The objective of this proposal is to understand the signal transduction pathway responsible for hypoxia-induced erythropoietin (Epo) production. The Hep3B cell line has previously been shown to regulate Epo production in a physiologic manner in response to hypoxia. It has also been used extensively as a model system to study the effects of inflammatory cytokines in the acute phase response. The addition of IL-1 alpha, IL-1 beta, or TNF-alpha results in a dose-dependent inhibition of hypoxia- induced Epo production. In contrast, the addition of IL/6 to hypoxic Hep3B cells results in a dose-dependent further stimulation of hypoxia-induced Epo production. Northern blot analyses indicate that these cytokines affect Epo production primarily at the mRNA level. In this proposal the applicant plans to investigate the molecular mechanisms by which the inflammatory cytokines exert their effects on hypoxia-induced Epo production. Experiments will be performed to characterize the cis and trans elements which are involved in the transcriptional and post- transcriptional regulation of these cytokine mediated events. This work will involve extensive analysis of the 5' and 3' flanking regions of the Epo gene. Methods to be employed include functional assays (nuclear run- off transcription assays, transient expression assays, in vitro RNA degradation analyses) and structural analyses (DNA and RNA mobility shift assays, in vitro DNAse I footprint analysis, protein purification and characterization). The applicant will test the hypothesis that the soluble form of guanylate cyclase is the heme protein oxygen sensor which mediates the hypoxia-induced increase in Epo production. In an effort to unravel the hypoxia-induced signal transduction pathway, changes in phosphorylation of cytosolic and membrane proteins will be investigated. Oxygen differs from the great majority of external cell signals by being a ubiquitous molecule that diffuses readily into cells independent of receptor binding. Accordingly, the signal transduction pathway from oxygen sensor to gene regulation may differ significantly from these associated with classic ligand-receptor binding. Investigation of the molecular basis for the observed effects of these cytokines on hypoxia-induced Epo production may provide detailed information on the molecular nature of the oxygen sensor. In addition, a better understanding of the molecular mechanisms governing the regulation of Epo production may enhance our understanding of the pathogenesis of the anemia of chronic disease and the nature of the acute phase response.
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