Regulation of Hypoxia Inducible Factor-1 by Nitric Oxide

University Of Virginia Charlottesville, Charlottesville VA

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Abstract

Hypoxia inducible factor-1 (HIF-1) is a transcription factor involved in the regulation of genes induced by low levels of oxygen. Regulation of HIF-1 is primarily determined by the stability of the HIF-1alpha subunit. In normoxia, HIF-1alpha mRNA is made but the protein is rapidly degraded by the ubiquitin-proteasome pathway. Nitric oxide (NO) has been shown to induce HIF-1 expression in normoxia. Our preliminary evidence suggests that S-nitrosylation reactions stabilize HIF-1alpha by inhibiting elements of the ubiquitin activating system. Because 1) the pulmonary vascular endothelium is not exposed to the profound levels of hypoxia often required to induce HIF-1 in vitro; 2) hemoglobin deoxygenation is associated with the transfer of nitrogen oxides to the vascular endothelium at pO2s more relevant to intravascular pathophysiology; and 3) downstream effects of HIF-1alpha stabilization on gene expression are implicated in the pathophysiology of pulmonary hypertension, we propose to clarify the mechanism by which NO activates HIF-1alpha in vitro and in vivo by testing the following hypotheses: Hypothesis number 1.: The expression of HIF-1 is regulated by nitric oxide (NO) in normoxia by S-nitrosylation of protein thiols. Hypothesis number 2. NO modifies HIF-1alpha stability in normoxia by modifying ubiquitin-dependent degradation through S- nitrosylation of HIF-1alpha and one or more enzymes of the ubiquitin activating pathway. Hypothesis number 3. S- nitrosoglutathione, and/or other related nitrogen oxides arising from hemoglobin deoxygenation induce HIF-1 expression in vivo. In testing this third hypothesis, we will control the effects of hypoxia on the gamma glutamyl transpeptidase knockout mouse. We have shown that this animal has attenuated responses to deoxyhemoglobin, that appear to involve decreased bioactivation of S-nitrosoglutathione. Taken together, these hypotheses represent a completely novel direction in the study of abnormal gene regulation in the pulmonary vascular endothelium. At the completion of this project, we believe we will have identified several new targets for the prevention and treatment of hypoxia- induced and primary pulmonary hypertension.