The major objective of this proposal is to study the regulatory mechanisms that control the production of erythropoietin in response to tissue hypoxia. The contribution of transcriptional and post-transcriptional regulation in the expression of the erythropoietin gene in response to hypoxia will be evaluated by """"""""run-on"""""""" analysis and by measurement of erythropoietin mRNA half-lives. For this latter purpose cells will be transfected with a plasmid construct which expresses the erythropoietin messenger at very high levels and whose transcriptional activity is independent of hypoxia. The location and limits of cis-acting regulatory regions of the erythropoietin gene will be determined by transient transfection assays in normal and hypoxic erythropoietin producing cells. For these experiments we will use a mini-gene construct which allows the differentiation between the endogenous and the transfected gene. Once the DNA regions containing tissue specific and/or hypoxic specific regulatory elements are determined, the trans-acting factors that interact with these regions will be studied by using DNA-protein interaction techniques and in vitro transcription systems. Attempts will be made to further characterize and purify these trans-acting factors with the use of DNA affinity chromatography. Furthermore, once purification is accomplished, attempts will be made to clone the corresponding genes utilizing expression libraries from erythropoietin producing cells. The elucidation of the mechanisms involved in the regulation of erythropoietin production will increase our understanding of the pathophysiology of anemias and polycythemias.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK034642-07
Application #
2139354
Study Section
Hematology Subcommittee 2 (HEM)
Project Start
1986-08-01
Project End
1996-11-30
Budget Start
1993-12-01
Budget End
1994-11-30
Support Year
7
Fiscal Year
1994
Total Cost
Indirect Cost
Name
Thomas Jefferson University
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
061197161
City
Philadelphia
State
PA
Country
United States
Zip Code
19107
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Srinivas, V; Zhu, X; Salceda, S et al. (1998) Hypoxia-inducible factor 1alpha (HIF-1alpha) is a non-heme iron protein. Implications for oxygen sensing. J Biol Chem 273:18019-22
Salceda, S; Beck, I; Srinivas, V et al. (1997) Complex role of protein phosphorylation in gene activation by hypoxia. Kidney Int 51:556-9
Salceda, S; Caro, J (1997) Hypoxia-inducible factor 1alpha (HIF-1alpha) protein is rapidly degraded by the ubiquitin-proteasome system under normoxic conditions. Its stabilization by hypoxia depends on redox-induced changes. J Biol Chem 272:22642-7
Salceda, S; Beck, I; Caro, J (1996) Absolute requirement of aryl hydrocarbon receptor nuclear translocator protein for gene activation by hypoxia. Arch Biochem Biophys 334:389-94
Minchenko, A; Bauer, T; Salceda, S et al. (1994) Hypoxic stimulation of vascular endothelial growth factor expression in vitro and in vivo. Lab Invest 71:374-9
Minchenko, A; Salceda, S; Bauer, T et al. (1994) Hypoxia regulatory elements of the human vascular endothelial growth factor gene. Cell Mol Biol Res 40:35-9
Schuster, S J; Caro, J (1993) Erythropoietin: physiologic basis for clinical applications. Vox Sang 65:169-79
Beck, I; Weinmann, R; Caro, J (1993) Characterization of hypoxia-responsive enhancer in the human erythropoietin gene shows presence of hypoxia-inducible 120-Kd nuclear DNA-binding protein in erythropoietin-producing and nonproducing cells. Blood 82:704-11
Beck, I; Ramirez, S; Weinmann, R et al. (1991) Enhancer element at the 3'-flanking region controls transcriptional response to hypoxia in the human erythropoietin gene. J Biol Chem 266:15563-6

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