Vitamin D decreases NFκB activity by increasing IκBα levels

Background. In a previous study we demonstrated the inhibitory effect of 1,25-dihydroxyvitamin D (1,25(OH)(2)D-3) and its less calcaemic analog 1,24(OH)(2)D-2 on the production of tumour necrosis factor alpha (TNF alpha) by human peritoneal macrophages. The aim of the present study is to examine whether this vitamin D inhibition of TNF alpha is mediated by its major transcription factor, nuclear factor-kappa B (NF kappa B). Methods. Murine macrophage cells (P388D1) were incubated with 10(-7) M 1,25(OH)(2)D-3 or 1,24(OH)(2)D-2 and then stimulated with lipopolysaccharide. NF kappa B activity was assayed using a reporter gene and by electrophoretic mobility shift assay (EMSA). In addition, we evaluated mRNA and protein levels of NF kappa B-p65 and of I kappa B alpha, a potent NF kappa B inhibitor, and phosphorylated I kappa B alpha. Results. Both 1,25(OH)(2)D-3 and 1,24(OH)(2)D-2 induced a 60% reduction of TNF alpha secretion. By using a reporter gene and EMSA we found that vitamin D markedly reduced NF kappa B activity. 1,25(OH)(2)D-3 or 1,24(OH)(2)D-2 decreased NF kappa B-p65 levels in the nucleus and increased NF kappa B-p65 levels in the cytosol; no changes were observed in the total levels of NF kappa B-p65 protein and mRNA. Concurrently, vitamin D induced a significant increase in mRNA and protein levels of I kappa B alpha (similar to 6.5- and 4.5-fold, respectively). Elevated levels of I kappa B alpha can be explained by the vitamin D-induced prolongation of I kappa B alpha-mRNA half-life from 110 to 190 min and by the decrease in I kappa B alpha phosphorylation. Conclusions. Vitamin D up-regulates I kappa B alpha levels by increasing mRNA stability and decreasing I kappa B alpha phosphorylation. The increase in I kappa B alpha levels reduces nuclear translocation of NF kappa B and thereby downgrades its activity. Since NF kappa B is a major transcription factor of inflammatory mediators, these findings suggest that the less-calcaemic analog, 1,24(OH)(2)D-2 may be effective as an anti-inflammatory therapeutic agent.