Kumar Seema, Coenen Michael, Iyer Seethalakshmi, Bahn Rebecca S
1 Division of Pediatric Endocrinology and Metabolism, Mayo Clinic , Rochester, Minnesota.
2 Division of Endocrinology, Diabetes, Metabolism and Nutrition, Mayo Clinic , Rochester, Minnesota.
Thyroid. 2015 Oct;25(10):1145-50. doi: 10.1089/thy.2015.0254. Epub 2015 Aug 19.
Activation of thyrotropin receptor (TSHR) and/or insulin-like growth factor (IGF-1) receptor (IGF-1R) enhances HA production and adipogenesis in orbital fibroblasts from patients with Graves' ophthalmopathy (GO) and recapitulates the tissue remodeling characteristic of the orbit in GO. A functional relationship between TSHR and IGF-1R has long been postulated, and recently bidirectional crosstalk between the receptors in GO fibroblasts was demonstrated. Because the transcription factor Forkhead box O-1 (FOXO1) was recently shown to be a critical downstream mediator of TSH and IGF-1 effects on thyrocyte proliferation, studies were designed to determine whether FOXO1 might similarly act as a common mediator of M22, a stimulatory TSHR antibody (TSAb), and IGF-1 in GO orbital fibroblasts.
FOXO1 mRNA and protein were measured in orbital tissue specimens derived from normal individuals and patients with GO. In addition, the control of FOXO1 cellular localization was investigated using quantitative Western blotting of fractionated cell lysates from orbital fibroblasts treated with M22 and/or IGF-1 with or without specific TSHR, IGF-1R, or PI3K/AKT1/2 inhibitors.
Significantly lower levels of both FOXO1 mRNA and protein were found in GO orbital tissue specimens compared with normal orbital tissues (M = 39%, p = 0.043; M = 46.4%; p = 0.028, respectively). In addition, treatment of GO orbital cultures with M22, IGF-1, or M22 plus IGF-1 increased cytoplasmic FOXO1 compared with control (1.63-fold, p = 0.008; 1.68-fold, p = 0.001; 1.61-fold, p ≤ 0.001, respectively) and decreased nuclear FOXO1 (M = 28%, p = 0.002; M = 38%, p ≤ 0.001; M = 35%, p = 0.007, respectively). These effects were inhibited by co-treatment with the respective, but not the opposite, receptor antagonist. AKT inhibition of M22 or IGF-1-treated cultures was found to increase nuclear (1.4-fold, p = 0.026; 1.3-fold, p = 0.001, respectively) and decrease cytoplasmic (24.2%, p = 0.001; 36%, p = 0.004, respectively) FOXO1 localization.
These data point to FOXO1 as an important mediator of TSAb and IGF-1 action via their cognate receptors in GO orbital fibroblasts. These findings provide a link between the low FOXO1 protein levels demonstrated in GO orbital tissue and the tissue remodeling characteristic of GO, and suggest novel therapy for GO aimed at increasing nuclear expression of FOXO1 in GO target cells.
促甲状腺激素受体（TSHR）和/或胰岛素样生长因子（IGF-1）受体（IGF-1R）的激活可增强Graves眼病（GO）患者眼眶成纤维细胞中的透明质酸生成和脂肪生成，并概括了GO眼眶组织重塑的特征。TSHR与IGF-1R之间的功能关系早已被推测，最近在GO成纤维细胞中证实了这两种受体之间的双向串扰。由于转录因子叉头框O-1（FOXO1）最近被证明是促甲状腺激素和IGF-1对甲状腺细胞增殖影响的关键下游介质，因此设计了研究来确定FOXO1是否可能同样作为刺激性TSHR抗体（TSAb）M22和IGF-1在GO眼眶成纤维细胞中的共同介质。
在来自正常个体和GO患者的眼眶组织标本中测量FOXO1 mRNA和蛋白质。此外，使用来自用M22和/或IGF-1处理的眼眶成纤维细胞的分级细胞裂解物的定量蛋白质印迹，研究了FOXO1细胞定位的控制，这些细胞裂解物使用或不使用特异性TSHR、IGF-1R或PI3K/AKT1/2抑制剂。
与正常眼眶组织相比，GO眼眶组织标本中FOXO1 mRNA和蛋白质水平均显著降低（分别为M = 39%，p = 0.043；M = 46.4%；p = 0.028）。此外，与对照相比，用M22、IGF-1或M22加IGF-1处理GO眼眶培养物会增加细胞质FOXO1（分别为1.63倍，p = 0.008；1.68倍，p = 0.001；1.61倍，p≤0.001）并降低细胞核FOXO1（分别为M = 28%，p = 0.002；M = 38%，p≤0.001；M = 35%，p = 0.007）。这些作用被与各自（而非相反）受体拮抗剂的联合处理所抑制。发现对M22或IGF-1处理的培养物进行AKT抑制可增加细胞核（分别为1.4倍，p = 0.026；1.3倍，p = 0.001）并降低细胞质（分别为24.2%，p = 0.001；36%，p = 0.004）FOXO1定位。
这些数据表明FOXO1是TSAb和IGF-1通过其在GO眼眶成纤维细胞中的同源受体发挥作用的重要介质。这些发现提供了GO眼眶组织中显示的低FOXO1蛋白水平与GO组织重塑特征之间的联系，并提示了旨在增加GO靶细胞中FOXO1核表达水平的GO新疗法。
