Rebecca K. Sawyer

Vitamin D Depletion Induces RANKL-Mediated Osteoclastogenesis and Bone Loss in a Rodent Model†

The association between increased risk of hip fracture and low vitamin D status has long been recognized. However, the level of vitamin D required to maintain bone strength is controversial. We used a rodent model of vitamin D depletion to quantify the 25‐hydroxyvitamin D (25D) levels required for normal mineralization. Six groups of 10‐wk‐old male Sprague‐Dawley rats (n = 42) were fed a diet containing 0.4% calcium and various levels of dietary vitamin D3 for 4 mo to achieve stable mean serum 25D levels ranging between 10 and 115 nM. At 7 mo of age, animals were killed, and the histomorphometry of distal and proximal femora and L2 vertebra was analyzed. Total RNA was extracted from whole femora for real‐time RT‐PCR analyses. In the distal femoral metaphysis, trabecular bone mineral volume (BV/TV) showed a significant positive association with circulating 25D levels (r2 = 0.42, p < 0.01) in the animals with serum 25D levels between 20 and 115 nM. Osteoclast surface (Oc.S) levels were positively associated with RANKL:OPG mRNA ratio, higher in groups with lower serum 25D levels, and were independent of serum 1,25D levels. Serum 25D levels <80 nM gave rise to osteopenia as a result of increased osteoclastogenesis, suggesting that levels of 25D >80 nM are needed for optimal bone volume. These data indicate that serum 25D levels are a major determinant of osteoclastogenesis and bone mineral volume and are consistent with the levels of 25D recommended to reduce the risk of fracture in humans.

25-Hydroxyvitamin D requirement for maintaining skeletal health utilising a Sprague-Dawley rat model

To study the role of vitamin D to optimise bone architecture, we have developed an animal model to investigate the effects of frank vitamin D-deficiency as well as graded depletion of circulating 25-hydroxyvitamin D(3) (25D) levels on the skeleton. Rats fed on dietary vitamin D levels from 0 to 500 ng/day achieved diet-dependent circulating levels of 25D ranging from 11 to 115 nmol/L. Levels of serum 1,25-dihydroxyvitamin D(3) (1,25D) increased as dietary vitamin D increased between 0 and 200 ng/day at which point a maximum level was achieved and retained with higher vitamin D intakes. The renal levels of 25-hydroxyvitamin D-1alpha-hydroxylase (CYP27B1) mRNA were highest in animal groups fed on vitamin D between 0 and 300 ng/day. In contrast, renal 25-hydroxyvitamin D 24-hydroxylase (CYP24) mRNA levels increased as dietary vitamin D increased achieving maximum levels in animals receiving 500 ng vitamin D/day. This animal model of vitamin D depletion is suitable to provide invaluable information on the serum levels of 25D and dietary calcium intake necessary for optimal bone structure. Such information is essential for developing nutritional recommendations to reduce the incidence of osteoporotic hip fractures.

The effect of dietary calcium on 1,25(OH)2D3 synthesis and sparing of serum 25(OH)D3 levels.

Vitamin D depletion in rats causes osteopenia in at least three skeletal sites. However it is unclear whether modulation of dietary calcium intake impacts on the relationship between the level of serum 25-hydroxyvitamin D (25D) and bone loss. Nine-month-old female Sprague-Dawley rats (n=5-6/group) were pair-fed a semi-synthetic diet containing either 0 or 20 IU vitamin D3/day with either low (0.1%) or high (1%) dietary Ca for 6 months. At 15 months of age, fasting bloods were collected for biochemical analyses. Serum 25D levels were lowest in the animals fed 0 IU vitamin D and 0.1% Ca. The animals fed 1% Ca had significantly higher serum 25D levels when compared to animals fed 0.1% Ca (P<0.05). The major determinants of serum 25D were dietary vitamin D and dietary calcium (Multiple R=0.75, P<0.05). Animals fed 0.1% Ca had higher renal CYP27B1 mRNA expression and 12-18-fold increased levels of serum 1,25D. Hence, the reported effects of low calcium diets on bone loss may be, in part, due to the subsequent effects of 25D metabolism leading to reduction in vitamin D status. Such an interaction has significant implications, given the recent evidence for local synthesis of active vitamin D in bone tissue.

The role of the calcitonin receptor in protecting against induced hypercalcemia is mediated via its actions in osteoclasts to inhibit bone resorption

Despite the therapeutic value of calcitonin in treating bone disease, a biological role of endogenous calcitonin is controversial. Having previously demonstrated that the CTR has a biological role in protecting against calcium stress using a global CTRKO mouse model, the purpose of this study was to determine whether the protection conferred by the CTR during induced hypercalcemia is mediated via CTR expression on osteoclasts. Mice were generated, in which the CTR was deleted specifically within osteoclasts (OCL-CTRKOs) and compared with mice in which the CTR was deleted globally (global CTRKOs). Significantly, peak serum calcium levels following induced hypercalcemia were >18% higher in global-CTRKOs and OCL-CTRKOs than controls (P<0.01) due to increased bone resorption (P<0.05). Peak serum calcium levels relative to controls were greater in global-CTRKO males than OCL-CTRKO males (P<0.001), which may, at least in part, be due to increased reabsorption of calcium in the kidney (P<0.01). Controls for all analyses were sex-matched littermates with normal CTR expression. In conclusion, the CTR protects against hypercalcemia predominantly via its inhibitory action on osteoclasts.

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

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.

Skeletal characterization of an osteoblast-specific vitamin D receptor transgenic (ObVDR-B6) mouse model.

BACKGROUND Overexpression of the human vitamin D receptor (hVDR) transgene under control of the human osteocalcin promoter in FVB/N mice (OSVDR) was previously demonstrated to exhibit increased cortical and trabecular bone volume and strength due to decreased bone resorption and increased bone formation. An important question to address is whether the OSVDR bone phenotype persists on an alternative genetic background such as C57Bl6/J. METHODS OSVDR mice (OSV3 line) were backcrossed onto the C57Bl6/J genetic background for at least 6 generations to produce OSVDR mice with 98.4% C57Bl6/J congenicity (ObVDR-B6 mice). Hemizygous male and female ObVDR-B6 and littermate wild-type (WT) mice were fed a standard laboratory chow diet and killed at 3, 9 and 20 weeks of age for analyses of biochemical and structural variables and dynamic indices of bone histomorphometry. RESULTS At 9 weeks of age, both cortical and trabecular femoral bone volumes were increased in both male and female ObVDR-B6 mice, when compared to WT levels (P<0.05), without systemic changes to calciotropic parameters. The increase in femoral trabecular bone volume was associated with increase in MAR (P<0.01) and reduced osteoclast size (P<0.05). However, in female mice trabecular bone volume was unchanged in femoral metaphysis of 20 weeks mice and in vertebra both at 9 and 20 weeks of age. Increased cortical bone in both male and female ObVDR-B6 mice was due largely to increased periosteal expansion and was associated with increased cortical strength at 20 weeks of age. CONCLUSION Overexpression of the human VDR gene in mature osteoblasts of C57Bl6/J mice increases cortical and trabecular bone volumes and confirms the previous reports of increased bone in OSVDR mice on the FVB/N background. However, site-specific and gender-related differences in bone volume suggest that the effects of osteoblast-specific VDR overexpression are more complex than hitherto recognised.