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Am J Physiol Endocrinol Metab 291: E315-E322, 2006. First published February 28, 2006; doi:10.1152/ajpendo.00590.2005
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Increased NF-{kappa}B activity in fibroblasts lacking the vitamin D receptor

Jun Sun,1 Juan Kong,2 Yingli Duan,1 Frances L. Szeto,3 Anne Liao,1 James L. Madara,1 and Yan Chun Li2,3

1Department of Pathology, 2Department of Medicine, and 3Committee on Molecular Metabolism and Nutrition, Biological Science Division, The University of Chicago, Chicago, Illinois

Submitted 28 November 2005 ; accepted in final form 23 February 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANT
 REFERENCES
 
1,25-Dihydroxyvitamin D [1,25(OH)2D3] is known to have anti-inflammatory activity; however, the molecular mechanism remains poorly defined. Here we show that the nuclear vitamin D receptor (VDR) is directly involved in the regulation of NF-{kappa}B activation, a pathway essential for inflammatory response. In mouse embryonic fibroblasts (MEFs) derived from VDR–/– mice, the basal level of {kappa}B inhibitor (I{kappa}B) {alpha} protein was markedly decreased compared with VDR+/– MEFs; however, degradation of I{kappa}B{alpha} and its phosphorylation in response to TNF-{alpha} treatment or Salmonella infection were not altered in VDR–/– cells, neither were the levels of I{kappa}B kinase-{alpha} and I{kappa}B kinase-beta proteins. Consistent with I{kappa}B{alpha} reduction, p65 accumulation in the nucleus was markedly increased in unstimulated VDR–/– cells. In addition, the physical interaction between VDR and p65 was absent in VDR–/– MEFs, which may free p65 and increase its activity. Consequently, these alterations combined led to a marked increase in nuclear p65 DNA binding and NF-{kappa}B transcriptional activity; consistently, induction of IL-6 by TNF-{alpha} or IL-1beta was much more robust in VDR–/– than in VDR+/– cells, indicating that VDR–/– cells are more susceptible to inflammatory stimulation. Therefore, cells lacking VDR appear to be more proinflammatory due to the intrinsic high NF-{kappa}B activity. The reduction of I{kappa}B{alpha} in VDR–/– MEFs may be partially explained by the lack of VDR-mediated stabilization of I{kappa}B{alpha} by 1,25(OH)2D3. This is supported by the observation that I{kappa}B{alpha} degradation induced by TNF-{alpha} was inhibited by 1,25(OH)2D3 in VDR+/– cells, but not in VDR–/– cells. Taken together, these data suggest that VDR plays an inhibitory role in the regulation of NF-{kappa}B activation.

inflammation; nuclear factor-{kappa}B; mouse embryonic fibroblasts

NUCLEAR FACTOR-{kappa}B (NF-{kappa}B) is a family of transcription factors that play an essential role in innate and adaptive immune responses (3). The NF-{kappa}B transcription factors are homo- or heterodimers formed by five proteins, including NF-{kappa}B1 (p105/p50), NF-{kappa}B2 (p100/p52), RelA (p65), RelB, and c-Rel. Different NF-{kappa}B dimers bind to specific DNA sequence in gene promoters to regulate transcription of a wide range of genes, including those involved in immune and inflammatory responses. Many proinflammatory cytokines and chemokines, such as IL-1, IL-6, IL-8, IL-12, and TNF-{alpha}, are targets of NF-{kappa}B regulation (3, 30).

NF-{kappa}B activation is tightly regulated. NF-{kappa}B is active in the nucleus, and its activity is inhibited by the inhibitor of {kappa}B (I{kappa}B). I{kappa}B binds to NF-{kappa}B to block the nuclear localization signal so that the NF-{kappa}B dimer is retained in the cytoplasm. Phosphorylation of I{kappa}B by I{kappa}B kinase (IKK) initiates the ubiquitylation and degradation of I{kappa}B by proteasome, leading to nuclear translocation and activation of NF-{kappa}B (3, 30). The IKK complex is activated by growth factors, proinflammatory cytokines (such as IL-1 and TNF-{alpha}), and hormones through TNF receptor and Toll-like receptor superfamily (3), which ultimately leads to the activation of the NF-{kappa}B pathway.

1,25-Dihydroxyvitamin D [1,25(OH)2D3], the hormonal form of vitamin D, is a multifunctional hormone that is involved in regulations ranging from calcium and phosphate homeostasis, electrolyte and blood pressure homeostasis, to immune response (29). Most activity of 1,25(OH)2D3 is mediated by the vitamin D receptor (VDR), a member of the nuclear receptor superfamily (14). As a ligand-activated transcription factor, VDR heterodimerizes with retinoid X receptor once activated by 1,25(OH)2D3 and binds to the vitamin D response element in the target gene promoter to regulate gene transcription. VDR has also been shown to physically interact with other regulatory proteins such as Smad3 (40), beta-catenin (32), NF-{kappa}B p65 (23), and cyclin D3 (15), but the physiological relevance of these protein-protein interactions remains to be determined.

The immunomodulatory role of vitamin D has been well documented in many studies in the last decades. 1,25(OH)2D3 inhibits T-cell activation and proliferation (2, 36) and suppresses production of IL-2 and IFN-{gamma} in Th1 cells (19, 35). On the other hand, 1,25(OH)2D3 promotes differentiation of Th2 cells and increases IL-4 production in these cells (4, 24). Thus 1,25(OH)2D3 modulates T-cell differentiation by driving cells toward the Th2 phenotype while inhibiting Th1 development. 1,25(OH)2D3 also regulates antigen-presenting cells such as macrophages and dendritic cells. It inhibits dendritic cell differentiation from peripheral blood monocytes and suppresses the production of IL-12, a cytokine crucial for the development of Th1 cells (1, 7, 33, 34). Consistent with its anti-inflammatory role, 1,25(OH)2D3 downregulates the expression of many other proinflammatory cytokines such as IL-1, IL-6, IL-8, and TNF-{alpha} in a variety of cell types (9, 12).

Previous works have suggested that 1,25(OH)2D3 directly modulates NF-{kappa}B activity. In dendritic cells, 1,25(OH)2D3 inhibits IL-12 expression through targeting the NF-{kappa}B pathway (7); 1,25(OH)2D3 also directly suppresses RelB transcription (8). In activated human lymphocytes, 1,25(OH)2D3 suppresses the increase in NF-{kappa}B p50 and its precursor p105 and c-rel proteins (41). 1,25(OH)2D3 has also been shown to decrease the DNA binding capacity of NF-{kappa}B in human fibroblasts (13). In human keratinocytes, 1,25(OH)2D3 is able to reduce NF-{kappa}B DNA binding activity by increasing I{kappa}B{alpha} protein levels, which contributes to the reduction in IL-8 production (37). In pancreatic islet cells, a vitamin D analog is reported to significantly downregulate proinflammatory chemokine production, which is associated with upregulation of I{kappa}B{alpha} transcription and arrest of NF-{kappa}B p65 nuclear translocation (11). However, it remains to be determined whether VDR is directly involved in the regulation of the NF-{kappa}B pathway.

In the present study, we investigated the effect of VDR ablation on NF-{kappa}B activation using mouse embryonic fibroblasts (MEFs) derived from VDR-null mice, because fibroblasts play an important role in inflammatory reactions and have readily inducible NF-{kappa}B activity. We found that cells lacking the VDR exhibit increased NF-{kappa}B activity due to the reduction in I{kappa}B{alpha} levels and the lack of VDR-p65 interaction. Our data suggest that VDR is directly involved in suppression of NF-{kappa}B activation, which may partially explain the VDR-mediated anti-inflammatory mechanism of vitamin D.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANT
 REFERENCES
 
Bacterial strains and growth condition. Salmonella typhimurium wild-type strain (WT-SL) and nonpathogenic strain PhoPc (28) were used in this study. Nonagitated microaerophilic bacterial cultures were prepared by inoculating 10 ml of Luria-Bertani broth with 0.01 ml of a stationary phase culture followed by overnight incubation (~18 h) at 37°C, as previously described (38).

Embryonic fibroblast isolation and culture. MEFs were isolated from E13.5 embryos generated from VDR+/– x VDR+/– mouse breeding (21). Briefly, the embryos were harvested and placed in PBS to remove the internal organs, head, and four limbs. The remaining embryo body was individually minced and digested with 0.5% trypsin and 10 mM EDTA for 0.5 h at 37°C. The digested materials were gently pipetted to single-cell suspension. The cells were cultured in DMEM (high glucose, 4.5 g/l) containing 10% fetal bovine serum, 50 µg/ml streptomycin, and 50 U/ml penicillin. Cells from each embryo were genotyped by PCR using genomic DNA isolated from the cells. VDR+/– and VDR–/– MEFs were used in experiments after more than 15 passages when they had been immortalized, as shown previously (39).

Western blot. Cells were rinsed twice in ice-cold HBSS, lysed in protein loading buffer (50 mM Tris, pH 6.8, 100 mM dithiothreitol, 2% SDS, 0.1% bromophenol blue, 10% glycerol), and sonicated. Equal volume of total cell lysates was separated by SDS-polyacrylamide gel electrophoresis, and proteins were transferred onto nitrocellulose membranes. The membranes were incubated with antibody against p65, I{kappa}B{alpha}, IKK{alpha}, or IKKbeta (Santa Cruz Biotechnology, Santa Cruz, CA), with anti-p-I{kappa}B{alpha}(Ser32/36) antibody (Cell Signaling Technology, Danvers, MA), or with anti-beta-actin antibody (Sigma, St. Louis, MO), and the antigen was visualized with enhanced chemiluminescent reagents (Amersham, Piscataway, NJ). The level of beta-actin served as an internal loading control.

Determination of p65 levels in cytosolic and nuclear compartments. VDR+/– and VDR–/– MEFs were untreated or treated with 20 ng/ml TNF-{alpha} for 30 min, and cells were lysed in a hypotonic buffer [10 mM HEPES, pH 7.9, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM DTT, and 1x Complete Protease Inhibitor Cocktail (Roche)]. After centrifugation, the cytosolic proteins in the supernatant were collected, and the nuclear pellets were extracted with a high salt buffer (20 mM HEPES, pH 7.9, 1.5 mM MgCl2, 20% glycerol, 0.2 mM EDTA, 300 mM KCl, 0.5 mM DTT, 1x Complete Protease Inhibitor Cocktail) for 30 min on ice, followed by centrifugation to collect the nuclear extracts. The level of p65 in the cytosolic and nuclear fractions was determined by Western blotting, and the membrane was reprobed with anti-Sp1 antibody (Santa Cruz Biotechnology) for nuclear protein loading control.

Coimmunoprecipitation assay. Cells were rinsed twice in ice-cold HBSS and lysed in cold immunoprecipitation buffer (1% Triton X-100, 150 mM NaCl, 10 mM Tris·HCl, pH 7.4, 1 mM EDTA, 1 mM EGTA, pH 8.0, 0.2 mM sodium orthovanadate) containing protease inhibitor cocktail (Boehringer Mannheim). Samples were precleared with protein A-agarose (Invitrogen). Precleared lysates were then incubated with 2 µg of anti-VDR antibody (Santa Cruz Biotechnology) for 1 h at 4°C. A 50% slurry of Protein A-agarose was added to the lysate and incubated for 30 min with agitation at 4°C and then washed with cold immunoprecipitation buffer. The pellet was resuspended in 0.1 M glycine, pH 2.5, and incubated with agitation for 10 min at 4°C and then centrifuged at 9,000 g for 2 min. The supernatant was removed and neutralized with 1 M Tris·HCl, pH 8.0. The samples were diluted with concentrated (5x) electrophoresis sample buffer (125 mM Tris, pH 6.8, 4% SDS, 10% glycerol, 0.006% bromophenol blue, 2% beta-mercaptoethanol), boiled for 5 min, separated by SDS-polyacrylamide gel electrophoresis, and transferred onto a nitrocellulose membrane. Membrane blots were probed with anti-p65 antibody and visualized by enhanced chemiluminescent.

Immunofluorescence analysis. MEFs were cultured in the presence or absence of Salmonella (1.6 x 1010 bacteria/ml) or TNF-{alpha} (20 ng/ml) for 1 h, washed, and incubated in media containing gentamicin for 3 h. The cells were rinsed three times in PBS, fixed for 10 min in 3.7% paraformaldehyde, and then permeabilized for 10 min with 0.2% Triton X-100, followed by rinse with PBS containing 10% bovine serum albumin for three times. The permeabilized cells were incubated with rabbit anti-NF-{kappa}B p65 antibody for 1 h at 37°C; after being rinsed three times with PBS, the cells were incubated with Alexa fluor 594 anti-goat secondary antibodies, Alexa fluor 488 anti-mouse FITC-conjugated secondary antibodies (Molecular Probes, Eugene, OR), or 4-diamidino-2-phenylindole (Jackson Immunoresearch Laboratories, West Grove, PA) for 1 h at 37°C. The samples were covered with SlowFade reagent (Molecular Probes) followed by a coverslip, and the edges were sealed to prevent drying. Specimens were examined with a Leica DMIRE2 scanning laser confocal microscope.

Cell transfection. MEFs were grown in 24-well plates in triplicates. At ~70–80% confluence, the cells were cotransfected with NF-{kappa}B reporter plasmid pNF-{kappa}B-Luc (Stratagene, La Jolla, CA), control plasmid pRL-TK (Promega, Madison, WI), and pcDNA3.1 or pcDNA-hVDR using LipofectAMINE (Invitrogen). After 24 h, the cells were lysed, and luciferase activity was determined using the Dual Luciferase Reporter Assay System (Promega) with a TD-20/20 luminometer (Turner Designs, Sunnyvale, CA). Firefly luciferase activity was normalized with Renilla luciferase activity, and the activity was expressed as relative units. In another experiment, MEFs were cotransfected with pNF-{kappa}B-Luc and pRL-TK. After 24 h, the cells were colonized with Salmonella (1.6 x 1010 bacteria/ml) or treated with TNF-{alpha} (20 ng/ml) for 30 min, washed with HBSS, and incubated in DMEM for 2 h before luciferase activity was measured.

NF-{kappa}B p65 DNA binding activity. Confluent MEFs were incubated with or without Salmonella (1.6 x 1010 bacteria/ml) or TNF-{alpha} (20 ng/ml) for 30 min, washed with HBSS three times, and incubated in DMEM for 2 h. The nuclear proteins were extracted using Nuclear Extract Kit (Active Motif, Carlsbad, CA). NF-{kappa}B activation was determined using TransAM NF-{kappa}B p65 Transcription Factor Assay Kit (Active Motif), which specifically measures the amount of NF-{kappa}B p65 bound to its consensus binding site (5'-GGGACTTTCC-3') with ELISA. All procedures were performed according to the manufacturer's instructions.

IL-6 assay. Confluent MEFs grown in 12-well plates were incubated with or without Salmonella (1.6 x 1010 bacteria/ml) or TNF-{alpha} (20 ng/ml) for 1 h. The cells were washed with HBSS three times and incubated in 500 µl of DMEM containing gentamicin at 37°C for 4 h or 24 h. The supernatant was collected and assayed for IL-6 using a mouse IL-6 enzyme immunometric assay kit (Assay Designs, Ann Arbor, MI), according to the manufacturer's instructions.

Northern blot. Northern blot analysis was performed as described previously (20). Briefly, total RNAs were isolated from MEFs using the TRIzol reagent (Invitrogen). The RNAs were separated on a 1.2% agarose gel containing 0.6 M formaldehyde and transferred onto a Nylon membrane (MSI, Westborough, MA), which was cross-linked with an UV cross-linker (Bio-Rad, Hercules, CA). The membrane was hybridized with 32P-labeled cDNA probes according to the method of Church and Gilbert (6). Variations in RNA loading were normalized with 36B4 cDNA probe.

Statistical analysis. Data are expressed as means ± SD. Differences between two samples were analyzed by Student's t-test. P values ≤0.05 were considered significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANT
 REFERENCES
 
We used MEFs to investigate the effect of VDR ablation on the NF-{kappa}B pathway. We first compared the protein level of the major components of the NF-{kappa}B pathway in VDR+/– and VDR–/– MEFs. In the present study, we used VDR+/– cells, as we wanted to compare the difference between VDR-null and one allele of the Vdr gene. Similar results were seen when VDR+/+ MEFs were used. As shown in Fig. 1, VDR+/– MEFs expressed the VDR protein, whereas no VDR was detected in VDR–/– cells (Fig. 1A). Interestingly, VDR ablation led to a marked reduction of I{kappa}B{alpha} protein (by >50%) in VDR–/– cells (Fig. 1, A and B). However, when the MEFs were treated with TNF-{alpha}, a proinflammatory cytokine, or incubated with WT-SL, a bacterial strain that induces inflammation (31), I{kappa}B{alpha} degradation followed a similar pattern in VDR+/– and VDR–/– MEFs (Fig. 1C); infection with a nonpathogenic Salmonella strain PhoPc caused no degradation, consistent with our previous observation in human epithelial cells (31). To further investigate the effect of VDR ablation on I{kappa}B{alpha} degradation, we monitored the change of I{kappa}B{alpha} protein levels in a time course of TNF-{alpha} or WT-SL treatment. As shown in Fig. 2, in both VDR+/– and VDR–/– MEFs, I{kappa}B{alpha} degradation peaked at ~20–30 min after TNF-{alpha} stimulation and recovered after ~60 min, even though its basal level was lower in VDR–/– cells (Fig. 2A). The pattern of I{kappa}B{alpha} phosphorylation following TNF-{alpha} treatment was also the same in VDR+/– and VDR–/– cells (Fig. 2B). Consistent with the protein degradation data, phosphorylation of I{kappa}B{alpha} peaked at 30 min and gradually decreased with time; at ~120 min the phosphorylation completely returned to the basal level (Fig. 2B). These data are consistent with the notion that I{kappa}B{alpha} phosphorylation leads to its degradation. Similar patterns of I{kappa}B{alpha} degradation and phosphorylation were also seen in VDR+/– and VDR–/– cells infected with WT-SL (data not shown). Therefore, despite the reduction in the basal level of I{kappa}B{alpha}, the pathway involved in I{kappa}B{alpha} degradation appears unaltered in VDR–/– cells.


Figure 1
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  		Fig. 1. Levels of {kappa}B inhibitor (I{kappa}B) {alpha} protein in mouse embryonic fibroblasts (MEFs). A: basal levels of I{kappa}B{alpha} protein in vitamin D receptor (VDR)+/– and VDR–/– MEFs determined by Western blotting (WB). Also shown is the VDR levels determined using anti-VDR antibody. B: quantitative data of I{kappa}B{alpha} protein levels in VDR+/– and VDR–/– cells. The data represent mean density of the I{kappa}B{alpha} band from four separate Western blot analyses. *P < 0.01. C: VDR+/– and VDR–/– MEFs were untreated (C) or treated with TNF-{alpha} (T), wild-type Salmonella (W), or nonpathogenic PhoPc Salmonella (P) for 30 min, and I{kappa}B{alpha} levels were determined by WB. The level of beta-actin serves as an internal loading control.

 

Figure 2
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  		Fig. 2. Changes of I{kappa}B{alpha} protein and its phosphorylation in the time course of TNF-{alpha} treatment. VDR+/– and VDR–/– MEFs were untreated (C) or treated with TNF-{alpha} (20 ng/ml) for the time periods as indicated (in minutes), and I{kappa}B{alpha} protein and phosphorylation were determined at each time point from total cell lysates. A: changes in the total protein levels of I{kappa}B{alpha} during TNF-{alpha} treatment (20–60 min). B: I{kappa}B{alpha} phosporylation levels in the course of TNF-{alpha} treatment (30–360 min), determined with an antibody specific for phosphorylated I{kappa}B{alpha}.

 
We then measured the protein levels of IKK{alpha} and IKKbeta, the kinases that phosphorylate I{kappa}B proteins to initiate the degradation by proteasome. As shown in Fig. 3, the same levels of IKK{alpha} and IKKbeta were seen in VDR+/– and VDR–/– MEFs under unstimulated or TNF-{alpha}-treated condition (Fig. 3, A and B, respectively), indicating that VDR ablation has no effects on IKKs.


Figure 3
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  		Fig. 3. Effect of VDR ablation on I{kappa}B kinase (IKK) {alpha} and IKKbeta. VDR+/– and VDR–/– MEFs were untreated (C) or treated with TNF-{alpha} for the time period as indicated. The levels of IKK{alpha} (A) and IKKbeta (B) protein in the cells were determined at each time point by Western blot using specific antibodies.

 
VDR has recently been shown to physically interact with p65 in human osteoblasts (23), but the functional relevance of this interaction in NF-{kappa}B signaling remains unclear. The level of p65 protein, the most abundant NF-{kappa}B species, was slightly reduced in VDR–/– cells (Fig. 4A); interestingly, p65 level appeared to be reduced by TNF-{alpha} treatment in a time-dependent manner in VDR–/– cells, and the reduction was undetected in VDR+/– cells (Fig. 4A). The level of p50, the heterodimeric partner of p65, was the same in VDR+/– and VDR–/– cells (data not shown). Coimmunoprecipitation assays showed that anti-VDR antibody was able to pull down p65 protein in VDR+/– MEFs, but not in VDR–/– cells (Fig. 4B), confirming the existence of VDR-p65 interaction in mouse fibroblasts. To address whether this interaction affects NF-{kappa}B transcriptional activity, VDR+/– and VDR–/– MEFs were cotransfected with a NF-{kappa}B reporter plasmid (pNF-{kappa}B-Luc) and a hVDR expression plasmid (pcDNA-hVDR). Interestingly, the basal NF-{kappa}B transcriptional activity was markedly increased in VDR–/– cells, and transfection of hVDR significantly reduced NF-{kappa}B activity in both VDR+/– and VDR–/– cells (by ~65% and 35%, respectively) (Fig. 4C).


Figure 4
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  		Fig. 4. Interaction of VDR and p65. A: effect of VDR ablation on p65 levels. VDR+/– and VDR–/– MEFs were untreated (C) or treated with TNF-{alpha} for the time as indicated, and p65 protein levels were determined at each time point by WB. B: protein-protein interaction between VDR and p65. VDR+/– and VDR–/– MEF lysates were immunoprecipitated with anti-VDR antibody (IP), and then the precipitated complex was probed with anti-p65 antibody by WB (left). Note that the background level of p65 was detectable in VDR–/– cells. An aliquot of the cell lysates was probed with antibodies against p65 and beta-actin to confirm equal input (right). C: suppression of NF-{kappa}B transcriptional activity by hVDR overexpression. VDR+/– and VDR–/– MEFs were cotransfected with pNF-{kappa}B-Luc reporter, pRL-TK, and pcDNA3.1 or pcDNA-hVDR, and luciferase activity was determined after 24 h as detailed in MATERIALS AND METHODS. *P < 0.05 vs. VDR+/– cells; **P < 0.05 vs. corresponding pcDNA control.

 
I{kappa}B inhibits NF-{kappa}B activity by retaining NF-{kappa}B in the cytoplasm and blocking its nuclear entrance; therefore, the level of I{kappa}B{alpha} directly determines the nuclear translocation of p65 and thus NF-{kappa}B transcriptional activity. Confocal microscopy showed that, in the unstimulated state, the majority of p65 was present in the cytoplasm in VDR+/– MEFs, and very little was seen in the nucleus (Fig. 5A, b); in contrast, in VDR–/– cells, p65 was distributed almost equally in the cytoplasm and nucleus, as the boundary between these two compartments was not detected (see Fig. 5A, d), suggesting that more p65 had translocated into the nucleus due to the reduced level of I{kappa}B{alpha}. When the cells were treated with TNF-{alpha} or Salmonella, increased translocation of p65 into the nucleus was clearly seen in both VDR+/– and VDR–/– cells (Fig. 5A, f, h, j, and l). The immunostaining results were confirmed by Western blot analysis of p65 levels in the cytosolic and nuclear compartments of MEFs. As shown in Fig. 5B, the basal level of p65 in the nuclear extracts was higher in VDR–/– cells than in VDR+/– cells (compare lane 2 vs. lane 6; equal loading of the nuclear proteins was confirmed by the level of Sp1, a nuclear protein absent in the cytosol); when the cells were stimulated with TNF-{alpha} for 30 min, marked nuclear translocation of p65 was seen in both VDR+/– and VDR–/– cells (Fig. 5B).


Figure 5
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  		Fig. 5. Translocation of p65 protein. A: confocal microscopy of immunostained MEFs. Unstimulated control (a–d), TNF-{alpha}-treated (e–h), and wild-type salmonella (WT-SL)-treated (i–l) VDR+/– (a, b, e, f, i, and j) and VDR–/– (c, d, g, h, k, and l) MEFs were stained with 4-diamidino-2-phenylindole to visualize the nucleus (a, c, e, g, i, and k) or with anti-p65 antibody (b, d, f, h, j, and l). The cells were observed under a confocal microscope. Note that, in unstimulated VDR+/– cells, p65 was mainly located in the cytoplasm (b), whereas, in VDR–/– cells, p65 was present in both the cytoplasm and nucleus (d). Arrows indicate the nucleus. B: Western blot analysis of p65 levels in cytosolic (C) and nuclear (N) extracts (25 µg/lane) isolated from unstimulated control and TNF-{alpha}-treated VDR+/– and VDR–/– MEFs. The nuclear protein Sp1, which is absent in the cytosolic fraction, serves as a nuclear protein loading control. Note the higher basal p65 level in VDR–/– nuclear extracts (lane 6) than in VDR+/– nuclear extracts (lane 2).

 
With increased nuclear accumulation of p65 and lack of VDR binding to p65 in the nucleus, VDR–/– MEFs were expected to have higher NF-{kappa}B activity. We, therefore, compared p65 DNA binding capacity and NF-{kappa}B transcriptional activity in VDR+/– and VDR–/– cells under unstimulated and stimulated conditions. NF-{kappa}B p65 DNA binding was determined by measuring the amount of nuclear p65 bound to the NF-{kappa}B DNA binding site, and NF-{kappa}B transcriptional activity was determine by luciferase reporter assays. As expected, the basal p65 DNA binding activity and NF-{kappa}B transcriptional activity were significantly increased in VDR–/– cells (Fig. 6, A and B, respectively; see untreated); when the cells were stimulated with TNF-{alpha} or WT-SL, the induction of both p65 DNA binding and NF-{kappa}B activity was much greater in VDR–/– cells than in VDR+/– cells (Fig. 6, A and B, respectively).


Figure 6
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  		Fig. 6. p65 DNA binding activity and NF-{kappa}B transcriptional activity. A: VDR+/– and VDR–/– MEFs were untreated or treated with TNF-{alpha} or WT-SL for 30 min. Nuclear extracts from both cell types were isolated and incubated with the NF-{kappa}B DNA binding sequence. The amount of p65 bound to the NF-{kappa}B site was determined by ELISA and expressed as optical density at 450 nm (OD450) values. B: VDR+/– and VDR–/– MEFs were cotransfected with pNF-{kappa}B-Luc and pRL-TK. The cells were then untreated or treated with TNF-{alpha} or WT-SL for 30 min, washed with HBSS, and incubated in DMEM for 2 h before the luciferase activities were determined. NF-{kappa}B transcriptional activity was expressed as relative luciferase activity normalized with the internal control activity. Results are representative of 4 separate experiments. Data are presented as the means ± SD from a single experiment assayed in triplicate. *P < 0.05 vs. corresponding VDR(+/–) value; **P < 0.05 vs. control values of the same genotype.

 
Since NF-{kappa}B is a key regulator involved in the synthesis of inflammatory cytokines, we measured the production of IL-6, a well-known NF-{kappa}B target gene, as a read-out of the downstream biological effect of NF-{kappa}B. The synthesis of IL-6 was compared in VDR+/– and VDR–/– MEFs under unstimulated and stimulated conditions. As shown in Fig. 7, in the basal state, while IL-6 secretion into the media by VDR+/– cells was undetectable, it was easily detectable and increased with time from 4 to 24 h in VDR–/– cells (Fig. 7A, see control). When the cells were stimulated with TNF-{alpha} or WT-SL, the secretion of IL-6 from VDR–/– cells was increased dramatically within 4 h, and even more after 24 h; in contrast, IL-6 levels in VDR+/– cells remained undetectable, except for the samples obtained at 24 h after TNF-{alpha} or Salmonella treatment (Fig. 7A). To confirm that VDR–/– cells are more susceptible to inflammatory stimuli, MEFs were treated with TNF-{alpha} or IL-1beta, and the level of IL-6 mRNA was determined. IL-6 mRNA was hardly detectable in unstimulated VDR+/– and VDR–/– MEFs. At a low dose of TNF-{alpha} (5 ng/ml), a dramatic upregulation of IL-6 mRNA was seen only in VDR–/– cells, but not in VDR+/– cells; at a high dose of TNF-{alpha} (20 ng/ml), a moderate induction of IL-6 mRNA was seen in VDR+/– cells, whereas the induction in VDR–/– cells was more robust (Fig. 7B). Similarly, the induction of IL-6 mRNA by IL-1beta (1 ng/ml) was also much more robust in VDR–/– cells than in VDR+/– cells (Fig. 7C). Therefore, the difference in IL-6 production between VDR+/– and VDR–/– cells is at the gene transcriptional level, which is known to be regulated by NF-{kappa}B.


Figure 7
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  		Fig. 7. IL-6 synthesis in MEFs. A: IL-6 secretion. VDR+/– and VDR–/– MEFs were untreated (C) or treated with TNF-{alpha} (20 ng/ml) or WT-SL for 1 h, washed, and then incubated in medium containing gentamicin for 4 or 24 h. IL-6 secretion into the medium was determined using a mouse IL-6 enzyme immunometric assay kit. Note that the basal IL-6 production in unstimulated VDR(+/–) cells was below the detectable limit. Results are representative of 3 separate experiments, and data are means ± SD from a single experiment assayed in triplicate. *P < 0.05, **P < 0.001 vs. corresponding VDR+/– value. B: IL-6 mRNA expression. VDR+/– and VDR–/– MEFs were untreated (C) or treated with 5 ng/ml (top) or 20 ng/ml (bottom) TNF-{alpha} (T) for 6 h. C: VDR+/– and VDR–/– MEFs were untreated (C) or treated with 1 ng/ml IL-1beta (T) for 6 h. Total cellular RNAs were extracted, and IL-6 mRNA levels were determined by Northern blotting. 36B4 is the internal loading control.

 
The increase in NF-{kappa}B activity in VDR–/– cells is attributed in part to the reduction in I{kappa}B{alpha}. To explain the I{kappa}B{alpha} reduction in VDR–/– MEFs, we determined the effect of vitamin D on I{kappa}B{alpha}. Unlike a recent report showing that 1,25(OH)2D3 can increase I{kappa}B{alpha} protein levels in keratinocytes (37), we did not observe changes in I{kappa}B{alpha} protein levels in either VDR+/– or VDR–/– MEFs after 24-h treatment with different doses of 1,25(OH)2D3 (from 10–10 to 10–7 M) (data not shown); instead, we found that 1,25(OH)2D3 was able to inhibit I{kappa}B{alpha} protein degradation induced by TNF-{alpha} treatment in VDR+/– MEFs, but not in VDR–/– cells (Fig. 8). The inhibition of I{kappa}B{alpha} degradation was apparent at a dose as low as 10–10 M of 1,25(OH)2D3 in VDR+/– cells, whereas no inhibition was seen in VDR–/– cells, regardless of the 1,25(OH)2D3 doses (Fig. 8). Similar results were seen when the cells were treated with IL-1beta (not shown). Therefore, VDR is required for vitamin D-mediated stabilization of I{kappa}B{alpha} protein in MEFs.


Figure 8
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  		Fig. 8. Stabilization of I{kappa}B{alpha} protein by vitamin D. VDR+/– and VDR–/– MEFs were pretreated with ethanol (–) or indicated doses of 1,25-dihydroxyvitamin D [1,25(OH)2D3] for 24 h, following by incubation with 20 ng/ml TNF-{alpha} for 30 min. Total cell lysates were then analyzed by WB with antibodies against I{kappa}B{alpha} and beta-actin. Note that no inhibition of I{kappa}B{alpha} degradation by 1,25(OH)2D3 was seen in VDR–/– cells.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANT
 REFERENCES
 
1,25(OH)2D3 has long been known to possess immunomodulatory activity, affecting both innate and adaptive immune responses. The main targets of vitamin D are thought to be T lymphocytes and antigen-presenting cells (macrophages and dendritic cells) (25). 1,25(OH)2D3 can suppress the production of a spectrum of inflammatory cytokines in the immune cells and other cells (such as keratinocytes), including IL-1, IL-2, IL-6, IL-8, INF-{gamma}, and TNF-{alpha} (12), and this action forms the basis for its anti-inflammatory mechanism. Since NF-{kappa}B plays a key role in inflammation and is involved in the regulation of a large number of inflammatory cytokines and chemokines (3), the effect of vitamin D on NF-{kappa}B pathway has been a focus of some previous investigations. 1,25(OH)2D3 has been shown to reduce NF-{kappa}B expression, NF-{kappa}B DNA binding, and transcriptional activity (7, 8, 11, 13, 41), but the role of VDR in NF-{kappa}B activation has not been directly studied.

In the present study, we set to address this question by examining the effect of VDR inactivation on the NF-{kappa}B pathway. We chose to use fibroblasts because they are easy to be established as immortalized cells and contain highly inducible NF-{kappa}B activity. We show here that mouse fibroblasts lacking VDR exhibit increased activity of NF-{kappa}B, leading to increased production of IL-6, and that VDR–/– cells are more susceptible to inflammatory stimuli in inflammatory response. Recently, we have also demonstrated that proinflammatory cytokines IL-1 and IL-18 are suppressed by 1,25(OH)2D3 and are markedly increased in VDR–/– keratinocytes (17). Collectively, these data suggest that cells that lack VDR appear to be in a preinflammatory or proinflammatory state. It will be very interesting to further investigate the inflammatory status of VDR-null mice. In this regard, it is interesting to note that VDR-null mice develop more severe inflammatory bowel disease than wild-type mice when crossed to the IL-10-null background (10).

Our data suggest that VDR is directly involved in the regulation of NF-{kappa}B activation. VDR–/– cells exhibit reduced I{kappa}B{alpha} levels, leading to increased nuclear translocation of p65, which is consistent with a previous observation that a vitamin D analog can block p65 nuclear translocation (11); on the other the hand, because of the lack of VDR, the physical interaction between VDR and p65 is absent in VDR–/– cells, which may free p65 and increase its activity. Consequently, these changes combined lead to a marked increase in NF-{kappa}B activity in VDR–/– cells. Therefore, VDR appears to regulate the NF-{kappa}B activation pathway by targeting I{kappa}B{alpha} and p65.

How VDR regulates I{kappa}B{alpha} remains unclear. In VDR–/– cells, the levels of IKK{alpha} and IKKbeta, the kinases critical for I{kappa}B phosphorylation and degradation, are unchanged; I{kappa}B{alpha} degradation in response to TNF-{alpha} stimulation or Salmonella colonization appears normal as well. Therefore, the events involved in phosphorylation of I{kappa}B{alpha} and the following proteasome-mediated I{kappa}B{alpha} degradation are unaltered in the absence of VDR. Since VDR is not involved in these events, the reduction of I{kappa}B{alpha} is unlikely due to altered IKK protein levels or impaired proteasome-mediated degradation; however, whether VDR inactivation alters IKK phosphorylation or IKK activity remains to be determined. We have excluded the possibility of protein-protein interaction between VDR and I{kappa}B{alpha} (data not shown). Interestingly, a recent study reports that 1,25(OH)2D3 can increase I{kappa}B{alpha} protein levels in keratinocytes (37), suggesting a direct involvement of VDR in the regulation of I{kappa}B{alpha} expression. However, we were not able to detect any changes in I{kappa}B{alpha} protein levels in both VDR+/– and VDR–/– MEFs after 1,25(OH)2D3 treatment. Instead, we found that 1,25(OH)2D3 can prevent or at least reduce the degradation of I{kappa}B{alpha} protein induced by inflammatory stimuli, and the inhibition of I{kappa}B{alpha} degradation is dependent on VDR. By inference, liganded VDR may help stabilize I{kappa}B{alpha} in fibroblasts, and this may partially explain why VDR ablation leads to a decrease in I{kappa}B{alpha} levels. Although the discrepancy between keratinocytes and fibroblasts may lie in the intrinsic property of different cell types, exactly how VDR affects I{kappa}B{alpha} stability needs to be further investigated.

A recent study demonstrates that VDR and p65 proteins physically bind to each other in osteoblasts (23). In the present study, we demonstrate, by coimmunoprecipitation assays, the presence of this interaction in fibroblasts and the absence of this interaction when VDR is ablated. In fact, the exact functional significance of VDR-p65 protein interaction remains to be determined. The previous report (23) provides evidence that p65 is integrated into the VDR transcriptional complex and affects VDR transcriptional activity, but whether this interaction affects NF-{kappa}B activity is not known. We speculate that the elevated basal NF-{kappa}B activity in VDR(–/–) cells results not only from the reduction in I{kappa}B{alpha}, which increases the nuclear accumulation of p65, but also from the disruption of the VDR-p65 interaction, which releases the restraint on p65 and thus increases its activity. In other words, under normal conditions, VDR-p65 interaction helps suppress p65 activity. This speculation seems to be supported by the cotransfection experiment that shows a reduction of NF-{kappa}B transcriptional activity in the presence of hVDR overexpression (Fig. 4C). However, whether VDR binding to p65 actually affects p65 DNA binding activity or its dimerization with p50 requires further investigations.

Given the crucial role of NF-{kappa}B in inflammatory response, and the therapeutic and pharmacological potentials of vitamin D and vitamin D analogs in treatment of autoimmune and inflammatory diseases, such as multiple sclerosis (5), type I diabetes (26, 27), inflammatory bowel disease (22), and psoriasis (16, 18), understanding the relationship and interaction between vitamin D, VDR, and NF-{kappa}B pathway has broad implications. The direct involvement of VDR in the regulation of NF-{kappa}B activation suggests an intrinsic inhibitory role of VDR in inflammation process, and this inhibitory action may likely be enhanced in the presence of vitamin D ligands. Thus the data presented in this report provide new information for understanding the anti-inflammatory mechanism of vitamin D and its analogs. Given the importance of this subject, more studies are warranted to further understand the functional significance as well as the molecular mechanism of VDR and NF-{kappa}B interaction.


   GRANT
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANT
REFERENCES
 
This work was supported in part by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-59327 (Y. C. Li), DK-47662 and DK-35932 (J. L. Madara), and Pilot and Feasibility Award DK-42086 (J. Sun).


   FOOTNOTES
 

Address for reprint requests and other correspondence: Y. C. Li, Dept. of Medicine, Univ. of Chicago, MC4080, 5841 S. Maryland Ave., Chicago, IL 60637 (e-mail: cyan{at}medicine.bsd.uchicago.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANT
 REFERENCES
 

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