell motility. PMID:24974583

  19. Smooth-muscle mitogen-activated protein (MAP) kinase: purification and characterization, and the phosphorylation of caldesmon.

    PubMed Central

    Childs, T J; Mak, A S

    1993-01-01

    A single 42 kDa isoform of mitogen-activated protein (MAP) kinase is expressed in both embryonic and adult chicken gizzard. The gizzard MAP kinase, which cross-reacts with anti-p44mpk antibody, has been purified from adult chicken gizzard and partially characterized. The purification protocol employs phenyl-Sepharose, polylysine-agarose, hydroxyapatite, Mono-Q and phenyl-Superose column chromatography. The purified enzyme phosphorylates myelin basic protein and gizzard high-molecular-mass (h-)caldesmon. Sea-star p44mpk and gizzard MAP kinase phosphorylate h-caldesmon at identical sites at the C-terminal domain, as revealed by tryptic-peptide mapping of the phosphorylated protein. Phosphorylation of h-caldesmon by gizzard MAP kinase abolishes its interaction with polymerized tubulin. The specific activity of the purified gizzard kinase toward myelin basic protein is similar to that of brain tau kinase, but is only a fraction of that of activated sea-star p44mpk. This suggests that, although a large amount of MAP kinase is present in the gizzard, only a small percentage of the enzyme is activated normally. Autophosphorylation of the gizzard kinase, at least in part on tyrosine residues, activates its kinase activity. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 6 PMID:8280072

  20. RNase activity of sialic acid-binding lectin from bullfrog eggs drives antitumor effect via the activation of p38 MAPK to caspase-3/7 signaling pathway in human breast cancer cells

    PubMed Central

    Kariya, Yukiko; Tatsuta, Takeo; Sugawara, Shigeki; Kariya, Yoshinobu; Nitta, Kazuo; Hosono, Masahiro

    2016-01-01

    Sialic acid-binding lectin obtained from bullfrog eggs (SBL) induces cell death in cancer cells but not in normal cells. This antitumor effect is mediated through its ribo-nuclease (RNase) activity. However, the underlying molecular mechanisms remain unclear. We found that the p38 mitogen-activated protein kinase (MAPK) signaling pathway was activated when SBL induced cell death in three human breast cancer cell lines: SK-BR-3, MCF-7, and MDA-MB231. The suppression of p38 MAPK phosphorylation by a p38 MAPK inhibitor as well as short interference RNA knockdown of p38 MAPK expression significantly decreased cell death and increased the cell viability of SBL-treated MDA-MB231 cells. H103A, an SBL mutant lacking in RNase activity, showed decreased SBL-induced cell death compared with native SBL. However, the loss of RNase activity of SBL had no effect on its internalization into cells. The H103A mutant also displayed decreased phosphorylation of p38 MAPK. Moreover, SBL promoted caspase-3/7 activation followed by a cleavage of poly (ADP-ribose)-polymerase, whereas the SBL mutant, H103A, lost this ability. The SBL-induced caspase-3/7 activation was suppressed by the p38 MAPK inhibitor, SB203580, as well as pan-caspase inhibitor, zVAD-fmk. In the presence of zVAD-fmk, the SBL-induced cell death was decreased. In addition, the cell viability of SBL-treated MDA-MB231 cells recovered by zVAD-fmk treatment. Taken together, our results suggest that the RNase activity of SBL leads to breast cancer cell death through the activation of p38 MAPK followed by the activation of caspase-3/7. PMID:27513956

  1. Gallic acid ameliorates renal functions by inhibiting the activation of p38 MAPK in experimentally induced type 2 diabetic rats and cultured rat proximal tubular epithelial cells.

    PubMed

    Ahad, Amjid; Ahsan, Haseeb; Mujeeb, Mohd; Siddiqui, Waseem Ahmad

    2015-10-01

    Diabetic nephropathy (DN) is one of the leading causes of morbidity and mortality in diabetic patients that accounts for about 40% of deaths in type 2 diabetes. p38 mitogen activated protein kinase (p38 MAPK), a serine-threonine kinase, plays an important role in tissue inflammation and is known to be activated under conditions of oxidative stress and hyperglycemia. The role of p38 MAPK has been demonstrated in DN, and its inhibition has been suggested as an alternative approach in the treatment of DN. In the present study, we investigated the nephroprotective effects of an anti-inflammatory phenolic compound, gallic acid (GA, 3,4,5-trihydroxybenzoic acid), in high fat diet/streptozotocin (HFD/STZ) induce type 2 diabetic wistar albino rats. GA (25 mg/kgbw and 50 mg/kgbw, p.o.) treatment for 16 weeks post induction of diabetes led to a significant reduction in the levels of blood glucose, HbA1c, serum creatinine, blood urea nitrogen and proteinuria as well as a significant reduction in the levels of creatinine clearance. GA significantly inhibited the renal p38 MAPK and nuclear factor kappa B (N-κB) activation as well as significantly reduced the levels of renal transforming growth factor beta (TGF-β) and fibronectin. Treatment with GA resulted in a significant reduction in the serum levels of proinflammatory cytokines viz. interleukin 1 beta (IL-1β), IL-6 and tumor necrosis factor alpha (TNF-α). Moreover, GA significantly lowered renal pathology and attenuated renal oxidative stress. In cultured rat NRK 52E proximal tubular epithelial cells, GA treatment inhibited high glucose induced activation of p38 MAPK and NF-κB as well as suppressed proinflammatory cytokine synthesis. The results of the present study provide in vivo and in vitro evidences that the p38 MAPK pathway plays an important role in the pathogenesis of DN, and GA attenuates the p38 MAPK-mediated renal dysfunction in HFD/STZ induced type 2 diabetic rats.

  2. Paeonol suppresses oxidized low-density lipoprotein induced endothelial cell apoptosis via activation of LOX-1/p38MAPK/NF-κB pathway.

    PubMed

    Bao, Mei-Hua; Zhang, Yi-Wen; Zhou, Hong-Hao

    2013-03-27

    Paeonol is an active compound isolated from traditional Chinese medicine, and has been shown to have anti-atherosclerosis, anti-inflammatory, antioxidant effects. The present investigation was undertaken to determine the suppression effects of paeonol on oxidized low-density lipoprotein (ox-LDL) induced endothelial cell line HUVEC apoptosis and to uncover some of the underlying mechanisms of these effects. Cell viability and lactate dehydrogenase (LDH) were measured to evaluate the cell injuries. Apoptosis was evaluated by Hoechst 33342 staining and flow cytometry. Intracellular reactive oxygen species (ROS) generation was detected by 2',7'-dichlorofluorescein diacetate (DCFH-DA). Real-time PCR was used to confirm the expression of LOX-1 mRNA. Western blotting was used to evaluate the protein expression of LOX-1 and Bcl-2, as well as caspase-3 cleavage, p38-mitogen-activated protein kinase (p38MAPK) phosphorylation. NF-κB nuclear translocation was detected by Western blotting and immunofluorescence. Caspase-3 activity was measured using a colorimetric protease assay kit. The results showed that ox-LDL significantly decreased cell viability and increased the LDH release, as well as the apoptotic rate (P<0.01). Pre-treatment of paeonol resulted in remarkable increase of cell viability, decrease of LDH release and cell apoptosis in a concentration-dependent manner. Besides, ox-LDL caused the up-regulation of LOX-1, the down-regulation of Bcl-2, the phosphorylation of p38MAPK, the translocation of NF-κB and the activation of caspase-3. Paeonol pre-treatment reversed these effects introduced by ox-LDL. Moreover, paeonol also showed its inhibition effects on ox-LDL induced ROS overproduction. These results indicate the preventive effects of paeonol on ox-LDL induced endothelial cell apoptosis. The effects might, at least partly, be obtained via inhibition of LOX-1-ROS- p38MAPK-NF-κB signaling pathway.

  3. 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

  4. 6-Hydroxydopamine activates the mitochondrial apoptosis pathway through p38 MAPK-mediated, p53-independent activation of Bax and PUMA.

    PubMed

    Gomez-Lazaro, Maria; Galindo, Maria F; Concannon, Caoimhín G; Segura, Miguel F; Fernandez-Gomez, Francisco J; Llecha, Nuria; Comella, Joan X; Prehn, Jochen H M; Jordan, Joaquin

    2008-03-01

    Mitochondrial alterations have been associated with the cytotoxic effect of 6-hydroxydopamine (6-OHDA), a widely used toxin to study Parkinson's disease. In previous work, we have demonstrated that 6-OHDA increases mitochondrial membrane permeability leading to cytochrome c release, but the precise mechanisms involved in this process remain unknown. Herein we studied the mechanism of increased mitochondrial permeability of SH-SY5Y neuroblastoma cells in response to 6-OHDA. Cytochrome c release induced by 6-OHDA occurred, in both SH-SY5Y cells and primary cultures, in the absence of mitochondrial swelling or a decrease in mitochondrial calcein fluorescence, suggesting little involvement of the mitochondrial permeability transition pore in this process. In contrast, 6-OHDA-induced cell death was associated with a significant translocation of the pro-apoptotic Bax protein from the cytosol to mitochondria and with a significant induction of the BH3-only protein PUMA. Experiments in mouse embryonic fibroblasts deficient in Bax or PUMA demonstrated a role for both proteins in 6-OHDA-induced apoptosis. Although 6-OHDA elevated both total and nuclear p53 protein levels, activation of p53 was not essential for subsequent cell death. In contrast, we found that p38 mitogen-activated protein kinase (MAPK) was activated early during 6-OHDA-induced apoptosis, and that treatment with the p38 MAPK inhibitor SKF86002 potently inhibited PUMA induction, green fluorescent protein-Bax redistribution and apoptosis in response to 6-OHDA. These data demonstrate a critical involvement of p38 MAPK, PUMA, and Bax in 6-OHDA-induced apoptosis.

  5. Competition between members of the tribbles pseudokinase protein family shapes their interactions with mitogen activated protein kinase pathways.

    PubMed

    Guan, Hongtao; Shuaib, Aban; Leon, David Davila De; Angyal, Adrienn; Salazar, Maria; Velasco, Guillermo; Holcombe, Mike; Dower, Steven K; Kiss-Toth, Endre

    2016-01-01

    Spatio-temporal regulation of intracellular signalling networks is key to normal cellular physiology; dysregulation of which leads to disease. The family of three mammalian tribbles proteins has emerged as an important controller of signalling via regulating the activity of mitogen activated protein kinases (MAPK), the PI3-kinase induced signalling network and E3 ubiquitin ligases. However, the importance of potential redundancy in the action of tribbles and how the differences in affinities for the various binding partners may influence signalling control is currently unclear. We report that tribbles proteins can bind to an overlapping set of MAPK-kinases (MAPKK) in live cells and dictate the localisation of the complexes. Binding studies in transfected cells reveal common regulatory mechanisms and suggest that tribbles and MAPKs may interact with MAPKKs in a competitive manner. Computational modelling of the impact of tribbles on MAPK activation suggests a high sensitivity of this system to changes in tribbles levels, highlighting that these proteins are ideally placed to control the dynamics and balance of activation of concurrent signalling pathways. PMID:27600771

  6. Competition between members of the tribbles pseudokinase protein family shapes their interactions with mitogen activated protein kinase pathways

    PubMed Central

    Guan, Hongtao; Shuaib, Aban; Leon, David Davila De; Angyal, Adrienn; Salazar, Maria; Velasco, Guillermo; Holcombe, Mike; Dower, Steven K.; Kiss-Toth, Endre

    2016-01-01

    Spatio-temporal regulation of intracellular signalling networks is key to normal cellular physiology; dysregulation of which leads to disease. The family of three mammalian tribbles proteins has emerged as an important controller of signalling via regulating the activity of mitogen activated protein kinases (MAPK), the PI3-kinase induced signalling network and E3 ubiquitin ligases. However, the importance of potential redundancy in the action of tribbles and how the differences in affinities for the various binding partners may influence signalling control is currently unclear. We report that tribbles proteins can bind to an overlapping set of MAPK-kinases (MAPKK) in live cells and dictate the localisation of the complexes. Binding studies in transfected cells reveal common regulatory mechanisms and suggest that tribbles and MAPKs may interact with MAPKKs in a competitive manner. Computational modelling of the impact of tribbles on MAPK activation suggests a high sensitivity of this system to changes in tribbles levels, highlighting that these proteins are ideally placed to control the dynamics and balance of activation of concurrent signalling pathways. PMID:27600771

  7. The inducible G protein-coupled receptor edg-1 signals via the G(i)/mitogen-activated protein kinase pathway.

    PubMed

    Lee, M J; Evans, M; Hla, T

    1996-05-10

    The edg-1 gene encodes an inducible G protein-coupled receptor (GPR) homologue that is induced during the in vitro differentiation of human endothelial cells. The aim of this study was to investigate the G protein-coupling and -signaling properties of the edg-1 polypeptide. The third cytosolic loop (i3) of edg-1 associates with G(i) alpha and G(o) alpha polypeptides in a guanosine 5'-O-(thiotriphosphate)-sensitive manner. Immunoprecipitation of the edg-1 polypeptide in transfected cells results in the co-precipitation of G(i) alpha 1 and G(i) alpha 3 polypeptides. These data strongly suggest that edg-1 is capable of coupling to the Gi pathway. Overexpression of the edg-1 GPR in human embryonic kidney 293 cells results in the sustained activation of the MAP kinase activity that is blocked by pertussis toxin treatment. Moreover, NIH3T3 cells permanently transfected with edg-1 exhibit enhanced MAP kinase and phospholipase A2 activities. These data suggest that the G(i)/mitogen-activated protein kinase pathway is a major signaling pathway regulated by the orphan receptor edg-1. PMID:8626678

  8. Dehydrocorydaline promotes myogenic differentiation via p38 MAPK activation.

    PubMed

    Yoo, Miran; Lee, Sang-Jin; Kim, Yong Kee; Seo, Dong-Wan; Baek, Nam-In; Ryu, Jae-Ha; Kang, Jong-Sun; Bae, Gyu-Un

    2016-10-01

    Muscle regeneration is a coordinated process that involves proliferation and differentiation of muscle progenitor cells. Activation of MyoD is a key event in myogenic differentiation, which is regulated by p38 mitogen‑activated protein kinases (MAPK). In a screen of natural compounds for the enhancement of MyoD activity, dehydrocorydaline (DHC) from the Corydalis tuber was identified. Treatment of C2C12 myoblasts with DHC increased the expression levels of muscle‑specific proteins, including MyoD, myogenin and myosin heavy chain. In addition, C2C12 myoblasts exhibited enhanced multinucleated myotube formation without any cytotoxicity. Treatment with DHC elevated p38 MAPK activation and the interaction of MyoD with an E protein, which is likely to result in activation of MyoD and promotion of myoblast differentiation. Furthermore, defects in differentiation‑induced p38 MAPK activation and myoblast differentiation induced by depletion of the promyogenic receptor protein Cdo in C2C12 myoblasts were restored by DHC treatment. In conclusion, these results indicated that DHC stimulates p38 MAPK activation, which can enhance heterodimerization of MyoD and E proteins, thus resulting in MyoD activation and myoblast differentiation. These findings suggested that DHC may be considered a potential therapeutic compound for the improvement of muscle stem cell regenerative capacity in injured muscle.

  9. Dehydrocorydaline promotes myogenic differentiation via p38 MAPK activation.

    PubMed

    Yoo, Miran; Lee, Sang-Jin; Kim, Yong Kee; Seo, Dong-Wan; Baek, Nam-In; Ryu, Jae-Ha; Kang, Jong-Sun; Bae, Gyu-Un

    2016-10-01

    Muscle regeneration is a coordinated process that involves proliferation and differentiation of muscle progenitor cells. Activation of MyoD is a key event in myogenic differentiation, which is regulated by p38 mitogen‑activated protein kinases (MAPK). In a screen of natural compounds for the enhancement of MyoD activity, dehydrocorydaline (DHC) from the Corydalis tuber was identified. Treatment of C2C12 myoblasts with DHC increased the expression levels of muscle‑specific proteins, including MyoD, myogenin and myosin heavy chain. In addition, C2C12 myoblasts exhibited enhanced multinucleated myotube formation without any cytotoxicity. Treatment with DHC elevated p38 MAPK activation and the interaction of MyoD with an E protein, which is likely to result in activation of MyoD and promotion of myoblast differentiation. Furthermore, defects in differentiation‑induced p38 MAPK activation and myoblast differentiation induced by depletion of the promyogenic receptor protein Cdo in C2C12 myoblasts were restored by DHC treatment. In conclusion, these results indicated that DHC stimulates p38 MAPK activation, which can enhance heterodimerization of MyoD and E proteins, thus resulting in MyoD activation and myoblast differentiation. These findings suggested that DHC may be considered a potential therapeutic compound for the improvement of muscle stem cell regenerative capacity in injured muscle. PMID:27573543

  10. 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

  11. Riboflavin-Induced Disease Resistance Requires the Mitogen-Activated Protein Kinases 3 and 6 in Arabidopsis thaliana

    PubMed Central

    Nie, Shengjun; Xu, Huilian

    2016-01-01

    As a resistance elicitor, riboflavin (vitamin B2) protects plants against a wide range of pathogens. At molecular biological levels, it is important to elucidate the signaling pathways underlying the disease resistance induced by riboflavin. Here, riboflavin was tested to induce resistance against virulent Pseudomonas syringae pv. Tomato DC3000 (Pst DC3000) in Arabidopsis. Results showed that riboflavin induced disease resistance based on MAPK-dependent priming for the expression of PR1 gene. Riboflavin induced transient expression of PR1 gene. However, following Pst DC3000 inoculation, riboflavin potentiated stronger PR1 gene transcription. Further was suggested that the transcript levels of mitogen-activated protein kinases, MPK3 and MPK6, were primed under riboflavin. Upon infection by Pst DC3000, these two enzymes were more strongly activated. The elevated activation of both MPK3 and MPK6 was responsible for enhanced defense gene expression and resistance after riboflavin treatment. Moreover, riboflavin significantly reduced the transcript levels of MPK3 and MPK6 by application of AsA and BAPTA, an H2O2 scavenger and a calcium (Ca2+) scavenger, respectively. In conclusion, MPK3 and MPK6 were responsible for riboflavin-induced resistance, and played an important role in H2O2- and Ca2+-related signaling pathways, and this study could provide a new insight into the mechanistic study of riboflavin-induced defense responses. PMID:27054585

  12. Characterization of a mitogen-activated protein kinase gene from cucumber required for trichoderma-conferred plant resistance.

    PubMed

    Shoresh, Michal; Gal-On, Amit; Leibman, Diana; Chet, Ilan

    2006-11-01

    The fungal biocontrol agent Trichoderma asperellum has been recently shown to induce systemic resistance in plants through a mechanism that employs jasmonic acid and ethylene signal transduction pathways. Mitogen-activated protein kinase (MAPK) proteins have been implicated in the signal transduction of a wide variety of plant stress responses. Here we report the identification and characterization of a Trichoderma-induced MAPK (TIPK) gene function in cucumber (Cucumis sativus). Similar to its homologs, wound-induced protein kinase, MPK3, and MPK3a, TIPK is also induced by wounding. Normally, preinoculation of roots with Trichoderma activates plant defense mechanisms, which result in resistance to the leaf pathogen Pseudomonas syringae pv lachrymans. We used a unique attenuated virus vector, Zucchini yellow mosaic virus (ZYMV-AGII), to overexpress TIPK protein and antisense (AS) RNA. Plants overexpressing TIPK were more resistant to pathogenic bacterial attack than control plants, even in the absence of Trichoderma preinoculation. On the other hand, plants expressing TIPK-AS revealed increased sensitivity to pathogen attack. Moreover, Trichoderma preinoculation could not protect these AS plants against subsequent pathogen attack. We therefore demonstrate that Trichoderma exerts its protective effect on plants through activation of the TIPK gene, a MAPK that is involved in signal transduction pathways of defense responses.

  13. Mitogen-Activated Protein Kinase Signaling in Plant-Interacting Fungi: Distinct Messages from Conserved Messengers[W

    PubMed Central

    Hamel, Louis-Philippe; Nicole, Marie-Claude; Duplessis, Sébastien; Ellis, Brian E.

    2012-01-01

    Mitogen-activated protein kinases (MAPKs) are evolutionarily conserved proteins that function as key signal transduction components in fungi, plants, and mammals. During interaction between phytopathogenic fungi and plants, fungal MAPKs help to promote mechanical and/or enzymatic penetration of host tissues, while plant MAPKs are required for activation of plant immunity. However, new insights suggest that MAPK cascades in both organisms do not operate independently but that they mutually contribute to a highly interconnected molecular dialogue between the plant and the fungus. As a result, some pathogenesis-related processes controlled by fungal MAPKs lead to the activation of plant signaling, including the recruitment of plant MAPK cascades. Conversely, plant MAPKs promote defense mechanisms that threaten the survival of fungal cells, leading to a stress response mediated in part by fungal MAPK cascades. In this review, we make use of the genomic data available following completion of whole-genome sequencing projects to analyze the structure of MAPK protein families in 24 fungal taxa, including both plant pathogens and mycorrhizal symbionts. Based on conserved patterns of sequence diversification, we also propose the adoption of a unified fungal MAPK nomenclature derived from that established for the model species Saccharomyces cerevisiae. Finally, we summarize current knowledge of the functions of MAPK cascades in phytopathogenic fungi and highlight the central role played by MAPK signaling during the molecular dialogue between plants and invading fungal pathogens. PMID:22517321

  14. Cdc42p-Interacting Protein Bem4p Regulates the Filamentous-Growth Mitogen-Activated Protein Kinase Pathway

    PubMed Central

    Pitoniak, Andrew; Chavel, Colin A.; Chow, Jacky; Smith, Jeremy; Camara, Diawoye; Karunanithi, Sheelarani; Li, Boyang; Wolfe, Kennith H.

    2014-01-01

    The ubiquitous Rho (Ras homology) GTPase Cdc42p can function in different settings to regulate cell polarity and cellular signaling. How Cdc42p and other proteins are directed to function in a particular context remains unclear. We show that the Cdc42p-interacting protein Bem4p regulates the mitogen-activated protein kinase (MAPK) pathway that controls filamentous growth in Saccharomyces cerevisiae. Bem4p controlled the filamentous-growth pathway but not other MAPK pathways (mating or high-osmolarity glycerol response [HOG]) that also require Cdc42p and other shared components. Bem4p associated with the plasma membrane (PM) protein, Sho1p, to regulate MAPK activity and cell polarization under nutrient-limiting conditions that favor filamentous growth. Bem4p also interacted with the major activator of Cdc42p, the guanine nucleotide exchange factor (GEF) Cdc24p, which we show also regulates the filamentous-growth pathway. Bem4p interacted with the pleckstrin homology (PH) domain of Cdc24p, which functions in an autoinhibitory capacity, and was required, along with other pathway regulators, to maintain Cdc24p at polarized sites during filamentous growth. Bem4p also interacted with the MAPK kinase kinase (MAPKKK) Ste11p. Thus, Bem4p is a new regulator of the filamentous-growth MAPK pathway and binds to general proteins, like Cdc42p and Ste11p, to promote a pathway-specific response. PMID:25384973

  15. Chitosan Controls Postharvest Decay on Cherry Tomato Fruit Possibly via the Mitogen-Activated Protein Kinase Signaling Pathway.

    PubMed

    Zhang, Danfeng; Wang, Hongtao; Hu, Yi; Liu, Yongsheng

    2015-08-26

    The inhibitive effects of chitosan on gray mold caused by Botrytis cinerea on cherry tomato fruit were evaluated. Decay incidence was tested on tomato stored at 22 °C. Hydrogen peroxide accumulation, malondialdehyde (MDA) production, peroxidase (POD) activity, and several related gene expressions (including MPK3, MPK6, PR1a1, and PR5) were determined. Results showed that 0.2% of chitosan solution significantly inhibited the tomato gray mold 3 days after inoculation. Hydrogen peroxide accumulated in the fruit epidermal peel along with chitosan treatment, while MDA production was not increased. POD activity was remarkably enhanced by the application of chitosan. The relative expressions of MPK3, MPK6, and PR1a1 were significantly induced in 10 min after chitosan treatment, while PR5 was induced in 20 min. These findings suggested that the effects of chitosan on inhibiting gray mold in cherry tomato fruit were probably associated with the mitogen-activated protein kinase (MAPK) signaling pathway. PMID:26223862

  16. Riboflavin-Induced Disease Resistance Requires the Mitogen-Activated Protein Kinases 3 and 6 in Arabidopsis thaliana.

    PubMed

    Nie, Shengjun; Xu, Huilian

    2016-01-01

    As a resistance elicitor, riboflavin (vitamin B2) protects plants against a wide range of pathogens. At molecular biological levels, it is important to elucidate the signaling pathways underlying the disease resistance induced by riboflavin. Here, riboflavin was tested to induce resistance against virulent Pseudomonas syringae pv. Tomato DC3000 (Pst DC3000) in Arabidopsis. Results showed that riboflavin induced disease resistance based on MAPK-dependent priming for the expression of PR1 gene. Riboflavin induced transient expression of PR1 gene. However, following Pst DC3000 inoculation, riboflavin potentiated stronger PR1 gene transcription. Further was suggested that the transcript levels of mitogen-activated protein kinases, MPK3 and MPK6, were primed under riboflavin. Upon infection by Pst DC3000, these two enzymes were more strongly activated. The elevated activation of both MPK3 and MPK6 was responsible for enhanced defense gene expression and resistance after riboflavin treatment. Moreover, riboflavin significantly reduced the transcript levels of MPK3 and MPK6 by application of AsA and BAPTA, an H2O2 scavenger and a calcium (Ca2+) scavenger, respectively. In conclusion, MPK3 and MPK6 were responsible for riboflavin-induced resistance, and played an important role in H2O2- and Ca2+-related signaling pathways, and this study could provide a new insight into the mechanistic study of riboflavin-induced defense responses. PMID:27054585

  17. Interplay between mitogen-activated protein kinase and nitric oxide in brassinosteroid-induced pesticide metabolism in Solanum lycopersicum.

    PubMed

    Yin, Yan-Ling; Zhou, Yue; Zhou, Yan-Hong; Shi, Kai; Zhou, Jie; Yu, Yunlong; Yu, Jing-Quan; Xia, Xiao-Jian

    2016-10-01

    Nitric oxide (NO) and mitogen-activated protein kinase (MPK) play important roles in brassinosteroid (BR)-induced stress tolerance, however, their functions in BR-induced pesticides metabolism remain unclear. Here, we showed that MPK activity and transcripts of SlMPK1 and SlMPK2 were induced by chlorothalonil (CHT), a widely used fungicide, in tomato leaves. However, cosilencing of SlMPK1/2 compromised the 24-epibrassinolide (EBR)-induced upregulation of detoxification genes and CHT metabolism in tomato leaves. In addition, cosilencing of SlMPK1/2 inhibited the accumulation of S-nitrosothiol (SNO), the reservoir of nitric oxide (NO) in plants, whereas tungstate, the inhibitor of nitrate reductase (NR), blocked EBR-induced SNO accumulation and MPK activity. Inhibiting the accumulation of NO by cPTIO, the specific scavenger and tungstate abolished the EBR-induced upregulation of detoxification genes, glutathione accumulation and CHT metabolism. The results showed that MPK and NR-dependent NO were involved in BR-induced CHT metabolism. Notably, there was a positive crosstalk between the MPK and NO production. PMID:27236431

  18. Silica nanoparticles induce cytokine responses in lung epithelial cells through activation of a p38/TACE/TGF-α/EGFR-pathway and NF-κΒ signalling

    SciTech Connect

    Skuland, Tonje Øvrevik, Johan; Låg, Marit; Schwarze, Per; Refsnes, Magne

    2014-08-15

    Amorphous silica nanoparticles (SiNPs) have previously been shown to induce marked cytokine (interleukin-6; IL-6 and interleukin-8; CXCL8/IL-8) responses independently of particle uptake in human bronchial epithelial BEAS-2B cells. In this study the involvement of the mitogen-activated protein kinases (MAP-kinases), nuclear factor-kappa Β (NF-κΒ) and in particular tumour necrosis factor-α converting enzyme (TACE) and—epidermal growth factor receptor (EGFR) signalling pathways were examined in triggering of IL-6 and CXCL8 release after exposure to a 50 nm silica nanoparticle (Si50). Exposure to Si50 increased phosphorylation of NF-κΒ p65 and MAP-kinases p38 and JUN-N-terminal protein kinase pathways (JNK), but not extracellular signal regulated kinases (ERK). Inhibition of NF-κΒ and p38 reduced the cytokine responses to Si50, whereas neither JNK- nor ERK-inhibition exerted any significant effect on the responses to Si50. Increases in membrane-bound transforming growth factor-α (TGF-α) release and EGFR phosphorylation were also observed after Si50 exposure, and pre-treatment with inhibitors of these pathways reduced the release of IL-6 and CXCL8, but did not affect the Si50-induced phosphorylation of p38 and p65. In contrast, p38-inhibition partially reduced Si50-induced TGF-α release, while the p65-inhibition was without effect. Overall, our results indicate that Si50-induced IL-6 and CXCL8 responses in BEAS-2B cells were regulated through combined activation of several pathways, including NF-κΒ and p38/TACE/TGF-α/EGFR signalling. The study identifies critical, initial events in the triggering of pro-inflammatory responses by nanoparticles. - Highlights: • Silica nanoparticles induce IL-6 and CXCL8 via NFκB and MAPKinase p38 in BEAS-2B • Silica nanoparticles induce release of the EGF-receptor ligand TGF-α • TGF-α release contributes to the IL-6 and CXCL8 release • Phosphorylation of p38 is involved in release of TGF-α.

  19. 3pK, a new mitogen-activated protein kinase-activated protein kinase located in the small cell lung cancer tumor suppressor gene region.

    PubMed Central

    Sithanandam, G; Latif, F; Duh, F M; Bernal, R; Smola, U; Li, H; Kuzmin, I; Wixler, V; Geil, L; Shrestha, S

    1996-01-01

    NotI linking clones, localized to the human chromosome 3p21.3 region and homozygously deleted in small cell lung cancer cell lines NCI-H740 and NCI-H1450, were used to search for a putative tumor suppressor gene(s). One of these clones, NL1G210, detected a 2.5-kb mRNA in all examined human tissues, expression being especially high in the heart and skeletal muscle. Two overlapping cDNA clones containing the entire open reading frame were isolated from a human heart cDNA library and fully characterized. Computer analysis and a search of the GenBank database to reveal high sequence identity of the product of this gene to serine-threonine kinases, especially to mitogen-activated protein kinase-activated protein kinase 2, a recently described substrate of mitogen-activated kinases. Sequence identitiy was 72% at the nucleotide level and 75% at the amino acid level, strongly suggesting that this protein is a serine-threonine kinase. Here we demonstrate that the new gene, referred to as 3pK (for chromosome 3p kinase), in fact encodes a mitogen-activated protein kinase-regulated protein serine-threonine kinase with a novel substrate specificity. PMID:8622688

  20. The CORM ALF-186 Mediates Anti-Apoptotic Signaling via an Activation of the p38 MAPK after Ischemia and Reperfusion Injury in Retinal Ganglion Cells

    PubMed Central

    Ulbrich, Felix; Kaufmann, Kai B.; Meske, Alexander; Lagrèze, Wolf A.; Augustynik, Michael; Buerkle, Hartmut; Ramao, Carlos C.; Biermann, Julia

    2016-01-01

    Purpose Ischemia and reperfusion injury may induce apoptosis and lead to sustained tissue damage and loss of function, especially in neuronal organs. While carbon monoxide is known to exert protective effects after various harmful events, the mechanism of carbon monoxide releasing molecules in neuronal tissue has not been investigated yet. We hypothesize that the carbon monoxide releasing molecule (CORM) ALF-186, administered after neuronal ischemia-reperfusion injury (IRI), counteracts retinal apoptosis and its involved signaling pathways and consecutively reduces neuronal tissue damage. Methods IRI was performed in rat´s retinae for 1 hour. The water-soluble CORM ALF-186 (10 mg/kg) was administered intravenously via a tail vein after reperfusion. After 24 and 48 hours, retinal tissue was harvested to analyze mRNA and protein expression of Bcl-2, Bax, Caspase-3, ERK1/2, p38 and JNK. Densities of fluorogold pre-labeled retinal ganglion cells (RGC) were analyzed 7 days after IRI. Immunohistochemistry was performed on retinal cross sections. Results ALF-186 significantly reduced IRI mediated loss of RGC. ALF-186 treatment differentially affected mitogen-activated protein kinases (MAPK) phosphorylation: ALF-186 activated p38 and suppressed ERK1/2 phosphorylation, while JNK remained unchanged. Furthermore, ALF-186 treatment affected mitochondrial apoptosis, decreasing pro-apoptotic Bax and Caspase-3-cleavage, but increasing anti-apoptotic Bcl-2. Inhibition of p38-MAPK using SB203580 reduced ALF-186 mediated anti-apoptotic effects. Conclusion In this study, ALF-186 mediated substantial neuroprotection, affecting intracellular apoptotic signaling, mainly via MAPK p38. CORMs may thus represent a promising therapeutic alternative treating neuronal IRI. PMID:27764224

  1. Src Tyrosine Kinase Activation by 4-Hydroxynonenal Upregulates p38, ERK/AP-1 Signaling and COX-2 Expression in YPEN-1 Cells

    PubMed Central

    Jang, Eun Ji; Jeong, Hyoung Oh; Park, Daeui; Kim, Dae Hyun; Choi, Yeon Ja; Chung, Ki Wung; Park, Min Hi; Yu, Byung Pal; Chung, Hae Young

    2015-01-01

    4-Hydroxynonenal (4-HNE), a major end product of lipid peroxidation, is highly reactive and involved in various cellular processes, such as inflammatory signaling. However, to date, the mechanistic roles of 4-HNE in inflammatory signaling related to protein tyrosine kinases have not been elucidated. In the present study, we investigated the interaction between 4-HNE and Src (a non-receptor tyrosine kinase) for its involvement in the molecular modulation of the inflammatory signaling pathway utilizing the YPEN-1 cell system. Immunoprecipitation experiments showed that 4-HNE phosphorylates (activates) Src at Tyr416 via adduct formation. In addition, LC-MS/MS and a docking simulation model revealed an addiction site at the Cys248 residue of Src, resulting in the stimulation of downstream p38, ERK/AP-1 and cyclooxygenase-2 (COX-2) signaling in YPEN-1 cells. The role of 4-HNE-activated Src in downstream inflammatory signaling was further investigated using dasatinib (a Src inhibitor) and by siRNA knockdown of Src. p38 and ERK were directly regulated by Src, as revealed by immunoblotting of the phosphorylated forms of mitogen-activated protein kinases (MAPKs), which are key elements in the signaling transduction pathway initiated by Src. The study also shows that Src modulates the HNE-enhanced activation of AP-1 and the expression of COX-2 (a target gene of AP-1). Together, the results of this study show that 4-HNE stimulates Src tyrosine kinase in activation of the inflammation process. PMID:26466383

  2. Anti-Food Allergic Activity of Sulfated Polysaccharide from Gracilaria lemaneiformis is Dependent on Immunosuppression and Inhibition of p38 MAPK.

    PubMed

    Liu, Qing-Mei; Yang, Yang; Maleki, Soheila J; Alcocer, Marcos; Xu, Sha-Sha; Shi, Chao-Lan; Cao, Min-Jie; Liu, Guang-Ming

    2016-06-01

    Polysaccharides from Gracilaria lemaneiformis in particular possess various bioactive functions, but their antiallergic activity remains incompletely defined. Sulfated polysaccharide from Gracilaria lemaneiformis (GLSP) was obtained by water extraction and ethanol precipitation followed by column chromatography. BALB/c mice, RBL-2H3, and KU812 cells were used for verifying the anti food allergic activity of GLSP. According to the results of mice experiment, GLSP was able to alleviate allergy symptoms, to reduce TM-specific IgE and IgG1, to suppress Th2 cell polarization, and to promote the function of regulatory T (Treg) cells. In addition, GLSP had the ability to inhibit the function of RBL-2H3 cells. Furthermore, GLSP inhibited the activation of KU812 via suppression of p38 mitogen-activated protein kinase (MAPK). In conclusion, immunosuppression as well as the reduction in the level of p38 MAPK may contribute to GLSP's putative activity against food allergy. GLSP may be used as a functional food component for allergic patients.

  3. Mitogen-activated protein kinase phosphatase-3 is a tumor promoter target in initiated cells that express oncogenic Ras.

    PubMed

    Warmka, Janel K; Mauro, Laura J; Wattenberg, Elizabeth V

    2004-08-01

    We have capitalized on the unique properties of the skin tumor promoter palytoxin, which does not activate protein kinase C, to investigate alternative mechanisms by which major signaling molecules can be modulated during carcinogenesis. We report here that palytoxin activates extracellular signal-regulated kinase (ERK) through a novel mechanism that involves inactivation of an ERK phosphatase in keratinocytes derived from initiated mouse skin (308 cells). Use of U0126 revealed that palytoxin requires the ERK kinase MEK to stimulate ERK activity, although palytoxin did not activate MEK. We found that 308 keratinocytes highly express mitogen-activated protein kinase phosphatase-3 (MKP-3), which selectively inactivates ERK. Palytoxin induced the loss of MKP-3 in a manner that corresponded to increased ERK phosphorylation. Complementary studies showed that sustained expression of exogenous MKP-3 inhibited palytoxin-stimulated ERK activation. As is characteristic of initiated keratinocytes, 308 cells express activated H-Ras. To investigate whether expression of oncogenic Ras is key to palytoxin-stimulated ERK activation, we determined how palytoxin affected ERK and MKP-3 in MCF10A human breast epithelial cells and in H-ras MCF10A cells, which stably express activated H-Ras. Palytoxin did not affect ERK activity in MCF10A cells, which had no detectable MKP-3. Like 308 cells, H-ras MCF10A cells highly express MKP-3. Strikingly, palytoxin stimulated ERK activity and induced a corresponding loss of MKP-3 in H-ras MCF10A cells. These studies indicate that in initiated cells palytoxin unleashes ERK activity by down-regulating MKP-3, an ERK inhibitor, and further suggest that MKP-3 may be a vulnerable target in cells that express oncogenic Ras.

  4. Mitogen-activated protein kinase phosphatase 1 is involved in tamoxifen resistance in MCF7 cells.

    PubMed

    Ma, Gang; Pan, Yixia; Zhou, Can; Sun, Ruifang; Bai, Jingjing; Liu, Peijun; Ren, Yu; He, Jianjun

    2015-11-01

    Tamoxifen resistance is a major clinical problem for ER-positive breast cancer, but the underlying mechanism is not completely elucidated. In the present study, we reported that mitogen-activated protein kinase (MAPK) phosphatase 1 (MKP-1), a member of the family of MKPs, is involved in tamoxifen resistance. We found that MKP1 expression increased in tamoxifen resistant MCF7 cells. To explore the possible role of MKP1 in tamoxifen resistance, siRNA targeting MKP1 was transfected into tamoxifen resistant MCF7 cells. To our surprise, knockdown of MKP-1 promoted cell death induced by tamoxifen. On the other hand, the MKP1 overexpressed MCF7 cell clone was established and MKP1 overexpression effectively attenuated MCF7 cell death induced by tamoxifen. In addition, we revealed that MKP1 inhibited tamoxifen‑mediated JNK activation in tamoxifen resistant MCF7 and MCF7 cells, and by this mechanism MKP1 was able to inhibit tamoxifen-induced cell death. We also showed that combined appliaction of MKP1 inhibitor triptolide and tamoxifen can effectively increase tamoxifen sensitivity in tamoxifen resistant MCF7 cells. Collectively, our results indicated that MKP-1 can attenuate tamoxifen-induced cell death through inhibiting the JNK signal pathway, which represents a novel mechanism of tamoxifen resistance in MCF7 cells.

  5. 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

  6. Microglial activation of p38 contributes to scorpion envenomation-induced hyperalgesia.

    PubMed

    Niu, Qing-Shan; Jiang, Feng; Hua, Li-Ming; Fu, Jin; Jiao, Yun-Lu; Ji, Yong-Hua; Ding, Gang

    2013-10-25

    Intraplantar (i.pl.) injection of BmK I, a receptor site 3-specific modulator of voltage-gated sodium channels (VGSCs) from the venom of scorpion Buthus martensi Karsch (BmK), was shown to induce long-lasting and spontaneous nociceptive responses as demonstrated through experiments utilizing primary thermal and mirror-imaged mechanical hypersensitivity with different time course of development in rats. In this study, microglia was activated on both sides of L4-L5 spinal cord by i.pl. injection of BmK I. Meanwhile, the activation of p38/MAPK in L4-L5 spinal cord was found to be co-expressed with OX-42, the cell marker of microglia. The unilateral thermal and bilateral mechanical pain hypersensitivity of rat induced by BmK I was suppressed in a dose-dependent manner following pretreatment with SB203580 (a specific inhibitor of p-p38). Interestingly, microglia activity was also reduced in the presence of SB203580, which suggests that BmK I-induced microglial activation is mediated by p38/MAPK pathway. Combined with previously published literature, the results of this study demonstrate that p38-dependent microglial activation plays a role in scorpion envenomation-induced pain-related behaviors. PMID:24064352

  7. 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.

  8. p38 MAPK Signaling in Pemphigus: Implications for Skin Autoimmunity

    PubMed Central

    Mavropoulos, Athanasios; Orfanidou, Timoklia; Liaskos, Christos; Smyk, Daniel S.; Spyrou, Vassiliki; Sakkas, Lazaros I.; Rigopoulou, Eirini I.; Bogdanos, Dimitrios P.

    2013-01-01

    p38 mitogen activated protein kinase (p38 MAPK) signaling plays a major role in the modulation of immune-mediated inflammatory responses and therefore has been linked with several autoimmune diseases. The extent of the involvement of p38 MAPK in the pathogenesis of autoimmune blistering diseases has started to emerge, but whether it pays a critical role is a matter of debate. The activity of p38 MAPK has been studied in great detail during the loss of keratinocyte cell-cell adhesions and the development of pemphigus vulgaris (PV) and pemphigus foliaceus (PF). These diseases are characterised by autoantibodies targeting desmogleins (Dsg). Whether autoantibody-antigen interactions can trigger signaling pathways (such as p38 MAPK) that are tightly linked to the secretion of inflammatory mediators which may perpetuate inflammation and tissue damage in pemphigus remains unclear. Yet, the ability of p38 MAPK inhibitors to block activation of the proapoptotic proteinase caspase-3 suggests that the induction of apoptosis may be a consequence of p38 MAPK activation during acantholysis in PV. This review discusses the current evidence for the role of p38 MAPK in the pathogenesis of pemphigus. We will also present data relating to the targeting of these cascades as a means of therapeutic intervention. PMID:23936634

  9. Cobalt oxide nanoparticles induced oxidative stress linked to activation of TNF-α/caspase-8/p38-MAPK signaling in human leukemia cells.

    PubMed

    Chattopadhyay, Sourav; Dash, Sandeep Kumar; Tripathy, Satyajit; Das, Balaram; Kar Mahapatra, Santanu; Pramanik, Panchanan; Roy, Somenath

    2015-06-01

    The purpose of this study was to determine the intracellular signaling transduction pathways involved in oxidative stress induced by nanoparticles in cancer cells. Activation of reactive oxygen species (ROS) has some therapeutic benefits in arresting the growth of cancer cells. Cobalt oxide nanoparticles (CoO NPs) are an interesting compound for oxidative cancer therapy. Our results showed that CoO NPs elicited a significant (P <0.05) amount of ROS in cancer cells. Co-treatment with N-aceyltine cystine (an inhibitor of ROS) had a protective role in cancer cell death induced by CoO NPs. In cultured cells, the elevated level of tumor necrosis factor-alpha (TNF-α) was noted after CoO NPs treatment. This TNF-α persuaded activation of caspase-8 followed by phosphorylation of p38 mitogen-activated protein kinase and induced cell death. This study showed that CoO NPs induced oxidative stress and activated the signaling pathway of TNF-α-Caspase-8-p38-Caspase-3 to cancer cells.

  10. SMALL GRAIN 1, which encodes a mitogen-activated protein kinase kinase 4, influences grain size in rice.

    PubMed

    Duan, Penggen; Rao, Yuchun; Zeng, Dali; Yang, Yaolong; Xu, Ran; Zhang, Baolan; Dong, Guojun; Qian, Qian; Li, Yunhai

    2014-02-01

    Although grain size is one of the most important components of grain yield, little information is known about the mechanisms that determine final grain size in crops. Here we characterize rice small grain1 (smg1) mutants, which exhibit small and light grains, dense and erect panicles and comparatively slightly shorter plants. The short grain and panicle phenotypes of smg1 mutants are caused by a defect in cell proliferation. The smg1 mutations were identified, using a map-based cloning approach, in mitogen-activated protein kinase kinase 4 (OsMKK4). Relatively higher expression of OsMKK4/SMG1 was detected in younger organs than in older ones, consistent with its role in cell proliferation. Green fluorescent protein (GFP)-OsMKK4/SMG1 fusion proteins appear to be distributed ubiquitously in plant cells. Further results revealed that OsMKK4 influenced brassinosteroid (BR) responses and the expression of BR-related genes. Thus, our findings have identified OsMKK4 as a factor for grain size, and suggest a possible link between the MAPK pathways and BRs in grain growth.

  11. The AngFus3 Mitogen-Activated Protein Kinase Controls Hyphal Differentiation and Secondary Metabolism in Aspergillus niger.

    PubMed

    Priegnitz, Bert-Ewald; Brandt, Ulrike; Pahirulzaman, Khomaizon A K; Dickschat, Jeroen S; Fleißner, André

    2015-06-01

    Adaptation to a changing environment is essential for the survival and propagation of sessile organisms, such as plants or fungi. Filamentous fungi commonly respond to a worsening of their growth conditions by differentiation of asexually or sexually produced spores. The formation of these specialized cell types is, however, also triggered as part of the general life cycle by hyphal age or density. Spores typically serve for dispersal and, therefore, translocation but can also act as resting states to endure times of scarcity. Eukaryotic differentiation in response to environmental and self-derived signals is commonly mediated by three-tiered mitogen-activated protein (MAP) kinase signaling cascades. Here, we report that the MAP kinase Fus3 of the black mold Aspergillus niger (AngFus3) and its upstream kinase AngSte7 control vegetative spore formation and secondary metabolism. Mutants lacking these kinases are defective in conidium induction in response to hyphal density but are fully competent in starvation-induced sporulation, indicating that conidiation in A. niger is triggered by various independent signals. In addition, the mutants exhibit an altered profile of volatile metabolites and secrete dark pigments into the growth medium, suggesting a dysregulation of the secondary metabolism. By assigning the AngFus3 MAP kinase pathway to the transduction of a potentially self-derived trigger, this work contributes to the unraveling of the intricate signaling networks controlling fungal differentiation. Moreover, our data further support earlier observations that differentiation and secondary metabolism are tightly linked in filamentous fungi.

  12. Gene-Environment Interactions Target Mitogen-activated Protein 3 Kinase 1 (MAP3K1) Signaling in Eyelid Morphogenesis*

    PubMed Central

    Mongan, Maureen; Meng, Qinghang; Wang, Jingjing; Kao, Winston W.-Y.; Puga, Alvaro; Xia, Ying

    2015-01-01

    Gene-environment interactions determine the biological outcomes through mechanisms that are poorly understood. Mouse embryonic eyelid closure is a well defined model to study the genetic control of developmental programs. Using this model, we investigated how exposure to dioxin-like environmental pollutants modifies the genetic risk of developmental abnormalities. Our studies reveal that mitogen-activated protein 3 kinase 1 (MAP3K1) signaling is a focal point of gene-environment cross-talk. Dioxin exposure, acting through the aryl hydrocarbon receptor (AHR), blocked eyelid closure in genetic mutants in which MAP3K1 signaling was attenuated but did not disturb this developmental program in either wild type or mutant mice with attenuated epidermal growth factor receptor or WNT signaling. Exposure also markedly inhibited c-Jun phosphorylation in Map3k1+/− embryonic eyelid epithelium, suggesting that dioxin-induced AHR pathways can synergize with gene mutations to inhibit MAP3K1 signaling. Our studies uncover a novel mechanism through which the dioxin-AHR axis interacts with the MAP3K1 signaling pathways during fetal development and provide strong empirical evidence that specific gene alterations can increase the risk of developmental abnormalities driven by environmental pollutant exposure. PMID:26109068

  13. Genome-Wide Identification and Expression Analysis of the Mitogen-Activated Protein Kinase Gene Family in Cassava.

    PubMed

    Yan, Yan; Wang, Lianzhe; Ding, Zehong; Tie, Weiwei; Ding, Xupo; Zeng, Changying; Wei, Yunxie; Zhao, Hongliang; Peng, Ming; Hu, Wei

    2016-01-01

    Mitogen-activated protein kinases (MAPKs) play central roles in plant developmental processes, hormone signaling transduction, and responses to abiotic stress. However, no data are currently available about the MAPK family in cassava, an important tropical crop. Herein, 21 MeMAPK genes were identified from cassava. Phylogenetic analysis indicated that MeMAPKs could be classified into four subfamilies. Gene structure analysis demonstrated that the number of introns in MeMAPK genes ranged from 1 to 10, suggesting large variation among cassava MAPK genes. Conserved motif analysis indicated that all MeMAPKs had typical protein kinase domains. Transcriptomic analysis suggested that MeMAPK genes showed differential expression patterns in distinct tissues and in response to drought stress between wild subspecies and cultivated varieties. Interaction networks and co-expression analyses revealed that crucial pathways controlled by MeMAPK networks may be involved in the differential response to drought stress in different accessions of cassava. Expression of nine selected MAPK genes showed that these genes could comprehensively respond to osmotic, salt, cold, oxidative stressors, and abscisic acid (ABA) signaling. These findings yield new insights into the transcriptional control of MAPK gene expression, provide an improved understanding of abiotic stress responses and signaling transduction in cassava, and lead to potential applications in the genetic improvement of cassava cultivars.

  14. Cloning of the mitogen-activated S6 kinase from rat liver reveals an enzyme of the second messenger subfamily

    SciTech Connect

    Kozma, S.C.; Ferrari, S. Bassand, P.; Siegmann, M.; Thomas, G. ); Totty, N. )

    1990-10-01

    Recently the authors reported the purification of a mitogen-activated S6 kinase from Swiss mouse 3T3 fibroblasts and rat liver. The rat liver protein was cleaved with cyanogen bromide or trypsin and 17 of the resulting peptides were sequenced. DNA primers were generated from 3 peptides that had homology to sequences of the conserved catalytic domain of protein kinases. These primers were used in the polymerase chain reaction to obtain a 0.4-kilobase DNA fragment. This fragment was either radioactively labeled and hybridized to Northern blots of poly(A){sup {sup plus}} mRNA or used to screen a rat liver cDNA library. Northern blot analysis revealed four transcripts of 2.5, 3.2, 4.0, and 6.0 kilobases, and five S6 kinase clones were obtained by screening the library. Only two of the clones, which were identical, encoded a full-length protein. This protein had a molecular weight of 56,160, which correlated closely to that of the dephosphorylated kinase determined by SDS/PAGE. The catalytic domain of the kinase resembles that of other serine/threonine kinases belonging to the second messenger subfamily of protein kinases.

  15. Genome-Wide Identification and Expression Analysis of the Mitogen-Activated Protein Kinase Gene Family in Cassava

    PubMed Central

    Yan, Yan; Wang, Lianzhe; Ding, Zehong; Tie, Weiwei; Ding, Xupo; Zeng, Changying; Wei, Yunxie; Zhao, Hongliang; Peng, Ming; Hu, Wei

    2016-01-01

    Mitogen-activated protein kinases (MAPKs) play central roles in plant developmental processes, hormone signaling transduction, and responses to abiotic stress. However, no data are currently available about the MAPK family in cassava, an important tropical crop. Herein, 21 MeMAPK genes were identified from cassava. Phylogenetic analysis indicated that MeMAPKs could be classified into four subfamilies. Gene structure analysis demonstrated that the number of introns in MeMAPK genes ranged from 1 to 10, suggesting large variation among cassava MAPK genes. Conserved motif analysis indicated that all MeMAPKs had typical protein kinase domains. Transcriptomic analysis suggested that MeMAPK genes showed differential expression patterns in distinct tissues and in response to drought stress between wild subspecies and cultivated varieties. Interaction networks and co-expression analyses revealed that crucial pathways controlled by MeMAPK networks may be involved in the differential response to drought stress in different accessions of cassava. Expression of nine selected MAPK genes showed that these genes could comprehensively respond to osmotic, salt, cold, oxidative stressors, and abscisic acid (ABA) signaling. These findings yield new insights into the transcriptional control of MAPK gene expression, provide an improved understanding of abiotic stress responses and signaling transduction in cassava, and lead to potential applications in the genetic improvement of cassava cultivars.

  16. In silico analysis reveals 75 members of mitogen-activated protein kinase kinase kinase gene family in rice.

    PubMed

    Rao, Kudupudi Prabhakara; Richa, Tambi; Kumar, Kundan; Raghuram, Badmi; Sinha, Alok Krishna

    2010-06-01

    Mitogen-Activated Protein Kinase Kinase Kinases (MAPKKKs) are important components of MAPK cascades, which are universal signal transduction modules and play important role in plant growth and development. In the sequenced Arabidopsis genome 80 MAPKKKs were identified and currently being analysed for its role in different stress. In rice, economically important monocot cereal crop only five MAPKKKs were identified so far. In this study using computational analysis of sequenced rice genome we have identified 75 MAPKKKs. EST hits and full-length cDNA sequences (from KOME or Genbank database) of 75 MAPKKKs supported their existence. Phylogenetic analyses of MAPKKKs from rice and Arabidopsis have classified them into three subgroups, which include Raf, ZIK and MEKK. Conserved motifs in the deduced amino acid sequences of rice MAPKKKs strongly supported their identity as members of Raf, ZIK and MEKK subfamilies. Further expression analysis of the MAPKKKs in MPSS database revealed that their transcripts were differentially regulated in various stress and tissue-specific libraries.

  17. Genome-Wide Identification and Expression Analysis of the Mitogen-Activated Protein Kinase Gene Family in Cassava

    PubMed Central

    Yan, Yan; Wang, Lianzhe; Ding, Zehong; Tie, Weiwei; Ding, Xupo; Zeng, Changying; Wei, Yunxie; Zhao, Hongliang; Peng, Ming; Hu, Wei

    2016-01-01

    Mitogen-activated protein kinases (MAPKs) play central roles in plant developmental processes, hormone signaling transduction, and responses to abiotic stress. However, no data are currently available about the MAPK family in cassava, an important tropical crop. Herein, 21 MeMAPK genes were identified from cassava. Phylogenetic analysis indicated that MeMAPKs could be classified into four subfamilies. Gene structure analysis demonstrated that the number of introns in MeMAPK genes ranged from 1 to 10, suggesting large variation among cassava MAPK genes. Conserved motif analysis indicated that all MeMAPKs had typical protein kinase domains. Transcriptomic analysis suggested that MeMAPK genes showed differential expression patterns in distinct tissues and in response to drought stress between wild subspecies and cultivated varieties. Interaction networks and co-expression analyses revealed that crucial pathways controlled by MeMAPK networks may be involved in the differential response to drought stress in different accessions of cassava. Expression of nine selected MAPK genes showed that these genes could comprehensively respond to osmotic, salt, cold, oxidative stressors, and abscisic acid (ABA) signaling. These findings yield new insights into the transcriptional control of MAPK gene expression, provide an improved understanding of abiotic stress responses and signaling transduction in cassava, and lead to potential applications in the genetic improvement of cassava cultivars. PMID:27625666

  18. Osmoregulation and fungicide resistance: the Neurospora crassa os-2 gene encodes a HOG1 mitogen-activated protein kinase homologue.

    PubMed

    Zhang, Yan; Lamm, Randy; Pillonel, Christian; Lam, Stephen; Xu, Jin-Rong

    2002-02-01

    Neurospora crassa osmosensitive (os) mutants are sensitive to high osmolarity and therefore are unable to grow on medium containing 4% NaCl. We found that os-2 and os-5 mutants were resistant to the phenylpyrrole fungicides fludioxonil and fenpiclonil. To understand the relationship between osmoregulation and fungicide resistance, we cloned the os-2 gene by using sib selection. os-2 encodes a putative mitogen-activated protein (MAP) kinase homologous to HOG1 and can complement the osmosensitive phenotype of a Saccharomyces cerevisiae hog1 mutant. We sequenced three os-2 alleles and found that all of them were null with either frameshift or nonsense point mutations. An os-2 gene replacement mutant also was generated and was sensitive to high osmolarity and resistant to phenylpyrrole fungicides. Conversely, os-2 mutants transformed with the wild-type os-2 gene could grow on media containing 4% NaCl and were sensitive to phenylpyrrole fungicides. Fludioxonil stimulated intracellular glycerol accumulation in wild-type strains but not in os-2 mutants. Fludioxonil also caused wild-type conidia and hyphal cells to swell and burst. These results suggest that the hyperosmotic stress response pathway of N. crassa is the target of phenylpyrrole fungicides and that fungicidal effects may result from a hyperactive os-2 MAP kinase pathway.

  19. The Cotton Mitogen-Activated Protein Kinase Kinase 3 Functions in Drought Tolerance by Regulating Stomatal Responses and Root Growth.

    PubMed

    Wang, Chen; Lu, Wenjing; He, Xiaowen; Wang, Fang; Zhou, Yuli; Guo, Xulei; Guo, Xingqi

    2016-08-01

    Mitogen-activated protein kinase (MAPK) cascades play critical roles in signal transduction processes in eukaryotes. The MAPK kinases (MAPKKs) that link MAPKK kinases (MAPKKKs) and MAPKs are key components of MAPK cascades. However, the intricate regulatory mechanisms that control MAPKKs under drought stress conditions are not fully understood, especially in cotton (Gossypium hirsutum) Here, we isolated and characterized the cotton group B MAPKK gene GhMKK3 Overexpressing GhMKK3 in Nicotiana benthamiana enhanced tolerance to drought, and the results of RNA sequencing (RNA-seq) and quantitative real-time PCR (qRT-PCR) assays suggest that GhMKK3 plays an important role in responses to abiotic stresses by regulating stomatal responses and root hair growth. Further evidence demonstrated that overexpressing GhMKK3 promoted root growth and ABA-induced stomatal closure. In contrast, silencing GhMKK3 in cotton using virus-induced gene silencing (VIGS) resulted in the opposite phenotypes. More importantly, we identified an ABA- and drought-induced MAPK cascade that is composed of GhMKK3, GhMPK7 and GhPIP1 that compensates for deficiency in the MAPK cascade pathway in cotton under drought stress conditions. Together, these findings significantly improve our understanding of the mechanism by which GhMKK3 positively regulates drought stress responses. PMID:27335349

  20. Genome-Wide Identification and Expression Analysis of the Mitogen-Activated Protein Kinase Gene Family in Cassava.

    PubMed

    Yan, Yan; Wang, Lianzhe; Ding, Zehong; Tie, Weiwei; Ding, Xupo; Zeng, Changying; Wei, Yunxie; Zhao, Hongliang; Peng, Ming; Hu, Wei

    2016-01-01

    Mitogen-activated protein kinases (MAPKs) play central roles in plant developmental processes, hormone signaling transduction, and responses to abiotic stress. However, no data are currently available about the MAPK family in cassava, an important tropical crop. Herein, 21 MeMAPK genes were identified from cassava. Phylogenetic analysis indicated that MeMAPKs could be classified into four subfamilies. Gene structure analysis demonstrated that the number of introns in MeMAPK genes ranged from 1 to 10, suggesting large variation among cassava MAPK genes. Conserved motif analysis indicated that all MeMAPKs had typical protein kinase domains. Transcriptomic analysis suggested that MeMAPK genes showed differential expression patterns in distinct tissues and in response to drought stress between wild subspecies and cultivated varieties. Interaction networks and co-expression analyses revealed that crucial pathways controlled by MeMAPK networks may be involved in the differential response to drought stress in different accessions of cassava. Expression of nine selected MAPK genes showed that these genes could comprehensively respond to osmotic, salt, cold, oxidative stressors, and abscisic acid (ABA) signaling. These findings yield new insights into the transcriptional control of MAPK gene expression, provide an improved understanding of abiotic stress responses and signaling transduction in cassava, and lead to potential applications in the genetic improvement of cassava cultivars. PMID:27625666

  1. Salt Stress in Arabidopsis: Lipid Transfer Protein AZI1 and Its Control by Mitogen-Activated Protein Kinase MPK3

    PubMed Central

    Pitzschke, Andrea

    2014-01-01

    A plant’s capability to cope with environmental challenges largely relies on signal transmission through mitogen-activated protein kinase (MAPK) cascades. In Arabidopsis thaliana, MPK3 is particularly strongly associated with numerous abiotic and biotic stress responses. Identification of MPK3 substrates is a milestone towards improving stress resistance in plants. Here, we characterize AZI1, a lipid transfer protein (LTP)-related hybrid proline-rich protein (HyPRP), as a novel target of MPK3. AZI1 is phosphorylated by MPK3 in vitro. As documented by co-immunoprecipitation and bimolecular fluorescence complementation experiments, AZI1 interacts with MPK3 to form protein complexes in planta. Furthermore, null mutants of azi1 are hypersensitive to salt stress, while AZI1-overexpressing lines are markedly more tolerant. AZI1 overexpression in the mpk3 genetic background partially alleviates the salt-hypersensitive phenotype of this mutant, but functional MPK3 appears to be required for the full extent of AZI1-conferred robustness. Notably, this robustness does not come at the expense of normal development. Immunoblot and RT–PCR data point to a role of MPK3 as positive regulator of AZI1 abundance. PMID:24214892

  2. Isolation of vulgin, a new antifungal polypeptide with mitogenic activity from the pinto bean.

    PubMed

    Ye, X Y; Ng, T B

    2003-02-01

    An antifungal polypeptide bearing an N-termnial sequence with some homology to chitinases was purified from an extract of pinto beans. The polypeptide, designated vulgin, exerted antifungal activity toward Mycosphaerella arachidicola, Coprinus cornatus, Fusarium oxysporum and Botrytis cinerea. Vulgin inhibited translation in a rabbit reticulocyte lysate system with an IC50 of 4.3 microM and HIV-1 reverse transcriptase activity with an IC50 of 58 microM. Vulgin stimulated in vitro incorporation of methyl [3H] thymidine into mouse splenocytes. PMID:12630696

  3. Positive feedback regulation of maize NADPH oxidase by mitogen-activated protein kinase cascade in abscisic acid signalling

    PubMed Central

    Lin, Fan; Ding, Haidong; Wang, Jinxiang; Zhang, Hong; Zhang, Aying; Zhang, Yun; Tan, Mingpu; Dong, Wen; Jiang, Mingyi

    2009-01-01

    In maize (Zea mays), abscisic acid (ABA)-induced H2O2 production activates a 46 kDa mitogen-activated protein kinase (p46MAPK), and the activation of p46MAPK also regulates the production of H2O2. However, the mechanism for the regulation of H2O2 production by MAPK in ABA signalling remains to be elucidated. In this study, four reactive oxygen species (ROS)-producing NADPH oxidase (rboh) genes (ZmrbohA–D) were isolated and characterized in maize leaves. ABA treatment induced a biphasic response (phase I and phase II) in the expression of ZmrbohA–D and the activity of NADPH oxidase. Phase II induced by ABA was blocked by pretreatments with two MAPK kinase (MPKKK) inhibitors and two H2O2 scavengers, but phase I was not affected by these inhibitors or scavengers. Treatment with H2O2 alone also only induced phase II, and the induction was arrested by the MAPKK inhibitors. Furthermore, the ABA-activated p46MAPK was partially purified. Using primers corresponding to the sequences of internal tryptic peptides, the p46MAPK gene was cloned. Analysis of the tryptic peptides and the p46MAPK sequence indicate it is the known ZmMPK5. Treatments with ABA and H2O2 led to a significant increase in the activity of ZmMPK5, although ABA treatment only induced a slight increase in the expression of ZmMPK5. The data indicate that H2O2-activated ZmMPK5 is involved in the activation of phase II in ABA signalling, but not in phase I. The results suggest that there is a positive feedback loop involving NADPH oxidase, H2O2, and ZmMPK5 in ABA signalling. PMID:19592501

  4. 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

  5. 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.

  6. Vesicular Stomatitis Virus Matrix Protein Impairs CD1d-Mediated Antigen Presentation through Activation of the p38 MAPK Pathway▿

    PubMed Central

    Renukaradhya, Gourapura J.; Khan, Masood A.; Shaji, Daniel; Brutkiewicz, Randy R.

    2008-01-01

    Natural killer T (NKT) cells are unique T lymphocytes that recognize CD1d-bound lipid antigens and play an important role in both innate and acquired immune responses against infectious diseases and tumors. We have already shown that a vesicular stomatitis virus (VSV) infection results in the rapid inhibition of murine CD1d-mediated antigen presentation to NKT cells. In the present study, it was found that the VSV matrix (VSV-M) protein is an important element in this decrease in antigen presentation postinfection. The VSV-M protein altered the intracellular distribution of murine CD1d molecules, resulting in qualitative (but not quantitative) changes in cell surface CD1d expression. The M protein was distributed throughout the infected cell, and it was found to activate the mitogen-activated protein kinase (MAPK) p38 very early postinfection. Infection of CD1d+ cells with a temperature-sensitive VSV-M mutant at the nonpermissive temperature both substantially reversed the inhibition of antigen presentation by CD1d and delayed the activation of p38. Thus, the VSV-M protein plays an important role in permitting the virus to evade important components of the innate immune response by regulating specific MAPK pathways. PMID:18815300

  7. Cathepsin S Activity Controls Injury-Related Vascular Repair in Mice via the TLR2-Mediated p38MAPK and PI3K−Akt/p-HDAC6 Signaling Pathway

    PubMed Central

    Wu, Hongxian; Hu, Lina; Takeshita, Kyosuke; Hu, Chen; Du, Qiuna; Li, Xiang; Zhu, Enbo; Huang, Zhe; Yisireyili, Maimaiti; Zhao, Guangxian; Piao, Limei; Inoue, Aiko; Jiang, Haiying; Lei, Yanna; Zhang, Xiaohong; Liu, Shaowen; Dai, Qiuyan; Kuzuya, Masafumi; Shi, Guo-Ping; Murohara, Toyoaki

    2016-01-01

    Objective— Cathepsin S (CatS) participates in atherogenesis through several putative mechanisms. The ability of cathepsins to modify histone tail is likely to contribute to stem cell development. Histone deacetylase 6 (HDAC6) is required in modulating the proliferation and migration of various types of cancer cells. Here, we investigated the cross talk between CatS and HADC6 in injury-related vascular repair in mice. Approach and Results— Ligation injury to the carotid artery in mice increased the CatS expression, and CatS-deficient mice showed reduced neointimal formation in injured arteries. CatS deficiency decreased the phosphorylation levels of p38 mitogen-activated protein kinase, Akt, and HDAC6 and toll-like receptor 2 expression in ligated arteries. The genetic or pharmacological inhibition of CatS also alleviated the increased phosphorylation of p38 mitogen-activated protein kinase, Akt, and HDAC6 induced by platelet-derived growth factor BB in cultured vascular smooth muscle cells (VSMCs), and p38 mitogen-activated protein kinase inhibition and Akt inhibition decreased the phospho-HDAC6 levels. Moreover, CatS inhibition caused decrease in the levels of the HDAC6 activity in VSMCs in response to platelet-derived growth factor BB. The HDAC6 inhibitor tubastatin A downregulated platelet-derived growth factor–induced VSMC proliferation and migration, whereas HDAC6 overexpression exerted the opposite effect. Tubastatin A also decreased the intimal VSMC proliferation and neointimal hyperplasia in response to injury. Toll-like receptor 2 silencing decreased the phosphorylation levels of p38 mitogen-activated protein kinase, Akt, and HDAC6 and VSMC migration and proliferation. Conclusions— This is the first report detailing cross-interaction between toll-like receptor 2–mediated CatS and HDAC6 during injury-related vascular repair. These data suggest that CatS/HDAC6 could be a potential therapeutic target for the control of vascular diseases that are

  8. The three-dimensional structure of MAP kinase p38[beta]: different features of the ATP-binding site in p38[beta] compared with p38[alpha

    SciTech Connect

    Patel, Sangita B.; Cameron, Patricia M.; O'Keefe, Stephen J.; Frantz-Wattley, Betsy; Thompson, Jed; O'Neill, Edward A.; Tennis, Trevor; Liu, Luping; Becker, Joseph W.; Scapin, Giovanna; Merck

    2010-10-18

    The p38 mitogen-activated protein kinases are activated in response to environmental stress and cytokines and play a significant role in transcriptional regulation and inflammatory responses. Of the four p38 isoforms known to date, two (p38{alpha} and p38{beta}) have been identified as targets for cytokine-suppressive anti-inflammatory drugs. Recently, it was reported that specific inhibition of the p38{alpha} isoform is necessary and sufficient for anti-inflammatory efficacy in vivo, while further inhibition of p38{beta} may not provide any additional benefit. In order to aid the development of p38{alpha}-selective compounds, the three-dimensional structure of p38{beta} was determined. To do so, the C162S and C119S,C162S mutants of human MAP kinase p38{beta} were cloned, expressed in Escherichia coli and purified. Initial screening hits in crystallization trials in the presence of an inhibitor led upon optimization to crystals that diffracted to 2.05 {angstrom} resolution and allowed structure determination (PDB codes 3gc8 and 3gc9 for the single and double mutant, respectively). The structure of the p38{alpha} C162S mutant in complex with the same inhibitor is also reported (PDB code 3gc7). A comparison between the structures of the two kinases showed that they are highly similar overall but that there are differences in the relative orientation of the N- and C-terminal domains that causes a reduction in the size of the ATP-binding pocket in p38{beta}. This difference in size between the two pockets could be exploited in order to achieve selectivity.

  9. Phosphorylation of mitogen-activated protein kinase by one-trial and multi-trial classical conditioning.

    PubMed

    Crow, T; Xue-Bian, J J; Siddiqi, V; Kang, Y; Neary, J T

    1998-05-01

    The pathway supporting the conditioned stimulus (CS) is one site of plasticity that has been studied extensively in conditioned Hermissenda. Several signal transduction pathways have been implicated in classical conditioning of this preparation, although the major emphasis has been on protein kinase C. Here we provide evidence for the activation and phosphorylation of a mitogen-activated protein kinase (MAPK) pathway by one-trial and multi-trial conditioning. A one-trial in vitro conditioning procedure consisting of light (CS) paired with the application of 5-HT results in the increased incorporation of 32PO4 into proteins detected with two-dimensional gel electrophoresis. Two of the phosphoproteins have molecular weights of 44 and 42 kDa, consistent with extracellular signal-regulated protein kinases (ERK1 and ERK2). Phosphorylation of the 44 and 42 kDa proteins by one-trial conditioning was inhibited by pretreatment with PD098059, A MEK1 (ERK-Activating kinase) inhibitor. Assays of ERK activity with brain myelin basic protein as a substrate revealed greater ERK activity for the group that received one-trial conditioning compared with an unpaired control group. Western blot analysis of phosphorylated ERK using antibodies recognizing the dually phosphorylated forms of ERK1 and ERK2 showed an increase in phosphorylation after one-trial conditioning compared with unpaired controls. The increased phosphorylation of ERK after one-trial conditioning was blocked by pretreatment with PD098059. Hermissenda that received 10 or 15 conditioning trials showed significant behavioral suppression compared with pseudo-random controls. After conditioning and behavioral testing, the conditioned animals showed significantly greater phosphorylation of ERK compared with the pseudo-random controls. These results show that the ERK-MAPK signaling pathway is activated in Pavlovian conditioning of Hermissenda.

  10. Mitogen-activated protein kinases with distinct requirements for Ste5 scaffolding influence signaling specificity in Saccharomyces cerevisiae.

    PubMed

    Flatauer, Laura J; Zadeh, Sheena F; Bardwell, Lee

    2005-03-01

    Scaffold proteins are believed to enhance specificity in cell signaling when different pathways share common components. The prototype scaffold Ste5 binds to multiple components of the Saccharomyces cerevisiae mating pheromone response pathway, thereby conducting the mating signal to the Fus3 mitogen-activated protein kinase (MAPK). Some of the kinases that Ste5 binds to, however, are also shared with other pathways. Thus, it has been presumed that Ste5 prevents its bound kinases from transgressing into other pathways and protects them from intrusions from those pathways. Here we found that Fus3MAPK required Ste5 scaffolding to receive legitimate signals from the mating pathway as well as misdirected signals leaking from other pathways. Furthermore, increasing the cellular concentration of active Ste5 enhanced the channeling of inappropriate stimuli to Fus3. This aberrant signal crossover resulted in the erroneous induction of cell cycle arrest and mating. In contrast to Fus3, the Kss1 MAPK did not require Ste5 scaffolding to receive either authentic or leaking signals. Furthermore, the Ste11 kinase, once activated via Ste5, was able to signal to Kss1 independently of Ste5 scaffolding. These results argue that Ste5 does not act as a barrier that actively prevents signal crossover to Fus3 and that Ste5 may not effectively sequester its activated kinases away from other pathways. Rather, we suggest that specificity in this network is promoted by the selective activation of Ste5 and the distinct requirements of the MAPKs for Ste5 scaffolding. PMID:15713635

  11. Purification and characterization of a galactose-specific lectin with mitogenic activity from pinto beans.

    PubMed

    Wong, Jack H; Wong, Clarence C T; Ng, T B

    2006-05-01

    A galactose-specific dimeric lectin from pinto beans was purified using a procedure that involved affinity chromatography on Affi-gel blue gel, anion exchange chromatography on Q-Sepharose, fast protein liquid chromatography (FPLC)-ion exchange chromatography on Mono S, and FPLC-gel filtration on Superdex 200. The molecular mass of this homodimeric lectin was 62 kDa and that of each of its subunits was 31 kDa. The hemagglutinating activity of pinto bean lectin was stable within the pH range of 3-12 and the temperature range of 0-70 degrees C. By using the [3H-methyl]-thymidine incorporation assay, it was shown that the lectin had the ability to evoke a mitogenic response from murine splenocytes but it did not inhibit proliferation of L1210 leukemia cells. The pinto bean lectin inhibited HIV-1 reverse transcriptase with an IC50 of 3 microM. PMID:16600511

  12. Phenotypic and transcriptional plasticity directed by a yeast mitogen-activated protein kinase network.

    PubMed

    Breitkreutz, Ashton; Boucher, Lorrie; Breitkreutz, Bobby-Joe; Sultan, Mujahid; Jurisica, Igor; Tyers, Mike

    2003-11-01

    The yeast pheromone/filamentous growth MAPK pathway mediates both mating and invasive-growth responses. The interface between this MAPK module and the transcriptional machinery consists of a network of two MAPKs, Fus3 and Kss1; two regulators, Rst1 and Rst2 (a.k.a. Dig1 and Dig2); and two transcription factors, Ste12 and Tec1. Of 16 possible combinations of gene deletions in FUS3, KSS1, RST1, and RST2 in the sigma1278 background, 10 display constitutive invasive growth. Rst1 was the primary negative regulator of invasive growth, while other components either attenuated or enhanced invasive growth, depending on the genetic context. Despite activation of the invasive response by lesions at the same level in the MAPK pathway, transcriptional profiles of different invasive mutant combinations did not exhibit a unified program of gene expression. The distal MAPK regulatory network is thus capable of generating phenotypically similar invasive-growth states (an attractor) from different molecular architectures (trajectories) that can functionally compensate for one another. This systems-level robustness may also account for the observed diversity of signals that trigger invasive growth. PMID:14668360

  13. Mitogen-activated protein kinase (MAPK) regulates the expression of progelatinase B (MMP-9) in breast epithelial cells.

    PubMed

    Reddy, K B; Krueger, J S; Kondapaka, S B; Diglio, C A

    1999-07-19

    Mitogen-activated protein kinases (MAPKs) play a major role in the mitogenic signal transduction pathway and are essential components of both growth and differentiation. Constitutive activation of the MAPK cascade is associated with the carcinogenesis and metastasis of human breast and renal cell carcinomas. The gelatinases B (MMP-9) and A (MMP-2) are 2 members of the matrix metalloproteinase (MMPs) family which are expressed in human cancers and thought to play a critical role in tumor cell invasion and metastasis. In a previous study, we have shown that EGF and amphiregulin upregulate MMP-9 in metastatic SKBR-3 cells but have no effect on MMP-2 secretion. We now investigated specific step(s) in EGF-induced signalling associated with regulation of cell proliferation and MMP-9 induction. EGF-induced signalling in SKBR-3 cells was blocked by relatively specific inhibitors either on ras (FPT inhibitor-1) or P13 kinase (Wortmannin) or by reduction in EGF-induced tyrosine kinase activity (RG 13022). Blocking these signalling pathways significantly inhibited of EGF-induced cell proliferation but only partially reduced in EGF-induced MMP-9 secretion. In contrast, when SKBR-3 cells were exposed to MEK inhibitor (PD 98059) or MAPK inhibitors (Apigenin or MAPK antisense phosphorothioate oligodeoxynucleotides), EGF-induced cell proliferation, MMP-9 induction and invasion through reconstituted basement membrane were significantly reduced. Our results suggest that interfering with MAPK activity may provide a novel means of controlling growth and invasiveness of tumors in which the signalling cascade is activated.

  14. Sphingosine kinase 1 inhibition improves lipopolysaccharide/D-galactosamine-induced acute liver failure by inhibiting mitogen-activated protein kinases pathway

    PubMed Central

    Tian, Tao; Tian, Weiliang; Yang, Fan; Zhao, Risheng; Huang, Qian

    2016-01-01

    Background Sphingosine kinase 1 (SphK1)/sphingosine-1-phosphate (S1P)/sphingosine-1-phosphate receptors (S1PRs) signaling plays a key role in inflammatory responses. Lei et al. showed that SphK1 inhibition presented a hepatoprotective effect on acute liver damage via decreasing hepatic high-mobility group box 1 (HMGB1) cytoplasmic translocation. Objective We aim to determine whether SphK1 or S1PRs inhibition improves lipopolysaccharide (LPS)/D-galactosamine (GalN)-induced acute liver failure by inhibiting the mitogen-activated protein kinases (MAPKs) pathway. Methods A mouse model of acute liver failure was induced by LPS/GalN. Male C57BL/6J mice (6–8 weeks) were randomly distributed into five groups: control group, LPS/GalN group, SphK1 inhibition group (LPS/GalN+SKI-5c), S1PR1 inhibition group (LPS/GalN+W146), and S1PR3 inhibition group (LPS/GalN+CAY10444). Results We confirmed the findings of Lei et al. that hepatic SphK1 expression was upregulated; serum transaminase activity (AST, ALT), as well as serum TNF-α and IL-6, were decreased by SphK1 inhibition. We further showed that the expression of S1PR1 and S1PR3 was augmented in response to LPS/GalN. SphK1 inhibition improves hepatic hemorrhage, and the activities of hepatic caspase-3 and myeloperoxidase (MPO). Furthermore, the activation of the MAPKs family (JNK, ERK and p38) was suppressed by SphK1 inhibition. However, S1PR1 or S1PR3 inhibition did not protect the mouse against liver damage, though S1PR1 or S1PR3 inhibition reduced serum TNF-α and IL-6, and partially attenuated the phosphorylation of the MAPKs signaling. Conclusions SphK1 inhibition improves LPS/GalN-induced liver injury by inhibiting activation of MAPKs signaling. PMID:27733910

  15. Intracellular labile iron determines H2O2-induced apoptotic signaling via sustained activation of ASK1/JNK-p38 axis.

    PubMed

    Mantzaris, M D; Bellou, S; Skiada, V; Kitsati, N; Fotsis, T; Galaris, D

    2016-08-01

    Hydrogen peroxide (H2O2) acts as a second messenger in signal transduction participating in several redox regulated pathways, including cytokine and growth factor stimulated signals. However, the exact molecular mechanisms underlying these processes remain poorly understood and require further investigation. In this work, using Jurkat T lymphoma cells and primary human umbilical vein endothelial cells, it was observed that changes in intracellular "labile iron" were able to modulate signal transduction in H2O2-induced apoptosis. Chelation of intracellular labile iron by desferrioxamine rendered cells resistant to H2O2-induced apoptosis. In order to identify the exact points of iron action, we investigated selected steps in H2O2-mediated apoptotic pathway, focusing on mitogen activated protein kinases (MAPKs) JNK, p38 and ERK. It was observed that spatiotemporal changes in intracellular labile iron, induced by H2O2, influenced the oxidation pattern of the upstream MAP3K ASK1 and promoted the sustained activation of JNK-p38 axis in a defined time-dependent context. Moreover, we indicate that H2O2 induced spatiotemporal changes in intracellular labile iron, at least in part, by triggering the destabilization of lysosomal compartments, promoting a concomitant early response in proteins of iron homeostasis. These results raise the possibility that iron-mediated oxidation of distinct proteins may be implicated in redox signaling processes. Since labile iron can be pharmacologically modified in vivo, it may represent a promising target for therapeutic interventions in related pathological conditions.

  16. p38alpha, but not p38beta, inhibits the phosphorylation and presence of c-FLIPS in DISC to potentiate Fas-mediated caspase-8 activation and type I apoptotic signaling.

    PubMed

    Tourian, Leon; Zhao, Hong; Srikant, Coimbatore B

    2004-12-15

    Pharmacological inhibitors of JNK (SP600125) and p38 (PD169316) sensitize tumor cells to Fas-mediated apoptosis. PD169316 is less potent than SP600125 and diminishes its effect when present together. Because the p38 isoforms that promote (p38alpha) or inhibit (p38beta) apoptosis are both suppressed by PD169316, we investigated their regulatory involvement in Fas-signaling. We report here, that p38alpha, but not p38beta, exerts its proapoptotic effect by inhibiting the phosphorylation and presence of c-FLIPS, but not c-FLIPL, in the DISC to promote caspase-8 activation and type I signaling in Fas-activated Jurkat cells. Its effect was enhanced by enforced expression of Flag-tagged p38alpha and was attenuated by its inactive mutant (p38alpha-AGF) or by translational silencing. By contrast, type II signaling was facilitated by p38alpha-dependent mitochondrial presence of tBid and inhibition of Bcl-2 (Ser70) phosphorylation as well as by p38alpha/beta-dependent mitochondrial localization of Bax and inhibition of phosphorylation of Bad (Ser112/Ser155). Potentiation of Fas-mediated apoptosis by the inhibition of JNK1/2 correlated with the loss of Bad (Ser136) phosphorylation and was dependent on the stimulatory effect of p38alpha on DISC and the downstream effects of both p38alpha and p38beta. These data underscore the need to reassess the findings obtained with pan-p38 inhibitors and suggest that activation of p38alpha coupled with targeted inhibition of p38beta and JNK1/2 should optimally sensitize tumor cells to Fas-mediated apoptosis.

  17. Upregulation of early growth response factor-1 by bile acids requires mitogen-activated protein kinase signaling

    SciTech Connect

    Allen, Katryn; Kim, Nam Deuk; Moon, Jeon-OK; Copple, Bryan L.

    2010-02-15

    Cholestasis results when excretion of bile acids from the liver is interrupted. Liver injury occurs during cholestasis, and recent studies showed that inflammation is required for injury. Our previous studies demonstrated that early growth response factor-1 (Egr-1) is required for development of inflammation in liver during cholestasis, and that bile acids upregulate Egr-1 in hepatocytes. What remains unclear is the mechanism by which bile acids upregulate Egr-1. Bile acids modulate gene expression in hepatocytes by activating the farnesoid X receptor (FXR) and through activation of mitogen-activated protein kinase (MAPK) signaling. Accordingly, the hypothesis was tested that bile acids upregulate Egr-1 in hepatocytes by FXR and/or MAPK-dependent mechanisms. Deoxycholic acid (DCA) and chenodeoxycholic acid (CDCA) stimulated upregulation of Egr-1 to the same extent in hepatocytes isolated from wild-type mice and FXR knockout mice. Similarly, upregulation of Egr-1 in the livers of bile duct-ligated (BDL) wild-type and FXR knockout mice was not different. Upregulation of Egr-1 in hepatocytes by DCA and CDCA was prevented by the MEK inhibitors U0126 and SL-327. Furthermore, pretreatment of mice with U0126 prevented upregulation of Egr-1 in the liver after BDL. Results from these studies demonstrate that activation of MAPK signaling is required for upregulation of Egr-1 by bile acids in hepatocytes and for upregulation of Egr-1 in the liver during cholestasis. These studies suggest that inhibition of MAPK signaling may be a novel therapy to prevent upregulation of Egr-1 in liver during cholestasis.

  18. Toward a comprehensive phylogenetic reconstruction of the evolutionary history of mitogen-activated protein kinases in the plant kingdom.

    PubMed

    Janitza, Philipp; Ullrich, Kristian Karsten; Quint, Marcel

    2012-01-01

    The mitogen-activated protein kinase (MAPK) pathway is a three-tier signaling cascade that transmits cellular information from the plasma membrane to the cytoplasm where it triggers downstream responses. The MAPKs represent the last step in this cascade and are activated when both tyrosine and threonine residues in a conserved TxY motif are phosphorylated by MAPK kinases, which in turn are themselves activated by phosphorylation by MAPK kinase kinases. To understand the molecular evolution of MAPKs in the plant kingdom, we systematically conducted a Hidden-Markov-Model based screen to identify MAPKs in 13 completely sequenced plant genomes. In this analysis, we included green algae, bryophytes, lycophytes, and several mono- and eudicotyledonous species covering >800 million years of evolution. The phylogenetic relationships of the 204 identified MAPKs based on Bayesian inference facilitated the retraction of the sequence of emergence of the four major clades that are characterized by the presence of a TDY or TEY-A/TEY-B/TEY-C type kinase activation loop. We present evidence that after the split of TDY- and TEY-type MAPKs, initially the TEY-C clade emerged. This was followed by the TEY-B clade in early land plants until the TEY-A clade finally emerged in flowering plants. In addition to these well characterized clades, we identified another highly conserved clade of 45 MAPK-likes, members of which were previously described as Mak-homologous kinases. In agreement with their essential functions, molecular population genetic analysis of MAPK genes in Arabidopsis thaliana accessions reveal that purifying selection drove the evolution of the MAPK family, implying strong functional constraints on MAPK genes. Closely related MAPKs most likely subfunctionalized, a process in which differential transcriptional regulation of duplicates may be involved. PMID:23230446

  19. Role of a mitogen-activated protein kinase cascade in ion flux-mediated turgor regulation in fungi.

    PubMed

    Lew, Roger R; Levina, Natalia N; Shabala, Lana; Anderca, Marinela I; Shabala, Sergey N

    2006-03-01

    Fungi normally maintain a high internal hydrostatic pressure (turgor) of about 500 kPa. In response to hyperosmotic shock, there are immediate electrical changes: a transient depolarization (1 to 2 min) followed by a sustained hyperpolarization (5 to 10 min) prior to turgor recovery (10 to 60 min). Using ion-selective vibrating probes, we established that the transient depolarization is due to Ca(2+) influx and the sustained hyperpolarization is due to H(+) efflux by activation of the plasma membrane H(+)-ATPase. Protein synthesis is not required for H(+)-ATPase activation. Net K(+) and Cl(-) uptake occurs at the same time as turgor recovery. The magnitude of the ion uptake is more than sufficient to account for the osmotic gradients required for turgor to return to its original level. Two osmotic mutants, os-1 and os-2, homologs of a two-component histidine kinase sensor and the yeast high osmotic glycerol mitogen-activated protein (MAP) kinase, respectively, have lower turgor than the wild type and do not exhibit the sustained hyperpolarization after hyperosmotic treatment. The os-1 mutant does not exhibit all of the wild-type turgor-adaptive ion fluxes (Cl(-) uptake increases, but net K(+) flux barely changes and net H(+) efflux declines) (os-2 was not examined). Both os mutants are able to regulate turgor but at a lower level than the wild type. Our results demonstrate that a MAP kinase cascade regulates ion transport, activation of the H(+)-ATPase, and net K(+) and Cl(-) uptake during turgor regulation. Other pathways regulating turgor must also exist.

  20. Mitogen-activated protein kinase is required for the behavioural desensitization that occurs after repeated injections of angiotensin II.

    PubMed

    Vento, Peter J; Daniels, Derek

    2012-12-01

    Angiotensin II (Ang II) acts on central angiotensin type 1 (AT(1)) receptors to increase water and saline intake. Prolonged exposure to Ang II in cell culture models results in a desensitization of the AT(1) receptor that is thought to involve receptor internalization, and a behavioural correlate of this desensitization has been shown in rats after repeated central injections of Ang II. Specifically, rats given repeated injections of Ang II drink less water than control animals after a subsequent test injection of Ang II. In the same conditions, however, repeated injections of Ang II have no effect on Ang II-induced saline intake. Given earlier studies indicating that separate intracellular signalling pathways mediate Ang II-induced water and saline intake, we hypothesized that the desensitization observed in rats may be incomplete, leaving the receptor able to activate mitogen-activated protein (MAP) kinases (ERK1/2), which play a role in Ang II-induced saline intake without affecting water intake. In support of this hypothesis, we found no difference in MAP kinase phosphorylation after an Ang II test injection in rats given prior treatment with repeated injections of vehicle, Ang II or Sar(1),Ile(4),Ile(8)-Ang II (SII), an Ang II analogue that activates MAP kinase without G protein coupling. In addition, we found that pretreatment with the MAP kinase inhibitor U0126 completely blocked the desensitizing effect of repeated Ang II injections on water intake. Furthermore, Ang II-induced water intake was reduced to a similar extent by repeated injections of Ang II or SII. The results suggest that G protein-independent signalling is sufficient to produce behavioural desensitization of the angiotensin system and that the desensitization requires MAP kinase activation.

  1. p38 MAPK Signaling in Osteoblast Differentiation

    PubMed Central

    Rodríguez-Carballo, Eddie; Gámez, Beatriz; Ventura, Francesc

    2016-01-01

    The skeleton is a highly dynamic tissue whose structure relies on the balance between bone deposition and resorption. This equilibrium, which depends on osteoblast and osteoclast functions, is controlled by multiple factors that can be modulated post-translationally. Some of the modulators are Mitogen-activated kinases (MAPKs), whose role has been studied in vivo and in vitro. p38-MAPK modifies the transactivation ability of some key transcription factors in chondrocytes, osteoblasts and osteoclasts, which affects their differentiation and function. Several commercially available inhibitors have helped to determine p38 action on these processes. Although it is frequently mentioned in the literature, this chemical approach is not always as accurate as it should be. Conditional knockouts are a useful genetic tool that could unravel the role of p38 in shaping the skeleton. In this review, we will summarize the state of the art on p38 activity during osteoblast differentiation and function, and emphasize the triggers of this MAPK. PMID:27200351

  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. [6]-Gingerol inhibits COX-2 expression by blocking the activation of p38 MAP kinase and NF-kappaB in phorbol ester-stimulated mouse skin.

    PubMed

    Kim, Sue Ok; Kundu, Joydeb Kumar; Shin, Young Kee; Park, Jin-Hong; Cho, Myung-Haing; Kim, Tae-Yoon; Surh, Young-Joon

    2005-04-01

    [6]-Gingerol, a pungent ingredient of ginger (Zingiber officinale Roscoe, Zingiberaceae), has a wide array of pharmacologic effects. The present study was aimed at unraveling the molecular mechanisms underlying previously reported antitumor promoting effects of [6]-gingerol in mouse skin in vivo. One of the well-recognized molecular targets for chemoprevention is cyclooxygenase-2 (COX-2) that is abnormally upregulated in many premalignant and malignant tissues and cells. In our present study, topical application of [6]-gingerol inhibited COX-2 expression in mouse skin stimulated with a prototype tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). Since the transcription factor nuclear factor-kappaB (NF-kappaB) is known to regulate COX-2 induction, we attempted to determine the effect of [6]-gingerol on TPA-induced activation of NF-kappaB. Pretreatment with [6]-gingerol resulted in a decrease in both TPA-induced DNA binding and transcriptional activities of NF-kappaB through suppression of IkappaBalpha degradation and p65 nuclear translocation. Phosphorylation of both IkappaBalpha and p65 was substantially blocked by [6]-gingerol. In addition, [6]-gingerol inhibited TPA-stimulated interaction of phospho-p65-(Ser-536) with cAMP response element binding protein-binding protein, a transcriptional coactivator of NF-kappaB. Moreover, [6]-gingerol prevented TPA-induced phosphorylation and catalytic activity of p38 mitogen-activated protein (MAP) kinase that regulates COX-2 expression in mouse skin. The p38 MAP kinase inhibitor SB203580 attenuated NF-kappaB activation and subsequent COX-2 induction in TPA-treated mouse skin. Taken together, our data suggest that [6]-gingerol inhibits TPA-induced COX-2 expression in mouse skin in vivo by blocking the p38 MAP kinase-NF-kappaB signaling pathway. PMID:15735738

  4. P38 activation is more important than ERK activation in lung injury induced by prolonged hyperbaric oxygen.

    PubMed

    Ma, Jun; Fang, Yi-Qun; Gu, Ai-Mei; Wang, Fang-Fang; Zhang, Shi; Li, Kai-Cheng

    2013-01-01

    Prolonged exposure to hyperbaric oxygen can cause pulmonary and nerve system toxicity. Although hyperbaric oxygen treatment has been used for a broad spectrum of ailments, the mechanisms of prolonged hyperbaric oxygen-induced lung injury are not fully understood. The purpose of the present work was to investigate the roles of ERK, p38, and caspase-3 in rat lung tissue exposed to hyperbaric oxygen at 2.3 atmospheres absolute (atm abs) for two, six and 10 hours. The results showed that the ERK and p38 were phosphorylated at two hours and reached a peak at six hours into exposure to hyperbaric oxygen. While the phosphorylation level of ERK decreased, p38 remained at a high level of activation at 10 hours. The activation of ERK and p38 was down-regulated when rats were exposed to normoxic hyperbaric nitrogen for 10 hours. However, caspase-3 was activated at six hours and 10 hours into exposure to hyperbaric oxygen. These results demonstrated different changes of activation of ERK and p38 during lung injury induced by prolonged exposure to hyperbaric oxygen. The time course changes of activated caspase-3 were similar to the process of p38 activation upon exposure to hyperbaric oxygen. In this way, activation of p38, not ERK, seems to be a mechanism associated with prolonged hyperbaric oxygen-induced lung injury.

  5. Role of Phosphatidylinositol Phosphate Signaling in the Regulation of the Filamentous-Growth Mitogen-Activated Protein Kinase Pathway

    PubMed Central

    Adhikari, Hema

    2015-01-01

    Reversible phosphorylation of the phospholipid phosphatidylinositol (PI) is a key event in the determination of organelle identity and an underlying regulatory feature in many biological processes. Here, we investigated the role of PI signaling in the regulation of the mitogen-activated protein kinase (MAPK) pathway that controls filamentous growth in yeast. Lipid kinases that generate phosphatidylinositol 4-phosphate [PI(4)P] at the Golgi (Pik1p) or PI(4,5)P2 at the plasma membrane (PM) (Mss4p and Stt4p) were required for filamentous-growth MAPK pathway signaling. Introduction of a conditional allele of PIK1 (pik1-83) into the filamentous (Σ1278b) background reduced MAPK activity and caused defects in invasive growth and biofilm/mat formation. MAPK regulatory proteins that function at the PM, including Msb2p, Sho1p, and Cdc42p, were mislocalized in the pik1-83 mutant, which may account for the signaling defects of the PI(4)P kinase mutants. Other PI kinases (Fab1p and Vps34p), and combinations of PIP (synaptojanin-type) phosphatases, also influenced the filamentous-growth MAPK pathway. Loss of these proteins caused defects in cell polarity, which may underlie the MAPK signaling defect seen in these mutants. In line with this possibility, disruption of the actin cytoskeleton by latrunculin A (LatA) dampened the filamentous-growth pathway. Various PIP signaling mutants were also defective for axial budding in haploid cells, cell wall construction, or proper regulation of the high-osmolarity glycerol response (HOG) pathway. Altogether, the study extends the roles of PI signaling to a differentiation MAPK pathway and other cellular processes. PMID:25724886

  6. Histamine activates p38 MAP kinase and alters local lamellipodia dynamics, reducing endothelial barrier integrity and eliciting central movement of actin fibers.

    PubMed

    Adderley, Shaquria P; Lawrence, Curtis; Madonia, Eyong; Olubadewo, Joseph O; Breslin, Jerome W

    2015-07-01

    The role of the actin cytoskeleton in endothelial barrier function has been debated for nearly four decades. Our previous investigation revealed spontaneous local lamellipodia in confluent endothelial monolayers that appear to increase overlap at intercellular junctions. We tested the hypothesis that the barrier-disrupting agent histamine would reduce local lamellipodia protrusions and investigated the potential involvement of p38 mitogen-activated protein (MAP) kinase activation and actin stress fiber formation. Confluent monolayers of human umbilical vein endothelial cells (HUVEC) expressing green fluorescent protein-actin were studied using time-lapse fluorescence microscopy. The protrusion and withdrawal characteristics of local lamellipodia were assessed before and after addition of histamine. Changes in barrier function were determined using electrical cell-substrate impedance sensing. Histamine initially decreased barrier function, lamellipodia protrusion frequency, and lamellipodia protrusion distance. A longer time for lamellipodia withdrawal and reduced withdrawal distance and velocity accompanied barrier recovery. After barrier recovery, a significant number of cortical fibers migrated centrally, eventually resembling actin stress fibers. The p38 MAP kinase inhibitor SB203580 attenuated the histamine-induced decreases in barrier function and lamellipodia protrusion frequency. SB203580 also inhibited the histamine-induced decreases in withdrawal distance and velocity, and the subsequent actin fiber migration. These data suggest that histamine can reduce local lamellipodia protrusion activity through activation of p38 MAP kinase. The findings also suggest that local lamellipodia have a role in maintaining endothelial barrier integrity. Furthermore, we provide evidence that actin stress fiber formation may be a reaction to, rather than a cause of, reduced endothelial barrier integrity. PMID:25948734

  7. Histamine activates p38 MAP kinase and alters local lamellipodia dynamics, reducing endothelial barrier integrity and eliciting central movement of actin fibers

    PubMed Central

    Adderley, Shaquria P.; Lawrence, Curtis; Madonia, Eyong; Olubadewo, Joseph O.

    2015-01-01

    The role of the actin cytoskeleton in endothelial barrier function has been debated for nearly four decades. Our previous investigation revealed spontaneous local lamellipodia in confluent endothelial monolayers that appear to increase overlap at intercellular junctions. We tested the hypothesis that the barrier-disrupting agent histamine would reduce local lamellipodia protrusions and investigated the potential involvement of p38 mitogen-activated protein (MAP) kinase activation and actin stress fiber formation. Confluent monolayers of human umbilical vein endothelial cells (HUVEC) expressing green fluorescent protein-actin were studied using time-lapse fluorescence microscopy. The protrusion and withdrawal characteristics of local lamellipodia were assessed before and after addition of histamine. Changes in barrier function were determined using electrical cell-substrate impedance sensing. Histamine initially decreased barrier function, lamellipodia protrusion frequency, and lamellipodia protrusion distance. A longer time for lamellipodia withdrawal and reduced withdrawal distance and velocity accompanied barrier recovery. After barrier recovery, a significant number of cortical fibers migrated centrally, eventually resembling actin stress fibers. The p38 MAP kinase inhibitor SB203580 attenuated the histamine-induced decreases in barrier function and lamellipodia protrusion frequency. SB203580 also inhibited the histamine-induced decreases in withdrawal distance and velocity, and the subsequent actin fiber migration. These data suggest that histamine can reduce local lamellipodia protrusion activity through activation of p38 MAP kinase. The findings also suggest that local lamellipodia have a role in maintaining endothelial barrier integrity. Furthermore, we provide evidence that actin stress fiber formation may be a reaction to, rather than a cause of, reduced endothelial barrier integrity. PMID:25948734

  8. Reversion of apoptotic resistance of TP53-mutated Burkitt lymphoma B-cells to spindle poisons by exogenous activation of JNK and p38 MAP kinases

    PubMed Central

    Farhat, M; Poissonnier, A; Hamze, A; Ouk-Martin, C; Brion, J-D; Alami, M; Feuillard, J; Jayat-Vignoles, C

    2014-01-01

    Defects in apoptosis are frequently the cause of cancer emergence, as well as cellular resistance to chemotherapy. These phenotypes may be due to mutations of the tumor suppressor TP53 gene. In this study, we examined the effect of various mitotic spindle poisons, including the new isocombretastatin derivative isoNH2CA-4 (a tubulin-destabilizing molecule, considered to bind to the colchicine site by analogy with combretastatin A-4), on BL (Burkitt lymphoma) cells. We found that resistance to spindle poison-induced apoptosis could be reverted in tumor protein p53 (TP53)-mutated cells by EBV (Epstein Barr virus) infection. This reversion was due to restoration of the intrinsic apoptotic pathway, as assessed by relocation of the pro-apoptotic molecule Bax to mitochondria, loss of mitochondrial integrity and activation of the caspase cascade with PARP (poly ADP ribose polymerase) cleavage. EBV sensitized TP53-mutated BL cells to all spindle poisons tested, including vincristine and taxol, an effect that was systematically downmodulated by pretreatment of cells with inhibitors of p38 and c-Jun N-terminal kinase (JNK) mitogen-activated protein kinases. Exogenous activation of p38 and JNK pathways by dihydrosphingosine reverted resistance of TP53-mutated BL cells to spindle poisons. Dihydrosphingosine treatment of TP53-deficient Jurkat and K562 cell lines was also able to induce cell death. We conclude that activation of p38 and JNK pathways may revert resistance of TP53-mutated cells to spindle poisons. This opens new perspectives for developing alternative therapeutic strategies when the TP53 gene is inactivated. PMID:24787013

  9. Histamine activates p38 MAP kinase and alters local lamellipodia dynamics, reducing endothelial barrier integrity and eliciting central movement of actin fibers.

    PubMed

    Adderley, Shaquria P; Lawrence, Curtis; Madonia, Eyong; Olubadewo, Joseph O; Breslin, Jerome W

    2015-07-01

    The role of the actin cytoskeleton in endothelial barrier function has been debated for nearly four decades. Our previous investigation revealed spontaneous local lamellipodia in confluent endothelial monolayers that appear to increase overlap at intercellular junctions. We tested the hypothesis that the barrier-disrupting agent histamine would reduce local lamellipodia protrusions and investigated the potential involvement of p38 mitogen-activated protein (MAP) kinase activation and actin stress fiber formation. Confluent monolayers of human umbilical vein endothelial cells (HUVEC) expressing green fluorescent protein-actin were studied using time-lapse fluorescence microscopy. The protrusion and withdrawal characteristics of local lamellipodia were assessed before and after addition of histamine. Changes in barrier function were determined using electrical cell-substrate impedance sensing. Histamine initially decreased barrier function, lamellipodia protrusion frequency, and lamellipodia protrusion distance. A longer time for lamellipodia withdrawal and reduced withdrawal distance and velocity accompanied barrier recovery. After barrier recovery, a significant number of cortical fibers migrated centrally, eventually resembling actin stress fibers. The p38 MAP kinase inhibitor SB203580 attenuated the histamine-induced decreases in barrier function and lamellipodia protrusion frequency. SB203580 also inhibited the histamine-induced decreases in withdrawal distance and velocity, and the subsequent actin fiber migration. These data suggest that histamine can reduce local lamellipodia protrusion activity through activation of p38 MAP kinase. The findings also suggest that local lamellipodia have a role in maintaining endothelial barrier integrity. Furthermore, we provide evidence that actin stress fiber formation may be a reaction to, rather than a cause of, reduced endothelial barrier integrity.