Remodeling of focal adhesions and actin regulatory proteins in trabecular meshwork under tensile stress requires TRPV4-dependent Rho signaling

Abstract

Purpose : The trabecular meshwork (TM), a highly mechanosensitive tissue within the iridocorneal angle of the eye, regulates fluid flow in part through dynamic actomyosin contractions. Tensile stress facilitates contractility in part through the Rho pathway but the transduction mechanism that links mechanical force to Rho signaling and remodeling of focal contacts with the extracellular matrix have not been identified. Here we test the hypothesis that the process requires activation of the TRPV4 channel, and investigate the contribution of TRPV4-mediated calcium signaling and Rho activation on stretch-activated remodeling of the TM cytoskeleton, focal adhesions and ECM.

Methods : Immortalized (juxtacanalicular) and primary (corneoscleral) human TM cells were transfected with mCherry:actin/mApple:actin,stimulated with radial strain (1-12%) for 1-24 hours in the presence/absence of selective TRPV4 modulators or shRNA. mRNA and protein levels of cytoskeletal and focal adhesion molecules were measured with qRT-PCR, Western blots, phalloidin fluorescence and antibody labeling. [Ca²⁺]_TM levels in stretched cells were measured with EMCCD cameras in the presence/absence of agonists/antagonists of intracellular Ca²⁺ signaling and Rho pathways

Results : Mechanical stretch or TRPV4 activation elevated [Ca²⁺]_i and induced time-dependent upregulation of genes and total proteins encoding focal adhesion kinase (FAK) and zyxin. Substantial upregulation of stress fibers was detected together with phosphorylation of paxillin, FAK, zyxin and vinculin. Mechanical stretch facilitated colocalization between different focal adhesion components and zyxin through a process that required Ca²⁺. This Ca²⁺ was associated with TRPV4 activation and was upstream of ROCK- and LMCK-dependent actin remodeling.

Conclusions : These results show that the reorganization of the TM cytoskeleton in the presence of tensile stress follows a highly choreographed sequence of steps associated with gene expression, protein translation and transport of cytoskeletal, focal adhesion and ECM proteins. Mechano-activated production of stress fibers and hosphorylatiuon of key actin adaptor proteins requires RhoA/ROCK and calcium ions however the Rho pathway is downstream of TRPV4 activation, and upstream of LMCK-dependent filament assembly that is initiated by paxilin and the focal adhesion kinase.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.