Yoshinobu Kaneko

Identification of Regulatory Sequences and Binding Proteins in the Type II Sodium/Phosphate Cotransporter NPT2 Gene Responsive to Dietary Phosphate

Dietary phosphate (Pi) is a most important regulator for renal Pi reabsorption. The type II sodium-dependent phosphate (Na/Pi) cotransporters (NPT2) are located at the apical membranes of renal proximal tubular cells and major functional transporters associated with renal Pi reabsorption. The consumption of a low-Pi diet induces the synthesis of NPT2, whereas a high Pi diet decreases it. The molecular mechanisms of regulation by dietary Pi are not yet known. In this report, in weaning mice fed a low-Pi diet for 4 days, the NPT2 mRNA level was increased 1.8-fold compared with mice fed a normal Pi diet. This increase was due to an elevation of the transcriptional activity. In theNPT2 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 Pi. 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 NPT2gene. Two cDNA clones that encoded protein of the mouse transcription factor μE3 (TFE3) were isolated. This is a DNA-binding protein that activates transcription through the μE3 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 Pi 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 Pi.