The Role Of Macrophage Migration Inhibitory Factor In Cell Proliferation Migration And Adhesion

The highly conserved and archetypical yet atypical cytokine macrophage migration inhibitory factor(MIF) fulfills pleiotropic immune functions in many acute and chronic inflammatory diseases.Recent evidence has emerged from both expression and functional studies to implicate MIF in various aspects of cardiovascular disease.MIF plays important role in the inflammatory pathogenesis of atherosclerosis and its consequences,namely unstable plaque formation, remodeling after arterial injury,aneurysm formation,myocardial infarction,or ischemia-reperfusion injury.The functional data are reconciled with recent progress in the identification of heptahelical(CXC chemokine) receptors for MIF,its prototypic role as their noncanonical ligand,and its signal transduction profile operative in atherogenic and inflammatory recruitment of mononuclear cells and in the oxidative damage and apoptosis of cardiomyocytes.Its unique features and functions clearly distinguish MIF from other cytokines implicated in atherogenesis and make it a prime target for achieving therapeutic regression of atherosclerosis.The potential of targeting or exploiting MIF for therapeutic strategies or as a diagnostic marker in the management of cardiovascular diseases or disorders is scrutinized.In this study,we found inactivation of MIF in HeLa cells resulted in suppression of migration ability.We further show that MIF is required for cells motility through activates Src and FAK signaling and provided a new target for improving the treatment efficiency of atherosclerosis through inactivation of the MIF gene.EXPERIMENTAL PROCEDURESVector construction.Among different MIF target sequences examined,the 19-nucleotide gene-specific sequence spanning from nucleotides 427 nt to 445 nt downstream of the gene transcription start site was selected to suppress MIF gene expression.After BLAST analysis,to ensure that there was no significant sequence homology with other human genes,the selected sequence was inserted into a Bglâ…¡/Hindâ…¢-cut pSuper puro vector to generate the pSuper-MIF vector.All constructs were confirmed by DNA sequence analysis.Cell culture,treatments,and transfections.HeLa cells were grown at 37℃in a 5%CO₂ atmosphere in Dulbecco's modified Eagle's medium(DMEM) supplemented with 10%FCS,2 mmol/L glutamine,and antibiotics.phoenix packaging cells and HeLa cells stably transfected with pSuper.puro and pSuper-MIF were maintained in DMEM supplemented with 10% FCS in the presence of puromycin(2 mg/ml).All transfections were done using LipofectAMINE 2000 reagent(Invitrogen) according to the manufacturer's protocols. Transfect phoenix packaging cells were done according to the manufacturer's protocols to produce ecotropic retroviral supernatants.48 hours posttransfection, select infected cells with puromycin(1-3μg/ml).After 20 days in selective medium, three pools,referred as phoenix-pSuper and phoenix- pSuper-MIF1,pSuper-MIF2, were isolated respectaly.Generation of stable MIF-silenced lines.To obtain HeLa cell lines stably expressing siRNAs,the ecotropic retroviral supernatants produced by phoenix packaging cells were collected and filter the tissue culture medium through a 0.45μm filter,and use the viral supernatant for infection of HeLa cell lines after addition of 4μg/ml polybrene.Infected HeLa cell lines for at least 6 hr and allow to recover for 24 hr with fresh medium.Select infected cells with puromycin(1-3μg/ml).After 15 days in selective medium,three pools,referred as HeLa-pSuper.puro,HeLa-pSuper-MIF1 SiRNAand HeLa-pSuper-MIF2 SiRNA were isolated.The HeLa- pSuper-MIF1 SiRNA was subjected to further cloning procedures by the limiting dilution method.The clones presenting a＞95%reduction in MIF protein levels compared with control.All stable cell lines obtained were cultured in DMEM supplemented with 2mg/ml puromycin.The puromycin selective pressure was removed 24 hours before experimental procedures.HeLa-pSuper.puro and HeLa-pSuper-MIF cells proliferation was measured by plating equal cell numbers onto six-well dishes in duplicate,followed by direct counting of trypsinized cells using a Coulter counter.Each growth curve represents at least two independent experiments.Cell Cycle Analysis HeLa-pSuper-MIF and HeLa-pSuper were treated with TNF-a and CHX10μg/ml for 12 hours.Trypsinized HeLa-pSuper.and HeLa-pSuper-MIF were washed with phosphate-buffered saline and fixed in 70%ethanol on ice for at least 24 h.The cells were washed with phosphate-buffered saline containing 1%FBS,resuspended in phosphate-buffered saline containing 1%FBS and 25μg/ml RNase A,and incubated for 30 min at 37℃.Prior to flow cytometry,100μl of propidium iodide solution(50μg/ml propidium iodide and 0.1%sodium citrate) was added to the cell suspension and incubated for 1 h on ice.The staining was assessed using the FACSC alibur flow cytometer and CellQuest software following calibration using DNA QCTM beads(BD Biosciences).Data were analyzed using ModFit software(BD Biosciences). SA-β-gal stainingCells were stained for SA-β-gal activity(Senescence-associatedβ-galactosidase) as described by Dimri.Briefly,1×10⁴ cells were seeded in 6-well plate for 24h and were washed twice with phosphate-buffered saline,fixed with 2%formaldehyde and 0.2%glutaraldehyde in PBS,and washed twice in PBS.Cells were stained at 37℃for 12h in X-gal staining solution(1mg/ml X-gal,40mmol/L citric acid/sodium phosphate(pH 6.0),5 mmol/L potassium ferrocyanide,150 mmol/L NaCl,2 mmol/L MgCl₂).Cells were examined under bright field illumination(Leica)Adhesion AssayCoat 96-well-plate with FN at 37℃for 1 hr Leave some wells uncoated as negative control.Wash with washing buffer for 2 times.Block plates with blocking buffer at 37℃in CO₂ incubator for 45-60 minutes.Wash with washing buffer.Chill the plates on ice.Count cell to 0.4×10⁶/ml.Add 50μl cells in each well. Incubate in CO₂ incubator at 37 C for 30 minutes.Shake the plate at 2000 rpm for 10-15 seconds.Wash with washing buffer 2-3 times.Fix with 4%paraformaldehyde. Incubate at RT for 10-15 minutes.Wash with washing buffer.Stain with Crystal Violet for 10 minutes.Wash with water.Turn the plates upside down.Let the plates dry up completely.Add 2%SDS.Incubate at RT for 30 min.Read plate at 570nm.Migration AssaysFor migration assays,modified Boyden chambers(Millicell-PCF,8-μm pore size;Millipore,Bedford,MA) were placed in a 24-well plate and coated with 10μg/ml rat tail collagen(Roche Applied Science) for 16 h at 37℃.After removal of collagen and washing with PBS,migration medium(DMEM with 0.5%bovine serum albumin)was added to lower chamber in 0.4 ml.HeLa pSuper or HeLa-pSuper-MIF RNAi cells were added to the upper compartment(0.2×10⁶ in 0.3 ml of migration medium).The plates were incubated at 37℃for 16 h for migration. The cells were removed from the upper membrane surface with a cotton tip applicator and washed with PBS,and cells on the lower membrane surface were fixed with 4% formaldehyde.The cells were then stained(0.1%Crystal Violet in 20%ethanol and enumerated by counting four(200x) fields/chamber).Wound closure assayA wound was induced on the confluent monolayer cells by scraping a gap using a micropipette tip and the speed of wound closure was monitored every 24 hours. Photographs were taken under 100×magnifications using phase-contrast microscopy immediately after wound incision and at later time points.Confocal microscopyCells grown on coverslips were fixed with 4%paraformaldehyde and permeabilized in 0.1%Triton X-100-PBS for 10 minutes.After blocking with 0.2% bovine serum albumin,cells were incubated with rabbit anti-p53 monoclonal antibody followed by decoration with fluorescein-conjugated anti-mouse IgG.Images were acquired on a Leica confocal microscope.Western blot analysisEqual amounts of protein(35μg/sample) from whole-cell extracts were separated by SDS-PAGE and blotted to PVDF membranes.After blocking with 5%skim milk solution,membranes were incubated with rabbit polyclonal anti-MIF, anti-cyclinD1,anti-Bcl-2,anti-Bax,anti-FAK,anti-p53,anti-p21,anti-ICAM-1, anti-Src,anti-AKT and anti-ERK1/2(Santa Cruz Biotechnology,Santa Cruz,USA) followed by decoration with peroxidase-labeled anti-rabbit or anti-mouse IgG, respectively(Super Signal detection kit,Pierce).StatisticsThe results are expressed as the means±S.E.The data were analyzed by one-way or two-way analysis of variance using SPSS17.0 statistical program.P values <0.05 were considered significant.RESULTSGrowth Properties of HeLa cells knockdown MIFTo test the hypothesis that MIF contributes in HeLa cell growth,we established stable knockdown of MIF cell line HeLa-pSuper-MIF using SiRNA technique, which displayed a significant decrease MIF at protein level compared with vector control.HeLa cells were derived from and assayed in several experimental settings. Under normal growth conditions,HeLa-pSuper-MIF proliferated slightly more slowly than the control HeLa-pSuper.Note morphologic changes induced by downregulation of MIF in HeLa cells.Silencing of MIF in HeLa Cells Results in the cell stay in G0/G1 phase and decrease the cyelin D1 expressionTo further confirm the MIF expression on HeLa cell growth,we then studied if downregulation of MIF could lead to any changes in the cell cycle distribution and the relative protein such as cyclin D1.Cell cycle analysis showed that silencing of MIF in HeLa cells results in the cell stay in G0/G1 phase and decrease the cyclin D1 expression.Down-regulation of MIF leads to suppression of p53 and p21As p53 play an important role in cell survivle,we then studied if there were any change in p53 and p21 expression,p53 and p21 were detectable in low level in HeLa-pSuper-MIF cell,whereas p53 protein level was high in HeLa-pSuper cell.In addition,we also used immunoflourecent staining to test p53 expression.These results show that MIF can affect the proappotosis gene p53.Silencing of MIF does not change the TNF-αinduced apoptosis pathwayHeLa-pSuper-MIF and HeLa-pSuper were treated with TNF-αand CHX 10μg/ml for 12 hours.We found that decreased Bax protein expression was found in the HeLa-pSuper-MIF.These results indicate that downregulation of MIF in HeLa cells has led to increased sensitivity to TNF-αthrough activation of induced apoptosis. Silencing of MIF in HeLa Cells Results in decrease cell senescenceAt the normal grown condition,silencing of MIF in HeLa cells results in decrease cell senescence,while the HeLa-pSuper cells showed a senescent phenotype that included flattening of cells and expression of senescence-associatedβ-galaetosidase(SA-βgal)MIF is required for cell migration potential of HeLa CellTo further study the association between MIF expression and metastatic ability of HeLa cells,we then tested the migration ability of the HeLa cells expressing.We analyzed the effect of MIF on the migratory capability of the HeLa cells by using several function assays.First,wound-healing assay showed that,24h after a wound was made on the monolayer of cells,the control cells extensively migrated into the denuded area,but this migratory capacity was significantly compromised by depletion of MIF in HeLa cells.Then,migration assay showed that compared with control cells,diminished MIF expression also reduced HeLa cell migration through a membrane by two-chamber assay.Overall,these data suggest that MIF expression is essential for the migratory behavior of HeLa cell.MIF-induced migration activity is associated with the activation of FAK signalingCell migration is a complex event that depends on the coordinated remodeling of the actin cytoskeleton as well as the regulated assembly and turnover of focal adhesions.The FAK acts as a protein-protein adapter critical to the formation of focal adhesions and mediates adhesion and growth factor-dependent signals into the cells. Tyrosine phosphorylation of FAK is one of the key signaling events during cell motility.Consistent with our observation above,HeLa cells with MIF knockdown showed decreased FAK expression.Consequently,Src,one of the upstream FAK, also showed reduced in MIF knockdown cells.These data suggest that MIF-induced migration activity is associated with the activation of FAK signaling through Src. Since ICAM-1 is the most important adhesion molecule involved in the extravasation of leukocytes into the surrounding tissue-it has been demonstrated to be both required as well as sufficient for the process of extravasation-most studies have been focused on the expression of ICAM-1.We also tested the expression of ICAM-1 in HeLa-pSuper-MIF and HeLa-pSuper.The data shows that compared with control cells,diminished MIF expression reduced ICAM-1,ERK1/2 and AKT in HeLa cells.Innovation of this study In this study,we used siRNA to knockdown MIF for evaluation of its function in HeLa cell line,which showed that MIF plays an important role in proliferation,migration and adhesion in vitro.We also investigated the potential molecular mechanism of the phenomenon.Together with prior studies of our group,these results strongly indicate that MIF may possibly promote the atherosclerotic lesions development through regulating the proteins that controlling cell adhesion,migration and proliferation.The present data suggest that MIF may represent an important new target for human atherosclerosis therapeutics.