Amyloid Precursor Protein Overexpression in Down Syndrome Trophoblast Reduces Cell Invasiveness and Interferes with Syncytialization
2018; Elsevier BV; Volume: 188; Issue: 10 Linguagem: Inglês
10.1016/j.ajpath.2018.07.004
ISSN1525-2191
AutoresOscar Gee‐Wan Wong, Claire Ling-Yang Cheung, Philip P.C. Ip, Hys Ngan, Any Cheung,
Tópico(s)Pregnancy and preeclampsia studies
ResumoThe placentas of Down syndrome (DS) pregnancies exhibit morphologic and functional abnormalities. Although the increase in dosage of certain genes on chromosome 21 has been associated with the DS phenotype, the effects on placenta have seldom been studied. Herein, we examine the expression of four dosage-sensitive genes (APP, ETS2, SOD1, and HMGN1) in normal and DS placentas. We demonstrated significant overexpression of amyloid precursor protein (APP) in DS placentas at RNA and protein levels by real-time quantitative PCR, Western blot analysis, and immunohistochemistry. Inducible APP overexpression trophoblast cell line models were established using a Tet-On system. APP induction in HTR-8/SVneo dose-dependently decelerated cell growth, enhanced apoptosis, and reduced cell migration and invasion when compared with the uninduced controls. Concomitantly, decreased β-human chorionic gonadotropin in the culture medium was also detected on induction. Moreover, although forskolin treatment induced α/β-human chorionic gonadotropin and syncytin expression in BeWo cells, such induction of syncytialization was inhibited by APP overexpression. E-cadherin immunofluorescence also demonstrated a decrease in syncytia formation in forskolin-treated BeWo-overexpressing APP. By liquid chromatography–tandem mass spectrometry, proteins related to cell-cell adhesion, protein translation, processing, and folding were found to be up-regulated in APP-induced HTR-8/SVneo clones. Our data demonstrated, for the first time, the effects of increased APP expression in DS placenta. The placentas of Down syndrome (DS) pregnancies exhibit morphologic and functional abnormalities. Although the increase in dosage of certain genes on chromosome 21 has been associated with the DS phenotype, the effects on placenta have seldom been studied. Herein, we examine the expression of four dosage-sensitive genes (APP, ETS2, SOD1, and HMGN1) in normal and DS placentas. We demonstrated significant overexpression of amyloid precursor protein (APP) in DS placentas at RNA and protein levels by real-time quantitative PCR, Western blot analysis, and immunohistochemistry. Inducible APP overexpression trophoblast cell line models were established using a Tet-On system. APP induction in HTR-8/SVneo dose-dependently decelerated cell growth, enhanced apoptosis, and reduced cell migration and invasion when compared with the uninduced controls. Concomitantly, decreased β-human chorionic gonadotropin in the culture medium was also detected on induction. Moreover, although forskolin treatment induced α/β-human chorionic gonadotropin and syncytin expression in BeWo cells, such induction of syncytialization was inhibited by APP overexpression. E-cadherin immunofluorescence also demonstrated a decrease in syncytia formation in forskolin-treated BeWo-overexpressing APP. By liquid chromatography–tandem mass spectrometry, proteins related to cell-cell adhesion, protein translation, processing, and folding were found to be up-regulated in APP-induced HTR-8/SVneo clones. Our data demonstrated, for the first time, the effects of increased APP expression in DS placenta. Down syndrome (DS) is the most common congenital abnormality in humans, affecting 1 in approximately 1000 live births worldwide. An extra chromosome 21 (trisomy 21) is the major cause, accounting for approximately 95% of all cases. DS manifests in a spectrum of phenotypes, including challenges in cognitive ability and physical growth, cardiac defects, craniofacial alterations, and muscle hypotonia.1Antonarakis S.E. Lyle R. Dermitzakis E.T. Reymond A. Deutsch S. Chromosome 21 and Down syndrome: from genomics to pathophysiology.Nat Rev Genet. 2004; 5: 725-738Crossref PubMed Scopus (488) Google Scholar Growth retardation in trisomy 21 fetuses is partly attributable to placental structural abnormalities.2Qureshi F. Jacques S.M. Johnson M.P. Hume Jr., R.F. Kramer R.L. Yaron Y. Evans M.I. Trisomy 21 placentas: histopathological and immunohistochemical findings using proliferating cell nuclear antigen.Fetal Diagn Ther. 1997; 12: 210-215Crossref PubMed Scopus (36) Google Scholar, 3Scifres C.M. Nelson D.M. Intrauterine growth restriction, human placental development and trophoblast cell death.J Physiol. 2009; 587: 3453-3458Crossref PubMed Scopus (187) Google Scholar Various developmental defects in the placentas of DS pregnancies have been reported.4Pidoux G. Gerbaud P. Cocquebert M. Segond N. Badet J. Fournier T. Guibourdenche J. Evain-Brion D. Human trophoblast fusion and differentiation: lessons from trisomy 21 placenta.Placenta. 2012; 33 Suppl: S81-S86Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar The chorionic villi in trisomy 21 were reported to be bigger and more asymmetric and irregular in shape than were normal villi.2Qureshi F. Jacques S.M. Johnson M.P. Hume Jr., R.F. Kramer R.L. Yaron Y. Evans M.I. Trisomy 21 placentas: histopathological and immunohistochemical findings using proliferating cell nuclear antigen.Fetal Diagn Ther. 1997; 12: 210-215Crossref PubMed Scopus (36) Google Scholar, 5Ricco R. Dalena A.M. Valente T. Lettini T. Sanguedolce F. Delfino A.R. Quantitative study of placental villi in trisomy by analytical morphometry.Anal Quant Cytol Histol. 2009; 31: 41-48PubMed Google Scholar Histomorphometrically, the villi in trisomy 21 placentas had an increased percentage of two-layered trophoblast and an increased proportion of villus capillaries with nucleated red blood cells.6Roberts L. Sebire N.J. Fowler D. Nicolaides K.H. Histomorphological features of chorionic villi at 10-14 weeks of gestation in trisomic and chromosomally normal pregnancies.Placenta. 2000; 21: 678-683Abstract Full Text PDF PubMed Scopus (71) Google Scholar The increased two-layered trophoblast phenotype of trisomy 21 placenta has been postulated to be the consequence of defective trophoblast fusion and differentiation,4Pidoux G. Gerbaud P. Cocquebert M. Segond N. Badet J. Fournier T. Guibourdenche J. Evain-Brion D. Human trophoblast fusion and differentiation: lessons from trisomy 21 placenta.Placenta. 2012; 33 Suppl: S81-S86Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar because it has been shown that cultured villous cytotrophoblast cells from placentas of trisomy 21 fused inefficiently to form multinucleated syncytiotrophoblast.7Pidoux G. Guibourdenche J. Frendo J.L. Gerbaud P. Conti M. Luton D. Muller F. Evain-Brion D. Impact of trisomy 21 on human trophoblast behaviour and hormonal function.Placenta. 2004; 25 Suppl A: S79-S84Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar The inefficient differentiation from cytotrophoblast into syncytiotrophoblast leads to the secretion of an abnormal and weakly bioactive human chorionic gonadotropin (hCG) and results in defective regulation of placental development.8Frendo J.L. Guibourdenche J. Pidoux G. Vidaud M. Luton D. Giovangrandi Y. Porquet D. Muller F. Evain-Brion D. Trophoblast production of a weakly bioactive human chorionic gonadotropin in trisomy 21-affected pregnancy.J Clin Endocrinol Metab. 2004; 89: 727-732Crossref PubMed Scopus (43) Google Scholar In addition, the number of mature hCG receptor (LHCGR) molecules expressed on the surface of trisomy 21–affected cytotrophoblasts has also been shown to reduce significantly compared with normal cytotrophoblasts.9Pidoux G. Gerbaud P. Marpeau O. Guibourdenche J. Ferreira F. Badet J. Evain-Brion D. Frendo J.L. Human placental development is impaired by abnormal human chorionic gonadotropin signaling in trisomy 21 pregnancies.Endocrinology. 2007; 148: 5403-5413Crossref PubMed Scopus (49) Google Scholar The molecular basis underlies these alterations in trophoblast differentiation, and hCG signaling in DS placentas is largely unknown. It is believed that the various DS phenotypes are the results of the extra copy of dosage-sensitive genes among the genes present on human chromosome 21. A list of genes with direct evidence that their increased dose or allelic variation induces one or more phenotypes of DS has been compiled.10Lana-Elola E. Watson-Scales S.D. Fisher E.M. Tybulewicz V.L. Down syndrome: searching for the genetic culprits.Dis Model Mech. 2011; 4: 586-595Crossref PubMed Scopus (85) Google Scholar Microarray analysis of genes dysregulated in cultured trisomy 21 trophoblasts also revealed highly up-regulated genes on chromosome 21 in DS trophoblasts, supporting the hypothesis that candidate dosage-sensitive genes are involved in DS pathogenesis.11Rozovski U. Jonish-Grossman A. Bar-Shira A. Ochshorn Y. Goldstein M. Yaron Y. Genome-wide expression analysis of cultured trophoblast with trisomy 21 karyotype.Hum Reprod. 2007; 22: 2538-2545Crossref PubMed Scopus (31) Google Scholar The gene APP encoding amyloid precursor protein (APP) is located on chromosome 21 and is one of the earliest genes known to be overexpressed in the brain of DS patients.12Neve R.L. Finch E.A. Dawes L.R. Expression of the Alzheimer amyloid precursor gene transcripts in the human brain.Neuron. 1988; 1: 669-677Abstract Full Text PDF PubMed Scopus (313) Google Scholar Pathologic cleavage of APP into amyloid-β protein is a hallmark of Alzheimer disease. Overexpression of APP in DS brain contributes to the high penetrance of early-onset Alzheimer disease commonly seen in DS patients.13Weksler M.E. Szabo P. Relkin N.R. Reidenberg M.M. Weksler B.B. Coppus A.M. Alzheimer's disease and Down's syndrome: treating two paths to dementia.Autoimmun Rev. 2013; 12: 670-673Crossref PubMed Scopus (20) Google Scholar Up-regulation of APP in other organs/tissues of DS patients has also been reported.14Moncaster J.A. Pineda R. Moir R.D. Lu S. Burton M.A. Ghosh J.G. Ericsson M. Soscia S.J. Mocofanescu A. Folkerth R.D. Robb R.M. Kuszak J.R. Clark J.I. Tanzi R.E. Hunter D.G. Goldstein L.E. Alzheimer's disease amyloid-beta links lens and brain pathology in Down syndrome.PLoS One. 2010; 5: e10659Crossref PubMed Scopus (111) Google Scholar, 15Pallister C. Jung S.S. Shaw I. Nalbantoglu J. Gauthier S. Cashman N.R. Lymphocyte content of amyloid precursor protein is increased in Down's syndrome and aging.Neurobiol Aging. 1997; 18: 97-103Abstract Full Text PDF PubMed Scopus (24) Google Scholar, 16Govoni S. Bergamaschi S. Gasparini L. Quaglia C. Racchi M. Cattaneo E. Binetti G. Bianchetti A. Giovetti F. Battaini F. Trabuechi M. Fibroblasts of patients affected by Down's syndrome oversecrete amyloid precursor protein and are hyporesponsive to protein kinase C stimulation.Neurology. 1996; 47: 1069-1075Crossref PubMed Scopus (24) Google Scholar It was also one of the most significantly overexpressed genes in cultured trisomy 21 trophoblasts.11Rozovski U. Jonish-Grossman A. Bar-Shira A. Ochshorn Y. Goldstein M. Yaron Y. Genome-wide expression analysis of cultured trophoblast with trisomy 21 karyotype.Hum Reprod. 2007; 22: 2538-2545Crossref PubMed Scopus (31) Google Scholar However, whether APP is contributing to the DS placental phenotypes is unclear. In this study, we explored the expression patterns and functional impact of one of the four previously suggested dosage-sensitive genes (APP, ETS2, SOD1, and HMGN1) in DS placentas.10Lana-Elola E. Watson-Scales S.D. Fisher E.M. Tybulewicz V.L. Down syndrome: searching for the genetic culprits.Dis Model Mech. 2011; 4: 586-595Crossref PubMed Scopus (85) Google Scholar APP was found to be significantly overexpressed in DS placentas. An in vitro inducible expression system was then established to study the functional impact of APP overexpression on trophoblast biology. Our results suggest that APP is contributing to the defective trophoblast phenotype of DS. Seventy-one formalin-fixed, paraffin-embedded (FFPE) placental tissues, including 37 placentas from normal pregnancies (4 first trimester, 31 second trimester, and 2 third trimester) and 34 placentas from DS pregnancies (2 first trimester, 30 second trimester, 2 third trimester) were retrieved from the archive of the Department of Pathology, Queen Mary Hospital (Pokfulam, Hong Kong). Each block was confirmed to contain only placental tissue by hematoxylin and eosin staining. The use of the clinical samples for this study has been reviewed and approved by the HKU/Hospital Authority Hong Kong West Cluster Institutional Review Board (reference UW 13-124). The trophoblast cell line HTR-8/SVneo was a gift from Dr. Peeyush K. Lala (University of Western Ontario, London, Canada)17Graham C.H. Hawley T.S. Hawley R.G. MacDougall J.R. Kerbel R.S. Khoo N. Lala P.K. Establishment and characterization of first trimester human trophoblast cells with extended lifespan.Exp Cell Res. 1993; 206: 204-211Crossref PubMed Scopus (821) Google Scholar and was cultured in RPMI 1640 medium supplemented with 10% heat-inactivated qualified fetal bovine serum (catalog number 16140071; Gibco, Waltham, MA) and 0.01% penicillin-streptomycin. The choriocarcinoma cell line, BeWo, was purchased from ATCC (Manassas, VA) and was cultured in F-12K supplemented with 10% heat-inactivated qualified fetal bovine serum and 0.01% penicillin-streptomycin. The cells were maintained at 37°C under 5% CO2. Five sections (10 μm thick) were cut from each placenta FFPE block, and the total RNA was extracted using RNeasy FFPE kit (Qiagen, Venlo, the Netherlands). The quality of the samples was analyzed by a Bioanalyzer (Agilent, Santa Clara, CA). The mean RNA integrity number was 2.497 (SD = 0.9592; mean RNA integrity number normal = 2.495; mean RNA integrity number DS = 2.500). RNA of such quality is considered acceptable for RNA extracted from FFPE tissue and has been used in previous gene expression studies using real-time quantitative RT-PCR (RT-qPCR).18Haynes H.R. Killick-Cole C.L. Hares K.M. Redondo J. Kemp K.C. Moutasim K.A. Faulkner C. Wilkins A. Kurian K.M. Evaluation of the quality of RNA extracted from archival FFPE glioblastoma and epilepsy surgical samples for gene expression assays.J Clin Pathol. 2018; 71: 695-701Crossref PubMed Scopus (8) Google Scholar At least 500 ng total RNA was reverse transcribed into cDNA using a PrimeScript RT kit (Clontech, Mountain View, CA), according to the manufacturer's protocol. Each RT-qPCR contains 1× HotStart SYBR Green qPCR master mix (Excell, Shanghai, China), 1 μmol/L forward primer, 1 μmol/L reverse primer, 0.5 μL cDNA, and 4.5 μL milli-Q water. RT-qPCRs were run using a 7900HT Fast Real-Time PCR System (Applied Biosystems, Foster City, CA). Sequences of the primers used were as follows: APP, 5′-ATCTCCGCGGGGATCCATGCTGCCCGGTTTGGCACTGCTC-3′ (forward) and 5′-GCGGATCGATGGATCCCTAGTTCTGCATCTGCTCAAAGAA-3′ (reverse); ETS2, 5′-ATCTCCGCGGGGATCCATGAATGATTTCGGAATCAAGAAT-3′ (forward) and 5′-GCGGATCGATGGATCCTCAGTCCTCCGTGTCGGGCTGGAC-3′ (reverse); SOD1, 5′-ATCTCCGCGGGGATCCATGGCGACGAAGGCCGTGTGCGTG-3′ (forward) and 5′-GCGGATCGATGGATCCTTATTGGGCGATCCCAATTACACC-3′ (reverse); HMGN1, 5′-ATCTCCGCGGGGATCCATGCCCAAGAGGAAGGTCAGCTCC-3′ (forward) and 5′-GCGGATCGATGGATCCTTAATCAGACTTGGCTTCTTTCTC-3′ (reverse); YWHAZ, 5′-ACTTTTGGTACATTGTGGCTTCAA-3′ (forward) and 5′-CCGCCAGGACAAACCAGTAT-3′ (reverse); glyceraldehyde-3-phosphate dehydrogenase, 5′-AGATCATCAGCAATGCCTCC-3′ (forward) and 5′-CATGAGTCCTCCCACGATAC-3′ (reverse); and TOP1, 5′-GATGAACCTGAAGATGATGGC-3′ (forward) and 5′-TCAGCATCATCCTCATCTCG-3′ (reverse). The method of choice for antigen retrieval in immunohistochemistry was by heating the FFPE sections bathed in TE buffer (10 mmol/L Tris, pH 8.0, and 5 mmol/L EDTA) in a pressure cooker. The sections were then incubated with anti-APP antibody (catalog number ab32136; Abcam, Cambridge, UK) (dilution, 1:250) at 4°C overnight, and the signal was visualized using the EnVision+ Dual Link System (Agilent, Santa Clara, CA).19Siu M.K. Wong E.S. Chan H.Y. Ngan H.Y. Chan K.Y. Cheung A.N. Overexpression of NANOG in gestational trophoblastic diseases: effect on apoptosis, cell invasion, and clinical outcome.Am J Pathol. 2008; 173: 1165-1172Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar Negative control was prepared by replacing the primary antibody with phosphate-buffered saline.20Mak V.C. Wong O.G. Siu M.K. Wong E.S. Ng W.Y. Wong R.W. Chan K.K. Ngan H.Y. Cheung A.N. FBI-1 is overexpressed in gestational trophoblastic disease and promotes tumor growth and cell aggressiveness of choriocarcinoma via PI3K/Akt signaling.Am J Pathol. 2015; 185: 2038-2048Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar Fetal and placental tissue found to be positive was used as positive control. The stained sections were scanned with an Aperio slide scanner (Leica Biosystems, Wetzlar, Germany). The representative regions of trophoblast on each section were marked by a pathologist (A.N.Y.C.), and the staining intensity of the regions was evaluated by the positive pixel count v9 algorithm included in the system. Western blot analysis was performed according to standard procedures. Antibodies used include the following: APP (1:1000; catalog number ab32136; Abcam), green fluorescent protein (1:1000; ZsGreen; catalog number 632598; Clontech), β-actin (1:10,000; catalog number A5060; Sigma-Aldrich, St. Louis, MO), caspase 3 (1:1000; catalog number 9662; Cell Signaling, Danvers, MA), phosphorylated extracellular signal-regulated kinase (ERK) 1/2 (1:1000; catalog number 4376; Cell Signaling), ERK1/2 (1:3000; catalog number 9102; Cell Signaling), and LHCGR (1:1000; catalog number 19968-1-AP; Proteintech, Rosemont, IL). The functional implication of APP overexpression was investigated in a previously established trophoblast cell line HTR-8/SVneo.17Graham C.H. Hawley T.S. Hawley R.G. MacDougall J.R. Kerbel R.S. Khoo N. Lala P.K. Establishment and characterization of first trimester human trophoblast cells with extended lifespan.Exp Cell Res. 1993; 206: 204-211Crossref PubMed Scopus (821) Google Scholar HTR-8/SVneo was stably transfected with a Tet-On expression plasmid harboring APP ORF pTRE3G-BI-APP. Two clones, B2 and B10, were selected. APP was induced in B2 and B10 by including the recombinant protein Tet-Express (Clontech) in the culture medium. For assessing the impact of APP overexpression on syncytialization, BeWo was first stably transfected with pCMV-TET3G, which expresses the Tet-On 3G transactivator protein. BeWo-tet3G was then transiently transfected with pTRE3G-BI-APP using PolyJet (Signagen, Rockville, MD), according to the manufacturer's protocol, and APP was induced with doxycycline. Growth of induced cells over a period of 6 days was evaluated and compared using MTT assay, according to standard protocol. The rates of cell proliferation of induced cells were evaluated using bromodeoxyuridine (BrdU) assay 2 days after induction (Cell Signaling), according to the protocol provided by the manufacturer. DNA fragmentation was measured by flow cytometry analysis of propidium iodide stained cells for sub-G1 fraction. Apoptotic cells in cultured cells and FFPE tissue sections were visualized by TdT-mediated dUTP nick-end labeling (TUNEL) assay using the DeadEnd Colorimetric TUNEL System (Promega, Madison, WI), according to the protocol provided by the manufacturer. Cell mobility and invasiveness were evaluated using transwell migration/invasion assays, as previously described.19Siu M.K. Wong E.S. Chan H.Y. Ngan H.Y. Chan K.Y. Cheung A.N. Overexpression of NANOG in gestational trophoblastic diseases: effect on apoptosis, cell invasion, and clinical outcome.Am J Pathol. 2008; 173: 1165-1172Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar Briefly, induced B2 and B10 cells were seeded into the upper chamber of transwell inserts at a density of 3 × 105 per insert. The inserts were uncoated for migration assay or coated with Matrigel (Corning Life Sciences, Oneonta, NY) for invasion assay. The cells were allowed to migrate through the membrane for 24 hours, after which the cells in the upper chamber were removed and migrated cells were stained with crystal violet. Syncytialization of BeWo treated with 100 μmol/L forskolin was visualized by immunofluorescence staining of E-cadherin (catalog number 610181; BD Biosciences, San Jose, CA). Syncytialization was quantified by dividing the number of nuclei belonging to fused cells by the total number of nuclei in a field. Five fields were counted for each condition. The concentration of total β-hCG in the conditioned medium of B2 and B10 with or without induction by Tet-Express was measured by ARCHITECT total β-hCG test (Abbott Ireland, Longford, Ireland), according to the manufacturer's instruction. The sample processing and liquid chromatography–tandem mass spectrometry was performed in the Li Ka Shing Faculty of Medicine, Proteomics and Metabolomics Core Facility, The University of Hong Kong. Proteins were extracted from cell lysates of uninduced and induced (with 4 ng/μL Tet-Express protein) HTR-8/SVneo-APP by bead beating using Precellys homogenizer (Bertin Technologies, Versailles, France). Three replicates of each protein sample were subjected to trypsin digestion, followed by reduction and alkylation using a filter-aided sample preparation method.21Wisniewski J.R. Zougman A. Nagaraj N. Mann M. Universal sample preparation method for proteome analysis.Nat Methods. 2009; 6: 359-362Crossref PubMed Scopus (5042) Google Scholar LysC-Tryptic peptides were extracted, cleaned, and reconstituted for liquid chromatography–tandem mass spectrometry analysis performed on an Orbitrap Fusion Lumos mass spectrometer (Thermo Fisher Scientific, Bremen, Germany) interfaced with a Dionex 3000RSLC nanoLC (Thermo Fisher Scientific, San Jose, CA). The high-resolution, high–mass accuracy mass spectrometry data obtained were processed using Maxquant version 1.5.3.30 (Max Planck Institute of Biochemistry, Munich, Germany), wherein data were searched using the Andromeda algorithm against National Center for Biotechnology Information human RefSeq protein database. Appropriate parameters were used to obtain data at 0.1% false discovery rate at peptide and protein levels. Proteins identified from both uninduced and induced conditions were quantified using the peptide label-free quantification intensities, and their ratio obtained was used for label-free quantitation to calculate the fold change. Data visualization and statistical data analysis were performed with Perseus software version 1.5.4.1 (Max Planck Institute of Biochemistry). To investigate the expression of the four candidate dosage-sensitive genes (APP, ETS2, SOD1, and HMGN1) in DS placentas, RT-qPCR was used to measure the relative expression levels of the genes in FFPE samples of placentas from normal and DS pregnancies. YWHAZ, which was previously shown to be a suitable reference gene for gene expression studies in placenta, was used as the reference gene.22Meller M. Vadachkoria S. Luthy D.A. Williams M.A. Evaluation of housekeeping genes in placental comparative expression studies.Placenta. 2005; 26: 601-607Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar Two additional reference genes (GAPDH and TOP1) were used for further validation and yielded similar observations.23Kaitu'u-Lino T.J. Hastie R. Cannon P. Lee S. Stock O. Hannan N.J. Hiscock R. Tong S. Stability of absolute copy number of housekeeping genes in preeclamptic and normal placentas, as measured by digital PCR.Placenta. 2014; 35: 1106-1109Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar Although all four genes are located on chromosome 21, not all of them are overexpressed in DS placenta. APP mRNA was, on average, overexpressed by approximately 2.3-fold in DS placenta than in normal placentas (P < 0.0009) (Figure 1A). SOD1 was slightly overexpressed, but did not reach statistical significance (P = 0.3155) (Figure 1A). ETS2 and HMGN1, on the other hand, were both statistically significantly down-regulated (Figure 1A). Because APP was shown to be overexpressed in DS placenta, it was chosen for further investigation. To characterize the spatial expression pattern of APP in placentas, the APP protein was stained in the FFPE tissue samples by immunohistochemistry. Cytoplasmic immunoreactivity for APP was found predominantly in villous cytotrophoblast and stromal cells as well as extravillous intermediate trophoblasts (Figure 1, B–E). To compare the APP protein expression level quantitatively, representative trophoblastic areas on all the slides were scanned by the Aperio ScanScope digital pathology system (Leica Biosystems). The APP immunoreactivity in chorionic villi of DS placenta was, on average, approximately 2.3-fold that of normal placentas (P < 0.0001) (Figure 1F). To investigate the functional impact of APP overexpression on trophoblast, inducible APP expression was established in the trophoblast cell line HTR-8/SVneo. HTR-8/SVneo was stably transfected with pTRE3G-BI-APP, and single clones were selected. Two clones, B2 and B10, were used for subsequent studies because of their responsiveness to induction by the recombinant inducer protein Tet-Express. Titration of Tet-Express showed that both B2 and B10 responded dose dependently by expressing APP at different levels (Figure 2A). When induced, pTRE3G-BI-APP also expresses ZsGreen1 Fluorescent Protein, which serves as an induction marker (Figure 2A). Adding Tet-Express to parental HTR-8/SVneo had no observable effect on the morphology or cell growth rate (data not shown). B2 and B10, induced to express APP, grew slightly slower than cells without induction (Figure 2B). This decelerated cell growth was not attributable to lower cell proliferation, because induced cells incorporated BrdU at the rate as uninduced cells (Figure 2C). There was increased apoptosis, however, in the APP-induced cells, as suggested by increased DNA fragmentation observed by flow cytometry (Figure 2, D and E), increased presence of cleaved caspase 3 (Figure 2F), and increased TUNEL signal (Figure 2G). To further examine whether APP overexpression is associated with higher apoptotic activity in DS placenta, TUNEL was performed in FFPE blocks of 10 normal and 10 DS placentas. Although only scanty TUNEL signals could be observed predominantly at the nuclei of the syncytiotrophoblasts of normal placental villi, more frequent TUNEL signals were found in DS placental villi, also prevalent at the nuclei of syncytiotrophoblasts (Figure 2H), concurring with the previous findings.24Wong S.Y. Ngan H.Y. Chan C.C. Cheung A.N. Apoptosis in gestational trophoblastic disease is correlated with clinical outcome and Bcl-2 expression but not Bax expression.Mod Pathol. 1999; 12: 1025-1033PubMed Google Scholar To evaluate the effect of APP overexpression on cell mobility and invasiveness, B2 and B10 cells were induced to express APP and used in transwell migration/invasion assays. Both clones displayed dose-dependent reduction in the number of cells migrated through the membrane (Figure 3A). A similar trend was observed in Matrigel-coated transwells (invasion assay), which suggested that B2 and B10 migrated and invaded through transwell slower when induced with APP (Figure 3A). Phosphorylation of ERK1/2 is a key event in activating the invasive ability of extravillous trophoblasts.25Gupta S.K. Malhotra S.S. Malik A. Verma S. Chaudhary P. Cell signaling pathways involved during invasion and syncytialization of trophoblast cells.Am J Reprod Immunol. 2016; 75: 361-371Crossref PubMed Scopus (109) Google Scholar The phosphorylation status of ERK1/2 was, therefore, investigated in the induced cells. Concurring with the reduced cell mobility and invasiveness, phosphorylation of ERK1/2 was suppressed by APP overexpression (Figure 3B). To examine whether APP overexpression interferes with hCG secretion, the conditioned medium of B2 and B10, induced to express APP, was collected, and total β-hCG concentration was measured. Over a period of 48 hours, the amount of hCG in the conditioned medium of uninduced B2 and B10 accumulated (Figure 4A). APP induction dose dependently reduced the amount of total β-hCG secreted by the cells (Figure 4A). Because hCG is mainly produced by syncytiotrophoblasts, we hypothesized that APP overexpression has an impact on the cytotrophoblast-syncytiotrophoblast differentiation. Cultured cytotrophoblasts can be induced to fuse by forskolin treatment.26Wice B. Menton D. Geuze H. Schwartz A.L. Modulators of cyclic AMP metabolism induce syncytiotrophoblast formation in vitro.Exp Cell Res. 1990; 186: 306-316Crossref PubMed Scopus (255) Google Scholar HTR-8/SVneo, however, could not undergo syncytialization (data not shown). Another inducible system, BeWo-Tet3G, transiently transfected with pTRE3G-BI-APP, induced by doxycycline, was therefore used to assess the effect of APP overexpression on syncytialization (Figure 4B).27Yoshie M. Kashima H. Bessho T. Takeichi M. Isaka K. Tamura K. Expression of stathmin, a microtubule regulatory protein, is associated with the migration and differentiation of cultured early trophoblasts.Hum Reprod. 2008; 23: 2766-2774Crossref PubMed Scopus (27) Google Scholar Syncytialization was observed through E-cadherin immunofluorescence of BeWo-Tet3G cells. Distinct immunoreactivity for E-cadherin at the cell membrane of individual cells was found in uninduced BeWo-Tet3G cells, highlighting the cell boundaries (Figure 4C). On forskolin treatment, multinucleated cells with no observable E-cadherin separating the nuclei, suggestive of syncytialization, were found (Figure 4C). Induced APP expression (marked by green fluorescent protein) reduced the number of such multinucleated cells (Figure 4, C and E). Forskolin treatment also led to increased α-hCG and β-hCG, as well as syncytin transcript, as assessed by RT-qPCR, again supportive of syncytialization (Figure 4D). Although APP induction slightly and insignificantly up-regulated syncytin, it significantly suppressed forskolin-mediated up-regulation of syncytin; however, it did not reach statis
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