HSP27 and HSP70: potentially oncogenic apoptosis inhibitors

Stress or heat shock proteins (HSPs) such as HSP27 and HSP70 are expressed in response to a wide variety of physiological and environmental insults including heat, reactive oxygen species or anticancer drugs. Their overexpression allows cells to survive to otherwise lethal conditions. Several different mechanisms may account for the cytoprotective activity of HSP27 and HSP70. First, both proteins are powerful chaperones. Second, both inhibit key effectors of the apoptotic machinery including the apoptosome, the caspase activation complex (both HSP27 and HSP70), and apoptosis inducing factor (only HSP70). Third, they both play a role in the proteasome-mediated degradation of apoptosis-regulatory proteins. HSP27 and HSP70 may participate in oncogenesis, as suggested by the fact that overexpression of heat shock proteins can increase the tumorigenic potential of tumor cells. The down-regulation or selective inhibition of HSP70 might constitute a valuable strategy for the treatment of cancer.     

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.     

Cell cycle-dependent association of HSP70 with specific cellular proteins

In asynchronous populations of HeLa cells maintained at control or heat shock temperatures, HSP70 levels and its subcellular distribution exhibit substantial heterogeneity as demonstrated by indirect immunofluorescence with HSP70-specific monoclonal antibodies. Of particular interest is a subpopulation of cells in which the characteristic nuclear accumulation and nucleolar association of HSP70 is not detected after heat shock treatment. This apparent variation in the heat shock response is not observed when synchronized cells are examined. In this study, we demonstrate that three monoclonal antibodies to HSP70, in particular, do not detect nucleolar-localized HSP70 in heat-shocked G2 cells. This is not due to an inability of G2 cells to respond to heat shock as measured by increased HSP70 mRNA and protein synthesis, or due to a lack of accumulation of HSP70 after heat shock in G2. Rather the epitopes recognized by the various antibodies appear to be inaccessible, perhaps due to the association of HSP70 with other proteins. Non-denaturing immunoprecipitations with these HSP70-specific antibodies suggest that HSP70 may interact with other cellular proteins in a cell cycle-dependent manner.     

Transfection of human HSP27 in rodent cells: Absence of compensatory regulation between small heat shock proteins

1. 1. We examined rodent cells transfected with an expression plasmid encoding a human small heat shock protein for possible compensatory expression of endogenous heat shock genes. For these investigations, human hsp27 was transfected into CHO cells which express endogenous HSP25.

2. 2. Both endogenous HSP25 and transfected HSP27 were expressed and multiple phosphorylated isoforms were detected upon exposure to thermal stress.

3. 3. Levels of endogenous HSP70 and HSP25 did not appear to be altered by expression of the heterologous heat shock protein.

4. 4. These results suggest that compensatory interactions are not exhibited in the expression of the heat shock genes examined, and that independent regulation may exist not only between the large and small heat shock proteins, but also between individual small heat shock proteins as well.

     

Apoptosis Versus Cell Differentiation: Role of Heat Shock Proteins HSP90, HSP70 and HSP27

Heat shock proteins HSP27, HSP70 and HSP90 are molecular chaperones whose expression is increased after many different types of stress. They have a protective function helping the cell to cope with lethal conditions. The cytoprotective function of HSPs is largely explained by their anti-apoptotic function. HSPs have been shown to interact with different key apoptotic proteins. As a result, HSPs can block essentially all apoptotic pathways, most of them involving the activation of cystein proteases called caspases. Apoptosis and differentiation are physiological processes that share many common features, for instance, chromatin condensation and the activation of caspases are frequently observed. It is, therefore, not surprising that many recent reports imply HSPs in the differentiation process. This review will comment on the role of HSP90, HSP70 and HSP27 in apoptosis and cell differentiation. HSPs may determine de fate of the cells by orchestrating the decision of apoptosis versus differentiation.Key Words: apoptosis, differentiation, heat shock proteins, chaperones, cancer cells, anticancer drugs     

Stressful preconditioning and HSP70 overexpression attenuate proteotoxicity of cellular ATP depletion

Rat H9c2 myoblasts were preconditioned by heat or metabolic stress followed by recovery under normal conditions. Cells were then subjected to severe ATP depletion, and stress-associated proteotoxicity was assessed on 1) the increase in a Triton X-100-insoluble component of total cellular protein and 2) the rate of inactivation and insolubilization of transfected luciferase with cytoplasmic or nuclear localization. Both heat and metabolic preconditioning elevated the intracellular heat shock protein 70 (HSP70) level and reduced cell death after sustained ATP depletion without affecting the rate and extent of ATP decrease. Each preconditioning attenuated the stress-induced insolubility among total cellular protein as well as the inactivation and insolubilization of cytoplasmic and nuclear luciferase. Transient overexpression of human HSP70 in cells also attenuated both the cytotoxic and proteotoxic effects of ATP depletion. Quercetin, a blocker of stress-responsive HSP expression, abolished the effects of stressful preconditioning but did not influence the effects of overexpressed HSP70. Analyses of the cellular fractions revealed that both the stress-preconditioned and HSP70-overexpressing cells retain the soluble pool of HSP70 longer during ATP depletion. Larger amounts of other proteins coimmunoprecipitated with excess HSP70 compared with control cells deprived of ATP. This is the first demonstration of positive correlation between chaperone activity within cells and their viability in the context of ischemia-like stress.     

Recombinant HSP70 and mild heat shock stimulate growth of aged mesenchymal stem cells

Heat shock proteins including the major stress protein HSP70 support intracellular homeostasis and prevent protein damage after a temperature increase and other stressful environmental stimuli, as well as during aging. We have shown earlier that prolonged administration of recombinant human HSP70 to mice exhibiting Alzheimer’s-like neurodegeneration as well as during sepsis reduces the clinical manifestations of these pathologies. Herein, we studied the action of recombinant human HSP70 on young and aged mouse mesenchymal stem cells (MSCs) in culture. The results obtained indicate that HSP70 at concentrations of 2 μg/ml and higher significantly stimulates growth of aged but not young MSCs. A similar effect is produced by application of a mild heat shock (42 °C 5 min) to the cells. Importantly, responses of young and aged MSCs to heat shock treatment of various durations differed drastically, and aged MSCs were significantly more sensitive to higher heat stress exposures than the young cells. Western blotting and protein labeling experiments demonstrated that neither mild heat shock nor exogenous HSP70 administration resulted in significant endogenous HSP70 induction in young and aged MSCs, whereas mild heat shock increased HSC70 levels in aged MSCs. The results of this study suggest that the administration of exogenous HSP70 and the application of mild heat stress may produce a certain “rejuvenating” effect on MSCs and possibly other cell types in vivo, and these interventions may potentially be used for life extension by delaying various manifestations of aging at the molecular and cellular level.     

The Effect of Manganese-induced Cytotoxicity on mRNA Expressions of HSP27, HSP40, HSP60, HSP70 and HSP90 in Chicken Spleen Lymphocytes in Vitro

The purpose of this study was to investigate the effect of manganese (Mn)-induced cytotoxicity on heat shock proteins in chicken spleen lymphocytes. Lymphocytes were cultured in medium in the absence and presence of MnCl₂ (2?×?10^?4, 4?×?10^?4, 6?×?10^?4, 8?×?10^?4, 10?×?10^?4, and 12?×?10^?4 mmol/L) for 12, 24, 36, and 48 h in vitro. Then, the mRNA levels of HSP27, HSP40, HSP60, HSP70, and HSP90 were examined by real-time quantitative PCR. The results showed that the mRNA levels of HSP27, HSP40, HSP60, HSP70, and HSP90 in all treatment groups at all time points, except mRNA levels of HSP27 at 48 h, had the same tendency. As manganese concentration increased, the mRNA expression of the heat shock proteins first increased and then decreased. In other words, we demonstrated that the mRNA expression of the heat shock proteins was induced at lower concentrations of manganese and was inhibited at higher concentrations. Mn had a dosage-dependent effect on HSP27, HSP40, HSP60, HSP70, and HSP90 mRNA expression in chicken spleen lymphocytes in vitro.     

Exosome-dependent trafficking of HSP70: a novel secretory pathway for cellular stress proteins

The heat shock proteins (HSPs) are a family of intracellular proteins found in all eukaryotes and prokaryotes. Their functions are well characterized and are central to maintaining cellular homeostasis and in promoting cell survival in response to stressful cellular conditions. However, several studies provide evidence that specific members of the HSP family might be secreted via an unidentified exocytotic pathway. Here we show that exosomes, small membrane vesicles that are secreted by numerous cell types, contribute to the release of HSP70 from human peripheral blood mononuclear cells (PBMCs) in both basal and stress-induced (heat shock at 40 or 43 degrees C for 1 h) states. HSP70 release from PBMCs is independent of the common secretory pathway because Brefeldin A, an inhibitor of the classical protein transport pathway, did not block HSP70 release. Furthermore, we show that HSP70 release from PBMCs does not occur via a lipid raft-dependent pathway, because treatment with methyl-beta-cyclodextrin, a raft-disrupting drug, had no affect on HSP70 release. To examine whether exosomes contributed to HSP70 release from PBMCs, exosomes were purified from PBMC cultures, and exosomal number and HSP70 content were determined. We demonstrate that although heat shock does not influence the exosomal secretory rate, the HSP70 content of exosomes isolated from heat shocked PBMCs is significantly higher than control. These data identify a novel secretory pathway by which HSP70 can be actively released from cells in both the basal and stress-induced state.     

Intracellular delivery of HSP70 using HIV-1 Tat protein transduction domain

Heat shock protein 70 (HSP70) is an intracellular stress protein that confers cytoprotection to a variety of cellular stressors. Several lines of evidence have suggested that augmentation of the heat shock response by increasing the expression of HSP70 represents a potential therapeutic strategy for the treatment of critically ill patients. The Tat protein of human immunodeficiency virus 1 (HIV-1) has been used previously to deliver functional cargo proteins intracellularly when added exogenously to cultured cells. We generated a Tat-HSP70 fusion protein using recombinant methods and treated HSF -/- cells with either Tat-HSP70 or recombinant HSP70 prior to exposure to hyperoxia or lethal heat shock. We showed that biologically active, exogenous HSP70 can be delivered into cells using the HIV-1 Tat protein, and that the Tat-mediated delivery of HSP70 confers cytoprotection against thermal stress and hyperoxia and may represent a novel approach to augmenting intracellular HSP70 levels.     

Heat shock proteins HSP70 and HSP27 in the cerebral spinal fluid of patients undergoing thoracic aneurysm repair correlate with the probability of postoperative paralysis

An understanding of the time course and correlation with injury of heat shock proteins (HSPs) released during brain and/or spinal cord cellular stress (ischemia) is critical in understanding the role of the HSPs in cellular survival, and may provide a clinically useful biomarker of severe cellular stress. We have analyzed the levels of HSPs in the cerebrospinal fluid (CSF) from patients who are undergoing thoracic aneurysm repair. Blood and CSF samples were collected at regular intervals, and CSF was analyzed by enzyme-linked immunosorbent assay for HSP70 and HSP27. These results were correlated with intraoperative somatosensory-evoked potentials measurements and postoperative paralysis. We find that the levels of these proteins in many patients are elevated and that the degree of elevation correlates with the risk of permanent paralysis. We hypothesize that sequential measurement intraoperatively of the levels of the heat shock proteins HSP70 and HSP27 in the CSF can predict those patients who are at greatest risk for paralysis during thoracic aneurysm surgery and will allow us to develop means of preventing or attenuating this severe and often fatal complication.     

Differences in preferential synthesis and redistribution of HSP70 and HSP28 families by heat or sodium arsenite in Chinese hamster ovary cells

Since both heat and sodium arsenite induce thermotolerance, we investigated the differences in synthesis and redistribution of stress proteins induced by these agents in Chinese hamster ovary cells. Five major heat shock proteins (HSPs; Mr 110, 87, 70, 28, and 8.5 kDa) were preferentially synthesized after heat for 10 min at 45.5 degrees C, whereas four major HSPs (Mr 110, 87, 70, and 28 kDa) and one stress protein (33.3 kDa) were preferentially synthesized after treatment with 100 microM sodium arsenite (ARS) for 1 hr. Two HSP families (HSP70a,b,c, and HSP28a,b,c) preferentially relocalized in the nucleus after heat shock. In contrast, only HSP70b redistributed into the nucleus after ARS treatment. Furthermore, the kinetics of synthesis of each member of HSP70 and HSP28 families and their redistribution were different after these treatments. The maximum rates of synthesis of HSP70 and HSP28 families, except HSP28c, were 6-9 hr after heat shock, whereas those of HSP70b and HSP28b,c were 0-2 hr after ARS treatment. In addition, the maximum rates of redistribution of HSP70 and HSP28 families occurred 3-6 hr after heat shock, whereas that of HSP70b occurred immediately after ARS treatment. The degree of redistribution of HSP70b after ARS treatment was significantly less than that after heat treatment. These results suggest that heat treatment but not sodium arsenite treatment stimulates the entry of HSP70 and HSP28 families into the nucleus.     

Heat shock resistance conferred by expression of the human HSP27 gene in rodent cells

Heat shock induces in cells the synthesis of specific proteins called heat shock proteins (HSPs) and a transient state of thermotolerance. The putative role of one of the HSPs, HSP27, as a protective molecule during thermal stress has been directly assessed by measuring the resistance to hyperthermia of Chinese hamster and mouse cells transfected with the human HSP27 gene contained in plasmid pHS2711. One- and two-dimensional gel electrophoresis of [3H]leucine- and [32P]orthophosphate-labeled proteins, coupled with immunological analysis using Ha27Ab and Hu27Ab, two rabbit antisera that specifically recognize the hamster and the human HSP27 protein respectively, were used to monitor expression and inducibility of the transfected and endogenous proteins. The human HSP27 gene cloned in pHS2711 is constitutively expressed in rodent cells, resulting in accumulation of the human HSP27 and all phosphorylated derivatives. No modification of the basal or heat-induced expression of endogenous HSPs is detected. The presence of additional HSP27 protein provides immediate protection against heat shock administered 48 h after transfection and confers a permanent thermoresistant phenotype to stable transfectant Chinese hamster and mouse cell lines. Mild heat treatment of the transfected cells results in an induction of the full complement of the endogenous heat shock proteins and a small increase in thermoresistance, but the level attained did not surpass that of heat-induced thermotolerant control cells. These results indicate that elevated levels of HSP27 is sufficient to give protection from thermal killing. It is concluded that HSP27 plays a major role in the increased thermal resistance acquired by cells after exposure to HSP inducers.     

Phosphorylation of HSP27 during development and decay of thermotolerance in Chinese hamster cells

The small molecular weight heat shock protein HSP27 was recently shown to confer a stable thermoresistant phenotype when expressed constitutively in mammalian cells after structural gene transfection. These results suggested that HSP27 may also play an important role in the development of thermotolerance, the transient ability to survive otherwise lethal heat exposure after a mild heat shock. In Chinese hamster O23 cells increased thermoresistance is first detected at 2 h after a triggering treatment of 20 min at 44 degrees C, attains a maximum at 5 hours, and decays thereafter with a half-life of 10 h. We found that the development and decay of transient thermotolerance cannot be solely explained on the basis of changes in the cellular concentration of HSP27. The cellular HSP27 concentration is not increased appreciably at 2 h after heat shock and attains a maximum at 14 h. Similar results were obtained in the case of another heat shock protein, HSP70. HSP70 follows slightly faster kinetics of accumulation (peaks at 10 h) and decays much more rapidly (ti/2 = 4h) than HSP27 (t1/2 = 13h). HSP27 has 3 isoelectric variants A, B, and C of which B and C are phosphorylated. In cells maintained at normal temperature, HSP27A represents more than 90% of all HSP27. Shifting the cell culture temperature from 37 to 44 degrees C induces the incorporation of 32P into the more acidic B and C forms, a process that occurs very rapidly since the reduction in the concentration of the A form and a corresponding increase in the level of B and C is detectable by immunoblot analysis within 2.5 min at 44 degrees C. Analyses performed at various times during development and decay of transient thermotolerance revealed a close relationship between the effect of heat shock on HSP27 phosphorylation and cell ability to survive. For example, fully thermotolerant cells (5 h post-induction) are refractory to induction of HSP27 phosphorylation by a 20-min heat shock. The induction of HSP27 phosphorylation was also studied in a family of clonal cell lines of O23 cells that are thermoresistant as a result of the constitutive expression of a transfected human HSP27 gene. In these thermoresistant cells, phosphorylation of the endogenous hamster HSP27 is induced to a level comparable to that found in the thermosensitive parental cells. However, phosphorylation of the exogenous human protein, which represents more than 80% of total HSP27 in these cells, was much less induced.(ABSTRACT TRUNCATED AT 250 WORDS)     

HSP27 inhibits cytochrome c-dependent activation of procaspase-9.

We have previously shown that the small heat shock protein HSP27 inhibited apoptotic pathways triggered by a variety of stimuli in mammalian cells. The present study demonstrates that HSP27 overexpression decreases U937 human leukemic cell sensitivity to etoposide-induced cytotoxicity by preventing apoptosis. As observed for Bcl-2, HSP27 overexpression delays poly(ADP-ribose)polymerase cleavage and procaspase-3 activation. In contrast with Bcl-2, HSP27 overexpression does not prevent etoposide-induced cytochrome c release from the mitochondria. In a cell-free system, addition of cytochrome c and dATP to cytosolic extracts from untreated cells induces the proteolytic activation of procaspase-3 in both control and bcl-2-transfected U937 cells but fails to activate procaspase-3 in HSP27-overexpressing cells. Immunodepletion of HSP27 from cytosolic extracts increases cytochrome c/dATP-mediated activation of procaspase-3. Overexpression of HSP27 also prevents procaspase-9 activation. In the cell-free system, immunodepletion of HSP27 increases LEDH-AFC peptide cleavage activity triggered by cytochrome c/dATP treatment. We conclude that HSP27 inhibits etoposide-induced apoptosis by preventing cytochrome c and dATP-triggered activity of caspase-9, downstream of cytochrome c release.     

HSP70 and Genomic Stability

The 70 kDa heat shock proteins (HSP70) were initially identified by their elevated expression following hyperthermic cell stress, however, these highly conserved proteins also protect critical cellular functions from a wider range of important environmental and physiological stresses. At least one result of HSP70 expression is inhibition of stress induced caspase activation as well as downstream events in the apoptotic cell death pathway. HSP70 have been reported upregulated in tumor cells, selective inhibition of such proteins might be valuable approach to treat cancer. A recent study revealed that cells with inactivated HSP70 displayed telomere instability and high frequency of spontaneous chromosomal aberrations, indicating a possible role for HSP70 proteins in the maintenance of genomic stability.     

HSP70 and genomic stability

The 70 kDa heat shock proteins (HSP70s) were initially identified by their elevated expression following hyperthermic cell stress, however, these highly conserved proteins also protect critical cellular functions from a wider range of important environmental and physiological stresses. At least one result of HSP70 expression is inhibition of stress induced caspase activation as well as downstream events in the apoptotic cell death pathway. HSP70 have been reported upregulated in tumor cells, selective inhibition of such proteins might be valuable approach to treat cancer. A recent study revealed that cells with inactivated HSP70 displayed telomere instability and high frequency of spontaneous chromosomal aberrations, indicating a possible role for HSP70 proteins in the maintenance of genomic stability.