Endothelin-1 stimulates tyrosine phosphorylation and the activities of two mitogen-activated protein kinases in cultured vascular smooth muscle cells.

OBJECTIVE: Endothelin-1 (ET-1) has been reported to stimulate the expression of the proto-oncogenes c-fos and c-myc, and to cause DNA synthesis in vascular smooth muscle cells (VSMC). The purpose of this study was to clarify the signalling pathway from ET receptors to the nucleus. DESIGN: Mitogen-activated protein (MAP) kinase, which is activated by various growth factors via phosphorylation of tyrosine and threonine residues, plays important roles as an intermediate in the signalling pathways from growth factor receptors to the ribosomes and nucleus. We examined the effect of ET-1 on the phosphorylation and activation of MAP kinase in cultured VSMC. METHODS: Extracts of ET-1-stimulated VSMC were analysed by one- and two-dimensional gel electrophoresis and anion-exchange column chromatography. Tyrosine-phosphorylated proteins and MAP kinases were detected by immunoblot analyses with anti-phosphotyrosine and anti-MAP kinase antibodies, respectively. The MAP kinase activity was measured using myelin basic protein as a substrate. The MAP kinases were isolated from 32P-labelled VSMC and subjected to phosphoamino acid analysis. RESULTS: ET-1 induced tyrosine phosphorylation of at least five proteins of about 79, 77, 73, 45 and 40 kDa in VSMC. The mobilities of the tyrosine-phosphorylated 45- and 40-kDa proteins were identical with those of the two proteins that were recognized by anti-MAP kinase antibody upon one- and two-dimensional gel electrophoresis. ET-1 stimulated MAP kinase activity in a time-course similar to that of the tyrosine phosphorylation of the 45- and 40-kDa proteins. The ET-1-stimulated MAP kinase activity was resolved almost equally into two peaks upon Mono Q column chromatography (kinase 1 and kinase 2). Kinase 1 and kinase 2 were co-eluted with the tyrosine-phosphorylated 40- and 45-kDa proteins, respectively. The apparent molecular masses of kinase 1 and kinase 2 estimated by MAP kinase assay in polyacrylamide gel were identical with those of tyrosine-phosphorylated 40- and 45-kDa proteins, respectively. Upon phosphoamino acid analysis, ET-1 stimulated phosphorylation of MAP kinases not only on tyrosine but also on threonine residues. CONCLUSIONS: ET-1 induces tyrosine and threonine phosphorylation and the activation of two species of MAP kinases of 40 and 45 kDa in VSMC.     

Lipopolysaccharide activation of human endothelial and epithelial cells is mediated by lipopolysaccharide-binding protein and soluble CD14.

Myeloid cell activation by lipopolysaccharides (LPS) involves two proteins, plasma LPS-binding protein (LBP) and cell-membrane CD14. Cell membrane CD14, anchored by a glycerophosphatidylinositol tail, is the cellular receptor for LPS-LBP complexes. Another form of CD14, without the lipid tail, circulates as a soluble plasma protein. In this work we show that soluble CD14 (sCD14) is required for activation of endothelial and epithelial cells by LPS. We propose that LPS-LBP complexes transfer LPS to sCD14, and the LPS-sCD14 complexes then bind to a cellular receptor. Support for this pathway comes from experiments in which LBP and CD14 in normal human serum are blocked by specific antibodies, experiments in which serum is replaced by purified LBP and sCD14, and experiments in which specific binding of [3H]LPS to epithelial cells is quantitated.     

Insulin enhances vascular cell adhesion molecule-1 expression in human cultured endothelial cells through a pro-atherogenic pathway mediated by p38 mitogen-activated protein-kinase

Aims/hypothesis Although hyperinsulinaemia in Type 2 diabetes in states of insulin resistance is a risk factor for atherosclerotic vascular disease, underlying mechanisms are poorly understood. We tested the hypothesis that insulin increases monocyte-endothelial interactions, which are implicated in atherosclerosis.Methods We treated human umbilical vein endothelial cells with insulin (10^–10 to 10^–7 mol/l) for 0 to 24 h. To dissect potentially implicated signal transduction pathways, we treated endothelial cells with known pharmacological inhibitors of two distinct insulin signalling pathways: the phosphatidylinositol-3-kinase (PI3-kinase) inhibitor wortmannin (3×10^–8 to 10^–6 mol/l), involved in insulin-induced endothelial nitric oxide synthase stimulation, and the p38 mitogen-activated protein (p38MAP) kinase inhibitor SB-203580 (10^–7 to 2×10^–6 mol/l). We measured adhesion molecule expression by cell surface enzyme immunoassays and U937 monocytoid cell adhesion in rotational adhesion assays.Results At pathophysiological concentrations (10^–9 to 10^–7 mol/l), insulin concentration-dependently induced vascular cell adhesion molecule (VCAM)-1 (average increase: 1.8-fold) peaking at 16 h. By contrast, the expression of intercellular adhesion molecule-1 and E-selectin were unchanged. The effect on VCAM-1 was paralleled by increased U937 cell adhesion. In the absence of cytotoxicity, wortmannin significantly potentiated the effect of insulin alone on VCAM-1 surface expression and monocytoid cell adhesion, whereas SB-203580 (10^–6 mol/l) completely abolished such effects.Conclusions/interpretation These observations indicate that insulin promotes VCAM-1 expression in endothelial cells through a p38MAP-kinase pathway, amplified by the PI3-kinase blockage. This could contribute to explaining the increased atherosclerosis occurring in subjects with hyperinsulinaemia, or in states of insulin resistance, which feature a defective PI3-kinase pathway.Abbreviations VCAM-1 vascular cell adhesion molecule-1 - ICAM-1 intercellular adhesion molecule-1 - PI3-kinase phosphatidylinositol 3-kinase - MAP mitogen-activated protein - NO nitric oxide - EIA enzyme immunoassays     

Tumor necrosis factor-alpha induces insulin resistance in endothelial cells via a p38 mitogen-activated protein kinase-dependent pathway

Chronic inflammation contributes to vascular insulin resistance and endothelial dysfunction. Systemic infusion of TNF-alpha abrogates insulin's action to enhance skeletal muscle microvascular perfusion. In skeletal muscle TNF-alpha induces insulin resistance via the p38 MAPK pathway. To examine whether p38 MAPK also regulates TNF-alpha-induced vascular insulin resistance, bovine aortic endothelial cells (bAECs) were incubated+/-TNF-alpha (5 ng/ml) for 6 h in the presence or absence of SB203580 (p38 MAPK specific inhibitor, 10 microM) after serum starvation for 10 h. For the last 30 min, cells were treated+/-1 nM insulin, and insulin receptor substrate (IRS)-1, Akt, endothelial nitric oxide synthase (eNOS), p38 MAPK, ERK1/2, c-Jun N-terminal kinase, and AMP-activated protein kinase (AMPK) phosphorylation, and eNOS activity were measured. TNF-alpha increased p38 MAPK phosphorylation, potently stimulated IRS-1 serine phosphorylation, and blunted insulin-stimulated IRS-1 tyrosine and Akt phosphorylation and eNOS activity. TNF-alpha also potently stimulated the phosphorylation of ERK1/2 and AMPK. Treatment with SB203580 decreased p38 MAPK phosphorylation back to the baseline and restored insulin sensitivity of IRS-1 tyrosine and Akt phosphorylation and eNOS activity in TNF-alpha-treated bAECs without affecting TNF-alpha-induced ERK1/2 and AMPK phosphorylation. We conclude that in cultured bAECs, TNF-alpha induces insulin resistance in the phosphatidylinositol 3-kinase/Akt/eNOS pathway via a p38 MAPK-dependent mechanism and enhances ERK1/2 and AMPK phosphorylation independent of the p38 MAPK pathway. This differential modulation of TNF-alpha's actions by p38 MAPK suggests that p38 MAPK plays a key role in TNF-alpha-mediated vascular insulin resistance and may contribute to the generalized endothelial dysfunction seen in type 2 diabetes mellitus and the cardiometabolic syndrome.     

Interleukin-12 induces tyrosine phosphorylation and activation of 44-kD mitogen-activated protein kinase in human T cells

Interleukin-12 (IL-12) is a novel cytokine that enhances numerous functional activities of human T cells and natural killer (NK) cells. The present studies were undertaken to characterize some of the early signaling events following IL-12 stimulation of mitogen-activated normal T cells. In these cells, IL-12 induces rapid tyrosine phosphorylation of proteins of 21, 44, and 54 kD. However, IL-12 does not induce tyrosine phosphorylation in normal resting T cells. In conjunction with increased tyrosine phosphorylation of several substrates, IL-12 stimulation resulted in increased in vitro kinase activity of immunoprecipitated tyrosine phosphorylated proteins. The 44- kD protein has been characterized as one isoform of the mitogen- activated protein (MAP) kinase family. Increased tyrosine phosphorylation of MAP kinase following IL-12 stimulation was also associated with enhanced enzymatic activity of this protein in vitro as measured by myelin basic protein phosphotransferase assay. These studies identify MAP kinase as one of the intracellular elements of the IL-12 signaling pathway in human T cells.     

Inhibition of p38 mitogen-activated protein kinase ameliorates cytokine up-regulated shigatoxin-1 toxicity in human brain microvascular endothelial cells

Brain injury in hemolytic-uremic syndrome (HUS) may be enhanced by inflammatory cytokine up-regulation of endothelial cell sensitivity to shigatoxin (Stx). The present study investigated whether inflammatory cytokine up-regulation of Stx toxicity could be ameliorated by inhibiting candidate signal transduction pathways. Exposure of human brain endothelial cells (HBECs) to tumor necrosis factor (TNF) greatly increased Stx-1 and Stx-2 cytotoxicity; this was reduced by inhibition of p38 mitogen-activated protein kinase (MAPK), but not c-Jun kinase. SB203580, a specific inhibitor of p38 MAPK, reduced TNF-stimulated Stx cytotoxicity in HBECs, TNF-stimulated (125)Stx-1 binding to intact HBECs, the cellular content of Gb3 (galactose alpha 1,4, galactose ss 1,4, glucose-ceramide) (the Stx receptor), and TNF-stimulated Gb3 synthase and glucosylceramide synthase activities but did not affect lactosylceramide synthase activities or mRNA content. Thus, inhibition of p38 MAPK substantially reduces inflammatory cytokine up-regulation of Stx-receptor synthesis and cell-surface expression, thereby decreasing Stx cytotoxicity. Inhibition of p38 MAPK may be of therapeutic benefit in HUS.     

Lysophosphatidylcholine activates mitogen-activated protein kinases by a tyrosine kinase-dependent pathway in bovine aortic endothelial cells

Lysophosphatidylcholine (lyso-PC) is a major component of an atherogenic lipoprotein. In this study, to investigate the involvement of mitogen-activated protein kinases in the signaling pathway by lyso-PC in endothelial cells, we measured the activity of extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) in bovine aortic endothelial cells. Lyso-PC activated ERK and JNK in a dose-dependent manner. However, the time courses of activation of these kinases were different. ERK and JNK activation by lyso-PC was inhibited by a tyrosine kinase inhibitor, herbimycin A, but not by a protein kinase C (PKC) specific inhibitor. We conclude, therefore, that lyso-PC-mediated ERK and JNK activation is caused by a tyrosine kinase-dependent mechanism, but not conventional types of PKC-dependent mechanisms.     

Lysophosphatidylcholine induces apoptosis in human endothelial cells through a p38-mitogen-activated protein kinase-dependent mechanism

Lysophosphatidylcholine (lysoPC) is a component of oxidized low density lipoprotein (LDL) and is involved in the pathogenesis of atherosclerosis and inflammation. Previous studies demonstrated that lysoPC can induce various protein kinases including tyrosine kinases, protein kinase C (PKC), and mitogen-activated protein kinases (MAPK) in vascular endothelial cells. However, the role of lysoPC-activated kinases remains undefined. In this study, we examined the effect of lysoPC on apoptosis and investigated the role of lysoPC-activated protein kinases in human umbilical vein endothelial cells (HUVEC). The presence of apoptosis was evaluated by morphological criteria, MTT assay, and electrophoresis of DNA fragments showing the characteristic apoptotic ladder, TUNEL analysis, and quantified as the proportion of hypodiploid cells by flow cytometry. The lysoPC induced apoptosis in a time- and dose-dependent manner. It stimulated the phosphorylation of extracellular signal-regulated kinase1/2 (ERK1/2) and p38-MAPK in HUVEC. The use of specific pharmacologic inhibitors indicated that the p38-MAPK-signaling pathway (SB203580) is required for lysoPC-induced apoptotic signals. Furthermore, lysoPC-induced apoptosis was inhibited by DEVD-FMK (a caspas-3/CPP32 inhibitor), suggesting involvement of an important segment in the apoptosis. These results demonstrate that lysoPC induces apoptosis in human endothelial cells through a p38-MAPK-dependent pathway.     

Eotaxin induces degranulation and chemotaxis of eosinophils through the activation of ERK2 and p38 mitogen-activated protein kinases

Eotaxin and other CC chemokines acting via CC chemokine receptor-3 (CCR3) are believed to play an integral role in the development of eosinophilic inflammation in asthma and allergic inflammatory diseases. However, little is known about the intracellular events following agonist binding to CCR3 and the relationship of these events to the functional response of the cell. The objectives of this study were to investigate CCR3-mediated activation of the mitogen-activated protein (MAP) kinases extracellular signal-regulated kinase-2 (ERK2), p38, and c-jun N-terminal kinase (JNK) in eosinophils and to assess the requirement for MAP kinases in eotaxin-induced eosinophil cationic protein (ECP) release and chemotaxis. MAP kinase activation was studied in eotaxin-stimulated eosinophils (more than 97% purity) by Western blotting and immune-complex kinase assays. ECP release was measured by radioimmunoassay. Chemotaxis was assessed using Boyden microchambers. Eotaxin (10(-11) to 10(-7) mol/L) induced concentration-dependent phosphorylation of ERK2 and p38. Phosphorylation was detectable after 30 seconds, peaked at about 1 minute, and returned to baseline after 2 to 5 minutes. Phosphorylation of JNK above baseline could not be detected. The kinase activity of ERK2 and p38 paralleled phosphorylation. PD980 59, an inhibitor of the ERK2-activating enzyme MEK (MAP ERK kinase), blocked phosphorylation of ERK2 in a concentration-dependent manner. The functional relevance of ERK2 and p38 was studied using PD98 059 and the p38 inhibitor SB202 190. PD98 059 and SB202 190 both caused inhibition of eotaxin-induced ECP release and chemotaxis. We conclude that eotaxin induces a rapid concentration-dependent activation of ERK2 and p38 in eosinophils and that the activation of these MAP kinases is required for eotaxin-stimulated degranulation and directed locomotion. (Blood. 2000;95:1911-1917)     

实验性结肠炎p38丝裂原活化蛋白激酶表达的研究

目的观察三硝基苯磺酸（TNBS）诱导的大鼠结肠炎结肠组织p38MAPK表达与激活及大鼠血清TNF-α、IL-10水平变化,探讨p38MAPK在实验性结肠炎中的作用与机制。方法20只SD大鼠随机分为正常对照组（N）与模型组（M）,TNBS／乙醇法构建结肠炎模型,观察炎症活动指数（DAI）、大体形态损伤指数（CMDI）、组织学损伤指数（TDI）,ELISA法检测血清TNF-α、IL-10水平,免疫组化染色检测大鼠结肠p38MAPK、p-p38MAPK表达。结果与N组相比,M组DAI、CMDI、TDI显著升高（P〈0．01）,血清TNF-α升高,IL-10水平下降（P〈0．01）,结肠组织p38MAPK表达增加（P〈0．05）,p-p38MAPK表达显著增加（P〈0．01）。结论p38MAPK在实验性结肠炎的表达与激活均有明显增加,可能通过调节TNF-α、IL-10等细胞因子的表达参与结肠炎的发病。     

Angiostatin diminishes activation of the mitogen-activated protein kinases ERK-1 and ERK-2 in human dermal microvascular endothelial cells

Angiostatin is an endogenous inhibitor of angiogenesis that was isolated from tumor-bearing mice. It has been established that angiostatin inhibits endothelial cell proliferation; however, the underlying mechanisms remain to be elucidated. Here we report that angiostatin reduces transiently the phosphorylation of the mitogen-activated protein kinases ERK-1 and ERK-2 in human dermal microvascular cells, but not in human vascular smooth muscle cells or human dermal fibroblasts. We demonstrate that angiostatin diminishes ERK activation by basic fibroblast growth factor and vascular endothelial growth factor. Dephosphorylation of ERK and other tyrosine-phosphorylated proteins was blocked by pretreatment of the cells with sodium meta-vanadate, an inhibitor of protein tyrosine phosphatases, indicating that angiostatin signaling may require the activity of a tyrosine phosphatase. Concentrations of angiostatin that inhibited ERK activation also inhibited basic fibroblast growth factor-stimulated collagen gel invasion by endothelial cells, but did not affect endothelial cell proliferation. We thus show that angiostatin inhibits primarily the invasion of endothelial cells and exerts minimal (if any) effects on their proliferation. Invasion is a process that involves proteolysis, adhesion and migration, all of which have been linked to ERK signaling.     

IL-1β activates p44/42 and p38 mitogen-activated protein kinases via different pathways in cat esophageal smooth muscle cells

AIM: To examine the pathway related to the IL-1β-induced activation of mitogen-activated protein (MAP)kinases in cat esophageal smooth muscle cells.METHODS: Culture of the esophageal smooth muscle cells from cat was prepared. Specific inhibitors were treated before applying the IL-1β. Western blot analysis was performed to detect the expressions of COX, iNOS and MAP kinases.RESULTS: In the primary cultured cells, although IL-1βfailed to upregulate the COX and iNOS levels, the levels of the phosphorylated forms of p44/42 MAP kinase and p38 MAP klnase increased in both concentration- and time-dependent manner, of which the level of activation reached a maximum within 3 and 18 h, respectively.The pertussis toxin reduced the level of p44/42 MAP kinase phosphorylation. Tyrphostin 51 and genistein also inhibited this activation. Neomycin decreased the density of the p44/42 MAP kinase band to the basal level.Phosphokinase C (PKC) was found to play a mediating role in the IL-1β-induced p44/42 MAP kinase activity.In contrast, the activation of p38 MAP kinase was inhibited only by a pretreatment with forskolin, and was unaffected by the other compounds.CONCLUSION: Based on these results, IL-1β-Induced p44/42 MAP kinase activation is mediated by the Gi protein, tyrosine kinase, phospholipase C (PLC) and PKC. The pathway for p38 MAP kinase phosphorylation is different from that of p44/42 MAP kinase, suggesting that it plays a different role in the cellular response to IL-1β.     

IL-1β activates p44/42 and p38 mitogen-activated protein kinases via different pathways in cat esophageal smooth muscle cells

AIM: To examine the pathway related to the IL-1beta-induced activation of mitogen-activated protein (MAP) kinases in cat esophageal smooth muscle cells. METHODS: Culture of the esophageal smooth muscle cells from cat was prepared. Specific inhibitors were treated before applying the IL-1beta. Western blot analysis was performed to detect the expressions of COX, iNOS and MAP kinases. RESULTS: In the primary cultured cells, although IL-1beta failed to upregulate the COX and iNOS levels, the levels of the phosphorylated forms of p44/42 MAP kinase and p38 MAP kinase increased in both concentration- and time-dependent manner, of which the level of activation reached a maximum within 3 and 18 h, respectively. The pertussis toxin reduced the level of p44/42 MAP kinase phosphorylation. Tyrphostin 51 and genistein also inhibited this activation. Neomycin decreased the density of the p44/42 MAP kinase band to the basal level. Phosphokinase C (PKC) was found to play a mediating role in the IL-1beta-induced p44/42 MAP kinase activity. In contrast, the activation of p38 MAP kinase was inhibited only by a pretreatment with forskolin, and was unaffected by the other compounds. CONCLUSION: Based on these results, IL-1beta-induced p44/42 MAP kinase activation is mediated by the Gi protein, tyrosine kinase, phospholipase C (PLC) and PKC. The pathway for p38 MAP kinase phosphorylation is different from that of p44/42 MAP kinase, suggesting that it plays a different role in the cellular response to IL-1beta.     

MCP-1 induces activation of MAP-kinases ERK,JNK and p38 MAPK in human endothelial cells

Activation of vascular endothelial cells (ECs) plays an important pathogenic role in the development of atherosclerosis. Monocyte chemoattractant protein-1 (MCP-1) is a potent chemoattractant of monocytes. Besides induction of monocyte recruitment, it has been suggested that MCP-1 can also affect the cellular responses of ECs. We investigated whether MCP-1 activated the three major mitogen activated protein (MAP)-kinases extracellular signal-regulated kinase (ERK), c-Jun amino terminal kinase (JNK) and p38 MAPK. Stimulation of ECs with MCP-1 induced a time- and concentration-dependent activation of all three MAP-kinases, concentrations as low as 0.1 ng/ml were sufficient for this mechanism. MCP-1 also induced secretion of matrix metalloproteinase (MMP)-2 which along with ERK activation was inhibited by PD098059. The results demonstrate that MCP-1 can lead to direct activation of MAP kinases together with induction of MMP2 in ECs. Our data thus propose a new mechanism for the proatherogenic effect of MCP-1.     

Lysophosphatidylcholine activates p38 and p42/44 mitogen-activated protein kinases in monocytic THP-1 cells, but only p38 activation is involved in its stimulated chemotaxis

Oxidized LDLs (OxLDLs) have been shown to be involved in recruitment of blood monocytes into the arterial subendothelial space, which is the earliest step in atherogenesis, but the underlying molecular mechanisms are poorly understood. The present study demonstrated that lysophosphatidylcholine (LPC), a major phospholipid component of OxLDL, strongly evoked phosphorylation and activation of p38 and p42/44 mitogen-activated protein kinases in monocytic cells. The stimulation of p38 and p42/44 occurred in a dose- and time-dependent manner, reaching the maximal activation at 25 microg/mL LPC within 5 minutes. Interestingly, inhibition of p38 activation by OxLDL or LPC, using its selective inhibitors (SB203580 and SKF86002), completely blocked OxLDL- or LPC-stimulated chemotaxis of THP-1 cells, which was measured in a transwell chemotaxis assay. In contrast, inhibition of p42/44 activation by its potent inhibitor (PD98059) did not block OxLDL- or LPC-stimulated chemotaxis. Moreover, expression of a p38 dominant-negative mutant (p38AF) reduced cell chemotaxis significantly. In addition, activation of p38 by LPC was apparently mediated neither by scavenger receptors nor by tyrosine kinase receptors. It was, however, effectively blocked by pertussis toxin and substantially reduced by phospholipase C inhibitor (U73122) and phosphatidylinositol 3-kinase inhibitors (wortmannin and LY294002). LPC also inhibited forskolin-stimulated cAMP accumulation in a pertussis toxin-sensitive manner, indicating that Gi/Go proteins likely mediated the effects of LPC. Our results suggested that OxLDL/LPC efficiently activated both p38 and p42/44, but only the activation of p38 was functionally associated with OxLDL-/LPC-induced chemotaxis in THP-1 cells.     

P38信号通路对人脐静脉内皮细胞单核细胞趋化蛋白-1表达的影响

目的 探讨P38信号通路(P38MAPK)和单核细胞趋化蛋白-1(MCP-1)的关系，以及P38MAPK在糖尿病(DM)动脉粥样硬化(AS)中的作用。方法 分别以高葡萄糖(HG)、糖基化终产物(AGEs)、高胰岛素(HIns)和过氧化氢(H2O2)孵育人脐静脉内皮细胞(HUVECs)，观察HUVECs P38MAPK的蛋白表达和MCP-1 mRNA表达；以P38MAPK特异抑制剂SB203580预处理，再用以上4种刺激因素孵育HUVECs，观察MCP-1 mRNA在HUVECs的表达。结果 HG、AGEs、HIns和H2O2均可独立激活P38MAPK，使磷酸化P38MAPK蛋白表达量及MCP-1 mRNA表达量增加；SB203580预处理后，MCP-1 mRNA表达被明显抑制。结论 P38MAPK调控MCP-1的表达，表明P38MAPK可能是DM致AS发生的始动信号之一。