HER1 Signaling Mediates Extravillous Trophoblast Differentiation in Humans: HBEGF-Stimulated

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). more
EGF and HBEGF Differentially Activate HER1
The time-dependent activation of HER1 signaling following stimulation with either EGF or HBEGF (10 ng/ml) was assessed by Western blotting using a panel of antibodies against phosphorylated tyrosine residues in HER1 (Fig. 5). Blots were probed with either anti-HER1 (Tyr992), anti-HER1 (Tyr1045), or anti-HER1 (Tyr1068); each of these respective sites is associated with activation of specific downstream signaling pathways. Blots were also stripped and reprobed with anti-HER1 antibody that detected a single 170-kDa band corresponding to total nonphosphorylated HER1 in all samples (Fig. 5A). Interestingly, EGF (black bars) and HBEGF (gray bars) stimulated differential and time-dependent phosphorylation of the three tyrosine residues. At the tyrosine 992 site, only HBEGF induced band intensity that peaked between 10 and 30 min of stimulation (P < 0.001 vs. EGF, Fig. 5B). In contrast, EGF was the stronger activator of the tyrosine 1045 site where  band intensity increased between 1 and 4 h (P < 0.001 vs. HBEGF, Fig. 5C). Though the tyrosine 1068 site was phosphorylated by both EGF and HBEGF, as demonstrated by an increase in band intensity above their respective time zero controls, HBEGF was the stronger activator and led to more sustained phosphorylation across the time course (15 min: P < 0.05, 1 h: P < 0.001, Fig. 5D; n = 3).


FIG. 4. A and B) HBEGF-stimulated JAR cell migration is mediated by HER1. A) JAR cells were exposed to increasing concentrations (1-100 ng/ ml) of HBEGF (gray bars) that stimulated a significant increase in numbers of migrated cells. B) Preincubation of the JAR cells for 30 min with either AG4178 (2 цМ, striped bar) or AG825 (5 цМ, shaded bar) prior to exposure to HBEGF (10 ng/ml gray bar) resulted in inhibition of HBEGF-mediated JAR cell invasion. ***P < 0.001, **P < 0.01, *P < 0.05, n = 4. C) Effect of the PI3K inhibitor on HBEGF-stimulated JAR cell migration. JAR cells were preincubated with either 5 iM PD90589 (PD), 100 nM and 50 nM Ro318220 (Ro), 10 iM and 1 iM LY294002 (Ly), or 1 iM SB203580 (SB) prior to 48-h exposure to HbEGF (10 ng/ml) in the lower chamber of a migration assay. HBEGF-mediated migration of JAR cells was significantly inhibited by the PI3K inhibitor LY294002 alone. ***P < 0.001, **P < 0.01, *P < 0.05, n = 4.


FIG. 5. Differential tyrosine phosphorylation of HER1 by EGF and HBEGF. JAR cells were stimlulated with either EGF (10 ng/ml) or HBEGF (10 ng/ml) across a 4-h time course. A) Representative photomicrographs of Western blotting using a anti-total HER1 antibody detected a 170-kDa band corresponding to the known molecular weight of HER1 that did not change in intensity across the time course in response to EGF or HBEGF treatment. In contrast, the anti-phosphotyrosine antibodies against the Tyr 992, Tyr 1045, or the Tyr 1068 phosphorylation sites of HER1 detected bands that displayed time-dependent phosphorylation patterns that differed between EGF (black bars) and HEGF (gray bars) treatment. B) When expressed as a ratio over the corresponding total nonphosphory-lated HER1 bands, only HBEGF induced a significant increase in phosphorylation of the Tyr 992 site between 10 and 30 min. C) At the Tyr 1045 site, only EGF increased phosphorylation status above the respective control at 1 h and 4 h. D) While both EGF and HBEGF mediated an increase in phosphorylation of the Tyr 1068 site above the time 0 control, HBEGF lead to a stronger and more sustained activation at  1 h. *P < 0.05, **P < 0.01, ***P < 0.001, n = 3.

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