Hic-5 Regulates Actin Cytoskeletal Reorganization and Expression of Fibrogenic Markers and Myocilin in Trabecular Meshwork Cells.

Homeostasis of aqueous humor (AH) drainage through the trabecular meshwork (TM) is essential for maintenance of normal intraocular pressure.1 Importantly, blockage or increased resistance to AH outflow through the TM leads to elevated IOP, which is a major risk factor for primary open-angle glaucoma,2,3 a leading cause of blindness in the United States.4 The conventional AH outflow pathway of human eyes through which most AH is drained consists of the TM, juxtacanalicular (JCT) tissue, and Schlemm's canal (SC).5 The cells derived from these different tissues of the AH outflow pathway exhibit endothelial and mesenchymal characteristics.5,6 Various physiological and etiological mechanisms have been reported to account for homeostatic and deregulated AH outflow, respectively, through the conventional or trabecular pathway.2,3,5,7–10 Importantly, abundant experimental evidence also points to a role for the cellular properties of TM and SC, including contractile characteristics, actin cytoskeletal organization, cell adhesive interactions, mechanical and mechanosensing properties, and extracellular matrix (ECM) organization and production, in regulating AH outflow through the TM.2 To explore additional intracellular molecular mechanisms regulating the TM cell contractile, cell adhesive, and biomechanical properties for a thorough understanding of both homeostatic and deregulated AH outflow and IOP, we hypothesized that Hic-5, a multidomain adaptor protein, may play a crucial role in regulation of TM cell integrin–matrix interactions, actin cytoskeletal organization, and glucocorticoid-induced gene expression, eventually influencing AH outflow through the TM. Hic-5 is an inducible focal adhesion (FA) protein that belongs to the family of paxillin proteins expressed abundantly in smooth muscle cells of various tissues.11–14 Hic-5 was originally isolated as a hydrogen peroxide (H2O2)- and TGF-β1-inducible (TGFB1I1) gene that encodes a 55-kDa protein.11,12,15 Interestingly, in separate studies, this protein has also been identified and characterized as an androgen or glucocorticoid receptor coactivator (ARA55) involved in transcriptional regulation and senescence.16–19 Hic-5 contains four LD (leucine-aspartate repeat) motifs at the amino terminus and four LIM domains at the carboxyl terminus and serves as a multiprotein interacting adaptor protein.11,12 The involvement of Hic-5 in both cytoskeletal organization and transcriptional regulation occurs primarily via the scaffolding properties of this protein.12,15,20,21 Hic-5 exhibits a significant homology to paxillin and binds to FA kinase, protein tyrosine kinase (PYK2), protein tyrosine phosphatase (PTP-PEST), and vinculin, and competes with paxillin to regulate integrin-mediated cell adhesion, actin cytoskeletal organization, contractile activity, and cell spreading.12,20,22–31 Moreover, Hic-5 has been considered as a therapeutic target since elevated levels of this protein have been reported in various diseases,32 and because Hic-5 induces cell plasticity, epithelial to mesenchymal transition (EMT) and fibrosis via Rho/Rho kinase, serum response factor/myocardin transcriptional regulators, and SMAD signaling.14,23 Considering the multiple characteristics of Hic-5 that appear to be relevant to TM cytoskeletal biology, TGF-β signaling, myocilin, Rho GTPase signaling, AH outflow, and steroid-induced glaucoma, we asked ourselves whether Hic-5 plays a critical role in regulation of AH outflow and IOP. To address this possibility, in this initial study we examined Hic-5 expression and distribution in the AH outflow pathway and its regulation and effects on actin cytoskeletal organization, FAs, integrins, myocilin, and fibrogenic activity in human TM (HTM) cells. To the best of our knowledge, this is the first study on Hic-5 demonstrating its regulation and effects in TM cells.