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Endocrinology, doi:10.1210/en.2006-0403
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Endocrinology Vol. 147, No. 10 4801-4810
Copyright © 2006 by The Endocrine Society

Exogenous 1,25-Dihydroxyvitamin D3 Exerts a Skeletal Anabolic Effect and Improves Mineral Ion Homeostasis in Mice that Are Homozygous for Both the 1{alpha}-Hydroxylase and Parathyroid Hormone Null Alleles

Yingben Xue, Andrew C. Karaplis, Geoffrey N. Hendy, David Goltzman and Dengshun Miao

The Research Center for Bone and Stem Cells (Y.X., D.M.), Department of Anatomy, Histology, and Embryology and Institute of Dental Research, Nanjing Medical University, Nanjing, Jiangsu, People’s Republic of China; Calcium Research Laboratory (Y.X., G.N.H., D.G., D.M.), McGill University Health Centre, and Department of Medicine, McGill University, Montréal, Québec H3A 1A1, Canada; and Lady Davis Research Institute (A.C.K.), Sir Mortimer B. Davis-Jewish General Hospital and Department of Medicine, McGill University, Montréal, Québec H3T 1E2, Canada

Address all correspondence and requests for reprints to: Dr. Dengshun Miao, The Research Center for Bone and Stem Cells, Department of Anatomy, Histology, and Embryology and Institute of Dental Research, Nanjing Medical University, Nanjing, Jiangsu 210029, The People’s Republic of China. E-mail: dsmiao{at}njmu.edu.cn.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
1,25-Dihydroxyvitamin D3 [1,25(OH)2D3] and PTH each modulate calcium and skeletal homeostasis. To identify 1,25(OH)2D3-mediated skeletal and mineral ion actions independent of PTH, double-knockout mice, which are homozygous for both the 1{alpha}-hydroxylase and PTH null alleles, were treated with 1,25(OH)2D3, sc, from d 4 to 14 and compared with vehicle-treated animals. Serum calcium rose in 1,25(OH)2D3-treated double-knockout mice, and messenger RNA and protein levels of the renal calcium transporters TRPV5, calbindin-D28K, calbindin-D9K, and Na+/Ca2+ exchanger 1 were up-regulated. Parameters of endochondral bone formation, including long bone length, epiphyseal volume, chondrocyte proliferation and differentiation, and cartilage matrix mineralization, were all increased by 1,25(OH)2D3, Exogenous 1,25(OH)2D3 also increased both trabecular and cortical bone; augmented both osteoblast number and type I collagen deposition in bone matrix; and up-regulated expression levels of the osteoblastic genes alkaline phosphatase, type I collagen, and osteocalcin. Furthermore, in 1,25(OH)2D3-treated double mutants, osteoclastic bone resorption appeared to decline. The results indicate that administered 1,25(OH)2D3 used intestinal and renal but not skeletal mechanisms to elevate serum calcium and that this sterol can promote endochondral and appositional bone increases independent of endogenous PTH.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
1,25-DIHYDROXYVITAMIN D3 [1,25(OH)2D3] and PTH directly affect calcium homeostasis, and each exerts important regulatory effects on the other. Whereas PTH is the principal hormone involved in the minute-to-minute regulation of ionized calcium levels in the extracellular fluid, 1,25(OH)2D3 plays a key role in the day-to-day maintenance of calcium balance. PTH stimulates the production of 1,25(OH)2D3 by activating the renal 25-hydroxyvitamin D-1{alpha}-hydroxylase [1{alpha}(OH)ase] (1, 2), and 1,25(OH)2D3 in turn suppresses the synthesis and secretion of PTH (3) and controls parathyroid cell growth (4). Vitamin D receptors are present in the parathyroid glands and act as sensors for the detection of adequate levels of 1,25(OH)2D3, thus regulating parathyroid hormone synthesis and release (5). Vitamin D deficiency, therefore, causes parathyroid hyperplasia and secondary hyperparathyroidism. 1,25(OH)2D3 suppression of PTH synthesis occurs through negative regulation of the rate of PTH gene transcription by the 1,25(OH)2D3-vitamin D receptor (VDR)/retinoid X receptor complex (6). Both 1,25(OH)2D3 and PTH have catabolic and anabolic actions in bone and they can interact to maintain ion and bone homeostasis.

Intermittent administration of PTH in vivo has been shown to increase bone mass (7, 8). Thus, once-a-day sc injection of PTH stimulates the formation of new bone and increases bone mass in ovariectomized monkeys (9) and patients with osteoporosis (10, 11). PTH is the first anabolic agent to be approved for clinical use in osteoporosis. 1,25(OH)2D3 has also been used in the treatment of postmenopausal osteoporosis but based primarily on the finding that these patients have impaired calcium absorption that can be corrected by small, apparently physiological, doses of 1,25(OH)2D3 (12). During the past 3 decades, 1,25(OH)2D3 has been used in clinical trials examining bone loss and osteoporosis, but the results have been inconsistent. Some trials have shown that 1,25(OH)2D3 is effective in preventing osteoporosis by increasing low bone mineral density (13, 14, 15) and decreasing fracture rates (16), whereas other trials could not detect any significant effect of 1,25(OH)2D3 on decreasing fractures (17).

Several studies have shown that bone volume and osteoblast numbers are increased in hypocalcemic-1{alpha}(OH)ase–/– (18, 19) and VDR–/– (20, 21) animals with secondary hyperparathyroidism. These increases most likely reflect the well-characterized anabolic activity of PTH (22). We recently found that in 1{alpha}(OH)ase–/–, VDR–/–, and 1{alpha}(OH)ase–/–VDR–/– mice on a rescue diet, which normalizes serum calcium, phosphorus and PTH levels, there was a reduction in osteoblast numbers, mineral apposition rate, and bone volume below levels seen in wild-type mice (23). These results demonstrated that in the presence of normal circulating PTH, if deficiency of 1,25(OH)2D3 action occurs, bone formation is reduced. These results therefore showed a physiologic anabolic role for endogenous 1,25(OH)2D3 and the VDR in vivo.

Nevertheless, because of the complex interactions of 1,25(OH)2D3 and PTH, it is difficult to determine the precise mechanism of each hormone in regulating skeletal and mineral homeostasis when both are present. Thus, we previously reported that treatment of 1{alpha}(OH)ase–/– mice that have secondary hyperparathyroidism with exogenous 1,25(OH)2D3 reduced bone volume, but this was almost certainly due to the reduction in circulating PTH levels that were induced by the 1,25(OH)2D3 treatment (23). Recently we generated double-knockout (KO) mice that are homozygous for both the 1{alpha}(OH)ase and PTH null alleles and compared them with mice null for each single gene and wild-type mice at 2 wk of age (24). Double-KO mice died 1–3 wk postnatally, before the end of weaning with severe tetany and hypocalcemia and skeletal defects that were more severe than in either type of single gene KO mice. We previously used this double-KO model to examine the effect of the amino-terminal regions of PTH and PTHrP on bone and mineral metabolism (25). In the present studies, we report the use of this double-KO model to determine the effects of exogenous 1,25(OH)2D3 on skeletal and mineral homeostasis independent of the confounding actions of PTH. 1,25(OH)2D3 was administered daily starting from d 4 until 14 d of age. At d 14, we compared the 1,25(OH)2D3-treated double-KO mice with vehicle-treated double-KO mice and vehicle-treated wild-type mice.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Derivation of PTH and 1{alpha}(OH)ase double-null mice and genotyping of mice
The derivation of the two parental strains of PTH–/– mice and 1{alpha}(OH)ase–/– mice by homologous recombination in embryonic stem cells and genotyping of mice was previously described by Xue et al. (24, 25). The PTH–/–1{alpha}(OH)ase–/– (double KO) mice have been reported by us to have undetectable circulating levels of PTH and 1,25(OH)2D3 (24).

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 light, 12-h dark cycle. Ordinarily, double-KO mice die with severe tetany, approximately 3 wk after birth, before the end of weaning. The wild-type pups and double mutants were injected sc with vehicle or 0.02 µg 1,25(OH)2D3/d starting at d 4 for 10 d. After 1,25(OH)2D3 administration for 10 d, the 1,25(OH)2D3-treated double-KO mice were compared with vehicle-treated wild-type and vehicle-treated double-KO mice.

Biochemistry
Serum concentrations of calcium and phosphorus, urine calcium, and creatinine were determined by routine methods using diagnostic reagents (Sigma, St. Louis, MO).

RT-PCR
RNA was isolated from mouse kidneys and long bones, using Trizol reagent (Invitrogen Inc., Carlsbad, CA) according to the manufacturer’s protocol. RT-PCR was performed by a one-step method using QIAGEN One-Step RT-PCR kit (QIAGEN, Mississauga, Canada) according to the manufacturer’s instructions (24, 25) with the primer sets shown in Table 1Go.


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  		TABLE 1. RT-PCR primers used with their name, orientation (S, sense; AS, antisense), sequence, annealing temperature (Tm), and length of amplicon (bp)

 
Western blot analysis
Proteins were extracted from kidneys and quantitated by a kit (Bio-Rad, Mississauga, Ontario, Canada). Thirty-microgram protein samples were fractionated by SDS-PAGE and transferred to nitrocellulose membranes. Immunoblotting was carried out as described (24, 25, 26) using antibodies against TRPV5 (ECaC1), calbindin-D28K, and calbindin-D9K, Na+/Ca2+ exchanger 1 (NCX1) (Swant, Bellinzona, Switzerland) and ß-tubulin (Santa Cruz Biotechnology Inc., Santa Cruz, CA). Bands were visualized using enhanced chemiluminescence (Amersham, Aylesbury, UK).

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

Microcomputed tomography (micro-CT)
Femurs obtained from 2-wk-old mice were dissected free of soft tissue, fixed overnight in 70% ethanol, and analyzed by micro-CT with a SkyScan 1072 scanner and associated analysis software (SkyScan, Antwerp, Belgium) as described (24, 25, 27). Briefly, image acquisition was performed at 100 kV and 98 µA with a 0.9° rotation between frames. During scanning, the samples were enclosed in tightly fitting plastic wrap to prevent movement and dehydration. Thresholding was applied to the images to segment the bone from the background. Two-dimensional images were used to generate three-dimensional (3D) renderings using the 3D Creator software supplied with the instrument. The resolution of the micro-CT images is 18.2 microns.

Histology
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 described (26). Proximal ends of tibiae were decalcified in EDTA glycerol solution for 5–7 d at 4 C. Decalcified right femurs and tibiae 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 (H&E) or histochemically for tartrate-resistant acid phosphatase (TRAP) activity or immunohistochemically as described below. Alternatively, undecalcified left tibiae were embedded in LR White acrylic resin (London Resin Co. 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.

Histochemical staining for TRAP
Enzyme histochemistry for TRAP was performed as previously described (28). Dewaxed sections were preincubated for 20 min in buffer containing 50 mM sodium acetate and 40 mM sodium tartrate (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.

Immunohistochemical staining
The proliferating cell nuclear antigen (PCNA) and type I and X collagens were determined by immunohistochemistry as described previously (24, 25, 26, 29). Mouse monoclonal antibody against PCNA (Medicorp Inc., Montréal, Canada), affinity-purified goat antihuman type I collagen antibody (Southern Biotechnology Associates, Birmingham, AL), and rabbit antiserum to type X collagen (a generous gift of Dr. A. R. Poole, Shriners Hospital, Montréal, Canada) were applied to dewaxed paraffin sections overnight at room temperature. As a negative control, 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 Tris-buffered saline, the sections were incubated with secondary antibody (biotinylated goat antirabbit IgG or biotinylated rabbit antigoat IgG; Sigma), washed as before, and incubated with the Vectastain ABC-AP kit or the Vectastain Elite ABC kit (Vector Laboratories, Inc., Ontario, Canada) for 45 min. After washing as before, red pigmentation to demarcate regions of immunostaining was produced by a 10- to 15-min treatment with Fast Red TR/Naphthol AS-MX phosphate [containing 1 mM levamisole as endogenous alkaline phosphatase (ALP) inhibitor; Sigma], or gray pigmentation was likewise produced using a Vector SG kit (Vector Laboratories, Inc.). After washing with distilled water, the sections were counterstained with methyl green and mounted with Kaiser’s glycerol jelly.

Computer-assisted image analysis
After H&E staining or histochemical or immunohistochemical staining of sections from six mice of each genotype, images of fields were photographed with a Leica (Cambridge, UK) DFC camera. Images of micrographs from single sections were digitally recorded using a rectangular template, and recordings were processed and analyzed using Northern Eclipse image analysis software as previously described (24, 25, 26, 29, 30).

Statistical analysis
Data from image analysis are presented as means ± SEM. Statistical comparisons were made using a one-way ANOVA, with P < 0.05 being considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effect of exogenous 1,25(OH)2D3 on calcium, phosphorus, and renal calcium transporters in vivo
Serum calcium levels rose, from 43% of wild-type mice in vehicle-treated double-KO mice, to 69% of wild-type mice in 1,25(OH)2D3-treated double-KO mice, at 2 wk of age (Fig. 1AGo). The vehicle-treated double-KO mice displayed elevated serum phosphorus levels, compared with wild-type mice; however, the serum phosphorus levels were decreased to normal in 1,25(OH)2D3-treated double-KO mice at 2 wk of age (Fig. 1BGo). The urine calcium to creatinine ratio was significantly elevated in vehicle-treated double-KO mice, compared with vehicle-treated wild-type mice. Urine calcium to creatinine ratio was significantly reduced in 1,25(OH)2D3-treated double-KO mice relative to vehicle-treated double-KO mice but was not normalized (Fig. 1CGo).


Figure 1
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  		FIG. 1. Effect of exogenous 1,25(OH)2D3 on serum calcium and phosphorus, urine calcium relative to creatinine, and renal calcium transporters in vivo. Serum calcium (A) and phosphorus (B) and urine calcium to creatinine ratio (C) were determined in 2-wk-old vehicle-treated wild-type (WT-vehicle), vehicle-treated PTH–/–1{alpha}(OH)ase–/– mice (DKO-vehicle), and 1,25(OH)2D3-treated PTH–/–1{alpha}(OH)ase–/– mice (DKO+1,25D) as described in Materials and Methods. Each value is the mean ± SEM of determinations in six mice of each group. D, Representative RT-PCR analysis of renal extracts for gene expression of TRPV5, calbindin-D9K (CB9K), calbindin-D28K (CB28K), and NCX1. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA was the loading control. E, Representative Western blots of renal extracts for expression of TRPV5, CB9K, CB28K, and NCX1. ß-Tubulin was used as a loading control for Western blots. F, TRPV5, CB28K, CB9,K and NCX1 mRNA levels relative to GAPDH mRNA level were assessed by densitometric analysis and expressed relative to respective levels of these transporters in wild-type mice. Each value is the mean ± SEM of triplicate determinations. G, TRPV5, CB28K, CB9K, and NCX1 protein levels relative to ß-tubulin protein level were assessed by densitometric analysis and expressed relative to respective levels of these transporters in wild-type mice. Each value is the mean ± SEM of triplicate determinations. *, P < 0.05; ***, P < 0.001, compared with vehicle-treated wild-type mice; #, P < 0.05; ###, P < 0.001, compared with vehicle-treated double-KO mice.

 
To determine whether the raised serum calcium levels and lower urine calcium levels induced by exogenous 1,25(OH)2D3 were contributed to by increased renal reabsorption of calcium, we examined the mRNA and protein expression levels of the renal calcium transporters TRPV5, calbindin-D28K, calbindin-D9K, and NCX1 by RT-PCR and Western blots. Although the mRNA and protein levels of these genes were down-regulated in the kidney of double-KO mice relative to wild-type, they were up-regulated by 1,25(OH)2D3 treatment (Fig. 1Go, D–G).

Effect of exogenous 1,25(OH)2D3 on skeletal phenotypes
To assess the effect of exogenous 1,25(OH)2D3 administration on postnatal skeletal development, long bones were analyzed by radiography (Fig. 2AGo) and micro-CT (Fig. 2BGo). The length of femurs was shorter in vehicle-treated double-KO mice than wild-type mice, but the length approached that of their wild-type littermates at 2 wk of age in 1,25(OH)2D3-treated mice (Fig. 2Go, A and C). Radiolucency in epiphyses and metaphyses was greater in vehicle-treated double-KO mice relative to wild-type mice; however, radiolucency was significantly lower at 2 wk of age in 1,25(OH)2D3-treated animals (Fig. 2AGo). From 3D reconstructed longitudinal sections and cross-sections of the distal ends of femurs (Fig. 2BGo), it can be seen that epiphyses were smaller, cortices were thinner, and cortical volumes were lower in vehicle-treated double-KO mice, compared with wild-type mice. After treatment with 1,25(OH)2D3, however, epiphyses were larger (Fig. 2DGo), and trabecular (Fig. 2EGo) and cortical (Fig. 2FGo) bone volumes were greater than in vehicle-treated double-KO mice.


Figure 2
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  		FIG. 2. Effect of exogenous 1,25(OH)2D3 on skeletal phenotypes. A, Representative contact radiographs of the femurs from 2-wk-old vehicle-treated wild-type (WT-vehicle), vehicle-treated PTH–/–1{alpha}(OH)ase–/– mice (DKO-vehicle), and 1,25(OH)2D3-treated PTH–/–1{alpha}(OH)ase–/– mice (DKO+1,25D). B, Longitudinal sections and cross-sections of 3D reconstructed proximal end of tibiae by micro-CT. Quantitation of femoral length (C), epiphyseal volume (D), trabecular bone volume (BV/TV) (E), and cortical volume (F) were determined in 2-wk-old vehicle-treated wild-type (WT-vehicle), vehicle-treated PTH–/–1{alpha}(OH)ase–/– mice (DKO-vehicle), and 1,25(OH)2D3-treated PTH–/–1{alpha}(OH)ase–/– mice (DKO+1,25D) as described in Materials and Methods. Each value is the mean ± SEM of determinations in six mice of each group. *, P < 0.05, compared with vehicle-treated wild-type mice; #, P < 0.05, compared with vehicle-treated DKO mice.

 
Effect of exogenous 1,25(OH)2D3 on chondrocyte proliferation and differentiation and cartilage mineralization
To assess the effect of exogenous 1,25(OH)2D3 administration on endochondral bone formation, we examined the histology of the cartilaginous growth plates, proliferation and differentiation of chondrocytes, and mineralization of the hypertrophic zone in 2-wk-old mice. The growth plate width (Fig. 3Go, A and B) was narrower, PCNA-positive chondrocytes (Fig. 3Go, C and D) were decreased, and the type X collagen-positive hypertrophic zone (Fig. 3Go, E and F) was less intensely stained, dramatically narrowed, and less mineralized (Fig. 3Go, G and H) in vehicle-treated double-KO mice, compared with wild-type littermates. However, after 10 d of 1,25(OH)2D3 administration, all these parameters were improved in the double-KO mice, although they were still below wild-type levels (Fig. 3Go, A–H).


Figure 3
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  		FIG. 3. Effect of exogenous 1,25(OH)2D3 on chondrocyte proliferation and differentiation and cartilage mineralization. Paraffin-embedded sections of tibiae from the vehicle-treated wild-type (WT-vehicle), vehicle-treated PTH–/–1{alpha}(OH)ase–/– mice (DKO-vehicle), and 1,25(OH)2D3-treated PTH–/–1{alpha}(OH)ase–/– mice (DKO+1,25D) at 2 wk of age were stained with H&E (A) and immunostained for PCNA (C) or type X collagen (Col X) (E) as described in Materials and Methods. G, Undecalcified sections of tibiae were stained with the von Kossa procedure as described in Materials and Methods. Scale bars (A–D), 100, 25, 25, and 50 µm, respectively. B, Width of growth plates (GPs) was determined as described in Materials and Methods. D, Numbers of PCNA-positive chondrocytes as a percent of total chondrocytes per field were determined by image analysis. F, The type X collagen immunopositive area as a percentage of the growth plate field was determined. H, The mineralized area as a percent of the cartilage matrix per field was determined by image analysis. Each value is the mean ± SEM of determinations in six animals of each group. *, P < 0.05, compared with vehicle-treated wild-type mice; #, P < 0.05, compared with vehicle-treated DKO mice.

 
Effect of exogenous 1,25(OH)2D3on osteoblastic bone formation parameters
To determine whether the alterations in bone volume were associated with alterations in osteoblastic bone formation, histomorphometric analysis for osteoblast number, type I collagen deposition, and osteoid volume in bone matrix were performed at 2 wk of age. The number of osteoblasts and matrix deposition of type I collagen (Fig. 4Go, A, B, E, and F) were increased in 1,25(OH)2D3-treated double mutants, compared with vehicle-treated double mutants, although they did not reach the levels observed in vehicle-treated wild-type mice. Consistent with the improvement of serum calcium levels, the mineralization in the trabecular and cortical bone matrix was also improved in 1,25(OH)2D3-treated double mutants, compared with vehicle-treated double mutants (Fig. 4Go, C, D, and G). Furthermore, in keeping with the histological observations, levels of expression of the osteoblastic-specific genes ALP, type I collagen, and osteocalcin were all up-regulated after1,25(OH)2D3 administration to double-KO mice as demonstrated by RT-PCR (Fig. 4HGo). These results indicate that stimulation of osteoblastic bone formation is a major factor contributing to the bone anabolic actions of exogenous 1,25(OH)2D3.


Figure 4
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  		FIG. 4. Effect of exogenous 1,25(OH)2D3 on osteoblastic parameters. Representative micrographs of decalcified paraffin-embedded sections stained with H&E (A) and immunostained for type I collagen (Col I) (B) and undecalcified LR White resin-embedded sections stained with the von Kossa procedure in the trabeculae (C) and cortex (D) of the proximal ends of tibiae of the vehicle-treated wild-type (WT-vehicle), vehicle-treated PTH–/–1{alpha}(OH)ase–/– mice (DKO-vehicle), and 1,25(OH)2D3-treated PTH–/–1{alpha}(OH)ase–/– mice (DKO+1,25D) at 2 wk of age. Scale bar, 25 µm. E, Numbers of osteoblasts per square millimeter tissue area (N.Ob/T.Ar) were counted in the primary spongiosa of H&E-stained tibiae of the mice and presented as mean ± SEM. F, The type I collagen-positive area as a percent of the tissue area was determined in the metaphyseal regions for each group. G, Osteoid volume was determined in undecalcified von Kossa-stained sections and is presented as a percent of bone volume [OV/BV (%)] of trabeculae and cortex. Each value is the mean ± SEM of determinations in six animals of each group. ***, P < 0.001 relative to vehicle-treated wild-type mice; ###, P < 0.001, compared with vehicle-treated DKO mice. H, Representative RT-PCR analysis of long-bone extracts for gene expression of ALP, type I collagen (Col I), and osteocalcin (OCN). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA was used as loading controls.

 
Effect of exogenous 1,25(OH)2D3 on osteoclastic parameters
To determine whether 1,25(OH)2D3 can also exert a catabolic effect on bone in PTH and 1,25(OH)2D3-deficient mice, histochemical staining for TRAP (Fig. 5AGo) was performed and the number of osteoclasts and the osteoclastic surface were measured by histomorphometric analysis. The results revealed that the number of TRAP-positive osteoclasts relative to bone perimeter [number per millimeter (N.Oc/B.Pm)] and osteoclastic surface relative to bone surface (Oc.S/BS, percent) were slightly decreased in vehicle-treated double-KO mice and significantly decreased in 1,25-(OH)2D3-treated double-KO mice, compared with, vehicle-treated wild-type mice (Fig. 5Go, A–C). It also was found that the number of TRAP-positive osteoclasts at the hypertrophic cartilage/bone interface was slightly decreased in vehicle-treated double-KO mice and significantly decreased in 1,25-(OH)2D3-treated double-KO mice, compared with vehicle-treated wild-type mice (Fig. 5DGo). Consistent with the alteration of osteoclast numbers, gene expression levels of receptor activator of nuclear factor-{kappa}B ligand (RANKL) and osteoprotegerin (OPG) were reduced in vehicle-treated double-KO mice and 1,25(OH)2D3-treated double-KO mice, compared with vehicle-treated double-KO mice (Fig. 5EGo). The ratio of RANKL to OPG was significantly decreased in vehicle-treated double-KO mice and 1,25(OH)2D3-treated double-KO mice, compared with vehicle-treated wild-type mice (Fig. 5FGo).


Figure 5
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  		FIG. 5. Effect of exogenous 1,25(OH)2D3 on osteoclastic parameters. A, Representative micrographs of sections of the tibial metaphysis stained histochemically for TRAP in the vehicle-treated wild-type (WT-vehicle), vehicle-treated PTH–/–1{alpha}(OH)ase–/– mice (DKO-vehicle), and 1,25(OH)2D3-treated PTH–/–1{alpha}(OH)ase–/– mice (DKO+1,25D) at 2 wk of age. Scale bar, 50 µm. B, Number of TRAP-positive osteoclasts related to bone perimeter [N.Oc/B.Pm (number per millimeter)]. C, Osteoclastic surface relative to bone surface (Oc.S/BS, percent). D, Number of TRAP-positive osteoclasts per cartilage/bone interface of the proximal end of tibiae. Each value is the mean ± SEM of determinations in six animals of each group. D, Representative RT-PCR analysis, performed on bone extracts for RANKL and OPG mRNAs as described in Materials and Methods. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA was a loading control. E, RANKL and OPG mRNA levels relative to GAPDH mRNA level were determined by densitometric analysis, and the ratios are expressed as a percent of respective ratios in wild-type mice. Each value is the mean ± SEM of triplicate determinations. *, P < 0.05: **, P < 0.01 relative to vehicle-treated wild-type mice; #, P < 0.01, compared with the vehicle-treated double-KO mice.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The current study demonstrates that exogenous 1,25(OH)2D3 administration increases serum calcium levels and exerts a skeletal anabolic role independent of circulating PTH. Although the 1,25(OH)2D3 was assessed only at a single dose and time point, this was nevertheless clearly sufficient to demonstrate an increase in cortical and trabecular bone. Inasmuch as only a single dose of 1,25(OH)2D3 was administered, however, the potency of the sterol could not be assessed.

There is general agreement and considerable evidence that 1,25(OH)2D3 can increase serum calcium and phosphate by increasing intestinal absorption of calcium (31). Our results reveal that besides increasing intestinal calcium absorption, exogenous 1,25(OH)2D3 up-regulated renal calcium transporters and appeared to increase renal reabsorption of calcium. This is consistent with a previous report that 1,25(OH)2D3 administration to 1{alpha}(OH)ase–/– mice raised serum calcium and increased the expression of renal calcium transporters (32). The exact mechanism whereby 1,25(OH)2D3 administration reduced serum phosphorus in blood is uncertain, but this was possibly a consequence, at least in part, of increased deposition of phosphorus in bone and cartilage during the rapid skeletal growth and mineralization that was induced by 1,25(OH)2D3. In addition, recent studies have shown that 1,25(OH)2D3 can increase levels of the phosphatonin, fibroblast growth factor 23 (33), which may have therefore augmented renal phosphate excretion. Consequently the net effect of 1,25(OH)2D3, which can also enhance phosphorus absorption from gut, was a reduction in serum phosphorus.

Our results demonstrate that many indices of endochondral bone formation were improved after 10 d of exogenous 1,25(OH)2D3 administration to the double-KO mice. The indices increased include epiphyseal volume, chondrocyte proliferation and differentiation, cartilaginous matrix mineralization, and long bone length. We previously reported similar increases in these parameters in double-KO mice receiving either PTH (1–34) or PTHrP (1–86); however, augmentations in epiphyseal size were less than were currently observed with 1,25(OH)2D3. These differences between PTH and 1,25(OH)2D3 actions on the growth plate are consistent with our previous findings that endogenous PTH deficiency in PTH–/– mice caused only a slight reduction in bone length, whereas endogenous 1,25(OH)2D3 deficiency in 1{alpha}(OH)ase–/– mice caused a highly significant decrease in bone length (24, 25) and support the concept that 1,25(OH)2D3 plays a more prominent role in enhancing endochondral bone formation than does the N-terminal region of PTH. The positive effect of exogenous PTH that we previously observed on the avascular growth plate of the double-KO mice was likely mainly due to increased extracellular calcium (34) induced by PTH. In contrast, the positive effect of 1,25(OH)2D3 on the growth plate was likely in part due to increased extracellular calcium induced by 1,25(OH)2D3 and in part due to a direct effect of 1,25(OH)2D3. Thus, we and others have reported that the cartilaginous growth plate was enlarged and distorted with a widened hypertrophic zone in 1{alpha}(OH)ase–/– (18, 19) and VDR–/– adult animals (20, 21). Although a rescue diet that normalized serum calcium did not completely reverse the abnormal growth plate in the 1{alpha}(OH)ase–/– mice (23, 35), the growth plate was completely restored after treatment with exogenous 1,25(OH)2D3, which normalized serum calcium but also raised circulating 1,25(OH)2D3 levels (23, 35). These studies therefore support the thesis that both extracellular calcium and 1,25(OH)2D3 are coregulators in optimal development of the growth plate.

A striking finding from the current study is that in addition to stimulating endochondral bone formation, exogenous 1,25(OH)2D3 administration also increased appositional bone at both trabeculae and cortices. Thus, trabecular and cortical bone volume, osteoblast numbers, and type I collagen deposition in bone matrix were all increased. In addition, osteoblastic gene expression levels of ALP, type I collagen, and osteocalcin were all up-regulated after 10 d of 1,25(OH)2D3 administration to the double-KO mice, although, the effects were weaker than were similar effects previously observed with PTH administration in the same model. Our results with 1,25(OH)2D3 also complement our previous observations in 2-wk-old 1{alpha}(OH)ase–/– mice (24), which, in the absence of endogenous 1,25(OH)2D3, had diminished trabecular bone volume, reduced osteoblastic bone formation parameters, and decreased gene expression levels of markers of the osteoblastic phenotype. Thus, whereas our previous results pointed to a physiologic skeletal anabolic role of endogenous 1,25(OH)2D3 in vivo (24), our current results demonstrate a skeletal anabolic role for exogenous 1,25(OH)2D3 in vivo in a PTH-independent manner.

These observations support the growing body of evidence that exogenous 1,25(OH)2D3 and analogs have anabolic effects on bone. Thus, 1,25(OH)2D3 treatment of cultures of mature osteoblasts in vitro up-regulates osteoblast-associated genes and enhances subsequent differentiation (36, 37) and 1,25(OH)2D3, and analogs have also recently been shown to increase collagen synthesis in a primary organ culture system in vitro (38). In vivo, in aged ovariectomized rats (39, 40, 41, 42) or a mouse model of accelerated senescence (43), intermittent oral administration of analogs of 1,25(OH)2D3 or iv infusion of 1,25(OH)2D3, respectively, have been reported to exhibit beneficial effects on cortical bone in long-bone diaphyses generally by stimulating periosteal and endocortical bone formation. Furthermore, transgenic mice overexpressing the VDR in mature osteoblastic cells displayed increased cortical bone due to periosteal bone formation and also increased trabecular bone volume (44). Nevertheless, because calcium levels were increased by 1,25(OH)2D3 administration in our studies, we cannot exclude the possibility that calcium per se, perhaps via the calcium receptor (45), may contribute to the skeletal anabolic effects of exogenous 1,25(OH)2D3.

Although increased osteoclastic bone resorption (38, 46, 47) by stimulating RANKL and inhibiting OPG (48) is a potential side effect of administering 1,25(OH)2D3, our results have shown that daily 1,25(OH)2D3, under the current experimental paradigm, reduced TRAP-positive osteoclasts and osteoclastic surface. This is compatible with previous studies in other models in which suppression of osteoclastic bone resorption was also observed (43, 44, 49). The role of calcium per se in potentially modifying the effect of 1,25(OH)2D3 on bone resorption remains to be elucidated.

In summary, exogenous 1,25(OH)2D3 increased serum calcium independent of PTH, not due to increased bone resorption, but likely due to increased intestinal calcium absorption in association with increased renal calcium reabsorption facilitated by enhanced expression of renal transporters. Endochondral bone formation was augmented, which emphasizes the important role for the active form of vitamin D in growth and which may indicate an important role for this sterol in facilitating the achievement of peak bone mass. Exogenous administration of 1,25(OH)2D3 also, independently of PTH, increased both cortical and trabecular bone. Whether the increased bone mass observed in hypoparathyroid patients treated with long-term 1,25(OH)2D3 reflects in part this growth-promoting action of the sterol remains to be determined. Nevertheless our findings reemphasize its potential therapeutic role in augmenting bone mass. Clearly the development of analogs that selectively activate the skeletal anabolic actions identified, in preference to activation of calcium transport mechanisms in the intestine and kidney, may provide important new agents for low bone mass therapy.


   Footnotes
 
This work was supported by start-up funding from Nanjing Medical University, China (to D.M.) and operating grants from the Canadian Institutes for Health Research (to A.C.K., G.N.H., and D.G.).

Disclosure statement: the authors have nothing to disclose.

First Published Online July 20, 2006

Abbreviations: ALP, Alkaline phosphatase; 3D, three-dimensional; H&E, hematoxylin and eosin; KO, knockout; micro-CT, microcomputed tomography; NCX1, Na+/Ca2+ exchanger 1; 1,25(OH)2D3, 1,25-dihydroxyvitamin D3; OPG, osteoprotegerin; PCNA, proliferating cell nuclear antigen; RANKL, receptor activator of nuclear factor-{kappa}B ligand; TRAP, tartrate-resistant acid phosphatase; VDR, vitamin D receptor.

Received March 30, 2006.

Accepted for publication July 10, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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