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BET Bromodomains Regulate Transforming Growth Factor-β-induced Proliferation and Cytokine Release in Asthmatic Airway Smooth Muscle*

  • Author Footnotes
    2 Members of Interuniversity Attraction Poles Program-Belgian State-Belgian Science Policy Project P7/30.
    Mark M. Perry
    Correspondence
    Imperial College Research Fellow. To whom correspondence should be addressed: National Heart and Lung Institute, Imperial College London, Dovehouse St., London, SW3 6LY, UK. Tel.: 44-20-7352-8121, Ext. 3027; E-mail: [email protected]
    Footnotes
    2 Members of Interuniversity Attraction Poles Program-Belgian State-Belgian Science Policy Project P7/30.
    Affiliations
    Experimental Studies, National Heart and Lung Institute, Imperial College London and Royal Brompton National Institute for Health Research Biomedical Research Unit, London SW3 6LY, United Kingdom
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  • Andrew L. Durham
    Affiliations
    Experimental Studies, National Heart and Lung Institute, Imperial College London and Royal Brompton National Institute for Health Research Biomedical Research Unit, London SW3 6LY, United Kingdom
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  • Philip J. Austin
    Affiliations
    Experimental Studies, National Heart and Lung Institute, Imperial College London and Royal Brompton National Institute for Health Research Biomedical Research Unit, London SW3 6LY, United Kingdom
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  • Author Footnotes
    2 Members of Interuniversity Attraction Poles Program-Belgian State-Belgian Science Policy Project P7/30.
    Ian M. Adcock
    Footnotes
    2 Members of Interuniversity Attraction Poles Program-Belgian State-Belgian Science Policy Project P7/30.
    Affiliations
    Experimental Studies, National Heart and Lung Institute, Imperial College London and Royal Brompton National Institute for Health Research Biomedical Research Unit, London SW3 6LY, United Kingdom
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  • Author Footnotes
    2 Members of Interuniversity Attraction Poles Program-Belgian State-Belgian Science Policy Project P7/30.
    ,
    Author Footnotes
    3 Senior Investigator of the National Institute for Health Research.
    Kian Fan Chung
    Footnotes
    2 Members of Interuniversity Attraction Poles Program-Belgian State-Belgian Science Policy Project P7/30.
    3 Senior Investigator of the National Institute for Health Research.
    Affiliations
    Experimental Studies, National Heart and Lung Institute, Imperial College London and Royal Brompton National Institute for Health Research Biomedical Research Unit, London SW3 6LY, United Kingdom
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  • Author Footnotes
    * This work was supported by Asthma UK Grant 08/041 and Wellcome Trust Grant 085935 (to K. F. C.). It was also supported by the National Institute for Health Research Respiratory Disease Biomedical Research Unit at the Royal Brompton National Health Service Foundation Trust and Imperial College London and the Innovative Medicines Initiative.
    2 Members of Interuniversity Attraction Poles Program-Belgian State-Belgian Science Policy Project P7/30.
    3 Senior Investigator of the National Institute for Health Research.
    4 The abbreviation used is: ASMairway smooth muscle.
Open AccessPublished:February 19, 2015DOI:https://doi.org/10.1074/jbc.M114.612671
      Airway smooth muscle (ASM) mass is increased in asthma, and ASM cells from patients with asthma are hyperproliferative and release more IL-6 and CXCL8. The BET (bromo- and extra-terminal) family of proteins (Brd2, Brd3, and Brd4) govern the assembly of histone acetylation-dependent chromatin complexes. We have examined whether they modulate proliferation and cytokine expression in asthmatic ASM cells by studying the effect of BET bromodomain mimics JQ1/SGCBD01 and I-BET762. ASM cells from healthy individuals and nonsevere and severe asthmatics were pretreated with JQ1/SGCBD01 and I-BET762 prior to stimulation with FCS and TGF-β. Proliferation was measured by BrdU incorporation. IL-6 and CXCL8 release was measured by ELISA, and mRNA expression was measured by quantitative RT-PCR. ChIP using a specific anti-Brd4 antibody and PCR primers directed against the transcriptional start site of IL-6 and CXCL8 gene promoters was performed. Neither JQ1/SGCBD01 nor I-BET762 had any effect on ASM cell viability. JQ1/SGCBD01 and I-BET762 inhibited FCS+TGF-β-induced ASM cell proliferation and IL-6 and CXCL8 release in healthy individuals (≥ 30 nm) and in nonsevere and severe asthma patients (≥100 nm), with the latter requiring higher concentrations of these mimics. JQ1/SGCBD01 reduced Brd4 binding to IL8 and IL6 promoters induced by FCS+TGF-β. Mimics of BET bromodomains inhibit aberrant ASM cell proliferation and inflammation with lesser efficiency in those from asthmatic patients. They may be effective in reducing airway remodeling in asthma.

      Introduction

      Chronic airflow obstruction and airway inflammation and remodeling are fundamental characteristics of asthma (
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      The abbreviation used is: ASM
      airway smooth muscle.
      mass is increased in asthma because of enhanced ASM hyperplasia and hypertrophy, resulting in subepithelial fibrosis and narrowing of the airways. ASM cells also play a key role in the chronic inflammatory response in the airways of asthmatics through the expression of cytokines, growth factors, and proteases (
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      Histone modifications such as acetylation, phosphorylation, and methylation are regulated in a temporal and spatial manner to control chromatin structure under a “histone code or language” (
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      ). Enhanced histone acetylation is associated with altered cellular function including the increased expression of inflammatory genes. Acetylated histones are detected by proteins that contain a bromodomain motif, such as the BET (bromo- and extra-terminal) family members that include Brd2, Brd3, and Brd4 and that govern the assembly of histone acetylation-dependent chromatin complexes. This epigenetic tag is linked to control RNA polymerase II activity via recruitment of transcriptional activators such as pTEFb (
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      ). BET family members such as Brd4 have been implicated in the regulation of NF-κB-induced inflammatory gene expression in monocytes and macrophages through recognition of acetylated histone tags (
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      ).
      We hypothesized that BET proteins play an important role in the regulation of proliferation and release of pro-inflammatory cytokines in ASM cells from patients with asthma. BET mimics JQ1/SGCBD01 and I-BET762 that prevent the association of bromodomain-containing proteins with acetylated lysines have recently been developed (
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      ). These mimics affect proliferation and the expression of some inflammatory genes in murine and human cell types (
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      ), but this is the first time that effects have been reported in these primary ASM cells from patients with asthma. We used these inhibitors to investigate the role of BET proteins in the regulation of TGF-β-induced proliferation and inflammation of ASM obtained from patients with different severities of asthma. Our results show that inhibition of BET bromodomains can reduce both aberrant ASM cell proliferation and expression of cytokines from patients with severe asthma by reducing Brd4 binding to the promoter regions of IL8 and IL6 genes.

      DISCUSSION

      We have shown that the BET mimics JQ1/SGCBD01 and I-BET762 inhibited FCS+TGF-β-induced cell proliferation and inflammation in ASM cells from healthy and asthmatic individuals. The concentrations required to inhibit proliferation were higher in cells from nonsevere and severe asthmatics, indicating that there is an innate resistance of asthmatic cells to the effect of these BET mimics. Inhibition of c-Myc affected ASM proliferation but not IL-6 or CXCL8 production. JQ1/SGCBD01 reduced the association of Brd4 to the IL6 and IL8 promoters, thereby modulating their transcription.
      We showed that BET proteins in primary ASM cells are important in controlling ASM cell proliferation both in healthy and asthmatic cells. Previous studies have mainly focused on cancerous cell lines, where JQ1/SGCBD01 inhibited cellular proliferation of NUT midline carcinoma (
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      Inhibition of BET bromodomain targets genetically diverse glioblastoma.
      ), lung cancer cells (
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      ), acute myeloid leukemia cells (
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      RNAi screen identifies Brd4 as a therapeutic target in acute myeloid leukaemia.
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      ). This mechanism is documented to occur via a decrease in either c-Myc expression or c-Myc association with a number of proliferative gene promoters. c-Myc is a regulatory transcription factor that regulates cell growth, and deregulation of c-Myc in cancer contributes to proliferation, tumorigenesis, and resistance to apoptosis. Furthermore, amplification of c-Myc is one of the most common genetic alterations in cancer genomes (
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      ). Although we show that inhibition of c-Myc inhibits proliferation, we saw no effect of JQ1/SGCBD01 upon the induced expression of c-Myc expression by FCS+TGFβ, demonstrating that the effect of JQ1+ upon c-Myc expression seen in cancerous cells and nonprimary cells is different from that seen in noncancerous asthmatic ASM cells.
      Our results provide evidence that the effect of JQ1/SGCBD01 and I-BET762 is independent of the induction of c-Myc, as has been demonstrated in glioblastomas where JQ1/SGCBD01 had a minimal effect upon c-Myc expression. JQ1/SGCBD01 had a significant effect upon p21WAF1 expression, returning it to basal levels (
      • Cheng Z.
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      Inhibition of BET bromodomain targets genetically diverse glioblastoma.
      ). We have previously shown that primary ASM proliferation is under the control, at least in part, of p21WAF1 and p27kip1 (
      • Perry M.M.
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      ,
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      Transforming growth factor-β and nuclear factor E2-related factor 2 regulate antioxidant responses in airway smooth muscle cells.
      ). Here we show that JQ1/SGCBD01 may mediate its response in asthmatic ASM by returning the reduced expression of these cyclin inhibitors to basal levels. The release of IL-6 and CXCL8 from ASM cells was not affected by pharmacological inhibition of c-Myc which corresponded with the failure to show marked physical association of c-Myc to the IL6 and IL8 promoters.
      We also demonstrate that BET bromodomain mimics JQ1/SGCBD01 and I-BET762 inhibited FCS+TGF-β-induced release and gene expression of IL-6 and CXCL8 in ASM cells from all three groups of subjects. However, these inhibitors were less effective on ASM cells from patients with asthma. Our chromatin immunoprecipitation data show that there was a greater amount of enrichment of Brd4 at the IL6 and IL8 promoters at baseline in ASM cells from severe asthma compared with nonasthmatics subjects. FCS+TGF-β increased Brd4 binding to the IL6 and IL8 promoters in the controls but only the IL8 promoter in the ASM cells isolated from severe asthmatics. JQ1/SGCBD01 did not significantly inhibit this enrichment of either the IL6 or the IL8 promoters.
      Increased Brd4 binding at baseline may allow greater IL6 and IL8 gene expression, explaining the increased inflammation in severe asthmatics compared with controls. Thus, inhibition of Brd4 binding to the gene promoters may explain the mechanism of action of JQ1/SGCBD01 and by extension of I-BET762 in inhibiting cytokine gene expression. The differential effect of JQ1/SGCBD01 and I-BET762 on IL-6 and CXCL8 expression and on basal Brd4 promoter association may reflect differences in the underlying mechanisms controlling these genes, with IL6 expression being regulated by Brd4 proteins at a paused promoter, whereas IL8 transcription requires Brd4 recruitment before transcription can occur. Promoter pausing is an increasingly identified as a key regulatory step in the transcription of rapidly transcribed genes, including those involved in stress and inflammation (
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      ). Brd4 may be bound to the IL6 promoter region prior to the TGF/FCS signal being received, allowing more rapid response to stimuli. Brd4 has previously been associated with recruitment of the pause release factor p-TEFB (
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      ), and we therefore believe that the role of transcriptional pausing in the regulation of inflammation will make an interesting area of future research.
      Similar findings have been reported with both JQ1/SGCBD01 and I-BET762. In murine bone marrow-derived macrophages exposed to bacterial endotoxin, I-BET762 inhibited the expression of pro-inflammatory cytokines such as IL-1β, IL-6, IL-12α, CXCL9, and CCL2, and I-BET762 displaced the BET proteins Brd2, Brd3, and Brd4 from the IL6 promoter (
      • Nicodeme E.
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      Suppression of inflammation by a synthetic histone mimic.
      ). I-BET762 was also shown to rescue mice from endotoxin-induced death. Similar results using JQ1/SGCBD01 were obtained for the endotoxin-induced production of IL-6 and TNF-α in mouse macrophages. In these experiments, Brd2 and c-Myc were shown to be physically associated with the promoter regions of these inflammatory genes (
      • Belkina A.C.
      • Nikolajczyk B.S.
      • Denis G.V.
      BET protein function is required for inflammation: Brd2 genetic disruption and BET inhibitor JQ1 impair mouse macrophage inflammatory responses.
      ). Both I-BET762 and JQ1/SGCBD01 have been reported to inhibit the release of IL-6 from human lung fibroblasts and their proliferation induced by TGF-β, by reducing Brd2 and Brd4 association with growth factor-responsive genes and enhanced transcription of these genes (
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      • Demartino J.A.
      • Kitson C.
      • Stevenson C.S.
      • Budd D.C.
      BET bromodomain proteins mediate downstream signaling events following growth factor stimulation in human lung fibroblasts and are involved in bleomycin-induced pulmonary fibrosis.
      ). The differential effect of JQ1/SGCBD01 and I-BET762 on IL-6 and CXCL8 expression and on basal Brd4 promoter association may reflect differences in the underlying mechanisms controlling these genes, with IL6 expression being regulated by Brd proteins at a paused promoter, whereas IL8 transcription requires Brd recruitment before transcription can occur.
      A higher concentration of both JQ1/SGCBD01 and I-BET762 was needed to inhibit proliferation and IL-6 and CXCL8 release from cells from patients with asthma compared with nonasthmatic subjects. Asthmatic ASM cells demonstrate a greater degree of proliferation and release of cytokines such as IL-6 when exposed to FCS+TGF-β compared with ASM cells from nonasthmatic subjects (
      • Perry M.M.
      • Baker J.E.
      • Gibeon D.S.
      • Adcock I.M.
      • Chung K.F.
      Airway smooth muscle hyperproliferation is regulated by microRNA-221 in severe asthma.
      ,
      • Chang P.J.
      • Bhavsar P.K.
      • Michaeloudes C.
      • Khorasani N.
      • Chung K.F.
      Corticosteroid insensitivity of chemokine expression in airway smooth muscle of patients with severe asthma.
      ). The increased Brd4 association observed in the asthmatic samples (and possible transcriptional pausing of the inflammatory gene promoters) may explain the higher levels of transcription of IL6 and IL8 after stimulation of the cells (FIGURE 6, FIGURE 7). This may also explain the need for a greater amount of the BET mimics to produce the same effect as in nonasthmatic ASM cells. Part of the explanation could lie with the greater association of Brd4 to the IL6 promoter observed in severe asthmatics at baseline compared with controls at baseline. Although not significant, the binding of Brd4 to the promoter regions of IL6 and IL8 increased when the cells were stimulated by FCS+TGF-β, an effect that was prevented by pretreatment of the cells with JQ1/SGCBD01.
      The relative insensitivity of ASM cells from asthma patients to the BET mimics reminds us of the similar insensitivity observed with corticosteroids (
      • Perry M.M.
      • Baker J.E.
      • Gibeon D.S.
      • Adcock I.M.
      • Chung K.F.
      Airway smooth muscle hyperproliferation is regulated by microRNA-221 in severe asthma.
      ,
      • Chang P.J.
      • Bhavsar P.K.
      • Michaeloudes C.
      • Khorasani N.
      • Chung K.F.
      Corticosteroid insensitivity of chemokine expression in airway smooth muscle of patients with severe asthma.
      ). The basis for corticosteroid insensitivity in asthma may be multifactorial (
      • Adcock I.M.
      • Ford P.A.
      • Bhavsar P.
      • Ahmad T.
      • Chung K.F.
      Steroid resistance in asthma: mechanisms and treatment options.
      ). A lack of recruitment of histone deacetylase 2 to the RNA polymerase compatible with histone deacetylase activity being reduced in peripheral blood mononuclear cells and alveolar macrophages from patients with asthma has been proposed (
      • Hew M.
      • Bhavsar P.
      • Torrego A.
      • Meah S.
      • Khorasani N.
      • Barnes P.J.
      • Adcock I.
      • Chung K.F.
      Relative corticosteroid insensitivity of peripheral blood mononuclear cells in severe asthma.
      ,
      • Cosío B.G.
      • Mann B.
      • Ito K.
      • Jazrawi E.
      • Barnes P.J.
      • Chung K.F.
      • Adcock I.M.
      Histone acetylase and deacetylase activity in alveolar macrophages and blood mononocytes in asthma.
      ). The gene promoters in cells from asthma versus nonasthma may have an enhanced acetylation status that may in part account for the reduced sensitivity to BET mimics in these cells.
      BET bromodomain mimics can inhibit both aberrant ASM cell proliferation and pro-inflammatory cytokine production from cells from patients with asthma including severe asthma. Interestingly, a pharmaceutical company, Tensha Therapeutics, is currently in phase I clinical trials evaluating a derivative of JQ1/SGCBD01 (TEN-010) in patients with solid tumors (
      • Rohn J.
      Tensha therapeutics.
      ), and although local delivery of these compounds has yet to be studied, systemic administration of JQ1/SGCBD01 significantly inhibits inflammatory cytokine expression in diseased gingival tissues (
      • Meng S.
      • Zhang L.
      • Tang Y.
      • Tu Q.
      • Zheng L.
      • Yu L.
      • Murray D.
      • Cheng J.
      • Kim S.H.
      • Zhou X.
      • Chen J.
      BET inhibitor JQ1 blocks inflammation and bone destruction.
      ) and demonstrates no affect upon systemic blood pressure (
      • Anand P.
      • Brown J.D.
      • Lin C.Y.
      • Qi J.
      • Zhang R.
      • Artero P.C.
      • Alaiti M.A.
      • Bullard J.
      • Alazem K.
      • Margulies K.B.
      • Cappola T.P.
      • Lemieux M.
      • Plutzky J.
      • Bradner J.E.
      • Haldar S.M.
      BET bromodomains mediate transcriptional pause release in heart failure.
      ). These compounds may therefore be effective in reducing features of airway remodeling in severe asthma, which often forms the basis of chronic airflow obstruction and greater inflammatory responses that are often insensitive to the effects of anti-inflammatory agents such as corticosteroids.

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