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First published online 30 September 2008
doi: 10.1242/jcs.029215

Journal of Cell Science 121, 3459-3467 (2008)
Published by The Company of Biologists 2008
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Research Article

Loss of protein kinase C{epsilon} results in impaired cutaneous wound closure and myofibroblast function

Andrew Leask1,*,{ddagger}, Xu Shi-wen2,*, Korsa Khan2, Yunliang Chen3, Alan Holmes2, Mark Eastwood3, Christopher P. Denton2, Carol M. Black2 and David J. Abraham2

1 CIHR Group in Skeletal Development and Remodeling, Division of Oral Biology and Department of Physiology and Pharmacology, Schulich School of Medicine and Dentistry, University of Western Ontario, London ON, Canada N6A 5C1
2 Centre for Rheumatology, Department of Medicine, Royal Free and University College Medical School, University College London (Royal Free Campus), Rowland Hill Street, London NW3 2PF, UK
3 Centre for Tissue Engineering Research, Department of Biomedical Sciences, University of Westminster, London W1W 6UW, UK

{ddagger} Author for correspondence (e-mail: andrew.leask{at}schulich.uwo.ca)

Accepted 20 July 2008


   Summary
Top
 Summary
Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Cutaneous wound repair requires the de novo induction of a specialized form of fibroblast, the {alpha}-smooth muscle actin ({alpha}-SMA)-expressing myofibroblast, which migrates into the wound where it adheres to and contracts extracellular matrix (ECM), resulting in wound closure. Persistence of the myofibroblast results in scarring and fibrotic disease. In this report, we show that, compared with wild-type littermates, PKC{epsilon}-/- mice display delayed impaired cutaneous wound closure and a reduction in myofibroblasts. Moreover, both in the presence and absence of TGFβ, dermal fibroblasts from PKC{epsilon}-/- mice cultured on fibronectin show impaired abilities to form `supermature' focal adhesions and {alpha}-SMA stress fibers, and reduced pro-fibrotic gene expression. Smad3 phosphorylation in response to TGFβ1 was impaired in PKC{epsilon}-/- fibroblasts. PKC{epsilon}-/- fibroblasts show reduced FAK and Rac activation, and adhesive, contractile and migratory abilities. Overexpressing constitutively active Rac1 rescues the defective FAK phosphorylation, cell migration, adhesion and stress fiber formation of these PKC{epsilon}-/- fibroblasts, indicating that Rac1 operates downstream of PKC{epsilon}, yet upstream of FAK. These results suggest that loss of PKC{epsilon} severely impairs myofibroblast formation and function, and that targeting PKC{epsilon} may be beneficial in selectively modulating wound healing and fibrotic responses in vivo.

Key words: Myofibroblasts, TGFβ, Wound healing


   Introduction
Top
 Summary
 Introduction
Results
 Discussion
 Materials and Methods
 References
 
Tissue repair requires a well-defined series of events, starting with the plugging of the wound by a fibrin clot, and ending with the reconstitution of the epithelial barrier and the underlying connective tissue (Martin, 1997Go). During normal wound healing in adult animals, fibroblasts initially migrate into the wound area and synthesize a collagen- and cellular fibronectin-rich ECM (Eckes et al., 1999Go). This migration involves the extension of lamellopodia and filopodia, accompanied by the assembly of focal adhesions at the leading edge of the cell, and disassembly of the focal adhesions at the base of the protrusion. Regulation of focal adhesion formation and turnover is intimately related to the ability of the focal adhesions, through transmembrane integrins, to mediate contact with the extracellular matrix and the actin cytoskeleton (Burridge and Chrzanowska-Wodnicka, 1996Go). These focal adhesions typically contain β- and {gamma}-cytoplasmic actins, {alpha}-SMA, {alpha}v integrin, vinculin, paxillin, {alpha}-actinin, talin, focal adhesion kinase (FAK) and tyrosine-phosphorylated proteins (Dugina et al., 2001Go; Zamir and Geiger, 2001Go). These specialized fibroblasts are termed myofibroblasts, and generate the tensile forces required for wound closure. Abnormal persistence of the myofibroblast results in scarring and fibrotic disease (Gabbiani, 2003Go; Chen et al., 2005Go). Thus, selective modulation of myofibroblast action is expected to have profound impact on controlling tissue repair and fibrosis.

Focal adhesion proteins serve as a point of convergence for signals that result from stimulation of growth factor receptors (Turner, 2000Go). For example, paxillin provides a platform for protein tyrosine kinases such as FAK and SRC, which are activated as a result of adhesion or growth factor stimulation (Hagel et al., 2002Go). Fibroblasts lacking FAK or paxillin spread poorly and cannot migrate (Ilic et al., 1995Go; Webb et al., 2004Go). FAK-deficient fibroblasts actually possess more focal adhesions than do control cells, suggesting that FAK recruitment may be involved with the turnover of existing focal adhesions (Ilic et al., 1995Go). Indeed, FAK, paxillin, Src and ERK are required for focal adhesion disassembly at the base of extruding lamellopodia (Webb et al., 2004Go). FAK recruitment to paxillin is also required for Rac activation (Ishibe et al., 2004Go), which is essential for cell migration (Klemke et al., 1998Go). FAK activation is also required for matrix contraction (Midwood and Schwarzbauer, 2002Go).

Many details still remain to be discovered about the regulation and spatiotemporal relationship of the activation of focal adhesion components. For example, formation of focal adhesions is associated with the stimulation of protein kinase C (PKC) (Woods and Couchman, 1992Go), yet the contribution of PKC to focal adhesion formation is not yet understood.

PKC is a family of serine/threonine kinases that regulate a variety of cell functions including proliferation, gene expression, cell cycle, differentiation, cytoskeletal organization cell migration and apoptosis (Carter and Kane, 2004Go). The existence of multiple isozymes of PKC has raised the issue of whether each PKC isozyme has a specific function. Indeed, differences in PKC isozyme protein structure and substrate preferences have allowed the family to be divided into three groups (Way et al., 2000Go). The conventional PKC isozymes ({alpha}, βI, βII, and {gamma}) are Ca2+-dependent and are activated by phospholipids and diacylglycerol. The novel isozymes ({delta}, {epsilon}, µ, and {theta}) are calcium insensitive, but phospholipid and diacylglycerol dependent. Finally, the atypical PKC isozymes ({eta} and {lambda}/{zeta}) are insensitive to both Ca2+ and diacylglycerol. Although the activation mechanism of the PKC isozyme family is clearly different among the three subgroups: conventional, novel and atypical PKC, whether or not each isozyme in a subgroup has a specific function or activation mechanism has not been clarified. Indeed, many PKCs display overlapping substrate specificities in vitro (Teicher, 2006Go). Overall, studies in which active or dominant-negative PKC isoforms are overexpressed in vitro have been imperfect in predicting the physiological consequences of loss of individual PKC isoforms in vivo (Teicher, 2006Go). There may be functional compensation by other PKC isoforms in vivo (Teicher, 2006Go). Moreover, data from dominant-negative or constitutively active studies can be misleading, as it is not known whether the overexpressed protein only impacts the isoform of interest or also affects other closely related PKC isoforms (Moscat et al., 2006Go). Furthermore, as PKC isozymes do not function in isolation but exist in complexes with other signaling molecules, overexpressed proteins may also impact the function of these additional molecules and hence indirectly affect cell function (Teicher, 2006Go). Thus, to identify the in vivo functions of individual PKC isoforms, the use of genetic models, such as knockout mice, is essential.


Figure 1
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  		Fig. 1. PKC{epsilon}-/- mice show significantly reduced wound closure and presence of {alpha}-SMA-expressing myofibroblasts. (A) A 4 mm dermal punch was placed in the back of PKC{epsilon}+/+ and PKC{epsilon}-/-, and wound closure was measured. A total of six animals were analyzed (four wounds/animal). Eight-week-old females were assessed (*P<0.05, closure in PKC{epsilon}-/- significantly different from PKC{epsilon}+/+ mice). (B) Anti-{alpha}-SMA ({alpha}-SMA) staining of 14 day wounds. Sectioning and staining revealed the reduced presence of {alpha}-SMA-positive myofibroblasts (arrow). Sections probed with the {alpha}-SMA antibody were counterstained with Hematoxylin. Scale bar: 100 µm. (C) Western blot analysis of lysates prepared from of PKC{epsilon}+/+ and PKC{epsilon}+/+ wounds (25 µg/lane). Densitometry represents mean±s.d. of eight independent experiments (i.e. extracts from eight separate wounds; *P<0.05).

 
Protein kinase C{epsilon} (PKC{epsilon}) contains an actin-binding motif that is unique to this individual member of the PKC gene family (Prekeris et al., 1998Go). In addition, thrombin-induced matrix contraction by fibroblasts is associated with the interaction of PKC{epsilon} with {alpha}-SMA (Bogatkevich et al., 2003Go). Moreover, PKC{epsilon} is required for the recycling of integrin β1, to which PKC{epsilon} is bound via RACK (Besson et al., 2002Go), from inside the cell to the cell surface (Ivaska et al., 2002Go). Finally, PKC{epsilon} is required for the ability of TNF{alpha} to cause phosphorylation of STAT1, a process mediated by integrins (Ivaska et al., 2003Go). These results suggest that PKC{epsilon} may play a crucial role in stress fiber formation at focal adhesions. However, this hypothesis has yet to be tested. Indeed, the precise contribution of PKC{epsilon} to focal adhesion formation in fibroblasts and in wound closure is not known. PKC{epsilon} knockout mice are viable and appear essentially normal, but show elevated susceptibility to bacterial infection (Castrillo et al., 2001Go) and subtle cardiovascular defects in response to hypoxia (Saurin et al., 2001; Littler et al., 2003Go). In this report, we have used PKC{epsilon} knockout mice to assess the contribution of protein kinase C{epsilon} to focal adhesion formation, adhesion, migration and ECM contraction and wound closure.


   Results
Top
 Summary
 Introduction
 Results
Discussion
 Materials and Methods
 References
 
Protein kinase C{epsilon} knockout mice show reduced wound closure and presence of {alpha}-SMA expressing myofibroblasts
To investigate whether PKC{epsilon}-/- mice displayed a delay in cutaneous wound closure, we subjected 8-week-old mice homozygous for deletions in the PKC{epsilon} gene, or their wild-type littermates, to the dermal punch wound model of wound healing. We monitored wound closure over a 14-day period, and found that PKC{epsilon}-/- animals displayed a marked reduction in the rate of wound closure (Fig. 1A). We examined sections taken from isolated tissue from animals 14 days-post wounding and found that PKC{epsilon}-/- animals showed reduced presence of {alpha}-SMA-expressing myofibroblasts therein (Fig. 1B). Decreased {alpha}-SMA expression was verified using Western blot analysis if tissue extracts of wounds from PKC{epsilon}+/+ and PKC{epsilon}-/- animals (Fig. 1C). These results indicate that the wound healing phenotype of PKC{epsilon}-/- animals may arise due to impaired myofibroblast activity and function.

PKC{epsilon} is required for `supermature' focal adhesion formation
To begin to investigate the molecular defects underlying the inability of PKC{epsilon}-/- to rapidly close dermal wounds, fibroblasts were cultured from the dermis of mice homozygous for deletions in the PKC{epsilon} gene and wild-type littermates. Genotypes of the fibroblasts were confirmed by western blot analysis and RT-PCR (not shown). Our in vivo data indicated that loss of PKC{epsilon} led to reduced tissue remodeling, wound closure and myofibroblast activity. To investigate this issue, we investigated whether loss of PKC{epsilon} affected the ability of fibronectin to induce the appearance of {alpha}-SMA stress fibers and intensely vinculin-positive `supermature' focal adhesions, key features of myofibroblasts (Hinz and Gabbiani, 2003Go). Fibronectin was used in our assays as this protein is a key component of the provisional matrix laid down in the initial phases of wound healing (Grinnell, 1984Go) and therefore provided an appropriate parallel approach to our in vivo wound healing studies. Intriguingly, we found that PKC{epsilon}-/- fibroblasts cultured on fibronectin were able to recruit actin to form stress, as visualized by staining cells with phalloidin (Fig. 2). However, we found that {alpha}-SMA stress fiber networks, as visualized by staining of cells with an anti-{alpha}-SMA antibody, did not form in PKC{epsilon} -/- fibroblasts (Fig. 2). To investigate whether PKC{epsilon} was required for optimal focal adhesion formation by fibroblasts, we subjected PKC{epsilon}+/+ and PKC{epsilon}-/- fibroblasts growing on fibronectin to indirect immunofluorescence with anti-focal adhesion kinase (FAK) and anti-vinculin antibodies. We found that vinculin and phospho-FAK staining were markedly reduced in intensity and number in PKC{epsilon}-/- fibroblasts (Fig. 2). That intense vinculin staining did not appear in PKC{epsilon} -/- fibroblasts is consistent with a failure of PKC{epsilon}-/- cells to support formation of {alpha}-SMA stress fibers, as highly vinculin-positive focal adhesions are correlated with the larger, so-called `supermature' {alpha}-SMA stress fiber-nucleating FAs (Dugina et al., 2001Go).


Figure 2
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  		Fig. 2. PKC{epsilon}-/- fibroblasts show reduced appearance of {alpha}-SMA stress fibers and `supermature' focal adhesions on fibronectin. PKC{epsilon}+/+ (WT) and PKC{epsilon}-/- fibroblasts cultured on fibronectin were fixed with paraformaldeyde and stained to detect phosphorylated FAK (pFAK), FAK, {alpha}-SMA, total actin, and vinculin. Cells were counterstained with DAPI to detect nuclei (blue stain). PKC{epsilon}+/+, but not PKC{epsilon}-/-, cells show foci of phospho-FAK staining (pFAK) or intense vinculin staining, which are characteristic of `supermature' focal adhesions.

 

To confirm the notion that PKC{epsilon} was required for focal adhesion and {alpha}-SMA stress fiber formation, cytoskeletal protein preparations were prepared from wild-type and PKC{epsilon} knockout fibroblasts grown on fibronectin. The resultant protein preparations were subjected to western blot analysis with anti-{alpha}-SMA, anti-actin, anti-paxillin, anti-FAK, anti-Rac and anti-vinculin antibodies. Although these components of stress fiber and focal adhesion complexes were readily detected in the insoluble fraction of normal fibroblasts, suggesting that {alpha}-SMA stress fibers and focal adhesions were able to be formed in wild-type fibroblasts, {alpha}-SMA, paxillin, moesin and vinculin were found at reduced levels the insoluble fraction of PKC{epsilon} knockout fibroblasts, giving further support to the notion that loss of PKC{epsilon} impaired the formation of {alpha}-SMA stress fibers and focal adhesions (Fig. 3A). Consistent with the notion that an actin network formed in PKC{epsilon}-/- fibroblasts, actin was recruited to the insoluble fraction of both PKC{epsilon}+/+ and PKC{epsilon}-/- fibroblasts (Fig. 3A). Interestingly, however, FAK was found in the insoluble protein fraction, although it displayed reduced phosphorylation (Fig. 3A). These results suggest that the defect in FAK activation lies not at the level of FAK recruitment, but rather at the level of FAK activation. Consistent with this notion, immunoprecipitation experiments with an anti-paxillin antibody, which binds activated FAK and is required for focal adhesion assembly (Turner, 2000Go), showed that PKC{epsilon}-/- fibroblasts showed markedly reduced binding between paxillin and FAK (Fig. 3B). Intriguingly, Rac, a protein activated and localized to focal adhesions upon adhesion and necessary for migration (Ishibe et al., 2004Go; Klemke et al., 1998Go), showed markedly reduced recruitment to the insoluble protein fraction (Fig. 3A).


Figure 3
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  		Fig. 3. PKC{epsilon}-/- fibroblasts show reduced recruitment of focal adhesion components on fibronectin. (A) Compartmentalization of focal adhesion components in PKC{epsilon}+/+ (WT) and PKC{epsilon}-/- fibroblasts. Cells were cultured until 80% confluence on fibronectin. Fibronectin is a major component of the provisional extracellular matrix deposited during wound healing (Grinnell, 1984Go). Triton-soluble and -insoluble protein extracts were prepared and subjected to SDS/PAGE as described in the Materials and Methods. Recruitment of FAK and β-actin is not affected. Western blot analyses (25 µg/lane) were then performed with anti-{alpha}-SMA, anti-β-actin, anti phospho-FAK, anti-FAK, anti-Rac1, anti-paxillin and anti-vinculin antibodies. There was reduced recruitment of Rac, paxillin, vinculin, phospho-FAK and {alpha}-SMA to the insoluble (cytoskeleton) fraction of the cell. Recruitment of FAK and β-actin was not affected. FAK phosphorylation was not completely impaired in the absence of PKC{epsilon}. Densitometry was performed (n=3, average±s.d. is shown, *P<0.05 relative to wild-type control). (B) Interaction between paxillin and FAK in PKC{epsilon}+/+ and PKC{epsilon}-/- fibroblasts. Cell extracts (25 µg) from cells cultured in an analogous fashion to those in A were immunoprecipitated with {alpha}-FAK or {alpha}-paxillin ({alpha}-PAX) or normal goat serum control (C), as indicated. The resultant protein extracts were subjected to SDS/PAGE, and immunoprecipitated FAK was detected with a {alpha}-FAK antibody by western analysis. FAK shows reduced interaction with paxillin in the absence of PKC{epsilon}.

 
To extend our analyses into the contribution of PKC{epsilon} to fibroblast biology, we assessed whether PKC{epsilon}-deficient fibroblasts showed an impaired response to the potent pro-fibrotic, myofibroblast-inducing cytokine TGFβ (Leask and Abraham, 2004Go). First, we used immunofluorescence and western blot analyses to show that the ability of TGFβ to induce {alpha}-SMA stress fiber formation was impaired in the absence of PKC{epsilon} (Fig. 4A). In addition, we used western blot analysis to show that the ability of TGFβ to induce phosphorylation of Smad3 was impaired in the absence of PKC{epsilon} (Fig. 4B). As these data suggested TGFβ-induced gene expression responses might be impaired in the absence of PKC{epsilon} we investigated whether loss of PKC{epsilon} affected the ability of fibroblasts to induce the pro-fibrotic proteins CCN2, type I collagen, β1 integrin, {alpha}4 integrin and paxillin. We found that induction in response to TGFβ of all of these were impaired in PKC{epsilon}-deficient fibroblasts (Fig. 4C). Finally, basal integrin, {alpha}-SMA and CCN2 expression were impaired (Fig. 4C). These results collectively imply that loss of PKC{epsilon} significantly affects TGFβ signaling and myofibroblast induction in response to this growth factor.


Figure 4
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  		Fig. 4. Loss of PKC{epsilon} reduces the ability of TGFβ to induce {alpha}-SMA stress fibers, Smad3 phosphorylation, pro-fibrotic protein expression and matrix contraction in fibroblasts. (A) Immunofluorescence analysis. PKC{epsilon}+/+ and PKC{epsilon}-/- fibroblasts were cultured on fibronectin, serum starved for 18 hours and treated for an additional 24 hours with or without added TGFβ1 (4 ng/ml). Cells were fixed with paraformaldeyde and stained to detect {alpha}-SMA. Cells were counterstained with DAPI to detect nuclei. (B) Western blot analysis and Smad phosphorylation. PKC{epsilon}+/+ and PKC{epsilon}-/- fibroblasts were cultured on fibronectin, serum starved for 18 hours and treated for an additional 0.5 hours with or without added TGFβ1 (4 ng/ml). Proteins were extracted, using 2% SDS, from cells. Equal amounts of protein (20 µg) were subjected to SDS/PAGE and western blot analyses with the antibodies indicated. (C) Western blot analysis and TGFβ-induced gene expression. PKC{epsilon}+/+ and PKC{epsilon}-/- fibroblasts were cultured on fibronectin, serum starved for 18 hours and treated for an additional 24 hours with or without added TGFβ1 (4 ng/ml). Proteins were extracted, using 2% SDS, from cells. Equal amounts of protein (20 µg) were subjected to SDS/PAGE and western blot analyses with the antibodies indicated. Note that basal protein expression is reduced in the absence of PKC{epsilon}. Moreover, expression of proteins in response to TGFβ is also impaired. (D) Floating collagen gel contraction assay. PKC{epsilon}+/+ and PKC{epsilon}-/- fibroblasts were seeded into a floating collagen gel matrix, and incubated for 24 hours in the presence and absence of added TGFβ1 as described in the Materials and Methods. The weight of the contracted collagen gel was then measured. Cells were assayed in triplicate; experiments were performed thrice. Values shown are average±s.d. *significantly different from control (P<0.05). (E) PKC{epsilon}+/+ and PKC{epsilon}-/- fibroblasts were seeded into a floating collagen gel matrix, in the presence or absence of the protein kinase C inhibitor calphostin C and analyzed as in C.

 
Protein kinase C{epsilon} knockout fibroblasts show reduced contraction and adhesion of ECM
To provide a functional context for our observations, we compared the abilities of PKC{epsilon}+/+ and PKC{epsilon} -/- fibroblasts to contract a collagen gel matrix, suspended in medium, over a 24 hour period (Grinnell, 2003Go; Shi-wen et al., 2004Go). We found that PKC{epsilon}-/- fibroblasts exhibited reduced ability to contract a floating collagen gel matrix both in the presence and absence of added TGFβ1 (Fig. 4D). Consistent with this observation, the ability of normal fibroblasts to contract a collagen gel was reduced by a general protein kinase C inhibitor, calphostin (Fig. 4E). To extend these data, we conducted a contraction assay using a fibroblast-embedded collagen gel matrix, which is fixed at one end and attached to a force monitor (Eastwood et al., 1994Go). Loss of PKC{epsilon} resulted in an impaired ability of fibroblasts to generate contractile force across a fixed collagen gel lattice containing fibroblasts (Fig. 5A). We then compared the abilities of PKC{epsilon}+/+ and PKC{epsilon} -/- fibroblasts to adhere to fibronectin, a key component of the provisional matrix deposited in the initial stages of wounding (Grinnell, 1984Go), and type I collagen. We found that PKC{epsilon}-/- fibroblasts, relative to PKC{epsilon}+/+ fibroblasts, displayed markedly reduced abilities to adhere to fibronectin and type I collagen (Fig. 5B). Collectively, these results are consistent with the wound healing phenotype of the PKC{epsilon}-/- mice, and suggest PKC{epsilon} is required for crucial features of wound closure, namely the ability of fibroblasts to adhere to and contract ECM.


Figure 5
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  		Fig. 5. Loss of PKC{epsilon} results in a reduced ability of fibroblasts to contract and adhere to matrix. (A) Contraction of a collagen gel matrix: FPCL analysis. The effect of loss of PKC{epsilon} expression on contractile force generated by fibroblasts in a fixed, tethered floating collagen gel lattice was investigated using a Culture Force Monitor (Eastwood et al., 1994Go). Force generated by PKC{epsilon}+/+ and PKC{epsilon}-/- fibroblasts was assessed over 24 hours. Traces are the result of using 5 ml of collagen gel at a density of 1 million cells per ml of collagen gel in 2% serum. A representative trace is shown (n=3). The higher contractile forces generated by cells derived from PKC{epsilon}+/+ in comparison with PKC{epsilon}-/- fibroblasts. (B) Loss of PKC{epsilon} results in a reduced ability of fibroblasts to adhere to extracellular matrix. Adhesion of PKC{epsilon}+/+ and PKC{epsilon}-/- fibroblasts on fibronectin (FN) and type I collagen (col) was assessed as described in the Materials and Methods. Cells were detached from plates with EDTA, and equal numbers of cells were placed into individual wells of 96-well plates. Adherent cells were detected (Absorbance 450) 45 minutes post-adhesion (average±s.d., n=3). *P<0.05 relative to wild-type control.

 


Figure 6
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  		Fig. 6. Loss of PKC{epsilon} results in a reduced ability of fibroblasts to migrate on extracellular matrix. (A) As described in the Materials and Methods, migration was measured using a scratch wound assay. PKC{epsilon}+/+ and PKC{epsilon}-/- fibroblasts were cultured on fibronectin until confluence, and a linear scrape was made across the cell layer. Migration was monitored for the times indicated. Three independent experiments were performed Gap size expressed as a percentage of the original wound is shown (average±standard deviation) (*P<0.05 relative to wild-type control). (B) Migration was monitored as in A, in the presence and absence of added TGFβ1 (4 ng/ml). Quantitiative densitometry data are indicated on the right. Gap size expressed as a percentage of the original wound is shown (average±s.d.), *P<0.05 relative to wild-type control. Three independent experiments were performed.

 
Protein kinase C{epsilon} knockout fibroblasts show reduced migratory ability that depends on Rac activation
To extend these studies, we compared the abilities of PKC{epsilon}+/+ and PKC{epsilon}-/- fibroblasts to migrate on fibronectin by employing a so-called scratch wound assay in which a `wound' is introduced, by a pipette, in a confluent cell monolayer (Kinsella and Wight, 1986Go). Both in the presence and absence of added TGFβ, we found that PKC{epsilon}-/- fibroblasts showed greatly reduced migratory ability compared with PKC{epsilon}+/+ fibroblasts (Fig. 6). Collectively, these results are consistent with the notion that loss of PKC{epsilon} results in impairment of the ability of fibroblasts to participate in wound healing responses. Our previous observation that FAK, but not Rac, was appropriately recruited to the cytoskeleton in PKC{epsilon}-/- fibroblasts (Fig. 3) led us to investigate further the role of Rac in the phenotype of PKC{epsilon}-/- fibroblasts. We then used a standard Rac activity assay to show that PKC{epsilon}-/- fibroblasts, while displaying equal levels of Rac expression to wild-type cells with and without a 1-hour treatment with TGFβ, displayed markedly reduced Rac activity both in the presence and absence of added TGFβ (Fig. 7A,B).


Figure 7
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  		Fig. 7. Loss of PKC{epsilon} results in impaired Rac signaling in fibroblasts. (A) Rac is expressed equally in PKC{epsilon}+/+ (WT) and PKC{epsilon}-/- fibroblasts. Cells were loaded with or without GTP{gamma}S (positive control, to assess maximal Rac activation) and in the presence or absence of TGFβ (4 ng/ml, 1 hour). Cell lysates were subjected to SDS/PAGE and western blot analysis with an anti-Rac antibody. Experiments were performed six independent times. Quantitiative densitometry data are indicated on the right. (B) Rac activity assay. Input protein assessed in parallel with A was used in the standard Rac activity assay, as described in the Materials and Methods. Rac-GTP was immunoprecipitated from cell lysates. The precipitated Rac-GTP was detected by immunoblot analysis using anti-Rac. GTP{gamma}S (lane 1), GDP (lane 2), PKC{epsilon}+/+ (WT, lane 3), PKC{epsilon}+/+ (WT)+ TGFβ (lane 4); PKC{epsilon}-/- (lane 5); PKC{epsilon}-/- + TGFβ (lane 6). Experiments were performed three independent times. Quantitative densitometry data are indicated on the right (*P<0.05 relative to wild-type control).

 

Based on these results, we reasoned that Rac might not be effectively activated in PKC{epsilon}-/- fibroblasts, and that the inability of FAK to be phosphorylated may be due to a markedly reduced level of Rac activity in PKC{epsilon} -/- fibroblasts. We used western blot analysis to show that PKC{epsilon}-/- fibroblasts cultured on fibronectin showed markedly endogenous reduced FAK phosphorylation (Fig. 8A). To assess whether the lower level of Rac contributed to the reduced FAK phosphorylation and to the reduced migration observed in PKC{epsilon}-/- fibroblasts, we transfected an expression vector encoding constitutively activated Rac into PKC{epsilon}-/- fibroblasts. Overexpression of constitutively activated Rac1 enhanced FAK phosphorylation and the reduced integrin β1 expression in PKC{epsilon}-/- fibroblasts (Fig. 8A). Moreover, constitutively active Rac1 restored the ability of PKC{epsilon}-/- fibroblasts to adhere to fibronectin and collagen, to possess abundant actin stress fibers (Fig. 8B,C) and to migrate (Fig. 8D). Thus, we concluded that PKC{epsilon} was required for the activation of Rac, which was in turn required for maximal activation of FAK. It was still possible that the defect in PKC{epsilon}-/- cells was due to the reduction of integrin expression (Fig. 4B) rather than to the inability of Rac to be activated. To investigate this possibility, we showed that Rac inhibition could reduce integrin β1 expression in PKC{epsilon}+/+ cells, indicating that Rac activation is located upstream of integrin β1 expression (Fig. 9A). Moreover, Rac inhibition had a similar effect on wild-type fibroblasts to the loss of PKC{epsilon}, namely Rac inhibition resulted in reduced {alpha}-SMA expression (Fig. 9A), ECM contraction (Fig. 9B) and cell migration (Fig. 9C). Collectively, our results are consistent with the notion that the markedly reduced ability of focal adhesions to be properly formed in PKC{epsilon}-/- fibroblasts (owing to an inability to properly activate Rac and, as a result, to phosphorylate FAK) contributes to defective wound healing observed in PKC{epsilon}-/- mice.


Figure 8
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  		Fig. 8. Phenotype of PKC{epsilon}-/- fibroblasts rescued by transfection with expression vector encoding constitutively active Rac1. (A) FAK phosphorylation and integrin β1 expression were analyzed as described in the Materials and Methods. Western blot analysis was used to detect phosphorylated FAK, total FAK, integrin β1 and total Rac1 in PKC{epsilon}+/+ and PKC{epsilon}-/- fibroblasts cultured on fibronectin. Transfection of constitutively active Rac (caRac), compared with empty expression vector (empty), increases phosphorylation of FAK and integrin β1 expression in PKC{epsilon}-/-, as revealed by western blot analysis. Cell extracts were prepared 24 hours post-transfection. FAK phosphorylation and integrin β1 expression were not completely impaired in the absence of PKC{epsilon}. Quantitation of three independent experiments was performed using densitometry. Average±s.d. is shown (n=3, *P<0.05). (B) Adhesion. Cells cultured on fibronectin were transfected with empty expression vector or expression vector encoding activated Rac1 (caRac). Adhesion of PKC{epsilon}+/+ (WT) and PKC{epsilon}-/- fibroblasts on fibronectin and type I collagen was monitored. Cells were detached from plates by using EDTA, and equal numbers of cells were placed into individual wells of 96-well plate. Adherent cells were detected (Absorbance 450) 45 minutes post-adhesion (average±s.d., n=3). (C) Stress fiber formation. PKC{epsilon}+/+ and PKC{epsilon}-/- fibroblasts cultured on fibronectin were fixed with paraformaldeyde and stained to detect {alpha}-SMA. Cells were counterstained with DAPI to detect nuclei (blue stain). PKC{epsilon}-/- cells transfected with caRac1 show stress fibers. (D) Migration of PKC{epsilon}+/+ (WT) and PKC{epsilon}-/- fibroblasts on fibronectin was monitored, using a scratch wound assay as described in the Materials and Methods. Photographs were taken 72 hours post-transfection, 48 hours after introduction of the scratch wound. Gap size expressed as a percentage of the original wound is shown (average±s.d.; n=3). Overexpression of Rac1 significantly increased migration of PKC{epsilon}-/- fibroblasts (Student's paired t-test, *P<0.05) compared with the appropriate control.

 

Figure 9
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  		Fig. 9. Rac inhibition reduces integrin β1 expression in PKC{epsilon}+/+ fibroblasts. (A) As described in the Materials and Methods, western blot analysis was used to detect integrin β1 expression in PKC{epsilon}+/+ and PKC{epsilon}-/- fibroblasts. Cells were incubated in the presence or absence of the Rac inhibitor NSC23766 (100 µM, 1 hour). Rac inhibition reduced integrin β1 expression in PKC{epsilon}+/+, but not in PKC{epsilon}-/-, cells, indicating Rac operates upstream of integrin β1 expression and, in this context, in a PKC{epsilon}-dependent fashion. Rac inhibition also reduced {alpha}-SMA protein expression in PKC{epsilon}+/+, but not PKC{epsilon}-/-, cells. (B) As described in the Materials and Methods, a floating collagen gel contraction assay was used to detect ECM contraction by PKC{epsilon}+/+ and PKC{epsilon}-/- fibroblasts over a 24-hour period. Cells were incubated in the presence or absence of the Rac inhibitor NSC23766 (100 µM). Contraction was observed only in untreated PKC{epsilon}+/+ cells (*P<0.05). (C) As described in the Materials and Methods, a cell migration assay was performed on PKC{epsilon}+/+ and PKC{epsilon}-/- fibroblasts over a 72-hour period in the presence or absence of the Rac inhibitor NSC23766 (100 µM). Gap size expressed as a percentage of the original wound is shown (average±s.d.). The presence of the Rac inhibitor reduced migration of PKC{epsilon}+/+ cells (*P<0.05).

 

   Discussion
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 Summary
 Introduction
 Results
 Discussion
Materials and Methods
 References
 
Although it has been shown that PKC{epsilon} is activated upon cell adhesion, the function of PKC{epsilon} in focal adhesion formation and function is unknown. In this report, we test the effect of loss of PKC{epsilon} in fibroblasts and show that recruitment of focal adhesion components to the cytoskeleton and phosphorylation of FAK was impaired in PKC{epsilon} knockout fibroblasts. Consistent with this idea, PKC{epsilon}-/- mice showed reduced rates of wound closure, cellularization and ECM deposition. In addition, isolated PKC{epsilon}-/- fibroblasts showed reduced focal adhesion formation and migration and contraction of ECM. PKC{epsilon} -/- fibroblasts showed reduced adhesive signaling, as visualized by FAK and Rac activation. As transfection of Rac at least partially restored the defects in FAK phosphorylation and migration in the PKC{epsilon}-/- fibroblasts, suggesting that activation of Rac is downstream of PKC{epsilon} but upstream of FAK. These results are consistent with recent findings that Rac is required for deformation-induced FAK phosphorylation (Chaturvedi et al., 2007Go) and that loss of Rac1 in keratinocytes results in delayed wound closure (Tscharntke et al., 2007Go). However, perhaps our most crucial finding was that although overall formation of the normal actin cytoskeleton was not impaired in PKC{epsilon} knockout fibroblasts, formation of the highly-contractile {alpha}-SMA cytoskeleton was markedly reduced in the absence of PKC{epsilon}. Similar to this observation, PKC{epsilon} -/- fibroblasts were able to form normal focal adhesions and actin fibers, but not the characteristic `supermature' focal adhesions, as visualized by intense vinculin staining, of myofibroblasts (Dugina et al., 2001Go). It should be pointed out that the overall rate of wound closure was impaired in the absence of PKC{epsilon}. Our results indicate that a crucial feature of this defect arises because of impaired myofibroblast formation. Although this defect is likely to have an indirect effect on the rate of re-epithelialization, we cannot exclude the possibility that loss of PKC{epsilon} may also directly affect keratinocytes function. Evaluating this latter hypothesis is beyond the scope of our current study.

Fibrotic diseases are characterized by the failure to terminate normal tissue repair and the persistence of myofibroblasts within lesions (Gabbiani, 2003Go; Shi-wen et al., 2004Go; Chen et al., 2005Go). Myofibroblast formation can be driven by many processes, including tension, TGFβ, thrombin, ET1 and CCN2 (Arora et al., 1999Go; Hinz et al., 2001Go; Shephard et al., 2004Go; Shi-wen et al., 2004Go; Shi-wen et al., 2006aGo; Shi-wen et al., 2006bGo; Chen et al., 2005Go; Uehta et al., 1997; Leask and Abraham, 2004Go; Leask and Abraham, 2006Go; Goffin et al., 2006Go; Kennedy et al., 2007Go). These processes cooperate in inducing myofibroblast persistence within the milieu of tissue repair and fibrosis (Arora et al., 1999Go; Hinz et al., 2001Go; Shephard et al., 2004Go; Shi-wen et al., 2006aGo; Shi-wen et al., 2006bGo). Thus, our results showing that PKC{epsilon} mediates myofibroblast formation downstream are a useful first step in identifying a target suitable for anti-fibrotic drug intervention. Moreover, several growth factors and cytokines that promote myofibroblast formation are pleiotropic, which is reflected in the phenotype of their respective knockout mice being lethal or displaying severe developmental defects (e.g. Ivkovic et al., 2003Go; Hines et al., 1994Go; Kurihara et al., 1994Go). Conversely, PKC{epsilon}-/- mice are viable and healthy, unless subjected to particular stresses, such as subcutaneous injection of bacteria or hypoxia, in which case they show increased susceptibility to infection and reduced vasoconstriction, respectively (Castrillo et al., 2001Go; Saurin et al., 2002Go; Littler et al., 2003Go). These data, combined with our observations that loss of PKC{epsilon} results in impaired tissue repair and myofibroblast function, suggest that compared to the other pro-fibrotic targets described above, PKC{epsilon} may be a suitable selective target for controlling tissue repair and in alleviating pathological scarring in vivo. Direct testing of this hypothesis using animal models of fibrosis is under way, but is beyond the scope of our current study. However, it is interesting to note that we have recently demonstrated that mice possessing a fibroblast-specific deletion of Rac1 are resistant to bleomycin-induced skin fibrosis (Liu et al., 2008Go).

In conclusion, our results showing that PKC{epsilon} is required for normal tissue repair and myofibroblast formation provide new insights into the function of specific PKC isoforms and into the integrated molecular mechanisms that underlie normal tissue repair.


   Materials and Methods
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 Results
 Discussion
 Materials and Methods
References
 
Punch wound assay
All animal protocols were approved by the local animal ethics committee at the Royal Free and University College Medical School. Female PKC{epsilon}+/+ and PKC{epsilon}-/- mice aged 8 weeks, a kind gift from Peter Parker (University College London) (Castrillo et al., 2001Go), were anesthetized with Avertin (500 mg/kg). The back was shaved and cleaned with alcohol. Four equidistant 4 mm3 full-thickness excisional wounds were made on either side of the midline of six animals/data set. Mice were sacrificed by CO2 euthanasia after 3, 7, 10 and 14 days. Immediately after sacrificing, the wound diameter was measured and wounds were photographed before samples were collected for histology, immunohistochemistry and protein extraction.

Cell culture, immunofluorescence and western analysis
Fibroblasts were isolated from explants (4- to 6-week-old animals) as previously described (Chen et al., 2004Go). To mimic conditions in the provisional matrix post-wounding (Grinnell, 1994), cells were cultured on fibronectin (4 mg/ml Sigma) (Chen et al., 2004Go). Cells were subjected to indirect immunofluorescence analysis as previously described (Chen et al., 2004Go) using anti-{alpha}-SMA, rhodamine phalloidin (Sigma), anti-vinculin or anti-phospho-FAK (Cell Signaling), followed by an appropriate secondary antibody (Jackson ImmunoResearch). Photomicrographs were taken (Zeiss Axiphot) using a digital camera and Adobe Photoshop. Where indicated, cells were stained with DAPI (Molecular Probes, Eugene, OR) to detect nuclei. Alternatively, cells were lysed in 2% SDS, proteins quantified (Pierce) and subjected to western blot analysis as previously described (Shi-wen et al., 2004Go; Shi-wen et al., 2006aGo) with anti-β1 integrin, anti-{alpha}4 integrin (Santa Cruz), anti-CCN2 (Abcam), anti-paxillin (Cell Signaling), anti-type I collagen (Biodesign), anti-phosphoo-Smad3 (Rockland), anti-{alpha}-SMA or β-actin (Sigma) antibodies. Where indicated, cells were pretreated with the Rac inhibitor NSC23766 (100 µM, 1 hour, Calbiochem). Where indicated, cell extraction was performed in Triton lysis buffer (to generate a Triton-soluble fraction) and the Triton insoluble (cytoskeletal) fraction was solubilized in 2% SDS prior to western blot analysis. For the Rac rescue experiment, an expression vector encoding constitutively active Rac (V12rac1) under the control of the cytomegalovirus promoter (courtesy of Alan Hall, University College London) or an empty expression vector was transfected into cells using Fugene (Roche) at a ratio of 2 µg DNA:3 µl Fugene.

Tissue sectioning
Specimens were fixed after 24 hours in 10% neutral buffered formalin after which they were embedded in paraffin. Paraffin-embedded sections were cut (3 µm) on a Leica RM 2135 microtome. For {alpha}-SMA staining, an anti-{alpha}-SMA antibody (Chemicon: 1:50 dilution, 1 hour) was used followed by detection using Vectastain (Vector Labs), as described by the manufacturer. Samples were counterstained with Hematoxylin.

Rac activity assay
A standard commercially available Rac-GTP pulldown assay was used (Upstate Biotechnology; Lake Placid, NY). Rac activity assays were performed as described by the manufacturer. Briefly, cells were grown in 6 cm dishes. Cells were lysed in a buffer containing NP-40. A PAK-GSH fusion protein bound to agarose beads was added, and active Rac, which binds PAK-GSH, was separated by repetitive centrifugation and washing. After the specimens were boiled in Laemmli buffer, they were subjected to SDS-PAGE and Rac was quantified by Western blot analysis. In some experiments, cell lysates were stimulated with GTPS (100 µmol/l) to obtain maximal activation of Rac.

Adhesion assay
Fibroblasts were isolated and cultured as described above. Adhesion assays were performed essentially as previously described (Chen et al., 2004Go). Wells of 96-well plates overnight, 4°C, with 10 µg/ml fibronectin (Sigma) or type I collagen (First Link) in 0.5% bovine serum albumin (BSA), 1 PBS. Wells were blocked for 1 hour in 10% BSA in PBS at room temperature. Fibroblasts were harvested with 2 mM EDTA in PBS (20 minutes, room temperature), washed twice with DMEM serum-free medium containing 1% BSA (Sigma, St Louis, MO), resuspended in the same medium at 2.5x105 cells/ml and 100 µl of suspension was incubated in each well for 1 hour. Non-adherent cells were subsequently removed by washing with PBS. To detect cell adhesion, an acid phosphatase assay was used, adherent cells were quantified by incubation with 100 µl substrate solution (0.1 M sodium acetate, pH 5.5; 10 mM -p-nitophenylphosphate and 0.1% Triton X-100) for 2 hours at 37°C. The reaction was stopped by the addition of 15 µl 1N NaOH/well and A450 was measured. Comparison of adhesive abilities was performed by using Student's unpaired t-test. P<0.05 was considered to be statistically significant.

Collagen gel contraction
Experiments were performed essentially as described previously (Shi-wen et al., 2004Go). Briefly, 24-well tissue culture plates were pre-coated with BSA. Cells were used at passage 3. Trypsinized fibroblasts were suspended in MCDB medium (Sigma) and mixed with collagen solution [one part of 0.2 M N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (HEPES), pH 8.0; four parts collagen (Vitrogen-100, 3 mg/ml) and five parts of MCDB X 2] yielding a final concentration of 80,000 cells per ml and 1.2 mg/ml collagen. Collagen/cell suspension (1 ml) was added to each well. After polymerization, gels were detached from wells by adding 1 ml of MCDB medium. Contraction of the gel was quantified by loss of gel weight and decrease in gel diameter over a 24-hour period

Fibroblast populated collagen lattices (FPCL)
Measurement of contractile force generated within a three-dimensional, tethered FPCL was performed as described previously (Eastwood et al., 1994Go; Shi-wen et al., 2004Go) Using 1106 cells/ml of collagen gel (First Link, UK), we measured the force generated across the collagen lattice with a culture force monitor, which measures forces exerted by cells within a collagen lattice over 24 hours as fibroblasts attach, spread, migrate and differentiate into myofibroblasts. In brief, a rectangular fibroblast seeded collagen gel was cast and floated in medium in 2% FCS in the presence or absence of TGFβ1 (4 ng/ml), while tethered to two flotation bars on either side of the long edges, in turn attached to a ground point at one end and a force transducer at the other. Cell-generated tensional forces in the collagen gel are detected by the force transducer and logged into a personal computer. Graphical readings are produced every 15 seconds providing a continuous output of force (Dynes: 110-5N) generated (Eastwood et al., 2004). The cells used in these experiments were passage matched; experiments were run in parallel and three independent times. A representative trace is shown.

Migration assays
For in vitro wounding (migration) experiments, cultured fibroblasts obtained from wild-type or PKC{epsilon} KO mice were grown on fibronectin in 12-well plates. Medium was removed and cells were once rinsed with serum-free medium + 0.1% BSA and were cultured for 24 hours in serum-free medium + 0.1% BSA. The monolayer was artificially injured by scratching across the plate with a blue pipette tip (~1.3 mm width) (Kinsella and Wight, 1986Go). The wells were washed twice to remove detached cells or cell debris. The cells were then cultured in serum-free medium with or without added TGFβ1 (4 ng/ml R&D Systems). Mitomycin C (10 µg/ml) was always included in the media to prevent cell proliferation. After 48 hours, five representative images of the scratched areas under each condition were photographed.


   Acknowledgments
 
This work is supported by grants from the Ontario Thoracic Society, the Scleroderma Society, the Arthritis Research Campaign, the Welton Foundation, the Reynaud's and Scleroderma Society, Gap B funds from the University of Western Ontario, the Canadian Foundation for Innovation, and the Canadian Institutes of Health Research. A.L. is a New Investigator of the Arthritis Society (Scleroderma Society of Ontario) and the recipient of an Early Researcher Award. We thank Peter Parker, Richard Whalen and Justin Mason (Cancer Research UK, London Research Institute, London, UK) for the PKC{epsilon} knockout mice and advice in their use, and Alan Hall (University College London, London, UK) for the expression vector encoding constitutively active Rac1.


   Footnotes
 
* These authors contributed equally to this work Back


   References
Top
 Summary
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 

Arora, P. D., Narani, N. and McCulloch, C. A. (1999). The compliance of collagen gels regulates transforming growth factor-beta induction of alpha-smooth muscle actin in fibroblasts. Am. J. Pathol. 154, 871-882.[Abstract/Free Full Text]

Besson, A., Wilson, T. L. and Yong, V. W. (2002). The anchoring protein RACK1 links protein kinase Cepsilon to integrin beta chains. Requirements for adhesion and motility. J. Biol. Chem. 277, 22073-22084.[Abstract/Free Full Text]

Bogatkevich, G. S., Tourkina, E., Abrams, C. S., Harley, R. A., Silver, R. M. and Ludwicka-Bradley, A. (2003). Contractile activity and smooth muscle alpha-actin organization in thrombin-induced human lung myofibroblasts. Am. J. Physiol. Lung Cell Mol. Physiol. 285, L334-L343.[Abstract/Free Full Text]

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

Carter, C. A. and Kane, C. J. (2004). Therapeutic potential of natural compounds that regulate the activity of protein kinase C. Curr. Med. Chem. 11, 2883-2902.[Medline]

Castrillo, A., Pennington, D. J., Otto, F., Parker, P. J., Owen, M. J. and Bosca, L. (2001). Protein kinase Cepsilon is required for macrophage activation and defense against bacterial infection. J. Exp. Med. 194, 1231-1242.[Abstract/Free Full Text]

Chaturvedi, L. S., Marsh, H. M., Shang, X., Zheng, Y. and Basson, M. D. (2007). Repetitive deformation activates focal adhesion kinase and ERK mitogenic signals in human Caco-2 intestinal epithelial cells through Src and Rac1. J. Biol. Chem. 282, 14-28.[Abstract/Free Full Text]

Chen, Y., Abraham, D. J., Shi-wen, X., Pearson, J. D., Black, C. M., Lyons, K. M. and Leask, A. (2004). CCN2 (connective tissue growth factor) promotes fibroblast adhesion to fibronectin. Mol. Biol. Cell 15, 5635-5646.[Abstract/Free Full Text]

Chen, Y., Shi-Wen, 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 transforming growth factor-beta/activin-linked kinase 5, heparan sulfate-containing proteoglycans, and MEK/ERK: insights into pathological scarring in chronic fibrotic disease. Am. J. Pathol. 167, 1699-1711.[Abstract/Free Full Text]

Dugina, V., Fontao, L., Chaponnier, C., Vasiliev, J. and Gabbiani, G. (2001). Focal adhesion features during myofibroblastic differentiation are controlled by intracellular and extracellular factors. J. Cell Sci. 114, 3285-3296.[Medline]

Eastwood, M., McGrouther, D. A. and Brown, R. A. (1994). Fibroblast responses to mechanical forces. Biochim. Biophys. Acta 1201, 186-192.[Medline]

Eckes, B., Kessler, D., Aumailley, M. and Krieg, T. (1999). Interactions of fibroblasts with the extracellular matrix: implications for the understanding of fibrosis. Springer Semin. Immunopathol. 21, 415-429.[Medline]

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

Goffin, J. M., Pittet, P., Csucs, G., Lussi, J. W., Meister, J. J. and Hinz, B. (2006). Focal adhesion size controls tension-dependent recruitment of alpha-smooth muscle actin to stress fibers. J. Cell Biol. 172, 259-268.[Abstract/Free Full Text]

Grinnell, F. (1984). Fibronectin and wound healing. J. Cell Biochem. 26, 107-116.[CrossRef][Medline]

Grinnell, F. (2003). Fibroblast biology in three-dimensional collagen matrices. Trends Cell Biol. 13, 264-269.[CrossRef][Medline]

Hagel, M., George, E. L., Kim, A., Tamimi, R., Opitz, S. L., Turner, C. E., Imamoto, A. and Thomas, S. M. (2002). The adaptor protein paxillin is essential for normal development in the mouse and is a critical transducer of fibronectin signaling. Mol. Cell. Biol. 22, 901-915.[Abstract/Free Full Text]

Hines, K. L., Kulkarni, A. B., McCarthy, J. B., Tian, H., Ward, J. M., Christ, M., McCartney-Francis, N. L., Furcht, L. T., Karlsson, S. and Wahl, S. M. (1994). Synthetic fibronectin peptides interrupt inflammatory cell infiltration in transforming growth factor beta 1 knockout mice. Proc. Natl. Acad. Sci. USA 91, 5187-5191.[Abstract/Free Full Text]

Hinz, B. and Gabbiani, G. (2003). Mechanisms of force generation and transmission by myofibroblasts. Curr. Opin. Biotechnol. 14, 538-546.[CrossRef][Medline]

Hinz, B., Mastrangelo, D., Iselin, C. E., Chaponnier, C. and Gabbiani, G. (2001). Mechanical tension controls granulation tissue contractile activity and myofibroblast differentiation. Am. J. Pathol. 159, 1009-1020.[Abstract/Free Full Text]

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 and enhanced focal adhesion contact formation in cells from FAK-deficient mice. Nature 377, 539-544.[CrossRef][Medline]

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.

Ivaska, J., Whelan, R. D., Watson, R. and Parker, P. J. (2002). PKC epsilon controls the traffic of beta1 integrins in motile cells. EMBO J. 21, 3608-3619.[CrossRef][Medline]

Ivaska, J., Bosca, L. and Parker, P. J. (2003). PKCepsilon is a permissive link in integrin-dependent IFN-gamma signalling that facilitates JAK phosphorylation of STAT1. Nat. Cell Biol. 5, 363-369.[CrossRef][Medline]

Ivkovic, S., Yoon, B. S., Popoff, S. N., Safadi, F. F., Libuda, D. E., Stephenson, R. C., Daluiski, A. and Lyons, K. M. (2003). Connective tissue growth factor coordinates chondrogenesis and angiogenesis during skeletal development. Development 130, 2779-2791.[Abstract/Free Full Text]

Kennedy, L., Liu, S., Shi-Wen, X., Chen, Y., Eastwood, M., Carter, D. E., Lyons, K. M., Black, C. M., Abraham, D. J. and Leask, A. (2007). CCN2 is necessary for the function of mouse embryonic fibroblasts. Exp. Cell Res. 313, 952-964.[CrossRef][Medline]

Kinsella, M. G. and Wight, T. N. (1986). Modulation of sulfated proteoglycan synthesis by bovine aortic endothelial cells during migration. J. Cell Biol. 102, 679-687.[Abstract/Free Full Text]

Klemke, R. L., Leng, J., Molander, R., Brooks, P. C., Vuori, K., Cheresh, D. A. (1998). CAS/Crk coupling serves as a "molecular switch" for induction of cell migration. J. Cell Biol. 140, 961-972.[Abstract/Free Full Text]

Kurihara, Y., Kurihara, H., Suzuki, H., Kodama, T., Maemura, K., Nagai, R., Oda, H., Kuwaki, T., Cao, W. H., Kamada, N. et al. (1994). Elevated blood pressure and craniofacial abnormalities in mice deficient in endothelin-1. Nature 368, 703-710.[CrossRef][Medline]

Leask, A. and Abraham, D. J. (2004). TGF-beta signaling and the fibrotic response. FASEB J. 18, 816-827.[Abstract/Free Full Text]

Leask, A. and Abraham, D. J. (2006). All in the CCN family: essential matricellular signaling modulators emerge from the bunker. J. Cell Sci. 119, 4803-4810.[Abstract/Free Full Text]

Littler, C. M., Morris, K. G., Jr, Fagan, K. A., McMurtry, I. F., Messing, R. O. and Dempsey, E. C. (2003). Protein kinase C-epsilon-null mice have decreased hypoxic pulmonary vasoconstriction. Am. J. Physiol. Heart Circ. Physiol. 284, H1321-H1331.[Abstract/Free Full Text]

Liu, S., Kapoor, M., Shi-wen, X., Kennedy, L., Chen. Y., Eastwood, M., Denton, C. P., Glogauer, M., Abraham, D. J., Leask, A. et al. (2008). Investigation of the role of Rac1 in a bleomycin-induced scleroderma model using fibroblast-specific Rac1 knockout mice. Arthritis Rheum. 58, 2189-2195.[CrossRef][Medline]

Martin, P. (1997). Wound healing-aiming for perfect skin regeneration. Science 276, 75-81.[Abstract/Free Full Text]

Midwood, K. S. and Schwarzbauer, J. E. (2002). Tenascin-C modulates matrix contraction via focal adhesion kinase- and Rho-mediated signaling pathways. Mol. Biol. Cell 13, 3601-3613.[Abstract/Free Full Text]

Moscat, J., Rennert, P. and Diaz-Meco, M. T. (2006). PKCzeta at the crossroad of NF-kappaB and Jak1/Stat6 signaling pathways. Cell Death Differ. 13, 702-711.[CrossRef][Medline]

Prekeris, R., Hernandez, R. M., Mayhew, M. W., White, M. K. and Terrian, D. M. (1998). Molecular analysis of the interactions between protein kinase C-epsilon and filamentous actin. J. Biol. Chem. 273, 26790-26798.[Abstract/Free Full Text]

Saurin, A. T., Pennington, D. J., Raat, N. J., Latchman, D. S., Owen, M. J. and Marber, M. S. (2002). Targeted disruption of the protein kinase C epsilon gene abolishes the infarct size reduction that follows ischaemic preconditioning of isolated buffer-perfused mouse hearts. Cardiovasc. Res. 55, 672-680.[Abstract/Free Full Text]

Shephard, P., Hinz, B., Smola-Hess, S., Meister, J. J., Krieg, T. and Smola, H. (2004). Dissecting the roles of endothelin, TGF-beta and GM-CSF on myofibroblast differentiation by keratinocytes. Thromb. Haemost. 92, 262-274.[Medline]

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 myofibroblast induction through the ETA receptor via a rac/phosphoinositide 3-kinase/Akt-dependent pathway and is essential for the enhanced contractile phenotype of fibrotic fibroblasts. Mol. Biol. Cell 15, 2707-2719.[Abstract/Free Full Text]

Shi-wen, X., Stanton, L. A., Kennedy, L., Pala, D., Chen, Y., Howat, S. L., Renzoni, E. A., Carter, D. E., Bou-Gharios, G., Stratton, R. J. et al. (2006a). CCN2 is necessary for adhesive responses to transforming growth factor-beta1 in embryonic fibroblasts. J. Biol. Chem. 281, 10715-10726.[Abstract/Free Full Text]

Shi-wen, X., Rodriguez-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. (2006b). Constitutive ALK5-independent c-Jun N-terminal kinase activation contributes to endothelin-1 overexpression in pulmonary fibrosis: evidence of an autocrine endothelin loop operating through the endothelin A and B receptors. Mol. Cell. Biol. 26, 5518-5527.[Abstract/Free Full Text]

Tscharntke, M., Pofahl, R., Chrostek-Grashoff, A., Smyth, N., Niessen, C., Niemann, C., Hartwig, B., Herzog, V., Klein, H. W., Krieg, T. et al. (2007). Impaired epidermal wound healing in vivo upon inhibition or deletion of Rac1. J. Cell. Sci. 120, 1480-1490.[Abstract/Free Full Text]

Teicher, B. A. (2006). Protein kinase C as a therapeutic target. Clin. Cancer Res. 12, 5336-5345.[Free Full Text]

Turner, C. E. (2000). Paxillin and focal adhesion signalling. Nat. Cell Biol. 2, E231-E236.[CrossRef][Medline]

Uehata, M., Ishizaki, T., Satoh, H., Ono, T., Kawahara, T., Morishita, T., Tamakawa, H., Yamagami, K., Inui, J., Maekawa, M. et al. (1997). Calcium sensitization of smooth muscle mediated by a Rho-associated protein kinase in hypertension. Nature 389, 990-994.[CrossRef][Medline]

Way, K. J., Chou, E. and King, G. L. (2000). Identification of PKC-isoform-specific biological actions using pharmacological approaches. Trends Pharmacol. Sci. 21, 181-187.[CrossRef][Medline]

Webb, D. J., Donais, K., Whitmore, L. A., Thomas, S. M., Turner, C. E., Parsons, J. T. and Horwitz, A. F. (2004). FAK-Src signalling through paxillin, ERK and MLCK regulates adhesion disassembly. Nat. Cell Biol. 6, 154-161.[CrossRef][Medline]

Woods, A. and Couchman, J. R. (1992). Protein kinase C involvement in focal adhesion formation. J. Cell Sci. 101, 277-290.[Abstract/Free Full Text]

Zamir, E. and Geiger, B. (2001). Molecular complexity and dynamics of cell-matrix adhesions. J. Cell Sci. 114, 3583-3590.[Medline]


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