Evidence that TRPC3 is a molecular component of the 1alpha,25(OH)2D3-activated capacitative calcium entry (CCE) in muscle and osteoblast cells

In chick skeletal muscle and in rat osteoblast-like cells (ROS 17/2.8), 1alpha,25-dihydroxy-Vitamin-D(3) [1alpha,25(OH)(2)D(3)] stimulates release of Ca(2+) from inner stores and extracellular cation influx through both voltage-dependent and capacitative Ca(2+) entry (CCE) channels. We investigated the involvement of TRPC proteins in CCE induced by 1alpha,25(OH)(2)D(3). Two fragments were amplified by RT-PCR, exhibiting >85% sequence homology with human TRPC3. Northern and Western blots employing TRPC3-probes and anti-TRPC3 antibodies, respectively, confirmed endogenous expression of a TRPC3-like protein. Both cell types transfected with anti-TRPC3 antisense oligodeoxynucleotides showed reduced CCE and Mn(2+) entry induced by either thapsigargin or 1alpha,25(OH)(2)D(3). In muscle cells, anti-VDR antisense inhibited steroid-induced Ca(2+) and Mn(2+) influx and co-immunoprecipitation of TRPC3 and VDR was observed, suggesting an association between both proteins and a functional role of the receptor in 1alpha,25(OH)(2)D(3) activation of CCE. In osteoblasts, two PCR fragments showing high homology with human INAD-like sequences were obtained. Northern blot and antisense functional assays suggested the involvement of the INAD-like protein in CCE regulation by the hormone. Therefore, we propose that an endogenous TRPC3 protein mediates 1alpha,25(OH)(2)D(3) modulation of CCE in muscle and osteoblastic cells, which seems to implicate VDR-TRPC3 association and the participation of a INAD-like scaffold protein.     

1alpha,25(OH)2D3 induces capacitative calcium entry involving a TRPC3 protein in skeletal muscle and osteoblastic cells

This work describes the involvement of TRPC proteins in capacitative calcium entry (CCE) induced by 1alpha,25-dihydroxy-vitamin-D3 [1alpha,25(OH)2D3] in chick skeletal muscle and in rat osteoblast-like cells (ROS 17/2.8) and the role of the vitamin D receptor (VDR) in this non-genomic rapid response mediated by the hormone. We propose that an endogenous TRPC3 protein mediates 1alpha,25(OH)2D3 modulation of CCE in these cells, which seems to implicate VDR-TRPC3 association and the participation of an INAD-like scaffold protein.     

Characterization of a 1,25(OH)2-vitamin D3-responsive capacitative Ca2+ entry pathway in rat osteoblast-like cells

We investigated the existence of a capacitative Ca2+ entry (CCE) pathway in ROS 17/2.8 osteoblast-like cells and its responsiveness to 1,25-dihydroxy-vitamin D3 [1,25(OH)2D3]. Depletion of inner Ca2+ stores with thapsigargin or 1,25(OH)2D3 in the absence of extracellular Ca2+ transiently elevated cytosolic Ca2+ ([Ca2+]i); after recovery of basal values, Ca2+ re-addition to the medium markedly increased Ca2+ entry, reflecting pre-activation of a CCE pathway. Recovery of the Ca2+ overshoot that followed the induced CCE was mainly mediated by the plasma membrane Ca2+-ATPase. Addition of 1,25(OH)2D3 to the declining phase of the thapsigargin-induced CCE did not modify further [Ca2+]i, indicating that steroid activation of CCE was dependent on store depletion. Pre-treatment with 1 microM Gd3+ inhibited 30% both thapsigargin- and 1,25(OH)2D3-stimulated CCE, whereas 2.5 microM Gd3+ was required for maximal inhibition ( approximately 85%). The activated CCE was permeable to both Mn2+ and Sr2+. Mn2+ entry sensitivity to Gd3+ was the same as that of the CCE. However, 1-microM Gd3+ completely prevented capacitative Sr2+ influx, whereas subsequent Ca2+ re-addition was reduced only 30%. These results suggest that in ROS 17/2.8 cells CCE induced by thapsigargin or 1,25(OH)2D3 is contributed by at least two cation entry pathways: a Ca2+/Mn2+ permeable route insensitive to very low micromolar (1 microM) Gd3+ accounting for most of the CCE and a minor Ca2+/Sr2+/Mn2+ permeable route highly sensitive to 1 microM Gd3+. The Ca2+-mobilizing agonist ATP also stimulated CCE resembling the Ca2+/Sr2+/Mn2+ permeable entry activated by 1,25(OH)2D3. The data demonstrates for the first time, the presence of a hormone-responsive CCE pathway in an osteoblast cell model, raising the possibility that it could be an alternative Ca2+ influx route through which osteotropic agents influence osteoblast Ca2+ homeostasis. Copyright Wiley-Liss, Inc.     

Protein kinase C alpha modulates the Ca2+ influx phase of the Ca2+ response to 1alpha,25-dihydroxy-vitamin-D3 in skeletal muscle cells.

Treatment of chick skeletal muscle cells with 1alpha,25-dihydroxy-vitamin D3 [1alpha,25(OH)2D3] triggers a rapid and sustained increase in cytosolic Ca2+ ([Ca2+]i), which depends on Ca2+ mobilization from inner stores and extracellular Ca2+ entry. Fluorimetric analysis of changes in [Ca2+]i in Fura-2-loaded cells revealed that the hormone significantly stimulates the Ca2+ influx phase within the concentration range of 10(-12)-10(-6) M, with maximal effects (3.5-fold increase) at 10(-9) M 1alpha,25(OH)2D3. The effects of the sterol on the Ca2+ entry pathway were abolished by the PKC inhibitors bisindolylmaleimide and calphostin. We have recently shown that, in these cells, 1alpha,25(OH)2D3 activates and translocates PKC alpha to the membrane, suggesting that this isozyme accounts for PKC-dependent 1alpha,25(OH)2D3 modulation of Ca2+ entry. The role of PKC alpha was specifically addressed here using antisense technology. When the expression of PKC alpha was selectively knocked out by intranuclear microinjection of an antisense oligonucleotide against PKC alpha mRNA, the Ca2+ influx component of the response to 1alpha,25(OH)2D3 was markedly reduced (-60%). These results demonstrate that 1alpha,25(OH)2D3-induced activation of PKC alpha enhances extracellular Ca2+ entry partially contributing to maintainance of the sustained phase of the Ca2+ response to the sterol.     

Receptor-operated Ca2+ entry mediated by TRPC3/TRPC6 proteins in rat prostate smooth muscle (PS1) cell line

Prostate smooth muscle cells predominantly express alpha1-adrenoceptors (alpha1-AR). alpha1-AR antagonists induce prostate smooth muscle relaxation and therefore they are useful therapeutic compounds for the treatment of benign prostatic hyperplasia symptoms. However, the Ca(2+) entry pathways associated with the activation of alpha1-AR in the prostate have yet to be elucidated. In many cell types, mammalian homologues of transient receptor potential (TRP) genes, first identified in Drosophila, encode TRPC (canonical TRP) proteins. They function as receptor-operated channels (ROCs) which are involved in various physiological processes such as contraction, proliferation, apoptosis, and differentiation. To date, the expression and function of TRPC channels have not been studied in prostate smooth muscle. In fura-2 loaded PS1 (a prostate smooth muscle cell line) which express endogenous alpha1A-ARs, alpha-agonists epinephrine (EPI), and phenylephrine (PHE) induced Ca(2+) influx which depended on the extracellular Ca(2+) and PLC activation but was independent of PKC activation. Thus, we have tested two membrane-permeable analogues of diacylglycerol (DAG), oleoyl-acyl-sn-glycerol (OAG) and 1,2-dioctanoyl-sn-glycerol (DOG). They initiated Ca(2+) influx whose properties were similar to those induced by the alpha-agonists. Sensitivity to 2-aminoethyl diphenylborate (2-APB), SKF-96365 and flufenamate implies that Ca(2+)-permeable channels mediated both alpha-agonist- and OAG-evoked Ca(2+) influx. Following the sarcoplasmic reticulum (SR) Ca(2+) store depletion by thapsigargin (Tg), a SERCA inhibitor, OAG and PHE were both still able to activate Ca(2+) influx. However, OAG failed to enhance Ca(2+) influx when added in the presence of an alpha-agonist. RT-PCR and Western blotting performed on PS1 cells revealed the presence of mRNAs and the corresponding TRPC3 and TRPC6 proteins. Experiments using an antisense strategy showed that both alpha-agonist- and OAG-induced Ca(2+) influx required TRPC3 and TRPC6, whereas the Tg-activated ("capacitative") Ca(2+) entry involved only TRPC3 encoded protein. It may be thus concluded that PS1 cells express TRPC3 and TRPC6 proteins which function as receptor- and store-operated Ca(2+) entry pathways.     

Involvement of tyrosine kinase activity in 1alpha,25(OH)2-vitamin D3 signal transduction in skeletal muscle cells

In cultured chick skeletal muscle cells loaded with Fura-2, the tyrosine kinase inhibitors herbimycin A and genistein abolished both the fast inositol 1,4,5-trisphosphatedependent Ca(2+) release from internal stores and extracellular Ca(2+) influx induced by 1alpha, 25(OH)(2)-vitamin D(3) (1alpha,25(OH)(2)D(3)). Daidzein, an inactive analog of genistein, was without effects. Tyrosine phosphatase inhibition by orthovanadate increased cytosolic Ca(2+). Anti-phosphotyrosine immunoblot analysis revealed that 1alpha, 25(OH)(2)D(3) rapidly (0.5-10 min) stimulates in a concentrationdependent fashion (0.1-10 nm) tyrosine phosphorylation of several myoblast proteins, among which the major targets of the hormone could be immunochemically identified as phospholipase Cgamma (127 kDa), which mediates intracellular store Ca(2+) mobilization and external Ca(2+) influx, and the growth-related proteins mitogen-activated protein (MAP) kinase (42/44 kDa) and c-myc (65 kDa). Genistein suppressed the increase in phosphorylation and concomitant elevation of MAPK activity elicited by the sterol. Both genistein and the MAPK kinase (MEK) inhibitor PD98059 abolished stimulation of DNA synthesis by 1alpha,25(OH)(2)D(3). The sterol-induced increase in tyrosine phosphorylation of c-myc, a finding not reported before for cell growth regulators, was totally suppressed by the specific Src inhibitor PP1. These results demonstrate that tyrosine phosphorylation is a previously unrecognized mechanism involved in 1alpha,25(OH)(2)D(3) regulation of Ca(2+) homeostasis in hormone target cells. In addition, the data involve tyrosine kinase cascades in the mitogenic effects of 1alpha, 25(OH)(2)D(3) on skeletal muscle cells.     

Involvement of calmodulin in 1alpha,25-dihydroxyvitamin D3 stimulation of store-operated Ca2+ influx in skeletal muscle cells

The steroid hormone 1alpha,25-dihydroxyvitamin D(3) (1, 25-(OH)(2)D(3)) rapidly modulates Ca(2+) homeostasis in avian skeletal muscle cells by driving a complex signal transduction mechanism, which promotes Ca(2+) release from inner stores and cation influx from the outside through both L-type and store-operated Ca(2+) (SOC) channels. In the present work, we evaluated the involvement of calmodulin (CAM) in 1,25-(OH)(2)D(3) regulation of SOC influx in chick skeletal muscle cells. Treatment with 10(-9) m 1,25-(OH)(2)D(3) in Ca(2+)-free medium resulted in a rapid but transient Ca(2+) rise correlated with the sterol-induced inositol 1,4,5-trisphosphate (IP(3)) production. The SOC influx stimulated by the hormone was insensitive to both CAM antagonists (fluphenazine, trifluoperazine, chlorpromazine, compound 48/80) and the CAM-dependent protein kinase II (CAMKII) inhibitor KN-62 when added after the sterol-dependent Ca(2+) transient, but it was completely abolished when added prior to the IP(3)-induced mobilization of Ca(2+) from endogenous stores. Moreover, in cells microinjected with antisense oligonucleotides directed against the CAM mRNA the sterol-stimulated SOC influx was reduced up to 60% respect to uninjected cells. The present results suggest that the 1, 25-(OH)(2)D(3)-induced (IP(3)-mediated) cytosolic Ca(2+) transient is required for CAM, activation which in turn activates SOC influx in a mechanism that seems to include CAMKII.     

Muscarinic acetylcholine receptors activate TRPC6 channels in PC12D cells via Ca2+ store-independent mechanisms

In this paper we report that stimulation of mAChRs in PC12D cells activates Ca2+ channels that are regulated independently of intracellular Ca2+ stores. In nominally Ca2+-free medium, exposure of PC12D cells to carbachol stimulates a robust influx of Ba2+, a Ca2+ substitute. This influx is blocked by atropine, but not by inhibitors of the nicotinic acetylcholine receptor or L-, N-, or T-type voltage-regulated Ca2+ channels. By contrast, depletion of intracellular Ca2+ stores with thapsigargin only weakly stimulates Ba2+ influx. Unlike store-operated Ca2+ channels (SOCCs), which close only after intracellular Ca2+ stores refill, channels mediating carbachol-stimulated Ba2+ influx rapidly close following the inactivation of mAChRs with atropine. Ba2+ influx is inhibited by extracellular Ca2+, by the Ca2+ channel blocker SKF-96365, and by activation of protein kinase C (PKC). Exogenous expression of antisense RNA encoding the rat canonical-transient receptor potential Ca2+ channel subtype 6 (TRPC6) or the N-terminal domain of TRPC6 blocks carbachol-stimulated Ba2+ influx in PC12D cells. Expression of TRPC6 antisense RNA or the TRPC6 N-terminal domain also blocks Ba2+ influx stimulated by 1-oleoyl-2-acetyl-sn-glycerol (OAG), a diacylglycerol analog previously shown to activate exogenously expressed TRPC6 channels. These data show that mAChRs in PC12D cells activate endogenous Ca2+ channels that are regulated independently of Ca2+ stores and require the expression of TRPC6.     

Sensitivity to growth suppression by 1alpha,25-dihydroxyvitamin D(3) among MCF-7 clones correlates with Vitamin D receptor protein induction

The antiproliferative effect of 1alpha,25-dihydroxyvitamin D(3) (1alpha,25(OH)(2)D(3)) has been studied for a decade in diverse model systems, but the signalling pathways linking 1alpha,25(OH)(2)D(3) to cell cycle arrest remains unclear. In our attempt to establish a model system which would allow further identification of important players in the process of the 1alpha,25(OH)(2)D(3) imposed cell cycle arrest, we have isolated derivatives of the human breast cancer cell line MCF-7 and chosen two nearly 1alpha,25(OH)(2)D(3) resistant and two hypersensitive sub-clones. Investigation of cell cycle proteins regulated by 1alpha,25(OH)(2)D(3) in these clones indicates that activation of one component/pathway is responsible for the linkage between 1alpha,25(OH)(2)D(3) and growth arrest. Protein levels of the Vitamin D receptor (VDR) were elevated in sensitive cells upon 1alpha,25(OH)(2)D(3) treatment, whereas resistant clones were unable to induce VDR upon 1alpha,25(OH)(2)D(3) treatment. Our data show that VDR protein levels and the ability of a cell to induce VDR upon 1alpha,25(OH)(2)D(3) treatment correlate with the antiproliferative effects of 1alpha,25(OH)(2)D(3), and suggest that the level of VDR in cancer cells might serve as a prognostic marker for treatment of cancer with 1alpha,25(OH)(2)D(3) analogues.     

Membrane actions of vitamin D metabolites 1alpha,25(OH)2D3 and 24R,25(OH)2D3 are retained in growth plate cartilage cells from vitamin D receptor knockout mice

1alpha,25(OH)(2)D(3) regulates rat growth plate chondrocytes via nuclear vitamin D receptor (1,25-nVDR) and membrane VDR (1,25-mVDR) mechanisms. To assess the relationship between the receptors, we examined the membrane response to 1alpha,25(OH)(2)D(3) in costochondral cartilage cells from wild type VDR(+/+) and VDR(-/-) mice, the latter lacking the 1,25-nVDR and exhibiting type II rickets and alopecia. Methods were developed for isolation and culture of cells from the resting zone (RC) and growth zone (GC, prehypertrophic and upper hypertrophic zones) of the costochondral cartilages from wild type and homozygous knockout mice. 1alpha,25(OH)(2)D(3) had no effect on [(3)H]-thymidine incorporation in VDR(-/-) GC cells, but it increased [(3)H]-thymidine incorporation in VDR(+/+) cells. Proteoglycan production was increased in cultures of both VDR(-/-) and VDR(+/+) cells, based on [(35)S]-sulfate incorporation. These effects were partially blocked by chelerythrine, which is a specific inhibitor of protein kinase C (PKC), indicating that PKC-signaling was involved. 1alpha,25(OH)(2)D(3) caused a 10-fold increase in PKC specific activity in VDR(-/-), and VDR(+/+) GC cells as early as 1 min, supporting this hypothesis. In contrast, 1alpha,25(OH)(2)D(3) had no effect on PKC activity in RC cells isolated from VDR(-/-) or VDR(+/+) mice and neither 1beta,25(OH)(2)D(3) nor 24R,25(OH)(2)D(3) affected PKC in GC cells from these mice. Phospholipase C (PLC) activity was also increased within 1 min in GC chondrocyte cultures treated with 1alpha,25(OH)(2)D(3). As noted previously for rat growth plate chondrocytes, 1alpha,25(OH)(2)D(3) mediated its increases in PKC and PLC activities in the VDR(-/-) GC cells through activation of phospholipase A(2) (PLA(2)). These responses to 1alpha,25(OH)(2)D(3) were blocked by antibodies to 1,25-MARRS, which is a [(3)H]-1,25(OH)(2)D(3) binding protein identified in chick enterocytes. 24R,25(OH)(2)D(3) regulated PKC in VDR(-/-) and VDR(+/+) RC cells. Wild type RC cells responded to 24R,25(OH)(2)D(3) with an increase in PKC, whereas treatment of RC cells from mice lacking a functional 1,25-nVDR caused a time-dependent decrease in PKC between 6 and 9 min. 24R,25(OH)(2)D(3) dependent PKC was mediated by phospholipase D, but not by PLC, as noted previously for rat RC cells treated with 24R,25(OH)(2)D(3). These results provide definitive evidence that there are two distinct receptors to 1alpha,25(OH)(2)D(3). 1alpha,25(OH)(2)D(3)-dependent regulation of DNA synthesis in GC cells requires the 1,25-nVDR, although other physiological responses to the vitamin D metabolite, such as proteoglycan sulfation, involve regulation via the 1,25-mVDR.     

Antagonistic action of a 25-carboxylic ester analogue of 1alpha, 25-dihydroxyvitamin D3 is mediated by a lack of ligand-induced vitamin D receptor interaction with coactivators

A 25-carboxylic ester analogue of 1alpha,25-dihydroxyvitamin D(3) (1alpha,25-(OH)(2)D(3)), ZK159222, was described as a novel type of antagonist of 1alpha,25-(OH)(2)D(3) signaling. The ligand sensitivity of ZK159222, in facilitating complex formation between 1alpha,25-(OH)(2)D(3) receptor (VDR) and the retinoid X receptor (RXR) on a 1alpha,25-(OH)(2)D(3) response element (VDRE), was approximately 7-fold lower when compared with 1alpha,25-(OH)(2)D(3). However, ZK159222 was not able to promote a ligand-dependent interaction of the VDR with the coactivator proteins SRC-1, TIF2, and RAC3, neither in solution nor in a complex with RXR on DNA. Functional analysis in HeLa and COS-7 cells demonstrated a 10-100-fold lower ligand sensitivity for ZK159222 than for 1alpha, 25-(OH)(2)D(3) and, most interestingly, a potency that was drastically reduced compared with 1alpha,25-(OH)(2)D(3). A cotreatment of 1alpha,25-(OH)(2)D(3) with a 100-fold higher concentration of ZK159222 resulted in a prominent antagonistic effect both in functional in vivo and in in vitro assays. These data suggest that the antagonistic action of ZK159222 is due to a lack of ligand-induced interaction of the VDR with coactivators with a parallel ligand sensitivity, which is sufficient for competition with the natural hormone for VDR binding.     

Epigenetic repression of transcription by the Vitamin D3 receptor in prostate cancer cells

Normal prostate epithelial cells are acutely sensitive to the antiproliferative action of 1alpha,25-dihydroxyvitamin D(3) (1alpha,25(OH)(2)D(3)), whilst prostate cancer cell lines and primary cultures display a range of sensitivities. We hypothesised that key antiproliferative target genes of the Vitamin D receptor (VDR) were repressed by an epigenetic mechanism in 1alpha,25(OH)(2)D(3)-insensitive cells. Supportively, we found elevated nuclear receptor co-repressor and reduced VDR expression correlated with reduced sensitivity to the antiproliferative action of 1alpha,25(OH)(2)D(3). Furthermore, the growth suppressive actions of 1alpha,25(OH)(2)D(3) can be restored by co-treatment with low doses of histone deacetylation inhibitors, such as trichostatin A (TSA) to induce apoptosis. Examination of the regulation of VDR target genes revealed that co-treatment of 1alpha,25(OH)(2)D(3) plus TSA co-operatively upregulated GADD45alpha. Similarly in a primary cancer cell culture, the regulation of appeared GADD45alpha repressed. These data demonstrate that prostate cancer cells utilise a mechanism involving deacetylation to suppress the responsiveness of VDR target genes and thus ablate the antiproliferative action of 1alpha,25(OH)(2)D(3).     

Chemopreventive efficacy of 25-hydroxyvitamin D3 in colon cancer

Recently, it has been reported that 25-hydroxyvitamin D3-1alpha-hydroxylase [1alpha(OH)ase, CYP27B1], required to convert non-toxic 25-hyxdroxyvitamin D3 [25(OH)D(3)] to its active metabolite [1alpha,25(OH)(2)D(3)], is present in the epithelial cells of the human colon. In the present study, the potential chemoprotective role of 25(OH)D(3) was evaluated for colon cancer using the HT-29, human colon cancer cell line. Colon cancer cells were treated with 25(OH)D(3) (500nM or 1muM), 1alpha,25(OH)(2)D(3) (500nM), cholecalciferol (D3, 1muM) or vehicle and cell number determined at days 2 and 5 post-treatment. Results showed that both 25(OH)D(3) and 1alpha,25(OH)(2)D(3) induced dose- and time-dependent anti-proliferative effects on the HT-29 cells, with maximum inhibition noted at day 5. Western blot analyses revealed an up-regulation of VDR and 1alpha(OH)ase expression following 24h of treatment with 25(OH)D(3), and 1alpha,25(OH)(2)D(3). These results are consistent with the expression of VDR and 1alpha(OH)ase in samples of normal colonic tissue, aberrant crypt foci (ACFs) and colon adenocarcinomas. The VDR expression was sequentially increased from normal to pre-cancerous lesions to well-differentiated tumors and then decreased in poorly differentiated tumors. Expression of 1alpha(OH)ase was equally expressed in normal, pre-cancerous lesions and malignant human colon tissues. The increased expression of 1alpha(OH)ase in colon cancer cells treated with the pro-hormone and its anti-proliferative effects, suggest that 25(OH)D(3) may offer possible therapeutic and chemopreventive option in colon cancer.     

Natural metabolites of 1alpha,25-dihydroxyvitamin D(3) retain biologic activity mediated through the vitamin D receptor

1alpha,25-dihydroxyvitamin D(3) (1alpha,25(OH)(2)D(3)), the active metabolite of vitamin D, mediates many of its effects through the intranuclear vitamin D receptor (VDR, NR1I1), that belongs to the large superfamily of nuclear receptors. Vitamin D receptor can directly regulate gene expression by binding to vitamin D response elements (VDREs) located in promoter or enhancer regions of various genes. Although numerous synthetic analogs of 1alpha,25(OH)(2)D(3) have been analysed for VDR binding and transactivation of VDRE-driven gene expression, the biologic activity of many naturally occurring metabolites has not yet been analyzed in detail. We therefore studied the transactivation properties of 1alpha,24R, 25-trihydroxyvitamin D(3) (1alpha,24R,25(OH)(3)D(3)), 1alpha, 25-dihydroxy-3-epi-vitamin D(3) (1alpha,25(OH)(2)-3-epi-D(3)), 1alpha,23S,25-trihydroxyvitamin D(3) (1alpha,23S,25(OH)(3)D(3)), and 1alpha-hydroxy-23-carboxy-24,25,26,27-tetranorvitamin D(3) (1alpha(OH)-24,25,26,27-tetranor-23-COOH-D(3); calcitroic acid) using the human G-361 melanoma cell line. Cells were cotransfected with a VDR expression plasmid and luciferase reporter gene constructs driven by two copies of the VDRE of either the mouse osteopontin promoter or the 1alpha,25(OH)(2)D(3) 24-hydroxylase (CYP24) promoter. Treatment with 1alpha,25(OH)(2)D(3) or the metabolites 1alpha,24R,25(OH)(3)D(3), 1alpha,25(OH)(2)-3-epi-D(3), and 1alpha,23S,25(OH)(3)D(3) resulted in transactivation of both constructs in a time- and dose-dependent manner, and a postitive regulatory effect was observed even for calcitroic acid in the presence of overexpressed VDR. The metabolites that were active in the reporter gene assay also induced expression of CYP24 mRNA in the human keratinocyte cell line HaCaT, although with less potency than the parent hormone. A ligand-binding assay based on nuclear extracts from COS-1 cells overexpressing human VDR demonstrated that the metabolites, although active in the reporter gene assay, were much less effective in displacing [(3)H]-labeled 1alpha,25(OH)(2)D(3) from VDR than the parent hormone. Thus, we report that several natural metabolites of 1alpha,25(OH)(2)D(3) retain significant biologic activity mediated through VDR despite their apparent low affinity for VDR.     

Central role of VDR conformations for understanding selective actions of vitamin D(3) analogues

The vitamin D(3) receptor (VDR) acts primarily as a heterodimer with the retinoid X receptor (RXR) on different types of 1alpha,25-dihydroxyvitamin D(3) (1alpha,25(OH)(2)D(3)) response elements (VDREs). Therefore, DNA-bound VDR-RXR heterodimers can be considered as the molecular switches of 1alpha,25(OH)(2)D(3) signalling. Functional conformations of the VDR within these molecular switches appear to be of central importance for describing the biologic actions of 1alpha,25(OH)(2)D(3) and its analogues. Moreover, VDR conformations provide a molecular basis for understanding the potential selective profile of VDR agonists, which is critical for a therapeutic application. This review discusses VDR conformations and their selective stabilization by 1alpha,25(OH)(2)D(3) and its analogues, such as EB1089 and Gemini, as a monomer in solution or as a heterodimer with RXR bound to different VDREs and complexed with coactivator or corepressor proteins.