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(Circulation Research. 2005;97:975.)
© 2005 American Heart Association, Inc.

Molecular Medicine

Dual Pathways for Nuclear Factor B Activation by Angiotensin II in Vascular Smooth Muscle

Phosphorylation of p65 by IB Kinase and Ribosomal Kinase

Liping Zhang, Jizhong Cheng, Yewei Ma, Walter Thomas, Jiqiang Zhang, Jie Du

From the Department of Medicine-Nephrology (L.Z., J.C., Y.M., J.Z., J.D.), Baylor College of Medicine, Houston, Tex; and Molecular Endocrinology (W.T.), Baker Heart Research Institute, Melbourne, Victoria, Australia.

Correspondence to Jie Du, BCM 285, One Baylor Plaza, Houston, TX 77030. E-mail jdu{at}bcm.edu

	Abstract

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Activation of nuclear factor (NF)-B by angiotensin II (Ang II) plays an essential role in stimulating expression of vascular adhesion molecules, which are essential for vascular inflammation. We report that Ang II activates NF-B by phosphorylating its p65 subunit via a pathway mediated partially by ribosomal S6 kinase (RSK). In investigating other pathway(s) that may be involved, we found that the ability of Ang II to activate NF-B in mouse embryonic fibroblast is suppressed (&70%) either by deletion of IB Kinase (IKK) or by inhibiting or knocking down IKK in vascular smooth muscle cells using a dominant-negative IKK adenovirus or small interference RNA to IKKß. Thus, Ang II also stimulates NF-B via IKK. In vitro, we found that Ang II stimulates IKK to phosphorylate myelin basic protein and the p65 subunit of NF-B. The mechanism by which Ang II activates IKK is to increase phosphorylation of IKKß in its activation loop (Ser181) rather than IB phosphorylation. Inhibiting both the RSK and IKK pathways completely blocks the Ang II–induced p65 phosphorylation and NF-B activation. These 2 pathways are independent: inhibiting IKK does not block Ang II–induced phosphorylation of RSK, whereas inhibiting mitogen-activated protein kinase 1 does not affect phosphorylation of IKK. Finally, we found that Ang II can induce expression of vascular adhesion molecules by 2 pathways; both IKK and RSK lead to phosphorylation of the p65 subunit of NF-B to increase vascular cell adhesion molecule-1 transcription. The 2 pathways are functionally important because inhibiting IKK and RSK in vascular smooth muscle cells blocks Ang II–induced expression of vascular cell adhesion molecule-1 and intercellular adhesion molecule-1 to limit vascular inflammation.

Key Words: angiotension II • cell signaling • nuclear factor B

	Introduction

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Nuclear factor (NF)-B activation by classic cytokines (eg, tumor necrosis factor [TNF]-) requires serine phosphorylation, ubiquitination, and degradation of IB by the proteasome, resulting in the release of NF-B.¹ This proceeds by phosphorylation of IB via an IB kinase (IKK).² The proinflammatory effect of angiotensin II (Ang II) also involves activation of NF-B in vascular smooth muscle cells (VSMCs),³ but the mechanism is different because Ang II induces minimal phosphorylation and degradation of IB.^4–6 Earlier, we reported that Ang II activates NF-B by phosphorylating its p65 subunit, rather than promoting IB degradation.⁷ This is important because others have shown that phosphorylation of p65 at multiple serine sites increases the transcriptional capacity of NF-B in the nucleus.^8–11 Specifically, phosphorylation of p65 at serine 536 increases transactivation of NF-B, whereas mutation of this serine to alanine impairs cytokine-induced stimulation of NF-B.¹²

Previously, we showed that Ang II induces phosphorylation of p65 in VSMCs partly by activating the Ras/mitogen-activated protein kinase (MAPK) pathway and its downstream effecter, ribosomal S6 kinase (RSK).⁷ However, when we inhibited the MAPK/RSK pathway, we found only partial blockade of the Ang II–induced phosphorylation of p65 and NF-B promoter activity. Therefore, another pathway(s) must mediate p65 phosphorylation and activation of NF-B.

In part, activation of NF-B by Ang II involves reactive oxygen species (ROS)-signaling cascades in VSMCs,¹³ although the redox-sensitive target(s) remains unclear. One possibility is IKK, because it was recently discovered that IKK phosphorylates substrates other than IB, such as insulin receptor substrate-1 and steroid receptor coactivator-3, as well as p65.^14–16 Therefore, we examined whether alternative kinase activities of IKK will stimulate NF-B. We found 2 pathways by which Ang II can activate NF-B. Both activated RSK, and IKK can stimulate phosphorylation of p65 and activate NF-B, and these pathways can cooperate to produce maximal NF-B–mediated responses.

	Materials and Methods

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Cell Culture and Antibodies
Both rat aortic VSMCs¹⁷ and mouse embryonic fibroblast (MEF) cells (derived from mice null in IKK and IKKß) were cultured in DMEM) supplemented with 10% FBS. Antibodies against phospho-IKKß (Ser181), phospho-IB (Ser32/36), phospho-p44/42 (Thr202/Tyr204) MAPK, phospho-NF-B p65 (Ser536), and IB were bought from Cell Signaling (Beverly, Mass). Antibodies against vascular cell adhesion molecule (VCAM), intracellular adhesion molecule (ICAM), IKK, and IKKß were purchased from Santa Cruz Biotechnology (Santa Cruz, Calif). The ß-actin antibody was purchased from Sigma Aldrich (St Louis, Mo).

NF-B Activity Assay
NF-B transcriptional activity was evaluated using a NF-B-luc reporter vector as described.¹⁸

In Vitro Kinase Assay
VSMCs were cultured to 95% confluence and then serum starved for 24 hours before adding 100 nmol/L Ang II for 5 minutes. IKK and IKKß were immunoprecipitated using antibodies from Santa Cruz Biotechnology. In vitro IKK activity was measured by incubating immunocomplexed IKK or IKKß with different substrates: recombinant myelin basic protein (MBP) (Upstate) or other substrates (glutathione S-transferase [GST]-IB, GST-IBß, GST-p65, or GST-p65 [Ser536A]) as described.^7,18

Measurement of Intracellular Levels of ROS
Ninety-five percent confluent VSMCs were infected with an adenovirus, Ad.catalase or Ad.ß-gal (as control), for 24 hours before being serum starved for 24 hours. The cells were then loaded with 50 µmol/L 2',7'-dichlorodihydro-fluorescein diacetate (H₂DCF-DA) (Sigma Aldrich) for 15 minutes at 37°C. A change in the DCF fluorescence of Ang II–treated and serum-free cells was recorded with an Flx-800 microplate fluorescence reader (Bio-Tek Instruments Inc, Winooski, Vt) at excitation/emission wavelengths of 485/528 nm. Changes in fluorescence are expressed as percentage increase relative to the intensity at time 0.

VSMC Transfection
VSMCs were transfected with 5 µg of plasmid per 2x10⁶ cells using Nucleofector reagent and electroporator (AMAXA, San Diego).

Electrophoretic Mobility-Shift Analysis
Nuclear extracts from VSMCs were prepared as described.⁷ The oligonucleotide containing the NF-B consensus sequence (–153 to –188) in VCAM promoter (5'-TGCCCTGGGTTTCCCCTTGAAGGGATTTCCCTCCG-3') was labeled by -³²dCTP. Specificity of binding was tested by incubating with 100-fold molar excess of unlabeled probe or a classic NF-B probe (Promega, Madison, Wis).

VCAM-1 Promoter Activity Assay
The VCAM-1-luc plasmid was constructed by PCR amplifying the human VCAM-1 promoter, a region spanning –294 to +12 bp. It was cloned into pGL3-basic vector (Promega). This short promoter fully responds to Ang II and proinflammatory stimuli.^19–21 The mutation of NF-B sites within –294 to +12 of VCAM promoter (VCAM-1-mut-luc) (GGGTTTCCCCGCCTTTCCGG and GGGATTTCCCGCCATTTCGG) were generated by PCR-mediated mutagenesis. Wild-type and mutated constructs were confirmed by automated DNA sequencing. VSMCs were transfected with 5 µg of VCAM-1-luc by electroporation (AMAXA) for 24 hours and then rendered quiescent by incubating cells in serum-free media for 24 hours. The quiescent cells were treated with Ang II for 6 hours, and luciferase activities were determined as described.⁷

Densitometry of the blots was analyzed using NIH ImageJ. All values are means±SE. Statistical significance was assessed by 2-tailed Student’s t test or 1-way ANOVA.

	Results

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Ang II Activates NF-B Through an IKK Pathway
To determine whether IKK is involved in Ang II activation of NF-B, we expressed NF-B-luc and a dominant-negative IKK (Ad.IKK KM) or IKKß (Ad.IKKß KA) in VSMCs using recombinant adenoviruses. A green fluorescent protein (GFP)-expressing adenovirus was used as a control. As shown in Figure 1A, Ang II–induced transcriptional activity of NF-B was significantly suppressed by expression of Ad.IKK KM (–60.2±2.3%; P<0.05), Ad.IKKß KA (–65.3±7.4%, P<0.05), or both (–71.4±2.8%, P<0.05) when compared with NF-B transcriptional activity measured in Ad.GFP (empty vector)-infected cells.

View larger version (15K): [in this window] [in a new window]   Figure 1. IKK is involved in Ang II–induced stimulation of NF-B. A, VSMCs were infected with adenoviruses containing dominant-negative IKK (KM), IKKß (KA), or both (IKK KM and IKKß KA) or a control GFP with an adenovirus containing luciferase reporter driven by the NF-B promoter. The cells were treated with or without Ang II (100 nmol/L) for 24 hours. The fold increases of NF-B activity over control, Ad.GFP (serum-free condition), are expressed as mean±SE of 4 separate experiments. *P<0.05 vs GFP (–); P<0.05 vs GFP group. B, MEFs expressing AT1 receptor or GFP were treated with 100 nmol/L Ang II for 10 minutes, and the level of phospho-extracellular signal–regulated kinase 1/2 (p-ERK) was detected by immunoblotting (lane 4). C, Ad.AT1– and Ad.NF-B-luc–infected MEF wild-type and IKK-null cells were treated with or without 100 nmol/L Ang II for 6 hours. NF-B activity was measured, and fold changes vs nontreated cells were calculated (*P<0.05; n=4). The data are expressed as mean±SE of 4 separate experiments. D, VSMCs were cotransfected SiRNA to IKKß or SiRNA control (siCONTROL, nontargeting SiRNA pool; Dharmacon, Lafayett, CO) with p-NF-B-luc and CMV renilla as internal control for 72 hours before cells were treated with 100 nmol/L Ang II for 6 hours. The fold changes in NF-B activity of Ang II–treated cells over cells incubated in serum-free media are indicated. The data are expressed as mean±SE of 4 separate experiments (D, bottom). Representative Western blot shows downregulation of IKKß levels by SiRNA to IKKß (D, top).

To confirm that IKK is involved in Ang II activation of NF-B, we measured NF-B activity in MEFs derived from mice that were null in IKK or IKKß (IKK^–/– or IKKß^–/–). Because MEFs express very low levels of Ang II type 1 (AT₁) receptor, we infected these cells with an adenovirus encoding the recombinant AT₁ receptor.²² The infection rate was 67%. We achieved successful reconstitution of the AT₁ receptor because we found that Ang II induced phosphorylation of extracellular signal–regulated kinase 1/2 in AT₁ virus-infected MEFs (Figure 1B). Ang II induced a 6-fold increase in NF-B promoter activity in cells expressing IKKs and the AT₁ receptor. In contrast, Ang II–induced NF-B promoter activities in AT₁-positive cells that did not express either IKK or IKKß were only 1.6±0.1- or 2.7±0.1-fold (Figure 1C). These results demonstrate that Ang II activation of NF-B depends on IKKs. Notably, activation of NF-B by Ang II is dependent on both IKK and IKKß. This is not surprising because IKK phosphorylates the catalytic subunit of IKKß in the IKK complex, and this complex can phosphorylate downstream effectors/targets.¹⁸

We also confirmed that Ang II can activate NF-B in VSMCs through an IKK pathway. When we knocked down the level of IKKß by using small interference RNA (SiRNA), we achieved &70% decrease in IKKß level. There was at least a 50% reduction in NF-B promoter activity when VSMCs were exposed to Ang II (Figure 1D)

Ang II Induces Phosphorylation of IKK to Activate NF-B
Inflammatory stimuli increase the phosphorylation of multiple serine/threonine sites on IKK to regulate its activity positively or negatively.²³ To understand the relationships among Ang II, IKK, and NF-B, we examined whether Ang II stimulated phosphorylation of IKKß at serine 181, because phosphorylation of serine 181 is essential for dimerization and activation of the IKK complex.²⁴ VSMCs were treated with different concentrations of Ang II (0 to 10 µmol/L) or for different times (0 to 60 minutes), and the cell lysates were used for immunoblotting with anti–phospho-IKKß Ser181. As shown in Figure 2A, even 1 nmol/L Ang II could increase IKKß phosphorylation at serine 181. The stimulation of IKK phosphorylation by 100 nmol/L Ang II was maximal at 10 minutes and declined after 30 minutes (Figure 2B). Interestingly, TNF also induced phosphorylation of serine 181 on IKKß, but the response to TNF was much smaller than the response to Ang II (Figure 2C). Next, we transfected MEFs that are null in IKKß with the inactive IKKß178/181 (AA)²⁴ and compared Ang II–induced NF-B promoter activity to this cell transfected with wild-type IKKß. Expression of the wild-type IKKß restored Ang II activation of NF-B in AT₁-expressing MEF IKKß-null cell (Figure 2D; 6.0±0.8-fold, P<0.05, n=4). NF-B activation was not restored in cells that expressed the mutated IKKß178/181 (AA) (1.8±0.3 fold, P<0.05, n=4). These results indicate that phosphorylation of IKKß at serine 181 is essential for Ang II–induced activation of NF-B.

View larger version (28K): [in this window] [in a new window]   Figure 2. Ang II stimulates IKK phosphorylation at Ser181 to mediate Ang II–induced NF-B activity. A, Quiescent VSMCs were treated for 10 minutes with different doses of Ang II, then immunoblotted with antibodies against phospho-IKKß (p-IKKß) Ser181. ß-Actin was used as a loading control. Bottom, Summary of densitometric analysis of phospho-IKKß normalized for ß-actin from 3 experiments. B and C, Quiescent VSMCs were treated with 100 nmol/L Ang II (B) or 10 ng/mL TNF (C) for the times indicated and then immunoblotted with antibodies against phospho-IKKß Ser181 or ß-actin (top) and subjected to densitometric analysis (bottom). D, IKKß-null MEF was cotransfected with pNF-B-luc, CMV-renilla luciferase, and different IKK-expressing plasmids for 24 hours and then treated with or without 100 nmol/L Ang II for 6 hours. The dual luciferase activity was measured according to the instructions of the manufacturer. Changes in NF-B activity are compared with empty vector transfection, serum-free condition (*P<0.05; n=4)

Ang II–Stimulated IKK Activity Directly Phosphorylates p65 but Not IB
We used MBP as a substrate to examine the activity of IKK that was immunoprecipitated from Ang II–treated VSMCs. As shown in Figure 3A, IKK activity was significantly increased after 5 and 10 minutes of exposure to Ang II. However, when the recombinant proteins GST-IB or GST-IBß were used as substrates, Ang II induced a minimal phosphorylation of these potential substrates (Figure 3B). Moreover, there were no significant changes in phosphorylation and degradation of IB in VSMCs that were exposed to Ang II.⁷ In contrast, TNF treatment yielded extensive phosphorylation of recombinant IBs in an in vitro kinase assay (Figure 3B), and it also increased phosphorylation and degradation of IB in VSMCs.⁷

View larger version (26K): [in this window] [in a new window]   Figure 3. Ang II stimulates IKK activity. A, Immunocomplexes (IP) of IKKß from Ang II–treated VSMCs were incubated with recombinant MBP protein (1 µg) in the presence of [-32P]ATP. Phosphorylated MBP was separated (10% SDS-PAGE) (top). Summary of percentage increases (vs control) of 3 repeated experiments is shown at the bottom. B, Immunocomplexes of IKKß from cells treated with 100 nmol/L Ang II (5 minutes) or 10 ng/mL TNF (5 minutes) and then incubated with recombinant IB or IBß in the presence of [-32P]ATP. Phosphorylation of IB or IBß (top) and summary of percentage increases (vs control) of 3 repeated experiments (bottom). C, AT1-expressing MEF (wild-type), IKK-, or IKKß-null were treated with or without Ang II for 5 minutes, and the phosphorylation of p65 at Ser536 was detected; ß-actin was used as a loading control (top). Bottom, Summary of densitometric analysis of band intensity normalized for ß-actin (n=3). *P<0.05 vs wild-type serum-free condition. D, Immunocomplex of IKKß from Ang II–treated or control, serum-free VSMCs were incubated with purified recombinant GST-p65 (WT) or mutated GST-p65 (Ser536A) in the presence of [-32P]ATP. Autoradiography of phosphorylation of the recombinant p65 is shown.

Earlier, we found that Ang II stimulates the phosphorylation of p65 on Ser536. As shown in Figure 3C, Ang II treatment of AT₁-expressing MEFs stimulated phosphorylation of p65 at Ser536, whereas cells that were null in IKK or IKKß had reduced p65 phosphorylation. Consistent with these results, we found that when recombinant GST-p65 (p65 WT) was used as an in vitro substrate, Ang II stimulated IKK activity to phosphorylate recombinant GST-p65 but did not phosphorylate a mutated version, p65Ser536A (Figure 3D). Therefore, Ang II stimulates IKK to change its substrate specificity from IB to p65 of NF-B in VSMCs.

Activation of IKK by Ang II Is Dependent on ROS
To elucidate a mechanism by which Ang II phosphorylates and activates IKK, we examined the role of ROS. As expected from earlier reports, we found that Ang II dramatically increased DCF-DA fluorescence (a measure of ROS activation) compared with VSMCs incubated without Ang II (Figure 4A). When catalase was overexpressed in VSMCs, the release of ROS was absent (Figure 4A).

View larger version (15K): [in this window] [in a new window]   Figure 4. Ang II increases intracellular ROS production, activating IKK and increasing NF-B promoter activity. A, VSMCs were infected with Ad.catalase or Ad.ß-gal and incubated with DCF diacetate for 30 minutes in the presence of 100 nmol/L Ang II. The DCF fluorescent signal is expressed as a percentage of that present in cells at time 0. ß-gal indicates ß-galactosidase. B, VSMCs infected with Ad.catalsae or Ad.GFP (control) were treated with or without Ang II for 10 minutes. Phospho-IKKß (p-IKKß) was detected by immunoblotting. C, VSMCs were infected with Ad.GFP or Ad.catalase with Ad.NF-B-luc and treated with or without Ang II for 24 hours before luciferase activity was measured. The fold change in luciferase activity was compared vs Ad.GFP-infected, untreated cells.

To determine whether the Ang II–induced increase in ROS production is responsible for IKK, and ultimately NF-B activation, we expressed catalase in VSMCs; catalase suppressed Ang II–induced phosphorylation of IKKß (Figure 4B), and Ang II stimulation of NF-B promoter activity was also partially blocked (Figure 4C).

Both IKK and MAPK Kinase-1 Are Involved in Ang II–Induced Activation of NF-B and Adhesion Molecule Expression
We compared the contributions of the IKK and MAPK kinase-1 (MEK1)/RSK pathways to Ang II–induced phosphorylation of p65 and activation of NF-B. Inhibition of MEK1 in MEF cells that are null in IKKß completely suppressed the NF-B response to Ang II (Figure 5A). In contrast, when we inhibited MEK1, by using the U0126 inhibitor alone, or IKK, by using dominant-negative IKKß KA alone, there was only partial inhibition of NF-B activation. Inhibition of both IKKß (by using a dominant-negative IKKß) and MEK1 (by adding U0126) at the same time will completely suppress Ang II–induced phosphorylation of p65 and the activation of NF-B (Figure 5B and 5C). These results indicate that both pathways coordinate Ang II–induced activation of NF-B in VSMCs.

View larger version (31K): [in this window] [in a new window]   Figure 5. Two signaling pathways are involved in activation of NF-B by Ang II. A, AT1-expressing wild-type and IKKß-null MEFs infected with Ad.NF-B-luc were pretreated with U0126 (10 µmol/L) for 30 minutes and then treated with or without Ang II 100 nmol/L for an additional 6 hours. The fold increases in NF-B reporter activities were compared with values from wild-type cells incubated in serum-free media. B, VSMCs coinfected with dominant-negative Ad.IKKß KA and Ad.NF-B-luc were treated with or without U0126 for 30 minutes before Ang II treatment (6 hours). Ad.GFP-infected cells were used as controls. The NF-B reporter activities are expressed as fold changes compared with Ad.GFP-infected cells incubated in serum-free condition (*P<0.05; n=3). C, VSMCs infected with dominant-negative Ad.IKKß KA were treated with or without U0126 for 30 minutes before Ang II treatment (5 minutes). Ad.GFP-infected cells were used as a control. Phosphorylation of IKK, p65, and RSK were detected by immunoblotting (top). ß-Actin was used as the loading control. Bottom, Summary of 3 experiments yielding band intensity normalized for ß-actin. D, VSMCs were treated for different times (1 to 3 days) with 100 nmol/L Ang II (top) or with different doses of Ang II (24 hours) (bottom). The level of ICAM-1 or VCAM-1 was detected. E, VSMCs were treated for 3 to 24 hours with 100 nmol/L Ang II, and the level of ICAM-1 or VCAM-1 was detected. Bottom, Densitometric analysis of Ang II–induced VCAM-1 expression that is normalized for ß-actin. F, VSMCs were infected with dominant-negative Ad.IKKß KA or Ad.GFP and then treated with or without U0126 for 30 minutes before Ang II treatment (24 hours). ICAM-1 or VCAM-1 was detected using anti–ICAM-1 or anti–VCAM-1 antibodies; ß-actin was used as the loading control. G, VSMCs were transfected with VCAM-luc or VCAM-mut-luc for 24 hours. The cells were treated with or without Ang II (100 nmol/L) for 6 hours. The data are expressed as mean±SE of 4 separate experiments.*P<0.05), Ang II–treated vs serum-free group. H, VSMCs were infected with dominant-negative adenovirus IKKß and GFP (as control) and treated with or without 100 nmol/L Ang II for 30 minutes. Nuclear extracts from these cells were subjected to electrophoretic mobility-shift analysis by using the NF-B consensus probe from VCAM promoter (see Material and Methods). NS indicates nonspecific binding.

To test the physiological relevance of the Ang II–induced IKK and MEK1 pathways, we examined the expression of both VCAM-1 and ICAM-1 in VSMCs that had been treated for 3 days with or without 100 nmol/L Ang II. Ang II increased the expression of VCAM-1 and ICAM-1 after 1 day, and their expression increased through 3 days (Figure 5D, top). Ang II induced the expression of VCAM-1 and ICAM-1 in a dose-dependent fashion (Figure 5D, bottom). We also exposed VSMCs to 100 nmol/L Ang II for a short period time (3 to 24 hours) and found that the earliest time of protein expression following Ang II exposure was 6 hours (24.3% increase; P=NS); after 18 hours, the increase was 228% (P<0.05) (Figure 5E). Inhibition of IKK or MEK1 partially blocked Ang II–induced expression of VCAM-1 and ICAM-1 in VSMCs, whereas inhibiting both IKK and MEK1 completely blocked expression of VCAM-1 and ICAM-1 (Figure 5F).

To determine whether Ang II regulates VCAM transcription via a NF-B mechanism, we cloned the VCAM promoter (–294/+12), which contains 2 NF-B–binding sites, into a luciferase vector. After 6 hours of exposure to Ang II, VCAM promoter activity increased &5-fold over control (Figure 5G). Site-specific mutation of both NF-B–binding sites within this VCAM promoter abolished the ability of Ang II to increase VCAM promoter activity (Figure 5G). In the nuclear extracts isolated from VSMC treated with Ang II, we found evidence for increased binding of NF-B to the NF-B sites in the VCAM promoter (Figure 5H). The Ang II–induced NF-B binding was partially blocked when cells were treated to express a dominant-negative IKKß. We conclude that Ang II increases VCAM expression through an IKK-regulated activation of NF-B. The mechanism is phosphorylation of p65.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
In response to cytokines such as TNF, NF-B is activated via a mechanism that involves activation of IKK and phosphorylation of IB, leading to IB degradation by the ubiquitin–proteasome system.¹ We recently found that Ang II can activate NF-B more directly by inducing phosphorylation of its p65 subunit in VSMCs.⁷ This new mechanism involves a MEK1/RSK pathway and accounts for &70% of the Ang II–induced phosphorylation of p65 and NF-B activation. We now provide evidence that Ang II–induced phosphorylation of p65 can also be mediated by stimulating IKK activity in VSMCs. Together, the RSK and IKK pathways interact and mediate Ang II–induced phosphorylation of the p65 component of NF-B and NF-B activation. The result is an increase in ICAM-1 and VCAM-1 expression in VSMCs.

Importantly, this second pathway that Ang II activates is dependent on IKK, because expression of a dominant-negative form of IKK or a deficiency of IKK reduces Ang II–induced (&70%) stimulation of NF-B (Figure 1A and 1C). Notably, inhibition of both of the RSK and IKK pathways completely suppressed Ang II activation of NF-B. Because blocking the RSK pathway only partially inhibited Ang II–induced NF-B transcriptional activity, we conclude that Ang II stimulates both IKK and RSK activities and that they act together to stimulate NF-B maximally and generate inflammatory responses in VSMCs (Figure 5A).

Interestingly, in VSMCs, neither the IKK nor RSK results in IB phosphorylation. Instead, the pathways result in stimulation of IKK to phosphorylate p65 preferentially. Unlike TNF-induced responses, Ang II does not stimulate phosphorylation or degradation of IB in cultured VSMCs.⁷ Second, TNF-induced IKK activity in VSMCs phosphorylates recombinant GST-IB (Figure 3B), but Ang II–stimulated IKK activity does not phosphorylate GST-IB, even though it phosphorylates MBP (Figure 3A and 3B). Third, Ang II–stimulated IKK activity in VSMCs directly phosphorylates recombinant p65-GST on Ser536 (Figure 3D). Lastly, in VSMCs stimulated by Ang II, neither IKK nor RSK phosphorylates recombinant GST-IB in vitro. Together, these results indicate that Ang II treatment of VSMCs changes the substrate specificity of IKK from the classical phosphorylation of IB to direct phosphorylation of p65. Our results also suggest that there is agonist-specific regulation of NF-B activity.

Our results are consistent with phosphorylation of IKKß at serine 181 as an agonist-specific event in VSMCs. Compared with TNF (Figure 2C), Ang II induces a more rapid and stronger phosphorylation of IKKß (Figure 2A and 2B). Based on these results, we speculate that agonist-specific phosphorylation of IKKß on serine 181 may explain the change in IKK substrate specificity. In fact, others have identified specific phosphorylation sites in IKK that can regulate its activity positively or negatively.²³ In support of this explanation, expression of IKKß-containing mutations in serine sites 179/181 prevented the stimulation of NF-B activity by Ang II in AT₁-expressing MEFs (Figure 2D). It will be interesting to determine how a difference in the IKK phosphorylation profiles induced by TNF and Ang II can change IKK substrate specificity.

What Ang II–stimulated pathways lead to phosphorylation and activation of IKK? We examined the influence of the antioxidant catalase and found that it partially suppresses Ang II–induced phosphorylation of IKK and p65, the activation of NF-B, and the expression of adhesion molecules. These results suggest that ROS must act upstream of IKK. Although we did not examine this possibility in depth, others^25,26 have pointed out that the apoptosis signal–regulating kinase-1 (ASK1) can be activated by Ang II via a generation of ROS and that activated ASK1 can cause NF-B activation. Because ASK1 is a member of the MEK kinase family, several reports have concluded that MEK kinases can phosphorylate IKK.²⁷ Thus, ASK1 could be a redox-sensitive upstream kinase that activates IKK in response to Ang II. When both IKK and RSK are inhibited, the effects of Ang II on IKK, p65, and NF-B and the expression of adhesion molecules are completely suppressed. These results suggest that activation of both IKK and RSK is physiologically important in regulating NF-B transcriptional activity by Ang II.

In summary, we have identified 2 pathways that activate NF-B in VSMCs. The redox-dependent activation of the IKK and MEK1/RSK pathways can act in a tissue-specific, coordinated fashion to phosphorylate and activate p65 directly, rather than acting through degradation of IB. This convergence of the IKK and MEK1/RSK pathways mediates the expression of adhesion molecules in VSMCs.

	Acknowledgments

 
This project was supported by NIH grants RO1 HL 70762 and 1P50-DK064233 and a Scientist Development Grant from the American Heart Association. We are indebted to Drs W.E. Mitch and D.A. Konkel for helpful discussions and critical reading of the manuscript. We thank E. Tamayo for technical assistance.

	Footnotes

 
Original received March 21, 2005; revision received September 1, 1005; accepted September 29, 2005.

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