Induction of heat shock protein 27 in rat embryos exposed to hyperthermia

Previously we reported that eight proteins were reproducibly induced in postimplantation rat embryos exposed to a brief heat shock (43°C, 15 min). The major heat-inducible rat embryo protein has now been identified as heat shock protein 72 (Hsp 72). In addition, the induction of Hsp 72 is temporally correlated with induction of thermotolerance. One of the other rat embryo proteins previously shown to be induced by elevated temperature is a heat shock protein of approximately 27 kilodaltons (Hsp 27). In this report we show that this protein is recognized by an antibody directed against a conserved peptide sequence of Hsp 27. Unlike Hsp 72, Hsp 27 is constitutively expressed in the rat embryo in the absence of any thermal stress; however, the level of Hsp 27 is increased approximately 2–3-fold after thermal stress (43°C, 10 min). Immunohistochemical analysis revealed that the constitutively expressed Hsp 27 is localized primarily to cells of the heart, cells that are uniquely resistant to the cytotoxic effects of hyperthermia. After thermal stress, Hsp 27 is expressed in all tissues of the embryo. Finally, our data show that Hsp 27 exists in the rat embryo as three major isoforms indicative of different phosphorylation states. Furthermore, most Hsp 27 in the heart is phosphorylated, whereas in the rest of the embryo, nonphosphorylated Hsp 27 predominates. After thermal stress, levels of phosphorylated isoforms increase dramatically in nonheart tissues of the embryo. Together, these results suggest that Hsp 27 may play a role in the development of thermotolerance in the postimplantation mammalian embryo. © 1996 Wiley-Liss, Inc.     

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

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

Effects of 1.8 GHz radiofrequency field on DNA damage and expression of heat shock protein 70 in human lens epithelial cells

To investigate the DNA damage, expression of heat shock protein 70 (Hsp70) and cell proliferation of human lens epithelial cells (hLEC) after exposure to the 1.8 GHz radiofrequency field (RF) of a global system for mobile communications (GSM). An Xc-1800 RF exposure system was used to employ a GSM signal at 1.8 GHz (217 Hz amplitude-modulated) with the output power in the specific absorption rate (SAR) of 1, 2 and 3 W/kg. After 2 h exposure to RF, the DNA damage of hLEC was accessed by comet assay at five different incubation times: 0, 30, 60, 120 and 240 min, respectively. Western blot and RT-PCR were used to determine the expression of Hsp70 in hLECs after RF exposure. The proliferation rate of cells was evaluated by bromodeoxyuridine incorporation on days 0, 1 and 4 after exposure. The results show that the difference of DNA-breaks between the exposed and sham-exposed (control) groups induced by 1 and 2 W/kg irradiation were not significant at any incubation time point (P > 0.05). The DNA damage caused by 3 W/kg irradiation was significantly increased at the times of 0 and 30 min after exposure (P < 0.05), a phenomenon that could not be seen at the time points of 60, 120 or 240 min (P > 0.05). Detectable mRNA as well as protein expression of Hsp70 was found in all groups. Exposure at SARs of 2 and 3 W/kg for 2 h exhibited significantly increased Hsp70 protein expression (P < 0.05), while no change in Hsp70 mRNA expression could be found in any of the groups (P > 0.05). No difference of the cell proliferation rate between the sham-exposed and exposed cells was found at any exposure dose tested (P > 0.05). The results indicate that exposure to non-thermal dosages of RF for wireless communications can induce no or repairable DNA damage and the increased Hsp70 protein expression in hLECs occurred without change in the cell proliferation rate. The non-thermal stress response of Hsp70 protein increase to RF exposure might be involved in protecting hLEC from DNA damage and maintaining the cellular capacity for proliferation.     

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     

Modulation of heat shock protein response in SH-SY5Y by mobile phone microwaves

AIM: To investigate putative biological damage caused by GSM mobile phone frequencies by assessing electromagnetic fields during mobile phone working. METHODS: Neuron-like cells, obtained by retinoicacid-induced differentiation of human neuroblastoma SH-SY5Y cells, were exposed for 2 h and 4 h to microwaves at 1800 MHz frequency bands. RESULTS: Cell stress response was evaluated by MTT assay as well as changes in the heat shock protein expression (Hsp20, Hsp27 and Hsp70) and caspase-3 activity levels, as biomarkers of apoptotic pathway. Under our experimental conditions, neither cell viability nor Hsp27 expression nor caspase-3 activity was significantly changed. Interestingly, a significant decrease in Hsp20 expression was observed at both times of exposure, whereas Hsp70 levels were significantly increased only after 4 h exposure. CONCLUSION: The modulation of the expression of Hsps in neuronal cells can be an early response to radiofrequency microwaves.     

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.     

烟蚜热激蛋白基因MpHsp70的克隆及在UV-B胁迫下的表达分析

【目的】为探讨烟蚜Myzus persicae适应UV-B胁迫的分子机制。【方法】采用RT-PCR和RACE技术克隆了烟蚜热激蛋白基因Hsp70的全长,利用生物信息学方法分析了其特征;采用实时荧光定量PCR检测了不同时长(0,15,30,60,90和120 min) UV-B胁迫下烟蚜成虫中该基因的相对表达量。【结果】克隆获得烟蚜Hsp70基因并命名为MpHsp70(GenBank登录号:MF509827),该基因全长为2 221 bp,开放阅读框(ORF) 1 965 bp,编码654个氨基酸,蛋白相对分子量为71. 41kD,等电点(p I)为5. 34,末端高度保守序列EEVD显示该蛋白属于胞质热激蛋白。系统进化关系分析表明,MpHsp70与多种昆虫的Hsp70的同源性较高,表现出Hsp70基因的高度保守性。实时荧光定量PCR分析表明,随着UV-B照射时间的延长,烟蚜成虫体内MpHsp70基因的表达量先上升后下降,当照射时间为30 min时表达量最大。【结论】烟蚜MpHsp70基因可以响应UV-B的胁迫,它可能在烟蚜适应UV-B胁迫过程中起到重要作用。     

Localization and Expression of Hsp27 and αB-Crystallin in Rat Primary Myocardial Cells during Heat Stress In Vitro

Neonatal rat primary myocardial cells were subjected to heat stress in vitro, as a model for investigating the distribution and expression of Hsp27 and αB-crystallin. After exposure to heat stress at 42°C for different durations, the activities of enzymes expressed during cell damage increased in the supernatant of the heat-stressed myocardial cells from 10 min, and the pathological lesions were characterized by karyopyknosis and acute degeneration. Thus, cell damage was induced at the onset of heat stress. Immunofluorescence analysis showed stronger positive signals for both Hsp27 and αB-crystallin from 10 min to 240 min of exposure compared to the control cells. According to the Western blotting results, during the 480 min of heat stress, no significant variation was found in Hsp27 and αB-crystallin expression; however, significant differences were found in the induction of their corresponding mRNAs. The expression of these small heat shock proteins (sHsps) was probably delayed or overtaxed due to the rapid consumption of sHsps in myocardial cells at the onset of heat stress. Our findings indicate that Hsp27 and αB-crystallin do play a role in the response of cardiac cells to heat stress, but the details of their function remain to be investigated.     

Selective stimulation of Hsp27 and αB-crystallin but not Hsp70 expression by p38 MAP kinase activation

The levels of Hsp27 and αB-crystallin in C6 rat glioma cells, that had been heated at 43°C for 30 min with a subsequent culture for 16 h at 37°C, were markedly increased. The exposure of the cells to a low concentration (0.1–3 µg/ml) of anisomycin for a few hours after heat stress stimulated the accumulation of the small stress proteins Hsp27 and αB-crystallin, but not that of Hsp70. The levels of mRNAs for Hsp27 and αB-crystallin but not that for Hsp70 increased in cells that had been exposed to heat and subsequently for 2 h to 0.1–3 µg/ml anisomycin. The results of a reporter assay, using an αB-crystallin promotor fused to a luciferase reporter gene, suggested that the increase in level of αB-crystallin mRNA was due to the production of new mRNA. The activation of the binding of heat shock factors to heat shock elements induced in cells that had been heat stressed was barely affected by subsequent exposure to anisomycin at 0.3 µg/ml. The stimulatory effects of anisomycin were also observed in cells that had been exposed to NaAsO₂, or CdCl₂. The active form of p38 mitogen activated protein (MAP) kinase was increased in cell that had been subjected to heat shock and subsequent exposure to 0.3 µg/ml of anisomycin. The heat-induced accumulations of Hsp27 and αB-crystallin were also stimulated by cycloheximide, another stimulator of p38 MAP kinase. SB202190, a specific inhibitor of p38 MAP kinase, suppressed the stimulation by anisomycin of the heat stress-induced expressions of Hsp27 and αB-crystallin. These results suggest that the signal transduction pathway of the stress-induced expressions of Hsp27 and αB-crystallin in C6 glioma cells includes a process that is sensitive to p38 MAP kinase.     

Insulin-induced myocardial protection in isolated ischemic rat hearts requires p38 MAPK phosphorylation of Hsp27

Six hours after insulin treatment, hearts express heat shock protein 70 (Hsp70) and have improved contractile function after ischemia-reperfusion injury. In this study we examined hearts 1 h after insulin treatment for contractile function and for expression of Hsp70 and Hsp27. Adult, male Sprague-Dawley rats were assigned to groups: 1) sham, 2) control, 3) insulin injected (200 microU/g body wt), 4) heat shock treated (core body temperature, 42 degrees C for 15 min), and 5) heat shock and insulin treated. At 1 h after these treatments, hearts were isolated, equilibrated to Langendorff perfusion for 30 min, and then subjected for 30 min no-flow global ischemia (37 degrees C) followed by 2 h of reperfusion. Insulin-treated hearts had significantly increased contractile function compared with control hearts. At 1 h after insulin treatment, a minimal change in Hsp70 and Hsp27 content were detected. By 3 h after insulin treatment, a significant increase in Hsp70, but not Hsp27, was detected by Western blot analysis. By immunofluorescence, minimal Hsp70 was detected in insulin-treated hearts, whereas Hsp27 was detected in all hearts, indicative of its constitutive expression. Phosphospecific isoforms of Hsp27 were detected in insulin-, heat shock-, and heat shock and insulin-treated hearts. After ischemia and reperfusion, the insulin-treated hearts had significantly elevated levels of phosphorylated Hsp27. Inhibition of p38 MAPK with SB-203580 blocked the insulin-induced phosphorylation of Hsp27 and the improved functional recovery. In conclusion, insulin induces an apparent rapid phosphorylation of Hsp27 that is associated with improved functional recovery after ischemia-reperfusion injury.     

The role of heat shock protein 70 in the thermoresistance of prostate cancer cell line spheroids

Heat shock protein 70 (Hsp70), a protein induced in cells exposed to sublethal heat shock, is present in all living cells and has been highly conserved during evolution. The aim of the current study was to determine the role of heat shock proteins in the resistance of prostate carcinoma cell line spheroids to hyperthermia. In vitro, the expression of Hsp70 by the DU 145 cell line, when cultured as monolayer or multicellular spheroids, was studied using Western blotting and enzyme-linked immunosorbent assay methods. The level of Hsp70 in spheroid cultures for up to 26 days at 37 degrees C remained similar to monolayer cultures. However, in samples treated with hyperthermia at 43 degrees C for 120 min, the spheroid cultures expressed a higher level of Hsp70 as compared to monolayer culture. Under similar conditions of heat treatment, the spheroids showed more heat resistance than monolayer cultures as judged by the number of colonies that they formed in suspension cultures. The results suggest that cells cultured in multicellular spheroids showed more heat resistance as compared to monolayer cultures by producing higher levels of Hsp70.     

Heat Shock Modulates Neutrophil Motility in Zebrafish

Heat shock is a routine method used for inducible gene expression in animal models including zebrafish. Environmental temperature plays an important role in the immune system and infection progression of ectotherms. In this study, we analyzed the impact of short-term heat shock on neutrophil function using zebrafish (Danio rerio) as an animal model. Short-term heat shock decreased neutrophil recruitment to localized Streptococcus iniae infection and tail fin wounding. Heat shock also increased random neutrophil motility transiently and increased the number of circulating neutrophils. With the use of the translating ribosome affinity purification (TRAP) method for RNA isolation from specific cell types such as neutrophils, macrophages and epithelial cells, we found that heat shock induced the immediate expression of heat shock protein 70 (hsp70) and a prolonged expression of heat shock protein 27 (hsp27). Heat shock also induced cell stress as detected by the splicing of X-box binding protein 1 (xbp1) mRNA, a marker for endoplasmic reticulum (ER) stress. Exogenous expression of Hsp70, Hsp27 and spliced Xbp1 in neutrophils or epithelial cells did not reproduce the heat shock induced effects on neutrophil recruitment. The effect of heat shock on neutrophils is likely due to a combination of complex changes, including, but not limited to changes in gene expression. Our results indicate that routine heat shock can alter neutrophil function in zebrafish. The findings suggest that caution should be taken when employing a heat shock-dependent inducible system to study the innate immune response.     

Heat shock protein 70 is translocated to lipid droplets in rat adipocytes upon heat stimulation

In mammalian cells, lipid storage droplets contain a triacylglycerol and cholesterol ester core surrounded by a phospholipid monolayer into which a number of proteins are imbedded. These proteins are thought to be involved in modulating the formation and metabolic functions of the lipid droplet. In this study, we show that heat stress upregulates several heat shock proteins (Hsps), including Hsp27, Hsp60, Hsp70, Hsp90, and Grp78, in primary and differentiated adipocytes. Immunostaining and immunoblotting data indicate that among the Hsps examined, only Hsp70 is induced to redirect to the lipid droplet surface in heat-stressed adipocytes. The thermal induction of Hsp70 translocation to lipid droplet does not typically happen in a temperature- or time-dependent manner and occurs abruptly at 30-40 min and rapidly achieves a steady state within 60 min after 40 degrees C stress of adipocytes. Though Hsp70 is co-localized with perilipin on the lipid droplets in stressed adipocytes, immunoprecipitation experiments suggest that Hsp70 does not directly interact with perilipin. Alkaline treatments indicate that Hsp70 associates with the droplet surface through non-hydrophobic interactions. We speculate that Hsp70 might noncovalently associate with monolayer microdomains of the lipid droplet in a manner similar to its interaction with lipid bilayer moieties composed of specific fatty acids. As an acute and specific cellular response to the heat stimulation, accumulation of Hsp70 on adipocytes lipid droplets might be involved in stabilizing the droplet monolayer, transferring nascent proteins to the lipid droplets, or chaperoning denatured proteins on the droplet for subsequent refolding.     

Hsp70 attenuates hypoxia/reoxygenation-induced activation of poly(ADP-ribose) synthetase in the nucleus of adult rat cardiomyocytes

Effects of heat shock protein 70 (Hsp70) translocated to nuclear fraction on hypoxia/reoxygenation injury was examined by using adult cardiomyocytes isolated from rats. Cardiomyocytes were exposed to heat shock at 42°C for 15 min (HS group), and then incubated at 37°C for 6–24 h. Hsp70 production increased and the protein translocated from cytosol to nucleus. The maximum level of Hsp70 in the nuclear fraction was observed 12 h after HS. When cardiomyocytes without exposure to HS (nHS group) were subjected to 120 min hypoxia/15 min reoxygenation (Hypo/Reoxy), post-hypoxic cell viability was approximately 25% of the pre-hypoxic value. A rise in poly(ADP-ribose) synthetase (PARS) activity in the nuclear fraction was observed in nHS group, associated with an increase in polyADP-ribosylated protein. In contrast, post-hypoxic cell viability of HS group was approximately 60% of the pre-hypoxic value. Hypo/reoxy-induced rise in PARS activity and increase in polyADP-ribosylated protein were attenuated in HS group. To confirm the relationship between an increase in cell viability after Hypo/Reoxy and attenuation of PARS activation, cardiomyocytes without exposure to HS were subjected to Hypo/Reoxy in the presence of 1 mM 3-aminobenzamide, an inhibitor of PARS. Treatment of cells with 3-aminobenzamide attenuated Hypo/Reoxy-induced decrease in cell viability. These results suggest that Hsp70 translocated into nucleus after HS may attenuate PARS activation during Hypo/Reoxy, leading to the cytoprotection of cardiomyocytes against Hypo/Reoxy injury.     

Deoxyribonucleic acid damage induced by doxorubicin in peripheral blood mononuclear cells: possible roles for the stress response and the deoxyribonucleic acid repair process

Doxorubicin is an antineoplastic drug widely used in cancer treatment. However, many tumors are intrinsically resistant to the drug or show drug resistance after an initial period of response. Among the different molecules implicated with doxorubicin resistance are the heat shock proteins (Hsps). At present we do not know with certainty the mechanism(s) involved in such resistance. In the present study, to advance our knowledge on the relationship between Hsps and drug resistance, we have used peripheral blood mononuclear cells obtained from healthy nonsmoker donors to evaluate the capacity of a preliminary heat shock to elicit the Hsp response and to establish the protection against the deoxyribonucleic acid (DNA) damage induced by doxorubicin. DNA damage and repair were determined using the alkaline comet assay. We also measured the expression of Hsp27, Hsp60, Hsp70, Hsp90, hMLH1, hMSH2, and proliferating cell nuclear antigen by immunocytochemistry. The damage induced by doxorubicin was more efficiently repaired when the cells were previously heat shocked followed by a resting period of 24 hours before drug exposure, as shown by (1) the increased number of undamaged cells (P < 0.05), (2) the increased DNA repair capacity (P < 0.05), and (3) the high expression of the mismatch repair (MMR) proteins hMLH1 and hMSH2 (P < 0.05). In addition, in the mentioned group of cells, we confirmed by Western blot high expression levels of Hsp27 and Hsp70. We also noted a nuclear translocation of Hsp27 and mainly of Hsp70. Furthermore, inducible Hsp70 was more expressed in the nucleus than Hsc70, showing a possible participation of Hsp70 in the DNA repair process mediated by the MMR system.     

Production of heat shock proteins, cytokines, and nitric oxide in toxic stress

Expression of heat shock proteins Hsp27, Hsp90, and Hsp70 and production of tumor necrosis factors (TNF-alpha, TNF-beta), interferon-gamma (IFN-gamma), interleukin-2, -3, -6, and nitric oxide (NO) were studied under conditions of acute and chronic intoxication of animals with lipopolysaccharides. Injection of endotoxin increased expression of heat shock proteins Hsp70 and Hsp90-alpha in mouse cells. Acute toxic stress also provoked a sharp increase in the production of TNF-alpha, TNF-beta, and NO in mouse cells. The production of other cytokines (interleukins and IFN-gamma) was changed insignificantly. In the model of chronic toxic stress, changes in the production of Hsp70, Hsp90, TNF, and NO were followed during 11 days after the beginning of the toxin injections. The expression of Hsp70 and Hsp90 in acute stress was significantly higher than at the final stage of the chronic exposure. The changes in the TNF and NO productions, on one hand, and the production of heat shock proteins, on the other hand, were synchronous. The findings indicate that repeated injections of increasing endotoxin doses result in a decreased ability of the body cells to respond to stress by overproduction of heat shock proteins, TNF, and NO.     

Heat shock response and acute lung injury

All cells respond to stress through the activation of primitive, evolutionarily conserved genetic programs that maintain homeostasis and assure cell survival. Stress adaptation, which is known in the literature by a myriad of terms, including tolerance, desensitization, conditioning, and reprogramming, is a common paradigm found throughout nature, in which a primary exposure of a cell or organism to a stressful stimulus (e.g., heat) results in an adaptive response by which a second exposure to the same stimulus produces a minimal response. More interesting is the phenomenon of cross-tolerance, by which a primary exposure to a stressful stimulus results in an adaptive response whereby the cell or organism is resistant to a subsequent stress that is different from the initial stress (i.e., exposure to heat stress leading to resistance to oxidant stress). The heat shock response is one of the more commonly described examples of stress adaptation and is characterized by the rapid expression of a unique group of proteins collectively known as heat shock proteins (also commonly referred to as stress proteins). The expression of heat shock proteins is well described in both whole lungs and in specific lung cells from a variety of species and in response to a variety of stressors. More importantly, in vitro data, as well as data from various animal models of acute lung injury, demonstrate that heat shock proteins, especially Hsp27, Hsp32, Hsp60, and Hsp70 have an important cytoprotective role during lung inflammation and injury.     

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.