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.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Clinical Investigator Award (CIA) (K08)
Project #
5K08HL003125-05
Application #
2685202
Study Section
Research Training Review Committee (RTR)
Project Start
1994-04-01
Project End
1999-03-31
Budget Start
1998-04-01
Budget End
1999-03-31
Support Year
5
Fiscal Year
1998
Total Cost
Indirect Cost
Name
Boston University
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
604483045
City
Boston
State
MA
Country
United States
Zip Code
02118
Graven, K K; Yu, Q; Pan, D et al. (1999) Identification of an oxygen responsive enhancer element in the glyceraldehyde-3-phosphate dehydrogenase gene. Biochim Biophys Acta 1447:208-18
Graven, K K; McDonald, R J; Farber, H W (1998) Hypoxic regulation of endothelial glyceraldehyde-3-phosphate dehydrogenase. Am J Physiol 274:C347-55
Graven, K K; Farber, H W (1998) Endothelial cell hypoxic stress proteins. J Lab Clin Med 132:456-63
Graven, K K; Farber, H W (1997) Endothelial hypoxic stress proteins. Kidney Int 51:426-37
Tucci, M; McDonald, R J; Aaronson, R et al. (1996) Specificity and uniqueness of endothelial cell stress responses. Am J Physiol 271:L341-8
Aaronson, R M; Graven, K K; Tucci, M et al. (1995) Non-neuronal enolase is an endothelial hypoxic stress protein. J Biol Chem 270:27752-7
Graven, K K; Farber, H W (1995) Hypoxia-associated proteins. New Horiz 3:208-18