Immunolocalization of stress proteins and extracellular matrix proteins in the rat tibia

Stress proteins (heat shock proteins [hsps]) serve a number of protective functions, including protection from apoptosis and acting as chaperones during protein biosynthesis. For example, hsp 27 has been defined as a chaperone for the G3 domain of aggrecan, while hsp 47 is the chaperone for type I collagen. Separate cytoprotective roles for hsp 27 and hsp 70 have been demonstrated. The aim of this study was to define the expression of hsps in osteoblastic and chondrocytic cells of the growing rat long bone in relationship to the immunohistochemical localization of aggrecan, type I collagen and the presence of fragmented DNA that defines apoptotic events. Tibiae were harvested from Fisher 344 rats (n=6) and fixed in 10% buffered formalin. Samples were decalcified in 10% EDTA, bisected, and processed for histologic examination. Sections (5 mm) were immunohistochemically stained using a streptavidin-biotin detection method. Co-localization of hsps with apoptosis was achieved using the TUNEL procedure. In the rat tibia growth plate, aggrecan was generally distributed throughout cartilage and chondrocytes. However, hsp 27 expression was observed only in the lower hypertrophic chondrocytes. hsp27 was present in osteoblasts lining newly formed bone. hsp 47 staining was also prominent within these osteoblasts where collagen type I immunolocalization occurred. The inducible form of hsp 70 was localized to the osteoblastic cells lining new bone in the primary spongiosa. In cartilage, DNA fragmentation was restricted to the hypertrophic, hsp27-positive, chondrocytes. In contrast, DNA fragmentation was not co-localized with hsp27-positive osteoblastic cells of the primary spongiosa, although occasional apoptotic cells were identified. These results indicate that apoptosis is a mechanism by which hypertrophic chondrocytes are eliminated from cartilage prior to calcification, but that other mechanisms are also likely to be involved. They also suggest that hsps have cytoprotective and biosynthetic functions within osteoblasts and chondrocytes, but apoptotic signals may override these effects in some instances, resulting in apoptosis.     

Heat shock protein induction and induced thermal tolerance are independent in adult salamanders

Ectothermic vertebrates become thermally tolerant (heat hardened) after exposure to heat shock. Eukaryotic cells show a similar response. Cellular thermal tolerance is correlated with the induction of heat shock proteins (hsps). We have investigated the relationship between heat hardening in salamanders and the induction of hsps in the tissues of these organisms. Although the synthesis of hsps can be induced in these animals by sublethal heat shocks, conditions required for hsp induction and heat hardening often do not coincide. We conclude that induced thermal tolerance in adult salamanders is independent of hsp induction in their tissues.     

Flavonoids inhibit the expression of heat shock proteins

Cells exposed to several forms of stress, such as heat shock, transiently synthesize a group of proteins called heat shock proteins (hsps). Although many stressors other than heat shock are known to induce hsps, inhibitors of hsp expression have never been reported. Here we show that quercetin and several other flavonoids inhibit the synthesis of hsps induced by heat shock in two human cell lines, Hela cells and COLO320 DM cells. Quercetin inhibited the induction of hsp70 at the level of mRNA accumulation. This is the first report to describe the inhibition of hsp expression by reagents.     

Dissociation of 68,000 Mr heat shock protein synthesis from thermotolerance expression in rat fibroblasts

Rat embryonic fibroblasts growing exponentially at either 35, 37, or 39 degrees C were exposed to 42 degrees C for times up to 6 hr. Cell survival was unaffected by this heat shock in cultures growing at 39 degrees C but survival was decreased in a temperature dependent manner in cells growing at 37 or 35 degrees C. Exposure to 42 degrees C of cells previously adapted to 35 or 37 degrees C resulted in the induction of heat shock proteins (hsps) with apparent molecular weights of 68,000 (hsp 68), 70,000 (hsp 70), and 89,000 (hsp 89); cells previously adapted to 39 degrees C expressed all hsps except hsp 68. Inasmuch as the synthesis of certain hsps may function to protect cells from thermal damage, these data indicate that hsp 68 may not be required for this adaptation-related thermotolerant survival response. Hsp 68 may only be expressed in cells destined to die.     

Heat Shock Proteins hsp90 and hsp70 Protect Neuronal Cells from Thermal Stress but Not from Programmed Cell Death

Abstract: Prior exposure to a mild thermal stress can protect neuronal cells from a subsequent more severe stress including high temperature, ischemia, glutamate toxicity, or stimuli inducing apoptosis. Although the protective effect of thermal stress correlates with the elevated expression of the heat shock proteins (hsps), the protective effect of individual hsps has never been directly demonstrated in neuronal cells. Here we show that the constitutive overexpression of either of the major hsps, hsp90 or hsp70, can protect neuronal cells from thermal stress but not from stimuli that induce apoptosis. The possible mechanisms by which thermal stress can protect neuronal cells from apoptosis are discussed.     

Induction of heat shock proteins during initiation of solvent formation inClostridium acetobutylicum

Summary The response to stresses produced by changes in the fermentation conditions ofClostridium acetobutylicum in continuous culture was determined under acid- and solvent-producing conditions. Using a phosphate-limited chemostat it was found that specificheatshockproteins (hsp 73, hsp 72 [Dnak], hsp 67 [GroEL], hsp 17 and hsp 14) were synthesized at elevated levels during the shift from acid to solvent formation. The induction of these stress proteins was observed before acetone and butanol were detected in the medium and was therefore not a response to these solvents present in the medium. Simultaneously with the induction of hsps, changes in the synthesis rates of other cellular proteins were observed. Synthesis of proteins associated with the acid production phase decreased and of proteins correlated with the solvent production phase increased. Some hsps, including the DnaK- and GroEL-similar proteins, hsp 73 and hsp 21, were also induced by a change in the growth rate and/or the pH. The analysis of the general regulation of the heat shock response inC. acetobutylicum revealed that the induction of at least 15 hsps after a temperature up-shift was transient and that two temporal classes of hsps could be distinguished. The synthesis of one group of hsps reached a maximum after 6 min and another around 11 min after the temperature upshift and returned to steady-state levels 30 to 40 min after the shock.     

Hsp27 protects mitochondria of thermotolerant cells against apoptotic stimuli

Enhanced cell survival and resistance to apoptosis during thermotolerance correlates with an increased expression of heat shock proteins (Hsps). Here we present additional evidence in support of the hypothesis that the induction of Hsp27 and Hsp72 during acquired thermotolerance in Jurkat T-lymphocytes prevents apoptosis. In thermotolerant cells, Hsp27 was shown to associate with the mitochondrial fraction, and inhibition of Hsp27 induction during thermotolerance in cells transfected with hsp27 antisense potentiated mitochondrial cytochrome c release after exposure to various apoptotic stimuli, despite the presence of elevated levels of Hsp72. Caspase activation and apoptosis were inhibited under these conditions. In vitro studies revealed that recombinant Hsp72 more efficiently blocked cytochrome c-mediated caspase activation than did recombinant Hsp27. A model is presented for the inhibition of apoptosis during thermotolerance in which Hsp27 preferentially blocks mitochondrial cytochrome c release, whereas Hsp72 interferes with apoptosomal caspase activation.     

Bacterial toxins induce heat shock proteins in human neutrophils

We studied the influence of different bacterial toxins (alveolysin; toxic shock syndrome toxin 1, TSST-1 and erythrogenic toxin A, ETA) on the expression of heat shock proteins (hsps) in isolated human polymorphonuclear granulocytes (PMNs). As was shown by Western blotting (anti-hsp72) ETA and TSST-1 were potent inducers of hsps at low toxin concentrations (10 ng/ml). Alveolysin led to the expression of hsps at hemolytic concentrations (1 HU; 700 ng/ml) whereas at subhemolytic concentrations (7 ng/ml) no heat shock response was observed. The induction of heat shock proteins was also accompanied by increased mRNA levels for hsp70 as was determined by PCR-analysis.     

Regulation of apoptotic signal transduction pathways by the heat shock proteins

The study about apoptotic signal transductions has become a project to reveal the molecular mechanisms of apoptosis. Heat shock proteins (hsps), which play an important role in cell growth and apoptosis, have attracted great attentions. A lot of researches have showed there is a hsps superfamily including hsp90, hsp70, hsp60 and hsp27, etc., which regulates the biological behaviors of cells, particularly apoptotic signal transduction in Fas pathway, JNK/SAPK pathway and caspases pathway at different levels, partly by the function of molecular chaperone.     

Regulation of apoptotic signal transduction pathways by the heat shock proteins

With progressing recognition of apoptosis in bio-logical and medical sciences, the apoptotic signal transduction has rapidly become a dominant project to reveal the molecular mechanisms of apoptotic process. A lot of researches about apoptotic signal transduction have showed the expression of heat shock proteins was closely correlated with cell growth and differen-tiation, and involved in the regulation of apoptosis in different signal transduction pathways. Here we re-view the effects of hsps…     

Plasminogen and angiostatin interact with heat shock proteins

Previous studies from this laboratory have demonstrated that plasminogen and angiostatin bind to endothelial cell (EC) surface-associated actin via their kringles in a specific manner. Heat shock proteins (hsps) like hsp 27 are constitutively expressed by vascular ECs and regulate actin polymerization, cell growth, and migration. Since many hsps have also been found to be highly abundant on cell surfaces and there is evidence that bacterial surface hsps may interact with human plasminogen, the purpose of this study was to determine whether human plasminogen and angiostatin would interact with human hsps. ELISAs were developed in our laboratory to assess these interactions. It was observed that plasminogen bound to hsps 27, 60, and 70. In all cases, binding was inhibited (85–90%) by excess (50 mM) lysine indicating kringle involvement. Angiostatin predominantly bound to hsp 27 and to hsp 70 in a concentration- and kringle-dependent manner. As observed previously for actin, there was concentration-dependent inhibition of angiostatin’s interaction with hsp 27 by plasminogen. In addition, 30-fold molar excess actin inhibited (up to 50%), the interaction of plasminogen with all hsps. However, 30-fold molar excess actin could only inhibit the interaction of angiostatin with hsp 27 by 15–20%. Collectively, these data indicate that (i) while plasminogen interacts specifically with hsp 27, 60, and 70, angiostatin interacts predominantly with hsp 27 and to some extent with hsp 70; (ii) plasminogen only partially displaces angiostatin’s binding to hsp 27 and (iii) actin only partially displaces plasminogen/angiostatin binding to hsps. It is conceivable therefore that surface-associated hsps could mediate the binding of these ligands to cells like ECs.     

Is heat shock protein re-induction during tolerance related to the stressor-specific induction of heat shock proteins?

The existence of stressor-specific induction programs of heat shock proteins (hsps) leads us to analyze the possible occurrence of a stressor-specific tolerance induced by either heat shock, arsenite, or cadmium. As a measure of this tolerance re-induction of hsps was studied. In this paper, we tested whether the refractory state is either valid for each specific hsp (implying independent regulation of every member of the heat shock protein family) or extends from small subsets of the hsp-family to even larger groups of proteins (indicating a more common denominator in their regulation). (Re-)induction of hsps does not seem to be regulated at the level of each individual hsp since differences in induced synthesis of hsps between two stressor conditions are not supplemented systematically upon the sequential application of the two stressors. The most notable example in this respect is hsp60. A pretreatment with cadmium, which hardly induces synthesis of this hsp, does induce a tolerance to (re)-induction by heat shock, which normally induces hsp60. This suggests the existence of a more common denominator regulating the coordinate expression of at least some hsps. From our data we conclude that the degree, but not the pattern, of hsp re-induction is influenced by the type of stressor used in the pretreatment. The pattern of hsps induced by a secondary applied stressor still shows most of its stressor-specificity and seems to be independent of any pretreatment. The possible implications of stressor-specificity are discussed. © 1996 Wiley-Liss, Inc.     

A comparison of sodium arsenite- and hyperthermia-induced stress responses and abnormal development in cultured postimplantation rat embryos.

Acute exposures to sodium arsenite (50 microM) were embryotoxic in day 10 rat embryos exposed in vitro. Sodium arsenite-induced embryotoxicity was characterized by decreased growth (crown-rump length, somite number, and embryo protein content) and abnormal development (hypoplastic prosencephalon, abnormal somites, and abnormal flexion of the tail). At embryotoxic exposures, sodium arsenite also induced the synthesis of three heat shock proteins (hsps), one of which is recognized by a monoclonal antibody specific for the heat-inducible hsp 72. In addition, sodium arsenite induced the accumulation of heat-inducible hsp 70 mRNA. Although the abnormal morphologies induced by sodium arsenite and hyperthermia appear to be different, the stress response as measured by the synthesis of hsps, the accumulation of hsp 72 protein, and the accumulation of hsp 70 mRNA is similar in embryos exposed to these two embryotoxic agents. Thus, sodium arsenite and hyperthermia both induce a stress response; however, the relationship between the induction of a stress response and the subsequent abnormal development that ensues is unclear.     

Modulation of the Stress-Induced Synthesis of hsp27 and αB-Crystallin by Cyclic AMP in C6 Rat Glioma Cells

Abstract: The possible participation of cyclic AMP in the stress-induced synthesis of two small stress proteins, hsp27 and αB-crystallin, in C6 rat glioma cells was examined by specific immunoassays, western blot analysis, and northern blot analysis. When C6 cells were exposed to arsenite (50–100 µM for 1 h) or heat (42°C for 30 min), expression of hsp27 and αB-crystallin was stimulated, with levels of the two proteins reaching a maximum after 10–16 h of culture. Induction of hsp27 was markedly enhanced when cells were exposed to arsenite in the presence of isoproterenol (20 µM) or epinephrine (20 µM) but not in the presence of phenylephrine. The stimulatory effects of isoproterenol and epinephrine were blocked completely by propranolol, an antagonist of β-adrenergic receptors. Cholera toxin (2 µg/ml), forskolin (20 µM), and dibutyryl cyclic AMP (2.5 mM), all of which are known to increase intracellular levels of cyclic AMP, also stimulated the arsenite- or heat-induced accumulation of hsp27. Treatment of cells with each of these modulators alone did not result in the induction of hsp27. The level of hsp70 in C6 cells, as estimated by western blot analysis, was also enhanced by arsenite or heat stress. However, induction of hsp70 by stress was barely stimulated by isoproterenol. By contrast, induction of αB-crystallin by heat or arsenite stress was suppressed when isoproterenol, cholera toxin, forskolin, or dibutyryl cyclic AMP was present during the stress period. Northern blot analysis of the expression of mRNAs for hsp70, hsp27, and αB-crystallin showed that the modulation of the stress-induced accumulation of the three hsps by the various agents was regulated at the level of the corresponding mRNA. These results indicate that stress responses of hsp70, hsp27, and αB-crystallin in C6 rat glioma cells are regulated differently and, moreover, that when the level of cyclic AMP increases in cells, the response to stress of hsp27 is stimulated but that of αB-crystallin is suppressed.     

Expression of Drosophila's 27 kDa heat shock protein into rodent cells confers thermal resistance.

The role of hsp27, one of Drosophila melanogaster's small heat shock proteins, in the process of thermotolerance was investigated. The coding sequence of hsp27 was subcloned downstream of the human hsp27 promoter which has been shown to be constitutively expressed in Chinese hamster O23 cells. Cellular resistance to a thermal stress was measured two days after transfection by a survival assay following a 3.5 h heat treatment at 44 degrees C. Expression of Drosophila hsp27 was shown to confer thermal resistance to O23 cells in a manner which was dependent on the level of expression of this hsp. Immunoblot analysis confirmed that the thermal resistance was related to the expression of Drosophila hsp27 as none of the endogeneous hsps showed an increased level under these conditions.     

Members of the Drosophila HSP 70 family share ATP-binding properties

In Drosophila, the hsp 70 family consists of a group of proteins of similar molecular masses (hsps 68, 70 and 72) that exist as multiple isoforms. In this report, it is shown that hsps 68, 70 and 72 from Drosophila cells can be purified by affinity chromatography on ATP-agarose. Furthermore it is demonstrated that the multiple members of the hsp 70 family, which accumulate in large amounts in the nucleus during a heat shock, can be specifically solubilized from the isolated nuclei fraction by ATP. One of the major cognate proteins (hsc 70) also shows similar behavior. These data suggest that most, if not all, of the related Drosophila hsps 70 possess, like their mammalian counterparts, an ATP-binding site which could be related to their function in the stress response.     

Estrogen-induced resistance to osteoblast apoptosis is associated with increased hsp27 expression

Estrogen has been shown to protect osteoblastic cells from apoptosis. Similarly, estrogen treatment preceding heat shock elevates heat shock protein 27 (hsp27) expression and increases thermoresistance in the murine estrogen receptor-transformed SMER14 osteoblastic cell line. Forced expression of hsp27 expression in other cell lines limits apoptosis. The purpose of this study was to examine the effects of estrogen on staurosporine-induced apoptosis in the context of hsp27 expression. Cell viability was measured by the MTT assay. Early apoptotic events were examined by fluorescent microscopy by using FITC-conjugated Annexin V staining. TUNEL labeling was used to compare the number of apoptotic nuclei following staurosporine treatment of estrogen pretreated or untreated cells. Estrogen treatment increased SMER14 cell viability, but not ROS17/2.8 cell viability, in the presence of staurosporine. Estrogen treatment also reduced annexin V staining and DNA fragmentation. Similar treatment increased SMER14 cell hsp27 levels. The concurrent reduction in induced apoptosis suggests a possible estrogenic mechanism for increasing and/or maintaining the number of viable osteoblasts in bone.