Hypoxia-inducible factor-1 (HIF-1) promotes its degradation by induction of HIF-α-prolyl-4-hydroxylases
Hypoxia-inducible factor-1 (HIF-1) promotes its degradation by induction of HIF-α-prolyl-4-hydroxylases
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DOI：
10.1042/bj20040620
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发表时间：
2004-08-01
影响因子：
4.1
通讯作者：
Metzen, E
中科院分区：
文献类型：
--
作者：
Marxsen, JH;Stengel, P;Metzen, E
参与氧依赖性基因表达的重要调节因子是转录因子HIF（缺氧诱导因子），其由氧敏感性α亚基（HIF-1 α或HIF-2 α）和组成性表达的β亚基组成。在常氧条件下，HIF-1 α通过氧敏感性双加氧酶家族对Pro-564和Pro-402的翻译后羟基化而不稳定。这三种HIF修饰的人类酶被称为含脯氨酰羟化酶结构域的蛋白质（PHD 1、PHD 2和PHD 3）。脯氨酰羟基化导致HIF-1 α的pVHL（von-Hippel-Lindau蛋白）依赖性泛素化和快速蛋白酶体降解。在本研究中，我们报告，人PHD 2和PHD 3诱导的原代和转化细胞系缺氧。在人骨肉瘤细胞系U2 OS中，通过RNA干扰选择性抑制HIF-1 α表达导致PHD 2和PHD 3的缺氧诱导完全丧失。在pVHL缺陷的RCC 4细胞中，缺氧对PHD 2的诱导作用丧失。这些结果表明，低氧诱导PHD 2和PHD 3是严重依赖于HIF-α。使用VHL捕获试验，我们表明，HIF-α脯氨酰-4羟化酶的细胞质和核蛋白提取物的能力，提高了长期暴露于缺氧。复氧后HIF-1 α的降解加速，这证明了本结果的功能相关性。我们提出了一种直接的负调控机制，它限制了缺氧时HIF-1 α的积累，并导致长期缺氧后复氧时加速降解。
An important regulator involved in oxygen-dependent gene expression is the transcription factor HIF (hypoxia-inducible factor), which is composed of an oxygen-sensitive alpha-subunit (HIF-1alpha or HIF-2alpha) and a constitutively expressed beta-subunit. In normoxia, HIF-1alpha is destabilized by post-translational hydroxylation of Pro-564 and Pro-402 by a family of oxygen-sensitive dioxygenases. The three HIF-modifying human enzymes have been termed prolyl hydroxylase domain containing proteins (PHD1, PHD2 and PHD3). Prolyl hydroxylation leads to pVHL (von-Hippel-Lindau protein)-dependent ubiquitination and rapid proteasomal degradation of HIF-1alpha. In the present study, we report that human PHD2 and PHD3 are induced by hypoxia in primary and transformed cell lines. In the human osteosarcoma cell line, U2OS, selective suppression of HIF-1alpha expression by RNA interference resulted in a complete loss of hypoxic induction of PHD2 and PHD3. Induction of PHD2 by hypoxia was lost in pVHL-deficient RCC4 cells. These results suggest that hypoxic induction of PHD2 and PHD3 is critically dependent on HIF-alpha. Using a VHL capture assay, we demonstrate that HIF-alpha prolyl-4hydroxylase capacity of cytoplasmic and nuclear protein extracts was enhanced by prolonged exposure to hypoxia. Degradation of HIF-1alpha after reoxygenation was Accelerated, which demonstrates functional relevance of the present results. We propose a direct, negative regulatory mechanism, which limits accumulation of HIF-1alpha in hypoxia and leads to accelerated degradation on reoxygenation after long-term hypoxia.