Characterization of the human type II Na/Pi-cotransporter promoter.
The type II Na/Pi-cotransporter is expressed preferentially in renal proximal tubular epithelial cells. Comparison of the 5′ flanking region of the human NPT-2 gene with the opossum cell line (OK cell) and the murine Npt2 promoters revealed two conserved regions, one representing a putative C/EBP alpha site, the other a consensus TATA-box. In contrast to the OK cell and murine Npt-2 gene, the human exon 1 is flanked by two Alu-repeats, a short 90-bp inverted Alu element which is located within the promoter region and a full-length forward repeat present in intron 1. A 497-bp human promoter fragment including the inverted Alu-repeat was cloned in front of a luciferase reporter gene. The construct was active in OK and HeLa-S3 cells but no activity could be detected in the human monocyte cell line U937, the murine renal cortex cell line MCT and the dog kidney cell line MDCK. A twofold increase in promoter activity was observed in HeLa-S3 cells for a 5′ truncated fragment of 253 bp missing the inverted Alu-repeat. In the OK cell system the absence of the Alu-repeat was unable to modify promoter activity. In electrophoretic mobility shift assays (EMSAs) with a 31-bp oligonucleotide representing the conserved region with homology to C/EBP alpha we could provide evidence for specific DNA/protein interactions with nuclear extracts derived from kidney and liver cell lines but not for HeLa-S3 and U937 nuclear extracts. Specific interactions could also be observed with nuclear extracts from renal cortex, medulla and rat liver but not from rat spleen, intestine and heart. Southern-Western blotting techniques suggest that a 31-kDa nuclear protein from kidney-derived cells binds to the C/EBP-like region of the NPT2 promoter.
Institute of Physiology, University of Zurich, Winterthurerstr. 190, CH-8057 Zurich, Switzerland.
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Hilfiker
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Kvietikova
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Hartmann
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Stange
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Murer
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D005091:Exons; D017930:Genes, Reporter; D006801:Humans; D007668:Kidney / Q000166:cytology / Q000378:metabolism; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D050610:Sodium-Phosphate Cotransporter Proteins, Type II / Q000235:genetics* / Q000378:metabolism*
D050610:Sodium-Phosphate Cotransporter Proteins, Type II