Vitamin D Promotes Odontogenic Differentiation of Human Dental Pulp Cells via ERK Activation

The active metabolite of vitamin D such as 1α,25-dihydroxyvitamin D₃ (1α,25(OH)₂D₃) is a well-known key regulatory factor in bone metabolism. However, little is known about the potential of vitamin D as an odontogenic inducer in human dental pulp cells (HDPCs) in vitro. The purpose of this study was to evaluate the effect of vitamin D₃ metabolite, 1α,25(OH)₂D₃, on odontoblastic differentiation in HDPCs. HDPCs extracted from maxillary supernumerary incisors and third molars were directly cultured with 1α,25(OH)₂D₃ in the absence of differentiation-inducing factors. Treatment of HDPCs with 1α,25(OH)₂D₃ at a concentration of 10 nM or 100 nM significantly upregulated the expression of dentin sialophosphoprotein (DSPP) and dentin matrix protein1 (DMP1), the odontogenesis-related genes. Also, 1α,25(OH)₂D₃ enhanced the alkaline phosphatase (ALP) activity and mineralization in HDPCs. In addition, 1α,25(OH)₂D₃ induced activation of extracellular signal-regulated kinases (ERKs), whereas the ERK inhibitor U0126 ameliorated the upregulation of DSPP and DMP1 and reduced the mineralization enhanced by 1α,25(OH)₂D₃. These results demonstrated that 1α,25(OH)₂D₃ promoted odontoblastic differentiation of HDPCs via modulating ERK activation.     

Structural Evidence for Enhancement of Sequential Vitamin D3 Hydroxylation Activities by Directed Evolution of Cytochrome P450 Vitamin D3 Hydroxylase

Vitamin D₃ hydroxylase (Vdh) isolated from actinomycete Pseudonocardia autotrophica is a cytochrome P450 (CYP) responsible for the biocatalytic conversion of vitamin D₃ (VD₃) to 1α,25-dihydroxyvitamin D₃ (1α,25(OH)₂VD₃) by P. autotrophica. Although its biological function is unclear, Vdh is capable of catalyzing the two-step hydroxylation of VD₃, i.e. the conversion of VD₃ to 25-hydroxyvitamin D₃ (25(OH)VD₃) and then of 25(OH)VD₃ to 1α,25(OH)₂VD₃, a hormonal form of VD₃. Here we describe the crystal structures of wild-type Vdh (Vdh-WT) in the substrate-free form and of the highly active quadruple mutant (Vdh-K1) generated by directed evolution in the substrate-free, VD₃-bound, and 25(OH)VD₃-bound forms. Vdh-WT exhibits an open conformation with the distal heme pocket exposed to the solvent both in the presence and absence of a substrate, whereas Vdh-K1 exhibits a closed conformation in both the substrate-free and substrate-bound forms. The results suggest that the conformational equilibrium was largely shifted toward the closed conformation by four amino acid substitutions scattered throughout the molecule. The substrate-bound structure of Vdh-K1 accommodates both VD₃ and 25(OH)VD₃ but in an anti-parallel orientation. The occurrence of the two secosteroid binding modes accounts for the regioselective sequential VD₃ hydroxylation activities. Moreover, these structures determined before and after directed evolution, together with biochemical and spectroscopic data, provide insights into how directed evolution has worked for significant enhancement of both the VD₃ 25-hydroxylase and 25(OH)VD₃ 1α-hydroxylase activities.     

Activity of 25-Hydroxylase in Human Gingival Fibroblasts and Periodontal Ligament Cells

Background

We previously demonstrated that 25-hydroxyvitamin D₃ concentrations in gingival crevicular fluid are 300 times higher than those in the plasma of patients with aggressive periodontitis. Here we explored whether 25-hydroxyvitamin D₃ can be synthesized by periodontal soft tissue cells. We also investigated which of the two main kinds of hydroxylases, CYP27A1 and CYP2R1, is the key 25-hydroxylase in periodontal soft tissue cells.

Methodology/Principal Findings

Primary cultures of human gingival fibroblasts and periodontal ligament cells from 5 individual donors were established. CYP27A1 mRNA, CYP2R1 mRNA and CYP27A1 protein were detected in human gingival fibroblasts and periodontal ligament cells, whereas CYP2R1 protein was not. After incubation with the 25-hydroxylase substrate vitamin D₃, human gingival fibroblasts and periodontal ligament cells generated detectable 25-hydroxyvitamin D₃ that resulted in the production of 1α,25-dihydroxyvitamin D₃. Specific knockdown of CYP27A1 in human gingival fibroblasts and periodontal ligament cells using siRNA resulted in a significant reduction in both 25-hydroxyvitamin D₃ and 1α,25-dihydroxyvitamin D₃ production. Knockdown of CYP2R1 did not significantly influence 25-hydroxyvitamin D₃ synthesis. Sodium butyrate did not influence significantly CYP27A1 mRNA expression; however, interleukin-1β and Porphyromonas gingivalis lipopolysaccharide strongly induced CYP27A1 mRNA expression in human gingival fibroblasts and periodontal ligament cells.

Conclusions

The activity of 25-hydroxylase was verified in human gingival fibroblasts and periodontal ligament cells, and CYP27A1 was identified as the key 25-hydroxylase in these cells.     

Functional significance of vitamin D receptor FokI polymorphism in human breast cancer cells

Background

The FokI vitamin D receptor (VDR) polymorphism results in different translation initiation sites on VDR. In the VDRff variant, initiation of translation occurs at the first ATG site, giving rise to a full length VDR protein of 427 amino acids. Conversely, in the VDRFF variant, translation begins at the second ATG site, resulting in a truncated protein with three less amino acids. Epidemiological studies have paradoxically implicated this polymorphism with increased breast cancer risk. 1α,25 (OH)₂D₃, the active metabolite of vitamin D, is known to inhibit cell proliferation, induce apoptosis and potentiate differentiation in human breast cancer cells. It is well documented that 1α,25 (OH)₂D₃ downregulates estrogen receptor α expression and inhibits estrogen mediated signaling in these cells. The functional significance of the VDR FokI polymorphism in vitamin D action is undefined.

Methods/Findings

To elucidate the functional role of FokI polymorphism in breast cancer, MCF-7-Vector, MCF-7-VDRff and MCF-7-VDRFF stable cell lines were established from parental MCF-7 cells as single-cell clones. In response to 1α,25 (OH)₂D₃ treatments, cell growth was inhibited by 60% in VDRFF cells compared to 28% in VDRff cells. The induction of the vitamin D target gene CYP24A1 mRNA was 1.8 fold higher in VDRFF cells than in VDRff cells. Estrogen receptor-α protein expression was downregulated by 62% in VDRFF cells compared to 25% in VDRff cells. VDR protein stability was greater in MCF-7-VDRFF cells in the presence of cycloheximide. PCR array analyses of VDRff and VDRFF cells revealed increased basal expression levels of pro-inflammatory genes Cyclooxygenase-2, Interleukin-8 and Chemokine (C-C Motif) Ligand 2 in MCF-7-VDRff cells by 14, 52.7 and 5 fold, respectively.

Conclusions/Significance

These results suggest that a VDRff genotype may play a role in amplifying aggressive breast cancer, paving the way for understanding why some breast cancer cells respond inefficiently to vitamin D treatment.     

Primary Human Osteoblasts in Response to 25-Hydroxyvitamin D3, 1,25-Dihydroxyvitamin D3 and 24R,25-Dihydroxyvitamin D3

The most biologically active metabolite 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃) has well known direct effects on osteoblast growth and differentiation in vitro. The precursor 25-hydroxyvitamin D₃ (25(OH)D₃) can affect osteoblast function via conversion to 1,25(OH)₂D₃, however, it is largely unknown whether 25(OH)D₃ can affect primary osteoblast function on its own. Furthermore, 25(OH)D₃ is not only converted to 1,25(OH)₂D₃, but also to 24R,25-dihydroxyvitamin D₃ (24R,25(OH)₂D₃) which may have bioactivity as well. Therefore we used a primary human osteoblast model to examine whether 25(OH)D₃ itself can affect osteoblast function using CYP27B1 silencing and to investigate whether 24R,25(OH)₂D₃ can affect osteoblast function. We showed that primary human osteoblasts responded to both 25(OH)D₃ and 1,25(OH)₂D₃ by reducing their proliferation and enhancing their differentiation by the increase of alkaline phosphatase, osteocalcin and osteopontin expression. Osteoblasts expressed CYP27B1 and CYP24 and synthesized 1,25(OH)₂D₃ and 24R,25(OH)₂D₃ dose-dependently. Silencing of CYP27B1 resulted in a decline of 1,25(OH)₂D₃ synthesis, but we observed no significant differences in mRNA levels of differentiation markers in CYP27B1-silenced cells compared to control cells after treatment with 25(OH)D₃. We demonstrated that 24R,25(OH)₂D₃ increased mRNA levels of alkaline phosphatase, osteocalcin and osteopontin. In addition, 24R,25(OH)₂D₃ strongly increased CYP24 mRNA. In conclusion, the vitamin D metabolites 25(OH)D₃, 1,25(OH)₂D₃ and 24R,25(OH)₂D₃ can affect osteoblast differentiation directly or indirectly. We showed that primary human osteoblasts not only respond to 1,25(OH)₂D₃, but also to 24R,25(OH)₂D₃ by enhancing osteoblast differentiation. This suggests that 25(OH)D₃ can affect osteoblast differentiation via conversion to the active metabolite 1,25(OH)₂D₃, but also via conversion to 24R,25(OH)₂D₃. Whether 25(OH)D₃ has direct actions on osteoblast function needs further investigation.     

25-Hydroxyvitamin D3 is an agonistic vitamin D receptor ligand

25-Hydroxyvitamin D₃ 1α-hydroxylase encoded by CYP27B1 converts 25-hydroxyvitamin D₃ into 1α,25-dihydroxyvitamin D₃, a vitamin D receptor ligand. 25-Hydroxyvitamin D₃ has been regarded as a prohormone. Using Cyp27b1 knockout cells and a 1α-hydroxylase-specific inhibitor we provide in four cellular systems, primary mouse kidney, skin, prostate cells and human MCF-7 breast cancer cells, evidence that 25-hydroxyvitamin D₃ has direct gene regulatory properties. The high expression of megalin, involved in 25-hydroxyvitamin D₃ internalisation, in Cyp27b1^?/? cells explains their higher sensitivity to 25-hydroxyvitamin D₃. 25-Hydroxyvitamin D₃ action depends on the vitamin D receptor signalling supported by the unresponsiveness of the vitamin D receptor knockout cells. Molecular dynamics simulations show the identical binding mode for both 25-hydroxyvitamin D₃ and 1α,25-dihydroxyvitamin D₃ with the larger volume of the ligand-binding pocket for 25-hydroxyvitamin D₃. Furthermore, we demonstrate direct anti-proliferative effects of 25-hydroxyvitamin D₃ in human LNCaP prostate cancer cells. The synergistic effect of 25-hydroxyvitamin D₃ with 1α,25-dihydroxyvitamin D₃ in Cyp27b1^?/? cells further demonstrates the agonistic action of 25-hydroxyvitamin D₃ and suggests that a synergism between 25-hydroxyvitamin D₃ and 1α,25-dihydroxyvitamin D₃ might be physiologically important. In conclusion, 25-hydroxyvitamin D₃ is an agonistic vitamin D receptor ligand with gene regulatory and anti-proliferative properties.     

Treatment with Vitamin D/MOG Association Suppresses Experimental Autoimmune Encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE) is an animal model to study multiple sclerosis (MS). Considering the tolerogenic effects of active vitamin D, we evaluated the therapeutic effect of myelin oligodendrocyte glycoprotein (MOG) associated with active vitamin D in EAE development. EAE was induced in female C57BL/6 mice by immunization with MOG emulsified with Complete Freund’s Adjuvant plus Mycobacterium tuberculosis. Animals also received two intraperitoneal doses of Bordetella pertussis toxin. One day after immunization, mice were treated with 0,1μg of 1α,25-dihydroxyvitamin D3 (1,25(OH)₂D₃) every other day during 15 days (on days 1, 3, 5, 7, 9, 11, 13 and 15). MOG (150μg) was co-administered on days 3 and 11. The administration of 1,25(OH) ₂D₃ or MOG determined significant reduction in EAE incidence and in clinical scores. When MOG was associated with 1,25(OH) ₂D₃ the animals did not develop EAE. Spleen and central nervous system (CNS) cell cultures from this group produced less IL-6 and IL-17 upon stimulation with MOG in comparison to the EAE control group. In addition, this treatment inhibited dendritic cells maturation in the spleen and reduced inflammatory infiltration in the CNS. The association of MOG with 1,25(OH) ₂D₃ was able to control EAE development.     

Regulation of Adipogenesis and Key Adipogenic Gene Expression by 1, 25-Dihydroxyvitamin D in 3T3-L1 Cells

The functions of 1, 25-dihydroxyvitamin D (1, 25-(OH)₂D₃) in regulating adipogenesis, adipocyte differentiation and key adipogenic gene expression were studied in 3T3-L1 preadipocytes. Five concentrations (0.01, 0.1, 1, 10, 100nM) of 1, 25-(OH)₂D₃ were studied and lipid accumulation measured by Oil Red O staining and expression of adipogenic genes quantified using quantitative real-time PCR. Adipogenic responses to 1, 25-(OH)₂D₃ were determined on 6, and 12 h, and days 1-10 after induction of adipogenesis by a hormonal cocktail with or without 1, 25-(OH)₂D₃. In response to 1, 25-(OH)₂D₃ (1, 10, and 100 nM), lipid accumulation and the expression of PPARγ, C/EBPα, FABP4 and SCD-1 were inhibited through day 10, and vitamin D receptor expression was inhibited in the early time points. The greatest inhibitory effect was upon expression of FABP4. Expression of SREBP-1c was only affected on day 2. The lowest concentrations of 1, 25-(OH)₂D₃ tested did not affect adipocyte differentiation or adipogenic gene expression. The C/EBPα promoter activity response to 1, 25-(OH)₂D₃ was also tested, with no effect detected. These results indicate that 1, 25-(OH)₂D₃ inhibited adipogenesis via suppressing adipogenic-specific genes, and is invoked either during PPARγ activation or immediately up-stream thereof. Gene expression down-stream of PPARγ especially FABP4 was strongly inhibited, and we suggest that the role of 1, 25-(OH)₂D₃ in regulating adipogenesis will be informed by further studies of adipogenic-specific gene promoter activity.     

Vitamin D Receptor Gene Expression and Function in a South African Population: Ethnicity,Vitamin D and FokI

Polymorphisms of the vitamin D receptor gene (VDR) have been associated inconsistently with various diseases, across populations of diverse origin. The T(f) allele of the functional SNP FokI, in exon 2 of VDR, results in a longer vitamin D receptor protein (VDR) isoform, proposed to be less active. Genetic association of VDR with disease is likely confounded by ethnicity and environmental factors such as plasma 25(OH)D₃ status. We hypothesized that VDR expression, VDR level and transactivation of target genes, CAMP and CYP24A1, depend on vitamin D, ethnicity and FokI genotype. Healthy volunteers participated in the study (African, n = 40 and White, n = 20). Plasma 25(OH)D₃ levels were quantified by LC-MS and monocytes cultured, with or without 1,25(OH)₂D₃. Gene expression and protein level was quantified using qRT-PCR and flow cytometry, respectively. Mean plasma 25(OH)D₃ status was normal and not significantly different between ethnicities. Neither 25(OH)D₃ status nor 1,25(OH)₂D₃ supplementation significantly influenced expression or level of VDR. Africans had significantly higher mean VDR protein levels (P<0.050), nonetheless transactivated less CAMP expression than Whites. Genotyping the FokI polymorphism by pyrosequencing together with HapMap data, showed a significantly higher (P<0.050) frequency of the CC genotype in Africans than in Whites. FokI genotype, however, did not influence VDR expression or VDR level, but influenced overall transactivation of CAMP and 1,25(OH)₂D₃-elicited CYP24A1 induction; the latter, interacting with ethnicity. In conclusion, differential VDR expression relates to ethnicity, rather than 25(OH)D₃ status and FokI genotype. Instead, VDR transactivation of CAMP is influenced by FokI genotype and, together with ethnicity, influence 1,25(OH)₂D₃-elicited CYP24A1 expression. Thus, the expression and role of VDR to transactivate target genes is determined not only by genetics, but also by ethnicity and environment involving complex interactions which may confound disease association.     

Maternal Vitamin D Status in Preeclampsia: Seasonal Changes Are Not Influenced by Placental Gene Expression of Vitamin D Metabolizing Enzymes

Preeclampsia, a hypertensive disorder in pregnancy develops in 2–8% of pregnancies worldwide. Winter season and vitamin D deficiency have been associated with its onset.

Objective

To investigate the influence of season on maternal vitamin D status and placental vitamin D metabolism.

Methods

25-OH vitamin D and 1,25-(OH)₂ vitamin D were measured in maternal serum obtained during the winter or summer months from 63 pregnant women at delivery (43 healthy, 20 preeclampsia). In a subgroup, mRNA expression of CYP24A1 (24-hydroxylase), CYP27B1 (1α-hydroxylase) and VDR (vitamin D receptor) were quantified by real time PCR in placental samples of 14 women with normal pregnancies and 13 with preeclampsia.

Results

In patients with preeclampsia,25-OH vitamin D levels were lower, but differed significantly from controls only in summer (18.21±17.1 vs 49.2±29.2 ng/mL, P<0.001), whereas 1,25-(OH)₂ vitamin D levels were significantly lower only in winter (291±217 vs 612.3±455 pmol/mL, P<0.05). A two-factorial analysis of variance produced a statistically significant model (P<0.0001) with an effect of season (P<0.01) and preeclampsia (P = 0.01) on maternal 25-OH vitamin D levels, as well as a significant interaction between the two variables (P = 0.02). Placental gene expression of CYP24A1, CYP27B1, and VDR did not differ between groups or seasons. A negative correlation between placental gene expression of CYP24A1 and CYP27B1 was observed only in healthy controls (r = −0.81, P<0.0001).

Summary

Patients with preeclampsia displayed lower vitamin D serum levels in response to seasonal changes.The regulation of placental CYP24A1, but not of the VDR or CYP27B1 might be altered in preeclampsia.     

Metabolism of 1α,25-dihydroxyvitamin D2 by human CYP24A1

The metabolism of 1α,25-dihydroxyvitamin D₂ (1α,25(OH)₂D₂) by human CYP24A1 was examined using the recombinant enzyme expressed in Escherichia coli cells. HPLC analysis revealed that human CYP24A1 produces at least 10 metabolites, while rat CYP24A1 produces only three metabolites, indicating a remarkable species-based difference in the CYP24A1-dependent metabolism of 1α,25(OH)₂D₂ between humans and rats. LC-MS analysis and periodate treatment of the metabolites strongly suggest that human CYP24A1 converts 1α,25(OH)₂D₂ to 1α,24,25,26(OH)₄D₂, 1α,24,25,28(OH)₄D₂, and 24-oxo-25,26,27-trinor-1α(OH)D₂ via 1α,24,25(OH)₃D₂. These results indicate that human CYP24A1 catalyzes the C₂₄-C₂₅ bond cleavage of 1α,24,25(OH)₂D₂, which is quite effective in the inactivation of the active form of vitamin D₂. The combination of hydroxylation at multiple sites and C-C bond cleavage could form a large number of metabolites. Our findings appear to be useful to predict the metabolism of vitamin D₂ and its analogs in the human body.     

The Effects of 1α, 25-dihydroxyvitamin D3 and Transforming Growth Factor-β3 on Bone Development in an Ex Vivo Organotypic Culture System of Embryonic Chick Femora

Transforming growth factor-beta3 (TGF-β3) and 1α,25-dihydroxyvitamin D₃ (1α,25 (OH) ₂D₃) are essential factors in chondrogenesis and osteogenesis respectively. These factors also play a fundamental role in the developmental processes and the maintenance of skeletal integrity, but their respective direct effects on these processes are not fully understood. Using an organotypic bone rudiment culture system the current study has examined the direct roles the osteotropic factors 1α,25 (OH)₂D₃ and TGF-β3 exert on the development and modulation of the three dimensional structure of the embryonic femur. Isolated embryonic chick femurs (E11) were organotypically cultured for 10 days in basal media, or basal media supplemented with either 1α,25 (OH) ₂D₃ (25 nM) or TGF-β3 (5 ng/mL & 15 ng/mL). Analyses of the femurs were undertaken using micro-computed tomography (μCT), histology and immunohistochemistry. 1α,25 (OH)2D3 supplemented cultures enhanced osteogenesis directly in the developing femurs with elevated levels of osteogenic markers such as type 1 collagen. In marked contrast organotypic femur cultures supplemented with TGF-β3 (5 ng/mL & 15 ng/mL) demonstrated enhanced chondrogenesis with a reduction in osteogenesis. These studies demonstrate the efficacy of the ex vivo organotypic embryonic femur culture employed to elucidate the direct roles of these molecules, 1α,25 (OH) ₂D₃ and TGF-β3 on the structural development of embryonic bone within a three dimensional framework. We conclude that 1α,25(OH)₂D and TGF-β3 modify directly the various cell populations in bone rudiment organotypic cultures effecting tissue metabolism resulting in significant changes in embryonic bone growth and modulation. Understanding the roles of osteotropic agents in the process of skeletal development is integral to developing new strategies for the recapitulation of bone tissue in later life.     

Clonal Differences in Expression of 25-Hydroxyvitamin D3-1α-hydroxylase, of 25-Hydroxyvitamin D3-24-hydroxylase, and of the Vitamin D Receptor in Human Colon Carcinoma Cells: Effects of Epidermal Growth Factor and 1α,25-Dihydroxyvitamin D3

Human colon carcinoma cells express 25-hydroxyvitamin D₃-1α-hydroxylase (CYP27B1) and thus produce the vitamin D receptor (VDR) ligand 1α,25-dihydroxyvitamin D₃ (1,25-D3), which can be metabolized by 25-hydroxyvitamin D₃-24-hydroxylase (CYP24). Expression of VDR, CYP27B1, and CYP24 determines the efficacy of the antimitotic action of 1,25-D3 and is distinctly related to the degree of differentiation of cancerous lesions. In the present study we addressed the question of whether the effects of epidermal growth factor (EGF) and of 1,25-D3 on VDR, CYP27B1, and CYP24 gene expression in human colon carcinoma cell lines also depend on the degree of cellular differentiation. We were able to show that slowly dividing, highly differentiated Caco-2/15 cells responded in a dose-dependent manner to both EGF and 1,25-D3 by up-regulation of VDR and CYP27B1 expression, whereas in highly proliferative, less differentiated cell lines, such as Caco-2/AQ and COGA-1A and -1E, negative regulation was observed. CYP24 mRNA was inducible in all clones by 1,25-D3 but not by EGF. From the observed clonal differences in the regulatory effects of EGF and 1,25-D3 on VDR and CYP27B1 gene expression we suggest that VDR-mediated growth inhibition by 1,25-D3 would be efficient only in highly differentiated carcinomas even when under mitogenic stimulation by EGF.     

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.