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

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Abstract
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.