Yemanyi Felix, Vranka Janice, Raghunathan Vijay Krishna
Department of Basic Sciences, College of Optometry, University of Houston, Houston, TX, United States.
Casey Eye Institute, Oregon Health and Science University, Portland, OR, United States.
Invest Ophthalmol Vis Sci. 2020 Aug 3;61(10):41. doi: 10.1167/iovs.61.10.41.
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.
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.
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.
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.
本研究旨在确定京尼平诱导的交联细胞衍生基质（XCDM）是否通过失调β-连环蛋白和Yes相关蛋白（YAP）/含PDZ结合基序的转录共激活因子（TAZ）信号通路，在人小梁网（hTM）细胞中诱导纤维化表型。
用对照或京尼平处理细胞衍生基质5小时，以获得各自的未交联（CDM）和XCDM，并对其进行表征。将hTM细胞接种在这些基质上，在无血清培养基中添加/不添加Wnt通路调节剂培养24小时。测定Wnt/β-连环蛋白和YAP/TAZ通路信号介质和靶点的弹性模量、基因和蛋白质（全细胞和亚细胞组分）表达。
在最高京尼平浓度（10% XCDM）下，XCDM的N-ε（γ-谷氨酰）-赖氨酸交联免疫染色增加，形态上出现融合，且更硬（5.3倍，P < 0.001）。在10% XCDM上，hTM细胞硬度增加7.8倍（P < 0.001），总β-连环蛋白不变，磷酸化β-连环蛋白升高，磷酸化糖原合成酶激酶3β被抑制。虽然10% XCDM对细胞质β-连环蛋白水平无影响，但它降低了细胞核β-连环蛋白、钙黏蛋白11以及关键Wnt靶基因/蛋白。10% XCDM增加了hTM细胞中的总TAZ，降低了磷酸化TAZ，并增加了细胞质TAZ水平。10% XCDM增加了总YAP，降低了细胞核YAP水平以及关键YAP/TAZ靶基因/蛋白。Wnt激活使hTM细胞免受10% XCDM诱导的与细胞核β-连环蛋白增加相关的硬化作用。
细胞质TAZ增加可能抑制β-连环蛋白的核穿梭或调节对房水稳态重要的钙黏蛋白11。细胞质TAZ升高可能抑制YAP在细胞核中可能的稳态功能。总之，TAZ的细胞质定位可能是小梁网细胞外基质（ECM）交联增加如何在体内导致硬度增加和眼压升高的重要下游事件。然而，Wnt通路激活可能改善交联ECM诱导的眼压升高表型。
