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 In unpublished Preliminary Studies, we demonstrate novel regulation of epo enhancer function by Early Growth Response (Egr) members, stress-responsive transcription factors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.
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.