Function And Regulatory Mechanisms Of YAP/TAZ During Uterine Decidualization In Mice

Decidualization,during which uterine stromal cells proliferate and differentiate into decidual cells,occurs in response to the implantating embryo,and is a key process for embryonic development and successful pregnancy.Defective decidualization might lead to a range of pregnancy disorders,including recurrent spontaneous abortion and early pregnancy loss.Although numerous transcription factors,cytokines as well as signaling pathways have been demonstrated to be potential participated in the process of implantation and decidualization,its underlying regulatory mechanisms still remain largely unknown.Plenty of studies have implicated yes-associated protein(YAP)and transcriptional co-activator with PDZ-binding motif(TAZ)in the modulating of organ size,stem cell function,regeneration and tumor development.Knock-out studies in mice have confirmed that YAP/TAZ play important roles in mammalian embryo development.YAP deletion is embryonic lethal and TAZ deletion results in viable mice with kidney defects.However,there is still very limited information available regarding the function and regulatory mechanisms of YAP/TAZ in the process of decidualization.This study was designed to investigate the function and regulation mechanisms of YAP/TAZ during decidualization in mouse uterus,and provide bases of further clarifying the mechanisms of mammalian embryo implantation and decidualization.In this study,we observed gradually increased YAP expression during mouse uterine decidualization,and demonstrated that inactivation of YAP impaired the proliferation and differentiation of stromal cells followed by the blockade of cell cycle transition.In uterine stromal cells,Bmp2 promoted YAP expression and nuclear sequestration via Bmp type I receptor Alk2,thereby led to elevated YAP-TEAD transcriptional activity.Inactivation of YAP by si RNA or inhibitor abrogated the induction of Bmp2 on stromal cell differentiation.Using luciferase reporter assays,we revealed that the YAP translocated in the nuclear could directly bind to TEAD in Rrm2 promoter and modulate its function in stromal cells.Overexpression of Rrm2 ameliorated the DNA damage of stromal cells induced by YAP inactivation as evidenced by decreased 8-OHd G content,and restored stromal differentiation.Further analysis revealed that YAP mediated the regulation of Bmp2 on Rrm2 expression whose suppression by Triapine blocked the promotion of Bmp2 on stromal cell differentiation.Moreover,inhibition of YAP significantly attenuated GR and GPX activities,which further led to decreased GSH content,however,the levels of the above parameters showed opposite trends in the Rrm2 overexpression group.By detecting the ROS content,ATP level,mitochondrial DNA copy number,mitochondrial membrane potential and the opening of mitochondrial permeability transition pores in stromal cells,we found that YAP inactivation caused oxidative stress in stromal cells which further led to mitochondrial dysfunction.Meanwhile,inactivation of YAP triggered apoptosis in stromal cells via promoting the expression of Caspase-3 and Bax and inhibiting Bcl-2 expression.However,the oxidative stress,mitochondrial dysfunction and apoptosis induced by YAP inactivation were prevented by GSH which effectively removed the elevated levels of ROS.The result showed that TAZ was localized in decidua where it promoted stromal cell proliferation followed by accelerated G1/S phase transition via Ccnd3 and Cdk4,and advanced the expression or activity of stromal differentiation markers Prl8a2,Prl3c1 and ALP,indicating the importance of TAZ in the initiation of decidualization.Knockdown of TAZ impeded HB-EGF induction of stromal cell proliferation and differentiation.Under oxidative stress,TAZ protected stromal cell differentiation against oxidative damage by reducing intracellular ROS and enhancing antioxidant capacity dependent of Nrf2/ARE/Foxo1 pathway.TAZ strengthened transcriptional activity of Nrf2 which directly bound to antioxidant response element(ARE)of Foxo1 promoter region.Additionally,silencing of TAZ caused an accumulation of intracellular ROS through the heightened NOX activity whose blockage by APO rescued the defect of stromal cell differentiation.Further analysis revealed that TAZ might restore mitochondrial function as indicated by the improvement of ATP level,mt DNA copy number and mitochondrial membrane potential with reduction for mitochondrial superoxide.Meanwhile,TAZ modulated the activities of mitochondrial respiratory chain complex I and III whose suppression by ROT and AA resulted in an incapability of TAZ in defensing oxidative injury of stromal differentiation.Moreover,TAZ prevented apoptosis of stromal cells via up-regulating Bcl2 expression and inhibiting Casp3 activity as well as Bax expression during oxidative stress.In conclusion,this study suggest an essential role of YAP/TAZ during mouse decidualization.Our results demonstrate that YAP exerts its effects on uterine decidualization via TEAD/Rrm2/GSH/ROS pathway in response of Bmp2.TAZ might mediate HB-EGF function in uterine decidualization through targeting Ccnd3 and ameliorate oxidative stress-mediated damage on stromal cell differentiation via Nrf2/ARE/Foxo1 pathway.