Methods: NIH3T3 cells were subjected to well-defined, physiologically relevant forces applied through collagen-coated magnetite beads attached to integrins. After force treatments, protein recruitment to beads was measured by immunofluorescence and confocal microscopy or by isolating magnetite bead-associated proteins and immunoblotting. Force-induced promoter activation was studied by transfection of luciferase promoter constructs.
Results: In NIH 3T3 cells treated with siRNA to knock down mDia and then subjected to tensile force using collagen-coated magnetite beads attached to b1 integrins, actin assembly was inhibited at bead contact sites. Force-induced nuclear translocation of MRTF-A, a transcriptional co-activator of SMA, was reduced 50% by mDia knockdown. The expression of another transcriptional co-activator of SMA, serum response factor, was reduced by 50% after siRNA knockdown of mDia or by 100% in cells transfected with catalytically inactive mDia. Force-induced activation of the SMA promoter and SMA expression were blocked by knock down of siRNA of mDia. In assays to measure myofibroblast-mediated contraction, knockdown of mDia reduced anchored collagen gel contraction by 50%.
Conclusions: mDia plays an important role in the development of force-induced transcriptional activation of SMA and in myofibroblast differentiation.