The ability to adapt to hypoxic stress is crucial to the survival of virtually all life forms. These adaptations are mediated largely by changes in gene expression. One family of hypoxia-inducible genes, including erythropoietin (Epo), vascular endothelial growth factor (VEGF), and the glucose transporters GLUT1 and GLUT3, has also been shown to be upregulated by the transition metals cobalt, nickel, and manganese. Little is known about the oxygen (02) sensing pathway(s) which mediate this regulation. The long term objective of this proposal is to clone and characterize the genes which play a significant role in the 02 sensing pathway(s) of mammalian cells. This has proven difficult to accomplish in diploid eukryotic cells. Given the paramount importance of the adaptive responses to hypoxia, it seemed reasonable to hypothesize that these responses are highly conserved. We have shown that when the yeast, Saccharomyces cerevisiae, is transformed with a reporter construct consisting of the promoter of the anaerobically-induced yeast gene, ANB1, attached to the lacZ gene and grown under hypoxic conditions or in the presence of the transition metals mentioned above, an inducible phenotype is observed which is similar to the hypoxia and transition metal induction of Epo, VBGF, GLUT1, and GLUT3. Moreover, disruption of a candidate 02 sensor in yeast recapitulates this hypoxic phenotype even under normoxic conditions. These findings provide a new strategy to identify the genes involved in the 02 sensing pathway.
The specific aims of this proposal are: 1) To identify the S. cerevisiae gene(s) responsible for the phenotype observed with the ANB1-lacZ reporter construct and confirm its hypoxia and cobalt inducibility, analogous to mammalian genes. 2) To use the gene(s) identified in Specific Aim #1 in assays designed to identify the genes involved in the 02 sensing pathway in & cerevisiae using standard yeast genetic techniques. 3) To further test the hypothesis that the recently cloned FHB gene encodes an 02 sensor in S. cerevisiae. 4) To identify the homologous genes involved in 02 sensing and signal transduction in mammalian cells using complementation strategies, low stringency hybridization of mammalian DNA libraries, and computer searches. Acquiring a better understanding of the molecular mechanisms governing the response to hypoxic stress is the crucial first step in the design and development of novel strategies to treat human conditions resulting from, or exacerbated by, a lack of 02. Likely applications would be aimed at improving the ability to treat anemias as well as vascular and neurological diseases.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
2R01DK045098-05A1
Application #
2016522
Study Section
Hematology Subcommittee 2 (HEM)
Project Start
1992-05-15
Project End
2001-03-31
Budget Start
1997-04-15
Budget End
1998-03-31
Support Year
5
Fiscal Year
1997
Total Cost
Indirect Cost
Name
Brigham and Women's Hospital
Department
Type
DUNS #
071723621
City
Boston
State
MA
Country
United States
Zip Code
02115
Jiang, Yide; Vasconcelles, Michael J; Wretzel, Sharon et al. (2002) Mga2p processing by hypoxia and unsaturated fatty acids in Saccharomyces cerevisiae: impact on LORE-dependent gene expression. Eukaryot Cell 1:481-90
Vasconcelles, M J; Jiang, Y; McDaid, K et al. (2001) Identification and characterization of a low oxygen response element involved in the hypoxic induction of a family of Saccharomyces cerevisiae genes. Implications for the conservation of oxygen sensing in eukaryotes. J Biol Chem 276:14374-84
Jiang, Y; Vasconcelles, M J; Wretzel, S et al. (2001) MGA2 is involved in the low-oxygen response element-dependent hypoxic induction of genes in Saccharomyces cerevisiae. Mol Cell Biol 21:6161-9
Huang, L E; Willmore, W G; Gu, J et al. (1999) Inhibition of hypoxia-inducible factor 1 activation by carbon monoxide and nitric oxide. Implications for oxygen sensing and signaling. J Biol Chem 274:9038-44
Levy, A P; Levy, N S; Iliopoulos, O et al. (1997) Regulation of vascular endothelial growth factor by hypoxia and its modulation by the von Hippel-Lindau tumor suppressor gene. Kidney Int 51:575-8
Levy, N S; Goldberg, M A; Levy, A P (1997) Sequencing of the human vascular endothelial growth factor (VEGF) 3' untranslated region (UTR): conservation of five hypoxia-inducible RNA-protein binding sites. Biochim Biophys Acta 1352:167-73
Iliopoulos, O; Levy, A P; Jiang, C et al. (1996) Negative regulation of hypoxia-inducible genes by the von Hippel-Lindau protein. Proc Natl Acad Sci U S A 93:10595-9
Levy, A P; Levy, N S; Goldberg, M A (1996) Hypoxia-inducible protein binding to vascular endothelial growth factor mRNA and its modulation by the von Hippel-Lindau protein. J Biol Chem 271:25492-7
Levy, A P; Levy, N S; Wegner, S et al. (1995) Transcriptional regulation of the rat vascular endothelial growth factor gene by hypoxia. J Biol Chem 270:13333-40
Levy, A P; Levy, N S; Loscalzo, J et al. (1995) Regulation of vascular endothelial growth factor in cardiac myocytes. Circ Res 76:758-66

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