Inhibitory effect of NF-kappa B 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 dimini shed in osteoporosis and inflammatory arthritis. The inflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) is produced in excess in these diso rders and has been shown to decrease osteoblast transcriptional responsiven ess 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 stu dies 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 retinoi d-stimulated transcription in the absence of known TNF-alpha-responsive DNA sequences. We tested the hypothesis that the TNF-alpha-stimulated transcri ption factor nuclear factor (NF)-kappa B could, in part, mediate TNF-alpha action by inhibiting the transcriptional potency of the VDR and RXR at thei r cognate cis regulatory sites. Osteoblastic ROS 17/2.8 cells transfected w ith a dose of NF-kappa B comparable to that stimulated by TNF-alpha decreas ed 1,25(OH)(2)D-3-stimulated transcription. This inhibitory effect of NF-ka ppa B was not observed on basal transcription of a heterologous reporter in the absence of the VDRE. The effects of NF-kappa B and TNF-alpha were comp arable but not additive. COS-7 cells were cotransfected with reporters unde r the regulation of VDRE or RXRE along with vectors expressing VDR, RXR, an d NF-kappa B nuclear proteins. Reconstituted NF-kappa B and the NF-kappa B subunit p65 alone, but not p50, dose dependently suppressed basal and ligan d-stimulated transcription. p65 overexpression completely abrogated enhance d VDRE-mediated transcriptional activity in response to 1,25( OH)(2)D-3. El ectrophoretic mobility shift experiments did not reveal a direct effect of recombinant NF-kappa B or its individual subunits on the binding of heterod imeric VDR-RXR to DNA. These results suggest that TNF-alpha inhibition of h ormone-stimulated transcriptional activation may be mediated by activation of NF-kappa B. In contrast, the inhibitory effect of TNF-alpha on binding o f receptors to DNA is unlikely to be mediated by NF-kappa B and is not nece ssary for inhibition of transcription.