Stress-induced platelet-activating factor synthesis in human neutrophils

Platelet-activating factor (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine; PAF) is a potent inflammatory mediator produced by cells in response to physical or chemical stress. The mechanisms linking cell injury to PAF synthesis are unknown. We used liquid chromatography-tandem mass spectrometry to investigate stress-induced PAF synthesis in human neutrophils. PAF synthesis induced by extracellular pH 5.4 correlated with the activation of a stress-activated kinase, p38 mitogen-activated protein kinase (MAPK), and was blocked by the p38 MAPK inhibitor SB 203580. A key enzyme of PAF synthesis, acetyl-CoA:lysoPAF acetyltransferase, which we have previously shown is a target of p38 MAPK, was also activated in an SB 203580-sensitive fashion. Another MAPK pathway, extracellular signal-regulated kinase-1/2 (ERK-1/2), was also activated. Surprisingly, the pharmacological blockade of the ERK-1/2 pathway with PD 98059 did not block, but rather enhanced, PAF accumulation. Two unexpected actions of PD 98059 may underlie this phenomenon: an augmentation of stress-induced p38 MAPK phosphorylation and an inhibition of PAF catabolism. The latter effect did not appear to be due to a direct inhibition of PAF acetylhydrolase. Finally, similar results were obtained using another form of cellular stress, hypertonic sodium chloride. These data are consistent with a model in which stress-induced PAF accumulation is regulated positively by p38 MAPK and negatively by ERK-1/2. Such a model contrasts with the PAF accumulation induced by other forms of stimulation, which we and others have found is up-regulated by both p38 MAPK and ERK-1/2.     

Involvement of MAP kinases in TGF-beta-stimulated vascular endothelial growth factor synthesis in osteoblasts

Transforming growth factor-beta (TGF-beta) reportedly induces vascular endothelial growth factor (VEGF) synthesis in osteoblast-like MC3T3-E1 cells. We have recently shown that TGF-beta activates p44/p42 mitogen-activated protein (MAP) kinase and p38 MAP kinase in these cells. In the present study, we investigated the exact mechanism of TGF-beta behind the synthesis of VEGF in MC3T3-E1 cells. PD98059 and U-0126, specific inhibitors of MEK, suppressed the VEGF synthesis induced by TGF-beta. U-0126 inhibited the TGF-beta-induced p44/p42 MAP kinase phosphorylation. SB203580 and PD169316, inhibitors of p38 MAP kinase, reduced the TGF-beta-stimulated VEGF synthesis. SB202474, a negative control for p38 MAP kinase inhibitor, did not affect the VEGF synthesis. A combination with PD98059 and SB203580 almost completely suppressed the TGF-beta-induced VEGF synthesis. Retinoic acid, which alone failed to affect VEGF synthesis, markedly enhanced the VEGF synthesis stimulated by TGF-beta. Retinoic acid enhanced the TGF-beta-increased levels of VEGF mRNA. The amplifications by retinoic acid of TGF-beta-increased VEGF synthesis and levels of VEGF mRNA were reduced by PD98059 or SB203580. The combination of PD98059 and SB203580 almost completely suppressed the enhancement by retinoic acid of VEGF synthesis induced by TGF-beta. Taken together, our results strongly suggest that both p44/p42 MAP kinase and p38 MAP kinase take part in TGF-beta-stimulated VEGF synthesis in osteoblasts, and that retinoic acid upregulates the VEGF synthesis.     

Basal cPLA(2) phosphorylation is sufficient for Ca(2+)-induced full activation of cPLA(2) in A549 epithelial cells

The release of [(3)H] arachidonic acid (AA) and its connection with the triggering of the MAP kinase cascade were studied in the human A549 epithelial cell line upon stimulation with thapsigargin. Thapsigargin can increase AA release along with the increase of intracellular calcium concentration, phosphorylation, and activation of extracellular regulated kinase (ERK) and cytosolic phospholipase A(2) (cPLA(2)). Both ERK and cPLA(2) phosphorylation in response to thapsigargin were inhibited by PD 98059, a specific inhibitor of MAP kinase kinase of the ERK group (MEK), and EGTA. cPLA(2) phosphorylation was not affected by Ro 31-8220 (an inhibitor of all PKC isoforms) or LY 379196 (a PKCbeta selective inhibitor), while both of them indeed attenuated ERK activation. On the other hand, rottlerin (the selective PKCdelta inhibitor), SB 203580 (the selective p38 MAPK inhibitor), and wortmannin (the PI 3-kinase inhibitor) can affect neither cPLA(2) nor ERK phosphorylation. In A549 cells, PKC activator PMA cannot increase either the basal or thapsigargin-induced (3)H-AA release, while it can induce the phosphorylation of ERK and cPLA(2.) The PMA-induced ERK phosphorylation was inhibited by Ro 31-8220, LY 379196, rottlerin, and PD 98059, but unaffected by SB 203580 and wortmannin. Moreover, the phosphorylation by PMA was non-additive with that of thapsigargin. This implies that intracellular Ca(2+) level is the key factor for induction of cPLA(2) activity and thapsigargin-elicited ERK activation itself is substantially sufficient for cPLA(2) activation upon intracellular Ca(2+) increase.     

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.     

Activation of arachidonate release and cytosolic phospholipase A2 via extracellular signal-regulated kinase and p38 mitogen-activated protein kinase in macrophages stimulated by bacteria or zymosan

The mitogen-activated protein kinases (MAP kinases), extracellular signal-regulated kinase (ERK) and p38, can both contribute to the activation of cytosolic phospholipase A2 (cPLA2). We have investigated the hypothesis that ERK and p38 together or independent of one another play roles in the regulation of cPLA2 in macrophages responding to the oral bacterium Prevotella intermedia or zymosan. Stimulation with bacteria or zymosan beads caused arachidonate release and enhanced in vitro cPLA2 activity of cell lysate by 1.5- and 1.7-fold, respectively, as well as activation of ERK and p38. The specific inhibitor of MAP kinase kinase, PD 98059, and the inhibitor of p38, SB 203580, both partially inhibited cPLA2 activation and arachidonate release induced by bacteria and zymosan. Together, the two inhibitors had additive effects and completely blocked cPLA2 activation and arachidonate release. The present results demonstrate that ERK and p38 both have important roles in the regulation of cPLA2 and together account for its activation in P. intermedia and zymosan-stimulated mouse macrophages.     

MEK-1/2 inhibition prevents 5-lipoxygenase translocation in N-formylpeptide-challenged human neutrophils

In order to elucidate the role of mitogen-activated protein kinase kinase (MEK-1/2) in 5-lipoxygenase (5-LO) activation we studied the N-formyl-methionyl-leucyl-phenylalanine (fMLP)-induced 5-LO translocation in human blood neutrophils (PMNs). In non-primed, Ca(2+)-repleted PMNs, fMLP consistently stimulated MEK-1/2 phosphorylation, but induced 5-LO translocation and product formation (430+/-128 pmol; SEM, n=13) only in 13 of 18 PMN preparations from different healthy donors. In fMLP-responsive cells, the MEK-1/2 inhibitor PD098059 (50 microM) attenuated MEK phosphorylation and abolished 5-LO activation at the translocation step. The fMLP-mediated 5-LO product formation was also sensitive to MEK inhibition by U0126 and to p38 inhibition by SB203580. But in contrast to PD098059, U0126 at 10 microM and SB203580 at 20-50 microM impaired 5-LO activity in the cell-free assay setting, suggesting direct actions of higher concentrations of U0126 and SB203580 on 5-LO apart from MEK and p38 inhibition, respectively. These data show that fMLP initiates 5-LO product formation in non-primed, Ca(2+)-repleted human blood PMNs from healthy donors, and that MEK signaling is pivotal, but not sufficient for 5-LO activation in response to the receptor agonist fMLP.     

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.     

Upregulation by retinoic acid of transforming growth factor-beta-stimulated heat shock protein 27 induction in osteoblasts: involvement of mitogen-activated protein kinases

We investigated whether transforming growth factor-beta (TGF-beta) stimulates the induction of heat shock protein (HSP) 27 and HSP70 in osteoblast-like MC3T3-E1 cells and the mechanism underlying the induction. TGF-beta increased the level of HSP27 but had no effect on the HSP70 level. TGF-beta stimulated the accumulation of HSP27 dose-dependently, and induced an increase in the level of mRNA for HSP27. TGF-beta induced the phosphorylation of p44/p42 mitogen-activated protein (MAP) kinase and p38 MAP kinase. The HSP27 accumulation induced by TGF-beta was significantly suppressed by PD98059, an inhibitor of the upstream kinase of p44/p42 MAP kinase, or SB203580, an inhibitor of p38 MAP kinase. PD98059 and SB203580 suppressed the TGF-beta-stimulated increase in the level of mRNA for HSP27. Retinoic acid, a vitamin A (retinol) metabolite, which alone had little effect on the HSP27 level, markedly enhanced the HSP27 accumulation stimulated by TGF-beta. Retinoic acid enhanced the TGF-beta-induced increase of mRNA for HSP27. The amplification of TGF-beta-stimulated HSP27 accumulation by retinoic acid was reduced by PD98059 or SB203580. Retinoic acid failed to affect the TGF-beta-induced phosphorylation of p44/p42 MAP kinase or p38 MAP kinase. These results strongly suggest that p44/p42 MAP kinase and p38 MAP kinase take part in the pathways of the TGF-beta-stimulated HSP27 induction in osteoblasts, and that retinoic acid upregulates the TGF-beta-stimulated HSP27 induction at a point downstream from p44/p42 MAP kinase and p38 MAP kinase.     

Role of mitogen-activated protein kinase-mediated cytosolic phospholipase A2 activation in arachidonic acid metabolism in human eosinophils.

The objective of this investigation was to determine the role of secretory and cytosolic isoforms of phospholipase A(2) (PLA(2)) in the induction of arachidonic acid (AA) and leukotriene synthesis in human eosinophils and the mechanism of PLA(2) activation by mitogen-activated protein kinase (MAPK) isoforms in this process. Pharmacological activation of eosinophils with fMLP caused increased AA release in a concentration (EC(50) = 8.5 nM)- and time-dependent (t(1/2) = 3.5 min) manner. Both fMLP-induced AA release and leukotriene C(4) (LTC(4)) secretion were inhibited concentration dependently by arachidonic trifluoromethyl ketone, a cytosolic PLA(2) (cPLA(2)) inhibitor; however, inhibition of neither the 14-kDa secretory phospholipase A(2) by 3-(3-acetamide-1-benzyl-2-ethylindolyl-5-oxy)propanephosphonic acid nor cytosolic Ca(2+)-independent phospholipase A(2) inhibition by bromoenol lactone blocked hydrolysis of AA or subsequent leukotriene synthesis. Pretreatment of eosinophils with a mitogen-activated protein/extracellular signal-regulated protein kinase (ERK) kinase inhibitor, U0126, or a p38 MAPK inhibitor, SB203580, suppressed both AA production and LTC(4) release. fMLP induced phosphorylation of MAPK isoforms, ERK1/2 and p38, which were evident after 30 s, maximal at 1-5 min, and declined thereafter. fMLP stimulation also increased cPLA(2) activity in eosinophils, which was inhibited completely by 30 microM arachidonic trifluoromethyl ketone. Preincubation of eosinophils with U0126 or SB203580 blocked fMLP-enhanced cPLA(2) activity. Furthermore, inhibition of Ras, an upstream GTP-binding protein of ERK, also suppressed fMLP-stimulated AA release. These findings demonstrate that cPLA(2) activation causes AA hydrolysis and LTC(4) secretion. We also find that cPLA(2) activation caused by fMLP occurs subsequent to and is dependent upon ERK1/2 and p38 MAPK activation. Other PLA(2) isoforms native to human eosinophils possess no significant activity in the stimulated production of AA or LTC(4).     

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.     

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.     

Ligation of the alpha2M* signaling receptor regulates synthesis of cytosolic phospholipase A2

We have studied the regulation of cytosolic phospholipase A2 (cPLA2) synthesis in macrophages stimulated with receptor-recognized forms of alpha2-macroglobulin (alpha2M*). [35S]methionine-labeled cells were stimulated with alpha2M* and [35S]cPLA2 was immunoprecipitated with a monoclonal antibody directed against cPLA2. The precipitates were electrophoresed, immunoblotted, cPLA2 detected by Enhanced Chemifluorescence, and its radioactivity determined. Stimulation of cells with alpha2M* caused a two- to threefold increase in cPLA2 synthesis compared to buffer-treated cells which was consistently maximal at 200 pM of alpha2M*. Actinomycin D or cycloheximide treatment of cells drastically reduced alpha2M*-induced cPLA2 synthesis. Likewise, inhibition of protein kinase C with chelerythrin, farnesyl transferase with manumycin A, MEK kinase with U0126, Erk1/2 kinases with PD98059, p38MAPK with SB203580, PI 3-kinase with wortmannin or LY294002, p70s6k with rapamycin, or depletion of [Ca2+]i with either BAPTA/AM or EGTA drastically reduced alpha2M* induction of cPLA2. Inhibition of NFKB activation with BAY11-7182 or PGA1 also abolished alpha2M* induction of cPLA2. We conclude that alpha2M*-induced cPLA2 synthesis is controlled by [Ca2+]i levels, tyrosine kinase activity, the p21ras-dependent MAPK and PI 3-kinase downstream signaling pathways, and regulation of NFkappaB.     

Evidence implicating a mid-region sequence of IGFBP-3 in its specific IGF-independent actions

In the present study, we investigated whether the mitogen-activated protein (MAP) kinase superfamily is involved in the bone morphogenetic protein (BMP)-4-stimulated synthesis of osteocalcin in osteoblast-like MC3T3-E1 cells. BMP-4 dose-dependently stimulated osteocalcin synthesis. BMP-4 markedly induced the phosphorylation of p44/p42 MAP kinase and p38 MAP kinase, while having little effect on SAPK (stress-activated protein kinase)/JNK (c-Jun N terminal kinase) phosphorylation. SB203580 and PD169316, specific inhibitors of p38 MAP kinase, significantly reduced the osteocalcin synthesis stimulated by BMP-4. In contrast, PD98059 and U0126, inhibitors of upstream kinase of p44/p42 MAP kinase, markedly enhanced the BMP-4-stimulated osteocalcin synthesis. The BMP-4-induced phosphorylation of p44/p42 MAP kinase was suppressed by PD98059, which did not, however, affect the BMP-4-induced phosphorylation of p38 MAP kinase. Taken together, our results strongly suggest that p38 MAP kinase takes part in BMP-4-stimulated osteocalcin synthesis as a positive regulator in osteoblasts, whereas p44/p42 MAP kinase acts as a negative regulator in the synthesis.     

Contribution of mitogen-activated protein kinase to stimulation of phospholipase D by the chemotactic peptide fMet-Leu-Phe in human neutrophils.

Phospholipase D (PLD) plays an important role in signaling through phosphatidylcholine (PC) and in the production of superoxide (respiratory burst) by polymorphonuclear leukocytes (PMN) stimulated by the chemoattractant fMet-Leu-Phe (fMLP). However, the regulation of PLD activity by protein kinases is not fully understood. In the present study, we have used a mitogen-activated protein (MAP) kinase inhibitor (PD 98059) to investigate a possible connection between extracellular signal-regulated kinase (ERK) and PLD activity and respiratory burst. Using a range of concentrations (3-20 microM) which inhibit ERK activity, PD 98059 inhibited PLD activity induced by fMLP in cytochalasin B-primed PMN, as assessed by production-tritiated phosphatidylethanol (PEt), phosphatidic acid (PA), and hydrolysis of PC. However, the inhibition was partial (approximately 50%), while inhibition of PC hydrolysis was almost complete, suggesting a concomitant inhibition of PLA2 activity. In addition, PD 98059 reduced fMLP-induced respiratory burst by 50%, an effect which was correlated with PLD inhibition of PLD (r = 0.981, P < 0.01), and neither did PD 98059 inhibit the PLD activity and respiratory burst induced by PKC upon its direct activation by phorbol myristate acetate. These data provide the first evidence for implication of the ERK cascade in the stimulation of PLD through Gi signaling. They further indicate that PLD stimulation by fMLP receptors occurs through two pathways, dependent and independent on MAP kinase, the former pathway being linked to superoxide production.     

Endothelin-1 induces vascular endothelial growth factor synthesis in osteoblasts: involvement of p38 mitogen-activated protein kinase

We previously reported that endothelin-1 (ET-1) stimulates p44/p42 mitogen-activated protein (MAP) kinase and p38 MAP kinase in osteoblast-like MC3T3-E1 cells. In the present study, we investigated the effect of ET-1 on the synthesis of vascular endothelial growth factor (VEGF) in these cells. ET-1 significantly stimulated VEGF secretion time-dependently 18 hours after the stimulation. The stimulatory effect was dose-dependent in the range between 0.1 nM and 0.1 micro;M. BQ123, an antagonist of endothelin(A) (ET(A)) receptor, inhibited the ET-1-induced VEGF secretion. The ET-1-induced VEGF secretion was suppressed by SB203580 and PD169316, inhibitors of p38 MAP kinase, but not PD98059, an inhibitor of the upstream kinase that activates p44/p42 MAP kinase. 12-O-Tetradecanoylphorbol-13-acetate (TPA), a protein kinase C (PKC)-activating phorbol ester, stimulated VEGF secretion. Calphostin C, a specific PKC inhibitor, suppressed the VEGF secretion by ET-1. TPA-induced VEGF secretion was suppressed by SB203580. Taken together, our results strongly suggest that ET-1 stimulates VEGF synthesis via ET(A) receptor in osteoblasts and that p38 MAP kinase is involved at a point downstream from PKC in the VEGF synthesis.     

Interference by leptin with Helicobacter pylori lipopolysaccharide-induced cytosolic phospholipase A2 activation in gastric mucosal cells.

Activation of cytosolic phospholipase A(2) (cPLA(2)) by bacterial LPS for the rapid release of arachidonic acid from membrane phospholipids is considered a key step in the generation of platelet-activating factor (PAF), recognized as the most proximal mediator of inflammatory events triggered by bacterial infection. In this study, we report on the role of leptin in modulation of the detrimental consequences of H. pylori LPS-induced cPLA(2) activation that result in the disturbances in gastric mucin synthesis. Employing gastric mucosal cells labeled with [(3)H] arachidonic acid, we show that H. pylori LPS-induced cPLA(2) activation, associated with up-regulation in apoptosis and PAF generation, and the impairment in gastric mucin synthesis, was subject to a dose-dependent suppression by leptin, as well as the inhibition by MAFP, a specific inhibitor of cPLA(2). A potentiation in the countering capacity of leptin on the LPS-induced up-regulation in apoptosis, arachidonic acid release and PAF generation was attained in the presence of ERK inhibitor, PD98059, while PI3K inhibitor, wortmannin had no effect. On the other hand, the prevention by leptin of the LPS detrimental effect on mucin synthesis was subject to suppression by wortmannin, an inhibitor of PI3K as well as the inhibitor of ERK, PD98059. Moreover, potentiation in the effect of leptin on the LPS-induced decrease in mucin synthesis was attained with cPLA(2) inhibitor, MAFP as well as PAF receptor antagonist, BN52020. The results of our findings point to H. pylori LPS-induced ERK-dependent cPLA(2) activation as a critical factor influencing the level of PAF generation, and hence the extent of pathological consequences of H. pylori infection on the synthesis of gastric mucin. Furthermore, we show that leptin counters the pathological consequences of H. pylori-induced cPLA(2) activation on gastric mucin synthesis through the involvement in signaling events controlled by MAPK/ERK and PI3K pathways.     

SAPK/JNK plays a role in transforming growth factor-beta-induced VEGF synthesis in osteoblasts.

We previously reported that transforming growth factor-beta (TGF-beta) activates p44/p42 mitogen-activated protein (MAP) kinase and p38 MAP kinase, resulting in the stimulation of vascular endothelial growth factor (VEGF) synthesis in osteoblast-like MC3T3-E1 cells. In the present study, we investigated the involvement of stress-activated protein kinase/c- Jun N-terminal kinase (SAPK/JNK), another member of the MAP kinase superfamily, in TGF-beta-induced VEGF synthesis in these cells. TGF-beta markedly induced SAPK/JNK phosphorylation. SP600125, a specific inhibitor of SAPK/JNK, markedly reduced TGF-beta-induced VEGF synthesis. SP600125 suppressed TGF-beta-induced SAPK/JNK phosphorylation. PD98059, an inhibitor of upstream kinase of p44/p42 MAP kinase and SB203580, an inhibitor of p38 MAP kinase, each failed to reduce TGF-beta-induced SAPK/JNK phosphorylation. A combination of SP600125 and PD98059 or SP600125 and SB203580 suppressed TGF-beta-stimulated VEGF synthesis in an additive manner. These results strongly suggest that TGF-beta activates SAPK/JNK in osteoblasts, and that SAPK/JNK plays a role in addition to p42/p44 MAP kinase and p38 MAP kinase in TGF-beta-induced VEGF synthesis.     

Sphingosine 1-Phosphate Regulates Heat Shock Protein 27 Induction by a p38 MAP Kinase-Dependent Mechanism in Aortic Smooth Muscle Cells

In an aortic smooth muscle cell line, A10 cells, we investigated the effect of sphingosine 1-phosphate on the induction of heat shock protein 27 (HSP27), a low-molecular-weight heat shock protein. Sphingosine 1-phosphate significantly induced the accumulation of HSP27 in a pertussis toxin-sensitive manner. The effect was dose-dependent in the range between 0.1 and 30 microM. Sphingosine 1-phosphate stimulated an increase in the levels of mRNA for HSP27. Sphingosine 1-phosphate stimulated both p42/p44 mitogen-activated protein (MAP) kinase and p38 MAP kinase activation. PD98059, an inhibitor of the upstream kinase that activates p42/p44 MAP kinase, did not affect sphingosine 1-phosphate-stimulated HSP27 induction. In contrast, SB203580, an inhibitor of p38 MAP kinase, reduced sphingosine 1-phosphate-induced HSP27 induction. SB203580 reduced the levels of mRNA for HSP27 induced by sphingosine 1-phosphate. These results indicate that sphingosine 1-phosphate stimulates the induction of HSP27 via p38 MAP kinase activation in aortic smooth muscle cells.