Pulmonary vascular endothelia cells regulate vascular tone, hemostasis, inflammation, angiogenesis and permeability within the lungs. In order to maintain lung homeostasis theses cells must adapt to acute and chronic changes in environmental metabolites, especially decreases in blood and tissue oxygen content. Our laboratory has shown that pulmonary artery endothelial cells in culture respond to hypoxia MWs, 34, 36, 39, 47 an 56kD. I have recently isolated and identified the 36kD protein as the glycolytic enzyme glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Preliminary studies demonstrating an increase in GAPDH transcripts by northern blot analysis and nuclear run-off assays suggest that GAPDH upregulation is transcriptionally regulated by hypoxia in endothelial cells. None of the other cell types we have studied unregulated GAPDH in response to hypoxia. In this proposal I plan to study in further detail the transcriptional and post-transcriptional regulation of endothelial GAPDH gene expression by hypoxia in pulmonary artery endothelial cells, and characterize the mechanisms that make this response potentially unique to endothelial cells. I will examine the time course and oxygen-concentration response of mRNA induction using northern blot analysis nuclear run-off assays. RNA stability studies, immunoprecipitation assays and western blots studies will be used to define potential post-transcriptional mechanism(s) of GAPDH upregulation. Since we and others have shown that , in comparison to other cell types, pulmonary artery endothelial cells are relatively hypoxia in endothelial cells to that of other, more hypoxia-sensitive cell types. To identify the DNA sequences which mediate induction of GAPDH by hypoxia in endothelial cells GAPDH-promoter-CAT plasmids will be constructed and transfected into pulmonary artery endothelial cells. The potential enhancer regions of the GAPDH gene will be assessed using deletion analysis. DNase footprinting and gel-mobility shift assays will be used to characterize the trans-acting factors involved in endothelial cell GAPDH expression. The same techniques will be use to examine these factors in other, more hypoxia-sensitive cells types. Finally, to explore the functional significance of GAPDH overexpression in hypoxia tolerance, I will analyze cell survival in hypoxia-sensitive cell types stably transfected with a full length GAPDH plasmid driven by a strong constitutive promoter. The studies outlined in this proposal will determine whether the mechanisms used to upregulate GAPDH are somehow distinct to endothelial cells. These studies will provide new insight into the endothelial cell's ability to tolerate hypoxia and may have broader implications for the regulation of other hypoxia-responsive genes.
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