FIGURE 5.

CD44 serves as a docking molecule for MMP-9 on the cell surface and facilitates MMP-9-mediated cell migration. Effect of specific peptides on cell migration. A, CD44 forms a complex with MMP-9 in co-transfected COS-1 cells examined by a co-immunoprecipitation (IP) assay. MMP-9 in the conditioned medium (CM) and CD44 in the cell lysate (CL) were used as control for protein expression. B, silencing of CD44 in COS-1 cells using a shRNA approach. COS-1 cells were infected with retrovirus containing shRNA against CD44 and luciferase control. Total RNAs were extracted followed by a real time RT-PCR analysis. The relative quantitative value of CD44 expression was normalized against housekeeping genes, HPRT1 and GAPDH. Each bar represents the mean ± S.E. C, MMP-9 enhancement of cell migration is dependent on CD44. CD44-silenced COS-1 cells were transfected with MMP-9 or vector control followed by a transwell migration assay (upper panel). Each data point was performed in triplicate and the experiment was repeated three times (*, p < 0.05; **, p < 0.01). Western blotting for MMP-9, CD44, and α/β-tubulin (lower panel) was performed to monitor protein expression in the transfected COS-1 cells. D, peptides mimicking the outermost b-stand of blade I interfere with MMP-9 heterodimer formation (upper panel). 20 μg of total cell lysates were examined by Western blotting using anti-MMP-9 antibody to determine equal expression of MMP-9 (lower panel). IS4 and IS4 scrambled peptides (100 μm) were incubated with the cell lysate for 24 h prior to a co-immunoprecipitation assay. E, dose-dependent inhibition (from 1 μm to 1 mm) of MMP-9-mediated cell migration by IS4 peptides. COS-1 cells transfected with MMP-9 cDNAs or vector control were preincubated with 1% DMSO, IS4 peptide (SRPQGPFL), or IS4 scrambled peptide (GLSQPRFP) at different doses for 30 min followed by a transwell chamber migration assay. Each data point was performed in triplicate and the experiment was repeated three times (*, p < 0.05). F, inhibition of cell migration by MMP-9-specific peptides in human cancer cells expressing endogenous MMP-9. Human fibrosarcoma HT-1080 cells and breast cancer MDA-MB-435 cells were preincubated with control and specific peptides (100 μm) for 30 min followed by a transwell chamber migration assay. Each data point was performed in triplicate and the experiment was repeated three times (*, p < 0.05).