Matrix stiffness promotes corneal fibrosis through the YAP/TAZ–β-catenin mechanotransduction pathway: Proteomic and functional validation

Corneal fibrosis, a major cause of corneal opacification and vision loss, is characterized by a progressive increase in extracellular matrix (ECM) stiffness. This heightened stiffness accelerates fibrosis, establishing a self-perpetuating vicious cycle. However, the central mechanotransduction mechanisms through which matrix stiffness drives fibrotic progression remain poorly understood. To investigate this, primary human corneal fibroblasts were cultured on hydrogels with varying stiffness. Label-free quantitative proteomics revealed that increased ECM stiffness triggered widespread proteomic reprogramming, with significant enrichment in pathways related to fibrosis, Hippo signaling, cytoskeletal remodeling, and glycolysis. Further experimental validation confirmed that matrix stiffness coordinately activated the Hippo pathway effectors YAP/TAZ (Yes-associated protein 1/Transcriptional coactivator with PDZ-binding motif) and the canonical Wnt component β-catenin. Importantly, in human fibrotic corneal tissues obtained from clinical specimens, YAP and β-catenin were highly expressed and spatially co-localized with the myofibroblast marker alpha-smooth muscle actin (α-SMA). Modulation of the YAP/TAZ-β-catenin axis through genetic silencing (siRNA) and pharmacological approaches, including pathway inhibitors and agonists, significantly altered stiffness-induced fibrotic phenotypes. Mechanistic studies revealed that YAP/TAZ functioned as key mechanotransducers upstream of β-catenin, and that YAP and β-catenin physically interacted. Collectively, this work identifies the YAP/TAZ-β-catenin pathway as one of the key mechanotransduction pathways of stiffness-induced corneal fibrosis. These findings not only deepen our understanding of the biomechanical mechanisms underlying fibrosis but also provide a mechanistic rationale for developing anti-fibrotic biomaterials and therapies that target mechanical sensing. STATEMENT OF SIGNIFICANCE: This study identifies the YAP/TAZ-β-catenin axis as one of the key mechanotransduction pathways in stiffness-driven corneal fibrosis. Label-free quantitative proteomics revealed widespread protein expression alterations in primary human corneal fibroblasts subjected to pathological matrix stiffness. Pharmacological and genetic perturbations confirmed that YAP/TAZ acts upstream of β-catenin to promote myofibroblast differentiation, supported by evidence of direct protein-protein interaction and co-localization of YAP and β-catenin in fibrotic human corneal tissue. These findings highlight the importance of the mechanical microenvironment in corneal fibrosis beyond classical biochemical stimuli and suggest that targeting this pathway may offer a therapeutic strategy for corneal fibrosis.