Treatment of Cell Lines and Fluorescent Immunocytochemistry
After a period of 24 h of serum starvation (above), JAR cells were preincubated with either vehicle (0.0025% DMSO) or AG1478 (2 iM in 0.0025% DMSO) for 30 min. JAR cells were then stimulated with either DCM + vehicle, EGF + vehicle, or +/- AG1478 (2 iM in 0.0025% DMSO) as indicated in each experiment for 24 h. DCM was collected from primary first trimester decidual cell cultures as previously described. Briefly, decidual cells were serum-starved in 0.2% BSA (Sigma) RPMI supplemented with Normocin (serum-free DCM) for 48 h. Following the treatment period, immunocytochemistry was performed on confluent JAR monolayers. JAR cells were fixed with 4% paraformaldehye, permeabilized using 0.02% triton X100, and quickly exposed to Sudan Black. All primary and secondary antibodies used in these procedures are detailed in Table 1.
Primary and secondary antibodies were prepared and used as described above. Slides were coverslipped using 90% glycerol. All incubations were performed in a light-protected incubation chamber. Fluorescent images were captured as described above.
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HER1 Signaling Mediates Extravillous Trophoblast Differentiation in Humans: JAR Cell Migration Assays
Treatment of Cell Lines and Fluorescent Immunocytochemistry
HER1 Signaling Mediates Extravillous Trophoblast Differentiation in Humans: Fluorescent Immunohistochemistry
Placental villous explants were fixed and processed to paraffin blocks from which 5-im sections were cut and adhered to Superfrost Plus glass slides (VWR, Mississauga, ON). Sections were deparaffinized in xylene and rehydrated through a descending concentration gradient of ethanol. Antigen retrieval was performed using either microwave pretreatment in 10 mM sodium citrate buffer (pH 6; Sigma), 0.02% Triton X100 (Sigma), 0.125% Trypsin (Sigma), or 10 ig/ml proteinase K (Roche, Montreal, QC, Canada) at 37°C (Table 1). Slides used for immunofluorescence detection were rapidly exposed to 0.1% Sudan Black in 70% ethanol to prevent autofluorescence (Sigma) and washed prior to blocking for nonspecific binding in serum-free DAKO protein block (DAKO, Mississauga, ON, Canada) for 1 h. All primary and secondary antibodies used in these procedures are detailed in Table 1. Primary antibodies were prepared in DakoCytomation Antibody Diluent with Background-Reducing Components (DAKO) and were incubated on sections overnight at 48C. In control experiments, primary antibodies were replaced with DAKO blocking solution or mouse or rabbit IgG at the same concentration as the primary antibody. Secondary biotinylated antibodies were prepared in PBS +
0.04% Azide (Sigma) + 0.008% gelatin (Sigma), used at 1:300, and detected using Streptavidin-Alexa488 (1:1000; Invitrogen); cells were counterstained with the nuclear stain Hoescht 33258 (1 lg/ml, 1 h; Sigma). All incubations were performed in a light-protected incubation chamber. Slides were washed in PBS and mounted in 50% glycerol/50% PBS for deconvolution microscopy. Immunofluorescent images were captured using a Sony Interline ICX285ER Progressive scan camera and an Olympus IX70 microscope (Olympus America Inc., Melville, NY). Images were collected using Resolve3D Image acquisition software and deconvolved using Deltavision softWoRx 2.50 software (Applied Precision, Issaquah, WA).
First trimester placentae and decidua were obtained at the time of elective terminations of pregnancy. Informed consent was obtained from each patient, and collections were approved by the Mount Sinai Hospital’s Review Committee on the Use of Human Subjects. Tissue was collected into ice-cold PBS for villous explant or decidual cell culture.
These data, together with the phenotype-specific expression profile of HER isoforms in EVT, suggests that different members of the EGF family may be capable of initiating distinct signaling events in proliferative and invasive EVT. A wealth of studies has established roles for HER signaling in a variety of cellular functions. In particular, HER1 has been reported to be an important signal mediator of mitotic events, and a growing number of studies report that HER2 signaling is involved in invasive processes such as those underlying breast cancer metastasis. The involvement of HER1 and HER2 in each of these respective processes, their EVT phenotype-specific expression pattern, and the detection of many EGF family ligands at the maternal/fetal interface, such as HBEGF, transforming growth factor-a (TGF), and amphiregulin (AREG), support a role for these proteins in the expression of EVT phenotype.
Extravillous trophoblast (EVT) migration and invasion into the maternal decidua are critical aspects of normal human placentation. The process of EVT invasion leads to the transformation of the maternal spiral arteries into large-diameter, low-resistance, high-flow vessels capable of supplying adequate blood into the intervillous space to nourish the growing conceptus. In normal pregnancies the depth of trophoblast invasion is strictly regulated to ensure adequate access of placental cells to the maternal spiral arteries. The importance of this regulation is underscored by the pathologies of pregnancy that arise from aberrant invasion; for instance, preeclampsia is marked by hypoinvasion of the trophoblast into the decidua and a failure of vascular remodeling, whereas choriocarcinoma, invasive moles, and placenta accreta are characterized by hyperinvasion of the trophoblast into maternal tissues.
The DSCs-Expressed CD82 Controls the Invasiveness of Trophoblast Cells via Integrinbeta1/MAPK/MAPK3/1 Signaling Pathway: Conclusion
Trophoblast invasion involves protelysis and remodeling of the uterine decidua. In addition to the MMPs, the integrin repertoire of the endometrium and decidua may play an important role in successful implantation. According to a timed expression correlating with embryo attachment, the avp3 and a4p1 integrins are considered markers of uterine receptivity. The avp3 integrin has been shown to be highly expressed at the time of embryo attachment, and aberrant expression of avp3 is associated with infertility. The miscarriage has been found to have a lower expression of a4p3 and a5p1 integrins in the endometrium during the implantation window than that of unexplained infertility. Moreover, the trophectoderm also express several integrins, a3, a5, p1, p3, p4, and p5, that are implicated in blastocyst attachment to the endometrial surface. In female mice lacking a functional integrinp1 gene, embryos develop normally to the blastocyst stage but fail to implant properly and die. In our study, CD82 in DSCs down-regulates the expression of integrinp1, which suggests a mechanism of CD82 in DSCs that controls the invasiveness of trophoblast cells.
The DSCs-Expressed CD82 Controls the Invasiveness of Trophoblast Cells via Integrinbeta1/MAPK/MAPK3/1 Signaling Pathway: DISCUSSION
Successful pregnancy depends on the ability of trophoblast cells to invade the uterine decidual stroma and to gain access to the maternal circulation, which is a mechanism similar to that of tumor cells. However, as opposed to malignant invasion, the trophoblast invasion is strictly limited in normal pregnancy. These events are regulated by the cross-talking of paracrine and autocrine factors between the trophoblast cells and DSCs at the maternal-fetal interface. DSCs secrete a lot of cytokines and express proteins, such as TIMP1, that control the invasiveness of the trophoblast cells. As a wide-spectrum tumor metastasis suppressor gene, CD82 is expressed in the primary DSCs but not in the primary trophoblast cells, so CD82 might be the media of cross-talking between DSCs and trophoblast cells. Consistent with transcription level, the decidua from the unexplained miscarriage had a much higher CD82 protein expression than that of the normal early pregnancy termination, based on immunohistochemistry and Western blot (P < 0.01; Fig. 8, b and c), which suggests that the CD82 overexpression in decidua restricted the appropriate invasion of trophoblasts, leading to early pregnancy wastage. Therefore, in the present study, we have investigated whether the DSCs-expressed CD82 regulates the invasion of trophoblast cells. As shown in Figure 3 and Figure 5, we have demonstrated that human DSCs from the first-trimester pregnancy express CD82 that inhibits the invasion of trophoblast cells through up-regulating the transcription and translation of TIMP1. DSCs and trophoblast cells produce TIMP1, which controls MMP secretion of DSCs and trophoblast cells. MMPs are partly responsible for placentation and spiral artery remodeling. MMPs are involved in pregnancy complications, including not only spontaneous abortion but also preeclampsia, fetal growth restriction, and so on, that result from an insufficient invasion of trophoblasts.
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.
The DSCs-Expressed CD82 Controls the Invasiveness of Trophoblast Cells via Integrinbeta1/MAPK/MAPK3/1 Signaling Pathway: Signaling Pathway
Overexpression 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.
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.