Laminin 1, fibronectin, and collagen type IV were obtained from Sigma. Elastase, MMP-2, and laminin 5 were obtained from Chemicon International. Rhodamine-labeled Lycopersicon esculentum lectin was obtained from Vector Laboratories. Horseradish peroxidase (HRP)–labeled Neutr-Avidin was obtained from Pierce. Antilaminin antibody was obtained from Chemicon International. Antibodies directed to the M13 phage was obtained from Pharmacia. Given the facts that proteases known to cleave laminin are thought to contribute to angiogenesis and tumor metastasis, the possibility exists that structural remodeling of laminin may facilitate exposure of cryptic sites that regulate both endothelial and tumor cell behavior ( 19– 24).Īntibodies, peptides, and reagents. Although a wealth of information is currently available concerning how interactions with native laminin regulates cellular behavior, relatively little is known concerning the response of cells to interactions with structurally modified laminin. Cellular interactions with laminin regulates cellular and molecular processes, including adhesion, migration, cell cycle control, proliferation, differentiation, and gene expression ( 15– 18). Distinct combinations of these three chains result in at least 12 different laminin isoforms ( 15, 16). At least five α, three β, and three γ chains have been identified. Laminin is a large heterotrimeric molecule composed of three chains designated as α, β, and γ. In addition to collagen type IV, a second component of basement membrane is laminin. Thus, a more complete understanding of the molecular mechanisms by which these cryptic elements function in vivo will provide important new insight into the regulation of tissue homeostasis and disease. In addition, many non-ECM molecules also contain cryptic regulatory sites including matrix metalloproteinase-2 (MMP-2 PEX), plasminogen (angiostatin), and calreticulin (vasostatin refs. Studies have provided examples of this concept from other ECM molecules, including collagens VII, XV, and XVIII thrombospondin vitronectin fibronectin and perlecan ( 6– 10). In fact, reports have documented new regulatory functions for noncollagenous domains of the basement membrane protein collagen type IV ( 4, 5). Recent studies have provided evidence that posttranslational modification of proteins as well as proteolytic release of fragments of larger molecules can result in the acquisition of unique functional properties ( 1– 3). Experimental evidence is accumulating that proteolytic remodeling of ECM proteins is not only a mechanism to facilitate destruction of physical barriers but also an active process required to expose cryptic regulatory information that can be selectively transferred to cells to help control complex cellular behavior. The importance of extracellular matrix (ECM) remodeling in regulating invasive cellular behavior is well known. These novel findings suggest the existence of functionally relevant cryptic laminin epitopes in vivo and that selective targeting of these laminin epitopes may represent an effective new strategy for the treatment of malignant tumors by affecting both the endothelial and tumor cell compartments. This inhibitory activity was associated with a selective up-regulation of the cyclin-dependent kinase inhibitor P27 KIP1 and induction of cellular senescence. Moreover, the STQ peptide selectively inhibited endothelial and tumor cell adhesion, migration, and proliferation in vitro and inhibited angiogenesis, tumor growth, and experimental metastasis in vivo. The cryptic laminin epitope recognized by this peptide was selectively exposed within malignant melanoma in vivo, whereas little if any was detected in normal mouse skin. The STQ peptide selectively bound denatured and proteolyzed laminin but showed little interaction with native laminin. Here, we report the isolation and characterization of a synthetic peptide that binds to a cryptic epitope in laminin. However, little is known concerning the existence and the functional significance of cryptic laminin epitopes in regulating angiogenesis and tumor cell metastasis. An in-depth understanding of how both endothelial and tumor cells use matrix-immobilized cryptic ECM epitopes to regulate invasive cell behavior may lead to the development of novel strategies for the treatment of human tumors. Angiogenesis and tumor metastasis depend on extracellular matrix (ECM) remodeling and subsequent cellular interactions with these modified proteins.
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