Characterization of the human type II Na/P i -cotransporter promoter

Abstract

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.

Molecular Characteristics of Phosphate Transporters and Their Regulation

“…For the murine gene, two transcription initiation and two polyadenylation sites have been identified. The NPT2 promoter was functional in cells from renal proximal origin (OK), but not from distal tubules (MDCK) [17]. The promoter activity is regulated by CO 2 /HCO 3 2– and vitamin D 3 [18, 19]; transcriptional regulation has also been proposed for thyroid hormone (T 3 ) [20].…”

An apical expression signal of the renal type IIc Na+-dependent phosphate cotransporter in renal epithelial cells

“…NaPi-IIa also shows a high degree of homology to another transporter, NaPi-IIb, at the level of primary structure (ϳ70% similarity), but these transporters show different expression patterns in vivo (9,15). NaPi-IIa is expressed on the apical membrane of renal proximal tubules, and NaPi-IIb is expressed in the intestine and lung epithelia.…”

Protein-DNA Interactions at the Opossum Npt2a Promoter are Dependent upon NHERF-1

“…The 5'-flanking regions of the rat (~4kb rNpt2a), human (~2.4kb hNpt2a), and opossum (~3.7kb oNpt2a) genes were active in reporter gene assays only in the OK cells but not HeLa, NIH-3T3, or HEK293 cells [6][7][8][9]. 5'-deletion analyses identified a region within 250bp upstream of the transcription start site as sufficient to drive cell-specific expression of the human and opossum promoters [6,10]. A transgenic mouse harboring a rat Npt2a promoter-driven LacZ gene showed localized renal cortical-specific expression in the kidney and no other tissue, confirming that 4.7kb of the rat 5'-flanking region is sufficient to control tissue-specific expression in vivo [11].…”