A2b adenosine signaling represses CIITA transcription via an epigenetic mechanism in vascular smooth muscle cells

Biochimica et Biophysica Acta 1849 (2015) 665–676

Contents lists available at ScienceDirect

Biochimica et Biophysica Acta journal homepage: www.elsevier.com/locate/bbagrm

A2b adenosine signaling represses CIITA transcription via an epigenetic mechanism in vascular smooth muscle cells Jun Xia a,b,1, Mingming Fang c,d,1, Xiaoyan Wu c,1, Yuyu Yang c,e,1, Liming Yu c, Huihui Xu c, Hui Kong a, Qi Tan a, Hong Wang a,⁎, Weiping Xie a,⁎, Yong Xu c,⁎⁎ a

Department of Respiratory Medicine, the First Affiliated Hospital of Nanjing Medical University, China Department of Respiratory Medicine, Jiangsu Province Hospital of Traditional Chinese Medicine, China Key Laboratory of Cardiovascular Disease and Molecular Intervention, Department of Pathophysiology, Nanjing Medical University, China d Department of Nursing, Jiangsu Jiankang Vocational University, Nanjing, China e State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing, China b c

a r t i c l e

i n f o

Article history: Received 22 November 2014 Received in revised form 5 February 2015 Accepted 3 March 2015 Available online 10 March 2015 Keywords: CIITA transcription VSMC Adenosine A2b signaling STAT1 PCAF/GCN5 WDR5

a b s t r a c t Chronic inflammation plays a major role in the pathogenesis of atherosclerosis. Vascular smooth muscle cells (VSMC), by expressing and presenting major histocompatibility complex II (MHC II) molecules, help recruit T lymphocyte and initiate the inflammatory response within the vasculature. We have previously shown that VSMCs isolated from mice with deficient adenosine A2b receptor (A2b-null) exhibit higher expression of class II transactivator (CIITA), the master regulator of MHC II transcription, compared to wild type littermates. Here we report that activation of A2b adenosine signaling suppresses CIITA expression in human aortic smooth muscle cells. Down-regulation of CIITA expression was largely attributable to transcriptional repression of type III and IV promoters. Chromatin immunoprecipitation (ChIP) analyses revealed that A2b signaling repressed CIITA transcription by attenuating specific histone modifications on the CIITA promoters in a STAT1-dependent manner. STAT1 interacted with PCAF/GCN5, histone H3K9 acetyltransferases, and WDR5, a key component of the mammalian H3K4 methyltransferase complex, to activate CIITA transcription. A2b signaling prevented recruitment of PCAF/GCN5 and WDR5 to the CIITA promoters in a STAT1-dependent manner. In conclusion, our data suggest that adenosine A2b signaling represses CIITA transcription in VSMCs by manipulating the interaction between STAT1 and the epigenetic machinery. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Atherosclerosis is a multistep pathology characterized by the formation in the vessels lipid-laden fibrous plaques [1]. The theories regarding the atherogenesis have evolved over the past century [2]. By now, it is clear that atherosclerosis is a disease of chronic inflammation [3]. Within the atherosclerotic lesion, there is a range of pro-inflammatory infiltrates including neutrophils, mast cells, monocytes/macrophages, and lymphocytes [4]. These subsets of immune cells play differential roles at various stages of atherogenesis and are directly responsible for plaque destabilization [5]. Abbreviations: CIITA, class II transactivator; GCN5, general control of amino acid synthesis protein 5; PCAF, p300-and-CBP-associated factor; STAT1, signal transducer and activator of transcription 1; VSMC, vascular smooth muscle cell; WDR5, WD repeat-containing protein 5 ⁎ Corresponding authors at: Department of Respiratory Medicine, First Affiliated Hospital of Nanjing Medical University, Nanjing, Jiangsu 210029, China. ⁎⁎ Correspondence to: Y. Xu, Department of Pathophysiology, Nanjing Medical University, Nanjing, Jiangsu 210029, China. E-mail addresses: [email protected] (H. Wang), [email protected] (W. Xie), [email protected] (Y. Xu). 1 These authors contributed equally to this work.

http://dx.doi.org/10.1016/j.bbagrm.2015.03.001 1874-9399/© 2015 Elsevier B.V. All rights reserved.

T lymphocytes are present in atherosclerotic plaques in humans [6] and promote atherogenesis in mice as evidenced by adaptive transfer experiments [7]. Engagement of T cells requires antigen presentation via the major histocompatibility complex (MHC) molecules. Seminal studies conducted by the Hansson laboratory have demonstrated that vascular smooth muscle cells (VSMCs) are the major source of MHC II synthesis thereby contributing to T cell activation [8]. Class II transactivator, or CIITA, is considered the master regulator of MHC II transcription [9]. CIITA can be induced by IFN-γ in VSMCs to drive MHC II expression [10,11]. The elucidation of the molecular networks modulating IFN-γ dependent CIITA induction would yield potential strategies for the intervention of atherosclerosis. CIITA transcription is controlled by four different promoters, of which type III and type IV are selectively activated by IFN-γ in human VSMCs [10,11]. The sequence-specific transcription factor STAT1 mediates both type III and type IV CIITA message expression [12]. A number of different signaling pathways fine-tune CIITA expression. For instance, toll-like receptor 2 (TLR2) dependent MAPK signaling dampens IFN-γ induced type IV transcription by evoking histone hypoacetylation on the promoter region [13]. Several anti-inflammatory cytokines, including TGF-β, IL-4, and IL-10, can all antagonize IFN-γ induced CIITA

666

J. Xia et al. / Biochimica et Biophysica Acta 1849 (2015) 665–676

expression although the mechanisms vary [14]. Therefore, CIITA transcription can be deemed as a converging point for different pathogenic factors in human diseases including atherosclerosis. The epigenetic manipulations in this process remain incompletely understood. Adenosine signaling through the A2b receptor is intimately associated with suppression of vascular inflammation [15,16]. Previously, we have shown that VSMCs isolated from A2b deficient mice exhibit enhanced response to IFN-γ with regard to CIITA induction. Continuing this line of investigation, we report here that A2b signaling downregulates CIITA transcription at least in part by modulating the interplay between STAT1 and histone modifying proteins, namely the histone acetyltransferases PCAF/GCN5 and WDR5, a component of the H3K4 methyltransferase complex. Thus, targeting members of this epigenetic complex on CIITA promoters may offer new solutions to modulate the immune response during atherogenesis.

2. Materials and methods 2.1. Cell culture and treatment Human aortic smooth muscle cell (HASMC) was purchased from Promocell (Heidelberg, Germany) and maintained in SMBM with supplements supplied by the vendor. Rat vascular smooth muscle cell (A10), and human embryonic kidney cell (293FT) were maintained in Dulbecco's modified Eagle's medium (DMEM) (Invitrogen) supplemented with 10% fetal bovine serum (FBS, Hyclone). Human and rat recombinant IFN-γ were from R&D. 5′-N-Ethylcarboxamidoadenosine (NECA), MRS-1754, and forskolin were all from Sigma. Treatment protocols were essentially as described before [17].

2.2. Plasmids, small interfering RNAs (siRNAs), transient transfection, and luciferase assay FLAG-tagged STAT1, HA-tagged WDR5, Myc-tagged PCAF, HAtagged GCN5, CIITA type III promoter luciferase construct, and CIITA type IV promoter luciferase construct have been described previously [11,12,18–23]. shRNA plasmid targeting human STAT1 and control shRNA plasmid were purchased from Sigma. Lentiviral particles were generated as previously described [24]. siRNA sequence for human PCAF, 5′-TCGCCGTGAAGAAAGCGCA-3′, for human GCN5, 5′-TCGCCG TGAAGAAAGCGCA-3′, and for human WDR5, 5′-GTGGAAGAGTGACT GCTAA-3′. Transient transfections were performed with Lipofectamine 2000 (Invitrogen). An EGFP expression construct was included in each well to monitor transfection efficiency. Luciferase activities were assayed 24–48 h after transfection using a luciferase reporter assay system (Promega). Luciferase activities were normalized by both protein concentration and GFP fluorescence. Experiments were routinely performed in triplicate wells and repeated three times.

2.3. Protein extraction, immunoprecipitation, and Western Whole cell lysates were obtained by re-suspending cell pellets in RIPA buffer (50 mM Tris pH7.4, 150 mM NaCl, 1% Triton X-100) with freshly added protease inhibitor (Roche). Specific antibodies or preimmune IgGs (P.I.I.) were added to and incubated with cell lysate overnight before being absorbed by Protein A/G-plus Agarose beads. Precipitated immune complex was released by boiling with 1 × SDS electrophoresis sample buffer. Alternatively, FLAG-conjugated beads (M2, Sigma) were added to and incubated with lysates overnight. Precipitated immune complex was eluted with 3× FLAG peptide (Sigma). Western blot analyses were performed with anti-CIITA, anti-PCAF, anti-GCN5, anti-Myc (Santa Cruz), anti-WDR5 (Bethyl Laboratories), anti-HA, anti-FLAG, and anti-β-actin (Sigma) antibodies.

2.4. Chromatin immunoprecipitation (ChIP) and re-ChIP ChIP assays were performed essentially as described before [25–27]. Aliquots of 100 μg nuclear protein were used for each reaction with antiCIITA, anti-STAT1, anti-PCAF, anti-GCN5, anti-WDR5 (Santa Cruz), antiacetyl H3K9, anti-acetyl H3K14, anti-acetyl H3K18, anti-acetyl H3K27, anti-dimethylated H3K4, anti-trimethylated H3K4 (Millipore/Upstate), or pre-immune IgG. For re-ChIP, immune complexes were eluted with the elution buffer (1% SDS, 100 mM NaCO3), diluted with the re-ChIP buffer (1% Triton X-100, 2 mM EDTA, 150 mM NaCl, 20 mM Tris pH 8.1), and subject to immunoprecipitation with a second antibody of interest. Precipitated genomic DNA was amplified by real-time PCR with primers that amplify the promoter regions of type III and type IV CIITA as previously described [28]. Serial dilutions of standard genomic DNA extracted from normal cells were used to calculate the amount of DNA being precipitated by a particular antibody. 10% of the starting material was also included as the input. Data were normalized to input and expressed as fold change compared to the control group. Experiments were routinely performed in triplicate wells and repeated three times.

2.5. RNA extraction and real-time PCR RNA was extracted using an RNeasy RNA isolation kit (Qiagen). Reverse transcriptase reactions were performed using a SuperScript First-strand synthesis system (Invitrogen). Real-time PCR reactions were performed on an ABI STEPONE Plus (Life Tech) with previously described primers [11,29]. Experiments were routinely performed in triplicate wells and repeated three times.

2.6. Statistical analysis One-way ANOVA with post-hoc Scheffe analyses were performed using an SPSS package. P values smaller than .05 were considered statistically significant (*).

3. Results 3.1. Adenosine A2b signaling down-regulates CIITA transcription in human smooth muscle cells We have previously shown that vascular smooth muscle cells isolated from A2b deficient mice exhibit enhanced response to IFN-γ by expressing more CIITA messages [17]. Activation of the A2b signaling pathway also down-regulated CIITA transcription in lung fibroblast cells [28]. To tackle whether a similar observation could be made in human vascular smooth muscle cells, we treated primary human aortic smooth muscle cells (HASMC) with a generic adenosine receptor agonist, NECA. Indeed, NECA treatment led to a decrease in message (Fig. 1A) and protein (Fig. 1B) levels of CIITA. This effect could be blocked by a specific A2b antagonist, MRS-1754 (Fig. 1C, D). Moreover, forskolin, an activator of adenylyl cyclase downstream of the A2b signaling, down-regulated the induction of CIITA expression by IFN-γ in HASMCs (Fig. 1E, F). CIITA transcription is mediated by type III and type IV promoters in human smooth muscle cells [10]. To examine whether decreased CIITA expression following activation of A2b signaling could be a result of repressed transcription rate, we performed reporter assays with CIITA type III and type IV promoter constructs. As shown in Fig. 1G and H, treatment with either NECA or forskolin dampened the activities of type III and type IV CIITA promoters. Therefore, adenosine signaling through the A2b receptor down-regulates CIITA transcription in human vascular smooth muscle cells.

J. Xia et al. / Biochimica et Biophysica Acta 1849 (2015) 665–676

667

Fig. 1. Adenosine A2b signaling down-regulates CIITA expression in human smooth muscle cells. (A, B) HASMCs were treated with FN-γ and NECA for 24 h. mRNA (A) and protein (B) levels of CIITA were probed by qPCR and Western, respectively. (C, D) HASMCs were treated with IFN-γ, NECA, and/or MRS-1754 for 24 h. mRNA (C) and protein (D) levels of CIITA were probed by qPCR and Western, respectively. (E, F) HASMCs were treated with IFN-γ and increasing doses of forskolin for 24 h. mRNA (E) and protein (F) levels of CIITA were probed by qPCR and Western, respectively. (G, H) Type III and type IV CIITA promoter-luciferase constructs were transfected into A10 cells followed by indicated treatments for 24 h. Luciferase activities were normalized by protein concentration and GFP fluorescence (for transfection efficiency) and expressed as relative luciferase activities compared to the control group.

668

J. Xia et al. / Biochimica et Biophysica Acta 1849 (2015) 665–676

Fig. 2. STAT1 coordinates histone modifications on the CIITA promoters. (A, B) HASMCs were treated with IFN-γ, NECA, and/or MRS-1754 for 24 h. ChIP assays were performed with indicated antibodies and precipitated DNA was amplified with primers spanning type III (A) and type IV (B) CIITA promoters. (C, D) HASMCs were treated with IFN-γ and forskolin for 24 h. ChIP assays were performed with indicated antibodies and precipitated DNA was amplified with primers spanning type III (C) and type IV (D) CIITA promoters.

3.2. A2b signaling manipulates histone modifications on the CIITA promoters by targeting STAT1 CIITA transcription is associated with the mobilization of the epigenetic machinery [30,31]. Following IFN-γ treatment, several histone markers synonymous with transcriptional activation, including overall acetylated histone H3, acetylated H3 lysine 9 (H3K9), acetylated H3K14, acetylated H3K18, acetylated H3K27, dimethylated and trimethylated H3K4, all started to accumulate on type III and type IV promoters (Fig. 2A, B). Treatment of NECA attenuated the enrichment of active histone marks while MRS-1754 blocked the effect of NECA. Treatment of forskolin had a similar effect as NECA (Fig. 2C, D). Thus, repression of CIITA transcription by A2b signaling can be partially explained by the loss of signature histone modifications from type III and type IV promoters. STAT1 is known to mediate IFN-γ induced CIITA transcription [12]. IFN-γ promoted STAT1 occupancies on the CIITA promoters, which is blocked by A2b signaling indicating that A2b signaling might influence the chromatin structure surrounding the CIITA promoters by targeting STAT1 (Fig. 2A, B). Indeed, when endogenous STAT1 in HASMCs was depleted by lentivirus carrying short hairpin

RNA (shRNA) for STAT1, neither NECA nor MRS-1754 was able to alter histone modifications on CIITA promoters (Fig. S1A-S1D). These data allude to a scenario wherein in response to IFN-γ, STAT1 brings histone modifying enzymes to the CIITA promoters, a process with which A2b signaling can interfere. 3.3. A2b signaling disrupts the interaction between STAT1 and PCAF/GCN5 It has been documented that CBP/p300, PCAF/GCN5, and WDR5 are responsible for H3K27 acetylation, H3K9 acetylation, and H3K4 methylation, respectively [32,33]. Since the interaction between STAT1 and CBP/p300 has been extensively studied [34,35], we decided to focus on the interplay between STAT1, PCAF/GCN5, and WDR5 as well as the potential impact of A2b signaling on these interactions. Co-immunoprecipitation assays performed in both HEK293 cells (Fig. S2A) and VSMCs (Fig. S2B) confirmed that STAT1 interacted with PCAF and GCN5. Occupancies of PCAF (Fig. 3A) and GCN5 (Fig. 3B) on the CIITA promoters were significantly stimulated by IFN-γ in HASMCs. More importantly, IFN-γ treatment led to the formation of both a STAT1-PCAF complex and a STAT1-GCN5 complex on CIITA promoters (Fig. 3C). In reporter assays, PCAF/GCN5 enhanced the activation of

Fig. 3. STAT1 interacts with PCAF/GCN5 to activate CIITA transcription. (A, B) HASMCs were treated with or without IFN-γ for 24 h. ChIP assays were performed with anti-PCAF (A) or antiGCN5 (B). (C) HASMCs were treated with or without IFN-γ for 24 h. Re-ChIP was performed with indicated antibodies. (D, E) A10 cells were transfected with type III or type IV CIITA promoter construct along with indicated expression constructs. (F, G) HASMCs were transfected with indicated siRNA followed by treatment with IFN-γ. CIITA mRNA (F) and protein (G) levels were examined by qPCR and Western.

J. Xia et al. / Biochimica et Biophysica Acta 1849 (2015) 665–676

669

670

J. Xia et al. / Biochimica et Biophysica Acta 1849 (2015) 665–676

CIITA transcription by STAT1 (Fig. 3D, E). PCAF and GCN5 silencing (Fig. S2C) additively attenuated the induction of CIITA type III and type IV mRNA levels by IFN-γ (Fig. 3F) and down-regulated CIITA protein levels (Fig. 3G). NECA markedly disrupted the binding of PCAF/GCN5 on the CIITA promoters while pre-treatment with MRS-1754 restored PCAF/GCN5 binding (Fig. 4A). Similarly, forskolin treatment also dampened the recruitment of PCAF/GCN5 (Fig. S3A, S3B). Of note, activation of A2b signaling directly targeted the interaction between STAT1 and PCAF/GCN5 (Fig. 4B, S3C). Finally, stimulation of CIITA promoter activities by the STAT1/PCAF complex (Fig. 4C) and the STAT/GCN5 complex (Fig. 4D) was inhibited by NECA treatment but could be brought back by MRS1754. Together, these data confirm that PCAF and GCN5 interact with STAT1 to activate CIITA transcription and that A2b signaling suppresses CIITA transcription likely via disrupting the STAT1/PCAF/ GCN5 complex.

3.4. A2b signaling disrupts the interaction between STAT1 and WDR5 Next, we probed the possibility that STAT1 and WDR5 might cooperate to activate CIITA transcription in VSMCs. As shown in Fig. 5A, IFN-γ treatment resulted in a robust enrichment of WDR5 on the CIITA promoters. Immunoprecipitation assays revealed that STAT1 and WDR5 interacted in HEK293 cells (Fig. S4A) and VSMCs (Fig. S4B). More importantly, the interaction between STAT1 and WDR5 was enhanced on the CIITA promoters in VSMCs in response to IFN-γ treatment (Fig. 5B). Reporter assay showed that co-transfection of WDR5 enhanced STAT1dependent activation of CIITA promoters (Fig. 5C). Finally, siRNAmediated silencing of WDR5 (Fig. S4C), but not ASH2 (Fig. S4D), another component of the H3K4 methyltransferase complex, blocked the induction of CIITA expression in HASMCs (Fig. 5D, E). Activation of A2b signaling by NECA significantly down-regulated the occupancy of WDR5 on the CIITA promoters but not the GAPDH promoter; pre-treatment of MRS-1754 normalized the binding of WDR5 (Fig. 6A). In addition, NECA attenuated the interaction between STAT1 and WDR5 on the CIITA promoters, which was rescued by MRS-1754 (Fig. 6B). Forskolin treatment also impaired the binding of WDR5 (Fig. S5A) as well as the interaction between WDR5 and STAT1 (Fig. S5B) on the CIITA promoters. In reporter assay, activation of CIITA transcription by STAT1 and WDR5 was blocked NECA but could be restored by MRS-1754 (Fig. 6C). Of interest, neither NECA nor MRS1754 impacted CIITA expression when WDR5 was knocked down (Fig. 6D), indicating that the A2b signaling could indeed target WDR5 to influence CIITA transcription.

3.5. A2b signaling disrupts the synergy between PCAF/GCN5 and WDR5 Finally, we examined the possibility that there might be a synergy between PCAF/GCN5 and WDR5 in the modulation of CIITA transcription in VSMC and that adenosine signaling might target this potential interaction between PCAF/GCN5 and WDR5 to inhibit CIITA induction. Co-expression of WDR5 enhanced the activation of CIITA promoter activities in the presence of PCAF (Fig. 7A) or GCN5 (Fig. 7B). IFN-γ stimulated the PCAF/WDR5 interaction and the GCN5/WDR5 interaction on the CIITA promoters in HASMCs (Fig. 7C). NECA disrupted these interactions while pre-treatment of MRS-1754 blocked the NECA effect. Finally, to examine whether A2b signaling disrupts the crosstalk between WDR5 and GCN5/PCAF by targeting STAT1, we silenced STAT1 expression by shRNA mediated knockdown. Indeed, in the absence of STAT1, A2b signaling was no longer able to influence the interaction between WDR5 and GCN5/PCAF on the CIITA promoters (Fig. S6). In conclusion, A2b signaling could potentially interfere with the corroboration between PCAF/GCN5 and WDR5, in a STAT1-dependent manner, to down-regulate CIITA transcription in VSMC.

4. Discussion Basic and clinical research in the past three decades has unequivocally established atherosclerosis as a human pathology of chronic inflammation [36,37]. CIITA expression is pivotal to the immune homeostasis and if aberrantly activated can contribute to inflammationrelated diseases including atherosclerosis [38]. The current report details the epigenetic regulation of CIITA transcription in vascular smooth muscle cells, the major source of CIITA within the atherosclerotic plaque [8], and unveils a potential mechanism by which activation of adenosine A2b signaling attenuates vascular inflammation [15–17]. The novel findings contained in this report provide renewed rationale for targeting the adenosine signaling pathway in the intervention of atherosclerosis. PCAF and GCN5 share a redundant role of acetylating histone H3K9 [32]. Here we demonstrate that both PCAF and GCN5 can be recruited to the CIITA promoters in response to IFN-γ treatment. In addition, silencing either PCAF or GCN5 results in a decrease in CIITA induction although the simultaneous loss of PCAF and GCN5 leads to a further down-regulation. These data indicate that PCAF and GCN5 may not possess selective affinities for gene promoters. Instead, it is the cumulative activities of PCAF and GCN5 combined in cells that determine the transcriptional outcome. These data indicate that a pan-histone acetyltransferase inhibitor will likely be sufficient to repress CIITA expression and therefore temper vascular inflammation. Using biochemical purification techniques, Wang et al. have identified both PCAF/GCN5 and WDR5 as components of the human ATAClike complex [39]. What left unanswered in that study was whether these proteins could actually interact with each other on individual gene promoters in response to specific stimuli. Here we demonstrate that the formation of a PCAF/WDR5 and a GCN5/WDR5 complex can be induced by IFN-γ on the CIITA promoters and that co-expression of WDR5 and PCAF/GCN5 additively activates CIITA transcription in reporter assays (Fig. 7). These data are consistent with a recent report by Tang et al. that depicts the crosstalk between p300, a histone acetyltransferase, and SET1, a histone methyltransferase, in modulating p53-dependent transcription [40]. Since epigenetic factors rely on sequence-specific transcription factors to be recruited to gene promoters to participate in transcriptional regulation, it is conceivable that STAT1 might be responsible for coordinating the dialogue between WDR5 and PCAF/GCN5. Several lines of evidence support this hypothesis. First, STAT1 silencing erases both histone acetylation and histone methylation at the same time (Fig. S1). Second, activation of A2b signaling blocks STAT1 nuclear accumulation [28] while concomitantly disrupting the interaction between WDR5 and PCAF/GCN5 on the CIITA promoters (Fig. 7). Future studies employing ChIP coupled with high-throughput sequencing techniques will shed additional light on the complex interplay between STAT1 and the various histone modifying enzymes in the process of context-specific transcriptional regulation. In our system, the acetylation of H3K14 and H3K18 surrounding the CIITA promoters was not altered by A2b signaling (Fig. 2). Although various reports have pinned PCAF or GCN5 as a mediator of promoterspecific H3K14 acetylation [41,42], two independent investigations have provided unequivocal evidence that the MYST histone acetyltransferase HBO1 is responsible for global H3K14 acetylation in mice [43,44]. On the other hand, CBP and p300 seem to play redundant roles in acetylating H3K18 [32]. These reports, however, do not take into account how H3K14/H3K18 acetylation is regulated at individual promoter regions and more important, how the expression of individual genes differentially responds specific lysine acetylation status. A few lingering issues need to be addressed. Histone acetyltransferases (HATs) usually reside in large mega-dalton complexes, the substrate specificity of which is determined not the catalytic subunits as studied here but rather the regulatory subunits [45,46]. For instance, HBO1 can be tailored to acetylate either histone H3 or H4 depending on its

J. Xia et al. / Biochimica et Biophysica Acta 1849 (2015) 665–676

671

Fig. 4. Adenosine A2b signaling disrupts the interaction between STAT1 and PCAF/GCN5. (A) HASMCs were treated IFN-γ, NECA, and/or MRS-1754 for 24 h. ChIP assays were performed with anti-PCAF or anti-GCN5. (B) HASMCs were treated IFN-γ, NECA, and/or MRS-1754 for 24 h. Re-ChIP assays were performed with indicated antibodies. (C, D) A10 cells were transfected with type III or type IV CIITA promoter construct along with indicated expression constructs followed by treatment with NECA and/or MRS-1754.

672

J. Xia et al. / Biochimica et Biophysica Acta 1849 (2015) 665–676

associated factors [47]. Therefore, the antagonism between A2b and IFN-γ signaling pathways might target the regulatory factors within the HAT complexes, which could lead to selective modulation of lysine

acetylation. Alternatively, the acetylation status of any given lysine residue is determined by the balance between HATs and histone deacetylases (HDACs). While the present study exclusively focuses on

Fig. 5. STAT1 interacts with WDR5 to activate CIITA transcription. (A) HASMCs were treated with or without IFN-γ for 24 h. ChIP assays were performed with anti-WDR5. (B) HASMCs were treated with or without IFN-γ for 24 h. Re-ChIP was performed with indicated antibodies. (C) A10 cells were transfected with type III or type IV CIITA promoter construct along with indicated expression constructs. (D, E) HASMCs were transfected with indicated siRNA followed by treatment with IFN-γ. CIITA mRNA (D) and protein (E) levels were examined by qPCR and Western.

J. Xia et al. / Biochimica et Biophysica Acta 1849 (2015) 665–676

673

Fig. 6. Adenosine A2b signaling attenuates the recruitment of WDR5 to the CIITA promoters. HASMCs were treated IFN-γ, NECA, and/or MRS-1754 for 24 h. ChIP assays were performed with anti-WDR5. (B) HASMCs were treated IFN-γ, NECA, and/or MRS-1754 for 24 h. Re-ChIP assays were performed with indicated antibodies. (C) A10 cells were transfected with type III or type IV CIITA promoter construct along with indicated expression constructs followed by treatment with NECA and/or MRS-1754. (D) HASMCs were transfected with indicated siRNA followed by treatment with IFN-γ NECA, and/or MRS-1754 for 24 h. CIITA expression was measured by Western.

674

J. Xia et al. / Biochimica et Biophysica Acta 1849 (2015) 665–676

the HATs, it is entirely possible that A2b signaling might target the HDACs to influence histone acetylation. For instance, SIRT2, SIRT7, and HDAC9 have all been shown to regulate H3K18 acetylation [48–50]. We propose that the cumulative activity of all the HATs and HDACs present on a given promoter site is subject to the regulation by converging signaling pathways and as such determines the transcriptional outcome at that site. The validation of this hypothesis, however, would demand a genome-wide survey of binding patterns of all the known HATs and HDACs under different circumstances. At this point, the possibility that H3K14/H3K18 acetylation may be fine-tuned by A2b and IFNγ signaling pathways on regions other than the CIITA promoter cannot be ruled out. Before any attempt to exploit the current findings in the development of novel interventional strategies can be launched, a few caveats should be taken into account. First, although activation of adenosine signaling clearly bestows anti-inflammatory effects to an organism [51], detrimental effects have been observed. For instance, A2b signaling contributes to the proliferation of bladder and breast cancer and deterioration of septic shock in mice [52,53]. As suggested by our previous report, a similar scenario, in which A2b signaling represses CIITA expression in a STAT1-dependent manner, taking place in lung fibroblast cells may account for the increased susceptibility to infection and/or cancer [28]. This apparent discrepancy creates a dilemma in terms of drug delivery: inhibition of A2b signaling in smooth muscle cells could potentially attenuate vascular inflammation while at the same time boosting the chances of microbe infection and/or cancer evasion. Our findings, past and present, which undoubtedly confirm the intricacy of A2b signaling in the pathogenesis of human diseases, call for the development of a “smart” drug delivery system that can differentiate one cell type from another, as highlighted by the recent advances

in material/surface science [54]. Second, both WDR5 and GCN5 have been implicated in maintaining the differentiation capability of cells within the vasculature [55–57]. Since the differentiation potential of vascular progenitor cells holds a key promise in the intervention of atherosclerosis [58,59], targeting WDR5 and/or GCN5 to suppress CIITA expression and hence vascular inflammation should be weighed against the potential loss of self-renewal ability of vascular cells. In conclusion, we present evidence to support a role for A2b signaling in repressing CIITA transcription in vascular smooth muscle cells by interfering with the recruitment of epigenetic factors to the CIITA promoters. Additional studies are warranted to delineate the interaction between STAT1 and PCAF/GCN5/WDR5 in VSMCs on a genomewide basis to allow a more comprehensive assessment of the role A2b signaling plays in regulating VSMC phenotype before the current findings can be extrapolated to aid the development of anti-atherosclerotic strategies. Conflict of interest None. Transparency document The Transparency document associated with this article can be found, in the online version. Acknowledgments This work was supported, in part, by grants from the National Basic Science Project of China (2012CB517503), the National Natural Science

Fig. 7. PCAF/GCN5 and WDR5 synergistically activate CIITA transcription. (A, B) A10 cells were transfected with type III or type IV CIITA promoter construct along with indicated expression constructs. (C) HASMCs were treated IFN-γ, NECA, and/or MRS-1754 for 24 h. Re-ChIP assays were performed with indicated antibodies.

J. Xia et al. / Biochimica et Biophysica Acta 1849 (2015) 665–676

Foundation of China (81273571, 81001427, 31270805, 31200645), the Program for New Century Excellent Talents in University of China (NCET-11-0991), the Jiangsu Provincial Special Program of Medical Science (BL2012012), the Natural Science Foundation of Jiangsu Province (BK2012043), the Ministry of Education (212059), and a Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD). YX is a Fellow at the Collaborative Innovation Center for Cardiovascular Disease Translational Medicine. Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.bbagrm.2015.03.001. References [1] K.J. Kingsbury, Concept of human atherosclerosis, Nature 224 (1969) 146–149. [2] G.K. Hansson, Atherosclerosis—an immune disease: The Anitschkov Lecture 2007, Atherosclerosis 202 (2009) 2–10. [3] Y. Mizuno, R.F. Jacob, R.P. Mason, Inflammation and the development of atherosclerosis, J. Atheroscler. Thromb. 18 (2011) 351–358. [4] C. Weber, A. Zernecke, P. Libby, The multifaceted contributions of leukocyte subsets to atherosclerosis: lessons from mouse models, Nat. Rev. Immunol. 8 (2008) 802–815. [5] B. Halvorsen, K. Otterdal, T.B. Dahl, M. Skjelland, L. Gullestad, E. Oie, P. Aukrust, Atherosclerotic plaque stability—what determines the fate of a plaque? Prog. Cardiovasc. Dis. 51 (2008) 183–194. [6] L. Jonasson, J. Holm, O. Skalli, G. Bondjers, G.K. Hansson, Regional accumulations of T cells, macrophages, and smooth muscle cells in the human atherosclerotic plaque, Arteriosclerosis 6 (1986) 131–138. [7] X. Zhou, A. Nicoletti, R. Elhage, G.K. Hansson, Transfer of CD4(+) T cells aggravates atherosclerosis in immunodeficient apolipoprotein E knockout mice, Circulation 102 (2000) 2919–2922. [8] L. Jonasson, J. Holm, O. Skalli, G. Gabbiani, G.K. Hansson, Expression of class II transplantation antigen on vascular smooth muscle cells in human atherosclerosis, J. Clin. Invest. 76 (1985) 125–131. [9] W. Reith, B. Mach, The bare lymphocyte syndrome and the regulation of MHC expression, Annu. Rev. Immunol. 19 (2001) 331–373. [10] G. Buttice, J. Miller, L. Wang, B.D. Smith, Interferon-gamma induces major histocompatibility class II transactivator (CIITA), which mediates collagen repression and major histocompatibility class II activation by human aortic smooth muscle cells, Circ. Res. 98 (2006) 472–479. [11] X. Kong, M. Fang, F. Fang, P. Li, Y. Xu, PPARgamma enhances IFNgamma-mediated transcription and rescues the TGFbeta antagonism by stimulating CIITA in vascular smooth muscle cells, J. Mol. Cell. Cardiol. 46 (2009) 748–757. [12] J.F. Piskurich, M.W. Linhoff, Y. Wang, J.P. Ting, Two distinct gamma interferoninducible promoters of the major histocompatibility complex class II transactivator gene are differentially regulated by STAT1, interferon regulatory factor 1, and transforming growth factor beta, Mol. Cell. Biol. 19 (1999) 431–440. [13] M.E. Pennini, R.K. Pai, D.C. Schultz, W.H. Boom, C.V. Harding, Mycobacterium tuberculosis 19-kDa lipoprotein inhibits IFN-gamma-induced chromatin remodeling of MHC2TA by TLR2 and MAPK signaling, J. Immunol. 176 (2006) 4323–4330. [14] G.M. O'Keefe, V.T. Nguyen, E.N. Benveniste, Class II transactivator and class II MHC gene expression in microglia: modulation by the cytokines TGF-beta, IL-4, IL-13 and IL-10, Eur. J. Immunol. 29 (1999) 1275–1285. [15] D. Yang, M. Koupenova, D.J. McCrann, K.J. Kopeikina, H.M. Kagan, B.M. Schreiber, K. Ravid, The A2b adenosine receptor protects against vascular injury, Proc. Natl. Acad. Sci. U. S. A. 105 (2008) 792–796. [16] D. Yang, Y. Zhang, H.G. Nguyen, M. Koupenova, A.K. Chauhan, M. Makitalo, M.R. Jones, C. St Hilaire, D.C. Seldin, P. Toselli, E. Lamperti, B.M. Schreiber, H. Gavras, D.D. Wagner, K. Ravid, The A2B adenosine receptor protects against inflammation and excessive vascular adhesion, J. Clin. Invest. 116 (2006) 1913–1923. [17] Y. Xu, K. Ravid, B.D. Smith, Major histocompatibility class II transactivator expression in smooth muscle cells from A2b adenosine receptor knock-out mice: crosstalk between the adenosine and interferon-gamma signaling, J. Biol. Chem. 283 (2008) 14213–14220. [18] G. Caignard, A.V. Komarova, M. Bourai, T. Mourez, Y. Jacob, L.M. Jones, F. Rozenberg, A. Vabret, F. Freymuth, F. Tangy, P.O. Vidalain, Differential regulation of type I interferon and epidermal growth factor pathways by a human Respirovirus virulence factor, PLoS Pathog. 5 (2009) e1000587. [19] F.J. Liddle, J.V. Alvarez, V. Poli, D.A. Frank, Tyrosine phosphorylation is required for functional activation of disulfide-containing constitutively active STAT mutants, Biochemistry 45 (2006) 5599–5605. [20] F. Antunes, A. Marg, U. Vinkemeier, STAT1 signaling is not regulated by a phosphorylation-acetylation switch, Mol. Cell. Biol. 31 (2011) 3029–3037. [21] M. Wu, P.F. Wang, J.S. Lee, S. Martin-Brown, L. Florens, M. Washburn, A. Shilatifard, Molecular regulation of H3K4 trimethylation by Wdr82, a component of human Set1/COMPASS, Mol. Cell. Biol. 28 (2008) 7337–7344. [22] T. Masatsugu, K. Yamamoto, Multiple lysine methylation of PCAF by Set9 methyltransferase, Biochem. Biophys. Res. Commun. 381 (2009) 22–26.

675

[23] R. Paolinelli, R. Mendoza-Maldonado, A. Cereseto, M. Giacca, Acetylation by GCN5 regulates CDC6 phosphorylation in the S phase of the cell cycle, Nat. Struct. Mol. Biol. 16 (2009) 412–420. [24] Y. Xu, L. Wang, G. Buttice, P.K. Sengupta, B.D. Smith, Major histocompatibility class II transactivator (CIITA) mediates repression of collagen (COL1A2) transcription by interferon gamma (IFN-gamma), J. Biol. Chem. 279 (2004) 41319–41332. [25] F. Fang, Y. Yang, Z. Yuan, Y. Gao, J. Zhou, Q. Chen, Y. Xu, Myocardin-related transcription factor A mediates OxLDL-induced endothelial injury, Circ. Res. 108 (2011) 797–807. [26] M. Fang, X. Kong, P. Li, F. Fang, X. Wu, H. Bai, X. Qi, Q. Chen, Y. Xu, RFXB and its splice variant RFXBSV mediate the antagonism between IFNgamma and TGFbeta on COL1A2 transcription in vascular smooth muscle cells, Nucleic Acids Res. 37 (2009) 4393–4406. [27] W. Tian, H. Xu, F. Fang, Q. Chen, Y. Xu, A. Shen, Brahma-related gene 1 bridges epigenetic regulation of proinflammatory cytokine production to steatohepatitis in mice, Hepatology 58 (2013) 576–588. [28] M. Fang, J. Xia, X. Wu, H. Kong, H. Wang, W. Xie, Y. Xu, Adenosine signaling inhibits CIITA-mediated MHC class II transactivation in lung fibroblast cells, Eur. J. Immunol. 43 (2013) 2162–2173. [29] P. Li, Y. Zhao, X. Wu, M. Xia, M. Fang, Y. Iwasaki, J. Sha, Q. Chen, Y. Xu, A. Shen, Interferon gamma (IFN-gamma) disrupts energy expenditure and metabolic homeostasis by suppressing SIRT1 transcription, Nucleic Acids Res. 40 (2012) 1609–1620. [30] N.T. Mehta, A.D. Truax, N.H. Boyd, S.F. Greer, Early epigenetic events regulate the adaptive immune response gene CIITA, Epigenetics 6 (2011) 516–525. [31] A.D. Truax, O.I. Koues, M.K. Mentel, S.F. Greer, The 19S ATPase S6a (S6'/TBP1) regulates the transcription initiation of class II transactivator, J. Mol. Biol. 395 (2010) 254–269. [32] Q. Jin, L.R. Yu, L. Wang, Z. Zhang, L.H. Kasper, J.E. Lee, C. Wang, P.K. Brindle, S.Y. Dent, K. Ge, Distinct roles of GCN5/PCAF-mediated H3K9ac and CBP/p300-mediated H3K18/27 ac in nuclear receptor transactivation, EMBO J. 30 (2011) 249–262. [33] J. Wysocka, T. Swigut, T.A. Milne, Y. Dou, X. Zhang, A.L. Burlingame, R.G. Roeder, A.H. Brivanlou, C.D. Allis, WDR5 associates with histone H3 methylated at K4 and is essential for H3 K4 methylation and vertebrate development, Cell 121 (2005) 859–872. [34] E. Korzus, J. Torchia, D.W. Rose, L. Xu, R. Kurokawa, E.M. McInerney, T.M. Mullen, C.K. Glass, M.G. Rosenfeld, Transcription factor-specific requirements for coactivators and their acetyltransferase functions, Science 279 (1998) 703–707. [35] A.E. Horvai, L. Xu, E. Korzus, G. Brard, D. Kalafus, T.M. Mullen, D.W. Rose, M.G. Rosenfeld, C.K. Glass, Nuclear integration of JAK/STAT and Ras/AP-1 signaling by CBP and p300, Proc. Natl. Acad. Sci. U. S. A. 94 (1997) 1074–1079. [36] G.K. Hansson, P. Libby, The immune response in atherosclerosis: a double-edged sword, Nat. Rev. Immunol. 6 (2006) 508–519. [37] P. Libby, Inflammation in atherosclerosis, Nature 420 (2002) 868–874. [38] X. Wu, X. Kong, L. Luchsinger, B.D. Smith, Y. Xu, Regulating the activity of class II transactivator by posttranslational modifications: exploring the possibilities, Mol. Cell. Biol. 29 (2009) 5639–5644. [39] Y.L. Wang, F. Faiola, M. Xu, S. Pan, E. Martinez, Human ATAC Is a GCN5/PCAFcontaining acetylase complex with a novel NC2-like histone fold module that interacts with the TATA-binding protein, J. Biol. Chem. 283 (2008) 33808–33815. [40] Z. Tang, W.Y. Chen, M. Shimada, U.T. Nguyen, J. Kim, X.J. Sun, T. Sengoku, R.K. McGinty, J.P. Fernandez, T.W. Muir, R.G. Roeder, SET1 and p300 act synergistically, through coupled histone modifications, in transcriptional activation by p53, Cell 154 (2013) 297–310. [41] I.M. Love, P. Sekaric, D. Shi, S.R. Grossman, E.J. Androphy, The histone acetyltransferase PCAF regulates p21 transcription through stress-induced acetylation of histone H3, Cell Cycle 11 (2012) 2458–2466. [42] A.M. Cieniewicz, L. Moreland, A.E. Ringel, S.G. Mackintosh, A. Raman, T.M. Gilbert, C. Wolberger, A.J. Tackett, S.D. Taverna, The bromodomain of Gcn5 regulates site specificity of lysine acetylation on histone H3, Mol. Cell. Proteomics 13 (2014) 2896–2910. [43] A.J. Kueh, M.P. Dixon, A.K. Voss, T. Thomas, HBO1 is required for H3K14 acetylation and normal transcriptional activity during embryonic development, Mol. Cell. Biol. 31 (2011) 845–860. [44] Y. Mishima, S. Miyagi, A. Saraya, M. Negishi, M. Endoh, T.A. Endo, T. Toyoda, J. Shinga, T. Katsumoto, T. Chiba, N. Yamaguchi, I. Kitabayashi, H. Koseki, A. Iwama, The Hbo1– Brd1/Brpf2 complex is responsible for global acetylation of H3K14 and required for fetal liver erythropoiesis, Blood 118 (2011) 2443–2453. [45] H.M. Chan, N.B. La Thangue, p300/CBP proteins: HATs for transcriptional bridges and scaffolds, J. Cell Sci. 114 (2001) 2363–2373. [46] X.J. Yang, The diverse superfamily of lysine acetyltransferases and their roles in leukemia and other diseases, Nucleic Acids Res. 32 (2004) 959–976. [47] M.E. Lalonde, N. Avvakumov, K.C. Glass, F.H. Joncas, N. Saksouk, M. Holliday, E. Paquet, K. Yan, Q. Tong, B.J. Klein, S. Tan, X.J. Yang, T.G. Kutateladze, J. Cote, Exchange of associated factors directs a switch in HBO1 acetyltransferase histone tail specificity, Genes Dev. 27 (2013) 2009–2024. [48] H.A. Eskandarian, F. Impens, M.A. Nahori, G. Soubigou, J.Y. Coppee, P. Cossart, M.A. Hamon, A role for SIRT2-dependent histone H3K18 deacetylation in bacterial infection, Science 341 (2013) 1238858. [49] M.F. Barber, E. Michishita-Kioi, Y. Xi, L. Tasselli, M. Kioi, Z. Moqtaderi, R.I. Tennen, S. Paredes, N.L. Young, K. Chen, K. Struhl, B.A. Garcia, O. Gozani, W. Li, K.F. Chua, SIRT7 links H3K18 deacetylation to maintenance of oncogenic transformation, Nature 487 (2012) 114–118. [50] K. Yan, Q. Cao, C.M. Reilly, N.L. Young, B.A. Garcia, N. Mishra, Histone deacetylase 9 deficiency protects against effector T cell-mediated systemic autoimmunity, J. Biol. Chem. 286 (2011) 28833–28843.

676

J. Xia et al. / Biochimica et Biophysica Acta 1849 (2015) 665–676

[51] H.A. Johnston-Cox, M. Koupenova, K. Ravid, A2 adenosine receptors and vascular pathologies, Arterioscler. Thromb. Vasc. Biol. 32 (2012) 870–878. [52] C. Cekic, D. Sag, Y. Li, D. Theodorescu, R.M. Strieter, J. Linden, Adenosine A2B receptor blockade slows growth of bladder and breast tumors, J. Immunol. 188 (2012) 198–205. [53] B.G. Belikoff, S. Hatfield, P. Georgiev, A. Ohta, D. Lukashev, J.A. Buras, D.G. Remick, M. Sitkovsky, A2B adenosine receptor blockade enhances macrophage-mediated bacterial phagocytosis and improves polymicrobial sepsis survival in mice, J. Immunol. 186 (2011) 2444–2453. [54] J. Zhou, J.J. Rossi, Cell-specific aptamer-mediated targeted drug delivery, Oligonucleotides 21 (2011) 1–10. [55] L. Li, J. Zhu, J. Tian, X. Liu, C. Feng, A role for Gcn5 in cardiomyocyte differentiation of rat mesenchymal stem cells, Mol. Cell. Biochem. 345 (2010) 309–316.

[56] M.R. Alexander, G.K. Owens, Epigenetic control of smooth muscle cell differentiation and phenotypic switching in vascular development and disease, Annu. Rev. Physiol. 74 (2012) 13–40. [57] Y.S. Ang, S.Y. Tsai, D.F. Lee, J. Monk, J. Su, K. Ratnakumar, J. Ding, Y. Ge, H. Darr, B. Chang, J. Wang, M. Rendl, E. Bernstein, C. Schaniel, I.R. Lemischka, Wdr5 mediates self-renewal and reprogramming via the embryonic stem cell core transcriptional network, Cell 145 (2011) 183–197. [58] W.T. Wong, N.F. Huang, C.M. Botham, N. Sayed, J.P. Cooke, Endothelial cells derived from nuclear reprogramming, Circ. Res. 111 (2012) 1363–1375. [59] Z. Raval, D.W. Losordo, Cell therapy of peripheral arterial disease: from experimental findings to clinical trials, Circ. Res. 112 (2013) 1288–1302.