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1.  Hsp90 Orchestrates Transcriptional Regulation by Hsf1 and Cell Wall Remodelling by MAPK Signalling during Thermal Adaptation in a Pathogenic Yeast 
PLoS Pathogens  2012;8(12):e1003069.
Thermal adaptation is essential in all organisms. In yeasts, the heat shock response is commanded by the heat shock transcription factor Hsf1. Here we have integrated unbiased genetic screens with directed molecular dissection to demonstrate that multiple signalling cascades contribute to thermal adaptation in the pathogenic yeast Candida albicans. We show that the molecular chaperone heat shock protein 90 (Hsp90) interacts with and down-regulates Hsf1 thereby modulating short term thermal adaptation. In the longer term, thermal adaptation depends on key MAP kinase signalling pathways that are associated with cell wall remodelling: the Hog1, Mkc1 and Cek1 pathways. We demonstrate that these pathways are differentially activated and display cross talk during heat shock. As a result ambient temperature significantly affects the resistance of C. albicans cells to cell wall stresses (Calcofluor White and Congo Red), but not osmotic stress (NaCl). We also show that the inactivation of MAP kinase signalling disrupts this cross talk between thermal and cell wall adaptation. Critically, Hsp90 coordinates this cross talk. Genetic and pharmacological inhibition of Hsp90 disrupts the Hsf1-Hsp90 regulatory circuit thereby disturbing HSP gene regulation and reducing the resistance of C. albicans to proteotoxic stresses. Hsp90 depletion also affects cell wall biogenesis by impairing the activation of its client proteins Mkc1 and Hog1, as well as Cek1, which we implicate as a new Hsp90 client in this study. Therefore Hsp90 modulates the short term Hsf1-mediated activation of the classic heat shock response, coordinating this response with long term thermal adaptation via Mkc1- Hog1- and Cek1-mediated cell wall remodelling.
Author Summary
Candida albicans is one of the most persistent yeast pathogens known to man, causing frequent mucosal infections (thrush) in otherwise healthy individuals, and potentially fatal bloodstream infections in immunocompromised patients. C. albicans colonises warm-blooded animals and occupies thermally buffered niches. Yet during its evolution this pathogen has retained the classic heat shock response whilst other stress responses have diverged significantly. We have established that the essential, evolutionarily conserved molecular chaperone, Hsp90, coordinates thermal adaptation. Hsp90 interacts with and modulates the activity of the heat shock transcription factor, Hsf1, thereby controlling the expression of heat shock proteins required for the clearance of proteins damaged by proteotoxic stresses. In addition, Hsp90 modulates the activities of key MAP kinase signalling pathways that mediate cell wall remodelling and long term adaptation to heat shock. Loss of any of these factors results in a significant reduction in thermotolerance.
PMCID: PMC3531498  PMID: 23300438
2.  Transcriptional profiling of Arabidopsis heat shock proteins and transcription factors reveals extensive overlap between heat and non-heat stress response pathways 
BMC Genomics  2007;8:125.
The heat shock response of Arabidopsis thaliana is dependent upon a complex regulatory network involving twenty-one known transcription factors and four heat shock protein families. It is known that heat shock proteins (Hsps) and transcription factors (Hsfs) are involved in cellular response to various forms of stress besides heat. However, the role of Hsps and Hsfs under cold and non-thermal stress conditions is not well understood, and it is unclear which types of stress interact least and most strongly with Hsp and Hsf response pathways. To address this issue, we have analyzed transcriptional response profiles of Arabidopsis Hsfs and Hsps to a range of abiotic and biotic stress treatments (heat, cold, osmotic stress, salt, drought, genotoxic stress, ultraviolet light, oxidative stress, wounding, and pathogen infection) in both above and below-ground plant tissues.
All stress treatments interact with Hsf and Hsp response pathways to varying extents, suggesting considerable cross-talk between heat and non-heat stress regulatory networks. In general, Hsf and Hsp expression was strongly induced by heat, cold, salt, and osmotic stress, while other types of stress exhibited family or tissue-specific response patterns. With respect to the Hsp20 protein family, for instance, large expression responses occurred under all types of stress, with striking similarity among expression response profiles. Several genes belonging to the Hsp20, Hsp70 and Hsp100 families were specifically upregulated twelve hours after wounding in root tissue, and exhibited a parallel expression response pattern during recovery from heat stress. Among all Hsf and Hsp families, large expression responses occurred under ultraviolet-B light stress in aerial tissue (shoots) but not subterranean tissue (roots).
Our findings show that Hsf and Hsp family member genes represent an interaction point between multiple stress response pathways, and therefore warrant functional analysis under conditions apart from heat shock treatment. In addition, our analysis revealed several family and tissue-specific heat shock gene expression patterns that have not been previously described. These results have implications regarding the molecular basis of cross-tolerance in plant species, and raise new questions to be pursued in future experimental studies of the Arabidopsis heat shock response network.
PMCID: PMC1887538  PMID: 17519032
3.  Heat shock factor 2 is required for maintaining proteostasis against febrile-range thermal stress and polyglutamine aggregation 
Molecular Biology of the Cell  2011;22(19):3571-3583.
HSF2 regulates proteostasis capacity against febrile-range thermal stress, which provides temperature-dependent mechanisms of cellular adaptation to thermal stress. Furthermore, HSF2 has a strong impact on disease progression of Huntington's disease R6/2 mice, suggesting that it could be a promising therapeutic target for protein misfolding diseases.
Heat shock response is characterized by the induction of heat shock proteins (HSPs), which facilitate protein folding, and non-HSP proteins with diverse functions, including protein degradation, and is regulated by heat shock factors (HSFs). HSF1 is a master regulator of HSP expression during heat shock in mammals, as is HSF3 in avians. HSF2 plays roles in development of the brain and reproductive organs. However, the fundamental roles of HSF2 in vertebrate cells have not been identified. Here we find that vertebrate HSF2 is activated during heat shock in the physiological range. HSF2 deficiency reduces threshold for chicken HSF3 or mouse HSF1 activation, resulting in increased HSP expression during mild heat shock. HSF2-null cells are more sensitive to sustained mild heat shock than wild-type cells, associated with the accumulation of ubiquitylated misfolded proteins. Furthermore, loss of HSF2 function increases the accumulation of aggregated polyglutamine protein and shortens the lifespan of R6/2 Huntington's disease mice, partly through αB-crystallin expression. These results identify HSF2 as a major regulator of proteostasis capacity against febrile-range thermal stress and suggest that HSF2 could be a promising therapeutic target for protein-misfolding diseases.
PMCID: PMC3183013  PMID: 21813737
4.  Transcription factor OsHsfC1b regulates salt tolerance and development in Oryza sativa ssp. japonica 
AoB Plants  2012;2012:pls011.
The paper describes the functional analysis of a class C heat shock transcription factor from rice (Oryza sativa). OsHsfC1b is shown to play a role in ABA-mediated salt stress tolerance and is required for plant growth under non-stress conditions.
Background and aims
Salt stress leads to attenuated growth and productivity in rice. Transcription factors like heat shock factors (HSFs) represent central regulators of stress adaptation. Heat shock factors of the classes A and B are well established as regulators of thermal and non-thermal stress responses in plants; however, the role of class C HSFs is unknown. Here we characterized the function of the OsHsfC1b (Os01g53220) transcription factor from rice.
We analysed the expression of OsHsfC1b in the rice japonica cultivars Dongjin and Nipponbare exposed to salt stress as well as after mannitol, abscisic acid (ABA) and H2O2 treatment. For functional characterization of OsHsfC1b, we analysed the physiological response of a T-DNA insertion line (hsfc1b) and two artificial micro-RNA (amiRNA) knock-down lines to salt, mannitol and ABA treatment. In addition, we quantified the expression of small Heat Shock Protein (sHSP) genes and those related to signalling and ion homeostasis by quantitative real-time polymerase chain reaction in roots exposed to salt. The subcellular localization of OsHsfC1b protein fused to green fluorescent protein (GFP) was determined in Arabidopsis mesophyll cell protoplasts.
Principal results
Expression of OsHsfC1b was induced by salt, mannitol and ABA, but not by H2O2. Impaired function of OsHsfC1b in the hsfc1b mutant and the amiRNA lines led to decreased salt and osmotic stress tolerance, increased sensitivity to ABA, and temporal misregulation of salt-responsive genes involved in signalling and ion homeostasis. Furthermore, sHSP genes showed enhanced expression in knock-down plants under salt stress. We observed retarded growth of hsfc1b and knock-down lines in comparison with control plants under non-stress conditions. Transient expression of OsHsfC1b fused to GFP in protoplasts revealed nuclear localization of the transcription factor.
OsHsfC1b plays a role in ABA-mediated salt stress tolerance in rice. Furthermore, OsHsfC1b is involved in the response to osmotic stress and is required for plant growth under non-stress conditions.
PMCID: PMC3357053  PMID: 22616023
5.  Exploring temporal transcription regulation structure of Aspergillus fumigatus in heat shock by state space model 
BMC Genomics  2009;10:306.
The thermotolerance of Aspergillus fumigatus plays a critical role in mammalian and avian infections. Thus, the identification of its adaptation mechanism to higher temperature is very important for an efficient anti-fungal drug development as well as fundamental understanding of its pathogenesis. We explored the temporal transcription regulation structure of this pathogenic fungus under heat shock conditions using the time series microarray data reported by Nierman et al. (Nature 2005, 438:1151-1156).
The estimated transcription regulation structure of A. fumigatus shows that the heat shock proteins are strongly negatively associated with central metabolic pathway genes such as the tricarboxylic acid cycle (TCA cycle) and carbohydrate metabolism. It was 60 min and 120 min, respectively, after the growth temperature changes from 30°C (corresponding to environments of tropical soil) to 37°C and 48°C (corresponding to temperatures in the human body and compost, respectively) that some of genes in TCA cycle were started to be upregulated. In these points, most of heat shock proteins showed lowest expression level after heat shocks. Among the heat shock proteins, the HSP30 (AFU6G06470), a single integral plasma membrane heat shock protein, presented most active role in transcription regulation structure in both heat shock conditions of 37°C and 48°C. The metabolic genes associated with multiple genes in the gene regulation network showed a tendency to have opposite expression patterns of heat shock proteins. The role of those metabolic genes was second regulator in the coherent feed-forward loop type of regulation structure having heat shock protein as its first regulator. This type of regulation structure might be very advantageous for the thermal adaptation of A. fumigatus under heat shock because a small amount of heat shock proteins can rapidly magnify their regulation effect on target genes. However, the coherent feed-forward loop type of regulation of heat shock proteins with metabolic genes became less frequent with increasing temperature. This might be the reason for dramatic increase in the expression of heat shock proteins and the number of heat shock response genes at heat shock of 48°C.
We systemically analysed the thermal adaption mechanism of A. fumigatus by state space model with times series microarray data in terms of transcription regulation structure. We suggest for the first time that heat shock proteins might efficiently regulate metabolic genes using the coherent feed-forward loop type of regulation structure. This type of regulation structure would also be efficient for adjustment to the other stresses requiring rapid change of metabolic mode as well as thermal adaptation.
PMCID: PMC2714559  PMID: 19586549
6.  The translation elongation factor eEF1A1 couples transcription to translation during heat shock response 
eLife  2014;3:e03164.
Translation elongation factor eEF1A has a well-defined role in protein synthesis. In this study, we demonstrate a new role for eEF1A: it participates in the entire process of the heat shock response (HSR) in mammalian cells from transcription through translation. Upon stress, isoform 1 of eEF1A rapidly activates transcription of HSP70 by recruiting the master regulator HSF1 to its promoter. eEF1A1 then associates with elongating RNA polymerase II and the 3′UTR of HSP70 mRNA, stabilizing it and facilitating its transport from the nucleus to active ribosomes. eEF1A1-depleted cells exhibit severely impaired HSR and compromised thermotolerance. In contrast, tissue-specific isoform 2 of eEF1A does not support HSR. By adjusting transcriptional yield to translational needs, eEF1A1 renders HSR rapid, robust, and highly selective; thus, representing an attractive therapeutic target for numerous conditions associated with disrupted protein homeostasis, ranging from neurodegeneration to cancer.
eLife digest
Living cells must be able to withstand changes in the environment. For example, if there is a sudden increase in temperature—which could damage proteins or other molecules—most cells can respond with the ‘heat shock response’. In humans and other mammals, a single protein called heat shock factor 1 triggers the production of numerous heat shock proteins that protect the cell from the detrimental effects of high temperatures. Most heat shock proteins protect cells by binding to, and stabilizing, other molecules in the cell; this prevents these molecules from being damaged or from aggregating and allows them to continue to function as normal.
A protein called eEF1A1 is involved in the final stages of protein production and also enhances the function of heat shock factor 1 during the heat shock response. To make a protein, an enzyme called RNA polymerase transcribes DNA in the nucleus of the cell into a messenger RNA molecule that then exits the nucleus and binds to a ribosome. This molecular machine then translates the messenger RNA sequence into a protein by joining together individual building blocks called amino acids in the correct order. Like other elongation factors, eEF1A1 helps to select the amino acids that match the sequence of the messenger RNA template. However, it was unclear how eEF1A1 helped to protect cells during the heat shock response.
Nudler et al. have now engineered cells—from humans and mice—that make less of the eEF1A1 protein than normal. These cells had enough of this protein to support their growth and development under normal conditions, but not enough to help during the heat shock response. When these cells are subjected to a sudden increase in temperature, they fail to produce a sufficient amount of major heat shock proteins. Heat shock factor 1 is needed to transcribe the genes that encode these heat shock proteins, and Nudler et al. found that eEF1A1 must bind to heat shock factor 1 and then to a moving RNA polymerase for these genes to be transcribed efficiently. Moreover, the eEF1A1 protein was shown to bind to and stabilize the heat shock proteins' messenger RNAs, and aid their export from the nucleus and their binding to the ribosome.
These newly discovered roles for eEF1A1 during the heat shock response highlight this elongation factor as a promising drug target for treating diseases where protein folding goes awry, for example in Alzheimer's or Parkinson's disease. In adults, neurons do not make enough eEF1A1, and Nudler et al. suggest that enabling these cells to make more of this protein could help to treat a range of neurodegenerative conditions.
PMCID: PMC4164936  PMID: 25233275
transcription factors; RNA stability; nuclear export; human; mouse
7.  Transcriptomic analysis of grape (Vitis vinifera L.) leaves during and after recovery from heat stress 
BMC Plant Biology  2012;12:174.
Grapes are a major fruit crop around the world. Heat stress can significantly reduce grape yield and quality. Changes at the molecular level in response to heat stress and subsequent recovery are poorly understood. To elucidate the effect of heat stress and subsequent recovery on expression of genes by grape leaves representing the classic heat stress response and thermotolerance mechanisms, transcript abundance of grape (Vitis vinifera L.) leaves was quantified using the Affymetrix Grape Genome oligonucleotide microarray (15,700 transcripts), followed by quantitative Real-Time PCR validation for some transcript profiles.
We found that about 8% of the total probe sets were responsive to heat stress and/or to subsequent recovery in grape leaves. The heat stress and recovery responses were characterized by different transcriptional changes. The number of heat stress-regulated genes was almost twice the number of recovery-regulated genes. The responsive genes identified in this study belong to a large number of important traits and biological pathways, including cell rescue (i.e., antioxidant enzymes), protein fate (i.e., HSPs), primary and secondary metabolism, transcription factors, signal transduction, and development. We have identified some common genes and heat shock factors (HSFs) that were modulated differentially by heat stress and recovery. Most HSP genes were upregulated by heat stress but were downregulated by the recovery. On the other hand, some specific HSP genes or HSFs were uniquely responsive to heat stress or recovery.
The effect of heat stress and recovery on grape appears to be associated with multiple processes and mechanisms including stress-related genes, transcription factors, and metabolism. Heat stress and recovery elicited common up- or downregulated genes as well as unique sets of responsive genes. Moreover, some genes were regulated in opposite directions by heat stress and recovery. The results indicated HSPs, especially small HSPs, antioxidant enzymes (i.e., ascorbate peroxidase), and galactinol synthase may be important to thermotolerance of grape. HSF30 may be a key regulator for heat stress and recovery, while HSF7 and HSF1 may only be specific to recovery. The identification of heat stress or recovery responsive genes in this study provides novel insights into the molecular basis for heat tolerance in grape leaves.
PMCID: PMC3497578  PMID: 23016701
8.  Inhibition of heat shock factor activity prevents heat shock potentiation of glucocorticoid receptor-mediated gene expression 
Cell Stress & Chaperones  1999;4(4):223-234.
Using mouse L929 cells stably transfected with a glucocorticoid receptor (GR)-responsive murine mammary tumor virus–chloramphenicol acetyltransferase (MMTV–CAT) reporter gene (LMCAT2 cells), we have shown that cellular stress (heat or chemical shock) can cause a dramatic increase in the levels of dexamethasone (Dex)-induced CAT gene expression. We refer to this response as the heat shock potentiation effect, or HSPE. As the cellular heat shock response also involves the activation of heat shock transcription factor (HSF), we have, in the present study, examined the role of HSF in the stress potentiation of GR by use of a flavonoid compound, quercetin, recently shown to selectively inhibit the stress response in a variety of human and murine cell lines. Analysis of the HSPE, as well as heat shock protein synthesis and activation of HSF during time-courses of recovery following heat shock, revealed a similar pattern for each response, with peak activities occurring about 16 h after stress. These data suggest a correlation between the activation of both GR and HSF in stressed cells. In L929 cells stably transfected with a CAT reporter plasmid under the control of the HSF-responsive hsp70 promoter (LHSECAT cells), pretreatment with quercetin was found to cause a dose- and time-dependent inactivation of HSF activity following heat shock, but only when added before the stress event. In LMCAT2 cells, quercetin similarly inhibited both heat and chemical shock potentiation of Dex-induced GR activity. This activity of quercetin was not the result of post-transcriptional or general cytotoxic properties, as quercetin (1) did not significantly affect GR or HSF activities when added after the stress event, (2) did not reduce CAT gene expression as controlled by the constitutive SV40 early promoter, and (3) did not alter normal (non-stress), Dex-induced MMTV–CAT expression. Thus, quercetin appears to be an effective and selective inhibitor of HSF stress-induced activation and its ability to prevent the stress potentiation of GR suggests either a direct or indirect involvement by stress-activated HSF in this process, or the existence of a regulatory step common to both the heat shock and HSPE responses.
PMCID: PMC312937  PMID: 10590836
9.  Interaction between heat shock factor and hsp70 is insufficient to suppress induction of DNA-binding activity in vivo. 
Molecular and Cellular Biology  1994;14(10):6552-6560.
The intracellular level of free heat shock proteins, in particular the 70-kDa stress protein family, has been suggested to be the basis of an autoregulatory mechanism by which the cell measures the level of thermal stress and regulates the synthesis of heat shock proteins. It has been proposed that the DNA-binding and oligomeric state of the heat shock transcription factor (HSF) is a principal step in the induction pathway that is responsive to the level of 70-kDa stress protein. To test this hypothesis, we investigated the association between HSF and 70-kDa stress protein by means of a coimmunoprecipitation assay. We found that 70-kDa stress proteins associate to similar extents with both latent and active forms of HSF, although unlike other 70-kDa stress protein substrates, the association with HSF was not significantly disrupted in the presence of ATP. Gel mobility shift assays indicated that active HSF trimers purified from a bacterial expression system could not be substantially deactivated in vitro with purified 70-kDa stress protein and ATP. In addition, elevated concentrations of hsp70 alone could not significantly inhibit induction of the DNA-binding activity of endogenous HSF in cultured rat cells, and the induction was also not inhibited in cultured rat cells or Drosophila cells containing elevated levels of all members of the heat shock protein family. However, the deactivation of HSF to the non-DNA-binding state after prolonged heat stress or during recovery could be accelerated by increased levels of heat shock proteins. Hence, the level of heat shock proteins may affect the rate of disassembly of HSF trimers, but another mechanism, as yet undefined, appears to control the onset of the oligomeric transitions.
PMCID: PMC359185  PMID: 7935376
10.  In Vivo Chaperone Activity of Heat Shock Protein 70 and Thermotolerance 
Molecular and Cellular Biology  1999;19(3):2069-2079.
Heat shock protein 70 (Hsp70) is thought to play a critical role in the thermotolerance of mammalian cells, presumably due to its chaperone activity. We examined the chaperone activity and cellular heat resistance of a clonal cell line in which overexpression of Hsp70 was transiently induced by means of the tetracycline-regulated gene expression system. This single-cell-line approach circumvents problems associated with clonal variation and indirect effects resulting from constitutive overexpression of Hsp70. The in vivo chaperone function of Hsp70 was quantitatively investigated by using firefly luciferase as a reporter protein. Chaperone activity was found to strictly correlate to the level of Hsp70 expression. In addition, we observed an Hsp70 concentration dependent increase in the cellular heat resistance. In order to study the contribution of the Hsp70 chaperone activity, heat resistance of cells that expressed tetracycline-regulated Hsp70 was compared to thermotolerant cells expressing the same level of Hsp70 plus all of the other heat shock proteins. Overexpression of Hsp70 alone was sufficient to induce a similar recovery of cytoplasmic luciferase activity, as does expression of all Hsps in thermotolerant cells. However, when the luciferase reporter protein was directed to the nucleus, expression of Hsp70 alone was not sufficient to yield the level of recovery observed in thermotolerant cells. In addition, cells expressing the same level of Hsp70 found in heat-induced thermotolerant cells containing additional Hsps showed increased resistance to thermal killing but were more sensitive than thermotolerant cells. These results suggest that the inducible form of Hsp70 contributes to the stress-tolerant state by increasing the chaperone activity in the cytoplasm. However, its expression alone is apparently insufficient for protection of other subcellular compartments to yield clonal heat resistance to the level observed in thermotolerant cells.
PMCID: PMC84000  PMID: 10022894
11.  Induction of physiological thermotolerance in MDCK monolayers: contribution of heat shock protein 70 
Cell Stress & Chaperones  2006;11(3):268-275.
Enhanced survival of both individual cells and whole organisms following a heat stress is termed thermotolerance. In organisms, the maintenance of tissue function rather than the survival of individual cells ultimately determines outcome following thermal challenge. We used MDCK kidney epithelial cells to compare alterations in chaperone activity (as a measure of cellular tolerance) and epithelial barrier function (as a measure of physiological tolerance) after thermal challenge. Quercetin, an inhibitor of heat shock factor–dependent transcriptional activity, both potentiated the effects of heat on naive monolayers and blocked conditioning of monolayers following moderate heat shock, suggesting a central role of heat shock protein (HSP) family members in the maintenance of epithelial integrity. We used MDCK cells that constitutively overexpressed HSP70 to demonstrate 2 functionally distinct components of the response of monolayers to thermal stress. The maintenance of epithelial barrier function during exposure to elevated temperatures is regulated by a complex network of processes that involve the actions of HSP70 but that are independent of alterations in chaperone activity as reflected by changes in the thermal inactivation/refolding of luciferase. In contrast, the restoration of barrier function following a heat stress is directly modulated by HSP70 in a manner that can be fully accounted for by changes in chaperone activity. This study demonstrates an important, albeit complex, protective role for heat shock proteins in the modulation of MDCK epithelial barrier function following a thermal stress.
PMCID: PMC1576477  PMID: 17009600
12.  Hyperthermia in the febrile range induces HSP72 expression proportional to exposure temperature but not to HSF-1 DNA-binding activity in human lung epithelial A549 cells 
Cell Stress & Chaperones  2009;14(5):499-508.
Expression of heat shock proteins (HSPs) is classically activated at temperatures above the physiologic range (≥42°C) via activation of the stress-activated transcription factor, heat shock factor-1 (HSF-1). Several studies suggest that less extreme hyperthermia, especially within the febrile range, as occurs during fever and exertional/environmental hyperthemia, can also activate HSF-1 and enhance HSP expression. We compared HSP72 protein and mRNA expression in human A549 lung epithelial cells continuously exposed to 38.5°C, 39.5°C, or 41°C or exposed to a classic heat shock (42°C for 2 h). We found that expression of HSP72 protein and mRNA increased linearly as incubation temperature was increased from 37°C to 41°C, but increased abruptly when the incubation temperature was raised to 42°C. A similar response in luciferase activity was observed using A549 cells stably transfected with an HSF-1-responsive luciferase reporter plasmid. However, activation of intranuclear HSF-1 DNA-binding activity was comparable at 38.5°C, 39.5°C, and 41°C and only modestly greater at 42°C but the mobility of HSF1 protein on a denaturing gel was altered with increasing exposure temperature and was distinctly different at 42°C. These findings indicate that the proportional changes in HSF-1-dependent HSP72 expression at febrile-range temperatures are dependent upon exposure time and temperature but not on the degree of HSF-1 DNA-binding activity. Instead, HSF-1-mediated HSP expression following hyperthermia and heat shock appears to be mediated, in addition to HSF-1 activation, by posttranslational modifications of HSF-1 protein.
PMCID: PMC2728283  PMID: 19221897
Hyperthermia; Fever; Heat shock; HSF-1; HRE promoter; HSP72
13.  Heat shock factor 1–mediated thermotolerance prevents cell death and results in G2/M cell cycle arrest 
Cell Stress & Chaperones  2001;6(4):326-336.
Mammalian cells respond to environmental stress by activating heat shock transcription factors (eg, Hsf1) that regulate increased synthesis of heat shock proteins (Hsps). Hsps prevent the disruption of normal cellular mitosis, meiosis, or differentiation by environmental stressors. To further characterize this stress response, transformed wild-type Hsf1+/+ and mutant Hsf1−/− mouse embryonic fibroblasts (MEFs) were exposed to (1) lethal heat (45°C, 60 minutes), (2) conditioning heat (43°C, 30 minutes), or (3) conditioning followed by lethal heat. Western blot analysis demonstrated that only Hsf1+/+ MEFs expressed inducible Hsp70s and Hsp25 following conditioning or conditioning and lethal heat. Exposure of either Hsf1+/+ or Hsf1−/− MEFs to lethal heat resulted in cell death. However, if conditioning heat was applied 6 hours before lethal heat, more than 85% of Hsf1+/+ MEFs survived, and cells in G2/M transiently increased 3-fold. In contrast, conditioned Hsf1−/− MEFs neither survived lethal heat nor exhibited this G2/M accumulation. Coinfection with adenoviral Hsp70 and Hsp25 constructs did not fully recreate thermotolerance in either Hsf1+/+ or Hsf1−/− MEFs, indicating other Hsf1-mediated gene expression is required for complete thermotolerance. These results demonstrate the necessity of Hsf1-mediated gene expression for thermotolerance and the involvement of cell cycle regulation, particularly the G2/M transition, in this thermotolerant response.
PMCID: PMC434415  PMID: 11795469
14.  An artificial HSE promoter for efficient and selective detection of heat shock pathway activity 
Cell Stress & Chaperones  2014;20(2):277-288.
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 material
The online version of this article (doi:10.1007/s12192-014-0540-5) contains supplementary material, which is available to authorized users.
PMCID: PMC4326385  PMID: 25168173
Heat shock; HSF1; Reporter; Burn
15.  HSP90 Interacts with and Regulates the Activity of Heat Shock Factor 1 in Xenopus Oocytes 
Molecular and Cellular Biology  1998;18(9):4949-4960.
Transcriptional activation of heat shock genes is a reversible and multistep process involving conversion of inactive heat shock factor 1 (HSF1) monomers into heat shock element (HSE)-binding homotrimers, hyperphosphorylation, and further modifications that induce full transcriptional competence. HSF1 is controlled by multiple regulatory mechanisms, including suppression by additional cellular factors, physical interactions with HSP70, and integration into different cellular signaling cascades. However, the signaling mechanisms by which cells respond to stress and control the HSF1 activation-deactivation pathway are not known. Here we demonstrate that HSP90, a cellular chaperone known to regulate several signal transduction molecules and transcription factors, functions in the regulation of HSF1. The existence of HSF1-HSP90 heterocomplexes was shown by coimmunoprecipitation of HSP90 with HSF1 from unshocked and heat-shocked nuclear extracts, recognition of HSF1-HSE complexes in vitro by using HSP90 antibodies (Abs), and recognition of HSF1 in vivo by HSP90 Abs microinjected directly into oocyte nuclei. The functional impact of HSP90-HSF1 interactions was analyzed by using two strategies: direct nuclear injection of HSP90 Abs and treatment of cells with geldanamycin (GA), an agent that specifically blocks the chaperoning activity of HSP90. Both HSP90 Abs and GA delayed the disassembly of HSF1 trimers during recovery from heat shock and specifically inhibited heat-induced transcription from a chloramphenicol acetyltransferase reporter construct under control of the hsp70 promoter. HSP90 Abs activated HSE binding in the absence of heat shock, an effect that could be reversed by subsequent injection of purified HSP90. GA did not activate HSE binding under nonshock conditions but increased the quantity of HSE binding induced by heat shock. On the basis of these findings and the known properties of HSP90, we propose a new regulatory model in which HSP90 participates in modulating HSF1 at different points along the activation-deactivation pathway, influencing the interconversion between monomeric and trimeric conformations as well as transcriptional activation. We also put forth the hypothesis that HSP90 links HSF1 to cellular signaling molecules coordinating the stress response.
PMCID: PMC109079  PMID: 9710578
16.  Membrane Fluidity and Temperature Sensing Are Coupled via Circuitry Comprised of Ole1, Rsp5, and Hsf1 in Candida albicans 
Eukaryotic Cell  2014;13(8):1077-1084.
Temperature is a ubiquitous environmental variable which can profoundly influence the physiology of living cells as it changes over time and space. When yeast cells are exposed to a sublethal heat shock, normal metabolic functions become repressed and the heat shock transcription factor Hsf1 is activated, inducing heat shock proteins (HSPs). Candida albicans, the most prevalent human fungal pathogen, is an opportunistic pathogen that has evolved as a relatively harmless commensal of healthy individuals. Even though C. albicans occupies thermally buffered niches, it has retained the classic heat shock response, activating Hsf1 during slow thermal transitions such as the increases in temperature suffered by febrile patients. However, the mechanism of temperature sensing in fungal pathogens remains enigmatic. A few studies with Saccharomyces cerevisiae suggest that thermal stress is transduced into a cellular signal at the level of the membrane. In this study, we manipulated the fluidity of C. albicans membrane to dissect mechanisms of temperature sensing. We determined that in response to elevated temperature, levels of OLE1, encoding a fatty acid desaturase, decrease. Subsequently, loss of OLE1 triggers expression of FAS2, encoding a fatty acid synthase. Furthermore, depletion of OLE1 prevents full activation of Hsf1, thereby reducing HSP expression in response to heat shock. This reduction in Hsf1 activation is attributable to the E3 ubiquitin ligase Rsp5, which regulates OLE1 expression. To our knowledge, this is the first study to define a molecular link between fatty acid synthesis and the heat shock response in the fungal kingdom.
PMCID: PMC4135801  PMID: 24951438
17.  Multiple Components of the HSP90 Chaperone Complex Function in Regulation of Heat Shock Factor 1 In Vivo 
Molecular and Cellular Biology  1999;19(12):8033-8041.
Rapid and transient activation of heat shock genes in response to stress is mediated in eukaryotes by the heat shock transcription factor HSF1. It is well established that cells maintain a dynamic equilibrium between inactive HSF1 monomers and transcriptionally active trimers, but little is known about the mechanism linking HSF1 to reception of various stress stimuli or the factors controlling oligomerization. Recent reports have revealed that HSP90 regulates key steps in the HSF1 activation-deactivation process. Here, we tested the hypothesis that components of the HSP90 chaperone machine, known to function in the folding and maturation of steroid receptors, might also participate in HSF1 regulation. Mobility supershift assays using antibodies against chaperone components demonstrate that active HSF1 trimers exist in a heterocomplex with HSP90, p23, and FKBP52. Functional in vivo experiments in Xenopus oocytes indicate that components of the HSF1 heterocomplex, as well as other components of the HSP90 cochaperone machine, are involved in regulating oligomeric transitions. Elevation of the cellular levels of cochaperones affected the time of HSF1 deactivation during recovery: attenuation was delayed by immunophilins, and accelerated by HSP90, Hsp/c70, Hip, or Hop. In immunotargeting experiments with microinjected antibodies, disruption of HSP90, Hip, Hop, p23, FKBP51, and FKBP52 delayed attenuation. In addition, HSF1 was activated under nonstress conditions after immunotargeting of HSP90 and p23, evidence that these proteins remain associated with HSF1 monomers and function in their repression in vivo. The remarkable similarity of HSF1 complex chaperones identified here (HSP90, p23, and FKBP52) and components in mature steroid receptor complexes suggests that HSF1 oligomerization is regulated by a foldosome-type mechanism similar to steroid receptor pathways. The current evidence leads us to propose a model in which HSF1, HSP90 and p23 comprise a core heterocomplex required for rapid conformational switching through interaction with a dynamic series of HSP90 subcomplexes.
PMCID: PMC84888  PMID: 10567529
18.  Hsp70 Inhibits Aminoglycoside-Induced Hair Cell Death and is Necessary for the Protective Effect of Heat Shock 
Sensory hair cells of the inner ear are sensitive to death from aging, noise trauma, and ototoxic drugs. Ototoxic drugs include the aminoglycoside antibiotics and the antineoplastic agent cisplatin. Exposure to aminoglycosides results in hair cell death that is mediated by specific apoptotic proteins, including c-Jun N-terminal kinase (JNK) and caspases. Induction of heat shock proteins (Hsps) is a highly conserved stress response that can inhibit JNK- and caspase-dependent apoptosis in a variety of systems. We have previously shown that heat shock results in a robust upregulation of Hsps in the hair cells of the adult mouse utricle in vitro. In addition, heat shock results in significant inhibition of both cisplatin- and aminoglycoside-induced hair cell death. In our system, Hsp70 is the most strongly induced Hsp, which is upregulated over 250-fold at the level of mRNA 2 h after heat shock. Therefore, we have begun to examine the role of Hsp70 in mediating the protective effect of heat shock. To determine whether Hsp70 is necessary for the protective effect of heat shock against aminoglycoside-induced hair cell death, we utilized utricles from Hsp70.1/3−/− mice. While heat shock inhibited gentamicin-induced hair cell death in wild-type utricles, utricles from Hsp70.1/3−/− mice were not protected. In addition, we have examined the role of the major heat shock transcription factor, Hsf1, in mediating the protective effect of heat shock. Utricles from Hsf1−/− mice and wild-type littermates were exposed to heat shock followed by gentamicin. The protective effect of heat shock on aminoglycoside-induced hair cell death was only observed in wild-type mice and not in Hsf1−/− mice. To determine whether Hsp70 is sufficient to protect hair cells, we have utilized transgenic mice that constitutively overexpress Hsp70. Utricles from Hsp70-overexpressing mice and wild-type littermates were cultured in the presence of varying neomycin concentrations for 24 h. The Hsp70-overexpressing utricles were significantly protected against neomycin-induced hair cell death at moderate to high doses of neomycin. This protective effect was achieved without a heat shock. Taken together, these data indicate that Hsp70 and Hsf1 are each necessary for the protective effect of heat shock against aminoglycoside-induced death. Furthermore, overexpression of Hsp70 alone significantly inhibits aminoglycoside-induced hair cell death.
PMCID: PMC2538150  PMID: 18512096
ototoxicity; vestibular; utricle; Hsf1 knockout; Hsp70 knockout; Hsp70 overexpressor
19.  Regulation of Thermotolerance by Stress-Induced Transcription Factors in Saccharomyces cerevisiae▿  
Eukaryotic Cell  2008;7(5):783-790.
The heat shock transcription factor Hsf1 and the general stress transcription factors Msn2 and Msn4 (Msn2/4) are major regulators of the heat shock response in Saccharomyces cerevisiae. Here, we show that transcriptional activation of their target genes, including HSP104, an antistress chaperone gene, is obligatory for thermotolerance. Although Hsf1 activity might be necessary before the exposure of cells to high temperature, severe heat shock induced the binding of hyperphosphorylated Hsf1 to its target promoters. However, promoter-bound, phosphorylated Hsf1 was inactive for transcription because RNA polymerase II was inactive at high temperatures. Rather, our results suggest that Hsf1 activates the transcription of most of its target genes during the recovery period following severe heat shock. This delayed upregulation by Hsf1, which would be induced by misfolded proteins that accumulate in severely heat-shocked cells, is required for the resumption of normal cell growth. In contrast, the factors Msn2/4 were not involved in the delayed upregulation of genes and were dispensable for cell growth during the recovery period, suggesting that they play a role before the exposure to high temperature. These results show that Hsf1 and Msn2/4 act differentially before and after exposure to extreme temperatures to ensure cell survival and growth.
PMCID: PMC2394977  PMID: 18359875
20.  Inducible and constitutive heat shock gene expression responds to modification of Hsp70 copy number in Drosophila melanogaster but does not compensate for loss of thermotolerance in Hsp70 null flies 
BMC Biology  2008;6:5.
The heat shock protein Hsp70 promotes inducible thermotolerance in nearly every organism examined to date. Hsp70 interacts with a network of other stress-response proteins, and dissecting the relative roles of these interactions in causing thermotolerance remains difficult. Here we examine the effect of Hsp70 gene copy number modification on thermotolerance and the expression of multiple stress-response genes in Drosophila melanogaster, to determine which genes may represent mechanisms of stress tolerance independent of Hsp70.
Hsp70 copy number in four strains is positively associated with Hsp70 expression and inducible thermotolerance of severe heat shock. When assayed at carefully chosen temperatures, Hsp70 null flies are almost entirely deficient in thermotolerance. In contrast to expectations, increasing Hsp70 expression levels induced by thermal pretreatment are associated with increasing levels of seven other inducible Hsps across strains. In addition, complete Hsp70 loss causes upregulation of the inducible Hsps and six constitutive stress-response genes following severe heat shocks.
Modification of Hsp70 copy number quantitatively and qualitatively affects the expression of multiple other stress-response genes. A positive association between absolute expression levels of Hsp70 and other Hsps after thermal pretreatment suggests novel regulatory mechanisms. Severe heat shocks induce both novel gene expression patterns and almost total mortality in the Hsp70 null strain: alteration of gene expression in this strain does not compensate for Hsp70 loss but suggests candidates for overexpression studies.
PMCID: PMC2257928  PMID: 18211703
21.  A Novel Mouse HSF3 Has the Potential to Activate Nonclassical Heat-Shock Genes during Heat Shock 
Molecular Biology of the Cell  2010;21(1):106-116.
HSF1 is a master regulator of the heat-shock response in mammalian cells, whereas in avian cells, HSF3, which was considered as an avian-specific factor, is required for the expression of classical heat-shock genes. Here, the authors identify mouse HSF3, and demonstrate that it has the potential to activate only nonclassical heat-shock genes.
The heat-shock response is characterized by the expression of a set of classical heat-shock genes, and is regulated by heat-shock transcription factor 1 (HSF1) in mammals. However, comprehensive analyses of gene expression have revealed very large numbers of inducible genes in cells exposed to heat shock. It is believed that HSF1 is required for the heat-inducible expression of these genes although HSF2 and HSF4 modulate some of the gene expression. Here, we identified a novel mouse HSF3 (mHSF3) translocated into the nucleus during heat shock. However, mHSF3 did not activate classical heat-shock genes such as Hsp70. Remarkably, overexpression of mHSF3 restored the expression of nonclassical heat-shock genes such as PDZK3 and PROM2 in HSF1-null mouse embryonic fibroblasts (MEFs). Although down-regulation of mHSF3 expression had no effect on gene expression or cell survival in wild-type MEF cells, it abolished the moderate expression of PDZK3 mRNA and reduced cell survival in HSF1-null MEF cells during heat shock. We propose that mHSF3 represents a unique HSF that has the potential to activate only nonclassical heat-shock genes to protect cells from detrimental stresses.
PMCID: PMC2801703  PMID: 19864465
22.  Core temperature correlates with expression of selected stress and immunomodulatory genes in febrile patients with sepsis and noninfectious SIRS 
Cell Stress & Chaperones  2009;15(1):55-66.
Environmental hyperthermia and exercise produce extensive changes in gene expression in human blood cells, but it is unknown whether this also happens during febrile-range hyperthermia. We tested the hypothesis that heat shock protein (HSP) and immunomodulatory stress gene expression correlate with fever in intensive care unit patients. Whole blood messenger RNA was obtained over consecutive days from 100 hospitalized patients suffering from sepsis or noninfectious systemic inflammatory response syndrome (SIRS) as defined by conventional criteria. The most abnormal body temperature in the preceding 24 h was recorded for each sample. Expression analysis was performed using the Affymetrix U133 chip. ANCOVA followed by correlation analysis was performed on a subset of 278 prospectively identified sequences of interest. Temperature affected expression of 60 sequences, either independently or as a function of clinical diagnosis. Forty-eight of these (representing 38 genes) were affected by temperature only, including several HSPs, transcription factors heat shock factor (HSF)-1 and HSF-4, cellular adhesion molecules such as ICAM1/CD54 and JAM3, toll receptors TLR-6 and TLR-7, ribosomal proteins, and a number of molecules involved in inflammatory pathways. Twelve sequences demonstrated temperature-dependent responses that differed significantly between patients with sepsis and noninfectious SIRS: CXCL-13; heat shock proteins DNAJB12 and DNAJC4; the F11 receptor; folate hydrolase 1; HSF-2; HSP 70 proteins HSPA1A, HSPA1B, and HSPA1L; interleukin 8; lipopolysaccharide binding protein; and prostaglandin E synthase. Febrile-range temperatures achieved during sepsis and noninfectious SIRS correlate with detectable changes in stress gene expression in vivo, suggesting that fever can activate HSP gene expression and modify innate immune responses. For some genes, it appears that clinical condition can alter temperature-sensitive gene expression. Collectively, these data underscore the potential importance of body temperature in shaping the immune response to infection and injury.
PMCID: PMC2866972  PMID: 19496026
Heat shock; Fever; Hyperthermia; Sepsis; SIRS; Microarray; mRNA; Whole blood; Gene expression; Gene chip
23.  Differential Expression of Heat Shock Transcription Factors and Heat Shock Proteins after Acute and Chronic Heat Stress in Laying Chickens (Gallus gallus) 
PLoS ONE  2014;9(7):e102204.
Heat stress due to high environmental temperature negatively influences animal performances. To better understand the biological impact of heat stress, laying broiler breeder chickens were subjected either to acute (step-wisely increasing temperature from 21 to 35°C within 24 hours) or chronic (32°C for 8 weeks) high temperature exposure. High temperature challenges significantly elevated body temperature of experimental birds (P<0.05). However, oxidation status of lipid and protein and expression of heat shock transcription factors (HSFs) and heat shock proteins (HSPs) 70 and 90 were differently affected by acute and chronic treatment. Tissue-specific responses to thermal challenge were also found among heart, liver and muscle. In the heart, acute heat challenge affected lipid oxidation (P = 0.05) and gene expression of all 4 HSF gene expression was upregulated (P<0.05). During chronic heat treatment, the HSP 70 mRNA level was increased (P<0.05) and HSP 90 mRNA (P<0.05) was decreased. In the liver, oxidation of protein was alleviated during acute heat challenge (P<0.05), however, gene expression HSF2, 3 and 4 and HSP 70 were highly induced (P<0.05). HSP90 expression was increased by chronic thermal treatment (P<0.05). In the muscle, both types of heat stress increased protein oxidation, but HSFs and HSPs gene expression remained unaltered. Only tendencies to increase were observed in HSP 70 (P = 0.052) and 90 (P = 0.054) gene expression after acute heat stress. The differential expressions of HSF and HSP genes in different tissues of laying broiler breeder chickens suggested that anti-heat stress mechanisms might be provoked more profoundly in the heart, by which the muscle was least protected during heat stress. In addition to HSP, HSFs gene expression could be used as a marker during acute heat stress.
PMCID: PMC4114549  PMID: 25072282
24.  Heat Shock Response in CHO Mammalian Cells Is Controlled by a Nonlinear Stochastic Process 
PLoS Computational Biology  2007;3(10):e187.
In many biological systems, the interactions that describe the coupling between different units in a genetic network are nonlinear and stochastic. We study the interplay between stochasticity and nonlinearity using the responses of Chinese hamster ovary (CHO) mammalian cells to different temperature shocks. The experimental data show that the mean value response of a cell population can be described by a mathematical expression (empirical law) which is valid for a large range of heat shock conditions. A nonlinear stochastic theoretical model was developed that explains the empirical law for the mean response. Moreover, the theoretical model predicts a specific biological probability distribution of responses for a cell population. The prediction was experimentally confirmed by measurements at the single-cell level. The computational approach can be used to study other nonlinear stochastic biological phenomena.
Author Summary
The structure of an unknown biological system is uncovered by experimentally perturbing the system with a series of input signals. The response to these perturbations is measured as output signals. Then, the mathematical relation between the input and the output signals constitutes a model for the system. As a result, a classification of biological molecular networks can be devised using their input–output functional relation. This article studies the input–output functional form for the response to heat shocks in mammalian cells. The Chinese hamster ovary (CHO) mammalian cells were perturbed with a series of heat pulses of precise duration and temperature. The experimental data, taken at the single-cell level, revealed a simple and precise mathematical law for the time evolution of the heat shock response. Parameters of the mathematical law can be experimentally measured and can be used by heat shock biologists to classify the heat shock response in different experimental conditions. Since the response to heat shock is the outcome of a transcriptional factor control, it is highly probable that the empirical law is valid for other biological systems. The mathematical model explains not only the mean value of the response but also the time evolution of its probability distribution in a cell population.
PMCID: PMC2000973  PMID: 17922567
25.  Regulation of Cyclooxygenase-2 Expression by Heat: A Novel Aspect of Heat Shock Factor 1 Function in Human Cells 
PLoS ONE  2012;7(2):e31304.
The heat-shock response, a fundamental defense mechanism against proteotoxic stress, is regulated by a family of heat-shock transcription factors (HSF). In humans HSF1 is considered the central regulator of heat-induced transcriptional responses. The main targets for HSF1 are specific promoter elements (HSE) located upstream of heat-shock genes encoding cytoprotective heat-shock proteins (HSP) with chaperone function. In addition to its cytoprotective function, HSF1 was recently hypothesized to play a more complex role, regulating the expression of non-HSP genes; however, the non-canonical role of HSF1 is still poorly understood. Herein we report that heat-stress promotes the expression of cyclooxygenase-2 (COX-2), a key regulator of inflammation controlling prostanoid and thromboxane synthesis, resulting in the production of high levels of prostaglandin-E2 in human cells. We show that heat-induced COX-2 expression is regulated at the transcriptional level via HSF1-mediated signaling and identify, by in-vitro reporter gene activity assay and deletion-mutant constructs analysis, the COX-2 heat-responsive promoter region and a new distal cis-acting HSE located at position −2495 from the transcription start site. As shown by ChIP analysis, HSF1 is recruited to the COX-2 promoter rapidly after heat treatment; by using shRNA-mediated HSF1 suppression and HSE-deletion from the COX-2 promoter, we demonstrate that HSF1 plays a central role in the transcriptional control of COX-2 by heat. Finally, COX-2 transcription is also induced at febrile temperatures in endothelial cells, suggesting that HSF1-dependent COX-2 expression could contribute to increasing blood prostaglandin levels during fever. The results identify COX-2 as a human non-classical heat-responsive gene, unveiling a new aspect of HSF1 function.
PMCID: PMC3275557  PMID: 22347460

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