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

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

Andrew Leask^1,*,, Xu Shi-wen^2,*, 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

View larger version (27K): [in this window] [in a new window]   Fig. 1. PKC-/- mice show significantly reduced wound closure and presence of -SMA-expressing myofibroblasts. (A) A 4 mm dermal punch was placed in the back of PKC+/+ and PKC-/-, 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-/- significantly different from PKC+/+ mice). (B) Anti--SMA (-SMA) staining of 14 day wounds. Sectioning and staining revealed the reduced presence of -SMA-positive myofibroblasts (arrow). Sections probed with the -SMA antibody were counterstained with Hematoxylin. Scale bar: 100 µm. (C) Western blot analysis of lysates prepared from of PKC+/+ and PKC+/+ wounds (25 µg/lane). Densitometry represents mean±s.d. of eight independent experiments (i.e. extracts from eight separate wounds; *P<0.05).

View larger version (53K): [in this window] [in a new window]   Fig. 2. PKC-/- fibroblasts show reduced appearance of -SMA stress fibers and `supermature' focal adhesions on fibronectin. PKC+/+ (WT) and PKC-/- fibroblasts cultured on fibronectin were fixed with paraformaldeyde and stained to detect phosphorylated FAK (pFAK), FAK, -SMA, total actin, and vinculin. Cells were counterstained with DAPI to detect nuclei (blue stain). PKC+/+, but not PKC-/-, cells show foci of phospho-FAK staining (pFAK) or intense vinculin staining, which are characteristic of `supermature' focal adhesions.

View larger version (67K): [in this window] [in a new window]   Fig. 3. PKC-/- fibroblasts show reduced recruitment of focal adhesion components on fibronectin. (A) Compartmentalization of focal adhesion components in PKC+/+ (WT) and PKC-/- fibroblasts. Cells were cultured until 80% confluence on fibronectin. Fibronectin is a major component of the provisional extracellular matrix deposited during wound healing (Grinnell, 1984). 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--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 -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. 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+/+ and PKC-/- fibroblasts. Cell extracts (25 µg) from cells cultured in an analogous fashion to those in A were immunoprecipitated with -FAK or -paxillin (-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 -FAK antibody by western analysis. FAK shows reduced interaction with paxillin in the absence of PKC.

View larger version (49K): [in this window] [in a new window]   Fig. 4. Loss of PKC reduces the ability of TGFβ to induce -SMA stress fibers, Smad3 phosphorylation, pro-fibrotic protein expression and matrix contraction in fibroblasts. (A) Immunofluorescence analysis. PKC+/+ and PKC-/- 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 -SMA. Cells were counterstained with DAPI to detect nuclei. (B) Western blot analysis and Smad phosphorylation. PKC+/+ and PKC-/- 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+/+ and PKC-/- 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. Moreover, expression of proteins in response to TGFβ is also impaired. (D) Floating collagen gel contraction assay. PKC+/+ and PKC-/- 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+/+ and PKC-/- 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.

View larger version (12K): [in this window] [in a new window]   Fig. 5. Loss of PKC 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 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., 1994). Force generated by PKC+/+ and PKC-/- 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+/+ in comparison with PKC-/- fibroblasts. (B) Loss of PKC results in a reduced ability of fibroblasts to adhere to extracellular matrix. Adhesion of PKC+/+ and PKC-/- 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.

View larger version (65K): [in this window] [in a new window]   Fig. 6. Loss of PKC 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+/+ and PKC-/- 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.

View larger version (30K): [in this window] [in a new window]   Fig. 7. Loss of PKC results in impaired Rac signaling in fibroblasts. (A) Rac is expressed equally in PKC+/+ (WT) and PKC-/- fibroblasts. Cells were loaded with or without GTPS (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. GTPS (lane 1), GDP (lane 2), PKC+/+ (WT, lane 3), PKC+/+ (WT)+ TGFβ (lane 4); PKC-/- (lane 5); PKC-/- + 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).

View larger version (37K): [in this window] [in a new window]   Fig. 8. Phenotype of PKC-/- 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+/+ and PKC-/- 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-/-, 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. 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+/+ (WT) and PKC-/- 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+/+ and PKC-/- fibroblasts cultured on fibronectin were fixed with paraformaldeyde and stained to detect -SMA. Cells were counterstained with DAPI to detect nuclei (blue stain). PKC-/- cells transfected with caRac1 show stress fibers. (D) Migration of PKC+/+ (WT) and PKC-/- 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-/- fibroblasts (Student's paired t-test, *P<0.05) compared with the appropriate control.

View larger version (26K): [in this window] [in a new window]   Fig. 9. Rac inhibition reduces integrin β1 expression in PKC+/+ fibroblasts. (A) As described in the Materials and Methods, western blot analysis was used to detect integrin β1 expression in PKC+/+ and PKC-/- 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+/+, but not in PKC-/-, cells, indicating Rac operates upstream of integrin β1 expression and, in this context, in a PKC-dependent fashion. Rac inhibition also reduced -SMA protein expression in PKC+/+, but not PKC-/-, cells. (B) As described in the Materials and Methods, a floating collagen gel contraction assay was used to detect ECM contraction by PKC+/+ and PKC-/- 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+/+ cells (*P<0.05). (C) As described in the Materials and Methods, a cell migration assay was performed on PKC+/+ and PKC-/- 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+/+ cells (*P<0.05).

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