BBB Seminar: Matthias Chiquet
Unraveling a RhoA-dependent mechanotransduction pathway in fibroblasts
Matthias Chiquet, Department of Anatomy, University of Bern, Switzerland
Mechanical stress is an important regulator of tissue homeostasis and in part controls the synthesis and turnover of extracellular matrix (ECM) in connective tissues. Strains within the ECM substrate are transmitted to a cell's cytoskeleton via integrin receptors in its matrix adhesion sites; these are known to trigger intracellular signals in response to mechanical cues. By culturing fibroblasts on elastic silicone membranes coated with fibronectin, we sought to elucidate the mechanotransduction pathway responsible for induction of the ECM protein, tenascin-C, by cyclic strain. We found that the RhoA/ROCK pathway, which primarily controls actin dynamics, was crucial for this response. Cyclic strain activated RhoA, enhanced stress fiber formation, and induced tenascin-C in fibroblasts. Inhibition of ROCK suppressed, whereas activators of RhoA enhanced the induction of tenascin-C by cyclic strain. Latrunculin A abolished all RhoA-triggered responses, indicating that they rely on an intact actin cytoskeleton.
Upstream of RhoA/ROCK, β-1-integrins and integrin-linked kinase (ILK) are required, since fibroblasts deficient for either of these focal adhesion components failed to up-regulate tenacin-C mRNA after cyclic strain. In wild-type but not ILK-/- fibroblasts, cyclic strain induced reorganization of actin stress fibers and focal adhesions, as well as nuclear translocation of MKL1/MAL, a transcriptional co-activator of serum response factor that links RhoA activation to gene expression. However, mechanosensation was not entirely abolished in ILK-deficient fibroblasts, since cyclic strain activated Erk-1/2 and induced c-fos mRNA in these cells. Our results indicated that ILK is selectively required for the induction of specific genes (among them tenascin-C) by mechanical stimulation via a RhoA- and MAL-mediated pathway.
ILK-deficient cells are unable to assemble a fibronectin (FN) matrix. To test the hypothesis that their defective mechanotransduction is linked to the absence of pericellular FN, we generated FN-deficient fibroblasts and plated them onto vitronectin-coated elastic membranes (in FN-depleted medium). Exactly as ILK-/- cells and contrary to FN-expressing control cells grown on vitronectin, FN-deficient fibroblasts subjected to cyclic strain exhibited neither an increase in actin stress fibers, nor translocation of MAL to the nucleus, nor induction of tenascin-C mRNA. However, when FN-deficient cells were plated on FN-coated membranes, their defects in RhoA-dependent mechanotransduction were restored. These results showed that the pericellular fibronectin matrix secreted by normal fibroblasts is a necessary component of the strain-sensing machinery that leads to activation of the RhoA pathway.
Host: Donald Gullberg, Department of Biomedicine