Fig. 4. Matrix-mediated signaling requires uPAR–β1-integrin association. (A) Matrix-induced MMP9 expression and ERK activation are dependent on uPAR–β1-integrin association. H1299 cells expressing WT, H, D and HD uPAR were serum-starved and cultured on poly-L-lysine (PL), fibronectin (Fn) or laminin-5 (Ln5), for 20 minutes or 24 hours in serum-free DMEM. The conditioned media after 24 hours on matrices were analyzed by gelatin zymography. Clear zones of degradation are seen at 92 kDa indicated as MMP9. Lysates from cells cultured for 20 minutes on matrices were immunoblotted with anti-ERK-P and anti-ERK antibodies. (B) Matrix-induced MMP1 expression is dependent on uPAR–β1-integrin association. Cells expressing WT and HD uPAR were serum-starved and cultured on poly-L-lysine, fibronectin or laminin-5 for 48 hours in serum-free DMEM. The conditioned media were immunoblotted for MMP1. Some WT cells were pretreated with the MEK inhibitor PD98059 (10 µM). Total ERK was detected as a loading control. (C) uPAR–β1-integrin blocking peptides inhibit matrix-induced MMP expression. Serum-starved cells expressing WT uPAR were pretreated with 0.4 mM peptide 325, scrambled 325, β1P1 or scrambled β1P1 and then plated on fibronectin or laminin-5 for 24 hours (for MMP9) or 48 hours (for MMP1) in serum-free DMEM. The conditioned media were analyzed by gelatin zymography for MMP9 or immunoblotted for MMP1. The volume of conditioned medium loaded to the gel was normalized to total protein in the lysate. All data shown are representative of three experiments with similar results.