Tan M, Gu Q, He H, Pamarthy D, Semenza G L, Sun Y
Department of Radiation Oncology, University of Michigan Comprehensive Cancer Center, Ann Arbor, MI 48109-096, USA.
Oncogene. 2008 Feb 28;27(10):1404-11. doi: 10.1038/sj.onc.1210780. Epub 2007 Sep 10.
SAG (sensitive to apoptosis gene) or ROC2/RBX2 is the second family member of ROC1/RBX1, a component of SCF (Skp1, Cullin, F-box protein) and VCB (von Hippel-Lindau (VHL), Cullin and Elongin B/C) E3 ubiquitin ligases. SAG protected cells from hypoxia-induced apoptosis when overexpressed. We report here that SAG was subjected to hypoxia induction at the levels of mRNA and protein. Hypoxia induction of SAG was largely HIF-1alpha dependent. A consensus HIF-1-binding site, GCGTG was identified in the first intron of the SAG gene. In response to hypoxia, HIF-1 bound to this site and transactivated SAG expression. SAG transactivation required both the intact binding site in cis and HIF-1alpha in trans. On the other hand, like its family member, ROC1, SAG promoted VHL-mediated HIF-1alpha ubiquitination and degradation, which was significantly inhibited upon small interfering RNA silencing of SAG or ROC1. Furthermore, the endogenous HIF-1alpha at both basal and hypoxia-induced levels was significantly increased upon SAG silencing. Finally, SAG forms in vivo complex with Cul-5 and VHL under hypoxia condition. These results suggest an HIF-1-SAG feedback loop in response to hypoxia, as follows: hypoxia induces HIF-1 to transactivate SAG. Induced SAG then promotes HIF-1alpha ubiquitination and degradation. This feedback loop may serve as a cellular defensive mechanism to reduce potential cytotoxic effects of prolonged HIF-1 activation under hypoxia.
SAG（凋亡敏感基因）或ROC2/RBX2是ROC1/RBX1的第二个家族成员，ROC1/RBX1是SCF（Skp1、Cullin、F-box蛋白）和VCB（冯·希佩尔-林道（VHL）、Cullin和延伸蛋白B/C）E3泛素连接酶的一个组成部分。过表达时，SAG可保护细胞免受缺氧诱导的凋亡。我们在此报告，SAG在mRNA和蛋白质水平上受到缺氧诱导。SAG的缺氧诱导在很大程度上依赖于HIF-1α。在SAG基因的第一个内含子中鉴定出一个共有HIF-1结合位点GCGTG。在缺氧反应中，HIF-1结合到该位点并反式激活SAG表达。SAG反式激活既需要顺式完整的结合位点，也需要反式的HIF-1α。另一方面，与它的家族成员ROC1一样，SAG促进VHL介导的HIF-1α泛素化和降解，在对SAG或ROC1进行小干扰RNA沉默后，这种促进作用受到显著抑制。此外，在沉默SAG后，基础水平和缺氧诱导水平的内源性HIF-1α均显著增加。最后，在缺氧条件下，SAG在体内与Cul-5和VHL形成复合物。这些结果表明存在一个响应缺氧的HIF-1-SAG反馈环，如下：缺氧诱导HIF-1反式激活SAG。诱导产生的SAG随后促进HIF-1α泛素化和降解。这个反馈环可能作为一种细胞防御机制，以减少缺氧条件下长时间HIF-1激活的潜在细胞毒性作用。
