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Originally published In Press as doi:10.1074/jbc.M310271200 on January 22, 2004

J. Biol. Chem., Vol. 279, Issue 16, 16754-16766, April 16, 2004
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Inactivation of the 25-Hydroxyvitamin D 1{alpha}-Hydroxylase and Vitamin D Receptor Demonstrates Independent and Interdependent Effects of Calcium and Vitamin D on Skeletal and Mineral Homeostasis*

Dibyendu K. Panda{ddagger}, Dengshun Miao{ddagger}, Isabel Bolivar, Jiarong Li, Rujuan Huo, Geoffrey N. Hendy, and David Goltzman§

From the Calcium Research Laboratory, Departments of Medicine, Physiology, and Human Genetics, McGill University Health Centre and McGill University, Montreal, Quebec H3A 1A1, Canada

Received for publication, September 16, 2003 , and in revised form, January 6, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
We employed a genetic approach to determine whether deficiency of 1,25-dihydroxyvitamin D (1,25(OH)2D) and deficiency of the vitamin D receptor (VDR) produce the same alterations in skeletal and calcium homeostasis and whether calcium can subserve the skeletal functions of 1,25(OH)2D and the VDR. Mice with targeted deletion of the 25-hydroxyvitamin D 1{alpha}-hydroxylase (1{alpha}(OH)ase-/-) gene, the VDR gene, and both genes were exposed to 1) a high calcium intake, which maintained fertility but left mice hypocalcemic; 2) this intake plus three times weekly injections of 1,25(OH)2D3, which normalized calcium in the 1{alpha}(OH)ase-/- mice only; or 3) a "rescue" diet, which normalized calcium in all mutants. These regimens induced different phenotypic changes, thereby disclosing selective modulation by calcium and the vitamin D system. Parathyroid gland size and the development of the cartilaginous growth plate were each regulated by calcium and by 1,25(OH)2D3 but independent of the VDR. Parathyroid hormone secretion and mineralization of bone reflected ambient calcium levels rather than the 1,25(OH)2D/VDR system. In contrast, increased calcium absorption and optimal osteoblastogenesis and osteoclastogenesis were modulated by the 1,25(OH)2D/VDR system. These studies indicate that the calcium ion and the 1,25(OH)2D/VDR system exert discrete effects on skeletal and calcium homeostasis, which may occur coordinately or independently.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Vitamin D plays a major role in modulating calcium and skeletal homeostasis and exerts a significant influence on the growth and differentiation of a variety of tissues (1-3). Vitamin D is absorbed from the diet and generated in skin by exposure to ultraviolet light. The secosteroid is transported in blood bound to vitamin D-binding protein (4) and hydroxylated in the liver at the 25-position by a vitamin D 25-hydroxylase (CYP27) (5). The metabolite 25-hydroxyvitamin D is further hydroxylated at the 1{alpha}-position to produce the active moiety, 1,25-dihydroxyvitamin D (1,25(OH)2D)1 (1-3). The enzyme catalyzing the production of 1,25(OH)2D is 25-hydroxyvitamin D 1{alpha}-hydroxylase (1{alpha}(OH)ase or CYP27B1). Both the cDNA and gene encoding this mitochondrial cytochrome P450 enzyme have been cloned from several species (6-12). A number of tissues can synthesize 1,25(OH)2D, but the kidney is the principal site generating the circulating hormone. The renal 1{alpha}(OH)ase is known to be tightly regulated by several factors including parathyroid hormone (PTH), calcium, phosphorus, and 1,25(OH)2D per se (1-3). An alternate site of hydroxylation of 25-hydroxyvitamin D can be catalyzed by the enzyme 25-hydroxyvitamin D 24-hydroxylase (24(OH)ase or CYP24), yielding the metabolite 24,25-dihydroxyvitamin D (13). In target tissues, 1,25(OH)2D is believed to exert most of its actions by binding to the vitamin D receptor (VDR), a member of the nuclear hormone receptor superfamily, and by regulating the transcription of vitamin D target genes (14). Nevertheless, nongenomic effects of 1,25(OH)2D have been reported in which 1,25(OH)2D interacts with a putative membrane receptor, mediating the opening of calcium and chloride voltage-gated channels and activating mitogen-activated protein kinase (15).

We (16) and others (17) have previously reported a mouse model deficient in 1,25(OH)2D by targeted ablation of the 1{alpha}(OH)ase gene (1{alpha}(OH)ase-/-). After weaning, mice, fed a diet of regular mouse chow, developed hyperparathyroidism, retarded growth, and the skeletal abnormalities characteristic of rickets. These abnormalities mimic those described in the human genetic disorder vitamin D-dependent rickets type I (also called pseudovitamin D deficiency rickets) (18, 19). Several laboratories have also reported mouse models with targeted ablation of the VDR gene (VDR-/-) (20-22). These animals develop manifestations similar to those with 1{alpha}(OH)ase ablation but also display alopecia. This constellation of abnormalities is observed in humans with VDR mutations in the inherited disorder vitamin D-dependent rickets type II (also called hereditary vitamin D-resistant rickets) (23). Rescue of this phenotype has been successfully accomplished with a high calcium, high phosphorus, high lactose diet administered for at least 1 month after weaning (22, 24). Consequently, it has been postulated that the major action of the VDR in skeletal growth, maturation, and remodeling is its role in intestinal calcium absorption (25).

If 1,25(OH)2D and VDR are both necessary and sufficient for the vitamin D endocrine system, then mutant animals deficient in either 1,25(OH)2D or VDR (1{alpha}(OH)ase-/- and VDR-/-, respectively) and the mutant animals deficient in both ligand and receptor (1{alpha}(OH)ase-/-VDR-/-) should exhibit the same phenotypic alternations in mineral and skeletal homeostasis and should respond in the same way to alterations in dietary calcium. Furthermore, if the major role of the VDR in skeletal function is to increase extracellular fluid calcium by increasing intestinal absorption, as has been postulated (26), then the three mutant animals should also exhibit similar skeletal phenotypic changes as the serum calcium is altered. To test these hypotheses, we mated heterozygous animals with deletion of the 1{alpha}(OH)ase and the VDR and compared siblings that were homozygous for deletion of the genes encoding 1{alpha}(OH)ase, VDR, and both genes. The use of the double mutants permitted us to explore whether the elevated endogenous 1,25(OH)2D levels seen in VDR-/- mice might play a role in defining the phenotypes observed. We exposed these mutants to environmental conditions that would alter concentrations of the calcium ion or of the 1,25(OH)2D3 ligand. The results demonstrate significant phenotypic differences that suggest discrete roles for the calcium ion and components of the 1,25(OH)2D/VDR endocrine system in modulating mineral and skeletal homeostasis.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Derivation of 1{alpha}(OH)ase and VDR Double Null Mice—The derivation of the two parental strains of 1{alpha}(OH)ase-/- mice and VDR-/- mice by homologous recombination in embryonic stem cells was previously described by Panda et al. (16) and Li et al. (20), respectively. VDR-/- mice were a generous gift of Dr. Marie Demay (Massachusetts General Hospital, Boston, MA). Briefly, a neomycin resistance gene was inserted in place of exons VI, VII, and VIII of the mouse 1{alpha}(OH)ase gene, replacing both the ligand binding and heme binding domains. RT-PCR of renal RNA from homozygous 1{alpha}(OH)ase-/- mice confirmed that no 1{alpha}(OH)ase mRNA is expressed from this allele (16). A neomycin resistance gene was inserted in place of exon III of the mouse VDR gene, replacing the second zinc finger of the DNA binding domain. RT-PCR of intestinal and renal RNA from homozygous VDR-/- mice confirmed that a truncated mRNA is expressed from this allele (20). Mice heterozygous for the null 1{alpha}(OH)ase allele and mice heterozygous for the VDR allele were fertile (16, 20). VDR+/- mice were mated with 1{alpha}(OH)ase+/- mice, and offspring heterozygous at both loci were then mated to one another to generate pups homozygous for both 1{alpha}(OH)ase and VDR null alleles [1{alpha}(OH)ase-/-VDR-/-]. Lines were maintained by mating 1{alpha}(OH)ase-/-VDR-/- males and 1{alpha}(OH)ase+/-VDR+/- females. These mice were maintained on a mixed genetic background with contributions from C57BL/6J and BALB/c strains. To enhance fertility of females, all breeders were maintained on a high calcium diet containing 1.5% calcium in the drinking water and autoclaved chow containing 1% calcium, 0.85% phosphorus, 0% lactose and 2.2 units/g vitamin D (Ralston Purina Co., St. Louis, MO).

In Vivo Experiments—All animal experiments were carried out in compliance with and approval by the Institutional Animal Care and Use Committee. Mutant mice and control littermates were maintained in a virus- and parasite-free barrier facility and exposed to a 12-h/12-h light/dark cycle. At 21 days of age, wild-type (WT) and mutant mice were weaned onto one of three different regimens and maintained on these until sacrifice at 4 months of age: 1) the high-calcium diet described above; 2) this same high-calcium diet plus three times weekly intraperitoneal injections of 1,25(OH)2D3, 0.0625 µg per mouse (27); or 3) a "rescue diet" (TD96348 Teklad, Madison, WI) of {gamma}-irradiated chow containing 2% calcium, 1.25% phosphorus, 20% lactose, and 2.2 units/g vitamin D.

Genotyping of Mice—Genomic DNA was isolated from tail fragments by standard phenol/chloroform extraction and isopropyl alcohol precipitation (16). To determine the genotype at both the 1{alpha}(OH)ase and VDR loci, four PCRs were conducted for each animal. To test for the presence of the wild-type 1{alpha}(OH)ase allele, DNA was amplified with forward primer 5'-AGACTGCACTCCACTCTGAG-3' and reverse primer 5'-GTTTCCTACACGGATGTCTC-3'. For the neomycin gene, the primers were neo-F 5'-ACAACAGACAATCGGCTGCTC-3' and neo-R 5'-CCATGGGTCACGACGAGATC-3'. The wild type VDR allele was detected using forward primer 5'-CTGCCCTGCTCCACAGTCCTT-3' and reverse primer 5'-CGAGACTCTCCAATGTGAAGC-3'. The disrupted VDR allele was assayed using the neo forward primer 5'-GCTGCTCTGATGCCGCCGTGTTC-3' and a neo reverse primer 5'-GCACTTCGCCCAATAGCAGCCAG-3'. PCR conditions were 30 cycles for all: 1{alpha}(OH)ase allele, 94 °C for 1 min, 58 °C for 1 min, and 72 °C for 1 min; VDR and disrupted VDR allele, 94 °C for 1 min, 65 °C for 1 min, and 72 °C for 1 min; and neomycin, 94 °C for 1 min, 60 °C for 1 min, and 72 °C for 1 min.

RT-PCR—RNA was isolated from mouse kidney and long bones, using Trizol reagent (Invitrogen) according to the manufacturer's protocol. The forward and the reverse primers used for amplification of the mouse 1{alpha}(OH)ase mRNA were 5'-GCAGAGGCTCCGAAGTCTTC-3' and 5'-TGTCTGGGACACGGGAATTC-3', and primers for 24(OH)ase mRNA were 5'-ACCGTGGACAGAACGCAATGG-3' and 5'-AAATCCAGAGCGTGCTGCCTG-3'. The forward and reverse primers for core binding factor a I (Cbfa I) mRNA were 5'-GTGACACCGTGTCAGCAAAG-3' and 5'-GGAGCACAGGAAGTTGGGAC-3'. For receptor activator of NF-{kappa}B ligand (RANKL) mRNA, the forward and the reverse primers were 5'-CACACCTCACCATCAATGCTGC-3' and 5'-GAAGGGTTGGACACCTGAATGC-3'. The forward and the reverse primers for GAPDH used as a loading control were 5'-CATGGAGAAGGCTGGGGCTC-3' and 5'-CACTGACACGTTGGCAGTGG-3'. The conditions for 32 cycles of PCRs were 94 °C for 1 min, 58 °C for 1 min, and 72 °C for 1 min.

Biochemical and Hormone Analyses—Serum calcium and alkaline phosphatase were determined by an autoanalyzer (Beckman Synchron 67; Beckman Instruments). Serum 1,25(OH)2D3 was measured by radioimmunoassay (ImmunoDiagnostic Systems, Bolden, UK), and intact PTH was measured by a two-site immunoradiometric assay (Immunotopics, San Clemente, CA).

Skeletal Radiography—Femurs were removed and dissected free of soft tissue. Contact radiographs were taken using a Faxitron model 805 (Faxitron Contact, Faxitron, Germany) radiographic inspection system (22-kV voltage and 4-min exposure time). Eastman Kodak Co. X-Omat TL film was used and processed routinely.

Western Blot Analysis—Proteins were extracted from long bones and quantitated by a protein assay kit (Bio-Rad). Protein samples (30 µg) were fractionated by SDS-PAGE and transferred to polyvinylidene difluoride membrane. Immunoblotting was carried out using monoclonal antibodies against Runx2/Cbfa I (MBL International, Woburn, MA) and tubulin (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Bands were visualized using the ECL chemiluminescence detection method (Amersham Biosciences).

Histology—Thyroparathyroidal tissue, femurs, and tibiae were removed and 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 previously described (28). The proximal ends of the tibiae were decalcified in EDTA glycerol solution for 5-7 days at 4 °C. Decalcified tibiae and other tissues were dehydrated and embedded in paraffin, after which 5-µm sections were cut on a rotary microtome. The sections were stained with hematoxylin and eosin or histochemically for collagen, alkaline phosphatase (ALP) activity, or tartrate-resistant acid phosphatase (TRAP) activity as described below. Alternatively, undecalcified tibiae were embedded in LR White acrylic resin (London Resin Company Ltd., London, UK) and 1-µm sections were cut on an ultramicrotome. These sections were stained for mineral with the von Kossa staining procedure and counterstained with toluidine blue.

Immunohistochemical Staining for Aggrecan and RANKL—The cartilage matrix protein, aggrecan, and the transcription factor, RANKL, were determined by immunohistochemistry as described previously (28). Briefly, rabbit antiserum to bovine aggrecan (R130; courtesy of Dr. A. R. Poole, Shriners Hospital, Montreal, Canada) or affinity-purified goat polyclonal antibody raised against a peptide mapping at the carboxyl terminus of RANKL (C-20; Santa Cruz Biotechnology Inc., Santa Cruz, CA) were applied to dewaxed paraffin sections overnight at room temperature. As a negative control, the preimmune serum was substituted for the primary antibody. After washing with high salt buffer (50 mM Tris-HCl, 2.5% NaCl, 0.05% Tween 20, pH 7.6) for 10 min at room temperature followed by two 10-min washes with PBS, the sections were incubated with secondary antibody (biotinylated goat anti-rabbit IgG or biotinylated rabbit anti-goat IgG; Sigma), washed as before, and processed using the Vectastain ABC-AP kit (Vector Laboratories, Inc.). Red pigmentation to demarcate regions of immunostaining was produced by a 10-15-min treatment with Fast Red TR/Naphthol AS-MX phosphate (Sigma; containing 1 mM levamisole as endogenous alkaline phosphatase inhibitor). The sections were then washed with distilled water, counterstained with methyl green, and mounted with Kaiser's glycerol jelly.

Histochemical Staining for Collagen, ALP, and TRAP—Total collagen was detected in paraffin sections using a modified method of Lopez-De Leon and Rojkind (29). Dewaxed sections were exposed to 1% sirius red in saturated picric acid for 1 h. After washing with distilled water, the sections were counterstained with hematoxylin and mounted with Biomount medium.

Enzyme histochemistry for ALP activity was performed as previously described (30, 31). Briefly, following preincubation overnight in 1% magnesium chloride in 100 mM Tris-maleate buffer (pH 9.2), dewaxed sections were incubated for 2 h at room temperature in a 100 mM Tris-maleate buffer containing naphthol AS-MX phosphate (0.2 mg/ml; Sigma) dissolved in ethylene glycol monomethyl ether (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) and mounted with Kaiser's glycerol jelly.

Enzyme histochemistry for TRAP was performed using a modification of a previously described protocol (32). Dewaxed sections were preincubated for 20 min in buffer containing 50 mM sodium acetate and 40 mM sodium tartrate at pH 5.0. Sections were then incubated for 15 min at room temperature in the same buffer containing 2.5 mg/ml naphthol AS-MX phosphate (Sigma) 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.

Double Calcein Labeling—Double calcein labeling was performed by intraperitoneal injection of mice with 10 µg of calcein/g of body weight (C-0875; Sigma) at 10 days and 3 days prior to sacrifice. Bones were harvested and embedded in LR White acrylic resin described as above. Serial sections were cut, and the freshly cut surface of each section was viewed and imaged using fluorescence microscopy. The double calceinlabeled width of cortex and trabeculae was measured using Northern Eclipse image analysis software version 6.0 (Empix Imaging Inc., Mississauga, Canada), and the mineral apposition rate was calculated as the interlabel width/labeling period.

Computer-assisted Image Analysis—After hematoxylin and eosin staining or histochemical staining of sections from six mice of each genotype on each dietary regimen, 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 using Northern Eclipse image analysis software (28, 33). To measure the size of the parathyroid glands, the border of the glands were traced on micrographs of hematoxylin and eosin stained sections and traced areas of parathyroid glands were recorded automatically by Northern Eclipse image analysis software. For measuring the width of growth plates of tibiae, the distances between the proximal (epiphyseal) and distal (metaphyseal) sides of the growth plate were traced on micrographs of hematoxylin and eosin-stained sections, and traced distances were recoded automatically by Northern Eclipse image analysis software. For determining the trabecular bone volume relative to the total volume (BV/TV) in collagen-stained sections, the osteoid volume relative to the bone volume (OV/BV) in von Kossa-stained sections, ALP-positive area and intensity (summary total gray) in ALP histochemical-stained sections, and the number and size of osteoclasts in TRAP histochemical-stained sections, thresholds were set using green and red channels. The thresholds were determined as described previously (28). The trabecular volume was measured in the metaphyseal region from 0.5 mm below the distal (metaphyseal) side of the growth plate to 1.5 mm toward the diaphysis, and ALP and TRAP parameters were measured in the fields of metaphyseal regions.

Bone Marrow Cell Cultures—Primary bone marrow cell cultures were performed as previously described (34). Tibiae and femurs of 4-month-old mice fed a rescue diet were removed under aseptic conditions, and bone marrow cells were flushed out with Dulbecco's modified Eagle's minimal essential medium containing 10% fetal calf serum, 50 µg/ml ascorbic acid, 10 mM {beta}-glycerophosphate, and 10-8 M dexamethasone. Cells were dispersed by repeated pipetting, and a single cell suspension was achieved by forcefully expelling the cells through a 22-gauge syringe needle. 106 bone marrow cells were cultured in 55-cm2 Petri dishes in 10 ml of the above mentioned medium. The medium was changed every 4 days. The nonadherent cells containing hematopoietic elements were removed by gently pipetting when the medium was changed for the first time. Cultures were maintained for 18 days. At the end of the culture period, cells were washed with PBS, fixed with PLP fixative, and then stained. For determination of total colonies formed, cells were first washed in borate buffer (10 mM; pH 8.8) and then stained with 1% methylene blue (w/v) in borate buffer for 30 min at room temperature. Cells were then washed three times in borate buffer alone and left to dry before the number of colonies was quantitated by image analysis as described. For determination of mineralized colonies, cells were exposed to a solution of Alizarin Red S, pH 6.2 (1 mg/ml), for 30 min at room temperature, after which the colonies were gently washed under running water and left to dry. After each staining, culture plates were photographed over a light box with a Sony chargecoupled device camera. Images were analyzed using Northern Eclipse image analysis software. The data were imported to a spreadsheet program and processed as previously described (34).

Statistical Analysis—Data from image analysis are presented as means ± S.E. Statistical comparisons were made using a two-way analysis of variance, with p < 0.05 being considered significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Genotypic Selection of Mutant Mice—Representative PCR profiles used for genotyping the mutant mice are shown in Fig. 1a. The neomycin cassette replaced the second zinc finger of the DNA binding domain of the VDR (upper panel) and replaced both the substrate binding and heme binding domains of the 1{alpha}(OH)ase enzyme (lower panel) in the VDR-/- and 1{alpha}(OH)ase-/- mice, respectively.



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  		FIG. 1.
Genotyping of mice, expression of 1{alpha}(OH)ase and 24(OH)ase genes, and serum chemistry. a, representative PCR profiles used for genotyping the mutant mice were obtained as described under "Experimental Procedures." A neomycin cassette (neo) replaced exon III of the VDR gene (upper panels) and exons VI, VII, and VIII of the 1{alpha}(OH)ase gene (bottom panels). b, comparison of 1{alpha}(OH)ase and 24(OH)ase expression in kidney of WT, 1{alpha}(OH)ase-/-, VDR-/-, and 1{alpha}(OH)ase-/-VDR-/- mice fed a high calcium diet, a rescue diet, or a high calcium diet with 1,25(OH)2D3 administration as described under "Experimental Procedures." Specific 1{alpha}(OH)ase and 24(OH)ase products were amplified from the tissue RNAs by RT-PCR. The GAPDH was used as a loading control. Serum calcium (c) 1,25(OH)2D3 (d), PTH (e), phosphorus (f), and ALP (g) were determined in WT, 1{alpha}(OH)ase-/-, VDR-/-, and 1{alpha}(OH)ase-/-VDR-/- mice fed a high calcium diet, a rescue diet, or a high calcium diet with 1,25(OH)2D3 administration as described under "Experimental Procedures." Each value is the mean ± S.E. of determinations in five mice of the same genotype. *, p < 0.05; **, p < 0.01; ***, p < 0.001 compared with wild-type on the same diet.

 
Expression of 1{alpha}(OH)ase and 24(OH)ase Genes—The 1{alpha}(OH)ase gene was expressed at higher levels in the VDR-/- mice than in wild-type mice when animals received a high calcium intake (Fig. 1b, left panel); this was not reduced by administering exogenous 1,25(OH)2D3 to these animals (Fig. 1b, right panel) but was reduced by eliminating hypocalcemia with the rescue diet (Fig. 1b, middle panel).

Expression of the 24(OH)ase gene was reduced in all mutant mice on the high calcium diet (Fig. 1b, upper panel) but was restored to wild-type levels in 1{alpha}(OH)ase-/- mice receiving exogenous 1,25(OH)2D3 (Fig. 1b, lower panel). On the rescue diet, 24(OH)ase gene expression was restored to normal or nearly normal (Fig. 1b, middle panel).

Biochemistry—On the high calcium intake, serum 1,25(OH)2D3 levels were undetectable in 1{alpha}(OH)ase-/- mice and the 1{alpha}(OH)ase-/-VDR-/- mice but were 9-fold elevated in the VDR-/- mice (Fig. 1c), consistent with the increased 1{alpha}(OH)ase activity and with diminished 1,25(OH)2D clearance as a result of decreased 24(OH)ase expression in VDR-/- mice. On the rescue diet, 1,25(OH)2D3 levels fell toward the normal range of wild-type mice in VDR-/- mice in which 1{alpha}(OH)ase fell and 24(OH)ase rose from their respective levels in corresponding hypocalcemic animals. With 1,25(OH)2D3 treatment, serum 1,25(OH)2D3 concentrations in 1{alpha}(OH)ase-/- mice were not significantly different from wild-type but remained highly elevated in VDR-/- mice; furthermore, in the 1{alpha}(OH)ase-/- VDR-/- mice, which had reduced 24(OH)ase expression, concentrations of 1,25(OH)2D3 rose sharply to a level approaching that of VDR-/- mice.

On the high calcium intake, all mutant animals were hypocalcemic (Fig. 1d), but when the rescue diet was administered, mean serum calcium levels in all mutant mice rose to wild-type levels. With exogenous 1,25(OH)2D3 treatment, serum calcium rose to wild-type levels in 1{alpha}(OH)ase-/- mice, but both the VDR-/- mice and the 1{alpha}(OH)ase-/-VDR-/- mice remained significantly hypocalcemic.

All of the hypocalcemic mutant mice had markedly elevated serum PTH concentrations on the high calcium intake (Fig. 1e), and these animals were also hypophosphatemic (Fig. 1f). Serum alkaline phosphatase levels, most likely reflecting osteoblast stimulation, paralleled the PTH levels (Fig. 1g). In mutant animals on the rescue diet, serum PTH concentrations as well as serum phosphorus and alkaline phosphatase all returned to the wild-type range (Fig. 1, e-g). Treatment with exogenous 1,25(OH)2D3 normalized serum PTH, phosphorus, and alkaline phosphatase concentrations in 1{alpha}(OH)ase-/- mice, but all parameters remained abnormal in VDR-/- mice and 1{alpha}(OH)ase-/-VDR-/- mice (Fig. 1, e-g).

Parathyroid Gland Size in Wild-type and Mutant Mice—Although the parathyroid glands in the VDR-/- mice were enlarged on the high calcium intake, they were even larger in the 1{alpha}(OH)ase-/- mice and the 1{alpha}(OH)ase-/-VDR-/- mice (Fig. 2, a and d). On the rescue diet, parathyroid gland size decreased into the normal range in the normocalcemic VDR-/- mice but still remained significantly enlarged in the two normocalcemic but 1,25(OH)2D-deficient models (i.e. the 1{alpha}(OH)ase-/- mice and the 1{alpha}(OH)ase-/-VDR-/- mice) (Fig. 2, b and d). After 1,25(OH)2D3 treatment, parathyroid gland size normalized in the 1{alpha}(OH)ase-/- animals but not in the two VDR-deficient models (i.e. the VDR-/- and the double mutant that remained hypocalcemic).



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  		FIG. 2.
Parathyroid gland size. Representative micrographs of parathyroid glands and adjacent thyroid tissue of wild-type (WT), 1{alpha}(OH)ase-/-, VDR-/-, and 1{alpha}(OH)ase-/-VDR-/- littermates fed a high calcium diet (a), a rescue diet (b), or a high calcium diet with 1,25(OH)2D3 injections (c) sections were stained with hematoxylin and eosin. Bar, 100 µm. d, parathyroid gland sizes (areas) were determined by computerassisted image analysis as described under "Experimental Procedures" and are presented as the mean ± S.E. in six mice of the same genotype on the same diet. *, p < 0.05; **, p < 0.01; ***, p < 0.001 compared with wild type on the same diet.

 
Skeletal Alterations—On the high calcium intake, femoral size was significantly reduced in all hypocalcemic mutant models, reflecting decreased long bone growth (Fig. 3a). On the rescue diet, femoral size of all three mutant models remained slightly decreased but approached that of the wild-type (Fig. 3b). Exogenous 1,25(OH)2D3 treatment only improved femoral growth of the 1{alpha}(OH)ase-/- mice and not that of the VDR-/- or the double mutants (Fig. 3c).



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  		FIG. 3.
Radiographs and femoral lengths. Representative contact radiographs of the femurs of WT, 1{alpha}(OH)ase-/-, VDR-/-, and 1{alpha}(OH)ase-/- VDR-/- mice fed a high calcium diet (a), a rescue diet (b), or a high calcium diet with 1,25(OH)2D3 administration (c). The right panels show the quantitation of femoral length from each mutant model fed each of the three diets. Each value is the mean ± S.E. of determinations in four mice of the same genotype on the same diet. ***, p < 0.001 relative to wild-type on the same diet.

 
The cartilaginous growth plates were enlarged and distorted in all three mutant models on the high calcium intake, but these alterations were most pronounced in the 1{alpha}(OH)ase-/- mice and the 1{alpha}(OH)ase-/-VDR-/- mice (Fig. 4, a and d) and less pronounced in the VDR-/- animals. The growth plate of the VDR-/- mice appeared normal on the rescue diet but still remained substantially altered in the 1{alpha}(OH)ase-/- and double mutant mice (Fig. 4, b and d). Treatment with exogenous 1,25(OH)2D3 normalized the growth plate of the 1{alpha}(OH)ase-/- mice but not that of the VDR-/- or the double mutant (Fig. 4, c and d).



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  		FIG. 4.
Size of the cartilaginous growth plates. Representative micrographs of the proximal end of the tibia of WT, 1{alpha}(OH)ase-/-, VDR-/-, and 1{alpha}(OH)ase-/-VDR-/- mice fed a high calcium diet (a), a rescue diet (b), or a high calcium diet with 1,25(OH)2D3 administration (c). Sections were stained with hematoxylin and eosin. Bar, 100 µm. The width of the cartilaginous growth plate in the mutants on the different diets was determined as described under "Experimental Procedures" and is shown in d. Each value is the mean ± S.E. of determinations in six mice of the same genotype on the same diet. *, p < 0.05; ***, p < 0.001 relative to wild type on the same diet.

 
Trabecular bone volume (BV/TV) was increased in all three hypocalcemic mutant models on a high calcium intake (Fig. 5, a and d), but after 4 months on a rescue diet, trabecular bone volume was reduced in all three mutants below the levels in wild-type animals (Fig. 5, b and d). In contrast, treatment with 1,25(OH)2D3 normalized trabecular bone volumes in the 1{alpha}(OH)ase-/- mice but not in the other two models (Fig. 5, c and d).



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  		FIG. 5.
Bone volume. Representative histology of the upper half of the tibia of WT, 1{alpha}(OH)ase-/-, VDR-/-, and 1{alpha}(OH)ase-/-VDR-/- mice fed either a high calcium diet (a), a rescue diet (b), or a high calcium diet with 1,25(OH)2D3 injections (c). Sections were stained for collagen. Bar, 400 µm. The trabecular bone volume (d) was determined as described under "Experimental Procedures" and is presented as a percentage of the tissue volume (BV/TV (%)) for each mutant on each diet. Each value is the mean ± S.E. of determinations in six animals of the same genotype. *, p < 0.05; ***, p < 0.001 compared with wild type on the same diet.

 
In the hypocalcemic mice on the high calcium intake, osteoid volume reflecting unmineralized bone matrix was increased in all mutant animals in both trabecular and cortical bone (Fig. 6, a and b). When hypocalcemia was eliminated by the rescue diet, osteoid volume was reduced to that of wild type in all mutants. In contrast, exogenous 1,25(OH)2D3 treatment normalized osteoid only in the 1{alpha}(OH)ase-/- mice (Fig. 6, a and b). Mineral apposition rate was increased in all three mutants on the high calcium intake and was reduced below normal in all three mutants on the rescue diet. Mineral apposition rate was normalized by 1,25(OH)2D3 treatment only in 1{alpha}(OH)ase-/- mice but remained elevated in the VDR-/- and 1{alpha}(OH)ase-/- VDR-/- mice (Fig. 6, c and d).



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  		FIG. 6.
Osteoid volume and mineral apposition rate. Osteoid volume was determined in undecalcified von Kossa-stained sections as described under "Experimental Procedures" and is presented as a percentage of bone volume (OV/BV (%)) of trabeculae (a) and of cortex (b) in WT, 1{alpha}(OH)ase-/-, VDR-/-, and 1{alpha}(OH)ase-/-VDR-/- mice fed a high calcium diet, a rescue diet, or a high calcium diet with 1,25(OH)2D3 injections. Mineral apposition rate (MAR) of trabeculae (c) and cortex (d) of the same animals was also determined as described under "Experimental Procedures." Each value is the mean ± S.E. of determinations in six animals of the same genotype on the same diet. **, p < 0.01; ***, p < 0.001 relative to wild-type mice on the same diet.

 
Osteoblast levels were significantly elevated in all three mutants with secondary hyperparathyroidism (Fig. 7, a and d) on the high calcium intake, and Cbfa I mRNA and protein expression in bone were also increased (Fig. 7, e and f). Osteoblasts were significantly reduced in the mutants on the rescue diet to levels below those in wild-type mice (Fig. 7, b and d), and Cbfa I mRNA or protein was also reduced (Fig. 7, e and f). After exogenous 1,25(OH)2D3 treatment, only the 1{alpha}(OH)ase-/- mice normalized their osteoblast levels, whereas these remained elevated in the VDR-/- and the double mutants (Fig. 7, c and d).



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  		FIG. 7.
Alkaline phosphatase and Cbfa I expression. Representative micrographs of tibial sections, stained for ALP activity, from wild-type (WT), 1{alpha}(OH)ase-/-, VDR-/-, and 1{alpha}(OH)ase-/-VDR-/- littermates fed a high calcium diet (a), a rescue diet (b), or a high calcium diet with 1,25(OH)2D3 injections (c). Bar, 100 µm. The ALP-positive area as a percentage of the tissue area and the relative intensity of the ALP positivity (summary total gray of ALP-positive production) were determined in the metaphyseal regions as described under "Experimental Procedures" for each mutant on each diet shown in the left and right panel, respectively, of d. Each value is the mean ± S.E. of determinations in six animals of the same genotype. ***, p < 0.001 relative to wild-type mice on the same diet. RT-PCR (e) and Western blots (f) of long bone extracts for expression of Cbfa I. GAPDH and tubulin were used as loading controls for RT-PCR and Western blots, respectively. Lane 1, WT; lane 2, 1{alpha}(OH)ase-/-; lane 3, VDR-/-; lane 4, 1{alpha}(OH)ase-/-VDR-/-.

 
To determine the cause of the reduced osteoblasts in the mutants on the rescue diet, primary bone marrow cultures were examined for the capacity to formed osteogenic colonies from marrow progenitors. Reduced bone forming colonies were observed in the marrow of all three mutants on the rescue diet compared with wild-type controls (Fig. 8).



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  		FIG. 8.
Bone marrow cell cultures and mineralized colony formation. Bone marrow cells from WT, 1{alpha}(OH)ase-/-, VDR-/-, and 1{alpha}(OH)ase-/-VDR-/- littermates fed a rescue diet were cultured in osteogenic differentiation medium for 18 days and examined for total number of colonies (Total CFU-f) by methylene blue staining (a) and for mineralized colonies (CFU-fob) by alizarin red S staining (b). Quantification by image analysis (c) as described under "Experimental Procedures." Values are the mean ± S.E. of triplicate determinations from three replicate experiments. *, p < 0.05; ***, p < 0.001 versus wild-type controls.

 
TRAP-positive osteoclast numbers were not significantly different from levels in wild-type animals in any of the mutants exposed to any of the dietary manipulations (Fig. 9). However, the average size of the TRAP-positive osteoclasts was reduced in all three mutants on the high calcium intake. On the rescue diet, TRAP-positive osteoclast size was no different in the mutant mice than in wild-type mice. After 1,25(OH)2D3 treatment, however, only the 1{alpha}(OH)ase-/- mice normalized, and average osteoclast size remained below wild-type in the VDR-/- and double mutants (Fig. 9, c and d).



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  		FIG. 9.
Acid phosphatase staining. Representative micrographs of sections stained histochemically for TRAP activity of the tibial metaphysis of WT, 1{alpha}(OH)ase-/-, VDR-/-, and 1{alpha}(OH)ase-/-VDR-/- littermates fed a high calcium diet (a), a rescue diet (b), or a high calcium diet with 1,25(OH)2D3 injections (c). Bar, 200 µm. The number of TRAP-positive osteoclasts per field of tissue and the average size of TRAP-positive osteoclasts are shown in the left and right panels, respectively, of d. Each value is the mean ± S.E. of determinations in six animals of the same genotype. **, p < 0.01; ***, p < 0.001 relative to wild-type mice on the same diet.

 
Expression of RANKL mRNA was diminished in bone of the mutants compared with wild-type bone, when the animals were on a high calcium intake (Fig. 10a). RANKL protein in osteoblastic cells was also found to be low in all three mutants on the high calcium intake (Fig. 10, b, e, and f) and in the two mutants treated with 1,25(OH)2D3 that were deficient in VDR (Fig. 10, d-f). On the rescue diet, RANKL mRNA (Fig. 10a) and protein (Fig. 10, c, e, and f) were markedly below wild-type levels.



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  		FIG. 10.
Expression of RANKL. RT-PCR (a) was performed on bone extracts for expression of mRNA encoding RANKL as described under "Experimental Procedures." GAPDH was employed as a loading control. Lane 1, WT; lane 2, 1{alpha}(OH)ase-/-; lane 3, VDR-/-; lane 4, 1{alpha}(OH)ase-/- VDR-/-. Representative micrographs (b-d) are shown of sections stained by immunohistochemistry for RANKL of tibial metaphysis of WT, 1{alpha}(OH)ase-/-, VDR-/-, and 1{alpha}(OH)ase-/-VDR-/- littermates fed a high calcium diet (b), a rescue diet (c), or a high calcium diet with 1,25(OH)2D3 injections (d). The RANKL-positive tissue area and RANKL staining intensity (summary total gray) are shown in e and f, respectively. Each value is the mean ± S.E. of determinations in six animals of the same genotype. *, p < 0.05; ***, p < 0.001 relative to wild-type mice on the same diet.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
We have analyzed mouse mutants that harbor deletions of the genes encoding the 1{alpha}(OH)ase, the VDR, or both genes and subjected them to three different nutritional regimens. The analysis of the double mutants allowed us to assess which phenotypic differences seen in these animals compared with the VDR-/- mice might be due to the elevated endogenous 1,25(OH)2D of the VDR-/- mice. This would indicate that the 1,25(OH)2D functions in the absence of an intact VDR. The high calcium intake facilitated breeding of the mutant mice, notably the double mutants, in view of the fact that both 1{alpha}(OH)ase-/- and VDR-/- animals display reduced fertility, at least in part because of severe hypocalcemia (35). Nevertheless, in the absence of dietary lactose, serum calcium concentrations remained subnormal in mutants fed the high calcium intake. The lactose in the rescue diet facilitates calcium entry across the intestine independent of the 1,25(OH)2D/VDR system, although the mechanism is unclear. Serum calcium was normalized on the rescue diet in the absence of 1,25(OH)2D or of the VDR or of both 1,25(OH)2D and VDR and thus was not due to a VDR-independent action of 1,25(OH)2D. In the absence of lactose, endogenous elevated 1,25(OH)2D could not normalize calcium in VDR-/- mice, and exogenous 1,25(OH)2D3 could only normalize calcium in the 1{alpha}(OH)ase-/- mice that retained an intact VDR. Consequently, these results confirm the importance of both 1,25(OH)2D and the VDR in intestinal calcium absorption. Indeed, 1,25(OH)2D-dependent increases in gut absorption of calcium (36) and in intestinal calbindin D9k are well documented, and effects on the duodenal epithelial calcium channels have recently been reported (37).

In the hypocalcemic mice on the high calcium intake, secondary hyperparathyroidism (based on serum PTH and alkaline phosphatase concentrations) appeared more severe in the 1{alpha}(OH)ase-/- and 1{alpha}(OH)ase-/-VDR-/- mice than in VDR-/- mice (i.e. in the mutants with 1,25(OH)2D deficiency). Nevertheless, when serum calcium was raised by the rescue diet, normalization of the elevated serum PTH concentrations occurred in all mutants. In 1{alpha}(OH)ase-/- mice treated with exogenous 1,25(OH)2D3, in which mean serum calcium levels were normalized, circulating PTH concentrations fell into the normal range, but PTH levels remained increased in 1,25(OH)2D3-treated VDR-/- and in 1{alpha}(OH)ase-/-VDR-/- mice whose ambient calcium levels remained low. Consequently, the ambient calcium concentration appears to suppress secretion of PTH independently of the 1,25(OH)2D/VDR system.

Increased parathyroid gland size was present in all hypocalcemic mutants and remained elevated in the 1{alpha}(OH)ase-/- and double mutants on the rescue diet when they were no longer hypocalcemic. However, parathyroid gland size was normalized in the VDR-/- animals on the rescue diet when these animals were no longer hypocalcemic. In these mice, the elevated endogenous circulating 1,25(OH)2D levels may have contributed, even in the absence of an intact VDR, to the reduction in gland size. Administration of exogenous 1,25(OH)2D3 normalized parathyroid gland size in the 1{alpha}(OH)ase-/- mice but not in the VDR-/- mutants or double mutants, where hypocalcemia persisted. Previous studies have demonstrated an in vivo role for extracellular calcium (38) in parathyroid cell growth via the calcium-sensing receptor, and previous in vitro studies have also suggested a role for 1,25(OH)2D (39). Our studies confirm both of these actions in vivo and demonstrate the cooperative nature of the calcium ion and 1,25(OH)2D in exerting this effect.

The cartilaginous growth plate was enlarged and distorted with a widened hypertrophic zone in all three mutants while they were hypocalcemic, but the growth plate abnormalities were more pronounced in the 1{alpha}(OH)ase-/- and the 1{alpha}(OH)ase-/-VDR-/- mutants, both of which had undetectable serum 1,25(OH)2D, than in the VDR-/- animals, which had markedly elevated endogenous serum 1,25(OH)2D. When hypocalcemia was eliminated on the rescue diet, the growth plate including the widened hypertrophic zone of the VDR-/- mice normalized as previously described (25) but still remained considerably enlarged in the 1{alpha}(OH)ase-/- mice and in the 1{alpha}(OH)ase-/-VDR-/- mice. As also reported by others (36), exogenous 1,25(OH)2D3 normalized the growth plate of 1{alpha}(OH)ase-/- mice. However, it did not normalize the growth plate of the double mutant where hypocalcemia persisted. Consequently, both ambient calcium and 1,25(OH)2D, even in the absence of an intact VDR, appear to exert a major effect on the cartilaginous growth plate and are co-operatively required for its normal development. The persistent abnormality of the growth plate in normocalcemic 1{alpha}(OH)ase-/- mice on the rescue diet has also been noted by others (40) and has been observed in VDR/retinoid X receptor {gamma} double null mutant mice (41). Consequently, this supports the possibility that 1,25(OH)2D interacts with a novel nuclear receptor in chondrocytes that heterodimerizes with retinoid X receptor {gamma} (41).

Bone volume and osteoblast numbers were increased in the mutant animals with secondary hyperparathyroidism as has been previously reported in studies with the 1{alpha}(OH)ase-/- (16, 17) and the VDR-/- models (20, 21). These increases most likely reflect the well characterized "anabolic" activity of PTH (42). Consistent with this observation, increases were observed in the transcription factor Cbfa I/Runx2, which is known to be essential for osteoblastic differentiation during embryogenesis and is also required for the anabolic effect of PTH in postnatal animals (43). The increased bone volume was, however, associated with increased osteoid volume in the animals with secondary hyperparathyroidism. In those animals who received a high calcium intake, serum calcium levels, although decreased, were higher than we and others have previously reported in mutant mice fed a normal calcium intake (16, 17, 20, 21). Osteoid volumes were therefore lower than in previous reports. In addition, mineral apposition could be detected and was, in fact, elevated in the mice with secondary hyperparathyroidism. The mineral apposition rate fell as PTH levels fell, reflecting the reduced bone formation. Unmineralized osteoid was no greater than in wild-type mice when hypocalcemia and hypophosphatemia were eliminated (i.e. in 1,25(OH)2D3-treated 1{alpha}(OH)ase-/- mice) but also in all mutants on the rescue diet. Consequently, once extracellular calcium and phosphorus levels are normalized, mineralization of osteoid does not appear to require the 1,25(OH)2D/VDR system.

Despite greatly increased PTH levels, osteoclast numbers in these mutants with deficiency of the 1,25(OH)2D/VDR system were not significantly increased above levels observed in normocalcemic vitamin D-replete wild-type controls whose PTH levels were normal. Bone turnover in the mutant animals was therefore uncoupled, and osteoclast numbers in the mutants could be considered inappropriately low. We did find, in the present studies, that average osteoclast size was decreased in the mutants with secondary hyperparathyroidism on the high calcium diet and in the 1,25(OH)2D3-treated VDR-/- and double mutants. Furthermore, levels of RANKL, the transcription factor stimulated by 1,25(OH)2D3 that is required for the normal differentiation and maintenance of osteoclasts (44), were also decreased in the mutants. Consequently, the 1,25(OH)2D/VDR system appears necessary for maximal PTH-induced osteoclast production, and the increase in bone volume appeared to reflect enhanced osteoblastic activity due to PTH that was dissociated from an increase in bone resorption.

The increase in the thickness of the cartilaginous growth plate in the mutants may also have been contributed to by impaired growth plate remodeling (i.e. impaired resorption of hypertrophic chondrocytes by chondroclasts/osteoclasts at the chondro-osseous junction). This has been previously noted in VDR-/- mutants (26). It has been reported that in co-cultures in vitro using wild-type spleen cells but osteoblasts from VDR-/- mice, 1,25(OH)2D3 was unable to stimulate osteoclast production, whereas PTH and other bone-resorbing agents could (45). Consequently, the "normal level" of osteoclasts seen in our in vivo models in the absence of the 1,25(OH)2D/VDR system may have reflected the action of PTH and other local bone resorbers. The more exuberant osteoclastic response generally expected with sustained increases in PTH of the magnitude seen in our mutants (42) may reflect increased production of 1,25(OH)2D and its action via the VDR in skeletal cells when these factors are not limiting.

Another striking finding in all three mutants of the 1,25(OH)2D/VDR system was the reduction in osteoblast numbers, mineral apposition rate, and bone volume below levels in wild-type mice that occurred on the rescue diet. This was associated with decreased production of mineralized colonies ex vivo, providing further evidence that osteogenesis was impaired. The issue of whether vitamin D directly induces bone formation has been controversial (46). However, our observation in animals with normal calcium levels but deficient vitamin D indicate that the presence of an intact 1,25(OH)2D/VDR system may be necessary for base-line bone formation. Such findings were not previously reported in 1{alpha}(OH)ase-/- and VDR-/- mice fed a rescue diet and may represent differences in the duration of exposure to this diet postweaning (i.e. about 100 days in our study versus ~39 days (40) and 54 days (26), respectively, in previous studies). Consequently, bone formation may be more vitamin D-dependent as the animals age. The failure of elevated endogenous 1,25(OH)2D in the VDR-/- mice to alleviate the reduction in bone volume suggests that both 1,25(OH)2D and the VDR are required for base-line bone formation. Our results are therefore consistent with a physiologic anabolic role for endogenous 1,25(OH)2D and the VDR in vivo.

In summary, our studies show that the calcium ion and the 1,25(OH)2D/VDR system may exert physiological effects in vivo independently or in concert. Thus, mineralization of bone and inhibition of elevated PTH secretion appear mainly dependent on ambient calcium levels rather than the presence of the 1,25(OH)2D/VDR system, whereas optimal intestinal absorption of calcium, maximal increases in PTH-induced osteoclastogenesis, and maintenance of osteoblastogenesis seem dependent on a functional 1,25(OH)2D/VDR system. In contrast, the calcium ion and 1,25(OH)2D in the absence of an intact VDR may act in concert to regulate parathyroid gland size and the normal development of the cartilaginous growth plate. Finally, our studies also show that the vitamin D system is required for both anabolic and catabolic effects on the skeleton, therefore mimicking the dual functions of parathyroid hormone.


   FOOTNOTES
 
* This work was supported by Canadian Institutes of Health Research Grants IMH-63263 (to D. M.), MOP-57730 (to G. N. H.), and MOP-5775 (to D. G.), a grant from the Kidney Foundation of Canada (to G. N. H.), and National Cancer Institute of Canada Grant 012243 (to D. G.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} These two authors contributed equally to this work. Back

§ To whom correspondence should be addressed: Calcium Research Laboratory, Dept. of Medicine, Royal Victoria Hospital, H4.67, 687 Pine Ave. W., Montreal, Quebec H3A 1A1, Canada. Tel.: 514-843-1632; Fax: 514-843-1712; E-mail: david.goltzman{at}mcgill.ca.

1 The abbreviations used are: 1,25(OH)2D, 1,25-dihydroxyvitamin D; Cbfa I, core binding factor a I; 1{alpha}(OH)ase, 25-hydroxyvitamin D-1{alpha}-hydroxylase; PTH, parathyroid hormone; 24(OH)ase, 25-hydroxyvitamin D-24-hydroxylase; VDR, vitamin D receptor; RANKL, receptor activator of nuclear factor {kappa} B ligand; RT, reverse transcriptase; ALP, alkaline phosphatase; TRAP, tartrate-resistant acid phosphatase; WT, wild-type; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. Back



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Bouillon, R., Okamura, W. H., and Norman, A. W. (1995) Endocr. Rev. 16, 200-257[Abstract/Free Full Text]
  2. Jones, G., Strugnell, S. A., and DeLuca, H. F. (1998) Physiol. Rev. 78, 1193-1231[Abstract/Free Full Text]
  3. Sutton, A. L., and MacDonald, P. N. (2003) Mol. Endocrinol. 17, 777-791[Abstract/Free Full Text]
  4. Bikle, D. D., Gee, E., Halloran, B., Kowalski, M. A., Ryzen, E., and Haddad, J. G. (1986) J. Clin. Endocrinol. Metab. 63, 954-959[Abstract/Free Full Text]
  5. Gascon-Barre, M. (1997) in Vitamin D (Feldman, D., Glorieux, F. H., and Pike, J. W., eds) pp. 41-55, Academic Press, Inc., San Diego
  6. Takeyama, K., Kitanaka, S., Sato, T., Kobori, M., Yanagisawa, J., and Kato, S. (1997) Science 277, 1827-1830[Abstract/Free Full Text]
  7. St-Arnaud, R., Messerlian, S., Moir, J. M., Omdahl, J. L., and Glorieux, F. H. (1997) J. Bone Miner. Res. 12, 1552-1559[CrossRef][Medline] [Order article via Infotrieve]
  8. Shinki, T., Shimada, H., Wakino, S., Anazawa, H., Hayashi, M., Saruta, T., DeLuca, H. F., and Suda, T. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 12920-12925[Abstract/Free Full Text]
  9. Fu, G. K., Lin, D., Zhang, M. Y., Bikle, D. D., Shackleton, C. H., Miller, W. L., and Portale, A. A. (1997) Mol. Endocrinol. 11, 1961-1970[Abstract/Free Full Text]
  10. Fu, G. K., Portale, A. A., and Miller, W. L. (1997) DNA Cell Biol. 16, 1499-1507[Medline] [Order article via Infotrieve]
  11. Kimmel-Jehan, C., and DeLuca, H. F. (2000) Biochim. Biophys. Acta 1475, 109-113[Medline] [Order article via Infotrieve]
  12. Panda, D. K., Al Kawas, S., Seldin, M. F., Hendy, G. N., and Goltzman, D. (2001) J. Bone Miner. Res. 16, 46-56[CrossRef][Medline] [Order article via Infotrieve]
  13. St-Arnaud, R., Arabian, A., Travers, R., Barletta, F., Raval-Pandya, M., Chapin, K., Depovere, J., Mathieu, C., Christakos, S., Demay, M. B., and Glorieux, F. H. (2000) Endocrinology 141, 2658-2666[Abstract/Free Full Text]
  14. Haussler, M. R., Whitfield, G. K., Haussler, C. A., Hsieh, J. C., Thompson, P. D., Selznick, S. H., Dominguez, C. E., and Jurutka, P. W. (1998) J. Bone Miner. Res. 13, 325-349[CrossRef][Medline] [Order article via Infotrieve]
  15. Norman, A. W., Okamura, W. H., Bishop, J. E., and Henry, H. L. (2002) Mol. Cell. Endocrinol. 197, 1-13[CrossRef][Medline] [Order article via Infotrieve]
  16. Panda, D. K., Miao, D., Tremblay, M. L., Sirois, J., Farookhi, R., Hendy, G. N., and Goltzman, D. (2001) Proc. Natl. Acad. Sci. U. S. A. 98, 7498-7503[Abstract/Free Full Text]
  17. Dardenne, O., Prud'homme, J., Arabian, A., Glorieux, F. H., and St-Arnaud, R. (2001) Endocrinology 142, 3135-3141[Abstract/Free Full Text]
  18. Fraser, D., Kooh, S. W., Kind, H. P., Holick, M. F., Tanaka, Y., and DeLuca, H. F. (1973) N. Engl. J. Med. 289, 817-822[Medline] [Order article via Infotrieve]
  19. Eil, C., Liberman, U. A., and Marx, S. J. (1986) Adv. Exp. Med. Biol. 196, 407-422[Medline] [Order article via Infotrieve]
  20. Li, Y. C., Pirro, A. E., Amling, M., Delling, G., Baron, R., Bronson, R., and Demay, M. B. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 9831-9835[Abstract/Free Full Text]
  21. Yoshizawa, T., Handa, Y., Uematsu, Y., Takeda, S., Sekine, K., Yoshihara, Y., Kawakami, T., Arioka, K., Sato, H., Uchiyama, Y., Masushige, S., Fukamizu, A., Matsumoto, T., and Kato, S. (1997) Nat. Genet. 16, 391-396[CrossRef][Medline] [Order article via Infotrieve]
  22. Erben, R. G., Soegiarto, D. W., Weber, K., Zeitz, U., Lieberherr, M., Gniadecki, R., Moller, G., Adamski, J., and Balling, R. (2002) Mol. Endocrinol. 16, 1524-1537[Abstract/Free Full Text]
  23. Beer, S., Tieder, M., Kohelet, D., Liberman, O. A., Vure, E., Bar-Joseph, G., Gabizon, D., Borochowitz, Z. U., Varon, M., and Modai, D. (1981) Clin. Endocrinol. 14, 395-402[Medline] [Order article via Infotrieve]
  24. Donohue, M. M., and Demay, M. B. (2002) Endocrinology 143, 3691-3694[Abstract]
  25. Amling, M., Priemel, M., Holzmann, T., Chapin, K., Rueger, J. M., Baron, R., and Demay, M. B. (1999) Endocrinology 140, 4982-4987[Abstract/Free Full Text]
  26. Li, Y. C., Amling, M., Pirro, A. E., Priemel, M., Meuse, J., Baron, R., Delling, G., and Demay, M. B. (1998) Endocrinology 139, 4391-4396[Abstract/Free Full Text]
  27. Vegesna, V., O'Kelly, J., Uskokovic, M., Said, J., Lemp, N., Saitoh, T., Ikezoe, T., Binderup, L., and Koeffler, H. P. (2002) Endocrinology 143, 4389-4396[Abstract/Free Full Text]
  28. Miao, D., Bai, X., Panda, D., McKee, M., Karaplis, A., and Goltzman, D. (2001) Endocrinology 142, 926-939[Abstract/Free Full Text]
  29. Lopez-De Leon, A., and Rojkind, M. (1985) J. Histochem. Cytochem. 33, 737-743[Abstract]
  30. Miao, D., and Scutt, A. (2002) J. Histochem. Cytochem. 50, 333-340[Abstract/Free Full Text]
  31. He, B., Deckelbaum, R. A., Miao, D., Lipman, M. L., Pollak, M., Goltzman, D., and Karaplis, A. C. (2001) Endocrinology 142, 2070-2077[Abstract/Free Full Text]
  32. Miao, D., and Scutt, A. (2002) BMC Musculoskelet. Disord. 3, 16[CrossRef][Medline] [Order article via Infotrieve]
  33. Miao, D., He, B., Karaplis, A. C., and Goltzman, D. (2002) J. Clin. Invest. 109, 1173-1182[CrossRef][Medline] [Order article via Infotrieve]
  34. Bai, X., Miao, D., Panda, D., Grady, S., McKee, M. D., Goltzman, D., and Karaplis, A. C. (2002) Mol. Endocrinol. 16, 2913-2925[Abstract/Free Full Text]
  35. Johnson, L. E., and DeLuca, H. F. (2001) J. Nutr. 131, 1787-1791[Abstract/Free Full Text]
  36. Dardenne, O., Prudhomme, J., Hacking, S. A., Glorieux, F. H., and St-Arnaud, R. (2003) J. Bone Miner. Res. 18, 637-643[CrossRef][Medline] [Order article via Infotrieve]
  37. Van Cromphaut, S. J., Dewerchin, M., Hoenderop, J. G., Stockmans, I., Van Herck, E., Kato, S., Bindels, R. J., Collen, D., Carmeliet, P., Bouillon, R., and Carmeliet, G. (2001) Proc. Natl. Acad. Sci. U. S. A. 98, 13324-13329[Abstract/Free Full Text]
  38. Brown, E. M., and MacLeod, R. J. (2001) Physiol. Rev. 81, 239-297[Abstract/Free Full Text]
  39. Kremer, R., Bolivar, I., Goltzman, D., and Hendy, G. N. (1989) Endocrinology 125, 935-941[Abstract/Free Full Text]
  40. Dardenne, O., Prud'homme, J., Hacking, S. A., Glorieux, F. H., and St-Arnaud, R. (2003) Bone 32, 332-340[Medline] [Order article via Infotrieve]
  41. Yagishita, N., Yamamoto, Y., Yoshizawa, T., Sekine, K., Uematsu, Y., Murayama, H., Nagai, Y., Krezel, W., Chambon, P., Matsumoto, T., and Kato, S. (2001) Endocrinology 142, 5332-5341[Abstract/Free Full Text]
  42. Tam, C. S., Heersche, J. N., Murray, T. M., and Parsons, J. A. (1982) Endocrinology 110, 506-512[Abstract/Free Full Text]
  43. Krishnan, V., Moore, T. L., Ma, Y. L., Helvering, L. M., Frolik, C. A., Valasek, K. M., Ducy, P., and Geiser, A. G. (2003) Mol. Endocrinol. 17, 423-435[Abstract/Free Full Text]
  44. Kitazawa, S., Kajimoto, K., Kondo, T., and Kitazawa, R. (2003) J. Cell. Biochem. 89, 771-777[CrossRef][Medline] [Order article via Infotrieve]
  45. Takeda, S., Yoshizawa, T., Nagai, Y., Yamato, H., Fukumoto, S., Sekine, K., Kato, S., Matsumoto, T., and Fujita, T. (1999) Endocrinology 140, 1005-1008[Abstract/Free Full Text]
  46. Suda, T., Ueno, Y., Fujii, K., and Shinki, T. (2003) J. Cell. Biochem. 88, 259-266[CrossRef][Medline] [Order article via Infotrieve]

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