All-trans retinoic acid reduces the transcriptional regulation of intestinal sodium-dependent phosphate co-transporter gene (Npt2b).

Inorganic phosphate (Pi) homeostasis is regulated by intestinal absorption via type II sodium-dependent co-transporter (Npt2b) and by renal reabsorption via Npt2a and Npt2c. Although we previously reported that vitamin A-deficient (VAD) rats had increased urine Pi excretion through the decreased renal expression of Npt2a and Npt2c, the effect of vitamin A on the intestinal Npt2b expression remains unclear. In this study, we investigated the effects of treatment with all-trans retinoic acid (ATRA), a metabolite of vitamin A, on the Pi absorption and the Npt2b expression in the intestine of VAD rats, as well as and the underlying molecular mechanisms. In VAD rats, the intestinal Pi uptake activity and the expression of Npt2b were increased, but were reduced by the administration of ATRA. The transcriptional activity of reporter plasmid containing the promoter region of the rat Npt2b gene was reduced by ATRA in NIH3T3 cells overexpressing retinoic acid receptor (RAR) and retinoid X receptor (RXR). On the other hand, CCAAT/enhancer-binding proteins (C/EBP) induced transcriptional activity of the Npt2b gene. Knockdown of the C/EBP gene and a mutation analysis of the C/EBP responsible element in the Npt2b gene promoter indicated that C/EBP plays a pivotal role in the regulation of Npt2b gene transcriptional activity by ATRA. EMSA revealed that the RAR/RXR complex inhibits binding of C/EBP to Npt2b gene promoter. Together, these results suggest that ATRA may reduce the intestinal Pi uptake by preventing C/EBP activation of the intestinal Npt2b gene.

Inorganic phosphate (Pi) homeostasis in mammals is strictly controlled through the balance 2 of intestinal absorption and renal excretion/reabsorption [1,2]. The uptake of Pi is mediated by 3 type II sodium-dependent phosphate co-transporters (Npt2) in the brush-border membrane of 4 the small intestine and renal proximal tubule [1,2]. Npt2a and Npt2c are responsible for most Pi excretion. The feces were first dried at 110˚C for 12 hr then micropulverized, from which 100 14 mg samples were ashed at 250˚C for 3 hr, at 350˚C for 3 hr, and 550˚C for 24 hr as previously 15

Preparation of brush border membrane vesicles (BBMVs) and Pi uptake 2
BBMVs were prepared from rat small intestine and kidney by the Ca 2+ precipitation method as 3 described previously [3]. The uptake of 32 P into BBMVs was measured by a rapid filtration 4 technique. Ten l of vesicle suspension was added to 90 l of incubation solution that was 5 composed of 100 mM NaCl or choline chloride, 100 mM mannitol, 20 mM HEPES/Tris, and 6 0.1 mM KH 2 32 PO 4 , and the preparation was incubated at 20˚C. Na + -dependent and 7 Na + -independent Pi uptake were measured as described previously [35]. 8 9

Quantitative PCR analysis 1
Extraction of total RNA, cDNA synthesis, and real-time PCR were performed as described 2 previously [4]. The primer sequences (Npt2b, C/EBP, and C/EBP) for PCR amplification are 3 shown in Supplementary Table S1. Other primer sequences (Npt2a, Npt2c, PiT1, PiT2, and 4 -actin) were described previously [4]. Amplification products were then analyzed by a melting 5 curve, which confirmed the presence of a single PCR product in all reactions (apart from 6 negative controls). The PCR products were quantified by fit-point analysis, and results were 7 normalized to that of -actin.

Statistical analysis 12
Data were collected from more than 2 independent experiments and were reported as the means 13 ± S.E.M. Statistical analysis for 2-group comparison was performed using a 2-tailed Student's t-14 test, or one-way ANOVA with a Student-Newman post-hoc test for multi-group 15 comparison. All data analysis was performed using GraphPad Prism 5 software 16 (Graphpad Software, San Diego, CA, USA). p < 0.05 was considered statistically Previously, we reported that VAD rats had decreased Pi uptake, however, the effect of ATRA on 4 the intestinal Pi uptake remains unclear [4]. Furthermore, we did not examine the effects of 5 ATRA treatment on phosphate homeostasis in VAD rats. We made VAD rats and these rats were 6 treated with a total of 1 mg/kg body weight of ATRA. Because ATRA cannot be converted to 7 retinol, plasma levels of retinol were undetectable in both VAD and VAD+ATRA rats, which 8 was in line with our expectations (control: 511 ± 12.2 g/dl). Although the plasma Pi and Ca 9 levels were not changed among three groups of rats, ATRA treatment significantly reduced the 10 urine Pi/Cr and Ca/Cr ratios, which had been increased by VAD ( Table 1). Levels of plasma 11 Pi-regulating hormones (1,25[OH] 2 D 3 , PTH, and FGF23) did not differ between VAD and 12 control rats. In VAD rats, the plasma PTH level was not affected by ATRA, whereas the plasma 13 1,25(OH) 2 D 3 and FGF23 levels were significantly reduced by ATRA ( Table 1). The 14 Na + -dependent Pi uptake activity-but not the Na + -independent Pi uptake activity-in renal 15 BBMVs was markedly decreased in VAD rats, which was significantly increased by ATRA 16 treatment ( Figure 1A). As we previously reported, Western blotting revealed that the expression 17 levels of renal Npt2a and Npt2c proteins in VAD rats were significantly decreased in 18 Downloaded from https://portlandpress.com/biochemj/article-pdf/doi/10.1042/BCJ20190716/867328/bcj-2019-0716.pdf by guest on 15 February 2020 Biochemical Journal. This is an Accepted Manuscript. You are encouraged to use the Version of Record that, when published, will replace this version. The most up-to-date-version is available at https://doi.org/10.1042/BCJ20190716 comparison to controls. Furthermore, the decreased expression of Npt2c protein-but not 1 Npt2a-in the kidney of VAD mice was partially restored by ATRA treatment ( Figure 1B). Next, 2 we performed real-time PCR to measure the renal Npt2a and Npt2c mRNA expression. The 3 decreased mRNA levels of renal Npt2a and Npt2c in VAD rats were significantly increased by 4 ATRA treatment ( Figure 1C). Because Pi homeostasis is strictly controlled by intestinal absorption and renal excretion, we 9 next investigated whether ATRA regulates the Pi absorption and Npt2b expression in the 10 intestine. We used the jejunum for this analysis because the levels of Npt2b protein and mRNA 11 in the jejunum are higher than those in the duodenum and ileum of rats [10]. As expected, the 12 fecal Pi excretion in VAD rats was significantly decreased in comparison to controls (Table 1). 13 The Na + -dependent Pi uptake activity in intestinal BBMVs was markedly increased in VAD rats, 14 and this was significantly reduced by ATRA treatment (  the Npt2b mRNA expression in the duodenum and ileum was low, and was not changed among 6 three groups of rats ( Figure 2C). In the lung, the Npt2b mRNA expression was considerably 7 high, but was not changed among three groups. The hepatic Npt2b mRNA expression was 8 regulated by ATRA, similarly to the jejunum. ATRA did not affect the reduced expression of the 9 renal Npt2b mRNA in VAD rats. The expression of Npt2b mRNA was almost undetectable in 10 the spleen. Npt2b gene promoters to ATRA using a luciferase assay. Because we thought that the effects 17 of ATRA on the Npt2b gene expression differ among tissues, we used NIH3T3 cells for a luciferase assay to eliminate tissue specific factor. prNp2b-1.8k, phNp2b-1.5k, and 1 pmNp2b-1.7k reporter constructs, which respectively contained the promoter and exon 1 2 fragments of the rat, human, and mouse Npt2b gene, were utilized for a luciferase assay in 3 NIH3T3 cells. While ATRA had little impact on the transcriptional activity of prNp2b-1.8k 4 without the overexpression of RARRXR in NIH3T3 cells, its activity was markedly inhibited 5 by co-overexpressing RARs (RAR RARor RAR)/RXR (Figurer 3A). Furthermore, ATRA 6 additively reduced the promoter activity of rat Npt2b that was reduced by the overexpression of 7 RARsRXR in NIH3T3 cells ( Figure 3A). Next, ATRA dose-dependently reduced the rat Npt2b 8 gene promoter activity in NIH3T3 cells overexpressing RAR/RXR, as well as TTNPB, an 9 RAR-specific agonist ( Figure 3B). The phNp2b-1.5k and pmNp2b-1.  In the search for conserved putative regulatory elements, the sequence of the rat Npt2b gene 3 promoter region (−207 to +33) relative to the transcriptional start site was compared to the 4 corresponding regions of the human (−146 to +83) and mouse (−172 to +49) sequences. As 5 shown in Figure 4(A), highly conserved nucleotide sequences were determined among human, 6 rat, and mouse Npt2b gene promoters with a minimal sequence similarity of 73%. Interestingly, 7 a search for transcription factor binding motifs within this region suggested some potential 8 consensus binding site such as GC-box, C/EBP, and E-box ( Figure 4A). In order to understand 9 the molecular mechanisms underlying the responsiveness of these Npt2b genes to ATRA, 10 several reporter constructs lacking portions of the 5'-promoter region of the human, rat, and 11 mouse Npt2b genes were tested in NIH3T3 cells overexpressing RAR/RXR, with or without 12 ATRA. These deletion analyses suggest that the C/EBP binding site in the Npt2b gene promoter 13 is involved in the downregulation of the transcriptional activity of the Npt2b gene by ATRA and 14 its receptors ( Figure 4B).  overexpression of C/EBP or C/EBP each increased the promoter activity of the rat Npt2b 8 construct to more than double the original level. However, ATRA additively diminished the 9 transcriptional activity of the Npt2b gene, which was reduced by the overexpression of 10 RAR/RXR ( Figure 5C). Next, we analyzed levels of mRNA expression of C/EBP and 11 C/EBP in NIH3T3 cells using qPCR analysis with the absolute standard curve method.

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Unlike C/EBP, C/EBP gene was not expressed at all in NIH3T3 cells. Therefore, we 13 selected C/EBPfor gene knockdown experiments, but not C/EBP. We generated NIH3T3 14 cells with the knockdown of C/EBP endogenous C/EBP mRNA levels of which were 15 reduced by more than 50%, using C/EBP-specific siRNA (data not shown). As shown in 16 the reduction of the Npt2b gene promoter activity by ATRA ( Figure 5E). Next, to elucidate how 1 ATRA and its receptors downregulate the transcriptional activity of the Npt2b gene through the 2 action of C/EBP, we examined whether RAR/RXR affects the binding of C/EBP to the Npt2b 3 gene promoter by an EMSA analysis. As shown in Figure 5(F), a radiolabeled 4 oligonucleotide containing an Npt2b-C/EBP probe detected a band in nuclear extracts prepared 5 from NIH3T3 cells overexpressing C/EBPbut not RAR/RXR. Although these complexes are 6 susceptible to competition with unlabeled Npt2b-C/EBP, consensus C/EBP, and an antibody 7 against C/EBP, unlabeled mutated oligonucleotide (Mut: mutated Npt2b-C/EBP) did not 8 compete with these complexes. The formation of these complexes with C/EBP was inhibited 9 in the presence of nuclear extracts prepared from NIH3T3 cells overexpressing RAR/RXR 10 ( Figure 5F). Likewise, although in vitro synthesized C/EBPrecombinant protein bound to this 11 probe, this DNA-protein complex was inhibited by RARRXR recombinant protein and an 12 antibody against C/EBP(Supplementary Figure S4). In the present study, we have determined that the reduction of the intestinal Pi uptake activity 2 and the Npt2b expression in VAD rats were ameliorated by ATRA treatment. Furthermore, we 3 revealed that ATRA reduced the transcriptional activity of the Npt2b gene by inhibiting the 4 binding of C/EBP to the Npt2b gene promoter. Previously, we reported that VAD induced 5 hyperphosphaturia through the downregulation of the Npt2a and Npt2c gene expression in the 6 kidney, without changing the plasma Pi levels [4]. From these inconsistent results, we 7 hypothesized that ATRA might affect not only renal Pi reabsorption but also intestinal Pi 8 absorption. In this study, we found that ATRA increased the renal Pi uptake through the 9 induction of the expression of the Npt2a and Npt2c genes, the levels of which are reduced by 10 VAD, whereas the VAD-induced uptake of Pi via the Npt2b gene expression in the jejunum of 11 VAD rats was reduced by the administration of ATRA. Together, these results suggested that 12 ATRA did not change the plasma Pi levels because of the opposite effects of ATRA on the 13 intestinal Pi uptake and the renal Pi uptake. This is the first report to demonstrate the presence as a negative regulator [9,11]. In the present study, the levels of plasma 1,25(OH) 2 D 3 and 17 FGF-23 did not differ between VAD and control rats, as previously reported [4], whereas the 18 although we have investigated whether RAR could displace C/EBP from binding to a 17 canonical C/EBP binding site, an EMSA analysis showed that RAR/RXR could not bind to oligonucleotide containing an Npt2b-C/EBP probe. Surprisingly, it has been reported that 1 C/EBP could bind to the glucocorticoid receptor and RAR [44,45]. We also demonstrated 2 that RAR/RXR inhibited the binding of C/EBP to the promoter of the Npt2b gene using an 3 EMSA analysis. These data suggest that liganded RAR/RXR inhibits the binding C/EBP or  Hyperphosphatemia, which is associated with the pathophysiology of CKD, can lead to 17 vascular calcification, which has been linked to increased cardiovascular morbidity and 18 of adipogenesis [48]. In other words, the molecular mechanism through which niacin diminishes 7 the expression of Npt2b may be associated with the reduction of the C/EBP expression by 8 niacin. In the present study, we indicated that ATRA reduces the transcriptional activity of the 9 Npt2b gene by inhibiting the binding of C/EBP to the Npt2b gene promoter. That is to say, 10 ATRA and C/EBP may become therapeutic targets for the prevention of hyperphosphatemia in 11 CKD. Interestingly, elevated plasma retinol, ATRA, and retinol binding protein 4 (RBP4) levels