Department of Medical Biotechnology and Laboratory Science, College of Medicine, Chang Gung University, Tao-Yuan, Taiwan, ROC.
Biochem Biophys Res Commun. 2013 Jan 11;430(2):774-9. doi: 10.1016/j.bbrc.2012.11.072. Epub 2012 Nov 30.
Chaperonic proteins, including inducible HSP70 (HSP70i) and constitutive HSP70 (HSC70), have been implicated as essential players in the cellular adaptive protection. Ensuing studies demonstrated that overexpression of either protein individually protects against thermal and oxidative challenges. The present study aimed to determine whether a concurrent overexpression of both HSC70 and HSP70i confers a better metabolic protection than the expression of each protein alone. Using a rat heart-derived H9c2 cardiac myoblast cell line, we found that HSP70i was rapidly induced within 2-8h following a mild thermal preconditioning (43°C for 20 min) in both parental cells and an established H9/70c clonal sub-line overexpressing HSC70. The level of HSP70i protein in heat pretreated H9/70c clonal cells reached only 50% of that in heat pretreated H9c2 parental cells. Nevertheless, protection against lethal hyperthermia, menadione (an oxidant) and hydrogen peroxide (H(2)O(2)) exposure in the pretreated H9/70c clonal cells was significantly higher than the sum of protection afforded by the early induction of HSP70i in the pretreated parental cells and protection afforded by the pre-existing HSC70 in the H9/70c cells without preconditioning. Using dosimetric analysis, we also found that menadione resistance in the pretreated parental cells increased linearly with cellular HSP70i level (10-300 ng/mg total protein). However, the resistance in the pretreated H9/70c cells showed a biphasic relationship with cellular HSP70i level; when HSP70i concentration reached >250 ng/mg protein, survivability after menadione exposure was markedly enhanced. Similar results were observed in H9c2 cells genetically manipulated to overexpress both HSC70 and HSP70i. The survival benefit against lethal hyperthermia, oxidant treatment, and hypoxia/reoxygenation conferred by a concerted HSC70 and HSP70i overexpression was greater than the sum of benefits contributed by individual protein overexpression. Together, these findings suggest that HSC70 and HSP70i may complement each other in a synergistic manner to preserve cellular integrity during metabolic challenges.
伴侣蛋白，包括诱导型 HSP70（HSP70i）和组成型 HSP70（HSC70），被认为是细胞适应性保护的重要参与者。随后的研究表明，单独过表达这两种蛋白中的任何一种都能对抗热和氧化应激。本研究旨在确定同时过表达 HSC70 和 HSP70i 是否比单独表达每种蛋白提供更好的代谢保护。使用大鼠心脏衍生的 H9c2 心肌成肌细胞系，我们发现轻度热预处理（43°C 20 分钟）后，在亲本细胞和过表达 HSC70 的已建立的 H9/70c 克隆亚系中，HSP70i 在 2-8 小时内迅速诱导。热预处理 H9/70c 克隆细胞中的 HSP70i 蛋白水平仅达到热预处理 H9c2 亲本细胞的 50%。然而，预处理的 H9/70c 克隆细胞对致死性高热、menadione（一种氧化剂）和过氧化氢（H2O2）暴露的保护作用明显高于预处理亲本细胞中 HSP70i 的早期诱导所提供的保护作用和预处理 H9/70c 细胞中预先存在的 HSC70 所提供的保护作用之和。通过剂量测定分析，我们还发现预处理亲本细胞对 menadione 的抗性与细胞内 HSP70i 水平呈线性关系（10-300ng/mg 总蛋白）。然而，预处理的 H9/70c 细胞中的抗性与细胞内 HSP70i 水平呈双相关系；当 HSP70i 浓度达到>250ng/mg 蛋白时，暴露于 menadione 后的存活率显著提高。在遗传操作过表达 HSC70 和 HSP70i 的 H9c2 细胞中观察到类似的结果。协同过表达 HSC70 和 HSP70i 赋予的抗致死性高热、氧化剂处理和缺氧/复氧的生存益处大于单独过表达每种蛋白所带来的益处之和。总之，这些发现表明 HSC70 和 HSP70i 可能以协同方式相互补充，在代谢应激期间维持细胞完整性。
