INTRODUCTIONThe calcium-sensing receptor (CaR; CASR – Mouse GenomeInformatics) is a plasma membrane G-protein-coupled receptor thatis activated by extracellular calcium. CaR plays a central role incontrolling systemic calcium homeostasis, predominately throughits effects on the regulation of parathyroid hormone (PTH) secretionby the parathyroid glands and on urinary calcium excretion by thekidney (Brown et al., 1993; Brown and MacLeod, 2001). Evidencefor its crucial role in parathyroid function came from theidentification of inactivating mutations in CaR in familialhypocalciuric hypercalcemia (FHH) and neonatal severehyperparathyroidism (NSHPT) (Pollak et al., 1993). Patients withFHH are heterozygous for inactivating mutations in CaR and exhibitmild to moderate hypercalcemia, normal or mildly increasedcirculating PTH levels, and parathyroid histology that ranges fromnormal to mild hyperplasia. Patients with NSHPT are homozygousfor inactivating CaR mutations and have severe, life-threateninghypercalcemia accompanied by very high circulating PTH levelsand marked parathyroid hyperplasia. Targeted inactivation of CaRin mice has resulted in the development of models of the human
syndromes (Ho et al., 1995). Thus, mice with a heterozygousdeletion of the CaR gene mimic FHH, whereas homozygotes mimicNSHPT and generally die within a few days to weeks after birth.Interestingly, crossing CaR-knockout (CaR–/–) mice with Pth–/– micerescues the lethal phenotype of the CaR–/– mice, indicating thatlethality is due to severe hypercalcemia caused by markedhyperparathyroidism (Kos et al., 2003).
CaR–/– mice exhibit classic features of rickets, including growthretardation, expanded growth plates with reduced calcification,widened metaphyses and impaired bone mineralization (Garner etal., 2001; Ho et al., 1995). These abnormalities are rescued byconcomitant knockout of Pth. It is unknown, however, whether lossof CaR impacts tooth formation and dental alveolar bonedevelopment. CaR is expressed in the mandible and developingtooth, and might provide a mechanism for sensing and respondingto the alterations in extracellular calcium concentrations that takeplace during the formation of the mandibles and teeth (Dvorak et al.,2004; Mathias et al., 2001). Calcium is, of course, a key componentof teeth. It is found in the enamel, dentin and the surroundingextracellular matrix. Moreover, the teeth and dental alveolar boneare highly active tissues that constantly undergo remodelingthroughout the life cycle (Marks and Schroeder, 1996; Roberts,1999). Thus, CaR could potentially play important roles in theformation and development of the teeth and dental alveolar bone.
The vitamin D-PTH axis plays a central role in calcium andphosphorus homeostasis and is essential for skeletal developmentand mineralization. PTH and 1,25-dihydroxyvitamin D3
[1,25(OH)2D3] directly affect calcium homeostasis and each exertsimportant regulatory effects on the other. PTH stimulates theproduction of 1,25(OH)2D3 by activating the renal 25-
1Institute of Dental Research, Stomatological College and 2The Research Center forBone and Stem Cells, Nanjing Medical University, Nanjing, Jiangsu 210029, P. R. ofChina . 3Department of Medicine, McGill University, Montreal, H3A 1A1 Quebec,Canada. 4Department of Medicine, Brigham and Women’s Hospital, HarvardMedical School, Boston, MA 02115, USA.
SUMMARYTo determine whether the calcium-sensing receptor (CaR) participates in tooth formation and dental alveolar bone development inmandibles in vivo, we examined these processes, as well as mineralization, in 2-week-old CaR-knockout (CaR–/–) mice. We alsoattempted to rescue the phenotype of CaR–/– mice by genetic means, in mice doubly homozygous for CaR and 25-hydroxyvitamin D1a-hydroxylase [1a(OH)ase] or parathyroid hormone (Pth). In CaR–/– mice, which exhibited hypercalcemia, hypophosphatemia andincreased serum PTH, the volumes of teeth and of dental alveolar bone were decreased dramatically, whereas the ratio of the areaof predentin to total dentin and the number and surface of osteoblasts in dental alveolar bone were increased significantly, ascompared with wild-type littermates. The normocalcemia present in CaR–/–; 1a(OH)ase–/– mice only slightly improved the defects indental and alveolar bone formation observed in the hypercalcemic CaR–/– mice. However, these defects were completely rescued bythe additional elimination of hypophosphatemia and by an increase in parathyroid hormone-related protein (PTHrP) expression inthe 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 indicatesthat CaR participates in the formation of teeth and in the development of dental alveolar bone in mandibles in vivo, although itappears to do so largely indirectly.
Alterations in phosphorus, calcium and PTHrP contribute todefects in dental and dental alveolar bone formation incalcium-sensing receptor-deficient miceWen Sun1, Weiwei Sun2, Jingning Liu2, Xichao Zhou1, Yongjun Xiao3, Andrew Karaplis3, Martin R. Pollak4,Edward Brown4, David Goltzman3 and Dengshun Miao1,2,*
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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 productionof PTH (Cantley et al., 1985; Chan et al., 1986) and controlsparathyroid cell growth (Szabo et al., 1989). Suppression of PTHsynthesis by 1,25(OH)2D3 occurs through negative regulation of Pthtranscription by a 1,25(OH)2D3-vitamin D receptor (VDR)-retinoidX 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 isdeficient in PTH by targeting the Pth gene in embryonic stemcells. Although adult Pth-null mice develop hypocalcemia,hyperphosphatemia and low circulating 1,25(OH)2D3 levelsconsistent with primary hypoparathyroidism (Miao et al., 2004a), thisphenotype is not lethal. We (Panda et al., 2001) and others (Dardenneet al., 2001) have also previously reported the generation of a mousemodel that is deficient in 1,25(OH)2D as a result of targeted ablationof the 1a(OH)ase gene. After weaning, 1a(OH)ase–/– mice thatare fed a diet of regular mouse chow developed secondaryhyperparathyroidism, retarded growth and the skeletal abnormalitiescharacteristic of rickets. These abnormalities mimic those describedin 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 apredominant role in appositional bone growth, whereas 1,25(OH)2D3
acts predominantly on endochondral bone formation, and both playcollaborative 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 andintramembranous bone formation remain unknown. We thereforeused CaR–/– mice, CaR–/–; 1a(OH)ase–/– and CaR–/–; Pth–/– double-homozygous mice to dissect the individual contributions of calcium(acting via CaR), phosphorus, 1,25(OH)2D and PTH to the formationof teeth and dental alveolar bone.
MATERIALS AND METHODSDerivation of CaR–/–, CaR–/–; 1a(OH)ase–/– and CaR–/–; Pth–/– miceThe derivation of the three parental strains of CaR–/–, 1a(OH)ase–/– andPth–/– mice by homologous recombination in embryonic stem cells has beendescribed (Ho et al., 1995; Panda et al., 2001; Miao et al., 2002). Briefly, aneomycin resistance gene was inserted into exon 5 of the mouse CaR gene.Western blot analysis of kidney protein membrane extracts from CaR–/– miceconfirmed that no detectable protein is expressed from this allele in thistissue (Ho et al., 1995). A neomycin resistance gene replaced exons 6-8 ofthe mouse 1a(OH)ase gene, removing both the ligand-binding and theheme-binding domains (Panda et al., 2001). RT-PCR of renal RNA from1a(OH)ase–/– mice confirmed the lack of 1a(OH)ase expression. Aneomycin resistance gene was inserted into exon 3 of the mouse Pth gene,resulting in replacement of the entire coding sequence of mature PTH. Lackof PTH expression in parathyroid glands was confirmed by immunostaining(Miao et al., 2002). CaR+/– mice and 1a(OH)ase+/– mice were fertile andwere mated to produce offspring heterozygous at both loci, which were thenmated to generate CaR+/–; 1a(OH)ase+/– pups. Lines were maintained bymating CaR+/–; 1a(OH)ase+/– males and females on a mixed geneticbackground with contributions from 129/SvJ and BALB/c strains. CaR+/–
mice and Pth+/– mice were fertile and were mated to produce offspringheterozygous at both loci, which were then mated to generate CaR+/–; Pth+/–
pups. These mice were maintained on a mixed genetic background withcontributions from 129/SvJ and C57BL/6J strains. Mutant mice and controllittermates were maintained in a virus- and parasite-free barrier facility andexposed to a 12-hour light/12-hour dark cycle. In the current study, 2-week-old wild-type, CaR–/–, CaR–/–; 1a(OH)ase–/– and CaR–/–; Pth–/– mice wereused. All animal experiments were carried out in compliance with, andapproval by, the Institutional Animal Care and Use Committee.
Genotyping of miceTail fragment genomic DNA was isolated by standard phenol-chloroformextraction and isopropanol precipitation. To determine the genotype at theCaR, Pth and 1a(OH)ase loci, six PCR amplification reactions wereconducted. To assay the presence of the wild-type CaR allele, samples wereamplified with CaR forward primer (5�-TCTGTTCTCTTTAGGTC -CTGAAACA-3�) and CaR reverse primer (5�-TCATTGATGAACAG -TCTTTC TCCCT-3�). To detect the presence of the null CaR allele, the Neoforward primer r-Neo-2 (5�-TCTTGATTCCCACTTTGTGGTTCTA-3�)was used with the CaR reverse primer. The presence of the wild-type Pthallele was detected using the PTH forward primer (5�-GATGTCT -GCAAACACCGTGGCTAA-3�) and PTH reverse primer (5�-TCCA -AAGTTTCATTACAGTAGAAG-3�). The null Pth allele was detectedusing the Neo forward primer r-Neo-2 and the PTH reverse primer (Kos etal., 2003). For the wild-type 1a(OH)ase allele, forward primer (5�-AGACTGCACTCCACTCTGAG-3�) and reverse primer (5�-GTTT -CCTACACGGATGTCTC-3�) were used. The neomycin gene was detectedwith primers neo-F (5�-ACAACAGACAATCGGCTGCTC-3�) and neo-R(5�-CCATGGGTCACGACGAGATC-3�) (Panda et al., 2004). All PCRreactions were performed with 1 cycle of 95°C for 4 minutes, followed by35 cycles of 94°C for 30 seconds, 55°C for 30 seconds, 72°C for 30 seconds.
Biochemical and hormone analysesSerum calcium and phosphorus levels were determined using anautoanalyzer (Synchron 67, Beckman Instruments). Serum intact PTH wasmeasured by a two-site immunoradiometric assay (Immutopics, SanClemente, CA, USA).
RadiographyMandibles were removed and dissected free of soft tissue. Contactradiographs were taken using a Faxitron model 805 radiographic inspectionsystem (Faxitron, München, Germany), at 22 kV voltage and with a 4-minute 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-CTwith a SkyScan 1072 scanner and associated analysis software (SkyScan,Antwerp, Belgium) as described (Xue et al., 2005). Briefly, imageacquisition was performed at 100 kV and 98 mA with a 0.98° rotationbetween frames. During scanning, the samples were enclosed in tightlyfitting plastic wrap to prevent movement and dehydration. Thresholding wasapplied to the images to segment the bone from the background. Two-dimensional images were used to generate three-dimensional renderingsusing the 3D Creator software supplied with the instrument. The resolutionof the micro-CT images is 18.2 mm.
HistologyMandibles were removed, fixed in PLP fixative (2% paraformaldehydecontaining 0.075 M lysine and 0.01 M sodium periodate) overnight at 4°Cand processed histologically as described (Miao et al., 2001). Mandibleswere decalcified in EDTA-glycerol solution for 5-7 days at 4°C. Decalcifiedright mandibles were dehydrated and embedded in paraffin, and 5 mmsections cut on a rotary microtome. The sections were stained withHematoxylin and Eosin (HE), or histochemically for total collagen, alkalinephosphatase (ALP) activity or tartrate-resistant acid phosphatase (TRAP),or immunohistochemically as described below. Alternatively, non-decalcified left mandibles were embedded in LR White acrylic resin(London Resin Company, London, UK) and 1-mm sections cut on anultramicrotome. These sections were stained for mineral by the von Kossastaining procedure and counterstained with Toluidine Blue.
Histochemical staining for collagen, ALP and TRAPTotal collagen was detected in paraffin-embedded sections using a modifiedmethod of Lopez-De Leon and Rojkind (Panda et al., 2004). Dewaxedsections were exposed to 1% Sirius Red in saturated picric acid for 1 hour.After washing with distilled water, the sections were counterstained withHematoxylin and mounted with Biomount medium (Canemco, Quebec,Canada).
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Enzyme histochemistry for ALP activity was performed as described (Miaoand Scutt, 2002a). Briefly, following preincubation overnight in 100 mMMgCl2 in 100 mM Tris-maleate buffer (pH 9.2), dewaxed sections wereincubated for 2 hours at room temperature in 100 mM Tris-maleate buffercontaining naphthol AS-MX phosphate (0.2 mg/ml, dissolved in ethyleneglycol monomethyl ether, both Sigma) as substrate and Fast Red TR (0.4mg/ml, Sigma) as a stain for the reaction product. After washing with distilledwater, the sections were counterstained with Vector Methyl Green nuclearcounterstain (Vector Laboratories, Burlington, Ontario, Canada) and mountedwith Kaiser’s glycerol jelly.
Enzyme histochemistry for TRAP was performed using a modification ofa previously described protocol (Miao and Scutt, 2002b). Dewaxed sectionswere preincubated for 20 minutes in buffer containing 50 mM sodiumacetate and 40 mM sodium tartrate (pH 5.0). Sections were then incubatedfor 15 minutes at room temperature in the same buffer containing 2.5 mg/mlnaphthol AS-MX phosphate in dimethylformamide as substrate and 0.5mg/ml Fast Garnet GBC (Sigma) as a color indicator for the reactionproduct. After washing with distilled water, the sections were counterstainedwith Methyl Green and mounted in Kaiser’s glycerol jelly.
Immunohistochemical stainingImmunohistochemical staining was carried out for biglycan, dentinsialoprotein (DSP), proliferating cell nuclear antigen (PCNA) and parathyroidhormone-related protein (PTHrP) using the avidin-biotin-peroxidase complextechnique with affinity-purified rabbit anti-mouse biglycan (LF-106) antibody(courtesy of Dr L. W. Fisher, NIDCR, NIH, Bethesda, MD, USA), affinity-purified rabbit anti-mouse dentin sialoprotein (Santa Cruz, CA, USA), mouseanti-PCNA monoclonal antibody (Medicorp, Montreal, Canada), and rabbitantiserum against PTHrP[1-34] as described previously (Bai et al., 2007).Briefly, dewaxed and rehydrated paraffin-embedded sections were incubatedwith methanol:hydrogen peroxide (1:10) to block endogenous peroxidaseactivity and then washed in Tris-buffered saline (pH 7.6). The slides were thenincubated with the primary antibodies overnight at room temperature. Afterrinsing with Tris-buffered saline for 15 minutes, tissues were incubated withsecondary antibody (biotinylated goat anti-rabbit or anti-mouse IgG, Sigma).Sections were then washed and incubated with the Vectastain Elite ABCreagent (Vector Laboratories) for 45 minutes. Staining was developed using3,3-diaminobenzidine (2.5 mg/ml) followed by counterstaining with Mayer’sHematoxylin.
Quantitative real-time PCRRNA was isolated from mouse mandible bodies using Trizol reagent(Invitrogen) according to the manufacturer’s protocol. Reverse transcriptionreactions were performed using the SuperScript First-Strand SynthesisSystem (Invitrogen) as described (Xue et al., 2005). To determine thenumber of cDNA molecules in the reverse-transcribed samples, real-timePCR analyses were performed using the LightCycler system (Roche,Indianapolis, IN, USA). PCR was performed using 2 ml LightCycler DNAMaster SYBR Green I (Roche), each 5� and 3� primer at 0.25 mM, and 2 mlof samples or water to a final volume of 20 ml. The MgCl2 concentration wasadjusted to 3 mM. Samples were denatured at 95°C for 10 seconds, with atemperature transition rate of 20°C/second. Amplification and fluorescencedetermination were carried out in four steps: denaturation at 95°C for 10seconds, with a temperature transition rate of 20°C/second; annealing for 5seconds, with a temperature transition rate of 8°C/second; extension at 72°Cfor 20 seconds, with a temperature transition rate of 4°C/second; anddetection of SYBR Green fluorescence, which reflects the amount ofdouble-stranded DNA, at 86°C for 3 seconds. Thirty-five amplificationcycles were performed. To discriminate specific from non-specific cDNAproducts, a melting curve was obtained at the end of each run: products weredenatured at 95°C for 3 seconds, and the temperature was then decreased to58°C for 15 seconds and raised slowly from 58 to 95°C using a temperaturetransition rate of 0.1°C/second. To determine the number of copies of thetargeted DNA in the samples, purified PCR fragments of knownconcentration were serially diluted to serve as external standards in eachexperiment. Data were normalized to Gapdh levels in the samples. Theprimer sequences used for the real-time PCR were as described (Miao et al.,2004b; Xue et al., 2005).
Western blot analysisProteins were extracted from mandibular bones and quantitated using aprotein assay kit (Bio-Rad, Mississauga, Ontario, Canada). Protein samples(30 mg) were fractionated by SDS-PAGE and transferred to nitrocellulosemembranes. 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 analysisAfter HE staining or histochemical or immunohistochemical staining ofsections from six mice of each genotype, images of fields werephotographed with a Sony digital camera. Images of micrographs fromsingle sections were digitally recorded using a rectangular template, andrecordings were processed and analyzed using Northern Eclipse imageanalysis software as described (Miao et al., 2001; Miao et al., 2002).
Statistical analysisData from image analysis are presented as mean ± s.e.m. Statisticalcomparisons were made using a two-way ANOVA, with P<0.05 consideredsignificant.
RESULTSChanges in serum biochemistry in wild-type andmutant miceFirst, we compared the changes in serum calcium, phosphorus andPTH in mutant and wild-type mice. At 2 weeks of age, the CaR–/–
mice displayed hypercalcemia, hypophosphatemia and increasedserum PTH. The CaR–/–; 1a(OH)ase–/– mice displayednormocalcemia, hypophosphatemia and more severe PTHelevations, whereas the CaR–/–; Pth–/– mice exhibitednormocalcemia, hyperphosphatemia and undetectable serum PTH(Fig. 1A-C).
Imaging changes in wild-type and mutant miceWe next examined the phenotypes of teeth and mandibles in 2-week-old wild-type, CaR–/–, CaR–/–; 1a(OH)ase–/– and CaR–/–; Pth–/– miceby radiography and micro-CT scanning. The teeth and mandibleswere smaller in CaR–/– than in wild-type mice. Radiolucency wasincreased in all teeth, including molars and incisors, and in themandibles of CaR–/– mice compared with their wild-type littermates.The teeth and mandible were enlarged and their mineral densityincreased in CaR–/–; 1a(OH)ase–/– mice compared with CaR–/–
littermates, but these parameters were still reduced significantlycompared 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 ofthe first molar, and through the first, second and third molars inmandibles, were compared between wild-type mice and the threemutant models. The mineralized tooth volume in incisor and molarsand the mineralized cortical and alveolar bone volume in mandibleswere decreased in CaR–/– mice compared with wild-type littermates.The mineralization defects in teeth and alveolar bone were slightlyimproved in CaR–/–; 1a(OH)ase–/– mice, but were almostcompletely rescued in CaR–/–; Pth–/– mice as compared with CaR–/–
littermates (Fig. 1E).
Tooth volume and dental alveolar bone volume inmandiblesWe next examined the tooth volume and dental alveolar bonevolume in mandibles, using paraffin-embedded sections through thefirst molars, which were stained with HE (Fig. 2A) andhistochemically for total collagen (Fig. 2B). The dental volume (Fig.
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2C,D) and dental alveolar bone volume (Fig. 2E) were decreaseddramatically in CaR–/– mice compared with wild-type littermates.Both parameters were increased in CaR–/–; 1a(OH)ase–/– micecompared with CaR–/– littermates, but were still reducedsignificantly 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 volumewere normalized (Fig. 2A-E).
Predentin maturation and dentin formationWe then determined predentin maturation and dentin formation inthe mutant and wild-type mice. The thickness of the predentin andmineralized dentin in the first molars and the immunoreactivity ofbiglycan and dentin sialoprotein (DSP) were assessed by histology
and immunohistochemistry, respectively. The ratio of the areas ofpredentin to dentin in sections stained with HE (Fig. 3A-E) and theareas of biglycan-immunopositive dentin (Fig. 3B,F) were increasedsignificantly, whereas the areas of mineralized dentin (Fig. 3C,G)and DSP immunoreactivity (Fig. 3D,H) were decreaseddramatically, in the first molars of CaR–/– mice compared with wild-type littermates. The ratio of predentin to dentin (Fig. 3A,E) and thebiglycan-immunopositive dentin areas (Fig. 3B,F) were notsignificantly different between CaR–/–and CaR–/–; 1a(OH)ase–/–
mice. The areas of mineralized dentin and of DSP immunoreactivityin the first molar were, however, increased in CaR–/–; 1a(OH)ase–/–
mice compared with CaR–/– littermates, but were still reducedsignificantly compared with wild-type littermates (Fig. 3C,D,G,H).In CaR–/–; Pth–/– mice, these parameters were all normalized (Fig.3A-H).
Cell proliferation and PTHrP expression in theapical pulp and Hertwig’s epithelial root sheathWe immunostained for proliferating cell nuclear antigen (PCNA) toassess alterations in cell proliferation, and also for changes in PTHrP(PTHLH – Mouse Genome Informatics) expression. In wild-typemice, immunoreactivity of both antigens was observed in the apicalpulp, 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 inPCNA- and PTHrP-positive cells in the apical pulp and HERs wereimproved in CaR–/–; 1a(OH)ase–/– as compared with CaR–/– mice(Fig. 4A-D). There was no significant difference in the numbers ofPCNA-positive cells in the CaR–/–; Pth–/– and wild-type mice (Fig.4A,C), whereas the number of PTHrP-positive cells was increasedmarkedly in CaR–/–; Pth–/– mice as compared with wild-typelittermates (Fig. 4B,D). The alterations in PTHrP gene and protein
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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 themean ± 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 themandibles from 2-week-old wild-type, CaR–/–, CaR–/–; 1a(OH)ase–/– andCaR–/–; Pth–/– mice. (E) Micro-CT-scanned sections through the incisorsin front of the first molar (In), as well as the first, second and thirdmolars, from wild-type, CaR–/–; 1a(OH)ase–/– and CaR–/–; Pth–/– mice.
Fig. 2. Tooth volume and dental alveolar bone volume inmandibles. (A,B) Decalcified sections through the first molars and theincisors from 2-week-old wild-type, CaR–/–; 1a(OH)ase–/– and CaR–/–;Pth–/– mice that were stained with Hematoxylin and Eosin (HE) (A) orSirius Red for total collagen (B). Magnification, 50�. (C-E) Dentalvolume of incisors (C) and of the first molars (D), and dental alveolarbone volume of the mandibles (E), presented as mean ± s.e.m. ofdeterminations in six animals of each group. *, P<0.05; **, P<0.01,compared with wild-type mice; #, P<0.05, compared with CaR–/– mice.
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expression in mandibular extracts, as demonstrated by real-time RT-PCR (Fig. 4E) and western blots (Fig. 4F,G), were consistent withthose observed by immunohistochemistry.
Osteoblastic dental alveolar bone formationWe next determined osteoblastic dental alveolar bone formation inparaffin-embedded sections that were stained with HE and thenhistochemically for ALP. In addition, non-decalcified plastic-embedded sections were stained with von Kossa, and the numberand surface area of osteoblasts, the ALP-positive area and osteoidvolume were determined by image analysis. All these parameterswere clearly increased in CaR–/– mice when compared with wild-type mice, were slightly increased in CaR–/–; 1a(OH)ase–/– micecompared with CaR–/– mice, and were normalized in CaR–/–; Pth–/–
mice (Fig. 5A-G). We also examined the expression of genesinvolved in bone formation. RNA was isolated from dental alveolar
bone and real-time RT-PCR performed. Expression of theosteoblastic genes Cbfa1 (Runx2 – Mouse Genome Informatics),ALP, type I collagen and osteocalcin (Bglap1– Mouse GenomeInformatics) 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 resorptionOsteoclastic bone resorption in dental alveolar bone was examinedin 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 thedental alveolar bone in CaR–/– and CaR–/–; 1a(OH)ase–/– micecompared with their wild-type littermates, but there was nosignificant difference in the TRAP-positive osteoclast surface (Fig.6A-C). TRAP-positive osteoclast number and surface area were not
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Fig. 3. Predentin maturation and dentin formation in teeth.(A-D) Sections through the root wall of the first molars and incisorsfrom 2-week-old wild-type, CaR–/–; 1a(OH)ase–/– and CaR–/–; Pth–/– micestained with HE (A), or immunohistochemically stained for biglycan (B)or dentin sialoprotein (DSP) (D). (C) Non-decalcified sections throughthe first molars and incisors stained by the von Kossa procedure.Magnification, 400�. (E-H) The quantitative ratio of predentin todentin (E), the biglycan-positive area (F), the mineralized dentin area (G)and the DSP-positive area (H) in the first molars were determined byimage 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.
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 apicalpulp and HERs, from 2-week-old wild-type, CaR–/–; 1a(OH)ase–/– andCaR–/–; Pth–/– mice stained immunohistochemically for PCNA (A) andPTHrP (B). Magnification, 400�. (C,D) Numbers of PCNA-positive (C) orPTHrP-positive (D) cells per field were determined by image analysis,and the percentages of immunopositive cells relative to total cells arepresented as the mean ± s.e.m. of determinations in six animals of eachgroup. (E) Comparison of Pthrp gene expression levels in mandibulartissue of wild-type, CaR–/–, CaR–/–; 1a(OH)ase–/– and CaR–/–; Pth–/– miceas quantified by real-time RT-PCR. mRNA expression, normalized to thatof Gapdh, is shown relative to levels in wild-type mice. (F) Western blotsof mandibular extracts were carried out for expression of PTHrP, with b-tubulin as a loading control. (G) PTHrP protein levels relative to those ofb-tubulin were assessed by densitometric analysis and are expressedrelative 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.
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altered significantly in CaR–/–; Pth–/– mice (Fig. 6A-C). The ratio ofRANKL/OPG (Tnfsf11/Tnfrsf11b – Mouse Genome Informatics)mRNA levels showed no significant differences among the threeknockout models and wild-type mice, as demonstrated by real-timeRT-PCR (Fig. 6D).
DISCUSSIONWe used CaR–/– mice, and CaR–/–; 1a(OH)ase–/– and CaR–/–; Pth–/–
double-mutant mice and analyzed them biochemically, byradiography, micro-CT imaging, histology, immunohistochemistry,real-time RT-PCR and western blotting, to gain insight into the roleof CaR and of calcium, phosphorus, 1,25(OH)2D, PTH and PTHrPin the formation of teeth and the development of dental alveolarbone.
We found that in CaR–/– mice, the area of biglycan-immunopositive predentin was increased, but the dental volume andDSP level were reduced. We also found that osteoid volume in
dental alveolar bone was increased, but that dental alveolar bonevolume was reduced dramatically, although the number of ALP-positive osteoblasts was increased. These results demonstrate thatCaR deficiency produces defects in dental and dental alveolar bonemineralization and formation.
CaR–/– mice develop primary hyperparathyroidism and arehypercalcemic. To determine whether the hypercalcemia in CaR–/–
mice contributed to the defects in dental and dental alveolar bonedevelopment in these animals, we bred them with mice in which1a(OH)ase had been deleted. We recently examined 1a(OH)ase–/–
mice, which are hypocalcemic and display secondaryhyperparathyroidism and resultant hypophosphatemia, to assess theeffects of 1,25(OH)2D deficiency on dental and dental alveolar boneformation and mineralization in the mandibles. These studiesrevealed defects in dental and dental alveolar bone formation andmineralization (Liu et al., 2009). These defects were, however, lesssevere than in CaR–/– mice. In the present studies, after deletion of1a(OH)ase in CaR–/– mice in order to correct the hypercalcemia, amild improvement in dental and dental alveolar bone formation wasobserved. This included slight increases in dental volume, DSPproduction and dental alveolar bone volume. By contrast,mineralization of the teeth and dental alveolar bone was notimproved. These results therefore suggest that correction of thehypercalcemia partly corrects dental and dental alveolar boneformation, but does not correct the abnormal mineralization. In viewof the failure to completely rescue the defects of dental and dentalalveolar bone formation and mineralization in CaR–/– mice byconcomitant deletion of 1a(OH)ase, we sought to determinewhether these abnormalities were related to the more markedhyperparathyroidism and the presence of hypophosphatemia in theCaR–/–; 1a(OH)ase–/– mice, by deleting Pth from CaR–/– mice.
Ablation of Pth in CaR–/– mice has previously been reported toreverse the hyperparathyroidism and the associated hypercalcemiaand hypophosphatemia (Kos et al., 2003; Tu et al., 2003), and torescue the rachitic skeletal phenotype (Kos et al., 2003; Tu et al.,2003). In the present study, ablation of Pth in CaR–/– mice
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Fig. 5. Osteoblasts and osteoblastic gene expression inmandibles. (A-C) Sections of mandibles from 2-week-old wild-type,CaR–/–, CaR–/–; 1a(OH)ase–/– and CaR–/–; Pth–/– mice were stained withHE (A) or histochemically for ALP (B). (C) Non-decalcified sections ofmandibles 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 ofbone surface (B.S) (E) were determined in the dental alveolar bone ofHE-stained mandibles. (F) ALP-positive area as a percentage of thetissue area in the dental alveolar bone. (G) Osteoid volume (OV) in non-decalcified 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 mandibularextracts. mRNA expression normalized to Gapdh is shown relative tolevels in wild-type mice as the mean ± s.e.m. of determinations in sixanimals 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<0.001compared with CaR–/– mice.
Fig. 6. Osteoclasts in mandibles. (A) Sections of mandibles stainedhistochemically 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 ofosteoclasts (Oc.S) as a percentage of the bone surface (B.S) (C) weredetermined in the dental alveolar bone of TRAP-stained mandibles.Each value is the mean ± s.e.m. of determinations in six animals of eachgroup. (D) RANKL and OPG mRNA levels in mandibular extracts asassessed by real-time RT-PCR, normalized to Gapdh and shown relativeto levels in wild-type mice. RANKL/OPG ratios are mean ± s.e.m. ofdeterminations in six animals of the same genotype. *, P<0.05;**, P<0.01, compared with wild-type mice.
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ameliorated the defects in dental and dental alveolar bone formationand mineralization. Our results therefore suggest thathyperparathyroidism is the major factor contributing to defects inthe dental and dental alveolar bone development caused by CaRdeficiency, 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 cellularproliferation, differentiation and differentiated function, as well ascell death, both during development and in adult life (Bisello et al.,2004; Fiaschi-Taesch and Stewart, 2003; Miao et al., 2008). BecausePTHrP can modulate bone development both by binding to the typeI PTH/PTHrP receptor (PTH1R – Mouse Genome Informatics)(Juppner et al., 1991) and by an intracrine mode of action (Miao etal., 2008), we assessed whether defects in teeth and mandibledevelopment are associated with any alteration of PTHrP expressionin teeth and mandibles. Our results showed that PTHrP-immunopositive cells in the apical pulp and HERs, as well as PTHrPmRNA and protein levels in mandibular tissues, including teeth,were clearly decreased in CaR–/– mice. These parameters were onlyslightly increased in CaR–/–; 1a(OH)ase–/– relative to CaR–/– mice,but were increased markedly in CaR–/–; Pth–/– mice compared withtheir wild-type littermates. Previous studies have demonstrated thatelevated extracellular calcium acting via CaR can increase PTHrPrelease (Ahlstrom et al., 2008; Tfelt-Hansen et al., 2003). Our resultssupport the possibility that extracellular calcium stimulates PTHrPproduction via CaR and that CaR deficiency contributed to the lowPTHrP 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 of1a(OH)ase in CaR-deficient mice upregulated PTHrP gene andprotein expression in teeth and mandibles. This result suggests that1,25(OH)2D3 modulates PTHrP production in a CaR-independentfashion. 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 greaterdegree in CaR–/–; Pth–/– than in CaR–/–; 1a(OH)ase–/– mice.Reduced 1,25(OH)2D might, therefore, mediate increased PTHrPexpression in these PTH-deficient mice, but other mechanismsmight be operative as well. In CaR–/– mice, the downregulation ofPTHrP expression in mandibular tissues, including teeth, wasassociated with the defects in teeth and mandible caused by CaRdeficiency. Conversely, upregulation of PTHrP expression wasassociated with either improvement of the teeth and mandiblephenotypes in CaR–/–; 1a(OH)ase–/– mice or rescue of thesephenotypes in CaR–/–; Pth–/– mice. We previously reported thatPTHrP is required for increased trabecular bone volume in Pth–/–
mice (Miao et al., 2004b). Our present studies suggest that PTHrPmight play a local anabolic role in teeth and mandibular tissues, aswell as in bone.
Our studies and the studies of others have shown that osteoblastnumbers and trabecular bone volume in long bones are increasedsignificantly in 1a(OH)ase–/– mice and Vdr–/– mice on a normal oreven on a high calcium intake (Panda et al., 2004). This increasedisappeared 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 themandibles and the expression levels of several osteoblast-relatedgenes were all increased in CaR–/– and CaR–/–; 1a(OH)ase–/– mice,but dental alveolar bone volume was not increased. Because alveolarbone volume was restored in CaR–/–; Pth–/– mice, the linkage ofosteoblast activity to alveolar bone volume did not appear to berelated directly to CaR deficiency. Consequently, hypophosphatemia
per se, low PTHrP, or other factors yet to be determined, might playa role in preventing the coupling of the enhanced osteoblast activityto the development of increased alveolar bone volume in CaR–/– andCaR–/–; 1a(OH)ase–/– mice.
In summary, our data suggest that CaR is crucial for thedevelopment of teeth and alveolar bone postnatally. It is uncertain,however, what direct role, if any, CaR has on dental and dentalalveolar bone formation. A recent study demonstrated that deletionof CaR in osteoblasts resulted in profound bone defects (Chang etal., 2008); however, the mechanism underlying any putative directaction of CaR on skeletal development remains unclear. The roleof CaR in teeth and alveolar bone formation may therefore be, atleast 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 indirectlyregulate phosphorus levels by altering PTH levels (Brown et al.,1993; Brown and MacLeod, 2001). These analytes, per se, maythen modulate the postnatal development of teeth and alveolarbone.
AcknowledgementsThis work was supported by Key Project grants from the National NaturalScience Foundation of China (No. 30830103) and by the Program forChangjiang Scholars and Innovative Research Team in University (to D.M.), agrant from the Canadian Institutes for Health Research (to D.G.), and a grantfrom the National Institutes of Health (DK078331to E.B. and DK070756 toM.R.P.). Deposited in PMC for release after 12 months.
Competing interests statementThe authors declare no competing financial interests.
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