Skip to main content
Advertisement
Browse Subject Areas
?

Click through the PLOS taxonomy to find articles in your field.

For more information about PLOS Subject Areas, click here.

  • Loading metrics

1α,25(OH)2-3-Epi-Vitamin D3, a Natural Physiological Metabolite of Vitamin D3: Its Synthesis, Biological Activity and Crystal Structure with Its Receptor

  • Ferdinand Molnár,

    Affiliations Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), Institut National de Santé et de Recherche Médicale (INSERM) U964/Centre National de Recherche Scientifique (CNRS) UMR 7104/Université de Strasbourg, Illkirch, France, School of Pharmacy, Faculty of Health Sciences, University of Eastern Finland, Kuopio, Finland

  • Rita Sigüeiro,

    Affiliation Departamento de Quimica Organica, Universidad de Santiago de Compostela and Unidad Asociada al CSIC, Santiago de Compostela, Spain

  • Yoshiteru Sato,

    Affiliation Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), Institut National de Santé et de Recherche Médicale (INSERM) U964/Centre National de Recherche Scientifique (CNRS) UMR 7104/Université de Strasbourg, Illkirch, France

  • Clarisse Araujo,

    Affiliation Departamento de Quimica Organica, Universidad de Santiago de Compostela and Unidad Asociada al CSIC, Santiago de Compostela, Spain

  • Inge Schuster,

    Affiliation Institute of Pharmaceutical Chemistry, University of Vienna, Vienna, Austria

  • Pierre Antony,

    Affiliation Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), Institut National de Santé et de Recherche Médicale (INSERM) U964/Centre National de Recherche Scientifique (CNRS) UMR 7104/Université de Strasbourg, Illkirch, France

  • Jean Peluso,

    Affiliation Faculty of Pharmacy, Institut Gilbert Laustriat, UMR 7175 CNRS, University of Strasbourg, Illkirch, France

  • Christian Muller,

    Affiliation Faculty of Pharmacy, Institut Gilbert Laustriat, UMR 7175 CNRS, University of Strasbourg, Illkirch, France

  • Antonio Mouriño,

    Affiliation Departamento de Quimica Organica, Universidad de Santiago de Compostela and Unidad Asociada al CSIC, Santiago de Compostela, Spain

  • Dino Moras,

    Affiliation Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), Institut National de Santé et de Recherche Médicale (INSERM) U964/Centre National de Recherche Scientifique (CNRS) UMR 7104/Université de Strasbourg, Illkirch, France

  • Natacha Rochel

    rochel@igbmc.fr

    Affiliation Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), Institut National de Santé et de Recherche Médicale (INSERM) U964/Centre National de Recherche Scientifique (CNRS) UMR 7104/Université de Strasbourg, Illkirch, France

Abstract

Background

The 1α,25-dihydroxy-3-epi-vitamin-D3 (1α,25(OH)2-3-epi-D3), a natural metabolite of the seco-steroid vitamin D3, exerts its biological activity through binding to its cognate vitamin D nuclear receptor (VDR), a ligand dependent transcription regulator. In vivo action of 1α,25(OH)2-3-epi-D3 is tissue-specific and exhibits lowest calcemic effect compared to that induced by 1α,25(OH)2D3. To further unveil the structural mechanism and structure-activity relationships of 1α,25(OH)2-3-epi-D3 and its receptor complex, we characterized some of its in vitro biological properties and solved its crystal structure complexed with human VDR ligand-binding domain (LBD).

Methodology/Principal Findings

In the present study, we report the more effective synthesis with fewer steps that provides higher yield of the 3-epimer of the 1α,25(OH)2D3. We solved the crystal structure of its complex with the human VDR-LBD and found that this natural metabolite displays specific adaptation of the ligand-binding pocket, as the 3-epimer maintains the number of hydrogen bonds by an alternative water-mediated interaction to compensate the abolished interaction with Ser278. In addition, the biological activity of the 1α,25(OH)2-3-epi-D3 in primary human keratinocytes and biochemical properties are comparable to 1α,25(OH)2D3.

Conclusions/Significance

The physiological role of this pathway as the specific biological action of the 3-epimer remains unclear. However, its high metabolic stability together with its significant biologic activity makes this natural metabolite an interesting ligand for clinical applications. Our new findings contribute to a better understanding at molecular level how natural metabolites of 1α,25(OH)2D3 lead to significant activity in biological systems and we conclude that the C3-epimerization pathway produces an active metabolite with similar biochemical and biological properties to those of the 1α,25(OH)2D3.

Introduction

The 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3 or calcitriol), is the most active form of vitamin D3 and mediates its pleiotropic effects through VDR activation, which heterodimerizes with retinoid X receptor (RXR). VDR-induced genomic action results in growth inhibition of lymphomas, breast or prostate primary tumor cells, renal osteodystrophy, osteoporosis, psoriasis or autoimmune diseases [1], [2]. Consequently, VDR is an exquisite therapeutic target to combat human metabolic diseases and uncontrolled cell proliferation in many tissues [3][5]. In addition 1α,25(OH)2D3 is a key regulator of calcium and phosphate homeostasis and bone metabolism but its intrinsic hypercalcemic effect prevents its use in therapeutical applications [6].

1α,25(OH)2D3 is subjected to enzymatic inactivation via two major pathways leading to C-24 and C-23 hydroxylated metabolites in various tissues [7][17]. While the side chain oxidation is a general pathway associated to inactivation, another metabolite modified at the A-ring, the 1α,25(OH)2-3-epi-D3, has been shown to retain significant biological activity compared to the natural hormone [18], [19]. The 1α,25(OH)2-3-epi-D3 was initially identified in the culture of human neonatal keratinocytes [20], [21]. Further in vivo studies have characterized the occurrence of a C-3 epimerization pathway [22]. Indeed, this natural vitamin D3 metabolite was detected in serum of rats treated with pharmacological doses of 1α,25(OH)2D3, and may therefore play an important physiological role by buffering the level of 1α,25(OH)2D3. In addition, significant accumulation of 1α,25(OH)2-3-epi-D3 was observed in different human adenocarcinoma cell lines such as colon-derived Caco-2 cells[23] or NCI-H441 pulmonary cells [24]. Moreover, 1α,25(OH)2-3-epi-D3 was readily quantified in bovine parathyroid cells, [25] rat osteoblastic UMR 106 and Ros17/2.8 cells [26].

The production of 1α,25(OH)2-3-epi-D3 is initiated via A-ring C3-epimerization (Figure 1), where the C-3 hydroxyl moiety is changed from position β to its diastereomer α. The enzymes responsible for the C3-epimerization have not been identified to present date. It was also proposed by Reddy et al. that this pathway might be used for metabolites that resist inactivation through C-24 oxidation [18] a phenomenon well characterized in the bile acid metabolism where the reaction is catalyzed by bile acid hydroxysteroid dehydrogenase [27]. This pathway plays also a major role in the activation and/or inactivation of steroid hormones such as androgens [28].

thumbnail
Figure 1. Proposed pathway of the 1α,25(OH)2-3-epi-D3 production [18].

The reaction is initiated via A-ring C3-epimerization, where the C-3 hydroxyl moiety is changed from β to its diastereomer α. Two distinct pathways may be employed by cells to generate 1α,25(OH)2-3-epi-D3. The first, more likely used pathway, starts with dehydrogenation catalyzed by yet unidentified enzyme leading to a keto-intermediate, which is converted most probably by the same enzyme to the final product 1α,25(OH)2-3-epi-D3. The second one uses dehydration and a subsequent hydroxylation at C-3 α position.

https://doi.org/10.1371/journal.pone.0018124.g001

Despite a lower binding affinity than calcitriol, 1α,25(OH)2-3-epi-D3 possess significant biological activity only in specific tissues where it is produced [29]. The transcriptional response of the 1α,25(OH)2-3-epi-D3 compound varies for different VDR-regulated genes in different tissues. For instance, it shows lower activation of osteocalcin gene and lower HL60 differentiation [30] but has almost equipotent activity to 1α,25(OH)2D3 in inhibiting cellular proliferation in keratinocytes [19] and in suppressing parathyroid secretion in bovine parathyroid cells [25]. These in vitro properties associated with its low calcemic activity [31], [32] assign potential therapeutic interest to this compound.

To further unveil, the structural mechanism and structure-activity relationships of 1α,25(OH)2-3-epi-D3/hVDR-LBD complex, we describe a more effective synthetic route to the synthesis of 1α,25(OH)2-3-epi-D3, some of its in vitro biological properties and the crystal structure of its complex with hVDR LBD.

Results and Discussion

Synthesis of the 1α,25(OH)2-3-epi-D3

The synthesis of the target 1α,25(OH)2-3-epi-D3 (1, Scheme S1) was first described by Okamura's group at Riverside from (R)-carvone using the dienyne approach (13 steps, 8.5%) [33]. We describe here an efficient and alternative convergent synthesis of 1 from (S)-carvone (9 steps, 13%) that features a palladium catalyzed tandem process that produces the vitamin D triene unit stereoselectively in one pot by coupling enol triflate 3 (A-ring fragment) with an alkenyl metal intermediate 2 (CD-side chain fragment) [34]. For reproducibility reasons we employed Indium intermediates (M = InR2) instead of Zinc intermediates [35][37].

Synthesis of the A-ring fragment 3

Our synthesis starts with commercial (S)-carvone (4, Scheme S2), which was reduced under Luche conditions [38] to alcohol 5a and its epimer 5b (9∶1 ratio as determined by 1H-NMR). The mixture of alcohols 5 was subjected to Sharpless epoxidation [39] to provide the desired epoxyalcohol 6a (58% yield, two steps) and the starting ketone 4 (28%). The formation of 4 can be explained by oxidation of 5a through the corresponding chair-like equatorially oriented vanadium ester intermediate. Tert-butyldimethylsilyl protection of 6a gave 6b in 96% yield. Side-chain degradation on 6b by Daniewski's method [40] afforded alcohol 7a (71%), which was protected to 7b in the usual way (91%). Epoxide 7b was converted in 77% yield to dibromide 8b by the two-step sequence: 1) oxidative cleavage with periodic acid; 2) Corey-Fuchs side-chain extension [41]. Finally, consecutive treatment of 8b with lithium diisopropylamide and n-butyllithium followed by trapping of the resulting enolate with N-(5-Cl-2-pyridil)bis(triflate) gave the desired enol triflate 3 in 76% yield [42].

Synthesis of the upper fragment 2 and 1α,25(OH)2-3-epi-D3 (1b)

Alkenyl bromide 10 was prepared from ketone 9 by a modified [43] Trost procedure [44]. Treatment of a mixture of bromide 10 and indium trichloride with tert-butyllithium, and coupling of the resulting indium intermediate 2a with enol triflate 3 in the presence of catalytic amounts of (Ph3P)4Pd and (dppf)PdCl2, gave, after desilylation, the desired metabolite 1b in 58% yield (Scheme S3). The detailed synthesis is described in the Methods S1.

1α,25(OH)2D3 and 1α,25(OH)2-3-epi-D3 show similar properties in coactivator peptide recruitment

The human transcriptional intermediary factor TIF2 coactivator (NCOA2) has been shown to interact with VDR [45]. The induced recruitment of TIF2 coactivator peptide bearing the 3rd LXXLL motif to the hVDR LBD was monitored in the presence of increasing concentrations of 1α,25(OH)2D3 or 1α,25(OH)2-3-epi-D3 using the luminescent oxygen channeling assays [46]. Our results show that EC50 value for both metabolites are in the lower nanomolar range, 1.2 and 2.5 nM for 1α,25(OH)2D3 and 1α,25(OH)2-3-epi-D3, respectively (Figure 2A).

thumbnail
Figure 2. 1α,25(OH)2D3 and 1α,25(OH)2-3-epi-D3 show similar biological properties.

(A) Coactivator peptide recruitment assay was performed using AlphaScreen method in the presence of increasing concentrations of either 1α,25(OH)2D3 (green circles) or 1α,25(OH)2-3-epi-D3 (blue circles). The data represents two independent measurements in triplicates for which the mean and the S.D. of the mean was calculated. (B) Transactivation assays were performed in human breast cancer cells MCF7 cells with subsequent treatments of the increasing concentrations of either 1α,25(OH)2D3 (green circles) or 1α,25(OH)2-3-epi-D3 (blue circles). For every triplicate the mean and the S.D. were calculated. (C) Metabolism of 3H-25(OH)D3 in human keratinocytes. Kinetics of the primary metabolite 1α,25(OH)2D3 and its 3-epimer, is shown. The time point 5 h, where the 1α,25(OH)2-3-epi-D3 is the major metabolite is highlighted with red arrow. Confluent keratinocytes derived from lid skin were incubated in KGM (0.06 mM calcium) with 20.6 nM 3H[26], [27]-25(OH)D3 for the indicated time periods. CHCl3-extracts of the incubations were analyzed on Zorbax-Sil and individual metabolites identified by matching with authentic reference compounds and quantified as described in Materials and Methods. Data (± SD) was calculated from duplicate experiment. (D) Anti-proliferative cellular effect of 1α,25(OH)2D3 and 1α,25(OH)2-3-epi-D3 in human keratinocytes. Keratinocytes in serum-free KGM (0.06 mM calcium) were seeded into 96-well plates and 24 h later the indicated metabolites (range 0.1–100 nM). After further 24 h, 1 µCi 3H-thymidine was applied to each well and its incorporation determined as described in Methods. Data are mean values (± SD) from a representative experiment out of two independent studies, each done in triplicates. For all experiments Student's unpaired t-test was performed and p-values were calculated between values obtained for 1α,25(OH)2D3 and 1α,25(OH)2-3-epi-D3 (* p<0.05, ** p<0.01, *** p<0.001).

https://doi.org/10.1371/journal.pone.0018124.g002

1α,25(OH)2D3 and 1α,25(OH)2-3-epi-D3 induce expression of vitamin D target genes in human breast cancer (MCF-7) cells with similar potency

The transactivation potency of 1α,25(OH)2-3-epi-D3 has been reported for several VDR target genes in different model cell lines such as MG-63 or ROS17/2.8 osteosarcoma cells [24], [30]. While the transcriptional activity in MG-63 cells using a vitamin D-responsive element (VDRE) from human osteocalcin (−848/+10) and rat CYP24 (−291/+9) gene promoters was lower upon stimulation with 1α,25(OH)2-3-epi-D3 compared to 1α,25(OH)2D3 [47], using 2xVDREs reporter from CYP24 gene promoter in human melanoma G-361 cells comparable transcriptional activity was observed [48]. This response is mainly achieved in cells in which the 1α,25(OH)2-3-epi-D3 metabolite is produced [29]. We monitored the dose-dependent VDR induced transcriptional activity in human breast cancer cells (MCF-7) cells transfected with human CYP24 promoter (−414 to −64) containing VDRE fused to reporter luciferase gene (Figure 2B). Here, we show that 1α,25(OH)2-3-epi-D3 is slightly less potent than 1α,25(OH)2D3 in directing transactivation assay as the EC50 induced by 1α,25(OH)2-3-epi-D3 is twice higher than that of 1α,25(OH)2D3 (5.9 nM vs 2.9 nM). This difference is in agreement with our results obtained from cell free coactivator peptide recruitment assays. Our transactivation assays show that the dose-dependent comparison between the 1α,25(OH)2-3-epi-D3 and 1α,25(OH)2D3 reveals that at 50% of the dose-response, the transcriptional activity of the 3-epimer is 65% of that obtained with 1α,25(OH)2D3. Statistical analysis revealed a significant correlation between both the induced-coactivator recruitment and transactivation assays (Pearson r = 0.961** and r = 0.986**, respectively), indicating the similarity in the course of the dose response curves for both 1α,25(OH)2D3 and 1α,25(OH)2-3-epi-D3. The reason for the discrepancy from the previously reported lower transactivation potential of 1α,25(OH)2-3-epi-D3 may have its origin in different CYP24 promoter fragment used in our experiments. Although, the EMSA assays with nuclear extracts and in vitro translated full length VDR and RXR reported by Nakagawa et al. [47] showed decreased DNA complex formation of VDR-RXR heterodimer in the presence of 1α,25(OH)2-3-epi-D3 compared to 1α,25(OH)2D3, the same authors showed using two-hybrid system that the strength of VDR-RXR heterodimerization in presence of 10nM of the 3-epimer is 40% compared to that observed for 1α,25(OH)2D3.

Cell specific effects of 1α,25(OH)2-3-epi-D3

The magnitude of 1α,25(OH)2-3-epi-D3-mediated specific biological outcomes versus that induced by 1α,25(OH)2D3 is cell line specific. As such, it is established based only on CD11b antigen positive cell numbers that 1α,25(OH)2-3-epi-D3 is biologically less potent than 1α,25(OH)2D3 in the human leukemia anti-proliferation and pro-differentiation cellular model (HL60), compared to 1α,25(OH)2D3 [47]. We monitor the precise dose-dependent study of 1α,25(OH)2-3-epi-D3 - directed HL60 cell anti-proliferation and differentiation by live cell enumeration and flow cytometry based on the expression of both CD11c and CD14 cell surface markers. In our experiments for both, 1α,25(OH)2D3 and 1α,25(OH)2-3-epi-D3, only the saturating 100 nM concentration of ligand reduced the numbers of HL60 cells (Figure S1 and Methods S2). Although for 1α,25(OH)2-3-epi-D3 the related percentage of single positive or double CD11c/CD14 sub-populations was higher compared to that observed in control incubations, it was markedly reduced compared to that induced with 100 nM 1α,25(OH)2D3, consistent with the previous study [47].

Further, we hypothesized about the absence of the 1α,25(OH)2-3-epi-D3 signaling in HL60 cellular model and thus turned to characterize some of the biological properties of 1α,25(OH)2-3-epi-D3 in cells where it is produced [20], [21]. We first determined the kinetics of CYP27B1- and CYP24A1-catalyzed oxidation by monitoring the major lipophilic metabolites arising from a single pulse of 3H[26], [27]-25(OH)D3 at physiological concentration (20.6 nM). During the first two hours, we observed a rapid appearance of 1α,25(OH)2D3, from which at a slower rate the 3-epimer was irreversibly formed. In total, some 60 independent incubation experiments were performed on the kinetics of 3H[26], [27]-25(OH)D3 using primary keratinocytes from various donors and skin sites. In all experiments, highly comparable time course of 1α,25(OH)2D3 and 1α,25(OH)2-3-epi-D3 were recorded with 3-epimer exceeding 1α,25(OH)2D3 after longer incubation as shown in Figure 2C and in the detailed HPLC analysis in Figure S2 and Methods S2. Since the 1α,25(OH)2-3-epi-D3 is present steadily up to 5 h in rather high concentration in this tissue and the fact that the primary genomic effects of hVDR ligands are exerted in first hours suggested that primary keratinocytes may be a good cellular model to investigate the anti-proliferative actions of this metabolite. Therefore we determined the dose-dependent anti-proliferative effects of 1α,25(OH)2D3 and 1α,25(OH)2-3-epi-D3 using 3H-thymidine incorporation assay (Figure 2D), and found that the IC50 values for 1α,25(OH)2D3 and 1α,25(OH)2-3-epi-D3 were highly similar (41.4 and 66.1 nM, respectively) with no significant statistical difference (using unpaired t-test p = 0.074). In addition, we correlated the course of the anti-proliferation data between the two epimers and find a strong correlation (Pearson r = 0.940**) between them indicating the similar anti-proliferative activity for 1α,25(OH)2D3 and 1α,25(OH)2-3-epi-D3. The anti-proliferative effects of the two metabolites are comparable and they are in close agreement with our coactivator peptide recruitment (Figure 2A) and reporter gene assays (Figure 2B). Although in this assay we cannot totally exclude the possibility that the potential cell type specific difference in the function of the two natural ligands may be partly due to the accumulation of 1α,25(OH)2-3-epi-D3 in 1α,25(OH)2D3 treated cells with C3-epimerization ability leading to additive effect, we consider this accumulation process as a naturally occurring in vivo physiological event when 1α,25(OH)2D3 is present in these cells.

Overall structure of the hVDR complexed to 1α,25(OH)2-3-epi-D3

The mechanistic action of analogues of 1α,25(OH)2D3 is unveiled by the determination at high resolution of the crystal structure of their complexes with the VDR LBD [49][53]. We solved the crystal structure of the complex formed by 1α,25(OH)2-3-epi-D3 with the hVDR LBD mutant previously used to solve the structures of hVDR LBD in complexes with 1α,25(OH)2D3 or several synthetic agonists [49][54]. The crystal was isomorphous and the structure of hVDR LBD bound to 1α,25(OH)2-3-epi-D3 determined at a resolution of 1.9 Å (PDB ID: 3A78). The crystallographic data are summarized in Table S1. After refinement of the protein alone, the map showed an unambiguous electron density where to fit the ligand (Figure 3B). The complex formed by the hVDR LBD bound to 1α,25(OH)2-3-epi-D3 adopts the canonical active conformation as described in all previously reported agonist-bound nuclear receptor LBDs (Figure 3A). The conformation of the activation helix 12 is strictly maintained. When compared to the structure of hVDR LBD-1α,25(OH)2D3 complex, the atomic coordinates of hVDR LBD bound to 1α,25(OH)2-3-epi-D3 show very small root-mean-square deviation (RMSD) of 0.17 Å for all 255 Cα atoms, reflecting its high structural homology.

thumbnail
Figure 3. Overall structure of the VDR-1α,25(OH)2-3-epi-D3 and conformation of the bound ligand.

(A) Superimposition of the hVDR LBD– 1α,25(OH)2-3-epi-D3 (blue) and the hVDR LBD–1α,25(OH)2D3 (white). The ligands are shown in stick representation in blue for the 1α,25(OH)2-3-epi-D3 and in green for the 1α,25(OH)2D3. (B) The 1α,25(OH)2-3-epi-D3 is shown in its Fo – Fc electron density omit map contoured at 3 σ. The ligand is shown in stick representation with carbon and oxygen atoms in blue and red, respectively. (C) Stereo view of the ligand 3D conformations of 1α,25(OH)2-3-epi-D3 (blue) and 1α,25(OH)2D3 (green) in their VDR ligand-binding pockets (LBP).

https://doi.org/10.1371/journal.pone.0018124.g003

Conformation of the 3α-epimer in the ligand-binding pocket of hVDR

The 1α,25(OH)2-3-epi-D3, is buried in the predominantly hydrophobic ligand-binding pocket (LBP) of the VDR. The conformation of the 3-epi-hydroxyl group does not modify the A-ring chair conformation of the ligand. Furthermore the seco B-, C-, D- rings, and the aliphatic side chain present conformations similar to those observed with 1α,25(OH)2D3 (Figure 3B and C).

In the complexes of hVDR LBD bound to 1α,25(OH)2D3 versus 1α,25(OH)2-3-epi-D3, the distance between the C1-OH and the C25-OH groups varies from 13.1 Å to 12.7 Å and between the C3-OH and the C25-OH groups from 15.3 Å to 16.0 Å, respectively. The adaptation of the hVDR's LBP to different ligands can be described with the differential changes in the volumes of LBPs and bound ligands. In addition the parameter representing the % of LBP filling with ligand can provide useful information about the activity of ligand [55]. All these parameters are summarized in Table 1. Although the two diastereomer have the same molecular weight and differ only in the position of the C3-OH group, the 1α,25(OH)2-3-epi-D3 takes a slightly more compact conformation in the LBP. The graphical 0.2 Å mesh representation of the superimposed LPBs presented in Figure 4A and B show the surface area which is enlarged in case of 1α,25(OH)2D3 (in green) and the one enlarged in case of 1α,25(OH)2-3-epi-D3 bound hVDR LBP (in blue). This suggests that the hydrophobic residues lining the LBP are closer to the 3-epimer and may compensate for the canonical hydrogen bonds. We observed a notable adaptation with the displacement of the side chain of the residue Tyr147 by 2.0 Å compared to the 1α,25(OH)2D3 bound complex and the reorientation of the Glu277 side chain away from the 1α,25(OH)2-3-epi-D3 due to the α-position of the C3-OH group (Figure 4B). These specific rearrangements lead to a more compact conformation resulting in a 5% decrease in the volume of the LBP compared to 1α,25(OH)2D3.

thumbnail
Figure 4. Adaptability of the hVDR LBP upon 1α,25(OH)2-3-epi-D3 binding.

(A) The adaptation of the LBP is depicted by mesh representation of the superimposed LBP volumes calculated with Voidoo software. The green surface represent the LBP area where the 1α,25(OH)2D3 bound pocket is larger. The blue area represents similar increase but for 1α,25(OH)2-3-epi-D3 and the two main expanded regions are highlighted with red circles. (B) Adaptation of the residues Tyr147 and Glu277 in the LBP of the 1α,25(OH)2-3-epi-D3 hVDR complex. The distances between the ligand-specific positions of the residues are displayed in Å.

https://doi.org/10.1371/journal.pone.0018124.g004

Specific interactions of the 1α,25(OH)2-3-epi-D3

The hydrophobic and electrostatic interactions between the receptor and the ligand are similar between the two structures except around the C3-OH group. While the C1-OH and C25-OH display the canonical hydrogen bonds, the 3-epi-OH of 1α,25(OH)2-3-epi-D3 interacts through hydrogen bonding only with Tyr143 instead of interacting with both Tyr143 and Ser278 (Figure 5). A significant feature of the 1α,25(OH)2-3-epi-D3 is the compensation of the loss of interaction with Ser278 by a water-mediated hydrogen bond with the water molecule H2O1 (W1 in [50]). As such, the position of water H2O1 is moved 0.7 Å towards 1α,25(OH)2-3-epi-D3, thereby facilitating the specific water-mediated contacts. This water molecule is part of the network connecting another water molecule H2O2 to Arg274. All these water molecules are also present in the 1α,25(OH)2D3–hVDR complex [50]. The C3-OH hydrogen bonds have longer distances in the 3-epimer (3.0 Å instead of 2.8 Å with Tyr143 and 3.1 Å with the water molecule instead of 2.9 Å with Ser278). A study on the mutations of the residues forming the hydrogen bonds with the hydroxyl groups of 1α,25(OH)2-3-epi-D3 revealed that mutated residues contacting the 3-hydroxyl group are the less affected in term of activity. Mutation of Ser278 in Ala may result in a lower binding affinity for 1α,25(OH)2D3 [56] while showing a similar potency to activate the transcription [57], [56]. Due to the shift of the side chain of Tyr147, a hydrophobic interaction with this residue is lost in the 3-epimer structure. These structural data agree well with the lower binding affinity of this compound for VDR and to its induced biological activity.

thumbnail
Figure 5. Specific interactions of 1α,25(OH)2-3-epi-D3 in the LBP of the hVDR.

The ligands and residues in the superimposed structures are highlighted in color (1α,25(OH)2-3-epi-D3 in blue and in 1α,25(OH)2D3 green) and the important water molecules are represented with colored dots.

https://doi.org/10.1371/journal.pone.0018124.g005

In conclusion, we described a more effective synthesis of the highly stable 1α,25(OH)2-3-epi-D3, a natural metabolite. We have solved the crystal structure of hVDR LBD in complex with 1α,25(OH)2-3-epi-D3, which provides a mechanistic insight for the specific recognition of the two naturally occurring 3-epimers by hVDR. Indeed, the crystal structure reveals that the 3-epimer metabolite maintains the number of H-bonds by an alternative water-mediated interaction. In MCF-7 cells, the 1α,25(OH)2-3-epi-D3 on CYP24 gene promoter retains significant transcriptional activity. In addition, the anti-proliferative action of 1α,25(OH)2-3-epi-D3 is cell specific and the IC50 values of 1α,25(OH)2D3 and 1α,25(OH)2-3-epi-D3 in primary keratinocytes are in the same nanomolar range. Therefore, we conclude that the C3-epimerization pathway produces an active metabolite with similar biochemical and biological properties to those of the 1α,25(OH)2D3. The physiological role of this pathway as the specific biological action of the 3-epimer remains unclear and needs further investigation. However, its high metabolic stability together with its significant biologic activity makes this natural metabolite an interesting ligand for clinical applications. Further study on its target specificity and selectivity is required to the design of selective analogues. Our new findings contribute to a better understanding at molecular level how natural metabolites of 1α,25(OH)2D3 lead to significant activity in biological systems.

Materials and Methods

Ligands

1α,25(OH)2D3 was purchased from Cayman Chemical (Tallinn, Estonia) and the synthesis of 1α,25(OH)2-3-epi-D3 is described in more details in the Methods S1. Additional ligands and reference compounds are described in Methods S2. IUPAC rules were used for the name of the compounds. In addition to NMR spectra (summarized in Methods S1), HPLC analysis was used to determine the purity (>95%) of the vitamin D analogues.

Protein expression vectors for transactivation assays

Full-length cDNAs for human VDR [58] was subcloned into the T7/SV40 promoter-driven pSG5 expression vector (Stratagene, Heidelberg, Germany) and full-length cDNAs for green fluorescent protein (GFP) [59] was subcloned into parent vector resulting the pEGFP-C2 mammalian expression vector (Clontech Laboratories, Inc., USA).

Luciferase reporter gene construct

The fragment of the proximal promoter region (−414 to −64) of the human CYP24A1 gene was fused with the thymidine kinase promoter driving the firefly luciferase reporter gene [60].

Coactivator peptide recruitment assays

Biochemical interaction between human VDR-LBD and the coactivator peptide in the presence of 1α,25(OH)2D3 or 1α,25(OH)2-3-epi-D3 was assayed using the AlphaScreen technology. The assay was performed in white opaque 384-well microplate (OptiPlate-384 Perkin Elmer) using a final volume of 15 µl containing final concentrations of 100 nM E. coli-expressed hexahistidine (6xHis)-tagged VDR-LBD protein, 20 nM of the human TIF2-3 biotinylated peptide (Btn-QEPVSPKKKENALLRYLLDKDDTKD), and 10 µg/ml of both AlphaLISA Ni2+-chelate acceptor beads and (AL108C) and AlphaScreen streptavidin coated donor beads (6760002S) in an assay buffer containing 50 mM MOPS pH = 7.4, 50 mM NaF, 50 mM CHAPS, and 100 µg/ml bovine serum albumin. Different concentrations of 1α,25(OH)2D3 or 1α,25(OH)2-3-epi-D3 dissolved in DMSO (maintained at a final concentration of 1%) were added as indicated. The experiment represents two independent measurements in triplicates for, which the mean and the S.D. of the mean was calculated.

Transient transfections and luciferase reporter gene assays

MCF-7 cells were seeded into 24-well plates (100,000 cells/well) and grown overnight in phenol red-free Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% charcoal-stripped fetal bovine serum (FCS) and 0.6 µg/ml insulin. Plasmid DNA containing liposomes were formed by incubating 40 ng of an expression vector for hVDR, 100 ng of reporter plasmid and 10 ng pEGF-C2 with Fugene 6 (Roche Diagnostics, Switzerland) transfection reagent according to the recommendation of the manufacturer for 15 min at room temperature. After dilution with 500 µl of phenol red-free DMEM, the liposomes were added to the cells. Phenol red-free DMEM supplemented with 500 µl of 20% charcoal-stripped FCS was added 4 h after transfection, in the presence of ligands or solvent. The cells were lysed 16 h after the onset of stimulation using reporter gene lysis buffer (Roche Diagnostics, Switzerland). The lysates were assayed for luciferase activity as recommended by the supplier (Perkin-Elmer, The Netherlands). The luciferase activities were normalized to GFP expression. Data represent one triplicate for which the mean and the S.D. of the mean was calculated.

Data analysis for dose response curves

A non-linear curve fit was performed for the AlphaScreen and reporter gene assay experimental dose response data and from sigmoidal dose response curve then the EC50 values for the respective ligands were calculated using GraphPad Prism 4 (GraphPad Software Inc., San Diego, CA). The Student's unpaired t-test and Pearson correlation were performed with the SPSS software (SPSS Inc., version 14.0, Chicago, IL, USA).

Keratinocyte cell cultures

Normal human keratinocytes were isolated from fresh adult skin obtained from surgery and immediately transported to the laboratory under sterile conditions. Isolation and culture under serum-free conditions and without a feeder layer followed a modified protocol as used by Bikle et al [11]. The isolated epidermis was incubated in a 0.25% trypsin solution for 45 min at 37°C. Thereafter, the cells were scraped off and put in 50 ml Hank's balanced salt solution (HBSS) containing 10% FCS to block further trypsin digestion and centrifuged at 2000 rpm/2 min. The resulting cell pellet was suspended in Keratinocyte Growth Medium (KGM, Clonetics Corp., San Diego), a defined serum-free medium at low (0.06 mM) calcium containing 0.1 ng/ml epidermal growth factor, 5 µg/ml insulin, 0.5 µg/ml hydrocortisone, bovine pituitary extract, antibiotics (gentamycin, amphothericin) gave the primary culture. After 24 h, the cells were incubated at 37°C in 95% air/5% CO2 and the attached cells were washed and provided with fresh KGM medium. The culture medium was changed every other day and the cells were passaged when they reached 80–90% confluency (usually 6–10 days after plating).

Incubations of primary keratinocytes with 3H-25(OH)D3

Confluent human keratinocytes in 1 ml KGM and in 6-well plates were incubated in duplicates at 37°C with 20.6 nM 3H-25(OH)D3 (around 600 000 dpm/ml) for 1–23 h. Incubations were stopped with 1 ml methanol/well, the cells were scraped off, transferred to a test tube together with the supernatant and two washings (with 1 ml methanol and 0.8 ml water). Unmodified 3H-25(OH)D3 and most of the products were totally extracted from the combined solutions plus cell pellet according to the method of Bligh and Dyer [61] by three subsequent extractions with 2, 1 and 1 ml volumes of CHCl3 at room temperature. 3H-activity in the CHCl3-phase, in the water and total 3H-yield were determined. The combined CHCl3 extracts were then evaporated under argon at 35°C, the residues dissolved in 0.4 ml ethanol and an aliquot (containing around 250 000 dpm 3H-activity) subjected to HPLC-analysis (see Methods S2).

3H-Thymidine incorporation (anti-proliferation assay in primary keratinocytes)

Keratinocytes (second passage) in 200 µl KGM (low calcium) were plated in 96-well plates at an initial density of 104 cells/well, kept 24 h at 37°C in an incubator with 95% air/5% CO2. Thereafter, the test compounds 1,25(OH)2D3 or its 3-epimer were added in 1 µl ethanol to give final concentrations ranging from 0 to 100 nM, each condition in triplicates. After further 24 h, 50 µl 3H-thymidine (1 µCi) were added and incubation continued for additional 7 h. Then, incubations were stopped by cell harvesting (Filtermate 196 Harvester, Packard-Canberra) and lysis: After removing the supernatant (see below), the adherent cells were released by 5 min treatment with 100 ml 0.125% trypsin in PBS at 37°C, harvested on a filterplate and washed 3 x with redistilled water. After drying the plates, their bottoms were sealed with a film and 50 µl scintillation cocktail (MicroScint O, Packard) were added. The whole plates were sealed with Packard Cover Film and 3H-activity counted on a Microplate Scintillation Counter (TopCount, Packard Canberra). To check whether proliferative (3H-thymidine incorporating) cells could have been shed off, the supernatants were soaked through a 96-well filterplate (Unifilter Plate GF/C) and 3 x washed with redistilled water: in all conditions, 3H-activity was undetectable on these filterplates (in order to roughly assess cell numbers and check for substance related morphological changes/toxic effects, photographs were taken prior to compound addition and immediately before harvesting.) Data - used as means ± SD – were normalized (incorporated 3H-activity sample vs. blank) and analyzed using the GRAFIT Erithacus 4.0.19 IC50 software.

Protein purification and Crystallization

Purification and crystallization of the hVDR LBD complexed with 1α,25(OH)2-3-epi-D3 were performed as previously described [49]. The LBD of the hVDR (residues 118-427 Δ166-216) was cloned in pET-28b expression vector (Novagen) to obtain an N-terminal 6xHis fusion protein and was overproduced in E. coli BL21 (DE3) strain. Cells were grown in Luria Bertani medium and subsequently incubated for 6 h at 20°C with 1 mM isopropyl thio-β-D-galactoside. The protein purification included a metal affinity chromatography step on a Co2+-chelating resin (Clontech). The 6xHis tag was removed by thrombin digestion overnight at 4°C, and the protein was further purified by gel filtration on a Superdex S200 16/60. The sample buffer prior to protein concentration contained 10 mM Tris, pH = 7.5, 100 mM NaCl, and 10 mM dithiothreitol. The protein was concentrated to 3.5 mg/ml and incubated in the presence of a 1.5-fold molar excess of ligand. The purity and homogeneity of the protein were assessed by SDS-PAGE. The protein crystals were obtained at 4°C by vapor diffusion method using crystals of hVDR LBD-1α,25(OH)2D3 as microseeds. The reservoir solution contained 0.1 M MES and 1.4 M ammonium sulfate pH = 6.0.

X-Ray data collection and structure determination

The crystal was mounted in fiber loop and flash cooled in liquid nitrogen after cryoprotection with a solution containing the reservoir plus 30% glycerol and 2% polyethylene glycol 400. Data collection from a single frozen crystal was performed at 100 K on the beamline ID29 of the European Synchrotron Radiation Facility (Grenoble, France). The crystal belongs to the orthorhombic space group P212121 with one monomer per asymmetric unit. Data were integrated and scaled using MOSFLM [62] (see statistics in Table S1). A rigid body refinement was used with the structure of the hVDR LBD complexed to 1α,25(OH)2D3 as a starting model. Refinement involved iterative cycles of manual building and refinement calculations. The programs Refmac [63] and COOT [64] were used throughout structure determination and refinement. The omit map from the refined atomic model of hVDR LBD was used to fit the ligand to its electron density, shown in Figure 2A. Individual B-atomic factors were refined isotropically. Solvent molecules were then placed according to unassigned peaks in the difference Fourier map. In the hVDR/1α,25(OH)2-3-epi-D3 complex, refined at 1.9 Å with no σ cutoff, the final model consists of residues 118-423 (Δ166–216), the ligand, two sulphate ions and 372 water molecules. According to PROCHECK [65] 92.6% of peptide lies in most favored regions and 7.4% in additional allowed regions. Data are summarized in Table S1. The volumes of the ligand-binding pockets and ligands were calculated as previously reported [49].

Supporting Information

Figure S1.

Biological properties of 1α,25(OH)2D3 and 1α,25(OH)2-3-epi-D3 in HL60 cellular model. (A) 1α,25(OH)2-3-epi-D3-mediated HL60 cell growth. 1α,25(OH)2D3 or 1α,25(OH)2-3-epi-D3-treated HL60 at 1 nM and 100 nM concentrations are counted. Data are presented as mean±S.D. of the mean (*, p<0.05; **, p<0.01; ***, p<0.001). (B) 1α,25(OH)2-3-epi-D3-mediated HL60 cell differentiation into monocyte-like cells. HL60 cells were treated with either ethanol or 1 nM and 100"nM concentration of 1α,25(OH)2D3 or 1α,25(OH)2-3-epi-D3. Cells were labeled with PElabeled anti-human CD11c and FITC-labeled anti-human CD14, and HL60 cell differentiation was estimated by the double-positive CD11c/CD14 population. Data are representative of three distinct experiments.

https://doi.org/10.1371/journal.pone.0018124.s001

(PDF)

Figure S2.

Dominant production of the 1α,25(OH)2-3-epi-D3 in keratinocytes after 5 h. HPLC profile of the CHCl3-extract from keratinocytes after 5 h incubation is shown. The amount of 1α,25(OH)2-3-epi-D3 (blue star) is the highest from all the metabolites detected with HPLC. The peak of 1α,25(OH)2D3 is highlighted with green star.

https://doi.org/10.1371/journal.pone.0018124.s002

(PDF)

Table S1.

Data collection and refinement statistics.

https://doi.org/10.1371/journal.pone.0018124.s003

(PDF)

Scheme S2.

Synthesis of enol triflate 3. Si = TBS =  Si(t-Bu)(CH3)2.TBHP =  t-BuOOH (a) NaBH4, CeCl3·7H2O, MeOH, 0°C, 30 min. (b) TBHP, VO(acac)2, PhH, reflux, 30 min. (c) TBSCl, Im, DMF, rt, 12 h. (d) O3, MeOH-CH2Cl2, −78°C; Ac2O, Et3N, DMAP, −35°C to −8°C, 2 h; NaOAc, MeOH, 37°C, 12 h, (e) H5IO6, Et2O, rt, 2 h. (f) CBr4, Zn, Ph3P, CH2Cl2, rt, 40 min. (g) LDA, THF, −78°C, 1 h; n-BuLi, 15 min; 5-Cl-Py-2NTf2, −78°C to rt, 12 h.

https://doi.org/10.1371/journal.pone.0018124.s007

(PDF)

Scheme S3.

Synthesis of metabolite 1. TES  =  Si(CH2CH3)3. (a) (Ph3PCH2Br)Br, KOt-Bu, toluene, −5°C to rt, 1 h, 80%. (b) TESCl, Im, DMAP, DMF, rt, 3 h, 91%. (c) InCl3, t-BuLi, THF, −78°C to 0°C, 2 h. (d) 3, (Ph3P)4Pd, Et3N, THF, (dppf)PdCl2, 0°C to rt, 12 h. (e) HF·Py, Et3N, CH2Cl2, CH3CN, rt, 4 h, 58%.

https://doi.org/10.1371/journal.pone.0018124.s008

(PDF)

Acknowledgments

We thank the beam-line staff at the ESRF (Grenoble, France) for help during data collection and T. Huet for critical reading. We thank N. Rouleau and J. Hyvärinen from Perkin Elmer for support in AlphaScreen Assays and V. Prantner for help with statistical analysis.

Author Contributions

Conceived and designed the experiments: AM DM NR. Performed the experiments: FM RS YS CA IS PA JP. Analyzed the data: FM IS AM NR. Contributed reagents/materials/analysis tools: CM. Wrote the paper: FM AM NR.

References

  1. 1. Adorini L, Penna G (2008) Control of autoimmune diseases by the vitamin D endocrine system. Nat Clin Pract Rheumatol 4: 404–412.
  2. 2. Bouillon R, Eelen G, Verlinden L, Mathieu C, Carmeliet G, et al. (2006) Vitamin D and cancer. J Steroid Biochem Mol Biol 102: 156–162.
  3. 3. Pinette KV, Yee YK, Amegadzie BY, Nagpal S (2003) Vitamin D receptor as a drug discovery target. Mini Rev Med Chem 3: 193–204.
  4. 4. Campbell MJ, Adorini L (2006) The vitamin D receptor as a therapeutic target. Expert Opin Ther Targets 10: 735–748.
  5. 5. DeLuca HF (2008) Evolution of our understanding of vitamin D. Nutr Rev 66: S73–S87.
  6. 6. Nagpal S, Na S, Rathnachalam R (2005) Noncalcemic actions of vitamin D receptor ligands. Endocr Rev 26: 662–687.
  7. 7. Garabedian M, Lieberherr M, N'Guyen TM, Corvol MT, Du Bois MB, et al. (1978) The in vitro production and activity of 24, 25-dihydroxycholecalciferol in cartilage and calvarium. Clin Orthop Relat Res 135: 241–248.
  8. 8. Howard GA, Turner RT, Sherrard DJ, Baylink DJ (1981) Human bone cells in culture metabolize 25-hydroxyvitamin D3 to 1,25-dihydroxyvitamin D3 and 24,25-dihydroxyvitamin D3. J Biol Chem 256: 7738–7740.
  9. 9. Jones G, Vriezen D, Lohnes D, Palda V, Edwards NS (1987) Side-chain hydroxylation of vitamin D3 and its physiological implications. Steroids 49: 29–53.
  10. 10. Ishizuka S, Norman AW (1987) Metabolic pathways from 1α,25-dihydroxyvitamin D3 to 1α,25-dihydroxyvitamin D3-26,23-lactone. Stereo-retained and stereo-selective lactonization. J Biol Chem 262: 7165–7170.
  11. 11. Bikle DD, Nemanic MK, Whitney JO, Elias PW (1986) Neonatal human foreskin keratinocytes produce 1,25-dihydroxyvitamin D3. Biochemistry 25: 1545–1548.
  12. 12. Horst RL (1979) 25-OHD3-26,23-lactone: a metabolite of vitamin D3 that is 5 times more potent than 25-OHD3 in the rat plasma competitive protein binding radioassay. Biochem Biophys Res Commun 89: 286–293.
  13. 13. Wichmann JK, DeLuca HF, Schnoes HK, Horst RL, Shepard RM, et al. (1979) 25-Hydroxyvitamin D3 26,23-lactone: a new in vivo metabolite of vitamin D. Biochemistry 18: 4775–4780.
  14. 14. DeLuca HF, Suda T, Schnoes HK, Tanaka Y, Holick MF (1970) 25,26-dihydroxycholecalciferol, a metabolite of vitamin D3 with intestinal calcium transport activity. Biochemistry 9: 4776–4780.
  15. 15. Reinhardt TA, Napoli JL, Praminik B, Littledike ET, Beitz DC, et al. (1981) 1α-25,26-trihydroxyvitamin D3: an in vivo and in vitro metabolite of vitamin D3. Biochemistry 20: 6230–6235.
  16. 16. Holick MF, Schnoes HK, DeLuca HF, Gray RW, Boyle IT, et al. (1972) Isolation and identification of 24,25-dihydroxycholecalciferol, a metabolite of vitamin D made in the kidney. Biochemistry 11: 4251–4255.
  17. 17. Kumar R, Schnoes HK, DeLuca HF (1978) Rat intestinal 25-hydroxyvitamin D3- and 1α,25-dihydroxyvitamin D3-24-hydroxylase. J Biol Chem 253: 3804–3809.
  18. 18. Reddy GS, Rao DS, Siu-Caldera ML, Astecker N, Weiskopf A, et al. (2000) 1α,25-dihydroxy-16-ene-23-yne-vitamin D3 and 1α,25-dihydroxy-16-ene-23-yne-20-epi-vitamin D3: analogs of 1α,25-dihydroxyvitamin D3 that resist metabolism through the C-24 oxidation pathway are metabolized through the C-3 epimerization pathway. Arch Biochem Biophys 383: 197–205.
  19. 19. Norman AW, Bouillon R, Farach-Carson MC, Bishop JE, Zhou LX, et al. (1993) Demonstration that 1 beta,25-dihydroxyvitamin D3 is an antagonist of the nongenomic but not genomic biological responses and biological profile of the three A-ring diastereomers of 1α,25-dihydroxyvitamin D3. J Biol Chem 268: 20022–20030.
  20. 20. Reddy GS, Muralidharan KR, Okamura WH, Tserng K-Y, McLane JA (1994) Metabolism of 1α,25-dihydroxyvitamin D3 and one of its A-ring diastereomer 1α,25-dihydroxy-3-epivitamin D3 in neonatal human keratinocytes. In: Norman AW, Bouillon R, Thomasset M, editors. pp. 172–173. Vitamin D a pluripotent steroid hormone: Structural studies, molecular endocrinology and clinical applications, Walter de Gruyter, NY, USA. Walter de Gruyter, NY, USA.
  21. 21. Reddy GS, Siu-Caldera M-L, Schuster I, Astecker N, Tserng K-Y, et al. (1997) Target tissue specific metabolism of 1α,25-dihydroxyvitamin D3 through A-ring modification. In: Norman WA, Bouillon R, Thomasset M, editors. pp. 139–146. Vitamin D, chemistry, biology and clinical applications of the steroid hormone, Riverside, CA, USA. Riverside, CA, USA.
  22. 22. Sekimoto H, Siu-Caldera ML, Weiskopf A, Vouros P, Muralidharan KR, et al. (1999) 1α,25-dihydroxy-3-epi-vitamin D3: in vivo metabolite of 1α,25-dihydroxyvitamin D3 in rats. FEBS Lett 448: 278–282.
  23. 23. Bischof MG, Siu-Caldera ML, Weiskopf A, Vouros P, Cross HS, et al. (1998) Differentiation-related pathways of 1α,25-dihydroxycholecalciferol metabolism in human colon adenocarcinoma-derived Caco-2 cells: production of 1α,25-dihydroxy-3epi-cholecalciferol. Exp Cell Res 241: 194–201.
  24. 24. Rehan VK, Torday JS, Peleg S, Gennaro L, Vouros P, et al. (2002) 1α,25-dihydroxy-3-epi-vitamin D3, a natural metabolite of 1α,25-dihydroxy vitamin D3: production and biological activity studies in pulmonary alveolar type II cells. Mol Genet Metab 76: 46–56.
  25. 25. Brown AJ, Ritter C, Slatopolsky E, Muralidharan KR, Okamura WH, et al. (1999) 1α,25-dihydroxy-3-epi-vitamin D3, a natural metabolite of 1α,25-dihydroxyvitamin D3, is a potent suppressor of parathyroid hormone secretion. J Cell Biochem 73: 106–113.
  26. 26. Siu-Caldera ML, Sekimoto H, Weiskopf A, Vouros P, Muralidharan KR, et al. (1999) Production of 1α,25-dihydroxy-3-epi-vitamin D3 in two rat osteosarcoma cell lines (UMR 106 and ROS 17/2.8): existence of the C-3 epimerization pathway in ROS 17/2.8 cells in which the C-24 oxidation pathway is not expressed. Bone 24: 457–463.
  27. 27. Hylemon PB, Sjövall HDAJ (1985) Chapter 12 Metabolism of bile acids in intestinal microflora. New Comprehensive Biochemistry Sterols and Bile Acids. Elsevier. pp. 331–343.
  28. 28. Penning TM, Bennett MJ, Smith-Hoog S, Schlegel BP, Jez JM, et al. (1997) Structure and function of 3α-hydroxysteroid dehydrogenase. Steroids 62: 101–111.
  29. 29. Reddy GS, Rao DS, Siu-Caldera ML (2000) Natural metabolites of 1α,25-dihydroxy-vitamin D3 and its analogs. In: Norman AW, Bouillon R, Thomasset , editors. pp. 139–146. Vitamin D Endocrine system, Structural, Biological, Genetic and Clinical Aspects, University of California, Printing and Reprographics, Riverside, CA, USA.
  30. 30. Kamao M, Tatematsu S, Hatakeyama S, Sakaki T, Sawada N, et al. (2004) C-3 epimerization of vitamin D3 metabolites and further metabolism of C-3 epimers: 25-hydroxyvitamin D3 is metabolized to 3-epi-25-hydroxyvitamin D3 and subsequently metabolized through C-1α or C-24 hydroxylation. J Biol Chem 279: 15897–15907.
  31. 31. Morrison NA, Eisman JA (1991) Nonhypercalcemic 1,25-(OH)2D3 analogs potently induce the human osteocalcin gene promoter stably transfected into rat osteosarcoma cells (ROSCO-2). J Bone Miner Res 6: 893–899.
  32. 32. Fleet JC, Bradley J, Reddy GS, Ray R, Wood RJ (1996) 1α,25-(OH)2-vitamin D3 analogs with minimal in vivo calcemic activity can stimulate significant transepithelial calcium transport and mRNA expression in vitro. Arch Biochem Biophys 329: 228–234.
  33. 33. Muralidharan KR, De Lera AR, Isaeff SD, Norman AW, Okamura WH (1993) Studies of vitamin D (calciferol) and its analogs. 45. Studies on the A-ring diastereomers of 1α,25-dihydroxyvitamin D3. J Org Chem 58: 1895–1899.
  34. 34. Gómez-Reino C, Vitale C, Maestro M, Mouriño A (2005) Pd-catalyzed carbocyclization-Negishi cross-coupling cascade: a novel approach to 1α,25-dihydroxyvitamin D3 and analogues. Org Lett 7: 5885–5887.
  35. 35. Zhu G, Okamura WH (1995) Synthesis of Vitamin D (Calciferol). Chem Rev 95: 1877–1952.
  36. 36. Krause S, Schmalz HG (2000) Palladium-Catalyzed Synthesis of Vitamin D-Active Compounds. Organic Synthesis Highlights IV. Germany: Wiley and VCH. pp. 212–217.
  37. 37. Posner GH, Kahraman M (2003) Organic chemistry of vitamin D analogues (deltanoids). Eur J Org Chem 2003 3889–3895.
  38. 38. Luche JL (1978) Lanthanides in organic chemistry. 1. Selective 1, 2 reductions of conjugated ketones. J Am Chem Soc 100: 2226–2227.
  39. 39. Sharpless KB, Michaelson RC (1973) High stereo-and regioselectivities in the transition metal catalyzed epoxidations of olefinic alcohols by tert-butyl hydroperoxide. J Am Chem Soc 95: 6136–6137.
  40. 40. Daniewski AR, Garofalo LM, Hutchings SD, Kabat MM, Liu W, et al. (2002) Efficient synthesis of the A-ring phosphine oxide building block useful for 1α,25-dihydroxy-vitamin D and analogues. J Org Chem 67: 1580–1587.
  41. 41. Corey EJ, Fuchs PL (1972) A synthetic method for formyl--> ethynyl conversion (RCHO--> RCCH or RCCR'). Tetrahedron Lett 13: 3769–3772.
  42. 42. Mouriño A, Torneiro M, Vitale C, Fernández S, Pérez-Sestelo J, et al. (1997) Efficient and versatile synthesis of A-ring precursors of 1α,25-dihydroxy-vitamin D3 and analogues. Applications to the synthesis of Lythgoe-Roche phospine oxide. Tetrahedron letters 38: 4713–4716.
  43. 43. Gogoi P, Sigüeiro R, Eduardo S, Mouriño A (2010) An expeditious route to 1 α,25-dihydroxyvitamin D(3) and its analogues by an aqueous tandem palladium-catalyzed a-ring closure and suzuki coupling to the C/D unit. Eur J Chem 16: 1432–1435.
  44. 44. Trost BM, Dumas J, Villa M (1992) New Strategies for the synthesis of vitamin D metabolites via Pd-catalyzed reactions. J Am Chem Soc 114: 9836–9845.
  45. 45. Takeyama K, Masuhiro Y, Fuse H, Endoh H, Murayama A, et al. (1999) Selective interaction of vitamin D receptor with transcriptional coactivators by a vitamin D analog. Mol Cell Biol 19: 1049–1055.
  46. 46. Ullman EF, Kirakossian H, Singh S, Wu ZP, Irvin BR, et al. (1994) Luminescent oxygen channeling immunoassay: measurement of particle binding kinetics by chemiluminescence. Proc Natl Acad Sci U S A 91: 5426–5430.
  47. 47. Nakagawa K, Sowa Y, Kurobe M, Ozono K, Siu-Caldera ML, et al. (2001) Differential activities of 1α,25-dihydroxy-16-ene-vitamin D(3) analogs and their 3-epimers on human promyelocytic leukemia (HL-60) cell differentiation and apoptosis. Steroids 66: 327–337.
  48. 48. Harant H, Spinner D, Reddy GS, Lindley IJ (2000) Natural metabolites of 1α,25-dihydroxyvitamin D(3) retain biologic activity mediated through the vitamin D receptor. J Cell Biochem 78: 112–120.
  49. 49. Rochel N, Wurtz JM, Mitschler A, Klaholz B, Moras D (2000) The crystal structure of the nuclear receptor for vitamin D bound to its natural ligand. Mol Cell 5: 173–179.
  50. 50. Hourai S, Fujishima T, Kittaka A, Suhara Y, Takayama H, et al. (2006) Probing a water channel near the A-ring of receptor-bound 1 α,25-dihydroxyvitamin D3 with selected 2α-substituted analogues. J Med Chem 49: 5199–5205.
  51. 51. Eelen G, Valle N, Sato Y, Rochel N, Verlinden L, et al. (2008) Superagonistic fluorinated vitamin D3 analogs stabilize helix 12 of the vitamin D receptor. Chem Biol 15: 1029–1034.
  52. 52. Tocchini-Valentini G, Rochel N, Wurtz JM, Mitschler A, Moras D (2001) Crystal structures of the vitamin D receptor complexed to superagonist 20-epi ligands. Proc Natl Acad Sci U S A 98: 5491–5496.
  53. 53. Hourai S, Rodrigues LC, Antony P, Reina-San-Martin B, Ciesielski F, et al. (2008) Structure-based design of a superagonist ligand for the vitamin D nuclear receptor. Chem Biol 15: 383–392.
  54. 54. Rochel N, Tocchini-Valentini G, Egea PF, Juntunen K, Garnier JM, et al. (2001) Functional and structural characterization of the insertion region in the ligand binding domain of the vitamin D nuclear receptor. Eur J Biochem 268: 971–979.
  55. 55. Molnár F, Peräkylä M, Carlberg C (2006) Vitamin D receptor agonists specifically modulate the volume of the ligand-binding pocket. J Biol Chem 281: 10516–10526.
  56. 56. Reddy MD, Stoynova L, Acevedo A, Collins ED (2007) Residues of the human nuclear vitamin D receptor that form hydrogen bonding interactions with the three hydroxyl groups of 1α,25-dihydroxyvitamin D3. J Steroid Biochem Mol Biol 103: 347–351.
  57. 57. Choi M, Yamamoto K, Masuno H, Nakashima K, Taga T, et al. (2001) Ligand recognition by the vitamin D receptor. Bioorg Med Chem 9: 1721–1730.
  58. 58. Carlberg C, Bendik I, Wyss A, Meier E, Sturzenbecker LJ, et al. (1993) Two nuclear signalling pathways for vitamin D. Nature 361: 657–660.
  59. 59. Cormack BP, Valdivia RH, Falkow S (1996) FACS-optimized mutants of the green fluorescent protein (GFP). Gene 173: 33–38.
  60. 60. Väisänen S, Dunlop TW, Sinkkonen L, Frank C, Carlberg C (2005) Spatio-temporal activation of chromatin on the human CYP24 gene promoter in the presence of 1α,25-Dihydroxyvitamin D3. J Mol Biol 350: 65–77.
  61. 61. Bligh EG, Dyer WJ (1959) A rapid method of total lipid extraction and purification. Can J Biochem Physiol 37: 911–917.
  62. 62. Leslie AGW (1992) Recent changes to the MOSFLM package for processing film and image plate data. Joint CCP4 + ESF-EAMCB Newsletter on Protein Crystallography No. 26.
  63. 63. Murshudov GN, Vagin AA, Dodson EJ (1997) Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr D Biol Crystallogr 53: 240–255.
  64. 64. Emsley P, Cowtan K (2004) Coot: model-building tools for molecular graphics. Acta Crystallogr D Biol Crystallogr 60: 2126–2132.
  65. 65. Laskowski RA, MacArthur MW, Moss DS, Thornton JM (1993) PROCHECK: A program to check the stereochemical quality of protein structures. J Appl Crystallogr 26: 283–291.