Mechanotransduction in trabecular meshwork cells requires obligatory interactions between TRPV4 channels, Rho signaling and focal adhesions

Abstract

PurposeSustained exposure to mechanical forces triggers cellular remodeling that is essential to protect cells against mechanical stress. While this process is adaptive in the short term, chronic increases in cellular stiffness are paradigmatic of heart, lung, brain and eye diseases, however, the molecular mechanisms are not well understood. Here, we investigate the reciprocal interactions between cellular mechanotransducers (mechanosensitive ion channels and integrin‐based focal adhesions), calcium signaling, Rho signaling and actin remodeling in trabecular meshwork (TM) cells, which control intraocular pressure (IOP) via a feedback loop between IOP‐induced tensile stretch, cell stiffness and contractility. This process drives vision loss in glaucoma but the mechanisms are unknown.MethodsJuxtacanalicular and corneoscleral human TM cells were stimulated with biaxial strain (6–12%) for 1–24 hours in the presence of agonists/blockers of mechanosensitive channels, calcium chelators and/or Rho kinase inhibitors. [Ca2+] levels in TM cells were measured using optical imaging or electrophysiology. Live imaging cells were transfected mApple:actin or mCherry:actin. mRNA and protein levels of cytoskeletal, ECM and focal adhesion molecules were determined with qRT‐PCR, Western blot and immunostaining.ResultsExposure to strain and targeted activation of TRPV4 resulted in dramatic increase in F‐actin polymerization, which required calcium influx via TRPV4 channels. Stretch time‐dependently upregulated the transcript and protein levels of multiple focal adhesion (FA) proteins that included paxillin, focal adhesion kinase (FAK) and zyxin, an effect that was associated with phosphorylation of FA adapter/anchoring proteins and their translocation between cytoskeletal and membrane locations. Stretch‐induced, Rho‐dependent actin polymerization was downstream from TRPV4 channel activation and visualized as increased formation of stress fibers, however, not all of the intermediary steps were Ca2+‐dependent. Moreover, mechanical stress increased the size of FAs, facilitated vinculin colocalization with pFAK, and triggered zyxin relocation in a Ca2+‐dependent manner. Suppression of mechanotransduction mechanisms reduced actin polymerization, decreased cell contractility and increased fluid outflow.Summary/conclusionWe identified TRPV4 channels as key drivers of strain‐dependent cellular remodeling in the TM, and showed that this process plays a fundamental role in IOP regulation. TRPV4‐mediated Ca2+ influx was required to drive mechanoreciprocity – force‐dependent remodeling of FA and actin – to adapt the cells to mechanical stress. This time‐dependent mechanism appears to be required for the dynamic sensing of membrane strain and its role in adjusting steady‐state IOP. In glaucoma, this process is derailed by chronic increases in actin polymerization and TM stiffness that compromise fluid outflow from the anterior eye.Support or Funding InformationAcknowledgmentsFunding sourceThis work was supported by NIH (RO1EY027920, RO1EY022076, P30EY014800), Glaucoma Research Foundation and unrestricted grants from Research to Prevent Blindness to the Moran Eye Institute at the University of Utah.