Crosslinked Extracellular Matrix Stiffens Human Trabecular Meshwork Cells Via Dysregulating β-catenin and YAP/TAZ Signaling Pathways

Abstract

Purpose The purpose of this study was to determine whether genipin-induced crosslinked cell-derived matrix (XCDM) precipitates fibrotic phenotypes in human trabecular meshwork (hTM) cells by dysregulating β-catenin and Yes-associated protein (YAP)/ transcriptional coactivator with PDZ-binding motif (TAZ) signaling pathways. Methods Cell-derived matrices were treated with control or genipin for 5 hours to obtain respective uncrosslinked (CDM) and XCDMs and characterized. hTM cells were seeded on these matrices with/without Wnt pathway modulators in serum-free media for 24 hours. Elastic modulus, gene, and protein (whole cell and subcellular fractions) expressions of signaling mediators and targets of Wnt/β-catenin and YAP/TAZ pathways were determined. Results At the highest genipin concentration (10% XCDM), XCDM had increased immunostaining of N-ε(γ-glutamyl)-lysine crosslinks, appeared morphologically fused, and was stiffer (5.3-fold, P < 0.001). On 10% XCDM, hTM cells were 7.8-fold ( P < 0.001) stiffer, total β-catenin was unchanged, pβ-catenin was elevated, and pGSK3β was suppressed. Although 10% XCDM had no effect on cytoplasmic β-catenin levels, it reduced nuclear β-catenin, cadherin 11, and key Wnt target genes/proteins. The 10% XCDM increased total TAZ, decreased pTAZ, and increased cytoplasmic TAZ levels in hTM cells. The 10% XCDM increased total YAP, reduced nuclear YAP levels, and critical YAP/TAZ target genes/proteins. Wnt activation rescued hTM cells from 10% XCDM-induced stiffening associated with increased nuclear β-catenin. Conclusions Increased cytoplasmic TAZ may inhibit β-catenin from its nuclear shuttling or regulating cadherin 11 important for aqueous homeostasis. Elevated cytoplasmic TAZ may inhibit YAP's probable homeostatic function in the nucleus. Together, TAZ's cytoplasmic localization may be an important downstream event of how increased TM extracellular matrix (ECM) crosslinking may cause increased stiffness and ocular hypertension in vivo. However, Wnt pathway activation may ameliorate ocular hypertensive phenotypes induced by crosslinked ECM.

Extracellular matrix stiffness and TGFβ2 regulate YAP/TAZ activity in human trabecular meshwork cells

“…Through this mechanism, YAP/TAZ signaling regulates critical cellular functions and normal tissue homeostasis; imbalance or failure of this process is at the core of various diseases [64]. Indeed, elevated YAP/TAZ transcriptional activity is associated with glaucomatous HTM cell dysfunction [20, 21, 24, 65-68]. YAP/TAZ activity is upregulated in GTM cells from patients with glaucoma compared to HTM cells isolated from normal HTM tissues ( Fig.…”

“…Through this mechanism, YAP/TAZ signaling regulates critical cellular functions and normal tissue homeostasis; imbalance or failure of this process is at the core of various diseases [64]. Indeed, elevated YAP/TAZ transcriptional activity is associated with glaucomatous HTM cell dysfunction [20,21,24,[65][66][67][68].…”

“…YAP was found to be reduced in HTM cells incubated on stiff two-dimensional (2D) polyacrylamide hydrogels, while TAZ levels were increased [23]. On the contrary, other research showed that both YAP and TAZ were significantly higher in HTM cells on stiffer polyacrylamide substrates or stiffened HTM cell-derived matrices [24, 25]. Together, this suggests that the precise role of YAP/TAZ in HTM mechanotransduction is far from clear.…”

Therapeutic Drugs and Devices for Tackling Ocular Hypertension and Glaucoma, and Need for Neuroprotection and Cytoprotective Therapies

“…Normal average IOP is 15 mmHg and >90% of human subject have IOPs between 10 and 21 mmHg. However, in POAG, it is the AQH outflow pathway that is compromised (due to ECM/cellular debris accumulation due to increased transforming growth factor-β (TGF-β) (Tripathi et al, 1994;Hasenbach et al, 2016;Rao and Stubbs, 2021), aberrant cross-linking of ECM which stiffens the TM cells (Yemanyi et al, 2020), possible defects in the SC pore/aquaporin system (Wang et al, 2020), increased resistance at the SC due to angiopoietin/Tie-2 pathway defects (Thomson et al, 2014;Thomson et al, 2019;Bernier-Latmani and Petrova, 2017) and the IOP continues to rise due to the constant addition of AQH to the ANC from the CB. It is estimated that the veinous blood vessel component of the AQH drainage system accounts for <25-50% of total outflow resistance whereas the juxtacanalicular region of the TM/SC area is the major contributor providing >50-75% of total outflow resistance in POAG.…”

“…In certain cases, topical ocular dexamethasome treatment for ocular surface or ANC inflammation causes protein complexing (Yemanyi et al, 2020) and formation of cross-linked actin networks (Bermudez et al, 2017) and results in steroid-induced glaucoma. Moreover, there's evidence that accumulation of mutant myocilin (Kasetti et al, 2016;Kasetti et al, 2021) and amyloid proteins (Orwig et al, 2012), and IL-6-induced release of TGF-β1, which promotes ECM deposition in the ANC (Liton et al, 2009) and causes secretion of endothelin (Von Zee et al, 2012), causes IOP elevation.…”