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

Abstract

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 E3 (TFE3) were isolated. This is a DNA-binding protein that activates transcription through the E3 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 .The regulation of inorganic phosphate (P i ) 1 in the human body is controlled mainly by reabsorption in the proximal tubules of the kidneys (1-3). Apical Na ϩ -dependent phosphate (NaP i ) cotransport is central to the renal proximal tubular reabsorption of P i (2, 3). Studies of isolated kidney tubules and of brush-border membranes have demonstrated that the physiological regulation of proximal tubule P i transport involves complex hormonal and metabolic factors that affect the activity or expression of the transporter molecules (1-3). A major regulator of the NaP i cotransporter is dietary P i (4 -7). Dietary P i restriction is associated with an adaptive increase of the overall proximal tubular capacity to reabsorb P i (4 -7). Alterations of the dietary intake of P i lead to an adaptation of renal P i transport activity independent of extrarenal factors such as parathyroid hormone, growth hormone, and vitamin D (1-3, 9, 10). The molecular mechanisms of this adaptation are unknown.Three types of Na ϩ -dependent P i cotransporter have been isolated from a kidney library (11,12). Recent studies suggest that the type II transporters (NPT2) may play an important role in P i homeostasis in the kidney, that they are controlled b...

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“…Thus it will be possible to conduct further in vivo studies using deletion and mutational constructs based on this promoter region to try to ascertain the cis-acting, and subsequently trans-acting, elements responsible for specifying proximal tubule expression and thereby gain insight with respect to differentiation of specialized nephron segments, during development, or in response to renal injury (such as in recovery from acute tubule necrosis). Although we and others have conducted such deletion and mutation analyses in cell culture (7,10,22,27), the transgenic model provides a superior opportunity for such analysis in an in vivo system.…”