Hic-5, a Focal Adhesion Scaffolding Protein and Co-activator of Glucocorticoid Receptor Induces Actin Cytoskeletal Reorganization, Focal Adhesions, Collagen-1 and Myocilin in Human Trabecular Meshwork Cells

Abstract

Purpose: To explore the role of Hic-5, a TGF-beta and H₂O₂ inducible focal adhesion protein and co-activator of glucocorticoid receptor, in regulation of actin cytoskeletal organization, fibrogenic response and myocilin expression in human trabecular meshwork (HTM) cells.

Methods: Expression and distribution of Hic-5 in human aqueous humor (AH) outflow tissues, and regulation of Hic-5 expression and redistribution by RhoA, TGFβ2 and dexamethasone was investigated. The effects of adenovirus-mediated overexpression and siRNA-mediated deficiency of Hic-5, on actin cytoskeletal organization, focal adhesion formation, induction of myocilin, α-smooth muscle actin (α-SMA) and collagen-1A in HTM cells were determined by immunofluorescence and immunoblotting analyses.

Results: Hic-5 is distributed throughout the AH outflow pathway in the human eye including TM, Schlemm’s canal and juxtacanalicular tissues. In HTM cells, Hic-5 co-distributes with vinculin to the tips of actin stress fibers. Constitutively active RhoA (RhoAV14) and TGFβ2 significantly increased Hic-5 expression and led to reorganization of Hic-5 distribution in HTM cells. SMAD2/3 inhibition decreased the effect of TGFβ2-mediated induction of Hic-5 expression. Dexamethasone treatment led to redistribution of Hic-5 predominantly to the focal points of cross linked actin network (CLAN) like structures. Expression of recombinant Hic-5 in HTM cells induced bundling and crosslinking of actin filament and caused significant increase in the levels of myocilin and α-SMA and collagen 1A relative to GFP expressing control cells. Deficiency of Hic-5 led to loss of TGFβ2 and Dexamethasone- induced reorganization of actin as well as significant decreases in protein levels of myocilin, α-SMA and collagen 1A in TM cells.

Conclusions: Taken together, the findings from this study suggest that Hic-5 likely plays a crucial role downstream of Rho GTPase and TGF-β, to influence TM cell actin cytoskeletal organization and focal adhesion formation, and regulate the expression of myocilin, α-SMA and collagen-1. Therefore, Hic-5 dysregulation in TM cells is expected to affect mechanotransduction, cell plasticity and glucocorticoid responses and ultimately drive resistance to AH outflow through the TM.