Trophoblastic Proliferation And Invasion Regulated By ACTN4 Is Impaired In Early-onset Preeclampsia

Background: Early-onset preeclampsia(EO-PE)is a pregnancy complication earlier than the 34 th week that has long been attributed to inadequate trophoblast invasion.Cellular invasion depends on the formation of cytoskeleton and invasive structures.ACTN4 maintain cytoskeleton integrity and regulate cell movement.We therefore hypothesized that ACTN4 expression in trophoblasts is associated with EO-PE.Methods: ACTN4 expression patterns in EO-PE placentas(n=7)and normal placentas(n=10)were detected by immunohistochemistry and Western blotting.CCK-8 assay,flow cytometry and EdU assay were used to detect the cell proliferation in ACTN4 knockdown HTR8/SVneo cells.The interaction of ACTN4 and AKT were detected by immunofluorence and Co-IP,the roles of ACTN4 in AKT translocation were assessed by Co-IP and plasma-membrane fractions isolation.AKT activities were detected by Western blotting.The regulation of E-cadherin in ACTN4/?-catenin complex was examined by immunofluorence and Co-IPassay.Actin filament in podosomes formation were stained by TRITC-phalloidin.Cell invasion and migration were performed by Matrigel invasion assay and Transwell migration assay.In vivo study,ACTN4-deficient mice were generated by intravenous injection of EGFP-tagged ACTN4 interfering adenovirus.The blood pressure was monitored,placenta and fetuses were weighted.ACTN4 and AKT,GSK3?protein in mouse placenta were detected by Western blotting.Results: Immunohistochemistry and Western blotting indicated that ACTN4 less detected in EO-PE placenta trophoblasts.Knockdown ACTN4 inhibits HTR8/SVneo cells proliferation.Immunofluorescence and Co-IP confirmed that ACTN4 interaction with AKT in trophoblast cells.Gain-and loss-of ACTN4 revealed that ACTN4 mediated AKT membrane translocation and phosphorylation in role of trophoblasts proliferation.Moreover,ACTN4 interaction with ?-catenin in cell junction of JEG3 cells.knockdown E-cadherin negatively regulated ACTN4 and ?-catenin co-localization in JEG3 cell podosomes,and ACTN4 downregulation suppressed the E-cadherin-induced cell invasion and migration increase via depolymerizing actin filaments.Furthermore,loss of ACTN4 in vivo elevated maternal blood pressure and reduced placentas and fetus weight,which were mainly because of a decreased junctional/labyrinth zone ratio and proliferation cell in placentas,as well as downregulated of AKT phosphorylation.Conclusion: ACTN4 regulates trophoblast proliferation and motility through modulating AKT translocation and interacting with ?-catenin,respectively.ACTN4 deficiency results in impaired proliferation and aberrant cytoskeletal remodeling in trophoblasts,both of which lead to abnormal placentation and PE development.