Studys On Relevance And Mechanism Of Egr-1 And Osteopontin In Rat Vascular Smooth Muscle Cells

ObjectiveVascular remodeling diseases,such as atherosclerosis,restenosis(RS)following reconstructive vascular operation,and hypertension has become an increasingly serious threat to human health.Currently,many studies have demonstrated that migration and proliferation of the vascular smooth muscle cells(VSMCs)in the vascular tunica media is one pathological basis of vascular remodeling.Studies have found that some transcription factors can regulate the expressions of several VSMC proliferation-associated genes,and thereby regulate VSMC proliferation. At the same time,under the influence of various external stimuli,some extracellular matrix can be used as extracellular signal reaches the nucleus and then through an intracellular signal transduction system to induces the expression of a series of VSMC proliferation-associated transcription factors,and thereby promotes VSMC migration and proliferation.With careful attention to the reciprocal relationship between transcription factors and extracellular matrix,which conduce to finding out the mechanism underlying vascular remodeling.Early growth response factor-1(Egr-1),a zinc finger transcription factor,regulates the expression of multiple proliferation-associated genes,and its expression level is closely correlated with cell proliferation.Studies found that Egr-1 could mediate the migration and proliferation of VSMCs.Osteopontin(OPN)is a functionally important protein in the extracellular matrix(ECM),and anti-OPN antibodies can inhibit the phenotypic modulation,proliferation and migration of VSMCs.Although OPN and Egr-1 both play a significant role in mediating the process of migration and proliferation of the VSMCs,relatively little is known about the relationship between them.We used Egr-1 and OPN cDNA to transfect VSMCs and then detected the expression changes of Egr-1 and OPN and observed the relationship between Egr-1 and OPN before and after transfection.Meanwhile,we performed a chromatin immunoprecipitation(CHIP)assay to examine whether Egr-1 could bind to the OPN promoter,and In order to clarify the mechanism of OPN-mediated changes in Egr-1 expression,extracellular signal-regulated kinase(ERK)inhibitor was added to OPN-transfected cells.These results of the interaction between Egr-1 and OPN will likely provide an important theoretical and experimental basis needed to control the inappropriate remodeling of vessel walls.Materials and Methods1.Rat A10 aortic VSMCs were culturedRat A10 aortic VSMCs were purchased from ATCC.Cells were cultured in Dulbecco's Modified Eagle Medium(DMEM)with 10%fetal bovine serum(FBS)at 37℃in a humid atmosphere of 5%CO₂.2.Vectors and stable transfectionpCMV-Egr-1/NEO and pCMV(-)/NEO were kindly provided by Dr.Lorraine E. Chalifour,Division of Experimental Medicine,McGill University,Montreal,Canada. pET-28-rOPN/NEO and pET-28(-)/NEO were kindly provided by Dr.Harvey A Goldberg,University of Western Ontario,London,Canada.The plasmids were purified, amplified,identified and the ET-28-rOPN and ET-28(-)cassette were placed under control of a CMV promoter to drive OPN expression by TaKaRa.To establish stable cell lines,cells were cultured at 80%confluence in 6-well dishes and then transfected with either pCMV-Egr-1/NEO or pCMV(-)/NEO or pCMV-ET-28-rOPN/NEO or pCMV-ET-28(-)/NEO,which both express Geneticin 418(G418)resistance.All transfections were performed using FuGENE6 according to the manufacturer's instructions.One day later,cells were subcultured and grown in the presence of 400μg/ml G418.After 2 weeks,single G418-resistant colonies were obtained by serial dilution in 96-well dishes.Colonies were maintained in a medium containing 200μg/ml of G418 and analyzed individually for expression of Egr-1 or OPN.3.ERK inhibitor to inhibit the ERK pathwayCells expressing OPN were cultured at 80%confluence and then treated with 10μMPD98059,the ERK pathway inhibitor.24h after PD98059 treatment,we analyzed Egr-1 expression via Western blot.4.Reverse transcriptase polymerase chain reaction(RT-PCR)for Egr-1,OPNmRNATotal RNA was extracted from VSMCs using Trizol Reagent according to the manufacturer's instructions.RT-PCR analysis was performed using TaKaRa RNA PCR Kit(AMV)Ver.3.0 according to the manufacturer's instructions.Products were resolved by 1%agarose gel and bands visualized by ethidium bromide staining. Densitometric analysis of bands was performed using BioImaging Systems.5.Western blot analysis for Egr-1,OPN,ERK,P-ERK proteinProtein lysate(80μg)from cells was resolved by 12%sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE)and transferred to PVDF membranes. The filters were blocked with TBST buffer containing 5%skim milk,incubated with primary antibodies(anti-Egr-1(1:300),anti-OPN(1:500),anti-ERK(1:200), anti-P-ERK(1:200),andα-tubulin(1:500)overnight at 4℃Samples were then incubated with HRP-IgG secondary antibody and enhanced chemiluminescence(ECL) was used to visualize the bands.Band quantification was performed using Quantity One.Experiments were performed in triplicate.6.Chromatin immunoprecipitation(ChIP)analysis to observe the Egr-1 binds to the OPN gene promoter. ChIP assays were performed using the ChIP assay kit according to the manufacturer's instructions.The procedure included DNA-protein cross-linking inchromatin,shearing DNA into smaller fragments,immunoprecipitation with anti-Egr-1 antibody,and PCR identification of associated DNA sequences.Input was used as the positive control.7.Statistical analysesAll values were expressed as mean±SD.SPSS11.0 software was used for all statistical analysis.The relationship between Egr-1 and OPN was analyzed using bivariate correlation analytical method(Spearman's correlation analysis).Data were analyzed using one-way analysis of variance followed by a least significant difference test(LSD)for multiple comparisons.Differences were considered significant If p＜0.05.Results1.RT-PCR results showed that Egr-1 mRNA was more highly expressed in VSMCs which were stably transfected with Egr-1 than in control cells and vector-transfected cells(p＜0.01),similarly,OPN mRNA was more highly expressed in cells stably transfected with OPN cDNA than controls group and vector-transfected group(p＜0.01).2.We analyzed Egr-1 and OPN expression via RT-PCR and Western blot.The results showed that OPN mRNA and protein expression levels in the Egr-1-transfected VSMCs were significantly increased(p＜0.01),compared to untransfected cells and vector-transfected cells.Similarly,Egr-1 mRNA and protein levels were increased in the OPN-transfected VSMCs(p＜0.01).3.ChIP results showed that the OPN promoter sequence can be amplified in both control and Egr-1-transfected cells.4.Western blot results showed that levels of phospho-ERK were decreased relative to ERK in the PD98059 treated group compared to controls(p＜0.01). Furthermore,we found that Egr-1 expression was reduced in PD98059 treated cells compared to control-treated cells(p＜0.01).Conclusion1.Egr-1 cDNA or OPN cDNA was successfully transfected into VSMC_S.2.Egr-1 can affect the expression of OPN,Similarly,OPN can also affect the expression of Egr-1,and Egr-1 and OPN expression were positively correlated.3.Egr-1 can bind to the OPN promoter,and is likely to directly regulate its transcription.4.OPN acts through the ERK pathway to upregulate Egr-1.5.Egr-1 and OPN factors are likely to operate in a positive feedback loop in VSMC.