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Epidermal growth factor receptor-dependent Akt activation by oxidative stress enhances cell survival
X Wang1, K D McCullough, T F Franke
1Cell Stress and Aging Section, Laboratory of Biological Chemistry, NIA, National Institutes of Health, Baltimore, Maryland 21224-6825, USA.
Abstract:
The serine/threonine kinase Akt (also known as protein kinase B) is activated in response to various stimuli by a mechanism involving phosphoinositide 3-kinase (PI3-K). Akt provides a survival signal that protects cells from apoptosis induced by growth factor withdrawal, but its function in other forms of stress is less clear. Here we investigated the role of PI3-K/Akt during the cellular response to oxidant injury. H(2)O(2) treatment elevated Akt activity in multiple cell types in a time- (5-30 min) and dose (400 microM-2 mm)-dependent manner. Expression of a dominant negative mutant of p85 (regulatory component of PI3-K) and treatment with inhibitors of PI3-K (wortmannin and LY294002) prevented H(2)O(2)-induced Akt activation. Akt activation by H(2)O(2) also depended on epidermal growth factor receptor (EGFR) signaling; H(2)O(2) treatment led to EGFR phosphorylation, and inhibition of EGFR activation prevented Akt activation by H(2)O(2). As H(2)O(2) causes apoptosis of HeLa cells, we investigated whether alterations of PI3-K/Akt signaling would affect this response. Wortmannin and LY294002 treatment significantly enhanced H(2)O(2)-induced apoptosis, whereas expression of exogenous myristoylated Akt (an activated form) inhibited cell death. Constitutive expression of v-Akt likewise enhanced survival of H(2)O(2)-treated NIH3T3 cells. These results suggest that H(2)O(2) activates Akt via an EGFR/PI3-K-dependent pathway and that elevated Akt activity confers protection against oxidative stress-induced apoptosis.
Insights
Hydrogen peroxide (H2O2) activates the Akt pathway through epidermal growth factor receptor (EGFR) and phosphoinositide 3-kinase (PI3-K). This activation protects cells from oxidative stress-induced apoptosis.
Area of Science:
- Cellular Biology
- Molecular Biology
- Biochemistry
Background:
- The serine/threonine kinase Akt (protein kinase B) is crucial for cell survival signaling, particularly against apoptosis induced by growth factor withdrawal.
- Its role in cellular responses to other stressors, such as oxidant injury, remains less understood.
Purpose of the Study:
- To investigate the involvement of the phosphoinositide 3-kinase (PI3-K)/Akt pathway in the cellular response to hydrogen peroxide (H2O2)-induced oxidant injury.
- To elucidate the signaling mechanisms, including the role of epidermal growth factor receptor (EGFR), upstream of Akt activation by H2O2.
- To determine the impact of modulating PI3-K/Akt signaling on H2O2-induced apoptosis.
Main Methods:
- Treatment of various cell types with H2O2 to assess Akt activity.
- Utilized dominant-negative mutants of p85 (PI3-K regulatory subunit) and PI3-K inhibitors (wortmannin, LY294002) to block PI3-K/Akt signaling.
- Investigated EGFR phosphorylation and its role in H2O2-induced Akt activation.
- Assessed apoptosis in HeLa cells and cell survival in NIH3T3 cells under H2O2 treatment with altered PI3-K/Akt signaling.
Main Results:
- H2O2 treatment dose- and time-dependently elevated Akt activity in multiple cell types.
- Inhibition of PI3-K (using dominant-negative p85, wortmannin, or LY294002) prevented H2O2-induced Akt activation.
- H2O2-induced Akt activation was dependent on EGFR signaling, as evidenced by EGFR phosphorylation and prevention of Akt activation upon EGFR inhibition.
- Inhibition of PI3-K/Akt signaling significantly enhanced H2O2-induced apoptosis, while expression of activated Akt conferred protection against oxidative stress-induced cell death.
Conclusions:
- Hydrogen peroxide activates the Akt pathway through an EGFR and PI3-K-dependent mechanism.
- Elevated Akt activity provides a protective effect against apoptosis induced by oxidative stress.
- The PI3-K/Akt pathway is a critical mediator of cellular survival in response to H2O2-induced damage.