Kinetics of thermally induced heat shock protein 27 and 70 expression by bone marrow-derived mesenchymal stem cells

Although bone marrow-derived mesenchymal stem cells (MSCs) are an attractive cell therapy candidate, their potential is limited by poor survival following transplantation. Over-expression of anti-apoptotic heat shock proteins using viral vectors can improve the survival of these cells under stressful conditions in vitro and in vivo. It is also possible to induce heat shock protein expression in many cell types by simply exposing them to a transient, nonlethal elevation in temperature. The response profile of MSCs to such a thermal stress has not yet been reported. Therefore, this study sought to determine the kinetics of thermally induced heat shock protein expression by MSCs in vitro. To determine if heat shock protein expression was a function of thermal stress exposure time, MSCs were exposed to 42°C for 15, 30, 45, and 60 min and were harvested 24 h later. To establish the time-course of heat shock protein expression, MSCs were heat shocked for 60 min and harvested 2, 24, 48, 72, 96, and 120 h later. The cells were then analyzed for Hsp27 and Hsp70 expression by Western blot. Densitometric analysis revealed that exposure to a thermal stress induced expression of both Hsp27 and Hsp70 and that the level of expression was dependant on stress exposure time. Following 60 min of heat stress, both Hsp27 and Hsp70 accumulated maximal expression after 48 h with both proteins returning to constitutive expression levels by 120 h. This study demonstrates that heat shock protein expression can be induced in MSCs by a simple thermal stress.     

Hyperthermia assists survival of astrocytes from oxidative-mediated necrotic cell death.

In response to many stresses and pathologic states, including different models of nervous system injury, cells synthesize a variety of proteins, most notably the inducible 72 kDa heat shock protein 70 (Hsp70), which plays important roles in maintaining cellular integrity and viability. We report here that cultured astrocytes from rat diencephalon express high levels of Hsp70 upon exposure to elevated temperatures, and are less vulnerable to a subsequent oxidative stress. Complex oxidative stress was induced by exposure of astrocytes to an aqueous extract of tobacco smoke. This resulted in both glutathione and ATP depletion, along with cell death that proceeded through a necrotic pathway. Pretreatment of cultures with the glutathione replenishing agent, N-acetyl-L-cysteine, prevented glutathione and ATP loss as well as necrotic cell death. Thermal stress also protected astrocytes from necrotic cell death but without affecting glutathione or ATP levels. We propose that heat shock protects astrocytes from necrosis induced by oxidative stress, probably as a result of Hsp70 synthesis, through an antioxidant-ATP independent mechanism. As Hsp70 may transfer from glial to neuronal cells, its synthesis by astrocytes may represent an important survival mechanism by which astrocytes protect neurons against oxidative-mediated cell death.     

Differential heat shock gene hsp70-1 response to toxicants revealed by in vivo study of lungs in transgenic mice

Members of heat shock proteins (Hsp70) family have been considered to respond to a large variety of stressful conditions. But it was suggested that, in pulmonary cells, Hsp response depends more closely on the type of stimulus. The lungs are critical organs potentially subjected to air pollution affecting respiratory function and, therefore, these organs are of particular interest with regard to the stress response. To investigate the stress dependence of Hsp70 response in lungs, we created transgenic mice where the firefly luciferase reporter gene is under the control of the murine hsp70-1 promoter and exposed them to different sublethal toxic conditions. For each condition, the level of transgene induction and pulmonary toxicity were assessed. We found that hsp70-1 promoter was stimulated by heat shock and cadmium but not by ozone, paraquat, and parathion, even if these chemicals induced respiratory distress and lung inflammation. Similar observations were made when expression of the endogenous hsp70-1 gene was analyzed, indicating that our transgenic model was accurately detecting hsp70-1 induction. Thereby, it appeared that hsp70-1 response is selective and depends on signaling pathways triggered by the toxicants rather than by their pathologic toxicity per se. Furthermore, because all the chemicals used in our study have been previously described to increase the level of oxidative stress, it indicates that there is no direct and simple correlation between hsp70-1 response and the level of oxidative stress, but more specific oxidative patterns should be involved in Hsp regulation.     

Human esophageal microvascular endothelial cells respond to acidic pH stress by PI3K/AKT and p38 MAPK-regulated induction of Hsp70 and Hsp27

The heat shock response maintains cellular homeostasis following sublethal injury. Heat shock proteins (Hsps) are induced by thermal, oxyradical, and inflammatory stress, and they chaperone denatured intracellular proteins. Hsps also chaperone signal transduction proteins, modulating signaling cascades during repeated stress. Gastroesophageal reflux disease (GERD) affects 7% of the US population, and it is linked to prolonged esophageal acid exposure. GERD is characterized by enhanced and selective leukocyte recruitment from esophageal microvasculature, implying activation of microvascular endothelium. We investigated whether phosphatidylinositol 3-kinase (PI3K)/Akt and MAPK regulate Hsp induction in primary cultures of human esophageal microvascular endothelial cells (HEMEC) in response to acid exposure (pH 4.5). Inhibitors of signaling pathways were used to define the contribution of PI3K/Akt and MAPKs in the heat shock response and following acid exposure. Acid significantly enhanced phosphorylation of Akt and MAPKs in HEMEC as well as inducing Hsp27 and Hsp70. The PI3K inhibitor LY-294002, and Akt small interfering RNA inhibited Akt activation and Hsp70 expression in HEMEC. The p38 MAPK inhibitor (SB-203580) and p38 MAPK siRNA blocked Hsp27 and Hsp70 mRNA induction, suggesting a role for MAPKs in the HEMEC heat shock response. Thus acidic pH exposure protects HEMEC through induction of Hsps and activation of MAPK and PI3 kinase pathway. Acidic exposure increased HEMEC expression of VCAM-1 protein, but not ICAM-1, which may contribute to selective leukocyte (i.e., eosinophil) recruitment in esophagitis. Activation of esophageal endothelial cells exposed to acidic refluxate may contribute to GERD in the setting of a disturbed mucosal squamous epithelial barrier (i.e., erosive esophagitis, peptic ulceration). esophagus; esophagitis; gastroesophageal reflux disease; microvasculature; phosphatidylinositol 3-kinase/Akt; VCAM-1     

Induction of the 72-kilodalton heat shock protein and protection from ultraviolet B-induced cell death in human keratinocytes by repetitive exposure to heat shock or 15-deoxy-delta(12,14)-prostaglandin J2

It has been demonstrated that hyperthermia protects keratinocytes from ultraviolet B (UVB)-induced cell death in culture and in vivo. This effect is mediated by the antiapoptotic effect of heat shock proteins that are transiently induced after exposure to heat at sublethal temperatures. Consequently, induction of Hsp has been proposed as a novel means of photoprotection. However, in the face of daily UVB exposure of human skin in vivo, this approach would not be useful if keratinocytes become less sensitive to Hsp induction with repeated exposure to the inducing agent. The aim of this study was to investigate whether repeated exposure to hyperthermia or to the stress protein activating cyclopentenone prostaglandin 15-deoxy-delta(12,14)-prostaglandin J2 (15dPGJ2) leads to adaptation of the cells, attenuation of the heat shock response, and abrogation of the protective effect. Normal human epidermal keratinocytes (NHEK) and the carcinoma-derived cell line A431 were exposed to either 42 degrees C or to 15dPGJ2 for 4 hours at 24-hour intervals for 4 consecutive days. The intracellular level of the 72-kDa heat shock protein (Hsp72) was determined by enzyme-linked immunosorbent assay (ELISA). Cells were exposed to UVB from a metal halide source after the last heat or 15dPGJ2 treatment, and survival was determined 24 hours after exposure by a MTT assay. Our results demonstrate that (1) heat shock and 15dPGJ2 are potent inducers of Hsp72 expression and lead to increased resistance to UVB-induced cell death in human keratinocytes; (2) re-exposure to heat shock leads to a superinduction without attenuation of the absolute increase in Hsp72 and of its UVB-protective effect; (3) the UVB tolerance induced by 15dPGJ2 is enhanced by repeated exposure without a further increase of Hsp72; (4) repeated heat shock and 15dPGJ2 up to a concentration of 1 microg/mL have no influence on cell growth over a period of 4 days. We conclude that through repeated exposure to Hsp-inducing factors, stress tolerance can be maintained without additional toxicity in human keratinocytes. These results provide a basis for the development of nontoxic Hsp inducers that can be repeatedly applied without loss of effect.     

Regulation of heat shock protein 70 release in astrocytes: role of signaling kinases

The ability to mount a successful stress response in the face of injury is critical to the long-term viability of individual cells and to the organism in general. The stress response, characterized in part by the upregulation of heat shock proteins, is compromised in several neurodegenerative disorders and in some neuronal populations, including motoneurons (MNs). Because astrocytes have a greater capacity than neurons to survive metabolic stress, and because they are intimately associated with the regulation of neuronal function, it is important to understand their stress response, so that we may to better appreciate the impact of stress on neuronal viability during injury or disease. We show that astrocytes subjected to hyperthermia upregulate Hsp/c70 in addition to intracellular signaling components including activated forms of extracellular-signal-regulated kinase (ERK1/2), Akt, and c-jun N-terminal kinase/stress activated protein kinase (JNK/SAPK). Furthermore, astrocytes release increasing amounts of Hsp/c70 into the extracellular environment following stress, an event that is abrogated when signaling through the ERK1/2 and phosphatidylinositol-3 kinase (PI3K) pathways is compromised and enhanced by inhibition of the JNK pathway. Last, we show that the Hsp/c70 is released from astrocytes in exosomes. Together, these data illustrate the diverse regulation of stress-induced Hsp/c70 release in exosomes, and the way in which the balance of activated signal transduction pathways affects this release. These data highlight how stressful insults can alter the microenvironment of an astrocyte, which may ultimately have implications for the survival of neighboring neurons.     

Molecular analysis of Hsp70 mechanisms in plants and their function in response to stress

Studying the strategies of improving abiotic stress tolerance is quite imperative and research under this field will increase our understanding of response mechanisms to abiotic stress such as heat. The Hsp70 is an essential regulator of protein having the tendency to maintain internal cell stability like proper folding protein and breakdown of unfolded proteins. Hsp70 holds together protein substrates to help in movement, regulation, and prevent aggregation under physical and or chemical pressure. However, this review reports the molecular mechanism of heat shock protein 70 kDa (Hsp70) action and its structural and functional analysis, research progress on the interaction of Hsp70 with other proteins and their interaction mechanisms as well as the involvement of Hsp70 in abiotic stress responses as an adaptive defense mechanism.     

Thermal biology of horseshoe crab embryos and larvae: A role for heat shock proteins

Eggs of the American horseshoe crab, Limulus polyphemus L., develop on sandy estuarine beaches during the spring and summer, and are potentially vulnerable to thermal stress during the 3-4 weeks of development to the first instar (trilobite) larval stage. In many marine taxa, heat shock (stress) proteins (Hsp's) help individuals acclimate to stresses by restoring the proper folding of cellular proteins whose shape has been altered by temperature shock or other forms of environmental stress. We examined the survival of embryos and first instar (trilobite) larvae following heat shock, and compared the levels of Hsp70 in heat shocked and control animals. Animals acclimated to 13 or 22 °C had close to 100% survival when heat shocked for 3 h at 35 or 40 °C, but exposure to 45 °C for 3 h was lethal. To study the effect of heat shock on Hsp70 production under environmentally realistic conditions, animals were acclimated to either 13 or 22 °C, heat-shocked at 35 °C for 3 h, and soluble proteins were extracted following 0, 2, 4, or 6 h recovery at 22 °C. The relative amounts of Hsp70 in horseshoe crab embryos and larvae were examined using SDS-PAGE and Western blotting. Relative to controls animals held at a constant temperature, there was a slight elevation of Hsp70 only among heat shocked trilobite larvae in the 6 h recovery treatment. Hsp70 levels did not differ significantly between control and heat shocked embryos. Horseshoe crabs have adapted to living in a thermally stressful environment by maintaining a high baseline (constitutive) level of cellular stress proteins such as Hsp70, rather than by synthesizing inducible Hsp's when stressful temperatures are encountered. This may be an effective strategy given that the heat shocks encountered by intertidal embryos and larvae occur regularly as a function of diurnal and tidal temperature changes.     

The molecular chaperone Hsp78 confers compartment-specific thermotolerance to mitochondria

Hsp78, a member of the family of Clp/Hsp100 proteins, exerts chaperone functions in mitochondria of S. cerevisiae which overlap with those of mitochondrial Hsp70. In the present study, the role of Hsp78 under extreme stress was analyzed. Whereas deletion of HSP78 does not affect cell growth at temperatures up to 39 decrees C and cellular thermotolerance at 50 degrees C, Hsp78 is crucial for maintenance of respiratory competence and for mitochondrial genome integrity under severe temperature stress (mitochondrial thermotolerance). Mitochondrial protein synthesis is identified as a thermosensitive process. Reactivation of mitochondrial protein synthesis after heat stress depends on the presence of Hsp78, though Hsp78 does not confer protection against heat-inactivation to this process. Hsp78 appears to act in concert with other mitochondrial chaperone proteins since a conditioning pretreatment of the cells to induce the cellular heat shock response is required to maintain mitochondrial functions under severe temperature stress. When expressed in the cytosol, Hsp78 can substitute for the homologous heat shock protein Hsp104 in mediating cellular thermotolerance, suggesting a conserved mode of action of the two proteins. Thus, proteins of the Clp/Hsp100-family located in the cytosol and within mitochondria confer compartment-specific protection against heat damage to the cell.     

Heat shock proteins: a history of study in Russia

This review describes a brief history of the discovery and studies in Russia and associated countries of the main stress protein (Hsp70) that plays important roles both in the normal function of the cell and body as well as under various stressful stimuli. Research on this protein at the Institute of Molecular Biology (Moscow) began with the elucidation of its adaptive functions at the cellular level and at the level of the whole organism. These studies examined the function of Hsp70 under normal and extreme conditions using a wide range of model and non-model animal species, from Leishmania and Drosophila to camels and humans. These analyses made it possible to elucidate the primary regulations in the evolution and function of heat shock (HS) genes in the studied organisms. Next, we studied the structure and characteristic features of heat shock genes and proteins in species with contrasting habitat temperatures. The systems of Hsp70 expression and isolation we developed using various research objects allowed us to proceed to study the protective properties of human recombinant Hsp70 in normal-aging animal models as well as animal models experiencing sepsis, Alzheimer’s disease, and stroke. The results obtained open the prospects of using recombinant Hsp70 for the treatment of various neuropathologies in humans. This review describes the logic and history of investigation of Hsp70 performed by one group of scientists from Engelhardt Institute of Molecular Biology, Russian Academy of Sciences. It was not the goal of this paper to give a comprehensive general picture of other similar studies carried out in Russia during this period.     

Heat shock proteins: endogenous modulators of apoptotic cell death

The highly conserved heat shock proteins (HSPs) accumulate in cells exposed to heat and a variety of other stressful stimuli. HSPs, which function mainly as molecular chaperones, allow cells to adapt to gradual changes in their environment and to survive in otherwise lethal conditions. The events of cell stress and cell death are linked and HSPs induced in response to stress appear to function at key regulatory points in the control of apoptosis. HSPs include antiapoptotic and proapoptotic proteins that interact with a variety of cellular proteins. Their expression level can determine the fate of the cell in response to a death stimulus, and apoptosis-inhibitory HSPs, in particular HSP27 and HSP70, may participate in carcinogenesis. This review summarizes apoptosis-regulatory function of HSPs.     

Neuronal differentiation in PC12 cells is accompanied by diminished inducibility of Hsp 70 and Hsp60 in response to heat and ethanol

The stress response of PC12 cells was characterized by evaluating the production of heat shock proteins of the 70 kDa (Hsp70), 60 kDa (Hsp60) and 90 kDa (Hsp90) families by western blot analysis. Induction of Hsp synthesis was elicited by brief exposure to elevated temperatures or by addition of ethanol to the cultures. Normal PC12 cells responded to stress with rapid up-regulation of Hsp70 and Hsp60 production. However, fully differentiated PC12 cells (induced by nerve growth factor, NGF) failed to produce Hsp70 or Hsp60 in response to heat or ethanol treatment. The disappearance of the heat shock response of the cells was directly related to the extent of neuronal differentiation. The cellular levels of the constitutive proteins, Hsc70 and Hsp90, were not altered by differentiation of the cells. Production of Hsps was restored in the differentiated cells by removal of NGF which coincided with the loss of neurite expression and retraction of processes.     

70-kDa heat shock protein expression in cultured rat astrocytes after hypoxia: regulatory effect of almitrine

Induction of heat shock proteins (Hsps), especially the 70-kDa family, is well observed in nervous tissues in response to various stressful conditions. By using rat astrocytes in primary culture, the expression of the inducible (Hsp70) and the constitutive (Hsc70) 70-kDa Hsps immunoreactivity of cells exposed to hypoxic conditions has been investigated. We observed that exposure of astroglial cells to an hypoxic-normoxic sequence induces a significant decrease of Hsc70 immunoreactivity. The presence of the heat inducible stress protein Hsp70 is never observed in hypoxic cells not in control. Hsc 70 lowering is associated with ultrastructural alterations characterized by mitochondria swelling, formation of vacuoles and accumulation of dense material in the cell cytoplasm. The effects of addition of almitrine to the culture medium before and during hypoxia on Hsps immunoreactivity have been examined. The presence of the drug prevents the decrease of Hsc 70 immunoreactivity induced by hypoxia. Furthermore, some ultrastructural improvement is observed in astroglial cells treated with almitrine suggesting some protecting role of Hsc70 on cell damage induced by hypoxia.     

Arabidopsis thaliana J-class heat shock proteins: cellular stress sensors

Plants are sessile organisms that have evolved a variety of mechanisms to maintain their cellular homeostasis under stressful environmental conditions. Survival of plants under abiotic stress conditions requires specialized group of heat shock protein machinery, belonging to Hsp70:J-protein family. These heat shock proteins are most ubiquitous types of chaperone machineries involved in diverse cellular processes including protein folding, translocation across cell membranes, and protein degradation. They play a crucial role in maintaining the protein homeostasis by reestablishing functional native conformations under environmental stress conditions, thus providing protection to the cell. J-proteins are co-chaperones of Hsp70 machine, which play a critical role by stimulating Hsp70s ATPase activity, thereby stabilizing its interaction with client proteins. Using genome-wide analysis of Arabidopsis thaliana, here we have outlined identification and systematic classification of J-protein co-chaperones which are key regulators of Hsp70s function. In comparison with Saccharomyces cerevisiae model system, a comprehensive domain structural organization, cellular localization, and functional diversity of A. thaliana J-proteins have also been summarized.     

Reduced Enzyme Activity Following Hsp70 Overexpression in Drosophila melanogaster

Acclimation to environmental change can impose costs to organisms. One potential cost is the change in cell metabolism that follows a physiological response, e.g., high expression of heat shock proteins may alter specific activity of important enzymes. We examined the significance of this cost in a pair of Drosophila melanogaster lines transformed with additional copies of a gene that encodes the heat shock protein, Hsp70. Heat shock induces Hsp70 expression in all lines, but lines with extra copies produce much more Hsp70 than do excision control strains. The consequence of this supranormal Hsp70 expression is to reduce specific activity of both enzymes analyzed, adult alcohol dehydrogenase (ADH), which is heat sensitive, and lactate dehydrogenase, which is not. Strain differences were most pronounced under those conditions where Hsp70 expression was maximized, and not where the heat stress denatured proteins. That result supported the idea that Hsp70 expression is constrained evolutionarily by its tendency to bind nascent peptides when overabundant within the cell.     

Thermolability of mouse oocytes is due to the lack of expression and/or inducibility of Hsp70

Eukaryotic and prokaryotic cells have been shown to respond to physical and chemical stress by the induction of proteins called heat shock proteins. Heat shock protein 70 (Hsp70), is the most ubiquitous of these proteins. Although heat shock proteins are generally thought to protect cells from physiologically stressful stimuli, it cannot be assumed that this is so, because several cases exist in which thermotolerance is acquired without the production of heat shock proteins, and in several other cases the hyperproduction of these heat shock proteins does not produce thermotolerance. In this study we show that unfertilized mouse oocytes are sensitive to elevated temperatures, and that the synthesis of Hsp70 cannot be induced in these oocytes. Furthermore, our data demonstrate that the expression of Hsp70 in mouse oocytes is sufficient for the acquisition of thermotolerance. Mouse oocytes were injected with mRNA for Hsp70, and the viability of these oocytes was determined after heating. The number of viable oocytes was significantly higher in the group injected with Hsp70 mRNA and then heated compared with oocytes injected with Hsp70 antisense mRNA and sham-injected controls treated in an identical manner. No significant differences in the number of viable oocytes were found between the group that had been injected with Hsp70 mRNA, heated, and then allowed to recover for 3 hr and the group maintained at 37 degrees C throughout.(ABSTRACT TRUNCATED AT 250 WORDS)