Increase In Hsp70 Research Articles

Increased proteolysis of diphtheria toxin by human monocytes after heat shock: a subsidiary role for heat‐shock protein 70 in antigen processing

The expression of heat-shock proteins (hsp) increases after exposure to various stresses including elevated temperatures, oxidative injury, infection and inflammation. As molecular chaperones, hsp have been shown to participate in antigen processing and presentation, in part through increasing the stability and expression of major histocompatibility complex molecules. Heat shock selectively increases human T-cell responses to processed antigen, but does not affect T-cell proliferation induced by non-processed antigens. Here, we have analysed the mechanisms by which stress such as heat shock, and the ensuing hsp over-expression affect the processing of diphtheria toxin (DT) in peripheral blood monocytes. We found that heat shock increased DT proteolysis in endosomes and lysosomes while the activities of the cathepsins B and D, classically involved in DT proteolysis, were decreased. These effects correlated with the heat-shock-mediated increase in hsp 70 expression observed in endosomes and lysosomes. Actinomycin D or blocking anti-hsp 70 antibodies abolished the heat-shock-mediated increase in DT proteolysis. These data indicate that the increased expression of hsp 70 constitutes a subsidiary mechanism that facilitates antigen proteolysis in stressed cells. Confirming these data, presentation by formaldehyde-fixed cells of DT proteolysates that were obtained with endosomes and lysosomes from heat-shocked peripheral blood monocytes showed higher stimulation of T cells than those generated with endosomes and lysosomes from control peripheral blood monocytes.

Induction of the 72-kilodalton heat shock protein and protection from ultraviolet B–induced cell death in human keratinocytes by repetitive exposure to heat shock or 15-deoxy-Δ12,14-prostaglandin J2

It has been demonstrated that hyperthermia protects keratinocytes from ultraviolet B (UVB)-induced cell death in culture and in vivo. This effect is mediated by the antiapoptotic effect of heat shock proteins that are transiently induced after exposure to heat at sublethal temperatures. Consequently, induction of Hsp has been proposed as a novel means of photoprotection. However, in the face of daily UVB exposure of human skin in vivo, this approach would not be useful if keratinocytes become less sensitive to Hsp induction with repeated exposure to the inducing agent. The aim of this study was to investigate whether repeated exposure to hyperthermia or to the stress protein activating cyclopentenone prostaglandin 15-deoxy-delta(12,14)-prostaglandin J2 (15dPGJ2) leads to adaptation of the cells, attenuation of the heat shock response, and abrogation of the protective effect. Normal human epidermal keratinocytes (NHEK) and the carcinoma-derived cell line A431 were exposed to either 42 degrees C or to 15dPGJ2 for 4 hours at 24-hour intervals for 4 consecutive days. The intracellular level of the 72-kDa heat shock protein (Hsp72) was determined by enzyme-linked immunosorbent assay (ELISA). Cells were exposed to UVB from a metal halide source after the last heat or 15dPGJ2 treatment, and survival was determined 24 hours after exposure by a MTT assay. Our results demonstrate that (1) heat shock and 15dPGJ2 are potent inducers of Hsp72 expression and lead to increased resistance to UVB-induced cell death in human keratinocytes; (2) re-exposure to heat shock leads to a superinduction without attenuation of the absolute increase in Hsp72 and of its UVB-protective effect; (3) the UVB tolerance induced by 15dPGJ2 is enhanced by repeated exposure without a further increase of Hsp72; (4) repeated heat shock and 15dPGJ2 up to a concentration of 1 microg/mL have no influence on cell growth over a period of 4 days. We conclude that through repeated exposure to Hsp-inducing factors, stress tolerance can be maintained without additional toxicity in human keratinocytes. These results provide a basis for the development of nontoxic Hsp inducers that can be repeatedly applied without loss of effect.

Vitamin E isoform-specific inhibition of the exercise-induced heat shock protein 72 expression in humans

Increased levels of reactive oxygen and nitrogen species, as seen in response to exercise, challenge the cellular integrity. Important protective adaptive changes include induction of heat shock proteins (HSPs). We hypothesized that supplementation with antioxidant vitamins C (ascorbic acid) and E (tocopherol) would attenuate the exercise-induced increase of HSP72 in the skeletal muscle and in the circulation. Using randomization, we allocated 21 young men into three groups receiving one of the following oral supplementations: RRR-alpha-tocopherol 400 IU/day + ascorbic acid (AA) 500 mg/day (CEalpha), RRR-alpha-tocopherol 290 IU/day + RRR-gamma-tocopherol 130 IU/day + AA 500 mg/day (CEalphagamma), or placebo (Control). After 28 days of supplementation, the subjects performed 3 h of knee extensor exercise at 50% of the maximal power output. HSP72 mRNA and protein content was determined in muscle biopsies obtained from vastus lateralis at rest (0 h), postexercise (3 h), and after a 3-h recovery (6 h). In addition, blood was sampled for measurements of HSP72, alpha-tocopherol, gamma-tocopherol, AA, and 8-iso-prostaglandin-F2alpha (8-PGF2alpha). Postsupplementation, the groups differed with respect to plasma vitamin levels. The marker of lipid peroxidation, 8-iso-PGF2alpha, increased from 0 h to 3 h in all groups, however, markedly less (P < 0.05) in CEalpha. In Control, skeletal muscle HSP72 mRNA content increased 2.5-fold (P < 0.05) and serum HSP72 protein increased 4-fold (P < 0.05) in response to exercise, whereas a significant increase of skeletal muscle HSP72 protein content was not observed (P = 0.07). In CEalpha, skeletal muscle HSP72 mRNA, HSP72 protein, and serum HSP72 were not different from Control in response to exercise. In contrast, the effect of exercise on skeletal muscle HSP72 mRNA and protein, as well as circulating HSP72, was completely blunted in CEalphagamma. The results indicate that gamma-tocopherol comprises a potent inhibitor of the exercise-induced increase of HSP72 in skeletal muscle as well as in the circulation.

Ischemic preconditioning inhibits development of edematous cerulein-induced pancreatitis: Involvement of cyclooxygenases and heat shock protein 70

To determine whether ischemic preconditioning (IP) affects the development of edematous cerulein-induced pancreatitis and to assess the role of cyclooxygenase-1 (COX-1), COX-2, and heat shock protein 70 (HSP 70) in this process. In male Wistar rats, IP was performed by clamping of celiac artery (twice for 5 min at 5-min intervals). Thirty minutes after IP or sham operation, acute pancreatitis was induced by cerulein. Activity of COX-1 or COX-2 was inhibited by resveratrol or rofecoxib, respectively (10 mg/kg). IP significantly reduced pancreatic damage in cerulein-induced pancreatitis as demonstrated by the improvement of pancreas histology, reduction in serum lipase and poly-C ribonuclease activity, and serum concentration of pro-inflammatory interleukin (IL)-1beta. Also, IP attenuated the pancreatitis-evoked fall in pancreatic blood flow and pancreatic DNA synthesis. Serum level of anti-inflammatory IL-10 was not affected by IP. Cerulein-induced pancreatitis and IP increased the content of HSP 70 in the pancreas. Maximal increase in HSP 70 was observed when IP was combined with cerulein-induced pancreatitis. Inhibition of COXs, especially COX-2, reduced the protective effect of IP in edematous pancreatitis. Our results indicate that IP reduces pancreatic damage in cerulein-induced pancreatitis and this effect, at least in part, depends on the activity of COXs and pancreatic production of HSP 70.

Life long calorie restriction increases heat shock proteins and proteasome activity in soleus muscles of Fisher 344 rats

Heat shock proteins (HSP's) closely interact with 20S proteasome and have been shown to maintain catalytic activity, responsible for the prevention of protein aggregation. A decrease in both proteasome activity and heat shock proteins (HSP's) has been observed with age. We investigated whether life-long calorie restriction (CR), a natural intervention, which prolongs life span, could prevent the age-associated decline in HSP's and restore the proteolytic activity of the 20S proteasome in skeletal muscle. Hence, we investigated HSP's and proteasome activity in the soleus muscle from 12-mo-old (Adult) and 26–28mo old ad libitum fed (Old), and 26–28mo old CR (Old-CR; fed 40% of ad libitum for their lifespan) male Fisher 344 rats. Trypsin-like proteasome activity in Old rats was significantly less than both Adult and Old-CR rats. Furthermore, no significant changes where found in chymotrypsin-like proteasome activity due to age or diet. Levels of HSP 72 and 25 were significantly less in Old animals when compared to both Adult and Old-CR rats. In contrast, HSP 90 was elevated in Old rats by 220% compared to adult animals and life-long calorie restriction caused a significant induction (150%) compared to age-matched ad libitum fed animals. Protein carbonyls were significantly elevated in Old when compared to Adult rats, but showed no significant decline due to life long CR. This study shows that HSP's may be largely responsible for the restoration of the trypsin-like activity of the 20S proteasome with age. The large increase in HSP 90 is intriguing and further studies are required to elucidate its role in maintaining 20S proteasome function.

Heat shock protein expression in adenosine triphosphate depleted renal epithelial cells.

BackgroundIn this study, the putative interactions between apoptosis and heat shock proteins disturbed as a result of ATP depletion were investigated as a hypoxia model.MethodsThe direct cellular damages were assessed by the release of LDH from the cytoplasm of the human tubular epithelial cells (HK-2 cells) following ATP depletion. The Bcl-2/Bax mRNA expression ratio, used as an index to assess to what extent apoptosis contributed to tubular cell damage, and the expressions of HSP 90, 72 and 27 in relation to the Bcl-2/Bax ratio in the ischemic model, as parameters of their functional contributions to tubule cell damage, were also studied. Heat preconditioning (HS) was performed at 43°C in a temperature-regulated water bath for 1 h.ResultsThe release of LDH due to ATP depletion was not significantly increased in HK-2 cells compared to the control, but was slightly increased in heat preconditioned cells compared to non heat preconditioned cells, but the difference was not statistically significant (6.33±0.57 U/L vs. 8.67±2.52 U/L, p>0.05). The Bcl-2/ Bax mRNA expression ratio increased progressively from the control to the heat preconditioned and ATP depleted cells (control; 100%, ATP depletion; 154±6%, heat preconditioning; 212±6%, heat preconditioning and ATP depletion; 421±8%). No contribution of heat preconditioning and ATP depletion was observed on the expressions of HSP90 and HSP27. However, HSP72 expression was prominent by ATP depletion, especially after heat preconditioning.ConclusionThere may be a possibility that the preservation of cytolytic damage and an increase in the Bcl-2/Bax mRNA expression ratio is related to the increase of HSP72 in ATP depletion as a hypoxia model.

Negotiating brain anoxia survival in the turtle.

The turtle brain's extraordinary ability to tolerate anoxia is based on constitutive and expressed factors. Constitutive factors that predispose for anoxia tolerance include enhanced levels of glycogen stores, increased densities of protective receptors, elevated antioxidant capacities and elevated heat shock protein. However, to survive an anoxic insult, three distinct phases must be negotiated successfully. (1) A coordinated downregulation of ATP demand processes to basal levels. This phase, which takes 1-2 h, includes a reduction in voltage-gated K(+) (Kv) channel transcription and a substantial increase in Hsp72 and Hsc73 levels. During this period, adenosine and K(ATP) channels mediate several key events including channel arrest initiation and a reduction in the release of excitatory amino acids (EAAs). (2) Long-term survival (days) at basal levels of ATP expenditure. Neuronal network integrity is preserved through the continued operation of core activities. These include periodic electrical activity, an increased release of GABA and a continued release of glutamate and dopamine. Adenosine and GABA modulate the glutamate release. There is a further increase in Hsc73, indicating a 'housekeeping' role for this protein during this period. (3) A rapid upregulation of neuronal processes when oxygen becomes available to restore full function, together with the activation of protection mechanisms against reperfusion-generated reactive oxygen species.

Estrogen, heat shock proteins, and NFkappaB in human vascular endothelium.

We hypothesized that estrogen would increase HSP72 in human coronary artery endothelial cells (HCAEC), and that these would be more sensitive to estrogen than our previous observations in myocytes. HCAEC were treated with 17beta-estradiol or tamoxifen, ranging from physiological to pharmacological(1 nM to 10 micromol/L) for either 24 hours (early) or 7 days (chronic). HSP expression was assessed by Western blots. Both early and chronic 17beta-estradiol and tamoxifen increased HSP72. Electromobility shift assays (EMSA) showed activation of HSF-1 with early, but not chronic, 17beta-estradiol. 17beta-Estradiol activated NFkappaB within 10 minutes, and the ER-alpha selective inhibitor, ICI 182 780, abolished this effect. Transcription factor decoys containing the heat shock element blocked HSP72 induction. Estrogen pretreatment decreased lactate dehydrogenase release with hypoxia. This protective effect persisted despite blockade of HSF-1 by decoys. However, an NF-kappaB decoy prevented the increase in HSP72 and abolished the estrogen-associated protection during hypoxia. 17beta-Estradiol upregulates HSP72 early and chronically via different mechanisms in HCAEC, and provides cytoprotection during hypoxia, independent of HSP72 induction. NF-kappaB mediates the early increase in HSP72, suggesting that estrogen activates NF-kappaB via a nongenomic, receptor-dependent mechanism, and this leads to activation of HSF-1. Activation of NF-kappaB was critical for estrogen-associated protection. Further studies are needed to elucidate the involved signaling pathways. We hypothesized that estrogen would increase HSP72 in human coronary artery endothelial cells (HCAEC). Both early and chronic treatment increased HSP72. EMSA showed activation of HSF-1 with early, but not chronic, 17beta-estradiol. Transcription factor decoys blocked estrogen-related HSP72 induction. Estrogen decreased LDH release with hypoxia. An NF-kappaB decoy blocked the HSP72 increase and estrogen-associated protection.

Developmental toxicity and stress protein responses in zebrafish embryos after exposure to diclofenac and its solvent, DMSO

One of the most frequently detected pharmaceuticals in environmental water samples is the anti-rheumatic drug, diclofenac. Despite its increasing environmental significance, investigations concerning the effects of this drug on the early developmental stages of aquatic species are lacking up to now. To determine the developmental toxicity and proteotoxicity of this drug on the growing fish embryos, eggs of zebrafish were exposed to six concentrations of diclofenac (0, 1, 20, 100, 500, 1000, and 2000 μg l −1) using DMSO as solvent. Early life stage parameters such as egg and embryo mortality, gastrulation, somite formation, movement and tail detachment, pigmentation, heart beat, and hatching success were noted and described within 48- and 96-h of exposure. After the 96-h exposure, the levels of stress proteins (hsp 70) were determined in both the diclofenac-treated and respective DMSO controls. Results showed no significant inhibition in the normal development until the end of 96 h for all exposure groups. However, there was a delay in the hatching time among embryos exposed to 1000 and 2000 μg l −1. Late-hatched embryos (108 h) did not differ morphologically from normally hatched embryos. The mortality and average heart rate data did not show significant differences for all embryos in both diclofenac-treated and DMSO control groups. No significant malformations were likewise noted among all developing embryos throughout the exposure period. The levels of heat shock proteins in diclofenac-treated and control embryos did not differ significantly. DMSO control embryos, on the other hand, showed a concentration-dependent increase in hsp 70 levels. We suggest possible modulating effect of diclofenac in DMSO-triggered expression of stress proteins and this might have a possible repercussion on the use of DMSO as solvent in any toxicity assay. Since the present data indicate no significant embryotoxicity and proteotoxicity induced by diclofenac and due to the fact that the concentrations of diclofenac used in the present study is up to 2000-fold higher than the concentrations detected in the environment, it is unlikely that this drug would pose a hazard to early-life stages of zebrafish.

Estrogen and regulation of heat shock protein expression in female cardiomyocytes: cross-talk with NFкB signaling

Estrogen is associated with increased heat shock protein (HSP)72 and protection during hypoxia-reoxygenation in cardiomyocytes from adult male rats, as previously reported. We have also reported that female rats have more cardiac HSP72 than males. We hypothesized that, despite higher endogenous estrogen levels and higher baseline HSP72, 17β-estradiol treatment would still result in increased HSP72 and protection during hypoxia-reoxygenation in cardiomyocytes from females. Methods/Results. – Cardiac cells isolated from adult female rats were treated for 12 hr with 17β-estradiol (0.1, 10, or 50 μM), tamoxifen, (10 or 25 μM; estrogen receptor agonist/antagonist), geldanamycin (2, 5, or 10 μg/ml; inactivates HSP90, preventing interaction with HSF1), or vehicle. Western blot analyses revealed that treatment with 17β-estradiol (10 or 50 μM), tamoxifen (25 μM), and geldanamycin (all doses) resulted in significant increases in HSP72. Electromobility shift assays revealed activation of HSF1 by 2 to 3 hr, and NFкB activation by 15 min. HSP72 induction via HSF1 activation was confirmed using transcription factor decoys containing the heat shock element, which prevented the estrogen-related HSP72 induction. Estrogen pretreatment resulted in decreased LDH release during 24 hr hypoxia. This protective effect persisted despite decoy-mediated blockade of nuclear HSF1 binding. However, transfection with an NFкB decoy not only prevented an estrogen-associated increase in HSP72, but also abolished the estrogenrelated protection during hypoxia. Conclusions. – Despite higher endogenous estrogen, 17β-estradiol and the selective estrogen receptor modulator, tamoxifen, upregulate HSP72 in cardiomyocytes from adult females, and provide cytoprotection during hypoxia, independent of HSP induction. NFкB activation is necessary for the increase in HSP72, suggesting that estrogen treatment activates NFкB, with subsequent HSF1 activation. NFкB activation is critical for estrogen-associated HSP induction, and protection during hypoxia in female cardiocytes. Treatment with 17β-estradiol and tamoxifen may provide a novel means of protecting both male and female cardiac myocytes against hypoxia-induced damage. Further studies are needed to define the cross-talk between HSF1 and NFкB signaling pathways.

Increased expression of heat shock proteins in rat brain during aging: relationship with mitochondrial function and glutathione redox state

It is generally recognized that lipid peroxides play an important role in the pathogenesis of several diseases and that sulfhydryl groups are critically involved in cellular defense against endogenous or exogenous oxidants. Recent evidence indicates that lipid peroxides directly participate in induction of cytoprotective proteins, such as heat shock proteins (Hsps), which play a central role in the cellular mechanisms of stress tolerance. Oxidative damage plays a crucial role in the brain aging process and induction of Hsps is critically utilized by brain cells in the repair process following various pathogenic insults. In the present study, we investigated, in rats 6, 12, and 28 months old, the role of heat shock expression on aging-induced changes in mitochondrial and antioxidant redox status. In the brain expression of Hsp72 and Hsc70 increased with age up to 28 months; at this age the maximum induction was observed in the hippocampus and substantia nigra followed by cerebellum, cortex, septum and striatum. Hsps induction was associated with significant changes in glutathione (GSH) redox state and HNE levels. Interestingly, a significant positive correlation between decrease in GSH and increase in Hsp72 was observed in all brain regions examined during aging. Analysis of mitochondrial complexes showed a progressive decrease of Complex I activity and mRNA expression in the hippocampus and a significant decrease of Complex I and IV activities in the substantia nigra and septum. Our results sustain a role for GSH redox state in Hsp expression. Increase of Hsp expression promotes the functional recovery of oxidatively damaged proteins and protects cells from progressive age-related cell damage. Conceivably, heat shock signal pathway by increasing cellular stress resistance may represent a crucial mechanism of defence against free radical-induced damage occurring in aging brain and in neurodegenerative disorders.

異なる速度での一過性の走運動とトレーニングに伴うラット骨格筋の熱ショックタンパク質発現量の変化

It has been demonstrated that exercise induces heat shock proteins (HSPs) . However, no study has investigated changes in HSPs following endurance training at different speeds. Therefore, this study was designed to investigate the effect of treadmill training at different running speeds on induction HSPs. One group of male Wistar rats was assigned as a sedentary control, three groups were assigned for exercise training (10 m/min, 20 m/min and 30 m/min) and another three groups for one acute bout of exercise (10 m/min, 20 m/min and 30 m/min) . Each training group ran at each speed for 30 min/day, 5 days a week for 8 weeks. The acute exercise group performed the exercise only once. Forty-eight hours after the last exercise session was completed, the rats were sacrificed and the plantaris (PLA) and soleus (SOL) muscles were dissected. In the acute exercise group, the content of HSP72 in both the PLA and SQL increased (p<0.05) at all speeds, and the content of HSP60 increased significantly (p<0.05) at all speeds for the PLA, but not for the SOL. On the other hand, in the endurance group, the content of HSP72 and HSP60 in both muscles increased in 30 m/min groups. These results indicate that an increase in HSP72 and HSP60 by endurance training is induced by high intensity training in both muscles. This was not found to be the case with the acute exercise groups.

Glutamine attenuates tumor necrosis factor-α release and enhances heat shock protein 72 in human peripheral blood mononuclear cells

Objectives Overexpression of pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α) can contribute to multiple organ dysfunction syndrome and septic shock in critically ill patients. We previously found that glutamine (GLN) can attenuate cytokine expression, induce heat shock protein 72 (HSP 72), and protect against endotoxin-induced mortality and organ injury in an in vivo rat model. However, data on the effect of GLN on direct attenuation of cytokine release and HSP 72 expression in human peripheral blood polymorphonuclear cells (PBMCs) is lacking. Methods In this study, we assessed the effect of GLN on TNF-α and HSP 72 expression in human PBMCs. After treating with various doses of GLN, human PBMCs were stimulated with lipopolysaccharide (LPS). TNF-α release was analyzed via enzyme-linked immunosorbent assay and HSP 72 via western blot. Results GLN at doses greater than 4 mM decreased TNF-α release at 4 and 24 h after LPS stimulation. Sublethal heating of PBMCs before LPS also markedly decreased TNF-α after LPS. Doses of GLN greater than 2 to 4 mM led to an increase in HSP 72 expression after LPS. Conclusions These results indicate that GLN, which may improve outcomes in critically ill patients, can directly attenuate pro-inflammatory cytokine release in PBMCs. This effect may be related to enhanced HSP 72 expression.

Differential regulation of spontaneous and heat-induced HSP 70 expression in developing zebrafish (Danio rerio).

A spontaneous high expression of heat shock protein 70 (HSP 70) was found to arise in zebrafish (Danio rerio) at the larval stage (84 hr after fertilization). The level of HSP 70 in 84-hr-old larvae was estimated to be six- to eightfold that of 12-hr-old embryos. As heat-induced HSP 70 synthesis in many eukaryotic organisms is known to be mediated by a transcriptional-dependent pathway activated by heat shock factor 1 (HSF-1), we then examined if the spontaneous and heat-induced HSP 70 synthesis in zebrafish were controlled by the same mechanism. Although the transient increase of a 62-kDa HSF-1-like polypeptide in 72- to 96-hr-old larvae seemed to correlate with the onset of the spontaneous HSP 70 production, an anti-HSF-1 antibody cocktail supershifted the heat shock element (HSE) binding complex induced by stressed but not by unstressed zebrafish extracts. Northern blot and quantitative RT-PCR analysis demonstrated the predominant presence of the cognate form of hsp 70 mRNA (hsc 70 mRNA) in developing zebrafish. The extent of heat-induced HSP 70 production in 84-hr-old larvae matched well with a dramatic increase in hsp 70 mRNA accumulation, while no apparent increase in total hsp 70 mRNA could be detected in 72- to 84-hr-old unstressed larvae by northern blot analysis. The stable expression of hsc 70 mRNA specific to beta-actin mRNA in normal zebrafish was confirmed by RT-PCR analysis. Hence, the spontaneous high expression of HSP 70 in zebrafish is believed to be controlled by a mechanism different from the HSF-1-dependent transcriptional activation of hsp 70 under heat stress. J. Exp. Zool. 293:349-359, 2002.

Modulation of heat-induced cell death in PC-3 prostate cancer cells by the antioxidant inhibitor diethyldithiocarbamate.

To examine the relationships between the form of cell death (apoptosis or necrosis), reactive oxygen species (ROS) generation, superoxide dismutase (SOD) activity and the level of heat-shock protein 70 (hsp 70) expression after thermotherapy of PC-3 prostate cancer cells; also assessed were the tumoricidal effects of combined treatment with both heat and the antioxidant inhibitor diethyldithiocarbamate (DDC). PC-3 cells were treated with thermotherapy at 42, 43 or 44 degrees C for 30, 60, 90 or 120 min. Cell proliferation, ROS generation, SOD activity and cellular hsp 70 level were determined using tetrazolium-based cytotoxicity, fluorescent dichlorofluorescein (DCF) and nitroblue tetrazolium assays, Western blot analysis and flow cytometry, respectively. The apoptotic and necrotic cells were determined by staining with propidium iodide and fluorescein isothiocyanate-labelled annexin V. These variable were also measured after combined treatment of PC-3 cells with 1 mmol/L DDC and thermotherapy at 43 or 44 degrees C for 60 min. Cell survival was significantly lower after heating cells at 43 degrees C for 60, 90 and 120 min and at 44 degrees C for all periods tested (P<0.05). At 43 degrees C apoptosis increased with the duration of heating and was similarly enhanced after heating at 44 degrees C for 30 min. Necrosis was not increased by heating at 42 or 43 degrees C, but was markedly enhanced after heating at 44 degrees C with both the duration of heating and with time after heating. Significant increases in DCF production were induced by heating at 43 degrees C for 60, 90 and 120 min (P<0.05) and at 44 degrees C at all times (P<0.010-0.005). There was a significant correlation between the level of ROS generation and necrosis (P<0.001) but no correlation between the ROS level and apoptosis. SOD activity increased in cells after heating at 43 degrees C, with significant differences among cells heated for 60, 90 and 120 min (P<0.05). After heating at 44 degrees C, SOD activity was maximal in cells heated for 30 min (P<0.005), by 30 min and then decreased with time after heating. There were significant increases in hsp 70 level in cells heated at 43 degrees C for 90 and 120 min (P<0.05) and at 44 degrees C for 30 and 60 min (P<0.05 and <0.025, respectively). Hsp 70 levels decreased after heating at 44 degrees C for 90 and 120 min. The combination of DDC and heating significantly increased ROS generation and the percentage of cell death, and decreased SOD activity (P<0.05). These findings show a qualitative change in the form of cell death induced by thermotherapy of PC-3 cells, which changed from apoptosis to necrosis according to the degree and duration of heating. Mild thermotherapy induced marginally low occurrence of apoptosis of PC-3 cells and DDC may represent a useful future strategy for the treatment of prostate carcinoma.

The effect of early-age food restriction on heat shock protein 70 response in heat-stressed female broiler chickens

1. This study was conducted to determine the effect of early-age food restriction on heat shock protein (hsp) 70 synthesis in the brains of female broiler chickens exposed to high ambient temperatures. 2. Chicks were brooded for 3 weeks and then maintained at 24 ± 1°C. 3. On d 0, chicks were assigned to one of 4 feeding regimens; each regimen was applied to 4 cages of chicks. The regimens were: (1) ad libitum feeding (AL); (2) 80% food restriction at 4, 5 and 6 d of age (F80); (3) 60% food restriction at 4, 5, and 6 d of age (F60); and (4) 40% food restriction at 4, 5 and 6 d of age (F40). From d 35 to d 41, all chicks were subjected to 38 ± 1°C for 2 h/d. 4. One day following food restriction (d 7), hsp 70 expression in the brain samples of F60 and F40 chicks was augmented but not those fed AL and F80. 5. Prior to the heat challenge (d 35), all chicks had similar hsp 70 response. Irrespective of feeding regimen, there was a marked increase in hsp 70 expression after 4 d of heat treatment (d 38). Following 7 d of heat exposure (d 41), except for the F60 chicks, the augmented hsp 70 expression in the brains of AL, F80 and F40 birds was not maintained. 6. Enhancement of hsp 70 expression was noted in birds subjected to F60, but not AL, F80 or F40, throughout the period of heat exposure.

Presence of Heat Shock Protein 72 in Cardiomyocytes After Heat Stress

To the Editor: It is well-known that prior in vivo heat stress induces the synthesis of heat shock protein (HSP) 72 in the rat heart, but the precise content in the various cell-types has not yet been measured. It is therefore of great importance to conduct experiments as performed in the paper by Leger et al. 1 However, the overall conclusion that heat pretreatment does not lead to HSP72 synthesis in cardiomyocytes is unjustified. In addition, the statement that blood vessels play a primary role in the heat shock-induced cardioprotection is preliminary. The double-labeling experiments of Leger et al, 1 staining HSP72, as well as HSP27, clearly indicates the presence of both proteins in the same area, which the authors define as cardiomyocytes in the case of HSP27. Indeed, other studies indicate that cardiomyocytes do overexpress HSP72 after stress: cardiomyocytes of intact hearts are capable of overexpressing HSP72 after heat treatment. 2 Although the increase of HSP72 normalized for protein content was …

EXPRESSION OF hsp 27, hsp 60, hsc 70, AND hsp 70 BY IMMORTALIZED HUMAN PROXIMAL TUBULE CELLS (HK-2) FOLLOWING EXPOSURE TO HEAT SHOCK, SODIUM ARSENITE, OR CADMIUM CHLORIDE

The expression of hsp 27, hsp 60, hsc 70, and hsp 70 mRNA and protein was determined in immortalized human proximal tubule cells (HK-2) exposed to heat shock, sodium arsenite, or cadmium chloride (CdCl2) under both acute and extended conditions of exposure. It was demonstrated that the HK-2 cells did not exhibit the classic heat-shock response when subjected to an acute physical (heat) or chemical stress (sodium arsenite or CdCl2). Heat stress, elevated temperature at 42.5°C for 1 h, caused a marked increase only in hsp 70 mRNA and protein, but not hsp 27 or hsp 60 mRNA and protein. Similar results were obtained when the cells were subjected to a classic chemical stress of exposure to 100 µ M sodium arsenite for 4 h or CdCl2 for 4 h. These findings were in contrast to those found previously with mortal human proximal tubule (HPT) cells, where acute stress by all three stimuli elicited marked increases in hsp 27, hsp 60, and hsp 70 mRNA and protein. It was shown that the basal levels of expression of hsp 27 and hsp 60 in the HK-2 cells were elevated when compared to those found in unstressed HPT cells and that the basal levels were similar to those found in HPT cells under stress conditions. These results suggest that the failure of the HK-2 cells to in crease hsp 27 and hsp 60 levels in response to physical and chemical stress is because they already possess elevated basal levels of these proteins. This would indicate that one or more of the genetic events that resulted in the immortalization of the HK-2 cells also elicited a stress response for hsp 27 and hsp 60, but not for hsp 70, stress response family members. Overall, the results suggest that although there are differences in the regulation of the stress response between the immortal HK-2 and mortal HPT cell lines, as long as these differences are recognized, the HK-2 cell line should be a valuable adjunct to study the stress response of the proximal tubule in general and when exposed to environmental pollutants such as cadmium.

Heat-shock factor-1, steroid hormones, and regulation of heat-shock protein expression in the heart.

Heat-shock proteins (HSPs) are an important family of endogenous, protective proteins. Overexpression of HSPs is protective against cardiac injury. Previously, we observed that dexamethasone activated heat-shock factor-1 (HSF-1) and induced a 60% increase in HSP72 in adult cardiac myocytes. The mechanism responsible for this effect of dexamethasone is unknown. Because HSP90 is known to bind the intracellular hormone receptors, we postulated that the interaction between HSP90, the receptors, and HSF was an important element in activation of HSF-1 by hormones. We hypothesized that there is an equilibrium between HSP90 and the various receptors/enzymes that it binds and that alteration in levels of certain hormones will alter the intracellular distribution of HSP90 and activate HSF-1. We report that, in adult cardiac myocytes, HSF-1 coimmunoprecipitates with HSP90. HSP90 redistributes in cardiac myocytes after treatment with 17beta-estradiol or progesterone. Estrogen and progesterone activate HSF-1 in adult male isolated cardiac myocytes, and this is followed by an increase in HSP72 protein. Testosterone had no effect on HSP levels; however, no androgen receptor was found in cardiac myocytes; therefore, testosterone would not be expected to effect binding of HSP90 to HSF. Geldanamycin, which inactivates HSP90 and prevents it from binding to receptors, activates HSF-1 and stimulates HSP72 synthesis. Activation of HSF-1 by steroid hormones, resulting from a change in the interaction of HSP90 and HSF-1, represents a novel pathway for regulating expression of HSPs. These findings may explain some of the gender differences in cardiovascular disease.

Surviving the Heat

Mild heat shock can allow cells to survive a subsequently more severe heat stress. Gabai et al . show that mild heat shock induces the heat shock protein Hsp72 . The increase in Hsp72 is required for thermotolerance and the ability to survive normally nontoxic heat stress in fibroblasts is impaired if Hsp72 expression is blocked by expression of Hsp72 antisense RNA. Whereas many protein kinases, such as Akt, extracellular signal-regulated kinase 1/2 (ERK1/2), p38, and c-Jun NH 2 -terminal kinase (JNK), are activated by heat shock, during subsequent heat stress, JNK activity is suppressed in thermotolerant cells. Pharmacological analysis or expression of dominant-negative proteins suggested that activation of ERK1/2 and Akt promoted cell survival following heat shock but that inhibition of JNK was associated with cell survival. Overexpression of Hsp72 led to a suppression of JNK activation and increased cell survival following mild heat shock and inhibition of Hsp72 with antisense RNA led to prolonged JNK activation and decreased viability. Gabai, V.L., Yaglom, J.A., Volloch, V., Meriin, A.B., Force, T., Koutroumanis, M., Massie, B., Mosser, D.D., and Sherman, M.Y. (2000) Hsp72-mediated suppression of c-Jun N-terminal kinase is implicated in development of tolerance to caspase-independent cell death. Mol. Cell. Biol. 20 : 6826-6836. [Abstract] [Full Text]