The Proinflammatory Actions of Angiotensin II Are Dependent on p65 Phosphorylation by the IκB Kinase Complex*

The vasoactive hormone angiotensin II (Ang II) probably triggers inflammatory cardiovascular diseases by activating transcription factors such as NF-κB. We describe here a novel mode of NF-κB activation in cultured vascular smooth muscle cells exposed to Ang II. Ang II treatment resulted in an increase in the phosphotransferase activity of the IKK complex, which was mediated through the AT1 receptor subtype. The typical phosphorylation and proteasome-dependent degradation of the NF-κB inhibitor IκBα were not observed. Rather, Ang II treatment of vascular smooth muscle cells led to the phosphorylation of p65 on serine 536, a signal detected in both the cytoplasm and the nuclear compartments. The use of pharmacological inhibitors that inhibit the activation of MEK by Ang II revealed that phosphorylation of p65 on serine 536 did not require the MEK-ERK-RSK signaling pathway. On the other hand, specifically targeting the IKKβ subunit of the IKK complex by overexpression of a dominant negative version of IKKβ (IKKβ K44A) or silencing RNA technology demonstrated that the IKKβ subunit of the IKK complex was responsible for the detected phosphoserine 536 signal in Ang II-treated cells. Characterization of the signaling pathway leading to activation of the IKK complex by Ang II revealed that neither epidermal growth factor receptor transactivation nor the phosphatidylinositol 3-kinase-AKT signaling cascade were involved. Collectively, our data demonstrate that the proinflammatory activity of Ang II is independent of the classical pathway leading to IκBα phosphorylation and degradation but clearly depends on the recruitment of an IKK complex signaling cascade leading to phosphorylation of p65 on serine 536.

II) 6 is the multifunctional effector of the renin-angiotensin system. It binds to two distinct receptor subtypes, designated AT1 and AT2 (7). Most of the known effects of Ang II are relayed through binding of the AT1 subtype, and studies in models of vascular injury have demonstrated that blockade of the renin-angiotensin system or gene disruption of the AT1 receptor can prevent the development of atherosclerosis (8 -12). In addition to its vasoconstrictive role, Ang II may also act locally as a growth factor. Indeed, it is a hypertrophic factor for vascular smooth muscle cells (VSMC) and cardiac myocytes and has mitogenic effects for different cell types, such as cardiac fibroblasts (for review, see Refs. [13][14][15]. Interestingly, other studies have demonstrated that Ang II has proinflammatory actions by promoting an increase in the expression level of vascular cell adhesion molecule 1 (VCAM-1) and intercellular adhesion molecule 1 (ICAM-1) and several chemokines such as monocyte chemoattractant protein (MCP)-1, regulated upon activation, normal T-cell expressed and secreted (RANTES), interleukin (IL)-8, IL-6, and interferon-␥-inducible protein 10 (for reviews, see Refs. 16 and 17). However, the molecular understanding of the proinflammatory actions of Ang II is still unclear.
Activation of the Ang II AT1 receptor (AT1R) in VSMC is functionally linked to the recruitment and activation of cytosolic kinases such as the classical Ras/mitogen-activated protein kinase kinase-1 (MEK1)/ extracellular signal-regulated kinase (ERK)/ribosomal S6 kinase (RSK), protein kinases C, calmodulin (CAM) kinases, and Janus kinases. These kinases can in turn lead to the activation of transcription factors involved in gene regulation and the growth promoting effect of Ang II (15,18). The transcription factor nuclear factor B (NF-B) is a key regulator of inflammation, immune response, and cellular survival (19) and the proinflammatory actions of Ang II have been attributed to its activation (16, 20 -26).
The NF-B family is represented by five members: p50, p65(RelA), c-Rel, p52, and RelB. In resting cells, they exist as homo-or heterodimers that are sequestered in the cytoplasm in an inactive form through their association with one of several inhibitory molecules, namely IB-␣, -␤, -⑀, p105, and p100. These inhibitors mask the nuclear localization sequence in the Rel homology domain of NF-B, thereby preventing it from accumulating in the nucleus (for recent reviews, see Refs. 27 and 28). IB␣ is the most characterized member in this family. It is composed of three domains: an N-terminal signal responsive domain, a central ankyrin repeat domain that interacts with NF-B, and a C-terminal PEST domain that is responsible for the basal turnover of the protein (29). In the classical pathway, the first phase of NF-B activation mainly consists of the regulated degradation of IB␣ and is triggered by prototypical activators such as tumor necrosis factor (TNF)-␣, lipopolysaccharide, IL-1␤, and phorbol 12-myristate 13-acetate. These stimuli induce the phosphorylation of IB␣ at Ser 32 and Ser 36 in the N-terminal signal responsive domain by the canonical IB kinase (IKK) complex, which is composed of two catalytic subunits called IKK␣ and -␤ and one regulatory subunit IKK␥. Phosphorylated IB␣ is subsequently polyubiquitinated and targeted to the 26 S proteasome complex, resulting in the release and nuclear accumulation of NF-B, which can now stimulate target gene transcription. Studies report that Ang II can stimulate degradation of IB␣, but the decrease in its expression level is very small suggesting the existence of other pathways to regulate NF-B activity (24,26).
Notably, several lines of evidence suggest the existence of a second phase of NF-B activation. Part of this second phase involves phosphorylation of the p65 subunit, which plays a key role in determining both the strength and duration of the NF-B-mediated transcriptional response (for recent reviews, see Refs. 30 and 31). The sites of phosphorylation reported to date are Ser 276 and Ser 311 in the Rel homology domain and three phosphoacceptor sites located in the transactivation domain, Ser 468 , Ser 529 , and Ser 536 . Several candidate kinases that phosphorylate each serine residue have been identified, such as protein kinase A and mitogen-and stress-activated protein kinase-1 for Ser 276 (32,33), protein kinase C-for Ser 311 , glycogen synthase kinase-3␤ for Ser 468 , casein kinase II (CKII) for Ser 529 (34), and IKK␣/␤ as well as RSK for Ser 536 (35)(36)(37). Importantly, phosphorylation at Ser 536 was recently shown to reduce the ability of p65 to bind IB␣ (36) and to allow the recruitment of TAFII31, a component of the basal transcriptional machinery (37). Phosphorylation at Ser 536 is also responsible for recruitment of coactivators such as p300 (38) and also triggers its rapid turnover in the nucleus (39). All these evidences support the importance of p65 phosphorylation at Ser 536 in the function of NF-B. Notably, a recent study proposed that Ang II induces NF-B through a RSK-dependent pathway leading to Ser 536 phosphorylation of p65 (40). In light of these interesting findings, we have set up experiments to identify more precisely the molecular mechanism by which Ang II induces proinflammatory action in VSMC and now describe a series of studies dissecting the novel mechanism by which Ang II activates NF-B. We show that following binding to the AT1R, Ang II leads to an increase in the phosphotransferase activity of the IKK complex, which leads to phosphorylation of p65 on Ser 536 . Because IB␣ is not degraded in response to Ang II, we propose a model where it is the phosphorylation of p65 on Ser 536 that is likely to be involved in the proinflammatory actions of Ang II in VSMC.
Cell Types and Transfection-Rat VSMC and VSMC overexpressing a dominant-negative version of EGFR (HERCD533) (42) were obtained from Dr. Sylvain Meloche (University of Montreal, Montreal, Quebec) and Darren Richard (Laval University, Quebec, Quebec) and grown in low glucose Dulbecco's modified Eagle's medium supplemented with 10% calf serum, 2 mM-glutamine, and antibiotics (50 units/ml penicillin and 50 g/ml streptomycin). Cultures were maintained at 37°C in a humidified atmosphere of 95% air, 5% CO. All experiments were conducted on cells at passage levels 9 -14. Quiescent VSMC were obtained by incubation of 95% confluent cell cultures in serum-free Dulbecco's modified Eagle's medium-Ham's F-12 (1:1) supplemented with 15 mM Hepes (pH 7.4), 0.1% bovine serum albumin, and 5 g/ml transferrin for 48 h. For experiments with pharmacological inhibitors, the cells were treated with vehicle alone or with the indicated concentrations of inhibitors for 30 min before addition of Ang II. 293T cells were purchased from American Type Culture Collection (ATCC), cultured in minimal essential medium containing 10% fetal bovine serum, and transfected using the calcium phosphate coprecipitation method. All cells tested negative for mycoplasm contamination.
Retrovector Construction, Transduction, and Generation of Rat VSMC Overexpressing IB␣2N⌬4-The pMSCVneo IB␣2N⌬4 retrovector was a kind gift of Dr. John Hiscott (McGill University) and has been described (43). The Phoenix Amphotropic packaging cell line was transiently transfected with plasmids pMSCVneo and pMSCVneo IB␣2N⌬4. At 48 h post-transfection, retrovirus-containing medium was harvested and used to infect rat VSMC. VSMC were infected twice at 24-h intervals in the presence of 10 g/ml Polybrene (Sigma); selection with 800 g/ml G418 (Invitrogen) was started 5 days later. After 14 days, a population of resistant VSMC was selected. For selection of clones from both rat VSMC (Neo) and rat VSMC (IB␣2N⌬4), cloning rings were used, and expression of the transgene was verified by immunoblot analysis.
In Vitro Kinase Assays-The phosphotransferase activity of the IKK complex was measured as described previously (41). Briefly, 250 to 400 g of whole cell extracts were incubated for 4 h at 4°C with specific antibody to IKK␥ (anti-IKK␥ (SC-8330)) preabsorbed to protein A-Sepharose beads. The immune complexes were washed three times with lysis buffer and once with kinase buffer (20 mM Hepes, pH 7.4, 20 mM MgCl 2 , and 2 mM dithiothreitol). IKK complex activities were assayed by resuspending the beads in 40 l of kinase buffer containing 1 g of GST substrates (GST-IB␣ or GST-p65) as indicated, 20 M ATP, and 20 Ci of [␥-32 P]ATP. The reactions were incubated at 30°C for 30 min and stopped by the addition of 5ϫ Laemmli's sample buffer. The samples were analyzed by SDS-gel electrophoresis. Following Coomassie staining, the gels were dried and exposed to a Gel Documentation device (Typhoon scanner 9410, Amersham Biosciences) for imaging and quantification. In some experiments, the upper part of the gel was electrophoretically transferred to Hybond-C nitrocellulose membranes and immunoprecipitated IKK complex was revealed by immunoblotting.
Phosphorus 32 Labeling and Immunoprecipitation-Quiescent VSMC in 100-mm Petri dishes were metabolically labeled for 4 h at 37°C in bicarbonate and phosphate-free Hepes-buffered Dulbecco's modified Eagle's medium containing 0.75 mCi/ml [ 32 P]phosphoric acid. The cells were then stimulated by the addition of 100 nM Ang II or 20 ng/ml TNF-␣ to the medium for the indicated periods of time. Following a quick wash with ice-cold phosphate-buffered saline, whole cell extracts were prepared as described above and precleared for 1 h with 10 l of normal rabbit serum and incubated for 4 h at 4°C with anti-p65 antibody preabsorbed to protein A-Sepharose beads. Immune complexes were washed six times with lysis buffer. Protein complexes were eluted by heating at 95°C for 5 min in denaturating sample buffer and analyzed by SDS-gel electrophoresis on 10% acrylamide gels. The proteins were then electrophoretically transferred to polyvinylidene difluoride membranes (Millipore) in 25 mM Tris, 192 mM glycine, and 20% methanol, and exposed to a gel documentation device (Typhoon scanner 9410, Amersham Biosciences) for imaging and quantification. The membranes were used in immunoblotting experiments using anti-p65 antibody to verify the immunoprecipitated p65.

FIGURE 1. A NF-B super-repressor antagonizes Ang II-induced IL-6 transcription in VSMC.
A, production of stable cell lines expressing NF-B super-repressor. VSMC were infected with a retroviral construct expressing the NF-B super-repressor (IB␣2N⌬4) or encoding for the neomycin cassette only (Neo). Following selection of a stable population, clones were isolated and analyzed for the transgene expression by Western blot analysis. B, the expression of the NF-B super-repressor significantly abrogates Ang II-induced IL-6 and MCP-1 transcription. Quiescent Neo and IB␣2N⌬4 clones were stimulated for different times with 100 nM Ang II. Total RNA was isolated and subjected to reverse transcriptase-PCR analysis with primers directed against rat IL-6 and MCP-1 transcripts. Normalization was accomplished using primers against the RPL32 housekeeping gene.
Biosynthetic Labeling Experiments-To examine the turnover of IB␣ protein, quiescent VSMC in 100-mm Petri dishes were pulselabeled for 1 h with 166 Ci/ml of [ 35 S]methionine and [ 35 S]cysteine and then chased for the indicated times in serum-free medium containing excess methionine and cysteine and either Ang II or TNF-␣. The cells were then washed twice with ice-cold phosphate-buffered saline and lysed in Triton X-100 lysis buffer. Lysates (500 g of protein) were precleared for 1 h with 5 l of normal rabbit serum and the resulting supernatants were incubated with protein A-Sepharose beads preabsorbed with 2 g of anti-IB␣ for 4 h at 4°C. Immune complexes were washed five times with Triton X-100 lysis buffer. Proteins were eluted by heating at 95°C for 5 min in denaturing sample buffer and analyzed by SDS-gel electrophoresis on 12% acrylamide gels. The IB␣ protein was detected by fluorography and visualized using a gel documentation device (Typhoon scanner 9410, Amersham Biosciences).
Electromobility Shift Assays-Preparation of cytosolic and nuclear extracts as well as electromobility shift assays was accomplished as described previously (44).

NF-B-dependent Chemokine Gene Induction by Ang II in VSMC-
Because controversy exists in the literature as to know whether Ang II has the ability to activate NF-B transcription factor (45,46), we first verified the state of activation of known NF-B-regulated chemokines, namely IL-6 and MCP-1, in VSMC stably overexpressing the superrepressor version of IB␣, IB␣2N⌬4. This mutant molecule has been shown to abrogate totally NF-B induction by different stimuli. It involves mutation of the IB serine phosphorylation sites to alanine (S32A/S36A) and removal of the C-terminal PEST domain to generate a form of IB (IB␣2N⌬4). The basal turnover of this mutated IB is diminished but most importantly, IB␣2N⌬4 is no longer responsive to inducer-mediated phosphorylation and degradation, and thus acts as a transdominant repressor of the NF-B pathway (by sequestering NF-B subunits in the cytoplasm) (43). Following selection of the stable transfectant, clones were isolated and analyzed for transgene expression in Western blot analysis (see Fig. 1A). Note that when the level of IB␣2N⌬4 is high, the expression of endogenous IB␣ is diminished due to the fact that the IB gene is itself regulated by the NF-B transcription factor (Fig. 1A, compare clones IB␣2N⌬4 number 1 with 4). This illustrates that overexpression of IB␣2N⌬4 titrates out any free NF-B subunits thus allowing reduction in basal NF-B transcription of target genes such as IB␣. Following Ang II stimulation, induction of both MCP-1 and IL-6 transcripts was observed in the two clones of rat VSMC expressing only the neomycin cassette (clones neo 3 and 6). However, the presence of a high level of IB␣2N⌬4 in clone 4 strongly abrogated the induction of these transcripts by Ang II (Fig. 1B). The clone expressing a moderate level of IB␣2N⌬4 (clone 1) had an intermediate effect on the induction of IL-6 and MCP-1. Together, these data clearly demonstrate a role of NF-B in Ang II-induced proinflammatory genes in VSMC.
IKK Complex Activation by Ang II in VSMC-Given the essential role of the IKK in the activation of NF-B, we next verified the phosphotransferase activity of this complex when VSMC were exposed to Ang II. Following immunoprecipitation of the scaffolding protein of the IKK complex, IKK␥, we observed that Ang II treatment of VSMC resulted in a time-dependent ( Fig. 2A) and dose-dependent (Fig. 2B) stimulation of the IKK complex as observed by an increase in phosphotransferase activity toward the in vitro substrate GST-IB␣. The maximal increase in the phosphotransferase activity was observed at 1 M Ang II and sustained for at least 60 min starting at 1 min post-stimulation (Fig. 2, A   and B). Importantly, the observed IKK complex stimulation was completely abrogated when cells were preincubated with the AT1 antagonist Irbezartan, demonstrating that activation of the IKK complex by Ang II is AT1R-dependent (Fig. 2B). The use of the recombinant protein GST-IB␣2N, where Ser 32 and Ser 36 were mutated to alanine, showed no phosphorylation under our conditions thus confirming the specificity of the immunoprecipitated IKK complex in VSMC (Fig. 2C).
Nonclassical Activation of NF-B by Ang II in VSMC-Through phosphorylation-dependent degradation of IB proteins by the proteasome, activation of the IKK complex is a rate-limiting step in the activation of the NF-B transcription factor (27,47). Notably, several groups have published data showing that Ang II induces weak or strong degradation of IB proteins in VSMC (22,24). However, another recent study reported no degradation of the IB␣/␤ NF-B inhibitors in the same model exposed to Ang II (40). Given that we observed a significant increase in the phosphotransferase activity of the IKK complex in VSMC stimulated with Ang II (Fig. 2), we wanted to revisit the possibility that the expression level of IB proteins might be reduced following Ang II treatment. Western blot analysis clearly demonstrated no degradation of the two NF-B inhibitors, IB␣ and IB␤, nor phosphorylation of IB␣ on Ser 32 when cells were exposed to Ang II (see Fig. 3A, panels a-c) as opposed to TNF-␣-treated cells where a specific IB␣ phosphoserine 32 signal was observed after 5 min of stimulation (Fig.  3A, panel b, lanes 11 and 12) followed by a net decrease of the IB␣ isoform after 10 min (Fig. 3A, panel a, lane 12). To confirm this observation, we next determined the rate of IB␣ turnover by pulse-chase experiments on VSMC treated with Ang II and TNF-␣. When com-FIGURE 2. Activation of the canonical IKK complex in Ang II-treated VSMC. A, quiescent VSMC were stimulated with 100 nM Ang II for different times or with 20 ng/ml TNF-␣ for 10 min. Whole cell extracts were prepared and subjected to immunoprecipitation with anti-IKK␥ antibody. The phosphotransferase activity of the immunoprecipitates was assayed as described under "Experimental Procedures" using GST-IB␣ as substrate. After the kinase assay, reactions were run on SDS gels, dried, and exposed to the Typhoon image analyzer for quantification. B, activation of the IKK complex is dose-dependent and mediated by the AT1R. Quiescent VSMC were pretreated for 30 min with 1 M Irbezartan or 0.01% vehicle dimethyl sulfoxide and then stimulated with the indicated concentrations of Ang II for 5 min. Whole cell extracts were prepared and subjected to the in vitro kinase assay as described in A. C, IKK complex in vitro kinase assay is specific for Ser 32/36 of IB␣. Quiescent VSMC were stimulated for the indicated times with 100 nM Ang II. Whole cell extracts were prepared and the immunoprecipitated IKK complex was either incubated in the presence of GST-IB␣ wild type of GST-IB␣ 2N in which the two phosphoacceptor sites were mutated to alanine. After the kinase assay, reactions were run on SDS gels, dried, and exposed to the Typhoon image analyzer for quantification.
pared with untreated cells, stimulation of cells with Ang II did not affect the stability of IB␣, as opposed to stimulation with TNF-␣ where 90% of NF-B inhibitor was degraded after 15 min of treatment (Fig. 3B). Albeit the lack of IB␣ degradation by Ang II, we observed a significant DNA binding activity of p50/p65 heterodimers in VSMC (see supplemental data Fig. 1). Ang II has pleiotropic actions at multiple points in the NF-B activation pathway in VSMC. Reports have indeed demonstrated that Ang II could lead to NF-B activation through an increased processing of p105 to p50 (24), which is considered to be a constitutive process (27). In addition, Ang II induces tyrosine phosphorylation of IB␣ in fetal VSMC (48), a process thought to promote its physical dissociation from NF-B. However, our own immunoprecipitation studies and Western blot analysis clearly demonstrated no tyrosine phosphorylation of IB␣ when VSMC were exposed to Ang II as opposed to pervanadate treatment (see supplemental data Fig. 2A). No processing of p105 to p50 was also observed over a 16-h treatment period with Ang II (see supplemental data Fig. 2B). Collectively, these data demonstrate that, albeit a significant activation of the IKK complex by Ang II and the increase in DNA binding activity of p65/p50 complexes, the classical pathway leading to degradation of IB proteins and atypical models of activation (tyrosine phosphorylation of IB␣ and p105 processing), are not involved in Ang II-induced activation of the NF-B transcription factor.
Ang II Induces Phosphorylation of p65 on Ser 536 in an IKK-dependent Manner-In addition to IB proteins, it is now well recognized that components of the IKK complex have several other substrates including histone H3, p100, and p65. Whereas p65 is a substrate for IKK␣ and -␤ (for recent reviews, see Refs. 30 and 31), histone H3 and p100 are phosphorylated only by IKK␣ (47,49,50). Because we observed a significant activation of the IKK complex by Ang II, we next analyzed the phosphorylation state of p65 after immunoprecipitation from 32 P-labeled VSMC stimulated with Ang II or TNF-␣. p65 is expressed as a phosphoprotein in quiescent VSMC (Fig. 4A, lane 1) and exposure to Ang II or TNF-␣ significantly increased its phosphorylation level (Fig. 4A). Using a phosphospecific antibody, we next addressed if p65 phosphorylation was occurring on an IKK phosphorylation site, namely Ser 536 . A specific phosphoserine 536 signal was detected when cells were exposed to Ang II (Fig. 4B, panel a). Interestingly, a treatment of 15 min with another growth factor for VSMC, EGF, which like Ang II induces the activation of AKT and ERK (see Fig. 4B, panels c and d, lane 6), failed to induce phosphorylation of p65 on Ser-536 thus demonstrating the specificity of the signaling cascades activated by Ang II and leading to p65 phosphorylation in VSMC. In addition to IKK␣ and -␤, RSK1 was recently shown to target Ser 536 of p65 (36). Because Ang II is a strong activator of the MEK-ERK-RSK signaling cascade in VSMC, we first verified if RSK was recruited by Ang II to induce phosphorylation of p65 on Ser 536 . The use of pharmacological inhibitors that target specifically MEK 1/2, namely PD98059 and UO126, completely abrogated Ang II-induced RSK1 activation in VSMC without affecting the specific phosphoserine 536 signal (Fig. 5A). Inhibition of the MEK-ERK-RSK signaling cascade for up to ]cysteine for 60 min after which the medium was changed to fresh medium containing an excess of non-radiolabeled methionine/cysteine and 100 nM Ang II or 20 ng/ml TNF-␣. At different times, cell extracts were prepared and subjected to immunoprecipitation with anti-IB␣ antibody. After extensive washing, the immunoprecipitated proteins were separated by electrophoresis on 12% acrylamide gels and analyzed by fluorography using a Typhoon image analyzer.

FIGURE 4. Ang II-induced rapid phosphorylation of p65 in VSMC.
A, in vivo phosphorylation of p65. Quiescent VSMC were pulse-labeled with [ 32 P]orthophosphate for 4 h after which the labeled cells were stimulated with 100 nM Ang II or 20 ng/ml TNF-␣. At different times, cells extracts were prepared and subjected to immunoprecipitation with anti-p65 antibody. After extensive washing, the immunoprecipitated proteins were separated by electrophoresis on 10% acrylamide gels and transferred to a polyvinylidene difluoride membrane. The membrane was then exposed to a gel documentation device for imaging and quantification (TY) or revealed by immunoblotting (IB) with an anti-p65 antibody to confirm equal amounts of immunoprecipitated proteins. B, Ser 536 phosphorylation of p65 by Ang II. Quiescent VSMC were stimulated for the indicated times with 100 nM Ang II, 20 ng/ml TNF-␣, and 100 ng/ml EGF. Whole cell extracts were prepared and subjected to immunoblotting analysis using anti-phospho-p65 (Ser 536 ) antibody (a), anti-p65 antibody (b), anti-phospho-AKT (Ser 473 ) antibody (c), and anti-phospho-ERK1/2 (Thr 202 -Tyr 204 ) antibody (d). Membranes were stripped between each blotting experiment. C, the kinetic of Ser 536 phosphorylation of p65 paralleled the activation of the IKK complex. Quiescent VSMC were stimulated for the indicated times with Ang II. Whole cell extracts were prepared and subjected to a IKK complex kinase assay (a) as described in the legend to Fig. 2. For immunoblotting (lower  panel), the upper part of the gel was probed with an anti-IKK␤ antibody to confirm equal amounts of the immunoprecipitated kinase complex (b). WCE were also analyzed by immunoblotting using anti-phospho-p65 (Ser536) antibody (c) and anti-p65 antibody (d). Membranes were stripped between each blotting experiment. D, Ang II treatment leads to phosphorylation of p65 on Ser 536 in the cytoplasm. Quiescent VSMC were stimulated for the indicated periods of time with Ang II. Cytoplasmic and nuclear extracts were prepared and analyzed by immunoblotting using anti-phospho-p65 (Ser536) antibody and anti-p65. The purity of the nuclear fractions was demonstrated by the virtual exclusion of p105. Using a higher sensitivity setting on the documentation device allowed us to better appreciate the presence of p65 phosphorylated on Ser 536 in the nucleus (lower right panel). E, overexpression of a dominant negative version of IKK␤ in 293T abrogates the phosphorylation of p65 on Ser 536 by Ang II. 293T cells were cotransfected with AT1R in the presence of either pFlag-IKK␤ (K44A) or the vector alone. 30 h post-transfection, cells were incubated in serum-free medium for the next 18 h before stimulation with Ang II (100 nM) for the indicated periods of time. Whole cell extracts were prepared and subjected to immunoblot analysis using anti-phospho-p65 (Ser536) antibody, anti-FLAG antibody, and anti-p65 antibody as indicated. F, reducing the expression level of IKK␤ inhibits the phosphorylation of p65 on Ser 536 by Ang II. VSMC were transfected with a siCONTROL non-targeting silencing RNA (control) or two different RNA duplexes designed to specifically target IKK␤ as indicated. 24 h post-transfection, cells were incubated in serum-free medium for 48 h and then stimulated with Ang II for the indicated times. Whole cell extracts were prepared and analyzed by immunoblotting using the indicated antibodies. Membranes were stripped between each blotting experiment.
1 h did not prevent the rapid induction nor the sustained phosphorylation of p65 on Ser 536 by Ang II (Fig. 5B). To verify the possible role of the IKK complex in Ang II-induced p65 phosphorylation on Ser 536 , we next verified if the activation of the IKK complex followed a kinetic similar to the phosphoserine 536 signal. A detailed kinetics in VSMC revealed that the activation of the IKK complex by Ang II was rapid and sustained for at least 24 h (Fig. 5C, panel a). Interestingly, the activation of the IKK complex followed the same kinetic of phosphorylation of p65 on Ser 536 (Fig. 5C, panel c). As predicted, stimulation of the IKK complex by Ang II also resulted in an increase in phosphotransferase activity toward the in vitro substrate GST-p65-(354 -551) (see supplemental data Fig. 3). Because IKK-induced p65 phosphorylation was previously shown to occur in the cytoplasm (51, 52), we next addressed in which cellular compartments phosphorylation of p65 occurred in Ang II-treated cells. Fig. 5D shows the presence of a strong inducible phosphoserine 536 signal that was predominantly detected in the cytoplasmic compartment. Interestingly, a significant amount was also observed in the nucleus. To further substantiate the hypothesis of a possible involvement of the IKK complex in Ang II-mediated p65 phosphorylation, we took advantage of a point mutant version of IKK␤ (IKK␤ K44A), which lacks the ATP binding site and therefore acts in a dominant negative fashion. When co-expressed with the AT1R in 293T cells, it totally prevented the phosphorylation of p65 by Ang II (Fig. 5E). To directly ask whether the IKK␤ subunit of the IKK complex was responsible for the detected p65 phosphoserine 536 signal in Ang II-treated cells, we next used RNA silencing technology. Upon transfection of the indicated siRNA duplexes, the expression levels of IKK␤ were down-regulated by 90% and correlated with a net decrease of the phosphoserine 536 signal in Ang II-treated cells (Fig. 5F). Silencing IKK␤ in VSMC did not, however, affect the phosphorylation of the ERK isoforms by Ang II. Altogether, these data demonstrate that the IKK␤ subunit of the IKK complex plays an essential role in Ser 536 phosphorylation of p65 by Ang II.
IKK␤ is a cytoplasmic kinase and the above results suggest that it is the cytoplasmic phosphorylation of p65 that triggers its nuclear accumulation, possibly through a process involving loss of affinity for IB␣ without the requirement for its degradation (36). Consequently, to further substantiate the hypothesis of a dispensable role of the IB inhibitors in Ang II-induced nuclear accumulation of phosphorylated p65, VSMC were pretreated with the proteasome inhibitor MG-132 and again a significant amount of phosphorylated p65 was found in the nucleus of Ang II-treated cells (see supplemental data Fig. 4).
Molecular Characterization of AT1 Receptor Signaling to the IKK Complex-Because we observed an important role of the IKK complex in the proinflammatory actions of Ang II, we next wanted to verify what might be the signaling pathways coupling the AT1R to the IKK complex. Using pharmacological as well as molecular approaches, we address the roles of the EGF receptor and the PI 3-kinase-AKT pathway in Ang II-mediated IKK complex activation in VSMC. Fig. 6A shows that pretreatment with the tyrphostin AG1478, a selective EGFR kinase inhibitor that efficiently blocks the activation of ERK1 by Ang II (Ref. 41 and data not shown), did not antagonize the activation of the IKK complex. This observation is in agreement with activation of the phosphotransferase activity of the IKK complex in rat VSMC overexpressing a dominant-negative version of EGFR (HERCD533) (42) (Fig. 6B). Inhibition of the PI 3-kinase pathway by use of the specific inhibitor LY294002 inhibited activation of AKT by Ang II (data not shown), but was also without any consequence on Ang II-induced activation of the IKK complex in VSMC (Fig. 6C). Together, these data show that AT1R-mediated transactivation of the EGFR and the PI 3-kinase-AKT pathway are not involved in the induction of the IKK complex by Ang II in VSMC.

DISCUSSION
After primary injury of the vasculature, the inflammation response that follows is characterized by three key events: 1) increment of vascular permeability; 2) infiltration of inflammatory cells; and 3) tissue repair and remodeling (for review, see Ref. 16). Through the induction of a repertoire of NF-B-regulated genes such as IL-6, MCP-1, IL-8, RAN-TES, VCAM-1, and ICAM-1, it is now well appreciated that Ang II participates in these key events of the inflammatory response leading to the development of cardiovascular diseases like atherosclerosis (16). Whereas the pathways leading to NF-B activation following treatment with prototypical activators, such as TNF-␣, lipopolysaccharide, or IL-1␤, is well characterized, the molecular understanding of the signaling pathways that are involved in the coupling of G protein-coupled receptors, such as AT1R or AT2R to NF-B, is still unclear. The results presented in this report show that Ang II activates NF-B in cultured VSMC, and independently of IB␣ phosphorylation and IB ␣/␤ degradation (see Fig. 3). These observations strongly suggest that the classical pathway of NF-B activation is not triggered by Ang II in VSMC. Moreover, other mechanisms described as being responsible for Ang II-induced NF-B activation, namely p105 processing and tyrosine phosphorylation of IB␣ (24,48), are not observed in Ang II-treated VSMC. Instead, we now propose a new model where Ang II treatment leads to an AT1R-dependent activation of the IKK complex. The activated IKK complex then phosphorylates Ser 536 of p65 (see Fig. 7 for the detailed mechanism). The extent to which the present mechanism can account for the known proinflammatory actions of Ang II in the cardiovascular system is unknown at the moment as other transcription factors are also involved in the proinflammatory actions of the peptide, such as CREB and AP-1 (ATF-2/c-Jun) (53,54). However, p65 phosphorylation on Ser 536 has also been observed in Ang II-treated cultured adipocytes as well as in pressure-dependent activation of NF-B in arterial segments (55,56). Thus, a possible molecular link may exist between two known risk factors for the development of atherosclerosis, namely obesity and hypertension, for the activation of NF-B through p65 phosphorylation. FIGURE 6. Activation of the IKK complex by Ang II is independent of EGFR transactivation and the PI 3-kinase pathway. A, quiescent VSMC were pretreated for 30 min with 250 nM AG1478 or 0.01% Me 2 SO (DMSO) and then stimulated for 10 min with 100 nM Ang II as indicated. In vitro IKK complex kinase activity was measured as described in the legend to Fig. 2. B, quiescent parental (wt) and HERCD533 (mut) cells were stimulated with 100 nM Ang II for the indicated times and the phosphotransferase activity of the IKK complex was assayed as described above. C, quiescent VSMC were pretreated for 30 min with 30 M LY294002, or 0.01% Me 2 SO, and then stimulated for 10 min with 100 nM Ang II. An IKK complex kinase assay was conducted as above.
No Degradation of IB␣ in Ang II-treated VSMC-Many studies have looked at IB degradation when VSMC are exposed to Ang II. Intriguingly, no studies have addressed if the canonical IKK complex was activated by Ang II. Using in vitro kinase assays, our report clearly demonstrates a rapid and sustained phosphotransferase activity of the IKK complex, which was mediated by the AT1R and dependent on the concentration of Ang II used (see Figs. 2 and 5C). In response to prototypical activators of NF-B, activation of the IKK complex normally leads to phosphorylation and degradation of IB␣. Why then does Ang II not induce the degradation of IB␣? One possible scenario that might account for this observation is the recent observation demonstrating recruitment of ␤-arrestin proteins to IB␣ when cells are exposed to ␤2-adrenergic receptors (57,58). Interestingly, this association between ␤-arrestin proteins and IB␣ was shown to abrogate TNF-␣-induced IB␣ phosphorylation and degradation (57). Thus, experiments are underway to try to verify if the stimulation of VSMC with Ang II promotes the recruitment of ␤-arrestin proteins to IB␣, a process that could explain the incapacity of Ang II to induce the degradation of IB␣.
Phosphorylation of p65 by IKK␤-A recent report by Zhang and colleagues (40) demonstrated phosphorylation of p65 on Ser 536 in VSMC exposed to Ang II. Using the MEK 1/2 inhibitor U0126 as well as silencing RNA technology, they suggested that this was essentially occurring in a RSK-dependent manner. However, several observations from our study suggest a more important contribution of the IKK complex in this process. First, our study clearly shows that the two MEK 1/2 inhibitors, PD98059 and U0126, significantly abrogated the activation of RSK by Ang II without affecting the phosphoserine 536 signal (Fig. 5, A and B). Second, the kinetic of the IKK complex activation follows the same kinetic as p65 Ser 536 phosphorylation (Fig. 5C). Third, it was shown that IKK␤-dependent phosphorylation of Ser 536 of p65 occurs in the cyto-plasm (51,52), similar to what we observed in Ang II-treated cells (Fig.  5D), whereas RSK-dependent phosphorylation of p65 happens exclusively in the nuclear compartment (36). Fourth, overexpression of a dominant negative version of IKK␤ completely abrogated Ang II-induced phosphorylation of p65 on Ser 536 in 293T cells expressing the AT1R (Fig. 5E). Fifth, down-regulation of IKK␤ expression by siRNA technology has allowed us to conclude that activation of the IKK complex is responsible for the detected phosphoserine 536 signal observed in Ang II-treated VSMC (Fig. 5F). We do not have an explanation for the difference between the report by Zhang and colleagues (40) and this study. Notably, the two MEK 1/2 inhibitors UO126 and PD98059 had no effect on the phosphorylation of p65 on Ser 536 by Ang II in VSMC derived from Sprague-Dawley or Wistar-Kyoto rats (data not shown).
Pleiotropic Roles for the Phosphorylation of p65 on Ser 536 -Although the exact roles of this covalent modification of p65 are not precisely characterized, it seems to affect several aspects of NF-B signaling. Thus, Ser 536 phosphorylation of p65 has been implicated in many nuclear effects cumulating to activation of NF-B target genes (37,38). Through an increased association of a phosphomimetic form of p65 with TAFII31 (37), and the role of this phosphoacceptor site in recruiting p300 (38), it has become clear that phosphorylation of p65 may plays a major role in NF-B-mediated transcriptional response. Ser 536 phosphorylation of p65 could also affect its ability to associate with the IB␣ inhibitor. Notably, p53 was recently reported to induce NF-B activity without IB␣ degradation (36) and a model has been proposed to explain the enhanced nuclear localization and increased DNA binding of NF-B. In this model, p65 phosphorylated at Ser 536 loses its affinity for IB␣ and alters the basal nucleocytoplasmic shuttling properties of the NF-B⅐IB␣ complex favoring nuclear retention. Based on this model, our data suggest that a subpopulation of p65 phosphorylated on FIGURE 7. Proposed model for the activation of NF-B by Ang II in VSMC. Following binding to AT1R, Ang II induces the transactivation of the EGFR that then transmits downstream signals to two different kinase modules: the Raf/MEK/ERK and the PI 3-kinase/PDK/AKT modules (42,59,60). These signaling cascades are not involved in the activation of the canonical IKK complex by Ang II. Activated IKK complex is likely not able to engage IB␣ because of the possible recruitment of ␤-arrestin to the latter (57,58). Instead, the IKK complex induces the phosphorylation of p65 at Ser 536 in a complex formed of IB␣/p50/p65 in the cytoplasm (52). This complex shuttles in and out from the nucleus. A subpopulation of p65 phosphorylated at Ser 536 losses its affinity for IB␣ (36) thereby allowing a significant nuclear accumulation of p65 and engagement of the latter with coactivators (CBP/p300) (38) to the B-response element found on NF-B-regulated genes. Phosphorylation of p65 on Ser 536 could also be involved in the increase turnover of the protein (39) thus regulating the overall NF-B response.
Ser 536 loose their affinity for IB␣ and accumulate into the nuclear compartment (see Fig. 5D and supplemental data Fig. 4). Finally, phosphorylation of p65 on Ser 536 also triggers its rapid turnover in the nucleus (39). Interestingly, we also noticed a prolonged nuclear signal of p65 phosphorylated on Ser 536 in the presence of MG132 (compare Figs. 5D and supplemental data Fig. 4). Thus, it is likely that the induction of phosphorylation of p65 by Ang II regulates the overall NF-B response through both positive inputs (increase in nuclear accumulation and p65 transcriptional activity) as well as negative inputs (increase in p65 turnover).
AT1R Coupling to the IKK Complex-The mechanisms by which G protein-coupled receptor are able to transduce a signal from the cellular environment into the cytoplasm have been studied for decades. Albeit the essential roles of heterotrimeric G proteins in this process, it has now become well appreciated that G protein-coupled receptors use tyrosine kinase receptors to activate multiple signaling pathways, most of which influence cell growth and survival. Thus, for AT1R, it has been clearly established that transactivation of the EGFR is essential for activation of the Raf/MEK/ERK as well as PI 3-kinase/PDK/AKT modules (42,59,60). In addition to ERKs and AKT, activation of NF-B is also linked to cellular growth and survival, and with the observations that both PDK1 and AKT can act as IKK-activating kinases (61,62), it was therefore important to address if EGFR transactivation and the PI 3-kinase/PDK/AKT module were involved in the activation of the IKK complex by Ang II. A pharmacological approach using EGFR kinase and PI 3-kinase inhibitors and a molecular approach using VSMC overexpressing a dominant negative version of EGFR have allowed us to conclude that neither EGFR transactivation nor the PI 3-kinase pathway were involved in AT1R-mediated activation of the IKK complex.
In conclusion, our data suggest that AT1R is coupled to a nonclassical mode of activation of NF-B in VSMC involving an IKK complex/p65 Ser 536 signaling cascade. Understanding the role of inflammation in atherosclerosis might pave the way for the development of new therapeutics to treat this cardiovascular disease such as IKK␤ inhibitors (28). While our paper was submitted, a related paper was published (63) indicating a role of IKK␤ in p65 phosphorylation by Ang II.