HepII Domain of Fibronectin Affects Focal Adhesion Formation in Undifferentiated Compared to Differentiated Human Trabecular Meshwork Cells

Abstract

Abstract: : Purpose: Determine the effect of the Heparin (Hep) II domain of fibronectin (FN) on focal adhesion formation in Human Trabecular Meshwork (HTM) cells. Methods: Undifferentiated HTM (uHTM), differentiated HTM (dHTM) and immortalized TM-1 cells were serum starved for 24h. Cells were trypsinized and cycloheximide treated in suspension for 1h in the presence or absence of recombinant HepII domains. Cells were plated for 3h (uHTM) or 5h (dHTM, TM-1) on coverslips precoated with 10nM FN. Cells were also incubated with HepII domains containing mutations in the heparin (PRARI) or α4ß1 integrin (IDAPS) binding site in the type III₁₄ repeat. HepII mediated signaling pathways were determined by incubating cells for 2h with the PKC inhibitor H-7 (6µM IC₅₀) or the Rho kinase inhibitor Y-27632 (5µM), 1h (uHTM) or 3h (dHTM, TM-1) after attachment. FA formation was determined by immunofluorescence microscopy by staining cells permeabolized and fixed (0.5% TX-100, 4% paraformaldehyde) with an anti-vinculin antibody followed by secondary antibody. Actin stress fibers were visualized using phalloidin conjugated to Alexa 488. Results: uHTM cells plated on FN in the absence of the HepII domain made few FAs and had a bipolar morphology. In contrast, cells incubated with the HepII domain were well spread and assembled extensive FAs. This differs from dHTM and TM-1 cells, which were able to make extensive FAs on FN in the absence of the HepII domain. Incubation with HepII domain did not increase or disrupt FAs in dHTM and TM-1 cells. Mutating the PRARI sequence did not significantly inhibit FA formation in uHTM cells, but mutating the α4ß1 site abolished the ability of HepII domain to induce FAs. FA formation in uHTM cells may be independent of the PKC signaling pathway, but dependent on Rho kinase. H-7 did not significantly decrease FAs in uHTM cells incubated with HepII domain, whereas Y-27632 caused a decrease in FAs. In contrast, both H-7 and Y-27632 caused a decrease in FAs in dHTM and TM-1 cells. Conclusion: The HepII domain of FN affects the formation of focal adhesions and stress fibers in uHTM cells plated on FN, but not dHTM cells indicating that the HepII domain may have different signaling roles in TM cells depending on their differentiation state. Mutational analysis suggests the HepII domain may be mediating this effect in uHTM cells through an α4ß1 signaling pathway that may be independent of PKC, but dependent on Rho kinase. In contrast, dHTM cells utilize a PKC and Rho kinase dependent pathway to assemble FAs.

Keywords: 403 extracellular matrix • 339 cell adhesions/cell junctions • 580 signal transduction