Nox4 mediates angiotensin II-induced activation of Akt/protein kinase B in mesangial cells

Yves Gorin,1 Jill M. Ricono,1 Nam-Ho Kim,1 Basant Bhandari,1 Goutam Ghosh Choudhury,1,2,3 and Hanna E. Abboud1,3

2Geriatrics Research and Education Center, 3South Texas Veterans Health Care System, Audie L. Murphy Memorial Hospital Division, and 1Department of Medicine, The University of Texas Health Science Center, San Antonio, Texas 78229-3900

Submitted 25 November 2002 ; accepted in final form 21 March 2003


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
ANG II induces protein synthesis through the serine-threonine kinase Akt/protein kinase B (PKB) in mesangial cells (MCs). The mechanism(s) of activation of Akt/PKB particularly by G protein-coupled receptors, however, is not well characterized. We explored the role of the small GTPase Rac1, a component of the phagocyte NADPH oxidase, and the gp91phox homologue Nox4/Renox in this signaling pathway. ANG II causes rapid activation of Rac1, an effect abrogated by phospholipase A2 inhibition and mimicked by arachidonic acid (AA). Northern blot analysis revealed high levels of Nox4 transcript in MCs and transfection with antisense (AS) oligonucleotides for Nox4 markedly decreased NADPH-dependent reactive oxygen species (ROS)-producing activity. Dominant negative Rac1 (N17Rac1) as well as AS Nox4 inhibited ROS generation in response to ANG II and AA, whereas constitutively active Rac1 stimulated ROS formation. Moreover, N17Rac1 blocked stimulation of NADPH oxidase activity by AA. N17Rac1 or AS Nox4 abolished ANG II- or AA-induced activation of the hypertrophic kinase Akt/PKB. In addition, AS Nox4 inhibited ANG II-induced protein synthesis. These data provide the first evidence that activation by AA of a Rac1-regulated, Nox4-based NAD(P)H oxidase and subsequent generation of ROS mediate the effect of ANG II on Akt/PKB activation and protein synthesis in MCs.

reactive oxygen species; Rac1; arachidonic acid; protein synthesis


ANG II ACTIVATES MESANGIAL cells (MCs) and contributes to the pathogenesis of fibrosis of the glomerular micro-vascular bed (3). The actions of ANG II are mediated through two types of G protein-coupled receptors, referred to as AT1 and AT2. The signal transduction pathways that mediate the biological activities of ANG II are not completely characterized. In addition to the activation of the heterotrimeric G proteins, recent studies showed that ANG II also activates tyrosine kinases and that these pathways mediate some of the biological effects of ANG II in target tissues (3, 20). ANG II also activates phospholipase A2 (PLA2) to generate arachidonic acid (AA), which plays a role in a wide array of cellular responses (29). We recently reported that AA/redox-dependent activation of the serine-threonine kinase Akt/protein kinase B (PKB) represents a critical signaling pathway that mediates protein synthesis and MC hypertrophy in response to ANG II (15). However, the source of reactive oxygen species (ROS) and the mechanisms by which ANG II and AA enhance ROS production are not known. Recent studies demonstrated that NAD(P)H oxidases are a major source of ROS not only in phagocytes but also in nonphagocytic cells (11, 16, 17, 19, 23, 46). The phagocyte respiratory burst oxidase, an NADPH-dependent multicomponent enzyme, generates superoxide anion (), with secondary generation of hydrogen peroxide (H2O2) (10, 27). The catalytic subunit gp91phox is dormant in resting cells, but it becomes activated by assembly with cytosolic regulatory proteins including the small G protein Rac (10, 27). Although nonphagocytic NAD(P)H oxidases are presumed to share similarities with the phagocyte oxidase, the apparent lack of gp91phox in nonphagocytic cells suggests that other homologues exist in these cells. Indeed, a novel family of gp91phox-like protein, termed Nox proteins (for NADPH oxidase), has recently been identified (25). The Nox family includes the homolog Nox4 also referred to as Renox, predominantly expressed in kidney epithelium (12, 32) but also found in vascular smooth muscle cells (26), osteoblasts (46), and melanocytes (7).

In this study, we explored the role of Rac1 activation and Nox4 in MCs and provide the first evidence that in these cells ANG II activates Akt/PKB via stimulation of the GTPase Rac1. We show that a Nox4-containing NAD(P)H oxidase is present in MCs and that ROS derived from Nox4 contribute to ANG II-induced protein synthesis. We propose that PLA2-mediated generation of AA is responsible for ANG II-induced Rac1 activation. In turn, Rac1 regulates Akt/PKB activity through generation of ROS by stimulation of a Nox4-based NAD(P)H oxidase.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Cell culture and cell transfection. Rat glomerular MCs were isolated and characterized as described (14). 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 (15 µg of vector alone, Myc-N17Rac1, Myc-L61Rac1) via electroporation (Gene pulser, Bio-Rad) as previously described (5). Myc epitope-tagged mammalian expression constructs Myc-N17Rac1 and Myc-L61Rac1 were kindly provided by Dr. A. Hall (University College London, London, UK).

Antisense (AS) oligonucleotides were designed near the ATG start codon of rat Nox4 (5'-AGCTCCTCCAGGACAGCGCC-3'). AS and the corresponding sense (S) oligonucleotides were synthesized as phosphorothiolated oligonucleotides and purified by high-performance liquid chromatography (Advanced Nucleic Acid Core Facility, University of Texas Health Science Center at San Antonio, TX). AS and sense oligonucleotides for Nox4 were transfected by electroporation as described above.

Northern blot analysis. Total RNA was isolated from MCs by using RNAzol B method (Cinna Biotex), separated electrophoretically on formaldehyde-agarose gels, transferred to a gene screen membrane, and hybridized with full-lengh mouse Nox4 cDNA as described (34). Nox4 cDNA was a kind gift of Dr. K.-H. Krause (Geneva University Hospitals, Geneva, Switzerland).

Subcellular fractionation and measurement of Rac1 distribution. Subcellular fractionation was performed as previously described (14). The cytosolic and membrane fractions were subjected to 12.5% SDS-polyacrylamide gel electrophoresis. The separated proteins were electrophoretically transferred onto a polyvinylidene difluoride membrane. The membrane was blocked with 5% low-fat milk in Tris-buffered saline and then incubated with a mouse monoclonal anti-Rac1 (1:1,000 dilution) antibody (Upstate Biotechnology). The Rac1 antibodies were detected using horseradish peroxidase-conjugated goat anti-mouse IgG, and bands were visualized by enhanced chemiluminescence.

Measurement of Rac1 activity. Measurement of Rac1 activity was performed by affinity precipitation according to the modified method of Benard et al. (4). MCs were grown to near confluency in 100-mm dishes and were serum deprived for 48 h. Cells were then stimulated with 1 µM ANG II or 30 µM AA for the indicated times and lysed with the ice-cold cell lysis buffer (25 mM HEPES, pH 7.5, 150 mM NaCl, 1% Igepal CA-630, 10 mM MgCl2, 10% glycerol, 1 mM phenylmethylsulfonyl fluoride, 20 µg/ml aprotinin, and 20 µg/ml leupeptin) at 4°C for 30 min. Cell lysates were then centrifuged for 30 min at 10,000 g at 4°C, and the supernatant was incubated with 30 µg of the p21-binding domain of p21-activated kinase (PAK-1) linked to glutathione S-transferase (GST-PAK-1-PBD) on glutathione-agarose beads (Upstate Biotechnology) for 30 min at 4°C. The beads were then washed in the cell lysis buffer, and the bound proteins were eluted in Laemmli sample buffer and separated by 12.5% SDS-polyacrylamide gel electrophoresis. Immunoblotting was performed as described above for the measurement of Rac1 distribution.

Immunoprecipitation, Akt/PKB activity assay, and immunoblotting. MCs were grown in 60- or 100-mm dishes and serum deprived for 48 h. All incubations were carried out in serum-free RPMI 1640 at 37°C for a specified duration. The cells were lysed in radioimmune precipitation buffer [20 mmol/l 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, 1% NP-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 method. Immunoprecipitation and Akt/PKB activity assay were performed as described (15). For immunoblotting, rabbit polyclonal anti-Akt1/PKB{alpha} (Cell Signaling Technology; 1:1,000) was used.

Measurement of production in intact MCs. Measurement of released into the media of MCs was performed by detection of ferricytochrome c reduction, as described by Johnston (22). Medium from growth-arrested MCs grown in six-well plates (2 x 106 cells/well) was aspirated and replaced with 1 ml of Hanks' balanced salt solution without phenol red containing 80 µM cytochrome c with or without 1 µM ANG II or 30 µM AA. At the end of the incubation, the medium was removed and centrifuged for 2 min at 10,000 g at 4°C to stop the reaction. The optical density was measured by spectrophotometry at 550 nm and converted to nanomoles of cytochrome c reduced using the extinction coefficient {Delta}E550 = 21.0 x 103 M/cm. The reduction of cytochrome c that was inhibitable by pretreatment with superoxide dismutase (SOD; 50 µg/ml) represents release.

NADPH oxidase assay. NADPH oxidase activity was measured by the lucigenin-enhanced chemiluminescence method (18). MCs were washed five times in ice-cold phosphate-buffered saline and were scraped from the plate in the same solution followed by centrifugation at 800 g at 4°C for 10 min. The cell pellets were resuspended in lysis buffer containing 20 mM KH2PO4, pH 7.0, 1 mM EGTA, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, and 0.5 µg/ml leupeptin. Cell suspensions were homogenized with 100 strokes in a Dounce homogenizer on ice, and aliquots of the homogenates were used immediately. To start the assay, 100 µl of homogenate were added into 900 µl of 50 mM phosphate buffer, pH 7.0, containing 1 mM EGTA, 150 mM sucrose, 5 µM lucigenin as the electron acceptor, and 100 µM NADPH as an electron donor in the presence or absence of 30 µM AA. Photon emission in terms of relative light units was measured either every minute for 12 min or every 30 s for 10 min in a luminometer. There was no measurable activity in the absence of NADPH. A buffer blank (<5% of the cell signal) was subtracted from each reading before calculation of the data. production was expressed as relative chemiluminescence (light) units per milligram of protein. Protein content was measured using the Bio-Rad protein assay reagent.

Detection of intracellular ROS. The peroxide-sensitive fluorescent probe 2',7'-dichlorodihydrofluorescin diacetate (Molecular Probes) was used to assess the generation of intracellular ROS as described previously (15). This compound is converted by intracellular esterases to 2',7'-dichlorodihydrofluorescin, which is then oxidized by H2O2 to the highly fluorescent 2',7'-dichlorodihydrofluorescein (DCF). Differential interference contrast images were obtained simultaneously using an Olympus inverted microscope with a x40 Aplanfluo objective and an Olympus fluoview confocal laser-scanning attachment. DCF fluorescence was measured with an excitation wavelength of 488 nm light, and its emission was detected using a 510- to 550-nm band-pass filter.

Protein synthesis. [3H]Leucine incorporation into trichloroacetic acid-insoluble material was used to assess protein synthesis as described (15).

Statistical analysis. Results are expressed as means ± SE. Statistical significance was assessed by Student's unpaired t-test. Significance was determined as probability (P) <0.05.


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
ANG II activates Rac1 in MCs. Rac1 activation is known to be associated with an increase in the amount of membrane-bound Rac1 (6). As shown in Fig. 1A, top, treatment of serum-starved MCs with ANG II led to a twofold increase in the amount of Rac1 in the membrane fraction with a concomitant decrease in Rac1 content in the cytosolic fraction. ANG II produced a maximal translocation of Rac1 to the membrane at 5 min. With the use of an affinity binding assay with the p21(Cdc42 and Rac)-binding domain from PAK-1 (PAK-1-PBD) as a probe for Rac1-GTP, we investigated GTP loading of Rac1 in MCs. One micromolar ANG II increased binding of Rac1 to PAK-1-PBD in a time-dependent manner, peaking at 2.5–5 min (2-fold increase over the untreated control) with sustained effect up to 30 min (Fig. 1A, bottom).



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Fig. 1. ANG II activates Rac1 in mesangial cells (MCs). A, top: MCs were treated with 1 µM ANG II for the time periods indicated and fractionated to soluble and membrane fractions. Equal amounts of protein were immunoblotted with anti-Rac1 antibody. Immunoblots are representative of 3 independent experiments. A, bottom: cells were treated with 1 µM ANG II for the time periods indicated and binding of activated GTP-Rac1 to p21-binding domain of PAK-1 (PAK-1-PBD) immobilized on agarose beads was visualized by immunoblotting with monoclonal anti-Rac1 antibody. Total amounts of Rac in cell lysates are also shown. B: MCs were preincubated with mepacrine (500 µM, 5 min) or aristolochic acid (Aris; 50 µM, 30 min) followed by 1 µM ANG II for 2.5 min and Rac1 activity was determined as in A. Total amounts of Rac in cell lysates are also shown. Immunoblots are representative of 3 independent experiments. C: same as A with 30 µM arachidonic acid (AA) instead of ANG II.

 

To evaluate the role of PLA2 in the activation of Rac1 by ANG II, we examined the effect of two structurally unrelated PLA2 inhibitors, mepacrine and aristolochic acid. Preincubation of MCs with the inhibitors markedly reduced Rac1 binding to PAK-1-PBD induced by ANG II (Fig. 1B). Furthermore, the direct addition of AA (30 µM) to MCs resulted in a twofold increase in Rac1 translocation to the membrane fraction with a maximum effect at 5 min (Fig. 1C, top). Treatment of MCs with AA induced a time-dependent increase in the amount of affinity-purified Rac1-GTP, which was detectable within 1 min and maximal at 2.5–5 min (Fig. 1C, bottom). The time course of activation of Rac1 by AA correlated well with the kinetics of Rac1 activation by ANG II. Collectively, these data indicate that the effect of ANG II on Rac1 activation is mediated by AA via activation of PLA2.

Rac1 mediates ANG II-induced Akt/PKB activation in MCs. The ability of ANG II to stimulate both Rac1 and Akt/PKB via an AA/PLA2-dependent mechanism suggests that the small GTPase Rac1 may mediate the effects of ANG II on Akt/PKB. Therefore, we assessed the effect of dominant negative form of Rac1 (N17Rac1) on Akt/PKB activation. Expression of Myc-tagged dominant negative N17Rac1 in MCs suppressed both ANG II- and AA-induced activation of Akt/PKB (Fig. 2A). The role of Rac1 in Akt/PKB activation is supported by the observation that constitutively active Rac1 (L61Rac1) is sufficient to fully activate Akt/PKB (Fig. 2B). Immunoblotting of the cell lysates using anti-Myc antibody confirms expression of the mutant protein. Collectively, these data demonstrate that the small GTPase Rac1 regulates Akt/PKB activation in response to ANG II and AA.



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Fig. 2. Rac1 mediates ANG II-induced Akt/protein kinase B (PKB) activation. A: MCs were transfected with a plasmid-encoding Myc-N17Rac1 or vector and treated with or without 1 µM ANG II (10 min) or 30 µM AA (5 min). B: MCs were transfected with Myc-L61Rac1 or vector. A and B, top: Akt/PKB immunoprecipitates were incubated with myelin basic protein (MBP) and phosphorylation of the substrate was assayed. Middle: immunoblot analysis of Akt/PKB. Bottom: immunoblot analysis of cell lysates using anti-Myc antibody to confirm mutant protein expression. The barograms represent the ratio of the radioactivity incorporated into the phosphorylated MBP quantified by PhosphorImager analysis factored by the densitometric measurement of Akt/PKB band. These data are expressed as percentage of control where the ratio in the untreated cells was defined as 100%. Values are means ± SE of 3 independent experiments. **P < 0.01 vs. control.

 

Rac1 mediates the increase in production of ROS in response to ANG II and AA. To position Rac1 and ROS in the signal transduction pathway engaged by ANG II to regulate Akt/PKB activity, we first compared the ability of ANG II and AA to influence the rate of generation in vector-transfected and dominant negative N17Rac1-transfected MCs by measuring the SOD-inhibitable reduction of ferricytochrome c. As shown in Fig. 3A, treatment of vector-transfected cells with 1 µM ANG II or 30 µM AA resulted in a rapid and time-dependent increase in generation, which reached a plateau at 5–10 min, corresponding to a four- to fivefold increase over control values. A significant increase in generation was observed as early as 1 min after exposure to ANG II or AA. The time course of ANG II- and AA-induced generation paralleled that of ANG II- and AA-induced Rac1 activation. Expression of N17Rac1 mutant in MCs markedly reduced ANG II- and AA-stimulated generation (Fig. 3A).



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Fig. 3. Rac1 is required for the increase in production of reactive oxygen species (ROS) in response to ANG II and AA. A: confluent MCs transfected with Myc-N17Rac1 ({triangleup},{square}) or vector alone ({circ},{blacktriangleup},{blacksquare}) were untreated ({circ}) or treated with 1 µM ANG II ({blacktriangleup},{triangleup}) or 30 µM AA ({blacksquare},{square}) for increasing time intervals at 37°C in Hanks' balanced salt solution containing 80 µM ferricytochrome c. Data represent means ± SE of 3 separate experiments. B: NADPH oxidase activity was measured by incubating homogenates from MCs transfected by Myc-N17Rac1 ({blacksquare}) or vector ({circ},{bullet},{triangleup},{triangledown}) with 100 µM NADPH and 5 µM lucigenin alone ({circ}) or lucigenin in the presence of 30 µM AA alone ({bullet}) or AA and 50 µg/ml superoxide dismutase ({triangleup}) or 10 µM diphenyliodonium ({triangledown}). Superoxide generation was determined by photoemission every minute for 12 min and expressed as relative light units (RLU)/mg protein. Data represent means ± SE of 3 separate experiments. C: representative photomicrographs of 2',7'-dichlorodihydrofluorescein (DCF) fluorescence in vector-transfected MCs under basal conditions, 5 min after addition of ANG II (1 µM) or AA (30 µM) in the presence or the absence of catalase (Cat; 1,000 U/ml), and in cells transfected with Myc-N17Rac1 after addition of ANG II (1 µM) or AA (30 µM). C, bottom: MCs were transfected with constitutively active mutant Myc-L61Rac1.

 

We next evaluated the effect of AA on NADPH-dependent superoxide-producing activity in MCs transfected with the N17Rac1 mutant. With the use of lucigenin-enhanced chemiluminescence, we found that in vector-transfected cells addition of 30 µM AA to MC homogenates induced a time-dependent eightfold increase in NADPH-driven generation (Fig. 3B). Expression of N17Rac1 nearly abolished the activation of NADPH oxidase by AA. Preincubation of homogenates with diphenyleneiodonium (DPI) abrogated AA-stimulated NADPH oxidase activity. In addition, SOD (50 µg/ml) inhibited AA-induced photoemission, thereby confirming identity of the product as (Fig. 3B). These data indicate that activation of superoxide-producing NADPH oxidase by AA is most likely mediated via Rac1.

Dismutation of spontaneously, or enzymatically, by SOD produces H2O2. The production of intracellular H2O2 by vector- or N17Rac1-transfected MCs in response to ANG II or AA treatment was demonstrated with a fluorescence-based assay. ANG II- and AA-induced H2O2 production was significantly blocked by the N17Rac1 mutant (Fig. 3C). The role of Rac1 in generation of ROS is confirmed by the observation that expression of constitutively active Rac1, L61Rac1, led to an increase in intracellular ROS (Fig. 3C). To determine whether H2O2 is the source of the fluorescence, cells were preincubated with catalase, an enzyme that specifically metabolizes H2O2 to H2O and O2. Catalase completely blocked the ANG II- and AA-stimulated increase in DCF fluorescence, suggesting that intracellular H2O2 is primarily responsible for the fluorescence signal (Fig. 3C). Collectively, these data demonstrate that the rapid release of ROS elicited by ANG II and AA is mediated by Rac1 activation of a superoxide-generating NAD(P)H oxidase in MCs.

A Nox4-containing NAD(P)H oxidase is implicated in ANG II- and AA-induced ROS generation in MCs. It has been proposed that superoxide-producing NAD(P)H oxidases similar to the phagocyte NADPH oxidase exist in nonphagocytic cells (11, 16, 17, 19, 23, 46). The subsequent search for nonphagocyte NAD(P)H oxidases led to the discovery of the Nox family of gp91phox homologues (25). Because the gp91phox homologue Nox4 is highly expressed in the kidney (12, 33), we determined whether a Nox4-based oxidase may account for the ROS generation in response to ANG II and AA. Northern blot analysis reveals that a 3.1-kb Nox4 transcript is highly expressed in rat MCs (Fig. 4A, left). Transfection of MCs with phosphorothioate-modified AS oligonucleotides but not S oligonucleotides for Nox4 markedly decreased Nox4 mRNA expression (Fig. 4A, right). AS also caused a significant decrease in basal NADPH-dependent superoxide-producing activity (Fig. 4B), suggesting that the NADPH oxidase activity in MCs is at least partly due to Nox4. We next assessed the role of Nox4 in ANG II- and AA-induced ROS production. Transfection of AS Nox4 completely inhibited both ANG II- and AA-stimulated superoxide-specific ferricytochrome c reduction, whereas S Nox4 had no effect (Fig. 4C). Of note, as described above for the NADPH-dependent generation, basal production of in intact cells was also decreased by AS Nox4 treatment, suggesting that the Nox4-containing oxidase is the major source of ROS in MCs. Similarly, ANG II- and AA-stimulated ROS generation as measured by 2',7'-dichlorodihydrofluorescin diacetate fluorescence-based assay was significantly reduced in MCs transfected with AS Nox4 (Fig. 4D). Conversely, fluorescence was not affected by transfection of MCs with S Nox4. Together, these results indicate that Nox4 is required for an increase of ROS production by ANG II and AA in MCs.



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Fig. 4. Nox4 mediates the increase in production of ROS in response to ANG II and AA. A, left: Northern blot analysis showing Nox4 mRNA expression in MCs. Ten micrograms of total RNA from rat MCs were hybridized with Nox4 cDNA. A, right: transfection by electroporation of antisense (AS) Nox4 (1 µM) but not sense (S) Nox4 (1 µM) decreased mRNA expression of Nox4. A, bottom: ethidium bromide staining of the same blots. B: transfection of AS Nox4 (1 µM) but not S Nox4 (1 µM) reduced basal NADPH oxidase activity in MC homogenate. NADPH oxidase activity was measured as in Fig. 3B. The initial rate of enzyme activity was calculated over the first 30 to 120 s of exposure to NADPH, and NADPH-driven superoxide production was expressed as RLU · min1 · mg protein1. Values are means ± SE of 3 independent experiments. **P < 0.01 vs. control. C: S Nox4- or AS Nox4-transfected MCs were exposed to ANG II (1 µM) or AA (30 µM) for 5 min and superoxide-specific reduction of ferricytochrome c was measured as described in Fig. 3A. Values are means ± SE of 3 independent experiments. ** And @@P < 0.01 vs. control; ##P < 0.01 vs. treatment with ANG II or AA. D: representative photomicrographs of DCF fluorescence in untransfected MCs under basal conditions, 5 min after addition of ANG II (1 µM) or AA (30 µM), and in cells transfected with AS Nox4 (1 µM) after addition of ANG II (1 µM) or AA (30 µM).

 

Nox4 mediates ANG II-induced redox-dependent Akt/PKB activation and protein synthesis in MCs. We examined the effect of Nox4 AS oligonucleotides on activation of the AA-mediated redox-dependent activation of Akt/PKB. Transfection of MCs with AS Nox4, but not S Nox4, prevented activation of Akt/PKB in response to ANG II or AA (Fig. 5A). Moreover, transfection of MCs with AS Nox4 but not S Nox4 blocked Rac1-induced Akt/PKB activation (Fig. 5B), indicating that Nox4 is a downstream target of Rac1. These data support a role for Nox4 in the redox signaling cascade triggered by ANG II and mediated by AA and Rac1 to activate Akt/PKB.



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Fig. 5. Nox4 mediates ANG II- and Rac1-induced Akt/PKB activation in MCs. A: MCs were transfected with S Nox4 (1 µM) or AS Nox4 (1 µM) and treated with 1 µM ANG II or 30 µM AA for 5 min. B: MCs were transfected with Myc-L61Rac1 or vector in the presence or absence of S Nox4 (1 µM) or AS Nox4 (1 µM). A and B, top: Akt/PKB was assayed by MBP phosphorylation. Middle: immunoblot analysis with Akt/PKB antibody. B, bottom: immunoblot analysis of cell lysates using anti-Myc antibody to confirm mutant protein expression. These data are expressed as in Fig. 2. Values are means ± SE of 3 independent experiments. **P < 0.01 vs. control; ##P < 0.01 vs. ANG II or AA.

 

To assess the role of Nox4 in protein synthesis, we tested the effect of AS Nox4 on ANG II- and AA-stimulated [3H]leucine incorporation. As shown in Fig. 6, AS Nox4 but not S Nox4 significantly reduced stimulation of protein synthesis by ANG II and AA.



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Fig. 6. Nox4 mediates ANG II-induced protein synthesis in MCs. Serum-deprived MCs were transfected with S Nox4 (1 µM) or AS Nox4 (1 µM) and treated with (filled bars) or without (open bars) 1 µM ANG II (left) or 30 µM AA (right) for 48 h. Protein synthesis was measured by [3H]leucine incorporation into TCA precipitable material. Values are means ± SE of 3 independent experiments. **P < 0.01 compared with control; ##P < 0.01 compared with treatment with ANG II or AA.

 


    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In this study, we provide the evidence that ANG II activates the serine-threonine protein kinase Akt/PKB through the small GTPase Rac1 and a Nox4-containing NAD(P)H oxidase. We demonstrate that Rac1 activation is mediated by a PLA2-coupled generation of AA and that Rac1 uses Nox4-derived ROS as downstream signal transducers to stimulate Akt/PKB. Furthermore, Nox4 regulates ANG II-stimulated protein synthesis in MCs.

The cellular mechanisms by which ANG II exerts its biological activities are not completely defined. Involvement of small GTPases of the Rho family in ANG II signaling is suggested by several studies that describe a critical role for Rho in ANG II-induced vascular smooth muscle cell hypertrophy (1, 45) and for Rac1 in PAK and c-Jun NH2-terminal kinase activation (28, 31). Although ANG II is known to activate Akt/PKB in many cell types (15, 38), the small G protein Rac has not been implicated in the activation of Akt/PKB by ANG II. Here, we demonstrate that ANG II stimulates Rac1 and that inhibition of Rac1 by expression of dominant negative Rac1 blocked ANG II-induced Akt/PKB activation, indicating a role for the small G protein Rac1. Our findings are in contrast to other studies in endothelial cells (41), COS cells (24), or neuronal cells (39), demonstrating that Rac1 and Akt/PKB are activated in parallel, downstream of a phosphoinositide 3-kinase (PI3-K)-regulated pathway. However, we recently showed that in MCs ANG II-induced activation of Akt/PKB is PI3-K independent (15). On the other hand, Rac has recently been implicated as an upstream activator of Akt/PKB with stimulation of the T cell antigen receptor (13) and the Toll-like receptor (2). These data demonstrating that PLA2 inhibitors mepacrine and aristolochic acid abrogated ANG II-induced Rac1 activation and that AA mimics the stimulatory effect of ANG II on Rac1 indicate that AA acts as an upstream activator of Rac1 in the cascade linking PLA2-coupled ANG II receptor to the small GTPase. In neutrophils, AA induces the translocation of the GTP-bound active form of Rac from cytosol to membrane (30). Furthermore, Chuang et al. (9) found that AA can disrupt the binding of Rac to the GDP dissociation inhibitor RhoGDI (which maintained Rac1 in its inactive form) leading to Rac activation. Rac1 activation appears to be agonist dependent as well as cell specific. In fibroblasts and in response to tumor necrosis factor-{alpha}, Rac acts upstream of cytosolic PLA2 (43, 44). It is reasonable to assume that differences in Rac1 effector pathways, as well as in the nature of the isoform of PLA2 implicated in MCs, at least in part, may account for these differences.

Akt/PKB is a target of ROS in MCs and ROS play a role as second messengers mediating the stimulatory effect of ANG II and AA on Akt/PKB (15). To determine if ROS act downstream of Rac1, we introduced a dominant negative N17Rac1 into MCs, which resulted in a marked decrease in the level of ROS produced in response to ANG II and AA. Moreover, constitutively active L61Rac1 markedly increases ROS generation even in the absence of ANG II or AA treatment. Therefore, one function of small G protein Rac is to regulate redox-dependent signal transduction pathways. Examples of such a sequential pathway in which an agonist or other stimuli lead to activation of small GTPase Rac and subsequently to the generation of ROS are well documented (32, 3537, 40, 43, 44). However, it is currently unclear exactly how Rac regulates the production of ROS in nonphagocytic cells. Importantly, we show that stimulation of superoxide-producing NADPH oxidase activity by AA occurs through Rac1, revealing the existence of a functional link between Rac1 and NADPH-dependent ROS generation. In phagocytic cells, Rac proteins are involved in the assembly of the NADPH oxidase system, responsible for transferring electrons from NADPH to molecular oxygen with the subsequent production of , which is then rapidly dismutated spontaneously or enzymatically to H2O2. The phagocyte oxidase consists of two plasma membrane-associated proteins, gp91phox (the catalytic subunit) and p22phox, which comprise flavocytochrome b558, and two cytosolic factors, p47phox and p67phox (10, 27). Because many of the components of the NADPH oxidase system, such as p47phox, p67phox, and p22phox, appear to be expressed in a variety of ROS-producing nonphagocytic cell types including MCs (19, 23), it is tempting to speculate that there exists an NAD(P)H oxidase enzyme complex similar to the phagocyte oxidase whose activity may be regulated by Rac. However, the apparent absence in these cells of gp91phox, the major electron transport subunit of the enzyme, has cast doubt on this presumption. The recent identification in nonphagocytic cells of novel gp91phox homologues termed Nox proteins provides a possible explanation for this paradox (25). The present study indicates that Nox4, the gp91phox homologue, is highly expressed in MCs and that impairment of its function with AS oligonucleotides inhibits NADPH oxidase activity, providing the first identification of the catalytic subunit of NAD(P)H oxidase in MCs. Moreover, Nox4 is clearly required for ANG II- and AA-induced superoxide generation and Akt/PKB activation. Furthermore, Nox4 mediates the stimulation of Akt/PKB by Rac1. These data support the concept that a Nox4-based oxidase is coupled to ANG II redox signaling in MCs.

Although the oxidases of the Nox family are proposed to play a role in a variety of biological processes such as cell growth and angiogenesis, hypoxic response, immune function, bone resorption, and proton transport (12, 19, 25, 33, 46), their bona fide functions are largely unknown. The molecular mechanisms implicated in MC growth are poorly understood. We previously described that stimulation of protein synthesis in response to ANG II is mediated via a DPI-sensitive redox-dependent signaling cascade involving Akt/PKB activation in MCs (15). In this study, we report that inhibition of Nox4 by AS oligonucleotide treatment markedly reduced ANG II-induced protein synthesis, a critical step in MC hypertrophy. We also find that impairment of Nox4 function inhibited protein synthesis in response to AA, indicating that Nox4 is also a downstream target of AA in this pathway. These observations provide the first evidence that Nox4 contributes to protein synthesis. Nox4 has been proposed to be a key actor in events as diverse as oxygen sensing in the kidney (12, 33), bone resorption (46), cell growth inhibition in Nox4-overexpressing fibroblasts (12, 33), and cell growth induction in melanoma cells (7). The incomplete inhibition of ANG II-stimulated protein synthesis, in contrast to abolition of ANG II-/AA-induced superoxide production and Akt/PKB activation to Nox4 AS oligonucleotides, suggests the involvement of additional redox-insensitive signaling mechanisms in the regulation of protein synthesis by ANG II. For instance, redox-dependent activation of p38-mitogen-activated protein kinase and Akt/PKB by ANG II is not sufficient for vascular smooth muscle cell hypertrophy, but rather requires parallel, independent activation of the redox-insensitive extracellular signal-regulated kinases 1 and 2 (19, 26). Alternatively, other non-Nox4 oxidases may be involved. Interestingly, in vascular smooth muscle cells, at least two NAD(P)H oxidases are expressed: a Nox1-based oxidase and a Nox4-based oxidase (26, 32). It has been proposed that the different Nox proteins mediate different phases of the response to ANG II (26, 32). This study and our recent observations are consistent with the existence of a Rac1- and Nox4-dependent Akt/PKB activation pathway leading to stimulation of protein synthesis: ANG II -> PLA2 -> AA -> Rac1 -> Nox4 -> ROS -> Akt/PKB -> protein synthesis (Fig. 7). The precise mechanism by which ANG II, Nox4, and Akt/PKB stimulate protein synthesis remains to be determined. Protein synthesis is a critical step during cell hypertrophy. Cell cycle proteins have been incriminated in cell hypertrophy (21, 42). The cyclin kinase inhibitor p27kip1 mediates ANG II-induced hypertrophy in tubular epithelial cells (21) and glucose-induced hypertrophy in MCs (42). In addition, platelet-derived growth factor (PDGF) inhibits p27kip1, resulting in enhanced DNA synthesis in MCs (8). The effect of PDGF on p27kip1 is due to sustained PI3-K-dependent activation of Akt/PKB (8). It is important to emphasize that activation of Akt/PKB by ANG II is not sufficient to induce proliferation of MCs (15), suggesting that the extent and temporal activation of this signaling pathway regulate a different biological activity in MCs. Thus activation of Akt/PKB may result in hypertrophy or proliferation depending on the nature of the agonist and target cells. The hypertrophic effect of ANG II more likely triggers additional specific signaling cascade(s) concomitantly with Akt/PKB activation. The nature and redox sensitivity of these pathways, as well as their impact on cell cycle events, remain to be elucidated.



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Fig. 7. Proposed model of Nox4-dependent Akt/PKB activation by ANG II in MCs.

 


    DISCLOSURES
 
This work was supported in part through a Veterans Affairs (VA) Research Enhancement Award Program (to G. G. Choudhury and H. E. Abboud); VA Merit Review Award (to G. G. Choudhury); and National Institutes of Health Grants DK-43988, DK-33665 (H. E. Abboud), and DK-55815 (to G. G. Choudhury). Y. Gorin was supported by a Research Fellowship from the National Kidney Foundation and South Texas Affiliate and a Scientist Development Grant from the American Heart Association.


    ACKNOWLEDGMENTS
 
We thank S. Garcia for help with the cell culture.


    FOOTNOTES
 

Address for reprint requests and other correspondence: H. E. Abboud, The Univ. of Texas Health Science Center, Dept. of Medicine, Division of Nephrology MC 7882, 7703 Floyd Curl Drive, San Antonio, TX 78229-3900 (E-mail: abboud{at}uthscsa.edu).

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.


    REFERENCES
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