doi:10.1093/ndt/gfi254 Original Article

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 (TNFa) by human peritoneal macro-phages. The aim of the present study is to examine whether this vitamin D inhibition of TNFa is mediated by its major transcription factor, nuclear factor-kB (NFkB). Methods. Murine macrophage cells (P388D1) were incubated with 107M 1,25(OH)2D3 or 1,24(OH)2D2 and then stimulated with lipopolysaccharide. NFkB activity was assayed using a reporter gene and by electrophoretic mobility shift assay (EMSA). In addition, we evaluated mRNA and protein levels of NFkB-p65 and of IkBa, a potent NFkB inhibitor, and phosphorylated IkBa. Results. Both 1,25(OH)2D3 and 1,24(OH)2D2 induced a 60 % reduction of TNFa secretion. By using a reporter gene and EMSA we found that vitamin D markedly reduced NFkB activity. 1,25(OH)2D3 or 1,24(OH)2D2 decreased NFkB-p65 levels in the nucleus and increased NFkB-p65 levels in the cytosol; no changes were observed in the total levels of NFkB-p65 protein and mRNA. Concurrently, vitamin D induced a significant increase in mRNA and protein levels of IkBa (6.5- and 4.5-fold, respectively). Elevated levels of IkBa can be explained by the vitamin D-induced prolongation of IkBa-mRNA half-life from 110 to 190min and by the decrease in IkBa phosphorylation. Conclusions. Vitamin D up-regulates IkBa levels by increasing mRNA stability and decreasing IkBa phosphorylation. The increase in IkBa levels reduces nuclear translocation of NFkB and thereby down-grades its activity. Since NFkB is a major transcrip-tion 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