fibroblast expression of α-smooth muscle actin, α2β1 integrin and αvβ3 integrin: influence of...
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Experimental and Molecular Pathology 91 (2011) 394–399
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Experimental and Molecular Pathology
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Fibroblast expression of α-smooth muscle actin, α2β1 integrin and αvβ3 integrin:Influence of surface rigidity
Christine Jones, H. Paul Ehrlich ⁎Division of Plastic Surgery, The Pennsylvania State University, College of Medicine, Hershey, PA 17033, USA
⁎ Corresponding author at: Division of Plastic SurgeryUniversity, College of Medicine, 500 University Drive, He717 531 4339.
E-mail addresses: [email protected] (C. Jones)(H.P. Ehrlich).
0014-4800/$ – see front matter © 2011 Elsevier Inc. Aldoi:10.1016/j.yexmp.2011.04.007
a b s t r a c t
a r t i c l e i n f oArticle history:Received 4 April 2011Available online 22 April 2011
Keywords:MyofibroblastTensionPolyacrylamide gelCollagenIntegrinα Smooth muscle actin
Open wound contraction necessitates cell and connective tissue interactions, that produce tension.Investigating fibroblast responses to tension utilizes collagen coated polyacrylamide gels with differencesin stiffness. Human foreskin fibroblasts were plated on native type I collagen-coated polyacrylamide gel coverslips with different rigidities, which were controlled by bis-acrylamide concentrations. Changes in alphasmooth muscle actin (αSMA), α2β1 integrin (CD49B) and αvβ3 integrin (CD-51) were documented byimmuno-histology and Western blot analysis. Cells plated on rigid gels were longer, and expressed αvβ3integrin and αSMA within cytoplasmic stress fibers. In contrast, cells on flexible gels were shorter, expressedα2β1 integrin and had fine cytoskeletal microfilaments without αSMA. Increased tension changed the actinmakeup of the cytoskeleton and the integrin expressed on the cell's surface. These in vitro findings are inagreement with the tension buildup as an open wound closes by wound contraction. It supports the notionthat cells under minimal tension in early granulation tissue express α2β1 integrin, required for organizingfine collagen fibrils into thick collagen fibers. Thicker fibers create a rigid matrix, generating more tension.With increased tension cytoskeletal stress fibers develop that contain αSMA and αvβ3 integrin that replacesα2β1 integrin, consistent with cell switching from collagen to non-collagen proteins interactions.
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Introduction
Fibroblasts play a central role in restoring the integrity of thedermis in response to dermal loss. The wound fibroblast performsmany activities: migration into the dermal defect, proliferation,synthesis of macromolecules, organization of the newly synthesizedmacromolecules into a new connective tissue matrix, and thendisappearance by apoptosis. In the initial stage of wound healing, thecavity caused by tissue loss is filled with a fibrin matrix that has twoprimary functions. First, a deposited fibrin clot controls bleeding.Secondly the fibrin matrix serves as the highway for the invasion ofinflammatory cells and wound fibroblasts into the wound site(Desmouliere et al., 2003). Chemotactic cytokines are secreted in thelocal wound environment by platelets and leukocytes, activatingquiescent fibroblasts to migrate into the wound (Hinz and Gabbiani,2003). Activated fibroblasts contain fine actin cytoplasmic filamentsassociated with cell locomotion, which enhances fibroblast migrationinto the wound site. Young fibroblasts produce and release macro-molecules, which includes collagen, the structural component of thenewly-generated granulation tissue matrix that replaces the fibrin
matrix. The reorganization of collagen fibrils into thicker collagen fiberbundles by fibroblast compacts the newly deposited connective tissuematrix within granulation tissue, leading to wound contraction.
The manner in which wound fibroblasts respond to surroundingtension influenceswound repair. The compaction of granulation tissuepulls on the surrounding dermis, generating tension. The woundfibroblast matures and transforms into a myofibroblast, characterizedby prominent cytoplasmic stress fibers with the alpha smooth muscleactin (αSMA) isoform of actin. Themyofibroblast is the icon of fibrosis,associated with granulation tissue and wound contraction, as well aschronic forms of fibrosis that include hypertrophic scar and cirrhosis(Desmouliere et al., 2005). Released soluble factors that direct cell–cellinteractions and cell–matrix interactions are involved in initiating andcontrolling numerous fibroblast activities. One of these factors,transforming growth factor β1 (TGF-β1), is released from fibroblastsas well as inflammatory cells and is a major factor directing thetransformation of fibroblasts into myofibroblasts. In this study, thefocus is on changing the fibroblast phenotype to the myofibroblastthrough ECM–fibroblast interactions, rather than by the exogenousintroduction of cytokines such as TGF-β1.
An in vitro model for studying fibroblast interaction with thesurrounding connective tissue matrix is the fibroblast populatedcollagen lattice (PCL), which was first introduced by Bell et al. (1979).The Bell fibroblast PCL is a three-dimensional, free-floating, cell-populated collagen matrix, which undergoes lattice contraction,containing exclusively fibroblasts, as evidenced by cells that retain
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an elongated morphology as the PCL is reduced in size and that fail toexpressαSMA (Arora et al., 1999; Eckes et al., 2006; Ehrlich, 1988). Analternate model of contracting fibroblast PCL is the attached-delayed-released PCL. Fibroblast PCL is anchored to the underlying surface of atissue culture dish for 4 days. At day 4 the entire cell populationconsists of myofibroblasts, all cells expressing αSMA in cytoskeletalstress fibers. The release of these attached lattices produces rapidlattice contraction through the contraction of the resident myofibro-blasts (Tomasek et al., 1992).
The expression of αSMA in myofibroblast stress fibers can beinduced by TGF-β1 and by placing cells under tension. Including TGF-β1withdermalfibroblasts inmonolayer culture increases theproportion ofcells expressing αSMA in stress fibers (Desmouliere et al., 1993).Mechanical tension also enhances αSMA expression within fibroblastsmaintained on a stiff collagen-coated surface. Increased mechanicaltension on cells increases the expression of αSMA (Arora et al., 1999).The α2β1 integrin on the surface of fibroblasts fixes fibroblasts tocollagen and is required for free-floating fibroblast PCL contraction(Gullberg et al., 1989). Increasedα2β1 expression is a common featureoffibroblasts suspended in free-floatingPCL (Eckes et al., 2006). Initially,expression of α2β1 integrin is increased in fibroblasts within contract-ing PCL, but the level of expression returns to baseline following thecompletion of compaction activities (Klein et al., 1991). Compared tocells growing on plastic culture dishes, fibroblasts on collagen-coateddishes express greater amounts of α2β1 integrin. In contrast, thosesame cells maintained on a rigid plastic surface express αSMA andexpress little α2β1 integrin, which suggests that myofibroblastattachment to collagen differs from fibroblasts (Ehrlich et al., 1998).
Thin, collagen-coated polyacrylamide gels adherent to glass coverslips is a technique for the study of substrate rigidity on fibroblastphysiology. Because of the inert nature of the polyacrylamide surface,cells interact with the substrate solely through the collagen-coatedsurface. For the study of mechanical tension on cell physiology, thesegels have advantages over the PCL model (Beningo et al., 2002; WangandPelham, 1998). Through the thin gel surface, cells are observed andimaged at high resolution. The polyacrylamide substrate displaysnearly ideal elastic properties over a wide range of stiffness. Substraterigidity can be varied in a controlled, sequential and consistentmannerby altering the concentration of the cross-linking element of the gel,bis-acrylamide. Such stiffness-controlled properties facilitate a moreprecise measurement of fibroblast response to substrate tension.
In this study, collagen-coated polyacrylamide gels of varyingflexibility or stiffness examine the effects of substrate rigidity onfibroblast morphology and the expression ofαSMA, α2β1 integrin, andαvβ3 integrin. We compare changes in human dermal fibroblastphenotype, growing on relatively flexible surfaces and on rigidsubstrates. Fibroblast responses to tensionare to increase the expressionof αSMA and αvβ3 integrin, while limiting the expression of α2β1integrin.
Methods
Preparation of polyacrylamide gel sheets
Thin polyacrylamide gel film was deposited on activated glasssurfaces by the technique of Wang and Pelham (1998). Briefly, glasscover slips were activated by cleaning with ethanol, treating withsodium hydroxide, followed with 3-aminopropyltrimethoxysilaneand fixing with glutaraldehyde. A 10% polyacrylamide solution witheither 0.4% bis-acrylamide to create a rigid substrate or 0.025% bis-acrylamide for a flexible substrate was pipetted on the activated glasssurface. The polyacrylamide was cross linked and coated with rat tailtendon acid extracted type I collagen (Ehrlich and Rittenberg, 2000).The stiff 0.4% and flexible 0.025% bis-acrylamide collagen coated glasssurfaces were available for the plating of human dermal fibroblasts.Primary derived human foreskin fibroblasts between their 4th and 9th
passages maintained in Dulbecco's modification of Eagle's medium(DMEM) supplemented with 10% fetal bovine serum and 10 μg/mlgentamicin, referred to as complete DMEM, were released from tissueculture dishes by trypsinization, transferred to cover slips withcollagen coated polyacrylamide surfaces and maintained in completeDMEM. Fibroblastswere observed by phase-contrastmicroscopy. Cellswere photographed on days 1, 2 and 3 and cell lengths weremeasuredwith ImageJ software (Abramoff et al., 2004).
Immuno-histology
On days 1 and 3, fibroblasts maintained on either 0.4% or 0.025%bis-acrylamide substrates were fixed with 4% paraformaldehyde incytoskeletal buffer (137 mM NaCl; 5 mM KCl; 4 mM NaHCO2; 2 mMMgCl2; 3.5 mM glucose; 2 mM EGTA, 1.5 mM phosphate buffer, and5 mM PIPES buffer pH 6.1). Fixed cells were washed and thenpermeabilized with 0.1% Triton X-100 in cytoskeletal buffer. Somefixed, permeabilized cell preparations were incubated with rabbitmonoclonal antibody directed to vinculin (Cat. # V4139 Sigma) andothers were incubated with mouse monoclonal antibody directed toαSMA (Cat. # A2547 Sigma). Unbound primary antibodywaswashedaway and the cell preparations were incubated with a secondaryantibody: an Oregon-green-tagged goat anti-rabbit IgG antibody or arhodamine-tagged donkey anti-mouse IgG antibody. All cell prepa-rations received AlexaFluor phalloidin to label filamentous actin andDAPI to stain nuclei fluorescent blue (Invitrogen). Stained prepara-tions were mounted on glass microscope slides and viewed with aninverted Zeiss fluorescent microscope. Photographs were taken,using a 40× water-immersion objective with a CoolSnap videocamera.
Western blot analysis
On day 2, media were replaced with complete DMEM supplemen-ted with 50 μg/ml ascorbic acid 1-phosphate sesquimagnesium salt(Sigma Aldrich, St. Louis, MO). On day 3, the fibroblasts were preparedfor Western blot analysis. The fibroblast monolayer on the collagen–polyacrylamide surfacewas coveredwith 100 μl of lysis buffer [2% SDSin Tris buffer pH 6.8], glycerol and proteinase inhibitors (RocheDiagnostics, Indianapolis, IN) for 5 min. While keeping the collagen–polyacrylamide surface intact, cell contents were collected byscrapping the cells in lysate buffer onto a paraffin sheet. The harvestedcell lysate was pipetted into a microcentrifuge tube, sonicated, thenboiled for 5 min and centrifuged to remove particulate matter. Equalvolumes of lysate-supernatant from cultured fibroblast lysates wereelectrophoresed on a 4–20% Tris–HCl gradient gel. The proteins weretransferred to nitrocellulose membranes by electrophoresis and theprotein bandswere prepared for immuno-precipitation detection. Themembranewas probedwithmousemonoclonal antibodies directed toαSMA (Cat. # A2547 Sigma Aldrich) and α-tubulin (Cat. # T5168Sigma Aldrich), followed by a horseradish-peroxidase-conjugatedsecondary antibody (Jackson ImmunoResearch, West Grove, PA).Detection of antibody–protein bands used chemiluminescence withSuperSignal West Dura Extended Duration Substrate (Thermo Scien-tific, Rockford, IL), and X-ray film. The antibody–protein complexbands were eluted from the membrane and the membrane wasreprobed with mouse monoclonal antibodies directed to α2β1integrin/CD49b (Upstate Biotechnology, Lake Placid, NY) and thevitronectin receptor/CD51/αv integrin (Zymed Laboratories, SanFrancisco, CA) and the protein bands were detected as describedabove.
Results
Morphological features of fibroblasts grown on the more rigidsubstrate, 0.4% bis-acrylamide, were different from fibroblasts grown
Table 1Comparison of fibroblast cell lengths grown on stiff compared to flexible substrate.
Day 0.025% bisMean length±SD (μm)
0.4% bisMean length±SD (μm)
DifferenceP value
% Increasein length
1 193 (54) 245 (90) b0.003 21%2 207 (74) 253 (111) b0.002 18%3 171 (77) 260 (103) b0.0001 34%
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on the more flexible substrate, 0.025% bis-acrylamide. When celllengths were measured from phase-contrast images, the cells on therigid substrate were more elongated (Figs. 1A and C), as compared tocells maintained on the flexible substrate (Figs. 1B and D). Themeasured mean cell length of fibroblasts plated on the rigid substratewas 260 μm at day 3, compared to a mean cell length on more flexiblesubstrate of 171 μm, an increase of 34%. These differences were highlysignificant with a p valueb0.0001 (Table 1). The increased differencesin length of cells on stiff substrate versus flexible substrates wereshown as early as day 1, where fibroblasts on stiff surfaces had anaverage length of 245 μm as compared to 193 μm for cells on theflexible surface, a difference of 21%. That significant difference wasfound on day 2, 18% difference, reaching the maximum difference onday 3.
Images taken on days 1 and 3with a fluorescentmicroscope showeddifferences in the organization of the fibroblast's cytoskeleton andαSMA expression. Fibroblasts grown on the more flexible substratefailed to express αSMA and had fine microfilaments arranged in stressfibers (Fig. 2B). At 1 day, fibroblasts plated and maintained on the rigidsubstrate had more prominent, thick, actin-rich microfilaments ar-ranged in stress fibers, but expressed minimal levels of αSMA (Fig. 2A).At 3 days, thick actin-rich filaments were a common feature on theplasma membrane of fibroblasts grown on either firm or flexiblecollagen coated substrates. However, actin filaments within thecytoplasmof these cells showeddistinct variations. At 3 days,fibroblastsmaintained on the rigid substrate had maintained their actin-richcytoplasmic stress fibers that were now enriched in αSMA (Fig. 2C).Fibroblasts on the flexible surface had developed actin rich stress fibers,but they were devoid ofαSMA (Fig. 2D). Based uponαSMA expression,the fibroblasts maintained on firmer 0.4% bis-acrylamide substrate for3 days had transformed intomyofibroblasts. Fibroblasts growing on the
Fig. 1.Morphology of fibroblasts by phase-contrast microscopy. (A) and (B) are cultured cell(C) are cells growing on 0.025% bis-acrylamide, the flexible substrate, and (B) and (D) are
moreflexible substrate at 3 dayshad retained theirfibroblast phenotypeand had not transformed into myofibroblasts. Western blot analysispresented in Fig. 3 confirms that fibroblasts maintained on the moreflexible substrate at 3 days failed to express αSMA within cytoplasmicstress fibers.
As shown in Fig. 3, differences in protein levels for αSMA, α2β1integrin of the collagen-binding integrin CD-49b receptor, and αvintegrin from the CD-51 receptor between fibroblasts were maintainedon a firm substrate compared to maintenance on a flexible substrate. At2 days, therewere clear differences in thedensity of theprotein bands ofthese three proteins depending uponwhether fibroblasts were growingon rigid collagen-coated surfaces or more flexible collagen-coatedsurfaces. The increasedexpressionofαSMA infibroblastsmaintainedonthe 0.4% bis-acrylamide identified by immuno-histologywas confirmedby Western blot analysis (Fig. 3A). In addition, fibroblast expression ofthe collagen receptor,α2β1 integrin,wasgreater infibroblasts platedonflexible substrate as compared to cells residing on rigid substrates(Fig. 3B). Theα2 integrin was characteristically expressed in fibroblastsandminimally expressed inmyofibroblasts. An integrin associatedwithmyofibroblasts is the CD-51 receptor or the αvβ3 integrin, whichshowed greater expression in myofibroblasts that developed on thefirmer collagen-coated substrate (Fig. 3C).
s as they appear on day 1. (C) and (D) are cultured cells as they appear on day 3. (A) andcells growing on 0.4% bis-acrylamide, the rigid substrate. The bar represents 100 μm.
Fig. 2. (A) Morphology of fibroblasts grown on 0.025% bis-acrylamide or (B) 0.4% bis-acrylamide and viewed on day 3. Phalloidin stained filamentous actin is green and nuclei areblue. The bar represents 10 μm (C) fibroblast morphology on day 1 of culture on 0.025% bis-acrylamide and (D) 0.4% bis-acrylamide. Vinculin within focal adhesions is immuno-stained red; phalloidin stained filamentous actin in microfilaments is green, and nuclei are blue. The bar represents 10 μm.
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Discussion
Changes in fibroblast morphology, where αSMA is expressed incytoskeletal stress fibers, are implicated in numerous disease process-es (Desmouliere et al., 2003). In vitro a proportion of fibroblastsgrowing in monolayer culture on plastic with serum supplementedculture medium develop cytoskeletal stress fibers expressing αSMA(Desmouliere et al., 1992). The inclusion of TGF-β1 with fibroblasts inmonolayer culture enhances the expression of αSMA within cyto-skeletal stress (Desmouliere et al., 1993). Plating human dermal
Fig. 3. Western blots of cell lysates were performed on fibroblasts maintained on 0.4%and 0.025% bis-acrylamide collagen coated substrates for 2 days: (A) αSMA, (B) α2β1integrin, and (C) αvβ3 integrin. Equivalent protein loading on gels was confirmed byequal band densities in both samples using α-tubulin (data not shown).
fibroblasts at low density in monolayer, generates greater than 95% ofthe cells expressing αSMA in stress fibers, while plating fibroblasts athigh density generates few cells expressing αSMA in stress fibers(Masur et al., 1996). Fibroblasts growing on collagen coated dishesexpress little αSMA as compared to fibroblasts growing on nakedplastic surfaces, which express more αSMA (Ehrlich et al., 1998). Themechanism for cell adhesion is influenced not only by the composi-tion of the surface on which they adhere, but also by the rigidity of thesubstrate beneath that surface on which those cells adhere (Aroraet al., 1999; Eckes et al., 2006). Here, fibroblasts on collagen coatedsurfaces of 0.4% bis-acrylamide (a ridged surface) express αSMA andαvβ3 integrin compared to fibroblasts residing on collagen coatedsurfaces of 0.025% bis-acrylamide, a flexible surface (Yeung et al.,2005).
An in vitro model for studying fibroblast interactions with theirsurrounding connective tissue matrix is the fibroblast populatedcollagen lattice (PCL), which was first introduced by Bell et al. (1979).The Bell fibroblast PCL is a free-floating PCL, which undergoes a slowsteady reduction in area, and exclusively contains fibroblasts, cellsthat fail to express αSMA and retain an elongated shape as PCLcontract (Arora et al., 1999; Eckes et al., 2006; Ehrlich, 1988). In a freefloating PCL, a major degree of lattice contraction is achieved by2 days in the absence of tension. Fibroblasts lack cytoskeletal stress
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fibers and fail to express αSMA (Ehrlich et al., 2006). By polarizedlight microscopy, contracting free floating PCL develops thick collagenfibers exhibiting birefringence. The collagen fiber birefringence runsparallel to the long axis of elongated fibroblasts (Ehrlich, 1988). Thosesame fibroblasts cast in an attached PCL for 4 days, where tensiondevelops; all the cells express αSMA in stress fibers, havingtransformed into myofibroblasts (Tomasek et al., 1992). Upon releaseat 4 days, in minutes this once attached PCL undergoes rapid latticecontraction via the contraction of the resident myofibroblast popula-tions. However, unlike free floating PCL, where there is minimaltension, no collagen fiber birefringence develops (Ehrlich et al., 2006).The compaction of a collagen lattice under tension through rapidmyofibroblast contraction fails to organize fine collagen fibrils intolonger, thicker parallel oriented collagen fibers. The α2β1 integrin,recognized as the fibroblast-collagen binding integrin (Klein et al.,1991) is required for free floating fibroblast PCL contraction (Gullberget al., 1989). In unpublished studies there is enhanced αvβ3 integrinexpression in attached PCL.
By Western blot analysis human dermal fibroblasts growing oncollagen coated rigid surfaces, experiencing mechanical tension,express increased levels of αSMA and αvβ3 integrin, and decreasedlevels of α2β1 integrin. CD-51 receptor, αvβ3 integrin, contributes tothe generation of αSMA expression. Treating cells with antibodiesdirected to the αv integrin, downregulates the expression αSMA(Lygoe et al., 2007). The αvβ3 integrin binds to a variety of matrixproteins that include vitronectin and fibronectin. Blocking theexpression of αv integrin reduces fibroblast migration (Lygoe et al.,2007). In the absence of αv integrin expression, TGF-β1 inducedexpression ofαSMA is impaired (Lygoe et al., 2004). Mechanical stressrenders fibroblasts more susceptible to growth factors. As an exampleED-A fibronectin in combination with TGF-β1 together transformsfibroblasts into myofibroblasts (Hinz et al., 2001). A proposedmechanism for controlling cell phenotype is through mechanicaltension (Jiang et al., 2006). The sensing of mechanical tension utilizesintracellular signaling through receptor-like protein tyrosine phos-phatase (RPTPα) andαvβ3 integrin, both localized at the leading edgewithin cells experiencing tension (von Wichert et al., 2003). In theabsence of tension, RPTPα cannot interact with the αvβ3 integrin atthe cell's periphery. Increasing αvβ1 expression reduces the rate anddegree of free floating PCL contraction (Boemi et al., 1999; Lygoe et al.,2004). The αvβ3 integrin RPTPα complex senses mechanical tensionat the leading edge of cells, passing on properties of the extracellularmatrix to intracellular signal transduction pathways (Jiang et al.,2006).
Besides tension there are other reports of influencing theexpression of αSMA in cultured cells in monolayer. Cells plated atlow density, upon reaching confluence, most express αSMA incytoskeletal stress fibers (Masur et al., 1996). Casting a collagen latticeover a confluent layer of low density plated cells, all expressingαSMAwithin stress fibers, eliminates the expression of αSMA (Ehrlich et al.,2006). In monolayer culture about 20% of fibroblasts plated on plasticexpress αSMA as well as fail to express α2β1 integrin. Plating thosesame fibroblasts on a polymerized type I collagen surface, upregulatesthe expression of α2β1 integrin, while knocking down the expressionof αSMA (Ehrlich et al., 1998).
Wound fibroblasts are subjected to changes in tension as the repairprocessproceeds (Tomaseket al., 2002).After 1 week,fibroblastswithinwound granulation tissue express αSMA in cytoskeletal stress fibers,becoming myofibroblasts. Tension within granulation tissue increasesas granulation tissue is compacted, which pulls the surrounding skininto the defect. The musculature of surrounding skin response to theseforces is pulling out, which contributes to generating tension. Themyofibroblasts are the wound fibroblast phenotype that undergoesapoptosis in the remodeling phase of repair (Desmouliere et al., 2005).Tension can interferewithmyofibroblasts undergoing programmed celldeath. It is proposed myofibroblast populations identified within
hypertrophic scars that result from mechanical stress as well asinhibiting their entrance into apoptosis (Aarabi et al., 2007;Desmouliereet al., 2005). In amousemodel a brief periodof applied stress, during theproliferative phase of repair, generates excess fibrosis and retardsmyofibroblast entrance into apoptosis by activating the pro-survival Aktpathway (Aarabi et al., 2007). Tension also increases the transcription oftype I and III collagens as well as the synthesis of tissue inhibitors ofmetalloproteinases (MMP), which favors connective tissue accumula-tion (Derderian et al., 2005). Further study in the area of fibroblastresponse to mechanical stress may elucidate molecular pathways thatwill provide targets for intervening in the control of fibrosis and scarcontracture.
Conflict of interest
The authors declare that there is no conflict of interest.
Acknowledgments
The authors wish to thank Greory Saggers and Gretchen Snavelyfor technical help and Kimberly Walker for editing. The work wassupported by NIH grant GM-056851.
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