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.     

Altered expression of key players in vitamin D metabolism and signaling in malignant and benign thyroid tumors

1,25-Dihydroxyvitamin D(3) (1,25(OH)(2)D(3)), the active form of vitamin D, mediates antitumor effects in various cancers. The expression of key players in vitamin D signaling in thyroid tumors was investigated. Vitamin D receptor (VDR) and CYP27B1 and CYP24A1 (respectively activating and catabolizing vitamin D) expression was studied (RT-PCR, immunohistochemistry) in normal thyroid, follicular adenoma (FA), differentiated thyroid cancer (DTC) consisting of the papillary (PTC) and follicular (FTC) subtype, and anaplastic thyroid cancer (ATC). VDR, CYP27B1, and CYP24A1 expression was increased in FA and DTC compared with normal thyroid. However, in PTC with lymph node metastasis, VDR and CYP24A1 were decreased compared with non-metastasized PTC. In ATC, VDR expression was often lost, whereas CYP27B1/CYP24A1 expression was comparable to DTC. Moreover, ATC with high Ki67 expression (>30%) or distant metastases at diagnosis was characterized by more negative VDR/CYP24A1/CYP27B1 staining. In conclusion, increased expression of key players involved in local 1,25(OH)(2)D(3) signaling was demonstrated in benign and differentiated malignant thyroid tumors, but a decrease was observed for local nodal and especially distant metastasis, suggesting a local antitumor response of 1,25(OH)(2)D(3) in early cancer stages. These findings advocate further studies with 1,25(OH)(2)D(3) and analogs in persistent and recurrent iodine-refractory DTC.     

Vitamin D receptor interaction with specific DNA. Association as a 1,25-dihydroxyvitamin D3-modulated heterodimer.

The vitamin D receptor (VDR) is a member of the steroid receptor gene family. In this report, we examine the nature of specific VDR DNA binding utilizing the vitamin D-responsive element derived from the human osteocalcin promoter. Association of the VDR with the human osteocalcin 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) responsive element (VDRE) in vitro was characterized on VDRE affinity columns by both weak and strong interactions. Weak interaction was a property of the VDR itself, monomeric in nature, and determined exclusively by the VDR's DNA-binding domain. Strong interaction, in contrast, was dependent upon an intact receptor molecule as well as a heterologous mammalian cell nuclear accessory factor (NAF). Heteromeric interaction between VDR and NAF was independent of the VDR DNA-binding domain, suggesting the presence of a functional dimerization domain separate from that for DNA binding. Direct association of NAF with immobilized VDR revealed that the interaction does not require the presence of DNA. Most importantly, while occupancy of the VDR by 1,25(OH)2D3 was not required for VDR interactions with either DNA or NAF, the presence of hormone increased the apparent relative affinity of the VDR for NAF approximately 10-fold. These studies suggest that high affinity association of the VDR with DNA requires both the DNA-binding domain as well as an additional independent structure located within the steroid-binding region. This protein subdomain interacts with NAF and is regulated by 1,25(OH)2D3.     

Vitamin D signaling in immune-mediated disorders: Evolving insights and therapeutic opportunities

1,25(OH)(2)D(3), the active form of vitamin D, is a central player in calcium and bone metabolism. More recently, important immunomodulatory effects have been attributed to this hormone. The widespread presence of the vitamin D receptor (VDR) in the immune system and the expression of the enzymes responsible for the synthesis of the active 1,25(OH)(2)D(3) regulated by specific immune signals, even suggest a paracrine immunomodulatory role for 1,25(OH)(2)D(3). Additionally, the different molecular mechanisms used by 1,25(OH)(2)D(3) to exert its immunomodulatory effects prove of a broad action radius for this compound. Both, the effects of vitamin D deficiency and/or absence of the VDR as well as intervention with pharmacological doses of 1,25(OH)(2)D(3) or one of its less-calcemic analogs, affects immune system behavior in different animal models of immune-mediated disorders, such as type 1 diabetes. This review aims to summarize the data as they stand at the present time on the role of vitamin D in the pathogenesis of immune-mediated disorders, with special focus on type 1 diabetes, and on the therapeutic opportunities for vitamin D in the prevention and treatment of this autoimmune disease in mouse models and humans.     

Vitamin D analogs: therapeutic applications and mechanisms for selectivity

The vitamin D endocrine system plays a central role in mineral ion homeostasis through the actions of the vitamin D hormone, 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)], on the intestine, bone, parathyroid gland, and kidney. The main function of 1,25(OH)(2)D(3) is to promote the dietary absorption of calcium and phosphate, but effects on bone, kidney and the parathyroids fine-tune the mineral levels. In addition to these classical actions, 1,25(OH)(2)D(3) exerts pleiotropic effects in a wide variety of target tissues and cell types, often in an autocrine/paracrine fashion. These biological activities of 1,25(OH)(2)D(3) have suggested a multitude of potential therapeutic applications of the vitamin D hormone for the treatment of hyperproliferative disorders (e.g. cancer and psoriasis), immune dysfunction (autoimmune diseases), and endocrine disorders (e.g. hyperparathyroidism). Unfortunately, the effective therapeutic doses required to treat these disorders can produce substantial hypercalcemia. This limitation of 1,25(OH)(2)D(3) therapy has spurred the development of vitamin D analogs that retain the therapeutically important properties of 1,25(OH)(2)D(3), but with reduced calcemic activity. Analogs with improved therapeutic indices are now available for treatment of psoriasis and secondary hyperparathyroidism in chronic kidney disease, and research on newer analogs for these indications continues. Other analogs are under development and in clinical trials for treatment of various types of cancer, autoimmune disorders, and many other diseases. Although many new analogs show tremendous promise in cell-based models, this article will limit it focus on the development of analogs currently in use and those that have demonstrated efficacy in animal models or in clinical trials.     

A novel mutation in helix 12 of the vitamin D receptor impairs coactivator interaction and causes hereditary 1,25-dihydroxyvitamin D-resistant rickets without alopecia

Hereditary vitamin D-resistant rickets (HVDRR) is a genetic disorder most often caused by mutations in the vitamin D receptor (VDR). The patient in this study exhibited the typical clinical features of HVDRR with early onset rickets, hypocalcemia, secondary hyperparathyroidism, and elevated serum concentrations of alkaline phosphatase and 1,25-dihydroxyvitamin D [1,25-(OH)(2)D(3)]. The patient did not have alopecia. Assays of the VDR showed a normal high affinity low capacity binding site for [(3)H]1,25-(OH)(2)D(3) in extracts from the patient's fibroblasts. However, the cells were resistant to 1,25-dihydroxyvitamin D action as demonstrated by the failure of the patient's cultured fibroblasts to induce the 24-hydroxylase gene when treated with either high doses of 1,25-(OH)(2)D(3) or vitamin D analogs. A novel point mutation was identified in helix H12 in the ligand-binding domain of the VDR that changed a highly conserved glutamic acid at amino acid 420 to lysine (E420K). The patient was homozygous for the mutation. The E420K mutant receptor recreated by site-directed mutagenesis exhibited many normal properties including ligand binding, heterodimerization with the retinoid X receptor, and binding to vitamin D response elements. However, the mutant VDR was unable to elicit 1,25-(OH)(2)D(3)-dependent transactivation. Subsequent studies demonstrated that the mutant VDR had a marked impairment in binding steroid receptor coactivator 1 (SRC-1) and DRIP205, a subunit of the vitamin D receptor-interacting protein (DRIP) coactivator complex. Taken together, our data indicate that the mutation in helix H12 alters the coactivator binding site preventing coactivator binding and transactivation. In conclusion, we have identified the first case of a naturally occurring mutation in the VDR (E420K) that disrupts coactivator binding to the VDR and causes HVDRR.     

Antiproliferative role of vitamin D and its analogs – a brief overview

The active metabolite of vitamin D, 1, 25-dihydroxyvitamin D₃ [1,25(OH)₂D₃] – a seco-steroid hormone is a pivotal regulator of cellular proliferation and differentiation those are independent of its classical function of calcium homeostasis and bone mineralization. The existence of the nuclear vitamin D receptor (VDR) has been found in numerous tissues in different organs, which are the so-called 'non-classical' targets of this seco-steroid hormone. Vitamin D has been documented as a potent antiproliferative agent in different tissues and cells. Epidemiological studies reveal a negative correlation between physiological level of vitamin and cancer risk. Studies using animal models clearly demonstrate protective role of vitamin D in different cancer types by the reduction in tumor progression and by monitoring biochemical parameters. Experiments with cultured human and animal cancer cell lines show similar antiproliferative role of vitamin D manifested by up or down regulations of crucial genes leading to inhibition of cellular growth. Hypercalcemia hinders broad-spectrum therapeutic uses of vitamin D in cancer chemotherapy. Application of vitamin D analogs having similar chemical structures or other compounds having vitamin D like actions but lacking calcemic adverse effects are getting significant attention towards rational therapeutics to treat cancer. The current review focuses on the application of vitamin D and its analogs in different forms of cancer and on the molecular mechanism involved in vitamin D mediated inhibition in cellular proliferation, cell cycle, induction of apoptosis and tumor suppression, which may eventually evolve as a meaningful cancer therapy.     

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.