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).
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EGF, a HER1 Ligand, Induces Partial Differentiation of EVT
The effect of EGF treatment on the expression of EVT phenotype markers was also assessed in the placental villous explant model. Serial sections of control outgrowths marked by HLAG (Fig. 3A) demonstrate high levels of Cx40 throughout the outgrowth (Fig. 3D), restriction of HER1 staining to the proximal EVT (Fig. 3G), and low levels of HER2 and a1 integrin staining throughout the outgrowth (Fig. 3, J and M). Treatment with EGF had little effect on Cx40 or HER1 expression (Fig. 3, E and H) but resulted in the strong upregulation of both HER2 and a1 integrin throughout the EVT outgrowth (Fig. 3, K and N). The EGF-treated EVT outgrowths extended across the matrigel surface as columns of EVT that maintained cell-cell contacts. Co-incubation of EGF with AG1478 prevented the upregulation of both HER2 and a1 integrin as compared to EGF alone (Fig. 3, L and O; n = 3).
FIG. 1. DCM and EGF induce a switch in HER isoform expression. Confluent, serum-starved JAR monolayers were treated with either (C) DCM, (E) EGF (10 ng/ml), (D) DCM + AG1478 (2 iM), or (F) EGF + AG1478 (2 iM) for 24 h. Under serum-free media conditions, JAR cells predominantly express (A) HER1 (red), whereas both DCM and EGF treatments resulted in the (C) downregulation of HER1 and (E) upregulation of HER2 (green). Co-incubation of treatment media with AG1478 prevented this switch in HER isoform expression (D and F). AG1478 treatment alone did not alter HER isoform expression (B) as compared with serum-free controls (A) (n = 3). Rabbit IgG and mouse IgG negative controls are displayed in panels G and H, respectively. Cell nuclei were stained with Hoescht. Bars = 25 iM.
FIG. 2. AG1478 inhibits DCM-mediated HER1/2 regulation. Representative immu-nofluorescent images from established first trimester placental villous outgrowths treated for 48 h with control SFM (left panel), DCM (middle panel), or DCM + AG1478 (2 lM;right panel). Serial sections were stained with a panel of antibodies identifying EVT phenotype markers. HLAG was expressed in explant outgrowths (A-C) marking the presence of EVT. DCM down-regulated HER1 in the EVT (E) as compared with control explants (D);these effects were prevented by co-incubation with AG1478 (F). Furthermore, DCM treatment upregu-lated EVT expression of HER2 (H) as compared with control explants (G), and AG1478 inhibited this upregulation (I). Cell nuclei were stained with Hoescht. Negative controls were performed using mouse IgG (J) or rabbit IgG (K) followed by anti-mouse/ anti-rabbit biotinylated antibody and Strep-tavidin-Alexa 488. Bars = 100 iM. EVT, extravillous trophoblast outgrowth;V, villous. n = 3.
FIG. 3. AG1478 inhibits the effects of EGF on the differentiation of EVT. Representative micrographs of established first trimester placental villous outgrowths treated for 48 h with either control SFM (left panel), EGF (10 ng/ml;middle panel), or EGF + AGl478 (2 lM;right panel). Serial sections were stained with a panel of antibodies identifying EVT phenotype marker proteins. HLAG was expressed in all explant outgrowths (AC), marking the presence of EVT. EGF upregulated EVT expression of HER2 (K) and a1 integrin (N) as compared with respective control explants (J and M). AG1478 inhibited this EGF-mediated upregulation (L and O). Neither EGF nor EGF + AG1478 treatments had significant effects on placental villous outgrowth expression of Cx40 or HER1 compared with control explants (D-I). Cell nuclei were stained with Hoescht. Negative controls were performed by omission of primary antibody followed by anti-mouse or anti-rabbit biotinylated antibody and Streptavidin-Alexa 488 (P and Q, respectively). Bars = 100 iM. EVT, extravillous trophoblast outgrowth;V, villous. n 3.