GENETIC APPROACH TO INSULIN SIGNALING

胰岛素治疗增加了GH细胞中内源性催乳素基因的转录和由催乳素基因5'-侧翼DNA连接到细菌氯霉素乙酰转移酶（CAT）基因的嵌合质粒的表达。因此，该细胞培养系统准确地反映了胰岛素对催乳素的生理调节。由于糖尿病导致的催乳素表达缺陷被认为可以解释包括婴儿呼吸窘迫综合征和阳痿在内的几种疾病。这项研究的长期目标是发现这种调节的所有组成部分，以及它们如何相互作用导致催乳素基因表达增加。迄今为止的结果已经确定了一致的胰岛素反应因子（IRE）。这个IRE是ets基序相关序列CGGAA，它介导了胰岛素引起的泌乳素- cat表达增加10倍的100%。GABP被确定为可能介导胰岛素引起的催乳素- cat表达增加的转录因子。本研究的重点是胰岛素信号对催乳素基因表达的影响。其他人和我们所做的研究都没有确定导致激素调节基因表达的信号通路。这些研究使用各种信号通路的抑制剂或使用显性阴性或野生型信号分子的过表达进行。这些协议永远不能完全证明一个信号通路参与了一个特定的过程，它们不能揭示未知的途径。因此，本研究采用了一种遗传方法，该方法a)不需要任何先前的信号通路知识，b)将明确地确定任何被分离的分子作为基因表达信号的参与者的作用。首先，在泌乳素启动子的控制下，建立表达可选择标记的细胞系。该启动子的激素反应与野生型催乳素启动子相同。这些细胞系将被诱变，产生对激素无反应的突变体。然后对这些突变体进行分析，以确定产生激素无反应表型的突变分子。最后，将进行互补以确认它们的信号作用。这种应用可能被认为是高风险的，但它是唯一有合理成功可能性的方案，潜在的好处也很高。另一种选择是费力地检查和排除每个已知的信号通路，而不能保证在研究结束时比在研究开始时了解更多。

Insulin treatment increases the transcription of the endogenous prolactin gene and the expression of chimeric plasmids consisting of 5'-flanking DNA from the prolactin gene ligated to the bacterial chloramphenicol acetyl transferase (CAT) gene in GH cells. Thus, this cell culture system accurately reflects the physiological regulation of prolactin by insulin. Defects in prolactin expression due to diabetes have been suggested to account for several disorders including infant respiratory distress syndrome and impotence. The long-term goal of this research is to discover all of the components of this regulation and how they interact to cause increased prolactin gene expression. Results to date have identified a consensus insulin response element (IRE). This IRE is the Ets-motif-related sequence CGGAA and it mediates 100% of the >10-fold increase in Prolactin-CAT expression caused by insulin. GABP was identified as the transcription factor that probably mediates the increase in prolactin-CAT expression due to insulin. The studies proposed in this application focus on insulin signaling to prolactin gene expression. Studies done by others and by us have not identified the signaling pathway that leads to hormone-regulated gene expression. These studies were performed with inhibitors of the various signaling pathways or used overexpression of dominant negative or wild type signaling molecules. These protocols can never completely prove that a signaling pathway is involved in a particular process and they can not reveal unknown pathways. Therefore, this proposal uses a genetic approach that a) does not require any previous knowledge of signaling pathways and b) that will unequivocally establish the role of any molecule that is isolated as a participant in signaling to gene expression. First, cell lines were established that express a selectable marker under control of the prolactin promoter. The hormonal response of this promoter is identical with the wild type prolactin promoter. These cell lines will be mutagenized to produce mutants that are hormonally unresponsive. These mutants will then be analyzed to identify the mutant molecules that produce the hormonally non-responsive phenotype. Finally, complementation will be performed to confirm their signaling role. This application may he perceived to be high risk, but it is the only protocol with a reasonable likelihood of success and the potential benefits are also high. The alternative is to laboriously examine and eliminate each known signaling pathway with no guarantee of knowing more at the end then at the beginning of the studies.