Bárdos Julia I, Chau Noan-Minh, Ashcroft Margaret
Cell Growth Regulation and Angiogenesis Laboratory, Cancer Research UK Centre for Cancer Therapeutics, Institute of Cancer Research, Sutton, Surrey SM2 5NG, United Kingdom.
Mol Cell Biol. 2004 Apr;24(7):2905-14. doi: 10.1128/MCB.24.7.2905-2914.2004.
The hypoxia-inducible factor 1 (HIF-1) transcriptional complex is regulated by cellular oxygen levels and growth factors. The phosphoinosotide 3-kinase (PI-3K)-Akt/protein kinase B (PKB) pathway has been shown to regulate HIF-1 activity in response to oncogenic signals and growth factors. We assessed whether the HDM2 oncoprotein, a direct target of Akt/PKB, could regulate HIF-1alpha expression and HIF-1 activity under normoxic conditions. We found that growth factor stimulation, overexpression of Akt/PKB, or loss of PTEN resulted in enhanced expression of both HIF-1alpha and HDM2. Growth factor-mediated induction of HIF-1alpha was ablated by transient expression of a dominant negative form of Akt/PKB or by treatment with LY294002. Transient expression of HDM2 led to increased expression of HIF-1alpha. Pulse-chase and cycloheximide experiments revealed that HDM2 did not significantly affect the half-life of HIF-1alpha. Growth factor-induced HIF-1alpha and HDM2 proteins were localized to the nucleus, and induction of both proteins was observed in both p53(+/+) and p53(-/-) HCT116 cells to comparable levels. Importantly, insulin-like growth factor 1-induced HIF-1alpha expression was observed in p53-null mouse embryo fibroblasts (MEFs) but was significantly impaired in p53 Mdm2 double-null MEFs, indicating a requirement for Mdm2 in this process. Finally, we showed that phosphorylation at Ser166 in HDM2 contributed in part to growth factor-mediated induction of HIF-1alpha. Our study has important implications for the role of the PI-3K-Akt/PKB-HDM2 pathway in tumor progression and angiogenesis.
缺氧诱导因子1（HIF-1）转录复合物受细胞氧水平和生长因子调控。磷酸肌醇3激酶（PI-3K）-Akt/蛋白激酶B（PKB）通路已被证明可响应致癌信号和生长因子来调节HIF-1活性。我们评估了Akt/PKB的直接靶点HDM2癌蛋白在常氧条件下是否能调节HIF-1α表达和HIF-1活性。我们发现生长因子刺激、Akt/PKB过表达或PTEN缺失均导致HIF-1α和HDM2表达增强。生长因子介导的HIF-1α诱导可通过瞬时表达显性负性形式的Akt/PKB或用LY294002处理而消除。HDM2的瞬时表达导致HIF-1α表达增加。脉冲追踪和放线菌酮实验表明，HDM2对HIF-1α的半衰期没有显著影响。生长因子诱导的HIF-1α和HDM2蛋白定位于细胞核，在p53(+/+)和p53(-/-) HCT116细胞中均观察到这两种蛋白的诱导，且水平相当。重要的是，在p53基因缺失的小鼠胚胎成纤维细胞（MEF）中观察到胰岛素样生长因子1诱导的HIF-1α表达，但在p53 Mdm2双基因缺失的MEF中显著受损，表明在此过程中需要Mdm2。最后，我们表明HDM2中Ser166位点的磷酸化部分促成了生长因子介导的HIF-1α诱导。我们的研究对PI-3K-Akt/PKB-HDM2通路在肿瘤进展和血管生成中的作用具有重要意义。
