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, alpha-smooth muscle actin (alpha-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-beta(2), known to increase resistance to aqueous humor outflow and activate Rho/Rho kinase signaling. RhoAV14, lysophosphatidic acid, and TGF-beta(2) stimulated significant increases in Erk1/2 phosphorylation, paralleled by profound increases in fibronectin, serum response factor (SRF), and alpha-SMA expression. Treatment of RhoA-activated TM cells with inhibitors of Rho kinase or Erk, on the other hand, decreased fibronectin and alpha-SMA levels. Although suppression of SRF expression (both endogenous and RhoA, TGF-beta(2)-stimulated) via the use of short hairpin RNA decreased alpha-SMA levels, fibronectin was unaffected. Conversely, fibronectin induced alpha-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 alpha-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.