Abstract
We have previously found that overexpression of the Rel protein p50 stimulated C-reactive protein (CRP) expression in Hep 3B cells and that p50 could bind to a nonconsensus kappaB site overlapping the CCAAT/enhancer binding protein (C/EBP) binding site centered at position -53 on the CRP promoter. Accordingly, we employed EMSA to investigate possible cooperation between p50 and C/EBP proteins using an oligonucleotide probe (-63/-41) derived from the CRP promoter and containing both C/EBP and p50 binding sites. Abs to p50, but not to p65, decreased formation of C/EBPbeta-containing complexes in nuclei of IL-6-treated cells, indicating that ternary complexes containing C/EBPbeta and p50 are formed on the CRP promoter. Depletion of free Rel proteins by pretreatment of nuclear extracts with a kappaB consensus oligonucleotide markedly decreased formation of C/EBP complexes, indicating that Rel proteins are required for formation of such complexes. Overexpression of p50 in transient cotransfection studies using the proximal CRP promoter (-125/+9) linked to a luciferase reporter caused a 3-fold increase of luciferase activity, while C/EBPbeta overexpression caused an 18-fold increase; simultaneous overexpression of both transcription factors increased luciferase activity approximately 600-fold. Mutation of either the C/EBP binding site or the p50 binding site drastically reduced the effects of overexpressed transcription factors. Taken together, our findings indicate that binding of Rel p50 to the nonconsensus kappaB site enhances and stabilizes binding of C/EBPbeta to the CRP promoter and that binding of both C/EBPbeta and p50 to their overlapping cognate sites is required for induction of CRP expression by IL-6.
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