Dexamethasone and Glucocorticoid-Induced Matrix Temporally Modulate Key Integrins, Caveolins, Contractility, and Stiffness in Human Trabecular Meshwork Cells

PURPOSE. To determine the temporal effects of dexamethasone (DEX) and glucocorticoidinduced matrix (GIM) on integrins/integrin adhesomes, caveolins, cytoskeletal-related proteins, and stiffness in human trabecular meshwork (hTM) cells. METHODS. Primary hTM cells were plated on plastic dishes (TCP), treated with vehicle (Veh) or 100 nM DEX in 1% serum media for 1, 3, 5, and 7 day(s). Concurrently, hTM cells were also plated on vehicle control matrices (VehMs) and GIMs for similar time points; VehMs and GIMs had been generated from chronic cultures of Veh-/DEX-stimulated hTM cells and characterized biochemically. Subsets of cells prior to plating on TCP or VehMs / GIMs served as baseline. Protein expression of mechanoreceptors, cytoskeletal-related proteins, and elastic moduli of hTM cells were determined. RESULTS. Compared with Veh, DEX temporally overexpressed alpha V, beta 3, and beta 5 integrins from day 3 to day 7, and integrin linked kinase at day 7, in hTM cells. However, DEX decreased beta 1 integrin at day 1 and day 7, while increasing Cavin1 at day 7, in a time-independent manner. Further, DEX temporally upregulated a-smooth muscle actin(alpha-SMA) and RhoA at day 7 and day 5, respectively; while temporally downregulating Cdc42 at day 3 and day 7 in hTM cells. Conversely, GIM showed increased immunostaining of fibronectin extra-domain A and B isoforms. Compared with VehM, GIM temporally increased aV integrin, Cavin1, and RhoA from day 3 to day 7, at day 3 and day 7, and at day 5, respectively, in hTM cells. Further, GIM overexpressed a-SMA at day 3 and day 7, and stiffened hTM cells from day 1 to day 7, in a time-independent fashion. CONCLUSIONS. Our data highlight crucial mechanoreceptors, integrin adhesomes, and actin-related proteins that may temporally sustain fibrotic phenotypes precipitated by DEX and/or GIM in hTM cells.