1,25-dihydroxyvitamin D3 pretreatment limits prostaglandin biosynthesis by cytokine-stimulated adult human osteoblast-like cells

The steroid derivative 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃) is a regulator of bone biology, and there is evidence that 1,25(OH)₂D₃ modulates arachidonic acid metabolism in osteoblastic cell model systems and in bone organ cultures. In the present studies, 1,25(OH)₂D₃ decreased prostaglandin (PG) biosynthesis by normal adult human osteoblast-like (hOB) cell cultures by about 30%. The decrease was observed under basal incubation conditions, or in specimens stimulated by transforming growth factor-β₁ (TGF-β) or by tumor necrosis factor-α (TNF). The inhibition of the TGF-β-stimulated PG production appeared to reflect a diminished efficiency of arachidonic acid conversion into PGs by the cells, while the efficiency of substrate utilization for PG biosynthesis was unaffected by 1,25(OH)₂D₃ pretreatment in the unstimulated samples, or in samples stimulated with TNF or with TNF plus TGF-β. Free arachidonic acid levels were decreased following 1,25(OH)₂D₃ pretreatment in the TNF stimulated samples. hOB cell phospholipase A₂ activity was measured in subcellular fractions, and this activity was decreased by 20–25% in the 1,25(OH)₂D₃ pretreated samples. The addition of the selective inhibitor AACOCF₃ to the phospholipase A₂ assays provided evidence that it was the cytoplasmic isoform of the enzyme that was affected by the 1,25(OH)₂D₃ pretreatment of the hOB cells. Thus, 1,25(OH)₂D₃ regulation of hOB cell biology includes significant effects on arachidonic acid metabolism. In turn, this could influence the effects of other hormones and cytokines whose actions include the stimulated production of bioactive arachidonic acid metabolites. J. Cell. Biochem. 68:237–246, 1998. © 1998 Wiley-Liss, Inc.     

Ketoconazole inhibits self-induced metabolism of 1,25-dihydroxyvitamin D3 and amplifies 1,25-dihydroxyvitamin D3 receptor up-regulation in rat osteosarcoma cells

Ketoconazole (an inhibitor of vitamin D-24 hydroxylase) was used to study the role of self-induced 1,25-dihydroxyvitamin D3 (1,25-D3) metabolism on cellular responsiveness to 1,25-D3. Eighteen hours of treatment with 1,25-dihydroxy-[26,27-methyl-3H]vitamin D3 (1,25-[3H]D3) increased total 1,25-D3 receptors (VDR) from 60 to 170 fmol mg/protein. In cells treated with both 1,25-[3H]D3 and ketoconazole, up-regulation of VDR was increased by 40% over that observed with cells receiving 1,25-[3H]D3 alone. Ketoconazole alone had no agonistic activity. Treatment of cells with 1 nM 1,25-[3H]D3 plus increasing doses of ketoconazole (0-30 microM) resulted in a dose-dependent increase in occupied VDR and total VDR. This up-regulation was associated with reduced 1,25-[3H]D3 catabolism. 1,25-[3H]D3-induced up-regulation of VDR typically peaked at 14 h and declined thereafter. Ketoconazole lengthened the time to reach peak VDR up-regulation to 20 h. The ability of ketoconazole to increase cell responsiveness (VDR up-regulation) was the result of both increased and prolonged occupancy of VDR by 1,25-[3H]D3. The t1/2 of occupied VDR was 2 h in the absence of ketoconazole and greater than 7 h when ketoconazole was present. Collectively, these results suggested that self-induced catabolism of 1,25-D3 is an important regulator of VDR occupancy and therefore cellular responsiveness to hormone. These data also demonstrate the usefulness of ketoconazole as an inhibitor of vitamin D hydroxylases in intact cells.     

Modulatory effects of 1,25-dihydroxyvitamin D3 on human B cell differentiation

1,25-Dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) can modulate immune responses, but whether it directly affects B cell function is unknown. Patients with systemic lupus erythematosus, especially those with antinuclear Abs and increased disease activity, had decreased 1,25(OH)(2)D(3) levels, suggesting that vitamin D might play a role in regulating autoantibody production. To address this, we examined the effects of 1,25(OH)(2)D(3) on B cell responses and found that it inhibited the ongoing proliferation of activated B cells and induced their apoptosis, whereas initial cell division was unimpeded. The generation of plasma cells and postswitch memory B cells was significantly inhibited by 1,25(OH)(2)D(3), although the up-regulation of genetic programs involved in B cell differentiation was only modestly affected. B cells expressed mRNAs for proteins involved in vitamin D activity, including 1 alpha-hydroxylase, 24-hydroxylase, and the vitamin D receptor, each of which was regulated by 1,25(OH)(2)D(3) and/or activation. Importantly, 1,25(OH)(2)D(3) up-regulated the expression of p27, but not of p18 and p21, which may be important in regulating the proliferation of activated B cells and their subsequent differentiation. These results indicate that 1,25(OH)(2)D(3) may play an important role in the maintenance of B cell homeostasis and that the correction of vitamin D deficiency may be useful in the treatment of B cell-mediated autoimmune disorders.     

Heterologous up-regulation of the 1,25-dihydroxyvitamin D3 receptor by parathyroid hormone (PTH) and PTH-like peptide in osteoblast-like cells

1,25-Dihydroxyvitamin D3 (1,25-(OH)2D3) receptor content in cultured osteogenic sarcoma cells (UMR-106) was found to be increased after treatment with both bovine and human PTH and human PTH-like peptide (hPLP). The dose dependent increase of receptors was preceded by a dose dependent stimulation of cAMP production. This suggests a role for cAMP as mediator of the PTH- and hPLP-induced 1,25-(OH)2D3 receptor up-regulation. Furthermore, evidence was obtained that new mRNA and de novo receptor synthesis is involved in this heterologous 1,25-(OH)2D3 receptor up-regulation.     

Regulation of type I collagen synthesis by 1,25-dihydroxyvitamin D3 in human osteosarcoma cells

Synthesis of type I and III collagens has been examined in MG-63 human osteosarcoma cells after treatment with the steroid hormone, 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3). Analysis of total [3H]proline-labeled proteins and pepsin-derived collagens revealed that 1,25-(OH)2D3 selectively stimulated synthesis of alpha 1I and alpha 2I components of type I collagen after 6-12 h. Consistent with previous reports (Franceschi, R. T., Linson, C. J., Peter, T. C., and Romano, P. R. (1987) J. Biol. Chem. 262, 4165-4171), parallel increases in fibronectin synthesis were also observed. Hormonal effects were maximal (2- to 2.5-fold versus controls) after 24 h and persisted for at least 48 h. In contrast, synthesis of the alpha 1III component of type III collagen was not appreciably affected by hormone treatment. Of several vitamin D metabolites (1,25-(OH)2D3, 25-dihydroxyvitamin D3, and 24R,25-dihydroxyvitamin D3) tested for activity in stimulating type I collagen synthesis, 1,25-(OH)2D3 was found to be the most active. Analysis of collagen mRNA abundance by Northern blot hybridization indicated that both types I and III procollagen mRNAs were increased 4-fold after a 24-h exposure to 1,25-(OH)2D3. Pro alpha 1I mRNA remained elevated through the 48-h time point while pro alpha 2I and pro alpha 1III mRNAs returned to control values. These results indicate that the regulation of collagen synthesis by 1,25-(OH)2D3 is complex and may involve changes in translational efficiency as well as mRNA abundance. 1,25-(OH)2D3 also caused at least a 20-fold increase in levels of the bone-specific calcium-binding protein, osteocalcin. These results are consistent with the hypothesis that 1,25-(OH)2D3 is stimulating partial differentiation to the osteoblast phenotype in MG-63 cells.     

Effect of 1,25-dihydroxyvitamin D3 on phospholipid metabolism in a clonal osteoblast-like rat osteogenic sarcoma cell line

The effect of 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) on phospholipid metabolism was examined in clonal rat osteogenic sarcoma cells, UMR 106, of osteoblastic phenotype. Treatment of UMR 106 cells with 10(-8)M 1,25-(OH)2D3 for 48 h caused an increase in [14C]serine incorporation into phosphatidylserine (PS) and a decrease in [3H]ethanolamine, [3H]linositol, and [14C]choline incorporation into phosphatidylethanolamine (PE), phosphatidylinositol, and phosphatidylcholine, respectively; the decrease in [3H]ethanolamine incorporation into PE was the largest. The total contents of phospholipids were similarly affected by 10(-8)M 1,25-(OH)2D3 treatment, suggesting that the effects of 1,25-(OH)2D3 are due largely to alterations in the synthesis of these phospholipids. The effects of 1,25-(OH)2D3 were evident at 10(-10) M 1,25-(OH)2D3, and 10(-8)M 1,25-(OH)2D3 caused a maximal stimulation of [14C]PS synthesis (167% of control) and a maximal reduction in the [3H]PE synthesis (41% of control). The [14C]PS/[3H]PE ratio increased gradually and reached a maximum after 70 h of treatment with 10(-8)M 1,25-(OH)2D3. When the cells were cultured in calcium-free medium containing 0.5 mM EGTA or when 5 microM cycloheximide was added to the medium, the effect of 1,25-(OH)2D3 on phospholipid metabolism was almost completely inhibited. Neither 25-hydroxyvitamin D3 nor 24,25-dihydroxyvitamin D3 caused significant changes in phospholipid metabolism. These results suggest that 1,25-(OH)2D3 alters phospholipid metabolism by enhancing PS synthesis through a calcium-dependent stimulation of the base exchange reaction of serine with other phospholipids and that the effect of 1,25-(OH)2D3 requires the synthesis of new proteins. Because PS is thought to be important for apatite formation and bone mineralization by binding calcium and phosphate to form calcium-PS-phosphate complexes, the present data suggest that 1,25-(OH)2D3 may stimulate bone mineralization by a direct effect on osteoblasts, stimulating PS synthesis.     

The effect of 1,25-dihydroxyvitamin D3 on hematopoiesis in long-term human bone marrow cultures

The modulatory effect of 1,25-dihydroxyvitamin D3 (vit D) on the growth of myeloid progenitors and on the composition of the stromal layer in human bone marrow long-term cultures was studied. Vit D (2 X 10(-8) M) caused an enhancement in myeloid progenitor cell (CFU-C) growth in the nonadherent and adherent layers during the entire 5-week incubation period. The vitamin did not alter the differentiation pattern of CFU-C (monocyte-macrophage progenitors CFU-M, granulocytic progenitors CFU-G, or monocyte-granulocyte progenitors CFU-GM). Vit D caused a marked increase in the percentage of lipid-containing cells in the adherent layer and an increase in the number of cells that specifically bound My4 monoclonal antibody (McAb), that reacted positively to fluoride-sensitive alpha-naphthyl acetate esterase, and that phagocytosed Candida albicans (CA). Concentrated supernatants harvested from control cultures showed significant levels of myeloid colony stimulating factor (CSF) activity. The addition of vit D to cultures for 5 weeks did not alter CSF levels. These results suggest that vit D may play a role in hematopoiesis by acting directly on the progenitor cells or via the stromal cell production of stimulatory factor(s).     

The effect of 1,25-dihydroxyvitamin D3 on in vitro immunoglobulin production in human B cells

1,25-Dihydroxyvitamin D3 (1,25(OH)2D3) dose-dependently suppressed immunoglobulin (Ig) production of human B cells, as evaluated by IgG-plaque-forming cells (IgG-PFC) in the culture of pokeweed mitogen (PWM)-activated B cells. Similar suppressive effect of 1,25(OH)2D3 on Ig production of B cells was observed in the Staphylococcus aureus Cowan I(SAC)-induced Ig-producing system. The mean percentage of inhibitions at a concentration of 10(-9) M were 60.0 +/- 8.2% (mean +/- SE, n = 6) and 65.1 +/- 4.7% (n = 10) in PWM- and SAC-stimulated cultures, respectively. The suppression was strongly exhibited only when 1,25(OH)2D3 was added at the start of the 6-day culture, accompanied by a decrease in DNA synthesis of B cells in both culture systems. On the other hand, the addition of 1,25(OH)2D3 on day 4, when DNA synthesis reached at plateau and IgG-PFC began to be detectable, had no noticeable affect on either the number of PFC or DNA synthesis of B cells. Furthermore, 1,25(OH)2D3 suppressed Ig production even when B cells were exposed to the agent for 4 hr after the activation with PWM or SAC, but not before the activation. These results indicate that 1,25(OH)2D3 inhibits B cell proliferation before differentiation to Ig-secreting cells, consequently reducing Ig production; and that its action appears to be mediated by the cytosol receptors expressed on activated B cells. Thus, the agent may serve as an immunoregulating hormone in vivo, as well as in vitro.     

Ligand-dependent regulation of the 1,25-dihydroxyvitamin D3 receptor in rat osteosarcoma cells

The specific binding of radiolabeled 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) to intact rat osteosarcoma (ROS 17/2) cells was followed for 24 h. In the presence of 0.5-1.5 nM 1,25(OH)2D3, hormone binding increased over a period of 12 h, from 1.1 X 10(4) to 1.3 X 10(5) receptors/cell. The elevated level of hormone binding persisted through 24 h provided that the initial concentration of hormone was maintained. The concentration dependence of this increase in receptor level was centered between 10 and 30 pM 1,25(OH)2D3, and the binding at 12 h exhibited the metabolite specificity expected for a 1,25(OH)2D3 receptor. The t 1/2 values for the disappearance of unoccupied and occupied receptors were roughly the same, approximately 2.7 h; therefore, the increase in hormone binding was not due to receptor stabilization. In comparison, hormone-receptor complexes appeared to dissociate with a t 1/2 of 1 h. alpha-Amanitin treatment reduced the magnitude of receptor accumulation by 50-60%, indicating that mRNA synthesis was required to achieve the maximal response. Ligand-dependent regulation of cellular receptor levels provides a mechanism for amplifying the primary hormonal signal and is predicted to influence the kinetics, magnitude, and dose dependence of cellular responses.     

Neonatal human foreskin keratinocytes produce 1,25-dihydroxyvitamin D3

Primary cultures of neonatal human foreskin keratinocytes converted 25-hydroxyvitamin D in high yield to a metabolite with the chromatographic behavior of 1,25-dihydroxyvitamin D3. The identity of this metabolite as 1,25-dihydroxyvitamin D3 was confirmed both by its potency in displacing 1,25-dihydroxyvitamin D3 in the chick cytosol receptor assay and by mass spectral analysis. These results suggest that 1,25-dihydroxyvitamin D3 may be formed in the epidermis to regulate vitamin D production by the epidermis and to provide an alternative to 1,25-dihydroxyvitamin D3 production by the kidneys.     

26,26,26,27,27,27-hexafluoro-1,25-dihydroxyvitamin D3: a highly potent, long-lasting analog of 1,25-dihydroxyvitamin D3

A new fluoro analog of 1,25-dihydroxyvitamin D3, i.e., 26,26,26,27,27,27-hexafluoro-1,25-dihydroxyvitamin D3, has been compared with the native hormone, 1,25-dihydroxyvitamin D3, in its biological potency, duration of action, and binding to the vitamin D transport protein and intestinal receptor protein. The fluoro analog is about 5 times more active than the native hormone in healing rickets and elevating serum inorganic phosphorus levels of rachitic rats. It is about 10 times more active than 1,25-dihydroxyvitamin D3 in increasing intestinal calcium transport and bone calcium mobilization of vitamin D-deficient rats fed a low-calcium diet. Furthermore, the higher biopotency is manifested in animals after oral dosing. Of great importance is that the action of the fluoro analog is longer lasting than that of 1,25-dihydroxyvitamin D3. This is especially apparent in the elevation of serum phosphorus and bone mineralization responses. The fluoro analog is only slightly less competent than 1,25-dihydroxyvitamin D3 in binding to the vitamin D transport protein in rat blood, and is one-third as competent as 1,25-dihydroxyvitamin D3 in binding to the chick intestinal cytosol receptor for 1,25-dihydroxyvitamin D3. These results suggest that the basis for increased potency of this analog is likely the result of less rapid metabolism.     

Effect of 1,25-dihydroxyvitamin D3 on pancreatic B and D cell function

To investigate the effects of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) on pancreatic B and D cell function in normal rats, 1 microgram of 1,25(OH)2D3 was administered intravenously 20 hours before the experiment. The plasma 1,25(OH)2D3 and calcium concentrations were significantly elevated, and plasma insulin levels also increased in 1,25(OH)2D3-administered rats compared with controls. Glucose-induced insulin and somatostatin release from the isolated pancreas perfused with lower calcium, however, was the same between the 1,25(OH)2D3-administered group and the controls. On the other hand, when the isolated pancreas was perfused with higher calcium, the glucose-induced insulin release was significantly increased in the 1,25(OH)2D3-administered group, while no significant difference in somatostatin release was observed in any group. These results suggest that the sensitivity of pancreatic B cells to glucose perfused with more calcium may increase when 1,25(OH)2D3 has been previously administered. In addition, 1,25(OH)2D3 does not seem to affect the somatostatin release from the pancreatic D cells.     

Hormonal responses to 1,25-dihydroxyvitamin D3 in cultured mouse osteoblast-like cells--modulation by changes in receptor level

The level of 1,25(OH)2D3 receptors in cultured mouse osteoblast-like (OB) cells is modulated by the rate of cell proliferation. We have studied two 1,25(OH)2D3-induced bioresponses to ascertain whether the changes in receptor levels during growth in culture alter cell responsiveness. Nuclear receptor levels were high (127 fmol/100 micrograms DNA) in rapidly dividing (log) cells and low (25 fmol/100 micrograms DNA) in quiescent (confluent) cells. The bioresponses we studied were induction of 25(OH)D3-24-hydroxylase activity (24-hydroxylase) and inhibition of collagen synthesis. The basal levels of 24-hydroxylase were low and similar in cells at log growth phase and confluence. At a maximal induction dose of 13 nM, 1,25(OH)2D3 induced a three-fold rise in enzyme activity at long growth phase, but only caused less than two-fold rise at confluence. The half-maximal dose (ED50) was slightly shifted from 0.6 nM to 0.8 nM. Daily measurement of 1,25(OH)2D3 receptor levels and maximal induction of 24-hydroxylase activity throughout the culture cycle showed a strong correlation between receptor abundance and enzyme induction. The basal level of collagen synthesized by cells in log growth phase was approximately 5% and increased to approximately 8% at confluence. Maximal inhibition of collagen synthesis by 1,25(OH)2D3 reached 80% of control levels in log cells, but was only 40% of control in confluent cells. The ED50 was approximately 0.1 nM in the log cells and increased to approximately 1 nM at confluence. Daily assay of 1,25(OH)2D3 receptor levels and 1,25(OH)2D3 responses during the culture cycle indicated a correlation between changes in receptor level and the extent of inhibition of collagen synthesis. These changes in bioresponse at various growth phases did not occur in rat OB cells where the 1,25(OH)2D3 receptor levels were independent of cell proliferation. The results indicate that cell proliferation rate, via change in receptor levels, determines the magnitude and sensitivity of the cellular responses to 1,25(OH)2D3.     

The effects of 1,25-dihydroxyvitamin D3 on human T lymphocyte activation and proliferation: a cell cycle analysis

Recent studies have demonstrated that 1,25-dihydroxyvitamin D3 (calcitriol), the most biologically active metabolite of vitamin D, is a potent inhibitor of both lectin- and antigen-driven human T lymphocyte proliferation. To better characterize this effect, we performed cell cycle analysis of both untreated and calcitriol-treated peripheral blood mononuclear cells after PHA stimulation. By using the metachromatic dye acridine orange and flow cytometry, we found that calcitriol blocks the transition from the early, low RNA compartment of G1 (G1A) to the late, higher RNA compartment of G1 (G1B). Consistent with this observation was the inability of exogenous IL 1 or phorbol myristic acetate to overcome calcitriol's suppression of DNA synthesis. Indomethacin slightly reversed calcitriol's inhibition of transition from early to late G1, suggesting a minor, prostaglandin-dependent component to calcitriol's antiproliferative activity. Finally, by using the monoclonal antibodies anti-Tac and OKT9, we found that calcitriol had no effect on IL 2 receptor expression, an early G1 event, but markedly inhibited transferrin receptor expression, an IL 2-dependent, late G1 event. Thus, analysis of calcitriol's effects on the expression of these T cell activation antigens provides further evidence of the cell cycle specificity of calcitriol's action in regulating human T lymphocyte proliferation.     

Stimulation of 1,25-dihydroxyvitamin D3 production by 1,25-dihydroxyvitamin D3 in the hypocalcaemic rat.

Serum 1,25-dihydroxyvitamin D3 concentration and renal 25-hydroxyvitamin D 1 alpha-hydroxylase activity were measured in rats fed various levels of calcium, phosphorus and vitamin D3. Both calcium deprivation and phosphorus deprivation greatly increased circulating levels of 1,25-dihydroxyvitamin D3. The circulating level of 1,25-dihydroxyvitamin D3 in rats on a low-calcium diet increased with increasing doses of vitamin D3, whereas it did not change in rats on a low-phosphorus diet given increasing doses of vitamin D3. In concert with these results, the 25-hydroxyvitamin D 1 alpha-hydroxylase activity was markedly increased by vitamin D3 administration to rats on a low-calcium diet, whereas the same treatment of rats on a low-phosphorus diet had no effect and actually suppressed the 1 alpha-hydroxylase in rats fed an adequate-calcium/adequate-phosphorus diet. The administration of 1,25-dihydroxyvitamin D3 to vitamin D-deficient rats on a low-calcium diet also increased the renal 25-hydroxy-vitamin D 1 alpha-hydroxylase activity. These results demonstrate that the regulatory action of 1,25-dihydroxyvitamin D3 on the renal 25-hydroxyvitamin D3 1 alpha-hydroxylase is complex and not simply a suppressant of this system.     

Alteration of the kinetics of type I procollagen synthesis in human osteosarcoma cells by 1,25-dihydroxyvitamin D3

The kinetics of type I procollagen synthesis in a human osteosarcoma cell line, MG 63, were investigated after treatment with 1,25-dihydroxyvitamin D₃ (1,25-(OH)₂ D₃), a hormonal inducer of phenotypic differentiation. Pulse label and chase experiments demonstrated greatly enhanced production and more rapid reduction of intracellular procollagen molecules in the 1,25-(OH)₂ D₃–treated cells as compared to the nontreated case. After a chase for 1 h, labeled procollagen was reduced by nine-tenths in 1,25-(OH)₂ D₃–treated cells, while half of the radioactivity still remained in nontreated cells. The expression rate of type I collagen, which was examined by pulse label experiment, was elevated in association with an increase in the mRNA coding for the type I collagen α1 chain by 1,25-(OH)₂ D₃ treatment. However, the amount of intracellular procollagen present after 4 h continuous labeling was almost the same, independent of the 1,25-(OH)₂ D₃ treatment. Thus, we conclude that strage of the molecule was not affected. The results therefore suggest an increase in both the synthesis and secretion of type I collagen. The 1,25-(OH)₂ D₃ treatment was also found to induce the α subunit of prolyl 4-hydroxylase and to be associated with an elevated level of hydroxyproline in the procollagen. Moreover, gelatinase B–resistant procollagen molecules, indicative of intracellular procollagen molecules in the stable triple helical form, were detected only in the 1,25-(OH)₂ D₃–treated cells. These data suggest more efficient proline hydroxylation is involved in rapid secretion of procollagen after hormone administration. The present evidence points to posttranslational control of procollagen synthesis. J. Cell. Biochem. 65:542–549. © 1997 Wiley-Liss Inc.     

Effect of 1,25-dihydroxyvitamin D3 on human cancer cells in vitro

1,25-Dihydroxyvitamin D3 (1,25(OH)2D3) dependent growth and differentiation of 6 tumor cell lines has been determined by the use of the monolayer proliferation assay. Cell lines of 4 gastro-intestinal carcinomas, 1 malignant schwannoma, and 1 malignant histiocytoma have been established and characterized. Cells were incubated for 4, 7, and 11 days in the presence of 0.8 or 8 nM 1,25(OH)2D3 and for control without addition of the hormone. Proliferation rates of 1,25(OH)2D3 treated cells were compared with cell growth in the untreated controls. Five out of 6 cell lines showed a 1,25(OH)2D3 dependent growth pattern. With 8 nM 1,25(OH)2D3 they were all inhibited. With 0.8 nM, 3 of them were inhibited at any time of the test period, whereas 1 was stimulated at day 4 and inhibited at days 7 and 11. One cell line was stimulated at days 4, 7, and 11 when incubated with 0.8 nM 1,25(OH)2D3. No striking morphological changes could be observed in the presence of 1,25(OH)2D3. We conclude that 1,25(OH)2D3 dependent cells in vitro are not necessarily growth-inhibited by this compound. Thus, 1,25(OH)2D3 is not an exclusively proliferation inhibiting agent.     

Effect of 24,25-dihydroxyvitamin D3 on 1,25-dihydroxyvitamin D3 metabolism in calcium-deficient rats.

The effect of 24,25-dihydroxyvitamin D3 [24,25(OH)2D3] on 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] metabolism was examined in rats fed on a low-calcium diet. These rats exhibit hypocalcaemia, high urinary cyclic AMP excretion, a markedly elevated serum 1,25(OH)2D concentration and low serum concentrations of both 24,25(OH)2D and 25(OH)D. When the rats are treated orally with 1, 5 or 10 micrograms of 24,25(OH)2D3/100 g every day, there is a dramatic decrease in serum 1,25(OH)2D concentration in a dose-dependent manner concomitant with an increase in serum 24,25(OH)2D concentration. Serum calcium concentration and urinary cyclic AMP excretion are not significantly affected by the 24,25(OH)2D3 treatment, which suggests that parathyroid function is not affected by the 24,25(OH)2D3 treatment. The 25(OH)D3 1 alpha-hydroxylase activity measured in kidney homogenates is markedly elevated in rats on a low-calcium diet but is not affected by any doses of 24,25(OH)2D3. In contrast, recovery of intravenously injected [3H]1,25(OH)2D3 in the serum is decreased in 24,25(OH)2D3-treated rats. Furthermore, when [3H]1,25(OH)2D3 is incubated in vitro with kidney or intestinal homogenates of 24,25(OH)2D3-treated rats there is a decrease in the recovery of radioactivity in the total lipid extract as well as in the 1,25(OH)2D3 fraction along with an increase in the recovery of radioactivity in the water-soluble phase. These results are consistent with the possibility that 24,25(OH)2D3 has an effect on 1,25(OH)2D3 metabolism, namely that of enhancing the degradation of 1,25(OH)2D3. However, because a considerable proportion of the injected 24,25(OH)2D3 is expected to be converted into 1,24,25(OH)3D3 by renal 1 alpha-hydroxylase in 24,25(OH)2D3-treated rats, at least a part of the decrease in serum 1,25(OH)2D concentration may be due to a competitive inhibition by 24,25(OH)2D3 of the synthesis of 1,25(OH)2D3 from 25(OH)D3. Thus the physiological importance of the role of 24,25(OH)2D3 in regulating the serum 1,25(OH)2D concentration as well as the mechanism and metabolic pathway of degradation of 1,25(OH)2D3 remain to be clarified.