Experimental Chemotherapy Laboratory, Regina Elena Cancer Institute, Rome, Italy.
PLoS One. 2010 Jul 27;5(7):e11772. doi: 10.1371/journal.pone.0011772.
Hypoxia-Inducible Factor 1 (HIF-1) is a transcription factor that is a critical mediator of the cellular response to hypoxia. Enhanced levels of HIF-1alpha, the oxygen-regulated subunit of HIF-1, is often associated with increased tumour angiogenesis, metastasis, therapeutic resistance and poor prognosis. It is in this context that we previously demonstrated that under hypoxia, bcl-2 protein promotes HIF-1/Vascular Endothelial Growth Factor (VEGF)-mediated tumour angiogenesis.
METHODOLOGY/PRINCIPAL FINDINGS: By using human melanoma cell lines and their stable or transient derivative bcl-2 overexpressing cells, the current study identified HIF-1alpha protein stabilization as a key regulator for the induction of HIF-1 by bcl-2 under hypoxia. We also demonstrated that bcl-2-induced accumulation of HIF-1alpha protein during hypoxia was not due to an increased gene transcription or protein synthesis. In fact, it was related to a modulation of HIF-1alpha protein expression at a post-translational level, indeed its degradation rate was faster in the control lines than in bcl-2 transfectants. The bcl-2-induced HIF-1alpha stabilization in response to low oxygen tension conditions was achieved through the impairment of ubiquitin-dependent HIF-1alpha degradation involving the molecular chaperone HSP90, but it was not dependent on the prolyl hydroxylation of HIF-1alpha protein. We also showed that bcl-2, HIF-1alpha and HSP90 proteins form a tri-complex that may contribute to enhancing the stability of the HIF-1alpha protein in bcl-2 overexpressing clones under hypoxic conditions. Finally, by using genetic and pharmacological approaches we proved that HSP90 is involved in bcl-2-dependent stabilization of HIF-1alpha protein during hypoxia, and in particular the isoform HSP90beta is the main player in this phenomenon.
CONCLUSIONS/SIGNIFICANCE: We identified the stabilization of HIF-1alpha protein as a mechanism through which bcl-2 induces the activation of HIF-1 in hypoxic tumour cells involving the beta isoform of molecular chaperone HSP90.
缺氧诱导因子 1（HIF-1）是一种转录因子，是细胞对缺氧反应的关键介质。HIF-1 的氧调节亚单位 HIF-1alpha 的水平升高通常与肿瘤血管生成增加、转移、治疗抵抗和预后不良有关。正是在这种情况下，我们之前证明了在缺氧下，bcl-2 蛋白促进 HIF-1/血管内皮生长因子（VEGF）介导的肿瘤血管生成。
方法/主要发现：通过使用人黑色素瘤细胞系及其稳定或瞬时衍生的 bcl-2 过表达细胞，本研究确定 HIF-1alpha 蛋白稳定化是 bcl-2 在缺氧下诱导 HIF-1 的关键调节剂。我们还证明，bcl-2 在缺氧下诱导的 HIF-1alpha 蛋白积累不是由于基因转录或蛋白质合成增加所致。事实上，它与 HIF-1alpha 蛋白表达的翻译后水平调节有关，事实上，在对照系中其降解速度比 bcl-2 转染细胞快。bcl-2 诱导的 HIF-1alpha 稳定化对低氧张力条件的反应是通过损害依赖泛素的 HIF-1alpha 降解来实现的，这涉及分子伴侣 HSP90，但不依赖于 HIF-1alpha 蛋白的脯氨酰羟化。我们还表明，bcl-2、HIF-1alpha 和 HSP90 蛋白形成三聚体复合物，可能有助于增强 bcl-2 过表达克隆在低氧条件下 HIF-1alpha 蛋白的稳定性。最后，通过使用遗传和药理学方法，我们证明 HSP90 参与 bcl-2 依赖性 HIF-1alpha 蛋白在缺氧下的稳定，并特别证明 HSP90beta 同工型是这种现象的主要参与者。
结论/意义：我们确定 HIF-1alpha 蛋白的稳定化是 bcl-2 在缺氧肿瘤细胞中诱导 HIF-1 激活的一种机制，涉及分子伴侣 HSP90 的β同工型。
