In vivo chaperone activity of heat shock protein 70 and thermotolerance

Heat shock protein 70 (Hsp70) is thought to play a critical role in the thermotolerance of mammalian cells, presumably due to its chaperone activity. We examined the chaperone activity and cellular heat resistance of a clonal cell line in which overexpression of Hsp70 was transiently induced by means of the tetracycline-regulated gene expression system. This single-cell-line approach circumvents problems associated with clonal variation and indirect effects resulting from constitutive overexpression of Hsp70. The in vivo chaperone function of Hsp70 was quantitatively investigated by using firefly luciferase as a reporter protein. Chaperone activity was found to strictly correlate to the level of Hsp70 expression. In addition, we observed an Hsp70 concentration dependent increase in the cellular heat resistance. In order to study the contribution of the Hsp70 chaperone activity, heat resistance of cells that expressed tetracycline-regulated Hsp70 was compared to thermotolerant cells expressing the same level of Hsp70 plus all of the other heat shock proteins. Overexpression of Hsp70 alone was sufficient to induce a similar recovery of cytoplasmic luciferase activity, as does expression of all Hsps in thermotolerant cells. However, when the luciferase reporter protein was directed to the nucleus, expression of Hsp70 alone was not sufficient to yield the level of recovery observed in thermotolerant cells. In addition, cells expressing the same level of Hsp70 found in heat-induced thermotolerant cells containing additional Hsps showed increased resistance to thermal killing but were more sensitive than thermotolerant cells. These results suggest that the inducible form of Hsp70 contributes to the stress-tolerant state by increasing the chaperone activity in the cytoplasm. However, its expression alone is apparently insufficient for protection of other subcellular compartments to yield clonal heat resistance to the level observed in thermotolerant cells.     

Induced thermotolerance in bovine two-cell embryos and the role of heat shock protein 70 in embryonic development

Induced thermotolerance is a phenomenon whereby exposure to a mild heat shock can induce heat shock proteins (HSP) and other cellular changes to make cells more resistant to a subsequent, more severe heat shock. Given that the 2-cell bovine embryo is very sensitive to heat shock, but can also produce HSP70 in response to elevated temperature, experiments were conducted to test whether 2-cell embryos could be made to undergo induced thermotolerance. Another objective was to test the role of the heat-inducible form of heat shock protein 70 (HSP70i) in development and sensitivity of bovine embryos to heat shock. To test for induced thermotolerance, 2-cell bovine embryos were first exposed to a mild heat shock (40 degrees C for 1 hr, or 41 degrees C or 42 degrees C for 80 min), allowed to recover at 38.5 degrees C and 5% (v/v) CO2 for 2 hr, and then exposed to a severe heat shock (41 degrees C for 4.5, 6, or 12 hr). Regardless of the conditions, previous exposure to mild heat shock did not reduce the deleterious effect of heat shock on development of embryos to the blastocyst stage. The role of HSP70i in embryonic development was tested in two experiments by culturing embryos with a monoclonal antibody to the inducible form of HSP70. At both 38.5 degrees C and 41 degrees C, the proportion of 2-cell embryos that developed to blastocyst was reduced (P < 0.05) by addition of anti-HSP70i to the culture medium. In contrast, sensitivity to heat shock was not generally increased by addition of antibody. In conclusion, bovine 2-cell embryos appear incapable of induced thermotolerance. Lack of capacity for induced thermotolerance could explain in part the increased sensitivity of 2-cell embryos to heat shock as compared to embryos at later stages of development. Results also implicate a role for HSP70i in normal development of bovine embryos.     

Overexpression of Zostera japonica heat shock protein gene ZjHsp70 enhances the thermotolerance of transgenic Arabidopsis

Molecular Biology Reports - Heat shock protein 70s (Hsp70s) are major members of the heat shock protein family and play a variety of roles to protect plants against stress. Plant Hsp70s are a...     

Induction of heat shock proteins and thermotolerance by ethanol in Saccharomyces cerevisiae

The temperature sensitivity of $\text{Saccharomyces}\text{cerevisiae}$ and the conditions of moderate heat pretreatment required to induce thermotolerance are established. Ethanol is identified as an inducer of heat shock proteins and an inducer of thermotolerance.     

Inhibition of heat shock protein synthesis and thermotolerance by cycloheximide

Chinese hamster ovary (CHO) cells were exposed to a 43 degrees C, 15-min heat shock to study the relationship between protein synthesis and the development of thermotolerance. The 43 degrees C heat shock triggered the synthesis of three protein families having molecular weights of 110,000, 90,000, and 65,000 (HSP). These proteins were synthesized at 37 and 46 degrees C. This heat shock also induced the development of thermotolerance, which was measured by incubating the cells at 46 degrees C 4 h after the 43 degrees C heat treatment. CHO cells were also exposed to 20 micrograms/ml of cycloheximide for 30 min at 37 degrees C, 15 min at 43 degrees C, and 4 h at 37 degrees C. This treatment inhibited the enhanced synthesis of the Mr 110,000, 90,000, and 65,000 proteins. The cycloheximide was then washed out and the cells were incubated at 46 degrees C. HSP synthesis did not recover during the 46 degrees C incubation. This cycloheximide treatment also partially inhibited the development of thermotolerance. These results suggest that for CHO cells to express thermotolerance when exposed to the supralethal temperature of 46 degrees C protein synthesis is necessary.     

Induction of the heat shock response and translational thermotolerance in day 15 ovine trophectoderm

The objectives of this study were to determine the ability of trophectoderm from preimplantation ovine embryos to synthesize hsp70 in response to heat shock and to identify conditions which induce translational thermotolerance in this tissue. Day 15 embryos were collected, and proteins synthesized in 1.5-mm sections of trophectoderm were radioactively labeled with (35)S-methionine. One-dimensional SDS-PAGE gels, two-dimensional gel electrophoresis and Western blots were utilized to characterize the heat shock response and to examine the induction of translational thermotolerance. Increased synthesis of the 70 kDa heat shock proteins and a protein with an approximate molecular weight of 15 to 20 kDa was observed with heat shock (> or = 42 degrees C). Total protein synthesis decreased (P < 0.05) with increased intensity of heat shock. At 45 degrees C, protein synthesis was suppressed with little or no synthesis of all proteins including hsp70. Recovery of protein synthesis following a severe heat shock (45 degrees C for 20 min) occurred faster (P < 0.05) in trophectoderm pretreated with a mild heat shock (42 degrees C for 30 min) than trophectoderm not pretreated with mild heat. In summary, trophoblastic tissue obtained from ovine embryos exhibit the characteristic "heatshock" response similar to that described for other mammalian systems. In addition, a sublethal heat shock induced the ability of the tissue to resume protein synthesis following severe heat stress. Since maintaining protein synthesis is crucial to embryonic survival, manipulation of the heat-shock response may provide a method to enhance embryonic survival.     

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.     

Induction of thermotolerance and enhanced heat shock protein synthesis in chinese hamster fibroblasts by sodium arsenite and by ethanol

Synthesis of a family of proteins called “heat shock” proteins is enhanced in cells in response to a wide variety of environmental stresses. This suggests that these proteins may have functions essential to cell survival under stressful conditions. A causative relationship between heat shock protein synthesis and development of thermotolerance would imply that agents known to induce heat shock protein synthesis, such as sodium arsenite, also induce thermotolerance. Conversely, agents known to induce thermotolerance, such as ethanol, would also enhance heat shock protein synthesis. To test this hypothesis, I have examined the effect of sodium arsenite or ethanol treatment on protein synthesis and cell survival in Chinese hamster ovary HA-1 cells. After either sodium arsenite or ethanol treatment, the synthesis of heat shock proteins was greatly enhanced over that of untreated cells. In parallel, cell survival was increased as much as 10⁴-fold when cells exposed to either agent were challenged by a subsequent heat treatment. The synthesis of heat shock proteins correlated well with the development of thermotolerance. A qualitative analysis of individual proteins suggests that the synthesis of 70,000 and 87,000 molecular weight proteins most closely mirrored the development of thermotolerance. The results, therefore, strongly reinforce the hypothesis that a causal relationship exists between the enhanced synthesis of heat shock protein and cell survival under specific stresses.     

An Arabidopsis heat shock protein complements a thermotolerance defect in yeast.

The heat shock protein Hsp104 of the yeast Saccharomyces cerevisiae plays a key role in promoting survival at extreme temperatures. We found that when diverse higher plant species are exposed to high temperatures they accumulate proteins that are antigenically related to Hsp104. We isolated a cDNA corresponding to one of these proteins from Arabidopsis. The protein, AtHSP101, is 43% identical to yeast Hsp104. DNA gel blot analysis indicated that AtHSP101 is encoded by a single- or low-copy number gene. AtHsp101 mRNA was undetectable in the absence of stress but accumulated to high levels during exposure to high temperatures. When AtHSP101 was expressed in yeast, it complemented the thermotolerance defect caused by a deletion of the HSP104 gene. The ability of AtHSP101 to protect yeast from severe heat stress strongly suggests that this HSP plays an important role in thermotolerance in higher plants.     

Induction of heat shock protein 70 by herbimycin A and cyclopentenone prostaglandins in smooth muscle cells

This study characterizes Hsp70 induction in human smooth muscle cells (SMC) by herbimycin A and cyclopentenone prostaglandins. The magnitude of Hsp70 induction by cyclopentenone prostaglandins was 8- to 10-fold higher than induction by herbimycin A. Hsp70 induction by delta12PGJ2 was first observed at 10 microM, rose to 4000-5000 ng/mL within one log unit and a maximum response was not observed; concentrations of delta12PGJ2 higher than 30 microM were toxic to the cells. A maximum response with herbimycin A (500 ng/mL) was reached at 0.05 microM and maintained to 1 microM without toxicity. Both, delta12PGJ2 and herbimycin A, were inhibited by dithiothreitol (DTT, 100 microM) at lower concentrations and became less sensitive to inhibition at higher concentrations. Hsp70 induction after incubation of SMC with delta12PGJ2 followed by addition of herbimycin A was significantly higher than Hsp70 induction after incubation with herbimycin A followed by addition of delta12PGJ2. When cells were incubated with [3H]-PGJ2, followed by protein denaturation, substantial radioactivity remained protein-bound suggesting that the prostaglandin must be covalently bound. Covalent binding was largely insensitive to DTT. Maximal Hsp70 induction was observed after 5 minutes of exposure of the cells to herbimycin A followed by a 20 hour recovery period in agent-free medium. Cells required 3-4 hours of exposure to delta12PGJ2 followed by a 20 hour recovery period in order to see high Hsp70 induction. Binding of the heat shock factor (HSF) to the heat shock element (HSE) in the presence of herbimycin A or delta12PGJ2, and the effects of DTT, mirrored the results of Hsp70 induction. The results suggest that probable differences between the 2 agents are at the level of the signal transduction prior to HSF activation.     

A small heat shock protein cooperates with heat shock protein 70 systems to reactivate a heat-denatured protein

Small heat shock proteins (sHsps) are a diverse group of heat-induced proteins that are conserved in prokaryotes and eukaryotes and are especially abundant in plants. Recent in vitro data indicate that sHsps act as molecular chaperones to prevent thermal aggregation of proteins by binding non-native intermediates, which can then be refolded in an ATP-dependent fashion by other chaperones. We used heat-denatured firefly luciferase (Luc) bound to pea (Pisum sativum) Hsp18.1 as a model to define the minimum chaperone system required for refolding of a sHsp-bound substrate. Heat-denatured Luc bound to Hsp18.1 was effectively refolded either with Hsc/Hsp70 from diverse eukaryotes plus the DnaJ homologs Hdj1 and Ydj1 (maximum = 97% Luc reactivation with k(ob) = 1.0 x 10(-2)/min), or with prokaryotic Escherichia coli DnaK plus DnaJ and GrpE (100% Luc reactivation, k(ob) = 11.3 x 10(-2)/min). Furthermore, we show that Hsp18.1 is more effective in preventing Luc thermal aggregation than the Hsc70 or DnaK systems, and that Hsp18.1 enhances the yields of refolded Luc even when other chaperones are present during heat inactivation. These findings integrate the aggregation-preventive activity of sHsps with the protein-folding activity of the Hsp70 system and define an in vitro system for further investigation of the mechanism of sHsp action.     

Effects of cycloheximide on thermotolerance expression, heat shock protein synthesis, and heat shock protein mRNA accumulation in rat fibroblasts.

A single hyperthermic exposure can render cells transiently resistant to subsequent high temperature stresses. Treatment of rat embryonic fibroblasts with cycloheximide for 6 h after a 20-min interval at 45 degrees C inhibits protein synthesis, including heat shock protein (hsp) synthesis, and results in an accumulation of hsp 70 mRNA, but has no effect on subsequent survival responses to 45 degrees C hyperthermia. hsp 70 mRNA levels decreased within 1 h after removal of cycloheximide but then appeared to stabilize during the next 2 h (3 h after drug removal and 9 h after heat shock). hsp 70 mRNA accumulation could be further increased by a second heat shock at 45 degrees C for 20 min 6 h after the first hyperthermic exposure in cycloheximide-treated cells. Both normal protein and hsp synthesis appeared increased during the 6-h interval after hyperthermia in cultures which received two exposures to 45 degrees C for 20 min compared with those which received only one treatment. No increased hsp synthesis was observed in cultures treated with cycloheximide, even though hsp 70 mRNA levels appeared elevated. These data indicate that, although heat shock induces the accumulation of hsp 70 mRNA in both normal and thermotolerant cells, neither general protein synthesis nor hsp synthesis is required during the interval between two hyperthermic stresses for Rat-1 cells to express either thermotolerance (survival resistance) or resistance to heat shock-induced inhibition of protein synthesis.     

Induction of heme oxygenase-1 and heat shock protein 70 in rat hepatocytes: The role of calcium signaling

Stress response genes including heat shock proteins are induced under a variety of conditions to confer cellular protection. This study investigated the role of calcium signaling in the induction of two stress response genes, heme oxygenase-1/hsp32 and hsp70, in isolated rat hepatocytes. Both genes were induced by cellular glutathione depletion. This induction could be inhibited by BAPTA-AM. Culturing in a calcium-free medium prevented the induction of hsp70 gene expression after glutathione depletion without affecting heme oxygenase-1 gene expression. Thapsigargin increased the gene expression of heme oxygenase-1 but not that of hsp70. Thapsigargin-induced heme oxygenase-1 induction was completely inhibited by BAPTA-AM. Incubation with the Ca²⁺-ionophore A23187 augmented heme oxygenase-1 (two-fold) and hsp70 (5.2-fold) mRNA levels. Our data suggests a significant role of Ca²⁺-dependent pathways in the induction of the two stress genes. An increase in the cytoplasmic Ca²⁺ activity seems to play a key role in the cascade of signaling leading to the induction of the two genes. However, the source of Ca²⁺ that fluxes into the cytoplasm seems to be different. Our data provides evidence for a compartmentalization of calcium fluxes, i.e. the Ca²⁺ flux from intracellular stores (e.g. the endoplasmic reticulum) plays a major role in the induction of heme oxygenase-1. By contrast, Ca²⁺ flux from the extracellular medium seems to be a mechanism initiating the cellular signaling cascade leading to hsp70 gene induction.     

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.     

The heat shock protein HSP70 and heat shock cognate protein HSC70 contribute to antimony tolerance in the protozoan parasite leishmania

Antimony-containing drugs are still the drugs of choice in the treatment of infections caused by the parasite Leishmania. Resistance to antimony is now common in some parts of the world, and several mechanisms of resistance have been described. By transfecting cosmid banks and selecting with potassium antimonyl tartrate (SbIII), we have isolated a cosmid associated with resistance. This cosmid contains 2 copies of the heat shock protein 70 (HSP70) and 1 copy of the heat shock cognate protein 70 (HSC70). Several data linked HSP70 to antimony response and resistance. First, several Leishmania species, both as promastigotes and amastigotes, increased the expression of their HSP70 proteins when grown in the presence of 1 or 2 times the Effect Concentration 50% of SbIII. In several mutants selected for resistance to either SbIII or to the related metal arsenite, the HSP70 proteins were found to be overexpressed. This increase was also observed in revertant cells grown for several passages in the absence of SbIII, suggesting that this increased production of HSP70 is stable. Transfection of HSP70 or HSC70 in Leishmania cells does not confer resistance directly, though these transfectants were better able to tolerate a shock with SbIII. Our results are consistent with HSP70 and HSC70 being a first line of defense against SbIII until more specific and efficient resistance mechanisms take over.     

Localization of heat shock protein 70 in rat mast cells

Heat shock proteins (Hsp) are intracellular chaperons, as well as extracellular molecules with immunomodulatory and signaling functions engaged in adaptation to stress on the cellular and organism levels. The presence of Hsp in secretory granules of mast cells (MCs) may be correlated with mast cells’ active participation in adaptation to stress. Using immunoelectron microscopy, we showed that Hsp70 was localized in secretory granules of rat pericardial and peritoneal mast cells. Localization of Hsp70 in rat peritoneal mast cells isolated by Percoll density gradient centrifugation was confirmed by immunoblotting. The possible involvement of mast cells in production of extracellular Hsp70, as well as Hsp70 functions inside the mast cells, is discussed.