RESUMEN
Expression of the acute phase protein C-reactive protein (CRP) is tightly regulated in hepatocytes. Although very little CRP mRNA is transcribed normally, inflammatory stimuli are followed by a dramatic increase in mRNA synthesis and accumulation. IL-6 and IL-1beta are believed to be the major cytokines responsible for induction of CRP and other acute phase proteins. Our previous studies, using transient transfection and EMSA experiments, implicated involvement of the transcription factors C/EBPbeta, STAT3, Rel p50, and c-Rel in CRP induction. In the current study we used chromatin immunoprecipitation assays to determine the kinetics of transcription factor occupancy of these transcription factors on the endogenous CRP promoter. All of these transcription factors were found bound to the endogenous CRP promoter in the absence of cytokines, but cytokine treatment markedly increased binding of only C/EBPbeta. In addition, c-Rel and TATA box-binding protein (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-1beta plus IL-6, peaking at 12 h with secondary peaks at 18 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.
Asunto(s)
Proteína C-Reactiva/genética , Proteína C-Reactiva/metabolismo , Proteína beta Potenciadora de Unión a CCAAT/metabolismo , Regiones Promotoras Genéticas , ARN Mensajero/biosíntesis , Transcripción Genética , Proteína C-Reactiva/biosíntesis , Línea Celular Tumoral , Cromatina/metabolismo , Hepatocitos/inmunología , Hepatocitos/metabolismo , Humanos , Inmunoprecipitación , Mediadores de Inflamación/metabolismo , Interleucina-1beta/farmacología , Interleucina-6/farmacología , Unión Proteica/inmunología , ARN Mensajero/metabolismoRESUMEN
C-reactive protein (CRP) is a plasma protein primarily synthesized in the liver following inflammatory stimuli as part of the acute phase response. Expression of CRP is tightly regulated in hepatocytes. Normally very little CRP mRNA is transcribed, but inflammatory stimuli are followed by a dramatic increase in mRNA synthesis and accumulation. Interleukins -6 and 1 (IL-6 and IL-1) are believed to be the major cytokines responsible for induction of acute phase protein biosynthesis. We previously demonstrated that in vivo c-Rel plays a novel regulatory role in that it appears to be in complex with C/EBPbeta when C/EBPbeta is bound to the CRP gene promoter following cytokine stimulation, but is not itself bound to DNA. In this study we found that recombinant c-Rel((1-300)) (truncated c-Rel protein missing the transactivation domain) increased the affinity of recombinant C/EBPbeta for a CRP-derived C/EBP site (-53) at least 10-fold. This effect was independent of a previously described p50 binding site at -43 and of binding of c-Rel to DNA. C/EBPbeta and c-Rel((1-300)) were found to physically interact in solution, and overexpression of c-Rel (either full length or truncated (1-300)) in the presence of overexpressed C/EBPbeta stimulated CRP transcription. We conclude that c-Rel((1-300)) binding to C/EBPbeta increases the affinity of C/EBPbeta for the C/EBP binding site at -53 on the CRP promoter, and that the transactivation domain of c-Rel is not necessary for this effect, which depends on protein: protein contacts with C/EBPbeta.