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P. P. Pattabiraman, P. V. Rao; Mechanistic Understanding of Rho GTPase-Induced Extracellular Matrix Synthesis and Organization in the Trabecular Meshwork Cells. Invest. Ophthalmol. Vis. Sci. 2009;50(13):4868.
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While abnormal activation of Rho/Rho kinase signaling is implicated in increased resistance to aqueous humor outflow, the mechanisms involved are not understood. Here we investigated the interaction between Rho-induced cytoskeletal tension and extracellular matrix (ECM) synthesis and assembly in trabecular meshwork cells.
TM cell cultures derived from human and porcine eyes were infected with adenoviral vectors expressing either a constitutively active RhoA (RhoAV14 mutant) and GFP (green fluorescence protein) or GFP alone. TM cells were then treated in separate experiments either with Rho GTPase activating physiological agents including LPA, TGFβ and Endothelin-1, or with pharmacological inhibitors of Rho Kinase (Y27632) and Rho GTPase (Lovastatin). Immunoblotting and immunostaining analyses were conducted for smooth muscle actin, vinculin, ECM proteins (e.g. Fibronectin, Tenascin C, and Laminin) and to detect activation of the MAP kinase pathway (e.g. ERK, P38 and JNK).
Expression of RhoAV14 resulted in significant increases in protein levels of smooth muscle actin, fibronectin, tenascin C and laminin in human TM cells by immunoblotting analyses. An increase in actin stress fibers, focal adhesions, as well as very compact fibronectin assembly was noted around cells expressing RhoAV14. Activation of Rho GTPase in TM cells also led to significant increases in ERK phosphorylation. Treatment of TM cells with the ERK kinase inhibitor, U0126, significantly lowered the ECM synthesis and actin levels. On the other hand, TM cells treated with the Rho Kinase inhibitor, Y27632, or Lovastatin, showed significantly decreased ERK phosphorylation and ECM synthesis and assembly.
Put together these data demonstrate ERK activation as a checkpoint under persistent RhoA activation leading to cytoskeletal contraction, increased ECM synthesis and assembly in TM cells. Importantly, these data reveal mechanistic interactions between Rho GTPase activation, ECM synthesis and remodeling, and ERK activation in TM cells, and suggest that these signaling pathways collectively regulate resistance to aqueous humor outflow via the trabecular meshwork.
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