Thyroid Research Unit, Mount Sinai School of Medicine, James J. Peters Veterans Affairs Medical Center, New York, New York 10468, USA.
Endocrinology. 2010 Nov;151(11):5537-49. doi: 10.1210/en.2010-0424. Epub 2010 Sep 15.
TSH receptor (TSHR) antibodies (Abs) may be stimulating, blocking, or neutral in their functional influences and are found in patients with autoimmune thyroid disease, especially Graves' disease (GD). Stimulators are known to activate the thyroid epithelial cells via both Gs- and Gq-coupled signaling pathways, whereas blockers inhibit the action of TSH and may act as weak agonists. However, TSHR neutral Abs do not block TSH binding and are unable to induce cAMP via Gsα. The importance of such neutral Abs in GD remains unclear because their functional consequence has been assumed to be zero. We hypothesized that: 1) neutral TSHR Abs are more common to GD than generally recognized; 2) they may induce distinct signaling imprints at the TSHR not seen with TSH itself; and 3) these signaling events may alter cellular function. To evaluate these hypotheses, we first confirmed the presence of neutral TSHR Abs in sera from patients with GD and then, using mouse and hamster neutral TSHR monoclonal Abs (N-mAbs) performed detailed signaling studies, including a proteomic Ab array, with rat thyrocytes (FRTL-5) as targets. This allowed us to examine a battery of signaling cascades and their downstream effectors. Neutral TSHR Abs were indeed frequently present in sera from patients with GD. Sixteen of 27 patients (59%) had detectable neutral TSHR Abs by competition assay with N-mAbs. On examining signaling cascades, we found that N-mAbs induced signal transduction, primarily via the protein kinase A II cascade. In addition to the activation of phosphatidylinositol 3K/Akt, N-mAbs, unlike TSH, had the ability to exclusively activate the mammalian target of rapamycin/p70 S6K, nuclear factor-κB, and MAPK-ERK1/2/p38α signaling cascades and their downstream effectors p90 ribosomal kinase/MAPK-interacting kinase-1/mitogen and stress-activated kinase-1 and N-mAbs activated all forms of protein kinase C isozymes. To define the downstream effector mechanisms produced by these signaling cascades, cytokine production, proliferation, and apoptosis in thyrocytes were investigated. Although N-mAbs produced less cytokines and proliferation compared with TSH, they had the distinction of inducing thyroid cell apoptosis under the experimental conditions used. When dissecting out possible mechanisms of apoptosis, we found that activation of multiple oxidative stress markers was the primary mechanism orchestrating the death signals. Therefore, using oxidative stress-induced apoptosis, N-mAbs may be capable of exacerbating the autoimmune response in GD via apoptotic cells inducing antigen-driven mechanisms. This may help explain the inflammatory nature of this common disorder.
促甲状腺激素受体 (TSHR) 抗体 (Abs) 在功能影响上可能具有刺激、阻断或中和作用，存在于自身免疫性甲状腺疾病患者中，尤其是 Graves 病 (GD)。已知刺激剂通过 Gs- 和 Gq 偶联信号通路激活甲状腺上皮细胞，而阻滞剂抑制 TSH 的作用并可能作为弱激动剂发挥作用。然而，TSHR 中性 Abs 不阻断 TSH 结合，并且不能通过 Gsα 诱导 cAMP。这些中性 Abs 在 GD 中的重要性尚不清楚，因为它们的功能后果被假定为零。我们假设：1）与普遍认为的相比，GD 中更常见的是中性 TSHR Abs；2）它们可能在 TSHR 上诱导与 TSH 本身不同的信号印记；3）这些信号事件可能改变细胞功能。为了验证这些假设，我们首先在 GD 患者的血清中证实了中性 TSHR Abs 的存在，然后使用小鼠和仓鼠中性 TSHR 单克隆 Abs (N-mAbs) 对大鼠甲状腺细胞 (FRTL-5) 进行了详细的信号研究。这使我们能够检查一系列信号级联及其下游效应器。中性 TSHR Abs 确实经常存在于 GD 患者的血清中。通过与 N-mAbs 的竞争测定，27 名患者中有 16 名（59%）可检测到中性 TSHR Abs。在检查信号级联时，我们发现 N-mAbs 主要通过蛋白激酶 A II 级联诱导信号转导。除了激活磷脂酰肌醇 3K/Akt 外，N-mAbs 与 TSH 不同，具有激活哺乳动物雷帕霉素靶蛋白/p70 S6K、核因子-κB 和 MAPK-ERK1/2/p38α 信号级联及其下游效应物 p90 核糖体激酶/MAPK 相互作用激酶-1/有丝分裂原和应激激活激酶-1 的能力，并且 N-mAbs 激活了所有形式的蛋白激酶 C 同工酶。为了定义这些信号级联产生的下游效应机制，研究了甲状腺细胞中的细胞因子产生、增殖和凋亡。尽管 N-mAbs 与 TSH 相比产生的细胞因子和增殖较少，但它们在使用的实验条件下具有诱导甲状腺细胞凋亡的区别。当剖析可能的凋亡机制时，我们发现激活多种氧化应激标志物是协调死亡信号的主要机制。因此，通过诱导细胞凋亡，N-mAbs 可能通过诱导抗原驱动机制使 GD 中的自身免疫反应恶化。这可以帮助解释这种常见疾病的炎症性质。
