Activation of plasminogen activator inhibitor-1 synthesis by phorbol esters in human promyelocyte HL-60--roles of PCKbeta and MAPK p42

HL-60 cells treated by PMA develop the monocyte adherent phenotype and synthesize plasminogen activator inhibitor type-1 (PAI-1). We focused our study on the identification of the PMA-activated protein kinase C (PKC) isoform and its downstream transduction pathway activating PAI-1 synthesis. Acquisition of the monocytic phenotype was evidenced by cell adherence (90-95%) and a sharp increase of CD 36 and receptor for urokinase plasminogen activator (uPAR) surface expression. Ro 31-8220, a specific inhibitor of PKC, prevented PMA-induced PAI-1 synthesis (mRNA and protein levels) and cell adhesion. To identify the PKC isoform, we took advantage of the HL-525 cell line, an HL-60 cell variant deficient in PKCbeta gene expression. This defect prevents PMA to induce the differentiation process. HL-525 stimulated by PMA did not synthesize PAI-1 nor become adherent. However, in HL-525 cells either pretreated by retinoic acid that reinduces PKCbeta gene expression or transfected with PKCbeta cDNA, PMA significantly activated PAI-1 synthesis and adhesion of cells. Immunoblotting of active Mitogen Activated Protein Kinase (MAPK) p42/p44 in HL-60 cells showed a preferential and sustained activation of the p42 isoform by PMA over the p44 isoform. Ro 31-8220 significantly attenuated this activation. PD 098059 and U0126, both highly specific MEK inhibitors, efficiently prevented PMA-induced PAI-1 synthesis (mRNA and protein levels) and cell adhesion whereas SB203580, a specific inhibitor of stress-activated MAPK p38, did not. Results obtained from HL-60 and HL-525 cells indicate that the PMA-activated transduction pathway of uPAR expression involves a PKC isoform other than PKCbeta. In conclusion, we propose that the pathway PKCbeta-MEK-MAPK p42 is a potential linear route for PAI-1 synthesis leading to morphological changes and adherence linked to PMA-induced differentiation in HL-60 cells.     

Cadmium-induced IL-6 and IL-8 expression and release from astrocytes are mediated by MAPK and NF-κB pathways

Chronic exposure to cadmium has been linked to brain cancers, learning disabilities and memory deficits. Previous studies of cadmium toxicity in the central nervous system report cadmium induces oxidative stress in neurons and astrocytes. In the peripheral system, cadmium promotes interleukin-6 (IL-6) and IL-8 production and release. Elevation of IL-6 expression is linked to the pathogenesis of neurodegenerative diseases and astrogliosis. IL-8 plays a role in angiogenesis of gliomas and neurodegenerative diseases. Herein, the effects of non-toxic concentrations of cadmium on the production of IL-6 and IL-8 and the underlying mechanisms were investigated. U-87 MG human astrocytoma cells and primary human astrocytes were exposed to cadmium chloride. At 24 h post-exposure to 1 and 10 μM, levels of intracellular cadmium in U-87 MG cells were 11.89 ± 3.59 and 53.08 ± 7.59 μg/g wet weight, respectively. These concentrations had minimal effects on cell morphology and viability. IL-6 and IL-8 mRNA levels and secretion increased in dose- and time-dependent manners post cadmium exposure. Acute exposure to cadmium increased phosphorylation of ERK1/2, p38 MAPK, and p65 NF-κB. Pretreatment with U0126–an inhibitor of MEK1 and MEK2 kinases–SB203580–a p38 MAPK inhibitor–and SC-514–an IKKβ inhibitor–suppressed cadmium-induced IL-8 expression and release. Upregulation of cadmium-induced IL-6 was inhibited by U0126 and SC-514, but not SB203580. On the other hand, SP600125–a JNK inhibitor–and celecoxib–a selective COX-2 inhibitor–had no effect on production of both cytokines. In conclusion, non-toxic concentrations of cadmium can stimulate IL-6 and IL-8 release through MAPK phosphorylation and NF-κB activation. Suppressing IL-6 and IL-8 production could be novel approaches to prevent cadmium-induced angiogenesis in gliomas and inflammation in the brain.     

Essential role for ERK mitogen-activated protein kinase in matrix metalloproteinase-9 regulation in rat cortical astrocytes

Matrix metalloproteinases (MMPs) contribute to the pathophysiology of brain injury and inflammation but little is known about their regulatory signaling pathways in brain cells. Here we examine the role of mitogen-activated protein (MAP) kinase pathways in MMP-9 regulation in cortical rat astrocytes. The protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA) induced MMP-9 but not MMP-2 secretion as measured by gelatin zymography. Northern blot and RT-PCR analysis showed that MMP-9 responses occurred at the mRNA level. Although PMA increased phosphorylation in all three major MAP kinase pathways (ERK, p38 MAP kinase, and JNK), only inhibition of the ERK pathway by the MEK/ERK inhibitor U0126 (0.1-10 microM) significantly reduced MMP-9 upregulation, even when treatment was delayed for 4 h after PMA exposure. Inhibitors of p38 MAP kinase (SB203580) and JNK (SP600125) had no effect. This PKC pathway was compared to a cytokine response by exposing astrocytes to TNFalpha, which also activated MAP kinase and induced MMP-9 upregulation. But in this case, all three MAP kinase inhibitors (U0126, SB203580, and SP600125) reduced TNFalpha-induced MMP-9 upregulation. Taken together, these results suggest that the ERK MAP kinase is essential for MMP-9 upregulation via PKC and cytokine pathways in astrocytes.     

Regulation of a Ca2+-activated K+ channel by calcium-sensing receptor involves p38 MAP kinase

By using pharmacological and molecular approaches, we previously showed that the G-protein-coupled, extracellular calcium (Ca2+(o))-sensing receptor (CaR) regulates a large-conductance (approximately 140 pS), Ca(2+)-activated K+ channel [IK(Ca); CAKC] in U87 astrocytoma cells. Here we show that elevated Ca2+(o) stimulates extracellular-signal-regulated kinase (ERK1/2) and p38 MAP kinase (MAPK). The effect of high Ca2+(o) on p38 MAPK but not ERK1/2 is CaR mediated, insofar as transduction with a dominant-negative CaR (R185Q) using recombinant adeno-associated virus (rAAV) attenuated the activation of p38 MAPK but not of ERK1/2. p38 MAPK activation by the CaR is likely to be protein kinase C (PKC) independent, in that the pan-PKC inhibitor GF109203X failed to abolish the high-Ca2+(o)-induced phosphorylation of p38 MAPK. Consistently with our data on the activation of this kinase, we observed that inhibiting p38 MAPK blocked the activation of the CAKC induced by the specific pharmacological CaR activator NPS R-467. In contrast, inhibiting MEK1 only transiently inhibited the activation of this K+ channel by NPS R-467, despite the continued presence of the antagonist. Similarly to the lack of any effect of the PKC inhibitor on the activation of ERK1/2 and p38 MAPK, inhibiting PKC had no effect on NPS R-467-induced activation of this channel. Therefore, our data show that the CaR, acting via p38 MAPK, regulates a large-conductance CAKC in U87 cells, a process that is PKC independent. Large-conductance CAKCs play an important role in the regulation of cellular volume, so our results have important implications for glioma cell volume regulation.     

Memory formation requires p38MAPK activity in the rat hippocampus

Using the specific inhibitor of p38MAPK, SB203580, we show a direct involvement of this protein kinase in short- and long-term memory. When given into the CA1 region of the rat dorsal hippocampus immediately, but not 30 or 120 min after training in a one-trial inhibitory avoidance task, SB203580 blocked short- and long-term memory formation. The SB203580 inactive analog, SB202474, had no effect whatsoever. Learning of the avoidance task was accompanied by an immediate and transient increase in hippocampal p38MAPK phosphorylation. No change in p38MAPK phosphorylation was detected in control animals that only received the electric foot-shock associated with the learning paradigm. Therefore, formation of short and long-term memory for inhibitory avoidance requires p38MAPK activation in the rat hippocampus.     

Transforming growth factor beta receptor family ligands inhibit hepatocyte growth factor synthesis and secretion from astrocytoma cells

Transforming growth factor beta (TGFbeta) and hepatocyte growth factor (HGF) promote glioma progression. Using U87human astrocytoma cells, which express TGFbeta receptors (TbetaRs), we show (1) mRNA expression of Smads (2, 3, 4), bone morphogenetic protein (BMP)- and activin-A receptors; (2) TGFbeta1 inhibits and HGF induces proliferation; (3) TGFbeta1 and activin-A equipotently inhibit HGF secretion more than BMP-2, but none alters c-Met expression. Because interfering with TbetaR signaling might nullify the beneficial inhibition of HGF secretion, activin-A should instead be considered for combination glioma therapy.     

Comparative study of vanadate- and phorbol ester-induced cyclo-oxygenase-2 expression in human endothelial cells

Our previous study showed that vanadate, an inhibitor of protein tyrosine phosphatases, induced the expression of cyclo-oxygenase (COX)-2 in a protein-tyrosine-kinase (PTK)-dependent manner in human umbilical vein endothelial cells (HUVEC). Here, we further compared the actions of vanadate and phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C (PKC), on induction of COX-2 with special reference to mitogen-activated protein kinases (MAPKs) in HUVEC. Vanadate induced activation of three families of MAPKs, extracellular signal-regulated kinases 1 and 2 (ERK1/2), p38, and c-Jun amino-terminal kinase (JNK) 1, while activation of ERK 1/2 alone was induced by PMA. The former activation by vanadate and the latter one by PMA were inhibited by tyrphostin-47, an inhibitor of PTKs, and by Ro31-8220, a PKC inhibitor, respectively. Either tyrphostin-47, PD98059, a specific inhibitor of the upstream kinase toward ERK1/2, or SB203580, a specific inhibitor of p38, completely suppressed vanadate-induction of COX-2 mRNA and protein. On the other hand, PMA-induction of COX-2 mRNA and protein was abolished by Ro31-8220 or PD98059 but not by SB203580. These data indicate that PMA-induced and PKC-dependent expression of COX-2 requires mainly activation of ERK 1/2 among MAPKs, while activation of both ERK1/2 and p38 or possibly of all three families of MAPKs is necessary for vanadate-induced and PTK-dependent expression of COX-2.     

Differential role of PTK,ERK and p38 MAPK in superoxide impairment of NMDA cerebrovasodilation

Previous studies in piglets have shown that the generation of oxygen free radicals (O(-)(2)) following traumatic brain injury and hypoxia/ischemia contribute to the reversal of N-methyl-D-aspartate (NMDA)-induced pial artery dilation to vasoconstriction. This study determined the contribution of protein tyrosine kinase (PTK) and mitogen-activated protein (MAPK) activation to impairment of NMDA cerebrovasodilation by O(-)(2) in piglets equipped with a closed window. Exposure of the cerebral cortex to a xanthine oxidase O(-)(2) generating system (OX) reversed NMDA (10(-8), 10(-6) M) dilation to vasoconstriction but such impairment was partially prevented by the PTK inhibitor, genistein, the MAPK (ERK isoform) inhibitor, U0126, and the MAPK (p38 isoform) inhibitor, SB203580 (9+/-1 and 15+/-1 vs. -1+/-1 and -1+/-1 vs. 5+/-1 and 9+/-1% for sham control, OX and OX in the presence of genistein, respectively). However, the p38 MAPK inhibitor, SB203580, prevented NMDA dilator impairment significantly less than the ERK MAPK inhibitor, U0126. Similar results were obtained for glutamate. These data show that PTK and MAPK activation by the presence of O(-)(2) contributes to the impairment of NMDA dilation. Furthermore, these data indicate a differential role for ERK and p38 MAPK activation in impairment of NMDA dilation by O(-)(2) in the brain.     

Anisomycin does not activate p38MAPK in human platelets.

Human platelets are known to contain three forms of mitogen-activated protein kinases; erk1, erk2, and p38MAPK. However the role(s) of mitogen-activated protein kinase cascades in platelet function remains to be determined. Evidence has been presented that suggests that these kinases are involved in the cytoskeleton and in the activation of phospholipase A2; however, other functions seem likely. The object of the present study was to examine the role of the p38MAPK in platelet function using anisomycin, a reported activator of p38MAPK, and SB203580, an inhibitor of p38MAPK. Thrombin and collagen caused the phosphorylation of p38MAPK and this was inhibited by SB203580. Anisomycin did not cause the aggregation of either intact or saponin-permeabilised platelets. In addition anisomycin failed to produce synergistic aggregation responses with submaximal concentrations of collagen, thrombin, the thromboxane mimetic U46619, or the calcium ionophore A23187. There was no detectable phosphorylation of p38MAPK in either intact platelets or platelet lysates incubated with anisomycin. Anisomycin also failed to modulate p38MAPK phosphorylation in response to submaximal concentrations of collagen, thrombin, U46619, or A23187. In contrast anisomycin did cause p38MAPK phosphorylation in rabbit lung and C3 fibroblasts and in rabbit lung fibroblast lysates. These data demonstrate that anisomycin has no detectable effect on either platelet function or p38MAPK phosphorylation and, therefore, that anisomycin has proven to be an ineffective tool to define the role that p38MAPK plays in platelet function.     

Biglycan,a Nitric Oxide-Downregulated Proteoglycan,Prevents Nitric Oxide-Induced Neuronal Cell Apoptosis via Targeting Erk1/2 and p38 Signaling Pathways

Nitric oxide (NO), a gaseous signaling molecule, induces apoptosis and mediates neurodegenerative diseases and brain injury. Biglycan (BGN), a member of the small leucine-rich proteoglycan family, was demonstrated to exert anti-apoptosis function in various disease models. However, little is known about the effect of BGN on NO-induced neurotoxicity. Here, for the first time, we reported that BGN protects against NO-induced apoptosis in human neuroblastoma SH-EP1 cells. This is supported by the finding that sodium nitroprusside (SNP), a NO donor, triggered downregulation of BGN in SH-EP1 cells, and over-expression of BGN strikingly attenuated NO-induced nuclear fragmentation and apoptosis of neuronal cells. More importantly, BGN remarkably blocked NO-induced phosphorylation of Erk1/2 and p38 signaling, but not JNK MAPK pathway in neuronal cells. Furthermore, inhibiting Erk1/2 by U0126 or p38 by SB203580 partially protected against NO-induced cell death. Conversely, downregulation of BGN by siRNA aggravated NO-induced neuronal cell death, which was not attenuated by U0126 or SB203580. These findings indicated that BGN, downregulated by NO, prevents NO-induced neuronal cell apoptosis via targeting Erk1/2 and p38 signaling pathways. Our results strongly suggest that BGN could be explored for the prevention of NO-induced neurodegenerative disorders.     

Inhibition of caspase-3 activation by SB 203580, p38 mitogen-activated protein kinase inhibitor in nitric oxide-induced apoptosis of PC-12 cells

Mitogen-activated protein kinase (MAPK) p38 plays pivotal role in cell proliferation, differentiation, and apoptosis when cysteine protease caspase induces apoptosis in different cell systems. SB 203580 (4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1 H-imidazole) is widely used as a specific inhibitor of p38 MAPK, and prevents apoptosis induced by various agents. The effect of SB 203580 on nitric oxide(NO)- or peroxynitrite-induced cell death is not known. Western blotting results indicate that p38 MAPK was activated significantly in NO- or peroxynitrite-induced cell death in a time-dependent manner, and subsequently this cell death was markedly inhibited by SB 203580, as determined by fluorescence-activated cell sorting (FACS)-can analyzer. Furthermore, NO/peroxynitrite-induced caspase-3 activation was notably inhibited by SB 203580, however, phosphorylation of either p38 MAPK or p44/42 was not influenced by SB 203580. Thus, it is likely that SB 203580 prevents NO/peroxynitrite-induced cell death by inhibiting caspase-3 activation in PC-12 cells.     

Mediation of BMP7 neuroprotection by MAPK and PKC IN rat primary cortical cultures

We have previously demonstrated that pretreatment with bone morphogenetic protein 7 (BMP7), a trophic factor in the TGFbeta superfamily, reduces ischemia-induced brain infarction induced by middle cerebral artery ligation in rats. Since the mitogen-activated protein kinase (MAPK) pathway is involved in many TGFbeta-mediated responses, we examined the interaction of BMP7 and MAPK in primary cultures obtained from the cerebral cortex of E16-17 rat embryos. Lactate dehydrogenase (LDH) in the media was used as an index of cell death. BMP7 did not alter LDH levels at low concentration (1.25 nM), but exhibited increased cellular toxicity at higher concentration (>12.5 nM). BMP7 at the low concentration significantly attenuated H2O2-induced increases in LDH activity and decreases in neuronal density. Pharmacological interactions were used to examine if MAPK was involved in this response. BMP7-induced protection was antagonized by the p42,44 MAPK kinase inhibitors PD98059 and U0125. The p38 MAPK antagonist SB203580, and their inactive analog SB202474, also attenuated BMP7-induced protection, suggesting that the interaction with p38 MAPK is nonspecific. Previous studies have indicated that SB202474 has inhibitory effects on other protein kinases. We found that the protein kinase C inhibitor chelerythrine antagonized BMP7-induced protection against H2O2. Western blot analysis indicated that BMP7 increased phosphorylation of p42,44 MAPK and PKC. Taken together, our data suggest that BMP7 is neuroprotective at low concentrations in primary cortical cell culture. The protective effects of BMP7 may involve the activation of p42,44 MAPK and PKC.     

脑缺血预处理对沙鼠脑缺血再灌注损伤 p38MAPK活化的作用

目的 探讨脑缺血预处理对脑缺血后p38MAPK信号的影响.方法 蒙古沙鼠分成对照组、脑缺血再灌注组、脑缺血预处理组和抑制剂SB203580组.制备脑缺血及脑缺血预处理动物模型.免疫组织化学法和蛋白质印迹法检测海马区磷酸化p38MAPK的表达,TUNEL 法检测神经细胞凋亡,TTC染色测定脑梗死体积.结果 与对照组比较,脑缺血再灌注组磷酸化p38MAPK表达增高,凋亡神经细胞数量增加(P<0.05),磷酸化p38MAPK阳性细胞与TUNEL阳性细胞为同一神经元.与脑缺血再灌注组比较,脑缺血预处理组凋亡神经细胞下降、磷酸化p38MAPK表达较少、梗死面积缩小(P<0.05);抑制剂SB203580组磷酸化p38MAPK表达水平与脑缺血预处理组相似,但两组神经细胞凋亡数量及脑梗死体积比较,差异有统计学意义(P<0.05).结论 缺血预处理对脑缺血性损伤具有保护作用,其机制并非完全依赖p38MAPK信号活化.

Abstract：

Objective To study the effect of ischemic preconditioning on p38MAPK pathway after ischemia - reperfusion injury in gerbils. Method Gerbils were divided randomly into control group, ischemia - reperfusion group ( I/R ) , ischemia preconditioning group ( IP ) and inhibitor SB203580 group. Transient ischemia - reperfusion model and ischemia preconditioning model were performed. The expression levels of p38MAPK phosphorylation were detected by immunohistochemistry and Western blot, the neurons apoptosis were detected by TUNEL method and the infarction volume assessments were performed by TTC staining. Results Compared with control group, there were higher expression levels of p38MAPK phosphorylation,more apoptotic neurons in I/R group. The p38MAPK phosphorylation \positive cells and TUNEL positive cells were located in the same neurons. Compared with I/R group, there were lower expression levels of p38MAPK phosphorylation,fewer apoptotic neurons and smaller infarction volumes in IP group( P <0.05). The levels of p38MAPK phosphorylation in SB203580 group were similar as those in IP group( P > 0. 05 ) . However, the number of apoptotic neurons and infarction volumes were obviously changed ( P <0.05). Conclusions IP has protective effect from I/R damage in gerbils, which might be not only involved p38MAPK pathway.     

Clozapine and the mitogen-activated protein kinase signal transduction pathway: implications for antipsychotic actions.

BACKGROUND: Mitogen-activated protein kinase (MAPK) signaling pathways respond to dopaminergic and serotonergic agents and mediate short- and long-term effects of intracellular signaling in neurons. Here we show that the antipsychotic agent, clozapine, selectively activates the MEK/ERK MAPK pathway, and inhibition of this pathway reverses clozapine's actions in the conditioned avoidance response (CAR) paradigm, a rodent behavioral assay of antipsychotic activity. METHODS: Phosphorylation patterns of MAPK pathway enzymes were determined by quantitative immunoblot analysis and immunohistochemistry of rat prefrontal cortex. Kinase inhibitors were used to assess the role of MAPK signaling pathways in mediating clozapine-induced suppression of CAR. RESULTS: Clozapine administration selectively increased phosphorylation of MEK1/2 but had no effect on p38 or JNK phosphorylation. Pretreatment with the 5-HT2A agonist (+/-)-2,5-dimethoxy-4-iodoamphetamine hydrochloride blocked the clozapine-induced increase in MEK1/2 phosphorylation. Immunohistochemistry revealed that clozapine treatment elevated the number of cells in the prefrontal cortex positive for phosphoERK, the downstream substrate of MEK1/2. Prior administration of MEK1/2 inhibitors U0126 or Sl327, or ERK inhibitor 5-iodotubercidin, reversed suppression of CAR induced by clozapine, whereas administration of vehicle, JNK or p38 inhibitors (L-JNK-1 and SB203580, respectively) had no effect. Inhibition of kinases upstream to MEK1/2 (PI-3K, PKC, and CaMKII) by administration of LY294002, bisindolylmaleimide, or KN-62, respectively, also reversed clozapine-induced suppression of CAR. CONCLUSIONS: These data support the hypothesis that the MEK/ERK signal transduction cascade participates in clozapine's antipsychotic actions.     

Characterization of the signal transduction pathways mediating noradrenaline-stimulated MAPK activation and c-fos expression in oligodendrocyte progenitors

In examining the signaling transduction pathway of adrenoceptors in oligodendrocyte progenitors, we have found that stimulation of alpha(1)-adrenoceptors with norepinephrine (NE), in the presence of 3 microM propranolol, increased the activity of mitogen-activated protein kinases (MAPKs). This stimulation was concentration- and time-dependent, with maximal response after 10 min of exposure to 10 microM NE. Pertussis toxin (PTX) blocked NE-mediated MAPK activation, suggesting that alpha(1)-adrenoceptor activates MAPK through a PTX-sensitive G-protein. In the presence of U73122, an inhibitor of phospholipase C (PLC), MAPK activation was blocked. In oligodendrocyte progenitor cultures, chronic treatment with phorbol-12-myristate-13-acetate (PMA) down-regulated protein kinase C (PKC) and blocked NE-mediated MAPK activation. The response to NE was also significantly decreased by the PKC inhibitors H7 and bisindolylmaleimide GF109203X. Similarly, the effect of NE on MAPK activation was not observed in a calcium-free medium. Furthermore, attenuation of MAPK activity was observed when cultures were pretreated with LY294002 and wortmannin, inhibitors of phosphatidylinositol-3 kinase (PI3K). These results suggest that alpha(1)-adrenoceptor-mediated activation of MAPK involves a PTX-sensitive G-protein, PLC, PI3K, and 1,2-diacyl glycerol (DAG)-dependent PKC isozyme. Stimulation of oligodendrocyte progenitors with NE also resulted in an increase in c-fos expression, which was mediated by both alpha(1)- and beta-adrenoceptor and was calcium-, PKC-, and protein kinase A (PKA)-dependent. Interestingly, in the presence of PD 098059, a specific inhibitor of MAPK kinase (MEK), both MAPK activity and c-fos expression were blocked. This suggests that MAPK is implicated in the transmission of the signal from alpha(1)-adrenoceptor to c-fos gene expression.     

p38MAPK在体外培养星形胶质细胞缺氧/复氧损伤中的表达

目的 建立SD大鼠星形胶质细胞缺氧复氧损伤模型,探讨p38MAPK活性变化与星形胶质细胞损伤的关系.方法 体外培养新生SD大鼠星形胶质细胞,实验设正常对照组（N）、SB203580组（SB组,10 μmol/L）、缺氧/复氧组（H/R组）和缺氧/复氧组＋SB203580阻断p38MAPK组（H/R＋SB组）.应用MTT法、WB法、ELISA法检测缺氧4 h、8 h、复氧6 h、12 h、24 h、48 h时细胞存活率,p38MAPK、p-p38（磷酸化p38MAPK）及TNF-α的变化.结果 培养星形胶质细胞GFAP阳性表达率大于97%.缺氧/复氧使星形胶质细胞活力降低,SB203580阻断p38MAPK细胞活力高于H/R组,各组星形胶质细胞总p38MAPK水平无显著变化,缺氧复氧干预后p-p38表达上调,TNF-α水平显著增高.用SB203580阻断p38MAPK通路后,SB＋H/R组较H/R组p-p38、TNF-α水平降低.SB组总p38MAPK、p-p38、TNF-α水平与N组比较无显著变化.结论 p38MAPK信号通路参与了星形胶质细胞缺氧复氧损伤过程.     

Increased expression of TF in BMP-7-treated human mononuclear cells depends on activation of select MAPK signaling pathways

Bone morphogenetic protein (BMP)-7 regulates atherosclerotic plaque calcification, and it contributes to increased thrombogenicity of lipid-rich lesions by enhancement of TF expression in monocytes/macrophages by unknown mechanism. Since Erk1/2, JNK and p38 mitogen activated protein kinases (MAPKs) regulate TF expression, we studied involvement of MAPK pathways in BMP-7-induced activation of TF in human mononuclear cells (MNCs). Whole blood from healthy volunteers was treated with BMP-7, MNCs were isolated, and TF expression was assessed by western blot (WB) and In-Cell Western assay. Phosphorylation and nuclear translocation of Smad1/5/8 in response to BMP-7 stimulation of MNCs was evaluated by WB and confocal microscopy. Activation of MAPKs was judged by measuring the levels of phoshorylated Erk1/2, JNK, and p38 in the lysates of MNCs. The impact of Erk1/2 and p38 activation was studied by use of PD98059 and SB203580 inhibitors, respectively. Stimulation of whole blood with BMP-7 increased the levels of TF in the lysates of MNCs by 7-fold as compared to 12-fold after LPS stimulation. It was followed by elevation in TF fuctional activity. It was accompanied by elevated levels of phosphorylated Smad 1/5/8 and nuclear translocation of Smad1/5/8 proteins. Treatment of whole blood with BMP-7 led to a phosphorylation of Erk1/2, JNK and p38 MAPKs. BMP-7-induced TF expression was partially inhibited by Erk1/2 inhibitor, whereas TF expression was completely abolished by p38 inhibitor. BMP-7-dependent activation of TF in human MNCs by BMP-7 is accompanied by activation of canonic Smad1/5/8 signaling pathway and depends on activation of Erk1/2 and p38.     

Ginkgolide B inhibits platelet release by blocking Syk and p38 MAPK phosphorylation in thrombin-stimulated platelets

Introduction

Atherosclerosis is a chronic vascular inflammatory disease. Platelets play a critic role in the initiation of vascular inflammation in atherosclerosis. In the present study, we investigated the effects of ginkgolide B on the inhibition of platelet release and the potential mechanisms.

Methods

Experiments were performed in freshly human platelets. Platelet aggregation and ATP release were measured with a Lumi-aggregometer. Thrombin (0.5 U/ml) was used to induce platelet activation. Protein expression and phosphorylation was examined by Western blotting.

Results

The results showed that ginkgolide B significantly suppressed ATP release by 50.8% in thrombin-activated platelets. Ginkgolide B completely abolished the expression of platelet factor 4 (PF4) and CD40 Ligand (CD40L). Moreover, ginkgolide B fully attenuated the phosphorylation of Syk and p38MAPK. Similarly, R788 (a syk inhibitor) and SB203580 (a p38 MAPK inhibitor) inhibited the expression PF4 and CD40L, respectively. Furthermore, the combination of low concentrations of ginkgolide B and R788 or SB203580 has synergistic inhibition on the expression of PF4 and CD40L. Ginkgolide B partially reduced calcium efflux by 52.7% in thrombin-stimulated platelets.

Conclusion

Ginkgolide B potently inhibited the expression of PF4 and CD40L in thrombin-activated platelets. Ginkgolide B partially decreased ATP release and Ca^2 + efflux. The mechanism might be associated with the inhibition of Syk and p38 MAPK phosphorylation. These results demonstrated that ginkgolide B might be a promising drug on inhibiting platelet function and reducing inflammation in atherosclerosis.