Category Archives: Trophoblast Cells : Part 2

The DSCs-Expressed CD82 Controls the Invasiveness of Trophoblast Cells via Integrinbeta1/MAPK/MAPK3/1 Signaling Pathway: The DSC-Expressed CD82

The DSC-Expressed CD82 Inhibits the Invasiveness of Trophoblast Cells in Coculture
To demonstrate the effects of CD82 in DSCs on the invasion of human first-trimester trophoblast cells, we used the matrigel-based transwell assay to evaluate the invasiveness of primary trophoblast cells in indirect or direct coculture with DSCs transfected by si-CD82 with si-negative control. The number of cells that migrated to the lower surface was counted after 48 h of incubation. As shown in Figure 6, the invasive index of human first-trimester trophoblast cells was significantly higher in coculture with DSCs in CD82 silence than in that of the si-negative control (P < 0.01), and there was no difference between the direct and indirect coculture (P > 0.05), which suggests that the DSC-expressed CD82 inhibited the invasion of human first-trimester trophoblast cells by way of soluble molecules.
The DSC-Expressed CD82 Inhibits the Invasiveness of Trophoblast Cells in Coculture Partly via the Integrinfi1/MAPK/MAPK3/1 Signaling Pathway
In contrast to the results of the CD82-overexpressed BeWo cells, the expression of integrinpl (P < 0.05) and the proportion of phospho-MAPK3/1 to total MAPK3/1 (P < 0.05) in DSCs were obviously higher after CD82 silence than that of the si-negative control (Fig. 7a), and the proportions of phospho-AKT to total AKT and phospho-p38 to total p38 were also not changed between the two groups.
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The DSCs-Expressed CD82 Controls the Invasiveness of Trophoblast Cells via Integrinbeta1/MAPK/MAPK3/1 Signaling Pathway: Signaling Pathway

The DSCs-Expressed CD82 Controls the Invasiveness of Trophoblast Cells via Integrinbeta1/MAPK/MAPK3/1 Signaling Pathway: Signaling PathwayOverexpression of CD82 Inhibits Invasiveness of BeWo Cells Partly via Integrinfi1/MAPK/MAPK3/1 Signaling Pathway
Integrinpl, PIK3, and MAPK signaling pathways are involved in modulation of migration and penetration of human cancer cells and trophoblast cells. Therefore, we determined whether CD82 inhibits the invasion of human first-trimester trophoblast cells via integrinpl, PIK3, or MAPK signaling pathways. First, we detected the expression of the critical molecules of these signaling pathways and found that CD82 overexpression in BeWo cells significantly decreased the expression of integrinpl (P < 0.05) and the proportion of phospho-MAPK3/1 to total MAPK3/1 (P < 0.05), but did not influence the proportions of phospho-AKT to total AKT and of phospho-p38 to total p38 when compared with the pcDNA3.1(+) vector (Fig. 5a), which suggests that the integrinpl and MAPK/MAPK3/1 signaling pathways were involved in the CD82-mediated suppression of human trophoblast cell invasiveness.
We next determined whether the CD82-controlled expression of integrinpl and TIMP1 in BeWo cells was through the integrinpl and MAPK/MAPK3/1 signaling pathways. As shown in Figure 5b, the TIMP1 expression of BeWo cells was also increased by U0126 or anti-integrinpl neutralizing antibody, but the expression of integrinpl was not effected by U0126.
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The DSCs-Expressed CD82 Controls the Invasiveness of Trophoblast Cells via Integrinbeta1/MAPK/MAPK3/1 Signaling Pathway: Primary Human DSCs

CD82 Up-Regulates TIMP1 and Down-Regulates Integrinfi1 in Primary Human DSCs
RT-PCR and in-cell Western were used to identify the CD82 expression in primary human DSCs. The CD82 mRNA (Fig. 3a, left) and protein levels (Fig. 3a, right) of DSCs were significantly decreased after the CD82 silence (P < 0.01).
The interaction between DSCs and trophoblast cells induces expression of invasion-relevant genes by activating the intracellular signaling pathway. MMP2 and MMP9 are two important protein enzymes that degrade the ECM and that are involved in human first-trimester trophoblast invasion. The silencing of CD82 in DSCs significantly decreased the mRNA (Fig. 3b, left) and protein (Fig. 3b, right) levels of TIMP1 compared with the si-negative control (P < 0.01 and P < 0.05, respectively). However, the mRNA and protein levels of MMP2, MMP9, and TIMP2 showed no statistical difference (P > 0.05) between the two groups. 
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The DSCs-Expressed CD82 Controls the Invasiveness of Trophoblast Cells via Integrinbeta1/MAPK/MAPK3/1 Signaling Pathway: In-Cell Western

The DSCs-Expressed CD82 Controls the Invasiveness of Trophoblast Cells via Integrinbeta1/MAPK/MAPK3/1 Signaling Pathway: In-Cell WesternWestern Blot Analysis
Total protein extracted from human decidual tissues from the early pregnancy termination (n = 6) and unexplained miscarriage (n = 6) were prepared using RIPA buffer. Then 60 ig protein was loaded onto a 10% polyacrylamide-SDS gel. The resolved proteins were transferred onto polyvinylidene difluoride membranes (Bio-Rad) and incubated with a 1:500 dilution of mouse anti-human CD82 monoclonal antibody and a 1:1000.
Statistics
All values are presented as the mean 6 SD. One-way ANOVA was used to detect the difference of invasion, CD82, and other molecule transcriptions and translations in human DSCs and BeWo cells. Differences were considered as statistically significant at P < 0.05.
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The DSCs-Expressed CD82 Controls the Invasiveness of Trophoblast Cells via Integrinbeta1/MAPK/MAPK3/1 Signaling Pathway: Immunofluorescence (IF)

Immunofluorescence (IF)
The CD82-transfected BeWo cells and CD82-silenced DSCs were plated on coverslips in 24-well plates (BD Biosciences) for 96 h. Monolayers were fixed with 4% (vol/vol) paraformaldehyde for 10 min at room temperature, followed by 10-min incubation with 0.2% (vol/vol) Triton X-100. After washing with PBS, nonspecific binding was blocked with 10% FBS in PBS. Anti-CD82 rabbit polyclonal antibody (1:50; SC-5540; Santa Cruz Biotechnology) and anti-TIMP1 and anti-integrinp1 mouse monoclonal antibodies diluted in PBS were added for 1 h, avoiding light, and then the slides were mounted in 0.1% (wt/vol) 4,6-diamidino-2-phenylindole (DAPI; 1:50; Invitrogen) for nuclear counterstaining for 5 min at room temperature and observed in an Olympus BX51 fluorescence microscope (Olympus). The secondary antibodies were Texas Red and fluorescein isothiocyanate (FITC)-conjugated anti-rabbit and anti-mouse IgG (1:250; Rockland). The experiments were carried out in triplicate and repeated three times.
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The DSCs-Expressed CD82 Controls the Invasiveness of Trophoblast Cells via Integrinbeta1/MAPK/MAPK3/1 Signaling Pathway: In-Cell Western

The DSCs-Expressed CD82 Controls the Invasiveness of Trophoblast Cells via Integrinbeta1/MAPK/MAPK3/1 Signaling Pathway: In-Cell WesternIn-Cell Western
According to the description by Egorina et al., we used a newly set up assay called in-cell Western to determine the in-cell protein level of CD82, min at room temperature and then incubated with mouse anti-human CD82, or with mouse anti-human MMP2, MMP9, TIMP1, TIMP2 (R&D Systems), titin (Chemicon), or integrinp1 (R&D Systems) primary antibody with actin (Santa Cruz Biotechnology) as control. After overnight treatment at 4°C, the wells were incubated with a corresponding second IRDye 700DX-conjugated affinity-purified (red fluorescence) anti-mouse and IRDye 800DX-conjugated affinity-purified (green fluorescence) anti-rabbit fluorescence antibody recommended by the manufacturer (Rockland, Inc.). This procedure was carried out in the dark. Images of the target gene were obtained using the Odyssey Infrared Imaging System (LI-COR Biosciences Gmbh). The protein expression level was calculated as the ratio of the intensity of the target gene to that of actin. The experiments were carried out in triplicate and repeated three times.
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The DSCs-Expressed CD82 Controls the Invasiveness of Trophoblast Cells via Integrinbeta1/MAPK/MAPK3/1 Signaling Pathway: Immunostaining

CD82 Transfection into BeWo Cells
An expression vector for the human CD82 gene (nucleotides 181 to 985 in GenBank accession no. NM_002231.2), pcDNA3.1(+)-CD82, was kindly provided by K. Milde-Langosch (University Clinic Hamburg-Eppendorf). The plasmid transient transfection was generated according to the transfection guidelines by Lipofectamine 2000 (Invitrogen). In brief, 1 X 105BeWo cells were plated in 96-well plates without antibiotics. When the cells were 90%-95% confluent, 1 ц1 Lipofectamine 2000, 50 ц1 OPTIMEM, and 0.4 ig plasmid pcDNA3.1(+) vector (Invitrogen) or pcDNA3.1(+)-CD82 were mixed and incubated for 20 min at room temperature and then added to each well. After 6 h of incubation, these cells were incubated in DMEM for a further 48 or 72 h in 5% CO2 at 37°C, until the gene overexpression was confirmed by RT-PCR (48 h, six-well plates) and in-cell Western (72 h, 96-well plates).
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The DSCs-Expressed CD82 Controls the Invasiveness of Trophoblast Cells via Integrinbeta1/MAPK/MAPK3/1 Signaling Pathway: Immunostaining

The DSCs-Expressed CD82 Controls the Invasiveness of Trophoblast Cells via Integrinbeta1/MAPK/MAPK3/1 Signaling Pathway: ImmunostainingFor immunohistochemistry, paraffin sections (5 mm) of human decidua and villi from unexplained miscarriage or early pregnancy termination were dehydrated in Tris-buffered saline (TBS) and incubated with hydrogen peroxide and 1% bovine serum albumin (BSA)/TBS to block endogenous peroxidase. The samples were then incubated with murine anti-human vimentin monoclonal antibody (1:100; ZA0511; Dingguo), cytokeratin-7 antibody (1:100; 18-0234; Zymed Laboratories), anti-human CD82 antibody (1:50; SC-17752; Santa Cruz Biotechnology), or mouse IgG isotype overnight at 4°C in a humid chamber. After washing three times with TBS, the sections were overlaid with peroxidase-conjugated goat anti-mouse IgG (SP-9002; Golden Bridge International, Inc.), and the reaction was developed with 3,3-diaminobenzidine (DAB) and counterstained with hematoxylin. The experiments were repeated five times.
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The DSCs-Expressed CD82 Controls the Invasiveness of Trophoblast Cells via Integrinbeta1/MAPK/MAPK3/1 Signaling Pathway: MATERIALS AND METHODS

Tissue Collection and Cell Culture
All procedures involving participants in this study were approved by the Human Research Ethics Committee of Obstetrics and Gynecology Hosptial, Fudan University, and all subjects completed an informed consent to collect tissue samples.
Decidual and placental tissues were from elective termination of a first-trimester pregnancy (gestational age 6-8 wk) for no medical reason or from an unexplained miscarriage in the first trimester. The tissues from first-trimester pregnancies were immediately put into ice-cold Dulbecco modified Eagle medium (DMEM high D-glucose; Gibco), transported to the laboratory within 30 min after surgery, and washed in Hanks balanced salt solution for isolation of DSCs and trophoblast cells.
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The DSCs-Expressed CD82 Controls the Invasiveness of Trophoblast Cells via Integrinbeta1/MAPK/MAPK3/1 Signaling Pathway

The DSCs-Expressed CD82 Controls the Invasiveness of Trophoblast Cells via Integrinbeta1/MAPK/MAPK3/1 Signaling PathwayIntroduction
Implantation of human conceptus involves invasion of trophoblast cells into the uterine epithelium and the underlying stroma, which undergo a complex process of proliferation, migration, and differentiation. A typical feature of placentation in humans is the high-intensity invasion of trophoblasts in order to gain access to the maternal circulation during the first trimester. An impaired endovascular trophoblast invasion has been confirmed to be associated not only with preeclampsia—fetal intrauterine growth restriction—but also human first-trimester or late miscarriage.
Trophoblast cells display the unique capability to physi-ologicallyinvade the surrounding tissue, similar to tumors. Trophoblast and tumor cells share the same biochemical mediators: the matrix metallopoteinases (MMPs) and tissue inhibitor of metalloproteinases (TIMPs). MMPs, such as MMP9 and MMP2, are critical for extracellular matrix (ECM) degradation and the invasion of the trophoblast. Moreover, MMPs are also involved in cell-cell communication via cell surface proteins to support adhesion and migration. Therefore, as cytotrophoblast cells differentiate, they change their repertoire of cell adhesion molecules, cadherins, and integrins. Aberration in the levels and proteolytic activities of MMP9 and MMP2 is one of the important factors affecting placentation and spiral artery remodeling. However, as opposed to malignant invasion, trophoblastic invasion during implantation and placentation is stringently controlled both in space and time. The decidua forms a dense cellular matrix believed to generate a local cytokine environment that promotes trophoblast attachment and acts as a physical barrier limiting trophoblast overinvasion. In addition, decidual cells express TIMPs, extracelluar matrix proteins, and adhesion molecules that directly control invasion of the trophoblast cells.
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