The Effect Of Rho/MRTF-A Signaling Pathway On The Migration Of Mesenchymal Stem Cells Induced By TNF-α And IL-1β

Mesenchymal stem cells can not only differentiate into multiple cells, but also targetly migrate to the sites of injury, chronic inflammation, and turner, repairing these parts in a certain mechanism. Having a comprehensive knowledge of migration mechanism of mesenchymal stem cells and improving the "homing" index are helpful for autologous repair and cell therapy.It is reported that inflammatory cytokines TNF-α and IL-1β can induce the migration of mesenchymal stem cells, but the details of migration mechanism remains unclear.In this study, migration modles of MSCs were established by treatment with 50ng/mL TNF-α and 50ng/mL IL-1β.After 72 hours, wound heal assay and transwell assay showed that TNF-α and IL-1β could induce the migration of mesenchymal stem cells. Then, migration markers MYL9 and CYR61 were tested by RT-PCR and Western Blot. The results showed that the expression of both MYL9 and CYR61 were increased. Next, the levels of MMP2, MMP9, TIMP1 and TIMP2 were detected by RT-PCR. The results showed that the expression of MMP9 was significantly upregulated.Rho/MRTF-A signal pathway is widely involved in cell migration. To determine whether Rho signaling pathway participated in the migration of mesenchymal stem cells induced by TNF-α and IL-1β, mesenchymal stem cells were pertreated with 10μmol/L the inhibitor of Rho signaling pathway-Y27632 for 0.5 hour before treated with TNF-α and IL-1β. Wound heal assay and transwell assay both showed that Y27632 could reduced the migration of mesenchymal stem cells caused by TNF-α and IL-1β. RT-PCR and Western Blot showed that Y27632 downregulated the expression of MYL9 and CYR61 in both mRNA levels and protein levels.It is reported that the activation of Rho signal pathway can promote MRTF-A to activate target genes. To further confirm whether MRTF-A can regulate expression of MYL9 and CYR61 in mesechymal stem cells, MSCs were transfected with MRTF-A and shRNA then the expression of MYL9 and CYR61 were detected by Western Blot. The results showed that the overexpression of MRTF-A could upregulate the expression of MYL9 and CYR61, while knockdown of MRTF-A could downregulate the expression of MYL9 and CYR61. Moreover, luciferase report assay showed MRTF-A could enhance transcriptional activity of MYL9 and CYR61 promoters in mouse MSC line C3H10T1/2 and model cell line COS7, and this action was dependented on CArG box in promoter of MYL9 and CYR61.