The superoxide-generating NADPH oxidase NOX1 is thought to be involved in signaling by the angiotensin II–receptor AT1R. However, underlying signaling steps are poorly understood. In this study, we investigated the effect of AngII on aortic smooth muscle from wild-type and NOX1-deficient mice. NOX1-deficient cells showed decreased basal ROS generation and did not produce ROS in response to AngII. Unexpectedly, AngII-dependent Ca2+ signaling was markedly decreased in NOX1-deficient cells. Immunostaining demonstrated that AT1R was localized on the plasma membrane in wild-type, but intracellularly in NOX1-deficient cells. Immunohistochemistry and immunoblotting showed a decreased expression of AT1R in the aorta of NOX1-deficient mice. To investigate the basis of the abnormal AT1R targeting, we studied caveolin expression and phosphorylation. The amounts of total caveolin and of caveolae were not different in NOX1-deficient mice, but a marked decrease occurred in the phosphorylated form of caveolin. Exogenous H2O2 or transfection of a NOX1 plasmid restored AngII responses in NOX1-deficient cells. Based on these findings, we propose that NOX1-derived reactive oxygen species regulate cell-surface expression of AT1R through mechanisms including caveolin phosphorylation. The lack cell-surface AT1R expression in smooth muscle could be involved in the decreased blood pressure in NOX1-deficient mice. Antioxid. Redox Signal. 11, 2371–2384.
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References
1.
AokiT, NomuraR, FujimotoT. Tyrosine phosphorylation of caveolin-1 in the endothelium. Exp Cell Res, 253:629–636. 1999.
2.
BanfiB, ClarkRA, StegerK, KrauseK-H. Two novel proteins activate superoxide generation by the NADPH oxidase NOX1. J Biol Chem, 278:3510–3513. 2003.
3.
BedardK, KrauseK-H. The NOX family of ROS-generating NADPH oxidases: physiology and pathophysiology. Physiol Rev, 87:245–313. 2007.
4.
ChenK, KirberMT, XiaoH, YangY, KeaneyJFJr.Regulation of ROS signal transduction by NADPH oxidase 4 localization. J Cell Biol, 181:1129–1139. 2008.
5.
CookJL, MillsSJ, NaquinRT, AlamJ, ReRN. Cleavage of the angiotensin II type 1 receptor and nuclear accumulation of the cytoplasmic carboxy-terminal fragment. Am J Physiol Cell Physiol, 292:C1313–C1322. 2007.
6.
CowleyAWJr.Long-term control of arterial blood pressure. Physiol Rev, 72:231–300. 1992.
DrabM, VerkadeP, ElgerM, KasperM, LohnM, LauterbachB, MenneJ, LindschauC, MendeF, LuftF, SchedlA, HallerH, KurzchaliaT. Loss of caveolae, vascular dysfunction, and pulmonary defects in caveolin-1 gene-disrupted mice. Science, 293:2449–2452. 2001.
10.
DubandJ, GimonaM, ScatenaM, SartoreS, SmallJ. Calponin and SM 22 as differentiation markers of smooth muscle: spatiotemporal distribution during avian embryonic development. Differentiation, 55:1–11. 1993.
11.
EggenaP, ZhuJH, CleggK, BarrettJD. Nuclear angiotensin receptors induce transcription of renin and angiotensinogen mRNA. Hypertension, 22:496–501. 1993.
12.
FellnerSK, ArendshorstWJ. Angiotensin II, reactive oxygen species, and Ca2+ signaling in afferent arterioles. Am J Physiol Renal Physiol, 289:F1012–F1019. 2005.
13.
FotiM, PhelouzatM-A, HolmA, RasmussonBJ, CarpentierJ-L. p56Lck anchors CD4 to distinct microdomains on microvilli. Proc Natl Acad Sci U S A, 99:2008–2013. 2002.
14.
Foyouzi-YoussefiR, ArnaudeauS, BornerC, KelleyWL, TschoppJ, LewDP, DemaurexN, KrauseK-H. Bcl-2 decreases the free Ca2+ concentration within the endoplasmic reticulum. Proc Natl Acad Sci U S A, 97:5723–5728. 2000.
15.
GarridoA, GriendlingK. NADPH oxidases and angiotensin II receptor signaling. Mol Cell Endocrinol, 2008, 10.1016/j.mce.2008.11.003.
16.
GavazziG, BanfiB, DeffertC, FietteL, SchappiM, HerrmannF, KrauseK. Decreased blood pressure in NOX1-deficient mice. FEBS Lett, 580:497–504. 2006.
17.
GavazziG, DeffertC, TrocmeC, SchappiM, HerrmannFR, KrauseK-H. NOX1 deficiency protects from aortic dissection in response to angiotensin II. Hypertension, 50:189–196. 2007.
18.
GriendlingK. Novel NAD(P)H oxidases in the cardiovascular system. Heart, 90:491–493. 2004.
19.
GriendlingK, DelafontaineP, RittenhouseS, GimbroneMJr, AlexanderR. Correlation of receptor sequestration with sustained diacylglycerol accumulation in angiotensin II-stimulated cultured vascular smooth muscle cells. J Biol Chem, 262:14555–14562. 1987.
20.
GriendlingK, MinieriC, OllerenshawJ, AlexanderR. Angiotensin II stimulates NADH and NADPH oxidase activity in cultured vascular smooth muscle cells. Circ Res, 74:1141–1148. 1994.
21.
HilenskiLL, ClempusRE, QuinnMT, LambethJD, GriendlingKK. Distinct subcellular localizations of Nox1 and Nox4 in vascular smooth muscle cells. Arterioscler Thromb Vasc Biol, 24:677–683. 2004.
22.
IshizakaN, GriendlingKK, LassegueB, AlexanderRW. Angiotensin II type 1 receptor: relationship with caveolae and caveolin after initial agonist stimulation. Hypertension, 32:459–466. 1998.
23.
KimuraA, MoraS, ShigematsuS, PessinJE, SaltielAR. The insulin receptor catalyzes the tyrosine phosphorylation of caveolin-1. J Biol Chem, 277:30153–30158. 2002.
24.
KurodaJ, NakagawaK, YamasakiT, NakamuraK, TakeyaR, KuribayashiF, Imajoh-OhmiS, IgarashiK, ShibataY, SueishiK, SumimotoH. The superoxide-producing NAD(P)H oxidase Nox4 in the nucleus of human vascular endothelial cells. Genes Cells, 10:1139–1151. 2005.
25.
LambethJD. Nox enzymes, ROS, and chronic disease: an example of antagonistic pleiotropy. Free Radic Biol Med, 43:332–347. 2007.
26.
LassegueB, GriendlingKK. Reactive oxygen species in hypertension: an update. Am J Hypertens, 17:852–860. 2004.
LeclercPC, Auger-MessierM, LanctotPM, EscherE, LeducR, GuillemetteG. A polyaromatic caveolin-binding-like motif in the cytoplasmic tail of the type 1 receptor for angiotensin II plays an important role in receptor trafficking and signaling. Endocrinology, 143:4702–4710. 2002.
29.
LyleAN, GriendlingKK. Modulation of vascular smooth muscle signaling by reactive oxygen species. Physiology, 21:269–280. 2006.
30.
MatsunoK, YamadaH, IwataK, JinD, KatsuyamaM, MatsukiM, TakaiS, YamanishiK, MiyazakiM, MatsubaraH, Yabe-NishimuraC. Nox1 is involved in angiotensin ii-mediated hypertension: a study in Nox1-deficient mice. Circulation, 112:2677–2685. 2005.
31.
PartonRG, Hanzal-BayerM, HancockJF. Biogenesis of caveolae: a structural model for caveolin-induced domain formation. J Cell Sci, 119:787–796. 2006.
32.
PartonRG, SimonsK. The multiple faces of caveolae. Nat Rev Mol Cell Biol, 8:185–194. 2007.
RojasA, FigueroaH, ReL, MoralesMA. Oxidative stress at the vascular wall. mechanistic and pharmacological aspects. Arch Med Res, 37:436–448. 2006.
35.
RubanyiGM, VanhouttePM. Superoxide anions and hyperoxia inactivate endothelium-derived relaxing factor. Am J Physiol Heart Circ Physiol, 250:H822–H827. 1986.
36.
SchmelterM, AteghangB, HelmigS, WartenbergM, SauerH. Embryonic stem cells utilize reactive oxygen species as transducers of mechanical strain-induced cardiovascular differentiation. FASEB J, 20:1182–1184. 2006.
37.
SerranderL, CartierL, BedardK, BanfiB, LardyB, PlastreO, SienkiewiczA, ForroL, SchlegelW, KrauseK. NOX4 activity is determined by mRNA levels and reveals a unique pattern of ROS generation. Biochem J, 406:105–114. 2007.
38.
SorescuD, WeissD, LassegueB, ClempusRE, SzocsK, SorescuGP, ValppuL, QuinnMT, LambethJD, VegaJD, TaylorWR, GriendlingKK. Superoxide production and expression of Nox family proteins in human atherosclerosis. Circulation, 105:1429–1435. 2002.
39.
TaniyamaY, Ushio-FukaiM, HitomiH, RocicP, KingsleyMJ, PfahnlC, WeberDS, AlexanderRW, GriendlingKK. Role of p38 MAPK and MAPKAPK-2 in angiotensin II-induced Akt activation in vascular smooth muscle cells. Am J Physiol Cell Physiol, 287:C494–C499. 2004.
40.
TouyzRM, HeG, DengL-Y, SchiffrinEL. Role of extracellular signal-regulated kinases in angiotensin II–stimulated contraction of smooth muscle cells from human resistance arteries. Circulation, 99:392–399. 1999.
41.
Ushio-FukaiM. Localizing NADPH oxidase-derived ROS. Sci STKE, 2006:re8. 2006.
42.
Ushio-FukaiM, AlexanderR. Reactive oxygen species as mediators of angiogenesis signaling: role of NAD(P)H oxidase. Mol Cell Biochem, 264:85–97. 2004.
43.
Ushio-FukaiM, AlexanderRW. Caveolin-dependent angiotensin II type 1 receptor signaling in vascular smooth muscle. Hypertension, 48:797–803. 2006.
44.
Ushio-FukaiM, GriendlingKK, BeckerPL, HilenskiL, HalleranS, AlexanderRW. Epidermal growth factor receptor transactivation by angiotensin II requires reactive oxygen species in vascular smooth muscle cells. Arterioscler Thromb Vasc Biol, 21:489–495. 2001.
45.
Ushio-FukaiM, ZuoL, IkedaS, TojoT, PatrushevNA, AlexanderRW. cAbl tyrosine kinase mediates reactive oxygen species- and caveolin-dependent AT1 receptor signaling in vascular smooth muscle: role in vascular hypertrophy. Circ Res, 97:829–836. 2005.
WyseBD, PriorIA, QianH, MorrowIC, NixonS, MunckeC, KurzchaliaTV, ThomasWG, PartonRG, HancockJF. Caveolin interacts with the angiotensin II type 1 receptor during exocytic transport but not at the plasma membrane. J Biol Chem, 278:23738–23746. 2003.
48.
ZhaoH, KalivendiS, ZhangH, JosephJ, NithipatikomK, Vásquez-VivarJ, KalyanaramanB. Superoxide reacts with hydroethidine but forms a fluorescent product that is distinctly different from ethidium: potential implications in intracellular fluorescence detection of superoxide. Free Radic Biol Med, 34:1359–1368. 2003.
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