The large secretory glycoprotein thyroglobulin is the primary translation product of thyroid follicular cells. This difficult-to-fold protein is susceptible to structural alterations that disable export of the misfolded thyroglobulin from the endoplasmic reticulum (ER), which is a known cause of congenital hypothyroidism characterized by severe chronic thyrocyte ER stress. Nevertheless, individuals with this disease commonly grow a goiter, indicating thyroid cell survival and adaptation. To model these processes, here we continuously exposed rat PCCL3 thyrocytes to tunicamycin, which causes a significant degree of ER stress that is specifically attributable to thyroglobulin misfolding. We found that, in response, PCCL3 cells down-regulate expression of the "tunicamycin transporter" (major facilitator superfamily domain containing-2A, Mfsd2a). Following CRISPR/Cas9-mediated Mfsd2a deletion, PCCL3 cells could no longer escape the chronic effects of high-dose tunicamycin, as demonstrated by persistent accumulation of unglycosylated thyroglobulin; nevertheless, these thyrocytes survived and grew. A proteomic analysis of these cells adapted to chronic ER protein misfolding revealed many hundreds of up-regulated proteins, indicating stimulation of ER chaperones, oxidoreductases, stress responses, and lipid biosynthesis pathways. Further, we noted increased phospho-AMP-kinase, suggesting up-regulated AMP-kinase activity, and decreased phospho-S6-kinase and protein translation, suggesting decreased mTOR activity. These changes are consistent with conserved cell survival/adaptation pathways. We also observed a less-differentiated thyrocyte phenotype with decreased PAX8, FOXE1, and TPO protein levels, along with decreased thyroglobulin mRNA levels. In summary, we have developed a model of thyrocyte survival and growth during chronic continuous ER stress that recapitulates features of congenital hypothyroid goiter caused by mutant thyroglobulin.

中文翻译：

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甲状腺细胞球蛋白折叠错误导致甲状腺细胞存活并适应慢性内质网应激。

大分泌型糖蛋白甲状腺球蛋白是甲状腺滤泡细胞的主要翻译产物。这种难以折叠的蛋白质易受结构改变的影响，从而无法将错误折叠的甲状腺球蛋白从内质网（ER）输出，这是先天性甲状腺功能低下的已知原因，其特征是严重的慢性甲状腺细胞ER应激。然而，患有这种疾病的个体通常会长出甲状腺肿，表明甲状腺细胞存活和适应。为了对这些过程进行建模，在这里我们连续将大鼠PCCL3甲状腺细胞暴露于衣霉素，从而引起显着程度的ER应激，具体是由于甲状腺球蛋白错折叠引起的。我们发现，作为回应，PCCL3细胞下调了“衣霉素转运蛋白”（主要的促进子超家族域，含有2A，Mfsd2a）。CRISPR / Cas9介导的Mfsd2a缺失后，PCCL3细胞不再能够逃脱高剂量衣霉素的慢性作用，这表现为未糖基化甲状腺球蛋白的持续积累。然而，这些甲状腺细胞存活并生长。对这些细胞进行蛋白质组学分析后发现，它们适应了慢性ER蛋白的错误折叠，揭示了数百种上调的蛋白，表明刺激了ER伴侣，氧化还原酶，应激反应和脂质生物合成途径。此外，我们注意到磷酸-AMP激酶增加，表明上调了AMP激酶活性，而磷酸-S6-激酶和蛋白质翻译减少，表明mTOR活性降低。这些变化与保守的细胞存活/适应途径一致。我们还观察到甲状腺细胞表型分化程度较低，PAX8，FOXE1，和TPO蛋白水平，以及甲状腺球蛋白mRNA水平下降。总而言之，我们已经建立了慢性持续内质网应激期间甲状腺细胞存活和生长的模型，该模型概括了由突变甲状腺球蛋白引起的先天性甲状腺功能低下甲状腺肿的特征。