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.     

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.     

The human heat-shock protein family. Expression of a novel heat-inducible HSP70 (HSP70B'') and isolation of its cDNA and genomic DNA.

The human heat-shock protein multigene family comprises several highly conserved proteins with structural and functional properties in common, but which vary in the extent of their inducibility in response to metabolic stress. We have isolated and characterized a novel human HSP70 cDNA, HSP70B' cDNA, and its corresponding gene sequence. HSP70B' cDNA hybrid-selected an mRNA encoding a more basic 70 kDa heat-shock protein that both the major stress-inducible HSP70 and constitutively expressed HSC70 heat-shock proteins, which in common with other heat-shock 70 kDa proteins bound ATP. The complete HSP70B' gene was sequenced and, like the major inducible HSP70 gene, is devoid of introns. The HSP70B' gene has 77% sequence similarity to the HSP70 gene and 70% similarity to HSC70 cDNA, with greatest sequence divergence towards the 3'-terminus. The HSP70B' gene represents a functional gene, as indicated by Northern-blot analysis with specific oligonucleotides, hybrid-selected translation with a specific 3' cDNA sequence and S1 nuclease protection experiments. In contrast with HSP70 mRNA, which is present at low concentrations in HeLa cells and readily induced by heat or CdCl2 treatment in both fibroblasts and HeLa cells, HSP70B' mRNA was induced only at higher temperature and showed no basal expression. The differences in patterns of induction may be due to the special features of the promoter region of the HSP70B' gene.     

Simulated diving after heat stress potentiates the induction of heat shock protein 70 and elevates glutathione in human endothelial cells

Heat stress prior to diving has been shown to confer protection against endothelial damage due to decompression sickness. Several lines of evidence indicate a relation between such protection and the heat shock protein (HSP)70 and HSP90 and the major cellular red-ox determinant, glutathione (GSH). The present study has used human endothelial cells as a model system to investigate how heat stress and simulated diving affect these central cellular defense molecules. The results demonstrated for the first time that a simulated dive at 2.6 MPa (26 bar) had a potentiating effect on the heat-induced expression of HSP70, increasing the HSP70 concentration on average 54 times above control level. In contrast, a simulated dive had no significant potentiating effect on the HSP90 level, which might be due to the higher baseline level of HSP90. Both 2 and 24-h dive had similar effects on the HSP70 and HSP90, suggesting that the observed effects were independent of duration of the dive. The rapid HSP response following a 2-h dive with a decompression time of 5 min might suggest that the effects were due to compression or pressure per se rather than decompression and may involve posttranslational processing of HSP. The exposure order seemed to be critical for the HSP70 response supporting the suggestion that the potentiating effect of dive was not due to de novo synthesis of HSP70. Neither heat shock nor a simulated dive had any significant effect on the intracellular GSH level while a heat shock and a subsequent dive increased the total GSH level approximately 62%. Neither of these conditions seemed to have any effect on the GSH red-ox status.     

Glutamine's protection against sepsis and lung injury is dependent on heat shock protein 70 expression

Glutamine (GLN) has been shown to protect against inflammatory injury and illness in experimental and clinical settings. The mechanism of this protection is unknown; however, laboratory and clinical trial data have indicated a relationship between GLN-mediated protection and enhanced heat shock protein 70 (HSP70) expression. The aim of this study was to examine the hypothesis that GLN's beneficial effect on survival, tissue injury, and inflammatory response after inflammatory injury is dependent on HSP70 expression. Mice with a specific deletion of the HSP70 gene underwent cecal ligation and puncture (CLP)-induced sepsis and were treated with GLN (0.75 g/kg) or a saline placebo 1 h post-CLP. Lung tissue NF-kappaB activation, inflammatory cytokine response, and lung injury were assessed post-CLP. Survival was assessed for 5 days post-CLP. Our results indicate that GLN administration improved survival in Hsp70(+/+) mice vs. Hsp70(+/+) mice not receiving GLN; however, GLN exerted no survival benefit in Hsp70(-/-) mice. This was accompanied by a significant decrease in lung injury, attenuation of NF-kappaB activation, and proinflammatory cytokine expression in GLN-treated Hsp70(+/+) mice vs. Hsp70(+/+) mice not receiving GLN. In the Hsp70(-/-) mice, GLN's attenuation of lung injury, NF-kappaB activation, and proinflammatory cytokine expression was lost. These results confirm our hypothesis that HSP70 expression is required for GLN's effects on survival, tissue injury, and the inflammatory response after global inflammatory injury.     

Impact of seasonal variation on HSP70 expression quantitated in stressed fish hepatocytes

Heat shock protein 70 (HSP70) is an effective molecular chaperone, playing a role in cell protection from damage in response to stress stimuli. Here, we report the impact of environmental stress on hepatocyte HSP70 expression in Mugil cephalus living in either a contaminated (Ennore) or uncontaminated (Kovalam) estuary over the course of two seasons. Oxidative and nitrative stress was determined along with quantification of HSP70 by enzyme-linked immunosorbent assay (ELISA) after electroelution from polyacrylamide gels. Fish from Ennore showed significantly higher levels of oxidative and nitrative stress and HSP70 expression than fish from Kovalam. Also, there was significant seasonal variation in all oxidative, nitrative stress marker levels and HSP70 expression which peaked during summer. These results provide direct evidence that HSP70 overexpression in fish hepatocytes under stress may aid cell survival by protecting against oxidative and nitrative stress-induced changes. In addition, seasonal variation may have a significant impact on HSP70 expression.     

The use of stress‐70 proteins in physiology: a re‐appraisal

There are few factors more important to the mechanisms of evolution than stress. The stress response has formed as a result of natural selection, improving the capacity of organisms to withstand situations that require action. The ubiquity of the cellular stress response suggests that effective mechanisms to counteract stress emerged early in the history of life, and their commonality proves how vital such mechanisms are to operative evolution. The cellular stress response (CSR) has been identified as a characteristic of cells in all three domains of life and consists of a core 44 proteins that are structurally highly conserved and that have been termed the ‘minimal stress proteome’ (MSP). Within the MSP, the most intensely researched proteins are a family of heat‐shock proteins known as HSP70. Superficially, correlations between the induction of stress and HSP70 differential expression support the use of HSP70 expression as a nonspecific biomarker of stress. However, we argue that too often authors have failed to question exactly what HSP70 differential expression signifies. Herein, we argue that HSP70 up‐regulation in response to stressors has been shown to be far more complex than the commonly accepted quasi‐linear relationship. In addition, in many instances, the uncertain identity and function of heat‐shock proteins and heat‐shock cognates has led to difficulties in interpretation of reports of inducible heat‐shock proteins and constitutive heat‐shock cognates. We caution against the broad application of HSP70 as a biomarker of stress in isolation and conclude that the application of HSP70 as a meaningful index of stress requires a higher degree of validation than the majority of research currently undertakes.     

High-molecular-mass complexes formed in vivo contain smHSPs and HSP70 and display chaperone-like activity.

Stress can have profound effects on the cell. The elicitation of the stress response in the cell is often accompanied by the synthesis of high-molecular-mass complexes, sometimes termed heat shock granules (HSGs). The presence of the complexes has been shown to be important for the survival of cells subjected to stress. We purified these complexes from heat-stressed BY-2 tobacco cells. HSG complexes formed in vivo contain predominantly smHSPs, HSP40 and HSP70 and display chaperone-like activity. Tubulins as well as other proteins may be part of the complex or its substrate. The proteins, except smHSPs and to some extent HSP70, were hypersensitive to proteolysis, suggesting that they were partially denatured and not an integral part of the HSG complexes. When citrate synthase was used as the substrate, in vivo generated HSG complexes exhibited strong nucleotide-dependent in vitro chaperone activity. Measurable ATP-mediated hydrolytic activity was detected. Isolated HSG complexes are stable until ATP is added, which leads to rapid dissociation of the complex into subunits. It is proposed that smHSPs form the core of the complex in association with ATP-dependent HSP70 and HSP40 cochaperones. Implications of these findings are discussed.     

Functional analysis of the Hikeshi-like protein and its interaction with HSP70 in Arabidopsis

Heat shock proteins (HSPs) refold damaged proteins and are an essential component of the heat shock response. Previously, the 70 kDa heat shock protein (HSP70) has been reported to translocate into the nucleus in a heat-dependent manner in many organisms. In humans, the heat-induced translocation of HSP70 requires the nuclear carrier protein Hikeshi. In the Arabidopsis genome, only one gene encodes a protein with high homology to Hikeshi, and we named this homolog Hikeshi-like (HKL) protein. In this study, we show that two Arabidopsis HSP70 isoforms accumulate in the nucleus in response to heat shock and that HKL interacts with these HSP70s. Our histochemical analysis revealed that HKL is predominantly expressed in meristematic tissues, suggesting the potential importance of HKL during cell division in Arabidopsis. In addition, we show that HKL regulates HSP70 localization, and HKL overexpression conferred thermotolerance to transgenic Arabidopsis plants. Our results suggest that HKL plays a positive role in the thermotolerance of Arabidopsis plants and cooperatively interacts with HSP70.