We studied whether bovine pituitary thyrotropin (bTSH) or human recombinant thyrotropin (rhTSH) stimulated p42/p44 mitogen-activated protein kinases (MAPKs) in Chinese hamster ovary cells expressing human thyrotropin receptor (CHO-hTSHR cells). We show that p42/p44 MAPK phosphorylation was induced by both TSH preparations at similar levels in CHO-hTSHR cells and in wild-type CHO cells. In contrast, cyclic adenosine monophosphate (cAMP) production was stimulated by TSH only in CHO-hTSHR cells, demonstrating that p42/p44 MAPK stimulation was independent of the TSH receptor. Moreover, similar results were obtained with two other cell lines: the FRTL-5 thyroid cell line and the CCL39 fibroblast cell line. Maximal stimulation of p42/p44 MAPK phosphorylation was observed after a 5- to 10-minute incubation with bTSH and rhTSH preparations. At this time, the phosphorylation of GST-Elk1 was also increased in a time- and concentration-dependent manner by bTSH preparations. The phosphorylation of p42/p44 MAPKs was abolished by PD 98059 and GF 109203X, indicating the involvement of MAPK kinases (MEK 1/2) and protein kinase C. In contrast, the activation of p42/p44 MAPKs was insensitive to H89, to cholera toxin and to pertussis toxin. These data suggest that the protein kinase A pathway was not implicated in p42/p44 MAPK activation by TSH preparations. Moreover, Gs or Gi/Go proteins do not appear to participate in p42/p44 MAPK activation. We also showed that these TSH preparations failed to induce activation of c-Jun NH2 terminal kinase. We therefore conclude that the commercial TSH preparations used in this study contained factor(s) responsible for the specific activation of p42/p44 MAPKs by a TSH receptor-independent mechanism.

译文

我们研究了牛垂体促甲状腺激素 (bTSH) 或人重组促甲状腺激素 (rhTSH) 是否刺激了表达人促甲状腺激素受体 (CHO-hTSHR细胞) 的中国仓鼠卵巢细胞中的p42/p44丝裂原活化蛋白激酶 (MAPKs)。我们显示,在CHO-hTSHR细胞和野生型CHO细胞中,两种TSH制剂均以相似水平诱导p42/p44 MAPK磷酸化。相反,仅在CHO-hTSHR细胞中,TSH刺激了环磷酸腺苷 (cAMP) 的产生,表明p42/p44 MAPK刺激独立于TSH受体。此外,用另外两种细胞系获得了类似的结果: FRTL-5甲状腺细胞系和CCL39成纤维细胞系。与bTSH和rhTSH制剂孵育5至10分钟后,观察到p42/p44 MAPK磷酸化的最大刺激。此时,GST-Elk1的磷酸化也通过bTSH制剂以时间和浓度依赖的方式增加。PD 98059和GF 109203X消除了p42/p44 MAPKs的磷酸化,表明MAPK激酶 (MEK 1/2) 和蛋白激酶C参与。相反,p42/p44 MAPKs的激活对H89,霍乱毒素和百日咳毒素不敏感。这些数据表明,蛋白激酶A途径与TSH制剂的p42/p44 MAPK激活无关。此外,Gs或Gi/Go蛋白似乎不参与p42/p44 MAPK激活。我们还表明,这些TSH制剂未能诱导c 6月NH2末端激酶的激活。因此,我们得出结论,本研究中使用的商业TSH制剂包含负责通过TSH受体非依赖性机制特异性激活p42/p44 MAPKs的因子。

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