Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.
Chem Res Toxicol. 2012 Oct 15;25(10):2194-202. doi: 10.1021/tx300274a. Epub 2012 Sep 25.
Hypoxia-inducible factor-1α (HIF-1α) is a critical regulator of cellular responses to hypoxia. Under normoxic conditions, the cellular HIF-1α level is regulated by hydroxylation by prolyl hydroxylases (PHDs), ubiquitylation, and proteasomal degradation. During hypoxia, degradation decreases, and its intracellular level is increased. Exogenously administered nitric oxide (NO)-donor drugs stabilize HIF-1α; thus, NO is suggested to mimic hypoxia. However, the role of low levels of endogenously produced NO generated during hypoxia in HIF-1α stabilization has not been defined. Here, we demonstrate that NO and reactive oxygen species (ROS) produced endogenously by human colon carcinoma HCT116 cells are responsible for HIF-1α accumulation in hypoxia. The antioxidant N-acetyl-L-cysteine (NAC) and NO synthase inhibitor N(G)-monomethyl L-arginine (L-NMMA) effectively reduced HIF-1α stabilization and decreased HIF-1α hydroxylation. These effects suggested that endogenous NO and ROS impaired PHD activity, which was confirmed by reversal of L-NMMA- and NAC-mediated effects in the presence of dimethyloxaloylglycine, a PHD inhibitor. Thiol reduction with dithiothreitol decreased HIF-1α stabilization in hypoxic cells, while dinitrochlorobenzene, which stabilizes S-nitrosothiols, favored its accumulation. This suggested that ROS- and NO-mediated HIF-1α stabilization involved S-nitrosation, which was confirmed by demonstrating increased S-nitrosation of PHD2 during hypoxia. Our results support a regulatory mechanism of HIF-1α during hypoxia in which endogenously generated NO and ROS promote inhibition of PHD2 activity, probably by its S-nitrosation.
缺氧诱导因子-1α（HIF-1α）是细胞对缺氧反应的关键调节因子。在常氧条件下，细胞 HIF-1α 水平受脯氨酰羟化酶（PHD）羟化、泛素化和蛋白酶体降解调节。在缺氧时，降解减少，其细胞内水平增加。外源性给予一氧化氮（NO）供体药物可稳定 HIF-1α；因此，NO 被认为模拟缺氧。然而，在缺氧期间内源性产生的低水平的 NO 对 HIF-1α 稳定的作用尚未确定。在这里，我们证明了人结肠癌细胞 HCT116 内源性产生的 NO 和活性氧（ROS）负责 HIF-1α 在缺氧时的积累。抗氧化剂 N-乙酰-L-半胱氨酸（NAC）和一氧化氮合酶抑制剂 N(G)-单甲基-L-精氨酸（L-NMMA）有效地减少了 HIF-1α 的稳定，并降低了 HIF-1α 的羟化。这些效应表明，内源性 NO 和 ROS 损害了 PHD 活性，这在 PHD 抑制剂二甲基乙二酰基甘氨酸存在的情况下，逆转了 L-NMMA 和 NAC 介导的作用得到证实。二硫苏糖醇的巯基还原降低了缺氧细胞中 HIF-1α 的稳定，而二硝基氯苯，稳定 S-亚硝基硫醇，有利于其积累。这表明 ROS 和 NO 介导的 HIF-1α 稳定涉及 S-亚硝化，这在缺氧期间 PHD2 的 S-亚硝化增加得到证实。我们的结果支持了 HIF-1α 在缺氧时的调节机制，其中内源性产生的 NO 和 ROS 促进 PHD2 活性的抑制，可能通过其 S-亚硝化。
