Impaired Trophoblastic Sphingosine-1-Phosphate Synthesis Induces Preeclampsia By Deactivating Yes-associated Protein

Background : Incomplete spiral artery remodeling,caused by impaired extravillous trophoblast invasion,is a fundamental pathogenic process associated with malplacentation and the development of preeclampsia.Nevertheless,the mechanisms controlling this regulation of trophoblast invasion are largely unknown.Sphingosinol-1-phosphate(S1P)is a bioactive metabolite,impaired metabolism of which may play an important role in the pathogenesis of PE.This study intends to clarify the metabolism and function of S1 P in trophoblast cells,so as to reveal the molecular mechanism of PE caused by impaired S1 P metabolism,thus provides in-depth insight for pathogenesis of PE,which may leads to better management for the PE patients.Methods:(1)S1P levels in normal and PE placentas were quantified by enzyme-linked immunosorbent assay(ELISA),the expression and localization of S1 P synthase 1(SPHK1)were quanfitied by real-time quantitative polymerase chain reaction(RT-PCR),western blot(WB)and immunofluorescence(IF),fluorescence labeled substrate(NBDSphingosine)was used for the measurement of the enzyme activity of SPHK1.(2)Pregnant CD1 mice were daily injected with SPHK1 inhibitor PF543 through tail vein during placentation;Blood pressure was monitored by tail-cuff plethysmography;The hemodynamics of uterine artery and umbilical vein were monitored by high frequency ultrasound;and the birth weight,crown-rump length(CRL)of the fetus from different gestation were recorded;H&E and IF staining were used to observe renal and placental structure and spiral artery remodeling.(3)Treat the HTR8/SVneo cells with PF543 and S1 P,cell viability was determined by CCK8 and ETHD-II staining;Matrigel based transwell was used to evaluate the invasive ability of HTR8/SVneo cell;F-actin polymerization and YAP subcellular localization were determined by co-staining of phalloidin and YAP immunofluorescence;Transcriptome sequencing combined with transcription factors(TF)prediction were used to determine downstream effector genes of YAP;the interaction between actin monomer(G-actin),polymerized actin(F-actin)and hippo pathway proteins(MST1,LATS1,YAP)was assessed with Co-IP;LATS phosphorylation site mutation YAP(YAP-5SA)was applied to identify the direct regulation of F-actin on YAP;S1PR1-5 were interfered by individual si RNA,then actin polymerization and YAP nuclear localization were detected by phalloidin and IF staining separately,cell invasion was assessed by Transwell;western blot(WB)and nuclear protein isolation were used to determine the activation of Rho AROCK pathway and YAP from normal and PE placentas.Results:(1)S1P metabolism is robust in trophoblasts as high expression of S1 P metabolic enzymes and receptors;SPHK1 expression and activity were significantly down-regulated in PE complicated placentas,along with reduced S1 P level.(2)Inhibition of SPHK1 activity during placentation in mice impaired the remodeling of spiral artery,placentation,and induced preeclampsia phenotypes,such as high blood pressure,kidney injury,and fetal growth restriction.(3)PF543 inhibited the invasion of HTR8/SVneo cells,while S1 P promoted the invasion of HTR8/SVneo cells;S1P promotes HTR8/SVneo invasion in YAP dependent manner,while S1 P activates YAP through Rho-ROCK induced actin polymerization;mutationbased YAP-5SA demonstrated that S1 P activation of YAP could be either dependent or independent of Hippo pathway;CTSD and EMILIN1,downstream molecules of YAP,may be involved in the YAP dependent regulation of trophoblast invasion ability;S1PR2 instead of other S1 PRs mediated the regulation of Rho-ROCK pathway by S1P;at last,PE placenta showed the same inhibition of Rho-ROCK pathway and deactivation of YAP.Conclusion: In summary,we found that the SPHK1-S1P-S1PR2 axis is downregulated in PE placentas,which impairs the Rho A/ROCK signaling pathway and actin polymerization,and that YAP activation mediates genes transcription and trophoblast invasion.Our novel findings highlight the importance of SPHK1-mediated S1 P synthesis in PE pathogenesis,thus providing an in-depth insight into the etiology of PE and potential intervention targets in the sphingolipid metabolism.