1,25 dihydroxyvitamin D3 alone or in combination with parathyroid hormone does not increase bone mass in young rats

Summary Parathyroid hormone (PTH) alone is known to increase bone mass, but clinical studies of osteoporotic men suggest that when 1,25 dihydroxyvitamin D₃ (1,25(OH)₂D₃) is given in combination with PTH, the effect on bone growth is enhanced. To determine if 1,25(OH)₂D₃ alone would stimulate bone growth, young male rats were given daily subcutaneous injections of either vehicle or 2.5, 5, 10, or 20 ng 1,25(OH)₂D₃ per 100 g body weight for 30 days. To determine if 1,25(OH)₂D₃ would augment the PTH anabolic response, rats were given daily subcutaneous injections of either vehicle for 12 days; or 4 μg/100 g hPTH alone or in combination with 5 ng/100 g 1,25(OH)₂D₃; or 8 μg/100 g hPTH alone or in combination with 5 ng/100 g 1,25(OH)₂D₃. Calcium (Ca), dry weight (DW), and hydroxyproline (Hyp) of the distal femur; the rate of mineralization in the metaphysis of the proximal tibia; and serum calcium and phosphate were measured. Low normocalcemic doses of 1,25(OH)₂D₃ did not significantly stimulate bone growth. 1,25(OH)₂D₃ did not augment the PTH-stimulated anabolic effect in young male rats. Low doses (2.5 and 5 ng) of 1,25(OH)₂D₃ were not hypercalcemic, and there was no increase in total bone calcium or dry weight although the 5 ng dose increased trabecular bone calcium. 1,25(OH)₂D₃ at 10 and 20 ng increased trabecular bone DW and Hyp, but mineralization was impaired and rats were hypercalcemic. 1,25(OH)₂D₃ in combination with PTH did not augment the PTH stimulation of bone growth as trabecular and cortical bone Ca, DW, and HYP were not increased in rats given both hPTH and 1,25(OH)₂D₃ compared with values for rats treated with hPTH alone.     

Human Osteoclast Formation from Blood Monocytes, Peritoneal Macrophages, and Bone Marrow Cells

Mononuclear precursors of the human osteoclast have been identified in both bone marrow and the circulation in man, but osteoclast membership of the mononuclear phagocyte system (MPS) and its precise cellular ontogeny remain controversial. We isolated human hematopoietic marrow cells, blood monocytes, and peritoneal macrophages and incubated each of these cell populations with UMR106 osteoblast-like cells on glass coverslips and dentine slices in both the presence and absence of 1,25 dihydroxyvitamin D₃ (1,25(OH)₂D₃), macrophage-colony stimulating factor (M-CSF), and dexamethasone. Cells isolated from peripheral blood and peritoneal dialysis fluid were positive only for monocyte/macrophage markers (CD11a, CD11b, CD14, and HLA-DR) and negative for osteoclast markers [tartrate-resistant acid phosphatase (TRAP), vitronectin reception (VNR), and calcitonin (CT) receptors and did not form resorption pits on dentine slices after 24 hours in culture. Similarly marrow cells did not form resorption pits on dentine slices after 24 hours in culture. However, after 14 days in co-culture with UMR106 cells, in the presence of 1,25(OH)₂D₃ and M-CSF, numerous TRAP, CT receptor, and VNR-positive multinucleated cells capable of extensive lacunar resorption were formed in co-cultures of all these preparations. The presence of 1,25 (OH)₂D₃, M-CSF, and UMR106 were absolute requirements for osteoclast differentiation. It is concluded that precursor cells capable of osteoclast differentiation are present in the marrow compartment, the monocyte fraction of peripheral blood, and in the macrophage compartment of extraskeletal tissues and that these cells are capable of differentiating into mature functional osteoclasts. These findings argue in favor of osteoclast membership of the human MPS. Received: 3 January 1997 / Accepted: 14 November 1997     

1,25-dihydroxyvitamin D3 causes an increase in the number of osteoclastlike cells in cat bone marrow cultures

Summary The direct effect of 1,25(OH)₂D₃ upon osteoclast formation from precursor cells is still unknown. In the present experiments we have tested the effects of 1,25(OH)₂D₃ on the generation of osteoclastlike cells in cat bone marrow cultures. These cultures contain proliferating nonattached mononuclear cells and precursor cells that subsequently attach to the culture flask surface and then fuse to form multinucleated osteoclastlike cells. After 7 days of culture we separated the nonattached precursor cells from the attached cells and studied the effects of 1,25(OH)₂D₃ (10⁻¹⁰ M–10⁻⁸ M) on multinucleated cell formation in these two cell populations. In cultures derived from the non-attached precursor cells, 7 days of treatment with 1,25(OH)₂D₃ (10⁻⁸ M) resulted in a 180% increase in the number of attached mononuclear cells and a 90% increase in the number of nuclei contained within multinucleated cells. These effects were dose-dependent. 1,25(OH)₂D₃ did not have a consistent effect on the number of nonattached precursor cells. In cultures derived from attached cells, 7 days of treatment with 1,25(OH)₂D₃ (10⁻⁸ M) induced a 50% increase in the number of mononuclear attached cells and a 40% increase in the number of nuclei within polykaryons. The most likely explanation for these results is that 1,25(OH)₂D₃ promotes the differentiation and subsequent adhesion of nonattached precursor cells, stimulates proliferation of attached mononuclear precursor cells, and possibly stimulates fusion of these attached precursor cells.     

Inhibition by aminohydroxypropylidene bisphosphonate (AHPrBP) of 1,25(OH)2 vitamin D3-induced stimulated bone turnover in the mouse

Summary The purpose of this study was to evaluate whether the 1,25(OH)₂D₃-induced increased bone mineralization in the mouse occurs in response to stimulation of bone resorption. In order to inhibit bone resorption, 35-day-old mice were given 16 μmol/kg/day of (3-amino-1-hydroxypropylidene)-1,1-bisphosphonate (AHPrBP) for 10 days, the first injection occurring 3 days prior to the continuous infusion of 0.06, 0.13, or 0.20 μg/kg/day of 1,25(OH)₂D₃ for 7 days. Two groups of mice were treated with AHPrBP or 1,25(OH)₂D₃ alone. The skeletal changes were assessed by histomorphometric study of caudal vertebrae after double³H-proline and double tetracycline labelings for evaluation of the matrix apposition rate (MaAR) and mineral apposition rate (MiAR), respectively. Treatment with AHPrBP alone or combined to 1,25(OH)₂D₃ decreased the number of acid phosphatase-stained osteoclasts and reduced the endosteal MaAR and MiAR and the amount of osteoid. When given alone, 1,25(OH)₂D₃ increased serum calcium above normal, enhanced the number of histochemically active osteoclasts, and stimulated the endosteal MiAR. Pretreatment with AHPrBP blocked both the increase in serum calcium and the stimulation of the MiAR induced by 1,25(OH)₂D₃ infusion though serum 1,25(OH)₂D₃ levels rose according to the dose given. The results show that 1) the serum calcium and the bone resorbing responses to 1,25(OH)₂D₃ infusion are prevented by pretreatment with AHPrBP, and 2) the stimulatory effect of 1,25(OH)₂D₃ on the mineralization rate is blocked when bone resorption is inhibited. The data indicate that 1,25(OH)₂D₃ promotes bone mineralization in the mouse mainly in response to stimulation of bone resorption.     

The influence of dihydroxylated vitamin D metabolites on bone formation in the chick

Summary The ability of 1,25(OH)₂D₃ and of 24,25(OH)₂D₃ to prevent or to heal rickets in chicks was evaluated by studies of plasma biochemistry, growth plate histology, bone morphometry and microradiography, and bone mineralization. 1,25(OH)₂D₃ at a dose of 100 ng/day produced fewest abnormalities compared with vitamin D₃-treated control chicks. Bone growth was slightly greater than vitamin D₃-treated controls in chicks given a lower dose of this metabolite; the reverse was observed in chicks given a higher dose. 24,25(OH)₂D₃ was less effective than 1,25(OH)₂D₃ in preventing rickets even at doses as high as 400 ng/day. Treatment of rachitic chicks with doses of 24,25(OH)₂D₃ up to 300 ng/day produced no healing effect on the bone lesions, in marked contrast to the beneficial effects observed with 1,25(OH)₂D₃.     

Avian medullary bone in organ culture: Effects of vitamin D metabolites on collagen synthesis

Summary A new organ culture system for the study of bone metabolism has been developed using chicken medullary bone. The presence of viable bone cells in culture was demonstrated by histological and histochemical techniques. Incorporation of³H-proline into collagenase-digestible protein (CDP) and noncollagen protein (NCP) was determined using purified bacterial collagenase. Collagen accounted for approximately 10–15% of the total protein labeled. The addition of 1,25-dihydroxycholecalciferol (1,25 (OH)₂D₃) resulted in a dose-dependent inhibition of³H-proline incorporation into CDP at doses from 10⁻¹⁰M to 10⁻⁷M, with maximal suppression reaching 30% of control. The effect was specific for collagen, since³H-proline incorporation into NCP was unaffected. Hydroxyproline analysis of bone explants and culture medium revealed a 1,25(OH)₂D₃-induced decrease in the³H-hydroxyproline content of the system (bone + medium), suggesting that the effect of 1,25(OH)₂D₃ is due to inhibition of collagen synthesis rather than enhanced collagen degradation, impaiored incorporation of collagen into bone matrix, or bone resorption Medullary bone collagen synthesis was not affected by 24,25(OH)₂D₃, either alone or in combination with 1,25(OH)₂D₃. Structure-activity studies of vitamin D metabolites showed that 1,25(OH)₂D₃ and 1,24,25(OH)₃D₃ were the most potent metabolites tested, followed by 1-alpha(OH)D₃. 25(OH)D₃ and 24,25(OH)₂D₃ had no effect at concentrations as high as 10⁻⁷M. These results indicate a possible role for vitamin D in the regulation of medullary bone formation during the reproductive cycle of the egg-laying hen, and suggest the potential utility of medullary bone as anin vitro model for the study of bone formation     

The effects of parathyroid hormone or 1,25-dihydroxyvitamin D3 on monocyte-osteoclast fusion

Summary ³H-thymidine-labeled blood monocytes were cultured with osteoclasts in the presence or absence of parathyroid hormone or 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃) in order to evaluate (1) the percentage of monocytes capable of fusing with osteoclasts, (2) if parathyroid hormone or 1,25(OH)₂D₃ influences the contribution of blood monocytes to osteoclast nuclear turnover. We found that within 24 hours of culture, about 8% of blood monocytes fuse with osteoclasts regardless of the presence of parathyroid hormone (PTH) or 1,25(OH)₂D₃. On the other hand, formation of nonosteoclastic giant cells by fusion of monocytes is enhanced by 5×10⁻⁹ M 1,25(OH)₂D₃ but only in the presence of the bone resorptive cells.     

Vitamin D3 analogs and salmon calcitonin partially reverse the development of renal osteodystrophy in rats

We have previously established an uremic rat model which is suitable for investigating the effect of various treatment modalities on the progression of renal osteodystrophy [1]. Four months subsequent to 5/6 nephrectomy, animals were treated three times a week for 3 months with either vehicle, 1,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃], 1,25(OH)₂D₃+24,25-dihydroxyvitamin D₃ [24,25(OH)₂D₃], 1,25(OH)₂D₃+calcitonin (CT), or 1,25(OH)₂D₃+ 24,25(OH)₂D₃+CT. At termination of the study, clinical chemistry, chemical composition, and mechanical properties of femurs, calvarial parathyroid hormone (PTH)-elicited adenylate cyclase (AC), and phospholipase C (PL-C) activities, femoral cross-sectional area, and bone histomorphometry were analyzed. The main findings were that 1,25(OH)₂D₃±24,25(OH)₂D₃ treatment enhanced elasticity as well as time to fracture at the femoral metaphysis. CT potentiated the increase in elasticity obtained by 1,25(OH)₂D₃±24,25(OH)₂D₃ treatment. Only 24,25(OH)₂D₃ administration rectified the supernormal PTH-stimulated uremic bone AC, and only 1,25(OH)₂D₃ medication normalized the diminished CT-elicited AC. The obliterated uremic bone PTH-sensitive PL-C was fully normalized by all drug regimens. Femoral shaft inner zone diameter was enhanced by uremia, however, all drug treatments normalized it. Ditto effect was registered with either drug treatment on the subnormal outer and inner zone widths. Histomorphometrical analyses showed that 1,25(OH)₂D₃ administration reduced both eroded and osteoid surfaces. Most prominently, adjuvant 24,25(OH)₂D₃ or CT administration potentiated the beneficial effect of 1,25(OH)₂D₃ on fibrosis and osteomalacia. We assert that vitamin D₃ treatment markedly reverses the development of renal osteodystrophy, and CT potentiates the effect of vitamin D₃.     

Vitamin K2 promotes 1α,25(OH)2 vitamin D3-induced mineralization in human periosteal osteoblasts

The effect of vitamin K on mineralization by human periosteal osteoblasts was investigated in the absence and presence of 1,25 dihydroxyvitamin D₃ (1,25(OH)₂D₃). Vitamin K₁ and K₂, but not vitamin K₃, at 2.5 M enhanced in vitro mineralization when cells were cultured with vitamin K for 20 days after reaching confluence in vitro. Vitamin K₂ (2-methyl-3-all-trans-tetraphenyl-1,4-naphthoquinone: menatetrenone) was the most potent of these vitamin K analogs; it slightly inhibited alkaline phosphatase (ALP) activity. Human osteoblasts were mineralized and showed the enhanced ALP activity on treatment with 10^-9 M of 1,25(OH)₂D₃ for 20 or 25 days after confluence. Vitamin K₂ promoted the 1,25(OH)₂D₃-induced mineralization, but slightly inhibited the 1,25(OH)₂D₃-induced ALP activity. Moreover, vitamin K₂ enhanced the 1,25(OH)₂D₃-induced osteocalcin accumulation in the cells and the extracellular matrix (cell layer), but inhibited the osteocalcin content in the medium produced by the 1,25(OH)₂D₃ treatment. However, vitamin K₂ alone did not induce osteocalcin production in the human osteoblasts. On Northern blot analysis, osteocalcin mRNA expression on 1,25(OH)₂D₃-treated cells was enhanced by vitamin K₂ treatment, but vitamin K₂ alone did not induce osteocalcin mRNA expression. Warfarin blocked both the 1,25(OH)₂D₃-induced osteocalcin production and the accumulation in the cell layer, and also blocked the 1,25(OH)₂D₃ plus vitamin K₂-induced osteocalcin production and the accumulation in the cell layer. The 1,25(OH)₂D₃-induced mineralization promoted by vitamin K₂ was probably due to the enhanced accumulation of osteocalcin induced by vitamin K₂ in the cell layer. However, we concluded that the mineralization induced by vitamin K₂ alone was due to the accumulation of osteocalcin in bovine serum on the cell layer, since osteocalcin extracted from the cell layer was not identified by specific antiserum against human osteocalcin, which does not cross-react with bovine osteocalcin. These results suggest that the mechanism underlying the mineralization induced by vitamin K₂ in the presence of 1,25(OH)₂D₃ was different from that of vitamin K₂ alone, and that osteocalcin plays an important role in mineralization by osteoblasts in vitro.     

Human parathyroid hormone (1–34) and salmon calcitonin do not reverse impaired mineralization produced by high doses of 1,25 dihydroxyvitamin D3

Summary We have reported recently that pharmacologic doses of 1,25 dihydroxyvitamin D₃ (1,25(OH)₂D₃) stimulated bone matrix formation but impaired mineralization. The objective of this study was to determine if parathyroid hormone (hPTH 1-34) or calcitonin (sCT) would mineralize the osteoid induced by 1,25(OH)₂D₃ in rat long bones. In one experiment, male Sprague-Dawley rats were given daily subcutaneous injections of vehicle: 8 μg hPTH(1-34); 125 ng 1,25(OH)₂D₃; or both 8 μg hPTH and 125 ng 1,25(OH)₂D₃ per 100 g body weight for 12 days. In a second experiment, rats received daily injections of vehicle: 2 U sCT; 125 ng 1,25(OH)₂D₃; or both 2 U sCT and 125 ng 1,25(OH)₂D₃ per 100 g body weight for 18 days. Calcium (Ca), hydroxyproline (Hyp), and dry weight (DW) of the distal femur and serum calcium, phosphate, and serum bone Gla protein (BGP) were measured. In rats given both 1,25(OH)₂D₃ and hPTH, total bone DW and Hyp increased (P<.01) without a corresponding increase in bone Ca so that Ca/Hyp decreased 47% (P<.01) from control and remained comparable to values for rats treated with 1,25(OH)₂D₃ alone. In rats treated with both 1,25(OH)₂D₃ and sCT, total bone DW and Hyp increased while Ca decreased so that Ca/Hyp decreased 38% from control (P<.05), and remained comparable to values for rats treated with 1,25(OH)₂D₃ alone. These results indicate that hPTH or sCT, given by intermittent injection to rats for 12 or 18 days respectively, failed to mineralize the osteoid induced by high doses of 1,25(OH)₂D₃.     

Comparison of 1,25-, 25-, and 24,25-hydroxylated vitamin D3 binding in fetal rat calvariae and osteogenic sarcoma cells

Summary The hormonal metabolite of vitamin D₃, 1,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃], exerts its biological effects by binding to a cytosolic receptor protein. Such a protein has been demonstrated in vitamin D₃ target organs including fetal rat calvariae and more recently in rat osteogenic sarcoma cells. In this study we have compared the binding of 25-hydroxyvitamin D₃ [25(OH)D₃] and 24,25-dihydroxyvitamin D₃ [24,25(OH)₂D₃] to that of 1,25-(OH)₂D₃ in fetal rat calvariae and osteogenic sarcoma (OS) cells. Sucrose density sedimentation, DNA-cellulose chromatography, and intracellular uptake studies have been employed to evaluate these interactions. In cytosol preparations from calvariae, [³H]-1,25(OH)₂D₃ bound to a 3.3S macromolecule and to a much greater extent to a 5.8S macromolecule while both [³H]25(OH)D₃ and [³H]24,25(OH)₂D₃ bound to the 5.8S macromolecule. By incubating intact calvariae and OS cells with labeled metabolites and thus establishing binding intracellularly prior to cell disruption, we have found that the 3.3S protein which has high specificity for 1,25(OH)₂D₃ occurs inside the cells; the 5.8S protein, however, does not occur inside the cells but is generated after cell disruption. The [³H]-1,25(OH)₂D₃-receptor complex adsorbed to DNA-cellulose and was eluted from this affinity resin at 0.28M KCl. In contrast, [³H]25(OH)D₃ and [³H]-24,25(OH)₂D₃ binding activity did not adsorb to DNA-cellulose. We conclude that, in contrast to the 3.3S protein, the 5.8S macromolecule does not fulfill receptor criteria but is rather generated by the experimental manipulation of the bone cells. Our data suggest that the vitamin D₃ actions on bone are mediated only via the 3.3S receptor, and hence quantitative but not qualitative differences of the effects of the various metabolites are feasible. With technical assistance by M. Larsen, D. Meler, and M. LaFrance.     

Effect of 24R,25-dihydroxyvitamin D3 on the formation and function of osteoclastic cells

Summary Previous reports demonstrated that the administration of large doses of 24R,25-dihydroxyvitamin D₃ [24R,25(OH)₂D₃] to animals with normal vitamin D supply causes an increase in bone volume with reduced bone resorption and decreased osteoclast number. The present study was undertaken to clarify if 24R,25(OH)₂D₃ has any inhibitory effect on the formation and function of osteoclasts. The effect of 24R,25(OH)₂D₃ on the formation of osteoclastic cells was examined by measuring the number of tartrate-resistant acid phosphatase-positive multinucleated cells (MNCs) formed from hemopoietic progenitor cells obtained from spleens of 5-fluorouracil-treated mice. Treatment with 1,25(OH)₂D₃ or parathyroid hormone fragment 1–34 [PTH(1–34)] stimulated osteoclast-like MNC formation in a dose-dependent manner. Addition of 24R,25(OH)₂D₃ alone showed a weak stimulatory effect on MNC formation at 10^-6 M, which appeared to be due to its binding to 1,25(OH)₂D₃ receptors. In contrast, when 24R,25(OH)₂D₃ was added together with 1,25(OH)₂D₃ or PTH(1–34), it inhibited osteoclast-like MNC formation stimulated by these hormones. A significant inhibition of MNC formation was observed with 10^-7M 24R,25(OH)₂D₃, and the stimulatory effect of 1,25(OH)₂D₃ or PTH(1–34) was almost completely eliminated with 10^-6 M 24R,25(OH)₂D₃. Neither 24S,25(OH)₂D₃ nor 25(OH)D₃ exhibited a similar inhibitory effect. The effect of 24R,25(OH)₂D₃ on the resorptive function of osteoclasts was examined by measuring the formation of resorption pits by mouse bone cells on dentine slices. Treatment with 24R,25(OH)₂D₃ also inhibited the resorption pit formation stimulated by 1,25(OH)₂D₃ or PTH(1–34) with similar dose response. These results demonstrate that 24R,25(OH)₂D₃ has a specific inhibitory effect on the formation and function of osteoclastic cells stimulated by 1,25(OH)₂D₃ or PTH, and suggest that these effects of 24R,25(OH)₂D₃ may play role in the regulation of bone metabolism by modulating the actions of osteotropic hormones on osteoclastic bone resorption.     

The effect of 1,25 dihydroxycholecalciferol on parathyroid hormone secretion by monolayer cultures of bovine parathyroid cells

Summary Controversy exists over a direct effect of 1,25(OH)₂D₃ on PTH secretion. To investigate the possibility that the suppressive effect of 1,25(OH)₂D₃ on PTH secretion may be demonstrable in 1,25(OH)₂D₃-depleted tissue and/or after prolonged periods of exposure to 1,25(OH)₂D₃, primary monolayer cultures of bovine parathyroid cells were established in 1∶1 DMEM/Ham's F-12 media supplemented with 2% calf serum but not 1,25(OH)₂D₃. Ionized calcium was maintained at 1.0 mM. Experiments were performed on 4-day-old culture cells. PTH concentration was measured using both a mid-region/carboxyl and an amino-terminal PTH antisera. 1,25(OH)₂D₃ at a concentration of 0.1 ng/ml suppressed PTH secretion by 32±7% after 48 hours. High calcium concentration (2.0 mM) suppressed PTH secretion by 37±10% and this effect was not additive over that of 1,25(OH)₂D₃. PTH secretion rate recovered fully 48 hours after normalization of the external calcium concentration but not after the removal of 1,25(OH)₂D₃. It is concluded that 1,25(OH)₂D₃ directly suppresses PTH secretion by monolayer culture of bovine parathyroid cells.     

1,25 dihydroxyvitamin D3 modifies cyclosporine-induced bone loss

Summary We have previously shown that cyclosporin A (CsA) produces high bone remodeling with resorption exceeding formation and loss of bone volume in the rat. This may have important clinical implications where CsA is widely used in organ transplantation. 1,25 dihydroxyvitamin D₃ (1,25(OH)₂D₃) is a bone mineralizing hormone which also has immune modifying properties. Consequently, we studied the effect of combined CsA and 1,25(OH)₂D₃ administration over 28 days in four groups of rats. Group A received vehicle (n=10), group B CsA (15 mg/kg) (n=10) alone, group C 1,25(OH)₂D₃ plus CsA (n=15), and group D 1,25(OH)₂D₃ alone (20 ng/100 g) (n=15). Rats were bled periodically at day 0, 7, 14, and 28 and Ca, parathyroid hormone (PTH), 1,25(OH)₂D, osteocalcin (bone Gla-protein, BGP), BUN, and creatinine were measured. Rats were sacrificed on day 28 and bones were examined histomorphometrically. Compared to controls, CsA resulted in significant elevation of BGP and a transient increase in 1,25(OH)₂D with excess bone remodeling and loss of bone volume. 1,25(OH)₂D₃ administration produced hypercalcemia, a significant rise in BGP, with suppression of PTH and increased osteoid volume. Combined therapy prevented the loss of bone volume probably due to increased osteoid tissue and enhanced osteoblast activity. Renal dysfunction, a side-affect of CsA, was not a factor. In conclusion, 1,25(OH)₂D₃ combined with CsA restores bone volume which is accompanied by increases in serum calcium and BGP.     

Effects of phosphate and 1,25(OH)2D3 on in vitro bone collagen synthesis in the hypophosphatemic mouse

Summary Calvarial bones from hypophosphatemic (Hyp) mice and normal littermates were cultured in a chemically defined medium to determine: (a) the effect of medium phosphate (Pi) concentration (1, 2, and 3 mM) on collagen synthesis; (b) the effect of 1,25-dihydroxycholecalciferol [1,25(OH)₂D₃] (10⁻¹²M–10⁻⁷M) on collagen synthesis; and (c) whether bone responsiveness to 1,25(OH)₂D₃ was affected by changes in medium Pi concentration. Bone collagen synthesis was evaluated by measuring [³H]hydroxyproline formation. The distribution of labeled hydroxyproline between bone explant and culture medium (total and dialyzable fraction) was studied. These experiments confirm that 1,25(OH)₂D₃ inhibits specifically bone collagen synthesis in vitro. We did not detect any effect of medium Pi concentration on basal collagen synthesis but were able to demonstrate that lowering medium Pi concentration increased the 1,25(OH)₂D₃-induced inhibition of collagen synthesis. Bones from both genotypes responded to 1,25(OH)₂D₃, but modulation of this response by changes in Pi concentration was altered in Hyp bone as, in contrast to normal bone, its response to 1,25(OH)₂D₃ was unaffected when medium Pi concentration was decreased from 3 to 2 mM. These findings support the hypothesis of an altered response of bone to 1,25(OH)₂D₃ in the Hyp mouse.     

Actions of parathyroid hormone and 1,25-dihydroxycholecalciferol on citrate decarboxylation in osteoblast-like bone cells differ in calcium requirement and in sensitivity to trifluoperazine

Summary The actions of PTH in OB bone cells appear to involve both calcium and cAMP. At present little information exists regarding the relationship, if any, between these two putative second messengers of hormone action in bone cells. In this report the molecular role of calcium in the actions of PTH and 1,25(OH)₂D₃ has been compared, since like PTH, the steroid 1,25(OH)₂D₃ is a potent bone resorbing hormone that exerts inhibition of citrate decarboxylation in OB cells, but unlike PTH does not activate adenylate cyclase. It was found that 1,25(OH)₂D₃ could initiate near maximum inhibition of citrate decarboxylation at extracellular calcium levels as low as 0.05 mM, whereas PTH effects began to be apparent only at 0.1 mM calcium, and maximum inhibition of citrate decarboxylation by PTH required 0.5 mM Ca. In addition, PTH-induced decrease in citrate decarboxylation was inhibited by low doses of TFP, an inhibitor of calmodulin and calcium-dependent, phospholipid-sensitive protein kinases, in contrast to 1,25(OH)₂D₃, whose effects were not reduced by this agent. These results suggest that: (a) the actions of 1,25(OH)₂D₃ may not be directly dependent on calcium influx; (b) in OB cell response to PTH a relationship probably exists between cAMP and calcium; and (c) this relationship may involve calmodulin, or calcium-dependent protein kinases that can be inhibited by TFP.     

Alkaline phosphatase inhibition by levamisole prevents 1,25-dihydroxyvitamin D3-stimulated bone mineralization in the mouse

Summary To determine the relationship between alkaline phosphatase (AP), 1,25(OD)₂D₃ and bone formationin vivo, we have examined the effects of levamisole, a stereospecific inhibitor of AP on bone formation and on 1,25(OH)₂D₃-stimulated bone mineralization in the mouse. Normal mice were injected daily with levamisole at doses of 40 and 80 mg/kg/b.w. The compound was given alone or in combination with 1,25(OH)₂D₃ infusion (0.05 μg/kg/d) for 7 days. Treatment with levamisole alone inhibited the serum AP activity (mainly of skeletal origin in mice) by 18.4 and 61.3% for the low and high dose respectively. No deleterious effect on body growth, tibia length, and bone cells population was detected. The moderate inhibition of AP activity produced by the lower dose of levamisole alone (18.4%) or in combination with 1,25(OH)₂D₃ (37.9%) was associated with a reduced endosteal matrix apposition rate (MaAR) determined by double³H-proline labeling method. This effect was related to a levamisole-induced fall in serum phosphate. Despite the moderate inhibition of AP activity, the mineral apposition rate (MiAR) determined by the double tetracycline labeling method remained normal. Moreover, 1,25(OH)₂D₃ infusion still resulted in increased MiAR which was stimulated to the same extent as in the absence of levamisole. By contrast, the more severe inhibition of AP activity induced by 80 mg/kg of levamisole alone (61.3%) or in combination with 1,25(OH)₂D₃ (45.8%) inhibited both the MaAR and the MiAR and prevented the stimulatory effect of 1,25(OH)₂D₃ on bone mineralization. The data show that AP activity affects the bone matrix and mineral apposition ratesin vivo and that severe inhibition of AP activity inhibits the 1,25(OH)₂D₃-induced stimulation of bone mineralization in the mouse.     

1,25-dihydroxyvitamin D3 inhibits directly human osteoclastogenesis by down-regulation of the c-Fms and RANK expression

Objective1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃) is a key molecule to maintain calcium homeostasis and bone metabolism. It was recently reported that 1,25(OH)₂D₃ directly inhibited osteoclast differentiation in mouse bone marrow cells and human bone marrow-derived colony-forming unit granulocyte macrophage (CFU-GM) cells. However, the direct effects of 1,25(OH)₂D₃ and its affecting mechanisms on the osteoclast differentiation of human osteoclast precursors remain largely unknown. In this study, we examined the direct effects of 1,25(OH)₂D₃ on the osteoclastogenesis of human peripheral blood (PB) osteoclast precursors.MethodsIn vitro osteoclastogenesis assays were performed using osteoclast precursors from normal PB. The gene expressions were analyzed using real-time PCR. The cell surface proteins, including c-Fms and RANK, were measured by flow cytometry.Results1,25(OH)₂D₃ strongly inhibited osteoclast differentiation and it suppressed the expression of RANK in the human PB osteoclast precursors. One mechanism of RANK inhibition by 1,25(OH)₂D₃ is down-regulation of the M-CSF receptor c-Fms, which is required for the expression of RANK. In contrast to the previous reports on mouse osteoclast precursors, 1,25(OH)₂D₃ did not affect the expression of c-Fos. Parallel to the inhibition of osteoclastogenesis, 1,25(OH)₂D₃ increased the expression and phosphorylation of CCAAT enhancer-binding protein β (C/EBPβ), which is a recently discovered inhibitor of osteoclastogenesis.ConclusionsOur results show that 1,25(OH)₂D₃ inhibits human osteoclastogenesis by decreasing the RANK+ osteoclast precursors, and we suggest that 1,25(OH)₂D₃ may be a powerful therapeutic agent for treating inflammation-induced bone disease that shows excessive osteoclast activation.     

Different Effects of Insulin and Insulin-Like Growth Factors I and II on Osteoprogenitors and Adipocyte Progenitors in Fetal Rat Bone Cell Populations

We investigated the effects of insulin (1–1,000 nM), insulin-like growth factor (IGF)-I, and IGF-II (3–100 nM each) alone or together with 10 nM dexamethasone (DEX) or 10 nM 1,25-dihydroxyvitamin D₃ (1,25[OH]₂D₃) on proliferation and differentiation of adipocyte and osteoblast progenitors in bone cell populations derived from fetal rat calvaria. The effects on differentiation were evaluated by counting the number of bone or osteoid nodules and adipocyte colonies and the effects on proliferation, by measuring their size by image analysis. The types of cells studied were 1,25(OH)₂D₃- and DEX-responsive adipocyte progenitors and DEX-dependent and independent osteoprogenitors. Both IGF-I and IGF-II stimulated osteoprogenitor differentiation both alone and in the presence of DEX, while insulin stimulated osteoprogenitor differentiation only in the absence of DEX. Neither IGF-I/-II nor insulin affected proliferation of osteoprogenitors. Insulin had little effect on adipocyte differentiation by itself but strongly stimulated differentiation in the presence of either 1,25(OH)₂D₃ or DEX, while IGF-II stimulated adipocyte differentiation in both the absence and presence of 1,25(OH)₂D₃ or DEX. IGF-I by itself or in the presence of DEX strongly stimulated adipocyte cell differentiation but had little effect in the presence of 1,25(OH)₂D₃. Our results demonstrate that insulin, IGF-II, and IGF-I have specific and different effects on the differentiation and proliferation of different groups of progenitor cells.     

Induction of matrix gla protein synthesis during prolonged 1,25-Dihydroxyvitamin D3 treatment of osteosarcoma cells

Summary The synthesis of matrix Gla protein (MGP) and bone Gla protein (BGP) have been shown to be mutually exclusive in all osteosarcoma cell lines investigated. In the cell lines that produce the respective proteins, synthesis is stimulated by 1,25-dihydroxyvitamin D₃(1,25(OH)₂D₃) within the first several hours of hormone treatment. In the present studies we have investigated the effects of longer-term treatment with 1,25(OH)₂D₃ in the ROS 17/2 cell line, a cell line that synthesizes BGP constitutively but does not synthesize MGP. In agreement with earlier studies, the rate of BGP synthesis increases within 8 hours of hormone treatment, is maximal by 24 hours, and remains at the maximal rate through 48 hours of 1,25(OH)₂D₃ treatment. The present study is the first to report that the rate of BGP secretion at times beyond 48 hours declines to that of control cultures despite the continued administration of 1,25(OH)₂D₃, and that MGP synthesis is induced in ROS 17/2 cells by 48 hours of 1,25(OH)₂D₃ treatment. At this time, MGP mRNA could be detected by northern blot analysis and MGP secretion could be demonstrated by radioimmunoassay of culture medium. Both the level of MGP message per unit total RNA and the rate of MGP secretion into culture medium increased steadily between 2 and 6 days of 1,25(OH)₂D₃ treatment. The MGP synthesized by the 1,25(OH)₂D₃-treated ROS 17/2 cells was identical to that found in bone by northern blot analysis of message and by western blot analysis of the media antigen. Halfmaximal induction of MGP synthesis was obtained with 0.3 nM 1,25(OH)₂D₃, a 60-fold higher dosage than was required for the half maximal stimulation of BGP synthesis in these cells. Treatment of ROS 17/2 cells with 24,24-F₂1,25(OH)₂D₃ suggests that the observed difference in dose dependence is not due to an increased rate of hormone catabolism.