Heat-induced Hsp70 Expression Research Articles

Expression of heat shock protein 70 and cell death kinetics after different thermal impacts on cultured retinal pigment epithelial cells

Recent technologies are broadening the possibility to treat the retinal pigment epithelium (RPE) with different thermal impacts, from sublethal to lethal ranges. Thus temperature-dependent subcellular molecular responses need to be elucidated in more detail. In this study, RPE cell viability and expression of heat shock protein 70 (Hsp70) were investigated after thermal irradiation with different temperature increase using an in-vitro model. Primary porcine RPE cell cultures were irradiated with different laser power of a thulium laser (λ = 1940 nm, beam-diameter 30 mm) for 10 s, such that the maximal temperatures at the center of the culture dish (Tmax) reach 40, 44, 47, 51 or 59 °C after 10-s irradiation. The temperature distribution across the culture dish shows a Gaussian decay from central position to the periphery of the dish. At 3, 24 and 48 h after irradiation cell viability was assessed with fluorescence microscopy using cell viability-indicating fluorescence dyes, followed by the determination of the threshold temperature for apoptotic change and death of RPE cells. Intracellular localization and amount of Hsp70 were investigated with immunofluorescence and western blotting, respectively. The threshold temperature (at the 10th second of irradiation: T10s) for cellular apoptosis and complete cell death showed a decrease over time after irradiation, suggesting a long-term process of thermally induced cell death. For complete cell death the threshold T10s was 52.1 ± 0.6 °C, 50.1 ± 1.4 °C, and 50.1 ± 0.8 °C, for 3, 24 and 48 h, respectively, whereas for the apoptotic changes 48.6 ± 1.8 °C, 47.2 ± 1.3 °C, and 46.7 ± 0.9 °C, respectively. Quantitative analysis of Hsp70 with western blotting showed a significant increase in intracellular Hsp70 at lethal irradiation with Tmax ≥ 51 °C, up to 19.6 ± 2.3 fold after 48 h at 59 °C, whereas sub-lethal irradiations with Tmax ≤ 44 °C led to a slight tendency of time-dependent increases (up to 1.8 ± 1.1 fold) over 48 h. Immunostainings for Hsp70 showed a circle- or ring-pattern of the Hsp70 staining during 3-48 h after irradiation, and the range of the 1st and 3rd quartiles of T10s for heat-induced Hsp70 expression over this time period was between 44.8 °C and 48.2 °C. A very strong staining of Hsp70 was observed at the border to the damaged zone, where many cells show the strong staining in the whole cytoplasmic space, while some cells in the nucleus, or some cells show the signs of cell migration and proliferation. Moreover, among the cells showing high intensity of Hsp70 staining, there are small round cells like apoptotic cells. Results suggest that RPE cell death after thermal irradiation may take time, and mostly undergoes through apoptosis, unless cells are immediately killed. Thermal irradiation-induced Hsp70 expression is not only temperature-dependent, but also depends largely on the existence of neighboring cell death, suggesting the crucial role of Hsp70 in apoptosis and wound healing processes of RPE cells. The increase of Hsp70 over 24-48 h indicates its long-term roles in cell responses both after sublethal and lethal thermal laser irradiations.

Suppressive Effects of Extract of Cedar Wood on Heat-induced Expression of Cellular Heat Shock Protein

In recent years, highly antimicrobial properties of cedar heartwood essential oil against the wood-rotting fungi and pathogenic fungi have been reported in several papers. Antimicrobial properties against oral bacteria by hinokitiol contained in Thujopsis have been also extensively studied. The relation of naturally derived components and human immune system has been studied in some previous papers. In the present study, we focused on Japanese cedar, which has the widest artificial afforestation site in the country among various tree species. Extract oil was obtained from mixture of sapwood and heartwood of about 40-year cedar grown in Oguni, Kumamoto, Japan. We examined the influence of extract components from Japanese cedar woods on the expression of heat shock protein 70 (Hsp70) during heating, and on the micronucleus formation induced by the treatment of bleomycin as a DNA damaging agent. Cell lines used in this study were human fetal glial cells (SVGp12) and human glioma cells (MO54). Remarkable suppression of the Hsp70 expression induced by heating at 43°C was detected by the treatment of cedar extract in both SVGp12 and MO54 cells. We also found that cedar extract had an inhibitory tendency to reduce the micronucleus formation induced by bleomycin. From these results, the extract components from Japanese cedar woods would have an inhibitory effect of the stress response as a suppression of the heat-induced Hsp70 expression, and might have a reductive effect on carcinogenicity.

RNA Interference Links Oxidative Stress to the Inhibition of Heat Stress Adaptation

Increased oxidative stress is associated with various diseases and aging, while adaptation to heat stress is an important determinant of survival and contributes to longevity. However, the impact of oxidative stress on heat resistance remains largely unclear. In this study we investigated how oxidative stress impinges on heat stress responses. We report that hydrogen-peroxide (H(2)O(2)) pretreatment inhibits both acquired thermotolerance and heat-induced Hsp70 expression in mammalian cells, as well as acquired thermotolerance in the nematode Caenorhabditis elegans, via RNA interference. Moreover, we demonstrate that elimination of RNA interference by silencing key enzymes in microRNA biogenesis, dcr-1 or pash-1, restores the diminished intrinsic thermotolerance of aged and H(2)O(2)-elimination compromised (catalase-2 and peroxiredoxin-2 deficient) worms. These results uncover a novel post-transcriptional element in the regulation of heat stress adaptation under oxidative conditions that may have implications in disease susceptibility and aging.

Adult heat tolerance variation in Drosophila melanogaster is not related to Hsp70 expression

Expression of heat-inducible Hsp70 is considered closely linked to thermotolerance in Drosophila melanogaster and other ectotherms. However, intra-specific variation of Hsp70 expression levels and its relationship to heat resistance has only been investigated in a few studies. Although in Drosophila larvae Hsp70 expression may be a key determinant of heat tolerance, the evidence for this in adults is equivocal. We therefore examined heat-induced Hsp70 expression and several measurements of adult heat tolerance in three independent collections of D. melanogaster, measured in three laboratories and using slightly different protocols. Expression levels of Hsp70 were quantified using ELISA or Western blots on extracts from adult females. Both Hsp70 and heat tolerance exhibited substantial within-population variation as previously reported. However, in all experiments there were no significant correlation between Hsp70 expression and laboratory assays of adult heat tolerance commonly used in Drosophila. When combining data across three studies we had high power to detect associations but the results showed that variation in Hsp70 expression is only likely to explain a small proportion of variation in adult heat tolerance. Therefore, although Hsp70 expression is a major component of the cellular heat stress response, its influence on intra-specific heat tolerance variation may be life-stage specific.

Heat stress-induced heat shock protein 70 expression is dependent on ERK activation in zebrafish ( Danio rerio) cells

Heat shock response is a common event that occurs in many species. Despite its evolutionary conservation, comparative studies of heat shock response have been largely unexplored. In mammals, heat shock response decreases with age through unclear mechanisms. Understanding how the age-related decline in heat shock response occurs may provide information to understanding the biology of aging. We have previously shown that heat shock response similarly declines with age in zebrafish. However, signaling pathways that regulate the heat shock response in zebrafish are unknown. In mammals there is evidence that mitogen-activated protein kinases (MAPKs) of the ERK family alter Hsp70 transcription, serving as a potential regulator of the heat shock response. We explored if heat stress-induced Hsp70 expression is altered by activation of ERK in the zebrafish Pac2 fibroblast cell line as occurs in mammalian cells. Heat stress induced both Hsp70 mRNA expression and phosphorylation of both ERK1 and ERK2 (ERK1/2) in Pac2 cells. ERK inhibitors PD98059 and U0126 blocked both heat stress-induced and plated-derived growth factor (PDGF)-induced ERK1/2 phosphorylation, and also diminished heat-induced Hsp70 expression. Pac2 cell viability was not affected by either the ERK inhibitors or heat stress. These results demonstrate that induction of Hsp70 in response to heat stress is dependent on ERK activation in Pac2 cells. This suggests that the heat shock response in zebrafish utilizes a similar signaling pathway to that of mammals and that zebrafish are a good model for comparative studies of heat shock response.

Knockdown resistance to heat stress and slow recovery from chill coma are genetically associated in a quantitative trait locus region of chromosome 2 in Drosophila melanogaster

In insects, two ecologically relevant traits of thermal adaptation are knockdown resistance to high temperature (KRHT) and chill-coma recovery (CCR). Chromosome 2 of Drosophila melanogaster was tested for quantitative trait loci (QTL) affecting both CCR and KRHT in backcrosses between homosequential lines that are fixed for the standard (noninverted) sequence of this autosome. These lines were obtained by artificial selection on KRHT and subsequent inbreeding from a stock that was derived from a single wild population. Heat-induced expression of the 70KD heat-shock protein (Hsp70) was also examined for variation between the lines. Composite interval mapping was performed for each trait on each reciprocal backcross, identifying one QTL region in the middle of chromosome 2 for both KRHT and CCR. The largest estimates of additive effects were found in pericentromeric regions of chromosome 2, accounting for 10-14% (CCR) and 10-17% (KRHT) of the phenotypic variance in BC populations. No QTL was found in the region of the heat-shock factor (hsf) gene. However, the two parental lines have diverged in the heat-induced Hsp70 expression. Distribution of KRHT QTL on chromosome 2 was similar between this study based on crosses between lines selected from a single wild population and previous work based on crosses between selection lines from different continents. Colocalized QTL showed a trade-off association between CCR and KRHT, which should be the result of either multiple, tightly linked trait-specific genes or a single gene with pleiotropic effects on the traits. We discuss candidate loci contained within the QTL regions.

Effect of glucocorticoid depletion on heat-induced Hsp70, IL-1β and TNF-α gene expression

When exposed to heat, conscious naive rats may develop lethal heatstroke, depending on heat load, i.e., time spent at high body core temperature. The occurrence of heatstroke was hypothesized to result from a defective glucocorticoid secretion related to altered heat-stress responses. We thus investigated the potential involvement of glucocorticoids in heat tolerance and its consequences on physiological responses, heat shock protein 70 (Hsp70), and cytokine mRNA expressions. Two hours before heat exposure, the animals were injected either with metyrapone, an inhibitor of corticosterone synthesis, or with its vehicle. Heat exposure lasted for 15, 30, 45 or 60 min. Thereafter, the rats were distributed into three groups according to their heat load: null, moderate (without any lethal risk) and intense (with lethal risk). Physiological responses were evaluated with colonic temperature, plasma lactate and hematocrit. Brain responses were assessed in frontal cortex through Hsp70, interleukin-1beta (IL-1β) and tumor necrosis factor-alpha (TNF-α) mRNA expressions. The animals with a severe heat load exhibited a high hematocrit, increased plasma lactate level and enhanced brain IL-1β and Hsp70 mRNA expressions. Independent of the heat load, Metyrapone rats showed the same thermophysiological responses and IL-1β and Hsp70 mRNA expressions when compared with vehicle rats. However, the Metyrapone rats experiencing an intense heat load exhibited an increased TNF-α mRNA expression. In conclusion, these data (i) confirm that heat load is important in the calibration of the risk attached to heat exposure; and (ii) suggest that corticosterone synthesis inhibition may favor TNF-α mRNA expression without any effect on Hsp70 mRNA expression.

Heat-induced hormesis in longevity of two sibling Drosophila species

Previous work showed that mild-heat stress induces longevity hormesis in a model organism, D. melanogaster. Here we compared the possible heat-induced hormesis in longevity of other species of Drosophila, D. buzzatii and its sibling species D. koepferae, in a single-sex environment. Possible correlations between longevity and heat-stress resistance were also tested by measuring longevity, heat-knockdown resistance and the heat-induced Hsp70 expression for each species in a common environment. D. buzzatii was longer lived than D. koepferae at benign temperature. Knockdown resistance to heat stress was positively correlated to longevity within species. However, the shorter-lived species was more resistant to knockdown by heat stress than the longer-lived species. The heat-induced Hsp70 expression was similar between species. A heat-shock treatment (37 degrees C for 1 h at 4 days of age) extended mean longevity in the longer lived species but not in the shorter lived species. In D. koepferae, the demographic rate of senescence decreased but the baseline mortality rate increased by heat-shock, resulting in no extension of mean longevity. Sympatric populations of closely related species can be differentially sensitive to temperature and exhibit different patterns of 37 degrees C-induced hormesis in demographic senescence and longevity. The results also show that positive correlations between stress resistance and life span within species can shift in sign across closely related species. Finally, this study shows that heat-induced hormesis in longevity can be found across different Drosophila species, as hormetic effects are not limited to the previously studied D. melanogaster.

Heat-induced expression of a molecular chaperone decreases by selecting for long-lived individuals

The 70 kDa heat-shock protein (Hsp70) exhibits a broad range of chaperone functions that respond both to internal and external stresses, and its heat-induced expression both declines with age and reduces age-specific mortality rates. Here we test for changes in both longevity and the level of Hsp70-induced expression as correlated responses to selection on both heat-stress resistance and longevity in D. melanogaster. Three replicated H lines were heat-stress selected and compared to their respective non-selected controls (C lines) in the 25th generation of heat-stress selection. The direct response in heat-stress resistance was 56% in males and 38% in females. Heat-stress selection improved longevity in males at normal temperatures. All lines were subsequently subjected to one generation of truncation selection on longevity (selection intensity, i=1.28 for H lines and 1.36 for C lines). The heat-induced Hsp70 expression seems to increase very weakly by heat-stress selection but shows a dramatic and persistent decline by selecting for long-lived flies. A mechanism of longevity selection, involving changes in Hsp70 regulation, is suggested.

Targeted disruption of hsf1 leads to lack of thermotolerance and defines tissue-specific regulation for stress-inducible Hsp molecular chaperones.

The rapid synthesis of heat shock proteins (Hsps) in cells subjected to environmental challenge is controlled by heat shock transcription factor-1 (Hsf1). Regulation of Hsps by Hsf1 is highly complex and, in the whole organism, remains largely unexplored. In this study, we have used mouse embryo fibroblasts and bone marrow progenitor cells from hsf1-/- mice as well as hsp70.3-lacZ knock-in mice bred on the hsf1deficient genetic background (hsf1-/--hsp70.3+/--lacZ), to further elucidate the function of Hsf1 and its participation as a transcriptional activator of Hsp70 synthesis under normal or heat-induced stress conditions in vitro and in vivo. The results revealed that heat-induced Hsp70 expression in mouse tissue is entirely controlled by Hsf1, whereas its activity is not required for tissue-specific constitutive Hsp70 expression. We further demonstrate that Hsf1 is critical for maintaining cellular integrity after heat stress and that cells from hsf1-/- mice lack the ability to develop thermotolerance. This deficiency is explained by the elimination of stress-inducible Hsp70 and Hsp25 response in the absence of Hsf1 activity, leading to a lack of Hsp-mediated inhibition of apoptotic cell death via both caspase-dependent and caspase-independent pathways. The pivotal role of the Hsf1 transactivator in regulating rapid synthesis of Hsps as a critical cellular defense mechanism against environmental stress-induced damage is underlined.

Pretransplant Induction of HSP-70 in Isolated Adult Pig Islets Decreases Early Islet Xenograft Survival

The heat-induced HSP-70 expression protects rat islet single cells against lysis mediated by nitric oxide (NO), reactive oxygen, and streptozotocin. The present study was performed to investigate the potential antiinflammatory effect of pretransplant heat shock in adult pig islets for subsequent early islet xenograft survival. Maximum HSP-70 expression in freshly isolated pig islets was induced by hyperthermia at 43 degrees C for 90 min prior to islet regeneration at 37 degrees C for 4-6 h. Heat-stressed and sham-treated islets were incubated in 0.6 mM H2O2 or 1.5 mM Na-nitroprusside at 37 degrees C for 20 h. Early graft survival was evaluated in normoglycemic Lewis rats after simultaneous, contralateral transplantation of heat-shocked islets and sham-treated islets into the renal subcapsular space of the same recipient. Prior hyperthermia significantly reduced specific lysis of islets exposed to NO or H2O2, although protection was only marginal. No differences were observed between viability of heat-shocked and sham-treated islets after NO exposure. In contrast, prior heat shock increased islet viability after H2O2 treatment. The finding that hyperthermia reduced recovery of initially grafted pig insulin 48 h after transplantation by 30% compared to controls contrasted significantly with an increased insulin recovery in heat-exposed islets at the end of simultaneous 37 degrees C culture. The observation, that the heat-induced HSP-70 expression decreases early islet xenograft survival as reflected by recovery of grafted insulin, implies an enhancement of islet immunogenicity and the induction of apoptosis. Future experiments aiming at augmentation of intrinsic defense mechanisms should consider detrimental effects associated with induction of heat shock proteins.

Suppression of heat-induced hsp-70 by simultaneous exposure to 50 mT magnetic field

Effect of extremely low frequency magnetic field (ELFMF) at 50 mT and 60 Hz on heat-induced expression of heat shock protein 70 (hsp-70) was examined in HL60RG cells. No increase in hsp-70 production was observed in the cells after exposure to 50 mT ELFMF alone. Simultaneous exposure to 50 mT ELFMF in combination with mild heat at 42 and 40 °C suppressed heat-induced hsp-70 expression. The suppression of hsp-70 occurred when cells were simultaneously exposed to both for longer periods of more than 5 h. However, the suppression of hsp-70 was not observed at a magnetic density of 5 and 0.5 mT. This result suggests that exposure to 50 mT ELFMF may act on a protection against the concomitant mild heat stress in HL60RG cells.

Proteasome Inhibitors Lactacystin and MG132 Inhibit the Dephosphorylation of HSF1 after Heat Shock and Suppress Thermal Induction of Heat Shock Gene Expression

Recently, we have shown that two proteasome inhibitors, MG132 and lactacystin, induce hyperphosphorylation and trimerization of HSF1, and transactivate heat shock genes at 37°C. Here, we examined the effects of these proteasome inhibitors and, in addition, a phosphatase inhibitor calyculin A (CCA) on the activation of HSF1 upon heat shock and during post-heat-shock recovery, with emphasis on HSF1 hyperphosphorylation and the ability of HSF1 to transactivate heat shock genes. When lactacystin, MG132, or CCA was present after heat shock, HSF1 remained hyperphosphorylated during post-heat-shock recovery at 37°C. Failure of HSF1 to recover to its preheated dephosphorylated state correlated well with the suppression of the heat-induced hsp70 expression. In vitro, HSF1 from heat-shocked cells, when dephosphorylated, showed an increase in HSE-binding affinity. Taken together, these data suggest that phosphorylation of HSF1 plays an important role in the negative regulation of heat-shock response. Specifically, during post-heat-shock recovery phase, prolonged hyperphosphorylation of HSF1 suppresses heat-induced expression of heat shock genes.

Potentiation of heat stress-induced hsp70 expression in vivo by aspirin.

Studies in cultured cells have demonstrated that non-steroidal anti-inflammatory agents can potentiate heat-induced hsp70 expression through activation of HSF1 to a DNA binding state. We investigated the influence of aspirin on hsp70 expression in intact rats subjected to heat stress. Rats were injected intraperitoneally either with aspirin (100 mg/kg) or vehicle alone, 60 min prior to their placement at 37 degrees C or room temperature for 30 min. hsp70 mRNA expression was analyzed in lung, liver and kidney isolated from animals assigned to one of four different treatment paradigms; untreated controls, heat, aspirin, and aspirin-plus-heat. Comparison of hsp70 expression in the treatment groups revealed that in all tissues examined, aspirin-plus-heat treatment resulted in 3-4 fold higher levels of hsp70 mRNA relative to those seen with heat treatment alone. Little or no hsp70 mRNA expression was detected in the unheated groups, regardless of aspirin treatment. In keeping with the mRNA expression, Hsp70 protein levels were also elevated in aspirin-plus-heat treated animals. Aspirin treatment did not alter hsp70 protein expression in the absence of heat. In contrast to in vitro observations, aspirin treatment in vivo did not alter HSF1 DNA binding properties. Core body temperature measurements revealed that aspirin pretreatment enhanced the rise in body temperature seen in response to heat treatment. This increased hyperthermic response to heat stress probably accounts for the potentiation of hsp70 expression observed in aspirin-plus-heat treated rats. Given the widespread use of aspirin in humans within a dose range comparable to that used here, our findings are likely to have important physiological consequences.

Modulation of Thermal Induction of hsp70 Expression by Ku Autoantigen or Its Individual Subunits

Previously, we proposed a dual control mechanism for the regulation of the heat shock response in mammalian cells: a positive control mediated by the heat shock transcription factor HSF1 and a negative control mediated by the constitutive heat shock element-binding factor (CHBF). To study the physiological role of CHBF in the regulation of heat shock response, we purified CHBF to apparent homogeneity and showed it to be identical to the Ku autoantigen, a heterodimer consisting of 70-kDa (Ku-70) and 86-kDa (Ku-80) polypeptides. To study further the functional significance of Ku/CHBF in the cellular response to heat shock, we established rodent cell lines that stably and constitutively overexpressed one or both subunits of the human Ku protein, and examined the thermal induction of hsp70 and other heat shock proteins in these Ku-overexpressing ing cells. We show that expression of the human Ku-70 and Ku-80 subunits jointly or of the Ku-70 subunit alone specifically inhibits heat-induced hsp70 expression. Conversely, expression of human Ku-80 alone does not have this effect. Thermal induction of other heat shock proteins in all of the Ku-overexpressing cell lines appears not to be significantly affected, nor is the state of phosphorylation or the DNA-binding ability of HSF1 affected. These findings support a model in which hsp70 expression is controlled by a second regulatory factor in addition to the positive activation of HSF1. The Ku protein, specifically the Ku-70 subunit, is involved in the regulation of hsp70 gene expression.

Suppression of heat-induced hsp70 expression by the 70-kDa subunit of the human Ku autoantigen.

Expression of the 70-kDa polypeptide of human Ku autoantigen in rat cells is shown to suppress specifically the induction of hsp70 upon heat shock. Thermal induction of other heat shock proteins is not significantly affected, nor is the state of phosphorylation or the DNA-binding ability of the heat shock transcription factor HSF1. These findings support a model in which hsp70 gene expression is controlled by a second regulatory factor in addition to the positive activator HSF1. The Ku autoantigen, or a protein closely related to it, is likely to be involved in the regulation of hsp70 expression.