Transactivation of c-reactive protein by IL-6 requires synergistic interaction of CCAAT/enhancer finding protein beta (C/EBP beta) and Rel p50

We have previously found that overexpression of the Rel protein p50 stimula ted C-reactive protein (CRP) expression in Hep 3B cells and that p50 could bind to a nonconsensus kappaB Site overlapping the CCAAT/enhancer binding p rotein (C/EBP) binding site centered at position -53 on the CRP promoter. A ccordingly, we employed ER-ISA to investigate possible cooperation between p50 and C/EBP proteins using an oligonucleotide probe (-63/-41) derived fro m the CRP promoter and containing both C/EBP and p50 binding sites. Abs to p50, but not to p65, decreased formation of C/EBP beta -containing complexe s in nuclei of IL-6-treated cells, indicating that ternary complexes contai ning C/EBP beta and p50 are formed on the CRP promoter. Depletion of free R el proteins by pretreatment of nuclear extracts with a kappaB consensus oli gonucleotide markedly decreased formation of C/EBP complexes, indicating th at Rel proteins are required for formation of such complexes. Overexpressio n of p50 in transient cotransfection studies using the proximal CRP promote r (-125/+9) linked to a luciferase reporter caused a 3-fold increase of luc iferase activity, while C/EBP beta overexpression caused an 18-fold increas e; simultaneous overexpression of both transcription factors increased luci ferase activity similar to 600-fold. Mutation of either the C/EBP binding s ite or the p50 binding site drastically reduced the effects of overexpresse d transcription factors. Taken together, our findings indicate that binding of Rel p50 to the nonconsensus kappaB site enhances and stabilizes binding of C/EBP beta to the CRP promoter and that binding of both C/EBP beta and p50 to their overlapping cognate sites is required for induction of CRP exp ression by IL-6.