- In a previous study we demonstrated a down-regulatory effect of vitamin D active metabolite (1,25(OH) 2 D 3 ) and its vitamin D 2 analog (1,24(OH) 2 D 2 ) on TNFa expression in macrophages. We also found an inhibitory effect in the physiological concentration (10 -10 M) of 1,25 (OH ) 2 D 5 which was dose-dependent. This down-regulation, caused by the decrease in NFKB activity by 1,25(OH ) 2 D 3 and 1,24(OH ) 2 D 2 , was demonstrated in P388D1 cells transfected with NFKB reporter gene (p NFKB-Luc) and by EMSA. In our present study we investigated the processes leading to reduced NFKB activity on P388D1 cells. A decrease in nuclei NFκB-p65 and an increase in cytosolic NFκB-p65, were measured, while no changes in total NFκB-p65 mRNA and protein levels were observed. Simultaneously, a significant increase in both mRNA and protein levels of the NFKB-cytosolic inhibitor, IκBα, were determined. The half-life of IκBα-mRNA increased, with a parallel decrease in the phosphorylation of its protein, as the first step of ubiquitinization and degradation. The present results demonstrate that 1,25(OH) 2 D 3 and 1,24(OH) 2 D 2 inhibit TNFa expression in macrophages, by increasing IκBα and decreasing NFKB activity. Since NFKB is a major transcription factor for TNFa and other inflammatory mediators, these findings suggest that 1,25(OH) 2 D 3 and 1,24(OH) 2 D 2 may be used therapeutically as anti-inflammatory agents.