Endoplasmic Reticulum stress and thyroid cell death

摘要 甲状腺激素甲状腺素（T4）的有效产生需要甲状腺球蛋白（Tg）的碘化， 在内质网（ER）中合成，并进行折叠和运输到甲状腺滤泡腔 其中发生碘化。内源性T4是100%由甲状腺细胞产生的，并且在Tg中T4的形成是不稳定的。 在进化过程中保持了5亿年以上。迄今为止，数百种不同的致病性突变， 已发现TG基因引起人类先天性甲状腺功能减退。遗传性甲状腺功能减退症 纯合（或复合杂合）TG突变是罕见的，但单一致病性TG的频率 等位基因在人群中非常常见（≥ 1：200个体）。迄今为止研究的所有Tg突变体都是错误折叠的 蛋白质被困在内质网，导致内质网应激。我们最近报道，在未经治疗的TG纯合子中， 内源性甲状腺激素合成仍然发生，尽管Tg从ER输出失败。机制 涉及甲状腺上皮细胞死亡，死亡的甲状腺细胞被挤出到甲状腺滤泡的管腔中，导致 毛囊腔中细胞的解体和碘化。Tg蛋白在大肠杆菌中的大量表达 甲状腺细胞，以及人群中极高频率的杂合TG突变，现在让我们 问甲状腺上皮细胞死亡是否也可能是一个（迄今未被怀疑的）普遍特征 在TG突变型的简单杂合子个体中。这可以很容易地在动物模型中进行测试。 此外，在纯合子rdw/rdw（Tg-G2298 R）大鼠中，甲状腺细胞死亡长期以来被认为是一个因素， 阻止甲状腺肿的生长。值得注意的是，我们现在发现，在先天性甲状腺肿小鼠（cog/cog，Tg-L2263 P）中， 也是普遍的甲状腺细胞死亡（尽管如此，甲状腺的生长速度超过了死亡）。在目前的提案中， 1)我们已经设计了一个rdw/rdw基因敲入小鼠，并将直接比较细胞生长和细胞死亡， 在cog/cog小鼠中观察到。2)我们将研究这两种模型的简单杂合子中的甲状腺细胞死亡， 具有完全正常的血清T4水平，但表现出甲状腺细胞ER应激。3)由于杂合突变体Tg错误折叠， 在ER中，它可以至少部分地通过ER相关蛋白降解（ERAD）而降解。我们发现 Tg的ERAD缺陷导致伴有炎性细胞浸润和卵泡退化的甲状腺炎。4)挤出 甲状腺细胞进入滤泡腔可能使细胞的非顶端部分暴露于周围的碘化， 滤泡细胞作为细胞毒性的贡献机制。为了测试这一点，我们将检查TG-KO小鼠模型， 其中促甲状腺激素刺激的过度生长（而不是ER应激）驱使甲状腺细胞进入卵泡腔， 基底外侧质膜对管腔的氧化/碘化环境敏感， 引发甲状腺细胞死亡总之，本提案的所有目标都将强调ER压力的问题- 甲状腺细胞死亡的依赖和独立机制。

ABSTRACT Efficient generation of the thyroid hormone thyroxine (T4) requires the iodination of thyroglobulin (Tg), which is synthesized in the endoplasmic reticulum (ER) and undergoes folding and trafficking to the thyroid follicle lumen wherein iodination takes place. Endogenous T4 is 100% produced by thyrocytes, and the formation of T4 in Tg has been conserved in evolution for ≥ 500 million years. To date, hundreds of different pathogenic mutations of the TG gene have been found to cause congenital hypothyroidism in humans. Genetic hypothyroidism from homozygous (or compound heterozygous) TG mutation is rare, but the frequency of a single pathogenic TG allele in the human population is very common (≥ 1:200 individuals). All Tg mutants studied to date are misfolded proteins trapped in the ER, causing ER stress. We recently reported that in untreated TG homozygotes, endogenous thyroid hormone synthesis still occurs despite failure of Tg export from the ER. The mechanism involves thyroid epithelial cell death, with extrusion of dead thyrocytes into the lumen of thyroid follicles, leading to the disintegration and iodination of those cells in the follicle lumen. Massive expression of Tg protein in thyrocytes, and the extremely high frequency of heterozygous TG mutations in the population, now leads us to ask whether thyroid epithelial cell death may also be a (heretofore unsuspected) widespread feature across individuals who are simple heterozygotes for mutant TG. This can be easily tested in animal models. Additionally, in homozygous rdw/rdw (Tg-G2298R) rats, thyroid cell death has long been recognized as a factor blocking goiter growth. Remarkably, we now find that in the congenital goiter mouse (cog/cog, Tg-L2263P), there is also widespread thyroid cell death (that is nevertheless outpaced by thyroid growth). In the current proposal, 1) we have engineered a rdw/rdw knockin mouse and will directly compare cell growth and cell death to that seen in cog/cog mice. 2) We will examine thyroid cell death in simple heterozygotes of these two models, which have perfectly normal serum T4 levels but exhibit thyrocyte ER stress. 3) As heterozygous mutant Tg is misfolded in the ER, it may be degraded at least in part by ER-associated protein degradation (ERAD). We find that defective ERAD of Tg leads to a thyroiditis with infiltration of inflammatory cells and follicle involuion. 4) Extrusion of thyrocytes into the follicle lumen may expose non-apical portions of the cell to iodination by surrounding follicular cells as a contributing mechanism of cytotoxicity. To test this, we will examine a TG-KO mouse model in which TSH-stimulated overgrowth (and not ER stress) drives thyrocytes into the follicle lumen, exposing the sensitive basolateral plasma membrane to the oxidative / iodination environment of the lumen, where it may trigger thyroid cell death. In summary, all of the Aims of this proposal will highlight questions of ER stress- dependent and independent mechanisms of thyrocyte death.