Abstract
Abstract: :
Purpose: Signaling mechanisms that regulate cell cycle progression/arrest could play a key role in determining the fate of lens epithelial cells to proliferate or differentiate. p21cip and p27kip are two important proteins that control the activity of cyclin–dependent kinase (CDK) and cyclin complexes during cell cycle. Studies from our laboratory show that PI–3K/Akt pathway modulates lens epithelial cell proliferation and differentiation. Here we investigated the regulatory role of PI–3K/Akt signaling on p21 and p27 expression during proliferation. Methods:Epithelial cells derived from rabbit lens capsules were grown in DMEM+10% FCS. To study the effect of IGF–1 or PI–3K inhibitors wortmannin (200 nM) or LY294002 (15 µM), cells were partially synchronized by serum deprivation and treated with desired factors. Overexpression of active Akt in epithelial cells was achieved by transfection of cells with a eukaryotic Akt cDNA plasmid. Cells were collected at different times (0–96 hr). Cell homogenates were analysed for various cell cycle components by immunoblotting and immunoprecipitation. Results:Significant differences in the kinetics of p21 and p27 expression were observed in exponentially growing lens epithelial cells. p21 protein level in resting cells or during early growth period (0–12 hr after seeding) was very low or barely detectable; its level increased by 3–4 fold at 24–48 hr and decreased thereafter. In contrast to p21, the level of p27 was very high in resting cells but decreased considerably between 0–48 hr, and elevated again at 72–96 hr when cell proliferation ceased. IGF–1, which enhanced lens cell proliferation, increased p21 and reduced the p27 expression between 24–48 hr. PI–3K pathway inhibitors wortmannin and LY294002 attenuated the effect of IGF–1 on p21 and p27 by more than 50%. Furthermore, overexpression of Akt in lens epithelial cells produced similar effects as IGF–1 on p21 and p27 expression, confirming the involvement of PI–3K/Akt signaling in their regulation. Expression of G1/S–phase components cyclin A and CDK2 in proliferating cells coincided with p21 expression. Immunoprecipitation experiments revealed the association of CDK2 with p21. Conclusions: Our studies suggest that in lens p21 positively regulates cell cycle progression, whereas up–regulation of p27 could lead to cell cycle arrest, a prerequisite for differentiation. Better understanding of these mechanisms may provide opportunities for potential therapies needed to suppress growth of epithelial cells that occurs during secondary cataract development.
Keywords: signal transduction • growth factors/growth factor receptors • proliferation