Nox4 mediates angiotensin II-induced activation of Akt/protein kinase B in mesan - 《American Journal of Physiology》美国生理学杂志干细胞之家



免疫细胞治疗专区	欢迎关注干细胞微信公众号

12 3 4 5 6 7 8 9 10 ... 26 下一页

返回列表

查看: 539622\|回复: 254	go Nox4 mediates angiotensin II-induced activation of Akt/protein kinase B in mesan [复制链接]

轻羽

新手上路

Rank: 1

积分: 0
威望: 0
包包: 0

楼主

发表于 2009-4-21 13:41 |只看该作者 |倒序浏览 |打印

作者：Yves Gorin, Jill M. Ricono, Nam-Ho Kim, Basant Bhandari, Goutam Ghosh Choudhury,  Hanna E. Abboud,作者单位：2 Geriatrics Research and Education Center, South Texas Veterans Health Care System, Audie L.Murphy Memorial Hospital Division, and Department ofMedicine, The University of Texas Health Science Center, San Antonio, Texas78229-3900
   3 c5 Y1 G& @6 P, O
   5 k( d. h" I. J: b/ H

                     * U6 l  m6 S4 C$ a4 m! @  c( J





      % B2 n9 ]; h' a! D

      4 ?' N% j, V' D+ V
      【摘要】" ]" C: b) T  N/ I) k
   ANG II induces protein synthesis through the serine-threonine kinaseAkt/protein kinase B (PKB) in mesangial cells (MCs). The mechanism(s) ofactivation of Akt/PKB particularly by G protein-coupled receptors, however, isnot well characterized. We explored the role of the small GTPase Rac1, acomponent of the phagocyte NADPH oxidase, and the gp91 phox homologue Nox4/Renox in this signaling pathway. ANG II causes rapid activation of Rac1, an effect abrogated by phospholipase A 2 inhibition andmimicked by arachidonic acid (AA). Northern blot analysis revealed high levelsof Nox4 transcript in MCs and transfection with antisense (AS)oligonucleotides for Nox4 markedly decreased NADPH-dependent reactive oxygenspecies (ROS)-producing activity. Dominant negative Rac1 (N17Rac1) as well asAS Nox4 inhibited ROS generation in response to ANG II and AA, whereasconstitutively active Rac1 stimulated ROS formation. Moreover, N17Rac1 blocked stimulation of NADPH oxidase activity by AA. N17Rac1 or AS Nox4 abolished ANGII- or AA-induced activation of the hypertrophic kinase Akt/PKB. In addition,AS Nox4 inhibited ANG II-induced protein synthesis. These data provide thefirst evidence that activation by AA of a Rac1-regulated, Nox4-based NAD(P)Hoxidase and subsequent generation of ROS mediate the effect of ANG II onAkt/PKB activation and protein synthesis in MCs.
      【关键词】 reactive oxygen species Rac arachidonic acid protein synthesis) [  P5 ~. P6 Z6 s7 q
   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 aremediated through two types of G protein-coupled receptors, referred to asAT 1 and AT 2. The signal transduction pathways thatmediate 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 pathwaysmediate some of the biological effects of ANG II in target tissues( 3, 20 ). ANG II also activatesphospholipase A 2 (PLA 2 ) to generate arachidonic acid(AA), which plays a role in a wide array of cellular responses( 29 ). We recently reportedthat AA/redox-dependent activation of the serine-threonine kinase Akt/proteinkinase B (PKB) represents a critical signaling pathway that mediates proteinsynthesis and MC hypertrophy in response to ANG II( 15 ). However, the source ofreactive oxygen species (ROS) and the mechanisms by which ANG II and AAenhance ROS production are not known. Recent studies demonstrated that NAD(P)Hoxidases are a major source of ROS not only in phagocytes but also innonphagocytic cells ( 11, 16, 17, 19, 23, 46 ). The phagocyte respiratoryburst oxidase, an NADPH-dependent multicomponent enzyme, generates superoxide anion ( ), with secondarygeneration of hydrogen peroxide (H 2 O 2 )( 10, 27 ). The catalytic subunitgp91 phox is dormant in resting cells, but it becomesactivated by assembly with cytosolic regulatory proteins including the small Gprotein Rac ( 10, 27 ). Although nonphagocyticNAD(P)H oxidases are presumed to share similarities with the phagocyteoxidase, the apparent lack of gp91 phox in nonphagocyticcells suggests that other homologues exist in these cells. Indeed, a novel family of gp91 phox -like protein, termed Nox proteins (for N ADPH ox idase), has recently been identified( 25 ). The Nox family includesthe homolog Nox4 also referred to as Renox, predominantly expressed in kidneyepithelium ( 12, 32 ) but also found in vascularsmooth muscle cells ( 26 ),osteoblasts ( 46 ), and melanocytes ( 7 ).

In this study, we explored the role of Rac1 activation and Nox4 in MCs andprovide the first evidence that in these cells ANG II activates Akt/PKB viastimulation of the GTPase Rac1. We show that a Nox4-containing NAD(P)H oxidaseis present in MCs and that ROS derived from Nox4 contribute to ANG II-induced protein synthesis. We propose that PLA 2 -mediated generation of AAis responsible for ANG II-induced Rac1 activation. In turn, Rac1 regulatesAkt/PKB activity through generation of ROS by stimulation of a Nox4-basedNAD(P)H oxidase.
0 I: ~) W$ T( C; n* Y) @& \
MATERIALS AND METHODS

Cell culture and cell transfection. Rat glomerular MCs were isolated and characterized as described( 14 ). Cells were maintained inRPMI 1640 tissue culture medium supplemented with antibiotic/antifungal solution and 17% fetal bovine serum. MCs were transiently transfected withplasmid DNA (15 µg of vector alone, Myc-N17Rac1, Myc-L61Rac1) viaelectroporation (Gene pulser, Bio-Rad) as previously described( 5 ). Myc epitope-taggedmammalian expression constructs Myc-N17Rac1 and Myc-L61Rac1 were kindlyprovided by Dr. A. Hall (University College London, London, UK).
2 d. i$ M/ D4 G' }) Z& i
Antisense (AS) oligonucleotides were designed near the ATG start codon ofrat Nox4 (5'-AGCTCCTCCAGGACAGCGCC-3'). AS and the correspondingsense (S) oligonucleotides were synthesized as phosphorothiolatedoligonucleotides and purified by high-performance liquid chromatography(Advanced Nucleic Acid Core Facility, University of Texas Health ScienceCenter at San Antonio, TX). AS and sense oligonucleotides for Nox4 weretransfected by electroporation as described above.

Northern blot analysis. Total RNA was isolated from MCs by using RNAzol B method (Cinna Biotex), separated electrophoretically onformaldehyde-agarose gels, transferred to a gene screen membrane, andhybridized with full-lengh mouse Nox4 cDNA as described( 34 ). Nox4 cDNA was a kindgift 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 andmembrane fractions were subjected to 12.5% SDS-polyacrylamide gelelectrophoresis. The separated proteins were electrophoretically transferredonto 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). TheRac1 antibodies were detected using horseradish peroxidase-conjugated goatanti-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 Benardet al. ( 4 ). MCs were grown tonear 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 andlysed with the ice-cold cell lysis buffer (25 mM HEPES, pH 7.5, 150 mM NaCl,1% Igepal CA-630, 10 mM MgCl 2, 10% glycerol, 1 mMphenylmethylsulfonyl fluoride, 20 µg/ml aprotinin, and 20 µg/mlleupeptin) 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 30µg of the p21-binding domain of p21-activated kinase (PAK-1) linked toglutathione S -transferase (GST-PAK-1-PBD) on glutathione-agarosebeads (Upstate Biotechnology) for 30 min at 4°C. The beads were thenwashed in the cell lysis buffer, and the bound proteins were eluted in Laemmlisample buffer and separated by 12.5% SDS-polyacrylamide gel electrophoresis. Immunoblotting was performed as described above for the measurement of Rac1distribution.& X, J4 F% _9 \1 t4 A  \
1 ?# s. P& ]$ a  k3 L
Immunoprecipitation, Akt/PKB activity assay, and immunoblotting. MCs were grown in 60- or 100-mm dishes and serum deprived for 48 h. Allincubations were carried out in serum-free RPMI 1640 at 37°C for aspecified duration. The cells were lysed in radioimmune precipitation buffer[20 mmol/l Tris · HCl, pH 7.5, 150 mM NaCl, 5 mM EDTA, 1 mMNa 3 VO 4, 1 mM phenylmethylsulfonyl fluoride, 20 µg/mlaprotinin, 20 µg/ml leupeptin, 1% NP-40] at 4°C for 30 min. The celllysates were centrifuged at 10,000 g for 30 min at 4°C. Proteinwas 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 (Cell Signaling Technology; 1:1,000)was used.7 A$ D$ k+ z* g( J( ~
5 m  ?/ W! Y8 `. s1 j6 L
Measurement of production in intact MCs. Measurement of released into the media ofMCs was performed by detection of ferricytochrome c reduction, asdescribed by Johnston ( 22 ).Medium from growth-arrested MCs grown in six-well plates (2 x 10 6 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 theincubation, the medium was removed and centrifuged for 2 min at 10,000 g at 4°C to stop the reaction. The optical density was measuredby spectrophotometry at 550 nm and converted to nanomoles of cytochrome c reduced using the extinction coefficient E 550 = 21.0 x 10 3 M/cm. The reduction of cytochrome c that was inhibitable by pretreatment withsuperoxide dismutase (SOD; 50 µg/ml) represents release.
$ ?7 y8 P8 ^" u" {9 t
NADPH oxidase assay. NADPH oxidase activity was measured by thelucigenin-enhanced chemiluminescence method( 18 ). MCs were washed fivetimes in ice-cold phosphate-buffered saline and were scraped from the plate inthe same solution followed by centrifugation at 800 g at 4°C for10 min. The cell pellets were resuspended in lysis buffer containing 20 mMKH 2 PO 4, pH 7.0, 1 mM EGTA, 1 mM phenylmethylsulfonylfluoride, 10 µg/ml aprotinin, and 0.5 µg/ml leupeptin. Cell suspensions were homogenized with 100 strokes in a Dounce homogenizer on ice, and aliquotsof the homogenates were used immediately. To start the assay, 100 µl ofhomogenate 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 absenceof 30 µM AA. Photon emission in terms of relative light units was measuredeither 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 ( beforecalculation of the data. production was expressed as relative chemiluminescence (light) units per milligram of protein. Protein content was measured using the Bio-Rad proteinassay reagent./ b# H3 p1 W9 l8 v: ?! k. j

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 isconverted by intracellular esterases to2',7'-dichlorodihydrofluorescin, which is then oxidized byH 2 O 2 to the highly fluorescent2',7'-dichlorodihydrofluorescein (DCF). Differential interferencecontrast images were obtained simultaneously using an Olympus invertedmicroscope with a x 40 Aplanfluo objective and an Olympus fluoviewconfocal laser-scanning attachment. DCF fluorescence was measured with anexcitation wavelength of 488 nm light, and its emission was detected using a510- to 550-nm band-pass filter." L8 P& \9 n# K: o2 i  M

Protein synthesis. [ 3 H]Leucine incorporation intotrichloroacetic acid-insoluble material was used to assess protein synthesis as described ( 15 ).: B) S# n1 a$ w2 Q0 a) e
5 i9 j( @" S+ ]' |! h
Statistical analysis. Results are expressed as means ± SE.Statistical significance was assessed by Student's unpaired t -test.Significance was determined as probability ( P )

RESULTS8 U/ s" n2 J/ U+ {' [
: S9 h8 }: V+ ]) B) S+ N( ^
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. 1 A, top,treatment of serum-starved MCs with ANG II led to a twofold increase in theamount of Rac1 in the membrane fraction with a concomitant decrease in Rac1content in the cytosolic fraction. ANG II produced a maximal translocation ofRac1 to the membrane at 5 min. With the use of an affinity binding assay withthe p21(Cdc42 and Rac)-binding domain from PAK-1 (PAK-1-PBD) as a probe forRac1-GTP, we investigated GTP loading of Rac1 in MCs. One micromolar ANG IIincreased binding of Rac1 to PAK-1-PBD in a time-dependent manner, peaking at2.5-5 min (2-fold increase over the untreated control) with sustainedeffect up to 30 min ( Fig.1 A, bottom ).8 |& Y$ j: ~; w  B/ \1 _
6 Z0 S/ E" i$ J5 l4 I, L5 a3 r
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 andfractionated to soluble and membrane fractions. Equal amounts of protein wereimmunoblotted with anti-Rac1 antibody. Immunoblots are representative of 3independent experiments. A, bottom : cells were treated with1 µM ANG II for the time periods indicated and binding of activatedGTP-Rac1 to p21-binding domain of PAK-1 (PAK-1-PBD) immobilized on agarosebeads was visualized by immunoblotting with monoclonal anti-Rac1 antibody.Total amounts of Rac in cell lysates are also shown. B : MCs werepreincubated with mepacrine (500 µM, 5 min) or aristolochic acid (Aris; 50µM, 30 min) followed by 1 µM ANG II for 2.5 min and Rac1 activity wasdetermined as in A. Total amounts of Rac in cell lysates are alsoshown. Immunoblots are representative of 3 independent experiments. C : same as A with 30 µM arachidonic acid (AA) instead ofANG II.

To evaluate the role of PLA 2 in the activation of Rac1 by ANG II, we examined the effect of two structurally unrelated PLA 2 inhibitors, mepacrine and aristolochic acid. Preincubation of MCs with theinhibitors markedly reduced Rac1 binding to PAK-1-PBD induced by ANG II( Fig. 1 B ).Furthermore, the direct addition of AA (30 µM) to MCs resulted in a twofoldincrease in Rac1 translocation to the membrane fraction with a maximum effect at 5 min ( Fig. 1 C, top ). Treatment of MCs with AA induced a time-dependent increase inthe amount of affinity-purified Rac1-GTP, which was detectable within 1 minand maximal at 2.5-5 min ( Fig.1 C, bottom ). The time course of activation ofRac1 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 activationis mediated by AA via activation of PLA 2.
1 q) i8 [8 d, y) T$ {7 C0 A! y
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/PLA 2 -dependent mechanism suggests that the small GTPase Rac1 maymediate the effects of ANG II on Akt/PKB. Therefore, we assessed the effect ofdominant negative form of Rac1 (N17Rac1) on Akt/PKB activation. Expression ofMyc-tagged dominant negative N17Rac1 in MCs suppressed both ANG II- andAA-induced activation of Akt/PKB ( Fig.2 A ). The role of Rac1 in Akt/PKB activation is supportedby the observation that constitutively active Rac1 (L61Rac1) is sufficient tofully activate Akt/PKB ( Fig. 2 B ). Immunoblotting of the cell lysates using anti-Mycantibody confirms expression of the mutant protein. Collectively, these datademonstrate that the small GTPase Rac1 regulates Akt/PKB activation inresponse to ANG II and AA.2 r% }/ a8 ]6 a, d
6 P: V! q% |3 R3 ^( ~# \; ]
Fig. 2. Rac1 mediates ANG II-induced Akt/protein kinase B (PKB) activation. A : MCs were transfected with a plasmid-encoding Myc-N17Rac1 or vectorand 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 withmyelin basic protein (MBP) and phosphorylation of the substrate was assayed. Middle : immunoblot analysis of Akt/PKB. Bottom : immunoblotanalysis of cell lysates using anti-Myc antibody to confirm mutant proteinexpression. The barograms represent the ratio of the radioactivityincorporated into the phosphorylated MBP quantified by PhosphorImager analysisfactored by the densitometric measurement of Akt/PKB band. These data areexpressed as percentage of control where the ratio in the untreated cells wasdefined as 100%. Values are means ± SE of 3 independent experiments.** P
/ m$ N. y) T% I, ^% Z9 B
Rac1 mediates the increase in production of ROS in response to ANG IIand AA. To position Rac1 and ROS in the signal transduction pathwayengaged by ANG II to regulate Akt/PKB activity, we first compared the abilityof ANG II and AA to influence the rate of generation invector-transfected and dominant negative N17Rac1-transfected MCs by measuring the SOD-inhibitable reduction of ferricytochrome c. As shown in Fig. 3 A, treatment ofvector-transfected cells with 1 µM ANG II or 30 µM AA resulted in arapid and time-dependent increase in generation, which reached aplateau at 5-10 min, corresponding to a four- to fivefold increase overcontrol values. A significant increase in generation was observed asearly as 1 min after exposure to ANG II or AA. The time course of ANG II- andAA-induced generationparalleled that of ANG II- and AA-induced Rac1 activation. Expression ofN17Rac1 mutant in MCs markedly reduced ANG II- and AA-stimulated generation( Fig. 3 A ).9 @4 H! \. V6 `  j0 C: h6 y/ c
% w2 @2 Z1 B+ \  h
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 withMyc-N17Rac1 (, ) or vector alone (,, ) wereuntreated ( ) or treated with 1 µM ANG II (, ) or 30 µMAA (, ) for increasing time intervals at 37°C in Hanks' balancedsalt solution containing 80 µM ferricytochrome c. Data representmeans ± SE of 3 separate experiments. B : NADPH oxidaseactivity was measured by incubating homogenates from MCs transfected byMyc-N17Rac1 ( ) or vector (,,, ) with 100 µMNADPH and 5 µM lucigenin alone ( ) or lucigenin in the presence of 30µM AA alone ( ) or AA and 50 µg/ml superoxide dismutase ( ) or10 µM diphenyliodonium ( ). Superoxide generation was determined byphotoemission 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 of2',7'-dichlorodihydrofluorescein (DCF) fluorescence invector-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 ANGII (1 µM) or AA (30 µM). C, bottom : MCs weretransfected with constitutively active mutant Myc-L61Rac1.
& d) G2 t7 _5 w& P
We next evaluated the effect of AA on NADPH-dependent superoxide-producing activity in MCs transfected with the N17Rac1 mutant. With the use oflucigenin-enhanced chemiluminescence, we found that in vector-transfectedcells addition of 30 µM AA to MC homogenates induced a time-dependenteightfold increase in NADPH-driven generation( Fig. 3 B ). Expression of N17Rac1 nearly abolished the activation of NADPH oxidase by AA.Preincubation of homogenates with diphenyleneiodonium (DPI) abrogatedAA-stimulated NADPH oxidase activity. In addition, SOD (50 µg/ml) inhibitedAA-induced photoemission, thereby confirming identity of the product as ( Fig. 3 B ). These dataindicate that activation of superoxide-producing NADPH oxidase by AA is mostlikely mediated via Rac1.
! v/ A/ c" x5 b8 Q; ^) \- a! [* O
Dismutation of spontaneously, or enzymatically, by SOD produces H 2 O 2.The production of intracellular H 2 O 2 by vector- orN17Rac1-transfected MCs in response to ANG II or AA treatment was demonstratedwith a fluorescence-based assay. ANG II- and AA-inducedH 2 O 2 production was significantly blocked by the N17Rac1mutant ( Fig. 3 C ). Therole of Rac1 in generation of ROS is confirmed by the observation thatexpression of constitutively active Rac1, L61Rac1, led to an increase in intracellular ROS ( Fig.3 C ). To determine whether H 2 O 2 is the source of the fluorescence, cells were preincubated with catalase, anenzyme that specifically metabolizes H 2 O 2 to H 2 O and O 2. Catalase completely blocked the ANG II- andAA-stimulated increase in DCF fluorescence, suggesting that intracellular H 2 O 2 is primarily responsible for the fluorescencesignal ( Fig. 3 C ).Collectively, these data demonstrate that the rapid release of ROS elicited byANG II and AA is mediated by Rac1 activation of a superoxide-generatingNAD(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 oxidaseexist in nonphagocytic cells( 11, 16, 17, 19, 23, 46 ). The subsequent search fornonphagocyte NAD(P)H oxidases led to the discovery of the Nox family ofgp91 phox homologues( 25 ). Because thegp91 phox 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 IIand AA. Northern blot analysis reveals that a 3.1-kb Nox4 transcript is highlyexpressed in rat MCs ( Fig.4 A, left ). Transfection of MCs withphosphorothioate-modified AS oligonucleotides but not S oligonucleotides forNox4 markedly decreased Nox4 mRNA expression( Fig. 4 A, right ). AS also caused a significant decrease in basalNADPH-dependent superoxide-producing activity ( Fig. 4 B ), suggestingthat the NADPH oxidase activity in MCs is at least partly due to Nox4. We nextassessed 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 hadno effect ( Fig. 4 C ).Of note, as described above for the NADPH-dependent generation, basal productionof in intact cells was alsodecreased by AS Nox4 treatment, suggesting that the Nox4-containing oxidase isthe major source of ROS in MCs. Similarly, ANG II- and AA-stimulated ROSgeneration as measured by 2',7'-dichlorodihydrofluorescin diacetate fluorescence-based assay was significantly reduced in MCstransfected with AS Nox4 ( Fig.4 D ). Conversely, fluorescence was not affected bytransfection of MCs with S Nox4. Together, these results indicate that Nox4 isrequired for an increase of ROS production by ANG II and AA in MCs.

Fig. 4. Nox4 mediates the increase in production of ROS in response to ANG II andAA. A, left : Northern blot analysis showing Nox4 mRNAexpression in MCs. Ten micrograms of total RNA from rat MCs were hybridizedwith Nox4 cDNA. A, right : transfection by electroporation ofantisense (AS) Nox4 (1 µM) but not sense (S) Nox4 (1 µM) decreased mRNAexpression of Nox4. A, bottom : ethidium bromide staining ofthe same blots. B : transfection of AS Nox4 (1 µM) but not S Nox4(1 µM) reduced basal NADPH oxidase activity in MC homogenate. NADPH oxidaseactivity was measured as in Fig.3 B. The initial rate of enzyme activity was calculatedover the first 30 to 120 s of exposure to NADPH, and NADPH-driven superoxideproduction was expressed as RLU · min - 1 · mg protein - 1. Values are means ±SE of 3 independent experiments. ** P C : S Nox4- or AS Nox4-transfected MCs were exposed to ANG II (1µM) or AA (30 µM) for 5 min and superoxide-specific reduction offerricytochrome c was measured as described in Fig. 3 A. Values aremeans ± SE of 3 independent experiments. ** And @@ P P D : representative photomicrographs of DCF fluorescence inuntransfected MCs under basal conditions, 5 min after addition of ANG II (1µM) or AA (30 µM), and in cells transfected with AS Nox4 (1 µM) afteraddition of ANG II (1 µM) or AA (30 µM).

Nox4 mediates ANG II-induced redox-dependent Akt/PKB activation andprotein synthesis in MCs. We examined the effect of Nox4 ASoligonucleotides on activation of the AA-mediated redox-dependent activationof Akt/PKB. Transfection of MCs with AS Nox4, but not S Nox4, preventedactivation of Akt/PKB in response to ANG II or AA( Fig. 5 A ). Moreover,transfection of MCs with AS Nox4 but not S Nox4 blocked Rac1-induced Akt/PKBactivation ( Fig. 5 B ),indicating that Nox4 is a downstream target of Rac1. These data support a rolefor Nox4 in the redox signaling cascade triggered by ANG II and mediated by AAand Rac1 to activate Akt/PKB.
0 k( X" H& l, k* I# L
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 weretransfected with Myc-L61Rac1 or vector in the presence or absence of S Nox4 (1µM) or AS Nox4 (1 µM). A and B, top : Akt/PKBwas assayed by MBP phosphorylation. Middle : immunoblot analysis withAkt/PKB antibody. B, bottom : immunoblot analysis of celllysates using anti-Myc antibody to confirm mutant protein expression. Thesedata are expressed as in Fig.2. Values are means ± SE of 3 independent experiments.** P P
# l  N% p1 y; }! W" K
To assess the role of Nox4 in protein synthesis, we tested the effect of ASNox4 on ANG II- and AA-stimulated [ 3 H]leucine incorporation. Asshown in Fig. 6, AS Nox4 butnot S Nox4 significantly reduced stimulation of protein synthesis by ANG IIand AA.

Fig. 6. Nox4 mediates ANG II-induced protein synthesis in MCs. Serum-deprived MCswere 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 30µM AA ( right ) for 48 h. Protein synthesis was measured by[ 3 H]leucine incorporation into TCA precipitable material. Valuesare means ± SE of 3 independent experiments. ** P P/ Z/ ~& D# P6 s& _

DISCUSSION

In this study, we provide the evidence that ANG II activates theserine-threonine protein kinase Akt/PKB through the small GTPase Rac1 and aNox4-containing NAD(P)H oxidase. We demonstrate that Rac1 activation ismediated by a PLA 2 -coupled generation of AA and that Rac1 usesNox4-derived ROS as downstream signal transducers to stimulate Akt/PKB.Furthermore, Nox4 regulates ANG II-stimulated protein synthesis in MCs.- v2 M& Z/ ^7 ^, Q4 r0 }" _) t9 C, p

The cellular mechanisms by which ANG II exerts its biological activitiesare not completely defined. Involvement of small GTPases of the Rho family inANG II signaling is suggested by several studies that describe a critical rolefor Rho in ANG II-induced vascular smooth muscle cell hypertrophy( 1, 45 ) and for Rac1 in PAK andc-Jun NH 2 -terminal kinase activation ( 28, 31 ). Although ANG II is knownto activate Akt/PKB in many cell types( 15, 38 ), the small G protein Rachas not been implicated in the activation of Akt/PKB by ANG II. Here, wedemonstrate that ANG II stimulates Rac1 and that inhibition of Rac1 byexpression of dominant negative Rac1 blocked ANG II-induced Akt/PKBactivation, indicating a role for the small G protein Rac1. Our findings arein contrast to other studies in endothelial cells( 41 ), COS cells( 24 ), or neuronal cells ( 39 ), demonstrating that Rac1and Akt/PKB are activated in parallel, downstream of a phosphoinositide3-kinase (PI3-K)-regulated pathway. However, we recently showed that in MCsANG II-induced activation of Akt/PKB is PI3-K independent( 15 ). On the other hand, Rachas recently been implicated as an upstream activator of Akt/PKB withstimulation of the T cell antigen receptor ( 13 ) and the Toll-likereceptor ( 2 ). These datademonstrating that PLA 2 inhibitors mepacrine and aristolochic acidabrogated 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 ofRac1 in the cascade linking PLA 2 -coupled ANG II receptor to thesmall GTPase. In neutrophils, AA induces the translocation of the GTP-boundactive form of Rac from cytosol to membrane( 30 ). Furthermore, Chuang etal. ( 9 ) found that AA candisrupt the binding of Rac to the GDP dissociation inhibitor RhoGDI (whichmaintained Rac1 in its inactive form) leading to Rac activation. Rac1activation appears to be agonist dependent as well as cell specific. Infibroblasts and in response to tumor necrosis factor-, Rac actsupstream of cytosolic PLA 2 ( 43, 44 ). It is reasonable toassume that differences in Rac1 effector pathways, as well as in the nature ofthe isoform of PLA 2 implicated in MCs, at least in part, mayaccount for these differences.7 F  j$ p6 \0 \' g# f+ B2 h3 Z# |

Akt/PKB is a target of ROS in MCs and ROS play a role as second messengersmediating the stimulatory effect of ANG II and AA on Akt/PKB( 15 ). To determine if ROS actdownstream of Rac1, we introduced a dominant negative N17Rac1 into MCs, whichresulted in a marked decrease in the level of ROS produced in response to ANGII and AA. Moreover, constitutively active L61Rac1 markedly increases ROSgeneration even in the absence of ANG II or AA treatment. Therefore, onefunction of small G protein Rac is to regulate redox-dependent signaltransduction pathways. Examples of such a sequential pathway in which anagonist or other stimuli lead to activation of small GTPase Rac andsubsequently to the generation of ROS are well documented( 32, 35 - 37, 40, 43, 44 ). However, it is currentlyunclear exactly how Rac regulates the production of ROS in nonphagocyticcells. Importantly, we show that stimulation of superoxide-producing NADPHoxidase activity by AA occurs through Rac1, revealing the existence of afunctional link between Rac1 and NADPH-dependent ROS generation. In phagocyticcells, 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 toH 2 O 2. The phagocyte oxidase consists of two plasmamembrane-associated proteins, gp91 phox (the catalyticsubunit) and p22 phox, which comprise flavocytochrome b 558, and two cytosolic factors, p47 phox and p67 phox ( 10, 27 ). Because many of thecomponents of the NADPH oxidase system, such as p47 phox,p67 phox, and p22 phox, appear to beexpressed in a variety of ROS-producing nonphagocytic cell types including MCs( 19, 23 ), it is tempting tospeculate that there exists an NAD(P)H oxidase enzyme complex similar to thephagocyte oxidase whose activity may be regulated by Rac. However, theapparent absence in these cells of gp91 phox, the majorelectron transport subunit of the enzyme, has cast doubt on this presumption.The recent identification in nonphagocytic cells of novelgp91 phox homologues termed Nox proteins provides apossible explanation for this paradox( 25 ). The present studyindicates that Nox4, the gp91 phox homologue, is highly expressed in MCs and that impairment of its function with AS oligonucleotidesinhibits NADPH oxidase activity, providing the first identification of thecatalytic subunit of NAD(P)H oxidase in MCs. Moreover, Nox4 is clearlyrequired for ANG II- and AA-induced superoxide generation and Akt/PKBactivation. Furthermore, Nox4 mediates the stimulation of Akt/PKB by Rac1. These data support the concept that a Nox4-based oxidase is coupled to ANG IIredox signaling in MCs.

Although the oxidases of the Nox family are proposed to play a role in avariety of biological processes such as cell growth and angiogenesis, hypoxicresponse, immune function, bone resorption, and proton transport( 12, 19, 25, 33, 46 ), their bona fide functionsare largely unknown. The molecular mechanisms implicated in MC growth arepoorly understood. We previously described that stimulation of proteinsynthesis in response to ANG II is mediated via a DPI-sensitiveredox-dependent signaling cascade involving Akt/PKB activation in MCs( 15 ). In this study, we reportthat inhibition of Nox4 by AS oligonucleotide treatment markedly reduced ANGII-induced protein synthesis, a critical step in MC hypertrophy. We also findthat impairment of Nox4 function inhibited protein synthesis in response toAA, indicating that Nox4 is also a downstream target of AA in this pathway. These observations provide the first evidence that Nox4 contributes to proteinsynthesis. Nox4 has been proposed to be a key actor in events as diverse asoxygen sensing in the kidney( 12, 33 ), bone resorption( 46 ), cell growth inhibitionin Nox4-overexpressing fibroblasts( 12, 33 ), and cell growth inductionin melanoma cells ( 7 ). Theincomplete inhibition of ANG II-stimulated protein synthesis, in contrast toabolition of ANG II-/AA-induced superoxide production and Akt/PKB activationto Nox4 AS oligonucleotides, suggests the involvement of additionalredox-insensitive signaling mechanisms in the regulation of protein synthesisby ANG II. For instance, redox-dependent activation of p38-mitogen-activated protein kinase and Akt/PKB by ANG II is not sufficient for vascular smoothmuscle cell hypertrophy, but rather requires parallel, independent activationof the redox-insensitive extracellular signal-regulated kinases 1 and 2( 19, 26 ). Alternatively, othernon-Nox4 oxidases may be involved. Interestingly, in vascular smooth musclecells, at least two NAD(P)H oxidases are expressed: a Nox1-based oxidase and aNox4-based oxidase ( 26, 32 ). It has been proposed thatthe different Nox proteins mediate different phases of the response to ANG II( 26, 32 ). This study and our recentobservations are consistent with the existence of a Rac1- and Nox4-dependentAkt/PKB activation pathway leading to stimulation of protein synthesis: ANG II PLA 2 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 acritical step during cell hypertrophy. Cell cycle proteins have beenincriminated in cell hypertrophy ( 21, 42 ). The cyclin kinaseinhibitor p27 kip1 mediates ANG II-induced hypertrophy intubular epithelial cells ( 21 )and glucose-induced hypertrophy in MCs( 42 ). In addition,platelet-derived growth factor (PDGF) inhibits p27 kip1,resulting in enhanced DNA synthesis in MCs( 8 ). The effect of PDGF onp27 kip1 is due to sustained PI3-K-dependent activation ofAkt/PKB ( 8 ). It is important toemphasize that activation of Akt/PKB by ANG II is not sufficient to induceproliferation of MCs ( 15 ), suggesting that the extent and temporal activation of this signaling pathwayregulate a different biological activity in MCs. Thus activation of Akt/PKBmay result in hypertrophy or proliferation depending on the nature of theagonist and target cells. The hypertrophic effect of ANG II more likelytriggers additional specific signaling cascade(s) concomitantly with Akt/PKBactivation. The nature and redox sensitivity of these pathways, as well astheir impact on cell cycle events, remain to be elucidated.

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

DISCLOSURES
. ?& K( n: w; k4 V, l/ i: N2 x' V
This work was supported in part through a Veterans Affairs (VA) ResearchEnhancement Award Program (to G. G. Choudhury and H. E. Abboud); VA MeritReview Award (to G. G. Choudhury); and National Institutes of Health GrantsDK-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 andSouth Texas Affiliate and a Scientist Development Grant from the AmericanHeart Association.2 a3 S! H9 Q- N3 o1 t% [+ J' O& r& x

ACKNOWLEDGMENTS5 T5 i) F. i: H' f& y1 N
: G) n7 H- z6 }. z  c
We thank S. Garcia for help with the cell culture.
      【参考文献】
Aikawa R,Komuro I, Nagai R, and Yazaki Y. Rho plays an important role inangiotensin II-induced hypertrophic responses in cardiac myocytes. Mol Cell Biochem 212:177-182, 2000.8 j4 h4 X. v* u. a9 @' ]; Y1 ^0 a5 _

7 p8 D: b) N9 m
Arbibe L, MiraJP, Teusch N, Kline L, Guha M, Mackman N, Godowski PJ, Ulevitch RJ, and KnausUG. Toll-like receptor 2-mediated NF- B activation requires aRac1-dependent pathway. Nat Immun 1: 533-540,2000.3 Z; I5 w' O/ y' \. r
  y; r. T2 k0 k$ p4 X$ t

: k/ W3 f! {) [6 ?3 T& Q
Ardaillou R,Chansel D, Chatziantoniou C, and Dussaule JC. Mesangial AT 1 receptors: expression, signaling, and regulation. J Am SocNephrol 10:S40-S46, 1999.

Benard V, BohlBP, and Bokoch GM. Characterization of rac and cdc42 activation inchemoattractant-stimulated human neutrophils using a novel assay for activeGTPases. J Biol Chem 274:13198-13204, 1999.
! \# c% P) t) i0 C  r+ M

Bhandari B,Wenzel UO, Marra F, and Abboud HE. Nuclear protein in mesangial cells thatbinds to the promoter region of the platelet-derived growth factor-A chaingene induction by phorbol ester. J Biol Chem 270: 5541-5548,1995.. p* E4 U: Z7 l# C( p
9 ?" r7 V/ H, |) k8 d
9 G* \' M1 M; p2 G0 q9 k  q
8 y" m' ~/ J, K. Q! G
Bokoch GM, BohlBP, and Chuang TH. Guanine nucleotide exchange regulates membranetranslocation of Rac/Rho GTP-binding proteins. J BiolChem 269:31674-31679, 1994.+ I; w9 v$ K9 _- z' v# a( M0 r% P

& h0 T) r! `' q; w  \: F

Brar SS,Kennedy TP, Sturrock AB, Huecksteadt TP, Quinn MT, Whorton AR, and HoidalJR. An NAD(P)H oxidase regulates growth and transcription in melanomacells. Am J Physiol Cell Physiol 282: C1212-C1224,2002.

% S  B6 ^* j% a/ T) Q) i+ {

Choudhury GG. Akt serine threonine kinase regulatesplatelet-derived growth factor-induced DNA synthesis in glomerular mesangialcells: regulation of c-fos and p27 kip1 gene expression. J Biol Chem 276:35636-35643, 2001.

8 n7 @) K! c; [5 }% G) W8 x. C
$ l4 U$ U9 p) ^! N: h1 [. g: e
Chuang TH, XuX, Knaus UG, Hart MJ, and Bokoch GM. GDP dissociation inhibitor preventsintrinsic and GTPase activating protein-stimulated GTP hydrolysis by the RacGTP-binding protein. J Biol Chem 268: 775-778,1993.
& x- ]* [( {. M" `6 M

( d+ a7 X+ ^, d+ e+ B
Clark RA. Activation of the neutrophil respiratory burst oxidase. J InfectDis 179:S309-S317, 1999.
9 k( M5 R$ [4 |5 Y! o, `5 Q
$ z" Z) Z! W0 p' o: a0 S! v: q% i

Cui XL andDouglas JG. Arachidonic acid activates c-jun N-terminal kinase throughNADPH oxidase in rabbit proximal tubular epithelial cells. ProcNatl Acad Sci USA 94:3771-3776, 1997./ A5 |1 E4 n: T" X" I$ G# r, [
+ X1 W8 I9 F( [  e6 K* U

) N4 Q! j; h- M! H/ o
Geiszt M, KoppJB, Varnai P, and Leto TL. Identification of renox, an NAD(P)H oxidase inkidney. Proc Natl Acad Sci USA 97: 8010-8014,2000.$ x  i& W3 J7 G
: D0 |4 l( b! D+ d. D
6 \6 w( t9 f! ^; n% N2 O
: U" s* F. F* |! Y+ _, t
Genot EM,Arrieumerlou C, Ku G, Burgering BM, Weiss A, and Kramer IM. The T-cellreceptor regulates Akt (protein kinase B) via a pathway involving Rac1 andphosphatidylinositide 3-kinase. Mol Cell Biol 20: 5469-5478,2000.3 A( I6 S+ D2 U7 L# G( Q

& c3 T' b# A# f- k9 d
' @. {0 h) S4 g2 m& i% F
Ghosh Choudhury G, Karamitsos C, Hernandez J, Gentilini A, BardgetteJ, and Abboud HE. PI-3-kinase and MAPK regulate mesangial cellproliferation and migration in response to PDGF. Am J Physiol RenalPhysiol 273:F931-F938, 1997.

( M% H6 d, `) C" [
. y6 d  ~/ ]# ^0 s9 H$ r# Y
Gorin Y, KimNH, Feliers D, Bhandari B, Ghosh Choudhury G, and Abboud HE. AngiotensinII activates Akt/protein kinase B by an arachidonic acid/redox-dependentpathway and independent of phosphoinositide 3-kinase. FASEBJ 15:1909-1920, 2001." m- n& A% L0 Y( `5 g$ g
/ A; V. ]( C- v7 D+ N. H* ?) t' h
3 Z& v" @7 r/ s' [% ~3 J

Gorin Y, OhayonR, Carvalho DP, Deme D, Leseney AM, Haye B, Kaniewski J, Pommier J, Virion A,and Dupuy C. Solubilization and characterization of a thyroidCa 2 -dependent and NADPH-dependentK 3 Fe(CN) 6 reductase. Relationship with theNADPH-dependent H 2 O 2 -generating system. Eur JBiochem 240:807-814, 1996.
9 a6 v" R3 T: D; p
0 T# Y. k2 T9 D

Greiber S,Munzel T, Kastner S, Muller B, Schollmeyer P, and Pavenstadt H. NAD(P)Hoxidase activity in cultured human podocytes: effects of adenosinetriphosphate. Kidney Int 53:654-663, 1998.

Griendling KK,Minieri CA, Ollerenshaw JD, and Alexander RW. Angiotensin II stimulatesNADH and NADPH oxidase activity in cultured vascular smooth muscle cells. Circ Res 74:1141-1148, 1994.7 t% G; ^: o: ~; p1 F) i, L

Griendling KK,Sorescu D, and Ushio-Fukai M. NAD(P)H oxidase: role in cardiovascularbiology and disease. Circ Res 86: 494-501,2000.4 W+ U* a* K2 [* V' j

2 b8 V/ X3 u( ]

Haendeler J andBerk BC. Angiotensin II mediated signal transduction. Important role oftyrosine kinases. Regul Pept 95: 1-7,2000.

  [* n3 n1 }8 h+ e
Hannken T,Schroeder R, Zahner G, Stahl RA, and Wolf G. Reactive oxygen speciesstimulate p44/42 mitogen-activated protein kinase and inducep27 Kip1 : role in angiotensin II-mediated hypertrophy ofproximal tubular cells. J Am Soc Nephrol 11: 1387-1397,2000.8 G9 \5 l2 x# ?3 h' O

- d4 C; P8 a$ c' d" n
Johnston RB Jr. Measurement of secreted bymonocytes and macrophages. Methods Enzymol 105: 365-369,1984.- l% F9 l$ _% L, C) j% Y

% C" q, |# [0 Z1 q" M1 u
- K9 g$ M( r* N* K
Jones SA,Hancock JT, Jones OT, Neubauer A, and Topley N. The expression of NADPHoxidase components in human glomerular mesangial cells: detection of proteinand mRNA for p47 phox, p67 phox, andp22 phox. J Am Soc Nephrol 5: 1483-1491,1995.
3 S  M: h+ @4 v) h% [% d" y2 U

8 B8 k0 M0 F9 |6 ]9 }0 b6 \
Klippel A,Kavanaugh WM, Pot D, and Williams LT. A specific product ofphosphatidylinositol 3-kinase directly activates the protein kinase Aktthrough its pleckstrin homology domain. Mol Cell Biol 17: 338-344,1997.' c4 L* r: P9 M$ M1 J+ d, M9 p6 p
: _5 Y& Z0 e1 Y$ g5 k

$ ~% e: n, A8 x% R- q
Lambeth JD. Nox/Duox family of nicotinamide adenine dinucleotide (phosphate) oxidases. Curr Opin Hematol 9:11-17, 2002.
# B" u5 I. F' Q5 U2 Z0 |" N
% U3 N+ E0 r  H0 {# V

Lassegue B,Sorescu D, Szocs K, Yin Q, Akers M, Zhang Y, Grant SL, Lambeth JD, andGriendling KK. Novel gp91 phox homologues in vascularsmooth muscle cells: nox1 mediates angiotensin II-induced superoxide formationand redox-sensitive signaling pathways. Circ Res 88: 888-894,2001.

) u3 Y- W  ]% {* l( V

Leusen JHW,Verhoeven AJ, and Roos D. Interactions between the components of the humanNADPH oxidase: a review about the intrigues in the phox family. Front Biosci 1:d72-d90, 1996.) c" X$ m0 a( p' R8 t# A5 C+ i8 A
9 R5 t& L* J. b! u

7 U9 T+ d7 Z# C1 ^2 W9 L
Murasawa S,Matsubara H, Mori Y, Masaki H, Tsutsumi Y, Shibasaki Y, Kitabayashi I, TanakaY, Fujiyama S, Koyama Y, Fujiyama A, Iba S, and Iwasaka T. Angiotensin IIinitiates tyrosine kinase Pyk2-dependent signalings leading to activation ofRac1-mediated c-Jun NH 2 -terminal kinase. J BiolChem 275:26856-26863, 2000.
3 e4 x( R6 `0 o" z0 I

% I; g* w, ?8 L4 l5 {
Piomelli D. Arachidonic acid in cell signaling. Curr Opin CellBiol 5:274-280, 1993.* h7 M: G. ]5 k  N. C! _. B- ]4 r

) _! ]& }; j% ?/ z6 \6 @4 Y+ Y

Sawai T, AsadaM, Nunoi H, Matsuda I, Ando S, Sasaki T, Kaibuchi K, Takai Y, and KatayamaK. Combination of arachidonic acid and guanosine5'- O -(3-thiotriphosphate) induce translocation of rac p21s tomembrane and activation of NADPH oxidase in a cell-free system. Biochem Biophys Res Commun 195:264-269, 1993.9 |+ k- ^6 c; t$ n& i3 Q7 e
. S' `/ t& P/ n. o5 H9 d7 J* d
0 K0 x( f" O) W3 Y0 V% V
' ~% A. G  o3 W2 p
Schmitz U,Thommes K, Beier I, Wagner W, Sachinidis A, Dusing R, and Vetter H. Angiotensin II-induced stimulation of p21-activated kinase and c-JunNH 2 -terminal kinase is mediated by Rac1 and Nck. J BiolChem 276:22003-22010, 2001.+ Y9 O' @" E. f( g
$ ^: \" O; |" Z$ Y/ ]7 `2 P. j/ G
  K; n. l& i# L% W
3 z+ y+ I0 G0 e0 v+ r; U4 Y' F
Seshiah PN,Weber DS, Rocic P, Valppu L, Taniyama Y, and Griendling KK. Angiotensin IIstimulation of NAD(P)H oxidase activity: upstream mediators. CircRes 91:406-413, 2002./ a/ o  ]7 d5 z0 Q! V

4 ]9 e# C9 R  _, a4 V& g* ]
Shiose A,Kuroda J, Tsuruya K, Hirai M, Hirakata H, Naito S, Hattori M, Sakaki Y, andSumimoto H. A novel superoxide-producing NAD(P)H oxidase in kidney. J Biol Chem 276:1417-1423, 2001./ A- d6 K; C+ Q! u# v
$ X. ?# j& ]! z) |1 c# `

Simon M, ClearyTG, Hernandez JD, and Abboud HE. Shiga toxin 1 elicits diverse biologicresponses in mesangial cells. Kidney Int 54: 1117-1127,1998.
% O6 X( C* l9 Y- M/ J, [
0 {/ \4 B) y, ]/ r) G& h" V) X* q0 F
6 f9 z8 ~( A, `6 Q& A
Sulciner DJ,Irani K, Yu ZX, Ferrans VJ, Goldschmidt-Clermont P, and Finkel T. Rac1regulates a cytokine-stimulated, redox-dependent pathway necessary forNF- B activation. Mol Cell Biol 16: 7115-7121,1996.
* p5 ?; }+ m+ R% ~

Sundaresan M,Yu ZX, Ferrans VJ, Sulciner DJ, Gutkind JS, Irani K, Goldschmidt-Clermont PJ,and Finkel T. Regulation of reactive-oxygen-species generation infibroblasts by Rac1. Biochem J 318: 379-382,1996.& f" n! v) g4 c

- q/ |$ Q* Z* d7 W- p! t
* q& a7 ?$ m+ _. `1 ?% D7 [: \. z
Suzukawa K,Miura K, Mitsushita J, Resau J, Hirose K, Crystal R, and Kamata T. Nervegrowth factor-induced neuronal differentiation requires generation ofRac1-regulated reactive oxygen species. J Biol Chem 275: 13175-13178,2000.7 q+ v; D  _; ~8 p6 \% M7 Q
- v( U- W0 C/ ?. h0 T

Ushio-Fukai M,Alexander RW, Akers M, Yin Q, Fujio Y, Walsh K, and Griendling KK. Reactive oxygen species mediate the activation of Akt/protein kinase B byangiotensin II in vascular smooth muscle cells. J BiolChem 274:22699-22704, 1999.& p0 G% g! ]/ t8 {. e
+ a* @* T. s+ z* }; V% ?

Van Weering DH,De Rooij J, Marte BM, Downward J, Bos JL, and Burgering BM. Protein kinaseB activation and lamel-lipodium formation are independent phosphoinositide3-kinase-mediated events differentially regulated by endogenous Ras. Mol Cell Biol 18:1802-1811, 1998.
, I) @! O& [0 n) H  N
  W; D9 q$ z' }, f3 z' y8 i

Wassmann S,Laufs U, Baumer AT, Muller K, Konkol C, Sauer H, Bohm M, and Nickenig G. Inhibition of geranylgeranylation reduces angiotensin II-mediated free radicalproduction in vascular smooth muscle cells: involvement of angiotensinAT 1 receptor expression and Rac1 GTPase. MolPharmacol 59:646-654, 2001.

- W9 ]& E/ V( h/ g2 {! v+ B9 F2 q1 X

Welch H,Eguinoa A, Stephens LR, and Hawkins PT. Protein kinase B and Rac areactivated in parallel within a phosphatidylinositide 3OH-kinase-controlledsignaling pathway. J Biol Chem 273: 11248-11256,1998.6 O1 u0 t& w8 N9 E& p. b# U
& @( S7 z; C1 E" C
/ `0 O" t. L" [# @
  J! F" a6 L0 I! O  z
Wolf G,Schroeder R, Zahner G, Stahl RA, and Shankland SJ. High glucose-inducedhypertrophy of mesangial cells requires p27 Kip1, aninhibitor of cyclin-dependent kinases. Am J Pathol 158: 1091-1100,2001.! b. j& ]  S  q  g* y0 P, {4 B
  I2 q& c2 O& G0 W

Woo CH, Eom YW,Yoo MH, You HJ, Han HJ, Song WK, Yo YJ, Chun JS, and Kim JH. Tumornecrosis factor- generates reactive oxygen species via a cytosolicphospholipase A 2 -linked cascade. J BiolChem 275:32357-32362, 2000.

) j' K. L' A, i+ @5 r
Woo CH, Lee ZW,Kim BC, Ha KS, and Kim JH. Involvement of cytosolic phospholipaseA 2, and the subsequent release of arachidonic acid, in signallingby rac for the generation of intracellular reactive oxygen species in rat-2fibroblasts. Biochem J 348:525-530, 2000." v( m8 t5 l1 V) u
4 D# H& g. O2 l, q) l

: ]3 H: q! z) K/ S* W$ I% \. T
Yamakawa T,Tanaka S, Numaguchi K, Yamakawa Y, Motley ED, Ichihara S, and Inagami T. Involvement of Rho-kinase in angiotensin II-induced hypertrophy of ratvascular smooth muscle cells. Hypertension 35: 313-318,2000.7 M+ a7 o5 D4 [0 P, e; Z0 @9 T6 F
; m. _, ^1 E) [2 F
4 {4 Z; B/ x9 I* z

Yang S,Madyastha P, Bingel S, Ries W, and Key L. A new superoxide-generatingoxidase in murine osteoclasts. J Biol Chem 276: 5452-5458,2001.

收藏0 分享0 顶0 踩0