Levels Of Inducible Hsp70 Research Articles

Heat shock protein 70 from a thermotolerant Diptera species provides higher thermoresistance to Drosophila larvae than correspondent endogenous gene

Heat shock proteins (Hsp70s) from two Diptera species that drastically differ in their heat shock response and longevity were investigated. Drosophila melanogaster is characterized by the absence of Hsp70 and other hsps under normal conditions and the dramatic induction of hsp synthesis after temperature elevation. The other Diptera species examined belongs to the Stratiomyidae family (Stratiomys singularior) and exhibits high levels of inducible Hsp70 under normal conditions coupled with a thermotolerant phenotype and much longer lifespan. To evaluate the impact of hsp70 genes on thermotolerance and longevity, we made use of a D.melanogaster strain that lacks all hsp70 genes. We introduced single copies of either S.singularior or D.melanogaster hsp70 into this strain and monitored the obtained transgenic flies in terms of thermotolerance and longevity. We developed transgenic strains containing the S.singularior hsp70 gene under control of a D.melanogaster hsp70 promoter. Although these adult flies did synthesize the corresponding mRNA after heat shock, they were not superior to the flies containing a single copy of D.melanogaster hsp70 in thermotolerance and longevity. By contrast, Stratiomyidae Hsp70 provided significantly higher thermotolerance at the larval stage in comparison with endogenous Hsp70.

Combined Pharmacological Induction of Hsp70 Suppresses Prion Protein Neurotoxicity in Drosophila

Prion diseases are rare and aggressive neurodegenerative disorders caused by the accumulation of misfolded, toxic conformations of the prion protein (PrP). Therapeutic strategies directed at reducing the levels of PrP offer the best chance of delaying or halting disease progression. The challenge, though, is to define pharmacologic targets that result in reduced PrP levels. We previously reported that expression of wild type hamster PrP in flies induces progressive locomotor dysfunction and accumulation of pathogenic PrP conformations, while co-expression of human Hsp70 delayed these changes. To validate the therapeutic potential of Hsp70, we treated flies with drugs known to induce Hsp70 expression, including the Hsp90 inhibitor 17-DMAG and the glucocorticoid dexamethasone. Although the individual treatment with these compounds produced no significant benefits, their combination significantly increased the level of inducible Hsp70, decreased the level of total PrP, reduced the accumulation of pathogenic PrP conformers, and improved locomotor activity. Thus, the combined action of two pharmacological activators of Hsp70 with distinct targets results in sustained high levels of inducible Hsp70 with improved behavioral output. These findings can have important therapeutic applications for the devastating prion diseases and other related proteinopathies.

Inducible HSP70 Antagonizes IL-1β Cytocidal Effects through Inhibiting NF-kB Activation via Destabilizing TAK1 in HeLa Cells

BackgroundDespite several reports describing the HSP70-mediated cytoprotection against IL-1, the precise mechanism for this phenomenon remains to be determined.Methods/Principal FindingsHere we used HeLa cells, a human epithelial carcinoma cell line, to evaluate the role of inducible HSP70 in response of IL-1β stimulation. We found that inducible HSP70 antagonized the cytotoxicity of IL-1β and improved the survival of HeLa cells. Further investigation demonstrated that increased expression level of inducible HSP70 reduced the complex of TAK1 and HSP90, and promoted the degradation of TAK1 protein via proteasome pathway. By overexpression and RNAi knockdown, we showed that inducible HSP70 modulated the NF-kB but not MAPKs signalings through influencing the stability of TAK1 protein in HeLa cells. Moreover, overexpression of HSP70 attenuated the production of iNOS upon IL-1β stimulation, validating that inducible HSP70 serves as a cytopretective factor to antagonize the cytocidal effects of IL-1β in HeLa cells.Conclusions/SignificanceOur observations provide evidence for a novel signaling mechanism involving HSP70, TAK1, and NF-κB in the response of IL-1β cytocidal effects. This research also provides insight into mechanisms by which HSP70 exerts its cytoprotective action upon toxic stimuli in tumor cells.

Cellular thermotolerance is associated with heat shock protein 70.1 genetic polymorphisms in Holstein lactating cows

Heat shock proteins (Hsp) are known to protect cells from several stressors. Nucleotide changes in the flanking regions [5'- and 3'-untranslated region (UTR)] of Hsp gene might affect inducibility, degree of expression, or stability of Hsp70 mRNA. The present study aimed to investigate the association between inducible Hsp70.1 single nucleotide polymorphisms (SNPs) and heat shock (HS) response of peripheral blood mononuclear cells (PBMC) in dairy cows. Four hundred forty-six Italian Holstein cows were genotyped for four Hsp70.1 SNPs: g895 C/- and g1128 G/T in 5'-UTR, and g2154 G/A and g64 G/T in 3'-UTR. Genetic polymorphisms in 3'-UTR of bovine Hsp70.1 gene resulted monomorphic. Distribution of alleles of the nucleotide sequence polymorphism within the 5'-UTR of the bovine Hsp70.1 gene were 81.2% and 18.8% for C and -, respectively, and 77.8% and 22.2% for G and T, respectively. Among the 446 genotyped animals, a group of cows balanced for days in milk and parity was selected to be representative of the following genotypes: CC (n = 8), C- (n = 7), and -- (n = 7) and GG (n = 8), GT (n = 11), and TT (n = 3) in 5'-UTR. PBMC were isolated from blood samples and heated at 43°C in thermal bath for 1h and then incubated at 39°C in atmosphere of 5% CO(2) for 1, 2, 4, 8, 16, and 24h (recovery times). Cell viability was determined by XTT assay. Gene and protein expression of Hsp70.1 was determined by real-time reverse transcription-polymerase chain reaction and by ELISA assay, respectively. For the two SNPs detected, one allele was the most frequent (C, 66.8% and G, 56.8%). Genotypes -- and TG showed higher (P < 0.05) viability compared with CC and GG, respectively. Genotypes C- and TT had intermediate viability. Gene expression of Hsp70.1 showed higher (P < 0.001) levels in -- and TG genotype compared with their counterparts. Genotypes -- and TG showed the higher level of inducible Hsp70.1 protein in respect to C-, TT and CC, GG. In conclusion, exposure to HS differently affected cell viability and gene and protein expression of Hsp70.1 in the selected genotypes. These results indicate that the presence of SNPs (C/- and G/T) in the 5'-UTR region of inducible Hsp70.1 ameliorates HS response and tolerance to heat of bovine PBMC. These mutation sites may be useful as molecular genetic markers to assist selection for heat tolerance.

α-Lipoic acid supplementation enhances heat shock protein production and decreases post exercise lactic acid concentrations in exercised standardbred trotters

Heat shock protein (HSP) expression is an adaptive mechanism against the disruption of cell homeostasis during exercise. Several antioxidant supplementation strategies have been used to enhance tissue protection. In this study, we examined the effects of a redox modulator, α-lipoic acid (LA) on HSP responses in six standardbred trotters following intense aerobic exercise. DL–LA supplementation (25 mg kg −1 d −1) for five weeks increased the resting levels of HSP90 (1.02 ± 0.155 in control and 1.26 ± 0.090 after supplementation in arbitrary units) and the recovery levels of inducible HSP70 (0.89 ± 0.056 in control and 1.05 ± 0.089 after supplementation in arbitrary units) in skeletal muscle. Furthermore, LA increased skeletal muscle citrate synthase activity at rest and lowered the blood lactate concentration during exercise without any changes in the heart rate. LA had no effect on concentrations of HSP60, HSP25 or GRP75 in skeletal muscle. LA decreased the exercise-induced increases in plasma aspartate aminotransferase and creatine kinase concentrations during recovery. Our results suggest that LA supplementation may enhance tissue protection and increase oxidative capacity of the muscle in horse.

Alpha-lipoic Acid Attenuates Exercise-induced Oxidative Stress And Enhances Heat Shock Protein Responses

Uncontrolled oxidative stress, a state where the increased production of ROS overwhelms endogenous antioxidant protection, may result in macromolecular damage and perturbation of redox control of signalling and cellular events. The heat shock proteins (HSPs) respond to a wide variety of stress, including oxidative stress. HSPs function primarily as molecular chaperones, but also control protein synthesis and trafficking, as well as regulate cellular adaptation and survival. PURPOSE: Several antioxidant supplementation strategies have been used to cope with exercise-induced oxidative stress and to facilitate adaptations to exercise. While antioxidant supplementation diminishes exercise-induced oxidative stress, there is also a risk of decreasing the normal response of tissues to exercise and blunting the adaptations. In this study we examined the protective role of natural thiol antioxidant, alpha-lipoic acid (LA) supplementation at rest, after exercise and at different recovery points in skeletal muscle in horses. METHODS: Six standardbred trotters were examined on the treadmill and exercised 75 min at individually defined aerobic level. Blood and muscle biopsy samples were taken before and immediately after treadmill exercise, as well as after 2-, 6-, 24- and 48-hour recovery. RESULTS: LA supplementation (25 mg kg-1 day-1) for five weeks increased the resting levels of HSP90 and the recovery levels of inducible HSP70 in gluteus medius muscle. In addition, following LA-supplementation, we observed a significant decrease in exercise-induced protein oxidation in plasma and free radical formation in muscle, measured by EPR signals. CONCLUSION: Our findings suggest that LA enhances HSP levels and decreases exercise-induced oxidative stress in skeletal muscle.

Transcription Inhibition of Heat Shock Proteins: A Strategy for Combination of 17-Allylamino-17-Demethoxygeldanamycin and Actinomycin D

The heat shock protein (HSP) 90 inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG) is currently in clinical trials because of its unique mechanism of action and antitumor activity. However, 17-AAG triggers the transcription and elevation of antiapoptotic HSP90, HSP70, and HSP27, which lead to chemoresistance in tumor cells. We hypothesized that inhibiting HSP90, HSP70, and HSP27 transcription may enhance 17-AAG-induced cell death in multiple myeloma cell lines. Actinomycin D (Act D), a clinically used agent and transcription inhibitor, was combined with 17-AAG. The concentrations for 17-AAG and Act D were selected based on the target actions and plasma levels during therapy. Inducible and constitutive HSP27, HSP70, and HSP90 mRNA and protein levels were measured by real-time reverse transcription-PCR and immunoblot assays. Compared with no treatment, Act D alone decreased HSP mRNA levels in MM.1S and RPMI-8226 cell lines. Combining Act D with 17-AAG did not attenuate 17-AAG-mediated increases in transcript levels of inducible HSP70; however, constitutive HSP mRNA levels were decreased. In contrast to its effect on mRNA levels, Act D was able to abrogate 17-AAG-mediated increases in all HSP protein levels. The cytotoxicity of combined Act D and 17-AAG was assessed. Treatment with Act D alone caused <40% cell death, whereas the combination of 17-AAG and Act D resulted in an increase of cell death in both multiple myeloma cell lines. In conclusion, these results indicate that 17-AAG-mediated induction of HSP70 and HSP27 expression can be attenuated by Act D and therefore can potentially improve the clinical treatment of multiple myeloma.

Lindane alters the levels of HSP70 and clusterin in adult rat testis

Lindane, an organochlorine pesticide, has been reported to induce reproductive abnormalities in male rats and induction of stress is considered to play an important role in the toxicity of lindane. To get more insight into the mechanism(s) involved in gonadal effect(s) of lindane, we sought to investigate whether treatment with lindane would alter the levels of stress proteins (heat shock proteins and clusterin) and change oxidative stress-related parameters (antioxidant enzymes and lipid peroxidation) in the testis of adult male rats. Animals used for experimentation were divided into six groups and four animals were maintained in each group. Animals were administered with a single dose of lindane (5 mg/kg body weight) and terminated at specific post-treatment intervals (0, 3, 6, 12, 24 and 72 h) to analyze the early stress response. Administration of lindane resulted in a sequential reduction in the activities of catalase and superoxide dismutase (SOD) with concomitant increase in the levels of lipid peroxidation. These changes elicited by lindane were significant between 6 and 24 h post-treatment. In case of heat shock protein, the level of the constitutive HSP70 remained constant at all post-treatment intervals. However, a significant induction in the level of inducible HSP70 was observed between 6 and 24 h post-treatment. Similarly, the levels of secretory clusterin (sCLU) was found be significantly elevated at 12 and 24 h after treatment. These findings indicate a transitory state of stress induced by lindane in rat testis accompanied by an alteration in the levels of stress proteins, which could be a part of protective mechanism mounted to reduce cellular damage.

Increased Levels of Inducible HSP70 in Cells Exposed to Electromagnetic Fields

Because reports in the literature on the effects of electromagnetic fields (EMFs) on expression of the 70-kDa heat-shock protein (HSP70) are somewhat contradictory, we studied the influence of low-frequency EMFs on the accumulation of inducible HSP70 in several cell models. Some of the cell types tested showed increased levels of HSP70 protein when exposed for 24 h to 50 Hz, 680 microT EMFs. In endothelial cells, EMFs alone induced only a poor and transient activation of the heat-shock transcription factor 1 (HSF1); however, neither the level of HSP70 mRNA nor the synthesis of HSP70 appeared to be altered significantly. Accordingly, transfection experiments involving HSP70 promoter showed that gene transcription was not affected. We also noted a marked reduction in proteasome activities in cell extracts exposed to EMFs. Interestingly, the heat-shock-induced levels of HSP70 mRNA and protein were increased by a concomitant weak stressor like EMFs. Taken together, our results indicate that in EMF-exposed endothelial cells, HSP70 gene transcription and translation are unaffected; however, EMFs alone promoted accumulation of the inducible HSP70 protein, probably by increasing its stability, and it enhanced accumulation and translation of the heat-induced HSP70 mRNA when applied in concert with heat shock.

Basal and inducible levels of Hsp70 in patients with acute heat illness induced during training.

Heat shock proteins (Hsps) or stress proteins, and, in particular, the inducible, cytosolic Hsp70, represent a highly conserved response to heat exposure and to a variety of noxious stimuli. Many investigations have shown correlations between the aberrant expression of Hsps and disease states. Whether the basal and inducible levels of Hsp70 are of any biological significance in patients with heat-induced diseases remains unknown. In the present study, we compared the basal and inducible levels of Hsp70 by flow cytometry in lymphocytes of patients with heat-induced diseases and after recovery from this disease, and in matched controls. Both groups comprised individuals who exercised by running in the same hot environment. The level of inducible Hsp70 was also measured after a heat treatment of lymphocytes in vitro. The results show that there is variation of basal and inducible Hsp70 levels among individuals. However, the group of patients suffering from heat-induced illnesses in May shows a significantly higher basal (P = 0.02) level of Hsp70 than does the control group. Individuals who have an increased level of Hsp70 may be more sensitive to heat or may respond differently. The level of Hsp70 may represent a biomarker to evaluate whether they are more susceptible to stresses than other individuals. Interestingly, the basal level of Hsp70 is higher in both the patient group and the control group in November than in May. In fact, the basal levels of Hsp70 in the patient and control groups are essentially the same in November, perhaps reflecting the successful stress conditioning of both groups.

Heat shock proteins refine the danger theory.

Deficiencies of the self–non-self paradigm in explaining immune phenomena, such as the existence and breaking of immune tolerance, have lead the likes of Janeway1 and Matzinger2 to suggest additional levels of control of antigen-specific immune responses. In simple terms, the immune system is thought to be turned on by ‘danger’ associated with certain molecules of infectious organisms, or cell products released during tissue damage or stress. Antigen-presenting cells (APC) possess pattern-recognition receptors that can recognize such molecules. This recognition is thought to cause upregulation of costimulatory molecules on local APC, which is the second signal [in addition to antigenic peptide with major histocompatibility complex (MHC)] that is required to initiate an antigen-specific immune response. Candidate danger signals from infectious organisms include lipopolysaccharide, mannans, glycans and CpG DNA motifs, whilst those from mammalian cells include heat shock proteins (hsp; inducible and constitutive), mitochondria, mannose, RNA and DNA. With respect to hsp, recent evidence now suggests that their role in signalling danger may be wide ranging and, not surprisingly for immunology, more complicated than first thought.

Variation of Heat Shock Protein 70 through the Cell Cycle in HL-60 Cells and Its Relationship to Apoptosis

Cells respond to a heat shock by synthesizing heat shock proteins, which help to protect the cells from further heating. Recent results indicate that heat shock protein 70 (hsp70) may help to protect cells from apoptosis. We have used flow cytometry to investigate the relationship between constitutive and inducible hsp70 and apoptosis through the cell cycle in HL-60 cells. Specific antibodies were used to measure hsp70 in cells costained with propidium iodide. In separate samples apoptosis was measured using the TdT assay. The apoptotic cells have a subdiploid DNA content, which allows them to be identified also in the bivariate histograms of heat shock protein vs DNA content. After HL-60 cells were heated at 45.0°C for 7.5 min and incubated for various times at 37°C, many of them underwent apoptosis. The level of inducible hsp70 (hsp72) was lower in the apoptotic cells than in the nonapoptotic population, but constitutive hsp70 (hsp73) was the same in both populations. Pretreatment with sodium vanadate increased the fraction of apoptotic cells twofold, slightly increased the level of hsp72 in the nonapoptotic cells, but did not affect hsp73. These results suggest that hsp72, but not hsp73, is involved in the development or prevention of apoptosis.

The effect of extract fromRhodiola rosea on the level of inducible HSP-70 in the myocardium during stress

It is demonstrated that the cardioprotective activity of extract fromRhodiola rosea in stress reaches the maximum 5 days after the first dose. Heat shock proteins appear in the myocardium 3 days after an 8-day administration of the extract. It is believed that these proteins are not an important factor in the cardioprotective effect of the extract.