TY - JOUR
T1 - Crosslinked Extracellular Matrix Stiffens Human Trabecular Meshwork Cells Via Dysregulating β-catenin and YAP/TAZ Signaling Pathways
AU - Yemanyi, Felix
AU - Vranka, Janice
AU - Raghunathan, Vijay Krishna
N1 - Funding Information:
The authors thank Alan R. Burns and Sam Hanlon for technical support with scanning electron microscopy, Hasna Baidouri with hTM cell isolation, and their funding sources: startup funding from UHCO (V.K.R.), NIH/NEI grants EY026048-01A1 (J.A.V. and V.K.R.), 5 P30 EY007551-30 (NEI Core grant to UHCO), and P30 EY010572 (NEI Core grant to OHSU), student Vision Research Support Grant (sVRSG), University of Houston College of Optometry (F.Y.), Sigma Xi’s Grant in Aid of Research (G2019100198492848; F.Y.), and by an unrestricted grant to the Casey Eye Institute from Research to Prevent Blindness, New York, NY. The authors also thank SavingSight (Kansas City, MO) eye bank for procuring all human donor eyes used in this work. Most importantly, we would like to thank the families of the organ donors without whose consent these experiments would be impossible.
Publisher Copyright:
Copyright 2020 The Authors
PY - 2020/8
Y1 - 2020/8
N2 - 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.
AB - 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.
KW - Crosslinking
KW - Extracellular matrix
KW - Mechanotransduction
KW - Trabecular meshwork
KW - Wnt
KW - YAP/TAZ
KW - β-catenin
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U2 - 10.1167/IOVS.61.10.41
DO - 10.1167/IOVS.61.10.41
M3 - Article
C2 - 32832971
AN - SCOPUS:85090074342
SN - 0146-0404
VL - 61
JO - Investigative Ophthalmology and Visual Science
JF - Investigative Ophthalmology and Visual Science
IS - 10
M1 - 41
ER -