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First published online 15 June 2004
doi: 10.1242/jcs.01188

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Crosslinking and G-protein functions of transglutaminase 2 contribute differentially to fibroblast wound healing responses

Phil Stephens^1,2,*, Pascale Grenard^6,*, Pascale Aeschlimann^3,6, Martin Langley³, Emma Blain⁶, Rachael Errington⁴, David Kipling^2,5, David Thomas^1,2 and Daniel Aeschlimann^2,3,6,

¹ Department of Oral Surgery, Medicine and Pathology, University of Wales College of Medicine, Heath Park, Cardiff CF14 4XY, UK
² Cardiff Institute of Tissue Engineering and Repair, University of Wales College of Medicine, Heath Park, Cardiff CF14 4XY, UK
³ Matrix Biology and Tissue Repair Research Unit, University of Wales College of Medicine, Heath Park, Cardiff CF14 4XY, UK
⁴ Department of Medical Biochemistry, University of Wales College of Medicine, Heath Park, Cardiff CF14 4XY, UK
⁵ Department of Pathology, University of Wales College of Medicine, Heath Park, Cardiff CF14 4XY, UK
⁶ Connective Tissue Biology Laboratories, School of Biosciences, Cardiff University, Museum Avenue, Cardiff CF10 3US, UK

View larger version (22K): [in a new window]   Fig. 1. Upregulation of TG2 expression in fibroblasts is linked to cell senescence and is independent of culture conditions. (A) Neonatal foreskin, senescent HCA2 and hTERT immortalised HCA2 fibroblasts were seeded into 0.5 mg/ml collagen type I lattices and diametric contraction was measured over a 14-day period. Values are the mean±s.d. (n=3). (B,C) Senescent and hTERT immortalised fibroblasts were grown in monolayer culture (Mono) or seeded into either attached (Attach) or unrestrained (Float) collagen I lattices for 2 days prior to RNA isolation. HCA2 fibroblasts were shown to express 4 iso-enzymes with TG activity (TG1, TG2, TG5 and TG7) by RT-PCR (B). mRNA levels were quantified with the 5' nuclease assay using the TaqMan system and are presented as a mean±s.d. (C). (D) hTERT immortalised HCA2, neonatal foreskin (from two individuals) and senescent HCA2 fibroblasts were extracted with 1% SDS/4 M urea-containing buffer. Cell extracts were separated in 4-20% SDS-PAGE gels and analysed for TG2 expression by immunoblotting with mAb CUB7402. Mol mass standards are indicated on the left. Re-probing of the membrane with antibodies to ß-tubulin (ß-tub) shows equal sample loading (bottom panel). Note, the comparison of pre-senescent to senescent HCA2 cells demonstrates the senescence-associated upregulation of TG2 for an individual fibroblast strain while the comparison of the pre-senescent HCA2 cells to neonatal cells from other individuals shows the level of variability in TG2 expression between individuals.

View larger version (60K): [in a new window]   Fig. 2. TG2 expression level modulates collagen lattice reorganisation by fibroblasts. hTERT immortalised HCA2 fibroblasts were stably transfected with mock, TG2 sense, TG2 antisense and TG2 C277S mutant expression constructs. (A) Transfected fibroblasts were extracted with 0.25 M sucrose/1% Triton X-100 to harvest the soluble TG fraction (S) and subsequently with 1% SDS/1 mM EDTA/1 mM DTT-containing buffer to solubilise the particulate TG fraction (P). Cell extracts were separated in 4-20% SDS-PAGE gels and analysed for TG2 expression by immunoblotting with mAb CUB7402. Mol mass standards are indicated on the right. Note, 5% and 25% of total protein extracted was applied for soluble and particulate fractions, respectively. (B) TG activity expressed by transfected fibroblasts was determined by incorporation of [3H]putrescine into N,N-dimethylcasein for 30 minutes at 37°C. The results from four independent measurements are presented as the mean±s.d. (C,D) Transfected fibroblasts were seeded into 0.5 mg/ml collagen type I lattices and diametric contraction was measured over a 14 day period. Values are the mean±s.d. (n=3). (D) Macroscopic images of lattices at day 3.

View larger version (47K): [in a new window]   Fig. 3. ECM crosslinking by TG2 affects production of active MMP-2. Mock, TG2 sense, TG2 antisense and TG2 C277S mutant transfected HCA2 fibroblasts were cultured in 0.5 mg/ml collagen I lattices, and conditioned media from day 7 and 14 cultures were analysed for gelatinase and TIMP activity by gelatin zymography (A) and reverse zymography (B), respectively. The position of the 72 kDa proform and 62 kDa activated forms of MMP-2 as well as TIMP-1, -2 and -3 are indicated on the right. An unidentified higher molecular mass band with TIMP activity, presumably a TIMP complex, is indicated with an asterisk. Molecular mass standards (kDa) are indicated on the left. (C) Densitometric analysis of the relative levels of pro- and active MMP-2 in conditioned medium after 14 day in culture. Values are the mean±s.e.m. of two independent experiments.

View larger version (49K): [in a new window]   Fig. 4. Altering TG2 expression affects cell-matrix interaction. (A) Fibroblasts (1.5x104) were seeded in wells of a 96-well plate coated with either 40 µg/ml collagen I (COL I) or plasma fibronectin (FN), or left uncoated (TCP). After 1 or 3 hours incubation, adherent cells were quantified by staining with crystal violet and measuring the cell-bound dye by determining the absorbance at 540 nm in Triton X-100 extracts. Values are the mean±s.d. of two independent experiments containing triplicate samples. (B) Phase contrast images of cells 5 hours after seeding on tissue culture plastic are shown. Scale bar: 100 µm.

View larger version (123K): [in a new window]   Fig. 5. TG2 is required for cell spreading. (A) Phase contrast images of fibroblasts cultured on top of collagen I lattices at different time points after seeding. (B) F-actin (FITC-phalloidin) and vinculin (TRITC immunolabelling) distribution in fibroblasts 6 hours after seeding on collagen I-coated glass coverslips. Images represent an extended focus view of five sections of a Z-series taken at 3 µm distance using confocal laser scanning microscopy. The area of the cell shown at high magnification has a blue frame in the insets. In the TG2-deficient cell, the lower inset shows a basal section with vinculin labelling only. Arrows indicate mature focal adhesions associated with stress fibres; arrowheads indicate focal complexes at cell periphery. Scale bars: 100 µm (A) and 10 µm (B).

View larger version (242K): [in a new window]   Fig. 6. Altering TG2 expression affects cell migration in an in vitro wound assay. (A) Confluent fibroblast monolayers were scratch wounded and repopulation of the denuded area followed over a 48-hour period using time-lapse microscopy (see Movies 1-4, http://jcs.biologists.org/supplemental/). (B) The migration paths of 10 cells were tracked over the first 20 hours using the Kinetic Imaging Lucida 4.0 software and the tracks overlaid (using an identical starting point) to illustrate the differences in migration profiles. A representative track is highlighted in colour. (C) An individual cell is highlighted in colour to illustrate the morphological changes in migrating cells. The line (arrowhead) indicates the position of the original edge of the wound. (D) 8 hours after wounding, F-actin was visualised with FITC-phalloidin. A lamellipodium (arrows) is formed in normal and TG2-overexpressing fibroblasts but not in TG2-deficient cells. Scale bars: 100 µm (A), 50 µm (C), and 45 µm (D).

View larger version (46K): [in a new window]   Fig. 7. TG2-deficient cells hyper-phosphorylate FAK but fail to activate PKC. Mock (lane 1), TG2 antisense (lane 2) and TG2 sense (lane 3) transfected HCA2 fibroblasts were extracted and equal amounts of protein were separated in 4-20% SDS-PAGE gels. (A) At the indicated timepoints after seeding, cells were analysed for pTyr (top), FAK (middle), and pTyr397 FAK (bottom) by immunoblotting with specific antibodies. Molecular mass standards (kDa) are indicated on the left. (B) Serum starved fibroblasts were extracted after 30 minutes in suspension or at the indicated timepoints after seeding on uncoated or collagen I-coated (ColI) dishes or uncoated dishes in the presence of serum (FCS) and analysed for FAK, pTyr, and Tyr397, Tyr576 or Tyr925 phosphorylated FAK. (C) Cytosolic (top) and membrane (bottom) fractions were probed with antibodies to PKC.

View larger version (112K): [in a new window]   Fig. 8. PKC activity is required for cell spreading. Phase contrast images (A-J) or vinculin immunolabelling (K,L) of fibroblasts 1 hour after seeding: mock transfected cells under normal culture conditions (A) or after treatment with 10 nM bisindolylmaleimide I (B), 10 nM bisindolylmaleimide V (C), 30 µM PD98059 (D), 1 nM TPA (E), 5 µM U-73122 (F) or 5 µM U-73343 (G); TG2-deficient cells under normal conditions (H,K) or after treatment with 1 nM TPA (I,L) or 5 µM U-73122 (J). Arrows indicate spread and polarized cells (I) and mature focal adhesions (L), respectively. Scale bars: 50 µm (A) and 9.5 µm (K).

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