Identification of novel mediators of Vitamin D signaling and 1,25(OH)2D3 resistance in mammary cells

Since the discovery of the Vitamin D receptor (VDR) in mammary cells, the role of the Vitamin D signaling pathway in normal glandular function and in breast cancer has been extensively explored. In vitro studies have demonstrated that the VDR ligand, 1,25(OH)₂D₃, modulates key proteins involved in signaling proliferation, differentiation and survival of normal mammary epithelial cells. Anti-proliferative and pro-differentiating effects of 1,25(OH)₂D₃ have also been observed in VDR positive breast cancer cells, indicating that transformation per se does not abolish Vitamin D signaling. However, many breast cancer cell lines are less sensitive to 1,25(OH)₂D₃ than normal mammary epithelial cells. Reduced sensitivity to 1,25(OH)₂D₃ has been linked to alterations in Vitamin D metabolizing enzymes as well as down regulation of VDR expression or function. In this report, we describe results from a proteomics screening approach used to search for proteins involved in dictating sensitivity or resistance to Vitamin D mediated apoptosis in breast cancer cells. Several proteins not previously linked to 1,25(OH)₂D₃ signaling were identified with this approach, and a distinct subset of proteins was linked to 1,25(OH)₂D₃ resistance. Follow-up studies to determine the relevance of these proteins to Vitamin D signaling in general are in progress.     

Vitamin D and cancer

Vitamin D, a steroid hormone and exerts its biological effects through its active metabolite 1alpha, 25 dihydroxyvitamin D3 [1,25(OH)2D3]. Like steroid hormones, 1,25(OH)2D3 is efficacious at very low concentrations and serves as a ligand for vitamin D receptors (VDR), associating with VDR very high affinity. Despite its potent property as a differentiating agent, its use in the clinical practice is hampered by the induction of hypercalcemia at a concentration required to suppress cancer cell proliferation. Therefore nearly 400 structural analogs of vitamin D3 have been synthesized and evaluated for their efficacy and toxicity. Among these analogs, relatively less toxic but highly efficacious analogs, EB1089, RO24-5531, 1alpha-hydroxyvitamin D5 and a few others have been evaluated in a preclinical toxicity and in Phase I clinical trials for dose tolerance in advanced cancer patients. Clinical trials using vitamin D analogs for prevention or therapy of cancer patients are still in their infancy. Vitamin D mediates its action by two independent pathways. Genomic pathway involves nuclear VDR and induces biological effects by interactions with hormone response elements and modulation of differential gene expressions. Evidence also suggests that vitamin D analogs also interact with steroid hormone(s) inducible genes. The non-genomic pathway is characterized by rapid actions of vitamin D. It involves interactions with membrane-VDR interactions and its interactions with protein kinase C and by altering intracellular calcium channels. Thus, the development of nontoxic analogs of vitamin D analogs and understanding of their molecular mechanism(s) of action are of significant importance in the prevention and treatment of cancer by vitamin D.     

Identification of novel mediators of Vitamin D signaling and 1,25(OH)2D3 resistance in mammary cells

Since the discovery of the Vitamin D receptor (VDR) in mammary cells, the role of the Vitamin D signaling pathway in normal glandular function and in breast cancer has been extensively explored. In vitro studies have demonstrated that the VDR ligand, 1,25(OH)₂D₃, modulates key proteins involved in signaling proliferation, differentiation and survival of normal mammary epithelial cells. Anti-proliferative and pro-differentiating effects of 1,25(OH)₂D₃ have also been observed in VDR positive breast cancer cells, indicating that transformation per se does not abolish Vitamin D signaling. However, many breast cancer cell lines are less sensitive to 1,25(OH)₂D₃ than normal mammary epithelial cells. Reduced sensitivity to 1,25(OH)₂D₃ has been linked to alterations in Vitamin D metabolizing enzymes as well as down regulation of VDR expression or function. In this report, we describe results from a proteomics screening approach used to search for proteins involved in dictating sensitivity or resistance to Vitamin D mediated apoptosis in breast cancer cells. Several proteins not previously linked to 1,25(OH)₂D₃ signaling were identified with this approach, and a distinct subset of proteins was linked to 1,25(OH)₂D₃ resistance. Follow-up studies to determine the relevance of these proteins to Vitamin D signaling in general are in progress.     

Altered Vitamin D receptor-coactivator interactions reflect superagonism of Vitamin D analogs

The active form of Vitamin D, 1alpha,25-dihydroxyvitamin D(3) [1,25-(OH)(2)D(3)], has potent antiproliferative actions on various normal and malignant cells. Calcemic effects, however, hamper therapeutic application of 1,25-(OH)(2)D(3) in hyperproliferative diseases. Two 14-epi-analogs of 1,25-(OH)(2)D(3) namely 19-nor-14-epi-23-yne-1,25-(OH)(2)D(3) (TX522) and 19-nor-14,20-bisepi-23-yne-1,25-(OH)(2)D(3) (TX527), display reduced calcemic effects coupled to an (at least 10-fold) increased antiproliferative potency when compared with 1,25-(OH)(2)D(3). Altered cofactor recruitment by the Vitamin D receptor (VDR) might underlie the superagonism of these 14-epi-analogs. Therefore, this study aims to evaluate their effects at the level of VDR-coactivator interactions. Mammalian two-hybrid assays with VDR and the coactivators TIF2 and DRIP205 showed the 14-epi-analogs to be more potent inducers of VDR-coactivator interactions than 1,25-(OH)(2)D(3). TX522 and TX527 require 30- and 40-fold lower doses to obtain the VDR-DRIP205 interaction induced by 1,25-(OH)(2)D(3) at 10(-8)M. Evaluation of additional 1,25-(OH)(2)D(3)-analogs and their impact on VDR-coactivator interactions revealed a strong correlation between the antiproliferative potency of an analog and its ability to induce VDR-coactivator interactions. In conclusion, these data show that altered coactivator binding by the VDR is one possible explanation for the superagonistic action of the two 14-epi-analogs TX522 and TX527.     

Vitamin D receptor interacts with DnaK/heat shock protein 70: identification of DnaK interaction site on vitamin D receptor

Vitamin D receptor (VDR) regulates the expression of vitamin D-dependent genes upon binding to its cognate ligand, 1alpha, 25-dihydroxyvitamin D3 (1,25(OH)2D3). This process represents a complex interaction of ligand-bound VDR with nuclear proteins like retinoid X receptor, nuclear accessory factors, and regulatory elements of the target gene. Expression of full-length VDR in Escherichia coli revealed that VDR binds DnaK, a member of heat-shock protein (Hsp) family, with high affinity. By systematic N-terminal truncation of VDR, the interaction site of DnaK on VDR was localized within a 17-amino-acid segment (105-122) representing the "hinge region" between the DNA-binding and hormone-binding domains of VDR. The putative DnaK-binding site was further localized between residues 105 to 109 of VDR by using binding-energy-minimization studies. The interaction of DnaK with VDR did not influence the binding of 1,25(OH)2D3 or nuclear accessory factor(s) to VDR. Furthermore, bovine brain Hsp 70, similar to DnaK, interacted with VDR-ligand-binding domain (105-427). These results suggest that DnaK/Hsp 70 may interact with VDR prior to the activation of the latter by 1,25(OH)2D3-binding.     

Functional and structural characterization of the insertion region in the ligand binding domain of the vitamin D nuclear receptor.

Vitamin D nuclear receptor mediates the genomic actions of the active form of vitamin D, 1,25(OH)2D3. This hormone is involved in calcium and phosphate metabolism and cell differentiation. Compared to other nuclear receptors, VDR presents a large insertion region at the N-terminal part of the ligand binding domain between helices H1 and H3, encoded by an additional exon. This region is poorly conserved in VDR in different species and is not well ordered as observed by secondary structure prediction. We engineered a VDR ligand binding domain mutant by removing this insertion region. Here we report its biochemical and biophysical characterization. The mutant protein exhibits the same ligand binding, dimerization with retinoid X receptor and transactivation properties as the wild-type VDR, suggesting that the insertion region does not affect these main functions. Solution studies by small angle X-ray scattering shows that the conformation in solution of the VDR mutant is similar to that observed in the crystal and that the insertion region in the VDR wild-type is not well ordered.     

Breast cancer cell regulation by high-dose Vitamin D compounds in the absence of nuclear vitamin D receptor

1alpha,25-dihydroxyvitamin D(3) (1,25D(3)) inhibits growth and induces apoptosis in breast cancer cells in vivo and in vitro. To examine the role of the Vitamin D receptor (VDR) in mediating the actions of 1,25D(3) at nanomolar and micromolar concentrations, mammary epithelial tumor cell lines generated in wild type (WT) and VDR knockout (VDRKO) mice were utilized. WT cells express VDR and are growth inhibited by 1,25D(3) and synthetic analogs EB1089 and CB1093 at 1nM concentrations, while VDRKO cells do not express VDR and are insensitive to Vitamin D compounds at concentrations up to 100nM. In the current studies, we have confirmed and extended these previous observations. At nanomolar concentrations of 1,25D(3) and all analogs tested, including EB1089, CB1093, MC1288, and KH1230, WT cells are growth inhibited and exhibit apoptotic morphology, while VDRKO cells show no growth inhibition or apoptosis. At concentrations of 1-10microM, however, 1,25D(3) and synthetic analogs induce growth inhibition and apoptotic morphology in both WT and VDRKO cell lines. These data indicate that nanomolar concentrations of 1,25D(3) and analogs mediate growth regulatory effects via mechanisms requiring the nuclear VDR, but that micromolar concentrations of Vitamin D compounds can exert non VDR-mediated effects.     

Ligand-specific structural changes in the vitamin D receptor in solution

Vitamin D receptor (VDR) is a member of the nuclear hormone receptor superfamily. When bound to a variety of vitamin D analogues, VDR manifests a wide diversity of physiological actions. The molecular mechanism by which different vitamin D analogues cause specific responses is not understood. The published crystallographic structures of the ligand binding domain of VDR (VDR-LBD) complexed with ligands that have differential biological activities have exhibited identical protein conformations. Here we report that rat VDR-LBD (rVDR-LBD) in solution exhibits differential chemical shifts when bound to three ligands that cause diverse responses: the natural hormone, 1,25-dihydroxyvitamin D(3) [1,25(OH)?D?], a potent agonist analogue, 2-methylene-19-nor-(20S)-1,25-dihydroxyvitamin D? [2MD], and an antagonist, 2-methylene-(22E)-(24R)-25-carbobutoxy-26,27-cyclo-22-dehydro-1α,24-dihydroxy-19-norvitamin D? [OU-72]. Ligand-specific chemical shifts mapped not only to residues at or near the binding pocket but also to residues remote from the ligand binding site. The complexes of rVDR-LBD with native hormone and the potent agonist 2MD exhibited chemical shift differences in signals from helix-12, which is part of the AF2 transactivation domain that appears to play a role in the selective recruitment of coactivators. By contrast, formation of the complex of rVDR-LBD with the antagonist OU-72 led to disappearance of signals from residues in helices-11 and -12. We present evidence that disorder in this region of the receptor in the antagonist complex prevents the attachment of coactivators.     

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.     

Lipopolysaccharide negatively modulates vitamin D action by down-regulating expression of vitamin D-induced VDR in human monocytic THP-1 cells

Vitamin D3, an important seco-steroid hormone for the regulation of body calcium homeostasis, promotes immature myeloid precursor cells to differentiate into monocytes/macrophages. Vitamin D receptor (VDR) belongs to a nuclear receptor super-family that mediates the genomic actions of vitamin D3 and regulates gene expression by binding with vitamin D response elements in the promoter region of the cognate gene. Thus by regulating gene expression, VDR plays an important role in modulating cellular events such as differentiation, apoptosis, and growth. Here we report lipopolysaccharide (LPS), a bacterial toxin; decreases VDR protein levels and thus inhibits VDR functions in the human blood monocytic cell line, THP-1. The biologically active form of vitamin D3, 1alpha,25-dihydroxy vitamin D3 [1,25(OH)2D3], induced VDR in THP-1 cells after 24 h treatment, and LPS inhibited 1,25(OH)2D3-mediated VDR induction. However, LPS and 1,25(OH)2D3 both increased VDR mRNA levels in THP-1 cells 20 h after treatment, as observed by real time RT-PCR. Moreover, LPS plus 1,25(OH)2D3 action on VDR mRNA level was additive and synergistic. A time course experiment up to 60 h showed an increase in VDR mRNA that was not preceded with an increase in VDR protein levels. Although the proteasome pathway plays an important role in VDR degradation, the proteasome inhibitor lactacystin had no effect on the LPS-mediated down-regulation of 1,25(OH)2D3 induced VDR levels. Reduced VDR levels by LPS were accompanied by decreased 1,25(OH)2D3/VDR function determined by VDR responsive 24-hydroxylase (CYP24) gene expression. The above results suggest that LPS impairs 1,25(OH)2D3/VDR functions, which may negatively affect the ability of 1,25(OH)2D3 to induce myeloid differentiation into monocytes/macrophages.     

Vitamin D and androgen regulation of prostatic growth

Vitamin D has been reported to inhibit the growth of prostate cancer cells and model systems. In this study, we examined the interaction between 1,25-dihydroxyvitamin D(3) (1,25 D) in the presence or absence of endogenous testosterone on the growth and development of the adult rat prostate. Male Sprague-Dawley rats (165 days old) were either kept intact or castrated. Seven days after castration, the rats were treated with vehicle (control) or 1,25 D for 3 weeks and then sacrificed. Both ventral and dorsal lateral prostates were harvested; whole tissue lysates were collected and AR and VDR protein levels were analyzed by immunoblot analyses. Administration of 1,25 D in the intact animals decreased the prostatic size by 40%, compared to control animals, whereas 1,25 D did not influence the size of the prostate in castrated rats. 1,25 D administration in intact groups also increased both the AR and VDR protein levels by approximately twofold, whereas in castrated groups, 1,25 D only increased the AR protein level by 1.5-2.5-fold. 1,25 D in the presence of endogenous testosterone inhibits prostatic growth, whereas 1,25 D in the absence of endogenous testosterone does not affect prostatic growth. The growth inhibitory activity of 1,25 D in the presence of testosterone may be mediated through the ligand activated AR and VDR pathways. These studies may reveal important information about the potential efficacy of 1,25 D as well as hormonal status in understanding the development of prostate diseases.