The dexamethasone-induced inhibition of proliferation, migration, and invasion in glioma cell lines is antagonized by macrophage migration inhibitory factor (MIF) and can be enhanced by specific MIF inhibitors.
ABSTRACT: Glioblastomas (GBMs) are the most frequent and malignant brain tumors in adults. Glucocorticoids (GCs) are routinely used in the treatment of GBMs for their capacity to reduce the tumor-associated edema. Few in vitro studies have suggested that GCs inhibit the migration and invasion of GBM cells through the induction of MAPK phosphatase 1 (MKP-1). Macrophage migration inhibitory factor (MIF), an endogenous GC antagonist is up-regulated in GBMs. Recently, MIF has been involved in tumor growth and migration/invasion and specific MIF inhibitors have been developed on their capacity to block its enzymatic tautomerase activity site. In this study, we characterized several glioma cell lines for their MIF production. U373 MG cells were selected for their very low endogenous levels of MIF. We showed that dexamethasone inhibits the migration and invasion of U373 MG cells, through a glucocorticoid receptor (GR)- dependent inhibition of the ERK1/2 MAPK pathway. Oppositely, we found that exogenous MIF increases U373 MG migration and invasion through the stimulation of the ERK1/2 MAP kinase pathway and that this activation is CD74 independent. Finally, we used the Hs 683 glioma cells that are resistant to GCs and produce high levels of endogenous MIF, and showed that the specific MIF inhibitor ISO-1 could restore dexamethasone sensitivity in these cells. Collectively, our results indicate an intricate pathway between MIF expression and GC resistance. They suggest that MIF inhibitors could increase the response of GBMs to corticotherapy.
Project description:Fibroblast-like synoviocytes mediate joint destruction in rheumatoid arthritis and exhibit sustained proinflammatory and invasive properties. CD44 is a polymorphic transmembrane protein with defined roles in matrix interaction and tumor invasion that is also a signaling coreceptor for macrophage migration inhibitory factor (MIF), which engages cell surface CD74. High-expression MIF alleles (rs5844572) are associated with rheumatoid joint erosion, but whether MIF signaling through the CD74/CD44 receptor complex promotes upstream autoimmune responses or contributes directly to synovial joint destruction is unknown. We report here the functional regulation of CD44 by an autocrine pathway in synovial fibroblasts that is driven by high-expression MIF alleles to up-regulate an inflammatory and invasive phenotype. MIF increases CD44 expression, promotes its recruitment into a functional signal transduction complex, and stimulates alternative exon splicing, leading to expression of the CD44v3-v6 isoforms associated with oncogenic invasion. CD44 recruitment into the MIF receptor complex, downstream MAPK and RhoA signaling, and invasive phenotype require MIF and CD74 and are reduced by MIF pathway antagonists. These data support a functional role for high-MIF expression alleles and the two-component CD74/CD44 MIF receptor in rheumatoid arthritis and suggest that pharmacologic inhibition of this pathway may offer a specific means to interfere with progressive joint destruction.
Project description:Macrophage migration Inhibitory factor (MIF) was one of the earliest pro-inflammatory cytokines to be identified. Increasing interest in this cytokine in recent decades has followed the cloning of human MIF and the generation of Mif(-/-) mice. Deepening understanding of signaling pathways utilized by MIF and putative receptor mechanisms have followed. MIF is distinct from all other cytokines by virtue of its unique induction by and counter regulation of glucocorticoids (GCs). MIF is further differentiated from other cytokines by its structural homology to specific tautomerase and isomerase enzymes and correlative in vitro enzymatic functions. The role of MIF in immune and inflammatory states, including a range of human autoimmune diseases, is now well established, as are the relationships between MIF polymorphisms and a number of inflammatory diseases. Here, we review the known pleiotropic activities of MIF, in addition to novel functions of MIF in processes including autophagy and autophagic cell death. In addition, recent developments in the understanding of the role of MIF in systemic lupus erythematosus (SLE) are reviewed. Finally, we discuss the potential application of anti-MIF strategies to treat human diseases such as SLE, which will require a comprehensive understanding of the unique and complex activities of this ubiquitously expressed cytokine.
Project description:<h4>Objective</h4>Glucocorticoids (GCs) are used as standard treatment for acute attacks of multiple sclerosis (MS). However, GCs eventually lose efficacy and do not prevent disease progression. Macrophage migration inhibitory factor (MIF) is the only known proinflammatory cytokine induced by GCs that inhibits their anti-inflammatory effects. Therefore, we investigated whether MIF plays a role in resistance to GC treatment in experimental autoimmune encephalomyelitis (EAE), an animal model of MS.<h4>Methods</h4>EAE was induced in wild-type (Wt) and MIF knockout (MIF(-/-)) mice followed by treatment with dexamethasone (Dex) before or upon disease onset. Splenocytes and brain mononuclear cells were harvested for cytokine ELISPOT assay and flow cytometry analysis.<h4>Results</h4>Treatment of EAE with Dex was substantially more efficacious in MIF(-/-) mice than Wt mice. Dex treatment decreased MOG35-55-induced cytokine production by Wt or MIF(-/-) CD4(+) T cells only at the onset of EAE but inhibited upregulation of T-bet during acute and chronic phases of disease, particularly in MIF(-/-) mice. Furthermore, passive EAE induced by adoptive transfer of T cells showed that Dex was highly effective in ameliorating disease induced by MIF(-/-) CD4(+) T cells but not by Wt CD4(+) T cells. The expression of T-bet and VLA-4 was decreased in CD4(+) T cells in MIF(-/-) mice compared with Wt mice.<h4>Conclusions</h4>Our data establish MIF as a key molecule in resistance of pathogenic CD4(+) T cells to GC treatment in EAE and as a potential target to enhance the effectiveness of steroid treatment in neuroinflammatory disorders.
Project description:Growth and invasion of metastatic colorectal cancer (CRC) cells in the liver depend on microenvironment. Here, we showed that human hepatic sinusoidal endothelial cells (HHSECs) induce chemotaxis and outgrowth of CRC cells. Macrophage migration inhibitory factor (MIF), released by HHSECs, stimulated chemotaxis of CRC cells. MIF secreted by HHSECs, but not by CRC cells themselves, promoted migration and epithelial-mesenchymal transition (EMT) and facilitated proliferation and apoptotic resistance of CRC cells. In orthotopic implantation models in nude mice, exogenous MIF stimulated growth of CRC cells and metastasis. Furthermore, MIF accelerated mobility of CRC cells by suppressing F-actin depolymerization and phosphorylating cofilin. Noteworthy, MIF levels were correlated with the size of hepatic metastases. We suggest that HHSECs and paracrine MIF promote initial migration and proliferation of CRC cells in the hepatic sinusoids to generate liver metastases.
Project description:c-Jun N-terminal kinase (JNK) is a member of the mitogen-activated protein kinase (MAPK) family and controls essential processes such as inflammation, cell differentiation, and apoptosis. JNK signalling is triggered by extracellular signals such as cytokines and environmental stresses. Macrophage migration inhibitory factor (MIF) is a pleiotropic pro-inflammatory cytokine with chemokine-like functions in leukocyte recruitment and atherosclerosis. MIF promotes MAPK signalling through ERK1/2, while it can either activate or inhibit JNK phosphorylation, depending on the cell type and underlying stimulation context. MIF activities are mediated by non-cognate interactions with the CXC chemokine receptors CXCR2 and CXCR4 or by ligation of CD74, which is the cell surface expressed form of the class II invariant chain. ERK1/2 signalling stimulated by MIF is dependent on CD74, but the receptor pathway involved in MIF activation of the JNK pathway is unknown. Here we comprehensively characterize the stimulatory effect of MIF on the canonical JNK/c-Jun/AP-1 pathway in fibroblasts and T cell lines and identify the upstream signalling components. Physiological concentrations of recombinant MIF triggered the phosphorylation of JNK and c-Jun and rapidly activated AP-1. In T cells, MIF-mediated activation of the JNK pathway led to upregulated gene expression of the inflammatory chemokine CXCL8. Activation of JNK signalling by MIF involved the upstream kinases PI3K and SRC and was found to be dependent on CXCR4 and CD74. Together, these data show that the CXCR4/CD74/SRC/PI3K axis mediates a rapid and transient activation of the JNK pathway as triggered by the inflammatory cytokine MIF in T cells and fibroblasts.
Project description:Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine. In addition to its known receptor-mediated biological activities, MIF possesses a catalytic site of unknown function between subunits of a homotrimer. Each subunit contributes three β-strands to adjacent subunits to form a core seven-stranded β-sheet for each monomer. MIF monomers, dimers, or trimers have been reported, but the active form that binds and activates the MIF receptor (CD74) is still a matter of debate. A cysteine mutant (N110C) that covalently locks MIF into a trimer by forming a disulfide with Cys-80 of an adjacent subunit is used to study this issue. Partial catalytic activity and receptor binding to CD74 are retained by N110C (locked trimer), but there is no cellular signaling. Wild-type MIF-induced cellular signaling, in vivo lung neutrophil accumulation, and alveolar permeability are inhibited with a fivefold excess of N110C. NMR and size-exclusion chromatography with light scattering reveal that N110C can form a higher-order oligomer in equilibrium with a single locked trimer. The X-ray structure confirms a local conformational change that disrupts the subunit interface and results in global changes responsible for the oligomeric form. The structure also confirms these changes are consistent for the partial catalytic and receptor binding activities. The absence of any potential monomer and the retention of partial catalytic and receptor binding activities despite changes in conformation (and dynamics) in the mutant support an endogenous MIF trimer that binds and activates CD74 at nanomolar concentrations. This conclusion has implications for therapeutic development.
Project description:D-dopachrome tautomerase (D-DT) is a newly described cytokine and a member of the macrophage migration inhibitory factor (MIF) protein superfamily. MIF is a broadly expressed pro-inflammatory cytokine that regulates both the innate and the adaptive immune response. MIF activates the MAP kinase cascade, modulates cell migration, and counter-acts the immunosuppressive effects of glucocorticoids. For many cell types, MIF also acts as an important survival or anti-apoptotic factor. Circulating MIF levels are elevated in the serum in different infectious and autoimmune diseases, and neutralization of the MIF protein via antibodies or small molecule antagonists improves the outcome in numerous animal models of human disease. Recently, a detailed investigation of the biological role of the closely homologous protein D-DT, which is encoded by a gene adjacent to MIF, revealed an overlapping functional spectrum with MIF. The D-DT protein also is present in most tissues and circulates in serum at similar concentrations as MIF. D-DT binds the MIF cell surface receptor complex, CD74/CD44, with high affinity and induces similar cell signaling and effector functions. Furthermore, an analysis of the signaling properties of the two proteins showed that they work cooperatively, and that neutralization of D-DT in vivo significantly decreases inflammation. In this review, we highlight the similarities and differences between MIF and D-DT, which we propose to designate "MIF-2", and discuss the implication of D-DT/MIF-2 expression for MIF-based therapies.
Project description:This study sought to investigate the biological effects of specific MIF inhibitor, ISO-1, on the proliferation, migration and invasion of PANC-1 human pancreatic cells in vitro, and on tumour growth in a xenograft tumour model in vivo. The effect of ISO-1 on PANC-1 cell proliferation was examined using CCK-8 cell proliferation assay. The effect of ISO-1 on collective cell migration and recolonization of PANC-1 cells was evaluated using the cell-wound closure migration assay. The effect of ISO-1 on the migration and invasion of individual PANC-1 cells in a 3-dimensional environment in response to a chemo-attractant was investigated through the use of Transwell migration/invasion assays. Quantitative real time PCR and western blot analyses were employed to investigate the effects of ISO-1 on MIF, NF-?B p65 and TNF-? mRNA and protein expression respectively. Finally, a xenograft tumor model in BALB/c nude mice were used to assess the in vivo effects of ISO-1 on PANC-1-induced tumor growth. We found high expression of MIF in pancreatic cancer tissues. We demonstrated that ISO-1 exerts anti-cancer effects on PANC-1 cell proliferation, migration and invasion in vitro, and inhibited PANC-1 cell-induced tumour growth in xenograft mice in vivo. Our data suggests that ISO-1 and its derivative may have potential therapeutic applications in pancreatic cancer.
Project description:Macrophage migration inhibitory factor (MIF) is an innate cytokine whose main actions include counter-regulating the immunosuppressive action of glucocorticoids and inhibiting activation-induced apoptosis. MIF is encoded in a functionally polymorphic locus and human genetic studies have shown significant relationships between high-expression MIF alleles, host inflammatory responses, and improved clinical outcome from infections. A recently completed candidate gene association study in the autoimmune disease systemic lupus erythematosus (SLE) indicates that individuals with a high-expression MIF allele have reduced incidence of SLE. Among patients with established disease however, those with end-organ complications have increased frequency of high-expression MIF alleles. Plasma MIF levels and Toll-like receptor (TLR) stimulated MIF production also reflect the underlying MIF genotype. These data suggest that MIF exerts a dual influence on the immunopathogenesis of SLE: high-expression MIF alleles are associated with a reduced susceptibility to SLE, perhaps by enhancing clearance of autoimmunogenic pathogens; once SLE develops however, low-expression MIF alleles protect from ensuing inflammatory end-organ damage. These data thus provide an example of the potential evolutionary advantage of maintaining an autoimmunity susceptibility gene in the population in that high-expression MIF alleles may allow for a maximal anti-infective response despite risk of autoimmunity. These results also support the clinical feasibility of pharmacologic MIF antagonism as such therapies may be most effectively applied in those individuals who, on the basis of their genotype, manifest a MIF dependent form of autoimmunity.
Project description:BACKGROUND: Macrophage migration inhibitory factor (MIF) is a pleiotropic cytokine and mediator of acute and chronic inflammatory diseases. MIF is overexpressed in various tumours and has been suggested as a molecular link between chronic inflammation and cancer. MIF overexpression is observed in breast cancer but its causal role in the development of this tumour entity is unclear. METHODS: MIF levels in breast cancer cell lines were determined by ELISA and Western blot. CD74 was measured by Western blot, fluorescence microscopy and flow cytometry. Cell proliferation was studied by BrdU incorporation, cell adhesion by Matrigel adhesion assay, and cell invasion by migration assay through Matrigel-coated filters using the Transwell system. MIF expression in primary human breast cancers was measured by tissue microarray and a semi-quantitative immunoreactivity score (IRS) and comparison with histopathological parameters and patient outcome data. RESULTS: MIF was abundantly expressed in the non-invasive breast cancer cell lines MDA-MB-468 and ZR-75-1, but not in invasive MDA-MB-231 cells, which in turn expressed higher levels of the MIF-receptor CD74. Stimulation with exogenous MIF led to a dramatic upregulation of MIF secretion (50-fold) in MDA-MB-231 cells. Autocrine MIF promoted tumour cell proliferation, as indicated by blockade of MIF or CD74 in MDA-MB-231 and MDA-MB-468, and MDA-MB-231 invasiveness was enhanced by exogenous MIF. We correlated the expression of MIF with histopathological parameters and patient outcome data, using a tissue microarray of 175 primary invasive breast cancers and 35 normal control tissues. MIF was upregulated in breast cancer versus normal tissue (median IRS = 8 versus 6). MIF expression showed positive correlations with progesterone (p = 0.006) and estrogen (p = 0.028) receptor expression, markers of a favourable prognosis and a negative correlation to tumour size (p = 0.007). In line with these data, disease-specific overall (OS) as well as recurrence-free (RFS) survival was significantly improved in breast cancer patients with abundant cytosolic MIF expression compared to MIF low expressers (5-year OS = 67% versus 50%, p = 0.0019; 5-year RFS = 52% versus 36%, p = 0.0327). CONCLUSION: We conclude that intracellular expression of MIF in breast cancer cells is beneficial, whereas extracellular MIF may play a pro-oncogenic role in promoting breast cancer cell-stroma interactions.