THE ANATOMICAL RECORD 291:183–190 (2008) Involvement of the Klotho Protein in Dentin Formation and Mineralization HIRONOBU SUZUKI,1* NORIO AMIZUKA,2 KIMIMITSU ODA,2,3 MASAKI NODA,4 HAYATO OHSHIMA,1 AND TAKEYASU MAEDA2,5 1 Divisions of Anatomy and Cell Biology of the Hard Tissue, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan 2 Center for Transdisciplinary Research, Niigata University, Niigata, Japan 3 Divisions of Oral Biochemistry, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan 4 Department of Molecular Pharmacology, Medical Research Institute, Tokyo Medical and Dental University, Tokyo, Japan 5 Divisions of Oral Anatomy, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan ABSTRACT Klotho-deﬁcient mice exhibit multiple pathological conditions resembling human aging. Our previous study showed alterations in the distribution of osteocytes and in the bone matrix synthesis in klotho-deﬁcient mice. Although the bone and tooth share morphological features such as mineralization processes and components of the extracellular matrix, little information is available on how klotho deletion inﬂuences tooth formation. The present study aimed to elucidate the altered histology of incisors of klothodeﬁcient mice–comparing the ﬁndings with those from their wild-type littermates, by using immunohistochemistry for alkaline phosphatase (ALP), osteopontin, and dentin matrix protein-1 (DMP-1), terminal deoxynucleotidyl transferase-mediated deoxyuridinetriphosphate nick end-labeling (TUNEL) detection for apoptosis, and electron probe microanalyzer (EPMA) analysis on calcium (Ca), phosphate (P), and magnesium (Mg). Klotho-deﬁcient incisors exhibited disturbed layers of odontoblasts, predentin, and dentin, resulting in an obscure dentin-predentinal border at the labial region. Several odontoblast-like cells without ALP activity were embedded in the labial dentin matrix, and immunopositivity for DMP-1 and osteopontin was discernible in the matrix surrounding these embedded odontoblast-like cells. TUNEL detection demonstrated an apoptotic reaction in the embedded odontoblast-like cells and pulpal cells in the klotho-deﬁcient mice. EPMA revealed lower concentrations of Ca, P, and Mg in the klotho-deﬁcient dentin, except for the dentin around abnormal odontoblast-like cells. These ﬁndings suggest the involvement of the klotho gene in dentinogenesis and its mineralization. Anat Rec, 291:183–190, 2008. Ó 2007 Wiley-Liss, Inc. Key words: klotho; incisor; dentin The klotho gene encodes secretory and membranebound forms of its protein, respectively, containing one and two b-glucosidase–like domains in both humans (Matsumura et al., 1998) and mice (Shiraki-Iida et al., 1998). Disruptions in the klotho gene are linked to multiple aging phenotypes and age-related disorders (Kuro-o et al., 1997; Korosu et al., 2005; Masuda et al., 2005). Deﬁciency of the klotho gene in mice causes osteoporosis, skin atrophy, ectopic calciﬁcation, pulmonary emphysema, gonadal dysplasia, and defective hearing, which are symptoms of aging in humans (Kuro-o et al., 1997). Ó 2007 WILEY-LISS, INC. Grant sponsor: Niigata University. *Correspondence to: Hironobu Suzuki, Division of Anatomy and Cell Biology of the Hard Tissue, Niigata University Graduate School of Medical and Dental Sciences, 2-5274, Gakkochodori, Niigata, 951-8514, Japan. Fax: 81-25-227-0804. E-mail: firstname.lastname@example.org Received 7 August 2007; Accepted 25 October 2007 DOI 10.1002/ar.20630 Published online 18 December 2007 in Wiley InterScience (www. interscience.wiley.com). 184 SUZUKI ET AL. Because klotho severely inﬂuences many organs without the klotho protein, this protein has been regarded as a circulating factor related to human aging (Xiao et al., 2004). Recently, a klotho protein and ﬁbroblast growth factor (FGF) receptor (FGFR) construct was developed as a speciﬁc receptor for FGF23 (Urakawa et al., 2006). The klotho protein directly binds FGFR, with the klothoFGFR complex binding to FGF23 with a higher afﬁnity than FGFR or the klotho protein alone (Kurosu et al., 2006). More recently, alpha-klotho has been reported to be involved in calcium homeostasis by mediating increased activity for Na1, K1-ATPase (Imura et al., 2007). Given the above, it appears that the klotho protein has a distinct function besides that of being an anti-aging factor. Dentinogenesis imperfecta (DGI) and dentin dysplasia (DD) are allelic disorders affecting dentin formation. Many researchers have reported a relation between DGI/DD and mutations in the dentin sialophosphoprotein (DSPP) gene in humans (Xiao et al., 2001; Zhang et al., 2001; Rajpar et al., 2002; Dong et al., 2005; Beattie et al., 2006). Gene mutations including DSPP (Sreenath et al., 2003; Ye et al., 2004; Lopez Franco et al., 2005; Goldberg et al., 2006) and gene overexpression (Thyagarajan et al., 2001; Savage et al., 2006) inﬂuence dentin formation in mice. Our previous study demonstrated that a klotho deﬁciency caused irregular distributions of osteocytes and bone matrix proteins as well as the accelerated aging of bone cells (Suzuki et al., 2005). Osteoblasts and odontoblasts share features: both cell types synthesize collagenous and noncollagenous proteins, promote mineralization, and differentiate from mesenchymal cells. Recently, a homozygous missense mutation in human klotho causes severe tumoral calcinosis (Ichikawa et al., 2007), which is rare autosomal recessive metabolic disorder characterized by extraosseous calcium phosphate deposition in the skin, muscle, joints, and visceral organs (Metzker et al., 1988). Although one may easily infer that the klotho gene is somehow involved in dentin formation in mouse, this hypothesis is yet to be proven. This study aimed to identify the morphological alterations in incisors of klotho-deﬁcient (klotho2/2) mice by using immunohistochemical technique. In addition, an electron probe microanalyzer (EPMA) analysis was performed for demonstrating the elemental mapping of calcium (Ca), phosphorus (P), and magnesium (Mg), major components of the mineralized dentin matrix. MATERIALS AND METHODS Tissue Preparation Animal experiments followed the Guiding Principles for the Care and Use of Animals, as approved by Niigata University. Klotho2/2 mice and wild-type littermates (aged 6 weeks old, n 5 3 each) were purchased from Japan CLEA (Tokyo, Japan). Under anesthesia (intraperitoneal injection of chloral hydrate, 400 mg/100 g of body weight), klotho2/2 mice and their wild-type littermates were perfused transcardially with 4% paraformaldehyde in a 0.1 M phosphate buffer (PB: pH 7.4). Mandibles were immediately removed and immersed in the same ﬁxative for an additional 8 hr at 48C. The specimens were decalciﬁed with 10% ethylenediaminetetraacetic acid–2Na solutions, and were dehydrated with ascending concentrations of ethanol before parafﬁn embedding. Serial sagittal sections were cut at a thickness of 4 mm. Immunohistochemistry for Alkaline Phosphatase, Osteopontin, Dentin Matrix Protein-1, and Type I Collagen Deparafﬁnized sections were primarily incubated for 2–3 hr at room temperature with rabbit polyclonal antisera against alkaline phosphatase (ALP; Oda et al., 1999), osteopontin (OPN; LSL, Tokyo, Japan), dentin matrix protein-1 (DMP-1, Takara BIO Inc., Ohtsu, Japan), or type I collagen (Cosmo BIO, Tokyo, Japan), diluted at 1:250, 1:2,500, 1:500, and 1:3,500, respectively. For DMP1 detection, sections were pretreated with 3% trypsin for 30 min. Sections were then reacted with horseradish peroxidase (HRP) -conjugated anti-rabbit IgG (Amersham Bui., Tokyo, Japan). The immunoreaction was visualized by incubation with 0.04% 3-30 -diaminobenidine and 0.003% hydrogen peroxidase. The sections were faintly counterstained with 0.03% methyl green. Detection of Apoptosis Reaction We used the ‘‘TACS 2TdT-Blue Label In Situ Apoptosis Detection Kit’’ (TREVIGEN Inc., Gaithersburg, MD) for the terminal deoxynucleotidyl transferase-mediated deoxyuridinetriphosphate nick end-labeling (TUNEL) method. Dewaxed sections were incubated with 1% proteinase K (TREVIGEN Inc.) diluted 1:200 at 378C for 15 min, followed with inhibition of the endogenous peroxidase activity at room temperature for 5 min. After incubation with the TdT enzyme diluted 1:50 at 378C for 1 hr, the sections were then reacted with HRP-conjugated streptavidin at room temperature for 15 min. The apoptosis reaction was made visible by incubation with a blue label solution. The sections were faintly counterstained with nuclear fast red. Elemental Mapping by EPMA An electron probe microanalyzer (EPMA; 8705, Shimazu, Co. Ltd, Kyoto, Japan) was used for the elemental mapping of Ca, P, and Mg. Undecalciﬁed 6-week-old mandibles were embedded in epoxy resin and trimmed with diamond disks until exposure to a sagittal plane. After polishing, the specimens were sputter-coated with carbon before elemental analysis. For each experiment, 256 3 256 pixels mapping were performed. The accelerating voltage and beam current were set to 15 kV and 0.03 mA, respectively, and integrating time was 0.05 seconds at each pixel. RESULTS Histological Observation Mandibular incisors in the wild-type mice had regularly distributed layers consisting of subodontoblasts, odontoblasts, predentin, and dentin in both their labial and lingual regions (Fig. 1a,b). Odontoblasts were regularly arranged along the surface of a uniformly thick predentin layer (Fig. 1b). In the klotho2/2 mice, these cell layers remained in the lingual region, while differing in their labial region (Fig. 1d,e). Although preodontobalsts showed same arrangement to those in wild-type, DEFECTIVE DENTIN FORMATION IN klotho DEFICIENCY 185 Fig. 1. a–f: Photographs of the mandibular incisors in 6-week-old wild-type (a–c) and klotho2/2 mice (d–f). Stained with hematoxylin– eosin (a,b,d,e), and immunostained with an alkaline phosphatase (ALP) antibody (c,f). Panels a and d are low-magniﬁcation images of the mandibular incisors in the wild-type (a) and klotho2/2 (d) mice. In contrast to the normal layer structure composed of odontoblasts (OB), predentin (PD), and dentin (D) in the wild-type mouse (b is the higher magniﬁcation of the boxed area in a), the predentin–dentin border at the labial region appears unclear in the klotho2/2 mouse (e is the higher magniﬁcation of the boxed area in d). The dentin matrix at the labial region surrounds some odontoblasts (e arrows). While odontoblasts and subodontoblastic-layer cells exhibit immunopositivity for ALP in both type of mice (c,f), the encircled odontoblasts are devoid of ALP immunopositivity in the klotho2/2 mouse (f arrows). Scale bars 5 500 mm in a,d, 50 mm in b,c,e,f. differentiated odontoblasts, located at more incisal position from matrix-forming stage, became to exhibit an irregularly arrangement; some odontoblast-like cells were embedded in the secreted dentin matrix (Fig. 1e, arrows). The dentin and predentin layers were not uniform in thickness. Additionally, the border between these two layers could not be easily identiﬁed (Fig. 1e). The mandible of the klotho2/2 mice had the hematoxylin-positive bone matrix and the empty lacunae (Fig. 1d) we have previously reported (Suzuki et al., 2005). stronger DMP-1 immunoreactivity in the klotho2/2 (Fig. 2k) rather than in the wild-type mice. When observing the cross-sectioned dentin and predentin of the incisor in the wild-type mice, these appeared immunonegative for OPN—except for the peritubular dentin with very faint OPN immunopositivity (Fig. 2e,g). The molar cementum expressed immunopositivity for OPN (Fig. 2f). The osteocytic canaliculi also revealed intense OPN immunoreactivity, although still relatively weak to that of the bone surface and walls of the osteocytic lacunae (Fig. 2f). In contrast, the dentin matrix at the periphery of the abnormally embedded odontoblast-like cells in the klotho2/2 mice displayed a faint immunoreaction for OPN (Fig. 2m,o, arrows). OPN immunopositivity along dentinal tubules appeared to spread out slightly (Fig. 2m). The molar cementum and the surface of alveolar bone showed a stronger OPN immunoreaction (Fig. 2n). The dentin and bone matrices showed uniform immunoreactivity for type I collagen in the wild-type mice. Compared with the dentin, the predentin matrix exhibited a more intense immunoreaction for type I collagen (Fig. 2h). In the klotho2/2 mice, however, type I collagen immunopositivity was irregularly distributed: the dentin matrix surrounding odontoblast-like cells showed an intense immunoreaction, while some parts of the predentin adjacent to authentic odontoblasts showed a weak immunoreaction (Fig. 2p). In contrast, no apparent difference in immunoexpression pattern of DMP-1, OPN, and type I collagen was recognizable at the lingual region of the klotho2/2 mice compared with the wild-type mice; cross-sectioned den- Immunohistochemical Observation We used the serial sections for immunohistochemical observation each other. Odontoblasts and subodontoblast cell layers of the wild-type incisors showed an intense ALP activity (Fig. 1c). In the klotho2/2 mice, the odontoblasts and subodontoblast layer cells were also positive for ALP; the odontoblasts-like cells, which were found embedded in the dentin matrix displayed no such immunopositivity (Fig. 1f, arrows). DMP-1 immunopositivity was discernible in the dentinal tubules, the walls of the osteocytic lacunae and canaliculi of the alveolar bone, and in the cementum of molar roots of the wild-type mice (Fig. 2a–d). Alternatively, the klotho2/2 mice showed diffusely distributed and unevenly intense DMP1 immunopositivity; the positivity for DMP-1 could be seen, in part, on the dentinal tubules (Fig. 2i,j) and the dentin matrix surrounding the embedded odontoblast-like cells (Fig. 2l, arrows). The cementum of molar roots, the surface of the alveolar bone, and the walls of the osteocytic lacunae expressed 186 SUZUKI ET AL. Fig. 2. a–p: Expression of immunoreactivity for dentin matrix protein-1 (DMP-1; a–d,i–l), osteopontin (OPN; e–g,m–o), and type I collagen (h,p) in the wild-type (a–h) and klotho2/2 mice (i–p). Panels a and i are low-magniﬁcation images of the mandibular incisor in the wildtype (a) and klotho2/2 (i) mice. Panels b–d and j–l are higher magniﬁcations of the boxed areas in panel a and i, respectively. Immunoreaction for DMP-1 is recognizable in the peritubular dentin (b), cementum of the molar root (TR in c), the walls of the osteocytic lacunae and canaliculi of the alveolar bone (AB in c), and the dentinal tubules (d) in the wild-type mouse. In the klotho2/2 mouse, the peritubular dentin shows a weak immunoreactivity for DMP-1 (j). The dentin matrix surrounding the odontoblasts (OB) at the labial region also displays DMP-1 activity (l arrows) in addition to an increase in immunointensity for DMP-1 at the osteocytic lacunae of the walls and the surface of the alveolar bone (k). The peritubular dentin also exhibits a faint immu- noreaction for OPN at the incisal position in the wild-type (e) and klotho2/2 (m) mice. The OPN immunoreaction is more intense at the surface of the alveolar bone, and walls of the osteocytic lacunae in addition to the cementum in both type of mice (f,n), and its reaction appears stronger in the klotho2/2 mouse than the wild-type. While the sagittally-cut dentin (D) and predentin (PD) lack any immunoreaction for OPN in the wild-type mouse (g), the dentin surrounding odontoblasts shows a slight OPN immunoreaction in the klotho2/2 mouse (o, arrows). Furthermore, the matrix of predentin and dentin exhibits a type-I collagen immunoreaction, whose expression in the former is stronger than in the latter (h). On the other hand, type-I collagen immunoreaction is not uniformly distributed in the dentin, with a more intense reaction in the dentin matrix, including some odontoblasts in the klotho2/2 mouse (p). Scale bars 5 500 mm in a,i, 50 mm in b,c,e,f,j,k,m,n, 25 mm in d,g,l,o, 20 mm in h,p. tin and cementum showed immunoreactivity for DMP-1, but not for OPN. The dentin and predentin matrices demonstrated uniform immunoreactivity for type I collagen at the lingual region of the klotho2/2 mice. layers (Fig. 3a,c). In contrast, in the klotho2/2 mice, TUNEL positivity was found in these odontoblasts-like cells embedded in the dentin, in pulpal cells located more incisially, and in the alveolar osteocytes (Fig. 3d–f). Apoptosis Reaction Elemental Mapping by EPMA The wild-type mice specimens had several bone marrow cells reactive for TUNEL, indicative of apoptosis (Fig. 3b). No apoptotic reaction existed in odontoblasts or the pulpal cells in the pulp core and subodontoblastic The incisor dentin of the wild-type mice demonstrated the gradient distribution of Ca (Fig. 4a) and Mg (Fig. 4c): Ca was at a higher level near the enamel while Mg was at a lower one. P distribution was almost uniform DEFECTIVE DENTIN FORMATION IN klotho DEFICIENCY 187 Fig. 3. a–f: Apoptosis detection on the mandible of the wild-type (a–c) and klotho2/2 mice (d–f) by terminal deoxynucleotidyl transferase-mediated deoxyuridinetriphosphate nick end-labeling (TUNEL) reaction. Odontoblasts and pulpal cells are nonreactive in the wild-type incisor (a,c), although some bone mallow cells show an apoptotic reaction (b in arrows). The klotho2/2 mouse demonstrates apoptotic reactions in some incisal–pulpal cells (d, arrows) and odontoblasts surrounded by the dentin matrix (f, arrows) in addition to some osteocytes in the alveolar bone (AB; e in arrows) in. D, dentin; TR, tooth root. Scale bars 5 50 mm in a,d,e, 25 mm in b, 100 mm in c,f. (Fig. 4b). In contrast, the klotho2/2 dentin had an irregular distribution of Ca (Fig. 4d), P (Fig. 4e), and Mg (Fig. 4f) concentrations. Interestingly, the portions with decreased concentrations of Ca and P (Fig. 4d,e) appeared to colocate with the area populated by embedded odontoblast-like cells, while demonstrating spotty, increased concentrations of Mg (Fig. 4f). histochemical ﬁndings of a different localization pattern for Ca21-Mg21-ATPase between the labial and lingual odontoblasts in the rat incisors (Suzuki, 1985). Malformations of the dentin have been classiﬁed into two major groups and categorized according to different phenotypic characteristics, for example, DGI types I–III, and DD types I and II. Mutation within DSPP, which encodes both dentin sialoprotein (DSP) and dentin phosphoprotein (DPP; MacDougall et al., 1997; Feng et al., 1998), has been associated with the pathogenesis of DGI type-II (Xiao et al., 2001; Zhang et al., 2001), DGI typeIII (Dong et al., 2005), and DD-II (Rajpar et al., 2002). To date, no mutation in any other gene besides DSPP has been shown to cause non-syndromic, heritable dentin defects. Our present observations showed that the klotho2/2 mice shared some pathomorphological features with DSPP knockout mice, which develop a defective mineralization that is similar to human DGI-III (Sreenath et al., 2003)—although we cannot classify the dental abnormality seen in the klotho2/2 mice as a proper dentin disease. However, the abnormal odontoblasts and dentin matrix in the klotho2/2 mice were limited to the labial region of their incisors: it is possible that this region-speciﬁc change in dentin is an exclusive abnormality of the klotho2/2 mice. Previous reports have shown that tumoral calcinosis occasionally displays dental abnormalities, including short bulbous roots, pulp stones, and partial obliteration of the pulp cavity (Burkes et al., 1991; Polykandriotis et al., 2004; Specktor et al., 2006). This disorder is caused by a homozygous missense mutation in human klotho (Ichikawa et al., 2007). Furthermore, the gene of FGF23 and GALNT3 (UDP-N-acetyl-a-D-galactosamine: polypeptide N-acetylgalactosaminyl-transferase) is also involved in tumoral calcinosis (Chefetz et al., 2005; Specktor et al., 2006). Recent studies have shown that FGF23 requires the klotho protein to bind and signal through FGFR with the klotho-FGFR complex binding to FGF23 with a higher afﬁnity than FGFR or the klotho protein alone (Kurosu et al., 2006). Furthermore, the phenotypes including ectopic calciﬁcation, emphysema, and skin atrophy of klotho2/2 mice overlap with those of FGF23-null mice (Razzaque and Lanske, 2006). Taken together with current observation of in incisor dentinogenesis, it is DISCUSSION Our histological and immunohistochemical observations of the klotho2/2 phenotype have revealed the abnormal distribution and morphology of odontoblasts and dentin matrix of the labial region of the mandibular incisor. In a normal phenotype, odontoblasts would never be embedded in the dentin. However, klotho-deﬁcient incisors had odontoblasts with nuclear pyknosis embedded in their dentin, which may represent a pace unbalanced between odontoblasts movement toward the pulpal side and dentin synthesis. In addition, the EPMA analysis revealed that the klotho2/2 dentin had signiﬁcantly lower concentrations of Ca, P, and Mg, when compared with wild-type counterparts. Consistent with our previous report (Suzuki et al., 2005), the klotho2/2 alveolar bone featured (1) an altered distribution of osteocytes and bone matrix proteins, (2) osteoblasts that were weakly reactive for ALP immunostaining, (3) many apoptotic osteoblasts and osteocytes and (4) abnormal distributions of DMP-1 and OPN. These ﬁndings suggest the involvement of klotho in the formation and mineralization of both dentin and the alveolar bone. It is interesting that the histological abnormality in the klotho2/2 incisor is restricted to its labial region. A similar change in the klotho2/2 mice has been reported in the rat incisor suffering from HHM (hypercalcemia of malignancy; Kato et al., 2003) or treated with hexachlorobenzen (Long et al., 2004). On the other hand, Savage et al. (2006) reported dentin malformations restricted to the lingual side in the mandibular incisor of p20C/EBPbeta transgenic mice. In our view, however, there is not enough data to explain the mechanism of such region-speciﬁc alterations for klotho-deﬁcient and other animal models. One possible explanation is that odontoblasts show different phenotypes at the labial and lingual regions, as suggested by previous 188 SUZUKI ET AL. Fig. 4. a–f: Elemental mapping of calcium (Ca; a,d), phosphorus (P; b,e), and magnesium (Mg; c,f) in mandibular incisors at the labial region of the 6-week-old wild-type (a–c) and klotho2/2 (d–f) mice. The portion colored red is the high elemental concentration, while the dark blue is the low one. The wild-type dentin (D) indicates that the Ca increases from the dental pulp to the enamel (E; a) in contrast to a decrease in Mg (c). P is uniformly deposited at the dentin in the wild-type mouse (b). In the klotho2/2 mouse, on the other hand, the three elements are uniformly distributed in the dentin except for the matrix—including odontoblasts (d–f), all with lower element distributions than in the wildtype mouse. The dentin matrix surrounding odontoblasts shows a low distribution of Ca and P, but a high level of Mg (d–f). Scale bars 5 200 mm in a–f. likely that the depletion of klotho gene inﬂuences on dentinogenesis with klotho protein binding FGF23. Dentin mineralization is mediated by phosphorylated extracellular matrix proteins localized within collagen gap areas. These proteins bind calcium and phosphate ions in an appropriate conformation, so as to become nuclei that would trigger the formation of apatite crystals (Glimcher, 1989; Landis, 1996). Many noncollage- DEFECTIVE DENTIN FORMATION IN klotho DEFICIENCY nous proteins secreted by odontoblasts play important roles in the process of mineralization, for example, OPN (Fujisawa et al., 1993), bone sialoprotein (Chen et al., 1992; Fujisawa et al., 1993), osteonectin (Papagerakis et al., 2002), osteocalcin (Gorski, 1998), BAG-75 (Gorski, 1998), DSPP (MacDougall et al., 1997; Feng et al., 1998), and DMP1 (Feng et al., 2003; 2006; Lu et al., 2007). Our ﬁndings indicated that klotho deletion altered the distributions of DMP-1 as well as Ca, P, and Mg in the labial dentin containing odontoblast-like cells, suggesting that DMP-1 is required for the transportation of Ca and P. DMP-1 has been shown to be involved in the initiation of bone and dentin mineralization (Narayanan et al., 2003; Lu et al., 2007). DMP-1 is capable of inducing the cytodifferentiation of dental pulp stem cells into odontoblasts (Almushayt et al., 2006) and regulating the formation of the dentin tubular system (Lu et al., 2007). Therefore, we can postulate that the altered distribution of DMP-1 compromises odontoblast functions in the klotho2/2 mice. In addition, DMP-1 acts as a transcriptional factor that targets the nucleus, besides being a mineralization regulator (Narayanan et al., 2003). DMP-1 controls the transcription of the DSPP gene (Ye et al., 2004; Narayanan et al., 2006), whose mutation and depletion induces DGI/DD in humans, and impaired dentin mineralization in mice that is similar to human DGI-III (Sreenath et al., 2003). These ﬁndings lead us to draw the hypothesis that the klotho gene affects odontoblastic functionality and dentin mineralization by indirectly mediating proteins such as DMP-1. However, the klotho function is not yet completely elucidated, and further investigation of this issue is required. ACKNOWLEDGMENT H.S. was funded by a grant for Promotion of Niigata University Research Projects. LITERATURE CITED Almushayt A, Narayanan K, Zaki AE, George A. 2006. Dentin matrix protein 1 induces cytodifferentiation of dental pulp stem cells into odontoblasts. Gene Ther 13:611–620. Beattie ML, Kim JW, Gong SG, Murdoch-Kinch CA, Simmer JP, Hu JC. 2006. Phenotypic variation in dentinogenesis imperfecta/dentin dysplasia linked to 4q21. J Dent Res 85:329–333. Burkes EJ Jr, Lyles KW, Dolan EA, Giammara B, Hanker J. 1991. Dental lesions in tumoral calcinosis. J Oral Pathol Med 20:222– 227. Chefetz I, Heller R, Galli-Tsinopoulou A, Richard G, Wollnik B, Indelman M, Koerber F, Topaz O, Bergman R, Sprecher E, Schoenau E. 2005. A novel homozygous missense mutation in FGF23 causes Familial Tumoral Calcinosis associated with disseminated visceral calciﬁcation. Hum Genet 118:261–266. Chen J, Shapiro HS, Sodek J. 1992. Development expression of bone sialoprotein mRNA in rat mineralized connective tissues. J Bone Miner Res 7:987–997. Dong J, Gu T, Jeffords L, MacDougall M. 2005. Dentin phosphoprotein compound mutation in dentin sialophosphoprotein causes dentinogenesis imperfecta type III. Am J Med Genet A 132:305– 309. Feng JQ, Luan X, Wallace J, Jing D, Ohshima T, Kulkarni AB, D’Souza RN, Kozak CA, MacDougall M. 1998. Genomic organization, chromosomal mapping, and promoter analysis of the mouse dentin sialophosphoprotein (Dspp) gene, which codes for both dentin sialoprotein and dentin phosphoprotein. J Biol Chem 273:9457–9464. 189 Feng JQ, Huang H, Lu Y, Ye L, Xie Y, Tsutsui TW, Kunieda T, Castranio T, Scott G, Bonewald LB, Mishina Y. 2003. The Dentin matrix protein 1 (Dmp1) is speciﬁcally expressed in mineralized, but not soft, tissues during development. J Dent Res 82:776– 780. Feng JQ, Ward LM, Liu S, Lu Y, Xie Y, Yuan B, Yu X, Rauch F, Davis SI, Zhang S, Rios H, Drezner MK, Quarles LD, Bonewald LF, White KE. 2006. Loss of DMP1 causes rickets and osteomalacia and identiﬁes a role for osteocytes in mineral metabolism. Nat Genet 38:1310–1315. Fujisawa R, Butler WT, Brunn JC, Zhou HY, Kuboki Y. 1993. Differences in composition of cell-attachment sialoproteins between dentin and bone. J Dent Res 72:1222–1226. Glimcher MJ. 1989. Mechanism of calciﬁcation: role of collagen ﬁbrils and collagen-phosphoprotein complexes in vitro and in vivo. Anat Rec 224:139–153. Goldberg M, Septier D, Oldberg A, Young MF, Ameye LG. 2006. Fibromodulin-deﬁcient mice display impaired collagen ﬁbrillogenesis in predentin as well as altered dentin mineralization and enamel formation. J Histochem Cytochem 54:525–537. Gorski JP. 1998. Is all bone the same? Distinctive distributions and properties of non-collagenous matrix proteins in lamellar vs. woven bone imply the existence of different underlying osteogenic mechanisms. Crit Rev Oral Biol Med 9:201–223. Ichikawa S, Imel EA, Kreiter ML, Yu X, Mackenzie DS, Sorenson AH, Goetz R, Mohammadi M, White KE, Econs MJ. 2007. A homozygous missense mutation in human KLOTHO causes severe tumoral calcinosis. J Clin Invest 117:2684–2691. Imura A, Tsuji Y, Murata M, Maeda R, Kubota K, Iwano A, Obuse C, Togashi K, Tominaga M, Kita N, Tomiyama K, Iijima J, Nabeshima Y, Fujioka M, Asato R, Tanaka S, Kojima K, Ito J, Nozaki K, Hashimoto N, Ito T, Nishio T, Uchiyama T, Fujimori T, Nabeshima Y. 2007. alpha-Klotho as a regulator of calcium homeostasis. Science 316:1615–1618. Kato A, Suzuki M, Karasawa Y, Sugimoto T, Doi K. 2003. Histopathological study on the PTHrP-induced incisor lesions in rats. Toxicol Pathol 31:480–485. Kuro-o M, Matsumura H, Aizawa H, Kawaguchi H, Suga T, Utsugi T, Ohyama Y, urabayashi M, Kaname T, Kume E, Iwasaki H, Iida A, Shiraki-Iida T, Nishikawa S, Nagai R, Nabeshima Y. 1997. Mutation of the mouse klotho gene leads to a syndrome resembling aging. Nature 390:45–51. Kurosu H, Yamamoto M, Clark JD, Pastor JV, Nandi A, Gurnani P, McGuinness OP, Chikuda H, Yamaguchi M, Kawaguchi H, Shimomura I, Takayama Y, Herz J, Kahn CR, Rosenblatt KP, Kuro-o M. 2005. Suppression of aging in mice by the hormone Klotho. Science 309:1829–1833. Kurosu H, Ogawa Y, Miyoshi M, Yamamoto M, Nandi A, Rosenblatt KP, Baum MG, Schiavi S, Hu MC, Moe OW, Kuro-o M. 2006. Regulation of ﬁbroblast growth factor-23 signaling by klotho. J Biol Chem 281:6120–6123. Landis WJ. 1996. Mineral characterization in calcifying tissues: atomic, molecular and macromolecular perspectives. Connect Tissue Res 34:239–246. Long PH, Herbert RA, Nyska A. 2004. Hexachlorobenzene-induced incisor degeneration in Sprague-Dawley rats. 2004. Toxicol Pathol 32:35–40. Lopez Franco GE, Huang A, Pleshko Camacho N, Blank RD. 2005. Dental phenotype of the col1a2(oim) mutation: DI is present in both homozygotes and heterozygotes. Bone 36:1039–1046. Lu Y, Ye L, Yu S, Zhang S, Xie Y, McKee MD, Li YC, Kong J, Eick JD, Dallas SL, Feng JQ. 2007. Rescue of odontogenesis in Dmp1deﬁcient mice by targeted re-expression of DMP1 reveals roles for DMP1 in early odontogenesis and dentin apposition in vivo. Dev Biol 303:191–201. MacDougall M, Simmons D, Luan X, Nydegger J, Feng J, Gu TT. 1997. Dentin phosphoprotein and dentin sialoprotein are cleavage products expressed from a single transcript coded by a gene on human chromosome 4. Dentin phosphoprotein DNA sequence determination. J Biol Chem 272:835–842. Masuda H, Chikuda H, Suga T, Kawaguchi H, Kuro-o M, 2005. Regulation of multiple ageing-like phenotypes by inducible klotho 190 SUZUKI ET AL. gene expression in klotho mutant mice. Mech Ageing Dev 126:1274–1283. Matsumura Y, Aizawa H, Shiraki-Iida T, Nagai R, Kuro-o M, Nabeshima Y. 1998. Identiﬁcation of human klotho gene and its two transcripts encoding membrane and secreted klotho protein. Biochem Biophys Res Commun 242:626–630. Metzker A, Eisenstein B, Oren J, Samuel R. 1988. Tumoral calcinosis revisited--common and uncommon features. Report of ten cases and review. Eur J Pediatr 147:128–132. Narayanan K, Ramachandran A, Hao J, He G, Park KW, Cho M, George A. 2003. Dual functional roles of dentin matrix protein 1. Implications in biomineralization and gene transcription by activation of intracellular Ca21 store. J Biol Chem 278:17500– 17508. Narayanan K, Gajjeraman S, Ramachandran A, Hao J, George A. 2006. Dentin matrix protein 1 regulates dentin sialophosphoprotein gene transcription during early odontoblast differentiation. J Biol Chem 281:19064–19071. Oda K, Amaya Y, Fukushi-Irie M, Kinameri Y, Ohsuye K, Kubota I, Fujimura S, Kobayashi J. 1999. A general method for rapid puriﬁcation of soluble versions of glycosylphosphatidylinositol-anchored proteins expressed in insect cells: an application for human tissue-nonspeciﬁc alkaline phosphatase. J Biochem (Tokyo) 126: 694–699. Papagerakis P, Berdal A, Mesbah M, Peuchmaur M, Malaval L, Nydegger J, Simmer J, Macdougall M. 2002. Investigation of osteocalcin, osteonectin, and dentin sialophosphoprotein in developing human teeth. Bone 30:377–385. Polykandriotis EP, Beutel FK, Horch RE, Grunert J. 2004. A case of familial tumoral calcinosis in a neonate and review of the literature. Arch Orthop Trauma Surg 124:563–567. Rajpar MH, Koch MJ, Davies RM, Mellody KT, Kielty CM, Dixon MJ. 2002. Mutation of the signal peptide region of the bicistronic gene DSPP affects translocation to the endoplasmic reticulum and results in defective dentine biomineralization. Hum Mol Genet 11:2559–2565. Razzaque MS, Lanske B. 2006. Hypervitaminosis D and premature aging: lessons learned from Fgf23 and Klotho mutant mice. Trends Mol Med 12:298–305. Savage T, Bennett T, Huang YF, Kelly PL, Durant NE, Adams DJ, Mina M, Harrison JR. 2006. Mandibular phenotype of p20C/EBPbeta transgenic mice: reduced alveolar bone mass and site-speciﬁc dentin dysplasia. Bone 39:552–564. Shiraki-Iida T, Aizawa H, Matsumura Y, Sekine S, Iida A, Anazawa H, Nagai R, Kuro-o M, and Nabeshima Y. 1998. Structure of the mouse klotho gene and its two transcripts encoding membrane and secreted protein. FEBS Lett 424:6–10. Specktor P, Cooper JG, Indelman M, Sprecher E. 2006. Hyperphosphatemic familial tumoral calcinosis caused by a mutation in GALNT3 in a European kindred. J Hum Genet 51:487–490. Sreenath T, Thyagarajan T, Hall B, Longenecker G, D’Souza R, Hong S, Wright JT, MacDougall M, Sauk J, Kulkarni AB. 2003. Dentin sialophosphoprotein knockout mouse teeth display widened predentin zone and develop defective dentin mineralization similar to human dentinogenesis imperfecta type III. J Biol Chem 278:24874–24880. Suzuki A. 1985. Ultrastructual and cytochemical studies on the dentinogenesis of rat incisors at the lingual side. Jpn J Oral Biol 27:215–253. Suzuki H, Amizuka N, Oda K, Li M, Yoshie H, Ohshima H, Noda M, Maeda T. 2005. Histological evidence of the altered distribution of osteocytes and bone matrix synthesis in klotho-deﬁcient mice. Arch Histol Cytol 68:371–381. Thyagarajan T, Sreenath T, Cho A, Wright JT, Kulkarni AB. 2001. Reduced expression of dentin sialophosphoprotein is associated with dysplastic dentin in mice overexpressing transforming growth factor-beta 1 in teeth. J Biol Chem 276:11016–11020. Urakawa I, Yamazaki Y, Shimada T, Iijima K, Hasegawa H, Okawa K, Fujita T, Fukumoto S, Yamashita K. 2006. Klotho converts canonical FGF receptor into a speciﬁc receptor for FGF23. Nature 444:770–774. Xiao S, Yu C, Chou X, Yuan W, Wang Y, Bu L, Fu G, Qian M, Yang J, Shi Y, Hu L, Han B, Wang Z, Huang W, Liu J, Chen Z, Zhao G, Kong X. 2001. Dentinogenesis imperfecta 1 with or without progressive hearing loss is associated with distinct mutations in DSPP. Nat Genet 27:201–204. Xiao NM, Zhang YM, Zheng Q, Gu J. 2004. Klotho is a serum factor related to human aging. Chin Med J 117:742–747. Ye L, MacDougall M, Zhang S, Xie Y, Zhang J, Li Z, Lu Y, Mishina Y, Feng JQ. 2004. Deletion of dentin matrix protein-1 leads to a partial failure of maturation of predentin into dentin, hypomineralization, and expanded cavities of pulp and root canal during postnatal tooth development. J Biol Chem 279:19141–19148. Zhang X, Zhao J, Li C, Gao S, Qiu C, Liu P, Wu G, Qiang B, Lo WH, Shen Y. 2001. DSPP mutation in dentinogenesis imperfecta Shields type II. Nat Genet 27:151–152.