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.
Pope
R
R
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.
Mungre
S
S
Liu
H
H
Thimmapaya
B
B
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