Tenascin-C suppresses Rho activation

Abstract

Cell binding to extracellular matrix (ECM) components changes cytoskeletal organization by the activation of Rho family GTPases. Tenascin-C, a developmentally regulated matrix protein, modulates cellular responses to other matrix proteins, such as fibronectin (FN). Here, we report that tenascin-C markedly altered cell phenotype on a three-dimensional fibrin matrix containing FN, resulting in suppression of actin stress fibers and induction of actin-rich filopodia. This distinct morphology was associated with complete suppression of the activation of RhoA, a small GTPase that induces actin stress fiber formation. Enforced activation of RhoA circumvented the effects of tenascin. Effects of active Rho were reversed by a Rho inhibitor C3 transferase. Suppression of GTPase activation allows tenascin-C expression to act as a regulatory switch to reverse the effects of adhesive proteins on Rho function. This represents a novel paradigm for the regulation of cytoskeletal organization by ECM.

Figure 1

Filopodia form in response to a fibrin-FN+tenascin-C matrix. Fibrin-FN (A and B), fibrin-FN+tenascin-C (C and D), and fibrin-FN+70Ten (F and G) matrices were formed on glass coverslips and cells were allowed to spread for 1 h. Cells were analyzed by phase microscopy or washed, fixed, permeabilized, and incubated with rhodamine-phalloidin to stain filamentous actin. (E) 70Ten contains the amino-terminal 70-kD region of FN including the fibrin cross-linking site (X) connected to all type III repeats and the terminal knob of tenascin-C containing adhesive and anti-adhesive domains. Bars: (A, C, and F) 10 μm; (B, D, and G) 100 μm.

Figure 2

Tenascin-C inhibits Rho activation. Relative amounts of activated (GTP-bound) Rho and Cdc42 were determined using an affinity assay. (A and B) NIH3T3 fibroblasts were serum starved, then plated on tissue culture plastic (−) or on matrices with the indicated components. Active GTPases were isolated and analyzed along side total cell lysates by immunoblotting with anti-Rho or anti-Cdc42 antibodies.

Figure 3

Rho inactivation causes restoration of filopodia. NIH3T3 cells were serum starved for 24 h either without (A and B) or with (C and D) 25 μg/ml C3 transferase added to the medium. Before plating on fibrin-FN+70Ten matrices, all cells were pretreated with 200 ng/ml LPA while in suspension for 30 min. Cells were allowed to spread for 1 h before examination by phase optics (A and C) or visualization of the actin cytoskeleton (B and D). Bars, 20 μm.

Figure 4

RhoA activity ablates effect of tenascin-C. Rat1 fibroblasts carrying active RhoA-V14 under the control of a tetracycline repressible promoter were allowed to spread on fibrin-FN (A and C) or fibrin-FN+ 70Ten matrix (B and D). Cells under repressed conditions (no active RhoA-V14 protein; A and B) or after induction to express active RhoA-V14 (C and D) were stained with rhodamine-phalloidin. Rat1 cells constitutively expressing active Cdc42-V12 were plated on fibrin-FN (E) or fibrin-FN+ 70Ten (F) matrix for 1 h. Bar, 20 μm.

Figure 5

Matrix induction of filopodia formation. Cells were fixed and stained with rhodamine-phalloidin at the indicated times after plating on fibrin-FN (left) or fibrin-FN+70Ten matrix (right). Bar, 20 μm.