Biomacromolecules 2003, 4, 766-771
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Alternative Intracellular Signaling Mechanism Involved in the Inhibitory Biological Response of Functionalized PMMA-Based Polymers Catharina Latz,† Graciela Pavon-Djavid,† Ge´ rard He´ lary,† Margaret DM Evans,‡ and Ve´ ronique Migonney*,† Laboratoire de Biomate´ riaux et Polyme` res de Spe´ cialite´ , Institut Galile´ e, Universite´ Paris 13, 99 Avenue Jean Baptiste Cle´ ment, 93430 Villetaneuse, France, and CSIRO Molecular Science, 11 Julius Avenue, North Ryde, Sydney, Australia 2113 Received December 16, 2002; Revised Manuscript Received March 10, 2003
A PMMA-based polymer previously shown to inhibit cell proliferation was compared to untreated PMMA. Conformation of adsorbed proteins, cell adhesion, cytoskeleton formation, and integrin activation were examined. Fibronectin adsorbed in a different conformation on the PMMA-based polymer exposing a different balance of the heparin-binding domains. Fibroblasts attached in equal numbers to both surfaces over a 4-h period, but the integrins involved in the adhesion process elicited different intracellular signaling pathways. Cells attached to PMMA showed activation of FAK and MAP as they spread using an assembled actin cytoskeleton. Cells attached to the polymer showed early and strong MAP activity that resulted in nonassembly of the actin cytoskeleton and sub-optimal cell spreading. We conclude that the chemistry of the polymer surface dictated a different conformation of the adsorbed proteins that resulted in alternative cell signaling and diminished cell spreading. This accounted for the biological inhibition previously reported on the PMMAbased polymer. Introduction Polymer surfaces that inhibit cell adhesion and/or proliferation are a useful tool for many applications in biomaterials. The substitution of cross-linked polystyrene with aspartic acid sulfamide groups PSSO2Asp has been shown to be suboptimal for cell growth compared with substitution of polystyrene with sulfonate groups, PSSO3Na.1 More recently, functionalized poly(methyl methacrylate) (PMMA) based polymers were synthesized to obtain linear polymers bearing sulfonate and carboxylate groups. Polymers were synthesized by radical copolymerization of the appropriate monomers in order to provide a random distribution of the ionic groups. These PMMA-based polymers substantially inhibit the proliferation of lens epithelial cells2 and fibroblasts3 compared with nonfunctionalized PMMA without any accompanying cytotoxicity. This work found that the biological inhibition was dependent on the chemical composition of the PMMA-based polymer and on the distribution of the anionic carboxylate and sulfonate groups. More specifically, the work identified the molar fraction of carboxylate groups on the total ionic groups to be the significant factor in the inhibitory biological response observed. Although the mechanism underlying the inhibitory capabilities of the PMMA-based polymers is not fully understood, it is thought to involve the conformation of the proteins, specifically fibronectin (Fn), adsorbed onto the functionalized * To whom correspondence should be addressed. † Universite ´ Paris 13. ‡ CSIRO Molecular Science.
surfaces.1-3 Previous data showed that the PMMA-based polymers bound fibronectin (Fn) with equal affinity to PMMA but yielded a different biological response.3 The adsorption of proteins onto the polymer surface is the primary event in the adhesion process of cells. The conformation of those protein molecules adsorbed on a surface has a significant effect on the binding of cells to that surface because of the exposure (or nonexposure) of the cell-binding sites on the protein molecule that occurs in response to the chemistry of the underlying polymer surface. Fn undergoes conformational changes that affect its biological activity when it is adsorbed onto polymer surfaces,4,5 and this is dictated by the chemistry of the substrate.6 Although the classic cell-binding region of the Fn molecule is known to be the (G)RGDS peptide sequence contained in the central portion of the molecule,7 there are independent cell typespecific sites including heparin binding regions I, II, and III respectively located in the NH2 terminal part of Fn for HBRI and near the COOH-terminal end of Fn for HBRII.8 Cell adhesion to a surface is mediated by cell surface receptors of the integrin family.9 Integrins span the plasma membrane of the cell, and each integrin receptor has a specific extracellular domain that identifies a defined sequence presented by proteins in the extracellular environment. An integrin is activated when it engages its complimentary ligand, and this triggers a series of events within the cell that result in integrin clustering and the formation of defined cell-substrate contact sites within the cell (focal adhesions or focal contacts).10 For the assembly of focal adhesion and stress fibers, at least, two adhesion-receptor-
10.1021/bm025764g CCC: $25.00 © 2003 American Chemical Society Published on Web 04/09/2003
Response of Functionalized PMMA-Based Polymers
mediated signals are required in primary fibroblasts cells cultured on Fn coated substrates.11-13 One is mediated through the interactions of integrins with the RGDS peptide sequence cell-binding domain of Fn. The other one is mediated through the interaction of cell-surface heparan sulfate proteoglycans with the Carboxy-terminal heparinbinding domain (HBRII) of Fn.14,15 Focal adhesions are linked to the ends of actin filament bundles by cytoskeletal proteins (e.g., paxillin, vinculin, and talin) and signaling molecules such as focal adhesion kinase (FAK). Interaction between fibronectin and integrin regulates not only cytoskeleton organization but also the activation of mitogenactivated protein kinase (MAPK).16,17 The phosphorylation of FAK is one of the earliest signaling events associated with integrin binding, and it mediates downstream signaling steps in the MAPK cascade.18 Activation of FAK and MAPK are important in the regulation of cell adhesion, migration, and spreading.19 In addition, signals mediated from ligand-bound integrins are recognized as regulators of cell survival and apoptosis in some cell types.20 The objective of the current study was to probe the biological cell proliferation inhibition known to exist with functionalized PMMA-based polymers of defined chemical composition. Several aspects of the cell adhesion process have been compared on nonfunctionalized PMMA and a representative functionalized PMMA-based polymer which was characterized by nuclear magnetic resonance (NMR) and acidimetric titration as described in a previous paper.3 The current study has examined the conformation of adsorbed Fn, the kinetics of the initial cell adhesion process, the formation of the actin cytoskeleton during the process of cell adhesion, and some markers of integrin activation (FAK and MAP) during that initial cell adhesion process. Experimental Section Polymer Synthesis. PMMA, methyl methacrylate (MMA), and methacrylic acid (MA) were purchased from Aldrich. Sodium styrene sulfonate (NaSS) was purchased from Fluka and was purified by recrystallization in ethanol/H2O (90/ 10) (v/v) and dried under vacuum. The thermal initiator 2,2azobis(isobutyronitrile) (AIBN) was from Aldrich and was purified by recrystallization in methanol and kept under argon atmosphere. The functionalized PMMA-based polymer, was synthesized by radical polymerization. Briefly, the polymerization of MMA, MA, and NaSS was carried out in dimethyl-sulfoxide (DMSO) at 70 °C under nitrogen until complete conversion. The polymer was precipitated in water and dried at 70 °C under vacuum. Polymer composition was determined by 1H NMR in deuterated DMSO using a Bruker ACE 200. The [COO-]/[COO-/ + SO3-] ratio of the polymer was 0.6. The two polymers assessed in this study were coded as follows: 1. nonfunctionalized PMMA (control) and 2. PMMA-based polymer (IP) (inhibitory polymer) DMSO solutions of PMMA and IP polymer (200 g/L) were molded into disks of 16 mm diameter and 150 µm thickness. Disks samples were dried at 70 °C under vacuum for 2 days then extensively washed with aqueous sodium
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chloride solutions (0.15 M) and water. Prior to experiments, samples were sterilized under UV light and then equilibrated at pH 7.4 by washings in sterile phosphate buffer saline (PBS). Conformation of Fibronectin on the Polymer Surfaces. The conformation of fibronectin (Fn) adsorbed to the PMMA and functionalized PMMA-based polymer surface was checked using radiolabeled antibodies.Antibodies were labeled with 125I-Na using the T-chloramine method. A total of 200 µL of antibody (1 mg/mL in PBS) was added to 10 µL of 125I-Na (100mCi/mL), 10 µL of T-chloramine (2.5 mg/mL in PBS), and 60 µL of PBS. After 45 s, the reaction was stopped by adding 10 µL of sodium metabisulfite (5 mg/mL in PBS). Radiolabeled antibodies were eluted with PBS on a Sephadex column (Pharmacia). Antibodies concentrations were determined by Bradford protein assay. Radiolabel yields higher than 90% were obtained. A range of antibodies sensitive to different domains of the Fn molecule was used. A polyclonal fibronectin antibody (F-3648 from Sigma, USA) was used to detect the presence of entire Fn molecule; a monoclonal antibody specific to the heparin domain of the Fn molecule known as heparin binding I (HBRI) (M009 from Takara Biomedicals, Japan) was used to detect the exposed heparin-HBRI domains located in the NH2-terminal end of the Fn molecule; a monoclonal antibody specific to the heparin domain of the Fn molecule known as heparin binding II (HBRII) (M115 from Takara Biomedicals, Japan) was used to detect the exposed heparin-HBRII domains located in COOH-terminal end of the Fn molecule, and a monoclonal antibody specific (Takara Biomedicals, Japan) was used to detect the RGDS cell-binding domain of the Fn molecule. Samples of PMMA and IP were incubated with 10-7M Fn solution (human Fn from Euromedex) in PBS or DMEM containing 10% normal serum (human from Etablissement Franc¸ ais du Sang) for 60 min at 37 °C. A time of 1 h of incubation corresponds to the equilibrium of adsorption. Specific radiolabeled antibodies were incubated with Fn preadsorbed polymers for 1 h at 37 °C. Bovine serum albumin (BSA) was used as a control to account for the nonspecific binding of antibodies. For each polymer and each antibody, data was expressed as a ratio of the amounts of antibodies bound to the surface measured respectively after incubation with the culture medium containing human serum 10% (v/v) and with pure Fn medium. Cell Culture. McCoy human fibroblasts were purchased from ATCC (CRL-1696, USA). Cells were cultured in 25 cm2 tissue culture flasks with DMEM supplemented with 10% (fœtal bovine serum) FBS, penicillin (0.1 unit/mL), streptomycin (100 µg/mL), and L-glutamine (2 mM). Cultures were incubated at 37 °C under a humidified atmosphere in a 5% CO2 incubator with twice weekly medium changes. Subcultures were made every 6 to 8 days. Experiments were performed using cells in the 16th and 17th passage. Cell Seeding onto the Polymer Surfaces. Cells were serum-starved in DMEM containing 0.5% FBS for 16 h and harvested by trypsin/EDTA treatment (10 mM EDTA in PBS, 0.25% trypsin, 1mM EDTA, in Hanks). The trypsin
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was inactivated by soybean trypsin inhibitor (0.5 mg/mL, Sigma) with 0.25% BSA (Fraction V, Sigma) in DMEM, and cells were collected by centrifugation, then resuspended in DMEM containing 0.1% BSA, and held in suspension for 1 h at 37 °C at a concentration of 3.5 × 105 cells/mL with gentle agitation. PMMA and IP disks precoated with Fn (10-7M in PBS) with seeded cells were incubated at 37 °C in a humidified atmosphere of 5% CO2 in air until predetermined times of 5, 15, 30, 60, 150, and 240 min after cell seeding, at which times the polymer disks with adherent cells were removed from the culture wells. Disks with adherent cells were then dedicated either to the quantification of cell adhesion, to the measurement of intracellular signaling activity or, to the visualization of actin cytoskeleton. Quantification of Cell Adhesion. The numbers of cells adherent to each polymer surface was quantified using a cell counter (Coulter ZM, Coultronics, France), and the resulting number was expressed as a percentage of those originally seeded on that surface. Experiments were performed with triplicate samples in three separate experiments. Statistical analysis was performed using SPSS for Windows 9.0 software. A two-tailed p
