Abstract:
Graves\' ophthalmopathy (GO) is an extra-thyroidal complication of Graves\' disease which can lead to vision loss in severe cases. Currently, treatments of GO are not sufficiently effective, so novel therapeutic strategies are needed. As platelet-derived growth factor (PDGF)-BB induces several effector mechanisms in GO orbital fibroblasts including cytokine production and myofibroblast activation, this study aims to investigate the roles of histone lysine methyltransferases (HKMTs) in PDGF-BB-activated GO orbital fibroblasts by screening with HKMTs inhibitors library. From the total of twelve selective HKMT inhibitors in the library, EZH2, G9a and DOT1L inhibitors, DZNeP, BIX01294 and Pinometostat, respectively, prevented PDGF-BB-induced proliferation and hyaluronan production by GO orbital fibroblasts. However, only EZH2 inhibitor, DZNeP, significantly blocked pro-inflammatory cytokine production. For the HKMTs expression in GO orbital fibroblasts, PDGF-BB significantly and time-dependently induced EZH2, G9a and DOT1L mRNA expression. To confirm the role of EZH2 in PDGF-BB-induced orbital fibroblast activation, EZH2 silencing experiments revealed suppression of PDGF-BB-induced collagen type I and α-SMA expression along with decreasing histone H3 lysine 27 trimethylation (H3K27me3) level. In a more clinically relevant model than orbital fibroblast culture experiments, DZNeP treated GO orbital tissues significantly reduced pro-inflammatory cytokine production while slightly reduced ACTA2 mRNA expression. Our data is the first to demonstrate that among all HKMTs EZH2 dominantly involved in the expression of myofibroblast markers in PDGF-BB-activated orbital fibroblast from GO presumably via H3K27me3. Thus, EZH2 may represent a novel therapeutics target for GO.
摘要:
Graves眼病(GO)是Graves病的甲状腺外并发症,严重时会导致视力丧失。目前,GO的治疗不够有效,所以需要新的治疗策略。由于血小板衍生生长因子(PDGF)-BB在GO轨道成纤维细胞中诱导几种效应机制,包括细胞因子产生和肌成纤维细胞活化,本研究旨在通过HKMTs抑制剂库筛选,探讨组蛋白赖氨酸甲基转移酶(HKMTs)在PDGF-BB激活的GO轨道成纤维细胞中的作用。从库中的十二种选择性HKMT抑制剂中,EZH2,G9a和DOT1L抑制剂,DZNeP,BIX01294和Pinometostat,分别,通过GO轨道成纤维细胞阻止PDGF-BB诱导的增殖和透明质酸的产生。然而,只有EZH2抑制剂,DZNeP,显著阻断促炎细胞因子的产生。对于GO眶成纤维细胞中的HKMTs表达,PDGF-BB显著且时间依赖性地诱导EZH2、G9a和DOT1LmRNA表达。为了证实EZH2在PDGF-BB诱导的眼眶成纤维细胞活化中的作用,EZH2沉默实验显示抑制PDGF-BB诱导的I型胶原蛋白和α-SMA表达,同时降低组蛋白H3赖氨酸27三甲基化(H3K27me3)水平。在比眼眶成纤维细胞培养实验更临床相关的模型中,DZNeP处理的GO眼眶组织显著降低促炎细胞因子产生,同时略微降低ACTA2mRNA表达。我们的数据首次证明,在所有HKMTs中,EZH2主要参与来自GO的PDGF-BB激活的眼眶成纤维细胞中肌成纤维细胞标志物的表达,大概是通过H3K27me3。因此,EZH2可以代表GO的新型治疗靶标。