Analysis of protein binding to heat shock protein 70 in pancreatic islet cells exposed to elevated temperatures or interleukin 1 beta

To elucidate a role for heat shock proteins in islet function, isolated pancreatic islets were labeled with [35S]methionine after control, heat shock, or interleukin 1 beta (IL-1 beta) treatment, extracted in the presence of detergent, and then passed over affinity columns with antibodies against heat shock protein 70 (hsp 70), hsp 70 itself, or ATP conjugated to the columns. In control or IL-1 beta-treated islets, the antibody column efficiently absorbed hsp 70 together with two other proteins of molecular masses 46 and 53 kDa. In extracts from heat-shocked cells, the binding of cellularly synthesized hsp 70 to the antibody column was inefficient but improved by the addition of unlabeled partially purified hsp 70 to the extracts. When assessing the binding of proteins in the extracts to the hsp 70 column, hsp 70 and the 46- and 53-kDa proteins among others all bound to the column. No differences in the patterns of binding to the hsp 70 column between extracts from the different islet exposures were noticed. The 46-kDa protein was identified as actin by immunoblot analysis. ATP-agarose column chromatography revealed a pattern of binding similar to that of the hsp 70 column. It is concluded that hsp 70 contains at least two functional domains, one adjacent to the epitope recognized by the antibody and active in restoring cellular function after heat shock, whereas the other has the ability to bind the 46- and 53-kDa and possibly other proteins. Furthermore, the stress induced by heat shock differs significantly from that after IL-1 beta treatment with respect to the functional behavior of hsp 70.     

Immunological evidence for the association of p53 with a heat shock protein, hsc70, in p53-plus-ras-transformed cell lines.

A rabbit antiserum was prepared against the C-terminal peptide of 21 amino acids from the human heat shock protein hsp70. These antibodies were shown to be specific for this highly inducible heat shock protein (72 kilodaltons [kDa] in rat cells), and for a moderately inducible, constitutively expressed heat shock protein, hsc70 (74 kDa). In six independently derived rat cell lines transformed by a murine cDNA-genomic hybrid clone of p53 plus an activated Ha-ras gene, elevated levels of p53 were detected by immunoprecipitation by using murine-specific anti-p53 monoclonal antibodies. In all cases, the hsc70, but not the hsp70, protein was coimmunoprecipitated with the murine p53 protein. Similarly, antiserum to heat shock protein coimmunoprecipitated p53. Western blot (immunoblot) analysis demonstrated that the hsc70 and p53 proteins did not share detectable antigenic epitopes. The results provide clear immunological evidence for the specific association of a single heat shock protein, hsc70, with p53 in p53-plus-ras-transformed cell lines. A p53 cDNA clone, p11-4, failed to produce clonable cell lines from foci of primary rat cells transfected with p11-4 plus Ha-ras. A mutant p53 cDNA clone derived from p11-4, SVKH215, yielded a 2- to 35-fold increase in the number of foci produced after transfection of rat cells with SVKH215 plus Ha-ras. When cloned, 87.5% of these foci produced transformed cell lines. SVKH215 encodes a mutant p53 protein that binds preferentially to the heat shock proteins of 70 kDa compared with binding by the parental p11-4 p53 gene product. These data suggest that the p53-hsc70 protein complex could have functional significance in these transformed cells.     

Heat shock protein 90beta1 is essential for polyunsaturated fatty acid-induced mitochondrial Ca2+ efflux

Nonesterified fatty acids may influence mitochondrial function by alterations in gene expression, metabolism, and/or mitochondrial Ca(2+) ([Ca(2+)](m)) homeostasis. We have previously reported that polyunsaturated fatty acids induce Ca(2+) efflux from mitochondria, an action that may deplete [Ca(2+)](m) and thus contribute to nonesterified fatty acid-responsive mitochondrial dysfunction. Here we show that the chaperone protein heat shock protein 90 beta1 (hsp90beta1) is required for polyunsaturated fatty acid-induced mitochondrial Ca(2+) efflux (PIMCE). Retinoic acid induced differentiation of human teratocarcinoma NT2 cells in association with attenuation of PIMCE. Proteomic analysis of mitochondrial proteins revealed that hsp90beta1, among other proteins, was reduced in retinoic acid-differentiated cells. Blockade of PIMCE in NT2 cells by 17-(dimethylaminoethylamino)-17-demethoxygeldanamycin, a known inhibitor of the chaperone activity of hsp90, and hsp90beta1 RNA interference demonstrated that hsp90beta1 is essential for PIMCE. We also show localization of hsp90beta1 in mitochondria by Western blot and immunofluorescence. Distinctive effects of inhibitors binding to the N or C terminus of hsp90 on PIMCE in isolated mitochondria suggested that the C terminus of hsp90beta1 plays a critical role in PIMCE.     

Differences in the expression of heat-shock proteins and antioxidant enzymes between human and rodent pancreatic islets: implications for the pathogenesis of insulin-dependent diabetes mellitus.

BACKGROUND: It has previously been observed that the insulin-producing cells of human pancreatic islets are more resistant to alloxan-, streptozotocin-, nitroprusside-, or cytokine-induced injury than those of mouse and rat islets. MATERIALS AND METHODS: Human pancreatic islets were obtained from heart-beating organ donors. The expression of the stress proteins heat shock protein 70 (hsp70) and heme oxygenase and the anti-apoptosis gene bcl-2 was determined in isolated rat, mouse, and human islets, either cultured in vitro or transplanted under the kidney capsule of nude mice, using immunoblot analysis. Rat and human islet sensitive hydrogen peroxide was assess by glucose oxidation measurements. Isolated islets were also analyzed for their catalase and superoxide dismutase activities, and the islet cell levels of reduced glutathione were determined in response to hydrogen peroxide and nitroprusside. Programmed cell death in human and rat islets in response to streptozotocin was evaluated using TUNEL staining. RESULTS: Cultured human islets expressed higher contents of hsp70 than mouse and rat islets at basal conditions. Also after 4 weeks under the kidney capsule of normoglycemic mice, the hsp70 levels were higher in human islets than in rat islets. The expression of another stress protein, heme oxygenase (HO), was strongly increased in cultured rat islets, but was not affected in human islets. Expression of the bcl-2 gene could not be detected in human islets. In spite of this, 0.5 mM streptozotocin induced apotosis in rat but not in human islet cells. Hydrogen peroxide (0.1 and 0.4 mM) decreased glucose oxidation rates in rat but not in human islets. The levels of reduced glutathione were moderately decreased in human and rat islet cells and sharply decreased in mouse islet cells in response to hydrogen peroxide. Moreover, the activities of catalase and superoxide dismutase (SOD) were markedly lower in mouse islets than in human islets. The activity of catalase was lower in rat islets than in human islets. CONCLUSION: Human islets differ clearly from mouse and rat islets in their increased expression of hsp70, catalase, and SOD, which may explain the increased resistance of human islets to beta cell toxins.     

Expression analysis of heat shock genes in the skin, spleen and blood of common carp (Cyprinus carpio) after cadmium exposure and hypothermia

Heat shock proteins are chaperones that play a pivotal role in controling multiple regulatory pathways such as stress defense, hormone signaling, cell cycle control, cell proliferation and differentiation, and apoptosis. In this study, the expression patterns of four well-known heat shock genes (hsp70, hsc70-1, hsc70-2 and hsp90α) were characterized in the skin, spleen and blood cells of the common carp, under unstressed conditions and after Cd2+ treatment or hypothermia. The examined genes were expressed in a tissue-specific manner: hsc70-2 was expressed constitutively, and was at best only slightly inducible; hsp90α exhibited a high basic expression in all three tissues, whereas hsc70-1 did so only in the blood cells, the expression of hsp70 proved to be below the level of detection in unstressed fish. Cold shock induced the expression of hsp genes in the spleen (hsp90α) and blood cells (hsp70, hsc70-1 and hsp90α), while Cd2+ treatment has no effect on the expression pattern. The highest inducibilities were detected in the skin: for hsp70 an induction of at least 20-fold after cadmium exposure, for hsc70-1 of at least 30-fold and for hsp90α of 3-fold after hypothermia.     

hsp70-DnaJ chaperone pairs prevent nitric oxide-mediated apoptosis in RAW 264.7 macrophages

Excess nitric oxide (NO) induces apoptosis in some cell types, including macrophages. Heat shock protein of 70 kDa (hsp70) has been reported to protect cells from various stresses, including apoptosis-inducing stimuli. Several mammalian cytosolic DnaJ homologs, partner chaperones of hsp70 family members, have been identified. We asked if a DnaJ homolog is required to prevent NO-mediated apoptosis. When mouse macrophage-like RAW 264.7 cells were treated with an NO donor, SNAP, apoptosis occurred. This apoptosis could be prevented by pretreatment of the cells with heat or a low dose of SNAP. Under these conditions, levels of hsc70 (an hsp70 member) remained unchanged, whereas hsp70 was markedly induced. Of the DnaJ homologs dj1 (hsp40/hdj-1) was strongly induced and dj2 (HSDJ/hdj-2) was moderately induced. In transfection experiments, hsp70, hsc70, dj1 or dj2 alone was ineffective in preventing NO-mediated apoptosis. In contrast, both dj1 and dj2, in combination with hsc70 or hsp70, prevented the cells from apoptosis. The hsp70-DnaJ chaperone pairs exerted their anti-apoptotic effects upstream of caspase 3 activation, and apparently upstream of cytochrome c release from mitochondria.     

Heat shock proteins gp96 and hsp70 activate the release of nitric oxide by APCs

NO is a cytotoxic and immunomodulatory cytokine produced by macrophages and dendritic cells. We show that stimulation of murine and human macrophages with the heat shock proteins gp96 and hsp70 results in induction of inducible NO synthase and the production of NO. The release of NO by monocytes exposed to hsp60 has been documented previously. Immature, but not mature, dendritic cells respond in the same manner. The activity of heat shock proteins is relatively unaffected by an antagonist of LPS, and is abrogated by heat denaturation. Macrophages have been shown previously to produce NO in response to stimulation with IFN-gamma; stimulation of macrophages with mixtures of IFN-gamma and gp96 or hsp70 leads to a synergistic production of NO. The present observations extend the roles of these heat shock proteins in innate immune responses to another potent and highly conserved function of APC.     

Probiotics potentiate IL-6 production in IL-1beta-treated Caco-2 cells through a heat shock-dependent mechanism

IL-6 may exert anti-inflammatory and protective effects in intestinal mucosa and enterocytes. The influence of probiotics on mucosal and enterocyte IL-6 production is not known. We tested the hypothesis that the probiotic bacteria Lactobacillus paracasei and Lactobacillus plantarum regulate IL-6 production in intestinal epithelial cells. Cultured Caco-2 cells were treated with 1 ng/ml of IL-1beta in the absence or presence of different concentrations of L. paracasei or L. plantarum followed by measurement of IL-6 production. The role of heat shock response was examined by determining the expression of heat shock protein 70 (hsp70) and hsp27, by downregulating their expression with small interfering RNA (siRNA), or by treating cells with quercetin. Treatment of the Caco-2 cells with IL-1beta resulted in increased IL-6 production, confirming previous reports from this laboratory. Probiotics alone did not influence IL-6 production, but the addition of probitoics to IL-1beta-treated cells resulted in a substantial augmentation of IL-6 production. Treatment of the Caco-2 cells with live L. paracasei increased cellular levels of hsp70 and hsp27 and the potentiating effect on IL-6 production was inhibited by quercetin and by hsp70 or hsp27 siRNA. Results suggest that probiotics may enhance IL-6 production in enterocytes subjected to an inflammatory stimulus and that this effect may, at least in part, be heat shock dependent.     

Major heat shock protein hsp70 protects tumor cells from tumor necrosis factor cytotoxicity.

Heat treatment and various other stresses render tumor cells resistant to cytotoxicity mediated by tumor necrosis factors (TNFs). Here, we elucidate the molecular basis of this phenomenon by demonstrating that the major heat shock protein, hsp70, protects tumor cells from TNF cytotoxicity even in the absence of stress. The human hsp70 gene was stably introduced into highly TNF-sensitive WEHI-S tumor cells both in the sense and antisense orientation. All clones constitutively expressing the exogenous human hsp70 gene were protected from TNF-mediated killing approximately 1000-fold. Remarkably, the growth of one clone was actually stimulated by low concentrations of TNF. Moreover, a clone expressing antisense hsp70 RNA was rendered extremely sensitive to TNFs. Hsp70-mediated protection from TNF cytotoxicity was confirmed in transient expression experiments employing retroviral vectors. Changes in cellular sensitivity to TNF were not associated with alterations in the binding of TNF to its receptors. Neither the transfection procedure itself nor overexpression of the low molecular weight heat shock protein, hsp27, had any effect on cellular susceptibility to TNFs. Our data suggest that hsp70 may increase the oncogenic potential of some tumor cells by providing them with an escape mechanism from immunological defense.     

Heat shock restores insulin secretion after injury by nitric oxide by maintaining glucokinase activity in rat islets

Heat shock protein (hsp), including hsp70, has been reported to restore the glucose-induced insulin release suppressed by nitric oxide (NO). However, the mechanism underlying this recovery remains unclear. In the present study, we examine the effects, in rat islets, of heat shock on insulin secretion inhibited by a small amount of NO and also on glucose metabolism, the crucial factor in insulin release. Exposure to a higher dose (15 U/ml) of interleukin-1beta (IL-1beta) abolished the insulin release by stimulation of glucose or KCl in both control and heat shocked islets. In rat islets exposed to a lower dose (1.5 U/ml) of IL-1beta, insulin secretion in response to glucose, but not to glyceraldehydes (GA), ketoisocaproate (KIC), or KCl, was selectively impaired, concomitantly with lower ATP concentrations in the presence of 16.7 mM glucose, while such suppression of insulin secretion and ATP content was not observed in heat shock-treated islets. NO production in islets exposed to 1.5 U/ml IL-1beta was significantly, but only partly, decreased by heat shock treatment. The glucose utilization rate measurement using [5-3H]-glucose and [2-3H]-glucose and the glucokinase activity in vitro were reduced in islets treated with 1.5 U/ml IL-1beta. In heat shock-treated islets, glucose utilization and glucokinase activity were not affected by 1.5 U/ml IL-1beta. These data suggest that heat shock restores glucose-induced insulin release inhibited by NO by maintaining glucokinase activity and the glucose utilization rate in islets in addition to reducing endogenous NO production.     

Adaptation of the body to stress effects increases resistance of nuclear DNA of cardiac cells due to accumulation of heat shock proteins in the nucleus]

It was shown that adaptation to stress exposure increased the resistance of nuclear DNA in myocardial cells to the damaging action of exogenous one-chain DNA (50 micrograms/ml). This protective effect was accompanied by a pronounced accumulation of heat shock proteins (hsp) 70 in nucleoplasm of myocardial cells from adapted animals. Possible mechanism of the DNA protective effect of adaptation and the role of hsp 70 are under discussion.     

Metal chelatorNNN’ N’-tetrakis-(2-pyridylmethyl)ethylene diamine inhibits the induction of heat shock protein 70 synthesis by heat in cultured keratinocytes

Heat shock protein (HSP) synthesis results from various types of injury, including heat shock (HS) and some oxidants. The intracellular signals leading to HSP synthesis are not yet fully elucidated. We have studied the influence ofNNN’N’-tetrakis(2-pyridylmethyl)ethylene diamine (TPEN), a metal chelator known to induce cellular zinc and copper deprivation, on resistance to heat and on hsp70 synthesis in HaCaT keratinocytes. TPEN was shown to sensitize HaCaT cells to heat shock. The effect of TPEN was neutralized by equimolar Zn²⁺. By the use of sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis and Western blotting characterization of hsp70, it was shown that cultured HaCaT cells constitutively express the inducible form of hsp70. The application of TPEN alone slightly increases the level of hsp70 but inhibits its induction by HS. This inhibitory effect is related to metal deprivation, because it is eliminated when Cu²⁺ or Zn²⁺ ions are supplied together with TPEN. These results suggest that these metals are involved in the expression by keratinocytes of a stress protein which has a protective action against environmental stress.     

High-level, heat-regulated synthesis of proteins in eukaryotic cells

Plasmids have been constructed in which promoters of 70-kDa heat-shock protein genes (hsp70) of human and Drosophila origin were linked to three different eukaryotic genes encoding human growth hormone (hGH), chicken lysozyme (cL) and a human influenza haemagglutinin (HA). Following transfection into widely divergent eukaryotic cells, the hybrid genes direct the transient, heat-regulated synthesis of the three proteins. hGH and cL are secreted into the medium. A human hsp70-hGH construct was used to establish stable mouse fibroblast lines that are capable of producing and secreting hGH at high levels following heat induction: hGH is secreted at a 500-1200-fold higher rate by heat-treated than by untreated cells.     

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

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