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)₂D₃) and its less calcaemic analog 1,24(OH)₂D₂ on the production of tumour necrosis factor alpha (TNFα) by human peritoneal macrophages. The aim of the present study is to examine whether this vitamin D inhibition of TNFα is mediated by its major transcription factor, nuclear factor-κB (NFκB). Methods. Murine macrophage cells (P388D1) were incubated with 10^-7 M 1,25(OH)₂D₃ or 1,24(OH)₂D₂ and then stimulated with lipopolysaccharide. NFκB activity was assayed using a reporter gene and by electrophoretic mobility shift assay (EMSA). In addition, we evaluated mRNA and protein levels of NFκB-p65 and of IκBα, a potent NFκB inhibitor, and phosphorylated IκBα. Results. Both 1,25(OH)₂D₃ and 1,24(OH)₂D₂ induced a 60% reduction of TNFα secretion. By using a reporter gene and EMSA we found that vitamin D markedly reduced NFκB activity. 1,25(OH)₂D₃ or 1,24(OH)₂D₂ decreased NFκB-p65 levels in the nucleus and increased NFκB-p65 levels in the cytosol; no changes were observed in the total levels of NFκB-p65 protein and mRNA. Concurrently, vitamin D induced a significant increase in mRNA and protein levels of IκBα (∼6.5- and 4.5-fold, respectively). Elevated levels of IκBα can be explained by the vitamin D-induced prolongation of IκBα-mRNA half-life from 110 to 190 min and by the decrease in IκBα phosphorylation. Conclusions. Vitamin D up-regulates IκBα levels by increasing mRNA stability and decreasing IκBα phosphorylation. The increase in IκBα levels reduces nuclear translocation of NFκB and thereby downgrades its activity. Since NFκB is a major transcription factor of inflammatory mediators, these findings suggest that the less-calcaemic analog, 1,24(OH)₂D₂ may be effective as an anti-inflammatory therapeutic agent.