Liu H, Bowes R C, van de Water B, Sillence C, Nagelkerke J F, Stevens J L
W. Alton Jones Cell Science Center, Lake Placid, New York 12946, USA.
J Biol Chem. 1997 Aug 29;272(35):21751-9. doi: 10.1074/jbc.272.35.21751.
Activation of stress response genes can impart cellular tolerance to environmental stress. Iodoacetamide (IDAM) is an alkylating toxicant that up-regulates expression of hsp70 (Liu, H., Lightfoot, D. L., and Stevens, J. L. (1996) J. Biol. Chem. 271, 4805-4812) and grp78 in LLC-PK1 renal epithelial cells. Therefore, we used IDAM to determine the role of these genes in tolerance to toxic chemicals. Prior heat shock did not protect cells from IDAM but pretreatment with trans-4,5-dihydroxy-1,2-dithiane (DTTox), thapsigargin, or tunicamycin enhanced expression of the endoplasmic reticulum (ER) chaperones GRP78 and GRP94 and rendered cells tolerant to IDAM. Cells expressing a 524-base pair antisense grp78 fragment (pkASgrp78) had a diminished capacity to up-regulate grp78 and grp94 expression after ER stress. Protection against IDAM due to prior ER stress was also attenuated in pkASgrp78 cells suggesting that ER chaperones of the GRP family are critical for tolerance. Covalent binding of IDAM to cellular macromolecules and depletion of cellular thiols was similar in tolerant and naïve cells. However, DTTox pretreatment blocked the increases in cellular Ca2+ and lipid peroxidation observed after IDAM treatment. Overexpressing the ER Ca2+-binding protein calreticulin prevented IDAM-induced cell death, the rise in cytosolic Ca2+, and oxidative stress. Although activation of the ER stress response did not prevent toxicity due to Ca2+ influx, EGTA-AM and ruthenium red both blocked cell death suggesting that redistribution of intracellular Ca2+ to the mitochondria may be important in toxicity. The data support a model in which induction of ER stress proteins prevents disturbances of intracellular Ca2+ homeostasis, thus uncoupling toxicant exposure from oxidative stress and cell death. Multiple ER stress proteins are likely to be involved in this tolerance response.
应激反应基因的激活可赋予细胞对环境应激的耐受性。碘乙酰胺（IDAM）是一种烷基化毒物，可上调LLC-PK1肾上皮细胞中hsp70（Liu, H., Lightfoot, D. L., and Stevens, J. L. (1996) J. Biol. Chem. 271, 4805 - 4812）和grp78的表达。因此，我们使用IDAM来确定这些基因在对有毒化学物质耐受性中的作用。预先的热休克并不能保护细胞免受IDAM的影响，但用反式-4,5-二羟基-1,2-二硫烷（DTTox）、毒胡萝卜素或衣霉素预处理可增强内质网（ER）伴侣蛋白GRP78和GRP94的表达，并使细胞对IDAM产生耐受性。表达524个碱基对反义grp78片段（pkASgrp78）的细胞在ER应激后上调grp78和grp94表达的能力减弱。由于预先的ER应激而对IDAM的保护在pkASgrp78细胞中也减弱了，这表明GRP家族的ER伴侣蛋白对耐受性至关重要。IDAM与细胞大分子的共价结合以及细胞内硫醇的消耗在耐受性细胞和未处理细胞中相似。然而，DTTox预处理可阻断IDAM处理后观察到的细胞内Ca2+增加和脂质过氧化。过表达ER Ca2+结合蛋白钙网蛋白可防止IDAM诱导的细胞死亡、胞质Ca2+升高和氧化应激。尽管ER应激反应的激活并不能防止由于Ca2+内流引起的毒性，但EGTA-AM和钌红均能阻断细胞死亡，这表明细胞内Ca+向线粒体的重新分布可能在毒性中起重要作用。数据支持一种模型，即ER应激蛋白的诱导可防止细胞内Ca2+稳态的紊乱，从而使毒物暴露与氧化应激和细胞死亡脱钩。多种ER应激蛋白可能参与这种耐受性反应。
