A 58-base-pair region of the human C3 gene confers synergistic inducibility by interleukin-1 and interleukin-6.
We have cloned the promoter for the human third component of complement (C3) gene and have identified sequences involved in its regulation during the acute-phase response. A construct linking 199 bp of the C3 promoter to the firefly luciferase gene was found to be very responsive to interleukin-1 (IL-1) and modestly responsive to interleukin-6 (IL-6) by transfection analysis in the human hepatoma line Hep3B2. Simultaneous treatment with the two cytokines showed a strong synergy between the actions of the two molecules. A 58-bp fragment (-127 to -70 bp) was shown by 5' and 3' deletional mutagenesis to contain cis-acting elements that mediated both the IL-1 response and the IL-1-plus-IL-6 synergistic response of this promoter. When coupled to a heterologous promoter, this fragment enabled the synergistic induction by IL-1 plus IL-6. Sequences homologous to the palindrome ACATTGCACAATCT, which mediates the induction of the IL-6 gene by IL-1 (S. Akira, H. Isshiki, T. Sugita, O. Tanabe, S. Kinoshita, Y. Nishio, T. Nakajima, T. Hirano, and T. Kishimoto, EMBO J. 9:1897-1906, 1990), and the core sequence of the IL-6-responsive element of the rat alpha 2-macroglobulin gene (CTGGGA; M. Hattori, L. J. Abraham, W. Northemann, and G. H. Fey, Proc. Natl. Acad. Sci. USA 87:2364-2368, 1990) are contained within this fragment in immediate juxtaposition and partially overlapping. Site-directed mutagenesis within this homology region drastically reduced the inducibility of the C3 promoter by either cytokine. DNase I footprinting analysis defined a binding site for the transcription factor CCAAT/enhancer-binding protein (C/EBP), which included the IL-1-responsive element-like sequence. No differences were seen between the footprints generated by using extracts from unstimulated and IL-1-stimulated Hep3B2 cells. However, gel retardation analyses revealed two IL-1-specific bands. The data suggest that the induction by IL-1 is mediated by a factor belonging to the family of C/EBP-related proteins.
Wilson
D R
DR
Department of Pathology, Baylor College of Medicine, Houston, Texas 77030.
Juan
T S
TS
Wilde
M D
MD
Fey
G H
GH
Darlington
G J
GJ
D000818:Animals; D001482:Base Composition; D001483:Base Sequence; D003001:Cloning, Molecular; D001517:Coleoptera / Q000201:enzymology / Q000235:genetics; D003176:Complement C3 / Q000096:biosynthesis / Q000235:genetics*; D003850:Deoxyribonuclease I; D004357:Drug Synergism; D006801:Humans; D007375:Interleukin-1 / Q000494:pharmacology*; D015850:Interleukin-6 / Q000494:pharmacology*; D008156:Luciferases / Q000235:genetics; D051379:Mice; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D015345:Oligonucleotide Probes; D011401:Promoter Regions, Genetic / Q000187:drug effects*; D011994:Recombinant Proteins / Q000494:pharmacology; D015183:Restriction Mapping; D012689:Sequence Homology, Nucleic Acid; D014158:Transcription, Genetic / Q000187:drug effects*
D003176:Complement C3; D007375:Interleukin-1; D015850:Interleukin-6; D015345:Oligonucleotide Probes; D011994:Recombinant Proteins; D008156:Luciferases; D003850:Deoxyribonuclease I