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PubMed2002

A 72 kDa heat shock protein is protective against the selective vulnerability of CA1 neurons and is essential for the tolerance exhibited by CA3 neurons in the hippocampus.

The correlation between the expression of a 72 kDa heat shock protein and vulnerability of hippocampal CA1, CA3, and dentate gyrus regions to glutamate toxicity was investigated using a highly specific antisense oligonucleotide technique. Glutamate (1 mM, 15 min) caused region-dependent neuronal damage in cultured hippocampal slices 24 h after exposure and the most severe damage was observed in CA1. When slices were heat-shocked (43.5 degrees C, 30 min) before exposure to glutamate, neuronal damage in CA1 was attenuated. The strongest protection was observed when the interval between the heat shock and the exposure to glutamate was 3 days, which coincided with the maximal induction of a 72 kDa heat shock protein in neurons. When the expression of a 72 kDa heat shock protein was suppressed by the antisense oligonucleotide, the protective effect of the heat shock was completely inhibited. Glutamate itself also induced a 72 kDa heat shock protein in neurons, region-dependently, 24 h after the exposure. The signal of a 72 kDa heat shock protein in CA3 and dentate gyrus was significantly stronger than that in CA1. When the antisense oligonucleotide was applied, the damage in CA3 and dentate gyrus was exaggerated dose-dependently, and this effect was more remarkable in CA3 than in the dentate gyrus. Based on these data, we concluded that: (i) a 72 kDa heat shock protein has a protective effect against the selective vulnerability of CA1 neurons, (ii) a 72 kDa heat shock protein is an essential factor for the tolerance exhibited by CA3 neurons, and (iii) dentate gyrus tolerance is based on mechanisms other than those mediated through a 72 kDa heat shock protein.

Animals↗
PubMed1999

A short lived protein involved in the heat shock sensing mechanism responsible for stress-activated protein kinase 2 (SAPK2/p38) activation.

The stress-activated protein kinase 2 (SAPK2/p38) is activated by various environmental stresses and also by a vast array of agonists including growth factors and cytokines. This implies the existence of multiple proximal signaling pathways converging to the SAPK2/p38 activation cascade. Here, we show that there is a sensing mechanism highly specific to heat shock for activation of SAPK2/p38. After mild heat shock, cells became refractory to reinduction of the SAPK2/p38 pathway by a second heat shock. This was not the result of a toxic effect because the cells remained fully responsive to reinduction by other stresses, cytokines, or growth factors. Neither the activity of SAPK2/p38 itself nor the accumulation of the heat shock proteins was essential in the desensitization process. The cells were not desensitized to heat shock by other treatments that activated SAPK2/p38. Moreover, inhibiting SAPK2/p38 activity during heat shock did not block desensitization. Also, overexpression of HSP70, HSP27, or HSP90 by gene transfection did not cause desensitization, and inhibiting their synthesis after heat shock did not prevent desensitization. Desensitization rather appeared to be linked closely to the turnover of a putative upstream activator of SAPK2/p38. Cycloheximide induced a progressive and eventually complete desensitization. The effect was specific to heat shock and minimally affected activation by other stress inducers. Inhibiting protein degradation with MG132 caused the constitutive activation of SAPK2/p38, which was blocked by a pretreatment with either cycloheximide or heat shock. The results thus indicate that there is a sensing pathway highly specific to heat shock upstream of SAPK2/p38 activation. The pathway appears to involve a short lived protein that is the target of rapid successive up- and down-regulation by heat shock.

Amino Acid Sequence↗
PubMedDate not supplied

Heat-induced apoptosis via caspase-3 activation in tumour cells carrying mutant p53.

Apoptosis plays an important role in heat-induced cell death. However, the mechanism of heat-induced apoptosis has not yet been elucidated. In the present study, the signal transduction pathway underlying heat-induced apoptosis was investigated in heat-resistant HeLa cells carrying mutant p53 gene and heat sensitive HeLa cells that had been transduced with an antisense TNF gene. Induction of mutant p53, but not p21/WAF-1, was observed after heat treatment of both the resistant and sensitive cells. Heat-induced cytotoxicity was not inhibited in either cells with interleukin-1beta-converting enzyme (ICE: caspase-1) like protease inhibitor Ac-YVAD-CHO. In contrast, there was 48% and 63% inhibition of cytotoxicity in HeLa and transfectants, respectively, with a caspase-3 inhibitor (Ac-DEVD-CHO). Heat-induced apoptosis was also prevented by administration of Ac-DEVD-CHO in both cells. In addition, an augmentation of heat-induced cytotoxicity in transfectants was almost completely inhibited by Ac-DEVD-CHO. Further, caspase-3 mRNA expression was increased remarkably in heat-treated HeLa cells and transfectants. Taken together, these results suggest that activation of caspase-3 is involved in the signal transduction pathway of heat-induced apoptosis of the tumour cells carrying mutant p53.

Base Sequence↗
PubMedDate not supplied

Selective enhancement of cytochrome p-450 activity in rat hepatocytes by in vitro heat shock.

We investigated the effect of heat shock on cytochrome P-450 activity in rat hepatocytes and report a significant, selective, and time-dependent enhancement of cytochrome P-450 activity in heatshocked hepatocytes. Stable long-term cultures of rat hepatocytes were heat shocked (42.5 degrees C) for 1 to 3 h and allowed to recover at 37 degrees C. Cytochrome P-450-dependent ethoxyresorufin O-dealkylase (EROD) and benzyloxyresorufin O-dealkylase (BROD) activities were measured up to 48 h after heat shock treatment. In general, the optimal heat shock exposure time was between 2 and 3 h. BROD activity (induced by sodium phenobarbital) increased approximately 6-fold in hepatocytes heat shocked for 3 h in comparison with hepatocytes maintained at 37 degrees C. EROD activity (induced by 3-methylcholanthrene) increased 2-fold on exposure to heat shock for 2 h. The expression of inducible heat shock proteins Hsp70 and Hsp32 was verified by Western immunoblot analyses. In the absence of the appropriate inducer, heat shock treatment did not enhance cytochrome P-450 activity. Furthermore, enhanced P-450 enzyme activity was delayed for heat-shocked hepatocytes. It is hypothesized that heat shock treatment attenuates the negative effects triggered by the addition of the toxic inducers and possibly stabilizes the levels of cytochrome P-450 proteins. These results suggest that heat shock treatment may be used to enhance the functionality of hepatocytes, specifically, in bioartificial liver assist devices.

Albumins↗
PubMed1999

Reduction of hepatic microcirculatory failure caused by normothermic ischemia/reperfusion-induced injury by means of heat shock preconditioning.

Transient sublethal hyperthermia and the recovery from this exposure to heat (heat shock preconditioning) provides a cytoprotective effect on oxidative insults through an intracellular protective response, heat shock response. The impact of heat shock preconditioning on hepatic microvascular failure, which is a causative determinant of ischemia/reperfusion-induced injury of the liver, was investigated by using intravital fluorescence microscopy. In Sprague-Dawley rats, normothermic ischemia was induced by totally clamping the hepatoduodenal ligament for 20 min, followed by 120 min of reperfusion. Heat shock preconditioning was performed by whole-body hyperthermia (42 degrees C for 15 min) and subsequent 48 h recovery. In accordance with the prominent induction of heat shock protein 70 in the liver tissue, the postischemic decrease in sinusoidal perfusion rate and sinusoidal diameter, and the postischemic increase in the number of stagnant leukocytes in sinusoids and adherent leukocytes in postsinusoidal venules were significantly attenuated in the heat shock-treated animals. Furthermore, liver enzyme release (glutamate pyruvate transaminase and alpha-glutathione S-transferase) was significantly reduced and postischemic deterioration of bile production was attenuated. The 7-day survival rate after 20-minute ischemia was significantly improved from 50% to 80% (heat shock-nontreated group vs. heat shock-treated group, P < 0.05). These results indicate that heat shock preconditioning attenuates ischemia/reperfusion-induced hepatic injury by preventing postischemic microvascular disturbances, and that its protective effect is circumstantially associated with the concomitant induction of heat shock protein 70.

Alanine Transaminase↗
PubMed2004

Heat shock inhibition of lipopolysaccharide-mediated tumor necrosis factor expression is associated with nuclear induction of MKP-1 and inhibition of mitogen-activated protein kinase activation.

OBJECTIVE: Application of heat shock before an inflammatory stimulus often results in an attenuated response to that stimulus. As a result, it has become increasingly appreciated that heat shock may induce cross-tolerance to a variety of stimuli based on in vitro and in vivo models. Circulating peripheral blood monocytes are key mediators of cytokine release following endotoxin challenge. The mitogen-activated protein kinases play a key role in the transcriptional regulation of this response including expression of tumor necrosis factor. As such, counterregulatory phosphatases that target mitogen-activated protein kinase may play a role in this heat shock-mediated effect. We hypothesized that prior heat shock to monocytes would induce a phosphatase, MKP-1, that regulated mitogen-activated protein kinase activity and subsequently conferred cross-tolerance to lipopolysaccharide stimulation. DESIGN: Experimental. SETTING: University research foundation laboratory. SUBJECTS: THP-1 human monocyte cell line. INTERVENTIONS: THP-1 cells were exposed to either heat shock (43 degrees C, 1 hr) or normothermia (37 degrees C, 1 hr) and allowed to recover before stimulation with endotoxin (lipopolysaccharide). MEASUREMENTS AND MAIN RESULTS: Induction of a heat shock response was determined by heat shock protein-70 expression. Tumor necrosis factor and interleukin-10 were measured by enzyme-linked immunosorbent assay to assess heat shock inhibition of lipopolysaccharide-induced gene expression. The effect of heat shock on lipopolysaccharide-mediated activation of the p38 and ERK kinases was examined by measuring phospho-specific isoforms of p38 and ERK1/2 and correlated to in vitro kinase activity. Confirmatory data were generated from experiments employing either pharmacologic inhibition or genetic deletion of MKP-1. Heat shock induced the nuclear localized phosphatase, MKP-1, that attenuated p38 and ERK kinase activity resulting in significantly diminished tumor necrosis factor expression in response to lipopolysaccharide. CONCLUSIONS: The effect of heat shock on decreasing the tumor necrosis factor response to lipopolysaccharide is conferred by induction of MKP-1, which negatively regulates p38 and ERK kinases. Modulation of phosphatase activity may be a potential strategy for attenuating acute inflammatory responses.

Animals↗
PubMed1997

Brief heat shock treatment induces a long-lasting alteration in the glycolipid receptor binding specificity and growth rate of Haemophilus influenzae.

After brief heat shock treatment, clinical strains of nontypeable Haemophilus influenzae show a long-lasting change in the binding specificity for glycolipids and a markedly increased growth rate in vitro. Non-heat-shocked H. influenzae specifically binds to phosphatidylethanolamine (PE), gangliotetraosylceramide (Gg4), and gangliotriosylceramide (Gg3) and binds minimally to sulfatoxygalactosylceramide (SGC; also called sulfatide). After a 5-min heat shock at 42 degrees C, strains of H. influenzae showed a marked increase in binding to SGC and acquired the ability to bind to sulfatoxygalactosylglycerol (SGG) in thin-layer chromatography overlays. Additionally, heat-shocked H. influenzae cells showed an increased growth rate (twofold). Increased sulfatide binding and growth rate were retained for approximately 60 generations, after which the heat-shocked organisms reverted to their original glycolipid binding pattern (i.e., PE, Gg3, and Gg4) and growth rate. Such organisms could then be reexposed to heat, and the heat shock phenotype would be reestablished. After exposure of the organisms to brief heat shock, Western blotting of a surface extract of H. influenzae with anti-bovine-brain hsp-70 monoclonal antibody showed an increase in two protein bands at 82 and 60 kDa. This antibody was a potent inhibitor of the binding of heat-shocked H. influenzae to SGC and SGG but had no effect on PE, Gg3, or Gg4 binding in vitro. In contrast, an antibody against an H. influenzae PE-Gg3-Gg4-binding adhesin that was recently identified (J. Busse, E. Hartmann, and C. A. Lingwood, J. Infect. Dis. 175:77-83, 1996) selectively inhibited the organism's binding to PE and Gg3. This indicates that cell surface hsp-70-related heat shock proteins can mediate H. influenzae attachment to sulfoglycolipids following heat shock. We suggest that such increased binding to sulfated glycolipids may be a response to fever following H. influenzae infection in humans.

Antibodies, Blocking↗
PubMed1992

Characterization of the Agrobacterium tumefaciens heat shock response: evidence for a sigma 32-like sigma factor.

We have characterized the heat shock response of Agrobacterium tumefaciens and compared it with the well-characterized Escherichia coli heat shock response. Four major heat shock proteins with apparent molecular masses of 98, 75, 65, and 20 kDa were identified by pulse-labelling cultures after temperature upshift. The three largest proteins comigrated with proteins that were antigenically related to the E. coli heat shock proteins sigma 70, DnaK, and GroEL, respectively. The heat shock proteins were also strongly induced by ethanol and cadmium chloride and were mildly induced by mitomycin C. To determine whether the A. tumefaciens heat shock regulatory system was similar to that of E. coli, we introduced the E. coli dnaK gene into A. tumefaciens. The E. coli DnK protein was expressed in A. tumefaciens, and its synthesis was induced after heat shock. Primer extension analysis of the E. coli dnaK gene in A. tumefaciens indicated that transcription initiated from one or possibly both of the E. coli heat shock promoters. We conclude that A. tumefaciens has a heat shock response similar to that of E. coli, in that (i) similar proteins are induced by heat shock, (ii) synthesis of these proteins is induced in response to similar stimuli, and (iii) A. tumefaciens can recognize an E. coli heat shock promoter, suggesting that A. tumefaciens has a sigma factor similar to sigma 32.

Agrobacterium tumefaciens↗
PubMed2002

Cold shock response in sporulating Bacillus subtilis and its effect on spore heat resistance.

Cold shock and ethanol and puromycin stress responses in sporulating Bacillus subtilis cells have been investigated. We show that a total of 13 proteins are strongly induced after a short cold shock treatment of sporulating cells. The cold shock pretreatment affected the heat resistance of the spores formed subsequently, with spores heat killed at 85 or 90 degrees C being more heat resistant than the control spores while they were more heat sensitive than controls that were heat treated at 95 or 100 degrees C. However, B. subtilis spores with mutations in the main cold shock proteins, CspB, -C, and -D, did not display decreased heat resistance compared to controls, indicating that these proteins are not directly responsible for the increased heat resistance of the spores. The disappearance of the stress proteins later in sporulation suggests that they cannot be involved in repairing heat damage during spore germination and outgrowth but must alter spore structure in a way which increases or decreases heat resistance. Since heat, ethanol, and puromycin stress produce similar proteins and similar changes in spore heat resistance while cold shock is different in both respects, these alterations appear to be very specific.

Bacillus subtilis↗
PubMed2003

Inactivation of dual-specificity phosphatases is involved in the regulation of extracellular signal-regulated kinases by heat shock and hsp72.

Extracellular signal-regulated kinase 1 (ERK1) and ERK2 (ERK1/2) dramatically enhance survival of cells exposed to heat shock. Using Cos-7 cells and primary human fibroblasts (IMR90 cells), we demonstrated that heat shock activates ERKs via two distinct mechanisms: stimulation of the ERK-activating kinases, MEK1/2, and inhibition of ERK dephosphorylation. Under milder heat shock conditions, activation of ERKs proceeded mainly through stimulation of MEK1/2, whereas under more severe heat shock MEK1/2 could no longer be activated and the inhibition of ERK phosphatases became critical. In Cos-7 cells, nontoxic heat shock caused rapid inactivation of the major ERK phosphatase, MKP-3, by promoting its aggregation, so that in cells exposed to 45 degrees C for 20 min, 90% of MKP-3 became insoluble. MKP-3 aggregation was reversible and, 1 h after heat shock, MKP-3 partially resolubilized. The redistribution of MKP-3 correlated with an increased rate of ERK dephosphorylation. Similar heat-induced aggregation, followed by partial resolubilization, was found with a distinct dual-specificity phosphatase MKP-1 but not with MKP-2. Therefore, MKP-3 and MKP-1 appeared to be critical heat-labile phosphatases involved in the activation of ERKs by heat shock. Expression of the major heat shock protein Hsp72 inhibited activation of MEK1/2 and prevented inactivation of MKP-3 and MKP-1. Hsp72DeltaEEVD mutant lacking a chaperone activity was unable to protect MKP-3 from heat inactivation but interfered with MEK1/2 activation similar to normal Hsp72. Hence, Hsp72 suppressed ERK activation by both protecting dual-specificity phosphatases, which was dependent on the chaperone activity, and suppressing MEK1/2, which was independent of the chaperone activity.

Animals↗
PubMed1995

Catalase inhibition with 3-amino-1,2,4-triazole does not abolish infarct size reduction in heat-shocked rats.

BACKGROUND: Recent studies have shown that improved myocardial salvage after heat-shock pretreatment correlates with the amount of induced cardiac heat-shock protein (HSP)72. However, heat shock also induces myocardial catalase activity, potentially reducing free radical-mediated ischemic injury. The aim of the present study was to determine whether catalase inhibition with 3-amino-1,2,4-triazole (3-AT) abolishes the reduction of infarct size conferred by heat-shock treatment in rats. METHODS AND RESULTS: Myocardial catalase activity was measured in both heat-shocked and control rats 60 minutes after either 3-AT (1000 mg/kg IV) or saline infusion. In separate experiments, heat-shocked and control rats were treated with 3-AT or saline 60 minutes before being subjected to 35 minutes of left coronary artery occlusion and 120 minutes of reperfusion. Infarct size was determined by dual perfusion with triphenyltetrazolium chloride and phthalocyanine blue dye. Heat-shock treatment significantly increased myocardial catalase compared with control animals (180.5 +/- 4.8, n = 6, versus 86.2 +/- 14.7, n = 5, units/g wet wt; P < .05). Treatment with 3-AT significantly reduced myocardial catalase activity in both heat-shocked and control animals (29.6 +/- 5.7, n = 5, and 36.4 +/- 15.3, n = 6, respectively). Heat-shock treatment significantly reduced infarct size in rats that were both treated and untreated with 3-AT compared with respective control groups (22.5 +/- 3.7%, n = 26, 28.2 +/- 4.0%, n = 22, 52.0 +/- 3.0%, n = 23, and 48.6 +/- 3.2%, n = 26, respectively; P < .0001 for both heat-shocked groups versus both control groups; infarct mass/risk area mass x 100). CONCLUSIONS: Catalase inhibition with 3-AT does not abolish the reduction of infarct size in heat-shocked rats.

Amitrole↗
PubMed1997

Potentiation of heat stress-induced hsp70 expression in vivo by aspirin.

Studies in cultured cells have demonstrated that non-steroidal anti-inflammatory agents can potentiate heat-induced hsp70 expression through activation of HSF1 to a DNA binding state. We investigated the influence of aspirin on hsp70 expression in intact rats subjected to heat stress. Rats were injected intraperitoneally either with aspirin (100 mg/kg) or vehicle alone, 60 min prior to their placement at 37 degrees C or room temperature for 30 min. hsp70 mRNA expression was analyzed in lung, liver and kidney isolated from animals assigned to one of four different treatment paradigms; untreated controls, heat, aspirin, and aspirin-plus-heat. Comparison of hsp70 expression in the treatment groups revealed that in all tissues examined, aspirin-plus-heat treatment resulted in 3-4 fold higher levels of hsp70 mRNA relative to those seen with heat treatment alone. Little or no hsp70 mRNA expression was detected in the unheated groups, regardless of aspirin treatment. In keeping with the mRNA expression, Hsp70 protein levels were also elevated in aspirin-plus-heat treated animals. Aspirin treatment did not alter hsp70 protein expression in the absence of heat. In contrast to in vitro observations, aspirin treatment in vivo did not alter HSF1 DNA binding properties. Core body temperature measurements revealed that aspirin pretreatment enhanced the rise in body temperature seen in response to heat treatment. This increased hyperthermic response to heat stress probably accounts for the potentiation of hsp70 expression observed in aspirin-plus-heat treated rats. Given the widespread use of aspirin in humans within a dose range comparable to that used here, our findings are likely to have important physiological consequences.

Animals↗
PubMed2003

[Expression of HSP70 and P-glycoprotein (P-gp) in human hepatocarcinoma HepG2 cells induced by heat shock and inhibiting effect of quercetin on them].

BACKGROUND & OBJECTIVE: Heat shock protein 70 (HSP70) has been thought to inhibit apoptosis and reduce the effectiveness of hyperthermal therapy and chemotherapy on cancer. However, the relationship between HSP70 and the drug resistance to chemotherapy has not been definited. P-glycoprotein (P-gp) was a kind of protein that could decrease the effectiveness of chemotherapy. In order to explore the relationship between HSP70 and P-gp, the human hepatocarcinoma line HepG2 cells was induced by heat shock in vitro, and the inhibiting effect of quercetin on them was observed at the same time to seek the method increasing the effectiveness of hyperthermal therapy on hepatocarcinoma. METHODS: HepG2 cells were bathed in water at 42 degrees C stably for 90 minutes. The expression of HSP70 and P-gp were detected at 0, 2, 4, 8, 12, and 24 hours after 42 degrees C heat shock using immunocytochemistry and flow cytometry (FCM). Furthermore, at 4 hours later after heat shock, they were also detected in HepG2 cells which had been pretreated by quercetin in different concentrations within an hour before 42 degrees C heat shock. RESULTS: (1) Immunocytochemistry showed that HSP70 overexpressed in the cells and "moved" from cytoplasm to nucleus and nucleolus after heat shock. The percentage of HSP70 positive cells came to the climax (11.47%) at 4th hour and increased about 2 folds compared with that of the control group (6.64%) (P< 0.01). At the 24th hour after heat shock, it declined to a low level. The percentage of cells with P-gp positive went up following HSP70 after heat shock. It came to the climax(96.31%) at the 12th hour. And it increased about 3 folds compared with that of the control group(33.95%) (P< 0.01). (2) The overexpression of HSP70 and P-gp induced by heat shock could be inhibited effectively by quercetin in a dose-dependent manner, especially by quercetin at the concentrations of 100- 200 micromol/L(P< 0.01). CONCLUSION: (1) The overexpression of HSP70 and P-gp in HepG2 cells can be induced by heat shock and P-gp maybe has a correlation with HSP70. (2) The overexpression of HSP70 and P-gp in HepG2 cells induced by heat shock can be inhibited by quercetin.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1997

The phorbol ester 12-O-tetradecanoylphorbol 13-acetate enhances the heat-induced stress response.

Induction of heat shock gene expression is mediated by specific heat shock transcription factors (HSFs), but the signaling pathways leading to activation of HSFs are poorly understood. To elucidate whether protein kinase C-responsive signaling pathways could be involved in the regulation of heat shock gene expression, we have examined the effects of the protein kinase C activator 12-O-tetradecanoylphorbol 13-acetate (TPA) on the heat-induced stress response in K562 cells. We demonstrate that TPA treatment markedly enhances heat shock gene expression during heat stress, although TPA alone does not induce the heat shock response. This TPA-mediated enhancement can initially be detected as an accelerated acquisition of DNA binding and transcriptional activity of HSF1 resulting in elevated Hsp70 protein concentrations. In the presence of TPA, the attenuation of HSF1 DNA binding activity during continuous exposure to heat shock occurs more rapidly and in concert with the appearance of newly synthesized Hsp70, which supports earlier studies on the autoregulatory role of Hsp70 in deactivation of HSF1. During heat stress, a correlation between the hyperphosphorylation of HSF1 and its transcriptional activity was observed, in both the presence and the absence of TPA. Our results show that the heat-induced stress response can be significantly modulated by activation of protein kinase C-responsive signaling pathways.

DNA↗
PubMed2005

Hsp27 enhances recovery of splicing as well as rephosphorylation of SRp38 after heat shock.

A heat stress causes a rapid inhibition of splicing. Exogenous expression of Hsp27 did not prevent that inhibition but enhanced the recovery of splicing afterward. Another small heat shock protein, alphaB-crystallin, had no effect. Hsp27, but not alphaB-crystallin, also hastened rephosphorylation of SRp38-dephosphorylated a potent inhibitor of splicing-after a heat shock, although it did not prevent dephosphorylation by a heat shock. The effect of Hsp27 on rephosphorylation of SRp38 required phosphorylatable Hsp27. A Hsp90 client protein was required for the effect of Hsp27 on recovery of spicing and on rephosphorylation of SRp38. Raising the Hsp70 level by either a pre-heat shock or by exogenous expression had no effect on either dephosphorylation of SRp38 during heat shock or rephosphorylation after heat shock. The phosphatase inhibitor calyculin A prevented dephosphorylation of SRp38 during a heat shock and caused complete rephosphorylation of SRp38 after a heat shock, indicating that cells recovering from a heat shock are not deficient in kinase activity. Together our data show that the activity of Hsp27 in restoring splicing is not due to a general thermoprotective effect of Hsp27, but that Hsp27 is an active participant in the (de)phosphorylation cascade controlling the activity of the splicing regulator SRp38.

Cell Cycle Proteins↗
PubMed2001

The effect of co-overproduction of DnaK/DnaJ/GrpE and ClpB proteins on the removal of heat-aggregated proteins from Escherichia coli DeltaclpB mutant cells--new insight into the role of Hsp70 in a functional cooperation with Hsp100.

The effect of overproduction of the Hsp70 system proteins (DnaK, DnaJ, GrpE) and/or ClpB (Hsp100) from plasmids on the process of formation and removal of heat-aggregated proteins from Escherichia coli cells (the S fraction) was investigated by sucrose density gradient centrifugation. Two plasmids were employed: pKJE7 carrying the dnaK/dnaJ/grpE genes under the control of the araB promoter and pClpB carrying the clpB gene under the control of its own promoter (sigma(32)-dependent). In the wild-type cells the S fraction after 15 min of heat shock amounted to 21% of cellular insoluble proteins (IP), and disappeared 10 min after transfer of the culture to 37 degrees C. In contrast to this, in the clpB mutant the S fraction was larger (35% IP) and its elimination was retarded, nearly 60% of the aggregated proteins remained stable 30 min after heat shock. This result points to the importance of ClpB in removal of the heat-aggregated proteins from cells. Overproduction of the Hsp70 system proteins (exceeding by about 1.5-fold that of wild-type) in wild-type and DeltaclpB cells completely prevented the formation of the S fraction during heat shock. Overproduction of ClpB (exceeding by about eight-fold that of wild-type) in the same background did not prevent protein aggregation after heat shock and only partly compensated for the effect of the mutation in the clpB gene. Monitoring the S fraction during co-production of DnaK/DnaJ/GrpE and ClpB in the DeltaclpB mutant revealed that both the levels of expression and the ratios of ClpB to Hsp70 system proteins had a significant effect on the formation and removal of protein aggregates in heat-shocked E. coli cells. In the presence of excess ClpB, an increase in the levels of DnaK, DnaJ and GrpE was required to prevent aggregate formation upon heat shock or to efficiently remove protein aggregates after heat shock. Therefore, it is supposed that a high level of ClpB under some conditions, especially at insufficient levels of Hsp70 system proteins, may support protein aggregation resulting from heat shock and may lead to stabilization of hydrophobic aggregates.

Bacterial Proteins↗
PubMed2002

Proteasome inhibitors induce heat shock response and increase IL-6 expression in human intestinal epithelial cells.

In previous studies, the heat shock response, induced by hyperthermia or sodium arsenite, increased interleukin (IL)-6 production in intestinal mucosa and cultured human enterocytes. A novel way to induce the heat shock response, documented in other cell types, is treatment with proteasome inhibitors. It is not known if proteasome inhibition induces heat shock in enterocytes or influences IL-6 production. Here we tested the hypothesis that treatment of cultured Caco-2 cells, a human intestinal epithelial cell line, with proteasome inhibitors induces the heat shock response and stimulates IL-6 production. Treatment of Caco-2 cells with one of the proteasome inhibitors MG-132 or lactacystin activated the transcription factor heat shock factors (HSF)-1 and -2 and upregulated cellular levels of the 72-kDa heat shock protein HSP-72. The same treatment resulted in increased gene and protein expression of IL-6, a response that was blocked by quercetin. Additional experiments revealed that the IL-6 gene promoter contains a HSF-responsive element and that the IL-6 gene may be regulated by the heat shock response. The present results suggest that proteasome inhibition induces heat shock response and IL-6 production in enterocytes and that IL-6 may be a heat shock-responsive gene, at least under certain circumstances. The observations are important considering the multiple biological roles of IL-6, both locally in the gut mucosa and systemically, and considering recent proposals in the literature to use proteasome inhibitors in the clinical setting to induce the heat shock response.

Acetylcysteine↗
PubMed1994

Inhibitors of tyrosine and Ser/Thr phosphatases modulate the heat shock response.

Following heat shock the expression of heat shock genes is regulated by the heat shock transcription factor, HSF, known to bind to arrays of the heat shock element, NGAAN, upstream of the heat shock genes. Phosphorylation of HSF is necessary for its activation. We report that the treatment of Chinese hamster HA-1 cells with 250 nM of okadaic acid (OA), a ser/thr phosphatase inhibitor, leads to an increase in activated HSF after heat shock. This is followed by the activation of the transcription of heat shock genes as assayed by the increase in the synthesis of beta-galactosidase in an HA-1 cell line containing the heat shock promoter ligated to the beta-galactosidase gene. To investigate the specificity of OA, we used other phosphatase inhibitors. We found that treatment of HA-1 cells with 500 microM of sodium vanadate, an inhibitor of tyr/phosphatases, resulted in a three to fivefold reduction in HSF activation and binding to the heat shock element following heat shock. Such reduction in HSF activation virtually abolished beta-galactosidase induction. Reduced HSP synthesis was further confirmed by SDS-PAGE and Western blot analysis using anti-HSP-70 and 28 antibodies. Sodium vanadate treatment of heat shocked cells greatly reduced levels of thermotolerance. These results show that ser/thr and specifically tyr/phosphatase inhibitors modulate the signal transduction pathway of HSF activation.

Animals↗