AUTOREHLATION OF TRANSCRIPTION OF G-PROTEIN MESANGIAL GELLS

G 蛋白系膜凝胶转录的自转录

• 批准号: 09671185
• 负责人: KAWASHIMA Akira
• 金额: $ 1.09万
• 依托单位: TOKYO WOMEN'S MEDICAL UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1997
• 资助国家: 日本
• 起止时间: 1997 至 1998
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09671185/
• 关键词: AUTOREGULATION; TRANSCRIPTION; G-PROTEIN; LLC-PK1 CELL; Sp1; 緊糸球体; メサンギウム細胞; 増殖; 転写因子

A previous report demonstrated that Gαi-2 is regulated by steroid hormone. A mutation of the Gαi-2 gene that decreases GTPase activity produces the oncogene gip2. Autoregulation of this gene should be critical to prevent overgrowth of the cells. We detected Gai-2 protein in glomerular cells by immunostaining and immunoblotting. Then, we tried to transfect Gαi-2 gene into mesangial cells using calcium phosphate precipitation method or lipofection method. However, it was insufficient for the following experiments. Thus, we used LLC-PK1 cells in place of mesangial cells. In the cells transfected with 5'-flanking sequences fused to firefly luciferase cDNA reporter gene, we demonstrated a 24 % transcriptional repression of Gαi-2 gene after overexpressing Gαi-2 protein and a 88 % repression of gip2 protein when gip2 protein is overexpressed. Deletion analysis of the reporter gene indicated that the site of repression in gip2 transfected cells occurred in the region of the Gαi-2 gene promoter. Utilizing the 27-bp DNA segment as a probe in mobility shift assay, we identified a complex with decreased binding in gip2 induced cells. This DNA segment containing tow GC boxes was recognized specifically by a transcription factor Sp1. Antibody to Sp1 added to the mobility shift assay supershifted the complex. These studies demonstrate that overexpression of Gαi-2 protein represses Gαi-2 gene transcription by a reduction of transcription factor Sp1, and suggest the possibility of an autoregulation mechanism of Gαi-2 transcription.

以前的研究表明Gαi-2受类固醇激素的调节。Gαi-2基因的突变降低了GT3活性，产生致癌基因gip 2。这种基因的自动调节对于防止细胞过度生长至关重要。免疫组化和免疫印迹法检测肾小球细胞中Gai-2蛋白的表达。然后，我们尝试用磷酸钙沉淀法和脂质体转染法将Gαi-2基因转染到系膜细胞中。但是，这对于接下来的实验来说是不够的。因此，我们使用LLC-PK 1细胞代替系膜细胞。在转染萤火虫荧光素酶cDNA报告基因5 '侧翼序列的细胞中，我们证明了在过表达Gα i-2蛋白后，Gα i-2基因的转录抑制率为24%，而在过表达gip 2蛋白时，gip 2蛋白的转录抑制率为88%。报告基因的缺失分析表明，gip 2转染细胞中的阻遏位点发生在Gαi-2基因启动子区域。利用27-bp的DNA片段作为探针在迁移率变动分析中，我们鉴定了在gip 2诱导的细胞中结合减少的复合物。该DNA片段含有两个GC盒，可被转录因子Sp1特异性识别。将Sp1抗体加入到迁移率变动测定中，使复合物超移。这些研究表明，Gαi-2蛋白的过表达通过减少转录因子Sp1的表达而抑制Gαi-2基因的转录，并提示Gαi-2基因转录的自调节机制的可能性。