Restoration of impaired p38 activation by insulin in insulin resistant skeletal muscle cells treated with thiazolidinediones

We have previously reported that thiazolidinediones (TZDs) are able to restore the tyrosine phosphorylation of insulin receptor and insulin receptor substrate-1, activation of phosphatidyl inositol 3-kinase and glucose uptake in insulin resistant skeletal muscle cells [21]. In this study, we investigated the effects of insulin stimulation and TZDs on the role of mitogen-activated protein kinase (MAPK) in insulin resistant skeletal muscle cells. All the three MAPKs [extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38 MAPK] were activated by insulin in the sensitive skeletal muscle cells. In contrast, activation of p38 MAPK was impaired in insulin resistant cells, where as ERK and JNK were activated by insulin. Treatment with TZDs resulted in the restoration of p38 MAPK activity in insulin resistant cells. The treatment of cells with p38 MAPK inhibitor, SB203580, blocked the insulin stimulated glucose uptake in sensitive as well as resistant cells and it also prevented the activation of p38 by insulin. These results suggest the potential involvement of p38 as well as the mechanistic role of TZDs in insulin resistance.     

Restoration of impaired p38 activation by insulin in insulin resistant skeletal muscle cells treated with thiazolidinediones

We have previously reported that thiazolidinediones (TZDs) are able to restore the tyrosine phosphorylation of insulin receptor and insulin receptor substrate-1, activation of phosphatidyl inositol 3-kinase and glucose uptake in insulin resistant skeletal muscle cells. In this study, we investigated the effects of insulin stimulation and TZDs on the role of mitogen-activated protein kinase (MAPK) in insulin resistant skeletal muscle cells. All the three MAPKs [extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38 MAPK] were activated by insulin in the sensitive skeletal muscle cells. In contrast, activation of p38 MAPK was impaired in insulin resistant cells, where as ERK and JNK were activated by insulin. Treatment with TZDs resulted in the restoration of p38 MAPK activity in insulin resistant cells. The treatment of cells with p38 MAPK inhibitor, SB203580, blocked the insulin stimulated glucose uptake in sensitive as well as resistant cells and it also prevented the activation of p38 by insulin. These results suggest the potential involvement of p38 as well as the mechanistic role of TZDs in insulin resistance.     

Enhancement of fibroblast collagenase-1 (MMP-1) gene expression by tumor promoter okadaic acid is mediated by stress-activated protein kinases jun N-terminal kinase and p38

Collagenase-1 (matrix metalloproteinase-1, MMP-1) is expressed by several types of cells, including fibroblasts, and apparently plays an important role in the remodeling of collagenous extracellular matrix in various physiologic and pathologic situations. Here, we have examined the molecular mechanisms of the activation of fibroblast MMP-1 gene expression by a naturally occurring non-phorbol ester type tumor promoter okadaic acid (OA), a potent inhibitor of serine/threonine protein phosphatase 2A. We show that in fibroblasts OA activates three distinct subgroups of mitogen activated protein kinases (MAPKs): extracellular signal-regulated kinase 1,2 (ERK 1,2), c-Jun N-terminal-kinase/stress-activated protein kinase (JNK/SAPK) and p38. Activation of MMP-1 promoter by OA is entirely blocked by overexpression of dual-specificity MAPK phosphatase CL100. In addition, expression of kinase-deficient forms of ERK 1,2, SAPKβ, p38, or JNK/SAPK kinase SEK1 strongly inhibited OA-elicited activation of MMP-1 promoter. OA-elicited enhancement of MMP-1 mRNA abundance was also strongly prevented by two chemical MAPK inhibitors: PD 98059, a specific inhibitor of the activation of ERK1,2 kinases MEK1,2; and SB 203580, a selective inhibitor of p38 activity. Results of this study show that MMP-1 gene expression in fibroblasts is coordinately regulated by ERK1,2, JNK/SAPK, and p38 MAPKs and suggest an important role for the stress-activated MAPKs JNK/SAPK and p38 in the activation of MMP-1 gene expression. Based on these observations, it is conceivable that specific inhibition of stress-activated MAPK pathways may serve as a novel therapeutic target for inhibiting degradation of collagenous extracellular matrix.     

Differential activation of stress-responsive signalling proteins associated with altered loading in a rat skeletal muscle

Skeletal muscle undergoes a significant reduction in tension upon unloading. To explore intracellular signalling mechanisms underlying this phenomenon, we investigated twitch tension, the ratio of actin/myosin filaments, and activities of key signalling molecules in rat soleus muscle during a 3-week hindlimb suspension and 2-week reloading. Twitch tension and myofilament ratio (actin/myosin) gradually decreased during unloading but progressively recovered to initial levels during reloading. To study the involvement of stress-responsive signalling proteins during these changes, the activities of protein kinase C alpha (PKCalpha) and three mitogen-activated protein kinases (MAPKs)--c-Jun NH2-terminal kinase (JNK), extracellular signal-regulated protein kinase (ERK), and p38 MAPK--were examined using immunoblotting and immune complex kinase assays. PKCalpha phosphorylation correlated positively with the tension (Pearson's r = 0.97, P < 0.001) and the myofilament ratio (r = 0.83, P < 0.01) over the entire unloading and reloading period. Treatment of the soleus muscle with a PKC activator resulted in a similar paralleled increment in both PKCalpha phosphorylation and the alpha-sarcomeric actin expression. The three MAPKs differed in the pattern of activation in that JNK activity peaked only for the first hours of reloading, whereas ERK and p38 MAPK activities remained elevated during reloading. These results suggest that PKCalpha may play a pivotal role in converting loading stress to intracellular changes in contractile proteins that determine muscle tension. Differential activation of MAPKs may also help alleviate muscle damage, modulate energy transport and/or regulate the expression of contractile proteins upon altered loading.     

Molecular cloning and characterization of a novel dual specificity phosphatase, MKP-5.

A group of dual specificity protein phosphatases negatively regulates members of the mitogen-activated protein kinase (MAPK) superfamily, which consists of three major subfamilies, MAPK/extracellular signal-regulated kinase (ERK), stress-activated protein kinase (SAPK)/c-Jun N-terminal kinase (JNK), and p38. Nine members of this group of dual specificity phosphatases have previously been cloned. They show distinct substrate specificities for MAPKs, different tissue distribution and subcellular localization, and different modes of inducibility of their expression by extracellular stimuli. Here we have cloned and characterized a novel dual specificity phosphatase, which we have designated MKP-5. MKP-5 is a protein of 482 amino acids with a calculated molecular mass of 52.6 kDa and consists of 150 N-terminal amino acids of unknown function, two Cdc25 homology 2 regions in the middle, and a C-terminal catalytic domain. MKP-5 binds to p38 and SAPK/JNK, but not to MAPK/ERK, and inactivates p38 and SAPK/JNK, but not MAPK/ERK. p38 is a preferred substrate. The subcellular localization of MKP-5 is unique; it is present evenly in both the cytoplasm and the nucleus. MKP-5 mRNA is widely expressed in various tissues and organs, and its expression in cultured cells is elevated by stress stimuli. These results suggest that MKP-5 is a novel type of dual specificity phosphatase specific for p38 and SAPK/JNK.     

MAPK signalling in rheumatoid joint destruction: can we unravel the puzzle?

Mitogen-activated protein kinases (MAPKs) have been associated with the pathogenesis of rheumatoid arthritis (RA), but the individual contributions of the three MAPK family members are incompletely understood. Although previous data have established a role for c-Jun N-terminal kinase (JNK) and extracellular signal-related kinase (ERK) in different animal models of arthritis, most recent data indicate that the stable activation of p38 MAPK and in part of ERK significantly contributes to destructive arthritis in mice transgenic for human tumour necrosis factor-alpha. These data highlight the complexity of MAPK signalling in arthritis and provide a basis for the design of novel strategies to treat human RA.     

Suppression of extracellular signal-regulated kinase activity in herpes simplex virus 1-infected cells by the Us3 protein kinase

Host mitogen-activated protein kinases (MAPKs) are deregulated by herpes simplex virus 1 (HSV-1). Unlike p38 MAPK and Jun N-terminal protein kinase (JNK), which require ICP27 for their activation early in infection, extracellular signal-regulated kinase (ERK) activity is suppressed by an unknown mechanism. Here, we establish that HSV-1-induced suppression of ERK activity requires viral gene expression, occurs with delayed-early kinetics, and requires the functional virus-encoded Us3 Ser/Thr protein kinase. Finally, Us3 expression in uninfected cells was necessary and sufficient to suppress ERK activity in the absence of any other virus-encoded gene products. This demonstrates that inhibition of ERK activity in HSV-1-infected cells is an intrinsic Us3 function and defines a new role for this alphaherpesvirus Us3 kinase in regulating MAPK activation in infected cells.     

Mitogen-activated protein kinases mediate stretch-induced c-fos mRNA expression in myometrial smooth muscle cells

Evidence indicates that stretch of theuterus imposed by the growing fetus contributes to the onset of labor.Previously we have shown that mechanically stretching rat myometrialsmooth muscle cells (SMCs) induces c-fos expression. Toinvestigate this stretch-induced signaling, we examined the involvementof the mitogen-activated protein kinase (MAPK) family. We show thatstretching rat myometrial SMCs induces a rapid and transientphosphorylation (activation) of MAPKs: extracellular signal-regulatedprotein kinase (ERK), c-Jun NH₂-terminal kinase (JNK), andp38. The use of selective inhibitors for the ERK pathway (PD-98059 andU-0126), p38 (SB-203580), and JNK pathway (curcumin) demonstrated that activation of all three MAPK signaling pathways was necessary foroptimal stretch-induced c-fos expression. We alsodemonstrate that upstream tyrosine kinase activity is involved in themechanotransduction pathway leading to stretch-induced MAPK activationand c-fos mRNA expression. To further examine the role ofMAPKs in vivo, we used a unilaterally pregnant rat model. MAPKs (ERKand p38) are expressed in the pregnant rat myometrium with maximal ERKand p38 phosphorylation occurring in the 24 h immediatelypreceding labor. Importantly, the rise in MAPK phosphorylation wasconfined to the gravid horn and was absent in the empty uterine horn,suggesting that mechanical strain imposed by the growing fetus controlsMAPK activation in the myometrium. Collectively, this data indicatethat mechanical stretch modulates MAPK activity in the myometriumleading to c-fos expression.

     

Novel crosstalk between ERK MAPK and p38 MAPK leads to homocysteine‐NMDA receptor‐mediated neuronal cell death

Hyperhomocysteinemia is an independent risk factor for both acute and chronic neurological disorders, but little is known about the underlying mechanisms by which elevated homocysteine can promote neuronal cell death. We recently established a role for NMDA receptor‐mediated activation of extracellular signal‐regulated kinase (ERK)‐MAPK in homocysteine‐induced neuronal cell death. In this study, we examined the involvement of the stress‐induced MAPK, p38 in homocysteine‐induced neuronal cell death, and further explored the relationship between the two MAPKs, ERK and p38, in triggering cell death. Homocysteine‐mediated NMDA receptor stimulation and subsequent Ca²⁺ influx led to a biphasic activation of p38 MAPK characterized by an initial rapid, but transient activation followed by a delayed and more prolonged response. Selective inhibition of the delayed p38 MAPK activity was sufficient to attenuate homocysteine‐induced neuronal cell death. Using pharmacological and RNAi approaches, we further demonstrated that both the initial and delayed activation of p38 MAPK is downstream of, and dependent on activation of ERK MAPK. Our findings highlight a novel interplay between ERK and p38 MAPK in homocysteine‐NMDA receptor‐induced neuronal cell death.     

MAPK mediates PKC-dependent contraction of cat esophageal and lower esophageal sphincter circular smooth muscle

Esophageal (ESO) circular muscle contraction and lower esophageal sphincter (LES) tone are PKC dependent. Because MAPKs may be involved in PKC-dependent contraction, we examined ERK1/ERK2 and p38 MAPKs in ESO and LES. In permeabilized LES muscle cells, ERK1/2 antibodies reduced 1,2-dioctanoylglycerol (DG)- and threshold ACh-induced contraction, which are PKC dependent, but not maximal ACh, which is calmodulin dependent. LES tone was reduced by the ERK1/2 kinase inhibitor PD-98059 and by the p38 MAPK inhibitor SB-203580. In permeable ESO cells, ACh contraction was reduced by ERK1/ERK2 and p38 MAPK antibodies and by PD-98059 and SB-203580. ACh increased MAPK activity and phosphorylation of MAPK and of p38 MAPK. The 27-kDa heat shock protein (HSP27) antibodies reduced ACh contraction. HSP27 and p38 MAPK antibodies together caused no greater inhibition than either one alone. p38 MAPK and HSP27 coprecipitated after ACh stimulation, suggesting that HSP27 is linked to p38 MAPK. These data suggest that PKC-dependent contraction in ESO and LES is mediated by the following two distinct MAPK pathways: ERK1/2 and HSP27-linked p38 MAPK.     

Activation of mitogen-activated protein kinases during natural freezing and thawing in the wood frog

The responses of mitogen-activated protein kinase (MAPK) family members, including ERK (extracellular signal-regulated kinase), JNK (c-Jun NH₂-terminal kinase), and p38, in the metabolic responses to whole animal freezing (up to 24 h frozen at –2.5°C) and thawing (up to 4 h at 5°C after a 12 h freeze) were examined in four organs (liver, kidney, heart, brain) of the freeze-tolerant wood frog Rana sylvatica. Levels of the active phosphorylated form of p38 increased within 20 min as an early response to freezing in liver and kidney but rose later (after 12 h) in heart. Both JNK and p38 were activated during thawing in liver, kidney and heart with temporally-distinct patterns in each organ. The only MAPK response to freeze/thaw in frog brain was a transient elevation of p38 after 90 min thawing. ERK activity did not respond to freeze/thaw in any organ. The levels of c-Fos increased during freezing in kidney and brain whereas c-Jun was unaffected by freeze/thaw. Organ-specific responses by MAPKs, particularly p38, suggest that these may have roles in regulating metabolic or gene expression responses that may be adaptive in dealing with freezing stress or metabolic recovery during thawing.     

Insight into skeletal muscle mechanotransduction: MAPK activation is quantitatively related to tension.

The mechanism by which mechanical forces acting through skeletal muscle cells generate intracellular signaling, known as mechanotransduction, and the details of how gene expression and cell size are regulated by this signaling are poorly understood. Mitogen-activated protein kinases (MAPKs) are known to be involved in mechanically induced signaling in various cell types, including skeletal muscle where MAPK activation has been reported in response to contraction and passive stretch. Therefore, the investigation of MAPK activation in response to mechanical stress in skeletal muscle may yield important information about the mechanotransduction process. With the use of a rat plantaris in situ preparation, a wide range of peak tensions was generated through passive stretch and concentric, isometric, and eccentric contractile protocols, and the resulting phosphorylation of c-Jun NH(2)-terminal kinase (JNK), extracellular regulated kinase (ERK), and p38 MAPKs was assessed. Isoforms of JNK and ERK MAPKs were found to be phosphorylated in a tension-dependent manner, such that eccentric > isometric > concentric > passive stretch. Peak tension was found to be a better predictor of MAPK phosphorylation than time-tension integral or rate of tension development. Differences in maximal response amplitude and sensitivity between JNK and ERK MAPKs suggest different roles for these two kinase families in mechanically induced signaling. A strong linear relationship between p54 JNK phosphorylation and peak tension over a 15-fold range in tension (r(2) = 0.89, n = 32) was observed, supporting the fact that contraction-type differences can be explained in terms of tension and demonstrating that MAPK activation is a quantitative reflection of the magnitude of mechanical stress applied to muscle. Thus the measurement of MAPK activation, as an assay of skeletal muscle mechanotransduction, may help elucidate mechanically induced hypertrophy.     

Creation of a stress-activated p90 ribosomal S6 kinase. The carboxyl-terminal tail of the MAPK-activated protein kinases dictates the signal transduction pathway in which they function

Mitogen-activated protein kinase-activated protein kinases (MAPKAPKs) lie immediately downstream of the mitogen-activated protein kinases (MAPKs), extracellular signal-regulated kinase (ERK), and p38 MAPK. Although the family of MAPKAPKs shares sequence similarity, it demonstrates selectivity for the upstream activator. Here we demonstrate that each of the ERK- and p38 MAPK-regulated MAPKAPKs contains a MAPK docking site positioned distally to the residue(s) phosphorylated by MAPKs. The isolated MAPK docking sites show specificity for the upstream activator similar to that reported for the full-length proteins. Moreover, replacement of the ERK docking site of p90 ribosomal S6 kinase with the p38 MAPK docking site of MAPKAPK2 converts p90 ribosomal S6 kinase into a stress-activated kinase in vivo. It is apparent that mechanisms controlling events downstream of the proline-directed MAPKs involve specific MAPK docking sites within the carboxyl termini of the MAPKAPKs that determine the cascade in which the MAPKAPK functions.     

Mitogen-activated protein kinases and their role in regulation of cellular processes

Mitogen-activated protein kinases (MAPKs) are evolutionary conserved enzymes connecting cell-surface receptors to critical regulatory targets within cells. The three major MAPK cascades are known, the extracellular signal-regulated protein kinase (ERK) cascade, c-Jun amino-terminal protein kinase/stress-activated protein kinase (JNK/SAPK) cascade and p38-MAPK cascade. This paper is focused on characterization of these MAPK cascades in terms of their distribution and biological role in some pathological processes (apoptosis, hypertrophy) with a special orientation on the role of MAPKs in cardiovascular system during ischemia/reperfusion.     

丝裂原活化蛋白激酶(MAPK)生物学功能的结构基础

丝裂原活化蛋白激酶 (MAPK)是生物体内重要的信号转导系统之一 ,能对广泛的细胞外刺激发生反应 .蛋白激酶的空间构象是其功能的重要决定因素 .对MAPK蛋白结构的研究表明 ,MAPK的结构与功能之间具有密切的关系 .尽管MAPK各亚族的结构非常相似 ,但也存在着一些差异 ,这些差异是不同亚族对不同的细胞外刺激产生特异性反应的结构基础 .某些关键性结构 ,例如Loop12 ,在MAPK对上游激酶的作用、下游底物的选择以及亚细胞定位中都具有重要作用 .进一步深入研究MAPK的空间结构 ,探讨MAPK的生物学功能与其空间构象之间的关系 ,对于开发新的MAPK通路抑制剂用于治疗某些严重疾病有着重要的临床意义     

Protein phosphatase 2A-mediated cross-talk between p38 MAPK and ERK in apoptosis of cardiac myocytes

Mitogen-activated protein kinases (MAPKs) play different regulatory roles in signaling oxidative stress-induced apoptosis in cardiac ventricular myocytes. The regulation and functional role of cross-talk between p38 MAPK and extracellular signal-regulated kinase (ERK) pathways were investigated in cardiac ventricular myocytes in the present study. We demonstrated that inhibition of p38 MAPK with SB-203580 and SB-239063 enhanced H(2)O(2)-stimulated ERK phosphorylation, whereas preactivation of p38 MAPK with sodium arsenite reduced H(2)O(2)-stimulated ERK phosphorylation. In addition, pretreatment of cells with the protein phosphatase 2A (PP2A) inhibitors okadaic acid and fostriecin increased basal and H(2)O(2)-stimulated ERK phosphorylation. We also found that PP2A coimmunoprecipitated with ERK and MAPK/ERK (MEK) in cardiac ventricular myocytes, and H(2)O(2) increased the ERK-associated PP2A activity that was blocked by inhibition of p38 MAPK. Finally, H(2)O(2)-induced apoptosis was attenuated by p38 MAPK or PP2A inhibition, whereas it was enhanced by MEK inhibition. Thus the present study demonstrated that p38 MAPK activation decreases H(2)O(2)-induced ERK activation through a PP2A-dependent mechanism in cardiac ventricular myocytes. This represents a novel cellular mechanism that allows for interaction of two opposing MAPK pathways and fine modulation of apoptosis during oxidative stress.