Molecular Mechanisms of the Hypoxic Response

缺氧反应的分子机制

• 批准号: 8434756
• 负责人: FRANK S LEE
• 金额: $ 24.39万
• 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/8434756
• 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.

描述（申请人提供）：哺乳动物对缺氧转录反应的主要调节因子是转录因子缺氧诱导因子（HIF），其亚基以氧敏感方式在蛋白质周转水平上受到调节。在含氧量正常的条件下，脯氨酰羟化酶结构域蛋白 (PHD) 位点特异性羟基化 HIF-(，进而通过泛素蛋白酶体途径靶向 HIF-( 进行降解。在缺氧条件下，这种本质上依赖于氧的翻译后修饰受到抑制，从而使 HIF-( 稳定。然后，HIF 上调一系列 涉及细胞、局部和全身对缺氧反应的基因。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，可调节红细胞质量以响应氧张力的变化。因此，了解 HIF 调节将对了解和治疗红细胞质量调节疾病（例如贫血）产生影响，而贫血又是一种重要的并发症 在许多临床环境中，包括终末期肾病和化疗。更一般地说，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，因此有关 HIF 调节的知识也将影响我们对这些疾病的理解。 存在三种 HIF-( 同工型（HIF-1(、HIF-2( 和 HIF-3())）和三种脯氨酰羟化酶结构域 蛋白质（PHD1、PHD2、PHD3）可以将它们羟基化，这就提出了一个关键问题：哪些亚型对人类生理学和病理生理学很重要。我们与 Terence Lappin 教授的团队合作，确定了一个因 HIF2A 基因 G537W 错义突变而患有遗传性红细胞增多症（红细胞质量增加）的家族，以及另一个患有遗传性红细胞增多症的家族。 PHD2 基因 P317R 错义突变导致红细胞增多。这些研究首次鉴定了任何 HIF 或任何 PHD 同种型的遗传突变，并确定了红细胞增多症的两个新的遗传原因。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) 研究新的红细胞增多症相关 HIF-2( 和 PHD2 突变) 为了支持我们的假设，即这些蛋白质关键控制 EPO，（2）使用 Hif2a 敲入小鼠来模拟人类 G537W 错义突变，并检查 Hif2-（的体内失调的功能后果，以及（3）使用 Phd2 敲入小鼠进行 P317R 突变，并使用全局条件性 Phd2 敲除小鼠来检查 Phd2 的机制 调节红细胞质量。总的来说，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，我们对哺乳动物氧传感途径的理解。