ROCK Inhibition Suppresses Thrombospondin-1 and Rewires DNA Methylation in the Trabecular Meshwork

Rho-associated protein kinase (ROCK) inhibitors reduce cytoskeletal tension in trabecular meshwork (TM) cells, but the mechanisms linking this acute response to sustained extracellular matrix remodelling remain incompletely understood. In this study, we investigated whether the ROCK inhibitor Y39983 regulates thrombospondin-1 (THBS1) and DNA methylation programs, elements known to modulate extracellular matrix (ECM) remodelling, in primary human trabecular meshwork (hTM) cells. Primary hTM cells were treated with Y39983, 5-azacytidine (5-Aza, a DNA methyltransferase inhibitor), or both in combination. Gene expression of THBS1, DNA methyltransferase, ECM, and cytoskeletal molecules was assessed using RT-qPCR. THBS1 promoter methylation was examined through methylation-specific PCR, while genome-wide methylation remodelling was profiled using whole-genome bisulphite sequencing (WGBS) followed by pathway enrichment analysis. 5-Azacytidine abolished Y39983-dependent effects on DNMT1 and DNMT3A and attenuated THBS1 regulation. Methylation-specific PCR suggested altered THBS1 promoter methylation after Y39983 treatment, while whole-genome bisulphite sequencing identified 8845 differentially methylated genes, including 5608 hypomethylated and 3237 hypermethylated genes, enriched in ErbB signalling, focal adhesion, and regulation of the actin cytoskeleton. Independently, Y39983 reduced the basal and stimulus-induced expression of THBS1, as well as ECM- and cytoskeleton-associated genes, including COL4A1, COL6A1, and ACTG1. These findings demonstrate that ROCK inhibition is associated with changes in DNMT-related gene expression and DNA methylation patterns in hTM cells. However, because 5-azacytidine has effects beyond selective DNMT inhibition, further studies using orthogonal approaches are required to establish whether DNA methylation directly mediates transcriptional responses to Y-39983.