Gene structure and functional analysis of the human Na+/phosphate co-transporter

Abstract

Three lambda phage clones encompassing the Na+/phosphate co-transporter (NaPi-3) gene and its 5' flanking region were isolated from a human genomic DNA library. The gene comprises 13 exons and 12 introns and spans approx. 14 kb. All exon-intron junctions conform to the GT/AG rule. The major transcription-initiation site was determined by primer-extension analysis and is an adenosine residue 57 bp upstream of the 3' end of the first exon. There is a typical TATA box 28 bp upstream of the major transcription-initiation site and various cis-acting elements, including a cAMP-responsive element, AP-1, AP-2 and SP-1 sites in the 5' flanking region. This region also contains three direct-repeat-like sequences that resemble the consensus binding sequence for members of the steroid-thyroid hormone receptor superfamily, including vitamin D. Deletion analysis suggests that the region from nt-2409 to nt-1259 in the 5' flanking region may be involved in kidney-specific gene expression. Vitamin D responsiveness of the NaPi-3 promoter was also detected in COS-7 cells co-transfected with a human vitamin D receptor expression vector. The presence of the three vitamin D receptor- responsive elements in the NaPi-3 promoter may be important in mediating the enhanced expression of the gene by 1,25-dihydroxyvitamin D3.

Increased circulating levels of FGF23: an adaptive response in primary hyperparathyroidism?

“…This suggests that in PHPT, factors other than PTH, FGF23, or their combined effect may play a role in phosphate homeostasis. A clear contender is 1,25(OH) 2 D. The net effect of 1,25(OH) 2 D on gut, kidney, bone, and parathyroids is to increase serum phosphate levels, by upregulating NaPi2b co-transporter expression in the intestinal tract (1, 7) and NaPi2 co-transporter (NaPi3) gene in the kidney (3,8) and by directly reducing PTH synthesis and secretion by the parathyroid (29 In our study, patients with PHPT had significantly increased 1,25(OH) 2 D levels compared with euparathyroid patients, but also demonstrated significantly increased FGF23 levels. A new hypothesis has been recently proposed to explain the need for two phosphaturic hormones, PTH and FGF23, with the former repressed and the latter induced by 1,25(OH) 2 D (36).…”

“…Parathyroid hormone (PTH) and the active metabolite of vitamin D (1,25(OH) 2 D) are prime regulators of calcium homeostasis but also have significant effects on phosphate homeostasis by downregulating or upregulating the sodium phosphate co-transporters in the proximal tubules of the kidneys and in enterocytes of the intestinal tract (1)(2)(3)(4)(5)(6)(7)(8). However, the major player of the bone-kidney axis controlling phosphate homeostasis has been shown to be fibroblast growth factor 23 (FGF23).…”

Regulation of Type II Renal Na+-dependent Inorganic Phosphate Transporters by 1,25-Dihydroxyvitamin D3

“…Plasmid Construction-Two luciferase reporter vectors (p3P2400 and p3P1260) containing the 5Ј-flanking region of the human NaPi-3 gene were described in a previous study (15). The luciferase reporter plasmids pTKDRCF, pTKDRCR, pTKDRCmt2, and pTKDRCmt3 containing the DR-C sequence of the NaPi-3 gene promoter in the forward and reverse directions, respectively, were constructed by placing synthetic double-stranded DNA (annealed oligonucleotides with XhoI cleavage sites (5Ј-TCGAGATCAGGGGCAGCAAGGGCAGAAATG-3Ј and 5Ј-TCGACATTTCTGCCCTTGCTGCCCCTGATC-3Ј) and with the mutation bases indicated in Table I for pTKDRCmt2 and pTKDRCmt3) corresponding to nucleotides Ϫ1982 to Ϫ1957 (relative to the transcription start site) of NaPi-3 upstream of the minimum promoter region of the herpes simplex virus-thymidine kinase gene (kindly provided by H. Kondo) (27) in the pGL3 vector (Promega, Madison, WI).…”

“…1,25-(OH) 2 D 3 was previously shown to stimulate the transcriptional activity of this promoter in COS-7 cells (15). The luciferase activity of COS-7 cells expressing VDR and transfected with the luciferase reporter vector p3P2400, which contains 2462 base pairs (nucleotides Ϫ2409 to ϩ53) of the NaPi-3 gene and all three direct repeatlike motifs (DR-A, DR-B, and DR-C) present in the promoter, increased markedly up on the exposure of the cells to 1,25-(OH) 2 D 3 .…”

“…We also characterized the promoter of the human type II phosphate transporter NaPi-3 gene with regard to transcriptional regulation by the vitamin D receptor (VDR), because we and another group demonstrated that the structures of the type II Na-P i transporters (rat NaPi-2, human NaPi-3 and mouse NaPi-7) gene are highly conserved (15,16). In addition, 1,25-(OH) 2 D 3 is known to affect renal Na-P i co-transport activity in these three species (2).…”