Stromal cell derived factor-2 is involved in the unfolded protein response in human placenta

Abstract

s / Placenta 35 (2014) A1eA112 A104 Objective: Depending on the receptor context the wingless ligand Wnt5a affects non-canonical as well as canonical Wnt signaling thereby controlling different developmental processes. The presence of Wnt5a in decidual stromal cells suggested that the ligand could regulate early placental development and trophoblast behavior. Hence, we herein studied the influence of Wnt5a on trophoblast proliferation and motility. Methods: Immunofluorescence staining and western blot analyses were used to determine Wnt5a-producing cell types in early placental tissues and cell lines. Gene silencing of Wnt5a using siRNAs in SGHPL-5 cells and treatment of placental explant cultures and primary isolated cytotrophoblasts (CTB) with recombinant Wnt5a were performed. Wnt5a-dependent effects on proliferationwere studied by counting cumulative cell numbers, cyclin D1 expression and BrdU incorporation, visualized by immunofluorescence staining. Tomeasure apoptosis, cleaved caspase-3 expressionwas analyzed bywestern blotting. Motility assayswere performed uponWnt5a treatment or gene silencing. Furthermore, Wnt5a dependent activation of signaling pathways was analyzed. Results:Wnt5awas detectable in mesenchymal cells and leucocytes of the villous core and the decidua, as well as in decidual glands, CTBs and the trophoblastic cell line SGHPL-5. Silencing of Wnt5a in SGHPL-5 cells decreased proliferation and cyclin D1 expression which could be reverted by supplementation of recombinant Wnt5a. Additionally, increased BrdU incorporation into villous and cell column trophoblasts of placental explant cultures was detectable upon Wnt5a treatment. Furthermore, knock down cells showed increased apoptosis. Wnt5a addition increased, and gene silencing decreased the motility of SGHPL-5 cells. Treatment of SGHPL-5 cells, CTBs and placental explant cultures with recombinant Wnt5a provoked induction of the MAP Kinase pathway. Conclusions: Expression data and functional studies suggest a major role of the Wnt ligand Wnt5a in maintaining proliferation of CTBs and protection from apoptosis via non canonical Wnt signaling and the MAPK pathway. Supported by grant P-25187 of the Austrian Science Funds (FWF) and Herzfelder'sche Familienstiftung. P2.139-N. STROMAL CELL DERIVED FACTOR-2 IS INVOLVED IN THE UNFOLDED PROTEIN RESPONSE IN HUMAN PLACENTA Aline R. Lorenzon-Ojea , Susan J. Fisher , Estela Bevilacqua a Cell and Developmental Biology University of Sao Paulo, Sao Paulo SP, Brazil; Department of Obstetrics, Gynecology & Reproductive Sciences, School of Medicine University of California San Francisco, San Francisco CA, USA Objetives: Adverse environmental conditions lead to disruption of endoplasmic reticulum homeostasis causing accumulation of unfolded/misfolded proteins in ER and activating the Unfolded Protein Response (UPR). UPR restores ER homeostasis, but if fails, cells go into apoptosis. SDF2 is expressed in humanplacenta; it is upregulated during cytotrophoblast differentiation and downregulated in hypoxia. The human SDF2 is similar to Arabidopsis thaliana SDF2-like protein, which is involved in ER stress response. In this study we investigated the SDF2 role in UPR in human placenta. Methods: Primary cell culture of villous cytotrophoblast and BeWo cells were induced to ER stress with tunicamycin. Protein concentrations of SDF2, GRP78/BiP and Caspase-3 were evaluated by Western Blot. Gene expression of SDF2, sXBP1 and CHOP were investigated through qPCR in SDF2-silenced or not BeWo cells. YWHAZ and b-actin was used as loading control for qPCR and Western Blot respectively. Results: SDF2 is downregulated in CTBs after 3h and 24h of tunicamycin exposure (2.5 and 5.0 mg/mL). In BeWo cells it is downregulated after 6h of treatment and it is restored after 24 h. GRP78/BiP is upregulated after 3h in CTBs, while in BeWo cells is upregulated only after 24 h. sXBP1 and CHOP mRNA confirmed ER stress condition. SDF2 mRNA did not change during tunicamycin treatment. In siRNA assays, CHOP was downregulated and GRP78/BiP activation was antecipated. We did not observed cleaved Caspase 3 for CTBs or BeWo cells. Conclusions: Downregulation of CHOP and antecipation of GRP78/BiP in siRNA treated cells, indicate a role of SDF2 in the control of survival/ apoptosis in trophoblast ER stress response. The placenta faces several changes in environmental conditions during development which could onset the UPR. Identifying new factors and key targets of this response could open a new field of investigation for gestation diseases. *Supported by FAPESP and CNPq P2.140-N. EFFECTS OF THE TRIIODOTHYRONINE IN MOUSE TROPHOBLAST CELLS CULTURE Juneo Silva, Natalia Ocarino, Rogeria Serakides Departamento de Clinica e Cirurgia Veterin arias, Universidade Federal de Minas Gerais, Belo Horizonte/Minas Gerais, Brazil Objectives: The objective of the present study was to evaluate the differentiation and gene expression in vitro of hormonal, immune and angiogenic factors in mouse trophoblast cells with different doses of T3. Methods: Twenty-five adult female mouse were sacrificed at 7,5 days of gestation. Ectoplacental cones were dissected from the maternal tissues and cultured during 24 and 48 hours in standard medium without T3 (control) andwith different doses of T3 (10-4M; 10-7M; 10-9M). The gene expression of Tpbp, Prl3b1, VEGF, PGF, PL-1, and INFy was evaluated in trophoblast cells by real-time RT-PCR. The data were analyzed using an SNK test. Results: The doses of 10-7 and 10-9 M of T3 increased the mRNA expression for Tpbp, PGF, INFy and PL-1 with 24 and/or 48 hours of culture. In addition, the dose of 10-7 M of T3 also increased the gene expression for Pl3b1 and VEGF with 24 hours of culture. The dose of 10-4 M of T3, on the contrary, reduced the mRNA expression for PL-1 and VEGF with 24 and 48 hours of culture, respectively. Conclusion: The doses of 10-7 and 10-9 M of T3 increased the differentiation and gene expression of hormonal, immune and angiogenic factors by mouse trophoblast cells and these effects were dependent of the culture time. In contrast, the dose of 10-4 M of T3 reduced the gene expression of VEGF and PL-1 by trophoblast cells. FUNDING: Fapemig, CNPq, PRPq P2.141-N. MIR-134 REGULATES INVASION AND PROLIFERATION IN HTR-8/SVNEO CELLS Stephanie Ospina-Prieto, Diana M. Morales-Prieto, Wittaya Chaiwangyen, Udo R. Markert University Hospital Jena. Department of Obstetrics. Placenta-Lab, Jena, Germany Background: Human placenta tissue exhibits a specific miRNA expression pattern that dynamically changes with the gestational age. This profile includes expression of a larger miRNA cluster located on the chromosome 14 (C14MC). Among these miRNAs, miR-134 is high expressed in first trimester but low in third trimester trophoblast cells. Recent reports suggested a role of miR-134 in the control of cell proliferation and invasion, but its function in trophoblast cells remains poorly understood. In this work, we aimed to investigate the role of miR-134 in the proliferation and invasion of HTR-8 cells. Material andmethods: Total RNAwas isolated fromHTR-8svneo cells using miRVana isolation kit. Expression of 43 miRNAs belonging to C14MC was quantitatively analysed by TaqMan Human MicroRNA Arrays. Expression of miR-134, miR-411 and miR-539 was confirmed by individual qPCR and normalized to RNU48. Silencing and overexpression of miR-134 was carried out by transfection with miR-134 miRIDIAN hairpin inhibitor, miR-134 mimic, or negative controls. Changes in cell viability, cell proliferation and invasion were assessed by MTS, BrdU and Matrigel assays, respectively. Results: C14MCmiRNAs are highly expressed in first trimester trophoblast cells and HTR-8 cells. Among them, 40miRNAs were down, and 6 were upregulated in third trimester trophoblast compared to first trimester. MiR134 was among the highest expressed miRNAs in HTR-8 cells. Silencing of