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

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.

Ligand-receptor binding has been demonstrated to induce production of ROS (2), and antioxidants block some aspects of receptor-coupled signal transduction (3,4), suggesting that ROS participate in transmission of the receptor signal to induce biological responses. In particular, in vascular smooth muscle cells (VSMCs), ROS have been shown to play an important role in regulating cell growth. We have previously reported that the peptide hormone angiotensin II (Ang II), which acts on G protein-coupled AT 1 receptors (5), induces a rapid increase in intracellular H 2 O 2 that is involved in its hypertrophic response (6). Similar results have been found for platelet-derived growth factor-induced cell proliferation, which was shown to be dependent on H 2 O 2 (3) .
In addition to activating mitogenic signals, growth factors stimulate anti-apoptotic/cell survival pathways, providing a delicate homeostatic balance between cell proliferation and cell death. Although many of the mechanisms involved in proliferation have been defined, much less is known about the signaling events leading to cell survival. Antioxidants have been shown to induce apoptosis in VSMCs (7), suggesting that the biochemical pathways involved in cell survival are additional potential targets for ROS. Growing evidence indicates that the serine-threonine kinase Akt/protein kinase B (PKB) is a critical enzyme in a cell survival pathway that protects cells from apoptosis (8). Akt/PKB can be activated by a wide variety of growth stimuli, including platelet-derived growth factor, epidermal growth factor, insulin, thrombin, and nerve growth factor (8). Recent work has shown that Akt/PKB is not only a cell survival kinase but may play an important role in protein synthesis, a crucial event in the hypertrophic response (9,10).
Upstream signaling pathways leading to Akt/PKB activation include phosphatidylinositol 3-kinase (PI3-K) and Ras (11). However, cellular stresses such as heat shock and hyperosmolarity (12) stimulate Akt/PKB through a pathway independent of PI3-K (13). The hypertrophic agent Ang II activates both PI3-K and production of ROS in VSMCs (4,14), but its ability to activate Akt/PKB and the relationship of PI3-K and ROS to Akt/PKB activation have not been investigated. We have previously shown that Ang II induces rapid production of superoxide and H 2 O 2 and that ROS are required for VSMC hypertrophy (4). These observations raise the possibility that Akt/ PKB is also activated by Ang II in a redox-sensitive manner and may be involved in the hypertrophic response. We show here that Akt/PKB is robustly stimulated by both Ang II and exogenous H 2 O 2 , via PI3-K-dependent mechanisms, and that Ang II-induced Akt/PKB activation is mediated through an increase in intracellular H 2 O 2 . Furthermore, Akt/PKB appears to play a significant role in hypertrophy. These results provide insight into the selective and highly organized signaling mech-anisms coordinated by ROS that mediate the hypertrophic response to Ang II in VSMCs.
Cell Culture-VSMCs were isolated from male Harlan Sprague-Dawley rat thoracic aortas by enzymatic digestion as described previously (15). Cells were grown in DMEM supplemented with 10% calf serum, 2 mM glutamine, 100 units/ml penicillin, and 100 g/ml streptomycin and were passaged twice a week by harvesting with trypsin:EDTA and seeding into 75-cm 2 flasks. For experiments, cells between passages 6 and 19 were used at confluence.
In some experiments, we used VSMCs that had been stably transfected with human catalase. In these cells, catalase mRNA and protein expression are significantly increased (4,6). Transfected cells were maintained in selection medium until they were plated into 35-or 100-mm dishes for experiments.
Detection of Akt/PKB Phosphorylation by Immunoblotting-VSMCs at 80 -90% confluence in 100-mm dishes were made quiescent by incubation with DMEM containing 0.1% calf serum for 24 h. Cells were stimulated with agonist at 37°C in serum-free DMEM for specified durations. After treatment, cells were lysed with 500 l of ice-cold lysis buffer, pH 7.4 ((mM) 50 HEPES, 5 EDTA, 50 NaCl), 1% Triton X-100, protease inhibitors (10 g/ml aprotinin, 1 mM phenylmethylsulfonyl fluoride, 10 g/ml leupeptin), and phosphatase inhibitors ((mM) 50 sodium fluoride, 1 sodium orthovanadate, 10 sodium pyrophosphate, 0.001 microcystin). Solubilized proteins were centrifuged at 14,000 ϫ g in a microfuge (4°C) for 30 min, and supernatant protein was quantified by the Bradford assay. Proteins (25 g) were separated using 9% SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. Membranes were blocked overnight at room temperature with phosphate-buffered saline containing 6% nonfat dry milk and 0.1% Tween 20. Blots were incubated with primary rabbit polyclonal phosphospecific Akt/PKB antibody (detects Akt/PKB only when activated by phosphorylation on Ser 473 ) at 1:1000. After incubation with secondary antibodies (horseradish peroxidase-conjugated goat anti-rabbit antibody, 1:2000), phosphorylated forms of proteins were detected by enhanced chemiluminescence. Band intensity was quantified by densitometry of immunoblots using NIH Image, version 1.61. Phosphorylation of Akt/PKB on Ser 473 is required for activation (16); therefore, phosphorylation at this site was routinely taken as a measure of Akt/ PKB enzymatic activity. In some experiments, Akt/PKB activity was verified directly in Akt/PKB immunoprecipitates.
The kinase reaction was carried out by incubating the beads in 50 l of kinase buffer containing 10 Ci [␥-32 P]ATP, 50 M ATP, 7.5 mM MgCl 2 , and 2 g of histone 2B for 30 min at 30°C. Anti-Akt/PKB immunoprecipitates were subjected to 15% SDS-polyacrylamide gel electrophoresis, and 32 P-labeled histone 2B was detected using a Phosphor-Imager and quantified by densitometry using NIH Image, version 1.61. In some experiments, GSK-3 fusion protein (1 g) was used as the substrate. Radiolabeled ATP was omitted from the reaction, and antiphospho-GSK-3 antibody was used to detect phosphorylated GSK-3.
Measurement of Intracellular H 2 O 2 Levels-H 2 O 2 levels were measured using the peroxide-sensitive fluorophore DCF-DA (5 M) as de-scribed previously (4,6). Although DCF-DA is oxidized by H 2 O 2 as well as other peroxides, the complete inhibition of fluorescence in Ang IIstimulated cells by the addition of catalase (350 units/ml) (data not shown) and by catalase overexpression (4) indicates that the fluorescence signal evoked by Ang II was derived predominantly from H 2 O 2 .
Construction of Dominant-Negative Akt/PKB Adenovirus and Infection of VSMCs-pcDNA HA-Akt(AA) was a kind gift from Dr. J. R. Testa (Fox Chase Cancer Center). HA-Akt(AA) is a cDNA encoding mouse Akt/PKB containing alanine mutations in the regulatory site (Thr 308 and Ser 473 ) fused to the hemagglutinin (HA) epitope (16). HA-Akt(AA) was inserted into the EcoRI/XbaI site of the pACCMVpLpA plasmid. pACCMVpLpA-HA-Akt(AA) was cotransfected into 293 cells with a vector modified from the Ad5 genome, which has a 4.3-kilobase PBRx insert and confers resistance to tetracyclin and ampicillin (pJM17). The resulting replication-defective recombinant adenoviruses were purified from isolated plaques and amplified in 293 cells. Viral preparations were purified by two CsCl gradient centrifugations as described previously (17) [ 3 H]Leucine Incorporation-To measure hypertrophy of VSMCs, cells were quiesced for 48 h in DMEM containing 0.1% calf serum. Cells were incubated with [ 3 H]leucine (0.5 Ci/ml) in the presence or absence of 100 nM Ang II for an additional 24 h, and [ 3 H]leucine incorporation was measured as described previously (6).
Statistical Analysis-Results are expressed as mean Ϯ S.E. Statistical significance was assessed by Student's unpaired two-tailed t test on untransformed data. A p value of Ͻ0.05 was considered to be statistically significant.  (Fig. 1A). Akt/PKB activation was still detectable at 60 min. H 2 O 2 -induced Akt/PKB phosphorylation was dose-de-pendent, with a threshold between 50 -100 M and a maximal effect occurring at 200 M (Fig. 1B). These concentrations are similar to those previously reported for H 2 O 2 -stimulated proliferation and p38 mitogen-activated protein kinase (p38 MAPK ) activation in VSMCs (4,18). These data suggest that Akt/PKB is a target of ROS in VSMCs.

Effect of
Ang II, an important hypertrophic/anti-apoptotic vasoactive substance, has been shown to utilize ROS as signaling molecules (4). Based on the above data, we assessed the ability of Ang II to activate Akt/PKB. Ang II caused a rapid, robust activation of Akt/PKB ( Fig. 2A), which peaked at 5 min (6-Ϯ 0.4-fold increase) and then gradually decreased, remaining above baseline for at least 30 min. Ang II-induced Akt/PKB phosphorylation was dose-dependent, with a threshold at 1 nM and a maximal effect occurring at 100 nM (Fig. 2B).
To confirm that Akt/PKB phosphorylation on Ser 473 reflects activation, we also quantified H 2 O 2 -and Ang II-induced Akt/ PKB activity using GSK-3 or histone 2B as a substrate. As shown in Fig. 3, both H 2 O 2 and Ang II increased Akt/PKB activity. The time course of activation correlated well with that of Akt/PKB phosphorylation (compare Figs. 1A, 2A, and 3), verifying that phosphorylation of Akt/PKB is a measure of activation.
Role of PI3-K in H 2 O 2 and Ang II-induced Akt/PKB Activation-As noted above, both PI3-K-dependent and PI3-K-independent pathways have been shown to be involved in Akt/PKB activation in other cell types (13,19). To assess the role of PI3-K in H 2 O 2 -and Ang II-induced Akt/PKB phosphorylation, VSMCs were pretreated with the PI3-K inhibitors wortmannin (0.001-0.1 M) and LY294002 (0.1-10 M). These concentrations have been previously shown to effectively abrogate PI3-K activity (14,20). As shown in Fig. 4  bility, we first examined the effect of DPI, an inhibitor of flavin-containing oxidative enzymes, on Akt/PKB phosphorylation. We have previously shown that DPI inhibits Ang IIstimulated production of ROS that are derived from the NADH/ NADPH oxidase in VSMCs (21). As shown in Fig. 5, DPI (10 M) partially, but significantly, inhibited Ang II-stimulated Akt/PKB phosphorylation by 50 Ϯ 2%. These data support the concept that Ang II-induced Akt/PKB activation is mediated in part by ROS.
To more directly assess the role of [H 2 O 2 ] i in Ang II-induced Akt/PKB activation, we used VSMCs in which catalase is stably overexpressed (4,6). We have previously used these cells to demonstrate a role for [H 2 O 2 ] i in p38 MAPK activation (4). In these cells, the Ang II-induced increase in [H 2 O 2 ] i was significantly inhibited by 75 Ϯ 1% (Fig. 6A). As shown in Fig. 6B, Akt/PKB phosphorylation by Ang II was dramatically inhibited in catalase-overexpressing VSMCs compared with cells transfected with vector alone (82 Ϯ 2% inhibition). Similar results were obtained with a second line of catalase-overexpressing cells. This effect was not due to differences in AT 1 receptor expression or nonspecific inhibition of signaling pathways caused by overexpression of catalase, because vector-and catalase-transfected cells were matched for receptor number, and p42/44 MAPK activation by Ang II was unaffected (4,6). These results strongly suggest that Ang II-induced Akt/PKB activation is mediated by intracellularly produced H 2 O 2 .
Role of Akt/PKB in Ang II-induced Hypertrophy-We have previously shown that ROS play an important role in Ang II-induced hypertrophy (4,6,22), raising the possibility that Akt/PKB is also involved in this response. To assess the role of Akt/PKB in hypertrophy, we tested the effect of dominantnegative Akt/PKB (HA-Akt(AA)) on Ang II-stimulated [ 3 H]leucine incorporation. This Akt/PKB mutant effectively inhibits endogenous Akt/PKB activity, as demonstrated by its ability to inhibit insulin-induced Akt/PKB activity in CHO cells (10). As shown in Fig. 7, infection of VSMCs with adenovirus encoding dominant-negative Akt/PKB (Ad-HA-Akt(AA)) inhibited Ang II-induced [ 3 H]leucine incorporation in a dose-dependent manner (100 -600 MOI) without inhibiting the basal levels. The extent of inhibition paralleled the expression of HA-Akt(AA), as determined by Western analysis (data not shown). The inhibitory effects of Ad-HA-Akt(AA) are not caused by nonspecific or toxic effect of viral infection, because infection of the cells with a control virus containing the ␤-galactosidase gene had no effect on the hypertrophic response up to 600 MOI (Fig. 7), and Ad-HA-Akt(AA) did not affect p38 MAPK phosphorylation (data not shown). Furthermore, the trypan blue exclusion test for cell viability indicated that cells infected with Ad-HA-Akt(AA) were Ͼ95% viable up to 600 MOI. These results suggest that Akt/PKB contributes to Ang II-induced hypertrophy.

DISCUSSION
It has become apparent that ROS play important roles as modulators of Ang II signal transduction in VSMCs (4, 6, 21, 22). We have previously found that generation of ROS is required for Ang II-induced hypertrophy and that one of the Here we extend these observations to demonstrate that Ang II activates the pivotal cell survival kinase Akt/PKB in a PI3-Kdependent manner. Importantly, Ang II-induced Akt/PKB phosphorylation is mediated by intracellular H 2 O 2 , indicating that Akt/PKB is part of a redox-sensitive signaling pathway. Our studies also demonstrate a previously unappreciated role for Akt/PKB in Ang II-induced hypertrophy of VSMCs.
Akt/PKB has been shown to be activated by various growth factors and by cellular stresses such as heat shock and hyperosmolarity (8,13). Consistent with our findings, the Akt/PKB pathway can also be activated by G protein-coupled receptor agonists, including thrombin in human platelets (23), isoproterenol in rat epididymal fat cells (24), and fMet-Leu-Phe in human neutrophils (25). More recently, Murga et al. (26) reported that stimulation of M 1 or M 2 muscarinic receptors transfected into COS-7 cells induces Akt/PKB activation. The upstream signaling mechanisms responsible for Akt/PKB activation by these various agonists have not been fully elucidated.
We have previously shown that Ang II stimulates superoxide generation in VSMCs by activating an NADH/NADPH oxidase (21,22). Superoxide is rapidly dismuted to H 2 O 2 , which may be the ROS that is most important in modulating biological responses (6). Indeed, Ang II-induced H 2 O 2 formation is detectable as early as 1 min after agonist stimulation (4), suggesting that it may mediate subsequent early signaling events. Previous experiments with catalase-overexpressing cells have demonstrated that intracellularly produced H 2 O 2 mediates activation of p38 MAPK and the hypertrophic response induced by Ang II (4,6), emphasizing the critical role of ROS as signaling molecules. In this study, we demonstrate that Ang II-induced Akt/PKB phosphorylation is significantly inhibited both by the NADH/NADPH oxidase inhibitor DPI and by overexpression of catalase (Figs. 5 and 6), suggesting that ROS act as potential signal transducers linking the AT 1 receptor to the Akt/PKB pathway in VSMCs. The redox sensitivity of Akt/PKB is further confirmed by the observation that exogenous H 2 O 2 stimulates Akt/PKB phosphorylation (Fig. 1). A similar effect of H 2 O 2 was found in COS-7 cells transfected with Akt/PKB, but the role of ROS in agonist-mediated Akt/PKB phosphorylation was not assessed (27).
Growing evidence suggests that PI3-K is involved in the activation of Akt/PKB by mitogens in various systems (28). PI3-K-independent mechanisms have also been documented; however, Akt/PKB activation by ␤ 3 -adrenergic receptor (24), cyclic AMP (29), and cellular stress such as heat shock and hyperosmolarity (13) are all mediated by pathways insensitive to PI3-K blockers. In this study, we found that PI3-K is a crucial upstream mediator for Ang II-induced Akt/PKB activation, because two structurally unrelated, specific PI3-K inhibitors, wortmannin and LY294002, dose-dependently blocked Akt/PKB phosphorylation (Fig. 4). These agents abrogated H 2 O 2 -induced Akt/PKB activation as well, suggesting that PI3-K may be involved in coupling Ang II-induced H 2 O 2 forma- tion to the Akt/PKB pathway. The differential sensitivity of Ang II-and H 2 O 2 -induced Akt/PKB activation to LY294002 presumably results from the involvement of additional signaling mechanisms stimulated by Ang II but not H 2 O 2 . This is supported by the fact that neither DPI nor catalase completely inhibited Ang II activation of Akt/PKB.
Although PI3-K appears to be both necessary and sufficient for Akt/PKB activation, our present data clearly indicate that Akt/PKB activation by Ang II is redox-sensitive. The molecular target of ROS involved in agonist-induced Akt/PKB phosphorylation remains to be defined. It has been reported that the PI3-K products phosphatidylinositol 3,4,5-trisphosphate and phosphatidylinositol 3,4-bisphosphate interact with the pleckstrin homology domain of Akt/PKB resulting in the translocation of Akt/PKB to the plasma membrane, where it is activated by phosphorylation on Thr 308 and Ser 473 (16). Although phosphatidylinositol-dependent kinase-1 (PDK1) has been shown to phosphorylate Thr 308 (30), the kinase responsible for Ser 473 phosphorylation has not been molecularly identified but is referred to as PDK2 (31). Either of these kinases could be redox-sensitive, or another upstream step might be sensitive to ROS, including PI3-K itself. In VSMCs, we have obtained preliminary evidence of a role for the redox-sensitive kinase p38 MAPK (4) in Akt/PKB activation. 2 The precise relationship between p38 MAPK , PI3-K, PDK, and Akt/ PKB requires further investigation.
Proposed functional roles for Akt/PKB include modulation of glycogen synthesis, cell cycle regulation and cell growth, cell survival, and protein synthesis (11). Many of these events are modulated by Ang II (32)(33)(34), suggesting that Akt/PKB may be a critical control point in the complex array of Ang II signaling pathways in VSMCs. GSK-3 is the most well studied substrate of Akt/PKB, and is inactivated by phosphorylation leading to increased glycogen synthesis. It is also involved in activation of the AP-1 transcription factor, which interestingly enough is redox-sensitive and activated by Ang II (35). Among the cell cycle proteins, the transcription factor E2F is activated by Akt/PKB (36), which may be involved in Ang II-induced cell cycle progression. Recent reports also revealed that Akt/PKB plays a major role in cell survival (8), another physiological effect of Ang II (32). In the present study, we found that dominant-negative Akt/PKB inhibited Ang II-induced protein synthesis, indicating that this kinase may be involved in VSMC hypertrophy. This result is supported by the previous observation that Akt/PKB is upstream of p70 S6K (70-kDa ribosomal S6 kinase) (37), a mediator of protein synthesis and an important substrate of Ang II (38). Thus, the present study suggests that Akt/PKB may be a critical point of convergence among multiple growth-related signaling pathways activated by Ang II in VSMCs and provides insight into a central role of ROS in preserving the delicate balance between VSMC hypertrophy, survival, and apoptosis.