Review
Sex-related differences in the skeletal phenotype of aged vitamin D receptor global knockout mice

https://doi.org/10.1016/j.jsbmb.2015.12.005Get rights and content

Highlights

  • Adult male Vdr−/− mice exhibit bone loss despite being fed the rescue diet.

  • Female Vdr−/− mice exhibit vertebral bone loss and yet increased femoral bone volume.

  • Rescue diet fed to Vdr−/− mice does not normalise bone volume.

  • In addition to the intestine, VDR may be required to directly regulate bone homeostasis.

Abstract

The role of the vitamin D receptor (VDR) in maintaining skeletal health appears to be complex and dependent on the physiological context. Global Vdr deletion in a mouse model (Vdr−/−) results in hypocalcemia, secondary hyperparathyroidism and bone features typical of vitamin D-dependent rickets type II. When weanling Vdr−/− mice are fed a diet containing high levels of calcium, phosphorus and lactose, termed the rescue diet, normalisation of serum calcium, phosphate and parathyroid hormone levels results in prevention of rickets at 10 weeks of age. However, 17 week old male Vdr −/− mice, fed the rescue diet, have been reported as osteopenic due to a decrease in bone formation when compared to wild type mice. We now report confirmation of this finding with further data on the effect of the rescue diet on appendicular and axial skeletal structures in male and female Vdr−/− mice at 26 weeks of age compared to Vdr+/− controls. All Vdr−/− mice were normocalcemic with no evidence of any mineralization defect. However, male Vdr−/− mice exhibited significantly reduced mineral in femoral and vertebral bones when compared to control littermate Vdr+ /− mice, consistent with the previously reported data. In contrast, 26-week-old female Vdr−/− mice demonstrated significantly increased femoral trabecular bone volume although there was decreased vertebral trabecular bone volume, similar to males, and femoral cortical bone volume was unchanged. Thus, the Vdr−/− mouse model displays sex- and site-specific differences in skeletal structures with long-term feeding of a rescue diet. Although the global Vdr−/− ablation does not permit the determination of skeletal mechanisms producing these differences, these data confirm skeletal changes even when fed the rescue diet.

Introduction

The vitamin D receptor (VDR) knockout (Vdr−/−) mouse model fed standard chow diet demonstrate features typical of the human disease vitamin D-dependent rickets type II [1], [2]. The inability for the 1,25 dihydroxyvitamin D3 (1,25D) ligand to bind to the VDR results in gross changes to mineral ion homeostasis as a result of impaired intestinal calcium absorption and actions at the kidney and bone. At weaning, Vdr−/− mice develop hypocalcemia and secondary hyperparathyroidism giving rise to rickets with severe defects in bone growth and mineralization. Importantly, when weanling Vdr−/− mice are fed a diet containing high levels of calcium (2%), phosphorus (1.25%) and lactose (20%), normalization of plasma calcium, phosphate and parathyroid hormone (PTH) levels occurs, preventing the rickets bone phenotype and resulting in normal bone structure at least up to 10 weeks of age [3], [4], [5]. These data clearly demonstrate that the actions of 1,25D, acting through the VDR, are critical for maintaining plasma calcium and phosphate homeostasis, in order to adequately mineralize the skeleton in young mice.

Longer-term feeding of the rescue diet to Vdr−/− mice until adulthood, however, appears to be insufficient to maintain normal bone mineral volume levels. When weanling male Vdr−/− mice were fed the rescue diet until 17 weeks of age, a marked reduction in bone volume was observed compared to wild type mice, without the features of osteomalacia and akin to osteopenia [6]. These animals maintained normal serum calcium, phosphate and PTH levels. Furthermore, the osteopenia occurred without increased bone resorptive activity and was, at least in part, due to a significant reduction in mineral apposition rate (MAR). These data raise the possibility that VDR plays additional roles in the maintenance of bone homeostasis in adulthood beyond intestinal and renal transport of calcium. VDR-mediated activities in bone have been shown through numerous in vitro and in vivo studies to include regulation of proliferation, differentiation and mineralisation of osteoblasts [7], [8], inhibition of mineralization in osteocytes [9] and the well-described bone resorption response via RANKL signalling [10], [11].

In an effort to clarify the benefits and/or inadequacies of the rescue diet on calcium and skeletal homeostasis in Vdr−/− mice, we examined the bone phenotype of Vdr−/−, compared to Vdr+/− mice following long-term feeding of rescue diet from weaning until 26 weeks of age using micro-computerised tomography to assess trabecular and cortical bone at axial and appendicular sites.

Section snippets

Animals

Male and female Vdr−/− and Vdr+/− littermate mice were bred by mating Vdr+/− females with Vdr/− males. Mice were group-housed, with 5 or fewer animals per cage. At 20 days of age, all mice were fed rescue diet containing 2% calcium, 1.25% phosphorus, 20% lactose diet based on the Teklad diet TD96348 (Specialty Feeds, WA, Australia). Vdr+/− littermate mice were used as control mice as it has been previously demonstrated that intestinal calcium absorption, serum PTH and serum 1,25D were all

Results

This study was designed to investigate the effect of the presence or absence of the VDR on bone mineral homeostasis on rescue diet-fed, litter-matched mice in which either one (Vdr+/−) or no intact wild-type VDR allele (Vdr−/−) was present. The earliest publications in this field analyzed the effect of ablation of both VDR alleles against both the heterozygous (Vdr+/−) and wild type (Vdr+/+) littermate mice and no differences between mice with either one or two VDR alleles were detected when

Discussion

We have studied the capacity of the high calcium and phosphorus rescue diet to maintain bone mineral when fed to weanling male and female Vdr−/− mice until 26 weeks of age. It is well known weanling Vdr−/− mice fed a standard calcium and phosphorus diet develop both hypocalcemia and secondary hyperparathyroidism with a profoundly under-mineralised skeleton consistent with rickets. However, when Vdr−/− mice are fed a rescue diet sufficient to maintain normocalcemia and normophosphatemia

Acknowledgements

We would like to thank Dr. Ian Parkinson for his assistance with individual trabecular segmentation analyses. PHA is supported by a NHMRC Career Development Award (GNT1051858). JWR is supported by an APA through the University of South Australia.

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