Angiotensin II impairs the insulin signaling pathway promoting production of nitric oxide by inducing phosphorylation of insulin receptor substrate-1 on Ser312 and Ser616 in human umbilical vein endothelial cells

It has been suggested that serine (Ser) phosphorylation of insulin receptor substrate-1 (IRS-1) decreases the ability of IRS-1 to be phosphorylated on tyrosine, thereby attenuating insulin signaling. There is evidence that angiotensin II (AII) may impair insulin signaling to the IRS-1/phosphatydilinositol 3-kinase (PI 3-kinase) pathway by enhancing Ser phosphorylation. Insulin stimulates NO production by a pathway involving IRS-1/PI3-kinase/Akt/endothelial NO synthase (eNOS). We addressed the question of whether AII affects insulin signaling involved in NO production in human umbilical vein endothelial cells and tested the hypothesis that the inhibitory effect of AII on insulin signaling was caused by increased site-specific Ser phosphorylation in IRS-1. Exposure of human umbilical vein endothelial cells to AII resulted in inhibition of insulin-stimulated production of NO. This event was associated with impaired IRS-1 phosphorylation at Tyr612 and Tyr632, two sites essential for engaging the p85 subunit of PI3-kinase, resulting in defective activation of PI 3-kinase, Akt, and eNOS. This inhibitory effect of AII was reversed by the type 1 receptor antagonist losartan. AII increased c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK) 1/2 activity, which was associated with a concomitant increase in IRS-1 phosphorylation at Ser312 and Ser616, respectively. Inhibition of JNK and ERK1/2 activity reversed the negative effects of AII on insulin-stimulated NO production. Our data suggest that AII, acting via the type 1 receptor, increases IRS-1 phosphorylation at Ser312 and Ser616 via JNK and ERK1/2, respectively, thus impairing the vasodilator effects of insulin mediated by the IRS-1/PI 3-kinase/Akt/eNOS pathway.     

p38 mitogen-activated protein kinase mediates palmitate-induced apoptosis but not inhibitor of nuclear factor-kappaB degradation in human coronary artery endothelial cells

Plasma free fatty acids are elevated in patients with type 2 diabetes and contribute to the pathogenesis of insulin resistance and endothelial dysfunction. The p38 MAPK mediates stress, inflammation, and apoptosis. Whether free fatty acids induce apoptosis and/or activate nuclear factor-kappaB inflammatory pathway in human coronary artery endothelial cells (hCAECs) and, if so, whether this involves the p38 MAPK pathway is unknown. hCAECs (passages 4-6) were grown to 70% confluence and then incubated with palmitate at concentrations of 0-300 microm for 6-48 h. Palmitate at 100, 200, or 300 microm markedly increased apoptosis after 12 h of incubation. This apoptotic effect was time (P=0.008) and dose (P=0.006) dependent. Palmitate (100 microm for 24 h) induced a greater than 2-fold increase in apoptosis, which was accompanied with a 4-fold increase in p38 MAPK activity (P<0.001). Palmitate did not affect the phosphorylation of Akt1 or ERK1/2. SB203580 (a specific inhibitor of p38 MAPK) alone did not affect cellular apoptosis; however, it abolished palmitate-induced apoptosis and p38 MAPK activation. Palmitate significantly reduced the level of inhibitor of nuclear factor-kappaB (IkappaB). However, treatment of cells with SB203580 did not restore IkappaB to baseline. We conclude that palmitate induces hCAEC apoptosis via a p38 MAPK-dependent mechanism and may participate in coronary endothelial injury in diabetes. However, palmitate-mediated IkappaB degradation in hCAECs is independent of p38 MAPK activity.     

p38MAPK-eNOS-N0信号通路在人脐静脉内皮细胞凋亡中的保护作用

目的探讨p38MAPK信号通路在胰高血糖素样肽1(GLP-1)拮抗人脐静脉内皮细胞凋亡中的作用。方法实验分为对照组、糖基化终末产物(AGE)组、GLP-1组、AGE+GLP-1组、AGE+SB203580组、AGE+GLP-1+SB203580组及AGE+GLP-1+L-NAME组,Western blot检测p-p38MAPK/p38MAPK、磷酸化内皮型一氧化氮合酶/内皮型一氧化氮合酶(p-eNOS/eNOS)蛋白表达情况,NO检测试剂盒(一步法)检测NO含量,DCFH-DA荧光探针检测细胞活性氧(ROS)含量,Annexin V/PI流式检测细胞凋亡率。结果与AGE组相比,GLP-1预处理可诱导p-p38MAPK蛋白表达下降(P=0.000);与对照组比较,GLP-1或p38 MAPK抑制剂(SB203580)预处理后,受AGE抑制的eNOS蛋白表达或诱导的ROS水平分别显著升高(P=0.004)或下降(P=0.000);GLP-1预处理后,因AGE诱导的细胞凋亡率显著降低(P=0.000),而加入L-NAME后,GLP-1的抗凋亡作用显著减弱(P=0.002);GLP-1预处理后,细胞NO含量较单纯AGE组明显升高(P=0.000),而予以L-NAME后,细胞NO含量显著降低(P=0.011)。结论GLP-1可抑制p38 MAPK信号通路的活化,拮抗AGE对血管内皮细胞的氧化损伤;上调eNOS蛋白的表达,拮抗AGE诱导的内皮细胞NO生成障碍及细胞凋亡,从而延缓糖尿病合并动脉粥样硬化的发生发展。     

PGE1 inhibits the expression of PAI-1 mRNA induced by TNF-alpha in human mesangial cells.

We examined the effect of PGE1 on the expression of plasminogen activator inhibitor-1 (PAI-1) mRNA induced by tumor necrosis factor-alpha (TNF-alpha) in human mesangial cells, because PAI-1 is one of major factors for the progression of glomerulosclerosis. The expression of PAI-1 mRNA was increased after stimulation with TNF-alpha, and it was diminished by pre-incubation with PGE1. Next, we examined the effect of PGE1 on the phosphorylation of mitogen activated protein kinase (MAPK) family and Akt. TNF-alpha activated the phosphorylation of p44/42 MAPK, p38 MAPK, SAPK/JNK and Akt in mesangial cells. PGE1 inhibited the TNF-alpha induced phosphorylation of SAPK/JNK and Akt, but not p44/42 MAPK and p38 MAPK. The TNF-alpha induced expression of PAI-1 mRNA was not affected by PD98059, an inhibitor of MEK, SB203580, an inhibitor of p38 MAPK, nor LY294002, an inhibitor of PI3 K. However, DMAP, an inhibitor of SAPK/JNK, inhibited the expression of PAI-1 mRNA, suggesting that the TNF-alpha induced expression of PAI-1 mRNA is regulated by the SAPK/JNK dependent pathway in human mesangial cells. By the incubation with H8, an inhibitor of PKA, the inhibitory effect of PGE1 on the expression of PAI-1 mRNA was abolished, suggesting that PGE1 inhibited the PAI-1 mRNA expression via the PKA pathway. Our results suggest that the inhibition of PAI-1 synthesis by PGE1 in human mesangial cells may have therapeutic implications for glomerulosclerosis such as occurs in diabetic nephropathy.     

Extracellular signal-regulated kinase and p38 mitogen-activated protein kinase mediate macrophage proliferation induced by oxidized low-density lipoprotein

We previously reported that oxidized low-density lipoprotein (Ox-LDL)-induced expression of granulocyte/macrophage colony-stimulating factor (GM-CSF) via PKC, leading to activation of phosphatidylinositol-3 kinase (PI-3K), was important for macrophage proliferation [J Biol Chem 275 (2000) 5810]. The aim of the present study was to elucidate the role of extracellular-signal regulated kinase 1/2 (ERK1/2) and of p38 MAPK in Ox-LDL-induced macrophage proliferation. Ox-LDL-induced proliferation of mouse peritoneal macrophages assessed by [3H]thymidine incorporation and cell counting assays was significantly inhibited by MEK1/2 inhibitors, PD98059 or U0126, and p38 MAPK inhibitors, SB203580 or SB202190, respectively. Ox-LDL-induced GM-CSF production was inhibited by MEK1/2 inhibitors but not by p38 MAPK inhibitors in mRNA and protein levels, whereas recombinant GM-CSF-induced macrophage proliferation was inhibited by p38 MAPK inhibitors but enhanced by MEK1/2 inhibitors. Recombinant GM-CSF-induced PI-3K activation and Akt phosphorylation were significantly inhibited by SB203580 but enhanced by PD98059. Our results suggest that ERK1/2 is involved in Ox-LDL-induced macrophage proliferation in the signaling pathway before GM-CSF production, whereas p38 MAPK is involved after GM-CSF release. Thus, the importance of MAPKs in Ox-LDL-induced macrophage proliferation was confirmed and the control of MAPK cascade could be targeted as a potential treatment of atherosclerosis.     

p38 mitogen-activated protein kinase activates eNOS in endothelial cells by an estrogen receptor alpha-dependent pathway in response to black tea polyphenols

Black tea has been shown to improve endothelial function in patients with coronary artery disease and recent data indicate the polyphenol fraction of black tea enhances endothelial nitric oxide synthase (eNOS) activity through p38 MAP kinase (p38 MAPK) activation. Because the mechanisms for this phenomenon are not yet clear, we sought to elucidate the signaling events in response to black tea polyphenols. Bovine aortic endothelial cells (BAECs) exposed to black tea polyphenols demonstrated eNOS activation that was inhibited by the estrogen receptor (ER) antagonist ICI 182,780, and siRNA-mediated silencing of ER expression. Consistent with this observation, black tea polyphenols induced time-dependent phosphorylation of ERalpha on Ser-118 that was inhibited by ICI 182,780. Phosphorylation of ERalpha on Ser-118 was due to p38 MAP kinase (p38 MAPK) as, it was inhibited by SB203580 and overexpression of dominant-negative p38alpha MAPK. Conversely, constitutively active MKK6 induced p38 MAPK activation that recapitulated the effects of polyphenols by inducing ERalpha phosphorylation and downstream activation of Akt, and eNOS. The key role of ERalpha Ser-118 phosphorylation was confirmed in eNOS-transfected COS-7 cells, as polyphenol-induced eNOS activation required cotransfection with ERalpha subject to phosphorylation at Ser-118. This residue appeared critical for functional association of ERalpha with p38 MAPK as ERalpha with Ser-118 mutated to alanine could not form a complex with p38 MAPK. These findings suggest p38 MAP kinase-mediated eNOS activation requires ERalpha and these data uncover a new mechanism of ERalpha activation that has broad implications for NO bioactivity and endothelial cell phenotype.     

Adiponectin inhibits endothelial synthesis of interleukin-8

Adiponectin is an antiatherogenic adipokine that inhibits inflammation by mechanisms that are not completely understood. We explored the effect of adiponectin on endothelial synthesis of interleukin-8 (IL-8), a pro-inflammatory chemokine that plays a role in atherogenesis. Adiponectin decreased the secretion of IL-8 from human aortic endothelial cells (HAEC) stimulated with tumor necrosis factor-alpha (TNF-alpha). Adiponectin also inhibited IL-8 mRNA expression induced by TNF-alpha. Phosphorylation of IkappaB-alpha was decreased by adiponectin, but phosphorylation of ERK, SAPK/JNK, and p38MAPK were unaffected. Adiponectin increased intra-cellular cAMP levels in HAEC in a dose-dependent manner; PKA activity was also increased. The inhibitory effect of adiponectin on TNF-alpha-induced IL-8 synthesis was inhibited by pretreatment with Rp-cAMP, a PKA inhibitor. These observations suggest that adiponectin inhibits IL-8 synthesis through inhibition of a PKA dependent NF-kappaB signaling pathway. We also showed that adiponectin enhances Akt phosphorylation. The inhibitory effect of adiponectin on TNF-alpha-induced IL-8 synthesis was abrogated in part by pretreatment with the PI3 kinase inhibitor LY294002 or by Akt siRNA transfection, suggesting that Akt activation might inhibit IL-8 synthesis induced by TNF-alpha. We conclude that inhibition of NF-kappaB and activation of Akt phosphorylation may mediate adiponectin inhibition of atherosclerosis.     

Inhibition of p38 MAPK activation via induction of MKP-1: atrial natriuretic peptide reduces TNF-alpha-induced actin polymerization and endothelial permeability

The atrial natriuretic peptide (ANP) is a cardiovascular hormone possessing antiinflammatory potential due to its inhibitory action on the production of inflammatory mediators, such as tumor necrosis factor-alpha (TNF-alpha). The aim of this study was to determine whether ANP is able to attenuate inflammatory effects of TNF-alpha on target cells. Human umbilical vein endothelial cells (HUVECs) were treated with TNF-alpha in the presence or absence of ANP. Changes in permeability, cytoskeletal alterations, phosphorylation of p38 MAPK and HSP27, and expression of MKP-1 were determined by macromolecule permeability assay, fluorescence labeling, RT-PCR, and immunoblotting. Antisense studies were done by transfecting cells with MKP-1 antisense oligonucleotides. Activation of HUVECs with TNF-alpha lead to a significant increase of macromolecule permeability and formation of stress fibers. Treatment of cells with ANP (10(-8) to 10(-6) mol/L) significantly reduced the formation of stress fibers and elevated permeability. Both TNF-alpha-induced effects were shown to be mediated via the activation of p38 using SB203580, a specific inhibitor of p38. ANP significantly reduced the TNF-alpha-induced activation of p38 and attenuated the phosphorylation of HSP27, a central target downstream of p38. ANP showed no effect on p38 upstream kinases MKK3/6. However, a significant induction of the MAPK phosphatase MKP-1 mRNA and protein could be observed in ANP-treated cells. Antisense experiments proved a causal role for MKP-1 induction in the ANP-mediated inhibition of p38. These data show the inhibitory action of ANP on TNF-alpha-induced changes in endothelial cytoskeleton and macromolecule permeability involving an MKP-1-induced inactivation of p38 MAPK. These effects point to an antiinflammatory and antiatherogenic potential of this cardiovascular hormone.     

Aberrant p38 mitogen-activated protein kinase signalling in skeletal muscle from Type 2 diabetic patients

Aims/hypothesis p38 mitogen activated protein kinase (MAPK) is generally thought to facilitate signal transduction to genomic, rather than metabolic responses. However, recent evidence implicates a role for p38 MAPK in the regulation of glucose transport; a site of insulin resistance in Type 2 diabetes. Thus we determined p38 MAPK protein expression and phosphorylation in skeletal muscle from Type 2 diabetic patients and non-diabetic subjects.Methods In vitro effects of insulin (120 nmol/l) or AICAR (1 mmol/l) on p38 MAPK expression and phosphorylation were determined in skeletal muscle from non-diabetic (n=6) and Type 2 diabetic (n=9) subjects.Results p38 MAPK protein expression was similar between Type 2 diabetic patients and non-diabetic subjects. Insulin exposure increased p38 MAPK phosphorylation in non-diabetic, but not in Type 2 diabetic patients. In contrast, basal phosphorylation of p38 MAPK was increased in skeletal muscle from Type 2 diabetic patients.Conclusion/interpretation Insulin increases p38 MAPK phosphorylation in skeletal muscle from non-diabetic subjects, but not in Type 2 diabetic patients. However, basal p38 MAPK phosphorylation is increased in skeletal muscle from Type 2 diabetic patients. Thus, aberrant p38 MAPK signalling might contribute to the pathogenesis of insulin resistance.Abbreviations AICAR 5-aminoimidazole-4-carboxamide ribonucleoside - AMPK 5-AMP activated protein kinase - ERK 1/2 extracellular regulated kinase - GIR glucose infusion rate - IRS-1 insulin receptor substrate 1 - MAPK mitogen-activated protein kinase - PI phosphatidylinositol - VO_2max maximal oxygen uptake     

Development of whole-body and skeletal muscle insulin resistance after one day of hindlimb suspension

Hindlimb suspension (HS) of rats is a model of simulated weightlessness and induces dynamic alterations in insulin action. In the present study, the effect of acute (1-day) HS on whole-body glucose tolerance and insulin action on skeletal muscle glucose transport was assessed in juvenile, female Sprague-Dawley rats. Compared to weight-bearing control rats, 1-day HS animals displayed significantly decreased glucose tolerance and diminished whole-body insulin sensitivity. Glucose transport activity in the 1-day unweighted soleus muscle was significantly decreased (P <.05) compared to weight-bearing control muscles both in the absence and presence of insulin (2 mU/mL). Insulin-mediated glucose transport activity in the extensor digitorum longus (EDL) muscles also tended (P =.09) to be lower. There was no change in the protein expression of insulin receptor beta-subunit (IR-beta), insulin receptor substrate-1 (IRS-1), IRS-2, the p85 subunit of phosphatidylinositol-3 kinase (PI3-kinase), Akt, and glucose transporter protein 4 (GLUT-4). The activities of these proteins were also unchanged, as insulin-stimulated IR-beta tyrosine phosphorylation, IRS-1 tyrosine phosphorylation, IRS-1-associated p85, and Akt serine phosphorylation were similar to controls. However, basal Akt phosphorylation was significantly depressed (P <.05) in the 1-day HS soleus. In addition, the protein expression and basal phosphorylation of the stress-activated p38 mitogen-activated protein kinase (p38 MAPK) were significantly elevated (P <.05) in the 1-day unweighted soleus. These results indicate that the development of insulin resistance in the 1-day unweighted soleus is not due to impaired functionality of elements involved in the IR/IRS-1/PI3-kinase/Akt signaling pathway. However, activation of p38 MAPK may play a role in this response.     

软脂酸通过丝裂原活化蛋白激酶通路促进血管内皮细胞凋亡

目的探讨软脂酸(PA)诱导的血管内皮细胞凋亡中丝裂原活化蛋白激酶(MAPK)通路的作用。方法将人脐静脉内皮细胞(HUVEC)分对照组、PA组、MAPK通路干预组[分别先用p38抑制剂SB203580、氨基末端激酶(JNK)抑制剂PD98059、细胞外信号调节激酶(ERK)抑制剂SP600125干预]再分为PA+SB组、PA+PD组、PA+SP组。流式细胞仪检测细胞凋亡率;Western blot法检测caspase-3、磷酸化p38、JNK和ERK1/2表达水平;分光光度法检测caspase-3的活性。结果与对照组比较,PA组、PA+SB组、PA+PD组、PA+SP组HUVEC凋亡及caspase-3表达和活性明显增加,PA组磷酸化p38MAPK表达明显增加(P<0.05)。与PA组比较,PA+SB组HUVEC细胞凋亡率、caspase-3表达和活性明显降低(P<0.05);而PA+PD组和PA+SP组HUVEC凋亡率、caspase-3表达和活性无明显变化(P>0.05)。结论 PA通过p38MAPK通路促进内皮细胞凋亡。     

Suppression of p38 mitogen-activated protein kinase inhibits hepatitis B virus replication in human hepatoma cell: the antiviral role of nitric oxide

Summary.  The role of the p38 mitogen-activated protein kinase (MAPK) pathway in hepatitis B virus (HBV) replication was investigated in this study. After transient transfection with HBV plasmid, p38 MAPK, but not JNK or ERK1/2, was significantly phosphorylated in human hepatoma cell Huh7. Interestingly, HBV proteins and RNA synthesis were significantly inhibited by a specific inhibitor of p38 MAPK, SB203580, in a dose-dependent manner. Intracellular core-associated DNA, extracellular virion-associated DNA and covalently closed circular DNA were also significantly inhibited by SB203580. Further results showed the antiviral role of nitric oxide (NO) on the suppression of HBV replication and downregulation of p38 MAPK phosphorylation. In conclusion, these results suggested that suppression of phosphorylation of p38 MAPK by inhibitor or NO could inhibit intracellular HBV replication.     

C-reactive protein impairs hepatic insulin sensitivity and insulin signaling in rats: role of mitogen-activated protein kinases

Plasma C-reactive protein (CRP) concentration is increased in the metabolic syndrome, which consists of a cluster of cardiovascular disease risk factors, including insulin resistance. It is not known, however, whether CRP is merely a marker of accompanying inflammation or whether it contributes causally to insulin resistance. The objective of this study is to investigate the role that CRP may play in the development of insulin resistance. We examined the effect of single-dose intravenous administration of purified human (h)CRP on insulin sensitivity in Sprague-Dawley rats using the euglycemic, hyperinsulinemic clamp technique. hCRP was associated with impaired insulin suppression of endogenous glucose production with no reduction in peripheral tissue glucose uptake, suggesting that hCRP mediated insulin resistance in the liver but not extrahepatic tissues. We further assessed components of the insulin signaling pathway and mitogen-activated protein kinases (MAPKs) in the liver. Liver tissues derived from hCRP-treated rats showed reduced insulin-stimulated insulin receptor substrate (IRS) tyrosine phosphorylation, IRS/phosphatidylinositol 3-kinase (PI3K) association, and Akt phosphorylation, consistent with hCRP-induced impairment of hepatic insulin signaling. Furthermore, hCRP enhanced phosphorylation of extracellular signal-regulated kinase (ERK)1/2 and p38 MAPK as well as IRS-1 Ser(612) . Finally, we observed in primary cultured rat hepatocytes that U0126 (a selective inhibitor of MAPK/ERK kinase1/2) corrected hCRP-induced impairment of insulin signaling. CONCLUSIONS: hCRP plays an active role in inducing hepatic insulin resistance in the rat, at least in part by activating ERK1/2, with downstream impairment in the insulin signaling pathway.     

Overexpression of the dual-specificity phosphatase MKP-4/DUSP-9 protects against stress-induced insulin resistance

Insulin resistance, a hallmark of type 2 diabetes and obesity, is associated with increased activity of MAP and stress-activated protein (SAP) kinases, which results in decreased insulin signaling. Our goal was to investigate the role of MAP kinase phosphatase-4 (MKP-4) in modulating this process. We found that MKP-4 expression is up-regulated during adipocyte and myocyte differentiation in vitro and up-regulated during fasting in white adipose tissue in vivo. Overexpression of MKP-4 in 3T3-L1 cells inhibited ERK and JNK phosphorylation and, to a lesser extent, p38MAPK phosphorylation. As a result, the phosphorylation of IRS-1 serine 307 induced by anisomycin was abolished, leading to a sensitization of insulin signaling with recovery of insulin-stimulated IRS-1 tyrosine phosphorylation, IRS-1 docking with phosphatidylinositol 3-kinase, and Akt phosphorylation. MKP-4 also reversed the effect of TNF-alpha to inhibit insulin signaling; alter IL-6, Glut1 and Glut4 expression; and inhibit insulin-stimulated glucose uptake in 3T3-L1 adipocytes. Overexpression of MKP-4 in the liver of ob/ob mice decreased ERK and JNK phosphorylation, leading to a reduction in fed and fasted glycemia, improved glucose intolerance, decreased expression of gluconeogenic and lipogenic genes, and reduced hepatic steatosis. Thus, MKP-4 has a protective effect against the development of insulin resistance through its ability to dephosphorylate and inactivate crucial mediators of stress-induced insulin resistance, such as ERK and JNK, and increasing MKP-4 activity might provide a therapy for insulin-resistant disorders.     

Ischemic preconditioning involves dual cardio-protective axes with p38MAPK as upstream target

The existing literature indicates a crucial role of p38 MAP (mitogen-activated protein) kinase (p38MAPK) and its downstream target MAPKAP kinase 2 (MK2) in ischemic preconditioning (IPC). Accordingly, deletion of MK2 gene should abolish the cardioprotective ability of IPC. Interestingly, we were able to partially precondition the hearts from MK2(-/-) knockout mice suggesting the existence of an as yet unknown alternative downstream target of p38MAPK. A recent study from our laboratory also determined a crucial role of CREB (cyclic AMP response element binding protein) in IPC. Since CREB is a downstream target of MSK-1 (mitogen- and stress-activated protein kinase-1) situated at the crossroad of ERK (extracellular receptor kinase) and p38MAPK signaling pathways, we reasoned that MSK-1 could be a downstream molecular target for p38MAPK and ERK signaling in the IPC hearts. To test this hypothesis, the rat hearts were subjected to IPC by four cyclic episodes of 5 min ischemia and 10 min reperfusion. As expected, IPC induced the activation of ERK1/2, p38MAPK, MK2 and HSP (heat shock protein) 27 as evidenced by their increased phosphorylation; and the inhibition of p38MAPK with SB203580 almost completely, and the inhibition of ERK1/2 with PD098059 partially, abolished cardioprotective effects of IPC. Inhibition of MSK-1 with short hairpin RNA (shRNA) also abolished the IPC-induced cardioprotection. SB203580 partially blocked the effects of MSK-1 suggesting that MSK-1 sits downstream of p38MAPK. shRNA-MSK-1 blocked the contribution of both p38MAPK and ERK1/2 as it is uniquely situated at the downstream crossroad of both of these MAP kinases. Although MSK-1 sits downstream of both ERK1/2 and p38MAPK, ERK1/2 activation appears to play less significant role compared to p38MAPK, since its inhibition blocked MSK activation only partially. Consistent with these results, shRNA-MSK-1 blocked the partial PC in MK2(-/-) hearts, and in combination with SB203580, completely abolished the PC effects in the wild-type hearts. The IPC-induced survival signaling was almost completely inhibited with SB203580, and only partially with PD 098059 as evidenced from the inhibition patterns of IPC induced activation of CREB, Akt and Bcl-2. Again SB203580 alone or in combination with shRNA-MSK-1 inhibited IPC induced survival signal comparatively, suggesting that MSK-1 exists downstream of p38MAPK. Taken together, these results indicate for the first time MSK-1 as an alternative (other than MK2) downstream target for p38MAPK, which also transmits survival signal through the activation of CREB.     

The p38 mitogen-activated protein kinase pathway plays a critical role in thrombin-induced endothelial chemokine production and leukocyte recruitment

Thrombin, the terminal serine protease in the coagulation cascade, is a proinflammatory molecule in vivo and induces endothelial activation in vitro. The cellular signaling mechanisms involved in this function are unknown. The role of the p38 mitogen-activated protein kinase (MAPK) signaling pathway in thrombin-induced chemokine production was studied. Phosphorylation of both p38 MAPK and its substrate, ATF-2, was observed in human umbilical vein endothelial cells (HUVECs) stimulated with thrombin, with a maximum after 5 minutes of stimulation. Using the selective p38 MAPK inhibitor SB203580, there was a significant decrease in thrombin-induced interleukin-8 (IL-8) and monocyte chemotactic protein-1 (MCP-1) protein production and messenger RNA steady-state levels. In addition, SB203580 decreased IL-8 and MCP-1 production induced by the thrombin receptor-1 agonist peptide (TRAP), suggesting functional links between the thrombin G protein-coupled receptor and the p38 MAPK pathway. Furthermore, endothelial activation in the presence of SB203580 decreased the chemotactic activity of thrombin-stimulated HUVEC supernatant on neutrophils and monocytic cells. In contrast, the p42/p44 MAPK pathway did not appear to be involved in thrombin- or TRAP-induced endothelial chemokine production, because there was no reduction in the presence of the p42/p44-specific inhibitor PD98059. These results demonstrate that the p38 rather than p42/44 MAPK signaling pathway plays an important role in thrombin-induced endothelial proinflammatory activation and suggest that inhibition of p38 MAPK may be an interesting target for anti-inflammatory strategies in vascular diseases combining thrombosis and inflammation. (Blood. 2001;98:667-673)     

Molecular mechanisms of ghrelin-mediated endothelial nitric oxide synthase activation

Metabolic syndrome accelerates the atherosclerotic process, and the earliest event of which is endothelial dysfunction. Ghrelin, a newly discovered gastric peptide, improves endothelial function and inhibits proatherogenic changes. In particular, low ghrelin concentration has been associated with several features of metabolic syndrome, including obesity, insulin resistance, and high blood pressure. However, the molecular mechanisms underlying ghrelin vascular actions remain largely unclear. Here, we showed that ghrelin activated endothelial nitric oxide (NO) synthase (eNOS) in cultured endothelial cells (ECs) and in intact vessels. Specifically, ghrelin rapidly induced phosphorylation of eNOS on an activation site and production of NO in human umbilical vein ECs and bovine aortic ECs. The eNOS phosphorylation was also observed in mouse aortas ex vivo perfused with ghrelin and in aortic tissues isolated from mice injected with ghrelin. Mechanistically, ghrelin stimulated AMP-activated protein kinase (AMPK) and Akt activation in cultured ECs and intact vessels. Inhibiting AMPK and Akt with their pharmacological inhibitors, small interference RNA and adenoviruses carried dominant-negative mutants, markedly attenuated ghrelin-induced eNOS activation, and NO production. Furthermore, ghrelin receptor/Gq protein/calcium-dependent pathway mediates activation of AMPK, Akt, and eNOS, and calmodulin-dependent kinase kinase is a potential convergent point to regulate Akt and AMPK activation in ghrelin signaling. Importantly, eNOS activation is critical for ghrelin inhibition of vascular inflammation. Together, both in vitro and in vivo data demonstrate a new role of ghrelin signaling for eNOS activation, and highlight the therapeutic potential for ghrelin to correct endothelial dysfunction associated with atherosclerotic vascular diseases and metabolic syndrome.