Transcription factor c-Rel enhances C-reactive protein expression by facilitating the binding of C/EBPbeta to the promoter.
Induction of C-reactive protein (CRP) synthesis in hepatocytes by cytokines occurs at the transcriptional level. In Hep3B cells, the transcription factors C/EBPbeta, STAT3, and Rel p50 have been shown to participate in this process. A C/EBP binding site centered at -53 and an overlapping nonconsensus kappaB site on the promoter are critical for CRP expression. We have previously found that an oligonucleotide containing a kappaB site diminished binding of C/EBPbeta to the C/EBP site, suggesting that unidentified Rel proteins present in Hep3B nuclei facilitate the formation of C/EBPbeta-complexes. The current studies were undertaken to determine which of the five Rel proteins, p50/p65/p52/c-Rel/RelB, play such a role. Mutation of the nonconsensus kappaB site did not abolish binding of C/EBPbeta to its binding site, indicating that this site was not necessary for the formation of C/EBPbeta-complexes. Depletion of Rel proteins from Hep3B nuclei led to decreased formation of C/EBPbeta-complexes on a CRP promoter-derived oligonucleotide that contained only the intact C/EBP binding site but not the nonconsensus kappaB site. This finding indicates that Rel proteins are involved in the binding of C/EBPbeta to its binding site by a kappaB site-independent mechanism. Electrophoretic mobility shift assays (EMSAs) revealed that it was c-Rel that facilitated formation of C/EBPbeta-complexes and that c-Rel bound directly to C/EBPbeta-complexes formed on the C/EBP site. Cotransfection of c-Rel enhanced the induction of CRP promoter-driven luciferase activity and enhanced endogenous CRP expression in cells transfected with C/EBPbeta. We conclude that c-Rel regulates CRP expression without the requirement of binding to a kappaB site, and binds directly to C/EBPbeta to facilitate the binding of C/EBPbeta to the CRP promoter.
Agrawal
Alok
A
Department of Pharmacology, East Tennessee State University, Johnson City, TN 37614, USA.
Samols
David
D
Kushner
Irving
I
D000818:Animals; D001665:Binding Sites; D002097:C-Reactive Protein / Q000096:biosynthesis / Q000235:genetics*; D022762:CCAAT-Enhancer-Binding Proteins / Q000378:metabolism*; D004247:DNA / Q000378:metabolism; D005786:Gene Expression Regulation / Q000502:physiology*; D006801:Humans; D011401:Promoter Regions, Genetic; D020569:Proto-Oncogene Proteins c-rel / Q000378:metabolism*
D022762:CCAAT-Enhancer-Binding Proteins; D020569:Proto-Oncogene Proteins c-rel; D002097:C-Reactive Protein; D004247:DNA