This Article Abstract Full Text (PDF) Methods Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Ruiz-Ortega, M. Articles by Egido, J. Search for Related Content PubMed PubMed Citation Articles by Ruiz-Ortega, M. Articles by Egido, J. Related Collections Mechanism of atherosclerosis/growth factors ACE/Angiotension receptors Cell signalling/signal transduction Gene regulation Hypertension - basic studies

(Circulation Research. 2000;86:1266.)
© 2000 American Heart Association, Inc.

Molecular Medicine

Angiotensin II Activates Nuclear Transcription Factor B Through AT₁ and AT₂ in Vascular Smooth Muscle Cells

Molecular Mechanisms

Marta Ruiz-Ortega, Oscar Lorenzo, Mónica Rupérez, Sven König, Burghardt Wittig, Jesús Egido

From the Vascular and Renal Research Laboratory (M.R.-O., O.L., M.R., J.E.), Fundación Jimenez Diaz, Universidad Autónoma Madrid, Spain, and Department of Molecular Biology and Bioinformatics (S.K., B.W.), Freie Universität Berlin, Germany.

Correspondence to Marta Ruiz-Ortega, PhD, Renal and Vascular Research Laboratory, Fundación Jiménez Díaz, Avda Reyes Católicos 2, 28040 Madrid, Spain. E-mail mruizo{at}fjd.es

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Abstract—Nuclear factor-B (NF-B) regulates many genes involved in vascular physiopathology. We have previously observed in vivo NF-B activation in injured vessels that diminished by angiotensin-converting enzyme inhibition. In the present work, we investigated the effect of angiotensin II (Ang II) on NF-B activity in rat vascular smooth muscle cells, evaluating the molecular mechanisms and the specific receptor subtype involved. Ang II increased NF-B DNA binding (5-fold, 10^-9 mol/L at 1 hour; electrophoretic mobility shift assay), nuclear translocation of p50/p65 subunits, and cytosolic inhibitor B (IB) degradation. Ang II elicited NF-B–mediated transcription (transfection of a reporter gene) and expression of NF-B–related genes (monocyte chemoattractant protein-1 and angiotensinogen). AT₁ (DUP753) and AT₂ (PD123319 and CGP42112) receptor antagonists inhibited Ang II–induced NF-B DNA binding in a dose-dependent manner (85% for each one; 10^-5 mol/L at 1 hour). The AT₂ agonist p-aminophenylalanine⁶–Ang II augmented NF-B binding (4.6-fold, 10^-9 mol/L at 1 hour), p65 nuclear levels, and transcription of an NF-B reporter gene. AT₁ antagonist markedly inhibited NF-B–mediated transcription and gene expression. Some differences between AT₁/AT₂ intracellular signals were found. Antioxidants and ceramide inhibitors, but not protein kinase C inhibitors, diminished NF-B activation elicited by both Ang II and the AT₂ agonist, while tyrosine kinase inhibitors only decreased Ang II–induced NF-B activity. Our results demonstrate that Ang II activates NF-B via AT₁ and AT₂, although NF-B–mediated transcription occurred mainly through AT₁. Both receptors share some signaling pathways (oxygen radicals and ceramide); however, tyrosine kinases only participate in AT₁/NF-B responses. These data provide novel insights into Ang II actions, suggesting a potential implication of the AT₂ in the pathobiology of vascular cells.

Key Words: angiotensin II • nuclear factor-B • receptors • vascular smooth muscle cell

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Angiotensin II (Ang II) is the main effector peptide of the renin-angiotensin system that plays an important role in several cardiovascular diseases associated with vascular smooth muscle cell (VSMC) growth and inflammation, including hypertension, atherosclerosis, restenosis after balloon injury, and myocardial infarction.¹ Ang II exerts its biological effects through the stimulation of specific receptors located on the cell surface. The use of selective antagonists has revealed the heterogeneity of Ang II receptors (AT-Rs),² and different cDNAs corresponding to each receptor have been isolated.^{2 3} AT₁ mediates many important cardiovascular responses, such as vasoconstriction, vascular and cardiac remodeling, and cell survival/death^{1 2 3} and evokes several intracellular signals such as calcium mobilization and activation of protein kinases, including protein kinase C (PKC) and mitogen-activated protein (MAP) cascade.⁴ AT₂ is involved in some Ang II actions, including apoptosis and inflammatory cell recruitment, and elicits different second messengers, such as MAP phosphatase activation and kinase inhibition.^{3 5}

Recent studies have shown that Ang II activates some nuclear transcription factors. In VSMCs, Ang II activates the signal transducer and activator transcription factor (STAT) and activator protein-1 (AP-1) through AT₁.^{6 7} We have recently demonstrated that Ang II activates nuclear factor-B (NF-B) in VSMCs and mesangial cells.^{8 9} However, AT-R subtype and molecular mechanisms of this process have not been elucidated. NF-B could play an important role in cardiovascular pathophysiology through the regulation of several genes, including cytokines, adhesion proteins, NO synthase, and angiotensinogen, as well as other products involved in atherosclerosis, inflammation, proliferation, and immune response.^{10 11} Elevated tissular NF-B activity has been described in an experimental model of atherosclerosis, correlated with increased macrophage infiltration and monocyte chemoattractant protein-1 (MCP-1) expression, which diminished by angiotensin-converting enzyme (ACE) inhibition,⁸ and also in a model of endothelial damage coincidentally with leukocyte adhesion molecule expression.¹² These data suggest that NF-B could be involved in the pathogenesis of several cardiovascular diseases, such as atherosclerosis and hypertension.

In the present study, we demonstrate that in cultured rat VSMCs, Ang II activates NF-B by a mechanism mediated by both AT₁ and AT₂. These results show new concepts of AT-R–mediated cell signaling in the cardiovascular system and could be important for a better understanding of the implication of Ang II in the pathogenesis of vascular damage.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Thoracic aortic rat VSMCs were serum starved for 48 hours and used for experiments. AT₂ and angiotensinogen mRNA expression were analyzed by reverse transcriptase–polymerase chain reaction (RT-PCR), and MCP-1 expression was analyzed by Northern blot.

Western Blot and Immunohistochemistry for AT-R
Total proteins were resolved in 12% SDS-PAGE gels, transferred, blocked, and incubated with specific AT₂ and AT₁ antibodies for 18 hours at 4°C. Detection was performed with peroxidase-conjugated secondary antibody and developed using an enhanced chemiluminescence kit (Amersham). For immunoperoxidase staining, cells were fixed in methanol/acetone at –20°C and incubated with primary antibodies and then with peroxidase-conjugated secondary antibody.

NF-B DNA Binding Activity
Nuclear and cytosolic extracts were prepared by homogenization and centrifugation.^{8 9} NF-B activity was determined in nuclear extracts by binding with labeled NF-B consensus and analyzed by electrophoretic mobility shift assay (EMSA). To quantify nuclear p50 and p65 levels and cytosolic IB and IBß, Western blot analyses were done. For immunofluorescence staining, cells were fixed in 3% paraformaldehyde for 10 minutes on ice followed by 0.1% Triton X-100 for 1 minute and then incubated with antibodies against p50/p65 subunits and with FITC-labeled IgG as secondary antibody.

Transient Transfections and Luciferase Assay
Double transient transfections of growth-arrested VSMCs with NF-B/luc and thymidine kinase (TK)–Renilla were performed by particle-mediated gene transfer, with the Biolistic PDS-1000/He System (Bio-Rad Laboratories) and gold microcarriers coated with DNA, into quiescent VSMCs.¹³ After transfection, cells were serum starved for 24 hours before stimulation. Lysates were assayed for luciferase and Renilla activities (Promega).

Statistical Analysis
Results are expressed as n-fold increase over control in densitometric arbitrary units and as mean±SEM of the experiments performed. Significance was established using the GraphPAD Instat program with the Student t test; differences were considered significant if the P value was <0.05.

An expanded Materials and Methods section is available online at http://www.circresaha.org.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Ang II Activates NF-B Through AT₁ and AT₂ in Cultured Rat VSMCs
Ang II increased NF-B DNA binding activity at as early as 30 minutes, peaked at 1 hour, and declined by 2 hours. The maximal response was observed with 10^-9 mol/L Ang II (5-fold over control, n=10, P<0.05), with an intensity similar to that of 100 U/mL tumor necrosis factor- (TNF-) (Figure 1A). VSMCs were preincubated with specific AT-R antagonists, DUP753 for AT₁ and PD123319 for AT₂. Both antagonists partially blocked the Ang II–induced NF-B DNA binding activity at all time points. The maximal inhibitory effect was found after 30 minutes (Figure 1B). The inhibitory effect of each antagonist was dose dependent, being maximal with 10^-5 mol/L (88% and 84% inhibition versus Ang II alone, for DUP753 and PD123319, respectively, after 1 hour; n=6, P<0.05) (Figure 1C). When both antagonists were added simultaneously, a marked inhibition of the Ang II effect was observed (96% inhibition, 10^-5 mol/L, n=3). Neither DUP753 nor PD123319 alone significantly affected NF-B activation in unstimulated cells (0.94- and 1-fold over control, respectively; n=6, P=NS) (Figure 1C). In VSMCs, CGP42112 has been used to demonstrate AT₂ binding sites.¹⁴ CGP42112 alone activated NF-B, maximal at 10^-5 mol/L (4.5-fold over control; n=4, P=0.05) showing an agonist effect, as previously reported.² CGP42112 blocked Ang II action (10^-5 mol/L, 82% inhibition versus Ang II alone, at 1 hour; n=4, P<0.05). To further demonstrate that the receptor subtype was associated with NF-B activation, we used an AT₂ agonist, p-aminophenylalanine⁶–Ang II (pNH₂FAII).¹⁵ Treatment for 1 hour with pNH₂FAII increased NF-B DNA binding activity in a dose-dependent manner (Figure 1D), with a maximal response at 10^-9 mol/L (4.6-fold, n=4, P<0.05). The specificity was demonstrated by the fact that only PD123319 blocked the pNH₂FAII-induced NF-B activation (Figure 1D). On the whole, these results suggest that Ang II–induced NF-B DNA binding activity was mediated by both AT₁ and AT₂.

View larger version (17K): [in this window] [in a new window]   Figure 1. A, Effect of Ang II on NF-B activity in VSMCs. VSMCs were incubated with Ang II (10-9 mol/L) for 30, 60, and 120 minutes. In parallel experiments, cells were treated for 60 minutes with Ang II (10-7 and 10-9 mol/L) and TNF- (100 U/mL). Competition assays with a 100-fold excess of unlabeled or mutant NF-B and unrelated (AP-1) oligonucleotides show specific NF-B complexes. Ang II activates NF-B through AT1 and AT2. B, Time course. Cells were preincubated for 30 minutes with the AT1 DUP753 (10-6 mol/L) and the AT2 PD123319 (10-5 mol/L) antagonists and then stimulated with 10-9 mol/L Ang II for 30 and 60 minutes. C, Dose response. Cells were pretreated for 30 minutes with DUP753 and PD123319 alone (10-5 to 10-7 mol/L) or in combination (10-5 mol/L), and then stimulated with 10-9 mol/L Ang II for 60 minutes. Both antagonists at 10-5 mol/L had no effect on NF-B activity in control cells. D, AT2 agonist activates NF-B. VSMCs were incubated with pNH2FAII (10-7 to 10-11 mol/L) for 60 minutes. At some points, cells were pretreated with DUP753 or PD123319 (10-5 mol/L). Upper panels show representative autoradiograms from 7 to 10 different EMSA experiments with similar results. Positions of specific NF-B complexes and free oligonucleotide are indicated (arrows). Lower panels show values of mean±SEM obtained by densitometric analysis. *P<0.05 vs control; §P=NS vs control; P<0.05 vs Ang II; P<0.05 vs pNH2FAII.

We have also observed that, in our experimental conditions, VSMCs express AT₂ at gene and protein levels (see online Materials and Methods, available at http://www.circresaha.org). These data are in agreement with previous studies showing detectable AT₂ binding sites in cultured VSMCs.¹⁴

Ang II Translocates p50/p65 NF-B Complexes Into the Nuclei and Degrades Cytosolic Inhibitor B (IB)
We have studied the composition of NF-B complexes induced by Ang II in rat VSMCs (see online Materials and Methods, available at http://www.circresaha.org). By supershift assays, we have observed that the NF-B complex activated is a p50/p65 heterodimer. After 1 hour of Ang II stimulation, a translocation of p50 and p65 subunits from cytosol to nuclei was observed (immunofluorescence and Western blot). pNH₂FAII upregulated nuclear p50 and p65 levels, with a maximal response at 10^-9 mol/L and with an intensity and kinetics similar to those of Ang II. These data suggest that AT₂ is involved in the transcriptional regulation of NF-B–controlled genes. NF-B activation involves dissociation of IB by phosphorylation and subsequent degradation.¹⁰ On Ang II stimulation, cytosolic IB was rapidly degraded, whereas IBß remained unchanged. This effect was closely correlated with the time course of Ang II on NF-B activation and with the translocation of p50/p65 to the nuclei. After 2 hours, Ang II treatment increased cytosolic IB levels, probably because of new protein synthesis. When cells were pretreated with either AT₁ or AT₂ antagonists, an inhibition of Ang II–induced IB degradation was observed (Figure 2), suggesting that both receptors participate in this process.

View larger version (44K): [in this window] [in a new window]   Figure 2. Role of AT-R antagonists on IB. Cells were preincubated for 30 minutes with DUP753 (10-6 mol/L) and PD123319 (10-5 mol/L) and then stimulated with 10-9 mol/L Ang II for 60 minutes. Upper panels, Representative experiment of 4 with comparable results; lower panel, mean±SEM. *P<0.05 vs control.

Molecular Mechanisms of Ang II–Induced NF-B Activation
We next investigated which intracellular signaling responses elicited by Ang II could be involved in NF-B activation in VSMCs, trying to elucidate differences between AT₁ and AT₂. For this reason, we have used different inhibitors (see online Materials and Methods, available at http://www.circresaha.org). PKC inhibitors did not modify the NF-B activation induced by Ang II or the AT₂ agonist (Figure 3A), suggesting that PKC is not involved in this process. Phosphotyrosine kinase (PTK) inhibitors caused a marked reduction in Ang II–induced NF-B DNA binding activity (genistein, 95% inhibition, at 10^-6 mol/L; n=4, P<0.05; Figure 3B). In contrast, they had no effect on the AT₂ agonist (Figure 3B), suggesting that activation of PTK could be involved in Ang II–induced NF-B response via AT₁. NF-B activation is also mediated by active oxygen radicals.¹⁶ Structurally diverse antioxidants markedly diminished the NF-B activation elicited by Ang II and the AT₂ agonist pNH₂FAII (Figure 3C). Another possible signaling pathway of NF-B activation could involve ceramide production.¹⁷ The inhibitor of ceramide synthase fumonisin B₁ inhibited the NF-B activation induced by Ang II and pNH₂FAII (Figure 3D), suggesting that ceramide could be a mediator of Ang II/AT₂–induced NF-B activation.

View larger version (21K): [in this window] [in a new window]   Figure 3. Molecular mechanisms of Ang II–induced NF-B activation. VSMCs were preincubated for 1 hour with inhibitors and then stimulated with 10-9 mol/L Ang II and/or pNH2FAII for 60 minutes. A, PKC inhibitors H-7 (10-5 mol/L) and bisindolylmaleimide (BIP) (10-7 mol/L). Effect on PKC agonist PMA (10-7 mol/L) was also evaluated. B, PTK inhibitor genistein (10-5 to 10-7 mol/L). C, Antioxidants superoxide dismutase (SOD; 10 g/L) and catalase (0.1 to 1 g/L). D, Ceramide production inhibitor fumonisin B1 (FB1; 10-5 to 10-6 mol/L). Shown in each panel is a representative EMSA of 3 performed. Lower panels, Mean±SEM obtained by densitometric analysis. *P<0.05 vs control; §P<0.05 vs PMA; P<0.05 vs Ang II; P<0.05 vs pNH2FAII.

Effect of Ang II on NF-B–Mediated Gene Transcription
To investigate the effects of the Ang II receptor antagonists on NF-B–mediated gene expression, two different strategies were followed; these were transient transfection with a reporter plasmid containing NF-B promoter binding sites and gene expression analysis of NF-B–related genes.

Growth-arrested VSMCs were cotransfected with NF-B/luc and TK-Renilla (internal control) by Biolistic gene transfer. Then, cells were serum starved for 24 hours before stimulation with 10^-7 mol/L Ang II or phorbol 12-myristate 13-acetate (PMA) for an additional 24 hours, and luciferase activity was measured. Ang II activated the expression of the reported NF-B/luc plasmid (6-fold versus control; n=5, P<0.05), with a similar response to PMA. No increase was seen with the control plasmid (not shown). pNH₂FAII also increased luciferase activity (3.7-fold, P<0.05; Figure 4). When cells were pretreated with either AT₁ or AT₂ antagonists, a partial decrease of Ang II–induced NF-B–mediated transcription was observed that was completely blocked when both antagonists were added together (Figure 4, 95% inhibition versus Ang II alone; n=4, P<0.05). Interestingly, the inhibitory effect of DUP753 was higher than that of PD123319, suggesting that although Ang II increases NF-B–mediated transcription through AT₁ or AT₂, the AT₁/NF-B pathway seems to be more active.

View larger version (18K): [in this window] [in a new window]   Figure 4. Transient transfection of the NF-B promoter in quiescent VSMCs. Quiescent cells were treated with 10-7 mol/L Ang II, PMA, or pNH2FAII for 24 hours. Cells were pretreated for 30 minutes with DUP753 and PD123319 alone (10-5 mol/L) or in combination and then stimulated with 10-7 mol/L Ang II for 24 hours. Data are expressed as n-fold increase vs control, corrected by the Renilla values. Data are mean±SEM of 4 experiments performed in triplicate. *P<0.05 vs control; P<0.05 vs Ang II.

We further evaluated the role of AT-R in some NF-B–controlled genes. Ang II stimulation increased MCP-1 mRNA levels at as early as 3 hours, being maximal at 6 hours (10^-7 mol/L; 5-fold versus control, n=5, P<0.05, Northern blot) and diminished after 24 hours, as previously shown.^{8 9} DUP753 significantly diminished Ang II–induced MCP-1 gene expression (Figure 5), whereas PD123319 only produced a slight decrease. pNH₂FAII also increased MCP-1 mRNA but to a lesser extent than Ang II (Figure 5). In addition, inhibitors of NF-B activation, MG132 (Figure 5B), gliotoxin, and pyrrolidine dithiocarbamate (not shown) diminished MCP-1 mRNA induction caused by Ang II. By RT-PCR, Ang II upregulated angiotensinogen mRNA at 6 hours, which was only diminished by DUP753 (Figure 5B). These data suggest that MCP-1 and angiotensinogen gene expression were mainly mediated by AT₁.

View larger version (45K): [in this window] [in a new window]   Figure 5. A, Analysis of MCP-1 mRNA expression in VSMCs. Quiescent cells were treated with 10-7 mol/L Ang II for 6 hours. TNF- (100 U/mL) was used as positive control. Cells were pretreated for 30 minutes with DUP753 and PD123319 and then stimulated with 10-7 mol/L Ang II for an additional 6 hours. Left panels, Effect of NF-B inhibitor (MG132; 10-5 mol/L) on MCP-1 mRNA expression induced by Ang II and AT2 agonist. MCP-1 (upper panels) and GAPDH (middle) mRNA expression was determined by Northern blot. Shown is a representative autoradiogram from 5 different experiments with identical results. Lower panels, Mean±SEM obtained by densitometric analysis. *P<0.05 vs control values. B, Ang II increased angiotensinogen gene expression via AT1. Quiescent VSMCs constitutively express angiotensinogen (Ao) mRNA as shown by RT-PCR. Cells were stimulated for 6 hours with 10-7 mol/L Ang II. Shown is a representative RT-PCR of 2 performed; GAPDH was used as internal control.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
Ang II acts through two specific receptor subtypes, AT₁ and AT₂. AT₁ predominates in vascular tissues and is responsible for most of the physiopathological actions of Ang II.^{2 3} AT₂ is mainly present in fetal tissues and is reexpressed in pathological conditions, such as myocardial infarction and cardiac hypertrophy,³ as well as in cultured VSMCs,¹⁴ as shown in this article. Pharmacological and molecular tools are contributing to the elucidation of the AT₁/AT₂ functions. Specific receptor antagonists have demonstrated that AT₁ is involved in cell proliferation, production of cytokines, and matrix proteins,^{2 3 18} and in the pathogenesis of Ang II–induced hypertension and cardiac hypertrophy.^{19 20} AT₂ participates in cell growth inhibition, neointimal formation after vascular injury, trophic effects of VSMCs, and blood pressure control.³ Some Ang II responses could be mediated by both receptors, including NO release, collagen synthesis and -2-adrenoreceptor activity.^{21 22 23} In VSMCs we have demonstrated that both AT₁ and AT₂ activate nuclear NF-B DNA binding activity that is functional in its ability to transactivate B-containing promoters. The AT₁ antagonist markedly inhibited NF-B–mediated gene transcription. Ang II via AT₁ increases interleukin (IL)–6 and angiotensinogen mRNA through an NF-B–mediated transcriptional mechanism.^{11 18} In the same way, Ang II–induced MCP-1 mRNA was diminished by AT₁ antagonist and by different NF-B inhibitors. However, AP-1 and NF-B cooperate in the MCP-1/IL-1ß response,²⁴ showing a potential role for other transcription factors. Ang II–induced hypertension in rats is characterized by marked monocyte infiltration and vascular cell adhesion molecule and MCP-1 expression in the aorta.^{25 26} ACE inhibitors reduce the presence of monocyte/macrophages in the vessel wall of hypertensive rats.²⁷ In an experimental model of atherosclerosis, ACE inhibition diminished NF-B activity and chemokine expression in the lesion.⁸ All of these data suggest that Ang II could contribute to inflammatory events in atherosclerosis and hypertension through vascular inflammatory genes, by the AT₁/NF-B pathway. In contrast, we have observed that an AT₂ agonist increased NF-B DNA binding and mediated gene transcription. AT₂ upregulates the chemokine RANTES and renal inflammatory cell recruitment,⁵ showing a possible gene target for the AT₂/NF-B pathway. These data suggest that Ang II, acting mainly via AT₁, and in particular conditions through AT₂, could regulate several NF-B–related genes involved in the pathogenesis of cardiovascular diseases.

NF-B has been implicated in the transcription of genes mediating cell growth control, but its role in growth regulation remains to be established. The development of atherosclerotic fibrous plaques is due to activation of VSMCs, which proliferate and increase matrix deposition. Activation of NF-B has been observed in human atherosclerotic lesions and in cultured VSMCs in a proliferative state.^{8 12} Apoptosis of VSMCs and macrophages has been found in atherosclerotic lesions.²⁸ In most cell types, NF-B mediates cell survival signals, protecting cells from apoptosis, but under certain conditions it may also induce apoptosis.²⁹ In vitro experiments suggest that Ang II may cause growth via AT₁ and apoptosis via AT₂. In vivo, stimulation of AT₁ or AT₂ causes apoptosis in the media of blood vessels.³⁰ Among the intracellular mechanisms elicited by AT₂, ceramide production seems to be involved in apoptosis³¹ and NF-B activation. A similar behavior was observed with TNF-, which increases ceramide production; activates NF-B; and, depending on cell culture conditions, causes cell proliferation or apoptosis. Although many studies have been done, future work is necessary to completely understand the in vivo relation between NF-B, cell growth/apoptosis, and the role of AT-R in these processes.

AT₁ and AT₂ are coupled to G proteins and belong to the 7-transmembrane-domain receptor family.² Although the intracellular signalings elicited after activation of the AT₁ and AT₂ are different, our data suggest that both receptors share a common molecular pathway: the activation of NF-B. Many agents activate NF-B, but the mechanisms are not well understood.¹⁰ Recently, 2 different pathways leading to NF-B activation have been suggested, because sanguinarine blocked NF-B activation caused by okadaic acid, PMA, TNF-, and IL-1ß, but not by ceramides and H₂O₂.³² Reactive oxygen metabolites (reactive oxygen species; ROS) serve as common intracellular agents for NF-B by a wide range of stimuli. We have demonstrated that a variety of structurally diverse antioxidants blocked the activation of NF-B elicited by Ang II and AT₂ agonist, suggesting that ROS act as intermediates of both AT₁/AT₂, as occurs with TNF- and IL-1ß.³² Antioxidants also mediate other Ang II effects, mainly through AT₁, such as AP-1 activation, cell proliferation, and protein synthesis.^{2 3 7} Some differences between AT₁ and AT₂ signaling systems were found. PTK inhibitors diminished Ang II–induced NF-B activation but had no effect on AT₂ agonist, showing that PTK mediates only the AT₁/NF-B pathway. In contrast, PKC is not involved in Ang II–induced NF-B activation. The role of PKC on NF-B seems to be cell/stimulus specific, given that it mediates H₂O₂ action³³ but has no effect on TNF- and IL-1ß.³⁴ Some signaling mechanisms of AT₁ are similar to those used by cytokines, such as activation of the PTK, PKC, MAP kinase (MAPK), Janus kinase (JAK)/STAT, and AP-1 pathways.⁴ The JAK/STAT system activates ROS-sensitive PTK,¹⁶ which could participate in NF-B activation. MAPK is required for the activation of several transcription factors, including NF-B,³⁵ but how p38 MAPK may affect NF-B function is unclear. The inhibitor of p38 MAPK SB203580 abolished TNF-–induced cytokine synthesis and blocked NF-B–mediated luciferase transactivation in response to TNF- without affecting NF-B translocation.³⁶ Ang II–induced MCP-1 gene expression via AT₁ is dependent on redox-sensitive signals and on activation of PTK and MAP.³⁷ All of these data suggest a potential mechanism of AT₁/NF-B/gene regulation, via ROS and PTK, that is common to proinflammatory cytokines as TNF- and IL-1ß. The AT₂ is linked to inhibition of MAP kinase, activation of protein-phosphotyrosine phosphatase, and changes in phospholipase A₂ activity.^{2 3} Phosphorylation of IB is a prerequisite for its degradation and subsequent liberation of active NF-B. For this reason, after stimulation of AT₂, we could expect activation of some phosphatases that may regulate IB degradation. However, AT₂ activates NF-B (DNA binding and gene transcription), suggesting that Ang II–induced phosphatases are not involved in this process. In addition, the phosphatase inhibitor okadaic acid activates NF-B by a process independent of phosphatase inhibition but dependent on ROS.²⁶ Ceramide production is a second messenger for AT₂, via G protein and phosphatase activation, and an essential step in programmed cell death.³¹ Moreover, ceramide generated by sphingomyelin breakdown by sphingomyelinase mediated TNF- and IL-1ß NF-B activation.¹⁷ Given that a ceramide inhibitor diminished Ang II and AT₂-induced NF-B activation, ceramide could be a potential mediator of the AT₂/NF-B pathway.

In summary, our results show that in cultured rat VSMCs, AT₁ and AT₂ mediate Ang II–induced NF-B activation. Both receptors share some intracellular signals in the Ang II /NF-B pathway, such as ROS and ceramide production, but PTK only mediates the AT₁/NF-B pathway. These signaling mechanisms are similar to those used by proinflammatory cytokines, such as IL-1ß and TNF-, providing a point for "cross talk" between Ang- and cytokine-activated second messenger pathways, and supporting the emerging idea of Ang II as a true cytokine. Ang II–mediated gene transcription occurred mainly through AT₁, because the AT₁ antagonist markedly inhibited NF-B–mediated gene transcription and Ang II–induced overexpression of related genes, such as angiotensinogen, chemokines (MCP-1), cytokines,¹⁸ and adhesion molecules,²⁶ involved in the development of atherosclerosis. Finally, our data also show a novel action of AT₂, the activation of NF-B, which suggests a potential involvement in the pathogenesis of cardiovascular diseases.

	Acknowledgments

 
This work has been supported by grants from Fondo de Investigación Sanitaria (95/0093, 96/2021, 99/0425), Comunidad Autonoma de Madrid (97/084/0003), and Ministerio de Educación y Ciencia (SAF 97/0055, PM97/0085); by European Union Concerted Action Grant BMH 4-CT98-3631 (DG 12-SSM1); and by Fudación Renal Iñigo Alvarez de Toledo. M.R.-O. is a postdoctoral fellow of CAM; O.L. is a fellow of FIS. We thank Drs C. Caramelo and M. Ortego for careful reading of the manuscript and L. Gulliksen for her secretarial assistance.

Received March 24, 2000; accepted May 1, 2000.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Baker KM, Booz GW, Dostal DE. Cardiac actions of angiotensin II: role of an intracardiac renin-angiotensin system. Annu Rev Physiol. 1992;54:227–241.[Medline] [Order article via Infotrieve]
2. Timmermans PBMWM, Wong PC, Chiu AT, Herblin WF, Benfield P, Carini DJ, Lee RJ, Wexler RW, Saye JAM, Smith RD. Angiotensin II receptors and angiotensin II receptor antagonist. Pharmacol Rev. 1993;45:205–251.[Medline] [Order article via Infotrieve]
3. Matsubara H. Pathophysiological role of angiotensin II type 2 receptor in cardiovascular and renal diseases. Circ Res. 1998;83:1182–1191.[Abstract/Free Full Text]
4. Griedling KK, Ushio-Fukai M, Lassègue B, Alexander RW. Angiotensin II signaling in vascular smooth muscle cells. Hypertension. 1997;29:366–373.[Abstract/Free Full Text]
5. Wolf G, Ziyadeh FN, Thaiss F, Tomaszewiski J, Caron RJ, Wenzel U, Zahner G, Helmchen U, Stahl RAK. Angiotensin II stimulates expression of the chemokine RANTES in rat glomerular endothelial cells: role of the angiotensin type 2 receptor. J Clin Invest. 1997;100:1047–1058.[Medline] [Order article via Infotrieve]
6. Marrero MB, Schieffer B, Paxton WG, Heerdt L, Berk BC, Delafontaine P, Bernstein KE. Direct stimulation of Jak/STAT pathway by the angiotensin II AT1 receptor. Nature. 1995;375:247–250.[Medline] [Order article via Infotrieve]
7. Takeuchi K, Nakemura N, Cook NS, Pratt RE, Dzau VJ. Angiotensin II can regulate gene expression by the AP-1 binding sequence via a protein kinase C-dependent pathway. Biochem Biophys Res Commun. 1990;172:1189–1194.[Medline] [Order article via Infotrieve]
8. Hernández-Presa M, Bustos C, Ortego M, Tuñón J, Ruiz-Ortega M, Egido J. Angiotensin converting enzyme inhibition prevents arterial NF-B activation, MCP-1 expression and macrophage infiltration in a rabbit model of early accelerated atherosclerosis. Circulation. 1997;95:1532–1541.[Medline] [Order article via Infotrieve]
9. Ruiz-Ortega M, Bustos C, Hernández-Presa MA, Lorenzo O, Plaza JJ, Egido J. Angiotensin II participates in mononuclear cell recruitment in the kidney through nuclear factor-B activation and monocyte chemoattractant protein-1 gene expression. J Immunol. 1998;161:430–439.[Abstract/Free Full Text]
10. Barnes PJ, Karin M. Nuclear factor-B: a pivotal transcription factor in chronic inflammatory diseases. N Engl J Med. 1997;336:1066–1071.[Free Full Text]
11. Brasier AR, Li J. Mechanisms for inducible control of angiotensinogen gene transcription. Hypertension. 1996;27:465–475.[Abstract/Free Full Text]
12. Linder V, Collins T. Expression of NF-B and IB by aortic endothelium in an arterial injury model. Am J Pathol. 1996;148:427–438.[Abstract]
13. Kalkbrenner F, Dippel E, Schroff M, Wittig B, Schultz G. Nuclear application of antisense oligonucleotides by microinjection and ballistomagnetic transfer to identify G protein heterotrimers activating phospholipase C. Methods Mol Biol. 1997;83:203–216.[Medline] [Order article via Infotrieve]
14. Kambayashi H, Bardham S, Inagami T. Peptide growth factors markedly decrease the ligand binding of angiotensin type-2 receptor in rat cultured vascular smooth muscle cells. Biochem Biophys Res Commun. 1993;194:478–482.[Medline] [Order article via Infotrieve]
15. Speth RC, Kim KH. Discrimination of two angiotensin II receptor subtypes with a selective agonist analogue of angiotensin II, p-aminophenylalanine6-angiotensin II. Biochem Biophys Res Commun. 1990;169:997–1006.[Medline] [Order article via Infotrieve]
16. Sen CK, Packer L. Antioxidants and redox regulation of gene transcription. FASEB J. 1996;10:709–720.[Abstract]
17. Schutze S, Potthoff K, Machleidt T, Berkovic D, Wiegmann K, Kronke M. TNF activates NF-B by phosphatidylcholine-specific phospholipase C-induced "acidic" sphingomyelin breakdown. Cell. 1992;71:765–776.[Medline] [Order article via Infotrieve]
18. Han Y, Runge MS, Brasier AR. Angiotensin II induces interleukin-6 transcription in vascular smooth muscle cells through pleiotropic activation of nuclear factor-B transcription factors. Circ Res. 1999;84:695–703.[Abstract/Free Full Text]
19. Crawford DC, Chobanian AV, Brecher P. Angiotensin II induces fibronectin expression associated to cardiac fibrosis in the rat. Circ Res. 1994;74:727–739.[Abstract]
20. Sadoshima J, Izumo S. Molecular characterization of angiotensin II-induced hypertrophy of cardiac myocytes and hyperplasia of cardiac fibroblasts: critical role of the AT1 receptor subtype. Circ Res. 1993;73:413–423.[Abstract]
21. Seyedi N, Xu X, Nasjletti A, Hintze TH. Coronary kinin generation mediates nitric oxide release after angiotensin receptor stimulation. Hypertension. 1995;26:164–170.[Abstract/Free Full Text]
22. Brilla CG, Zhou G, Matsubara L, Weber KT. Collagen metabolism in cultured adult rat cardiac fibroblasts: response to angiotensin II and aldosterone. J Mol Cell Cardiol. 1994;26:809–820.[Medline] [Order article via Infotrieve]
23. Yang CH, Shyr MH, Tan PP, Chan SH. Participation of AT1 and AT2 receptors in the differential interaction between angiotensin II or III and -2 adrenoceptors in the nucleus reticularis gigantocellularis in cardiovascular regulation and antinociception in rats. J Pharmacol Exp Ther. 1996;279:795–802.[Abstract/Free Full Text]
24. Martin T, Cardarelli PM, Parry GC, Felts KA, Cobb RR. Cytokine induction of monocyte chemoattractant protein-1 gene expression in human endothelial cells depends on the cooperative action of NF-B and AP-1. Eur J Immunol. 1997;27:1091–1097.[Medline] [Order article via Infotrieve]
25. Capers Q 4th, Alexander RW, Lou P, De Leon H, Wilcox JN, Ishizaka N, Howard AB, Taylor WR Monocyte chemoattractant protein-1 expression in aortic tissues of hypertensive rats. Hypertension. 1997;30:1397–1402.[Abstract/Free Full Text]
26. Tummala PE, Chen XL, Sundell CL, Laursen JB, Hammes CP, Alexander RW, Harrison DG, Medford RM. Angiotensin II induces vascular cell adhesion molecule-1 expression in rat vasculature: a potential link between the renin-angiotensin system and atherosclerosis. Circulation. 1999;100:1223–1229.[Medline] [Order article via Infotrieve]
27. Clozel M, Kuhn H, Hefti F, Baumgartner HR. Endothelial dysfunction and subendothelial monocyte macrophages in hypertension: effect of angiotensin converting enzyme inhibition. Hypertension. 1991;18:132–141.[Abstract]
28. Blanco-Colio LM, Hernandez-Presa MA, Ortego M, Martin-Ventura JL, Tuñon J, Egido J. Potential role of apoptosis in atherosclerosis. Cardiovasc Risk Factors. 1999;9:169–174. Available at http://www.crf.medynet.com.
29. Barinaga M. Life-death balance within the cell. Science. 1996;274:724.[Free Full Text]
30. Diep QN, Li JS, Schiffrin EL. In vivo study of AT(1) and AT(2) angiotensin receptors in apoptosis in rat blood vessels. Hypertension. 1999;34:617–624.[Abstract/Free Full Text]
31. Lehtonen JYA, Horiuchi M, Daviet L, Akishita M, Dzau VJ. Activation of the de novo biosynthesis of sphingolipids mediates angiotensin II type 2 receptor-induced apoptosis. J Biol Chem. 1999;274:16901–16906.[Abstract/Free Full Text]
32. Chaturvedi MM, Kumar A, Darnay BG, Chainy GBN, Agarwal S, Agarwal BB. Sanguinarine (pseudochelerythrine) is a potent inhibitor of NF-B activation, I-B phosphorylation and degradation. J Biol Chem. 1997;272:30129–30134.[Abstract/Free Full Text]
33. Meichle A, Schutze S, Hensel G, Brunsing D, Kronke M. Protein kinase C-independent activation of nuclear factor-B by tumor necrosis factor. J Biol Chem. 1990;265:8339–8343.[Abstract/Free Full Text]
34. Barchowsky A, Munro SR, Morana SJ, Vincenti MP, Treadwell M. Oxidant-sensitive and phosphorylation-dependent activation of NF-B and AP-1 in endothelial cells. Am J Physiol. 1995;269:L829–L836.[Abstract/Free Full Text]
35. Garcia J, Lemercier B, Roman-Roman S, Rawadi G. A Mycoplasma fermentans-derived synthetic lipopeptide induces AP-1, and NF-B activity, and cytokine secretion in macrophages via the activation of mitogen-activated protein kinase pathways. J Biol Chem. 1998;273:34391–34398.[Abstract/Free Full Text]
36. Bergmann M, Hart L, Lindsay M, Barnes PJ, Newton R. IB degradation and nuclear factor-B DNA binding are insufficient for interleukin-1ß and tumor necrosis factor-–induced B-dependent transcription: requirement for an additional activation pathway. J Biol Chem. 1998;273:6607–6610.[Abstract/Free Full Text]
37. Chen XL, Tummala PE, Olbrych MT, Alexander RW, Medford RM. Angiotensin II induces monocyte chemoattractant protein-1 gene expression in rat vascular smooth muscle cells. Circ Res. 1998;83:952–959.[Abstract/Free Full Text]

This article has been cited by other articles:

				A. Yaren, I. Oztop, S. Turgut, G. Turgut, S. Degirmencioglu, and M. Demirpence Angiotensin-Converting Enzyme Gene Polymorphism Is Associated with Anemia in Non Small-Cell Lung Cancer Experimental Biology and Medicine, January 1, 2008; 233(1): 32 - 37. [Abstract] [Full Text] [PDF]

				N. D. Vaziri, Y. Bai, Z. Ni, Y. Quiroz, R. Pandian, and B. Rodriguez-Iturbe Intra-Renal Angiotensin II/AT1 Receptor, Oxidative Stress, Inflammation, and Progressive Injury in Renal Mass Reduction J. Pharmacol. Exp. Ther., October 1, 2007; 323(1): 85 - 93. [Abstract] [Full Text] [PDF]

				N. Henke, R. Schmidt-Ullrich, R. Dechend, J.-K. Park, F. Qadri, M. Wellner, M. Obst, V. Gross, R. Dietz, F. C. Luft, et al. Vascular Endothelial Cell Specific NF-{kappa}B Suppression Attenuates Hypertension-Induced Renal Damage Circ. Res., August 3, 2007; 101(3): 268 - 276. [Abstract] [Full Text] [PDF]

				L. Pattacini, B. Casali, L. Boiardi, N. Pipitone, L. Albertazzi, and C. Salvarani Angiotensin II protects fibroblast-like synoviocytes from apoptosis via the AT1-NF-{kappa}B pathway Rheumatology, August 1, 2007; 46(8): 1252 - 1257. [Abstract] [Full Text] [PDF]

				Y. Ozawa and H. Kobori Crucial role of Rho-nuclear factor-{kappa}B axis in angiotensin II-induced renal injury Am J Physiol Renal Physiol, July 1, 2007; 293(1): F100 - F109. [Abstract] [Full Text] [PDF]

				M. T. Coughlan, V. Thallas-Bonke, J. Pete, D. M. Long, A. Gasser, D. C. K. Tong, M. Arnstein, S. R. Thorpe, M. E. Cooper, and J. M. Forbes Combination Therapy with the Advanced Glycation End Product Cross-Link Breaker, Alagebrium, and Angiotensin Converting Enzyme Inhibitors in Diabetes: Synergy or Redundancy? Endocrinology, February 1, 2007; 148(2): 886 - 895. [Abstract] [Full Text] [PDF]

				P. K. Mehta and K. K. Griendling Angiotensin II cell signaling: physiological and pathological effects in the cardiovascular system Am J Physiol Cell Physiol, January 1, 2007; 292(1): C82 - C97. [Abstract] [Full Text] [PDF]

				Y. Ozawa, H. Kobori, Y. Suzaki, and L. G. Navar Sustained renal interstitial macrophage infiltration following chronic angiotensin II infusions Am J Physiol Renal Physiol, January 1, 2007; 292(1): F330 - F339. [Abstract] [Full Text] [PDF]

				J. L. ZHUO, O. A. CARRETERO, and X. C. LI Effects of AT1 Receptor-Mediated Endocytosis of Extracellular Ang II on Activation of Nuclear Factor-{kappa}B in Proximal Tubule Cells Ann. N.Y. Acad. Sci., December 1, 2006; 1091(1): 336 - 345. [Abstract] [Full Text] [PDF]

				G. Fernandez-Juarez, V. Barrio, S. G. de Vinuesa, M. Goicoechea, M. Praga, and J. Luno Dual Blockade of the Renin-Angiotensin System in the Progression of Renal Disease: The Need for More Clinical Trials J. Am. Soc. Nephrol., December 1, 2006; 17(12_suppl_3): S250 - S254. [Abstract] [Full Text] [PDF]

				H. Okada, T. Inoue, T. Kikuta, Y. Watanabe, Y. Kanno, S. Ban, T. Sugaya, M. Horiuchi, and H. Suzuki A Possible Anti-Inflammatory Role of Angiotensin II Type 2 Receptor in Immune-Mediated Glomerulonephritis during Type 1 Receptor Blockade Am. J. Pathol., November 1, 2006; 169(5): 1577 - 1589. [Abstract] [Full Text] [PDF]

				P. M Ridker, E. Danielson, N. Rifai, R. J. Glynn, and for the Val-MARC Investigators Valsartan, Blood Pressure Reduction, and C-Reactive Protein: Primary Report of the Val-MARC Trial Hypertension, July 1, 2006; 48(1): 73 - 79. [Abstract] [Full Text] [PDF]

				V Teplitsky, Y Shoenfeld, and A Tanay The renin-angiotensin system in lupus: physiology, genes and practice, in animals and humans Lupus, June 1, 2006; 15(6): 319 - 325. [Abstract] [PDF]

A. Douillette, A. Bibeau-Poirier, S.-P. Gravel, J.-F. Clement, V. Chenard, P. Moreau, and M. J. Servant The Proinflammatory Actions of Angiotensin II Are Dependent on p65 Phosphorylation by the I{kappa}B Kinase Complex J. Biol. Chem., May 12, 2006; 281(