Dual Pathways for Nuclear Factor κB Activation by Angiotensin II in Vascular Smooth Muscle
Phosphorylation of p65 by IκB Kinase and Ribosomal Kinase
Activation of nuclear factor (NF)-κB 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-κB 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-κB in mouse embryonic fibroblast is suppressed (&70%) either by deletion of IκB 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 IKKβ. Thus, Ang II also stimulates NF-κB via IKK. In vitro, we found that Ang II stimulates IKK to phosphorylate myelin basic protein and the p65 subunit of NF-κB. The mechanism by which Ang II activates IKK is to increase phosphorylation of IKKβ in its activation loop (Ser181) rather than IκB phosphorylation. Inhibiting both the RSK and IKK pathways completely blocks the Ang II–induced p65 phosphorylation and NF-κB 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-κB 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.
Nuclear factor (NF)-κB activation by classic cytokines (eg, tumor necrosis factor [TNF]-α) requires serine phosphorylation, ubiquitination, and degradation of IκB by the proteasome, resulting in the release of NF-κB.1 This proceeds by phosphorylation of IκB via an IκB kinase (IKK).2 The proinflammatory effect of angiotensin II (Ang II) also involves activation of NF-κB in vascular smooth muscle cells (VSMCs),3 but the mechanism is different because Ang II induces minimal phosphorylation and degradation of IκB.4–6 Earlier, we reported that Ang II activates NF-κB by phosphorylating its p65 subunit, rather than promoting IκB degradation.7 This is important because others have shown that phosphorylation of p65 at multiple serine sites increases the transcriptional capacity of NF-κB in the nucleus.8–11 Specifically, phosphorylation of p65 at serine 536 increases transactivation of NF-κB, whereas mutation of this serine to alanine impairs cytokine-induced stimulation of NF-κB.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-κB promoter activity. Therefore, another pathway(s) must mediate p65 phosphorylation and activation of NF-κB.
In part, activation of NF-κB 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 IκB, 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-κB. We found 2 pathways by which Ang II can activate NF-κB. Both activated RSK, and IKK can stimulate phosphorylation of p65 and activate NF-κB, and these pathways can cooperate to produce maximal NF-κB–mediated responses.
Materials and Methods
Cell Culture and Antibodies
Both rat aortic VSMCs17 and mouse embryonic fibroblast (MEF) cells (derived from mice null in IKKα and IKKβ) were cultured in DMEM) supplemented with 10% FBS. Antibodies against phospho-IKKβ (Ser181), phospho-IκBα (Ser32/36), phospho-p44/42 (Thr202/Tyr204) MAPK, phospho-NF-κB p65 (Ser536), and IκBα were bought from Cell Signaling (Beverly, Mass). Antibodies against vascular cell adhesion molecule (VCAM), intracellular adhesion molecule (ICAM), IKKα, and IKKβ were purchased from Santa Cruz Biotechnology (Santa Cruz, Calif). The β-actin antibody was purchased from Sigma Aldrich (St Louis, Mo).
NF-κB Activity Assay
NF-κB transcriptional activity was evaluated using a NF-κB-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. IKKα and IKKβ were immunoprecipitated using antibodies from Santa Cruz Biotechnology. In vitro IKK activity was measured by incubating immunocomplexed IKKα or IKKβ with different substrates: recombinant myelin basic protein (MBP) (Upstate) or other substrates (glutathione S-transferase [GST]-IκBα, GST-IκBβ, 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.β-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.
VSMCs were transfected with 5 μg of plasmid per 2×106 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-κB consensus sequence (−153 to −188) in VCAM promoter (5′-TGCCCTGGGTTTCCCCTTGAAGGGATTTCCCTCCG-3′) was labeled by α-32dCTP. Specificity of binding was tested by incubating with 100-fold molar excess of unlabeled probe or a classic NF-κB 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-κB sites within −294 to +12 of VCAM promoter (VCAM-1-mut-luc) (GGGTTTCCCC→GCCTTTCCGG and GGGATTTCCC→GCCATTTCGG) 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.
Ang II Activates NF-κB Through an IKK Pathway
To determine whether IKK is involved in Ang II activation of NF-κB, we expressed NF-κB-luc and a dominant-negative IKKα (Ad.IKKα KM) or IKKβ (Ad.IKKβ 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-κB was significantly suppressed by expression of Ad.IKKα KM (−60.2±2.3%; P<0.05), Ad.IKKβ KA (−65.3±7.4%, P<0.05), or both (−71.4±2.8%, P<0.05) when compared with NF-κB transcriptional activity measured in Ad.GFP (empty vector)-infected cells.
To confirm that IKK is involved in Ang II activation of NF-κB, we measured NF-κB activity in MEFs derived from mice that were null in IKKα or IKKβ (IKKα−/− or IKKβ−/−). 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-κB promoter activity in cells expressing IKKs and the AT1 receptor. In contrast, Ang II–induced NF-κB promoter activities in AT1-positive cells that did not express either IKKα or IKKβ were only 1.6±0.1- or 2.7±0.1-fold (Figure 1C). These results demonstrate that Ang II activation of NF-κB depends on IKKs. Notably, activation of NF-κB by Ang II is dependent on both IKKα and IKKβ. This is not surprising because IKKα phosphorylates the catalytic subunit of IKKβ in the IKK complex, and this complex can phosphorylate downstream effectors/targets.18
We also confirmed that Ang II can activate NF-κB in VSMCs through an IKK pathway. When we knocked down the level of IKKβ by using small interference RNA (SiRNA), we achieved &70% decrease in IKKβ level. There was at least a 50% reduction in NF-κB promoter activity when VSMCs were exposed to Ang II (Figure 1D)
Ang II Induces Phosphorylation of IKK to Activate NF-κB
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-κB, we examined whether Ang II stimulated phosphorylation of IKKβ 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-IKKβ Ser181. As shown in Figure 2A, even 1 nmol/L Ang II could increase IKKβ 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, TNFα also induced phosphorylation of serine 181 on IKKβ, but the response to TNFα was much smaller than the response to Ang II (Figure 2C). Next, we transfected MEFs that are null in IKKβ with the inactive IKKβ178/181 (AA)24 and compared Ang II–induced NF-κB promoter activity to this cell transfected with wild-type IKKβ. Expression of the wild-type IKKβ restored Ang II activation of NF-κB in AT1-expressing MEF IKKβ-null cell (Figure 2D; 6.0±0.8-fold, P<0.05, n=4). NF-κB activation was not restored in cells that expressed the mutated IKKβ178/181 (AA) (1.8±0.3 fold, P<0.05, n=4). These results indicate that phosphorylation of IKKβ at serine 181 is essential for Ang II–induced activation of NF-κB.
Ang II–Stimulated IKK Activity Directly Phosphorylates p65 but Not IκB
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-IκBα or GST-IκBβ 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 IκBα in VSMCs that were exposed to Ang II.7 In contrast, TNFα treatment yielded extensive phosphorylation of recombinant IκBs in an in vitro kinase assay (Figure 3B), and it also increased phosphorylation and degradation of IκBα in VSMCs.7
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 IKKα or IKKβ 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 IκB to p65 of NF-κB 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).
To determine whether the Ang II–induced increase in ROS production is responsible for IKK, and ultimately NF-κB activation, we expressed catalase in VSMCs; catalase suppressed Ang II–induced phosphorylation of IKKβ (Figure 4B), and Ang II stimulation of NF-κB promoter activity was also partially blocked (Figure 4C).
Both IKK and MAPK Kinase-1 Are Involved in Ang II–Induced Activation of NF-κB 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-κB. Inhibition of MEK1 in MEF cells that are null in IKKβ completely suppressed the NF-κB response to Ang II (Figure 5A). In contrast, when we inhibited MEK1, by using the U0126 inhibitor alone, or IKK, by using dominant-negative IKKβ KA alone, there was only partial inhibition of NF-κB activation. Inhibition of both IKKβ (by using a dominant-negative IKKβ) and MEK1 (by adding U0126) at the same time will completely suppress Ang II–induced phosphorylation of p65 and the activation of NF-κB (Figure 5B and 5C). These results indicate that both pathways coordinate Ang II–induced activation of NF-κB in VSMCs.
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-κB mechanism, we cloned the VCAM promoter (−294/+12), which contains 2 NF-κB–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-κB–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-κB to the NF-κB sites in the VCAM promoter (Figure 5H). The Ang II–induced NF-κB binding was partially blocked when cells were treated to express a dominant-negative IKKβ. We conclude that Ang II increases VCAM expression through an IKK-regulated activation of NF-κB. The mechanism is phosphorylation of p65.
In response to cytokines such as TNFα, NF-κB is activated via a mechanism that involves activation of IKK and phosphorylation of IκB, leading to IκB degradation by the ubiquitin–proteasome system.1 We recently found that Ang II can activate NF-κB 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-κB 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-κB and NF-κB 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-κB (Figure 1A and 1C). Notably, inhibition of both of the RSK and IKK pathways completely suppressed Ang II activation of NF-κB. Because blocking the RSK pathway only partially inhibited Ang II–induced NF-κB transcriptional activity, we conclude that Ang II stimulates both IKK and RSK activities and that they act together to stimulate NF-κB maximally and generate inflammatory responses in VSMCs (Figure 5A).
Interestingly, in VSMCs, neither the IKK nor RSK results in IκB phosphorylation. Instead, the pathways result in stimulation of IKK to phosphorylate p65 preferentially. Unlike TNFα-induced responses, Ang II does not stimulate phosphorylation or degradation of IκB in cultured VSMCs.7 Second, TNFα-induced IKK activity in VSMCs phosphorylates recombinant GST-IκB (Figure 3B), but Ang II–stimulated IKK activity does not phosphorylate GST-IκB, 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-IκB in vitro. Together, these results indicate that Ang II treatment of VSMCs changes the substrate specificity of IKK from the classical phosphorylation of IκB to direct phosphorylation of p65. Our results also suggest that there is agonist-specific regulation of NF-κB activity.
Our results are consistent with phosphorylation of IKKβ at serine 181 as an agonist-specific event in VSMCs. Compared with TNFα (Figure 2C), Ang II induces a more rapid and stronger phosphorylation of IKKβ (Figure 2A and 2B). Based on these results, we speculate that agonist-specific phosphorylation of IKKβ 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 IKKβ-containing mutations in serine sites 179/181 prevented the stimulation of NF-κB 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 TNFα 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-κB, 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, others25,26 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-κB 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-κB 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-κB transcriptional activity by Ang II.
In summary, we have identified 2 pathways that activate NF-κB 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 IκB. This convergence of the IKK and MEK1/RSK pathways mediates the expression of adhesion molecules in VSMCs.
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.
Original received March 21, 2005; revision received September 1, 1005; accepted September 29, 2005.
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