From the Turku Centre for Biotechnology, University of Turku and Åbo Akademi University, Tykistökatu 6, 20520 Turku, Finland.
the Department of Medical Biochemistry, Faculty of Medicine, University of Turku, Kiinamyllynkatu 10, 20520 Turku, Finland.
J Biol Chem. 2019 Mar 8;294(10):3760-3771. doi: 10.1074/jbc.RA118.004902. Epub 2019 Jan 7.
Most clear cell renal cell carcinomas (ccRCCs) have inactivation of the von Hippel-Lindau tumor suppressor protein (pVHL), resulting in the accumulation of hypoxia-inducible factor α-subunits (HIF-α) and their downstream targets. HIF-2α expression is particularly high in ccRCC and is associated with increased ccRCC growth and aggressiveness. In the canonical HIF signaling pathway, HIF-prolyl hydroxylase 3 (PHD3) suppresses HIF-2α protein by post-translational hydroxylation under sufficient oxygen availability. Here, using immunoblotting and immunofluorescence staining, qRT-PCR, and siRNA-mediated gene silencing, we show that unlike in the canonical pathway, PHD3 silencing in ccRCC cells leads to down-regulation of HIF-2α protein and mRNA. Depletion of other PHD family members had no effect on HIF-2α expression, and PHD3 knockdown in non-RCC cells resulted in the expected increase in HIF-2α protein expression. Accordingly, PHD3 knockdown decreased HIF-2α target gene expression in ccRCC cells and expression was restored upon forced HIF-2α expression. The effect of PHD3 depletion was pinpointed to mRNA stability. In line with these results, a strong positive correlation of and mRNA expression in ccRCC tumors was detected. Our results suggest that in contrast to the known negative regulation of HIF-2α in most cell types, high PHD3 expression in ccRCC cells maintains elevated HIF-2α expression and that of its target genes, which may enhance kidney cancer aggressiveness.
大多数透明细胞肾细胞癌（ccRCC）存在抑癌基因 von Hippel-Lindau 蛋白（pVHL）失活，导致缺氧诱导因子α亚基（HIF-α）及其下游靶基因积累。HIF-2α在 ccRCC 中的表达特别高，与 ccRCC 的生长和侵袭性增加有关。在经典的 HIF 信号通路中，在氧气充足的情况下，HIF-脯氨酰羟化酶 3（PHD3）通过翻译后羟化作用抑制 HIF-2α 蛋白。在这里，我们通过免疫印迹、免疫荧光染色、qRT-PCR 和 siRNA 介导的基因沉默实验，表明与经典通路不同，在 ccRCC 细胞中沉默 PHD3 会导致 HIF-2α 蛋白和 mRNA 的下调。其他 PHD 家族成员的耗竭对 HIF-2α 的表达没有影响，而非 RCC 细胞中 PHD3 的敲低导致 HIF-2α 蛋白表达的预期增加。因此，PHD3 敲低降低了 ccRCC 细胞中 HIF-2α 靶基因的表达，而强制表达 HIF-2α 则恢复了其表达。PHD3 耗竭的影响主要在于 mRNA 稳定性。与这些结果一致，在 ccRCC 肿瘤中检测到 和 mRNA 表达的强烈正相关。我们的研究结果表明，与大多数细胞类型中已知的 HIF-2α 负调控相反，ccRCC 细胞中高 PHD3 表达维持了 HIF-2α 及其靶基因的高表达，这可能增强了肾癌的侵袭性。
