Complement factor B gene regulation: synergistic effects of TNF-alpha and IFN-gamma in macrophages.
Complement factor B (Bf) plays an important role in activating the alternative complement pathway. The inflammatory cytokines, in particular TNF-alpha and IFN-gamma, are critical in the regulation of Bf gene expression in macrophages. In this study, we investigated the mechanisms of Bf gene regulation by TNF-alpha and IFN-gamma in murine macrophages. Northern analysis revealed that Bf mRNA expression was synergistically up-regulated by TNF-alpha and IFN-gamma in MH-S cells. Truncations of the 5' Bf promoter identified a region between -556 and -282 bp that mediated TNF-alpha responsiveness as well as the synergistic effect of TNF-alpha and IFN-gamma on Bf expression. Site-directed mutagenesis of a NF-kappaB-binding element in this region (-433 to -423 bp) abrogated TNF-alpha responsiveness and decreased the synergistic effect of TNF-alpha and IFN-gamma on Bf expression. EMSAs revealed nuclear protein binding to this NF-kappaB cis-binding element on TNF-alpha stimulation. Supershift analysis revealed that both p50 and p65 proteins contribute to induction of Bf by TNF-alpha. An I-kappaB dominant negative mutant blocked Bf induction by TNF-alpha and reduced the synergistic induction by TNF-alpha and IFN-gamma. In addition, the proteasome inhibitor MG132, which blocks NF-kappaB induction, blocked TNF-alpha-induced Bf promoter activity and the synergistic induction of Bf promoter activity by TNF-alpha and IFN-gamma. LPS was found to induce Bf promoter activity through the same NF-kappaB cis-binding site. These findings suggest that a NF-kappaB cis-binding site between -433 and -423 bp is required for TNF-alpha responsiveness and for TNF-alpha- and IFN-gamma-stimulated synergistic responsiveness of the Bf gene.
Department of Medicine, University of Calgary, Alberta, Canada.
Yong
Y
Huang
Peter M
PM
Krein
Daniel A
DA
Muruve
Brent W
BW
Winston
D020121:5' Untranslated Regions / Q000502:physiology; D000818:Animals; D002460:Cell Line; D002467:Cell Nucleus / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D011415:Complement Factor B / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism*; D004357:Drug Synergism; D005786:Gene Expression Regulation / Q000276:immunology*; D020672:I-kappa B Proteins / Q000378:metabolism / Q000502:physiology; D007371:Interferon-gamma / Q000502:physiology*; D007375:Interleukin-1 / Q000494:pharmacology; D008070:Lipopolysaccharides / Q000494:pharmacology; D008262:Macrophage Activation / Q000235:genetics; D016676:Macrophages, Alveolar / Q000276:immunology* / Q000378:metabolism*; D051379:Mice; D016297:Mutagenesis, Site-Directed; D016328:NF-kappa B / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D052002:NF-kappa B p50 Subunit; D010766:Phosphorylation; D011401:Promoter Regions, Genetic / Q000276:immunology; D011485:Protein Binding / Q000235:genetics / Q000276:immunology; D012333:RNA, Messenger / Q000096:biosynthesis; D020218:Response Elements / Q000276:immunology; D051996:Transcription Factor RelA; D014409:Tumor Necrosis Factor-alpha / Q000502:physiology*
D020121:5' Untranslated Regions; D020672:I-kappa B Proteins; D007375:Interleukin-1; D008070:Lipopolysaccharides; D016328:NF-kappa B; D052002:NF-kappa B p50 Subunit; D012333:RNA, Messenger; D051996:Transcription Factor RelA; D014409:Tumor Necrosis Factor-alpha; D007371:Interferon-gamma; D011415:Complement Factor B