Araki E, Haag B L, Matsuda K, Shichiri M, Kahn C R
Joslin Diabetes Center, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02215, USA.
Mol Endocrinol. 1995 Oct;9(10):1367-79. doi: 10.1210/mend.9.10.8544845.
To evaluate the potential for regulation of the insulin receptor substrate IRS-1, we have cloned the mouse IRS-1 gene, identified its promoter, and analyzed promoter activity in the basal state and in response to stimulation. The 5'-region of the mouse IRS-1 gene lacks typical CAAT and TATA boxes but contains nine potential Sp1 binding sites consistent with a housekeeping gene. The 5'-region of the IRS-1 gene also has significant regions of homology with the promoters of the progesterone receptor gene, the insulin-like growth factor I receptor gene, and the androgen receptor gene. Multiple transcription start sites were identified 0.4-1.2 kilobases (kb) upstream from the start codon. Using a chloramphenicol acetyl transferase assay in Chinese hamster ovary (CHO) cells, basal promoter activity was present in the 3.2 kb 5'-flanking region of IRS-1 gene. Within this region, there were 184-base pair and 60-base pair negative regulatory elements at -3.2 kb and -1.6 kb surrounded by positive elements. By gel shift assay, a nuclear factor was identified in CHO cells which binds to -1606 and -1586 sequence in the negative regulatory element and appears to be distinct from C/EBP, CREB, and AP-1. In 3T3-F442A adipocytes dexamethasone treatment significantly decreased IRS-1 mRNA and IRS-1 protein. This was due to a decrease in the half-life of IRS-1 mRNA, with no change in IRS-1 promoter-chloramphenicol acetyl transferase activity. Insulin also decreased IRS-1 protein by approximately 60% within 9 h but did so without altering IRS-1 mRNA levels or chloramphenicol acetyl transferase activity. Thus, both insulin and dexamethasone down-regulate IRS-1 expression at the posttranscriptional level; with insulin this is probably due to an effect on protein half-life, whereas with dexamethasone the effect is due to a change in the half-life of IRS-1 mRNA.
为了评估胰岛素受体底物IRS-1的调控潜力，我们克隆了小鼠IRS-1基因，鉴定了其启动子，并分析了基础状态下以及刺激反应中的启动子活性。小鼠IRS-1基因的5'区域缺乏典型的CAAT和TATA框，但含有9个与管家基因一致的潜在Sp1结合位点。IRS-1基因的5'区域还与孕酮受体基因、胰岛素样生长因子I受体基因和雄激素受体基因的启动子有显著的同源区域。在起始密码子上游0.4 - 1.2千碱基（kb）处鉴定出多个转录起始位点。使用中国仓鼠卵巢（CHO）细胞中的氯霉素乙酰转移酶测定法，IRS-1基因的3.2 kb 5'侧翼区域存在基础启动子活性。在该区域内， - 3.2 kb和 - 1.6 kb处有184碱基对和60碱基对的负调控元件，周围环绕着正调控元件。通过凝胶迁移试验，在CHO细胞中鉴定出一种核因子，它与负调控元件中的 - 1606和 - 1586序列结合，并且似乎与C/EBP、CREB和AP-1不同。在3T3 - F442A脂肪细胞中，地塞米松处理显著降低了IRS-1 mRNA和IRS-1蛋白水平。这是由于IRS-1 mRNA半衰期的缩短，而IRS-1启动子 - 氯霉素乙酰转移酶活性没有变化。胰岛素在9小时内也使IRS-1蛋白水平降低了约60%，但没有改变IRS-1 mRNA水平或氯霉素乙酰转移酶活性。因此，胰岛素和地塞米松均在转录后水平下调IRS-1的表达；胰岛素可能是通过影响蛋白质半衰期起作用，而地塞米松则是通过改变IRS-1 mRNA的半衰期起作用。
