TY - JOUR
T1 - Ksp-cadherin gene promoter. I. Characterization and renal epithelial cell-specific activity
AU - Whyte, Dilys A.
AU - Li, Congyi
AU - Thomson, R. Brent
AU - Nix, Stagey L.
AU - Zanjani, Reza
AU - Karp, Sharon L.
AU - Aronson, Peter S.
AU - Igarashi, Peter
PY - 1999/10
Y1 - 1999/10
N2 - Kidney-specific cadherin (Ksp-cadherin, cadherin 16) is a novel, kidney- specific member of the cadherin superfamily that is expressed exclusively in the basolateral membrane of renal tubular epithelial cells. To characterize the Ksp-cadherin gene promoter, a λ bacteriophage clone containing 3.7 kb of the proximal 5' flanking region of the mouse Ksp-cadherin gene was isolated. The transcription initiation site was mapped by RNase protection assays and 5' rapid amplification of cDNA ends, and a 709-bp intron was identified within the 5' untranslated region. The proximal 5' flanking region was 'TATA- less' but contained other consensus promoter elements including an initiator (Inr), GC boxes, and a CAAT box. Potential binding sites were identified for transcription factors that are involved in tissue-specific gene expression including activator protein-2 (AP-2), hepatocyte nuclear factor-3 (HNF-3), basic helix-loop-helix (bHLH) proteins, CCAAT/enhancer-binding protein (C/EBP), and GATA factors. Transfection of luciferase reporter plasmids containing 2.6 kb of the 5' flanking region markedly increased luciferase activity in renal epithelial cells (MDCK and mIMCD-3) but not in mesenchymal cells (NIH 3T3 and MMR1). Deletion analysis identified an 82-bp region 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 promoter. Mutational analysis showed that sequences within the HNF-3 consensus site and CAAT box were involved in protein binding and promoter activity. We conclude that the proximal 5' flanking region of the mouse Ksp-cadherin gene contains an orientation-dependent promoter that is kidney epithelial cell specific. The region of the promoter from -113 to -31 is required for transcriptional activity and contains binding sites for nuclear proteins that are specifically expressed in renal epithelial cells.
AB - Kidney-specific cadherin (Ksp-cadherin, cadherin 16) is a novel, kidney- specific member of the cadherin superfamily that is expressed exclusively in the basolateral membrane of renal tubular epithelial cells. To characterize the Ksp-cadherin gene promoter, a λ bacteriophage clone containing 3.7 kb of the proximal 5' flanking region of the mouse Ksp-cadherin gene was isolated. The transcription initiation site was mapped by RNase protection assays and 5' rapid amplification of cDNA ends, and a 709-bp intron was identified within the 5' untranslated region. The proximal 5' flanking region was 'TATA- less' but contained other consensus promoter elements including an initiator (Inr), GC boxes, and a CAAT box. Potential binding sites were identified for transcription factors that are involved in tissue-specific gene expression including activator protein-2 (AP-2), hepatocyte nuclear factor-3 (HNF-3), basic helix-loop-helix (bHLH) proteins, CCAAT/enhancer-binding protein (C/EBP), and GATA factors. Transfection of luciferase reporter plasmids containing 2.6 kb of the 5' flanking region markedly increased luciferase activity in renal epithelial cells (MDCK and mIMCD-3) but not in mesenchymal cells (NIH 3T3 and MMR1). Deletion analysis identified an 82-bp region 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 promoter. Mutational analysis showed that sequences within the HNF-3 consensus site and CAAT box were involved in protein binding and promoter activity. We conclude that the proximal 5' flanking region of the mouse Ksp-cadherin gene contains an orientation-dependent promoter that is kidney epithelial cell specific. The region of the promoter from -113 to -31 is required for transcriptional activity and contains binding sites for nuclear proteins that are specifically expressed in renal epithelial cells.
KW - Cell adhesion
KW - Gene regulation
KW - Kidney specific
KW - Transcription factor
UR - http://www.scopus.com/inward/record.url?scp=0032750545&partnerID=8YFLogxK
U2 - 10.1152/ajprenal.1999.277.4.f587
DO - 10.1152/ajprenal.1999.277.4.f587
M3 - Article
C2 - 10516284
AN - SCOPUS:0032750545
SN - 1931-857X
VL - 277
SP - F587-F598
JO - American Journal of Physiology - Renal Fluid and Electrolyte Physiology
JF - American Journal of Physiology - Renal Fluid and Electrolyte Physiology
IS - 4 46-4
ER -