TRPV4 mediates the Ca2+ 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 MYH9) at cell-matrix adhesion sites. As Ca²⁺ plays a central role in controlling actomyosin-dependent functions, we examined how Ca²⁺ controls the generation of cell extensions and collagen remodeling. Ratio fluorimetry demonstrated localized Ca²⁺ influx at the extensions of fibroblasts. Western blotting and quantitative (q)PCR showed high expression levels of the Ca²⁺-permeable transient receptor potential vanilloid-4 (TRPV4) channel, which co-immunoprecipitated with β1 integrin and localized to adhesions. Treatment with β2β1-integrin-blocking antibody or the TRPV4-specific antagonist AB159908, as well as reduction of TRPV4 expression through means of siRNA, blocked Ca²⁺ 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 Ca²⁺ depletion inhibited the interaction of purified FliI with NMMIIA filaments. Fluorescence resonance energy transfer experiments showed that FliI-NMMIIA interactions require Ca²⁺ influx.We conclude that Ca²⁺ influx through the TRPV4 channel regulates FliI-NMMIIA interaction, which in turn enables generation of the cell extensions essential for collagen remodeling. [ABSTRACT FROM AUTHOR]