Abstract
Abstract: :
Purpose: Lens epithelial cell migration is an important event during lens development and growth. While actomyosin dynamics and cell–ECM interactions are critical to cell migration, very little is known, regarding the regulation of cell migration in the ocular lens. Since myosin light chain (MLC) phosphorylation plays an important role in the regulation of actomyosin and cell–ECM interactions, we have addressed the regulation of MLC phosphorylation in lens and in lens epithelial cells in this study. Methods: Distribution of MLC in micro–dissected layers from porcine lens and cryo–sectioned mouse lenses (1–, 3–, 7–day old) were analyzed by Western blot analysis and immuofluorescence, respectively, using polyclonal antibody against phospho–MLC. Serum–starved porcine primary lens epithelial cells were treated with EGF, FGF, TGF–ß, LPA or TNF–α, followed by the determination of MLC phosphorylation status by Western blot analysis. Selective pharmacological inhibitors of Rho kinase (Y–27632), MLC kinase (ML–7) and PKC (GF109203X), were used to assess the potential involvement of these kinases in growth factor–mediated regulation of MLC phosphorylation. Mouse lens organ culture experiments were performed to explore the role of Rho kinase in MLC phosphorylation and in the maintenance of lens transparency. Results: Immunofluorescence analysis showed positive staining for MLC throughout the mouse lens (days 1–7), with intense staining in the posterior lens fibers attached to the lens capsule. Porcine lens epithelium and outer and inner cortical regions contained readily detectable levels of MLC. Growth factor treatment of porcine lens epithelial cells stimulated clear increases in MLC phosphorylation, with TGF–ß and LPA exerting stronger effects relative to EGF, b–FGF and TNF–α. Y–27632 (10 µM) caused a complete inhibition of MLC phosphorylation, while the MLCK inhibitor–ML–7 (50 µM) yielded a noticeable decrease in the basal level of MLC phosphorylation. The PKC inhibitor–GF–109203X (50 µM) was without effect. Y–27632 also blocked growth factor–induced stimulation of MLC phosphorylation in lens epithelial cells. In preliminary studies, treatment of organ cultured lenses with 25 µM Y–27632, led to the development of sutural nuclear opacity. This latter effect was associated with decreased basal levels of MLC phosphorylation. Conclusions: This data demonstrates a potential role for Rho kinase in regulating growth factor–stimulated MLC phosphorylation in the lens, and indicate the importance of MLC phosphorylation in maintaining lens function.
Keywords: growth factors/growth factor receptors • cytoskeleton • cataract