Transcriptomic analysis of vitamin D responses in uterine and peripheral NK cells.
ABSTRACT: Vitamin D deficiency is prevalent in pregnant women and is associated with adverse pregnancy outcomes, in particular disorders of malplacentation. The active form of vitamin D, 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), is a potent regulator of innate and adaptive immunity, but its immune effects during pregnancy remain poorly understood. During early gestation, the predominant immune cells in maternal decidua are uterine natural killer cells (uNK), but the responsivity of these cells to 1,25(OH)2D3 is unknown despite high levels of 1,25(OH)2D3 in decidua. Transcriptomic responses to 1,25(OH)2D3 were characterised in paired donor uNK and peripheral natural killer cells (pNK) following cytokine (CK) stimulation. RNA-seq analyses indicated 911 genes were differentially expressed in CK-stimulated uNK versus CK-stimulated pNK in the absence of 1,25(OH)2D3, with predominant differentially expressed pathways being associated with glycolysis and transforming growth factor ? (TGF?). RNA-seq also showed that the vitamin D receptor (VDR) and its heterodimer partner retinoid X receptor were differentially expressed in CK-stimulated uNK vs CK-stimulated pNK. Further analyses confirmed increased expression of VDR mRNA and protein, as well as VDR-RXR target in CK-stimulated uNK. RNA-seq analysis showed that in CK-stimulated pNK, 1,25(OH)2D3 induced 38 and suppressed 33 transcripts, whilst in CK-stimulated uNK 1,25(OH)2D3 induced 46 and suppressed 19 genes. However, multiple comparison analysis of transcriptomic data indicated that 1,25(OH)2D3 had no significant overall effect on gene expression in either CK-stimulated pNK or uNK. These data indicate that CK-stimulated uNK are transcriptionally distinct from pNK and, despite expressing abundant VDR, neither pNK nor uNK are sensitive targets for vitamin D.
Project description:This study investigated the effects of 1,25(OH)2D3 and 24R,25(OH)2D3 on corneal epithelial cell proliferation, migration, and on the vitamin D activating enzyme CYP27B1 (produces 1,25(OH)2D3) and inactivating enzyme CYP24A1 (produces 24R,25(OH)2D3). The role of the vitamin D receptor (VDR) was also examined. In VDR wildtype mouse corneal epithelial cells (WT), 1,25(OH)2D3 increased CYP24A1 protein expression and decreased CYP27B1 expression. In VDR knockout mouse epithelial cells (KO), 1,25(OH)2D3 increased CYP24A1 and CYP27B1 protein expression. 1,25(OH)2D3 did not affect WT cell proliferation, but did stimulate VDR KO cell proliferation. In a human corneal epithelial cell line (HCEC), 1,25(OH)2D3 increased CYP24A1 mRNA and protein expression. 1,25(OH)2D3 increased CYP27B1 mRNA levels in HCEC, but had no effect on CYP27B1 protein levels. 1,25(OH)2D3 inhibited HCEC proliferation and stimulated cell migration in primary human epithelial cells. 24,25(OH)2D3, on the other hand, increased both CYP24A1 and CYP27B1 protein expression in WT and VDR KO cells, and stimulated cell proliferation in both WT and KO cells. In HCEC, 24,25(OH)2D3 increased CYP24A1 and CYP27B1 mRNA and protein expression, and stimulated cell migration. In human primary corneal epithelial cells, 24,25(OH)2D3 stimulated migration. We conclude that 24R,25(OH)2D3 is likely involved in corneal epithelial cell regulation independent of 1,25(OH)2D3 or VDR.
Project description:The potential functional significance of human 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] receptor (hVDR) phosphorylation at Ser-208 was evaluated by cotransfecting COS-7 kidney cells with hVDR constructs and the catalytic subunit of human casein kinase 11 (CK-11). Under these conditions, hVDR is intensely phosphorylated in a reaction that depends on both CK-II and the presence of Ser-208. The resulting hyperphosphorylated receptor is unaltered in its kinetics for binding the 1,25(OH)2D3 ligand, its partitioning into the nucleus, and its ability to associate with a vitamin D responsive element. Replacement of Ser-208 with glycine or alanine indicates that phosphorylation of hVDR at Ser-208 is not obligatory for 1,25(OH)2D3 action, but coexpression of wild-type hVDR and CK-11 elicits a dose-dependent enhancement of 1,25(OH)2D3-stimulated transcription of a vitamin D responsive element reporter construct. This enhancement by CK-II is abolished by mutating Ser-208 to glycine or alanine and does not occur with glucocorticoid receptor-mediated transcription. Therefore, phosphorylation of hVDR by CK-11 at Ser-208 specifically modulates its transcriptional capacity, suggesting that this covalent modification alters the conformation of VDR to potentiate its interaction with the machinery for DNA transcription.
Project description:Renal vitamin D receptor (VDR) is required for 1,25-dihydroxyvitamin D3-[1,25(OH)2D3]-induced renal reabsorption of calcium and for 1,25(OH)2D3-induced 1,25(OH)2D3 24-hydroxylase. The long-term effect of vitamin D and dietary calcium on the expression of renal VDR was examined in the nonobese diabetic mouse. Vitamin D-deficient and vitamin D-replete mice were maintained on diets containing 0.02%, 0.25%, 0.47%, and 1.20% calcium with or without 50 ng of 1,25(OH)2D3 per day. Vitamin D-replete mice on a 1.20% calcium diet had renal VDR levels of 165 fmol/mg protein. Calcium restriction caused renal VDR levels to decrease to <30 fmol/mg protein in vitamin D-deficient mice and to approximately 80 fmol/mg protein in vitamin D-replete mice. When dietary calcium was present, 50 ng of 1,25(OH)2D3 elevated the VDR levels 2- to 10-fold, depending on vitamin D status and the level of calcium. In the absence of either vitamin D or calcium, the VDR mRNA was expressed at a basal level. 1,25(OH)2D3 supplementation caused relative VDR mRNA to increase 8- to 10-fold in the vitamin D-deficient mouse when dietary calcium was available. This increase was completely absent in the calcium-restricted mice. This in vivo study demonstrates that 1,25(OH)2D3 and calcium are both required for renal VDR mRNA expression above a basal level, furthering our understanding of the complex regulation of renal VDR by 1,25(OH)2D3 and calcium.
Project description:The active form of vitamin D3, 1,25(OH)2D3, has significant immunomodulatory properties and is an important determinant in the differentiation of CD4+ effector T cells. The biological actions of 1,25(OH)2D3 are mediated by the vitamin D receptor (VDR) and are believed to correlate with the VDR protein expression level in a given cell. The aim of this study was to determine if and how 1,25(OH)2D3 by itself regulates VDR expression in human CD4+ T cells. We found that activated CD4+ T cells have the capacity to convert the inactive 25(OH)D3 to the active 1,25(OH)2D3 that subsequently up-regulates VDR protein expression approximately 2-fold. 1,25(OH)2D3 does not increase VDR mRNA expression but increases the half-life of the VDR protein in activated CD4+ T cells. Furthermore, 1,25(OH)2D3 induces a significant intracellular redistribution of the VDR. We show that 1,25(OH)2D3 stabilizes the VDR by protecting it from proteasomal degradation. Finally, we demonstrate that proteasome inhibition leads to up-regulation of VDR protein expression and increases 1,25(OH)2D3-induced gene activation. In conclusion, our study shows that activated CD4+ T cells can produce 1,25(OH)2D3, and that 1,25(OH)2D3 induces a 2-fold up-regulation of the VDR protein expression in activated CD4+ T cells by protecting the VDR against proteasomal degradation.
Project description:Uterine fibroids (leiomyomas) are the most common benign tumors associated with excessive deposition of extracellular matrix (ECM)-associated proteins that increase fibroid tumorigenicity. Herein, we determined the expression levels of vitamin D receptor (VDR) protein in human uterine fibroids and compared these levels to those in adjacent normal myometrium. Using Western blot analysis, we found that more than 60% of uterine fibroids analyzed (25 of 40) expressed low levels of VDR. We also found that the biologically active 1,25-dihydroxyvitamin D3 (1,25[OH]2D3), which functions via binding to its nuclear VDR, induced VDR in a concentration-dependent manner and reduced ECM-associated fibrotic and proteoglycans expression in immortalized human uterine fibroid cell line (HuLM). At 1-10 nM concentrations, 1,25(OH)2D3 significantly induced (P < 0.05) nuclear VDR, which was further stimulated by higher concentrations of 1,25(OH)2D3 in HuLM cells. 1,25(OH)2D3 at 10 nM also significantly reduced (P < 0.05) the protein expression of ECM-associated collagen type 1, fibronectin, and plasminogen activator inhibitor-1 (PAI-1) in HuLM cells. We also found that 1,25(OH)2D3 reduced mRNA and protein expressions of proteoglycans such as fibromodulin, biglycan, and versican in HuLM cells. Moreover, the aberrant expression of structural smooth muscle actin fibers was reduced by 1,25(OH)2D3 treatment in a concentration-dependent manner in HuLM cells. Taken together, our results suggest that human uterine fibroids express reduced levels of VDR compared to the adjacent normal myometrium and that treatment with 1,25(OH)2D3 can potentially reduce the aberrant expression of major ECM-associated proteins in HuLM cells. Thus, 1,25(OH)2D3 might be an effective, safe, nonsurgical treatment option for human uterine fibroids.
Project description:OBJECTIVE:Colorectal cancer (CRC) is a major health concern. Vitamin D deficiency is associated with high CRC incidence and mortality, suggesting a protective effect of vitamin D against this disease. Given the strong influence of tumour stroma on cancer progression, we investigated the potential effects of the active vitamin D metabolite 1?,25-dihydroxyvitamin D3 (1,25(OH)2D3) on CRC stroma. DESIGN:Expression of vitamin D receptor (VDR) and two 1,25(OH)2D3 target genes was analysed in 658 patients with CRC with prolonged clinical follow-up. 1,25(OH)2D3 effects on primary cultures of patient-derived colon normal fibroblasts (NFs) and cancer-associated fibroblasts (CAFs) were studied using collagen gel contraction and migration assays and global gene expression analyses. Publicly available data sets (n=877) were used to correlate the 1,25(OH)2D3-associated gene signature in CAFs with CRC outcome. RESULTS:High VDR expression in tumour stromal fibroblasts was associated with better overall survival (OS) and progression-free survival in CRC, independently of its expression in carcinoma cells. 1,25(OH)2D3 inhibited the protumoural activation of NFs and CAFs and imposed in CAFs a 1,25(OH)2D3-associated gene signature that correlated with longer OS and disease-free survival in CRC. Furthermore, expression of two genes from the signature, CD82 and S100A4, correlated with stromal VDR expression and clinical outcome in our cohort of patients with CRC. CONCLUSIONS:1,25(OH)2D3 has protective effects against CRC through the regulation of stromal fibroblasts. Accordingly, expression of VDR and 1,25(OH)2D3-associated gene signature in stromal fibroblasts predicts a favourable clinical outcome in CRC. Therefore, treatment of patients with CRC with VDR agonists could be explored even in the absence of VDR expression in carcinoma cells.
Project description:Heterogeneous nuclear ribonucleoprotein (hnRNP) C1/C2 plays a pivotal role in vitamin D receptor (VDR) signaling by acting as a vitamin D response element (VDRE)-binding protein (VDRE-BP). Transcriptional regulation by active 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) involves occupancy of VDRE by VDRE-BP or 1,25(OH)2D3 bound-VDR. This relationship is disrupted by over-expression of VDRE-BP and can cause a form of human hereditary vitamin D-resistant rickets (HVDRR). DNA array analyses using B-cells from an HVDRR patient and matched control defined a sub-cluster of genes where 1,25(OH)2D3-regulated transcription was abrogated by over-expression of VDRE-BP. Amongst these, the DNA-damage-inducible transcript 4 (DDIT4), an inhibitor of mammalian target of rapamycin (mTOR) signaling, was also induced by 1,25(OH)2D3 in human osteoblasts. Chromatin immunoprecipitation using 1,25(OH)2D3-treated osteoblasts confirmed that liganded VDR and VDRE-BP compete for binding to the proximal promoter of the DDIT4 gene in a similar fashion to other known 1,25(OH)2D3-target genes. Treatment of osteoblasts with 1,25(OH)2D3 induced DDIT4 expression and suppressed phosphorylated S6K1T389 protein (a downstream target of mTOR). The functional importance of this for 1,25(OH)2D3 responses in osteoblasts was underlined by the fact that siRNA knockdown of DDIT4 expression suppressed antiproliferative and cell growth responses to 1,25(OH)2D3. These data confirm that VDRE-BP is required for normal 1,25(OH)2D3-mediated transcription and cell function in osteoblasts. Conversely over-expression of VDRE-BP exerts a dominant-negative effect on transcription of 1,25(OH)2D3-target genes. Characterization of VDRE-BP action in 1,25(OH)2D3-treated osteoblasts highlights an entirely novel role for vitamin D as a regulator of mTOR – a known ‘master regulator’ of cell function. We performed gene expression microarray analysis in HVDRR EBV-transformed B-cells and control cells in the presence or absence of vitamin D.
Project description:The biologically active metabolite of vitamin D (cholecalciferol), i.e. 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], is a secosteroid hormone whose mode of action involves stereospecific interaction with an intracellular receptor protein (vitamin D receptor; VDR). 1,25(OH)2D3 is known to be a principal regulator of calcium homeostasis, and it has numerous other physiological functions including inhibition of proliferation of cancer cells, effects on hormone secretion and suppression of T-cell proliferation and cytokine production. Although the exact mechanisms involved in mediating many of the different effects of 1,25(OH)2D3 are not completely defined, genomic actions involving the VDR are clearly of major importance. Similar to other steroid receptors, the VDR is phosphorylated; however, the exact functional role of the phosphorylation of the VDR remains to be determined. The VDR has been reported to be regulated by 1,25(OH)2D3 and also by activation of protein kinases A and C, suggesting co-operativity between signal transduction pathways and 1,25(OH)2D3 action. The VDR binds to vitamin D-responsive elements (VDREs) in the 5' flanking region of target genes. It has been suggested that VDR homodimerization can occur upon binding to certain VDREs but that the VDR/retinoid X receptor (RXR) heterodimer is the functional transactivating species. Other factors reported to be involved in VDR-mediated transcription include chicken ovalbumin upstream promoter (COUP) transcription factor, which is involved in active silencing of transcription, and transcription factor IIB, which has been suggested to play a major role following VDR/RXR heterodimerization. Newly identified vitamin D-dependent target genes include those for Ca2+/Mg(2+)-ATPase in the intestine and p21 in the myelomonocytic U937 cell line. Elucidation of the mechanisms involved in the multiple actions of 1,25(OH)2D3 will be an active area of future research.
Project description:Purpose:We have observed noticably weak epithelial attachment in vitamin D receptor knockout mice (VDR KO) undergoing epithelial debridement. We hypothesized that VDR KO negatively affects corneal epithelial cell desmosomes and/or hemidesmosomes. Methods:Transcript levels of desmosome and hemidesmosome proteins in VDR KO corneas were assessed by qPCR. Western blotting and immunochemistry were used to detect proteins in cultured cells exposed to 1,25(OH)2D3 and 24R,25(OH)2D3. Results:VDR KO resulted in decreased corneal desmosomal desmoglein 1 (DSG1) and desmocollin 2 (DSC2) mRNA, and hemidesmosomal plectin mRNA. DSG1 and plectin protein expression were reduced in VDR KO corneas. DSG1 protein expression increased in VDR wild types (VDR WT) and VDR KO mouse primary epithelial cells (MPCEC) treated with 1,25(OH)2D3 and 24R,25(OH)2D3. 24R,25(OH)2D3 treatment resulted in increased plectin and integrin ?4 levels in VDR WT MPCEC, and decreased levels in VDR KO MPCEC. Treatment of human corneal epithelial cells (HCEC) with 1,25(OH)2D3 and 24R,25(OH)2D3 resulted in increased DSC2 and DSG1 protein expression. Plectin and integrin ?4 were only increased in 24R,25(OH)2D3 treated HCEC. Conclusions:VDR KO results in reduced desmosomal and hemidesmosomal mRNA and protein levels. 1,25(OH)2D3 and 24R,25(OH)2D3 increased DSG1 protein in all cells tested. For hemidesmosome proteins, 24R,25(OH)2D3 increased plectin and integrin ?4 protein expression in VDR WT and HCEC, with decreased expression in VDR KO MPCEC. Thus, vitamin D3 is involved in desmosome and hemidesmosome junction formation/regulation, and their decreased expression likely contributes to the loosely adherent corneal epithelium in VDR KO mice. Our data indicate the presence of a VDR-independent pathway.
Project description:The pandemic increase in obesity is inversely associated with vitamin D levels. While a higher BMI was causally related to lower 25-hydroxyvitamin D (25(OH)D), no evidence was obtained for a BMI lowering effect by higher 25(OH)D. Some of the physiological functions of 1,25(OH)2D3 (1,25-dihydroxycholecalciferol or calcitriol) via its receptor within the adipose tissue have been investigated such as its effect on energy balance, adipogenesis, adipokine, and cytokine secretion. Adipose tissue inflammation has been recognized as the key component of metabolic disorders, e.g., in the metabolic syndrome. The adipose organ secretes more than 260 different proteins/peptides. However, the molecular basis of the interactions of 1,25(OH)2D3, vitamin D binding proteins (VDBPs) and nuclear vitamin D receptor (VDR) after sequestration in adipose tissue and their regulations are still unclear. 1,25(OH)2D3 and its inactive metabolites are known to inhibit the formation of adipocytes in mouse 3T3-L1 cell line. In humans, 1,25(OH)2D3 promotes preadipocyte differentiation under cell culture conditions. Further evidence of its important functions is given by VDR knock out (VDR(-/-)) and CYP27B1 knock out (CYP27B1 (-/-)) mouse models: Both VDR(-/-) and CYP27B1(-/-) models are highly resistant to the diet induced weight gain, while the specific overexpression of human VDR in adipose tissue leads to increased adipose tissue mass. The analysis of microarray datasets from human adipocytes treated with macrophage-secreted products up-regulated VDR and CYP27B1 genes indicating the capacity of adipocytes to even produce active 1,25(OH)2D3. Experimental studies demonstrate that 1,25(OH)2D3 has an active role in adipose tissue by modulating inflammation, adipogenesis and adipocyte secretion. Yet, further in vivo studies are needed to address the effects and the effective dosages of vitamin D in human adipose tissue and its relevance in the associated diseases.