Vitamin D receptor deletion leads to reduced level of IκBα protein through protein translation, protein–protein interaction, and post-translational modification

https://doi.org/10.1016/j.biocel.2009.11.012Get rights and content

Abstract

Vitamin D receptor plays an essential role in the regulation of inflammation. Previous studies demonstrate that vitamin D receptor negatively modulates the proinflammatory NF-κB pathway. However, it is unknown how vitamin D receptor regulates IκBα, the endogenous inhibitor of NF-κB. Here we investigated the molecular mechanism of vitamin D receptor deletion and IκBα expression. We found that cells lacking vitamin D receptor had significantly increased levels of IκBα mRNA and simultaneously decreased levels of IκBα protein. Lacking vitamin D receptor abolished its binding to the IκBα promoter. Moreover, the levels of protein translation regulators and the rate of protein synthesis were both decreased in cells lacking vitamin D receptor. At the post-translational level, IκBα ubiquitination was enhanced, indicating increased degradation of IκBα in the absence of vitamin D receptor. We further transfected cells with a plasmid carrying either wild-type or mutant IκBα. The expression of wild-type IκBα was much higher in the cells with vitamin D receptor than in the cells without vitamin D receptor, whereas the expression of exogenous IκBα was equally high in both cell lines. In summary, vitamin D receptor deletion affects IκBα through mRNA transcription, protein translation, protein–protein interaction, post-translational modification, and protein degradation, thus reducing the level of IκBα protein. Cells lacking vitamin D receptor are known in a proinflammatory state with activation of NF-κB. Our study provides new insight into vitamin D receptor regulation of an inhibitor of NF-κB in inflammation. Deletion of vitamin D receptor contributes to the activation of NF-κB on multiple levels.

Introduction

The active form of vitamin D, 1,25-dihydroxyvitamin D (1,25(OH)2D3), is known to have anti-inflammatory activity. For example, vitamin D is an environmental factor that influences the course and severity of Inflammatory Bowel Disease (IBD) (Lim et al., 2005). Low levels of vitamin D have been reported in patients with IBD (Sentongo et al., 2002). In animal models, 1,25(OH)2D3 suppressed the development of IBD (Cantorna et al., 2004). The vitamin D receptor (VDR) is required for all known biological effects of vitamin D. Accumulated evidences suggest that VDR signaling plays an essential role in the regulation of inflammation. Therefore, extensive studies are investigating the mechanism and potential application of 1,25(OH)2D3, analogs and VDR agonists in the autoimmune diseases, such as type 1 diabetes, IBD, and multiple sclerosis (Giarratana et al., 2004, Gregori et al., 2002, Adorini and Penna, 2008, Nagpal et al., 2001, Hewison, 2008).

The nuclear factor-κB (NF-κB) family is a group of transcription factors that plays an essential role in inflammation. NF-κB is active in the nucleus, and its activity is inhibited by the inhibitor of κBα (IκBα). IκBα binds to NF-κB and blocks the nuclear localization signal so that the NF-κB dimer (p50 and p65) is retained in the cytoplasm. Phosphorylation of IκBα by IκB kinase (IKK) initiates the ubiquitination and degradation of IκBα, leading to nuclear translocation and activation of NF-κB (Bonizzi and Karin, 2004). VDR has been shown to interact physically with NF-κB p65 in human osteoblasts (Lu et al., 2004) and mouse embryonic fibroblast cells (Sun et al., 2006), and VDR expression negatively regulates NF-κB activity (Sun et al., 2006). Of interest, the expression of IκBα is also affected by the status of VDR. In mouse embryonic fibroblast cells (MEF) lacking VDR, the total level of IκBα protein is only 40% of that in VDR+/− cells (Sun et al., 2006). However, the functional relevance of VDR and IκBα in regulating the activity of NF-κB remains unclear.

It is reported that 1,25(OH)2D3 increases IκBα levels by stabilizing IκBα mRNA and decreasing the level of IκBα phosphorylation, thus decreasing NF-κB activity in macrophages and keratinocytes (Cohen-Lahav et al., 2007, Cohen-Lahav et al., 2006, Riis et al., 2004). The vitamin D analog significantly down-regulates proinflammatory chemokine production by islet cells. Giarratana et al. (2004) found that the inhibition of islet chemokine is associated with the up-regulation of IκBα transcription and with arrest of NF-κB p65 nuclear translocation. Our data demonstrate that VDR ablation leads to a marked reduction in IκBα protein in fibroblasts (Sun et al., 2006) and intestinal epithelial cells (Sun's unpublished data). By inference, 1,25(OH)2D3-bound VDR may help stabilize IκBα in fibroblasts and epithelial cells. This may partially explain why VDR ablation leads to a decrease in IκBα levels (Sun et al., 2006). However, the molecular mechanism of VDR regulation of IκBα expression levels requires further investigation.

In the current study, we investigated the molecular mechanism by which VDR ablation leads to the reduction of the total IκBα protein level. We used VDR+/− and VDR−/− cells as our experimental models to explore VDR regulation of IκBα in terms of mRNA and protein expression levels, post-translational modification, and degradation. We chose VDR+/− and VDR−/− MEF cells because we wanted to compare the difference between VDR-null cells and those carrying one allele of the Vdr gene. We found that the VDR regulates IκBα at multiple levels including mRNA transcription, protein translation, post-translational modification, and degradation by the proteasome. Results in this study may provide new insight into vitamin D receptor regulation of an inhibitor of NF-κB in inflammation.

Section snippets

Mouse embryonic fibroblast (MEF) isolation and culture

MEFs were isolated from E13.5 embryos generated from VDR+/− × VDR+/− mouse breeding (Sun et al., 2006). Briefly, the embryos were harvested and placed in PBS during the removal of the internal organs, head, and four limbs. The remaining embryo bodies were minced individually 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

Cells lacking VDR have decreased total IκBα protein and increased IκBα mRNA

As measured by Western blotting, the total level of IκBα protein in the VDR−/− MEF cells was reduced significantly compared to the VDR+/− MEFs (Fig. 1A). The total amount of IκBα in VDR−/− cells is about 40% that of the cells containing VDR. After determining that deletion of VDR reduces the amount of IκBα protein in intestinal epithelial cells, the effect of VDR on IκBα RNA levels was examined using RT-PCR. We observed significantly higher levels of IκBα mRNA in VDR−/− MEFs compared to the VDR

Discussion

The aim of the present study is to examine the cause of the reduction in the level of the IκBα protein induced by the deletion of VDR. We examined the regulation of IκBα at multiple levels including mRNA expression, protein expression, modification, and degradation. We found that VDR knockout cells have significantly increased levels of IκBα mRNA and decreased levels of IκBα protein. In the VDR knockout cells, the binding of VDR and the IκBα promoter is abolished. The level of translation of

Acknowledgements

We thank Dr. Sanjay B. Maggirwar for providing wild-type and mutant IκBα vectors and Lester Liu for his technical support.

Grants: This work was supported by NIDDK KO1 DK075386 to J.S.

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