Molecular Mechanisms of the Hypoxic Response

描述（由申请人提供）：哺乳动物对缺氧的转录反应的主要调节因子是转录因子缺氧诱导因子（HIF），其亚基以氧敏感的方式在蛋白质周转水平上进行调节。在常氧条件下，脯氨酸羟化酶结构域蛋白（PHD）位点-特异性地羟化HIF-(，从而靶向HIF-（通过泛素-蛋白酶体途径进行降解）。在缺氧条件下，这种固有的氧依赖性的翻译后修饰被抑制，从而使HIF-（）稳定。然后，HIF上调一系列参与细胞、局部和全身缺氧反应的基因。典型的HIF靶基因是编码促红细胞生成素（EPO）的基因，EPO是一种糖蛋白激素，根据氧张力的变化调节红细胞质量。因此，了解HIF调节将有助于理解和治疗红细胞质量调节障碍，如贫血，贫血是许多临床情况下的重要并发症，包括终末期肾病和化疗。更普遍的是，缺氧是许多人类疾病的核心特征，包括冠状动脉、脑血管和肿瘤疾病，因此关于HIF调节的知识也将影响我们对这些疾病的理解。有三种HIF-（异构体）(HIF-1（, HIF-2（和HIF-3））和三种脯氨酸羟化酶结构域蛋白（PHD1, PHD2, PHD3）可以羟基化它们，这就提出了哪些异构体对人体生理和病理生理重要的关键问题。在与Terence Lappin教授团队的合作中，我们发现了一个由于HIF2A基因中G537W错义突变而导致遗传性红细胞增多（红细胞数量增加）的家族，以及另一个由于PHD2基因中P317R错义突变而导致红细胞增多的家族。这些研究首次确定了任何HIF或任何PHD亚型的遗传突变，并建立了两种新的红细胞增多症遗传原因。我们随后在这两个基因中发现了额外的突变。我们的具体目标是：(1)使用体外实验研究新的红细胞增生相关的HIF-2（和PHD2）突变，以支持我们的假设，即这些蛋白质对EPO有关键的控制作用；(2)使用Hif2a敲入小鼠来模拟人类G537W错义突变，并检查Hif2-(的失调在体内的功能后果；(3)使用PHD2敲入小鼠来研究P317R突变。以及一个全局条件敲除Phd2小鼠来研究Phd2调节红细胞质量的机制。总的来说，我们预计这些研究将大大增加我们对EPO调控的理解，更广泛地说，我们对哺乳动物氧感应途径的理解。

DESCRIPTION (provided by applicant): The master regulator of the mammalian transcriptional response to hypoxia is the transcription factor Hypoxia Inducible Factor (HIF), the subunit of which is regulated at the level of protein turnover in an oxygen-sensitive manner. Under normoxic conditions, Prolyl Hydroxylase Domain protein (PHD) site- specifically hydroxylates HIF-(, which in turn targets HIF-( for degradation by the ubiquitin-proteasome pathway. Under hypoxic conditions, this posttranslational modification, which is inherently oxygen dependent, is inhibited, thereby allowing stabilization of HIF-(. HIF then upregulates a battery of genes involved in cellular, local, and systemic responses to hypoxia. The prototypical HIF target gene is that encoding for Erythropoietin (EPO), a glycoprotein hormone that regulates red blood cell mass in response to changes in oxygen tension. Thus, understanding HIF regulation will have implications for understanding and treating disorders of red blood cell mass regulation, such as anemia, which in turn is a significant complication seen in many clinical settings, including end stage renal disease and chemotherapy. More generally, hypoxia is a central feature of many human diseases, including coronary artery, cerebrovascular, and neoplastic disease, and therefore knowledge regarding HIF regulation will also impact our understanding of these diseases. There are three HIF-( isoforms (HIF-1(, HIF-2(, and HIF-3() and three Prolyl Hydroxylase Domain proteins (PHD1, PHD2, PHD3) that can hydroxylate them, raising the critical question of which isoforms are important for human physiology and pathophysiology. In collaboration with Professor Terence Lappin's group, we have identified a family with hereditary erythrocytosis (increased red blood cell mass) due to a G537W missense mutation in the HIF2A gene, and another family with erythrocytosis due to a P317R missense mutation in the PHD2 gene. These studies provide the first identification of hereditary mutations in any HIF or in any PHD isoform, and establish two new genetic causes of erythrocytosis. We have subsequently identified additional mutations in both genes. Our Specific Aims are to (1) study new erythrocytosis-associated HIF-2( and PHD2 mutations using in vitro assays in order to bolster our hypothesis that these proteins critically control EPO, (2) employ a Hif2a knockin mouse to model the human G537W missense mutation and examine functional consequences in vivo of dysregulation of Hif2-(, and (3) employ both a Phd2 knockin mouse for the P317R mutation, and a global conditional Phd2 knockout mouse to examine the mechanism by which Phd2 regulates red cell mass. Collectively, we anticipate that these studies will substantially increase our understanding of EPO regulation and, more broadly, our understanding of the mammalian oxygen sensing pathway. PUBLIC HEALTH RELEVANCE: This project seeks to identify and characterize the molecular pathway that leads to the control of red blood cell mass, and more generally, the response to low oxygen tension. The proposed studies focus on two proteins named Hypoxia Inducible Factor-2 and Prolyl Hydroxylase Domain protein 2 that have been implicated in controlling the hormone, Erythropoietin, that determines red cell mass. The proposed experiments will have implications for treating diseases such as anemia, in which red blood cell mass is abnormally low.