The type II sodium-dependent phosphate co-transporters Npt2a and Npt2c play critical roles in the reabsorption of Pi by renal proximal tubular cells. The vitamin A metabolite ATRA (all-trans-retinoic acid) is important for development, cell proliferation and differentiation, and bone formation. It has been reported that ATRA increases the rate of Pi transport in renal proximal tubular cells. However, the molecular mechanism is still unknown. In the present study, we observed the effects of a VAD (vitamin A-deficient) diet on Pi homoeostasis and the expression of Npt2a and Npt2c genes in rat kidney. There was no change in the plasma levels of Pi, but VAD rats significantly increased renal Pi excretion. Renal brush-border membrane Pi uptake activity and renal Npt2a and Npt2c expressions were significantly decreased in VAD rats. The transcriptional activity of a luciferase reporter plasmid containing the promoter region of human Npt2a and Npt2c genes was increased markedly by ATRA and a RAR (retinoic acid receptor)-specific analogue TTNPB {4-[E-2-(5,6,7,8-tetrahydro-5,5,8,8-tetra-methyl-2-naphtalenyl)-1-propenyl] benzoic acid} in renal proximal tubular cells overexpressing RARs and RXRs (retinoid X receptors). Furthermore, we identified RAREs (retinoic acid-response elements) in both gene promoters. Interestingly, the half-site sequences (5′-GGTTCA-3′: −563 to −558) of 2c-RARE1 overlapped the vitamin D-responsive element in the human Npt2c gene and were functionally important motifs for transcriptional regulation of human Npt2c by ATRA and 1,25(OH)2D3 (1α,25-dihydroxyvitamin D3), in both independent or additive actions. In summary, we conclude that VAD induces hyperphosphaturia through the down-regulation of Npt2a and Npt2c gene expression in the kidney.

INTRODUCTION

Pi plays a critical role in skeletal development, mineral metabolism, and diverse cellular functions involving intermediary metabolism and energy-transfer mechanisms [1,2]. The serum Pi level is maintained within a narrow range through a complex interplay between intestinal absorption, exchange with intracellular and bone storage pools, and renal tubular reabsorption [1,2]. The crucial regulated step in Pi homoeostasis is the transport of Pi in the brush-border membrane of the renal proximal tubule. Pi transport is mediated by several Npts (sodium-dependent phosphate co-transporters), which have been classified in three categories: type I (Npt1), type II (Npt2a and Npt2c) and type III (PiT1 and PiT2) [1,2].

Npt1 is not regulated by dietary Pi. In fact, studies in Npt1cRNA-injected oocytes have revealed that it may also function as a channel for Cl and organic anions [3,4]. PiT1 and PiT2 are ubiquitously expressed and serve housekeeping functions [5]. However, it has been reported that PiT2 is expressed in the apical membrane of renal proximal tubules, is regulated by dietary Pi and may contribute to the phosphaturia of dietary potassium deficiency [6,7]. Npt2a and Npt2c are responsible for most of the Pi reabsorption in the kidney [8,9]. Npt2c was originally identified as a growth-related Pi transporter expressed in the kidney [10]. Although Npt2c has been reported to play only a minor role in renal phosphate reuptake in rodents, defects in this gene have recently been reported to underlie HHRH (hereditary hypophosphatemic rickets with hypercalciuria) [11,12]. This suggests that both Npt2a and Npt2c are important in renal reabsorption of phosphate. Npt2a and Npt2c are strictly regulated by dietary Pi and Pi-regulating hormones such as PTH (parathyroid hormone), 1,25(OH)2D3 (1α,25-dihydroxyvitamin D3) and FGF23 (fibroblast growth factor 23) [3,10,1318]. Recently, we discovered that thyroid hormones regulate Pi homoeostasis through transcriptional control of the renal Npt2a gene [19]. However, the mechanisms that regulate Pi homoeostasis, including the roles of Npt2a and Npt2c, have not been fully elucidated and the presence of other Pi-regulating factors can be assumed.

ATRA (all-trans-retinoic acid), a metabolite of vitamin A, stimulates Pi uptake in OK (opossum kidney) cells, a process regulated by a genomic mechanism [20]. Furthermore, there are several reports about the effects of vitamin A on the metabolism of Pi and calcium in rats [2123]. However, the effects of ATRA on renal Pi homoeostasis and the expression of Npt2a and Npt2c in kidney remain unclear, both in vitro and in vivo. ATRA plays an important role in the development and differentiation of epithelial tissues and immunity [2426]. The physiological actions of retinoids are mediated by specific nuclear receptors, such as RARs (retinoic acid receptors) and RXRs (retinoid X receptors). These receptors are members of the steroid/thyroid hormone nuclear receptor superfamily and act as ligand-dependent transcriptional factors. RARs and RXRs regulate the transcription of target genes by binding to RAREs (retinoic acid-response elements) composed typically of two DRs (direct repeats) of a core hexameric motif, PuG(G/T)TCA in their promoters [27,28]. The classical RARE is a 5-bp-spaced DR (DR-5), but RAR–RXR heterodimers also bind to DRs separated by 1 bp (DR-1) or 2 bp (DR-2). RXRs also bind to DR-1 as RXR–RXR homodimers [29].

In the present study, we have used VAD (vitamin A-deficient) rats to determine the effects of vitamin A on the expression of Npt2a and Npt2c in kidney. We also characterized the promoter of human Npt2a and Npt2c genes with regard to transcriptional regulation through RARs.

MATERIALS AND METHODS

VAD animals

Male Wistar rats (3 weeks old, 30–50 g) were purchased from Japan SLC (Shizuoka, Japan). All rats were randomly divided into two groups and housed in metal cages at 22 °C with a 12h light/12h dark cycle and given free access to food and drinking water. The control group was fed on an altered AIN93-G diet (Oriental Yeast, Osaka, Japan) containing 0.6% Pi and 0.6% Ca2+, whereas the VAD group was fed on an altered vitamin A-deficient AIN-93G diet (Oriental Yeast) containing 0.6% Pi and 0.6% Ca2+ for 7 weeks. Both groups of rats fasted for 24 h in metabolic cages with water ad libitum before being killed and tissue extraction. Composition of the diets is shown in Supplementary Table S1 (at http://www.BiochemJ.org/bj/429/bj4290583add.htm). Rats were maintained under pathogen-free conditions and handled in accordance with the Guidelines for Animal Experimentation of the Tokushima University School of Medicine.

Plasma and urine parameters

Concentrations of Pi, Ca2+ and Cr (creatinine) were determined as described previously [9]. Concentrations of plasma vitamin A (retinol) were determined by HPLC assays (Mitsubishi Chemical Medience, Tokyo, Japan). Concentrations of plasma 1,25(OH)2D, PTH and FGF23 were determined as described previously [9]. Metabolic cages were used for 24 h urine collection. The FEIs (fractional excretion indexes) for Pi (FEI Pi) and for Ca2+ (FEI Ca2+) were calculated as urine Pi or Ca2+/(urine Cr×plasma Pi or Ca2+).

Preparation of BBMVs (brush-border membrane vesicles) and Pi uptake

BBMVs were prepared from rat kidney by the Ca2+ precipitation method as described previously [30]. The uptake of 32P into BBMVs was measured by a rapid filtration technique. Vesicle suspension (10 μl) was added to 90 μl of incubation solution (100 mM NaCl, 100 mM mannitol, 20 mM Hepes/Tris, and 0.1 mM KH232PO4, pH 7.5), and the preparation was incubated at 20 °C. Sodium-dependent Pi uptake was measured as described previously [30].

Western blot analysis

Protein samples were heated at 95 °C for 5 min in sample buffer in the presence of 5% 2-mercaptoethanol and subjected to SDS/PAGE. The separated proteins were transferred by electrophoresis on to PVDF (Immobilon-P, Millipore). The membranes were treated with diluted anti-Npt2a antibody (1:5000) and anti-Npt2c antibody (1:500). Mouse anti-actin monoclonal antibody (Sigma) was used as an internal control. Goat anti-rabbit IgG(H+L)–horseradish peroxidase conjugate (Bio-Rad) was utilized as the secondary antibody, and signals were detected using the ECL Plus system (GE Healthcare).

Quantitative PCR analysis

Extraction of total RNA, cDNA synthesis and real-time PCR were performed as described previously [19]. The primer sequences for PCR amplification are shown in Supplementary Table S2 (at http://www.BiochemJ.org/bj/429/bj4290583add.htm). Amplification products were then analysed by a melting curve, which confirmed the presence of a single PCR product in all reactions (apart from negative controls). The PCR products were quantified by fit-point analysis, and results were normalized to that of β-actin.

Reporter plasmid construction

Luciferase reporter plasmids pNp2a-2.4k and pNp2a-600 were described previously [31]. Reporter plasmids pNp2a-1.8k, pNp2a-1008, pNp2a-800 and pNp2c-900 were constructed by PCR amplification of pNp2a-2.4k or human genomic DNA as the template using gene-specific primers (see Supplementary Table S3 at http://www.BiochemJ.org/bj/429/bj4290583add.htm). These PCR products were subcloned into a pGL-2 or pGL-3 vector (Promega). Mutated reporter plasmids pNp2a-1.8k-Mut (Mut-A), pNp2c-900-Mut1 (Mut-C1), pNp2c-900-Mut2 (Mut-C2) and pNp2c-900-Mut3 (Mut-C3) were constructed with the QuikChange® site-directed mutagenesis kit (Stratagene) using the oligonucleotides shown in Supplementary Table S3. Reporter plasmids pNp2c-600, pNp2c-400, pNp2c-150 and pNp2c+14 were cloned by the digestion of pNp2c-900 using NheI, SmaI, KpnI, SacI and BglII restriction enzymes respectively. The β-gal (β-galactosidase) expression vector pCMV-β (Clontech) was used as an internal control. Each plasmid was purified with the PureYield™ Plasmid Midiprep System (Promega).

Transfection and luciferase assay

OK-P cells were a gift from Dr Judith A. Cole (Department of Biology, University of Memphis, Memphis, TN, U.S.A.). OK-P cells were cultured as described previously [19]. Mouse RARα, RARβ and RARγ expression vectors, pSG5-RARα, pSG5-RARβ and pSG5-RARγ respectively, and mouse RXRα expression vector pSG5-RXRα were provided by Dr P. Chambon (Institute de Génétique et de Biologie Moléculaire et Cellulaire, Centre National de la Recherche Scientifique, Université Louis Pasteur, Strasbourg, France). Luciferase reporter plasmids (0.4 μg) were transfected into cells with expression vectors (0.2 μg) by Lipofectamine™ reagent (Invitrogen). The DNA–Lipofectamine™ mixture was removed after 4 h of incubation, and the cells were fed with DMEM (Dulbecco's modified Eagle's medium) containing 10% FBS (fetal bovine serum). Then cells were treated with various concentrations of ATRA (Sigma), TTNPB {4-[E-2-(5,6,7,8-tetrahydro-5,5,8,8-tetra-methyl-2-naphtalenyl)-1-propenyl] benzoic acid} (Biomol Research Laboratories) or ethanol as a vehicle control for an additional 16 h. The normalized transfection efficiency for luciferase activity was determined by co-transfecting with 0.2 μg of the β-gal expression vector pCMV-β (Stratagene). Cells were harvested in a lysis buffer supplied with the Pica-gene luciferase assay kit (Toyo Ink, Tokyo, Japan) and the lysates were assayed for luciferase activity and β-gal activity.

Extraction of nuclear protein

Nuclear extracts were prepared according to a mini-extraction protocol with minor modifications [32]. Briefly, cells were cultured on 35-mm dishes to 90% confluence and transfected with pSG5-RARβ and pSG5-RXRα. After washing, cells were harvested by scraping into ice-cold PBS and collected by centrifugation at 500 g for 5 min. Cells were lysed with buffer A (10 mM Hepes, pH 7.9, containing 10 mM KCl, 0.1 mM EDTA, 0.5% Nonidet P40, 1 mM dithiothreitol and 0.5 mM PMSF) on ice for 20 min and then centrifuged at 14 000 g for 15 min at 4 °C. The nuclear pellets were washed three times with buffer A and resuspended in buffer C (20 mM Hepes, pH 7.9, 0.5 M KCl, 1 mM EDTA, 1 mM dithiothreitol and 1 mM PMSF) for 30 min at 4 °C on a rotating wheel and then centrifuged at 14 000 g for 15 min at 4 °C.

Coupled transcription/translation assays

Each RARβ and the RXRα proteins were synthesized with the TNT® Quick Coupled Transcription/Translation System (Promega) at 30 °C for 90 min in the presence of 20 μM methionine. Generated proteins were used for EMSAs (electrophoretic mobility-shift assays).

EMSAs

EMSAs were performed as described previously [30]. Double-stranded nucleotides for 2a-RARE (2a), 2a-M, 2c-RARE1 (2c-1), 2c-M1, 2c-RARE2 (2c-2) and 2c-M2 were synthesized (see Supplementary Table S4 at http://www.BiochemJ.org/bj/429/bj4290583add.htm). Purified DNA fragments were radiolabelled with [γ-32P]ATP (110 TBq/mmol; ICN, Costa Mesa, CA, U.S.A.) using T4 polynucleotide kinase (Takara, Shiga, Japan). Prepared nuclear protein and the in vitro translated protein (15 μg and 2 μl respectively) were incubated with the radiolabelled probe in binding buffer [10 mM Tris/HCl, pH 7.5, 1 mM dithiothreitol, 1 mM EDTA, 10% glycerol, 1 mM MgCl2, 0.25 mg/ml BSA, 2.5 μg/ml salmon sperm DNA and 2 μg poly(dI-dC)·(dI-dC) (GE Healthcare)] in a final volume of 20 μl for 30 min at room temperature (20 °C). Specificity of the binding reaction was determined with a 100-fold molar excess of the indicated cold competitor oligonucleotide. DR-5 and NF-κB (nuclear factor κB) consensus oligonucleotides were purchased from Santa Cruz Biotechnology (catalogue numbers sc-2559 and sc-2505 respectively). RAR components of protein–DNA complexes were analysed using a RARβ-specific antibody (sc-552, Santa Cruz Biotechnology). The reaction mixture was then subjected to electrophoresis on a 5% polyacrylamide gel with 0.25× TBE running buffer for 2 h at 150 V. The gel was dried and analysed with a Fujix Bio-imaging analyser (BAS-1500, Fujifilm, Tokyo, Japan).

Statistical analysis

Data are expressed as means±S.E.M. Results were analysed for statistical significance using an unpaired Student's t test. P<0.05 was considered statistically significant.

RESULTS

Plasma and urine parameters in VAD rats

Plasma retinol levels were significantly decreased in VAD rats (15.6±2.59 μg/dl) compared with control rats (50.3±1.78 μg/dl, P<0.001). Although plasma Pi and Ca2+ levels did not differ between VAD and control rats, the urine Pi/Cr ratio (but not the Ca2+/Cr ratio) was significantly increased in VAD rats compared with controls (Table 1). However, plasma phosphate-regulating hormones, 1,25(OH)2D3, PTH and FGF23 levels did not differ between VAD and control rats [1,25(OH)2D3 (pg/ml): 498±18.7 and 539±33.6; PTH (pg/ml): 129±2.45 and 123±5.95; FGF23 (pg/ml): 218±2.45 and 241±9.32 respectively].

Table 1
Effects of vitamin A deficiency on plasma and urine Pi levels

Values are means±S.E.M. (n=5–7). *P<0.01.

 Control VAD 
Plasma   
 Pi (mg/dl) 5.51±0.17 5.67±0.20 
 Ca2+ (mg/dl) 9.80±0.21 10.4±0.40 
Urine   
 Pi/Cr 2.15±0.13 3.08±0.21 * 
 Ca2+/Cr 0.086±0.022 0.050±0.011 
 FEI Pi 0.39±0.02 0.54±0.03 * 
 FEI Ca2+ 0.016 0.015 
 Control VAD 
Plasma   
 Pi (mg/dl) 5.51±0.17 5.67±0.20 
 Ca2+ (mg/dl) 9.80±0.21 10.4±0.40 
Urine   
 Pi/Cr 2.15±0.13 3.08±0.21 * 
 Ca2+/Cr 0.086±0.022 0.050±0.011 
 FEI Pi 0.39±0.02 0.54±0.03 * 
 FEI Ca2+ 0.016 0.015 

Effects of VAD diets on Pi uptake and the expression of the Npt2 gene in rat kidney

Pi uptake activity in renal BBMVs was significantly decreased in VAD rats compared with control rats (Figure 1A). Western blot analysis revealed that expression of both Npt2a and Npt2c proteins was markedly decreased in VAD rats compared with controls (Figure 1B). In contrast, there was no difference in Npt1 protein expression between the groups (results not shown). Next, we performed real-time PCR analysis to measure renal Npt2a and Npt2c mRNA expression. Figure 1(C) shows that renal Npt2a and Npt2c mRNA levels were significantly decreased in VAD rats compared with control rats. However, mRNA levels of renal Npt1, PiT1 and PiT2 did not change between the two groups [VAD (relative mRNA levels compared with control 100%) compared with control; Npt1: 116±7.4 compared with 100±4.6; PiT1: 113±11.9 compared with 100±7.0; PiT2: 91±12.4 compared with 100±7.3; n=6–7].

Effects of vitamin A deficiency on Pi uptake and expression of Npt2a and Npt2c in the rat kidney

Figure 1
Effects of vitamin A deficiency on Pi uptake and expression of Npt2a and Npt2c in the rat kidney

(A) Sodium-dependent Pi transport activity was assessed by measurement of Pi uptake into renal BBMVs (n=4). Values are means±S.E.M. (n=4). *P<0.01. (B) Western blotting analysis of Npt2a and Npt2c in BBMVs. Each lane was loaded with 25 μg of BBMVs. β-actin was used as an internal control. Values are means±S.E.M. (n=4). *P<0.05. (C) Npt2a and Npt2c mRNA levels in renal BBMVs were quantified by quantitative PCR, as detailed in the Materials and methods section. β-actin was used as an internal control. Values are means±S.E.M. (n=6–7). *P<0.001, **P<0.0001.

Figure 1
Effects of vitamin A deficiency on Pi uptake and expression of Npt2a and Npt2c in the rat kidney

(A) Sodium-dependent Pi transport activity was assessed by measurement of Pi uptake into renal BBMVs (n=4). Values are means±S.E.M. (n=4). *P<0.01. (B) Western blotting analysis of Npt2a and Npt2c in BBMVs. Each lane was loaded with 25 μg of BBMVs. β-actin was used as an internal control. Values are means±S.E.M. (n=4). *P<0.05. (C) Npt2a and Npt2c mRNA levels in renal BBMVs were quantified by quantitative PCR, as detailed in the Materials and methods section. β-actin was used as an internal control. Values are means±S.E.M. (n=6–7). *P<0.001, **P<0.0001.

Transcriptional regulation of human Npt2a and Npt2c gene promoters by ATRA

We previously identified the human Npt2a gene, and characterized its promoter contained within a 2.4-kb region 5′ of exon 1 [31] (Figure 2A). In the present study, we have now determined that the 1751-bp sequence in 5′ of exon 1 and intron 1 of the human Npt2c gene (GenBank® accession number AB546724) also contains a number of possible binding sites for regulatory proteins (Supplementary Figure S1 at http://www.BiochemJ.org/bj/429/bj4290583add.htm). To understand the molecular mechanisms behind the regulation of Npt2a and Npt2c gene expression by vitamin A, we examined the responsiveness of human Npt2a and Npt2c gene promoters to ATRA by luciferase assay. The pNp2a-2.4k reporter construct containing the promoter, exon 1 fragments of the human Npt2a gene, and the pNp2c-900 reporter construct, which contained the promoter, exon 1, intron 1 and exon 2 fragments of the human Npt2c gene, were utilized in OK-P cells. ATRA stimulated the transcriptional activity of both reporter constructs in OK-P cells expressing RARα and RARβ, but not in OK-P cells expressing RARγ (Figures 2A and 2B). It has been reported that TTNPB is a RAR-specific agonist and has greater affinity for RARs than ATRA [33]. Figures 2(C) and 2(D) illustrate that the promoter activities of human Npt2a and Npt2c genes were increased by ATRA or TTNPB in a dose-dependent fashion and the stimulating activity of TTNPB on the human Npt2a and Npt2c gene promoters was approx. 10-fold greater than that of ATRA.

Activation of human Npt2a and Npt2c gene promoters by ATRA and its receptors in OK-P cells

Figure 2
Activation of human Npt2a and Npt2c gene promoters by ATRA and its receptors in OK-P cells

OK-P cells were seeded into 35-mm-diameter dishes and transfected 24 h later with pSG5-RAR (α, β, γ), pSG5-RXRα, pCMV-β and either pNp2a-2.4k (A) or pNp2c-900 (B). (C and D) OK-P cells were seeded into 35-mm-diameter dishes and transfected 24 h later with pSG5-RXRα, pCMV-β and either pNp2a-2.4k, pSG5-RARβ (C) or pNp2c-900, pSG5-RARα (D). Cells were treated with vehicle (NT, ethanol) or the indicated concentrations of either ATRA or TTNPB in 10% FBS-supplemented medium, and cell lysates were assessed for β-gal and luciferase activities 16 h later. Each point represents the average of quadruplicate analysis±S.E.M. normalized for β-gal activity. Similar results were obtained for three independent experiments. *P<0.001, **P<0.0001 compared with NT.

Figure 2
Activation of human Npt2a and Npt2c gene promoters by ATRA and its receptors in OK-P cells

OK-P cells were seeded into 35-mm-diameter dishes and transfected 24 h later with pSG5-RAR (α, β, γ), pSG5-RXRα, pCMV-β and either pNp2a-2.4k (A) or pNp2c-900 (B). (C and D) OK-P cells were seeded into 35-mm-diameter dishes and transfected 24 h later with pSG5-RXRα, pCMV-β and either pNp2a-2.4k, pSG5-RARβ (C) or pNp2c-900, pSG5-RARα (D). Cells were treated with vehicle (NT, ethanol) or the indicated concentrations of either ATRA or TTNPB in 10% FBS-supplemented medium, and cell lysates were assessed for β-gal and luciferase activities 16 h later. Each point represents the average of quadruplicate analysis±S.E.M. normalized for β-gal activity. Similar results were obtained for three independent experiments. *P<0.001, **P<0.0001 compared with NT.

Deletion analysis of human Npt2a and Npt2c gene promoters

In order to understand the molecular mechanisms underlying the responsiveness of human Npt2a and Npt2c genes to ATRA, several reporter constructs lacking portions of the 5′-promoter region of human Npt2a and Npt2c genes were tested in OK-P cells expressing RAR and RXR, with or without ATRA. Reporter constructs, pNp2a-2.4k, pNp2a-1.8k and pNp2a-1008 markedly increased luciferase activity 4–8-fold in response to ATRA, whereas pNp2a-800 and pNp2a-600 exhibited little increase (Figure 3A). These data suggested that the sequence from −1008 to −800 is important for ATRA-dependent activation of the human Npt2a gene promoter in OK-P cells. In deletion analysis of the human Npt2c gene promoter, pNp2c-900 and pNp2c-600 increased luciferase activity 3-fold in response to ATRA but pNp2c-400 increased less than 2-fold. In contrast, pNp2c-150 and pNp2c+14 exhibited no increase (Figure 3B). These data suggest that the sequence from −600 to −150 is important for ATRA-dependent activation of the human Npt2c gene promoter in OK-P cells.

Identification of RAREs in Npt2a and Npt2c genes

Figure 3
Identification of RAREs in Npt2a and Npt2c genes

Transcriptional activity of deletion constructs of human Npt2a and human Npt2c gene promoters in OK-P cells. Deletion constructs were generated by PCR amplification or restriction enzyme digestion as described in the Materials and methods section and are illustrated on the left-hand side of (A) and (B). The RAREs are shown as black boxes. Either pNp2a-2.4k, pSG5-RARβ (A) or pNp2c-900, pSG5-RARα (B) was transfected with pSG5-RXRα and pCMV-β into OK-P cells. Cells were treated with vehicle (NT, ethanol) or 100 nM ATRA in 10% FBS-supplemented medium, and cell lysates were assessed for β-gal and luciferase activities 16 h later. Each point represents the average of quadruplicate analysis±S.E.M. normalized for β-gal activity. Similar results were obtained for three independent experiments. *P<0.001, **P<0.0001 compared with NT.

Figure 3
Identification of RAREs in Npt2a and Npt2c genes

Transcriptional activity of deletion constructs of human Npt2a and human Npt2c gene promoters in OK-P cells. Deletion constructs were generated by PCR amplification or restriction enzyme digestion as described in the Materials and methods section and are illustrated on the left-hand side of (A) and (B). The RAREs are shown as black boxes. Either pNp2a-2.4k, pSG5-RARβ (A) or pNp2c-900, pSG5-RARα (B) was transfected with pSG5-RXRα and pCMV-β into OK-P cells. Cells were treated with vehicle (NT, ethanol) or 100 nM ATRA in 10% FBS-supplemented medium, and cell lysates were assessed for β-gal and luciferase activities 16 h later. Each point represents the average of quadruplicate analysis±S.E.M. normalized for β-gal activity. Similar results were obtained for three independent experiments. *P<0.001, **P<0.0001 compared with NT.

Identification of the RAREs in the Npt2a and Npt2c gene promoters

We previously reported the sequence of the 5′ flanking region and the identification of the VDRE (vitamin D-responsive element) in the human Npt2a gene [30]. The sequences of the RAREs in human Npt2a (2a-RARE: −855 to −839) and Npt2c (2c-RARE1: −578 to −558 and 2c-RARE2: −338 to −322) genes are shown in Figure 4(A). Although both 2a-RARE and 2c-RARE2 had a 5-nt spacer, 2c-RARE1 had an uncommon 6-nt spacer. We further investigated 2a-RARE, 2c-RARE1 and 2c-RARE2 by EMSA with various oligonucleotides as probes and competitors. As shown in Figure 4(B), a radiolabelled consensus DR-5 oligonucleotide probe detected a band in protein extracts prepared from OK-P cells overexpressing RARβ and RXRα. These complexes were susceptible to competition with unlabelled DR-5, 2a-RARE (2a), 2c-RARE1 (2c-1) and 2c-RARE2 (2c-2) (Figure 4B), but not with mutated probes (2a-M, 2c-M1 and 2c-M2) (see Figure 4A). The NF-κB oligonucleotide, used as a non-specific competitor, showed no inhibition. Similar results were also obtained in another competition experiment using a radiolabelled oligonucleotide containing 2a-RARE, 2c-RARE1 and 2c-RARE2, with in vitro synthesized RARβ and RXRα. As for 2a-RARE, the formation of this complex with the RAR–RXR heterodimer was inhibited in the presence of unlabelled 2a-RARE and DR5, but not unlabelled 2a-M. In addition, an antibody against RARβ inhibited complex formation with 2a-RARE (Figure 4C). Likewise, two oligonucleotides containing 2c-RARE1 and 2c-RARE2 also formed a complex, which was inhibited in the presence of unlabelled 2c-RARE1, 2c-RARE2 and DR5, but not 2c-M1 and 2c-M2. Furthermore, an antibody against RARβ inhibited complex formation with 2c-RARE1 and 2c-RARE2 (Figure 4D).

The RAR–RXR heterodimer binds to RAREs of human Npt2a and Npt2c genes

Figure 4
The RAR–RXR heterodimer binds to RAREs of human Npt2a and Npt2c genes

(A) The DNA sequences of putative RAREs in the upstream regions of human Npt2a and Npt2c genes. The nucleotide numbering represents the boundaries of RARE relative to each transcriptional start site of human Npt2a and Npt2c genes. Each of the half-sites of RAREs are indicated by left arrows. The down arrows represent the doublet altered nucleotide bases introduced by site-directed mutagenesis into each of the half-sites of the three RAREs (2a-RARE, 2c-RARE1 and 2c-RARE2). (B) EMSAs using 32P-labelled consensus DR-5 as probes. EMSAs were performed with nuclear extracts (N.E.) from OK-P cells overexpressing RARβ and RXRα, with the addition of unlabelled competitor oligonucleotides as indicated. A 100-fold molar excess of each competitor was used. (C and D) EMSAs using 32P-labelled 2a-RARE, 2c-RARE1 and 2c-RARE2 as probes. EMSAs were performed with in vitro synthesized RARβ and RXRα, with the addition of unlabelled competitor oligonucleotides as indicated. A 100-fold molar excess of each competitor was used. The location of the DNA–protein complex band is indicated by arrowheads. 2a, 2a-RARE; 2a-M, mutated 2a-RARE; 2c-1, 2c-RARE1; 2c-2, 2c-RARE2; 2c-M1, mutated 2c-RARE1; 2c-M2, mutated 2c-RARE2; αRARβ, RARβ-specific antibody; DR5, consensus DR-5; NFkB, NF-κB-binding sequence.

Figure 4
The RAR–RXR heterodimer binds to RAREs of human Npt2a and Npt2c genes

(A) The DNA sequences of putative RAREs in the upstream regions of human Npt2a and Npt2c genes. The nucleotide numbering represents the boundaries of RARE relative to each transcriptional start site of human Npt2a and Npt2c genes. Each of the half-sites of RAREs are indicated by left arrows. The down arrows represent the doublet altered nucleotide bases introduced by site-directed mutagenesis into each of the half-sites of the three RAREs (2a-RARE, 2c-RARE1 and 2c-RARE2). (B) EMSAs using 32P-labelled consensus DR-5 as probes. EMSAs were performed with nuclear extracts (N.E.) from OK-P cells overexpressing RARβ and RXRα, with the addition of unlabelled competitor oligonucleotides as indicated. A 100-fold molar excess of each competitor was used. (C and D) EMSAs using 32P-labelled 2a-RARE, 2c-RARE1 and 2c-RARE2 as probes. EMSAs were performed with in vitro synthesized RARβ and RXRα, with the addition of unlabelled competitor oligonucleotides as indicated. A 100-fold molar excess of each competitor was used. The location of the DNA–protein complex band is indicated by arrowheads. 2a, 2a-RARE; 2a-M, mutated 2a-RARE; 2c-1, 2c-RARE1; 2c-2, 2c-RARE2; 2c-M1, mutated 2c-RARE1; 2c-M2, mutated 2c-RARE2; αRARβ, RARβ-specific antibody; DR5, consensus DR-5; NFkB, NF-κB-binding sequence.

To test further whether 2a-RARE, 2c-RARE1 and 2c-RARE2 sequences were the target sequence of the candidate RARE, we determined the luciferase activity of pNp2a-1.8k-Mut (Mut-A), pNp2c-900-Mut1 (Mut-C1), pNp2c-900-Mut2 (Mut-C2) and pNp2c-900-Mut3 (Mut-C3), whose sequences and constructs are indicated in Figures 4(A) and 5(A). Figure 5(B) showed that 2a-RARE was essential for activation of the Npt2a gene promoter by ATRA, and 2c-RARE1 was more important for the Npt2c gene promoter to respond to ATRA compared with 2c-RARE2.

Mutation analysis of the human Npt2a and Npt2c gene promoters in OK-P cells

Figure 5
Mutation analysis of the human Npt2a and Npt2c gene promoters in OK-P cells

(A) Schematic diagram showing the wild-type (WT-A) human Npt2a reporter plasmid with RARE as well as reporter plasmid with mutation (Mut-A) in one half-site and wild-type (WT-C) human Npt2c reporter plasmid with RARE as well as reporter plasmid with mutation (Mut-C1, Mut-C2 and Mut-C3) in one or two half-sites, shown with an X through the mutant half-site. The mutated sequences of these reporter plasmids are shown in Figure 6(A). (B) Each human Npt2a reporter plasmid (WT-A and Mut-A), pSG5-RARβ or Npt2c reporter plasmid (WT-C, Mut-C1, Mut-C2 and Mut-C3), pSG5-RARα was transfected with pSG5-RXRα and pCMV-β into OK-P cells. Cells were treated with vehicle (NT, ethanol) or 100 nM ATRA in 10% FBS-supplemented medium, and cell lysates were assessed for β-gal and luciferase activities 16 h later. Each point represents the average of quadruplicate analysis±S.E.M. normalized for β-gal activity. Similar results were obtained in three independent experiments. *P<0.001, **P<0.0001 compared with NT.

Figure 5
Mutation analysis of the human Npt2a and Npt2c gene promoters in OK-P cells

(A) Schematic diagram showing the wild-type (WT-A) human Npt2a reporter plasmid with RARE as well as reporter plasmid with mutation (Mut-A) in one half-site and wild-type (WT-C) human Npt2c reporter plasmid with RARE as well as reporter plasmid with mutation (Mut-C1, Mut-C2 and Mut-C3) in one or two half-sites, shown with an X through the mutant half-site. The mutated sequences of these reporter plasmids are shown in Figure 6(A). (B) Each human Npt2a reporter plasmid (WT-A and Mut-A), pSG5-RARβ or Npt2c reporter plasmid (WT-C, Mut-C1, Mut-C2 and Mut-C3), pSG5-RARα was transfected with pSG5-RXRα and pCMV-β into OK-P cells. Cells were treated with vehicle (NT, ethanol) or 100 nM ATRA in 10% FBS-supplemented medium, and cell lysates were assessed for β-gal and luciferase activities 16 h later. Each point represents the average of quadruplicate analysis±S.E.M. normalized for β-gal activity. Similar results were obtained in three independent experiments. *P<0.001, **P<0.0001 compared with NT.

Additive actions of ATRA and 1,25(OH)2D3 on the transcriptional regulation of the human Npt2c gene

The existence of a consensus VDRE (−549 to −563) in the human Npt2c gene promoter has been reported previously [34]. Interestingly, the half-site sequences (5′-GGTTCA-3′: −563 to −558) of 2c-RARE1 overlapped the VDRE in the human Npt2c gene as described above. Therefore we examined the responsiveness of the human Npt2c gene promoter to ATRA and/or 1,25(OH)2D3 in OK-P cells overexpressing RAR and/or VDR (vitamin D receptor) and RXR. In Figure 6(B), ATRA and 1,25(OH)2D3 stimulated the transcriptional activity of the human Npt2c gene. Moreover, the two ligands exhibited additive action on the transcriptional regulation of the human Npt2c gene. In addition, Mut-C1, as described in Figure 5(B), abrogated the ability to respond to 1,25(OH)2D3 in a dose-dependent manner compared with pNp2c-900 (Figure 6C).

Additive action of ATRA and 1,25(OH)2D3 on the transcriptional regulation of the human Npt2c gene

Figure 6
Additive action of ATRA and 1,25(OH)2D3 on the transcriptional regulation of the human Npt2c gene

(A) The sequences of the RARE and VDRE were found in the upstream regions of the human Npt2c gene. The nucleotide numbering represents the boundaries of RARE and VDRE relative to the transcriptional start site of human Npt2c gene. The RARE (−558 to −575) and VDRE (−549 to −563) is indicated by arrows and boxes. (B) pNp2c-900 was transfected with pSG5-RARα and/or pSG5-VDR and pSG5-RXRα and pCMV-β into OK-P cells. Cells were treated with 100 nM ATRA and/or 100 nM 1,25(OH)2D3 or vehicle (NT, ethanol) in 10% FBS-supplemented medium, and cell lysates were assessed for β-gal and luciferase activities 16 h later. (C) WT-C or Mut-C1 was transfected into OK-P cells with pSG5-VDR, pSG5-RXRα and pCMV-β. Cells were treated with the indicated concentrations of 1,25(OH)2D3 or vehicle (NT, ethanol) in 10% FBS-supplemented medium, and cell lysates were assessed for β-gal and luciferase activities 16 h later. Each point represents the average of quadruplicate analysis±S.E.M. normalized for β-gal activity. Similar results were obtained for three independent experiments. *P<0.001, **P<0.0001 compared with NT.

Figure 6
Additive action of ATRA and 1,25(OH)2D3 on the transcriptional regulation of the human Npt2c gene

(A) The sequences of the RARE and VDRE were found in the upstream regions of the human Npt2c gene. The nucleotide numbering represents the boundaries of RARE and VDRE relative to the transcriptional start site of human Npt2c gene. The RARE (−558 to −575) and VDRE (−549 to −563) is indicated by arrows and boxes. (B) pNp2c-900 was transfected with pSG5-RARα and/or pSG5-VDR and pSG5-RXRα and pCMV-β into OK-P cells. Cells were treated with 100 nM ATRA and/or 100 nM 1,25(OH)2D3 or vehicle (NT, ethanol) in 10% FBS-supplemented medium, and cell lysates were assessed for β-gal and luciferase activities 16 h later. (C) WT-C or Mut-C1 was transfected into OK-P cells with pSG5-VDR, pSG5-RXRα and pCMV-β. Cells were treated with the indicated concentrations of 1,25(OH)2D3 or vehicle (NT, ethanol) in 10% FBS-supplemented medium, and cell lysates were assessed for β-gal and luciferase activities 16 h later. Each point represents the average of quadruplicate analysis±S.E.M. normalized for β-gal activity. Similar results were obtained for three independent experiments. *P<0.001, **P<0.0001 compared with NT.

DISCUSSION

In the present study, we have determined that renal Pi uptake activity and renal Npt2a and Npt2c expression are decreased in VAD rats and that the transcriptional activity of human Npt2a and Npt2c genes are up-regulated by ATRA and its receptors. VAD rats showed decreased plasma retinol levels and increased urinary Pi excretion compared with control rats, but showed no change in plasma Pi levels (Table 1). A similar observation was made by Webb et al. [35], who showed that vitamin A-deprived sheep significantly increased urinary Pi excretion without affecting plasma Pi levels. Indeed, VAD rats showed a decrease in renal Pi and both renal Npt2a and Npt2c mRNA and protein levels compared with controls (Figure 1). Recently, Villa-Bellosta et al. [6] reported that PiT2 is expressed in the apical membrane of rat renal proximal tubules and is regulated by dietary Pi. Breusegem et al. [7] reported that renal Npt2c and PiT2 might contribute to the phosphaturia of dietary potassium deficiency. However, there was no difference in Npt1, PiT1 and PiT2 mRNA expression in the two rat groups (results not shown). These results suggest that VAD induces hyperphosphaturia through down-regulated expression of Npt2a and Npt2c genes in the kidney. It is now unclear why VAD did not alter plasma Pi levels despite inducing hyperphosphaturia. In an effort to explain these data, we analysed urinary deoxypryridinoline excretion, a bone resorption marker. However, urinary deoxypyridinoline excretion was not changed (results not shown). Because it has been recently reported that Npt2b plays a critical role in intestinal Pi absorption and contributes to the maintenance of systemic Pi homoeostasis [36], further study is necessary to understand whether intestinal Pi uptake activity and Npt2b expression are affected by VAD.

Renal Pi reabsorption is regulated by a variety of Pi-regulating hormones, 1,25(OH)2D3, PTH and FGF23 [1417,28]. The effects of vitamin A on plasma 1,25(OH)2D3 and PTH, but not plasma FGF23 levels, have been previously investigated [22,23]. In the results from the present study, the levels of these hormones in VAD rats did not change compared with control rats. These observations suggest that VAD-mediated down-regulation of Npt2a and Npt2c gene expression was independent of plasma 1,25(OH)2D3, PTH and FGF23 levels (Figures 1B and 1C). In the past, it was reported that ATRA stimulates Pi uptake through the regulation of a genomic mechanism in OK cells [20]. Therefore we hypothesized that ATRA might increase the expression of Npt2a and Npt2c through regulated transcription.

We previously isolated human Npt2a, mouse Npt2a and mouse Npt2c genes [13,31,37], characterized their promoters, and identified the VDRE in the human Npt2a gene [30]. In the present study, we identified the sequence 5′ upstream of exon 1 and intron 1 in the human Npt2c gene. In addition, to understand the molecular mechanism by which ATRA or TTNPB (the RAR-specific analogue) modulated human Npt2a and Npt2c gene promoters, luciferase assays were performed in OK-P cells overexpressing RARβ and RXRα. The transcriptional activities of the two gene promoters were increased by ATRA or TTNPB in a dose-dependent manner (Figure 2).

The results shown in Figure 5 indicated that all of the RAREs we identified in these genes were essential. Barthel et al. [34] reported the existence of a consensus VDRE in the human Npt2c gene on the basis of in silico analysis. Interestingly, unlike 2a-RARE and 2c-RARE2, 2c-RARE1 had an uncommon 6-nt spacer and the half-site sequences (5′-GGTTCA-3′: −563 to −558) of 2c-RARE1 overlapped the VDRE in the human Npt2c gene as described above (Figure 6A). Both ATRA and 1,25(OH)2D3 are fat-soluble vitamins and require RXR protein to function as a partner in heterodimer formation for transcriptional action. For example, in vitro and in vivo studies have variously indicated antagonistic, additive or synergistic interactions between these vitamins [3840]. In the present study, we showed that ATRA and 1,25(OH)2D3 exhibited additive action on the transcriptional regulation of the human Npt2c gene (Figure 6B). In mutation analysis, Mut-C1 abrogated the ability to respond to not only ATRA but also 1,25(OH)2D3 compared with WT-C (Figures 5D and 6C). These results suggested that the overlapped half-site sequences (5′-GGTTCA-3′) of both 2c-RARE1 and VDRE were functionally important motifs for transcriptional regulation of human Npt2c by ATRA and 1,25(OH)2D3 in both independent or additive actions. Thus the present paper is the first report characterizing the human Npt2c promoter region and assessing the unique transcriptional regulation of the promoter by ATRA and 1,25(OH)2D3. The physiological implications of the ATRA and 1,25(OH)2D3 additive action for the transcriptional control of human Npt2c gene has not been clear, however. Our results from the present study suggest that the fat-soluble vitamins A and D may co-ordinately activate renal Pi absorption to supply the sufficient Pi required for development and growth in humans.

In summary, we have revealed that vitamin A regulates the renal expression of Npt2a and Npt2c genes in rats. Furthermore, the transcriptional mechanisms by which RAR- and RXR-regulated human Npt2a and Npt2c gene promoters were clarified in proximal tubular cells. It is now clear that ATRA regulates renal Pi homoeostasis through transcriptional control of Npt2a and Npt2c genes.

Abbreviations

     
  • ATRA

    all-trans-retinoic acid

  •  
  • BBMV

    brush-border membrane vesicle

  •  
  • β-gal

    β-galactosidase

  •  
  • Cr

    creatinine

  •  
  • 1,25(OH)2D3

    1α,25-dihydroxyvitamin D3

  •  
  • DR

    direct repeat

  •  
  • EMSA

    electrophoretic mobility-shift assay

  •  
  • FBS

    fetal bovine serum

  •  
  • FEI

    fractional excretion index

  •  
  • FGF23

    fibroblast growth factor 23

  •  
  • Npt

    sodium-dependent phosphate co-transporter

  •  
  • NF-κB

    nuclear factor κB

  •  
  • OK cell

    opossum kidney cell

  •  
  • PTH

    parathyroid hormone

  •  
  • RAR

    retinoic acid receptor

  •  
  • RARE

    retinoic acid-responsive element

  •  
  • RXR

    retinoid X receptor

  •  
  • TTNPB

    4-[E-2-(5,6,7,8-tetrahydro-5,5,8,8-tetra-methyl-2-naphtalenyl)-1-propenyl] benzoic acid

  •  
  • VAD

    vitamin A-deficient

  •  
  • VDR

    vitamin D receptor

  •  
  • VDRE

    vitamin D-responsive element

AUTHOR CONTRIBUTION

Masashi Masuda and Hironori Yamamoto were responsible for the experimental work. Mina Kozai and Takashi Uebanso helped with the EMSA and luciferase reporter assays. Mariko Ishiguro, Yuichiro Takei, Sarasa Tanaka, Otoki Nakahashi and Shoko Ikeda helped with the animal studies, including the measurement of plasma mineral levels and the RNA and protein experiments. Hironori Yamamoto, Yutaka Taketani, Hiroko Segawa, Ken-ichi Miyamoto and Eiji Takeda were responsible for the planning of the work and writing the manuscript.

We thank A. Edazawa, A. Otani, C. Urakawa, H. Kikuchi (Department of Clinical Nutrition, Institute of Health Biosciences, University of Tokushima Graduate School, Tokushima, Japan) for technical assistance and Dr M. Iwata (Laboratory of Biodefense Research, Faculty of Pharmaceutical Sciences at Kagawa Campus, Tokushima Bunri University, Tokushima, Japan), Dr T. Nikawa (Department of Nutrition, Institute of Health Biosciences, University of Tokushima Graduate School, Tokushima, Japan), Dr S. Kato (Institute of Molecular and Cellular Biosciences, University of Tokyo, Tokyo, Japan) for helpful discussions and comments.

FUNDING

This work was supported by the Ministry of Education, Science, Sports and Culture of Japan [grant numbers 16790526 (to H.Y.), 13470013 (to E.T.)]; and by the Human Nutritional Science on Stress Control 21st Century Center of Excellence Program (COE).

References

References
1
Murer
H.
Hernando
N.
Forster
I.
Biber
J.
Proximal tubular phosphate reabsorption: molecular mechanisms
Physiol. Rev.
2000
, vol. 
80
 (pg. 
1373
-
1409
)
2
Takeda
E.
Yamamoto
H.
Nashiki
K.
Sato
T.
Arai
H.
Taketani
Y.
Inorganic phosphate homeostasis and the role of dietary phosphorus
J. Cell. Mol. Med.
2004
, vol. 
8
 (pg. 
191
-
200
)
3
Verri
T.
Markovich
D.
Perego
C.
Norbis
F.
Stange
G.
Sorribas
V.
Biber
J.
Murer
H.
Cloning of a rabbit renal Na-Pi cotransporter, which is regulated by dietary phosphate
Am. J. Physiol. Renal Physiol.
1995
, vol. 
268
 (pg. 
F626
-
F633
)
4
Busch
A. E.
Schuster
A.
Waldegger
S.
Wagner
C. A.
Zempel
G.
Broer
S.
Biber
J.
Murer
H.
Lang
F.
Expression of a renal type I sodium/phosphate transporter (NaPi-1) induces a conductance in Xenopus oocytes permeable for organic and inorganic anions
Proc. Natl. Acad. Sci. U.S.A.
1996
, vol. 
93
 (pg. 
5347
-
5351
)
5
Kavanaugh
M. P.
Miller
D. G.
Zhang
W.
Law
W.
Kozak
S. L.
Kabat
D.
Miller
A. D.
Cell-surface receptors for gibbon ape leukemia virus and amphotropic murine retrovirus are inducible sodium-dependent phosphate symporters
Proc. Natl. Acad. Sci. U.S.A.
1994
, vol. 
91
 (pg. 
7071
-
7075
)
6
Villa-Bellosta
R.
Ravera
S.
Sorribas
V.
Stange
G.
Levi
M.
Murer
H.
Biber
J.
Forster
I. C.
The Na+-Pi cotransporter PiT-2 (SLC20A2) is expressed in the apical membrane of rat renal proximal tubules and regulated by dietary Pi
Am. J. Physiol. Renal Physiol.
2009
, vol. 
296
 (pg. 
F691
-
F699
)
7
Breusegem
S. Y.
Takahashi
H.
Giral-Arnal
H.
Wang
X.
Jiang
T.
Verlander
J. W.
Wilson
P.
Miyazaki-Anzai
S.
Sutherland
E.
Caldas
Y.
, et al. 
Differential regulation of the renal sodium-phosphate cotransporters NaPi-IIa, NaPi-IIc, and PiT-2 in dietary potassium deficiency
Am. J. Physiol. Renal Physiol.
2009
, vol. 
297
 (pg. 
F350
-
F361
)
8
Miyamoto
K.
Ito
M.
Tatsumi
S.
Kuwahata
M.
Segawa
H.
New aspect of renal phosphate reabsorption: the type IIc sodium-dependent phosphate transporter
Am. J. Nephrol.
2007
, vol. 
27
 (pg. 
503
-
515
)
9
Segawa
H.
Onitsuka
A.
Furutani
J.
Kaneko
I.
Aranami
F.
Matsumoto
N.
Tomoe
Y.
Kuwahata
M.
Ito
M.
Matsumoto
M.
, et al. 
Npt2a and Npt2c in mice play distinct and synergistic roles in inorganic phosphate metabolism and skeletal development
Am. J. Physiol. Renal Physiol.
2009
, vol. 
297
 (pg. 
F671
-
F678
)
10
Segawa
H.
Kaneko
I.
Takahashi
A.
Kuwahata
M.
Ito
M.
Ohkido
I.
Tatsumi
S.
Miyamoto
K.
Growth-related renal type II Na/Pi cotransporter
J. Biol. Chem.
2002
, vol. 
277
 (pg. 
19665
-
19672
)
11
Bergwitz
C.
Roslin
N. M.
Tieder
M.
Loredo-Osti
J. C.
Bastepe
M.
Abu-Zahra
H.
Frappier
D.
Burkett
K.
Carpenter
T. O.
Anderson
D.
, et al. 
SLC34A3 mutations in patients with hereditary hypophosphatemic rickets with hypercalciuria predict a key role for the sodium-phosphate cotransporter NaPi-IIc in maintaining phosphate homeostasis
Am. J. Hum. Genet.
2006
, vol. 
78
 (pg. 
179
-
192
)
12
Lorenz-Depiereux
B.
Benet-Pages
A.
Eckstein
G.
Tenenbaum-Rakover
Y.
Wagenstaller
J.
Tiosano
D.
Gershoni-Baruch
R.
Albers
N.
Lichtner
P.
Schnabel
D.
, et al. 
Hereditary hypophosphatemic rickets with hypercalciuria is caused by mutations in the sodium-phosphate cotransporter gene SLC34A3
Am. J. Hum. Genet.
2006
, vol. 
78
 (pg. 
193
-
201
)
13
Ohkido
I.
Segawa
H.
Yanagida
R.
Nakamura
M.
Miyamoto
K.
Cloning, gene structure and dietary regulation of the type-IIc Na/Pi cotransporter in the mouse kidney
Pflugers Arch.
2003
, vol. 
446
 (pg. 
106
-
115
)
14
Nashiki
K.
Taketani
Y.
Takeichi
T.
Sawada
N.
Yamamoto
H.
Ichikawa
M.
Arai
H.
Miyamoto
K.
Takeda
E.
Role of membrane microdomains in PTH-mediated down-regulation of NaPi-IIa in opossum kidney cells
Kidney Int.
2005
, vol. 
68
 (pg. 
1137
-
1147
)
15
Segawa
H.
Yamanaka
S.
Onitsuka
A.
Tomoe
Y.
Kuwahata
M.
Ito
M.
Taketani
Y.
Miyamoto
K.
Parathyroid hormone-dependent endocytosis of renal type IIc Na-Pi cotransporter
Am. J. Physiol. Renal Physiol.
2007
, vol. 
292
 (pg. 
F395
-
F403
)
16
Segawa
H.
Kawakami
E.
Kaneko
I.
Kuwahata
M.
Ito
M.
Kusano
K.
Saito
H.
Fukushima
N.
Miyamoto
K.
Effect of hydrolysis-resistant FGF23-R179Q on dietary phosphate regulation of the renal type-II Na/Pi transporter
Pflugers Arch.
2003
, vol. 
446
 (pg. 
585
-
592
)
17
Inoue
Y.
Segawa
H.
Kaneko
I.
Yamanaka
S.
Kusano
K.
Kawakami
E.
Furutani
J.
Ito
M.
Kuwahata
M.
Saito
H.
, et al. 
Role of the vitamin D receptor in FGF23 action on phosphate metabolism
Biochem. J.
2005
, vol. 
390
 (pg. 
325
-
331
)
18
Segawa
H.
Kaneko
I.
Yamanaka
S.
Ito
M.
Kuwahata
M.
Inoue
Y.
Kato
S.
Miyamoto
K.
Intestinal Na-Pi cotransporter adaption to dietary Pi content in vitamin D receptor null mice
Am. J. Physiol. Renal Physiol.
2004
, vol. 
287
 (pg. 
F39
-
F47
)
19
Ishiguro
M.
Yamamoto
H.
Masuda
M.
Kozai
M.
Takei
Y.
Sato
T.
Segawa
H.
Taketani
Y.
Arai
H.
Miyamoto
K.
Takeda
E.
Thyroid hormones regulate phosphate homeostasis through transcriptional control of the renal type IIa sodium-dependent phosphate co-transporter (Npt2a) gene
Biochem. J.
2010
, vol. 
427
 (pg. 
161
-
169
)
20
de Toledo
F. G.
Beers
K. W.
Dousa
T. P.
Pleiotropic upregulation of Na+-dependent cotransporters by retinoic acid in opossum kidney cells
Am. J. Physiol. Renal Physiol.
1997
, vol. 
273
 (pg. 
F438
-
F444
)
21
Nerurkar
M. K.
Sahasrabudhe
M. B.
Metabolism of calcium, phosphorus and nitrogen in hypervitaminosis A in young rats
Biochem. J.
1956
, vol. 
63
 (pg. 
344
-
349
)
22
Frankel
T. L.
Seshadri
M. S.
McDowall
D. B.
Cornish
C. J.
Hypervitaminosis A and calcium-regulating hormones in the rat
J. Nutr.
1986
, vol. 
116
 (pg. 
578
-
587
)
23
Hough
S.
Avioli
L. V.
Muir
H.
Gelderblom
D.
Jenkins
G.
Kurasi
H.
Slatopolsky
E.
Bergfeld
M. A.
Teitelbaum
S. L.
Effects of hypervitaminosis A on the bone and mineral metabolism of the rat
Endocrinology
1988
, vol. 
122
 (pg. 
2933
-
2939
)
24
Ross
S. A.
McCaffery
P. J.
Drager
U. C.
De Luca
L. M.
Retinoids in embryonal development
Physiol. Rev.
2000
, vol. 
80
 (pg. 
1021
-
1054
)
25
De Luca
L. M.
Retinoids and their receptors in differentiation, embryogenesis, and neoplasia
FASEB J.
1991
, vol. 
5
 (pg. 
2924
-
2933
)
26
Stephensen
C. B.
Vitamin A, infection, and immune function
Annu. Rev. Nutr.
2001
, vol. 
21
 (pg. 
167
-
192
)
27
Chambon
P.
A decade of molecular biology of retinoic acid receptors
FASEB J.
1996
, vol. 
10
 (pg. 
940
-
954
)
28
Mangelsdorf
D. J.
Evans
R. M.
The RXR heterodimers and orphan receptors
Cell
1995
, vol. 
83
 (pg. 
841
-
850
)
29
Bastien
J.
Rochette-Egly
C.
Nuclear retinoid receptors and the transcription of retinoid-target genes
Gene
2004
, vol. 
328
 (pg. 
1
-
16
)
30
Taketani
Y.
Segawa
H.
Chikamori
M.
Morita
K.
Tanaka
K.
Kido
S.
Yamamoto
H.
Iemori
Y.
Tatsumi
S.
Tsugawa
N.
, et al. 
Regulation of type II renal Na+-dependent inorganic phosphate transporters by 1,25-dihydroxyvitamin D3
J. Biol. Chem.
1998
, vol. 
273
 (pg. 
14575
-
14581
)
31
Taketani
Y.
Miyamoto
K.
Tanaka
K.
Katai
K.
Chikamori
M.
Tatsumi
S.
Segawa
H.
Yamamoto
H.
Morita
K.
Takeda
E.
Gene structure and functional analysis of the human Na+/phosphate co-transporter
Biochem. J.
1997
, vol. 
324
 (pg. 
927
-
934
)
32
Schreiber
E.
Matthias
P.
Müller
M.
Schaffner
W.
Rapid detection of octamer binding proteins with ‘mini-extracts’, prepared from a small number of cells
Nucleic Acids Res.
1989
, vol. 
17
 pg. 
6419
 
33
Pignatello
M. A.
Kauffman
F. C.
Levin
A. A.
Multiple factors contribute to the toxicity of the aromatic retinoid, TTNPB (Ro 13-7410): binding affinities and disposition
Toxicol. Appl. Pharmacol.
1997
, vol. 
142
 (pg. 
319
-
327
)
34
Barthel
T. K.
Mathern
D. R.
Whitfield
G. K.
Haussler
C. A.
Hopper
H. A.
4th
Hsieh
J. C.
Slater
S. A.
Hsieh
G.
Kaczmarska
M.
Jurutka
P. W.
, et al. 
1,25-Dihydroxyvitamin D3/VDR-mediated induction of FGF23 as well as transcriptional control of other bone anabolic and catabolic genes that orchestrate the regulation of phosphate and calcium mineral metabolism
J. Steroid Biochem. Mol. Biol.
2007
, vol. 
103
 (pg. 
381
-
388
)
35
Webb
K. E.
Jr
Mitchell
G. E.
Jr
Little
C. O.
Schmitt
G. H.
Polyuria in vitamin A-deficient sheep
J. Anim. Sci.
1968
, vol. 
27
 (pg. 
1657
-
1662
)
36
Sabbagh
Y.
O'Brien
S.
Song
W.
Boulanger
J.
Stockmann
A.
Arbeeny
C.
Schiavi
S.
Intestinal npt2b plays a major role in phosphate absorption and homeostasis
J. Am. Soc. Nephrol.
2009
, vol. 
20
 (pg. 
2348
-
2358
)
37
Yamamoto
H.
Tani
Y.
Kobayashi
K.
Taketani
Y.
Sato
T.
Arai
H.
Morita
K.
Miyamoto
K.
Pike
J. W.
Kato
S.
Takeda
E.
Alternative promoters and renal cell-specific regulation of the mouse type IIa sodium-dependent phosphate cotransporter gene
Biochim. Biophys. Acta
2005
, vol. 
1732
 (pg. 
43
-
52
)
38
Reinhardt
T. A.
Koszewski
N. J.
Omdahl
J.
Horst
R. L.
1,25-dihydroxyvitamin D3 and 9-cis-retinoids are synergistic regulators of 24-hydroxylase activity in the rat and 1,25-dihydroxyvitamin D3 alters retinoic acid metabolism in vivo
Arch. Biochem. Biophys.
1999
, vol. 
368
 (pg. 
244
-
248
)
39
Rohde
C. M.
DeLuca
H. F.
All-trans retinoic acid antagonizes the action of calciferol and its active metabolite, 1,25-dihydroxycholecalciferol, in rats
J. Nutr.
2005
, vol. 
135
 (pg. 
1647
-
1652
)
40
Wang
K.
Chen
S.
Xie
W.
Wan
Y.
Retinoids induce cytochrome P450 3A4 through RXR/VDR-mediated pathway
Biochem. Pharmacol.
2008
, vol. 
75
 (pg. 
2204
-
2213
)

Author notes

1

These authors contributed equally to this work.

Supplementary data