Elsevier

Molecular Immunology

Volume 47, Issue 9, May 2010, Pages 1728-1738
Molecular Immunology

1α,25-Dihydroxyvitamin D3 inhibits transcriptional potential of nuclear factor kappa B in breast cancer cells

https://doi.org/10.1016/j.molimm.2010.03.004Get rights and content

Abstract

1α,25-Dihydroxyvitamin D3 (VD3), the biologically active form of vitamin D, may have either pro- or anti-inflammatory activities because of its diverse actions on nuclear factor kappa B (NF-κB). Previous studies indicated that VD3 can either activate or inhibit NF-κB via Akt-induced IκBα phosphorylation and increase in IκBα synthesis respectively. At present, the relevant contribution of each mechanism has not been fully explored. We observed a VD3-mediated NF-κB inhibitory effect in vitamin D receptor (VDR)-positive MCF-7 breast cancer cells. We showed that VD3 induced VDR-dependent IκBα expression but still able to lead on transient NF-κB p65 nuclear translocation through Akt-induced IκBα phosphorylation. Upon TNFα stimulation, VD3 was not capable to inhibit IκBα degradation, p65 nuclear translocation and p65/p50-DNA binding. Here, we found that VD3 strongly repressed p65 transactivation in MCF-7 cells using Gal4-p65 chimeras system. VDR was required for the VD3-mediated transrepression and mutations in VDR affected its suppressive ability. We also demonstrated that neither inhibition of p65 phosphorylation nor acetylation was responsible for the transrepression. In fact, we found that treatment of MCF-7 cells with histone deacetylase inhibitors abrogated VD3-induced p65 transrepression. In addition, knockdown of two nuclear corepressors HDAC3 and SMRT relieved p65 transactivation and particular TNFα-triggered gene expression. In conclusion, the reduction of gene activation by VD3 in breast cancer cells was caused by the interference of the transactivation potential of NF-κB p65 subunit. Our studies provide a scientific background for rational use of vitamin D in the prevention and treatment of inflammatory diseases.

Introduction

Nuclear factor kappa B (NF-κB) is an ubiquitous nuclear transcription factor regulating dozens of genes involved in inflammation, and also in growth regulation, apoptosis, cancer invasion/metastasis, tumor promotion, carcinogenesis (Aggarwal, 2004). NF-κB consists of a family of transcription factors including p65 (RelA), p105/p50, p100/p52, RelB and c-Rel. The classic form of NF-κB is the p65/p50 heterodimer that contains the transcriptional activation domain and is sequestered in the cytoplasm as an inactive complex by IκB (Baldwin, 1996). Acute stimuli such as TNF-α, LPS or PMA led to the activation of IκB kinases (IKK) which in turn phosphorylate Ser32 and Ser36 within the N-terminal response domain of IκB (Karin and Ben-Neriah, 2000). Phosphorylated IκB would undergo ubiquitination-dependent proteolysis and the release of IκB unmasks the nuclear localization signal and results in the translocation of NF-κB to the nucleus, followed by the activation of specific target genes (Karin and Ben-Neriah, 2000). Furthermore, NF-κB activation can be positively or negatively regulated by p65 posttranslational modification, in which phosphorylation and acetylation at various amino acid residues in p65 were found to contribute to its transcriptional activities by affecting the transcription regulators recruitment (Hoffmann et al., 2006).

Nuclear hormone receptors have been reported to inhibit NF-κB responses by several different mechanisms. To all appearances the most extensively studied receptor is the glucocorticoid receptor (GR). Dexamethasone, a synthetic glucocorticoid, was shown to induce up-regulation of IκBα, thus sequestrates NF-κB in the cytoplasm (Auphan et al., 1995, Scheinman et al., 1995). Interestingly, no canonical GR responsive element has been found in the IκBα proximal promoter (Deroo and Archer, 2001). At a later time, conflicting results have been documented that GR-induced IκBα expression is either not involved or only accounting part of the hormone-mediated down-regulation of NF-κB activity (Brostjan et al., 1996, Heck et al., 1996). In addition, De Bosscher et al. (1997) showed that activated GR represses transactivation potential of NF-κB p65 subunit, as evidenced by the repressive effect of GR on Gal4-p65-mediated luciferase system. Over a decade, the roles of a number of proposed nuclear models including coactivator competition, disruption of coactivator complex, blocking of RNA polymerase activity, histone modification and inhibition of corepressor clearance have been suggested in hormone receptors-mediated NF-κB transrepression (Pascual and Glass, 2006).

1α,25-Dihydroxyvitamin D3 (VD3) is a steroid hormone and vitamin D receptor (VDR) is the only nuclear protein that binds to this biologically most active vitamin D metabolite. The immunomodulatory actions of VD3 have been well documented in recent studies, however, its role on NF-κB activity remains controversy. We and other researchers have previously demonstrated that VD3 can trigger NF-κB activity through PI3K/Akt pathways (Adams and Teegarden, 2004, Tse et al., 2007). Also, treatment of NB4 cells with VD3 causes a rapid phosphorylation of IκBα (Berry et al., 2002). Contrary to these observations, VD3 has been suggested to inhibit NF-κB activity by increasing IκBα expression in different cell lines (Riis et al., 2004, Cohen-Lahav et al., 2006, Tse et al., 2007, Sun et al., 2008). VD3 was also found to suppress proinflammatory chemokine production, such as MCP-1 and IL-8, via targeting NF-κB (Harant et al., 1997, Zhang et al., 2007). Sun et al. (2006), using mouse embryonic fibroblasts (MEFs) derived from VDR−/− mice, demonstrated that VDR plays an inhibitory role in NF-κB activation by regulating IκBα levels and VDR-p65 interaction. Taken together, the role of VDR in the relation of NF-κB pathway remains to be elucidated.

Here we investigate the molecular mechanism of NF-κB suppressive activity by VD3. We show that VD3 inhibits basal and various stimulus induced NF-κB gene expressions in VDR-positive breast cancer cells. VD3 alone induces both VDR-dependent IκBα expression and transient p65 nuclear translocation via PI3K/Akt-dependent IκBα phosphorylation in MCF-7 cells, however, those effects cannot account for the basal repression of NF-κB-driven promoter activity. Under TNFα stimulation, VD3 does not alter IκBα degradation, p65 nuclear translocation and p65/p50-DNA binding activity. Instead, VD3 causes down-regulation of transactivation potential of p65 through the activated VDR, with the involvement of two nuclear corepressors HDAC3 and SMRT.

Section snippets

Materials

The following antibodies and reagents were used in this study: anti-p65, anti-p50, anti-IκBα, anti-Akt (Ser473), anti-lamin B, anti-GAPDH and anti-VDR (Santa Cruz Inc., CA, USA); anti-p-IκBα (Ser32/36) (Cell Signaling Technology, MA, USA); anti-p-p65 (Ser276) (Millipore Cooperation, MA, USA); VD3 and LY294002 (Alexis Biochemicals, CA, USA); TNFα (Wako pure Chemical Industries Ltd., Japan); trichostatin A (TSA), sodium butyrate (NaBT) (Sigma); Lipofectamine 2000 and cell culture reagents

VD3 down-regulators NF-κB activity in MCF-7 breast cancer cells

To determine whether VD3 treatment can alter NF-κB activity, the effect of VD3 on TNFα-induced gene expression in MCF-7 was determined. We found that co-treatment of VD3 suppressed TNFα-induced NF-κB-regulated gene products MCP-1 and MMP-9 (Fig. 1A). To test whether VD3 could inhibit NF-κB activation induced by different stimulus, MCF-7 cells were transiently transfected with 3x NF-κB-ConA luciferase reporter. Addition of VD3 was capable to inhibit basal and NF-κB signals triggered by various

Discussion

VD3 and its synthetic analogs possess potent immunomodulatory activities, however, the molecular mechanism of VD3-mediated inflammatory gene alteration is not yet fully understood. The NF-κB pathway, which is demonstrated to play a key role in modulating inflammation, was served as a target for VD3's action in this study. Our data show that VD3 can affect NF-κB activity by multiple parameters include cell types, dose of VD3, ability of triggering PI3K/Akt-inducded IκBα phosphorylation, IκBα

Disclosures

The authors have no financial of interest.

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    This project is supported by RGC/CERG grant of HKSAR Government (HKBU 1488/06).

    1

    Present address: Departments of Pathology and Cell Biology, University of South Florida College of Medicine, 12901 Bruce B. Downs Blvd., Tampa, FL 33612–4799, USA.

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