Extracellular matrix (ECM) fragments or cryptic sites unmasked by proteinases have been postulated to affect tissue remodeling and cancer progression. Therefore, the elucidation of their identities and functions is of great interest. Here, we show that matrix metalloproteinases (MMPs) generate a domain (DIII) from the ECM macromolecule laminin-5. Binding of a recombinant DIII fragment to epidermal growth factor receptor stimulates downstream signaling (mitogen-activated protein kinase), MMP-2 gene expression, and cell migration. Appearance of this cryptic ECM ligand in remodeling mammary gland coincides with MMP-mediated involution in wild-type mice, but not in tissue inhibitor of metalloproteinase 3 (TIMP-3)–deficient mice, supporting physiological regulation of DIII liberation. These findings indicate that ECM cues may operate via direct stimulation of receptor tyrosine kinases in tissue remodeling, and possibly cancer invasion.

Background: The immense diagnostic potential of human plasma has prompted great interest and effort in cataloging its contents, exemplified by the Human Proteome Organization (HUPO) Plasma Proteome Project (PPP) pilot project. Due to challenges in obtaining a reliable blood plasma protein list, HUPO later re-analysed their own original dataset with a more stringent statistical treatment that resulted in a much reduced list of high confidence (at least 95%) proteins compared with their original findings. In order to facilitate the discovery of novel biomarkers in the future and to realize the full diagnostic potential of blood plasma, we feel that there is still a need for an ultra-high confidence reference list (at least 99% confidence) of blood plasma proteins.

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Laminins are a family of extracellular matrix glycoproteins involved in cell adhesion and migration. A major obstacle to understanding their structure-function relationships is the lack of small laminin domains capable of replicating integrin-binding, cell-adhesive, and migratory functions of the intact molecule. Here, we show that the recombinant LG3 (rLG3) module (26 kDa) of laminin-5 (Ln-5) ␣ 3 chain replicated key Ln-5 activities. rLG3 but not rLG1 or rLG2 supported cell adhesion and migration of at least two distinct cell lines, in an integrin ␣ 3 ␤ 1 -dependent manner. Cell adhesion to rLG3 was regulated by divalent cations and accompanied by cell spreading and tyrosine phosphorylation of FAK focal adhesion kinase. The integrin binding activity of rLG3 was confirmed by rLG3 affinity chromatography of detergent cell lysates, which resulted in specific purification of integrin ␣ 3 ␤ 1 . To our knowledge, this is the first report directly demonstrating that a recombinant laminin LG module is an active domain capable of supporting integrin-dependent cell adhesion and migration.

The extracellular matrix macromolecule laminin-5 (Ln-5) is converted by matrix metalloproteinases (MMP) MT1-MMP and MMP-2 into a migration-promoting substrate in vitro. We now report that cleavage of Ln-5 by MT1-MMP occurs in vivo and affects epithelial tissue organization and probably Ln-5 turnover. In MT1-MMP knockout (KO) mice, the kidneys showed increased levels of total Ln-5 gamma2 subunit, but significantly reduced amounts of gamma2', an amino-terminal truncated proteolytic form of gamma2. The kidney tubular epithelia of KO animals were poorly differentiated, a phenotype reminiscent of human congenital mixed hypoplastic/dysplastic disorders. To establish a better link between Ln-5 proteolytic cleavage and epithelial morphology, MT1-MMP expression was reconstituted by transfection of MT1-MMP into a Ln-5 positive, MT1-MMP deficient epithelial cell line. MT1-MMP transfectants demonstrated increased levels of processed Ln-5 gamma2 chain and enhanced spreading on Ln-5, but not fibronectin. Recombinant MT1-MMP cleaved gamma2 constructs in vitro at a known in vivo gamma2 gamma2' processing site. These results strongly indicate that Ln-5 is a physiological substrate of MT1-MMP in vivo. Proteolytic processing of gamma2 subunit by MT1-MMP may influence Ln-5 turnover in epithelial basement membranes and affect epithelial morphogenesis.