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.

The regulation of inorganic phosphate (P i ) 1 in the human body is controlled mainly by reabsorption in the proximal tu-bules of the kidneys (1)(2)(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)(2)(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 by parathyroid hormone, and by the dietary intake of P i (11)(12)(13). In a previous study, we investigated the cellular mechanism of the up-regulation of the NaP i cotransporter in mice induced by the intake of a low P i diet and found that the administration of a low P i diet to the mice clearly stimulated the elevation of NPT2 mRNA and protein (7). We have been studying the NPT2 gene expression using dietary P i feeding in mice. The NPT2 genes respond at the transcriptional and post-transcriptional level to an increased P i concentration in the diet (6,7). The DNA sequences responsible for the P i response in the mouse kidney, which we have designated the phosphate response elements (PRE), have been mapped. The PRE of the NPT2 gene promoter shares a region with 9 of 10 bp identity to yeast phosphate-responsive transcription factor Pho4 binding element. At the center of this region is a CACGTG motif, the core recognition site for the helix-loop-helix of transcription factors. This segment contains a 5Ј-CACGTG-3Ј motif that is sufficient to confer the transactivation by dietary P i deprivation. We also isolated a transcription factor (a helix-loop-helix protein), which has structural features very similar to those of yeast P i regulon Pho 4.

EXPERIMENTAL PROCEDURES
Animals and Diets-Male ICR mice (3 weeks after birth) were purchased from SLC (Shizuoka, Japan). They were housed in plastic cages and the animals were fed standard mouse chow (Oriental, Osaka, Japan) ad libitum. For the first week, they were fed the diet. After the period, they received a diet containing 1.2% calcium, 0.6% phosphorus and 4.4 IU vitamin D 3 /g for 5 days. Thereafter, they were fed a diet with a normal P i level (0.6%) for 7 days, given between 11:00 a.m. and 1:00 p.m. (6,7). On the 8th day, the following three groups of six mice each were established the normal P i group, mice that were chronically fed a diet containing 0.6% P i ; the low P i group, mice that received a diet containing a low percentage (0.02%) of P i ; and the high P i group, in which the mice received a high percentage (1.2%) P i diet. After 4 days of the given diet, all of the mice were anesthetized with intraperitoneal pentobarbital, and the kidneys were rapidly removed. One-half of each kidney was used for RNA isolation and the other half was used for the isolation of brush-border membrane vesicles (7).
Northern Blot Analysis--Total RNA was isolated from the kidney by extraction with acid guanidine thiocyanate/phenol/chloroform using the method of Chomczynski et al. (13). A NaP i -7 cDNA probe (2.4 kilobase pairs) was obtained from a mouse kidney cDNA library (5). The internal standard was GAPDH cDNA.
Cloning of DNA-binding Proteins Using the Yeast One-hybrid System-A reporter gene, NPT2 PRE-CYC1-HIS3, for yeast one-hybrid study (14) was constructed as follows. The Saccharomyces cerevisiae HIS3 coding region connected downstream of the UAS (upstream activating sequence)-less S. cerevisiae CYC1 promoter was constructed on a pUC19-based plasmid containing the S. cerevisiae ADE2 gene fragment. This plasmid was designed as pCHNaPi0. The five tandem copies of 36-bp double-stranded oligonucleotide, which originates from the sequences corresponding to the nucleotide positions from Ϫ1010 to Ϫ985 in human NPT2 promoter, including PRE, were inserted into upstream of the CYC1 promoter on pCHNaPi0 as its UAS sequences. The resulting plasmid, pCHNaPi5, was integrated into a ade2 locus on chromosome of S. cerevisiae strain HYP100 (MATa ura3-52 leu2-3,112 trp1⌬ his3⌬ ade2-101 lys2-801; our stock strain), and this integrant strain was designated as YBH5. The other yeast strain YBH0 was constructed by the similar integration using pCHNaPi0 instead of pCHNaPi5.
More than 2 ϫ 10 6 colonies of YBH5 transformants by mouse kidney cDNA library (mouse kidney MATCHMAKER cDNA, CLONTECH Inc., Palo Alto, CA) were screened by their growth on the histidine omission plates with 30 mM of 3-amino-1,2,4-triazole as described in the CLON-TECH manual. Eighty-five of positive colonies were obtained on the original plates, and two colonies out of them showed reproducible phenotypes on the above condition. Plasmids carrying the cDNA clones were obtained by extraction from the two yeast transformants as described (15) followed by transformation of E. coli.
Reporter Plasmid Construction-A 2450-bp BamHI-EcoRI DNA fragment containing the 5Ј-flanking region of the NPT2 gene was subcloned into pBluescript II SK(ϩ) (16). A BamHI-HindIII fragment from the resulting plasmid was then subcloned upstream of the coding region of the luciferase gene in the vector Pica-Basic (Toyo Ink, Tokyo, Japan) to generate the reporter plasmid p3P2400. Mouse transcription factor E3 (TFE3) expression vectors were constructed by subcloning on an EcoRI DNA fragment containing the full-length TFE3 cDNA into pcDL-SR␣-296 (kindly provided by N. Arai) (17). The internal control vector pCMV␤, which expresses ␤-galactosidase, was obtained from CLON-TECH. Each plasmid was purified with a plasmid kit (Qiagen, Hilden, Germany).
Cell Culture and Transient Transfection-COS-7 cells (Riken Cell Bank, Tokyo) were cultured at 37°C and under 5% CO 2 in Dulbecco's modified Eagle's medium (Sanko-junyaku, Tokyo) with 10% fetal bovine serum (Sigma). OK cells (ATCC:CRL1840) were maintained in F-12/ Dulbecco's modified Eagle's medium (1:1, v/v) containing 10% fetal bovine serum (16). Cells were transfected using the DEAE-dextran method with 5 g of the NPT2 gene promoter-luciferase reporter plasmid, 0.5 g of mouse TFE3L, or TFE3S expression plasmid and 5 g of pCMV␤ per 5 ϫ 10 5 cells, as described previously (16 -18). After transfection, the cells were incubated under standard conditions for 48 h and then exposed to various agents for 15 h. Cells were then harvested in cell lysis buffer, and the lysate was assayed for luciferase activity, ␤-galactosidase activity, and protein concentration (19).
Electrophoretic Mobility Shift Assay (EMSA) and DNase Footprint Assays-Nuclear extracts were prepared from kidney or COS-7 cells as described previously (9). The 32 P-labeled Ϫ1045 to Ϫ1035 probe and wild-type and mutant promoter probes were purified from a nondenaturing acrylamide gel. EMSA was carried out as described previously (9) with the following modifications. The binding buffer for the probes was 4 mM HEPES, pH 7.9, 10% glycerol, 50 mM KCl, 50 M EDTA, and 0.1 mM dithiothreitol, and the gel was run in a low ionic strength buffer (6.4 mM Tris, pH 7.5, 3.3 mM sodium acetate, and 1 mM EDTA) at room temperature. Supershift assays were carried out as described previously (9) using available antibodies to TFE3 from Dr. K. Calame (20). A DNase I footprinting analysis was performed using a commercial kit (SureTrac Footprinting Kit, Amersham Pharmacia Biotech, Uppsala, Sweden).
Shift-Western Blotting-Shift-Western blotting was performed according to the method of Demczuk et al. (21). This method was developed for identification and analysis of protein and DNA components of gel-shift assays. Approximately 20 g of nuclear protein was used in a preparative gel shift. After transfer onto DE81 paper, the protein-DNA complexes were identified by autoradiography. Proteins were eluted from excised filter pieces with 0.4 M acetic acid, 1 M NaCl at 65°C for 40 min and then precipitated for 30 min with 10% cold CCl 3 COOH, washed once in acetone and twice in cold 100% ethanol, vacuum-dried, resuspended, denatured in sample buffer, and subjected to SDS/10% polyacrylamide gel electrophoresis and Western blotting (21).
Nuclear Run-on Transcription Assay-Nuclei were prepared from the kidney of mice fed a low P i diet, and nuclear run-on transcription assays were performed as described previously (22). RNA was extracted and resuspended in 300 l of hybridization buffer (7% SDS, 10% polyethylene glycol (8,000), 1.5% saline/sodium phosphate/EDTA). Aliquots of RNA from treated and untreated samples were counted in a scintillation counter, and an equal number of counts from each condition (1-2 ϫ 10 6 cpm) were hybridized to linearized cDNAs (5 g) for NPT1, NPT2, GAPDH, pBluescript II DNA, which were immobilized to Hybond filters using a slot blot apparatus.
Stastical Analysis-Data are expressed as the mean Ϯ S.E. Differences between experimental groups were determined by analysis of variance, and p values Ͻ 0.05 were accepted as indicating a significant difference.

Effects of Low P i Diet on the Expression of the Type II Trans-
porter Gene-The brush-border membrane vesicles isolated from renal proximal tubules of mice fed a diet low in P i for 4 days were prepared and used for the assay of P i transport activity. The analysis of the Na ϩ -dependent P i uptake at 1 min revealed an approximately 1.7-fold increase in these mice compared with mice that received a control P i diet (p Ͻ 0.01) (Fig.  1A). As shown in Fig. 1B, the NPT2 mRNA levels were significantly increased (by about 1.8-fold) in the mice fed the low P i diet for 4 days. In addition, the amounts of the NPT2 protein FIG. 1. Effects of dietary P i on P i transport activity, type II transporter mRNA, and protein levels in mouse kidney. brushborder membrane vesicles were isolated from mice fed the test diet (a low P i diet (LP), a normal P i diet (CP), or a high P i diet (HP)) for 4 days. Na ϩ -dependent P i transport activity (A) and the amounts of NPT2 protein (C) were determined as described under "Experimental Procedures." The levels of NPT2 mRNA were determined using Northern blot analysis (B). The density of the signals was measured using an imaging analyzer (Fuji-BAS2000). The relative intensity was based on the control mRNA (GAPDH). *, p Ͻ 0.01. The data are mean Ϯ S.E. of six mice per group. D, in vitro transcription in isolated nuclei of renal cells from mice fed the low P i diet. Nuclei were isolated from the renal cortex of mice fed a normal P i diet (CP), or 4 days after the change to a low P i diet (LP), and were assayed for transcription in vivo. 32 P-Labeled transcripts were hybridized to NPT1, NPT2, and GAPDH cDNA. Lane CP, the normal P i diet; lane LP, the low P i diet.
(the 80 -90-kDa bands) were significantly increased (by about 2.5-fold for the 90-kDa band) compared with those in mice fed the control diet (Fig. 1C). In contrast, the high P i diet significantly suppressed these three parameters (transport activity, mRNA, and protein).
We studied the in vitro transcription in isolated nuclei of renal cortex cells from mice fed the low P i diet. As shown in Fig.  1D, the transcriptional activity in the NPT2 is significantly increased in the mice fed the low P i diet compared with those in the mice fed the control P i . The transcriptional activity of the type I NaP i cotransporter NPT1 gene was not significantly different between the normal P i and low P i groups. Thus, the elevation (1.8-fold) of the NPT2 mRNA levels in the mice fed the low P i diet was, at least in part, due to the increase in the transcription of the NPT2 gene.
An E Box at Ϫ1010 to Ϫ985 Plays an Important Role in the Response to Dietary P i -To clarify the protein binding region in the NPT-2 gene promoter induced by the feeding of a low P i FIG. 2. In vitro DNase I footprint analysis in NPT2 promoter. The NPT2 gene fragments were incubated with DNase I and 20 g of kidney extracts isolated from mice fed a low P i diet. Nucleotide sequence of a portion of NPT2 and exon 1. Footprints are underlined by a black bar (FP-1 to FP-6). The major transcription start site is marked by an arrow. diet, we performed DNase footprint assays using nuclear extracts from the renal cortex isolated from mice fed a low P i diet (Fig. 2). The footprints in the nuclear extract isolated from mouse renal proximal tubular cells had a series of hypersensitive sites in common and a protected region extending from Ϫ2018 to Ϫ1996 nt (FP-1), Ϫ1556 to Ϫ1535 nt (FP-2), Ϫ 1010 to Ϫ985 nt (FP-3) nt relative to the transcription start site of the NPT2 gene that may indicate the binding of a factor in mouse kidneys (Fig. 2). There was also additional protection within the regions of Ϫ779 to Ϫ757 nt (FP-4), Ϫ635 to Ϫ612 nt (FP-5) and Ϫ321 to Ϫ298 nt (FP-6), indicating that an additional factor binds the DNA in mice kidney (data not shown). In the comparison of these elements between the mice fed the low P i and high P i diets, we found that the extent of the protection at Ϫ1010 to Ϫ985 nt (FP-3) was significantly different between these two groups of animals (Fig. 3A). However, the other protected regions were not changed by the deprivation of dietary P i (data not shown). We designed the FP-3 position as PRE.
Mutation Analysis of the PRE Sequence of the NPT2 Gene Promoter-Next, we used the oligonucleotide of PRE (Ϫ1010/ Ϫ985 nt) and performed EMSA. In nuclear extract isolated from mice fed a low P i diet, the increased DNA-protein complex was observed in EMSA (Fig. 4), but the increase in the DNA/ protein complex was not observed with FP-1, FP-2, FP-4, FP-5, and FP-6 probes (data not shown). The protein-DNA complex was completely inhibited unlabeled PRE oligonucleotide.
Interestingly, the sequence of PRE was very similar to those of the P i responsible element for the promoter of the P i transporter gene PHO84 and acid phosphatase gene PHO5 in yeast (23). This element is known to be a Pho4 binding site. Pho4 is a helix-loop-helix transcription factor for the genes associated with yeast P i metabolism and binds the E box sequence 5Ј-CACGTG-3Ј. To confirm that the E box sequence is the target sequence of the putative binding protein, we performed EMSAs with oligonucleotides containing specific mutations of this sequence (Fig. 3B). The PRE-MT1 oligonucleotide, in which GG in the 5Ј half-site of the PRE sequence was changed to TT, showed binding activity similar to that of the PRE-WT (Figs. 3B and 4B). The mutation of TG in the E-box to AA (PRE-MT2) abolished the ability to interact with PRE-WT (Fig. 3B). Mutation of the second and third nucleotides (PRE-MT3) of the E-box also abolished the binding activity (Fig. 4B), suggesting that the putative binding protein recognized the sequence 5Ј-CACGTG-3Ј in the promoter of the NPT2 gene.
Functional Role of PRE in the NPT2 Promoter-We investigated the role of the PRE in the basic promoter activity of the NPT2 gene. The vector (p3P1170) was constructed with the NPT2 gene promoter (Ϫ1289 to ϩ54 nt), which contains the PRE (Ϫ110/Ϫ985 nt), linked to the luciferase reporter gene in OK cells. Transfection of p3P1170 into OK cells showed a 6-fold increase in the activity of control vector-transfected OK cells. Mutation of the E box in the PRE (p3P1170MT) markedly decreased the basic promoter activity, suggesting that the PRE is important for the expression of the NPT2 gene in OK cells (Fig. 5).
Relationship between Plasma P i Levels and PRE Binding Activity-To further analyze whether the binding activity in the PRE is regulated by plasma P i concentration, we measured plasma P i levels and PRE binding activity in the nuclei isolated from mice fed a low P i , normal P i , or a high P i diet. As shown in Fig. 6, the binding activity in the PRE-WT oligonucleotide decreased in parallel with the elevation of plasma P i levels. There was a good correlation with both parameters (PRE binding activity and plasma P i concentration).
Cloning of PRE-binding Protein by the Yeast One-hybrid System-To begin to identify some of the proteins that bind this segment, we used this DNA (nucleotide positions from Ϫ1010 to Ϫ985) as a UAS of the reporter gene's promoter in the yeast one-hybrid system and screened a mouse kidney cDNA library. The yeast strain YBH5 is histidine auxotroph and 3-amino-1,2,4-triazole-sensitive phenotypes depending on the expression of the reporter gene, NPT2 PRE-CYC1-HIS3 on its chromosome. More than 2 ϫ 10 6 colonies of YBH5 transformants by mouse kidney cDNA library which produced fusion proteins between cDNA-encoding protein and the transcriptional activation domain of GAL4 were screened. Two positive colonies reproducibly showed histidine prototroph and 30 mM 3-amino-1,2,4-triazole resistance phenotypes. The plasmids obtained from the two colonies didn't give histidine prototrophy phenotype to YBH0 strain which has a UAS-less CYC1-HIS3 reporter gene on its chromosome instead of NPT2 PRE-CYC1-HIS3. It was speculated that the cDNA on the two plasmids encode the binding proteins to the NPT2 PRE sequence on the reporter gene. One cDNA contained an almost full-length coding sequence for TFE3 and the other was a shorter partial cDNA for TFE3. TFE3 is a DNA-binding protein that activates transcription through the E3 site of the immunoglobulin heavy chain enhancer (17,18). To isolate the functional TFE3 full-length cDNA clone, we screened the mouse kidney cDNA library (from mice fed a low P i diet). We isolated TFE3L and TFE3S cDNA clones. The full-length TFE3 was termed TFE3L, and TFE3S is the isoform for the TFE3L and is truncated at the N-terminal region (transactivation domain) of TFE3L (32) (Fig. 7A).
Functional Analysis of the Effects of TFE3L and TFE3S on the Transactivation of the NPT2 Gene Promoter-To determine whether TFE3 can activate the transcription of the NPT2 gene, the TFE3L cDNA was cloned in a mammalian expression vector, and the vector was cotransfected with the NPT2 gene promoter (Ϫ1289 to ϩ54) linked to the luciferase reporter gene (p3P1170WT) in COS-7 cells. TFE3L stimulated the luciferase activity 6-fold compared with that of the control vector (Fig.  7B). TFE3S also stimulated the transcription (2.8-fold). TFE3L induced the luciferase activity in the COS-7 cells transfected with the NPT2 gene promoter with the mutation of the PRE (p3P1170MT), but the induction was lower compared with that in the p3P1170WT clone (Fig. 7B).
Binding of TFE3 to the PRE-To further confirm the specific binding to TFE3L and TFE3S in the promoter of the NPT2 gene, EMSAs were performed using a 32 P-labeled Ϫ1010 to Ϫ985 oligonucleotide as a probe (PRE-WT) and several competitor DNA oligonucleotides (PRE-MT1, PRE-MT2, and PRE-MT3) corresponding to the E box (Fig. 8). In this experiment, the mouse TFE3L expression vector was transfected into COS-7 cells, and the nuclear extract expressing mouse TFE3L was used for EMSA. The PRE-WT oligonucleotide was in competition for the binding. In addition, the PRE-MT1 oligonucleotide partially prevented the binding. The PRE-MT2 and PRE-MT3 oligonucleotides were unable to compete for the DNAprotein binding (Fig. 8, lanes 4 and 5). In addition, TFE3specific antibodies completely prevented the DNA-protein binding in an EMSA (Fig. 8, lane 6). It suggests that TFE3 OK cells were transfected with luciferase reporter genes constructed with the wild-type (p3P1170WT) or the indicated mutation (p3P1170MT) of the NPT2 promoter. Luciferase activity was determined as described previously (19). Each bar represents the mean Ϯ S.E. of five independent transfections.
FIG. 6. Relationship between the PRE binding activity and serum P i concentration. The PRE binding activity were measured in nuclei isolated from mice fed a high P i , a normal P i , or a low P i diet. Nuclear extracts were prepared from the kidneys of mice fed a low P i for 3 days, control P i diet for 5 days, or a high P i diet for 8 days. Serum P i concentration was measured as described previously (7). The binding activity was measured by densitometric analysis. Data represent a relative activity for the binding activity of the nuclei isolated from normal diet fed mouse (serum P i ϭ 6.4 mg/dl).
protein bound to the Ϫ1010 to Ϫ985 oligonucleotide.
To further confirm that the DNA-binding protein is TFE3, we performed the shift-Western analysis. The nuclear extract isolated from mice fed a low P i diet was incubated with 32 Plabeled PRE oligonucleotide and then performed the shift-Western analysis (Fig. 8B, lanes 1 and 2). This method was developed for identification and analysis of protein and DNA components of gel-shift assays. The protein-DNA complexes, separated in polyacrylamide gels, were transferred onto stacked nitrocellulose and anion-exchange membranes. The proteins bound to nitrocellulose were identified by immunoblotting (Fig. 8B, lane 2), while the DNA, which bound only to the anion-exchange membrane, was detected by autoradiography (Fig. 8B, lane 1).
This experiment clearly indicated that the DNA binding protein is TFE3. Moreover, we determined whether TFE3 an- The protein-DNA complexes, separated in polyacrylamide gels, were transferred onto stacked nitrocellulose and anion-exchange membranes. The proteins bound to nitrocellulose were identified by immunoblotting by TFE3-specific antibody (lane 2), while the DNA, which bound only to the anion-exchange membrane, was detected by autoradiography (lane 1). Nuclear proteins were prepared from the kidneys of mice fed a low P i diet. C, effect of TFE3 antibody on nuclear protein binding to PRE. The nuclear extract from kidney cortex of mice fed a low P i diet incubated with 32 P-labeled PRE-WT oligonucleotide. The TFE3 antibody was added to EMSA reaction medium. Lane 1, DNA protein complex in EMSA; lane 2, added TFE3 antibody. tibody inhibits the binding of PRE-WT and renal nuclear extract isolated from mice fed a low P i diet. As shown in Fig. 8C, TFE3 antibody completely blocked the binding of PRE-WT and the nuclear protein (Fig. 8C, lane 2).
Expression of TFE3 mRNA-To further clarify the role of TFE3 on the expression of mouse NPT2, the amounts of TFE3 mRNA were determined in the kidney of the mice fed a low P i diet. The levels of TFE3 mRNA were markedly increased 12 h after mice were changed to the low P i diet and had slightly decreased by 72 h (Fig. 9A). At 72 h, the levels of TFE3 mRNA was 2.1-fold the control (zero time) (Fig. 9B). In contrast, the levels of mouse NPT2 mRNA were increased at 48 h and significantly elevated at 72 h. DISCUSSION Chronic dietary P i restriction leads to an increased NaP i cotransport rate, along with increased NPT2 protein and mRNA (3)(4)(5)(6)(7)(8). In the present study, the transcription rate was significantly increased in nuclei isolated from the kidney cortex of mice fed the low P i diet. Recent reports suggest that the up-regulation is due to the elevation of the type II transporter synthesis by P i deprivation and is the elevation of the stability for the type II transporter mRNA, but not transcription (24,25). However, in the present study, we concluded that the elevation of the NPT2 mRNA level is due, at least in part, to an increase in the transcription rate. The difference in the findings of run-on assay may be based on the feeding schedule or animal age, because our feeding schedule used meal feeding. In this schedule, the animals can feed only during one period of 2 h in a day. This schedule is useful to investigate the effect of dietary P i on the regulation of NPT2 synthesis (6). In the low P i group, the plasma P i levels were suddenly decreased compared with those in the normal P i group. When the animals were fed a low P i diet ad libitum, the plasma P i levels were gradually decreased. The differences of the feeding schedule might have affected the regulation of NPT2 in the kidney.
In a nuclear run-on assay, we analyzed the four marker genes used: neutral basic amino acid transporter (NBAT), peptide transporters (PepT1), type I NaP i cotransporter (Npt1), and GAPDH (data not shown). In these conditions, we clearly found that all cDNA did not respond to dietary P i in a run-on assay. We also observed the increase in the stability of the NPT2 transcripts in vitro assay (26). This step may also be an important regulatory point as proposed by the Murer and Ghishan studies (24,25).
The present DNA footprinting analysis showed that six regions of the NPT2 gene promoter were masked by the nuclear protein isolated from the mice fed a low P i diet. In addition, the gel-shift mobility assay demonstrated that the binding for the element was markedly increased in the nuclei isolated from the kidney cortex of the mice fed the low P i diet. This binding protein recognized the consensus sequence 5Ј-CACGTG-3Ј known as the E box. The PRE of the NPT2 gene was further investigated by EMSA with various oligonucleotides as probes and competitors.
Interestingly, the sequence of the PRE was very similar to those in the P i -response element for the promoter of the P i transporter gene PHO84 and acid phosphatase gene PHO5 (23,27,28) in the yeast S. cerevisiae. This element is known to be a Pho4 binding site. Pho4 is a helix-loop-helix transcription factor for the genes associated with yeast P i metabolism (23,27,28). An EMSA demonstrated the formation of two complexes between oligonucleotides containing PRE and nuclear extract. The binding of the sequence with isolated nuclei was detected in the mice fed the low-P i diet, but not in those fed the normal diet. The formation of the protein-DNA complex was inhibited in the presence of an oligonucleotide containing the Pho4 binding site of the yeast PHO84 gene promoter.
We isolated cDNAs for the protein TFE3L/S by the yeast one-hybrid system. The coexpression of TFE3 markedly stimulated the promoter activity in the NPT2 gene, but not in the NPT2 gene with the mutation sequence in the E box. This suggested the possibility that TFE3L/S might bind specifically to the Ϫ1010 to Ϫ985-bp segment of the human NPT2 gene in P i deprivation. Indeed, the incubation with antibodies for mouse TFE3 completely inhibited DNA/nuclear protein binding, suggesting that protein-DNA complex in EMSA using the renal nuclear extract are TFE3.
In addition, we identified a similar PRE in the NPT2 gene of opossum kidney (29) cells. The similar sequences are located at position Ϫ2453 to Ϫ2441 relative to the transcription start site of the opossum NPT2 gene promoter. The sequence is 5Ј-CAC-NNTGC-3Ј, and TFE3 can bind this E box sequence. In addition, 25-hydroxyvitamin D 3 1␣-hydroxylase (1␣-hydroxylase) catalyzes hydroxylation, mainly in the kidney, of 25-hydroxyvitamin D 3 into 1␣,25-dihydroxyvitamin D 3 , a hormonal form of vitamin D, acting as a key enzyme of vitamin D biosynthesis (30). Dietary P i restriction increases 1␣-hydroxylase activity, while high P i diet decreases it (31). In the mouse and human 1␣-hydroxylase gene promoters, the similar sequence is located at position Ϫ660 to Ϫ655 relative to the transcription start site of the mouse 1␣-hydroxylase gene (32,33). It is possible that the PRE sequence in the 1␣-hydroxylase gene promoter may also be important for dietary P i regulation (data not shown). To further clarify the role of the PRE in the NPT2 gene promoter, we are now cloning mouse and rat NPT2 gene promoter.
What is the nature of the P i -responsive factor? Factors binding to the CACGTG motif have been shown to belong to the c-Myc family. While many members of this family have been identified, TFE3 is the predominant factor in renal extracts that binds to the PRE in vitro. A recent study demonstrated FIG. 9. Time course of TFE3 and NPT2 mRNA expression in mice fed a low P i diet. A, mice were fed a diet with a normal P i level (0.6%) for 7 days, given between 11:00 a.m. and 1:00 p.m. On the 8th day, mice received a diet containing a low percentage (0.02%) of P i . At 0, 12, 24, 48, and 72 h after receiving the diet, total RNA (20 g) was prepared as described under "Experimental Procedures." Northern blot analysis was performed using mouse TFE3 and NPT2 cDNA probes. that the TFE3 gene was a candidate for papillary cell carcinoma (34), suggesting that the gene may function as the tumor repressor in renal cells. In addition, Hua et al. (35) reported that TFE3 is an important transcription factor in at least one TGF-␤-activated signal transduction pathway. TGF-␤ is known to have widespread regulatory effects on extracellular matrix and has been implicated as a major cause of increased extracellular matrix synthesis and buildup of pathological matrix within glomeruli in experimental glomerulonephritis (36). A mechanism of the rapid therapeutic effect of a low protein diet on experimental glomerulonephritis is through suppression of TGF-␤ expression and prevention of the induction of extracellular matrix synthesis within the injured glomeruli (36). It is possible that TFE3 regulated the expression of TGF-␤ in this model. Furthermore, in uremic animals, a low P i diet prevented hyperparathyroidism, while a high P i diet produced hyperplasia of the parathyroid glands (37). These data suggest that the P i response factor TFE3 may regulate cell proliferation and matrix synthesis.
Finally, we used the yeast one-hybrid system to clone a transcription factor (TFE3) that binds to a specific sequence in the promoter of the NPT2 gene. TFE3 is known to activate transcription through the E3 site of the immunoglobulin heavy chain enhancer. The characterization of TFE3L and TFE3S-interacting transcription factors and investigations of their regulation may provide further insights into the molecular mechanisms involved in the regulation of the NPT2 gene by dietary P i .