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.     

Regulation of the vitamin D receptor and cornifin beta expression in vaginal epithelium of the rats through vitamin D3

The aim of the present study was to determine the respective role of 1,25-dihydroxyvitamin D3 on vaginal epithelium and 1,25-dihydroxyvitamin D3 receptor expression in ovariectomized rats and vitamin D3 treated rats. Bilateral ovariectomies were performed in 20 mature, non-pregnant Wistar female rats. All the animals were divided into 2 groups consisting of 10 rats each. Group I served as control. In group II, animals were injected intramuscularly with vitamin D3 (50, 00 IU/kg). Two weeks after the injections, vaginas of animals in group I and group II were removed removed and processed for immunohistochemistry. Epithelial differentiation, 1,25-dihydroxyvitamin D3 receptor and cornifin beta expression were investigated in vaginal epithelium of control group (ovariectomized) and vitamin D3 treated rats. Vaginal epithelial cells from vitamin D3 treated animals changed into highly- stratified keratinizing layers. 1,25-dihydroxyvitamin D3 receptor and cornifin beta as a marker of squamous differentiation were present in ovariectomized rats treated with 1,25-dihydroxyvitamin D3. In contrast, cornifin beta and 1,25-dihydroxyvitamin D3 receptor were absent in all layers of vaginal epithelium in control group. We demonstrated for the first time that 1,25-dihydroxyvitamin D3 induced proliferation of vaginal epithelium consistent with the cornifin beta expression and 1,25-dihydroxyvitamin D3 up-regulated 1,25-dihydroxyvitamin D3 receptor expression in vaginal epithelium.     

Increased nuclear expression and transactivation of vitamin D receptor by the cardiotonic steroid bufalin in human myeloid leukemia cells

The active form of vitamin D₃, 1α,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃], is a potent ligand for the nuclear receptor vitamin D receptor (VDR) and induces myeloid leukemia cell differentiation. The cardiotonic steroid bufalin enhances vitamin D-induced differentiation of leukemia cells and VDR transactivation activity. In this study, we examined the combined effects of 1,25(OH)₂D₃ and bufalin on differentiation and VDR target gene expression in human leukemia cells. Bufalin in combination with 1,25(OH)₂D₃ enhanced the expression of VDR target genes, such as CYP24A1 and cathelicidin antimicrobial peptide, and effectively induced differentiation phenotypes. An inhibitor of the Erk mitogen-activated protein (MAP) kinase pathway partially inhibited bufalin induction of VDR target gene expression. 1,25(OH)₂D₃ treatment induced transient nuclear expression of VDR in HL60 cells. Interestingly, bufalin enhanced 1,25(OH)₂D₃-induced nuclear VDR expression. The MAP kinase pathway inhibitor increased nuclear VDR expression induced by 1,25(OH)₂D₃ and did not change that by 1,25(OH)₂D₃ plus bufalin. A proteasome inhibitor also enhanced 1,25(OH)₂D₃-induced CYP24A1 expression and nuclear VDR expression. Bufalin-induced nuclear VDR expression was associated with histone acetylation and VDR recruitment to the CYP24A1 promoter in HL60 cells. Thus, the Na⁺,K⁺-ATPase inhibitor bufalin modulates VDR function through several mechanisms, including Erk MAP kinase activation and increased nuclear VDR expression.     

Lithocholic acid derivatives act as selective vitamin D receptor modulators without inducing hypercalcemia

1alpha,25-Dihydroxyvitamin D(3) [1,25(OH)(2)D(3)], a vitamin D receptor (VDR) ligand, regulates calcium homeostasis and also exhibits noncalcemic actions on immunity and cell differentiation. In addition to disorders of bone and calcium metabolism, VDR ligands are potential therapeutic agents in the treatment of immune disorders, microbial infections, and malignancies. Hypercalcemia, the major adverse effect of vitamin D(3) derivatives, limits their clinical application. The secondary bile acid lithocholic acid (LCA) is an additional physiological ligand for VDR, and its synthetic derivative, LCA acetate, is a potent VDR agonist. In this study, we found that an additional derivative, LCA propionate, is a more selective VDR activator than LCA acetate. LCA acetate and LCA propionate induced the expression of the calcium channel transient receptor potential vanilloid type 6 (TRPV6) as effectively as that of 1alpha,25-dihydroxyvitamin D(3) 24-hydroxylase (CYP24A1), whereas 1,25(OH)(2)D(3) was more effective on TRPV6 than on CYP24A1 in intestinal cells. In vivo experiments showed that LCA acetate and LCA propionate effectively induced tissue VDR activation without causing hypercalcemia. These bile acid derivatives have the ability to function as selective VDR modulators.     

Role of vitamin D3 receptor in the synergistic differentiation of WEHI-3B leukemia cells by vitamin D3 and retinoic acid.

WEHI-3B D- cells differentiate in response to 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) but not to all-trans-retinoic acid (RA) or other inducing agents. Combinations of RA with 1,25-(OH)2D3 interact to produce synergistic differentiation of WEHI-3B D- cells. To determine factors involved in the synergistic interaction, expression of the 1,25-(OH)2D3 receptor (VDR) and retinoid receptors, RARalpha and RXRalpha, was measured. No VDR was detected in untreated WEHI-3B D- cells; however, RA and 1,25-(OH)2D3 when used as single agents caused a slight induction of the VDR and in combination produced a marked increase in the VDR. In contrast, no changes in RARalpha and RXRalpha were initiated by these compounds. An RAR-selective agonist combined with 1,25-(OH)2D3 produced synergistic differentiation of WEHI-3B D- cells, whereas an RXR-selective agonist did not. To gain information on the role of the VDR in the synergistic interaction, the VDR gene was transferred into WEHI-3B D+ cells, in which no VDR was detected and no synergism was produced. Expression of the VDR conferred differentiation responsiveness to 1,25-(OH)2D3 in WEHI-3B D+ cells. These findings suggest that (a) induction of VDR expression is a key component in the synergistic differentiation induced by 1,25-(OH)2D3 and RA and (b) RAR and not RXR must be activated for enhanced induction of the VDR and for the synergistic differentiation produced by RA and 1, 25-(OH)2D3.