Mechanistic basis of Rho GTPase-induced extracellular matrix synthesis in trabecular meshwork cells

Abstract

Pattabiraman PP, Rao PV. Mechanistic basis of Rho GTPaseinduced extracellular matrix synthesis in trabecular meshwork cells. Am J Physiol Cell Physiol 298: C749 -C763, 2010. First published November 25, 2009 doi:10.1152/ajpcell.00317.2009.-Elevated intraocular pressure arising from impaired aqueous humor drainage through the trabecular pathway is a major risk factor for glaucoma. To understand the molecular basis for Rho GTPase-mediated resistance to aqueous humor drainage, we investigated the possible interrelationship between actomyosin contractile properties and extracellular matrix (ECM) synthesis in human trabecular meshwork (TM) cells expressing a constitutively active form of RhoA (RhoAV14). TM cells expressing RhoAV14 exhibited significant increases in fibronectin, tenascin C, laminin, ␣-smooth muscle actin (␣-SMA) levels, and matrix assembly in association with increased actin stress fibers and myosin light-chain phosphorylation. RhoAV14-induced changes in ECM synthesis and actin cytoskeletal reorganization were mimicked by lysophosphatidic acid and TGF-␤2, known to increase resistance to aqueous humor outflow and activate Rho/Rho kinase signaling. RhoAV14, lysophosphatidic acid, and TGF-␤ 2 stimulated significant increases in Erk1/2 phosphorylation, paralleled by profound increases in fibronectin, serum response factor (SRF), and ␣-SMA expression. Treatment of RhoA-activated TM cells with inhibitors of Rho kinase or Erk, on the other hand, decreased fibronectin and ␣-SMA levels. Although suppression of SRF expression (both endogenous and RhoA, TGF-␤ 2-stimulated) via the use of short hairpin RNA decreased ␣-SMA levels, fibronectin was unaffected. Conversely, fibronectin induced ␣-SMA expression in an SRF-dependent manner. Collectively, data on RhoA-induced changes in actomyosin contractile activity, ECM synthesis/assembly, and Erk activation, along with fibronectin-induced ␣-SMA expression in TM cells, reveal a potential molecular interplay between actomyosin cytoskeletal tension and ECM synthesis/assembly. This interaction could be significant for the homeostasis of aqueous humor drainage through the pressure-sensitive trabecular pathway. fibronectin; actomyosin; extracellular signal-regulated kinases; serum response factor GLAUCOMA IS A SECOND MAJOR cause of blindness in the United States. A major risk factor for primary open-angle glaucoma is elevated intraocular pressure caused by increased resistance to aqueous humor outflow localized within the trabecular pathway (15,56). Abnormal accumulation of extracellular matrix (ECM) and/or extracellular material, which increases resistance to drainage of aqueous humor through the trabecular pathway, is believed to be partly responsible for the elevated intraocular pressure and primary open-angle glaucoma (24,28,49). The cellular mechanisms that regulate the production and turnover of ECM within the outflow pathway and how ECM turnover is linked to regulation of aqueous humor outflow through the trabecular meshwork (TM) are far from clearly understood (13,...

Bioactive Lysophospholipids: Role in Regulation of Aqueous Humor Outflow and Intraocular Pressure in the Context of Pathobiology and Therapy of Glaucoma

“…43,82,83 In addition, these bioactive lipids have been shown to play a critical role in the expression of ECM and a-smooth muscle actin. 84 Therefore, the rest of this discussion will focus on the biological effects of LPA, S1P, ATX, and PLA2, and other lipids in the regulation of AH outflow and IOP in both normal and glaucoma subjects.…”

“…87 There is, however, overwhelming evidence that LPA influences cell adhesive, actin cytoskeletal organization, and contractile properties of TM cells derived from different species, including human and porcine. 42,80,84,88 Moreover, both TM cells and TM tissue derived from human eyes have been shown to express various LPA-specific GPCR receptors that are specific to LPA. 42 In serum-starved TM and SC cells, LPA has been shown to induce a robust increase in actin stress fibers, focal adhesions (vinculin and paxillin based), and contraction in association with increased myosin light chain (MLC) phosphorylation.…”

“…42 These effects were found to be mediated predominantly by LPA-induced activation of Rho GTPase signaling. 42,84 LPA was one of the earliest biological agents recognized to be a potent activator of Rho GTPase signaling in various endothelial and epithelial cell types. 89 In TM cells, LPA also has been shown to increase the expression of various ECM proteins and CTGF.…”

“…89 In TM cells, LPA also has been shown to increase the expression of various ECM proteins and CTGF. 84,90 In addition, LPA has been reported to increase SC cell stiffness and intracellular calcium levels. 19,42 Interestingly, myocilininduced anti or de-adhesive interactions in TM cells have been found to be suppressed in the presence of LPA.…”

Involvement of RhoA/ROCK signaling for alteration of stress fiber via lymphotoxin β receptor stimulation in fibroblastic reticular cell isolated from lymph node

“…RhoA activity associated with agonistic anti-LTβR antibody-treated FRC by Rho pull-down assay Stress fiber assembly is regulated by a signal transduction involving the RhoA small GTPase (Pattabiraman & Rao 2010). To address the possibility that the stress fiber alteration of agonistic anti-LTβR antibody-treated FRC is involved in RhoA signaling, we checked the level of Rho-guanosine diphosphate (GDP)/guanosine triphosphate (GTP) exchange activity associated with FRCs.…”