The long term objective of this project is to elucidate the molecular mechanism of growth hormone action in the liver. This application will focus primarily on two (designated Spi-1(S3) and Spi-2(S20)) interrelated gene products of 6 which have been identified as growth hormone responsive. These products are induced 5 fold within four hours of growth hormone administration to a hypophysectomized animal and respond to growth hormone in hepatocyte cultures. The response of these mRNA in hepatocyte cultures and the rapid response suggests that these products are primary products of growth hormone action. The two products are highly homologous with members of the serine protease inhibitor(Spi) gene family as revealed by cDNA sequencing and peptide prediction. To further understand the regulation of Spi-1 and Spi-2, specific oligonucleotide probes will be constructed for each product. The respective genes will be isolated and their structure evaluated by endonuclease restriction mapping and limited DNA sequencing. The site of initiation of RNA synthesis and identification of introns will be determined by primer extention and S1 nuclease mapping. Second, whether the site of regulation of these gene products is at the level of gene transcription will be determined by in vitro nuclear transcription run-on assays, measurement of nuclear precursors and mature mRNA. The steady state and the time course of changes will be evaluated and compared. Third, studies will be performed to determine whether these gene products are primarily responsive to growth hormone. The effect of protein synthesis inhibitors on growth hormone induction and the effect of insulin like growth factor I on these products in hepatocyte cultures will be examined. Fourth, a cell culture system will be developed to evaluate the regulation of the isolated Spi-1 or Spi-2 gene by DNA-mediated gene transfer. The tentative plan is to insert the gene for either Spi-1 or Spi-2 into pSV40 neo and then transfect this DNA into a host cell line which contains functional growth hormone receptors. The expression and regulation of the newly introduced DNA will be examined by the transient expression assay and after stable integration, using a quantitative S1 nuclease assay. This approach should provide an excellent model system for investigation of growth hormone action and provide a clearer understanding the processes involved. These studies may also provide important information as to the mechanism of polypeptide hormone action on gene expression. Finally, insight may be gained about additional physiological mechanisms of growth hormone action by investigation of these two serine protease inhibitor like polypeptides.
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