Protective Effect Of Heat Shock Research Articles

Exosomes mediate sensory hair cell protection in the inner ear.

Hair cells, the mechanosensory receptors of the inner ear, are responsible for hearing and balance. Hair cell death and consequent hearing loss are common results of treatment with ototoxic drugs, including the widely used aminoglycoside antibiotics. Induction of heat shock proteins (HSPs) confers protection against aminoglycoside-induced hair cell death via paracrine signaling that requires extracellular heat shock 70-kDa protein (HSP70). We investigated the mechanisms underlying this non-cell-autonomous protective signaling in the inner ear. In response to heat stress, inner ear tissue releases exosomes that carry HSP70 in addition to canonical exosome markers and other proteins. Isolated exosomes from heat-shocked utricles were sufficient to improve survival of hair cells exposed to the aminoglycoside antibiotic neomycin, whereas inhibition or depletion of exosomes from the extracellular environment abolished the protective effect of heat shock. Hair cell-specific expression of the known HSP70 receptor TLR4 was required for the protective effect of exosomes, and exosomal HSP70 interacted with TLR4 on hair cells. Our results indicate that exosomes are a previously undescribed mechanism of intercellular communication in the inner ear that can mediate nonautonomous hair cell survival. Exosomes may hold potential as nanocarriers for delivery of therapeutics against hearing loss.

Heat shock protein-mediated protection against Cisplatin-induced hair cell death.

Cisplatin is a highly successful and widely used chemotherapy for the treatment of various solid malignancies in both adult and pediatric patients. Side effects of cisplatin treatment include nephrotoxicity and ototoxicity. Cisplatin ototoxicity results from damage to and death of cells in the inner ear, including sensory hair cells. We showed previously that heat shock inhibits cisplatin-induced hair cell death in whole-organ cultures of utricles from adult mice. Since heat shock protein 70 (HSP70) is the most upregulated HSP in response to heat shock, we investigated the role of HSP70 as a potential protectant against cisplatin-induced hair cell death. Our data using utricles from HSP70 (-/-) mice indicate that HSP70 is necessary for the protective effect of heat shock against cisplatin-induced hair cell death. In addition, constitutive expression of inducible HSP70 offered modest protection against cisplatin-induced hair cell death. We also examined a second heat-inducible protein, heme oxygenase-1 (HO-1, also called HSP32). HO-1 is an enzyme responsible for the catabolism of free heme. We previously showed that induction of HO-1 using cobalt protoporphyrin IX (CoPPIX) inhibits aminoglycoside-induced hair cell death. Here, we show that HO-1 also offers significant protection against cisplatin-induced hair cell death. HO-1 induction occurred primarily in resident macrophages, with no detectable expression in hair cells or supporting cells. Depletion of macrophages from utricles abolished the protective effect of HO-1 induction. Together, our data indicate that HSP induction protects against cisplatin-induced hair cell death, and they suggest that resident macrophages mediate the protective effect of HO-1 induction.

Inner ear supporting cells protect hair cells by secreting HSP70

Mechanosensory hair cells are the receptor cells of hearing and balance. Hair cells are sensitive to death from exposure to therapeutic drugs with ototoxic side effects, including aminoglycoside antibiotics and cisplatin. We recently showed that the induction of heat shock protein 70 (HSP70) inhibits ototoxic drug-induced hair cell death. Here, we examined the mechanisms underlying the protective effect of HSP70. In response to heat shock, HSP70 was induced in glia-like supporting cells but not in hair cells. Adenovirus-mediated infection of supporting cells with Hsp70 inhibited hair cell death. Coculture with heat-shocked utricles protected nonheat-shocked utricles against hair cell death. When heat-shocked utricles from Hsp70-/- mice were used in cocultures, protection was abolished in both the heat-shocked utricles and the nonheat-shocked utricles. HSP70 was detected by ELISA in the media surrounding heat-shocked utricles, and depletion of HSP70 from the media abolished the protective effect of heat shock, suggesting that HSP70 is secreted by supporting cells. Together our data indicate that supporting cells mediate the protective effect of HSP70 against hair cell death, and they suggest a major role for supporting cells in determining the fate of hair cells exposed to stress.

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.

Heat Shock Induces Barotolerance in Listeria monocytogenes

The aim of this study was to investigate the effect of heat shock on the resistance of Listeria monocytogenes to high pressure processing (HPP). L. monocytogenes ATCC 19115 was grown to stationary phase at 15°C and inoculated into whole ultrahigh-temperature milk at ~107 CFU/ml. Milk samples (5 ml) were placed into plastic transfer pipettes, which were heat sealed and then heated in a water bath at 48°C for 10 min. Immediately after heat shock, the milk was cooled in water (20°C) for 25 min and then placed on ice. The samples were high pressure processed at ambient temperature (~23°C) at 400 MPa for various times up to 150 s. Following HPP, the samples were spread plated on tryptic soy agar supplemented with yeast extract. Heat shock significantly increased the D400 MPa-value of L. monocytogenes from 35 s in non–heat-shocked cells to 127 s in heat-shocked cells (P < 0.05). Addition of chloramphenicol before heat shock eliminated the protective effect of heat shock (P < 0.05). Heat shock for 5, 10, 15, or 30 min at 48°C resulted in maximal barotolerance (P < 0.05); increasing the time to 60 min significantly decreased survival compared with that at 5, 10, 15, or 30 min (P < 0.05). These results indicate that prior heat shock significantly increases the barotolerance of L. monocytogenes and that de novo protein synthesis during heat shock is required for this enhanced barotolerance.

Worms with Fevers

Ever wondered whether taking medicine to reduce a fever might inhibit a protective response of the body to infection? New evidence for such a possibility is presented by Singh and Aballay, who report studies of the effects of increased temperature on immunity of the nematode C. elegans to bacterial infections. Worms don't have fevers, of course, but such poikilotherms are known to respond to infection by migrating toward a warmer environment. Exposure of C. elegans to heat shock (incubation at 32°C for 2 hours) increased resistance and delayed the death of animals exposed to the human pathogen Pseudomonas aerugninosa . The p38 MAPK (mitogen-activated protein kinase) pathway was not required for this effect, but the heat-shock transcription factor HSF-1 was. No protective effect of heat shock was evident in animals treated with hsf-1 RNAi, and overexpression of HSF-1 made animals more resistant to infection. Genes encoding the heat-shock protein (HSP) chaperones are controlled by HSF-1, and RNAi to HSP-encoding genes increased susceptibility of the worms to infection. The forkhead transcription factor DAF-16 regulates genes that influence longevity as well as stress responses and is positively regulated by heat shock. Animals expressing additional gene copies of daf-16 or with mutations in daf-2 (a gene encoding an insulin-like negative regulator of DAF-16) showed increased resistance to pathogens that was reduced in animals in which function of HSF-1 was inhibited by RNAi. Thus, HSF-1 appears to be part of a protective heat-induced response to infection. The authors propose that therapeutic agents aimed at activation of the HSF-1 pathway (without the additional dangerous effects of high fevers) might be effective for treatment of bacterial infections. V. Singh, A. Aballay, Heat-shock transcription factor (HSF)-1 pathway required for Caenorhabditis elegans immunity. Proc. Natl. Acad. Sci. U.S.A. 103 , 13092-13097 (2006). [Abstract] [Full Text]

Heat Shock–Induced Protection of Renal Proximal Tubular Epithelial Cells from Cold Storage and Rewarming Injury

Cold storage and reperfusion injury to transplanted kidneys contributes to increased incidence of delayed graft function and may have a negative impact on graft survival. This study examined the mechanisms by which previous heat shock protects against cell death in an in vitro model of kidney storage. Cold storage is mimicked by incubating human renal proximal tubular epithelial (HK-2) cells in University of Wisconsin solution at 4 degrees C with and without subsequent rewarming. Heat shock was induced by incubation of cells at 42 degrees C for 1 h. Altered protein expression was measured by Western blot, and cell viability and apoptosis were measured by propidium iodide DNA staining using flow cytometry. The specific role of heat-shock protein 70 (HSP-70) was determined both by siRNA knockdown and by stable overexpression approaches. Cold storage and rewarming-induced cell death was associated with decreased expression of HSP-70, HSP-90, HSP-27, and Bcl-2. Previous heat shock significantly reduced HK-2 cell death after cold storage and rewarming and was associated with the maintenance of HSP-70, HSP-27, and Bcl-2 protein levels. Blocking heat stress-induced HSP-70 with siRNA did not significantly block the protective effect of heat stress against cold storage and rewarming cell death; however, overexpression of HSP-70 protected HK-2 cells from this stress. It is concluded that previous heat shock protects HK-2 cells from cold storage and rewarming injury. siRNA inhibition of HSP-70 induction did not block the protective effect of heat shock, indicating that HSP-70 is not essential to the heat stress-induced protective effect reported in this study.

Heat shock prevents simulated ischemia-induced apoptosis in renal tubular cells via a PKC-dependent mechanism.

Heat shock produces cellular tolerance to a variety of adverse conditions; however, the protective effect of heat shock on renal cell ischemic injury remains unclear. Protein kinase C (PKC) has been implicated in the signaling mechanisms of acute preconditioning, yet it remains unknown whether PKC mediates heat shock-induced delayed preconditioning in renal cells. To study this, renal tubular cells (LLC-PK1) were exposed to thermal stress (43 degrees C) for 1 h and heat shock protein (HSP) 72 induction was confirmed by Western blot analysis. Cells were subjected to simulated ischemia 24 h after thermal stress, and the effect of heat shock (delayed preconditioning) on ischemia-induced apoptosis (terminal deoxynucleotidyl transferase dUTP nick-end labeling) and B cell lymphoma 2 (Bcl(2)) expression (Western) was determined. Subsequently, the effect of PKC inhibition on HSP72 induction and heat stress-induced ischemic tolerance was evaluated. Thermal stress induced HSP72 production, increased Bcl(2) expression, and prevented simulated ischemia-induced renal tubular cell apoptosis. PKC inhibition abolished thermal induction of HSP72 and prevented heat stress-induced ischemic tolerance. These data demonstrate that thermal stress protects renal tubular cells from simulated ischemia-induced apoptosis through a PKC-dependent mechanism.

Induction of heat shock protein 70 protects mesangial cells against oxidative injury

The heat shock response is an immediate cellular response to elevated temperatures and other types of injury that consists of the synthesis of so-called heat shock protein (hsp). This study was designed to investigate the production and the protective role of the 70 kDa hsp (hsp70) in cultured rat mesangial cells. When mesangial cells undergo thermal (45 degrees C, 15 min) stimulation, they express hsp70 mRNA expression and increased hsp70 protein production. Following this, Northern blots show an enhanced gene expression of hsp70 at one hour that reached a maximum by 12 hours after heat shock. The hsp70 protein production, estimated by Western blots, was detectable 12 hours after heat shock and reached a maximum by 36 hours. Oxidative injury generated by xanthine and xanthine oxidase inhibited cell survival and cellular proliferation, as measured by trypan blue exclusion and [3H]-labeled thymidine uptake. It did not affect hsp70 mRNA expression. Furthermore, when mesangial cells were preconditioned by heat shock, subsequent oxidative injury caused less inhibition of cell survival and cellular proliferation. Pretreatment of cells with quercetin, a transcription inhibitor, abolished the protective effect of heat shock on subsequent oxidative injury. We conclude that heat shock, not oxidative injury, induces hsp70 in mesangial cells, and this induction of hsp70 protects mesangial cells against subsequent oxidative injury.

Heat-shocked monocytes are resistant to Staphylococcus aureus-induced apoptotic DNA fragmentation due to expression of HSP72.

Human peripheral blood monocytes became apoptotic following phagocytosis of Staphylococcus aureus. The consequences of heat stress for monocytes were studied with regard to the effect on S. aureus-induced apoptosis. Exposure of monocytes to 41.5 degrees C for 1 h resulted in HSP72 expression and had no influence on phagocytosis of bacteria; moreover, phagocytosis of S. aureus immediately or shortly after heat shock had no effect on the S. aureus-induced monocyte apoptosis, as evidenced by DNA fragmentation assay. In contrast, cells which recovered from heat shock for 18 to 24 h, although active as phagocytes, were resistant to the S. aureus-induced apoptosis. The observed protective effect was related to the induction of HSP72, since blocking of HSP72 synthesis by an antisense oligomer abolished the protective effect of heat shock on bacterium-induced monocyte apoptosis.

Heat shock protects endotheliocyte actin cytoskeleton from destruction under conditions of ATP depletion: Role of heat shock protein HSP27

Heat shock followed by 12–18-hour recovery induced tolerance of cultured endothelial cells to ATP depletion preventing degradation of actin filaments and HSP27 dephosphorylation/redistribution. HSP27 accumulates during this period, while inhibition of protein synthesis abolishes the protective effect of heat shock.

Dissociation of Heat Shock Proteins Expression with Ischemic Tolerance by Whole Body Hyperthermia in Rat Heart

Heat shock (HS) results in the expression of heat shock proteins (hsp) and confers tolerance against subsequent ischemic injury. We examined the extent of myocardial protectionin vivo, and determined the level of hsp expression induced by HS as a function of time. Anesthetized rats were subjected to HS by raising core temperature to 42°C for 15 min and they were then allowed to recover from 2 to 30 h (n=8–11 for each time point). At the appropriate time, animals were subjected to 30 min of ischemia via ligation of the LAD, followed by 90 min of reperfusion. Infarct size was determined by tetrazolium staining and hsp expression was assessed by Western blots. Following ischemia/reperfusion, the infarct sizes (% risk area) were 51.3±3.7, 41.0±7.7, 48.0±6.9 after 2, 4 and 12 h of HS, which were not significantly different from 39.2±2.75 in non heat-shocked animals (P>0.05). In contrast, the infarct size was reduced significantly to 11.0±3.1% in 24 h HS group (P<0.01vnon-heat-shocked control, 2, 4 and 12 h HS groups), but increased back to 40.0±3.2% (P<0.01) by 30 h after HS. No major significant differences in the mean arterial blood pressure, heart rate or rate pressure product was observed between different groups. The synthesis of 72- and 27-kD hsp in HS groups was rapid, reaching >80% of maximum within 4 h of initial insult and peaked by 12 h, whereas the protective effect of HS was absent at these time points. We conclude that ischemic tolerance afforded by HS cannot be solely explained on the basis of hsp expression, and may be dependent on factors such as post-translational modifications, translocation of hsps or some other as yet unidentified factors.

Astrocyte survival and HSP70 heat shock protein induction following heat shock and acidosis

Although severe acidosis is an important mediator of brain infarction, recent evidence suggests that mild acidosis may protect ischemic cells. The HSP70 heat shock protein is induced by acidosis in cultured cells and in ischemic brain and protects cells against many types of injury. Therefore, this study determined whether induction of heat shock proteins protects cultured astrocytes against acidosis. Brief exposure of cultured cortical astrocytes to acid (pH 5.2 for 40 min) or heat shock (45°C for 40 min) markedly induced hsp70 mRNA and HSP70 protein. HSP70 protein was detected with the C92 monoclonal antibody (Welch and Suhan: J Cell Biol 103:2035, 1986), which has been shown to recognize the protein product of the full-length rat hsp70 cDNA (Longo et al: J Neurosci Res 36:325, 1993). Heat shock of the cultured cortical astrocytes completely protected the astrocytes from an otherwise lethal heat exposure 24 h later (45°C for 4 h). In contrast, heat pretreatment sensitized the astrocytes to injury from acidosis 24 h later. Acid pretreatment, which markedly induced the HSP70 protein without producing astrocytic cell death, similarly sensitized the cells to injury from acidosis 24 h later (60% survival following pH 5.2 for 3 h versus 90% survival in controls; P < 0.0001). Surprisingly, heat shock pretreatment protected astrocytes against exposure to acid 48 h later (P < 0.05, 1.5–3 h), whereas acid pretreatment had no effect on astrocyte survival 48 h later. Since heat shock did not protect against acidosis at 24 h when HSP70 induction was maximal but did protect at 48 h when HSP70 was markedly diminished, the protective effect of heat shock at 48 h may be related to stress proteins present at 48 h. It is concluded that induction of HSP70 and other heat shock proteins by heat shock protects astrocytes against subsequent lethal heat shock. However, heat shock and acid treatment increase the vulnerability of astrocytes to acidosis 24 h later in spite of the induction of HSP70 heat shock proteins. The finding that heat shock protected astrocytes against acidosis 2 days later may suggest that delayed induction of stress proteins partially protects the astrocytes against damage produced by high concentrations of hydrogen ions. © 1996 Wiley-Liss, Inc.

Astrocyte survival and HSP70 heat shock protein induction following heat shock and acidosis.

Although severe acidosis is an important mediator of brain infarction, recent evidence suggests that mild acidosis may protect ischemic cells. The HSP70 heat shock protein is induced by acidosis in cultured cells and in ischemic brain and protects cells against many types of injury. Therefore, this study determined whether induction of heat shock proteins protects cultured astrocytes against acidosis. Brief exposure of cultured cortical astrocytes to acid (pH 5.2 for 40 min) or heat shock (45 degrees C for 40 min) markedly induced hsp70 mRNA and HSP70 protein. HSP70 protein was detected with the C92 monoclonal antibody (Welch and Suhan: J Cell Biol 103:2035, 1986), which has been shown to recognize the protein product of the full-length rat hsp70 cDNA (Longo et al: J Neurosci Res 36:325, 1993). Heat shock of the cultured cortical astrocytes completely protected the astrocytes from an otherwise lethal heat exposure 24 h later (45 degrees C for 4 h). In contrast, heat pretreatment sensitized the astrocytes to injury from acidosis 24 h later. Acid pretreatment, which markedly induced the HSP70 protein without producing astrocytic cell death, similarly sensitized the cells to injury from acidosis 24 h later (60% survival following pH 5.2 for 3 h versus 90% survival in controls; P < 0.0001). Surprisingly, heat shock pretreatment protected astrocytes against exposure to acid 48 h later (P < 0.05, 1.5-3 h), whereas acid pretreatment had no effect on astrocyte survival 48 h later. Since heat shock did not protect against acidosis at 24 h when HSP70 induction was maximal but did protect at 48 h when HSP70 was markedly diminished, the protective effect of heat shock at 48 h may be related to stress proteins present at 48 h. It is concluded that induction of HSP70 and other heat shock proteins by heat shock protects astrocytes against subsequent lethal heat shock. However, heat shock and acid treatment increase the vulnerability of astrocytes to acidosis 24 h later in spite of the induction of HSP70 heat shock proteins. The finding that heat shock protected astrocytes against acidosis 2 days later may suggest that delayed induction of stress proteins partially protects the astrocytes against damage produced by high concentrations of hydrogen ions.

NO as a physiological signal molecule that triggers thymocyte apoptosis.

Nitric oxide (NO) produced at high concentrations by the inducible NO synthase (iNOS) is an important effector molecule involved in immune regulation and defence. In in vitro experiments we could show that NO represents a signal for triggering apoptosis in 30% of thymocytes with a concomittant decrease in CD4+CD8+ cells. In addition it protects the remaining cell population from apoptosis induced by glucocorticoids comparable to the protective effect of heat shock.

Nitric oxide induces apoptosis in mouse thymocytes.

Nitric oxide (NO) produced at high concentrations by the inducible NO synthase is an important effector molecule involved in immune regulation and defense. We have examined whether NO represents a signal for triggering apoptosis in thymocytes. Freshly isolated thymocytes were incubated with different chemical NO donors for various intervals. Apoptosis was determined by detection of DNA strand breaks with in situ nick translation. All NO donors induced thymocyte apoptosis with 30% positive thymocytes vs 10% in controls after 8 h. Apoptosis was prevented by addition of ZnSO4. Short-term pre-exposure to NO resulted in protection from apoptosis induced by glucocorticoids comparable with the protective effect of heat shock. Flow cytometry revealed that NO treatment as well as heat shock or dexamethasone incubation is accompanied by reduction in the CD4+ CD8+ thymocyte subpopulation. Apoptosis induction was accompanied by increased expression of p53, as detected by PCR analysis 2 h after NO donor addition. In vivo treatment of mice with endotoxin results in increased thymic apoptosis. Focal apoptosis was found to occur in close proximity to blood vessels 18 h after LPS treatment. Capillary endothelium and dendritic cells adjacent to apoptotic foci were found to stain strongly for inducible NO synthase expression. Furthermore, in an in vitro experiment using cocultures of thymocytes with LPS/cytokine-activated endothelial cells expressing inducible NO synthase, a significantly increased rate of thymocyte apoptosis was found, and this could be prevented completely by inhibiting NO production. Addition of dexamethasone to these cocultures did not lead to a further increase in the percentage of apoptotic thymocytes, underlining the protective effect of NO on dexamethasone-induced apoptosis.

The protective effect of heat shock proteins against photoinhibition under heat shock in barley ( Hordeum vulgare)

Barley leaves, when analyzed by variable fluorescence measurements of PS II activity, have been found to possess a considerably higher resistance against photoinhibition under heat stress conditions after a pretreatment by heat shock. This preconditioning, protecting 2 h heat shock treatment could be applied either 24 h or immediately prior to the combination of heat- and photoinhibitory light stress at a light intensity of 1000 W m −2. Surprisingly, resistance against light stress under heat shock of heat-pretreated plants was even somewhat higher than in unshocked plants which were photoinhibited under normal growth temperature. The preformed, accumulated heat shock proteins (HSP) did not protect the leaves against photoinhibition under normal growth temperatures. Heat shock treatments under light intensities used for normal growth did not impair activity of PS II. Inhibition of protein synthesis by cycloheximide during the first heat shock abolished almost completely the protective effect of the conditioning heat shock. Plants pretreated with heat in the presence of chloramphenicol were as resistant against light stress during the first hour as were the controls which were heat treated in the absence of the drug; however, under prolonged conditions of light stress the chloramphenicol treated plantlets showed a rapid decline in PS II activity.