HSF1 mediated stress response of heavy metals

Abstract

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.

Quantitative Comparison of HSF1 Activators

“…Here, we used an artificial promoter exclusively reacting to HSF activity, thus, providing high specificity by excluding any other transcription factors or signaling pathways taking part in stress-induced Hsp expression. We previously used such cell lines for analyzing kinetics of different heat treatments [ 40 ] and heavy metal treatments [ 42 ]. For this work, we created a novel, optimized HSF reporter cell line (Fig.…”

“…To achieve high signal intensity, the very stable wild-type NanoLuc (Nluc) was used (X9-12H). For kinetic analyses, an Nluc with a very short half-life (NlucPAU) was integrated into the reporter construct (X8-12H) [ 42 ]. Furthermore, a cell line containing an HSPA1A promoter [ 40 ] in combination with NlucPAU (X8-72) was generated.…”

“…1 a) backbones containing either the HSPA1A promoter or 12 × repeated HSE sites [ 40 ] into HEK293 cells with the PiggyBac transposon system [ 41 ]. For pGVL9, the reporter luciferase NlucPAU was changed to a non-degrading version of NanoLuc luciferase [ 42 ]. Due to poor transfection efficiency of fibroblast cell lines, the WI-rep2-12H (single clone #5) cells were generated with lentiviral transduction of immortalized WI38 cells with pLVrep2 12HSE where the pGVL9 12HSE dual luc + puromycin resistance cassette (see Fig.…”

“…Cell lysis, RNA isolation, cDNA synthesis, and qPCR reaction were described before [ 42 ]. The qPCRs for HSPA1 (detecting HSPA1A and HSPA1B coding for the Hsp72) and GAPDH were TaqMan assays, for HSP90AA (coding for Hsp90) and HSPB1 (coding for Hsp27) that were performed as SybrGreen assays.…”

HepGentox: a novel promising HepG2 reportergene-assay for the detection of genotoxic substances in complex mixtures

“…A six times multimerized p53 binding site was introduced upstream of an Nluc reporter gene. We chose the short-lived NlucPAU (NanoLuc containing mRNA and protein destabilizing sequences Steurer et al, 2018) to reduce the background signal (= no accumulation) and obtain high induction rates after a short incubation time at lower cytotoxic side effects. The construct was stably integrated into HepG2 cells and one clone was selected as the HepGentox cell line.…”