Abstract
Abstract: :
Purpose:Factors such as radiation, chemicals and oxidative stress, which induce cataract, are known to cause apoptosis. Here we investigated the role of IGF–1 in preventing apoptosis in lens epithelial cells and involvement of PI–3K/Akt signaling in the cell–survival process. Methods: Apoptosis in rabbit lens epithelial cell cultures was induced by staurosporine (10 ng/ml). Cellular apoptosis was detected by identifying the characteristic ladde–like fragmentation of DNA in agarose gels, and intense blue fluorescence with Hoechst 33258 exhibited by nuclei of live cells. Cell proliferation was measured with a DNA binding fluorescent dye, CyQuant GR. Overexpression of constitutively active Akt (cAkt) in epithelial cells was achieved by the transfection of cells with an eukaryotic plasmid carrying Akt cDNA using Fugene 6. Immunoblotting was performed to identify proteins. Results: IGF–1 (5–50 nM) and insulin (100–400 nM) suppressed staurosporine–induced apoptosis in a dose–and time–dependent manner as evidenced by a significant decrease in DNA fragmentation and number of intense blue fluorescent Hoechst stain–positive nuclei in live cultures. DNA degradation was almost completely inhibited in the presence of 50 nM IGF–1. PI–3K inhibitors wortmannin (200 nM) and LY294002 (10 µM) blocked IGF–1–mediated rescue of cells. Treatment of epithelial cells with IGF–1 for 10 min to 24 h resulted in the sustained activation of PI–3K downstream kinases Akt and p70 S6 kinase (p70 S6K). IGF–1 also induced the phosphorylation and inactivation of Bad, a proapoptotic member of the Bcl–2 family. While rapamycin, the specific inhibitor of p70 S6K, reduced IGF–1–promoted lens epithelial cell proliferation, its presence showed no significant effect on the cell–survival function of IGF–1. Further, wortmannin and LY294002, but not rapamycin, blocked the IGF–1–mediated phosphorylation of Bad and suppressed the degradation of caspase–3 substrate protein poly(ADP–ribose) polymerase–1 (PARP–1) during apoptosis. Lens epithelial cells overexpressing cAkt were not susceptible to apoptosis, and PARP–1 degradation did not occur in these cells in the presence of staurosporine. Conclusions: Our studies demonstrate that IGF–1 is very potent in preventing apoptosis of lens epithelial cells and plays an important role in epithelial cell survival through the activation of the PI–3K/Akt/Bad signaling pathway while promoting cell proliferation through PI–3K/p70 S6K signaling. Akt is a pivotal component in the cell–survival mechanism initiated by PI–3K activation.
Keywords: growth factors/growth factor receptors • apoptosis/cell death • signal transduction