Regulation of heat shock protein 70 synthesis by heat shock in the preimplantation murine embryo

Induced thermotolerance in murine embryos occurs at the 8-cell stage when embryos are maintained in vitro but not until the blastocyst stage if development proceeds in vivo. Present results indicate that ability of embryos to undergo induced thermotolerance is not limited by heat shock protein 70 (HSP70) synthesis. Exposure of 8-cell embryos to 40 degrees C enhanced synthesis of 2 constitutive HSP70 proteins (HSC70 and HSC72) and induced another protein, HSP68; exposure of 43 degrees C was required to induce similar responses in expanded blastocysts. Unlike induced thermotolerance, increased synthesis of HSP70 molecules did not depend on whether embryos were cultured or developed in vivo. Thus, other biochemical mechanisms in addition to HSP70 confer thermotolerance in the preimplantation-stage murine embryo. The observation that the temperature threshold for induction of HSP70 synthesis increased from the 8-cell to the blastocyst stage is indicative of these other biochemical processes.     

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.     

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.     

Molecular identification and expression of heat shock cognate 70 (HSC70) in the Pacific white shrimp Litopenaeus vannamei

Heat shock protein 70s (HSP70s) are fundamental chaperone proteins that are indispensable to most living organisms. In order to investigate the function of HSP70 and heat shock response in shrimp, a heat shock cognate (HSC70) gene of the white shrimp (Litopenaeus vannamei), containing a 1959-bp open reading frame, was cloned and characterized. The amino acid sequence, 71.5 kDa of molecular weight, shares 80-99.6% homology with 12 diverse species' HSP70s and HSC70s. In fact, some segments of the eukaryotic HSC70 sequence, such as ATP/GTP-binding site, cytoplasmic HSP70 C-terminal sequence, and GGMP/GAP repeats, are also found in the putative shrimp HSC70. Moreover, multi-tissue RT-PCR was performed to assay the basal expressions of HSC70 in the heart, gill, hepatopancreas, stomach, gut, and muscle. The results demonstrate that the basal expressions of HSC70 in theses organs are similar to that of beta-actin. Furthermore, quantitative real-time experiments showed that HSC70 was up-regulated in hepatopancreas (4.6-fold), stomach (5.9-fold), gut (2.6-fold), and muscle (3.5-fold) but not in the heart (1.7-fold) and gill (1.6-fold) after 2 h of heat shock. Nevertheless, the HSC70 was found to be highly expressed in the heart and gill following 6 h of heat shock. This suggests that HSC70 in white shrimp possess both short-term and long-term responses to heat shock stress, indicating this HSC70 may be a heat-dependent HSC70 member. Finally, we constructed an expression vector to generate HSC70 in Escherichia coli BL21, which displayed immune cross-reactivity with mouse HSP70 antibody. In conclusion, the identification and expression of white shrimp HSC70 gene present useful data for studying the molecular mechanism of heat shock response and the effect of heat shock proteins in shrimps' cytoprotection.     

The neuroprotective potential of heat shock protein 70 (HSP70)

In response to many metabolic disturbances and injuries, including stroke, neurodegenerative disease, epilepsy and trauma, the cell mounts a stress response with induction of a variety of proteins, most notably the 70-kDa heat shock protein (HSP70). Whether stress proteins are neuroprotective has been hotly debated, as these proteins might be merely an epiphenomenon unrelated to cell survival. Only recently, with the availability of transgenic animals and gene transfer, has it become possible to overexpress the gene encoding HSP70 to test directly the hypothesis that stress proteins protect cells from injury. A few groups have now shown that overproduction of HSP70 leads to protection in several different models of nervous system injury. This review will cover these studies, along with the potential mechanisms by which HSP70 might mediate cellular protection.     

Changes in the heat shock 70 kDa protein level in human neutrophils induced by heat shock

Changes in the content of constitutive and inducible proteins of the family of heat shock 70 kDa proteins (HSP70) caused by heat shock in human neutrophils, white blood cells with an atypically short lifespan, which provide a nonspecific defense of the organism against bacterial pathogens, have been studied. An analysis of the intracellular content of the constitutive and inducible HSP70 proteins by flow cytometry revealed a biphasic dynamics of changes in the protein level, which was characterized by an increase in the protein level immediately after heat shock followed by a decrease within 15–30 min after the termination of heat treatment. Because the inhibitor of protein synthesis cycloheximide did not change the dynamics profile, it was assumed that the increase in the HSP70 level is related not to the de novo synthesis of these proteins but to conformational changes of HSP70 molecules and an increased accessibility of some epitopes for antibody binding. Using a panel of antibodies specific to the N-terminal ATP-binding or the C-terminal substrate-binding domains of the protein, it was shown by cell immunofluorescence and flow cytometry that the heat shock-associated increase in the intracellular HSP70 level results from an increased efficiency of the binding of antibodies recognizing the substrate-binding domain. It was also demonstrated that the decrease in the intracellular HSP70 level after the heat shock, may be partially due to a release into the extracellular space of both the constitutive and inducible HSP70 proteins, which is regulated with the involvement of ABC-transporters.     

Proteomic profiling of heat shock protein 70 family members as biomarkers for hepatitis C virus-related hepatocellular carcinoma

To identify proteins linked to the pathogenesis of hepatocellular carcinoma (HCC) associated with hepatitis C virus (HCV), we profiled protein expression levels in samples of HCC. To identify essential proteins, ten samples of HCV-related HCC were analyzed by two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization-time of flight mass spectrometry. These experiments revealed increased levels of nine proteins in cancerous tissues compared to levels in corresponding noncancerous liver tissues. We focused on four members of the heat shock protein 70 family: 78 kDa glucose-regulated protein (GRP78), heat shock cognate 71 kDa protein (HSC70), 75 kDa glucose-regulated protein (GRP75), and heat shock 70 kDa protein 1 (HSP70.1). These results were confirmed by immunoblot analysis. In an additional 11 samples, the same expression patterns of these four proteins were observed. In total, 21 samples showed statistically significant up-regulation of GRP78, GRP75 and HSP70.1 in cancerous tissues. HSC70 showed a tendency toward overexpression. There has been no report describing overexpression of these four proteins simultaneously in HBV-related HCC as well as nonviral HCC. Our results suggest that these four proteins play important roles in the pathogenesis of HCV-related HCC and could be molecular targets for diagnosis and treatment of this disease.     

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.     

The maximal cytoprotective function of the heat shock protein 27 is dependent on heat shock protein 70

Endogenous heat shock proteins (HSPs) 70 and 25/27 are induced in renal cells by injury from energy depletion. Transfected over-expression of HSPs 70 or 27 (human analogue of HSP25), provide protection against renal cell injury from ATP deprivation. This study examines whether over-expressed HSP27 depends on induction of endogenous HSPs, in particular HSP70, to afford protection against cell injury. LLC-PK1 cells transfected with HSP27 (27OE cells) were injured by ATP depletion for 2 h and recovered for 4 h in the presence of HSF decoy, HSP70 specific siRNA (siRNA-70) and their respective controls. Injury in the presence of HSF decoy, a synthetic oligonucleotide identical to the heat shock element, the nuclear binding site of HSF, decreased HSP70 induction by 80% without affecting the over-expression of transfected HSP27. The HSP70 stress response was completely ablated in the presence of siRNA-70. Protection against injury, provided by over-expression of HSP27, was reduced by treatment with HSF decoy and abolished by treatment with siRNA-70. Immunoprecipitation studies demonstrated association of HSP27 with actin that was not affected by either treatment with HSF decoy or siRNA. Therefore, HSP27 is dependent on HSP70 to provide its maximal cytoprotective effect, but not for its interaction with actin. This study suggests that, while it has specific action on the cytoskeleton, HSP 25/27 must have coordinated activity with other HSP classes, especially HSP70, to provide the full extent of resistance to injury from energy depletion.     

Expression of heat shock protein70 in pig oocytes: heat shock response during oocyte growth

The heat shock response of growing and fully-grown pig oocytes was analyzed in vitro by determining heat shock protein70 (HSP70) synthesis under both normal conditions (39 degrees C; 0 and 6h) and after heat shock (43 degrees C; 1, 4 and 6h). The expression of HSP70 in oocytes was detected by immunoblotting analysis. Growing oocytes measuring 80-99 microm synthesized a high number of HSP70 without heat shock effect, and these were capable of increasing the synthesis of HSP70 after heat shock to a maximum after 1h. Growing oocytes measuring 100-115 microm also synthesized HSP70 without heat shock and after it, but the HSP70 synthesis was not statistically changed by increasing duration of heat shock. In fully-grown oocytes, great amounts of HSP70 were found without heat shock treatment, and the contents of HSP70 significantly decreased after heat shock. These results indicate that growing oocytes are able to synthesize HSP70 after heat shock. This ability declines at the end of the growth period, and fully-grown oocytes are unable to induce HSP70 synthesis after heat shock. HSP70 is synthesized and stored during oocyte growth. The high HSP70 synthesis in non-heat-treated growing oocytes and a great amount of HSP70 in fully-grown oocytes support the hypothesis that HSP70 is important for oocyte growth and maturation.     

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.     

Light-inducible geneHSP70B encodes a chloroplast-localized heat shock protein inChlamydomonas reinhardtii

The nuclear heat shock geneHSP70B ofChlamydomonas reinhardtii is inducible by heat stress and light. Induction by either environmental cue resulted in a transient elevation in HSP70B protein. Here we describe the organization and nucleotide sequence of theHSP70B gene. The deduced protein exhibits a distinctly higher homology to prokaryotic HSP70s than to those of eukaryotes, including the cytosolic HSP70A ofChlamydomonas reinhardtii. The HSP70B protein, as previously demonstrated by in vitro translation, is synthesized with a cleavable presequence. Using an HSP70B-specific antibody, this heat shock protein was localized to the chloroplast by cell fractionation experiments. A stromal location was suggested by the presence of a conserved sequence motif used for cleavage of presequences by a signal peptidase of the stroma. Amino acid alignments of HSP70 proteins from various organisms and different cellular compartments allowed the identification of sequence motifs, which are diagnostic for HSP70s of chloroplasts and cyanobacteria.     

Antarctic marine molluscs do have an HSP70 heat shock response

The success of any organism depends not only on niche adaptation but also the ability to survive environmental perturbation from homeostasis, a situation generically described as stress. Although species-specific mechanisms to combat “stress” have been described, the production of heat shock proteins (HSPs), such as HSP70, is universally described across all taxa. Members of the HSP70 gene family comprising the constitutive (HSC70) and inducible (HSP70) members, plus GRP78 (glucose-regulated protein, 78 kDa), a related HSP70 family member, were cloned using degenerate polymerase chain reaction (PCR) from two evolutionary divergent Antarctic marine molluscs (Laternula elliptica and Nacella concinna), a bivalve and a gastropod, respectively. The expression of the HSP70 family members was surveyed via quantitative PCR after an acute 2-h heat shock experiment. Both species demonstrated significant up-regulation of HSP70 gene expression in response to increased temperatures. However, the temperature level at which these responses were induced varied with the species (+6–8°C for L. elliptica and +8–10°C for N. concinna) compared to their natural environmental temperature). L. elliptica also showed tissue-specific expression of the genes under study. Previous work on Antarctic fish has shown that they lack the classical heat shock response, with the inducible form of HSP70 being permanently expressed with an expression not further induced under higher temperature regimes. This study shows that this is not the case for other Antarctic animals, with the two molluscs showing an inducible heat shock response, at a level probably set during their temperate evolutionary past.     

Cell signaling and heat shock protein expression

Exposure of cells and organs to heat shock is associated with numerous changes in various cellular metabolic parameters and overexpression of proteins collectively known as heat shock proteins (HSP). In this communication we review the cell-signaling events that are altered in response to heat shock as they relate to the subsequent induction of HSP 70 kd (HSP-70) expression. We also review the mechanisms by which HSP-70 is involved in conferring cytoprotective effects. The possibility of altering HSP expression through manipulations of the cell-signal process has clinical importance.The opinions or assertions contained herein are the private views of the authors and are not to be construed as official or reflecting the views of the Department of the Army or Department of Defense.     

Heat shock protein 25 or inducible heat shock protein 70 activates heat shock factor 1: dephosphorylation on serine 307 through inhibition of ERK1/2 phosphorylation

The expression of heat shock proteins (HSPs) is known to be increased via activation of heat shock factor 1 (HSF1), and excess expression of HSPs exerts feedback inhibition of HSF1. However, the molecular mechanism to modulate such relationships between HSPs and HSF1 is not clear. In the present study, we show that stable transfection of either Hsp25 or inducible Hsp70 (Hsp70i) increased expression of endogenous HSPs such as HSP25 and HSP70i through HSF1 activation. However, these phenomena were abolished when the dominant negative Hsf1 mutant was transfected to HSP25 or HSP70i overexpressed cells. Moreover, the increased HSF1 activity by either HSP25 or HSP70i was found to result from dephosphorylation of HSF1 on serine 307 that increased the stability of HSF1. Either HSP25 or HSP70i inhibited ERK1/2 phosphorylation because of increased MKP1 phosphorylation by direct interaction of these HSPs with MKP1. Treatment of HOS and NCI-H358 cells, which showed high expressions of endogenous HSF1, with small interfering RNA (siRNA) of either HSP27 (siHSP27)or HSP70i (siHSP70i) inhibited both HSP27 and HSP70i proteins; this was because of increased ERK1/2 phosphorylation and serine phosphorylation of HSF1. The results, therefore, suggested that when the HSF1 protein level was high in cancer cells, excess expression of HSP27 or HSP70i strongly facilitates the expression of HSP proteins through HSF1 activation, resulting in severe radio- or chemoresistance.