Cell growth regulation by fibronectin

4287

Journal of Cell Science 113, 4287-4299 (2000) Printed in Great Britain © The Company of Biologists Limited 2000 JCS1610

Fibronectin polymerization stimulates cell growth by RGD-dependent and -independent mechanisms Jane Sottile1,*, Denise C. Hocking2 and Kurt J. Langenbach3,‡ 1Department of Medicine, Center for Cardiovascular Research and 2Department of Pharmacology and Physiology, University of Rochester Medical Center, 601 Elmwood Ave, Box 679, Rochester, NY 14642, USA 3Department of Physiology and Cell Biology, Albany Medical College, Albany, NY 12208, USA

*Author for correspondence (e-mail: [email protected]) ‡Present address: Department of Neurology and Neurological Sciences, Stanford University School of Medicine, Stanford, CA 94305, USA

Accepted 26 September; published on WWW 7 November 2000

SUMMARY Many aspects of cell behavior are regulated by cellextracellular matrix interactions, including cell migration and cell growth. We previously showed that the addition of soluble fibronectin to collagen-adherent fibronectin-null cells enhances cell growth. This growth-promoting effect of fibronectin depended upon the deposition of fibronectin into the extracellular matrix; occupancy and clustering of fibronectin-binding integrins was not sufficient to trigger enhanced cell growth. To determine whether the binding of integrins to fibronectin’s RGD site is required for fibronectin-enhanced cell growth, the ability of fibronectin lacking the integrin-binding RGD site (FN∆RGD) to promote cell growth was tested. FN∆RGD promoted cell growth when used as an adhesive substrate or when added in solution to collagen-adherent fibronectin-null cells. Addition of FN∆RGD to collagen-adherent fibronectin-null cells resulted in a 1.6-1.8× increase in cell growth in comparison with cells grown in the absence of fibronectin. The growth-promoting effects of FN∆RGD and wild-type fibronectin were blocked by inhibitors of fibronectin polymerization, including the anti-fibronectin antibody, L8. In addition, FN∆RGD-induced cell growth was

completely inhibited by the addition of heparin, and was partially blocked by either heparitinase-treatment or by addition of recombinant fibronectin heparin-binding domain. Heparin and heparitinase-treatment also partially blocked the growth-promoting effects of wild-type fibronectin, as well as the deposition of wild-type fibronectin into the extracellular matrix. These data suggest that cell surface heparan-sulfate proteoglycans contribute to the growth-promoting effects of FN∆RGD and wild-type fibronectin. Addition of heparin, treatment with heparitinase, or incubation with monoclonal antibody L8 all inhibited the formation of short linear FN∆RGD fibrils on the cell surface. Inhibitory β1 integrin antibodies had no effect on FN∆RGD fibril formation, FN∆RGDinduced cell growth, or cell adhesion on FN∆RGD-coated substrates. These data suggest that fibronectin fibril formation can promote cell growth by a novel mechanism that is independent of RGD-integrin binding, and that involves cell surface proteoglycans.

INTRODUCTION

Francis, 1994; Meredith et al., 1993; Ruoslahti and Reed, 1994). Integrins and integrin-dependent signalling events also regulate the deposition of fibronectin into the extracellular matrix (Giancotti and Ruoslahti, 1990; Wu et al., 1993; Wu et al., 1998). Fibronectin also binds to cell surface proteoglycans, including syndecans (Carey, 1997; Saunders and Bernfield, 1988) and CD44 (Naor et al., 1997). Binding of fibronectin to syndecan involves the interaction of fibronectin’s carboxylterminal heparin-binding domain with heparan sulfate glycosaminoglycan chains on syndecan (Saunders and Bernfield, 1988). Much data have suggested that the heparinbinding domain of fibronectin contributes to cell spreading and stress fiber formation in a variety of cell types (Bloom et al., 1999; Izzard et al., 1986; Woods et al., 1986). The addition of heparin-binding fragments of fibronectin in solution or

The interaction of cells with the extracellular matrix is important in regulating cell growth, differentiation, migration and survival (Hynes, 1990). Integrins are a major class of transmembrane receptors that mediate cell adhesion to fibronectin (Hynes, 1992; Pytela et al., 1985) as well as to other extracellular matrix proteins (Hynes, 1992; Ruoslahti, 1988; Yamada, 1989). Binding of extracellular matrix proteins to integrins leads to the generation of intracellular signals, many of which are similar to intracellular signals generated by growth factor stimulation (Assoian, 1997; Juliano, 1996; Schwartz, 1997). Integrin-extracellular matrix interactions are also important for cell survival, as disruption of integrinmediated attachment to the extracellular matrix induces apoptosis in epithelial and endothelial cells (Frisch and

Key words: Fibronectin, Extracellular matrix, Cell growth

4288 J. Sottile, D. C. Hocking and K. J. Langenbach adsorbed to the substrate can restore stress fiber formation in cells adherent to the cell-binding fragment of fibronectin (Bloom et al., 1999; Woods et al., 1986). In some systems, the requirement for the heparin-binding domain of fibronectin can be bypassed by treatment of cells with activators of protein kinase C (PKC) (Woods and Couchman, 1992) or Rho (Saoncella et al., 1999). These data suggest that cell surface proteoglycans cooperate with integrins in mediating maximal cell adhesion and spreading through PKC- and/or Rhodependent mechanisms. Our recent data demonstrate that deposition of fibronectin into the extracellular matrix stimulates adhesion-dependent cell growth, and that ligation and clustering of fibronectinbinding integrins are not sufficient to promote enhanced cell growth (Sottile et al., 1998). These data are consistent with other studies showing that inhibition of fibronectin deposition, or disruption of a preformed fibronectin matrix, decreases cell proliferation (Bourdoulous et al., 1998; Clark et al., 1997; Mercurius and Morla, 1998). Others have shown that cell growth in response to fibronectin can also be regulated by the three-dimensional organization of fibronectin fibrils within the matrix (Sechler and Schwarzbauer, 1998). Taken together, these data indicate that extracellular matrix fibronectin plays an important role in regulating adhesion-dependent cell growth. We previously demonstrated that integrin ligation is not sufficient to promote fibronectin-dependent cell growth (Sottile et al., 1998). To determine whether RGD-integrin binding is required for fibronectin-induced cell growth, we examined the effects of fibronectin lacking the RGD sequence (FN∆RGD) on the growth of fibronectin-null cells. Our data demonstrate that cells adhere, spread and grow on substrates coated with FN∆RGD, and that addition of FN∆RGD to substrate-adherent fibronectin-null cells results in a 1.6-1.8× increase in cell growth in comparison with cells grown in the absence of fibronectin. FN∆RGD stimulated cell growth to approximately 50-60% of the levels induced by wild-type fibronectin, indicating that both RGD-dependent and -independent mechanisms are involved in the cell growth response to fibronectin. The ability of FN∆RGD to enhance growth of collagen-adherent cells was completely inhibited by the addition of heparin, and was partially blocked by heparitinase treatment, or by addition of recombinant fibronectin heparinbinding domain. These treatments also blocked the formation of short linear FN∆RGD fibrils on the cell surface. Interestingly, our data also demonstrate that αv and β3 integrin antibodies inhibit cell adhesion to FN∆RGD. However, αv and β3 integrin antibodies did not inhibit either FN∆RGD fibril formation or the ability of FN∆RGD to induce growth of collagen-adherent cells. These data indicate that fibronectin fibril formation and fibronectin-induced cell growth can occur in the absence of RGD, and suggest a role for proteoglycans in the cell growth response to fibronectin polymerization.

al., 1991). Rabbit polyclonal antibody to α5 integrin cytoplasmic domain (a gift of Dr Susan LaFlamme, Albany Medical College, Albany, NY, USA) and polyclonal anti-fibronectin antibody were previously described (LaFlamme et al., 1992; Sottile and Mosher, 1993). Polyclonal anti-vinculin antibody was from Sigma (St Louis, MO, USA). FITC-phalloidin was purchased from Molecular Probes (Eugene, OR, USA). Monoclonal anti-integrin antibodies to α5, αv, β1 and β3 subunits, and control hamster IgG and IgM were purchased from Pharmingen (San Diego, CA, USA). Proteins Human fibronectin was purified from Cohn’s fractions I and 2 (a generous gift from Dr Ken Ingham, American Red Cross, Bethesda, MD, USA) as previously described (Miekka et al., 1982). Rat plasma fibronectin was purified on columns of gelatin-Sepharose. Full-length recombinant rat fibronectin, and fibronectin lacking the RGD site (FN∆RGD) were expressed in insect cells and purified from insect cell conditioned medium as described (Hocking et al., 2000). Production and purification of recombinant rat 70 kDa and 40 kDa fibronectin fragments have been previously described (Sottile and Mosher, 1997). Recombinant III12-13 was produced in bacteria and purified as described (Hocking et al., 1999). Cell culture Mouse embryo cells were derived from fibronectin-null embryos and adapted to grow under serum-free conditions in defined medium (a 1:1 mixture of Cellgro (Mediatech, Herndon, VA, USA) and Aim V (Life Technologies, Gaithersburg, MD, USA) as described (Sottile et al., 1998). These media do not require serum supplementation. Thus, the cells are cultured under conditions where no exogenous source of fibronectin or other extracellular matrix proteins is present. The insect cell line IPLB-SF-21, adapted to grow in the serum-free medium SF900-II, was obtained from Life Technologies (Gaithersburg, MD, USA). SF21 cells do not produce any detectable endogenous fibronectin. Thus, recombinant proteins produced by these cells do not contain any contaminating fibronectin. Immunofluorescence Fibronectin-null cells were plated onto 18 mm glass coverslips precoated with vitronectin (5 µg/ml), recombinant fibronectin (10 µg/ml) or FN∆RGD (10 µg/ml). Cells were seeded in defined medium, and incubated at 37°C for various lengths of time. Defined medium contains 1.5 mg/ml albumin, which would effectively block any remaining protein binding sites on the dishes. After allowing cells to attach and spread for 6 hours, some wells were supplemented with fibronectin, FN∆RGD or heparin. Cells were then fixed with paraformaldehyde, permeabilized with 0.5% Triton X-100, and incubated with the indicated primary antibodies for 60 minutes. After washing, cells were incubated with fluorescein isothiocyanate (FITC)or Texas Red-conjugated antibodies for 30 minutes. Following washing, cells were mounted in glycerol gel (Sigma) and examined using an Olympus BX60 microscope equipped with epifluorescence.

MATERIALS AND METHODS

Cycloheximide treatment Fibronectin-null cells were preincubated for 2 hours with 20 µg/ml cycloheximide (Sigma). Cells were then trypsinized, washed twice with medium containing soybean trypsin inhibitor (Sigma) and cycloheximide, then replated onto vitronectin-coated 18mm glass coverslips in defined medium in the presence of cycloheximide (20 µg/ml). Control cells were cultured in the absence of cycloheximide. Cells were allowed to attach and spread for 1-3 hours before being processed for immunofluorescence.

Immunological reagents Monoclonal antibody L8, which recognizes an epitope in fibronectin’s I-9 and III-1 modules, was a generous gift of Dr Michael Chernousov (Penn State College of Medicine, Danville, PA, USA) (Chernousov et

Cell adhesion assays Tissue culture dishes (96-well) were coated with wild-type fibronectin or FN∆RGD at 10 µg/ml in PBS at 4°C overnight. Collagen was coated onto dishes in 0.02 N acetic acid at 50 µg/ml at 4°C overnight.

Cell growth regulation by fibronectin 4289 Wells were washed with phosphate-buffered saline (PBS), then blocked with 1% bovine serum albumin (BSA) in PBS for 60 minutes at 37°C. Cells were preincubated with anti-integrin or control antibodies at 25-50 µg/ml, or with various fibronectin fragments for 30 minutes at room temperature prior to plating at 5×105 cell/ml in 0.1 ml of defined medium. The cells were allowed to attach for 3060 minutes at 37°C. Cells were washed with PBS, then fixed with 1% paraformaldehyde for 30 minutes at room temperature. Cells were stained with 0.5% Crystal Violet, then air dried. The dye was solubilized with 1% sodium dodecyl sulfate, and the absorbance at 592 nm was determined using a Wallac Victor2 (Gaithersberg, MD, USA) plate reader. Background absorbance of protein-coated wells in the absence of cells was subtracted from each data point. Cell growth assays on fibronectin- and FN∆RGD-coated dishes Tissue culture dishes (24 well; Corning, Cambridge, MA, USA) were coated with recombinant fibronectin or recombinant FN∆RGD in PBS at 2.5-20 µg/ml at 37°C overnight. Wells were washed with PBS before seeding cells at 0.5×104 cell/cm2 in 1 ml of defined medium. The cells were allowed to grow for various lengths of time at 37°C. Cells were washed with PBS, then fixed with 1% paraformaldehyde for 30 minutes at room temperature. Cells were stained with 0.5% Crystal Violet, and the absorbance determined on a spectrophotometer as described (Hocking et al., 1998; Sottile et al., 1998). Cell growth assays on collagen-coated dishes Collagen was coated onto dishes in 0.02 N acetic acid at 50 µg/ml at 4°C overnight. Fibronectin-null cells cultured on collagen-coated dishes have a doubling time of approximately 20 hours (Sottile et al., 1998). Collagen-coated wells were washed with PBS before seeding cells at 0.5×104 cell/cm2 in 1 ml of defined medium. 4-6 hours after seeding, plasma fibronectin, recombinant wild-type fibronectin or recombinant FN∆RGD (20 nM) were added to some of the wells. For inhibition experiments, heparin, recombinant fibronectin fragments, purified L8 IgG or integrin antibodies were added at the time of addition of soluble fibronectin. Control wells received PBS,

nonimmune mouse IgG (Cappel), hamster IgG or IgM (Pharmingen) or recombinant 70 kDa amino-terminal or 40 kDa gelatin-binding fragments of fibronectin. Cells were allowed to grow for the indicated times, then assayed for cell number using Crystal Violet, as described above. Heparitinase and chondroitinase treatment Fibronectin-null cells were seeded in defined medium onto collagencoated dishes. Following a 4 hour incubation, wells were either supplemented with 0.005 U/ml heparitinase or 0.6 U/ml chondroitinase ABC (Seikagaku America, Falmouth, MA, USA) (Chen et al., 1996a; Minden et al., 1995; Shishido et al., 1995). Following a 2 hour incubation, some wells were supplemented with 20 nM wild-type (WT) fibronectin or 20 nM FN∆RGD. Fresh heparitinase or chondroitinase were added daily. Cells were allowed to grow for 3 days, then processed for immunofluorescence microscopy or assayed for cell number using Crystal Violet, as described above.

RESULTS Cell spreading and focal contact formation on FN∆RGD Identifying the role of fibronectin and fibronectin polymerization in regulating adhesion-dependent cell growth has been complicated by the presence of fibronectin in the serum used to culture cells, and by the ability of most adherent cells to constitutively synthesize and deposit fibronectin into the extracellular matrix. To circumvent these problems, we established fibronectin-null cell lines that do not make fibronectin, but are capable of polymerizing exogenously added fibronectin (Sottile et al., 1998). In many cell types, the α5β1 integrin plays a major role in mediating cell attachment to fibronectin. We previously

Fig. 1. Cell spreading on wild-type and ∆RGD fibronectin. Tissue culture dishes were precoated with 10 µg/ml of wild-type (WT) or ∆RGD fibronectin. Fibronectin-null cells were seeded onto coated wells in defined medium. 1 hour (A) or 3 hours (B) after seeding, the cells were fixed with 2.5% paraformaldehyde, permeabilized with 0.5% Triton X-100, then incubated with FITC-phalloidin (to detect actin) and a mouse monoclonal antibody to vinculin. After washing, cells were incubated with Texas Red-anti mouse antibody to detect vinculin. Actin (Panels 1,3,5,7) and vinculin (Panels 2,4,6,8) staining was detected using an Olympus microscope equipped with epifluorescence. Photographs were taken with a Spot digital camera. Bar, 10 µm.

4290 J. Sottile, D. C. Hocking and K. J. Langenbach showed that fibronectin-null cells have α5- and αv-containing integrins on their cell surface, but no detectable cell surface α4 integrins (Sottile et al., 1998). The major binding site in fibronectin for α5β1 is the Arg-Gly-Asp (RGD) sequence that is contained within the III-10 module (Pierschbacher and Ruoslahti, 1984a; Pierschbacher and Ruoslahti, 1984b). Cell adhesion to fibronectin can be disrupted by addition of RGD peptides, demonstrating the importance of the RGD sequence in mediating cell adhesive events (D’Souza et al., 1991; Pierschbacher and Ruoslahti, 1984a). To determine whether regions of fibronectin other than the RGD sequence are able to promote cell attachment and growth, recombinant fibronectin that lacks the RGD sequence (FN∆RGD) was coated onto tissue culture dishes, and the ability of fibronectin-null cells to attach, spread and grow was monitored. As shown in Fig. 1, cells attached, spread and formed vinculin-containing focal contacts (top panels) on FN∆RGD-coated substrates. Cell spreading and stress fiber formation on FN∆RGD was delayed in comparison with spreading on wild-type fibronectin (compare Fig. 1A,B). However, by 3 hours, cells seeded on FN∆RGD were well spread and contained prominent stress fibers (Fig. 1, Panel 5). Others have shown that the presence of α5β1 in focal contacts depends upon ligand occupancy (LaFlamme et al., 1992). The α5β1 integrin is not present in focal contacts of fibroblasts (LaFlamme et al., 1992) or fibronectin-null cells (Sottile et al., 1998) adherent to surfaces coated with extracellular matrix proteins other than fibronectin, but redistributes to focal contacts following addition of RGDcontaining fibronectin fragments or peptides (LaFlamme et al.,

1992; Sottile et al., 1998). Although RGD is the major binding site in fibronectin for α5β1 (Pierschbacher and Ruoslahti, 1984a; Pierschbacher and Ruoslahti, 1984b), α5β1 has also been shown to interact with the amino-terminal 70 kDa portion of fibronectin (Hocking et al., 1998). Therefore, we asked whether adhesion to FN∆RGD resulted in the clustering of α5β1 in focal contacts in an RGD-independent manner. As shown in Fig. 2, α5-integrin was not detected in focal contacts of cells seeded on FN∆RGD (Fig. 2F,H). As expected, cells adherent to wild-type fibronectin contained α5 integrin in their focal contacts (Fig. 2B,D). Since cell spreading was delayed on FN∆RGD in comparison with spreading on wild-type fibronectin (Fig. 1), we next tested whether cell spreading on FN∆RGD was mediated by production of cell-derived adhesive molecules other than fibronectin. Fibronectin-null cells cultured in the presence of the protein synthesis inhibitor, cycloheximide, spread (Fig. 2C,D,G,H) and formed vinculin-containing focal contacts (Fig. 2C,G) when seeded onto FN∆RGD (Fig. 2G,H) or wild-type (Fig. 2C,D) fibronectin. Cells did not spread on uncoated tissue culture wells in the absence (Fig. 2I,J) or presence (not shown) of cycloheximide. Taken together, these data indicate that cell spreading on FN∆RGD does not require the production of cell-derived adhesive factors, and that fibronectin sequences other than the integrin-binding RGD site are able to mediate cell spreading and focal contact formation. Cell adhesion to FN∆RGD is αv-integrin dependent The α5β1 integrin has been shown to bind to the 70 kDa amino terminal region of fibronectin (Hocking et al., 1998).

Fig. 2. Ability of FN∆RGD to promote cell spreading and focal contact formation. Fibronectin-null cells were seeded in defined medium onto uncoated coverslips (I,J) or onto coverslips coated with wild-type fibronectin (WT) or FN∆RGD in the absence (−CHX: A,B,E,F,I,J) or presence (+CHX: C,D,G,H) of cycloheximide. 3 hours after seeding, cells were fixed then incubated with a polyclonal antibody to α5 integrin and a monoclonal antibody to vinculin, followed by a Texas Red-conjugated anti-rabbit antibody to visualize α5 (B,D,F,H, J) and a FITC-antimouse antibody to visualize vinculin (A,C,E,G,I). Few cells attached to uncoated coverslips; to visualize cells that did attach, the cells shown in I and J were not washed prior to fixation. Cells were examined using an Olympus microscope equipped with epifluorescence, and photographed with a Spot digital camera. Bar, 10 µm.

Cell growth regulation by fibronectin 4291 A

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Fig. 3. Effect of integrin antibodies on cell adhesion to fibronectin and ∆RGD fibronectin. Fibronectin-null cells were incubated with antibodies (25 µg/ml) or with the 70 kDa or 40 kDa fragments (1.42 µM) of fibronectin for 30 minutes at room temperature prior to seeding onto tissue culture dishes precoated with 10 µg/ml wild-type fibronectin (WT FN) or FN∆RGD (∆RGD). Cells were allowed to attach for 30 (WT) or 60 minutes (∆RGD) and were then washed, fixed with 1% paraformaldehyde, then stained with 0.5% Crystal Violet. The absorbance was determined using a plate reader as described in Materials and Methods. Similar results were seen when the adhesion assays on FN∆RGD were done for 30 minutes. Data are presented as percentage adhesion of cells plated in the absence of competitor proteins. Values are means ± s.e.m. of triplicate determinations.

Cell growth in response to FN∆RGD Cell spreading is critical for cell cycle progression (Chen et al., 1997; Hansen et al., 1994; Huang et al., 1998). However, spreading by itself is not sufficient to promote cell growth (Davey et al., 1999). Therefore, to determine whether FN∆RGD was able to promote cell growth in addition to cell spreading, fibronectin-null cells were seeded onto tissue culture plates coated with increasing concentrations of FN∆RGD or wild-type fibronectin. As shown in Fig. 4, cells grown on dishes coated with FN∆RGD achieved cell densities approximately 60% of those observed with cells seeded on wild-type fibronectin. This cell density is similar to those achieved by fibronectin-null cells adherent to type I collagen (Sottile et al., 1998). Maximal cell growth on both fibronectin

and FN∆RGD occurred at a coating concentration of approximately 10 µg/ml. Adhesion and growth of cells on FN∆RGD-coated substrates was not blocked by function-blocking anti-β1 integrin antibodies (Fig. 5), indicating that cell growth in response to FN∆RGD is β1 integrin-independent. In contrast, cell growth on collagen I-coated substrates was drastically reduced by β1 antibodies. The effect of β3 antibodies on cell growth could not be assessed, since β3 antibodies inhibit cell adhesion to FN∆RGD (Fig. 3A). We previously showed that addition of soluble fibronectin to collagen, fibronectin, or laminin-adherent fibronectin-null cells 3 Absorbance (540nm)

Therefore, to determine whether adhesion of fibronectin-null cells to FN∆RGD is mediated by 70 kDa-integrin interactions, we tested whether addition of the 70 kDa fragment inhibited cell adhesion to FN∆RGD. As shown in Fig. 3, the 70 kDa amino-terminal fragment and the control 40 kDa gelatinbinding fragment of fibronectin did not inhibit adhesion to FN∆RGD or wild-type fibronectin. To determine whether cell adhesion to FN∆RGD is integrin mediated, we tested the ability of integrin antibodies to block adhesion to FN∆RGD. As shown in Fig. 3A, inhibitory α5 and β1 integrin antibodies did not block adhesion to FN∆RGD. In contrast, adhesion to collagen I-coated dishes was blocked by β1 antibodies (not shown). Surprisingly, β3 integrin antibodies partially inhibited (40%) cell adhesion to FN∆RGD (Fig. 3A), but had little effect on adhesion to wild-type fibronectin (Fig. 3B). When antibodies to αv and β3 integrins were added together, >70% of adhesion to FN∆RGD was inhibited (Fig. 3A). Control antibodies had no effect on cell adhesion to wild-type fibronectin or FN∆RGD. These data indicate that in the absence of RGD-integrin binding, αv- and β3-containing integrins mediate cell adhesion to fibronectin.

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Fig. 4. Comparison of cell growth on wild-type and ∆RGD fibronectin-coated dishes. Tissue culture dishes were precoated with various amounts of wild-type (WT, 䊊) or ∆RGD (䊉) fibronectin. Fibronectin-null cells were seeded onto coated wells in defined medium. Cells were incubated for 4 days, then washed, fixed with 1% paraformaldehyde, stained with 0.5% Crystal Violet and the absorbance at 540 nm determined. Values are means of duplicate determinations, and error bars the range.

4292 J. Sottile, D. C. Hocking and K. J. Langenbach

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Fig. 5. Effect of β1 integrin antibodies on cell growth. Fibronectinnull cells in defined medium were seeded onto dishes precoated with collagen type I (white bars) or FN∆RGD (black bars). After allowing cells to attach and spread for 2 (FN∆RGD) or 6 (collagen) hours, the cell culture medium was supplemented with β1 integrin or control IgM antibodies at 25 µg/ml. Cells were allowed to grow for 4 days and were then processed as described in Fig. 4. Data are presented as percentage growth of cells incubated in the absence of antibodies. Values are means of duplicate determinations, and error bars the range.

resulted in a 2-5× increase in cell growth (Sottile et al., 1998). This fibronectin-enhanced cell growth depended upon deposition of fibronectin into the extracellular matrix (Sottile et al., 1998). Fibronectin deposition is a cell-mediated process (McDonald, 1988; McKeown-Longo and Mosher, 1983) in

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Fig. 6. Effect of FN∆RGD on cell growth. Fibronectin-null cells were seeded on collagen-coated wells in defined medium. 6 hours after seeding, cells were supplemented with 20 nM human plasma fibronectin (pFN, 䊉), wild-type recombinant fibronectin (WTFN, 䊊), recombinant FN∆RGD (䊐) or were given an equivalent volume of PBS (−FN, 䊏). Various times after seeding, cells were processed as described in Fig. 4. Values are means of duplicate determinations, and error bars the range.

which binding of fibronectin to cell surface receptors triggers homophilic binding interactions between fibronectin molecules (Aguirre et al., 1994; Chernousov et al., 1991; Hocking et al., 1994; Morla and Ruoslahti, 1992). To determine whether fibronectin can enhance cell growth of collagen-adherent cells in the absence of RGD-integrin interactions, collagen-adherent

Fig. 7. Deposition of wild-type and FN∆RGD into the extracellular matrix. Fibronectin-null cells were seeded in defined medium onto vitronectin-coated dishes. Following a 6 hour incubation, wells were either supplemented with 20 nM fibronectin (FN; A-D) or FN∆RGD (EH). At the time of fibronectin addition, some wells were supplemented with the 350 nM 70 kDa amino-terminal fragment of fibronectin (70K) or the 40 kDa gelatin-binding fragment of fibronectin (40K). Following a 3 day incubation, cells were fixed, then permeabilized with 0.5% Triton-X-100. Cells were incubated with a polyclonal antibody to fibronectin followed by incubation with fluorescein-conjugated goat antirabbit IgG (A,C-E,G,H). Cells were examined using an Olympus microscope equipped with epifluorescence. (B,F) The corresponding phase pictures to A and E, respectively. Bar, 10 µm.

Cell growth regulation by fibronectin 4293 fibronectin-null cells were incubated in the presence of FN∆RGD, and cell growth was monitored over the course of 4 days. Fig. 6 demonstrates that addition of FN∆RGD to fibronectin-null cells resulted in a 1.8× increase in cell growth over that observed in cells adherent to collagen alone. Wildtype recombinant fibronectin and plasma fibronectin increased cell growth 3.5 and 4×, respectively, over that observed in cells adherent to collagen alone (Fig. 6). These data indicate that fibronectin can stimulate the growth of collagen-adherent cells in the absence of RGD-integrin interactions. Monoclonal antibody L8 inhibits cell surface staining of FN∆RGD and inhibits FN∆RGD-induced growth Previous studies have shown that fibronectin lacking the RGD site is impaired in its ability to form fibronectin fibrils; in CHO cells, short linear fibrils formed at the periphery of cells, mostly in areas of cell-cell contact (Sechler et al., 1996). Addition of FN∆RGD to fibronectin-null cells results in the formation of short stitch-like fibrils on the cell surface (Fig. 7E). In contrast, cells incubated with wild-type fibronectin (Fig. 7A) elaborate an extensive fibronectin matrix. Formation of FN∆RGD fibrils was blocked by agents known to inhibit formation of wild-type fibronectin matrix, including the 70 kDa amino-terminal fragment of fibronectin (Fig. 7G) and the anti-fibronectin antibody, L8 (see below). The III-1 module of fibronectin is thought to be involved in fibronectin-fibronectin interactions that are important during fibronectin fibrillogenesis (Chernousov et al., 1991; Hocking et al., 1994; Morla and Ruoslahti, 1992; Sechler et al., 1996).

Previous studies have shown that antibodies that bind to III-1, such as 9D2 and L8, inhibit fibronectin polymerization (Chernousov et al., 1987; Chernousov et al., 1991). The inhibition of wild-type fibronectin polymerization by 9D2 blocks the ability of fibronectin to promote cell growth (Sottile et al., 1998). To determine whether III-1 plays a role in the formation of FN∆RGD fibrils and in the ability of FN∆RGD to enhance cell growth, we asked whether these events could be blocked by monoclonal antibody L8. As shown in Fig. 8, addition of L8 to fibronectin-null cells blocked the deposition of FN∆RGD (Fig. 8E) and wild-type fibronectin (Fig. 8B) on the cell surface, as detected by indirect immunofluorescence microscopy; control IgG had no effect (Fig. 8C,F). Addition of L8 did not result in any change in cell morphology (not shown). To determine whether L8 could block the ability of FN∆RGD to promote cell growth, collagen-adherent fibronectin-null cells were incubated with FN∆RGD in the presence or absence of L8. As shown in Fig. 9, addition of L8 blocked >80% of FN∆RGD-induced cell growth. Addition of L8 resulted in a similar inhibition of wild-type fibronectin-induced cell growth (Fig. 9). Taken together, these data indicate that the organization of FN∆RGD into short linear fibrils depends upon the 70 kDa amino-terminal domain, as well as III-1-fibronectin interactions, and thus occurs by a mechanism similar to that used to polymerize wild-type fibronectin. These data also indicate that III-1-fibronectin interactions are critical for the cell growth response to FN∆RGD. Integrins are known to participate in fibronectin fibril formation (Akiyama et al., 1989; Fogerty et al., 1990; Giancotti

Fig. 8. Effect of L8 on fibronectin and FN∆RGD deposition. Fibronectin-null cells were seeded in defined medium onto vitronectin-coated dishes. Following a 6 hour incubation, wells were either supplemented with 20 nM wild-type fibronectin (WT; A-C) or FN∆RGD (D-F). At the time of fibronectin addition, some wells were also supplemented with 50 µg/ml monoclonal antibody L8 (B,E) or mouse IgG (C,F). Control wells received an equal volume of PBS (A,D). Following a 3 day incubation, cells were fixed, then permeabilized with 0.5% Triton-X-100. Cells were incubated with a polyclonal antibody to fibronectin followed by incubation with fluorescein-conjugated goat anti-rabbit IgG. Cells were examined using an Olympus microscope equipped with epifluorescence to visualize fibronectin and FN∆RGD. Bar, 10 µm.

4294 J. Sottile, D. C. Hocking and K. J. Langenbach 2.5

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Fig. 9. Effect of L8 on fibronectin- and FN∆RGD-induced growth. Fibronectin-null cells were seeded in defined medium onto collagencoated dishes. Following a 6 hour incubation, wells were supplemented with either 20 nM wild-type fibronectin (WT) or FN∆RGD. At the time of fibronectin addition, some wells were also supplemented with 50 µg/ml monoclonal antibody L8 or mouse IgG. Following a 5 day incubation, cells were fixed with 1% paraformaldehyde, then stained with 0.5% Crystal Violet and the absorbance at 540 nm determined. Data is expressed as fold increase in growth; growth of control wells (cells incubated in the absence of fibronectin) was set equal to 1. Values are means of duplicate determinations, and error bars the range.

and Ruoslahti, 1990; Zhang et al., 1993), and antibodies to α5 and β1 integrins partially inhibit fibronectin polymerization

(Akiyama et al., 1989; Fogerty et al., 1990). To compare the effects of anti-integrin antibodies on fibronectin and FN∆RGD fibril formation, and fibronectin- and FN∆RGD-induced growth, function-blocking β1 and β3 antibodies were added to fibronectin-null cells that were incubated with either FN∆RGD or with non-mutant fibronectin. β1 and β3 antibodies did not prevent the deposition of FN∆RGD on the cell surface (Fig. 10B). Similarly, addition of αv and β3 antibodies did not inhibit FN∆RGD fibril formation (not shown). In contrast, fibril formation of non-mutant fibronectin was partially blocked by the presence of β1 and β3 antibodies, resulting in the formation of shorter, less extensive fibrils (Fig. 10E). To determine whether β3 integrin antibodies inhibit cell growth, we asked whether FN∆RGD-induced growth on collagencoated dishes was blocked by addition of β3 antibodies. As shown in Fig. 11, β3 integrin antibodies partially blocked cell growth both in the absence (29%) and presence (22%) of FN∆RGD. However, the relative increase in cell growth induced by FN∆RGD was unchanged by β3 integrin antibody treatment (without anti-β3=1.7×; with anti-β3=1.8×). No further decrease in cell growth was observed when αv and β3 integrin antibodies were added together (not shown). Heparin and heparitinase-treatment inhibit FN∆RGD induced cell growth Antibodies to β3 integrins did not prevent enhanced cell growth induced by FN∆RGD (Fig. 11). These data suggest that binding of FN∆RGD to a non-integrin receptor may contribute to the cell growth response. Since fibronectin is known to bind to proteoglycans, we asked whether heparin could block the

Fig. 10. Effect of integrin antibodies on fibronectin and FN∆RGD deposition. Fibronectin-null cells were seeded in defined medium onto vitronectin-coated dishes. The day after seeding, wells were supplemented with 20 nM nonmutant fibronectin (D-F) or FN∆RGD (A-C). At the time of fibronectin addition, some wells were also supplemented with β1 and β3 (25 µg/ml each) antibodies (B,E) or with control IgG and IgM antibodies (25 µg/ml each; C,F). Control wells were supplemented with an equal volume of PBS (A,D). Following a 4 day incubation, cells were fixed, then permeabilized with 0.5% Triton-X-100. Cells were incubated with a polyclonal antibody to fibronectin followed by incubation with fluorescein-conjugated goat anti-rabbit IgG. Cells were examined using an Olympus microscope equipped with epifluorescence to visualize fibronectin or FN∆RGD. Bar, 10 µm.

Cell growth regulation by fibronectin 4295 type fibronectin by 45% (Fig. 12A). Lower doses of heparin (10 µg/ml) partially inhibited growth induced by FN∆RGD (70%) and wild-type fibronectin (30%). Heparin (1 mg/ml) had little effect (<15% decrease) on cell growth in the absence of added fibronectin (data not shown). The ability of heparin and heparin-binding fragments of fibronectin to inhibit cell growth suggests that fibronectin-proteoglycan binding may be a critical component of fibronectin- and FN∆RGD-induced growth. To further explore the possibility that cell surface proteoglycans may be involved in fibronectin-induced cell growth, we examined the effect of heparitinase and chondroitinase treatment on fibronectin-induced cell growth. As shown in Fig. 12B, heparitinase treatment (+H) of fibronectin-null cells attenuated the growth-promoting effects of both wild-type fibronectin and FN∆RGD, causing a 43% reduction in the growth-promoting effects of FN∆RGD and a 34% decrease in the growth-promoting effects of wild-type fibronectin. Chondroitinase treatment (+C) had no effect on FN∆RGD or fibronectin induced cell growth (Fig. 12B). Taken together, these data support a role for cell surface heparan sulfate proteoglycans in mediating the growth-promoting effects of fibronectin.

300

% Control Growth

250 200 150 None

100

+∆RGD

50

+FN

0 None

β3

IgG

Fig. 11. Effect of β3 integrin antibodies on fibronectin- and FN∆RGD-induced cell growth. Fibronectin-null cells were seeded on collagen-coated wells in defined medium. 6 hours after seeding, cells were supplemented with 20 nM rat plasma fibronectin (+FN, hatched boxes), FN∆RGD (white boxes) or were given an equivalent volume of PBS (none; black boxes). At the time of fibronectin addition, some wells were also supplemented with β3 integrin antibodies (25 µg/ml) or with control IgG (25 µg/ml). 3 days after seeding, cells were processed as described in Fig. 4. Growth of cells incubated in the absence of antibodies and in the absence of added fibronectin was set equal to 100%. Values are means of duplicate determinations, and error bars the range.

Cell surface staining of FN∆RGD is attenuated by heparin and heparitinase treatment Many studies have shown that clustering of cell surface receptors, including growth factor receptors and integrins, is necessary for transducing downstream signalling events (Akiyama et al., 1994; Clark and Brugge, 1995; Heldin, 1995; Miyamoto et al., 1995; Weiss and Schlessinger, 1998). To determine whether the heparin-binding activity of fibronectin is important for the clustering of FN∆RGD into fibrils on the cell surface, we asked whether FN∆RGD fibril formation could

ability of FN∆RGD to promote growth. As shown in Fig. 12A, the growth-promoting effects of FN∆RGD were completely abolished by the addition of 1 mg/ml heparin (H1), and were partially (45%) inhibited by addition of the recombinant fibronectin heparin-binding domain containing modules III12,13 (HBD). The addition of heparin (1 mg/ml) to fibronectin-null cells also decreased growth induced by wild-

B

A

3.0

2.0

Fold Increase

1.8 2.5 1.6 2.0

1.4 1.2

1.5

1.0 1.0 - H.01 H1 HBD - H.01 H1 HBD WT ∆RGD

+

+

-

-

+ -

+ -

+

+

+

+

+ -

+H

+C

+ -

+ -

-

+

+H

+C

+

+

Fig. 12. Effect of heparin and heparitinase on fibronectin- and FN∆RGD-induced cell growth. (A) Fibronectin-null cells were seeded in defined medium onto collagen-coated dishes. Following a 6 hour incubation, wells were either supplemented with 10 nM wild-type fibronectin (WT) or FN∆RGD. At the time of fibronectin addition, some wells were also supplemented with 0.01 (H.01) or 1 mg/ml heparin (H1), 0.5 µM III12,13 (HBD), or with an equivalent volume of PBS (−). Cells were allowed to grow for 5 days, then processed as described in Fig. 4. (B) Fibronectinnull cells were seeded onto collagen-coated dishes and allowed to attach for 4 hours. Wells were then either supplemented with 0.005 U/ml heparitinase (H) or 0.6 U/ml chondroitinase ABC (C). Following a 2 hour incubation, some wells were supplemented with 20 nM wild-type (WT) fibronectin or 20 nM FN∆RGD. Fresh heparitinase or chondroitinase were added daily. Cells were allowed to grow for 3 days, then processed as described in Fig. 4. Data are expressed as fold increase in growth; growth of control wells (cells incubated in the absence of fibronectin) was set equal to 1. Values are means of duplicate determinations, and error bars the range.

4296 J. Sottile, D. C. Hocking and K. J. Langenbach

Fig. 13. Effect of heparin and heparitinase on fibronectin and FN∆RGD deposition. Fibronectin-null cells were seeded in defined medium onto vitronectin-coated dishes. Following a 6 hour incubation, wells were either supplemented with 20 nM wild-type fibronectin (A,B) or FN∆RGD (F,G) in the presence (+Hep; B,G) or absence (−Hep; A,F) of 1 mg/ml heparin. For cells treated with heparitinase and condroitinase, wells were either untreated (C,H), or supplemented with 0.005 U/ml heparitinase (D,I) or 0.6 U/ml chondroitinase ABC (E,J) 4 hours after seeding. Following a 2 hour incubation, some wells were supplemented with 20 nM wild-type (WT) fibronectin (C-E) or 20 nM FN∆RGD (H-J). Control wells received an equivalent volume of PBS. Fresh heparitinase or chondroitinase were added daily. Following a 3 day incubation, cells were fixed, then permeabilized with 0.5% Triton-X-100. Cells were incubated with a polyclonal antibody to fibronectin followed by a fluorescein-conjugated goat anti-rabbit IgG. Cells were examined using an Olympus microscope equipped with epifluorescence. Bars, 10 µm.

be blocked by either the addition of heparin or by heparitinase treatment (Fig. 13). FN∆RGD staining was eliminated when cells were incubated in the presence of heparin (Fig. 13G), or when cells were treated with heparitinase (Fig. 13I). Both treatments also attenuated fibril formation in cells incubated with wild-type fibronectin (Fig. 13B,D). Addition of heparin or treatment with heparitinase did not cause any noticeable decrease in cell attachment or spreading (not shown). In addition, chondroitinase treatment did not affect the deposition of either wild-type fibronectin (Fig. 13E) or FN∆RGD (Fig. 13J). These data suggest that heparan sulfate proteoglycans may participate in FN∆RGD fibril formation on the cell surface. DISCUSSION We previously demonstrated that fibronectin polymerization into the extracellular matrix promotes adhesion-dependent growth, and that integrin-ligation and clustering are not sufficient to promote enhanced cell growth in response to fibronectin (Sottile et al., 1998). In this study, we have extended these findings by demonstrating that fibronectin can stimulate cell growth in the absence of RGD-integrin ligation by a mechanism that remains dependent on fibronectin polymerization. FN∆RGD promoted cell growth to approximately 50-60% of the levels induced by wild-type fibronectin (Figs 4, 6). Thus, it is likely that both RGDdependent and -independent mechanisms are involved in the cell growth response to wild-type fibronectin. We previously showed that fibronectin-null cells do not express α4 integrins on their cell surface (Sottile et al., 1998).

In addition, all of the remaining fibronectin-binding integrins, including those that contain αv (αvβ1, αvβ3, αvβ5, αvβ6) have been shown to interact with fibronectin via the RGD site in III-10 (Chen et al., 1996b; Plow et al., 2000). Therefore, it was surprising to find that cell adhesion to FN∆RGD could be blocked with antibodies to αv and β3 integrins (Fig. 3). To our knowledge, this is the first demonstration that binding of fibronectin to αv-containing integrins can occur independently of the RGD sequence. αvβ3 binds to a surprisingly large number of molecules, by both RGD-dependent and independent mechanisms (Plow et al., 2000). In addition, binding of fibronectin to the platelet receptor, αIIbβ3, can occur by RGD-dependent and −independent mechanisms (Bowditch et al., 1991). The site in fibronectin that mediates cell attachment to αv-containing integrins in the absence of RGD is not currently known. Our previous data indicate that the ability of fibronectin to enhance cell growth depends upon its ability to become deposited into the extracellular matrix (Sottile et al., 1998). FN∆RGD does not polymerize into robust fibrils in the extracellular matrix of fibronectin-null cells, but forms short stitch-like fibrils on the cell surface (Fig. 7). Others have shown that FN∆RGD can be induced to form fibrils by an α4β1dependent mechanism following integrin activation (Sechler et al., 2000). The FN∆RGD fibrils reported here are generated by a distinct mechanism, since fibronectin-null cells do not have cell surface α4 integrins (Sottile et al., 1998). The short linear FN∆RGD fibrils are abolished by the exogenous addition of heparin, and by heparitinase-treatment, which also inhibited the growth-promoting effects of both FN∆RGD and wild-type fibronectin (Figs 12, 13). These data suggest that cell surface heparan sulfate proteoglycans may play an important role in

Cell growth regulation by fibronectin 4297 fibronectin fibril formation, as well as in mediating fibronectin’s growth-promoting effects. Interestingly, both the formation of FN∆RGD fibrils on the cell surface, and the ability of FN∆RGD to induce cell growth were blocked by the presence of monoclonal antibody L8. L8 recognizes an epitope in fibronectin’s I9 and III1 modules (Chernousov et al., 1991), and inhibits fibronectin deposition into the extracellular matrix, presumably by blocking fibronectin-fibronectin interactions (Chernousov et al., 1987; Chernousov et al., 1991). It is possible that binding of FN∆RGD to the cell surface exposes fibronectin-interactive sites within III-1, as has previously been proposed for wildtype fibronectin (Hocking et al., 1994; Morla and Ruoslahti, 1992; Sechler et al., 1996). The ability of L8 to block both FN∆RGD fibril formation and the growth-promoting effects of FN∆RGD suggests that exposure of self-interactive sites that lead to clustering of fibronectin into fibrils is a critical event during fibronectin-stimulated cell growth and can occur in the absence of RGD-integrin interactions. The loss of FN∆RGD fibrils with heparitinase treatment suggests the possibility that heparan sulfate proteoglycans may play a role in exposing fibronectin self-interactive sites on the cell surface. Our data are consistent with a model in which fibronectin can trigger cell growth by two mechanisms: (1) by serving as an adhesive substate for cells and thus allowing for cell spreading and adhesion-dependent cell cycle progression; and (2) as a consequence of its polymerization into fibrils in the extracellular matrix. αv-containing integrins may promote cell growth via effects on cell adhesion and spreading, since cell adhesion to FN∆RGD is dependent upon αv and β3 integrins (Fig. 3). β3 integrin antibodies partially inhibited the growth of collagen-adherent cells in the presence and absence of FN∆RGD. However, the relative increase in growth induced by FN∆RGD was not affected by antibody treatment (Fig. 11). The effect of β3 integrin antibodies on cell growth could be due to the ability of these antibodies to partially block adhesion (not shown) and growth (Fig. 11) on collagen I in the absence of FN∆RGD. In contrast, our data suggest that heparan sulfate proteoglycans participate in the cell growth response to fibronectin by a mechanism that depends upon fibronectin deposition into the extracellular matrix. The ability of heparitinase to inhibit FN∆RGD cell surface staining and to attenuate FN∆RGDinduced growth suggests that binding of fibronectin to heparan sulfate proteoglycans contributes to the growthpromoting effects of FN∆RGD. It is likely that proteoglycans also contribute to the growth-promoting effects of wild-type fibronectin, as treatment of cells with heparitinase, or addition of soluble heparin decreased deposition of wild-type fibronectin into the extracellular matrix (Fig. 13) and partially inhibited fibronectin-induced cell growth (Fig. 12). Several heparin-binding domains have been identified within the fibronectin molecule. Heparin-binding activity has been localized to modules I1-5 (the 27 kDa amino-terminal region), III1, and the carboxyl-terminal heparin-binding domain containing III12-14 (Hynes, 1990; Litvinovich et al., 1992). Previous studies have demonstrated a role for fibronectin heparin-binding domains in fibronectin deposition into the extracellular matrix. The amino-terminal 27-kDa fragment of fibronectin is important for binding of soluble fibronectin to the surface of substrate-attached cells (McKeown-Longo and

Mosher, 1985; Quade and McDonald, 1988), while the carboxyl-terminal heparin-binding domain has been shown to partially inhibit fibronectin binding and deposition into the extracellular matrix (Bultmann et al., 1998). Our data demonstrate that recombinant III12,13 partially inhibited FN∆RGD-induced cell growth (Fig. 12); the 70 kDa aminoterminal fragment (which contains modules I1-5) also partially blocks FN∆RGD-induced growth (data not shown). The ability of III12,13 and the 70 kDa fragment to inhibit FN∆RGDinduced growth could be due to the direct involvement of these domains in fibronectin-proteoglycan interactions. Others have proposed that heparan sulfate proteoglycans act as coreceptors with integrins to promote maximal cell spreading on fibronectin (Saoncella et al., 1999; Woods et al., 1986). Our data demonstrate that fibronectin can promote adhesion and focal contact formation in the absence of fibronectin-RGD interactions, and that this adhesion depends upon binding to αv- and β3-containing integrins. Our data also suggest an important role for fibronectin-proteoglycan interactions in mediating FN∆RGD’s growth-promoting effects, and in promoting the formation of FN∆RGD fibrils on the cell surface. Although FN∆RGD can form short stitch-like fibrils on the cell surface, its inability to form extensive fibrils suggests that exposure of additional fibronectin-fibronectin interactive sites critical for fibril growth may depend upon integrin interactions with fibronectin’s RGD site. Our data also suggest that proteoglycans are important regulators of wildtype fibronectin function, since heparin and heparitinase treatment attenuated both fibronectin deposition into the extracellular matrix and the growth-promoting effects of wildtype fibronectin. Fibronectin is known to interact with a number of cell surface and cell-associated proteoglycans that have been shown to regulate cell migration and cell growth, including syndecan, perlecan and CD44 (Carey, 1997; Mathiak et al., 1997; Naor et al., 1997). Our data suggest that one mechanism whereby proteoglycans affect cell growth may be by regulating fibronectin deposition into the extracellular matrix. This hypothesis is supported by a recent study showing that transfection of a mutant syndecan 2 into CHO cells can inhibit fibronectin deposition into the extracellular matrix (Klass et al., 2000). Together, these data suggest that fibronectin fibril formation can promote cell growth by a novel mechanism that is independent of RGD-integrin binding, and that involves cell surface proteoglycans. This research was supported by grants HL50549 and HL03971 (to J.S.), and HL60181 and HL64074 (to D.H.) from the National Institutes of Health. K.L. was supported by National Institutes of Health Predoctoral Training Grant T32-HL07194. The authors thank Dr Jean Schwartzbauer for providing fibronectin cDNAs, and for help in expressing full length fibronectins; Drs Susan LaFlamme, Deane Mosher, and Michael Chernousov for providing antibodies; Ms Michelle Arquiett for technical assistance; and Dr Susan LaFlamme for critically reading this manuscript.

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