Title: Inactivation of the 25-Hydroxyvitamin D 1α-Hydroxylase and Vitamin D Receptor Demonstrates Independent and Interdependent Effects of Calcium and Vitamin D on Skeletal and Mineral Homeostasis
Abstract: 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 1α-hydroxylase (1α(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 1α(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. 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 1α-hydroxylase (1α(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 1α(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. 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 (1Bouillon R. Okamura W.H. Norman A.W. Endocr. Rev. 1995; 16: 200-257Crossref PubMed Google Scholar, 2Jones G. Strugnell S.A. DeLuca H.F. Physiol. Rev. 1998; 78: 1193-1231Crossref PubMed Scopus (1040) Google Scholar, 3Sutton A.L. MacDonald P.N. Mol. Endocrinol. 2003; 17: 777-791Crossref PubMed Scopus (263) Google Scholar). 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 (4Bikle D.D. Gee E. Halloran B. Kowalski M.A. Ryzen E. Haddad J.G. J. Clin. Endocrinol. Metab. 1986; 63: 954-959Crossref PubMed Scopus (482) Google Scholar) and hydroxylated in the liver at the 25-position by a vitamin D 25-hydroxylase (CYP27) (5Gascon-Barre M. Feldman D. Glorieux F.H. Pike J.W. Vitamin D. Academic Press, Inc., San Diego1997: 41-55Google Scholar). The metabolite 25-hydroxyvitamin D is further hydroxylated at the 1α-position to produce the active moiety, 1,25-dihydroxyvitamin D (1,25(OH)2D) 1The abbreviations used are: 1,25(OH)2D, 1,25-dihydroxyvitamin D; Cbfa I, core binding factor a I; 1α(OH)ase, 25-hydroxyvitamin D-1α-hydroxylase; PTH, parathyroid hormone; 24(OH)ase, 25-hydroxyvitamin D-24-hydroxylase; VDR, vitamin D receptor; RANKL, receptor activator of nuclear factor κ B ligand; RT, reverse transcriptase; ALP, alkaline phosphatase; TRAP, tartrate-resistant acid phosphatase; WT, wild-type; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. (1Bouillon R. Okamura W.H. Norman A.W. Endocr. Rev. 1995; 16: 200-257Crossref PubMed Google Scholar, 2Jones G. Strugnell S.A. DeLuca H.F. Physiol. Rev. 1998; 78: 1193-1231Crossref PubMed Scopus (1040) Google Scholar, 3Sutton A.L. MacDonald P.N. Mol. Endocrinol. 2003; 17: 777-791Crossref PubMed Scopus (263) Google Scholar). The enzyme catalyzing the production of 1,25(OH)2D is 25-hydroxyvitamin D 1α-hydroxylase (1α(OH)ase or CYP27B1). Both the cDNA and gene encoding this mitochondrial cytochrome P450 enzyme have been cloned from several species (6Takeyama K. Kitanaka S. Sato T. Kobori M. Yanagisawa J. Kato S. Science. 1997; 277: 1827-1830Crossref PubMed Scopus (468) Google Scholar, 7St-Arnaud R. Messerlian S. Moir J.M. Omdahl J.L. Glorieux F.H. J. Bone Miner. Res. 1997; 12: 1552-1559Crossref PubMed Scopus (275) Google Scholar, 8Shinki T. Shimada H. Wakino S. Anazawa H. Hayashi M. Saruta T. DeLuca H.F. Suda T. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 12920-12925Crossref PubMed Scopus (177) Google Scholar, 9Fu G.K. Lin D. Zhang M.Y. Bikle D.D. Shackleton C.H. Miller W.L. Portale A.A. Mol. Endocrinol. 1997; 11: 1961-1970Crossref PubMed Google Scholar, 10Fu G.K. Portale A.A. Miller W.L. DNA Cell Biol. 1997; 16: 1499-1507Crossref PubMed Scopus (110) Google Scholar, 11Kimmel-Jehan C. DeLuca H.F. Biochim. Biophys. Acta. 2000; 1475: 109-113Crossref PubMed Scopus (8) Google Scholar, 12Panda D.K. Al Kawas S. Seldin M.F. Hendy G.N. Goltzman D. J. Bone Miner. Res. 2001; 16: 46-56Crossref PubMed Scopus (50) Google Scholar). A number of tissues can synthesize 1,25(OH)2D, but the kidney is the principal site generating the circulating hormone. The renal 1α(OH)ase is known to be tightly regulated by several factors including parathyroid hormone (PTH), calcium, phosphorus, and 1,25(OH)2D per se (1Bouillon R. Okamura W.H. Norman A.W. Endocr. Rev. 1995; 16: 200-257Crossref PubMed Google Scholar, 2Jones G. Strugnell S.A. DeLuca H.F. Physiol. Rev. 1998; 78: 1193-1231Crossref PubMed Scopus (1040) Google Scholar, 3Sutton A.L. MacDonald P.N. Mol. Endocrinol. 2003; 17: 777-791Crossref PubMed Scopus (263) Google Scholar). 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 (13St-Arnaud R. Arabian A. Travers R. Barletta F. Raval-Pandya M. Chapin K. Depovere J. Mathieu C. Christakos S. Demay M.B. Glorieux F.H. Endocrinology. 2000; 141: 2658-2666Crossref PubMed Scopus (202) Google Scholar). 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 (14Haussler M.R. Whitfield G.K. Haussler C.A. Hsieh J.C. Thompson P.D. Selznick S.H. Dominguez C.E. Jurutka P.W. J. Bone Miner. Res. 1998; 13: 325-349Crossref PubMed Scopus (1241) Google Scholar). 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 (15Norman A.W. Okamura W.H. Bishop J.E. Henry H.L. Mol. Cell. Endocrinol. 2002; 197: 1-13Crossref PubMed Scopus (103) Google Scholar). We (16Panda D.K. Miao D. Tremblay M.L. Sirois J. Farookhi R. Hendy G.N. Goltzman D. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 7498-7503Crossref PubMed Scopus (549) Google Scholar) and others (17Dardenne O. Prud'homme J. Arabian A. Glorieux F.H. St-Arnaud R. Endocrinology. 2001; 142: 3135-3141Crossref PubMed Scopus (286) Google Scholar) have previously reported a mouse model deficient in 1,25(OH)2D by targeted ablation of the 1α(OH)ase gene (1α(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) (18Fraser D. Kooh S.W. Kind H.P. Holick M.F. Tanaka Y. DeLuca H.F. N. Engl. J. Med. 1973; 289: 817-822Crossref PubMed Scopus (335) Google Scholar, 19Eil C. Liberman U.A. Marx S.J. Adv. Exp. Med. Biol. 1986; 196: 407-422Crossref PubMed Scopus (15) Google Scholar). Several laboratories have also reported mouse models with targeted ablation of the VDR gene (VDR-/-) (20Li Y.C. Pirro A.E. Amling M. Delling G. Baron R. Bronson R. Demay M.B. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 9831-9835Crossref PubMed Scopus (829) Google Scholar, 21Yoshizawa 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. Kato S. Nat. Genet. 1997; 16: 391-396Crossref PubMed Scopus (1001) Google Scholar, 22Erben R.G. Soegiarto D.W. Weber K. Zeitz U. Lieberherr M. Gniadecki R. Moller G. Adamski J. Balling R. Mol. Endocrinol. 2002; 16: 1524-1537Crossref PubMed Scopus (208) Google Scholar). These animals develop manifestations similar to those with 1α(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) (23Beer S. Tieder M. Kohelet D. Liberman O.A. Vure E. Bar-Joseph G. Gabizon D. Borochowitz Z.U. Varon M. Modai D. Clin. Endocrinol. 1981; 14: 395-402Crossref PubMed Scopus (58) Google Scholar). 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 (22Erben R.G. Soegiarto D.W. Weber K. Zeitz U. Lieberherr M. Gniadecki R. Moller G. Adamski J. Balling R. Mol. Endocrinol. 2002; 16: 1524-1537Crossref PubMed Scopus (208) Google Scholar, 24Donohue M.M. Demay M.B. Endocrinology. 2002; 143: 3691-3694Crossref PubMed Scopus (92) Google Scholar). 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 (25Amling M. Priemel M. Holzmann T. Chapin K. Rueger J.M. Baron R. Demay M.B. Endocrinology. 1999; 140: 4982-4987Crossref PubMed Google Scholar). 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 (1α(OH)ase-/- and VDR-/-, respectively) and the mutant animals deficient in both ligand and receptor (1α(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 (26Li Y.C. Amling M. Pirro A.E. Priemel M. Meuse J. Baron R. Delling G. Demay M.B. Endocrinology. 1998; 139: 4391-4396Crossref PubMed Google Scholar), 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 1α(OH)ase and the VDR and compared siblings that were homozygous for deletion of the genes encoding 1α(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. Derivation of 1α(OH)ase and VDR Double Null Mice—The derivation of the two parental strains of 1α(OH)ase-/- mice and VDR-/- mice by homologous recombination in embryonic stem cells was previously described by Panda et al. (16Panda D.K. Miao D. Tremblay M.L. Sirois J. Farookhi R. Hendy G.N. Goltzman D. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 7498-7503Crossref PubMed Scopus (549) Google Scholar) and Li et al. (20Li Y.C. Pirro A.E. Amling M. Delling G. Baron R. Bronson R. Demay M.B. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 9831-9835Crossref PubMed Scopus (829) Google Scholar), 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 1α(OH)ase gene, replacing both the ligand binding and heme binding domains. RT-PCR of renal RNA from homozygous 1α(OH)ase-/- mice confirmed that no 1α(OH)ase mRNA is expressed from this allele (16Panda D.K. Miao D. Tremblay M.L. Sirois J. Farookhi R. Hendy G.N. Goltzman D. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 7498-7503Crossref PubMed Scopus (549) Google Scholar). 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 (20Li Y.C. Pirro A.E. Amling M. Delling G. Baron R. Bronson R. Demay M.B. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 9831-9835Crossref PubMed Scopus (829) Google Scholar). Mice heterozygous for the null 1α(OH)ase allele and mice heterozygous for the VDR allele were fertile (16Panda D.K. Miao D. Tremblay M.L. Sirois J. Farookhi R. Hendy G.N. Goltzman D. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 7498-7503Crossref PubMed Scopus (549) Google Scholar, 20Li Y.C. Pirro A.E. Amling M. Delling G. Baron R. Bronson R. Demay M.B. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 9831-9835Crossref PubMed Scopus (829) Google Scholar). VDR+/- mice were mated with 1α(OH)ase+/- mice, and offspring heterozygous at both loci were then mated to one another to generate pups homozygous for both 1α(OH)ase and VDR null alleles [1α(OH)ase-/-VDR-/-]. Lines were maintained by mating 1α(OH)ase-/-VDR-/- males and 1α(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 (27Vegesna V. O'Kelly J. Uskokovic M. Said J. Lemp N. Saitoh T. Ikezoe T. Binderup L. Koeffler H.P. Endocrinology. 2002; 143: 4389-4396Crossref PubMed Scopus (38) Google Scholar); or 3) a “rescue diet” (TD96348 Teklad, Madison, WI) of γ-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 (16Panda D.K. Miao D. Tremblay M.L. Sirois J. Farookhi R. Hendy G.N. Goltzman D. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 7498-7503Crossref PubMed Scopus (549) Google Scholar). To determine the genotype at both the 1α(OH)ase and VDR loci, four PCRs were conducted for each animal. To test for the presence of the wild-type 1α(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: 1α(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 1α(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-κB 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 (28Miao D. Bai X. Panda D. McKee M. Karaplis A. Goltzman D. Endocrinology. 2001; 142: 926-939Crossref PubMed Scopus (139) Google Scholar). 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 (28Miao D. Bai X. Panda D. McKee M. Karaplis A. Goltzman D. Endocrinology. 2001; 142: 926-939Crossref PubMed Scopus (139) Google Scholar). 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 (29Lopez-De Leon A. Rojkind M. J. Histochem. Cytochem. 1985; 33: 737-743Crossref PubMed Scopus (565) Google Scholar). 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 (30Miao D. Scutt A. J. Histochem. Cytochem. 2002; 50: 333-340Crossref PubMed Scopus (183) Google Scholar, 31He B. Deckelbaum R.A. Miao D. Lipman M.L. Pollak M. Goltzman D. Karaplis A.C. Endocrinology. 2001; 142: 2070-2077Crossref PubMed Scopus (35) Google Scholar). 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 (32Miao D. Scutt A. BMC Musculoskelet. Disord. 2002; 3: 16Crossref PubMed Scopus (54) Google Scholar). 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 (28Miao D. Bai X. Panda D. McKee M. Karaplis A. Goltzman D. Endocrinology. 2001; 142: 926-939Crossref PubMed Scopus (139) Google Scholar, 33Miao D. He B. Karaplis A.C. Goltzman D. J. Clin. Invest. 2002; 109: 1173-1182Crossref PubMed Scopus (237) Google Scholar). 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 (28Miao D. Bai X. Panda D. McKee M. Karaplis A. Goltzman D. Endocrinology. 2001; 142: 926-939Crossref PubMed Scopus (139) Google Scholar). 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 (34Bai X. Miao D. Panda D. Grady S. McKee M.D. Goltzman D. Karaplis A.C. Mol. Endocrinol. 2002; 16: 2913-2925Crossref PubMed Scopus (92) Google Scholar). 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 β-glycerophosphate, and 10-8m 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 (34Bai X. Miao D. Panda D. Grady S. McKee M.D. Goltzman D. Karaplis A.C. Mol. Endocrinol. 2002; 16: 2913-2925Crossref PubMed Scopus (92) Google Scholar). 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. 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 1α(OH)ase enzyme (lower panel) in the VDR-/- and 1α(OH)ase-/- mice, respectively. Expression of 1α(OH)ase and 24(OH)ase Genes—The 1α(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 r