expression of integrin 1 by fibroblasts is required for ... · in tissue repair and fibrogenic...

3674 Research Article

IntroductionDuring normal healing of skin, connective tissue is repairedexclusively through the action of fibroblasts, which migrate intothe wound site where they synthesize and remodel a newextracellular matrix (ECM) (Martin, 1997; Eckes et al., 1999;Gurtner et al., 2008). The ability of cells to migrate, attach to andremodel ECM is mediated by specialized cell surface structurestermed focal adhesions (FAs), which mediate adhesion betweenthe ECM and the actin cytoskeleton through integrin cell surfacereceptors (Burridge and Chrzanowska-Wodnicka, 1996; Zaidel-Bar et al., 2007). The fibroblasts that generate the adhesive andbiomechanical forces required for wound closure are termedmyofibroblasts, because they express highly contractile proteinsincluding -smooth muscle actin (-SMA) (Dugina et al., 2001;Chen et al., 2005; Hinz et al., 2007). Although myofibroblastsnormally disappear from the healed wound, the persistence of themyofibroblast appears to be a major contributor to the phenotypeof excessive scarring and fibrotic disorders (Dugina et al., 2001;Chen et al., 2005; Hinz et al., 2007; Varga and Abraham, 2007).Controlling myofibroblast action is therefore essential to controlnormal tissue repair and to develop novel, effective and rationalanti-fibrotic therapies.

Several growth factors and cytokines contribute to wound closurein vivo (Werner and Grose, 2003; Barrientos et al., 2008); however,it is now increasingly clear that proteins of the focal adhesionsmight act as a common integration point for signals emanatingfrom growth factors and their receptors (Burridge and

Chrzanowska-Wodnicka, 1996; Zamir and Geiger, 2001) and frominteractions with the external environment (Zaidel-Bar et al., 2007;Geiger et al., 2009). As an example, fibroblasts lacking focaladhesion kinase (FAK) spread poorly, cannot migrate, and cannotproperly respond to TGF (Ilic et al., 1995; Liu et al., 2007).Recently, we have shown that fibroblast expression of Rac1, asmall GTPase that is expressed ubiquitously and is recruited toFAs by paxillin (Burridge and Wennerberg, 2004; Ishibe et al.,2004), is required for fibrogenesis and cutaneous tissue repair invivo (Liu et al., 2008; Liu et al., 2009a) and for induction ofmyofibroblast formation by endothelin-1 (Shi-wen et al., 2004).Moreover, we have shown that activation of cell adhesion issufficient to cause increased expression of mRNAs encodingfibrogenic proteins (Kennedy et al., 2008). Collectively, theseresults suggest that adhesion and adhesive signaling has a key rolein tissue repair and fibrogenic responses in vivo.

Given their role in cell adhesion and adhesive signaling, integrinexpression by fibroblasts must be important for tissue repair in vitro,although the contribution of individual integrins to this process isstill being elucidated (Hynes, 2002; Schultz and Wysocki, 2009).Integrin 1 is a common mediator of fibroblast attachment to collagentype I and fibronectin (Lafrenie and Yamada, 1996; Gabbiani, 2003),suggesting that expression of integrin 1 by fibroblasts has a keyrole in cutaneous tissue repair. However, this hypothesis has not yetbeen tested. As integrin-1-knockout mice die in utero (Stephens etal., 1995), conditional-knockout mouse models are necessary toaddress this question. Mice harboring the integrin 1 allele flanked

Expression of integrin 1 by fibroblasts is required fortissue repair in vivoShangxi Liu1, Xu Shi-wen2, Katrin Blumbach3, Mark Eastwood4, Christopher P. Denton2, Beate Eckes3,Thomas Krieg3, David J. Abraham2,* and Andrew Leask1,*,‡

1The Canadian Institute of Health Research Group in Skeletal Development and Remodeling, Division of Oral Biology and Department ofPhysiology and Pharmacology, Schulich School of Medicine and Dentistry, University of Western Ontario, Dental Sciences Building, London,ON N6A 5C1, Canada2Centre for Rheumatology, Department of Inflammation, Division of Medicine, UCL-Medical School (Royal Free Campus), University CollegeLondon, London NW3 2PF, UK3Department of Dermatology, University of Cologne, Kerpener Street 62, D-50937 Cologne, Germany4Division of Biosciences, University of Westminster, 115 New Cavendish Street, London W1W 6UW, UK*These authors contributed equally to this work‡Author for correspondence ([email protected])

Accepted 9 July 2010Journal of Cell Science 123, 3674-3682 © 2010. Published by The Company of Biologists Ltddoi:10.1242/jcs.070672

SummaryIn tissue repair, fibroblasts migrate into the wound to produce and remodel extracellular matrix (ECM). Integrins are believed to becrucial for tissue repair, but their tissue-specific role in this process is poorly understood. Here, we show that mice containing afibroblast-specific deletion of integrin 1 exhibit delayed cutaneous wound closure and less granulation tissue formation, includingreduced production of new ECM and reduced expression of -smooth muscle actin (-SMA). Integrin-1-deficient fibroblasts showedreduced expression of type I collagen and connective tissue growth factor, and failed to differentiate into myofibroblasts as a result ofreduced -SMA stress fiber formation. Loss of integrin 1 in adult fibroblasts reduced their ability to adhere to, to spread on and tocontract ECM. Within stressed collagen matrices, integrin-1-deficient fibroblasts showed reduced activation of latent TGF. Additionof active TGF alleviated the phenotype of integrin-1-deficient mice. Thus integrin 1 is essential for normal wound healing, whereit acts, at least in part, through a TGF-dependent mechanism in vivo.

Key words: -SMA, Myofibroblast, Matrix contraction, Integrins

Jour

nal o

f Cel

l Sci

ence

by loxP sites have been generated, and these mice have been usedsuccessfully to delete integrin 1 in vivo using mice expressing Crerecombinase under the control of a tissue-specific promoter(Raghavan et al., 2000; Piwko-Czuchra et al., 2009; Bauer et al.,2009). Recently, we have shown that mice containing a fibroblast-specific deletion of integrin 1 show resistance to bleomycin-inducedskin fibrosis (Liu et al., 2009b). In this report, we study the influenceof deletion of integrin 1 in adult fibroblasts on cutaneous woundhealing and the underlying cell and molecular processes involved.Our data provide new and valuable insights into the contribution ofintegrin 1 to fibroblast biology.

ResultsDeletion of integrin 1 causes delayed cutaneous woundrepairTo test whether integrin-1-deficient mice showed impairedcutaneous wound closure, we subjected 8-week-old micehomozygous for deletions in the integrin 1 gene (Itgb1), or wild-

3675Integrin 1 and tissue repair

type littermate counterparts, to dermal punch wounding. Loss of1 integrin was shown by real-time PCR and western blot analysisof fibroblasts cultured from control (C/C) and integrin-1-deficient(K/K) animals (Fig. 1A,B). Compared with wounded controlanimals, integrin-1-deficient animals showed a significantlyreduced rate of wound closure (Fig. 1C). Studies using wild-typemice treated with or without tamoxifen revealed that tamoxifenalone did not affect wound closure (not shown). Integrin-1-deficient animals were examined 7 days after wounding. Theydisplayed reduced collagen production and less granulation tissue(Fig. 2), as well as myofibroblasts with decreased -SMA andPCNA expression at the site of injury (Fig. 3) compared withcontrol animals.

Deletion of integrin 1 causes impaired migration,adhesion, proliferation and spreadingTo study the molecular mechanism(s) of failed wound repair, wecultured primary dermal fibroblasts from explants of animals

Fig. 1. Conditional deletion of integrin 1 infibroblasts results in impaired cutaneous woundhealing. Mice deleted for integrin 1 (K/K) andcontrol mice (C/C) that were otherwise geneticallyidentical were generated and genotyped.(A)Fibroblasts from integrin 1 and control micewere cultured. RNA was extracted from cells andsubjected to real-time PCR analysis to detectmRNA encoding integrin 1 and GAPDH, andrelative expression was calculated by the Ct

method. Six mice per group were analyzed. Datarepresent means + s.d. (*P<0.05; Student’s t-test).(B)Fibroblasts from integrin 1 and control micewere cultured. Cell extracts were subjected toSDS-PAGE and western blot analysis with anti-integrin-1 antibody. Before hybridization withantibody, blots were stained using Ponceau Red toverify equal loading. (C)Mice were wounded andwound closure was measured on day 0, day 3, day7 and day 10. Six mice per group (four wounds permouse) were analyzed. Data represent means + s.d.(*P<0.05; Student’s t-test).

Fig. 2. Conditional deletion of integrin 1 in fibroblastsresults in impaired cutaneous wound healing. Mice werewounded and quality of wound closure was measured.Histological sections of mouse wounds 7 days afterwounding were investigated using hematoxylin and eosin(H&E, top) and trichrome (bottom) stain to detect collagen.Area of granulation tissue and hydroxyproline levels weremeasured and results are shown in graphs on right. For allassays, six mice per group were analyzed. Data representmeans + s.d. (*P<0.05, **P<0.01; Student’s t-test). Note thatintegrin-1-deficient wounds had defects in wound closureas revealed by reduced granulation tissue in the wound. epi,epidermis; der, dermis.

Jour

nal o

f Cel

l Sci

ence

deleted for integrin 1 or not. Using a scratch wound assay of invitro wound repair, we showed that integrin-1-deficient cellsmigrated more slowly (Fig. 4A). Single-cell tracking confirmedthat persistence of migration in integrin-1-deficient fibroblasts oncollagen I and on tissue culture plastic was significantly diminished(Fig. 4B). Integrin-1-deficient cells also showed reducedproliferation (Fig. 5A) and an impaired ability to adhere to afibronectin substrate (Fig. 5B) than observed with control cells.Cell spreading was also monitored microscopically after adhesion,using antibodies against Rhodamine-phalloidin and vinculin. Lossof integrin 1 also resulted in abnormal spreading on fibronectin,as revealed with anti-vinculin antibody (green) and Rhodamine-phalloidin (red) staining to detect actin (Fig. 5C).

Deletion of integrin 1 causes impaired myofibroblastdifferentiation and ECM contractionMyofibroblasts are the major cell type responsible for ECMremodeling during wound healing (Dugina et al., 2001; Hinz et al.,

3676 Journal of Cell Science 123 (21)

2007). We showed that integrin-1-deficient fibroblasts containedreduced numbers of -SMA-containing stress fibers (Fig. 6A)compared with control cells, suggesting that integrin-1-deficientcells had impaired myofibroblast differentiation. Consistent withthis observation, integrin-1-deficient fibroblasts also had reduced-SMA protein expression (Fig. 6B). Moreover, loss of integrin 1resulted in cells that displayed reduced expression of mRNAsencoding profibrotic proteins, including -SMA and type I collagen(Fig. 6C). Finally, integrin-1-deficient fibroblasts were less ableto migrate and thus to generate contractile forces to contract free-floating and tethered collagen gel matrices (Fig. 7A,B). These datasuggest that integrin 1 deficiency results in a dramatic failure offibroblast attachment to collagen and differentiation intomyofibroblasts.

Loss of integrin 1 results in reduced TGF activationIt has been proposed that integrins (in particular integrin v)mediate the activation of latent TGF during myofibroblast

Fig. 3. Conditional deletion of integrin 1 infibroblasts results in impaired cutaneous woundhealing. Wounds in mice 7 days after wounding weremeasured using PCNA (red) to detect proliferation and-SMA antibody (red) to detect myofibroblasts. Nucleiare stained with DAPI (blue). For all assays, six miceper group were analyzed. Data represent means + s.d.(*P<0.05, **P<0.01; Student’s t-test).

Fig. 4. Loss of integrin 1 results in areduced ability of fibroblasts to migrateon ECM. Fibroblasts were isolated byexplant culture from mice containing theintegrin 1 gene (C/C) or not (K/K).(A)Cells were subjected to scratch woundassay of cell migration. Fibroblasts from sixmice per group were analyzed at 24 and 48hours. Red lines represent the cell front.Graphs show means + s.d. (*P<0.05;Student’s t-test). (B)Control (and integrin1-deficient fibroblasts (both passage 3)were seeded at low density onto collagen Ior tissue culture plastic. Motility of singlecells was tracked over 5 hours as soon ascells had settled down; each dot representsone cell. Persistence of migration wascalculated. Red bars indicate the means ofn117 control and n72 mutant fibroblastson collagen I; n123 control, n69 mutantcells on plastic. Persistent migration issignificantly reduced in mutant fibroblastson both substrates (P0.02 on collagen I;P0.01 on plastic). Note that in all cases,the same number of cells were initiallyplated.

Jour

nal o

f Cel

l Sci

ence

contraction (Wipff et al., 2007). To test whether loss of integrin 1resulted in impaired activation of latent TGF, we co-cultured incollagen gel matrices, fibroblasts and mink lung reporter cellsstably transfected with plasminogen activator inhibitor-1 promoter-luciferase construct, which responds to TGF (Abe et al., 1994;Wipff et al., 2007). As anticipated, luciferase activity was elevated


in gels populated with wild-type cells, but not in gels populatedwith integrin-1-deficient cells (Fig. 8). The activity observed inthe presence of wild-type cells was significantly inhibited by theaddition of the small molecule inhibitor of the TGF type I receptor(ALK5) or the neutralizing anti-TGF antibody 1D11, indicatingthe involvement of TGF in this process (Fig. 8A,B). These datastrongly suggest that integrin-1-knockout fibroblasts possessdecreased TGF activation in bio-mechanically loaded gels, whichis characterized by the formation of myofibroblasts. To test themechanism underlying integrin-1-mediated TGF activation,assays were performed in the presence of DMSO and the myosinATPase contraction inhibitor blebbistatin. Matrix remodeling andECM contraction in floating gel matrices involves actin or myosincontraction (Daniels et al., 2003; Mirastschijski et al., 2004;Eastwood et al., 1998); thus blebbistatin blocked collagen gelcontraction mediated by wild-type integrin 1 cells in floatingcollagen gel matrices (Fig. 8C). Blebbistatin reduced TGFactivation in gels embedded with wild-type cells (Fig. 8C). ThusTGF activation in floating collagen gel matrices depends on bothmyosin ATPase and contraction. Addition of TGF1 rescued thecontractile and TGF activation defects of integrin-1-knockoutcells (Fig. 8C).

Western blot analyses of wild-type (C/C) and integrin-1-deficient (K/K) cells showed no difference in levels of TGFtypeI or TGFtype II; however, integrin-1-deficient cells showedelevated integrin 3 expression (Fig. 9), an integrin that haspreviously been associated with decreased rates of tissue repair(Reynolds et al., 2005).

Based on these results, we investigated whether phenotype ofintegrin-1-deficient mice could be rescued by addition ofexogenous active TGF1 to mice subjected to the dermal punchmodel of tissue repair. By day 3 after wounding, addition of TGF1resulted in increased wound closure in both wild-type and integrin-1-deficient mice (Fig. 10A). By day 5 and day 7 after wounding,TGF1 increased wound closure in integrin-1-deficient mice tothe level observed in wild-type mice (Fig. 10A). In addition,application of TGF1 to day 7 wounds of integrin-1-deficient

Fig. 5. Loss of integrin 1 results in a reduced ability of fibroblasts toadhere to ECM, proliferate and spread on ECM. Fibroblasts were isolatedby explant culture from mice containing the integrin 1 gene (C/C) or not(K/K). For all assays, fibroblasts from six mice per group were analyzed. Cellswere subjected to (A) a proliferation assay and (B) a fibronectin adhesionassay (means ± s.d. are shown; *P<0.05; Student’s t-test). (C)Cell spreadingon fibronectin was monitored for 2 hours. Cells were fixed and stained withanti-vinculin antibody (green) to detect focal adhesions and Rhodamine-phalloidin to detect actin (red). Nuclei are stained with DAPI (blue).Representative images are shown.

Fig. 6. Loss of integrin 1 results in a reduction inmyofibroblast formation. Fibroblasts were isolatedby explant culture from mice containing the integrin1 gene (C/C) or not (K/K). (A)Cells were fixed andstained with anti-vinculin antibody to detect focaladhesions (green) and rhodamine-phalloidin to detectactin and -SMA (red). Colocalization of vinculinand actin is seen as yellow staining. Intensity ofstress fibers was assessed by linear scans acrossfibroblasts (white lines) using image analysissoftware (Northern Eclipse, Empix); representativetraces are shown below. Integrin-1-deficient cellshave a reduced number of actin stress fibers,indicating reduced myofibroblast formation. Nucleiare stained with DAPI (blue). (B)Cells weresubjected to western blot analysis with an anti--SMA antibody. (C)Real-time PCR analysis. mRNAharvested from cells (ten mice total per group, eachassay performed in triplicate, means + s.d. areshown) was subjected to real-time PCR analysis todetect the mRNAs indicated (*P<0.05; Student’st-test).

Jour

nal o

f Cel

l Sci

ence

mice resulted in levels of granulation tissue, collagen, and -SMAproduction that were similar to those observed with the control-injected wild-type mice (Fig. 10B–D). Taken together, these dataindicate that the addition of exogenous active TGF effectivelyrescued the wound-healing defects of integrin-1-deficient mice.These data strongly suggest that expression of integrin 1 byfibroblasts is required for efficient wound closure, specificallyproper granulation tissue formation and dermal repair, via a TGF-dependent mechanism.


DiscussionIntegrin 1 is essential for embryonic development and themaintenance of several tissues (Brakebusch and Fässler, 2005;Fässler and Meyer, 1995). Integrin-1-knockout mice die in utero(Stephens et al., 1995), indicating an essential role for this integrinin development. Cell-type-specific deletions of the integrin 1gene has revealed a key role for integrin 1 in supporting stem andprogenitor cell properties, such as adhesion to their niche and theappropriate orientation of the mitotic spindle to control thesymmetry of cell division (Lechler and Fuchs, 2005; Kuang et al.,2007). Mutant mice lacking integrin 1 in skin epidermis possessedsevere blistering as a result of impaired attachment of basalkeratinocytes to the basement membrane, impaired keratinocyteproliferation, dermal fibrosis and severely delayed wound healing(Raghavan et al., 2000; Brakebush et al., 2000; Grose et al., 2002).However, the role of integrin 1 in fibroblasts and myofibroblast-

Fig. 7. Loss of integrin 1 results in a reduction in ECM contraction. Lossof integrin 1 results in a reduced ability of fibroblasts to contract a collagengel matrix. (A)FPCL analysis reveals the effect of loss of integrin 1expression on contractile force generated by fibroblasts in a fixed, tetheredcollagen gel lattice investigated using a culture force monitor. Force (dynes)generated by fibroblasts was assessed over a 24 hour period. Representativetraces are shown (n3). Note the reduced contractile forces generated by cellsderived from integrin-1-knockout (K/K) cells in comparison with normalfibroblasts (C/C). (B)Floating gel analysis reveals the effect of loss of integrin1 expression on ECM contraction generated by fibroblasts embedded in afloating collagen gel matrix assessed over 24 hours. Contraction was assessedphotographically and by measuring the weight of contracted gels (fibroblastsfrom three animals were used, and experiments were performed in triplicate;means + s.d. are indicated). Note that wild-type fibroblasts were able tocontract a collagen gel matrix (*P<0.05; Student’s t-test), relative to integrin-1-deficient cells.

Fig. 8. Loss of integrin 1 results in a reduction in activation of latent TGF. Mink lung epithelial cells stably transfected with a PAI-1 promoter and luciferaseconstruct were cultured alone or were co-cultured for 24 hours with wild-type (C/C) or integrin-1-knockout (K/K) cells that were embedded within a collagen gelmatrix. Mink lung cells were lysed, and luciferase activity determined. Data are presented as means + s.d. of one representative experiment performed in triplicate.Experiments were performed a minimum of three times. Cells were incubated in the presence or absence of (A) ALK5 inhibitor (10M) or DMSO control,(B) ID11 or IgG (10g/ml) control or (C) DMSO control or Bbst (blebbistatin; 100M). (D)The phenotype of integrin-1-knockout cells is rescued by addition ofTGF. Cells were incubated in the presence or absence of TGF1 (4 ng/ml) for 2 hours before conducting assays (*P<0.05; Student’s t-test).

Fig. 9. Loss of integrin 1 results in an increase in integrin 3 expression.Fibroblasts were isolated by explant culture from mice containing the integrin1 gene (C/C) or not (K/K). Cell extracts were subjected to SDS-PAGE andwestern blot analysis with antibodies against integrin 3, TGF type Ireceptor, TGF type II receptor and GAPDH. Densitometry measurements ofthe gels are shown on the right. Error bars represent means + s.d. (n3;P<0.05; Student’s t-test).

Jour

nal o

f Cel

l Sci

ence

like cells in vivo is largely unknown, as is the precise cell andmolecular mechanism underlying integrin 1 action via fibroblastsin tissue repair.

Integrin 1 is a key mediator of fibroblast adhesion to type Icollagen and fibronectin (Lafrenie and Yamada, 1996; Gabbiani,2003). In this study, we show that loss of integrin 1, specificallyin adult fibroblasts, results in impaired cutaneous tissue repair invivo. Integrin-1-deficient mice showed reduced rates of woundclosure, and decreased myofibroblast production and collagendeposition. Consistent with these results, cultured fibroblastsderived from integrin-1-deficient mice possessed decreased levelsof -SMA mRNA and protein expression. Consistent with thesedata, integrin-1-deficient fibroblasts exhibited reduced -SMAstress fiber formation and reduced expression of mRNA encodingtype I collagen and connective tissue growth factor (CTGF).Moreover, integrin 1 was required for optimal cell migration,adhesion and ECM contraction. These results are in sharp contrastto the conventional knock-out mice lacking integrin 3, which


show enhanced wound healing that is complete several days earlierthan in wild-type mice, and enhanced dermal fibroblast infiltrationinto wound sites (Reynolds et al., 2005). Interestingly, we foundthat loss of integrin 1 resulted in an increase in expression ofintegrin 3, providing a possible mechanistic basis for our study.It is also interesting to note that wound healing in the integrin-2-knockout mouse was similar to that in our fibroblast-specificknockout of 1 integrin (Peters et al., 2005). Here, the mechanismsunderlying the severely impaired wound healing were associatedwith a severe reduction of neutrophils and macrophages into thewounds, causing a lack of secreted TGF1. As our mice are onlydeficient in integrin 1 in fibroblasts, the molecular mechanismunderlying impaired healing is likely to be different. Indeed, it waspreviously suggested that mechanical contraction of ECM byfibroblasts activates TGF in a fashion requiring integrin V andan intact cytoskeleton (Wipff et al., 2007). To determine whethersuch a mechanism might be responsible for the phenotype ofintegrin-1-knockout fibroblasts, we showed that wild-type

Fig. 10. TGF1 rescues the in vivo phenotype ofintegrin-1-deficient mice. Mice were treated withor without TGF1. Wounds and sections (day 7wounds) were analyzed as in Figs 1–3. Three miceeach with four wounds were analyzed. Data representmeans + s.d. (P-values are as indicated; one-wayANOVA). (A)Wound closure measured over 7 days.(B)Area of granulation tissue at day 7 afterwounding. (C)Trichrome collagen stain (blue) andcollagen score at day 7 after wounding. Nucleistained with trichrome appear as black.(D)Myofibroblast formation measured by -SMAstaining (red) at day 7. Nuclei stained with DAPI(blue).

Jour

nal o

f Cel

l Sci

ence


fibroblasts, but not integrin-1-deficient fibroblasts, embeddedwithin a collagen gel were able to support high levels of activityof a TGF-responsive promoter in a fashion that depended onmyosin-mediated contraction. TGF rescued the phenotype ofintegrin-1-deficient mice. These results collectively suggest thatexpression of integrin-1 by fibroblasts is essential for woundhealing, and that the function of integrin 1 occurs partly througha TGF-dependent mechanism probably through a defect inactivation of latent TGF. Our results demonstrate for the firsttime that integrin 1 expression by fibroblasts is involved with thisprocess and is required for formation of granulation tissue in skin.

Fibrosis is characterized by the persistence of myofibroblastswithin scars (Dugina et al., 2001; Chen et al., 2005; Gabbiani,2003). Although myofibroblasts can form through the presence ofextracellular signaling molecules including TGF, ET-1 and CCN2,it is now increasingly appreciated that adhesive signaling alsocontributes to their formation (Werner and Grose, 2003; Leask,2006; Shi-wen et al., 2006a,b; Shi-wen et al., 2007). Our datashowing that integrin 1, a protein that mediates cell attachmentof ECM, was required for tissue repair in vivo is consistent withthis latter notion. Taken together with observations that thepersistent fibrotic phenotype of lung scleroderma fibroblasts is atleast partially mediated by integrin 1 (Shi-wen et al., 2007),which is upregulated in scleroderma fibroblasts (Chen et al., 2005;Chen et al., 2008), our results strongly suggest that the targeting ofadhesion signaling is a novel, appropriate strategy for anti-fibroticdrug intervention. In summary, the observations presented hereindicate that integrin 1 is essential for proper granulation tissueformation and thus for wound closure and might have futureimplications for the treatment of non-healing ulcers and fibrosis.

Materials and MethodsGeneration of integrin 1 conditional-knockout miceIntegrin 1 conditional-knockout mice were generated as described (Liu et al.,2009b). Briefly, mice possessing a tamoxifen-dependent Cre recombinase under thecontrol of a fibroblast-specific regulatory sequence from the pro2(I) collagen gene(Zheng et al., 2002) were crossed with mice homozygous for a conditional Itgb1(integrin 1) allele (Raghavan et al., 2000) (Jackson Laboratories, Bar Harbor, ME)to generate Cre and integrin 1 heterozygous mice, which were mated to generateCre integrin 1 homozygous mice. Animals used for experiments were genotypedby polymerase chain reaction (Raghavan et al., 2000; Zheng et al., 2002). To deleteintegrin 1, 3-week-old mice were given intraperitoneal injections of tamoxifensuspension (0.1 ml of 10 mg/ml 4-hydroxytamoxifen, Sigma, St Louis, MO) over 10days. Deletion of integrin 1 was tested by PCR genotyping. All animal protocolswere approved by the appropriate regulatory authority.

Dermal punch woundingLittermate mice homozygous for the loxP Itgb1 allele and heterozygous for type ICol1A2-Cre were treated with tamoxifen (‘knockout integrin 1’, K/K) or corn oil(‘conditional integrin 1’, C/C). Three weeks after cessation of tamoxifen injection,wounding experiments were conducted. Mice were anesthetized (90 g ketamineplus 10 g xylazine/g), shaved, depilated with Nair and cleaned with alcohol. Permouse, four bilateral full-thickness skin wounds were created, using a sterile 4 mmbiopsy punch on the dorsorostral back skin without injuring the underlying muscle.Wounds were separated by a minimum of 6 mm of uninjured skin, and digitallyphotographed at 0, 3, 7 and 10 days after wounding using a Sony D-9 digital camera.The wound area was determined using Northern Eclipse (Empix) software, andwound closure was expressed as percentage of initial wound size. In addition, micewere sacrificed using CO2 at 3, 7 and 10 days after wounding for histological,immunohistochemical and hydroxyproline analyses.

Immunofluorescence staining, histology and assessmentWound tissue sections (0.5 m) were cut using a microtome (Leica, Richmond Hill,ON) and collected on Superfrost Plus slides (Fisher Scientific, Ottawa, ON). Sectionswere then de-waxed in xylene and rehydrated by successive immersion in descendingconcentrations of alcohol. The sections were then subjected for immunofluorescencestaining. Briefly, tissue sections were blocked by incubated with 5% donkey serumfor 1 hour and washed with phosphate-buffered saline (PBS). Sections were thenincubated with primary antibodies for 1 hour at room temperature under humidified

conditions. Primary antibodies used were: mouse anti--smooth muscle actin (anti--SMA; 1:500 dilution, Sigma), rabbit anti-proliferating cell nuclear antigen (anti-PCNA; 1:500 dilution, Abcam, Cambridge, MA). After primary antibody incubation,sections were washed with PBS and incubated with appropriate fluorescent secondaryantibodies (Jackson ImmunoResearch, West Grove, PA) for 1 hour at roomtemperature. Sections were then washed with PBS, mounted with mounting mediumcontaining DAPI and photographed using a Zeiss fluorescence microscope andNorthern Eclipse software (Empix, Missassagua, ON). The tissue expression of -SMA was graded on a scale of 0–3 by three blinded observers; 0 signifies nostaining, 1 signifies very little staining, 2 signifies moderate staining, 3 signifiesextensive staining. For PCNA staining, the percentage of positive cell staining wascalculated per mm2 using image analysis software (Northern Eclipse, Empix). Toassess the effects of integrin 1 deletion on wound collagen synthesis, trichromecollagen stain was used. To assess the amount of granulation tissue, hematoxylin andeosin staining and Northern Eclipse software (Empix) was used.

Subcutaneous injection of TGF1 at wound sites of miceTo locally supply additional TGF1 in the wound vicinity, carrier-free recombinanthuman TGF1 (R&D Systems, Wiesbaden, Germany) was injected at four sitesaround the wound, allowing to infiltrate the wound margins, at a total dose of 0.40g as described (Peters et al., 2005). Mock injections were made using only thecarrier PBS. First injections were made on day 1 after wounding, followed by furtherinjections every second day until wounds were harvested for paraffin embedding at7 days after wounding.

Hydroxyproline assayHydroxyproline assay was performed essentially as described previously (Liu et al.,2008; Liu et al., 2009a; Reddy and Enwemeka, 1996). Wound tissues werehomogenized in saline, hydrolyzed with 2N NaOH for 30 minutes at 120°C, followedby the determination of hydroxyproline by modification of the Neumann and Logan’sreaction using Chloramine T and Ehrlich’s reagent using a hydroxyproline standardcurve and measuring at 550 nm. Values were expressed as g hydroxyproline permg protein.

Cell culture, immunofluorescence and western analysisPrimary dermal fibroblasts were isolated from explants (4- to 6-week-old animals)as described (Chen et al., 2005). Cells were subjected to indirect immunofluorescenceanalysis as described (Chen et al., 2005; Kennedy et al., 2007) using anti--SMA,Rhodamine-phalloidin (Sigma) and anti-vinculin (Sigma) antibodies, followed by anappropriate secondary antibody (Jackson ImmunoResearch). Cells were photographed(Zeiss Axiphot B-100, Empix). Alternatively, cells were lysed in 2% SDS, proteinsquantified (Pierce) and subjected to western blot analysis as described (Shi-wen etal., 2004; Chen et al., 2005; Kennedy et al., 2007). Anti-integrin-1 antibody wasfrom R&D Systems; anti-integrin-3, anti-TGFtype I receptor, anti-TGFtype IIreceptor and anti-GAPDH antibodies were from Santa Cruz Biotechnology.Fluorescence intensity of -SMA fibers was quantified by line scan measurementusing Northern Eclipse (Empix) software (Liu et al., 2007). MLEC-PAI/Luc, a minklung epithelial cell line stably transfected with an expression plasmid containing aTGF-responsive region (–799 to +71) of human plasminogen activator inhibitor-1(PAI1) gene fused to the firefly luciferase reporter gene. Cells were maintained inDMEM supplemented with 5% fetal calf serum.

Real-time polymerase chain reaction (RT-PCR)RT-PCR was performed essentially as described (Kennedy et al., 2007; Kennedy etal., 2008; Pala et al., 2008). A total of ten control and conditional-knockout micewere analyzed independently for each data point (mean ± s.d. shown from these tenindependent animals; Student’s paired t-test). Cells were cultured until 80%confluence, serum starved for 24 hours and total RNA was isolated (Qiagen).Integrity of the RNA was verified by gel electrophoresis. Total RNA (25 ng) wasreverse transcribed and amplified (TaqMan Assays on Demand; Applied Biosystems)as described (One-step Mastermix; Applied Biosystems) using the ABI Prism 7900HT sequence detector (Perkin-Elmer-Cetus, Vaudreuil, QC). Triplicates of eachsamples were run, and expression values were standardized to values obtained withcontrol 18S RNA primers using the Ct method.

Adhesion assayFibroblasts were isolated and cultured as described above. Adhesion assays wereperformed essentially as previously described (Chen et al., 2004; Chen et al., 2005).Wells of 96-well plates were incubated overnight at 4°C with 10 g/ml fibronectin(Sigma) in 0.5% bovine serum albumin (BSA), 1� PBS. Subsequently, cells wereblocked for 1 hour in 10% BSA in PBS, room temperature. Fibroblasts wereharvested with 2 mM EDTA in PBS (20 minutes, room temperature), washed twicewith DMEM serum-free medium containing 1% BSA (Sigma), resuspended in thesame medium at 2.5�105 cells/ml and 100 l suspension was incubated in each wellfor the times indicated. Non-adherent cells were removed by washing with PBS. Todetect cell adhesion, an acid phosphatase assay was used; adherent cells werequantified by incubation with 100 l substrate solution (0.1 M sodium acetate, pH5.5; 10 mM p-nitrophenylphosphate and 0.1% Triton X-100) for 2 hours at 37°C.The reaction was stopped by the addition of 15 l of 1 N NaOH per well and A450

Jour

nal o

f Cel

l Sci

ence


was measured. Comparison of adhesive abilities was performed by using Student’sunpaired t-test. P<0.05 was considered statistically significant.

Collagen gel contractionExperiments were performed essentially as described (Shi-wen et al., 2004). For afloating gel assay, 24-well tissue culture plates were pre-coated with BSA. Cellswere used at passage three. Trypsinized fibroblasts were suspended in MCDBmedium (Sigma) and mixed with collagen solution [one part 0.2 M HEPES, pH 8.0,four parts 3 mg/ml collagen (Nutragen, Inamed) and five parts of 2� MCDB] for afinal concentration of 80,000 cells per ml in 1.2 mg/ml collagen. Collagen cellsuspension (1 ml) was then added to each well to polymerize. Gels were thendetached from wells by adding 1 ml MCDB medium. Gel contraction was quantifiedby measuring changes in weight. When indicated, contraction assays were performedin the presence of DMSO and 100 M blebbistatin (myosin ATPase/contractioninhibitor, Calbiochem).

Fibroblast-populated collagen latticesMeasurement of contractile forces generated within a three-dimensional FPCL wasperformed as described (Shi-wen et al., 2004). Briefly, using 1�106 cells/ml ofcollagen gel (First Link, UK), we measured the force generated across a collagenlattice using a culture force monitor which measures forces exerted over 24 hours.A rectangular FPCL was cast, floated in medium in 2% FCS and attached to aground point at one end and a force transducer at the other. Cell-generated tensionalforces in the collagen gel are detected by the force transducer and logged into apersonal computer (t-CFM) (Denton et al., 2009). Graphical readings are producedevery 15 seconds providing a continuous output of force (Dynes: 1�10–5 N)generated. Experiments were run in parallel and three independent times, and arepresentative trace is shown.

Migration and proliferation assaysFor in vitro wounding (migration) experiments, fibroblasts obtained from integrin1 conditional (C/C) and knockout (K/K) mice were cultured in 12-well plates(2�105 cells/well). The next day, the cells were confluent. Medium was removed,and cells rinsed with serum-free medium with 0.1% BSA and cultured for anadditional 24 hours in serum-free medium plus 0.1% BSA. The monolayer wasartificially injured by scratching across the plate with a blue pipette tip (approximately1.3 mm width). Cells were washed twice to remove detached cells or cell debris, andcultured in serum-free medium in the presence of mitomycin C (10 g/ml, Sigma)to prevent cell proliferation. After 24 and 48 hours, images of the scratched areasunder each condition were photographed. Migration distance was measured (mean± s.d.) using Northern Eclipse software (Empix). For path trajectory studies, cellswere plated at low density (5000 cells/cm2) on type I collagen (30 g/ml in PBS,acid-extracted from newborn calf skin) or tissue culture plastic. Cells were observedfor 5 hours in an incubator chamber attached to an IX81 microscope (OlympusEuropa, Hamburg) at 37°C, 5% CO2 and 60% humidity (n69–123). Frames weretaken every 30 minutes using an OBS CCD FV2T camera (Olympus). To quantifycell movements, trajectories were measured using OBS Cell R software (Olympus).Persistence was calculated as the ratio of net length to total length, where net lengthis distance between start and end point, and total length is length of the entire path(Danen et al., 2005). For the cell proliferation assay, cells were seeded in 96-wellplates at 2000 cells/well. Cell number was determined after 24, 48 and 72 hours ofincubation using the MTT kit (Roche).

TGF bioassayFibroblasts (80,000 cells per ml collagen) co-cultured by embedding within a 3Dcollagen gel matrix with mink lung cells to produce which luciferase under controlof the PAI-1 promoter in response to TGF- (Abe et al., 1994; Wipff et al., 2007).Activation of TGF was assessed 24 hours later by lysing mink lung cells, andluciferase activity was assessed by light production from a luciferin substrate(Promega) using a luminometer (Wallac). Data are presented as mean ± s.d. of onerepresentative experiment performed in triplicate. Experiments were performed aminimum of three times. Co-cultures were performed in the absence and presenceof a TGF neutralizing antibody [1D11; R&D Systems (10 g/ml)], ALK-5 inhibitor[SB431542; Tocris (10 M) a specific inhibitor for TGF- type I receptor(TRI)/ALK-5] or blebbistatin (100 M; Calbiochem). As controls, cells wereincubated with IgG (Jackson ImmunoResearch) or DMSO.

This work was supported by grants from the Canadian Foundationfor Innovation, the Canadian Institutes of Health Research and theOntario Thoracic Society (to A.L.), the Arthritis Research Campaign(UK) and the Scleroderma Society (to D.J.A. and C.P.D.), and theDeutsche Forschungsgemeinschaft (SFB829 to B.E. and T.K.). A.L. isa New Investigator of the Arthritis Society (Scleroderma Society ofOntario), the recipient of an Early Researcher Award and a member ofthe Canadian Scleroderma Research Group New Emerging Team.

ReferencesAbe, M., Harpel, J. G., Metz, C. N., Nunes, I., Loskutoff, D. J. and Rifkin, D. B. (1994).

An assay for transforming growth factor-beta using cells transfected with a plasminogenactivator inhibitor-1 promoter-luciferase construct. Anal. Biochem. 216, 276-284.

Barrientos, S., Stojadinovic, O., Golinko, M. S., Brem, H. and Tomic-Canic, M.(2008). Growth factors and cytokines in wound healing. Wound Repair Regen. 16, 585-601.

Bauer, M., Brakebusch, C., Coisne, C., Sixt, M., Wekerle, H., Engelhardt, B. andFässler, R. (2009). Beta1 integrins differentially control extravasation of inflammatorycell subsets into the CNS during autoimmunity. Proc. Natl. Acad. Sci. USA 106, 1920-1925.

Brakebusch, C. and Fässler, R. (2005). 1 integrin function in vivo: adhesion, migrationand more. Cancer Metastasis Rev. 24, 403-411.

Brakebusch, C., Grose, R., Quondamatteo, F., Ramirez, A., Jorcano, J. L., Pirro, A.,Svensson, M., Herken, R., Sasaki, T., Timpl, R. et al. (2000). Skin and hair follicleintegrity is crucially dependent on beta 1 integrin expression on keratinocytes. EMBOJ. 19, 3990-4003.

Burridge, K. and Chrzanowska-Wodnicka, M. (1996). Focal adhesions, contractility,and signaling. Annu. Rev. Cell Dev. Biol. 12, 463-518.

Burridge, K. and Wennerberg, K. (2004). Rho and Rac take center stage. Cell 116, 167-179.

Chen, Y., Shiwen, X., van Beek, J., Kennedy, L., McLeod, M., Renzoni, E. A., Bou-Gharios, G., Wilcox-Adelman, S., Goetinck, P. F., Eastwood, M. et al. (2005).Matrix contraction by dermal fibroblasts requires TGFbeta/ALK5, heparan sulfatecontaining proteoglycans and MEK/ERK: Insights into pathological scarring in chronicfibrotic disease. Am. J. Pathol. 167, 1699-1711.

Chen, Y., Leask, A., Abraham, D. J., Pala, D., Shiwen, X., Khan, K., Liu, S., Carter,D. E., Wilcox-Adelman, S., Goetinck, P. et al. (2008). Heparan sulfate-dependentERK activation contributes to the overexpression of fibrotic proteins and enhancedcontraction by scleroderma fibroblasts. Arthritis Rheum. 58, 577-585.

Danen, E. H., van Rheenen, J., Franken, W., Huveneers, S., Sonneveld, P., Jalink, K.and Sonnenberg, A. (2005). Integrins control motile strategy through a Rho-cofilinpathway. J. Cell Biol. 169, 515-526.

Daniels, J. T., Schultz, G. S., Blalock, T. D., Garrett, Q., Grotendorst, G. R., Dean, N.M. and Khaw, P. T. (2003). Mediation of transforming growth factor-beta(1)-stimulatedmatrix contraction by fibroblasts: a role for connective tissue growth factor in contractilescarring. Am. J. Pathol. 163, 2043-2052.

Denton, C. P., Khan, K., Hoyles, R. K., Shiwen, X., Leoni, P., Chen, Y., Eastwood, M.and Abraham, D. J. (2009). Inducible lineage-specific deletion of TbetaRII in fibroblastsdefines a pivotal regulatory role during adult skin wound healing. J. Invest. Dermatol.129, 194-204.

Dugina, V., Fontao, L., Chaponnier, C., Vasiliev, J. and Gabbiani, G. (2001). Focaladhesion features during myofibroblastic differentiation are controlled by intracellularand extracellular factors. J. Cell Sci. 114, 3285-3296.

Eastwood, M., McGrouther, D. A. and Brown, R. A. (1998). Fibroblast responses tomechanical forces. Proc. Inst. Mech. Eng. H. 212, 85-92.

Eckes, B., Kessler, D., Aumailley, M. and Krieg, T. (1999). Interactions of fibroblastswith the extracellular matrix: implications for the understanding of fibrosis. SpringerSemin. Immunopathol. 21, 415-429.

Fässler, R. and Meyer, M. (1995). Consequences of lack of 1 integrin gene expressionin mice. Genes Dev. 9, 1896-1908.

Gabbiani, G. (2003). The myofibroblast in wound healing and fibrocontractive diseases.J. Pathol. 200, 500-503.

Geiger, B., Spatz, J. P. and Bershadsky, A. D. (2009). Environmental sensing throughfocal adhesions. Nat. Rev. Mol. Cell Biol. 10, 21-33.

Grose, R., Hutter, C., Bloch, W., Thorey, I., Watt, F. M., Fässler, R., Brakebusch, C.and Werner, S. (2002). A crucial role of beta 1 integrins for keratinocyte migration invitro and during cutaneous wound repair. Development 129, 2303-2315.

Gurtner, G. C., Werner, S., Barrandon, Y. and Longaker, M. T. (2008). Wound repairand regeneration. Nature 453, 314-321.

Hinz, B., Phan, S. H., Thannickal, V. J., Galli, A., Bochaton-Piallat, M. L. andGabbiani, G. (2007). The myofibroblast: one function, multiple origins. Am. J. Pathol.170, 1807-1816.

Hynes, R. O. (2002). Integrins: bidirectional, allosteric signaling machines. Cell 110, 673-687.

Ilic, D., Furuta, Y., Kanazawa, S., Takeda, N., Sobue, K., Nakatsuji, N., Nomura, S.,Fujimoto, J., Okada, M., Yamamoto, T. et al. (1995). Reduced cell motility andenhanced focal adhesion contact formation in cells from FAK-deficient mice. Nature377, 539-544.

Ishibe, S., Joly, D., Liu, Z. X. and Cantley, L. G. (2004). Paxillin serves as an ERK-regulated scaffold for coordinating FAK and Rac activation in epithelial morphogenesis.Mol. Cell 6, 257-267.

Kennedy, L., Liu, S., Shi-wen, X., Carter, D., Lyons, K., Black, C. M., Abraham, D.J. and Leask, A. (2007). CCN2 is essential for fibroblast function. Exp. Cell Res. 313,952-964.

Kennedy, L., Shi-wen, X., Carter de Abraham, D. J. and Leask, A. (2008). Adhesionof cells to fibronectin induces a tissue repair gene expression program. Matrix Biol. 27,274-281.

Kuang, S., Kuroda, K., Le Grand, F. and Rudnicki, M. A. (2007). Asymmetric self-renewal and commitment of satellite stem cells in muscle. Cell 129, 999-1010.

Lafrenie, R. M. and Yamada, K. M. (1996). Integrin-dependent signal transduction. J.Cell Biochem. 61, 543-553.

Leask, A. (2006). Scar wars: Is TGF the phantom menace in scleroderma? Arthritis Res.Ther. 8, 213.

Jour

nal o

f Cel

l Sci

ence


Lechler, T. and Fuchs, E. (2005). Asymmetric cell divisions promote stratification anddifferentiation of mammalian skin. Nature 437, 275-280.

Liu, S., Shi-wen, X., Kennedy, L., Pala, D., Carter de Black, C. M., Abraham, D. J.and Leask, A. (2007). FAK is required for TGF-induced JNK phosphorylation infibroblasts: implications for acquisition of a matrix remodeling phenotype. Mol. Biol.Cell 18, 2169-2178.

Liu, S., Kapoor, M., Shi-wen, X., Kennedy, L., Denton, C. P., Glogauer, M., Abraham,D. J. and Leask A. (2008). Investigation of the role of Rac1 in a bleomycin-inducedscleroderma model using fibroblast-specific Rac1 knockout mice. Arthritis Rheum. 58,2189-2195.

Liu, S., Kapoor, M. and Leask, A. (2009a). Rac1 expression by fibroblasts is requiredfor tissue repair in vivo. Am J. Pathol. 174, 1847-1856.

Liu, S., Kapoor, M., Denton, C. P., Abraham, D. J. and Leask, A. (2009b). Loss of 1integrin in mouse fibroblasts results in resistance to a mouse model of skin scleroderma.Arthritis Rheum. 60, 2871-2821.

Martin, P. (1997). Wound healing-aiming for perfect skin regeneration. Science 276, 75-81.

Mirastschijski, U., Haaksma, C. J., Tomasek, J. J. and Ågren, M. S. (2004). Matrixmetalloproteinase inhibitor GM 6001 attenuates keratinocyte migration, contraction andmyofibroblast formation in skin wounds. Exp. Cell Res. 299, 465-475.

Pala, D., Kapoor, M., Woods, A., Kennedy, L., Liu, S., Chen, S., Bursell, L., Lyons,K., Carter, D. and Beier, F. (2008). FAK/src suppresses early chondrogenesis: centralrole of CCN2. J. Biol. Chem. 283, 9239-9247.

Peters, T., Sindrilaru, A., Hinz, B., Hinrichs, R., Menke, A., Al-Azzeh, E. A.,Holzwarth, K., Oreshkova, T., Wang, H. and Kess, D. (2005). Wound-healing defectof CD18(–/–) mice due to a decrease in TGF-beta1 and myofibroblast differentiation.EMBO J. 24, 3400-3410.

Piwko-Czuchra, A., Koegel, H., Meyer, H., Bauer, M., Werner, S., Brakebusch, C. andFässler, R. (2009). Beta1 integrin-mediated adhesion signalling is essential for epidermalprogenitor cell expansion. PLoS ONE 4, e5488.

Raghavan, S., Bauer, C., Mundschau, G., Li, Q. and Fuchs, E. (2000). Conditionalablation of beta1 integrin in skin. Severe defects in epidermal proliferation, basementmembrane formation, and hair follicle invagination. J. Cell Biol. 150, 1149-1160.

Reddy, G. K. and Enwemeka, C. S. (1996). A simplified method for the analysis ofhydroxyproline in biological tissues. Clin. Biochem. 29, 225-229.

Reynolds, L. E., Conti, F. J., Lucas, M., Grose, R., Robinson, S., Stone, M., Saunders,G., Dickson, C., Hynes, R. O., Lacy-Hulbert, A. et al. (2005). Accelerated re-

epithelialization in beta3-integrin-deficient- mice is associated with enhanced TGF-beta1 signaling. Nat. Med. 11, 167-174.

Schultz, G. S. and Wysocki, A. (2009). Interactions between extracellular matrix andgrowth factors in wound healing. Wound Repair. Regen. 17, 153-162.

Shi-wen, X., Chen, Y., Denton, C. P., Eastwood, M., Renzoni, E. A., Bou-Gharios, G.,Pearson, J. D., Dashwood, M., du Bois, R. M., Black, C. M. et al. (2004). Endothelin-1 promotes mechanoregulation and myofibroblast formation in fibroblasts through theETA receptor via Akt/PI3 kinase: implications for lung fibrosis. Mol. Biol. Cell 15,2707-2719.

Shi-wen, X., Rodrigues-Pascual, F., Lamas, S., Holmes, A., Howat, S., Pearson, J. D.,Dashwood, M. R., du Bois, R. M., Denton, C. P., Black, C. M. et al. (2006a).Constitutive ALK5-indepenent JNK activation contributes to endothelin-1 over-expression in pulmonary fibrosis. Mol. Cell. Biol. 26, 5518-5527.

Shi-wen, X., Stanton, L., Kennedy, L., Pala, D., Chen, Y., Howat, S. L., Renzoni, E.A., Carter, D. E., Bou-Gharios, G., Stratton, R. J. et al. (2006b). CCN2 is necessaryfor adhesive responses to TGFß1 in embryonic fibroblasts. J. Biol. Chem. 281, 10715-10726.

Shi-wen, X., Renzoni, E. A., Kennedy, L., Howat, S., Chen, Y., Pearson, J. D., Bou-Gharios, G., Dashwood, M. R., du Bois, R. M., Black, C. M. et al. (2007). Endogenousendothelin-1 signaling contributes to type I collagen and CCN2 overexpression infibrotic fibroblasts. Matrix Biol. 26, 625-632.

Stephens, L. E., Sutherland, A. E., Klimanskaya, I. V., Andrieux, A., Meneses, J.,Pedersen, R. A. and Damsky, C. H. (1995). Deletion of beta 1 integrins in mice resultsin inner cell mass failure and peri-implantation lethality. Genes Dev. 9, 1883-1895.

Varga, J. and Abraham, D. (2007). Systemic sclerosis: a prototypic multisystem fibroticdisorder. J. Clin. Invest. 117, 557-567.

Werner, S. and Grose, R. (2003). Regulation of wound healing by growth factors andcytokines. Physiol. Rev. 83, 835-870.

Wipff, P. J., Rifkin, D. B., Meister, J. J. and Hinz, B. (2007). Myofibroblast contractionactivates latent TGF-beta1 from the extracellular matrix. J. Cell Biol. 179, 1311-1323.

Zaidel-Bar, R., Itzkovitz, S., Ma’ayan, A., Iyengar, R. and Geiger, B. (2007). Functionalatlas of the integrin adhesome. Nat. Cell Biol. 9, 858-867.

Zamir, E. and Geiger, B. (2001). Molecular complexity and dynamics of cell-matrixadhesions. J. Cell Sci. 114, 3583-3590.

Zheng, B., Zhang, Z., Black, C. M., de Crombrugghe, B. and Denton, C. P. (2002).Ligand-dependent genetic recombination in fibroblasts: a potentially powerful techniquefor investigating gene function in fibrosis. Am. J. Pathol. 160, 1609-1617.

Jour

nal o

f Cel

l Sci

ence

expression of integrin 1 by fibroblasts is required for ... · in tissue repair and fibrogenic...

Documents