CDB20:0002953 TNC — ITGB3

ABSTRACT: Human umbilical vein endothelial cells were found to attach and partially spread on human tenascin. The attachment of endothelial cells to tenascin results in elongated cells with interconnecting processes and is distinct from the flattened appearance of endothelial cells on fibronectin, collagen, vitronectin or laminin substrata, suggesting a role for tenascin in modulating cell adhesion and motility. Endothelial attachment to tenascin was partially inhibitable by the SRRGDMS peptide derived from human tenascin and completely inhibitable by anti-integrin antibodies to alpha 2 beta 1 and alpha v beta 3. Endothelial cell attachment to tenascin could be inhibited up to 80% with anti-alpha 2 and anti-beta 1 monoclonal antibodies P1E6 and P4C10, respectively, and this was associated with a complete loss in cell spreading. In contrast, pretreatment of endothelial cells with the anti-alpha v beta 3 monoclonal antibody LM609, resulted in a 35% inhibition in cell attachment but did not alter cell spreading. In combination the anti-alpha 2 and anti-alpha v beta 3 antibodies, could completely abrogate cell spreading and attachment to tenascin-coated surfaces. Affinity purification of 125I-labeled endothelial cell extract on a tenascin matrix column followed by immunoprecipitation with monoclonal antibodies to different integrin alpha and beta subunits resulted in the identification of alpha 2 beta 1 and alpha v beta 3 integrins, respectively, as tenascin binding receptors. Collagen affinity-purified alpha 2 beta 1 receptor from endothelial cells bound not only to collagen and laminin but also to tenascin in a radio receptor binding assay. The results demonstrate that alpha 2 beta 1 and alpha v beta 3 mediate distinct endothelial cell interactions with tenascin; cell spreading and cell binding, respectively. Binding by alpha v beta 3 is mediated by the SRRGDMS site on tenascin, whereas the alpha 2 beta 1 binding site remains undefined. The interaction of alpha 2 beta 1 and alpha v beta 3 with tenascin may be regulated in a cell type-specific manner as evidenced by the binding of endothelial cell alpha 2 beta 1 and alpha v beta 3 to tenascin, and the lack of binding by the same receptors on osteosarcoma MG63 to tenascin.

ABSTRACT: Tenascin-C (TN-C) is induced in pulmonary vascular disease, where it colocalizes with proliferating smooth muscle cells (SMCs) and epidermal growth factor (EGF). Furthermore, cultured SMCs require TN-C for EGF-dependent growth on type I collagen. In this study, we explore the regulation and function of TN-C in SMCs. We show that a matrix metalloproteinase (MMP) inhibitor (GM6001) suppresses SMC TN-C expression on native collagen, whereas denatured collagen promotes TN-C expression in a beta 3 integrin- dependent manner, independent of MMPs. Floating type I collagen gel also suppresses SMC MMP activity and TN-C protein synthesis and induces apoptosis, in the presence of EGF. Addition of exogenous TN-C to SMCs on floating collagen, or to SMCs treated with GM6001, restores the EGF growth response and "rescues" cells from apoptosis. The mechanism by which TN-C facilitates EGF-dependent survival and growth was then investigated. We show that TN-C interactions with alpha v beta 3 integrins modify SMC shape, and EGF- dependent growth. These features are associated with redistribution of filamentous actin to focal adhesion complexes, which colocalize with clusters of EGF-Rs, tyrosine-phosphorylated proteins, and increased activation of EGF-Rs after addition of EGF. Cross-linking SMC beta 3 integrins replicates the effect of TN-C on EGF-R clustering and tyrosine phosphorylation. Together, these studies represent a functional paradigm for ECM-dependent cell survival whereby MMPs upregulate TN-C by generating beta 3 integrin ligands in type I collagen. In turn, alpha v beta 3 interactions with TN-C alter SMC shape and increase EGF-R clustering and EGF-dependent growth. Conversely, suppression of MMPs downregulates TN-C and induces apoptosis.