Effects of chemical inhibition of N-WASP, a critical regulator of actin polymerization on aqueous humor outflow through the conventional pathway.

The integrity of actin cytoskeletal organization in aqueous humor outflow pathway is thought to play a critical role in modulation of aqueous humor outflow through the trabecular meshwork. Our understanding of the regulation of actin cytoskeletal dynamics in outflow pathway, however, is very limited. To explore the potential importance of Neural Wiskott-Aldrich syndrome protein (N-WASP), a critical regulator of actin polymerization/nucleation in aqueous humor outflow pathway, the effects of Wiskostatin, a selective pharmacological inhibitor of N-WASP, on aqueous humor outflow facility were evaluated using enucleated porcine eyes and a constant pressure perfusion system. Further, drug induced effects on actin cytoskeletal organization, cell adhesions, myosin II phosphorylation, matrix metalloproteinase (MMP) activity, and cytoskeletal protein profile in porcine trabecular meshwork (TM) cells were determined by immunofluorescence, zymography, and mass spectrometry. Aqueous humor outflow facility was increased significantly and progressively in the Wiskostatin perfused porcine eyes. The Wiskostatin perfused eyes appear to exhibit increased giant vacuoles in the inner wall of aqueous plexi and deformation of aqueous plexi. The Wiskostatin treated TM cells demonstrated extensive vacuoles in their cytosol, and both actin stress fibers and focal adhesions were decreased in a reversible manner. The drug-treated TM cells also revealed decreased myosin II and actin in the cytoskeletal enriched triton insoluble fraction but did not affect myosin II phosphorylation or MMP-2 activity. These data demonstrate that the chemical inhibition of N-WASP increases aqueous humor outflow facility in association with decreased actomyosin interaction and cell adhesive interactions revealing the importance of N-WASP in homeostasis of aqueous humor outflow.

The role of protein kinase C in modulation of aqueous humor outflow facility.

The elevated intraocular pressure that is commonly associated with glaucoma is believed to arise due to impairment of trabecular meshwork (TM) function. Although the TM and Schlemm's canal (SC) comprise the major route for aqueous humor outflow, little is known about the potential signaling mechanisms involved in the regulation of aqueous outflow. Based on knowledge regarding the role of protein kinase C (PKC) in vascular biology, we sought to understand the contribution of the PKC pathway towards outflow function by studying the modulation of contractile and morphological characteristics of TM and SC cells. We investigated the involvement of PKC in regulation of myosin light chain (MLC) phosphorylation, formation of actin stress fibers and integrin-ECM adhesions (focal adhesions) in human TM and SC cells and correlated these changes with aqueous outflow facility measured in an enucleated porcine whole eye perfusion model. Expression and distribution of PKC isoforms (alpha and epsilon ) in TM and SC cells and tissues was confirmed by Western blot and immunohistochemical analysis, respectively. Both, pharmacological activators (phorbol-12-myristate 13-acetate (PMA) and phorbol-12,13-dibutyrate (PDBu)) and inhibitors (staurosporine and GF109203X) of PKC were found to induce changes in cell shape (retraction and rounding up) and cytoskeletal organization in human TM and SC cells. While PMA and PDBu produced an increase in formation of actin stress fibers and focal adhesions and in MLC phosphorylation, PKC inhibitors were observed to induce contrasting effects in these cells. Intriguingly, both PDBU and GF109203X caused increases in aqueous outflow facility in the perfusion model. The PKC inhibitor (GF109203X) increased outflow by 46% while the PKC activator (PDBu) only increased outflow by 27%. These results suggest that PKC might play an important role in modulation of aqueous outflow facility by regulating MLC phosphorylation and thereby, the morphological and cytoskeletal characteristics of TM and SC cells.

Inhibition of Rho-kinase induces alphaB-crystallin expression in lens epithelial cells.

The small heat shock protein, alphaB-crystallin, has been shown to interact with actin and intermediate filament proteins. However, little is known regarding the cellular mechanisms regulating such interactions. In this study, we explored the role of the Rho/Rho-kinase pathway in alphaB-crystallin distribution and expression in porcine lens epithelial cells. alphaB-crystallin was distributed uniformly throughout the cytoplasm and did not exhibit any unique redistribution in response to actin depolymerization induced by Rho/Rho-kinase inhibitors (C3-exoenzyme or Y-27632) or by overexpression of the dominant negative mutant of Rho-kinase (DNRK) in porcine lens epithelial cells. Interestingly, alphaB-crystallin levels markedly increased in lens epithelial cells treated with the inhibitors of Rho/Rho-kinase proteins (lovastatin, Y-27632 or DNRK) while a protein kinase C inhibitor (GF109203x) was found to have no effect. Further, Y-27632 showed a dose (2-50 microM) response effect on alphaB-crystallin induction. Nocodazole, a microtubule-depolymerizing agent, elicited an increase in alphaB-crystallin levels but latrunculin, an actin depolymerizing agent, did not show any significant effect. Pretreatment with cycloheximide or genistein blocked the Rho-kinase inhibitor-induced increase in alphaB-crystallin protein levels. Rho-kinase inhibitor-induced increases in alphaB-crystallin levels were found to be associated with activation of P38 mitogen-activated protein kinase (MAPK). These results suggest that Rho/Rho-kinase negatively regulates alphaB-crystallin expression, and this response appears to be dependent on tyrosine-protein kinase and P38 MAPK function. Finally, alphaB-crystallin induction appears to be better correlated with the direct inhibition of Rho/Rho-kinase than with actin depolymerization per se.