RhoA-induced changes in fibroblasts cultured on organic monolayers

Biomaterials 20 (1999) 2435}2446

RhoA-induced changes in "broblasts cultured on organic monolayers Kristin B. McClary!, David W. Grainger",* !Department of Biochemistry, Colorado State University, Ft. Collins, CO 80523-1872, USA "Department of Chemistry, Colorado State University, Ft. Collins, CO 80523-1872, USA

Abstract Substantial previous work indicates that adherent cell morphology in culture is modulated by surface chemistry. Activation of the intracellular small molecular weight GTPase, RhoA, has recently been shown to play an essential role in controlling initiation of key integrin-mediated events in surface adhesion and proliferation. RhoA is interconvertible between an active, membrane-bound form and an inactive, cytosolic RhoGDI-bound form in response to integrin stimulation. This study reports the use of self-assembled functionalized organic alkylthiol monolayers (SAMs) as well-de"ned cell culture substrates to investigate the relationships between surface chemistry, RhoA activation and subsequent cell morphological and molecular level signal transduction responses in cells attaching to derivatized SAMs. Well-controlled alkylthiol surface chemistries were used to monitor and modulate the activation state of RhoA in attaching cells. Activation states were determined indirectly by fractionating cell lysates into membrane and cytosolic fractions by ultracentrifugation. Western blots were then performed, showing RhoA localization to be surface chemistry-dependent. RhoGDI levels and its intracellular localization were also shown to be surface-chemistry dependent. Cells cultured on }CH terminated SAMs, which 3 normally exhibit a low-growth phenotype, were transfected with a constitutively active mutant form of RhoA. Subsequent cell morphological changes were observed on SAM surfaces by #uorescence microscopy. Results support surface chemistry in#uences on the activation state of RhoA mediated by adsorbed proteins and distinct changes in adherent cell morphology resulting from modulation of this activation state. ( 1999 Elsevier Science Ltd. All rights reserved. Keywords: Self-assembled organic monolayers; RhoA; GTPase; Cell culture; Biomaterials; Signal transduction; Surface chemistry; Integrin; Fibroblasts; Fibronectin

1. Introduction In the presence of serum, cells attach to surfaces through interactions with an adsorbed extracellular matrix. This process has been extensively studied and remains an issue of primary importance to biomaterials development [1}3]. Cells are well-known to interact with extracellular matrices primarily through cell surface integrin receptors [4]. Integrin binding to speci"c sequences within extracellular matrix proteins (ECMs) is responsible for initiating multiple downstream signals that regulate many important aspects of cell proliferation and di!erentiation [5}7]. Surfaces have been shown to directly in#uence adsorbed ECM protein composition and conformation

* Corresponding author. E-mail address: [email protected] (D.W. Grainger)

[8}12]. Subsequent signal transduction cascades are affected by the resultant ECM conformations at cell-contacting surfaces. For this reason, numerous studies have examined the e!ects of biomaterials surface properties on protein adsorption and its resulting in#uence on cell adhesion, attachment, and proliferation. Very few studies, however, have investigated important intracellular, downstream signaling processes responsible for cell phenotypic regulation or genetic expression as a function of surface-induced changes in ECM protein conformation or composition. Several key regulators of integrin}ECM mediated signal transduction events in various cell types have been identi"ed [5,13}16]. The family of small molecular weight GTPases has recently been shown to be early upstream players in cell physiological responses to surfaces. These enzymatic cell signal regulators are subdivided into "ve classes: Ras, Rab, Arf, Ran and Rho [17]. Rho family members*CDC42, Rac and Rho*have been shown to play dynamic roles in regulating the actin

0142-9612/99/$ - see front matter ( 1999 Elsevier Science Ltd. All rights reserved. PII: S 0 1 4 2 - 9 6 1 2 ( 9 9 ) 0 0 1 7 1 - 4

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cytoskeleton. Rac is important in actin polymerization associated with membrane ru%ing and lamellipodia formation [18,19]. CDC42 is responsible for cell "lapodia formation [18]. Rho activity is required for focal contact and stress "ber formation that facilitates cell spreading and adhesion on substrates [18,20,21]. Three di!erent speci"c Rho protein sub-families (A, B and C) have been detected in all cell types examined to date [22,23]. All Rho family members, like most G-proteins, cycle between GDP-bound inactive and GTP-bound active forms, aided by a number of intracellular regulatory proteins. Activation of RhoA has been shown to be an integrin- and growth factor-dependent event [15]. Although the exact mechanism of RhoA activation remains unclear, many have been proposed. Current models depict RhoA in the GDP-bound, inactive form, complexed with a guanine nucleotide dissociation inhibitor (Rho}GDI) and found in the cytoplasm of unstimulated, quiescent cells. Upon integrin receptor or growth factor stimulation, RhoA is activated by conversion to GTP}RhoA by a guanine nucleotide exchange factor (GEX) [24]. Because this activation is substrate-dependent for anchorage-dependent cells, RhoA modulation should exhibit a surface dependence, cued by integrin} surface coupling events. RhoA is primarily known for its regulation of cellsurface-induced focal contact and stress "ber formation [18,20,21]. Formation of stress "bers is linked to the Ser}Thr kinase, p160ROCK that interacts with RhoA in a GTP-dependent manner [25]. Two substrates for this kinase, myosin light chain phosphatase and myosin light chain, are known to regulate the assembly of actin} myosin "lament bundles, and recent work has shown that Rho-induced stress "ber formation occurs primarily through bundling of preexisting "laments [26]. These signaled responses, in tandem with others, are important for determining phenotypic expression of cells on surfaces. Clari"cation of how surfaces in#uence or direct these responses together with speci"c biological cues, is a signi"cant issue that should prove useful in understanding and controlling cell}biomaterials interactions, as well as preserving cell phenotype in both cultured and tissue engineered cells. Focal contact formation, creating adhesion plaques that regulate cell attachment to surfaces, is initiated following stress "ber formation. RhoA activation is known to be upstream of activation of focal adhesion kinase (FAK). RhoA-stimulated bundling and contractility of actin}myosin complexes leads to the clustering of integrin receptors, thereby stimulating the activation of FAK [27]. As FAK activation is a precursor to complete focal contact formation, RhoA is essential for inducing focal contact formation and, thereby, also signi"cant in cell}surface interactions. Culture surfaces comprising self-assembled monolayers (SAMs) of terminally derivatized alkylthiols on

Scheme 1. Well-controlled model culture surfaces: functionalized organic self-assembled monolayer "lms (SAMs) on gold-coated supports. Terminal organic monolayer surface functional groups form a densepacked array at the cell-contacting surface while stable surface anchoring is mediated via sulfur-metal epitaxy and chemisorption. Well-organized, su$ciently stable organic adlayers on metal supports provide a convenient route to well-ordered functionalized coatings of consistent surface chemistry for cell culture experiments.

gold (see Scheme 1) modulate cell morphological and growth characteristics [12,28}32]. Speci"cally, varying terminal chemistries present on SAM surfaces modify extracellular matrix protein ("bronectin) adsorption composition, conformation and subsequent cell adhesion, spreading and growth. SAMs terminated in }COOH groups exhibit high levels of "broblast attachment, spreading and growth, while these responses are minimized on }CH terminated SAM culture surface 3 [12,28]. Fibroblasts plated on }COOH terminated SAM surfaces are capable of forming well-de"ned focal contacts and stress "bers, indicating e!ective communication between ECMs adsorbed on the SAM surface from serum and integrin-mediated intracellular signaling mechanisms. Cells attached to }CH terminated SAM 3 surfaces lack focal contact and stress "bers [12]. These results included some assessment of ECM composition and conformation, speci"cally for "bronectin, on di!erent SAM chemistries in producing these cell responses. Signi"cantly, the evidence implicates a possible role for Rho in observed surface chemistry-in#uenced di!erences in cell behavior. Rho activation may be absent or ine$cient in cells plated on the }CH terminated SAMs, 3 leading to the observed lack of stress "ber and focal contact formation observed in cells cultured on these SAM surfaces. While a great deal of evidence implicates RhoA-mediated, integrin-dependent regulation of numerous intracellular signaling pathways as a gatekeeper for induction of cell growth phenotypes in response to surface chemistry, no systematic study has been reported that determines changes in these pathways as a function of cell-exposed surface chemistry. Such an integrated approach is a potentially valuable strategy to evaluate biomaterial surface-cell behavior at a level never before studied. This study probes the relationship between well-controlled cell culture surface chemistry and RhoA

K.B. McClary, D.W. Grainger / Biomaterials 20 (1999) 2435}2446

in attachment dependent "broblasts. The approach attempts to provide a better understanding of essential speci"c surface requirements that prompt cell growth and proliferation, and begins to link key internal cell signaling events to extracellular determinants, including surface chemistry.

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tion was determined using standard Bradford assays and equivalent amounts of total protein were then ultracentrifuged at 100 000]g for 60 min. Supernatants (cytosolic fractions) were poured o! and the pellet (membrane fraction) was washed 2 times in bu!er prior to blotting analysis. Blots were scanned and then quantitated by densitometry using Phoretix (Nonlinear Dynamics Ltd., Ann Arbor, MI).

2. Materials and methods 2.3. Western blot analysis 2.1. Cells and culture substrates Swiss 3T3 "broblasts (American Type Culture Collection, Manassas, VA) were grown in Dulbecco's Modi"ed Eagle's Medium (DMEM, Life Technologies Inc., Rockville, MD) with 100 U ml~1 penicillin and 100 lg ml~1 streptomycin (Sigma, St. Louis, MO), supplemented with 10% fetal calf serum (FBS, complete medium, Life Technologies Inc., Rockville, MD) [12]. Cells were "rst cultured on tissue culture polystyrene, harvested using trypsin treatment, and plated at a density of 105 cells ml~1 onto various cell culture substrates in the described culture conditions. Asymmetric u-functionalized alkylthiols bearing terminal methyl (}CH ) and carboxylate (}COOH) func3 tional groups were synthesized by well-known protocols as previously described [33}35]. Self-assembled monolayer (SAM) "lms of these compounds were fabricated on gold-coated Mylar surfaces (Courtalds Performance Films, Canoga Park, CA, FM-1 gold coating, 99.99% purity, deposited to 70}90 As thickness by evaporative deposition onto 0.0007 gauge tin-coated PET substrate) by a standard method [33}35]. These clean, gold-coated substrates were immersed in 1 mM solutions of the alkylthiol compounds in nitrogen-purged ethanol for 24 h. Substrates were then rinsed in pure ethanol, blown dry in a stream of nitrogen gas and characterized by surface analysis techniques as previously described [12]. 2.2. Cytosolic/membrane fractionation The activation state of RhoA was indirectly determined by examining the relative levels of RhoA proteins located in the cytosolic and membrane fractions, respectively, in cells plated on di!erent SAM terminal chemistries. Cell lysates were obtained from culture on various SAM surfaces and fractionated into cytosolic and membrane fractions. Speci"cally, "broblasts cultured on SAM surfaces for 24 h as described above were washed 3 times with PBS (0.1 M phosphate, 0.15 M NaCl, pH 7.2) and lysed in hypotonic bu!er (10 mM Tris, pH 7.5, 5 mM MgCl , 1 mM PMSF, 1 mM apropotin, 100 nM okadaic 2 acid) on ice for 5 min. Plates were then scraped and resulting cell lysates were transferred to a microfuge tube. Lysates were frozen/thawed 3 times and then centrifuged at 15 000 rpm for 5 min at 43C. Total protein concentra-

Either whole cell lysates or fractionated lysates were chloroform/methanol precipitated as described by Wessel and Flugge [37]. Total protein levels were determined using a "lter dye binding assay as previously described [38]. Equivalent amounts of total protein were separated by electrophoresis on 10% polyacrylamide gels and transferred electrophoretically to polyvinylidene di#uoride (PVDF, NEN Life Science Products, Boston, MA). PVDF was "rst blocked with 4% bovine serum albumin (BSA, Sigma, St. Louis, MO) in PBS overnight. The PVDF was then incubated for 1 h with either anti-RhoA or anti-Rho}GDI (both from Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Following incubation, blots were extensively washed with TTBS (150 mM NaCl, 50 mM Tris}Cl, 0.05% Tween, pH 7.6) and then incubated with secondary antibody conjugated with alkaline phosphatase (Santa Cruz Biotechnology Inc.) for 1 h at room temperature. Following extensive washing with TTBS, enhanced chemiluminescence (Dupont NEN, Wilmington, DE) was used to develop the blots. 2.4. Mutant RhoA expression Swiss 3T3 "broblasts were plated on }CH terminated 3 SAMs precoated with ECM proteins from culture in DMEM#10% FBS. The }CH chemistry was chosen 3 due to its demonstrated inability to support cell spreading under normal serum culture conditions [12]. Following 24 h of attachment in complete media as described above, cells were transfected using LipofectAMINETM Reagent (Life Technologies Inc.) with 7.5 lg each of pINDmycRhoAVal14, which expresses a myc-tagged constitutively active form of RhoA under control of an ecdysone-inducible expression system (Invitrogen, Carlsbad, CA) and pRXR, which produces the operator protein for the inducible system. The system is based on an insect regulatory mechanism [39]. Control transfections were also performed with the pIND vector alone. Following transfections, cells were rinsed and expression was induced with Ponasterone A (Invitrogen) for 48 h. Cells were then "xed and stained for (1) mutant RhoA expression using an anti-PE10 (myc) polyclonal antibody (Santa Cruz Biotechnology) and a #uorescein-labeled secondary antibody (Molecular Probes, Eugene, OR), (2) "lamentous actin formation using rhodamine-labeled

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phalloidin (Molecular Probes), or (3) paxillin localization to focal contacts using an anti-paxillin monoclonal antibody (Transduction Laboratories) and an Alexa 350labeled secondary antibody (Molecular Probes) as previously described [12]. Cells cultured on }COOH terminated SAM surfaces were also transfected as a control to understand the morphological e!ects of constitutively active RhoA expression in cells containing active RhoA plated on }COOH terminated SAM surfaces known to support cell attachment and proliferation [12]. C3 transferase irreversibly ADP-ribosylates RhoA on amino acid Asn-41, e!ectively inactivating RhoA [36]. This enzyme was used as a tool to determine dependence of various cell functions on activated RhoA. Experiments were performed to con"rm whether enhanced cell spreading demonstrated on }COOH terminated SAMs is RhoA dependent. To accomplish this, Swiss 3T3 "broblasts were cultured as described above on }COOH terminated SAM surfaces in complete media. Following 24 h of attachment, complete media was supplemented with 5 lg ml~1 recombinant C3 exoenzyme (Calbiochem, La Jolla, CA) and cells were incubated for 12 h. Cells were then imaged using phase contract microscopy.

3. Results 3.1. Intracellular location of RhoA Fig. 1 represents a RhoA Western blot of whole cell lysates from Swiss 3T3 "broblasts plated on }CH and 3 }COOH terminated SAMs. Blot quantitation by den-

Fig. 1. RhoA levels are reduced in "broblasts cultured on }CH ter3 minated SAM surfaces: Swiss 3T3 "broblasts were plated in DMEM#10% FBS on }COOH or }CH SAM surfaces overnight. 3 Native whole cell lysates were then prepared; total protein was normalized. Lysate samples were separated using a 10% denaturing PAGE. A Western blot was performed using a monoclonal antibody for RhoA and blots were developed by standard chemiluminescence.

Fig. 2. Inactive, cytosolic RhoA is enriched in cells cultured on }CH 3 terminated SAM surfaces: Swiss 3T3 "broblasts were plated in DMEM#10% FBS on }COOH or }CH SAM surfaces overnight. 3 Native whole cell lysates were isolated, normalized for total protein, and ultracentrifuged at 33 000 rpm to fractionate the cellular cytosolic and membrane protein fractions. Fractionated samples were then separated using a 10% denaturing PAGE. A Western blot was performed using monoclonal antibodies speci"c for RhoA; blots were developed by standard chemiluminescence. Duplicate lanes show cytosolic (C) and membrane (M) fractions with bands marked for RhoA detection as a function of }COOH or }CH SAM surface chemistry. 3

sitometry indicates that the overall levels of RhoA present are two times less in cells plated on }CH termin3 ated SAMs than in cells plated on }COOH terminated SAMs. This is consistent with previously documented increased "broblast attachment and growth, and enhanced focal adhesions and actin "lament formation on }COOH over }CH SAMs [12] and associated in3 creased RhoA}GTPase activity, as well as with other results described below. Results for the RhoA Western blot of cytosolic and membrane fractions from cells plated on derivatized SAM surfaces are shown in Fig. 2. Cytosolic lanes were loaded with 1 th of the total cytosolic protein fraction 20 and the membrane lanes were loaded with the entire membrane protein fraction obtained, as membrane fraction levels equivalent to cytosolic levels are undetectable by Western blot analysis. Bands representing RhoA were evident in both cytosolic and membrane fractions in cells plated on both }CH and }COOH terminated SAMs. 3 However, roughly half (44% by densitometry) as much RhoA is localized to the membrane fraction in cells plated on }CH terminated SAMs. This indicates that 3 RhoA may be ine$ciently activated and transported to the membrane in these cells, consistent with the low adherence and growth results reported on these same surfaces [12].

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3.2. Rho}GDI levels are higher in cells plated on }CH3 terminated SAMs A Western blot from whole cell lysates of cells on both surfaces probed with a monoclonal antibody speci"c for Rho}GDI is displayed in Fig. 3. The di!erent band intensities indicate that cells plated on the }CH termin3 ated SAM surface have 22% more Rho}GDI than cells plated on }COOH terminated SAM surfaces. This correlates directly with the higher level of inactivated, GDPbound RhoA demonstrated to be present in these cells in Figs. 1 and 2. Fig. 4 shows results from the Western blot for the same cell lysates separated into RhoA cytosolic and membrane fractions by ultracentrifugation. Densitometry analysis indicates that the cytosolic fractions of lysates from both SAMs comprise larger proportions of Rho}GDI. However, cells plated on }CH terminated SAM surfaces have 3 four times as much RhoA in the cytosolic fraction (inactive) as in the membrane fraction (active), whereas this ratio is only three in cells plated on }COOH terminated SAM surfaces (densitometry, n"6, t-test, P(0.05). This is consistent with Rho}GDI's known role in binding to cytosolic, inactive RhoA present in higher levels in the cytosol in cells plated on }CH terminated SAM 3 surfaces.

Fig. 3. Rho}GDI levels are enhanced in "broblasts cultured on }CH 3 terminated SAM surfaces: Swiss 3T3 "broblasts were plated in DMEM#10% FBS on }COOH or }CH SAM surfaces overnight. 3 Native whole cell lysates were isolated and normalized for total protein. Lysate samples were separated using a 10% denaturing PAGE. Western blotting was performed using monoclonal antibodies speci"c for Rho}GDI; blots were developed by standard chemiluminescence. Lanes 1 and 2, and lanes 3 and 4, are duplicates, respectively, from cells cultured on }COOH and }CH SAM surfaces. Dark bands represent 3 Rho}GDI levels expressed in cells on each SAM chemistry.

3.3. C3 toxin inactivation of RhoA in cells plated on }COOH terminated SAM surfaces Phase contrast microscope photos of cells in Fig. 5 show that exposure to the cytotoxin, C3, causes adherent, well-spread "broblasts on }COOH terminated SAM surfaces to retract cellular processes and to begin to adopt a non-spread, low growth phenotype. This response is consistent with the inactivation of intracellular RhoA known to result from irreversible ADP-ribosylation of RhoA by C3 [36]. 3.4. Mutant RhoA expression in transfected xbroblasts plated on derivatized SAM surfaces Fig. 2 above shows low levels of both RhoA activation and its subsequent transport to the membrane. This result is supported by previous evidence that cells plated on }CH terminated SAMs fail to attach and spread 3 [12,28], and suggests that such surface activation of RhoA, mediated through adsorbed ECM and integrinsignaling, might be by-passed using an inducible, expressed constitutively active form of RhoA that requires no surface induction. Fig. 6A}C display #uorescence microscopy images from immunostaining experiments on mutant RhoA transfected cells plated on }COOH terminated SAM surfaces. Fig. 6A represents cells stained with polyclonal anti-myc and a #uorescein-labeled secondary antibody,

Fig. 4. Rho}GDI is enriched in the cytosolic fraction of cells cultured on }CH terminated SAM surfaces: Swiss 3T3 "broblasts were plated 3 in DMEM#10% FBS on }COOH or }CH SAM surfaces overnight. 3 Native whole cell lysates were isolated, normalized for total protein, and ultracentrifuged at 33 000 rpm to fractionate the cellular cytosolic and membrane protein fractions. Fractionated samples were then separated using a 10% denaturing PAGE. A Western blot was performed using monoclonal antibodies speci"c for Rho}GDI; blots were developed by standard chemiluminescence. Duplicate lanes show dark bands for cytosolic (C) and membrane (M) Rho}GDI as a function of }COOH or }CH SAM surface chemistry. 3

demonstrating that Swiss 3T3 "broblasts plated on }COOH terminated SAM surfaces are capable of expressing the myc-tagged form of this protein. Fig. 6B shows the same cells stained with rhodamine-labeled

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Fig. 5. RhoA inactivation leads to loss of growth phenotype in "broblasts cultured on }COOH terminated SAM surfaces: Swiss 3T3 "broblasts were plated on }COOH derivatized SAM surfaces in DMEM#10% FBS. Following overnight incubation, cells were rinsed and exposed to 4 lg ml~1 of C3 transferase in complete medium for 12 h. Cells were then "xed as described and imaged using phase contrast microscopy. Images are of cells plated on }COOH terminated SAM surfaces (A) before and (B) after exposure to C3 transferase. Exposure to C3 transferase blocks RhoA activation irreversibly, causing initially adherent, spread cells on this surface chemistry to revert to a rounded, retracted morphology. Bar, 20 lm.

within the total cell population decreased over time as a function of mutant RhoA expression on this surface. By contrast, Fig. 7 shows the morphological e!ects of constitutively active RhoA expression in cells cultured on }CH terminated SAM surfaces that lack a spread 3 phenotype. Fig. 7A and B indicate that cells expressing mutant RhoA, as evidenced by positive staining with a #uorescently tagged myc antibody, are capable of achieving a spread morphology on }CH terminated 3 SAM surfaces*a result opposite to the morphological response observed with non-transfected cells on this surface. Staining observed in non-induced cells was a result of non-speci"c #uorescent antibody staining. Due to the compact nature of unspread, non-induced cells, nonspeci"c staining makes staining levels di$cult to interpret in rounded, retracted cells. Similar to other results [15], rhodamine phalloidin staining, shown in Fig. 7B, indicates the presence of F-actin swirls, an intermediate actin organization response that is distinctly di!erent from the complete lack of actin polymerization seen in non-transfected cells on this surface. By 26 h, however, these cells retract and begin to adopt the characteristic Rho phenotype, as described above for cells plated on }COOH terminated SAM surfaces. Fig. 7C shows that although cells are capable of attaining a spread morphology, no discernible focal contacts were formed during the process. This correlates with the noted lack of available ECM integrin binding adhesion sites on this surface [12]. Fig. 7D summarizes average cell length over time for "broblasts plated-CH terminated SAM surfaces 3 transfected with constitutively active RhoA. An overall increase in cell spreading is observed up to 22 h. After this time, cells begin to retract, similar to the observed response in transfected cells spread on }COOH terminated SAM surfaces. This response results from the lack of intrinsic controls on RhoA expression in this system and is characteristic of cells over-expressing active Rho protein [40].

4. Discussion phalloidin*a toxin marker that binds to "lamentous actin. Fig. 7C shows the same cells as in Fig. 6A and B, but stained with a monoclonal antibody for paxillin, a focal adhesion protein, and an Alexa 350-labeled secondary antibody. Results reported above in Figs. 1 and 2 showed that RhoA is e!ectively activated in normal, non-transfected cells cultured on }COOH terminated SAM surfaces. When constitutively active RhoA is expressed in these adherent, spread cells on }COOH SAMs, they begin to retract and adopt a rounded morphology, lacking organized stress "bers. This response is commonly referred to as the &Rho phenotype' [40]. All "broblasts in the "gures are transfected with constitutively active RhoA, with the exception of the cell indicated by an arrow. Fig. 6D shows that the average cell length

In this work, we have attempted to address initial aspects of how RhoA contributes to surface chemistryinduced modulation of cell attachment and morphology. Correlation of RhoA behavior on two di!erent culture chemistries correlates with a number of cell morphological features reported for these surfaces [12]. In addition, we show that arti"cially overriding suboptimal surfaceinduced RhoA activation observed for cells cultured on }CH terminated SAMs by expressing a constitutively 3 active form of RhoA is not entirely su$cient to induce a growth morphology in adherent cells, although interesting in#uences are notable. These studies were motivated initially by our observations of di!erential focal contact and stress "ber

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Fig. 6. Expression of constitutively active RhoA in "broblasts cultured on }COOH terminated SAM surfaces leads to a loss of growth morphology: Swiss 3T3 "broblasts were cultured in DMEM#10% FBS on }COOH derivatized SAM surfaces and transfected with an inducible expression vector for c-mycRhoAVal14 that expresses a constitutively active RhoA. Following overnight incubation, cells were rinsed and expression was induced with Ponasterone A for 26 h, with representative samples taken before induction and at 18, 22 and 26 h after induction. Cells shown in (A) were "xed and stained with a monoclonal antibody for the presence of expression using a polyclonal anti-c-myc and then a #uorescein-labeled secondary antibody. Cells shown in (B) were stained for the presence of stress "bers using rhodamine-labeled phalloidin. Cells shown in (C) were stained for the presence of paxillin in focal contacts using a monoclonal anti-paxillin and then an Alexa 350-labeled secondary antibody. Cells were imaged by #uorescence microscopy. Cell lengths of transfected and untransfected cells was measured using the Metamorph imaging program and average length (in lm) as a function of time was determined. (D) Represents average cell length as a function of mutant RhoA gene expression over time. Bar, 20 lm.

formation in cells plated on derivatized SAM surfaces [12]. Fibroblasts cultured in serum on }CH terminated 3 SAMs lack the formation of stress "bers and focal contacts. These structures are commonly used as indicators of e!ective outside-in signaling between surface-adsorbed ECM proteins (e.g. "bronectin) and intracellular, growth-stimulating signal transduction mechanisms.

These observations complement previous "ndings that "bronectin SAM surface adsorption and its RGD cell binding domain surface availability are reduced on }CH SAMs when compared to that on }COOH termin3 ated SAMs [12], and led to the hypothesis that surfacedependent outside-in signaling fails to su$ciently activate RhoA on these culture surfaces. Signi"cantly,

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Fig. 7. Expression of constitutively active RhoA in "broblasts cultured on }CH terminated SAM surfaces causes an initial increase in cell spreading 3 followed by cell retraction: Swiss 3T3"broblasts were cultured in DMEM#10% FBS on }CH derivatized SAM surfaces and transfected with an 3 inducible expression vector for c-mycRhoAVal14 that expresses a constitutively active RhoA. Following overnight incubation, cells were rinsed and expression was induced with Ponasterone A for 26 h, with representative samples taken before induction and at 18, 22 and 26 h after induction. Cells shown in (A) were "xed and stained with a monoclonal antibody for the presence of expression using a polyclonal anti-c-myc and then a #uorescein-labeled secondary antibody. (B) Shows cells stained for the presence of stress "bers using rhodamine-labeled phalloidin. (C) Shows cells stained for the presence of paxillin in focal contacts using a monoclonal anti-paxillin and then an Alexa 350-labeled secondary antibody. Cells were imaged by #uorescence microscopy. Cell lengths of transfected and untransfected cells was measured using the Metamorph imaging program and average length (in lm) as a function of time was determined. (D) Represents average cell length as a function of mutant RhoA gene expression over time. Bar, 20 lm.

proof of this hypothesis could establish an important link between cell biological processes responsible for phenotypic expression and speci"c surface chemistry. As described above, RhoA's activation state is believed to be modulated by both integrin and growth factordependent events [41]. RhoA mediates several cell at-

tachment and spreading events investigated herein. RhoA's activation state is known to be directly in#uenced by integrin-speci"c events governed by ECM protein conformation and under surface chemistry control [41]. We contend that RhoA is e!ectively activated and found in the GTP bound-active form in cells

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cultured on the }COOH terminated SAM surfaces. Conversely, due to the low-growth phenotype observed in cells on }CH terminated SAMs, RhoA is ine$ciently 3 activated in these cells and therefore found in the GDPbound inactive state. At the present time, no anti-Rhospeci"c antibody is available to e!ectively precipitate Rho in its native form, thus allowing an analysis of the GTP/GDP state of the protein. However, the GTP}Rho form is thought to be produced in response to extracellular signals, including critical levels of integrin binding. Following this activation, RhoA is believed to be transported to the membrane [42,43]. All known Rho proteins contain a carboxy-terminal CAAX box motif (C"cysteine, A"aliphatic amino acid, and X"any amino acid) shown to be geranyl}gernaylated on the cysteine residue by the enzyme geranyl}geranyl transferase Type I [44]. This action promotes plasma membrane localization [43]. After completing its action, GTP}Rho is hydrolyzed to GDP}Rho by Rho}GTPase activating protein (Rho}GAP) [45] then bound by Rho}GDI, which inhibits GDP dissociation [46] and GTP binding [47], released from the membrane, and translocated back to the cytosol in its inactive form. The activation state of RhoA was indirectly determined by examining the relative RhoA levels located in cytosolic and membrane fractions in cells cultured on di!erent terminal SAM surface chemistries. Translocation of RhoA to the membrane fraction, presumably following activation, correlates with several of the observed cell phenotypes indicating e!ective outside-in signaling between the cell and the extracellular environment. Consistent with this result was the observation that minimal amounts of RhoA are activated and localized to the membrane fractions in cells cultured on }CH ter3 minated SAMs due to the absence of these events. Total RhoA levels were reduced in cells cultured on }CH 3 terminated SAMs and signi"cantly less RhoA was localized to the isolated membrane fraction, indicating ine$cient RhoA activation and translocation. Inactive RhoA is known to be sequestered in the cytoplasm by Rho}GDI. To strengthen the connection between RhoA's observed inactive state and the }CH 3 terminated SAM surface chemistry, relative levels of Rho}GDI and its localization in cells on the two di!erent derivatized SAM surfaces were compared. Consistent with the observed presence of a larger amount of inactive, GDP-bound, cytoplasmic RhoA in cells adherent to }CH terminated SAM surfaces, Rho}GDI is also pres3 ent in higher concentrations in the cytoplasm of these cells compared to lysates from cells on }COOH SAMs. The connection between SAM surface chemistry, ECMintegrin binding, cell growth phenotype, and control of RhoA activation is proposed based on this evidence. The role of RhoA in modulating surface-induced changes in adherent cell morphology was further addressed by arti"cially altering the activation state of RhoA in

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cells cultured on derivatized SAM surfaces. Rho protein activity is readily modi"ed through the use of an exoenzyme produced by Clostridium botulinum, the C3 transferase, which irreversibly ADP-ribosylates Rho on amino acid Asn-41 [36]. When C3 transferase was introduced into a variety of cell types, actin-stress "bers were lost and cells rounded up [40,48], thus implicating RhoA in these intracellular events. C3 transferase was used to correlate RhoA activation with adhesion and spreading observed in cells cultured on }COOH terminated SAM surfaces [12]. Inactivation of endogenous RhoA in spread cells plated on }COOH terminated SAM surfaces results in loss of the growth phenotype, indicating a dependence on RhoA activation. An expression vector for constitutively active RhoA introduced into "broblasts plated on }COOH terminated SAM surfaces e!ectively activated and transported RhoA to the membrane in these cells. Cell morphology changes monitored as a function of over-expression of mutant RhoA indicates that over-expression of constitutively active RhoA in cells that already contain adequate amounts of activated RhoA leads to a distinct morphology (the Rho phenotype [40]). The cell body begins to retract and round up in this phenotype and has also been shown to be induced after micro-injection of normal Rho protein into cells, but higher concentrations are required [40]. An inducible, constitutively active, mutant form of RhoA was expressed in transfected "broblasts plated on }CH SAMs to attempt to &force the hand' of RhoA in 3 modulating surface-dependent cell behavior. Fibroblasts expressing this constitutively active RhoA mutant are capable of achieving a spread morphology on }CH 3 terminated SAM surfaces that is otherwise not observed on this surface. However, the rhodamine phalloidin stress "ber staining indicated the presence of F-actin swirls, an intermediate actin organization response distinctly di!erent from the fully organized stress "bers characteristic of cells plated on }COOH terminated SAMs. In the presence of this mutant active RhoA, actin "laments appeared to lack contact with sites on the cell plasma membrane. This was con"rmed by the lack of focal contact formation observed in these cells [12] and are consistent with previous work with mutant active RhoA in adherent cells in culture. Hotchin and Hall [15] showed that "broblasts plated on surface-adsorbed poly-L-lysine in the absence of extracellular matrix proteins formed actin swirls similar to those reported here when microinjected with constitutively active recombinant Rho protein. In addition, they found that functionally active Rho alone was not su$cient to trigger normal, complete focal contact assembly in the absence of extracellular matrix. Our results indicate that not only is the presence of extracellular matrix essential for RhoA activation, but the presentation of surface-adsorbed ECM protein binding motifs to cell integrin receptors is also linked

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and critical. Speci"cally, extracellular matrix proteins responsible for integrin signaling must be present in the appropriate levels (density) and su$ciently integrin-recognizable conformation to produce normal cell phenotypic responses in culture. Our previous research [12] has demonstrated that "bronectin adsorbs from serum or albumin mixtures to }CH terminated SAMs at lower densities and in a less 3 biologically active conformation than observed on }COOH terminated SAMs. While other ECM proteins have also been implicated in roles governing cell adhesion (e.g. collagen, laminin and vitronectin), "bronectin has a well-documented role in several critical cell}surface events. Once adsorbed, "bronectin is known to be actively restructured by receptors on attached cells at the surface into an organized "brillar matrix. This reorganization and restructuring plays an essential role in the biological activity of "bronectin [49]. The intracellular actin cytoskeleton is known to a!ect the assembly of "bronectin into an organized ECM. Newly assembled extracellular "bronectin "brils are aligned with intracellular actin}myosin bundles and focal adhesions [50]. In addition, disruption of actin "laments with the cytotoxin, cytochalasin, inhibits extracellular "bronectin matrix assembly [50]. Additionally, Rho-mediated actomyosin contractility is known to expose a cryptic assembly site in "bronectin [51]. Tension produced by receptors in attached cells at surfaces results in increased exposure of a self-assembly site in adsorbed "bronectin molecules. Exposure of this site has been demonstrated to induce "bronectin matrix reorganization and assembly. Taken together, surface chemistry in#uences on ECM formation directly a!ect cell-surface behavior via integrin signaling, and by implication, RhoA activation. Results presented here support the contention that RhoA is not e$ciently activated in cells adhering to }CH terminated SAM surfaces. Lack of RhoA activa3 tion leads to the observed absence of actin "lament bundling and subsequent reduction in cell tension and contractility. Taken together, the sequence of events that emerges involves a surface chemistry dependence and regulation of these processes. Reduced levels of biologically active "bronectin adsorb on }CH terminated 3 SAM surfaces from serum; poorly recognized integrin binding sites (e.g. "bronectin's 10th Type III RGD motif ) compromise initial cell attachment. Signaling of RhoA on the intracellular side of the membrane is thus ine$cient in this case, leading to subsequent ine$cient activation and translocation of RhoA to generate focal contacts and actin "lament response. This then inhibits cell spreading, "lament-induced tension, integrin clustering and resultant extracellular "bronectin remodeling necessary for further phenotypic expression and cell growth responses. Such an interpretation adds new molecular details involving the role of the GTPase RhoA as a &gatekeeper'

regulating cell-surface behavior in the new, developing molecular model for complex outside-in signal transduction processes in adherent, anchorage-dependent cells. That Rho is highly conserved across a wide range of cells has profound implications for the generalized process of surface chemistry-modulated responses involving Rho proteins. Signi"cantly, the capabilities demonstrated here to interrogate and manipulate the expression and activation of RhoA on surfaces should enhance further understanding of how cells precisely respond to adsorbed proteins and functional chemistry on surfaces. Correlations of molecular biological events associated with cellsurface signaling should facilitate development of new biomaterials that integrate tissues using surface-tailored biological cues. Such rational approaches to needed materials improvements require full knowledge and control of signaling pathways to desired phenotypic endpoints. Study of RhoA modulation using well-controlled, albeit relatively simple surface chemistry, provides a new basis for assessing cell}surface interactions that contribute to such approaches.

5. Conclusions The important intracellular signaling protein, RhoA, has been found to be in#uenced by cell culture surface chemistry. Using well-de"ned model organic surfaces in serum-based cell attachment experiments, the following conclusions are drawn: 1. RhoA becomes activated and membrane-localized in "broblasts cultured on hydrophilic }COOH terminated surfaces, consistent with its known role in integrin-mediated cell signaling and growth responses. Such a result supports previous observations of greater cell attachment, proliferation and spreading, enhanced "bronectin adsorption and greater integrin binding motif availability on these surfaces over the more hydrophobic }CH terminated SAM surfaces 3 [12]. 2. Disabling the RhoA signaling process using C3 transferase (cytotoxin) produces an approximation of a low growth, rounded cell phenotype on surfaces, consistent with RhoA's known in#uence as a determinant for cell}surface association. 3. Transfection of low-growth phenotype, rounded cells on the poorly supportive }CH terminated culture 3 surface with a constitutively active RhoA mutant produces a transformation to a spread cell phenotype with partial actin organization, which is insu$cient to facilitate a growth phenotype in these cells. De"ciencies in the attachment process are consistent with low cell attachment, spreading and proliferation, low "bronectin ECM densities and lack of RGD}integrin binding sites found previously on these surfaces [12].

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F-actin assembly prompted by activated RhoA cannot mature without su$cient integrin involvement. Acknowledgements Support from NIH grant R01 GM56751-01 and from the Colorado Advanced Technology Initiative are gratefully appreciated. Criticism and technical guidance from J.R. Bamburg, B. Bernstein and T. Kuhn have proven extremely helpful. References [1] Werb Z, Tremble PM, Behrendtsen O, Crowley E. Damsky CH. Signal transduction through the "bronectin receptor induces collagenase and stromelysin gene expression. J Cell Biol 1989; 109(2):877}89. [2] Adams JC, Watt FM. Regulation of development and di!erentiation by the extracellular matrix. Development 1993;117:1183}98. [3] Streuli CH, Bailey N, Bissell MJ. Control of mammary epithelial di!erentiation: basement membrane induced tissue-speci"c gene expression in the absence of cell}cell interaction and morphological polarity. J Cell Biol 1991;115:1383}95. [4] Hynes RO. Integrins: versatility, modulation, and signaling in cell adhesion. Cell 1992;69(1):11}25. [5] Clark EA, Brugge JS. Integrins and signal transduction pathways: the road taken. Science 1995;268(5208):233}9. [6] Damsky C, Tremble P, Werb Z. Signal transduction via the "bronectin receptor: do integrins regulate matrix remodeling? Matrix Suppl 1992;1:184}91. [7] Ruoslahti E, Pierschbacher MD. New perspectives in cell adhesion: RGD and integrins. Science 1987;238(4826):491}7. [8] Garcia AJ, Vega MD, Boettiger D. Modulation of cell proliferation and di!erentiation through substrate-dependent changes in "bronectin conformation. Mol Biol Cell 1999;10:785}98. [9] Grinnell F, Feld MK. Adsorption characteristics of plasma "bronectin in relationship to biological activity. J Biomed Mater Res 1981;15:363. [10] Juliano DJ, Saavedra SS, Truskey GA. E!ect of the conformation and orientation of adsorbed "bronectin on endothelial cell spreading and the strength of adhesion. J Biomed Mater Res 1993;27(8):1103}13. [11] Underwood PA, Steele JG, Dalton BA. E!ects of polystyrene surface chemistry on the biological activity of solid phase "bronectin and vitronectin, analyzed with monoclonal antibodies. J Cell Sci 1993;104(Pt 3):793}803. [12] McClary KB, Ugarova T, Grainger DG. Modulating "broblast adhesion, spreading and proliferation with self-assembled monolayers of alkylthiolates on gold. J Biomed Mater Res 1999, in press. [13] Haas TA, Plow EF. Integrin-ligand interactions: a year in review. Curr Opin Cell Biol 1994;6(5):656}62. [14] Hansen LK, Mooney DJ, Vacanti JP, Ingber DE. Integrin binding and cell spreading on extracellular matrix act at di!erent points in the cell cycle to promote hepatocyte growth. Mol Biol Cell 1994;5(9):967}75. [15] Hotchin NA, Hall A. Regulation of the actin cytoskeleton, integrins and cell growth by the Rho family of small GTPases. Cancer Surv 1996;27:311}22. [16] Schwartz MA, Lechene C, Ingber DE. Insoluble "bronectin activates the Na/H antiporter by clustering and immobilizing integrin a5b1, independent of cell shape. Proc Natl Acad Sci USA 1991; 88:7849}53.

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[17] Boguski MS, McCormick F. Proteins regulating Ras and its relatives. Nature 1993;366:643}54. [18] Nobes CD, Hall A. Rho, rac, and cdc42 GTPases regulate the assembly of multimolecular focal complexes associated with actin stress "bers, lamellipodia, and "lopodia. Cell 1995;81(1):53}62. [19] Ridley AJ, Paterson HF, Johnston CL, Diekmann D, Hall A. The small GTP-binding protein rac regulates growth factor-induced membrane ru%ing. Cell 1992;70:401}10. [20] Ridley AJ, Hall A. The small GTP-binding protein rho regulates the assembly of focal adhesion and actin stress "bers in response to growth factors. Cell 1992;70:389}99. [21] Miura Y, Kikuchi A, Musha T, Kuroda S, Yaku H, Sasaki T, Takai Y. Regulation of morphology by rho p21 and its inhibitory GDP/GTP exchange protein (rhoGDI) in Swiss 3T3 cells. J Biol Chem 1993;268:510}5. [22] Maduale P, Axel R. A novel ras-related gene family. Cell 1985; 41:31}40. [23] Olo!son B, Chardin P, Touchot N, Zahraoui A, Tavitan A. Expression of the ras-related ra1A, rho12 and rab genes in adult mouse tissues. Oncogene 1988;3:231}4. [24] Cerione RA, Zheng Y. The Dbl family of oncogenes. Curr Opin Cell Biol 1996;8:216}22. [25] Leung T, Chen Q, Manser EL. The p160 RhoA-binding kinase ROK alpha is a member of a kinase family and is involved in the reorganization of the cytoskeleton. Mol Cell Biol 1996;16:5313. [26] Machesky LM, Hall A. Role of actin polymerization and adhesion to extracellular matrix in Rac- and Rho-induced cytoskeletal reorganization. J Cell Biol 1997;138(4):913}26. [27] Chrzanowska-Wodnicka M, Burridge K. Rho-stimulated contractility drives the formation of stress "bers and focal contacts. J Cell Biol 1996;133:1403}15. [28] Tidwell CD, Ertel SI, Ratner BD. Endothelial cell growth and protein adsorption on terminally functionalized, self-assembled monolayers of alkanethiolates on gold. Langmuir 1997;13: 3404}13. [29] Lewandowska K, Balachander N, Sukenik CN. Culp LA. Modulation of "bronectin adhesive functions for "broblasts and neural cells by chemically derivatized substrata. J Cell Physiol 1989; 141(2):334}45. [30] Healy KE, Thomas CH, Rezania A, Kim JE, McKeown PJ, Lom B, Hockberger PE. Kinetics of bone cell organization and mineralization on materials with patterned surface chemistry. Biomaterials 1996;17(2):195}208. [31] Lopez G, Albers M, Schreiber S, Carroll R, Peralta E, Whitesides G. Convenient methods for patterning the adhesion of mammalian cells to surfaces using self-assembled monolayers of alkanethiolates on gold. J Am Chem Soc 1993;115:5877}8. [32] Mrksich M, Chen CS, Xia Y, Dike LE, Ingber DE, Whitesides GM. Controlling cell attachment on contoured surfaces with self-assembled monolayers of alkanethiolates on gold. Proc Natl Acad Sci USA 1996;93(20):10775}8. [33] Ulman A. An introduction to ultrathin organic "lms: from Langmuir}Blodgett to self-assembly. Boston: Academic Press, 1991. p. 442. [34] Ulman A. Characterization of organic thin "lms. Boston, Greenwich: Butterworth-Heinemann, Manning, 1995. p. 276. [35] Dubois LH, Nuzzo RG. Synthesis, structure and properties of model organic surfaces. Ann Rev Phys Chem 1992;43:437}63. [36] Aktories K, Hall A. Botulinum ADP-ribosyltransferase C3: a new tool to study low molecular weight GTP-binding proteins. Trends Pharmacol Sci 1989;10(10):415}8. [37] Wessel D. Flugge UI. A method for the quantitative recovery of protein in dilute solution in the presence of detergents and lipids. Anal Biochem 1984;138(1):141}3. [38] Minamide LS, Bamburg JR. A "lter paper dye-binding assay for quantitative determination of protein without interference from reducing agents or detergents. Anal Biochem 1990;190(1):66}70.

2446

K.B. McClary, D.W. Grainger / Biomaterials 20 (1999) 2435}2446

[39] No D, Yao T-P. Evans RM. ecdysone-inducible gene expression in mammalian cells and transgenic mice. Proc Natl Acad Sci USA 1996;93:3346}51. [40] Paterson HF, Self AJ, Garrett MD, Just I, Aktories K, Hall A. Microinjection of recombinant p21rho induces rapid changes in cell morphology. J Cell Biol 1990;111(3):1001}7. [41] Hotchin NA, Hall A. The assembly of integrin adhesion complexes requires both extracellular matrix and intracellular rho/rac GTPases. J Cell Biol 1995;131(6):1857}65. [42] Fleming IN, Elliott CM, Exton JH. Di!erential translocation of Rho family GTPases by lysophosphatidic acid, endothelin-1, and platelet-derived growth factor. J Biol Chem 1996;271(51): 33067}73. [43] Adamson P, Paterson H, Hall A. Intracellular localization of the p21rho proteins. J Cell Biol 1952;119(3):617}27. [44] Moores SL, Schaber MD, Mosser SD, Rands E, O'Hara MB, Garski VM, Marshall MS, Pompliano DL, Gibbs JD. Sequence dependence of protein prenylation. J Biol Chem 1991;266: 14603}10. [45] Lamarche N, Hall A. GAPs for rho-related GTPases. Trends Genet 1994;10(12):436}40.

[46] Ueda T, Kikuchi A, Ohga N, Takai Y. Puri"cation and characterization from bovine brain cytosol of a novel regulatory protein inhibiting the dissociation of GDP from and the subsequent binding of GTP to rhoB p20, a ras p21-like GTP-binding protein. J Biol Chem 1990;265:9373}80. [47] Hart MJ, Maru Y, Leonard D, Wittle ON, Evans T, Cerione RA. A GDP dissociation inhibitor that serves as a GTPase inhibitor for the Ras-like protein CDC42Hs. Science 1992;258:812}5. [48] Rubin EJ, Gill DM, Boquet P, Popo! MR. Functional modi"cation of a 21-kilodalton G protein when ADP-ribosylated by exoenzyme C3 of clostridium botulinum. Mol Cell Biol 1988;8:418}26. [49] Mosher DF. Organization of the provisonal "bronectin matrix: control of products of blood coagulation. Thromb Haemostasis 1995;74:529}33. [50] Wu CY, Keivens VM, Otoole TE, McDonald JA, Ginsberg MH. Integrin activation and cytoskeletal interaction are essential for the assembly of a "bronectin matrix. Cell 1995;83:715}24. [51] Zhong C, Chrzanowska-Wodnicka M, Brown J, Shaub A, Belkin AM, Burridge K. Rho-mediated contractility exposes a cryptic site in "bronectin and induces "bronectin matrix assembly. J Cell Biol 1998;141(2):539}51.