Dataset Information

Crosslinked Extracellular Matrix Stiffens Human Trabecular Meshwork Cells Via Dysregulating ?-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 ?-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/?-catenin and YAP/TAZ pathways were determined. Results:At the highest genipin concentration (10% XCDM), XCDM had increased immunostaining of N-?(?-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 ?-catenin was unchanged, p?-catenin was elevated, and pGSK3? was suppressed. Although 10% XCDM had no effect on cytoplasmic ?-catenin levels, it reduced nuclear ?-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 ?-catenin. Conclusions:Increased cytoplasmic TAZ may inhibit ?-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.

SUBMITTER: Yemanyi F

PROVIDER: S-EPMC7452853 | biostudies-literature | 2020 Aug

REPOSITORIES: biostudies-literature

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

Yemanyi Felix F Vranka Janice J Raghunathan Vijay Krishna VK

Investigative ophthalmology & visual science 20200801 10

<h4>Purpose</h4>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.<h4>Methods</h4>Cell-derived matrices were treated with control or genipin for 5 hours to obtain respective uncrosslinked (CDM) and XCDMs and characterized. hTM cells ...[more]

PMID: 32832971

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Project description:PurposeBiophysical and biochemical attributes of the extracellular matrix are major determinants of cell fate in homeostasis and disease. Ocular hypertension and glaucoma are diseases where the trabecular meshwork tissue responsible for aqueous humor egress becomes stiffer accompanied by changes in its matrisome in a segmental manner with regions of high or low flow. Prior studies demonstrate these alterations in the matrix are dynamic in response to age and pressure changes. The underlying reason for segmentation or differential response to pressure and stiffening are unknown. This is largely due to a lack of appropriate models (in vitro or ex vivo) to study this phenomena.MethodsPrimary trabecular meshwork cells were isolated from segmental flow regions, and cells were cultured for 4 weeks in the presence or absence or dexamethasone to obtain cell derived matrices (CDM). The biomechanical attributes of the CDM, composition of the matrisome, and incidence of crosslinks were determined by atomic force microscopy and mass spectrometry.ResultsData demonstrate that matrix deposited by cells from low flow regions are stiffer and exhibit a greater number of immature and mature crosslinks, and that these are exacerbated in the presence of steroid. We also show a differential response of high or low flow cells to steroid via changes observed in the matrix composition. However, no correlations were observed between elastic moduli and presence or absence of mature and immature crosslinks in the CDMs.ConclusionRegardless of a direct correlation between matrix stiffness and crosslinks, we observed distinct differences in the composition and mechanics of the matrices deposited by segmental flow cells. These results suggest distinct differences in cellular identify and likely a basis for mechanical memory post isolation and culture. Nevertheless, we conclude that although a mechanistic basis for matrix stiffness was undetermined in this study, it is a viable tool to study cell-matrix interactions and further our understanding of trabecular meshwork pathobiology.

Project description:PurposeLysophosphatidic acid (LPA) and soluble interleukin-6 receptor (sIL6R) are elevated in primary open angle glaucoma (POAG). LPA and IL6 modulate in response to biomechanical stimuli and converge on similar fibrotic phenotypes. Thus, we determined whether LPA and IL6 trans-signaling (IL6/sIL6R) interact via Yes-associated protein (YAP)/Transcriptional coactivator with a PDZ-binding motif (TAZ) or Signal transducer and activator of transcription 3 (STAT3) pathways in human trabecular meshwork (hTM) cells.MethodsConfluent primary hTM cells were serum starved for 24 hours, and treated with vehicle, LPA (20 µM), IL6 (100 ng/mL)/sIL6R (200 ng/mL), or both (LPA + IL6/sIL6R) for 24 hours, with or without a YAP inhibitor (verteporfin; 2 µM) or STAT3 inhibitor (2 µM). Expression of key receptors and ligands, signaling mediators, actomyosin machinery, cell contractility, and extracellular matrix (ECM) targets of both signaling pathways was determined by immunocytochemistry, RT-qPCR, and Western blotting.ResultsLPA and IL6 trans-signaling coupling overexpressed/activated receptors and ligands, glycoprotein-130, IL6, and autotaxin; signaling mediators, YAP, TAZ, Pan-TEAD, and phosphorylated STAT3 (pSTAT3); actomyosin and contractile machinery components, myosin light chain 2 (MLC2), phosphorylated MLC2, rho-associated protein kinase 1, filamentous actin, and α-smooth muscle actin; and fibrotic ECM proteins, collagen I and IV, fibronectin, laminin, cysteine-rich angiogenic inducer 61, and connective tissue growth factor in hTM cells; mostly beyond LPA or IL6 trans-signaling alone. Verteporfin inhibited YAP, TAZ, and pSTAT3, with concomitant abrogation of aforementioned fibrotic targets; the STAT3 inhibitor was only partially effective.ConclusionsThese data suggest synergistic crosstalk between LPA and IL6 trans-signaling, mediated by YAP, TAZ, and pSTAT3. By completely inhibiting these mediators, verteporfin may be more efficacious in ameliorating LPA and/or IL6 trans-signaling-induced ocular hypertensive phenotypes in hTM cells.

Dataset Information

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

Publications

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

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