FoxO1 transcription factor promotes integrin β3 expression in trophoblasts

Abstract

s / Placenta 35 (2014) A1eA112 A67 provide novel insights toward understanding of the molecular processes regulating EVT cell invasion. P2.17. FOXO1 TRANSCRIPTION FACTOR PROMOTES INTEGRIN b3 EXPRESSION IN TROPHOBLASTS Chie-Pein Chen , Yi-Hsin Wu , Hungwen Chen c Division of High Risk Pregnancy, Mackay Memorial Hospital, Taipei, Taiwan; Department of Medical Research, Mackay Memorial Hospital, Taipei, Taiwan; c Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan Objective: The forkhead O1 transcription factor (FoxO1) involves in a range of extracellular signals, including growth factor signaling, inflammation, oxidative stress, cell survival and cell cycle progression in multiple cell types, but its effect in integrin ®3 expression of trophoblasts has not been reported previously. Methods: An extravillous trophoblast cell line (HTR-8/SVneo) was transfected with pcDNA3 expression vectors containing FLAG-tagged fulllength FoxO1 constructs (FoxO1-WT) or the constitutively nuclear mutant in three Akt phosphorylation sites replaced by alanine (FoxO1-AAA ) using Lipofectamine 2000 reagent to generate stable expression clones. The trophoblast JEG-3 cell line and HEK-293 cells were transiently transfected for the study. The Western blots, flow cytometry, adhesion and migration assay, and MTT assay were used in the experiments. Results: FoxO1 and integrin b3 were expressed in HTR-8, JEG-3 trophoblasts and HEK-293 cells. FoxO1 factor is negatively regulated by Akt phosphorylation followed by its nuclear export. FoxO1-AAA is not subject to Akt phosphorylation. The expression of integrin b3 was up-regulated in cells transfected by FoxO1-WT, which was further enhanced by cells transfected by FoxO1-AAA. The adhesion and migration of trophoblast cells was increased by cells transfected by FoxO1-WT, and was further promoted by cells transfected by FoxO1-AAA without increasing the cell apoptosis. The LY294002, a PI3K/Akt inhibitor, increased integrin b3 expression in trophoblasts. siRNA for FoxO1 inhibited the integrin b3 expression in these cells, but not control scrambled siRNA. Conclusion: These data demonstrate that FoxO1 enhances trophoblast integrin b3 expression. Integrin b3 expression in trophoblasts involves a novel regulatory cascade through the PI3K/Akt/FoxO1 pathway. P2.18-N. CHANGES IN EXTRAVILLOUS TROPHOBLAST (EVT) GENE EXPRESSION IN CASES OF ABNORMALLY INVASIVE PLACENTA SUPPORT AN ENHANCED EPITHELIAL-TO-MESENCHYMAL TRANSITION Sonia DaSilva-Arnold, Stacy Zamudio, Abdulla Al-Khan, Nicholas Illsley Hackensack University Medical Center, Hackensack, NJ, USA Objectives: Abnormally invasive placenta (AIP) occurs when trophoblast invades deeper than normal into the myometrium and, in severe cases (placenta percreta), completely through the uterine serosa. Consequences can be severe, involving major maternal hemorrhage and requiring peripartum hysterectomy. During normal placental development, villous cytotrophoblast differentiate into more invasive EVT, a process marked by an epithelial-to-mesenchymal transition (EMT), similar to that observed in cancer metastasis. We asked whether EVT involved in AIP pathologies were driven further down the EMT pathway compared to normal EVT. Methods: We compared EVTs derived from placental basal plate tissue of normal, term pregnancies (n1⁄44) and uteroplacental tissue from placenta percreta (n1⁄43). Following tissue digestion, cell isolation employed immunomagnetic affinity separation targeting HLA-G, an EVT cell surface marker. Quantification of cells expressing HLA-G was performed by flow cytometry. Cellular RNA was analyzed on targeted PCR arrays containing EMT-related genes (Qiagen). Results: Analysis of EVT RNA (average RIN score1⁄49.7) from normal placenta and placenta percreta (HLA-G purity 95% & 73%) revealed upregulation of vimentin (VIM; 8.7-fold, p1⁄40.08, Student’s t-test), matrix metalloproteinase-9 (MMP-9; 9.4-fold, p1⁄40.03), secreted phosphoprotein 1 (SPP1; 13.9-fold, p1⁄40.02), and transforming growth factor 2 (TGFb2; 2.2fold, p1⁄40.01). We also observed down-regulation of keratin-19 (KRT19; -1.9-fold, p1⁄40.006), and tissue inhibitor of metalloproteinase 1 (TIMP1; -2.4-fold, p1⁄40.09). Conclusion: Multiple genes that are known markers of the EMT were altered, including VIM, KRT19, MMP-9, SPP1 and TIMP1, supporting the hypothesis that progression down the EMT is enhanced in AIP. Moreover increased expression of MMP-9 combined with down-regulation of TIMP1 would be expected where over-invasion is present, and are likely functionally involved in AIP. There are also changes, such as increased TGFb2, which differ from the metastatic profile. These data will provide clues to the regulatory pathways driving AIP and possibly biomarkers that could be detected early in the disease process. P2.19-N. DEVELOPING A QUANTITATIVE SCORING SYSTEM TO ASSESS THE ‘CLINICAL SEVERITY’ OF THE ABNORMALLY INVASIVE PLACENTA (AIP) Sally Collins , Stacey Zamudio , Nicholas Illsley , Abdulla AlKhan , Lawrence Impey b University of Oxford, Oxford, UK; b John Radcliffe Hospital, Oxford, UK; Hackensack University Medical Center,