Ksp-cadherin gene promoter. I. Characterization and renal epithelial cell-specific activity

Kidney-specific cadherin (Ksp-cadherin, cadherin 16) is a novel, kidney-spe cific member of the cadherin superfamily that is expressed exclusively in t he basolateral membrane of renal tubular epithelial cells. To characterize the Ksp-cadherin gene promoter, a lambda bacteriophage clone containing 3.7 kb of the proximal 5' flanking region of the mouse Ksp-cadherin gene was i solated. The transcription initiation site was mapped by RNase protection a ssays and 5' rapid amplification of cDNA ends, and a 709-bp intron was iden tified within the 5' untranslated region. The proximal 5' flanking region w as "TATA-less" but contained other consensus promoter elements including an initiator (Inr), GC boxes, and a CAAT box. Potential binding sites were id entified for transcription factors that are involved in tissue-specific gen e expression including activator protein-2 (AP-2), hepatocyte nuclear facto r-3 (HNF-3), basic helix-loop-helix (bHLH) proteins, CCAAT/enhancer-binding protein (C/EBP), and GATA factors. Transfection of luciferase reporter pla smids containing 2.6 kb of the 5' flanking region markedly increased lucife rase activity in renal epithelial cells (MDCK and mIMCD-3) but not in mesen chymal cells (NIH 3T3 and MMR1). Deletion analysis identified an 82-bp regi on from -31 to -113 that was essential for promoter activity in transfected renal epithelial cells. Electrophoretic mobility-shift assays showed that mIMCD-3 cells contain nuclear proteins that bind to this region of the prom oter. Mutational analysis showed that sequences within the HNF-3 consensus site and CAAT box were involved in protein binding and promoter activity. W e conclude that the proximal 5' flanking region of the mouse Ksp-cadherin g ene contains an orientation-dependent promoter that is kidney epithelial ce ll specific. The region of the promoter from -113 to -31 is required for tr anscriptional activity and contains binding sites for nuclear proteins that are specifically expressed in renal epithelial cells.