Decay of the oocyte-type heat shock response of Xenopus laevis

Xenopus oocytes have a complex heat shock response. During transition of the oocyte into fertilized egg, the heat shock response undergoes several qualitative and quantitative changes culminating in its complete extinction. Heat shock induces oocytes to synthesize four heat shock proteins (hsps): 83, 76, 70, and 57. After ovulation, two additional proteins (hsps 22 and 16) are inducible. The heat shock response of spawned eggs can be modified by changing the ionic configuration of the external medium and by adding pyruvate and oxaloacetate to the media. Since Xenopus eggs do not synthesize mRNA, these modifications to the external medium apparently alter the utilization of preexisting messenger RNAs in protein synthesis. Artificial activation terminates inducibility of hsps 76, 57, and 16 and diminishes the hsp 70 response. Two new heat shock proteins-66 and 48-are also inducible in artificially activated eggs. Fertilization, on the other hand, terminates the heat shock response; no hsps can be induced. However, hsp 70 appears to be made constitutively in fertilized eggs. RNA blot analyses reveal that oogenic hsp 70 messenger RNA is retained in eggs and early embryos. This messenger is apparently used for heat-induced synthesis of hsp 70 before fertilization and for constitutive synthesis of hsp 70 in zygotes.     

Glutamine is a powerful effector of heat shock protein expression in Drosophila Kc cells.

We have investigated the effects of extracellular anions on the regulation of expression of the heat shock response in Drosophila Kc cells incubated in defined balanced salt solutions. Widely varying chloride concentrations had no effect on normal or heat shock protein (hsp) expression. Increasing glutamate concentrations from zero to 15 mM increased hsp expression more than 100-fold while affecting expression of non-heat-shock proteins minimally. Glutamine was 20-100-fold more potent than glutamate in supporting hsp expression, while other amino acids were less effective or supported no detectable hsp synthesis in heat shock. Inhibition of glutamine synthetase with methionine-sulfoximine resulted in very low hsp expression with glutamate and normal high level expression with glutamine, confirming the importance of glutamine. The absence of glucose and treatment with 2-deoxyglucose did not change the requirement for adequate glutamine for hsp expression. Cells heat shocked under conditions which gave very low hsp expression resumed growth when returned to normal medium as well as cells which expressed normal levels of hsps. Measurements of free amino acid levels in cells heat shocked in the presence and absence of glutamine showed a correlation between glutamine levels and amount of hsp expression. We conclude that a physiological process regulated by glutamine or a glutamine metabolite is important for normal hsp expression in heat shock conditions in Drosophila.     

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.     

Factors influencing the heat shock response of Xenopus laevis embryos

We have further characterized the heat shock response of Xenopus laevis embryos. Xenopus embryos respond to heat shock by consistently synthesizing four major heat shock proteins (hsps) of 62, 70, 76, and 87 kilodaltons. In addition to these hsps, heat-shocked embryos also exhibit the synthesis of several minor hsps. The synthesis of these hsps is often variable. We have monitored the effects of different temperatures and lengths of heat shock on the pattern and intensity of hsp synthesis. In general, the four major hsps are induced more strongly at higher temperatures and during increasing intervals of heat shock. The temperature and duration of heat shock can affect the synthesis of the minor hsps, however. Some hsps are synthesized at lower temperatures only (i.e., below 37 degrees C), whereas others are synthesized only at higher temperatures (i.e., above 37 degrees C). We have extensively examined the characteristics of hsp 35 synthesis, one of the most variably synthesized hsps. This hsp is characteristically synthesized at temperatures above 35 degrees C and usually during the first 40 min of heat shock, after which it becomes undetectable. In some experiments, its synthesis is restimulated during later intervals of heat shock. Hsp 35 is also under developmental regulation. It is not synthesized by heat-shocked embryos until the late blastula to early gastrula stage. After this brief period of inducibility, its synthesis is dramatically reduced in mid- to late gastrulae, but reappears in heat-shocked neurulae. We have previously demonstrated that hsp 35 is related to the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The induction of hsp 35 synthesis is inversely correlated with the constitutive levels of GAPDH specific activity. In this paper we document further correlations between the synthesis of hsp 35 and GAPDH specific activity during early Xenopus development.     

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.     

Suppression of heat- and polyglutamine-induced cytotoxicity by nonsteroidal anti-inflammatory drugs.

We have shown that sodium salicylate activates the heat shock promoter and induces the expression of heat shock proteins (hsps), with a concomitant increase in the thermotolerance of cells. To determine whether these effects are generally displayed by nonsteroidal anti-inflammatory drugs (NSAIDs), we examined the effects of a cyclooxygenase inhibitor, indomethacin, and a lipoxygenase inhibitor, nordihydroguaiaretic acid. Both inhibitors up-regulated the hsp promoter at 37 degrees C through the activation of heat shock factors, and increased cellular levels of hsps in mammalian cells, although the degree of the expression of hsps and thermotolerance of cells differed depending on the drugs. Furthermore, NSAIDs such as sodium salicylate and indomethacin suppressed the protein aggregation and apoptosis caused by an expanded polyglutamine tract in a cellular model of polyglutamine disease. These findings suggest that NSAIDs generally induce the expression of hsps in mammalian cells and may be used for the protection of cells against deleterious stressors and neurodegenerative diseases.     

Heat Shock Proteins and Their mRNAs in Dry and Early Imbibing Embryos of Wheat

Two-dimensional gels of in vitro translation products of mRNAs isolated from quiescent wheat (Triticum aestivum) embryos demonstrate the presence of mRNAs encoding heat shock proteins (hsps). There were no detectable differences in the mRNAs found in mature embryos from field grown, from 25°C growth chamber cultivated, or from plants given 38°C heat stresses at different stages of seed development. The mRNAs encoding several developmentally dependent (dd) hsps were among those found in the dry embryos. Stained two-dimensional gels of proteins extracted from 25°C growth chamber cultivated wheat embryos demonstrated the presence of hsps, including dd hsps. A study of the relationship of preexisting hsp mRNAs and the heat shock response during early imbibition was undertaken. Heat shocks (42°C, 90 minutes) were administered following 1.5, 16, and 24 hours of 25°C imbibition. While the mRNAs encoding the low molecular weight hsps decayed rapidly upon imbibition, the mRNAs for dd hsps persisted longer and were still detectable following 16 hours of imbibition. After 1.5 hours of imbibition, the mRNAs for the dd hsps did not accumulate in response to heat shock, even though the synthesis of the proteins was enhanced. Thus, an applied heat shock appeared to lead to the preferential translation of preexisting dd hsp mRNAs. The mRNAs for the other hsps, except hsp 70, were newly transcribed at all of the imbibition times examined. The behavior of the hsp 70 group of proteins during early imbibition was examined by RNA gel blot analysis. The mRNAs for the hsp 70 group were detectable at moderate levels in the quiescent embryo. The relative level of hsp 70 mRNA increased after the onset of imbibition at 25°C and remained high through 25.5 hours of prior imbibition. The maximal levels of these mRNAs at 25°C was reached at 17.5 hours of imbibition. Heat shock caused modest additional accumulation of hsp70 mRNA at later imbibition times.     

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.     

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.     

Effect of diadenosine tetraphosphate microinjection on heat shock protein synthesis in Xenopus laevis oocytes.

Bisnucleosides polyphosphates are thought to be chemical messengers signalling to the cell the onset of various stresses. Diadenosine tri- and tetraphosphates (respectively, Ap3A and Ap4A) accumulate in prokaryotic and eukaryotic cells under heat shock conditions, suggesting they could trigger the synthesis of heat shock proteins (hsps). In this study, Ap4A, Ap3A and, as a control, Ap4 (adenosine tetraphosphate) were injected into Xenopus oocytes. Whereas none of these compounds is able to trigger the synthesis of hsps in the absence of hyperthermic treatment, nuclear microinjection of Ap4A after a mild heat shock specifically enhances the synthesis of the 70-kd hsp, which is involved in the regulation and possibly the termination of the heat shock response. The microinjection of Ap4A prior to the hyperthermic treatment results in a strong inhibition of hsps synthesis (with the exception of the 70-kd hsp) suggesting that Ap4A is involved in the regulation and/or termination of the heat shock response. Ap3A and Ap4 do not induce any detectable modification of hsps expression.     

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.     

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.