Promoter activity of carbonic anhydrase II regulatory regions in cultured renal proximal tubular cells.
Carbonic anhydrase II (CAII) plays an important role in the acid-base homeostasis of the body and its deficiency results in renal tubular acidosis. In order to identify the regulatory regions in the CAII gene for the future development of kidney-targeted gene therapy, we investigated the 5' region of the gene for its promoter activity. Deletion constructs with various lengths of the 5' flanking region of the human CAII promoter were ligated to the CAT reporter gene and lipofected in primary cultures of mouse proximal renal tubular cells and in cells of the established porcine proximal tubular cell line, LLC-PK1. The CAT activity was measured 48 hours after gene transfection. The -12000/CAT and -1300/CAT constructs expressed the highest CAT activity in both types of renal tubular cells (143- and 180-fold increase, respectively, in mouse proximal tubular cells; 50- and 70-fold increase, respectively, in LLC-PK1 cells) but not the -420/CAT, -270/CAT, or -180/CAT constructs (9, 12, and 9% of that of -1300/CAT construct, respectively, in mouse proximal tubular cells and, 23, 9, and 8%, respectively, in LLC-PK1 cells, all p <0.01 vs. -1300/CAT construct). No cytotoxicity was detected in the transfected cells. A computer search identified multiple putative transcription factor binding elements including Ap1 and Ap2 binding elements, which are present in the -1300/CAT construct but not in the shorter constructs. In conclusion, we demonstrate that the human CAII 5' sequence of proximal 1.3 kb contains strong promoter sequence(s) for renal tubular cells.
Department of Medicine, University of Arizona Health Sciences Center, Tucson 85724, USA.
L W
LW
Lai
R P
RP
Erickson
P J
PJ
Venta
R E
RE
Tashian
Y H
YH
Lien
D000818:Animals; D001483:Base Sequence; D002256:Carbonic Anhydrases / Q000235:genetics*; D002470:Cell Survival / Q000502:physiology; D002478:Cells, Cultured; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic; D017930:Genes, Reporter; D005822:Genetic Vectors; D006801:Humans; D007687:Kidney Tubules, Proximal / Q000201:enzymology* / Q000502:physiology; D051379:Mice; D011401:Promoter Regions, Genetic / Q000502:physiology*; D012045:Regulatory Sequences, Nucleic Acid; D013552:Swine; D014162:Transfection
D015500:Chloramphenicol O-Acetyltransferase; D002256:Carbonic Anhydrases