CD40 induces interleukin-6 gene transcription in dendritic cells: regulation by TRAF2, AP-1, NF-kappa B, AND CBF1.
CD40-induced activation of cytokine gene expression in dendritic cells (DC) is an important process in the initiation of primary immune responses. We have determined the intracellular signaling events that lead to CD40 ligation-induced activation of interleukin-6 (IL-6) gene transcription in a murine DC line, FSDC, that is phenotypically representative of bone marrow-derived DC. IL-6 reverse transcriptase-PCR and promoter assays established the responsiveness of FSDC to anti-CD40 ligation. Further promoter assays showed that the transcription factors NF-kappaB and AP-1 are downstream transcriptional mediators of CD40-induced IL-6 gene expression. Anti-CD40 treatment of FSDC stimulated increased expression of specific NF-kappaB (p50:p65) and AP-1 (c-Jun, JunB, JunD, and c-Fos) DNA-protein complexes. Overexpression of an IkappaB-alpha super-repressor or a dominant negative JunD resulted in a strong inhibition of CD40-inducible IL-6 promoter activity supporting a role for both transcription factors. Upstream signal transduction events were studied by transfection of wild type and mutant human CD40 expression constructs into FSDC followed by stimulation with an anti-human CD40 antibody. These experiments revealed that anti-CD40 stimulation of NF-kappaB and IL-6 gene transcription requires specific amino acid residues in the cytoplasmic region of CD40 involved in the recruitment of TRAF2. Induction of IL-6 mRNA by anti-CD40 treatment was found to be a transient event (24 h) and was followed by a diminution of IL-6 transcript to levels below those found in unstimulated cells. This loss of IL-6 expression was associated with reduced p50:p65 NF-kappaB DNA binding and elevated binding of CBF1 to a site overlapping the NF-kappaB site. Overexpression of CBF1 resulted in a profound inhibition of basal and anti-CD40-induced IL-6 promoter activities indicating that prolonged induction of CBF1 may contribute to the transient nature of the IL-6 response. The physiological relevance of these molecular events to DC function is discussed.
Mann
Jelena
J
Division of Infection, Inflammation and Repair and Division of Cancer Sciences, University of Southampton, Southampton General Hospital, Southampton SO16 6YD, United Kingdom.
Oakley
Fiona
F
Johnson
Peter W M
PW
Mann
Derek A
DA
D020816:Amino Acid Motifs; D000818:Animals; D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; D001665:Binding Sites; D019013:CD40 Antigens / Q000096:biosynthesis* / Q000378:metabolism; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D003593:Cytoplasm / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D003713:Dendritic Cells / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005656:Fungal Proteins / Q000378:metabolism*; D017930:Genes, Reporter; D006801:Humans; D015151:Immunoblotting; D015850:Interleukin-6 / Q000096:biosynthesis* / Q000235:genetics* / Q000378:metabolism; D008156:Luciferases / Q000378:metabolism; D051379:Mice; D009154:Mutation; D016328:NF-kappa B / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000378:metabolism*; D016755:Proto-Oncogene Proteins c-jun / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction; D029701:Saccharomyces cerevisiae Proteins; D015398:Signal Transduction; D047992:TNF Receptor-Associated Factor 2; D013997:Time Factors; D018808:Transcription Factor AP-1 / Q000378:metabolism*; D014158:Transcription, Genetic; D014162:Transfection
D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; C064042:CBF1 protein, S cerevisiae; D019013:CD40 Antigens; D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D015850:Interleukin-6; D016328:NF-kappa B; D011506:Proteins; D016755:Proto-Oncogene Proteins c-jun; D012333:RNA, Messenger; D029701:Saccharomyces cerevisiae Proteins; D047992:TNF Receptor-Associated Factor 2; D018808:Transcription Factor AP-1; D004247:DNA; D008156:Luciferases