Expression of the acute phase protein C-reactive protein (CRP) is tightly regulated in hepatocytes. While very little CRP mRNA is transcribed normally, inflammatory stimuli are followed by a dramatic increase in mRNA synthesis and accumulation. Interleukins -6 and 1β (IL-6 and IL-1β) are believed to be the major cytokines responsible for induction of CRP and other acute phase proteins.
We previously demonstrated that in vitro c-Rel plays a novel regulatory role by forming a complex with C/EBPβ when C/EBPβ is bound to the CRP gene promoter following cytokine stimulation. c-Rel does not by itself bind to the DNA. In these studies we found that recombinant c-Rel(1-300) (lacks transactivation domain) increased the affinity of recombinant C/EBPβ for a CRP-derived C/EBP site (-53) at least 10 fold. C/EBPβ and c-Rel(1-300) were found to physically interact in solution, and overexpression of c-Rel in the presence of overexpressed C/EBPβ stimulated CRP transcription. We concluded that c-Rel(1-300) binding to C/EBPβ increased the affinity of C/EBPβ for the CRP-C/EBP(-53) site, and that the transactivation domain of c-Rel is not necessary for this effect, which depends on protein: protein contacts with C/EBPβ.
We also employed chromatin immunoprecipitation (ChIP) assays to determine the kinetics of transcription factor occupancy of these transcription factors on the endogenous CRP promoter. C/EBPβ, STAT3, p50, and c-Rel were found bound to the endogenous CRP promoter in the absence of cytokines, and cytokine treatment markedly increased binding of only C/EBPβ. In addition, c-Rel and TBP appeared to occupy the promoter in parallel in the presence of cytokines. In the absence of cytokines, CRP mRNA accumulation was not measurable but began to increase by 3 h after exposure of cells to IL-1β + IL-6, peaking at 12 h with secondary peaks at 18 h and 24 h. The secondary peaks in mRNA expression paralleled the pattern of binding of c-Rel and TBP to the CRP promoter. We conclude that the CRP promoter has a low level of transcription factor occupancy in the absence of cytokines and induction occurs with binding of C/EBP, and that c-Rel and TBP are important for modulating CRP expression.