Actin-dependent Regulation of a-Smooth Muscle Actin Expression

Background: Fibroblasts adhere to extracellular matrices through integrins, cell surface receptors that provide sites for force transfer to the actin cytoskeleton. During the development of fibrosis in periodontal tissue responding to chronic pressure or volume overload, fibroblasts can become activated to become myofibroblasts. Myofibroblasts strongly express a-smooth muscle actin (SMA) and secrete abundant, disorganized collagen that forms hypertrophic scars. Objective: To determine the actin-dependent regulatory steps in the mechanical-induction of SMA. Methods: NIH3T3 cells were subjected to well-defined, physiologically relevant forces applied by a standardized system through collagen-coated magnetite beads attached to integrins. After force treatments with collagen, FAK, gelsolin and actin cytoskeleton recruitment were measured by isolating magnetite beads and immunoblotting. Force-induced SMA promoter activation were studied by transfections of luciferase SMA promoter construct and siRNA for FAK knockdown. Results: Upon stimulation by mechanical force, FAK and gelsolin were recruited to magnetite beads and increased phosphorylation of FAK at Tyr397 was detected. In FAK knockdown cells, mechanical-induction of the a-SMA promoter activity was reduced compared to wild-type. Dominant negative PIP5Kg inhibited phosphorylation of FAK at Tyr397 following force stimulation. Conclusions: FAK appeared to be a mechanosensor protein to cell stretching. These data suggest a novel pathway where FAK regulates actin assembly through gelsolin by the second messenger, PIP2.