Laboratory of Endocrinology and Receptor Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health Bethesda, MD, United States.
Metabolic Disease Branch, National Institutes of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD, United States.
Front Endocrinol (Lausanne). 2020 Apr 30;11:276. doi: 10.3389/fendo.2020.00276. eCollection 2020.
Thyrotropin hormone (TSH) was reported to exhibit biphasic regulation of cAMP production in human thyroid slices; specifically, upregulation at low TSH doses transitioning to inhibition at high doses. We observed this phenomenon in HEK293 cells overexpressing TSH receptors (TSHRs) but in only 25% of human thyrocytes (hThyros) . Because TSHR expression in hThyros was low, we tested the hypothesis that high, levels of TSHRs were needed for biphasic cAMP regulation. We increased expression of TSHRs by infecting hThyros with adenoviruses expressing human TSHR (AdhTSHR), measured TSH-stimulated cAMP production and TSHR homodimerization. TSHR mRNA levels in hThyros were 100-fold lower than in human thyroid tissue. AdhTSHR infection increased TSHR mRNA expression to levels found in thyroid tissue and flow cytometry showed that cell-surface TSHRs increased more than 15-fold. Most uninfected hThyro preparations exhibited monotonic cAMP production. In contrast, most hThyro preparations infected with AdhTSHR expressing TSHR at levels exhibited biphasic TSH dose responses. Treatment of AdhTSHR-infected hThyros with pertussis toxin resulted in monotonic dose response curves demonstrating that lower levels of cAMP production at high TSH doses were mediated by G/G proteins. Proximity ligation assays confirmed that AdhTSHR infection markedly increased the number of TSHR homodimers. We conclude that levels of TSHRs as homodimers are needed for hThyros to exhibit biphasic TSH regulation of cAMP production.
促甲状腺激素（TSH）被报道在人甲状腺切片中表现出 cAMP 产生的双相调节；具体来说，在低 TSH 剂量下上调，在高剂量下抑制。我们在过表达 TSH 受体（TSHRs）的 HEK293 细胞中观察到了这种现象，但在仅 25%的人甲状腺细胞（hThyros）中观察到了这种现象。由于 hThyros 中的 TSHR 表达较低，我们测试了高浓度 TSHRs 对于双相 cAMP 调节是必需的假设。我们通过感染表达人 TSHR 的腺病毒（AdhTSHR）来增加 hThyros 中的 TSHR 表达，测量 TSH 刺激的 cAMP 产生和 TSHR 同源二聚体化。hThyros 中的 TSHR mRNA 水平比甲状腺组织低 100 倍。AdhTSHR 感染增加了 TSHR mRNA 表达水平，达到甲状腺组织中的水平，流式细胞术显示细胞表面 TSHR 增加了 15 倍以上。大多数未感染的 hThyro 制剂表现出单调的 cAMP 产生。相比之下，大多数用表达 TSHR 的 AdhTSHR 感染的 hThyro 制剂以双相 TSH 剂量反应表现出双相 TSH 剂量反应。用百日咳毒素处理感染 AdhTSHR 的 hThyros 导致单调的剂量反应曲线，表明在高 TSH 剂量下较低的 cAMP 产生是由 G/G 蛋白介导的。接近连接测定证实 AdhTSHR 感染显著增加了 TSHR 同源二聚体的数量。我们得出结论，作为同源二聚体的 TSHR 水平是 hThyro 表现出双相 TSH 调节 cAMP 产生所必需的。
