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。     

MAPKs信号通路在内皮素-1诱导的牙周膜干细胞成骨分化过程中的作用研究

目的:研究丝裂原激活蛋白激酶(Mitogen-activated protein kinases,MAPKs)信号通路在内皮素-1(Endothelin-1,ET-1)诱导的牙周膜干细胞(Periodontal ligament stem cells,PDLSCs)成骨分化中的调控机制。方法:体外培养牙周膜干细胞,于ET-1处理前分别加入ERK通路抑制剂PD98059,JNK通路抑制剂SP600125,p38α通路抑制剂SB203580。以未作任何处理的牙周膜干细胞为空白对照,以单独加入抑制剂的牙周膜干细胞为阴性对照,用100nMET-1干预不同组牙周膜干细胞14d,通过实时定量PCR及Western blot技术检测细胞Runx2和OCN基因和蛋白的表达情况。结果:ET-1对牙周膜干细胞Runx2和OCN基因及蛋白表达具有显著促进作用,但PD98059抑制了ET-1对牙周膜干细胞Runx2和OCN表达的促进效应,而SP600125和SB203580的抑制作用不显著。结论:ET-1通过ERK信号通路调控牙周膜干细胞Runx2和OCN的表达。     

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。     

丝裂原活化蛋白激酶类和蛋白激酶C在转化生长因子β1诱导肾小管细胞结缔组织生长因子表达中的作用

目的了解转化生长因子β1（TGF-β1）诱导肾小管细胞结缔组织生长因子（CTGF）表达的机制，特别是蛋白激酶C（PKC）和丝裂原活化蛋白激酶（MAPK）在CTGF基因表达中的作用及其对Smad磷酸化的影响。方法分别应用PKC抑制剂G06850以及MAPK的3个组成成分ERK、JNK和p38MAPK的抑制剂PD98059、U0126、SP600125和SB203580阻断相应通路，观察其对TGF．131诱导的CTGF表达以及Smad2／Smad3磷酸化的影响。结果TGF-β1（5μg／L）以时间依赖方式诱导HK-2细胞中Smad2／Smad3的磷酸化，从基础值0．87±0．09上升至2h时高峰2．350±0.11。PKC抑制剂G06850（5μmol／L）和ERK抑制剂PD98059（10μmol／L）、U0126（10μmol／L）可部分抑制TGF-β1诱导的CTGF表达，而p38MAPK抑制剂SB203580（20μmol／L）和JNK抑制剂SP600125（10μmol／L）对TGF-β1诱导的CTGF的表达无影响。PKC抑制剂G06850（5μmol／L）可减少TGF-β1诱导的Smad2／Smad3磷酸化，而ERK抑制剂PD98059（10μmol／L）和U0126（10μmol／L）对Smad2／Smad3的磷酸化没有影响。结论在肾小管上皮细胞中，TGF-β1诱导CTGF的表达需要PKC和Ras／MEK／ERK的参与。PKC以Smad依赖的方式参与肾小管上皮细胞中TGF-β1诱导的CTGF的表达，而Ras／MEK／ERK对CTGF表达的调节不依赖于Smads。     

核酶基因抑制肝癌细胞低氧诱导因子-1α表达的实验研究

目的探讨靶向HIF-1α核酶基因对肝癌细胞HIF-1α表达的调控。方法采用脂质体介导的方法，将靶向HIF-1α核酶基因真核表达载体转染肝癌细胞Hep3B2，并予低氧条件诱导。于转染后48h采用Western Blotting检测Hep3B2细胞中HIF-1α蛋白的表达水平；荧光报告基因方法检测HIF-1转录活性。结果Hep3B2细胞低氧诱导后，HIF-1α蛋白表达水平、HIF-1转录活性增高（1．0±0．02），转染核酶基因400μmol／L后48h，Hep3B2细胞低氧诱导的HIF-1α蛋白表达水平明显下降，HIF-1转录活性下调（0．12±0．025，P〈0．05）。结论核酶基因可特异性抑制低氧诱导的肝癌细胞HIF-1α表达，降低其转录活性。     

缺氧诱导对人结肠癌SW480细胞缺氧诱导因子-1α和Survivin基因表达的影响

目的 构建缺氧诱导模型和观察缺氧对人结肠癌SW480细胞系缺氧诱导因子-1α（HIF-1α）和Survivin基因表达的影响。方法 利用氯化钴（CoCl2）构建缺氧诱导模型；利用逆转录-聚合酶链反应（RT—PCR）分别测定CoCl2缺氧诱导与SW480细胞HIF—1α和Survivin基因mRNA表达的时效关系（0、2、4、8、24h）和量效关系（0、50、100、150、200／μmol／L）。结果HIF-1α和Survivin基因mRNA表达与氯化钴浓度呈明显的剂量依赖关系；HIF-1α基因表达随诱导时间的延长而逐渐增强，4h亚组出现一个表达高峰，Survivin基因在缺氧诱导4h呈现表达最高峰；HIF-1α与survivin基因mRNA的表达在缺氧诱导量效关系（r=0．521，P＜0．05）和时效关系（r=0．693，P＜0．05）中均呈显著正相关。结论 缺氧可以诱导HIF—1α和Survivin基因的表达增加；HIF—1α与Survivin基因的表达均呈显著相关，提示Survivin基因可能与HIF—1α存在某种调控关系并在缺氧抗凋亡机制中发挥作用。     

两种不同病理类型肾病患者尿蛋白诱导肾小管上皮细胞上调表达趋化因子及其分子信号机制研究

目的 观察不同病理类型肾病患者尿蛋白对人近端肾小管上皮细胞（HK-2）分泌趋化因子的影响并探讨其分子信号机制。 方法 所有患者均经临床和肾穿刺活检确诊为原发性局灶节段性肾小球硬化（FSGS）或微小病变性肾病（MCD）。以超滤法浓缩提取患者尿中总蛋白，分别刺激体外培养的HK-2细胞。RT-PCR检测调节正常T细胞表达和分泌的细胞因子（RANTES）及巨噬细胞移动抑制因子（MIF）的mRNA水平。免疫荧光、ELISA及流式细胞仪检测蛋白水平的表达。Western 印迹法检测p38 MAPK和胞外信号调节激酶（ERK）水平。特异性抑制剂SB203580抑制p38磷酸化；PD98059则用于抑制ERK磷酸化。 结果 两种尿蛋白成分有差异，FSGS患者尿蛋白中含有更多的大分子量蛋白。两种尿蛋白均刺激HK-2细胞RANTES及MIF表达上调，而FSGS患者尿蛋白刺激作用更强。两种尿蛋白均显著激活HK-2细胞内ERK和p38 MAPK信号通路。特异性抑制剂分别抑制ERK或p38 MAPK的活化后，FSGS患者尿蛋白介导的RANTES和MIF的上调表达作用可被SB203580或PD98059抑制，而MCD患者尿蛋白的刺激作用却仅能被SB203580所阻断。 结论 FSGS肾病患者的尿蛋白比MCD患者的尿蛋白有更强的上调HK-2细胞RANTES及MIF表达的作用。两种尿蛋白介导趋化因子上调表达的分子信号机制存在差异。     

二甲基乙二酰基甘氨酸对缺氧复氧诱导的肾小管上皮细胞损伤的保护作用及其机制

目的 通过脯氨酸羟化酶抑制剂二甲基乙二酰基甘氨酸（DMOG）稳定缺氧诱导因子1α（HIF-1α）表达,探讨其对缺氧复氧诱导的肾小管上皮细胞（HKC）损伤的保护作用及其机制。 方法 制作无糖缺氧复氧细胞损伤模型,用不同浓度的DMOG预处理,锥虫蓝染色和乳酸脱氢酶（LDH）活性方法检测细胞活力及损伤;Annexin V和PI染色流式细胞仪技术检测细胞凋亡;实时荧光定量PCR方法检测红细胞生成素（EPO）、热休克蛋白70（HSP70）和血红素氧合酶1（HO-1） mRNA的表达;Western印迹法检测HIF-1α、活性caspase-3和Bcl-2蛋白表达。 结果 正常情况下HKC细胞内几乎无HIF-1α蛋白表达,DMOG刺激6 h后HIF-1α蛋白及其靶基因EPO、HSP70和HO-1 mRNA表达均显著上调（均P ＜ 0.01）,且呈浓度依赖性。500 μmol/L或1 mmol/L DMOG预处理可明显改善缺氧复氧诱导的细胞损伤,表现为细胞存活率升高（95.6%±1.8%、96.1%±1.0%比 83.3%±3.1%）;培养上清液中LDH 活性下降;细胞凋亡减少（8.6%±2.7%、6.1%±2.3%比19.2%±4.0%）（均P ＜ 0.05）。另外,细胞内活性caspase-3蛋白表达显著下调,而Bcl-2蛋白表达则显著上调（均P ＜ 0.05）。 结论 DMOG预处理可稳定肾小管上皮细胞内HIF-1α表达,对缺氧复氧诱导的肾小管上皮细胞损伤具有一定保护作用。其机制可能与促进EPO、HSP70和HO-1表达,抑制caspase-3活化,上调Bcl-2表达有关。     

丝裂原蛋白激酶信号转导通路在机械通气相关性肺损伤中的作用

目的观察机械通气介导肺损伤（VILI）过程中丝裂原蛋白激酶（MAPK）的活性变化以及对细胞因子的影响，从中探讨VILI发生机制和MAPK的作用。方法72只SD大鼠随机分为未处理的对照组（不行机械通气）、正常通气组、过度通气组和采用MAPK抑制剂SP600125（JNK）、SB203580（p38）、PD98059（ERK）分别预处理上述3组。机械通气4h后取大鼠肺组织采用Western blot方法测定各组的总JNK、ERK、p-38蛋白激酶的表达及其磷酸化水平变化。同时以酶联免疫吸附试验（EUSA）方法测定大鼠肺组织、支气管肺泡灌洗液（BALF）和血浆中的肿瘤坏死因子-α（TNF-α）、巨噬细胞炎性蛋白-2（MIP-2）浓度。结果正常和过度机械通气4h后均能激活JNK、ERK、p38激酶，但以过度通气组为著（P〈0．01）。过度通气组大鼠肺组织、BALF、血浆中的TNF-α、MIP-2含量显著高于其他组（P〈0．01）。JNK、ERK、p38抑制剂显著降低肺组织、BALF中的TNF-α、MIP-2含量（P〈0．05或0．01），且JNK和ERK抑制剂作用强于p38抑制剂。结论过度机械通气激活了肺细胞中的JNK、ERK、p38激酶，且JNK、ERK、p38参与了VILI细胞因子的产生，即MAPK信号转导通路的激活可能是VILI发生机制之一。     

ERK/MAPK通路参与肝癌产生多药耐药的胞内信号传导

目的探讨微环境诱导肝癌产生多药耐药的胞内信号传导途径。方法分别使HepG2细胞在缺氧、低糖环境下生长或稳定整合HBX基因,运用Western蛋白印迹法检测这些细胞内ERK／MAPK的活性。用ERK／MAPK特异性阻断剂U0126处理这些细胞后,用Western蛋白印迹法检测缺氧诱导因子-1α（HIF—1α）和多药耐药相关蛋白的表达变化,逆转录聚合酶链反应和免疫细胞化学技术分别检测HIF-1α在mRNA水平表达量和部位的改变。结果不同环境下生长的HepG2细胞中,磷酸肜非磷酸化ERK／MAPK比例均有不同程度的增高。用U0126处理12h后,这些细胞中HIF-1α和多药耐药相关蛋白的表达下降,且HIF-1α表达由胞核向胞质转位,其mRNA水平无显著变化。结论ERK／MAPK信号通路是微环境诱导肝癌产生多药耐药的重要胞内信号传导途径。     

Immune suppression in polymicrobial sepsis: differential regulation of Th1 and Th2 responses by p38 MAPK

BACKGROUND: Studies have indicated that a shift from a Th1 to a Th2 response occurs that contributes to the late immunosuppression seen during sepsis. However, the mechanism by which this occurs is unknown. In this regard, mediators released in response to sepsis are thought to upregulate a family of stress-induced mitogen-activated protein kinases (MAPKs), such as JNK, ERK, and p38 MAPK, which may play a role in this process. MATERIALS AND METHODS: To determine the role of MAPK in immune suppression, we induced polymicrobial sepsis in C3H/HeN male mice using cecal ligation and puncture (CLP). Splenic lymphocytes were harvested 24 h post-CLP and stimulated with the T-cell mitogen concanavalin A, and the expression and activation of these MAPKs were assessed by Western analysis. To determine the extent to which these MAPKs may have an impact on splenic immune function, cells were pretreated with a 10 microM concentration of the p38 MAPK inhibitor SB203580 or the MEK inhibitor PD98059 or with DMSO vehicle. The cells were then stimulated with 2.5 microg/ml of the T-cell mitogen concanavalin A, and cytokine release was then determined (by ELISA). RESULTS: In the lymphocytes from CLP mice no JNK signal was detected, however, p38 expression and activation were markedly (P < 0.05, n = 6) increased. In contrast, the expression of activated ERK markedly decreased following septic challenge. The results indicate that p38 MAPK inhibition with SB203580 suppressed the sepsis-induced augmentation of interleukin-10 release while restoring the suppressed Th1 cytokine interleukin-2 release typically encountered following sepsis. Inhibition of ERK had no effect on cytokine release. Neither PD98059 nor SB203580 had an effect on interferon gamma release or on proliferative capacity. CONCLUSION: This would indicate that the induction of p38 MAPK activation in splenocytes contributes to the immunosuppression seen in late sepsis.     

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.     

Effect of cysteine-rich protein 61 on oxidative stress in human kidney tubular epithelial cell line after anoxia

Objective To investigate the effect and mechanism of cysteine-rich protein 61(Cyr61) on oxidative stress in human kidney tubular epithelial cell line after anoxia. Methods Human kidney tubular epithelial cell line (HK-2 cells) were divided into 5 groups：control group, Cyr61 group, MAPK inhibitor group (Cyr61+PD98059), p38 inhibitor group (Cyr61+SB203580) and PI3K inhibitor group (Cyr61+Wortmannin). Each group was pretreated for 12 h and then injured by anoxia．The cell viability was determined by MTT assay and the apoptosis rate of HK-2 cells was determined by flow-cytometry. The cellular ROS level was measured by spectro-fluorometry. The cellular superoxide dismutase (SOD) and catalase (CAT) were measured by nephelometry test. The expression of Nrf2 in HK-2 cells was detected by Western blotting. Results Anoxia enhanced the expression of ROS and Nrf2, decreased the expression of SOD and CAT significantly,meanwhile decreased HK-2 viability and increased HK-2 apoptosis (all P＜0.05). Cyr61 increased the expression of p-Akt, Nrf2, SOD and CAT in HK-2, and decreased the expression of ROS, at the same time increased HK-2 viability and decreased HK-2 apoptosis (all P＜0.05). Wortmannin inhibited the expression of p-Akt,Nrf2, SOD and CAT, meanwhile decreased HK-2 viability and increased HK-2 apoptosis (P＜0.05). PD98059 and SB203580 had no affect on HK-2 compared to Cyr61 group (P＞0.05). Conclusions Cyr61 promotes the expression of Nrf2 through PI3K pathway in HK-2, which enhances the expression of SOD and CAT, and decreases the expression of ROS. Cyr61 exhibits protective effects on HK-2 cells injured by oxidative stress after anoxia.     

Mitogen-activated protein kinases signal inhibition of apoptosis in lipopolysaccharide-stimulated neutrophils.

BACKGROUND: Neutrophil (PMN) apoptosis is critical to the resolution of infection and the limitation of inflammation. Bacterial endotoxin (lipopolysaccharide [LPS]) inhibits PMN apoptosis and activates the p38 mitogen-activated protein kinase (MAPK) signal cascade. The role of p38 and other MAPKs (ERK and SAPK/JNK) in regulating PMN apoptosis after LPS stimulation is unknown. We hypothesize that MAPK activation by LPS signals inhibition of PMN apoptosis. METHODS: PMNs were isolated from the blood of healthy human volunteers and incubated with PD98059 (ERK inhibitor), SB203580 (p38 inhibitor), or 0.1% dimethyl sulfoxide (vehicle) for 1 hour before treatment with LPS (0, 10, or 1000 ng/mL). Neutrophil MAPK activation was determined by Western blot analysis for phosphorylated p38, ERK, and SAPK/JNK. Apoptosis was quantified by flow cytometry with use of propidium iodide and annexin V. RESULTS: LPS inhibited PMN apoptosis and activated p38 and ERK in a dose- and time-dependent fashion. SAPK/JNK was not activated by LPS. Treatment of cells with ERK inhibitor before LPS stimulation abrogated LPS signaled inhibition of PMN apoptosis. Conversely, p38 inhibition with SB203580 augmented inhibition of apoptosis by LPS. CONCLUSIONS: These data demonstrate opposing roles of MAPKs in mediating PMN apoptosis after LPS stimulation. We conclude that LPS signal transduction by ERK inhibits PMN apoptosis while activation of p38 promotes apoptosis.     

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.     

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.     

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. CONCLUSIONS: Although MAPKs may orchestrate cardioprotection as triggers and mediators in IPC, they are devoid of triggering, but they may have mediator effects in APC.