Lee Wing-Kee, Chakraborty Prabir K, Roussa Eleni, Wolff Natascha A, Thévenod Frank
Institute of Physiology & Pathophysiology, ZBAF, University of Witten/Herdecke, Witten, Germany.
Biochim Biophys Acta. 2012 Oct;1823(10):1864-76. doi: 10.1016/j.bbamcr.2012.06.003. Epub 2012 Jun 13.
Upon endoplasmic reticulum (ER) stress induction, cells endeavor to survive by engaging the adaptive stress response known as the unfolded protein response or by removing aggregated proteins via autophagy. Chronic ER stress culminates in apoptotic cell death, which involves induction of pro-apoptotic CHOP. Here, we show that bestrophin-3 (Best-3), a protein previously associated with Ca(2+)-activated Cl(-) channel activity, is upregulated by the ER stressors, thapsigargin (TG), tunicamycin (TUN) and the toxic metal Cd(2+). In cultured rat kidney proximal tubule cells, ER stress, CHOP and cell death were induced after 6h by Cd(2+) (25μM), TG (3μM) and TUN (6μM), were associated with increased cytosolic Ca(2+) and downstream formation of reactive oxygen species and attenuated by the Ca(2+) chelator BAPTA-AM (10μM), the antioxidant α-tocopherol (100μM), or overexpression of catalase (CAT). Immunofluorescence staining showed Best-3 expression in the plasma membrane, nuclei and intracellular compartments, though not in the ER, in cultured cells and rat kidney cortex sections. Best-3 mRNA was augmented by ER stress and signaled through increased Ca(2+), oxidative stress and ERK1/2 phosphorylation, because it was attenuated by α-tocopherol, CAT expression, BAPTA-AM, calmodulin kinase inhibitor calmidazolium (40μM), ERK1/2 inhibitor U0126 (10μM), and ERK1/2 RNAi. Knockdown of Best-3 resulted in decreased cell number consequentially of cell death, as determined by nuclear staining and PARP-1 cleavage. Furthermore, reduced ER stress-cell death by Best-3 overexpression is attributed to diminished CHOP. Since Best-3 overexpression did not affect upstream signaling pathways, we hypothesize that Best-3 possibly interferes with CHOP transcription. From our novel observations, we conclude that ERK1/2-dependent Best-3 activation regulates cell fate decisions during ER stress by suppressing CHOP induction and death.
在内质网（ER）应激诱导下，细胞通过启动称为未折叠蛋白反应的适应性应激反应或通过自噬清除聚集蛋白来努力存活。慢性内质网应激最终导致凋亡性细胞死亡，这涉及促凋亡因子CHOP的诱导。在此，我们表明，Bestrophin-3（Best-3），一种先前与Ca(2+)激活的Cl(-)通道活性相关的蛋白质，被内质网应激剂毒胡萝卜素（TG）、衣霉素（TUN）和有毒金属Cd(2+)上调。在培养的大鼠肾近端小管细胞中，Cd(2+)（25μM）、TG（3μM）和TUN（6μM）在6小时后诱导内质网应激、CHOP和细胞死亡，这与胞质Ca(2+)增加以及下游活性氧的形成有关，并被Ca(2+)螯合剂BAPTA-AM（10μM）、抗氧化剂α-生育酚（100μM）或过氧化氢酶（CAT）的过表达所减弱。免疫荧光染色显示，在培养细胞和大鼠肾皮质切片中，Best-3在质膜、细胞核和细胞内区室中表达，但在内质网中不表达。内质网应激使Best-3 mRNA增加，并通过增加Ca(2+)、氧化应激和ERK1/2磷酸化发出信号，因为它被α-生育酚、CAT表达、BAPTA-AM、钙调蛋白激酶抑制剂卡咪唑（40μM）、ERK1/2抑制剂U0126（10μM）和ERK1/2 RNAi所减弱。敲低Best-3导致细胞数量减少，这是由核染色和PARP-1裂解所确定的细胞死亡的结果。此外，Best-3过表达减少内质网应激诱导的细胞死亡归因于CHOP的减少。由于Best-3过表达不影响上游信号通路，我们推测Best-3可能干扰CHOP转录。根据我们的新观察结果，我们得出结论，ERK1/2依赖性Best-3激活通过抑制CHOP诱导和死亡来调节内质网应激期间的细胞命运决定。
