Control of Intracellular Signaling by Modulation of Fibronectin

Langmuir 2003, 19, 1723-1729

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Control of Intracellular Signaling by Modulation of Fibronectin Conformation at the Cell-Materials Interface† Todd Miller and David Boettiger* Department of Microbiology, University of Pennsylvania, Philadelphia, Pennsylvania 19104-6076 Received June 28, 2002. In Final Form: September 27, 2002 Fibronectin adsorbs to a variety of surfaces. Adsorption on different biomaterial substrates can result in different conformations for the adsorbed fibronectin. The adsorption isotherms of fibronectin to either neutral polystyrene or negatively charged polystyrene are indistinguishable. Sulfonation was used to vary the level of negative charge on polystyrene surfaces, and adhesion strength of HT1080 cells to these surfaces following fibronectin coating was measured using a spinning disk device. Adhesion to the different fibronectin surfaces was R5β1 integrin dependent, and charge variation had little effect on overall adhesion strength. Ligation of R5β1 integrin to surface-bound fibronectin resulted in a fibronectin-density-dependent increase in phosphorylation of FAK Y397 when negatively charged substrates were used but resulted in no FAK phosphorylation when uncharged substrates were used. Similarly, cell spreading was dependent on the level of negative charge. Thus, the use of specific biomaterial substrates can separate the adhesion and signaling functions of R5β1 integrin. Polylysine coating was used to analyze the effect of positive charge. On the polylysine substrate, there is an increase in the level of nonspecific adhesion following coating with fibronectin and/or bovine serum albumin (BSA). On BSA substrates, there was no stimulation of FAK Y397 phosphorylation. Addition of fibronectin to the polylysine surfaces produced a weak stimulation of FAK Y397 phosphorylation indicating that the positively charged surface was also unfavorable for supporting R5β1-mediated signals.

Introduction Medical devices for implantation are constructed from a variety of different materials concentrating in materials thought to be relatively biologically inert. Nevertheless, the process of implantation generates a materialsbiological interface. While cells may interact directly with the material, unless there is integrin receptor engagement this adhesive interaction does not generate the necessary survival signals and is expected to lead to cellular apoptosis.1 However, this interface is often modulated by extracellular matrix molecules which adhere to the materials face and are able to bind to integrin receptors to provide for cellular survival and function.2,3 The recognition of this issue has led to the investigation of means to add chemically simplified binding sites to materials in order to replace the need for the extracellular matrix component. Polystyrene dishes have provided the standard for the growth of cells in culture for the past 40 years and have provided the basis for the enormous expansion in cell culture based analyses. The natural polystyrene surface, however, provides a very poor substrate for the culture of cells as anyone who has mistakenly attempted to culture cells on bacterial polystyrene dishes can attest. The initial development of the “tissue culture” surface was essentially empirical (and highly proprietary at the time). The surfaces in use today are generated using glow discharge resulting principally in the addition of oxygen and nitrogen * Corresponding author. David Boettiger, Department of Microbiology, 3610 Hamilton Walk, Philadelphia, PA 19104-6076. † Part of the Langmuir special issue entitled The Biomolecular Interface. (1) Frisch, S. M.; Screaton, R. A. Curr. Opin. Cell Biol. 2001, 13, 555-562. (2) Meredith, J. E., Jr.; Fazeli, B.; Schwartz, M. A. Mol. Biol. Cell 1993, 4, 953-961. (3) Weaver, V. M.; Bissell, M. J. J. Mammary Gland Biol. Neoplasia 1999, 4, 193-201.

derivatives to the polystyrene producing a net negative charge. A negatively charged polystyrene surface can also be generated by the controlled sulfonation of the polystyrene surfaces using concentrated sulfuric acid.4 The advantage of this approach is that the level of negative charge can be modulated by altering the sulfonation conditions. Because the tissue-culture-treated polystyrene dishes were developed using primary fibroblasts and fibroblastlike cell lines, they are particularly adept for the culture of these cell types. Many of these cells use R5β1 integrin as their primary adhesion receptor when the cells are grown in the presence of serum, although other integrins including Rvβ3 contribute significantly to adhesion of some of these cultured cells. Since fibronectin serves as the primary ligand for R5β1 integrin,5 the negative charge on tissue-culture-treated polystyrene dishes may provide an optimal presentation of fibronectin. This hypothesis is supported by the finding that fibronectin adsorbed to different surfaces has different conformations as demonstrated by electron spin resonance,6 infrared spectroscopy,7 and antibody binding.8-10 Furthermore, in a myogenic differentiation system, fibronectin on bacterial polystyrene dishes supported cell attachment and cell proliferation but did not support differentiation or expression of muscle specific markers. In contrast, fibronectin adsorbed to tissue culture polystyrene or to collagencoated polystyrene supported differentiation with minimal (4) Martin, G. R.; Rubin, H. Exp. Cell Res. 1974, 85, 319-333. (5) Ruoslahti, E. Annu. Rev. Biochem. 1988, 57, 375-413. (6) Narasimhan, C.; Lai, C. S. Biochemistry 1989, 28, 5041-5046. (7) Pitt, W. G.; Spiegelberg, S. H.; Cooper, S. L. In Proteins at Interfaces; Horbett, T. A., Brash, J. L., Eds.; American Chemical Society: Washington, DC, 1987; pp 324-338. (8) Grinnell, F.; Feld, M. K. J. Biol. Chem. 1982, 257, 4888-4893. (9) Underwood, P. A.; Steele, J. G.; Dalton, B. A. J. Cell Sci. 1993, 104, 793-803. (10) Garcia, A. J.; Vega, M. D.; Boettiger, D. Mol. Biol. Cell 1999, 10, 785-798.

10.1021/la0261500 CCC: $25.00 © 2003 American Chemical Society Published on Web 11/05/2002

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cell proliferation. Since in all cases the cells were dependent on R5β1 for adhesion and survival, it appears that R5β1-mediated signaling was modulated by the conformation of, or the substrate used for, fibronectin.10 Thus, R5β1-fibronectin binding can result in a multiplicity of different signals. Similar multifunctional signaling for R5β1 has been reported for control of tumor cell proliferation,11 and analogous signaling paradigms are proposed for Rvβ3 integrin.12 The engineering of materials for biological interfaces may need to address issues both of adhesion and of adhesion-mediated signaling. The phosphorylation of FAK has been shown to be a common consequence of integrin-ligand binding or integrin clustering and hence appears to be an early event in the integrin signaling cascade.13,14 The FAK protein becomes colocalized to integrin structures depending on sequences in its C-terminal (FAT) domain that bind to paxillin.15 Binding of R5β1 integrin to substrate-attached fibronectin results in the phosphorylation of Y397 which in turn provides a binding site for the SH2 domains of src family kinases and PI-3 kinase.16-18 The initial phosphorylation of Y397 is thought to be catalyzed by FAK itself, whereas the other sites including Y407, Y576/77, Y861, and Y925 may be phosphorylated by the associated src family kinases and serve to propagate downstream signals. There is also integrin signaling which appears to be independent of FAK.19 Thus, while FAK phosphorylation provides a convenient starting point for analysis of R5β1mediated signaling, it may not represent the full impact of this signaling. Materials and Methods Cells and Reagents. Human fibrosarcoma HT1080 cells (CCL-121) (ATCC, Manassas, VA) were grown and maintained in Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal calf serum. The cells were grown for 20 h in the absence of serum using DMEM before use. The cells were dissociated with EDTA, washed, and plated on the prepared fibronectin substrates as indicated. Human plasma fibronectin and cell culture reagents were purchased from Life Technologies (Grand Island, NY). All other reagents were purchased from Sigma (St. Louis, MO). Sulfation and Analysis of Substrates. Untreated polystyrene culture dishes (Falcon 1007) were treated using concentrated sulfuric acid diluted to 0%, 80%, 85%, 90%, 95%, 97%, and 100% acid for 2 h and 100% overnight treatment. Following acid treatment, dishes were incubated in 20% sodium carbonate for 20 min at room temperature, washed with sterile water, and air-dried. To determine the level negative charge introduced by the sulfonation, a subset of plates were incubated with 0.01% crystal violet solution for 10 min at room temperature. Unbound dye was washed off with distilled water, the plates were airdried, and the bound dye was eluted with 2 mL of 100% ethanol for 10 min at room temperature. Dye concentration was determined by measurement of O.D600. Spinning Disk Analysis of Adhesion Strength. To analyze the adhesion to polystyrene surfaces, 25 mm disks were cut from Corning Suspension culture dishes and the edges were smooth(11) Varner, J. A.; Emerson, D. A.; Juliano, R. L. Mol. Biol. Cell 1995, 6, 725-740. (12) Cheresh, D. A.; Stupack, D. G. Nat. Med. (NY) 2002, 8, 193194. (13) Kornberg, L. J.; Earp, H. S.; Turner, C. E.; Prockop, C.; Juliano, R. L. Proc. Natl. Acad. Sci. U.S.A. 1991, 88, 8392-8396. (14) Guan, J. L.; Trevithick, J. E.; Hynes, R. O. Cell. Regul. 1991, 2, 951-964. (15) Sieg, D. J.; Hauck, C. R.; Schlaepfer, D. D. J. Cell Sci. 1999, 112, 2677-2691. (16) Cobb, B. S.; Schaller, M. D.; Leu, T. H.; Parsons, J. T. Mol. Cell Biol. 1994, 14, 147-155. (17) Hanks, S. K.; Polte, T. R. BioEssays 1997, 19, 137-145. (18) Calalb, M. B.; Polte, T. R.; Hanks, S. K. Mol. Cell Biol. 1995, 15, 954-963. (19) Giancotti, F. G.; Ruoslahti, E. Science 1999, 285, 1028-1032.

Miller and Boettiger ened using a Dremel. The disks were washed with ethanol and then distilled water and placed in PBS. Disks were treated with sulfuric acid as above to give a range of negative surface charge, washed, and coated with fibronectin in complete PBS, pH 7.4, at room temperature for 30 min followed by blocking with heatinactivated 1% bovine serum albumin (BSA) in complete PBS, pH 7.4, for 30 min immediately prior to the addition of cells. Conversion of coating concentrations to coating densities was determined using 125I-labeled fibronectin as described.20 For polylysine-coated surfaces, the disks were incubated in 100 µg/ mL poly-L-lysine for 30 min before incubation with fibronectin. For collagen coating, surfaces were treated with 100 µg/mL bovine collagen solution and allowed to dry overnight. The spinning disk analysis was performed as described.20 Briefly, about 5 × 104 cells were plated on a 25 mm disk in 400 µL of adhesion buffer and incubated at room temperature for 60 min. The disks were spun for 5 min at a set speed; cells were fixed with formalin and stained with ethidium homodimer (Molecular Probes). Disks were analyzed on a Nikon Optiphot microscope with a 10× objective using an XY stage controlled by Stage Pro to position the stage at 61 preset positions, and the cells in the microscope field were counted using a Photometrics SensSys camera and ImagePro version 3.1 software. Data processing and curve fitting are done with SigmaPlot version 7.0. Analysis of FAK Phosphorylation. Cells were incubated for 24 h in serum-free DMEM prior to dissociation with EDTA. Cell suspensions were washed and resuspended in adhesion buffer (complete PBS plus 200 mM glucose), and 1 × 106 cells were seeded on 60 mm fibronectin-coated culture dishes (Falcon 1007) for 60 min at room temperature. Fibronectin coating of untreated polystyrene culture dishes was done by incubation with fibronectin in concentrations of 1, 3, 5, or 10 µg/mL for 1 h at room temperature followed by incubation with 1% BSA for 1 h. Concentrated RIPA buffer containing vanadate was added to solubilize the cells without prior washing to avoid loss of loosely attached cells. Extracts were concentrated by size-exclusion filtration using Centricon YM-10 filters (Millipore), and protein concentration was determined by BCA protein assay (Pierce). Samples were separated by 7% SDS-PAGE, transferred to nitrocellulose membranes (Amersham-Pharmacia), and analyzed by Western blotting. Polyclonal anti-FAKpY397 was obtained from Biosource (Camarillo, CA), and monoclonal anti-FAK antibody was purchased from Transduction Laboratories (Lexington, KY). Blots were developed using ECL detection reagents (Amersham-Pharmacia) and quantified using a Fuji-LAS-1000 image analysis system. The extent of FAK tyrosine phosphorylation was normalized to the amount of total FAK protein recovered from each sample.

Results Generation and Analysis of Substrates. Limited treatment of untreated polystyrene culture dishes with sulfuric acid was used to sulfonate the phenyl ring. The reaction was limited by manipulating both sulfuric acid concentration and time of treatment to give a range of sulfonation levels that provided a range of negative charge densities on the surface. The level of charge was monitored by the binding of the positively charged dye, crystal violet,4 which was proportional to the surface charge density. Figure 1 shows that a range of surface charge densities from the untreated polystyrene level to about 3 times the charge density for tissue-culture-treated dishes were achieved. The isotherms for the adsorption of purified fibronectin to untreated bacterial polystyrene and tissue-culturetreated polystyrene have been published previously.10 On both surfaces, the level of adsorption increased to a saturation value of 360 ng/cm2 at a coating concentration of about 10 µg/mL. Given the size of a fibronectin molecule based on electron microscopic analysis of 6 × 60 nm,21 (20) Garcia, A. J.; Huber, F.; Boettiger, D. J. Biol. Chem. 1998, 273, 10988-10993.

Control of Intracellular Signaling

Figure 1. Sulfonation of polystyrene surfaces. Untreated culture dishes were treated with surfuric acid at the concentrations shown for 2 h or overnight. Untreated and tissueculture-treated dishes were used for reference points. Levels of charge on the dish were assayed by the binding of crystal violet. OD600 measures the level of crystal violet adsorbed and is proportional to surface negative charge.

this coating density would approximately produce a fibronectin monolayer. Some studies have shown that there is a break in the adsorption isotherm around the 10 µg/mL coating concentration and a slower rise at higher concentrations.22 This continued rise is likely to be due to the ability of fibronectin to bind to fibronectin and would result in multimolecular layers. There were no distinguishable differences in the adsorption of fibronectin to polystyrene surfaces with different surface charges. Control of FAK Phosphorylation by Substrate Surface Charge. We have found that Y397 phosphorylation levels are directly proportional to R5β1-fibronectin binding levels for several cell types including HT1080 cells.23,24 These phosphorylation measurements are likely to be the result of stabilization of the phosphorylated form by its binding to an SH2 domain and hence its protection from phosphatase. The other phosphorylation sites on FAK responded to other treatments such as integrin clustering, but only Y397 showed a consistent direct correlation with the level of bound R5β1. Therefore, these studies have focused on phosphorylation of Y397. To analyze the control of FAK signaling by substrate surface charge, HT1080 cells were plated on treated polystyrene surfaces precoated with 250 ng/cm2 human fibronectin and incubated for 60 min at 22 °C. For FAK phosphorylation, this is equivalent to 15 min at 37 °C (data not shown). The lower temperature was used to give greater synchrony in the biochemical reactions associated with the adhesion process. Figure 2 shows the specific phosphorylation of FAK Y397 as a function of substrate charge. For comparison, the initial point represents untreated polystyrene and “tc” marks the values for the tissue culture dish control. The level of phosphorylated FAK Y397 was very sensitive to low levels of charge between the untreated and tissue culture levels, showed a broad peak at intermediate negative charge densities, and decreased at higher negative charge densities. Analysis of Adhesion Strength and Cell Spreading. For the HT1080 cells, adhesion to a fibronectin (21) Hynes, R. O. Fibronectins; Springer-Verlag: New York, 1990. (22) DiMilla, P. A.; Stone, J. A.; Quinn, J. A.; Albelda, S. M.; Lauffenburger, D. A. J. Cell. Biol. 1993, 122, 729-737. (23) Datta, A.; Shi, Q.; Boettiger, D. Mol. Cell. Biol. 2001, 21, 72957306. (24) Shi, Q.; Boettiger, D. Manuscript submitted.

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Figure 2. FAK phosphorylation as a function of charge. HT1080 cells were plated for 60 min on dishes treated as in Figure 1 plus fibronectin at 5 µg/mL and blocked with BSA. Blots show FAK phosphorylated on Y397 (pFAK) and total FAK (FAK). The plot shows the quantified ratio of phosphorylated to total FAK.

substrate is mediated by R5β1 integrin as determined by the reduction in adhesion strength following the addition of blocking antibodies to either R5, β1, or the R5β1 binding domain of fibronectin.10,20 This was verified for each of the surfaces used here (data not shown). Cell spreading requires integrin-mediated adhesion as well as cytoskeletal rearrangement. Cell spreading on substrates that have different charge densities was evaluated by phase contrast microscopy at 1 h after plating on fibronectincoated surfaces as shown in Figure 3, and the relative increase in spread area was measured and statistically evaluated as shown in Table 1. There was a