Macrophage migration inhibitory factor (MIF) but not its homologue D-dopachrome tautomerase (D-DT) promotes fibroblast motility in a CD44/CD74-independent manner
Macrophage migration inhibitory factor (MIF) and D-dopachrome tautomerase (D-DT) are paralogous pro-inflammatory cytokines. Whereas cytosolic MIF plays a role in cell cycle progression, secreted MIF and D-DT activate second messenger signalling through a CD44/CD74 receptor complex. Using an interactome screen, cytosolic MIF/D-DT were found to interact with many actin cytoskeleton-associated proteins in NIH3T3 fibroblasts suggesting a direct involvement of MIF/D-DT in modulating cell motility, but whether the receptors are indispensable for mediating motility remains elusive. To differentiate between receptor- and non-receptor-mediated events, COS-7/M6 fibroblasts deficient in CD44/CD74 (WT) and stable cell lines expressing CD44 and CD74 were generated. Chemokinesis (random single-cell motility) was assessed in cells stimulated with recombinant MIF/D-DT, mutated forms of MIF, inhibitors of MIF s tautomerase activity, and inhibitors of clathrin- and non-clathrin-dependent endocytosis. Due to low D-DT s tautomerase activity, ISO-1/4-IPP were not tested with D-DT. In the presence of CD44/CD74, both MIF and D-DT stimulated chemokinesis but only MIF enhanced chemokinesis in the absence of receptors. The stimulatory effect of MIF on migration depended on its tautomerase activity and lipid raft/caveolae-mediated but not clathrin-mediated endocytosis. To observe a direct effect of MIF and D-DT on actin dynamics, actin polymerization was measured in an in vitro actin assembly assay. MIF but not D-DT decreased the rate of F-actin assembly. By decreasing the rate of cell extract-driven actin assembly, MIF phenocopies the function of F-actin capping proteins. Moreover, stimulation with MIF increased the number of prominent F-actin stress fibres in COS-7/M6 WT. Taken together, MIF stimulates fibroblast chemokinesis independent of the status of its cell-surface receptors, likely by directly modulating the actin cytoskeleton. MIF-mediated chemokinesis appears to depend on lipid raft/caveolae-mediated endocytosis. D-DT triggers chemokinesis only in the presence of MIF/D-DT receptors CD44/CD74 implying D-DT requires the classical receptor-driven second-messenger transduction pathway.
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