Cooperation of SRC-1 and p300 With NF- B and CREB in Angiotensin II-Induced IL-6 Expression in Vascular Smooth Muscle Cells

【摘要】  Objective- The purpose of this study was to evaluate the role of coactivator histone acetyltransferases (HATs) p300 and SRC-1 in angiotensin II (Ang II)-induced interleukin-6 (IL-6) gene expression in vascular smooth muscle cells (VSMCs).

Methods and Results- Ang II increased IL-6 mRNA expression via NF- B and CREB in an extracellular signal-regulated kinase (ERK)-dependent manner in rat VSMCs. It was also significantly enhanced by the histone deacetylase inhibitor, Trichostatin A. Chromatin immunoprecipitation (ChIP) assays showed that Ang II increased Histone H3 Lysine (K9/14) acetylation on the IL-6 promoter. Ang II-induced IL-6 promoter transactivation was significantly enhanced by p300 and SRC-1, with maximal activation in cells cotransfected with NF- B (p65) and SRC-1. Nucleofection of VSMCs with either an ERK phosphorylation site mutant of SRC-1 or p300/CBP HAT deficient mutants significantly blocked Ang II-induced IL-6 expression. ChIP assays revealed that Ang II enhanced coordinate occupancy of p65, CREB, p300, and SRC-1 at the IL-6 promoter. An ERK pathway inhibitor blocked Ang-induced IL-6 promoter SRC-1 occupancy and histone acetylation.

Conclusions- Ang II-induced IL-6 expression requires NF- B and CREB as well as ERK-dependent histone acetylation mediated by p300 and SRC-1. These results provide new insights into nuclear chromatin mechanisms by which Ang II regulates inflammatory gene expression.

In vascular smooth muscle cells Angiotensin II-induced IL-6 expression involves histone H3 lysine acetylation mediated by histone acetyl transferases p300 and SRC-1 and their cooperation with transcription factors NF- B and CREB. ERK signaling plays a key role by regulating the activation of these transcription factors and histone acetyl transferases.

【关键词】  angiotensin II histone acetylation IL NF B CREB SRC CBP/p ERK vascular smooth muscle cells

Introduction

Several lines of evidence demonstrate that Angiotensin II (Ang II) plays a crucial role in vascular inflammation and atherosclerosis. This has been attributed not only to its pressor responses but also to its growth promoting and proinflammatory actions. 1-4 Ang II can provoke inflammatory responses in the vessel wall as exemplified by the induction of oxidant stress, 5 proinflammatory cytokines and chemokines such as interleukin-6 (IL-6), 6,7 IL-8, 8 monocyte chemoattractant protein-1, 9 and receptors for cytokines, such as IL-18 receptor 10 in vascular smooth muscle cells (VSMCs). Ang II effects are mostly mediated by Ang II type 1 (AT-1) receptor in VSMCs. The signal transduction and in vitro regulatory mechanisms involved in Ang II signaling leading to VSMC hypertrophy, migration, and inflammatory gene expression in VSMCs have been quite extensively examined. 11,12 However, the subtle nuclear transcriptional and chromatin remodeling events occurring in VSMCs in response to Ang II are not very clear.

The contributions of inflammatory cytokines and chemokines, including IL-6, to the pathogenesis of atherosclerosis have been well documented. 13-17 IL-6 has growth-promoting and chemotactic effects in VSMCs. 18,19 Ang II-mediated hypertension was attenuated in IL-6 knockout mice. 20 Ang II-induced IL-6 gene expression was shown to be mediated by NADPH oxidase and the JAK-STAT pathway in VSMCs. 21 The IL-6 promoter sequence is highly conserved among human, rat, and mouse species and has consensus-binding sites for the transcription factors nuclear factor B (NF- B) and cAMP response element binding protein (CREB). 22 Mutation of either NF- B or CREB binding sites can inhibit Ang II-induced IL-6 gene expression in VSMCs. 6,7 Recent studies suggest that Ang II can induce IL-6 expression via extracellular regulated kinase (ERK)-Ribosomal S6 kinase (RSK)-dependent phosphorylation of p65, the active subunit of NF- B. 23 However, it is not clear whether ERK regulates both CREB and NF- B activity.

Chromatin remodeling events, such as nuclear histone lysine (K) acetylation, can lead to active transcription by rendering gene promoters more accessible to the transcription machinery. Coactivator histone acetyltransferases (HATs) such as CBP/p300, steroid receptor coactivator-1 (SRC-1), and P/CAF 24,25 mediate these acetylation reactions. However, the roles of such chromatin events and transcriptional mechanisms in Ang II-induced IL-6 expression are not known. Here, we show for the first time that Ang II-induced IL-6 expression in VSMCs involves chromatin histone acetylation, in vivo promoter recruitment of coactivator HATs (SRC-1 and p300), as well as transcription factors (CREB and NF- B) and cooperation between these proteins. We also demonstrate that ERK is a key upstream regulator of Ang II-induced NF- B, CREB, and SRC-1 activation and histone acetylation during IL-6 expression.

Materials and Methods

Complete details of Materials and Methods are available online at http://atvb.ahajournals.org. Briefly, primary cultures of rat VSMCs (RVSMCs) were obtained from thoracic aortas of male Sprague-Dawley rats by enzymatic digestion. RVSMCs were serum depleted for 48 hours before stimulation with Ang II. In some experiments CHO-AT 1a cell line stably expressing AT-1R was used. IL-6 mRNA levels were quantified by RT-PCR using IL-6 specific primers and 18S RNA as internal control. Activation of CREB, NF- B, and ERK1/2 was examined by immunoblotting with phosphospecific antibodies. IL-6 promoter transactivation was examined by transient transfection of cells with IL-6 promoter (wild-type or mutants) fused to luciferase reporter gene along with indicated expression vectors, and luciferase activities determined 24 to 48 hours later. Histone acetylation and promoter recruitment of transcription factors or coactivator HATs were examined by chromatin immunoprecipitation (ChIP) assays using indicated antibodies. ChIP-enriched samples were analyzed by real time qPCR using primers that amplify a 101-bp IL-6 promoter region surrounding NF- B and CREB binding sites. QPCR data were analyzed by the 2 - Ct method and results expressed as fold over control after normalizing with input samples.

Results

ERK Mediates Ang II-Induced IL-6 Gene Expression via Activation of CREB and NF- B

To understand the detailed molecular mechanisms involved in Ang II-induced IL-6 gene expression, we first performed a time-course by stimulating quiescent RVSMCs with Ang II (100 nmol/L) for the indicated time periods. RT-PCR analysis showed that Ang II increased IL-6 mRNA expression as early as 15 minutes, peaking around 1 hour, and returning to basal by 3 hours ( Figure 1A and 1 B). Next we examined the roles of oxidant stress, ERK, and p38 MAPKs using pharmacological inhibitors. RVSMCs were pretreated with an ERK pathway inhibitor U0126, or p38 MAPK inhibitor SB202190, or NADPH oxidase inhibitor Apocynin, and stimulated with Ang II for 1 hour. U0126 completely inhibited Ang II-induced IL-6 mRNA expression, whereas, in contrast, the p38 inhibitor (SB) and NADPH oxidase inhibitor had no effect ( Figure 1 C). These results demonstrate that ERK activation plays a key role in Ang II-induced IL-6 gene expression in VSMCs. We next examined the role of ERK in Ang II-induced activation of the transcription factors, NF- B (p65), and CREB, known to regulate IL-6 gene expression. Ang II-induced rapid phosphorylation of ERK, CREB (at Ser 133), and NF- B p65 (at Ser 536) by 5 minutes and U0126 clearly abolished these phosphorylation events ( Figure 1 D), indicating that ERK signaling activates NF- B and CREB, which in turn may regulate Ang-induced IL-6 expression.

Figure 1. Involvement of ERK MAPK in Ang II-induced IL-6 expression in VSMCs. A and B, Time course of IL-6 expression. A, Serum-depleted RVSMCs were either left untreated ("O") or treated with Ang II (100 nmol/L) for indicated time periods and IL-6 mRNA expression analyzed by relative RT-PCR with 18S as internal control. B, Line graph representing IL-6 mRNA levels normalized to 18S RNA (mean±SE, * P <0.001 vs 0 time point, n=3). C, Role of ERK in IL-6 expression. Serum-depleted RVSMCs were pretreated for 45 minutes with no inhibitor, U0126 (10 µmol/L), Apocynin (30 µmol/L), SB202190 (1 µmol/L), or vehicle DMSO (0.1%) and then stimulated with Ang II (100 nmol/L) for 1 hour. IL-6 mRNA levels were analyzed by relative RT-PCR with 18S as internal control. D, Involvement of ERK in CREB and NF- B activation. Serum-depleted RVSMCs were pretreated for 45 minutes with or without U0126 (10 µmol/L) and stimulated with Ang II (100 nmol/L) for indicated time periods. Cell lysates were immunoblotted with respective antibodies to detect levels of phospho-CREB, phospho-p65, phospho-ERK1/2, or total CREB (loading control). Results shown are representative of 3 independent experiments.

CREB and NF- B Mediate Ang II-Induced IL-6 Gene Expression

Next we examined the roles of NF- B and CREB binding sites in the IL-6 promoter. Although NF- B and CREB have each been implicated in Ang II-induced IL-6 expression, 6,7 their specific contributions and nuclear roles are not fully understood. Hence, we transfected RVSMCs with reporter plasmids containing luciferase gene under the control of wild-type IL-6 promoter (-596 to +14) 26 or those with NF- B or CREB binding sites mutated. Transfected cells were stimulated with Ang II and luciferase activity measured. Results showed that mutation of NF- B binding site inhibits both basal and Ang II-induced transcription from the IL-6 promoter ( Figure 2 A). However, mutation of the CRE site blocked only Ang II effects ( Figure 2 A). Thus, whereas NF- B p65 is required for both basal and Ang II-induced IL-6 expression, CREB is only required for Ang II effects.

Figure 2. Roles of NF- B and CREB. A, RVSMCs were transiently transfected with either wild-type (WT), CRE binding site ( CRE), or NF- B binding site ( NF- B) mutant IL-6-luciferase constructs and luciferase activities (RLU) determined 6 hours after Ang II treatment (mean±SE * P <0.05 vs WT without Ang II; ** P <0.001 vs Ang II-treated WT activity, n=3). B, Cross talk between NF- B and CREB. CHO-AT 1a cells were cotransfected with either wild-type (WT), CRE mutant, or NF-kB binding site mutant IL-6-luciferase constructs along with an empty vector or expression vectors for either p65 or CREB, and luciferase activities measured in control (Ctrl) and Ang II (100 nmol/L)-stimulated cells. Data represents mean±SE of 3 experiments performed in triplicate (# P <0.05 vs WT control, * P <0.05 vs corresponding WT treatment, ** P <0.05 vs Ang II-treated WT luc activity). C, Opposing effects of p65 and p50 subunits of NF- B on IL-6 transcription: CHO-AT 1a cells were cotransfected with IL-6 promoter-luciferase constructs along with either an empty vector pCR3.1 or expression vectors for either p65 or p50, and luciferase activities measured in control and Ang II (100 nmol/L)-stimulated cells. Results are expressed as fold over control for Ang II stimulated samples (mean±SE, * P <0.001 vs p65, n=3).

Next we studied the potential cooperation between transcription factors NF- B and CREB, by examining the effects of overexpressing p65 and CREB on transcriptional activation of wild-type and mutant IL-6 promoter-luciferase reporters. A CHO cell line stably overexpressing AT 1a receptor (CHO-AT 1a ) was used in these experiments, because they can be transfected with multiple plasmids with high efficiency and are also Ang II responsive. Results are shown in Figure 2 B. Ang II significantly induced activation of WT IL-6 promoter (open bars) but failed to activate both NF- B mutant (checked bars) and CRE mutant (solid bars) promoters, similar to that observed in RVSMCs ( Figure 2 A). Cotransfection of p65 or CREB alone significantly increased WT IL-6 promoter activity, and Ang II further increased this (open bars). Furthermore, p65 or CREB overexpression failed to transactivate NF- B mutant promoter (checked bars) or CRE mutant promoter (solid bars), respectively. But, interestingly, the NF- B mutation also blocked the effects of CREB and conversely, the CRE mutation blocked the stimulatory effects of p65. These observations of a cross-talk indicate that both p65 and CREB are required for maximal induction of Ang II-induced transcription from the IL-6 promoter.

To examine whether the p50 subunit of NF- B also plays a role, we cotransfected CHO-AT 1a cells with IL-6 promoter-luciferase and a p50 expression vector, with or without p65 expression vector, in presence or absence of Ang II. Although p50 did not alter Ang II-induced IL-6 transactivation, it significantly inhibited the stimulatory effects of p65 ( Figure 2 C).

Role of Histone Acetylation in Ang II-Induced IL-6

To examine the role of histone acetylation in Ang II-induced IL-6 expression, RVSMCs were pretreated with 300 nmol/L trichostatin A (TSA), an inhibitor of histone deacetylases, for 24 hours and then stimulated with Ang II for 30 minutes, the time point at which Ang II induction of IL-6 is sub-maximal. Results showed that TSA significantly enhanced both basal and Ang II-induced IL-6 expression ( Figure 3A and 3 B), confirming the involvement of histone acetylation in these events.

Figure 3. Role of histone acetylation in Ang II-induced IL-6. A, Serum-depleted RVSMCs were pretreated with or without 300 nmol/L Trichostatin A (TSA) for 24 hours and then stimulated with Ang II for 30 minutes. IL-6 mRNA levels were then analyzed by relative RT-PCR. B, Bar graphs representing of IL-6 mRNA levels (mean±SE) normalized to 18S RNA (# P <0.05 vs untreated control; * P <0.001 vs Ang II, 30 minutes, n=3). C, Serum-depleted RVSMCs were either untreated (Ctrl) or stimulated with Ang II (100 nmol/L) for 30 minutes. ChIP assays were performed with antibody against acetyl histone H3-K9/14. PCR was performed on immunoprecipitated DNA and Input DNA using rat IL-6 promoter primers spanning NF- B and CREB binding sites. D, Bar graphs representing relative levels of acetylated H3-K9/14 on IL-6 promoter normalized to Input (quantified by densitometry) for gel shown in C (mean±SE, * P <0.04 vs control, n=3). E, Time-course of histone acetylation. Same as in C, except RVSMCs were stimulated with Ang II for indicated time periods and ChIP samples analyzed by real-time qPCRs. Results shown are mean±SE of triplicate qPCRs from 2 independent experiments.

We next performed ChIP assays to examine whether Ang II can induce histone lysine acetylation in vivo at the IL-6 promoter. ChIP assays using anti-acetyl histone H3 ( -AcH3-K9/14) followed by relative PCR analysis showed that Ang II can significantly increase H3-K9/14 acetylation at NF- B and CREB sites on the IL-6 promoter in 30 minutes ( Figure 3C and 3 D). We further confirmed this by performing ChIP assays followed by real time quantitative PCR (qPCR), which showed that the time course of H3-K9/14-acetylation correlates closely with the time at which Ang II significantly increases IL-6 transcription ( Figure 3 E). These results show for the first time that key chromatin modification events associated with active gene transcription are stimulated during Ang II-induced IL-6 transcription.

Role of HATs in the Induction of IL-6

Having demonstrated the requirement of histone acetylation in Ang II-induced IL-6 expression, next we examined the role of coactivators with HAT activity. CHO-AT 1a cells were transiently transfected with the IL-6-promoter luciferase constructs along with either an empty plasmid vector or vectors encoding the HATs CBP, p300, P/CAF, or SRC-1. Luciferase activities were measured 6 hours after Ang II treatment. As shown in Figure 4 A, CBP, p300, P/CAF, and SRC-1 could each significantly induce IL-6 promoter transactivation in the basal state (white bars) to about the same extent. Furthermore, Ang II-induced transactivation of the IL-6 promoter was also significantly augmented by all four HATs (black bars). Interestingly, among these, SRC-1 displayed maximal effect on IL-6 transcriptional activity.

Figure 4. Involvement of HATs and cooperation with NF- B or CREB. A, CHO-AT 1a cells were cotransfected with wild-type IL-6-luciferase construct along with an empty vector or expression vectors for CBP, p300, P/CAF, or SRC-1. Cells were serum depleted for 24 hours and luciferase activities measured in control (white bars) and Ang II (100 nmol/L) stimulated (black bars) cells. Data represents mean±SE of 3 experiments performed in triplicate (# P <0.001 vs untreated control, * P <0.001 vs Ang II-treated WT luc activity). B, Same as in A, except that cells were transfected with p65 along with an empty expression vector or vectors expressing indicated HATs. Data represents mean±SE of 3 experiments performed in triplicate (# P <0.001 vs p65 alone [bar 3], ** P <0.001 vs p65+Ang II [bar 4], * P <0.05 vs corresponding pCR3.1 control [bars 1 and 2]). C, Same as in A, except that cells were transfected with CREB expression vector along with an empty expression vector or vectors expressing indicated HAT proteins. Data represents mean±SE of 3 experiments performed in triplicate (# P <0.05 vs CREB alone [bar 3], ** P <0.001 vs CREB+Ang II [bar 4], * P <0.05 vs corresponding pCR3.1 control [bars 1 and 2].

Cooperation of HATs With NF- B and CREB

Evidence shows that NF- B and CREB can each independently cooperate with several coactivators. However, very little is known about such nuclear events during Ang II-induced gene regulation. Hence, we first cotransfected p65 (NF- B active subunit) expression vector alone, or in combination with expression vectors for CBP, p300, P/CAF, or SRC-1 in CHO-AT 1a cells. Results showed that p300, CBP, P/CAF, and SRC-1 could each cooperate and synergize with p65 to various degrees and this synergy was maintained during Ang II stimulation ( Figure 4 B, bars 5 to 12). Interestingly, among the HATs examined, the cooperation and synergistic effects between SRC-1 and p65 on IL-6 transcription was most dramatic (nearly 17-fold without Ang II and 40-fold with Ang II, compared with p65 alone [ Figure 4 B, last two bars]). These new results reveal a novel role for SRC-1 in basal and Ang II-induced IL-6 expression in VSMCs.

We next examined whether these HATs also display such cooperation with CREB. Results showed that cotransfection of p300, P/CAF, and SRC-1 but not CBP could enhance CREB-induced IL-6 promoter transactivation to almost similar extents ( Figure 4 C). Taken together, these results demonstrate that HATs in collaboration with CREB and NF- B are required for the maximal activation of IL-6 promoter by Ang II in CHO-AT 1a cells.

HAT Activity of p300 and CBP, but not of P/CAF, Is Essential for Ang II-induced IL-6 Expression

Having demonstrated the role of HATs in Ang II-induced IL-6 transactivation in CHO-AT 1a cells, we next evaluated whether their HAT activities are required for IL-6 mRNA induction by Ang II by using dominant negative (D/N) mutants. RVSMCs were nucleofected with either a control vector expressing EGFP or vectors expressing p300 or P/CAF mutants lacking HAT domains 27 ( Figure 5A and 5 B) or CBP HAT mutant 28 ( Figure 5C and 5 D). Ang II-induced IL-6 mRNA expression was analyzed by relative RT-PCR. The D/N mutants of p300 and CBP, but not of P/CAF, significantly blocked IL-6 induction by Ang II compared with control EGFP vector in RVSMCs ( Figure 5A through 5 D). Thus the HAT activities of p300 and CBP, and not P/CAF, are necessary for Ang II-mediated IL-6 expression in VSMCs.

Figure 5. Requirement of p300, CBP, and SRC-1 for Ang II-mediated IL-6 mRNA induction in VSMCs. A, C, and E, RVSMCs were nucleofected with either control pEGFP plasmid or dominant negative (D/N) mutants of p300 or P/CAF lacking HAT domain (A), or HAT-deficient CBP (C) or mutant SRC-1 lacking ERK phosphorylation sites (E). After overnight recovery, cells were serum starved for 24 hours, stimulated with Ang II (100 nmol/L for 1 hour), and IL-6 mRNA expression analyzed by RT-PCR. B, D, and F, Ang II-induced IL-6 expression normalized to 18S RNA expressed as fold over control (mean±SE, * P <0.01 vs control pEGFP transfected cells, n=3).

Role of SRC-1 in Ang II-induced IL-6 Gene Expression

Transient transfection assays in CHO-AT 1a cells showed that SRC-1 could be an important player in IL-6 induction by Ang II ( Figure 4A and 4 B). This prompted us to further examine the role of SRC-1 in Ang II effects by using a D/N mutant of SRC-1 in which the ERK phosphorylation sites, Thr 1179 and Ser 1185, are mutated to Ala. 29 Phosphorylation of these sites by ERK is important for the coactivator role of SRC-1. 29,30 Nucleofection of this D/N SRC-1 mutant in RVSMCs significantly abolished Ang II-induced IL-6 mRNA relative to EGFP vector ( Figure 5E and 5 F). These results clearly demonstrate that SRC-1 is a key player in Ang II-induced IL-6 expression, and suggest that Ang II-mediated ERK activation can promote coactivators like SRC-1 to regulate inflammatory genes.

Recruitment of Transcription Factors and Coactivators to the IL-6 Promoter by Ang II: Role of ERK

We next examined whether Ang II can enhance the occupancy of NF- B, CREB, SRC-1, and p300 to the IL-6 promoter. ChIP analysis with the appropriate antibodies followed by real time qPCRs showed for the first time that CREB, p65 ( Figure 6 A), and p300 ( Figure 6 B) are recruited to the IL-6 promoter by 15 minutes, followed by enrichment of SRC-1 at 30 minutes ( Figure 6 B) in Ang II-stimulated RVSMCs.

Figure 6. ChIP assays: Recruitment of transcription factors and HATs to IL-6 promoter and role of ERK. A and B, Recruitment of transcription factors and HATs: Serum-depleted RVSMCs were either left untreated or stimulated with Ang II (100 nmol/L) for indicated time periods. ChIP assays were performed with antibodies against p65 and CREB (A) or coactivators SRC-1 and p300 (B). Real-time qPCRs were performed in triplicate on immunoprecipitated DNA and Input DNA using rat IL-6 promoter primers (mean±SE, * P <0.001 vs control, n=3). C and D, Regulation of SRC-1 recruitment and histone acetylation by ERK: Serum-depleted RVSMCs were pretreated with vehicle DMSO or ERK inhibitor UO126 (10 µmol/L) and treated with or without Ang II (100 nmol/L) for indicated time periods. ChIP assays were performed with antibodies against SRC-1(C) or acetyl histone H3-K9/14 (D). Real-time qPCRs were performed in triplicate with IL-6 promoter primers (mean±SE, * P <0.001, # P <0.05; **## P <0.01 vs control, n=3).

Our data above shows that mutation of the ERK target sites on SRC-1 completely abolishes Ang II-induced IL-6 induction. Therefore, we further examined the role of ERK on SRC-1 recruitment and histone acetylation at IL-6 promoter. ChIP analysis showed that pretreatment with the ERK inhibitor U0126 not only inhibits Ang II-induced recruitment of SRC-1 ( Figure 6 C), but also completely abolishes histone H3 K9/14 acetylation at the IL-6 promoter ( Figure 6 D). These results clearly demonstrate that Ang II-activated ERK regulates SRC-1 recruitment, thereby augmenting histone acetylation and IL-6 induction.

Discussion

Ang II is known to have inflammatory effects on the vasculature 3 but the nuclear events at the level of chromatin and signaling mechanisms regulating them are not known. In the present study we have demonstrated for the first time that Ang II can induce IL-6 expression via novel nuclear chromatin modification events in an ERK-dependent manner in VSMCs. Transcription factors NF- B and CREB are involved in the induction of several proinflammatory cytokines. Our results also demonstrate that, whereas NF- B is required for both basal as well as Ang II-mediated IL-6 induction, CREB is required only for Ang II effects. Furthermore, NF- B and CREB appeared to cross talk because mutation of NF- B site could block CREB effects and vice versa. On the other hand, the p50 subunit of NF- B had an inhibitory effect. The p50 subunit does not have the transcription activation domain and acts as a repressor in several systems by virtue of its association with histone deacetylases. 31 Further studies are required to dissect the specific mechanistic roles of these 2 NF- B subunits in Ang II-mediated gene transcription.

Because we observed hyperacetylation of histones at the IL-6 promoter in response to Ang II, we envisaged that coactivators with HAT activity and chromatin remodeling might play important roles in IL-6 induction. In support of this, we found that SRC-1, CBP/p300, and P/CAF increased IL-6 promoter transcriptional activation in CHO-AT 1a cells. We also observed cooperation between these HATs and NF- B or CREB on basal and Ang II-induced IL-6 transactivation. Interestingly, there was a marked synergy between SRC-1 and p65 on IL-6 promoter transactivation. Further studies in VSMCs demonstrated that only SRC-1 and CBP/p300, but not P/CAF, are required for Ang II-induced IL-6 mRNA expression. Although SRC-1 was identified as a steroid receptor coactivator, it can also coactivate NF- B. 32 Our results show for the first time the involvement of SRC-1 and its role as a NF- B coactivator in Ang II actions in VSMCs.

ERK mediates the growth promoting effects of Ang II on VSMCs. 33 ERK can phosphorylate p65, the transcriptionally active subunit of NF- B. Our results demonstrate involvement of ERK in the phosphorylation of both NF- B and CREB. Furthermore, the ERK inhibitor also blocked Ang II-induced IL-6 expression. Thus, our results confirm that NF- B and CREB are the major transcription factors regulating IL-6 expression in an ERK-dependent manner. Recent studies implicate RhoA in Ang II-induced p65 phosphorylation and IL-6 expression. 34 Further studies are needed to examine the role of RhoA in ERK activation in VSMCs. We also observed that inhibitors of NADPH oxidase apocynin ( Figure 1 C) and DPI (10 µmol/L, data not shown) did not significantly block Ang II-mediated IL-6 induction, suggesting that reactive oxygen species (ROS) are not involved. Although ROS produced by NAD(P)H oxidases (Noxes) mediate Ang II signaling leading to VSMC growth, migration, and hypertrophy, 35 ERK was reported to be redox-insensitive. 36 Our current study further confirms this and also demonstrates that Ang II-induced IL-6 in VSMCs is ROS independent.

Our findings suggest that ERK is a potential master regulator of Ang II actions on chromatin events regulating IL-6 expression. This is supported by the complete inhibition of Ang II-induced IL-6 promoter histone acetylation and SRC-1 recruitment by the ERK inhibitor U0126. Further, a SRC-1 D/N mutant harboring mutations at ERK target sites also completely blocked Ang II-induced IL-6. Phosphorylation by ERK is required for the transactivation potential of SRC-1. 29,30 These novel observations suggest that Ang II-induced ERK activation promotes SRC-1 phosphorylation, leading to its recruitment to the IL-6 promoter and subsequent histone acetylation. Interestingly, CBP/p300 can also be phosphorylated by ERK in vitro. 37 p300 recruitment to the keratin 16 promoter in response to epidermal growth factor is dependent on ERK. 38 However, it is unclear whether ERK actually phosphorylates p300.

In summary, we have shown the operation of previously unrecognized nuclear events in Ang II-induced transcriptional activation of proinflammatory genes in VSMCs. Ang II activates ERK, which in turn can activate p65, CREB, and SRC-1. Cooperation between them at the IL-6 promoter induces transcription. We also identified SRC-1 and CBP/p300 as novel mediators of Ang II action. These results could lead to the identification of key nuclear factors as therapeutic targets for Ang II-induced cardiovascular disorders.

Acknowledgments

We are deeply grateful to all those who generously provided plasmid expression vectors and reagents.

Sources of Funding

These studies were supported by grants from the National Institutes of Health (NIDDK and NHLBI; to R.N.), and a predoctoral fellowship from the American Heart Association, Western States Affiliate (to S.S.).

Disclosures

None.

S.S. and MA.R. contributed equally to this study.

Original received January 8, 2007; final version accepted April 23, 2007.

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作者单位：Graduate School of Biological Sciences (S.S., C.W., R.N.) and the Department of Diabetes (S.S., M.A.R., L.M., M.W., R.N.), Beckman Research Institute of City of Hope, Duarte, Calif.