Dynamic Regulation of Erythropoietin Gene Expression in Mammals

描述(由申请人提供)： 项目摘要/摘要促红细胞生成素(EPO)分泌受损是大多数慢性贫血患者贫血的根本原因。在过去的一个世纪里，研究人员发现，对贫血或全身缺氧做出反应的红细胞聚集的有效诱因是循环促红细胞生成素，这是一种由肾脏产生的促红细胞生成细胞因子。大约三分之一的慢性贫血患者肾脏正常，但EPO水平过低。为什么EPO在这些患者中的表达减弱？这个问题的答案需要首先了解EPO基因在正常个体中的表达是如何调节的。在过去的十年中，对EPO调节的研究主要集中在低氧信号转导方面。利用以氧依赖的方式产生EPO的肝癌细胞系，研究人员定义了EPO基因的缺氧反应增强子区。这进而导致在EPO增强子区域鉴定出一个顺式作用的DNA增强子元件，缺氧反应元件(HRE)，然后纯化了HRE结合的氧敏感转录因子缺氧诱导因子1α(HIF-11)。研究人员最初假设HIF-11调节内源性EPO基因的表达。然而，小鼠模型研究表明，第二个和相关的HIF成员HIF-21对体内EPO基因的表达至关重要。我们的长期目标是确定调控哺乳动物EPO表达的分子机制。EPO的合成通常会随着组织的缺氧而迅速增加，然后在几个小时内恢复到基线水平。虽然目前已认识到HIF-21在EPO调节中的重要作用，但导致体内短暂EPO基因表达或贫血患者EPO基因表达异常抑制的因素仍然知之甚少。在未发表的初步研究中，我们展示了早期生长反应(Egr)成员对EPO增强子功能的新调节，Egr是应激反应转录因子，其活性也因缺氧而改变。我们进一步证明，已知的抑制Egr信号的因子也以HIF-21依赖的方式抑制EPO增强子的活性。我们假设，低氧最初触发Egr和HIF-2的激活，导致EPO基因表达的协同激活。随着持续的低氧暴露，我们认为诱导了Egr抑制因子，从而减弱了EPO基因的表达。在这个方案中，我们将定义Egr/HIF-2信号调节EPO增强子活性的分子和生化机制，并将使用细胞培养和小鼠基因敲除模型来确定Egr/HIF-2信号在EPO调控中的生物学作用。破译Egr/HIF-2信号如何调节EPO增强子活性将提供对正常EPO调节的机械性见解，并将识别调控慢性贫血患者内源性EPO基因表达的新的分子靶点。 公共卫生相关性： 项目简介对退伍军人保健的潜在影响是确定可以被新的药物疗法靶向的特定分子途径，从而在我们的大部分患有慢性贫血的退伍军人患者中恢复正常的促红细胞生成素的产生。

DESCRIPTION (provided by applicant): PROJECT SUMMARY/ABSTRACT Impaired erythropoietin (epo) production is the root cause of anemia in a majority of chronic anemia patients. Over the past century, investigators discovered that the potent inducer of red blood cell mass in response to anemia or systemic hypoxia is circulating epo, a pro-erythrogenic cytokine produced by the kidney. Approximately one-third of chronic anemia patients have normal kidneys, but inappropriately low epo levels. Why is epo expression blunted in these patients? The answer to this question requires first understanding how epo gene expression is regulated in normal individuals. Studies of epo regulation over the past decade have focused on hypoxia signaling. Using hepatoma cell lines producing epo in an oxygen-dependent manner, investigators defined the hypoxia- responsive enhancer region of the epo gene. This in turn led to identification of a cis-acting DNA enhancer element, the Hypoxia Responsive Element (HRE), in the epo enhancer region followed by purification of an HRE-binding oxygen-sensitive transcription factor, Hypoxia Inducible Factor 1 alpha (HIF-11). Investigators initially assumed that HIF-11 regulates endogenous epo gene expression. However, mouse model studies reveal that the second and related HIF member, HIF-21, is critical for in vivo epo gene expression. Our long-term goal is to define the molecular mechanisms regulating mammalian epo expression. Epo synthesis normally increases rapidly with tissue hypoxia, and then returns to baseline within hours. While an essential role of HIF-21 in epo regulation is now recognized, the factors responsible for temporal epo gene expression in vivo, or for abnormal repression of epo gene expression in anemia patients, remain poorly understood. In unpublished Preliminary Studies, we demonstrate novel regulation of epo enhancer function by Early Growth Response (Egr) members, stress-responsive transcription factors whose activity is also altered by hypoxia. We further demonstrate that factors known to repress Egr signaling also repress epo enhancer activity in a HIF-21 dependent manner. We hypothesize that hypoxia initially triggers Egr and HIF-2 activation, leading to synergistic activation of epo gene expression. With continued hypoxia exposure, we propose Egr repressive factors are induced that attenuate epo gene expression. In this proposal, we will define molecular and biochemical mechanisms whereby Egr/HIF-2 signaling regulates epo enhancer activity, and we will define the biological role of Egr/HIF-2 signaling in epo regulation using cell culture and mouse knockout models. Deciphering how Egr/HIF-2 signaling regulates epo enhancer activity will provide mechanistic insights into normal epo regulation and will identify novel molecular targets for modulation of endogenous epo gene expression in chronic anemia patients. PUBLIC HEALTH RELEVANCE: PROJECT NARRATIVE The Potential Impact on Veterans Health Care is identifying specific molecular pathways that can be targeted by novel drug therapies, thereby restoring normal erythropoietin production in a large segment of our veteran patients with chronic anemia.