Activation of the Canonical Wingless/T-Cell Factor Signaling Pathway Promotes Invasive Differentiation of Human Trophoblast
2006; Elsevier BV; Volume: 168; Issue: 4 Linguagem: Inglês
10.2353/ajpath.2006.050686
ISSN1525-2191
AutoresJürgen Pollheimer, Thomas Loregger, Stefan Sonderegger, Leila Saleh, Sandra Bauer, Martin Bilban, K. Czerwenka, Peter Husslein, Martin Knöfler,
Tópico(s)Prenatal Screening and Diagnostics
ResumoThe molecular mechanisms governing invasive differentiation of human trophoblasts remain largely elusive. Here, we investigated the role of Wnt-β-catenin-T-cell factor (TCF) signaling in this process. Reverse transcriptase-polymerase chain reaction and Western blot analyses demonstrated expression of Wnt ligands, frizzled receptors, LRP-6, and TCF-3/4 transcription factors in total placenta and different trophoblast cell models. Immunohistochemistry of placental tissues and differentiating villous explant cultures showed that expression of TCF-3/4 strongly increased in invading trophoblasts. Some of these cells also accumulated dephosphorylated β-catenin in the nucleus. Wnt3A treatment of primary cytotrophoblasts and SGHPL-5 cells induced activity of TCF-luciferase reporters. Accordingly, the ligand provoked interaction of TCF-3/4 with β-catenin as assessed in electrophoretic mobility shift assays (EMSAs) and up-regulation of Wnt/TCF target genes as observed by Western blot analyses. Wnt3A stimulated trophoblast migration and invasion through Matrigel, which could be blocked by addition of Dickkopf-1, mediating in-hibition of canonical Wnt signaling. Dickkopf-1 also reduced basal migration, invasion, and proliferation of cytotrophoblasts, suggesting expression of endogenous Wnt ligand(s). Immunohistochemistry revealed that the percentage of extravillous trophoblasts containing nuclear β-catenin was significantly higher in placentas of complete hydatidiform mole pregnancies as compared to normal placentas. Thus, canonical Wnt signaling may promote invasive trophoblast differentiation, and exaggerated activation of the path-way could contribute to trophoblastic hyperplasia and local invasion. The molecular mechanisms governing invasive differentiation of human trophoblasts remain largely elusive. Here, we investigated the role of Wnt-β-catenin-T-cell factor (TCF) signaling in this process. Reverse transcriptase-polymerase chain reaction and Western blot analyses demonstrated expression of Wnt ligands, frizzled receptors, LRP-6, and TCF-3/4 transcription factors in total placenta and different trophoblast cell models. Immunohistochemistry of placental tissues and differentiating villous explant cultures showed that expression of TCF-3/4 strongly increased in invading trophoblasts. Some of these cells also accumulated dephosphorylated β-catenin in the nucleus. Wnt3A treatment of primary cytotrophoblasts and SGHPL-5 cells induced activity of TCF-luciferase reporters. Accordingly, the ligand provoked interaction of TCF-3/4 with β-catenin as assessed in electrophoretic mobility shift assays (EMSAs) and up-regulation of Wnt/TCF target genes as observed by Western blot analyses. Wnt3A stimulated trophoblast migration and invasion through Matrigel, which could be blocked by addition of Dickkopf-1, mediating in-hibition of canonical Wnt signaling. Dickkopf-1 also reduced basal migration, invasion, and proliferation of cytotrophoblasts, suggesting expression of endogenous Wnt ligand(s). Immunohistochemistry revealed that the percentage of extravillous trophoblasts containing nuclear β-catenin was significantly higher in placentas of complete hydatidiform mole pregnancies as compared to normal placentas. Thus, canonical Wnt signaling may promote invasive trophoblast differentiation, and exaggerated activation of the path-way could contribute to trophoblastic hyperplasia and local invasion. The invasive differentiation program of human trophoblasts represents an essential process of placentation and therefore plays a critical role in fetal growth and survival. Extravillous trophoblasts invading uterine tissue originate from proliferative trophoblast cell columns that attach to the decidualized endometrium. Between the 10th and 18th weeks of pregnancy, these cells establish the vascular connection between mother and fetus, ensuring continuous supply of nutrients and oxygen. They transform maternal spiral arteries into vessels of low resistance by replacing endothelial and vascular smooth muscle cells, thereby increasing blood flow to the placenta.1Pijnenborg R Bland JM Robertson WB Brosens I Uteroplacental arterial changes related to interstitial trophoblast migration in early human pregnancy.Placenta. 1983; 4: 397-413Abstract Full Text PDF PubMed Scopus (567) Google Scholar Analyses of anchoring villi in situ and of differentiating villous explant cultures in vitro suggest that the molecular processes controlling invasive trophoblast differentiation could have similarities with the mechanisms governing tumor invasion and metastasis. 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Moreover, we examined nuclear expression of β-catenin in normal placentas and tissues from CHM. The data suggest that canonical Wnt signaling regulates invasive trophoblast differentiation. In gestational diseases such as CHMs, elevated nuclear localization of dephosphorylated β-catenin may indicate abnormal activation of the signal transduction pathway. Placental tissues of early (between the 6th and 12th weeks of gestation, n = 38) and mid pregnancy (between the 18th and 22nd weeks of gestation, n = 12) were obtained from legal abortions with the permission of the ethical committee of the Medical University of Vienna. Informed consent was obtained from women donating their placentas. Tissue samples were used for immunohistochemistry, reverse transcriptase-polymerase chain reaction (RT-PCR), and Western blotting. Paraffin-embedded placentas of CHM pregnancies (n = 13) between the 7th and 14th weeks of gestation (7th week, n = 1; 8th week, n = 2; 9th week, n = 4; 10th week, n = 3; 11th week, n = 2; 14th week, n = 1) were retrieved from the archive of the Department of Clinical Pathology, Medical University of Vienna. Based on the original report of the Department of Obstetrics and Gynecology, which included data from high-resolution ultrasound and determination of hCG levels, histological evaluation was performed. All cases were further investigated by immunohistochemical p57KIP2 staining as previously mentioned.40Jun SY Ro JY Kim KR p57kip2 is useful in the classification and differential diagnosis of complete and partial hydatidiform moles.Histopathology. 2003; 43: 17-25Crossref PubMed Scopus (79) Google Scholar, 41Fukunaga M Immunohistochemical characterization of p57(KIP2) expression in early hydatidiform moles.Hum Pathol. 2002; 33: 1188-1192Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar JEG-3 choriocarcinoma cells and trophoblastic SGHPL-5 were cultivated in Dulbecco's modified Eagle's medium (DMEM) and in a 1:1 mixture of DMEM and Ham's F-12, respectively, supplemented with 10% fetal calf serum, 2 mmol/L glutamine, and 0.05 mg/ml gentamicin (all purchased from Invitrogen, Carlsbad, CA) as previously mentioned.42Knofler M Meinhardt G Bauer S Loregger T Vasicek R Bloor DJ Kimber SJ Husslein P Human hand1 basic helix-loop-helix (bHLH) protein: extra-embryonic expression pattern, interaction partners and identification of its transcriptional repressor domains.Biochem J. 2002; 361: 641-651Crossref PubMed Scopus (54) Google Scholar, 43Pollheimer J Bauer S Huber A Husslein P Aplin JD Knofler M Expression pattern of collagen XVIII and its cleavage product, the angiogenesis inhibitor endostatin, at the fetal-maternal interface.Placenta. 2004; 25: 770-779Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar SGHPL-5 cells exhibit features of invasive, extravillous trophoblasts because they were shown to express HLA class I, cytokeratin 7, and hCG.44Choy MY Manyonda IT The phagocytic activity of human first trimester extravillous trophoblast.Hum Reprod. 1998; 13: 2941-2949Crossref PubMed Scopus (106) Google Scholar All experiments using SGHPL-5 cells were performed between passages two and five. Preparation and cultivation of villous explants of different first trimester placentas (n = 15) between the 8th and 10th weeks of gestation were performed as described elsewhere.45Genbacev O Schubach SA Miller RK Villous culture of first trimester human placenta—model to study extravillous trophoblast (EVT) differentiation.Placenta. 1992; 13: 439-461Abstract Full Text PDF PubMed Scopus (154) Google Scholar Briefly, mesenchymal villi were dissected under the microscope and grown on Matrigel-coated 24-well plates (Becton Dickinson, Bedford, MA) for 24 and 72 hours. On extracellular matrix contact explant cultures formed anchoring villi and expressed invasive cell markers of the extravillous trophoblast, ie, α1β1 and α51 integrins and HLA-G1.5Vicovac L Jones CJ Aplin JD Trophoblast differentiation during formation of anchoring villi in a model of the early human placenta in vitro.Placenta. 1995; 16: 41-56Abstract Full Text PDF PubMed Scopus (141) Google Scholar, 46Bauer S Pollheimer J Hartmann J Husslein P Aplin JD Knöfler M TNF inhibits trophoblast migration through elevation of PAI-1 in first trimester villous explant cultures.J Clin Endocrinol Metab. 2004; 89: 812-822Crossref PubMed Scopus (226) Google Scholar In addition, extravillous trophoblast outgrowths were obtained 5 days after seeding of dissected villi on plastics. At the end of the culture period, invaded/migrated extravillous trophoblasts were mechanically separated from attaching villi, and protein/mRNA were isolated using the TRI-Reagent method as described elsewhere.43Pollheimer J Bauer S Huber A Husslein P Aplin JD Knofler M Expression pattern of collagen XVIII and its cleavage product, the angiogenesis inhibitor endostatin, at the fetal-maternal interface.Placenta. 2004; 25: 770-779Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar Both mRNA and protein were tested for the presence of the extravillous trophoblast-specific markers cytokeratin 7 and HLA-G1 using specific PCR primer sets46Bauer S Pollheimer J Hartmann J Husslein P Aplin JD Knöfler M TNF inhibits trophoblast migration through elevation of PAI-1 in first trimester villous explant cultures.J Clin Endocrinol Metab. 2004; 89: 812-822Crossref PubMed Scopus (226) Google Scholar or the following antibodies: anti-cytokeratin 7 (clone OV-TL 12/30, mouse, 10 μg/ml; DAKO, Glostrup, Denmark) or anti-HLA-G1 (clone MEM-G/1, mouse, 2 μg/ml; Exbio, Praha, Czech Republic), respectively (data not shown). Villous cytotrophoblasts were isolated at early gestation (between the 10th and 12th weeks, n = 10) by enzymatic dispersion and Percoll (5 to 70%) density gradient centrifugation as described.47Knofler M Saleh L Bauer S Galos B Rotheneder H Husslein P Helmer H Transcriptional regulation of the human chorionic gonadotropin β gene during villous trophoblast differentiation.Endocrinology. 2004; 145: 1685-1694Crossref PubMed Scopus (49) Google Scholar, 48Kliman HJ Nestler JE Sermasi E Sanger JM Strauss III, JF Purification, characterization, and in vitro differentiation of cytotrophoblasts from human term placentae.Endocrinology. 1986; 118: 1567-1582Crossref PubMed Scopus (1395) Google Scholar Furthermore, cells were immunopurified by depleting CD45-positive cells and fibroblast by using monoclonal anti-CD45 antibody (CD45RB, clone PD7/26, 0.2 μg/106 cells; DAKO) and anti-fibroblast-specific antigen antibodies (1:100, clone ASO2; Dianova, Hamburg, Germany), respectively, as described elsewhere.49Fisher SJ Cui TY Zhang L Hartman L Grahl K Zhang GY Tarpey J Damsky CH Adhesive and degradative properties of human placental cytotrophoblast cells in vitro.J Cell Biol. 1989; 109: 891-902Crossref PubMed Scopus (401) Google Scholar, 50Blaschitz A Weiss U Dohr G Desoye G Antibody reaction patterns in first trimester placenta: implications for trophoblast isolation and purity screening.Placenta. 2000; 21: 733-741Abstract Full Text PDF PubMed Scopus (129) Google Scholar Cell preparations were routinely checked by immunocytochemistry using anti-cytokeratin 7 and anti-vimentin antibodies (clone Vim 3B4, 1.2 μg/ml; DAKO) to detect trophoblasts (99.8%) and contaminating stromal cells (0.2%), respectively. Pure trophoblasts were resuspended in DMEM containing 10% fetal calf serum and cultivated in uncoated or Matrigel-coated 24-well plates (Costar, Corning, NY). Villous fibroblasts of first trimester placentas (n = 5) were isolated after gradient centrifugation of trypsinized placental material (between 25% and 35% Percoll) and passaged two times in DMEM supplemented with 10% fetal calf serum. Fibroblasts were characterized by vimentin immunocytochemistry (100% of cells), and the absence of contaminating trophoblasts was confirmed by cytokeratin 7 staining. For cryosectioning placental tissues or explant cultures were fixed in 2% paraformaldehyde, soaked in 0.5 mol/L sucrose/phosphate-buffered saline (PBS), covered with OCT compound (Sakura, Zoetermonde, The Netherlands), frozen in liquid nitrogen, and stored at −80°C as described recently.46Bauer S Pollheimer J Hartmann J Husslein P Aplin JD Knöfler M TNF inhibits trophoblast migration through elevation of PAI-1 in first trimester villous explant cultures.J Clin Endocrinol Metab. 2004; 89: 812-822Crossref PubMed Scopus (226) Google Scholar For immunohistochemistry, tissues (4-μm serial sections) were postfixed with 1% paraformaldehyde (10 minutes) and treated with 0.1% Triton X-100/PBS (5 minutes). After incubation in blocking solution (NEN, Boston, MA), slides were incubated overnight with primary antibodies followed by 1-hour treatment with fluorescein isothiocyanate-conjugated goat anti-mouse antibody (5 μg/ml; Molecular Probes, Eugene, OR) or fluorescein isothiocyanate-conjugated goat anti-rabbit antibody (5 μg/ml; Molecular Probes). The following primary antibodies were used: anti-active β-catenin (anti-ABC, clone 8E7, mouse, 1.5 μg/ml; R&D Systems, Minneapolis, MN), anti-Ki-67 (clone Ki-S5, mouse, 5 μg/ml; Chemicon, Temecula, CA), anti-p57KIP2, (C-20, rabbit, 2 μg/ml; Santa Cruz Biotechnology, Santa Cruz, CA), anti-TCF-3/4, (clone 6F12-3, mouse, 10 μg/ml; Upstate, Lake Placid, NY), anti-TCF-4 (clone 6H5-3, mouse, 5 μg/ml; Upstate) and anti-LEF-1 (anti-LEF, clone 2D12, mouse, 5 μg/ml; Upstate). Finally, all sections were counterstained with 1 μg/ml of DAPI (4′6 diamidine-2′-phenylindole dihydrochloride) from Roche (Mannheim, Germany) and covered with fluoromount G (Soubio, Birmingham, AL). Sections were analyzed by fluorescence microscopy (Olympus BX50, Hamburg, Germany) and digitally photographed. For paraffin embedding, placental tissue was prepared as previously described.51Knofler M Mosl B Bauer S Griesinger G Husslein P TNF-alpha/TNFRI in primary and immortalized first trimester cytotrophoblasts.Placenta. 2000; 21: 525-535Abstract Full Text PDF PubMed Scopus (62) Google Scholar Briefly, after fixation (4% formaldehyde, 24 hours at 4°C), samples were dehydrated and embedded in paraffin (Merck). Serial sections (4 to 5 μm) were prepared, deparaffinized, and finally heated in a microwave oven (2 × 5 minutes, 850 W). Blocking procedures and antibody staining were performed as described above. Total RNA isolation using TRI-Reagent was performed as suggested by the manufacturer (Molecular Research Center Inc., OH). Before supplementation of TRI-Reagent, tissue samples were first minced using a Braun microdismembrator (Mikro-Dismembrator S; B. Braun Biotech International, Melsungen, Germany). For RT-PCR analysis, first-strand cDNA synthesis using 2 μg of total RNA and SuperScript (10 U/μl; Invitrogen) was performed. Semiquantitative PCR amplification (45 seconds at 96°C, 1 minute at 56°C, 1 minute at 72°C) was performed with PCR reagent system (Invitrogen) in a RoboCycler Gradient 96 (Stratagene, Amsterdam, The Netherlands) using 0.5 U Taq polymerase (Invitrogen). Cycle numbers were optimized within the linear range of individual PCR reactions. In all experiments, possible DNA contamination was assessed by negative control RT-PCR in which reverse transcriptase was omitted in the RT step. Sequences of the forward and reverse primers to identify mRNA expression were 5′-TAAAGTGCCCGTGGTGCAG-3′ and 5′-TCTGTTCATGCTGAGGCTTCAC-3′ for LEF-1 (485 bp DNA fragment, 30 cycles), 5′-CATCTGCAGCTCTGCCATTGTGAC-3′ and 5′-AGGGATGATCGCCACTGGCAAG-3′ for TCF-3 (328 bp, 30 cycles), 5′-CGAGACGCCAAGTCACAGAC-3′ and 5′-TTGACCAATGAACTCGATAAAC-3′ for TCF-4 (451 bp, 30 cycles), 5′-AAGATGGTGCCAACTTCACCG-3′ and 5′-CTGCCTTCTTGGGGGCTTTGC-3′ for Wnt2b (321 bp, 30 cycles), 5′-GGGCCACCTGCTGAAGGAGAA-3′ and 5′-TTGACG AAGCAGCACCAGTGGAA-3′ for Wnt7b (333 bp, 30 cycles), 5′-AAGGCTTCCACAGTGACACAAGG-3′ and 5′-AGAGGAGAGAAACCCCAACTACCAC-3′ for fzd3 (330 bp, 30 cycles), 5′-AGTCTTCAGCGGCTTGTATCTTGT-3 and 5′-GCTCCGTCCGCTTTCACCTCT-3′ for fzd6 (561 bp, 30 cycles), and 5′-CATCCTCGTCTTTCACTCATC-3′ and 5′-GGCTCGAGGTCTGTCCTGCT-3′ for LRP-6 (550 bp, 30 cycles). GAPDH (sense primer, 5′-CCATGGAGAAGGCTGGGG-3′, and anti-sense primer, 5′-CAAAGTTGTCATGGATGACC-3′) was used as loading control (185 bp, 20 cycles). The PCR products were analyzed on 1.5% agarose gels containing ethidium bromide and photographed under UV radiation. All PCR fragments were sequence verified on a 16-capillary sequencer by using the nonradioactive ABI PRISM Terminator cycle sequencing ready reaction kit as specified by the supplier (Applied Biosystems, Foster City, CA). Cells were lysed in a buffer containing 10 mmol/L Tris-HCl (pH 7.8), 1 mmol/L KCl, 0.3% Triton X-100, and protease inhibitor cocktail (1:100; Sigma, St. Louis, MO). Protein quantity was evaluated by Bradford assay (Pierce, Rockford, IL), and quality was monitored by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Coomassie blue staining. A portion (20 μg) of total protein lysates was separated on denaturing polyacrylamide gels (10%), transferred to filters, and incubated overnight (4°C) with primary antibodies in Tris-buffered saline-0.3% Tween-20 (TBST) containing 0.5% nonfat dry milk as described recently.43Pollheimer J Bauer S Huber A Husslein P Aplin JD Knofler M Expression pattern of collagen XVIII and its cleavage product, the angiogenesis inhibitor endostatin, at the fetal-maternal interface.Placenta. 2004; 25: 770-779Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar Monoclonal anti-TCF-3/4 and anti-TCF-4 antibodies (Upstate) were used at a concentration of 2 or 1 μg/ml, respectively. Anti-LRP-6 antibody (Santa Cruz Biotechnology, Santa Cruz, CA) was used at a concentration of 0.4 μg/ml. Anti-β-catenin (clone 14, mouse; Transduction Laboratories, Lexington, KY) and anti-cyclin D1 (M20, rabbit; Santa Cruz Biotechnology) antibodies were used at 0.5 μg/ml and 0.2 μg/ml, respectively. After three washing steps in TBST, blots were incubated with peroxidase-linked anti-mouse IgG (1:80,000, NA 931; Amersham Pharmacia Biotech, Buckinghamshire, UK) or with peroxidase-linked anti-rabbit IgG (1:50,000; Amersham). Signals were developed using the Enhanced Chemiluminescence System (Amersham Pharmacia Biotech) according to the manufacturer's instructions. To monitor equal protein loading, blots were stripped in a buffer containing 100 mmol/L β-mercaptoethanol, 2% sodium dodecyl sulfate
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