TRPV4 mediates the Ca influx required for the interaction between flightless-1 and non-muscle myosin, and collagen remodeling.

Fibroblasts remodel extracellular matrix collagen, in part, through phagocytosis. This process requires formation of cell extensions, which in turn involves interaction of the actin-binding protein flightless-1 (FliI) with non-muscle myosin IIA (NMMIIA; heavy chain encoded by ) at cell-matrix adhesion sites. As Ca plays a central role in controlling actomyosin-dependent functions, we examined how Ca controls the generation of cell extensions and collagen remodeling. Ratio fluorimetry demonstrated localized Ca influx at the extensions of fibroblasts. Western blotting and quantitative (q)PCR showed high expression levels of the Ca-permeable transient receptor potential vanilloid-4 (TRPV4) channel, which co-immunoprecipitated with β1 integrin and localized to adhesions. Treatment with α2β1-integrin-blocking antibody or the TRPV4-specific antagonist AB159908, as well as reduction of TRPV4 expression through means of siRNA, blocked Ca influx. These treatments also inhibited the interaction of FliI with NMMIIA, reduced the number and length of cell extensions, and blocked collagen remodeling. Pulldown assays showed that Ca depletion inhibited the interaction of purified FliI with NMMIIA filaments. Fluorescence resonance energy transfer experiments showed that FliI-NMMIIA interactions require Ca influx. We conclude that Ca influx through the TRPV4 channel regulates FliI-NMMIIA interaction, which in turn enables generation of the cell extensions essential for collagen remodeling.