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.     

Activation of extracellular signal-related kinases 1 and 2 in Sertoli cells in experimentally cryptorchid rhesus monkeys

目的:观察恒河猴隐睾(热刺激)模型中睾丸细胞外信号调节激酶1和2(ERK1/2),c-Jun N 末端激酶(JNKs)和 p38有丝分裂原激活蛋白激酶(MAPK)的时空表达变化并探讨其参与支持细胞去分化的可能调节作用。方法:通过免疫组化和 Western blot 方法观察 ERK1/2、p38以及 JNK 在处于隐睾不同时期的睾丸中的表达变化。结果:腹部温度没有明显改变隐睾的睾丸细胞中的 ERK1/2表达量,但明显活化了磷酸化 ERK1/2在支持细胞中的表达。腹腔内的热压明显增加睾丸细胞中 JNK 总体水平的表达,但没有活化磷酸化 JNK 的表达。睾丸细胞内磷酸化 p38以及非磷酸化的 p38的表达不受热刺激的影响。不成熟或未分化的支持细胞的标志分子(CK-18)的时空表达变化与 ERK1/2在隐睾睾丸内的活化存在一致性。结论:隐睾睾丸内 ERK1/2的活化可能参与支持细胞受热刺激后发生去分化的调节过程。     

The impact of altered annexin I protein levels on apoptosis and signal transduction pathways in prostate cancer cells

BACKGROUND: Although reduced expression levels of annexin I (ANX I) protein is a common finding in all stages of prostate cancer a causative relationship between ANX I dysregulation and prostate cancer development has yet to be established. METHODS: Annexin I expression was restored in LNCaP and MDA PCa 2b that normally express low or undetectable levels of ANX I protein. The impact of restoring ANX I expression on cell viability, colony formation in soft agar, apoptosis, and extracellular signal-regulated kinases (ERK), p38, c-Jun N-terminal kinases (JNK) activation was examined. RESULTS: Restoring ANX I expression reduced cell viability, colony formation, in addition to inducing apoptosis. The proliferative response of epidermal growth factor was blocked by restoring ANX I expression. Furthermore, increasing basal and induced levels of phosphorylated p38 and JNK were observed in prostate cancer cells following restoration of ANX I expression. CONCLUSIONS: Annexin I may have tumor suppressor functions in prostate cancer. The pro-apoptotic effect of ANX I involves the activation of p38 and JNK, which appears to shift the balance of signal transduction away from proliferation and toward apoptosis.     

Transforming growth factor-beta1 induces apoptosis in vascular endothelial cells by activation of mitogen-activated protein kinase

BACKGROUND: Vascular endothelial cell apoptosis is central in atherosclerosis and intimal hyperplasia. Transforming growth factor (TGF)-beta1 induces endothelial cell apoptosis through unidentified mechanism(s). Although TGF-beta1 signals through the Smad proteins, in some nonendothelial cell types it also activates the mitogen-activated protein kinase (MAPK) (extracellular signal-regulated kinase, c-Jun N-terminal kinase, and p38 MAPK [p38(MAPK)]). p38(MAPK) relays apoptotic signals in several cell types. We hypothesized that TGF-beta1 activates endothelial cell MAPKs and induces apoptosis through p38(MAPK) activation. METHODS: Human umbilical vein or bovine capillary endothelial cells were incubated with TGF-beta1 for 0.5 to 12 hours. MAPK activation was characterized by Western blotting with antibodies to phosphorylated extracellular signal-regulated kinase 1/2, p38(MAPK), or c-Jun N-terminal kinases 1/2. To study apoptosis, extracts of cells incubated with TGF-beta1 for 6 hours with or without MAPK inhibitors were characterized by Western blotting analysis of poly (ADP-Ribose) polymerase degradation. RESULTS: TGF-beta1 induced p38(MAPK), extracellular signal-regulated kinase 1/2, and c-Jun N-terminal kinase 1/2 activation and increased apoptosis. Inhibition of p38(MAPK) significantly reduced TGF-beta1-induced apoptosis. In contrast, inhibition of other signaling pathways was ineffective. CONCLUSIONS: TGF-beta1 induces endothelial cell apoptosis through p38(MAPK) activation. Because TGF-beta1 is upregulated in vascular remodeling, p38(MAPK) is a potential target to prevent endothelial cell apoptosis during this process.     

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.     

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.     

晚期糖基化产物受体信号传导在羧甲赖氨酸诱导足细胞表达单核细胞趋化因子中的作用

目的 了解足细胞上晚期糖基化产物受体（RAGE）激活后的细胞内信号的传导途径.方法 激光共聚焦显微镜观察细胞内反应性氧自由基（ROS）的产生.Western印迹方法检测足细胞内丝裂原激活的蛋白激酶（MAPK）家族磷酸化,RT-PCR方法检测单核细胞趋化因子-1（MCP-1）的mRNA表达.结果 足细胞细胞核内存在基础性的ROS,AGE和羧甲赖氨酸（carboxymethyllysine,CML）分别诱导细胞浆内ROS增加2.2和2.6倍.N-乙酰-L-半胱氨酸（NAC）抑制基础性和诱导性ROS形成,RAGE中和抗体则完全抑制诱导性的ROS产生.NAC也可直接抑制CML以及外源性H2O2诱导的MCP-1的表达.使用CML刺激足细胞10 min后,磷酸化细胞外信号调节激酶（ERK）增加3.8倍.而CML刺激足细胞120 min仍没有发现磷酸化的p38MAPK和应激活化蛋白激酶（SAPK）/氨基末端激酶（JNK）表达或上调.使用NAC,7氨4三氟甲基香豆素（AFC）可以完全防止ERK磷酸化并抑制MCP-1的mRNA表达.PD98059阻断了ERK磷酸化却并不能完全抑制MCP-1的mRNA的表达.结论 足细胞上RAGE激活后,通过ROS-p21Ras-ERK信号途径诱导足细胞表达MCP-1.     

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。     

Parathyroid hormone-related protein induces insulin expression through activation of MAP kinase-specific phosphatase-1 that dephosphorylates c-Jun NH2-terminal kinase in pancreatic beta-cells

Parathyroid hormone-related protein (PTHrP) increases the content and mRNA level of insulin in a mouse beta-cell line, MIN6, and primary-cultured mouse islets. We examined the mechanism of PTHrP-induced insulin expression. The PTHrP effect was markedly augmented by SB203580, a mitogen-activated protein (MAP) kinase inhibitor, and SB203580 itself increased insulin expression extensively, even without PTHrP. Because SB203580 inhibits both p38 and c-jun NH(2)-terminal kinases (JNKs), we investigated the JNK-specific inhibitor SP600125. SP600125 also increased insulin content and its mRNA level. PTHrP induced dephosphorylation of JNK1/2, and PTHrP-induced insulin expression was blocked by a dominant-negative type JNK-APF. We suspected that dual specificity MAP kinase phosphatases (MKPs) may be involved in the PTHrP-induced insulin expression by inactivating JNK1/2. MIN6 cells contained at least five MKPs, among which only MKP-1 was inducible by PTHrP. PTHrP-induced insulin expression was blocked by the MKP-1 expression inhibitor Ro-31-8220, indicating that the PTHrP effect is mediated by MKP-1. Indeed, adenoviral MKP-1 expression increased insulin expression by decreasing a phosphorylation form of JNKs and a resulting phosphorylated form of c-jun in MIN6 cells. The phosphorylated form of c-jun is known to repress cAMP-dependent insulin gene promoter activity. Thus, MKP-1 controls the insulin expression by downregulating a JNK/c-jun pathway.     

Apoptosis induced by chemotherapeutic agents involves c-Jun N-terminal kinase activation in sarcoma cell lines.

Molecular mechanisms underlying chemotherapeutic agent-induced apoptosis in sarcoma cells are not well known. Induction of apoptosis is regulated by several components including mitogen-activated protein kinases (MAPKs) comprising ERK, p38MAPKs, and c-Jun N-terminal kinase (JNK). In the present study, we examined whether activation of JNK is induced by the chemotherapeutic agents cis-diaminedichloroplatinum (cisplatin, CDDP) or doxorubicin (DXR), and whether the ectopic expression of constitutively active (MKK7-JNK1) or dominant-negative form of JNK (dnJNK) influenced apoptosis in response to the CDDP or DXR in sarcoma cell lines MG-63 and SaOS-2. The CDDP or DXR induced JNK activation in the both cell lines, as assessed by Western blotting using phosphospecific antibodies. A transient expression of the activated form of JNK sensitized the MG-63 and SaOS-2 cells to the drug-induced apoptosis, while dnJNK1 reduced the proportion of apoptotic cell death. Apoptosis was determined by flow cytometry using annexin-V Cy5. Collectively, our results indicate that JNK activation is involved in apoptotic cell death in sarcoma cell lines following stimulation with CDDP or DXR.     

Cholinergic receptor induction and JNK activation in acute pancreatitis

BACKGROUND: Cholecystokinin-A (CCK-A) and cholinergic receptor pathways, capable of activating stress kinases p38 mitogen-activated protein kinase (p38(MAPK)) and cJUN N-terminal kinase (JNK), are implicated in the pathogenesis of ligation-induced acute pancreatitis in rats. As ligation-induced acute pancreatitis in rats is associated with CCK-A receptor induction and p38(MAPK) activation, and as receptor induction could amplify acinar hyperstimulation and exacerbate cell stress, we tested the hypothesis that the cholinergic M3 receptor is induced and JNK is activated in this model. METHODS: Cholinergic M3 receptor expression and JNK activation was compared in rats 1, 3, or 24 hours after sham operation or duct ligation. RESULTS: Immunoblot analysis of pancreatic homogenates showed a time-dependent increase in cholinergic M3 receptor protein, total JNK, and phospho-JNK after duct ligation. CONCLUSIONS: There is a rapid and progressive cholinergic M3 receptor induction and JNK activation in ligation-induced acute pancreatitis in rats. These findings may have significance in the mechanism of disease pathogenesis.     

Cardiopulmonary bypass reduces peripheral microvascular contractile function by inhibition of mitogen-activated protein kinase activity

BACKGROUND: Mitogen-activated protein kinases (MAPK) have been implicated in pathophysiologic responses to cardiopulmonary bypass (CPB). MAPK are deactivated by phosphatases, such as MAPK phosphatase-1 (MKP-1). We hypothesized that MAPK mediate peripheral microvascular contractile dysfunction caused by CPB in humans. METHODS: Skeletal muscle was harvested before and after CPB. Protein levels of MKP-1 and activated extracellular signal-regulated kinase 1/2 (ERK1/2) and p38 were measured. MKP-1 gene expression was measured. Peripheral microvessel responses to vasopressors were studied by videomicroscopy. Contractile function also was measured after MAPK inhibition with PD98059 (ERK1/2) and SB203580 (p38). ERK1/2, p38, and MKP-1 were localized by immunohistochemistry and in situ hybridization. RESULTS: ERK1/2 and p38 activity was decreased in peripheral tissue after CPB. MKP-1 was increased after CPB. Contractile responses of peripheral arterioles to phenylephrine and vasopressin were decreased after CPB. Microvessel reactivity also was reduced after treatment with PD98059 and SB203580. ERK1/2, p38, and MKP-1 localized to peripheral arterioles in tissue sections. CONCLUSIONS: CPB reduces ERK1/2 and p38 activity in peripheral tissue, potentially by MKP-1. Contractile responses of peripheral arterioles to phenylephrine and vasopressin are dependent on ERK1/2 and p38 and are decreased after CPB. These results suggest that alterations in MAPK pathways in part regulate peripheral microvascular dysfunction after CPB in humans.     

Fas and Fas-ligand expression in human pancreatic cancer

OBJECTIVE: To investigate Fas and FasL expression in pancreatic tissues and cultured pancreatic cancer cell lines, and to assess the ability of anti-Fas antibodies to induce apoptosis. SUMMARY BACKGROUND DATA: Activation of the Fas receptor by Fas-ligand (FasL) results in apoptosis, and dysregulation of this pathway may contribute to abnormal cell proliferation. METHODS: Northern blotting and immunohistochemistry were used to compare Fas and FasL expression in normal and cancerous tissues. DNA 3'-OH end labeling was used to detect apoptotic cells. The effects of Fas activation on cell growth and signaling pathways were investigated in culture. RESULTS: Pancreatic cancers exhibited increased Fas RNA levels, whereas FasL mRNA levels were similar in both groups. Despite the colocalization of Fas and FasL in the cancer cells, an apoptotic signal was present in approximately 10% of these cells in only 2 of 16 cancer samples. Fas and FasL were coexpressed in all four cell lines, whereas Fas-associated phosphatase 1 was below the level of detection in all cell lines. Only COLO-357 cells underwent apoptosis after Fas activation. Apoptosis was associated with enhanced activation of jun kinase (JNK) and p38 mitogen-activated protein kinase (MAPK). In the presence of actinomycin D, Fas antibody also induced apoptosis in the other three cell lines. CONCLUSIONS: These results suggest that pancreatic cancer cells are resistant to Fas-mediated apoptosis by mechanisms excluding receptor downregulation or Fas-associated phosphatase upregulation and raise the possibility that Fas-mediated apoptosis may be dependent on the activation of the JNK/p38 MAPK pathway in these cells.     

Vein graft arterialization causes differential activation of mitogen-activated protein kinases

OBJECTIVE: Vascular injury results in activation of the mitogen-activated protein kinases-extracellular-signal regulated kinases, c-jun N-terminal kinase, and p38(MAPK)-which have been implicated in cell proliferation, migration, and apoptosis. The goal of this study was to characterize mitogen-activated protein kinase activation in arterialized vein grafts. METHODS: Carotid artery bypass using reversed external jugular vein was performed in 29 dogs. Vein grafts were harvested after 30 minutes and 3, 8, and 24 hours, and 4, 7, 14, and 28 days. Contralateral external jugular vein and external jugular vein interposition vein-to-vein grafts were used as controls. Vein graft extracts were analyzed for extracellular-signal regulated kinases, c-jun N-terminal kinase, and p38(MAPK) activation. Proliferating cell nuclear antigen expression was investigated as a parameter of cell proliferation. Apoptosis was assessed by terminal deoxynucleotidyl transferase-mediated 2'-deoxyuridine 5'-triphosphate nick end labeling staining and intimal hyperplasia by morphometric examination of tissue sections. RESULTS: Significant intimal hyperplasia was observed at 28 days. Over the time points studied, vein graft arterialization resulted in bimodal activation of both extracellular-signal regulated kinase and p38(MAPK) (30 minutes through 3 hours; 4 days) but did not induce activation of c-jun N-terminal kinase. Proliferating cell nuclear antigen expression increased from days 1 through 28, and apoptosis increased between 8 and 24 hours. CONCLUSION: Vein graft arterialization induces bimodal activation of extracellular-signal regulated kinase and p38(MAPK); however, in contrast with what is described in arterial injury, it does not induce c-jun N-terminal kinase activation. These results provide the first comprehensive characterization of the mitogen-activated protein kinase signaling pathways activated in vein graft arterialization and identify mitogen-activated protein kinases as potential mediators of vein graft remodeling and subsequent intimal hyperplasia.     

MAPK信号通路在骨关节炎发病机制中的研究进展

丝裂原活化蛋白激酶（mitogen—activated proteinkinase,MAPK）真核细胞信号传递的重要途径之一,在调节控制细胞结构和功能活动中发挥关键作用。在真核生物中MAPK信号通路包括p38、ERK、JNK、ERK5等多个亚家族。随着研究的不断深入,发现p38、ERK、JNK信号转导途径的活化与骨关节炎（osteoarthritis,OA）软骨损伤密切相关,诱导软骨细胞产生基质金属蛋白酶,加速关节软骨病理性降解,并参与软骨细胞增殖、凋亡与分化等一系列反应,明确MAPK信号通路在OA中的发生发展机制已成为研究的新热点。     

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.     

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.