Torban Elena, Dziarmaga Alison, Iglesias Diana, Chu Lee Lee, Vassilieva Tatiana, Little Melissa, Eccles Michael, Discenza Maria, Pelletier Jerry, Goodyer Paul
Department of Experimental Medicine, McGill University, Montreal, Quebec, Canada.
J Biol Chem. 2006 May 5;281(18):12705-12. doi: 10.1074/jbc.M513181200. Epub 2005 Dec 19.
The transcription factor PAX2 is expressed during normal kidney development and is thought to influence outgrowth and branching of the ureteric bud. Mice with homozygous null Pax2 mutations have developmental defects of the midbrain-hindbrain region, optic nerve, and ear and are anephric. During nephrogenesis, PAX2 is also expressed by mesenchymal cells as they cluster and reorganize to form proximal elements of each nephron, but the function of PAX2 in these cells is unknown. In this study we hypothesized that PAX2 activates expression of WNT4, a secreted glycoprotein known to be critical for successful nephrogenesis. PAX2 protein was identified in distal portions of the "S-shaped" body, and the protein persists in the emerging proximal tubules of murine fetal kidney. PAX2 activated WNT4 promoter activity 5-fold in co-transfection assays with JTC12 cells derived from the proximal tubule. Inspection of the 5'-flanking sequence of the human WNT4 gene identified three novel PAX2 recognition motifs; each exhibited specific PAX2 protein binding in electromobility shift assays. Two motifs were contained within a completely duplicated 0.66-kb cassette. Transfection of JTC12 cells with a PAX2 expression vector was associated with a 7-fold increase in endogenous WNT4 mRNA. In contrast, Wnt4 mRNA was decreased by 60% in mesenchymal cell condensates of fetal kidney from mice with a heterozygous Pax2 mutation. We speculated that a key function of PAX2 is to activate WNT4 gene expression in metanephric mesenchymal cells as they differentiate to form elements of the renal tubules.
转录因子PAX2在正常肾脏发育过程中表达，被认为会影响输尿管芽的生长和分支。Pax2基因纯合缺失的小鼠存在中脑-后脑区域、视神经和耳朵的发育缺陷，并且无肾。在肾发生过程中，间充质细胞在聚集并重新组织形成每个肾单位的近端成分时也表达PAX2，但PAX2在这些细胞中的功能尚不清楚。在本研究中，我们假设PAX2激活WNT4的表达，WNT4是一种已知对成功肾发生至关重要的分泌糖蛋白。在“s形”小体的远端部分鉴定出PAX2蛋白，该蛋白在小鼠胎儿肾脏新出现的近端小管中持续存在。在与源自近端小管的JTC12细胞的共转染实验中，PAX2使WNT4启动子活性提高了5倍。对人WNT4基因5'侧翼序列的检查发现了三个新的PAX2识别基序；在电泳迁移率变动分析中，每个基序都表现出特异性的PAX2蛋白结合。两个基序包含在一个完全重复的0.66 kb盒式结构中。用PAX2表达载体转染JTC12细胞与内源性WNT4 mRNA增加7倍相关。相反，在Pax2基因杂合突变小鼠的胎儿肾脏间充质细胞凝集体中，Wnt4 mRNA减少了60%。我们推测，PAX2的一个关键功能是在中肾间充质细胞分化形成肾小管成分时激活WNT4基因表达。
