Macrophage migration inhibitory factor (MIF) and its homologue D-dopachrome tautomerase (D-DT) are widely expressed pro-inflammatory cytokines with chemokine-like functions that coordinate a wide spectrum of biological activities, such as migration. Here, we biotin-tagged intracellular MIF/D-DT in vivo to identify important cytosolic interactors and found a plethora of actin cytoskeleton-associated proteins. Although the receptor complex between CD74 and CD44 (CD74/CD44) is essential for signalling transduction in fibroblasts via extracellular MIF/D-DT, our interactome data suggested direct effects. We, thus, investigated whether MIF/D-DT can modulate cell migration independently of CD74/CD44. To distinguish between receptor- and non-receptor-mediated motility, we used fibroblasts that are either deficient or that express CD74/CD44 proteins, and treated them with recombinant MIF/D-DT. Interestingly, only MIF could stimulate chemokinesis in the presence or absence of CD74/CD44. The pro-migratory effects of MIF depended on lipid raft/caveolae-mediated but not clathrin-mediated endocytosis, on its tautomerase activity and, probably, on its thiol protein oxidoreductase activity. As MIF treatment restrained actin polymerisation in vitro, our findings establish a new intracellular role for MIF/D-DT in driving cell motility through modulation of the actin cytoskeleton.

巨噬细胞迁移抑制因子 (MIF) 及其同系物 D-多巴色素互变异构酶 (D-DT) 是广泛表达的促炎细胞因子，具有协调广泛的生物活性（如迁移）的趋化因子样功能。在这里，我们在体内用生物素标记细胞内 MIF/D-DT以确定重要的细胞溶质相互作用物，并发现了大量的肌动蛋白细胞骨架相关蛋白。尽管 CD74 和 CD44 (CD74/CD44) 之间的受体复合物对于通过细胞外 MIF/D-DT 在成纤维细胞中进行信号转导至关重要，但我们的相互作用组数据表明了直接影响。因此，我们研究了 MIF/D-DT 是否可以独立于 CD74/CD44 调节细胞迁移。为了区分受体介导和非受体介导的运动，我们使用了缺乏或表达 CD74/CD44 蛋白的成纤维细胞，并用重组 MIF/D-DT 处理它们。有趣的是，在存在或不存在 CD74/CD44 的情况下，只有 MIF 可以刺激趋化作用。MIF 的促迁移作用取决于脂筏/小窝介导的而非网格蛋白介导的内吞作用，取决于其互变异构酶活性，并且可能，其硫醇蛋白氧化还原酶活性。由于 MIF 处理抑制了肌动蛋白聚合在体外，我们的研究结果确立了 MIF/D-DT 通过调节肌动蛋白细胞骨架在驱动细胞运动中的新细胞内作用。