Molecular Biology of the Cell Vol. 10, 785���798, March 1999 Modulation of Cell Proliferation and Differentiation through Substrate-dependent Changes in Fibronectin Conformation Andre ��s J. Garc�� ��a,* Mar�� ��a D. Vega, and David Boettiger Department of Microbiology, University of Pennsylvania, Philadelphia, Pennsylvania 19104 Submitted September 28, 1998 Accepted January 4, 1999 Monitoring Editor: Joan Brugge Integrin-mediated cell adhesion to extracellular matrices provides signals essential for cell cycle progression and differentiation. We demonstrate that substrate-dependent changes in the conformation of adsorbed fibronectin (Fn) modulated integrin binding and controlled switching between proliferation and differentiation. Adsorption of Fn onto bacterial polystyrene (B), tissue culture polystyrene (T), and collagen (C) resulted in differences in Fn conformation as indicated by antibody binding. Using a biochemical method to quantify bound integrins in cultured cells, we found that differences in Fn conformation altered the quantity of bound a5 and b1 integrin subunits but not av or b3. C2C12 myoblasts grown on these Fn-coated substrates proliferated to different levels (B . T . C). Immunostaining for muscle-specific myosin revealed minimal differentia- tion on B, significant levels on T, and extensive differentiation on C. Differentiation required binding to the RGD cell binding site in Fn and was blocked by antibodies specific for this site. Switching between proliferation and differentiation was controlled by the levels of a5b1 integrin bound to Fn, and differentiation was inhibited by anti-a5, but not anti-av, antibodies, suggesting distinct integrin-mediated signaling pathways. Control of cell proliferation and differentiation through conformational changes in ex- tracellular matrix proteins represents a versatile mechanism to elicit specific cellular responses for biological and biotechnological applications. INTRODUCTION The adhesion of cells to their substrate through an extracellular matrix provides signals that influence their ability to survive, proliferate, and express spe- cific developmental phenotypes (Menko and Boetti- ger, 1987 Werb et al., 1989 Adams and Watt, 1990 Streuli et al., 1991 Zhu et al., 1996 Chen et al., 1997). One of the early examples was the development of in vitro culture conditions that permitted the differenti- ation of avian myogenic cells into contracting myo- tubes (Hauschka and Konigsberg, 1966 Bischoff and Holtzer, 1968). The critical element for this system was the precoating of tissue culture surfaces with rat tail collagen. This general principle of providing an ap- propriate substrate to permit the expression of devel- opmental phenotypes has been applied to a wide va- riety of cells. These include systems that allow the maintenance of neurons and outgrowth of growth cones (Westerfield, 1987) and the recapitulation of the stages of mammary gland development and involu- tion (Li et al., 1987 Barcellos-Hoff et al., 1989). These findings indicate that critical elements of the message directing the expression of a differentiated phenotype are encoded in the extracellular matrix. Cells interact with extracellular matrices primarily through integrins, a widely expressed family of cell surface receptors (Hynes, 1987), and integrin binding to its extracellular ligand is responsible for the down- stream effects of the matrix on cell function. For ex- ample, in the muscle differentiation system, antibodies to b1 integrin reversibly block differentiation and re- tain cells in a proliferating state (Menko and Boettiger, 1987). This fundamental principle of regulation of de- * Corresponding author: Woodruff School of Mechanical Engineer- ing, Georgia Institute of Technology, Atlanta, GA 30332-0405. E-mail address: andres.garcia@me.gatech.edu. �� 1999 by The American Society for Cell Biology 785

velopmental phenotype through binding of integrin receptors has been demonstrated for a variety of other systems, including mammary (Streuli et al., 1991) and kidney (Sorokin et al., 1990) epithelial cells and kera- tinocytes (Adams and Watt, 1990). This interaction is governed by the surface densities of integrin receptors and their ligands and the receptor���ligand binding af- finities. Integrin receptors undergo changes in confor- mation in response to intracellular signals that are capable of modulating their ligand binding affinity (Shattil et al., 1985). This modulation of integrin bind- ing has been shown to play roles in epithelial and muscle differentiation (Adams and Watt, 1990 Boetti- ger et al., 1995). Fibronectin (Fn)1 is one of the most intensively stud- ied components of the extracellular matrix, particu- larly in terms of its effects on cells. Fn plays a central role in the adhesion of many cell types to extracellular matrices and artificial substrata, including tissue cul- ture plastic dishes. Fn is an essential component for normal development, and Fn knockout mice fail to develop beyond embryonic day 10 or 11 (George et al., 1993). The Fn molecule is folded into globular do- mains specialized for particular functions, such as binding to integrins, collagen, heparan sulfate, hyal- uronic acid, and itself to form self-assembled fibrils (Engvall and Ruoslahti, 1977 Hayman et al., 1982 Laterra et al., 1983 Morla and Ruoslahti, 1992). Fn exhibits multiple, complex interactions both in vitro and in vivo. Upon adsorption to surfaces, Fn under- goes conformational changes that affect its biological activity (Grinnell and Feld, 1981 Iuliano et al., 1993 Underwood et al., 1993 Pettit et al., 1994 Garc�� ��a et al., 1998a). For example, Grinnell and Feld (1981, 1982) demonstrated that Fn adsorbed onto tissue culture polystyrene supports higher cell-spreading rates and Fn antibody binding compared with bacterial polysty- rene. In vivo, Fn is found in many sites of extracellular matrix deposition and in association with different matrix components (Hynes, 1990). In addition, it is expressed in different splice variants (Norton and Hynes, 1987), and recent evidence suggests that these variants affect the conformation of the molecule and modulate its interaction with other proteins (Manabe et al., 1997). Thus, its role as an adapter molecule for binding different elements in the extracellular space may be analogous to the growing collection of adapter molecules, such as Grb2 and cas, which are thought to participate in intracellular signaling pathways (Schlaepfer et al., 1997). In this study, we demonstrate that Fn adsorption onto different surfaces results in conformational changes that lead to differences in integrin receptor binding and modulate the switch between cell prolif- eration and myogenic differentiation. This demon- strates that the conformation of the extracellular ma- trix ligand, like the conformation of the integrin receptor, can be modified to regulate the integrin��� ligand interaction and integrin-mediated signaling. This may be particularly important in the case of Fn because of the large variety of processes that it con- trols, its widespread expression in different tissues, and its ability to associate with a variety of other extracellular molecules. In addition, control of inte- grin-ligand interactions and signaling through sub- strate-dependent conformational changes in the extra- cellular matrix represents a versatile approach to manipulate cellular responses in biomaterial and tis- sue engineering applications. MATERIALS AND METHODS Cells and Reagents Mouse C2C12 myoblasts (ATCC CRL-1772) were kindly provided by C. Emerson (University of Pennsylvania) and grown in Dulbec- co���s modified Eagle���s medium (DMEM), 15% FBS, and 1% penicil- lin-streptomycin. Human IMR-90 fibroblasts (ATCC CCL-186) were grown in DMEM, 10% FBS, and antibiotics. Fn- and vitronectin- depleted serum was prepared by sequential affinity chromatogra- phy through gelatin, Fn antibody, and glass columns. Human plasma Fn and tissue culture reagents were obtained from Life Technologies (Grand Island, NY). Bacterial (B, number 1007 Falcon, Lincoln Park, NJ) and tissue culture grade (T, number 25000 Corn- ing, Corning, NY) polystyrene plates were used. Collagen (C) plates were prepared by drying 0.1% collagen type I (Vitrogen-100 Celtrix Laboratories, Palo Alto, CA) from a dilute acetic acid solution onto T plates. Ethidium homodimer was obtained from Molecular Probes (Eugene, OR). All other reagents were obtained from Sigma (St. Louis, MO). Antibodies HFN7.1 and MF20 hybridomas were obtained from American Type Culture Collection (Manassas, VA). HFN7.1 antibody was affinity purified on a protein G-Sepharose column. Adhesion-blocking poly- clonal antibody against Fn was obtained from Cappel (Durham, NC). mAbs 3E1 and 4B2 were purchased from Life Technologies. Adhesion-blocking hamster anti-mouse integrin a5 and av mAbs were obtained from Pharmigen (San Diego, CA). For Western blot- ting, polyclonal antibodies against a5, av, and b3 integrin subunits were purchased from Chemicon (Temecula, CA), whereas antibod- ies against a3 and b1 were raised in this laboratory by standard procedures (Enomoto-Iwamoto et al., 1993). Alkaline phosphatase��� conjugated antibodies were obtained from Jackson ImmunoRe- search (West Grove, PA). Characterization of Fn Adsorption and Conformation Lyophilized Fn was reconstituted with sterile distilled H2O to 1 mg/ml. Substrates (B, T, and C) were coated with Fn diluted in Dulbecco���s PBS (137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4z7H2O, 1.5 mM KH2PO4, 0.9 mM CaCl2z2H2O, 1 mM MgCl2z6H2O, pH 7.4) for 30 min at 22��C and blocked in 1% BSA for 30 min. Adsorbed Fn for different coating concentrations was mea- sured using Fn iodinated with the Bolton���Hunter reagent (DuPont NEN, Boston, MA). 1 Abbreviations used: B, bacterial grade polystyrene C, collagen type I DMEM, Dulbecco���s modified Eagle���s medium Fn, fi- bronectin T, tissue culture polystyrene. A.J. Garc�� ��a et al. Molecular Biology of the Cell 786