Abstract
Abstract: :
Purpose: Lysophosphatidic acid (LPA) is a phospholipid growth factor released into the corneal microenvironment in response to injury. It typically stimulates cell signaling through LPA receptors coupled to Gαi, Gαq, and Gα12/13 G–proteins, leading to cell migration, survival, and proliferation. In stromal keratocytes these activities contribute towards stromal remodeling in response to corneal injury. In this study, we have investigated MLC phosphorylation (i.e., regulatory light chain of myosin II), critical for cell migration and cytokinesis, in corneal fibroblasts exposed to LPA. Methods: Experiments were conducted with cultured bovine stromal fibroblasts. Cells, grown to confluence on Petri dishes and serum starved for 12–16 hrs, were challenged with LPA with and without concomitant exposure to the rho kinase inhibitor, Y–27632 (10 µM). Protein extracts were assayed for MLC phosphorylation in two steps: (a) separation of phosphorylated and unphosphorylated forms by urea–glycerol gel electrophoresis, and (b) their subsequent identification by immunoblotting. As a positive control for MLC phosphorylation, cells were exposed to histamine (100 µM). Results: (1) LPA (10 µM) induced MLC phosphorylation (28.2 + 5.9%, n = 6) within 10 min of exposure. Y–27632 (10 µM) blocked basal MLC phosphorylation by 79.5 + 3.9% (n=4). LPA–induced phosphorylation was also blocked by Y–27632 (69 + 2.7% ; n=4). Exposure to histamine also induced MLC phosphorylation within 2 min (34%; n = 3). Conclusions: LPA induces MLC phosphorylation consistent with LPA receptor activation of Gα12/13, which are typically associated with rho activation. This phosphorylation may be important to cytokinesis and migration of keratocytes.
Keywords: cornea: stroma and keratocytes • cytokines/chemokines • growth factors/growth factor receptors