Effects of in vitro heat shock on immune cells in diet-induced obese mice

Obesity has been associated with impaired immune responses and inflammation. The mechanisms underlying these immune disturbances in obesity are not yet clarified. This study investigated the effects of in vitro heat shock (HS) on immune cells from the point of view of thymocyte apoptosis and T-cell mitogen-stimulated splenocyte cytokine production as well as the heat shock protein 70 (HSP70) protein levels in diet-induced obese mice to explore a possible association between the disturbance of T cell immunity and HS response in obesity. Obese mice had increased apoptotic and necrotic thymocytes populations and increased splenocyte cytokine production of both proinflammatory and anti-inflammatory cytokines compared with lean mice. The in vitro HS at 42 °C decreased the rate of live cells in thymocytes, and the degree of the decrease was larger in obese mice compared with lean mice. The in vitro HS increased the intracellular and extracellular HSP70 protein levels in thymocytes and splenocytes, while the effects of obesity on the HSP70 protein levels were not obvious. The in vitro HS prior to T cell mitogen stimulation decreased IFN-γ and IL-10 production by mitogen-stimulated splenocytes. This change in cytokine production due to HS was not affected by obesity. The obvious alteration of the HSP70 protein levels and association between cytokine production and the HS response in obesity were not found in this obesity model; however, our results indicate an association between the viability of thymocytes and an altered HS response in obesity and provide evidence that the increase in thymocyte apoptosis and acceleration of thymus involution in obesity could be, in part, due to the alteration of the HS response.     

Reversibility of heat shock in Chlamydia trachomatis

The heat shock effect on chlamydia development was studied. We report here that the reversibility of the heat shock response did not depend on the stage of chlamydial morphogenesis at which transfer to high temperature occurred, and the infectivity of the particles produced was not affected significantly, so long as the heat shock exposure was not prolonged. Exposure to heat shock for more than 9 h resulted in stagnation of the growth cycle, appearance of aberrant reticulate body particles and loss of infectivity. SDS-PAGE analysis of proteins synthesized under prolonged heat shock showed increased relative abundance of heat shock proteins in common with other procaryotic organisms.     

A heat shock protein 90 inhibitor that modulates the immunophilins and regulates hormone receptors without inducing the heat shock response

When a cell encounters external stressors, such as lack of nutrients, elevated temperatures, changes in pH or other stressful environments, a key set of evolutionarily conserved proteins, the heat shock proteins (hsps), become overexpressed. Hsps are classified into six major families with the hsp90 family being the best understood; an increase in cell stress leads to increased levels of hsp90, which leads to cellular protection. A hallmark of hsp90 inhibitors is that they induce a cell rescue mechanism, the heat shock response. We define the unique molecular profile of a compound (SM145) that regulates hormone receptor protein levels through hsp90 inhibition without inducing the heat shock response. Modulation of the binding event between heat shock protein 90 and the immunophilins/homologs using SM145, leads to a decrease in hormone receptor protein levels. Unlike N-terminal hsp90 inhibitors, this hsp90 inhibitor does not induce a heat shock response. This work is proof of principle that controlling hormone receptor expression can occur by inhibiting hsp90 without inducing pro-survival protein heat shock protein 70 (hsp70) or other proteins associated with the heat shock response. Innovatively, we show that blocking the heat shock response, in addition to hsp90, is key to regulating hsp90-associated pathways.     

Not changes in membrane fluidity but proteotoxic stress triggers heat shock protein expression in Chlamydomonas reinhardtii

A conserved reaction of all organisms exposed to heat stress is an increased expression of heat shock proteins (HSPs). Several studies have proposed that HSP expression in heat‐stressed plant cells is triggered by an increased fluidity of the plasma membrane. Among the main lines of evidence in support of this model are as follows: (a) the degree of membrane lipid saturation was higher in cells grown at elevated temperatures and correlated with a lower amplitude of HSP expression upon a temperature upshift, (b) membrane fluidizers induce HSP expression at physiological temperatures, and (c) membrane rigidifier dimethylsulfoxide dampens heat‐induced HSP expression. Here, we tested whether this holds also for Chlamydomonas reinhardtii. We show that heat‐induced HSP expression in cells grown at elevated temperatures was reduced because they already contained elevated levels of cytosolic HSP70A/90A that apparently act as negative regulators of heat shock factor 1. We find that membrane rigidifier dimethylsulfoxide impaired translation under heat stress conditions and that membrane fluidizer benzyl alcohol not only induced HSP expression but also caused protein aggregation. These findings support the classical model for the cytosolic unfolded protein response, according to which HSP expression is induced by the accumulation of unfolded proteins. Hence, the membrane fluidity model should be reconsidered.     

Dobutamine mediates cytoprotection by induction of heat shock protein 70 in vitro

Aims

Dobutamine is cytoprotective when applied before a subsequent stress. However, the underlying molecular mechanism is unknown. Dobutamine also inhibits nuclear factor (NF)-κB in human T lymphocytes. Other inhibitors of NF-κB induce a so-called heat shock response. We hypothesized that dobutamine mediates protection from apoptotic cell death by the induction of a heat shock response.

Main methods

Jurkat T lymphoma cells were preincubated with dobutamine (0.1, 0.5 mM) before the induction of apoptosis (staurosporine, 2 μM). DNA-binding of heat shock factor (HSF)-1 was analyzed by electrophoretic mobility shift assay, mRNA-expression of heat shock protein (hsp)70 and hsp90 by Northern Blot, activity of caspase-3 by fluorogenic caspase activity assay and cleavage of pro-caspase-3 by Western Blot. Apoptosis was assessed by flow cytometry after annexin V-fluorescein isothiocyanate staining. Hsp70 and hsp90 were inhibited using N-formyl-3,4-methylenedioxy-benzylidene-gamma-butyrolaetam and 17-allylamino-17-demethoxygeldana-mycin, respectively. All data are given as median and 25/75% percentile.

Key findings

Pre-incubation with dobutamine inhibited staurosporine-induced annexin V-fluorescence (28 [20–32] % vs. 12 [9–15] % for dobutamine 0.1 mM and 7 [5–12] % for dobutamine 0.5 mM, p < 0.001), cleavage of pro-caspase-3 as well as caspase-3-like activity (0.46 [0.40–0.48] vs. 0.32 [0.27–0.39] for Dobutamine 0.1 mM and 0.20 [0.19–0.23] for Dobutamine 0.5 mM, p < 0.01). Dobutamine induced DNA-binding of HSF-1 and mRNA-expression of hsp70 and hsp90. While inhibition of Hsp90 had no effect, inhibition of Hsp70 increased the number of annexin V-positive cells (33 [32–36] % vs. 18 [16–24] %) and caspase-3-like activity (0.21 [0.19–0.23] vs. 0.16 [0.13–0.17], p < 0.05).

Significance

Dobutamine protects from apoptotic cell death via the induction of Hsp70.     

心血管系统热休克蛋白的研究进展

多种应激因素如热应激,缺血,血流动力学变化能引起细胞内代谢异常,细胞骨架紊乱等一系列的病理改变,同时细胞亦相应合成一系列分子量不同的热休克蛋白分子（HSPs)。研究表明,HSPs通过其分子伴侣功能,对细胞产生保护作用。近年来的研究发现,在心肌缺血,缺血预处理,心肌肥大和血管损伤等病理生理条件下,HSP70,HSP90,HSP47,HSP32,HSP27等热休克蛋白分子均参与心血管系统的保护作用。     

Gambogic acid and gambogenic acid induce a thiol-dependent heat shock response and disrupt the interaction between HSP90 and HSF1 or HSF2

Cancer cells rely on heat shock proteins (HSPs) for growth and survival. Especially HSP90 has multiple client proteins and plays a critical role in malignant transformation, and therefore different types of HSP90 inhibitors are being developed. The bioactive natural compound gambogic acid (GB) is a prenylated xanthone with antitumor activity, and it has been proposed to function as an HSP90 inhibitor. However, there are contradicting reports whether GB induces a heat shock response (HSR), which is cytoprotective for cancer cells and therefore a potentially problematic feature for an anticancer drug. In this study, we show that GB and a structurally related compound, called gambogenic acid (GBA), induce a robust HSR, in a thiol-dependent manner. Using heat shock factor 1 (HSF1) or HSF2 knockout cells, we show that the GB or GBA-induced HSR is HSF1-dependent. Intriguingly, using closed form ATP-bound HSP90 mutants that can be co-precipitated with HSF1, a known facilitator of cancer, we show that also endogenous HSF2 co-precipitates with HSP90. GB and GBA treatment disrupt the interaction between HSP90 and HSF1 and HSP90 and HSF2. Our study implies that these compounds should be used cautiously if developed for cancer therapies, since GB and its derivative GBA are strong inducers of the HSR, in multiple cell types, by involving the dissociation of a HSP90-HSF1/HSF2 complex.     

Expression of heat shock protein in host macrophages correlates with a protective potential against infection with Leishmania major in mice

We studied the relationship between the expression of 65-kDa heat shock protein (HSP65) and resistance of mice to infection with Leishmania major (L. major), an obligate intracellular protozoan. C57BL/6 (B6) mice, a strain genetically resistant to L. major infection, expressed high level of HSP65 in their peritoneal and draining lymph node macrophages after infection, whereas susceptible BALB/c mice expressed only slightly at the early stage of infection. This protein was not expressed in the parasite itself and macrophages of non-infected mice. We examined the role of T cells in the expression of HSP65 by using SCID mice grafted with the fetal thymus from B6 or BALB/c mice (B6-TG or BALB-TG mice, respectively). Either BALB-TG or B6-TG mice were reconstituted with T cells derived from each grafted fetal thymus. B6-TG mice were markedly resistant against infection with L. major, compared with BALB-TG mice. Furthermore, the HSP65 expression in macrophages of thymus-grafted mice was similar to that of the thymus-donor type. That is, B6-TG mice expressed a high level of this protein, whereas BALB-TG mice did in lower level than B6-TG mice. These results show that T cells are necessary to express HSP65 and the expression correlates with a protective potential of T cells against infection with L. major.     

Altered expression of a heat shock protein in the mammalian nervous system in the presence of agents which affect microtubule stability

In vitro incubation of the isolated rabbit retina at elevated temperature results in the synthesis of a heat shock protein of M.W. 74,000 (hsp74). Recently we have demonstrated that this protein is associated with preparations of purified retinal microtubules and intermediate filaments. In order to examine the possibility that hsp74 synthesis is related to cytoskeletal stability, the effects of agents known to specifically affect microtubules were examined using an in vitro retinal system. Taxol, an antimitotic agent which stabilizes microtubules, was found to reduce the level of hsp74 synthesized in response to elevated temperature. Colchicine, a potent microtubule de-stabilizing agent, did not induce hsp74 synthesis in the absence of elevated temperature, however, under heat shock conditions, hsp74 synthesis was elevated in the presence of colchicine. Kinetics of microtubule assembly were similar in preparations isolated from cerebral hemispheres of control and hyperthermic animals however, microtubules from the latter were altered in appearance and exhibited a higher degree of crosslinking.     

DnaK expression in response to heat shock of Streptococcus mutans

Abstract The oral pathogen, Streptococcus mutans , persistently colonizes human hosts and initiates oral disease despite extreme variations in environmental conditions. To begin to investigate the role of the stress protein, DnaK (Hsp70), in environmental stress responses by S. mutans , pulse—chase experiments were initially used to establish that a functional heat shock response existed in this organism. A C-terminal fragment of the S. mutans dnaK gene was cloned and engineered to be expressed with a histidine tag. Using the recombinant DnaK protein that had been purified by nickel affinity chromatography, an antibody specific for the S. mutans DnaK protein was generated to analyse DnaK expression under homeostatic and heat shock conditions. Western blot analysis indicated that the anti-recombinant DnaK antibody specifically recognized a protein (molecular mass approx. 68 kDa) which was induced in response to thermal stress. Elucidating the role of DnaK in responses by S. mutans to various environmental Stressors will provide a better understanding of how DnaK is involved in survival of extreme environments and the contribution of the DnaK protein to the virulence of S. mutans .     

热休克蛋白27在肿瘤中的研究进展

热休克蛋白27（HSP27）作为热应激蛋白表达于各种生理或是环境损伤之后,保护细胞生存,其具有多种功能包括：分子伴侣,抗凋亡,参与细胞运动等。近年来发现HSP27在多种肿瘤中过度表达,参与肿瘤的发生发展,分化,耐药以及转移等方面,因而抑制HSP27成为一种新的肿瘤治疗策略。本文就相关研究进展进行综述。     

Discovery of novel heat shock protein (Hsp90) inhibitors based on luminespib with potent antitumor activity

A series of isosteric surrogates of the 4-phenyl group in luminespib were investigated as new scaffolds of the Hsp90 inhibitor for the discovery of novel antitumor agents. Among the synthesized surrogates of isoxazole and pyrazole, compounds 4a, 5e and 12b exhibited potent Hsp90 inhibition in ATPase activity and Her2 degradation assays and significant antitumor activity in A2780 and HCT116 cell lines. Animal studies indicated that compared to luminespib, their activities were superior in A2780 or NCI-H1975 tumor xenograft models. A molecular modeling study demonstrated that compound 4a could fit nicely into the N-terminal ATP binding pocket.     

Heat shock proteins as an aid in the treatment and diagnosis of heat stroke

It is now well established that induction of heat shock protein 72 (HSP72) protects the cell or tissue against a second otherwise lethal exposure to heat, a phenomenon known as thermotolerance. Because of this protective role, HSP72 is potentially useful in the treatment of heat illnesses, which range from relatively benign disorders such as heat cramps to heat stroke, which can be life threatening. This review discusses various ways in which HSP72 might be used in the diagnosis and treatment of the heat illnesses. This includes methods to induce HSP72, analysis of HSP72 in the cells and tissues of heat stroke patients, and screening methods to detect individuals who may be heat intolerant.     

The maximal cytoprotective function of the heat shock protein 27 is dependent on heat shock protein 70

Endogenous heat shock proteins (HSPs) 70 and 25/27 are induced in renal cells by injury from energy depletion. Transfected over-expression of HSPs 70 or 27 (human analogue of HSP25), provide protection against renal cell injury from ATP deprivation. This study examines whether over-expressed HSP27 depends on induction of endogenous HSPs, in particular HSP70, to afford protection against cell injury. LLC-PK1 cells transfected with HSP27 (27OE cells) were injured by ATP depletion for 2 h and recovered for 4 h in the presence of HSF decoy, HSP70 specific siRNA (siRNA-70) and their respective controls. Injury in the presence of HSF decoy, a synthetic oligonucleotide identical to the heat shock element, the nuclear binding site of HSF, decreased HSP70 induction by 80% without affecting the over-expression of transfected HSP27. The HSP70 stress response was completely ablated in the presence of siRNA-70. Protection against injury, provided by over-expression of HSP27, was reduced by treatment with HSF decoy and abolished by treatment with siRNA-70. Immunoprecipitation studies demonstrated association of HSP27 with actin that was not affected by either treatment with HSF decoy or siRNA. Therefore, HSP27 is dependent on HSP70 to provide its maximal cytoprotective effect, but not for its interaction with actin. This study suggests that, while it has specific action on the cytoskeleton, HSP 25/27 must have coordinated activity with other HSP classes, especially HSP70, to provide the full extent of resistance to injury from energy depletion.     

Bicyclol: a novel antihepatitis drug with hepatic heat shock protein 27/70-inducing activity and cytoprotective effects in mice

Heat shock proteins (HSPs) are the best-known endogenous factors that protect against cell injury under various pathological conditions and that can be induced by various physical, chemical, and biological stressors. New research seeks to discover a compound that is clinically safe and can induce the accumulation of HSPs in patients. This paper reports that the oral administration of three doses of bicyclol, a novel antihepatitis drug, induced hepatic HSP27 and HSP70 expression in a time- and dose-dependent manner, and that bicyclol treatment stimulated heat shock factor 1 (HSF1) activation in mice. The inducing effects of bicyclol on HSP27, HSP70 and HSF1 were all blocked by quercetin, an inhibitor of HSP biosynthesis. The cytoprotective effect of HSP27/70 induced by bicyclol against hepatotoxicity of acetaminophen (AP) was assessed in mice. The prior administration of bicyclol markedly suppressed AP-induced liver injury as indicated by the reduction in the elevation of serum alanine aminotransferase and aspartate aminotransferase, in liver necrosis, in the release of cytochrome c and apoptosis-inducing factor from mitochondria, as well as in hepatic deoxyribonucleic acid fragmentation in mice. However, all the above actions of bicyclol against AP-induced mouse liver injuries were significantly attenuated by quercetin. This is the first report to show that bicyclol induces hepatic HSP27/70 expression via activation of HSF1 and that the cytoprotective action of bicyclol against liver injury is mediated by its induction of HSP27/70. These results provide new evidence for elucidating the mechanism of the hepatoprotective action of bicyclol in animals and patients.     

热休克蛋白27 在肿瘤中的研究进展

热休克蛋白27(HSP27)作为热应激蛋白表达于各种生理或是环境损伤之后,保护细胞生存,其具有多种功能包括:分子伴侣,抗凋亡,参与细胞运动等。近年来发现HSP27在多种肿瘤中过度表达,参与肿瘤的发生发展,分化,耐药以及转移等方面,因而抑制HSP27成为一种新的肿瘤治疗策略。本文就相关研究进展进行综述。