Human skeletal muscle cell differentiation is associated with changes in myogenic markers and enhanced insulin-mediated MAPK and PKB phosphorylation

AIM: We hypothesized that myogenic differentiation of HSMC would yield a more insulin responsive phenotype. METHODS: We assessed expression of several proteins involved in insulin action or myogenesis during differentiation of primary human skeletal muscle cultures (HSMC). RESULTS: Differentiation increased creatine kinase activity and expression of desmin and myocyte enhancer factor (MEF)2C. No change in expression was observed for big mitogen-activated protein kinase (BMK1/ERK5), MEF2A, insulin receptor (IR), hexokinase II, and IR substrates 1 and 2, while expression of glycogen synthase, extracellular signal-regulated kinase 1 and 2 (ERK1/2 MAP kinase) and the insulin responsive aminopeptidase increased after differentiation. In contrast to protein kinase B (PKB)a, expression of (PKB)b increased, with differentiation. Both basal and insulin-stimulated PI 3-kinase activity increased with differentiation. Insulin-mediated phosphorylation of PKB and ERK1/2 MAP kinase increased after differentiation. CONCLUSION: Components of the insulin-signalling machinery are expressed in myoblast and myotube HSMC; however, insulin responsiveness to PKB and ERK MAP kinase phosphorylation increases with differentiation.     

Differential activation of mitogen‐activated protein kinase signalling pathways by isometric contractions in isolated slow‐ and fast‐twitch rat skeletal muscle

Activation of mitogen‐activated protein (MAP) kinases has been implicated in the signal transduction pathways linking exercise to adaptive changes of muscle protein expression. In the present study, we investigated whether contractions of isolated muscles induced phosphorylation of extracellular signal‐regulated kinase 1 and 2 (ERK1/2) and p38 MAPK in a fibre‐type dependent manner. Slow‐twitch (soleus) and fast‐twitch (epitrochlearis, extensor digitorum longus) rat skeletal muscles were exposed to intermittent tetanic stimulation. Compared with the contralateral non‐stimulated muscle, contractions increased ERK1/2 phosphorylation to the same extent in fast‐ and slow‐twitch muscles. Significant increase in phosphorylation of p38 MAPK was observed in the fast‐twitch muscles only. The total amount of ERK1/2 and p38 MAPK proteins was higher in the slow‐twitch soleus muscle. In conclusion, MAP kinase signalling pathways are differentially activated and expressed in slow‐ and fast‐twitch muscles. In addition, this activation is owing to muscle contraction per se and do not demand additional external influence.     

H2O2-induced phosphorylation of ERK1/2 and PKB requires tyrosine kinase activity of insulin receptor and c-Src

Hydrogen peroxide (H2O2) mimics many physiological responses of insulin, and increased H2O2 generation via the Nox-4 subunit of NAD(P)H oxidase was recently demonstrated to serve as a critical early step in the insulin signaling pathway. Exogenously added H2O2 has also been shown to activate several key components of the insulin signaling cascade. H2O2-induced signaling responses have been found to be associated with the activation of receptor and nonreceptor protein tyrosine kinases (PTK), including the insulin receptor (IR)-beta subunit. Therefore, in the present studies on Chinese hamster ovary cells overexpressing wild-type IR-PTK (CHO-IR) or a PTK-inactive form of IR (CHO-1018), we investigated whether IR-PTK plays a role in H2O2-induced signaling events. Treatment of CHO-IR cells with H2O2 increased the phosphorylation of extracellular signal-regulated kinases 1 and 2 (ERK1/2), protein kinase B (PKB), and glycogen synthase kinase-3beta while enhancing tyrosine phosphorylation of the IR-beta subunit and the p85 subunit of phosphatidylinositol 3-kinase (PI3K). Compared with CHO-IR cells, the stimulatory effect of H2O2 on ERK1/2 and PKB was partially reduced in CHO-1018 cells. However, pharmacological inhibition of Src family PTK by 4-amino-5-(4-chlorophenyl)-7-(tert-butyl)pyrazolo[3,4-d]pyrimidine (PP-2) almost completely blocked H2O2-stimulated phosphorylation of the p85 subunit of PI3K, ERK1/2, and PKB. Moreover, H2O2, but not insulin, induced Tyr-418 phosphorylation of Src, which was also suppressed by PP-2. Taken together, these data suggest that both IR-PTK and Src family PTKs contribute to H2O2-induced signaling in CHO-IR cells albeit IR-PTK has a less dominant role in this process.     

白细胞介素6导致脂肪细胞胰岛素抵抗机制的初步探讨

目的： 观察IL-6对3T3-L1脂肪细胞胰岛素敏感性的影响并初步探讨其机制。 方法： 用IL-6处理3T3-L1脂肪细胞48h后，观察胰岛素刺激的葡萄糖摄取，IRS-1蛋白表达和酪氨酸磷酸化以及PKB磷酸化水平。同时观察mTOR抑制剂那巴霉素对IL-6作用的影响。结果： IL-6使胰岛素刺激的葡萄糖摄取和PKB磷酸化下降约50％，同时明显降低IRS-1蛋白表达（约35％）和酪氨酸磷酸化（约40％）水平。IL-6的上述作用可被那巴霉素逆转。结论： IL-6导致的脂肪细胞胰岛素抵抗与IRS-1表达减少和酪氨酸磷酸化水平下降有关，那巴霉素可以逆转IL-6的作用，mTOR可能参与IL-6导致的胰岛素抵抗的发生。     

Post‐ischaemic activation of kinases in the pre‐conditioning‐like cardioprotective effect of the platelet‐activating factor

Aim: Platelet‐activating factor (PAF) triggers cardiac pre‐conditioning against ischemia/reperfusion injury. The actual protection of ischaemic pre‐conditioning occurs in the reperfusion phase. Therefore, we studied in this phase the kinases involved in PAF‐induced pre‐conditioning. Methods: Langendorff‐perfused rat hearts underwent 30 min of ischaemia and 2 h of reperfusion (group 1, control). Before ischaemia, group 2 hearts were perfused for 19 min with PAF (2 × 10^?11 m ); groups 3–5 hearts were co‐infused during the initial 20 min of reperfusion, with the protein kinase C (PKC) inhibitor chelerythrine (5 × 10^?6 m ) or the phosphoinositide 3‐kinase (PI3K) inhibitor LY294002 (5 × 10^?5 m ) and atractyloside (2 × 10^?5 m ), a mitochondrial permeability transition pore (mPTP) opener respectively. Phosphorylation of PKCε, PKB/Aκt, GSK‐3β and ERK1/2 at the beginning of reperfusion was also checked. Left ventricular pressure and infarct size were determined. Results: PAF pre‐treatment reduced infarct size (33 ± 4% vs. 64 ± 5% of the area at risk of control hearts) and improved pressure recovery. PAF pre‐treatment enhanced the phosphorylation/activation of PKCε, PKB/Aκt and the phosphorylation/inactivation of GSK‐3β at reperfusion. Effects on ERK1/2 phosphorylation were not consistent. Infarct‐sparing effect and post‐ischaemic functional improvement induced by PAF pre‐treatment were abolished by post‐ischaemic infusion of either chelerythrine, LY294002 or atractyloside. Conclusions: The cardioprotective effect exerted by PAF pre‐treatment involves activation of PKC and PI3K in post‐ischaemic phases and might be mediated by the prevention of mPTP opening in reperfusion via GSK‐3β inactivation.     

Editor's Choice

Aim: The purpose of this experiment was to investigate the role of extracellular signal‐regulated kinase 1/2 (ERK1/2) signalling in the contraction‐induced increase in muscle FA uptake. Methods: Male Wistar rats (n = 41) were randomly assigned to either a resting or stimulated group. Within each group, animals were randomly assigned to receive PD‐98059, an inhibitor of MAP/ERK kinase 1/2 (MEK1/2), a kinase upstream of ERK1/2 and perfused with 550 μm palmitate, [¹⁴C]palmitate, 7 mm glucose, and no insulin. In the stimulated group, electrical stimulation (ES) of supramaximal trains of 100 ms was delivered every 2 s for 20 min. Results: ERK1/2 phosphorylation was increased by 50% (P < 0.05) during ES but the contraction‐induced increase was prevented by the addition of PD‐98059. Glucose uptake increased by 3.6‐fold (P < 0.05) from rest to ES in muscle perfused without PD‐98059 and was not affected by the addition of PD‐98059 either at rest (P > 0.05) or during ES (P > 0.05). For a matched palmitate delivery, ES increased palmitate uptake by 35% (P < 0.05). PD‐98059 had no effect on palmitate uptake at rest but completely abolished the increase in palmitate uptake during ES. Plasma membrane FAT/CD36 protein content was increased by 38% during ES (P < 0.05) but the contraction‐induced increase was prevented by the addition of PD‐98059. AMPK activity was increased by ES (P < 0.05) but was unaffected by PD‐98059. Conclusion: These results show for the first time that the increase in FA uptake and in plasma membrane FAT/CD36 protein content is mediated, at least in part, by the ERK1/2 signalling pathway during muscle contraction.     

Distinct roles of Ca2+, calmodulin, and protein kinase C in H2O2-induced activation of ERK1/2, p38 MAPK, and protein kinase B signaling in vascular smooth muscle cells

We have shown earlier that extracellular signal-regulated kinases 1 and 2 (ERK1/2) and protein kinase B (PKB), two key mediators of growth-promoting and proliferative responses, are activated by hydrogen peroxide (H(2)O(2)) in A10 vascular smooth muscle cells (VSMC). In the present studies, using a series of pharmacological inhibitors, we explored the upstream mechanisms responsible for their activation in response to H(2)O(2). H(2)O(2) treatment of VSMC stimulated ERK1/2, p38 mitogen-activated protein kinase (MAPK), and PKB phosphorylation in a dose- and time-dependent fashion. BAPTA-AM and EGTA, chelators of intracellular and extracellular Ca(2+), respectively, inhibited H(2)O(2)-stimulated ERK1/2, p38 MAPK, and PKB phosphorylation. Fluphenazine, an antagonist of the Ca(2+)-binding protein calmodulin, also suppressed the enhanced phosphorylation of ERK1/2, p38 MAPK, and PKB. In contrast, the protein kinase C (PKC) inhibitors G? 6983 and R? 31-8220 attenuated H(2)O(2)-induced ERK1/2 phosphorylation, but had no effect on p38 MAPK and PKB phosphorylation. Taken together, these data demonstrate that the activation of Ca(2+)/calmodulin-dependent pathways represents a key component mediating the stimulatory action of H(2)O(2) on ERK1/2, p38 MAPK, and PKB phosphorylation. On the other hand, PKC appears to be an upstream modulator of the increased ERK1/2 phosphorylation, but not of p38 MAPK and PKB in response to H(2)O(2) in VSMC.     

Normal p21Ras/MAP kinase pathway expression and function in PBMC from patients with polycystic ovary disease

Polycystic ovary disease (PCOD) is associated with insulin resistance and increased prevalence of type II diabetes mellitus (T2DM). The p21Ras/MAP kinase is a major intracellular signaling pathway mediating insulin signaling in insulin responsive tissues. The expression, regulation and function of the p21Ras/MAP kinase pathway in PCOD patients were examined. Peripheral blood mononuclear cells (PBMC) were isolated from ten patients with PCOD and ten controls. The expression of p21Ras and its regulatory proteins; hSOS1 and p120GAP were studied. The basal and phytohemaglutinin (PHA) or insulin stimulated phosphorylation of MAP kinase was determined. Expression of p21Ras, and its regulatory proteins hSOS1 and p120GAP were similar in PCOD patients and controls. Basal, PHA and insulin stimulated phosphorylation of MAP kinase, were also comparable in the two groups as well as their PBMC proliferative response. These data indicate that the expression and overall function of the p21Ras/MAP kinase pathway remain intact in non-diabetic patients with PCOD.     

Stress induces the expression of heterotrimeric G protein beta subunits and the phosphorylation of PKB/Akt and ERK1/2 in rat brain

Various heterotrimeric G protein betagamma subunits (Gbetagamma) are region-specifically expressed in brain where associated with "stress-axis", however, the role of Gbetagamma-mediated signaling in regulating stress is unknown. This study was designed to examine the changes of Gbetagamma expression and Gbetagamma-mediated signaling in rat brain by stress. Experimental stress was induced by immobilization (2h/day for 7 days) and the level of mRNAs and proteins for Gbeta(1-5), and the phosphorylation of PKB/Akt (phosphatidylinositol 3-kinase-linked protein kinase B) and ERK1/2 (extracellular signal-regulated kinase 1/2) were measured in five different regions of rat brain including frontal cortex, striatum, hypothalamus, hippocampus, and cerebellum. As compared in not-handled non-stressed animals, the expression of both mRNAs and proteins for Gbeta(1-5) in brain regions associated with stress was increased in stressed animals. Especially, a significant increase in Gbetas immunoreactivity in the caudate putamen, the paraventricular nucleus of the hypothalamus (PVN), and the dentate gyrus of the hippocampus (DG) of stressed rats was observed. Stress significantly induced the phosphorylation of PKB/Akt and ERK1/2 in striatum, hypothalamus and hippocampus. Therefore, these results suggest that stress may activate, at least in part, the Gbetagamma-mediated PKB/Akt and ERK1/2 signaling pathway by increasing the expression of Gbetas to regulate the physiological responses.     

PKB在类胰蛋白酶诱导基因表达中的作用

目的：探讨蛋白激酶B(PKB)在类胰蛋白酶（tryptase）诱导基因表达中的作用。 方法： 采用RT-PCR和Western blotting 方法，检测tryptase对ECV304细胞PKB（Akt）的表达及其活性和对转录因子AP-1、NF-κB p65亚单位、JNK、p38MAPK、趋化因子IL-8表达的影响。 结果： 在ECV304中1 μg/L tryptase可使PKB蛋白质磷酸化水平增加并促进PKB、转录因子NF-κB P65亚单位、AP-1和趋化因子IL-8的表达，但对JNK、p38MAPK表达影响不大。PI3K特异性抑制剂LY294002可抑制PKB的表达增加，同时可抑制NF-κB P65亚单位和IL-8的表达增加；反义PKB质粒瞬时转染ECV304，可抑制PKB、AP1、NF-κB P65亚单位和IL-8的表达增加；PAR2的抗体可抑制PKB的磷酸化，但不能阻断PKB表达。 结论： 在ECV304细胞tryptase经其膜受体PAR2通过PI3K促进PKB的磷酸化而激活之，通过其下游途径促进趋化因子IL-8、转录因子AP-1、NF-κB P65亚单位和PKB本身的表达增加。     

IQGAP1在人骨髓瘤细胞中过表达且与ERK相互作用

 目的：探讨含IQ模序的GTP酶活化蛋白1(IQ motif-containing GTPase-activating protein 1,IQGAP1)在骨髓瘤细胞中的过表达及其机制。方法：RT-PCR和Western blotting 检测RPMI8226和U266细胞IQGAP1表达;使用不同的寡核苷酸序列构建沉默人IQGAP1的shRNA质粒（clone 1、clone 2、clone 3和clone 4）、转染RPMI8226细胞,RT-PCR和Western blotting检测其IQGAP1表达;Western blotting检测RPMI8226-shIQGAP1组 (clone 1)、RPMI8226-shRNA阴性对照组和RPMI8226未转染组细胞p-ERK1/2、ERK1/2、Akt、p-AKT、STAT3和p-STAT3水平,免疫共沉淀确定IQGAP1和ERK的关系。结果：IQGAP1在RPMI8226和U266骨髓瘤细胞株中过表达,使用shRNA表达质粒沉默RPMI8226细胞IQGAP1 后,IQGAP1表达减少,在有或无血管内皮生长因子（VEGF）/白细胞介素6（IL-6）作用下检测RPMI8226-shIQGAP1组 (clone 1) 细胞增殖明显下降。进一步检测3组细胞p-ERK1/2、ERK1/2、AKT、p-AKT、STAT3和p-STAT3蛋白水平,与RPMI8226-shRNA阴性对照组和RPMI8226未转染组相比,RPMI8226-shIQGAP1组ERK1/2磷酸化水平下降70.2%,免疫共沉淀法明确在RPMI8226细胞中IQGAP1和ERK存在相互作用。结论：IQGAP1在骨髓瘤细胞中的过表达可能与MAPK途径的ERK相互作用有关。     

Reversal of Multidrug Resistance by Gefitinib Via RAF1/ERK Pathway in Pancreatic Cancer Cell Line

Pancreatic cancer is a devastating malignancy, characterized by intrinsic or acquired resistance to conventional chemotherapies. Recent evidences suggest an involvement of tyrosine kinase pathway in the regulation of multidrug resistance (MDR) protein gene expression. The aim of this study was to test whether gefitinib, an epidermal growth factor receptor tyrosine kinase inhibitor could regulate the MDR protein gene expression and sensitize the resistant cancer cells to chemotherapy. The gene expression of MDR proteins (MRP1, MRP2, MRP3, and PGP) were evaluated by quantitative RT‐PCR, and expression levels of various tyrosine kinases were investigated by quantitative RT‐PCR and Western Blot in pancreatic cancer cell line. MTT assay was used for evaluating the effect of chemotherapeutic agents. Chemotherapeutics induced drug resistance by regulating the gene expression of MDR proteins (MRP1, MRP2, and MRP3), and increased the gene expression of RAF1/ERK and the phosphorylation of ERK in pancreatic cancer Bxpc‐3 cells. Gefitinib caused an inhibition of p‐ERK tyrosine kinase activation in a dose‐dependent manner, and reversed gemcitabine‐induced RAF1/ERK gene expression and p‐ERK activation. In addition, a reversal of MDR proteins gene expression was achieved by gefitinib, which sensitized resistant cells to gemcitabine. This study demonstrated that MDR of Bxpc‐3 cell is involved in the RAF1/ERK tyrosine kinase pathway. Gefitinib reverses the MDR protein gene expression and restores sensitivity of resistant cells to gemcitabine via RAF1/ERK signaling pathway. Combination of gefitinib with conventional chemotherapeutic agents may offer a new approach for the treatment of patients with pancreatic cancer. Anat Rec, 2012. © 2012 Wiley Periodicals, Inc.     

Mechanisms of H4/ICOS costimulation: effects on proximal TCR signals and MAP kinase pathways

H4/ICOS is a costimulatory molecule related to CD28. Its effects on early TCR signals have been analyzed in mouse CD4(+) Th2 cells, expressing H4/ICOS at higher levels than Th1 clones. Anti-H4/ICOS antibodies strongly enhanced CD3-mediated tyrosine phosphorylation of ZAP-70, zeta, or Vav, as well as extracellular signal-regulated kinase (ERK), Jun N-terminal kinase (JNK) and p38 MAP kinase activation in these cells. The association of phosphoinositide 3-kinase (PI-3K) to H4/ICOS was enhanced by H4/ICOS cross-linking, and PI-3K inhibitors inhibited ERK and JNK activation and IL-4/IL-10 secretion, but not p38 MAP kinase or ZAP-70 activation. H4/ICOS-mediated activation of JNK, but not ERK or p38, is partially dependent on the expression of CD4 by the cells, whereas H4/ICOS costimulation is partially independent on CD28 expression. Cytochalasin D, an inhibitor of actin polymerization, inhibited ZAP-70, MAP kinase activation, or IL-4/IL-10 secretion. Neither cyclosporin A nor inhibitors of PKC produced detectable inhibition of ZAP-70 phosphorylation or MAP kinase activation in these Th2 cells. Cyclosporin A strongly inhibited IL-4, but not IL-10 secretion. ERK or JNKinhibitors partially inhibited IL-4 and IL-10 secretion, while PKC or p38 inhibitors had no significant effects on IL-4 or IL-10 secretion. Taken together, our data show clear similarities of costimulation mechanisms between H4/ICOS and CD28 during the early steps of TCR activation.     

ERK5对体外血小板活化及在体血栓的影响

目的:探讨细胞外信号调节激酶5(ERK5)对体外血小板聚集及在体血栓的影响及机制。方法:采用Western blot对人血小板中ERK5的表达及其在血小板活化后的磷酸化水平进行检测;采用血小板聚集仪检测ERK5特异性抑制剂XMD8-92对血小板聚集及致密颗粒释放的影响;采用Fe Cl3颈动脉血栓模型检测ERK5对在体血栓的影响;采用Western blot检测XMD8-92对蛋白激酶B(Akt)和人第10号染色体缺失的磷酸酶及张力蛋白同源蛋白(PTEN)磷酸化的影响。结果:人血小板中存在ERK5的稳定表达,其磷酸化水平在血小板活化后显著升高(P0.05)。XMD8-92可抑制多种血小板激活剂引起的血小板聚集和致密颗粒释放(P0.05)。Western blot结果表明,XMD8-92可通过下调PTEN Ser370位点磷酸化而增强PTEN的活性,从而抑制Akt的磷酸化,这种抑制效果也通过血小板特异PTEN基因敲除小鼠得到了验证。在体血栓研究表明,XMD8-92经尾静脉给药,可显著延长小鼠第一次颈动脉血栓的形成时间。结论:ERK5可通过影响PTEN的磷酸化调节Akt的活化,进而影响到体外血小板的聚集和在体血栓的形成。     

p38 mitogen-activated protein kinase regulates growth factor-induced mitogenesis of rat pulmonary myofibroblasts

Myofibroblast proliferation is a central feature of pulmonary fibrogenesis. Several growth factors, including platelet-derived growth factor (PDGF) and epidermal growth factor (EGF), stimulate myofibroblast growth by activating extracellular signal regulated kinases 1 and 2 (ERK1/2). In this report, we demonstrate that PDGF-BB and EGF also activate the p38 mitogen-activated protein (MAP) kinase. Inhibition of p38 activity with the pyridinylimidazole compound SB203580 enhanced both PDGF-BB and EGF-stimulated DNA synthesis in rat lung myofibroblasts. ERK1/2 phosphorylation in response to either PDGF-BB or EGF treatment was significantly increased by pretreatment of cells with SB203580. We also demonstrated that ERK1/2-induced phosphorylation of PHAS-1 substrate was enhanced by inhibition of p38 MAP kinase with SB203580. However, SB203580 did not significantly increase growth factor-induced activation of MEK, the upstream kinase that phosphorylates ERK1/2. p38 MAP kinase was co-immunoprecipitated with ERK-1/2 following growth factor stimulation. Collectively, these data demonstrate that p38 MAP kinase activation negatively regulates PDGF- and EGF-mediated growth responses by directly interacting with ERK1/2 and suppressing its phosphorylation.     

Decrease in Akt/PKB signalling in human skeletal muscle by resistance exercise

We analysed the effects of resistance exercise upon the phosphorylation state of proteins associated with adaptive processes from the Akt/PKB (protein kinase B) and the mitogen-activated protein kinase (MAPK) pathways. Nine healthy young men (21.7 +/- 0.55 year) performed 10 sets of 10 leg extensions at 80% of their 1-RM (repetition maximum). Muscle biopsies were taken from the vastus lateralis at rest, within the first 30 s after exercise and at 24 h post-exercise. Immediately post exercise, the phosphorylation states of Akt/PKB on Thr308 and Ser473 and 4E-BP1 on Thr37/46 (eukaryotic initiation factor 4E-binding protein 1) were decreased (-60 to -90%, P < 0.05). Conversely, the phosphorylation of p70(s6k) (p70 ribosomal S6 kinase) on Thr421/Ser424 was increased more than 20-fold (P < 0.05), and this was associated with a 10- to 50-fold increase in the phosphorylation of p38 and ERK1/2 (extracellular signal-regulated kinase) (P < 0.05). Twenty-four hours post-exercise the phosphorylation state of Akt/PKB on Thr308 was depressed, whereas the phosphorylation of p70(s6k) on Thr421/Ser424 and sarcoplasmic ERK1/2 were elevated. The present results indicate that high-intensity resistance exercise in the fasted state inhibits Akt/PKB and 4E-BP1 whilst concomitantly augmenting MAPK signalling and p70(s6k) on Thr421/Ser424.     

The mitogen-activated/extracellular signal-regulated kinase kinase 1/2 inhibitor U0126 induces glial fibrillary acidic protein expression and reduces the proliferation and migration of C6 glioma cells

The extracellular signal-regulated kinase (ERK) signaling pathway has been implicated in diverse cellular functions. ERK and its activating kinase, mitogen-activated/extracellular signal-regulated kinase kinase (MEK), are downstream of cell surface receptors known to be up-regulated in many malignant gliomas. We sought to investigate the role of ERK in glioma cell migration, proliferation and differentiation using the rat-derived C6 glioma cell line and the MEK inhibitor, U0126. Treatment of C6 cells with U0126 caused a significant concentration-dependent reduction in cell proliferation and migration and also induced expression of glial fibrillary acidic protein, a marker of astrocytic differentiation. These results suggest that the ERK pathway regulates glioma cell proliferation, migration and differentiation.     

Differential activation of mitogen-activated protein kinase signalling pathways by isometric contractions in isolated slow- and fast-twitch rat skeletal muscle

Activation of mitogen-activated protein (MAP) kinases has been implicated in the signal transduction pathways linking exercise to adaptive changes of muscle protein expression. In the present study, we investigated whether contractions of isolated muscles induced phosphorylation of extracellular signal-regulated kinase 1 and 2 (ERK1/2) and p38 MAPK in a fibre-type dependent manner. Slow-twitch (soleus) and fast-twitch (epitrochlearis, extensor digitorum longus) rat skeletal muscles were exposed to intermittent tetanic stimulation. Compared with the contralateral non-stimulated muscle, contractions increased ERK1/2 phosphorylation to the same extent in fast- and slow-twitch muscles. Significant increase in phosphorylation of p38 MAPK was observed in the fast-twitch muscles only. The total amount of ERK1/2 and p38 MAPK proteins was higher in the slow-twitch soleus muscle. In conclusion, MAP kinase signalling pathways are differentially activated and expressed in slow- and fast-twitch muscles. In addition, this activation is owing to muscle contraction per se and do not demand additional external influence.     

Regulation and signal transduction of toll-like receptors in human chorioncarcinoma cell lines

PROBLEM: Toll-like receptors (TLRs) are expressed on placental cells. The aim of this study is to analyze signaling components activated in placenta cells after TLR ligand engagement. METHODS OF STUDY: In chorioncarcimoma cell lines the regulation of TLRs was determined by real time polymerase chain reaction as well as by fluorescence-activated cell sorter analysis. Activation of NF-kappaB was determined in a reporter assay system and the activation of the mitogen-activated protein kinase signaling pathways by immunoblot analysis. RESULTS: Both lipopolysaccharide (LPS) and DNA oligonucleotides containing unmethylated CpG motifs (CpG) induced the enhanced expression of TLR2 mRNA as well as a TLR2 surface protein expression. Functionally, incubation of JAR cells with microbial stimuli such as LPS activated NF-kappaB, as well as the phosphorylation of ERK1/2 and p38 MAP kinases and secretion of interleukin-8. CONCLUSION: The functional expression of TLRs on placental cells may play an important role in the initiation of an immune response in the developing fetus.