HBEGF-Stimulated JAR Cell Migration Is Mediated by HER1 and the PI3K Pathway
JAR cell migration assays were conducted using increasing concentrations of HBEGF as chemoattractant in the lower well of a transwell migration assay. HBEGF stimulated a dose-dependent and significant 3- to 6-fold increase in migrated JAR cells at 10 and 100 ng/ml (Fig. 4A, P < 0.001; n = 4). Preincubation of the JAR cells with AG1478 resulted in complete inhibition of HBEGF-induced JAR cell migration (Fig. 4B, striped bar, P < 0.001). In contrast, preincubation of JAR cells with AG825 only had a partial inhibitory effect on HBEGF-mediated migration (Fig. 4B, shaded bar, P < 0.01; n = 4). To further assess the downstream effects of the signaling pathways involved in HBEGF-stimulated JAR cell migration, we preincubated JAR cells with a panel of signaling pathway inhibitors against the ERK and MAPK14 (p38), pKc, and PIK3 pathways for 30 min prior to and during 48-h exposure to HBEGF as the chemotactic stimulus in a migration assay. Only inhibition of the PIK3 pathway using 1 iM LY294002 (Fig. 4 C, bold striped bar, P < 0.001; n = 3) had a significant inhibitory effect on HBEGF-mediated JAR cell migration (gray bar; Fig. 4C). Continue reading
Tag Archives: HER1
HBEGF-Stimulated JAR Cell Migration Is Mediated by HER1 and the PI3K Pathway
DCM and EGF Promote EVT Differentiation Through HER1-Dependent Mechanisms
The effect of DCM and 10 ng/ml EGF on HER isoform expression in JAR cells was examined using dual immunofluorescence. These data demonstrate that 24-h incubation with either DCM (Fig. 1C) or EGF (Fig. 1E) resulted in downregulation of HER1 (red) and upregulation of HER2 (green) expression as compared to SFM control (Fig. 1A). Coincubation of either DCM (Fig. 1D) or EGF (Fig. 1F) with the HER1 antagonist AG1478 inhibited this switch in HER expression. Incubation of the cells with AG1478 alone did not influence HER isoform expression (Fig. 2B; n = 3).
Using the placental villous explant model we show that after 4 days, culture control explants possess extensive EVT outgrowths that are marked by HLAG expression (Fig. 2A). These explants express high levels of HER1 in the villous cytotrophoblast and early proliferative EVT (Fig. 2D), while HER2 is undetectable (Fig. 2G). Treatment of placental villous explants with DCM resulted in the loss of HER1 expression in the EVT outgrowth (Fig. 2E) and upregulation of HER2 (Fig. 2H). Co-incubation of the explants with DCM plus AG1478 inhibited the switch in HER isoform such that cells of the placental villous outgrowth retained HER1 (Fig. 2F) and did not upregulate HER2 (Fig. 2I) as compared to DCM treatment alone. Negative controls using either mouse IgG or rabbit IgG showed no staining (Fig. 2, J and K; n = 3).
Western Blot Analysis
Serum-starved JAR cells were stimulated with either EGF or HBEGF (10 ng/ml) over a 4-h time course. Following treatment, cells were washed with ice-cold PBS and lysed using a Bio-Rad cell lysis kit according to the manufacturer’s instructions (Bio-Rad, Mississauga, ON). Equal amounts of JAR cell proteins per lane were analyzed by Western blot analysis. Briefly, 60 ig of total protein per sample were added to NUPAGE LDS sample buffer (Invitrogen) with 2.5% p-mercaptoethanol and boiled for 5 min. Proteins were run on precast 3%—8% Tris-Glycein gels (Invitrogen) and transferred to polyvinylidene fluoride membrane (Millipore) at 4°C overnight. Membranes were blocked with 5% nonfat milk in Tris-buffered saline with Tween (TBS-T; 10 mM Tris [pH 7.5], 100 mM NaCl, and 0.1% Tween 20) for 1 h at room temperature. Membranes were incubated with either rabbit polyclonal anti-phospho HER1, Tyr992, Tyr1045, Tyr1068, or anti-phospho p85 PIK3 (New England Biolabs, ON) at 4°C overnight, and then washed and incubated with anti-rabbit horseradish peroxidase (1:2000)-linked secondary antibody (New England Biolabs) for 1 h at room temperature. Membranes were then stripped and reprobed with antibodies against the respective nonphosphorylated antibody (anti-HER1 and Anti p85 PIK3, 1:1000; New England Biolabs). To further assess equal loading of protein samples, blots were stripped for a second time and reprobed with an antibody against the housekeeping protein p-actin (Abcam, Cambridge, MA). Antibody reactions were detected using Bio-Rad WesternC chemiluminescence detection kit (Bio-Rad), followed by detection of chemiluminescence and band analysis using the VersaDoc 6 gel documentation system and Quantity One Software (Bio-Rad).
HER1 Signaling Mediates Extravillous Trophoblast Differentiation in Humans: JAR Cell Migration Assays
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.
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.