title: Aberrant TGFβ Signaling Contributes to Altered Trophoblast Differentiation in Preeclampsia.
abstract: TGFβ has been implicated in preeclampsia, but its intracellular signaling via phosphorylated mothers against decapentaplegic (SMADs) and SMAD-independent proteins in the placenta remains elusive. Here we show that TGFβ receptor-regulated SMAD2 was activated (Ser(465/467) phosphorylation) in syncytiotrophoblast and proliferating extravillous trophoblast cells of first-trimester placenta, whereas inhibitory SMAD7 located primarily to cytotrophoblast cells. SMAD2 phosphorylation decreased with advancing gestation, whereas SMAD7 expression increased and shifted to syncytiotrophoblasts toward term. Additionally, we found that the TGFβ SMAD-independent signaling via partitioning defective protein 6 (PARD6)/Smad ubiquitylation regulatory factor was activated at approximately 10-12 weeks of gestation in cytotrophoblast and extravillous trophoblast cells comprising the anchoring column. Placentae from early-onset, but not late-onset, preeclampsia exhibited elevated SMAD2 phosphorylation and SMAD7 levels. Whereas PARD6 expression increased and SMURF1 levels decreased in preeclamptic placentae, their association increased. SMAD2 phosphorylation by TGFβ in villous explants and BeWo cells resulted in a reduction of Glial cell missing-1 (GCM1) and fusogenic protein syncytin-1 while increasing cell cycle regulators cyclin E-1 (CCNE1) and cyclin-dependent kinase 4. SMAD7 abrogated the proliferative effects of TGFβ. CCNE1 levels were increased in preeclamptic placentae, whereas GCM1 was markedly reduced. In addition, TGFβ treatment increased the association of PARD6 and SMURF1 and down-regulated Ras homolog gene family, member A (RHOA) GTPase in JEG3 cells. In a wound assay, TGFβ treatment increased the association of PARD6 and SMURF1 and triggered JEG3 cell migration through increased cellular protrusions. Taken together, our data indicate that TGFβ signaling via both SMAD2/7 and PARD6/SMURF1 pathways plays a role in trophoblast growth and differentiation. Altered SMAD regulation of GCM1 and CCNE1 and aberrant expression/activation of PARD6/SMURF1 may contribute to the pathogenesis of preeclampsia by affecting cellular pathways associated with this disorder.
authors.affiliation: Lunenfeld-Tanenbaum Research Institute (J.X., T.S., A.T., J.R., I.C.), Mt Sinai Hospital, Toronto, Ontario, Canada M5T 3H7; Departments of Obstetrics and Gynecology (Y.Y., O.N., I.C.), Physiology (T.S., J.R., M.P., I.C.), and Pediatrics (M.P.), University of Toronto, Toronto, Ontario, Canada; and Institute of Medical Science (M.P.), The Hospital for Sick Children, Toronto, Ontario, Canada M5G 1X8.
authors.forename: Jing
authors.initials: J
authors.lastname: Xu
authors.affiliation: Lunenfeld-Tanenbaum Research Institute (J.X., T.S., A.T., J.R., I.C.), Mt Sinai Hospital, Toronto, Ontario, Canada M5T 3H7; Departments of Obstetrics and Gynecology (Y.Y., O.N., I.C.), Physiology (T.S., J.R., M.P., I.C.), and Pediatrics (M.P.), University of Toronto, Toronto, Ontario, Canada; and Institute of Medical Science (M.P.), The Hospital for Sick Children, Toronto, Ontario, Canada M5G 1X8.
authors.forename: Tharini
authors.initials: T
authors.lastname: Sivasubramaniyam
authors.affiliation: Lunenfeld-Tanenbaum Research Institute (J.X., T.S., A.T., J.R., I.C.), Mt Sinai Hospital, Toronto, Ontario, Canada M5T 3H7; Departments of Obstetrics and Gynecology (Y.Y., O.N., I.C.), Physiology (T.S., J.R., M.P., I.C.), and Pediatrics (M.P.), University of Toronto, Toronto, Ontario, Canada; and Institute of Medical Science (M.P.), The Hospital for Sick Children, Toronto, Ontario, Canada M5G 1X8.
authors.forename: Yoav
authors.initials: Y
authors.lastname: Yinon
authors.affiliation: Lunenfeld-Tanenbaum Research Institute (J.X., T.S., A.T., J.R., I.C.), Mt Sinai Hospital, Toronto, Ontario, Canada M5T 3H7; Departments of Obstetrics and Gynecology (Y.Y., O.N., I.C.), Physiology (T.S., J.R., M.P., I.C.), and Pediatrics (M.P.), University of Toronto, Toronto, Ontario, Canada; and Institute of Medical Science (M.P.), The Hospital for Sick Children, Toronto, Ontario, Canada M5G 1X8.
authors.forename: Andrea
authors.initials: A
authors.lastname: Tagliaferro
authors.affiliation: Lunenfeld-Tanenbaum Research Institute (J.X., T.S., A.T., J.R., I.C.), Mt Sinai Hospital, Toronto, Ontario, Canada M5T 3H7; Departments of Obstetrics and Gynecology (Y.Y., O.N., I.C.), Physiology (T.S., J.R., M.P., I.C.), and Pediatrics (M.P.), University of Toronto, Toronto, Ontario, Canada; and Institute of Medical Science (M.P.), The Hospital for Sick Children, Toronto, Ontario, Canada M5G 1X8.
authors.forename: Jocelyn
authors.initials: J
authors.lastname: Ray
authors.affiliation: Lunenfeld-Tanenbaum Research Institute (J.X., T.S., A.T., J.R., I.C.), Mt Sinai Hospital, Toronto, Ontario, Canada M5T 3H7; Departments of Obstetrics and Gynecology (Y.Y., O.N., I.C.), Physiology (T.S., J.R., M.P., I.C.), and Pediatrics (M.P.), University of Toronto, Toronto, Ontario, Canada; and Institute of Medical Science (M.P.), The Hospital for Sick Children, Toronto, Ontario, Canada M5G 1X8.
authors.forename: Ori
authors.initials: O
authors.lastname: Nevo
authors.affiliation: Lunenfeld-Tanenbaum Research Institute (J.X., T.S., A.T., J.R., I.C.), Mt Sinai Hospital, Toronto, Ontario, Canada M5T 3H7; Departments of Obstetrics and Gynecology (Y.Y., O.N., I.C.), Physiology (T.S., J.R., M.P., I.C.), and Pediatrics (M.P.), University of Toronto, Toronto, Ontario, Canada; and Institute of Medical Science (M.P.), The Hospital for Sick Children, Toronto, Ontario, Canada M5G 1X8.
authors.forename: Martin
authors.initials: M
authors.lastname: Post
authors.affiliation: Lunenfeld-Tanenbaum Research Institute (J.X., T.S., A.T., J.R., I.C.), Mt Sinai Hospital, Toronto, Ontario, Canada M5T 3H7; Departments of Obstetrics and Gynecology (Y.Y., O.N., I.C.), Physiology (T.S., J.R., M.P., I.C.), and Pediatrics (M.P.), University of Toronto, Toronto, Ontario, Canada; and Institute of Medical Science (M.P.), The Hospital for Sick Children, Toronto, Ontario, Canada M5G 1X8.
authors.forename: Isabella
authors.initials: I
authors.lastname: Caniggia
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics / Q000378:metabolism; D000293:Adolescent; D000328:Adult; D016022:Case-Control Studies; D002454:Cell Differentiation / Q000235:genetics*; D049109:Cell Proliferation / Q000235:genetics; D019927:Cyclin E / Q000235:genetics / Q000378:metabolism; D051358:Cyclin-Dependent Kinase 4 / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins; D005260:Female; D015686:Gene Products, env / Q000235:genetics / Q000378:metabolism; D005865:Gestational Age; D006801:Humans; D007231:Infant, Newborn; D008297:Male; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism; D015513:Oncogene Proteins / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D010920:Placenta / Q000378:metabolism*; D011225:Pre-Eclampsia / Q000235:genetics* / Q000378:metabolism; D011247:Pregnancy; D011257:Pregnancy Proteins / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism*; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction; D051899:Smad2 Protein / Q000235:genetics / Q000378:metabolism; D051906:Smad7 Protein / Q000235:genetics / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D016212:Transforming Growth Factor beta / Q000235:genetics* / Q000378:metabolism; D014327:Trophoblasts / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases / Q000235:genetics / Q000378:metabolism; D055815:Young Adult; D020742:rhoA GTP-Binding Protein / Q000235:genetics / Q000378:metabolism
chemical_list: D048868:Adaptor Proteins, Signal Transducing; C475820:CCNE1 protein, human; D019927:Cyclin E; D004268:DNA-Binding Proteins; C491089:GCM1 protein, human; D015686:Gene Products, env; D009687:Nuclear Proteins; D015513:Oncogene Proteins; C499657:PARD6A protein, human; D011257:Pregnancy Proteins; D012333:RNA, Messenger; C497782:SMAD2 protein, human; C497806:SMAD7 protein, human; D051899:Smad2 Protein; D051906:Smad7 Protein; D014157:Transcription Factors; D016212:Transforming Growth Factor beta; C405335:syncytin; C066737:RHOA protein, human; C498342:SMURF1 protein, human; D044767:Ubiquitin-Protein Ligases; C495900:CDK4 protein, human; D051358:Cyclin-Dependent Kinase 4; D020742:rhoA GTP-Binding Protein