The heat shock response (HSR) pathway is a highly conserved cellular stress response and mediated by its master regulator HSF1. Activation of the pathway results in the expression of chaperone proteins (heat shock proteins; HSP) to maintain protein homeostasis. One of the genes strongest upregulated upon stress is HSPA1A (HSP72). Heavy metals are highly toxic to living organisms and known as environmental contaminants, due to industrialisation. Furthermore, many of them are well-described inducers of the HSR pathway. Here we compare the effect of different heavy metals, concerning their potential to activate HSF1 with a sensitive artificial heat shock reporter cell line, consisting of heat shock elements (HSE). In general the responses of the artificial promoter to heavy metal stress were in good agreement with those of well-established HSF1 target genes, like HSPA1A. Nevertheless, differences were observable when effects of heat and heavy metal stress were compared. Whereas heat stress preferentially activated the HSE promoter, heavy metals more strongly induced the HSPA1A promoter. We therefore analysed the HSPA1A promoter in more detail, by isolating and mutating the HSEs. The results indicate that the importance of the individual binding sites for HSF1 is determined by their sequence similarity to the consensus sequence and their position relative to the transcription start site, but they were not differentially affected by heat or heavy metal stress. In contrast, we found that other parts of the HSPA1A promoter have different impact on the response under different stress conditions. In this work we provide deeper insights into the regulation of HSP72 expression as a well as a method to quantitatively and sensitively evaluate different stressor on their potential to activate HSF1.

The heat shock response (HSR) pathway is a highly conserved rescue mechanism, which protects the cells from harmful insults disturbing the cellular protein homeostasis via expression of chaperones. Furthermore, it was demonstrated to play crucial roles in various diseases like neurodegeneration and cancer. For neurodegenerative diseases, an overexpression of chaperones is a potential therapeutic approach to clear the cells from non-functional protein aggregates. Therefore, activators of the HSR pathway and its master regulator HSF1 are under close observation. There are numerous HSR activators published in the literature using different model systems, experimental designs, and readout assays. The aim of this work was to provide a quantitative comparison of a broad range of published activators using a newly developed HSF responsive dual-luciferase cell line. Contrary to natural target genes, which are regulated by multiple input pathways, the artificial reporter exclusively reacts to HSF activity. In addition, the results were compared to endogenous heat shock protein expression. As a result, great differences in the intensity of pathway activation were observed. In addition, a parallel viability assessment revealed high variability in the specificity of the drugs. Furthermore, the differences seen compared to published data indicate that some activators exhibit tissue-specific differences leading to interesting assumptions about the regulation of HSF1.

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