Hsp70 inhibits aminoglycoside-induced hearing loss and cochlear hair cell death

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) can inhibit JNK- and caspase-dependent apoptosis in a variety of systems. We have previously shown that heat shock results in 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 this system, Hsp70 is the most strongly induced Hsp, which is upregulated over 250-fold at the level of mRNA 2 h after heat shock. Hsp70 overexpression inhibits aminoglycoside-induced hair cell death in vitro. In this study, we utilized Hsp70-overexpressing mice to determine whether Hsp70 is protective in vivo. Both Hsp70-overexpressing mice and their wild-type littermates were treated with systemic kanamycin (700 mg/kg body weight) twice daily for 14 days. While kanamycin treatment resulted in significant hearing loss and hair cell death in wild-type mice, Hsp70-overexpressing mice were significantly protected against aminoglycoside-induced hearing loss and hair cell death. These data indicate that Hsp70 is protective against aminoglycoside-induced ototoxicity in vivo.     

Expression of the molecular chaperone Hsp70 in detergent-resistant microdomains correlates with its membrane delivery and release

Accumulating evidence suggests that some heat shock proteins (Hsps), in particular the 72-kDa inducible Hsp70, associate to the cell membrane and might be secreted through an unknown mechanism to exert important functions in the immune response and signal transduction. We speculated that specialized structures named lipid rafts, known as important platforms for the delivery of proteins to the cell membrane, might be involved in the unknown mechanism ensuring membrane association and secretion of Hsp70. Lipid rafts are sphingolipid-cholesterol-rich structures that have been mainly characterized in polarized epithelial cells and can be isolated as detergent-resistant microdomains (DRMs). Analysis of soluble and DRM fractions prepared from unstressed Caco-2 epithelial cells revealed that Hsp70, and to a lesser extent calnexin, were present in DRM fractions. Increased expression of Hsps, through heat shock or by using drugs acting on protein trafficking or intracellular calcium level, induced an efficient translocation to DRM. We also found that Hsp70 was released by epithelial Caco-2 cells, and this release dramatically increased after heat shock. Drugs known to block the classical secretory pathway were unable to reduce Hsp70 release. By contrast, release of the protein was affected by the raft-disrupting drug methyl-beta-cyclodextrin. Our data suggest that lipid rafts are part of a mechanism ensuring the correct functions of Hsps and provide a rational explanation for the observed membrane association and release of Hsp70.     

Functional stabilization of Kv1.5 protein by Hsp70 in mammalian cell lines

The aim of this study was to elucidate the mechanisms for regulations of cardiac Kv1.5 channel expression. We particularly focused on the role of heat shock proteins (Hsps). We tested the effects of Hsps on the stability of Kv1.5 channels using biochemical and electrophysiological techniques: co-expression of Kv1.5 and Hsp family proteins in mammalian cell lines, followed by Western blotting, immunoprecipitation, pulse-chase analysis, immunofluorescence and whole-cell patch clamp. Hsp70 and heat shock factor 1 increased the expression of Kv1.5 protein in HeLa and COS7 cells, whereas either Hsp40, 27 or 90 did not. Hsp70 prolonged the half-life of Kv1.5 protein. Hsp70 was co-immunoprecipitated and co-localized with Kv1.5-FLAG. Hsp70 significantly increased the immunoreactivity of Kv1.5 in the endoplasmic reticulum, Golgi apparatus and on the cell membrane. Hsp70 enhanced Kv1.5 current of transfected cells, which was abolished by pretreatment with brefeldin A or colchicine. Thus, Hsp70, but not other Hsps, stabilizes functional Kv1.5 protein.     

Expression of inducible Hsp70 enhances the proliferation of MCF-7 breast cancer cells and protects against the cytotoxic effects of hyperthermia

Heat shock proteins (Hsps) are ubiquitous proteins that are induced following exposure to sublethal heat shock, are highly conserved during evolution, and protect cells from damage through their function as molecular chaperones. Some cancers demonstrate elevated levels of Hsp70, and their expression has been associated with cell proliferation, disease prognosis, and resistance to chemotherapy. In this study, we developed a tetracycline-regulated gene expression system to determine the specific effects of inducible Hsp70 on cell growth and protection against hyperthermia in MCF-7 breast cancer cells. MCF-7 cells expressing high levels of Hsp70 demonstrated a significantly faster doubling time (39 hours) compared with nonoverexpressing control cells (54 hours). The effect of elevated Hsp70 on cell proliferation was characterized further by 5-bromo-2'deoxyuridine labeling, which demonstrated a higher number of second and third division metaphases in cells at 42 and 69 hours, respectively. Estimates based on cell cycle analysis and mean doubling time indicated that Hsp70 may be exerting its growth-stimulating effect on MCF-7 cells primarily by shortening of the G0/G1 and S phases of the cell cycle. In addition to the effects on cell growth, we found that elevated levels of Hsp70 were sufficient to confer a significant level of protection against heat in MCF-7 cells. The results of this study support existing evidence linking Hsp70 expression with cell growth and cytoprotection in human cancer cells.     

Acclimation of the photosynthetic machinery to high temperature in Chlamydomonas reinhardtii requires synthesis de novo of proteins encoded by the nuclear and chloroplast genomes

The mechanism responsible for the enhancement of the thermal stability of the oxygen-evolving machinery of photosystem II during acclimation of Chlamydomonas reinhardtii to high temperatures such as 35 degrees C remains unknown. When cells that had been grown at 20 degrees C were transferred to 35 degrees C, the thermal stability of the oxygen-evolving machinery increased and within 8 h it was equivalent to that in cells grown initially at 35 degrees C. Such enhancement of thermal stability was prevented by cycloheximide and by lincomycin, suggesting that the synthesis de novo of proteins encoded by both the nuclear and the chloroplast genome was required for this process. No increase in thermal stability was observed when cells that had been grown at 35 degrees C were exposed to heat shock at 41 degrees C, optimum conditions for the induction of the synthesis of homologs of three heat shock proteins (Hsps), namely, Hsp60, Hsp70, and Hsp22. Moreover, no synthesis of these homologs of Hsps was induced at 35 degrees C. Thus it appears likely that Hsps are not involved in the enhancement of the thermal stability of the oxygen-evolving machinery.     

Neuronal differentiation in PC12 cells is accompanied by diminished inducibility of Hsp 70 and Hsp60 in response to heat and ethanol

The stress response of PC12 cells was characterized by evaluating the production of heat shock proteins of the 70 kDa (Hsp70), 60 kDa (Hsp60) and 90 kDa (Hsp90) families by western blot analysis. Induction of Hsp synthesis was elicited by brief exposure to elevated temperatures or by addition of ethanol to the cultures. Normal PC12 cells responded to stress with rapid up-regulation of Hsp70 and Hsp60 production. However, fully differentiated PC12 cells (induced by nerve growth factor, NGF) failed to produce Hsp70 or Hsp60 in response to heat or ethanol treatment. The disappearance of the heat shock response of the cells was directly related to the extent of neuronal differentiation. The cellular levels of the constitutive proteins, Hsc70 and Hsp90, were not altered by differentiation of the cells. Production of Hsps was restored in the differentiated cells by removal of NGF which coincided with the loss of neurite expression and retraction of processes.     

Expression and distribution of heat shock proteins in the heart of transported pigs

The expression and localization of four heat shock proteins (Hsp70, Hsp86, Hsp90, and Hsp27) were shown in the heart tissue of pigs transported for 6 h. Immunostaining detected the consistent presence of all Hsps in the pig myocardial cells under both transported and normal housing conditions. Immunohistochemical analysis revealed predominance of Hsp70 (significantly highest levels) and Hsp27 in the cytoplasm of myocardial cells. Hsp90 and Hsp86 were expressed both in the cytoplasm and in the nucleus, preferentially in the cytoplasm, of the myocardial cells. In view of their abundant and uniform distributions in the myocardial cells, the expression and distribution patterns of all detected Hsps within the myocardial cells, mostly limited to the cytoplasm, could be related to their chaperone function for cells with important special activities in this study. The identification of all four Hsps in the blood vessel endothelial cells possibly implies that endothelial cells react to ischemia and hypoxia by expressing Hsps. Immunoblot findings suggest that the level of all Hsps decreased in response to stress due to a 6 h journey. The decrease in Hsp levels in the myocardial cells may indicate that the transport stress may have overcharged the repair mechanisms of the cells. Whether this distinct depletion of Hsps contributes to an increased susceptibility to acute heart failure and the sudden death syndrome in transported pigs should be elucidated in future experiments.     

Tissue-specific variations in the induction of Hsp70 and Hsp64 by heat shock in insects

The patterns of heat-induced synthesis (37 degrees C to 45 degrees C) of heat shock proteins (Hsps) in different tissues of grasshoppers and cockroaches from natural populations and in laboratory-reared gram-pest (Heliothis armigera) were examined by 35S-methionine labeling and sodium dodecyl sulfate-polyacrylamide gel electrophoresis fluorography. Whereas 45 degrees C was lethal in most cases, optimal induction of Hsp synthesis was seen between 37 degrees C and 42 degrees C. The ongoing protein synthesis was not much affected at these temperatures, except in the tissues of adult H. armigera exposed to 42 degrees C. The profiles of the Hsps induced in the tissues of the insects, however, were different. From the relative abundance of the synthesis of 70-kDa (Hsp70) and 64-kDa (Hsp64) polypeptides, three categories of heat shock response were identified: (1) induction of abundant Hsp70 but little Hsp64 (malpighian tubules, male accessory glands, and ovaries of adult grasshoppers), (2) abundant Hsp64 but little Hsp70 (testes of adult grasshoppers, testes and malpighian tubules of adult cockroaches, and testes, malpighian tubules, and fat bodies of H. armigera larvae), and (3) induction of both Hsp70 and Hsp64 in more or less equal abundance (ovaries of adult cockroaches, salivary glands of H. armigera larvae, and malpighian tubules, male accessory glands, testes, and ovaries of adult H. armigera). Cockroaches collected from storerooms showed detectable synthesis of Hsp64 and/or Hsp70 only after heat shock, but those collected from drains showed detectable synthesis of both Hsp70 and Hsp64 in different tissues without heat stress. Western blotting showed that the 64-kDa polypeptide in these insects is a member of the Hsp60 family. Grasshopper testes, which synthesized negligible Hsp70 but abundant Hsp64 after heat shock, developed thermotolerance. Thus, heat shock response is modulated by developmental and environmental factors in different tissues of insects.     

棉花粉蚧热休克蛋白基因的鉴定

热休克蛋白(heat shock proteins,Hsps)是生物体或细胞受到热胁迫后新合成的一类遗传上高度保守的蛋白,在昆虫应对外界环境因子胁迫时起着重要作用。为了系统研究棉花粉蚧Phenacoccus solenopsis Hsp基因家族,对棉花粉蚧转录组基因注释信息进行分析、获得目标序列,并应用NCBI上Blast X等软件进行比对、共鉴定出24条热激蛋白(Hsp)基因,包括3个Hsp90、8个Hsp70、2个Hsp60和11个s Hsp(small heat shock protein,s Hsp)基因。对棉花粉蚧与模式昆虫家蚕Bombyx mori、黑腹果蝇Drosophila melanogaster、赤拟谷盗Tribolium castaneum系统进化关系分析显示,昆虫的小分子量热休克蛋白s Hsp具有很强的种属特异性,Hsp70家族的保守性比s Hsp强。棉花粉蚧热激蛋白基因的鉴定为深入研究该虫Hsp与生长发育、抗逆境的相互关系奠定了基础。     

Translational thermotolerance provided by small heat shock proteins is limited to cap-dependent initiation and inhibited by 2-aminopurine

Heat shock results in inhibition of general protein synthesis. In thermotolerant cells, protein synthesis is still rapidly inhibited by heat stress, but protein synthesis recovers faster than in naive heat-shocked cells, a phenomenon known as translational thermotolerance. Here we investigate the effect of overexpressing a single heat shock protein on cap-dependent and cap-independent initiation of translation during recovery from a heat shock. When overexpressing alphaB-crystallin or Hsp27, cap-dependent initiation of translation was protected but no effect was seen on cap-independent initiation of translation. When Hsp70 was overexpressed however, both cap-dependent and -independent translation were protected. This finding indicates a difference in the mechanism of protection mediated by small or large heat shock proteins. Phosphorylation of alphaB-crystallin and Hsp27 is known to significantly decrease their chaperone activity; therefore, we tested phosphorylation mutants of these proteins in this system. AlphaB-crystallin needs to be in its non-phosphorylated state to give protection, whereas phosphorylated Hsp27 is more potent in protection than the unphosphorylatable form. This indicates that chaperone activity is not a prerequisite for protection of translation by small heat shock proteins after heat shock. Furthermore, we show that in the presence of 2-aminopurine, an inhibitor of kinases, among which is double-stranded RNA-activated kinase, the protective effect of overexpressing alphaB-crystallin is abolished. The synthesis of the endogenous Hsps induced by the heat shock to test for thermotolerance is also blocked by 2-aminopurine. Most likely the protective effect of alphaB-crystallin requires synthesis of the endogenous heat shock proteins. Translational thermotolerance would then be a co-operative effect of different heat shock proteins.     

Increased ubiquitin-dependent degradation can replace the essential requirement for heat shock protein induction

Serine palmitoyltransferase, the first enzyme in ceramide biosynthesis, is required for resistance to heat shock. We show that increased heat shock sensitivity in the absence of serine palmitoyltransferase activity correlates with a lack of induction of the major heat shock proteins (Hsps) at high temperature. Normal heat shock resistance can be restored, without restoration of ceramide synthesis or induction of Hsps, by overexpression of ubiquitin. This function of ubiquitin requires the proteasome. These data imply that the essential function of Hsp induction is the removal of misfolded or aggregated proteins, not their refolding. This suggests that cells stressed by heat shock do not die because of the loss of protein activity due to their denaturation, but because of the inherent toxicity of the denatured and/or aggregated proteins.     

Deoxyribonucleic acid damage induced by doxorubicin in peripheral blood mononuclear cells: possible roles for the stress response and the deoxyribonucleic acid repair process

Doxorubicin is an antineoplastic drug widely used in cancer treatment. However, many tumors are intrinsically resistant to the drug or show drug resistance after an initial period of response. Among the different molecules implicated with doxorubicin resistance are the heat shock proteins (Hsps). At present we do not know with certainty the mechanism(s) involved in such resistance. In the present study, to advance our knowledge on the relationship between Hsps and drug resistance, we have used peripheral blood mononuclear cells obtained from healthy nonsmoker donors to evaluate the capacity of a preliminary heat shock to elicit the Hsp response and to establish the protection against the deoxyribonucleic acid (DNA) damage induced by doxorubicin. DNA damage and repair were determined using the alkaline comet assay. We also measured the expression of Hsp27, Hsp60, Hsp70, Hsp90, hMLH1, hMSH2, and proliferating cell nuclear antigen by immunocytochemistry. The damage induced by doxorubicin was more efficiently repaired when the cells were previously heat shocked followed by a resting period of 24 hours before drug exposure, as shown by (1) the increased number of undamaged cells (P < 0.05), (2) the increased DNA repair capacity (P < 0.05), and (3) the high expression of the mismatch repair (MMR) proteins hMLH1 and hMSH2 (P < 0.05). In addition, in the mentioned group of cells, we confirmed by Western blot high expression levels of Hsp27 and Hsp70. We also noted a nuclear translocation of Hsp27 and mainly of Hsp70. Furthermore, inducible Hsp70 was more expressed in the nucleus than Hsc70, showing a possible participation of Hsp70 in the DNA repair process mediated by the MMR system.     

Heat-shock proteins are induced in unstressed leaves of Nicotiana attenuata (Solanaceae) when distant leaves are stressed

In Nicotiana attenuata, systemic induction of heat-shock proteins (Hsps) was detected in response to the treatment of single leaves by either heat shock, mechanical damage, or exogenous application of methyl jasmonate (MJ). All treatments increased the abundance of members of the 70-kD Hsp (Hsp70) family and induced synthesis of one or more of the small Hsps (sHsp) (16-23 kD) in both treated and untreated leaves. These results provide the first evidence that Hsps can be systemically induced in plants and suggest that systemic induction of Hsps may be important in pre-adapting leaves to stress.     

Destabilization and recovery of a yeast prion after mild heat shock

Yeast prion [PSI⁺] is a self-perpetuating amyloid of the translational termination factor Sup35. Although [PSI⁺] propagation is modulated by heat shock proteins (Hsps), high temperature was previously reported to have little or no effect on [PSI⁺]. Our results show that short-term exposure of exponentially growing yeast culture to mild heat shock, followed by immediate resumption of growth, leads to [PSI⁺] destabilization, sometimes persisting for several cell divisions after heat shock. Prion loss occurring in the first division after heat shock is preferentially detected in a daughter cell, indicating the impairment of prion segregation that results in asymmetric prion distribution between a mother cell and a bud. Longer heat shock or prolonged incubation in the absence of nutrients after heat shock led to [PSI⁺] recovery. Both prion destabilization and recovery during heat shock depend on protein synthesis. Maximal prion destabilization coincides with maximal imbalance between Hsp104 and other Hsps such as Hsp70-Ssa. Deletions of individual SSA genes increase prion destabilization and/or counteract recovery. The dynamics of prion aggregation during destabilization and recovery are consistent with the notion that efficient prion fragmentation and segregation require a proper balance between Hsp104 and other (e.g., Hsp70-Ssa) chaperones. In contrast to heat shock, [PSI⁺] destabilization by osmotic stressors does not always depend on cell proliferation and/or protein synthesis, indicating that different stresses may impact the prion via different mechanisms. Our data demonstrate that heat stress causes asymmetric prion distribution in a cell division and confirm that the effects of Hsps on prions are physiologically relevant.     

Components of a mammalian protein disaggregation/refolding machine are targeted to nuclear speckles following thermal stress in differentiated human neuronal cells

Heat shock proteins (Hsps) are a set of highly conserved proteins involved in cellular repair and protective mechanisms. They counter protein misfolding and aggregation that are characteristic features of neurodegenerative diseases. Hsps act co-operatively in disaggregation/refolding machines that assemble at sites of protein misfolding and aggregation. Members of the DNAJ (Hsp40) family act as “holdases” that detect and bind misfolded proteins, while members of the HSPA (Hsp70) family act as “foldases” that refold proteins to biologically active states. HSPH1 (Hsp105α) is an important additional member of the mammalian disaggregation/refolding machine that acts as a disaggregase to promote the dissociation of aggregated proteins. Components of a disaggregation/refolding machine were targeted to nuclear speckles after thermal stress in differentiated human neuronal SH-SY5Y cells, namely: HSPA1A (Hsp70-1), DNAJB1 (Hsp40-1), DNAJA1 (Hsp40-4), and HSPH1 (Hsp105α). Nuclear speckles are rich in RNA splicing factors, and heat shock disrupts RNA splicing which recovers after stressful stimuli. Interestingly, constitutively expressed HSPA8 (Hsc70) was also targeted to nuclear speckles after heat shock with elements of a disaggregation/refolding machine. Hence, neurons have the potential to rapidly assemble a disaggregation/refolding machine after cellular stress using constitutively expressed Hsc70 without the time lag needed for synthesis of stress-inducible Hsp70. Constitutive Hsc70 is abundant in neurons in the mammalian brain and has been proposed to play a role in pre-protecting neurons from cellular stress.