Role of release of membrane-bound ligand in EGFR transactivation.
Involvement of membrane-bound EGFR ligand in LPA signal transduction was examined by use of MMP-inhibitor GM6001. Subconfluent cell cultures were pre-treated with 10 μM GM6001 for 30 min, then stimulated with 10 μM LPA for 1, 3 or 5 min as indicated. Cells were lysed for Western blotting or isoelectric focusing with NanoPro detection system as described in Methods. A: Western blots show the inhibition of LPA-induced EGFR, Akt and ERK phosphorylation in E10 and D2 cells, but no inhibition in SCC-9 cells. Graphs showing blot quantifications for E10 and D2 at 5 min are shown below the blots. B: NanoPro detection confirmed the partial ERK inhibition in E10 cells, however not statistically signinficant. C: Role of membrane-bound ligand in cell migration. Cell migration was detected after scratch wounding in a confluent cell layer. Inhibitor, solvent (DMSO) and/or LPA was added to the wells with serum-free medium as indicated. Digital images were obtained immediately after stimulation and after 17 h (D2), 24 h (E10) and 48 h (SCC-9) and wound closure was measured. Bars represent mean ± SEM, n = 3 or 4. LPA 10 μM, GM6001 10 μM. *indicates p ≤ 0.05, NS = Not significant.