Tubulin Acetylation Enhances Microtubule Stability in Trabecular Meshwork Cells Under Mechanical Stress

Abstract

PURPOSE. To study the roles of tubulin acetylation and cyclic mechanical stretch (CMS) in trabecular meshwork (TM) cells and their impact on outflow pathway physiology and pathology. METHODS. Primary TM cell cultures were subjected to CMS (8% elongation, 24 hours), and acetylated a-tubulin at lysine 40 (Ac-TUBA4) was assessed by western blotting and immunofluorescence. Enzymes regulating tubulin acetylation were identified via siRNA-mediated knockdowns of ATAT1, HDAC6, and SIRT2. Ac-TUBA4 levels were compared between glaucomatous (GTM) and non-glaucomatous (NTM) TM cells and in frozen sections of human cadaver eyes. The effect of tubulin acetylation on substrate stiffness and cell contractility was evaluated by culturing cells on substrates with varying stiffness and by collagen gel contraction assays, respectively. Microtubule stability was examined by monitoring resistance to nocodazole-induced depolymerization. The in vivo effect on intraocular pressure (IOP) was evaluated following intracameral injections of tubacin in mice. RESULTS. CMS induced tubulin acetylation in human TM cells by downregulating the deacetylase HDAC6. Elevated Ac-TUBA4 levels were observed in GTM compared NTM cells and tissues. Tubulin acetylation was not affected by substrate stiffness and did not show a direct effect on TM cell contractility. Tubulin acetylation was found to provide protection against microtubule destabilization induced by nocodazole. Importantly, intracameral injection of tubacin, an HDAC6 inhibitor, significantly lowered IOP in mice. CONCLUSIONS. Our study highlights a critical role of tubulin acetylation in TM cell response to mechanical stress and its potential impact on IOP regulation. Tubulin acetylation could represent a therapeutic target for glaucoma.