ROCK Inhibition Suppresses Thrombospondin-1 and Rewires DNA Methylation in the Trabecular Meshwork
6 affiliations
- 1School of Optometry, The Hong Kong Polytechnic University, Hong Kong, China
- 2Centre for Eye and Vision Research (CEVR), 17W Hong Kong Science Park, Hong Kong, China
- 3Research Centre for SHARP Vision (RCSV), The Hong Kong Polytechnic University, Hong Kong, China
- 4Ophthalmology, Boston University Chobanian & Avedisian School of Medicine, Boston, MA 02118, USA
- 5Department of Chemistry, Hong Kong Baptist University, Hong Kong, China
- 6School of Optometry and Vision Sciences, Cardiff University, Cardiff, UK
Abstract
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.
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BibTeX
@article{shan2026rock,
title = {{ROCK Inhibition Suppresses Thrombospondin-1 and Rewires DNA Methylation in the Trabecular Meshwork}},
author = {Sze-wan Shan and Kit-ying Choy and Choi-ying Ling and Hoi-lam Li and Hong-lok Lung and Chi-ho To and Chi-wai Do},
journal = {International Journal of Molecular Sciences},
year = {2026},
month = oct,
doi = {10.3390/ijms27209248},
url = {https://www.mdpi.com/1422-0067/27/20/9248},
note = {CC BY 4.0},
}
RIS
TY - JOUR
TI - ROCK Inhibition Suppresses Thrombospondin-1 and Rewires DNA Methylation in the Trabecular Meshwork
AU - Sze-wan Shan
AU - Kit-ying Choy
AU - Choi-ying Ling
AU - Hoi-lam Li
AU - Hong-lok Lung
AU - Chi-ho To
AU - Chi-wai Do
PY - 2026
DA - 2026/10/01
JO - International Journal of Molecular Sciences
DO - 10.3390/ijms27209248
UR - https://www.mdpi.com/1422-0067/27/20/9248
AB - 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.
SN - 1422-0067
ER -