Extracellular Matrix–Induced Myosin Light Chain Phosphorylation and Cell Adhesion in Trabecular Meshwork Cells

Abstract

Abstract: : Purpose: The extracellular matrix (ECM) of the aqueous humor outflow pathway is believed to be critical for maintenance of normal aqueous outflow through the trabecular meshwork (TM). The influence of ECM on TM cell physiology, however, is not thoroughly understood. The objective of this study was to investigate the effects of ECM proteins on contractile and adhesion properties of TM cells since these characteristics are thought to influence aqueous outflow facility. Methods: Serum starved porcine TM cells were tested for their responses to different ECM proteins including collagen IV, fibronectin and laminin, by evaluating cell morphology, actomyosin organization, focal adhesion formation and myosin light chan (MLC) phosphorylation. These studies were carried out using either glass coverslips or plastic petriplates precoated with different ECM proteins. To characterize the participation of different signaling pathways in the ECM–induced effects, serum starved porcine TM cells were pretreated with the inhibitors of Rho kinase (Y–27632), Protein Kinase C (GF109203X) and Src kinase (PP2) prior to evaluating cells for ECM–dependent changes in actomyosin organization, focal adhesions and MLC phosphorylation. Results: Fibronectin, laminin and collagen IV, in order of decreasing relative strength, were each noted to exert dramatic effects on cell morphology, actin stress fibers and focal adhesion formation in serum starved porcine TM cells. Additionally, these three different ECM proteins significantly increased MLC phosphorylation with the strongest response observed for fibronectin and laminin. While fibronectin–induced MLC phosphorylation was maximally suppressed by the Rho kinase inhibitor, laminin–induced MLC phosphorylation was maximally inhibited by the PKC inhibitor, with the Src kinase inhibitor revealing a moderate inhibition of both, fibronectin– and laminin–induced MLC phosphorylation. Conclusions:These data demonstrate that ECM proteins influence contractile properties, cell adhesions and actomyosin organization in TM cells, most likely via Rho kinase and PKC–dependent signaling pathways. This study suggests a potential mechanistic role for the ECM in modulation of aqueous humor outflow through the TM pathway.

Keywords: extracellular matrix • signal transduction • trabecular meshwork