Achuthan Adrian, Elsegood Caryn, Masendycz Paul, Hamilton John A, Scholz Glen M
Arthritis and Inflammation Research Centre and Cooperative Research Centre for Chronic Inflammatory Diseases, Department of Medicine, Royal Melbourne Hospital, University of Melbourne, Victoria 3050, Australia.
Cell Signal. 2006 Dec;18(12):2252-61. doi: 10.1016/j.cellsig.2006.05.007. Epub 2006 May 23.
Macrophages are an important component of the innate immune response to infection by microbial pathogens. The activation of macrophages by pathogens is largely mediated by Toll-like receptors (TLRs). Bacterial DNA, which contains unmethylated CpG dinucleotide motifs, is specifically recognised by TLR9 and triggers the activation of a complex network of intracellular signalling pathways that orchestrates the ensuing inflammatory responses of macrophages to the pathogen. Here, we have established that CpG DNA promotes reorganisation of the actin cytoskeleton and enhances cell spreading by primary mouse bone marrow macrophages. CpG DNA stimulation resulted in an approximately 70% increase in cell size. Notably, CpG DNA-induced cell spreading was dependent on the activity of Src-family kinases. Tyrosine phosphorylation of several proteins was increased in a Src-family kinase-dependent manner following CpG DNA stimulation of bone marrow macrophages, including the cytoskeletal protein paxillin. Paxillin was phosphorylated both in vitro and in vivo by the Src-family kinase Hck. Significantly, paxillin from CpG DNA-stimulated bone marrow macrophages had a greater capacity to bind the SH2 domain of the adapter protein Crk than did paxillin from unstimulated bone marrow macrophages. Furthermore, phosphorylation of paxillin by Hck created a binding site for Crk. We propose that the formation of paxillin-Crk complexes may mediate the cytoskeletal changes that underlie the increased cell spreading of macrophages following their activation by CpG DNA.
巨噬细胞是对微生物病原体感染的固有免疫反应的重要组成部分。病原体对巨噬细胞的激活很大程度上由Toll样受体（TLR）介导。含有未甲基化CpG二核苷酸基序的细菌DNA被TLR9特异性识别，并触发细胞内信号通路复杂网络的激活，该网络协调巨噬细胞对病原体随后的炎症反应。在这里，我们已经确定CpG DNA促进原代小鼠骨髓巨噬细胞的肌动蛋白细胞骨架重组并增强细胞铺展。CpG DNA刺激导致细胞大小增加约70%。值得注意的是，CpG DNA诱导的细胞铺展依赖于Src家族激酶的活性。在骨髓巨噬细胞受到CpG DNA刺激后，几种蛋白质的酪氨酸磷酸化以Src家族激酶依赖的方式增加，包括细胞骨架蛋白桩蛋白。桩蛋白在体外和体内均被Src家族激酶Hck磷酸化。重要的是，与未受刺激的骨髓巨噬细胞中的桩蛋白相比，来自CpG DNA刺激的骨髓巨噬细胞的桩蛋白与衔接蛋白Crk的SH2结构域结合的能力更强。此外，Hck对桩蛋白的磷酸化产生了一个Crk的结合位点。我们提出，桩蛋白-Crk复合物的形成可能介导了巨噬细胞在被CpG DNA激活后细胞铺展增加所依赖的细胞骨架变化。
