p38 MAPK信号转导通路参与NO诱导兔关节软骨细胞凋亡

目的探讨p38 MAPK信号转导通路在软骨细胞凋亡中的作用。方法体外培养兔关节软骨细胞,一氧化氮(NO)供体NOC-18和p38 MAPK抑制剂SB203580作用于细胞24 h,用AnnexinV-FITC/PI流式细胞术检测软骨细胞凋亡率,W estern b lot测定p38、磷酸化p38蛋白的表达水平。结果与对照组比较,SB203580显著降低了NOC-18诱导的软骨细胞凋亡率(P<0.05);NOC-18以浓度依赖的方式促进p38 MAPK的磷酸化,而SB203580能抑制其磷酸化(P<0.05)。结论p38 MAPK通路参与了NO诱导的兔关节软骨细胞凋亡的信号转导。     

p38 mitogen‐activated protein kinase impairment of innate immune cells is a characteristic feature of HBeAg‐negative chronic hepatitis B

The mitogen‐activated protein kinase p38 (MAPK) is implicated in the induction of immune responses by regulating the differentiation of T lymphocytes and production of cytokines. Our aim was to investigate p38MAPK phosphorylation in different stages of the natural history of hepatitis B virus (HBV) infection. Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll‐Hypaque density‐based centrifugation from 10 patients with HBeAg‐negative chronic hepatitis B [HBeAg(?) CHB;HBV‐DNA>2000IU/mL], eight patients with HBeAg‐negative chronic HBV infection [HBeAg(?) CI;undetectable HBV‐DNA] and 8 healthy controls (HCs). p38MAPK phosphorylation was assessed by phospho‐specific flow cytometry in PBMCs and cell subsets (CD3+,CD3?,CD56+,CD56?) after stimulation with cytokines (IL‐12+IL‐2 and IL‐12+IL‐18) or nonspecific stimuli [arsenite, phorbol 12‐myristate 13‐acetate (PMA) and ionomycin] at 0,30,60,120 and 240 minutes using p38 phospho‐specific conjugated antibodies. ΙFN‐γ was determined by ELISA in PBMCs culture supernatants after stimulation with rhIL‐2, rhIL‐12 and rhIL‐18, with and without pre‐treatment with the p38 MAPK inhibitor, SB203580. HBeAg(?) CI patients showed the highest expression of phosphor‐p38 MAPK in total PBMCs and subpopulations compared to HBeAg(?) CHB and HCs. A striking impairment in p38 phosphorylation was noted in CD56+ cells and in especially in NK cells (CD3‐CD56+). SB203580‐induced inhibition of p38MAPK phosphorylation was associated with suppression of IFN‐γ production in all groups. The universal lack of p38 MAPK activation in CD56+ and in particular in NK cells from HBeAg(?) CHB patients during viremia suggests a potential cell‐dependent implication of this pathway in the natural history of HBV infection.     

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。     

Src family kinases and nitric oxide production are required for hepatocyte growth factor-stimulated endothelial cell growth

Hepatocyte growth factor (HGF) is a potent mitogen for vascular endothelial cells (EC); however, signal transduction pathways for HGF-stimulated EC growth remain unclear. In the present study we investigated the role of Src family kinases and nitric oxide (NO) in HGF-stimulated EC growth. Human umbilical vein endothelial cells (HUVEC) were stimulated with HGF and NO was measured by an NOx analyzing HPLC system. Activation of ERK1/2 and p38 MAPK was assessed by Western blot. NO production in HUVEC increased 1.8-fold by HGF. A Src family kinases inhibitor PP1 inhibited HGF-stimulated NO production by 71%. HUVEC growth increased 1.9-fold in cell number by HGF. PP1 and Nitro-L-arginine methylester (L-NAME) inhibited HGF-stimulated HUVEC growth by 51 and by 71%. ERK1/2 and p38 MAPK were phosphorylated by HGF and a MEK inhibitor PD98059 and a p38 MAPK inhibitor SB203580 inhibited HGF-stimulated HUVEC growth by 66% and by 58%; however, HGF-induced phosphorylation of ERK1/2 and p38 MAPK was not inhibited by L-NAME, indicating that NO is not an upstream activator of ERK1/2 and p38 MAPK. These findings demonstrated that Src family kinases regulate HGF-stimulated NO production in HUVEC and that HGF stimulates HUVEC growth through NO-dependent and NO-independent pathways.     

p38 MAPK-mediated signals are required for inducing osteoclast differentiation but not for osteoclast function

Receptor activator of nuclear factor-kappaB ligand (RANKL)-induced signals play critical roles in osteoclast differentiation and function. SB203580, an inhibitor of p38 MAPK, blocked osteoclast formation induced by 1alpha,25-dihydroxyvitamin D(3) and prostaglandin E(2) in cocultures of mouse osteoblasts and bone marrow cells. Nevertheless, SB203580 showed no inhibitory effect on RANKL expression in osteoblasts treated with 1alpha,25-dihydroxyvitamin D(3) and prostaglandin E(2). RANKL-induced osteoclastogenesis in bone marrow cultures was inhibited by SB203580, suggesting a direct effect of SB203580 on osteoclast precursors, but not on osteoblasts, in osteoclast differentiation. However, SB203580 inhibited neither the survival nor dentine-resorption activity of osteoclasts induced by RANKL. Lipopolysaccharide (LPS), IL-1, and TNFalpha all stimulated the survival of osteoclasts, which was not inhibited by SB203580. Phosphorylation of p38 MAPK was induced by RANKL, IL-1, TNFalpha, and LPS in osteoclast precursors but not in osteoclasts. LPS stimulated phosphorylation of MAPK kinase 3/6 and ATF2, upstream and downstream signals of p38 MAPK, respectively, in osteoclast precursors but not in osteoclasts. Nevertheless, LPS induced degradation of IkappaB and phosphorylation of ERK in osteoclasts as well as in osteoclast precursors. These results suggest that osteoclast function is induced through a mechanism independent of p38 MAPK-mediated signaling.     

p38 mitogen-activated protein kinase contributes to cell cycle regulation by cAMP in FRTL-5 thyroid cells

OBJECTIVE: Thyrotropin activates the cAMP pathway in thyroid cells, and stimulates cell cycle progression in cooperation with insulin or insulin-like growth factor-I. Because p38 mitogen-activated protein kinases (p38 MAPKs) were stimulated by cAMP in the FRTL-5 rat thyroid cell line, we investigated (i) the effect of the specific inhibition of p38 MAPKs on FRTL-5 cell proliferation and (ii) the mechanism of action of p38 MAPKs on cell cycle control, by studying the expression and/or the activity of several cell cycle regulatory proteins in FRTL-5 cells. METHODS: DNA synthesis was monitored by incorporation of [(3)H]thymidine into DNA and the cell cycle distribution was assessed by fluorescence-activated cell sorter analysis. Expression of cell cycle regulatory proteins was determined by Western blot analysis. Cyclin-dependent kinase 2 (Cdk2) activity associated to cyclin E was immunoprecipitated and was measured by an in vitro kinase assay. RESULTS: SB203580, an inhibitor of alpha and beta isoforms of p38 MAPKs, but not its inactive analog SB202474, inhibited DNA synthesis and the G1-S transition induced by forskolin plus insulin. SB203580 inhibited specifically p38 MAPK activity but not other kinase activities such as Akt and p70-S6 kinase. Treatment of FRTL-5 cells with SB203580 decreased total and cyclin E-associated Cdk2 kinase activity stimulated with forskolin and insulin. However, inhibition of p38 MAPKs by SB203580 was without effect on total cyclin E and Cdk2 levels. The decrease in Cdk2 kinase activity caused by SB203580 treatment was not due to an increased expression of p21(Cip1) or p27(Kip1) inhibitory proteins. In addition, SB203580 affected neither Cdc25A phosphatase expression nor Cdk2 Tyr-15 phosphorylation. Inhibition of p38 MAPKs decreased Cdk2-cyclin E activation by regulating the subcellular localization of Cdk2 and its phosphorylation on Thr-160. CONCLUSIONS: These results indicate that p38 MAPK activity is involved in the regulation of cell cycle progression in FRTL-5 thyroid cells, at least in part by increasing nuclear Cdk2 activity.     

Cellular events during arthritis-induced hyperalgesia are mediated by Interleukin-6 and p38 MAPK and their effects on the expression of spinal mu-opioid receptors

Activation of mitogen-activated protein kinase (MAPK) enzymes in nociceptive plasticity has been extensively studied. P38 MAPK enzyme, which can be activated by cytokines, acts as a crucial intracellular regulator of environmental changes. The aim of this study was to elucidate the cellular events during arthritis-induced hyperalgesia that are mediated by interleukin-6 and p38 MAPK, and their effects on the expression of spinal mu-opioid receptors (MORs), in different stages of arthritis in male Wistar rats. Complete Freund’s adjuvant (CFA)-induced arthritis (AA) was caused by subcutaneous injection of CFA into the rats’ hindpaw. Anti-IL-6 antibody and p38 MAPK phosphorylation inhibitor were administered during 21 days of study. Spinal MOR, p38, and phosphorylated-p38 (pp38) proteins expressions were detected by Western blotting. Daily treatment with anti-IL-6 antibody and p38 MAPK phosphorylation inhibitor, SB203580, significantly decreased paw edema in AA group. Daily anti-IL-6 and SB203580 administration caused a significant reduction in hyperalgesia in the first week of the study, but increased hyperalgesia in the next 2 weeks in experimental groups compared to the AA control group. Expression of pp38 MAPK protein significantly decreased on the 3, 7, 14, and 21 days in AA+SB203580 and AA+anti-IL6 groups compared to AA group. Additionally, daily treatment with anti-IL6 antibody and SB203580 in AA group caused significantly decrease in spinal MOR expression compared to AA control group. The results of our study can confirm that activated spinal p38 MAPK enzyme may play an important role in cellular IL-6 signaling pathways in hyperalgesia variation during different stages of AA inflammation. Also, it can be suggested that at least a part of p38 MAPK effects on hyperalgesia is mediated by spinal MOR expression variation.     

联合应用p38MAPK抑制剂对降低结肠癌阿霉素耐药性的影响

目的:探讨p38 MAPK抑制剂通过Akt(蛋白激酶B)有关的信号途径对结肠癌细胞阿霉素(Doxorubicin)化疗敏感性的影响.方法:应用阿霉素(Dox)和p38 MAPK特异性抑制剂SB203580及两者联合应用去处理结肠癌HCT-116细胞株.采用MTT[3-(4,5-二甲基噻唑-2)-2,5-二苯基四氮唑溴盐]法检测化疗药物对肿瘤细胞的生存抑制率,Western blot蛋白免疫印迹技术检测化疗药物处理后癌细胞Akt和磷酸化Akt的表达水平.结果:MTT显示阿霉素和抑制剂SB203580对结肠癌细胞均有抑制作用(26.60%,33.87%),后者大于前者,两者相同用量联合后癌细胞抑制率更明显(56.04%).Western blot显带说明阿霉素处理组较联合组的Akt表达水平并无明显差异,而磷酸化的Akt水平较SB203580组和联合组显著升高.亦即抑制剂组和联合组方案可降低结肠癌细胞磷酸化Akt的表达水平.结论:p38 MAPK抑制剂SB203580可能通过阻断P13K/Akt信号途径或其他有关Akt的通路以提高结肠癌细胞阿霉素化疗的敏感性.     

p38 mitogen-activated protein kinase and c-Jun-NH2-terminal kinase regulate interleukin-8 and RANTES production in hyperosmolarity stimulated human bronchial epithelial cells

OBJECTIVE: We have previously shown that p38 mitogen-activated protein kinase (MAPK) regulates, at least in part, hyperosmolarity induced interleukin (IL)-8 expression in human bronchial epithelial cells (BEC). In the previous study, hyperosmolarity also activated c-Jun-NH2-terminal kinase (JNK); however, the role of the JNK signalling pathway has not been determined. In the present study, we examined the role of the JNK signalling pathway in hyperosmolarity induced IL-8 and RANTES production by BEC using the novel inhibitor of the JNK signalling pathway CEP 11004 in order to clarify these issues. METHODS: Bronchial epithelial cells that had been pre-incubated with SB 203580, CEP 11004 or a combination of these were exposed to a hyperosmolar medium and then the p38 MAPK and JNK phosphorylation activity in these cells and IL-8 and RANTES concentrations in the culture supernatants were determined. RESULTS: The results showed that: (i) hyperosmolarity induced the threonine and tyrosine phosphorylation of p38 MAPK and JNK; (ii) SB 203580, as the specific inhibitor of p38 MAPK activity, and CEP 11004 attenuated hyperosmolarity induced p38 MAPK and JNK activity, respectively; (iii) SB 203580 and CEP 11004, but not PD 98059, partially attenuated IL-8 and RANTES production; and (iv) a combination of SB 203580 and CEP 11004 attenuated IL-8 and RANTES production in an additive fashion. CONCLUSION: These results indicate that p38 MAPK and the JNK pathway regulate hyperosmolarity induced IL-8 and RANTES production by BEC.     

p38MAPK信号转导通路在EGF诱导食管腺癌SEG-1细胞表达u-PA中的作用

目的:探讨表皮生长因子(epidermal growth factor,EGF)对人食管腺癌SEG-1细胞尿激酶型纤溶酶原激活物(urokinase-type plasminogen activator,u-PA)mRNA和蛋白表达的影响及p38MAPK信号转导通路在其中的作用.方法:以相同浓度的EGF(100g/L)按时间梯度刺激SEG-1细胞,应用Western blot法测定各时间点总p38MAPK蛋白、磷酸化p38MAPK蛋白、u-PA蛋白表达,并应用RT-PCR方法检测各时间点u-PAmRNA表达.用p38MAPK特异抑制剂SB203580预处理细胞后,观察上述指标变化.结果:EGF可明显增强SEG-1细胞(u-PA)mRNA和蛋白的表达,并可激活p38MAPK蛋白的磷酸化,具有时间依赖性.SB203580能明显抑制EGF诱导的p38MAPK蛋白的磷酸化,用其阻断p38MAPK信号转导通路后,EGF对u-PAmRNA和蛋白表达的诱导作用受到显著抑制,并且具有剂量依赖性.结论:EGF可通过p38MAPK信号转导通路诱导SEG-1细胞表达u-PA.     

P38 MAPK信号通路在金黄色葡萄球菌诱导U937细胞凋亡中的作用

目的建立金黄色葡萄球菌(Staphylococcus aureus)诱导人巨噬细胞系U937细胞凋亡模型,探讨p38 MAPK信号转导通路在此凋亡过程中的作用。方法采用Annexin V FITC/PI双染流式细胞仪检测金黄色葡萄球菌感染不同时间时U937细胞的凋亡率;利用Western blotting检测p38MAPK的磷酸化水平;预先用不同浓度的SB203580(p38 MAPK途径抑制剂)处理U937细胞,采用Annexin V FITC/PI双染分析感染30min后U937细胞的凋亡情况。结果金黄色葡萄球菌可以诱导U937细胞凋亡,并且随着感染时间的延长,细胞凋亡率也逐渐增高。p38 MAPK在加入金黄色葡萄球菌15min后表达明显增高,30min时达高峰,随后,逐渐降低。总的p38 MAPK水平在整个实验期间内几乎无变化。用SB203580抑制p38 MAPK通路,引起抑制剂浓度依赖性的细胞凋亡率降低。结论金黄色葡萄球菌呈时间依赖性诱导U937细胞凋亡,p38 MAPK信号转导通路的激活在金黄色葡萄球菌诱导的U937细胞凋亡过程中起到了重要的作用。     

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.     

Inhibition of p38 mitogen-activated protein kinase attenuates experimental autoimmune hepatitis： Involvement of nuclear factor kappa B

To investigate the role of p38 mitogen-activated protein kinase （p38MAPK） in murine experimental autoimmune hepatitis （EAH）.METHODS： To induce EAH, the syngeneic S-100 antigen emulsified in complete Freud＇s adjuvant was injected intraperitoneally into adult male C57BI/6 mice. Liver injury was assessed by serum ALT and liver histology. The expression and activity of p38 MAPK were measured by Western blot and kinase activity assays. In addition, DNA binding activities of nuclear factor kappa B （NF-KB） were analyzed by electrophoretic mobility shift assay. The effects of SB203580, a specific p38 MAPK inhibitor, on liver injuries and expression of proinflammatory cytokines （interferon-y, IL-12, IL-1β and TNF-α） were observed.RESULTS： The activity of p38 MAPK and NF-~：B was increased and reached its peak 14 or 21 d after the first syngeneic S-100 administration. Inhibition of p38 MAPK activation by SB203580 decreased the activation of NF-~：B and the expression of proinflammatory cytokines. Moreover, hepatic injuries were improved significantly after SB203580 administration.     

Syk contributes to PDGF-BB-mediated migration of rat aortic smooth muscle cells via MAPK pathways

OBJECTIVE: Here we investigated the role of spleen tyrosine kinase (Syk) in the migration induced by platelet-derived growth factor (PDGF) in rat aortic smooth muscle cells (RASMC). METHODS: Cell migration was determined using a Boyden chamber, by wound-healing, and by aortic ring assays. Activity of Syk, mitogen-activated protein kinase (MAPK), and heat shock protein 27 (HSP27) were tested using immunoblotting with kinase inhibitors and small interference RNAs. RESULTS: PDGF-BB induced binding of Syk to the PDGFbeta receptor and increased the phosphorylation of Syk and migration in RASMC. These effects of PDGF-BB were inhibited by piceatannol, an inhibitor of Syk. PDGF-BB increased the phosphorylation of extracellular signal-regulated kinase (ERK) 1/2, p38 MAPK, and HSP27, which were significantly inhibited by piceatannol and in Syk-knockdown cells. The p38 MAPK inhibitor SB203580 and ERK1/2 inhibitor PD98059 inhibited the migration, which was further inhibited by the combination of these inhibitors. SB203580, but not PD98059, inhibited the phosphorylation of HSP27 induced by PDGF-BB in RASMC. PDGF-BB-induced migration was attenuated in HSP27-knockdown cells. Kinase inhibitors and Syk-knockdown diminished PDGF-BB-induced sprout outgrowth in the aortic ring assay. CONCLUSIONS: These results imply that Syk is an upstream signal of the p38 MAPK/HSP27 and ERK1/2 pathways that contributes to PDGF-BB-mediated migration in RASMC.     

Dichotomal role of inhibition of p38 MAPK with SB 203580 in experimental colitis

BACKGROUND: Crohn's disease is characterised by a chronic relapsing inflammation of the bowel in which proinflammatory cytokines play an important perpetuating role. Mitogen activated protein kinase p38 (p38 MAPK) has been established as a major regulator of the inflammatory response, especially with regard to production of proinflammatory cytokines, but its role in inflammatory bowel disease is unexplored. In this paper we describe the effects of a specific p38 MAPK inhibitor, SB 203580, in trinitrobenzene sulphonic acid (TNBS) induced colitis in mice. RESULTS: SB 203580 had a dichotomal effect in TNBS mice. Weight loss of TNBS mice treated with SB 203580 was significantly worse and colon weight on sacrifice was significantly increased in MAPK inhibitor treated TNBS mice (229.2 mg and 289.1 mg, respectively). However, the total number of cells in the caudal lymph node decreased to 188.8 x 10(4) cells in SB 203580 treated TNBS mice compared with 334 x 10(4) cells in vehicle treated mice. CD3/CD28 double stimulated caudal lymph node cells of SB 203580 treated mice showed decreased interferon gamma production but increased tumour necrosis factor alpha production. The concentration of interleukin 12p70 in colon homogenates was significantly decreased in SB 203580 treated mice whereas concentrations of interleukin 12p40, tumour necrosis factor alpha, and interleukin 10 were similar in vehicle and SB 203580 treated TNBS mice. CONCLUSION: Our results reveal a dichotomy in p38 MAPK action during experimental colitis.     

Inhibition of p38 mitogen-activated protein kinase decreases cardiomyocyte apoptosis and improves cardiac function after myocardial ischemia and reperfusion

BACKGROUND: Activation of p38 mitogen-activated protein kinase (MAPK) plays an important role in apoptotic cell death. The role of p38 MAPK in myocardial injury caused by ischemia/reperfusion, an extreme stress to the heart, is unknown. METHODS AND RESULTS: Studies were performed with isolated, Langendorff-perfused rabbit hearts. Ischemia alone caused a moderate but transient increase in p38 MAPK activity (3.5-fold increase, P<0.05 versus basal). Ischemia followed by reperfusion further activated p38 MAPK, and the maximal level of activation (6.3-fold, P<0.01) was reached 10 minutes after reperfusion. Administration of SB 203580, a p38 MAPK inhibitor, decreased myocardial apoptosis (14.7+/-3.2% versus 30.6+/-3.5% in vehicle, P<0.01) and improved postischemic cardiac function. The cardioprotective effects of SB 203580 were closely related to its inhibition of p38 MAPK. Administering SB 203580 before ischemia and during reperfusion completely inhibited p38 MAPK activation and exerted the most cardioprotective effects. In contrast, administering SB 203580 10 minutes after reperfusion (a time point when maximal MAPK activation had already been achieved) failed to convey significant cardioprotection. Moreover, inhibition of p38 MAPK attenuated myocardial necrosis after a prolonged reperfusion. CONCLUSIONS: These results demonstrate that p38 MAPK plays a pivotal role in the signal transduction pathway mediating postischemic myocardial apoptosis and that inhibiting p38 MAPK may attenuate reperfusion injury.     

Reduction of insulin-stimulated glucose uptake in L6 myotubes by the protein kinase inhibitor SB203580 is independent of p38MAPK activity

Insulin increases glucose uptake through translocation of the glucose transporter GLUT4 to the plasma membrane. We previously showed that insulin activates p38MAPK, and inhibitors of p38MAPKalpha and p38MAPKbeta (e.g. SB203580) reduce insulin-stimulated glucose uptake without affecting GLUT4 translocation. This observation suggested that insulin may increase GLUT4 activity via p38alpha and/or p38beta. Here we further explore the possible participation of p38MAPK through a combination of molecular strategies. SB203580 reduced insulin stimulation of glucose uptake in L6 myotubes overexpressing an SB203580-resistant p38alpha (drug-resistant p38alpha) but barely affected phosphorylation of the p38 substrate MAPK-activated protein kinase-2. Expression of dominant-negative p38alpha or p38beta reduced p38MAPK phosphorylation by 70% but had no effect on insulin-stimulated glucose uptake. Gene silencing via isoform-specific small interfering RNAs reduced expression of p38alpha or p38beta by 60-70% without diminishing insulin-stimulated glucose uptake. SB203580 reduced photoaffinity labeling of GLUT4 by bio-LC-ATB-BMPA only in the insulin-stimulated state. Unless low levels of p38MAPK suffice to regulate glucose uptake, these results suggest that the inhibition of insulin-stimulated glucose transport by SB203580 is likely not mediated by p38MAPK. Instead, changes experienced by insulin-stimulated GLUT4 make it susceptible to inhibition by SB203580.