Synthesis and biological evaluation of a 3-positon epimer of 1alpha,25-dihydroxy-2beta-(3-hydroxypropoxy)vitamin D3 (ED-71)

1alpha,25-Dihydroxy-2beta-(3-hydroxypropoxy)vitamin D(3) (ED-71), an analog of active vitamin D(3), 1alpha,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)], possesses a hydroxypropoxy substituent at the 2beta-position of 1,25(OH)(2)D(3). ED-71 has potent biological effects on bone and is currently under phase III clinical studies for bone fracture prevention. It is well-known that the synthesis and secretion of parathyroid hormone (PTH) is regulated by 1,25(OH)(2)D(3). Interestingly, during clinical development of ED-71, serum intact PTH in osteoporotic patients did not change significantly upon treatment with ED-71. The reason remains unclear, however. Brown et al. reported that 3-epi-1,25(OH)(2)D(3), an epimer of 1,25(OH)(2)D(3) at the 3-position, shows equipotent and prolonged activity compared to 1,25(OH)(2)D(3) at suppressing PTH secretion. Since ED-71 has a bulky hydroxypropoxy substituent at the 2-position, epimerization at the adjacent and sterically hindered 3-position might be prevented, which may account for its weak potency in PTH suppression observed in clinical studies. We have significant interest in ED-71 epimerization at the 3-position and the biological potency of 3-epi-ED-71 in suppressing PTH secretion. In the present studies, synthesis of 3-epi-ED-71 and investigations of in vitro suppression of PTH using bovine parathyroid cells are described. The inhibitory potency of vitamin D(3) analogs were found to be 1,25(OH)(2)D(3)>ED-71> or =3-epi-1,25(OH)(2)D(3)>3-epi-ED-71. ED-71 and 3-epi-ED-71 showed weak activity towards PTH suppression in our assays.     

Synthesis and biological evaluation of a 3-positon epimer of 1α,25-dihydroxy-2β-(3-hydroxypropoxy)vitamin D3 (ED-71)

1α,25-Dihydroxy-2β-(3-hydroxypropoxy)vitamin D₃ (ED-71), an analog of active vitamin D₃, 1α,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃], possesses a hydroxypropoxy substituent at the 2β-position of 1,25(OH)₂D₃. ED-71 has potent biological effects on bone and is currently under phase III clinical studies for bone fracture prevention. It is well-known that the synthesis and secretion of parathyroid hormone (PTH) is regulated by 1,25(OH)₂D₃. Interestingly, during clinical development of ED-71, serum intact PTH in osteoporotic patients did not change significantly upon treatment with ED-71. The reason remains unclear, however. Brown et al. reported that 3-epi-1,25(OH)₂D₃, an epimer of 1,25(OH)₂D₃ at the 3-position, shows equipotent and prolonged activity compared to 1,25(OH)₂D₃ at suppressing PTH secretion. Since ED-71 has a bulky hydroxypropoxy substituent at the 2-position, epimerization at the adjacent and sterically hindered 3-position might be prevented, which may account for its weak potency in PTH suppression observed in clinical studies. We have significant interest in ED-71 epimerization at the 3-position and the biological potency of 3-epi-ED-71 in suppressing PTH secretion. In the present studies, synthesis of 3-epi-ED-71 and investigations of in vitro suppression of PTH using bovine parathyroid cells are described. The inhibitory potency of vitamin D₃ analogs were found to be 1,25(OH)₂D₃ > ED-71 ≥ 3-epi-1,25(OH)₂D₃  3-epi-ED-71. ED-71 and 3-epi-ED-71 showed weak activity towards PTH suppression in our assays.     

Regulatory activities of 2 beta-(3-hydroxypropoxy)-1 alpha, 25-dihydroxyvitamin D3, a novel synthetic vitamin D3 derivative, on calcium metabolism

Regulatory activities of 2 beta-(3-hydroxypropoxy)-1 alpha, 25-dihydroxyvitamin D3 [ED-71], a novel synthetic vitamin D3 derivative, on calcium metabolism were investigated. The compound behaved similar to 1 alpha, 25-dihydroxyvitamin D3 [1,25(OH)2D3] in the ex vivo intestinal calcium transport using rat everted gut sac and the in vivo bone mobilization using vitamin D-deficient rats. By means of Raisz's assay method, 45Ca releasing activity of ED-71 was not greater than that of 1,25(OH)2D3. The time course curve of ED-71 in plasma made a mild round shape compared with that of 1,25(OH)2D3 and the former's plasma concentration remained increased longer than the latter's. The therapeutic effect of ED-71 for the animal models with osteoporosis seemed to be better than that of 1,25(OH)2D3. The results suggest that ED-71 may be a promising drug for therapy of osteoporosis.     

Synthesis of putative metabolites of 1alpha,25-dihydroxy-2beta-(3-hydroxypropoxy)vitamin D(3) (ED-71)

1alpha,25-Dihydroxy-2beta-(3-hydroxypropoxy)vitamin D(3) (ED-71), an analog of active vitamin D(3), 1alpha,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] is under phase III clinical trials in Japan for the treatment of osteoporosis and bone fracture prevention. Since ED-71 has a substituent at the 2beta-position of the A-ring, it is recognized that the metabolic pathway of ED-71 might be more complicated than 1,25(OH)(2)D(3) because of metabolism at the 2beta-position substituent in addition to the inherent metabolism of the side chain. To clarify the metabolism of hydroxypropoxy substituent of the 2beta-positon and a combination of metabolism between side chain and 2beta-positon, four putative metabolites of ED-71 have been prepared as authentic samples. The metabolites at the 2beta-positon, the methyl ester derivative considered as an ester standard of the oxidized metabolite and the tetraol derivative as the truncated metabolite were synthesized from alpha-epoxide, a key intermediate of ED-71 synthesis. The combination metabolites between side chain and 2beta-positon, the 24(S)- and 24(R)-pentaols were synthesized using Trost's convergent method.     

The effect of active vitamin D3 analogs and dexamethasone on the expression of osteocalcin gene in rat tibiae in vivo.

We tested the effects of 1 alpha,25-dihydroxyvitamin D3 (1,25-(OH)2D3), 2 beta-(3-hydroxypropoxy)-1 alpha,25-dihydroxyvitamin D3 (ED-71) and dexamethasone on osteocalcin mRNA levels in rat tibiae in vivo. Northern blot analysis showed that both 1,25-(OH)2D3 and ED-71 caused an increase in osteocalcin mRNA levels in bone: 1,25-(OH)2D3 induced a transient increase in the mRNA levels followed by a decrease in the control level by 12 h post administration. In contrast, ED-71 caused a persistent increase in osteocalcin mRNA level for seven days post administration. Serum osteocalcin levels paralleled the osteocalcin mRNA level in bone in both groups. Dexamethasone caused a marked reduction in both osteocalcin mRNA and serum osteocalcin levels. Suppressive effect of dexamethasone on osteocalcin expression was persistent for seven days at higher dose. Our results represent the first demonstration of the effect of active vitamin D and corticosteroid on the expression of osteocalcin mRNA in bone in vivo.     

ED-71, a new active vitamin D3, increases bone mineral density regardless of serum 25(OH)D levels in osteoporotic subjects

A previous randomized placebo-controlled double-blinded clinical trial revealed that treatment of osteoporotic subjects supplemented with 200 or 400 IU/day vitamin D3 with 0.75 μg/day ED-71 for 12 months increased lumbar and hip bone mineral density (BMD) by 3.4 and 1.5%, respectively, compared to placebo group (JCE&M 90:5031,2005). These effects on BMD were stronger than any previous results using 1(OH)D3 or 1,25(OH)2D3. However, there still was a concern that the effect of ED-71 could be observed because serum 25(OH)D in many of these subjects were below its optimal level. In order to address this issue, we performed post hoc analysis to compare the effect of ED-71 on lumbar and hip BMD between subjects with upper (>29 ng/mL) and lower tertiles (<25 ng/mL) of serum 25(OH)D. Lumbar BMD after 12-month treatment with 0.5, 0.75 and 1.0 μg/day ED-71 increased similarly in both lower and upper tertile groups of serum 25(OH)D. In addition, hip BMD also showed a tendency to increase when 0.75 and 1.0 μg/day ED-71 groups were combined together in both upper and lower serum 25(OH)D tertile groups, although the increase was not statistically significant. These results demonstrate that the effect of ED-71 on bone is independent of supplementary effect for nutritional vitamin D insufficiency, and suggest that ED-71 may exert its effect as a unique VDR ligand with stronger effect on bone compared to the natural ligand, 1,25(OH)2D3.     

Mechanistic studies to evaluate the enhanced antiproliferation of human keratinocytes by 1alpha,25-dihydroxyvitamin D(3)-3-bromoacetate, a covalent modifier of vitamin D receptor, compared to 1alpha,25-dihydroxyvitamin D(3).

1alpha,25-Dihydroxyvitamin D(3)-3-bromoacetate (1, 25(OH)(2)D(3)-3-BE), an affinity labeling analog of 1alpha, 25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)), displayed stronger antiproliferative activities than 1,25(OH)(2)D(3) at 10(-10)-10(-6) M dose levels in cultured human keratinocytes (CHK). Additionally, preincubation of the cells with 10(-6) M 1,25(OH)(2)D(3), followed by treatment with various doses of 1,25(OH)(2)D(3)-3-BE, resulted in a significantly stronger antiproliferative activity by the mixture than individual reagents at every dose level. To search for a mechanism of this observation, we determined that [(14)C]1, 25(OH)(2)D(3)-3-BE covalently labeled human recombinant 1alpha, 25-dihydroxyvitamin D(3) receptor (reVDR) swiftly (<1 min) with a 1:1 stoichiometry and induced conformational changes (in VDR) that are different from 1,25(OH)(2)D(3), by limited tryptic digestion. Furthermore, a protein band, corresponding to reVDR, was specifically labeled by [(14)C]1,25(OH)(2)D(3)-3-BE in CHK extract, indicating that VDR is the main target of [(14)C]1, 25(OH)(2)D(3)-3-BE. The above-mentioned observations suggest that a rapid covalent labeling of VDR in CHK might alter the interaction between the holo-VDR and 1,25(OH)(2)D(3)-controlled genes. Furthermore, we observed that 1,25(OH)(2)D(3)-3-BE significantly decreased the binding of VDR to human osteocalcin vitamin D responsive element (hOCVDRE), as well as the dissociation rate of VDR from hOCVDRE, compared with 1,25(OH)(2)D(3) in COS-1 cells, transiently transfected with a VDR construct. Additionally, 1, 25(OH)(2)D(3)-3-BE was found to be more potent in inducing 1alpha, 25-dihydroxyvitamin D(3)-24-hydroxylase (24-OHase) promoter activity and mRNA expression in keratinocytes. The accumulation of 24-OHase message was also prolonged by the analog. Collectively these results indicated that rapid covalent labeling of VDR in keratinocytes (by 1, 25(OH)(2)D(3)-3-BE) might result in the conversion of apo-VDR to a holo-form, with a conformation that is different from that of the 1, 25(OH)(2)D(3)-VDR complex. This resulted in an enhanced stability of the 1,25(OH)(2)D(3)-3-BE/VDR-VDRE complex and contributed to the amplified antiproliferative effect of 1,25(OH)(2)D(3)-3-BE in keratinocytes.     

Human male gamete endocrinology: 1alpha, 25-dihydroxyvitamin D3 (1,25(OH)2D3) regulates different aspects of human sperm biology and metabolism

Background  

A wider biological role of 1alpha,25-Dihydroxyvitamin D3 (1,25(OH)2D3), the active metabolite of vitamin D3, in tissues not primarily related to mineral metabolism was suggested. Recently, we evidenced the ultrastructural localization the 1,25(OH)2D3 receptor in the human sperm. However, the 1,25(OH)2D3 action in human male reproduction has not yet been clarified.     

Synthesis and biological activity of (22E,25R)- and (22,25S)-22-dehydro- 1 alpha,25-dihydroxy-26-methylvitamin D3

Both 25-epimers of (22E)-22-dehydro-1 alpha,25-dihydroxy-26-methylvitamin D3 [22-dehydro-26-methyl-1,25-(OH)2D3] were synthesized. The biological activity of these compounds was tested in binding affinity to chick intestinal receptor protein of 1 alpha,25-dihydroxy-vitamin D3 [1,25-(OH)2D3] and in stimulating for intestinal calcium transport and bone calcium mobilization with vitamin D-deficient rats. The relative potency of (25R)- and (25S)-22-dehydro-26-homo-1,25-(OH)2D3 and 1,25-(OH)2D3 in competing for the intestinal cytosolic binding was 1.7:1.5:1. A similar order of activity was observed on intestinal calcium transport and bone calcium mobilization. In the ability for stimulation of intestinal calcium transport, (25R)- and (25S)-22-dehydro-26-methyl-1,25-(OH)2D3 were about 3.6 and 2.1 times as active as 1,25-(OH)2D3, respectively. In bone calcium mobilization tests, (25R)- and (25S)-22-dehydro-26-methyl-1,25-(OH)2D3 were estimated to be 2.2 and 1.6 times as potent as 1,25-(OH)2D3, respectively.     

C-6 functionalized analogs of 25-hydroxyvitamin D3 and 1alpha,25-dihydroxyvitamin D3: synthesis and binding analysis with vitamin D-binding protein and vitamin D receptor.

In this article, we describe the development of a general synthetic strategy to functionalize the C-6 position of vitamin D3 and its biologically important metabolites, i.e. 25-hydroxyvitamin D3 (25-OH-D3) and 1alpha,25-dihydroxyvitamin D3 [1,25(OH)2D3]. We employed Mazur's cyclovitamin D method to synthesize vitamin D3 analogs with several functionalities at the C-6 position. In addition, we synthesized 6-(3-hydroxypropyl) and 6-[(2-bromoacetoxy)propyl] derivatives of 25-OH-D3 15 and 16, respectively, and 6-(3-hydroxypropyl) derivative of 1,25(OH)2D3 17. Competitive binding assays of 15-17 with human serum vitamin D-binding protein showed that all these analogs specifically bound to this protein, although with significantly lower affinity than the 25-OH-D3, the strongest natural binder, but with comparable affinity with 1,25(OH)2D3, the hormone. On the other hand, 6-[3-hydroxypropyl], 1alpha,25-dihydroxyvitamin D3 17 did not show any specific binding for recombinant nuclear vitamin D receptor. These results indicated that the region containing the C-6 position of the parent seco-steroid [1,25(OH)2D3] may be an important recognition marker towards vitamin D receptor binding. Information, delineated in this article, will be important for evaluating structure-activity relationship in synthetic analogs of vitamin D and its metabolites.     

Human leukemic (HMC-1) mast cells are responsive to 1alpha, 25-dihydroxyvitamin D(3): selective promotion of ICAM-3 expression and constitutive presence of vitamin D(3) receptor

Expression levels of adhesion molecules on HMC-1 mast cells were examined prior to and following administration of 1alpha, 25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)]. While most receptors (including ICAM-1) remained unchanged by the treatment, solely ICAM-3 expression was promoted in a dose- and time-dependent fashion, peaking at 50 nM of 1,25(OH)(2)D(3) and 72 h, illustrating that like other myeloid cells, human mast cells are 1,25(OH)(2)D(3) responsive, yet in a highly selective manner. Flow cytometric results were confirmed by ELISA, by semiquantitative RT-PCR, and functionally by showing enhanced anti-ICAM-3 mediated homotypic aggregation of 1,25(OH)(2)D(3) pretreated cells. Since cellular responsiveness is conferred by the vitamin D(3) receptor (VDR), we examined human mast cells for its expression. VDR was constitutively present in both HMC-1 and skin mast cells by RT-PCR technique and in nuclear extracts of HMC-1 cells by Western blot analysis. Our data thus suggest that human mast cells are direct targets of 1, 25(OH)(2)D(3) action.     

Membrane actions of vitamin D metabolites 1alpha,25(OH)2D3 and 24R,25(OH)2D3 are retained in growth plate cartilage cells from vitamin D receptor knockout mice

1alpha,25(OH)(2)D(3) regulates rat growth plate chondrocytes via nuclear vitamin D receptor (1,25-nVDR) and membrane VDR (1,25-mVDR) mechanisms. To assess the relationship between the receptors, we examined the membrane response to 1alpha,25(OH)(2)D(3) in costochondral cartilage cells from wild type VDR(+/+) and VDR(-/-) mice, the latter lacking the 1,25-nVDR and exhibiting type II rickets and alopecia. Methods were developed for isolation and culture of cells from the resting zone (RC) and growth zone (GC, prehypertrophic and upper hypertrophic zones) of the costochondral cartilages from wild type and homozygous knockout mice. 1alpha,25(OH)(2)D(3) had no effect on [(3)H]-thymidine incorporation in VDR(-/-) GC cells, but it increased [(3)H]-thymidine incorporation in VDR(+/+) cells. Proteoglycan production was increased in cultures of both VDR(-/-) and VDR(+/+) cells, based on [(35)S]-sulfate incorporation. These effects were partially blocked by chelerythrine, which is a specific inhibitor of protein kinase C (PKC), indicating that PKC-signaling was involved. 1alpha,25(OH)(2)D(3) caused a 10-fold increase in PKC specific activity in VDR(-/-), and VDR(+/+) GC cells as early as 1 min, supporting this hypothesis. In contrast, 1alpha,25(OH)(2)D(3) had no effect on PKC activity in RC cells isolated from VDR(-/-) or VDR(+/+) mice and neither 1beta,25(OH)(2)D(3) nor 24R,25(OH)(2)D(3) affected PKC in GC cells from these mice. Phospholipase C (PLC) activity was also increased within 1 min in GC chondrocyte cultures treated with 1alpha,25(OH)(2)D(3). As noted previously for rat growth plate chondrocytes, 1alpha,25(OH)(2)D(3) mediated its increases in PKC and PLC activities in the VDR(-/-) GC cells through activation of phospholipase A(2) (PLA(2)). These responses to 1alpha,25(OH)(2)D(3) were blocked by antibodies to 1,25-MARRS, which is a [(3)H]-1,25(OH)(2)D(3) binding protein identified in chick enterocytes. 24R,25(OH)(2)D(3) regulated PKC in VDR(-/-) and VDR(+/+) RC cells. Wild type RC cells responded to 24R,25(OH)(2)D(3) with an increase in PKC, whereas treatment of RC cells from mice lacking a functional 1,25-nVDR caused a time-dependent decrease in PKC between 6 and 9 min. 24R,25(OH)(2)D(3) dependent PKC was mediated by phospholipase D, but not by PLC, as noted previously for rat RC cells treated with 24R,25(OH)(2)D(3). These results provide definitive evidence that there are two distinct receptors to 1alpha,25(OH)(2)D(3). 1alpha,25(OH)(2)D(3)-dependent regulation of DNA synthesis in GC cells requires the 1,25-nVDR, although other physiological responses to the vitamin D metabolite, such as proteoglycan sulfation, involve regulation via the 1,25-mVDR.     

1 alpha,25-dihydroxyvitamin D3 inhibits productive infection of human monocytes by HIV-1.

Pretreatment of freshly isolated human peripheral blood monocytes with the steroid hormone, 1 alpha,25-dihydroxyvitamin D3 (1,25(OH)D), markedly reduced (by 95%) productive infection of human monocytes by HIV-1. Equivalent concentrations (10nM) of 25-hydroxyvitamin D3 (25(OH)D), the biologic precursor of 1,25(OH)D, were ineffective at reducing either CD4 expression or HIV-1 production. Pretreatment was required for modulation of HIV-1 infection by 1,25(OH)D. Interestingly, 1,25(OH)D-mediated decreases in p24 antigen production were observed prior to any observed reduction in CD4 expression, suggesting that 1,25(OH)D treatment may modulate HIV-1 infection of monocytes through additional factors besides decreased HIV-1 binding. These data raise the possibility that 1,25(OH)D compounds may be important in host resistance to HIV-1.     

Autoradiographic studies with 3H 1,25 (OH)2 vitamin D3 and 3H 25 (OH) vitamin D3 in rat parathyroid glands

Summary After injection of radiolabeled 1,25 (OH)₂ vitamin D₃, nuclear concentration of radioactivity is observed in parenchymal cells of the parathyroid gland in pregnant, adult male, and 10-day male neonatal rats. In competition studies with unlabeled 1,25 (OH)₂ vitamin D₃, but not with 25 (OH) vitamin D₃, nuclear uptake is prevented. Experiments with ³H 25 (OH) vitamin D₃, in contrast to ³H 1,25 (OH)₂ vitamin D₃, do not show nuclear concentration in cells of the parathyroid. The results of the autoradiographic studies suggest the presence of receptors for a direct effect of 1,25 (OH)₂ vitamin D₃ on the parathyroid gland for modulation of parathyroid hormone secretion.     

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.     

Synthesis and biological activity of 1 alpha,23,25,26-tetrahydroxyvitamin D3

The metabolic pathway from 1 alpha,25-dihydroxyvitamin D3 [1 alpha,25-(OH)2D3] to 1 alpha,25-dihydroxyvitamin D3-26,23-lactone includes the formation of 1 alpha,23,25-26-tetrahydroxyvitamin D3 [1 alpha,23,25,26-(OH)4D3]. The aim of the current study was to explore the as yet unknown biological properties of this vitamin D3 sterol. The four diastereoisomers of 1 alpha,23,25,26-(OH)4D3 were chemically synthesized. They were compared to 1 alpha,25-(OH)2D3 in terms of their affinity for the chick intestinal 1 alpha,25-(OH)2D3 receptor and their biologic activity in vivo (stimulation of intestinal calcium absorption and mobilization of calcium from bone in vitamin D-deficient rats). The 1,25-(OH)2D3 receptor binding affinities of 1 alpha,23(R)25(R)26-(OH)4D3, 1 alpha,23(S)25(S)26-(OH)4 D3, 1 alpha,23(S)25(R)26-(OH)4D3, and 1 alpha,23(R)25(S)26-(OH)4D3 were 11, 100, 216, and 443 times weaker than the binding affinity of 1 alpha,25-(OH)2D3, respectively. Compared to 1 alpha,25-(OH)2D3, the relative capacities of the 1 alpha,23,25,26-(OH)4D3 compounds to stimulate intestinal calcium absorption were 1/4 for 1 alpha,23(R)25(R)26-(OH)4D3; 1/19 for 1 alpha,23(S)25(S)26-(OH)4D3; 1/90 for 1 alpha,23(S)25(R)26-(OH)4D3; and 1/136 for 1 alpha,23(R)25(S)26-(OH)4D3. Maximal stimulation of intestinal calcium transport occurred 8 h after administration of vitamin D3 metabolites. Mobilization of calcium from bone was quantitated by serum calcium concentration measurements. The activities of 1 alpha,23(R)25(R)26-(OH)4D3, 1 alpha,23(S)25(S)26-(OH)4D3, 1 alpha,23(S)25(R)26-(OH)4D3, and 1 alpha,23(R)25(S)26-(OH)4D3 to increase serum calcium were estimated to be 4, 13, 43, and 69 times weaker than that of 1 alpha,25-(OH)2D3, respectively. These results illustrate the stereospecificity of the chicken intestine 1 alpha,25-(OH)2D3 receptor for binding of 1 alpha,23,25,26-(OH)4D3 and suggest that the 1 alpha,23,25,26-(OH)4D3 exerts its biological activity in the rat through an interaction with 1,25-(OH)2D3 receptors. In summary, the 1 alpha,23,25,26-(OH)4D3 had a markedly lower biological activity than 1 alpha,25-(OH)2D3.     

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.     

Modulation of 1alpha,25-dihydroxyvitamin D3-membrane associated, rapid response steroid binding protein expression in mouse odontoblasts by 1alpha,25-(OH)2D3

The rapid, nongenomic effects of 1alpha,25-dihydroxyvitamin D3 (1alpha,25-(OH)2D3 have been related to a 1,25D3-membrane associated, rapid response steroid binding protein or 1,25D3-[MARRS]bp, with a molecular weight of 65 kDa, in several tissues and species. Currently, no information is available concerning the nongenomic responses to 1alpha,25-(OH)2D3 in dental tissues. In order to investigate the expression of 1,25D3-[MARRS]bp in dental cells, in the presence or absence of 1alpha,25-(OH)2D3, we have used rabbit polyclonal antibodies directed against the N-terminus of the 1,25D3-[MARRS]bp (Ab099) that recognizes the 1alpha,25-(OH)2D3 binding protein in chick intestinal basolateral membranes and a mouse odontoblast-like cell line (MO6-G3). Western blotting and flow cytometric analyses with Ab099 specifically detected 1,25D3-[MARRS]bp in MO6-G3 cells. Moreover, 1,25D3-[MARRS]bp was up-regulated, in vivo, in differentiated dental cells. Electron microscopic analysis confirmed the plasma membrane localization of this binding protein and also showed its intracellular presence. Incubation of MO6-G3 cells with different doses of 1alpha,25-(OH)2D3 for 36 h resulted in an inhibition of 1,25D3-[MARRS]bp expression with a maximal effect at 50 nM steroid. In addition, the culture media of MO6-G3 cells contains immunoreactive 1,25D3-[MARRS]bp. Immunogold positive membrane vesicle-like structures are present in the extracellular matrix of MO6-G3 cells. Altogether, these results indicate that the 1,25D3-[MARRS]bp expression in MO6-G3 cells is modulated by 1alpha,25-(OH)2D3. In conclusion, this 1alpha,25-(OH)2D3 binding protein could play an important role in the rapid, nongenomic responses to 1alpha,25-(OH)2D3 in dental cells.