Laboratory of Endocrinology and Receptor Biology, National Institute of Diabetes and Digestive and Kidney Diseases , National Institutes of Health, Bethesda, Maryland.
Thyroid. 2013 Nov;23(11):1484-9. doi: 10.1089/thy.2013.0089. Epub 2013 Sep 20.
It is controversial whether persistent signaling by the thyrotropin (TSH) receptor (TSHR) is cell-type specific. We reported persistent TSHR signaling in human embryonic kidney 293 (HEK293) cells expressing human TSHRs (HEK-TSHRs), whereas another group reported persistent signaling in mouse thyroid follicles but not in HEK293 cells. Herein, we test this hypothesis directly.
We used two methods to measure persistent signaling in HEK-TSHRs and confirm our previous observations. In Method 1, we used a chemiluminescent immunoassay to measure intracellular cAMP accumulation over 30-60 min by adding a phosphodiesterase inhibitor to the incubation medium. In Method 2, we used an intracellular biosensor to record cAMP levels continuously.
Using Method 1, we show that TSHR signals persistently in human thyrocytes and human osteosarcoma U2OS-TSHR cells. Using Method 1 in HEK-TSHRs, we show that after 5 min, the phosphodiesterase inhibitor isobutylmethylxanthine (IBMX) increases cAMP to 2.5 pmol/well, TSH increases cAMP to 1.6 pmol/well, but IBMX added 30 min after TSH withdrawal increases cAMP to 105 pmol/well. Using Method 2 in HEK-TSHRs, we confirm that without IBMX, TSH causes a transient increase in cAMP and 30 min after TSH withdrawal, IBMX increases cAMP in cells pretreated with TSH more rapidly and to a higher level than IBMX added to cells not pre-exposed to TSH. Lastly, using Method 2, we show that in HEK-TSHRs phosphodiesterases types 3 and 4 are involved in degrading cAMP as the specific inhibitors Rolipram and Milrinone expose persistent TSHR signaling.
We conclude that persistent TSHR activation occurs in human thyrocytes, U2OS-TSHR cells and HEK-TSHRs; it is not cell-type specific but is revealed by inhibiting phosphodiesterases.
促甲状腺激素受体（TSHR）的持续信号是否具有细胞类型特异性存在争议。我们报道了在表达人 TSHR 的人胚肾 293（HEK293）细胞（HEK-TSHR）中存在持续的 TSHR 信号，而另一组则报道在小鼠甲状腺滤泡中存在持续的信号，但在 HEK293 细胞中不存在。在此，我们直接检验这一假说。
我们使用两种方法来测量 HEK-TSHR 中的持续信号，并证实了我们之前的观察结果。在方法 1 中，我们使用化学发光免疫测定法通过在孵育培养基中添加磷酸二酯酶抑制剂来测量 30-60 分钟内的细胞内 cAMP 积累。在方法 2 中，我们使用细胞内生物传感器连续记录 cAMP 水平。
使用方法 1，我们表明 TSHR 在人甲状腺细胞和人骨肉瘤 U2OS-TSHR 细胞中持续发出信号。在 HEK-TSHR 中使用方法 1，我们表明在 5 分钟后，磷酸二酯酶抑制剂异丁基甲基黄嘌呤（IBMX）将 cAMP 增加到 2.5 pmol/孔，TSH 将 cAMP 增加到 1.6 pmol/孔，但在 TSH 去除 30 分钟后添加 IBMX 将 cAMP 增加到 105 pmol/孔。在 HEK-TSHR 中使用方法 2，我们确认在没有 IBMX 的情况下，TSH 会导致 cAMP 的短暂增加，并且在 TSH 去除 30 分钟后，IBMX 会比 IBMX 预先添加到未暴露于 TSH 的细胞中更快地增加细胞内 cAMP 的水平。最后，使用方法 2，我们表明在 HEK-TSHR 中，磷酸二酯酶 3 和 4 型参与 cAMP 的降解，因为特异性抑制剂 Rolipram 和 Milrinone 暴露了持续的 TSHR 信号。
我们得出结论，持续的 TSHR 激活发生在人甲状腺细胞、U2OS-TSHR 细胞和 HEK-TSHR 中；它不是细胞类型特异性的，而是通过抑制磷酸二酯酶来揭示的。
