Purpose: To investigate the ability of insulin-like growth factor (IGF)-1 to prevent apoptosis in lens epithelial cells and the involvement of phosphatidylinositol 3-kinase (PI-3K)/Akt and PI-3K/p70 S6 kinase (p70 S6K) 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 ladder-like fragmentation of genomic DNA in agarose gels and the intense blue fluorescence exhibited by apoptotic nuclei of cells in live cultures in the presence of Hoechst 33,258 dye. Proliferation of lens epithelial cells grown in culture was measured with a DNA-binding fluorescent dye. Overexpression of the constitutively active Akt (CA-Akt) in epithelial cells was achieved by the transfection of cells using Fugene 6 reagent with a plasmid carrying Akt cDNA. Western immunoblotting was performed to identify various proteins of interest.
Results: IGF-1 (5 to 50 nM) and insulin (100 to 400 nM) suppressed lens epithelial cell apoptosis in a dose-dependent manner, as determined by a significant inhibition of genomic DNA fragmentation and the decreased number of intense blue fluorescent Hoechst stain-positive apoptotic nuclei in live cultures. DNA degradation was almost completely inhibited in the presence of 50 nM IGF-1 or 400 nM insulin. PI-3K inhibitors wortmannin and LY294002 blocked the IGF-1 effect on cell survival. Stimulation of lens epithelial cells with IGF-1 for 10 minutes to 24 hours resulted in the sustained activation of both Akt and p70 S6K. IGF-1 also induced the phosphorylation of Bad (a pro-apoptotic protein of the Bcl-2 family), which was inhibited by PI-3K inhibitors, but not by the p70 S6K inhibitor rapamycin. Furthermore, activation of Akt but not p70 S6K signaling by IGF-1 resulted in the inhibition of caspase-3 endogenous substrate poly (ADP-ribose) polymerase (PARP) degradation and apoptosis. The overexpression of CA-Akt in lens epithelial cells inhibited PARP breakdown and suppressed apoptosis. Inhibition of p70 S6K activation by rapamycin blocked IGF-1-promoted lens epithelial cell proliferation but not the cell-survival effect.
Conclusions: These studies demonstrated a role for IGF-1 in the prevention of the lens epithelial cell apoptosis process. Furthermore, these studies indicated that anti-apoptotic and proliferative signals from IGF-1 bifurcate downstream of PI-3K. Whereas IGF-1-mediated PI-3K/Akt signaling plays a pivotal role in cell survival by inactivating proapoptotic Bad protein and suppressing caspase activation, its stimulation of the PI-3K/p70 S6K cascade promotes proliferation.