The HSP70 heat shock response in the Antarctic fish <Emphasis Type="Italic">Harpagifer antarcticus</Emphasis>

Members of the HSP70 gene family comprising the constitutive (HSC70) and inducible (HSP70) genes, plus GRP78 (Glucose-regulated protein 78 kDa) were surveyed for expression levels via Q-PCR after both an acute 2-h heat shock experiment and a time course assay in the Antarctic plunderfish Harpagifer antarcticus. In general, down regulation of all genes was observed during the course of the heat shock experiments. This thermally induced down regulation was particularly acute for the GRP78 gene, which at one time point was more than 100-fold down regulated. These results demonstrate the loss of the heat shock response in H. antarcticus, a basal member of the Notothenioidei. This finding is discussed with reference to the survival of Notothenioids during observed ocean warming and also the reorganisation of cellular protein mechanisms of species living in extreme environments.     

Delayed cytoprotection induced by hypoxic preconditioning in cultured neonatal rat cardiomyocytes: role of GRP78

Hypoxic preconditioning (HPC) has been well demonstrated to have potent protective effects in many cell types; however, the mechanisms responsible for this phenomenon are not fully understood. Recently, glucose-regulated protein 78 (GRP78), an inducible molecular chaperon, was indicated to be associated with ischemic preconditioning. We hypothesized that HPC protects cardiomyocytes against hypoxia by inducing GRP78 in cultured neonatal rat cardiomyocytes. HPC was induced by exposing cardiomyocytes to brief hypoxia (1% O(2), 30 min) followed by reoxygenation. GRP78 was expressed constitutively in cultured cardiomyocytes and its expression was enhanced at 12 h, peaked at 24 h (207.3+/-23.6% of the baseline), and was sustained for up to 72 h after HPC. Twenty-four hours after HPC, the myocytes were subjected to prolonged hypoxia (1% O(2), 12 h). The lactic dehydrogenase (LDH) release and malondialdehyde (MDA) content were reduced, while cell viability and superoxide dismutase (SOD) activity were increased in the preconditioned cells compared with the non-HPC cells. The GRP78 protein level was higher in cells exposed to both HPC and hypoxia than in the cells exposed to HPC alone or hypoxia alone. Heat shock protein 70 (HSP70) was induced in parallel by late HPC. Transfection of GRP78 antisense oligonucleotides blocked GRP78 expression but not HSP70, resulting in attenuated cardioprotection afforded by late HPC. Furthermore, inducing GRP78 by gene transfer protected cardiomyocytes from hypoxic injury. These findings demonstrate that the induction of GRP78 partially mediates the late HPC, suggesting that GRP78 is a novel mechanism responsible for the late cytoprotection of HPC.     

A high throughput substrate binding assay reveals hexachlorophene as an inhibitor of the ER-resident HSP70 chaperone GRP78

Glucose-regulated protein 78 (GRP78) is the ER resident 70 kDa heat shock protein 70 (HSP70) and has been hypothesized to be a therapeutic target for various forms of cancer due to its role in mitigating proteotoxic stress in the ER, its elevated expression in some cancers, and the correlation between high levels for GRP78 and a poor prognosis. Herein we report the development and use of a high throughput fluorescence polarization-based peptide binding assay as an initial step toward the discovery and development of GRP78 inhibitors. This assay was used in a pilot screen to discover the anti-infective agent, hexachlorophene, as an inhibitor of GRP78. Through biochemical characterization we show that hexachlorophene is a competitive inhibitor of the GRP78-peptide interaction. Biological investigations showed that this molecule induces the unfolded protein response, induces autophagy, and leads to apoptosis in a colon carcinoma cell model, which is known to be sensitive to GRP78 inhibition.     

Continuous high expression of XBP1 and GRP78 is important for the survival of bone marrow cells in CCl4-treated cirrhotic liver

We have previously shown that infusion of bone marrow cells (BMC) improves CCl₄-induced cirrhosis. However, it is unclear why the injected BMC are resistant to CCl₄ damage and subsequently improve the local microenvironment in damaged liver. To analyze the cellular phenomena involved in this process, we studied the damaged liver using electron microscopy. We found that CCl₄ caused rough endoplasmic reticula to swell in hepatocytes. To analyze the gene expression patterns associated with this process, we conducted PCR-selected suppressive subtractive hybridization. We found that expression levels of HSP84, HSP40, and XBP1 differed markedly between control liver and liver infused with BMC. Immunohistochemical staining revealed that expression levels of HSP84 and HSP40 were markedly higher in the early phase of differentiation immediately after BMC infusion, but decreased over time. XBP1 expression remained high during the late phase, and GRP78 expression increased with XBP1 activation. We also found that GFP-positive BMC expressed XBP1 and GRP78. XBP1 and GRP78 are associated with ER stress. Thus, continuous high XBP1 and GRP78 expression might be essential for the survival and proliferation of BMC in a CCl₄-induced persistent liver damage environment.     

Evidence of prognostic relevant expression profiles of heat-shock proteins and glucose-regulated proteins in oesophageal adenocarcinomas

A high percentage of oesophageal adenocarcinomas show an aggressive clinical behaviour with a significant resistance to chemotherapy. Heat-shock proteins (HSPs) and glucose-regulated proteins (GRPs) are molecular chaperones that play an important role in tumour biology. Recently, novel therapeutic approaches targeting HSP90/GRP94 have been introduced for treating cancer. We performed a comprehensive investigation of HSP and GRP expression including HSP27, phosphorylated (p)-HSP27^(Ser15), p-HSP27^(Ser78), p-HSP27^(Ser82), HSP60, HSP70, HSP90, GRP78 and GRP94 in 92 primary resected oesophageal adenocarcinomas by using reverse phase protein arrays (RPPA), immunohistochemistry (IHC) and real-time quantitative RT-PCR (qPCR). Results were correlated with pathologic features and survival. HSP/GRP protein and mRNA expression was detected in all tumours at various levels. Unsupervised hierarchical clustering showed two distinct groups of tumours with specific protein expression patterns: The hallmark of the first group was a high expression of p-HSP27^{(Ser15, Ser78, Ser82)} and low expression of GRP78, GRP94 and HSP60. The second group showed the inverse pattern with low p-HSP27 and high GRP78, GRP94 and HSP60 expression. The clinical outcome for patients from the first group was significantly improved compared to patients from the second group, both in univariate analysis (p = 0.015) and multivariate analysis (p = 0.029). Interestingly, these two groups could not be distinguished by immunohistochemistry or qPCR analysis. In summary, two distinct and prognostic relevant HSP/GRP protein expression patterns in adenocarcinomas of the oesophagus were detected by RPPA. Our approach may be helpful for identifying candidates for specific HSP/GRP-targeted therapies.     

Triggers of the HSP70 stress response: environmental responses and laboratory manipulation in an Antarctic marine invertebrate (Nacella concinna)

The Antarctic limpet, Nacella concinna, exhibits the classical heat shock response, with up-regulation of duplicated forms of the inducible heat shock protein 70 (HSP70) gene in response to experimental manipulation of seawater temperatures. However, this response only occurs in the laboratory at temperatures well in excess of any experienced in the field. Subsequent environmental sampling of inter-tidal animals also showed up-regulation of these genes, but at temperature thresholds much lower than those required to elicit a response in the laboratory. It was hypothesised that this was a reflection of the complexity of the stresses encountered in the inter-tidal region. Here, we describe a further series of experiments comprising both laboratory manipulation and environmental sampling of N. concinna. We investigate the expression of HSP70 gene family members (HSP70A, HSP70B, GRP78 and HSC70) in response to a further suite of environmental stressors: seasonal and experimental cold, freshwater, desiccation, chronic heat and periodic emersion. Lowered temperatures (−1.9°C and −1.6°C), generally produced a down-regulation of all HSP70 family members, with some up-regulation of HSC70 when emerging from the winter period and increasing sea temperatures. There was no significant response to freshwater immersion. In response to acute and chronic heat treatments plus simulated tidal cycles, the data showed a clear pattern. HSP70A showed a strong but very short-term response to heat whilst the duplicated HSP70B also showed heat to be a trigger, but had a more sustained response to complex stresses. GRP78 expression indicates that it was acting as a generalised stress response under the experimental conditions described here. HSC70 was the major chaperone invoked in response to long-term stresses of varying types. These results provide intriguing clues not only to the complexity of HSP70 gene expression in response to environmental change but also insights into the stress response of a non-model species.     

Lack of an HSP70 heat shock response in two Antarctic marine invertebrates

Members of the HSP70 gene family comprising the inducible (HSP70) genes and GRP78 (glucose-regulated protein 78 kDa) were identified in an Antarctic sea star (Odontaster validus) and an Antarctic gammarid (Paraceradocus gibber). These genes were surveyed for expression levels via Q-PCR after an acute 2-hour heat shock experiment in both animals and a time course assay in O. validus. No significant up-regulation was detected for any of the genes in either of the animals during the acute heat shock. The time course experiment in O. validus produced slightly different results with an initial down regulation in these genes at 2°C, but no significant up-regulation of the genes either at 2 or 6°C. Therefore, the classical heat shock response is absent in both species. The data is discussed in the context of the organisms’ thermal tolerance and the applicability of HSP70 to monitor thermal stress in Antarctic marine organisms.     

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.     

The Constitutive Heat Shock Protein-70 Is Required for Optimal Expression of Myelin Basic Protein During Differentiation of Oligodendrocytes

To further elucidate the role of the constitutive heat shock protein-70 (HSC70) as a chaperone for the synthesis of myelin basic protein (MBP), HSC70 content was decreased in oligodendrocyte precursor cells prior to MBP expression either by transfection with an antisense oligonucleotide specific for HSC70, or by exposure to low levels of quercetin, a bioflavonoid known to decrease synthesis of HSC70. As these cells underwent differentiation in vitro, antisense treatment decreased HSC70 levels to 66% of controls. At the same time, a sharp induction resulted in the stress-inducible heat shock protein-70 (HSP70). Levels of two other stress proteins increased as well, namely, the 25-kDa heat shock protein (HSP25) and the 78-kDa glucose regulated protein (GRP78). MBP synthesis proceeded over a normal time course, but at only 50% of control values. As HSC70 content returned to normal, MBP synthesis was also restored to normal levels. Quercetin reduced the expression of HSC70 to an even greater extent than transfection, and prevented the induction of HSP70. In contrast to antisense-treated cells, MBP synthesis was essentially blocked in quercetin-treated cells even though levels of HSP25 and GRP78 increased. Taken together, these observations (a) indicate that HSP70 partially compensates for decreased chaperoning of nascent MBP by HSC70 (HSC70 and HSP70 are closely related and perform similar functions); (b) preclude the involvement of HSP25 and GRP78 in MBP synthesis; and (c) emphasize the requirement of HSC70 for optimal synthesis of MBP.     

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.     

Molecular identification of glucose-regulated protein 78 (grp78) gene from the Indian meal moth,Plodia interpunctella,and its regulation by nutrient uptake

Glucose-regulated protein 78 (GRP78) is a member of the HSP70 family of proteins and is localized in the endoplasmic reticulum (ER) within cells. GRP78 and its gene has been identified in only a few species of insects, and its role is not clear. Here, we identified full-length grp78 cDNA from the Indian meal moth, Plodia interpunctella, and demonstrated the role of grp78 in developmental and physiological processes of the insect. The deduced amino acid sequence of GRP78 contained highly conserved functional motifs of the HSP70 family and the C-terminal motif of KDEL, which is characteristic of ER-localized HSP70. It also showed high identity (93–94%) with GRP78 and related HSP70 proteins of lepidopteran species. Gene expression analysis showed that grp78 mRNA levels were high in the egg, feeding larval, and adult stages, but low in the molting, wandering larval, and pupal stages of development. In a tissue comparison of fifth instar P. interpunctella, grp78 level was higher in the gut than in the integument or fat bodies. Grp78 level decreased greatly when fifth-instar larvae were starved for 48 h, but recovered within 3–6 h after re-feeding. Our data suggest that grp78 is highly associated with dietary energy conditions during development and may play an important role in the nutritional physiology of insects.     

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.     

Analysis of the expression of a glucose-regulated protein (GRP78) promoter/CAT fusion gene during early Xenopus laevis development

Developmental regulation of the expression of a glucose-regulated gene encoding a 78 kd protein, GRP78, has been characterized by microinjection of a rat GRP78/CAT chimeric gene into early Xenopus embryos. Tunicamycin-induced expression of the chimeric gene during Xenopus development was similar to the pattern of endogenous GRP78 protein synthesis, with expression first being detected at gastrula and increasing at least until the tailbud stage. Deletion analysis of the rat GRP78 promoter revealed that sequences between -154 and -130 were necessary for full tunicamycin-inducible and constitutive expression of the fusion gene. These results suggest that there is conservation of regulatory elements of the GRP78 promoter between rat and Xenopus.     

Hypoxic preconditioning reinforces HIF-alpha-dependent HSP70 signaling to reduce ischemic renal failure-induced renal tubular apoptosis and autophagy

AimsRepetitive hypoxic preconditioning (RHP) may provide more efficient protection than single hypoxic preconditioning against renal ischemia/reperfusion-induced injury via hypoxia-induced factor 1α (HIF-1α)-dependent heat shock protein 70 (HSP70) pathways.Main methodsWistar rats were subjected to intermittent hypoxic exposure (15 h/day), whereas controls were kept at sea level. We evaluated renal expression of HIF-1α, HSP70, the endoplasmic reticulum stress protein GRP78, caspase 12, Beclin-1, and poly-(ADP-ribose)-polymerase (PARP) with western blotting. Renal apoptosis determined by terminal transferase dUTP nick end labeling (TUNEL), Beclin-1-dependent autophagy, and monocyte/macrophage (ED-1) infiltration were evaluated by immunocytochemistry. Renal function was determined with blood urea nitrogen (BUN) and plasma creatinine levels. HIF-1α inhibitors and Deoxyribonucleotide (DNA) or Ribonucleotide (RNA) interference of HSP70 were used to evaluate their possible roles in this process.Key findingsRenal HIF-1α and HSP70 expression were enhanced by hypoxic preconditioning and inhibited by the HIF-1α inhibitor, YC-1, as well as phosphatidylinositol 3-kinase (PI3K)/Akt inhibitors. After the return to normoxia, renal HSP70 protein levels were maintained for one week in the RHP group, but they decayed after one day in the single hypoxic preconditioning group. Ischemia/reperfusion significantly increased renal TUNEL-apoptosis, Beclin-1-dependent autophagy, ED-1 infiltration, expression of GRP78, caspase 12, Beclin-1, PARP, and BUN and plasma creatinine levels in control rats. RHP significantly decreased all ischemia/reperfusion-enhanced parameters. Intraperitoneal pretreatment with YC-1 and quercetin (an inhibitor of HSP70 induction) eliminated RHP-induced protection. Anti-sense oligodeoxyribonucleotides or interference RNA targeting HSP70 abrogated the protection against hypoxia/reoxygenation-induced oxidative injury in RHP-treated proximal tubules.SignificanceWe demonstrate that RHP promotes HIF-1α-dependent HSP70 signaling to reduce renal ischemia/reperfusion injury.