Stress-specific activation and repression of heat shock factors 1 and 2.

Vertebrate cells express a family of heat shock transcription factors (HSF1 to HSF4) that coordinate the inducible regulation of heat shock genes in response to diverse signals. HSF1 is potent and activated rapidly though transiently by heat shock, whereas HSF2 is a less active transcriptional regulator but can retain its DNA binding properties for extended periods. Consequently, the differential activation of HSF1 and HSF2 by various stresses may be critical for cells to survive repeated and diverse stress challenges and to provide a mechanism for more precise regulation of heat shock gene expression. Here we show, using a novel DNA binding and detection assay, that HSF1 and HSF2 are coactivated to different levels in response to a range of conditions that cause cell stress. Above a low basal activity of both HSFs, heat shock preferentially activates HSF1, whereas the amino acid analogue azetidine or the proteasome inhibitor MG132 coactivates both HSFs to different levels and hemin preferentially induces HSF2. Unexpectedly, we also found that heat shock has dramatic adverse effects on HSF2 that lead to its reversible inactivation coincident with relocalization from the nucleus. The reversible inactivation of HSF2 is specific to heat shock and does not occur with other stressors or in cells expressing high levels of heat shock proteins. These results reveal that HSF2 activity is negatively regulated by heat and suggest a role for heat shock proteins in the positive regulation of HSF2.

The loop domain of heat shock transcription factor 1 dictates DNA-binding specificity and responses to heat stress.

Eukaryotic heat shock transcription factors (HSF) regulate an evolutionarily conserved stress-response pathway essential for survival against a variety of environmental and developmental stresses. Although the highly similar HSF family members have distinct roles in responding to stress and activating target gene expression, the mechanisms that govern these roles are unknown. Here we identify a loop within the HSF1 DNA-binding domain that dictates HSF isoform specific DNA binding in vitro and preferential target gene activation by HSF family members in both a yeast transcription assay and in mammalian cells. These characteristics of the HSF1 loop region are transposable to HSF2 and sufficient to confer DNA-binding specificity, heat shock inducible HSP gene expression and protection from heat-induced apoptosis in vivo. In addition, the loop suppresses formation of the HSF1 trimer under basal conditions and is required for heat-inducible trimerization in a purified system in vitro, suggesting that this domain is a critical part of the HSF1 heat-stress-sensing mechanism. We propose that this domain defines a signature for HSF1 that constitutes an important determinant for how cells utilize a family of transcription factors to respond to distinct stresses.

Phosphorylation of serine 230 promotes inducible transcriptional activity of heat shock factor 1.

Heat shock factor 1 (HSF1) is a serine-rich constitutively phosphorylated mediator of the stress response. Upon stress, HSF1 forms DNA-binding trimers, relocalizes to nuclear granules, undergoes inducible phosphorylation and acquires the properties of a transactivator. HSF1 is phosphorylated on multiple sites, but the sites and their function have remained an enigma. Here, we have analyzed sites of endogenous phosphorylation on human HSF1 and developed a phosphopeptide antibody to identify Ser230 as a novel in vivo phosphorylation site. Ser230 is located in the regulatory domain of HSF1, and promotes the magnitude of the inducible transcriptional activity. Ser230 lies within a consensus site for calcium/calmodulin-dependent protein kinase II (CaMKII), and CaMKII overexpression enhances both the level of in vivo Ser230 phosphorylation and transactivation of HSF1. The importance of Ser230 was further established by the S230A HSF1 mutant showing markedly reduced activity relative to wild-type HSF1 when expressed in hsf1(-/-) cells. Our study provides the first evidence that phosphorylation is essential for the transcriptional activity of HSF1, and hence for induction of the heat shock response.

Odorants as cell-type specific activators of a heat shock response in the rat olfactory mucosa.

Heat shock, or stress, proteins (HSPs) are induced in response to conditions that cause protein denaturation. Activation of cellular stress responses as a protective and survival mechanism is often associated with chemical exposure. One interface between the body and the external environment and chemical or biological agents therein is the olfactory epithelium (OE). To determine whether environmental odorants affect OE HSP expression, rats were exposed to a variety of odorants added to the cage bedding. Odorant exposure led to transient, selective induction of HSP70, HSC70, HSP25, and ubiquitin immunoreactivities (IRs) in supporting cells and subepithelial Bowman's gland acinar cells, two OE non-neuronal cell populations involved with inhalant biotransformation, detoxification, and maintenance of overall OE integrity. Responses exhibited odor specificity and dose dependency. HSP70 and HSC70 IRs occurred throughout the apical region of supporting cells; ubiquitin IR was confined to a supranuclear cone-shaped region. Electron microscopic examination confirmed these observations and, additionally, revealed odor-induced formation of dense vesicular arrays in the cone-like regions. HSP25 IR occurred throughout the entire supporting cell cytoplasm. In contrast to classical stress responses, in which the entire array of stress proteins is induced, no increases in HSP40 and HSP90 IRs were observed. Extended exposure to higher odorant doses caused prolonged activation of the same HSP subset in the non-neuronal cells and severe morphological damage in both supporting cells and olfactory receptor neurons (ORNs), suggesting that non-neuronal cytoprotective stress response mechanisms had been overwhelmed and could no longer adequately maintain OE integrity. Significantly, ORNs showed no stress responses in any of our studies. These findings suggest a novel role for these HSPs in olfaction and, in turn, possible involvement in other normal neurophysiological processes.

Bag1-Hsp70 mediates a physiological stress signalling pathway that regulates Raf-1/ERK and cell growth.

Survival after stress requires the precise orchestration of cell-signalling events to ensure that biosynthetic processes are alerted and cell survival pathways are initiated. Here we show that Bag1, a co-chaperone for heat-shock protein 70 (Hsp70), coordinates signals for cell growth in response to cell stress, by downregulating the activity of Raf-1 kinase. Raf-1 and Hsp70 compete for binding to Bag1, such that Bag1 binds to and activates Raf-1, subsequently activating the downstream extracellular signal-related kinases (ERKs). When levels of Hsp70 are elevated after heat shock, or in cells conditionally overexpressing Hsp70, Bag1-Raf-1 is displaced by Bag1-Hsp70, and DNA synthesis is arrested. Mutants Bag1C204A and Bag1E208A, which cannot bind Hsp70, constitutively activate Raf-1/ERK kinases but are unaffected by Hsp70; consequently neither Bag1-Raf-1 nor DNA synthesis is negatively affected during heat shock. Likewise, mutants Hsp70F245S, Hsp70R262W and Hsp70L282R, which retain chaperone activity but do not bind to Bag1, fail to repress Bag1 activation of Raf-1/ERK kinase. We propose that Bag1 functions in the heat-shock response to coordinate cell growth signals and mitogenesis, and that Hsp70 functions as a sensor in stress signalling.

Reversible inhibition of Hsp70 chaperone function by Scythe and Reaper.

Protein folding mediated by the Hsp70 family of molecular chaperones requires both ATP and the co-chaperone Hdj-1. BAG-1 was recently identified as a bcl-2-interacting, anti-apoptotic protein that binds to the ATPase domain of Hsp70 and prevents the release of the substrate. While this suggested that cells had the potential to modulate Hsp70-mediated protein folding, physiological regulators of BAG-1 have yet to be identified. We report here that the apoptotic regulator Scythe, originally isolated through binding to the potent apoptotic inducer Reaper, shares limited sequence identity with BAG-1 and inhibits Hsp70- mediated protein refolding. Scythe-mediated inhibition of Hsp70 is reversed by Reaper, providing evidence for the regulated reversible inhibition of chaperone activity. As Scythe functions downstream of Reaper in apoptotic induction, these findings suggest that Scythe/Reaper may signal apoptosis, in part through regulating the folding and activity of apoptotic signaling molecules.

Role of the heat shock response and molecular chaperones in oncogenesis and cell death.

Exposure of cells to conditions of environmental stress-including heat shock, oxidative stress, heavy metals, or pathologic conditions, such as ischemia and reperfusion, inflammation, tissue damage, infection, and mutant proteins associated with genetic diseases-results in the inducible expression of heat shock proteins that function as molecular chaperones or proteases. Molecular chaperones are a class of proteins that interact with diverse protein substrates to assist in their folding, with a critical role during cell stress to prevent the appearance of folding intermediates that lead to misfolded or otherwise damaged molecules. Consequently, heat shock proteins assist in the recovery from stress either by repairing damaged proteins (protein refolding) or by degrading them, thus restoring protein homeostasis and promoting cell survival. The events of cell stress and cell death are linked, such that molecular chaperones induced in response to stress appear to function at key regulatory points in the control of apoptosis. On the basis of these observations-and on the role of molecular chaperones in the regulation of steroid aporeceptors, kinases, caspases, and other protein remodeling events involved in chromosome replication and changes in cell structure-it is not surprising that the heat shock response and molecular chaperones have been implicated in the control of cell growth. In this review, we address some of the molecular and cellular events initiated by cell stress-the interrelationships between stress signaling, cell death, and oncogenesis-and chaperones as potential targets for cancer diagnosis and treatment.

The chaperone function of hsp70 is required for protection against stress-induced apoptosis.

Cellular stress can trigger a process of self-destruction known as apoptosis. Cells can also respond to stress by adaptive changes that increase their ability to tolerate normally lethal conditions. Expression of the major heat-inducible protein hsp70 protects cells from heat-induced apoptosis. hsp70 has been reported to act in some situations upstream or downstream of caspase activation, and its protective effects have been said to be either dependent on or independent of its ability to inhibit JNK activation. Purified hsp70 has been shown to block procaspase processing in vitro but is unable to inhibit the activity of active caspase 3. Since some aspects of hsp70 function can occur in the absence of its chaperone activity, we examined whether hsp70 lacking its ATPase domain or the C-terminal EEVD sequence that is essential for peptide binding was required for the prevention of apoptosis. We generated stable cell lines with tetracycline-regulated expression of hsp70, hsc70, and chaperone-defective hsp70 mutants lacking the ATPase domain or the C-terminal EEVD sequence or containing AAAA in place of EEVD. Overexpression of hsp70 or hsc70 protected cells from heat shock-induced cell death by preventing the processing of procaspases 9 and 3. This required the chaperone function of hsp70 since hsp70 mutant proteins did not prevent procaspase processing or provide protection from apoptosis. JNK activation was inhibited by both hsp70 and hsc70 and by each of the hsp70 domain mutant proteins. The chaperoning activity of hsp70 is therefore not required for inhibition of JNK activation, and JNK inhibition was not sufficient for the prevention of apoptosis. Release of cytochrome c from mitochondria was inhibited in cells expressing full-length hsp70 but not in cells expressing the protein with ATPase deleted. Together with the recently identified ability of hsp70 to inhibit cytochrome c-mediated procaspase 9 processing in vitro, these data demonstrate that hsp70 can affect the apoptotic pathway at the levels of both cytochrome c release and initiator caspase activation and that the chaperone function of hsp70 is required for these effects.

Heat-shock protein 70 inhibits apoptosis by preventing recruitment of procaspase-9 to the Apaf-1 apoptosome.

The cellular-stress response can mediate cellular protection through expression of heat-shock protein (Hsp) 70, which can interfere with the process of apoptotic cell death. Stress-induced apoptosis proceeds through a defined biochemical process that involves cytochrome c, Apaf-1 and caspase proteases. Here we show, using a cell-free system, that Hsp70 prevents cytochrome c/dATP-mediated caspase activation, but allows the formation of Apaf-1 oligomers. Hsp70 binds to Apaf-1 but not to procaspase-9, and prevents recruitment of caspases to the apoptosome complex. Hsp70 therefore suppresses apoptosis by directly associating with Apaf-1 and blocking the assembly of a functional apoptosome.

Polyglutamine aggregates alter protein folding homeostasis in Caenorhabditis elegans.

Expansion of polyglutamine repeats in several unrelated proteins causes neurodegenerative diseases with distinct but related pathologies. To provide a model system for investigating common pathogenic features, we have examined the behavior of polyglutamine expansions expressed in Caenorhabditis elegans. The expression of polyglutamine repeats as green fluorescent protein (GFP)-fusion proteins in body wall muscle cells causes discrete cytoplasmic aggregates that appear early in embryogenesis and correlates with a delay in larval to adult development. The heat shock response is activated idiosyncratically in individual cells in a polyglutamine length-dependent fashion. The toxic effect of polyglutamine expression and the formation of aggregates can be reversed by coexpression of the yeast chaperone Hsp104. The altered homeostasis associated with polyglutamine aggregates causes both the sequestration of an otherwise soluble protein with shorter arrays of glutamine repeats and the relocalization of a nuclear glutamine-rich protein. These observations of induced aggregation and relocalization have implications for disorders involving protein aggregation.

Bag1 functions in vivo as a negative regulator of Hsp70 chaperone activity.

Studies on the Hsp70 chaperone machine in eukaryotes have shown that Hsp70 and Hsp40/Hdj1 family proteins are sufficient to prevent protein misfolding and aggregation and to promote refolding of denatured polypeptides. Additional protein cofactors include Hip and Bag1, identified in protein interaction assays, which bind to and modulate Hsp70 chaperone activity in vitro. Bag1, originally identified as an antiapoptotic protein, forms a stoichiometric complex with Hsp70 and inhibits completely Hsp70-dependent in vitro protein refolding of an unfolded polypeptide. Given its proposed involvement in multiple cell signaling events as a regulator of Raf1, Bcl2, or androgen receptor, we wondered whether Bag1 functions in vivo as a negative regulator of Hsp70. In this study, we demonstrate that Bag1, expressed in mammalian tissue culture cells, has pronounced effects on one of the principal activities of Hsp70, as a molecular chaperone essential for stabilization and refolding of a thermally inactivated protein. The levels of Hsp70 and Bag1 were modulated either by transient transfection or conditional expression in stably transfected lines to achieve levels within the range detected in different mammalian tissue culture cell lines. For example, a twofold increase in the concentration of Bag1 reduced Hsp70-dependent refolding of denatured luciferase by a factor of 2. This effect was titratable, and higher levels of wild-type but not a mutant form of Bag1 further inhibited Hsp70 refolding by up to a factor of 5. The negative effects of Bag1 were also observed in a biochemical analysis of Bag1- or Hsp70-overexpressing cells. The ability of Hsp70 to maintain thermally denatured firefly luciferase in a soluble state was reversed by Bag1, thus providing an explanation for the in vivo chaperone-inhibitory effects of Bag1. Similar effects on Hsp70 were observed with other cytoplasmic isoforms of Bag1 which have in common the carboxyl-terminal Hsp70-binding domain and differ by variable-length amino-terminal extensions. These results provide the first formal evidence that Bag1 functions in vivo as a regulator of Hsp70 and suggest an intriguing complexity for Hsp70-regulatory events.

A method for the quantitative analysis of human heat shock gene expression using a multiplex RT-PCR assay.

A quantitative multiplex RT-PCR assay is described to measure the levels of messenger RNAs for eight human genes encoding the heat shock proteins (HSP) and molecular chaperones hsp90alpha, hsp90beta, hsp70, hsc70, mtHsp75, Grp78 (BiP), hsp60 and hsp27. The basis of this assay is reverse transcription of total RNA isolated from human cells followed by amplification with PCR. By the careful selection of pairs of oligonucleotide primers corresponding to unique regions of each heat shock gene, selectivity can be attained such that messenger RNAs of multiple heat shock genes can be analyzed simultaneously in a single reaction. This method provides both the absolute and relative levels of each heat shock message by including in the reaction, reference control RNAs corresponding to in vitro transcripts of heat shock gene plasmids carrying small internal deletions.

Stress and the cell nucleus: dynamics of gene expression and structural reorganization.

A growing number of experimental observations reveal that the cell nucleus is functionally compartmentalized yet organized to ensure a dynamic response to events that influence nuclear activities. The cellular and molecular response to physiological and environmental stress induces a rapid and transient change in gene expression associated with major changes in nuclear architecture that impacts on signals involved in cell growth. In this review, we will address the effects of stress on the functional compartmentation of the cell nucleus and the dynamic reorganization of nuclear structures and function.

Fine mapping of the friend retrovirus resistance gene, Rfv3, on mouse chromosome 15.

Rfv3 is a host resistance gene that operates through an unknown mechanism to control the development of the virus-neutralizing antibody response required for recovery from infection with Friend retrovirus. The Rfv3 gene was previously mapped to an approximately 20-centimorgan (cM) region of chromosome 15. More refined mapping was not possible, due to a lack of microsatellite markers and leakiness in the Rfv3 phenotype, which prevented definitive phenotyping of individual recombinant mice. In the present study, we overcame these difficulties by taking advantage of seven new microsatellite markers in the Rfv3 region and by using progeny tests to accurately determine the Rfv3 phenotype of recombinant mice. Detailed linkage analysis of relevant crossovers narrowed the location of Rfv3 to a 0.83-cM region. Mapping of closely linked genes in an interspecific backcross panel allowed us to exclude two previous candidate genes, Ly6 and Wnt7b. These studies also showed for the first time that the Hsf1 gene maps to the Rfv3-linked cluster of genes including Il2rb, Il3rb, and Pdgfb. This localization of Rfv3 to a region of less than 1 cM now makes it feasible to attempt the cloning of Rfv3 by physical methods.

Intron-independent association of splicing factors with active genes.

The cell nucleus is organized as discrete domains, often associated with specific events involved in chromosome organization, replication, and gene expression. We have examined the spatial and functional relationship between the sites of heat shock gene transcription and the speckles enriched in splicing factors in primary human fibroblasts by combining immunofluorescence and fluorescence in situ hybridization (FISH). The hsp90alpha and hsp70 genes are inducibly regulated by exposure to stress from a low basal level to a high rate of transcription; additionally the hsp90alpha gene contains 10 introns whereas the hsp70 gene is intronless. At 37 degrees C, only 30% of hsp90alpha transcription sites are associated with speckles whereas little association is detected with the hsp70 gene, whose constitutive expression is undetectable relative to the hsp90alpha gene. Upon exposure of cells to heat shock, the heavy metal cadmium, or the amino acid analogue azetidine, transcription at the hsp90alpha and hsp70 gene loci is strongly induced, and both hsp transcription sites become associated with speckles in >90% of the cells. These results reveal a clear disconnection between the presence of intervening sequences at specific gene loci and the association with splicing factor-rich regions and suggest that subnuclear structures containing splicing factors are associated with sites of transcription.

Rapid and reversible relocalization of heat shock factor 1 within seconds to nuclear stress granules.

Heat shock factor 1 (HSF1) is essential for the stress-induced expression of heat shock genes. On exposure to heat shock, HSF1 localizes within seconds to discrete nuclear granules. On recovery from heat shock, HSF1 rapidly dissipates from these stress granules to a diffuse nucleoplasmic distribution, typical of unstressed cells. Subsequent reexposure to heat shock results in the rapid relocalization of HSF1 to the same stress granules with identical kinetics. Although the appearance of HSF1 stress granules corresponds to the hyperphosphorylated, trimeric DNA-binding state of HSF1 and correlates temporally with the inducible transcription of heat shock genes, they are also present in heat-shocked mitotic cells that are devoid of transcription. This finding suggests a role for HSF1 stress granules as a nuclear compartment for the temporal regulation and spatial organization of HSF1 activity and reveals new features of the dynamics of nuclear organization.

Structure of a new crystal form of human Hsp70 ATPase domain.

Hsp70 proteins are highly conserved proteins induced by heat shock and other stress conditions. An ATP-binding domain of human Hsp70 protein has been crystallized in two major morphological forms at pH 7.0 in the presence of PEG 8000 and CaCl2. Both crystal forms belong to the orthorhombic space group P212121, but show no resemblance in unit-cell parameters. Analysis of the crystal structures for both forms shows a 1-2 A shift of one of the subdomains of the protein. This conformational change could reflect a 'natural' flexibility of the protein which might be relevant to ATP binding and may facilitate the interaction of other proteins with Hsp70 protein.

Stress-induced activation of the heat-shock response: cell and molecular biology of heat-shock factors.

Exposure of cells to environmental and physiological stress leads to an imbalance in protein metabolism, which challenges the cell to respond rapidly and precisely to the deleterious effects of stress on protein homoeostasis. The heat-shock response, through activation of heat-shock transcription factors (HSFs) and the elevated expression of heat-shock proteins and molecular chaperones, protects the cell against the accumulation of non-native proteins. Activation of HSF1 involves a complex multi-step pathway in which the inert monomer oligomerizes to a DNA-binding, transcriptionally active, trimer which relocalizes within the the nucleus to form stress-induced HSF1 granules. Attenuation of the heat-shock response involves molecular chaperones which repress the HSF1 transactivation domain and HSF-binding protein 1 (HSBP1), which interacts with the HSF1 oligomerization domain of HSF1 to negatively regulate its activity, thus insuring that the expression of chaperones is precisely determined.