(A) Changes in fibronectin-induced MMP-2 and MMP-9 production achieved by manipulating integrin-mediated signal transduction pathways. 107 CEM cells were exposed to the above-described chemicals 30 minutes before exposure to fibronectin. Conditioned medium was obtained after 6 hours, concentrated, and subjected to gelatin zymography. Graph shows the variation in the intensity of the fibronectin-induced gelatinolytic signal induced by treatment with the different products and their simultaneous effect on cell adhesion, both expressed as a percentage of those obtained by fibronectin alone (labeled basal). (B) Intensity of MMP-2 and MMP-9 production induced by exposure to soluble or solid-phase fibronectin. 107 CEM cells were exposed to soluble (10 μg/mL) or solid-phase (plastic surfaces coated with 100 μg/mL) fibronectin for 24 hours. Conditioned media were concentrated and subjected to gelatin zymography. (C) Fibronectin-induced MMP-2 and MMP-9 production by cells transfected with a dominant negative mutant of Ha-Ras. 107CEM cells transfected with Ha-Ras-Asn 17, cloned in the dexamethasone-inducible vector pMMTV, were exposed to soluble fibronectin for 24 hours. Conditioned medium from transfected cells was concentrated and subjected to gelatin zymography. Lane 1 shows absence of gelatinolytic activity in conditioned medium from transfected cells stimulated with 0.5 μmol/L dexamethasone without exposure to fibronectin. Lanes 2 and 3 display gelatinolytic signals provided by conditioned medium from untreated transfected cells (lane 2) and from transfected cells treated with 0.5 μmol/L dexamethasone (lane 3), both exposed to fibronectin. (D) Effect of PI-3K, MEK-1/2, and p38 MAPK inhibition on fibronectin-induced MMP-2 and MMP-9 production. 2 × 107 CEM cells were preincubated with several very specific inhibitors of PI-3K (wortmannin), ERK-1/2 kinase (PD 98059), or p38 MAPK (SB 202190) for 30 minutes before exposure to soluble fibronectin at 10 μg/mL. Conditioned medium was obtained 6-hours later, concentrated, and subjected to gelatin zymography. The picture shows the gelatinolytic signals provided by conditioned medium from untreated cells exposed to fibronectin (lane 1), or pretreated with PD 98059 (lane 2), wortmannin (lane 3), or SB 202190 (lane 4).
