Blanc Antoine, Pandey Nihar R, Srivastava Ashok K
Centre de recherche, Centre hospitalier de l'Université de Montréal (CHUM)-Hôtel-Dieu and Department of Medicine, Université de Montréal, Montréal, Québec, Canada.
Antioxid Redox Signal. 2004 Apr;6(2):353-66. doi: 10.1089/152308604322899422.
We have shown earlier that extracellular signal-regulated kinases 1 and 2 (ERK1/2) and protein kinase B (PKB), two key mediators of growth-promoting and proliferative responses, are activated by hydrogen peroxide (H(2)O(2)) in A10 vascular smooth muscle cells (VSMC). In the present studies, using a series of pharmacological inhibitors, we explored the upstream mechanisms responsible for their activation in response to H(2)O(2). H(2)O(2) treatment of VSMC stimulated ERK1/2, p38 mitogen-activated protein kinase (MAPK), and PKB phosphorylation in a dose- and time-dependent fashion. BAPTA-AM and EGTA, chelators of intracellular and extracellular Ca(2+), respectively, inhibited H(2)O(2)-stimulated ERK1/2, p38 MAPK, and PKB phosphorylation. Fluphenazine, an antagonist of the Ca(2+)-binding protein calmodulin, also suppressed the enhanced phosphorylation of ERK1/2, p38 MAPK, and PKB. In contrast, the protein kinase C (PKC) inhibitors Gö 6983 and Rö 31-8220 attenuated H(2)O(2)-induced ERK1/2 phosphorylation, but had no effect on p38 MAPK and PKB phosphorylation. Taken together, these data demonstrate that the activation of Ca(2+)/calmodulin-dependent pathways represents a key component mediating the stimulatory action of H(2)O(2) on ERK1/2, p38 MAPK, and PKB phosphorylation. On the other hand, PKC appears to be an upstream modulator of the increased ERK1/2 phosphorylation, but not of p38 MAPK and PKB in response to H(2)O(2) in VSMC.
我们之前已经表明，细胞外信号调节激酶1和2（ERK1/2）以及蛋白激酶B（PKB）是促进生长和增殖反应的两个关键介质，在A10血管平滑肌细胞（VSMC）中，它们会被过氧化氢（H₂O₂）激活。在本研究中，我们使用一系列药理学抑制剂，探索了它们响应H₂O₂而被激活的上游机制。用H₂O₂处理VSMC会以剂量和时间依赖性方式刺激ERK1/2、p38丝裂原活化蛋白激酶（MAPK）和PKB磷酸化。BAPTA-AM和EGTA分别是细胞内和细胞外Ca²⁺的螯合剂，它们抑制了H₂O₂刺激的ERK1/2、p38 MAPK和PKB磷酸化。氟奋乃静是Ca²⁺结合蛋白钙调蛋白的拮抗剂，它也抑制了ERK1/2、p38 MAPK和PKB增强的磷酸化。相比之下，蛋白激酶C（PKC）抑制剂Gö 6983和Rö 31-8220减弱了H₂O₂诱导的ERK1/2磷酸化，但对p38 MAPK和PKB磷酸化没有影响。综上所述，这些数据表明Ca²⁺/钙调蛋白依赖性途径的激活是介导H₂O₂对ERK1/2、p38 MAPK和PKB磷酸化刺激作用的关键组成部分。另一方面，PKC似乎是VSMC中响应H₂O₂时ERK1/2磷酸化增加的上游调节剂，但不是p38 MAPK和PKB的上游调节剂。
