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Felix Yemanyi, Janice A Vranka, Vijaykrishna Raghunathan; Crosslinked ECM modulates β-Catenin and YAP/TAZ pathways in human trabecular meshwork cells. Invest. Ophthalmol. Vis. Sci. 2020;61(7):1434.
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© ARVO (1962-2015); The Authors (2016-present)
Increased ECM crosslinking enzymes and stiffness are implicated in ocular hypertension, but the underlying mechanisms are unknown. We investigated whether crosslinked cell-derived matrix (XCDM) modulates β-Catenin and YAP/TAZ pathways or stiffness in human trabecular meshwork (hTM) cells.
CDMs were generated from hTM cells after 4 wks of culture. Then, CDMs were crosslinked with 1%, 2% and 10% genipin/PBS for 5 h to obtain respective XCDMs. XCDMs were validated via immunostaining and atomic force microscopy (AFM). hTM cells from the same donor (n=4) were seeded on control (CDM) and XCDMs in serum-free media for 24 h. Western blotting was done for β-catenin and YAP/TAZ signaling molecules using whole cell lysates or cytoplasmic/nuclear fractions. Cell mechanics was done by AFM.
Compared to CDM, XCDMs had increased immunostaining of N-ε(γ-glutamyl) lysine crosslinks and were markedly stiffer. Whole cell lysates: While there were no differences in K-cadherin among groups, OB-cadherin was decreased (p<0.01, ANOVA) in hTM cells on 10% XCDM. There were no differences in β-Catenin among groups, though pGSK3β was reduced (p<0.001) on 2% and 10% XCDMs; while pβ-Catenin was increased (p<0.001) on 10% XCDM. TAZ was increased (p<0.01) on all XCDMs with pTAZ reduced (p<0.001) on 10% XCDM. YAP was increased (p<0.001) on 2% and 10% XCDMs with no differences in pYAP among groups. Cytoplasmic/Nuclear Fractions: β-Catenin was retained in the cytoplasm, though there were no differences among groups; in the nucleus, it was reduced in hTM cells on 10% XCDM (p<0.05). TAZ was also cytoplasmic, being increased (p<0.01) on 2% and 10% XCDMs, with no differences among groups in the nucleus. YAP showed complete shuttling into the nucleus but was reduced (p<0.01) on 10% XCDM. Cell stiffness: hTM cells were stiffer (p<0.001) on 2% (2.7-fold) and 10% (7.8-fold) XCDMs.
Increased cytoplasmic TAZ in response to a crosslinked ECM suggests it may sequester β-Catenin, thereby hindering the latter’s regulation of OB-cadherin or subsequent nuclear translocation, which is important for aqueous outflow. Also, elevated cytoplasmic TAZ could prevent YAP from functioning in the nucleus especially in the event of increased ECM crosslinking in vivo. Together, TAZ’s cytoplasmic localization could be an important downstream event of how ECM crosslinking enzymes cause increased cell stiffness and ocular hypertension.
This is a 2020 ARVO Annual Meeting abstract.
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