VDR Ligands Research Articles

Development of nanoluciferase-based sensing system that can specifically detect 1α,25-dihydroxyvitamin D in living cells

Previously, we reported a FLucN-LXXLL+LBD-FLucC system that detects VDR ligands using split firefly luciferase techniques, ligand binding domain (LBD) of VDR, and LXXLL sequences that interact with LBD after VDR ligand binding. In vivo, 25-hydroxyvitamin D3 (25(OH)D3) and 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3) act as VDR ligands that bind to VDR, and regulate bone-related gene expression. Therefore, the amount of 25(OH)D3 and 1α,25(OH)2D3 are indicators of bone-related diseases such as rickets and osteoporosis. In this study, we have developed a novel LgBiT-LXXLL+LBD-SmBiT system using NanoLuc Binary Technology (NanoBiT), which has an emission intensity several times higher than that of the split-type firefly luciferase. Furthermore, by using genetic engineering techniques, we attempted to construct a novel system that can specifically detect 1α,25(OH)2D3. Because histidine residues at positions 305 and 397 play important roles in forming a hydrogen bond with a hydroxyl group at position C25 of 25(OH)D3 and 1α,25(OH)2D3, His305 and His397 were each substituted by other amino acids. Consequently, the three mutant VDRs, H305D, H397N, and H397E were equally useful to detect 1α,25(OH)2D3 specifically. In addition, among the 58 variants of the LXXLL sequences, LPYEGSLLLKLLRAPVEE showed the greatest increase in luminescence upon the addition of 25(OH)D3 or 1α,25(OH)2D3. Thus, our novel system using NanoBiT appear to be useful for detecting native vitamin D or its derivatives.

The Centennial Collection of VDR Ligands: Metabolites, Analogs, Hybrids and Non-Secosteroidal Ligands.

Since the discovery of vitamin D a century ago, a great number of metabolites, analogs, hybrids and nonsteroidal VDR ligands have been developed. An enormous effort has been made to synthesize compounds which present beneficial properties while attaining lower calcium serum levels than calcitriol. This structural review covers VDR ligands published to date.

Development of a split luciferase biosensor that dramatically increases light intensity in response to VDR ligands

Development of a split luciferase biosensor that dramatically increases light intensity in response to VDR ligands

Nuclear Receptors Regulate Intestinal Inflammation in the Context of IBD.

Gastrointestinal (GI) homeostasis is strongly dependent on nuclear receptor (NR) functions. They play a variety of roles ranging from nutrient uptake, sensing of microbial metabolites, regulation of epithelial intestinal cell integrity to shaping of the intestinal immune cell repertoire. Several NRs are associated with GI pathologies; therefore, systematic analysis of NR biology, the underlying molecular mechanisms, and regulation of target genes can be expected to help greatly in uncovering the course of GI diseases. Recently, an increasing number of NRs has been validated as potential drug targets for therapeutic intervention in patients with inflammatory bowel disease (IBD). Besides the classical glucocorticoids, especially PPARγ, VDR, or PXR-selective ligands are currently being tested with promising results in clinical IBD trials. Also, several pre-clinical animal studies are being performed with NRs. This review focuses on the complex biology of NRs and their context-dependent anti- or pro-inflammatory activities in the regulation of gastrointestinal barrier with special attention to NRs already pharmacologically targeted in clinic and pre-clinical IBD treatment regimens.

Structural development of non-secosteroidal vitamin D receptor (VDR) ligands without any asymmetric carbon.

Non-secosteroidal VDR ligands without any assymmetric carbon were designed and synthesized based on the structure of the previously reported non-secosteroidal VDR agonist LG190178. The VDR-agonistic activity of all synthesized compounds was evaluated, and 7b emerged as a potent agonist activity with an EC50 value of 9.26 nM. Moreover, a docking simulation analysis was also performed to determine the binding mode of 7b with VDR-LBD.

Strategies and limitations associated with in vitro characterization of vitamin D receptor activators

In vitro cell-based assays are common screening tools used for the identification of new VDR ligands. For 25-hydroxyvitamin D3 [25(OH)D3] and 1α-hydroxyvitamin D3 [1α(OH)D3], protein expressions of CYP2R1 and CYP27B1, respectively, that form the active 1α,25-dihydroxyvitamin D3 [1,25(OH)2D3] ligand were detected in human embryonic kidney (HEK293) cells expressing the GAL4-hVDR, the human brain microvessel endothelial (hCMEC/D3) and adenocarcinoma colonic (Caco-2) cells. The impact of bioactivation enzymes was shown upon the addition of ketoconazole (10 μM KTZ), a pan-CYP inhibitor, which reduced the apparent potency of 25(OH)D3 and increased the EC50 from 272 to 608 nM in HEK293 cells. EIA assays verified that 1,25(OH)2D3 was formed and contributed to VDR activity independently of its precursors. In hCMEC/D3 cells where enzyme protein levels were lowest, changes in MDR1/P-gp expression with KTZ were minimal. In Caco-2 cells, the induction of TRPV6 (calcium channel), CYP24A1, CYP3A4, OATP1A2 and MDR1 mRNA expression was 1,25(OH)2D3 > 1α(OH)D3 > 25(OH)D3, with the magnitude of change being blunted by KTZ. Upon inclusion of KTZ in the cell-based assays, high transcriptional activities were observed for synthetic VDR activators from Teijin Pharma. Cyclopentanone derivatives: TPD-003, TPD-005, TPD-006, TPD-008 and TPD-009 (EC50s 0.06 to 67 nM, unchanged with KTZ) were found more potent over straight chain and lactone derivatives (antagonists). Most TPD compounds activated OATP1A2, CYP24A1, CYP3A4, and MDR1 (28–67%) and TRPV6 transcriptionally in Caco-2 cells. The results identified that cell-based assays with added KTZ could accurately identify new VDR activators, although these may be hypercalcemic with strong TRPV6 inducing properties.

Aromatic-Based Design of Highly Active and Noncalcemic Vitamin D Receptor Agonists.

We report the design, synthesis, biological evaluation, and structural analysis of a new class of vitamin D analogues that possess an aromatic m-phenylene D-ring and an alkyl chain replacing the C-ring. A key feature of the synthetic strategy is a stereoselective Pd-catalyzed construction of the triene system in aqueous medium that allows the rapid preparation of small amounts of VDR ligands for biological screening. Analogues with the shorter (2a) and longer (2d, 2e) side chains attached to the triene system have no calcemic activity. Compound 2a binds to VDR with the same order of magnitude than calcipotriol and oxacalcitriol. It also reduces proliferation in normal and tumor cells similarly to the natural hormone 1α,25-dihydroxyvitamin D3, calcipotriol, and oxacalcitriol, suggesting preclinical studies related to hyperproliferative disorders such as psoriasis and cancer.

Update on recent progress in vitamin D research. Development of novel Vitamin D Derivatives.

Vitamin D3 plays important roles in many physiological processes, including calcium and phosphate homeostasis, bone metabolism, and immune regulation. An active form of vitamin D3, 1α,25(OH)2D3, binds to vitamin D nuclear receptor(VDR, vitamin D receptor), and regulates expression of specific target genes. Thousands of vitamin D3 analogs have been synthesized, and the structure-activity relationships of secosteroidal compounds have been examined in detail. On the other hand, development of non-secosteroidal VDR ligands remains limited, and only a few non-secosteroidal VDR ligands with potent activity have been reported so far. Development of novel structures with unique biological profiles would enable the new clinical application of vitamin D function.

Development and Characterization of a Human Reporter Cell Line for the Assessment of Thyroid Receptor Transcriptional Activity: A Case of Organotin Endocrine Disruptors.

We developed and characterized the human luciferase reporter cell line PZ-TR for the assessment of thyroid receptor (TR) transcriptional activity. Triiodothyronine (T3) induced luciferase activity in a dose-dependent manner, and the sensitivity of assay allowed for the detection of nanomolar T3 concentrations. The luciferase activity was induced by a maximum of (2.42 ± 0.14)-(2.73 ± 0.23)-fold after 24 h of exposure to 10 nM T3. We did not observe a nonspecific induction of luciferase activity by other steroid hormones and VDR ligands, with the exception of partial activation by retinoic acids. Cryopreservation of PZ-TR cells did not influence their functionality, responsivity to T3, or cell morphology, even after long-term cultivation. PZ-TR cells were used to evaluate the effects of organic tin compounds on TR. We found that the tributyltin and triphenyltin derivatives induced luciferase activity and that application of organotins in combination with T3 enhanced the effect of T3. These findings indicate that organic tin compounds have potential to interfere with TR-mediated regulation of gene expression and influence the physiological activity of thyroid hormones.

Vitamin D receptor agonist EB1089 is a potent regulator of prostatic “intracrine” metabolism

A contributing factor to the emergence of castrate resistant prostate cancer (CRPC) is the ability of the tumor to circumvent low circulating levels of testosterone during androgen deprivation therapy (ADT), through the production of "intracrine" tumoral androgens from precursors including cholesterol and dehydroepiandrosterone (DHEA). As these processes promote AR signaling and prostate cancer progression their modulation is required for disease prevention and treatment. We evaluated the involvement of the vitamin D receptor ligand EB1089 in the regulation of genes with a role in androgen metabolism using the androgen dependent cell lines LNCaP and LAPC-4. EB1089 regulation of androgen metabolism was assessed using QRT-PCR, luciferase promoter assays, western blotting, enzyme activity assays, and LC-MS analyses. EB1089 induced significant expression of genes involved in androgen metabolism in prostate cancer cells. Real-Time PCR analysis revealed that VDR mediated significant regulation of CYP3A4, CYP3A5, CYP3A43, AKR1C1-3, UGT2B15/17, and HSD17B2. Data revealed potent regulation of CYP3A4 at the level of mRNA, protein expression and enzymatic activity, with VDR identified as the predominant regulator. Inhibition of CYP3A activity using the specific inhibitor ritonavir resulted in alleviation of the anti-proliferative response of VDR ligands in prostate cancer cells. Mass spectrometry revealed that overexpression of CYP3A protein in prostate cancer cells resulted in a significant increase in the oxidative inactivation of testosterone and DHEA to their 6-β-hydroxy-testosterone and 16-α-hydroxy-DHEA metabolites, respectively. These data highlight a potential application of VDR-based therapies for the reduction of growth-promoting androgens within the tumor micro-environment.

Development of novel non-secosteroidal VDR ligands based on spherical hydrophobic pharmacophores

Development of novel non-secosteroidal VDR ligands based on spherical hydrophobic pharmacophores

The role of lithocholic acid in the regulation of bile acid detoxication, synthesis, and transport proteins in rat and human intestine and liver slices

The effects of the secondary bile acid, lithocholic acid (LCA), a VDR, FXR and PXR ligand, on the regulation of bile acid metabolism (CYP3A isozymes), synthesis (CYP7A1), and transporter proteins (MRP3, MRP2, BSEP, NTCP) as well as nuclear receptors (FXR, PXR, LXRα, HNF1α, HNF4α and SHP) were studied in rat and human precision-cut intestine and liver slices at the mRNA level. Changes due to 5 to 10 μM of LCA were compared to those of other prototype ligands for VDR, FXR, PXR and GR. LCA induced rCYP3A1 and rCYP3A9 in the rat jejunum, ileum and colon, rCYP3A2 only in the ileum, rCYP3A9 expression in the liver, and CYP3A4 in the human ileum but not in liver. LCA induced the expression of rMRP2 in the colon but not in the jejunum and ileum but did not affect rMRP3 expression along the length of the rat intestine. In human ileum slices, LCA induced hMRP3 and hMRP2 expression. In rat liver slices, LCA decreased rCYP7A1, rLXRα and rHNF4α expression, induced rSHP expression, but did not affect rBSEP or rNTCP expression; whereas in the human liver, a small but significant decrease was found for hHNF1α expression. These data suggests profound species differences in the effects of LCA on bile acid transport, synthesis and detoxification. An examination of the effects of prototype VDR, PXR, GR and FXR ligands showed that these pathways are all intact in precision cut slices and that LCA exerted VDR, PXR and FXR effects. The LCA-induced altered enzymes and transporter expressions in the intestine and liver would affect the disposition of drugs.

Functionality of unliganded VDR in breast cancer cells: repressive action on CYP24 basal transcription

It is well-established that CYP24, an immediate target gene of VDR is upregulated by VDR ligands. This study is focused on the functional role of unliganded VDR by investigating the correlation between the expression of VDR protein and basal mRNA levels of CYP24 in breast cancer cell lines. Analyses of multiple breast cancer cell lines demonstrated an inverse correlation between VDR protein expression and CYP24 mRNA expression levels; while in the presence of ligand, VDR protein level was positively correlated with CYP24 expression. In MCF-7 cells, VDR was mainly distributed in the nuclei in the absence of ligand. VDR overexpression in MCF-7 cells and MDA-MB231 cells decreased CYP24 mRNA expression levels and CYP24 promoter activity. Conversely, knock-down of VDR using siRNA techniques in MCF-7 and T47D cells significantly increased CYP24 mRNA expression. We also found that overexpression of VDR with a polymorphic site (FokI-FF) at its AF-1 domain, which makes VDR shorter by three amino acids, failed to repress CYP24 promoter activity. This report provides conclusive evidence for the repressive action of unliganded VDR on the expression of its target gene CYP24 and the importance of an intact VDR AF-1 domain for its repressive action.

A Novel Bile Acid-Activated Vitamin D Receptor Signaling in Human Hepatocytes

ABSTRACT Vitamin D receptor (VDR) is activated by natural ligands, 1α, 25-dihydroxy-vitamin D3 (1α, 25(OH)2-D3) and lithocholic acid (LCA). Our previous study shows that VDR is expressed in human hepatocytes, and VDR ligands inhibit bile acid synthesis and transcription of the gene encoding cholesterol 7α-hydroxylase (CYP7A1). Primary human hepatocytes were used to study LCA and 1α, 25(OH)2-D3 activation of VDR signaling. Confocal immunofluorescent microscopy imaging and immunoblot analysis showed that LCA and 1α, 25(OH)2-D3 induced intracellular translocation of VDR from the cytosol to the nucleus, and also plasma membrane where VDR co-localized with caveolin-1. VDR ligands induced tyrosine phosphorylation of c-Src and VDR, and their interaction. Inhibition of c-Src abrogated VDR ligand-dependent inhibition of CYP7A1 mRNA expression. Kinase assays showed that VDR ligands specifically activated the c-Raf/MEK1/2/ERK1/2 pathway, which stimulates serine phosphorylation of VDR and HNF4α, and their interaction. Mammalian two-hybrid assays showed a VDR ligand dependent interaction of nuclear receptor corepressor-1 (NCoR-1) and silencing mediator of retinoid and thyroid (SMRT) with VDR/RXRα. Chromatin immunoprecipitation assays revealed that an ERK1/2 inhibitor reversed VDR ligand-induced recruitment of VDR, RXRα and corepressors to human CYP7A1 promoter. In conclusion, VDR ligands activate membrane VDR signaling to activate the MEK1/2/ERK1/2 pathway, which stimulates nuclear VDR/RXRα recruitment of co-repressors to inhibit CYP7A1 gene transcription in human hepatocytes. This membrane VDR signaling pathway may be activated by bile acids to inhibit bile acid synthesis as a rapid response to protect hepatocytes from cholestatic liver injury.

Cellular and Molecular Mechanisms Involved in the Action of Vitamin D Analogs Targeting Vitiligo Depigmentation

The active metabolite of vitamin D3 - 1,25-(OH)2D3 - exerts most of its physiological and pharmacological actions through its nuclear receptor (VDR), regulating the transcriptional machinery of a variety of cell types. Basic research motivated by the detection of VDR in numerous target cells, has indicated potential therapeutic applications of VDR ligands in osteoporosis, cancer, secondary hyperparathyroidism and autoimmune diseases such as psoriasis, systemic lupus erythematosus, rheumatoid arthritis, type 1 diabetes and multiple sclerosis. In recent years vitamin D analogs, particularly calcipotriol and tacalcitol, have been used as topical therapeutic agents in vitiligo, an autoimmune pigmentary disorder characterized by aberrant loss of functional melanocytes from involved epidermis. The presence of cytotoxic T cells targeting melanocyte antigens and imbalance of the cytokine network were described as characteristics of the disease, eventually leading to melanocyte damage and death. Vitamin D ligands are designed to target the local immune response in vitiligo, acting on specific T cell activation, mainly by inhibiting the transition of T cells from early to late G1 phase and by inhibiting the expression of several pro-inflammatory cytokines genes, such as those encoding tumor necrosis factor alpha (TNF-alpha) and interferon gamma (IFN-gamma). Vitamin D(3) compounds are known to influence melanocyte maturation and differentiation and also to up-regulate melanogenesis through pathways activated by specific ligand receptors, such as endothelin receptor and c-kit. In this review we summarize the complex pathogenetic rationale of vitamin D analogs in vitiligo depigmentation. Understanding the cellular and molecular mechanisms through which vitamin D targets the epidermal melanin unit is of great interest for identification of new effective therapeutic combination(s) that might induce repigmentation in vitiligo.

Identification and characterization of a novel nonsecosteroidal vitamin D receptor ligand

AbstractVitamin D receptor (VDR) and its ligands play important roles in mineral/skeletal homeostasis, cell proliferation/differentiation, and modulation of immune responses. VDR ligands make attractive candidates for the treatment/prevention of osteoporosis, psoriasis, and cancer. The major issue with current analogs is hypercalcemia. As nonsteroidal therapeutic agents with fewer side effects have been made for androgen and estrogen receptors, nonsecosteroidal VDR ligands possessing beneficial effects may also be possible. Here we present a novel podocarpic acid‐based nonsecosteroidal VDR ligand (VDRL‐1). VDRL‐1 bound to VDR with a Ki of ∼1.3 µM in competition binding assays. Data from binding and molecular docking suggest that VDRL‐1 interacts with the same binding pocket as 1α,25‐(OH)2 vitamin D3 (1,25‐D3). In cell‐based transcription assays, VDRL‐1 reached the same maximal activity as 1,25‐D3, albeit with an EC50 ∼1.0 µM. Likewise, VDRL‐1 was a full agonist in suppressing proliferation of several human cancer cells. Microarrays demonstrated that VDRL‐1 exerted a near identical expression pattern to that of 1,25‐D3 in several human cancer cells. Taqman analysis of known and novel VDR‐regulated genes showed that VDRL‐1 was a full agonist on osteocalcin (OC), CYP24A1, and other genes. It exerted partial agonist activity on G0/G1 switch gene 2 (G0S2). Furthermore, VDRL‐1 could be a full or partial agonist of calcium transporter 1 (TRPV6) expression and partially antagonized 1,25‐D3 in cells where it partially regulated TRPV6. Thus, the current study describes a novel nonsecosteroidal VDR ligand that behaves mostly like 1,25‐D3, but possesses unique characteristics. Drug Dev Res 68:51‐60, 2007. © 2007 Wiley‐Liss, Inc.

Ligand Recognition by Vitamin D Receptor: Total Alanine Scanning Mutational Analysis of the Residues Lining the Ligand Binding Pocket of Vitamin D Receptor

We performed total alanine scanning mutational analysis (ASMA) of the residues lining the ligand binding pocket (LBP) of the human vitamin D receptor (hVDR) to investigate allosteric effects of ligands in the function of nuclear receptors (NRs). This was accomplished for the first time in the NR superfamily. The effects of ligand structure were also examined in this system (termed 2D-ASMA) using 8 representative VDR ligands. The results clearly revealed the role and importance of all amino acid residues lining the LBP and the relationships between ligand binding and transcriptional potency. 2D-ASMA indicated ligand-specific ligand-protein interactions, which are essential in determining the transactivation potency of the ligand. Taking the results as a whole, we suggest a ligand-mediated allosteric network, which allows transmission of information from ligands to the interfaces of the VDR in association with protein cofactors and was shown to be linked to a part of the network identified by statistical coupling analysis (SCA).

Tolerogenic dendritic cells induced by vitamin D receptor ligands enhance regulatory T cells inhibiting allograft rejection and autoimmune diseases.

Dendritic cells (DCs) not only induce but also modulate T cell activation. 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] induces DCs with a tolerogenic phenotype, characterized by decreased expression of CD40, CD80, and CD86 costimulatory molecules, low IL-12 and enhanced IL-10 secretion. We have found that a short treatment with 1,25(OH)(2)D(3) induces tolerance to fully mismatched mouse islet allografts that is stable to challenge with donor-type spleen cells and allows acceptance of donor-type vascularized heart grafts. This effect is enhanced by co-administration of mycophenolate mofetil (MMF), a selective inhibitor of T and B cell proliferation that has also effects similar to 1,25(OH)(2)D(3) on DCs. Graft acceptance is associated with an increased percentage of CD4(+)CD25(+) regulatory cells in the spleen and in the draining lymph node that can protect 100% of syngeneic recipients from islet allograft rejection. CD4(+)CD25(+) cells, able to inhibit the T cell response to a pancreatic autoantigen and to significantly delay disease transfer by pathogenic CD4(+)CD25(-) cells, are also induced by treatment of adult nonobese diabetic (NOD) mice with 1,25-dihydroxy-16,23Z-diene-26,27-hexafluoro-19-nor vitamin D(3) (BXL-698). This treatment arrests progression of insulitis and Th1 cell infiltration, and inhibits diabetes development at non-hypercalcemic doses. The enhancement of CD4(+)CD25(+) regulatory T cells, able to mediate transplantation tolerance and to arrest type 1 diabetes development by a short oral treatment with VDR ligands, suggests possible clinical applications of this approach.

Vitamin D analogs: mechanism of action and therapeutic applications.

The physiological VDR ligand, 1 alpha,25-dihydroxyvitamin D3, acts upon a wide variety of tissues and cells, both related to and unrelated to calcium and phosphate homeostasis. The noncalcemic actions of natural and synthetic VDR ligands are exemplified by their potent anti-proliferative, prodifferentiative and immunomodulatory activities. As a result, a VDR ligand is an approved drug for the topical treatment of psoriasis. A plethora of actions of 1 alpha,25-dihydroxyvitamin D3 in various systems have suggested wide clinical applications of VDR ligands in such diverse disease states as inflammation (rheumatoid arthritis, psoriatic arthritis), dermatological indications (psoriasis, photoaging and skin rejuvenation), osteoporosis, cancers (breast, prostate, colon, leukemia and myelodysplastic syndrome) and autoimmune diseases (multiple sclerosis, type I diabetes and systemic lupus erythematosus). VDR ligands have shown therapeutic potential in limited human clinical trials as well as in animal models of these diseases. Some of the VDR ligands have shown not only potent preventive but also therapeutic anabolic activities in animal models of osteoporosis. However, the use of VDR in above mentioned indications as well as in oral therapy for psoriasis and even topical therapy for severe psoriasis is hampered by its associated toxicity, namely hypercalcemia. New VDR ligands have been synthesized which exhibit greater specificity by retaining desirable properties, but with reduced calcemic potential. The discovery of novel vitamin D3 analogs along with an increased understanding of the biological functions and mechanisms of action of VDR are likely to result in improved treatments for responsive indications.

Selective recognition of vitamin D receptor conformations mediates promoter selectivity of vitamin D analogs.

The transcription factor VDR is the nuclear receptor for 1alpha, 25-dihydroxyvitamin D3 (VD) and the mediator of all genomic actions of the nuclear hormone and its synthetic analogs. The sharp biological profile of the model VD analog 1(S), 3(R)-dihydroxy-20(R)-(5'-ethyl-5'-hydroxy-hepta-1'(E), 3'(E)-dien-1'-yl)-9,10-secopregna-5(Z),7(E),10(19)-triene (EB1089) (i.e., its high antiproliferative effect combined with low calcemic actions) has been correlated with the selectivity of EB1089 to activate heterodimeric complexes of VDR with its partner retinoid X receptor (RXR) on VD response elements (VDREs). These VDREs are formed by an inverted palindromic arrangement of two hexameric core binding motifs spaced by nine nucleotides (IP9) rather than VDREs that are formed by direct repeats with three intervening nucleotides (DR3). In this report, ligand-dependent gel-shift assays were used for a comparison of the ability of VD and EB1089 to stabilize VDR-RXR heterodimers on these two VDRE types. The gel-shift assays revealed EB1089 to be more sensitive for complexes on IP9-type VDREs than on DR3-type VDREs. In addition, a gel-shift clipping method was established to identify and compare complexes of ligand-stabilized VDR-RXR heterodimers on different VDREs. On each VDRE, two complexes could be discriminated that seemed to contain different functional conformations of the VDR and allowed a more differential view on DNA-complexed VDR-RXR heterodimers. The VDR-RXR conformation (which was more ligand-sensitive) gained through EB1089 a higher affinity (7-fold) for DNA binding and a more sensitive (9-fold) activation of an IP9-type VDRE than of a DR3-type VDRE, whereas with the natural hormone VD, no VDRE-type preference could be observed. This indicates that promoter selectivity of VDR ligands is based on their property to selectively increase affinity for VDREs and very sensitively stabilize VDR conformations in VDR-RXR-VDRE complexes.