Involvement of Phosphatidylinositol-3Kinase (PI-3K) in Lens Epithelial Cell Differentiation

Abstract

Abstract: : Purpose: Growth factors that bind to tyrosine kinase receptors promote lens epithelial cell proliferation and their differentiation. We have shown that insulin and IGF-1 stimulate PI-3K in lens epithelial cells and PI-3K inhibitors block the growth factor promoted cell proliferation (ARVO Abs No 4955, 2001). In this study we investigated whether PI-3K signaling regulates epithelial cell differentiation. Methods: Epithelial cells from 10-day-old chicken embryo lenses were isolated and cultured in DMEM supplemented with 10% FCS or with insulin (200 nM), IGF-1(50 nM), PDGF (40 ng/ml) and PI-3K inhibitors, wortmannin (200 nM) or LY294002 (20 µM). Synthesis of δ-crystallin protein in cultures was determined by metabolic labeling of the cells with [₃₅S]-methionine followed by SDS-PAGE and autoradiography. PI-3K activity was assayed after immunoprecipitation in the presence of phosphatidylinositol and [ γ₃₂P]-ATP. Activation of the Akt was determined by immunoblotting with anti-phospho Akt antibody. Results: Stimulation of the chicken lens cultures for 10 min with insulin, IGF-1, and PDGF increased the PI-3K activity. The presence of wortmannin and LY294002 for 48-72 h in cultures resulted in about 40-50% increase in the synthesis of δ-crystallin (the chicken lens differentiation marker protein) when compared to untreated cultures. The ability of insulin and IGF-1 to activate the PI-3K downstream target, Akt, was also changed significantly as the differentiation progressed. In the early cultures without any lentoid bodies, IGF-1 induced the phosphorylation of Akt by about 10 fold, whereas in cultures that started developing lentoids Akt phosphorylation decreased to 5 fold. In cultures with extensively spread lentoids the Akt phosphorylation was 1- 2 fold. Conclusion: Our current and previous studies indicate that proliferation and differentiation processes are regulated by PI-3K. While inhibition of PI-3K signaling induces the differentiation, its activation by growth factors promotes the proliferation. Akt is known to protect the cells from apoptosis. Decreased Akt activation during differentiation correlates with the current view that cellular events that take place during lens epithelial differentiation are similar to programmed cell death/apoptosis process.

Keywords: 423 growth factors/growth factor receptors • 580 signal transduction • 515 phosphorylation