A New Cellular Signaling Mechanism for Angiotensin II Activation of NF-B
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动脉硬化血栓血管生物学 2005年第6期
From the Departments of Internal Medicine (L.Z., Y.M., J.Z., J.C., J.D.) and Human Biological Chemistry and Genetics (J.D.), The University of Texas Medical Branch, Galveston.
Correspondence to Jie Du, Department of Internal Medicine, 9.138 Medical Research Building, 301 University Blvd, University of Texas Medical Branch, Galveston, TX 77555-1064. E-mail jidu@utmb.edu
Abstract
Objective— Angiotensin II (Ang II) promotes vascular inflammation and remodeling via activation of nuclear factor B (NF-B)–mediated transcription of proinflammatory genes such as interleukin-6 (IL-6). We examined the signaling mechanism whereby Ang II activates NF-B in vascular smooth muscle cells (VSMCs).
Methods and Results— Ang II treatment did not increase phosphorylation of inhibitor of B (IB) or IB? or decrease their levels. In contrast, mitogen-activated protein kinase kinase-1 (MEK1) inhibition (dominant-negative MEK1 adenovirus or inhibitor U0126) suppressed Ang II–induced NF-B promoter activity, NF-B DNA-binding activity, p65 phosphorylation, and led to 70% reduction in IL-6 transcription/production. The mechanism involved Ang II activation of Ras and MEK1. Signaling distal to MEK1 involved extracellular signal-regulated kinase (ERK) because inhibition of MEK1 suppressed the Ang II–induced activation of ribosomal S6 kinase (RSK), a substrate of ERK. Downregulation of RSK by small interfering RNA (SiRNA) in VSMCs was found to suppress Ang II–induced activation of NF-B and p65 phosphorylation. Immunopurified RSK from Ang II–treated VSMCs phosphorylated recombinant glutathione S-transferase–p65 in vitro.
Conclusion— We uncovered a nonclassical signaling pathway (Ras/MEK1/ERK/RSK) from Ang II to activation of NF-B, a mechanism by which Ang II stimulates RSK-mediated phosphorylation of p65 to participate in vascular inflammation.
We examined an intracellular mechanism whereby Ang II activates NF-B. Ang II caused no significant increase in IB phosphorylation or protein levels. Instead, we found a nonclassical signaling pathway, Ras/MEK1/ERK/RSK, that activated NF-B via phosphorylation of p65, leading to IL-6 production and suggesting a mechanism by which Ang II causes vascular inflammation.
Key Words: angiotensin II ? signaling pathway ? NF-B ? MAP kinase ? inflammation
Introduction
Angiotensin II has proinflammatory actions that increase the expression of chemokines and cellular adhesion molecules in vascular abnormalities, such as atherosclerosis,1,2 transplant vasculopathy,3,4 or restenosis injury.5 These actions of angiotensin II (Ang II) are attributed to activation of nuclear factor B (NF-B), a key transcription factor that stimulates the transcription of chemokines, as well as other genes. Several groups have demonstrated that Ang II activates NF-B in vivo and in vitro after binding to the Ang II type 1 (AT1) or AT2 receptor.6–8 Moreover, in an experimental model of atherosclerosis exhibiting increased inflammation, NF-B activity is increased in vascular tissue; this response was diminished by an angiotensin-converting enzyme inhibitor.9
The mechanism by which Ang II activates NF-B is unclear. The classical NF-B activation model involves sequestration of NF-B in the cytoplasm by inhibitor of B (IB), preventing NF-B from migrating to the nucleus. When IB is phosphorylated, it is degraded, permitting NF-B to translocate to the nucleus. However, several reports suggest that Ang II can stimulate degradation of IB, but the change in the level of IB was small.7,8,10 A nonclassical model suggests that Ang II induces tyrosine phosphorylation of IB to promote its physical dissociation from NF-B.11 In fact, there are observations suggesting that this or other nonclassical pathways are active. For example, the induction of NF-B by UV light does not require phosphorylation of IB.12 There are also reports that NF-B can be activated by phosphorylation of the NF-B p65 subunit.13,14 A study by Bohuslav et al demonstrated that p65 can be phosphorylated in the nucleus. This change reduces the ability of IB to bind to either a p65 homodimer or the p65/p50 heterodimer.15 Consequently, phosphorylated p65 can bind to NF-B consensus sequences in a promoter.
In exploring how Ang II regulates the transcriptional activity of NF-B, we found that inhibition of mitogen-activated protein kinase kinase-1 (MEK1) suppressed the transcriptional activity of NF-B. We explored the cellular signaling pathway linking Ang II to MEK and then to stimulation of NF-B transcriptional activity. This pathway involves Ras/MEK1/extracellular signal-regulated kinase (ERK)/ribosomal S6 kinase (RSK) and activates NF-B in vascular smooth muscle cells (VSMCs) by phosphorylating the p65 subunit of NF-B. Biologically, activation of this pathway contributes to Ang II–induced interleukin-6 (IL-6) expression that has been implicated in vascular inflammation.
Methods
Cell Culture
VSMCs were isolated from the thoracic aortas of male Sprague-Dawley rats by enzymatic digestion and then cultured in DMEM supplemented with 10% FBS. VSMCs were identified by positive staining with monoclonal antibody to smooth muscle -actin (Sigma-Aldrich).
Animals
Three-month-old male Sprague-Dawley rats were implanted with osmotic pumps (model 2001; Alza Corp). Animals in the Ang II–treated group received a pump filled with Ang II and delivered at a dosage of 500 ng/kg per minute in Ringer’s solution (0.01 mol/L acetic acid in saline). Another Ang II–treated group received antihypertensive therapy hydralazine (10 mg/kg per day) in drinking water. Control animals received a pump with Ringer’s solution without Ang II. Systolic blood pressure (SBP) was measured by tail-cuff plethysmography method (Visitech Systems Inc). SBP values were derived from an average of 6 to 8 measurements per animal at each time point. Each group of 6 rats was pair fed for 7 days and perfuse-fixed with 4% paraformaldehyde. The thoracic aorta was embedded in paraffin and examined.
NF-B Luciferase Reporter Assay
NF-B transcriptional activity was evaluated by using an adenovirus NF-B-Luc luciferase reporter (Ad.NF-B-Luc). Briefly, cells were infected with Ad.NF-B-Luc at a multiplicity of infection of 5 for 24 hours before 100 nmol/L Ang II treatment; from each sample, 5 μg of total protein was assayed for luciferase activity using the manufacturer protocol (Promega).
Electrophoretic Mobility Shift Analysis
Nuclear extracts were prepared as described16 and incubated with an NF-B oligonucleotide (Promega). The gel shift analysis was performed at least 3x.
Agarose Oligonucleotide Pull-Down Assay
The NF-B sequence from -192/-172 of intercellular adhesion molecule (ICAM) with 3-terminal biotinylation (5' GTTCCACGGCACCCCCTG3') and its complementary strand (5' CCTGCAGGGGGTGCCGTG 3') were synthesized (BioSource International). After annealing, the double-stranded oligonucleotide was incubated with streptavidin-conjugated agarose beads (Pierce) for 1 hour at 4°C and then washed twice with capsid lysis buffer (CLB) buffer (20 mmol/L Tris-HCI, pH 7.5, 2 mmol/L EDTA, 2 mmol/L EGTA, 100 μg/mL aprotinin, 10 mmol/L benzamidine, 5 mmol/L dithioerythritol [DTT], 1 mmol/L phenylmethylsulfonyl fluoride (PMSF), 100 μg/mL leupeptin, 50 mmol/L NaF, 5 mmol/L Na4P2O7, 1 mmol/L Na3VO4, and 1% Nonidet P-40). Nuclear protein extract (20 μg per sample) was suspended in 300 μL of CLB after being precleared with agarose beads for 1 hour at 4°C to remove nonspecific binding to the beads. Lysates were incubated with the -192/-172 ICAM beads for 1 hour at 4°C, and beads were washed 3x with CLB buffer and eluted by boiling in Laemmli buffer for 10 minutes. Proteins were separated using a sodium dodecyl sulfate (SDS)–polyacrylamide gel, transferred to a polyvinylidene fluoride membrane, and immunoblotted with anti-p65 antibody (1:1000).
Ras Activation
Ras activity was determined by measuring binding of only the activated form of Ras to the protein binding domain (PBD) of Raf (Upstate). A total of 200 μg of cell lysates from control or Ang II (100 nmol/L)–treated VSMCs were incubated with glutathione S-transferase (GST)–Raf-1–PBD agarose beads to precipitate activated Ras. After separation using SDS-PAGE, activation of Ras by Ang II was detected by Western blotting using an anti-Ras monoclonal antibody (Upstate).
RSK Kinase Assay
VSMCs were cultured to 95% confluence, serum starved for 24 hours, and then treated for 5 minutes by adding 100 nmol/L Ang II. RSKs were immunoprecipitated by phospho-RSK antibody (Cell Signaling), and its kinase activity in this immune complex was measured by using recombinant p65 as substrate. Briefly, kinase activity was determined in 50 μL by incubating RSK immune complex with GST-p65 for 30 minutes at 30°C in a mixture (20 mmol/L 3-(N-morpholino)propanesulfonic acid, pH 7.2, 25 mmol/L ?-glycerol phosphate, 5 mmol/L EGTA, 1 mmol/L Na3VO4, 1 mmol/L DTT, 2 μmol/L protein kinase A inhibitor peptide, 1 μmol/L microcystin-LR, 15 mmol/L MgCl2, 100 μmol/L cold ATP, and 10 μmol/L -32P ATP). The reaction was stopped by adding 2x loading buffer and immediately boiled before being separated on a 10% SDS-PAGE.
RNase Protection Assay
Expression of IL-6 mRNA was investigated by RNase protection assay (RPA) by using a rat cytokine probe set (rCK-1; Pharmingen). A total of 5 μg of total RNA, prepared by Tri Reagent (Sigma-Aldrich), was hybridized with 5x105 cpm of -P32–labeled probe.17
Statistical Analysis
Statistical comparisons were made using 2-tailed Student t test. Experimental values were reported as means±SE. Differences in mean values were considered significant at P<0.05.
Results
Ang II–Induced IL-6 mRNA Expression and Production in VSMCs and Rat Arteries
IL-6 is a proinflammatory chemokine in blood vessels. Previous studies have shown that Ang II increases expression of IL-6 in VSMCs, and this event is dependent on NF-B,10,18 after mutating the IL-6 promoter, and thus eliminating an NF-B site in an IL-6 promoter luciferase construct, Ang II no longer stimulates IL-6 luciferase reporter activity.10 We also found that Ang II can increase IL-6 mRNA expression 3-fold above control within 1 hour in VSMCs, measured by RPA (Figure 1A). To ask whether Ang II regulates IL-6 in vivo, we infused Ang II into rats via osmotic minipumps for 7 days. Infusion of Ang II (500 ng/kg per minute) increased the SBPs compared with sham infusion at day 3 (153±9 mm Hg versus 135±7 mm Hg; n=6; P<0.05) and day 7 (178±14 mm Hg versus 132±8 mm Hg; n=6; P<0.01). At day 7, the thoracic aorta were perfuse-fixed and processed for immumohistochemical staining of IL-6. The immunostaining indicated that IL-6 expression in aorta is increased (Figure IA, available online at http://atvb.ahajournals.org) compared with control rats (Figure IB). We then tested whether the increased IL-6 expression is caused by hypertension. Ang II–infused rats were treated with the antihypertensive drug hydralazine, and blood pressure dropped from 178±14 mm Hg (Ang II infusion) to 138±9 mm Hg (n=6; P<0.01; Ang II with hydralazine) at day 7, but the increase in IL-6 expression in the aorta was essentially unchanged (Figure IC). Thus, Ang II elicits an inflammatory response in vascular cells and artery.
Figure 1. Ang II induces IL-6 mRNA expression and NF-B activity. A, RPA of total RNA from VSMCs. Lanes are: (1) control (serum free); (2) 100 nmol/L Ang II for 1 hour; and (3) 20 μmol/L U0126 for 30 minutes before treating with 100 nmol/L Ang II for 1 hour in VSMCs (the rat cytokine multiprobe set rCK-1 on left). B and C, NF-B activity was determined after incubating VSMCs with 100 nmol/L Ang II or 10 ng/mL TNF- for 24 hours (B) or different concentrations of Ang II (C). Error bars represent the SD of NF-B luciferase activity performed in triplicate. *P<0.01 vs control. D, Nuclear extracts from VSMCs treated for 10 to 30 minutes with 100 nmol/L Ang II were subjected to EMSA. F indicates free probe; B, bound complex; U, unbound probe.
Ang II Induces NF-B Transcriptional Activity
Several reports suggest that Ang II–induced IL-6 expression is mediated by activation of NF-B.10,18 We examined this pathway and found that Ang II increases NF-B luciferase activity in VSMCs that were infected with an NF-B–Luc reporter minigene (Figure 1B). Luciferase activity increased in an Ang II dose-dependent fashion (Figure 1C), but the ability of Ang II to stimulate NF-B–dependent transcription was less than that of tumor necrosis factor- (TNF-; Figure 1B). Analysis of NF-B binding activity by electrophoretic mobility-shift assay (EMSA) using nuclear extracts from VSMCs that had been treated with Ang II for 10 to 30 minutes revealed a significant increase in NF-B binding activity versus the basal activity in nonstimulated cells (Figure 1D). The specificity of NF-B binding activity was confirmed because either competition with a 100-fold molar excess of unlabeled probe or use of a mutated probe eliminated binding (data not shown).
Ang II–Induced Activation of NF-B Does Not Associate With Significant Downregulation of IB
The classical pathway for NF-B activation involves phosphorylation and proteolytic degradation of the IB isoforms IB and IB?. We found that incubating VSMCs with 100 nmol/L Ang II caused minimal phosphorylation and degradation of IB, and we also detected very little decrease in IB? level (Figure 2A). In sharp contrast, incubation with TNF- (10 ng/mL) caused significant IB phosphorylation at ser32 and decreased the IB protein level (Figure 2B). These results indicate that the classical pathway of phosphorylation and degradation of IB is not a major pathway by which Ang II activates NF-B.
Figure 2. Ang II stimulates minimal phosphorylation or degradation of IB. A, Quiescent VSMCs were treated with 100 nmol/L Ang II for different times and then immunoblotted with antibodies against IB, IB?, or phosphorylated IB. ?-actin was used as a loading control. B, The same experiment was performed using VSMCs treated with TNF-.
Ang II Induces p65 Binding to NF-B Consensus Site and p65 Phosphorylation at Ser536
NF-B binding to its consensus sequence stimulated by Ang II could arise from increased nuclear translocation of NF-B or from reduced binding of IB with NF-B. Treating of VSMCs with Ang II caused a minimal increase in nuclear translocation of p65 (Figure II, available online at http://atvb.ahajournals.org) compared with TNF- stimulation; this finding is consistent with a minimal decrease in cytoplasmic IB (Figure 2A). However, we did find a significant increase in p65 from nuclear proteins that bound to an NF-B consensus site (Figure 3A). Others have demonstrated that phosphorylation of p65 reduces the ability of IB to bind a p65 homodimer or the p65/p50 heterodimer in cell nuclei with enhanced binding of p65 to NF-B consensus sequences.15 As shown in Figure 3B, Ang II increased phosphorylation of p65 at ser536 in VSMC nuclei; this phosphorylation was time- and Ang II concentration–dependent (Figure 3B and 3C). These results suggest that Ang II causes phosphorylation of p65 that increase NF-B transcriptional activity.
Figure 3. Ang II induces binding of p65 to an NF-B consensus site and phosphorylation of p65. A, Nuclear proteins from Ang II–stimulated VSMCs were isolated and incubated with the NF-B consensus sequence from ICAM. Bound p65 was detected by immunoblotting. B, Nuclear proteins from Ang II–treated VSMCs were immunoblotted with an antibody against phospho-p65 (p-p65) at ser536, and ?-tubulin was used for a loading control. C, Nuclear proteins were isolated from VSMCs after treatment with different concentration of Ang II. p65 protein was detected by immunoblotting with a phospho-p65–ser536.
Mitogen-Activated Protein Kinase Participates in the Ang II–Induced NF-B Activity and Increases IL-6 mRNA Expression
What is the signaling system by which Ang II induces NF-B transcriptional activity? Ang II treatment induced phosphorylation of ERK in <5 minutes (Figure IIIA, available online at http://atvb.ahajournals.org). Inhibition of MEK1 with 20 μmol/L U0126 blocked Ang II–mediated activation of NF-B reporter activity by 60.9±9.05% (n=4; P<0.01; Figure 4A). U0126 also suppressed Ang II–induced p65 phosphorylation (Figure 4B). To examine the specificity of MEK1 in this process, we used an adenovirus vector containing a dominant-negative MEK1 to block MEK1 activity in VSMCs. Under these conditions, NF-B transcriptional activity stimulated by Ang II was reduced by 54±7.15% (n=4; P<0.001) versus infection with Ad.? gal (empty vector control; Figure IIIB). Inhibition of MEK1 also blocked binding of NF-B to DNA, nuclear p65 binding (data not shown), and p65 phosphorylation (Figure IIIC). Finally, blocking MEK1 with U0126 blocked 70% of Ang II–induced IL-6 mRNA in VSMCs (Figure 1A). These data indicate that the MEK1 pathway is involved in Ang II–induced NF-B activation and IL-6 transcription.
Figure 4. MEK1 pathway mediates NF-B activation by Ang II. A, VSMCs were infected with Ad.NF-B-luc, incubated with different concentrations of U0126 for 30 minutes, and then treated with 100 nmol/L Ang II for 24 hours. The fold change (mean±SD) in NF-B was compared with results from cells incubated in serum-free media (*P<0.01 vs control; n=4). B, Phosphorylation of p65 (p-p65) was detected in VSMCs that were incubated with different concentrations of U0126 for 30 minutes and then treated with 100 nmol/L Ang II for 10 minutes.
Ras Plays a Role in Ang II–Induced NF-B Activity
To link Ang II to MEK1 activation, we examined the role of Ras, which can activate MEK1. First, we evaluated Ang II–induced Ras activation by a nonradioactive assay that measures binding of activated Ras to the PBD domains of Raf. Compared with values from nonstimulated VSMCs, Ang II stimulated activation of Ras as early as 1 minute (Figure 5A). We also infected VSMCs with a recombinant adenovirus that encodes a dominant-negative form of Ras (Ad.Ras Y57). As expected, infection with Ad.Ras Y57 resulted in overexpression of a mutant Ras that blocked the ability of Ang II to activate the downstream target of Ras: ERK (Figure 5B). It also blocked Ang II–stimulated NF-B promoter activity (Figure 5C) and serine phosphorylation of p65 (Figure 5B).
Figure 5. Ras is involved in Ang II–induced NF-B activity. A, VSMCs were treated with 100 nmol/L Ang II. A total of 200 μg of cell lysates was affinity precipitated with GST-Raf-PBD agarose beads to collect activated Ras. After SDS-PAGE, Ras was detected with an anti-Ras antibody. B, Ad.Ras Y57–infected VSMCs were treated with Ang II. The p65 phosphorylation (p-p65) and Ras were detected by immunoblotting. C, Ad.Ras Y57– and Ad.NF-B-luc–infected VSMCs were treated with 100 nmol/L Ang II for 24 hours, and NF-B activity was determined. Shown is the mean±SD of normalized NF-B luciferase reporter activity performed in triplicate. *P<0.01 vs control.
RSK Mediates NF-B Activation in Response to Ang II Treatment
To identify the kinase that is downstream of the MEK1 pathway, we examined the downstream target of ERK: RSK. RSK has been shown to activate NF-B after phorbol ester treatment.19 Ang II treatment induced phosphorylation of RSK at ser380 (Figure IVA, available online at http://atvb.ahajournals.org) in a dose-dependent manner (Figure IVB). Inhibition of MEK1 activity by U0126 blocked phosphorylation of RSK and p65 (Figure 6A). Next, we knocked down 80% of RSK by transfecting VSMCs with 4 small interfering RNA (SiRNA) vectors for RSK1 and RSK220 (Figure 6B). There was a 63.9±3.3% (n=5; P<0.01) reduction of NF-B activation after Ang II treatment and an almost complete inhibition of p65 serine phosphorylation (Figure 6C). This result indicates that RSK activation participates in the phosphorylation of p65 and ultimately increased NF-B activity. To determine whether RSK can directly phosphorylate p65, we immunoprecipitated RSK from Ang II–treated or –untreated VSMCs and incubated it with a wild-type GST-p65 fusion protein or an ser536-mutated GST-p65 protein. As depicted in Figure 6D, RSK from Ang II–treated cells caused a 3- to 4-fold induction of p65 phosphorylation over control values. In contrast, the serine536-mutated p65 was not phosphorylated in response to Ang II.
Figure 6. RSK mediates NF-B activation after Ang II treatment. A, The influence of U0126 30 minutes before Ang II treatment is shown. B, VSMCs were transfected with a mixture of 4 SiRNAs against RSK1 and RSK2 and then treated with/without Ang II. The RSK level, its phosphorylation, and the phosphorylation of p65 (p-p65) were measured by immunoblotting. Phospho-ERK was a positive control for the effects of Ang II. C, VSMCs that were transfected with SiRNAs were then infected with Ad.NF-B-luc, and NF-B activity (mean±SD) was measured after 24-hour Ang II treatment (*P<0.01 vs control; n=6). D, To measure the phosphorylation of p65, the cell lysates of VSMCs treated with Ang II for 10 minutes were immunoprecipitated with an RSK antibody. The immunocomplex was then incubated with wild-type or ser536A mutant p65 protein in the presence of [-32P]ATP.
Discussion
Ang II increases the expression of proinflammatory cytokines through activation of NF-B in VSMCs.21 Because inflammation plays a role in the development of atherosclerosis, we decided to study mechanisms that increase activation of NF-B and the expression of proinflammatory cytokines. We chose to study the proinflammatory cytokine IL-6 because results from in vivo and in vitro studies have demonstrated that IL-6 is present in atherosclerotic plaques22 and that Ang II, a mediator of atherosclerosis, can increase IL-6 expression through activation of NF-B.10,18 Our results show that Ang II–induced NF-B transcriptional activity is mediated by a nonclassical pathway. NF-B–mediated transcription rises in response to stimulation of the Ras/MEK1/ERK/RSK pathway. RSK phosphorylates p65 of NF-B in nuclei of Ang II–treated VSMCs, leading to increased binding of p65 to NF-B binding sequence.
We explored this nonclassical pathway because we found that Ang II–induced activation of NF-B transcription in VSMCs could not be attributed to phosphorylation and degradation of IB (Figure 2A). Others have reported that there is weak degradation of IB in response to Ang II,7,8,10 and there are reports that other stimuli such as UV light, hypoxia, and reoxygenation will activate NF-B via nonclassical pathways.12,23 For example, induction of NF-B by UV light does not require activation of IB kinases (IKKs) or phosphorylation of IB on serines 32 and 36;12 hypoxia, reoxygenation, or treatment with the tyrosine phosphatase inhibitor pervanadate causes tyrosine phosphorylation of IB, which promotes its physical dissociation from NF-B and consequent activation of NF-B.23
An interesting result was the requirement for involvement of the Ras/MEK1/ERK signaling pathway in the induction of NF-B transcriptional activity by Ang II. For example, when MEK1 was inhibited, there was a significant reduction in NF-B transcriptional activity and expression of IL-6 in VSMCs treated with Ang II (Figures 1A and 4A). Presumably, a similar pathway is activated in airway inflammation, where stimulation of MAP kinase plays an important role in activation of NF-B transcriptional activity.24
Although the pathway from MEK1 to ERK is defined, it was not clear initially how ERK could activate NF-B without phosphorylating IB. We found that Ang II increases the kinase activity of RSK, leading to direct phosphorylation of p65 and increased NF-B transcriptional activity. In support of this sequence of events, we found that RSK phosphorylates the p65 component of NF-B in an in vitro biochemical assay (using recombinant GST-p65; Figure 6D). Phosphorylation of the p65 subunit occurs in other settings. For example, p53-induced, RSK1-mediated phosphorylation of p65 reduces the affinity of p65 for the NF-B inhibitor IB; it also decreases IB-mediated export of shuttling forms of NF-B from the nucleus, thereby promoting binding of NF-B to cognate B enhancers.15 Indeed, our data show that Ang II does not increase translocation (Figure II) but increases p65 phosphorylation and p65 binding to NF-B consensus sequence in the nuclei (Figure 3A and 3B)
Our evidence for a nonclassical Ras/MEK1/ERK pathway that is activated by Ang II includes a suppression of RSK and NF-B transcriptional activity when we inhibited components of the pathway (Ras and MEK1). Specifically, inhibition of components of the Ras/MEK1/ERK pathway led to a decrease in RSK phosphorylation, p65 phosphorylation, and NF-B luciferase reporter activity, plus suppressed expression of IL-6. The importance of p65 phosphorylation was also demonstrated when we downregulated RSK in VSMCs by SiRNAs; the serine phosphorylation of p65 and Ang II–activated NF-B activity were reduced (Figure 6B and 6C).
Notably, suppression of MEK1/RSK inhibited Ang II–induced NF-B activation and expression of IL-6 by only 70%. The remaining 30% of Ang II–induced NF-B activity may involve another pathway, such as production of reactive oxygen species (ROS) or other kinases. This possibility is raised because Pueyo et al found that antioxidants will diminish Ang II–induced NF-B activation,25 and we have found that Ang II could activate IKK but did not phosphorylate IB by the classic pathway (Zhang et al, unpublished data, 2005). The contribution of ROS to activation of p65 and NF-B is presently unknown. Previous studies have shown that AT1 and AT2 receptors are involved in Ang II activation of NF-B.6,7 The role of these receptors in Ras/MEK1/RSK-mediated phosphorylation of p65 remains to be determined.
In summary, we have uncovered a new pathway by which Ang II activates NF-B in VSMCs. It is IB independent and proceeds through a MEK1-dependent RSK1-mediated p65 phosphorylation. This signaling pathway differs from the pathways stimulated by proinflammatory cytokines such as TNF- and, hence, elucidates a mechanism for Ang II–induced inflammatory responses. The actions of MEK1/RSK could provide a potential therapeutic target for cardiovascular diseases.
Acknowledgments
This project was supported by the National Institutes of Health through grant RO1 HL 70762 and a scientist development grant to J.D. from the American Heart Association. The authors thank Dr Ping Li at Dr Feng’s laboratory (Baylor College of Medicine) for help in RPA. We are indebted to Drs W.E. Mitch and D.A. Konkel for helpful discussions and critical reading of this manuscript.
References
Printseva O, Peclo MM, Gown AM. Various cell types in human atherosclerotic lesions express ICAM-1. Further immunocytochemical and immunochemical studies employing monoclonal antibody 10F3. Am J Pathol. 1992; 140: 889–896.
Davies MJ, Gordon JL, Gearing AJ, Pigott R, Woolf N, Katz D, Kyriakopoulos A. The expression of the adhesion molecules ICAM-1, VCAM-1, PECAM, and E-selectin in human atherosclerosis. J Pathol. 1993; 171: 223–229.
Alpers CE, Hudkins KL, Davis CL, Marsh CL, Riches W, McCarty JM, Benjamin CD, Carlos TM, Harlan JM, Lobb R. Expression of vascular cell adhesion molecule-1 in kidney allograft rejection. Kidney Int. 1993; 44: 805–816.
Ardehali A, Laks H, Drinkwater DC, Ziv E, Drake TA. Vascular cell adhesion molecule-1 is induced on vascular endothelia and medial smooth muscle cells in experimental cardiac allograft vasculopathy. Circulation. 1995; 92: 450–456.
Plow EF, D’Souza SE. A role for intercellular adhesion molecule-1 in restenosis. Circulation. 1997; 95: 1355–1356.
Esteban V, Lorenzo O, Ruperez M, Suzuki Y, Mezzano S, Blanco J, Kretzler M, Sugaya T, Egido J, Ruiz-Ortega M. Angiotensin II, via AT1 and AT2 receptors and NF-kappaB pathway, regulates the inflammatory response in unilateral ureteral obstruction. J Am Soc Nephrol. 2004; 15: 1514–1529.
Wolf G, Wenzel U, Burns KD, Harris RC, Stahl RA, Thaiss F. Angiotensin II activates nuclear transcription factor-kappaB through AT1 and AT2 receptors. Kidney Int. 2002; 61: 1986–1995.
Wu L, Iwai M, Li Z, Shiuchi T, Min LJ, Cui TX, Li JM, Okumura M, Nahmias C, Horiuchi M. Regulation of inhibitory protein-kappaB and monocyte chemoattractant protein-1 by angiotensin II type 2 receptor-activated Src homology protein tyrosine phosphatase-1 in fetal vascular smooth muscle cells. Mol Endocrinol. 2004; 18: 666–678.
Hernandez-Presa M, Bustos C, Ortego M, Tunon J, Renedo G, Ruiz-Ortega M, Egido J. Angiotensin-converting enzyme inhibition prevents arterial nuclear factor-kappa B activation, monocyte chemoattractant protein-1 expression, and macrophage infiltration in a rabbit model of early accelerated atherosclerosis. Circulation. 1997; 95: 1532–1541.
Han Y, Runge MS, Brasier AR. Angiotensin II induces interleukin-6 transcription in vascular smooth muscle cells through pleiotropic activation of nuclear factor-kappa B transcription factors. Circ Res. 1999; 84: 695–703.
Wu L, Iwai M, Li Z, Shiuchi T, Min LJ, Cui TX, Li JM, Okumura M, Nahmias C, Horiuchi M. Regulation of I{kappa}B and MCP-1 by angiotensin type 2 receptor-activated SHP-1 in fetal vascular smooth muscle cells. Mol Endocrinol. 2003.
Wu S, Tan M, Hu Y, Wang JL, Scheuner D, Kaufman RJ. Ultraviolet light activates NFkappaB through translational inhibition of IkappaBalpha synthesis. J Biol Chem. 2004; 279: 34898–34902.
Hu J, Nakano H, Sakurai H, Colburn NH. Insufficient p65 phosphorylation at S536 specifically contributes to the lack of NF-kappaB activation and transformation in resistant JB6 cells. Carcinogenesis. 2004; 25: 1991–2003.
Mattioli I, Sebald A, Bucher C, Charles RP, Nakano H, Doi T, Kracht M, Schmitz ML. Transient and selective NF-kappa B p65 serine 536 phosphorylation induced by T cell costimulation is mediated by I kappa B kinase beta and controls the kinetics of p65 nuclear import. J Immunol. 2004; 172: 6336–6344.
Bohuslav J, Chen LF, Kwon H, Mu Y, Greene WC. p53 induces NF-kappaB activation by an IkappaB kinase-independent mechanism involving phosphorylation of p65 by ribosomal S6 kinase 1. J Biol Chem. 2004; 279: 26115–26125.
Scheidegger KJ, Du J, Delafontaine P. Distinct and common pathways in the regulation of insulin-like growth factor-1 receptor gene expression by angiotensin II and basic fibroblast growth factor. J Biol Chem. 1999; 274: 3522–3530.
Feng L, Sun W, Xia Y, Tang WW, Chanmugam P, Soyoola E, Wilson CB, Hwang D. Cloning two isoforms of rat cyclooxygenase: differential regulation of their expression. Arch Biochem Biophys. 1993; 307: 361–368.
Kranzhofer R, Schmidt J, Pfeiffer CA, Hagl S, Libby P, Kubler W. Angiotensin induces inflammatory activation of human vascular smooth muscle cells. Arterioscler Thromb Vasc Biol. 1999; 19: 1623–1629.
Kim KW, Kim SH, Lee EY, Kim ND, Kang HS, Kim HD, Chung BS, Kang CD. Extracellular signal-regulated kinase/90-KDA ribosomal S6 kinase/nuclear factor-kappa B pathway mediates phorbol 12-myristate 13-acetate-induced megakaryocytic differentiation of K562 cells. J Biol Chem. 2001; 276: 13186–13191.
Arthur JS, Fong AL, Dwyer JM, Davare M, Reese E, Obrietan K, Impey S. Mitogen- and stress-activated protein kinase 1 mediates cAMP response element-binding protein phosphorylation and activation by neurotrophins. J Neurosci. 2004; 24: 4324–4332.
Suzuki Y, Ruiz-Ortega M, Lorenzo O, Ruperez M, Esteban V, Egido J. Inflammation and angiotensin II. Int J Biochem Cell Biol. 2003; 35: 881–900.
Saadeddin SM, Habbab MA, Ferns GA. Markers of inflammation and coronary artery disease. Med Sci Monit. 2002; 8: RA5–RA12.
Natarajan R, Fisher BJ, Jones DG, Fowler AA III. Atypical mechanism of NF-kappaB activation during reoxygenation stress in microvascular endothelium: a role for tyrosine kinases. Free Radical Biol Med. 2002; 33: 962.
Duan W, Chan JH, Wong CH, Leung BP, Wong WS. Anti-inflammatory effects of mitogen-activated protein kinase kinase inhibitor U0126 in an asthma mouse model. J Immunol. 2004; 172: 7053–7059.
Pueyo ME, Gonzalez W, Nicoletti A, Savoie F, Arnal JF, Michel JB. Angiotensin II stimulates endothelial vascular cell adhesion molecule-1 via nuclear factor-kappaB activation induced by intracellular oxidative stress. Arterioscler Thromb Vasc Biol. 2000; 20: 645–651.(Liping Zhang; Yewei Ma; J)
Correspondence to Jie Du, Department of Internal Medicine, 9.138 Medical Research Building, 301 University Blvd, University of Texas Medical Branch, Galveston, TX 77555-1064. E-mail jidu@utmb.edu
Abstract
Objective— Angiotensin II (Ang II) promotes vascular inflammation and remodeling via activation of nuclear factor B (NF-B)–mediated transcription of proinflammatory genes such as interleukin-6 (IL-6). We examined the signaling mechanism whereby Ang II activates NF-B in vascular smooth muscle cells (VSMCs).
Methods and Results— Ang II treatment did not increase phosphorylation of inhibitor of B (IB) or IB? or decrease their levels. In contrast, mitogen-activated protein kinase kinase-1 (MEK1) inhibition (dominant-negative MEK1 adenovirus or inhibitor U0126) suppressed Ang II–induced NF-B promoter activity, NF-B DNA-binding activity, p65 phosphorylation, and led to 70% reduction in IL-6 transcription/production. The mechanism involved Ang II activation of Ras and MEK1. Signaling distal to MEK1 involved extracellular signal-regulated kinase (ERK) because inhibition of MEK1 suppressed the Ang II–induced activation of ribosomal S6 kinase (RSK), a substrate of ERK. Downregulation of RSK by small interfering RNA (SiRNA) in VSMCs was found to suppress Ang II–induced activation of NF-B and p65 phosphorylation. Immunopurified RSK from Ang II–treated VSMCs phosphorylated recombinant glutathione S-transferase–p65 in vitro.
Conclusion— We uncovered a nonclassical signaling pathway (Ras/MEK1/ERK/RSK) from Ang II to activation of NF-B, a mechanism by which Ang II stimulates RSK-mediated phosphorylation of p65 to participate in vascular inflammation.
We examined an intracellular mechanism whereby Ang II activates NF-B. Ang II caused no significant increase in IB phosphorylation or protein levels. Instead, we found a nonclassical signaling pathway, Ras/MEK1/ERK/RSK, that activated NF-B via phosphorylation of p65, leading to IL-6 production and suggesting a mechanism by which Ang II causes vascular inflammation.
Key Words: angiotensin II ? signaling pathway ? NF-B ? MAP kinase ? inflammation
Introduction
Angiotensin II has proinflammatory actions that increase the expression of chemokines and cellular adhesion molecules in vascular abnormalities, such as atherosclerosis,1,2 transplant vasculopathy,3,4 or restenosis injury.5 These actions of angiotensin II (Ang II) are attributed to activation of nuclear factor B (NF-B), a key transcription factor that stimulates the transcription of chemokines, as well as other genes. Several groups have demonstrated that Ang II activates NF-B in vivo and in vitro after binding to the Ang II type 1 (AT1) or AT2 receptor.6–8 Moreover, in an experimental model of atherosclerosis exhibiting increased inflammation, NF-B activity is increased in vascular tissue; this response was diminished by an angiotensin-converting enzyme inhibitor.9
The mechanism by which Ang II activates NF-B is unclear. The classical NF-B activation model involves sequestration of NF-B in the cytoplasm by inhibitor of B (IB), preventing NF-B from migrating to the nucleus. When IB is phosphorylated, it is degraded, permitting NF-B to translocate to the nucleus. However, several reports suggest that Ang II can stimulate degradation of IB, but the change in the level of IB was small.7,8,10 A nonclassical model suggests that Ang II induces tyrosine phosphorylation of IB to promote its physical dissociation from NF-B.11 In fact, there are observations suggesting that this or other nonclassical pathways are active. For example, the induction of NF-B by UV light does not require phosphorylation of IB.12 There are also reports that NF-B can be activated by phosphorylation of the NF-B p65 subunit.13,14 A study by Bohuslav et al demonstrated that p65 can be phosphorylated in the nucleus. This change reduces the ability of IB to bind to either a p65 homodimer or the p65/p50 heterodimer.15 Consequently, phosphorylated p65 can bind to NF-B consensus sequences in a promoter.
In exploring how Ang II regulates the transcriptional activity of NF-B, we found that inhibition of mitogen-activated protein kinase kinase-1 (MEK1) suppressed the transcriptional activity of NF-B. We explored the cellular signaling pathway linking Ang II to MEK and then to stimulation of NF-B transcriptional activity. This pathway involves Ras/MEK1/extracellular signal-regulated kinase (ERK)/ribosomal S6 kinase (RSK) and activates NF-B in vascular smooth muscle cells (VSMCs) by phosphorylating the p65 subunit of NF-B. Biologically, activation of this pathway contributes to Ang II–induced interleukin-6 (IL-6) expression that has been implicated in vascular inflammation.
Methods
Cell Culture
VSMCs were isolated from the thoracic aortas of male Sprague-Dawley rats by enzymatic digestion and then cultured in DMEM supplemented with 10% FBS. VSMCs were identified by positive staining with monoclonal antibody to smooth muscle -actin (Sigma-Aldrich).
Animals
Three-month-old male Sprague-Dawley rats were implanted with osmotic pumps (model 2001; Alza Corp). Animals in the Ang II–treated group received a pump filled with Ang II and delivered at a dosage of 500 ng/kg per minute in Ringer’s solution (0.01 mol/L acetic acid in saline). Another Ang II–treated group received antihypertensive therapy hydralazine (10 mg/kg per day) in drinking water. Control animals received a pump with Ringer’s solution without Ang II. Systolic blood pressure (SBP) was measured by tail-cuff plethysmography method (Visitech Systems Inc). SBP values were derived from an average of 6 to 8 measurements per animal at each time point. Each group of 6 rats was pair fed for 7 days and perfuse-fixed with 4% paraformaldehyde. The thoracic aorta was embedded in paraffin and examined.
NF-B Luciferase Reporter Assay
NF-B transcriptional activity was evaluated by using an adenovirus NF-B-Luc luciferase reporter (Ad.NF-B-Luc). Briefly, cells were infected with Ad.NF-B-Luc at a multiplicity of infection of 5 for 24 hours before 100 nmol/L Ang II treatment; from each sample, 5 μg of total protein was assayed for luciferase activity using the manufacturer protocol (Promega).
Electrophoretic Mobility Shift Analysis
Nuclear extracts were prepared as described16 and incubated with an NF-B oligonucleotide (Promega). The gel shift analysis was performed at least 3x.
Agarose Oligonucleotide Pull-Down Assay
The NF-B sequence from -192/-172 of intercellular adhesion molecule (ICAM) with 3-terminal biotinylation (5' GTTCCACGGCACCCCCTG3') and its complementary strand (5' CCTGCAGGGGGTGCCGTG 3') were synthesized (BioSource International). After annealing, the double-stranded oligonucleotide was incubated with streptavidin-conjugated agarose beads (Pierce) for 1 hour at 4°C and then washed twice with capsid lysis buffer (CLB) buffer (20 mmol/L Tris-HCI, pH 7.5, 2 mmol/L EDTA, 2 mmol/L EGTA, 100 μg/mL aprotinin, 10 mmol/L benzamidine, 5 mmol/L dithioerythritol [DTT], 1 mmol/L phenylmethylsulfonyl fluoride (PMSF), 100 μg/mL leupeptin, 50 mmol/L NaF, 5 mmol/L Na4P2O7, 1 mmol/L Na3VO4, and 1% Nonidet P-40). Nuclear protein extract (20 μg per sample) was suspended in 300 μL of CLB after being precleared with agarose beads for 1 hour at 4°C to remove nonspecific binding to the beads. Lysates were incubated with the -192/-172 ICAM beads for 1 hour at 4°C, and beads were washed 3x with CLB buffer and eluted by boiling in Laemmli buffer for 10 minutes. Proteins were separated using a sodium dodecyl sulfate (SDS)–polyacrylamide gel, transferred to a polyvinylidene fluoride membrane, and immunoblotted with anti-p65 antibody (1:1000).
Ras Activation
Ras activity was determined by measuring binding of only the activated form of Ras to the protein binding domain (PBD) of Raf (Upstate). A total of 200 μg of cell lysates from control or Ang II (100 nmol/L)–treated VSMCs were incubated with glutathione S-transferase (GST)–Raf-1–PBD agarose beads to precipitate activated Ras. After separation using SDS-PAGE, activation of Ras by Ang II was detected by Western blotting using an anti-Ras monoclonal antibody (Upstate).
RSK Kinase Assay
VSMCs were cultured to 95% confluence, serum starved for 24 hours, and then treated for 5 minutes by adding 100 nmol/L Ang II. RSKs were immunoprecipitated by phospho-RSK antibody (Cell Signaling), and its kinase activity in this immune complex was measured by using recombinant p65 as substrate. Briefly, kinase activity was determined in 50 μL by incubating RSK immune complex with GST-p65 for 30 minutes at 30°C in a mixture (20 mmol/L 3-(N-morpholino)propanesulfonic acid, pH 7.2, 25 mmol/L ?-glycerol phosphate, 5 mmol/L EGTA, 1 mmol/L Na3VO4, 1 mmol/L DTT, 2 μmol/L protein kinase A inhibitor peptide, 1 μmol/L microcystin-LR, 15 mmol/L MgCl2, 100 μmol/L cold ATP, and 10 μmol/L -32P ATP). The reaction was stopped by adding 2x loading buffer and immediately boiled before being separated on a 10% SDS-PAGE.
RNase Protection Assay
Expression of IL-6 mRNA was investigated by RNase protection assay (RPA) by using a rat cytokine probe set (rCK-1; Pharmingen). A total of 5 μg of total RNA, prepared by Tri Reagent (Sigma-Aldrich), was hybridized with 5x105 cpm of -P32–labeled probe.17
Statistical Analysis
Statistical comparisons were made using 2-tailed Student t test. Experimental values were reported as means±SE. Differences in mean values were considered significant at P<0.05.
Results
Ang II–Induced IL-6 mRNA Expression and Production in VSMCs and Rat Arteries
IL-6 is a proinflammatory chemokine in blood vessels. Previous studies have shown that Ang II increases expression of IL-6 in VSMCs, and this event is dependent on NF-B,10,18 after mutating the IL-6 promoter, and thus eliminating an NF-B site in an IL-6 promoter luciferase construct, Ang II no longer stimulates IL-6 luciferase reporter activity.10 We also found that Ang II can increase IL-6 mRNA expression 3-fold above control within 1 hour in VSMCs, measured by RPA (Figure 1A). To ask whether Ang II regulates IL-6 in vivo, we infused Ang II into rats via osmotic minipumps for 7 days. Infusion of Ang II (500 ng/kg per minute) increased the SBPs compared with sham infusion at day 3 (153±9 mm Hg versus 135±7 mm Hg; n=6; P<0.05) and day 7 (178±14 mm Hg versus 132±8 mm Hg; n=6; P<0.01). At day 7, the thoracic aorta were perfuse-fixed and processed for immumohistochemical staining of IL-6. The immunostaining indicated that IL-6 expression in aorta is increased (Figure IA, available online at http://atvb.ahajournals.org) compared with control rats (Figure IB). We then tested whether the increased IL-6 expression is caused by hypertension. Ang II–infused rats were treated with the antihypertensive drug hydralazine, and blood pressure dropped from 178±14 mm Hg (Ang II infusion) to 138±9 mm Hg (n=6; P<0.01; Ang II with hydralazine) at day 7, but the increase in IL-6 expression in the aorta was essentially unchanged (Figure IC). Thus, Ang II elicits an inflammatory response in vascular cells and artery.
Figure 1. Ang II induces IL-6 mRNA expression and NF-B activity. A, RPA of total RNA from VSMCs. Lanes are: (1) control (serum free); (2) 100 nmol/L Ang II for 1 hour; and (3) 20 μmol/L U0126 for 30 minutes before treating with 100 nmol/L Ang II for 1 hour in VSMCs (the rat cytokine multiprobe set rCK-1 on left). B and C, NF-B activity was determined after incubating VSMCs with 100 nmol/L Ang II or 10 ng/mL TNF- for 24 hours (B) or different concentrations of Ang II (C). Error bars represent the SD of NF-B luciferase activity performed in triplicate. *P<0.01 vs control. D, Nuclear extracts from VSMCs treated for 10 to 30 minutes with 100 nmol/L Ang II were subjected to EMSA. F indicates free probe; B, bound complex; U, unbound probe.
Ang II Induces NF-B Transcriptional Activity
Several reports suggest that Ang II–induced IL-6 expression is mediated by activation of NF-B.10,18 We examined this pathway and found that Ang II increases NF-B luciferase activity in VSMCs that were infected with an NF-B–Luc reporter minigene (Figure 1B). Luciferase activity increased in an Ang II dose-dependent fashion (Figure 1C), but the ability of Ang II to stimulate NF-B–dependent transcription was less than that of tumor necrosis factor- (TNF-; Figure 1B). Analysis of NF-B binding activity by electrophoretic mobility-shift assay (EMSA) using nuclear extracts from VSMCs that had been treated with Ang II for 10 to 30 minutes revealed a significant increase in NF-B binding activity versus the basal activity in nonstimulated cells (Figure 1D). The specificity of NF-B binding activity was confirmed because either competition with a 100-fold molar excess of unlabeled probe or use of a mutated probe eliminated binding (data not shown).
Ang II–Induced Activation of NF-B Does Not Associate With Significant Downregulation of IB
The classical pathway for NF-B activation involves phosphorylation and proteolytic degradation of the IB isoforms IB and IB?. We found that incubating VSMCs with 100 nmol/L Ang II caused minimal phosphorylation and degradation of IB, and we also detected very little decrease in IB? level (Figure 2A). In sharp contrast, incubation with TNF- (10 ng/mL) caused significant IB phosphorylation at ser32 and decreased the IB protein level (Figure 2B). These results indicate that the classical pathway of phosphorylation and degradation of IB is not a major pathway by which Ang II activates NF-B.
Figure 2. Ang II stimulates minimal phosphorylation or degradation of IB. A, Quiescent VSMCs were treated with 100 nmol/L Ang II for different times and then immunoblotted with antibodies against IB, IB?, or phosphorylated IB. ?-actin was used as a loading control. B, The same experiment was performed using VSMCs treated with TNF-.
Ang II Induces p65 Binding to NF-B Consensus Site and p65 Phosphorylation at Ser536
NF-B binding to its consensus sequence stimulated by Ang II could arise from increased nuclear translocation of NF-B or from reduced binding of IB with NF-B. Treating of VSMCs with Ang II caused a minimal increase in nuclear translocation of p65 (Figure II, available online at http://atvb.ahajournals.org) compared with TNF- stimulation; this finding is consistent with a minimal decrease in cytoplasmic IB (Figure 2A). However, we did find a significant increase in p65 from nuclear proteins that bound to an NF-B consensus site (Figure 3A). Others have demonstrated that phosphorylation of p65 reduces the ability of IB to bind a p65 homodimer or the p65/p50 heterodimer in cell nuclei with enhanced binding of p65 to NF-B consensus sequences.15 As shown in Figure 3B, Ang II increased phosphorylation of p65 at ser536 in VSMC nuclei; this phosphorylation was time- and Ang II concentration–dependent (Figure 3B and 3C). These results suggest that Ang II causes phosphorylation of p65 that increase NF-B transcriptional activity.
Figure 3. Ang II induces binding of p65 to an NF-B consensus site and phosphorylation of p65. A, Nuclear proteins from Ang II–stimulated VSMCs were isolated and incubated with the NF-B consensus sequence from ICAM. Bound p65 was detected by immunoblotting. B, Nuclear proteins from Ang II–treated VSMCs were immunoblotted with an antibody against phospho-p65 (p-p65) at ser536, and ?-tubulin was used for a loading control. C, Nuclear proteins were isolated from VSMCs after treatment with different concentration of Ang II. p65 protein was detected by immunoblotting with a phospho-p65–ser536.
Mitogen-Activated Protein Kinase Participates in the Ang II–Induced NF-B Activity and Increases IL-6 mRNA Expression
What is the signaling system by which Ang II induces NF-B transcriptional activity? Ang II treatment induced phosphorylation of ERK in <5 minutes (Figure IIIA, available online at http://atvb.ahajournals.org). Inhibition of MEK1 with 20 μmol/L U0126 blocked Ang II–mediated activation of NF-B reporter activity by 60.9±9.05% (n=4; P<0.01; Figure 4A). U0126 also suppressed Ang II–induced p65 phosphorylation (Figure 4B). To examine the specificity of MEK1 in this process, we used an adenovirus vector containing a dominant-negative MEK1 to block MEK1 activity in VSMCs. Under these conditions, NF-B transcriptional activity stimulated by Ang II was reduced by 54±7.15% (n=4; P<0.001) versus infection with Ad.? gal (empty vector control; Figure IIIB). Inhibition of MEK1 also blocked binding of NF-B to DNA, nuclear p65 binding (data not shown), and p65 phosphorylation (Figure IIIC). Finally, blocking MEK1 with U0126 blocked 70% of Ang II–induced IL-6 mRNA in VSMCs (Figure 1A). These data indicate that the MEK1 pathway is involved in Ang II–induced NF-B activation and IL-6 transcription.
Figure 4. MEK1 pathway mediates NF-B activation by Ang II. A, VSMCs were infected with Ad.NF-B-luc, incubated with different concentrations of U0126 for 30 minutes, and then treated with 100 nmol/L Ang II for 24 hours. The fold change (mean±SD) in NF-B was compared with results from cells incubated in serum-free media (*P<0.01 vs control; n=4). B, Phosphorylation of p65 (p-p65) was detected in VSMCs that were incubated with different concentrations of U0126 for 30 minutes and then treated with 100 nmol/L Ang II for 10 minutes.
Ras Plays a Role in Ang II–Induced NF-B Activity
To link Ang II to MEK1 activation, we examined the role of Ras, which can activate MEK1. First, we evaluated Ang II–induced Ras activation by a nonradioactive assay that measures binding of activated Ras to the PBD domains of Raf. Compared with values from nonstimulated VSMCs, Ang II stimulated activation of Ras as early as 1 minute (Figure 5A). We also infected VSMCs with a recombinant adenovirus that encodes a dominant-negative form of Ras (Ad.Ras Y57). As expected, infection with Ad.Ras Y57 resulted in overexpression of a mutant Ras that blocked the ability of Ang II to activate the downstream target of Ras: ERK (Figure 5B). It also blocked Ang II–stimulated NF-B promoter activity (Figure 5C) and serine phosphorylation of p65 (Figure 5B).
Figure 5. Ras is involved in Ang II–induced NF-B activity. A, VSMCs were treated with 100 nmol/L Ang II. A total of 200 μg of cell lysates was affinity precipitated with GST-Raf-PBD agarose beads to collect activated Ras. After SDS-PAGE, Ras was detected with an anti-Ras antibody. B, Ad.Ras Y57–infected VSMCs were treated with Ang II. The p65 phosphorylation (p-p65) and Ras were detected by immunoblotting. C, Ad.Ras Y57– and Ad.NF-B-luc–infected VSMCs were treated with 100 nmol/L Ang II for 24 hours, and NF-B activity was determined. Shown is the mean±SD of normalized NF-B luciferase reporter activity performed in triplicate. *P<0.01 vs control.
RSK Mediates NF-B Activation in Response to Ang II Treatment
To identify the kinase that is downstream of the MEK1 pathway, we examined the downstream target of ERK: RSK. RSK has been shown to activate NF-B after phorbol ester treatment.19 Ang II treatment induced phosphorylation of RSK at ser380 (Figure IVA, available online at http://atvb.ahajournals.org) in a dose-dependent manner (Figure IVB). Inhibition of MEK1 activity by U0126 blocked phosphorylation of RSK and p65 (Figure 6A). Next, we knocked down 80% of RSK by transfecting VSMCs with 4 small interfering RNA (SiRNA) vectors for RSK1 and RSK220 (Figure 6B). There was a 63.9±3.3% (n=5; P<0.01) reduction of NF-B activation after Ang II treatment and an almost complete inhibition of p65 serine phosphorylation (Figure 6C). This result indicates that RSK activation participates in the phosphorylation of p65 and ultimately increased NF-B activity. To determine whether RSK can directly phosphorylate p65, we immunoprecipitated RSK from Ang II–treated or –untreated VSMCs and incubated it with a wild-type GST-p65 fusion protein or an ser536-mutated GST-p65 protein. As depicted in Figure 6D, RSK from Ang II–treated cells caused a 3- to 4-fold induction of p65 phosphorylation over control values. In contrast, the serine536-mutated p65 was not phosphorylated in response to Ang II.
Figure 6. RSK mediates NF-B activation after Ang II treatment. A, The influence of U0126 30 minutes before Ang II treatment is shown. B, VSMCs were transfected with a mixture of 4 SiRNAs against RSK1 and RSK2 and then treated with/without Ang II. The RSK level, its phosphorylation, and the phosphorylation of p65 (p-p65) were measured by immunoblotting. Phospho-ERK was a positive control for the effects of Ang II. C, VSMCs that were transfected with SiRNAs were then infected with Ad.NF-B-luc, and NF-B activity (mean±SD) was measured after 24-hour Ang II treatment (*P<0.01 vs control; n=6). D, To measure the phosphorylation of p65, the cell lysates of VSMCs treated with Ang II for 10 minutes were immunoprecipitated with an RSK antibody. The immunocomplex was then incubated with wild-type or ser536A mutant p65 protein in the presence of [-32P]ATP.
Discussion
Ang II increases the expression of proinflammatory cytokines through activation of NF-B in VSMCs.21 Because inflammation plays a role in the development of atherosclerosis, we decided to study mechanisms that increase activation of NF-B and the expression of proinflammatory cytokines. We chose to study the proinflammatory cytokine IL-6 because results from in vivo and in vitro studies have demonstrated that IL-6 is present in atherosclerotic plaques22 and that Ang II, a mediator of atherosclerosis, can increase IL-6 expression through activation of NF-B.10,18 Our results show that Ang II–induced NF-B transcriptional activity is mediated by a nonclassical pathway. NF-B–mediated transcription rises in response to stimulation of the Ras/MEK1/ERK/RSK pathway. RSK phosphorylates p65 of NF-B in nuclei of Ang II–treated VSMCs, leading to increased binding of p65 to NF-B binding sequence.
We explored this nonclassical pathway because we found that Ang II–induced activation of NF-B transcription in VSMCs could not be attributed to phosphorylation and degradation of IB (Figure 2A). Others have reported that there is weak degradation of IB in response to Ang II,7,8,10 and there are reports that other stimuli such as UV light, hypoxia, and reoxygenation will activate NF-B via nonclassical pathways.12,23 For example, induction of NF-B by UV light does not require activation of IB kinases (IKKs) or phosphorylation of IB on serines 32 and 36;12 hypoxia, reoxygenation, or treatment with the tyrosine phosphatase inhibitor pervanadate causes tyrosine phosphorylation of IB, which promotes its physical dissociation from NF-B and consequent activation of NF-B.23
An interesting result was the requirement for involvement of the Ras/MEK1/ERK signaling pathway in the induction of NF-B transcriptional activity by Ang II. For example, when MEK1 was inhibited, there was a significant reduction in NF-B transcriptional activity and expression of IL-6 in VSMCs treated with Ang II (Figures 1A and 4A). Presumably, a similar pathway is activated in airway inflammation, where stimulation of MAP kinase plays an important role in activation of NF-B transcriptional activity.24
Although the pathway from MEK1 to ERK is defined, it was not clear initially how ERK could activate NF-B without phosphorylating IB. We found that Ang II increases the kinase activity of RSK, leading to direct phosphorylation of p65 and increased NF-B transcriptional activity. In support of this sequence of events, we found that RSK phosphorylates the p65 component of NF-B in an in vitro biochemical assay (using recombinant GST-p65; Figure 6D). Phosphorylation of the p65 subunit occurs in other settings. For example, p53-induced, RSK1-mediated phosphorylation of p65 reduces the affinity of p65 for the NF-B inhibitor IB; it also decreases IB-mediated export of shuttling forms of NF-B from the nucleus, thereby promoting binding of NF-B to cognate B enhancers.15 Indeed, our data show that Ang II does not increase translocation (Figure II) but increases p65 phosphorylation and p65 binding to NF-B consensus sequence in the nuclei (Figure 3A and 3B)
Our evidence for a nonclassical Ras/MEK1/ERK pathway that is activated by Ang II includes a suppression of RSK and NF-B transcriptional activity when we inhibited components of the pathway (Ras and MEK1). Specifically, inhibition of components of the Ras/MEK1/ERK pathway led to a decrease in RSK phosphorylation, p65 phosphorylation, and NF-B luciferase reporter activity, plus suppressed expression of IL-6. The importance of p65 phosphorylation was also demonstrated when we downregulated RSK in VSMCs by SiRNAs; the serine phosphorylation of p65 and Ang II–activated NF-B activity were reduced (Figure 6B and 6C).
Notably, suppression of MEK1/RSK inhibited Ang II–induced NF-B activation and expression of IL-6 by only 70%. The remaining 30% of Ang II–induced NF-B activity may involve another pathway, such as production of reactive oxygen species (ROS) or other kinases. This possibility is raised because Pueyo et al found that antioxidants will diminish Ang II–induced NF-B activation,25 and we have found that Ang II could activate IKK but did not phosphorylate IB by the classic pathway (Zhang et al, unpublished data, 2005). The contribution of ROS to activation of p65 and NF-B is presently unknown. Previous studies have shown that AT1 and AT2 receptors are involved in Ang II activation of NF-B.6,7 The role of these receptors in Ras/MEK1/RSK-mediated phosphorylation of p65 remains to be determined.
In summary, we have uncovered a new pathway by which Ang II activates NF-B in VSMCs. It is IB independent and proceeds through a MEK1-dependent RSK1-mediated p65 phosphorylation. This signaling pathway differs from the pathways stimulated by proinflammatory cytokines such as TNF- and, hence, elucidates a mechanism for Ang II–induced inflammatory responses. The actions of MEK1/RSK could provide a potential therapeutic target for cardiovascular diseases.
Acknowledgments
This project was supported by the National Institutes of Health through grant RO1 HL 70762 and a scientist development grant to J.D. from the American Heart Association. The authors thank Dr Ping Li at Dr Feng’s laboratory (Baylor College of Medicine) for help in RPA. We are indebted to Drs W.E. Mitch and D.A. Konkel for helpful discussions and critical reading of this manuscript.
References
Printseva O, Peclo MM, Gown AM. Various cell types in human atherosclerotic lesions express ICAM-1. Further immunocytochemical and immunochemical studies employing monoclonal antibody 10F3. Am J Pathol. 1992; 140: 889–896.
Davies MJ, Gordon JL, Gearing AJ, Pigott R, Woolf N, Katz D, Kyriakopoulos A. The expression of the adhesion molecules ICAM-1, VCAM-1, PECAM, and E-selectin in human atherosclerosis. J Pathol. 1993; 171: 223–229.
Alpers CE, Hudkins KL, Davis CL, Marsh CL, Riches W, McCarty JM, Benjamin CD, Carlos TM, Harlan JM, Lobb R. Expression of vascular cell adhesion molecule-1 in kidney allograft rejection. Kidney Int. 1993; 44: 805–816.
Ardehali A, Laks H, Drinkwater DC, Ziv E, Drake TA. Vascular cell adhesion molecule-1 is induced on vascular endothelia and medial smooth muscle cells in experimental cardiac allograft vasculopathy. Circulation. 1995; 92: 450–456.
Plow EF, D’Souza SE. A role for intercellular adhesion molecule-1 in restenosis. Circulation. 1997; 95: 1355–1356.
Esteban V, Lorenzo O, Ruperez M, Suzuki Y, Mezzano S, Blanco J, Kretzler M, Sugaya T, Egido J, Ruiz-Ortega M. Angiotensin II, via AT1 and AT2 receptors and NF-kappaB pathway, regulates the inflammatory response in unilateral ureteral obstruction. J Am Soc Nephrol. 2004; 15: 1514–1529.
Wolf G, Wenzel U, Burns KD, Harris RC, Stahl RA, Thaiss F. Angiotensin II activates nuclear transcription factor-kappaB through AT1 and AT2 receptors. Kidney Int. 2002; 61: 1986–1995.
Wu L, Iwai M, Li Z, Shiuchi T, Min LJ, Cui TX, Li JM, Okumura M, Nahmias C, Horiuchi M. Regulation of inhibitory protein-kappaB and monocyte chemoattractant protein-1 by angiotensin II type 2 receptor-activated Src homology protein tyrosine phosphatase-1 in fetal vascular smooth muscle cells. Mol Endocrinol. 2004; 18: 666–678.
Hernandez-Presa M, Bustos C, Ortego M, Tunon J, Renedo G, Ruiz-Ortega M, Egido J. Angiotensin-converting enzyme inhibition prevents arterial nuclear factor-kappa B activation, monocyte chemoattractant protein-1 expression, and macrophage infiltration in a rabbit model of early accelerated atherosclerosis. Circulation. 1997; 95: 1532–1541.
Han Y, Runge MS, Brasier AR. Angiotensin II induces interleukin-6 transcription in vascular smooth muscle cells through pleiotropic activation of nuclear factor-kappa B transcription factors. Circ Res. 1999; 84: 695–703.
Wu L, Iwai M, Li Z, Shiuchi T, Min LJ, Cui TX, Li JM, Okumura M, Nahmias C, Horiuchi M. Regulation of I{kappa}B and MCP-1 by angiotensin type 2 receptor-activated SHP-1 in fetal vascular smooth muscle cells. Mol Endocrinol. 2003.
Wu S, Tan M, Hu Y, Wang JL, Scheuner D, Kaufman RJ. Ultraviolet light activates NFkappaB through translational inhibition of IkappaBalpha synthesis. J Biol Chem. 2004; 279: 34898–34902.
Hu J, Nakano H, Sakurai H, Colburn NH. Insufficient p65 phosphorylation at S536 specifically contributes to the lack of NF-kappaB activation and transformation in resistant JB6 cells. Carcinogenesis. 2004; 25: 1991–2003.
Mattioli I, Sebald A, Bucher C, Charles RP, Nakano H, Doi T, Kracht M, Schmitz ML. Transient and selective NF-kappa B p65 serine 536 phosphorylation induced by T cell costimulation is mediated by I kappa B kinase beta and controls the kinetics of p65 nuclear import. J Immunol. 2004; 172: 6336–6344.
Bohuslav J, Chen LF, Kwon H, Mu Y, Greene WC. p53 induces NF-kappaB activation by an IkappaB kinase-independent mechanism involving phosphorylation of p65 by ribosomal S6 kinase 1. J Biol Chem. 2004; 279: 26115–26125.
Scheidegger KJ, Du J, Delafontaine P. Distinct and common pathways in the regulation of insulin-like growth factor-1 receptor gene expression by angiotensin II and basic fibroblast growth factor. J Biol Chem. 1999; 274: 3522–3530.
Feng L, Sun W, Xia Y, Tang WW, Chanmugam P, Soyoola E, Wilson CB, Hwang D. Cloning two isoforms of rat cyclooxygenase: differential regulation of their expression. Arch Biochem Biophys. 1993; 307: 361–368.
Kranzhofer R, Schmidt J, Pfeiffer CA, Hagl S, Libby P, Kubler W. Angiotensin induces inflammatory activation of human vascular smooth muscle cells. Arterioscler Thromb Vasc Biol. 1999; 19: 1623–1629.
Kim KW, Kim SH, Lee EY, Kim ND, Kang HS, Kim HD, Chung BS, Kang CD. Extracellular signal-regulated kinase/90-KDA ribosomal S6 kinase/nuclear factor-kappa B pathway mediates phorbol 12-myristate 13-acetate-induced megakaryocytic differentiation of K562 cells. J Biol Chem. 2001; 276: 13186–13191.
Arthur JS, Fong AL, Dwyer JM, Davare M, Reese E, Obrietan K, Impey S. Mitogen- and stress-activated protein kinase 1 mediates cAMP response element-binding protein phosphorylation and activation by neurotrophins. J Neurosci. 2004; 24: 4324–4332.
Suzuki Y, Ruiz-Ortega M, Lorenzo O, Ruperez M, Esteban V, Egido J. Inflammation and angiotensin II. Int J Biochem Cell Biol. 2003; 35: 881–900.
Saadeddin SM, Habbab MA, Ferns GA. Markers of inflammation and coronary artery disease. Med Sci Monit. 2002; 8: RA5–RA12.
Natarajan R, Fisher BJ, Jones DG, Fowler AA III. Atypical mechanism of NF-kappaB activation during reoxygenation stress in microvascular endothelium: a role for tyrosine kinases. Free Radical Biol Med. 2002; 33: 962.
Duan W, Chan JH, Wong CH, Leung BP, Wong WS. Anti-inflammatory effects of mitogen-activated protein kinase kinase inhibitor U0126 in an asthma mouse model. J Immunol. 2004; 172: 7053–7059.
Pueyo ME, Gonzalez W, Nicoletti A, Savoie F, Arnal JF, Michel JB. Angiotensin II stimulates endothelial vascular cell adhesion molecule-1 via nuclear factor-kappaB activation induced by intracellular oxidative stress. Arterioscler Thromb Vasc Biol. 2000; 20: 645–651.(Liping Zhang; Yewei Ma; J)