Identification of regulatory sequences and binding proteins in the type IIsodium/phosphate cotransporter NPT2 gene responsive to dietary phosphate

Dietary phosphate (P-i) is a most important regulator for renal P-i reabsor ption. The type II sodium-dependent phosphate (Na/P-i) cotransporters (NPT2 ) are located at the apical membranes of renal proximal tubular cells and m ajor functional transporters associated with renal P-i reabsorption. The co nsumption 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 mRN A level was increased 1.8-fold compared with mice fed a normal P-i diet. Th is increase was due to an elevation of the transcriptional activity. In the NPT2 gene promoter, the DNA footprint analysis showed that six regions wer e masked by the binding protein, but at the position -1010 to -985 upstream of the transcription start site, the binding clearly responded to the leve ls of dietary P-i, The phosphate response element (PRE) of the NPT2 gene wa s 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 t he PRE sequences in the proximal promoter of the NPT2 gene. Two cDNA clones that encoded protein of the mouse transcription factor mu E3 (TFE3) were i solated. This is a DNA-binding protein that activates transcription through the mu E3 site of the immunoglobulin heavy chain enhancer. TFE3 antibody c ompletely inhibited the binding to the PRE. The coexpression of TFE3 in COS -7 cells transfected with the NPT2 gene promoter markedly stimulated the tr anscriptional activity. The feeding of a low P-i diet significantly increas ed the amount of TFE3 mRNA in the kidney. These findings suggest that TFE3 may participate in the transcriptional regulation of the NPT2 gene by dieta ry P-i.