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RESEARCH REPORT |
Department of Pediatric Dentistry, Osaka University Graduate School of Dentistry, 1-8, Yamadaoka, Suita, Osaka 565-0871, Japan
* corresponding author, ooshima{at}dent.osaka-u.ac.jp
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: Hyp mouse • sodium-dependent phosphate co-transporter • tooth development • X-linked hypophosphatemic vitamin-D-resistant rickets • hypophosphatemia
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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X-linked hypophosphatemia (XLH) is a heritable type of vitamin-D-resistant rickets and is related to hypomineralization in dentin, which is characterized by interglobular dentin, widened predentin, and irregular dentinal tubules (Abe et al., 1998). In addition, several XLH patients have been shown to have enamel defects (Goodman et al., 1998). Previously, many types of loss-of-function mutations in PHEX (phosphate regulating gene homologies to endopeptidase on the X chromosome) were reported in XLH patients (The Hyp Consortium, 1995; Dixon et al., 1998). The Hyp mouse is a murine homolog of human XLH and has been used as an animal model for human XLH rickets (Eicher et al., 1976; Tenenhouse et al., 1978), while the Phex gene in Hyp mice has a deletion of the 3' end (Beck et al., 1997; Strom et al., 1997). In Hyp mice, a reduced expression of Npt2a and 2c occurs in the kidneys, which results in a reduction in serum phosphate concentration, due to a defect of phosphate re-absorption in the renal proximal tubules (Tenenhouse and Beck, 1996; Tenenhouse et al., 2003). Npt2b expression is also reduced in the small intestines of Hyp mice (Tenenhouse et al., 2002).
Phosphate homeostasis during the formation of teeth, which are highly calcified, remains unclear. The purpose of the present study was to evaluate the influence of a defect of Phex on the expression of Npt2 in Hyp mouse teeth, which may be important in phosphate homeostasis of developing teeth in humans.
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MATERIALS & METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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Animals
C57BL/6J Hyp mice (Hyp mice) were bred in our laboratory and used in the present study. C57BL/6J mice (wild-type mice) were purchased from CLEA JAPAN (Tokyo, Japan) and used as control animals.
RT-PCR and Northern Blot Analysis
Total RNA was prepared from the tooth germs, kidneys, and small intestines of wild-type mice at the ages of 2, 6, and 10 days, by means of an RNAgents Kit (Promega, Madison, WI, USA). First-strand cDNA synthesis from total RNA was performed by superscript III reverse transcriptase (Invitrogen, Carlsbad, CA, USA) and oligo (dT) primer. Incubation in the reverse-transcription reaction mixture was performed at 42°C for 50 min, and then at 70°C for 15 min. Next, polymerase chain-reaction (PCR) amplifications were performed with the primer pairs for Npt2a, 2b, and 2c, and for ß-actin. The nucleotide sequences of the primer pairs were 5'-GGTG ACAGTTCTGGTGCAGAGTT-3' and 5'-GGGCAGGAAAAGTCTGTG TTG-3' for Npt2a, 5'-GACCTGCCT GAACTCCAGGAC-3' and 5'-AGTCAGCCAAGCAAAGGGAA-3' for Npt2b, 5'-CTGGTGGACCGGAG TCTGAG-3' and 5'-GCGTTCTTCA GCGGAGAGAA-3' for Npt2c, and 5'-GCTCTTTTCCAGCCTTCCTT-3' and 5'-AGGTCTTTACGGATGTCAACG-3' for '-actin.
Fragments of around 400 to 450 base pairs, including Npt2a, 2b, 2c, and ß-actin cDNA, were inserted separately into PCR II TOPO vectors (Invitrogen). Digoxigenin (DIG)-labeled antisense riboprobes were then synthesized with the use of a DIG RNA Labeling Kit (Roche Diagnostics, Indianapolis, IN, USA) and each plasmid.
Samples of 20 µg of total RNA from tooth germs, kidneys, and small intestines of wild-type mice at the age of 6 days were fractioned on 1% formaldehyde-agarose gels, then transferred to Hybond N nylon membranes (Amersham Biosciences, Piscataway, NJ, USA), after which the membranes were hybridized with each DIG-labeled probe. After hybridization, the membranes were incubated with alkaline-phosphatase-conjugated anti-DIG antibody (Roche Diagnostics) and detected with the use of a DIG Nucleic Acid Detection Kit (Roche Diagnostics).
In situ Hybridization
Heads from wild-type mice at the age of 6 days (n = 4) were dissected out, embedded in paraffin, and cut into sections at a thickness of 6 µm. In situ hybridization was performed with the use of a DIG-labeled probe for Npt2b mRNA and In situ Hybridization Reagents (Nippon Gene, Tokyo, Japan), as recommended by the manufacturer. Hybridization was performed for 16 hrs at 37°C and detected with the use of a DIG Nucleic Acid Detection Kit.
Organ Culture
Tooth germs of the lower first molars were surgically removed from both Hyp and wild-type mice on embryonic day 17, and cultured for 14 days in a modified Trowell’s system in BGJb medium (Gibco, Grand Island, NY, USA) containing 100 µg/mL of ascorbic acid in a humidified atmosphere (5% CO2/95% air at 37°C) (Yamada et al., 1980). Some of the tooth germs derived from wild-type mice were cultured in the medium supplemented with 20 µM of antisense phosphorothioate-oligo-deoxynucleotide for the 15-mer cDNA sequence of mouse Phex, to arrest translation of the gene. The nucleotide sequences were 5'-GCTCCCTGTTTCTGC-3' for antisense and 5'-GCAGAAACAGGGAGC-3' for sense oligo-deoxynucleotide, which was used as a control. Some of the tooth germs were embedded in paraffin, and used for histological analysis.
Quantitative RT- PCR Analysis
Total RNA was isolated from tooth germs of the lower first molars and small intestines of wild-type and Hyp mice (n = 10 of each) at the age of 6 days, and also from cultured tooth germs (n = more than 8 of each), as described above. Following first-strand cDNA synthesis, real-time quantitative PCR assays were performed with an SYBR Green Master Mix (Applied Biosystems, Foster City, CA, USA), along with the primer pairs for Npt2b and Phex, as well as ß-actin as an internal control. The nucleotide sequences of the primer pairs were 5'-GAGACACCAAAGGGAAGACGCTC-3' and 5'-TACATCCAGGGAGCACACGA-3' for Npt2b, 5'-GAAAGACATTGGTCCCTCGG-3' and 5'-TGGCAATGGTTTT CTTCCTCTC-3' for phex, and 5'-GCTCTTTTCCAGCC TTCCTT-3' and 5'-AGGTCTTTACGGATGTCAACG-3' for '-actin. The cycle profile was as follows: 50°C for 2 min and 95°C for 1 min, followed by 40 cycles at 95°C for 15 sec and 60°C for 1 min. Thereafter, a dissociation protocol was performed as follows: 95°C for 15 sec, and 60 C for 20 sec, followed by a slow ramp (20 min) from 60°C to 95°C.
Statistical Analysis
Ratios for the quantity of mRNA for Npt2b or Phex and ß-actin were calculated, and the results were considered to reflect the relative quantities of Npt2b and Phex mRNA. Inter-group differences were estimated by a Mann-Whitney U test.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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). Northern blot analysis revealed a strong expression of Npt2b mRNA by the tooth germ specimens, whereas bands for Npt2a and 2c mRNA were not detectable (Fig. 1b). Further, Npt2a mRNA expression was observed in only the kidneys, while that of Npt2c was found in the kidneys and small intestines.
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), and ameloblasts near the cusp tip had differentiated into mature ameloblasts (Fig. 1g). Our in situ hybridization analysis showed specific signals for Npt2b mRNA in both secretory and mature ameloblasts (Figs. 1c, 1d, 1f). Odontoblasts had specific signals for Npt2b mRNA (Figs. 1c, 1f), although the signals became weaker as they matured (Fig. 1d). No signals were observed for pre-odontoblasts (Fig. 1f).
Quantitative RT-PCR analysis revealed that the amounts of Npt2b mRNA in both tooth germs and small intestines of Hyp mice at the age of 6 days were significantly lower than those from wild-type mice in vivo (Fig. 2a). Further, the expression level of Npt2b mRNA in cultured tooth germs from Hyp mice was also significantly lower in the in vitro experiments (Fig. 2b).
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). The morphological features were similar to those of tooth germs cultured in medium without oligo-deoxynucleotide (Figs. 3a, 3b). Quantitative RT-PCR analysis showed that the expression level of the Phex gene in the tooth germs cultured with antisense oligo-deoxynucleotide was reduced (Fig. 4a). Further, the amount of Npt2b mRNA in tooth germs cultured with antisense oligo-deoxynucleotide was significantly lower than that in those cultured with sense oligo-deoxynucleotide and without oligo-deoxynucleotide (Fig. 4b).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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In a previous Northern blot analysis, Npt2c mRNA was not found to be expressed in the intestines of nine-week-old mice (Ohkido et al., 2003). However, in the present study, we found evidence of Npt2c mRNA expression in the intestines of two-to 10-day-old mice. Since age-dependence was observed with regard to the level of Npt2c expression in the kidneys (Segawa et al., 2002), it may also be age-dependent in the intestines.
Previously, it has been considered that the hypomineralization seen in Hyp mouse teeth is caused by a low level of serum phosphate. However, a recovery of serum phosphate levels in Hyp mice, achieved by a diet high in calcium and phosphorus, did not adequately improve hypomineralization (Masatomi et al., 1996). Further, siblings of Hyp mice, which exhibited low serum phosphate levels but had normal phex gene expression, did not have the features characteristic of Hyp mice, such as widened predentin and interglobular dentin (Ogawa et al., 2006). Together, those results suggested the presence of factors that induce abnormal mineralization independent of serum phosphate level. The present study revealed a reduced expression of Npt2b mRNA in the developing teeth of Hyp mice in vivo, which was also observed in cultured tooth germs derived from Hyp mice in vitro. We concluded that this reduced expression is an intrinsic defect of Hyp mouse teeth.
An important genetic defect of Hyp mice is the loss-of-function mutation of Phex (Beck et al., 1997; Strom et al., 1997). Phex is mainly distributed in osteoblasts and odontoblasts (Ruchon et al., 1998), and is not found in the kidneys or intestines. Hence, it has been proposed that phosphate homeostasis defects in kidneys and intestines may be regulated by one or more circulating factors, known as phosphatonins, which are down-regulated by Phex (Saito et al., 2003; Schiavi and Kumar, 2004; Miyamoto et al., 2005). Patients with autosomal-dominant hypophosphatemic rickets (ADHR) have mutations of fibroblast growth factor 23 (FGF23) that prevent its proteolysis. XLH is characterized by deformities similar to those associated with ADHR, such as low serum phosphorus concentration, rickets, osteomalacia, short stature, and dental abscesses (ADHR Consortium, 2000). Further, serum FGF23 concentrations are elevated in XLH and Hyp mice (Yamazaki et al., 2002; Liu et al., 2003). Therefore, FGF23 is proposed as a candidate phosphatonin. Indeed, the deletion of FGF23 from Hyp mice reversed hypophosphatemia, abnormal 1,25(OH)2D3 levels, rickets, and osteomalacia (Sitara et al., 2004; Liu et al., 2006), suggesting that FGF23 is, at least partially, a cause of the renal and hard-tissue phenotypes seen in Hyp mice. Moreover, FGF23 was shown to down-regulate serum 1,25(OH)2D3 levels, as well as inorganic phosphate uptake and the expression of Npt2a, 2b, and 2c in both kidneys and intestines as a circulating factor (Saito et al., 2003; Tenenhouse et al., 2003; Miyamoto et al., 2005). In intestines, Npt2b expression is regulated by 1,25(OH)2D3 (Xu et al., 2002; Miyamoto et al., 2005). Therefore, the effects of FGF23 on the reduction of Npt2b expression in intestines may be related to the change of serum 1,25(OH)2D3 levels. In contrast, a reduced expression of Npt2b mRNA was observed in cultured tooth germs derived from Hyp mice in the present in vitro experiments. Further, tooth germs that were cultured in medium supplemented with antisense oligonucleotide for Phex also showed reduced levels of Npt2b mRNA expression. These results suggest that the reduction of Npt2b expression in Hyp mouse teeth may be independent of hormonal regulation. In previous studies, over-expression of Phex in Hyp mice rescued the bone phenotype nearly completely, while it did not have an effect on phosphate homeostasis (Bai et al., 2002; Erben et al., 2005), suggesting that different mechanisms contribute to renal phosphate wasting, as well as to hypomineralization of bone and dentin in Hyp mice. FGF23 mRNA is highly expressed in Hyp mouse bones (Liu et al., 2003). Therefore, it is possible that FGF23 also has a role as a direct inhibitory factor of mineralization in Hyp mice, although the details remain unclear. Based on these results, we speculate the presence of an inhibitory factor of Npt2b expression, which is regulated by Phex, functions in an autocrine and/or paracrine manner, and also has hormonal regulation activities.
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ACKNOWLEDGMENTS |
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Received January 23, 2006; Last revision October 19, 2006; Accepted October 23, 2006
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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ADHR Consortium (2000). Autosomal dominant hypophosphataemic rickets is associated with mutations in FGF23. Nat Genet 26:345–348.[ISI][Medline]
Bai X, Miao D, Panda D, Grady S, McKee MD, Goltzman D, et al. (2002). Partial rescue of the Hyp phenotype by osteoblast-targeted PHEX (phosphate-regulating gene with homologies to endopeptidases on the X chromosome) expression. Mol Endocrinol 16:2913–2925.
Beck L, Soumounou Y, Martel J, Krishnamurthy G, Gauthier C, Goodyer CG, et al. (1997). Pex/PEX tissue distribution and evidence for a deletion in the 3' region of the Pex gene in X-linked hypophosphatemic mice. J Clin Invest 99:1200–1209.[ISI][Medline]
Dixon PH, Christie PT, Wooding C, Trump D, Grieff M, Holm I, et al. (1998). Mutational analysis of PHEX gene in X-linked hypophosphatemia. J Clin Endocrinol Metab 83:3615–3623.
Eicher EM, Southard JL, Scriver CR, Glorieux FH (1976). Hypophosphatemia: mouse model for human familial hypophosphatemic (vitamin D-resistant) rickets. Proc Natl Acad Sci USA 73:4667–4671.
Erben RG, Mayer D, Weber K, Jonsson K, Juppner H, Lanske B (2005). Overexpression of human PHEX under the human beta-actin promoter does not fully rescue the Hyp mouse phenotype. J Bone Miner Res 20:1149–1160.[ISI][Medline]
Goodman JR, Gelbier MJ, Bennett JH, Winter GB (1998). Dental problems associated with hypophosphataemic vitamin D resistant rickets. Int J Paediatr Dent 8:19–28.[Medline]
Hilfiker H, Hattenhauer O, Traebert M, Forster I, Murer H, Biber J (1998). Characterization of a murine type II sodium-phosphate cotransporter expressed in mammalian small intestine. Proc Natl Acad Sci USA 95:14564–14569.
HYP Consortium (1995). A gene (PEX) with homologies to endopeptidases is mutated in patients with X-linked hypophosphatemic rickets. The HYP Consortium. Nat Genet 11:130–136.[ISI][Medline]
Liu S, Guo R, Simpson LG, Xiao ZS, Burnham CE, Quarles LD (2003). Regulation of fibroblastic growth factor 23 expression but not degradation by PHEX. J Biol Chem 278:37419–37426.
Liu S, Zhou J, Tang W, Jiang X, Roue DW, Quarles LD (2006). Pathogenic role of FGF23 in Hyp mice. Am J Physiol Endocrinol Metab 291:38–49.
Lundquist P, Ritchie HH, Moore K, Lundgren T, Linde A (2002). Phosphate and calcium uptake by rat odontoblast-like MRPC-1 cells concomitant with mineralization. J Bone Miner Res 17:1801–1813.[ISI][Medline]
Masatomi Y, Nakagawa Y, Kanamoto Y, Sobue S, Ooshima T (1996). Effects of serum phosphate level on formation of incisor dentine in hypophosphatemic mice. J Oral Pathol Med 25:182–187.[ISI][Medline]
Miyamoto K, Ito M, Kuwahata M, Kato S, Segawa H (2005). Inhibition of intestinal sodium-dependent inorganic phosphate transport by fibroblast growth factor 23. Ther Apher Dial 9:331–335.[ISI][Medline]
Murer H, Lotscher M, Kaissling B, Levi M, Kempson SA, Biber J (1996). Renal brush border membrane Na/Pi-cotransport: molecular aspects in PTH-dependent and dietary regulation. Kidney Int 49:1769–1773.[ISI][Medline]
Ogawa T, Onishi T, Hayashibara T, Sakashita S, Okawa R, Ooshima T (2006). Dentinal defects in Hyp mice not caused by hypophosphatemia alone. Arch Oral Biol 51:58–63.[ISI][Medline]
Ohkido I, Segawa H, Yanagida R, Nakamura M, Miyamoto K (2003). Cloning, gene structure and dietary regulation of the type-IIc Na/Pi cotransporter in the mouse kidney. Pflugers Arch 446:106–115.[ISI][Medline]
Ruchon AF, Marcinkiewicz M, Siegfried G, Tenenhouse HS, DesGroseillers L, Crine P, et al. (1998). Pex mRNA is localized in developing mouse osteoblasts and odontoblasts. J Histochem Cytochem 46:459–468.
Saito H, Kusano K, Kinosaki M, Ito H, Hirata M, Segawa H, et al. (2003). Human fibroblast growth factor-23 mutants suppress Na+-dependent phosphate co-transport activity and 1alpha, 25-dihydroxyvitamin D3 production. J Biol Chem 278:2206–2211.
Schiavi SC, Kumar R (2004). The phosphatonin pathway: new insights in phosphate homeostasis. Kidney Int 65:1–14.[ISI][Medline]
Segawa H, Kaneko I, Takahashi A, Kuwahata M, Ito M, Ohkido I, et al. (2002). Growth-related renal type II Na/Pi cotransporter. J Biol Chem 277:19665–19672.
Sitara D, Razzaque MS, Hesse M, Yoganathan S, Taguchi T, Erben RG, et al. (2004). Homozygous ablation of fibroblast growth factor-23 results in hyperphosphatemia and impaired skeletogenesis, and reverses hypophosphatemia in Phex-deficient mice. Matrix Biol 23:421–432.[ISI][Medline]
Strom TM, Francis F, Lorenz B, Boddrich A, Econs MJ, Lehrach H, et al. (1997). Pex gene deletions in Gy and Hyp mice provide mouse models for X-linked hypophosphatemia. Hum Mol Genet 6:165–171.
Tenenhouse HS, Beck L (1996). Renal Na(+)-phosphate cotransporter gene expression in X-linked Hyp and Gy mice. Kidney Int 49:1027–1032.[ISI][Medline]
Tenenhouse HS, Scriver CR, McInnes RR, Glorieux FH (1978). Renal handling of phosphate in vivo and in vitro by the X-linked hypophosphatemic male mouse: evidence for a defect in the brush border membrane. Kidney Int 14:236–244.[ISI][Medline]
Tenenhouse HS, Gauthier C, Martel J, Hoenderop JG, Hartog A, Meyer MH, et al. (2002). Na/P(i) cotransporter (Npt2) gene disruption increases duodenal calcium absorption and expression of epithelial calcium channels 1 and 2. Pflugers Arch 444:670–676.[ISI][Medline]
Tenenhouse HS, Martel J, Gauthier C, Segawa H, Miyamoto K (2003). Differential effects of Npt2a gene ablation and X-linked Hyp mutation on renal expression of Npt2c. Am J Physiol Renal Physiol 285:F1271–F1278.
Traebert M, Hattenhauer O, Murer H, Kaissling B, Biber J (1999). Expression of type II Na-P(i) cotransporter in alveolar type II cells. Am J Physiol 277(5 Pt 1):L868–L873.
Xu H, Bai L, Collins JF, Ghishan FK (2002). Age-dependent regulation of rat intestinal type IIb sodium-phosphate cotransporter by 1,25-(OH)(2) vitamin D(3). Am J Physiol Cell Physiol 282:C487–C493.
Xu Y, Yeung CH, Setiawan I, Avram C, Biber J, Wagenfeld A, et al. (2003). Sodium-inorganic phosphate cotransporter NaPi-IIb in the epididymis and its potential role in male fertility studied in a transgenic mouse model. Biol Reprod 69:1135–1141.
Yamada M, Bringas P Jr, Grodin M, MacDougall M, Cummings E, Grimmett J, et al. (1980). Chemically-defined organ culture of embryonic mouse tooth organs: morphogenesis, dentinogenesis and amelogenesis. J Biol Buccale 8:127–139.[ISI][Medline]
Yamazaki Y, Okazaki R, Shibata M, Hasegawa Y, Satoh K, Tajima T, et al. (2002). Increased circulatory level of biologically active full-length FGF-23 in patients with hypophosphatemic rickets/osteomalacia. J Clin Endocrinol Metab 87:4957–4960.
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