Biswas Sudipta, Gupta Manveen Kaur, Chattopadhyay Debasis, Mukhopadhyay Chinmay K
Special Centre for Molecular Medicine, Jawaharlal Nehru University, New Delhi 110-067, India.
Am J Physiol Heart Circ Physiol. 2007 Feb;292(2):H758-66. doi: 10.1152/ajpheart.00718.2006. Epub 2006 Nov 3.
Hypoxia-inducible factor (HIF)-1 activation in response to hypoxia requires mitochondrial generation of reactive oxygen species (ROS). In contrast, the requirement of ROS for HIF-1 activation by growth factors like insulin remains unexplored. To explore that, insulin-sensitive hepatic cell HepG2 or cardiac muscle cell H9c2 cells were pretreated with NADPH oxidase inhibitor diphenyleneiodonium chloride (DPI) or apocynin and HIF-1 activation was tested by electrophoretic mobility shift and reporter gene assay. Antioxidants DPI or apocynin completely blocked insulin-stimulated HIF-1 activation. The restoration of HIF-1 activation by H(2)O(2) in DPI-pretreated cells not only confirmed the role of ROS but also identified H(2)O(2) as the responsible ROS. The role of NADPH oxidase was further confirmed by greater stimulation of HIF-1 during simultaneous treatment of suboptimal concentration of insulin along with NADPH but not by NADH. The role of oxidant generated by insulin is found to inhibit the protein tyrosine phosphatase as suggested by the following observations. First, tyrosine phosphatase-specific inhibitor sodium vanadate compensates DPI-inhibited HIF-1 activity. Second, sodium vanadate stimulates HIF-1 activation with suboptimal concentration of insulin. Third, DPI and pyrrolidene dithiocarbamate (PDTC) blocks insulin-receptor tyrosine kinase activation. The activity of phosphatidylinositol 3-kinase as evidenced by Akt phosphorylation, involved in HIF-1 activation, is also dependent on ROS generation by insulin. Finally, DPI pretreatment blocked insulin-stimulated expression of genes like VEGF, GLUT1, and ceruloplasmin. Overall, our data provide strong evidence for the essential role of NADPH oxidase-generated ROS in insulin-stimulated activation of HIF-1.
缺氧诱导因子（HIF）-1对缺氧的激活反应需要线粒体产生活性氧（ROS）。相比之下，ROS在胰岛素等生长因子激活HIF-1过程中的作用尚未得到探索。为了探究这，用NADPH氧化酶抑制剂二苯基碘鎓氯化物（DPI）或载脂蛋白预处理胰岛素敏感的肝细胞HepG2或心肌细胞H9c2，然后通过电泳迁移率变动分析和报告基因检测来检测HIF-1的激活情况。抗氧化剂DPI或载脂蛋白完全阻断了胰岛素刺激的HIF-1激活。DPI预处理细胞中H₂O₂对HIF-1激活的恢复不仅证实了ROS的作用，还确定H₂O₂是起作用的ROS。同时用次优浓度的胰岛素和NADPH而非NADH处理时，HIF-1受到更大刺激，这进一步证实了NADPH氧化酶的作用。如下观察结果表明，胰岛素产生的氧化剂的作用是抑制蛋白酪氨酸磷酸酶。首先，酪氨酸磷酸酶特异性抑制剂钒酸钠可补偿DPI抑制的HIF-1活性。其次，钒酸钠在次优浓度胰岛素作用下刺激HIF-1激活。第三，DPI和吡咯烷二硫代氨基甲酸盐（PDTC）阻断胰岛素受体酪氨酸激酶激活。参与HIF-1激活的Akt磷酸化所证明的磷脂酰肌醇3激酶活性也依赖于胰岛素产生的ROS。最后，DPI预处理阻断了胰岛素刺激的VEGF、GLUT1和铜蓝蛋白等基因的表达。总体而言，我们的数据为NADPH氧化酶产生的ROS在胰岛素刺激的HIF-1激活中的重要作用提供了有力证据。
