Petronini P G, Alfieri R, Campanini C, Borghetti A F
Istituto di Patologia Generale, Università degli Studi di Parma, Italy.
J Cell Physiol. 1995 Mar;162(3):322-9. doi: 10.1002/jcp.1041620304.
The simultaneous exposure of WI-38 human fibroblasts (HF) to a heat shock (45 degrees C, 30 min) and an alkaline shift (> or = pH 8.0) in the incubation medium increased and extended the expression of heat shock proteins (hsps). Hsp70 was the most prominent inducible hsp synthesized during the recovery phase after the double shock, and the increase in synthesis depended on the degree of alkalinization during the heat shock. The accumulation of inducible hsp70, which was shown by Western blotting to occur in the late part of the recovery period, was more pronounced in the cells exposed to alkaline medium during the heat shock. Northern blotting did not reveal any increase in hsp70 mRNA, although time course studies following the double shock indicated a more prolonged presence of mRNA. Hsp70 gene activation was evaluated by a gel retardation assay using a 32P-labelled DNA oligonucleotide containing the heat shock consensus element (HSE) and a heat shock-induced specific binding protein (heat shock transcription factor, HSTF) from the cell extract. Heat shock activated HSTF-DNA binding and induced hsp70 mRNA expression as well as the synthesis and accumulation of hsp70. Alkaline shift, which by itself did not induce hsps expression, activated HSTF DNA-binding. However, in combination with heat shock, alkaline shift enhanced and prolonged HSTF-HSE complex association and hsp expression at both mRNA and protein levels. Since the alkaline shift-induced activation of hsp gene does not allow full transcription, these results provide further support for the multistep nature of the heat shock transcriptional response.
将WI-38人成纤维细胞（HF）同时暴露于热休克（45摄氏度，30分钟）以及孵育培养基中的碱性转变（pH≥8.0），可增加并延长热休克蛋白（hsps）的表达。热休克蛋白70（Hsp70）是双休克后恢复阶段合成的最显著的可诱导热休克蛋白，其合成增加取决于热休克期间的碱化程度。通过蛋白质免疫印迹法显示，可诱导的Hsp70在恢复期后期积累，在热休克期间暴露于碱性培养基的细胞中更为明显。Northern印迹法未显示Hsp70 mRNA有任何增加，尽管双休克后的时间进程研究表明mRNA存在的时间更长。使用含有热休克共有元件（HSE）的32P标记DNA寡核苷酸和来自细胞提取物的热休克诱导特异性结合蛋白（热休克转录因子，HSTF），通过凝胶阻滞试验评估Hsp70基因激活。热休克激活了HSTF-DNA结合，并诱导了Hsp70 mRNA表达以及Hsp70的合成和积累。碱性转变本身不诱导热休克蛋白表达，但激活了HSTF与DNA的结合。然而，与热休克相结合时，碱性转变在mRNA和蛋白质水平上增强并延长了HSTF-HSE复合物的结合以及热休克蛋白的表达。由于碱性转变诱导的热休克蛋白基因激活不允许完全转录，这些结果为热休克转录反应的多步骤性质提供了进一步的支持。
