Mammalian tumor suppressor Int6 specifically targets hypoxia inducible factor 2α for degradation by hypoxia- and pVHL-independent regulation
Mammalian tumor suppressor Int6 specifically targets hypoxia inducible factor 2α for degradation by hypoxia- and pVHL-independent regulation
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DOI：
10.1074/jbc.m700423200
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发表时间：
2007-04-27
影响因子：
4.8
通讯作者：
Shibasaki, Futoshi
中科院分区：
文献类型：
--
作者：
Chen, Li;Uchida, Kazuyo;Shibasaki, Futoshi
缺氧诱导因子HIF-1 α和HIF-2 α在其DNA结合和二聚化结构域方面结构相似，但在其反式激活结构域方面不同，并且如使用hif-1 α（-/-）和hif-2 α（-/-）小鼠的实验所示，在其功能方面不同。这意味着HIF-1 α和HIF-2 α可能具有独特的靶基因。为了解决这一矛盾，并确定HIF-2 α特异性靶基因，我们进行了酵母双杂交分析，并确定了肿瘤抑制基因Int 6/eIF 3e/p48作为一种新的靶基因产物参与HIF-2 α的调控。int 6基因首先从筛选中鉴定，其中小鼠乳腺肿瘤病毒被用作插入诱变剂以鉴定其功能对乳腺肿瘤形成至关重要的基因。在这里，通过使用双杂交分析，在哺乳动物细胞中的免疫沉淀，和HRE报告分析，我们报告的HIF-2 α（但不是HIF-1 α或HIF-3 α）与Int 6的特异性相互作用。结果表明，Int 6的直接相互作用诱导蛋白酶体转运蛋白敏感的HIF-2 α降解。这种降解在肾细胞癌786-O细胞中清楚地观察到，并且被发现是缺氧和pVHL独立的。此外，通过int 6-siRNA载体或显性阴性突变体Int 6-Delta C敲低Int 6蛋白增加了内源性HIF-2 α表达，即使在常氧下也是如此，并诱导了一系列关键的血管生成因子，包括血管内质生长因子、血管生成素和碱性成纤维细胞生长因子mRNA。这些结果表明，Int 6是HIF-2-α依赖性血管生成以及癌症形成的一种新的和关键的决定因素，并且int 6-siRNA转移可能是诸如心脏和脑缺血、肝硬化和阻塞性血管疾病等病理状况中的有效治疗策略。
The hypoxia-inducible factors HIF-1 alpha and HIF-2 alpha are structurally similar as regards their DNA-binding and dimerization domains, but differ in their transactivation domains and, as is shown by experiments using hif-1 alpha(-/-) and hif-2 alpha(-/-) mice, in their functions. This implies that HIF-1 alpha and HIF-2 alpha may have unique target genes. To address this discrepancy and identify HIF-2 alpha-specific target genes, we performed yeast two-hybrid analysis and identified the tumor suppressor Int6/eIF3e/p48 as a novel target gene product involved in HIF-2 alpha regulation. The int6 gene was first identified from a screen in which the mouse mammary tumor virus was employed as an insertional mutagen to identify genes whose functions are critical for breast tumor formation. Here, by using two-hybrid analysis, immunoprecipitation in mammalian cells, and HRE-reporter assays, we report the specific interaction of HIF-2 alpha( but not HIF-1 alpha or HIF-3 alpha) with Int6. The results indicate that the direct interaction of Int6 induces proteasome inhibitor-sensitive HIF-2 alpha degradation. This degradation was clearly observed in renal cell carcinoma 786-O cells, and was found to be both hypoxia-and pVHL-independent. Furthermore, Int6 protein knockdown by int6-siRNA vectors or the dominant-negative mutant Int6-Delta C increased endogenous HIF-2 alpha expression, even under normoxia, and induced sets of critical angiogenic factors comprising vascular endoplasmic growth factor, angiopoietin, and basic fibroblast growth factor mRNA. These results indicate that Int6 is a novel and critical determinant of HIF-2-alpha dependent angiogenesis as well as cancer formation, and that int6-siRNA transfer may be an effective therapeutic strategy in pathological conditions such as heart and brain ischemia, hepatic cirrhosis, and obstructive vessel diseases.