Defining the involvement of p38alpha MAPK in the production of anti- and proinflammatory cytokines using an SB 203580-resistant form of the kinase

Despite its lack of specificity, the inhibitor SB 203580 has been widely used to implicate p38 mitogen-activated protein kinase (MAPK) in the synthesis of many cytokines. Here we show unequivocally that the production of interleukin (IL)-1beta, IL-6, IL-10, and tumor necrosis factor alpha (TNFalpha) requires p38 MAPK activity by demonstrating that the inhibitory effects of SB 203580 were reversed by expression of an SB 203580-resistant form of p38alpha (SBR-p38alpha) that fails to bind to SB 203580. This strategy established the requirement for p38 activity for the lipopolysaccharide-stimulated production of IL-10, IL-1beta, and IL-6 by the monocytic cell WEHI 274 and the production of IL-6 and TNFalpha stimulated by ligation of the Fc-gamma receptor of the mast cell MC/9. Expression of SBR-p38alpha in primary macrophages abrogated the ability of SB 203580 to inhibit the lipopolysaccharide-stimulated production of TNFalpha but not of IL-10. Expression of SBR-p38alpha in primary T lymphocytes abrogated the ability of SB 203580 to inhibit the production of interferon-gamma induced by co-ligation of CD3 and CD28 but not the production of interferon-gamma or IL-10 induced by IL-12. These results suggest that the levels of p38 MAPK activity required for maximal cytokine production vary with different cytokines and stimuli.     

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

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

Involvement of p38 mitogen-activated protein kinase in the cell growth inhibition by sodium arsenite.

It is well-known that p38 mitogen-activated protein kinase (p38MAPK) participates in cellular responses to mitogenic stimuli, environmental and genotoxic stresses, and apoptotic agents. Although there are several reports on p38MAPK in relation to cell growth and apoptosis, the exact mechanism of p38MAPK-mediated cell growth regulation remains obscure. Here, we examined possible roles of p38MAPK in the sodium arsenite-induced cell growth inhibition in NIH3T3 cells. Sodium arsenite induced transient cell growth delay with marked activation of p38MAPK. In addition, arsenite induced CDK inhibitor p21(CIP1/WAF1) and enhanced its binding to the CDK2, which resulted in inhibition of CDK2 activity. The levels of cyclin D1 expression and the CDK4 kinase activity were also significantly reduced. pRB was hypophosphorylated by sodium arsenite. SB203580, a specific inhibitor of p38MAPK, blocked arsenite-induced growth inhibition as well as the arsenite-induced p21(CIP1/WAF1) expression. Expression of dominant negative p38MAPK also blocked arsenite-induced p21(CIP1/WAF1) expression. Inhibited-CDK2 activity was also completely reversed by SB203580 or expression of dominant negative p38MAPK, while the decreased-cyclin D1 protein by the compound was not restored. These data demonstrate a possible link between the activation of p38MAPK and induction of p21(CIP1/WAF1), suggesting that the activation of p38MAPK is, at least in part, related to the cell growth inhibition by sodium arsenite.     

The Mycobacterium tuberculosis early secreted antigenic target of 6 kDa inhibits T cell interferon-γ production through the p38 mitogen-activated protein kinase pathway

We reported previously that the early secreted antigenic target of 6 kDa (ESAT-6) from Mycobacterium tuberculosis directly inhibits human T cell IFN-γ production and proliferation in response to stimulation with anti-CD3 and anti-CD28. To determine the mechanism of this effect, we treated T cells with kinase inhibitors before stimulation with ESAT-6. Only the p38 MAPK inhibitor, SB203580, abrogated ESAT-6-mediated inhibition of IFN-γ production in a dose-dependent manner. SB203580 did not reverse ESAT-6-mediated inhibition of IL-17 and IL-10 production, suggesting a specific effect of SB203580 on IFN-γ production. SB203580 did not act through inhibition of AKT (PKB) as an AKT inhibitor did not affect ESAT-6 inhibition of T cell IFN-γ production and proliferation. ESAT-6 did not reduce IFN-γ production by expanding FoxP3(+) T regulatory cells. Incubation of T cells with ESAT-6 induced phosphorylation and increased functional p38 MAPK activity, but not activation of ERK or JNK. Incubation of peripheral blood mononuclear cells with ESAT-6 induced activation of p38 MAPK, and inhibition of p38 MAPK with SB203580 reversed ESAT-6 inhibition of M. tuberculosis-stimulated IFN-γ production by peripheral blood mononuclear cells from subjects with latent tuberculosis infection. Silencing of p38α MAPK with siRNA rendered T cells resistant to ESAT-6 inhibition of IFN-γ production. Taken together, our results demonstrate that ESAT-6 inhibits T cell IFN-γ production in a p38 MAPK-dependent manner.     

Geldanamycin, an inhibitor of Hsp90 increases cytochrome P450 2E1 mediated toxicity in HepG2 cells through sustained activation of the p38MAPK pathway

Cytochrome P450 2E1 (CYP2E1) can mediate reactive oxygen species (ROS) induced cell death through its catalytic processes. Heat shock protein 90 (Hsp90) is an important molecular chaperone which is essential for cellular integrity. We previously showed that inhibition of Hsp90 with Geldanamycin (GA), an inhibitor of Hsp90 increased CYP2E1 mediated toxicity in CYP2E1 over-expressing HepG2 cells (E47 cells) but not in C34-HepG2 cells devoid of CYP2E1 expression. The aim of the present study was to test the hypothesis that the potentiation of CYP2E1 toxicity in E47 cells with GA may involve changes in mitogen activated protein kinase signal transduction pathways. GA was toxic to E47 cells and SB203580, an inhibitor of p38 MAPK prevented this decrease in viability. The protective effects of SB203580 were effective only when SB203580 was added before GA treatment. GA activated p38 MAPK in E47 cells and this activation was an early and a sustained event. GA elevated ROS levels and lipid peroxidation and lowered GSH levels in E47 cells and these changes were blunted or prevented by treatment with SB203580. Apoptosis was increased by GA and prevented by pre-treatment with SB203580. The loss in mitochondrial membrane potential in E47 cells after GA treatment was also decreased significantly with SB203580 treatment. The activity and expression of CYP2E1 and Hsp90 levels were not altered by SB203580. In conclusion, the inhibition of Hsp90 with GA increases the toxicity of CYP2E1 in HepG2 cells through an early and sustained activation of the p38 MAPK pathway.     

Role of p38 mitogen-activated protein kinase in cardiac myocyte secretion of the inflammatory cytokine TNF-alpha

This study examined the hypothesis that burn trauma promotes cardiac myocyte secretion of inflammatory cytokines such as tumor necrosis factor (TNF)-alpha and produces cardiac contractile dysfunction via the p38 mitogen-activated protein kinase (MAPK) pathway. Sprague-Dawley rats were divided into four groups: 1) sham burn rats given anesthesia alone, 2) sham burn rats given the p38 MAPK inhibitor SB203580 (6 mg/kg po, 15 min; 6- and 22-h postburn), 3) rats given third-degree burns over 40% total body surface area and treated with vehicle (1 ml of saline) plus lactated Ringer solution for resuscitation (4 ml x kg(-1). percent burn(-1)), and 4) burn rats given injury and fluid resuscitation plus SB203580. Rats from each group were killed at several times postburn to examine p38 MAPK activity (by Western blot analysis or in vitro kinase assay); myocardial function and myocyte secretion of TNF-alpha were examined at 24-h postburn. These studies showed significant activation of p38 MAPK at 1-, 2-, and 4-h postburn compared with time-matched shams. Burn trauma impaired cardiac mechanical performance and promoted myocyte secretion of TNF-alpha. SB203580 inhibited p38 MAPK activity, reduced myocyte secretion of TNF-alpha, and prevented burn-mediated cardiac deficits. These data suggest p38 MAPK activation is one aspect of the signaling cascade that culminates in postburn secretion of TNF-alpha and contributes to postburn cardiac dysfunction.     

The regulation of Th1 responses by the p38 MAPK

IL-12 is a dimeric cytokine that is produced primarily by APCs. In this study we examined the role that the p38 MAPKs (MAPK/p38) play in regulating IL-12 production. We show that inhibition of p38 dramatically increased IL-12 production upon stimulation, while decreasing TNF-α. This reciprocal effect on these two cytokines following MAPK/p38 inhibition occurred in many different APCs, following a variety of different stimuli. IL-12 production was also increased in macrophages treated with small interfering RNA to limit p38α expression, and in macrophages deficient in MKK3, a kinase upstream of p38. The increase in IL-12 production following MAPK/p38 inhibition appears to be due to enhanced IL-12 (p40) mRNA stability. We show that MAPK/p38 inhibition can promote Th1 immune responses and thereby enhance vaccine efficacy against leishmaniasis. In a mouse model of Leishmania major infection, vaccination with heat-killed L. major plus CpG and SB203580 elicited complete protection against infection compared with heat-killed L. major plus CpG without SB203580. Thus, this work suggests that MAPK/p38 inhibitors may be applied as adjuvants to bias immune responses and improve vaccinations against intracellular pathogens.     

A specific inhibitor of the p38 mitogen activated protein kinase affects differentially the production of various cytokines by activated human T cells: dependence on CD28 signaling and preferential inhibition of IL-10 production

Cytokine production upon T cell activation results from the integration of multiple signaling pathways from TCR/CD3 and from costimulatory molecules such as CD28. Among these pathways, the possible role of p38 mitogen activated protein kinase (MAPK) is the least understood. Here, we used a highly specific p38 MAPK inhibitor, the SB203580 compound, to examine the role of this enzyme in the induction of various cytokines in human T cells stimulated with anti-CD3 and anti-CD28 mAb together or in combination with PMA. Cytokine induction was monitored by ELISA and at the mRNA level. While SB203580 had little effect on IL-2 production and proliferation, it significantly reduced the production of several other cytokines. The secretion of IL-4, IL-5, IL-13, and TNF-alpha was inhibited by 20-50% with modes of T cell activation involving the CD28 pathway, whereas their mRNA expression was little affected. In contrast, IFN-gamma induction via CD28/PMA or CD3/CD28, but not CD3/PMA, was markedly diminished both at the protein and at the mRNA levels. Most interestingly, SB203580 also suppressed IL-10 secretion and mRNA induction via CD28-dependent activation by 75-85% (IC50 approximately 0.2 microM). Subset analysis suggested that this inhibition did not reflect a differential effect on T cell subsets. Therefore, p38 MAPK activity appears to contribute to cytokine production, mostly via CD28-dependent signaling. Moreover, IL-10 seems to rely more on this activity than other cytokines for its induction in T cells.     

Intratracheal administration of mitochondrial DNA directly provokes lung inflammation through the TLR9–p38 MAPK pathway

An increasing number of studies have focused on the phenomenon that mitochondrial DNA (mtDNA) activates innate immunity responses. However, the specific role of mtDNA in inflammatory lung disease remains elusive. This study was designed to examine the proinflammatory effects of mtDNA in lungs and to investigate the putative mechanisms. C57BL/6 mice were challenged intratracheally with mtDNA with or without pretreatment with chloroquine. Changes in pulmonary histopathology, cytokine concentrations, and phosphorylation levels of p38 MAPK were assayed at four time points. In in vitro experiments, THP-1 macrophages were pretreated or not pretreated with chloroquine, TLR9 siRNA, p38 MAPK siRNA, or SB203580 and then incubated with mtDNA. The levels of cytokines and p-p38 MAPK were detected by ELISA and Western blot, respectively. The intratracheal administration of mtDNA induced infiltration of inflammatory cells, production of proinflammatory cytokines (including IL-1β, IL-6, and TNF-α), and activation of p38 MAPK. The chloroquine pretreatment resulted in an abatement of mtDNA-induced local lung inflammation. In vitro experiments showed that the exposure of THP-1 macrophages to mtDNA also led to a significant upregulation of IL-1β, IL-6, and TNF-α and the activation of p38 MAPK. And these responses were inhibited either by chloroquine and TLR9 siRNA or by SB203580 and p38 MAPK siRNA pretreatment. The intratracheal administration of mtDNA induced a local inflammatory response in the mouse lung that depended on the interactions of mtDNA with TLR9 and may be correlated with infiltrating macrophages that could be activated by mtDNA exposure via the TLR9–p38 MAPK signal transduction pathway.     

Antiproliferative effect of IL-1 is mediated by p38 mitogen-activated protein kinase in human melanoma cell A375.

The role of p38 mitogen-activated protein kinase (MAPK) in IL-1-induced growth inhibition was investigated using IL-1-sensitive human melanoma A375-C2-1 cells and IL-1-resistant A375-R8 cells. In both cells, p38 MAPK was activated by IL-1. A selective inhibitor for p38 MAPK, SB203580, almost completely recovered the IL-1-induced growth inhibition in A375-C2-1 cells. IL-1-induced IL-6 production was also suppressed by SB203580. However, the reversal effect of SB203580 was not due to the suppression of IL-6 production because the SB203580 effect was still observed in the presence of exogenous IL-6. Down-regulation of ornithine decarboxylase (ODC) activity as well as its protein level has been shown to be essential for IL-1-induced growth inhibition. SB203580 also reversed the IL-1-induced down-regulation of ODC activity and intracellular polyamine levels without affecting ODC mRNA levels in A375-C2-1 cells. In IL-1-resistant R8 cells, however, IL-1 only slightly suppressed ODC activity. In A375-C2-1 cells, the mRNA expression level of antizyme (AZ), a regulatory factor of ODC activity, has been shown to be up-regulated by IL-1. IL-1-induced up-regulation of AZ mRNA level was not affected by SB203580. These findings demonstrate that p38 MAPK plays an important role in IL-1-induced growth inhibition in A375 cells through down-regulating ODC activity without affecting the level of ODC mRNA and AZ mRNA. In IL-1-resistant A375-R8 cells, IL-1 signaling pathway is deficient between p38 MAPK activation and down-regulation of ODC activity.     

Raf-1 is activated by the p38 mitogen-activated protein kinase inhibitor, SB203580

SB203580 (4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imi dazole) is widely used as a specific inhibitor of p38 mitogen-activated protein kinase (MAPK). Here, we report that SB203580 activates the serine/threonine kinase Raf-1 in quiescent smooth muscle cells in a dose-dependent fashion. The concentrations of SB203580 required lie above those necessary to inhibit p38 MAPK and we were unable to detect basal levels of active p38 MAPK. SB203580 does not directly activate Raf-1 in vitro, and fails to activate Ras, MEK, and ERK in intact cells. In vitro, however, SB203580-stimulated Raf-1 activates MEK1 in a coupled assay. We conclude that activation of Raf-1 by SB203580 is not mediated by an inhibition of p38 MAPK, is Ras-independent, and is uncoupled from MEK/ERK signaling.     

Activin A induces erythroid gene expressions and inhibits mitogenic cytokine-mediated K562 colony formation by activating p38 MAPK

Activin A, a member of the transforming growth factor (TGF)-beta superfamily, is involved in the regulation of erythroid differentiation. Previous studies have shown that activin A inhibited the colony-forming activity of mouse Friend erythroleukemia cells, however, the mechanism remains unknown. First, we show herein that activin A induced the expression and activated the promoters of alpha-globin and zeta-globin in K562 cells, confirming that activin A induces erythroid differentiation in K562 cells. The p38 mitogen activated protein kinase (MAPK) inhibitor, SB203580, inhibited and the extracellular signal regulated kinase (ERK) inhibitor, PD98059, enhanced the expression and promoter activities of alpha-globin and zeta-globin by activin A, indicating that p38 MAPK and ERK are crucial for activin A-induced erythroid genes expression. Second, SB203580 inhibited the inhibitory effect of activin A on the colony-forming activity of K562 cells using the methylcellulose colony assay, indicating that activin A inhibits K562 colony formation by activating p38 MAPK. In addition, mitogenic cytokines SCF, IL-3, and GM-CSF induced colony formation of K562 cells that could be inhibited by PD98059 or enhanced by SB203580, respectively, indicating that these mitogenic cytokines induce K562 colony formation by activating ERK and inactivating p38 MAPK. Furthermore, activin A reduced the induction effect of these mitogenic cytokines on K562 colony formation in a dose-dependent manner. The inhibition of p38 MAPK reverted the inhibitory effect of activin A on mitogenic cytokine-mediated K562 colony formation. We conclude that activin A can regulate the same pathway via p38 MAPK to coordinate cell proliferation and differentiation of K562 cells.     

Thrombin activates p38 mitogen-activated protein kinase in vascular smooth muscle cells

Thrombin is a potent mitogen for vascular smooth muscle cells. However, the signaling pathways by which thrombin mediates its mitogenic response are not fully understood. The ERK (extracellular signal-regulated protein kinase) and JNK (c-Jun N-terminal kinase) members of the mitogen-activated protein kinase (MAPK) family are reported to be activated by thrombin. We have investigated the response to thrombin of another member of the MAPK family, p38 MAPK, which has been suggested to be activated by both stress and inflammatory stimuli in vascular smooth muscle cells. We found that thrombin induced time- and dose-dependent activation of p38 MAPK. Maximal stimulation of p38 MAPK was observed after a 10-min incubation with 1 unit ml(-1) thrombin. GF109203X, a protein kinase C inhibitor, and prolonged treatment with phorbol 12-myristate 13-acetate partially inhibited p38 MAPK activation. A tyrosine kinase inhibitor, genistein, also inhibited p38 MAPK activation in a dose-dependent manner. p38 MAPK activation was inhibited by overexpression of betaARK1ct (beta-adrenergic receptor kinase I C-terminal peptide). p38 MAPK activation was also inhibited by expression of dominant-negative Ras, not by dominant-negative Rac. We next examined the effect of a p38 MAPK inhibitor, SB203580, on thrombin-induced proliferation. SB203580 inhibited thrombin-induced DNA synthesis in a dose-dependent manner. These results suggest that thrombin activates p38 MAPK in a manner dependent on Gbetagamma, protein kinase C, a tyrosine kinase, and Ras, that p38 MAPK has a role in thrombin-induced mitogenic response in the cells.     

IL-1- and TNF-induced bone resorption is mediated by p38 mitogen activated protein kinase

We have previously shown that p38 mitogen-activated protein kinase (MAPK) inhibitors, which block the production and action of inflammatory cytokines such as tumor necrosis factor (TNF) and interleukin-1 (IL-1), are effective in models of bone and cartilage degradation. To further investigate the role of p38 MAPK, we have studied its activation in osteoblasts and chondrocytes, following treatment with a panel of proinflammatory and osteotropic agents. In osteoblasts, significant activation of p38 MAPK was observed following treatment with IL-1 and TNF, but not parathyroid hormone, transforming growth factor-beta (TGF-beta), 1,25(OH)(2)D(3), insulin-like growth factor-1 (IGF-1), or IGF-II. Similar results were obtained using primary bovine chondrocytes and an SV40-immortalized human chondrocyte cell line, T/C28A4. SB 203580, a selective inhibitor of p38 MAPK, inhibited IL-1 and TNF-induced p38 MAPK activity and IL-6 production (IC(50)s 0.3--0.5 microM) in osteoblasts and chondrocytes. In addition, IL-1 and TNF also activated p38 MAPK in fetal rat long bones and p38 MAPK inhibitors inhibited IL-1- and TNF-stimulated bone resorption in vitro in a dose-dependent manner (IC(50)s 0.3--1 microM). These data support the contention that p38 MAPK plays a central role in regulating the production of, and responsiveness to, proinflammatory cytokines in bone and cartilage. Furthermore, the strong correlation between inhibition of kinase activity and IL-1 and TNF-stimulated biological responses indicates that selective inhibition of the p38 MAPK pathway may have therapeutic utility in joint diseases such as rheumatoid arthritis (RA).     

Relationship between p38 mitogen-activated protein kinase and small GTPase Rac for the activation of NADPH oxidase in bovine neutrophils

Superoxide production by NADPH oxidase is essential for bactericidal properties of neutrophils. However, molecular mechanisms underlying the activation of this enzyme remain largely unknown. Here, using bovine neutrophils we examined the role of p38 mitogen-activated protein kinase (p38 MAPK) in the signaling pathways of the NADPH oxidase activation. Superoxide production was induced by stimulation with serum-opsonized zymosan (OZ) and attenuated by p38 MAPK inhibitor, SB203580. OZ stimulation induced the translocation of p47(phox) and Rac to the plasma membrane and SB203580 completely blocked the translocation of Rac, but only partially blocked that of p47(phox). Furthermore, SB203580 abolished the OZ-elicited activation of Rac, which was assessed by detecting the GTP-bound form of this protein. Phosphatidylinositol 3-kinase (PI3K) inhibitors, wortmannin and LY294002, blocked not only p38 MAPK activation but also Rac activation. However, SB203580 showed no effect on the PI3K activity. These results suggested that PI3K/p38 MAPK/Rac pathway was present in the activation of NADPH oxidase in bovine neutrophils.