Figure 3

IGF-1 regulates the activity of MMP-9 and MMP-2 by different mechanisms. A, B) DU145 cells were treated with IGF-1 for varying amounts of time and both cell lysate and conditioned media were collected. A) Immunoblot of cell lysates indicates that IGF-1 induces an increase in the cellular protein expression of MMP-9 in a time-dependent manner at 8 and 24 hours of treatment, then a decrease at 32 and 48 hours of treatment. B) Immunoblot of conditioned media indicates that IGF-1 induces an increase in the secreted protein expression of MMP-9 in a time-dependent manner seen after 32 hours of treatment. The amount of protein in each lane is normalized to cell number. C, D) DU145 cells were pre-treated with the PI3-K inhibitor wortmannin or the MEK inhibitor PD98059, then treated with IGF-1 for 8 hours and both cell lysate and conditioned media were collected. The control consisted of adding a similar amount of the vehicle (1× PBS for IGF-1, DMSO for wortmannin or PD98059) for each addition in the test conditions. C) Immunoblot of lysates indicates that the IGF-1-induced increase in the cellular protein expression of MMP-9 is attenuated in the presence of PD98059 and even more so in the presence of wortmannin. No change in MMP-2 expression is seen in any of the conditions. D) Protein expression of MMP-2 in conditioned media is unaltered compared to mock-treated control cells, whereas that of MMP-9 follows the same pattern seen in cell lysates. The amount of protein in each lane is normalized to cell number. All blots are representative of three separate experiments in which a similar trend was observed.