Abstract
1,25-Dihydroxyvitamin D (1,25(OH)2D3) is known to suppress NF-κB activity, but the underlying mechanism remains poorly understood. Here we show that the vitamin D receptor (VDR) physically interacts with IκB kinase β (IKKβ) to block NF-κB activation. 1,25(OH)2D3 rapidly attenuates TNFα-induced p65 nuclear translocation and NF-κB activity in a VDR-dependent manner. VDR overexpression inhibits IKKβ-induced NF-κB activity. GST pull-down assays and coimmunoprecipitation experiments demonstrated that VDR physically interacts with IKKβ and that this interaction is enhanced by 1,25(OH)2D3. Protein mapping reveals that VDR-IKKβ interaction occurs between the C-terminal portions of the VDR and IKKβ proteins. Reconstitution of VDR(-/-) cells with the VDR C terminus restores the ability to block TNFα-induced NF-κB activation and IL-6 up-regulation. VDR-IKKβ interaction disrupts the formation of the IKK complex and, thus, abrogates IKKβ phosphorylation at Ser-177 and abolishes IKK activity to phosphorylate IκBα. Consequently, stabilization of IκBα arrests p65/p50 nuclear translocation. Together, these data define a novel mechanism whereby 1,25(OH)2D3-VDR inhibits NF-κB activation.
Highlights
NF-B is a family of transcription factors consisting of five proteins: NF-B1 (p105/p50), NF-B2 (p100/p52), RelA (p65), RelB, and c-Rel
Anti-HA antibodies coprecipitated FLAG-IKK only in cells cotransfected with HA-vitamin D receptor (VDR) or HA-VDR-C and not in cells transfected with HA-VDR-N (Fig. 3D). These results indicate that the C-terminal hinge and ligand-binding domain (LBD) fragment of VDR protein interacts with IKK
This was not surprising because VDR-C has a LBD for 1,25(OH)2D3 binding. These results demonstrate that reconstitution of VDRϪ/Ϫ cells with the C terminus of human VDR (hVDR) to a physiological level is sufficient to block TNF␣ induction of NF-B activity and IL-6 expression
Summary
NF-B is a family of transcription factors consisting of five proteins: NF-B1 (p105/p50), NF-B2 (p100/p52), RelA (p65), RelB, and c-Rel. VDR and IKK Interaction nuclear translocation, blocks NF-B DNA binding, increases IB␣ levels, or stabilizes IB␣ protein [10, 12, 14, 15, 17, 18]. Immunostaining showed that TNF␣-induced p65 nuclear translocation was blocked by overnight 1,25(OH)2D3 pretreatment in VDRϩ/Ϫ MEF but not in VDRϪ/Ϫ MEF cells (Fig. 1, B and C), confirming the requirement of VDR for inhibition of NF-B activation.
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