MG53 Inhibits Angiogenesis Through Regulating Focal Adhesion Kinase Signalling

Objective:MG53,which belongs to the tripartite motif(TRIM)protein family,is also termed as TRIM72.MG53 is composed of a typical TRIM domain at the amino terminus and a PRY-SPRY domain at the carboxyl terminus.The TRIM structure comprises a RING domain,one or two B-box domains and a coiled-coil domain.MG53 is predominantly expressed in striated muscle(cardiac and skeletal muscle)and has been reported to be involved in the regulation of a variety of physiological and pathophysiological functions in striated muscle.MG53 in skeletal muscle can be recruited to the plasma membrane injury site and promotes the repair of acute membrane damage as an essential component in the membrane repair machinery.MG53 in cardiac muscle has been demonstrated to play a crucial role in protecting myocardium from ischemia/reperfusion injury through participating in ischemic preconditioning and ischemic postconditioning.Although there is no expression of native MG53 in a variety of tissues,previous reports have demonstrated that MG53 is a myokine/cardiokine secreted from cardiac and skeletal muscle into circulation,suggesting MG53 may participate in the regulation of multiple biological processes in non-striated muscle tissues.Furthermore,growing evidence has also indicated systematic delivery of recombinant human MG53(rhMG53)can play widely protective roles in lung,kidney,liver,brain,skin and retina.However,the effects of MG53 on endothelial cell function and angiogenesis have not been reported.Angiogenesis is a physiological process including the development of new blood vessels from pre-existing vessels and the subsequent formation of a vascular network.The functional responses of endothelial cells,such as proliferation,migration and tube formation,are of great importance during angiogenesis.Focal adhesion kinase(FAK)is a cytoplasmic tyrosine kinase which is involved in integrin-and other cell surface receptor-mediated signal transductions.Some studies have suggested that excessive angiogenesis induced by diabetic retinopathy can be prevented by down-regulating FAK signaling and the activation of FAK signaling can enhance the angiogenesis in human chondrosarcoma,which indicate that FAK plays a crucial role in angiogenesis.Previous studies have demonstrated that MG53 can directly bind to FAK to regulate myogenesis in myoblast cells,indicating that FAK can serve as an essential target of MG53.In this study,we intend to investigate the effects of MG53 on endothelial cell function and angiogenesis and to explore the molecular mechanisms involved.Methods:1.Human umbilical vein endothelial cells(HUVECs)cultured in vitro were stimulated with rhMG53.Then,rhMG53 uptake,cell migration and tube formation were determined.2.The binding of rhMG53 to FAK and its effects on FAK phosphorylation were firstly detected.The effects of rhMG53 on FAK-Src complex formation and downstream Src/Akt/ERK1/2 signaling activation were further determined.3.Postnatal mice(5 days after birth)were injected intraperitoneally with rhMG53(6 mg/kg)every day for 1 week.Then,the whole retina was isolated to detect the effects of rhMG53 on vessel density.Results:1.rhMG53 directly enters endothelial cells.HUVECs were treated with rhMG53(0,5,10 and 20 μg/m L)for 24 hours.Then,the rhMG53 uptake by endothelial cells was detected with Western blotting.The results demonstrated that MG53 was not expressed in endothelial cells,but intracellular MG53 significantly increased in a concentration-dependent manner.To determine the time course of rhMG53 uptake,HUVECs were exposed to rhMG53(10 μg/m L)for 0,0.25,0.5,1,2,4,8,12 and 24 hours and the results showed that the entry of rhMG53 into endothelial cells occurred 15 minutes after it was added to the extracellular space and increased gradually in a time-dependent manner.Consistent with Western blotting results,the experiments involving immunofluorescence staining also confirmed that 15-min incubation of HUVECs with rhMG53 resulted in protein uptake.Taken together,these results indicate rhMG53 can directly enter into endothelial cells.2.rhMG53 enters endothelial cells in a cholesterol-dependent manner.To investigate the specific mechanism of rhMG53 uptake,HUVECs were pre-treated with MβCD(5 m M)for 1 h,followed by stimulation with rhMG53(10 μg/m L)for 3 h.Western blotting and immunofluorescence staining were used to detect rhMG53 uptake.The results showed that MβCD significantly decreased the uptake of rhMG53 by endothelial cells,suggesting that rhMG53 may enter endothelial cells in a cholesterol-dependent manner.3.rhMG53 binds to FAK and inhibits FAK phosphorylation.In order to investigate the roles of rhMG53 uptake by endothelial cells,HUVECs were stimulated with rhMG53(10 μg/m L)for 24 h and the cell lysates were immunoprecipitated with anti-FAK antibody or Ig G then blotted with anti-MG53 antibody.The results showed that rhMG53 directly bound to FAK.Then HUVECs were stimulated with rhMG53(0,5,10 and 20 μg/m L)for 24 h and the concentration course of rhMG53-induced phosphorylation of FAK was detected by Western blotting.To determine the time course of rhMG53-induced dephosphorylation of FAK,HUVECs were exposed to rhMG53(10 μg/m L)for 0,2,4,6,8,12 and 24 hours.The results displayed that rhMG53 remarkably decreased the phosphorylation of FAK in a concentration-and time-dependent manner.All of the results suggest that rhMG53 directly binds to FAK and simultaneously inhibits the activation of FAK.4.rhMG53 uncouples the FAk-Src interaction and inhibits the activation of Src/Akt/ERK1/2 signaling.We further investigate the effects of rhMG53 on Src/Akt/ERK1/2,the downstream signaling of FAK.HUVECs were stimulated with rhMG53(10 μg/m L)for 24 h and the interaction between FAK and Src was firstly determined by immunoprecipitation.The results showed that rhMG53 significantly inhibited the FAK-Src complex formation.Then,HUVECs were stimulated with rhMG53(0,5,10 and 20μg/m L)for 24 h to detect the effects of rhMG53 on Src/Akt/ERK1/2phosphorylation.The results displayed rhMG53 also significantly suppressed the phosphorylation of Src,Akt and ERK1/2 in a concentration-dependent manner.As a whole,all of these results indicate that rhMG53 uncouples FAK-Src interaction and subsequently suppresses the activation of FAK/Src/Akt/ERK1/2 signaling pathways.5.rhMG53 inhibits endothelial cell migration and tube formation.HUVECs were stimulated with rhMG53(0,5,10 and 20 μg/m L)for 24 h,then cell migration was detected by Transwell migration assay.The results demonstrated that rhMG53 significantly decreased the number of migrated cells.We further investigated the effects of rhMG53 on tube formation.The results showed rhMG53 significantly inhibited endothelial cell tube formation,indicating rhMG53 markedly inhibited angiogenesis in vitro.6.rhMG53 inhibits angiogenesis in mouse retina.To examine the significance of our findings on animal level,postnatal mice were injected intraperitoneally with rhMG53(6 mg/ kg)every day for 1 week.Then,the whole retina was cut into four radial incisions and the vessels were labeled by IB4 staining.The results demonstrated that the vessel density in rhMG53-treated mouse retina significantly decreased.These data clarify the significance of anti-angiogenesis effects of rhMG53 in mouse retina.Conclusion:rhMG53 can enter endothelial cells in a cholesterol-dependent manner,directly bind to FAK,inhibit FAK phosphorylation,uncouple the FAK-Src interaction,further inactivate Src/Akt/ERK1/2 signaling pathways,and consequently inhibit angiogenesis.