Cell density-dependent vitamin D effects on calcium accumulation in rat osteogenic sarcoma cells (ROS 17/2)

Summary Rat osteogenic sarcoma cells have been used widely as a model system to study actions of 1,25(OH)₂D₃ and other hormones in osteoblastlike cells. However, some of the pleiotypic manifestations of hormones in these cells vary greatly dependent upon the cell population density and other conditions of culture. Therefore, we have studied the effect of cell density on the relationship between 1,25(OH)₂D₃ and the initial⁴⁵Ca accumulation in ROS 17/2 cells in order to establish conditions suitable for studying the effect of 1,25(OH)₂D₃ on calcium fluxes in these cells. Cells were grown in the presence and absence of 1,25(OH)₂D₃ for 48 hours and then incubated for 4 min in the culture medium containing 0.5 μCi/ml of⁴⁵CaCl₂. In high population density cultures, 0.25–1.0 pg/ml of 1,25(OH)₂D₃ stimulated the intracellular accumulation of⁴⁵Ca (cpm/mg protein), whereas 80 pg/ml or higher concentrations inhibited accumulation of⁴⁵Ca. In low density cultures, concentrations less than 80 pg/ml had no effect, 80–120 pg/ml increased the intracellular accumulation, and as much as 200 pg/ml failed to show the inhibitory effect. These results indicate that the ROS 17/2 cell responses to 1,25(OH)₂D₃ are biphasic—low concentrations stimulating and high concentrations inhibiting⁴⁵Ca accumulation. The sensitivity of the cells to 1,25(OH)₂D₃ increases as the cell population density increases. These observations suggest that the culture density and dose-response relationship must be carefully defined inin vitro studies utilizing osteogenic cell culture systems.     

Glucocorticoids increase the 1,25(OH)2D3 receptor concentration in rat osteogenic sarcoma cells

Summary We have used cultured osteoblastlike rat osteogenic sarcoma cells (ROS 17/2) which have receptors for 1,25(OH)₂D₃ and for glucocorticoids, and have examined the modulation of the 1,25(OH)₂D₃ receptor by the potent glucocorticoid triamcinolone acetonide. We report that triamcinolone acetonide caused an increase of the 1,25(OH)₂D₃ receptor concentration in these cells but it did not affect the affinity of the receptor to 1,25(OH)₂D₃; this phenomenon occurred in a dosedependent fashion for triamcinolone (10⁻⁹ to 10⁻⁷ M) with a maximum increase of 1,25(OH)₂D₃ receptor concentration of ⋍twofold. During the culture period, the 1,25(OH)₂D₃ receptor concentration was altered both in untreated as well as in triamcinolone-treated cells, being highest at the early logarithmic phase and diminished progressively as cells approached confluence. However, throughout the culture period, the 1,25(OH)₂D₃ receptor concentration was higher in the triamcinolone-treated cells.     

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.     

Effects of vitamin D and parathyroid hormone on cyclic AMP production by bone cells isolated from rat calvariae

Summary Studies presented here were designed to investigate further the basis for an impaired cAMP response to parathyroid hormone (PTH) in osteoblastlike calvarial bone cells isolated from vitamin D-deficient rat pups. The goal was to perturb Ca, PTH, and vitamin Din vivo in order to see which factors might be responsible for the impairedin vitro bone cell cAMP response. Pups either were parathyroidectomized (PTX) 3–5 days, implanted with osmotic minipumps delivering high doses of PTH, given repeated, high doses of 1,25(OH)₂D₃, or were D-deficient (-D, i.e., born and suckled by D-deficient mothers). Osteoblastlike bone cells, isolated by sequential enzyme digestion and centrifugation, were exposed to PTH for 5 min in the presence of a phosphodiesterase inhibitor. In bone cells isolated from -D rat pups, both basal and PTH-induced cAMP accumulation were significantly lower than in +D bone cells. Earlier, we had shown that two daily injections of -D pups with 50 ng 1,25(OH)₂D₃ restores this reduced bone cAMP response of -D pups toward normal. In the present study, neither basal nor PTH-induced bone cell cAMP accumulation was affected by subjecting D-replete pups to PTX, PTH infusion, or repeated high doses of 1,25(OH)₂D₃ despite the fact that each treatment markedly changed serum Ca or serum immunoreactive PTH. The results indicate that the impaired bone cell cAMP response seen in -D pups is not a direct result of chronic hypocalcemia and that the “heterologous desensitization” seenin vitro with added 1,25(OH)₂D₃ could not be duplicated byin vivo treatment of +D pups with supraphysiologic doses of 1,25(OH)₂D₃. Finally the lack of alteration in the bone cell cAMP response to PTHin vitro after chronic PTH infusionin vivo fails to support the notion that the impaired response in -D bone cells can be explained entirely by “homologous desensitization” induced by high circulating levels of PTH in the hypocalcemic, -D rat pup.     

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.     

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.     

Modulation of PTH-stimulated cyclic AMP in cultured rodent bone cells: The effects of 1,25(OH)2 vitamin D3 and its interaction with glucocorticoids

Summary Parathyroid hormone (PTH)-stimulated cyclic adenosine monophosphate (cAMP) in rat osteoblastlike (OB) cells has been shown to be modulated by steroid hormones; glucocorticoids are known to increase the level, while the effects of 1,25(OH)₂D₃ are inhibitory. In the present study, we found that the PTH-stimulated cAMP responses are similar in neonatal mouse and fetal rat OB cells. Dexamethasone (0.13–13nM) augmented PTH-stimulated cAMP in both species. Mouse cells showed a higher maximal response to dexamethasone (100% increment) than rat cells (60–70% increment) with similar sensitivity to dexamethasone (ED₅₀ ∼ 1.0 nm). On the other hand, 1,25(OH)₂D₃ decreased PTH-stimulated cAMP, but the effect required pharmacological levels of hormone; mouse cells responded at a lower dose (1.3 nM) and were more sensitive than rat cells (responded at 13 nM) to 1,25(OH)₂D₃ treatment. Introduction of physiological concentrations of 1,25(OH)₂D₃ (0.013–1.3 nm) in addition to dexamethasone (13 nM) resulted in a synergistic enhancement of PTH-stimulated cAMP in rat cells. In contrast, a dose-dependent antagonistic effect was observed in mouse cells. In summary, our findings demonstrate species and concentration-dependent differences in hormonal responses to 1,25(OH)₂D₃ and a complex interplay among PTH, dexamethasone, and 1,25(OH)₂D₃.     

Simulated acidosis does not impair 1,25-dihydroxyvitamin D3 production by cultured kidney cells

Summary Cultured mouse kidney cells grown in serum-free medium were used to assess the metabolism of 25-hydroxyvitamin D₃ in the presence of simulated metabolic acidosis. Kidney epithelial cells isolated from 4–6 week old mice were grown to confluence in a defined serum-free medium at pH 7.4. The confluent monolayers were incubated with tritiated 25-hydroxyvitamin D₃ for 6 hours, the samples were extracted, and vitamin D metabolites were separated and quantitated by high pressure liquid chromatography (HPLC). The pH of the incubation medium was set at 6.9, 7.1, 7.4, or 7.7 by adjusting the bicarbonate concentration, using chloride as the balancing anion at constant Pco₂. When pH was altered at the beginning of the 6 hour assay, production of 1,25-dihydroxyvitamin D₃ was the same at each pH. More prolonged pH perturbation for a total of 30 hours likewise had no influence on 1,25-dihydroxyvitamin D₃ production. These results confirm that intact mammalian kidney cells in serum-free culture possess an active 25-hydroxyvitamin D₃-1-hydroxylase and that the activity of the enzyme is unaffected by pH over the range 6.8–7.7. In experiments where acidosis has been shown to alter 1,25-dihydroxyvitamin D₃ production, the mechanism was probably indirect.     

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.     

Effects of vitamin D deficiency in the chicken embryo

Summary Vitamin D-deficient chicken embryos were obtained by feeding laying hens a diet in which 5 μg 1,25(OH)₂D₃/kg feed were substituted for the vitamin D₃ supplement in the control diet. Hatchability, total Ca and inorganic P concentration in blood, and tibial ash/dry weight ratio were determined in the vitamin D-deficient embryos and in embryos obtained from hens fed the control diet supplemented with 1100 IU vitamin D₃/kg feed. After 5 weeks on the substituted diet the hens laid eggs that showed decreased hatchability in spite of excellent shell quality. All determinations in blood and bones were made on embryos of eggs laid after 6–12 weeks on the diets. On the 17th day of incubation the embryos derived from hens fed the substituted diet showed significant hypocalcemia and hyperphosphatemia and a low tibial ash/dry weight ratio. Injection of 1,25(OH)₂D₃ 3 days before killing corrected the hypocalcemia of the deficient embryos. Those chicks that managed to hatch had normal levels of calcium and inorganic phosphate 1 day after hatching. These findings support previous suggestions by us and other authors that vitamin D metabolites are required by the embryo in order to mobilize calcium from the shell, and decreased hatchability in vitamin D-deficient embryos is related to a defect in calcium mobilization from the shell. While in previous studies a decrease in hatchability was the only parameter used to judge D deficiency of the embryos in our present studies, the deficiency is confirmed by demonstrating a deficit in mineral metabolism which is a more specific sign of D deficiency.     

Acquired alterations in vitamin D metabolism in the acidotic state

Summary Classic (type I) renal tubular acidosis in children is attended by growth retardation and rickets, abnormalities that can be corrected by alkali therapy alone. We have employed the NH₄Cl-treated rachitic chick as a model to investigate vitamin D metabolism in the acidotic state. NH₄Cl ingestion for 96 h was associated with a rise in serum calcium, a significant decrease in blood pH (7.42+0.08 vs 7.30±0.08,P<0.005), decreased [³H]1,25(OH)₂D₃ following [³H]25OHD D₃ injections, and enhanced metabolic clearance of administered [³H]1,25(OH)₂D₃. The data collectively suggest that metabolic acidosis in the chick alters the production and degradation of 1,25(OH)₂D₃.     

Effects of vitamin D-binding protein on bone resorption stimulated by 1,25 dihydroxyvitamin D3

Summary Vitamin D and its metabolites are tightly bound to the serum vitamin D-binding protein (DBP) and only the free hormone is considered to be physiologically active. On the other hand, DBP could interact with cell membranes and even favor its intracellular entry. The present study was undertaken to examine the effects of DBP on bone resorption stimulated by 1,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃]. Forelimb bones from 19-day-old fetal rats were cultured for 5 days in the presence of purified human or rat serum albumin (hSAP or rSAP) and 1,25(OH)₂D₃, with or without human or rat DBP (hDBP or rDBP). Bone resorption was assessed by measuring the release of previously incorporated⁴⁵Ca. We found that the resorptive response to 1,25(OH)₂D₃ was minimally altered by hDBP (5 μM). The minimal effects of hDBP on 1,25(OH)₂D₃ activity on rat bones might be explained by a 6-fold lower affinity of hDBP (1.1×10⁷ M⁻¹) than rDBP (5.9×10⁷ M⁻¹) for 1,25(OH)₂D₃ or by species differences in cellular recognition of DBP. In a homologous rat system, however, rDBP at low (0.5 μM) or physiological (5 μM) concentration significantly decreased 1,25(OH)₂D₃-induced bone resorption. These data therefore support the hypothesis that free rather than DBP-bound 1,25(OH)₂D₃ is physiologically important.     

Lack of influence of 24,25-dihydroxyvitamin D3 on parathyroid hormone secretion from normal or hyperplastic glands

Summary The role of 24,25(OH)₂D₃ on parathyroid gland function remains controversial. The present studies were performedin vitro using (a) dispersed normal bovine parathyroid cells (bPTC) and (b) dispersed canine PTC (cPTC) prepared from glands of normal dogs, dogs with chronic renal failure (CRF), and dogs with CRF treated with 24,25(OH)₂D₃, 2.5 μg orally every day for more than 6 months. Bovine parathyroid cells were incubated for up to 180 min at 0.5, 1.0, and 3.0 mM external calcium in the presence or absence of 24,25(OH)₂D₃ (100 or 1000 nM). Similar experiments were conducted with cells incubated for 24 h in the presence of either the ethanol vehicle or 24,25(OH)₂D₃ (1000 nM). Parathyroid hormone secretion, measured in the supernatant by both C-terminal and N-terminal assays, did not show any differences between control and experimental groups at any time interval. Canine parathyroid cells obtained from uremic animals showed an average threefold increase in the total amount of PTH secreted, on a per cell basis over 180 min at 0.5 mM Ca²⁺, when compared with normal controls. However, there was no significant difference in PTH secretion at any level of calcium concentration between the cells obtained from parathyroid glands of CRF dogs and 24,25(OH)₂D₃-treated CRF dogs. Acute exposure to 24,25(OH)₂D₃ (1000 nM)in vitro of the cells obtained from the glands of CRF dogs also had no effect on PTH secretion. We conclude that 24,25(OH)₂D₃ has no direct effect on PTH secretion from dispersed parathyroid cells of either normal or uremic animals.     

Demonstration of 1,25(OH)2 vitamin D3 receptors and actions in vascular smooth muscle cellsIn vitro

Summary Binding of [³H] 1,25 (OH)₂D₃ and effects of 1,25 (OH)₂D₃ on cell ultrastructure were evaluated in vascular smooth muscle cells (VSMC) primary cultures (aortic media). Specific reversible binding of [³H] 1,25 (OH)₂D₃ by a 3.5 S macromolecule with DNA binding, K_D 6.2×10⁻¹⁰M and N_max 16 fmol/mg protein was demonstrated. Incubation of VSMC with 10⁻⁸ M 1,25 (OH)₂D₃, but not 25 (OH)D₃, in the presence of 10% FCS for up to three weeks caused rapid reversible appearance in the cytoplasm of membrane-bounded electron-dense lysosomal particles which on electronspectroscopic imaging contained Ca and Pi. VSMC are targets for vitamin D.     

1,25 Dihydroxyvitamin D3 is required for growth-independent expression of alkaline phosphatase in cultured rat osteoblasts

Summary Osteoblastic cells were isolated from periosteum-stripped parietal bones of neonatal rat calvaria, seeded at low density (5,000 cells/35 mm of Falcon dish), and cultured for 6 days in BGJ medium supplemented with 20% of vitamin D-depleted FCS or vitamin D and calcium-depleted FCS, with daily addition of 1,25 dihydroxyvitamin D₃ (10⁻⁹ M) or 24,25-dihydroxyvitamin D₃ (10⁻⁹ M). Plating efficiency, clonal growth (number and size distribution of the colonies formed), and the alkaline phosphatase phenotype were evaluated on days 2 and 6 of culture. (1) Culture for 6 days in media not supplemented with 1,25(OH)₂D₃ led to a significant (P<0.001) loss of the alkaline phosphatase phenotype of the osteoblastic cells; the loss was greater in proliferating cells than in nonproliferating ones and occurred in both 0.12 mM or 1.1 mM ionized calcium concentrations. (2) Daily addition of 1,25(OH)₂D₃ (10⁻⁹ M) but not 24,25(OH)₂D₃ maintained the basal percentage of Alk Pase positive cell units in nonproliferating cells and significantly reduced the loss of this phenotype in proliferating colonies. (3) This effect did not stem from an action of the hormone on cell growth. 1,25(OH)₂D₃ was also found to enhance the adhesiveness of the seeded osteoblasts, irrespective of the medium calcium concentration.     

Effect of 1,25(OH)2D3 and 24,25(OH)2D3 on calcium ion fluxes in costochondral chondrocyte cultures

Summary Vitamin D₃ metabolites have been shown to affect proliferation, differentiation, and maturation of cartilage cells. Previous studies have shown that growth zone chondrocytes respond primarily to 1,25(OH)₂D₃ whereas resting zone chondrocytes respond primarily to 24,25(OH)₂D₃. To examine the role of calcium in the mechanism of hormone action, this study examined the effects of the Ca ionophore A23187, 1,25(OH)₂D₃, and 24,25(OH)₂D₃ on Ca influx and efflux in growth zone chondrocytes and resting zone chondrocytes derived from the costochondral junction of 125 g rats. Influex was measured as incorporation of⁴⁵Ca. Efflux was measured as release of⁴⁵Ca from prelabeled cultures into fresh media. The pattern of⁴⁵Ca influx in unstimulated (control) cells over the incubation period was different in the two chondrocyte populations, whereas the pattern of efflux was comparable. A23187 induced a rapid influx of⁴⁵Ca in both types of chondrocytes which peaked by 3 minutes and was over by 6 minutes. Influx was greatest in the growth zone chondrocytes. Addition of 10⁻⁸–10⁻⁹ M 1,25(OH)₂D₃ to growth zone chondrocyte cultures results in a dose-dependent increase in⁴⁵Ca influx after 15 minutes. Efflux was stimulated by these concentrations of hormone throughout the incubation period. Addition of 10⁻⁶–10⁻⁷ M 24,25(OH)₂D₃ to resting zone chondrocytes resulted in an inhibition in ion efflux between 1 and 6 minutes, with no effect on influx during this period. Efflux returned to control values between 6 and 15 minutes.⁴⁵Ca influx was inhibited by these concentrations of hormone from 15 to 30 minutes. These studies demonstrate that changes in Ca influx and efflux are metabolite specific and may be a mechanism by which vitamin D metabolites directly regulated chondrocytes in culture.     

Calcium,vitamin D-endocrine system,and parathyroid hormone in black and white males

Summary The serum and urinary calcium, 25-hydroxyvitamin D (25OHD), 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃), and parathyroid hormone (PTH) were studied in healthy black and white males living in Belgium, and the results were compared to data in blacks of similar age living in Za?re. Dietary calcium and vitamin D were estimated in a subsample of blacks and whites examined in Belgium. Compared to whites (9.51±0.28 mg%) serum calcium was somewhat lower in blacks (9.26±0.27 mg% in Belgium; 9.19±0.48 mg% in Za?re). The 24 hour urinary calcium excretion averaged 215.0±16.7 mg% in whites and was higher (P<0.05 or less) than in blacks (115±71 mg% in Belgium; 36±33 mg% in Za?re). The serum 25OHD levels were similar in whites and blacks evaluated in Za?re, both being higher (P<0.05 or less) than in blacks living in Belgium. In the latter blacks, an inverse correlation was observed between the 25OHD level and the duration of the stay in a temperate climate. Parathyroid hormone levels were slightly higher in blacks living in Belgium than in the other two groups of subjects. The serum levels of 1,25(OH)₂D₃ and human vitamin D-binding protein were similar in the three groups of subjects. Dietary calcium averaged 541±152 mg/day in blacks and was significantly (P<0.001) less than in whites (1,203±508 mg/day), whereas no significant difference was observed in dietary vitamin D intake between blacks and whites. It is concluded that calcium intake is low in blacks but stimulation of parathyroid hormone and 1,25(OH)₂D₃ required to achieve normocalcemia does not occur.     

Regulation of creatine kinase activity in rat osteogenic sarcoma cell clones by parathyroid hormone,prostaglandin E2, and vitamin D metabolites

Summary We have previously shown that both parathyroid hormone (PTH) and prostaglandin E₂ (PGE₂) stimulate the activity of creatine kinase BB (CKBB) in rat bone cells in culture. Therefore, morphologically distinct rat osteogenic sarcoma cells in culture were tested for stimulation of CKBB activity by hormones that regulate skeletal tissues. PTH stimulated CKBB in the osteoblast-like clone ROS 17/2; 1α,25(OH)₂D₃ inhibited this activity while PGE₂, CT and 24R,25(OH)₂D₃ had no significant effect. PGE₂ stimulated CKBB activity in the fibroblast-like clone ROS 24/1, which was unresponsive to PTH, CT and Vitamin D metabolites. 24R,25(OH)₂D₃ as well as PGE₂ (but not PTH, CT or 1α25(OH)₂D₃) stimulated CKBB in clone ROS 25/1, suggesting that this fibroblast-like clone has some chondroblast-like character. Both PTH and PGE₂ stimulated the brain type isoenzyme of CK (CKBB), although the osteogenic sarcoma cell clones contain a significant proportion of the muscle type of CK (CKMM). Thus, increased CKBB activity can serve as an additional characteristic marker for the action of steroid and polypeptide hormones and for prostaglandins.     

Inhibitory effects of 9-cis and all-trans retinoic acid on 1,25(OH)2 vitamin D3-induced bone resorption

The effects of retinoic acid (RA), and calcitriol are mediated by specific nuclear receptors (RARs and VDR, respectively). Induction of RAR and VDR responsive elements in target genes requires a cofactor, the retinoid-X-receptor (RXR), with its ligand 9-cis RA. We have previously demonstrated the expression of RARs and RXRs in osteoblasts, and herein investigated the effects of the retinoids all-trans RA and 9-cis RA alone and combined with calcitriol on bone resorption in vitro, measured by ⁴⁵Ca-release from prelabeled neonatal mouse calvarial bones. All-trans RA and 9-cis RA were powerful stimulators of bone resorption and essentially equipotent. At threshold concentrations (1 nM) both 9-cis RA and at-RA markedly inhibited the resorption induced by calcitriol (1 pM). The findings are compatible with a physiological role for retinoids in bone metabolism.