Depletion of intracellular ascorbate by the carcinogenic metals nickel and cobalt results in the induction of hypoxic stress
Depletion of intracellular ascorbate by the carcinogenic metals nickel and cobalt results in the induction of hypoxic stress
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DOI：
10.1074/jbc.m403057200
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发表时间：
2004-09-24
影响因子：
4.8
通讯作者：
Kasprzak, KS
中科院分区：
文献类型：
--
作者：
Salnikow, K;Donald, SP;Kasprzak, KS
将细胞暴露于镍（II）和钴（II）的致癌化合物会导致HIF-1转录因子的激活，并上调一组缺氧诱导基因。然而，这些金属激活HIF-1的机制尚不清楚。最近的研究表明，HIF-1的HIF α亚基中脯氨酸的羟基化是HIF-1与von Hippel-Lindau肿瘤抑制蛋白结合以及随后的蛋白酶体破坏所必需的。在这里，我们表明，负责任的脯氨酰羟化酶的镍（II）和钴（II）的目标，因为降解的报告蛋白含有氧依赖性降解结构域（Pro-402/564）的HIF α被取消在冯希佩尔-林道依赖的方式在细胞暴露于镍（II）或钴（II）。脯氨酰羟化酶的酶活性取决于铁作为活化金属，2-酮戊二酸作为共底物，和抗坏血酸作为辅因子。羟化酶活性可因这些因子中任一种的消耗而受损。我们发现，暴露于镍（II）或钴（II）的细胞不影响细胞内铁的水平。相反，镍（II）或钴（II）暴露大大耗尽细胞内抗坏血酸。共同暴露的细胞金属和抗坏血酸导致细胞内抗坏血酸的增加和逆转金属诱导的稳定HIF-1 α和HIF-1依赖的基因转录。由于抗坏血酸是必不可少的保持铁在脯氨酰羟化酶的活性铁（II）的状态，我们认为，所观察到的抗坏血酸镍（II）或钴（II）的耗竭有利于铁的氧化，从而灭活酶。
Exposure of cells to carcinogenic compounds of nickel( II) and cobalt( II) causes activation of the HIF-1 transcription factor and up-regulates a battery of hypoxia-inducible genes. However, the mechanism of HIF-1 activation by these metals is not known. It was shown recently that hydroxylation of prolines in the HIFalpha subunit of HIF-1 is required for its binding with the von Hippel-Lindau tumor suppressor protein and the subsequent proteasomal destruction. Here we show that responsible prolyl hydroxylases are targets for both nickel( II) and cobalt( II) because degradation of a reporter protein containing the oxygen-dependent degradation domain (Pro-402/564) of HIFalpha was abolished in a von Hippel-Lindau-dependent manner in cells exposed to nickel( II) or cobalt( II). The enzymatic activity of prolyl hydroxylases depends on iron as the activating metal, 2-oxoglutarate as a co-substrate, and ascorbic acid as a cofactor. Hydroxylase activity can be impaired by the depletion of any of these factors. We found that exposure of cells to nickel( II) or cobalt( II) did not affect the level of intracellular iron. Instead, nickel( II) or cobalt( II) exposure greatly depleted intracellular ascorbate. Co-exposure of cells to metals and ascorbate resulted in the increase of intracellular ascorbate and reversed both metal-induced stabilization of HIF-1alpha and HIF-1-dependent gene transcription. Because ascorbate is essential for maintaining iron in prolyl hydroxylases in the active iron( II) state, we suggest that the observed depletion of ascorbate by nickel( II) or cobalt( II) favors iron oxidation and thus inactivation of the enzyme.