Effects of chronic heat stress on the expressions of heat shock proteins 60, 70, 90, A2, and HSC70 in the rabbit testis

Few studies have focused on the expression of heat shock proteins (HSPs) after chronic heat stress. The objective of this study was to investigate the effect of chronic high temperature–humidity index treatment on the expressions of HSP60, HSP70, HSP90, HSPA2 and HSC70, in the Rex rabbit testis and the expressions of these proteins after recovery from the chronic heat shock. Thirty mature male rabbits of the same age were randomly divided into three groups: control, heat stress, and recovery. The western blot results showed that the expressional levels of HSP60, HSP90, and HSC70 increased significantly and HSPA2 was elevated slightly after a 9-week heat treatment. HSP70 was absent in the control testis and had a high level of expression after heat stress. All of these proteins partially reverted back to normal levels after a 9-week recovery. The immunohistochemical results indicated that the expression patterns of HSP60, HSP90, HSPA2, and HSC70 did not change.     

Differential localization of heat shock proteins 90, 70, 60 and 27 in human decidua and placenta during pregnancy

Little is known about the localization of heat shock proteins (HSPs) in the decidua and placenta during the course of normal pregnancy. In this study, we have examined the localization of the HSPs in decidual and placental tissues obtained from women during the first, second and third trimesters of pregnancy (five in each trimester) by immunohistochemistry using highly specific antisera. HSPs 90, 70, 60 and 27 were detected in decidual stromal cells during each trimester. The intensity of staining did not change during gestation for HSPs 60 and 27, whereas it decreased with advancing gestation for HSPs 90 and 70. HSPs 90 and 60 were localized primarily in the nucleus, whereas HSP 70 was present equally in the nucleus and the cytoplasm; HSP 27 was primarily in the cytoplasm. In the placenta, HSPs 90, 70 and 60 were localized in cytotrophoblast, syncytiotrophoblast, intermediate trophoblast, Hofbauer and endothelial cells. HSPs 90 and 60 were localized primarily in the nucleus, while HSP 70 was in the nucleus and the cytoplasm. In the placenta, HSP 27 was detected only in the intermediate trophoblast and syncytiotrophoblast cells and only in the first two trimesters. These results indicate that there are striking differences in the subcellular localization of HSPs in the decidua and the placenta during normal pregnancy.     

Developmental changes of heat-shock proteins in porcine testis by a proteomic analysis

Heat-shock proteins (HSPs) are important in spermatogenesis. This study investigated developmental changes in the expression of major HSPs in porcine testis. The testis from five immature (mean age 2.9+/-0.1 months) and five mature boars (35.7+/-14.0 months) were examined. Two-dimensional polyacrylamide gel electrophoresis was conducted and proteins were identified by Western blotting and/or matrix-assisted laser desorption/ionization mass spectrometry. Moreover, the 90, 70, and 60 kDa HSPs, 70 kDa heat-shock cognate protein (HSC 70), tubulin, and actin were quantified on two-dimensional gels. Protein spots were quantified by densitometry, combined with a computer-assisted image analysis system. Immunohistochemistry was performed to analyze the expression pattern of major HSPs and beta-tubulin in testis. One isoform of HSP 90 (HSP 90 alpha), two isoforms of HSC 70 (HSC 70a and HSC 70c), one isoform of HSP70 (HSP 70e), and tubulin increased after sexual maturation (P<0.05). A testis-specific HSP70 (P70t) was markedly increased in the testes of sexually mature boars. Meanwhile, levels of actin and some isoforms of HSPs including 60 kDa HSP remained similar in both groups. These observations were further confirmed by immunohistochemistry; therefore, the upregulation of protein expression in the adult testis could be attributed to a higher level of protein expression and the number of cells that were HSPs-positive already resided in the immature testis. The differential expression of major HSPs suggested that they may be important in porcine spermatogenesis.     

Quantitative proteomics of heat-treated human cells show an across-the-board mild depletion of housekeeping proteins to massively accumulate few HSPs

Classic semiquantitative proteomic methods have shown that all organisms respond to a mild heat shock by an apparent massive accumulation of a small set of proteins, named heat-shock proteins (HSPs) and a concomitant slowing down in the synthesis of the other proteins. Yet unexplained, the increased levels of HSP messenger RNAs (mRNAs) may exceed 100 times the ensuing relative levels of HSP proteins. We used here high-throughput quantitative proteomics and targeted mRNA quantification to estimate in human cell cultures the mass and copy numbers of the most abundant proteins that become significantly accumulated, depleted, or unchanged during and following 4 h at 41 °C, which we define as mild heat shock. This treatment caused a minor across-the-board mass loss in many housekeeping proteins, which was matched by a mass gain in a few HSPs, predominantly cytosolic HSPCs (HSP90s) and HSPA8 (HSC70). As the mRNAs of the heat-depleted proteins were not significantly degraded and less ribosomes were recruited by excess new HSP mRNAs, the mild depletion of the many housekeeping proteins during heat shock was attributed to their slower replenishment. This differential protein expression pattern was reproduced by isothermal treatments with Hsp90 inhibitors. Unexpectedly, heat-treated cells accumulated 55 times more new molecules of HSPA8 (HSC70) than of the acknowledged heat-inducible isoform HSPA1A (HSP70), implying that when expressed as net copy number differences, rather than as mere “fold change” ratios, new biologically relevant information can be extracted from quantitative proteomic data. Raw data are available via ProteomeXchange with identifier PXD001666.

Electronic supplementary material

The online version of this article (doi:10.1007/s12192-015-0583-2) contains supplementary material, which is available to authorized users.     

Secretion of the heat shock proteins HSP70 and HSC70 by baby hamster kidney (BHK‐21) cells

The HSPs (heat‐shock proteins) of the 70‐kDa family, the constitutively expressed HSC70 (cognate 70‐kDa heat‐shock protein) and the stress‐inducible HSP70 (stress‐inducible 70‐kDa heat‐shock protein), have been reported to be actively secreted by various cell types. The mechanisms of the release of these HSPs are obscure, since they possess no consensus secretory signal sequence. We showed that baby hamster kidney (BHK‐21) cells released HSP70 and HSC70 in a serum‐free medium and that this process was the result of an active secretion of HSPs rather than the non‐specific release of the proteins due to cell death. It was found that the secretion of HSP70 and HSC70 is independent of de novo protein synthesis. BFA (Brefeldin A) did not inhibit the basal secretion of HSPs, indicating that the secretion of HSP70 and HSC70 from cells occurs by a non‐classical pathway. Exosomes did not contribute to the secretion of HSP70 and HSC70 by cells. MBC (methyl‐β‐cyclodextrin), a substance that disrupts the lipid raft organization, considerably reduced the secretion of both HSPs, indicating that lipid rafts are involved in the secretion of HSP70 and HSC70 by BHK‐21 cells. The results suggest that HSP70 and HSC70 are actively secreted by BHK‐21 cells in a serum‐free medium through a non‐classical pathway in which lipid rafts play an important role.     

Differential regulation of HSC70, HSP70, HSP90 alpha, and HSP90 beta mRNA expression by mitogen activation and heat shock in human lymphocytes

A subset of heat shock proteins, HSP90 alpha, HSP90 beta, and a member of the HSP70 family, HSC70, shows enhanced synthesis following mitogenic activation as well as heat shock in human peripheral blood mononuclear cells. In this study, we have examined expression of mRNA for these proteins, including the major 70-kDa heat shock protein, HSP70, in mononuclear cells following either heat shock or mitogenic activation with phytohemagglutinin (PHA), ionomycin, and the phorbol ester, tetradecanoyl phorbol acetate. The results demonstrate that the kinetics of mRNA expression of these four genes generally parallel the kinetics of enhanced protein synthesis seen following either heat shock or mitogen activation and provide clear evidence that mitogen-induced synthesis of HSC70 and HSP90 is due to increased mRNA levels and not simply to enhanced translation of preexisting mRNA. Although most previous studies have focused on cell cycle regulation of HSP70 mRNA, we found that HSP70 mRNA was only slightly and transiently induced by PHA activation, while HSC70 is the predominant 70-kDa heat shock protein homologue induced by mitogens. Similarly, HSP90 alpha appears more inducible by heat shock than mitogens while the opposite is true for HSP90 beta. These results suggest that, although HSP70 and HSC70 have been shown to contain similar promoter regions, additional regulatory mechanisms which result in differential expression to a given stimulus must exist. They clearly demonstrate that human lymphocytes are an important model system for determining mechanisms for regulation of heat shock protein synthesis in unstressed cells. Finally, based on kinetics of mRNA expression, the results are consistent with the hypothesis that HSC70 and HSP90 gene expression are driven by an IL-2/IL-2 receptor-dependent pathway in human T cells.     

The small heat shock proteins family: the long forgotten chaperones

Small heat shock proteins are a rather heterogeneous family of ATP-independent chaperones, some of which have been proven to block protein aggregation and help the cells to survive stressful conditions. Although much less studied than high molecular weight HSPs like HSP70/HSPA or HSP90/HSPC, their implication in physio-pathological processes and human diseases is now well evidenced, as it will be discussed in the different reviews of this special issue. In this mini-review we will just present a general introduction about the small heat shock proteins family. This article is part of a Directed Issue entitled: Small HSPs in physiology and pathology.     

Seasonal variation in expression pattern of genes under HSP70

Heat shock protein 70 (HSP70) is one of the most abundant and best characterized heat shock protein family that consists of highly conserved stress proteins, expressed in response to stress, and plays crucial roles in environmental stress tolerance and adaptation. The present study was conducted to identify major types of genes under the HSP70 family and to quantify their expression pattern in heat- and cold-adapted Indian goats (Capra hircus) with respect to different seasons. Five HSP70 gene homologues to HSPA8, HSPA6, HSPA1A, HSPA1L, and HSPA2 were identified by gene-specific primers. The cDNA sequences showed high similarity to other mammals, and proteins have an estimated molecular weight of around 70 kDa. The expression of HSP70 genes was observed during summer and winter. During summer, the higher expression of HSPA8, HSPA6, and HSPA1A was observed, whereas the expression levels of HSPA1L and HSPA2 were found to be lower. It was also observed that the expression of HSPA1A and HSPA8 was higher during winter in both heat- and cold-adapted goats but downregulates in case of other HSPs. Therefore, both heat and cold stress induced the overexpression of HSP70 genes. An interesting finding that emerged from the study is the higher expression of HSP70 genes in cold-adapted goats during summer and in heat-adapted goats during winter. Altogether, the results indicate that the expression pattern of HSP70 genes is species- and breed-specific, most likely due to variations in thermal tolerance and adaptation to different climatic conditions.     

Myocardial heat shock proteins during the development of heart failure

When cardiomyocytes are exposed to stresses, production of heat shock proteins (HSPs) in the cells is enhanced. Such increase in cellular HSP production is considered to bring about tolerance against stress-induced cell damage. The exact role of the cellular HSPs remains unclear. In the present study, HSPs in the viable left ventricular myocardium were determined during the development of heart failure following coronary artery ligation (CAL). The rats after CAL showed symptoms of chronic heart failure (CHF) at the 8th week, but not at the 1st and 2nd weeks. Myocardial HSP27, which may bind to cytoskeletal protein, at the 1st, 2nd, and 8th weeks after CAL was approximately 180, 160, and 125% of the control, respectively. Myocardial HSP60, one of mitochondrial proteins, at the 8th week increased to 140% of the control, whereas those at the 1st and 2nd weeks did not change. Myocardial HSP72, an inducible form of HSP70 family, at the 1st week after CAL increased to 180% of the control, whereas that at the 2nd or 8th week was similar to control. Myocardial heat shock constitutive protein 73 (HSC73), a constitutively expressed form of HSP70 family, and HSP90, which may bind to steroid hormone receptor and actin fiber, of CAL rats did not alter throughout the experiment. These findings show that diverse changes in the production of myocardial HSPs occur during the development of heart failure. Only the increase in myocardial HSP60 production was associated with the development of CHF.     

Muscle type-specific response of HSP60, HSP72, and HSC73 during recovery after elevation of muscle temperature.

An original method to induce heat stress was used to clarify the time course of changes in heat shock proteins (HSPs) in rat skeletal muscles during recovery after a single bout of heat stress. One hindlimb was inserted into a stainless steel can and directly heated by raising the air temperature inside the can via a flexible heater twisted around the steel can. Muscle temperature was increased gradually and maintained at 42 degrees C for 60 min. Core rectal and contralateral muscle temperatures were increased <1.5 degrees C during the heat stress. HSP60, HSP72, and heat shock cognate (HSC) 73 content in the slow soleus and fast plantaris in both limbs were determined immediately (0 h) and 2, 4, 8, 12, 24, 36, 48, or 60 h after heat stress. Within 0-4 h, all HSPs were approximately 1.5- to 2.2-fold higher in heat-stressed than contralateral soleus. Compared with the contralateral plantaris, the heat-stressed plantaris had a higher (1.5-fold) HSP60 content immediately and 2 h after heat stress and a higher (2.5- to 6.8-fold) HSP72 content between 24 and 48 h after heat stress. Plantaris HSC73 content was not affected by heat stress. This unique heat-stress method provides advantages over existing systems; muscle temperature can be controlled precisely during heating and the HSP response can be compared between muscles in heat-stressed and contralateral limbs of individual rats. Results show a differential response of HSPs in the soleus and plantaris during recovery after heat stress; soleus demonstrated a more rapid and broader HSP response to heat stress than plantaris.     

Identification of two molecular chaperons (HSX70, HSC70) in mature human erythrocytes

Two-dimensional gel electrophoresis of cytosolic proteins from mature human erythrocytes combined with immunoblotting revealed the presence of a group of heat shock proteins (HSPs) that included two molecular chaperons of the HSP70 family (HSX70, inducible; HSC70, constitutively expressed) and HSP90. As expected for cells devoid of organelles, erythrocytes do not contain stress proteins that are localized either in the mitochondria (HSP60, glucose-regulated protein (GRP 75) or in the endoplasmic reticulum (GRP78 or Ig heavy chain-binding protein, endoplasmin). Since red cells are unable to replace proteins whose structure has been damaged by environmental changes the results are taken to imply a role for chaperons in monitoring, protecting, and maintaining the structure and stability of erythrocyte proteins.     

Time course and differential responses of the major heat shock protein families in human skeletal muscle following acute nondamaging treadmill exercise.

The exercise-induced expression of heat shock proteins (HSPs) in rodent models is relatively well defined. In contrast, comparable data from human studies are limited and the exercise-induced stress response of human skeletal muscle is far from understood. This study has characterized the time course and magnitude of the HSP response in the skeletal muscles of a healthy active, but untrained, young male population following a running exercise protocol. Eight subjects performed 45 min of treadmill running at a speed corresponding to their lactate threshold (11.7 +/- 0.5 km/h; 69.8 +/- 4.8% maximum O2 uptake). Muscle biopsies were obtained from the vastus lateralis muscle immediately before and at 24 h, 48 h, 72 h, and 7 days postexercise. Exercise induced a significant (P < 0.05) but variable increase in HSP70, heat shock cognate (HSC) 70, and HSP60 expression with peak increases (typically occurring at 48 h postexercise) to 210, 170, and 139% of preexercise levels, respectively. In contrast, exercise did not induce a significant increase in either HSP27, alphaB-crystallin, SOD 2 (MnSOD) protein content, or the activity of SOD and catalase. When examining baseline protein levels, HSC70, HSP27, and alphaB-crystallin appeared consistently expressed between subjects, whereas HSP70 and MnSOD displayed marked individual variation of up to 3- and 1.5-fold, respectively. These data are the first to define the time course and extent of HSP production in human skeletal muscle following a moderately demanding and nondamaging running exercise protocol. Data demonstrate a differential effect of aerobic exercise on specific HSPs.     

The regulation of heat shock proteins in response to dehydration in <Emphasis Type="Italic">Xenopus laevis</Emphasis>

African clawed frogs (Xenopus laevis) endure bouts of severe drought in their natural habitats and survive the loss of approximately 30% of total body water due to dehydration. To investigate molecular mechanisms employed by X. laevis during periods of dehydration, the heat shock protein response, a vital component of the cytoprotective stress response, was characterized. Using western immunoblotting and multiplex technology, the protein levels of HSP27, HSP40, HSP60, HSP70, HSC70, and HSP90 were quantified in the liver, skeletal muscle, kidney, lung, and testes from control frogs and those that underwent medium or high dehydration (~16 or ~30% loss of total body water). Dehydration increased HSP27 (1.45–1.65-fold) in the kidneys and lungs, and HSP40 (1.39–2.50-fold) in the liver, testes, and skeletal muscle. HSP60 decreased in response to dehydration (0.43–0.64 of control) in the kidneys and lungs. HSP70 increased in the liver, lungs, and testes (1.39–1.70-fold) during dehydration, but had a dynamic response in the kidneys (levels increased 1.57-fold with medium dehydration, but decreased to 0.56 of control during high dehydration). HSC70 increased in the liver and kidneys (1.20–1.36-fold), but decreased in skeletal muscle (0.27–0.55 of control) during dehydration. Lastly, HSP90 was reduced in the kidney, lung, and skeletal muscle (0.39–0.69 of control) in response to dehydration, but rose in the testes (1.30-fold). Overall, the results suggest a dynamic tissue-specific heat shock protein response to whole body dehydration in X. laevis.     

Developmental regulation of heat shock protein synthesis and HSP 70 RNA accumulation during postimplantation rat embryogenesis

Exposure of postimplantation rat embryos on days 9, 10, 11, and 12 of gestation to an in vitro heat shock of 43 degrees C for 30 min results in the induction of heat shock proteins (HSPs) in day 9 and 10 embryos, a severely attenuated response in day 11 embryos, and no detectable response in day 12 embryos. The heat shock response in day 9 embryos (presomite stage) is characterized by the synthesis of HSPs with molecular weights of 28-78 kDa. In heat shocked day 10 embryos, two additional HSPs are induced (34 and 82 kDa). In addition, two HSPs present on day 9 are absent on day 10. In day 11 heat shocked embryos, only three HSPs (31, 39, and 69 kDa) are induced, while in day 12 embryos no detectable HSPs are induced. Northern blot analysis of HSP 70 RNA levels indicates that the accumulation of this RNA, but not actin RNA, varies depending on developmental stage at the time of exposure to heat as well as the duration of the heat shock. Day 9 embryos exhibit the most pronounced accumulation of HSP 70 RNA while embryos on days 10-12 exhibit an increasingly attenuated accumulation of HSP 70 RNA, particularly after the more acute exposures (43 degrees C for 30 or 60 min). Thus, the ability to synthesize HSP 70 and to accumulate HSP 70 RNA changes dramatically as rat embryos develop from day 9 to day 12 (presomite to 31-35 somite stages).     

Heat stress facilitates stretch-induced hypertrophy of cultured muscle cells

Increased mechanical stress induced by stretch is an important growth stimulus in skeletal muscle. Heat shock proteins (HSPs) are an important family of endogenous, protective proteins. HSP90 and HSP70 families show elevated levels under beat stress. Mechanical stress, such as physical exercise, is known to induce not only muscular hypertrophy but also the elevation of HSPs expression in skeletal muscle. The purpose of this study was to determine whether heat stress facilitates the stretch-induced hypertrophy of skeletal muscle cells. Cultured rat myotubes (L6) were plated on collagenized Silastic membranes and incubated at 41 degrees C for 60 and 75 minutes (heat shock). Following the incubation, the cells were subjected two-second stretching and four-second releasing for 4 days at 37 degrees C. Protein concentrations in the homogenates and pellets of the cultured skeletal muscle cells increased under heat shock and/or mechanical stretching. The protein concentration of cells following mechanical stretching following heat shock was significantly higher than that following either heat shock or mechanical stretching alone. HSP72 in supernatants and HSP90 in pellets increased under heat shock and/or mechanical stretching. HSP90 in supernatants decreased following heat shock and/or mechanical stretching. Changes in HSPs and cellular protein concentrations in stressed cells suggest that the expression of HSPs may be closely related with muscular hypertrophy.     

Bacterial Lipopolysaccharide Augments Febrile-Range Hyperthermia-Induced Heat Shock Protein 70 Expression and Extracellular Release in Human THP1 Cells

Sepsis, a devastating and often lethal complication of severe infection, is characterized by fever and dysregulated inflammation. While infections activate the inflammatory response in part through Toll-like receptors (TLRs), fever can partially activate the heat shock response with generation of heat shock proteins (HSPs). Since extracellular HSPs, especially HSP70 (eHSP70), are proinflammatory TLR agonists, we investigated how exposure to the TLR4 agonist, bacterial lipopolysaccharide (LPS) and febrile range hyperthermia (FRH; 39.5°C) modify HSP70 expression and extracellular release. Using differentiated THP1 cells, we found that concurrent exposure to FRH and LPS as well as TLR2 and TLR3 agonists synergized to activate expression of inducible HSP72 (HSPA1A) mRNA and protein via a p38 MAP kinase-requiring mechanism. Treatment with LPS for 6 h stimulated eHSP70 release; levels of eHSP70 released at 39.5°C were higher than at 37°C roughly paralleling the increase in intracellular HSP72 in the 39.5°C cells. By contrast, 6 h exposure to FRH in the absence of LPS failed to promote eHSP70 release. Release of eHSP70 by LPS-treated THP1 cells was inhibited by glibenclamide, but not brefeldin, indicating that eHSP70 secretion occurred via a non-classical protein secretory mechanism. Analysis of eHSP70 levels in exosomes and exosome-depleted culture supernatants from LPS-treated THP1 cells using ELISA demonstrated similar eHSP70 levels in unfractionated and exosome-depleted culture supernatants, indicating that LPS-stimulated eHSP70 release did not occur via the exosome pathway. Immunoblot analysis of the exosome fraction of culture supernatants from these cells showed constitutive HSC70 (HSPA8) to be the predominant HSP70 family member present in exosomes. In summary, we have shown that LPS stimulates macrophages to secrete inducible HSP72 via a non-classical non-exosomal pathway while synergizing with FRH exposure to increase both intracellular and secreted levels of inducible HSP72. The impact of increased macrophage intracellular HSP70 levels and augmented secretion of proinflammatory eHSP70 in the febrile, infected patient remains to be elucidated.     

Proteasome inhibition induces differential heat shock protein response but not unfolded protein response in HepG2 cells

Liver, a central organ responsible for the metabolism of carbohydrates, proteins, and lipoproteins, is exposed to various kinds of physiological, pathological, and environmental stresses. We hypothesized that blockage of proteasome degradation pathway induces heat shock protein (HSP) response and unfolded protein response in the liver cells. In this study, we have characterized cellular responses to proteasome inhibition in HepG2 cells, a well-differentiated human hepatoma cells. We found that proteasome inhibition induced differential response among cytosolic HSPs, that is, increased expression of HSP70, but no change in HSP40, HSC70, and HSP90. However, proteasome inhibition did not induce typical unfolded protein response as indicated by absence of stimulation of GRP78 and GRP94 proteins. Upon proteasome inhibition, inclusion bodies were accumulated, and ubiquitin-conjugated proteins appeared in insoluble fraction, together with HSP40, HSP70, HSC70, and HSP90. After proteasome inhibition, misfolded proteins were increased in the cytosol and in the ER compartment as evaluated by examining ubiquitin-conjugated proteins. However, essentially all ER-associated ubiquitin-conjugated proteins were located on the surface of the ER, which explains why proteasome inhibition does not induce unfolded protein response. In conclusion, proteasome inhibition induces differential HSP response, but not unfolded protein response in HepG2 cells. Our study also suggests that HSPs play important roles in directing proteasomal degradation and protein aggregate formation.