Hypoxia-inducible factor hydroxylases are oxygen sensors in the brain /

by Dalgard, Clifton Lee.

Title of Dissertation: “Hypoxia-Inducible Factor Hydroxylases Are Oxygen Sensors in the Brain” Name: Clifton Lee Dalgard, Ph.D. 2005 Dissertation directed by: Ajay Verma, M.D., Ph.D. Associate Professor Uniformed Services University of the Health Sciences In mammalian cells, molecular oxygen is a requirement for normal growth, metabolism, and survival. Tissues in which oxygen demand surpasses oxygen supply become hypoxic and cannot maintain normal function. Thus, the ability to sense oxygen levels is necessary for an organism to survive and thrive in changing environmental and physiological conditions. HIF-1 is a transcription factor complex that is essential and central to several cellular and systemic adaptations to hypoxia. For example, vascular endothelial growth factor and erythropoietin are HIF-1 target genes that are important in angiogenesis and erythropoiesis, respectively. HIF-1 consists of two subunits, alpha and beta, and control of HIF-1 function is accomplished through the hydroxylation of proline residues and an asparagine residue on the ?-subunit of HIF-1. Under normoxic conditions, hydroxylated HIF-1? is constantly and rapidly degraded, thus HIF-1 is inactivated. Additionally, undegraded HIF-1? is hydroxylated at an asparagine residue in the c-terminal region, which prevents it from binding to the co-transcriptional activator p300. The post-translational modifications of HIF-1? are performed by four oxygendependent enzymes, the three HIF-1? prolyl hydroxylases (HPH-1, HPH-2, and HPH-3) and the asparaginyl hydroxylase FIH-1 (Factor Inhibiting HIF). Since these enzymes iv modify HIF-1? in an oxygen-dependent manner, they have been suggested to function as oxygen sensors in vivo. No studies of these oxygen sensors have been conducted in the mammalian brain or brain derived cells. This dissertation describes biochemistry, cellular and molecular biology, and whole animal physiology of these oxygen sensors. Using human glioma cell lines, we demonstrate that HPHs are themselves induced by hypoxia, thus suggesting the presence of a negative feedback system to modulate hypoxic gene expression. For the three HPHs, we found differential distribution of expression between different brain cell types and different brain regions. The same HPH homologues that are regulated in permanent cell lines are regulated in brain cells in culture and in vivo. We found that different brain regions induce HPH expression to different extents and hypoxic induction of the oxygen sensors was more prominent in young animals than in old and was manifested by increases in protein expression and enzymatic activity. We also found in addition to oxygen availability, the HIF hydroxylases are also regulated by certain glycolytic metabolites. We specifically identified pyruvate and oxaloacetate as the regulatory metabolites and demonstrated that their mode of action involves a reversible inactivation of HIF hydroxylation. Pyruvate and oxaloacetate induced HIF-1 in cells and also resulted in upregulation of HPH-1 and HPH-2. These results suggest HIF prolyl hydroxylases are sensor of oxygen tensions as well as glycolytic metabolite accumulation. Moreover, both of these stimuli increase expression of these hydroxylases which may serve as a negative feedback system for these sensing mechanisms. Given that the brain is highly sensitive to low oxygen tensions, these studies may provide valuable insight to develop novel tools and therapies for oxygen-associated brain diseases like stroke, heart failure, and brain cancer. v