Analysis of ARD1 function in hypoxia response using retroviral RNA interference
Analysis of ARD1 function in hypoxia response using retroviral RNA interference
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DOI：
10.1074/jbc.m412055200
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发表时间：
2005-05-06
影响因子：
4.8
通讯作者：
Li, BY
中科院分区：
文献类型：
--
作者：
Fisher, TS;Des Etages, S;Li, BY
细胞缺氧反应在缺氧诱导因子（HIF）活性水平上受到调节。最近发现的许多氧传感器都是 HIF 修饰酶，它们通过改变 HIF 修饰来响应低氧，从而导致其激活。除了 HIF 脯氨酸羟化酶和天冬酰胺羟化酶之外，ARD1 最近被描述为调节其稳定性的 HIF-1 α 乙酰化酶。我们发现 ARD1 在许多细胞系中因缺氧和缺氧模拟化合物而下调。在调查了这些细胞系的促红细胞生成素产生和逆转录病毒转染效率后，我们选择使用 HepG2 细胞来研究 ARD1 的功能。由逆转录病毒载体传递的 ARD1 短发夹 RNA 导致 ARD1 信息减少 80% 以上。我们观察到促红细胞生成素和血管内皮生长因子蛋白的产生减少，而 HIF-1 α 蛋白水平没有变化。在常氧和缺氧条件下用表达 ARD1 短发夹 RNA 的病毒转导的 HepG2 细胞或用过表达重组 ARD1 的病毒转导的 HepG2 细胞的基因芯片分析证实，抑制 ARD1 不会引起 HIF 和下游靶基因的激活。然而，该分析表明，ARD1 参与细胞增殖和调节缺氧反应期间调节的一系列细胞代谢途径。使用细胞分裂的荧光标记分析证实了 ARD1 在细胞增殖中的作用。从这些研究中，我们得出结论，ARD1 不需要抑制 HIF，但需要维持哺乳动物细胞的细胞增殖。
Cellular hypoxia response is regulated at the level of hypoxia-inducible factor (HIF) activity. A number of recently identified oxygen sensors are HIF-modifying enzymes that respond to low oxygen by altering HIF modification and thus lead to its activation. In addition to the HIF proline hydroxylases and asparagine hydroxylases, ARD1 is recently described as a HIF-1 alpha acetylase that regulates its stability. We found that ARD1 is down-regulated in a number of cell lines in response to hypoxia and hypoxia mimic compounds. After surveying these lines for erythropoietin production and retroviral transfection efficiency, we chose to use HepG2 cells to study the function of ARD1. ARD1 short hairpin RNA delivered by a retroviral vector caused >80% reduction in ARD1 message. We observed decreases in erythropoietin and vascular endothelial growth factor protein production, whereas there was no change in the HIF-1 alpha protein level. A gene chip analysis of HepG2 cells transduced with virus expressing ARD1 short hairpin RNA under normoxia and hypoxia conditions or with virus overexpressing recombinant ARD1 confirmed that inhibition of ARD1 does not cause activation of HIF and downstream target genes. However, this analysis revealed that ARD1 is involved in cell proliferation and in regulating a series of cellular metabolic pathways that are regulated during hypoxia response. The role of ARD1 in cell proliferation is confirmed using fluorescence labeling analysis of cell division. From these studies we conclude that ARD1 is not required to suppress HIF but is required to maintain cell proliferation in mammalian cells.