Regulation of ERK-mediated signal transduction by p38 MAP kinase in human monocytic THP-1 cells

SB 203580 has been widely used to specifically shut down the p38 MAP kinase-dependent pathway, although it is capable of inducing c-Raf kinase activity in cells. The present study demonstrates that SB 203580 activates members of the ERK cascade, c-Raf, MEK, and ERK, in human monocytic THP-1 cells. The activation of these kinases was sustained for at least 24 h after SB 203580 treatment and was also observed in U937 cells, suggesting that c-Raf efficiently transduces the signal even in the presence of the inhibitor in these cells. However, the expression of ERK cascade-dependent genes, such as c-fos and IL-1beta, was extremely limited. Analysis of the cellular distribution of ERK in SB 203580-treated cells indicated that nuclear translocation of phosphorylated ERK was impaired. Also, nuclear translocation of ERK induced by 12-O-tetradecanoyl-phorbol-13-acetate (TPA) was inhibited by SB 239063, which does not associate with c-Raf and is highly selective for p38 MAP kinase. In addition, the forced expression of the dominant negative mutant of p38 MAP kinase suppressed serum responsive element-dependent transactivation induced by TPA. These results suggest that the steady-state level of p38 MAP kinase activity modulates ERK signaling.     

Differential activation of mitogen-activated protein kinases in AGS gastric epithelial cells by cag+ and cag- Helicobacter pylori.

The aim of this study was to determine whether Helicobacter pylori activates mitogen-activated protein (MAP) kinases in gastric epithelial cells. Infection of AGS cells with an H. pylori cag+ strain rapidly (5 min) induced a dose-dependent activation of extracellular signal-regulated kinases (ERK), p38, and c-Jun N-terminal kinase (JNK) MAP kinases, as determined by Western blot analysis and in vitro kinase assay. Compared with cag+ strains, cag- clinical isolates were less potent in inducing MAP kinase, particularly JNK and p38, activation. Isogenic inactivation of the picB region of the cag pathogenicity island resulted in a similar loss of JNK and p38 MAP kinase activation. The specific MAP kinase inhibitors, PD98059 (25 microM; MAP kinase kinase (MEK-1) inhibitor) and SB203580 (10 microM; p38 inhibitor), reduced H. pylori-induced IL-8 production in AGS cells by 78 and 82%, respectively (p < 0.01 for each). Both inhibitors together completely blocked IL-8 production (p < 0.001). However, the MAP kinase inhibitors did not prevent H. pylori-induced IkappaBalpha degradation or NF-kappaB activation. Thus, H. pylori rapidly activates ERK, p38, and JNK MAP kinases in gastric epithelial cells; cag+ isolates are more potent than cag- strains in inducing MAP kinase phosphorylation and gene products of the cag pathogenicity island are required for maximal MAP kinase activation. p38 and MEK-1 activity are required for H. pylori-induced IL-8 production, but do not appear to be essential for H. pylori-induced NF-kappaB activation. Since MAP kinases regulate cell proliferation, differentiation, programmed death, stress, and inflammatory responses, activation of gastric epithelial cell MAP kinases by H. pylori cag+ strains may be instrumental in inducing gastroduodenal inflammation, ulceration, and neoplasia.     

Role of mitogen-activated protein kinases and tyrosine kinases on IL-1-Induced NF-kappaB activation and iNOS expression in bovine articular chondrocytes.

Nitric oxide (NO), produced by the inducible isoform of the NO synthase (iNOS), plays an important role in the pathophysiology of arthritic diseases. This work aimed at elucidating the role of the mitogen-activated protein kinases (MAPK), p38MAPK and p42/44MAPK, and of protein tyrosine kinases (PTK) on interleukin-1beta (IL-1)-induced iNOS expression in bovine articular chondrocytes. The specific inhibitor of the p38MAPK, SB 203580, effectively inhibited IL-1-induced iNOS mRNA and protein synthesis, as well as NO production, while the specific inhibitor of the p42/44MAPK, PD 98059, had no effect. These responses to IL-1 were also inhibited by treatment of the cells with the tyrosine kinase inhibitors, genistein and tyrphostin B42, which also prevented IL-1-induced NF-kappaB activation. The p38MAPK inhibitor, SB 203580, had no effect on IL-1-induced NF-kappaB activation. Finally, the p42/44MAPK inhibitor, PD 98059, prevented IL-1-induced AP-1 activation in a concentration that did not inhibit iNOS expression. In conclusion, this study shows that (1) PTK are part of the signaling pathway that leads to IL-1-induced NF-kappaB activation and iNOS expression; (2) the p38MAPK cascade is required for IL-1-induced iNOS expression; (3) the p42/44MAPK and AP-1 are not involved in IL-1-induced iNOS expression; and (4) NF-kappaB and the p38MAPK lie on two distinct pathways that seem to be independently required for IL-1-induced iNOS expression. Hence, inhibition of any of these two signaling cascades is sufficient to prevent iNOS expression and the subsequent production of NO in articular chondrocytes.     

Involvement of p42/p44 MAPK, JNK, and NF-kappaB in IL-1beta-induced ICAM-1 expression in human pulmonary epithelial cells

Interleukin-1beta (IL-1beta) has been shown to induce the expression of intercellular adhesion molecule-1 (ICAM-1) on airway epithelial cells and contributes to inflammatory responses. However, the mechanisms regulating ICAM-1 expression by IL-1beta in human A549 cells was not completely understood. Here, the roles of mitogen-activated protein kinases (MAPKs) and NF-kappaB pathways for IL-1beta-induced ICAM-1 expression were investigated in A549 cells. IL-1beta induced expression of ICAM-1 protein and mRNA in a time- and concentration-dependent manner. The IL-1beta induction of ICAM-1 mRNA and protein were partially inhibited by U0126 and PD98059 (specific inhibitors of MEK1/2) and SP600125 [a specific inhibitor of c-Jun-N-terminal kinase (JNK)]. U0126 was more potent than other inhibitors to attenuate IL-1beta-induced ICAM-1 expression. Consistently, IL-1beta stimulated phosphorylation of p42/p44 MAPK and JNK which was attenuated by pretreatment with U0126 or SP600125, respectively. Moreover, transfection with dominant negative mutants of MEK1/2 (MEK K97R) or ERK2 (ERK2 K52R) also attenuated IL-1beta-induced ICAM-1 expression. The combination of PD98059 and SP600125 displayed an additive effect on IL-1beta-induced ICAM-1 gene expression. IL-1beta-induced ICAM-1 expression was almost completely blocked by a specific NF-kappaB inhibitor helenalin. Consistently, IL-1beta stimulated translocation of NF-kappaB into the nucleus and degradation of IkappaB-alpha which was blocked by helenalin, U0126, or SP600125. Taken together, these results suggest that activation of p42/p44 MAPK and JNK cascades, at least in part, mediated through NF-kappaB pathway is essential for IL-1beta-induced ICAM-1 gene expression in A549 cells. These results provide new insight into the mechanisms of IL-1beta action that cytokines may promote inflammatory responses in the airway disease.     

Activation of extracellular signal-regulated kinase (ERK)1/2 contributes to cytokine-induced apoptosis in purified rat pancreatic beta-cells

Cytokines may contribute to beta-cell apoptosis in the early stages of type 1 diabetes mellitus. It has been reported recently that interleukin-1 beta (IL-1 beta) induces activation of the mitogen-activated protein kinases (MAPK) p38 and ERK1/2 in neonatal rat islets. Since these kinases may participate in cytokine-induced apoptosis, we evaluated whether cytokines induce activation of MAPKs in FACS-purified primary rat beta-cells, and whether blockers of p38 and/or ERK1/2 prevent beta-cell death. IL-1 beta, but not interferon-gamma (IFN-gamma), caused phosphorylation of the substrates Elk-1, ATF-2 and hsp25, and the phosphorylation of both Elk-1 and hsp25 were decreased by the p38 blocker SB203580 (p38i) and the MAPK/ERK blocker PD 098059 (MEKi). When added together, p38i and MEKi decreased IL-1 beta-induced nitrite production over 24 hours by 60%, but did not affect IL-1 beta-induced manganese superoxide dismutase (MnSOD) mRNA expression. To test the effects of MAPK inhibitors on beta-cell death by necrosis or apoptosis, these cells were exposed for 6 or 9 days to IL-1 beta + IFN-gamma. This treatment induced cell death, mostly by apoptosis. The MEKi, but not the p38i, significantly decreased cytokine-induced apoptosis, thus decreasing the total number of dead cells. This protection was only partial, suggesting that ERK1/2 activation is not the only mechanism by which cytokines induce beta-cell apoptosis. We conclude that IL-1 beta induces activation of both p38 and ERK1/2, and that ERK1/2 contributes to the pro-apoptotic effects of the cytokine in primary beta-cells.     

Nuclear factor-kappa B activation by the CXC chemokine melanoma growth-stimulatory activity/growth-regulated protein involves the MEKK1/p38 mitogen-activated protein kinase pathway

Melanoma growth stimulatory activity/growth-regulated protein (MGSA/GRO), a CXC chemokine, plays an important role in inflammation, wound healing, growth regulation, angiogenesis, and tumorigenesis. Constitutive expression of MGSA/GROalpha in melanoma tumors is associated with constitutive nuclear factor (NF)-kappaB activity. We show here that either exogenous addition or continuous expression of MGSA/GROalpha in immortalized melanocytes enhances NF-kappaB activation, as well as mitogen-activated protein (MAP) kinase kinase kinase (MEKK) 1, MAP kinase kinase (MEK) 3/6, and p38 MAP kinase activation. Expression of dominant negative M-Ras (S27N), dominant negative MEKK1 (K432M), or specific chemical inhibitors for p38 MAP kinase (SB202190 and SB203580) block MGSA/GROalpha-induced NF-kappaB transactivation, demonstrating that Ras, MEKK1, and p38 are involved in the signal pathways of MGSA/GROalpha activation of NF-kappaB. Expression of dominant active Ras or dominant active MEKK1 alone can also stimulate NF-kappaB activation. The expression of dominant negative MEKK1 inhibits the Ras-induced NF-kappaB activation, suggesting that MEKK1 is a downstream target of Ras. Moreover, MGSA/GROalpha induction of NF-kappaB is independent of the MEK1/ERK cascade, because MGSA/GROalpha failed to increase ERK and ELK activation, and specific chemical inhibitors for MEK1 (PD98059) had no effect on MGSA/GROalpha-enhanced NF-kappaB activation. These data demonstrate that NF-kappaB activation is required for MGSA/GROalpha-induced melanocyte transformation through a Ras/MEKK1/p38 cascade in melanocytes.     

Selective activation of p38 mitogen-activated protein kinase cascade in human neutrophils stimulated by IL-1beta.

We investigated activation of mitogen-activated protein kinase (MAPK) subtype cascades in human neutrophils stimulated by IL-1beta. IL-1beta induced phosphorylation and activation of p38 MAPK and phosphorylation of MAPK kinase-3/6 (MKK3/6). Maximal activation of p38 MAPK was obtained by stimulation of cells with 300 U/ml IL-1beta for 10 min. Extracellular signal-regulated kinase (ERK) was faintly phosphorylated and c-Jun N-terminal kinase (JNK) was not phosphorylated by IL-1beta. IL-1beta primed neutrophils for enhanced release of superoxide (O(2)(-)) stimulated by FMLP in parallel with increased phosphorylation of p38 MAPK. IL-1beta also induced O(2)(-) release and up-regulation of CD11b and CD15, and both responses were inhibited by SB203580 (p38 MAPK inhibitor), suggesting that p38 MAPK activation mediates IL-1beta-induced O(2)(-) release and up-regulation of CD11b and CD15. Combined stimulation of neutrophils with IL-1beta and G-CSF, a selective activator of the ERK cascade, resulted in the additive effects when the priming effect and phosphorylation of p38 MAPK and ERK were assessed. IL-1beta induced phosphorylation of ERK and JNK as well as p38 MAPK in human endothelial cells. These findings suggest that 1) in human neutrophils the MKK3/6-p38 MAPK cascade is selectively activated by IL-1beta and activation of this cascade mediates IL-1beta-induced O(2)(-) release and up-regulation of CD11b and CD15, and 2) the IL-1R-p38 MAPK pathway and the G-CSF receptor-ERK pathway work independently for activation of neutrophils.     

The neuroprotective agents chlomethiazole and SB203580 inhibit IL-1beta signalling but not its biosynthesis in rat cortical glial cells

Chlomethiazole and pyridinyl imidazole compounds, exemplified by SB203580, are structurally distinct p38 mitogen-activated protein kinase inhibitors with neuroprotective properties in models of cerebral ischaemia. We have examined their effects in interleukin-1beta (IL-1beta) synthesis, release and signalling in rat cortical glial cells, given the important role of IL-1beta in cerebral ischaemia. We analysed (i) IL-1beta mRNA expression by northern blot, (ii) IL-1beta protein precursor levels within the cells by western blot, and (iii) the levels of the mature IL-1beta protein secreted into the medium by enzyme-linked immunosorbent assay (ELISA) after treatment of rat cortical glial cells with lipopolysaccharide. While the induction of IL-1beta expression by lipopolysaccharide or by IL-1beta itself was very sensitive to nuclear factor kappa B (NF-kappaB) inhibitors, chlomethiazole or SB203580 were nearly without effect, indicating a differential regulation as compared to peripheral cells, e.g. monocytes. In contrast, chlomethiazole and SB203580 potently inhibited the IL-1beta-induced expression of c-fos and inducible nitric oxide synthase, as monitored by northern blot and quantitative RT-PCR, respectively. Because IL-1beta-induced expression of c-fos and inducible nitric oxide synthase is believed to directly contribute to the pathology of cerebral ischaemic injury, the results suggest a direct mechanism for the neuroprotective effects of chlomethiazole and SB203580, and further establish the anti-inflammatory properties of chlomethiazole.     

Angiotensin II differentially regulates interleukin-1-beta-inducible NO synthase (iNOS) and vascular cell adhesion molecule-1 (VCAM-1) expression: role of p38 MAPK

Angiotensin II is implicated in pathophysiological processes associated with vascular injury and repair, which include regulating the expression of numerous NF-kappaB-dependent genes. The present study examined the effect of angiotensin II on interleukin-1beta-induced NF-kappaB activation and the subsequent expression of inducible NO synthase (iNOS) and vascular cell adhesion molecule-1 (VCAM-1) in cultured rat vascular smooth muscle cells. Neither NF-kappaB activation nor iNOS or VCAM-1 expression was induced in cells treated with angiotensin II alone. However, when added together with interleukin-1beta, angiotensin II, through activation of the AT(1) receptor, inhibited iNOS expression and enhanced VCAM-1 expression induced by the cytokine. The inhibitory effect of angiotensin II on iNOS expression was associated with a down-regulation of the sustained activation of extracellular signal-regulated kinase (ERK) and NF-kappaB by interleukin-1beta, whereas the effect on VCAM-1 was independent of ERK activation. The effect of angiotensin II on iNOS was abolished by inhibition of p38 mitogen-activated protein kinase (MAPK) with SB203580, but not by inhibition of PI3 kinase with wortmannin or stress-activated protein kinase/c-Jun NH(2)-terminal kinase (JNK) with JNK inhibitor II. Thus, angiotensin II, by a mechanism that requires the participation of p38 MAPK, differentially regulates the expression of NF-kappaB-dependent genes in response to interleukin-1beta stimulation by controlling the duration of activation of ERK and NF-kappaB.