EGF stimulates proliferation in the bovine placental trophoblast cell line F3 via Ras and MAPK

Contour diagram of FITC-Annexin V/PI flow cytometry of cotyledonary cells after digestions with collagenase 1 (A) or trypsin (B). The lower left quadrant of each panel (R1) shows the viable cells, which exclude PI and are negative for FITC-Annexin V binding. The right quadrants (R2+R3) contain non-viable cells which are either in later stages of apoptosis or necrotic (upper right, positive for Annexin V and PI) or early apoptotic (lower right, positive for Annexin V and negative for PI). One representative experiment out of three is shown.

Bovine caruncular cell line BCEC-1 (A, E) and cotyledonary trophoblast cell line F3 on different substrata (B–D) in culture. (A) BCEC-1 at passage 21 (P21) shows curve-like morphology when grown on uncoated tissue flasks. (B) F3 cells (P24) exhibit a cobblestone-like phenotype with weakly confined colonies and some cells that display an outspread morphology. (C) After 13 days on collagen gel F3 (P24) colonies are clearly defined with no outspread cells present. (D, E) Scanning electron microscopy (SEM) of F3 (D, P24) and BCEC-1 cells (E, P21) grown on uncoated tissue flasks. Each image shows a single cell with dense apical microvilli (arrowheads).

Characterisation of F3 via immunofluorescence at different time points in culture. Directly after isolation, around 80% of yielded cells stain positive with an antibody against ovine placental lactogen (A, red). On cryosections of placentomes (B, in vivo control) this antibody stains exclusively TGC (red). The nuclei are stained with Hoechst (A–H, blue). Double labelling of F3 cells in culture (C–F) with cytokeratin (C–D, green) and ovine placental lactogen antibody (C–D, red) shows binucleate F3 cells (C, P2) still expressing TGC specific products while no cytokeratin is detectable. At higher passages (D, P18) similar cells express cytokeratin, but no TGC specific products are present anymore. F3 cells constantly express beta-tubulin (E, green) and tight junctional zonula occludens-2 (Zo-2) protein (E, red; P20). Representative isotype control (F; P20). Low density lipoprotein (Dil-AC-LDL, red) labelling shows no uptake of acetylated LDL in F3 cells (G; P20), whereas bovine umbilical vein endothelial cells (H, positive control) were positive. Scale bar is 50μm.

Identification of F3 cells according to genomic DNA or mRNA expression. (A) The presence of a Y-chromosome specific sequence (163bp) in F3 cells (lane 3, passage 3) confirmed the fetal origin. The liver of a male fetus (lane 2) served as positive control and maternal BCEC-1 cells as negative control (lane 1). Band sizes (lane M, top to bottom) are 500, 400, 300, 200 and 100bp. Lane 4 represents the negative PCR control. (B) Trophoblast F3 cells (lane 3) and bovine cotyledonary homogenate (lane 2) showed specific bands for PL at 230bp, while cultured fetal mesenchyme cells were negative (lane 1). GAPDH was used as a housekeeping gene. Specific products (197bp) for GAPDH were amplified from cultured fetal mesenchymal cells, cotyledonary homogenate and F3 cells (lanes 5–7, respectively). Lane 4 shows the negative PCR control.

(A) Daily growth curve of F3 trophoblast cells. 1×10⁵ cells were seeded in 25cm² culture flasks and grown in medium containing 10% FCS, 2mM Glutamine and antibiotics (full supplemented medium). The medium was changed every 48h and cell number was determined every 24h by counting four random fields per time on a haemacytometer. The experiment was repeated 8 times (n=8). (B) Growth response of F3 and BCEC-1 cells after incubation with EGF (50ng/ml) for 48h. Proliferation was determined by MTT assay. Results were normalized relative to the control (SF=serum-free medium) and then averaged (means±SEM, F3 n=20, BCEC-1 n=5). Asterisks represent p<0.001 compared to control. 10% FCS=SF+10% FCS; SF+EGF=SF+EGF 50ng/ml; SF+EGF+PD=SF+EGF 50ng/ml+50μM PD98059 MEK inhibitor.

Ras activation and MAPK phosphorylation in F3 or BCEC-1 cells upon stimulation with EGF. (A) Cells were stimulated for the indicated times with 50ng/ml EGF and analyzed for activated Ras (using a pull-down assay with Raf-RBD as bait, 21kDa). A representative western immunoblot probed with anti-Ras antibody shows that EGF leads to Ras activation only in F3 cells, while no activation is induced in maternal BCEC-1 cells. (B) Representative western blot of F3 and BCEC-1 protein extracts after stimulation with EGF (50ng/ml) for indicated times. Analysis of probes for MAPK phosphorylation (pMAPK42/44, 42 and 44kDa) revealed that a phosphorylation occurred in F3 cells, in contrast BCEC-1 cells showed no significant phosphorylation. Actin (42kDa) was used as loading control. (C) Densitometric data from five experiments (mean±SD) describing the time course of EGF induced MAPK phosphorylation in F3 cells. Optical densities were evaluated with the program Bio 1D (Vilber Lourmat) and corrected for actin as loading control. Asterisks represent p<0.001 compared to control (time 0). A maximum stimulation is present after 5min and decreases until 30min.