Effects of insulin, contraction, and phorbol esters on mitogen-activated protein kinase signaling in skeletal muscle from lean and ob/ob mice

Effects of diverse stimuli, including insulin, muscle contraction, and phorbol 12-myristate-13-acetate (PMA), were determined on phosphorylation of mitogen-activated protein kinase (MAPK) signaling modules (c-Jun NH(2)-terminal kinase [JNK], p38 MAPK, and extracellular signal-related kinase [ERK1/2]) in skeletal muscle from lean and ob/ob mice. Insulin increased phosphorylation of JNK, p38 MAPK, and ERK1/2 in isolated extensor digitorum longus (EDL) and soleus muscle from lean mice in a time- and dose-dependent manner. Muscle contraction and PMA also elicited robust effects on these parallel MAPK modules. Insulin action on JNK, p38 MAPK, and ERK1/2 phosphorylation was significantly impaired in EDL and soleus muscle from ob/ob mice. In contrast, muscle contraction-mediated JNK, p38 MAPK, and ERK1/2 phosphorylation was preserved. PMA effects on phosphorylation of JNK and ERK1/2 were normal in ob/ob mice, whereas effects on p38 MAPK were abolished. In conclusion, insulin, contraction, and PMA activate MAPK signaling in skeletal muscle. Insulin-mediated responses on MAPK signaling are impaired in skeletal muscle from ob/ob mice, whereas the effect of contraction is generally well preserved. In addition, PMA-induced phosphorylation of JNK and ERK1/2 are preserved, whereas p38 MAPK pathways are impaired in skeletal muscle from ob/ob mice. Thus, appropriate MAPK responses can be elicited in insulin-resistant skeletal muscle via an insulin-independent mechanism.     

Role of reactive oxygen species in TGF-beta1-induced mitogen-activated protein kinase activation and epithelial-mesenchymal transition in renal tubular epithelial cells

Epithelial-mesenchymal transition (EMT) plays an important role in renal tubulointerstitial fibrosis and TGF-beta1 is the key inducer of EMT. Phosphorylation of Smad proteins and/or mitogen-activated protein kinases (MAPK) is required for TGF-beta1-induced EMT. Because reactive oxygen species (ROS) are involved in TGF-beta1 signaling and are upstream signaling molecules to MAPK, this study examined the role of ROS in TGF-beta1-induced MAPK activation and EMT in rat proximal tubular epithelial cells. Growth-arrested and synchronized NRK-52E cells were stimulated with TGF-beta1 (0.2 to 20 ng/ml) or H(2)O(2) (1 to 500 microM) in the presence or absence of antioxidants (N-acetylcysteine or catalase), inhibitors of NADPH oxidase (diphenyleneiodonium and apocynin), mitochondrial electron transfer chain subunit I (rotenone), and MAPK (PD 98059, an MEK [MAP kinase/ERK kinase] inhibitor, or p38 MAPK inhibitor) for up to 96 h. TGF-beta1 increased dichlorofluorescein-sensitive cellular ROS, phosphorylated Smad 2, p38 MAPK, extracellular signal-regulated kinases (ERK)1/2, alpha-smooth muscle actin (alpha-SMA) expression, and fibronectin secretion and decreased E-cadherin expression. Antioxidants effectively inhibited TGF-beta1-induced cellular ROS, phosphorylation of Smad 2, p38 MAPK, and ERK, and EMT. H(2)O(2) reproduced all of the effects of TGF-beta1 with the exception of Smad 2 phosphorylation. Chemical inhibition of ERK but not p38 MAPK inhibited TGF-beta1-induced Smad 2 phosphorylation, and both MAPK inhibitors inhibited TGF-beta1- and H(2)O(2)-induced EMT. Diphenyleneiodonium, apocynin, and rotenone also significantly inhibited TGF-beta1-induced ROS. Thus, this data suggest that ROS play an important role in TGF-beta1-induced EMT primarily through activation of MAPK and subsequently through ERK-directed activation of Smad pathway in proximal tubular epithelial cells.     

Stimulation of MAPK cascades by insulin and osmotic shock: lack of an involvement of p38 mitogen-activated protein kinase in glucose transport in 3T3-L1 adipocytes

Osmotic shock and insulin stimulate GLUT4 translocation and glucose transport via mechanisms that are for the most part distinct yet convergent. In this article, we investigated the effect of osmotic shock and insulin on the activation of the mitogen-activated protein kinase (MAPK) cascades in differentiated 3T3-L1 adipocytes. The MAPKs are activated by phosphorylation on conserved tyrosine and threonine residues. Both sorbitol and insulin strongly stimulated extracellular regulated kinase (ERK) 1 and 2 phosphorylation (8- and 18-fold, respectively). In contrast, c-jun-NH2-terminal kinase (JNK)/stress-activated protein kinase (SAPK) phosphorylation was stimulated only by sorbitol (sevenfold) and not by insulin. Phosphorylation of p38 MAPK was stimulated strongly by sorbitol (22-fold) but weakly by insulin (2.7-fold). Measurement of intrinsic JNK and p38 MAPK activity confirmed the phosphorylation studies. JNK and p38 MAPK were activated only significantly by sorbitol. The MAPKs are phosphorylated by dual-specificity kinases (mitogen-activated ERK-activating kinase [MEK] or MAPK kinase [MKK]). As expected, sorbitol and insulin both stimulated MEK phosphorylation. MKK4 was phosphorylated only in response to sorbitol, and neither of the stimuli caused phosphorylation of MKK3 or 6. To determine the functional significance of the observed activation of p38 MAPK in response to insulin and osmotic shock, we used three pyridinyl imidazole p38 MAPK inhibitors, SB203580, SB202190, and PD169316. Insulin and osmotic shock-stimulated glucose transport was not inhibited by any inhibitor at concentrations that were shown to block p38 MAPK activity. Furthermore, activation of the p38 MAPK pathway by treatment of cells with anisomycin did not stimulate glucose transport. These results suggest that activation of the p38 MAPK pathway is not involved in the stimulation of glucose transport.     

Evidence for JNK-dependent up-regulation of proteoglycan synthesis and for activation of JNK1 following cyclical mechanical stimulation in a human chondrocyte culture model

OBJECTIVE: To examine the expression of mitogen-activated protein kinases (MAPKs) in human chondrocytes, to investigate whether selective activation of MAPKs is involved in up-regulation of proteoglycan (PG) synthesis following cyclical mechanical stimulation (MS), and to examine whether MS is associated with integrin-dependent or independent activation of MAPKs. METHODS: The C-28/I2 and C-20/A4 human chondrocyte cell lines were mechanically stimulated in monolayer cell culture. PG synthesis was assessed by [(35)S]-sulphate incorporation in the presence and absence of the p38 inhibitor SB203580, and the extracellular-regulated kinase (ERK1/2) inhibitor PD98059. Kinase expression and activation were assessed by Western blotting using phosphorylation status-dependent and independent antibodies, and by kinase assays. The Jun N-terminal kinase (JNK) inhibitor SP600125 and the anti-beta(1) integrin (CD29) function-blocking antibody were used to assess JNK activation and integrin dependence, respectively. RESULTS: Increased PG synthesis following 3 h of cyclic MS was abolished by pretreatment with 10 microM SB203580, but was not affected by 50 microM PD98059. The kinases p38, ERK1/ERK2 and JNKs were expressed in both stimulated and unstimulated cells. Phosphorylated p38 was detected at various time points following 0.5, 1, 2 and 3 h MS in C-28/I2, but not detected in C-20/A4 cell lines. Phosphorylation of ERK1 and ERK2 was not significantly affected by MS. Phosphorylation of the 54 and 46 kDa JNKs increased following 0.5, 1, 2 and 3 h of MS, and following CO(2) deprivation. MS-induced JNK phosphorylation was inhibited by SB203580 at concentrations > or =5 microM and activation of JNK1 following MS was blocked by SP600125 and partially inhibited by anti-CD29. CONCLUSIONS: The data suggest JNK, rather than p38 or ERK dependent increases in PG synthesis, and selective, partially integrin-dependent, activation of JNK kinases in human chondrocyte cell lines following cyclical MS. JNK activation is also very sensitive to changes in CO(2)/pH in this chondrocyte culture model.     

Anti-apoptotic effect of quercetin: intervention in the JNK- and ERK-mediated apoptotic pathways

BACKGROUND: Bioflavonoid quercetin inhibits hydrogen peroxide (H2O2)-induced apoptosis via intervention in the activator protein 1 (AP-1)-mediated apoptotic pathway. In this report, we investigated molecular events involved in the anti-apoptotic effect of quercetin, focusing especially on its effects on the family of mitogen-activated protein (MAP) kinases. METHODS: Cultured mesangial cells were exposed to H2O2, and activation of c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinases (ERKs), and p38 MAP kinase was evaluated in the presence or absence of quercetin. Using pharmacological and genetic inhibitors, the roles for individual MAP kinases in H2O2-induced apoptosis were examined. Involvement of ERKs in the induction and activation of AP-1 was also investigated using Northern blot analysis and a reporter assay. RESULTS: Mesangial cells exposed to H2O2 exhibited rapid phosphorylation of JNK, ERKs, and p38 MAP kinase. Quercetin abrogated the activation of all three MAP kinases in response to H2O2. Pretreatment with MAP kinase kinase inhibitor PD098059 or JNK-c-Jun/AP-1 inhibitor curcumin attenuated the H2O2-induced apoptosis. In contrast, the p38 MAP kinase inhibitor SB203580 did not improve the cell survival. Consistently, transfection with dominant-negative mutants of ERK1 and ERK2 or a dominant-negative mutant of JNK inhibited H2O2-induced apoptosis. Transfection with a dominant-negative p38 MAP kinase did not attenuate the apoptotic process. Inhibition of ERKs by PD098059 suppressed induction of c-fos without affecting early induction of c-jun, leading to attenuated activation of AP-1 in response to H2O2. CONCLUSIONS: These results suggested that (1) activation of JNK and ERKs, but not p38 kinase, is required for the H2O2-induced apoptosis; and (2) suppression of the JNK-c-Jun/AP-1 pathway and the ERK-c-Fos/AP-1 pathway is involved in the anti-apoptotic effect of quercetin.     

The SOD mimetic tempol ameliorates glomerular injury and reduces mitogen-activated protein kinase activity in Dahl salt-sensitive rats

It was shown recently that renal injury in Dahl salt-sensitive (DS) hypertensive rats is accompanied by mitogen-activated protein kinase (MAPK) activation. The present study was conducted to elucidate the contribution of reactive oxygen species to MAPK activities and renal injury in DS rats. DS rats were maintained on high salt (H; 8.0% NaCl; n = 7) or low salt (L; 0.3% NaCl; n = 6) diets; H + a superoxide dismutase mimetic, tempol (3 mmol/L in drinking water; n = 8); or H + hydralazine (0.5 mmol/L in drinking water; n = 8) for 4 wk. Mean BP (MBP) in DS/H and DS/L rats was 185 +/- 7 and 113 +/- 3 mmHg, respectively. DS/H rats showed a higher ratio of urinary protein excretion and creatinine (U(protein)V/U(cr)V; 20.3 +/- 1.1) and a higher cortical collagen content (22 +/- 1 micro g/mg) than in DS/L rats (2.4 +/- 0.1 and 13 +/- 1 micro g/mg, respectively). The expression of p22-phox and Nox-1, essential components of NAD(P)H oxidase, in renal cortical tissue was approximately threefold higher in DS/H rats than in DS/L rats. Increased activities of renal cortical MAPK, including extracellular signal-regulated kinases (ERK) 1/ERK2 and c-Jun NH(2)-terminal kinases (JNK) were also observed in DS/H rats by 7.0 +/- 0.7- and 4.3 +/- 0.2-fold, respectively. Tempol treatment significantly decreased MBP (128 +/- 3 mmHg), U(protein)V/U(cr)V (4.8 +/- 0.4), and cortical collagen content (14 +/- 1 micro g/mg) and normalized ERK1/ERK2 and JNK activities in DS/H rats. Histologically, tempol markedly ameliorated progressive sclerotic and proliferative glomerular changes in DS/H rats. Hydralazine-treated DS/H rats showed similar MBP (127 +/- 5 mmHg) to tempol-treated DS/H rats. Hydralazine also decreased U(protein)V/U(cr)V (16.2 +/- 1.5) and cortical collagen content (19 +/- 1 micro g/mg) in DS/H rats. However, these values were significantly higher than those of tempol-treated rats. Furthermore, although hydralazine significantly reduced JNK activity (-56 +/- 3%), ERK1/ERK2 activities were unaffected. These data suggest that reactive oxygen species, generated by NAD(P)H oxidase, contribute to the progression of renal injury through ERK1/ERK2 activation in DS/H hypertensive rats.     

Involvement of the mitogen-activated protein kinase family in tetracaine-induced PC12 cell death

BACKGROUND: To explore whether cytotoxicity of local anesthetics is related to apoptosis, the authors examined how local anesthetics affect mitogen-activated protein kinase (MAPK) family members, extracellular signal-regulated kinases (ERKs), c-Jun N-terminal kinases (JNKs)-stress-activated protein kinases, and p38 kinase, which are known to play important roles in apoptosis. METHODS: Cell death was evaluated using PC12 cells. Morphologic changes of cells, cellular membrane, and nuclei were observed. DNA fragmentation was electrophoretically assayed. Western blot analysis was performed to analyze phosphorylation of the MAPK family, cleavage of caspase-3 and poly(adenosine diphosphate-ribose) polymerase. Intracellular Ca2+ concentration was measured using a calcium indicator dye. RESULTS: Tetracaine-induced cell death was shown in a time- and concentration-dependent manner and characterized by nuclear condensation or fragmentation, membrane blebbing, and internucleosomal DNA fragmentation. Caspase-3 activation and phosphorylation of ERK, JNK, and p38 occurred in the cell death. PD98059, an inhibitor of ERK, enhanced tetracaine-induced cell death and JNK phosphorylation, whereas ERK phosphorylation was inhibited. Curcumin, an inhibitor of JNK pathway, attenuated the cell death. Increase of intracellular Ca2+ concentration was detected. In addition to the increase of ERK phosphorylation and the decrease of JNK phosphorylation, two Ca2+ chelators protected cells from death. Neither cell death nor phosphorylation of the MAPK family was caused by tetrodotoxin. Nifedipine did not affect tetracaine-induced apoptosis. CONCLUSIONS: Tetracaine induces apoptosis of PC12 cells via the MAPK family. ERK activation protects cells from death, but JNK plays the opposite role. Toxic Ca2+ influx caused by tetracaine seems to be responsible for the cell death, but blocking of Na+ channels or L-type Ca2+ channels is unlikely involved in the tetracaine's action for apoptosis.     

Angiotensin-(1-7) inhibits angiotensin II-stimulated phosphorylation of MAP kinases in proximal tubular cells

Angiotensin-converting enzyme 2 (ACE2) is a homolog of ACE, which is not blocked by ACE inhibitors. High amounts of ACE2 are present in the proximal tubule, and ACE2 catalyzes generation of angiotensin 1-7 (Ang-(1-7)) by this segment. Ang-(1-7) binds to a receptor distinct from the AT1 or AT2 Ang II receptor, identified as the mas receptor. We studied the effects of Ang-(1-7) on Ang II-mediated cell signaling pathways in proximal tubule. In primary cultures of rat proximal tubular cells, activation of mitogen-activated protein kinases (MAPK) was detected by immunoblotting, in the presence or absence of agonists/antagonists. Transforming growth factor-beta1 (TGF-beta1) was measured by enzyme-linked immunosorbent assay. Ang II (5 min, 10(-7) M) stimulated phosphorylation of the three MAPK (p38, extracellular signal-related kinase (ERK 1/2), and c-Jun N-terminal kinase (JNK)). While incubation of proximal tubular cells with Ang-(1-7) alone did not significantly affect MAPK phosphorylation, Ang-(1-7) (10(-7) M) completely inhibited Ang II-stimulated phosphorylation of p38, ERK 1/2, and JNK. This inhibitory effect was reversed by the Ang-(1-7) receptor antagonist, D-Ala7-Ang-(1-7). Ang II significantly increased production of TGF-beta1 in proximal tubular cells, an effect that was partly inhibited by Ang-(1-7). Ang-(1-7) had no significant effect on cyclic 3',5'-adenosine monophosphate production in these cells. In summary, Ang-(1-7) inhibits Ang II-stimulated MAPK phosphorylation in proximal tubular cells. Generation of Ang-(1-7) by proximal tubular ACE2 could thereby serve a protective role by counteracting the effects of locally generated Ang II.     

Involvement of the Mitogen-activated Protein Kinase Family in Tetracaine-induced PC12 Cell Death

Background: To explore whether cytotoxicity of local anesthetics is related to apoptosis, the authors examined how local anesthetics affect mitogen-activated protein kinase (MAPK) family members, extracellular signal-regulated kinases (ERKs), c-Jun N-terminal kinases (JNKs)-stress-activated protein kinases, and p38 kinase, which are known to play important roles in apoptosis.

Methods: Cell death was evaluated using PC12 cells. Morphologic changes of cells, cellular membrane, and nuclei were observed. DNA fragmentation was electrophoretically assayed. Western blot analysis was performed to analyze phosphorylation of the MAPK family, cleavage of caspase-3 and poly(adenosine diphosphate-ribose) polymerase. Intracellular Ca2+ concentration was measured using a calcium indicator dye.

Results: Tetracaine-induced cell death was shown in a time- and concentration-dependent manner and characterized by nuclear condensation or fragmentation, membrane blebbing, and internucleosomal DNA fragmentation. Casepase-3 activation and phosphorylation of ERK, JNK, and p38 occurred in the cell death. PD98059, an inhibitor of ERK, enhanced tetracaine-induced cell death and JNK phosphorylation, whereas ERK phosphorylation was inhibited. Curcumin, an inhibitor of JNK pathway, attenuated the cell death. Increase of intracellular Ca2+ concentration was detected. In addition to the increase of ERK phosphorylation and the decrease of JNK phosphorylation, two Ca2+ chelators protected cells from death. Neither cell death nor phosphorylation of the MAPK family was caused by tetrodotoxin. Nifedipine did not affect tetracaine-induced apoptosis.     

The effects of a novel resuscitation strategy combining pentoxifylline and hypertonic saline on neutrophil MAPK signaling

BACKGROUND: The combination of hypertonic saline (HS) and pentoxifylline (PTX) has been shown to synergistically downregulate neutrophil oxidative burst in vitro. We investigated the effects of HS/PTX on human neutrophil mitogen-activated protein kinase (MAPK) signaling and the role of Protein kinase A (PKA) in this process. METHODS: Isolated neutrophils were treated with PTX (2 mmol/L), HS10 (10 mmol/L above isotonicity), and HS40 (40 mmol/L above isotonicity) alone or in combination for determination of intracellular cyclic adenosine monophosphate (cAMP) concentrations. Human neutrophils were stimulated with f-methionyl-leucyl-phenylalanine (fMLP) (1 micromol/L) before the treatments above in both the presence and the absence of PKA inhibition for Western blot analysis of MAPK p38 and extracellular signal-related kinase 1/2 (ERK 1/2) phosphorylation. RESULTS: Concomitant exposure to HS/PTX results in an additive increase in intracellular cAMP. fMLP-induced ERK 1/2 phosphorylation was synergistically attenuated by HS/PTX. Both PTX and HS reduced p38MAPK phosphorylation. No additive effect was observed with combined treatment. Although PKA inhibition abrogated the effects of PTX, HS retained some capacity to attenuate MAPK phosphorylation. CONCLUSION: HS/PTX is more effective in attenuating neutrophil ERK signaling than either component alone, whereas both components alone or in combination produced comparable results with p38MAPK. Although PTX functions primarily through PKA activation, HS may suppress neutrophils through a partially PKA-independent mechanism.     

Arachidonic acid directly activates members of the mitogen-activated protein kinase superfamily in rabbit proximal tubule cells

BACKGROUND: To explore the roles of eicosanoids in arachidonic acid-induced mitogen-activated protein kinase (MAPK) signal transduction, we have shown that exposure of proximal tubular cells to arachidonic acid induces phosphorylation of c-Jun NH2-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK), two members of the MAPK superfamily. We observed that ketoconazole, an inhibitor of the cytochrome P450 pathway, blocked ERK but not JNK activation. METHODS: Direct regulation of arachidonic acid on mitogen-activated protein kinase (MAPK) signaling pathways was evaluated more directly by utilizing specific enzyme inhibitors of the cytochrome P450 metabolic pathway and by comparing the relative efficacy of arachidonic acid versus its cytochrome P450 metabolites (exogenous and endogenous), eicosatetraynoic acid (ETYA), and other fatty acids on the phosphorylation of members of the MAPK superfamily (ERKs, JNK, and p38(MAPK)), by utilizing early passage rabbit proximal tubular epithelial cells. RESULTS: Arachidonic acid activated p38(MAPK), a third member of the MAPK superfamily, in a time- and concentration-dependent manner. Studies designed to evaluate the ability of arachidonic acid and its cytochrome P450 metabolites (endogenously and exogenously) to stimulate ERKs, JNK, and p38(MAPK) found four conclusions. First, the metabolites of arachidonic acid generated endogenously by cytochrome P450 2C1 significantly augmented basal ERK activity, whereas the metabolites generated by the 2C2 isozyme significantly augmented basal p38(MAPK) activity. However, their effects were less profound than arachidonic acid itself. In contrast, there were no significant effects with transfection of either isozyme on basal JNK activity. Second, a variety of exogenous cytochrome P450 products were less potent than arachidonic acid on a molar basis in stimulating the activity of all three MAPKs. Third, ketoconazole and 17-octadecynoic acid, inhibitors of the cytochrome P450 pathway, as well as PPOH and DDMS, inhibitors of the epoxygenase and omega-hydroxylase pathways, respectively, failed to significantly reduce the effects of arachidonic acid to activate ERK and p38(MAPK) (JNK was not evaluated). Finally, arachidonic acid, its inactive analog ETYA, and other fatty acids with differing chain lengths and degrees of saturation stimulated the activity of all three MAPKs. CONCLUSIONS: These observations substantiate a role for arachidonic acid and other fatty acids in signaling linked to the MAPK superfamily in rabbit proximal tubular epithelium without the necessity of conversion to cytochrome P450 metabolites.     

p38MAPK与 ERK1/2的协同效应及对成骨细胞分化的调控

 目的探讨骨髓间质干细胞(bone marrow mesenchymal stem cells,BMSCs)向成骨细胞分化过程中 p38MAPK与 ERK1/2的协同效应及其机制。方法以成骨细胞分化添加剂诱导小鼠 BMSCs向成骨细胞分化,测定碱性磷酸酶活性和钙沉积量。检测磷酸化 p38MAPK和磷酸化 ERK1/2(p-ERK1/2)的表达水平评估通路的激活状况。以 SB203580或 PD98059阻断 p38MAPK或 ERK1/2通路,观察对成骨细胞分化的影响。以 SB203580或亚砷酸钠阻断或激活 p38MAPK通路,观察 p-ERK1/2的变化。以冈田酸抑制蛋白磷酸酯酶 2A(protein phosphatases type 2A,PP2A)活性,观察 p-ERK1/2的变化及对成骨细胞分化的影响。通过免疫共沉淀实验观察 PP2A和 ERK1/2间的结合及 SB203580对结合的影响。结果成骨细胞分化添加剂诱导 BMSCs向成骨细胞分化的过程伴有 ERK1/2和 p38MAPK通路的激活, SB203580剂量±赖性抑制成骨细胞分化,PD98059剂量±赖性增强成骨细胞分化。 SB203580使 p-ERK1/2表达增加,亚砷酸钠减弱其表达。冈田酸使 p-ERK1/2表达增加,并使成骨细胞分化受到抑制。 PP2A可直接与 ERK1/2结合,SB203580使 PP2A与 ERK1/2的结合减弱。结论 p38MAPK可通过 PP2A与 ERK1/2产生协同效应,并调节 BMSCs向成骨细胞分化。     

Modulation of renal epithelial barrier function by mitogen-activated protein kinases (MAPKs): mechanism of cyclosporine A-induced increase in transepithelial resistance

BACKGROUND: Cyclosporine A (CsA) has been shown to increase transepithelial resistance in Madin-Darby canine kidney (MDCK) cells, and the mechanism may involve altered phosphorylation of junctional proteins. In this study, we examine the effect of the extracellular signal-regulated protein kinase (ERK) 1/2 and p38 mitogen-activated protein kinase (MAPK) pathways on the basal transepithelial resistance (TER) and on the CsA-induced increase in TER across MDCK monolayers. Here we present evidence that CsA may be mediating some of its effects through activation of the ERK 1/2 MAPK pathway. METHODS: MDCK cells were treated with CsA (4.2 micromol/L) and paracellular permeability was assessed by measuring TER. The role of the ERK 1/2 and the p38 MAPK pathways in modulating TER was investigated using the inhibitors PD98059 and U0126 for ERK 1/2 and SB203580 for p38. ERK 1/2 and p38 phosphorylation/activation was also examined by Western blot analysis. RESULTS: CsA (4.2 micromol/L) increased the TER of MDCK monolayers. The ERK 1/2 inhibitor PD98059 decreased basal TER and also ameliorated the CsA-induced increase in TER. Similar results were found with the U0126 inhibitor of ERK 1/2. The p38 inhibitor SB203580 had no effect on the basal TER of the monolayers, however, SB203580 significantly augmented the CsA-induced increase in TER. CsA was shown to significantly activate ERK 1/2 and this activation by CsA was prevented by PD98059. Inhibition of the p38 pathway by SB203580 also resulted in activation of ERK 1/2 and this activation of ERK 1/2 was further enhanced by CsA. No effect of CsA or the inhibitors PD98059 or SB203580 on p38 phosphorylation was detected. CONCLUSION: The results presented here suggest that activation of the ERK 1/2 MAPK cascade is important in the regulation of the paracellular permeability in MDCK cells. Activation of this pathway appears to be pivotal to the CsA-induced increase in TER.     

Sphingosine-1-phosphate-induced smooth muscle cell migration involves the mammalian target of rapamycin

BACKGROUND: Vascular smooth muscle cell (SMC) migration is an important component of the development of intimal hyperplasia. Sphingosine-1-phosphate (S-1-P) is a lipid released from activated platelets with numerous cellular effects including the stimulation of SMC migration in vitro. We examined the role of the mammalian target of rapamycin and ribosomal p70S6 kinase (p70S6K) in S-1-P-induced SMC migration . METHODS: Rat arterial SMCs were cultured in vitro. Linear wound and Boyden microchemotaxis assays of migration were performed in the presence of S-1-P (0.01 to 100 micromol/L) with and without rapamycin (10 nmol/L). Western blotting was performed for phosphorylated and total p70S6K, ERK1/2, and p38(MAPK) after stimulation with S-1-P (0.1 micromol/L), with and without rapamycin pretreatment. Phosphorylation of p70S6K was also assayed after S-1-P treatment in the presence and absence of inhibitors of PI3 kinase (wortmannin, WN, and LY294002, LY), Akt (AktI), p38(MAPK) (SB203580), and MEK1 (PD98059). RESULTS: S-1-P stimulated migration of SMCs in both linear wound and Boyden chamber assays compared to control (P < .05); these responses were inhibited by rapamycin to below the level of control (P < .05 vs S-1-P alone for both assays) in a dose-dependent manner (inhibitory concentration of 50%, 10 nmol/L). S-1-P stimulated phosphorylation of ERK1/2, p38(MAPK), and p70S6K, which peaked at 5 minutes for ERK1/2 and p38(MAPK) and10 minutes for p70S6K (2-fold increase over control for each, P < .05). Rapamycin prevented the phosphorylation of p70S6K at the Thr 389 site (which correlates with enzyme activity), reduced ERK1/2 phosphorylation, but had no effect on the Thr 421/Ser 424 site or on p38(MAPK) phosphorylation. Wortmannin and LY294002 inhibited phosphorylation of the Thr 389 site of p70S6K. AktI and SB203580 had no effect on p70S6K, whereas PD98059 had a marginal effect. CONCLUSIONS: S-1-P-induced SMC migration was completely inhibited by rapamycin, indicating that the p70S6K pathway is involved. This mechanism likely involves modulation of the ERK1/2 pathway. S-1-P stimulates phosphorylation of p70S6K in a MEK1-dependent, PI3 kinase-dependent, but Akt-independent manner.     

肾病综合征氧化低密度脂蛋白脂质肾损伤的实验研究

目的 通过观察氧化低密度脂蛋白（oxidized low-density lipoprotein,ox-LDL）对系膜细胞（Mesangial Cells,MCs）分泌炎症介质功能的影响及MAPK信号通路和核因子-κB（nuclear factor kappa B,NF-κB）活性的改变,进一步阐明脂质在肾损伤中的作用机制.方法 利用ox-LDL诱导大鼠系膜细胞增殖,分别采用ELISA、real-time PCR、western blot技术检测MCs炎症介质、MAPK通路相关蛋白（p38、JNK、ERK）及NF-κB的表达水平.结果 利用ox-LDL诱导大鼠系膜细胞增殖并加入CXCR6受体后,其表面炎症因子[CXCL16、CD36、ADAM10、ADAM17、干扰素（IFN）、白细胞介素（IL6）、肿瘤坏死因子（TNF-α）]的表达水平以及MAPK信号通路（p38、JNK、ERK）、NF-κB的磷酸化水平显著升高（P〈0.01）.结论 ox-LDL可促使系膜细胞释放CXCL16、CD36、ADAM10、ADAM17、IFN、IL6、TNF-α等炎症介质,CXCR6可介导这一途径.ox-LDL激活MAPK信号转导通路,使p38、ERK1/2、SAPK/JNK的磷酸化水平升高,激活了NF-κB p65的活性,CXCR6-CXCL16介导MAPK信号途径.     

戊乙奎醚预处理对脓毒症小鼠肺损伤时MAPK信号转导通路的影响

目的 探讨戊乙奎醚(PHC)预处理对脓毒症小鼠肺损伤时丝裂原活化蛋白激酶(MAPK)信号转导通路的影响.方法 健康雌性昆明小鼠105只,体重20～25 g,随机分为3组(n=35):假手术组(S组)、脓毒症(CLP)组和戊乙奎醚(PHC)组.采用盲肠结扎并穿孔法制备脓毒症模型.PHC组于造模前1 h腹腔注射戊乙奎醚0.45 mg/kg,s组和CLP组于造模前1 h注射等容量生理盐水.于造模后即刻测定肺微血管通透性;造模后12 h时进行动脉血气分析,观察肺组织病理结果,测定肺组织丙二醛(MDA)含量、超氧化物歧化酶(SOD)活性和磷酸化的p38丝裂原活化蛋白激酶(p38MAPK)、细胞外信号调节激酶(ERK1/ERK2)和c-jun氨基末端蛋白激酶(JNK)表达.结果 与S组比较,CLP组PaO2、PaO2/FiO2和pH值降低,肺微血管通透性和肺组织MDA含量升高,SOD活性降低,磷酸化的p38MAPK、ERK1/ERK2和JNK表达上调(P<0.05或0.01);与CLP组比较,PHC组PaO2、PaO2/FiO2和pH值升高,肺微血管通透性和肺组织MDA含量降低,SOD活性升高,磷酸化的p38MAPK和ERK1/ERK2表达下调(P<0.05或0.01).结论 戊乙奎醚预处理可通过抑制MAPK信号转导通路(p38MAPK和ERK1/ERK2)的激活,从而减轻脓毒症小鼠肺损伤.