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.     

Metabolism of 20-epimer of 1alpha,25-dihydroxyvitamin D3 by CYP24: species-based difference between humans and rats

The 20-epi form of 1alpha,25-dihydroxyvitamin D(3) (1alpha,25(OH)(2)-20-epi-D(3)) is expected as drugs for leukemia, other cancers or psoriasis, because it shows several-hundred fold enhanced ability to induce cell differentiation and growth inhibition than 1alpha,25-dihydroxyvitamin D(3) while its calcemic activity is only slightly elevated. In this study, we compared the human and rat CYP24-dependent metabolism of 1alpha,25(OH)(2)-20-epi-D(3) by using the Escherichia coli expression system. The HPLC and LC-MS analyses of the metabolites revealed that rat CYP24 converted 1alpha,25(OH)(2)-20-epi-D(3) to 25,26,27-trinor-1alpha(OH)-24(COOH)-20-epi-D(3) through 1alpha,24,25(OH)(3)-20-epi-D(3) and 1alpha,25(OH)(2)-24-oxo-20-epi-D(3). The binding affinity of trinor-1alpha(OH)-24(COOH)-20-epi-D(3) for vitamin D receptor (VDR) was less than 1/4000 of that of 1alpha,25(OH)(2)-20-epi-D(3). These results suggest that rat CYP24 can almost completely inactivate 1alpha,25(OH)(2)-20-epi-D(3). On the other hand, human CYP24 mainly converted 1alpha,25(OH)(2)-20-epi-D(3) to its putative demethylated compound with a hydroxyl group, via 1alpha,24,25(OH)(3)-20-epi-D(3), 1alpha,25(OH)(2)-24-oxo-20-epi-D(3), and 1alpha,23,25(OH)(3)-24-oxo-20-epi-D(3). All of these metabolites showed considerable affinity for vitamin D receptor. These results clearly demonstrate the species-based difference between human and rat on the CYP24-dependent metabolism of 1alpha,25(OH)(2)-20-epi-D(3).     

Clonal differences in expression of 25-hydroxyvitamin D(3)-1alpha-hydroxylase,of 25-hydroxyvitamin D(3)-24-hydroxylase,and of the vitamin D receptor in human colon carcinoma cells: effects of epidermal growth factor and 1alpha,25-dihydroxyvitamin D(3)

Human colon carcinoma cells express 25-hydroxyvitamin D(3)-1alpha-hydroxylase (CYP27B1) and thus produce the vitamin D receptor (VDR) ligand 1alpha,25-dihydroxyvitamin D(3) (1,25-D3), which can be metabolized by 25-hydroxyvitamin D(3)-24-hydroxylase (CYP24). Expression of VDR, CYP27B1, and CYP24 determines the efficacy of the antimitotic action of 1,25-D3 and is distinctly related to the degree of differentiation of cancerous lesions. In the present study we addressed the question of whether the effects of epidermal growth factor (EGF) and of 1,25-D3 on VDR, CYP27B1, and CYP24 gene expression in human colon carcinoma cell lines also depend on the degree of cellular differentiation. We were able to show that slowly dividing, highly differentiated Caco-2/15 cells responded in a dose-dependent manner to both EGF and 1,25-D3 by up-regulation of VDR and CYP27B1 expression, whereas in highly proliferative, less differentiated cell lines, such as Caco-2/AQ and COGA-1A and -1E, negative regulation was observed. CYP24 mRNA was inducible in all clones by 1,25-D3 but not by EGF. From the observed clonal differences in the regulatory effects of EGF and 1,25-D3 on VDR and CYP27B1 gene expression we suggest that VDR-mediated growth inhibition by 1,25-D3 would be efficient only in highly differentiated carcinomas even when under mitogenic stimulation by EGF.     

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.