Abstract
Angiogenesis promotes tumor growth and metastasis. Cell adhesion molecules interact with the extracellular matrix (ECM) and increase cell adhesion and migration during angiogenesis. Thrombomodulin (TM) is a cell surface transmembrane glycoprotein expressed in endothelial cells. However, the function and significance of TM in cell-matrix interactions and angiogenesis remain unclear. Here, we first demonstrated that recombinant lectin-like domain of TM interacts with an ECM protein, fibronectin, and identified the N-terminal 70-kDa domain of fibronectin as the TM-binding site. Exogenous expression of TM in TM-deficient A2058 melanoma cells enhanced cell adhesion and migration on fibronectin and invasion on Matrigel. In addition, TM increased focal adhesion kinase (FAK) phosphorylation and matrix metalloproteinase-9 production. In mice bearing subcutaneous B16F10 melanoma tumors, immunofluorescence analysis indicated that TM was highly expressed and co-localized with fibronectin on the tumor vasculature. The interaction between TM and fibronectin in tumor blood vessels was also validated by the proximity ligation assay. In human umbilical vein endothelial cells, up-regulation of TM by vascular endothelial growth factor (VEGF), a tumor angiogenic factor, promoted cell adhesion and tube formation, whereas TM knockdown by RNA interference attenuated VEGF-induced cell adhesion and tube formation. In summary, TM promotes angiogenesis by enhancing cell adhesion, migration, and FAK activation through interaction with fibronectin. TM may represent a novel target for inhibiting tumor angiogenesis.
Highlights
Cell surface adhesion molecules interact with the extracellular matrix (ECM) to regulate cell adhesion and migration during embryonic development, organ morphogenesis, and angiogenesis [1, 2]
To investigate whether the lectin-like domain of TM directly interacts with ECM proteins, we first performed solid-phase binding assays. rTMD1 (Ala1 through Ala155) with c-Myc and His tags was prepared with the mammalian protein expression system
vascular endothelial growth factor (VEGF) treatment markedly induced tube formation of shLuc-expressing cells on Matrigel by 2.6-fold, while TM knockdown reduced VEGF-stimulated tube formation (Figure 9D and Supplementary Figure S6). These results indicate that TM mediates VEGF-induced endothelial cell adhesion and tube formation, implying that TM has a role in VEGFdependent tumor angiogenesis
Summary
Cell surface adhesion molecules interact with the extracellular matrix (ECM) to regulate cell adhesion and migration during embryonic development, organ morphogenesis, and angiogenesis [1, 2]. Integrins can mediate cell-ECM interactions and regulate cell adhesion, migration, gene expression, and matrix remodeling [3, 4]. After binding to the ECM, integrins initiate signal transduction by recruiting various signaling and adaptor proteins via their cytoplasmic domains [3]. In response to integrin-mediated cell adhesion, FAK is phosphorylated at several tyrosine residues, including Tyr397, Tyr576, and Tyr577 [5]. Other cell surface molecules, including syndecans and CD44, have been reported to mediate cell-ECM interactions [6, 7]
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