Arcand Julie, Robitaille Geneviève, Koenig Martial, Senécal Jean-Luc, Raymond Yves
Laboratory for Research in Autoimmunity, Notre-Dame Hospital, Centre Hospitalier de l'Université de Montréal, and Université de Montréal, Montréal, Québec, Canada.
Arthritis Rheum. 2012 Mar;64(3):826-34. doi: 10.1002/art.33377.
Previous studies have demonstrated that, once released into the extracellular environment, the systemic sclerosis (SSc)-associated autoantigen DNA topoisomerase I (topo I) binds specifically to the surface of fibroblasts via an unknown receptor. We extended these results by identifying topo I-mediated cellular effects and characterizing the specific target of topo I on fibroblast surfaces.
Purified topo I was used to investigate intracellular signaling pathway activation and tested for cell migration. To demonstrate the expression of specific chemokine receptors on fibroblasts, we performed immunoblotting and flow cytometry. To evaluate the direct interaction between chemokine receptor and topo I, a protein-protein based enzyme-linked immunosorbent assay (ELISA) was used. Finally, topo I coupled to the fluorochrome phycoerythrin (PE) was used to investigate competition of topo I specific binding on fibroblast surfaces with chemokine ligand.
Topo I stimulated the phosphorylation of phospholipase Cγ1, c-Raf, ERK-1/2, and p38 MAPK, intracellular signaling pathways that stimulated fibroblast migration via a G(αi) protein-coupled receptor. CCR7 was found to interact directly with topo I. Furthermore, its ligand, CCL21, competed in vitro for this interaction and in vivo with the binding of PE-coupled topo I to fibroblast surfaces.
These new roles of topo I in fibroblast physiology and the identification of its target on the cell surface demonstrate that topo I is a bifunctional autoantigen and open up new perspectives of study in the field of SSc-associated anti-topo I autoantibodies.
以往研究表明，系统性硬化症（SSc）相关自身抗原DNA拓扑异构酶I（拓扑异构酶I）一旦释放到细胞外环境中，会通过一种未知受体特异性结合成纤维细胞表面。我们通过确定拓扑异构酶I介导的细胞效应并表征拓扑异构酶I在成纤维细胞表面的特定靶点，扩展了这些研究结果。
使用纯化的拓扑异构酶I研究细胞内信号通路激活情况，并检测细胞迁移。为了证明成纤维细胞上特定趋化因子受体的表达，我们进行了免疫印迹和流式细胞术检测。为了评估趋化因子受体与拓扑异构酶I之间的直接相互作用，使用了基于蛋白质-蛋白质的酶联免疫吸附测定（ELISA）。最后，使用与荧光染料藻红蛋白（PE）偶联的拓扑异构酶I来研究趋化因子配体对拓扑异构酶I在成纤维细胞表面特异性结合的竞争作用。
拓扑异构酶I刺激了磷脂酶Cγ1、c-Raf、ERK-1/2和p38丝裂原活化蛋白激酶（MAPK）的磷酸化，这些细胞内信号通路通过G（αi）蛋白偶联受体刺激成纤维细胞迁移。发现CCR7与拓扑异构酶I直接相互作用。此外，其配体CCL21在体外竞争这种相互作用，并在体内竞争PE偶联的拓扑异构酶I与成纤维细胞表面的结合。
拓扑异构酶I在成纤维细胞生理学中的这些新作用及其在细胞表面靶点的鉴定表明，拓扑异构酶I是一种双功能自身抗原，并为SSc相关抗拓扑异构酶I自身抗体领域的研究开辟了新的视角。
