Department of Ophthalmology, Severance Hospital, Institute of Vision Research, Yonsei University College of Medicine, Seoul, Korea.
Division of Endocrinology and Metabolism, Department of Internal Medicine, Yonsei University College of Medicine, Seoul, Korea.
J Mol Endocrinol. 2021 May 11;66(4):313-323. doi: 10.1530/JME-21-0057.
We examined endoplasmic reticulum (ER) stress-related gene expression in orbital tissues from patients with Graves' orbitopathy (GO) and the effects of silencing protein kinase RNA-like endoplasmic reticulum kinase (PERK) in primary orbital fibroblast cultures to demonstrate the therapeutic potential of PERK-modulating agents in GO management. The expression of ER stress-related genes in orbital tissue harvested from individuals with or without GO was studied using real-time PCR. The role of PERK in GO pathogenesis was examined through small-interfering RNA (siRNA)-mediated silencing in cultured primary orbital fibroblasts. Intracellular reactive oxygen species (ROS) levels induced in response to cigarette smoke extract (CSE) or hydrogen peroxide were measured using 5-(and 6)-carboxy-20,70-dichlorodihydrofluorescein diacetate staining and flow cytometry. Cells were stained with Oil Red O, and adipogenesis-related transcription factor expression was evaluated through Wwestern blotting after adipogenic differentiation. PERK, activating transcription factor 4 (ATF4), and CCAAT-enhancer-binding protein (C/EBP)-homologous protein (CHOP) mRNA levels were significantly higher in GO orbital tissues than in non-GO orbital tissues. PERK silencing inhibited CSE- or hydrogen peroxide-induced ROS generation. After adipogenic differentiation, GO orbital fibroblasts revealed decreased lipid droplets and downregulation of C/EBPα, C/EBPβ, and peroxisome proliferator-activator gamma (PPARγ) in PERK siRNA-transfected cells. The orbital tissues of patients with GO were exposed to chronic ER stress and subsequently exhibited enhanced unfolded protein response (especially through the PERK pathway). PERK silencing reduced oxidative stress and adipogenesis in GO orbital fibroblasts in vitro. Our results imply that PERK-modulating agents can potentially be used to manage GO.
我们研究了格雷夫斯眼病（GO）患者眼眶组织中内质网（ER）应激相关基因的表达，并在原代眼眶成纤维细胞培养物中沉默蛋白激酶 RNA 样内质网激酶（PERK），以证明 PERK 调节剂在 GO 管理中的治疗潜力。使用实时 PCR 研究了从患有或不患有 GO 的个体中收获的眼眶组织中 ER 应激相关基因的表达。通过 siRNA 介导的培养的原代眼眶成纤维细胞中 PERK 的沉默，研究了 PERK 在 GO 发病机制中的作用。使用 5-（和 6）-羧基-20，70-二氯二氢荧光素二乙酸酯染色和流式细胞术测量细胞内活性氧（ROS）水平，以响应香烟烟雾提取物（CSE）或过氧化氢而诱导。在用 CSE 或过氧化氢诱导成脂分化后，用油红 O 染色，并通过 Western blot 评估脂肪生成相关转录因子的表达。GO 眼眶组织中的 PERK、激活转录因子 4（ATF4）和 CCAAT 增强子结合蛋白（C/EBP）-同源蛋白（CHOP）mRNA 水平明显高于非 GO 眼眶组织。PERK 沉默抑制了 CSE 或过氧化氢诱导的 ROS 生成。在成脂分化后，GO 眼眶成纤维细胞显示出减少的脂滴，并且在 PERK siRNA 转染细胞中下调 C/EBPα、C/EBPβ 和过氧化物酶体增殖物激活受体γ（PPARγ）。GO 患者的眼眶组织暴露于慢性 ER 应激下，随后表现出增强的未折叠蛋白反应（特别是通过 PERK 途径）。PERK 沉默减少了 GO 眼眶成纤维细胞中的氧化应激和脂肪生成。我们的结果表明，PERK 调节剂可能可用于治疗 GO。
