Double-stranded RNA-dependent Protein Kinase (pkr) Is Essential for Thermotolerance, Accumulation of HSP70, and Stabilization of ARE-containing HSP70 mRNA during Stress

Zhao, Ming-Lang; Tang, Dan; Lechpammer, Stanislav; Hoffman, Alexander; Asea, Alexzander; Stevenson, Mary Ann; Calderwood, Stuart K.

doi:10.1074/jbc.m208408200

Cited by 51 publications

(51 citation statements)

References 41 publications

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“…Alfieri et al reported that the stability of mouse hsp70 mRNA can be altered by hypertonicity, and Zhao et al showed that the cis-acting A + U rich elements (ARE) in the 3'-untranslated region (UTR) of hsp70.1 controls the mRNA stability (Alfieri et al, 2002;Zhao et al, 2002). We found that human hsp70-2 mRNA had two AUUUA sequences in its 3' UTR.…”

Section: Discussionmentioning

confidence: 69%

The role of tonicity responsive enhancer sites in the transcriptional regulation of human hsp70-2 in response to hypertonic stress

Heo

Lee²,

Kim³

et al. 2006

Exp Mol Med

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The inducible 70 kDa heat shock proteins (Hsp70) in mice are encoded by two almost identical genes, hsp70.1 and hsp70.3. Studies have found that only hsp70.1 is induced by hypertonic stress while both hsp70.1 and hsp70.3 genes are expressed in response to heat shock stress. It is unclear if the human counterparts, hsp70-2 and hsp70-1, are differentially regulated by heat shock and osmotic stress. This study found that only hsp70-2 was induced by hypertonic stress in human embryonic kidney epithelial cells and fibroblasts, while heat shock stress induced both hsp70-1 and hsp70-2. The human hsp70-2 promoter region contains three TonE (tonicity-responsive enhancer) sites, which were reported to play an important role in the response to hypertonicity. When the reporter plasmids containing different parts of the 5' flanking region of hsp70-2 were transfected into human embryonic kidney epithelial cells or fibroblasts, one TonE site at -135 was found to play a key role in the response to hypertonicity. The inactivation of the TonE site using site-directed mutagenesis led to the complete loss of induction by hypertonicity, which demonstrates the essential role of the TonE site. This suggests that the TonE site and the TonEBP (TonE binding protein) are the major regulators for the cellular response against high osmolarity in human kidney tissue.

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Section: Discussionmentioning

confidence: 69%

The role of tonicity responsive enhancer sites in the transcriptional regulation of human hsp70-2 in response to hypertonic stress

Heo

Lee²,

Kim³

et al. 2006

Exp Mol Med

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“…To test this possibility, we sought a means of inhibiting the synthesis of endogenous heat shock proteins. It has been shown previously (51) that 2-aminopurine inhibits the synthesis of Hsp70, a finding in agreement with the fact that one of the kinases inhibited by 2-aminopurine, PKR, is required for the heat shock response (52,53). Therefore, we tested the effect of 2-aminopurine.…”

Section: Shsps Protect Cap-dependent Translation After Heat Shockmentioning

confidence: 76%

“…The nature of the cellular damage sensors is still unclear, but one recently elucidated mechanism involves the activation of the double-stranded RNA-activated kinase through activation of RAX/PACT (56,57). In the absence of double-stranded RNAactivated kinase, Hsp70 is not induced (53). Hsp70 induction is also blocked by 2-aminopurine (Ref.…”

Section: Discussionmentioning

confidence: 99%

Translational Thermotolerance Provided by Small Heat Shock Proteins Is Limited to Cap-dependent Initiation and Inhibited by 2-Aminopurine

Doerwald

Onnekink

Genesen

et al. 2003

Journal of Biological Chemistry

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Heat shock results in inhibition of general protein synthesis. In thermotolerant cells, protein synthesis is still rapidly inhibited by heat stress, but protein synthesis recovers faster than in naive heat-shocked cells, a phenomenon known as translational thermotolerance. Here we investigate the effect of overexpressing a single heat shock protein on cap-dependent and cap-independent initiation of translation during recovery from a heat shock. When overexpressing ␣B-crystallin or Hsp27, cap-dependent initiation of translation was protected but no effect was seen on cap-independent initiation of translation. When Hsp70 was overexpressed however, both cap-dependent and -independent translation were protected. This finding indicates a difference in the mechanism of protection mediated by small or large heat shock proteins. Phosphorylation of ␣B-crystallin and Hsp27 is known to significantly decrease their chaperone activity; therefore, we tested phosphorylation mutants of these proteins in this system. ␣B-crystallin needs to be in its non-phosphorylated state to give protection, whereas phosphorylated Hsp27 is more potent in protection than the unphosphorylatable form. This indicates that chaperone activity is not a prerequisite for protection of translation by small heat shock proteins after heat shock. Furthermore, we show that in the presence of 2-aminopurine, an inhibitor of kinases, among which is double-stranded RNA-activated kinase, the protective effect of overexpressing ␣B-crystallin is abolished. The synthesis of the endogenous Hsps induced by the heat shock to test for thermotolerance is also blocked by 2-aminopurine. Most likely the protective effect of ␣B-crystallin requires synthesis of the endogenous heat shock proteins. Translational thermotolerance would then be a co-operative effect of different heat shock proteins.

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“…Transcription decays rapidly after initiation, while HSF continues to bind to HSE for several hours after transcription is ceased 18,19 . HSP mRNA is then observed within 1 hr of activation and is maintained at these high levels for up to 24 hr due to enhanced stabilization after stress 20 . After acute stress, HSP protein expression is delayed due to initial translational inhibition but Hsp70, Hsp90 and Hsp110 are observed by 2-6 hr after a 43°C heat shock and can persist in cells for up to 100 hr 21 ( Figure 1).…”

Section: Nih Public Accessmentioning

confidence: 99%

Role of Heat Shock Factors in Stress-Induced Transcription

Murshid¹,

Prince²,

Lang³

et al. 2017

Methods in Molecular Biology

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Heat shock proteins (HSP) are rapidly induced after stresses such as heat shock and accumulate at high concentrations in cells. HSP induction involves a family of heat shock transcription factors (HSF) that bind the heat shock elements of the HSP genes and mediate transcription in trans. We discuss methods for the study of HSP binding to HSP promoters and the consequent increases in HSP gene expression in vitro and in vivo. Keywordsheat; shock; factor; binding; transcription (1) IntroductionHeat shock factor (HSF) was first discovered in yeast as a sequence specific transcription factor that binds to the promoters of heat shock protein (HSP) genes 1 . HSF was shown to bind as a trimer to three inverted repeats of the sequence nGAAn at high affinity, an activity that was later shown in Drosophila HSF and human HSF1 2-5 . In more complex organisms, there at least 4 members of the family in avian and mammalian species and multiple members in higher plants [6][7][8][9][10][11] . The current consensus in mammalian cells is that HSF1 is the 2 Communicating author: Stuart K Calderwood, scalderw@bidmc.harvard.edu; telephone 617-667-0970. 1 Equal contributor(1) The EMSA technique has the advantages that it is rapid, sensitive and straightforward to carry out. For assessing the significance of the transcription factor-response element interaction, it is however lacking in that response elements in chromatin are wound along nucleaomes and may not be available for binding. The EMSA reaction is carried out using naked DNA. In addition as ChIP on CHIP and ChIP-seq studies begin to accumulate it is evident that response elements for particular factors are more flexible than suspected from early studies.(2) Some of these problems can be avoided using the ChIP assay that measures HSF binding to chromosomal DNA in vivo. This technique is highly dependent on availability of high affinity and specific antibodies for transcription factors. This can be overcome by overexpression of the factor with a seuqence tag and carrying out ChIP with anti-TAG antibody. However this can introduce potential artifacts involved with protein overexpression. (3) To assess the results of HSF-DNA binding we have used two approaches. We have used transfection of reporter constructs containing either HSP promoters or HSE coupled to reporter genes CAT or luciferase. The assays have the advantages of being rapid and permitting accumulation of plentiful data. The promoter portion of the construct can be tailored to assess the activity of a single transcription factor such as HSF1. The assay is however indirect and does not measure the transcription of the native, chromosomallyembedded gene. There are other potential complications, as reporters require to be translated and yield enzymatically active proteins. (4) Transcriptional rate of HSP genes such can be assess directly by run-on assay. This assay indicates that joint activities of all response elements in the HSP gene promoters. Although genes such as HSP70B respond only to HSF1 or heat shock, ot...

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Double-stranded RNA-dependent Protein Kinase (pkr) Is Essential for Thermotolerance, Accumulation of HSP70, and Stabilization of ARE-containing HSP70 mRNA during Stress

Cited by 51 publications

References 41 publications

The role of tonicity responsive enhancer sites in the transcriptional regulation of human hsp70-2 in response to hypertonic stress

The role of tonicity responsive enhancer sites in the transcriptional regulation of human hsp70-2 in response to hypertonic stress

Translational Thermotolerance Provided by Small Heat Shock Proteins Is Limited to Cap-dependent Initiation and Inhibited by 2-Aminopurine

Role of Heat Shock Factors in Stress-Induced Transcription

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