Department of Rheumatology, Dublin Academic Medical Centre, Conway Institute of Biomolecular and Biomedical Research, University College Dublin, Ireland.
Ann Rheum Dis. 2011 Jul;70(7):1296-303. doi: 10.1136/ard.2010.142240. Epub 2011 Apr 10.
Serum amyloid A (A-SAA) is an acute-phase protein with cytokine-like properties implicated in the pathogenesis of rheumatoid arthritis (RA), atherosclerosis, diabetes and Alzheimer's disease. This study characterises the mechanism of A-SAA-induced cytoskeletal rearrangement and migration in synovial fibroblasts and microvascular endothelial cells (human dermal endothelial cells; HDEC).
Immunohistology and immunofluorescence were used to examine αvβ3 and β1-integrins, filamentous actin (F-actin) and focal adhesion expression in rheumatoid arthritis synovial tissue (RAST) and rheumatoid arthritis synovial fibroblast cells (RASFC). A-SAA-induced αvβ3 and β1-integrin binding was measured by adhesion assay. Cytoskeletal rearrangement and ρ-GTPase activation following A-SAA stimulation was examined using dual immunofluorescent staining for F-actin/vinculin staining, pull down assays and immunoblotting for Cdc42 and RhoA. Cell growth, invasion/migration, angiogenesis and actin formation were examined in the presence or absence of specific Rac1 and Cdc42 inhibitors (NSC23766 and 187-1).
αvβ3, β1-integrin and F-actin predominantly localised to vascular endothelium and lining layer cells in RAST, compared with osteoarthritis and normal control synovial tissue. A-SAA significantly increased αvβ3 and β1 binding in RASFC. A-SAA induced cytoskeletal disassembly, loss of focal adhesions and filopodia formation in RASFC and HDEC. A-SAA significantly induced Cdc42 activation but failed to promote RhoA activation in HDEC and synovial fibroblast cells. Blockade of Rac-1 and Cdc42 inhibited A-SAA-induced cell growth, invasion/migration, actin cytoskeletal rearrangement and angiogenesis.
These data show a novel mechanism for A-SAA-induced cell migrational events in RA mediated via cytoskeletal signalling pathways.
血清淀粉样蛋白 A（A-SAA）是一种急性期蛋白，具有细胞因子样特性，与类风湿关节炎（RA）、动脉粥样硬化、糖尿病和阿尔茨海默病的发病机制有关。本研究描述了 A-SAA 诱导滑膜成纤维细胞和微血管内皮细胞（人真皮内皮细胞；HDEC）细胞骨架重排和迁移的机制。
免疫组织化学和免疫荧光法检测 RA 滑膜组织（RAST）和 RA 滑膜成纤维细胞（RASFC）中αvβ3 和β1-整合素、丝状肌动蛋白（F-actin）和黏着斑的表达。通过黏附试验测定 A-SAA 诱导的αvβ3 和β1-整合素结合。用 F-actin/vinculin 双重免疫荧光染色、下拉试验和 Cdc42 和 RhoA 免疫印迹检测 A-SAA 刺激后细胞骨架重排和ρ-GTPase 激活。在存在或不存在特异性 Rac1 和 Cdc42 抑制剂（NSC23766 和 187-1）的情况下，检测细胞生长、侵袭/迁移、血管生成和肌动蛋白形成。
与骨关节炎和正常对照滑膜组织相比，αvβ3、β1 整合素和 F-actin 主要定位于 RAST 的血管内皮细胞和衬里层细胞。A-SAA 显著增加了 RASFC 中的αvβ3 和β1 结合。A-SAA 诱导 RASFC 和 HDEC 细胞骨架解体、黏附斑丢失和丝状伪足形成。A-SAA 显著诱导 Cdc42 激活，但未能促进 HDEC 和滑膜成纤维细胞中的 RhoA 激活。Rac-1 和 Cdc42 的阻断抑制了 A-SAA 诱导的细胞生长、侵袭/迁移、细胞骨架重排和血管生成。
这些数据显示了 A-SAA 诱导 RA 中细胞迁移事件的新机制，该机制通过细胞骨架信号通路介导。
