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(The FASEB Journal. 2001;15:1909-1920.)
© 2001 FASEB

Angiotensin II activates Akt/protein kinase B by an arachidonic acid/redox-dependent pathway and independent of phosphoinositide 3-kinase

YVES GORIN*, NAM-HO KIM*, DENIS FELIERS*, BASANT BHANDARI*, GOUTAM GHOSH CHOUDHURY*,{dagger} and HANNA E. ABBOUD*,{dagger}1

* Department of Medicine, The University of Texas Health Science Center, and
{dagger} South Texas Veterans Health Care System, Audie L. Murphy Memorial Hospital Division, San Antonio, Texas 78229-3900,USA

1Correspondence: Department of Medicine, Division of Nephrology MC 7882, The University of Texas Health Science Center, 7703 Floyd Curl Dr., San Antonio, TX 78229-3900, USA. E-mail: abboud{at}uthscsa.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DICUSSION
 REFERENCES
 
Angiotensin II (Ang II) exerts contractile and trophic effects in glomerular mesangial cells (MCs). One potential downstream target of Ang II is the protein kinase Akt/protein kinase B (PKB). We investigated the effect of Ang II on Akt/PKB activity in MCs. Ang II causes rapid activation of Akt/PKB (5–10 min) but delayed activation of phosphoinositide 3-kinase (PI3-K) (30 min). Activation of Akt/PKB by Ang II was not abrogated by the PI3-K inhibitors or by the introduction of a dominant negative PI3-K, indicating that in MCs, PI3-K is not an upstream mediator of Akt/PKB activation by Ang II. Incubation of MCs with phospholipase A2 inhibitors also blocked Akt/PKB activation by Ang II. AA mimicked the effect of Ang II. Inhibitors of cyclooxygenase-, lipoxyogenase-, and cytochrome P450-dependent metabolism did not influence AA-induced Akt/PKB activation. However, the antioxidants N-acetylcysteine and diphenylene iodonium inhibited both AA- and Ang II-induced Akt/PKB activation. Dominant negative mutant of Akt/PKB or antioxidants, but not the dominant negative form of PI3-K, inhibited Ang II-induced protein synthesis and cell hypertrophy. These data provide the first evidence that Ang II induces protein synthesis and hypertrophy in MCs through AA/redox-dependent pathway and Akt/PKB activation independent of PI3-K.—Gorin, Y., Kim, N.-H., Feliers, D., Bhandari, B., Choudhury, G. G., Abboud, H. E. Angiotensin II activates Akt/protein kinase B by an arachidonic acid/redox-dependent pathway and independent of phosphoinositide 3-kinase.


Key Words: kidney • mesangium • hypertrophy • Akt/PKB • Ang II


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DICUSSION
 REFERENCES
 
MESANGIAL CELLS (MCS) are vascular pericytes that play a role in regulating blood flow and filtration surface area of the glomerular microvascular bed. In disease states, MCs are activated by hormones and cytokines and acquire a myofibroblast phenotype. Activated MCs secrete a variety of autocoids such as reactive oxygen species (ROS), bioactive lipids, growth factors, and extracellular matrix proteins (1) . The octapeptide hormone angiotensin II (Ang II) activates MCs, modulates the glomerular filtration rate via contraction of these cells, and stimulates MC growth and hypertrophy and the synthesis of extracellular matrix components. These effects of Ang II contribute to the pathogenesis of fibrosis of the glomerular microvascular bed (1) .

The actions of Ang II are mediated through two types of G-protein-coupled receptors, referred to AT1 and AT2. Most of the biological effects of Ang II on MCs are mediated via AT1 receptors (1) . In addition to activation of the heterotrimeric G-proteins, studies that are more recent have shown that Ang II also activates tyrosine kinases and that the interaction between these pathways determines the biological effects of Ang II in target tissues (1 , 2) . Ang II also activates phospholipase A2 to generate arachidonic acid (AA), which plays a role in a wide array of cellular responses such as proliferation, chemotaxis, and inflammation (3) .

One of the potential targets for Ang II is the serine-threonine kinase Akt/protein kinase B (PKB). Akt/PKB contains a pleckstrin homology (PH) domain that is part of a slightly larger portion in the NH2 terminus, called the Akt homology domain. The phosphoinositide 3-kinase (PI3-K) product phosphatidylinositol-3,4-bisphosphate binds in vitro directly to the PH domain and increases enzyme activity (4) . Akt/PKB has been shown to be activated by factors that stimulate PI3-K, including thrombin, platelet-derived growth factor, and insulin (4) . Ang II activates PI3-K in vascular smooth muscle cells (5) , in which Ang II has recently been reported to activate Akt/B in a PI3-K-dependent manner (6 , 7) . Activated Akt/PKB may mediate several biological responses including cell survival, protein synthesis, cell hypertrophy, and modulation of vascular tone by regulating nitric oxide production (7 8 9 10) .

This study provides the first evidence that in MCs Ang II activates Akt/PKB via the generation of AA and ROS and independent of PI3-K activation. Acting through AA, ROS, and Akt/PKB, but not through PI3-K, Ang II stimulates protein synthesis and cell hypertrophy. Collectively, the data demonstrate that Akt/PKB is regulated in a cell type-specific manner.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DICUSSION
 REFERENCES
 
Reagents
Tissue culture materials were obtained from Life Technologies (Carlsbad, CA). Nonidet P-40, phenylmethylsulfonyl fluoride, aprotinin, leupeptin, Na3VO4, N-acetylcysteine, diphenylene iodonium, hydrogen peroxide, wortmannin, mepacrine, indomethacin, nordihydroguaiaretic acid, ketoconazole, myelin basic protein, phosphatidylinositol, angiotensin II, and arachidonic acid were purchased from Sigma (St. Louis, MO). Recombinant platelet-derived growth factor BB was obtained from R&D Systems (Minneapolis, MN). Sheep anti-Akt1/PKB{alpha} antibody was purchased from Upstate Biotechnology (Lake Placid, NY); LY294002 and aristolochic acid were obtained from Calbiochem (La Jolla, CA); 2',7'-dichlorodihydrofluorescin diacetate was from Molecular Probes (Eugene, OR). Rabbit polyclonal antibody against the regulatory subunit of PI3-K (p85{alpha}) was obtained from Santa Cruz Biotechnology (Santa Cruz, CA). [{gamma}-32P]ATP was from Dupont/NEN (Boston, MA). Mammalian expression construct SR{alpha}-{Delta}p85 (11) was kindly provided by Drs. W. Ogawa and M. Kasuga (Kobe University Medical School, Osaka, Japan). Hemagglutinin (HA) epitope-tagged expression construct of HA-Akt(K179M) (12) was a generous gift of Dr. T. F. Franke (Harvard Institutes of Medicine, Boston, MA).

Cell culture and transfections
Rat glomerular MCs were isolated and characterized as described (13) . These cells were used between the 15th and 30th passages. Selected experiments were performed in primary and early passaged MCs to confirm the data obtain with late passages. Cells were maintained in RPMI 1640 tissue culture medium supplemented with antibiotic/antifungal solution and 17% fetal bovine serum. MCs were transiently transfected with plasmid DNA via electroporation (Gene pulser, Bio-Rad, Hercules, CA) as described previously (14) . Briefly, cells (10x106/ml) were resuspended in 1 ml of medium containing serum and 15 µg of vector alone, SR{alpha}-{Delta}p85, or HA-Akt(K179M) plasmids. Electroporation was then performed at 960 µF and 300 V. The cells were placed on ice for 10 min and plated at approximatively 80% confluence. After 48 h of transfection, the cells were made quiescent by incubation in serum-free RPMI 1640 for 48 h before treatment.

Immunoprecipitation and Akt/PKB activity assay
MCs were grown to near confluency in 60 or 100 mm dishes and made quiescent by serum-deprivation for 48 h. All incubations were carried out in serum-free RPMI 1640 at 37°C for specified duration. The cells were lysed in radioimmune precipitation buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5 mM EDTA, 1 mM Na3VO4, 1 mM phenylmethylsulfonyl fluoride, 20 µg/ml aprotinin, 20 µg/ml leupeptin, and 1% Nonidet P-40) at 4°C for 30 min. The cell lysates were centrifuged at 10,000 g for 30 min at 4°C. Protein was determined in the cleared supernatant using the Bio-Rad protein assay reagent. For immunoprecipitation, equal amounts of protein (50–100 µg) were incubated with sheep anti-Akt1/PKB{alpha} antibody for 3 h. Protein G-Sepharose beads were added and the resulting mixture was further incubated at 4°C for 1 h on a rotating device. The beads were washed three times with radioimmune precipitation buffer and twice with PBS. The kinase reaction was carried out by incubating the immunobeads in kinase assay buffer (50 mM Tris-HCl, pH 7.4, 10 mM MgCl2, 0.5 mM dithiothreitol, and 0.5 mM Na3VO4) in the presence of 5 µg/ml myelin basic protein and 50 µM cold ATP plus 1 µCi of [{gamma}-32P]ATP for 30 min at 30°C. This reaction was stopped by the addition of 2x sample buffer, after which the samples were subjected to 12.5% SDS-PAGE and phosphorylated myelin basic protein was visualized by autoradiography or a PhosphorImager. The bands were quantitated by densitometry and/or PhosphorImager analysis.

Immunoblotting
MCs lysates were prepared as described above for Akt/PKB activity assays. For immunoblotting, proteins (25–50 µg) were separated using 12.5% SDS-PAGE and transferred to polyvinylidene difluoride membranes. Blots were incubated with sheep anti-Akt1/PKB{alpha} antibody at 1:1000 and the primary antibodies were detected using horseradish peroxidase-conjugated anti-sheep IgG at 1:2000. Bands were visualized by enhanced chemiluminescence.

Phosphoinositide 3-kinase activity
MCs in 60 mm dishes were serum-deprived for 48 h and incubated in the same medium with various agents as specified. Cells were lysed in radioimmune precipitation buffer and equivalent amounts of protein were incubated with an anti-p85{alpha} antibody for 3 h. Protein G-Sepharose beads were added and the resulting mixture was further incubated at 4°C for 1 h on a rotating device. The immunoprecipitates were washed three times with radioimmune precipitation buffer, once with PBS, once in H2O, and once with 500 mM LiCl. After washing, the immunobeads were resuspended in 50 µl of PI3-K assay buffer (20 mM Tris-HCl, pH 7.5, 100 mM NaCl, and 0.5 mM EGTA); 0.5 µl of 20 mg/ml phosphatidylinositol was added, mixed, and incubated at 25°C for 10 min; 1 µl of 1 M MgCl2 and 5 µCi of [{gamma}32P]ATP were then added simultaneously and incubated at 25°C for additional 10 min. The reaction was stopped by the addition of 150 µl of chloroform/methanol/37% HCl 10:20:0.2. The samples were extracted with chloroform and dried. Radioactive lipids were separated by thin-layer chromatography and developed in chloroform/methanol/30% ammonium hydroxide/water 46:41:5:8. After drying, the plates were autoradiographed.

Detection of intracellular hydrogen peroxide
The H2O2-sensitive fluorescent probe 2',7'-dichlorofluorescin diacetate was used to assess the generation of intracellular H2O2. This compound is converted by intracellular esterases to 2',7'-dichlorofluorescin, then oxidized by H2O2 to the highly fluorescent 2',7'-dichlorofluorescen (DCF). MCs were grown to near confluency in coverglass chambers and were made quiescent by serum-deprivation for 48 h. Cells were then incubated with 10 µM 2',7'-dichlorofluorescin diacetate for 30 min at 37°C. The supernatant was removed and replaced with fresh media before treatment of MCs with 1 µM Ang II or 30 µM AA for different periods of time. Differential interference contrast images were obtained simultaneously using an Olympus inverted microscope with x40 Aplanfluo objective and an Olympus fluoview confocal laser scanning attachment. The DCF fluorescence was measured with an excitation wavelength of 488 nm light and its emission was detected using a 510–550 nm bandpass filter.

Measurement of DNA synthesis
DNA synthesis is measured as incorporation of [3H]thymidine into trichloroacetic acid (TCA) -insoluble material as described previously (15) . Briefly, confluent MCs were washed with PBS and incubated in serum-free medium for 48 h. Ang II or AA were then added for additional 48 h before pulsing with 1 µCi/ml [3H]thymidine for 4 h. The medium was removed and the cells were washed twice with ice-cold 5% TCA to remove unincorporated [3H]thymidine. Cells were solubilized by adding 0.75 ml of 0.25 N NaOH and 0.1% SDS. A 0.5 ml volume of this cell lysate was neutralized and counted in a scintillation counter.

[3H]leucine incorporation
[3H]leucine incorporation was measured by using the procedure of Jaimes et al. (16) . MCs were grown is 6-well dishes, made quiescent in serum-free medium for 48 or 72 h. MCs were then incubated with or without Ang II or AA for 48 h. Six hours before harvesting, MCs were pulsed with 2 µCi/ml [3H]leucine. At the end of this incubation period, cells were washed three times with PBS and solubilized overnight with 1.5 ml of 0.1% SDS. The contents of two wells were pooled and transferred to a plastic tube containing 60 µl of 10% bovine serum albumin. Proteins were precipitated with 300 µl 20% TCA and left overnight at 4°C. Samples were then centrifuged at 2000 g for 30 min at 4°C, the supernatant was discarded, and the pellet was resuspended in 0.5 N NaOH. Duplicate aliquots (0.5 ml) were removed and counted in a scintillation counter.

Statistical analysis
Results are expressed as mean + SE. Statistical significance was assessed by Student’s unpaired t test. Significance was determined as probability (P) less than 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DICUSSION
 REFERENCES
 
Effects of Ang II on Akt/PKB activity in MCs
Ang II activates multiple serine-threonine kinases in MCs. To examine the effect of Ang II on the activity of Akt/PKB, cultured MCs were incubated with either 1 µM Ang II for different periods of time or with various concentrations of Ang II (0.001–1 µM) for 5 min. Anti Akt/PKB immunoprecipitates were used in an immune complex kinase assay using myelin basic protein as a substrate. As shown in Fig. 1A , Ang II caused a rapid increase of Akt/PKB activity in a time-dependent manner, an effect that peaked at 5–10 min and subsided by 30 min. Stimulation of MCs with different concentrations of Ang II showed a dose-dependent increase in Akt/PKB activity with a threshold at 0.001 µM and a maximal effect occurring at 0.1 µM to 1 µM (Fig. 1B ). Ang II had no significant effect on Akt/PKB protein levels as determined by immunoblotting (Fig. 1A , B , middle panels).



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  		Figure 1. Effects of Ang II on Akt/PKB activity in MCs. A) Time course of Akt/PKB activation by Ang II. Serum-deprived MCs were treated with 1 µM Ang II for the periods indicated. B) Dose response of Akt/PKB activation by Ang II. Cells were treated with various concentrations of Ang II (0.001–1 µM) for 5 min. Akt/PKB immunoprecipitates were incubated with myelin basic protein and phosphorylation of the substrate was assayed. The top panels are representative images of myelin basic protein phosphorylation by Ang II. The middle panels show the immunoblot analysis of cell lysates with Akt/PKB antibody. In the lower panels, each barogram represents the ratio of the radioactivity incorporated into the phosphorylated myelin basic protein quantified by PhosphorImager analysis factored by the densitometric measurement of Akt/PKB band. The data are expressed as % of control where the ratio in the untreated cells was defined as 100%. Values are the means ± SE of 3 independent experiments. *P < 0.05; **P < 0.01 vs. control.

Ang II-stimulated Akt/PKB activation is PI3-K-independent
Activation of PI3-K has been shown to be necessary and sufficient for growth factor-induced increase in Akt/PKB activity. However, PI3-K-independent activation of Akt/PKB has been demonstrated in response to heat shock, hyperosmolarity, ß-adrenergic receptor activation, and cAMP (17 18 19) . To assess whether PI3-K is involved in Ang II-induced Akt/PKB activation in MCs, we first investigated the effect of Ang II on PI3-K activity. MCs were treated with 1 µM Ang II for various periods (0–120 min). Cell lysates were immunoprecipitated using an antibody to p85{alpha} subunit of PI3-K, and PI3-K activity in the immunoprecipitates was measured by immune complex kinase assay. As shown in Fig. 2A , Ang II stimulates PI3-K activity in a time-dependent manner. Maximum activation was observed at 30 min. Note that the time course of PI3-K activation by Ang II does not correlate with that of Akt/PKB activation (compare Fig. 1A and Fig. 2A ). Ang II activated Akt/PKB within 2.5 min of incubation. In contrast, maximum stimulation of PI3-K by Ang II was not observed until 30 min. This difference in the kinetics of activation of these enzymes indirectly suggests that Ang II-induced activation of Akt/PKB is not mediated through PI3-K. MCs were then pretreated with two structurally dissimilar PI3-K inhibitors—wortmannin (250 and 500 nM) and LY294002 (50 and 100 µM)—before exposure to Ang II. We have previously shown that these concentrations of the compounds completely inhibit PI3-K activity in MCs (13) . As shown in Fig. 2B , Ang II-induced Akt/PKB activation is not prevented by either inhibitor. Moreover, we measured Akt/PKB activation by Ang II in MCs transiently transfected with a dominant negative form of the p85{alpha} subunit of PI3-K lacking the p110 binding site ({Delta}p85). We observed that expression of {Delta}p85 had no effect on Ang II-induced Akt/PKB activation (Fig. 2C ). In contrast, {Delta}p85 expression in cells incubated in parallel nearly abolished the stimulation of Akt/PKB by platelet-derived growth factor (PDGF) -BB, an agonist that acts via a receptor tyrosine kinase and is known to activate the PI3-K/Akt/PKB pathway in many cell types (Fig. 2C ). Collectively, these data indicate that in MCs, PI3-K is not an upstream mediator of Akt/PKB activation in response to Ang II.



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  		Figure 2. Role of PI3-K in Akt/PKB activation by Ang II in MCs. A) Time course of PI3-K activation by Ang II. Serum-deprived MCs were treated with 1 µM Ang II for the indicated periods. Cell lysates were immunoprecipitated with an antibody against the p85{alpha} subunit of PI3-K, and the lipid kinase activity of the immunoprecipitates was assayed as described in Materials and Methods representative of 3 other chromatograms. B) Effect of PI3-K inhibitors on Ang II-induced Akt/PKB activation. MCs were preincubated with either wortmannin or LY294002 for 2 h before exposure to Ang II (1 µM, 5 min). C) MCs were transiently transfected with either the empty vector as control or a dominant negative mutant of p85 ({Delta}p85) and stimulated with 1 µM Ang II or 10 ng/ml PDGF-BB for 5 min. B, C) Measurement of activity and data are expressed as in Fig. 1 . Values are the means ± SE of 3 independent experiments. **P < 0.01 vs. control.

Role of AA and phospholipase A2 in Ang II-induced Akt/PKB activation in MCs
AA is a potent second messenger that elicits many biological responses. Ang II stimulates phospholipase A2 (PLA2) and rapid release of AA in MCs (20) . Although members of mitogen-activated protein kinase and protein kinase C families are known downstream targets of AA (21 22 23 24 25 26) , the role, if any, of AA in the activation of Akt/PKB has not been investigated. Since Ang II-induced Akt/PKB activation appears to be PI3-K independent and to elucidate an alternative mechanism of activation, we examined the effect of AA on Akt/PKB activity. As shown in Fig. 3A , exposure of MCs to 30 µM AA caused significant activation of Akt/PKB. AA-induced Akt/PKB activation was dose dependent, with a maximal effect occurring at 30 µM (Fig. 3B ). The time course of activation of Akt/PKB by AA correlated well with the kinetics of Akt/PKB activation by Ang II, with activation seen as early as 2.5 min after treatment (compare Fig. 1A and Fig. 3A ). This observation is consistent with the contention that AA may mediate Akt/PKB activation in response to Ang II. A major mechanism of increase in AA production by Ang II is hydrolysis of phospholipids by PLA2. To evaluate the role of PLA2 in the activation of Akt/PKB by Ang II, we examined the effect of two structurally unrelated PLA2 inhibitors, mepacrine and aristolochic acid. Preincubation of MCs with mepacrine (500 µM, 5 min) or aristolochic acid (50 µM, 30 min) dramatically reduced Akt/PKB activity induced by Ang II (Fig. 3C ). These data indicate that the effect of Ang II on Akt/PKB is most likely mediated by AA via activation of PLA2.



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  		Figure 3. A) Time-dependent activation of Akt/PKB by AA. Serum-deprived MCs were exposed to 30 µM AA for the times indicated. B) Dose response of Akt/PKB activation by AA. Cells were treated with various concentrations of AA (5–30 µM) for 5 min. C) Effect of mepacrine and aristolochic acid on Ang II-induced Akt/PKB activation. Serum-deprived MCs were preincubated with mepacrine (500 µM, 5 min) or aristolochic acid (50 µM, 30 min), followed by Ang II for 5 min. D) Effects of inhibitors of AA metabolism on activation of Akt/PKB induced by AA. Quiescent MCs were preincubated with indomethacin (indo, 100 µM), nordihydroguaiaretic acid (NDGA, 10 µM), or ketoconazole (keto, 20 µM) for 30 min before exposure to 30 µM AA for 5 min. Measurement of activity and data are expressed as in Fig. 1 . Values are the means ± SE of 3 independent experiments. **P < 0.01 vs. control.

AA metabolism to eicosanoids is not necessary for Akt/PKB activation
In most mammalian cells, AA is oxidized through the cyclooxygenase, lipoxygenase, and/or cytochrome P450 pathways to yield eicosanoids that mediate diverse biological effects. To determine whether these metabolic pathways mediate the effects of AA on Akt/PKB activity, MCs were treated with the cyclooxygenase inhibitor indomethacin (100 µM), the lipoxygenase inhibitor nordihydroguaiaretic acid (NDGA, 10 µM), or the cytochrome P450 inhibitor ketoconazole (20 µM) for 30 min, followed by incubation with 30 µM arachidonic acid. Concentrations of the inhibitors used are known to abrogate eicosanoid biosynthesis in mammalian cells, including MCs. None of the AA oxygenation inhibitors significantly influenced the stimulatory effect of AA-induced on Akt/PKB (Fig. 3D ). These findings suggest that the effect of AA on Akt/PKB activity is not dependent on subsequent eicosanoid biosynthesis.

Effect of exogenous H2O2 on Akt/PKB activation in MCs
We next investigated a potential role for ROS on Akt/PKB activation. H2O2 has recently been shown to activate Akt/PKB in other cell types (7 , 17 , 27 , 28) . However, this effect has not been investigated in MCs. To assess the effect of H2O2 on Akt/PKB in MCs, cells were treated with H2O2. The maximum concentration used (200 µM) is similar to those previously reported for H2O2-induced activation of two known redox-sensitive kinases in MCs, the mitogen-activated protein kinases ERK and JNK (29) . As shown in Fig. 4A , H2O2 induced a rapid activation of Akt/PKB with an effect seen as early as 1 min and a peak effect occurring 5 min after the addition of H2O2. The effect of H2O2 was dose dependent starting at 50 µM, with a maximal effect occurring at 200 µM (Fig. 4B ). These data demonstrate that Akt/PKB is a target of ROS in MCs.



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  		Figure 4. Effects of H2O2 on Akt/PKB activity in MCs. A) Time course of Akt/PKB activation by H2O2. Serum-deprived MCs were treated with 200 µM H2O2 for the periods indicated. B) Dose response of Akt/PKB activation by H2O2. Cells were treated with various concentrations of H2O2 (50–200 µM) for 5 min. Measurement of activity and data are expressed as in Fig. 1 . Values are the means ± SE of 3 independent experiments. *P < 0.05; **P < 0.001 vs. control.

Effect of Ang II and AA on reactive oxygen species production in MCs
Ang II and AA increase NADPH-driven O2-· production in vascular smooth cells (30) , aortic adventitial fibroblasts (31) , endothelial cells (32) , and renal tubular epithelial cells (24) and Ang II increases the generation of ROS in MCs (16 , 33) . To evaluate the role of Ang II and AA as signals leading to an oxidative stress in MCs, we examined their effect on the generation of ROS. The production of intracellular H2O2 by MCs in response to Ang II or AA treatment was demonstrated with a fluorescence-based assay using peroxide-sensitive fluorophore 2',7'-dichlorodihydrofluorescin diacetate and laser-scanning confocal microscopy. Stimulation of MCs with 1 µM Ang II or 30 µM AA resulted in a rapid and time-dependent increase in DCF fluorescence, with the maximal effect (a threefold increase over control) apparent 5–10 min after treatment (Fig. 5A ). Ang II- and AA-induced H2O2 production was almost completely blocked by diphenylene iodonium (DPI, 10 µM), an inhibitor of flavoprotein-containing enzymes, such as the NAD(P)H oxidase systems (Fig. 5B --G ). Moreover, the role of AA as a mediator of Ang II effects was further supported by the observation that previous incubation (5 min) with the PLA2 inhibitor, mepacrine (500 µM) dramatically reduced Ang II-induced increase in H2O2 production (Fig. 5H -J ). Collectively, these results demonstrate that Ang II and AA elicit a burst of H2O2 production in MCs, which is mediated by an AA-dependent mechanism. Furthermore, it appears that the time courses of intracellular H2O2 generation in response to Ang II or AA are consistent with a potential role for these ROS in downstream signaling events, particularly regulation of redox-sensitive protein kinases.



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  		Figure 5. Effect of Ang II and AA on the production of reactive oxygen species in MCs. A) MCs were serum-starved and treated with 1 µM Ang II or 30 µM AA for the indicated periods. DCF fluorescence reflecting the relative levels of ROS (arbitrary units) was imaged with a confocal laser scanning fluorescence microscope and quantified as described in Materials and Methods. Values are the means ± SE of 3 independent experiments. *P < 0.05; **P < 0.001 vs. control. B–J), Representative photomicrographs of DCF fluorescence in MCs under basal conditions (B, E, H), 10 min after addition of Ang II (1 µM, C and I) or AA (30 µM, F), and in cells pretreated with DPI (10 µM, 30 min) before incubation for 10 min with either 1 µM Ang II (D) or 30 µM AA (G). J) MCs were stimulated for 10 min with 1 µM Ang II in the presence of mepacrine (500 µM, 5 min).

Role of reactive oxygen species in Ang II- and AA-induced Akt/PKB activation in MCs
Taken together, the stimulation of Akt/PKB by exogenous H2O2, the correlation between the time courses of H2O2-, Ang II-, and AA-induced Akt/PKB activation (compare Figs. 1A , 3A, and 4A ), and the ability of Ang II and AA to stimulate H2O2 generation indicate that ROS may mediate the effects of Ang II and AA on Akt/PKB. To test this hypothesis, we examined the effect of N-acetylcysteine (NAC), a ROS scavenger, and DPI on Ang II- and AA-induced Akt/PKB activation. As shown in Fig. 6A , NAC (20 mM) and DPI (10 µM) significantly inhibited both Ang II- and AA-induced Akt/PKB activation. These data strongly suggest that Ang II- and AA-induced Akt/PKB activation is mediated by ROS.



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  		Figure 6. Effects of antioxidants on Akt/PKB and PI3-K activation by Ang II and AA in MCs. A) Serum-deprived MCs were preincubated with or without 20 mM NAC or 10 µM DPI for 30 min before treatment with or without 1 µM Ang II and with or without 30 µM AA for 5 min. Measurement of activity and data are expressed as in Fig. 1 . Values are the means ± SE of 3 independent experiments. **P < 0.01 vs. control. B) Serum-deprived MCs were preincubated with or without 20 mM N-acetylcysteine or 10 µM DPI for 30 min before treatment with or without Ang II (1 µM, 30 min). Lysates were prepared and immunoprecipitated with an antibody directed against p85{alpha} subunit of PI3-K, and the lipid kinase activity of immunoprecipitated PI3-K was assayed as described in Materials and Methods. Data are from a single experiment representative of at least 3 others.

If ROS mediate the effects of Ang II and AA on Akt/PKB independent of PI3-K, antioxidants would not be expected to interfere with Ang II-induced PI3-K activation. Incubation of MCs with 20 mM NAC, 10 µM DPI, concentrations that inhibit Ang II- and AA-induced Akt/PKB activation, did not influence Ang II-induced PI3-K (Fig. 6B ), indicating that Ang II-induced PI3-K activation is not mediated by ROS. Moreover, treatment of MCs with exogenous H2O2 did not stimulate PI3-K (data not shown). Collectively, these data indicate that Ang II-induced Akt/PKB activation occurs in a manner independent of PI3-K.

Role of Akt/PKB and ROS in Ang II- and AA-induced MC hypertrophy
To study MC hypertrophy, the incorporation of [3H]leucine (a measure of protein synthesis) was compared with the incorporation of [3H]thymidine (a measure of DNA synthesis). Exposure of quiescent confluent MCs to various concentrations of Ang II (0.05–5 µM) dose-dependently stimulated [3H]leucine incorporation. The maximal effect occurred at 1 µM and represented a 1.5- to 1.9-fold increase in [3H]leucine incorporation compared with untreated cells (Fig. 7A ). Ang II had no significant effect on [3H]thymidine incorporation (Fig. 7A ), suggesting that Ang II induces MC hypertrophy. As shown in the right panel of Fig. 7A, AA mimicked the effect of Ang II on protein synthesis. Indeed, AA significantly increased [3H]leucine incorporation into MCs in a dose-dependent manner (7.5–60 µM), with a maximum response at 30–60 µM. As observed with Ang II, AA treatment did not cause significant increase in DNA synthesis, indicating that AA may also act as a potent mediator of MC hypertrophy. Of note, the dose responses of Ang II- and AA-induced [3H]leucine incorporation paralleled the dose responses of Ang II- and AA-induced Akt/PKB activation, respectively.



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  		Figure 7. Role of Akt/PKB and ROS in Ang II- and AA-induced protein synthesis in MCs. A) Effect of Ang II and AA on protein and DNA synthesis in MCs. Serum-deprived MCs were treated with or without various concentrations (µM) of Ang II or AA for 48 h and protein (open bars) or DNA synthesis (filled bars) were measured respectively by [3H]leucine and [3H]thymidine incorporation, as described in Materials and Methods. B) MCs were transfected with HA-tagged kinase-inactive Akt/PKB mutant [HA-Akt(K179M)] or vector as control and treated with (filled bars) or without (open bars) 1 µM Ang II or 30 µM AA for 48 h. Ang II- and AA-induced [3H]leucine incorporation were then assayed as described in Materials and Methods. The bottom panels show immunoblots of cells transfected with empty vector or the dominant negative form of Akt using anti-HA antibody. C) MCs were transfected with the {Delta}p85 mutant or vector as control and treated with (filled bars) or without (open bars) 1 µM Ang II for 48 h. Ang II-induced [3H]leucine incorporation was then assayed as described in Materials and Methods. D) Serum-deprived MCs were treated with (filled bars) and without (open bars) 1 µM Ang II (left panel) or 30 µM AA (right panel) for 48 h in the presence or absence of the indicated inhibitors (mepacrine, 500 µM for 5 min; indo, 100 µM for 30 min; NDGA, 10 µM for 30 min; keto, 20 µM for 30 min; NAC, 20 mM for 30 min; DPI, 5 µM for 15 min). Protein synthesis was measured by [3H]-leucine incorporation as described in Materials and Methods. Values are the means ± SE of 3 independent experiments. *P < 0.05; **P < 0.01 compared with control. @@,P < 0.01 vs. treatment with Ang II or AA alone.

The above studies suggest that Ang II and AA are strong activators for Akt/PKB; therefore, we postulated that this kinase might be involved in Ang II- and AA-induced protein synthesis. Inhibition of Akt/PKB by transient transfection of the cells with HA-tagged kinase-inactive Akt/PKB with a point mutation in the ATP binding site [HA-Akt(K179M)] dramatically reduced Ang II- and AA-induced [3H]leucine incorporation (Fig. 7B ). On the other hand, expression of {Delta}p85 in MCs did not prevent stimulation of protein synthesis by Ang II (Fig. 7D ). Furthermore, exposure of MCs to 20 mM NAC or 5 µM DPI, concentrations that blocked the activation of Akt/PKB, inhibited the increase in [3H]leucine incorporation induced by Ang II or AA (Fig. 7C ). Pretreatment of MCs with mepacrine also abolished the hypertrophic effect of Ang II (Fig. 7C ). In addition, we found that none of the inhibitors of AA oxidation by cyclooxygenase, lipoxygenase, and cytochrome P-450 epoxygenase altered Ang II- or AA-induced protein synthesis (Fig. 7C ). Moreover, these data provide additional evidence for the involvement of an AA- and ROS-dependent mechanism in the Ang II-induced Akt/PKB activation and implicate AA and ROS as mediators of the signaling pathway activated by Ang II leading to MC hypertrophy via Akt/PKB and independent of PI3-K.


   DICUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DICUSSION
REFERENCES
 
In this study, we demonstrate that in MCs, Ang II activates the serine-threonine protein kinase Akt/PKB that mediates MC protein synthesis and hypertrophy through a novel AA-dependent and redox-sensitive signaling pathway, independent of PI3-K. We propose that in MCs, Akt/PKB activation by Ang II involves AA generation via PLA2; AA then results in increased production of ROS, which increases Akt/PKB activity.

Akt/PKB acts in multiple signaling pathways as a downstream target of PI3-K upon activation of tyrosine kinase and G-protein-coupled receptors (4) . PI3-K-independent activation of Akt/PKB has been reported in response to cellular stress such as heat shock and hyperosmolarity (17) , ß3-adrenergic receptor activation (18) , and cAMP (19) . However, the precise mechanisms by which PI3-K-independent activation of Akt/PKB occurs remain to be determined. In this study, the mechanism by which Ang II activates Akt/PKB does not involve PI3-K as 1) Ang II-induced Akt/PKB activation precedes activation of PI3-K, and 2) two structurally unrelated PI3-K inhibitors—wortmannin and LY294002–and direct inhibition of PI3-K by {Delta}p85, a dominant negative mutant of the enzyme, had no effect on Akt/PKB activation by Ang II. Ang II stimulates the release of AA on PLA2 activation in a variety of cell types including MCs (20 , 23 , 34) . Many protein kinases that include protein kinase C, ERK, JNK, and p38 MAPK have been identified as intracellular targets for AA (21 22 23 24 25 26) . In rabbit renal proximal tubular epithelial cells, AA induces JNK activation and mediates the action of Ang II on ERK (23 , 24) . The data presented in this study demonstrate that AA mimics the stimulatory effect of Ang II on Akt/PKB with a striking parallel in time course, suggesting that Akt/PKB activation by Ang II is mediated via AA release. Use of the PLA2 inhibitors mepacrine and aristolochic acid provides further evidence that AA acts as a second messenger in mediating the stimulatory effect of Ang II on Akt/PKB. The present study, to our knowledge, is the first report that a polyunsaturated fatty acid, AA, results in the activation of Akt/PKB. MCs express the three known isoforms of PLA2, which include secretory, cytoplasmic, and Ca2+-independent PLA2 (35) . A membrane-associated and G-protein-linked PLA2 have been implicated as a prime second messenger of Ang II in renal proximal tubule epithelium (36) . The specific isoforms of PLA2 involved in Ang II-induced Akt/PKB activation in MCs remain to be determined.

In most mammalians cells, AA is oxidized predominantly through cyclooxygenases, lipoxygenases, and/or cytochrome P450 pathways to yield eicosanoids that mediate most of its biological effects. Although metabolites of this lipid exert important effects by influencing many cellular functions, a direct role of AA has been implicated in certain cellular responses via activation of protein kinases and/or phosphatases (24 , 25 , 37 , 38) . The failure of three eicosanoid biosynthesis inhibitors to modify AA-induced Akt/PKB activation makes it very unlikely that these metabolites mediate the effect of AA. In MCs, AA release in response to interleukin 1 has been shown to activate JNK by a mechanism that does not require prostanoid production (22) . Our data indicate that AA can activate Akt/PKB without the requirement of eicosanoid biosynthesis.

We also show that millimolar concentrations of H2O2 lead to activation of Akt/PKB in MCs, indicating that Akt/PKB is a target of ROS. Exposure of MCs to Ang II and AA elicit a rapid increase in intracellular H2O2. The generation of intracellular H2O2 in response to Ang II and AA is abolished by DPI, an inhibitor of flavin-containing oxidases, such as NAD(P)H oxidase. These early effects of H2O2 generation support the contention that ROS mediate early signaling events in response to Ang II and AA. Moreover, the effect of Ang II and AA on Akt/PKB is markedly reduced by the antioxidants NAC and DPI. Collectively, these findings indicate that ROS are potential signaling molecules responsible for Akt/PKB activation by Ang II and AA. The observation that inhibitors of cyclooxygenase, lipoxygenase, and cytochrome P450 had no effect on AA-induced Akt/PKB activity provides further evidence that AA-stimulated ROS production rather than ROS-mediated modification of AA is responsible for the activation of Akt/PKB. The precise mechanism by which AA results in the generation of ROS remains to be determined. It has been shown that AA generated through PLA2 triggers the activation of the phagocyte NADPH oxidase (39) . Furthermore, one target of AA appears to be the GTPase Rac, which constitutes part of the NADPH oxidase complex and is required for oxidase activation (40) . Although the NADPH oxidase components p47phox, p67phox, and p22phox are known to be expressed in MCs (41) , the precise structure and the mechanism of activation of the various oxidases responsible for generation of ROS in these cells remain to be determined.

The finding that Ang II-induced PI3-K stimulation is not affected by antioxidants provides further evidence of a PI3-K-independent activation of Akt/PKB by Ang II in MCs. A recent study in vascular smooth muscle cells that relied on PI3-K inhibitors concluded that activation of Akt/PKB by Ang II in these cells is mediated by PI3-K and that PI3-K activation is redox sensitive (7) . Although MCs may share few properties with smooth muscle cells, they more closely resemble pericytes in other microvascular beds. Indeed, the genetic program involved in development of MCs is different from that in smooth muscle cells (42 , 43) . Thus, the mechanism of Akt/PKB activation by Ang II is cell type specific.

Growing evidence indicates that Akt/PKB plays various roles in regulating cell function. For example, recent reports reveal that Akt/PKB mediates protein synthesis, phosphorylation of nitric oxide synthase, and cell survival (4 , 7 8 9 , 44) . We also report that inhibition of Akt/PKB activation in response to Ang II by expression of dominant negative Akt/PKB markedly reduced Ang II-induced protein synthesis in MCs. On the other hand, the PI3-K dominant negative mutant had no effect on Ang II-induced protein synthesis and cell hypertrophy, indicating that this biological effect of Akt/PKB in MCs is also PI3-K-independent. Moreover, we show that AA mimics the effects of Ang II on protein and DNA synthesis, demonstrating that this polyunsaturated fatty acid is a modulator of MC hypertrophy. Furthermore, the observation that a dominant negative mutant of Akt/PKB also significantly inhibits the stimulation of protein synthesis by AA positions Akt/PKB as a downsteam target of AA in this pathway. Our findings also provide evidence that ROS play an important role in both Ang II- and AA-induced hypertrophy and that AA metabolism to eicosanoids is not necessary for the stimulation of protein synthesis. Thus, our study is consistent with the existence of a PI3-K-independent Akt/PKB activation pathway leading to MC hypertrophy: Ang II -> PLA2 -> AA -> ROS -> Akt/PKB -> protein synthesis and cell hypertrophy. Understanding the precise signaling pathways that mediate the effects of Ang II is of significant biological relevance. ROS induce apoptosis of MCs through activation of activator protein 1 (AP-1) (45 , 46) . AP-1 is generally regarded as a redox-sensitive transcription factor and is activated by Ang II (47) . Moreover, glycogen synthase kinase 3, one of the best-studied downstream substrates of Akt/PKB, is involved in modulation of AP-1 activity (48) . Akt/PKB also regulates nuclear factor kappa B (NF-{kappa}B), another redox-sensitive nuclear factor (49) . Ang II modulates NF-{kappa}B in several cell types, including MCs (50) . Blockage of NF-{kappa}B can selectively sensitize MCs to apoptosis (51) . Thus, Akt/PKB and its regulation in response to Ang II may play a central role in preserving the delicate balance between hypertrophy, survival, and apoptosis in MCs. Selective targeting of signaling pathways activated by Ang II receptors may have therapeutic implications in glomerular diseases characterized by activation of the renin-angiotensin system.


   ACKNOWLEDGMENTS
 
We wish to thank Dr. J. Zhang for imaging studies and Sergio Garcia for help with the cell culture. This work was supported in part by the Veterans Administration Medical Research Service and National Institutes of Health grants DK43988 (H.E.A.) and DK 50190 (G.G.C.). G.G.C. and H.E.A. are supported by Veterans Affairs Medical Research Service Merit Award and Veterans Affairs REAP Award. Y.G. is supported by a Research Fellowship from the National Kidney Foundation.

Received for publication March 20, 2001. Accepted for publication May 15, 2001.


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DICUSSION
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