C-reactive protein inhibits chemotactic peptide-induced p38 mitogen-activated protein kinase activity and human neutrophil movement.

Serum levels of the acute-phase reactant, C-reactive protein (CRP), increase dramatically during acute inflammatory episodes. CRP inhibits migration of neutrophils toward the chemoattractant, f-Met-Leu-Phe (fMLP) and therefore acts as an anti-inflammatory agent. Since tyrosine kinases are involved in neutrophil migration and CRP has been shown to decrease phosphorylation of some neutrophil proteins, we hypothesized that CRP inhibits neutrophil chemotaxis via inhibition of MAP kinase activity. The importance of p38 MAP kinase in neutrophil movement was determined by use of the specific p38 MAP kinase inhibitor, SB203580. CRP and SB203580 both blocked random and fMLP-directed neutrophil movement in a concentration-dependent manner. Additionally, extracellular signal-regulated MAP kinase (ERK) was not involved in fMLP-induced neutrophil movement as determined by use of the MEK-specific inhibitor, PD98059. Blockade of ERK with PD98059 did not inhibit chemotaxis nor did it alter the ability of CRP or SB203580 to inhibit fMLP-induced chemotaxis. More importantly, CRP inhibited fMLP-induced p38 MAP kinase activity in a concentration-dependent manner as measured by an in vitro kinase assay. Impressively, CRP-mediated inhibition of p38 MAP kinase activity correlated with CRP-mediated inhibition of fMLP-induced chemotaxis (r = -0.7144). These data show that signal transduction through p38 MAP kinase is necessary for neutrophil chemotaxis and that CRP intercedes through this pathway in inhibiting neutrophil movement.     

Regulation of glucose transport and glycogen synthesis in L6 muscle cells during oxidative stress. Evidence for cross-talk between the insulin and SAPK2/p38 mitogen-activated protein kinase signaling pathways

We have investigated the cellular mechanisms that participate in reducing insulin sensitivity in response to increased oxidant stress in skeletal muscle. Measurement of glucose transport and glycogen synthesis in L6 myotubes showed that insulin stimulated both processes, by 2- and 5-fold, respectively. Acute (30 min) exposure of muscle cells to hydrogen peroxide (H(2)O(2)) blocked the hormonal activation of both these processes. Immunoblot analyses of cell lysates prepared after an acute oxidant challenge using phospho-specific antibodies against c-Jun N-terminal kinase (JNK), p38, protein kinase B (PKB), and p42 and p44 mitogen-activated protein (MAP) kinases established that H(2)O(2) induced a dose-dependent activation of all five protein kinases. In vitro kinase analyses revealed that 1 mM H(2)O(2) stimulated the activity of JNK by approximately 8-fold, MAPKAP-K2 (the downstream target of p38 MAP kinase) by approximately 12-fold and that of PKB by up to 34-fold. PKB activation was associated with a concomitant inactivation of glycogen synthase kinase-3. Stimulation of the p38 pathway, but not that of JNK, was blocked by SB 202190 or SB203580, while that of p42/p44 MAP kinases and PKB was inhibited by PD 98059 and wortmannin respectively. However, of the kinases assayed, only p38 MAP kinase was activated at H(2)O(2) concentrations (50 microM) that caused an inhibition of insulin-stimulated glucose transport and glycogen synthesis. Strikingly, inhibiting the activation of p38 MAP kinase using either SB 202190 or SB 203580 prevented the loss in insulin-stimulated glucose transport, but not that of glycogen synthesis, by oxidative stress. Our data indicate that activation of the p38 MAP kinase pathway plays a central role in the oxidant-induced inhibition of insulin-regulated glucose transport, and unveils an important biochemical link between the classical stress-activated and insulin signaling pathways in skeletal muscle.     

Protein kinase B/Akt mediates cAMP- and cell swelling-stimulated Na+/taurocholate cotransport and Ntcp translocation

Cyclic AMP and cell swelling stimulate hepatic Na+/TC cotransport and Ntcp translocation via the phosphoinositide 3-kinase signaling pathway. To determine the downstream target of the phosphoinositide 3-kinase action, we examined the role of protein kinase B (PKB)/Akt using SB203580 in hepatocytes as well as by transfection with a dominant negative (DN-PKB) or a constitutively active (CA-PKB) form of PKB in HuH-Ntcp cells. Both cAMP and cell swelling stimulated p38 mitogen-activated protein (MAP) kinase as well as PKB activity. Although 100 microm SB203580 inhibited cell swelling- and 8-chlorophenylthio-cAMP-induced activation of both p38 MAP kinase and PKB, 1 microm SB203580 inhibited activation of p38 MAP kinase, but not of PKB, in hepatocytes. 100 microm, but not 1 microm SB203580, inhibited cell swelling- and cAMP-induced increases in taurocholate (TC) uptake and Ntcp translocation in hepatocytes. TC uptake in HuH-Ntcp cells was more than 90% dependent on extracellular Na+. Cyclic AMP and cell swelling increased TC uptake by 50-100% and PKB activity 2-4-fold in HuH-Ntcp cells transfected with the empty vector and failed to increase PKB activity, TC uptake, and Ntcp translocation in DN-PKB-transfected HuH-Ntcp cells. Transfection with CA-PKB increased PKB activity, TC uptake, and Ntcp translocation in HuH-Ntcp cells compared with cells transfected with the empty vector. In contrast, transfection with DN-PKB did not affect basal PKB activity, TC uptake, or Ntcp translocation. Taken together, these results strongly suggest that cell swelling and cAMP-mediated stimulation of hepatic Na+/TC cotransport and Ntcp translocation requires activation of PKB and is mediated at least in part via a phosphoinositide 3-kinase/PKB-signaling pathway.     

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.     

Activation of p90RSK and cAMP response element binding protein in stimulated neutrophils: novel effects of the pyridinyl imidazole SB 203580 on activation of the extracellular signal-regulated kinase cascade.

Neutrophils stimulated with the chemoattractant FMLP or the phorbol ester PMA are known to exhibit activation of a 90-kDa renaturable protein kinase. Activation of this kinase was maximal at approximately 1-3 min after cell stimulation and the time course for activation was similar to that of the extracellular-regulated kinases (ERKs) and p38-mitogen activated protein kinase (p38MAPK). Compounds that block activation of ERK-1/2 (PD 98059) or that inhibit the activity of p38MAPK (SB 203580) blocked activation of this 90-kDa kinase. SB 203580 is a highly selective inhibitor of p38MAPK in vitro and is under intense study as a lead compound for developing novel anti-inflammatory agents. However, we demonstrate that SB 203580 at concentrations >/=10 microM can also inhibit activation of ERK-1/2 in neutrophils. An Ab to the protein kinase p90RSK2 (also referred to as MAPKAP-K1b, or p90rsk) immunoprecipitated the active 90-kDa kinase from lysates of stimulated neutrophils. No activity was observed for this enzyme in immunoprecipitates obtained from unstimulated cells, and the amounts of activity were markedly reduced if the cells were treated with PD 98059 or SB 203580 before stimulation. Neutrophils stimulated with FMLP exhibited phosphorylation of the cAMP response element binding protein (CREB), and this reaction was inhibited by SB 203580 and PD 98059. These data establish that the renaturable 90-kDa protein kinase is p90RSK2 and that CREB may be a substrate for this enzyme in these cells. Novel effects of compound SB 203580 on stimulated neutrophils are also described.     

Stress-activated protein kinase/c-Jun N-terminal kinase (JNK) plays a part in endothelin-1-induced vascular endothelial growth factor synthesis in osteoblasts

We previously reported that endothelin-1 (ET-1) activates both p44/p42 mitogen-activated protein (MAP) kinase and p38 MAP kinase in osteoblast-like MC3T3-E1 cells, and that not p44/p42 MAP kinase but p38 MAP kinase participates in the ET-1-induced vascular endothelial growth factor (VEGF) synthesis. In the present study, we investigated the involvement of stress-activated protein kinase/c-Jun N-terminal kinase (JNK) in ET-1-induced VEGF synthesis in these cells. ET-1 significantly induced the phosphorylation of JNK in a dose-dependent manner in the range between 0.1 and 100 nM. SP600125, an inhibitor of JNK, markedly reduced the ET-1-induced VEGF synthesis. A combination of SP600125 and SB203580 additively reduced the ET-1-stimulated VEGF synthesis. SP600125 suppressed the ET-1-induced phosphorylation of JNK, while having no effect on the phosphorylation of p38 MAP kinase elicited by ET-1. SB203580, an inhibitor of p38 MAP kinase, hardly affected the ET-1-induced phosphorylation of JNK. These results strongly suggest that JNK plays a role in ET-1-induced VEGF synthesis in addition to p38 MAP kinase in osteoblasts.     

An inhibitor of p38 mitogen-activated protein kinase prevents insulin-stimulated glucose transport but not glucose transporter translocation in 3T3-L1 adipocytes and L6 myotubes

The precise mechanisms underlying insulin-stimulated glucose transport still require investigation. Here we assessed the effect of SB203580, an inhibitor of the p38 MAP kinase family, on insulin-stimulated glucose transport in 3T3-L1 adipocytes and L6 myotubes. We found that SB203580, but not its inactive analogue (SB202474), prevented insulin-stimulated glucose transport in both cell types with an IC50 similar to that for inhibition of p38 MAP kinase (0.6 microM). Basal glucose uptake was not affected. Moreover, SB203580 added only during the transport assay did not inhibit basal or insulin-stimulated transport. SB203580 did not inhibit insulin-stimulated translocation of the glucose transporters GLUT1 or GLUT4 in 3T3-L1 adipocytes as assessed by immunoblotting of subcellular fractions or by immunofluorescence of membrane lawns. L6 muscle cells expressing GLUT4 tagged on an extracellular domain with a Myc epitope (GLUT4myc) were used to assess the functional insertion of GLUT4 into the plasma membrane. SB203580 did not affect the insulin-induced gain in GLUT4myc exposure at the cell surface but largely reduced the stimulation of glucose uptake. SB203580 had no effect on insulin-dependent insulin receptor substrate-1 phosphorylation, association of the p85 subunit of phosphatidylinositol 3-kinase with insulin receptor substrate-1, nor on phosphatidylinositol 3-kinase, Akt1, Akt2, or Akt3 activities in 3T3-L1 adipocytes. In conclusion, in the presence of SB203580, insulin caused normal translocation and cell surface membrane insertion of glucose transporters without stimulating glucose transport. We propose that insulin stimulates two independent signals contributing to stimulation of glucose transport: phosphatidylinositol 3-kinase leads to glucose transporter translocation and a pathway involving p38 MAP kinase leads to activation of the recruited glucose transporter at the membrane.     

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

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.     

Involvement of mitogen-activated protein kinase homologues in the regulation of lipopolysaccharide-mediated induction of cyclo-oxygenase-2 but not nitric oxide synthase in RAW 264.7 macrophages.

In RAW 264.7 macrophages lipopolysaccharide (LPS) stimulated the activation of p42 and p44 MAP kinases and their upstream activator mitogen-activated protein (MAP) kinase kinase (MAPKK), and induced the 69-kDa isoform of cyclo-oxygenase-2 (COX-2) and the 130-kDa isoform of nitric oxide synthase (iNOS). PD 098059, a specific inhibitor of the activation of MAPKK, prevented LPS-mediated activation of MAPKK (IC50 = 3.0 +/- 0.1 microM, n = 3) and p42/44 MAP kinases and substantially reduced the induction of COX-2 by approximately 40%-70%, but was without effect upon the induction of iNOS. In parallel, LPS also stimulated the activation of p38 MAP kinase and the MAPKAP kinase-2, a downstream target of p38 MAP kinase. SB 203580, a specific inhibitor of p38 MAP kinase prevented the activation of p38 MAP kinase (IC50 = 3.3 +/- 1.4 microM, n = 3) and MAPKAP kinase-2 by LPS and reduced the induction of COX-2 by approximately 50-90%, with no significant effect upon iNOS expression. These studies indicate the involvement of both the classical p42/44 MAP kinases and p38 MAP kinase in the regulation of COX-2 but not iNOS induction following exposure to LPS.     

p38 mitogen-activated protein kinase mediates adenosine-induced alterations in myocardial glucose utilization via 5'-AMP-activated protein kinase

Adenosine-induced acceleration of glycolysis in hearts stressed by transient ischemia is accompanied by suppression of glycogen synthesis and by increases in activity of adenosine 5'-monophosphate-activated protein kinase (AMPK). Because p38 mitogen-activated protein kinase (MAPK) may regulate glucose metabolism and may be activated downstream of AMPK, this study determined the effects of the p38 MAPK inhibitors SB202190 and SB203580 on adenosine-induced alterations in glucose utilization and AMPK activity. Studies were performed in working rat hearts perfused aerobically following stressing by transient ischemia (2 x 10-min ischemia followed by 5-min reperfusion). Phosphorylation of AMPK and p38 MAPK each were increased fourfold by adenosine, and these effects were inhibited by either SB202190 or SB203580. Neither of these inhibitors directly affected AMPK activity. Attenuation of the adenosine-induced increase in AMPK and p38 MAPK phosphorylation by SB202190 and SB203580 occurred independently of any change in tissue ATP-to-AMP ratio and did not alter glucose uptake, but it was accompanied by an increase in glycogen synthesis and glycogen content and by inhibition of glycolysis and proton production. There was a significant inverse correlation between the rate of glycogen synthesis and AMPK activity and between AMPK activity and glycogen content. These data demonstrate that AMPK is likely downstream of p38 MAPK in mediating the effects of adenosine on glucose utilization in hearts stressed by transient ischemia. The ability of p38 MAPK inhibitors to relieve the inhibition of glycogen synthesis and to inhibit glycolysis and proton production suggests that these agents may restore adenosine-induced cardioprotection in stressed hearts.     

p38 mitogen-activated protein kinase and c-jun-NH2-terminal kinase regulate RANTES production by influenza virus-infected human bronchial epithelial cells

Airway epithelial cells which are the initial site of influenza virus (IV) infection are suggested to participate in airway inflammatory response by expressing various cytokines including RANTES; however, the intracellular signal that regulates RANTES expression has not been determined. In the present study, we examined the role of p38 mitogen-activated protein (MAP) kinase, extracellular signal-regulated kinase (Erk), and c-Jun-NH2-terminal kinase (JNK) in RANTES production by IV-infected human bronchial epithelial cells. The results showed that IV infection induced increases in p38 MAP kinase, and Erk and JNK phosphorylation and activity. SB 203580, PD 98059, and CEP-1347 attenuated IV-infection induced p38 MAP kinase activity, Erk activity, and JNK activity, respectively. SB 203580 and CEP-1347 attenuated RANTES production by 45.3% and 45.2%, respectively, but a combination of these inhibitors additively attenuated by 69.1%. In contrast, PD 98059 did not attenuate. Anti-IL-1alpha mAb, anti-IL-1beta mAb, anti-TNF-alpha mAb, anti-IL-8 mAb, anti-IFN-beta mAb, anti-RANTES mAb, and a combination of these mAbs did not affect IV infection-induced increases in p38 MAP kinase, Erk, and JNK phosphorylation, indicating that each cytokine neutralized by corresponding Ab was not involved in IV infection-induced phosphorylation of MAP kinases. N-acetylcysteine (NAC) did not affect IV infection-induced increases in MAP kinase phosphorylation, whereas NAC attenuated RANTES production by 18.2%, indicating that reactive oxygen species may act as a second messenger leading to RANTES production via p38 MAP kinase- and JNK-independent pathway. These results indicate that p38 MAP kinase and JNK, at least in part, regulate RANTES production by bronchial epithelial cells.     

The involvement of p38 mitogen-activated protein kinase in the alpha-melanocyte stimulating hormone (alpha-MSH)-induced melanogenic and anti-proliferative effects in B16 murine melanoma cells

Activation of p38 or p44/42 mitogen-activated protein (MAP) kinases has been shown to trigger differentiation in a number of cell types. The present study has investigated the roles of these kinases in the alpha-melanocyte stimulating hormone (alpha-MSH)-induced melanogenic and proliferative responses in B16 melanoma cells. Treatment of cells with alpha-MSH led to the time-dependent phosphorylation of both p38 and p44/42 MAP kinases. However, only inhibition of p38 MAP kinase activity with SB 203580 blocked both the alpha-MSH-induced melanogenic and anti-proliferative effects. It therefore appears that activation of the p38 pathway can promote melanogenesis and inhibit growth of B16 melanoma cells.     

Specific induction of heat shock protein 27 by glucocorticoid in osteoblasts

It is generally recognized that osteoporosis is a common complication of patients with glucocorticoid excess and that glucocorticoid receptor is associated with heat shock protein (HSP) 70 and HSP90 in a heterocomplex. In the present study, we investigated whether glucocorticoid induces HSP27, HSP70, and HSP90 in osteoblast-like MC3T3-E1 cells. Dexamethasone time-dependently increased the levels of HSP27, while having no effect on the levels of HSP70 or HSP90. The effect of dexamethasone was dose-dependent in the range between 0.1 nM and 0.1 microM. Dexamethasone induced an increase of the levels of mRNA for HSP27. Dexamethasone induced the phosphorylation of p38 mitogen-activated protein (MAP) kinase. SB203580 and PD169316, inhibitors of p38 MAP kinase, suppressed the HSP27 accumulation by dexamethasone. In addition, SB203580 reduced the dexamethasone-stimulated increase of the mRNA levels for HSP27. The dexamethasone-induced phosphorylation of p38 MAP kinase was reduced by SB203580. These results strongly suggest that glucocorticoid stimulates the induction of neither HSP70 nor HSP90, but HSP27 in osteoblasts, and that p38 MAP kinase is involved in the induction of HSP27.     

Involvement of p38 mitogen-activated protein kinase in basic fibroblast growth factor-induced interleukin-6 synthesis in osteoblasts.

We previously showed that basic fibroblast growth factor (bFGF)-induced activation of protein kinase C (PKC) via phosphatidylinositol-hydrolyzing phospholipase C and phosphatidylcholine-hydrolyzing phospholipase D suppresses interleukin-6 (IL-6) synthesis by bFGF itself in osteoblast-like MC3T3-E1 cells. In the present study, we further investigated the mechanism underlying the bFGF-induced IL-6 synthesis in MC3T3-E1 cells. bFGF time-dependently induced the phosphorylation of p38 mitogen-activated protein (MAP) kinase. SB203580, a specific inhibitor of p38 MAP kinase, suppressed the bFGF-induced IL-6 synthesis dose-dependently. The phosphorylation of p38 MAP kinase by bFGF was suppressed by TMB-8, an inhibitor of intracellular Ca(2+) mobilization, or the depletion of extracellular Ca(2+) with EGTA. A23187, a Ca-ionophore, stimulated the phosphorylation of p38 MAP kinase. SB203580 inhibited the A23187-induced synthesis of IL-6. 1-Oleoyl-2-acetyl-sn-glycerol, a synthetic diacylglycerol activating PKC, reduced the bFGF-induced IL-6 synthesis. 12-O-Tetradecanoylphorbol-13-acetate, an activator of PKC, attenuated the phosphorylation of p38 MAP kinase by bFGF, but did not affect the A23187-induced phosphorylation. These results strongly suggest that bFGF-induced IL-6 synthesis is mediated via p38 MAP kinase activation in osteoblasts, and that PKC acts at a point upstream from p38 MAP kinase.     

MAP kinases, phosphatidylinositol 3-kinase, and p70 S6 kinase mediate the mitogenic response of human endothelial cells to vascular endothelial growth factor

Although the significance of vascular endothelial growth factor (VEGF) and its receptors in angiogenesis is well established, the signal transduction cascades activated by VEGF and their involvement in mediating the mitogenic response of endothelial cells to VEGF are incompletely characterized. Here we demonstrate that VEGF activates mitogen-activated protein (MAP) kinases, including the extracellular signal-regulated protein kinase (ERK) and p38 MAP kinase, phosphatidylinositol 3-kinase (PI 3-kinase), and p70 S6 kinase in human umbilical vein endothelial cells (HUVEC). The activation of these enzymes was assayed by kinase phosphorylation and by kinase activity towards substrates. Studies with PI 3-kinase inhibitors revealed that activation of p70 S6 kinase was mediated by PI 3-kinase. Selective inhibition of ERK, PI 3-kinase, and p70 S6 kinase with the inhibitors PD098059, LY294002, and rapamycin, respectively, inhibited VEGF-stimulated HUVEC proliferation. In marked contrast, the p38 MAP kinase inhibitor SB203580 not only failed to inhibit but actually enhanced HUVEC proliferation; this effect was associated with the phosphorylation of Rb protein. Rb phosphorylation resulted from a decrease in the level of the cdk inhibitor p27KiP1. These results indicate that the activities of ERK, PI 3-kinase, and p70 S6 kinase are essential for VEGF-induced HUVEC proliferation. p38 MAP kinase suppresses endothelial cell proliferation by regulating cell-cycle progression.     

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.     

Mechanism of prostaglandin D(2)-stimulated heat shock protein 27 induction in osteoblasts

We previously showed that prostaglandin D(2) (PGD(2)) stimulates activation of protein kinase C (PKC). We investigated whether PGD(2) stimulates the induction of heat shock protein (HSP) 27 and HSP70 in osteoblast-like MC3T3-E1 cells and the mechanism underlying the induction. PGD(2) increased the levels of HSP27 while having little effect on HSP70 levels. PGD(2) stimulated the accumulation of HSP27 dose dependently in the range between 10 nM and 10 microM. PGD(2) induced an increase in the levels of mRNA for HSP27. The PGD(2)-stimulated accumulation of HSP27 was reduced by staurosporine or calphostin C, inhibitors of PKC. PGD(2) induced the phosphorylation of p44/p42 mitogen-activated protein (MAP) kinase and p38 MAP kinase. The HSP27 accumulation induced by PGD(2) was significantly suppressed by PD98059, an inhibitor of the upstream kinase of p44/p42 MAP kinase, or SB203580, an inhibitor of p38 MAP kinase. Calphostin C suppressed the PGD(2)-induced phosphorylation of p44/p42 MAP kinase and p38 MAP kinase. PD98059 or SB203580 suppressed the PGD(2)-increased levels of mRNA for HSP27. These results strongly suggest that PGD(2) stimulates HSP27 induction through p44/p42 MAP kinase activation and p38 MAP kinase activation in osteoblasts and that PKC acts at a point upstream from both the MAP kinases.     

The mitogen-activated protein kinase p38 is necesssary for interleukin 1beta-induced monocyte chemoattractant protein 1 expression by human mesangial cells

Mitogen-activated protein (MAP) kinases have been suggested as potential mediators for interleukin 1beta (IL-1beta)-induced gene activation. This study investigated the role of the MAP kinases p38 and ERK2 in IL-1beta-mediated expression of the chemokine MCP-1 by human mesangial cells. Phosphorylation of p38 kinase, which is necessary for activation, increased significantly after IL-1beta treatment. p38 kinase immunoprecipitated from IL-1beta-treated cells phosphorylated target substrates to a greater extent than p38 kinase from controls. SB 203580, a selective p38 kinase inhibitor, was used to examine the role of p38 kinase in MCP-1 expression. SB 203580 decreased IL-1beta-induced MCP-1 mRNA and protein levels, but did not affect MCP-1 mRNA stability. Because NF-kappaB is necessary for MCP-1 gene expression, the effect of p38 kinase inhibition on IL-1beta induction of NF-kappaB was measured. SB 203580 (up to 25 microM) had no effect on IL-1beta-induced NF-kappaB nuclear translocation or DNA binding activity. Our previous work showed that IL-1beta also activates the MAP kinase ERK2 in human mesangial cells. PD 098059, a selective inhibitor of the ERK activating kinase MEK1, had no effect on IL-1beta-induced MCP-1 mRNA or protein levels, or on IL-1beta activation of NF-kappaB. These data indicate that p38 kinase is necessary for the induction of MCP-1 expression by IL-1beta, but is not involved at the level of cytoplasmic activation of NF-kappaB. In contrast, ERK2 does not mediate IL-1beta induced MCP-1 gene expression.