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Hyper-expression of Osteocalcin mRNA in Odontoblasts of Hyp Mice
1 Department of Pediatric Dentistry, Osaka University Graduate School of Dentistry, 1-8, Yamadaoka, Suita, Osaka 565-0871, Japan; and Correspondence: * corresponding author, ooshima{at}dent.osaka-u.ac.jp
The Hyp mouse is a murine homologue of human X-linked hypophosphatemia that displays hypo-mineralization in bone and dentin. In this study, we tested the hypothesis that the defect in Hyp mice leads to alterations in the expression of dentin matrix proteins that may be associated with the hypo-mineralization changes in the tissues. Quantitative RT-PCR analyses showed that expression of the osteocalcin gene in Hyp mice tooth germ samples was significantly higher than in wild-type mice, whereas the gene expressions of osteonectin, osteopontn, dentin matrix protein 1, and type I collagen in both types of mice were similar. Further, cultured Hyp mice tooth germ samples exhibited a higher expression of the osteocalcin gene than did those from wild-type mice, which was in accord with the results of our in vivo analysis. These findings suggest that osteocalcin mRNA is highly expressed in Hyp mice odontoblasts and may be associated with dentin hypo-mineralization.
Key Words: dentinogenesis • osteocalcin • real-time RT-PCR • X-linked hypophosphatemic vitamin-D-resistant rickets
X-linked hypophosphatemia (XLH) is a heritable type of vitamin-D-resistant rickets that has a relationship with hypo-mineralization in dentin, characterized by interglobular dentin, widened predentin, and irregular dentinal tubules (Abe et al., 1988). The murine Hyp homologue is used as an animal model of human XLH rickets (Eicher et al., 1976). Hypo-mineralization in the bones and dentin of Hyp mice is considered to be caused by low serum phosphate levels, rising from a defect in phosphate transport in the renal brush border membrane (Tenenhouse et al., 1978). However, a recovery of serum phosphate levels in Hyp mice, accomplished by a diet high in calcium and phosphorus, could not sufficiently improve dentin defects (Masatomi et al., 1996). Further, osteoblast-like cells derived from Hyp mice have an impaired capacity to mineralize extracellular matrix in vitro, suggesting the presence of an intrinsic defect that induces abnormal mineralization in osteoblasts (Xiao et al., 1998). The serum concentration of osteocalcin (OC), which is one of the most abundant proteins among non-collagenous bone and dentin matrix proteins, has been shown to be higher in Hyp mice than in wild-type (WT) mice (Gundberg et al., 1992). Further, Hyp mice may have some aberrations in the expression of OC in osteoblasts (Ruchon et al., 2000; Miao et al., 2001), although the presence of an aberration of OC expression in Hyp mice odontoblasts has not been reported. In this study, we tested the hypothesis that the defect in Hyp mice leads to alterations in expression of dentin matrix proteins that may be associated with the hypo-mineralization changes in the tissues.
Prior to the study, all experiments were reviewed and approved by the Osaka University Graduate School of Dentistry Intramural Animal Use and Care Committee.
Animals
Organ Culture
Total RNA Preparation and Reverse Transcription
Real-time PCR
In situ Hybridization
Statistical Analysis
On post-natal day 2, dentin calcification had started, and faint OC mRNA reactivity was observed in odontoblasts from the lower first molars (data not shown). On post-natal day 6, formation of dentin and enamelhad progressed in the lower first molars (Figs. 1a  , 1b). No morphological discrepancies were observed between Hyp and WT mice, except for the occurrence of widened predentin in Hyp mice (Fig. 1b). Furthermore, OC mRNA reactivity was observed in the odontoblasts alone, though they weakened as the cytodifferentiation and mineralization processes progressed (Figs. 1c, 1d). The distributions of OC mRNA in both types of mice were similar.
RT-PCR analysis with the primer pairs for OC, OSN, OPN, DMP1, ColI, and β-actin yielded a single band of the expected size (101, 101, 77, 101, 101, and 95 bp, respectively) for each of the amplification products (Fig. 2A ). Quantities of OSN, OPN, DMP1, and ColI mRNA in the tooth germs from Hyp mice were similar to those from WT mice (Fig. 2B). However, the OC gene expression in Hyp mice was significantly higher than that in WT mice (P < 0.05) on both post-natal days 2 and 6 (Fig. 2).
The mandibular first molars from E17d Hyp and WT mice were in the late bell stage of development, and had no polarized odontoblasts (Figs. 3a , 3b, 3d, 3e). In the tooth germs, OC expression was not observed by RT-PCR (data not shown) or in situ hybridization (Figs. 3c, 3f). In tooth germs (from both types of mice) cultured for 10 days after E17d, most of the odontoblasts were elongated and had differentiated into the secretory phase (Figs. 3g, 3h, 3j, 3k), and also showed OC mRNA reactivity during in situ hybridization (Figs. 3i, 3l). Furthermore, expression of the OC gene in explants derived from the Hyp mice specimens was significantly higher than that in those from WT mice (P < 0.01) (Fig. 4), whereas the expressions of OSN, OPN, DMP1, and ColI were similar in both types of mice.
Results from the present quantitative RT-PCR analysis revealed that the amount of OC mRNA in the tooth germs of Hyp mice was significantly higher than that in age-matched WT mice at both the initial (PN2d) and active (PN6d) phases of dentin formation. This is the first known report of an aberration in expression of the OC gene in Hyp mouse teeth. The amounts of mRNA for other matrix proteins, OSN, OPN, DMP1, and ColI in both types of mice were similar, suggesting that the increase in quantity of OC in Hyp mice is not caused by over-secretion of all dentin matrix proteins. Furthermore, OC was expressed in odontoblasts alone, and its distribution in Hyp mice was similar to that in WT mice, which led us to conclude that odontoblasts in Hyp mice show a hyper-expression of OC during crown calcification. In a previous in situ hybridization study, stronger OC mRNA signals were observed in Hyp mouse skeletons on E17d as compared with those from WT mice, although post-natal 4 day Hyp and WT mice displayed similar levels of OC mRNA (Ruchon et al., 2000). Those findings agree with the present quantitative RT-PCR results for odontoblasts. We could not detect different OC mRNA signal densities in the present in situ hybridization study. OC expression weakens as dentinogenic processes progress, such as in skeletogenesis, as shown in a previous study (Ruchon et al., 2000); therefore, it may be difficult to distinguish OC mRNA signal densities by in situ hybridization during the progression phases of skeletogenesis and dentinogenesis. In contrast to our findings, Miao et al.(2001) found that immuno-positive areas of OC reactivity, in relation to total trabecular and cortical bone areas, were decreased in Hyp mice as compared with WT mice, while the density of the band related to OC in a Western blot analysis was also weaker in Hyp. The dentin in Hyp mice teeth is narrower than that in WT mice, because of the widened predentin; thus, it has been speculated that the ratio of OC immuno-positive areas to total tooth area may decrease in Hyp mice, since dentin has been shown to be immuno-positive and predentin immuno-negative for OC (de Vries et al., 1988). In the present in vitro experiments, cultures of tooth germs derived from Hyp mice, whose morphologic features and developmental phases were similar to those of the first molars of PN2d mice in vivo, showed a higher expression of OC than those from WT mice, which was similar to the results of our in vivo analysis. These findings suggest the independence of OC hyper-expression in a hypophosphatemic state, as well as the presence of hereditary defects in the mineralization capacity of odontoblasts in Hyp mice. In the present in situ hybridization analysis, OC reactivity was first detected in polarizing odontoblasts and was found to accumulate in secretory odontoblasts, after which it weakened as the processes of cytodifferentiation and mineralization progressed, results which agree with the findings of a previous study (Bleicher et al., 1999). OC contains 3 gamma-carboxyglutamic acid residues that bind to hydroxyapatite and may inhibit hydroxyapatite crystallization (Romberg et al., 1986), although the functional significance of OC in mineralization remains to be partly clarified. These results suggest that hyper-expression of OC may delay dentin mineralization and may be one of the causes of the hypo-mineralization of dentin in Hyp mice.
This study was supported by a Grant-in-Aid for Scientific Research (No. 15791204) from the Ministry of Education, Science, Culture, and Sports of Japan. Received for publication July 24, 2003. Revision received October 26, 2004. Accepted for publication October 27, 2004.
Journal of Dental Research, Vol. 84, No. 1,
84-88 (2005) This article has been cited by other articles:
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