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.     

Structural determinants for vitamin D receptor response to endocrine and xenobiotic signals

The vitamin D receptor (VDR), initially identified as a nuclear receptor for 1alpha,25-dihydroxyvitamin D3 [1alpha,25(OH)2D3], regulates calcium metabolism, cellular proliferation and differentiation, immune responses, and other physiological processes. Recently, secondary bile acids such as lithocholic acid (LCA) were identified as endogenous VDR agonists. To identify structural determinants required for VDR activation by 1alpha,25(OH)2D3 and LCA, we generated VDR mutants predicted to modulate ligand response based on sequence homology to pregnane X receptor, another bile acid-responsive nuclear receptor. In both vitamin D response element activation and mammalian two-hybrid assays, we found that VDR-S278V is activated by 1alpha,25(OH)2D3 but not by LCA, whereas VDR-S237M can respond to LCA but not to 1alpha,25(OH)2D3. Competitive ligand binding analysis reveals that LCA, but not 1alpha,25(OH)2D3, effectively binds to VDR-S237M and both 1alpha,25(OH)2D3 and LCA bind to VDR-S278V. We propose a docking model for LCA binding to VDR that is supported by mutagenesis data. Comparative analysis of the VDR-LCA and VDR-1alpha,25(OH)2D3 structure-activity relationships should be useful in the development of bile acid-derived synthetic VDR ligands that selectively target VDR function in cancer and immune disorders without inducing adverse hypercalcemic effects.     

1alpha,25-dihydroxyvitamin D(3)-26,23-lactone analogs antagonize differentiation of human leukemia cells (HL-60 cells) but not of human acute promyelocytic leukemia cells (NB4 cells).

We examined the effects of two novel 1alpha,25-dihydroxyvitamin D(3)-26,23-lactone (1alpha,25-(OH)(2)D(3)-26,23-lactone) analogs on 1alpha,25(OH)(2)D(3)-induced differentiation of human leukemia HL-60 cells thought to be mediated by the genomic action of 1alpha, 25-dihydroxyvitamin D(3) (1alpha,25-(OH)(2)D(3)) and of acute promyelocytic leukemia NB4 cells thought to be mediated by non-genomic actions of 1alpha,25-(OH)(2)D(3). We found that the 1alpha,25-(OH)(2)D(3)-26,23-lactone analogs, (23S)-25-dehydro-1alpha-hydroxyvitamin D(3)-26,23-lactone (TEI-9647) and (23R)-25-dehydro-1alpha-hydroxyvitamin D(3)-26,23-lactone (TEI-9648), inhibited differentiation of HL-60 cells induced by 1alpha,25-(OH)(2)D(3). However, 1beta-hydroxyl diastereomers of these analogs, i.e. (23S)-25-dehydro-1beta-hydroxyvitamin D(3)-26, 23-lactone (1beta-TEI-9647) and (23R)-25-dehydro-1beta-hydroxyvitamin D(3)-26,23-lactone (1beta-TEI-9648), did not inhibit differentiation of HL-60 cells caused by 1alpha,25-(OH)(2)D(3). A separate study showed that the nuclear vitamin D receptor (VDR) binding affinities of the 1-hydroxyl diastereomers were about 200 and 90 times weaker than that of 1alpha-hydroxyl diastereomers, respectively. Moreover, none of these lactone analogs inhibited NB4 cell differentiation induced by 1alpha,25-(OH)(2)D(3). In contrast, 1beta,25-dihydroxyvitamin D(3) (1beta,25-(OH)(2)D(3)) and 1beta,24R-dihydroxyvitamin D(3) (1beta,24R-(OH)(2)D(3)) inhibited NB4 cell differentiation but not HL-60 cell differentiation. Collectively, the results suggested that 1-hydroxyl lactone analogs, i.e. TEI-9647 and TEI-9648, are antagonists of 1alpha,25-(OH)(2)D(3), specifically for the nuclear VDR-mediated genomic actions, but not for non-genomic actions.     

An evaluation of the biologic activity and vitamin D receptor binding affinity of the photoisomers of vitamin D3 and previtamin D3

Skin is in the site of previtamin D3 and vitamin D3 synthesis and their isomerization in response to ultraviolet irradiation. At present, little is known about the function of the photoisomers of previtamin D3 and the vitamin D3 in skin cells. In this study we investigated the antiproliferative activity of the major photoisomers and their metabolites in the cultured human keratinocytes by determining their influence on 3H-thymidine incorporation into DNA. Our results demonstrated at both 10(-8) and 10(-6) M in a dose-dependent manner. Lumisterol, tachysterol3, 5,6-trans-vitamin D3, and 25-hydroxy-5,6-trans-vitamin D3 only induced significant inhibition at 10(-6) M. 25-Hydroxytachysterol3 was approximately 10- to 100-fold more active than tachysterol3. 7-Dehydrocholesterol was not active even at 10(-6) M. The dissociation constants of vitamin D receptor (VDR) for 25-hydroxytachysterol3, 25-hydroxy-5,6-trans-vitamin D3, and 5,6-trans-vitamin D3 were 22, 58, and 560 nM, respectively. The dissociation constants for 7-dehydrocholesterol, tachysterol, and lumisterol were greater than 20 microM. In conclusion, vitamin D3, its photoisomers and the photoisomers of previtamin D3 have antiproliferative activity in cultured human keratinocytes. However, the antiproliferative activity did not correlate with their binding affinity for VDR. The results suggest that some of the photoproducts may be metabolized to their 25-hydroxylated and 1 alpha,25-dihydroxylated counterparts before acting on VDR. Alternatively, a different receptor may recognize these photoproducts or another mechanism may be involved in modulating the antiproliferative activity of the photoisomers examined.     

Effects of vitamin D-binding proteins on HL-60 cell differentiation induced by 26,26,26,27,27,27-hexafluoro-1 alpha,25-dihydroxyvitamin D.

Monocytic differentiation-inducing activity of 26,26,26,27,27,27-hexafluoro-1 alpha,25-dihydroxyvitamin D3 [26,27-F6-1 alpha,25-(OH)2D3] was re-evaluated in human promyelocytic leukemia (HL-60) cells in serum-supplemented or serum-free culture. The order of in vitro potency for reducing nitroblue tetrazolium (NBT) was 26,27-F6-1 alpha,25-(OH)2D3 greater than 1 alpha, 25-dihydroxyvitamin D3 [1 alpha,25-(OH)2D3] = 26,26,26,27,27,27-F6-1 alpha,23(S), 25-trihydroxyvitamin D3 [26,27-F6-1 alpha,23(S), 25-(OH)3D3] under serum-supplemented culture conditions, whereas the order was 1 alpha, 25-(OH)2D3 = 26,27-F6-1 alpha,25-(OH)2D3 greater than 26,27-F6-1 alpha,23(S), 25-(OH)3D3 under serum-free culture conditions. This rank order for differentiation-inducing activity under serum-free culture conditions correlated well with the binding affinity of these analogs for vitamin D3 receptor of HL-60 cells. The order of relative % binding affinity for the vitamin D-binding protein in fetal calf serum was 1 alpha,25-(OH)2D3 (100%) much greater than 26,27-F6-1 alpha,25-(OH)2D3 (5.1%) greater than 26,27-F6-1 alpha,23(S), 25-(OH)3D3 (less than 1%). These results suggest that serum vitamin D-binding proteins apparently modulate monocytic differentiation of HL-60 cells by 26,27-F6-1 alpha,25-(OH)2D3 under serum-supplemented culture conditions.     

Efficient synthesis and biological evaluation of all A-ring diastereomers of 1alpha,25-dihydroxyvitamin D3 and its 20-epimer

An improved synthesis of the diastereomers of 1alpha,25-dihydroxyvitamin D3 (1) was accomplished utilizing our practical route to the A-ring synthon. We applied this procedure to synthesize for the first time all possible A-ring diastereomers of 20-epi-1alpha,25-dihydroxyvitamin D3 (2). Ten-step conversion of 1-(4-methoxyphenoxy)but-3-ene (6), including enantiomeric introduction of the C-3 hydroxyl group to the olefin by the Sharpless asymmetric dihydroxylation, provided all four possible stereoisomers of A-ring enynes (3). i.e., (3R,5R)-, (3R,5S)-, (3S,5R)- and (3S,5S)-bis[(tert-butyldimethylsilyl)oxy]oct-1-en-7-yne, in good overall yield. Palladium-catalyzed cross-coupling of the A-ring synthon with the 20-epi CD-ring portion (5), (E)-(20S)-de-A,B-8-(bromomethylene)cholestan-25-ol, followed by deprotection, afforded the requisite diastereomers of 20-epi-1alpha,25-dihydroxyvitamin D3 (2). The biological profiles of the synthesized stereoisomers were assessed in terms of affinities for vitamin D receptor (VDR) and vitamin D binding protein (DBP). HL-60 cell differentiation-inducing activity and in vivo calcium-regulating potency in comparison with the natural hormone.     

Mechanistic studies to evaluate the enhanced antiproliferation of human keratinocytes by 1alpha,25-dihydroxyvitamin D(3)-3-bromoacetate, a covalent modifier of vitamin D receptor, compared to 1alpha,25-dihydroxyvitamin D(3).

1alpha,25-Dihydroxyvitamin D(3)-3-bromoacetate (1, 25(OH)(2)D(3)-3-BE), an affinity labeling analog of 1alpha, 25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)), displayed stronger antiproliferative activities than 1,25(OH)(2)D(3) at 10(-10)-10(-6) M dose levels in cultured human keratinocytes (CHK). Additionally, preincubation of the cells with 10(-6) M 1,25(OH)(2)D(3), followed by treatment with various doses of 1,25(OH)(2)D(3)-3-BE, resulted in a significantly stronger antiproliferative activity by the mixture than individual reagents at every dose level. To search for a mechanism of this observation, we determined that [(14)C]1, 25(OH)(2)D(3)-3-BE covalently labeled human recombinant 1alpha, 25-dihydroxyvitamin D(3) receptor (reVDR) swiftly (<1 min) with a 1:1 stoichiometry and induced conformational changes (in VDR) that are different from 1,25(OH)(2)D(3), by limited tryptic digestion. Furthermore, a protein band, corresponding to reVDR, was specifically labeled by [(14)C]1,25(OH)(2)D(3)-3-BE in CHK extract, indicating that VDR is the main target of [(14)C]1, 25(OH)(2)D(3)-3-BE. The above-mentioned observations suggest that a rapid covalent labeling of VDR in CHK might alter the interaction between the holo-VDR and 1,25(OH)(2)D(3)-controlled genes. Furthermore, we observed that 1,25(OH)(2)D(3)-3-BE significantly decreased the binding of VDR to human osteocalcin vitamin D responsive element (hOCVDRE), as well as the dissociation rate of VDR from hOCVDRE, compared with 1,25(OH)(2)D(3) in COS-1 cells, transiently transfected with a VDR construct. Additionally, 1, 25(OH)(2)D(3)-3-BE was found to be more potent in inducing 1alpha, 25-dihydroxyvitamin D(3)-24-hydroxylase (24-OHase) promoter activity and mRNA expression in keratinocytes. The accumulation of 24-OHase message was also prolonged by the analog. Collectively these results indicated that rapid covalent labeling of VDR in keratinocytes (by 1, 25(OH)(2)D(3)-3-BE) might result in the conversion of apo-VDR to a holo-form, with a conformation that is different from that of the 1, 25(OH)(2)D(3)-VDR complex. This resulted in an enhanced stability of the 1,25(OH)(2)D(3)-3-BE/VDR-VDRE complex and contributed to the amplified antiproliferative effect of 1,25(OH)(2)D(3)-3-BE in keratinocytes.     

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.     

Molecular basis of the selective activity of vitamin D analogues

More than 2,000 synthetic analogues of the biological active form of vitamin D, 1alpha,25-dihydroxyvitamin D(3) (1alpha,25(OH)(2)D(3)), are presently known. Basically, all of them interfere with the molecular switch of nuclear 1alpha,25(OH)(2)D(3) signaling, which is the complex of the vitamin D receptor (VDR), the retinoid X receptor (RXR), and a 1alpha,25(OH)(2)D(3) response element (VDRE). Central element of this molecular switch is the ligand-binding domain (LBD) of the VDR, which can be stabilized by a 1alpha,25(OH)(2)D(3) analogue either in its agonistic, antagonistic, or non-agonistic conformation. The positioning of helix 12 of the LBD is of most critical importance for these conformations. In each of the three conformations, the VDR performs different protein-protein interactions, which then result in a characteristic functional profile. Most 1alpha,25(OH)(2)D(3) analogues have been identified as agonists, a few are antagonists (e.g., ZK159222 and TEI-9647), and only Gemini and some of its derivatives act under restricted conditions as non-agonists. The functional profile of some 1alpha,25(OH)(2)D(3) analogues, such as EB1089 and Gemini, can be modulated by protein and DNA interaction partners of the VDR. This provides them with some selectivity for DNA-dependent and -independent signaling pathways and VDRE structures.