Molecular Mechanisms of the Hypoxic Response

缺氧反应的分子机制

• 批准号: 7790056
• 负责人: FRANK S LEE
• 金额: $ 26.71万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-04-01 至 2015-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7790056
• 关键词: Adult; Anemia; Biological Models; Bone Marrow; Cardiovascular system; Chronic Kidney Failure; Clinical; Collaborations; Complex; Complication; Coronary artery; Coupled; Disease; End stage renal failure; Enhancers; Erythrocytes; Erythrocytoses; Erythropoietin; Family; Functional disorder; Gene Expression Regulation; Gene Targeting; Genes; Genetic; Genetic Transcription; Genetically Engineered Mouse; Germ-Line Mutation; Glycolysis; Glycoproteins; Hormones; Human; Hydroxylation; Hypoxia; Hypoxia Inducible Factor; In Vitro; Inherited; Kidney; Knockout Mice; Knowledge; Lead; Life; Liver; Mammalian Cell; Mediating; Methods; Missense Mutation; Modeling; Modification; Molecular; Mus; Mutation; Names; Oxygen; Oxygen measurement, partial pressure, arterial; Pathway interactions; Patients; Physiology; Post-Translational Protein Processing; Procollagen-Proline Dioxygenase; Production; Protein Isoforms; Proteins; Rare Diseases; Red Cell Mass result; Regulation; Role; Site; Tertiary Protein Structure; Therapeutic; Ubiquitin; angiogenesis; bHLH-PAS factor HLF; base; cerebrovascular; chemotherapy; egg; glucose uptake; human disease; hypoxia inducible factor 1; in vitro Assay; in vivo; interest; mouse model; multicatalytic endopeptidase complex; neoplastic; oxygen-regulated proteins; professor; protein degradation; public health relevance; research study; response; tissue oxygenation; transcription factor

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.

描述(申请人提供)：哺乳动物对低氧的转录反应的主要调节因子是转录因子低氧诱导因子(HIF)，其亚单位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，Pro羟基酶结构域蛋白(PHD)定点羟化HIF-(，进而靶向HIF-)，通过泛素-蛋白酶体途径降解。在低氧条件下，这种固有的氧依赖的翻译后修饰被抑制，从而使HIF-(。然后，HIF上调一系列基因，这些基因涉及细胞、局部和全身对低氧的反应。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，可以调节红细胞质量，以应对氧分压的变化。因此，了解HIF的调节对于理解和治疗红细胞量调节的紊乱具有重要意义，例如贫血，而贫血又是许多临床环境中常见的重大并发症，包括终末期肾脏疾病和化疗。更广泛地说，缺氧是许多人类疾病的中心特征，包括冠状动脉、脑血管和肿瘤疾病，因此关于HIF调控的知识也将影响我们对这些疾病的理解。有三种HIF-β亚型(HIF-1、HIF-2和HIF-3)和三种脯氨酸羟基酶结构域蛋白(PhD1、PHD2、PHD3)可以羟化它们，这就提出了哪些亚型对人体生理和病理生理学具有重要意义的关键问题。与Terence Lappin教授的团队合作，我们发现了一个由于HIF2A基因G537W错义突变导致的遗传性红细胞增多症(红细胞质量增加)家族，以及另一个由于PHD2基因P317R错义突变导致的红细胞增多症家族。这些研究首次鉴定了任何HIF或任何PHD亚型的遗传性突变，并建立了两个新的红细胞增多症的遗传原因。我们随后发现了这两个基因的额外突变。我们的具体目标是(1)利用体外实验研究新的红细胞增多症相关HIF-2(和PHD2)突变，以支持我们的假设，即这些蛋白质对EPO具有关键控制作用；(2)利用HIF2A敲除小鼠来模拟人类G537W错义突变，并检测HIF2-()失调在体内的功能后果；以及(3)利用PHD2敲除小鼠和全局条件性PHD2基因敲除小鼠来研究PHD2调节红细胞质量的机制。总而言之，我们预计这些研究将大大增加我们对EPO调节的理解，更广泛地说，我们对哺乳动物氧气感应途径的理解。 与公共健康相关：该项目试图识别和描述导致控制红细胞质量的分子途径，更广泛地说，是对低氧分压的反应。拟议的研究集中在两种蛋白质上，即缺氧诱导因子-2和Pro羟基酶结构域蛋白2，这两种蛋白质与控制决定红细胞质量的激素-红细胞生成素有关。拟议中的实验将对治疗贫血等疾病产生影响，在这些疾病中，红细胞质量异常低。