Signaling Mechanism Of Rho A Regulates Epidermal Growth Factor-induced Human Osteosarcoma MG63 Cell Migration

Osteosarcoma,the most common primary bone tumor,occurs most frequently in children and adolescents and has a 5-year survival rate that remains unsatisfactory.It is well known that epidermal growth factor receptor(EGFR)positively correlates with osteosarcoma TNM stage,which suggests that EGFR plays an important role in osteosarcoma progression.The purpose of this study was to explore the potential mechanisms underlying this correlation.To this end,we found that EGF promotes MG63 cell migration and invasion as well as stress fiber formation via Rho A activation and that these effects can be reversed by inhibiting Rho A expression.In addition,molecules that are downstream of Rho A,including ROCK,LIMK2,and Cofilin,are also activated by EGF in MG63 cells,leading to actin stress fiber formation and cell migration.The inhibition of ROCK,LIMK2,or Cofilin in MG63 cells using known inhibitors or shRNA prevents actin stress fiber formation and cell migration.Thus,we conclude that the signaling pathway Rho A/ROCK/LIMK2/Cofilin mediates actin microfilament formation in MG63 cells upon EGFR activation.This novel signaling pathway provides a promising target for preventing osteosarcoma progression and for treating osteosarcoma.1.EGF activates EGFR expression in MG63 cells and promotes cell migration by increasing actin stress fiber formation(1)BackgroundThe epidermal growth factor receptor(EGFR)family of receptor tyrosine kinases lies at the head of a complex signal transduction cascade that modulates cell proliferation,survival,adhesion,migration and differentiation.While growth-factor-induced EGFR signaling is essential for many normal morphogenic processes and involved in numerous additional cellular responses,the aberrant activity of members of this receptor family has been shown to play a key role in the development and growth of tumor cells.(2)ObjectiveThe purpose of this section is to explore whether EGF can activates EGFR expression in MG63 cells,and the effects of EGF treatments on actin stress fiber formation.(3)Methods and ResultsThe EGFR phosphorylation increased significantly after 2 hours of treatment with EGF and reached a peak at 4 hours after which EGFR phosphorylation decreased.Ten ng/ml EGF induced significantly increased invasion compared to the control group(p<0.05).In addition,the wound scratch assay showed that the migration of MG63 cells was dramatically increased in the 10 ng/ml EGF-treated group compared to the control group at 12,24,36,and 48 hours(p<0.05).Furthermore,the scratch was completely filled with migrated MG63 cells at 48 hours.Actin stress fibers have a fundamental role in providing force for several vital cellular processes,such as migration,cytokinesis,and morphogenesis.Therefore,we tested the formation and distribution of actin stress fibers in MG63 cells that were treated with EGF.From 12 to 18 h,the distribution and formation of actin stress fibers reached a peak around the nucleus in MG63 cells.(4)ConclusionEGF activates EGFR expression in MG63 cells,and it is able to promote cell migration by increasing actin stress fiber formation.2.Rho A is involved in the EGF-induced migration of MG63 cells(1)BackgroundRhoA is the most extensively studied member of the Rho GTPase family which belongs to the Ras super family of small G proteins.It plays an important role in signal transduction and has been reported to regulate many biological activities including the formation of stress fibers(1),gene transcription(2),membrane transport and focal adhesions(3)and tumor progression(4).(2)ObjectiveThe purpose of this section is to make sure whether Rho A is involved in the EGF-induced migration of MG63 cells.(3)Methods and ResultsTo determine the role of Rho A in MG63 cell migration,a pull-down assay was performed.We found that GTP-bound Rho A increased significantly 6 to 12 h after treatment with EGF.To further explore the role of Rho A in the EGF-induced migration of MG63 cells,Rho A shRNA interference was explored.The efficiency of Rho A shRNA was approximately 80%.The western results showed that the expression of Rho A and its downstream molecule ROCK was significantly reduced compared to the mCherry and control groups in MG63 cells that were treated with Rho A shRNA.Next,we conducted a transwell assay and found that Rho A shRNA significantly reduced MG63 cell migration compared to the control and mCherry groups.Moreover,the reorganization of actin stress fibers was also inhibited in the Rho A shRNA-treated group.To further explore the role of Rho A in MG63 cell migration,Rho inhibitor exoenzyme C3 was employed to specifically inhibit the expression of Rho A and its downstream molecule ROCK.The phosphorylation of Rho A and ROCK was inhibited by exoenzyme C3 in a concentration-dependent manner.Previously,we demonstrated that EGF increases actin stress fiber formation,while exoenzyme C3 treatment decreases it.The wound scratch assay showed that MG63 cells treated with EGF gradually migrated to the scratch and completely filled the scratch after 48 hours.By comparison,MG63 cells treated with EGF plus exoenzyme C3 showed reduced migration compared to the EGF-alone-treated group at 24,36 and 48 hours.(4)ConclusionRho A is involved in the EGF-induced migration of MG63 cells,and the inhibition of RhoA is able to inhibit the formation of stress fibers and migration of MG63 cells.3.ROCK promotes MG63 cell migration and stress fiber reorganization(1)BackgroundRho-associated protein kinase(ROCK)is a kinase belonging to the AGC(PKA/PKG/PKC)family of serine-threonine kinases.It is involved mainly in regulating the shape and movement of cells by acting on the cytoskeleton.ROCK plays a role in a wide range of different cellular phenomena,as ROCK is a downstream effector protein of the small GTPase Rho,which is one of the major regulators of the cytoskeleton.(2)ObjectiveThe purpose of this section is to make sure whether ROCK is involved in the EGF-induced migration of MG63 cells,and its stress fiber reorganization.(3)Methods and ResultsROCK is a main downstream effector of Rho A,and its primary role involves regulating the shape and movement of cells by acting on the cytoskeleton.Thus,we tested whether Rho A promoted EGF-induced stress fiber organization and migration in MG63 cells via ROCK.MG63 cells were pretreated with ROCK inhibitor Y27632 for 2 hours and then treated with EGF.We found that ROCK phosphorylation efficiently increased from 2 to 6 h and reached a maximum at 6 h.After 12 h,ROCK phosphorylation returned to the baseline level.The reorganization of stress fibers in the Y27632-pretreated group was significantly suppressed compared to the group without Y27632 pretreatment.Furthermore,the wound scratch assay showed no significant difference in MG63 cell migration between the control and Y27632-treated groups.However,the number of migrated MG63 cells in the EGF-treated group was clearly increased compared to the control and Y27632-treated groups(p<0.01).Interestingly,the number of migrated MG63 cells in the EGF +Y27632 group decreased compared to the group with EGF alone(p<0.05).(4)ConclusionROCK,the main downstream effector of Rho A,is able to promote MG63 cell migration and stress fiber reorganization.And the suppression of ROCK expression,resulting in the decreased of MG63 cell migration and stress fiber reorganization.4.ROCK mediates MG63 cell migration through the activation of LIMK2/Cofilinl signaling(1)BackgroundCofilin is a widely distributed intracellular actin-modulating protein that binds and depolymerizes filamentous F-actin and inhibits the polymerization of monomeric G-actin in a pH-dependent manner.It is involved in the translocation of actin-cofilin complex from cytoplasm to nucleus.(2)ObjectiveWe sought to identify whether ROCK mediated MG63 cell migration through activated LIMK2/Cofilinl signaling.(3)Methods and ResultsRho A activates downstream LIMK2 through ROCK,leading to the inactivation and phosphorylation of the actin-depolymerizing factor Cofilinl and subsequent cytoskeletal reorganization.Therefore,we sought to identify whether ROCK mediated MG63 cell migration through activated LIMK2/Cofilinl signaling.LIMK2 phosphorylation significantly increased and reached its peak at 6 h and then declined 12 h post EGF stimulation.Cofilinl phosphorylation showed a similar trend,increasing after 2 h and reaching its peak at 6 h.Moreover,the EGF-induced phosphorylation of LIMK2 and Cofilinl was suppressed by ROCK inhibitor Y27632,suggesting that EGF induced LIMK2 and Cofilinl activation through ROCK.We also explored whether LIMK2 and Cofilinl regulated EGF-induced stress fiber formation and reorganization.We designed siRNAs for LIMK2 and Cofilinl and found that the expression of LIMK2 and Cofilinl in MG63 cells was clearly inhibited with their respective siRNAs.siLIMK2 and siCofilinl inhibited stress fiber formation.(4)ConclusionLIMK2 and Cofilinl act downstream of Rho A/ROCK to regulate EGF-induced stress fiber formation in MG63 cells.