To investigate the effect of VDR inactivation on NF-κB activation, the protein level of the major components of the NF-κB pathway in VDR(+/-) and VDR(-/-) MEFs was compared. We found that VDR ablation led to a marked reduction of IκBα protein (by more than 50%) in VDR(-/-) cells ().However, IκBα degradation induced by TNFα treatment or Salmonella infection followed a similar time-course pattern in VDR(+/-) and VDR(-/-) MEFs, so is the pattern of IκBα phosphorylation following TNFα treatment. The same levels of IKKα and IKKβ were seen in VDR(+/-) and VDR(-/-) MEFs under unstimulated or TNFα-treated condition. Therefore, despite the reduction, the pathway involved in IκBα degradation appears unaltered in VDR(-/-) cells.
Comparison of NF-κB pathway between VDR(+/-) and VDR(-/-) MEFs
By confocal microscopy we found that, due to the reduced level of IκBα, more NF-κB p65 was translocated into the nucleus in VDR(-/-) cells than in VDR(+/-) cells in unstimulated state (), and this immunostaining observation was confirmed by Western blot analysis of p65 levels in the cytosolic and nuclear compartments of VDR(+/-) and VDR(-/-) MEFs. Consistently, the basal p65 DNA binding activity and NF-κB transcriptional activity were significantly increased in VDR(-/-) cells; when the cells were stimulated with TNFα or Salmonella, the induction of both p65 DNA binding and NF-κB activity was much greater in VDR(-/-) cells than in VDR(+/-) cells.
By co-immunoprecipitation assays we were able to pull down p65 protein in VDR(+/-) MEFs, but not in VDR(-/-) cells, using anti-VDR antibody (), confirming the existence of VDR-p65 interaction in mouse fibroblasts. Interestingly, the basal NF-κB transcriptional activity was markedly increased in VDR(-/-) cells, and transfection of hVDR significantly reduced NF-κB activity in both VDR(+/-) and VDR(-/-) cells (), suggesting VDR-p65 interaction affects NF-κB transcriptional activity. Therefore, it appears that increased nuclear accumulation of p65 and lack of VDR binding to p65 in the nucleus lead to higher NF-κB activity in VDR(-/-) cells.
Since NF-κB is a key regulator involved in the synthesis of inflammatory cytokines, we measured the production of IL-6, a well-known NF-κB target gene, as a read-out of the downstream biological effect of NF-κB. In the basal state, IL-6 secretion by VDR(+/-) cells was undetectable, but it was easily detected in VDR(-/-) cells; under the stimulation of TNFα or Salmonella, the secretion of IL-6 was much more robust in VDR(-/-) cells than in VDR(+/-) cells (). Similarly, Northern blot analyses showed that IL-6 mRNA expression induced by TNFα or IL-1β was more robust in VDR(-/-) cells than in VDR(+/-) cells ( ), confirming that VDR(-/-) cells are more susceptible to inflammatory stimuli.
The increase in NF-κB activity in VDR(-/-) cells is attributed in part to the reduction in IκBα. To explain the IκBα reduction in VDR(-/-) MEFs, we determined the effect of vitamin D on IκBα. We found that 1,25(OH)2D3 was able to inhibit IκBα protein degradation induced by TNFα or IL-1β treatment in VDER(+/-) MEFs, but not in VDR(-/-) cells, and the inhibition was apparent at a dose as low as 10-10 M of 1,25(OH)2D3. Therefore, VDR is required for vitamin D-mediated stabilization of IκBα protein in MEFs.