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Involvement of the Klotho Protein in Dentin Formation and Mineralization.

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THE ANATOMICAL RECORD 291:183–190 (2008)
Involvement of the Klotho Protein in
Dentin Formation and Mineralization
Divisions of Anatomy and Cell Biology of the Hard Tissue, Niigata University Graduate
School of Medical and Dental Sciences, Niigata, Japan
Center for Transdisciplinary Research, Niigata University, Niigata, Japan
Divisions of Oral Biochemistry, Niigata University Graduate School of Medical and
Dental Sciences, Niigata, Japan
Department of Molecular Pharmacology, Medical Research Institute, Tokyo Medical and
Dental University, Tokyo, Japan
Divisions of Oral Anatomy, Niigata University Graduate School of Medical and
Dental Sciences, Niigata, Japan
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).
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.
Received 7 August 2007; Accepted 25 October 2007
DOI 10.1002/ar.20630
Published online 18 December 2007 in Wiley InterScience (www.
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
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.
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.
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,
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
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
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
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
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-
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.
H.S. was funded by a grant for Promotion of Niigata
University Research Projects.
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dentin, involvement, formation, protein, klotho, mineralization
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