Laminin-5 (Ln-5) is an extracellular matrix substrate for cell adhesion and migration, which is found in many epithelial basement membranes. Mechanisms eliciting migration on Ln-5 need to be elucidated because of their relevance to tissue remodeling and cancer metastasis. We showed that exogenous addition of activated matrix metalloprotease (MMP) 2 stimulates migration onto Ln-5 in breast epithelial cells via cleavage of the γ2 subunit. To investigate the biological scope of this proteolytic mechanism, we tested a panel of cells, including colon and breast carcinomas, hepatomas, and immortalized hepatocytes, selected because they migrated or scattered constitutively in the presence of Ln-5. We found that constitutive migration was inhibited by BB94 or TIMPs, known inhibitors of MMPs. Limited profiling by gelatin zymography and Western blotting indicated that the ability to constitutively migrate on Ln-5 correlated with expression of plasma membrane bound MT1-MMP metalloprotease, rather than secretion of MMP2, since MMP2 was not produced by three cell lines (one breast and two colon carcinomas) that constitutively migrated on Ln-5. Moreover, migration on Ln-5 was reduced by MT1-MMP antisense oligonucleotides both in MMP2+ and MMP2− cell lines. MT1-MMP directly cleaved Ln-5, with a pattern similar to that of MMP2. The hemopexin-like domain of MMP2, which interferes with MMP2 activation, reduced Ln-5 migration in MT1-MMP+, MMP2+ cells, but not in MT1-MMP+, MMP2− cells. These results suggest a model whereby expression of MT1-MMP is the primary trigger for migration over Ln-5, whereas MMP2, which is activated by MT1-MMP, may play an ancillary role, perhaps by amplifying the MT1-MMP effects. Codistribution of MT1-MMP with Ln-5 in colon and breast cancer tissue specimens suggested a role for this mechanism in invasion. Thus, Ln-5 cleavage by MMPs may be a widespread mechanism that triggers migration in cells contacting epithelial basement membranes.
Membrane-type 1 matrix metalloproteinase (MT1-MMP) localizes at the front of migrating cells and degrades the extracellular matrix barrier during cancer invasion. However, it is poorly understood how the polarized distribution of MT1-MMP at the migration front is regulated. Here, we demonstrate that MT1-MMP forms a complex with CD44H via the hemopexin-like (PEX) domain. A mutant MT1-MMP lacking the PEX domain failed to bind CD44H and did not localize at the lamellipodia. The cytoplasmic tail of CD44H, which comprises interfaces that associate with the actin cytoskeleton, was important for its localization at lamellipodia. Overexpression of a CD44H mutant lacking the cytoplasmic tail also prevented MT1-MMP from localizing at the lamellipodia. Modulation of F-actin with cytochalasin D revealed that both CD44H and MT1-MMP co-localize closely with the actin cytoskeleton, dependent on the cytoplasmic tail of CD44H. Thus, CD44H appears to act as a linker that connects MT1-MMP to the actin cytoskeleton and to play a role in directing MT1-MMP to the migration front. The PEX domain of MT1-MMP was indispensable in promoting cell migration and CD44H shedding.
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