VDR in Osteoblast‐Lineage Cells Primarily Mediates Vitamin D Treatment‐Induced Increase in Bone Mass by Suppressing Bone Resorption

Long‐term treatment with active vitamin D [1α,25(OH)₂D₃] and its derivatives is effective for increasing bone mass in patients with primary and secondary osteoporosis. Derivatives of 1α,25(OH)₂D₃, including eldecalcitol (ELD), exert their actions through the vitamin D receptor (VDR). ELD is more resistant to metabolic degradation than 1α,25(OH)₂D₃. It is reported that ELD treatment causes a net increase in bone mass by suppressing bone resorption rather than by increasing bone formation in animals and humans. VDR in bone and extraskeletal tissues regulates bone mass and secretion of osteotropic hormones. Therefore, it is unclear what types of cells expressing VDR preferentially regulate the vitamin D–induced increase in bone mass. Here, we examined the effects of 4‐week treatment with ELD (50 ng/kg/day) on bone using osteoblast lineage‐specific VDR conditional knockout (Ob‐VDR‐cKO) and osteoclast‐specific VDR cKO (Ocl‐VDR‐cKO) male mice aged 10 weeks. Immunohistochemically, VDR in bone was detected preferentially in osteoblasts and osteocytes. Ob‐VDR‐cKO mice showed normal bone phenotypes, despite no appreciable immunostaining of VDR in bone. Ob‐VDR‐cKO mice failed to increase bone mass in response to ELD treatment. Ocl‐VDR‐cKO mice also exhibited normal bone phenotypes, but normally responded to ELD. ELD‐induced FGF23 production in bone was regulated by VDR in osteoblast‐lineage cells. These findings suggest that the vitamin D treatment‐induced increase in bone mass is mediated by suppressing bone resorption through VDR in osteoblast‐lineage cells. © 2017 The Authors. Journal of Bone and Mineral Research Published by Wiley Periodicals Inc.     

Compensatory Changes in Calcium Metabolism Accompany the Loss of Vitamin D Receptor (VDR) From the Distal Intestine and Kidney of Mice

1,25 Dihydroxyvitamin D₃ (1,25(OH)₂D) increases intestinal Ca absorption when dietary Ca intake is low by inducing gene expression through the vitamin D receptor (VDR). 1,25(OH)₂D‐regulated Ca absorption has been studied extensively in the small intestine, but VDR is also present in the large intestine. Our goal was to determine the impact of large intestinal VDR deletion on Ca and bone metabolism. We used transgenic mice expressing Cre‐recombinase driven by the 9.5‐kb human caudal type homeobox 2 (CDX2) promoter to delete floxed VDR alleles from the caudal region of the mouse (CDX2‐KO). Weanling CDX2‐KO mice and control littermates were fed low (0.25%) or normal (0.5%) Ca diets for 7 weeks. Serum and urinary Ca, vitamin D metabolites, bone parameters, and gene expression were analyzed. Loss of the VDR in CDX2‐KO was confirmed in colon and kidney. Unexpectedly, CDX2‐KO had lower serum PTH (–65% of controls, p < 0.001) but normal serum 1,25(OH)₂D and Ca levels. Despite elevated urinary Ca loss (eightfold higher in CDX2‐KO) and reduced colonic target genes TRPV6 (–90%) and CaBPD_9k (–80%) mRNA levels, CDX2‐KO mice had only modestly lower femoral bone density. Interestingly, duodenal TRPV6 and CaBPD_9k mRNA expression was fourfold and threefold higher, respectively, and there was a trend toward increased duodenal Ca absorption (+19%, p = 0.076) in the CDX2‐KO mice. The major finding of this study is that large intestine VDR significantly contributes to whole‐body Ca metabolism but that duodenal compensation may prevent the consequences of VDR deletion from large intestine and kidney in growing mice. © 2015 American Society for Bone and Mineral Research.     

The renin‐angiotensin system,blood pressure,and heart structure in patients with hereditary vitamin D–resistance rickets (HVDRR)

Vitamin D deficiency has been linked to hypertension and an increased prevalence of cardiovascular risk factors and disease. Studies in vitamin D receptor knockout (VDR KO) mice revealed an overstimulated renin‐angiotensin system (RAS) and consequent high blood pressure and cardiac hypertrophy. VDR KO mice correspond phenotypically and metabolically to humans with hereditary 1,25‐dihydroxyvitamin D–resistant rickets (HVDRR). There are no data on the cardiovascular system in human HVDRR. To better understand the effects of vitamin D on the human cardiovascular system, the RAS, blood pressure levels, and cardiac structures were examined in HVDRR patients. Seventeen patients (9 males, 8 females, aged 6 to 36 years) with hereditary HVDRR were enrolled. The control group included age‐ and gender‐matched healthy subjects. Serum calcium, phosphorous, creatinine, 25‐hydroxyvitamin D [25(OH)D],1,25‐dihydroxyvitamin D₃ [1,25(OH)₂D₃], parathyroid hormone (PTH), plasma rennin activity (PRA), aldosterone, angiotensin II (AT‐II), and angiotensin‐converting enzyme (ACE) levels were determined. Ambulatory 24‐hour blood pressure measurements and echocardiographic examinations were performed. Serum calcium, phosphorus, and alkaline phosphatase values were normal. Serum 1,25(OH)₂D₃ and PTH but not PRA and ACE levels were elevated in the HVDRR patients. AT‐II levels were higher than normal in the HVDRR patients but not significantly different from those of the controls. Aldosterone levels were normal in all HVDRR patients. No HVDRR patient had hypertension or echocardiographic pathology. These findings reveal that 6‐ to 36‐year‐old humans with HVDRR have normal renin and ACE activity, mild but nonsignificant elevation of AT‐II, normal aldosterone levels, and no hypertension or gross heart abnormalities. © 2011 American Society for Bone and Mineral Research     

Compound Heterozygous Mutations in the Vitamin D Receptor in a Patient With Hereditary 1,25‐Dihydroxyvitamin D‐Resistant Rickets With Alopecia

Hereditary vitamin D‐resistant rickets (HVDRR) is a rare recessive genetic disorder caused by mutations in the vitamin D receptor (VDR). In this study, we examined the VDR in a young girl with clinical features of HVDRR including rickets, hypophosphatemia, and elevated serum 1,25(OH)₂D. The girl also had total alopecia. Two mutations were found in the VDR gene: a nonsense mutation (R30X) in the DNA‐binding domain and a unique 3‐bp in‐frame deletion in exon 6 that deleted the codon for lysine at amino acid 246 (ΔK246). The child and her mother were both heterozygous for the 3‐bp deletion, whereas the child and her father were both heterozygous for the R30X mutation. Fibroblasts from the patient were unresponsive to 1,25(OH)₂D₃ as shown by their failure to induce CYP24A1 gene expression, a marker of 1,25(OH)₂D₃ responsiveness. [³H]1,25(OH)₂D₃ binding and immunoblot analysis showed that the patient's cells expressed the VDRΔK246 mutant protein; however, the amount of VDRΔK246 mutant protein was significantly reduced compared with wildtype controls. In transactivation assays, the recreated VDRΔK246 mutant was unresponsive to 1,25(OH)₂D₃. The ΔK246 mutation abolished heterodimerization of the mutant VDR with RXRα and binding to the coactivators DRIP205 and SRC‐1. However, the ΔK246 mutation did not affect the interaction of the mutant VDR with the corepressor Hairless (HR). In summary, we describe a patient with compound heterozygous mutations in the VDR that results in HVDRR with alopecia. The R30X mutation truncates the VDR, whereas the ΔK246 mutation prevents heterodimerization with RXR and disrupts coactivator interactions.     

17β-Estradiol increases the receptor number and modulates the action of 1,25-dihydroxyvitamin D3 in human osteosarcoma-derived osteoblast-like cells

It is well known that 17-estradiol (E₂) and 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃) have important roles in bone metabolism. This study was undertaken to examine E₂ regulation of 1,25(OH)₂D₃ receptor (VDR) expression and the biological action of 1,25(OH)₂D₃ in human osteoblast-like cells. When human osteosarcoma-derived osteoblast-like cells were treated with varying concentrations of E₂, the VDR levels increased by up to 100% in a dose-dependent manner. VDR levels significantly increased at 10 nM E₂ and this increase plateaued at 100 nM E₂. E₂-dependent increase of VDR was time dependent, plateauing, at 24 hours and was maintained for at least 48 hours in human osteoblast-like cells. Scatchard analysis showed that E₂ increased the number of VDR (12.3±0.4 versus 26.5±0.3 fmol/mg protein; mean ±SE of three independent experiments) rather than the Kd (0.15±0.02 versus 0.16±0.01 nM; mean±SE of three independent experiments). Tamoxifen (50 nM), a specific competitor with E₂, completely abolished the E₂-induced increase of VDR. The levels of VDR mRNA (4.5 kb) from the cells increased in a dose-dependent manner after E₂ treatment. With regard to the biological effects, E₂ increased by 10–25% the inhibitory effect of 1,25(OH)₂D₃ on cell growth. However, E₂ did not increase the stimulation of alkaline phosphatase activity by 1,25(OH)₂D₃. The present study suggests that E₂ modulates the biological action of 1,25(OH)₂D₃ through VDR levels in bone cells.     

Extra-intestinal calcium handling contributes to normal serum calcium levels when intestinal calcium absorption is suboptimal

The active form of vitamin D, 1,25(OH)₂D, is a crucial regulator of calcium homeostasis, especially through stimulation of intestinal calcium transport. Lack of intestinal vitamin D receptor (VDR) signaling does however not result in hypocalcemia, because the increased 1,25(OH)₂D levels stimulate calcium handling in extra-intestinal tissues. Systemic VDR deficiency, on the other hand, results in hypocalcemia because calcium handling is impaired not only in the intestine, but also in kidney and bone. It remains however unclear whether low intestinal VDR activity, as observed during aging, is sufficient for intestinal calcium transport and for mineral and bone homeostasis. To this end, we generated mice that expressed the Vdr exclusively in the gut, but at reduced levels. We found that ~ 15% of intestinal VDR expression greatly prevented the Vdr null phenotype in young-adult mice, including the severe hypocalcemia. Serum calcium levels were, however, in the low-normal range, which may be due to the suboptimal intestinal calcium absorption, renal calcium loss, insufficient increase in bone resorption and normal calcium incorporation in the bone matrix. In conclusion, our results indicate that low intestinal VDR levels improve intestinal calcium absorption compared to Vdr null mice, but also show that 1,25(OH)₂D-mediated fine-tuning of renal calcium reabsorption and bone mineralization and resorption is required to maintain fully normal serum calcium levels.     

Improvement of impaired calcium and skeletal homeostasis in vitamin D receptor knockout mice by a high dose of calcitriol and maxacalcitol

Vitamin D plays a major role in mineral and skeletal homeostasis through interaction with the nuclear vitamin D receptor (VDR) of target cells. Recent reports have indicated that some cellular effects of vitamin D may occur via alternative signaling pathways, but concrete evidence for mineral homeostasis has not been shown in vivo. To investigate this issue, the actions of calcitriol (1,25D) and maxacalcitol (OCT), which were developed for treatment of uremia-induced secondary hyperparathyroidism, were analyzed in VDR knockout (VDR^−/−) mice. The VDR^−/− mice were fed a rescue diet immediately after weaning. 1,25D, OCT or a control solution was administered intraperitoneally to these mice three times a week for eight weeks. Biological markers and bone growth were measured and bone histomorphometric analysis of the calcein-labeled tibia was performed 24 h after the final administration. Significantly higher levels of serum Ca²⁺ were observed in 1,25D- and OCT-treated mice, but the serum parathyroid hormone level was unchanged by both agents. Impaired bone growth, enlarged and distorted cartilaginous growth plates, morphological abnormalities of cancellous and cortical bones; a morbid osteoid increase, lack of calcein labeling, and thinning of cortical bone, were all significantly improved by 1,25D and OCT. The significance of these effects was confirmed by bone histomorphometrical analysis. Upregulation of the calbindin D_9k mRNA expression level in the duodenum may explain these findings, since this protein is a major modulator of Ca transport in the small intestine. We conclude that 1,25D and OCT both at a high dose exert significant effects on Ca and skeletal homeostasis with the principal improvement of Ca status in VDR^−/− mice, and some of these effects may occur through an alternative vitamin D signaling pathway.