Science in China Series C: Life Sciences © 2009
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Article
Dynamic change of Adamalysin 19 (ADAM19) in human placentas and its effects on cell invasion and adhesion in human trophoblastic cells ZHAO MeiRong1,2, QIU Wei1, LI YuXia1, SANG QingXiang Amy3 & WANG YanLing1† 1
State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China; Research Center of Environmental Science, Zhejiang University of Technology, Hangzhou 310032, China; 3 Department of Chemistry and Biochemistry and Institute of Molecular Biophysics, Florida State University, Tallahassee, FL 32306, USA 2
Human ADAM19 is a recently identified member of the ADAM family. It is highly expressed in human placentas, but its dynamic change and function at the human feto-maternal interface during placentation remain to be elucidated. In this present study, the spatial and temporal expression and cellular localization of ADAM19 in normal human placentas were first demonstrated, and the effects of ADAM19 on trophoblast cell adhesion and invasion were further investigated by using a human choriocarcinoma cell line (JEG-3) as an in vitro model. The data demonstrated that ADAM19 was widely distributed in villous cytotrophoblast cells, syncytiotrophoblast cells, column trophoblasts, and villous capillary endothelial cells during early pregnancy. The mRNA and protein level of ADAM19 in placentas was high at gestational weeks 8—9, but diminished significantly at mid- and term pregnancy. In JEG-3 cells, the overexpression of ADAM19 led to diminished cell invasion, as well as increases in cell adhesiveness and the expression of E-cadherin, with no changes in β-catenin expression observed. These data indicate that ADAM19 may participate in the coordinated regulation of human trophoblast cell behaviors during the process of placentation. ADAM19, human trophoblast cells, invasion, adhesion
A disintegrin and metalloproteinase (ADAMs) are a family of transmembrane proteins belonging to the metzincin clan of metalloendopeptidases. More than thirty ADAMs have already been identified[1], and participate in many physiological processes including sperm-egg binding and fusion[2,3], neurogenesis[4], and the development of various epithelial tissues[5], as well as pathological events such as the invasion and metastasis of some tumors[1]. Human ADAM19 (hADAM19), also referred to as adamalysin-19 and meltrin, was identified by Fritsche et al.[6] and Wei et al.[7] from a primary dendritic cell cDNA library. To date, ADAM19 expression has been demonstrated in multiple human tissues and cells, as well as several human cancer cell lines. In particular,
high levels of hADAM19 mRNA were detected in the placenta, heart, brain, lungs, bladder, spleen, appendix, and colon[7]. Similar to other ADAMs, hADAM19 has a signal sequence, a pro-domain with an unpaired cysteine residue (the “cysteine-switch”)[8], a metalloproteinase domain with a zinc-binding site, a disintegrin domain, a cysteine-rich domain, an epidermal growth factor (EGF)-like domain, a transmembrane domain, and a cytoplasmic domain with putative SH3 ligand binding sites. Received January 23, 2009; accepted March 4, 2009 doi: 10.1007/s11427-009-0102-8 † Corresponding author (email:
[email protected]) Supported by the National Natural Science Foundation of China (Grant No. 30530760), the Knowledge Innovation Program of Chinese Academy of Sciences (Grant No. KSCX2-YW-R-53) and a Program Enhancement Grant and Developing Scholar Award from Florida State University to Dr. QXA Sang.
Citation: Zhao M R, Qiu W, Li Y X, et al. Dynamic change of Adamalysin 19 (ADAM19) in human placentas and its effects on cell invasion and adhesion in human trophoblastic cells. Sci China Ser C-Life Sci, 2009, 52(8): 710-718, doi: 10.1007/s11427-009-0102-8
Data from Wei et al.[7] demonstrated that ADAM19 functions as an active metalloproteinase, cleaving α-2 macroglobulin in vitro. A null deficiency of ADAM19 in mice led to perinatal death, most likely because of cardiac defects[9]. Further investigation indicated that ADAM19 exerted essential roles in heart development via the proteolytic regulation of ErbB ligands[10]. Recent data from Huang et al.[11] demonstrated that ADAM19 could also inhibit cell migration mediated by both the α4 β1 and α5 β6 integrins, providing evidence for ADAM-integrin interactions in a cellular context. In mammals, a successful pregnancy is the result of complex maternal-fetal interactions. The adhesion of trophoblast cells to endometrial cells and the extracellular matrix (ECM), along with their invasion of the endometrium, are the key events for embryo implantation and placentation. It is well accepted that various proteinases and adhesive molecules coordinately participate in the control of trophoblast cell behaviors[12]. Recent data from Wang et al.[13], demonstrating a temporal change in ADAM19 expression at the fetomaternal interface in the rhesus monkey during early gestational stages, suggests the involvement of ADAM19 in various cellular events during embryonic implantation in primates. Therefore, considering the high expression of ADAM19 in human placentas, as well as its multifunctional potentials on account of its domain structure, we propose that ADAM19 may participate in regulating trophoblast cell functions in human placentas. In this present study, the spatial and temporal localization of ADAM19 in normal human placentas at different gestational stages was demonstrated. By using the choriocarcinoma cell line JEG-3, we further elucidated the effects of hADAM19 on trophoblast cell adhesion and invasion. These findings will aid in understanding the functions of ADAM19 during embryonic implantation and placentation.
1
Materials and methods
1.1 Human placental tissue preparation Human chorionic villi at gestational weeks 6—9 and 26 as well as term placentas were obtained at Beijing Haidian Hospital (Beijing, China) from patients who underwent therapeutic termination of pregnancy or term delivery. The patient’s informed consent and permission
from the Local Ethical Committee were obtained prior to tissue collection. There was no medical treatment prior to the termination of pregnancy. All placental tissues were normal by pathological exam. The gestational weeks of specimens at early pregnancies were determined by morphological observations of villi and pathological examinations, with the patient’s records of their menstrua as a reference. Half of each tissue sample was either flash frozen in liquid nitrogen or washed twice with PBS buffer and immediately fixed in 4% paraformaldehyde (PFA) at 4℃ for 10 h. Fixed tissues were then gradually dehydrated in ethanol and embedded in paraffin. Sections of 6 μm thickness were collected on SuperFrost Plus glass slides (Menzel-Gläser, Braunschweig, Germany)[14]. At least three specimens from each gestational stage were collected. 1.2 Culture of human choriocarcinoma (JEG-3) cells JEG-3 cells were cultured in DMEM medium (Gibco BRL, Grand Island, NY, USA) supplemented with 10% of fetal bovine serum (FBS; Gibco BRL), 2 mmol/L glutamine, and 1 mmol/L pyruvic sodium (Sigma, St. Louis, MO, USA). Cells were maintained at 37℃ in a humidified atmosphere of 5% CO2 and 95% air. The culture media were refreshed every day, and subculture, at a ratio of 1︰5, was performed with routine trypsinization every 4 days. 1.3 Transient transfection of hADAM19 The pCR3.1/hADAM19 plasmid with cDNA encoding full-length hADAM19 was constructed as previously described[15]. JEG-3 cells were cultured in 60 cm dishes and transfected with 5.0 µg of either the pCR3.1/ hADAM19 plasmid or the original pCR3.1 vector according to the manufacturer’s instructions for Lipofectamine 2000 Transfection Reagent (Invitrogen, Carlsbad, CA, USA). The transfected cells were named JEG/ hADAM19 and JEG/pCR3.1 respectively. Forty-eight hours after transfection, the cells were subjected to further assays including the protein expression of ADAM19, the invasion and adhesion of trophoblasts. 1.4 RNA isolation and semi-quantitative RT-PCR Tissue specimens were homogenized with a polytron homogenizer (Kinematika AG, Littau, Switzerland). Total RNA from tissues or cells was isolated using TRIZOl reagent (Gibco BRL) according to the manufacturer’s instructions. One microgram of total RNA was reverse transcribed in a 20 µL reaction mixture with
ZHAO M R et al. Sci China Ser C-Life Sci | Aug. 2009 | vol. 52 | no. 8 | 710-718
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random hexamer primers (Promega, Madison, WI, USA) by M-MuLV reverse transcriptase (Fermentas, Vilnius, Lithuania) as specified by the manufacturer. One microliter aliquots from the reverse transcription were amplified by PCR with specific primers (Runbio Biotechnology, Beijing, China) designed in accordance with cDNA sequences from the NCBI database (Table 1). The 25-µL PCR system contained 2 μL RT products, 200 μmol/L dNTPs, 2 mmol/L MgCl2, 1 IU Taq polymerase, and 10 pmol of each primer. The cycling number was determined by preliminary experiments to ensure that the amplification was within the reaction’s exponential phase. Amplification using total RNA as a template was included as a negative control to ensure the absence of genomic DNA contamination. PCR products were then subjected to electrophoresis on a 2% agarose gel and analyzed using the Gel-Pro Analyzer (software version 4.0; United Bio, Marlton, NJ, USA). The relative densities of detected genes were normalized against the value of GAPDH amplified with the same set of templates. 1.5 Immunohistochemistry (IHC) IHC was performed as previously described[16]. Briefly, paraffin sections were routinely rehydrated and retrieved in 20 µmol/L EDTA buffer (pH 8.0) prior to immersion in 1% hydrogen peroxide. The sections were incubated overnight at 4℃ with rabbit anti-hADAM19 IgG (10 µg/mL)[17]. The sections were further incubated with biotin-labeled goat anti-rabbit IgG (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and streptavidin peroxidase (Santa Cruz), and final visualization of positive staining was achieved by adding DAB substrate (Dako, Carpinteria, CA, USA). Counterstaining with haematoxylin was performed before the slides were mounted. Negative controls were performed by replacing the primary antibody with pre-immune IgG at the same concentration. Each slide was examined using a Nikon Eclipse 80i microscope (Nikon, Tokyo, Japan) with a SPOT digital camera system (Diagnostic Instruments Inc., Sterling Heights, MI, USA). Results were assessed based upon the evaluations of three indepenTable 1
dent examiners. 1.6 Immunofluorescent assays Cultured cells were fixed in 2% formaldehyde for 20 min and incubated at room temperature with 10% FBS for 1 h. The pre-treated cells were then incubated overnight at 4℃ with rabbit IgG against β-catenin (1︰500; Santa Cruz). Further incubation with FITC-labeled goat anti-rabbit IgG (Santa Cruz) followed at 37℃ for 1 h. The cells were counterstained with 0.1 mg/mL propidium iodide (PI; Sigma) for 5 min to visualize the nuclei. After mounting, the cells were observed using a Leica TCS NT confocal system (Leica, Wetzlar, Germany). 1.7 Western blot analysis Tissue specimens were homogenized with a polytron homogenizer (Kinematika) in a lysis buffer (20 mmol/L Tris-HCl, pH 8.0, 1 mmol/L DTT, 0.2% NP40, 100 μmol/L PMSF, 5 μg/mL aprotinin, chymostatin, leupeptin, pristine, and trypsin inhibitor). The cultured cells were lysed with lysis buffer on ice for 20 min. The lysates were centrifuged at 13000 r/min and the supernatants were collected. After measuring the protein concentration by Bradford assay, 25 μg of protein was subjected to 10% SDS-PAGE and transferred onto a polyvinylidene difluoride (PVDF) membrane. The membrane was blocked with 5% non-fat milk in PBS containing 0.1% Tween-20, incubated with rabbit anti-human ADAM19 IgG (1 µg/mL), and finally incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (1 ︰ 2000; Santa Cruz). Final visualization was achieved by the ECL Western Blotting Analysis System (Pierce, Rockford, IL, USA). The membrane was exposed to X-ray film (Fuji, Tokyo, Japan) and analyzed by the Gel-Pro Analyzer (software version 4.0; United Bio). 1.8 Transwell invasion assays Transwell invasion assays were conducted in 24-well fitted inserts with membranes (8 µm pore size; Millipore Corp., Bedford, MA, USA). Briefly, 2×104 JEG-3 cells in 200 μL DMEM medium with 10% FBS were plated
The primer sequences and reaction conditions for semi-quantitative RT-PCR
Gene name
Sequence of primers
Annealing temperature(℃)
Length of PCR product (bp)
ADAM19
Forward: CCA GAA GTA CCA TGA CAA CG
55
419
55
452
Reverse: ACA CTC TTC CCC ATC TTC C GAPDH
Forward: ACC ACA GTC CAT GCC ATC AC Reverse: TCC ACC ACC CTG TTG CTG TA
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ZHAO M R et al. Sci China Ser C-Life Sci | Aug. 2009 | vol. 52 | no. 8 | 710-718
in Transwell inserts pre-coated with collagen I (8 µg/insert; Cellmatrix Type I-A; Nitta Gelatin Inc., Osaka, Japan). Lower chambers were loaded with the same media. After incubating for 24 h, cells on the upper surface of the membranes were completely removed, and the migrated cells were fixed with 4% PFA and stained with hematoxylin. The membranes were then cut from the inserts and mounted onto glass slides. Cell invasion indices were determined by counting the number of stained cells in 5 randomly selected non-overlapping areas under a light microscope. 1.9 Cell adhesion analysis Cell adhesion analysis was performed as previous reported[18]. Briefly, JEG-3 cells were cultured in 96-well plates until 100% confluence was achieved. The JEG-3 cells transfected with pCR3.1/hADAM19 or pCR3.1 vectors were then seeded at 1×104 cells/well onto the attached bottom layer of cells in the 96-well plates. Two hours later, the non-adherent cells were discarded by washing with PBS buffer, and the remaining cells were fixed with 4% paraformaldehyde (PFA) and subjected to Giemsa staining. Cell amounts were measured by reading the absorbance at 655 nm, and the value of adherent cells was calculated by deducting that of the attached bottom cells.
1.10 Statistical analysis All experiments were repeated three times, each with at least three independent tissue specimens per developmental stage, or with at least three dishes of cells per treatment. The RT-PCR and Western blotting data were measured by comparing the densitometry value of detected molecules with that of GAPDH or actin in the same experimental set. The relative density was reported as the mean±SD according to the results of three independent experiments. Comparison of the values between groups was performed by ANOVA and P
