Induction Of Hsp Synthesis Research Articles

Phenotypic plasticity of HSP70s gene expression during diapause: signs of evolutionary responses to cold stress among Soybean Pod Borer populations (Leguminivora glycinivorella) in Northeast of China.

The soybean pod borer (Leguminivora glycinivorella Matsumura) successfully survives the winter because of its high expression of 70-kDa heat shock proteins (HSP70s) during its overwintering diapause. The amount of HSP70s is different under different environmental stresses. In this study, inducible heat shock protein 70 and its constitutive heat shock cognate 70 were cloned by RT-PCR and RACE. These genes were named Lg-hsp70 and Lg-hsc70, respectively. Gene transcription and protein expression after cold stress treatment (5°C to −5°C) were analyzed by western blotting and by qRT-PCR for four populations that were sampled in the northeast region of China, including Shenyang, Gongzhuling, Harbin and Heihe, when the soybean pod borer was in diapause. As the cold shock temperature decreased, the levels of Lg-HSP70s were significantly up-regulated. The amount of cold-induced Lg-HSP70s was highest in the southernmost population (Shenyang, 41°50′N) and lowest in the northernmost population (Heihe, 50°22′N). These results support the hypothesis that the soybean pod borer in the northeast region of China displays phenotypic plasticity, and the accumulation of Lg-HSP70s is a strategy for overcoming environmental stress. These results also suggest that the induction of HSP70 synthesis, which is a complex physiological adaptation, can evolve quickly and inherit stability.

The induction of Hsp70 synthesis by non-lethal heat shock confers thermotolerance and resistance to lethal ammonia stress in the common carp,Cyprinus carpio(Linn)

Exposure to heat-shock protein (Hsp) stimulating factors induces Hsp accumulation and confers tolerance to lethal ammonia stress on the common carp Cyprinus carpio. This study investigated whether a non-lethal heat shock bestowed similar protective effects against ammonia and induced thermotolerance, both thought to be rendered by increased amounts of Hsps. The 30-min lethal temperature (30 min LHT) and 1-h lethal ammonia concentration (1 h LCT) for this species occurred at 41°C and 14.2 mg/L NH3 respectively. Heating juvenile carp (5 cm) from 28°C to 32, 34 and 38°C, with a subsequent 8-h recovery period augmented tolerance to lethal heat and ammonia perturbation by two to threefold as compared with animals held at 28°C. Protection occurred in conjunction with Hsp70 accumulation in gills, substantiating the role of this Hsp in enhancing the stress tolerance of common carp.

Heat Shock Proteins and Disease Control in Aquatic Organisms

Four families of heat shock proteins (Hsps), including the small heat shock proteins (sHsps), Hsp70, Hsp90 and Hsp60, are synthesized under normal physiological conditions and in response to stress. sHsps protect proteins from irreversible denaturation independently of ATP. The remaining ATP-responsive Hsps fold nascent proteins, shield proteins from irreversible denaturation during stress and aid protein refolding. Several observations indicate that Hsps contribute to disease resistance in aquatic organisms, the first being that these proteins are produced in finfish, shellfish and bivalves upon infection with viral and bacterial pathogens. Induction of Hsp synthesis by heat shock and incubation with chemicals such as Pro-Tex® boosts resistance to pathogens, as does administration of exogenous Hsps to host organisms. The extent of Hsp accumulation and the increase in disease tolerance are generally correlated with one another. Not only do Hsps protect against pathogens by functioning as molecular chaperones, but they are thought to mediate humoral and cellular innate immune responses. Hsp70 is highly immunogenic and serves as a ligand for Toll-like receptors. Hsps elicit cytokine production and they deliver peptides to antigen presenting cells via major histocompatibility complexes (MHC). Vaccines have been produced for use in aquaculture by employing Hsps, either alone or fused to antigens obtained from pathogens. Hsps offer several advantages over current methods for the treatment of disease in commercially important organisms and they are being increasingly exploited as their roles in protein chaperoning and immune modulation are better understood.

Application of heat shock proteins as stress markers in aquatic organisms using endemic Baikal amphipods as an example

When heat shock proteins (HSPs) are used as biomarkers in monitoring studies of aquatic ecosystems, it is necessary to take into account the specificity of synthesis of these proteins in various organisms. This especially applies to endemic species and species with narrow ranges of adaptation for specific conditions in certain water bodies. In this study, we assessed the possibility to use HSPs as molecular stress markers in species with a narrow niche breadth using endemic Baikal amphipods (Crustacea, Amphipoda) as an example. The effect of stress induced by toxicants and temperature has been assessed. Proteins of families HSP70 and low-molecular-weight HSP related to alpha-crystallins were used as biomarkers. Temperature- and toxicant-induced stresses induced low-molecular-weight HSP synthesis in the endemic amphipod species studied. However, induction of HSP70 synthesis in the same species after temperature stress has not been detected. The specificity of synthesis of HSP70 is discussed. The results obtained in this study suggest that low-molecular-weight HSPs can be used as stress markers in Baikal species and species with a narrow niche breadth.

Stressor-specific induction of heat shock protein 70 in the freshwater prawn Macrobrachium malcolmsonii (H. Milne Edwards) exposed to the pesticides endosulfan and carbaryl

Agricultural run off that is contaminated with pesticides enters water bodies, thereby polluting the aquatic environment. The sensitivity of the freshwater prawn, Macrobrachium malcolmsonii, to such pesticides is well-documented. However, the stress response to sublethal concentrations of pesticides has scarcely been investigated. In the present study, the effect of two different sublethal concentrations (1/5th and 1/10th of LC 50 value) of endosulfan and carbaryl on the synthesis of hsp70 in various tissues of the prawn M. malcolmsonii was evaluated by Western blotting analysis. Among the tissues analyzed (gill, skeletal muscle, and hepatopancreas), induction of hsp70 synthesis was recorded only in the gill tissue of the prawns that had been exposed to either the high or low sublethal concentration of endosulfan. Induction of synthesis of hsp70 was not observed in any of the tissues sampled from the prawns that had been exposed to either the low or the high sublethal concentration of carbaryl. Pesticides interfere with the hydrophobic core of protein, thus leading to denaturation of the protein by exposing the lipophilic moieties to the aqueous environment. Specific induction of hsp70 in the gill possibly occurred since the gill is the route of exposure for pesticides in the prawns. The absence of induction of hsp70 synthesis in prawns that had been exposed to carbaryl suggests that induction of hsp70 synthesis is stressor-specific.

Heat shock proteins do not provide thermoprotection to normal cellular protein synthesis, α‐amylase mRNA and endoplasmic reticulum lamellae in barley aleurone layers

Heat shock in barley (Hordeum vulgare L. cv. Himalaya) aleurone layers induces the synthesis of heat shock proteins (hsps) and suppresses the synthesis and secretion of α‐amylase, the principal secretory protein. This is accompanied by the destabilization of α‐amylase mRNA and a concomitant dissociation of ER lamellae. In the absence of heat shock α‐amylase mRNA is extremely stable (Belanger et al. 1986. Proc. Natl. Acad. Sci. USA 83: 1354–1358). In most organisms there is a direct correlation between the synthesis of hsps and thermotolerance. The ability of hsps to provide thermoprotection to secretory protein synthesis, α‐amylase mRNA and ER lamellae was analyzed. Sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) of pulse‐chased, [35S]‐methionine‐labeled proteins revealed that the half‐life of hsps in barley aleurone cells recovering from heat shock was approximately 12 h. Within approximately 6 h, there was a recovery of α‐amylase mRNA and a reformation of ER lamellae. Heat shock protein synthesis was induced by either heat shock (40°C) or arsenite, the cells were allowed to recover for 8 h, then were re‐exposed to heat shock. Results from SDS‐PAGE showed that, despite the presence of hsps, α‐amylase synthesis was suppressed. Northern blot hybridizations showed that α‐amylase mRNA levels were reduced in heat‐shocked tissues. Transmission electron microscopy demonstrated that ER lamellar structures were dissociated. The synthesis of hsps did not enable barley aleurone cells to sustain the synthesis of any proteins at lethal temperature. In contrast, similar conditions established thermotolerance and provided thermoprotection to protein synthesis in germinating barley embryos. Our findings suggest that the aleurone layer does not become thermotolerant following the induction of hsp synthesis.

Cytokine induction of heat shock protein in human granulosa-luteal cells.

The infiltration of leukocytes is a characteristic feature of luteolysis in humans. Leukocytes are known to generate physiological inducers of cell stress such as cytokines which have been implicated as mediators of functional luteal regression. In cells exposed to stress, a response characterized by an increase in heat shock protein (HSP) synthesis occurs. Recently, the induction of HSP-70 in rat luteal cells has been shown to inhibit luteinizing hormone (LH) and cAMP-sensitive progesterone production, possibly by interfering with the translocation of cholesterol to the mitochondrial cytochrome P450scc. We therefore investigated whether HSP-70 is induced in human granulosa-luteal cells and its relationship to steroidogenesis. [35S]Methionine labelling showed an increase in a 70 kDa protein after heat treatment which was demonstrated to be HSP-70 by Western analysis using monoclonal antibodies against the constitutive and inducible forms of HSP-70. Induction of HSP-70 in human granulosa-luteal cells was also seen with interferon (IFN) gamma (10 ng/ml), tumour necrosis factor (TNF)-alpha (100 ng/ml) and a combination of IFN gamma/TNF-alpha (10/50 ng/ml). Interleukin-1 beta (IL-1 beta) (30 ng/ml) showed minimal induction of HSP-70 above control values. An increase in activated heat shock factor, which binds to the heat shock transcriptional control element, was detected after heat shock, IFN/TNF, and IFN treatment. Coincident with the induction of HSP-70 by heat shock was the inhibition of progesterone production compared with non-shocked granulosa-luteal cells. Heat shock inhibition of progesterone synthesis was partially reversed by the cell- and mitochondria-permeant cholesterol analogue, 22R-hydroxycholesterol. Cell viability was unaffected by heat treatment. White blood cell-depleted granulosa-luteal cell cultures treated with IFN demonstrated a significant reduction in progesterone production. Treatment with IFN/TNF, TNF, and IL-1 also decreased progesterone secretion, although statistical significance was not achieved. These findings provide evidence that a stress response occurs in human granulosa-luteal cells in response to heat and cytokines. The inhibition of gonadotrophin-sensitive steroidogenesis coincident with the induction of HSP-70 synthesis by physiological agents which are present in the corpus luteum implicates HSP-70 as a potential mediator of luteolysis in the human.

Inhibition mechanism of HSP70 induction in murine FM3A cells maintained at low culture temperature

We have shown previously that induction of HSP70 synthesis in murine FM3A and its mutant ts85 cells by heat shock is somehow modulated by culture temperature. In this study, we further examined activation of heat shock transcription factor (HSF) and induction kinetics of HSP70 synthesis and HSP70 mRNA in FM3A and ts85 cells maintained at 37°C (37°C-cells) and 33°C (33°C-cells). Upon exposure to heat shock, HSF was activated to a high level in 37°C-FM3A cells, whereas HSF was activated only to a low level in the 33°C-cells. The induction of HSP70 mRNA and HSP70 synthesis occurred successively in the 37°C-cells but not in the 33°C-cells. On the other hand, in both 37 and 33°C-ts85 cells, activation of HSF, induction of HSP70 mRNA, and HSP70 synthesis occurred successively. Characteristically, protein synthesis in both 33°C-FM3A and ts85 cells was significantly lower than in the respective 37°C-cells, but constitutive HSP73 levels were similar among both the 37 and 33°C-cells. Furthermore, inhibition of protein synthesis of FM3A cells did not influence the activation of HSF, but accelerated inactivation of the activated HSF. We discuss the possible inhibition mechanisms of activation of HSF in 33°C-FM3A cells, regarding the function of HSP70 in both protein synthesis and repression of HSF.

Inhibition mechanism of HSP70-induction in murine FM3A cells maintained at low culture temperature

Abstract We have shown previously that induction of HSP70 synthesis in murine FM3A and its mutant ts85 cells by heat shock is somehow modulated by culture temperature. In this study, we further examined activation of heat shock transcription factor (HSF) and induction kinetics of HSP70 synthesis and HSP70 mRNA in FM3A and ts85 cells maintained at 37°C (37°C-cells) and 33°C (33°C-cells). Upon exposure to heat shock, HSF was activated to a high level in 37°C-FM3A cells, whereas HSF was activated only to a low level in the 33°C-cells. The induction of HSP70 mRNA and HSP70 synthesis occurred successively in the 37°C-cells but not in the 33°C-cells. On the other hand, in both 37 and 33°C-ts85 cells, activation of HSF, induction of HSP70 mRNA, and HSP70 synthesis occurred successively. Characteristically, protein synthesis in both 33°C-FM3A and ts85 cells was significantly lower than in the respective 37°C-cells, but constitutive HSP73 levels were similar among both the 37 and 33°C-cells. Furthermore, inhibition of protein synthesis of FM3A cells did not influence the activation of HSF, but accelerated inactivation of the activated HSF. We discuss the possible inhibition mechanisms of activation of HSF in 33°C-FM3A cells, regarding the function of HSP70 in both protein synthesis and repression of HSF.

Synthesis of heat shock proteins in cultured chick pineal cells

Chick pineal cells in static culture display a persistent, photosensitive, circadian rhythm of melatonin synthesis and release. Melatonin synthesis and its rhythm are also sensitive to temperature changes. By SDS-PAGE autoradiography, we found that several heat shock proteins (HSPs 25, 70, and 90) are synthesized under temperature conditions which also cause changes in melatonin production and rhythms. We did not see substantial phase-dependent differences in the induction of HSP synthesis, nor did we detect any clear effect of light on the synthesis of these proteins.

Antiviral effect of short hyperthermic treatment at specific stages of vesicular stomatitis virus replication cycle.

Starting from the observation that the antiviral activity of cyclopentenone prostaglandins is associated with the synthesis of a 70K heat shock protein (HSP70), we have analysed the effect of short hyperthermic treatment (HT) on HSP70 induction and virus production in monkey epithelial cells during the replication of vesicular stomatitis virus (VSV). The heat shock response, as determined by HSP70 synthesis, appeared to be unaltered in VSV-infected cells in the first 4 h following virus infection, after which time it started to decline. No induction of HSP70 synthesis was observed when HT was applied 8 h after VSV infection. A 20 min HT at 45 degrees C was effective in suppressing VSV multiplication by more than 90% when applied at specific stages of the virus replication cycle. Synthesis of virus proteins was not affected by HT, indicating that the target for the treatment is a post-translational event. The HT-induced block of virus replication appeared to be associated with inhibition of VSV G protein maturation and HSP70 induction.

Expression of heat shock protein 70 is altered by age and diet at the level of transcription.

Because heat shock proteins have been shown to play a critical role in protecting cells from hyperthermia and other types of physiological stresses, it was of interest to determine what effect age and caloric restriction have on the ability of cells to regulate the expression of heat shock protein 70 (hsp70), the most prominent and most evolutionarily conserved of the heat shock proteins. Caloric restriction is the only experimental manipulation known to retard aging and increase survival of mammals. The ability of hepatocytes isolated from young/adult (4- to 7-month-old) and old (22- to 28-month-old) male Fischer F344 rats fed ad libitum or a caloric restriction diet (60% of the content of the ad libitum diet) to express hsp70 was determined after a mild heat shock (42.5 degrees C for 30 min). We found that the induction of hsp70 synthesis and mRNA levels by heat shock was 40 to 50% lower in hepatocytes isolated from old rats than in hepatocytes isolated from young rats. Using in situ hybridization, we found that essentially all hepatocytes from the young/adult and old rats expressed hsp70 in response to heat shock; therefore, the age-related decrease in the induction of hsp70 expression was not due to an age-related accumulation of cells that do not respond to heat shock. Measurements of hsp70 mRNA stability and hsp70 transcription demonstrated that the age-related decline in hsp70 expression arose from a decline in hsp70 transcription. Interestingly, the age-related decline in the induction of hsp70 expression was reversed by caloric restriction; e.g., the induction of hsp70 synthesis, mRNA levels, and nuclear transcription were significantly higher in hepatocytes isolated from old rats fed the caloric restricted diet than in hepatocytes isolated from old rats fed ad libitum. The levels of the heat shock transcription factor in nuclear extracts isolated from heat-shocked hepatocytes were measured in a gel shift assay. Binding of the heat shock transcription factor to the heat shock element decreased with age and was significantly higher in hepatocyte extracts isolated from old rats fed the caloric restriction diet than in those from old rats fed ad libitum. Thus, our study demonstrates that the ability of hepatocytes to respond to hyperthermia and express hsp70 decreases significantly with age and that this decrease occurs at the transcriptional level. In addition, caloric restriction, which retards aging, reversed the age-related decline in the induction of hsp70 transcription in hepatocytes.

Expression of Heat Shock Protein 70 is Altered by Age and Diet at the Level of Transcription

Because heat shock proteins have been shown to play a critical role in protecting cells from hyperthermia and other types of physiological stresses, it was of interest to determine what effect age and caloric restriction have on the ability of cells to regulate the expression of heat shock protein 70 (hsp70), the most prominent and most evolutionarily conserved of the heat shock proteins. Caloric restriction is the only experimental manipulation known to retard aging and increase survival of mammals. The ability of hepatocytes isolated from young/adult (4- to 7-month-old) and old (22- to 28-month-old) male Fischer F344 rats fed ad libitum or a caloric restriction diet (60% of the content of the ad libitum diet) to express hsp70 was determined after a mild heat shock (42.5 degrees C for 30 min). We found that the induction of hsp70 synthesis and mRNA levels by heat shock was 40 to 50% lower in hepatocytes isolated from old rats than in hepatocytes isolated from young rats. Using in situ hybridization, we found that essentially all hepatocytes from the young/adult and old rats expressed hsp70 in response to heat shock; therefore, the age-related decrease in the induction of hsp70 expression was not due to an age-related accumulation of cells that do not respond to heat shock. Measurements of hsp70 mRNA stability and hsp70 transcription demonstrated that the age-related decline in hsp70 expression arose from a decline in hsp70 transcription. Interestingly, the age-related decline in the induction of hsp70 expression was reversed by caloric restriction; e.g., the induction of hsp70 synthesis, mRNA levels, and nuclear transcription were significantly higher in hepatocytes isolated from old rats fed the caloric restricted diet than in hepatocytes isolated from old rats fed ad libitum. The levels of the heat shock transcription factor in nuclear extracts isolated from heat-shocked hepatocytes were measured in a gel shift assay. Binding of the heat shock transcription factor to the heat shock element decreased with age and was significantly higher in hepatocyte extracts isolated from old rats fed the caloric restriction diet than in those from old rats fed ad libitum. Thus, our study demonstrates that the ability of hepatocytes to respond to hyperthermia and express hsp70 decreases significantly with age and that this decrease occurs at the transcriptional level. In addition, caloric restriction, which retards aging, reversed the age-related decline in the induction of hsp70 transcription in hepatocytes.

The effect of age on the synthesis of two heat shock proteins in the hsp70 family.

Hepatocytes isolated from male F344 rats were exposed to elevated temperatures (40 degrees C to 45 degrees C), and the incorporation of [35S]-L-methionine into proteins was measured from fluorograms of two-dimensional polyacrylamide gels. The synthesis of two proteins was induced by temperatures of 40 degrees C to 42.5 degrees C; however, 45 degrees C inhibited the synthesis of all proteins. Based on their apparent molecular weights and pI values and their recognition by a monoclonal antibody to the HSP70 gene family, the two proteins induced by hyperthermia were found to be the highly heat-inducible hsp70 and the constitutive hsc70. Because a heat shock of 42.5 degrees C for 30 minutes had very little effect on cell viability and induced the synthesis of hsp70 and hsc70, this heat shock was used to study the effect of age on the ability of hepatocytes to respond to the stress of hyperthermia. Neither hepatocytes isolated from young adult (5-7 months) nor old (25-27 months) rats synthesized detectable amounts of hsp70 when incubated at 37 degrees C. However, heat shock induced the synthesis of both hsp70 and hsc70 in hepatocytes isolated from young adult and old rats. The induction of hsp70 synthesis was significantly lower (37%) for hepatocytes isolated from old rats compared to hepatocytes isolated from young adult rats. However, neither the basal level nor the induced level of hsc70 synthesis changed significantly with age. Thus, aging resulted in a decrease in the ability of hepatocytes to synthesize hsp70 in response to hyperthermia; this effect, however, was specific for hsp70.

Characteristic induction of 70,000 da-heat shock protein and metallothionein by zinc in HeLa cells.

The synthesis of a 70,000 dalton-heat shock protein (hsp70) is one of several heat shock proteins induced in HeLa cells during the incubation in medium containing zinc sulphate. The synthesis of hsp70 was increased in the presence of 200 microM zinc sulphate and above, but not at 100 microM zinc sulphate. On the other hand, the synthesis of metallothionein was activated in the presence of 100 microM zinc sulphate and above. Uptake of zinc into the cells depended on the concentration of zinc sulphate in the medium. The separation of intracellular zinc into three fractions by gel filtration chromatography; high molecular, metallothionein, and low molecular fractions, showed that zinc in the low molecular weight and metallothionein fractions was elevated in the presence of 100 microM zinc sulphate in the medium, whereas increase in the zinc content of the high molecular weight fraction occurred at 200 microM zinc sulphate and above. Inhibition of cell growth and cellular protein synthesis was also observed at 200 microM zinc sulphate and above, but not at 100 microM. From these findings, since the induction of hsp70 synthesis and inhibition of cell growth occurred concomitantly with the increase of zinc in the high and low molecular weight fractions, hsp70 seemed not to function in the detoxification of zinc, but it may participate in the repair of zinc-induced damage.

Suppression of virus replication by prostaglandin A is associated with heat shock protein synthesis.

The antiviral action of cyclopentenone prostaglandins (PGs) is generally associated with alterations in the synthesis and/or maturation of specific virus proteins. In particular, inhibition of Sendai virus (SV) replication in African green monkey kidney cells by PGA1 has been shown to be a cell-mediated event, due to alterations in SV protein glycosylation and accompanied by the induction of a cellular polypeptide of Mr 74K. In this report we identify this protein as a heat shock protein (HSP) related to the major 70K HSP group (HSP70). Induction of HSP70 synthesis by PGA1 was found to be dose-dependent, and an accumulation of HSP70 comparable to that occurring after heat shock could be obtained at concentrations of PGA1 that did not inhibit macromolecular synthesis in uninfected cells, but caused a dramatic block of virus replication in SV-infected cells. Induction of HSP70 by PGA1 occurred at the transcriptional level and was not affected by SV infection. HSP70 synthesis was evident between 2 and 3 h after PGA1 treatment, maximal at 12 h and went back to control levels by 26 h after the addition of PGA1, thus preceding virus protein synthesis. Finally, of several PGs tested, only those which possess antiviral activity induced the synthesis of HSP70. These results, together with the observation that suppression of HSP70 synthesis by actinomycin D also abolishes the PGA1-induced alteration of SV glycoproteins, suggest that HSP70 could play a role in the block of virus replication by cyclopentenone PGs.

Differential heat sensitivity of normal and transformed Syrian golden hamster embryo cells in confluence

The heat sensitivities of normal and transformed cells derived from Syrian golden hamster embryo (SHE) cells were investigated. There were no differences in heat sensitivities between normal and transformed cells when they were exponentially growing. When confluent cultures were heated in situ, only normal cells showed a reduced heat sensitivity, 4-5 times less than that of exponentially growing cells. However, when these confluent cultures were trypsinized, diluted in medium and heated in suspension, the difference disappeared. When mixed cultures of normal and transformed S11 cells in confluence were heated at 43 degrees C, transformed S11 cells were selectively killed. The induction of HSP70 synthesis by heating in normal SHE cells and transformed counterparts were investigated in confluence using monoclonal antibody. The rate of enhanced HSP70 synthesis after heat treatment did not correlate with the difference in heat sensitivities between normal and transformed SHE cells in confluence as measured by cell survival. The results that normal SHE cells become more resistant to heat than their transformed counterparts only in confluence suggest that cell-to-cell contact plays an important role in the heat resistance of normal SHE cells in vivo.