Matrix Biology 21 (2002) 547–558 0945-053X/02/$ - see front matter  2002 Elsevier Science B.V. and International Society of Matrix Biology. All rights reserved. PII: S0945-053X Ž 02 . 00068-9 Rhodocetin antagonizes stromal tumor invasion in vitro and other a2b1 integrin-mediated cell functions Johannes A. Eble *, Stephan Niland , Andre Dennes , Alletta Schmidt-Hederich , Peter Bruckner , a, a a a a ´ Georg Brunner b Institute of Physiological Chemistry and Pathobiochemistry, University of Munster, Waldeyerstraße 15, 48149 Munster, Germany a ¨ ¨ Fachklinik Hornheide, Department of Cancer Research, University of Munster, 48157 Munster, Germany b ¨ ¨ Received 6 February 2002; received in revised form 24 July 2002; accepted 31 July 2002 Abstract The pleiotropic effects of Calloselasma rhodostoma venom is caused by various toxins, among them kistrin and ancrod, which block platelet activation triggered by RGD-dependent integrins and the blood clotting cascade, respectively. Here, we demonstrate that rhodocetin, another component of this venom, acts as a2b1 integrin inhibiting disintegrin and antagonizes important cellular responses to type I collagen. Cell adhesion, migration, and collagen lattice contraction in vitro were specifically inhibited by rhodocetin, whereas expression of collagen-degrading matrix metalloproteases was differently modulated. Moreover, cell invasion of HT1080 fibrosarcoma cells into a type I collagen matrix, but not into a fibrin gel or a basement membrane-extracted matrigel was efficiently blocked by rhodocetin. Unlike its natural ligand collagen, rhodocetin failed to cluster a2b1 integrin, despite similar binding affinities. Hence, in the absence of focal adhesions cells do not attach firmly to rhodocetin and do not respond with any of a2b1-triggered cell reactions, except for MMP-1 production. Therefore, this disintegrin may be a valuable tool to specifically target stromal tumor invasion and to manipulate other a2b1 integrin-mediated functions, such as excessive scar contraction and fibrosis. Rhodocetin might be therapeutically useful because of its lack of interference with RGD-dependent integrins, low molecular mass, high solubility, and biochemical stability.  2002 Elsevier Science B.V. and International Society of Matrix Biology. All rights reserved. Keywords: Disintegrin; Cell-matrix interaction; Adhesion; Migration; Tumor invasion; Focal adhesion 1. Introduction Integrins are cell adhesion molecules consisting of two subunits a and b (for reviews see Eble, 1997; Humphries, 2000; Hynes, 1992). Approximately 25 integrins are known so far, which differ in their ligand binding specificities. Integrin ligands are extracellular matrix (ECM) proteins or cell-surface receptors. As a transmembrane linkage of ECM with the cytoskeleton, integrins convey both mechanical forces and signals Abbreviations: ECM, extracellular matrix; IGF-1, insulin-like growth factor-1; MMP, matrix metalloproteinase; MTT, 3-w4,5-dime- thylthiazol-2-ylx-2,5-diphenyltetrazolium bromide; PBS, phosphate- buffered saline (136 mM NaCl – 8.1 mM Na HPO Ø7H O – 1.5 mM 2 4 2 KH PO – 2.7 mM KCl, pH 7.4); TBS, Tris-buffered saline (50 mM 2 4 Tris–HCl (pH 7.4) – 150 mM NaCl). *Corresponding author. Tel.: q49-251-835-2289; fax: q49-251- 835-5596. E-mail address: eble@uni-muenster.de (J.A. Eble). from the outside to the inside of the cell and vice versa. Upon ligand binding, integrins cluster within the plasma membrane, representing the initial step in the formation of focal adhesions (Burridge and Chrzanowska-Wodni- cka, 1996; Miyamoto et al., 1995). Non-receptor tyro- sine kinases and cytoskeletal proteins are recruited to specific domains of clustered integrins and are assem- bled into supramolecular complexes of both structural and signaling molecules (Burridge and Chrzanowska- Wodnicka, 1996; Giancotti, 1997; Yamada and Geiger, 1997). Thus, ligand occupancy triggers assembly of focal adhesions, which eventually leads to various cel- lular responses, such as gene activation, cell adhesion, spreading, migration, differentiation, cell growth and proliferation. Various collagen binding integrins have been identi- fied: a1b1, a2b1, a10b1 and a11b1 integrin (Eble et al., 2001). Integrin a2b1 is the prominent collagen