Ha-RasV12-overexpression induces YAP nuclear translocation in MDCK cells: mechanism and function

Abstract

Our previous study showed that Ha-RasV12 overexpression induced nuclear translocation of hippo effector Yes-associated protein (YAP) in MDCK cells via hippo-independent pathway even at confluent stage Ha-RasV12 overexpression leads to downregulation of caveolin-1 (Cav1) and disruption of junction integrity Cav1 was reported to recruit E-cadherin/β-catenin complex and stabilize cell junction Furukawa et al showed that disruption of actomyosin tension in cell junction caused YAP nuclear localization in epithelial cells under high density We hypothesize that Ha-RasV12-decreased Cav1 leads to the lessened of actomyosin tension which subsequently facilitates YAP nuclear localization MK4 cells exhibits typical epithelial colonies with well-organized junctional actin belts lined with myosin IIB Upon IPTG administration Ha-RasV12 overexpression resulted in YAP nuclear translocation and loosened cell junction with disturbed distribution of myosin IIB which is prevented by overexpression of Cav1 Cav1 knockdown showed loosened cell junction with disturbed distribution of myosin IIB and the augmentation of YAP nuclear localization Treatment of actomyosin tension inhibitors including ML7 (myosin light chain kinase inhibitor) blebbistatin (myosin II inhibitor) induced YAP nuclear translocation and disturbed distribution of myosin IIB in confluent MK4 cells The regulation of ERK activation has been found related to Cav1 U0126 (MEK inhibitor) abolished Ha-RasV12 overexpression- and Cav1 absence-induced YAP nuclear translocation suggesting that MEK signaling is required for Ha-RasV12-induced YAP nuclear translocation Ha-RasV12 overexpressing cells exhibited apically cellular aggregates in overconfluent MK4 cells Inhibition of YAP activity by verteporfin did not abolish Ha-RasV12-induced cellular aggregates Immunostaining results showed that cells in aggregate area displayed cytosolic YAP but not nuclear YAP Although inhibition of Rac activity by EHT1864 inhibited Ha-RasV12-induced cellular aggregate it did not change RasV12-induced YAP nuclear translocation Taken together these data suggest that Cav1 functions to stabilize cell junction integrity which tends to retains YAP in the cytosol in confluent MDCK cells Moreover Rac activity but not nuclear YAP seems to be more important for multilayer cellular aggregates formation

Date of Award	2019
Original language	English
Supervisor	Ming-Jer Tang (Supervisor)