Hamel L, Kenney M, Jayyosi Z, Ardati A, Clark K, Spada A, Zilberstein A, Perrone M, Kaplow J, Merkel L, Rojas C
Department of Cardiovascular Biology, Rh ne Poulenc Rorer Central Research, Collegeville, PA 19426, USA.
Cell Stress Chaperones. 2000 Apr;5(2):121-31. doi: 10.1379/1466-1268(2000)005<0121:iohspb>2.0.co;2.
This study characterizes Hsp70 induction in human smooth muscle cells (SMC) by herbimycin A and cyclopentenone prostaglandins. The magnitude of Hsp70 induction by cyclopentenone prostaglandins was 8- to 10-fold higher than induction by herbimycin A. Hsp70 induction by delta12PGJ2 was first observed at 10 microM, rose to 4000-5000 ng/mL within one log unit and a maximum response was not observed; concentrations of delta12PGJ2 higher than 30 microM were toxic to the cells. A maximum response with herbimycin A (500 ng/mL) was reached at 0.05 microM and maintained to 1 microM without toxicity. Both, delta12PGJ2 and herbimycin A, were inhibited by dithiothreitol (DTT, 100 microM) at lower concentrations and became less sensitive to inhibition at higher concentrations. Hsp70 induction after incubation of SMC with delta12PGJ2 followed by addition of herbimycin A was significantly higher than Hsp70 induction after incubation with herbimycin A followed by addition of delta12PGJ2. When cells were incubated with [3H]-PGJ2, followed by protein denaturation, substantial radioactivity remained protein-bound suggesting that the prostaglandin must be covalently bound. Covalent binding was largely insensitive to DTT. Maximal Hsp70 induction was observed after 5 minutes of exposure of the cells to herbimycin A followed by a 20 hour recovery period in agent-free medium. Cells required 3-4 hours of exposure to delta12PGJ2 followed by a 20 hour recovery period in order to see high Hsp70 induction. Binding of the heat shock factor (HSF) to the heat shock element (HSE) in the presence of herbimycin A or delta12PGJ2, and the effects of DTT, mirrored the results of Hsp70 induction. The results suggest that probable differences between the 2 agents are at the level of the signal transduction prior to HSF activation.
本研究对除草菌素A和环戊烯酮前列腺素在人平滑肌细胞（SMC）中诱导热休克蛋白70（Hsp70）的情况进行了表征。环戊烯酮前列腺素诱导Hsp70的幅度比除草菌素A高8至10倍。δ12前列腺素J2（delta12PGJ2）诱导Hsp70最早在10微摩尔时观察到，在一个对数单位内升至4000 - 5000纳克/毫升，且未观察到最大反应；高于30微摩尔的δ12PGJ2浓度对细胞有毒性。除草菌素A（500纳克/毫升）在0.05微摩尔时达到最大反应，并在1微摩尔时保持，且无毒性。δ12PGJ2和除草菌素A在较低浓度下均被二硫苏糖醇（DTT，100微摩尔）抑制，在较高浓度下对抑制的敏感性降低。SMC先用δ12PGJ2孵育然后添加除草菌素A后诱导的Hsp70显著高于先用除草菌素A孵育然后添加δ12PGJ2后诱导的Hsp70。当细胞用[3H] - 前列腺素J2孵育，随后进行蛋白质变性时，大量放射性仍与蛋白质结合，这表明前列腺素必须共价结合。共价结合在很大程度上对DTT不敏感。细胞暴露于除草菌素A 5分钟后，在无试剂培养基中恢复20小时后观察到最大Hsp70诱导。细胞需要暴露于δ12PGJ2 3 - 4小时，随后恢复20小时才能观察到高Hsp70诱导。在存在除草菌素A或δ12PGJ2的情况下，热休克因子（HSF）与热休克元件（HSE）的结合以及DTT的作用反映了Hsp70诱导的结果。结果表明，这两种试剂之间可能的差异在于HSF激活之前的信号转导水平。
