The effects of a Rho-associated protein kinase (ROCK) inhibitor (Y39983) on human trabecular meshwork cells – a morphological and proteomic study

Abstract

Purpose : Rho-associated coiled coil-forming protein kinase (ROCK) inhibitors are a new anti-glaucoma drug class that lower intraocular pressure (IOP) by relaxing the trabecular meshwork (TM). In this study, the effect of Y39983, a ROCK inhibitor, on cellular morphology, motility and protein expression of human TM (hTM) cells was investigated. Methods : Primary cultures of hTM cell from normal donors were incubated with or without Y39983 (0.01-1 mM). Cellular motility and morphological changes were observed under a light microscope after 2 to 3 days treatment. Differentially expressed proteins were quantified by liquid chromatography tandem mass spectrometry (LC-MS/MS) using SWATHTM technologies. Results : hTM cells treated with 0.1 mM and 1 mM Y39983, but not controls or 0.01 mM Y39983 displayed stellate morphology. At 1 mM, treatment with Y39983 suppressed the migratory capacity of hTM cells and 20 proteins were differentially regulated in 1 mM treated cells. For example, GUSB, which facilitates the glycosaminoglycan breakdown, was upregulated while TSP1 and COL1A1 were downregulated. Using the online PANTHER classification system, three potential pathways and four molecular functions were identified. The pathways included p53, Integrin signaling, and Rho GTPase Cytoskeletal regulation. The molecular functions identified included binding, and catalytic, structural molecule, and transporter activities. One of the differentially expressed proteins, thrombospondin-1 (TSP1), was validated using semi-quantitative PCR and western blot analysis and found to be downregulated, consistent with the results in SWATH. Conclusions : Y39983 was found to alter hTM intracellular cell architecture and cellular motility. The majority of the protein changes observed in response to Y39983 are novel. Upregulation of TSP1 has previously been reported in corticosteroid-treated TM cells. The downregulation of TSP1 after Y39983 treatment suggests it may be potential target for understanding the mechanistic action of ROCK inhibitors on TM cells.