Mechanoreciprocity requires interactions between TRPV4 channels, the Rho pathway and actin cytoskeleton

Abstract

Intraocular pressure (IOP) is dynamically regulated by the trabecular meshwork (TM), a mechanosensitive tissue that protects the eye from injury through dynamic regulation of aqueous humor outflow from the anterior chamber of the eye. IOP-dependent increases in TM stiffness and contractility drive open angle glaucoma but the mechanotransduction mechanisms that regulate these processes remain poorly understood. We used fluorescence imaging and biochemical analyses to investigate cytoskeletal and focal adhesion remodeling in human TM cells stimulated with cyclic strain. The cells showed enhanced F-actin polymerization, increased number and size of focal adhesions, and activation of the Rho-associated protein kinase (ROCK). Stretch-induced activation of the small GTPase RhoA, and tyrosine phosphorylations of focal adhesion proteins paxillin, focal adhesion kinase (FAK), vinculin and zyxin were time-dependently inhibited by HC-067047, an antagonist of transient receptor potential vanilloid 4 (TRPV4) channels, and the ROCK inhibitor Y-27632. TRPV4 and ROCK activation were required for zyxin translocation and increase in the number/size of focal adhesions in stretched cells. Y-27632 blocked actin polymerization without affecting calcium influx induced by membrane stretch and the TRPV4 agonist GSK1016790A. These results reveal that mechanical tuning of TM cells requires parallel activation of TRPV4, integrins and ROCK, with chronic stress leading to sustained remodeling of the cytoskeleton and focal complexes.