Identification of regulatory sequences and binding proteins in the type II sodium/phosphate cotransporter NPT2 gene responsive to dietary phosphate.
Dietary phosphate (P(i)) is a most important regulator for renal P(i) reabsorption. The type II sodium-dependent phosphate (Na/P(i)) cotransporters (NPT2) are located at the apical membranes of renal proximal tubular cells and major functional transporters associated with renal P(i) reabsorption. The consumption of a low-P(i) diet induces the synthesis of NPT2, whereas a high P(i) diet decreases it. The molecular mechanisms of regulation by dietary P(i) are not yet known. In this report, in weaning mice fed a low-P(i) diet for 4 days, the NPT2 mRNA level was increased 1.8-fold compared with mice fed a normal P(i) diet. This increase was due to an elevation of the transcriptional activity. In the NPT2 gene promoter, the DNA footprint analysis showed that six regions were masked by the binding protein, but at the position -1010 to -985 upstream of the transcription start site, the binding clearly responded to the levels of dietary P(i). The phosphate response element (PRE) of the NPT2 gene was found to consist of the motif related to the E box, 5'-CACGTG-3'. A yeast one-hybrid system was used to clone a transcription factor that binds to the PRE sequences in the proximal promoter of the NPT2 gene. Two cDNA clones that encoded protein of the mouse transcription factor muE3 (TFE3) were isolated. This is a DNA-binding protein that activates transcription through the muE3 site of the immunoglobulin heavy chain enhancer. TFE3 antibody completely inhibited the binding to the PRE. The coexpression of TFE3 in COS-7 cells transfected with the NPT2 gene promoter markedly stimulated the transcriptional activity. The feeding of a low P(i) diet significantly increased the amount of TFE3 mRNA in the kidney. These findings suggest that TFE3 may participate in the transcriptional regulation of the NPT2 gene by dietary P(i).
Kido
S
S
Department of Clinical Nutrition, School of Medicine, Tokushima University, Kuramoto-Cho 3, Tokushima City 770, Japan.
Miyamoto
K
K
Mizobuchi
H
H
Taketani
Y
Y
Ohkido
I
I
Ogawa
N
N
Kaneko
Y
Y
Harashima
S
S
Takeda
E
E
D000818:Animals; D001483:Base Sequence; D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; D019556:COS Cells; D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D004247:DNA; D004268:DNA-Binding Proteins / Q000235:genetics; D004032:Diet; D005786:Gene Expression Regulation; D008297:Male; D051379:Mice; D008813:Mice, Inbred ICR; D008969:Molecular Sequence Data; D010710:Phosphates / Q000008:administration & dosage*; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012045:Regulatory Sequences, Nucleic Acid; D050608:Sodium-Phosphate Cotransporter Proteins; D050609:Sodium-Phosphate Cotransporter Proteins, Type I; D050610:Sodium-Phosphate Cotransporter Proteins, Type II; D050611:Sodium-Phosphate Cotransporter Proteins, Type III; D027981:Symporters; D014157:Transcription Factors / Q000235:genetics; D015533:Transcriptional Activation
D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; D002352:Carrier Proteins; D004268:DNA-Binding Proteins; D010710:Phosphates; D012333:RNA, Messenger; C493549:Slc17a2 protein, mouse; D050608:Sodium-Phosphate Cotransporter Proteins; D050609:Sodium-Phosphate Cotransporter Proteins, Type I; D050610:Sodium-Phosphate Cotransporter Proteins, Type II; D050611:Sodium-Phosphate Cotransporter Proteins, Type III; D027981:Symporters; C069203:TFE3 protein, human; D014157:Transcription Factors; D004247:DNA