REGULATION OF HEPATOCELLULAR FUNCTION BY GROWTH HORMONE

本项目的长期目标是阐明 生长激素在肝脏中作用的分子机制。 这 应用程序将主要集中在两个（指定的Spi-1（S3）和 Spi-2（S20））相关基因产物的6种， 被鉴定为生长激素敏感型 这些产品 在给予生长激素4小时内诱导5倍 对垂体切除的动物， 肝细胞培养 这些mRNA在肝细胞中的反应 文化和快速反应表明，这些产品是 生长激素作用的主要产物。 两款产品 与丝氨酸蛋白酶的成员高度同源 抑制剂（Spi）基因家族，如通过cDNA测序所揭示的， 肽预测 为了进一步了解Spi-1和Spi-2的调控， 将为每个产品构建寡核苷酸探针。 的 将分离各个基因，并通过以下方法评价它们的结构： 核酸内切酶限制性作图和有限的DNA测序。 RNA合成的起始位点和鉴定 内含子将通过引物延伸和S1核酸酶确定 映射. 第二，这些基因的调控位点是否 产物是在基因转录水平上确定的 通过体外核转录连续测定，测量 核前体和成熟mRNA。 稳态和 将对变化的时间进程进行评估和比较。 第三、 将进行研究以确定这些基因是否 产品主要对生长激素有反应。 效果 蛋白质合成抑制剂对生长激素诱导的影响， 胰岛素样生长因子I对这些产品的作用， 将检查肝细胞培养物。 第四，细胞培养 将开发一个系统，以评估 通过DNA介导的基因转移分离Spi-1或Spi-2基因。 的 初步计划是将Spi-1或Spi-2的基因插入到 pSV 40 neo，然后将该DNA转染到宿主细胞系中， 含有功能性生长激素受体。 表达 新引入的DNA的调节将通过以下方式进行检查： 瞬时表达测定和稳定整合后，使用 定量S1核酸酶测定。 这种方法应该提供一个 研究生长激素的优良模型系统 行动，并提供一个更清晰的理解所涉及的过程。 这些研究也可能提供重要的信息， 多肽激素对基因表达的作用机制。 最后，可以获得关于额外的生理学的见解。 生长激素的作用机制，通过调查这些 两种丝氨酸蛋白酶抑制剂样多肽。

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.