Vitamin D antagonist, TEI-9647, inhibits osteoclast formation induced by 1alpha,25-dihydroxyvitamin D3 from pagetic bone marrow cells

(23S)-25-Dehydro-1alpha-hydroxyvitamin D(3)-26,23-lactone (TEI-9647) functions an antagonist of the 1alpha,25-dihydroxyvitamin D(3) (1alpha,25-(OH)(2)D(3)) nuclear receptor (VDR)-mediated differentiation of human leukemia (HL-60) cells [J. Biol. Chem. 274 (1999) 16392]. We examined the effect of vitamin D antagonist, TEI-9647, on osteoclast formation induced by 1alpha,25-(OH)(2)D(3) from bone marrow cells of patients with Paget's disease. TEI-9647 itself never induced osteoclast formation even at 10(-6)M, but dose-dependently (10(-10) to 10(-6)M) inhibited osteoclast formation induced by physiologic concentrations of 1alpha,25-(OH)(2)D(3) (41 pg/ml, 10(-10)M) from bone marrow cells of patients with Paget's disease. At the same time, 10(-8)M of TEI-9647 alone did not cause 1alpha,25-(OH)(2)D(3) dependent gene expression, but almost completely suppressed TAF(II)-17, a potential coactivator of VDR and 25-hydroxyvitamin D(3)-24-hydroxylase (25-OH-D(3)-24-hydroxylase) gene expression induced by 10(-10)M 1alpha,25-(OH)(2)D(3) in bone marrow cells of patients with Paget's disease. Moreover, TEI-9647 dose-dependently inhibited bone resorption induced by 10(-9)M 1alpha,25-(OH)(2)D(3) by osteoclasts produced by RANKL and M-CSF treatment of measles virus nucleocapsid gene transduced bone marrow cells. These results suggest that TEI-9647 acts directly on osteoclast precursors and osteoclasts, and that TEI-9647 may be a novel agent to suppress the excessive bone resorption and osteoclast formation in patients with Paget's disease.     

Lack of bone resorption in osteosclerotic (oc/oc) mice is due to a defect in osteoclast progenitors rather than the local microenvironment provided by osteoblastic cells.

In a co-culture system of mouse spleen cells and osteoblastic cells, we have demonstrated that a suitable microenvironment must be provided by osteoblastic cells in order for osteoclast-like multinucleated cell (MNC) formation. Using this co-culture system, we examined the pathogenetic mechanism underlying the lack of bone resorption in osteosclerotic oc/oc mice. Numerous tartrate-resistant acid phosphatase (TRAP, an osteoclast marker enzyme)-positive MNCs were formed in response to 1 alpha,25-dihydroxyvitamin D3 [1 alpha,25(OH)2D3] both in co-cultures of oc/oc spleen cells and normal osteoblastic cells and in those of normal spleen cells and oc/oc osteoblastic cells. TRAP-positive MNCs derived from normal spleen cells tended to spread out on culture dishes, whereas those from oc/oc spleen cells remained as small, compact MNCs. When TRAP-positive MNCs enriched from co-cultures of normal spleen cells and oc/oc osteoblastic cells were cultured on dentine slices, they formed numerous resorption pits with ruffled borders and clear zones. In contrast, none of the TRAP-positive MNCs derived from oc/oc spleen cells formed either ruffled borders or resorption pits. These results indicate that the lack of bone resorption in oc/oc mice is due to a defect in osteoclast progenitors rather than the local microenvironment provided by osteoblastic cells.     

Species difference exists in the effects of 1alpha,25(OH)(2)D(3) and its analogue 2-methylene-19-nor-(20S)-1,25-dihydroxyvitamin D(3) (2MD) on osteoblastic cells

The direct effect of 1alpha,25(OH)(2)D(3) on osteoblasts remains unclear. In this study, we evaluated the in vitro effects of 1alpha,25(OH)(2)D(3) and its analogue, 2-methylene-19-nor-(20S)-1,25-dihydroxyvitamin D(3) (2MD), on osteoblasts from three different species, i.e. bone marrow stromal cells from the Sprague-Dawley (SD) rat, from the C57BL/6 mouse, as well as human osteoblast NHOst cells and human osteosarcoma derived MG-63 cells. We found that in rat cells, both compounds increased cell proliferation, inhibited cell apoptosis and increased alkaline phosphatase (ALP) activity. In mouse cells, however, both compounds initiated cell apoptosis and inhibited ALP activity. In human cells, although cell proliferation was inhibited by both compounds, cell apoptosis was inhibited and ALP activity was enhanced. In each species, 2MD was much more potent than 1alpha,25(OH)(2)D(3). To summarize, species differences should be taken into account in studies of vitamin D effects. However, in all tested species - rat, mouse and human - 2MD is considerably more potent in its effects on osteoblastic cells in vitro than 1alpha,25(OH)(2)D(3).     

Inhibitory effect of NPY on isoprenaline-induced osteoclastogenesis in mouse bone marrow cells

The effect of neuropeptide Y (NPY), a co-transmitter with noradrenaline in peripheral sympathetic nerve fibers, on the osteoclastogenesis in mouse bone marrow cell cultures treated with isoprenaline, a beta-adrenergic receptor (beta-AR) agonist, was examined. The mouse bone marrow cells constitutively expressed mRNAs for the NPY-Y1 receptor and beta2-AR. NPY inhibited the formation of osteoclast-like cells induced by isoprenaline but not that by 1alpha,25-dihydroxyvitamin D3 (1alpha,25(OH)2D3) or soluble receptor activator of nuclear factor-kappaB ligand (RANKL); and it suppressed the production of RANKL and cyclic AMP (cAMP) increased by isoprenaline but not those increased by 1alpha,25(OH)2D3. NPY also inhibited osteoclastogenesis induced by forskolin, an activator of adenylate cyclase; however, it did not inhibit that induced by exogenously supplied dibutyryl cAMP, a cell-permeable cAMP analog that activates cAMP-dependent protein kinase. These results demonstrate that NPY inhibited the isoprenaline-induced osteoclastogenesis by blocking the agonist-elicited increases in the production of cAMP and RANKL in mouse bone marrow cells, suggesting an interaction between NPY and beta-AR agonist in bone resorption.     

Role of vitamin D in bone resorption.

The idea that vitamin D must function at the bone site to promote bone mineralization has long existed since its discovery as an anti-rachitic agent. However, the definite evidence for this is still lacking. In contrast, much evidence has accumulated that 1 alpha,25(OH)2D3 in involved in bone resorption. 1 alpha,25(OH)2D3 tightly regulates differentiation of osteoclast progenitors into osteoclasts. Osteoclast progenitors have been thought to belong to the monocyte-macrophage lineage. 1 alpha,25(OH)2D3 greatly stimulates differentiation and activation of mononuclear phagocytes. Recent reports have indicated that differentiation of mononuclear phagocytes into osteoclasts is strictly regulated by osteoblastic cells, the process of which is also stimulated by 1 alpha,25(OH)2D3. In the differentiation of mononuclear phagocytes into osteoclasts, the target cells for 1 alpha,25(OH)2D3 appear to be osteoblastic stromal cells. Osteoblastic cells produce several proteins such as BGP, MGP, osteopontin and the third component of complement (C3) in response to the vitamin. They appear to be somehow involved in osteoclast differentiation and functions. Thus, 1 alpha,25(OH)2D3 seems to be involved in the differentiation of osteoclast progenitors into osteoclasts directly and also by an indirect mechanism involving osteoblastic cells. The precise role of osteoblastic cells in osteoclast development has to be elucidated in the future.     

1 alpha, 25-Dihydroxyvitamin D3 and analogues of vitamin D3 induce alkaline phosphatase activity in osteoblastic cells derived from newborn mouse calvaria

In order to study the effects of vitamin D metabolites on bone metabolism, clone MC3T3-E1 cells, which have retained osteoblastic activity, were cultured with various concentrations of the hormone, 1 alpha, 25-dihydroxyvitamin D3 [1 alpha, 25 (OH)2D3]. A physiological concentration of 1 alpha, 25 (OH)2D3 stimulated alkaline phosphatase (ALP) activity in the cells. Other metabolites--1 alpha, 24-dihydroxyvitamin D3 [1 alpha, 24 (OH)2D3], 1 alpha-hydroxyvitamin D3 [1 alpha (OH)D3], and 24R,25-dihydroxyvitamin D3 [24R,25 (OH)2D3]--also induced increases in ALP activity in a dose-dependent fashion. However, their effective concentrations were 100 or 1,000 times greater than that of 1 alpha, 25 (OH)2D3. Hormone-induced and native ALP activities in the cells were of the same type as that found in newborn mouse calvaria; that is, they were heat-labile, L-homoarginine- and levamisole-sensitive, and L-phenylalanine-insensitive (liver-bone-kidney type). These results show that vitamin D metabolites stimulate bone formation in vitro and that they may be involved in bone formation in vivo as well.     

Proliferation and differentiation of osteoblasts and adipocytes in rat bone marrow stromal cell cultures: effects of dexamethasone and calcitriol

During bone loss, osteoblast population can be replaced by adipose tissue. This apparent reciprocal relationship between decreased bone density and increased fat formation can be explained by an imbalance in the production of bone-forming and fat-forming cells in the marrow cavity. Thus, osteoblast and adipocyte pathways seem more closely and inversely related. In the present study, we investigated the effects of dexamethasone (dex) and calcitriol [1,25(OH)(2)D(3)] on proliferation and differentiation of osteoblasts and adipocytes in rat bone marrow stromal cell cultures. Stromal cells were grown in primoculture in presence of dex and subcultivated in presence of dex and/or 1,25(OH)(2)D(3). Total cell proliferation, osteoblast and adipocyte-cells number, and -mRNA specific markers were used to study the effects of hormonal treatment on stromal cells. Total cell proliferation was stimulated by dex and inhibited by 1,25(OH)(2)D(3). Dex increased osteoblast and adipocyte cell population whereas calcitriol decreased bone-forming cell number and increased fat cell population. The presence of both hormones led to a strong decrease in osteoblastic cells and to a strong increase in adipocytic cell number. Dex induced mRNA osteoblastic markers expression like bone sialoprotein (BSP) and osteocalcin (OC) and an adipocyte marker expression, the fatty acid binding protein aP2. Calcitriol decreased the dex-induced BSP expression but stimulated slightly OC and aP2 mRNA. The effects of both hormones was to increase strongly OC and aP2 mRNA. These results support that, in rat bone marrow, adipocyte proliferation and differentiation are stimulated by glucocorticoids and calcitriol which act synergically, whereas osteoblastic cell proliferation and differentiation are increased by dex and inhibited by 1,25(OH)(2)D(3).     

A novel interaction between thyroid hormones and 1,25(OH)(2)D(3) in osteoclast formation

Thyroid hormones enhance osteoclast formation and their excess is an important cause of secondary osteoporosis. 3,5,3' -Triiodo-L-thyronine (T3) induced the mRNA expression of receptor activator of nuclear factor-kappa B ligand (RANKL), which is a key molecule in osteoclast formation, in primary osteoblastic cells (POB). This effect was amplified in the copresence of 1 alpha,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)). Although T3 alone did not induce octeoclasts in coculture of bone marrow cells with POB, T3 enhanced 1,25(OH)(2)D(3)-induced osteoclast formation. Thyroxine (T4) also enhanced 1,25(OH)(2)D(3)-induced osteoclast formation. These data suggested that T4 was locally metabolized to T3 for its action, since T4 is a prohormone with little hormonal activity. The mRNA expression of type-2 iodothyronine deiodinase (D2), which is responsible for maintaining local T3 concentration, was induced by 1,25(OH)(2)D(3) dose- and time-dependently. Our data would facilitate our understanding of the mechanism of osteoclast formation by thyroid hormones and suggest a novel interaction between thyroid hormones and 1,25(OH)(2)D(3).     

Bioactive analogs that simulate subsets of biological activities of 1alpha,25(OH)(2)D(3) in osteoblasts

1alpha,25-Dihydroxyvitamin D(3) [1alpha,25(OH)(2)D(3)] treatment of osteoblastic cells elicits a series of measurable responses that include both rapid, membrane-initiated effects and longer-term nuclear receptor-mediated effects. Structural analogs have been identified and characterized that selectively activate subsets of these pathways. Two analogs from over 35 that have been tested were chosen for this comparison because they activate non-overlapping response pathways, presumably representing either membrane-initiated or nuclear receptor-initiated activities. Compound AT [25(OH)-16ene-23yne-D(3)] lacks the 1-hydroxyl essential for interacting with the nuclear receptor, but triggers Ca(2+) influx through plasma membrane Ca(2+) channels, augments parathyroid hormone (PTH)-induced Ca(2+) signals, dephosphorylates the matrix protein osteopontin (OPN), and along with PTH stimulates release of calcium from calvaria in organ culture. Compound BT [1alpha,24(OH)(2)-22ene-24cyclopropyl-D(3)] does not elicit any of the rapid responses or enhance PTH-induced bone resorption, but binds to the nuclear receptor for 1alpha,25(OH)(2)D(3) and increases steady state mRNA levels of both OPN and osteocalcin over a 48 h period. Together, these two analogs recapitulate all of the known actions of 1alpha,25(OH)(2)D(3) on osteoblasts. Based on these findings, we conclude that Ca(2+) release from bone stimulated by 1alpha,25(OH)(2)D(3) and PTH is related to the rapid, membrane-initiated actions and is not likely to involve binding to the nuclear receptor for 1alpha,25(OH)(2)D(3). Longer term stimulation of bone formation by 1alpha,25(OH)(2)D(3), however, appears to involve solely the nuclear receptor-mediated effects. These findings support our model of 1alpha,25(OH)(2)D(3) as a coupling factor for bone resorption and formation during bone remodeling.     

Vitamin D compounds exert anti-apoptotic effects in human osteosarcoma cells in vitro

Several studies have demonstrated that vitamin D regulates growth and differentiation in bone cells in vitro. In addition, in vivo studies have shown that vitamin D stimulates bone formation, increases the number of osteoblast precursor cells and prevents bone mineral loss. These observations indicate that vitamin D may have anabolic effects on bone, and thus therapeutic potential in the treatment of osteoporosis. However, little is known about the effects of vitamin D on apoptosis in bone cells and about the contribution of this process to the effect of vitamin D on bone mineral loss. To investigate this aspect in more detail, we studied the effect of 1alpha,25(OH)(2)D(3) and a series of analogues on apoptosis in human osteosarcoma cells. No significant induction of apoptosis was observed with any of the compounds after a 5 day treatment period. In contrast, some of the analogues showed a tendency to protect the cells from undergoing apoptosis. This anti-apoptotic effect of vitamin D was further confirmed by the ability of 1alpha,25(OH)(2)D(3) to suppress camptothecin- and staurosporin-induced DNA fragmentation in the cells. In cultures treated simultaneously with 1alpha,25(OH)(2)D(3) in combination with camptothecin or staurosporin, the level of DNA fragmentation was markedly reduced compared with cultures treated with camptothecin or staurosporin alone. On the basis of the present results, it is therefore concluded that vitamin D displays anti-apoptotic effects in human osteoblast-like osteosarcoma cells in vitro. This observation suggests that besides regulating growth and differentiation, vitamin D exerts its anabolic effects on bone by protecting osteoblastic cells from undergoing apoptosis.     

The unique action for bone metabolism of 1 alpha,25-(OH)2D3-26,23-lactone

[23 (S), 25 (R)]-1 alpha,25-Dihydroxyvitamin D3-26,23-lactone [( 23 (S),25 (R)]-1 alpha,25-(OH) 2D3-26,23-lactone) increased dose-dependently alkaline phosphatase activity in osteoblastic cells, clone MC3T3-E1, in medium containing 0.1% bovine serum albumin. The maximal stimulated enzyme activity per mg protein was 1.6-fold over that of control cultures at 250 pg/ml. The metabolite also increased collagen synthesis in a dose-related fashion. On the other hand, [23 (S),25 (R)]-1 alpha,25-(OH)2D3-26,23-lactone decreased slightly but significantly 45Ca mobilization, and blocked the resorptive action of 1 alpha,25-dihydroxyvitamin D3 but not that of parathyroid hormone, in mouse calvaria in organ culture. These results indicate that [23 (S),25 (R)]-1 alpha, 25-(OH)2D3-26,23-lactone stimulates the differentiation of osteoblasts and inhibits bone resorption in vitro.     

Effect of 1alpha,25(OH)2-vitamin D3 on TNF alpha-mediated apoptosis of human primary osteoblast-like cells in vitro.

1alpha,25(OH)2-vitamin D3 is a hormone which potentially stimulates bone cell growth and differentiation. TNFalpha is one possible inductor for apoptosis; apoptosis being an important regulatoring factor for bone modelling and remodelling. We examined the influence of physiological levels (0.1 nM) 1alpha,25(OH)2-vitamin D3 on TNFalpha-mediated apoptosis in human osteoblast-like cells. These human cells were obtained from bone fragments obtained during orthopedic operations on patients without systemic bone disease. Treatment with 1alpha,25(OH)2-vitamin D3 for 8 weeks resulted in a significant reduction (30%) of viable cell number compared to untreated cells. Incubation with TNFalpha (100 ng/ml for 4 hours) only had limited effects on the rate of apoptosis in control cells. After pretreatment with 1alpha,25(OH)2-vitamin D3, induction of apoptosis increased up to 10% in human osteoblast-like cells. In parallel to the induction of apoptosis, 1alpha,25(OH)2-vitamin D3 stimulated osteocalcin and alkaline phosphatase as markers of mature osteoblasts. Our data suggest that 1alpha,25(OH)2-vitamin D3 has a stimulatory effect on TNFalpha-induced apoptosis in human osteoblast-like cells as a result of 1alpha,25(OH)2-vitamin D3-induced cell differentiation.     

1 alpha,25-dihydroxyvitamin D3 modulation in lipid metabolism in established bone marrow-derived stromal cells, MC3T3-G2/PA6.

MC3T3-G2/PA6 (PA6) cells established from newborn mouse calvaria are preadipocytic stromal cells, which differentiate into adipocytes in response to glucocorticoids. We examined the effects of 1 alpha,25-dihydroxyvitamin D3[1 alpha,25(OH)2D3] on adipogenesis in PA6 cells. When PA6 cells were cultured with 10(-8) M dexamethasone, adipocytes containing oil red O-positive droplets first appeared on day 7 (3 days after confluence was attained) and the maximal synthesis of neutral lipids occurred on day 12. Simultaneous addition of 1 alpha,25(OH)2D3 at 10(-9)M completely blocked this dexamethasone-induced neutral lipid synthesis throughout the 14-day culture period. Dose-response studies of vitamin D3 derivatives showed that 1 alpha,25(OH)2D3 was the most potent in inhibiting neutral lipid synthesis in PA6 cells, followed by 1 alpha-hydroxyvitamin D3, 25-hydroxyvitamin D3, and 24R,25-dihydroxyvitamin D3, in that order. Dexamethasone greatly enhanced incorporation of [14C]-acetic acid into triacylglycerol in PA6 cells. The incorporation was markedly inhibited by the addition of 10(-9) M 1 alpha,25(OH)2D3. Instead, 1 alpha,25(OH)2D3 greatly increased incorporation of [14C]-acetic acid into phospholipids, such as phosphatidylcholine and phosphatidylethanolamine, irrespective of the presence or absence of dexamethasone. These results suggest that 1 alpha,25(OH)2D3 modulation of lipid metabolism in bone marrow stromal cells is receptor mediated.     

Basic fibroblast growth factor inhibits osteoclast-like cell formation

Basic fibroblast growth factor (bFGF) inhibited osteoclast-like cell formation in co-cultures of mouse bone marrow cells either with the mouse stromal cell line, ST2, or with primary osteoblastic cells. Basic FGF significantly inhibited the osteoclast-like cell formation, induced by 1α,25-dihydroxyvitamin D₃[1α, 25(OH)₂D₃] when the cytokine was added to the culture, at an intermediate stage, suggesting that bFGF inhibits the differentiation of the osteoclast progenitors. With regard to target cells, bFGF directly affected ST2; it increased [³H]thymidine uptake and decreased the number of alkaline phosphatase-positive cells. In contrast, bFGF had no inhibitory effect on the colony formation of bone marrow cells induced by macrophage colony stimulating factor in methylcellulose culture. In addition, ST2 cells treated with bFGF produced similar amounts of colony forming activity to those without the cytokine. These findings indicated that the bFGF is not involved in the proliferation of progenitor cells even in the presence of ST2 cells. Furthermore, bFGF inhibited osteoclast-like cell formation induced not only by 1α,25(OH)₂D₃, but also by prostaglandin E₂ and by interleukin-11. These results suggest that bFGF inhibits the common site of osteoclast-like cell formation, as induced by different mechanisms. Our data also indicated that the target cells for bFGF in inhibiting osteoclast formation are not osteoclast progenitors but stromal cells such as ST2 and osteoblastic cells, which support osteoclast development. © 1996 Wiley-Liss, Inc.     

Synthesis and activity of oleanolic acid derivatives, a novel class of inhibitors of osteoclast formation

Two series of oleanolic acid derivatives were synthesized and their inhibitory activity on the formation of osteoclast-like multinucleated cells (OCLs) induced by 1alpha,25-dihydroxy vitamin D3 was evaluated in a co-culture assay system. The structure-activity relationships, together with electronic structure based on the frontier molecular orbitals, for example, HOMO and LUMO, related to different amino acid substituents were studied. Derivatives with proline or phenylalanine showed a tendency to enhance the inhibitory activity.     

Establishment of murine macrophage-like mutant and hybrid cell lines: comparative analysis of the differentiation induced by 1 alpha,25-dihydroxyvitamin D3 and recombinant murine interferon-gamma

The purpose of this study is to establish the monocyte/macrophage-like cell lines which are sensitive to potent systemic and local factors, 1 alpha,25-dihydroxyvitamin D3 (1 alpha,25(OH)2 VD3) and interferon-gamma (IFN-gamma). We established two variant mouse macrophage-like cell lines, whose responses to 1 alpha,25(OH)2 VD3 and IFN-gamma differed from one another. The AH-sensitive mutant cell line (G3) was induced by allowing P388D1 tolerant to 8-azaguanine. G3 mutant cells were then fused with the 1 alpha,25(OH)2 VD3-stimulated bone marrow cells isolated from DBA/2 mice. After AH selection the hybrid cell line (XC) was established. The G3 mutant cell line and the XC hybrid cell line had macrophage-like characteristics, such as surface antigens, Fc receptor, C3 receptor, and lysosomal enzymes. The treatment of G3 mutant cells with 1 alpha,25(OH)2 VD3 inhibited cell proliferation with morphological changes, and increased acid phosphatase activity, phagocytic activity, and F4/80 antigen expression on the cell surface. In contrast, IFN-gamma inhibited cell proliferation without effect on acid phosphatase activity and phagocytic activity but increased F4/80 antigen expression. In XC hybrid cells, on the other hand, IFN-gamma, but not 1 alpha,25(OH)2 VD3, inhibited cell proliferation with morphological changes but increased phagocytic activity and F4/80 antigen expression. In addition, IFN-gamma, but not 1 alpha,25(OH)2 VD3, dose-dependently increased multinucleated cell formation of both cells. These findings suggest that the G3 mutant cell line with macrophage-like characteristics is 1 alpha,25(OH)2 VD3- and IFN-gamma-sensitive, and that the XC hybrid cell line is, despite its macrophage-like characteristics, only IFN-gamma-sensitive. Therefore, these newly established cell lines will provide useful systems in studying the differentiation of monocyte/macrophage lineage.     

Effect of 24,25-dihydroxyvitamin D3 in osteoclasts.

Previous results demonstrated that the administration of pharmacological doses of 24,25-dihydroxyvitamin D3 (24,25(OH)2D3) to animals reduces bone resorption and increases bone volume with a decrease in osteoclast number. In order to clarify whether 24,25(OH)2D3 has an effect to inhibit osteoclastic bone resorption, the effect of 24,25(OH)2D3 on the formation and function of osteoclastic cells was examined in vitro. Treatment of hemopoietic blast cells, which are progenitors of osteoclasts, with parathyroid hormone (PTH) or 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) stimulated the formation of osteoclast-like multinucleated cells in a dose-dependent manner. Although 24,25(OH)2D3 in itself had little effect on osteoclast-like multinucleated cells formation, it inhibited the stimulatory effect of PTH on the formation of osteoclastic cells. In addition, 24,25(OH)2D3 also inhibited the stimulation of resorption pit formation by osteoclasts under stimulation with PTH. In contrast, 1,25(OH)2D3 stimulated the formation and function of osteoclastic cells even at low concentrations, and the effect was additive to PTH. These results could not be explained by either an agonistic or antagonistic effect of 24,25(OH)2D3 on 1,25(OH)2D3, and are consistent with the assumption that 24,25(OH)2D3 has a unique inhibitory effect on the formation and function of osteoclasts. Because 24,25(OH)2D3 is shown to stimulate the degradation of 1,25(OH)2D3 and because the formation of 24,25(OH)2D3 is stimulated by 1,25(OH)2D3 not only in the kidney but also in many of its target tissues, including bone, the inhibitory effect of 24,25(OH)2D3 on osteoclastic bone resorption may play a role in the local modulation of the actions of osteotropic hormones in bone.