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. 2002 Oct;13(10):3601-13.
doi: 10.1091/mbc.e02-05-0292.

Tenascin-C modulates matrix contraction via focal adhesion kinase- and Rho-mediated signaling pathways

Kim S Midwood  1 , Jean E Schwarzbauer
Affiliations

Tenascin-C modulates matrix contraction via focal adhesion kinase- and Rho-mediated signaling pathways

Kim S Midwood et al. Mol Biol Cell. 2002 Oct.

Abstract

A provisional matrix consisting of fibrin and fibronectin (FN) is deposited at sites of tissue damage and repair. This matrix serves as a scaffold for fibroblast migration into the wound where these cells deposit new matrix to replace lost or damaged tissue and eventually contract the matrix to bring the margins of the wound together. Tenascin-C is expressed transiently during wound repair in tissue adjacent to areas of injury and contacts the provisional matrix in vivo. Using a synthetic model of the provisional matrix, we have found that tenascin-C regulates cell responses to a fibrin-FN matrix through modulation of focal adhesion kinase (FAK) and RhoA activation. Cells on fibrin-FN+tenascin-C redistribute their actin to the cell cortex, downregulate focal adhesion formation, and do not assemble a FN matrix. Cells surrounded by a fibrin-FN+tenascin-C matrix are unable to induce matrix contraction. The inhibitory effect of tenascin-C is circumvented by downstream activation of RhoA. FAK is also required for matrix contraction and the absence of FAK cannot be overcome by activation of RhoA. These observations show dual requirements for both FAK and RhoA activities during contraction of a fibrin-FN matrix. The effects of tenascin-C combined with its location around the wound bed suggest that this protein regulates fundamental processes of tissue repair by limiting the extent of matrix deposition and contraction to fibrin-FN-rich matrix in the primary wound area.

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Figures

Figure 1
Figure 1
Tenascin-C induces a distinct cytoskeletal organization. NIH 3T3 fibroblasts were allowed to interact with fibrin-FN (A–C) or fibrin-FN+tenascin-C (D–F) matrices for 1 h before fluorescent staining for actin with rhodamine-labeled phalloidin (A and D) and for cortactin with an anticortactin mAb (B and E). Sites of colocalization are shown in yellow (C and F). Bar, 10 μm.
Figure 2
Figure 2
Tenascin-C inhibits the formation of focal adhesions. NIH 3T3 fibroblasts were allowed to interact with fibrin-FN (A and B) or fibrin-FN + 70Ten (C and D) matrices for 1 h before staining for actin (A and C) and for vinculin with an antivinculin mAb (B and D). Bar, 10 μm.
Figure 3
Figure 3
Transient FAK phosphorylation in the presence of tenascin-C. NIH 3T3 fibroblasts were allowed to adhere to fibrin-FN+/−70Ten matrices for 15, 30, and 60 min before lysis and immunoprecipitation with anti-FAK antibodies. Proteins were separated by SDS-PAGE, and immunoblots were probed with an anti-FAK mAb to detect total cellular FAK and an antiphosphotyrosine mAb to detect active FAK.
Figure 4
Figure 4
Tenascin-C inhibits cell contractility. NIH 3T3 fibroblasts were mixed together with fibrin-FN and fibrin-FN+tenascin-C, 70Ten, 70TenS, BSA, or 70 kDa, and the matrices were allowed to polymerize in a 48-well dish. The matrix was detached from the dish, and the area of the matrix was measured after 4 h, subtracted from the starting area, and expressed as a percentage of the starting area. The data are expressed as the mean ± SEM of triplicate experiments.
Figure 5
Figure 5
The role of RhoA in the contraction of fibrin-FN matrices. NIH 3T3 fibroblasts treated with LPA, simvastatin, C3 transferase (C3), or Y27632 were mixed together with fibrin-FN and allowed to polymerize in a 48-well dish. Matrix contraction was measured as described in the legend to Figure 4. The data are expressed as the mean ± SEM of duplicate experiments. Contraction of matrices by cells treated with LPA and Y27632 was significantly different from that of untreated cells (★p < 0.05, Student's paired t test).
Figure 6
Figure 6
RhoA activation reverses inhibition by 70Ten. NIH 3T3 fibroblasts treated with LPA and Rat-1 fibroblasts expressing constitutively active RhoA or control DNA were incorporated into a fibrin-FN + 70Ten matrix. Contraction data are expressed as the mean ± SEM of duplicate experiments. Compared with untreated cells, the addition of LPA or expression of active RhoA significantly increased contraction of matrices (★p < 0.05; Student's paired t test).
Figure 7
Figure 7
Tenascin-C inhibits RhoA localization to the cell membrane. NIH 3T3 fibroblasts were allowed to adhere to fibrin-FN+/−70Ten matrices for 1 h before lysis and ultracentrifugation to separate cytoplasmic (C) and membrane (M) fractions. Proteins were separated by SDS-PAGE, and immunoblots were probed with an anti-Rho mAb.
Figure 8
Figure 8
Tenascin-C inhibits matrix assembly. Untreated NIH 3T3 fibroblasts (A) or fibroblasts treated with 70Ten (B) or both 70Ten and LPA (C) were allowed to adhere to fibrin-FN matrices in the absence of serum and in the presence of exogenous human pFN for 2 h, before immunofluorescence staining with an anti-human FN antibody 7.1 (Bar, 50 μm). In parallel experiments, matrix assembly was assessed by isolation of DOC-insoluble material. Proteins were separated by SDS-PAGE, and immunoblots were probed with antihuman FN antibody 7.1 (D).
Figure 9
Figure 9
FAK is required for matrix contraction. Untreated fibroblasts derived from wild-type (+/+) or FAK-deficient (−/−) mouse embryos or fibroblasts treated with LPA (+LPA) were mixed together with fibrin-FN (A) or fibrin-FN + 70Ten (B) and allowed to polymerize in a 48-well dish. Matrix contraction data are expressed as the mean ± SEM of duplicate experiments. The addition of LPA significantly stimulated contraction of FAK-deficient cells, compared with untreated cells (★p < 0.05; Student's paired t test).
Figure 10
Figure 10
FAK-deficient fibroblasts form stress fibers and focal adhesions. Fibroblasts derived from FAK-deficient mouse embryos were allowed to interact with fibrin-FN (A and B) or fibrin-FN + 70Ten (C and D) matrices for 1 h before staining for actin (A and C) and for vinculin (B and D). Bar, 10 μm.
Figure 11
Figure 11
Sustained FAK phosphorylation rescues contraction of fibrin-FN + 70Ten matrices. (A) NIH 3T3 fibroblasts were treated with phenylarsine oxide or pervanadate and then allowed to adhere to fibrin-FN + 70Ten matrices for 60 min before lysis and immunoprecipitation with anti-FAK antibodies. Proteins were separated by SDS-PAGE, and immunoblots were probed with an anti-FAK mAb to detect total cellular FAK and an antiphosphotyrosine mAb to detect active FAK. (B) NIH 3T3 fibroblasts treated with phenylarsine oxide (PAO), pervanadate (PV), LPA, PAO+LPA or PV+LPA were mixed together with fibrin-FN + 70Ten and allowed to polymerize in a 48-well dish. Matrix contraction was measured as described in the legend to Figure 4. The data are expressed as the mean ± SEM of duplicate experiments. The addition of both LPA and PAO or PV significantly stimulated contraction of matrices, compared with cells treated with LPA alone (★p < 0.05; Student's paired t test).
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