Paul H. Anderson

Mineralization and bone resorption are regulated by the androgen receptor in male mice.

Androgens play a key role in skeletal growth and bone maintenance; however, their mechanism of action remains unclear. To address this, we selectively deleted the androgen receptor (AR) in terminally differentiated, mineralizing osteoblasts using the Cre/loxP system in mice (osteocalcin‐Cre AR knockouts [mOBL‐ARKOs]). Male mOBL‐ARKOs had decreased femoral trabecular bone volume compared with littermate controls because of a reduction in trabecular number at 6, 12, and 24 wk of age, indicative of increased bone resorption. The effects of AR inactivation in mineralizing osteoblasts was most marked in the young mutant mice at 6 wk of age when rates of bone turnover are high, with a 35% reduction in trabecular bone volume, decreased cortical thickness, and abnormalities in the mineralization of bone matrix, characterized by increased unmineralized bone matrix and a decrease in the amount of mineralizing surface. This impairment in bone architecture in the mOBL‐ARKOs persisted throughout adulthood despite an unexpected compensatory increase in osteoblast activity. Our findings show that androgens act through the AR in mineralizing osteoblasts to maintain bone by regulating bone resorption and the coordination of bone matrix synthesis and mineralization, and that this action is most important during times of bone accrual and high rates of bone remodeling.

Osteoclastic Metabolism of 25(OH)-Vitamin D3: A Potential Mechanism for Optimization of Bone Resorption

The extrarenal synthesis of 1α,25 dihydroxyvitamin D3 (1,25D) has been demonstrated in a number of cell types including osteoblasts and cells of the monocyte/macrophage lineage. The skeleton appears responsive to serum levels of the 1,25D precursor, 25 hydroxyvitamin D3 (25D), in terms of bone mineralization parameters. The effect of metabolism of 25D into active 1,25D by osteoclast lineage cells is unknown. We found that CYP27B1 mRNA expression increased with exposure of human peripheral blood mononuclear cells (PBMCs) to macrophage colony-stimulating factor in the presence or absence of receptor activator of nuclear factor-κB ligand. Consistent with this, human osteoclast cultures incubated with 25D produced measurable quantities of 1,25D. Osteoclast formation from either mouse RAW264.7 cells or human PBMCs in the presence of physiological concentrations of 25D resulted in significant up-regulation of the key osteoclast transcription factor, nuclear factor of activated T cells-c1 in PBMCs and a number of key osteoclast marker genes in both models. The expression of the osteoblast coupling factor, ephrin-b2, was also increased in the presence of 25D. Levels of CYP27B1 and nuclear factor of activated T cells-1 mRNA correlated during osteoclastogenesis and also in a cohort of human bone samples. CYP27B1 short-hairpin RNA knockdown in RAW264.7 cells decreased their osteoclastogenic potential. 25D dose dependently reduced the resorptive capacity of PBMC-derived osteoclasts without compromising cell viability. 25D also reduced resorption by RAW264.7- and giant cell tumor-derived osteoclasts. Conversely, osteoclasts formed from vitamin D receptor-null mouse splenocytes had increased resorptive activity compared with wild-type cells. We conclude that 25D metabolism is an important intrinsic mechanism for optimizing osteoclast differentiation, ameliorating osteoclast activity, and potentially promoting the coupling of bone resorption to formation.

The skeleton as an intracrine organ for vitamin D metabolism.

The endocrine hormone, 1alpha,25-dihydroxyvitamin D(3) (1,25D) is an important regulator of calcium and phosphorus homeostasis. In this context, 1,25D is generally recognized as necessary for the maintenance of a healthy skeleton through its actions on the small intestine. In this review, we highlight the direct effects of 1,25D on the constituent cells of the bone, actions that are independent of effects on the intestine and kidney. We also consider the evidence that 25D levels, not 1,25D levels, correlate best with parameters of bone health, and that the bone itself is a site of metabolic conversion of 25D into 1,25D, by virtue of its expression of the 25-hydroxyvitamin D 1alpha-hydroxylase, CYP27B1. We review the evidence that at least osteoblasts and chondrocytes, and possibly also bone resorbing osteoclasts, are capable of such metabolic conversion, and therefore that these cells likely participate in autocrine and paracrine loops of vitamin D metabolism. We conclude that the skeleton is an intracrine organ for vitamin D metabolism, challenging the long-held notion that 1,25D is solely an endocrine hormone.

Sclerostin Regulates Release of Bone Mineral by Osteocytes by Induction of Carbonic Anhydrase 2

The osteocyte product sclerostin is emerging as an important paracrine regulator of bone mass. It has recently been shown that osteocyte production of receptor activator of NF‐κB ligand (RANKL) is important in osteoclastic bone resorption, and we reported that exogenous treatment of osteocytes with sclerostin can increase RANKL‐mediated osteoclast activity. There is good evidence that osteocytes can themselves liberate mineral from bone in a process known as osteocytic osteolysis. In the current study, we investigated sclerostin‐stimulated mineral dissolution by human primary osteocyte‐like cells (hOCy) and mouse MLO‐Y4 cells. We found that sclerostin upregulated osteocyte expression of carbonic anhydrase 2 (CA2/Car2), cathepsin K (CTSK/Ctsk), and tartrate‐resistant acid phosphatase (ACP5/Acp5). Because acidification of the extracellular matrix is a critical step in the release of mineral from bone, we further examined the regulation by sclerostin of CA2. Sclerostin stimulated CA2 mRNA and protein expression in hOCy and in MLO‐Y4 cells. Sclerostin induced a decrease in intracellular pH (pHi) in both cell types as well as a decrease in extracellular pH (pHo) and the release of calcium ions from mineralized substrate. These effects were reversed in the co‐presence of the carbonic anhydrase inhibitor, acetozolamide. Car2‐siRNA knockdown in MLO‐Y4 cells significantly inhibited the ability of sclerostin to both reduce the pHo and release calcium from a mineralized substrate. Knockdown in MLO‐Y4 cells of each of the putative sclerostin receptors, Lrp4, Lrp5 and Lrp6, using siRNA, inhibited the sclerostin induction of Car2, Catk and Acp5 mRNA, as well as pHo and calcium release. Consistent with this activity of sclerostin resulting in osteocytic osteolysis, human trabecular bone samples treated ex vivo with recombinant human sclerostin for 7 days exhibited an increased osteocyte lacunar area, an effect that was reversed by the co‐addition of acetozolamide. These findings suggest a new role for sclerostin in the regulation of perilacunar mineral by osteocytes.

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.

Calcitonin Receptor Plays a Physiological Role to Protect Against Hypercalcemia in Mice

It is well established that calcitonin is a potent inhibitor of bone resorption; however, a physiological role for calcitonin acting through its cognate receptor, the calcitonin receptor (CTR), has not been identified. Data from previous genetically modified animal models have recognized a possible role for calcitonin and the CTR in controlling bone formation; however, interpretation of these data are complicated, in part because of their mixed genetic background. Therefore, to elucidate the physiological role of the CTR in calcium and bone metabolism, we generated a viable global CTR knockout (KO) mouse model using the Cre/loxP system, in which the CTR is globally deleted by >94% but <100%. Global CTRKOs displayed normal serum ultrafiltrable calcium levels and a mild increase in bone formation in males, showing that the CTR plays a modest physiological role in the regulation of bone and calcium homeostasis in the basal state in mice. Furthermore, the peak in serum total calcium after calcitriol [1,25(OH)2D3]‐induced hypercalcemia was substantially greater in global CTRKOs compared with controls. These data provide strong evidence for a biological role of the CTR in regulating calcium homeostasis in states of calcium stress.

The metabolism of 25-(OH)vitamin D3 by osteoclasts and their precursors regulates the differentiation of osteoclasts.

Current evidence suggests that levels of 25-(OH)vitamin D3 (25D), rather than 1alpha,25-(OH)2vitamin D3 (1,25D), directly affect bone mineralization and that the skeleton is a site of extra-renal synthesis of 1,25D. Since cells of the monocyte lineage can also metabolise 25D, it is possible that osteoclasts participate in local production of, and the response to, 1,25D. In this study, we investigated the effects of vitamin D metabolism on osteoclastogenesis using both the murine RAW 264.7 cell line and the human peripheral blood mononuclear cell (PBMC) models. PBMC-derived osteoclasts expressed cytoplasmic cyp27b1 and nuclear vdr proteins. PBMC expressed CYP27B1 mRNA, levels of which increased during RANKL induced differentiation into osteoclasts in both cell types. While 1,25D elicited a robust CYP24 transcriptional response in PBMC, the response to 25D was approximately 100-fold less at the concentrations used. Using media devoid of pre-existing vitamin D metabolites, we found that 25D was metabolised by RAW 264.7 cells to 1,25D and resulted in significant elevation in the numbers of TRAP-positive, multinucleated osteoclasts when present in the cultures for the first 3-5 days. These results suggest that vitamin D metabolism by osteoclast lineage cells is an important regulator of osteoclast formation.

Mechanisms of vitamin D3 metabolite repression of IgE-dependent mast cell activation

BACKGROUND Mast cells have gained notoriety based on their detrimental contributions to IgE-mediated allergic disorders. Although mast cells express the vitamin D receptor (VDR), it is not clear to what extent 1α,25-dihydroxyvitamin D3 (1α,25[OH]2D3) or its predominant inactive precursor metabolite in the circulation, 25-hydroxyvitamin D3 (25OHD3), can influence IgE-mediated mast cell activation and passive cutaneous anaphylaxis (PCA) in vivo. OBJECTIVE We sought to assess whether the vitamin D3 metabolites 25OHD3 and 1α,25(OH)2D3 can repress IgE-dependent mast cell activation through mast cell-25-hydroxyvitamin D-1α-hydroxylase (CYP27B1) and mast cell-VDR activity. METHODS We measured the extent of vitamin D3 suppression of IgE-mediated mast cell degranulation and mediator production in vitro, as well as the vitamin D3-induced curtailment of PCA responses in WBB6F1-Kit(W/W-v) or C57BL/6J-Kit(W-sh/W-sh) mice engrafted with mast cells that did or did not express VDR or CYP27B1. RESULTS Here we show that mouse and human mast cells can convert 25OHD3 to 1α,25(OH)2D3 through CYP27B1 activity and that both of these vitamin D3 metabolites suppressed IgE-induced mast cell-derived proinflammatory and vasodilatory mediator production in a VDR-dependent manner in vitro. Furthermore, epicutaneously applied vitamin D3 metabolites significantly reduced the magnitude of skin swelling associated with IgE-mediated PCA reactions in vivo; a response that required functional mast cell-VDRs and mast cell-CYP27B1. CONCLUSION Taken together, our findings provide a mechanistic explanation for the anti-inflammatory effects of vitamin D3 on mast cell function by demonstrating that mast cells can actively metabolize 25OHD3 to dampen IgE-mediated mast cell activation in vitro and in vivo.

Determinants of circulating 1,25-dihydroxyvitamin D3 levels: the role of renal synthesis and catabolism of vitamin D.

Details of the molecular mechanisms determining levels of the secosteroid, 1,25-dihydroxyvitamin D(3) (1,25D) remain to be elucidated. The current paradigm for the control of serum 1,25D levels is the tight regulation of renal 25-hydroxyvitamin D-1alpha-hydroxlase (CYP27B1) activity by a number of physiological factors. 1,25D production is also regulated by the cytochrome P450 enzyme, 25-hydroxyvitamin D-24-hydroxylase (CYP24), which through side chain hydroxylation reactions, inactivates 1,25D. We have recently demonstrated that renal CYP27B1 and CYP24 expression contribute equally to regulating serum 1,25D levels. We now describe the contribution of renal Vitamin D receptor (VDR) expression in determining serum 1,25D levels. Serum 1,25D levels were decreased when the dietary calcium intake was increased. We measured mRNA levels for CYP27B1, CYP24 and VDR receptor in kidney RNA extracts from animals fed diets containing different levels of calcium, ranging from 0.05 to 1%. Serum 1,25D levels were negatively correlated with renal CYP24 mRNA levels (R2 = 0.35, P < 0.01) while renal VDR is positively correlated with renal CYP24 mRNA (R2 = 0.80, P < 0.001). However, only renal VDR mRNA remained a significant determinant of renal CYP24 expression when both these variables were included in multiple linear regression analysis (multiple R2 = 0.89, P < 0.001). These findings suggest that kidney CYP24 activity acts in concert with kidney CYP27B1 to control serum 1,25D levels and that serum 1,25D stimulates renal CYP24 expression by acting through the renal VDR.

Vitamin D metabolism within bone cells: Effects on bone structure and strength

The endocrine activity of 1,25-dihydroxyvitamin D (1,25(OH)(2)D(3)) contributes to maintaining plasma calcium and phosphate homeostasis through actions on the intestine, kidney and bone. A significant body of evidence has been published over the last 10 years indicating that all major bone cells have the capacity to metabolise 25-hydroxyvitamin D (25(OH)D(3)) to 1,25(OH)(2)D(3), which in turn exerts autocrine/paracrine actions to regulate bone cell proliferation and maturation as well as bone mineralisation and resorption. In vivo and in vitro studies indicate that these autocrine/paracrine activities of 1,25(OH)(2)D(3) in bone tissue contribute to maintaining bone mineral homeostasis and enhancing skeletal health.