Abstract

Background and Objectives: Myofibroblastic differentiation is an important event in gingival wound healing and chronic inflammation. Transforming frowth factor-β 1 (TGF-β1) is a potent growth factor that has been implicated in this process. Gingival myofibroblasts have an increased ability to remodel the extracellular matrix and this feature has been associated with changes in the distribution of F-actin and the expression of the myofibroblast marker α-smooth muscle actin. In the present study we have analyzed the role of TGF-β1 and the signaling routes activated by this factor in the cytoskeletal changes that characterize the myofibroblastic differentiation process in human gingival fibroblasts. Materials and methods: The signalling pathways involved in myofibroblastic differentiation were studied in primary cultures of human gingival fibroblasts using several signal transduction inhibitors. RhoA activation was analyzed through a pull-down assay. Distribution of focal adhesions and actin cytoskeleton was assessed by means of immunofluorescence and western blot. A cell adhesion assay was performed in TGF-β1-stimulated cells. Smooth muscle actin expression was studied through western blot and imunofluorescence. c-Jun N-terminal kinase phosphorylation was assessed through western blot. Results: Our observations show that TGF-β1 activated the GTPase RhoA, a key regulator of the actin cytoskeleton. As a consequence of this event, this growth factor stimulated the generation of actin stress fibers and the reinforcement of vinculin-enriched focal adhesions. These responses were blocked after inhibiting ROCK, the main target of RhoA activation. TGF-β1 also stimulated the adhesion of fibroblasts over fibronectin, an extracellular matrix molecule involved in myofibroblastic differentiation. Finally, induction of the myofibroblast marker α-smooth muscle actin by TGF-β1 was abolished by the c-Jun N-terminal protein kinase inhibitor SP600125, suggesting a role for this signaling pathway during the induction of this phenotype. Conclusions: We propose that TGF-β1 may promote the differentiation of myofibroblasts through the stimulation of cell spreading and adhesion, the reinforcement of focal adhesions, the maturation of the actin cytoskeleton, and the induction of α-smooth muscle actin. Activity of RhoA-ROCK and c-Jun N-terminal protein kinase signaling pathways are probably involved in these cellular events. © Blackwell Munksgaard 2006.