RESEARCH ARTICLE
985
Development 137, 985-992 (2010) doi:10.1242/dev.045898
© 2010. Published by The Company of Biologists Ltd
Alterations in phosphorus, calcium and PTHrP contribute to
defects in dental and dental alveolar bone formation in
calcium-sensing receptor-deficient mice
Wen Sun1, Weiwei Sun2, Jingning Liu2, Xichao Zhou1, Yongjun Xiao3, Andrew Karaplis3, Martin R. Pollak4,
Edward Brown4, David Goltzman3 and Dengshun Miao1,2,*
SUMMARY
To determine whether the calcium-sensing receptor (CaR) participates in tooth formation and dental alveolar bone development in
mandibles in vivo, we examined these processes, as well as mineralization, in 2-week-old CaR-knockout (CaR–/–) mice. We also
attempted to rescue the phenotype of CaR–/– mice by genetic means, in mice doubly homozygous for CaR and 25-hydroxyvitamin D
1a-hydroxylase [1a(OH)ase] or parathyroid hormone (Pth). In CaR–/– mice, which exhibited hypercalcemia, hypophosphatemia and
increased serum PTH, the volumes of teeth and of dental alveolar bone were decreased dramatically, whereas the ratio of the area
of predentin to total dentin and the number and surface of osteoblasts in dental alveolar bone were increased significantly, as
compared with wild-type littermates. The normocalcemia present in CaR–/–; 1a(OH)ase–/– mice only slightly improved the defects in
dental and alveolar bone formation observed in the hypercalcemic CaR–/– mice. However, these defects were completely rescued by
the additional elimination of hypophosphatemia and by an increase in parathyroid hormone-related protein (PTHrP) expression in
the apical pulp, Hertwig’s epithelial root sheath and mandibular tissue in CaR–/–; Pth–/– mice. Therefore, alterations in calcium,
phosphorus and PTHrP contribute to defects in the formation of teeth and alveolar bone in CaR-deficient mice. This study indicates
that CaR participates in the formation of teeth and in the development of dental alveolar bone in mandibles in vivo, although it
appears to do so largely indirectly.
INTRODUCTION
The calcium-sensing receptor (CaR; CASR – Mouse Genome
Informatics) is a plasma membrane G-protein-coupled receptor that
is activated by extracellular calcium. CaR plays a central role in
controlling systemic calcium homeostasis, predominately through
its effects on the regulation of parathyroid hormone (PTH) secretion
by the parathyroid glands and on urinary calcium excretion by the
kidney (Brown et al., 1993; Brown and MacLeod, 2001). Evidence
for its crucial role in parathyroid function came from the
identification of inactivating mutations in CaR in familial
hypocalciuric hypercalcemia (FHH) and neonatal severe
hyperparathyroidism (NSHPT) (Pollak et al., 1993). Patients with
FHH are heterozygous for inactivating mutations in CaR and exhibit
mild to moderate hypercalcemia, normal or mildly increased
circulating PTH levels, and parathyroid histology that ranges from
normal to mild hyperplasia. Patients with NSHPT are homozygous
for inactivating CaR mutations and have severe, life-threatening
hypercalcemia accompanied by very high circulating PTH levels
and marked parathyroid hyperplasia. Targeted inactivation of CaR
in mice has resulted in the development of models of the human
1
Institute of Dental Research, Stomatological College and 2The Research Center for
Bone and Stem Cells, Nanjing Medical University, Nanjing, Jiangsu 210029, P. R. of
China . 3Department of Medicine, McGill University, Montreal, H3A 1A1 Quebec,
Canada. 4Department of Medicine, Brigham and Women’s Hospital, Harvard
Medical School, Boston, MA 02115, USA.
*Author for correspondence ([email protected])
Accepted 21 January 2010
syndromes (Ho et al., 1995). Thus, mice with a heterozygous
deletion of the CaR gene mimic FHH, whereas homozygotes mimic
NSHPT and generally die within a few days to weeks after birth.
Interestingly, crossing CaR-knockout (CaR–/–) mice with Pth–/– mice
rescues the lethal phenotype of the CaR–/– mice, indicating that
lethality is due to severe hypercalcemia caused by marked
hyperparathyroidism (Kos et al., 2003).
CaR–/– mice exhibit classic features of rickets, including growth
retardation, expanded growth plates with reduced calcification,
widened metaphyses and impaired bone mineralization (Garner et
al., 2001; Ho et al., 1995). These abnormalities are rescued by
concomitant knockout of Pth. It is unknown, however, whether loss
of CaR impacts tooth formation and dental alveolar bone
development. CaR is expressed in the mandible and developing
tooth, and might provide a mechanism for sensing and responding
to the alterations in extracellular calcium concentrations that take
place during the formation of the mandibles and teeth (Dvorak et al.,
2004; Mathias et al., 2001). Calcium is, of course, a key component
of teeth. It is found in the enamel, dentin and the surrounding
extracellular matrix. Moreover, the teeth and dental alveolar bone
are highly active tissues that constantly undergo remodeling
throughout the life cycle (Marks and Schroeder, 1996; Roberts,
1999). Thus, CaR could potentially play important roles in the
formation and development of the teeth and dental alveolar bone.
The vitamin D-PTH axis plays a central role in calcium and
phosphorus homeostasis and is essential for skeletal development
and mineralization. PTH and 1,25-dihydroxyvitamin D3
[1,25(OH)2D3] directly affect calcium homeostasis and each exerts
important regulatory effects on the other. PTH stimulates the
production of 1,25(OH)2D3 by activating the renal 25-
DEVELOPMENT
KEY WORDS: Calcium-sensing receptor (CaR; CASR), 25-hydroxyvitamin D 1a-hydroxylase [1a(OH)ase; CYP27B1], Parathyroid hormone
(PTH), Parathyroid hormone-related protein (PTHrP; PTHLH), Teeth, Mandible, Mouse
RESEARCH ARTICLE
hydroxyvitamin D 1a-hydroxylase [1a(OH)ase; CYP27B1 –
Mouse Genome Informatics] (Brenza et al., 1998; Murayama et al.,
1998), and increased 1,25(OH)2D3 in turn suppresses the production
of PTH (Cantley et al., 1985; Chan et al., 1986) and controls
parathyroid cell growth (Szabo et al., 1989). Suppression of PTH
synthesis by 1,25(OH)2D3 occurs through negative regulation of Pth
transcription by a 1,25(OH)2D3-vitamin D receptor (VDR)-retinoid
X receptor (RXR) complex (Liu et al., 1996) in parathyroid cells
(Beckerman and Silver, 1999).
We have previously reported the creation of a mouse model that is
deficient in PTH by targeting the Pth gene in embryonic stem
cells. Although adult Pth-null mice develop hypocalcemia,
hyperphosphatemia and low circulating 1,25(OH)2D3 levels
consistent with primary hypoparathyroidism (Miao et al., 2004a), this
phenotype is not lethal. We (Panda et al., 2001) and others (Dardenne
et al., 2001) have also previously reported the generation of a mouse
model that is deficient in 1,25(OH)2D as a result of targeted ablation
of the 1a(OH)ase gene. After weaning, 1a(OH)ase–/– mice that
are fed a diet of regular mouse chow developed secondary
hyperparathyroidism, retarded growth and the skeletal abnormalities
characteristic of rickets. These abnormalities mimic those described
in the human disease vitamin D-dependent rickets type I (Fraser et al.,
1973). By comparing Pth–/– or 1a(OH)ase–/– mice with Pth–/–;
1a(OH)ase–/– double-null mice, we found that PTH plays a
predominant role in appositional bone growth, whereas 1,25(OH)2D3
acts predominantly on endochondral bone formation, and both play
collaborative roles in modulating skeletal and calcium homeostasis
(Xue et al., 2005). However, the relative contributions of calcium,
phosphorus, 1,25(OH)2D3 and PTH to dental formation and
intramembranous bone formation remain unknown. We therefore
used CaR–/– mice, CaR–/–; 1a(OH)ase–/– and CaR–/–; Pth–/– doublehomozygous mice to dissect the individual contributions of calcium
(acting via CaR), phosphorus, 1,25(OH)2D and PTH to the formation
of teeth and dental alveolar bone.
MATERIALS AND METHODS
Derivation of CaR–/–, CaR–/–; 1a(OH)ase–/– and CaR–/–; Pth–/– mice
The derivation of the three parental strains of CaR–/–, 1a(OH)ase–/– and
Pth–/– mice by homologous recombination in embryonic stem cells has been
described (Ho et al., 1995; Panda et al., 2001; Miao et al., 2002). Briefly, a
neomycin resistance gene was inserted into exon 5 of the mouse CaR gene.
Western blot analysis of kidney protein membrane extracts from CaR–/– mice
confirmed that no detectable protein is expressed from this allele in this
tissue (Ho et al., 1995). A neomycin resistance gene replaced exons 6-8 of
the mouse 1a(OH)ase gene, removing both the ligand-binding and the
heme-binding domains (Panda et al., 2001). RT-PCR of renal RNA from
1a(OH)ase–/– mice confirmed the lack of 1a(OH)ase expression. A
neomycin resistance gene was inserted into exon 3 of the mouse Pth gene,
resulting in replacement of the entire coding sequence of mature PTH. Lack
of PTH expression in parathyroid glands was confirmed by immunostaining
(Miao et al., 2002). CaR+/– mice and 1a(OH)ase+/– mice were fertile and
were mated to produce offspring heterozygous at both loci, which were then
mated to generate CaR+/–; 1a(OH)ase+/– pups. Lines were maintained by
mating CaR+/–; 1a(OH)ase+/– males and females on a mixed genetic
background with contributions from 129/SvJ and BALB/c strains. CaR+/–
mice and Pth+/– mice were fertile and were mated to produce offspring
heterozygous at both loci, which were then mated to generate CaR+/–; Pth+/–
pups. These mice were maintained on a mixed genetic background with
contributions from 129/SvJ and C57BL/6J strains. Mutant mice and control
littermates were maintained in a virus- and parasite-free barrier facility and
exposed to a 12-hour light/12-hour dark cycle. In the current study, 2-weekold wild-type, CaR–/–, CaR–/–; 1a(OH)ase–/– and CaR–/–; Pth–/– mice were
used. All animal experiments were carried out in compliance with, and
approval by, the Institutional Animal Care and Use Committee.
Development 137 (6)
Genotyping of mice
Tail fragment genomic DNA was isolated by standard phenol-chloroform
extraction and isopropanol precipitation. To determine the genotype at the
CaR, Pth and 1a(OH)ase loci, six PCR amplification reactions were
conducted. To assay the presence of the wild-type CaR allele, samples were
amplified with CaR forward primer (5⬘-TCTGTTCTCTTTAGGTCCTGAAACA-3⬘) and CaR reverse primer (5⬘-TCATTGATGAACAGTCTTTCTCCCT-3⬘). To detect the presence of the null CaR allele, the Neo
forward primer r-Neo-2 (5⬘-TCTTGATTCCCACTTTGTGGTTCTA-3⬘)
was used with the CaR reverse primer. The presence of the wild-type Pth
allele was detected using the PTH forward primer (5⬘-GATGTCTGCAAACACCGTGGCTAA-3⬘) and PTH reverse primer (5⬘-TCCAAAGTTTCATTACAGTAGAAG-3⬘). The null Pth allele was detected
using the Neo forward primer r-Neo-2 and the PTH reverse primer (Kos et
al., 2003). For the wild-type 1a(OH)ase allele, forward primer (5⬘AGACTGCACTCCACTCTGAG-3⬘) and reverse primer (5⬘-GTTTCCTACACGGATGTCTC-3⬘) were used. The neomycin gene was detected
with primers neo-F (5⬘-ACAACAGACAATCGGCTGCTC-3⬘) and neo-R
(5⬘-CCATGGGTCACGACGAGATC-3⬘) (Panda et al., 2004). All PCR
reactions were performed with 1 cycle of 95°C for 4 minutes, followed by
35 cycles of 94°C for 30 seconds, 55°C for 30 seconds, 72°C for 30 seconds.
Biochemical and hormone analyses
Serum calcium and phosphorus levels were determined using an
autoanalyzer (Synchron 67, Beckman Instruments). Serum intact PTH was
measured by a two-site immunoradiometric assay (Immutopics, San
Clemente, CA, USA).
Radiography
Mandibles were removed and dissected free of soft tissue. Contact
radiographs were taken using a Faxitron model 805 radiographic inspection
system (Faxitron, München, Germany), at 22 kV voltage and with a 4minute exposure time. X-Omat TL film (Eastman Kodak, Rochester, NY,
USA) was used and processed routinely.
Micro-computed tomography (micro-CT)
Mandibles were fixed overnight in 70% ethanol and analyzed by micro-CT
with a SkyScan 1072 scanner and associated analysis software (SkyScan,
Antwerp, Belgium) as described (Xue et al., 2005). Briefly, image
acquisition was performed at 100 kV and 98 mA with a 0.98° rotation
between frames. During scanning, the samples were enclosed in tightly
fitting plastic wrap to prevent movement and dehydration. Thresholding was
applied to the images to segment the bone from the background. Twodimensional images were used to generate three-dimensional renderings
using the 3D Creator software supplied with the instrument. The resolution
of the micro-CT images is 18.2 mm.
Histology
Mandibles were removed, fixed in PLP fixative (2% paraformaldehyde
containing 0.075 M lysine and 0.01 M sodium periodate) overnight at 4°C
and processed histologically as described (Miao et al., 2001). Mandibles
were decalcified in EDTA-glycerol solution for 5-7 days at 4°C. Decalcified
right mandibles were dehydrated and embedded in paraffin, and 5 mm
sections cut on a rotary microtome. The sections were stained with
Hematoxylin and Eosin (HE), or histochemically for total collagen, alkaline
phosphatase (ALP) activity or tartrate-resistant acid phosphatase (TRAP),
or immunohistochemically as described below. Alternatively, nondecalcified left mandibles were embedded in LR White acrylic resin
(London Resin Company, London, UK) and 1-mm sections cut on an
ultramicrotome. These sections were stained for mineral by the von Kossa
staining procedure and counterstained with Toluidine Blue.
Histochemical staining for collagen, ALP and TRAP
Total collagen was detected in paraffin-embedded sections using a modified
method of Lopez-De Leon and Rojkind (Panda et al., 2004). Dewaxed
sections were exposed to 1% Sirius Red in saturated picric acid for 1 hour.
After washing with distilled water, the sections were counterstained with
Hematoxylin and mounted with Biomount medium (Canemco, Quebec,
Canada).
DEVELOPMENT
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Role of CaR in mandibular development
Immunohistochemical staining
Immunohistochemical staining was carried out for biglycan, dentin
sialoprotein (DSP), proliferating cell nuclear antigen (PCNA) and parathyroid
hormone-related protein (PTHrP) using the avidin-biotin-peroxidase complex
technique with affinity-purified rabbit anti-mouse biglycan (LF-106) antibody
(courtesy of Dr L. W. Fisher, NIDCR, NIH, Bethesda, MD, USA), affinitypurified rabbit anti-mouse dentin sialoprotein (Santa Cruz, CA, USA), mouse
anti-PCNA monoclonal antibody (Medicorp, Montreal, Canada), and rabbit
antiserum against PTHrP[1-34] as described previously (Bai et al., 2007).
Briefly, dewaxed and rehydrated paraffin-embedded sections were incubated
with methanol:hydrogen peroxide (1:10) to block endogenous peroxidase
activity and then washed in Tris-buffered saline (pH 7.6). The slides were then
incubated with the primary antibodies overnight at room temperature. After
rinsing with Tris-buffered saline for 15 minutes, tissues were incubated with
secondary antibody (biotinylated goat anti-rabbit or anti-mouse IgG, Sigma).
Sections were then washed and incubated with the Vectastain Elite ABC
reagent (Vector Laboratories) for 45 minutes. Staining was developed using
3,3-diaminobenzidine (2.5 mg/ml) followed by counterstaining with Mayer’s
Hematoxylin.
Quantitative real-time PCR
RNA was isolated from mouse mandible bodies using Trizol reagent
(Invitrogen) according to the manufacturer’s protocol. Reverse transcription
reactions were performed using the SuperScript First-Strand Synthesis
System (Invitrogen) as described (Xue et al., 2005). To determine the
number of cDNA molecules in the reverse-transcribed samples, real-time
PCR analyses were performed using the LightCycler system (Roche,
Indianapolis, IN, USA). PCR was performed using 2 ml LightCycler DNA
Master SYBR Green I (Roche), each 5⬘ and 3⬘ primer at 0.25 mM, and 2 ml
of samples or water to a final volume of 20 ml. The MgCl2 concentration was
adjusted to 3 mM. Samples were denatured at 95°C for 10 seconds, with a
temperature transition rate of 20°C/second. Amplification and fluorescence
determination were carried out in four steps: denaturation at 95°C for 10
seconds, with a temperature transition rate of 20°C/second; annealing for 5
seconds, with a temperature transition rate of 8°C/second; extension at 72°C
for 20 seconds, with a temperature transition rate of 4°C/second; and
detection of SYBR Green fluorescence, which reflects the amount of
double-stranded DNA, at 86°C for 3 seconds. Thirty-five amplification
cycles were performed. To discriminate specific from non-specific cDNA
products, a melting curve was obtained at the end of each run: products were
denatured at 95°C for 3 seconds, and the temperature was then decreased to
58°C for 15 seconds and raised slowly from 58 to 95°C using a temperature
transition rate of 0.1°C/second. To determine the number of copies of the
targeted DNA in the samples, purified PCR fragments of known
concentration were serially diluted to serve as external standards in each
experiment. Data were normalized to Gapdh levels in the samples. The
primer sequences used for the real-time PCR were as described (Miao et al.,
2004b; Xue et al., 2005).
987
Western blot analysis
Proteins were extracted from mandibular bones and quantitated using a
protein assay kit (Bio-Rad, Mississauga, Ontario, Canada). Protein samples
(30 mg) were fractionated by SDS-PAGE and transferred to nitrocellulose
membranes. Immunoblotting was carried out as described (Xue et al., 2005)
using antibodies against PTHrP[1-34] (Upstate, NY, USA) and b-tubulin
(Santa Cruz). Bands were visualized using ECL chemiluminescence
(Amersham) and quantitated by Scion Image Beta 4.02 (Scion Corporation,
NIH).
Computer-assisted image analysis
After HE staining or histochemical or immunohistochemical staining of
sections from six mice of each genotype, images of fields were
photographed with a Sony digital camera. Images of micrographs from
single sections were digitally recorded using a rectangular template, and
recordings were processed and analyzed using Northern Eclipse image
analysis software as described (Miao et al., 2001; Miao et al., 2002).
Statistical analysis
Data from image analysis are presented as mean ± s.e.m. Statistical
comparisons were made using a two-way ANOVA, with P<0.05 considered
significant.
RESULTS
Changes in serum biochemistry in wild-type and
mutant mice
First, we compared the changes in serum calcium, phosphorus and
PTH in mutant and wild-type mice. At 2 weeks of age, the CaR–/–
mice displayed hypercalcemia, hypophosphatemia and increased
serum PTH. The CaR–/–; 1a(OH)ase–/– mice displayed
normocalcemia, hypophosphatemia and more severe PTH
elevations, whereas the CaR–/–; Pth–/– mice exhibited
normocalcemia, hyperphosphatemia and undetectable serum PTH
(Fig. 1A-C).
Imaging changes in wild-type and mutant mice
We next examined the phenotypes of teeth and mandibles in 2-weekold wild-type, CaR–/–, CaR–/–; 1a(OH)ase–/– and CaR–/–; Pth–/– mice
by radiography and micro-CT scanning. The teeth and mandibles
were smaller in CaR–/– than in wild-type mice. Radiolucency was
increased in all teeth, including molars and incisors, and in the
mandibles of CaR–/– mice compared with their wild-type littermates.
The teeth and mandible were enlarged and their mineral density
increased in CaR–/–; 1a(OH)ase–/– mice compared with CaR–/–
littermates, but these parameters were still reduced significantly
compared with wild-type littermates. These parameters were,
however, normalized in CaR–/–; Pth–/– mice (Fig. 1D).
Four micro-CT-scanned sections through the incisors in front of
the first molar, and through the first, second and third molars in
mandibles, were compared between wild-type mice and the three
mutant models. The mineralized tooth volume in incisor and molars
and the mineralized cortical and alveolar bone volume in mandibles
were decreased in CaR–/– mice compared with wild-type littermates.
The mineralization defects in teeth and alveolar bone were slightly
improved in CaR–/–; 1a(OH)ase–/– mice, but were almost
completely rescued in CaR–/–; Pth–/– mice as compared with CaR–/–
littermates (Fig. 1E).
Tooth volume and dental alveolar bone volume in
mandibles
We next examined the tooth volume and dental alveolar bone
volume in mandibles, using paraffin-embedded sections through the
first molars, which were stained with HE (Fig. 2A) and
histochemically for total collagen (Fig. 2B). The dental volume (Fig.
DEVELOPMENT
Enzyme histochemistry for ALP activity was performed as described (Miao
and Scutt, 2002a). Briefly, following preincubation overnight in 100 mM
MgCl2 in 100 mM Tris-maleate buffer (pH 9.2), dewaxed sections were
incubated for 2 hours at room temperature in 100 mM Tris-maleate buffer
containing naphthol AS-MX phosphate (0.2 mg/ml, dissolved in ethylene
glycol monomethyl ether, both Sigma) as substrate and Fast Red TR (0.4
mg/ml, Sigma) as a stain for the reaction product. After washing with distilled
water, the sections were counterstained with Vector Methyl Green nuclear
counterstain (Vector Laboratories, Burlington, Ontario, Canada) and mounted
with Kaiser’s glycerol jelly.
Enzyme histochemistry for TRAP was performed using a modification of
a previously described protocol (Miao and Scutt, 2002b). Dewaxed sections
were preincubated for 20 minutes in buffer containing 50 mM sodium
acetate and 40 mM sodium tartrate (pH 5.0). Sections were then incubated
for 15 minutes at room temperature in the same buffer containing 2.5 mg/ml
naphthol AS-MX phosphate in dimethylformamide as substrate and 0.5
mg/ml Fast Garnet GBC (Sigma) as a color indicator for the reaction
product. After washing with distilled water, the sections were counterstained
with Methyl Green and mounted in Kaiser’s glycerol jelly.
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Development 137 (6)
Fig. 2. Tooth volume and dental alveolar bone volume in
mandibles. (A,B) Decalcified sections through the first molars and the
incisors from 2-week-old wild-type, CaR–/–; 1a(OH)ase–/– and CaR–/–;
Pth–/– mice that were stained with Hematoxylin and Eosin (HE) (A) or
Sirius Red for total collagen (B). Magnification, 50⫻. (C-E) Dental
volume of incisors (C) and of the first molars (D), and dental alveolar
bone volume of the mandibles (E), presented as mean ± s.e.m. of
determinations in six animals of each group. *, P<0.05; **, P<0.01,
compared with wild-type mice; #, P<0.05, compared with CaR–/– mice.
2C,D) and dental alveolar bone volume (Fig. 2E) were decreased
dramatically in CaR–/– mice compared with wild-type littermates.
Both parameters were increased in CaR–/–; 1a(OH)ase–/– mice
compared with CaR–/– littermates, but were still reduced
significantly compared with wild-type littermates (Fig. 2A-E).
Ablation of Pth in CaR–/– mice was sufficient to rescue the CaR–/–
phenotypes as the dental volume and dental alveolar bone volume
were normalized (Fig. 2A-E).
Predentin maturation and dentin formation
We then determined predentin maturation and dentin formation in
the mutant and wild-type mice. The thickness of the predentin and
mineralized dentin in the first molars and the immunoreactivity of
biglycan and dentin sialoprotein (DSP) were assessed by histology
Cell proliferation and PTHrP expression in the
apical pulp and Hertwig’s epithelial root sheath
We immunostained for proliferating cell nuclear antigen (PCNA) to
assess alterations in cell proliferation, and also for changes in PTHrP
(PTHLH – Mouse Genome Informatics) expression. In wild-type
mice, immunoreactivity of both antigens was observed in the apical
pulp, and in the nuclei of cells of Hertwig’s epithelial root sheath
(HERs). In CaR–/– mice, cells positive for both PCNA (Fig. 4A,C)
and PTHrP (Fig. 4B,D) were clearly decreased. The reductions in
PCNA- and PTHrP-positive cells in the apical pulp and HERs were
improved in CaR–/–; 1a(OH)ase–/– as compared with CaR–/– mice
(Fig. 4A-D). There was no significant difference in the numbers of
PCNA-positive cells in the CaR–/–; Pth–/– and wild-type mice (Fig.
4A,C), whereas the number of PTHrP-positive cells was increased
markedly in CaR–/–; Pth–/– mice as compared with wild-type
littermates (Fig. 4B,D). The alterations in PTHrP gene and protein
DEVELOPMENT
Fig. 1. Concentrations of serum calcium, phosphorus and PTH,
and imaging of teeth and mandibles. (A-C) Serum calcium (A),
phosphorus (B) and PTH (C) were determined in sex-matched wild-type
(WT), CaR–/–, CaR–/–; 1a(OH)ase–/– and CaR–/–; Pth–/– mice. Bars show the
mean ± s.e.m. of determinations in five mice of each genotype. **,
P<0.01; ***, P<0.001, compared with wild-type mice; #, P<0.05; ###,
P<0.001, compared with CaR–/– mice. (D) Contact radiographs of the
mandibles from 2-week-old wild-type, CaR–/–, CaR–/–; 1a(OH)ase–/– and
CaR–/–; Pth–/– mice. (E) Micro-CT-scanned sections through the incisors
in front of the first molar (In), as well as the first, second and third
molars, from wild-type, CaR–/–; 1a(OH)ase–/– and CaR–/–; Pth–/– mice.
and immunohistochemistry, respectively. The ratio of the areas of
predentin to dentin in sections stained with HE (Fig. 3A-E) and the
areas of biglycan-immunopositive dentin (Fig. 3B,F) were increased
significantly, whereas the areas of mineralized dentin (Fig. 3C,G)
and DSP immunoreactivity (Fig. 3D,H) were decreased
dramatically, in the first molars of CaR–/– mice compared with wildtype littermates. The ratio of predentin to dentin (Fig. 3A,E) and the
biglycan-immunopositive dentin areas (Fig. 3B,F) were not
significantly different between CaR–/–and CaR–/–; 1a(OH)ase–/–
mice. The areas of mineralized dentin and of DSP immunoreactivity
in the first molar were, however, increased in CaR–/–; 1a(OH)ase–/–
mice compared with CaR–/– littermates, but were still reduced
significantly compared with wild-type littermates (Fig. 3C,D,G,H).
In CaR–/–; Pth–/– mice, these parameters were all normalized (Fig.
3A-H).
Fig. 3. Predentin maturation and dentin formation in teeth.
(A-D) Sections through the root wall of the first molars and incisors
from 2-week-old wild-type, CaR–/–; 1a(OH)ase–/– and CaR–/–; Pth–/– mice
stained with HE (A), or immunohistochemically stained for biglycan (B)
or dentin sialoprotein (DSP) (D). (C) Non-decalcified sections through
the first molars and incisors stained by the von Kossa procedure.
Magnification, 400⫻. (E-H) The quantitative ratio of predentin to
dentin (E), the biglycan-positive area (F), the mineralized dentin area (G)
and the DSP-positive area (H) in the first molars were determined by
image analysis, and the percentages are presented as the mean ±
s.e.m. of determinations in six animals of each group. *, P<0.05;
**, P<0.01; ***, P<0.001, compared with wild-type mice; #, P<0.05;
##, P<0.01; ###, P<0.001, compared with CaR–/– mice.
expression in mandibular extracts, as demonstrated by real-time RTPCR (Fig. 4E) and western blots (Fig. 4F,G), were consistent with
those observed by immunohistochemistry.
Osteoblastic dental alveolar bone formation
We next determined osteoblastic dental alveolar bone formation in
paraffin-embedded sections that were stained with HE and then
histochemically for ALP. In addition, non-decalcified plasticembedded sections were stained with von Kossa, and the number
and surface area of osteoblasts, the ALP-positive area and osteoid
volume were determined by image analysis. All these parameters
were clearly increased in CaR–/– mice when compared with wildtype mice, were slightly increased in CaR–/–; 1a(OH)ase–/– mice
compared with CaR–/– mice, and were normalized in CaR–/–; Pth–/–
mice (Fig. 5A-G). We also examined the expression of genes
involved in bone formation. RNA was isolated from dental alveolar
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Fig. 4. Cell proliferation and PTHrP expression in the apical pulp,
Hertwig’s epithelial root sheath (HERs) and mandibular tissue.
(A,B) Sections through the first molars and incisors, showing the apical
pulp and HERs, from 2-week-old wild-type, CaR–/–; 1a(OH)ase–/– and
CaR–/–; Pth–/– mice stained immunohistochemically for PCNA (A) and
PTHrP (B). Magnification, 400⫻. (C,D) Numbers of PCNA-positive (C) or
PTHrP-positive (D) cells per field were determined by image analysis,
and the percentages of immunopositive cells relative to total cells are
presented as the mean ± s.e.m. of determinations in six animals of each
group. (E) Comparison of Pthrp gene expression levels in mandibular
tissue of wild-type, CaR–/–, CaR–/–; 1a(OH)ase–/– and CaR–/–; Pth–/– mice
as quantified by real-time RT-PCR. mRNA expression, normalized to that
of Gapdh, is shown relative to levels in wild-type mice. (F) Western blots
of mandibular extracts were carried out for expression of PTHrP, with btubulin as a loading control. (G) PTHrP protein levels relative to those of
b-tubulin were assessed by densitometric analysis and are expressed
relative to levels in wild-type mice. *, P<0.05; **, P<0.01;
***, P<0.001, compared with wild-type mice; #, P<0.05; ##, P<0.01;
###, P<0.001, compared with CaR–/– mice.
bone and real-time RT-PCR performed. Expression of the
osteoblastic genes Cbfa1 (Runx2 – Mouse Genome Informatics),
ALP, type I collagen and osteocalcin (Bglap1– Mouse Genome
Informatics) was increased dramatically in CaR–/– and CaR–/–;
1a(OH)ase–/– mice and was normalized in CaR–/–; Pth–/– mice (Fig.
5H-K).
Osteoclastic dental alveolar bone resorption
Osteoclastic bone resorption in dental alveolar bone was examined
in paraffin-embedded sections stained histochemically for TRAP.
Osteoclast number and surface were determined by image analysis.
The number of TRAP-positive osteoclasts was increased in the
dental alveolar bone in CaR–/– and CaR–/–; 1a(OH)ase–/– mice
compared with their wild-type littermates, but there was no
significant difference in the TRAP-positive osteoclast surface (Fig.
6A-C). TRAP-positive osteoclast number and surface area were not
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Role of CaR in mandibular development
990
RESEARCH ARTICLE
Development 137 (6)
Fig. 5. Osteoblasts and osteoblastic gene expression in
mandibles. (A-C) Sections of mandibles from 2-week-old wild-type,
CaR–/–, CaR–/–; 1a(OH)ase–/– and CaR–/–; Pth–/– mice were stained with
HE (A) or histochemically for ALP (B). (C) Non-decalcified sections of
mandibles stained by the von Kossa procedure. Magnification, 400⫻.
(D,E) The number of osteoblasts (# N.Ob) per mm bone perimeter
(B.Pm) (D) and the surface of osteoblasts (Ob.S) as a percentage of
bone surface (B.S) (E) were determined in the dental alveolar bone of
HE-stained mandibles. (F) ALP-positive area as a percentage of the
tissue area in the dental alveolar bone. (G) Osteoid volume (OV) in nondecalcified von Kossa-stained sections as a percentage of bone volume
(BV) of trabeculae. (H-K) Expression of Cbfa1 (H), ALP (I), type I collagen
(J) and osteocalcin (K) as assessed by real-time RT-PCR of mandibular
extracts. mRNA expression normalized to Gapdh is shown relative to
levels in wild-type mice as the mean ± s.e.m. of determinations in six
animals of the same genotype. *, P<0.05; **, P<0.01; ***, P<0.001,
compared with wild-type mice; #, P<0.05; ##, P<0.01; ###, P<0.001
compared with CaR–/– mice.
altered significantly in CaR–/–; Pth–/– mice (Fig. 6A-C). The ratio of
RANKL/OPG (Tnfsf11/Tnfrsf11b – Mouse Genome Informatics)
mRNA levels showed no significant differences among the three
knockout models and wild-type mice, as demonstrated by real-time
RT-PCR (Fig. 6D).
DISCUSSION
We used CaR–/– mice, and CaR–/–; 1a(OH)ase–/– and CaR–/–; Pth–/–
double-mutant mice and analyzed them biochemically, by
radiography, micro-CT imaging, histology, immunohistochemistry,
real-time RT-PCR and western blotting, to gain insight into the role
of CaR and of calcium, phosphorus, 1,25(OH)2D, PTH and PTHrP
in the formation of teeth and the development of dental alveolar
bone.
We found that in CaR–/– mice, the area of biglycanimmunopositive predentin was increased, but the dental volume and
DSP level were reduced. We also found that osteoid volume in
dental alveolar bone was increased, but that dental alveolar bone
volume was reduced dramatically, although the number of ALPpositive osteoblasts was increased. These results demonstrate that
CaR deficiency produces defects in dental and dental alveolar bone
mineralization and formation.
CaR–/– mice develop primary hyperparathyroidism and are
hypercalcemic. To determine whether the hypercalcemia in CaR–/–
mice contributed to the defects in dental and dental alveolar bone
development in these animals, we bred them with mice in which
1a(OH)ase had been deleted. We recently examined 1a(OH)ase–/–
mice, which are hypocalcemic and display secondary
hyperparathyroidism and resultant hypophosphatemia, to assess the
effects of 1,25(OH)2D deficiency on dental and dental alveolar bone
formation and mineralization in the mandibles. These studies
revealed defects in dental and dental alveolar bone formation and
mineralization (Liu et al., 2009). These defects were, however, less
severe than in CaR–/– mice. In the present studies, after deletion of
1a(OH)ase in CaR–/– mice in order to correct the hypercalcemia, a
mild improvement in dental and dental alveolar bone formation was
observed. This included slight increases in dental volume, DSP
production and dental alveolar bone volume. By contrast,
mineralization of the teeth and dental alveolar bone was not
improved. These results therefore suggest that correction of the
hypercalcemia partly corrects dental and dental alveolar bone
formation, but does not correct the abnormal mineralization. In view
of the failure to completely rescue the defects of dental and dental
alveolar bone formation and mineralization in CaR–/– mice by
concomitant deletion of 1a(OH)ase, we sought to determine
whether these abnormalities were related to the more marked
hyperparathyroidism and the presence of hypophosphatemia in the
CaR–/–; 1a(OH)ase–/– mice, by deleting Pth from CaR–/– mice.
Ablation of Pth in CaR–/– mice has previously been reported to
reverse the hyperparathyroidism and the associated hypercalcemia
and hypophosphatemia (Kos et al., 2003; Tu et al., 2003), and to
rescue the rachitic skeletal phenotype (Kos et al., 2003; Tu et al.,
2003). In the present study, ablation of Pth in CaR–/– mice
DEVELOPMENT
Fig. 6. Osteoclasts in mandibles. (A) Sections of mandibles stained
histochemically for tartrate-resistant acid phosphatase (TRAP) activity.
Magnification, 200⫻. (B,C) The number of TRAP-positive osteoclasts
(N.Oc) per mm bone perimeter (B.Pm) (B) and the surface area of
osteoclasts (Oc.S) as a percentage of the bone surface (B.S) (C) were
determined in the dental alveolar bone of TRAP-stained mandibles.
Each value is the mean ± s.e.m. of determinations in six animals of each
group. (D) RANKL and OPG mRNA levels in mandibular extracts as
assessed by real-time RT-PCR, normalized to Gapdh and shown relative
to levels in wild-type mice. RANKL/OPG ratios are mean ± s.e.m. of
determinations in six animals of the same genotype. *, P<0.05;
**, P<0.01, compared with wild-type mice.
ameliorated the defects in dental and dental alveolar bone formation
and mineralization. Our results therefore suggest that
hyperparathyroidism is the major factor contributing to defects in
the dental and dental alveolar bone development caused by CaR
deficiency, at least in part via the accompanying hypophosphatemia.
PTHrP is a ubiquitously produced local paracrine/autocrine/
intracrine factor, the role of which is to regulate cellular
proliferation, differentiation and differentiated function, as well as
cell death, both during development and in adult life (Bisello et al.,
2004; Fiaschi-Taesch and Stewart, 2003; Miao et al., 2008). Because
PTHrP can modulate bone development both by binding to the type
I PTH/PTHrP receptor (PTH1R – Mouse Genome Informatics)
(Juppner et al., 1991) and by an intracrine mode of action (Miao et
al., 2008), we assessed whether defects in teeth and mandible
development are associated with any alteration of PTHrP expression
in teeth and mandibles. Our results showed that PTHrPimmunopositive cells in the apical pulp and HERs, as well as PTHrP
mRNA and protein levels in mandibular tissues, including teeth,
were clearly decreased in CaR–/– mice. These parameters were only
slightly increased in CaR–/–; 1a(OH)ase–/– relative to CaR–/– mice,
but were increased markedly in CaR–/–; Pth–/– mice compared with
their wild-type littermates. Previous studies have demonstrated that
elevated extracellular calcium acting via CaR can increase PTHrP
release (Ahlstrom et al., 2008; Tfelt-Hansen et al., 2003). Our results
support the possibility that extracellular calcium stimulates PTHrP
production via CaR and that CaR deficiency contributed to the low
PTHrP expression in the CaR–/– mice. The secosteroid 1,25(OH)2D3
has been reported to downregulate PTHrP (Kremer et al., 1996;
Tovar Sepulveda and Falzon, 2002), and we found that deletion of
1a(OH)ase in CaR-deficient mice upregulated PTHrP gene and
protein expression in teeth and mandibles. This result suggests that
1,25(OH)2D3 modulates PTHrP production in a CaR-independent
fashion. Although 1,25(OH)2D levels are reduced in CaR–/–; Pth–/–
mice, the levels are still higher than in CaR–/–; 1a(OH)ase–/– mice,
yet PTHrP gene and protein expression were upregulated to a greater
degree in CaR–/–; Pth–/– than in CaR–/–; 1a(OH)ase–/– mice.
Reduced 1,25(OH)2D might, therefore, mediate increased PTHrP
expression in these PTH-deficient mice, but other mechanisms
might be operative as well. In CaR–/– mice, the downregulation of
PTHrP expression in mandibular tissues, including teeth, was
associated with the defects in teeth and mandible caused by CaR
deficiency. Conversely, upregulation of PTHrP expression was
associated with either improvement of the teeth and mandible
phenotypes in CaR–/–; 1a(OH)ase–/– mice or rescue of these
phenotypes in CaR–/–; Pth–/– mice. We previously reported that
PTHrP is required for increased trabecular bone volume in Pth–/–
mice (Miao et al., 2004b). Our present studies suggest that PTHrP
might play a local anabolic role in teeth and mandibular tissues, as
well as in bone.
Our studies and the studies of others have shown that osteoblast
numbers and trabecular bone volume in long bones are increased
significantly in 1a(OH)ase–/– mice and Vdr–/– mice on a normal or
even on a high calcium intake (Panda et al., 2004). This increase
disappeared when circulating PTH was normalized and is, therefore,
attributable to the anabolic effect of PTH. In the present study,
however, circulating PTH levels, osteoblast numbers in the
mandibles and the expression levels of several osteoblast-related
genes were all increased in CaR–/– and CaR–/–; 1a(OH)ase–/– mice,
but dental alveolar bone volume was not increased. Because alveolar
bone volume was restored in CaR–/–; Pth–/– mice, the linkage of
osteoblast activity to alveolar bone volume did not appear to be
related directly to CaR deficiency. Consequently, hypophosphatemia
RESEARCH ARTICLE
991
per se, low PTHrP, or other factors yet to be determined, might play
a role in preventing the coupling of the enhanced osteoblast activity
to the development of increased alveolar bone volume in CaR–/– and
CaR–/–; 1a(OH)ase–/– mice.
In summary, our data suggest that CaR is crucial for the
development of teeth and alveolar bone postnatally. It is uncertain,
however, what direct role, if any, CaR has on dental and dental
alveolar bone formation. A recent study demonstrated that deletion
of CaR in osteoblasts resulted in profound bone defects (Chang et
al., 2008); however, the mechanism underlying any putative direct
action of CaR on skeletal development remains unclear. The role
of CaR in teeth and alveolar bone formation may therefore be, at
least in part, to directly regulate the levels of ambient calcium
(Brown et al., 1993; Brown and MacLeod, 2001) and PTHrP
(Ahlstrom et al., 2008; Tfelt-Hansen et al., 2003) and to indirectly
regulate phosphorus levels by altering PTH levels (Brown et al.,
1993; Brown and MacLeod, 2001). These analytes, per se, may
then modulate the postnatal development of teeth and alveolar
bone.
Acknowledgements
This work was supported by Key Project grants from the National Natural
Science Foundation of China (No. 30830103) and by the Program for
Changjiang Scholars and Innovative Research Team in University (to D.M.), a
grant from the Canadian Institutes for Health Research (to D.G.), and a grant
from the National Institutes of Health (DK078331to E.B. and DK070756 to
M.R.P.). Deposited in PMC for release after 12 months.
Competing interests statement
The authors declare no competing financial interests.
References
Ahlstrom, M., Pekkinen, M., Riehle, U. and Lamberg-Allardt, C. (2008).
Extracellular calcium regulates parathyroid hormone-related peptide expression
in osteoblasts and osteoblast progenitor cells. Bone 42, 483-490.
Bai, X., Miao, D., Goltzman, D. and Karaplis, A. C. (2007). Early lethality in Hyp
mice with targeted deletion of Pth gene. Endocrinology 148, 4974-4983.
Beckerman, P. and Silver, J. (1999). Vitamin D and the parathyroid. Am. J. Med.
Sci. 317, 363-369.
Bisello, A., Horwitz, M. J. and Stewart, A. F. (2004). Parathyroid hormonerelated protein: an essential physiological regulator of adult bone mass.
Endocrinology 145, 3551-3553.
Brenza, H. L., Kimmel-Jehan, C., Jehan, F., Shinki, T., Wakino, S., Anazawa,
H., Suda, T. and DeLuca, H. F. (1998). Parathyroid hormone activation of the
25-hydroxyvitamin D3-1alpha-hydroxylase gene promoter. Proc. Natl. Acad. Sci.
USA 95, 1387-1391.
Brown, E. M. and MacLeod, R. J. (2001). Extracellular calcium sensing and
extracellular calcium signaling. Physiol. Rev. 81, 239-297.
Brown, E. M., Gamba, G., Riccardi, D., Lombardi, M., Butters, R., Kifor, O.,
Sun, A., Hediger, M. A., Lytton, J. and Hebert, S. C. (1993). Cloning and
characterization of an extracellular Ca(2+)-sensing receptor from bovine
parathyroid. Nature 366, 575-580.
Cantley, L. K., Russell, J., Lettieri, D. and Sherwood, L. M. (1985). 1,25Dihydroxyvitamin D3 suppresses parathyroid hormone secretion from bovine
parathyroid cells in tissue culture. Endocrinology 117, 2114-2119.
Chan, Y. L., McKay, C., Dye, E. and Slatopolsky, E. (1986). The effect of 1,25
dihydroxycholecalciferol on parathyroid hormone secretion by monolayer
cultures of bovine parathyroid cells. Calcif. Tissue Int. 38, 27-32.
Chang, W., Tu, C., Chen, T. H., Bikle, D. and Shoback, D. (2008). The
extracellular calcium-sensing receptor (CaSR) is a critical modulator of skeletal
development. Sci. Signal. 1, ra1.
Dardenne, O., Prud’homme, J., Arabian, A., Glorieux, F. H. and St-Arnaud, R.
(2001). Targeted inactivation of the 25-hydroxyvitamin D(3)-1(alpha)-hydroxylase
gene (CYP27B1) creates an animal model of pseudovitamin D-deficiency rickets.
Endocrinology 142, 3135-3141.
Dvorak, M. M., Siddiqua, A., Ward, D. T., Carter, D. H., Dallas, S. L., Nemeth,
E. F. and Riccardi, D. (2004). Physiological changes in extracellular calcium
concentration directly control osteoblast function in the absence of calciotropic
hormones. Proc. Natl. Acad. Sci. USA 101, 5140-5145.
Fiaschi-Taesch, N. M. and Stewart, A. F. (2003). Parathyroid hormone-related
protein as an intracrine factor-trafficking mechanisms and functional
consequences. Endocrinology 144, 407-411.
Fraser, D., Kooh, S. W., Kind, H. P., Holick, M. F., Tanaka, Y. and DeLuca, H. F.
(1973). Pathogenesis of hereditary vitamin-D-dependent rickets. An inborn error
DEVELOPMENT
Role of CaR in mandibular development
RESEARCH ARTICLE
of vitamin D metabolism involving defective conversion of 25-hydroxyvitamin D
to 1 alpha,25-dihydroxyvitamin D. N. Engl. J. Med. 289, 817-822.
Garner, S. C., Pi, M., Tu, Q. and Quarles, L. D. (2001). Rickets in cation-sensing
receptor-deficient mice: an unexpected skeletal phenotype. Endocrinology 142,
3996-4005.
Ho, C., Conner, D. A., Pollak, M. R., Ladd, D. J., Kifor, O., Warren, H. B.,
Brown, E. M., Seidman, J. G. and Seidman, C. E. (1995). A mouse model of
human familial hypocalciuric hypercalcemia and neonatal severe
hyperparathyroidism. Nat. Genet. 11, 389-394.
Juppner, H., Abou-Samra, A. B., Freeman, M., Kong, X. F., Schipani, E.,
Richards, J., Kolakowski, L. F., Jr, Hock, J., Potts, J. T., Jr, Kronenberg, H. M.
et al. (1991). A G protein-linked receptor for parathyroid hormone and
parathyroid hormone-related peptide. Science 254, 1024-1026.
Kos, C. H., Karaplis, A. C., Peng, J. B., Hediger, M. A., Goltzman, D.,
Mohammad, K. S., Guise, T. A. and Pollak, M. R. (2003). The calcium-sensing
receptor is required for normal calcium homeostasis independent of parathyroid
hormone. J. Clin. Invest. 111, 1021-1028.
Kremer, R., Sebag, M., Champigny, C., Meerovitch, K., Hendy, G. N., White,
J. and Goltzman, D. (1996). Identification and characterization of 1,25dihydroxyvitamin D3-responsive repressor sequences in the rat parathyroid
hormone-related peptide gene. J. Biol. Chem. 271, 16310-16316.
Liu, H., Guo, J., Wang, L., Chen, N., Karaplis, A., Goltzman, D. and Miao, D.
(2009). Distinctive anabolic roles of 1,25-dihydroxyvitamin D3 and parathyroid
hormone in teeth and mandible versus long bones. J. Endocrinol. 203, 203213.
Liu, S. M., Koszewski, N., Lupez, M., Malluche, H. H., Olivera, A. and Russell,
J. (1996). Characterization of a response element in the 5⬘-flanking region of
the avian (chicken) PTH gene that mediates negative regulation of gene
transcription by 1,25-dihydroxyvitamin D3 and binds the vitamin D3 receptor.
Mol. Endocrinol. 10, 206-215.
Marks, S. C., Jr and Schroeder, H. E. (1996). Tooth eruption: theories and facts.
Anat. Rec. 245, 374-393.
Mathias, R. S., Mathews, C. H., Machule, C., Gao, D., Li, W. and Denbesten,
P. K. (2001). Identification of the calcium-sensing receptor in the developing
tooth organ. J. Bone Miner. Res. 16, 2238-2244.
Miao, D. and Scutt, A. (2002a). Histochemical localization of alkaline
phosphatase activity in decalcified bone and cartilage. J. Histochem. Cytochem.
50, 333-340.
Miao, D. and Scutt, A. (2002b). Recruitment, augmentation and apoptosis of rat
osteoclasts in 1,25-(OH)2D3 response to short-term treatment with 1,25dihydroxyvitamin D3 in vivo. BMC Musculoskelet. Disord. 3, 16.
Miao, D., Bai, X., Panda, D., McKee, M., Karaplis, A. and Goltzman, D.
(2001). Osteomalacia in hyp mice is associated with abnormal phex expression
and with altered bone matrix protein expression and deposition. Endocrinology
142, 926-939.
Miao, D., He, B., Karaplis, A. C. and Goltzman, D. (2002). Parathyroid hormone
is essential for normal fetal bone formation. J. Clin. Invest. 109, 1173-1182.
Development 137 (6)
Miao, D., He, B., Lanske, B., Bai, X. Y., Tong, X. K., Hendy, G. N., Goltzman,
D. and Karaplis, A. C. (2004a). Skeletal abnormalities in Pth-null mice are
influenced by dietary calcium. Endocrinology 145, 2046-2053.
Miao, D., Li, J., Xue, Y., Su, H., Karaplis, A. C. and Goltzman, D. (2004b).
Parathyroid hormone-related peptide is required for increased trabecular bone
volume in parathyroid hormone-null mice. Endocrinology 145, 3554-3562.
Miao, D., Su, H., He, B., Gao, J., Xia, Q., Zhu, M., Gu, Z., Goltzman, D. and
Karaplis, A. C. (2008). Severe growth retardation and early lethality in mice
lacking the nuclear localization sequence and C-terminus of PTH-related protein.
Proc. Natl. Acad. Sci. USA 105, 20309-20314.
Murayama, A., Takeyama, K., Kitanaka, S., Kodera, Y., Hosoya, T. and Kato, S.
(1998). The promoter of the human 25-hydroxyvitamin D3 1 alpha-hydroxylase
gene confers positive and negative responsiveness to PTH, calcitonin, and 1
alpha,25(OH)2D3. Biochem. Biophys. Res. Commun. 249, 11-16.
Panda, D. K., Miao, D., Tremblay, M. L., Sirois, J., Farookhi, R., Hendy, G. N.
and Goltzman, D. (2001). Targeted ablation of the 25-hydroxyvitamin D
1alpha-hydroxylase enzyme: evidence for skeletal, reproductive, and immune
dysfunction. Proc. Natl. Acad. Sci. USA 98, 7498-7503.
Panda, D. K., Miao, D., Bolivar, I., Li, J., Huo, R., Hendy, G. N. and Goltzman,
D. (2004). Inactivation of the 25-hydroxyvitamin D 1alpha-hydroxylase and
vitamin D receptor demonstrates independent and interdependent effects of
calcium and vitamin D on skeletal and mineral homeostasis. J. Biol. Chem. 279,
16754-16766.
Pollak, M. R., Brown, E. M., Chou, Y. H., Hebert, S. C., Marx, S. J.,
Steinmann, B., Levi, T., Seidman, C. E. and Seidman, J. G. (1993). Mutations
in the human Ca(2+)-sensing receptor gene cause familial hypocalciuric
hypercalcemia and neonatal severe hyperparathyroidism. Cell 75, 1297-1303.
Roberts, W. E. (1999). Bone dynamics of osseointegration, ankylosis, and tooth
movement. J. Indiana Dent. Assoc. 78, 24-32.
Szabo, A., Merke, J., Beier, E., Mall, G. and Ritz, E. (1989). 1,25(OH)2 vitamin
D3 inhibits parathyroid cell proliferation in experimental uremia. Kidney Int. 35,
1049-1056.
Tfelt-Hansen, J., MacLeod, R. J., Chattopadhyay, N., Yano, S., Quinn, S., Ren,
X., Terwilliger, E. F., Schwarz, P. and Brown, E. M. (2003). Calcium-sensing
receptor stimulates PTHrP release by pathways dependent on PKC, p38 MAPK,
JNK, and ERK1/2 in H-500 cells. Am. J. Physiol. Endocrinol. Metab. 285, E329E337.
Tovar Sepulveda, V. A. and Falzon, M. (2002). Regulation of PTH-related protein
gene expression by vitamin D in PC-3 prostate cancer cells. Mol. Cell. Endocrinol.
190, 115-124.
Tu, Q., Pi, M., Karsenty, G., Simpson, L., Liu, S. and Quarles, L. D. (2003).
Rescue of the skeletal phenotype in CasR-deficient mice by transfer onto the
Gcm2 null background. J. Clin. Invest. 111, 1029-1037.
Xue, Y., Karaplis, A. C., Hendy, G. N., Goltzman, D. and Miao, D. (2005).
Genetic models show that parathyroid hormone and 1,25-dihydroxyvitamin D3
play distinct and synergistic roles in postnatal mineral ion homeostasis and
skeletal development. Hum. Mol. Genet. 14, 1515-1528.
DEVELOPMENT
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