Leonardi Antonio, Vito Pasquale, Mauro Claudio, Pacifico Francesco, Ulianich Luca, Consiglio Eduardo, Formisano Silvestro, Di Jeso Bruno
Dipartimento di Biologia e Patologia Cellulare e Molecolare, Federico II, University of Naples, 80100 Naples, Italy.
Endocrinology. 2002 Jun;143(6):2169-77. doi: 10.1210/endo.143.6.8825.
Perturbing the endoplasmic reticulum homeostasis of thyroid cell lines with thapsigargin, a specific inhibitor of the sarcoendoplasmic reticulum Ca(2+) adenosine triphosphatases, and tunicamycin, an inhibitor of the N-linked glycosylation, blocked Tg in the endoplasmic reticulum. This event was signaled outside the endoplasmic reticulum and resulted in activation of the c-Jun N-terminal kinase (JNK)/stress-activated protein kinase and nuclear factor-kappa B (NF-kappa B) stress response pathways. Activation of the JNK/stress-activated protein kinase signaling pathway was assessed by measuring the amount of phospho-JNK and the activity of JNK by kinase assays. Activation of the NF-kappa B signaling pathway was assessed by measuring the level of inhibitory subunit I kappa B alpha, DNA binding, and transcriptional activity of NF-kappa B. Cycloheximide treatment, at a dose able to profoundly inhibit protein synthesis in FRTL-5 cells, obliterated the decrease in the level of the inhibitory subunit I kappa B alpha produced by thapsigargin and tunicamycin. Therefore, protein synthesis was required to generate a signal from stressed endoplasmic reticulum. This substantiates the hypothesis that endoplasmic reticulum retention of newly synthesized Tg and other cargo (secretory and membrane) proteins functions upstream of signal activation. Dominant negative TNF receptor-associated factor 2 (TRAF2) inhibited activation of NF-kappa B, which was also inhibited in embryonic fibroblasts derived from TRAF2(-/-) mice, respect to their normal counterpart. These data extend the recent demonstration that TRAF2 mediated JNK activation in response to endoplasmic reticulum stress and strongly strengthened the idea that endogenous stress signals initiated in the endoplasmic reticulum proceed by a pathway similar to that initiated by plasma membrane receptors in response to extracellular signals.
用毒胡萝卜素（肌浆内质网Ca(2+)三磷酸腺苷酶的特异性抑制剂）和衣霉素（N-连接糖基化抑制剂）扰乱甲状腺细胞系的内质网稳态，会使甲状腺球蛋白（Tg）在内质网中滞留。这一事件在内质网外发出信号，并导致c-Jun氨基末端激酶（JNK）/应激激活蛋白激酶和核因子-κB（NF-κB）应激反应途径的激活。通过激酶测定法测量磷酸化JNK的量和JNK的活性来评估JNK/应激激活蛋白激酶信号通路的激活。通过测量抑制性亚基IκBα的水平、DNA结合以及NF-κB的转录活性来评估NF-κB信号通路的激活。用能深刻抑制FRTL-5细胞中蛋白质合成的剂量的环己酰亚胺处理，消除了毒胡萝卜素和衣霉素所导致的抑制性亚基IκBα水平的降低。因此，蛋白质合成是从应激内质网产生信号所必需的。这证实了新合成的Tg和其他货物（分泌性和膜性）蛋白在内质网中的滞留作用于信号激活上游的假说。显性负性肿瘤坏死因子受体相关因子2（TRAF2）抑制了NF-κB的激活，在源自TRAF2(-/-)小鼠的胚胎成纤维细胞中相对于其正常对应物也观察到了NF-κB激活的抑制。这些数据扩展了最近关于TRAF2介导内质网应激反应中JNK激活的证明，并有力地强化了这样一种观点，即内质网中引发的内源性应激信号通过一条类似于质膜受体响应细胞外信号所引发的途径进行传递。
