RhoA Mediates Angiotensin II–Induced Phospho-Ser536 Nuclear Factor B/RelA Subunit Exchange on the Interleukin-6 Promoter in VSMCs
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Ruwen Cui, Brian Tieu, Adrian Recinos, R
参见附件。
the Department of Medicine (R.C., B.T., A.R., R.G.T., A.R.B.), Department of Biochemistry and Molecular Biology (B.T.), Stark Diabetes Center (R.G.T.)
Sealy Center for Molecular Medicine (A.R.B.), University of Texas Medical Branch, Galveston.
Abstract
The vasoconstrictor angiotensin II (Ang II) accelerates atherosclerosis by inducing vascular gene expression programs, producing monocyte recruitment, and vascular remodeling. In vascular smooth muscle cells (VSMCs), Ang II signaling activates interleukin (IL)-6 expression, a cytokine producing acute-phase inflammation, mediated by the transcription factor nuclear factor B (NF-B). The classical NF-B activation pathway involves cytoplasmic-to-nuclear translocation of the potent RelA transactivating subunit; however, because nuclear RelA is present in VSMCs, the mechanism by which NF-B activity is controlled is incompletely understood. In this study, we focus on early activation steps controlling RelA activation. Although Ang II only weakly induces 1.5-fold RelA nuclear translocation, RelA is nevertheless required because short interfering RNA–mediated RelA knockdown inhibits inducible IL-6 expression. We find instead that Ang II stimulation rapidly induces RelA phosphorylation at serine residue 536, a critical regulatory site in its transactivating domain. Chromatin immunoprecipitation assays indicate no significant changes in total RelA binding to the native IL-6 promoter, but an apparent increase in fractional binding of phospho-Ser536 RelA. Inactivation of RhoA by treatment with Clostridium botulinum exoenzyme C3 exotoxin or expression of dominant negative RhoA blocks Ang II–inducible RelA Ser536 phosphorylation and IL-6 expression. Finally, enhanced phospho-Ser536 RelA formation in the aortae of rats chronically infused with Ang II was observed. Together, these data indicate a novel mechanism for Ang II–induced NF-B activation in VSMCs, mediated by RhoA-induced phospho-Ser536 RelA formation, IL-6 expression, and vascular inflammation.
Key Words: RhoA nuclear factor B vascular inflammation atherosclerosis
Introduction
Atherosclerosis (AS) is a chronic inflammatory disease of the macrovasculature whose appearance and rate of progression are accelerated by angiotensin II (Ang II)–induced signaling.1,2 Ang II accelerates AS by inducing gene expression programs that influence monocyte recruitment, remodel the vascular wall, induce reactive oxygen species formation, accelerate a prothrombotic state, and activate the hepatic acute-phase response.3 Ang II induces gene expression programs by binding the high affinity type 1 angiotensin receptor (AT1) expressed by the major cellular components of the vessel wall, including vascular smooth muscle cells (VSMCs).4 Activated AT1 converges on nuclear factor-B (NF-B), a transcription factor that plays an important role in inflammatory gene expression.
In most cells, NF-B is a transcription factor inactivated in the cytoplasm by association with the inhibitory molecules known as IBs.5 In the classical NF-B activation pathway, cytokines, viral particles, and bacterial cell wall components activate the multiprotein IB kinase (IKK), a kinase that phosphorylates IB in its NH2-terminal regulatory domain, targeting the inhibitor for proteolytic degradation.6,7 IB destruction releases NF-B, a heterodimer of the DNA-binding subunit, NF-B1, and the transcriptional activating subunit, RelA, to enter the nucleus. Liberated NF-B binds to specific regulatory motifs in target genes, activating their expression by forming a multiprotein complex with transcriptional coactivators.8 Although the classical NF-B activation pathway operates in many cells, VSMCs, interestingly, have constitutive nuclear NF-B complexes.9 Whether these nuclear NF-B isoforms are controlled by stimulus-induced regulatory pathways has not been fully resolved.
In VSMCs, NF-B controls a genetic network of cytokines, chemokines, and adhesion molecules that promote mononuclear infiltration and inflammation.10 Of relevance, NF-B mediates Ang II–induced transcriptional activation of IL-6,11,12 a pleiotropic cytokine that induces elevated circulating acute-phase reactants, such as C-reactive protein, characteristically found in response to vascular inflammation produced by advanced AS.3,13 In this study, we have sought to determine the mechanism for the early activation of NF-B in VSMCs by focusing on regulatory phosphorylation modifications of the RelA transcriptional activator. We report that Ang II induces RelA phosphorylation at serine residue 536, at a key regulatory site in its COOH-terminal transactivation domain. Formation of phospho-Ser536 RelA rapidly peaks within minutes of Ang II stimulation and occurs before detectable changes in IB proteolysis. We further demonstrate that Ang II induces GTP exchange of inactive GDP-bound RhoA and that activated RhoA is upstream of phospho-Ser536 RelA formation because expression of a dominant negative RhoA mutation blocks phospho-Ser536 RelA formation and IL-6 expression. These studies, for the first time, indicate a RhoA- phospho-Ser536 RelA pathway controlling Ang II–induced cytokine expression in VSMCs and may be a target for anti-inflammatory therapies of the vascular wall.
Materials and Methods
An expanded Materials and Methods section, with information on VSMC culture, IL-6 expression, Western immunoblot, electrophoretic gel mobility shift assay, and immunofluorescence, is in the online data supplement, available at http://circres.ahajournals.org.
Short Interfering RNA Transfection
Short interfering RNAs (siRNAs) for rat RelA and control siRNA (Dharmacon Smart Pools; catalog nos. M-080033-00 and D-001206-1305, respectively) were transfected by TransIT-siQUEST Transfection Reagent (Mirus) at a final concentration of 50 nmol/L. Cells were stimulated 72 hours later.
RhoA Assay and Inactivation
GTP-bound RhoA was measured by binding 40 μg of VSMC lysate to immobilized glutathione S-transferase (GST)-Rhotekin RhoA binding domain (residues 7 to 89; Upstate Lake Placid, NY) in binding buffer (150 mmol/L NaCl, 1% IGEPAL CA-630, 1 mmol/L EDTA, 2% glycerol, 25 mmol/L HEPES, pH 7.5) for 45 minutes at 4°C.14 After washing in binding buffer, GST-bound RhoA was detected by Western immunoblot. For experiments to inhibit RhoA signaling, 5 μg of Clostridium botulinum exoenzyme C3 (C3) (CalBiochem) was introduced into 2x106 VSMCs in 400 μL by electroporation (450 V at 25 μF in 0.4 cm cuvette; Bio-Rad).15 Cells were iced, replated, and stimulated 48 hours later.
Chromatin Immunoprecipitation Assay
VSMCs (2 to 4x106) were sequentially crosslinked, and sheared chromatin was immunoprecipitated with 4 μg of antibody (Ab).16 Immunoprecipitates were captured with protein-A magnetic beads (Dynal Inc), and DNA was eluted.16 De-crosslinked DNA was phenol-chloroform purified, and IL-6 promoter was amplified using forward (5'-CCCCCTCCTA GCTGTGATTC-3') and reverse (5'-GAAGGGCAGAT GGAGTTGAC-3') primers. PCR products were identified by agarose gel electrophoresis staining with ethidium bromide.
Adenovirus Generation and Manipulation
Adenovirus (Ad)-NF-B luciferase was described.17 Recombinant Ad expressing a 2x Myc-tagged dominant negative (DN) RhoA containing Thr-to-Asn substitution at residue 19 (T19N) was obtained (UMR cDNA Resource Center) and cloned into pAdTrack-CMV.18 Recombinant Ad-DN RhoA Thr-to-Asn mutant (Ad-DNRhoA[T19N]) was generated in Escherichia coli BJ5183 cells, using pAdEasy1, and transfected into HEK 293 cells.18 CsCl gradient-purified virus was used to infect VSMC for 60 hours before Ang II stimulation.
Ang II Infusion and Phospho-Ser536 RelA Assay
Sprague–Dawley rats (250 g) were infused with saline (sham) or Ang II (0.5 μg/kg per minute in saline) for 10 days by osmotic minipump (Alzet, Durect Corp) in accordance with our protocol, which was approved by the Institutional Animal Care and Use Committee of the University of Texas Medical Branch. After 10 days, animals were euthanized, aortae were dissected, and lysates were prepared in radioimmunoprecipitation assay (RIPA) buffer. Phospho-Ser536 was detected by sandwich ELISA (Cell Signaling Inc) according to the instructions of the supplier. Data are represented as fold change over control.
Results
Ang II Activates NF-B–Dependent Transcription
Ang II activates IL-6 expression in VSMC by a time- and dose-dependent transcriptional mechanism mediated by type 1 receptor AT1, being sensitive to the inhibitory effects of Dup753.12 To establish the kinetics of IL-6 expression and secretion, a time series of Ang II stimulation was performed on early passage primary rat VSMC. Ang II rapidly induced expression of 2 major IL-6 mRNA transcripts, 1.8 and 2.4 kb in size, within 30 minutes. IL-6 expression peaked at 60 minutes and subsequently fell 120 minutes after stimulation (Figure 1A). This rapid induction of IL-6 mRNA preceded a gradual increase in IL-6 protein secretion, detectable by ELISA (Figure 1B). Here, IL-6 protein accumulated in the conditioned medium increasing by 2.5-fold at 3 hours (relative to unstimulated concentrations), reaching a plateau of a 90 pg/mL 4 hours after stimulation (5-fold over spontaneous IL-6 release). Together, these data confirm that Ang II induces coupled IL-6 expression/secretion and indicate that the genomic response is rapid and transient.
Previously we have shown that the IL-6 response was transcriptional because it was sensitive to the effects of actinomycin D and required a functional NF-B response element in its proximal promoter.12 However, because VSMCs have constitutive NF-B–dependent reporter activity,9 our findings could be interpreted to mean that the NF-B site was permissive for IL-6 expression, but not being directly Ang II induced. To confirm that NF-B transcriptional activity is directly induced by Ang II, VSMCs were transduced with a multimeric NF-B–dependent luciferase reporter gene17 and stimulated with Ang II (Figure 1C). Here, we found that Ang II activated luciferase reporter gene activity by 3.3-fold (n=3, P<0.01).
To further characterize the mechanism for Ang II–induced RelA·NF-B1 activation, EMSA assays were performed on nuclear extracts prepared from Ang II–stimulated VSMCs. Primary VSMCs were confirmed to be homogenously smooth muscle cell -actin positive (online data supplement). Although Ang II weakly induced RelA·NF-B translocation, the effect was more dramatic in early passage (P4) cells than late passage cells (P17) (Figure 2A). Quantitation by phosphorimage analysis showed that Ang II stimulation only induced 2-fold changes in RelA complex binding in VSMCs at times preceding the rapid IL-6 mRNA expression. This VSMC complex exactly comigrated with RelA complex from tumor necrosis factor (TNF)-stimulated HepG2 cells, a well-characterized RelA complex demonstrated by supershift and affinity complex analyses (not shown).19,20 Moreover, the VSMC complex primarily contained the RelA transactivator, as demonstrated by Ab interference assay, where the addition of anti-RelA Ab disrupted the constitutive VSMC complex (Figure 2A, top right). Finally the RelA complex specifically bound NF-B binding site, being competed with unlabeled wild type, but not mutant, NF-B DNA binding sites (Figure 2A, bottom right).
Consistent with our microaffinity DNA-binding studies reported earlier, Western immunoblots of nuclear extracts indicated that Ang II produced only 1.5-fold nuclear translocation of RelA (Figure 2B) and Ang II induced only a 40% reduction in cytoplasmic IB proteolysis at 30 minutes, consistent with our earlier findings (Figure 2C).12 Moreover, the amount of RelA DNA-binding activity per microgram of nuclear protein in Ang II–stimulated VSMCs was approximately one-fifteenth that of TNF-induced HeLa cells (online data supplement). Together, we conclude that VSMCs have constitutive nuclear RelA binding activity that is induced by Ang II to, at most, 2-fold. This finding led us to examine whether Ang II might affect alternative activation modes of RelA.
Ang II Induces Rapid Phosphorylation of NF-B/RelA at Ser536
Because a number of studies have implicated an important role of COOH-terminal serine phosphorylation in licensing the transactivation function of RelA,21 Ang II–induced changes in phospho-Ser536 RelA abundance were measured by Western immunoblot. Interestingly, phospho-Ser536 RelA was strongly and rapidly induced, peaking 1 to 15 minutes after Ang II exposure (Figure 3A, top). From 30 to 60 minutes after Ang II stimulation, phospho-Ser536 RelA was reduced to background, indicating RelA phosphorylation was a rapid and transient effect, before the weak IB proteolysis. Importantly, these changes were independent of detectable effect on the abundance of total RelA (Figure 3A, bottom). In addition, we did not observe Ang II–inducible Ser276 phosphorylation, another RelA activating event (not shown), indicating that induction of Ser536 phosphorylation was relatively selective.
Estimates from cytoplasmic and nuclear fractions from equivalent cell numbers indicate the amount of cytoplasmic RelA is 40-fold greater than nuclear for both control and Ang II–stimulated VSMCs (not shown). To confirm the nuclear localization of the phospho-Ser536 RelA, semiquantitative immunofluorescence microscopy was performed. Control or Ang II–stimulated VSMCs (for 30 minutes) were fixed and incubated with anti-pan RelA Ab. Five fields were randomly photographed, and 100 individual cells were scored for the presence of nuclear RelA by an observer masked to the treatment. In control VSMCs, 11% of cells were scored as RelA positive, increasing to 15% after Ang II stimulation (Figure 3B, top). By contrast, a different staining pattern was seen with anti–phospho-Ser536 RelA Ab. In the absence of stimulation, only 3% of nuclei were phospho-Ser536 RelA positive, increasing to 14% after Ang II stimulation (Figure 3B, bottom). We concluded that a small fraction of VSMCs activate the NF-B pathway in response to Ang II and that its effect is greatest on nuclear phospho-Ser536 RelA, rather than affecting total nuclear abundance of RelA.
To establish the requirement of RelA in Ang II–induced IL-6 expression, we inhibited RelA expression by siRNA transfection. Under optimized conditions, we found that RelA siRNA reduced RelA protein expression by 80% relative to control siRNA-transfected VSMCs (Figure 4A). Control or anti-RelA siRNA-transfected VSMCs were next stimulated in the absence or presence of Ang II and changes in IL-6 measured by quantitative real-time PCR (Q-RT-PCR). Relative to control cells, VSMCs lacking RelA expression had significantly reduced basal and Ang II–stimulated IL-6 mRNA expression (Figure 4B). Specifically, RelA siRNA-transfected cells stimulated with Ang II had a 75% reduced level of IL-6 mRNA relative to control transfected Ang II–stimulated cells. Together, these findings indicated that the rapid effects of Ang II on IL-6 expression is RelA dependent, is preceded by phospho-Ser536 RelA formation, and occurs without producing large changes in steady-state RelA nuclear abundance.
Ang II Induces an Increased Fraction of Phospho-Ser536 RelA Binding to the Native IL-6 Promoter
Recent modifications of the chromatin immunoprecipitation (ChIP) assay by our laboratory have allowed for quantitative measurement of NF-B binding to target gene networks.10,16 In this assay, cells are treated with soluble DNA- and protein-crosslinking reagents and the chromatin solubilized and subjected to specific immunoprecipitation. After reversal of the DNA crosslinks, the target gene is detected by PCR. We applied the ChIP assay to measure the effect of Ang II stimulation on RelA binding to the native IL-6 gene promoter, reasoning that the binding of phospho-Ser536 may be Ang II inducible. We first performed ChIP in a time course of Ang II stimulation by using a non–phosphorylation-sensitive immunoprecipitating RelA Ab. Expectedly, even in the absence of stimulation, RelA was detected as binding to the native IL-6 gene, because the IL-6 PCR product was greater in the anti-RelA immunoprecipitates versus the IgG controls (Figure 5, top). We noted that total RelA binding to the IL-6 gene did not increase after Ang II stimulation. We next determined whether differences in phospho-Ser536 RelA binding could be detected. In this experiment, low background levels of phospho-Ser536 RelA were associated with the IL-6 gene in the absence of Ang II stimulation and at 5 minutes of stimulation. By contrast, a sharp induction of phospho-Ser536 RelA inducibly bound IL-6, peaking 30 minutes after Ang II stimulation (Figure 5, bottom), indicating that Ang II induces the fraction of phospho-Ser536 RelA binding on the native IL-6 promoter.
Ang II–Induced RelA Ser536 Phosphorylation and IL-6 Formation Are Dependent on the G Protein RhoA
We noted previous studies suggesting that NF-B is induced by the effects of the small GTP-binding protein (G protein), RhoA, where expression of constitutively active RhoA is associated with NF-B activation in fibroblasts22 and treatment with the C botulinum C3 exotoxin, a highly selective RhoA inhibitor,23 blocks NF-B reporter activity in VSMCs.24 However, the mechanism by which G proteins mediate Ang II–induced NF-B activation in VSMCs is not understood.
To determine whether Ang II activated RhoA in VSMCs at times consistent with IL-6 production, measurement of activated GTP-bound RhoA was performed by a immobilized GST-Rhotekin binding assay.14 For this experiment, a time course of VSMC extracts were captured by immobilized GST-Rhotekin beads and the amount of activated RhoA was measured by Western immunoblot (Figure 6A). A significant enrichment of activated (GTP-bound) RhoA was seen within 5 to 30 minutes of stimulation, times consistent with its potential involvement in IL-6 expression and phospho-Ser536 RelA formation. These data confirmed earlier work in cardiomyocytes that Ang II rapidly (within minutes) activates RhoA in VSMCs by inducing GTP exchange.25
To confirm earlier studies that RhoA may mediate Ang II–inducible NF-B activation,24 we determined the effects of C3 exotoxin on Ang II–inducible RhoA activation, phospho-Ser536 RelA formation, and IL-6 expression. Exposure of VSMCs to C3 potently inhibited formation of phospho–myosin light chain (MLC) kinase, a well-established target of RhoA-dependent signaling (Figure 6B). Ang II–induced formation of phospho-Ser536 RelA formation was completely inhibited in C3 exotoxin–treated VSMCs (Figure 6C). The ability to block phospho-Ser536 RelA formation by C3 exotoxin allowed us to test whether phospho-Ser536 RelA binding was required for Ang II–induced IL-6 gene expression. Under these conditions, Ang II–induced IL-6 expression was significantly inhibited; in this experiment, a 12-fold induction of IL-6 expression observed by Q-RT-PCR was reduced to 1.5 fold by C3 toxin treatment (Figure 6D). Importantly, because C3 has no effect on the related G proteins Rac or cdc42,26 these data strongly implicate RhoA as an essential mediator in AT1/NF-B/IL-6 signaling pathway.
To separately confirm the role of RhoA in phospho-Ser536 RelA–mediated signaling in VSMCs, the effects of transducing VSMCs with a recombinant adenovirus expressing a dominant-negative RhoA mutation (Ad-DNRhoA[T19N]) were explored. VSMCs were transduced with control adenovirus (Ad-EGFP) or Ad-DNRhoA(T19N) at similar multiplicities of infection. Western immunoblots were performed to determine the level of DNRhoA(T19N) fusion protein using anti-RhoA and anti–C-Myc Abs. The relative migration of DNRhoA(T19N) was determined by anti–c-Myc staining of the epitope tag, where we found the fusion protein migrated more slowly than endogenous RhoA. In all cells expressing DNRhoA(T19N), we noted that DNRhoA(T19N) expression reduced endogenous RhoA expression by 50%, suggesting RhoA may be under autoregulatory control (Figure 7A). Formation of phospho-Ser536 RelA was next measured in response to Ang II stimulation; similar to the effect of C3 exotoxin, Ad-DNRhoA(T19N)–transduced VSMCs had a complete inhibition of phospho-Ser536 RelA formation (Figure 7B), and inducible IL-6 expression was strongly and significantly inhibited (80%) (Figure 7C). Together, these data indicate that RhoA mediates Ang II–induced phospho-Ser536 formation and IL-6 expression by a novel mechanism inducing RelA subunit exchange on the IL-6 gene promoter.
Although cultured VSMCs have been extensively used for investigation of signaling events in smooth muscle, we sought to confirm that this pathway was also active in vivo. For this, we examined phospho-Ser536 RelA formation in aortae from chronically Ang II–infused rats. Rats were infused with Ang II at modest pressor doses, and abundance of phospho-Ser536 RelA was quantitated by sandwich ELISA. We observed a 1.6-fold increase in phospho-Ser536 RelA formation, which was confirmed by Western blot (Figure 8), confirming that Ang II induces phospho-Ser536 RelA formation in vivo.
Discussion
Ang II is the primary mediator of the activated renin angiotensin system, a homeostatic endocrine system controlling fluid and electrolyte balance and long-term control of blood pressure.27 Recently, Ang II has been demonstrated to accelerate AS in experimental animal models1,2 and has been linked to premature cardiovascular mortality in large-scale epidemiological studies in humans.3,28 The demonstration that Ang II activates proinflammatory intracellular signaling pathways in VSMCs that converge on NF-B11,12,29 has provided fundamental insight on how Ang II induces vascular chemokine,30 cytokine,12 and adhesion molecule expression.31 Surprisingly, NF-B pathway regulation is under tissue-specific control, where in VSMCs, a fraction of NF-B is nuclear, and the classical activation pathway involving highly inducible NF-B nuclear translocation is not strongly activated. Instead, we observe that Ang II more rapidly induces formation of phospho-Ser536 RelA, resulting in increased fraction of Ser536 phosphorylated RelA binding the endogenous IL-6 promoter. We further implicate the requirement for a RhoA-mediated pathway in phospho-Ser536 RelA formation and IL-6 expression using 2 independent methods for affecting RhoA signaling and establish its relevance in a chronic model of Ang II infusion. These studies indicate that Ang II induces a RhoA/phospho-Ser536 RelA pathway to control inducible cytokine expression in VSMCs (schematically illustrated in the online data supplement).
The Rho GTP-binding proteins are well-established mediators of G protein–coupled receptor (GPCR) signaling pathways.32 Activated GPCRs stimulate inactive RhoA association with guanine nucleotide exchange factors (GEFs) to exchange GDP for GTP. Activated (GTP-bound) RhoA mediates signals that induce cytoskeletal reorganization, actin stress fiber formation, and focal adhesion organization, explaining the effects of Ang II on cardiovascular hypertrophy.12 Recently, RhoA has been found to mediate inducible gene expression, where it is involved in Ang II–induced upregulation of atrial natriuretic factor (ANF) in cardiomyocytes25 and monocyte chemoattractant protein (MCP)-1 expression in VSMCs.30 However, the detailed mechanisms for ANF- and MCP-1 upregulation have not been further elucidated. Our study indicates that Ang II–activated RhoA is upstream of NF-B phosphorylation, and this mediates cytokine gene transcription.
Ectopic expression of the G proteins RhoA, Rac-1, and cdc42 induce NF-B translocation in fibroblasts.22 However, our data are not consistent with this mechanism in VSMCs, where we find that RhoA rapidly mediates RelA Ser536 phosphorylation without strongly inducing changes in RelA nuclear abundance or proteolysis of the cytoplasmic IB inhibitor. These findings suggest, in contrast to the fibroblast studies, that activated RhoA is not coupled to IKK in VSMCs. In both VSMCs and liver cells, we previously demonstrated Ang II–induced NF-B transcriptional activity without strong effects on IB proteolysis.12,20 In earlier studies, we did not have the tools to dissect the relevant transcriptional activator or determine its changes in subunit phosphorylation. Here, we demonstrate that specific downregulation of the RelA transactivating subunit blocks Ang II–induced IL-6 expression, demonstrating the requirement for RelA. Furthermore, inhibition of Ang II–inducible Ser536 phosphorylation by either C3 exotoxin or expressing DN-RhoA inhibits IL-6 activation. These findings strongly suggest that the major transcriptional activator mediating Ang II–induced IL-6 expression is Ser536-phosphorylated RelA. Finally, it is known that thrombin stimulates phospho-Ser536 RelA formation in a RhoA/RhoA kinase–dependent manner in endothelial cells, mediating intercellular adhesion molecule (ICAM)-1 expression.33 This suggests that the RhoA pathway mediates RelA activation in multiple cell types and in response to a diverse set of stimuli. It will be of interest to dissect the downstream mediators of RhoA, particularly RhoA kinase in the RelA activation pathway.
Inducible Ser536 phosphorylation of RelA in its COOH terminus induces its transcriptional activity without affecting its DNA-binding ability. The mechanism by which Ser536 phosphorylation controls RelA transcriptional activity has been shown in other cell types to be the result of enhanced binding of the TATA-binding protein-associated factor II-31, a rate-limiting component of the TFIID complex.34 Enhanced TAFII-31 recruitment to target genes results in stronger RNA Pol II–dependent transcription. It will be interesting to determine whether Ang II induces TAFII-31 recruitment in VSMCs. RelA Ser536 phosphorylation is mediated by a spectrum of unrelated kinases controlled by distinct signal-transduction pathways. These include IKK itself,34 ribosomal S6 kinase,35,36 the IKK-related TANK-binding kinase TBK/T2K,37 and others. Because IB proteolysis is not observed in VSMCs when Ser536 is phosphorylated, it is doubtful that IKK mediates Ang II–induced RelA phosphorylation. Which of these other kinases control Ser536 phosphorylation in Ang II–stimulated VSMCs, and how they may be activated by RhoA, will require further study.
Our observations indicate that phospho-Ser536 RelA rapidly exchanges with inactive nonphosphorylated RelA on the target IL-6 gene in its native chromatin context. This finding is consistent with how NF-B interacts with target chromatin. Recent work using fluorescence microscopy has shown that NF-B transiently binds its chromatin targets with an exchange rate of seconds.38 Interpreted together, these findings suggest that chromatin-bound NF-B is in dynamic exchange with non–DNA-associated nucleoplasmic pool. This behavior allows for the rapid exchange of nonphosphorylated RelA with the Ang II–induced phospho-Ser536 RelA binding to the IL-6 gene.
In summary, we report here that Ang II activates the latent transcriptional activation potential of RelA by phosphorylation at a critical Ser536 residue in a RhoA-dependent manner. Activated phospho-Ser536 RelA, in turn, exchanges with chromatin-associated nonphosphorylated RelA to activate IL-6 expression, producing a novel activation pathway governing NF-B transcription factor activation. Therapeutic targeting of this pathway may have important implications for inflammatory diseases of the cardiovascular system.
Acknowledgments
Sources of Funding
This project was supported by National Heart, Lung, and Blood Institute grants R01 HL40218 and HL70925 (to A.R.B.). Core Laboratory support was from National Institute of Environmental Health Sciences (NIEHS) grant P30 ES06676 (to J. Halpert, University of Texas Medical Branch). B.T. is a recipient of an NIEHS predoctoral training grant (M. Moslen, Director) and a McLaughlin Fund Fellowship in Infection and Immunity (S. Lemon, Director).
Disclosures
None.
Footnotes
Original received October 25, 2005; resubmission received July 19, 2006; accepted August 24, 2006.
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Buss H, Dorrie A, Schmitz ML, Hoffmann E, Resch K, Kracht M. Constitutive and interleukin-1-inducible phosphorylation of p65 NF-B at serine 536 is mediated by multiple protein kinases including IB kinase (IKK)-, IKK, IKK, TRAF family member-associated (TANK)-binding kinase 1 (TBK1), and an unknown kinase and couples p65 to TATA-binding protein-associated factor II31-mediated interleukin-8 transcription. J Biol Chem. 2004; 279: 55633–55643.
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the Department of Medicine (R.C., B.T., A.R., R.G.T., A.R.B.), Department of Biochemistry and Molecular Biology (B.T.), Stark Diabetes Center (R.G.T.)
Sealy Center for Molecular Medicine (A.R.B.), University of Texas Medical Branch, Galveston.
Abstract
The vasoconstrictor angiotensin II (Ang II) accelerates atherosclerosis by inducing vascular gene expression programs, producing monocyte recruitment, and vascular remodeling. In vascular smooth muscle cells (VSMCs), Ang II signaling activates interleukin (IL)-6 expression, a cytokine producing acute-phase inflammation, mediated by the transcription factor nuclear factor B (NF-B). The classical NF-B activation pathway involves cytoplasmic-to-nuclear translocation of the potent RelA transactivating subunit; however, because nuclear RelA is present in VSMCs, the mechanism by which NF-B activity is controlled is incompletely understood. In this study, we focus on early activation steps controlling RelA activation. Although Ang II only weakly induces 1.5-fold RelA nuclear translocation, RelA is nevertheless required because short interfering RNA–mediated RelA knockdown inhibits inducible IL-6 expression. We find instead that Ang II stimulation rapidly induces RelA phosphorylation at serine residue 536, a critical regulatory site in its transactivating domain. Chromatin immunoprecipitation assays indicate no significant changes in total RelA binding to the native IL-6 promoter, but an apparent increase in fractional binding of phospho-Ser536 RelA. Inactivation of RhoA by treatment with Clostridium botulinum exoenzyme C3 exotoxin or expression of dominant negative RhoA blocks Ang II–inducible RelA Ser536 phosphorylation and IL-6 expression. Finally, enhanced phospho-Ser536 RelA formation in the aortae of rats chronically infused with Ang II was observed. Together, these data indicate a novel mechanism for Ang II–induced NF-B activation in VSMCs, mediated by RhoA-induced phospho-Ser536 RelA formation, IL-6 expression, and vascular inflammation.
Key Words: RhoA nuclear factor B vascular inflammation atherosclerosis
Introduction
Atherosclerosis (AS) is a chronic inflammatory disease of the macrovasculature whose appearance and rate of progression are accelerated by angiotensin II (Ang II)–induced signaling.1,2 Ang II accelerates AS by inducing gene expression programs that influence monocyte recruitment, remodel the vascular wall, induce reactive oxygen species formation, accelerate a prothrombotic state, and activate the hepatic acute-phase response.3 Ang II induces gene expression programs by binding the high affinity type 1 angiotensin receptor (AT1) expressed by the major cellular components of the vessel wall, including vascular smooth muscle cells (VSMCs).4 Activated AT1 converges on nuclear factor-B (NF-B), a transcription factor that plays an important role in inflammatory gene expression.
In most cells, NF-B is a transcription factor inactivated in the cytoplasm by association with the inhibitory molecules known as IBs.5 In the classical NF-B activation pathway, cytokines, viral particles, and bacterial cell wall components activate the multiprotein IB kinase (IKK), a kinase that phosphorylates IB in its NH2-terminal regulatory domain, targeting the inhibitor for proteolytic degradation.6,7 IB destruction releases NF-B, a heterodimer of the DNA-binding subunit, NF-B1, and the transcriptional activating subunit, RelA, to enter the nucleus. Liberated NF-B binds to specific regulatory motifs in target genes, activating their expression by forming a multiprotein complex with transcriptional coactivators.8 Although the classical NF-B activation pathway operates in many cells, VSMCs, interestingly, have constitutive nuclear NF-B complexes.9 Whether these nuclear NF-B isoforms are controlled by stimulus-induced regulatory pathways has not been fully resolved.
In VSMCs, NF-B controls a genetic network of cytokines, chemokines, and adhesion molecules that promote mononuclear infiltration and inflammation.10 Of relevance, NF-B mediates Ang II–induced transcriptional activation of IL-6,11,12 a pleiotropic cytokine that induces elevated circulating acute-phase reactants, such as C-reactive protein, characteristically found in response to vascular inflammation produced by advanced AS.3,13 In this study, we have sought to determine the mechanism for the early activation of NF-B in VSMCs by focusing on regulatory phosphorylation modifications of the RelA transcriptional activator. We report that Ang II induces RelA phosphorylation at serine residue 536, at a key regulatory site in its COOH-terminal transactivation domain. Formation of phospho-Ser536 RelA rapidly peaks within minutes of Ang II stimulation and occurs before detectable changes in IB proteolysis. We further demonstrate that Ang II induces GTP exchange of inactive GDP-bound RhoA and that activated RhoA is upstream of phospho-Ser536 RelA formation because expression of a dominant negative RhoA mutation blocks phospho-Ser536 RelA formation and IL-6 expression. These studies, for the first time, indicate a RhoA- phospho-Ser536 RelA pathway controlling Ang II–induced cytokine expression in VSMCs and may be a target for anti-inflammatory therapies of the vascular wall.
Materials and Methods
An expanded Materials and Methods section, with information on VSMC culture, IL-6 expression, Western immunoblot, electrophoretic gel mobility shift assay, and immunofluorescence, is in the online data supplement, available at http://circres.ahajournals.org.
Short Interfering RNA Transfection
Short interfering RNAs (siRNAs) for rat RelA and control siRNA (Dharmacon Smart Pools; catalog nos. M-080033-00 and D-001206-1305, respectively) were transfected by TransIT-siQUEST Transfection Reagent (Mirus) at a final concentration of 50 nmol/L. Cells were stimulated 72 hours later.
RhoA Assay and Inactivation
GTP-bound RhoA was measured by binding 40 μg of VSMC lysate to immobilized glutathione S-transferase (GST)-Rhotekin RhoA binding domain (residues 7 to 89; Upstate Lake Placid, NY) in binding buffer (150 mmol/L NaCl, 1% IGEPAL CA-630, 1 mmol/L EDTA, 2% glycerol, 25 mmol/L HEPES, pH 7.5) for 45 minutes at 4°C.14 After washing in binding buffer, GST-bound RhoA was detected by Western immunoblot. For experiments to inhibit RhoA signaling, 5 μg of Clostridium botulinum exoenzyme C3 (C3) (CalBiochem) was introduced into 2x106 VSMCs in 400 μL by electroporation (450 V at 25 μF in 0.4 cm cuvette; Bio-Rad).15 Cells were iced, replated, and stimulated 48 hours later.
Chromatin Immunoprecipitation Assay
VSMCs (2 to 4x106) were sequentially crosslinked, and sheared chromatin was immunoprecipitated with 4 μg of antibody (Ab).16 Immunoprecipitates were captured with protein-A magnetic beads (Dynal Inc), and DNA was eluted.16 De-crosslinked DNA was phenol-chloroform purified, and IL-6 promoter was amplified using forward (5'-CCCCCTCCTA GCTGTGATTC-3') and reverse (5'-GAAGGGCAGAT GGAGTTGAC-3') primers. PCR products were identified by agarose gel electrophoresis staining with ethidium bromide.
Adenovirus Generation and Manipulation
Adenovirus (Ad)-NF-B luciferase was described.17 Recombinant Ad expressing a 2x Myc-tagged dominant negative (DN) RhoA containing Thr-to-Asn substitution at residue 19 (T19N) was obtained (UMR cDNA Resource Center) and cloned into pAdTrack-CMV.18 Recombinant Ad-DN RhoA Thr-to-Asn mutant (Ad-DNRhoA[T19N]) was generated in Escherichia coli BJ5183 cells, using pAdEasy1, and transfected into HEK 293 cells.18 CsCl gradient-purified virus was used to infect VSMC for 60 hours before Ang II stimulation.
Ang II Infusion and Phospho-Ser536 RelA Assay
Sprague–Dawley rats (250 g) were infused with saline (sham) or Ang II (0.5 μg/kg per minute in saline) for 10 days by osmotic minipump (Alzet, Durect Corp) in accordance with our protocol, which was approved by the Institutional Animal Care and Use Committee of the University of Texas Medical Branch. After 10 days, animals were euthanized, aortae were dissected, and lysates were prepared in radioimmunoprecipitation assay (RIPA) buffer. Phospho-Ser536 was detected by sandwich ELISA (Cell Signaling Inc) according to the instructions of the supplier. Data are represented as fold change over control.
Results
Ang II Activates NF-B–Dependent Transcription
Ang II activates IL-6 expression in VSMC by a time- and dose-dependent transcriptional mechanism mediated by type 1 receptor AT1, being sensitive to the inhibitory effects of Dup753.12 To establish the kinetics of IL-6 expression and secretion, a time series of Ang II stimulation was performed on early passage primary rat VSMC. Ang II rapidly induced expression of 2 major IL-6 mRNA transcripts, 1.8 and 2.4 kb in size, within 30 minutes. IL-6 expression peaked at 60 minutes and subsequently fell 120 minutes after stimulation (Figure 1A). This rapid induction of IL-6 mRNA preceded a gradual increase in IL-6 protein secretion, detectable by ELISA (Figure 1B). Here, IL-6 protein accumulated in the conditioned medium increasing by 2.5-fold at 3 hours (relative to unstimulated concentrations), reaching a plateau of a 90 pg/mL 4 hours after stimulation (5-fold over spontaneous IL-6 release). Together, these data confirm that Ang II induces coupled IL-6 expression/secretion and indicate that the genomic response is rapid and transient.
Previously we have shown that the IL-6 response was transcriptional because it was sensitive to the effects of actinomycin D and required a functional NF-B response element in its proximal promoter.12 However, because VSMCs have constitutive NF-B–dependent reporter activity,9 our findings could be interpreted to mean that the NF-B site was permissive for IL-6 expression, but not being directly Ang II induced. To confirm that NF-B transcriptional activity is directly induced by Ang II, VSMCs were transduced with a multimeric NF-B–dependent luciferase reporter gene17 and stimulated with Ang II (Figure 1C). Here, we found that Ang II activated luciferase reporter gene activity by 3.3-fold (n=3, P<0.01).
To further characterize the mechanism for Ang II–induced RelA·NF-B1 activation, EMSA assays were performed on nuclear extracts prepared from Ang II–stimulated VSMCs. Primary VSMCs were confirmed to be homogenously smooth muscle cell -actin positive (online data supplement). Although Ang II weakly induced RelA·NF-B translocation, the effect was more dramatic in early passage (P4) cells than late passage cells (P17) (Figure 2A). Quantitation by phosphorimage analysis showed that Ang II stimulation only induced 2-fold changes in RelA complex binding in VSMCs at times preceding the rapid IL-6 mRNA expression. This VSMC complex exactly comigrated with RelA complex from tumor necrosis factor (TNF)-stimulated HepG2 cells, a well-characterized RelA complex demonstrated by supershift and affinity complex analyses (not shown).19,20 Moreover, the VSMC complex primarily contained the RelA transactivator, as demonstrated by Ab interference assay, where the addition of anti-RelA Ab disrupted the constitutive VSMC complex (Figure 2A, top right). Finally the RelA complex specifically bound NF-B binding site, being competed with unlabeled wild type, but not mutant, NF-B DNA binding sites (Figure 2A, bottom right).
Consistent with our microaffinity DNA-binding studies reported earlier, Western immunoblots of nuclear extracts indicated that Ang II produced only 1.5-fold nuclear translocation of RelA (Figure 2B) and Ang II induced only a 40% reduction in cytoplasmic IB proteolysis at 30 minutes, consistent with our earlier findings (Figure 2C).12 Moreover, the amount of RelA DNA-binding activity per microgram of nuclear protein in Ang II–stimulated VSMCs was approximately one-fifteenth that of TNF-induced HeLa cells (online data supplement). Together, we conclude that VSMCs have constitutive nuclear RelA binding activity that is induced by Ang II to, at most, 2-fold. This finding led us to examine whether Ang II might affect alternative activation modes of RelA.
Ang II Induces Rapid Phosphorylation of NF-B/RelA at Ser536
Because a number of studies have implicated an important role of COOH-terminal serine phosphorylation in licensing the transactivation function of RelA,21 Ang II–induced changes in phospho-Ser536 RelA abundance were measured by Western immunoblot. Interestingly, phospho-Ser536 RelA was strongly and rapidly induced, peaking 1 to 15 minutes after Ang II exposure (Figure 3A, top). From 30 to 60 minutes after Ang II stimulation, phospho-Ser536 RelA was reduced to background, indicating RelA phosphorylation was a rapid and transient effect, before the weak IB proteolysis. Importantly, these changes were independent of detectable effect on the abundance of total RelA (Figure 3A, bottom). In addition, we did not observe Ang II–inducible Ser276 phosphorylation, another RelA activating event (not shown), indicating that induction of Ser536 phosphorylation was relatively selective.
Estimates from cytoplasmic and nuclear fractions from equivalent cell numbers indicate the amount of cytoplasmic RelA is 40-fold greater than nuclear for both control and Ang II–stimulated VSMCs (not shown). To confirm the nuclear localization of the phospho-Ser536 RelA, semiquantitative immunofluorescence microscopy was performed. Control or Ang II–stimulated VSMCs (for 30 minutes) were fixed and incubated with anti-pan RelA Ab. Five fields were randomly photographed, and 100 individual cells were scored for the presence of nuclear RelA by an observer masked to the treatment. In control VSMCs, 11% of cells were scored as RelA positive, increasing to 15% after Ang II stimulation (Figure 3B, top). By contrast, a different staining pattern was seen with anti–phospho-Ser536 RelA Ab. In the absence of stimulation, only 3% of nuclei were phospho-Ser536 RelA positive, increasing to 14% after Ang II stimulation (Figure 3B, bottom). We concluded that a small fraction of VSMCs activate the NF-B pathway in response to Ang II and that its effect is greatest on nuclear phospho-Ser536 RelA, rather than affecting total nuclear abundance of RelA.
To establish the requirement of RelA in Ang II–induced IL-6 expression, we inhibited RelA expression by siRNA transfection. Under optimized conditions, we found that RelA siRNA reduced RelA protein expression by 80% relative to control siRNA-transfected VSMCs (Figure 4A). Control or anti-RelA siRNA-transfected VSMCs were next stimulated in the absence or presence of Ang II and changes in IL-6 measured by quantitative real-time PCR (Q-RT-PCR). Relative to control cells, VSMCs lacking RelA expression had significantly reduced basal and Ang II–stimulated IL-6 mRNA expression (Figure 4B). Specifically, RelA siRNA-transfected cells stimulated with Ang II had a 75% reduced level of IL-6 mRNA relative to control transfected Ang II–stimulated cells. Together, these findings indicated that the rapid effects of Ang II on IL-6 expression is RelA dependent, is preceded by phospho-Ser536 RelA formation, and occurs without producing large changes in steady-state RelA nuclear abundance.
Ang II Induces an Increased Fraction of Phospho-Ser536 RelA Binding to the Native IL-6 Promoter
Recent modifications of the chromatin immunoprecipitation (ChIP) assay by our laboratory have allowed for quantitative measurement of NF-B binding to target gene networks.10,16 In this assay, cells are treated with soluble DNA- and protein-crosslinking reagents and the chromatin solubilized and subjected to specific immunoprecipitation. After reversal of the DNA crosslinks, the target gene is detected by PCR. We applied the ChIP assay to measure the effect of Ang II stimulation on RelA binding to the native IL-6 gene promoter, reasoning that the binding of phospho-Ser536 may be Ang II inducible. We first performed ChIP in a time course of Ang II stimulation by using a non–phosphorylation-sensitive immunoprecipitating RelA Ab. Expectedly, even in the absence of stimulation, RelA was detected as binding to the native IL-6 gene, because the IL-6 PCR product was greater in the anti-RelA immunoprecipitates versus the IgG controls (Figure 5, top). We noted that total RelA binding to the IL-6 gene did not increase after Ang II stimulation. We next determined whether differences in phospho-Ser536 RelA binding could be detected. In this experiment, low background levels of phospho-Ser536 RelA were associated with the IL-6 gene in the absence of Ang II stimulation and at 5 minutes of stimulation. By contrast, a sharp induction of phospho-Ser536 RelA inducibly bound IL-6, peaking 30 minutes after Ang II stimulation (Figure 5, bottom), indicating that Ang II induces the fraction of phospho-Ser536 RelA binding on the native IL-6 promoter.
Ang II–Induced RelA Ser536 Phosphorylation and IL-6 Formation Are Dependent on the G Protein RhoA
We noted previous studies suggesting that NF-B is induced by the effects of the small GTP-binding protein (G protein), RhoA, where expression of constitutively active RhoA is associated with NF-B activation in fibroblasts22 and treatment with the C botulinum C3 exotoxin, a highly selective RhoA inhibitor,23 blocks NF-B reporter activity in VSMCs.24 However, the mechanism by which G proteins mediate Ang II–induced NF-B activation in VSMCs is not understood.
To determine whether Ang II activated RhoA in VSMCs at times consistent with IL-6 production, measurement of activated GTP-bound RhoA was performed by a immobilized GST-Rhotekin binding assay.14 For this experiment, a time course of VSMC extracts were captured by immobilized GST-Rhotekin beads and the amount of activated RhoA was measured by Western immunoblot (Figure 6A). A significant enrichment of activated (GTP-bound) RhoA was seen within 5 to 30 minutes of stimulation, times consistent with its potential involvement in IL-6 expression and phospho-Ser536 RelA formation. These data confirmed earlier work in cardiomyocytes that Ang II rapidly (within minutes) activates RhoA in VSMCs by inducing GTP exchange.25
To confirm earlier studies that RhoA may mediate Ang II–inducible NF-B activation,24 we determined the effects of C3 exotoxin on Ang II–inducible RhoA activation, phospho-Ser536 RelA formation, and IL-6 expression. Exposure of VSMCs to C3 potently inhibited formation of phospho–myosin light chain (MLC) kinase, a well-established target of RhoA-dependent signaling (Figure 6B). Ang II–induced formation of phospho-Ser536 RelA formation was completely inhibited in C3 exotoxin–treated VSMCs (Figure 6C). The ability to block phospho-Ser536 RelA formation by C3 exotoxin allowed us to test whether phospho-Ser536 RelA binding was required for Ang II–induced IL-6 gene expression. Under these conditions, Ang II–induced IL-6 expression was significantly inhibited; in this experiment, a 12-fold induction of IL-6 expression observed by Q-RT-PCR was reduced to 1.5 fold by C3 toxin treatment (Figure 6D). Importantly, because C3 has no effect on the related G proteins Rac or cdc42,26 these data strongly implicate RhoA as an essential mediator in AT1/NF-B/IL-6 signaling pathway.
To separately confirm the role of RhoA in phospho-Ser536 RelA–mediated signaling in VSMCs, the effects of transducing VSMCs with a recombinant adenovirus expressing a dominant-negative RhoA mutation (Ad-DNRhoA[T19N]) were explored. VSMCs were transduced with control adenovirus (Ad-EGFP) or Ad-DNRhoA(T19N) at similar multiplicities of infection. Western immunoblots were performed to determine the level of DNRhoA(T19N) fusion protein using anti-RhoA and anti–C-Myc Abs. The relative migration of DNRhoA(T19N) was determined by anti–c-Myc staining of the epitope tag, where we found the fusion protein migrated more slowly than endogenous RhoA. In all cells expressing DNRhoA(T19N), we noted that DNRhoA(T19N) expression reduced endogenous RhoA expression by 50%, suggesting RhoA may be under autoregulatory control (Figure 7A). Formation of phospho-Ser536 RelA was next measured in response to Ang II stimulation; similar to the effect of C3 exotoxin, Ad-DNRhoA(T19N)–transduced VSMCs had a complete inhibition of phospho-Ser536 RelA formation (Figure 7B), and inducible IL-6 expression was strongly and significantly inhibited (80%) (Figure 7C). Together, these data indicate that RhoA mediates Ang II–induced phospho-Ser536 formation and IL-6 expression by a novel mechanism inducing RelA subunit exchange on the IL-6 gene promoter.
Although cultured VSMCs have been extensively used for investigation of signaling events in smooth muscle, we sought to confirm that this pathway was also active in vivo. For this, we examined phospho-Ser536 RelA formation in aortae from chronically Ang II–infused rats. Rats were infused with Ang II at modest pressor doses, and abundance of phospho-Ser536 RelA was quantitated by sandwich ELISA. We observed a 1.6-fold increase in phospho-Ser536 RelA formation, which was confirmed by Western blot (Figure 8), confirming that Ang II induces phospho-Ser536 RelA formation in vivo.
Discussion
Ang II is the primary mediator of the activated renin angiotensin system, a homeostatic endocrine system controlling fluid and electrolyte balance and long-term control of blood pressure.27 Recently, Ang II has been demonstrated to accelerate AS in experimental animal models1,2 and has been linked to premature cardiovascular mortality in large-scale epidemiological studies in humans.3,28 The demonstration that Ang II activates proinflammatory intracellular signaling pathways in VSMCs that converge on NF-B11,12,29 has provided fundamental insight on how Ang II induces vascular chemokine,30 cytokine,12 and adhesion molecule expression.31 Surprisingly, NF-B pathway regulation is under tissue-specific control, where in VSMCs, a fraction of NF-B is nuclear, and the classical activation pathway involving highly inducible NF-B nuclear translocation is not strongly activated. Instead, we observe that Ang II more rapidly induces formation of phospho-Ser536 RelA, resulting in increased fraction of Ser536 phosphorylated RelA binding the endogenous IL-6 promoter. We further implicate the requirement for a RhoA-mediated pathway in phospho-Ser536 RelA formation and IL-6 expression using 2 independent methods for affecting RhoA signaling and establish its relevance in a chronic model of Ang II infusion. These studies indicate that Ang II induces a RhoA/phospho-Ser536 RelA pathway to control inducible cytokine expression in VSMCs (schematically illustrated in the online data supplement).
The Rho GTP-binding proteins are well-established mediators of G protein–coupled receptor (GPCR) signaling pathways.32 Activated GPCRs stimulate inactive RhoA association with guanine nucleotide exchange factors (GEFs) to exchange GDP for GTP. Activated (GTP-bound) RhoA mediates signals that induce cytoskeletal reorganization, actin stress fiber formation, and focal adhesion organization, explaining the effects of Ang II on cardiovascular hypertrophy.12 Recently, RhoA has been found to mediate inducible gene expression, where it is involved in Ang II–induced upregulation of atrial natriuretic factor (ANF) in cardiomyocytes25 and monocyte chemoattractant protein (MCP)-1 expression in VSMCs.30 However, the detailed mechanisms for ANF- and MCP-1 upregulation have not been further elucidated. Our study indicates that Ang II–activated RhoA is upstream of NF-B phosphorylation, and this mediates cytokine gene transcription.
Ectopic expression of the G proteins RhoA, Rac-1, and cdc42 induce NF-B translocation in fibroblasts.22 However, our data are not consistent with this mechanism in VSMCs, where we find that RhoA rapidly mediates RelA Ser536 phosphorylation without strongly inducing changes in RelA nuclear abundance or proteolysis of the cytoplasmic IB inhibitor. These findings suggest, in contrast to the fibroblast studies, that activated RhoA is not coupled to IKK in VSMCs. In both VSMCs and liver cells, we previously demonstrated Ang II–induced NF-B transcriptional activity without strong effects on IB proteolysis.12,20 In earlier studies, we did not have the tools to dissect the relevant transcriptional activator or determine its changes in subunit phosphorylation. Here, we demonstrate that specific downregulation of the RelA transactivating subunit blocks Ang II–induced IL-6 expression, demonstrating the requirement for RelA. Furthermore, inhibition of Ang II–inducible Ser536 phosphorylation by either C3 exotoxin or expressing DN-RhoA inhibits IL-6 activation. These findings strongly suggest that the major transcriptional activator mediating Ang II–induced IL-6 expression is Ser536-phosphorylated RelA. Finally, it is known that thrombin stimulates phospho-Ser536 RelA formation in a RhoA/RhoA kinase–dependent manner in endothelial cells, mediating intercellular adhesion molecule (ICAM)-1 expression.33 This suggests that the RhoA pathway mediates RelA activation in multiple cell types and in response to a diverse set of stimuli. It will be of interest to dissect the downstream mediators of RhoA, particularly RhoA kinase in the RelA activation pathway.
Inducible Ser536 phosphorylation of RelA in its COOH terminus induces its transcriptional activity without affecting its DNA-binding ability. The mechanism by which Ser536 phosphorylation controls RelA transcriptional activity has been shown in other cell types to be the result of enhanced binding of the TATA-binding protein-associated factor II-31, a rate-limiting component of the TFIID complex.34 Enhanced TAFII-31 recruitment to target genes results in stronger RNA Pol II–dependent transcription. It will be interesting to determine whether Ang II induces TAFII-31 recruitment in VSMCs. RelA Ser536 phosphorylation is mediated by a spectrum of unrelated kinases controlled by distinct signal-transduction pathways. These include IKK itself,34 ribosomal S6 kinase,35,36 the IKK-related TANK-binding kinase TBK/T2K,37 and others. Because IB proteolysis is not observed in VSMCs when Ser536 is phosphorylated, it is doubtful that IKK mediates Ang II–induced RelA phosphorylation. Which of these other kinases control Ser536 phosphorylation in Ang II–stimulated VSMCs, and how they may be activated by RhoA, will require further study.
Our observations indicate that phospho-Ser536 RelA rapidly exchanges with inactive nonphosphorylated RelA on the target IL-6 gene in its native chromatin context. This finding is consistent with how NF-B interacts with target chromatin. Recent work using fluorescence microscopy has shown that NF-B transiently binds its chromatin targets with an exchange rate of seconds.38 Interpreted together, these findings suggest that chromatin-bound NF-B is in dynamic exchange with non–DNA-associated nucleoplasmic pool. This behavior allows for the rapid exchange of nonphosphorylated RelA with the Ang II–induced phospho-Ser536 RelA binding to the IL-6 gene.
In summary, we report here that Ang II activates the latent transcriptional activation potential of RelA by phosphorylation at a critical Ser536 residue in a RhoA-dependent manner. Activated phospho-Ser536 RelA, in turn, exchanges with chromatin-associated nonphosphorylated RelA to activate IL-6 expression, producing a novel activation pathway governing NF-B transcription factor activation. Therapeutic targeting of this pathway may have important implications for inflammatory diseases of the cardiovascular system.
Acknowledgments
Sources of Funding
This project was supported by National Heart, Lung, and Blood Institute grants R01 HL40218 and HL70925 (to A.R.B.). Core Laboratory support was from National Institute of Environmental Health Sciences (NIEHS) grant P30 ES06676 (to J. Halpert, University of Texas Medical Branch). B.T. is a recipient of an NIEHS predoctoral training grant (M. Moslen, Director) and a McLaughlin Fund Fellowship in Infection and Immunity (S. Lemon, Director).
Disclosures
None.
Footnotes
Original received October 25, 2005; resubmission received July 19, 2006; accepted August 24, 2006.
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