Abstract

Hypoxia-inducible factor-1alpha (HIF-1alpha) is important for adaptation to hypoxia. Hypoxia is a common feature of cancer and inflammation, by which HIF-1alpha increases. However, prolonged hypoxia decreases HIF-1alpha, and the underlying mechanisms currently remain unclear. Cellular reactive oxygen species (ROS) increases in cancer and inflammation. In the present study, we demonstrated that prolonged hypoxia increased ROS, which induced prolyl hydroxylase domain-containing protein 2 (PHD2) and factor inhibiting HIF-1 (FIH-1), major regulators of HIF-1alpha. Cellular stress response (CSR) increased HIF-1alpha transcriptional activity by scavenging endogenous ROS. PHD2 and FIH-1 were induced by external hydrogen peroxide (H₂O₂) but were suppressed by ROS-scavenging catalase. We investigated the mechanisms by which PHD2 and FIH-1 are regulated by ROS. The knockdown of HIF-1alpha decreased PHD2 and FIH-1 mRNA levels, suggesting their regulation by HIF-1alpha. We then focused on redox factor-1 (Ref-1), which is a regulator of HIF-1alpha transcriptional activity. The knockdown of Ref-1 decreased PHD2 and FIH-1. Ref-1 was regulated by ROS. Prolonged hypoxia and the addition of H₂O₂ induced the expression of Ref-1. Furthermore, the knockdown of p65, a component of kappa-light-chain enhancer of activated B cells (NF-κB), efficiently inhibited the induction of Ref-1 by ROS. Collectively, the present results showed that prolonged hypoxia or increased ROS levels induced Ref-1, leading to the activation of HIF-1alpha transcriptional activity, while the activation of HIF-1alpha via Ref-1 induced PHD2 and FIH-1, causing the feedback of HIF-1alpha. To the best of our knowledge, this is the first study to demonstrate the regulation of HIF-1alpha via Ref-1 by ROS.

摘要

缺氧诱导因子-1α（HIF-1alpha）对于适应缺氧很重要。缺氧是癌症和炎症的共同特征，HIF-1alpha升高。但是，长时间缺氧会降低HIF-1alpha，目前尚不清楚其潜在机制。细胞活性氧（ROS）在癌症和炎症中增加。在本研究中，我们证明了长时间缺氧会增加ROS，从而诱导含脯氨酰羟化酶结构域的蛋白2（PHD2）和抑制HIF-1alpha的主要调节因子HIF-1（FIH-1）。细胞应激反应（CSR）通过清除内源性ROS来提高HIF-1alpha转录活性。PHD2和FIH-1由外部过氧化氢（H ₂ O ₂），但被ROS清除过氧化氢酶抑制。我们研究了ROS调控PHD2和FIH-1的机制。HIF-1alpha的降低降低了PHD2和FIH-1 mRNA的水平，表明它们受到HIF-1alpha的调节。然后，我们重点研究氧化还原因子1（Ref-1），它是HIF-1alpha转录活性的调节剂。Ref-1的敲低降低了PHD2和FIH-1。Ref-1受ROS调节。长时间缺氧和H ₂ O _2的添加诱导Ref-1的表达。此外，激活的B细胞（NF-κB）κ轻链增强子的组成部分p65的敲低有效抑制了ROS对Ref-1的诱导。总的来说，目前的结果表明，长时间缺氧或活性氧水平升高诱导Ref-1，从而导致HIF-1alpha转录活性的激活，而通过Ref-1激活HIF-1alpha则诱导PHD2和FIH-1，从而引起反馈HIF-1alpha。据我们所知，这是第一个证明ROS通过Ref-1调节HIF-1alpha的研究。