MECHANISM OF INDUCTION OF C/EBPδ BY INTERLEUKIN-6 DURING THE ACUTE PHASE RESPONSE

Abstract

C/EBPδ is a member of the C/EBP (CCAAT/enhancer binding protein) family of transcription factors whose expression has been shown to increase dramatically in liver during the acute phase response (APR). C/EBPδ expression is activated in response to APR mediators such as interleukin-6 (IL- 6). C/EBPδ is one of the major regulatory proteins involved in the induction of a number of acute phase (AP) genes in the liver by interacting with IL-6 responsive elements (IL-6REs) in the gene promoters. To better understand the mechanism by which C/EBPδ expression is activated by IL-6 during the APR, a study was initiated to map any potential IL-6REs within the C/EBPδ promoter and to determine the regulatory proteins that bind to these sites. IL-6 induction of C/EBPδ transcription was confirmed by Northern blot analysis in a hepatoma cell line, Hep3B. Cycloheximide inhibition experiments suggested that the IL-6 induction of C/EBPδ transcripts occurs at the transcriptional level and not through stabilization of the C/EBPδ mRNA. To identify regions within the C/EBPδ promoter responsible for the IL-6 induction, progressive 5' deletion mutants were constructed. These constructs were transiently transfected into Hep3B cells and tested for IL-6 inducibility. An IL- 6 responsive region was identified within the first 120 bases of the promoter sequences. A region between -114 and -100 contained sequences that closely resembles the acute phase responsive element (APRE), a motif found in the promoters of many IL-6 inducible genes that binds members of the STAT family of transcription factors. Mutational analysis of the APRE revealed that this site is essential for IL-6 inducibility of the C/EBPδ promoter. In addition, multiple copies of the C/EBPδ APRE inserted in a heterologous promoter are capable of conferring IL-6 inducibility. Electrophoresis mobility shift assay (EMSA) experiments demonstrated preferential binding of STAT3 compared to STAT1, to the C/EBPδ APRE site in response to IL-6. Additional cotransfection experiments with STAT expression vectors showed that STAT3, but not STAT1 or STAT5b, can transactivate the C/EBPδ promoter in an IL-6 responsive manner. Two Sp1 sites were also identified within the C/EBPδ proximal promoter region. Mutations introduced into either of these Sp1 sites decreased the IL-6 inducibility of the C/EBPδ promoter as well as transactivation by STAT3. These findings indicate that Sp1 proteins function cooperatively with STAT3 to activate transcription from the C/EBPδ promoter. Replacement of the C/EBPδ APRE with STAT sites from two IFN-y inducible genes conferred IFN-y responsiveness to the C/EBPδ promoter. This change in STAT specificity appears to be due to a single base difference within the STAT binding motifs. These results indicate that the C/EBPδ gene is designed to respond to certain cytokines, such as IL-6 and G-CSF, but not to others such as IFN-y.