- 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 (TNFalpha) by human peritoneal macrophages. The aim of the present study is to examine whether this vitamin D inhibition of TNFalpha is mediated by its major transcription factor, nuclear factor-kappaB (NFkappaB). 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. NFkappaB activity was assayed using a reporter gene and by electrophoretic mobility shift assay (EMSA). In addition, we evaluated mRNA and protein levels of NFkappaB-p65 and of IkappaBalpha, a potent NFkappaB inhibitor, and phosphorylated IkappaBalpha. Both 1,25(OH)(2)D(3) and 1,24(OH)(2)D(2) induced a 60% reduction of TNFalpha secretion. By using a reporter gene and EMSA we found that vitamin D markedly reduced NFkappaB activity. 1,25(OH)(2)D(3) or 1,24(OH)(2)D(2) decreased NFkappaB-p65 levels in the nucleus and increased NFkappaB-p65 levels in the cytosol; no changes were observed in the total levels of NFkappaB-p65 protein and mRNA. Concurrently, vitamin D induced a significant increase in mRNA and protein levels of IkappaBalpha (approximately 6.5- and 4.5-fold, respectively). Elevated levels of IkappaBalpha can be explained by the vitamin D-induced prolongation of IkappaBalpha-mRNA half-life from 110 to 190 min and by the decrease in IkappaBalpha phosphorylation. Vitamin D up-regulates IkappaBalpha levels by increasing mRNA stability and decreasing IkappaBalpha phosphorylation. The increase in IkappaBalpha levels reduces nuclear translocation of NFkappaB and thereby downgrades its activity. Since NFkappaB 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.