Absence Of Vitamin D Receptor Research Articles

1,25-Dihydroxyvitamin D3 regulates furin-mediated FGF23 cleavage.

Intact fibroblast growth factor 23 (iFGF23) is a phosphaturic hormone that is cleaved by furin into N-terminal and C-terminal fragments. Several studies have implicated vitamin D in regulating furin in infections. Thus, we investigated the effect of 1,25-dihydroxyvitamin D3 [1,25(OH)2D] and the vitamin D receptor (VDR) on furin-mediated iFGF23 cleavage. Mice lacking VDR (Vdr-/-) had a 25-fold increase in iFGF23 cleavage, with increased furin levels and activity compared with wild-type (WT) littermates. Inhibition of furin activity blocked the increase in iFGF23 cleavage in Vdr-/- animals and in a Vdr-knockdown osteocyte OCY454 cell line. Chromatin immunoprecipitation revealed VDR binding to DNA upstream of the Furin gene, with more transcription in the absence of VDR. In WT mice, furin inhibition reduced iFGF23 cleavage, increased iFGF23, and reduced serum phosphate levels. Similarly, 1,25(OH)2D reduced furin activity, decreased iFGF23 cleavage, and increased total FGF23. In a post hoc analysis of a randomized clinical trial, we found that ergocalciferol treatment, which increased serum 1,25(OH)2D, significantly decreased serum furin activity and iFGF23 cleavage, compared with placebo. Thus, 1,25(OH)2D inhibits iFGF23 cleavage via VDR-mediated suppression of Furin expression, thereby providing a mechanism by which vitamin D can augment phosphaturic iFGF23 levels.

Effects of 1,25 and 24,25 Vitamin D on Corneal Fibroblast VDR and Vitamin D Metabolizing and Catabolizing Enzymes

ABSTRACT Purpose: To investigate the effects of 1,25-Vit D3 and 24,25-Vit D3 on corneal fibroblast expression of the vitamin D-associated enzymes CYP27B1 and CYP24A1 and the roles of the vitamin D receptor (VDR) and protein disulfide isomerase, family A, member 3 (Pdia3) in these cells. Methods: CYP24A1, CYP27B1, VDR, and Pdia3 expression in corneas was detected using immunohistochemistry. Western blotting was used to measure protein expression in human and mouse fibroblasts, including VDR KO mouse cells, treated with 1,25-Vit D3 (20 nM) and 24,25-Vit D3 (100 nM). The Pdia3 inhibitor LOC14 was used to explore the role of Pdia3 as a Vit D3 receptor in these cells. Results: CYP24A1, CYP27B1, VDR, and Pdia3 were all expressed in mouse and human corneal fibroblasts. 1,25-Vit D3 significantly increased VDR expression in human and mouse fibroblasts. 1,25-Vit D3 and 24,25-VitD3 significantly increased CYP24A1 and CYP27B1 expression level in human, VDR WT mouse, and VDR KO mouse corneal fibroblasts. CYP24A1 and CYP27B1 expression was unchanged in VDR KO mouse fibroblasts treated with 1,25-Vit D3 or 24,25-Vit D3 plus LOC14. Human fibroblast VDR, CYP24A1, and CYP27B1 expression were unaffected by LOC14. Conclusions: Vitamin D metabolic enzymes, VDR, and Pdia3 are all expressed in mouse and human corneal fibroblasts. 1,25-Vit D3 modulates fibroblast vitamin D enzymes through both the VDR and Pdia3 pathways in a species-dependent manner. 24,25-Vit D3 can increase expression of fibroblast CYP24A1 and CYP27B1 in the absence of VDR and is likely involved in fibroblast regulation independent of 1,25-Vit D3 or VDR.

Vitamin D receptor promotes healthy microbial metabolites and microbiome

Microbiota derived metabolites act as chemical messengers that elicit a profound impact on host physiology. Vitamin D receptor (VDR) is a key genetic factor for shaping the host microbiome. However, it remains unclear how microbial metabolites are altered in the absence of VDR. We investigated metabolites from mice with tissue-specific deletion of VDR in intestinal epithelial cells or myeloid cells. Conditional VDR deletion severely changed metabolites specifically produced from carbohydrate, protein, lipid, and bile acid metabolism. Eighty-four out of 765 biochemicals were significantly altered due to the Vdr status, and 530 significant changes were due to the high-fat diet intervention. The impact of diet was more prominent due to loss of VDR as indicated by the differences in metabolites generated from energy expenditure, tri-carboxylic acid cycle, tocopherol, polyamine metabolism, and bile acids. The effect of HFD was more pronounced in female mice after VDR deletion. Interestingly, the expression levels of farnesoid X receptor in liver and intestine were significantly increased after intestinal epithelial VDR deletion and were further increased by the high-fat diet. Our study highlights the gender differences, tissue specificity, and potential gut-liver-microbiome axis mediated by VDR that might trigger downstream metabolic disorders.

Vitamin D Receptor Inhibits NLRP3 Activation by Impeding Its BRCC3-Mediated Deubiquitination.

The NLRP3 inflammasome is a multiprotein oligomer responsible for activation of the inflammatory response by promoting the maturation and secretion of the pro-inflammatory cytokines IL-1β and IL-18. Dysregulation of this inflammasome has been linked to several autoimmune diseases, indicating that NLRP3 is tightly regulated to prevent aberrant activation. The regulation of NLRP3 activation remains unclear. Here, we report the identification of vitamin D receptor (VDR) as a negative regulator of NLRP3 oligomerization and activation. VDR can physically bind NLRP3 and block the association of NLRP3 with BRCC3. When BRCC3-mediated deubiquitination of NLRP3 is inhibited by VDR, NLRP3 activation is subsequently inhibited. In the absence of VDR, caspase-1 activation and IL-1β release are increased in response to LPS-induced inflammation or alum-induced peritoneal inflammation, indicating that VDR is a negative regulator of NLRP3 inflammasome activation in vivo. In addition, vitamin D negatively regulates the NLRP3 inflammasome via VDR signaling to effectively inhibit IL-1β secretion. These studies demonstrate that VDR signaling constrains NLRP3 inflammasome activation and might be a potential treatment target for NLRP3 inflammasome-related diseases.

UV light suppression of EAE (a mouse model of multiple sclerosis) is independent of vitamin D and its receptor

Vitamin D and sunlight have each been reported to protect against the development of experimental autoimmune encephalomyelitis (EAE), a mouse model of multiple sclerosis (MS). To date, the contribution of each has been unclear as ultra violet (UV) exposure also causes the generation of vitamin D in the skin. To examine whether the UV based suppression of EAE results, at least, in part from the production of vitamin D, we studied the effect of UV light on EAE in mice unable to produce 7-dehydroxycholesterol (7-DHC), the required precursor of vitamin D. Furthermore, we examined UV suppression of EAE in mice devoid of the vitamin D receptor (VDR). Our results demonstrate that UV light suppression of EAE occurs in the absence of vitamin D production and in the absence of VDR. Future investigations will focus on identifying the pathway responsible for the protective action of UV in EAE and presumably human MS.

Absence of vitamin D receptor in mature osteoclasts results in altered osteoclastic activity and bone loss

Mature osteoclasts express the vitamin D receptor (VDR) and are able to synthesise and respond to 1,25(OH)2D3 via CYP27B1 enzyme activity. Whether vitamin D signalling within osteoclasts is necessary for the regulation of osteoclastic bone resorption in an in vivo setting is unclear. To determine the requirement for the VDR- and CYP27B1-mediated activity in mature osteoclasts, conditional deletion mouse models were created whereby either Vdr or Cyp27b1 gene was inactivated by breeding either Vdrfl/fl or Cyp27b1fl/fl mice with Cathepsin K-Cre transgenic mice (CstkCre) to generate CtskCre/Vdr−/− and CtskCre/Cyp27b1−/− mice respectively. To account for potential CtskCre-meaited off-target deletion of Vdr, Dmp1Cre were also used determine the effect of Vdr deletion in osteocytes. Furthermore, CtskCre/Vdr−/− mice were ovariectomised (OVX) to assess the role of VDR in osteoclasts under bone-loss conditions and bone marrow precursor cells were cultured under osteoclastogenic conditions to assess osteoclast formation. Six-week-old CtskCre/Vdr−/− female mice demonstrated a 15% decrease in femoral BV/TV (p<0.05). In contrast, BV/TV remained unchanged in CtskCre/Cyp27b1−/− mice as well as in Dmp1Cre/VDR−/− mice. When CtskCre/Vdr−/− mice were subjected to OVX, the bone loss that occurred in CtskCre/Vdr−/− was predominantly due to a diminished volume of thinner trabeculae when compared to control levels. These changes in bone volume in CtskCre/Vdr−/− mice occurred without an observable histological change in osteoclast numbers or size. However, while cultured bone marrow-derived osteoclasts from CtskCre/Vdr−/− mice were marginally increased when compared to VDRfl/fl mice, elevated expression of genes such as Cathepsin K, Nfatc1 and VATPase was observed. Collectively, these data indicate that the absence of VDR in mature osteoclasts causes exacerbated bone loss in young mice and during OVX which is associated with enhanced osteoclastic activity and without increased osteoclastogenesis.

Vitamin D receptor deficit induces activation of renin angiotensin system via SIRT1 modulation in podocytes

Vitamin D receptor (VDR) deficient status has been shown to be associated with the activation of renin angiotensin system (RAS). We hypothesized that lack of VDR would enhance p53 expression in podocytes through down regulation of SIRT1; the former would enhance the transcription of angiotensinogen (Agt) and angiotensinogen II type 1 receptor (AT1R) leading to the activation of RAS. Renal tissues of VDR mutant (M) mice displayed increased expression of p53, Agt, renin, and AT1R. In vitro studies, VDR knockout podocytes not only displayed up regulation p53 but also displayed enhanced expression of Agt, renin and AT1R. VDR deficient podocytes also displayed an increase in mRNA expression for p53, Agt, renin, and AT1R. Interestingly, renal tissues of VDR-M as well as VDR heterozygous (h) mice displayed attenuated expression of deacetylase SIRT1. Renal tissues of VDR-M mice showed acetylation of p53 at lysine (K) 382 residues inferring that enhanced p53 expression in renal tissues could be the result of ongoing acetylation, a consequence of SIRT1 deficient state. Notably, podocytes lacking SIRT1 not only showed acetylation of p53 at lysine (K) 382 residues but also displayed enhanced p53 expression. Either silencing of SIRT1/VDR or treatment with high glucose enhanced podocyte PPAR-y expression, whereas, immunoprecipitation (IP) of their lysates with anti-retinoid X receptor (RXR) antibody revealed presence of PPAR-y. It appears that either the deficit of SIRT1 has de-repressed expression of PPAR-y or enhanced podocyte expression of PPAR-y (in the absence of VDR) has contributed to the down regulation of SIRT1.

Vitamin D3-dependent VDR signaling delays ron-mediated breast tumorigenesis through suppression of β-catenin activity.

The Ron receptor is upregulated in human breast cancers and correlates with enhanced metastasis and reduced patient survival. Ron overexpression drives mammary tumorigenesis through direct β-catenin activation and augmented tumor cell proliferation, migration and invasion. Ron and β-catenin are also coordinately elevated in breast cancers. The vitamin D receptor (VDR) antagonizes β-catenin signaling. Herein, we examined mammary tumor onset and progression using a Ron-driven murine model of breast tumorigenesis crossed with VDR deficient mice. VDR ablation accelerated mammary tumor onset and led to tumors that exhibited a desmoplastic phenotype and enhanced metastases. Tumor levels of active β-catenin were markedly increased in the absence of VDR. In vitro, VDR activation in breast cancer cells reduced β-catenin activation and transcriptional activity leading to elevated expression of the extracellular Wnt inhibitor dickkopf-related protein 1, and a reduction in the interaction of β-catenin with the cyclin D1 promoter. Expression of a stabilized form or β-catenin ablated the protective effects of VDR activation.Collectively, these studies delineate a protective role for VDR signaling in Ron-induced mammary tumorigenesis through disruption of β-catenin activation.

The Protective Role of Vitamin D Signaling in Non-Melanoma Skin Cancer

Although the epidemiologic evidence that adequate vitamin D nutrition protects against non-melanoma skin cancer (NMSC) is limited, recent evidence that the vitamin D receptor (VDR) is protective is compelling. The role of vitamin D signaling in limiting the proliferation while promoting the differentiation of keratinocytes, the major cell in the epidermis from which NMSC are derived, is well known. However, recent findings that mice lacking the VDR are predisposed to skin cancer has brought to the fore the question of how the VDR is protective. In this review we will look first at the role of vitamin D signaling in regulating the proliferation and differentiation of keratinocytes. We will examine two pathways, β-catenin (CTNNB) and hedgehog (HH), that are regulated by vitamin D signaling and may contribute to the dysregulated proliferation and differentiation in the absence of VDR. We will then examine the failure of VDR deficient keratinocytes to repair DNA damaged by UVB. Finally we will examine the change in long non-coding RNA (LncRNA) expression in VDR null keratinocytes that in other cells is associated with malignant transformation, a potential newly appreciated mechanism by which vitamin D signaling is protective against NMSC.

Vitamin D Receptor Signaling Inhibits Atherosclerosis in Mice

Although vitamin D has been implicated in cardiovascular protection, few studies have addressed the role of vitamin D receptor (VDR) in atherosclerosis. Here we investigate the effect of inactivation of the VDR signaling on atherogenesis and the antiatherosclerotic mechanism of vitamin D. Low density lipoprotein receptor (LDLR)(-/-)/VDR(-/-) mice exhibited site-specific accelerated atherogenesis, accompanied by increases in adhesion molecules and proinflammatory cytokines in the aorta and cholesterol influx in macrophages. Macrophages showed marked renin up-regulation in the absence of VDR, and inhibition of renin by aliskiren reduced atherosclerosis in LDLR(-/-)/VDR(-/-) mice, suggesting that the renin-angiotensin system (RAS) promotes atherosclerosis in the absence of VDR. LDLR(-/-) mice receiving LDLR(-/-)/VDR(-/-) BMT developed larger lesions than LDLR(-/-) BMT controls. Moreover, LDLR(-/-) mice receiving Rag-1(-/-)/VDR(-/-) BMT, which were unable to generate functional T and B lymphocytes, still had more severe atherosclerosis than Rag-1(-/-) BMT controls, suggesting a critical role of macrophage VDR signaling in atherosclerotic suppression. Aliskiren treatment eliminated the difference in lesions between Rag-1(-/-)/VDR(-/-) BMT and Rag-1(-/-) BMT recipients, indicating that local RAS activation in macrophages contributes to the enhanced atherogenesis seen in Rag-1(-/-)/VDR(-/-) BMT mice. Taken together, these observations provide evidence that macrophage VDR signaling, in part by suppressing the local RAS, inhibits atherosclerosis in mice.

Vitamin D receptor-independent FGF23 actions in regulating phosphate and vitamin D metabolism

FGF23 suppresses both serum phosphate and 1,25-dihydroxyvitamin D [1,25D] levels in vivo. Because 1,25D itself is a potent regulator of phosphate metabolism, it has remained unclear whether FGF23-induced changes in phosphate metabolism were caused by a 1,25D-independent mechanism. To address this issue, we intravenously administered recombinant FGF23 to vitamin D receptor (VDR) null (KO) mice as a rapid bolus injection and evaluated the early effects of FGF23. Administration of recombinant FGF23 further decreased the serum phosphate level in VDR KO mice, accompanied by a reduction in renal sodium-phosphate cotransporter type IIa (NaPi2a) protein abundance and a reduced renal 25-hydroxyvitamin D-1alpha-hydroxylase (1alphaOHase) mRNA level. Thus FGF23-induced changes in NaPi2a and 1alphaOHase expression are independent of the 1,25D/VDR system. However, 24-hydroxylase (24OHase) mRNA expression remained undetectable by the treatment with FGF23. We also analyzed the regulatory mechanism for FGF23 expression. The serum FGF23 level was almost undetectable in VDR KO mice, whereas dietary calcium supplementation significantly increased circulatory levels of FGF23 and its mRNA abundance in bone. This finding indicates that calcium is another determinant of FGF23 production that occurs independently of the VDR-mediated mechanism. In contrast, dietary phosphate supplementation failed to induce FGF23 expression in the absence of VDR, whereas marked elevation in circulatory FGF23 was observed in wild-type mice fed with a high-phosphate diet. Taken together, FGF23 works, at least in part, in a VDR-independent manner, and FGF23 production is also regulated by multiple mechanisms involving VDR-independent pathways.