Thyroid Research Unit, Icahn School of Medicine at Mount Sinai, James J, Peters VA Medical Center, New York, NY, USA.
Thyroid Research Unit, Icahn School of Medicine at Mount Sinai, James J, Peters VA Medical Center, New York, NY, USA.
J Autoimmun. 2022 Jan;126:102746. doi: 10.1016/j.jaut.2021.102746. Epub 2021 Nov 18.
Graves' disease (GD) is associated with thyroid stimulating hormone (TSH) receptor (TSHR) antibodies of variable bioactivity. We have previously characterized "neutral" TSHR antibodies (N-TSHR-Abs) that bind to the hinge region of the TSHR ectodomain. We showed that an N-TSHR monoclonal antibody (mAb) failed to induce any G proteins to sustain survival signaling and lead to excessive stress and apoptosis. Furthermore, the addition of TSH, or the antioxidant N-acetyl-l-cysteine (NAC), rescued N-TSHR-mAb-induced apoptotic death. However, the detailed mechanisms of this rescue remained unclear.
Autophagy is activated in response to diverse stress related stimuli so we have, therefore, studied the autophagy response in rat thyroid cells (FRTL-5) during N-TSHR-mAb induced thyrocyte stress and apoptosis using the In Cell Western technique for quantitation along with immunocytochemistry.
Under starvation conditions with N-TSHR-mAb the addition of TSH or NAC prevented thyroid cell death by enhancing autophagy. This was evidenced by elevated levels of autophagy related proteins including beclin 1, LC3A, LC3B, ULK1, p62, and also activated pink and perkin mitophagy related proteins. The phenomenon was further confirmed by image analyses using Cyto-ID and Mito-ID autophagy detection systems. We also found that either TSH or NAC enhanced PKA, Akt, mTORC, AMPK, Sirtuins, PGC1α, NRF-2, mitofusin-2, TFAM and catalase in the N-TSHR-mAb stressed cells. Thus TSH or NAC restored cell survival signaling which reduced cell stress and enhanced mitochondrial biogenesis. The N-TSHR-mAb also activated cytochrome-C, Bax, caspase-9, caspase-3A, and had less effect on FADD or caspase-8 indicating activation of the intrinsic pathway for apoptosis.
These findings indicated that TSH or antioxidant can rescue thyroid cells from N-TSHR-mAb induced apoptosis via enhanced autophagy. These observations signify that N-TSHR-mAb in GD under low TSH conditions caused by the hyperthyroidism could be detrimental for thyrocyte survival which would be another factor able to precipitate ongoing autoinflammation.
Graves 病（GD）与甲状腺刺激激素（TSH）受体（TSHR）的各种生物活性抗体有关。我们之前已经描述了结合 TSHR 细胞外结构域铰链区的“中性”TSHR 抗体（N-TSHR-Abs）。我们表明，一种 N-TSHR 单克隆抗体（mAb）未能诱导任何 G 蛋白来维持存活信号，导致过度应激和细胞凋亡。此外，添加 TSH 或抗氧化剂 N-乙酰-l-半胱氨酸（NAC）可挽救 N-TSHR-mAb 诱导的凋亡死亡。然而，这种挽救的详细机制尚不清楚。
自噬是在应对各种与应激相关的刺激时被激活的，因此，我们使用 In Cell Western 技术定量检测和免疫细胞化学方法，研究了 N-TSHR-mAb 诱导的甲状腺细胞（FRTL-5）应激和凋亡过程中的自噬反应。
在 N-TSHR-mAb 饥饿条件下，添加 TSH 或 NAC 通过增强自噬来防止甲状腺细胞死亡。这一点可以通过升高自噬相关蛋白的水平来证明，包括 beclin 1、LC3A、LC3B、ULK1、p62，以及激活的 pink 和 perkin 线粒体自噬相关蛋白。这一现象还通过 Cyto-ID 和 Mito-ID 自噬检测系统的图像分析得到进一步证实。我们还发现，TSH 或 NAC 增强了 N-TSHR-mAb 应激细胞中的 PKA、Akt、mTORC、AMPK、Sirtuins、PGC1α、NRF-2、融合蛋白-2、TFAM 和过氧化氢酶。因此，TSH 或 NAC 恢复了细胞存活信号，降低了细胞应激并增强了线粒体生物发生。N-TSHR-mAb 还激活了细胞色素 C、Bax、半胱天冬酶-9、半胱天冬酶-3A，对 FADD 或半胱天冬酶-8 的影响较小，表明激活了细胞凋亡的内在途径。
这些发现表明，TSH 或抗氧化剂可以通过增强自噬来挽救 N-TSHR-mAb 诱导的甲状腺细胞凋亡。这些观察结果表明，GD 中的 N-TSHR-mAb 在由甲状腺功能亢进引起的低 TSH 条件下可能对甲状腺细胞的存活有害，这将是另一个能够引发持续自身炎症的因素。
