Dominant Role of the Second Heat Shock Element in Expression of the HSP70–1 Gene in Rat Liver after Whole Body Hyperthermia

We investigated the regulatory expression of the HSP70–1 gene in rat liver after whole body hyperthermia (rectal temperature of 42°C for 30 min). After heat treatment, HSP70–1 mRNA showed a peak at 1 h, and its protein product began to increase at 3 h. Under these conditions, we identified cis-acting elements involved in the regulatory expression of the HSP70–1 gene by in vivo genomic footprinting. We observed that the second heat shock element (HSE-2; −201 to −178) was clearly protected from methylation by dimethyl sulfate in parallel with the increase in HSP70–1 mRNA. On the contrary, the most proximal HSE-1 (−120 to −101) was protected only slightly. Furthermore, the flanking regions of HSE-2, that is, an NF-xB-like element on the upstream side and a GC box on the down stream side were also protected simultaneously in accordance with the induced expression of the HSP70–1 gene. Gel mobility shift assays with nuclear extracts from rat liver at 30 min after heat treatment showed maximal DNA binding activity equally with oligonucleotides containing HSE-1 and HSE-2, respectively, and also demonstrated the participation of heat shock factor-1 (HSF1) in both cases. These results suggest that, in addition to HSF1, other regulatory factors might be involved in the dominant protection of HSE-2 in the promoter of the HSP70–1 gene in rat liver after whole body hyperthermia.