Reactive Oxygen Species Mediate the Activation of Akt/Protein Kinase B by Angiotensin II in Vascular Smooth Muscle Cells

doi:10.1074/jbc.274.32.22699

Reactive Oxygen Species Mediate the Activation of Akt/Protein Kinase B by Angiotensin II in Vascular Smooth Muscle Cells*

From the ^‡Department of Medicine, Division of Cardiology, Emory University, Atlanta, Georgia 30322 and the ^¶Division of Cardiovascular Research, St. Elizabeth’s Medical Center and Tufts University School of Medicine, Boston, Massachusetts 02135

Abstract

Angiotensin II, a hypertrophic/anti-apoptotic hormone, utilizes reactive oxygen species (ROS) as growth-related signaling molecules in vascular smooth muscle cells (VSMCs). Recently, the cell survival protein kinase Akt/protein kinase B (PKB) was proposed to be involved in protein synthesis. Here we show that angiotensin II causes rapid phosphorylation of Akt/PKB (6- ± 0.4-fold increase). Exogenous H₂O₂ (50–200 μm) also stimulates Akt/PKB phosphorylation (maximal 8- ± 0.2-fold increase), suggesting that Akt/PKB activation is redox-sensitive. Both angiotensin II and H₂O₂stimulation of Akt/PKB are abrogated by the phosphatidylinositol 3-kinase (PI3-K) inhibitors wortmannin and LY294002 (2(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one), suggesting that PI3-K is an upstream mediator of Akt/PKB activation in VSMCs. Furthermore, diphenylene iodonium, an inhibitor of flavin-containing oxidases, or overexpression of catalase to block angiotensin II-induced intracellular H₂O₂production significantly inhibits angiotensin II-induced Akt/PKB phosphorylation, indicating a role for ROS in agonist-induced Akt/PKB activation. In VSMCs infected with dominant-negative Akt/PKB, angiotensin II-stimulated [³H]leucine incorporation is attenuated. Thus, our studies indicate that Akt/PKB is part of the remarkable spectrum of angiotensin II signaling pathways and provide insight into the highly organized signaling mechanisms coordinated by ROS, which mediate the hypertrophic response to angiotensin II in VSMCs.

Footnotes

↵* This work was supported by National Institutes of Health Grants HL38206, HL60728, and HL58000.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵§ To whom correspondence should be addressed: Div. of Cardiology, Emory University School of Medicine, 1639 Pierce Dr., Rm. 319, Atlanta, GA 30322. Tel: 404-727-8142; Fax: 404-727-3330; E-mail: mfukai@emory.edu.
↵2 M. Ushio-Fukai, R. W. Alexander, and K. K. Griendling, unpublished observations.
Abbreviations:

ROS

reactive oxygen species

VSMCs

vascular smooth muscle cells

Ang II

angiotensin II

PKB

protein kinase B

PI3-K

phosphatidylinositol 3-kinase

DCF-DA

2′,7′-dichlorofluorescein diacetate

LY294002

2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one)

DPI

diphenylene iodonium

DMEM

Dulbecco’s modified Eagle’s medium

HA

hemagglutinin

Ad-HA-Akt(AA)

dominant-negative HA-tagged double alanine mutant of Akt/PKB in adenoviral vector

Ad-β-Gal

bacterial β-galactosidase in adenoviral vector

MOI

multiplicity of infection

GSK-3

glycogen synthase kinase-3

p38^MAPK

p38 mitogen-activated protein kinase

PDK

phosphatidylinositol-dependent kinase

MOPS

4-morpholinepropanesulfonic acid
- Received January 25, 1999.
- Revision received April 14, 1999.
The American Society for Biochemistry and Molecular Biology, Inc.