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.     

Previtamin D3 with a trans-fused decalin CD-ring has pronounced genomic activity

Deletion of C19 in the structure of 1 alpha,25-dihydroxyvitamin D3 [1,25(OH)2D3] does not substantially alter the biological potency but prevents the conversion between the vitamin and the previtamin form. Hence, this modification allows the study of locked previtamin and vitamin forms. The locked 19-nor-1,25(OH)2-previtamin D3 analog (19-nor-previtamin D) had a low biological activity and was a rather weak activator of the genomic signal transduction pathway. 19-Nor-trans-decalin-1,25(OH)2-vitamin D3 (19-nor-TD-vitamin D), characterized by the presence of a trans-fused decalin CD-ring system, was 10-fold more potent than the parent compound and was a potent activator of the genomic signal transduction pathway. Surprisingly, the previtamin, 19-nor-trans-decalin-1,25(OH)2-previtamin D3 (19-nor-TD-previtamin D), was as potent as 1,25(OH)2D3 in inhibiting cell proliferation and inducing cell differentiation and represents the first previtamin structure with pronounced vitamin D-like activity. Furthermore, this compound interacted as efficiently as 1,25(OH)2D3 with the vitamin D receptor (VDR), retinoid X receptor (RXR), coactivators, and DNA, which illustrated its potent ability to activate the genomic signal transduction pathway. Analysis of the transactivation potency of 12 VDR point mutants after stimulation with 19-nor-TD-previtamin D revealed that this analog used the same contact points within the receptor as did 1,25(OH)2D3. This could be confirmed by modeling analysis of this compound in the ligand binding pocket of VDR. In conclusion, a previtamin D3 analog is presented with genomic activities equivalent to 1,25(OH)2D3.     

A perspective on how the Vitamin D sterol/Vitamin D receptor (VDR) conformational ensemble model can potentially be used to understand the structure-function results of A-ring modified Vitamin D sterols

The steroid hormone 1alpha,25(OH)(2)-Vitamin D(3) (1,25D) activates both genomic and non-genomic intracellular signaling cascades. It is also well recognized that co-incubation of 1,25D with its C-1 epimer, 1beta,25D (HL), suppresses the efficiency of the non-genomic signal activated by 1,25D alone and that its C-3 epimer, 3alpha-1,25D (HJ) is nearly as potent as 1,25D in suppressing PTH secretion, believed to be propagated by 1,25D's genomic signaling. Both these sterols lack the hypercalcemic effect induced by pharmacological doses of 1,25D and have reduced VDR affinity compared to 1,25D, as measured in a steroid competition assay. Recent functional studies suggest that the VDR is required for both non-genomic and genomic signaling. Along these lines we have recently proposed a Vitamin D sterol/VDR conformational ensemble model that posits the VDR contains two distinct, yet overlapping ligand binding sites, and that the potential differential stabilities of 1,25D and HL in these two pockets can be used to explain their different non-genomic signaling properties. The overlapping region is predominantly occupied by the sterol's A-ring when it is bound to either the genomic ligand binding pocket (G-pocket), defined by X-ray crystallography, or the alternative ligand binding pocket (A-pocket), discovered using in silico techniques (directed docking). Therefore, to gain further insight into the potential application of this model we docked the other A-ring diastereomer [(1beta,3alpha)=HH] of 1,25D and its 1- and 3-deoxy forms (25D and CF, respectively) to the A- and G-pockets to assess their potential stabilities in the pockets, relative to 1,25D. The models were then used to provide putative mechanistic arguments for their known structure-function experimental results. This model may provide new insights into how Vitamin D sterols that uncouple the unwanted hypercalcemic effect from attractive growth inhibitory/differentiation properties can do so by differentially stabilizing different subpopulations of VDR conformational ensemble members.     

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.     

Activation of Src kinase in skeletal muscle cells by 1, 1,25-(OH(2))-vitamin D(3) correlates with tyrosine phosphorylation of the vitamin D receptor (VDR) and VDR-Src interaction

The rapid effect of 1 alpha,25(OH(2))-vitamin D(3) [1 alpha, 25(OH(2))D(3)] on tyrosine kinase Src and its relationship to the vitamin D receptor (VDR) was investigated to further characterize the hormone signaling mechanism in chick muscle cells. Exposure of cultured myotubes to 1 alpha,25(OH(2))D(3) caused a time-dependent increase in Src activity, which was evident at 1 min (one-fold) and reached a maximum at 5 min (15-fold). Immunoblotting with anti-phosphotyrosine antibody of immunoprecipitated Src showed that the hormone decreased Src tyrosine phosphorylation state with maximal effects at 5 min. Using a database for protein consensus motifs we found a putative tyrosine phosphorylation site (amino acids 164-170: KTFDTTY) within the primary sequence of the chick VDR. When the myotube VDR was immunoprecipitated it appeared onto SDS-PAGE gels as a single band of 58 kDa recognized by an anti-phosphotyrosine antibody. Prior treatment of cells with (1)alpha,25(OH(2))D(3) significantly increased tyrosine phosphorylation of the VDR (two- to three-fold above basal levels). In agreement with Src being a SH2-domain containing protein involved in recognition of tyrosine-phosphorylated targets, immunoprecipitation with anti-Src antibody under native conditions followed by blotting with anti-VDR antibody, or using the antibodies in inverse order, showed that the VDR co-precipitates with Src, thus indicating the existence of a VDR/Src complex. Stimulation with the cognate VDR ligand significantly increased formation of the complex with respect to basal conditions. These results altogether provide the first evidence to date for 1 alpha,25(OH(2))D(3) activation involving Src association to tyrosine phosphorylated VDR.     

The rapid effects of 1,25-dihydroxyvitamin D3 require the vitamin D receptor and influence 24-hydroxylase activity: studies in human skin fibroblasts bearing vitamin D receptor mutations

If both rapid and genomic pathways may co-exist in the same cell, the involvement of the nuclear vitamin D receptor (VDR) in the rapid effects of 1,25-dihydroxyvitamin D(3) (1,25-(OH)(2)D(3)) remains unclear. We therefore studied rapid and long term effects of 1,25-(OH)(2)D(3) in cultured skin fibroblasts from three patients with severe vitamin D-resistant rickets and one age-matched control. Patients bear homozygous missense VDR mutations that abolished either VDR binding to DNA (patient 1, mutation K45E) or its stable ligand binding (patients 2 and 3, mutation W286R). In patient 1 cells, 1,25-(OH)(2)D(3) (1 pm-10 nm) had no effect on either intracellular calcium or 24-hydroxylase (enzyme activity and mRNA expression). In contrast, cells bearing the W286R mutation had calcium responses to 1,25-(OH)(2)D(3) (profile and magnitude) and 24-hydroxylase responses to low (1 pm-100 pm) 1,25-(OH)(2)D(3) concentrations (activity, CYP24, and ferredoxin mRNAs) similar to those of controls. The blocker of Ca(2+) channels, verapamil, impeded both rapid (calcium) and long term (24-hydroxylase activity, CYP24, and ferredoxin mRNAs) responses in patient and control fibroblasts. The MEK 1/2 kinase inhibitor PD98059 also blocked the CYP24 mRNA response. Taken together, these results suggest that 1,25-(OH)(2)D(3) rapid effects require the presence of VDR and control, in part, the first step of 1,25-(OH)(2)D(3) catabolism via increased mRNA expression of the CYP24 and ferredoxin genes in the 24-hydroxylase complex.