Perry Mark M, Durham Andrew L, Austin Philip J, Adcock Ian M, Chung Kian Fan
From the 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
From the 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.
J Biol Chem. 2015 Apr 3;290(14):9111-21. doi: 10.1074/jbc.M114.612671. Epub 2015 Feb 19.
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.
哮喘患者的气道平滑肌（ASM）质量增加，哮喘患者的ASM细胞增殖过度，且释放更多的白细胞介素-6（IL-6）和CXC趋化因子配体8（CXCL8）。BET（含溴结构域和额外末端结构域）蛋白家族（Brd2、Brd3和Brd4）调控组蛋白乙酰化依赖性染色质复合物的组装。我们通过研究BET溴结构域模拟物JQ1/SGCBD01和I-BET762的作用，来检测它们是否调节哮喘ASM细胞的增殖和细胞因子表达。来自健康个体以及非重度和重度哮喘患者的ASM细胞在用胎牛血清（FCS）和转化生长因子-β（TGF-β）刺激之前，先用JQ1/SGCBD01和I-BET762进行预处理。通过掺入5-溴脱氧尿嘧啶核苷（BrdU）来检测增殖情况。通过酶联免疫吸附测定（ELISA）检测IL-6和CXCL8的释放，通过定量逆转录聚合酶链反应（RT-PCR）检测信使核糖核酸（mRNA）表达。使用特异性抗Brd4抗体进行染色质免疫沉淀（ChIP），并使用针对IL-6和CXCL8基因启动子转录起始位点的聚合酶链反应（PCR）引物。JQ1/SGCBD01和I-BET762对ASM细胞活力均无任何影响。JQ1/SGCBD01和I-BET762抑制FCS+TGF-β诱导的健康个体（≥30纳摩尔）以及非重度和重度哮喘患者（≥100纳摩尔）的ASM细胞增殖以及IL-6和CXCL8释放，重度哮喘患者需要更高浓度的这些模拟物。JQ1/SGCBD01减少了FCS+TGF-β诱导的Brd4与IL8和IL6启动子的结合。BET溴结构域模拟物抑制哮喘患者ASM细胞的异常增殖和炎症，但其效率较低。它们可能对减轻哮喘中的气道重塑有效。
