Chen Run-Qiang, Yang Qing-Kai, Chen Yan-Ling, Oliveira Vasco A, Dalton William S, Fearns Colleen, Lee Jiing-Dwan
From the Departments of Immunology and Microbial Science, La Jolla, California 92037.
Chemistry, The Scripps Research Institute, La Jolla, California 92037.
J Biol Chem. 2009 Jun 19;284(25):16752-16758. doi: 10.1074/jbc.M109.014316. Epub 2009 Apr 30.
Hypoxia-inducible factor-1 (HIF-1) plays a central role in tumor progression by regulating genes involved in proliferation, glycolysis, angiogenesis, and metastasis. To improve our understanding of HIF-1 regulation by kinome, we screened a kinase-specific small interference RNA library using a hypoxia-response element (HRE) luciferase reporter assay under hypoxic conditions. This screen determined that depletion of cellular SMG-1 kinase most significantly modified cellular HIF-1 activity in hypoxia. SMG-1 is the newest and least studied member of the phosphoinositide 3-kinase-related kinase family, which consists of ATM, ATR, DNA-PKcs, mTOR, and SMG-1. We individually depleted members of the phosphoinositide 3-kinase-related kinase family, and only SMG-1 deficiency significantly augmented HIF-1 activity in hypoxia. We subsequently discovered that SMG-1 kinase activity was activated by hypoxia, and depletion of SMG-1 up-regulated MAPK activity under low oxygen. Suppressing cellular MAPK by silencing ERK1/2 or by treatment with U0126, a MAPK inhibitor, partially blocked the escalation of HIF-1 activity resulting from SMG-1 deficiency in hypoxic cells. Increased expression of SMG-1 but not kinase-dead SMG-1 effectively inhibited the activity of HIF-1alpha. In addition, cellular SMG-1 deficiency increased secretion of the HIF-1alpha-regulated angiogenic factor, vascular epidermal growth factor, and survival factor, carbonic anhydrase IX (CA9), as well as promoted the hypoxic cell motility. Taken together, we discovered that SMG-1 negatively regulated HIF-1alpha activity in hypoxia, in part through blocking MAPK activation.
缺氧诱导因子-1（HIF-1）通过调节参与增殖、糖酵解、血管生成和转移的基因，在肿瘤进展中发挥核心作用。为了加深我们对激酶组对HIF-1调控的理解，我们在缺氧条件下使用缺氧反应元件（HRE）荧光素酶报告基因检测法，筛选了一个激酶特异性小干扰RNA文库。该筛选确定，细胞SMG-1激酶的缺失在缺氧条件下对细胞HIF-1活性的影响最为显著。SMG-1是磷酸肌醇3激酶相关激酶家族中最新且研究最少的成员，该家族由ATM、ATR、DNA-PKcs、mTOR和SMG-1组成。我们分别敲除了磷酸肌醇3激酶相关激酶家族的成员，只有SMG-1的缺失在缺氧条件下显著增强了HIF-1活性。随后我们发现，SMG-1激酶活性在缺氧时被激活，而SMG-1的缺失在低氧条件下上调了MAPK活性。通过沉默ERK1/2或用MAPK抑制剂U0126处理来抑制细胞MAPK，可部分阻断缺氧细胞中因SMG-1缺失导致的HIF-1活性升高。SMG-1而非激酶失活的SMG-1的表达增加有效抑制了HIF-1α的活性。此外，细胞SMG-1的缺失增加了HIF-1α调控的血管生成因子血管内皮生长因子和存活因子碳酸酐酶IX（CA9）的分泌，并促进了缺氧细胞的迁移。综上所述，我们发现SMG-1在缺氧时负向调节HIF-1α活性，部分是通过阻断MAPK激活来实现的。
