Iontcheva Iveta, Amar Salomon, Zawawi Khalid H, Kantarci Alpdogan, Van Dyke Thomas E
Department of Periodontology and Oral Biology, Boston University Goldman School of Dental Medicine, Boston, Massachusetts 02118, USA.
Infect Immun. 2004 Apr;72(4):2312-20. doi: 10.1128/IAI.72.4.2312-2320.2004.
Moesin is a 78-kDa protein with diverse functions in linking the cytoskeleton to the membrane while controlling cell shape, adhesion, locomotion, and signaling. The aim of this study was to characterize the expression and localization of moesin in mononuclear phagocytes by using confocal microscopy, flow cytometry, immunoprecipitation, and Western blotting and to analyze the function of moesin as a lipopolysaccharide receptor, utilizing an antisense oligonucleotide approach to knock down the moesin gene. Results revealed that moesin is expressed on the surface of monocytes/macrophages and surface expression is increased after lipopolysaccharide stimulation. The total protein mass of moesin is increased in monocytes after lipopolysaccharide stimulation. Immunoprecipitation showed that moesin coprecipitates with TLR4, a well-known lipopolysaccharide receptor, suggesting an early role of moesin in the formation of the initiation complex for lipopolysaccharide signaling. Two antisense and two control sense oligonucleotides were synthesized and introduced every 4 h for 48 h in adherent macrophage-like cells. Cells were then stimulated with lipopolysaccharide for 4 h, and the supernatants were assayed for tumor necrosis factor alpha (TNF-alpha) production. Cell lysates were assayed for moesin expression by Western blotting immediately after the 48-h treatment period and also after 116 h of recovery to assess the return of moesin expression and function. Moesin gene expression was completely suppressed after 48 h of incubation with antisense oligonucleotides. The antisense elimination of moesin gene expression led to a significant reduction of lipopolysaccharide-induced TNF-alpha secretion. Restoration of moesin gene expression led to restoration of TNF-alpha production. These data suggest an important role for moesin in lipopolysaccharide-induced TNF-alpha production, highlighting its importance in lipopolysaccharide-mediated signal transduction.
膜突蛋白是一种78 kDa的蛋白质，在将细胞骨架与细胞膜连接的同时，还能控制细胞形状、黏附、运动和信号传导，具有多种功能。本研究的目的是通过共聚焦显微镜、流式细胞术、免疫沉淀和蛋白质印迹法来表征膜突蛋白在单核吞噬细胞中的表达和定位，并利用反义寡核苷酸方法敲除膜突蛋白基因，分析膜突蛋白作为脂多糖受体的功能。结果显示，膜突蛋白在单核细胞/巨噬细胞表面表达，脂多糖刺激后表面表达增加。脂多糖刺激后，单核细胞中膜突蛋白的总蛋白量增加。免疫沉淀显示，膜突蛋白与著名的脂多糖受体Toll样受体4（TLR4）共沉淀，表明膜突蛋白在脂多糖信号起始复合物形成中起早期作用。合成了两条反义寡核苷酸和两条对照正义寡核苷酸，每4小时向贴壁的巨噬细胞样细胞中导入一次，持续48小时。然后用脂多糖刺激细胞4小时，检测上清液中肿瘤坏死因子α（TNF-α）的产生。在48小时处理期结束后以及恢复116小时后，立即通过蛋白质印迹法检测细胞裂解物中膜突蛋白的表达，以评估膜突蛋白表达和功能的恢复情况。用反义寡核苷酸孵育48小时后，膜突蛋白基因表达被完全抑制。膜突蛋白基因表达的反义消除导致脂多糖诱导的TNF-α分泌显著减少。膜突蛋白基因表达的恢复导致TNF-α产生的恢复。这些数据表明膜突蛋白在脂多糖诱导的TNF-α产生中起重要作用，突出了其在脂多糖介导的信号转导中的重要性。
