YAP Signaling Pathway Mediates LPS-induced Tissue Factor Expression And Cellular Apoptosis In Endothelial Cells

OBJECTIVES: 1.To explore the effects of ROCK/YAP/Egr-1 signaling pathway on LPS-induced tissue factor expression in human endothelial cells 2.To investigate the roles of YAP/P73/(Bax and Bcl-2)/Caspase-3 signaling pathway on LPS-induced human endothelial cells apoptosis 3.To explore the effects of pulmonary YAP protein on acute lung injury under endotoxemia in miceMETHODS: 1.The expression of P-MYPT1?YAP?P-YAP(Ser127)?Egr-1 and TF in LPS-stimulated endothelial cells were measured by western blot assays.Human endothelial cells incubated with LPS were pretreated with or without the ROCK inhibitor Y-27632,a YAP si RNA or an Egr-1 si RNA.ROCK?YAP and Egr-1 signaling-induced down stream proteins expression was investigated by western blot.The LPS-induced activation of YAP and intracellular translocation were analyzed by an immunofluorescent assay.The interaction between YAP and Egr-1 was analyzed by immunoprecipitation.2.The expression of P-YAP(Tyr357)?P73?Bax?Bcl-2 and active Caspase-3 proteins in LPS-stimulated endothelial cells were measured by immunoblotting analysis.Activation and intracellular distribution of YAP protein were detected by western blot,immunofluorescence and immunocytochemistry staining.The interaction between YAP and P73 was analyzed by immunoprecipitation.Human endothelial cells incubated with LPS were pretreated with or without a YAP si RNA or a P73 si RNA.YAP and P73 signaling-mediated apoptosis was analyzed by flow cytometry and expression of apoptotic proteins was investigated by western blot.3.Furthermore,we intratrachealy injected YAP si RNA to assess LPS-induced acute lung injury(ALI)in mice by hematoxylin and eosin staining.RESULTS: 1.LPS rapidly induced ROCK activation and increased TF expression in human endothelial cells.LPS caused YAP shuttling into the nuclei of endothelial cells and combined with Egr-1 via the activation of ROCK.Furthermore,the LPS-mediated TF expression increase was prevented by ROCK inactivation,YAP knockdown and Egr-1 depletion,suggesting that LPS-induced TF expression is closely associated with the ROCK/YAP/Egr-1 signaling pathway in human endothelial cells.2.LPS-induced apoptosis of human endothelial cells in vitro required the presence of cycloheximide(CHX).P73 is an important transcription factor implicated in LPS-induced human endothelial cells apoptosis via the upregulation of BAX and the downregulation of BCL-2.LPS-induced activation and nuclear translocation of YAP in human endothelial cells promoted the formation of a signaling complex with P73.LPS-induced activation of YAP/P73 signaling led to human endothelial cells apoptosis by regulating BCL-2 family proteins and Caspase-3 activation.Moreover,LPS-induced human endothelial cells apoptosis was prevented by YAP depletion and P73 knockdown.3.Finally,an intratracheal injection of YAP si RNA relieved lung injury in LPS-stimulated mice.CONCLUSIONS: 1.Our findings indicate that ROCK/YAP/Egr-1 signaling pathway regulates tissue factor expression after stimulation with LPS in human endothelial cells.2.YAP/P73/(Bax and Bcl-2)signaling pathway is involved in LPS-induced human endothelial cells apoptosis through activation of Caspase-3.3.The lung YAP signaling plays an important role in LPS-induced ALI in mice.