Regulation of 25-hydroxyvitamin D3 metabolism in a human promyelocytic leukemia cell line (HL-60): 1,25-dihydroxyvitamin D3 stimulates the synthesis of 24,25-dihydroxyvitamin D3

The human promyelocytic leukemia cell line HL-60 undergoes macrophage-like differentiation after exposure to 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], the biologically active metabolite of vitamin D3. In the current study, we demonstrate that 1,25(OH)2D3 also regulates 25-hydroxyvitamin D3 [25(OH)D3] metabolism in HL-60 cells. The presence of 1,25(OH)2D3 in the culture medium of HL-60 cells stimulated the conversion of 7-10% of the substrate [25(OH)D3] to a more polar metabolite, which was identified as 24,25-dihydroxyvitamin D3 [24,25(OH)2D3] from the elution positions on sequential HPLC systems and the sensitivity to periodate treatment. The HL-60 subclone HL-60 blast, which is unresponsive to 1,25(OH)2D3 in terms of differentiation, also responded to 1,25(OH)2D3 treatment with the production of 24,25(OH)2D3. Maximal stimulation of 24,25(OH)2D3-synthesis (approximately 7 pmol/5 X 10(6) cells) in HL-60 cells was noted with a 12-h exposure to 10(-9) M 1,25(OH)2D3. The ability of vitamin D3 metabolites other than 1,25(OH)2D3 to induce the synthesis of 24,25(OH)2D3 in HL-60 cells was, with the exception of 1 alpha-hydroxyvitamin D3, in correlation with their reported affinities for the specific 1,25(OH)2D3 receptor which is present in HL-60 cells. Treatment of HL-60 cells with phorbol diesters abolished the 1,25(OH)2D3 responsiveness, while treatment with dimethylsulfoxide and interferon gamma did not markedly alter the 25(OH)D3 metabolism of HL-60 cells. Small amounts (approximately 1% of substrate) of two 25(OH)D3 metabolites, which comigrated with 5(E)- and 5(Z)-19-nor-10-keto-25-hydroxyvitamin D3 on two HPLC solvent systems, were synthesized by HL-60 cells, independently from 1,25(OH)2D3 treatment or stage of cell differentiation. Our results indicate that 1,25(OH)2D3 influences 25(OH)D3 metabolism of HL-60 cells independently from its effects on cell differentiation.     

gamma-Interferon stimulates production of 1,25-dihydroxyvitamin D3 by normal human macrophages

We show for the first time that normal human pulmonary alveolar macrophages (PAM) markedly enhance their basal rate of the production of [3H]-1,25(OH)2D3 when cultured in the presence of recombinant gamma-interferon (gamma-IFN). The rate of conversion of [3H]-25(OH)D3 to [3H]-1,25(OH)2D3 was dose-dependent in a linear fashion. A maximal production of 1,25(OH)2D3 by PAM occurred after exposure of PAM to gamma-IFN for one day. This maximum plateau-level was sustained for at least five days. The authenticity of the putative 1,25(OH)2D3 obtained from PAM was tested by demonstrating the exact comigration of [3H]-1,25(OH)2D3 with chemically synthesized 1,25(OH)2D3 in four different HPLC-systems.     

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.     

Metabolism of 25-hydroxyvitamin D3 to 24,25-dihydroxyvitamin D3 by blood derived macrophages from a patient with alveolar rhabdomyosarcoma during short-term culture and 1 alpha,25-dihydroxyvitamin D3 after long-term culture

We have examined the ability of blood-derived monocytes and macrophages isolated from a patient with alveolar rhabdomyosarcoma and hypercalcaemia, to form 24,25-dihydroxyvitamin D3 (24,25(OH)2D3) or 1 alpha,25-dihydroxyvitamin D3 (1 alpha,25(OH)2D3) from 25-hydroxyvitamin D3 (25(OH)D3). Adherent monocyte-macrophage cells incubated with 25(OH)D3 over the initial 2 days in culture synthesized 1.9 pmol 24,25(OH)2D3/h/incubation (representing 0.63 pmol/h/10(6) cells), whereas macrophages synthesized 1.03 and 1.15 pmol 1 alpha,25(OH)2D3/h/incubation after 1 and 4 weeks in culture respectively. In a further experiment synthesis of 1 alpha,25(OH)2D3 by long-term cultured macrophages fell from 2.25 to 0.04 pmol/h/incubation following exposure to 10 nM 1 alpha,25(OH)2D3 for 7 days, whereas 24,25(OH)2D3 synthesis was induced (0.46 pmol/h/incubation). The vitamin D3 metabolites were identified by co-chromatography with authentic 24,25(OH)2D3 or 1 alpha,25(OH)2D3 in three different high-performance liquid chromatography systems. Serum 1 alpha,25(OH)2D3 in the patient was markedly suppressed at 5 pg/ml (normal 20-50 pg/ml) indicating that raised 1 alpha,25(OH)2D3 was not the cause of the hypercalcaemia, but rather, that raised calcium may have suppressed renal 1 alpha,25(OH)2D3 synthesis. Administration of APD (3-amino-1-hydroxypropylidine-1,1-bisphosphonate) corrected the hypercalcaemia in the patient suggesting that increased bone resorption was responsible for the raised calcium. The results of this study show for the first time that immature blood derived monocyte-macrophage cells can synthesize 24,25(OH)2D3 before they mature into macrophages able to synthesize 1 alpha,25(OH)2D3.     

24,25-dihydroxyvitamin D3 binds to catalase

There is increasing evidence that the vitamin D metabolite, 24,25-dihydroxyvitamin D3 (24,25(OH)2D3) has endocrine actions. In the current work, we report that an endogenous binding protein for 24,25(OH)2D3 is catalase, based on sequence analysis of the isolated protein. An antibody (Ab 365) generated against equivalent protein recognized bovine catalase and a 64 kDa band in subcellular fractions of chick intestine. A commercially available anti-catalase antibody reduced specific [3H]24,25(OH)2D3 binding in subcellular fractions of chick intestine by greater than 65%, relative to the same fractions treated with an unrelated antibody (Ab 099). The same commercially available anti-catalase was able to block the inhibitory actions of 24,25(OH)2D3 on 32P uptake in isolated intestinal epithelial cell suspensions. We subsequently characterized binding of steroid to commercially available catalase, and found that between 0 and 5 nM of enzyme added to subcellular fraction P2 (20,000g, 10-min post-nuclear pellet) resulted in a linear increase in the amount of [3H]24,25(OH)2D3 specifically bound. Additional studies indicated that 25(OH)D3 was an effective competitor for binding, whereas 1,25(OH)2D3 only poorly displaced [3H]24,25(OH)2D3. Saturation analyses with added catalase yielded a physiologically relevant affinity constant (KD=5.6+/-2.7 nM) and a Bmax=209+/-34 fmols/mg protein, comparable to previous studies using purified basal lateral membranes or vesicular fractions. Moreover, in a study on subcellular fractions isolated from chickens of varying ages, we found that in females, both specific [3H]24,25(OH)2D3 binding and catalase activity increased from 7- to 58-week-old birds, whereas in males, elevated levels of both parameters were expressed in preparations of 7- and 58-week-old birds. The data suggest that signal transduction may occur through modulation of hydrogen peroxide production.     

Contributions of pro-oxidant and anti-oxidant conditions to the actions of 24,25-dihydroxyvitamin D3 and 1,25-dihydroxyvitamin D3 on phosphate uptake in intestinal cells

The steroid hormone 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] rapidly stimulates the uptake of phosphate in isolated chick intestinal cells, while the steroid 24,25-dihydroxyvitamin D3 [24,25(OH)2D3] inhibits the rapid stimulation by 1,25(OH)2D3. Earlier work in this laboratory has indicated that a cellular binding protein for 24,25(OH)2D3 is the enzyme catalase. Since binding resulted in decreased catalase activity and increased H2O2 production, studies were undertaken to determine if pro-oxidant conditions mimicked the inhibitory actions of 24,25(OH)2D3, and anti-oxidant conditions prevented the inhibitory actions of 24,25(OH)2D3. An antibody against the 24,25(OH)2D3 binding protein was found to neutralize the inhibitory effect of the steroid on 1,25(OH)2D3-mediated 32P uptake. Incubation of cells in the presence of 50 nM catalase was also found to alleviate inhibition. In another series of experiments, isolated intestinal epithelial cells were incubated as controls or with 1,25(OH)2D3, each in the presence of the catalase inhibitor 3-amino-1,2,4-triazole, or with 1,25(OH)2D3 alone. Cells exposed to hormone alone again showed an increased accumulation of 32P, while cells treated with catalase inhibitor and hormone had uptake levels that were indistinguishable from controls. We tested whether inactivation of protein kinase C (PKC), the signaling pathway for 32P uptake, occurred. Incubation of cells with phorbol-13-myristate (PMA) increased 32P uptake, while cells pretreated with 50 microM H2O2 prior to PMA did not exhibit increased uptake. Likewise, PMA significantly increased PKC activity while cells exposed to H2O2 prior to PMA did not. It is concluded that catalase has a central role in mediating rapid responses to steroid hormones.     

1,25-dihydroxyvitamin D-3 and 24,25-dihydroxyvitamin D-3 affect parathormone (PTH) -sensitive adenylate cyclase activity and alkaline phosphatase secretion of osteoblastic cells through different mechanisms of action

In UMR 106 rat osteosarcoma cells, parathormone (1-34hPTH) and calcitonin (sCT) stimulated adenylate cyclase (AC) activity 5.5-and 2.8-fold, respectively. AC in osteoblasts (OB) from collagenase-treated calvaria of 3-day-old rats responded similarly to 1-34hPTH. In contrast, fibroblasts (mouse fibroblastomas) displayed a marginal 1-34hPTH sensitive AC. Osteoclasts (OC) of collagenase-treated rat calvariae, rat monocytes and mouse macrophages did not demonstrate 1-34hPTH inducable AC activity. Physiological concentrations of 24,25-dihydroxyvitamin D-3 attenuated PTH-sensitive AC in OB and UMR 106 cells within 20 min, while 1,25-dihydroxyvitamin D-3 showed no such immediate effect. In contrast, the AC response to Gpp(NH)p was unaffected by 24,25-(OH)2D3, indicating that 24,25-(OH)2D3 interrupts the coupling of the PTH receptor to the GTP binding protein Gs. OB and UMR 106 cells were also subjected to long-term (48 h) incubation with vitamin D-3 metabolites, 1-34hPTH or 20% serum from patients with secondary hyperparathyroidism (sHBT-serum), respectively. PTH-sensitive AC was markedly attenuated by pre-exposure to both 1-34hPTH and 1,25-(OH)2D3, while minimally affected by corresponding 24,25-(OH)2D3 and 20% sHPT-serum treatment. The secretion of alkaline phosphatase (Alphos) from the two cell types was strongly increased by 1-34hPTH, the effect being abolished by the presence of 24,25-(OH)2D3. Iliac crest biopsies of normal individuals exhibited a clear negative correlation between PTH-sensitive AC and corresponding serum 24,25-(OH)2D3 levels. Basal AC activity was, however, negatively correlated to serum 1,25-(OH)2D3 concentrations. In summary, the results show that 24,25-(OH)2D3 reduces PTH-stimulated AC activity in and Alphos secretion from osteoblastic bone cells by rapidly and directly interfering with the plasma membrane. These data reinforce the probable in vivo significance of 24,25-(OH)2D3. Moreover, the negative correlation between basal AC activity and serum 1,25-(OH)2D3 levels indicates a possible role for 1,25-(OH)2D3 in regulating bone cell synthesis of AC components in vivo.     

The mechanism of 1,25-dihydroxyvitamin D(3) autoregulation in keratinocytes

The synthesis of 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) from its precursor, 25-dihydroxyvitamin D(3) (25(OH)D(3)), is catalyzed by the mitochondrial cytochrome P450 enzyme 25-hydroxyvitamin D(3)-1alpha-hydroxylase (1alpha-hydroxylase). It has been generally assumed that 1,25(OH)(2)D(3) inhibits the activity of this enzyme by regulating its expression at the genomic level. We confirmed that 1,25(OH)(2)D(3) reduced the apparent conversion of 25(OH)D(3) to 1,25(OH)(2)D(3) while stimulating the conversion of 1,25(OH)(2)D(3) and 25(OH)D(3) to 1,24,25(OH)(3)D(3) and 24,25(OH)(2)D(3), respectively. However, 1,25(OH)(2)D(3) failed to reduce the abundance of its mRNA or its encoded protein in human keratinocytes. Instead, when catabolism of 1,25(OH)(2)D(3) was blocked with a specific inhibitor of the 25-hydroxyvitamin D(3)-24-hydroxylase (24-hydroxylase) all apparent inhibition of 1alpha-hydroxylase activity by 1,25(OH)(2)D(3) was reversed. Thus, the apparent reduction in 1alpha-hydroxylase activity induced by 1,25(OH)(2)D(3) is due to increased catabolism of both substrate and product by the 24-hydroxylase. We believe this to be a unique mechanism for autoregulation of steroid hormone synthesis.     

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.     

Specific binding of 24R,25-dihydroxyvitamin D3 to chick intestinal mucosa: 24R,25-dihydroxyvitamin D3 is an allosteric effector of 1,25-dihydroxyvitamin D3 binding

We have previously described a significant decrease in the positive cooperativity level and affinity of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] binding to its chick intestinal chromatin receptor induced in vitro by a physiological 10-fold molar excess of (24R)-25-dihydroxyvitamin D3 [24R,25(OH)2D3] [F. Wilhelm and A. W. Norman (1985) Biochem. Biophys. Res. Commun. 126, 496-501]. In this report, we have initiated a comparative study of the binding of 24R,25(OH)2[3H]D3 and 1,25(OH)2[3H]D3 to the the intestinal chromatin fraction obtained from vitamin D-replete birds. 24R,25(OH)2[3H]D3 specific binding to this chromatin fraction was characterized by a dissociation constant (Kd) of 34.0 +/- 6.4 nM, a positive cooperativity level (nH) of 1.40 +/- 0.13, and a capacity (Bmax) of 47 +/- 8 fmol/mg protein. The very low relative competitive index (RCI) of 24R,25(OH)2D3 (0.11 +/- 0.03%) for the 1,25(OH)2D3 binding site/receptor, as well as the inability of 1,25(OH)2D3 to displace 24R,25(OH)2D3 from its binding site at a physiological molar ratio of 1:10, strongly suggest the independence of 24R,25(OH)2D3 and 1,25(OH)2D3 binding sites. Stereospecificity of the 24R,25(OH)2D3 binding sites was attested by the displacement of only 45 +/- 6% of 24R,25(OH)2D3 specific binding by equimolar concentrations of 24S,25(OH)2D3. Collectively these results suggest the existence of a binding domain/receptor for 24,25(OH)2D3 in the chick intestine which is independent of the 1,25(OH)2D3 receptor.     

Determination of vitamin D and its metabolites in plasma from normal and anephric man

A multiple assay capable of reliably determining vitamins D(2) and D(3) (ergocalciferol and cholecalciferol), 25(OH)D(2) (25-hydroxyvitamin D(2)) and 25(OH)D(3) (25-hydroxyvitamin D(3)), 24,25(OH)(2)D (24,25-dihydroxyvitamin D), 25,26(OH)(2)D (25,26-dihydroxyvitamin D) and 1,25(OH)(2)D (1,25-dihydroxyvitamin D) in a single 3-5ml sample of human plasma was developed. The procedure involves methanol/methylene chloride extraction of plasma lipids followed by separation of the metabolites and purification from interfering contaminants by batch elution chromatography on Sephadex LH-20 and Lipidex 5000 and by h.p.l.c. (high-pressure liquid chromatography). Vitamins D(2) and D(3) and 25(OH)D(2) and 25(OH)D(3) are quantified by h.p.l.c. by using u.v. detection, comparing their peak heights with those of standards. 24,25(OH)(2)D and 25,26(OH)(2)D are measured by competitive protein-binding assay with diluted plasma from vitamin D-deficient rats. 1,25(OH)(2)D is measured by competitive protein-binding assay with diluted cytosol from vitamin D-deficient chick intestine. Values in normal human plasma samples taken in February are: vitamin D 3.5+/-2.5ng/ml; 25(OH)D 31.6+/-9.3ng/ml; 24,25(OH)(2)D 3.5+/-1.4ng/ml; 25,26(OH)(2)D 0.7+/-0.5ng/ml; 1,25(OH)(2)D 31+/-9pg/ml (means+/-s.d.). Values in two normal human plasma samples taken in February after 1 week of high sun exposure are: vitamin D 27.1+/-7.9ng/ml; 25(OH)D 56.8+/-4.2ng/ml; 24,25(OH)(2)D 4.3+/-1.6ng/ml; 25,26(OH)(2)D 0.5+/-0.2ng/ml. Values in anephric-human plasma are: vitamin D 2.7+/-0.8ng/ml; 25(OH)D 36.4+/-16.5ng/ml; 24,25(OH)(2)D 1.9+/-1.3ng/ml; 25,26(OH)(2)D 0.6+/-0.3ng/ml; 1,25(OH)(2)D was undetectable.     

Effects of 24,25(OH)2D3, 1,25(OH)2D3 and 25(OH)D3 on alkaline and tartrate-resistant acid phosphatase activities in fetal rat calvaria

The aim of this work was to evaluate the effects of 24,25-dihydroxyvitamin D3, 24,25(OH)2D3, on alkaline phosphatase (AP) and tartrate-resistant acid phosphatase (TRAP) activities in fetal rat calvaria cultures. These actions were compared with those of 1,25-dihydroxyvitamin D3, 1,25(OH)2D3, and 25-hydroxyvitamin D3, 25(OH)D3, in similar experimental conditions. At 10 min, 30 min and at 24 h incubation time, 1,25(OH)2D3 (10(-10)M) and 25(OH)D3 (10(-7) M) produced a significant increase in AP and TRAP activities compared to control group (without vitamin D metabolites). However, 24,25(OH)2D3 (10(-7) M) only produced effects on phosphatase activities similar to those produced by 1,25(OH)2D3 and 25(OH)D3, after 24 h incubation time. These findings suggest that 1,25(OH)2D3 and 25(OH)2D3 could carry out actions in minutes (nongenomic mechanism), while 24,25(OH)2D3 needs longer periods of time to perform its biological actions (genomic mechanism).     

Regulation of rat yolk sac 25-hydroxy- and 1,25-dihydroxyvitamin D3 24-hydroxylase activities

The yolk sac of the pregnant rat which functions as a true placenta is a target organ for vitamin D. This tissue can hydroxylate in position 24 both 25-hydroxy- and 1,25-dihydroxyvitamin D3 (25-OHD3 and 1,25-(OH)2D3). The present report describes an in vitro model for the study of 1,25-(OH)2D3 action on the further metabolism of 25-OH[3H]D3 and 1,25-(OH)2[3H]D3 by yolk sac. The tissue explants were preincubated with 1,25-(OH)2D3 for 18 h in a serum-free culture medium. Physiological concentrations of 1,25-(OH)2D3 were the most effective in stimulating (7.5-fold) the 1,25-(OH)2D3 24-hydroxylase, while the 25-OHD3 24-hydroxylase stimulation (4-fold) required a 1,25-(OH)2D3 concentration of 10(-7) M. The stimulating effect of 1,25-(OH)2D3 on the 1,25-(OH)2D3 24-hydroxylase was temperature-dependent, and, since its was inhibited by actinomycin D and cycloheximide, required de novo protein synthesis. 1,24,25-(OH)3D3, 25-OHD3, and 24,25-(OH)2D3 were 10- to 1000-fold less potent than 1,25-(OH)2D3 in inducing the 1,25-(OH)2D3 hydroxylase. Our results strongly suggest that 1,25-(OH)2D3 regulated the 1,25-(OH)2D3 24-hydroxylase by a receptor-mediated process. Furthermore, 1,25-(OH)2D3 at 10(-9) M induced within 4 h an increase of its own degradation and the formation of an as yet unidentified major 1,25-(OH)2[3H]D3 metabolite. We conclude that the yolk sac can participate in the regulation of 1,25-(OH)2D3 concentration in the fetoplacental unit.     

Side-chain oxidation of vitamin D3 in mouse kidney mitochondria: effect of the Hyp mutation and 1,25-dihydroxyvitamin D3 treatment

Side-chain oxidation of vitamin D is an important degradative pathway. In the present study we compared the enzymes involved in side-chain oxidation in normal and Hyp mouse kidney. Homogenates of normal mouse kidney catalyze the conversion of 25-hydroxyvitamin D3 to 24,25-dihydroxyvitamin D3, 24-oxo-25-hydroxyvitamin D3 and 24-oxo-23,25-dihydroxyvitamin D3. After subcellular fractionation, total side-chain oxidative activity, estimated by the sum of the three products synthesized per milligram protein under initial rate conditions, coincided with the mitochondrial enzyme marker succinate-cytochrome-c reductase. Treatment of normal mice with 1,25-dihydroxyvitamin D3 (1.5 ng/g) resulted in an eightfold increase in mitochondrial enzyme activity, with no change in apparent Km but a significant rise in Vmax. With 24,25-dihydroxyvitamin D3 as the substrate, normal renal mitochondria produced 24-oxo-25-hydroxyvitamin D3 and 24-oxo-23,25-dihydroxyvitamin D3, and the synthesis of these metabolites could be increased sixfold by pretreatment with 1,25-dihydroxyvitamin D3. In the Hyp mouse, the side-chain oxidation pathway showed similar subcellular distribution of enzyme activity. However, product formation from 25-hydroxyvitamin D3 and 24,25-dihydroxyvitamin D3 was twofold greater in mutant than in normal mitochondria. Furthermore, 1,25-dihydroxyvitamin D3 pretreatment of Hyp mice resulted in a 3.4-fold increase over basal metabolism of both 25-hydroxyvitamin D3 and 24,25-dihydroxyvitamin D3. These results demonstrate that (i) kidneys from normal and Hyp mice possess basal and 1,25-dihydroxyvitamin D3 inducible enzyme system(s) in the mitochondrial fraction, which catalyze the side-chain oxidation of 25-hydroxyvitamin D3 and 24,25-dihydroxyvitamin D3, and (ii) the Hyp mutation appears to perturb the renal metabolism of both substrates only in the basal state.     

Demonstration of 1 alpha,25-dihydroxyvitamin D3 receptor-like molecule in ST 13 and 3T3 L1 preadipocytes and its inhibitory effects on preadipocyte differentiation

The active metabolite of vitamin D3, 1 alpha,25-dihydroxyvitamin D3 (1,25(OH)2D3), inhibited morphologic and enzymatic expression during differentiation of preadipocyte to adipocyte. In the presence of approximately 6.4-20 X 10(-10) M 1,25(OH)2D3, the triacylglycerol accumulation was only 50% of that of fully differentiated control cells. High-affinity binding sites for 1,25-dihydroxyvitamin D3 were detected in two preadipose cell lines. The 1,25(OH)2D3 binding component sediments at 3.3 S in 4-24% (w/v) sucrose gradients prepared in hypertonic buffer. Binding assay revealed that Nmax was 70 fmol/mg protein and 90 fmol/mg protein, and Kd value was 170 pM and 37 pM in cell lines ST 13 and 3T3 L1, respectively. We also found that differentiated adipocytes did not contain specific receptors for 1,25(OH)2D3. 1,25(OH)2D3, 1(OH)D3, 24,25(OH)2D3, and 24(OH)D3 all suppressed differentiation of preadipocytes to adipocytes, and the dose required closely reflected the affinities of the various metabolites and the synthetic derivative for 1,25(OH)2D3 receptor. It is suggested that the action of vitamin D3 on preadipocyte differentiation may result from a receptor-mediated event.     

On the specificity of vitamin D compounds binding to chick pig intestinal 1,25-dihydroxyvitamin D3 receptor

The binding activity of four vitamin D metabolites and/or analogs for the intestinal 1,25-dihydroxyvitamin D3 receptor was evaluated after incubation at 25 degrees C for 1 h or at 0-4 degrees C for 18 h. The incubation conditions, which had no effect on the binding of 1,25-dihydroxyvitamin D3, had a dramatic effect on the binding of 25-hydroxyvitamin D3 and 1 alpha-hydroxyvitamin D3 and a small but reproducible effect on 24,25-dihydroxyvitamin D3 binding to receptor. Affinities 10- to 20-fold higher were obtained for 25-hydroxyvitamin D3 and 1 alpha-hydroxyvitamin D3, and affinities 3-fold higher were obtained for 24,25-dihydroxyvitamin D3 at the 0-4 degrees C/18-h incubation. A comparison of intestinal receptor from chick and pig with nine vitamin D compounds showed no major differences between the two species. The relative affinity of the vitamin D analogs to compete with tritiated 1,25-dihydroxyvitamin D3 for the receptor in pig nuclear extract, expressed as ratios of the molar concentration required for 50% binding of the tritiated 1,25-dihydroxyvitamin D3 compared to nonradioactive 1,25-dihydroxyvitamin D3, are as follows: 1,25-dihydroxyvitamin D3 (1) = 1,25-dihydroxyvitamin D2 = 24-homo-1,25-dihydroxyvitamin D3 greater than 1,24,25-trihydroxyvitamin D3 (4) greater than 25-hydroxyvitamin D3 (21) = 10-oxo-19-nor-25-hydroxyvitamin D3 = 1 alpha-hydroxyvitamin D3 (37) greater than 24,25-dihydroxyvitamin D2 (257) much much greater than vitamin D3 (greater than 10(6)).     

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.     

Differential effects of 1,25-dihydroxyvitamin D3 on human lymphocytes and monocyte/macrophages: inhibition of interleukin-2 and augmentation of interleukin-1 production

Human peripheral blood monocytes and activated, but not resting, lymphocytes possess specific intracellular receptors for the active metabolite of vitamin D3, 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3). The effects of 1,25-(OH)2D3 on the function of these cells was therefore examined. The addition of physiologic concentrations of the hormone (0.001-0.1 nM) to lectin- or antigen-activated lymphocytes resulted in inhibition of lymphocyte proliferation. Supernatants from lectin-activated lymphocytes incubated with 1,25-(OH)2D3 had reduced interleukin-2 (IL-2) activity. The immediate biological precursor of 1,25-(OH)2D3, 25-hydroxyvitamin D3, did not affect function of lymphocytes or monocytes. The ability of exogenous recombinant IL-2 to reverse the inhibitory effects of the hormone on lymphocyte proliferation suggest that 1,25-(OH)2D3 does not alter the generation of IL-2 receptors. In contrast to its effects on IL-2 production, 1,25-(OH)2D3 caused a dose-dependent increase in the production of interleukin-1 (IL-1) by monocyte/macrophages. These results suggest that immune cells and their products can be regulated in a specific but diverse fashion by the vitamin D3-endocrine system.     

Increased placental concentrations of 25-hydroxyvitamin D3 and 24,25-dihydroxyvitamin D3 in pregnant rats treated with human chorionic gonadotropin

Pregnant rats were injected intrajugularly with 2500 i.u. human chorionic gonadotropin (HCG) toward the end of gestation (days 18-19) and 7.0 pmoles of tritiated 25-hydroxyvitamin D3 [( 3H]25(OH)D3) the following day. They were sacrificed ten to 24 hours later. [3H]25(OH)D3 and the in vivo produced [3H]24,25-dihydroxyvitamin D3 [( 3H]24,25(OH)2D3) in lipid extracts from maternal serum, kidneys, placenta and fetal tissues were separated by Sephadex LH-20 chromatography, and high performance liquid chromatography (HPLC). HCG treatment of pregnant rats increased significantly 25(OH)D3 levels in the placenta and kidneys and 24,25(OH)2D3 level in the placenta. Fetal metabolites levels were unaffected by HCG treatment. Serum and kidney levels of 25(OH)D3 and 24,25(OH)2D3 in pregnant rats were significantly lower than in non-pregnant rats. Serum and kidney levels of both metabolites in non-pregnant female rats treated with HCG did not differ from the untreated controls. HCG may, therefore, be involved in regulation of fetoplacental vitamin D metabolism.     

Responses of rachitic cartilage cells to metabolites of vitamin D3

Responses of cultured cartilage cells to metabolites of vitamin D3 were studied. Cells were obtained from the epiphyseal growth plate of rachitic chicks and were exposed to physiological and pharmacological concentrations of three metabolites of vitamin D3, 25 hydroxyvitamin D3 (25(OH)D3), 24,25-dihydroxyvitamin D3 (24,25(OH)2D3) and 1,25-dihydroxyvitamin D3 (1,25(OH)2D3). 1,25(OH)2D3 was found to reduce L-[U-14C]leucine incorporation into proteins and Na2 35SO4 incorporation into proteoglycans. The synthesis of 24,25(OH)2D3 from 25(OH)D3 was stimulated upon addition of 1,25(OH)2D3 to the cultures. Physiological concentrations of 24,25(OH)2D3 stimulated protein and proteoglycan synthesis. These findings support the notion that vitamin D3, through its active dihydroxylated metabolites, is directly involved in cartilage cells metabolism and healing of rickets.