Purchase this article with an account.
D Sailaja, G Chandrasekher, HE P Bazan; H2O2 Stimulates Phosphatidylinositol-3 Kinase (PI-3K) Signaling Through p70 S6 Kinase (p70 S6K) and Akt in Lens Epithelial Cells . Invest. Ophthalmol. Vis. Sci. 2002;43(13):2371.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
Purpose: Studies from our laboratory indicate that in lens epithelial cells PI-3K plays an important role in growth factor-mediated cellular processes such as proliferation and differentiation. Reactive oxygen species such as superoxides and H2O2 have been shown to serve as second messengers and mimic growth factor-induced stimulation of various signaling pathways in a wide variety of cell types. However, in lens H2O2 is considered to be involved in cataract formation. In the current study we have investigated the effect of H2O2 on PI-3K signaling, cell proliferation, and cell survival. Methods: Confluent cultures of rabbit lens epithelial cells were treated with 0-200 µM H2O2 or insulin (100 nM), IGF-1 (50 nM) and PDGF (40 ng/ml) at 37 C for different time periods. PI-3K (p85 subunit), phosphorylated forms of p70 S6K, and Akt proteins were identified by immunoblotting using specific antibodies. PI-3K activity after immunoprecipitation was assayed using phosphatidylinositol as substrate and [γ 32P]-ATP. Cell proliferation was measured by estimating the DNA content with fluorescent dye CyQuant GR. Apoptotic cells in cultures were idenfied by staining with Hoechst 33342 reagent. Results: Treatment of epithelial cells with H2O2 resulted in the activation of both p70 S6K and Akt, the downstream targets of PI-3K. The characteristic shift in p70S6K protein band migration due to phosphorylation was observed as early as 10 min and maximum shift was observed at 30 min. A 5-to-6 fold increase in phospho-Akt occured between 30-60 min. Phosphorylation of p70 S6K and Akt proteins decreased after 60 min but persisted even at 4 and 24 h. PI-3K activity was stimulated by 2-3 fold after 10-30 min. Stimulation with the growth factors also resulted in the activation of p70 S6K and Akt. Specific inhibitors of PI-3K, wortmannin (200 nM) and LY294002 (10 µM), blocked the H2O2-induced activation of p70 S6K and Akt. Presence of H2O2 for 24-48 h in proliferating cultures did not result in increased proliferation. No significant Hoechst 33342 positive staining for apoptotic cells was observed in H2O2treated cells after 24h. Conclusion: The current studies show that like growth factors, H2O2 also induces the activation of p70 S6K (a proliferation factor) and Akt (an anti-apoptotic factor) through PI-3K but it does not significantly affect cellular events such as proliferation nor cause apoptosis of lens epithelial cells. These results suggest that at moderate levels H2O2 could benefit the lens by protecting the epithelial layer.
This PDF is available to Subscribers Only