Staurosporine Targets The Hippo Pathway To Inhibit Cell Growth And VGLL4 Plays A Critical Role In Heart Valve Development

The Hippo signaling pathway regulates cell proliferation,organ size and tumorigenesis,and small molecules targeting the Hippo pathway have therapeutical potential in controlling tissue repair or malignancies.Here we report that Staurosporine(STS)can inhibit cell growth through regulating the activity of YAP,an effector of the Hippo pathway.Mechanistically,STS promotes phosphorylation of Hippo pathway kinases MST1/2 and LATS1/2,which in turn leads to YAP inactivation.Knockout MST1/2 partially blocks the effect of STS on YAP cytoplasmic localization,phosphorylation and target genes expression,whereas knockout LATS1/2 totally inhibits STS effect.In addition,the inhibitory effects of STS on cell growth and colony formation were partially blocked in LATS1/2 double knockout(dKO)cell.Together,our results demonstrate that STS is a positive regulator of the Hippo pathway and the inhibitory effect of STS on cell growth is at least partially mediated by the Hippo pathway.The Hippo pathway is not only essential for tumorigenesis,but also for organ development.YAP is an effector of the Hippo pathway and is mainly regulated by the upstream of Hippo kinase.Recent studies have found that YAP is regulated by VGLL4 that binds to TEAD4 and directly competes with YAP for binding to TEAD4.Although VGLL4 acts as a tumor suppressor to inhibit tumor growth in a variety of tumors,its role in development remains unknown.To investigate the developmental function of VGLL4,we construct the Vgll4 constitutive knockout mice.The homozygous mice show overall mortality shortly after birth and enlarged heart.VGLL4-eGFP reporter mouse shows that VGLL4 is mainly expressed in endothelial cells and valve interstitial cells at the post-EMT stage and is maintained till the adult stage.Genetic loss-of-function study indicates that only loss of Vgll4 in endothelial cells leads to arterial valve malformation accompanying expanded expression of YAP targets.Besides,our lineage trace data unequivocally showed that robustly enhanced proliferation of endothelial cells derived VICs is the main cause of valve thickening.Then we further semi-knockout Yap and found that arterial valves were rescued from malformation induced by Vgll4 deletion.Together,our results demonstrate that the Hippo pathway plays a role in valve development and homeostasis and suggest that manipulation of Hippo components would be a potential therapy for preventing the progression of congenital valve disease.