巨噬细胞中脂多糖诱导的原肌球蛋白诱导激酶信号通路。
Moesin-induced signaling in response to lipopolysaccharide in macrophages.
机构信息
Department of Preventive Dental Science, Division of Orthodontics, Faculty of Dentistry, King Abdulaziz University, Jeddah, Saudi Arabia.
出版信息
J Periodontal Res. 2010 Oct;45(5):589-601. doi: 10.1111/j.1600-0765.2010.01271.x. Epub 2010 Jun 10.
BACKGROUND AND OBJECTIVE
Many physiological and pathophysiological conditions are attributable in part to cytoskeletal regulation of cellular responses to signals. Moesin (membrane-organizing extension spike protein), an ERM (ezrin, radixin and moesin) family member, is involved in lipopolysaccharide (LPS)-mediated events in mononuclear phagocytes; however, its role in signaling is not fully understood. The aim of this study was to investigate the LPS-induced moesin signaling pathways in macrophages.
MATERIAL AND METHODS
Macrophages were stimulated with 500 ng/mL LPS in macrophage serum-free medium. For blocking experiments, cells were pre-incubated with anti-moesin antibody. Moesin total protein and phosphorylation were studied with western blotting. Moesin mRNA was assessed using quantitative real-time PCR. To explore binding of moesin to LPS, native polyacrylamide gel electrophoresis (PAGE) gel shift assay was performed. Moesin immunoprecipitation with CD14, MD-2 and Toll-like receptor 4 (TLR4) and co-immunoprecipitation of MyD88-interleukin-1 receptor-associated kinase (IRAK) and IRAK-tumor necrosis factor receptor-activated factor 6 (TRAF6) were analyzed. Phosphorylation of IRAK and activities of MAPK, nuclear factor kappaB (NF-kappaB) and IkappaBalpha were studied. Tumor necrosis factor alpha, interleukin-1beta and interferon beta were measured by ELISA.
RESULTS
Moesin was identified as part of a protein cluster that facilitates LPS recognition and results in the expression of proinflammatory cytokines. Lipopolysaccharide stimulates moesin expression and phosphorylation by binding directly to the moesin carboxyl-terminus. Moesin is temporally associated with TLR4 and MD-2 after LPS stimulation, while CD14 is continuously bound to moesin. Lipopolysaccharide-induced signaling is transferred downstream to p38, p44/42 MAPK and NF-kappaB activation. Blockage of moesin function interrupts the LPS response through an inhibition of MyD88, IRAK and TRAF6, negatively affecting subsequent activation of the MAP kinases (p38 and ERK), NF-kappaB activation and translocation to the nucleus.
CONCLUSION
These results suggest an important role for moesin in the innate immune response and TLR4-mediated pattern recognition in periodontal disease.
背景与目的
许多生理和病理状况部分归因于细胞对信号的反应的细胞骨架调节。膜突蛋白（膜组织延伸刺突蛋白），ERM（埃兹蛋白、根蛋白和膜突蛋白）家族成员，参与单核吞噬细胞中的脂多糖（LPS）介导的事件；然而，其在信号转导中的作用尚不完全清楚。本研究旨在探讨巨噬细胞中 LPS 诱导的膜突蛋白信号通路。
材料与方法
用 500ng/ml LPS 在无巨噬细胞血清培养基中刺激巨噬细胞。为了阻断实验，细胞先用抗膜突蛋白抗体孵育。用 Western blot 研究膜突蛋白总蛋白和磷酸化。用定量实时 PCR 评估膜突蛋白 mRNA。为了探讨膜突蛋白与 LPS 的结合，进行了天然聚丙烯酰胺凝胶电泳（PAGE）凝胶迁移分析。用 CD14、MD-2 和 Toll 样受体 4（TLR4）进行膜突蛋白免疫沉淀，并用 MyD88-白细胞介素-1 受体相关激酶（IRAK）和 IRAK-肿瘤坏死因子受体激活因子 6（TRAF6）进行共免疫沉淀。研究 IRAK 的磷酸化以及 MAPK、核因子 kappaB（NF-kappaB）和 IkappaBalpha 的活性。通过 ELISA 测定肿瘤坏死因子 alpha、白细胞介素-1beta 和干扰素 beta。
结果
膜突蛋白被鉴定为促进 LPS 识别并导致促炎细胞因子表达的蛋白质簇的一部分。LPS 通过直接与膜突蛋白羧基末端结合刺激膜突蛋白的表达和磷酸化。LPS 刺激后，膜突蛋白与 TLR4 和 MD-2 暂时相关，而 CD14 则持续与膜突蛋白结合。LPS 诱导的信号传递到 p38、p44/42MAPK 和 NF-kappaB 的激活。阻断膜突蛋白功能通过抑制 MyD88、IRAK 和 TRAF6 阻断 LPS 反应，从而负性影响 MAP 激酶（p38 和 ERK）、NF-kappaB 激活和核转位的后续激活。
结论
这些结果表明膜突蛋白在牙周病中的固有免疫反应和 TLR4 介导的模式识别中具有重要作用。
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