Regulation of the macrophage migration inhibitory factor gene expression by specific transcription factors in the cellular infection model

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Clinically, the severe sepsis or septic shock in patients was caused by the immune imbalance by endotoxin, which induces a large number of cytokine release in the early infection, resulting in the subsequent multiple organ damage, failure and death. The macrophage migration inhibitory factor (MIF) gene plays an important role in regulating the cytokine release in the early stage of infection. The regulation of MIF gene expression and the importance of transcription factors on its promoter in the bacterial infection model is not clear. Therefore, we focused on the promoter activity of MIF gene and the role of different transcription factors in the cellular infection model in this study. We first constructed different lengths of MIF gene’s promoter fragments into PGL4.10 vectors to investigate the MIF gene promoter activities. Then, we used daul-luciferase assaies and electrophoretic mobility shift assaies (EMSA) to investigate the effects of the putative transcription factor binding sites on MIF gene expression. Treatment of lipopolysaccharide (LPS) induced the promoter activity and dexamethasone (Dex) reversed this effect. We also performed the site-directed mutagenesis of the predicted binding sites for the transcription factors on the MIF promoter and promoter studies to investigate the importance of these sites. Mutating the binding site of hypoxia‐inducible factor 1 alpha (HIF-1α) on MIF promoter, the promoter activities were significantly decreased and LPS-induced effects were also inhibited. Mutations of the cAMP-response element (CRE) and specific protein 1 (Sp1) binding sites deacreased the MIF promoter activities, whereas, the effects of LPS and Dex were not altered. Mutating the predicted GR binding site at -540 (GR540) also significantly inhibited the promoter activities of MIF gene and LPS effects. However, it did not showed any shifted bands in the EMSA, suggesting that glucocorticoid receptor regulates the MIF promoter activity induced by LPS may be through an indirect pathway, such as the glucocorticoid-induced leucine zipper (GILZ) pathway, rather than bind to the promoter region directly. These findings indicate that the transcription factor binding sites of GR540 and HIF-1α may play important roles in the basal MIF gene promoter activity, as well as the effects of LPS and Dex treatments. The results of the study help us further understand the transcriptional mechanisms of MIF by GRs.