TGF(31-Induced (3 1-Induced Fibrotic Responses of Conjunctival Fibroblasts through the Wnt/(3-Catenin/CRYAB (3-Catenin/CRYAB Signaling Pathway

Conjunctival fi brosis is a common postoperative complication of glaucoma fi ltration surgery, resulting in uncontrolled intraocular pressure and surgery failure. Therefore, there is an urgent need to understand the molecular mechanisms underlying conjunctival fi brosis and to explore novel pharmacologic anti-fibrosis fi brosis therapies for glaucoma fi ltration surgery. Herein, the 4-dimensional data-independent acquisition (4DDIA) quantitative proteomic results, coupled with experimental data, revealed the activation of the Wnt/ (3-catenin pathway in transforming growth factor (TGF)-(31-induced human conjunctival fi broblasts (HConFs). Treatment with ICG-001, a Wnt/(3-catenin inhibitor, effectively inhibited cell proliferation and migration in TGF(31-treated HConFs. ICG-001 treatment alleviated the increased generation of extracellular matrix proteins induced by TGF(31. In addition, ICG-001 reduced the expression level of a smooth muscle actin (a-SMA) and inhibited cell contractility in TGF(31-treated HConFs. Proteomics data further suggested that aB-crystallin (CRYAB) was a downstream target of Wnt/(3-catenin, which was up-regulated by TGF(31 and down-regulated by ICG-001. Immunoblotting assay also indicated that ICG-001 reduced the expressions of ubiquitin and (3-catenin in TGF(31-treated HConFs, implying that CRYAB stabilized (3-catenin by inhibiting its ubiquitination degradation. Exogenous CRYAB promoted cell viability, increased extra- cellular matrix protein levels, and up-regulated a-SMA expression of HConFs under TGF(31 stimulation. CRYAB rescued TGF(31-induced fi brotic responses that were suppressed by ICG-001. In conclusion, this study elucidates the regulatory mechanism of the Wnt/(3-catenin/CRYAB pathway in conjunctival fi brosis, offering promising therapeutic targets for mitigating bleb scarring after glaucoma fi ltration surgery. (Am J Pathol 2024, 194: 1764-1779; https://doi.org/10.1016/j.ajpath.2024.05.002)