Stress gene induction in mammalian cells

本研究旨在评估哺乳动物对重金属损伤的应激反应及其在适应环境化学物质不利条件中的作用。我们的重点将是在哺乳动物细胞中通过醋酸铅（Pb）和盐酸诱导内质网（ER）伴侣糖调节蛋白78 （GRP78）。这些金属与超级基金场址存在的其他污染物一起，对人类和动物的健康构成严重风险。由于复杂化学混合物中毒理学相互作用的性质仍然不明确，因此将进行实验以检查对环境伤害的适应性反应的分子基础。具体来说，我们将验证两个假设：GRP78在金属诱导的信号转导改变后受到转录调节，并且这种反应提供细胞保护以抵抗随后的化学损伤。参与细胞保护的基因调控机制将在大鼠系膜克隆细胞系SCC-1和大鼠胶质瘤细胞系C6中进行研究。治疗方案将模拟人体中与毒理学相关的暴露，在一段时间内以亚细胞毒性浓度反复给药醋酸铅和氯化汞。在第一个目标中，我们将确定金属挑战是否会扰乱Ca2+稳态、氧化还原状态和/或蛋白质磷酸化，从而导致GRP78的激活。将探讨基因诱导的转录和转录后机制的贡献，以及基因5'调控区内外源调控的顺式作用元件的作用。增加GRP78量的细胞保护作用。转录和转录后机制对基因诱导的贡献，以及外源调控的顺式作用元件在基因5'调控区内的作用将被检查。在金属处理的细胞中，增加GRP78量的细胞保护作用，通过测量Ca2+稳态、错误折叠的蛋白质积累、细胞因子释放和金属结合来证明。将监测超级基金场址化学混合物对后续损伤的细胞耐受性，以确定相关的毒理学相互作用。我们的方法将与成像、现场服务和分析核心以及该计划的项目2、5和6联系在一起。拟议的研究将共同提供有关超级基金地点的关键化学-生物相互作用的信息。

Studies are proposed in this application to evaluate the mammalian stress response to heavy metal injury and its role in the adaptation to adverse conditions posed by environmental chemicals. Our focus will be the induction of the endoplasmic reticulum (ER) chaperone glucose-regulated protein 78 (GRP78) in mammalian cells by lead (Pb) acetate and HgCl2. These metals in combination with other contaminants present at Superfund sites pose serious risks to human and animal health. Because the nature of toxicological interactions in complex chemical mixture remains poorly defined, experiments will be conducted to examine the molecular basis of the adaptive response to environmental injury. Specifically, we will test two hypothesis: that GRP78 is regulated transcriptionally following metal-induced alterations in signal transduction, and that this response affords cytoprotection against subsequent chemical insult. Gene regulatory mechanisms involved in cytoprotection will be examined in SCC-1, a rat mesangial clonal cell line, and C6, a rat glioma cell line. Treatment regimens will model toxicologically relevant exposures in humans by use of repeated dosing with Pb acetate and HgCl2 at sub-cytotoxic concentration over a period of days. In the first aim, we will determine if metal challenge perturbs Ca2+ homeostasis, redox status and/or protein phosphorylation, leading to activation of GRP78. The contribution of transcriptional and post- transcriptional mechanisms of gene induction, and the role of xenobiotic- regulated cis-acting elements within the 5' regulatory region of the gene will be examined. The cytoprotective effects of increased amounts of GRP78. The contribution of transcriptional and post-transcriptional mechanisms to gene induction, and the role of xenobiotic-regulated cis- acting elements within the 5' regulatory region of the gene will be examined. The cytoprotective effects of increased amounts of GRP78 in metal-treated cells, as evidenced by measurements of Ca2+ homeostasis, misfolded protein accumulation, cytokine release, and metal binding will be examined. Cellular tolerance to subsequent injury by chemical mixtures present at Superfund sites will be monitored to define relevant toxicologic interactions. Our approach will be tied to the imaging, field services and analytical cores, as well as Projects 2, 5 and 6 of this Program. Together the proposed studies will provide information about critical chemico-biologic interactions at Superfund sites.