Tajinda K, Carroll J, Roberts C T
Department of Pediatrics, Oregon Health Sciences University, Portland 97201, USA.
Endocrinology. 1999 Oct;140(10):4713-24. doi: 10.1210/endo.140.10.7065.
The insulin-like growth factor I (IGF-I) receptor is a transmembrane tyrosine kinase that mediates the growth-promoting effects of IGF-I and IGF-II. Changes in IGF-I receptor messenger RNA levels are reflected in cell surface receptor number, and modulation of IGF-I receptor levels affects tumorigenicity in numerous cellular models; thus, control of IGF-I receptor gene expression appears to be an important level at which cellular proliferation and tumorigenic potential may be regulated. We have previously shown that the product of the WT1 Wilms' tumor suppressor gene represses IGF-I receptor gene expression both in vitro and in vivo, and that decreased WT1 levels are correlated with up-regulation of IGF-I receptor gene expression in Wilms' tumor, benign prostatic hyperplasia, and breast cancer. Gene regulation by WT1 is complex, in that the WT1 gene encodes a variety of products as a result of alternative splicing and RNA editing, and a number of missense point mutations have been characterized in Wilms' tumor-associated syndromes. Additionally, the WT1 protein has been demonstrated to self-associate through its N-terminal domain, although the role of this intermolecular interaction in transcriptional regulation by WT1 is unclear. In this report, we analyze the relative activity of wild-type and mutant versions of the WT1 protein with respect to IGF-I receptor promoter activity in transient transfection assays and assess the potential contribution of WT1 self-association to IGF-I receptor regulation using the yeast two-hybrid system. Of the naturally occurring variations in WT1 structure, only the presence of a three-amino acid KTS insert in the zinc finger domain introduced by alternative splicing of exon 9 had a significant effect on WT1 repression of IGF-I receptor promoter activity. The N- and C-terminal domains of WT1 also exhibited partial repression, as did the most common mutant version of the WT1 protein associated with Denys-Drash syndrome. Mutations in the WT1 N-terminus attenuated WT1 self-association in the yeast two-hybrid system, but did not impair transcriptional repression. Our results suggest that 1) the DNA-binding capacity of WT1 is critical for maximal repression of the IGF-I receptor promoter, but some effects may be mediated through protein-protein interactions involving the N-terminal domain; 2) WT1 self-association may not be required for repression of the IGF-I receptor promoter; and 3) the Denys-Drash syndrome version of the WT1 protein may exhibit residual or possible gain of function activity in some contexts rather than exerting dominant negative effects, as has been proposed previously.
胰岛素样生长因子I（IGF-I）受体是一种跨膜酪氨酸激酶，介导IGF-I和IGF-II的促生长作用。IGF-I受体信使核糖核酸水平的变化反映在细胞表面受体数量上，IGF-I受体水平的调节在众多细胞模型中影响肿瘤发生；因此，控制IGF-I受体基因表达似乎是调节细胞增殖和肿瘤发生潜能的一个重要层面。我们先前已表明，WT1威尔姆斯瘤抑癌基因的产物在体外和体内均抑制IGF-I受体基因表达，且在威尔姆斯瘤、良性前列腺增生和乳腺癌中，WT1水平降低与IGF-I受体基因表达上调相关。WT1对基因的调控很复杂，因为WT1基因通过可变剪接和RNA编辑编码多种产物，且在威尔姆斯瘤相关综合征中已鉴定出一些错义点突变。此外，WT1蛋白已被证明通过其N端结构域进行自我结合，尽管这种分子间相互作用在WT1转录调控中的作用尚不清楚。在本报告中，我们在瞬时转染实验中分析了WT1蛋白野生型和突变型相对于IGF-I受体启动子活性的相对活性，并使用酵母双杂交系统评估WT1自我结合对IGF-I受体调控的潜在贡献。在WT1结构的自然变异中，只有外显子9可变剪接引入的锌指结构域中一个三氨基酸KTS插入的存在对WT1抑制IGF-I受体启动子活性有显著影响。WT1的N端和C端结构域也表现出部分抑制作用，与迪尼-德兰综合征相关的WT1蛋白最常见突变型也是如此。WT1 N端的突变在酵母双杂交系统中减弱了WT1的自我结合，但不损害转录抑制。我们的结果表明：1）WT1的DNA结合能力对于最大程度抑制IGF-I受体启动子至关重要，但某些效应可能通过涉及N端结构域的蛋白质-蛋白质相互作用介导；2）WT1自我结合可能不是抑制IGF-I受体启动子所必需的；3）WT1蛋白的迪尼-德兰综合征版本在某些情况下可能表现出残余或可能的功能获得活性，而不是像先前提出的那样发挥显性负效应。
