Selective requirement for c-Rel during IL-12 P40 gene induction in macrophages

A major challenge in the study of gene regulation by NF-kappaB/Rel transcri ption factors is to understand, at the biological and mechanistic levels, t he selective functions of individual Rel family members. To study selectivi ty, we have examined the NF-kappaB/Rel protein binding site (Rel site) with in the IL-12 p40 promoter. IL-12 is a proinflammatory cytokine expressed by activated macrophages that serves as an essential inducer of T helper 1 ce ll development. In nuclear extracts from lipopolysaccharide-activated macro phages, the predominant Rel dimers capable of binding the IL-12 p40 Rel sit e were the p50/p65 and p50/c-Rel heterodimers and p50/p50 homodimer. The tw o heterodimers bound the site with comparable affinities and exhibited comp arable transactivation activities. In striking contrast, p40 mRNA and prote in concentrations were reduced dramatically in c-Rel(-/-) macrophages and o nly modestly in p65(-/-) macrophages. Other proinflammatory cytokine mRNAs and proteins were not significantly reduced in c-Rel(-/-) macrophages. Thes e results reveal that a c-Rel-containing complex is an essential and select ive activator of p40 transcription, which may reflect unique regulatory mec hanisms or biological functions of IL-12. Furthermore, because selectivity was not observed in vitro or in transient transactivation experiments, thes e findings suggest that an understanding of the selectivity mechanism may r equire an analysis of the endogenous p40 locus.