Division of Pediatric Endocrinology and Metabolism, Mayo Clinic, 200 First Street SW, Rochester, Minnesota 55902, USA.
J Clin Endocrinol Metab. 2012 May;97(5):1681-7. doi: 10.1210/jc.2011-2890. Epub 2012 Mar 7.
Graves' ophthalmopathy (GO) is characterized by expanded volume of the orbital fat and extraocular muscle tissues and elevated levels of TSH receptor autoantibodies (TRAb). The expansion of orbital tissues involves accumulation of hyaluronic acid (HA) within the orbit.
The objective of the study was to determine whether a monoclonal stimulatory TRAb (M22) impacts HA synthesis in GO orbital cells and, if so, whether this might be blocked by an IGF-I receptor (IGF-IR)-blocking antibody (1H7) or inhibitors of various downstream signaling cascades.
GO orbital fibroblast cultures (n = 6) were treated with M22, bovine TSH (bTSH), or IGF-I in serum-free medium. Some cultures also received 1H7, LY294002, rapamycin, or protein kinase A inhibitor.
HA production and phosphorylated Akt levels in media or immunoblotting for phosphorylated Akt were measured.
M22 or bTSH stimulated HA synthesis (2.1-fold with 100 ng/ml M22 and 1.9-fold with 10 U/liter bTSH; P < 0.05 each). M22-induced HA synthesis was inhibited by LY294002 or rapamycin but not by protein kinase inhibitor. HA synthesis stimulated by M22 or IGF-I was inhibited by 1H7 (mean 36.6 ± 5.6% and mean 45.8 ± 7.6%, respectively; P < 0.05 each). Similarly, M22- or IGF-I-stimulated Akt phosphorylation was inhibited by 1H7 (mean 54 ± 9.6 and 36.1 ± 8.8%, respectively; P = 0.01 each).
The stimulatory TRAb M22 increases HA production in undifferentiated GO orbital fibroblasts via phosphoinositide 3-kinase/phosphorylated AKT/mammalian target of rapamycin activation. Blockade of IGF-IR inhibits both HA synthesis and Akt phosphorylation induced by M22 or IGF-I in these cells, suggesting that TSH receptor and IGF-IR signaling may be closely linked in the GO orbit.
格雷夫斯眼病（GO）的特征是眼眶脂肪和眼外肌组织体积增大，促甲状腺激素受体自身抗体（TRAb）水平升高。眼眶组织的扩张涉及到透明质酸（HA）在眼眶内的积累。
本研究旨在确定单克隆刺激性 TRAb（M22）是否影响 GO 眼眶细胞中的 HA 合成，如果是，IGF-IR 阻断抗体（1H7）或各种下游信号转导途径的抑制剂是否可以阻断这种作用。
GO 眼眶成纤维细胞培养物（n = 6）在无血清培养基中用 M22、牛 TSH（bTSH）或 IGF-I 处理。一些培养物还接受了 1H7、LY294002、雷帕霉素或蛋白激酶 A 抑制剂的处理。
测量培养基中的 HA 产量和磷酸化 Akt 水平，或用磷酸化 Akt 的免疫印迹法测量。
M22 或 bTSH 刺激 HA 合成（100ng/ml M22 时增加 2.1 倍，10U/l bTSH 时增加 1.9 倍；均 P < 0.05）。M22 诱导的 HA 合成可被 LY294002 或雷帕霉素抑制，但不能被蛋白激酶抑制剂抑制。M22 或 IGF-I 诱导的 HA 合成被 1H7 抑制（分别为 36.6 ± 5.6%和 45.8 ± 7.6%；均 P < 0.05）。同样，M22 或 IGF-I 诱导的 Akt 磷酸化也被 1H7 抑制（分别为 54 ± 9.6%和 36.1 ± 8.8%；均 P = 0.01）。
刺激性 TRAb M22 通过磷酸肌醇 3-激酶/磷酸化 Akt/雷帕霉素靶蛋白的激活增加未分化的 GO 眼眶成纤维细胞中的 HA 产生。IGF-IR 阻断剂抑制 M22 或 IGF-I 诱导的这些细胞中的 HA 合成和 Akt 磷酸化，表明 TSH 受体和 IGF-IR 信号可能在 GO 眼眶中密切相关。
