H2O2 Stimulates Phosphatidylinositol-3 Kinase (PI-3K) Signaling Through p70 S6 Kinase (p70 S6K) and Akt in Lens Epithelial Cells

Abstract

Abstract: : 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.

Keywords: 580 signal transduction • 592 stress response • 515 phosphorylation