Departamento de Análises Clínicas e Toxicológicas, Faculdade de Farmácia, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais 31270-901, Brazil; Programa de Pós-Graduação em Biologia Celular, Departamento de Morfologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais 31270-901, Brazil; Laboratório de Imunofarmacologia, Departamento de Bioquímica e Imunologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais 31270-901, Brazil;
Departamento de Análises Clínicas e Toxicológicas, Faculdade de Farmácia, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais 31270-901, Brazil; Laboratório de Imunofarmacologia, Departamento de Bioquímica e Imunologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais 31270-901, Brazil;
J Immunol. 2014 Oct 1;193(7):3654-63. doi: 10.4049/jimmunol.1400334. Epub 2014 Aug 27.
The plasminogen (Plg)/plasmin (Pla) system is associated with a variety of biological activities beyond the classical dissolution of fibrin clots, including cell migration, tissue repair, and inflammation. Although the capacity of Plg/Pla to induce cell migration is well defined, the mechanism underlying this process in vivo is elusive. In this study, we show that Pla induces in vitro migration of murine fibroblasts and macrophages (RAW 264.7) dependent on the MEK/ERK pathway and by requiring its proteolytic activity and lysine binding sites. Plasmin injection into the pleural cavity of BALB/c mice induced a time-dependent influx of mononuclear cells that was associated with augmented ERK1/2 and IκB-α phosphorylation and increased levels of CCL2 and IL-6 in pleural exudates. The inhibition of protease activity by using a serine protease inhibitor leupeptin or two structurally different protease-activated receptor-1 antagonists (SCH79797 and RWJ56110) abolished Pla-induced mononuclear recruitment and ERK1/2 and IκB-α phosphorylation. Interestingly, inhibition of the MEK/ERK pathway abolished Pla-induced CCL2 upregulation and mononuclear cell influx. In agreement with a requirement for the CCL2/CCR2 axis to Pla-induced cell migration, the use of a CCR2 antagonist (RS504393) prevented the Plg/Pla-induced recruitment of mononuclear cells to the pleural cavity and migration of macrophages at transwell plates. Therefore, Pla-induced mononuclear cell recruitment in vivo was dependent on protease-activated receptor-1 activation of the MEK/ERK/NF-κB pathway, which led to the release of CCL2 and activation of CCR2.
纤溶酶原（Plg）/纤溶酶（Pla）系统与多种生物学活性相关，不仅限于经典的纤维蛋白凝块溶解，还包括细胞迁移、组织修复和炎症。尽管 Plg/Pla 诱导细胞迁移的能力已得到充分证实，但该过程在体内的机制仍难以捉摸。在本研究中，我们表明 Pla 依赖于 MEK/ERK 途径并通过需要其蛋白水解活性和赖氨酸结合位点诱导体外培养的鼠成纤维细胞和巨噬细胞（RAW 264.7）迁移。纤溶酶注射到 BALB/c 小鼠的胸腔中会引起单核细胞的时间依赖性流入，这与 ERK1/2 和 IκB-α 的磷酸化增加以及胸腔渗出物中 CCL2 和 IL-6 水平的增加有关。使用丝氨酸蛋白酶抑制剂亮抑蛋白酶或两种结构不同的蛋白酶激活受体-1 拮抗剂（SCH79797 和 RWJ56110）抑制蛋白酶活性，可消除 Pla 诱导的单核细胞募集以及 ERK1/2 和 IκB-α 的磷酸化。有趣的是，抑制 MEK/ERK 途径可消除 Pla 诱导的 CCL2 上调和单核细胞流入。与 Pla 诱导细胞迁移需要 CCL2/CCR2 轴相一致，使用 CCR2 拮抗剂（RS504393）可防止 Plg/Pla 诱导的单核细胞募集到胸腔和巨噬细胞在 Transwell 小室中的迁移。因此，体内 Pla 诱导的单核细胞募集依赖于蛋白酶激活受体-1 激活 MEK/ERK/NF-κB 途径，从而导致 CCL2 的释放和 CCR2 的激活。
