Feedback regulation of the heat shock response.

The heat shock response is triggered primarily by nonnative proteins accumulating in a stressed cell and results in increased expression of heat shock proteins (Hsps), i.e., of chaperones capable of participating in the refolding or elimination of nonnative proteins. Best known is the transcriptional part of this response that is mediated predominantly by heat shock factor 1 (HSF1). HSF1 activity is regulated at different levels by Hsps and co-chaperones and is modulated further by a number of mechanisms involving other stress-regulated aspects of cell metabolism.

Induction of cell stress through gene transfer of an engineered heat shock transcription factor enhances tumor immunogenicity.

Heat shock protein expression and release is closely associated with immunogenic forms of cell death. We show that activation of the stress response within tumor cells during cell death, using an engineered form of the heat shock transcription factor, leads to an immunogenic death. Cells dying through 'stressful death' show decreased phagocytosis by macrophages in vitro. Moreover, cells expressing heat shock proteins during cell death are significantly more protective against subsequent tumor challenge. These data demonstrate the utility of activating cellular stress programs over the course of cytotoxic therapies to enhance immune responses to dying cells.

Evidence for a mechanism of repression of heat shock factor 1 transcriptional activity by a multichaperone complex.

In the absence of stress, human heat shock factor 1 (hHSF1) is in its unactivated form. hHSF1 polypeptide is in a dynamic heterocomplex with Hsp90 and is incapable of specifically binding DNA. When cells are stressed, heterocomplex assembly is disrupted. Unbound hHSF1 homotrimerizes, acquires DNA binding activity, and concentrates in the nucleus, but remains transcriptionally inactive. A subsequent reaction converts this inactive, trimeric form into the active, hyperphosphorylated transcription factor. Subsequent to the stressful event, hHSF1 is deactivated and eventually returned to its unactivated form. Evidence is presented herein that trimeric hHSF1 has the propensity to dynamically associate with an Hsp90-immunophilin-p23 complex through its regulatory domain. Formation of this heterocomplex results in repression of the transcriptional activity of trimeric hHSF1. Stress-denatured proteins effectively compete with trimeric hHSF1 for Hsp90-immunophilin-p23 complex, counteracting assembly of the heterocomplex and repression of hHSF1 transcriptional activity. This repression mechanism may be required for a proportional transcriptional response to stress. Formation of the heterocomplex may also represent the first step toward returning the hHSF1 to its unactivated form.

Geldanamycin restores a defective heat shock response in vivo.

Induced expression of heat shock proteins (Hsps) plays a central role in promoting cellular survival after environmental and physiological stress. We have previously shown that scrapie-infected mouse neuroblastoma (ScN2a) cells fail to induce the expression of Hsp72 and Hsp28 after various stress conditions. Here we present evidence that this impaired stress response is due to an altered regulation of HSF1 activity. Upon stress in ScN2a cells, HSF1 was converted into hyperphosphorylated trimers but failed to acquire transactivation competence. A kinetic analysis of HSF1 activation revealed that in ScN2a cells trimer formation after stress was efficient, but disassembly of trimers proceeded much faster than in the uninfected cell line. Geldanamycin, a Hsp90-binding drug, significantly delayed disassembly of HSF1 trimers after a heat shock and restored stress-induced expression of Hsp72 in ScN2a cells. Heat-induced Hsp72 expression required geldanamycin to be present; following removal of the drug ScN2a cells again lost their ability to mount a stress response. Thus, our studies show that a defective stress response can be pharmacologically restored and suggest that the HSF1 deactivation pathway may play an important role in the regulation of Hsp expression.

A transcriptional feedback loop for tissue-specific expression of highly cytotoxic genes which incorporates an immunostimulatory component.

Transcriptional targeting of cytotoxic genes is an important way to control toxicity associated with gene transfer therapies, but supposedly, tissue-specific promoters are often either very weak and/or leaky. In addition, the phenotypic leakiness of such tissue-specific promoters is dependent upon the toxicity of the gene being used. Therefore, we devised a transcriptional feedback loop to restrict gene expression of very potent genes to melanoma cells. We screened different elements of the human tyrosinase promoter to find one which gave no detectable expression in non-melanoma cells but was active in melanoma cell lines. This weak, but highly tissue specific, element (Tyr-300) was then used as the basis for a transcriptional amplification feedback loop in which a consensus heat shock element (HSE) was cloned upstream of Tyr-300. The cytotoxic gene was cloned downstream of the HSE-Tyr-300 element along with a mutated form of the heat shock factor-1 (HSF-1) transcription factor, which no longer requires cellular stress to activate its trimerisation, nuclear localisation and transcriptional activation properties. Low levels of expression from Tyr-300 initiated expression of both the cytotoxic and the HSF-1 genes in melanoma cells. Gradual build up of HSF-1 amplified expression through binding to the HSE to give levels of cytotoxicity similar to that provided by a CMV promoter. However, no leakiness was observed in multiple non-melanoma cell lines tested. In addition to amplifying low levels of weak tissue-specific expression, the use of HSF-1 also leads to activation of endogenous stress-related genes such as hsp70. Induction of these genes, in the presence of cell killing by the cytotoxic gene, is a highly immunostimulatory event which enhances the antitumour vaccination effects of direct tumour cell destruction. Having demonstrated the compatibility of the component elements in plasmid form, we incorporated the feedback loop into a hybrid LTR-modified retroviral vector and confirmed that the system can be effective in the form of a viral vector. The format of the feedback loop described here could be exploited for any tissue type in which a highly tissue-specific element can be identified but which is itself too weak to be effective therapeutically.

Sensitization of tumor cells to fas killing through overexpression of heat-shock transcription factor 1.

Activation of the heat-shock or stress response is generally considered a cytoprotective response to heat or other proteotoxic stresses. In mammalian cells, stress-induced transcription of heat-shock genes is regulated by heat-shock transcription factor 1 (HSF1). We now show that activation of the Fas death receptor transactivates HSF1 in HeLa cells, a Fas-expressing cervical carcinoma line. Whereas HSF1 is constitutively expressed in a non-DNA-binding, transcriptionally inactive state, activation of Fas leads to enhanced transcription of a heat-shock reporter gene. The effects of Fas on heat-shock-gene transcription do not appear to be a consequence of cell death as they (1) precede apoptotic changes and (2) are not abrogated by YVAD-CMK, an inhibitor of Fas apoptosis that acts by blocking downstream effector proteases. Despite expressing Fas, HeLa cells are relatively insensitive to Fas-mediated killing, indicating that Fas expression alone, although necessary, is not sufficient for apoptosis. By overexpressing a constitutively activated form of HSF1, we sensitize HeLa cells to Fas-mediated killing. These findings shed new light on the interaction between two of the most evolutionarily conserved cell programs in nature, the Fas death pathway and the heat-shock response. Strategies designed to upregulate HSF1 in tumor cells, either through pharmacologic or gene-therapy approaches will hopefully provide a means with which to sensitize tumors to the killing effects of cancer therapies operating through the Fas receptor.

Modulation of tolerance by mutant heat shock transcription factors.

It ought to be possible to recruit normal cellular defenses to mitigate ischemia/reperfusion damage and to reduce toxicity of chemotherapeutic drugs. Stress-preconditioned cells acquire a tolerant state characterized by increased resistance to such insults. This state is widely believed to be mediated, partially, by heat shock proteins (Hsps). Indirect evidence suggests that stress-induced Hsp expression is controlled by heat shock transcription factor 1 (Hsf1), which factor may therefore represent a preferred target for therapeutic modulation of tolerance. In support, positively acting (Hsf1(+)) and negatively acting (Hsf1(-)) mutants of Hsf1 were identified. Inhibition of endogenous Hsf1 activity by Hsf1(-) prevents stress-induced Hsp synthesis and development of tolerance. Hsf1(+) drastically enhances expression of major Hsps in the absence of stress and induces tolerance against heat, simulated ischemia and toxicity by cyclophosphamide. Where compared, tolerance induced was slightly better than that produced by heat preconditioning. Thus, development of the tolerant state is dependent on increased levels of the cohort of Hsps induced by stress preconditioning, and Hsf1 can induce accumulation of a typical set of Hsps, which proteins are alone capable of providing tolerance at a similar level as heat preconditioning. These findings make Hsf1 a preferred target for pharmacological intervention to deliberately induce tolerance.

Delivery of a constitutively active form of the heat shock factor using a virus vector protects neuronal cells from thermal or ischaemic stress but not from apoptosis.

The heat shock proteins (HSPs) are induced by stressful stimuli and have a protective effect. Different HSPs protect with different efficiencies against different stresses indicating that optimal protection would be obtained with a non-stressful agent which induced a range of HSPs. We have prepared a herpesvirus vector expressing a constitutively active mutant form of heat shock factor 1 (HSF1) which, unlike the wild-type form of this transcription factor, does not require stress for its activation. Upon infection of neuronal cells, this virus induced a more restricted range of HSPs than in non-neuronal cells. Infection with the virus protected neuronal cells against subsequent thermal or ischaemic stress in accordance with its ability to induce HSP70 expression but did not protect them against apoptotic stimuli. The mechanisms of these effects and their significance for the use of HSF to manipulate HSP gene expression is discussed.

Repression of heat shock transcription factor HSF1 activation by HSP90 (HSP90 complex) that forms a stress-sensitive complex with HSF1.

Heat shock and other proteotoxic stresses cause accumulation of nonnative proteins that trigger activation of heat shock protein (Hsp) genes. A chaperone/Hsp functioning as repressor of heat shock transcription factor (HSF) could make activation of hsp genes dependent on protein unfolding. In a novel in vitro system, in which human HSF1 can be activated by nonnative protein, heat, and geldanamycin, addition of Hsp90 inhibits activation. Reduction of the level of Hsp90 but not of Hsp/c70, Hop, Hip, p23, CyP40, or Hsp40 dramatically activates HSF1. In vivo, geldanamycin activates HSF1 under conditions in which it is an Hsp90-specific reagent. Hsp90-containing HSF1 complex is present in the unstressed cell and dissociates during stress. We conclude that Hsp90, by itself and/or associated with multichaperone complexes, is a major repressor of HSF1.

Effect of N-acetylcysteine on heat shock protein induction by acetaminophen in mouse liver.

It was previously shown that a necrogenic dose of acetaminophen (APAP) induced the 25- and 70-kDa heat shock proteins (hsp25 and hsp70i) in mouse liver, whereas nonnecrogenic doses failed to alter the level of either hsp. A strong correlation between the intralobular sites of APAP arylation of protein and hsp induction suggested that APAP-induced protein denaturation may play a role in triggering hsp induction. This study was conducted to determine whether APAP arylation of protein without concurrent toxicity could cause hsp induction. APAP (250 mg/kg i.p.) hepatotoxicity was eliminated using N-acetylcysteine (NAC, 300 mg/kg i.p.) or the cytochrome P-450 inhibitor diallyl sulfide (200 mg/kg p.o.). NAC did not inhibit APAP arylation of protein when administered 1 or 3 hr after the APAP dose but decreased binding by approximately 50% when administered at the same time as the APAP dose. Even though APAP hepatotoxicity was blocked by NAC administered 0 or 1 hr after the APAP dose, NAC did not inhibit the induction of hsp25 or hsp70i, indicating that APAP arylation of protein may play a key role in triggering hsp induction. Diallyl sulfide blocked APAP arylation of protein, hepatotoxicity, and induction of both hsps. These data are consistent with the hypothesis that toxicant adduction of protein triggers hsp induction.

Correlation between glutathione oxidation and trimerization of heat shock factor 1, an early step in stress induction of the Hsp response.

The heat shock protein (Hsp) response is induced by heat shock and a large variety of different chemicals. Searching for a common denominator of these different inducers, we and others developed the notion that all inducers may generate abnormally folded, i.e. non-native, proteins, and that such non-native proteins may trigger the Hsp response. Experimentation prompted by this notion resulted, for example, in the demonstration that chemically denatured proteins, introduced in cells by microinjection, can activate the response. Based on the chemical nature of inducers and on results reported from several studies, we hypothesized that inducers of the Hsp response may be generally capable of triggering oxidation of non-protein thiols, particularly glutathione. Such oxidation is known to lead to formation of glutathione-protein mixed disulfides and protein-protein disulfides. Presumably, thiol adduction and cross-linking would affect the structure of proteins involved, resulting in unfolding of a fraction of these proteins, causing heat shock factor (Hsf) activation. To test the feasibility of this hypothesis, thirteen different inducers were selected, and it was shown that all chemical inducers as well as heat shock cause drastic oxidation of glutathione under conditions under which they induce HSE DNA-binding activity. Under the same conditions, all chemical inducers and heat shock also cause trimerization of Hsf1. For several inducers, it was also shown that they enhance thiol oxidation of proteins. Finally, in vitro experiments support the notion that activation of Hsf1 does not require oxidation of the factor itself or of its coregulators. These results are in complete agreement with the above hypothesis.

Transcriptional activation of heat shock factor HSF1 probed by phosphopeptide analysis of factor 32P-labeled in vivo.

Mapping of tryptic phosphopeptides of heat shock factor 1 (HSF1) from non-stressed or moderately heat-stressed HeLa cells, labeled in vivo by [32P]orthophosphate, revealed four major phosphopeptides A to D. Heat stress drastically increased phosphopeptide signals. To identify target peptides and amino acids and to correlate phosphorylation and transactivation function, phosphopeptide maps were produced of LexA-human HSF1 chimeras and mutant derivatives thereof, and transactivation activities of original and mutant chimeras were compared. LexA-HSF1 chimeras were previously shown to be regulated identically to HSF1, except that they transactivate promoters with LexA-binding sites instead of hsp promoters. The patterns of phosphopeptides of LexA-HSF1 and endogenous HSF1 were similar. Analysis of single residue substitutions suggested that phosphopeptide C is peptide VKEEPPSPPQSPR (297-309) phosphorylated on Ser-307 but not Ser-303. Substitution of Ser-307 but not Ser-303 caused deregulation of factor activity. Mapping of several constitutively active chimeras associated unphosphorylated peptide C with the transcriptionally active HSF1 conformation, suggesting that dephosphorylation of this peptide (at Ser-307) may either be an integral step in the activation process or serve to maintain the active conformation of HSF1. Exploiting this correlation, indirect evidence was obtained that activation domains of HSF1 interact with the distantly located regulatory domain to maintain the factor in an inactive state.

Abnormalities in stress proteins in prion diseases.

1. Prion diseases include kuru, Creutzfeldt-Jakob disease (CJD), Gerstmann-Sträussler-Scheinker disease (GSS), and fatal familia insomnia (FFI) of humans, as well as scrapie and bovine spongiform encephalopathy (BSE) of animals. 2. All these disorders involve conversion of the normal, cellular prion protein (PrPC) into the corresponding scrapie isoform (PrPSc). PrPC adopts a structure rich in alpha-helices and devoid of beta-sheet, in contrast to PrPSc, which has a high beta-sheet content and is resistant to limited digestion by proteases. That a conformational transition features in the conversion of PrPC into PrPSc implies that prion diseases are disorders of protein conformation. 3. This concept has been extended by our studies with heat shock proteins (Hsp), many of which are thought to function as molecular chaperones. We found that the induction of some Hsps but not others was profoundly altered in scrapie-infected cells and that the distribution of Hsp73 is unusual in these cells. 4. Whether the conversion of PrPC into PrPSc is assisted by molecular chaperones, or if the accumulation of the abnormally folded PrPSc is complexed with Hsps remains to be established.

Differential heat shock protein induction by acetaminophen and a nonhepatotoxic regioisomer, 3'-hydroxyacetanilide, in mouse liver.

The effect of acetaminophen (APAP) and 3'-hydroxyacetanilide (AMAP) on heat shock protein (hsp) induction in mouse liver was examined using Western blotting and immunohistochemistry. Western blots from APAP (200 mg/kg i.p.)-treated mice showed increased hsp25 levels at 6 and 24 hr and increased hsp70i levels at 3, 6 and 24 hr. No apparent induction was observed for other hsps (hsp60, hsc70, or hsp90). No increase in the levels of any of the hsps was apparent in Western blots from AMAP (1000 mg/kg i.p.)-treated mice. Immunohistochemical localization of hsp25 and hsp70i in the liver after APAP treatment showed increases in the levels of both hsps within the zone of affected cells at early time points (3 and 6 hr), but at 24 hr, elevated hsp25 levels were observed primarily in cells on the periphery of the lesions. Hepatocytes with increased hsp25 or hsp70i levels also had detectable reactive metabolite binding from APAP, as determined using immunostaining. No hepatotoxicity was observed in liver sections from AMAP treated mice, even though immunostaining indicated widespread reactive metabolite binding. Immunostaining for hsps confirmed that no increase in hsp25 or hsp70i levels occurred in response to this binding. Differences in hsp expression after APAP vs. AMAP may be due to differences in protein targets adducted by their respective reactive metabolites, in the concentrations of adducted proteins or perhaps in some other differential effect necessary for hsp upregulation.

Heat shock protein induction in murine liver after acute treatment with cocaine.

The effect of cocaine on heat shock protein (hsp) induction in murine liver was examined using Western blotting and immunohistochemistry. A single dose of cocaine (50 mg/kg, intraperitoneal [i.p.]) was administered to naive, phenobarbital (PB)-induced or beta-naphthoflavone (betaNF)-induced mice, and the level of hsps in the liver analyzed 3, 6, and 24 hours after the cocaine dose. As measured by Western blotting, hsp70i levels were increased at all time points, and hsp25 levels at the 6- and 24-hour time points. Levels of hsp60, hsc70, and hsp90 remained unchanged. Pretreatment of mice with the cytochrome P-450 inhibitor SKF-525A eliminated both cocaine hepatotoxicity and the induced accumulation of hsp25 and hsp70i. Immunohistochemical localization of hsp25 and hsp70i in the liver showed that concentrations of both hsps were elevated only in cells with altered morphology. As has been observed previously, hepatic enzyme induction with PB or betaNF shifted the location of the necrotic lesion within the lobule from zone 2, as observed in naive mice of this strain, toward zone 1 (PB) or zone 3 (betaNF), respectively. Localization of induced accumulation of hsp25 and hsp70i was found to shift within the lobule in parallel with the necrotic lesion in these animals. Immunostaining of cocaine reactive metabolites bound to proteins was superimposable on the areas with hsp accumulation and cells with altered morphology. Our observations indicate a strong spatial correlation within the lobule between cocaine reactive metabolite formation, induced accumulation of hsp25 and hsp70i, and cytotoxicity (necrosis).

Activation signals regulate heat shock transcription factor 1 in human B lymphocytes.

We previously showed that the ability of human B lymphocytes to elicit a cytoprotective heat shock response when confronted by heat or other stresses was dependent upon the state of cell activation. This was unexpected, considering the highly conserved nature of the heat shock response and the widely held belief that all nonmutated mature cells were capable of eliciting a heat shock response when stressed. To elucidate the mechanism by which activation primes B cells to respond to stresses, we examined heat shock transcription factor 1 (hHSF1) in B cells since this factor appears to be solely responsible for stress-induced transcription of heat shock genes in human cells. In the current report, we show that hHSF1-DNA binding complexes are undetectable in extracts of unactivated B cells. In fact, hHSF1 protein is not constitutively expressed in unactivated B cells, nor is its synthesis stress-inducible. However, following activation, hHSF1 can be found in either a transcriptionally active or an inactive state, depending upon whether the cell has been stressed or not. Thus, activation pathways play an important role in enabling B cells to survive and function properly in the context of physiologic stresses by regulating hHSF1.

Hyperphosphorylation of heat shock transcription factor 1 is correlated with transcriptional competence and slow dissociation of active factor trimers.

In the course of its activation by heat and other stresses, the inactive monomer of human heat shock transcription factor 1 (HSF1) is converted to a DNA-binding homotrimer and is hyperphosphorylated. At least four Ser/Thr residues in HSF1 appeared to be inducibly phosphorylated during heat shock. Ser/Thr protein kinase inhibitors inhibited, and protein phosphatase inhibitor calyculin A and phorbol ester enhanced, hsp70-CAT reporter gene expression but not heat shock element DNA binding activity in HeLa cells undergoing a moderate heat shock. Calyculin A (5-20 nM) caused hyperphosphorylation of HSF1, the extent of which was comparable to that produced by moderate to severe heat shock. Upon recovery from a 42 degrees C/30 min-heat shock, HSF1 trimers disassembled quantitatively within 2 h. Calyculin A interfered with the dissociation of HSF1 trimers. Thus, hyperphosphorylation increases the effective half-life of the HSF1 trimer, which may prolong factor activity subsequent to heat shock. Hyperphosphorylation also dramatically stimulated the transactivation function of HSF1: exposure to calyculin A of cells induced to form inactive HSF1 trimers resulted in the conversion of the inactive to active trimers. Given that deletion of certain sequences renders HSF1 constitutively active, these results suggested that the activation of HSF1 trimers by calyculin A was a consequence of hyperphosphorylation of HSF1 rather than of a downstream factor.

Protection against hepatotoxicity by a single dose of amphetamine: the potential role of heat shock protein induction.

Amphetamine has been shown previously to increase levels of the inducible 70-kDa heat shock protein (hsp70i) in mouse liver. In the present study, the hepatic concentrations of a variety of hsps in livers of mice pretreated with amphetamine (15 mg/kg, i.p.) were evaluated, and the time course of hsp induction was examined. Amphetamine treatment caused an acute rise in core body temperature to 40 degrees C for at least 1 hr and increased hsp25 and hsp70i levels, as measured by Western blotting, at 6, 24, 48, and 72 hr with no apparent induction of other hsps (hsp60, hsc70, or hsp90). A 72-hr amphetamine pretreatment lowered the hepatotoxicity of an acute dose of acetaminophen (350 mg/kg, i.p.) or bromobenzene (0.45 ml/kg, i.p.), but had no effect on the toxicity of carbon tetrachloride (0.04 ml/kg, i.p.) or cocaine (50 mg/kg, i.p.), as measured by serum alanine aminotransferase activity and histopathological analysis. No protection from acetaminophen or bromobenzene hepatotoxicity was observed when hepatotoxicant administration was delayed until hsp levels had returned to control values (144 hr after amphetamine pretreatment). Amphetamine pretreatment did not reduce in vivo covalent binding to proteins of radiolabeled [3H]acetaminophen, [14C]bromobenzene, [14C]carbon tetrachloride, or [3H]cocaine, indicating that the protective effects were not due to inhibition of reactive metabolite formation from these toxicants. These results suggest that elevated levels of hsp25 and hsp70i provide protection against acetaminophen and bromobenzene hepatotoxicity.

Induction of hsp 70 in HepG2 cells in response to hepatotoxicants.

The objective of this study was to determine if a variety of hepatotoxicants could induce the level of heat shock protein 70I, and whether or not elevated levels of heat shock proteins (hsp's) could provide cytoprotection from those hepatotoxicants. Exposure of HepG2 cells to cytotoxic concentrations of bromobenzene, cadmium, cyclophosphamide, or diethylnitrosamine increased the level of hsp 70I protein and mRNA, while carbon tetrachloride and cocaine had no effect on hsp 70I or mRNA levels. To determine if induction of hsp 70I might afford protection against cytotoxicity, HepG2 cells were given a prior sublethal heat shock (sub-LHS) (43 degrees C for 1 hr) to induce hsp's and then challenged 24 hr later with the hepatotoxicants. Sub-LHS pretreatment diminished toxicity from bromobenzene, cadmium, cyclophosphamide, or diethyl-nitrosamine, but not carbon tetrachloride or cocaine. In cells treated with [14C]carbon tetrachloride or [3H]cocaine, no detectable covalent binding to proteins was observed; whereas, [14C]-bromobenzene treatment resulted in substantial covalent binding to cellular protein. The apparent absence of formation of reactive metabolite adducted proteins from cocaine and carbon tetrachloride may explain why no hsp 70I induction was observed with these agents. The correlation between hepatotoxicant induction of hsp 70I and cytoprotection afforded by sub-LHS pretreatment suggests that hsp 70I induction may represent an important cellular defense mechanism in the liver.