Background: In Graves' orbitopathy (GO), localized orbital inflammation within the fixed orbit often leads to a fibrotic phenotype resulting in restrictive myopathy or refractory proptosis. However, the molecular pathways related to the transition from inflammation to fibrosis in GO are less understood. Yes-associated protein (YAP) and its homolog, transcriptional coactivator with PDZ-binding motif (TAZ; a Hippo pathway effector), are critical mechanosensors of mechanical stimuli and activate signaling cascades for cell proliferation, differentiation, and transformation. In this study, we aimed to examine the role of YAP in both inflammatory and fibrotic GO pathogenesis. Methods: Based on RNA sequencing performed on freshly obtained orbital adipose tissue from patients with GO and healthy individuals, Gene Ontology analysis and gene set-enrichment analysis were performed to analyze gene-expression differences between GO and normal orbital tissues. The role of YAP in GO-related inflammation and fibrosis was studied in primary cultured orbital fibroblasts. The effects of interleukin-1β (IL-1β)-induced inflammation and transforming growth factor-beta (TGF-β)-induced fibrosis on YAP expression were evaluated using real-time polymerase chain reaction and Western blotting analyses. The effects of YAP on inflammatory and fibrotic responses were also examined by YAP silencing or treatment with pharmacological YAP inhibitors. Results: RNA sequencing revealed enhanced YAP expression in GO orbital tissues. Gene Ontology analysis indicated that "response to mechanical stimulus"-related genes were overexpressed in GO orbital tissues, along with those enriched for the "adipose proliferation," "inflammatory responses," and "hormone stimulus responses" terms. IL-1β did not enhance YAP expression, and YAP silencing decreased IL-1β-induced IL-6 expression while increasing prostaglandin-endoperoxide synthase 2 expression, leading to paradoxical pro-inflammatory effects. Conversely, TGF-β enhanced YAP expression, and YAP silencing and pharmacological YAP inhibitor (cerivastatin, verteporfin, TED-347, and CA3) treatment significantly reduced TGF-β-induced myofibroblast differentiation and collagen formation. Conclusion: YAP, a mechanotransducer responding to mechanical stimuli, was strongly expressed in GO orbital tissues, and YAP was induced by TGF-β in orbital fibroblasts. Our study establishes YAP as a novel mediator of GO pathobiology, potentially mediating the transition from early inflammation to chronic fibrosis in GO. The finding that YAP inhibition suppressed TGF-β-induced fibrotic response suggests YAP as a therapeutic target against the fibrotic mechanism of GO.

中文翻译：

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Yes相关蛋白介导格雷夫斯眼眶病从炎症到纤维化的转变。

背景：在格雷夫斯眼眶病（GO）中，固定眼眶内的局部眼眶炎症通常会导致纤维化表型，从而导致限制性肌病或难治性突眼。然而，人们对 GO 从炎症向纤维化转变相关的分子途径知之甚少。Yes 相关蛋白 (YAP) 及其同源物、具有 PDZ 结合基序的转录共激活因子（TAZ；Hippo 通路效应子）是机械刺激的关键机械传感器，可激活细胞增殖、分化和转化的信号级联。在本研究中，我们旨在研究 YAP 在炎症和纤维化 GO 发病机制中的作用。方法：基于对 GO 患者和健康个体新鲜获得的眼眶脂肪组织进行 RNA 测序，进行基因本体分析和基因集富集分析，以分析 GO 和正常眼眶组织之间的基因表达差异。在原代培养的眼眶成纤维细胞中研究了 YAP 在 GO 相关炎症和纤维化中的作用。使用实时聚合酶链反应和蛋白质印迹分析评估白细胞介素-1β (IL-1β) 诱导的炎症和转化生长因子-β (TGF-β) 诱导的纤维化对 YAP 表达的影响。还通过 YAP 沉默或药理学 YAP 抑制剂治疗来检查 YAP 对炎症和纤维化反应的影响。结果：RNA 测序显示 GO 眼眶组织中 YAP 表达增强。基因本体分析表明，“对机械刺激的反应”相关基因在 GO 眼眶组织中过度表达，以及那些富含“脂肪增殖”、“炎症反应”和“激素刺激反应”术语的基因。IL-1β 不会增强 YAP 表达，而 YAP 沉默会降低 IL-1β 诱导的 IL-6 表达，同时增加前列腺素内过氧化物合酶 2 表达，从而导致矛盾的促炎作用。相反，TGF-β 增强 YAP 表达，YAP 沉默和药理学 YAP 抑制剂（西立伐他汀、维替泊芬、TED-347 和 CA3）治疗显着降低 TGF-β 诱导的肌成纤维细胞分化和胶原形成。结论：YAP是一种对机械刺激作出反应的机械传感器，在GO眼眶组织中强烈表达，并且YAP是由眼眶成纤维细胞中的TGF-β诱导的。我们的研究将 YAP 确立为 GO 病理学的新型介质，可能介导 GO 从早期炎症到慢性纤维化的转变。YAP 抑制抑制 TGF-β 诱导的纤维化反应的发现表明 YAP 作为针对 GO 纤维化机制的治疗靶点。