Knockdown of Akt Family Members Reveals Their Differentiation Functions against Stress-Induced Apoptosis Mediated by Various Signaling Pathways

Abstract

Purpose: : The Akt kinase signaling pathway plays a key role in regulating survival of ocular cells in both retina and lens. However, the relative importance of each member in regulating stress-induced apoptosis remains to be further elucidated. In the present study, we have knocked down expression of each individual isoform and explored their role against stress-induced apoptosis in both retinal pigment epithelial cells and human lens epithelial cells.

Methods: : The knockdown constructed obtained from Santa Cruz Biotechnology were introduced into both ARPE-19 cells and HLE cells. Stable clones were established under selection of purimycin and used for various experimentation. Western-blot analysis was conducted to confirm the knockdown. Live-dead and MTT assays were used to detect apoptosis. Western blot analysis and kinase assays were used to determine the expression and activity levels of various kinases and other signaling molecules.

Results: : Knockdown of Akt1 leads to upregulation of Akt2 and enhancement of the resistance against oxidative stress-induced apoptosis. Konckdown of Akt2, however, caused enhanced apoptosis. These results suggest that Akt2 plays a key in resisting oxidative stress-induced apoptosis.

Conclusions: : Akt2 is a major player responding to oxidative stress in the ocular tissues.

Keywords: signal transduction • oxidation/oxidative or free radical damage • apoptosis/cell death