Extracellular Matrix Stiffness and TGFβ2 Regulate YAP/TAZ Activity in Human Trabecular Meshwork Cells

Abstract

Primary open-angle glaucoma progression is associated with increased human trabecular meshwork (HTM) stiffness and elevated transforming growth factor beta 2 (TGFβ2) levels in the aqueous humor. Increased transcriptional activity of Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ), central players in mechanotransduction, are implicated in glaucomatous HTM cell dysfunction. Yet, the detailed mechanisms underlying YAP/TAZ modulation in HTM cells in response to alterations in extracellular matrix (ECM) stiffness and TGFβ2 levels are not well understood. Using biomimetic ECM hydrogels with tunable stiffness, here we show that increased ECM stiffness elevates YAP/TAZ nuclear localization potentially through modulating focal adhesions and cytoskeletal rearrangement. Furthermore, TGFβ2 increased nuclear YAP/TAZ in both normal and glaucomatous HTM cells, which was prevented by inhibiting extracellular-signal-regulated kinase and Rho-associated kinase signaling pathways. Filamentous (F)-actin depolymerization reversed TGFβ2-induced YAP/TAZ nuclear localization. YAP/TAZ depletion using siRNA or verteporfin decreased focal adhesions, ECM remodeling and cell contractile properties. Similarly, YAP/TAZ inactivation with verteporfin partially blocked TGFβ2-induced hydrogel contraction and stiffening. Collectively, our data provide evidence for a pathologic role of aberrant YAP/TAZ signaling in glaucomatous HTM cell dysfunction, and may help inform strategies for the development of novel multifactorial approaches to prevent progressive ocular hypertension in glaucoma.

Effects of Netarsudil-Family Rho Kinase Inhibitors on Human Trabecular Meshwork Cell Contractility and Actin Remodeling Using a Bioengineered ECM Hydrogel

“…Importantly, this can be accomplished under both normal and simulated glaucomatous conditions (32)(33)(34). We previously demonstrated the bidirectional responsiveness of the HTM hydrogel to pathologic TGFb2 induction (35) and ROCKi rescue using standard Y27632 (14) for correlative analyses of TM cell cytoskeletal organization with tissue-level functional changes (32).…”

“…Human donor eye tissue use was approved by the SUNY Upstate Medical University Institutional Review Board (protocol #1211036), and all experiments were performed in accordance with the tenets of the Declaration of Helsinki for the use of human tissue. Primary human TM (HTM) cells were isolated from healthy donor corneal rims discarded after transplant surgery, as previously described (32)(33)(34), and cultured according to established protocols (37,38). Three normal HTM cell strains (HTM05 [Male/57], HTM07 [Male/39], HTM36 [Female/56]) were used for the experiments in this study.…”

“…HTM cells (1.0 × 10 6 cells/ml) were thoroughly mixed with MA-COL (3.6 mg/ml), SH-HA (0.5 mg/ml, 0.025% (w/v) photoinitiator) and SH-ELP (2.5 mg/ml) on ice (Supplementary Figure 2A), followed by pipetting 10 ml droplets of the HTM cell-laden hydrogel precursor solution onto polydimethylsiloxane-coated (PDMS; Sylgard 184; Dow Corning) 24-well culture plates (Supplementary Figure 2B), according to our established protocols (32)(33)(34). Alternatively, 30 µl droplets of the HTM cellladen hydrogel precursor solution were pipetted onto Surfasilcoated (Fisher Scientific) 18 × 18-mm square glass coverslips followed by placing a regular 12-mm round glass coverslip onto the hydrogels to facilitate even spreading of the polymer solution.…”

Small Nucleolar RNAs in Pseudoexfoliation Glaucoma

“…During the onset of glaucoma, the elevation in IOP causes ischemia, hypoxia, and oxidative stress accumulation in the retina, all of which contribute to the apoptosis of RGCs [ 60 ]; AH outflow resistance is determined in particular by the ECM [ 64 ]. Along with increased protein deposition (mainly collagen and elastin), the ECM becomes more dense and stiff, which leads to an increase in IOP [ 65 ]. Many ECM-stimulating factors are found to be upregulated in glaucoma, including TGF-β2 in the AH and actin secreted by trabecular cells [ 65 , 66 ].…”

YAP/TAZ Promote Fibrotic Activity in Human Trabecular Meshwork Cells by Sensing Cytoskeleton Structure Alternation

“…YAP/TAZ transcriptional activity elevation is associated with TM cell dysfunction. It has been found that the YAP/TAZ nuclear localization was increased in the glaucomatous HTM cells compared to the normal HTM cells [18]. This study aimed to investigate the regulatory effect of YAP/TAZ on the fibrotic activity of HTM cells and their potential as biomarkers for fibrotic activity.…”