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JaeSang Ko Department of Ophthalmology, Severance Hospital, Institute of Vision Research, Yonsei University College of Medicine, Seoul, Korea

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Ji-Young Kim Department of Ophthalmology, Severance Hospital, Institute of Vision Research, Yonsei University College of Medicine, Seoul, Korea

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Eun Jig Lee Department of Endocrinology, Severance Hospital, Yonsei University of Medicine, Seoul, Korea

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Jin Sook Yoon Department of Ophthalmology, Severance Hospital, Institute of Vision Research, Yonsei University College of Medicine, Seoul, Korea

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Inflammation and adipogenesis represent the main pathogenic mechanisms of Graves’ orbitopathy (GO), and oxidative stress is a well-known inducer of GO pathology. Endoplasmic reticulum (ER) stress has been suggested as a major contributor to inflammation and reactive oxygen species (ROS) generation. In this study, we investigated the role of the ER-stress chaperone protein, binding immunoglobulin protein (BiP), in GO pathogenesis. Using primary cultures of orbital fibroblasts from patients with GO, we examined the role of BiP in GO pathogenesis by silencing its expression with small-interfering RNA (siRNA). Inflammatory cytokine expression was analysed by Western blotting and ELISA. Intracellular ROS levels induced by hydrogen peroxide or cigarette smoke extract were measured by 5-(and 6)-carboxy-20,70-dichlorodihydrofluorescein diacetate staining and flow cytometry. After adipogenic differentiation in BiP siRNA-transfected cells, the cells were stained with Oil Red O, and the levels of adipogenic transcription factors were determined by Western blot analysis. BiP mRNA expression levels were significantly higher in GO orbital tissues than in non-GO orbital tissues. Silencing BiP attenuated the expression of pro-inflammatory cytokines (interleukin-6, intercellular adhesion molecule-1, and monocyte chemotactic protein-1) in primary cultured GO orbital fibroblasts. Silencing BiP also reduced ROS generation, hyaluronan production, and adipocyte differentiation. These findings suggest that ER stress is involved in the aetiology of GO and that modulation of ER stress has therapeutic potential for GO.

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JaeSang Ko Department of Ophthalmology, Severance Hospital, Institute of Vision Research, Yonsei University College of Medicine, Seoul, Korea

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Ji-Young Kim Department of Ophthalmology, Severance Hospital, Institute of Vision Research, Yonsei University College of Medicine, Seoul, Korea

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Min Kyoung Chae Department of Ophthalmology, Severance Hospital, Institute of Vision Research, Yonsei University College of Medicine, Seoul, Korea

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Eun Jig Lee Division of Endocrinology and Metabolism, Department of Internal Medicine, Yonsei University College of Medicine, Seoul, Korea

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Jin Sook Yoon Department of Ophthalmology, Severance Hospital, Institute of Vision Research, Yonsei University College of Medicine, Seoul, Korea

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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.

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Min Seok Kim M Kim, College of Medicine, Yonsei University College of Medicine, Seodaemun-gu, Korea (the Republic of)

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Hyun Young Park H Park, Department of Ophthalmology, Institute of Vision Research, Yonsei University College of Medicine, Severance Hospital, Seodaemun-gu, Korea (the Republic of)

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Soo Hyun Choi S Choi, Department of Ophthalmology, Institute of Vision Research, Yonsei University College of Medicine, Severance Hospital, Seodaemun-gu, Korea (the Republic of)

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Eun-Ju Chang E Chang, Department of Biochemistry and Molecular Biology, University of Ulsan College of Medicine, Songpa-gu, Korea (the Republic of)

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JaeSang Ko J Ko, Department of Ophthalmology, Institute of Vision Research, Yonsei University College of Medicine, Severance Hospital, Seodaemun-gu, Korea (the Republic of)

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Jin Sook Yoon J Yoon, Department of Ophthalmology, Institute of Vision Research, Yonsei University College of Medicine, Severance Hospital, Seodaemun-gu, Korea (the Republic of)

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Pentraxin 3 (PTX3) is a prototypic humoral soluble pattern-recognition molecule known to function in immunity-related inflammation. Given the lack of information on the precise functions of PTX3 in the pathogenesis of Graves’ orbitopathy (GO), this study investigated the role of PTX3 in the inflammation and adipogenesis mechanism of GO. We first compared the PTX3 expression between orbital tissues from patients with GO and normal controls, using real-time polymerase chain reaction, which estimated significantly higher PTX3 transcript levels in the GO tissues than in the normal tissues. In addition, PTX3 production was markedly increased upon interleukin (IL)-1β and adipogenic stimulation. We then evaluated the effects of silencing PTX3 in primary orbital fibroblast cultures by analyzing the expression levels of pro-inflammatory cytokines, adipogenesis-related proteins, and downstream transcription factors in cells transfected with or without a small interfering RNA against PTX3, using western blot. Silencing PTX3 attenuated the IL-1β-induced secretion of pro-inflammatory cytokines, including IL-6, IL-8, monocyte chemotactic protein-1, intercellular adhesion molecule-1, and cyclooxygenase-2, and suppressed the IL-1β-mediated activation of p38 kinase, nuclear factor-κB, and extracellular signal-regulated kinase. Moreover, PTX3 knockdown suppressed adipogenic differentiation, as assessed using Oil Red O staining, as well as the expression of adipogenesis-associated transcription factors including peroxisome proliferator activator-γ, CCAAT/enhancer-binding proteins α and β, adipocyte protein 2, adiponectin, and leptin. Thus, this study suggests that PTX3 plays a significant role in the pathogenesis of GO and may serve as a novel therapeutic target for the condition.

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