Abstract
Abstract: :
Purpose: Fibroblasts and myofibroblasts both participate in corneal stroma repair. After wounding, fibroblasts are activated and migrate into the wound, degrade and replace damaged matrix. Subsequently, myofibroblast expressing smooth muscle alpha actin (SMαA) appear and promote wound closure. Whereas fibroblast growth factors (FGF) 1 or 2 with heparin induces myofibroblasts to dedifferentiate into fibroblasts, transforming growth factor beta (TGFß) induces the fibroblast to myofibroblast transition. Integrin–dependent signals from fibronectin have been implicated in TGFß–mediated SMαA expression. Since focal adhesion kinase (FAK) is downstream from fibronectin–integrin interaction, we asked whether FAK plays a role in the phenotype switching between fibroblasts and myofibroblasts. Methods: Wild–type (FAK +/+) or FAK –/– immortalized mouse embryonic fibroblasts (MEFs) and rabbit corneal fibroblasts were evaluated for expression of SMαA and FGF–R and FGF–R signal transduction in cell culture. Results: Surprisingly we found that mouse embryo fibroblasts (MEFs) lacking FAK expressed SMαA in their microfilaments. Furthermore 97% of the FAK –/– MEFs were SMαA stress fiber positive even after FGF–2/heparin treatment. This is contrary to FGF–2/heparin treated FAK +/+ MEFs in which only 2% of the cells expressed SMαA. Next we measured the cell surface expression of FGF–R by incubation with 125I–FGF–2. FAK +/+ MEFs bound 100% more 125I–FGF–2 when compared to FAK (–/–) MEFs. Third we measured the activation of downstream effectors of the FGF receptor (FGF–R): FGF–R substrate 2 (FRS2) and extracellular regulated kinase (ERK). Whereas FGF–2/heparin treatment induced phosphorylation of FRS2 and ERK in FAK +/+, there was no FGF–induced FRS2 or ERK phosphorylation in FAK –/– MEFs. Finally, inhibiting rabbit corneal fibroblast expression of FAK by transfection with FAK RNAi induced the myofibroblast phenotype. Treatment with FGF/heparin did not reverse the myofibroblast phenotype in rabbit cells that lacked FAK. Conclusions: FAKs absence results in myofibroblast differentiation. Furthermore FAK is necessary for FGF–2 signaling and for inhibition of myofibroblast differentiation. We propose a new role for FAK in positively regulating FGF–R expression and signaling, and negatively regulating SMαA expression.
Keywords: cornea: stroma and keratocytes • signal transduction • growth factors/growth factor receptors