Study Of Gadd45α Regulating Trophoblast Functions And Its Role In The Pathogenesis Of Preeclampsia

Objectives: To determine the Gadd45α expression and function in thehuman first trimester placenta and identify the underlying mechanisms thatregulate trophoblast migration and invasion. To investigate the roles ofGadd45α and its downstream signaling pathway p38MAPK in abnormaltrophoblast functions in preeclampsia utilizing the hypoxia/reoxygenationmodel to mimic the oxidative stress, and to identify the molecularmechanisms underlined, to provide new targets for treatment ofpreeclampsia, including Gadd45α knockdown and p38inhibitors.Methods:(1) The expression of Gadd45α in human placental sampleswas determined using immunohistochemistry.(2) Gadd45α was knockdownthrough transfecting human extravillous trophoblast (EVT) cell line,HTR8/SVneo or placental villous explants with lentiviral vector-basedshort-hairpin RNA (shRNA).(3) The transfection efficiency wasidentified by PCR and western blotting.(4) The role of Gadd45α in regulation of trophoblast invasion was determined by transwell migrationassay and Matrigel invasion assay.(5) Placental villious explants culturemodel was employed to further verify the effect of Gadd45α on outgrowthand migration of extravillous trophoblast cells.(6) Matrix metalloproteinase(MMP)2/9activities and tissue inhibitor of metalloproteinase (TIMP)1/2expressions were tested using gelatin gel zymography and western blotting,respectively.(7) Hypoxia/reoxygenation (H/R) model in HTR8/SVneo cellswas employed to mimic placental oxidative stress insult in this study.Moreover, Gadd45α silencing with lentiviral vector-based shRNA andSB203580, the specific p38inhibitor, were undertaken to further investigatethe possible molecular of Gadd45α and its downsteam p38signalingpathway in oxidative stress injury, apoptosis, and capabilities of trophoblastmigration/invasion and angiogenesis, and secretion of sFlt-1&sEng,activities of MMPs in H/R-induced extravillous trophoblast cells.Results:(1) Gadd45α was demonstrated to be strongly expressed insyncytiotrophoblasts and trophoblast columns of human placental villi, EVTcells and glandular epithelium within the maternal decidua during the firsttrimester. No differences in Gadd45a staining were observed between thegestational weeks.(2) Gadd45α knockdown significantly promoted themigration and invasion potentials of HTR8/SVneo cells, whereas it did notaffect cell proliferation or apoptosis.(3) These effects were related to increased activities of MMP2/9and the decreased expression of TIMP1/2.(4)H/R treatment induced the over-expression of Gadd45α and increasedactivation of p38MAPK, decreased activities of MMPs, and increasedsecretions of sFlt-1&sEng in HTR8/SVneo cells, which provides an in vitrosimple cell model to investigate preeclampsia.(5) Both knockdown ofGadd45α with lentiviral RNA interference and blockage of p38MAPKwith SB203580can effectively improve trophoblast migration and invasionof H/R-exposed HTR8/SVneo cells, and effectively decrease the secretionsof sFlt-1&sEng, possibly through directly or undirectely regulatingactivities of MMP2/9or deducing oxidative stress.Conclusion:(1) Gadd45α may be involved in human trophoblastmigration and invasion and may function as an important negative regulatorat the foetal-maternal interface during early pregnancy by directly orindirectly regulating MMP2/9activities.(2) There is a regulatory signallingpathway in extravillous trophoblast cells that H/R-exposure resultes ininduction of Gadd45α and over-activation of p38MAPK, ultimately injurestrophoblast functions and increases secretions of sFlt-1&sEng. Our studyunveiled the underlined mechanism of Gadd45α regulating trophoblastfunctions via p38MAPK in preeclampsia.(3) Gadd45α knockdown or p38blockage promotes trophoblast migration/invasion and angiogenesis inH/R-exposed extravillous trophoblast cells, and then improves the insufficient trophoblast invasion and shallow spiral artery remodelling inpreeclampsia, providing a novel way for the treatment of preeclampsia.