Werner H, Bach M A, Stannard B, Roberts C T, LeRoith D
Section on Molecular and Cellular Physiology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892.
Mol Endocrinol. 1992 Oct;6(10):1545-58. doi: 10.1210/mend.6.10.1448110.
The insulin-like growth factor I receptor (IGF-I-R) gene is expressed in most body tissues. The levels of IGF-I-R mRNA, however, are regulated by a number of physiological conditions (development, differentiation, and hormonal milieu) as well as in certain pathological states (diabetes and tumors). To understand the molecular mechanisms which control the transcription of the IGF-I-R gene, we have cloned the promoter of the rat receptor gene and have characterized its activity by transient expression assays. Different fragments of the 5'-flanking region (subcloned upstream of a luciferase reporter gene) were transfected into buffalo rat liver 3A cells (a cell line with a low number of IGF-I binding sites) and Chinese hamster ovary cells (a cell line with a higher number of cell-surface receptors). In both cell lines, most of the promoter activity was located in the proximal 416 base pairs of 5'-flanking region. However, further dissection of this proximal fragment revealed a cell type-specific pattern of promoter activity. Thus, in buffalo rat liver 3A cells, subfragments of this region each contributed to total activity, suggesting that contiguous cis-elements can act together to activate transcription. In Chinese hamster ovary cells, on the other hand, subfragments of the proximal promoter region partially substituted for the proximal 416 base pairs of 5'-flanking region. Coexpression studies using an IGF-I-R promoter reporter construct together with an Sp1 expression vector (under the control of an ADH promoter) were performed in SL2 cells, a Drosophila cell line which lacks endogenous Sp1. The results obtained showed that Sp1 can trans-activate the IGF-I-R promoter in vivo. Transient transfection assays were complemented with gel-retardation assays and DNase I footprinting experiments, which showed that transcription factor Sp1 is potentially an important regulator of IGF-I-R gene expression.
胰岛素样生长因子I受体（IGF-I-R）基因在大多数身体组织中表达。然而，IGF-I-R mRNA的水平受多种生理状况（发育、分化和激素环境）以及某些病理状态（糖尿病和肿瘤）的调节。为了了解控制IGF-I-R基因转录的分子机制，我们克隆了大鼠受体基因的启动子，并通过瞬时表达分析对其活性进行了表征。将5'-侧翼区的不同片段（亚克隆到荧光素酶报告基因的上游）转染到水牛大鼠肝3A细胞（一种IGF-I结合位点数量较少的细胞系）和中国仓鼠卵巢细胞（一种细胞表面受体数量较多的细胞系）中。在这两种细胞系中，大部分启动子活性位于5'-侧翼区近端的416个碱基对中。然而，对该近端片段的进一步剖析揭示了启动子活性的细胞类型特异性模式。因此，在水牛大鼠肝3A细胞中，该区域的亚片段各自对总活性有贡献，这表明相邻的顺式元件可以共同作用来激活转录。另一方面，在中国仓鼠卵巢细胞中，近端启动子区域的亚片段部分替代了5'-侧翼区近端的416个碱基对。使用IGF-I-R启动子报告构建体与Sp1表达载体（在ADH启动子的控制下）进行共表达研究，在缺乏内源性Sp1的果蝇细胞系SL2细胞中进行。获得的结果表明，Sp1可以在体内反式激活IGF-I-R启动子。瞬时转染分析辅以凝胶阻滞分析和DNase I足迹实验，结果表明转录因子Sp1可能是IGF-I-R基因表达的重要调节因子。
