Figure 3

EGF plus IL-1β synergistically upregulate MMP-9 in IBDC cells via the ERK1/2 pathway. A, RT-PCR showing that MMP-9 mRNA was expressed in BT474 cells and increased after EGF treatment. Knockdown of ERK1/2 using siRNA or pre-treatment with U0126 dose-dependently attenuated EGF-induced MMP-9 mRNA upregulation in the cells. B, Bars indicate the relative expression levels of MMP-9 mRNA normalized to GAPDH mRNA. ** P < 0.05 vs. control siRNA-transfected cells stimulated with EGF; ^ΔΔP < 0.05 vs. untransfected cells stimulated with EGF. C, IL-1β also increased MMP-9 mRNA expression in BT474 cells; EGF plus IL-1β synergistically increased MMP-9 expression compared to either EGF or IL-1β stimulation alone; this effect could be inhibited by ERK1/2 siRNA or pre-treatment with U0126. D, Bars indicate the relative expression levels of MMP-9 mRNA normalized to GAPDH mRNA. ** and ^▼▼, P < 0.05 vs. siRNA negative control-transfected cells stimulated with EGF plus IL-1β; ^ΔΔ and ^◇◇, P < 0.05 vs. untransfected cells stimulated with EGF plus IL-1β. E, EGF plus IL-1β synergistically increased MMP-9 expression compared to either EGF or IL-1β stimulation alone as analyzed by Western blotting. F to H, MMP-9 activity was analyzed using zymography. F, EGF increased MMP-9 activity; this effect could be attenuated by both ERK1/2 siRNA and U0126 in the cells. G, IL-1β also increased MMP-9 activity in BT474 cells; H, EGF plus IL-1β synergistically increased MMP-9 activity compared to either EGF or IL-1β stimulation alone.