Llewellyn D H, Kendall J M, Sheikh F N, Campbell A K
Department of Medical Biochemistry, University of Wales College of Medicine, Health Park, Cardiff, Wales, U.K.
Biochem J. 1996 Sep 1;318 ( Pt 2)(Pt 2):555-60. doi: 10.1042/bj3180555.
Calreticulin is now considered to be a multifunctional Ca(2+)-binding protein. Its primary role is as a Ca2+ storage protein within the lumen of the endoplasmic reticulum (ER), where it also seems to assist in the correct folding and assembly of proteins. We have investigated whether agents that affect these processes can alter calreticulin expression in HeLa cells. Perturbation of intracellular Ca2+ levels by prolonged exposure to either thapsigargin or ionomycin induced calreticulin mRNA, both in the presence and absence of extracellular Ca2+, consistent with the proposal that sustained depletion of the ER Ca2+ store can trigger these increases. The mechanism underlying the induction seems to be transcriptional up-regulation as both agents increased calreticulin promoter-driven firefly luciferase expression in transfected cells to the same degree as the observed increases in calreticulin mRNA. Experiments with a truncated promoter construct showed that the sequences that confer this inducibility reside within the 225 bp immediately upstream of the putative major transcriptional start site. We also examined the effect of tunicamycin, which inhibits N-linked glycosylation in the ER thereby interfering with protein processing. This caused increases in calreticulin mRNA greater than those with either thapsigargin or ionomycin, but failed to transactivate the calreticulin promoter. Thus either additional cis sequences that reside outside our promoter region are necessary for transcriptional activation by tunicamycin, or the increases in calreticulin mRNA occur post-transcriptionally. This suggests that there are probably different mechanisms by which calreticulin expression can be induced in response to agents that affect normal ER functioning.
钙网蛋白现在被认为是一种多功能的钙离子结合蛋白。它的主要作用是作为内质网（ER）腔内的一种钙离子储存蛋白，在那里它似乎还协助蛋白质的正确折叠和组装。我们研究了影响这些过程的试剂是否能改变HeLa细胞中钙网蛋白的表达。长时间暴露于毒胡萝卜素或离子霉素会扰乱细胞内钙离子水平，无论细胞外有无钙离子，都会诱导钙网蛋白mRNA的产生，这与内质网钙离子储存持续耗竭可引发这些增加的观点一致。诱导的潜在机制似乎是转录上调，因为这两种试剂在转染细胞中均使钙网蛋白启动子驱动的萤火虫荧光素酶表达增加，且增加程度与观察到的钙网蛋白mRNA增加程度相同。用截短的启动子构建体进行的实验表明，赋予这种诱导性的序列位于假定的主要转录起始位点上游紧邻的225 bp内。我们还研究了衣霉素的作用，它抑制内质网中的N-连接糖基化，从而干扰蛋白质加工。这导致钙网蛋白mRNA的增加幅度大于毒胡萝卜素或离子霉素，但未能激活钙网蛋白启动子。因此，要么衣霉素转录激活需要位于我们的启动子区域之外的其他顺式序列，要么钙网蛋白mRNA的增加发生在转录后。这表明，对于影响内质网正常功能的试剂，可能存在不同的机制来诱导钙网蛋白的表达。
