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Background: 1,25-Dihydroxycholecalciferol (1,25(OH)(2)D(3)) has been shown to mitigate epithelial inflammatory responses after antigen exposure. Patients with cystic fibrosis (CF) are at particular risk for vitamin D deficiency. This may contribute to the exaggerated inflammatory response to pulmonary infection in CF., Methods: CF respiratory epithelial cell lines were exposed to Pseudomonas aeruginosa lipopolysaccharide (LPS) and Pseudomonas conditioned medium (PCM) in the presence or absence of 1,25(OH)(2)D(3) or a range of vitamin D receptor (VDR) agonists. Levels of IL-6 and IL-8 were measured in cell supernatants, and cellular total and phosphorylated IκBα were determined. Levels of human cathelicidin antimicrobial peptide (hCAP18) mRNA and protein were measured in cells after treatment with 1,25(OH)(2)D(3)., Results: Pretreatment with 1,25(OH)(2)D(3) was associated with significant reductions in IL-6 and IL-8 protein secretion after antigen exposure, a finding reproduced with a range of low calcaemic VDR agonists. 1,25(OH)(2)D(3) treatment led to a decrease in IκBα phosphorylation and increased total cellular IκBα. Treatment with 1,25(OH)(2)D(3) was associated with an increase in hCAP18/LL-37 mRNA and protein levels., Conclusions: Both 1,25(OH)(2)D(3) and other VDR agonists significantly reduce the pro-inflammatory response to antigen challenge in CF airway epithelial cells. VDR agonists have significant therapeutic potential in CF., (Copyright © 2011 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.)

Signaling of cell differentiation is one of the important physiological functions of the activated vitamin D receptor (VDR). Activation of the VDR can be achieved not only by 1alpha,25-dihydroxyvitamin D(3) (1,25D), the natural ligand, but also by a large number of its analogs. These include a category containing two side chains emanating at C-20, generally referred to as Gemini. The introduction of a cyclopropyl moiety as part of the pro-R side chain provides modified Gemini compounds with increased steric requirement and decreased chain flexibility; the biological consequences of this novel structural variant are subject of this investigation. In general, the resulting 1alpha,25-dihydroxy-(4-hydroxy-4-methyl-pentyl)-21,22-cis-cyclo-cholecalciferols reduced had differentiation and transcriptional potency and induced cell cycle arrest less efficiently, as shown by a decrease in G1/S ratio, when compared to 1,25D. Modifying their calcitriol side chain in the form of a 4-hydroxy-4-trifluoromethyl-5,5,5-trifluoropent-2-ynyl moiety, however, resulted in pronounced induction of differentiation in 1,25D-sensitive and moderate level of differentiation in 1,25D-resistant leukemia cells.

Purpose: We hypothesized that deregulated corepressor actions, with associated histone deacetylation activity, epigenetically suppressed vitamin D receptor (VDR) responsiveness and drives resistance towards 1alpha,25-dihydroxyvitamin D(3)., Experimental Design: Profiling, transcriptional, and proliferation assays were undertaken in 1alpha,25(OH)(2)D(3)-sensitive MCF-12A nonmalignant breast epithelial cells, a panel of breast cancer cell lines, and a cohort of primary breast cancer tumors (n = 21)., Results: Elevated NCoR1 mRNA levels correlated with suppressed regulation of VDR target genes and the ability of cells to undergo arrest in G(1) of the cell cycle. A similar increased ratio of corepressor mRNA to VDR occurred in matched primary tumor and normal cells, noticeably in estrogen receptor alpha-negative (n = 7) tumors. 1alpha,25(OH)(2)D(3) resistance in cancer cell lines was targeted by cotreatments with either 1alpha,25(OH)(2)D(3) or a metabolically stable analogue (RO-26-2198) in combination with either trichostatin A (TSA; histone deacetylation inhibitor) or 5-aza-2'-deoxycytidine (DNA methyltransferase inhibitor). Combinations of vitamin D(3) compounds with TSA restored VDR antiproliferative signaling (target gene regulation, cell cycle arrest, and antiproliferative effects in liquid culture) to levels which were indistinguishable from MCF-12A cells., Conclusions: Increased NCoR1 mRNA is a novel molecular lesion in breast cancer cells, which acts to suppress responsiveness of VDR target genes, resulting in 1alpha,25(OH)(2)D(3) resistance and seems to be particularly associated with estrogen receptor negativity. This lesion provides a novel molecular diagnostic and can be targeted by combinations of vitamin D(3) compounds and low doses of TSA.

We previously synthesized calcitriol derivatives with two identical side chains emanating at C-20, also known as gemini. In view of the evidence identifying C-24 hydroxylation as the first step in the in the metabolic cascade of calcitriol and gemini, stereochemical differentiation between the possible epimeric 20R- and 20S side-chain hydroxylated gemini became of interest. We now report the stereoselective synthesis of these compounds. Of these, 1,24(R),25-trihydroxy-21-(3-hydroxy-3-methyl-butyl)-20(R)-19-nor-cholecalciferol was identified as the main metabolic product of 19-nor-gemini. In general, higher doses of the 24-hydroxylated gemini compounds were required to increase blood calcium levels in mice and to suppress INF-gamma release in MLR.

The vitamin D analog, 1alpha-fluoro-16-ene-20-epi-23-ene-26,27-bishomo-25-hydroxyvitamin D(3) (Ro-26-9228) is tissue selective, with a gene regulation preference for bone over duodenum in vivo. In the human osteoblast-like cells, hFOB, the vitamin D receptor (VDR)-mediated transcriptional potencies of Ro-26-9228 and 1,25-dihydroxyvitamin D(3) (1,25D(3)) were similar, but in the intestinal cells, Caco-2, transcriptional potency of Ro-26-9228 was 10-50 times lower. We hypothesized that transcriptional activation of the VDR by Ro-26-9228 in the two cell types is regulated differently, and compared VDR extracted from hFOB or Caco-2 cells for their abilities to interact with a p160 coactivator [glucocorticoid receptor-interacting protein (GRIP)] and with retinoid X receptor (RXR) by pull-down assays. 1,25D(3) had similar potencies to induce interactions of VDR from the two cell types with these partners of transcription. In contrast, Ro-26-9228 induced interaction of osteoblastic VDR with RXR and GRIP but did not induce these interactions with VDR from Caco-2 cells. Further studies revealed that in hFOB cells the unoccupied VDR was cytoplasmic and proteasome sensitive, and that ligand treatment caused a rapid accumulation of the VDR in the chromatin. Both cytoplasmic and chromatin-associated ligand-bound VDR from hFOB cells had the abilities to interact with GRIP. In contrast, in Caco-2 cells, unoccupied VDR was localized in both the cytoplasm (70%) and the chromatin (30%). In Caco-2 cells, the cytoplasmic VDR was proteasome resistant, and neither 1,25D(3) nor Ro-26-9228 induced its binding to GRIP. Only a small fraction of the chromatin-associated VDR was proteasome sensitive, and this fraction was distinguishable by a faster electrophoretic mobility. 1,25D(3) induced an accumulation of the proteasome-sensitive VDR in the chromatin of Caco-2 cells and binding to GRIP. Ro-26-9228 failed to induce accumulation of the proteasome-sensitive VDR in the chromatin or binding to GRIP, but a coincubation of Caco-2 cells with the analog and a proteasome inhibitor restored these abilities. These results suggest that Ro-26-9228 has poor ability to promote the accumulation of a proteasome-sensitive, transcriptionally active VDR isoform in Caco-2 cells, whereas it does not have this limitation in hFOB cells.

The vitamin D receptor (VDR) is one of the endocrine members of the nuclear receptor superfamily and has a characteristic high affinity for its natural ligand 1alpha,25-dihydroxyvitamin D3 [1alpha,25(OH)2D3]. From a mechanistic point of view, the most interesting analog of 1alpha,25(OH)2D3 is the one that carries two side chains, referred to as Gemini. In this study, molecular dynamics (MD) simulations of the Gemini-VDR complex were performed that demonstrated that the binding of a ligand with a 25% increased volume does not disturb the overall structure of the ligand-binding domain (LBD). It was found that one of the two side chains takes exactly the same position as the single side chain of the natural ligand, which suggests that the molecular mechanism of the agonism of Gemini is identical to that of 1alpha,25(OH)2D3. VDR single and double point mutants at L227, A303, I313, and L397 and in vitro and ex vivo assessment of their agonistic action confirmed the predictions of the MD simulations. Moreover, it was found that the second side chain of Gemini can choose between two binding positions within the ligand-binding pocket of the VDR. These two newly identified "corners" were characterized most specifically by the amino acids pairs L227/A303 and I313/L397. Therefore, Gemini is an important model compound that allows further insight into the molecular actions of the VDR but is, in parallel, also a promising precursor for the design of even more potent 1alpha,25(OH)2D3 analogs.

Our recent studies have shown that the vitamin D analog Ro-26-9228 restores bone mineral density without inducing hypercalcemia in osteopenic rats. Our ex vivo experiments demonstrated that the analog upregulated gene expression in trabecular bone but not in the duodenum of female rats. We examined the mechanism for the tissue selectivity of Ro-26-9228 in Caco-2, a human cell line of intestinal origin, and hFOB, and a human fetal osteoblast cell line. We found that the abilities of Ro-26-9228 and the natural hormone, 1,25-dihydroxyvitamin D(3) (1,25D(3)) to induce VDRE-reporter gene expression in transiently transfected human osteoblasts are similar. In contrast, in Caco-2 cells, Ro-26-9228 induces 40-fold less reporter gene expression than 1,25D(3) does. We also examined the abilities of the vitamin D receptor (VDR)-ligand complexes from these two cell lines to interact with partners of transcription (glucocorticoid receptor-interacting protein, VDR-interacting protein, and retinoid X receptor), in pull-down assays. These assays revealed that 1,25D(3) induces similar levels of interaction of these co-factors with VDR from both osteoblasts and intestinal cells. In contrast, Ro-26-9228 induces significant interaction of VDR from osteoblast cells with these co-factors, but less of VDR from Caco-2 cells. These results suggest that the cellular environment of intestinal cells, unlike that of osteoblasts, represses the ability of VDR-Ro-26-9228 complexes to interact with transcription partners., (Copyright 2002 Wiley-Liss, Inc.)

Exposure of leukemia cells to the physiologically active form of vitamin D3, 1,25-dihydroxyvitamin D3 (1,25D3) normalizes their phenotype to cells that resemble mature monocytes. One of the earliest detectable events in this process is an upregulation of the nuclear receptor for 1,25D3, the vitamin D receptor (nVDR). In contrast, the novel analog of 1,25D3, 1,25-dihydroxy-5,6 trans-16-ene-vitamin D3 (5,6-16D3), which has recently been reported to have low calcium-mobilizing activity in vivo, rapidly induced the expression of CD14, CD11b, and monocyte-specific esterase (MSE), classical markers of the mature monocyte, but upregulated nVDR expression less than 1,25D3. This upregulation was shown to be the result of altered degradation of the nVDR protein, while the levels of nVDR mRNA were constant. Knock-out of nVDR transcriptional activity by a decoy VDRE double-stranded deoxyoligonucleotide, markedly abrogated 1,25D3-induced differentiation, but incompletely inhibited 5,6-16D3-induced differentiation. These findings suggest that the unique ability of 5,6-16D3 to induce cell differentiation but not systemic hypercalcemia, may be due to the activation of pathways which initiate differentiation independently of nVDR.

The nuclear hormone 1alpha,25-dihydroxyvitamin D(3) (1alpha,25(OH)(2)D(3)) acts through the transcription factor vitamin D receptor (VDR) via combined contact with the retinoid X receptor (RXR), coactivator proteins, and specific DNA binding sites (VDREs). Ligand-mediated conformational changes of the VDR are the core of the molecular switch of nuclear 1alpha,25(OH)(2)D(3) signalling. Studying the interaction of 1alpha,25(OH)(2)D(3) analogues with this molecular switch should allow the characterization of their potential selective biological profile. A 1alpha,25(OH)(2)D(3) analogue with two side chains (Ro27-2310 or Gemini) was found to stabilize functional VDR conformations and VDR-RXR heterodimers on a VDRE with a slightly lower sensitivity than the natural hormone. A 19-nor derivative of Gemini (Ro27-5646) showed similar sensitivity whereas 5,6-trans (Ro27-6462) 3-epi (Ro27-5840) and 1alpha-fluoro (Ro27-3752) derivatives were equal to each other, but approximately 30-times less sensitive than Gemini. A des-C,D derivative of Gemini (Ro28-1909) showed only residual activity at maximal concentrations. In contrast to 1alpha,25(OH)(2)D(3), Gemini and its derivatives showed a differential preference in stabilizing VDR conformations which was found to be modulated by DNA coactivator and corepressor proteins. An analysis of the gene regulatory potential of the VDR agonists in cellular reporter gene systems demonstrated the same ranking as in the in vitro systems, but Gemini and its 19-nor derivative were found to be more sensitive than 1alpha,25(OH)(2)D(3) which indicates that the natural hormone is selectively metabolized. This study used straightforward methods for the in vitro and ex vivo evaluation of the gene regulatory potential of 1alpha,25(OH)(2)D(3) analogues. Gemini was highlighted as an interesting drug candidate which could not be optimized through obvious chemical modifications in its A-ring. J. Cell. Biochem. Suppl. 36: 179-190, 2001., (Copyright 2001 Wiley-Liss, Inc.)

The hormone 1alpha,25(OH)(2)-vitamin D(3) (125D) binds to its nuclear receptor (VDR) to stimulate gene transcription activity. Inversion of configuration at C-20 of the side chain to generate 20-epi-1alpha,25(OH)(2)D(3) (20E-125D) increases transcription 200-5000-fold over 125D with its 20-normal (20N) side chain. This enhancement has been attributed to the VDR ligand-binding domain (LBD) having different contact sites for 20N and 20E side chains that generate different VDR conformations. We synthesized 1alpha, 25-dihydroxy-21-(3-hydroxy-3-methylbutyl)vitamin D(3) (Gemini) with two six-carbon side chains (both 20N and 20E orientations). Energy minimization calculations indicate the Gemini side chain possesses significantly more energy minima than either 125D or 20E-125D (2346, 207, and 127 minima, respectively). We compared activities of 125D, 20E-125D, and Gemini, respectively, in several assays: binding to wild-type (100%, 147%, and 38%) and C-terminal-truncated mutant VDR; transcriptional activity (of the transfected osteopontin promoter in ROS 17/2.8 cells: ED(50) 10, 0.005, and 1.0 nM); mediation of conformational changes in VDR assessed by protease clipping (major trypsin-resistant fragment of 34, 34, and 28 kDa). For inhibition of cellular clonal growth of human leukemia (HL-60) and breast cancer (MCF7) cell lines, the ED(50)(125D)/ED(50)(Gem) was respectively 380 and 316. We conclude that while Gemini readily binds to the VDR and generates unique conformational changes, none of them is able to permit a superior gene transcription activity despite the presence of a 20E side chain.

The vitamin D receptor (VDR) is the nuclear receptor for 1, 25-dihydroxyvitamin D(3) [1alpha,25(OH)(2)D(3)] that acts as a ligand-dependent transcription factor via combined contact with coactivator proteins (steroid receptor coactivator-1, transcriptional intermediary factor 2, and receptor associated coactivator 3) and specific DNA binding sites [vitamin D response elements (VDREs)]. Ligand-mediated conformational changes of the VDR contribute to the key mechanisms in this nuclear hormone signaling process. 1alpha,25(OH)(2)D(3), MC1288 [20-epi-1alpha,25(OH)(2)D(3)], ZK161422 [20-methyl-1alpha,25(OH)(2)D(3)], and Ro27-2310 (also called Gemini, having two side chains at carbon 20) were used as model VDR agonists. The analysis of agonist-induced VDR conformations and coactivator interactions were found to be insufficient for extrapolating in vivo activities. In DNA-independent assays, such as classical limited protease digestions and glutathione S-transferase pull downs, Gemini seemed to be up to 10,000-fold and the other VDR agonists 10- to 100-fold weaker than in functional in vivo assays. A more accurate description of the gene regulatory potential of VDR agonists was obtained with all tested VDR agonists by analyzing VDR conformations in the context of VDRE-bound VDR-retinoid X receptor heterodimers, in such assays as gel supershift, gel shift clipping, and limited protease digestion in the presence of DNA and cofactor. Coactivators were found to shift the ligand sensitivity (by a factor of 4 for Gemini) and the ratio of VDR conformations in the presence of DNA toward the high-affinity ligand binding conformation (c1(LPD)). In conclusion, the induction of response element- and coactivator-modulated VDR conformations appears to be a key step for the gene regulatory function of a VDR agonist. The quantification of these effects would be of central importance for the evaluation of the cell-specific efficacy of systemically applied 1alpha, 25(OH)(2)D(3) analogs.

Numerous studies have indicated that the secosteroid hormone 1alpha, 25-dihydroxyvitamin D3 protects against the development of clinical prostate cancer (PC). Whether this hormone also has therapeutic potential for patients with advanced PC has not yet been evaluated. Several synthetic vitamin D analogues are now available that have reduced hypercalcemic effects and yet effectively induce differentiation in some cell types. For these reasons, these analogues may be safer and more effective for cancer therapy than the natural hormone. In the current study, 13 such analogues were screened for their abilities to inhibit the growth of PC cell lines. Three of the most consistently effective analogues (Ro 23-7553, Ro 24-5531, and Ro 25-6760) were then chosen for further analysis. Growth studies using clones of the JCA-1 cell line that were transfected with the vitamin D receptor cDNA indicate that the antiproliferative effects of these analogues require vitamin D receptor expression. Furthermore, these three analogues induce the secretion of prostate-specific acid phosphatase and prostate-specific antigen (two markers of the differentiated prostatic phenotype) in the cell line LNCaP. These in vitro studies suggest that Ro 23-7553, Ro 24-5531, and Ro 25-6760 should be further evaluated as therapeutic agents for the treatment of PC.