ReviewSex-related differences in the skeletal phenotype of aged vitamin D receptor global knockout mice
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.
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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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