Analysis of the Mechanisms of Cell and Tissue Specific Expression of the Insulin Receptor Substrate, IRS-1 Gene.

结果表明，IRS-1是胰岛素受体酪氨酸激酶的主要底物之一，在下游介导多种胰岛素信号，阐明了IRS-1组织特异性调控的分子机制，我们研究了CHO和HepG 2细胞中的顺式作用元件和反式作用因子。用IRS-1启动子-CAT融合质粒的各种缺失突变体进行氯霉素乙酰转移酶测定，鉴定了在每个细胞中负责正或负调控的几个区域，从-1645到-1585bp的区域在CHO细胞中负调控表达而在HepG 2细胞中阳性，在此区域内，-1645 ~-1605bp的片段仅在HepG 2细胞中上调IRS-1启动子的表达，而-1605 ~-1585bp的片段仅在CHO细胞中下调IRS-1启动子的表达。两种核蛋白结合到一个潜在的E盒上（nt-1635- 1630）和两个与潜在的C/EBP结合位点结合的核蛋白（nt-1599--1591）在HepG 2和CHO细胞中鉴定，使用在E盒或在C/C区突变的启动子的CAT测定。EBP结合位点分析表明，这两个序列对IRS-1基因的表达具有细胞特异性调控作用，因此我们认为，在HepG 2细胞中，这两个与Ebox结合的中性蛋白对IRS-1基因的表达具有正调控作用。而与C/EBP结合位点结合的两种核蛋白在CHO细胞中负调控C/EBP结合位点。

The results are summarized as follows.IRS-1 is one of the major substrates of insulin receptor tyrosine kinase and mediates multiple insulin signals downstream.Tp elucidate the molecular mechanisms of the tissue specific regulation of IRS-1, we have studied the cis-acting elements and trans-acting factors in CHO and HepG2 cells.Using the CAT (Chloramphenicol acetyltransferase) assay with the various deletion mutants of the IRS-1 promoter-CAT fusion plasmids, several regions responsible for positive or negative regulation in each cells were identified.A region from-1645 to-1585 bp which regulated expression negatively in CHO cells and positivity in HepG2 cells, was further analyzed.Within this region a fragment from-1645 to-1605bp up-regulated the IRS-1 promotor only in HepG2 cells, whereas a fragment from-1605 to-1585bp down-regulated only in CHO cells.In the gel mobility shift assay, several nucler proteins which bind to these fragments were detected, and among them, two nuclear proteins which bind to a potential E box (nt-1635--1630) , and two nuclear proteins which bind to a potential C/EBP binding site (nt-1599--1591) were identified in HepG2 and CHO cells, respectively.CAT assays using promoters mutated at the E box or at the C/EBP binding site revealed that these sequences were responsible for cell spcific regulation of the IRS-1 gene.We therefore concluded that the two nulclear proteins which bind to the E box regulate IRS-1 gene expression positively in HepG2 cells, and the two nuclear proteins which bind to the C/EBP binding site regulate it negatively in CHO cells.