Study On Function And Signal Transduction Of Macrophage Migration Inhibitory Factor

Macrophage migration inhibitory factor (MIF) is a pluripotent cytokine involved in inflammation and immune responses as well as in growth factor-dependent cell proliferation, cell cycle, angiogenesis, and tumorigenesis.MIF originally described as a T-lymphocyte product in 1966, has been well described as a potent pro-inflammatory mediator. As an important modulator of both T cell and macrophage , MIF has emerged as an important regulator of innate and adaptive immunity.In addtion , MIF is induced by glucocorticoids, and it counter-regulates the effects of glucocorticoids on inflammation and immunity, emerging as a key integrator of the immunoneuroendocrine interface. MIF is an upstream activator of monocytes/macrophages, and it is centrally involved in the pathogenesis of septic shock, arthritis, and other inflammatory diseases. It attracted more and more attention as therapeutic target. Although recent work has begun to illuminate the signal transduction pathways activated by MIF, but until now it's not well understood how MIF exerts its inflammatory effect on target cells .There are two distinct hypothetic pathways for MIF function: receptor-mediated signaling by MIF and signaling through JAB1/CSN5 following endocytosis of MIF. But which pathway is more important, and when does MIF work through one of the pathways are still unknown. We try to dissect the signal pathway systematically, which may help us strengthening our acknowledge of basic mechanism of immune system regulation, and may offer some clues to new therapeutic strategies targeting immune diseases.First we expressed and purified recombinant human MIF protein from E.coli. Using the recombinant MIF to stimulate RAW264.7, we established a serial of systems to detect our rhuMIF' function, e.g. NO production, TNF-a secretion, COX-2 induction, apoptosis and cell cycle changes. Also using cytosol-nuclear isolation and western blot, we can detect changes in cell-cycle related factors' nuclear localization.On the bases of the above functional assays, we studied mif s signal transduction pathways through special endocytosis and enzyme inhibitors. Our result suggested that production of NO was independent of endocytosis, but might be dependent on membrane receptor-mediated signaling. MIF could cause Gl arrest at high concentration. Following endocytosis, MIF could bind to JUN-activation domain binding protein 1 (Jabl), and then inhibit nuclear export of Jab 1, preventing Jabl induced degradation of the cell-proliferation inhibitor KIP1, thereby leading to cell-cycle regulation. MIF promotes cell growth, through PKC-, MAPK-, c-jun-AP-1-dependent and endocytosis-independent process, MIF could induce the generation of cyclooxygenase 2 (COX-2), and prevent activation-induced apoptosis mediated by the oxidative burst and by p53.