Structural Studies of Vitamin D Nuclear Receptor Ligand-Binding Properties.

The vitamin D nuclear receptor (VDR) and its natural ligand, 1α,25-dihydroxyvitamin D3 hormone (1,25(OH)2D3, or calcitriol), classically regulate mineral homeostasis and metabolism but also much broader range of biological functions, such as cell growth, differentiation, antiproliferation, apoptosis, adaptive/innate immune responses. Being widely expressed in various tissues, VDR represents an important therapeutic target in the treatment of diverse disorders. Since ligand binding is a key step in VDR-mediated signaling, numerous 1,25(OH)2D3 analogs have been synthesized in order to selectively modulate the receptor activity. Most of the synthetic analogs have been developed by modification of a parental compound and some of them mimic 1,25(OH)2D3 scaffold without being structurally related to it. The ability of ligands that have different size and conformation to bind to VDR and to demonstrate biological effects is intriguing, and therefore, ligand-binding properties of the receptor have been extensively investigated using a variety of biochemical, biophysical, and computational methods. In this chapter, we describe different aspects of the structure-function relationship of VDR in complex with natural and synthetic ligands coming from structural analysis. With the emphasis on the binding modes of the most promising compounds, such as secosteroidal agonists and 1,25(OH)2D3 mimics, we also highlight the action of VDR antagonists and the evidence for the existence of an alternative ligand-binding site within the receptor. Additionally, we describe the crystal structures of VDR mutants associated with hereditary vitamin D-resistant rickets that display impaired ligand-binding function.

1α,25-dihydroxyvitamin D3 and rosiglitazone synergistically enhance osteoblast-mediated mineralization.

Both vitamin D receptor (VDR) and peroxisome proliferator-activated receptor γ (PPAR-γ) are ligand-activated nuclear transcription factors that are instrumental for bone health. While 1α,25-dihydroxyvitamin D3 (1,25D3), the ligand for VDR, is essential for the development and maintenance of healthy bone, PPAR-γ agonists cause detrimental skeletal effects. Recent studies have revealed evidence for a cross-talk between 1,25D3- and PPAR-α/-δ ligand-mediated signaling but there is a current lack of knowledge regarding cross-talk between signaling of 1,25D3 and the PPAR-γ ligand mediated signaling. In this study, we investigated the cross-talk between 1,25D3- and PPAR-γ agonist rosiglitazone-mediated signaling in human osteoblasts. 1,25D3 slightly but significantly induced expression of the primary PPAR-γ target gene ANGPTL4 but did not influence FABP4. 1,25D3 did not change rosiglitazone regulation of ANGPTL4 and FABP4. The other way around, rosiglitazone reduced CYP24A1 gene expression but this did not change CYP24A1 induction by 1,25D3. The findings regarding CYP24A1 gene expression are in line with the observation that 1,25D3 levels in medium were not affected by rosiglitazone. Furthermore, rosiglitazone significantly inhibited 1,25D3-induction of BGLAP while rosiglitazone alone did not change BGLAP. Additionally, 1,25D3 and rosiglitazone increase osteoblast alkaline phosphatase activity and synergistically stimulated extracellular matrix mineralization. In conclusion, these data provide evidence for a cross-talk between rosiglitazone- and 1,25D3-mediated signaling leading to an acceleration of extracellular matrix mineralization. The data suggest that the reduction of the mineralization inhibitor BGLAP and the increased differentiation status underlie the increased mineralization.

Hydrogen/deuterium exchange reveals distinct agonist/partial agonist receptor dynamics within vitamin D receptor/retinoid X receptor heterodimer.

Regulation of nuclear receptor (NR) activity is driven by alterations in the conformational dynamics of the receptor upon ligand binding. Previously, we demonstrated that hydrogen/deuterium exchange (HDX) can be applied to determine novel mechanism of action of PPARγ ligands and in predicting tissue specificity of selective estrogen receptor modulators. Here, we applied HDX to probe the conformational dynamics of the ligand binding domain (LBD) of the vitamin D receptor (VDR) upon binding its natural ligand 1α,25-dihydroxyvitamin D3 (1,25D3), and two analogs, alfacalcidol and ED-71. Comparison of HDX profiles from ligands in complex with the LBD with full-length receptor bound to its cognate receptor retinoid X receptor (RXR) revealed unique receptor dynamics that could not be inferred from static crystal structures. These results demonstrate that ligands modulate the dynamics of the heterodimer interface as well as provide insight into the role of AF-2 dynamics in the action of VDR partial agonists.

Inhibition of mTORC1 by RAD001 (everolimus) potentiates the effects of 1,25-dihydroxyvitamin D(3) to induce growth arrest and differentiation of AML cells in vitro and in vivo.

OBJECTIVE: Differentiation-inducing therapy by agents such as 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) represents a useful approach for the treatment of acute myelogenous leukemia (AML). We previously showed that Gemini-23-yne-26,27-hexafluoro-D(3) inhibited the proliferation of MCF-7 breast cancer cells in association with inhibition of the mammalian target of rapamycin (mTOR) signaling. This study explored the drug interaction of 1,25(OH)(2)D(3) and rapamycin analog RAD001 (everolimus) in AML cells. MATERIALS AND METHODS: Effects of RAD001 and 1,25-(OH)(2)D(3) on the proliferation and differentiation of U937 cells were assessed by colony-forming assay and quantification of CD11b cell surface antigens and their endocytic capability, respectively. Effects of RAD001 and 1,25-(OH)(2)D(3) on Akt/mTOR complex-1 (mTORC1) signaling and cell-cycle-related molecules were explored by Western blot analysis. The reporter gene and chromatin immunoprecipitation assays were employed to examine the effects of RAD001 and 1,25-(OH)(2)D(3) on the promoter of the p21(waf1) gene. U937 murine xenograft model was utilized to explore the effects of RAD001 and 1,25-(OH)(2)D(3) in vivo. RESULTS: RAD001 potentiated the ability of 1,25(OH)(2)D(3) to induce growth arrest and differentiation of AML cells in parallel with downregulation of the levels of p-S6K and p-4E-BP1, substrates of mTORC1. In addition, RAD001 significantly enhanced 1,25(OH)(2)D(3)-mediated transcriptional activity of p21(waf1) in association with increased levels of the acetylated forms of histone H3 and vitamin D receptor bound to the p21(waf1) promoter in U937 cells. Moreover, RAD001 (3 mg/kg, every another day) significantly enhanced 1,25(OH)(2)D(3)-induced growth inhibition of U937 tumor xenografts in nude mice without adverse effects. CONCLUSIONS: Concomitant administration of 1,25(OH)(2)D(3) and the mTORC1 inhibitor may be a promising treatment strategy for individuals with AML.

Direct transcriptional regulation of RelB by 1alpha,25-dihydroxyvitamin D3 and its analogs: physiologic and therapeutic implications for dendritic cell function.

The nuclear factor-kappaB (NF-kappaB) protein RelB plays a unique role in dendritic cell (DC) function and, as such, is an important regulator of antigen presentation and immune regulation. In this study, inhibition of RelB expression in DCs exposed to an analog of the active form of vitamin D3 (1alpha,25-dihydroxyvitamin D3 (1alpha,25-(OH)2D3)) was observed and shown to be mediated by the vitamin D receptor (VDR). Potential vitamin D response elements were identified within promoter regions of human and mouse relB genes. In gel shift experiments, these motifs specifically bound VDR.retinoid X receptor-alpha complexes. Reporter assays confirmed that transcriptional activity of human and mouse relB promoters was inhibited by 1alpha,25-(OH)2D3 agonists in a DC-derived cell line. The inhibition was abolished by mutagenesis of the putative vitamin D response elements and was enhanced by overexpression of VDR. Mutagenesis of NF-kappaB response elements within the relB promoter did not affect the magnitude of 1alpha,25-(OH)2D3 analog-mediated inhibition, ruling out an indirect effect on NF-kappaB signaling. Glucocorticoid caused additional inhibition of relB promoter activity when combined with the 1alpha,25-(OH)2D3 analog. This effect was dependent on the integrity of the NF-kappaB response elements, suggesting separate regulatory mechanisms for the two steroid pathways on this promoter. We conclude that relB is a direct target for 1alpha,25-(OH)2D3-mediated negative transcriptional regulation via binding of VDR.retinoid X receptor-alpha to discrete DNA motifs. This mechanism has important implications for the inhibitory effect of 1alpha,25-(OH)2D3 on DC maturation and for the potential immunotherapeutic use of 1alpha,25-(OH)2D3 analogs alone or combined with other agents.

Regulation of NRAMP1 gene expression by 1alpha,25-dihydroxy-vitamin D(3) in HL-60 phagocytes.

The natural resistance-associated macrophage protein 1 (Nramp1) is a proton-dependent transporter of divalent metals. We studied NRAMP1 expression during HL-60 differentiation induced by VD and VD agonists. NRAMP1 and CD14 gene expression differed in kinetics of induction, mRNA levels and stability, and response to VD combined with PMA, whereas a combination of VD and IFN-gamma induced similar up-regulation. NRAMP1 protein expression paralleled the accumulation of mRNA and was localized in the phagosomal membrane after phagocytosis. A promoter construct extending 647 bp upstream of NRAMP1 ATG showed myeloid-specific transcription in transient transfection assays, which was up-regulated by VD in HL-60. In HL-60 clones stably transfected with this construct, transcription was apparently induced through indirect VD genomic effects, and there was accordance between the levels of reporter transcription and endogenous NRAMP1 mRNA in response to VD but not to IFN-gamma. Thus, VD genomic effects stimulate NRAMP1 transcription and protein expression in maturing phagocytes.