Inhibitory effect of NF-kappaB on 1,25-dihydroxyvitamin D(3) and retinoid X receptor function.
Responsiveness to 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] may be diminished in osteoporosis and inflammatory arthritis. The inflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) is produced in excess in these disorders and has been shown to decrease osteoblast transcriptional responsiveness to vitamin D and to inhibit the binding of the vitamin D receptor (VDR) and its nuclear partner the retinoid X receptor (RXR) to DNA. Previous studies have shown that a vitamin D (VDRE) or retinoid X DNA response element (RXRE) is sufficient to confer TNF-alpha inhibition of vitamin D or retinoid-stimulated transcription in the absence of known TNF-alpha-responsive DNA sequences. We tested the hypothesis that the TNF-alpha-stimulated transcription factor nuclear factor (NF)-kappaB could, in part, mediate TNF-alpha action by inhibiting the transcriptional potency of the VDR and RXR at their cognate cis regulatory sites. Osteoblastic ROS 17/2.8 cells transfected with a dose of NF-kappaB comparable to that stimulated by TNF-alpha decreased 1,25(OH)(2)D(3)-stimulated transcription. This inhibitory effect of NF-kappaB was not observed on basal transcription of a heterologous reporter in the absence of the VDRE. The effects of NF-kappaB and TNF-alpha were comparable but not additive. COS-7 cells were cotransfected with reporters under the regulation of VDRE or RXRE along with vectors expressing VDR, RXR, and NF-kappaB nuclear proteins. Reconstituted NF-kappaB and the NF-kappaB subunit p65 alone, but not p50, dose dependently suppressed basal and ligand-stimulated transcription. p65 overexpression completely abrogated enhanced VDRE-mediated transcriptional activity in response to 1,25(OH)(2)D(3). Electrophoretic mobility shift experiments did not reveal a direct effect of recombinant NF-kappaB or its individual subunits on the binding of heterodimeric VDR-RXR to DNA. These results suggest that TNF-alpha inhibition of hormone-stimulated transcriptional activation may be mediated by activation of NF-kappaB. In contrast, the inhibitory effect of TNF-alpha on binding of receptors to DNA is unlikely to be mediated by NF-kappaB and is not necessary for inhibition of transcription.
Farmer
P K
PK
Division of Endocrinology and Metabolism, Department of Medicine, Emory University School of Medicine and Veterans Affairs Medical Center, Atlanta, Georgia, 30033, USA. pfarmer@emory.edu
He
X
X
Schmitz
M L
ML
Rubin
J
J
Nanes
M S
MS
D000818:Animals; D019556:COS Cells; D004247:DNA / Q000378:metabolism; D019281:Dimerization; D004305:Dose-Response Relationship, Drug; D017930:Genes, Reporter / Q000502:physiology; D006801:Humans; D016328:NF-kappa B / Q000494:pharmacology*; D018167:Receptors, Calcitriol / Q000737:chemistry / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D018168:Receptors, Retinoic Acid / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D011994:Recombinant Proteins / Q000494:pharmacology; D020218:Response Elements / Q000187:drug effects / Q000502:physiology; D047488:Retinoid X Receptors; D014157:Transcription Factors / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
D016328:NF-kappa B; D018167:Receptors, Calcitriol; D018168:Receptors, Retinoic Acid; D011994:Recombinant Proteins; D047488:Retinoid X Receptors; D014157:Transcription Factors; D014409:Tumor Necrosis Factor-alpha; D004247:DNA