Regulation of TNF-alpha expression in normal macrophages: the role of C/EBPbeta.
C/EBPbeta is present in monocytes and macrophages, binds to the proximal region of the TNF-alpha promoter, and contributes to its regulation. This study was performed to characterize the ability of C/EBPbeta to regulate the TNF-alpha gene in myelomonocytic cells and primary macrophages. In transient transfection assays, overexpression of wild type C/EBPbeta resulted in a 3-4-fold activation of a 120 base pair TNF-alpha promoter-reporter construct, while overexpression of a dominant negative (DN) C/EBPbeta inhibited LPS-induced activation. In vitro monocyte-differentiated macrophages, infected with an adenoviral vector expressing the DN C/EBPbeta (AdDNC/EBPbeta) or the control Adbetagal, expressed their transgenes weakly, however expression was greatly enhanced in the presence of PMA. Infection with AdDNC/EBPbeta resulted in 60% suppression of LPS induced TNFalpha secretion compared to Adbetagal infection (P<0.001) in PMA-treated macrophages. Northern blot analysis demonstrated approximately a 40% reduction of the TNF-alpha mRNA in the presence of the DN C/EBPbeta, suggesting that the effect of the DN C/EBPbeta was at the transcriptional level. In contrast, AdDNC/EBPbeta infection did not result in inhibition of LPS-induced TNF-alpha secretion in the absence of PMA. Further, DN versions of both C/EBPbeta and c-Jun, but not NF-kappaB p65, suppressed PMA-induced TNF-alpha secretion in macrophages. These observations demonstrate that, C/EBPbeta and c-Jun contribute to the regulation of the TNF-alpha gene in normal macrophages following treatment with PMA.
Department of Medicine, Division of Arthritis and Connective Tissue Diseases and the Department of Microbiology and Immunology, Northwestern University Medical School, Chicago, IL 60611, USA.
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Pope
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Mungre
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Liu
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Thimmapaya
D000256:Adenoviridae / Q000235:genetics; D022762:CCAAT-Enhancer-Binding Proteins; D002454:Cell Differentiation; D004268:DNA-Binding Proteins / Q000502:physiology*; D005786:Gene Expression Regulation; D005822:Genetic Vectors; D006801:Humans; D066298:In Vitro Techniques; D048031:JNK Mitogen-Activated Protein Kinases; D008264:Macrophages / Q000502:physiology*; D020928:Mitogen-Activated Protein Kinases / Q000502:physiology; D009000:Monocytes / Q000166:cytology / Q000502:physiology; D009687:Nuclear Proteins / Q000502:physiology*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014158:Transcription, Genetic; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism
D022762:CCAAT-Enhancer-Binding Proteins; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D014409:Tumor Necrosis Factor-alpha; D048031:JNK Mitogen-Activated Protein Kinases; D020928:Mitogen-Activated Protein Kinases; D013755:Tetradecanoylphorbol Acetate