Role of interleukin (IL)-2 receptor beta-chain subdomains and Shc in p38 mitogen-activated protein (MAP) kinase and p54 MAP kinase (stress-activated protein Kinase/c-Jun N-terminal kinase) activation. IL-2-driven proliferation is independent of p38 and p54 MAP kinase activation

We have shown recently that interleukin (IL)-2 activates the mitogen-activated protein (MAP) kinase family members p38 (HOG1/stress-activated protein kinase II) and p54 (c-Jun N-terminal kinase/stress-activated protein kinase I). Furthermore, the p38 MAP kinase inhibitor SB203580 inhibited IL-2-driven T cell proliferation, suggesting that p38 MAP kinase might be involved in mediating proliferative signals. In this study, using transfected BA/F3 cell lines, it is shown that both the acidic domain and the membrane-proximal serine-rich region of the IL-2Rbeta chain are required for p38 and p54 MAP kinase activation and that, as for p42/44 MAP kinase, this activation requires the Tyr338 residue of the acidic domain, the binding site for Shc. It is well established that the acidic domain of the IL-2Rbeta chain is dispensable for IL-2-driven proliferation, and thus our observations suggest that neither p38 nor p54 MAP kinase activation is required for IL-2-driven proliferation of BA/F3 cells. In addition, the tetravalent guanylhydrazone inhibitor of proinflammatory cytokine production, CNI-1493, can block the activation of p54 and p38 MAP kinases by IL-2 but has no effect on IL-2-driven proliferation of BA/F3 cells, activated primary T cells, or a cytotoxic T cell line. Furthermore, our observations provide evidence for the existence of an additional, unknown target of the p38 MAP kinase inhibitor SB203580, the activation of which is essential for mitogenic signaling by IL-2.     

P2X7 receptor mediated phosphorylation of p38MAP kinase in the hippocampus

This study was designed to explore the effect of P2X7 receptor (P2X7R) activation on the expression of p38 MAP kinase (p38 MAPK) enzyme in hippocampal slices of wild-type (WT) and P2X7R^−/− mice using the Western blot technique and to clarify its role in P2X7 receptor mediated [³H]glutamate release. ATP (1 mM) and the P2X7R agonist BzATP (100 μM) significantly increased p38 MAPK phosphorylation in WT mice, and these effects were absent in the hippocampal slices of P2X7R^−/− mice. Both ATP- and BzATP-induced p38 MAPK phosphorylations were sensitive to the p38 MAP kinase inhibitor, SB203580 (1 μM). ATP elicited [³H]glutamate release from hippocampal slices, which was significantly attenuated by SB203580 (1 μM) but not by the extracellular signal-regulated kinase (ERK1/2) inhibitor, PD098095 (10 μM). Consequently, we suggest that P2X7Rs and p38 MAPK are involved in the stimulatory effect of ATP on glutamate release in the hippocampal slices of WT mice.     

Inhibition of p38 MAP kinase activity up-regulates multiple MAP kinase pathways and potentiates 1,25-dihydroxyvitamin D(3)-induced differentiation of human leukemia HL60 cells

Differentiation therapy for neoplastic diseases has potential for supplementing existing treatment modalities but its implementation has been slow. One of the reasons is the lack of full understanding of the complexities of cellular pathways through which signals for differentiation lead to cell maturation. This was addressed in this study using HL60 cells, a well-established model of differentiation of neoplastic cells. SB 203580 and SB 202190, specific inhibitors of a signaling protein p38 MAP kinase, were found to markedly accelerate monocytic differentiation of HL60 cells induced by low concentrations of 1,25-dihydroxyvitamin D(3) (1,25D(3)). Surprisingly, inhibition of p38 activity resulted in sustained enhancement of p38 phosphorylation and of its in vitro activity in the absence of the inhibitor, indicating up-regulation of the upstream components of the p38 pathway. In addition, SB 203580 or SB 202190 treatment of HL60 cells resulted in a prolonged activation of the JNK and, to a lesser extent, the ERK pathways. The data are consistent with the hypothesis that in HL60 cells an interruption of a negative feedback loop from a p38 target activates a common regulator of multiple MAPK pathways. The possibility also exists that JNK and/or ERK pathways amplify a differentiation signal provided by 1,25D(3).     

A single amino acid substitution makes ERK2 susceptible to pyridinyl imidazole inhibitors of p38 MAP kinase.

Mitogen-activated protein (MAP) kinases are serine/threonine kinases that mediate intracellular signal transduction pathways. Pyridinyl imidazole compounds block pro-inflammatory cytokine production and are specific p38 kinase inhibitors. ERK2 is related to p38 in sequence and structure, but is not inhibited by pyridinyl imidazole inhibitors. Crystal structures of two pyridinyl imidazoles complexed with p38 revealed these compounds bind in the ATP site. Mutagenesis data suggested a single residue difference at threonine 106 between p38 and other MAP kinases is sufficient to confer selectivity of pyridinyl imidazoles. We have changed the equivalent residue in human ERK2, Q105, into threonine and alanine, and substituted four additional ATP binding site residues. The single residue change Q105A in ERK2 enhances the binding of SB202190 at least 25,000-fold compared to wild-type ERK2. We report enzymatic analyses of wild-type ERK2 and the mutant proteins, and the crystal structure of a pyridinyl imidazole, SB203580, bound to an ERK2 pentamutant, I103L, Q105T, D106H, E109G. T110A. These ATP binding site substitutions induce low nanomolar sensitivity to pyridinyl imidazoles. Furthermore, we identified 5-iodotubercidin as a potent ERK2 inhibitor, which may help reveal the role of ERK2 in cell proliferation.     

Cell survival signalling in heart derived myofibroblasts induced by preconditioning and bradykinin: The role of p38 MAP kinase

Fibroblasts possess receptors for compounds released during ischemia, including bradykinin. The aims of the present study were to investigate tyrosine kinase and p38 MAP kinase signalling in heart derived myofibroblasts in response to bradykinin and preconditioning ischemia. Fibroblasts from neonatal rat hearts were subjected to pharmacological agents and/or simulated ischemia. Cell viability was measured by the conversion of a tetrazolium salt to its formazan derivative. Preconditioning with 30 min of simulated ischemia followed by 30 min recovery resulted in an 85.4% +/- 7.8% increase in cell survival above that of cells treated with prolonged ischemia alone. Cells treated with bradykinin showed a 35% +/- 7.9 increase in cell survival after lethal ischemia. The B2 receptor antagonist Hoe 140 blocked the protective effect of bradykinin, but did not block preconditioning. The K(ATP) channel blocker glibenclamide and the mitochondria specific K(ATP) blocker 5, hydroxydecanoate, abolished the cytoprotection induced by both preconditioning and bradykinin. The non specific tyrosine kinase inhibitor genistein also abolished the cytoprotection. Effective blockade of cytoprotection was obtained with K(ATP) channel blockers and the tyrosine kinase inhibitor when these compounds were given prior to the preconditioning stimulus and not during the lethal insult. The stress activated protein kinase p38 MAP kinase was investigated by Western blotting and by the use of a specific inhibitor (SB203580). Preconditioning reduced phospho-p38 MAP kinase; in contrast, bradykinin administration markedly increased phosphorylation of p38 MAP kinase. SB203580 protected cells from lethal simulated ischemia. In conclusion, cell survival-signalling pathways activated by bradykinin or simulated ischemia in heart fibroblasts protect via the opening of K(ATP) channels and are independent of the stress-activated p38 MAP kinase and/or related to inhibition of this kinase.     

Smad and p38 MAP kinase-mediated signaling of proteoglycan synthesis in vascular smooth muscle

Atherosclerosis is the underlying pathological process of most cardiovascular disease. A critical component of the "response to retention" hypothesis of atherogenesis is proteoglycan/low density lipoprotein (LDL) binding. Transforming growth factor beta (TGF-beta) is present in atherosclerotic lesions, regulates vascular smooth muscle cell (VSMC) proteoglycan synthesis via an unknown signaling pathway, and increases proteoglycan/LDL binding. This pathway was investigated using the activin receptor-like kinase 5 (ALK5) inhibitor SB431542 and inhibitors of p38 MAP kinase as a possible downstream or alternative mediator. TGF-beta stimulated and SB431542 inhibited the phosphorylation of Smad2/3. In human VSMC, TGF-beta increased [(35)S]sulfate incorporation into proteoglycans associated with a 19% increase in glycosaminoglycan (GAG) chain size by size exclusion chromatography. SB431542 caused a concentration-dependent decrease in TGF-beta-mediated [(35)S]sulfate incorporation with 92% inhibition at 3 mum. Two different p38 MAP kinase inhibitors, SB203580 and SB202190, but not the inactive analogue SB202474, concentration-dependently blocked TGF-beta-mediated [(35)S]sulfate incorporation. TGF-beta increased [(3)H]glucosamine incorporation into glycosaminoglycans by 180% and [(35)S]Met/Cys incorporation into proteoglycan core proteins by 35% with both effects completely inhibited by SB431542. Blocking both Smad2/3 and p38 MAP kinase pathways prevented the effect of TGF-beta to increase proteoglycan to LDL binding. TGF-beta mediates its effects on proteoglycan synthesis in VSMCs via the ALK5/Smad2/3 phosphorylation pathway as well as via the p38 MAP kinase signaling cascade. Further studies of downstream pathways controlling proteoglycan synthesis may identify potential therapeutic targets for the prevention of atherosclerosis and cardiovascular disease.     

Involvement of p38 MAP kinase in TGF-beta-stimulated VEGF synthesis in aortic smooth muscle cells

Although it is known that transforming growth factor (TGF)-beta induces vascular endothelial growth factor (VEGF) synthesis in vascular smooth muscle cells, the underlying mechanisms are still poorly understood. In the present study, we examined whether the mitogen-activated protein (MAP) kinase superfamily is involved in TGF-beta-stimulated VEGF synthesis in aortic smooth muscle A10 cells. TGF-beta stimulated the phosphorylation of p42/p44 MAP kinase and p38 MAP kinase, but not that of SAPK (stress-activated protein kinase)/JNK (c-Jun N-terminal kinase). The VEGF synthesis induced by TGF-beta was not affected by PD98059 or U0126, specific inhibitors of the upstream kinase that activates p42/p44 MAP kinase. We confirmed that PD98059 or U0126 did actually suppress the phosphorylation of p42/p44 MAP kinase by TGF-beta in our preparations. PD169316 and SB203580, specific inhibitors of p38 MAP kinase, significantly reduced the TGF-beta-stimulated synthesis of VEGF (each in a dose-dependent manner). PD169316 or SB203580 attenuated the TGF-beta-induced phosphorylation of p38 MAP kinase. These results strongly suggest that p38 MAP kinase plays a part in the pathway by which TGF-beta stimulates the synthesis of VEGF in aortic smooth muscle cells.     

Inhibition of p38MAP kinase potentiates the JNK/SAPK pathway and AP-1 activity in monocytic but not in macrophage or granulocytic differentiation of HL60 cells

Monocytic differentiation of HL60 cells induced by 1,25-dihydroxyvitamin D(3) (1,25 D(3)) has been recently shown (Exp Cell Res 258, 425, 2000) to be enhanced by an exposure to SB203580 or to SB202190, specific inhibitors of p38MAP kinase, with concomitant up-regulation of the c-jun N terminal kinase (JNK) pathway. In the present study we inquired if this enhancement and the JNK up-regulation are limited to 1,25 D(3)-induced differentiation, or if they occur more generally in HL60 cell differentiation. We found that dimethylsulfoxide (DMSO)-induced differentiation, and to a lesser extent tetradecanoylphorbol acetate (TPA)-induced macrophage differentiation were also potentiated by the p38MAPK inhibitors, but that granulocytic differentiation in response to all-trans retinoic acid (RA) was not. The enhancement of differentiation by p38MAPK inhibitors was accompanied by an activation of the JNK MAPK pathway, as shown by the phosphorylation levels of these kinases and by AP-1 binding, but only in 1,25 D(3)-treated cells. This shows that an up-regulation of the JNK stress pathway during 1,25 D(3)-induced monocytic differentiation occurs selectively in this lineage of differentiation and is not necessary for the expression of the differentiated phenotype.     

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.     

The molecular basis for coxib inhibition of p38alpha MAP kinase

In this work, we present the results of two combined approaches, molecular docking and comparative molecular field analysis (CoMFA), to propose how the selective cyclooxygenase-2 inhibitor celecoxib could act as a p38 mitogen-activated protein (MAP) kinase inhibitor. The docking analysis revealed why celecoxib has a less favorable binding energy (DeltaG= -12.4kcal/mol) than the selective p38 MAP kinase (p38 MAPK) inhibitor, SB203580 (DeltaG= -22.2kcal/mol). The CoMFA results revealed unfavorable steric effects that can be related to the predicted lower p38 MAP kinase inhibitory activity of celecoxib. Additionally, FlexX and CoMFA results also suggested that etoricoxib, another selective COX-2 inhibitor, could inhibit p38 MAP kinase.     

Differential effects of p38 MAP kinase inhibitors SB203580 and SB202190 on growth and migration of human MDA-MB-231 cancer cell line

p38 mitogen-activated protein kinase (MAPK) belongs to the MAPK superfamily, phosphorylating serine and/or threonine residues of the target proteins. The activation of p38 MAPK leads to cell growth, differentiation, inflammation, survival or apoptosis. In this study, we tested the effect of two highly specific and potent inhibitors of p38 MAPK (namely, SB203580 and SB202190) on human breast cancer cell line MDA-MB-231 to elucidate the controversial role of p38 MAPK on cell proliferation and/or cell migration/metastasis further. It was determined that the IC₅₀ value of SB203580 was 85.1 µM, while that of SB202190 was 46.6 µM, suggesting that SB202190 is slightly more effective than SB203580. To verify the effect of each inhibitor on cell proliferation and cytotoxicity, the cells were treated with various doses of SB203580 and SB202190 and examined using iCELLigence system. No significant effect of 1 and 5 µM of both inhibitors were seen on cell proliferation as compared to the DMSO-treated control cells for up to 96 h. On the other hand, both SB203580 and SB202190 significantly prevented cell proliferation at a concentration of 50 µM. SB202190 was again more effective than SB203580. Afterwards, we tested the effect of each inhibitor on cell migration using wound assay. Both SB203580 and SB202190 significantly reduced cell migration in a time-dependent manner at a concentration of 50 µM. However, interestingly it was observed that a low and noncytotoxic dose of 5 µM of SB203580 and SB202190 also did cause significant cell migration inhibition at 48 h of the treatment, corroborating the fact that p38 MAPK pathway has a critical role in cell migration/metastasis. Then, we tested whether each p38 MAPK inhibitor has any effect on cell adhesion during a treatment period of 3 h using iCELLigence system. A concentration of only 50 µM of SB202190 reduced cell adhesion for about 1.5 h (p < 0.001); after that period of time, cell adhesion in 50 µM SB202190-treated cells returned to the level of the control cells. To determine the mechanism of growth and cell migration inhibitory effects of p38 MAPK inhibitors, the activation/inactivation of various proteins and enzymes was subsequently analyzed by PathScan^® Intracellular Signaling Array kit. The ERK1/2 phosphorylation level was not modified by low concentrations (1 or 5 µM) of SB202190 and SB203580; while a high concentration (50 µM) of both inhibitors caused significant reductions in the ERK1/2 phosphorylation. In addition, it was determined that both p38 MAPK inhibitors caused significant increases on the Ser15 phosphorylation of mutant p53 in MDA-MB-231 under these experimental conditions; while SB202190 was more potent than SB203580.     

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

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.     

Involvement of p38 mitogen-activated protein kinase signaling pathway in osteoclastogenesis mediated by receptor activator of NF-kappa B ligand (RANKL)

The receptor activator of NF-kappaB ligand (RANKL) induces osteoclast differentiation from bone marrow cells in the presence of macrophage colony-stimulating factor. We found that treatment of bone marrow cells with SB203580 inhibited osteoclast differentiation via inhibition of the RANKL-mediated signaling pathway. To elucidate the role of p38 mitogen-activated protein (MAP) kinase pathway in osteoclastogenesis, we employed RAW264 cells which could differentiate into osteoclast-like cells following treatment with RANKL. In a dose-dependent manner, SB203580 but not PD98059, inhibited RANKL-induced differentiation. Among three MAP kinase families tested, this inhibition profile coincided only with the activation of p38 MAP kinase. Expression in RAW264 cells of the dominant negative form of either p38alpha MAP kinase or MAP kinase kinase (MKK) 6 significantly inhibited RANKL-induced differentiation of the cells. These results indicate that activation of the p38 MAP kinase pathway plays an important role in RANKL-induced osteoclast differentiation of precursor bone marrow cells.     

Kinetics of small molecule inhibitor binding to p38 kinase.

p38 mitogen-activated protein kinase (MAPK) (p38/p38-alpha/CSBP2/RK) has been implicated in the regulation of many proinflammatory pathways. Because of this, it has received much attention as a potential drug target for controlling diseases such as rheumatoid arthritis, endotoxic shock, inflammatory bowel disease, osteoporosis, and many others. A number of small molecule inhibitors of this kinase have been described, and in this paper we have used surface plasmon resonance to directly measure and quantitate their binding to p38. Despite the relatively low molecular mass (approximately 400 Da) of these inhibitors, specific binding can be observed. For the two most potent inhibitors studied, SB 203580 and RWJ 67657, dissociation constants, K(d)'s, of 22 and 10 nm, respectively, were obtained. These values closely match the IC(5)0 values observed in a cell-based TNF alpha release assay implying that p38 plays a major role in TNF alpha release. The association and dissociation rates for the binding of these inhibitors to p38 have also been quantitated. SB 203580 and RWJ 67657 have very similar association rates of around 8 x 10(5) m(-1) x s(-1), and the differences in affinity are determined by different dissociation rates. The weaker binding compounds have dissociation rates similar to SB 203580, but the association rates vary by an order of magnitude or more. The direct measurement of compounds binding to p38 may help in understanding the difference between potency and efficacy for these inhibitors. This in turn may yield clues on how to develop better inhibitors.     

p38 MAP kinase regulates BMP-4-stimulated VEGF synthesis via p70 S6 kinase in osteoblasts

We previously reported that p70 S6 kinase takes part in bone morphogenetic protein-4 (BMP-4)-stimulated vascular endothelial growth factor (VEGF) synthesis in osteoblast-like MC3T3-E1 cells. Recently, we showed that BMP-4-induced osteocalcin synthesis is regulated by p44/p42 MAP kinase and p38 MAP kinase in these cells. In the present study, we investigated whether the MAP kinases are involved in the BMP-4-stimulated synthesis of VEGF in MC3T3-E1 cells. PD-98059 and U-0126, inhibitors of the upstream kinase of p44/p42 MAP kinase, failed to affect BMP-4-stimulated VEGF synthesis. SB-203580 and PD-169316, inhibitors of p38 MAP kinase, significantly reduced VEGF synthesis, whereas SB-202474, a negative control for p38 MAP kinase inhibitor, had little effect on VEGF synthesis. The BMP-4-stimulated phosphorylation of p38 MAP kinase was not affected by rapamycin, an inhibitor of p70 S6 kinase. On the contrary, SB-203580 and PD-169316 reduced the BMP-4-stimulated phosphorylation of p70 S6 kinase. In addition, anisomycin, an activator of p38 MAP kinase, phosphorylates p70 S6 kinase, and the phosphorylation was suppressed by SB-203580. LY-294002, an inhibitor of phosphatidylinositol 3-kinase, failed to suppress the phosphorylation of p38 MAP kinase induced by BMP-4. Not BMP-4 but anisomycin weakly induced the phosphorylation of phosphoinositide-dependent kinase-1. However, anisomycin had little effect on phosphorylation of either Akt or the mammalian target of rapamycin. Taken together, our results suggest that p38 MAP kinase functions in BMP-4-stimulated VEGF synthesis as a positive regulator at a point upstream from p70 S6 kinase in osteoblasts.