Department of Ophthalmology, University Hospital Essen, University of Duisburg-Essen, 45147 Essen, Germany.
J Clin Endocrinol Metab. 2011 Nov;96(11):E1727-36. doi: 10.1210/jc.2011-1156. Epub 2011 Sep 14.
Excessive orbital fibroblast (OF) proliferation and extracellular matrix production, as well as inflammation resulting in the expansion and remodeling of orbital tissue, are characteristic of Graves ophthalmopathy (GO). Our aim was to analyze and inhibit signaling pathways in resident OF that are involved in GO. METHODS/MAIN OUTCOME MEASURES: Primary human OF were obtained from 12 patients with active, severe GO and from 12 healthy control subjects. The cells were characterized by immunofluorescence assay and flow cytometry. Tyrosine phosphorylation of cellular proteins was determined by Western blot techniques, immunoprecipitation, and protein identity with mass spectrometry. Cell proliferation was determined by 5-bromo-2-deoxyuridine incorporation, hyaluronan (HA) production was assessed by a HA-binding protein based assay, and intracellular reactive oxygen species (ROS) were determined by the dichlorofluorescein assay. Clathrin heavy-chain (CHC) expression was inhibited with small interfering RNA technology.
Tyrosine phosphorylation of CHC is constitutively increased in vitro in GO-derived OF, independent of serum or other stimulating factors. The proliferative and biosynthetic capabilities (production of HA, ROS) of GO-derived OF are significantly higher than those of OF from healthy control subjects. Down-regulation of CHC expression leads to a normalization of pathologically increased proliferation and production of HA and ROS in GO-derived OFs in vitro.
Our findings strongly suggest that clathrin and clathrin-mediated signaling pathways are involved in the inflammatory signal transduction of OF in GO. With the identification of clathrin, we report a new potential targeting molecule for specific pharmacological inhibition of the local inflammatory response characteristic of GO.
过度的眶成纤维细胞（OF）增殖和细胞外基质产生，以及炎症导致眶组织的扩张和重塑，是格雷夫斯眼病（GO）的特征。我们的目的是分析和抑制参与 GO 的驻留 OF 的信号通路。
方法/主要观察指标：从 12 例活动性、严重 GO 患者和 12 例健康对照者中获得原代人 OF。通过免疫荧光法和流式细胞术对细胞进行鉴定。通过 Western blot 技术、免疫沉淀和质谱鉴定细胞蛋白的酪氨酸磷酸化。通过 5-溴-2-脱氧尿苷掺入法测定细胞增殖，通过基于 HA 结合蛋白的测定法评估透明质酸（HA）的产生，通过二氯荧光素测定法测定细胞内活性氧（ROS）。使用小干扰 RNA 技术抑制网格蛋白重链（CHC）的表达。
GO 衍生的 OF 中 CHC 的酪氨酸磷酸化在体外持续增加，与血清或其他刺激因子无关。GO 衍生的 OF 的增殖和生物合成能力（HA、ROS 的产生）明显高于健康对照组的 OF。下调 CHC 表达可使 GO 衍生的 OF 体外病理性增加的增殖和 HA 及 ROS 的产生正常化。
我们的研究结果强烈表明网格蛋白和网格蛋白介导的信号通路参与了 GO 中 OF 的炎症信号转导。通过鉴定网格蛋白，我们报告了一种新的潜在靶向分子，可用于特异性抑制 GO 特征的局部炎症反应。
