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

Haiyan Li,Preethi S. Ganapathy,Samuel Herberg,W. Daniel Stamer,VijayKrishna Raghunathan

doi:10.3389/fcell.2022.844342

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.

Highlights

Primary open-angle glaucoma (POAG) is a leading cause of irreversible vision loss worldwide, and elevated intraocular pressure (IOP) is the primary modifiable risk factor (Quigley, 1993; Quigley and Broman, 2006; Kwon et al, 2009; Tham et al, 2014; Tamm et al, 2015)
We demonstrated that mRNA levels of Yesassociated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ) were both significantly upregulated in GTM cells compared to normal Human trabecular meshwork (HTM) cells (Figure 1B)
TGM2 and connective tissue growth factor (CTGF) have been shown to play a role in HTM cell pathobiology in glaucoma (Tovar-Vidales et al, 2008; Junglas et al, 2012); here we showed that mRNA of TGM2, CTGF and ankyrin repeat domain 1 (ANKRD1) were significantly upregulated in GTM cells vs. normal HTM cells, whereas no significant difference was observed for cysteine-rich angiogenic inducer 61 (CYR61) (Figure 1B; Supplementary Figures S2B,C)

Summary

INTRODUCTION

Primary open-angle glaucoma (POAG) is a leading cause of irreversible vision loss worldwide, and elevated intraocular pressure (IOP) is the primary modifiable risk factor (Quigley, 1993; Quigley and Broman, 2006; Kwon et al, 2009; Tham et al, 2014; Tamm et al, 2015). Other research showed that both YAP and TAZ were significantly higher in HTM cells on stiffer polyacrylamide substrates or stiffened HTM cell-derived matrices (Thomasy et al, 2013; Yemanyi et al, 2020) Whilst it appears that these findings may be contradictory, it is important to note that functional outcomes of YAP and TAZ are governed by their subcellular localization which is substrate and context dependent. Together, these highlight the importance of studying YAP/TAZ signaling in a microenvironment mimicking the native tissue to further our understanding of HTM cell mechanobiology with an emphasis on cell-ECM interactions. In this tissue-mimetic environment, we investigated whether YAP/TAZ inhibition would alleviate ECM stiffness- or TGFβ2induced HTM cell pathobiology

MATERIALS AND METHODS

RESULTS

DISCUSSION

DATA AVAILABILITY STATEMENT