Transfection of Myocilin Reduces Cell–Matrix Adhesiveness and Fibronectin Deposition in Human Trabecular Meshwork Cells

Abstract

Abstract: : Purpose: To assess the effects of myocilin overexpression on the cell–matrix adhesiveness and fibronectin deposition in human trabecular meshwork (TM) cells in culture. Myocilin is a gene linked to juvenile and chronic open angle glaucomas. Methods: Myocilin construct was transfected into TM cells. Cell adhesion assays were performed. The cohesiveness of cells to their underlying matrix was measured by the susceptibility to trypsin. The fibronectin deposition before and after treatment of lyosphosphatidic acid (LPA) was examined by immunofluorescence. The amount of fibronectin secreted was evaluated by Western blotting and the level of fibronectin mRNA was determined by relative quantitative RT–PCR. Activities of gelatinases were examined by zymography. Results: The myocilin transfected cells showed a dramatic loss of actin stress fibers and focal adhesions. The adhesion of myocilin transfectants to fibronectin, collagens and vitronectin was compromised and the trypsinization time needed to liberate cells from plates was shorter for myocilin transfectants compared to mock controls. The levels of both fibronectin protein and mRNA were reduced in myocilin transfectants but the activities of gelatinases were unaltered. The fibronectin deposition could be restored by treatment of LPA. Conclusions: These results indicated that myocilin has a de–adhesive activity, similar to that reported extensively with a group of matricellular proteins. The myocilin–mediated effects on actin structure and fibronectin deposition may involve signaling via the small GTPase Rho family.

Keywords: trabecular meshwork • gene/expression • cell adhesions/cell junctions