Erk and PI-3 kinase are necessary for collagen binding and actin reorganization in corneal epithelia.

PURPOSE: It was recently shown that phosphatidylinositol-(PI)3 kinase is upregulated in wounded rabbit corneal epithelia. Extracellular signal-regulated kinase (erk)-1 and -2 proteins and PI-3 kinase were activated in embryonic corneal epithelia after 1-hour stimulation by type I collagen. In the current investigation specific inhibitors of PI-3 kinase and mitogen-activated kinase-kinase (MEK-1 kinase) were used to determine the role of these signaling molecules in actin reorganization and collagen binding to isolated sheets of corneal epithelial tissue. METHODS: Effects of specific PI-3 kinase and MEK-1 inhibitors (LY294002, PD98059, respectively) were investigated in embryonic corneal epithelial tissues. Avian embryonic corneal epithelia were isolated as tissue sheets, organ cultured in the presence of these specific inhibitors, and stimulated with type I collagen. The tissues were evaluated for collagen-stimulated actin reorganization, erk-1 and -2 and PI-3 kinase activity, total filamentous actin accumulation, and collagen binding. RESULTS: The MEK-1 inhibitor PD98059 decreased erk-1 and -2 phosphorylation and blocked actin reorganization in a dose-dependent manner. The PI-3 kinase 85-kDa subunit was decreased 25% in LY294002-treated tissue, and collagen binding also decreased significantly in tissues treated with MEK-1 and PI-3 kinase inhibitors compared with control tissues. In addition, both inhibitors blocked actin cortical mat reorganization. CONCLUSIONS; PI-3 kinase and erk-1 and -2 signaling pathways are activated and necessary for collagen binding and integrin-mediated actin reorganization in embryonic avian corneal epithelium.

Zyxin and vinculin distribution at the cell-extracellular matrix attachment complex (CMAX) in corneal epithelial tissue are actin dependent.

Avian embryonic corneal epithelia are two cell layers thick. If isolated without (-) basal lamina, the basal cells have unorganized actin and project cytoplasmic protrusions termed blebs. The actin-based cytoskeleton at the cell-extracellular matrix junction (termed the actin cortical mat) is disrupted. These epithelia respond to soluble extracellular matrix molecules by reorganizing the actin cortical mat. Sheets of epithelia were isolated + or -basal lamina. Epithelia isolated -basal lamina were cultured +/- laminin-1 and/or +/- cytochalasin D (CD). The intracellular localization of zyxin, vinculin, paxillin, focal adhesion kinase, and tensin was determined using indirect immunohistochemistry. Protein levels were determined by Western blot analysis. Zyxin and vinculin were concentrated in two areas of the tissue. The interface between the upper cell layer (periderm) and the basal cells. The second area of concentration was at the inferior 1-4 microns of the basal cells in an area with multiple actin bundles termed the actin cortical mat. The actin bundles align toward zyxin and vinculin that were located near basal lateral membranes. Zyxin was displaced from the basal compartment of blebbing basal cells. In contrast tensin, vinculin and focal adhesion kinase were found diffusely throughout the blebs. Zyxin and vinculin redistributed to the basal-lateral membranes as actin bundles reorganized in laminin-stimulated epithelia. In contrast to the altered protein distribution, extractable protein levels were similar in blebbing and laminin-stimulated epithelia. Zyxin, vinculin, and other associated proteins were disrupted in the CD-treated tissues and do not colocalize with each other or CD-induced actin aggregates. The intracellular localization of zyxin and vinculin were concentrated in distinct areas along the inferior basolateral membranes of basal cells termed the cell-extracellular matrix attachment complex (CMAX). The distribution of CMAX proteins was dependent upon actin bundle organization.

ECM-stimulated actin bundle formation in embryonic corneal epithelia is tyrosine phosphorylation dependent.

Previous studies demonstrated that corneal epithelial cells isolated without basal lamina respond to extracellular matrix (ECM) in an actin dependent manner; the basal cell surface flattens and the actin cortical mat reorganizes. We hypothesize that the actin reorganization is initiated by intracellular signaling mechanisms that includes tyrosine phoshporylation and activation of the Rho, MAP kinase, and PI3 kinase signal transduction pathways. Our goals were to develop a morphological assay to test this hypothesis by answering the following questions: 1) Do the actin bundle formations in the cortical mat have the same configuration in response to different ECM molecules? 2) What is the minimum time ECM molecules need to be in contact with the tissue for the actin to reorganize? 3) Will blocking tyrosine phosphorylation inhibit reorganization of the actin? 4) Are known signal transduction proteins phosphorylated in response to soluble matrix molecules? The actin cortical mat demonstrated distinct bundle configurations in the presence of different ECM molecules. Soluble fibronectin accumulated at the basal cell surfaces 75-fold over 30 min in a clustered pattern. The cells need contact with ECM for a minimum of 10 min to reform the actin bundles at 2 hr. In contrast, two substances that bind to heptahelical receptors to stimulate the Rho pathway, bombesin and lysophosphatidic acid, reorganized the actin bundles in 15-30 min. Focal adhesion kinase, p190 Rho-GAP, tensin, and paxillin were tyrosine phosphorylated in response to soluble fibronectin, type I collagen, or laminin 1. Erk-1, erk-2, and PI3 kinase were activated after 1 hr stimulation by type I collagen. Herbimycin A blocked actin reorganization induced by ECM molecules. In conclusion, we have developed two morphological assays to examine the response of corneal epithelial cells to ECM molecules. In addition, actin bundle reorganization involved tyrosine phosphorylation, MAP kinase, and PI3 kinase activation.