Figure 4.

Protease-activated receptor-1 (PAR1)-stimulated epidermal growth factor (EGF) receptor (EGFR) and ErbB2 transactivation contributes to sustained extracellular signal-regulated kinase-1 and -2 (ERK1/2) signaling and promotes breast carcinoma cell invasion. (a) MDA-MB-231 cells were pretreated with 0.1% dimethyl sulfoxide (DMSO) or 2 µM AG1478 for 2 h at 37 °C and then incubated with 100 µM TFLLRNPNDK for various times at 37 °C. Cell lysates were immunoblotted with anti-phospho-p44/42 mitogen-activated protein kinase (MAPK) (ERK1/2) antibody. Membranes were stripped and reprobed with anti-p44/42 MAPK (ERK1/2) to control for loading. The data (mean ± s.e.) shown are expressed as fold increase over basal from at least three independent experiments. (b) MDA-MB-231 cells were electroporated with 600 nM ErbB2-specific or nonspecific siRNA. After 48 h, serum-deprived cells were incubated with or without 100 µM TFLLRNPNDK for various times at 37 °C and ERK1/2 activity was measured and quantified as described above. The inset confirms the loss of ErbB expression in ErbB2 siRNA-treated cells as detected by immunoblotting. (c) Serum-starved MDA-MB-231 cells were left untreated or treated with varying concentrations of thrombin at 37 °C and then added to the upper well and cellular invasion toward NIH 3T3-conditioned medium (CM) was assessed. Representative data from one experiment is shown. (d) MDA-MB-231 cells were pretreated with 0.1% DMSO or 2 µM AG1478 for 30 min at 37 °C. After the treatment, cells were incubated in the absence or presence of 1 pM thrombin and then added the upper well and cellular invasion toward NIH 3T3 fibroblast-CM present in the lower well with or without AG1478 was assessed. The data (mean ± s.e.) shown are expressed as fold increase over untreated control from three independent experiments. The difference between thrombin-stimulated cellular invasion in control and AG1478-treated cells was significant (***P<0.005).