Thrombospondin‐1 mediates Rho‐kinase inhibitor‐induced increase in outflow‐facility

• Published in: Journal of cellular physiology 2021-12, Vol.236 (12), p.8226-8238
• Main Authors: Shan, Sze‐Wan, Do, Chi‐Wai, Lam, Thomas Chuen, Li, Hoi‐Lam, Stamer, W. Daniel, To, Chi‐Ho
• Format: Article
• Language: English
• Subjects: Actin; Animals; Antiglaucoma Agents - pharmacology; Aqueous Humor - metabolism; Aqueous humour; Cell adhesion & migration; Cell migration; Cytoskeleton; Cytoskeleton - metabolism; Enzyme inhibitors; Enzyme Inhibitors - pharmacology; Glaucoma; Intraocular Pressure - drug effects; Kinases; Leucine; Lysine; Mice; Outflow; outflow facility; p53 Protein; Phosphorylation; Protein Kinase Inhibitors - pharmacology; Proteins; Proteomics; rho GTP-Binding Proteins - drug effects; rho GTP-Binding Proteins - metabolism; rho-Associated Kinases - metabolism; ROCK inhibitor; Rocks; Signal transduction; Signal Transduction - drug effects; Signaling; siRNA; Thrombospondin; Thrombospondins - metabolism; thrombospondin‐1; trabecular meshwork; Trabecular Meshwork - drug effects
• ISSN: 0021-9541; 1097-4652
• DOI: 10.1002/jcp.30492

Rho‐kinase (ROCK) inhibitors, a novel class of anti‐glaucoma agents, act by increasing the aqueous humor outflow through the conventional trabecular meshwork pathway. However, the downstream signaling consequences of the ROCK inhibitor are not completely understood. Our data show that Y39983, a selective ROCK inhibitor, could induce filamentous actin remodeling, reduced cell motility (as measured by cell migration), and transepithelial resistance in primary human TM (hTM) cells. After 2 days Y39983 treatment of hTM cells, a proteomic study identified 20 proteins whose expression was significantly altered. Pathway analysis of those proteins revealed the involvement of the p53 pathway, integrin signaling pathway, and cytoskeletal pathway regulation by Rho GTPase. Thrombospondin‐1 (TSP1), a matricellular protein that is increased in glaucoma patients, was downregulated fivefold following Y39983 treatment. More importantly, both TSP1 antagonist leucine–serine–lysine–leucine (LSKL) and small interfering RNA (siRNA) reduced TSP1 gene and protein expressions as well as hTM cell migration. In the presence of Y39983, no further inhibition of cell migration resulted after LSKL and TSP1 siRNA knockdown. Likewise, LSKL triggered a dose‐dependent increase in outflow facility in ex vivo mouse eyes, to a similar extent as Y39983 (83.8% increase by Y39983 vs. 71.2% increase by LSKL at 50 µM). There were no additive effects with simultaneous treatment with LSKL and Y39983, supporting the notion that the effects of ROCK inhibition were mediated by TSP1. This study provides the first evidence for reduced thrombospondin‐1 (TSP1) expression as a potential downstream target in ROCK inhibition. Considering that TSP1 is increased in glaucomatous eyes and TSP1 genetic ablation lowers intraocular pressure, our results support the notion that TSP1, via ROCK inhibition, represents a potential therapeutic target for enhancing outflow facility.