Integration of the NfκB p65 subunit into the vitamin D receptor transcriptional complex: Identification of p65 domains that inhibit 1,25-dihydroxyvitamin D3-stimulated transcription

Resistance to the action of vitamin D (D) occurs in response to tumor necrosis factor‐α (TNF‐α), an effect mediated by nuclear factor kappa B (NfκB). To determine the mechanism of NfκB inhibition of D‐stimulated transcription, chromatin immunoprecipitation assays (CHIP) were done in osteoblastic ROS 17/2.8 cells that had been treated with TNF‐α or transfected with the p65 subunit of NfκB. These treatments caused stable incorporation of p65 into the transcription complex bound to the vitamin D response element (VDRE) of the osteocalcin promoter. Deletion analysis of p65 functional domains revealed that the p65 N‐terminus and a midmolecular region were both required for the inhibitory action of p65. Pull‐down assays were done using an immobilized glutathione S‐transferase (GST)‐VDR fusion protein to study the effect of p65 on VDR binding to steroid coactivator‐1 (SRC‐1), a major D‐dependent coactivator. p65 inhibited VDR‐SRC‐1 binding in a dose‐dependent manner. Mutations of p65 that abrogated the inhibitory effect on D‐stimulated transcription also failed to inhibit VDR‐SRC‐1 binding. The inhibitory effect of p65 on VDR transactivation was not due to recruitment of a histone deacetylase (HDAC), since inhibition was not relieved by the HDAC inhibitors sodium butyrate or trichostatin A. Overexpression of SRC‐1 or the general coactivators, Creb binding protein or SRC‐3, also failed to relieve p65 inhibition of transcription. In addition, Chip assays revealed that TNF‐α treatment prevented D recruitment of SRC‐1 to the transcription complex. These results show that TNF‐α inhibition of vitamin D‐action includes stable integration of p65 in the VDR transcription complex. Once anchored to proteins within the complex, p65 disrupts VDR binding to SRC‐1, thus decreasing the efficiency of D‐stimulated gene transcription. © 2004 Wiley‐Liss, Inc.