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. 2004 Sep;15(9):4310-20.
doi: 10.1091/mbc.e04-05-0386. Epub 2004 Jul 7.

Myofibroblast development is characterized by specific cell-cell adherens junctions

B Hinz  1 , P Pittet, J Smith-Clerc, C Chaponnier, J-J Meister
Affiliations

Myofibroblast development is characterized by specific cell-cell adherens junctions

B Hinz et al. Mol Biol Cell. 2004 Sep.

Abstract

Myofibroblasts of wound granulation tissue, in contrast to dermal fibroblasts, join stress fibers at sites of cadherin-type intercellular adherens junctions (AJs). However, the function of myofibroblast AJs, their molecular composition, and the mechanisms of their formation are largely unknown. We demonstrate that fibroblasts change cadherin expression from N-cadherin in early wounds to OB-cadherin in contractile wounds, populated with alpha-smooth muscle actin (alpha-SMA)-positive myofibroblasts. A similar shift occurs during myofibroblast differentiation in culture and seems to be responsible for the homotypic segregation of alpha-SMA-positive and -negative fibroblasts in suspension. AJs of plated myofibroblasts are reinforced by alpha-SMA-mediated contractile activity, resulting in high mechanical resistance as demonstrated by subjecting cell pairs to hydrodynamic forces in a flow chamber. A peptide that inhibits alpha-SMA-mediated contractile force causes the reorganization of large stripe-like AJs to belt-like contacts as shown for enhanced green fluorescent protein-alpha-catenin-transfected cells and is associated with a reduced mechanical resistance. Anti-OB-cadherin but not anti-N-cadherin peptides reduce the contraction of myofibroblast-populated collagen gels, suggesting that AJs are instrumental for myofibroblast contractile activity.

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Figures

Figure 1.
Figure 1.
AJ development during myofibroblast differentiation in wound granulation tissue. (A–F) Sections of 6-d- (A and D), 9-d- (B and E), and 12-d-old (C) granulation tissue from full thickness rat wounds were costained for α-SMA (red) and β-catenin (green) (A–C) or N- (red) and OB-cadherin (green) (D and E); images were reconstructed from three laser scanning optical sections of 0.2 μm. Bar, 10 μm (A–E), 20 μm (inset, B). (G) Equal protein amounts from extracts from 3-to 12-d-old granulation tissue were blotted, and vimentin expression was quantified by densitometric analysis and used to equilibrate protein levels accordingly, followed by blotting again for cytoskeletal and AJ proteins. Protein loads and cadherin antibodies according to Table 1: 30 μg (1, 8, 12, 14, and 18), and 15 μg (all others). Presented blots are generated from one series of wound samples (3–12 d) and are representative for three independently collected series.
Figure 2.
Figure 2.
AJs are modified during myofibroblast differentiation in culture. (A–D) SCFs were grown for 5 d without (A and C) and with TGFβ (B and D) and costained for β-catenin (red) and F-actin (phalloidin, green) (A and B) or N- (green) and OB-cadherin (red) (C and D). Differentiated myofibroblasts were identified by expression of α-SMA (blue); arrowheads indicate colocalization of N- and OB-cadherin. Bars, 25 μm. (E and F) Total lysates of SCFs, LFs, and REF-52, grown for 5 d in control medium (CO), in the presence of TGFβ (TGF) or of TGFβ-sRII (RII) and vascular SMCs were blotted for cytoskeletal and AJ proteins. Anti-cadherin antibodies used in E: 9 and 11; in F, similar to Figure 1; protein loads: 5 μg (1, 8, 9, 11, 12, and 14), and 2.5 μg (all others).
Figure 3.
Figure 3.
α-SMA–positive and –negative fibroblasts segregate in suspension. TGFβ-treated and control SCFs were suspended, mixed, and plated for 2 h either immediately (A) or after 60-min rotated incubation (B–D) and stained for α-SMA (red), β-catenin (green), and nuclei (DAPI, blue). Bar, 50 μm (C and D), 150 μm (A and B). Stained samples were used to calculate the number (E) and mean size (F) of aggregates, classified by the percentage of α-SMA–positive cells per aggregate. Data are presented from three independent pooled experiments; error bars indicate SD of mean. Green bars indicate that maximum 30% of cells per aggregate are α-SMA–negative (green), red bars signify maximum 30% of α-SMA–positive cells per aggregate and gray bars indicate “mixed” aggregates, where 31–70% of the cells per aggregate are α-SMA positive.
Figure 4.
Figure 4.
Stabilization of AJ proteins with the cytoskeleton increases with myofibroblast differentiation. (A) SCFs and LFs were grown for 5 d in control medium (CO), in the presence of TGFβ (TGF) and TGFβ-sRII (RII). TX-100–insoluble fractions were prepared and blotted for AJ proteins and α-SMA; vimentin was used as loading control. (B) TX-100–insoluble protein was quantified by densitometry and normalized to the pooled TX-100–insoluble and –soluble fractions (=total protein) of TGFβ- (α-SMA–positive) and TGFβ-sRII treated (α-SMA–negative) SCFs.
Figure 5.
Figure 5.
Myofibroblast AJs provide mechanical resistance. SCFs were grown for 5 d with TGFβ (M) and without (F) on the bottom of a parallel-plate flow chamber; similarly treated SCFs were seeded onto this cell monolayer and adhered for 30 min. (A) Cells were stained for α-SMA (red) and β-catenin (green), and series of optical sections of 0.2 μm were produced using laser scanning confocal microscopy. Formation of AJs is demonstrated between a suspended myofibroblast (encircled area) and at the dorsal membrane of a plated myofibroblast, reconstructed from three optical sections at the cell-cell interface and in z-scans performed along the indicated lines. Bar, 10 μm. (B) Reinforcement of the cortical actin of a suspended cell and insertion of stress fibers of a plated cell at contact sites is schematized. (C and D) Cell pairs were subjected to hydrodynamic shear forces, ranging from 1 to 4 Nm–2 and the percentage of remaining suspended cells compared with the initially seeded cell number was determined before and after steps of 1 Nm–2 min–1 in control conditions (C and D, dotted lines) and in the presence of SMA-FP (5 μg/ml) (D). Note that homotypic myofibroblast pairs (M on M) exhibit higher intercellular adhesion compared with homotypic pairs of fibroblasts (F on F). SMA-FP reduces cell-cell attachment when fibroblasts were seeded onto myofibroblasts (F on M) but not in the reverse setup (M on F).
Figure 6.
Figure 6.
AJs are reinforced by α-SMA–mediated contractile activity. (A) REF-52 myofibroblasts grown on glass were transfected with EGFP-α-catenin, live recorded 50 min in control medium, and then treated with SMA-FP. Changes in AJ morphology are demonstrated for one selected cell-cell contact area (arrowheads) every 10 min in inverted fluorescence images. (B–E) LFs were treated for 2 h with control SKA-FP (B), SMA-FP (C), Y27632 (D), and LPA (E) and stained for β-catenin. Note that SMA-FP leads to reorganization of stripe-like AJs to a belt-like contact area within 30 min and induces a significant reduction of AJ formation at longer treatment, comparable with general inhibition of cell contractile activity. Bars, 25 μm. (F) Western blots compare association of AJ proteins with the TX-100–insoluble fractions of LFs after 2-h treatment with control SKA-FP (SKA) and SMA-FP (SMA).
Figure 7.
Figure 7.
Formation of OB-cadherin–containing AJs increases the contraction of myofibroblast-populated collagen gels. (A) Control and (B) TGFβ-treated SCF were transfected with EGFP-α-catenin and live treated for 2 h with function-blocking peptides directed against N- (A) and OB-cadherin (B). (C) SCFs grown in three-dimensional attached collagen gels form β-catenin–positive AJs. Bars, 50 μm. (D) After 5-d culture without (α-SMA–negative) and with TGFβ (α-SMA–positive), SCFs were treated for 3 h with anti-cadherin peptides, gels were detached, and contraction was measured after 30 min; error bars indicate SD of mean (*p ≤ 0.01, n = 3).
Figure 8.
Figure 8.
Model of AJ development during fibroblast to myofibroblast transition. (A) Fibroblasts in normal dermis and in mechanically unloaded three-dimensional collagen matrices exhibit a dendritic phenotype and cytoplasmic actin filaments are organized in a submembranous cortex; cells do not develop AJs but communicate via gap junctions (Salomon et al., 1988; Grinnell et al., 2003). (B) Mechanical activation by growth on rigid culture surfaces or tissue matrix reorganization during wound healing leads to the formation of α-SMA–negative stress fibers, i.e., to the development of the proto-myofibroblast (Tomasek et al., 2002; Hinz and Gabbiani, 2003b). Stress fibers of this migratory phenotype are connected to the ECM at sites of FAs and between cells at sites of AJs that predominantly express N-cadherin; few AJs coexpress N-cadherin and OB-cadherin. (C) TGFβ, in the presence of mechanical stress, leads to the further differentiation of myofibroblasts by inducing de novo expression of α-SMA and by increasing expression of OB-cadherin in AJs. The enhanced contractile activity mediated by α-SMA incorporation into stress fibers reinforces AJs and FAs to large supermature contact sites; AJs of differentiated myofibroblasts predominantly express OB-cadherin either exclusively or in conjunction with N-cadherin.
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