An artificial HSE promoter for efficient and selective detection of heat shock pathway activity.

Viktoria Ortner,Alfred Ludwig,Sarah Dunzinger,Elisabeth Riegel,Thomas Czerny

doi:10.1007/s12192-014-0540-5

Viktoria Ortner, Alfred Ludwig + Show 3 more

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https://doi.org/10.1007/s12192-014-0540-5

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Abstract

Detection of cellular stress is of major importance for the survival of cells. During evolution, a network of stress pathways developed, with the heat shock (HS) response playing a major role. The key transcription factor mediating HS signalling activity in mammalian cells is the HS factor HSF1. When activated it binds to the heat shock elements (HSE) in the promoters of target genes like heat shock protein (HSP) genes. They are induced by HSF1 but in addition they integrate multiple signals from different stress pathways. Here, we developed an artificial promoter consisting only of HSEs and therefore selectively reacting to HSF-mediated pathway activation. The promoter is highly inducible but has an extreme low basal level. Direct comparison with the HSPA1A promoter activity indicates that heat-dependent expression can be fully recapitulated by isolated HSEs in human cells. Using this sensitive reporter, we measured the HS response for different temperatures and exposure times. In particular, long heat induction times of 1 or 2 h were compared with short heat durations down to 1 min, conditions typical for burn injuries. We found similar responses to both long and short heat durations but at completely different temperatures. Exposure times of 2 h result in pathway activation at 41 to 44 °C, whereas heat pulses of 1 min lead to a maximum HS response between 47 and 50 °C. The results suggest that the HS response is initiated by a combination of temperature and exposure time but not by a certain threshold temperature.Electronic supplementary materialThe online version of this article (doi:10.1007/s12192-014-0540-5) contains supplementary material, which is available to authorized users.

Highlights

The heat shock (HS) response is a highly conserved stress response of all cells from bacteria to humans but with differences in the involved proteins and their regulation
HSPA1A is one of the strongest induced genes upon activation of the HSF-mediated HS response (Trinklein and Murray 2004). It has repeatedly been used as a marker gene for HS pathway activity
It is affected by multiple other pathways like the hypoxia pathway (Gogate et al 2012), Keap1-Nrf2-ARE pathway (Hensen et al 2013), MAPK/AP1 pathway (Mendillo et al 2012), TGF-ß pathway (Takenaka and Hightower 1992), NFB and CREB-mediated signalling (Sasi et al 2014)

Summary

Introduction

The heat shock (HS) response is a highly conserved stress response of all cells from bacteria to humans but with differences in the involved proteins and their regulation. The HS pathway was initially discovered as a reaction to higher temperatures, it was later shown that cells use this response when exposed to different kinds of cellular stress (Morimoto 1993). When exposed to heat stress, native proteins in the cells start to partially unfold. Stress acts on the cell membrane initialising hyperfluidisation and rearrangement of microdomains (Török et al 2013). To prevent cells from further destruction, the HS signalling pathway is induced (Morimoto 1993)

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