title: Integration of the NfkappaB p65 subunit into the vitamin D receptor transcriptional complex: identification of p65 domains that inhibit 1,25-dihydroxyvitamin D3-stimulated transcription.
abstract: Resistance to the action of vitamin D (D) occurs in response to tumor necrosis factor-alpha (TNF-alpha), an effect mediated by nuclear factor kappa B (NfkappaB). To determine the mechanism of NfkappaB inhibition of D-stimulated transcription, chromatin immunoprecipitation assays (CHIP) were done in osteoblastic ROS 17/2.8 cells that had been treated with TNF-alpha or transfected with the p65 subunit of NfkappaB. 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-alpha treatment prevented D recruitment of SRC-1 to the transcription complex. These results show that TNF-alpha 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.
authors.affiliation: Department of Medicine, Division of Endocrinology and Metabolism, Emory University School of Medicine and VA Medical Center, Atlanta, Georgia 30033, USA.
authors.forename: Xianghuai
authors.initials: X
authors.lastname: Lu
authors.forename: Paul
authors.initials: P
authors.lastname: Farmer
authors.forename: Janet
authors.initials: J
authors.lastname: Rubin
authors.forename: Mark S
authors.initials: MS
authors.lastname: Nanes
mesh_terms: D002117:Calcitriol / Q000494:pharmacology*; D002120:Calcium Channel Agonists / Q000494:pharmacology*; D002478:Cells, Cultured; D047369:Chromatin Immunoprecipitation; D017362:Cyclic AMP Response Element-Binding Protein / Q000378:metabolism; D051548:Histone Acetyltransferases; D006655:Histone Deacetylases / Q000378:metabolism; D006801:Humans; D016328:NF-kappa B / Q000235:genetics* / Q000378:metabolism*; D056920:Nuclear Receptor Coactivator 1; D010006:Osteoblasts / Q000378:metabolism; D015675:Osteocalcin / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics; D021122:Protein Subunits; D018167:Receptors, Calcitriol / Q000378:metabolism*; D017384:Sequence Deletion; D051996:Transcription Factor RelA; D014157:Transcription Factors / Q000378:metabolism; D014158:Transcription, Genetic / Q000187:drug effects*; D015533:Transcriptional Activation; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology; D030181:Vitamin D Response Element / Q000235:genetics*
chemical_list: D002120:Calcium Channel Agonists; D017362:Cyclic AMP Response Element-Binding Protein; D016328:NF-kappa B; D021122:Protein Subunits; D018167:Receptors, Calcitriol; D051996:Transcription Factor RelA; D014157:Transcription Factors; D014409:Tumor Necrosis Factor-alpha; D015675:Osteocalcin; D051548:Histone Acetyltransferases; C538859:NCOA1 protein, human; D056920:Nuclear Receptor Coactivator 1; D006655:Histone Deacetylases; D002117:Calcitriol