TRPV4 mediates the Ca2+ influx required for the interaction between flightless-1 and non-muscle myosin, and collagen remodeling

Abstract

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 Ca 2+ plays a central role in controlling actomyosin-dependent functions, we examined how Ca 2+ controls the generation of cell extensions and collagen remodeling. Ratio fluorimetry demonstrated localized Ca 2+ influx at the extensions of fibroblasts. Western blotting and quantitative (q)PCR showed high expression levels of the Ca 2+ -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 2+ 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 2+ depletion inhibited the interaction of purified FliI with NMMIIA filaments. Fluorescence resonance energy transfer experiments showed that FliI-NMMIIA interactions require Ca 2+ influx. We conclude that Ca 2+ influx through the TRPV4 channel regulates FliI-NMMIIA interaction, which in turn enables generation of the cell extensions essential for collagen remodeling.

Collagen Organization Within the Cartilage of Trpv4−/− Mice Studied with Two‐Photon Microscopy and Polarized Second Harmonic Generation

“…D,E). This observation could be correlated with other results obtained at the cellular level, showing the requirement of active TRPV4 channels for collagen compaction .…”

Blebbistatin reveals beneficial effects on the cystometric parameters in an animal model of detrusor overactivity

“…Studies of the lower urinary tract have indicated that TRPV channels, including TRPV1, TRPV2, TRPV4, TRPM8, and TRPA1, are expressed in the bladder and may act as sensors of stretch and/or chemical irritation (Andersson 2016). Furthermore, it was shown that Ca 2+ influx through TRPV4 channels regulates the interaction between nonmuscle myosin and actin binding protein Flightless I (Arora et al 2017). Taken together, it is plausible that the mechanism underlying DO development in models induced by the use of retinoids as well as the beneficial effects of BLEB in these models is mediated by their effects on TRPV channels.…”

TRPV4—A Missing Link Between Mechanosensation and Immunity

“…Finally, substrate stiffness is regulated by the composition of the ECM which changes as a function of physiological (development, aging) and pathophysiological (atherosclerosis, hypertension, fibrosis) processes (90)(91)(92)(93). TRPV4 has been identified as a major mechanosensor for substrate stiffness, but so far this function has been exclusively attributed to parenchymal cells (30,31,(94)(95)(96)). Yet, it is fair to speculate that changes in substrate stiffness will similarly affect the mechanical forces that act upon immune cells during the processes of adhesion and transmigration, which accordingly may affect TRPV4-dependent cellular responses.…”