Mammalian Heat Shock Response Research Articles

Mammalian Heat Shock Response and Mechanisms Underlying Its Genome-wide Transcriptional Regulation

The heat shock response (HSR) is critical for survival of all organisms. However, its scope, extent, and the molecular mechanism of regulation are poorly understood. Here we show that the genome-wide transcriptional response to heat shock in mammals is rapid and dynamic and results in induction of several hundred and repression of several thousand genes. Heat shock factor 1 (HSF1), the "master regulator" of the HSR, controls only a fraction of heat shock-induced genes and does so by increasing RNA polymerase II release from promoter-proximal pause. Notably, HSF2 does not compensate for the lack of HSF1. However, serum response factor appears to transiently induce cytoskeletal genes independently of HSF1. The pervasive repression of transcription is predominantly HSF1-independent and is mediated through reduction of RNA polymerase II pause release. Overall, mammalian cells orchestrate rapid, dynamic, and extensive changes in transcription upon heat shock that are largely modulated at pause release, and HSF1 plays a limited and specialized role.

Heat shock induces expression of OSTC/DC2, a novel subunit of oligosaccharyltransferase, in vitro and in vivo

Mammalian oligosaccharyltransferase complex subunit OSTC/DC2 protein has recently been shown to be a new subunit of the oligosaccharyltransferase; however, its physiological role is still unclear. Here, we report the expression pattern of OSTC/DC2 protein in the context of heat shock stress. Its upregulation was detected both in cells treated with heat shock in vitro and in an animal model of heat shock in vivo. Northern blot analysis indicated that OSTC/DC2 mRNA is ubiquitously expressed in various human tissues, with abundant expression in the placenta and liver. The temporal changes of OSTC/DC2 protein expression following acute heat shock in human malignant glioblastoma cell line U87MG and mice were analyzed by Western blot assay. In general, expression of OSTC/DC2 protein was elevated after heat shock; however, the time courses of the change of OSTC/DC2 protein expression varied in different tissues. In the cerebellum, heat shock induction of OSTC/DC2 protein and activation of AKT, a key regulator of stress response, followed a similar time course. These results suggest that the upregulation of OSTC/DC2, a novel component of the oligosaccharyltransferase complex, is part of the mammalian heat shock response.

Non-coding RNAs turn up the heat: An emerging layer of novel regulators in the mammalian heat shock response

The field of non-coding RNA (ncRNA) has expanded over the last decade following the discoveries of several new classes of regulatory ncRNA. A growing amount of evidence now indicates that ncRNAs are involved even in the most fundamental of cellular processes. The heat shock response is no exception as ncRNAs are being identified as integral components of this process. Although this area of research is only in its infancy, this article focuses on several classes of regulatory ncRNA (i.e., miRNA, lncRNA, and circRNA), while summarizing their activities in mammalian heat shock. We also present an updated model integrating the traditional heat shock response with the activities of regulatory ncRNA. Our model expands on the mechanisms for efficient execution of the stress response, while offering a more comprehensive summary of the major regulators and responders in heat shock signaling. It is our hope that much of what is discussed herein may help researchers in integrating the fields of heat shock and ncRNA in mammals.

Heat Shock and Caloric Restriction Have a Synergistic Effect on the Heat Shock Response in a sir2.1-dependent Manner in Caenorhabditis elegans

The heat shock response (HSR) is responsible for maintaining cellular and organismal health through the regulation of proteostasis. Recent data demonstrating that the mammalian HSR is regulated by SIRT1 suggest that this response may be under metabolic control. To test this hypothesis, we have determined the effect of caloric restriction in Caenorhabditis elegans on activation of the HSR and have found a synergistic effect on the induction of hsp70 gene expression. The homolog of mammalian SIRT1 in C. elegans is Sir2.1. Using a mutated C. elegans strain with a sir2.1 deletion, we show that heat shock and caloric restriction cooperate to promote increased survivability and fitness in a sir2.1-dependent manner. Finally, we show that caloric restriction increases the ability of heat shock to preserve movement in a polyglutamine toxicity neurodegenerative disease model and that this effect is dependent on sir2.1.

The effect of coupled stochastic processes in a two-state biochemical switch

Cell signaling pathways consist of multiple connections of different types of gene, mRNA and protein networks. It is not a trivial task to follow the signals flowing through these networks. The difficulty comes from considering the entire biological structure as a single network without breaking it into connected modules. The study of these networks simplifies if the complex system is reduced to a hierarchy of interconnected modules. Out of many potential modules, a specific one, namely the Goldbeter-Koshland switch, was encountered by the authors during their study of the Mammalian Heat Shock Response Network (MHSRN) where the switch acts as a stress sensor. Usually, only the steady state behavior of the switch is studied, in which the phosphorylated protein is given as a function of the enzyme concentration. Experimental results show that the heat shock response is still present 20h after the temperature stress had ended. Thus, it is useful to analyze the transient behavior of the switch that couples the environment to the MHSRN. A stochastic model for the switch is proposed using the Master Equation which is subsequently transformed into an equation for the factorial cumulant generating function. This generating function can be easily read from a graphical representation of the stochastic switch. The second order approximation of the equation for the factorial cumulant generating function is solved and the time dependence of the transient regime of the mean and standard deviation is readily obtained. Using the mean and standard deviation of the switch's output as a function of the stochastic input signals that represent the environment, we classify the switches according to different criteria. The switches differ by the numerical values of the parameters that characterize the switch's chemical reactions. The classifying criteria will distinguish the switches by the levels of the response for a given transition time and by the sensitivity of the response to the enzyme levels. It is also found that the environment can drastically change the response of the switch, which has important biological consequences.

The identification of protein kinase C iota as a regulator of the Mammalian heat shock response using functional genomic screens.

BackgroundThe heat shock response is widely used as a surrogate of the general protein quality control system within the cell. This system plays a significant role in aging and many protein folding diseases as well as the responses to other physical and chemical stressors.Methods/Principal FindingsIn this study, a broad-based functional genomics approach was taken to identify potential regulators of the mammalian heat shock response. In the primary screen, a total of 13724 full-length genes in mammalian expression vectors were individually co-transfected into human embryonic kidney cells together with a human HSP70B promoter driving firefly luciferase. A subset of the full-length genes that showed significant activation in the primary screen were then evaluated for their ability to hyper-activate the HSP70B under heat shock conditions. Based on the results from the secondary assay and gene expression microarray analyses, eight genes were chosen for validation using siRNA knockdown. Of the eight genes, only PRKCI showed a statistically significant reduction in the heat shock response in two independent siRNA duplexes compared to scrambled controls. Knockdown of the PRKCI mRNA was confirmed using quantitative RT-PCR. Additional studies did not show a direct physical interaction between PRKCI and HSF1.Conclusions/SignificanceThe results suggest that PRKCI is an indirect co-regulator of HSF1 activity and the heat shock response. Given the underlying role of HSF1 in many human diseases and the response to environmental stressors, PRKCI represents a potentially new candidate for gene-environment interactions and therapeutic intervention.

Evidence for bacterial origin of heat shock RNA-1

The heat shock RNA-1 (HSR1) is a noncoding RNA (ncRNA) reported to be involved in mammalian heat shock response. HSR1 was shown to significantly stimulate the heat-shock factor 1 (HSF1) trimerization and DNA binding. The hamster HSR1 sequence was reported to consist of 604 nucleotides (nt) plus a poly(A) tail and to have only a 4-nt difference with the human HSR1. In this study, we present highly convincing evidence for bacterial origin of the HSR1. No HSR1 sequence was found by exhaustive sequence similarity searches of the publicly available eukaryotic nucleotide sequence databases at the NCBI, including the expressed sequence tags, genome survey sequences, and high-throughput genomic sequences divisions of GenBank, as well as the Trace Archive database of whole genome shotgun sequences, and genome assemblies. Instead, a putative open reading frame (ORF) of HSR1 revealed strong similarity to the amino-terminal region of bacterial chloride channel proteins. Furthermore, the 5' flanking region of the putative HSR1 ORF showed similarity to the 5' upstream regions of the bacterial protein genes. We propose that the HSR1 was derived from a bacterial genome fragment either by horizontal gene transfer or by bacterial infection of the cells. The most probable source organism of the HSR1 is a species belonging to the order Burkholderiales.

Transcriptional repression by non‐coding RNA regulators of the mammalian heat shock response

We have found that mouse B2 RNA and human Alu RNA are transacting transcriptional repressors of RNA polymerase II. In response to heat shock, these ncRNAs are transcribed by RNA polymerase III from short interspersed elements (SINEs) that are abundant in the genomes and prototypically considered ‘junk’ DNA. B2 RNA and Alu RNA bind to core RNA polymerase II with high affinity and enter stable complexes at promoters of specific genes, where they repress transcription. Structure/function studies revealed that B2 RNA and Alu RNA contain two types of functionally distinct domains: Pol II binding domains and transcriptional repression domains. Our studies lead to a model in which Pol II binding domains in ncRNA transcriptional repressors bind a high affinity docking site on RNA polymerase II, thereby tethering distinct repression domains to the polymerase in order to block transcription. Surprisingly, the functional domains in SINE ncRNAs, much like those of classical protein transcriptional regulators, are modular and can be mixed and matched to create chimeric ncRNA transcriptional repressors. Thus, SINE‐encoded ncRNAs resemble prototypical protein regulators of eukaryotic transcription. Our work provides an explanation for how RNA polymerase II transcription is repressed during heat shock in mouse and human cells, and provides an evolutionary rationale for the maintenance of SINEs in the mouse and human genomes.

Beating the Heat: A Translation Factor and an RNA Mobilize the Heat Shock Transcription Factor HSF1

Cells respond to stress via orchestrated modifications in gene expression; in a recent publication in Nature, Shamovsky et al. (2006) report that an RNA and a translation factor regulate the activity of a transcriptional activator essential to the mammalian heat shock response.

The role of heat shock transcription factor 1 in the genome-wide regulation of the mammalian heat shock response.

Previous work has implicated heat shock transcription factor 1 (HSF1) as the primary transcription factor responsible for the transcriptional response to heat stress in mammalian cells. We characterized the heat shock response of mammalian cells by measuring changes in transcript levels and assaying binding of HSF1 to promoter regions for candidate heat shock genes chosen by a combination of genome-wide computational and experimental methods. We found that many heat-inducible genes have HSF1 binding sites (heat shock elements, HSEs) in their promoters that are bound by HSF1. Surprisingly, for 24 heat-inducible genes, we detected no HSEs and no HSF1 binding. Furthermore, of 182 promoters with likely HSE sequences, we detected HSF1 binding at only 94 of these promoters. Also unexpectedly, we found 48 genes with HSEs in their promoters that are bound by HSF1 but that nevertheless did not show induction after heat shock in the cell types we examined. We also studied the transcriptional response to heat shock in fibroblasts from mice lacking the HSF1 gene. We found 36 genes in these cells that are induced by heat as well as they are in wild-type cells. These results provide evidence that HSF1 does not regulate the induction of every transcript that accumulates after heat shock, and our results suggest that an independent posttranscriptional mechanism regulates the accumulation of a significant number of transcripts.

Role of BRCA1 in heat shock response.

The heat shock response is an evolutionarily conserved response to heat and other stresses that promotes the maintenance of key metabolic functions and cell survival. We report that exposure of human prostate (DU-145) and breast (MCF-7) cancer cells to heat (42 degrees C) caused a rapid disappearance of the breast cancer susceptibility gene-1 (BRCA1) protein, starting at approximately 1 h after the onset of heating and slightly lagging behind the increase in heat shock protein 70 (HSP70) levels. The heat-induced loss of BRCA1 occurred at the protein level, since: (1) BRCA1 mRNA expression was unaffected; and (2) the BRCA1 protein loss was also observed in DU-145 cells that expressed exogenous wild-type BRCA1 (wtBRCA1). In addition to heat regulation of BRCA1 protein levels, we also found that BRCA1 could modulate the heat shock response. Thus, wtBRCA1 overexpressing DU-145 cell clones showed significantly decreased sensitivity to heat-induced cytotoxicity; and Brca1 mutant mouse embryo fibroblasts showed increased sensitivity to heat. The DU-145 wtBRCA1 clones also showed increased expression of the small heat shock protein HSP27; and reporter assays revealed that wtBRCA1 stimulated a two to four-fold increase in HSP27 promoter activity, consistent with its ability to upregulate HSP27 mRNA and protein levels. In studies using epitope-tagged truncated BRCA1 proteins, the ability to stimulate the HSP27 promoter and to mediate heat-induced degradation required the amino-terminus but not the carboxyl-terminus of BRCA1. Although the heat-induced loss of BRCA1 appeared to be due to protein degradation, various protein metabolic agents (or combinations) failed to block this event, including: MG132 (a 26S proteasomal inhibitor), N-acetyl-leucyl-leucyl-norleucinal (a calpain inhibitor), z-VAD-fmk (a pan-caspase inhibitor), and ammonium chloride and chloroquine (which stabilize lysosomes). These findings suggest that in addition to its other functions, BRCA1 may participate in mammalian heat shock response pathways.

Analysis of the mammalian heat-shock response. Inducible gene expression and heat-shock factor activity.

The evolutionarily conserved heat-shock response has been extensively studied as a model for transcriptional regulation. In eukaryotic cells, the regulation of heat-shock gene expression is mediated by a family of related proteins, the heat-shock transcription factors (HSFs) (, , , , , , , ). Smaller eukaryotes such as yeast and Drosophila melanogaster usually express single members of the HSF family (, , ), while larger eukaryotes express multiple HSFs. At least four HSF family members have been identified in vertebrate systems (, , , , ). Multiple HSFs may have arisen to allow expression of heat shock proteins under different conditions, such that divergent signaling pathways converge to result in the production of a common class of proteins, the heat-shock proteins.

Effects of neurohormonal stress and aging on the activation of mammalian heat shock factor 1.

The mammalian heat shock response has been investigated extensively using tissue culture cells with only a limited amount of information available on animals and intact tissues. The neurohormonal stress response mediated by the hypothalamic-pituitary-adrenal axis leads to the activation of heat shock factor (HSF) in rat adrenal tissue. Here we show through the use of antibodies specific to each member of the HSF family that restraint-induced stress in intact Wistar rats and adrenocorticotropic hormone treatment of hypophysectomized animals leads to the activation of HSF1 monomers to trimers with DNA-binding activity. Because HSF1 is also the target factor for metabolic and environmental stress, these data reveal an intersection of pathways leading to HSF1 activation. Comparison of the biochemical properties and levels of HSF1 in the Wistar and Fischer 344 rat strains reveals that HSF1 is constitutively present in an activated DNA-binding state in the adrenals of Fischer 344 rats. During aging, the levels of HSF1 remain constant, yet the transcription factor from aged animals exhibits a decreased ability to bind DNA.

Inducers of the heat shock response stimulate phospholipase C and phospholipase A2 activity in mammalian cells.

Although the mammalian heat shock response has been well characterized, the processes that mediate the induction of the response and the regulation of heat shock protein function are not completely understood. We have investigated the potential role in heat-shocked cells of phosphoinositide-specific phospholipase C (PLC), a membrane enzyme activity involved in transmembrane signal transduction. Our studies indicate that heat shock activates PLC in each of seven cell lines, including cells of human, rat, mouse, and hamster origin. Heat shock produced increases in inositol phosphate concentrations comparable in magnitude to those achieved after simulation with growth factors, indicating that heat shock might initiate transmembrane signaling cascades of potential importance in cellular regulation. Common cellular responses to heat and growth factors also included feedback modulation of PLC by its products and the parallel stimulation of phospholipase A2 activity. In addition to heat shock, other agents that induce the stress response stimulated PLC activity. The data indicate a close correlation between expression of the mammalian heat shock response and stimulation of PLC activity and indicate a possible role for this enzyme activity in the regulation of some aspects of the stress response.