Purpose: To determine the mechanisms of action of phosphatidylinositol 3-kinase (PI3K) in lens cell differentiation and survival.
Methods: Primary quail lens cell cultures were treated at different stages of differentiation with the PI3K inhibitor LY294002, and expression of survival proteins and differentiation markers were determined by immunoblot analysis. The connection between PI3K regulation of lens differentiation and actin cytoskeleton reorganization was examined by fluorescent-phalloidin staining and Rac activity assay. Survival in the absence of PI3K signaling was examined by TUNEL and DAPI staining. Phosphorylation of the PI3K effector glycogen synthase kinase-3 (GSK3) in the absence of PI3K signaling was induced with lithium chloride.
Results: Exposure to LY294002 blocked lens epithelial cell differentiation initiation. This result was linked to attenuation of Rac activity and inhibition of actin filament reorganization from stress fibers to cortical fibers, which has been shown to signal lens differentiation initiation. The survival of lens epithelial cells in the absence of PI3K signaling correlated with induction of numerous survival factors, including Bcl-2. In contrast, inhibition of PI3K signaling in differentiating lens fiber cells induced apoptosis by blocking inactivation of GSK3, showing that PI3K/GSK3 signaling has a protective role in the late stages of differentiation as nuclei and organelles are lost.
Conclusions: PI3K signaling regulates lens cell differentiation initiation through its ability to signal reorganization of the actin cytoskeleton from stress fibers to cortical fibers. In differentiating lens fiber cells, PI3K has a protective function, signaling survival through inactivation of its downstream effector GSK3.