Molecular Mechanisms of the Hypoxic Response

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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.