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RhoA/ROCK-YAP/TAZ Axis Regulates the Fibrotic Activity in Dexamethasone-Treated Human Trabecular Meshwork Cells.

ABSTRACT: High intraocular pressure (IOP) is a major risk factor for glaucoma, a leading cause of irreversible blindness. Abnormal fibrotic activity in the human trabecular meshwork (HTM) cells is considered to be partly responsible for the increased resistance of aqueous humor outflow and IOP. This study aimed to identify the fibrotic pathways using integrated bioinformatics and further elucidate their mechanism of regulating fibrotic activity in dexamethasone (DEX)-treated HTM cells. Microarray datasets from the GEO database were obtained and analyzed by GEO2R. Bioinformatics analyses, including GO and KEGG analyses, were performed to explore biological functions and signaling pathways of differentially expressed genes (DEGs). The fibrotic pathways and targets were determined by western blot, RT-qPCR, or immunofluorescence staining. The cellular elastic modulus was measured using an atomic force microscope. A total of 204 DEGs, partly enriched in fibrotic activity (collagen-containing ECM, fibroblast activation) and Rap1, Ras, TGF-β, and Hippo pathways, were identified. Experimental results showed that DEX induced fibrotic activity and regulated the expression of RhoA/ROCK in HTM cells. Similarly, the constitutively active RhoA (RhoAG14V) also promoted the fibrotic activity of HTM cells. Mechanistically, RhoAG14V induced the expression and nuclear translocation of YAP/TAZ to produce CTGF. Moreover, inhibition of ROCK or YAP decreased the expression of Collagen I and α-SMA proteins induced by DEX or RhoAG14V in HTM cells. In conclusion, these results indicate that RhoA/ROCK-YAP/TAZ axis plays a crucial role in regulating the fibrotic activity of DEX-treated HTM cells.

SUBMITTER: Liu Z

PROVIDER: S-EPMC8450533 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:PurposeThe 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.MethodsCell-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.ResultsAt 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.ConclusionsIncreased 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.

Project description:Changes in the actin cytoskeleton, especially the formation of cross-linked actin networks (CLANs) are thought to contribute to the increased intraocular pressure observed in primary open-angle and steroid-induced glaucoma. To better understand the effects of glucocorticoids, we employed a shotgun method to analyze global changes in the cytoskeleton and integrin signaling pathways following dexamethasone (DEX) treatment of human trabecular meshwork (HTM) cells. RNA and cell lysates were obtained from HTM cells incubated with or without DEX. Changes in protein expression were determined by mass spectrometry (MS) following differential centrifugation of cell lysates to enrich for low-abundance cytoskeletal and signaling proteins, proteolytic digestion, and a titanium dioxide column to enrich for phosphopeptides. Results were validated by Western blots. Changes in RNA levels were determined with gene arrays and RT-PCR. Overall, MS identified 318 cytoskeleton associated proteins. Five of these proteins (PDLIM1, FGFR1OP, leiomodin-1, ZO-2 and LRP16A) were only detected in DEX-treated cells by MS. However, only PDLIM1 showed a statistically significant increase at the RNA level. Other proteins with differences at both the RNA and protein levels included ?3 integrin, caveolin-1, Borg2, raftlin1, PI-3 kinase regulatory subunit ?, transgelin, and filamin B. By immunofluorescence microscopy filamin B and PDLIM1 showed enhanced expression in human trabecular meshwork cells, but only PDLIM1 demonstrated significant localization within CLANs. Finally, MS showed that some of the cytoskeleton proteins (Borg2, leiomodin-1, LRP16A, raftlin1 and CKAP4) contained phosphorylated residues. This study suggests that DEX affects the expression of cytoskeleton proteins at the transcriptional and translational level and shows that a combined genomic and proteomic approach can be used for rapid analysis of proteins in the TM. It also shows that DEX altered the expression of components (PDLIM1 and ?3 integrins) involved in CLAN formation and provides new findings into the effects of glucocorticoids on the cytoskeleton.

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.

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RhoA/ROCK-YAP/TAZ Axis Regulates the Fibrotic Activity in Dexamethasone-Treated Human Trabecular Meshwork Cells.

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