1α,25-dihydroxyvitamin D3 rapidly alters phospholipid metabolism in the nuclea envelope of osteoblasts

1α,25-Dihydroxyvitamin D₃ (1α, 25-(OH)₂D₃) has been shown to increase cytosolic calcium and inositol trophosphate levels in rat osteosarcoma cells (ROS 17/2.8) and to increase nuclear calcium in these cells. To determine the mechanism(s) of 1α, (OH)₂D₃-induced changes in the calcium, the effect of the hormone on phospholipid metabolism in isolated osteoblast nuclei wa assessed. 1α,25 (OH)₂D₃, 20 nM, increased inositol triphosphate levels in the nuclei after 5 min of treatment. The biologically inactive epimer, 1β,25-(OH)₂D₃, had no significant effect on inositol triphosphate levels. ATP, 1 mM, also increased inositol triphosphate levels in the isolated nuclei after 5 min. 1α,25-(OH)₂D₃, 20 nM, increased calcium in the isolated nuclei in the presence but not in the absence of extranuclear calcium with 5 min. Nuclear calcium was also increased within 5 min by ATP, 1 mM, and inositol triphosphate, 1 mM. The effects of ATP on nuclear calcium was not additive with 1α, 25-(OH)₂D₃, suggesting that these two agents increase nuclear calcium in these osteoblast-like cells by similar mechanisms. In summary, 1α,25-(OH)₂D₃ amd ATP rapidly increase inositol triphosphate levels in isolated from ROS 17/2.8 cells. The hormone, the nucleotide, and the inositol phospholipid nuclear calcium. Thus, the 1α,25-(OH)₂D₃ and ATP effects of nuclear calcium may be mediated by changes in phospholipid metabolism in the nuclei of these osteoblastlike cells. © Wiley-Liss, Inc.     

Effects of the vitamin D3 analog 1α,25-dihydroxyvitamin D3-3β-bromoacetate on rat osteosarcoma cells: Comparison with 1α,25-dihydroxyvitamin D3

The actions of the hormonal form of vitamin D, 1α,25-dihydroxyvitamin D₃ [1α,25-(OH)₂D₃], are mediated by both genomic and nongenomic mechanisms. Several vitamin D synthetic analogs have been developed in order to identify and characterize the site(s) of action of 1α,25-(OH)₂D₃ in many cell types including osteoblastic cells. We have compared the effects of 1α,25-(OH)₂D₃ and a novel 1α,25-(OH)₂D₃ bromoester analog (1,25-(OH)₂-BE) that covalently binds to vitamin D receptors. Rat osteosarcoma cells that possess (ROS 17/2.8) or lack (ROS 24/1) the classic intracellular vitamin D receptor were studied to investigate genomic and nongenomic actions. In ROS 17/2.8 cells plated at low density, the two vitamin D compounds (1 × 10⁻⁸ M) caused increased cell proliferation, as assessed by DNA synthesis and total cell counts. Northern blot analysis revealed that the mitogenic effect of both agents was accompanied by an increase in steady-state osteocalcin mRNA levels, but neither agent altered alkaline phosphatase mRNA levels in ROS 17/2.8 cells. ROS 17/2.8 cells responded to 1,25-(OH)₂-BE but not the natural ligand with a significant increase in osteocalcin secretion after 72, 96, 120, and 144 hr of treatment. Treatment of ROS 17/2.8 cells with the bromoester analog also resulted in a significant decrease in alkaline phosphatase-specific activity. To compare the nongenomic effects of 1α,25-(OH)₂D₃ and 1,25-(OH)₂-BE, intracellular calcium was measured in ROS 24/1 cells loaded with the fluorescent calcium indicator Quin 2. At 2 × 10⁻⁸ M, both 1α,25-(OH)₂D₃ and 1,25-(OH)₂-BE increased intracellular calcium within 5 min. Both the genomic and nongenomic actions of 1,25-(OH)₂-BE are similar to those of 1α,25-(OH)₂D₃, and since 1,25-(OH)₂-BE has more potent effects on osteoblast function than the naturally occurring ligand due to more stable binding, this novel vitamin D analog may be useful in elucidating the structure and function of cellular vitamin D receptors. © 1996 Wiley-Liss, Inc.     

1α,25-Dihydroxyvitamin D3 receptor as a mediator of transrepression of retinoid signaling

The receptors for retinoic acid (RA) and for 1α,25-dihydroxyvitamin D₃ (VD), RAR, RXR, and VDR are ligand-inducible members of the nuclear receptor superfamily. These receptors mediate their regulatory effects by binding as dimeric complexes to response elements located in regulatory regions of hormone target genes. Sequence scanning of the tumor necrosis factor-α type I receptor (TNFαRI) gene identified a 3′ enhancer region composed of two directly repeated hexameric core motifs spaced by 2 nucleotides (DR2). On this novel DR2-type sequence, but not on a DR5-type RA response element, VD was shown to act through its receptor, the vitamin D receptor (VDR), as a repressor of retinoid signalling. The repression appears to be mediated by competitive protein–protein interactions between VDR, RAR, RXR, and possibly their cofactors. This VDR-mediated transrepression of retinoid signaling suggests a novel mechanism for the complex regulatory interaction between retinoids and VD. J. Cell. Biochem. 67:287–296, 1997. © 1997 Wiley-Liss, Inc.     

Evidence that annexin II is not a putative membrane receptor for 1alpha,25(OH)2-vitamin D3

The seco-steroid hormone 1alpha,25(OH)(2)-vitamin D(3) (1,25-D(3)) is known to generate biological responses via both genomic and non-genomic rapid signal transduction pathways. The calcium regulated annexin II/p11 heterotetramer (AII(2)/p11(2)] was proposed by Baran and co-authors to be the membrane receptor responsible for mediating non-genomic, rapid actions of 1,25-D(3), based on ligand affinity labeling, competition, and saturation analysis experiments. Given the cytosolic presence of both the monomeric and heterotetrameric form of AII and their functional regulation by intracellular calcium concentrations, which are known to be affected by 1,25-D(3) rapid, non-genomic activities, we investigated in vitro the affinity of [(3)H]1,25-D(3) for the AII monomer and AII(2)/p11(2) in the absence and presence of calcium using saturation analysis and gel-filtration chromatography. Using two different techniques for separating bound from free ligand (perchlorate and hydroxylapatite (HAP)) over a series of 30 experiments, no evidence for specific binding of [(3)H]1,25-D(3) was obtained with or without the presence of 700 nM exogenous calcium, using either the AII monomer or AII(2)/p11(2). However saturable binding of [(3)H]1,25-D(3) to the lipid raft/caveolae enriched rat intestinal fraction was consistently observed (K(d) = 3.0 nM; B(max) = 45 fmols/mg total protein). AII was detected in lipid raft/caveolae enriched fractions from rat and mouse intestine and ROS 17/2.8 and NB4 cells by Western blot, but incubation in the presence of exogenous calcium did not ablate 1,25-D(3) binding as reported by Baran et al. Our results suggest that AII does not bind 1,25-D(3) in a physiologically relevant manner; however, recent studies linking AII(2)/p11(2) phosphorylation to vesicle fusion and its calcium regulated localization may make AII a possible down-stream substrate for 1,25-D(3) induced rapid cellular effects.     

Model of three-dimensional structure of vitamin D receptor and its binding mechanism with 1alpha,25-dihydroxyvitamin D(3).

Comparative modeling of the vitamin D receptor three-dimensional structure and computational docking of 1alpha,25-dihydroxyvitamin D(3) into the putative binding pocket of the two deletion mutant receptors: (207-423) and (120-422, Delta [164-207]) are reported and evaluated in the context of extensive mutagenic analysis and crystal structure of holo hVDR deletion protein published recently. The obtained molecular model agrees well with the experimentally determined structure. Six different conformers of 1alpha,25-dihydroxyvitamin D(3) were used to study flexible docking to the receptor. On the basis of values of conformational energy of various complexes and their consistency with functional activity, it appears that 1alpha,25-dihydroxyvitamin D(3) binds the receptor in its 6-s-trans form. The two lowest energy complexes obtained from docking the hormone into the deletion protein (207-423) differ in conformation of ring A and orientation of the ligand molecule in the VDR pocket. 1alpha,25-Dihydroxyvitamin D(3) possessing the A-ring conformation with axially oriented 1alpha-hydroxy group binds receptor with its 25-hydroxy substituent oriented toward the center of the receptor cavity, whereas ligand possessing equatorial conformation of 1alpha-hydroxy enters the pocket with A ring directed inward. The latter conformation and orientation of the ligand is consistent with the crystal structure of hVDR deletion mutant (118-425, Delta [165-215]). The lattice model of rVDR (120-422, Delta [164-207]) shows excellent agreement with the crystal structure of the hVDR mutant. The complex obtained from docking the hormone into the receptor has lower energy than complexes for which homology modeling was used. Thus, a simple model of vitamin D receptor with the first two helices deleted can be potentially useful for designing a general structure of ligand, whereas the advanced lattice model is suitable for examining binding sites in the pocket.     

Metabolism of A-ring diastereomers of 1alpha,25-dihydroxyvitamin D3 by CYP24A1

The metabolism of 1alpha,25(OH)(2)D(3) (1alpha,3beta) and its A-ring diastereomers, 1beta,25(OH)(2)D(3) (1beta,3beta), 1alpha,25(OH)(2)-3-epi-D(3) (1alpha,3alpha), and 1beta,25(OH)(2)-3-epi-D(3) (1beta,3alpha), was examined to compare the substrate specificity and reaction specificity of CYP24A1 between humans and rats. The ratio between C-23 and C-24 oxidation pathways in human CYP24A1-dependent metabolism of (1alpha,3alpha) and (1beta,3alpha) was 1:1, although the ratio for (1alpha,3beta) and (1beta,3beta) was 1:4. These results indicate that the orientation of the hydroxyl group at the C-3 position determines the ratio between C-23 and C-24 oxidation pathways. A remarkable increase of metabolites in the C-23 oxidation pathway was also observed in rat CYP24A1-dependent metabolism. The binding affinity of human CYP24A1 for A-ring diastereomers was (1alpha,3beta)>(1alpha,3alpha)>(1beta,3beta)>(1beta,3alpha), indicating that both hydroxyl groups at C-1 and C-3 positions significantly affect substrate-binding. The information obtained in this study is quite useful for understanding substrate recognition of CYP24A1 and designing new vitamin D analogs.     

Cloning of a functional 25‐hydroxyvitamin D‐1α‐hydroxylase in zebrafish (Danio rerio)

Activation of precursor 25‐hydroxyvitamin D₃ (25D) to hormonal 1,25‐dihydroxyvitamin D₃ (1,25D) is a pivotal step in vitamin D physiology, catalysed by the enzyme 25‐hydroxyvitamin D‐1α‐hydroxylase (1α‐hydroxylase). To establish new models for assessing the physiological importance of the 1α‐hydroxylase‐25D‐axis, we used Danio rerio (zebrafish) to characterize expression and biological activity of the gene for 1α‐hydroxylase (cyp27b1). Treatment of day 5 zebrafish larvae with inactive 25D (5–150 nM) or active 1,25D (0.1–10 nM) induced dose responsive expression (15–95‐fold) of the vitamin D‐target gene cyp24a1 relative to larvae treated with vehicle, suggesting the presence of Cyp27b1 activity. A full‐length zebrafish cyp27b1 cDNA was then generated using RACE and RT‐PCR methods. Sequencing of the resulting clone revealed an open reading frame encoding a protein of 505 amino acids with 54% identity to human CYP27B1. Transfection of a cyp27b1 expression vector into HKC‐8, a human kidney proximal tubular epithelial cell line, enhanced intracrine metabolism of 25D to 1,25D resulting in greater than twofold induction of CYP24A1 mRNA expression and a 25‐fold increase in 1,25D production compared to empty vector. These data indicate that we have cloned a functional zebrafish CYP27B1, representing a phylogenetically distant branch from mammals of this key enzyme in vitamin D metabolism. Further analysis of cyp27b1 expression and activity in zebrafish may provide new perspectives on the biological importance of 25D metabolism. Copyright © 2014 John Wiley & Sons, Ltd.     

Metabolism of 3H-1α,25-dihydroxyvitamin D3 in cultured human keratinocytes

In the present investigation we studied the metabolism of 1α,25-dihydroxy-[1β-³H] vitamin D₃ (³H-1,25(OH)₂D₃) in culture-grown human keratinocytes (CHK). Our results showed that the cellular uptake of ³H-1,25(OH)₂D₃, upon incubation with CHK, occurred very rapidly; and it paralleled a decrease in the concentration of ³H-1,25(OH)₂D₃ in the medium. The amount of ³H-calcitroic acid, on the other hand, increased slowly in the medium, while the concentration of ³H-calcitroic acid in the cell remained undetectable during the whole period of incubation. When the cells were preincubated with 1,25(OH)₂D₃ (10^?8M), conversion of ³H-1,25(OH)₂D₃ to ³H-calcitroic acid increased almost twofold, indicating that 1,25(OH)₂D₃ catalyzed its own catabolism. © 1995 Wiley-Liss, Inc.     

Clonal Differences in Expression of 25-Hydroxyvitamin D3-1α-hydroxylase, of 25-Hydroxyvitamin D3-24-hydroxylase, and of the Vitamin D Receptor in Human Colon Carcinoma Cells: Effects of Epidermal Growth Factor and 1α,25-Dihydroxyvitamin D3

Human colon carcinoma cells express 25-hydroxyvitamin D₃-1α-hydroxylase (CYP27B1) and thus produce the vitamin D receptor (VDR) ligand 1α,25-dihydroxyvitamin D₃ (1,25-D3), which can be metabolized by 25-hydroxyvitamin D₃-24-hydroxylase (CYP24). Expression of VDR, CYP27B1, and CYP24 determines the efficacy of the antimitotic action of 1,25-D3 and is distinctly related to the degree of differentiation of cancerous lesions. In the present study we addressed the question of whether the effects of epidermal growth factor (EGF) and of 1,25-D3 on VDR, CYP27B1, and CYP24 gene expression in human colon carcinoma cell lines also depend on the degree of cellular differentiation. We were able to show that slowly dividing, highly differentiated Caco-2/15 cells responded in a dose-dependent manner to both EGF and 1,25-D3 by up-regulation of VDR and CYP27B1 expression, whereas in highly proliferative, less differentiated cell lines, such as Caco-2/AQ and COGA-1A and -1E, negative regulation was observed. CYP24 mRNA was inducible in all clones by 1,25-D3 but not by EGF. From the observed clonal differences in the regulatory effects of EGF and 1,25-D3 on VDR and CYP27B1 gene expression we suggest that VDR-mediated growth inhibition by 1,25-D3 would be efficient only in highly differentiated carcinomas even when under mitogenic stimulation by EGF.     

The development of CD-ring modified analogs of 1α,25-dihydroxyvitamin D

During a 20-year collaboration the laboratories of UGent and KU Leuven have developed different series of Vitamin D analogs characterized by structural modifications in the central CD-ring system. Modifications have first involved the introduction of substituents at C11 and the epimerization at C14, and subsequently more drastic changes consisting in both ring deletion and enlargement relative to the natural CD-ring system. Lately, the focus has shifted towards the synthesis of analogs featuring a symmetrical CD-ring core. As an illustration two different series are presented.     

Selective analogs of 1α,25-dihydroxyvitamin D3 for the study of specific functions of Vitamin D

New analogs of 1α,25-dihydroxyvitamin D₃ synthesized in our research group that show selective activity in vivo are presented along with supporting biological results. Compounds that act preferentially on intestine are 2-(3′-propylidene-19-nor-(20S or 20R))-1α,25-dihydroxyvitamin D₃ and 2-methylene-19-21-dinor-1α,25-dihydroxyvitamin D₃. Compounds that act anabolically to induce bone formation are 2-methylene-19-nor-(20S)-1α,25-dihydroxyvitamin D₃ (2MD), the 2α-methyl derivative, the 26,27-dimethyl derivative, and the 26-dimethylene derivative. Compounds that act preferentially on parathyroid glands are 2-methylene-19-nor-1α-hydroxy-homopregnacalciferol, the 20S-bishomo derivative and the 2-methylene-19,26,27-trinor-1α,25-dihydroxyvitamin D₃. These latter compounds do not elevate serum calcium until doses of the order of >300 μg/kg body weight are used, while parathyroid hormone levels are suppressed at much lower doses. Some of these novel analogs may ultimately be useful as new and safer therapeutic agents. Regardless of their clinical utility, they represent valuable research tools that can be used to study the specific functions of the Vitamin D hormone in vivo.     

Clonal differences in expression of 25-hydroxyvitamin D(3)-1alpha-hydroxylase,of 25-hydroxyvitamin D(3)-24-hydroxylase,and of the vitamin D receptor in human colon carcinoma cells: effects of epidermal growth factor and 1alpha,25-dihydroxyvitamin D(3)

Human colon carcinoma cells express 25-hydroxyvitamin D(3)-1alpha-hydroxylase (CYP27B1) and thus produce the vitamin D receptor (VDR) ligand 1alpha,25-dihydroxyvitamin D(3) (1,25-D3), which can be metabolized by 25-hydroxyvitamin D(3)-24-hydroxylase (CYP24). Expression of VDR, CYP27B1, and CYP24 determines the efficacy of the antimitotic action of 1,25-D3 and is distinctly related to the degree of differentiation of cancerous lesions. In the present study we addressed the question of whether the effects of epidermal growth factor (EGF) and of 1,25-D3 on VDR, CYP27B1, and CYP24 gene expression in human colon carcinoma cell lines also depend on the degree of cellular differentiation. We were able to show that slowly dividing, highly differentiated Caco-2/15 cells responded in a dose-dependent manner to both EGF and 1,25-D3 by up-regulation of VDR and CYP27B1 expression, whereas in highly proliferative, less differentiated cell lines, such as Caco-2/AQ and COGA-1A and -1E, negative regulation was observed. CYP24 mRNA was inducible in all clones by 1,25-D3 but not by EGF. From the observed clonal differences in the regulatory effects of EGF and 1,25-D3 on VDR and CYP27B1 gene expression we suggest that VDR-mediated growth inhibition by 1,25-D3 would be efficient only in highly differentiated carcinomas even when under mitogenic stimulation by EGF.     

Metabolism of 1α-hydroxyvitamin D3 by cytochrome P450scc to biologically active 1α,20-dihydroxyvitamin D3

Cytochrome P450scc (CYP11A1) metabolizes vitamin D3 to 20-hydroxyvitamin D3 as the major product, with subsequent production of dihydroxy and trihydroxy derivatives. The aim of this study was to determine whether cytochrome P450scc could metabolize 1α-hydroxyvitamin D3 and whether products were biologically active. The major product of 1α-hydroxyvitamin D3 metabolism by P450scc was identified by mass spectrometry and NMR as 1α,20-dihydroxyvitamin D3. Mass spectrometry of minor metabolites revealed the production of another dihydroxyvitamin D3 derivative, two trihydroxy-metabolites made via 1α,20-dihydroxyvitamin D3 and a tetrahydroxyvitamin D3 derivative. The K_m for 1α-hydroxyvitamin D3 determined for P450scc incorporated into phospholipid vesicles was 1.4 mol substrate/mol phospholipid, half that observed for vitamin D3. The k_cat was 3.0 mol/min/mol P450scc, 6-fold lower than that for vitamin D3. 1α,20-Dihydroxyvitamin D3 inhibited DNA synthesis by human epidermal HaCaT keratinocytes propagated in culture, in a time- and dose-dependent fashion, with a potency similar to that of 1α,25-dihydroxyvitamin D3. 1α,20-Dihydroxyvitamin D3 (10 μM) enhanced CYP24 mRNA levels in HaCaT keratinocytes but the potency was much lower than that reported for 1α,25-dihydroxyvitamin D3. We conclude that the presence of the 1-hydroxyl group in vitamin D3 does not alter the major site of hydroxylation by P450scc which, as for vitamin D3, is at C20. The major product, 1α,20-dihydroxyvitamin D3, displays biological activity on keratinocytes and therefore might be useful pharmacologically.