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.     

Activation pattern of mitogen-activated protein kinases in early phase of different size liver isografts in rats.

Mitogen-activated protein kinases (MAPK) play a pivotal role in ischemia reperfusion injuries of heart and liver, but the activation pattern of MAPKs in the early phase of different size liver isografts remains unclear. The experiment is designed to investigate the activation pattern and role of MAPKs in isografts of the rat with different size liver transplantation. The animal models of different size graft liver transplantation (whole graft, 50% size, or 30% size, respectively) were established and the sham operation group served as a control. The recipients were sacrificed at 0.5-, 2-, 6-, and 24-hour time points after transplantation to harvest the graft specimens and blood samples. The serum aspartate amino transferase (AST), alanine amino transferase (ALT) and tumor necrosis factor-alpha (TNF-alpha) levels, and histological findings were evaluated. The expressions of the total and phosphorylated p46/p54 JNKs, p38 MAPK, and p42/p44 ERKs were detected by Western blot. The serum ALT and AST levels increased significantly at the 0.5-hour time point and maintained high with the peak levels at the 6-hour time point after liver transplantation. The different sizes of liver isografts did not change the expressions of total p46/p54JNKs, p38MAPK, and p42/p44 ERKs. While the expressions of phosphorylated p46/p54JNKs, p38 MAPK, and p42/p44 ERKs were either negative or mildly up-regulated in the sham operation group, they were significantly activated in the transplanted liver at the 0.5-hour time point, especially in the 30% size liver transplantation group. In conclusion, the activation of three MAPKs in liver isografts correlates with graft size and the JNK and p38 MAPK are responsible for the graft injury while the ERK signal pathway maybe participate in the regulation of cell growth and differentiation after small-for-size liver transplantation.     

p38 MAP kinase mediates endotoxin-induced expression of cyclooxygenase-2 in enterocytes

BACKGROUND: Necrotizing enterocolitis (NEC) occurs only after bacterial colonization of the intestine, suggesting that bacterial products, including lipopolysaccharide (endotoxin,) interact with enterocytes in the pathogenesis of this disease. Inflammatory molecules such as cyclooxygenase-2 (COX-2) are important mediators of the septic response leading to NEC. We therefore hypothesized that endotoxin activates production of COX-2 in enterocytes and explored the relative contributions of known mitogen-activated protein kinases (MAPK) pathways in this process. METHODS: IEC-6 enterocytes were treated with 5 microg/mL endotoxin, or various stresses, or media alone, and COX-2 protein levels were assayed by immunoblots with anti-COX-2 antibodies. Activation of MAPK was examined by immunoblots with phospho-MAPK antibodies. MAPK activity was blocked by treatment with pharmacologic inhibitors or transfection with dominant-negative MAPK constructs. RESULTS: Endotoxin treatment caused increased expression of the COX-2 protein 24 hours after treatment. This was preceded by rapid and transient activation of the 3 major MAPKs: extracellular-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38. SB203580, a specific inhibitor of p38, but not U0126 (ERK inhibitor) or SP600125 (JNK inhibitor), blocked endotoxin-induced accumulation of COX-2 protein. This response was also blocked by expression of dominant-negative p38 but not by the dominant-negative ERK construct. Genotoxic stress that activated p38 but not ERK was an effective inducer of COX-2, whereas stresses that activated both p38 and ERK were not effective. ERK inhibition by U1026 enhanced endotoxin-induced production of COX-2, consistent with negative regulation of COX-2 by ERK. These data point to p38 as the MAPK that mediates endotoxin-induced production of COX-2 in enterocytes. CONCLUSIONS: Endotoxin may be capable of inducing the production of COX-2 in enterocytes via the p38 MAPK pathway, which may be relevant to the development of NEC.     

The Comparison of Mitogen-Activated Protein Kinases That Become Activated Within the Left Ventricular and Right Atrial Tissues Following Heart Transplantation in Canine Model

The activation of p38 mitogen-activated protein kinase (MAPK) plays an important role in ischemia/reperfusion injury. Some reports have documented MAPKs activation of the myocardium in human models, using right atrial (RA) tissue for samples. This study compared the activation of MAPKs in left ventricle (LV) and RA tissues in canine heart transplantation. Four dogs were used as baseline data at two points, before and 20 min after warm ischemia (baseline model), and eight dogs (four pairs of donor and recipient) were used at other points: 4 h after cold ischemia, and at 10, 60, and 180 min after reperfusion (transplantation model). In the transplantation model, donor hearts were left in situ for 20 min after cardiac arrest, and were immersed in Celsior solution for 4 h after coronary flushing. Orthotopic heart transplantation was then performed. Two groups were created: the LV and RA groups (n = 4 in each group). Heart tissue was harvested from the left ventricular wall in the LV group and from the right atrial appendage in the RA group. The activation of MAPKs, including p38 MAPK, c-Jun N-terminal protein kinase (JNK), and extracellular signal-regulated protein kinase (ERK), was evaluated at each point. The activation patterns of p38 MAPK and ERK were similar in the RA and LV groups, but JNK activation was different in the two groups, after ischemia and reperfusion. Thus, RA tissue may be deliberately used as a substitute for LV tissue when investigating the activation of MAPKs in a human model.     

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.     

Modulation of vascular smooth muscle cell alignment by cyclic strain is dependent on reactive oxygen species and P38 mitogen-activated protein kinase

PURPOSE: The aim of this study was to investigate the molecular targets of reactive oxygen species (ROS) and to determine whether cyclic strain induces smooth muscle cell (SMC) alignment via the ROS system. We assessed stretch-induced nicotinamide adenine dinucleotide phosphate (NAD(P)H) oxidase activation and the redox sensitivity of cyclic strain-stimulated activation of the mitogen-activated protein kinase (MAPK) family. METHODS: SMCs were seeded on flexible collagen I-coated plates and exposed to cyclic strain. NAD(P)H oxidase activation was measured with lucigenin-enhanced chemiluminescent detection of superoxide. Activation of MAPK was detected by determining phosphorylation of extracellular signal-regulated protein kinase (ERK1/2), c-jun N-terminal kinase (JNK1/2), and p38 MAPK with immunoblotting. In other experiments, SMCs were exposed to diphenylene iodonium (DPI), an NAD(P)H inhibitor, 30 minutes before stretch. MAPK activation and cell orientation were then assessed. RESULTS: Cyclic strain elicits a rapid increase in intracellular NADH/NADPH oxidase in SMCs. There was also a rapid and robust phosphorylation of ERK1/2, JNK1/2, and p38 MAPK. Cyclic strain-induced intracellular NAD(P)H generation was almost completely blocked with DPI. DPI also inhibited the strain-induced phosphorylation of ERK1/2, JNK1/2, and p38 MAPK. Both the p38 MAPK specific inhibitor, SB 202190, and DPI blocked cyclic strain-induced cell alignment, but PD98059, an ERK1/2-specific inhibitor, and SP600125, an anthrazolone inhibitor of JNK, did not. CONCLUSION: Our results provide evidence that p38 MAPK is a critical component of the oxidant stress ROS-sensitive signaling pathway and plays a crucial role in vascular alignment induced by cyclic stain.     

Differential Activation of Mitogen-activated Protein Kinases in Ischemic and Anesthetic Preconditioning

Background: Accumulating evidence pinpoints to the pivotal role of mitogen-activated protein kinases (MAPKs) in the signal transduction underlying cardiac preconditioning.

Methods: PD98059, an inhibitor of extracellular signal-regulated protein kinase (MEK-ERK1/2), and SB203580, an inhibitor of p38 MAPK, were used to evaluate the role of MAPKs with respect to postischemic functional recovery in isolated perfused rat hearts subjected to ischemic preconditioning (IPC) and anesthetic preconditioning (APC). Western blot analyses were used to determine the degree of ERK1/2 and p38 MAPK activation after the application of the preconditioning stimulus and after ischemia-reperfusion. Immunohistochemical staining served to visualize subcellular localization of activated MAPKs.

Results: PD98059 and SB203580 abolished postischemic functional recovery in IPC but not in APC. IPC but not APC markedly activated ERK1/2 and p38 MAPK, which were abrogated by coadministration of the specific blockers. Conversely, IPC and APC enhanced ERK1/2 activity after ischemia-reperfusion as compared to nonpreconditioned hearts, and IPC in addition enhanced p38 MAPK activity. Coadministration of PD98059 and SB203580 during IPC but not during APC inhibited postischemically enhanced MAPK activities. Moreover, chelerythrine and 5-hydroxydecanoate, effective blockers of IPC and APC, annihilated IPC- and APC-induced enhanced postischemic responses of MAPKs. Finally, administration of PD98059 during ischemia-reperfusion diminished the protective effects of IPC and APC. Immunohistochemistry revealed increased ERK1/2 activity primarily in intercalated discs and nuclei and increased p38 MAPK activity in the sarcolemma and nuclei of IPC-treated hearts.     

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.     

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.     

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.     

转化生长因子β1对肾小管上皮细胞中结缔组织生长因子基因启动子活性的影响

目的探讨转化生长因子[β1（TCF-β1）对人近端肾小管上皮细胞系HK-2中结缔组织生长因子（CTGF）基因启动子活性的调控作用，以及丝裂原激活蛋白激酶（MAPK）途径对该生长因子作用的影响。方法构建含有人类CTGF基因启动子的报告基因pCTGF-luc，将其瞬时转染HK-2细胞。通过检测荧光素酶的活性观察TGF-β1和MAPK途径抑制剂对CTGF基因启动子活性的影响。结果TGF-β1以剂量和时间依赖方式上调HK-2中CTGF基因启动子的活性。最佳刺激浓度是5ng／ml，最佳刺激时间为12h，荧光素酶相对活性分别为对照组的1．82倍和2．10倍（P〈0．05）。应用PD98059、SB203580和SP600125分别特异性抑制MAPK途径的胞外信号调节蛋白激酶（ERK）、蛋白激酶p38（p38MAPK）和c-Jun-氨基末端激酶（JNK）通路，对TGF-β1上调CTGF启动子活性的作用有不同影响。PD98059显著增加HK-2中pCTGF-luc的基础活性．并在一定浓度范围内（0．5～10μmol／L）促进TGF-β1的上调作用。SB203580对pCTGF-luc基础活性无影响，但以剂量依赖方式显著抑制TGF-β1的激活效应。而SP600125对基础状态和TGF-β1刺激下CTGF基因启动子活性无影响。结论TGF-β1以剂量和时间依赖方式上调HK-2中CTGF基因启动子活性，在转录水平调节CTGF表达。MAPK途径的ERK和p38MAPK通路可影响TGF-β1的这一调控作用。     

Cytochrome P450-dependent arachidonic acid metabolism in human kidney

Cytochrome P450-dependent arachidonic acid metabolism in human kidney cortex from several postmortem subjects has been characterized. Using HPLC and GC/MS, four cytochrome P450-arachidonic acid metabolites were tentatively but not unequivocally identified as epoxyeicosatrienoic acid (EET), dihydroxyeicosatrienoic acid (DHT) and 19- and 20-hydroxyeicosatetraenoic acids, suggesting the involvement of two major cytochrome P450 enzymes, epoxygenase and omega/omega-1 hydroxylases. This pattern of metabolism was similar to that found in rabbit and rat kidneys. The formation of these metabolites was dependent on the presence of NADPH and inhibited by IgG of NADPH-cytochrome P450 (c) reductase. Immunologic studies of renal cytochrome P450 epoxygenase demonstrated that antibodies prepared against human-purified hepatic cytochrome P450 epoxygenase recognized renal enzyme protein and inhibited the enzyme activity by 92%. In contrast, control immunoglobulin did not inhibit renal cytochrome P450 epoxygenase. Antibody inhibition of renal cytochrome P450 epoxygenase demonstrated a degree of conservation of both enzyme proteins between liver and kidney. Antibodies against lauric acid omega/omega-1 hydroxylases (P450 omega) inhibited the formation of omega/omega-1 hydroxylase products, 19- and 20-HETEs. Identical qualitative patterns of arachidonic acid metabolites were observed in all cortical microsomes studied. Interindividual variations were observed in the cytochrome P450-dependent arachidonic acid metabolism, and the activities ranged from 0.031 to 5.027 nmol arachidonic acid converted/mg protein/30 min. which is about a 150-fold difference. However, when the specific activities for total cytochrome P450-dependent arachidonic acid metabolism were calculated, two separate groups could be distinguished, high and low metabolizers of arachidonic acid.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mycophenolic acid induces islet apoptosis by regulating mitogen-activated protein kinase activation

Mycophenolic acid (MPA), an inosine monophosphate dehydrogenase inhibitor, is widely used as an immunosuppressive drug after transplantations including those of pancreas islet cells. However, recent reports have indicated that MPA has apoptotic effects on islet cells in vitro. To study the effect of MPA on islet cells and determine its mechanism, we used an insulin secreting cell-line, HIT-T15. We examined mitogen-activated protein kinase (MAPK) activation after MPA treatment, and determining cell death levels using methylthiazdetetrazolium assays. The activations of extracellular signal-regulated protein kinase (ERK), c-jun N-terminal kinase (JNK), and p38 MAPK and caspase-3 cleavage were measured by Western blotting. MPA (1, 10, 30 micromol/L) increased cell death and caspase-3 cleavage within 24 hours. Exogenous 500 micromol/L guanosine reversed the MPA-induced islet cell death, but exogenous adenosine did not. MPA 10 micromol/L induced cell apoptosis and increased the activations of JNK, ERK, and p38 MAPK. Furthermore, exogenous guanosine, but not exogenous adenosine, reversed these effects induced by MPA. This study demonstrated that MPA may induce islet apoptosis in HIT-T15 cells by increasing activations of JNK, ERK, and p38 MAPK in a guanosine-dependent manner.     

Expression of mitogen-activated protein kinase family in rat renal development

BACKGROUND: Among mitogen-activated protein kinase (MAPK) family members, extracellular signal-regulated kinase (ERK) promotes proliferation or differentiation, whereas c-Jun N-terminal kinase (JNK) and p38 MAPK (p38) are thought to inhibit cell growth and induce apoptosis. MAPK phosphatase-1 (MKP-1) inactivates and modulates MAPKs. During renal development, large scale proliferation and apoptosis occur. We investigated the temporal and spatial expression patterns of MAPKs and MKP-1 in rat kidney during development. METHODS: Western blot analysis and immunohistochemistry were performed in the developing and mature kidney of the rat. RESULTS: The expression of ERK, p38, and MKP-1 were high in developing kidney. On the other hand, JNK was abundantly expressed in adult kidney. Active forms of ERK, p38, and JNK correlated with the protein expression levels. Immunohistochemical studies revealed that ERK was strongly expressed by blastema cells, mesenchymal cells, and ureteric bud tips in nephrogenic zone of embryonic kidney. In neonatal kidney, ERK was more abundant in the deep cortex and the medulla corresponding to tubule maturation. p38 and MKP-1 were detected uniformly in mesenchymal cells, mesangial cells, and ureteric bud epithelia of fetal kidney without an obvious correlation with the occurrence of apoptosis. JNK was expressed by tubular cells and podocytes of adult kidney. CONCLUSIONS: ERK, p38, and MKP-1 are strongly expressed in developing kidney, and JNK is detected predominantly in adult kidney. Both the temporal and spatial expression of ERK coincides with the maturation of the kidney.     

烫伤大鼠心肌细胞丝裂素活化蛋白激酶的活化及胞内分布规律的研究

目的　观察大鼠严重烫伤后早期心肌细胞中丝裂素活化蛋白激酶 [MAPKs,包括 p38激酶、细胞外信号调节激酶 (ERKs)和应激活化蛋白激酶 (JNK) ]的活化及胞内分布规律 ,探讨其与心肌损伤的关系。　方法　制作严重烫伤大鼠模型 ,于伤后 1、3、6、12、2 4h取其全血及心肌组织标本 ,并取正常大鼠的相应标本为对照。常规检测各血清标本中肌酸激酶同工酶 MB(CK MB)的活力 ;采用Western印迹法 ,检测各心肌组织标本中MAPKs各成员的活化情况 ,并对其组织切片行免疫组化染色。　结果　伤后 p38激酶、ERK均发生活化并伴核转位 ,以 1、3、6h最明显 (P <0 .0 1) ;伤后 1～ 2 4hJNK均未见活化。血清CK MB含量于伤后 3h升高 ,12h达高峰 (P <0 .0 5～ 0 .0 1)。　结论　p38激酶和ERK信号通路可能在严重烧伤后早期心肌细胞发生的损伤性反应中起重要作用 ,而前者可能是烧伤引发心肌损伤的主要信号转导通路之一。     

Activation of mitogen-activated protein kinase cascades is involved in regulation of bone morphogenetic protein-2-induced osteoblast differentiation in pluripotent C2C12 cells

Bone morphogenetic protein (BMP)-2, a member of the transforming growth factor-beta (TGF-beta) superfamily, is able to induce osteoblastic differentiation of C2C12 cells. Both Smad and mitogen-activated protein kinase (MAPK) pathways are essential components of the TGF-beta superfamily signaling machinery. Although Smads have been demonstrated to participate in the BMP-2-induced osteoblastic differentiation of C2C12 cells, the role of MAPK has not been addressed. This report shows that BMP-2 activates ERK and p38, but not JNK, in C2C12 cells. Pretreatment of cells with the p38 inhibitor, SB203580, dramatically reduced BMP-2-induced expression of the osteoblast markers alkaline phosphatase (ALP) and osteocalcin (OC). Nevertheless, overexpression of MKK3, a protein kinase that phosphorylates and activates p38, failed to induce ALP or OC expression in the absence of BMP-2, indicating that p38 activation is necessary but not sufficient for the acquisition of the osteoblast phenotype by these cells. Although ALP induction was increased slightly in the presence of PD-98059, a selective inhibitor of the ERK cascade, this compound significantly inhibited both steady-state and BMP-2-induced OC RNA levels. Our results indicate that p38 and ERK cascades play a crucial role in the osteoblast differentiation of C2C12 cells mediated by BMP-2.     

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.     

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.