Arsenic trioxide induces Hsp70 expression via reactive oxygen species and JNK pathway in MDA231 cells

In the present study, we determined the molecular pathways that induce the heat shock proteins (Hsps) after treatment of cells with arsenic trioxide. Administration of arsenic trioxide to MDA231 cells leads to induce Hsp70, which is accompanied by generation of reactive oxygen species (ROS) and activation of c-Jun N-terminal kinase (JNK). We showed that arsenic trioxide-induced Hsp70 expression was caused by activation of ROS and prevented by the antioxidant N-Acetyl-Cysteine (NAC). SP600125 and dominant-negative SEK suppressed Hsp70 promoter-driven reporter gene expression, suggesting that JNK would be preferentially associated with the protective heat shock response against arsenic trioxide stress. In addition, SP600125, a specific JNK inhibitor, significantly reduced the amount of phosphorylated HSF1 upon administration of arsenic trioxide. It is likely that Hsp70 expression against arsenic trioxide exposure protects cells from oxidative injury and apoptotic cell death by means of JNK activity.     

Whole body hyperthermia in mice confers heat shock protein-dependent radioresistance of their bone marrow and thymocytes

Here we studied how whole body hyperthermia in mice affects radiosensitivity of their tissues and cells. To examine a contribution of heat shock proteins (Hsps) we used Quercetin, an inhibitor of the Hsp induction. Bone marrow and thymocytes did become more radioresistant in heat-preconditioned mice. The radioprotection was well manifested in 18–26 h after hyperthermia and was strongly suppressed in Quercetin-injected mice. The revealed correlation between the intracellular Hsp content and radioresistance suggests the radioprotective potential of stress-inducible Hsps. Thus, our finding points out new ways for artificial modulation of the in vivo radiation effects by manipulating the Hsp expression.     

Heat shock protein 70 purification and characterization from Cyprinion macrastomus macrastomus and Garra rufa obtusa

Organisms resist stress factors by common mechanisms at cellular and physiological levels. Heat shock proteins (Hsps) play an essential role as a protective mechanism against stressors, and Hsp70 has a central role in the heat shock response. Although several investigations have been made on the cellular functions of Hsps, very little is known about extreme temperature effects on organisms like fish. To address this point, we have studied two fish species, Cyprinion macrostomus macrostomus and Garra rufa obtuse, whose habitat is the Kangal hot spring in Turkey. The hot spring has multiple stress factors; extremely hot temperature, food deprivation, infectious medium and low levels of oxygen. Like other Hsp70s, two Cyprinidae species Hsp70 showed similar ATPase and substrate protein folding activities in vitro. These proteins also showed relatively higher thermal stability. Biochemical characterization of two Hsp70, suggested a Hsp mechanism capable of protecting the cell against stressors even under adverse conditions and in the presence of multiple stress factors.     

Functional stabilization of Kv1.5 protein by Hsp70 in mammalian cell lines

The aim of this study was to elucidate the mechanisms for regulations of cardiac Kv1.5 channel expression. We particularly focused on the role of heat shock proteins (Hsps). We tested the effects of Hsps on the stability of Kv1.5 channels using biochemical and electrophysiological techniques: co-expression of Kv1.5 and Hsp family proteins in mammalian cell lines, followed by Western blotting, immunoprecipitation, pulse-chase analysis, immunofluorescence and whole-cell patch clamp. Hsp70 and heat shock factor 1 increased the expression of Kv1.5 protein in HeLa and COS7 cells, whereas either Hsp40, 27 or 90 did not. Hsp70 prolonged the half-life of Kv1.5 protein. Hsp70 was co-immunoprecipitated and co-localized with Kv1.5-FLAG. Hsp70 significantly increased the immunoreactivity of Kv1.5 in the endoplasmic reticulum, Golgi apparatus and on the cell membrane. Hsp70 enhanced Kv1.5 current of transfected cells, which was abolished by pretreatment with brefeldin A or colchicine. Thus, Hsp70, but not other Hsps, stabilizes functional Kv1.5 protein.     

Theiler’s murine encephalomyelitis virus infection induces a redistribution of heat shock proteins 70 and 90 in BHK-21 cells,and is inhibited by novobiocin and geldanamycin

Theiler’s murine encephalomyelitis virus (TMEV) is a positive-sense RNA virus belonging to the Cardiovirus genus in the family Picornaviridae. In addition to other host cellular factors and pathways, picornaviruses utilise heat shock proteins (Hsps) to facilitate their propagation in cells. This study investigated the localisation of Hsps 70 and 90 in TMEV-infected BHK-21 cells by indirect immunofluorescence and confocal microscopy. The effect of Hsp90 inhibitors novobiocin (Nov) and geldanamycin (GA) on the development of cytopathic effect (CPE) induced by infection was also examined. Hsp90 staining was uniformly distributed in the cytoplasm of uninfected cells but was found concentrated in the perinuclear region during late infection where it overlapped with the signal for non-structural protein 2C within the viral replication complex. Hsp70 redistributed into the vicinity of the viral replication complex during late infection, but its distribution did not overlap with that of 2C. Inhibition of Hsp90 by GA and Nov had a negative effect on virus growth over a 48-h period as indicated by no observable CPE in treated compared to untreated cells. 2C was detected by Western analysis of GA-treated infected cell lysates at doses between 0.01 and 0.125 μM, suggesting that processing of viral precursors was not affected in the presence of this drug. In contrast, 2C was absent in cell lysates of Nov-treated cells at doses above 10 μM, although CPE was evident 48 hpi. This is the first study describing the dynamic behaviour of Hsps 70 and 90 in TMEV-infected cells and to identify Hsp90 as an important host factor in the life cycle of this virus.     

Heat shock protein synthesis during development in Caulobacter crescentus.

Caulobacter crescentus cells respond to a sudden increase in temperature by transiently inducing the synthesis of several polypeptides. Two of the proteins induced, Hsp62 and Hsp70, were shown to be analogous to the heat shock proteins of Escherichia coli, GroEL and DnaK, respectively, by immunological cross-reactivity with antibodies raised against the E. coli proteins. Two-dimensional gel electrophoretic resolution of extracts of cells labeled with [35S]methionine during heat shock led to the identification of 20 distinct Hsps in C. crescentus which are coordinately expressed, in response to heat, at the various stages of the cell division cycle. Thus, a developmental control does not seem to be superimposed on the transient activation of the heat shock genes. Nonetheless, under normal temperature conditions, four Hsps (Hsp70, Hsp62, Hsp24b, and Hsp23a) were shown to be synthesized, and their synthesis was cell cycle regulated.     

Overexpression of inducible 70-kDa heat shock protein in mouse attenuates skeletal muscle damage induced by cryolesioning

Heat shock protein expression is elevated upon exposure to a variety of stresses and limits the extent of stress-induced damage. To investigate the putative role of inducible 70-kDa heat shock protein (HSP70) in skeletal muscle damage and regeneration, soleus and tibialis anterior (TA) muscles from HSP70-overexpressing transgenic mice were subjected to cryolesioning and analyzed after 1, 10, and 21 days. Histological analysis showed that the muscles from both HSP70 and wild-type mice treated with radicicol (a HSP inducer) had decreased necrosis after cryolesioning compared with controls. The decrease in muscle fiber cross-sectional area in both soleus and TA muscles in 10 days postlesioning was attenuated in HSP70 mice compared with wild-type mice. Glutathione peroxidase activity was increased 1 day after cryolesioning in both HSP70 and control mice and remained elevated for up to 21 days. Immunodetection of neuronal cell adhesion molecule (a satellite cell marker) and developmental/neonatal MHC were significantly lower in cryolesioned HSP70-overexpressing mice than in cryolesioned controls. These results suggest that HSP70 protects skeletal muscle against injury and radicicol might be useful as a skeletal muscle protective agent. regeneration; radicicol; transgenic mouse; myoprotection     

Expression of the molecular chaperone Hsp70 in detergent-resistant microdomains correlates with its membrane delivery and release

Accumulating evidence suggests that some heat shock proteins (Hsps), in particular the 72-kDa inducible Hsp70, associate to the cell membrane and might be secreted through an unknown mechanism to exert important functions in the immune response and signal transduction. We speculated that specialized structures named lipid rafts, known as important platforms for the delivery of proteins to the cell membrane, might be involved in the unknown mechanism ensuring membrane association and secretion of Hsp70. Lipid rafts are sphingolipid-cholesterol-rich structures that have been mainly characterized in polarized epithelial cells and can be isolated as detergent-resistant microdomains (DRMs). Analysis of soluble and DRM fractions prepared from unstressed Caco-2 epithelial cells revealed that Hsp70, and to a lesser extent calnexin, were present in DRM fractions. Increased expression of Hsps, through heat shock or by using drugs acting on protein trafficking or intracellular calcium level, induced an efficient translocation to DRM. We also found that Hsp70 was released by epithelial Caco-2 cells, and this release dramatically increased after heat shock. Drugs known to block the classical secretory pathway were unable to reduce Hsp70 release. By contrast, release of the protein was affected by the raft-disrupting drug methyl-beta-cyclodextrin. Our data suggest that lipid rafts are part of a mechanism ensuring the correct functions of Hsps and provide a rational explanation for the observed membrane association and release of Hsp70.     

Expression and localization of Hsps in the heart and blood vessel of heat-stressed broilers

The objective of this study was to investigate the kinetics of Hsp60, Hsp70, Hsp90 protein, and messenger RNA (mRNA) expression levels and to correlate these heat shock protein (Hsp) levels with tissue damage resulting from exposure to high temperatures for varying amounts of time. One hundred broilers were heat-stressed for 0, 2, 3, 5, and 10 h, respectively, by rapidly increasing the ambient temperature from 22 +/- 1 degrees C to 37 +/- 1 degrees C. Obvious elevations of plasma creatine kinase indicate damage to myocardial cells after heat stress. Hsp70 and Hsp90, and their corresponding mRNAs in the heart tissue of heat-stressed broilers, elevated significantly after 2 h of heat exposure and decreased quickly with continued heat stress. However, the levels of hsp60 mRNA in the heart of heat-stressed broilers increased sharply (P < 0.01) at 2 h of heat stress but then decreased quickly after 3 h, while the level of Hsp60 protein in the heart increased (P < 0.01) at 2 h of heat stress and maintained a high level throughout heat exposure. The results indicate that the elevation of the three Hsps, especially Hsp60 in heart, may be important markers at the beginning of heat stress and act as protective proteins in adverse environments. The reduction of Hsp signals in the cytoplasm of myocardial cells implies that myocardial cell lesions may have an adverse impact on the function of Hsps during heat stress. Meanwhile, the localization of Hsp70 in blood vessels of broiler hearts suggests another possible mechanism for protection of the heart after heat exposure.     

Administration of Hsp70 in vivo inhibits motor and sensory neuron degeneration

The induction of heat shock proteins (Hsps) serves not only as a marker for cellular stress but also as a promoter of cell survival, which is especially important in the nervous system. We examined the regulation of the constitutive and stress-induced 70-kD Hsps (Hsc70 and Hsp70, respectively) after sciatic nerve (SN) axotomy in the neonatal mouse. Additionally, the prevention of axotomy-induced SN cell death by administration of several preparations of exogenous Hsc70 and Hsp70 was tested. Immunohistochemistry and Western blot analyses showed that endogenous levels of Hsc70 and Hsp70 did not increase significantly in lumbar motor neurons or dorsal root ganglion sensory neurons up to 24 hours after axotomy. When a variety of Hsc70 and Hsp70 preparations at doses ranging from 5 to 75 microg were applied to the SN stump after axotomy, the survival of both motor and sensory neurons was significantly improved. Thus, it appears that motor and sensory neurons in the neonatal mouse do not initiate a typical Hsp70 response after traumatic injury and that administration of exogenous Hsc/Hsp70 can remedy that deficit and reduce the subsequent loss of neurons by apoptosis.     

Destabilization and recovery of a yeast prion after mild heat shock

Yeast prion [PSI⁺] is a self-perpetuating amyloid of the translational termination factor Sup35. Although [PSI⁺] propagation is modulated by heat shock proteins (Hsps), high temperature was previously reported to have little or no effect on [PSI⁺]. Our results show that short-term exposure of exponentially growing yeast culture to mild heat shock, followed by immediate resumption of growth, leads to [PSI⁺] destabilization, sometimes persisting for several cell divisions after heat shock. Prion loss occurring in the first division after heat shock is preferentially detected in a daughter cell, indicating the impairment of prion segregation that results in asymmetric prion distribution between a mother cell and a bud. Longer heat shock or prolonged incubation in the absence of nutrients after heat shock led to [PSI⁺] recovery. Both prion destabilization and recovery during heat shock depend on protein synthesis. Maximal prion destabilization coincides with maximal imbalance between Hsp104 and other Hsps such as Hsp70-Ssa. Deletions of individual SSA genes increase prion destabilization and/or counteract recovery. The dynamics of prion aggregation during destabilization and recovery are consistent with the notion that efficient prion fragmentation and segregation require a proper balance between Hsp104 and other (e.g., Hsp70-Ssa) chaperones. In contrast to heat shock, [PSI⁺] destabilization by osmotic stressors does not always depend on cell proliferation and/or protein synthesis, indicating that different stresses may impact the prion via different mechanisms. Our data demonstrate that heat stress causes asymmetric prion distribution in a cell division and confirm that the effects of Hsps on prions are physiologically relevant.