Pope R, Mungre S, Liu H, Thimmapaya B
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.
Cytokine. 2000 Aug;12(8):1171-81. doi: 10.1006/cyto.2000.0691.
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.
C/EBPβ存在于单核细胞和巨噬细胞中，与TNF-α启动子的近端区域结合，并参与其调控。本研究旨在表征C/EBPβ在骨髓单核细胞和原代巨噬细胞中调节TNF-α基因的能力。在瞬时转染试验中，野生型C/EBPβ的过表达导致120个碱基对的TNF-α启动子-报告基因构建体激活3-4倍，而显性负性（DN）C/EBPβ的过表达抑制LPS诱导的激活。在体外单核细胞分化的巨噬细胞中，用表达DN C/EBPβ（AdDNC/EBPβ）或对照Adβgal的腺病毒载体感染后，其转基因表达较弱，但在PMA存在下表达大大增强。与Adβgal感染相比，用AdDNC/EBPβ感染导致PMA处理的巨噬细胞中LPS诱导的TNFα分泌抑制60%（P<0.001）。Northern印迹分析表明，在存在DN C/EBPβ的情况下，TNF-α mRNA减少约40%，这表明DN C/EBPβ的作用是在转录水平。相反，在没有PMA的情况下，AdDNC/EBPβ感染不会导致LPS诱导的TNF-α分泌受到抑制。此外，C/EBPβ和c-Jun的DN版本，但不是NF-κB p65，抑制巨噬细胞中PMA诱导的TNF-α分泌。这些观察结果表明，C/EBPβ和c-Jun在PMA处理后的正常巨噬细胞中参与TNF-α基因的调控。
