Vitamin D Receptor Signaling Pathway Research Articles

Stanniocalcin 1 and 1,25-dihydroxyvitamin D3 cooperatively regulate bone mineralization by osteoblasts

Stanniocalcin 1 (STC1) is a calcium- and phosphate-regulating hormone that is expressed in all tissues, including bone tissues, and is involved in calcium and phosphate homeostasis. Previously, STC1 expression was found to be increased by 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] administration in renal proximal tubular cells. In this study, we investigated whether STC1 directly regulates osteoblast differentiation or reciprocally controls the effects of 1,25(OH)2D3 on osteoblasts to contribute to bone homeostasis. We found that STC1 inhibited osteoblast differentiation in vitro and bone morphogenetic protein 2 (BMP2)-induced ectopic bone formation in vivo. Moreover, 1,25(OH)2D3 increased STC1 expression through direct binding to the Stc1 promoter of the vitamin D receptor (VDR). STC1 activated the 1,25(OH)2D3–VDR signaling pathway through the upregulation of VDR expression mediated by the inhibition of Akt phosphorylation in osteoblasts. STC1 further increased the effects of 1,25(OH)2D3 on receptor activator of nuclear factor-κB ligand (RANKL) secretion and inhibited osteoblast differentiation by exhibiting a positive correlation with 1,25(OH)2D3. The long-bone phenotype of transgenic mice overexpressing STC1 specifically in osteoblasts was not significantly different from that of wild-type mice. However, compared with that in the wild-type mice, 1,25(OH)2D3 administration significantly decreased bone mass in the STC1 transgenic mice. Collectively, these results suggest that STC1 negatively regulates osteoblast differentiation and bone formation; however, the inhibitory effect of STC1 on osteoblasts is transient and can be reversed under normal conditions. Nevertheless, the synergistic effect of STC1 and 1,25(OH)2D3 through 1,25(OH)2D3 administration may reduce bone mass by inhibiting osteoblast differentiation.

Clinical efficacy of moxibustion for ulcerative colitis and its influence on vitamin D receptor

ObjectiveTo observe the clinical efficacy of herbal cake-partitioned moxibustion for ulcerative colitis (UC) and elucidate its mechanism by targeting the vitamin D receptor (VDR) signaling pathway.MethodsA total of 63 patients with UC were randomly divided into an observation group (30 cases, treated with herbal cake-partitioned moxibustion) and a control group (33 cases, treated with sham herbal cake-partitioned moxibustion). Moxibustion treatment was performed at Qihai (CV6) and bilateral Tianshu (ST25) and Shangjuxu (ST37), 3 times per week for 12 weeks. The total effective rate, visual analog scale (VAS) score for abdominal bloating and pain, and hospital anxiety and depression scale (HADS) score were compared between the two groups. Enzyme-linked immunosorbent assay was used to detect the concentrations of serum C-reactive protein (CRP), 25-hydroxyvitamin D [25(OH)D], and interleukin-12 (IL-12)/interleukin-23 (IL-23) p40. Immunohistochemistry was used to observe the expression levels of VDR and regenerating gene IV (Reg IV) proteins in colonic mucosa. The expression levels of VDR, cytochrome p450 27B1 (CYP27B1), and Reg IV mRNAs were detected by real-time fluorescence quantitive polymerase chain reaction.ResultsAfter treatment, the total effective rate in the observation group was 86.7%, which was significantly higher than 51.5% in the control group (P<0.05). After treatment, the VAS scores for abdominal bloating and pain in the observation group were significantly decreased (P<0.01), as well as the HADS-depression subscale (HADS-D) and HADS-anxiety subscale (HADS) scores (P<0.05), while only the VAS score for abdominal pain in the control group was reduced (P<0.05), and the improvements of the scores in the observation group were more significant than those in the control group (P<0.05). After treatment, the serum CRP concentrations in both groups and the IL-12/IL-23 p40 concentration in the observation group were significantly decreased (P<0.05), and the concentrations in the observation group were lower than those in the control group (P<0.05). The expression levels of VDR protein and mRNA in the colon in both groups were all increased (P<0.01), and the expression levels of Reg IV protein and mRNA and CYP27B1 mRNA were all decreased in the two groups (P<0.05 or P<0.01); the improvements in the observation group were more notable than those in the control group (P<0.05 or P<0.01).ConclusionHerbal cake-partitioned moxibustion can effectively alleviate abdominal pain and diarrhea in patients with UC, improve depression and anxiety disorders, and regulate the expression of related proteins in the VDR signaling pathway. The mechanism may be related to inhibiting intestinal inflammation by reducing the release of the proinflammatory cytokine IL-12/IL-23 p40.

P021 Vitamin D receptor-dependent protective effect of moderate hypoxia in a mouse colitis model Running title: Hypoxia prevents mouse colitis through VDR

Abstract Background Although physiological hypoxia is important for maintaining the intestinal barrier, its effect on the barrier during acute colitis and the underlying mechanisms are not fully understood. Here,we aim to explore the influence of hypoxia in dextran sulfate sodium (DSS)-induced colitis mice and the role of hypoxia-inducible factor (HIF) and vitamin D receptor (VDR) in the process Methods Colitis mice were subjected to hypoxia to detect colitis symptoms and intestinal barrier function changes. Finally, the mechanisms underlying the changes were explored in a colon cell line. Results First, compared with colitis mice without hypoxia stimulation, those with hypoxia stimulation showed significantly decreased pathological damage and improved permeability of the intestinal barrier. The expression of tight junction proteins (including occludin, claudin-1 and ZO-1), HIF-1α as well as VDR was up-regulated in colitis mice with hypoxia stimulation. However, in VDR gene knockout (VDR-KO) colitis mice, hypoxia treatment showed no protective effect, suggesting the VDR dependency of this effect. Second, we identified that although hypoxia stimulation could enhance the single-layer epithelial transmembrane electrical resistance in DLD-1 cells, these effects disappeared in VDR-knockdown cells. Furthermore, over-expression of HIF-1α in DLD-1 can increase the expression of VDR, then chromatin immunoprecipitation and luciferase assays confirmed that HIF-1α can bind to the promoter region of the VDR gene under hypoxia. Finally, compared with their wild-type siblings, VDR-KO mice showed reduced abundance of anaerobic bacteria and SCFA-producing bacteria. Conclusion Moderate hypoxia was protective against DSS-induced colitis, and VDR is instrumental in it. Furthermore, HIF-1α mediates the effect of hypoxia on the VDR signaling pathway. Moreover, intestinal flora may be an important link between hypoxia and VDR.

Role of vitamin D-vitamin D receptor signaling on hyperoxia-induced bronchopulmonary dysplasia in neonatal rats.

Vitamin D exerts therapeutic effects on bronchopulmonary dysplasia (BPD), but its underlying mechanisms remain unclear. The present study was designed to investigate the effects of vitamin D on hyperoxia-induced BPD and elucidate the underlying mechanisms. Neonatal rats were exposed to either room air (control) or 75% O2 (hyperoxia) and intraperitoneally injected with vitamin D3. After 14 days, a histopathological examination was performed in the lungs of rats. Serum 25-hydroxyvitamin D (25OHD) was measured by liquid chromatography-tandom mass spectrometry (LC-MS)/MS. Interleukin 1 beta (IL-1β) and interferon gamma (IFN-γ) were measured by specificenzyme-linked immunosorbent assays. The messenger RNA and protein levels of vitamin D receptor (VDR), vascular endothelial growth factor (VEGF), VEGF receptor 2 (VEGFR2), and hypoxia-inducible factor 1α (HIF-1α) were determined by real-time quantitative reverse transcription polymerase chain reaction and immunoblot analysis, respectively. Treatment with vitamin D3 increased serum 25OHD and upregulated VDR in lung tissues with or without hyperoxia. In addition, treatment with vitamin D3 attenuated alveolar simplification, increased VEGF and VEGFR2, and protected alveolar simplification induced by hyperoxia. Furthermore, treatment with vitamin D3 resulted in a decrease of IL-1β and IFN-γ and an increase of HIF-1α in lung tissues under hyperoxia conditions. Vitamin D exerts protective effects on hyperoxia-induced BPD in neonatal rats by regulating vitamin D-VDR signaling pathways.

The effects of mitotane and 1α,25-dihydroxyvitamin D3 on Wnt/beta-catenin signaling in human adrenocortical carcinoma cells.

Mitotane is the only chemotherapeutic agent available for the treatment of adrenocortical carcinoma (ACC), however, the anti-neoplastic efficacy is limited due to several side-effects in vivo. There is, therefore, a need of exploring for new anti-tumoral agents which can be used either alone or in combination with mitotane. The active vitamin D metabolite 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3) acts as an anti-proliferative agent in human cancer by inhibiting the Wnt/beta-catenin pathway through the vitamin D receptor (VDR). The aim of this study was to study the effects of mitotane and 1α,25(OH)2D3, individually or in combination, in an in vitro model with H295R ACC cells, and to elucidate the molecular events behind their effects involving the Wnt/beta-catenin signaling. Multiple concentrations of mitotane and 1α,25(OH)2D3, individually or in combination, were tested on H295R cells for 24-96h, and the effects analysed by MTT. A reduction in cell growth was observed in a dose/time-dependent manner for both mitotane and 1α,25(OH)2D3. In addition, a combination of clinically sub-therapeutic concentrations of mitotane with 1α,25(OH)2D3, had an additive anti-proliferative effect (Combination Index = 1.02). In a wound healing assay, individual treatments of both mitotane and 1α,25(OH)2D3 reduced the migration ability of H295R cells, with the effect further enhanced on combining both the agents. Western blotting and qRT-PCR analysis showed a modulation of the Wnt/beta-catenin and VDR signaling pathways. Our results show an additive effect of mitotane and 1α,25(OH)2D3 on the inhibition of H295R ACC cell growth and viability, and suggest that molecular mechanisms of their effects involve a functional link between VDR and Wnt/beta-catenin pathways.

Accumulation of hyaluronic acid in stromal cells modulates osteoclast formation by regulation of receptor activator of nuclear factor kappa-B ligand expression

Hyaluronic acid (HA) has a pivotal role in bone and cartilage metabolism. In this study, we investigated the effect and underlying mechanisms of HA accumulation on the expression of receptor activator of nuclear factor kappa-B ligand (RANKL) induced by 1α,25(OH)2D3 and dexamethasone in stromal cells, which support osteoclastogenesis. Degradation of HA by hyaluronidase (HA'ase) treatment enhanced the expression of RANKL in ST2 cells stimulated with 1α,25(OH)2D3 and dexamethasone. Down-regulation of hyaluronan synthase 2 (HAS2) expression by siRNA also stimulated RANKL expression induced by 1α,25(OH)2D3 and dexamethasone. Results from a cell co-culture system with bone marrow cell showed that 1α,25(OH)2D3 and dexamethasone-induced RANKL expression in HA'ase treated- and HAS2 siRNA transfected-ST2 cells was down-regulated by treatment of cells with high molecular weight HA. In contrast, transforming growth factor-β1 (TGF-β1), which stimulates HAS2 expression and HA synthesis, down-regulated RANKL expression induced by 1α,25(OH)2D3 and dexamethasone. Interestingly, knockdown of has2 gene enhanced the expression of vitamin D receptor (VDR) and phosphorylation of signal transducers and activator of transcription 3 (STAT3) in ST2 cells stimulated by 1α,25(OH)2D3 and dexamethasone. These results indicate that accumulation of HA in bone marrow cells may affect RANKL-mediated osteoclast-supporting activity via regulation of VDR and STAT3 signaling pathways.

Are long non-coding RNAs involved in the interaction circuit between estrogen receptor and vitamin D receptor?

Breast cancer as a molecularly heterogeneous disorder harbors dysregulation of several signaling pathways. Estrogen receptor (ER) and vitamin D receptor (VDR) are tow signaling pathways whose fundamental roles in breast cancer have been documented. The presence of a negative vitamin D response element in the Estrogen receptor 1 (ESR1) gene promoter has provided evidences for interactions between ER and VDR signaling pathways. Moreover, recent data indicate that certain long non-coding RNAs (lncRNAs) regulate expression of ER and VDR. In the present study, we conducted an in silico approach for identification of lncRNAs that might affect these two pathways in opposite manners. Subsequently, we assessed the interaction of these lncRNAs with tumor suppressor/oncogenic miRNAs and transcription factors to highlight their importance in the pathogenesis of cancer. The results of the current study propose certain lncRNAs with regulatory roles on both ER and VDR signaling pathways to assess their expression pattern, functional roles and possible therapeutic application in future experiments.

A Combined Bioinformatics and Literature Based Approach for Identification of Long Non-coding RNAs That Modulate Vitamin D Receptor Signaling in Breast Cancer.

Long non-coding RNAs (lncRNAs) as an important fraction of human transcriptome have been shown to exert fundamental role in regulation of signaling pathways implicated in carcinogenesis. Among them is vitamin D receptor (VDR) signaling whose participation in various cancers including breast cancer (BC) is evident. In spite of the presence of several evidences for participation of lncRNAs as well as VDR signaling in BC pathogenesis, no comprehensive study has evaluated the link between lncRNA dysregulation and VDR signaling in BC. To introduce a bioinformatics approach for identification of lncRNAs that modulate VDR signaling in BC. This approach includes co-expression analysis, in silico identification of lncRNAs that target VDR and literature search. Tens of lncRNAs are predicted to affect VDR signaling. Among them are some lncRNAs such as MALAT1 which has prominent role in BC pathogenesis. Identification of the lncRNAs that influence VDR gene expression is possible through in silico analysis. Considering the prominent role of VDR in BC pathogenesis as well as availability of VDR modulating agents, evaluation of VDR signaling pathway and related networks are of practical significance and bioinformatics tools are expected to facilitate such action. Key words: vitamin D receptor - long non-coding RNAs - co-expression - bioinformatics - calcitriol receptor - computational biology.

Abstract 5215: Vitamin D receptor regulates intestinal barriers in colon cancer

Abstract Background Vitamin D and Vitamin D receptor (VDR) play important roles in the development of colon cancer. Tight junction structures are essential in intestinal barrier integrity, inflammation, and cancer. The disruption of tight junctions is a common manifestation of various diseases including cancers. Claudin-5 is a tight junction protein that is expressed in epithelia and endothelia and form paracellular barriers and pores that determine permeability. It is downregulated in colon cancer. Although VDR and Claudin-5 are all involved in colon cancer, it remains unclear whether they are closely related or function independently. Methods In the current study, we investigate the novel role of VDR in regulating Claudin-5, using whole body VDR knockout mice (VDR-/-) and intestinal epithelial VDR knockout mice (VDRΔIEC). In vitro, VDR-/- embryonic fibroblasts (MEF) cells, cultured human intestinal epithelial cells, organoids, and human colon cancer samples were used to determine the molecular mechanism. Results In intestinal samples of colon cancer patients, VDR expression is low, whereas the expression of Claudin-5 is also decreased. In the colon of VDR-/- mice and VDRΔIEC mice, deletion of VDR led to lower claudin-5 at the protein and mRNA levels. This observation was also confirmed by immunostaining of Claudin-5. In VDR-/- MEF cells, both Western blot and real time PCR revealed that VDR deletion led to lower protein and mRNA levels of claudin-5 in cells without any treatment. Vitamin D3 is known to increase VDR expression and activate VDR signaling. Protein and mRNA levels of Claudin-5 were significantly elevated in mice, mouse organoids, and human intestinal epithelial SKCO15 cells treated with 1, 25 vitamin D3. In contrast, knockdown of Claudin-5 using siRNA did not change the expression level of VDR protein in human colonic epithelial cells. Our data suggest that the Claudin-5 gene is a downstream target of the VDR signaling pathway. Conclusion Taken together, we show that VDR is important for the maintenance of physiological level of Claudin-5. This study also reveals a complex role for vitamin D/VDR regulation of intestinal barrier functions in colon cancer. Citation Format: Yong-guo Zhang, Rong Lu, Danika Bakke, Yinglin Xia, Jun Sun. Vitamin D receptor regulates intestinal barriers in colon cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5215.

Knockdown of Snail inhibits epithelial-mesenchymal transition of human laryngeal squamous cell carcinoma Hep-2 cells through the vitamin D receptor signaling pathway.

It has been well documented that Snail plays a decisive role in various tumors. However, the direct effect of Snail on laryngeal squamous cell carcinoma (LSCC) has not been elaborated. In this study, we firstly detected the expression of Snail in 14 samples of patients with LSCC and found that its content was high in cancer tissues compared with adjacent tissues. Then we established LSCC Hep-2 cells with Snail silencing and validated the knockdown efficiency by Western blotting and real-time PCR. Results showed that silencing of Snail significantly inhibited the ability of adhesion, migration, and invasion of Hep-2 cells. Further study revealed that knockdown of Snail suppressed the epithelial-mesenchymal transition (EMT) process of Hep-2 cells, as evidenced by downregulation of matrix metallopeptidase (MMP)-2, MMP-9, integrin subunit beta 1 (ITGβ1), β-catenin, vimentin, N-cadherin, and fibronectin and upregulation of vitamin D receptor (VDR) and E-cadherin. Additionally, transfection with the small interfering RNA of VDR reversed the effect induced by Snail silencing in Hep-2 cells. Taken together, these results demonstrate that knockdown of Snail can inhibit the EMT process of LSCC cells through the VDR signaling pathway in vitro.

Vitamin D receptor is a novel transcriptional regulator for Axin1

BackgroundAxin1 is a scaffold protein in the β-catenin destruction complex, which, if disrupted, contributes to pathogenesis of various human diseases, including colorectal carcinogenesis and inflammatory bowel diseases (IBD). We have previously demonstrated that Salmonella infection promotes the degradation and plasma sequestration of Axin1, leading to bacterial invasiveness and inflammatory responses. Vitamin D and the vitamin D receptor (VDR) appear to be important regulators of IBD and colon cancer. Although VDR and Axin1 are all involved in intestinal inflammation, it remains unclear whether these processes are related or function independently. In the current study, we hypothesize that VDR is an important regulator for the maintenance of physiological level of Axin1. MethodsUsing the intestinal epithelial conditional VDR knockout mouse model (VDRΔIEC) and cultured cell lines, influences of VDR status on the expression of Axin1 was evaluated by Western blots and real-time PCR. Loss- and gain-of-function assays were used to investigate the regulation of VDR on Axin1 at the transcriptional and translational levels. Cells were treated with cycloheximide or actinomycin for molecular mechanistic studies. Candidate genomic VDR binding sites for Axin1 were tested by chromatin immunoprecipitation (ChIP) assay. Physical interactions among VDR, Axin1, and β-catenin were tested by immunoprecipitation. Cellular localization of Axin1 with different VDR status was determined by fractionation and immunohistochemistry. ResultsWe found that VDR deletion led to lower protein and mRNA levels of Axin1, whereas knockdown of Axin1 did not change the expression level of VDR protein. Immunoprecipitation data did not support physical interaction between VDR and Axin1. The VDR regulation of Axin1 was through a VDR genomic binding site for Axin1 gene on the regulatory region. Fractionation data showed that cytosolic Axin1 was significantly reduced due to VDR deletion, leaving the nuclear fraction unchanged. In ileum, Axin1 was distributed in the cytosol of apical epithelium and crypts. ConclusionVDR is important for the maintenance of physiological level of Axin1. The discovery of Axin1 as a VDR target gene provides novel and fundamental insights into the interactions between the VDR and β-catenin signaling pathways.

Peroxisome Proliferator-Activated Receptor and Vitamin D Receptor Signaling Pathways in Cancer Cells

Peroxisome proliferator-activated receptors (PPARs) are members of the superfamily of nuclear hormone receptors, which respond to specific ligands such as polyunsaturated fatty acids by altering gene expression. Three subtypes of this receptor have been discovered, each evolving to achieve different biological functions. Like other nuclear receptors, the transcriptional activity of PPARs is affected not only by ligand-stimulation, but also by cross-talk with other molecules. For example, both PPARs and the RXRs are ligand-activated transcription factors that coordinately regulate gene expression. In addition, PPARs and vitamin D receptor (VDR) signaling pathways regulate a multitude of genes that are of importance for cellular functions including cell proliferation and cell differentiation. Interaction of the PPARs and VDR signaling pathways has been shown at the level of molecular cross-regulation of their transcription factor. A variety of ligands influencing the PPARs and VDR signaling pathways have been shown to reveal chemopreventive potential by mediating tumor suppressive activities in human cancers. Use of these compounds may represent a potential novel strategy to prevent cancers. This review summarizes the roles of the PPARs and the VDR in pathogenesis and progression of cancer.

Vitamin D receptor activation enhances Benzo[a]pyrene metabolism via CYP1A1 expression in macrophages

Benzo[a]pyrene (BaP) activates the aryl hydrocarbon (AHR) and induces the expression of xenobiotic metabolic genes, including cytochrome P450 (CYP) 1A1. CYP1A1 is involved not only in BaP detoxification but also in metabolic activation, which results in DNA adduct formation. Vitamin D receptor (VDR) also regulates expression of other xenobiotic metabolism genes, including CYP3A. We investigated the cross‐talk between AHR and VDR signaling pathways and found that 1α,25‐ dihydroxyvitamin D3 (1,25(OH)2D3) enhanced BaP‐induced transcription of CYP1A1 in human monocytic U937 cells and THP‐1 cells. 1,25(OH)2D3 alone did not induce CYP1A1 and 1,25(OH)2D3 plus BaP did not increase other CYP1 family (CYP1A2 or CYP1B1) mRNA expression in U937 cells. Combination of 1,25(OH)2D3 and BaP increased CYP1A1 protein levels, BaP hydroxylation activity and BaP‐DNA adduct formation in U937 cells and THP‐1 cells more effectively than BaP alone. The combined effect of 1,25(OH)2D3 and BaP on CYP1A1 mRNA expression in U937 cells and/or THP‐1 cells was inhibited by VDR knockdown and VDR or AHR antagonists. Electrophoretic mobility shift assays and chromatin immunoprecipitation assays showed that VDR directly bound to an everted repeat 8 motif in the human CYP1A1 promoter. Thus, induction of CYP1A1 by the activation of VDR and AHR may contribute to BaP‐mediated toxicity and the physiological function of this enzyme.

Vitamin D Receptor Activation Enhances Benzo[a]pyrene Metabolism via CYP1A1 Expression in Macrophages

Benzo[a]pyrene (BaP) activates the aryl hydrocarbon (AHR) and induces the expression of genes involved in xenobiotic metabolism, including CYP1A1. CYP1A1 is involved not only in BaP detoxification but also in metabolic activation, which results in DNA adduct formation. Vitamin D receptor (VDR) belongs to the NR1I subfamily of the nuclear receptor superfamily, which also regulates expression of xenobiotic metabolism genes. We investigated the cross-talk between AHR and VDR signaling pathways and found that 1α,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)], a potent physiological VDR agonist, enhanced BaP-induced transcription of CYP1A1 in human monocytic U937 cells and THP-1 cells, breast cancer cells, and kidney epithelium-derived cells. 1,25(OH)(2)D(3) alone did not induce CYP1A1, and 1,25(OH)(2)D(3) plus BaP did not increase CYP1A2 or CYP1B1 mRNA expression in U937 cells. The combination of 1,25(OH)(2)D(3) and BaP increased CYP1A1 protein levels, BaP hydroxylation activity, and BaP-DNA adduct formation in U937 cells and THP-1 cells more effectively than BaP alone. The combined effect of 1,25(OH)(2)D(3) and BaP on CYP1A1 mRNA expression in U937 cells and/or THP-1 cells was inhibited by VDR knockdown, VDR antagonists, and α-naphthoflavone, an AHR antagonist. Electrophoretic mobility shift assays and chromatin immunoprecipitation assays showed that VDR directly bound to an everted repeat (ER) 8 motif in the human CYP1A1 promoter. Thus, CYP1A1 is a novel VDR target gene involved in xenobiotic metabolism. Induction of CYP1A1 by the activation of VDR and AHR may contribute to BaP-mediated toxicity and the physiological function of this enzyme.

Effects of 1,25-(OH)2D3 on the expressions of vitamin D receptor, STAT5 and cytoskeletal rearrangement in human monocytes incubated with sera from type 2 diabetes patients and diabetic nephropathy patients with uremia

To explore the effects of 1,25-(OH)(2)D(3) and lipopolysaccharide (LPS) plus human recombinant interleukin-15 (IL-15) on expression of vitamin D receptor (VDR) and STAT5, and cytoskeletal rearrangement in human monocytes incubated with sera from type 2 diabetes (T2DM) patients and diabetic nephropathy (DN) patients with uremia. Peripheral sera were isolated from healthy volunteers (control group, T2DM patients and DN uremic non-dialysis patients). After incubation with or without 1,25(OH)(2)D(3), THP-1 monocytes were treated with LPS plus IL-15 prior to the collection of cells and supernatants. VDR mRNA transcription was examined by RT-PCR, whilst THP-1 monocytic VDR, STAT5 and p-STAT5 expressions were investigated by Western blotting. Concentrations of IL-6 and monocyte chemoattractant protein-1 (MCP-1) in supernatants were assessed by ELISA. Immunofluorescence and a laser confocal microscopy was used to examine the expression of VDR and cytoskeletal proteins. Compared to the normal control, LPS and IL-15 down-regulate monocytic VDR expression in T2DM patients and DN uremic patients, whilst with cytoskeletal rearrangement, they up-regulate p-STAT5 expression as well as IL-6 and MCP-1 activity. Such effects could be in part blocked by 1,25-(OH)(2)D(3). The above results suggest that the anti-inflammatory mechanism of 1,25-(OH)(2)D(3) may be related to cytoskeletal proteins, VDR and STAT5 signaling pathway.

Vitamin D and mucosal immune function

Significant advances have been made in the characterization of Vitamin D and the Vitamin D receptor (VDR) in immune function. The studies of signaling pathways involved in the response to infection and inflammation have led to a more detailed understanding of the cellular response to Vitamin D through VDR. This review summarizes recent progress in understanding how Vitamin D contributes to mucosal immune function, particularly in relation to the molecular mechanisms by which Vitamin D and VDR influence mucosal immunity, bacterial infection, and inflammation. Recently, it was shown that Vitamin D modulates the T cell antigen receptor, further demonstrating that Vitamin D has a nonclassical role in immunoregulation. The anti-inflammation and anti-infection functions for Vitamin D are newly identified and highly significant activities. Vitamin D/VDR have multiple critical functions in regulating the response to intestinal homeostasis, tight junctions, pathogen invasion, commensal bacterial colonization, antimicrobe peptide secretion, and mucosal defense. Interestingly, microorganisms modulate the VDR signaling pathway. Vitamin D is known as a key player in calcium homeostasis and electrolyte and blood pressure regulation. Recently, important progress has been made in understanding how the noncanonical activities of Vitamin D influence the pathogenesis and prevention of human disease. Vitamin D and VDR are directly involved in T cell antigen receptor signaling. The involvement of Vitamin D/VDR in anti-inflammation and anti-infection represents a newly identified and highly significant activity for VDR. Studies have indicated that the dysregulation of VDR may lead to exaggerated inflammatory responses, raising the possibility that defects in Vitamin D and VDR signaling transduction may be linked to bacterial infection and chronic inflammation. Further characterization of Vitamin D/VDR will help elucidate the pathogenesis of various human diseases and in the design of new approaches for prevention and treatment.

Elevated phosphorus modulates vitamin D receptor-mediated gene expression in human vascular smooth muscle cells

Clinical observations show that an increase in serum inorganic phosphorus (Pi) is linked to higher cardiovascular (CV) mortality, while vitamin D receptor (VDR) agonist therapy is associated with survival benefit in stage 5 chronic kidney disease. Smooth muscle cells (SMCs) play an important role in CV pathophysiology, but the interaction between Pi and the VDR signaling pathway in SMCs is not known. Real-time RT-PCR studies revealed that elevated Pi (2.06 mM) modulated VDR-mediated regulation of a panel of genes including thrombomodulin and osteopontin in SMCs. DNA microarray results demonstrated that increasing Pi from 0.9 to 2.06 mM exerted a widespread modulating effect on VDR-mediated gene expression. A total of 325 target genes were affected by paricalcitol at 0.9 mM Pi, with 195 up- and 130 downregulated. The number of target genes affected by paricalcitol at 2.06 mM Pi decreased to 86, with 55 up- and 31 downregulated. VDR-mediated gene expression in As4.1 cells (a juxtaglomerular cell-like cell line derived from kidney tumors in SV40 T-antigen transgenic mice) and peroxisome proliferator-activated receptor (PPAR)gamma-mediated gene expression in SMCs were also altered by elevated Pi, suggesting that the observation is not unique to VDR in SMCs. Mechanism analysis showed that elevated Pi had no significant effect on VDR or PPARgamma protein level but altered the cytosolic vs. nuclear distribution of NF-kappaB or nuclear receptor corepressor 1 (NCoR1). Our results demonstrate for the first time that elevated Pi affects VDR-mediated gene expression in human coronary artery SMCs and the effect is not limited to VDR in SMCs.

Epigenetic Silencing of CYP24 in Tumor-derived Endothelial Cells Contributes to Selective Growth Inhibition by Calcitriol

Calcitriol (1,25-dihydroxycholecalciferol), the most active form of vitamin D, has selective anti-proliferative effects on tumor-derived endothelial cells (TDEC) compared with Matrigel-derived endothelial cells (MDEC). Although both cell types have an intact vitamin D receptor-signaling axis, this study demonstrates that upon treatment with calcitriol, 24-hydroxylase (CYP24) mRNA, protein and enzymatic activity were markedly induced in MDEC in a time-dependent manner but not in TDEC. Furthermore, treatment of MDEC with a CYP24 small interfering RNA restored sensitivity to calcitriol. To investigate the lack of CYP24 induction in TDEC, we examined methylation patterns in the promoter regions of the CYP24 gene in these two cell types. We identified two putative CpG island regions located at the 5' end. Using methylation-specific PCR and bisulfite sequencing, we determined that these CpG islands were hypermethylated in TDEC but not in MDEC. These data may explain the recruitment of vitamin D receptor to the promoter region in MDEC but not TDEC, as revealed by chromatin immunoprecipitation analyses. Treatment of TDEC with the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine restored calcitriol-mediated induction of CYP24, which led to loss of sensitivity to calcitriol growth inhibitory effects. CYP24 promoter hypermethylation was also observed in endothelial cells isolated from other tumors but not in endothelial cells isolated from normal mouse tissues. These observations indicate that the methylation status of the CYP24 promoter differs in endothelial cells isolated from different microenvironments (tumor versus normal) and that methylation silencing of CYP24 contributes to selective calcitriol-mediated growth inhibition in endothelial cells.

Complex Role of the Vitamin D Receptor and Its Ligand in Adipogenesis in 3T3-L1 Cells

The vitamin D receptor (VDR) and its ligand 1,25-OH2-VD3 (calcitriol) play an essential role in mineral homeostasis in mammals. Interestingly, the VDR is expressed very early in adipogenesis in 3T3-L1 cells, suggesting that the VDR signaling pathway may play a role in adipocyte biology and function. Indeed, it has been known for a number of years that calcitriol is a potent inhibitor of adipogenesis in this model but with no clear mechanism identified. In this study, we have further defined the molecular mechanism by which the unliganded VDR and calcitriol-liganded VDR regulate adipogenesis. In the presence of calcitriol, the VDR blocks adipogenesis by down-regulating both C/EBPbeta mRNA expression and C/EBPbeta nuclear protein levels at a critical stage of differentiation. In addition, calcitriol allows for the up-regulation of the recently described C/EBPbeta corerepressor, ETO, which would further inhibit the action of any remaining C/EBPbeta, whose action is required for adipogenesis. In contrast, in the absence of calcitriol, the unliganded VDR appears necessary for lipid accumulation, since knock-down of the VDR using siRNA both delays and prevents this process. Taken together, these data support the notion that the intracellular concentrations of calcitriol can play an important role in either promoting or inhibiting adipogenesis via the VDR and the transcriptional pathways that it targets. Further examination of this hypothesis in vivo may shed new light on the biology of adipogenesis.

Expression of VDR and CYP24A1 mRNA in human tumors

1,25-dihydroxyvitamin D3 (1,25(OH)2D3) and its analogues have been shown to inhibit proliferation of human cancer cells mediated by vitamin D receptor (VDR). The over-expression of 25-hydroxyvitamin D-24-hydroxylase (CYP24A1), an enzyme involved in the metabolism of 1,25(OH)2D3 and its analogues, is associated with poor prognosis of some human cancers. In this study, we employed real-time reverse transcription PCR to examine the expression of VDR and CYP24A1 mRNA in a cohort of human breast, lung, colon and ovary tumor samples. We found that CYP24A1 mRNA was significantly up-regulated in colon, ovary and lung tumors, but down-regulated in breast tumor relative to the analogous normal tissues. As a comparison, VDR mRNA was modestly down-regulated in colon, breast and lung tumors, but highly up-regulated in ovarian tumors. Treatment of two breast cancer cell lines, SW-620 and MCF-7, and one colon cancer cell line, HT-29, by 1,25(OH)2D3 for 48 h profoundly stimulated CYP24A1 mRNA expression (EC50=0.6, 0.8 and 29.5 nM in SW-620, HT-29 and MCF-7, respectively), but did not significantly affect VDR mRNA expression. Growth as assessed by DNA synthesis was modestly arrested by 1,25(OH)2D3 after 72 h of incubation, but was not altered after a 5-day incubation period. These data suggest that the VDR signaling pathway may be compromised via the modulation of CYP24A1 and VDR in human tumors.