Differential temperature dependency of chemical stressors in HSF1-mediated stress response in mammalian cells

Expression of stress proteins is generally induced by a variety of stressors. To gain a better understanding of the sensing and induction mechanisms of stress responses, we studied the effects of culture temperature on responses to various stressors, since the induction of hsp70 in mammalian cells by heat shock is somehow modulated by culture temperature. Hsp70 was not induced by treatment with sodium arsenite, azetidine-2-carboxylic acid, or zinc sulfate at the level of heat shock factor (HSF) 1 activation in cells incubated at low temperature, although these treatments induced hsp70 in cells incubated at 37 degrees C. The repression of sodium arsenite or zinc sulfate-induced HSF1 activation by low temperature was not simply due to the inhibition of protein synthesis. On the other hand, heat shock and iodoacetamide induced HSF 1 activation in cells incubated at either temperature. Thus, there seem to be two kinds of stressors that induce HSF1 activation independently of or dependent on culture temperature. Furthermore, the reduction of glutathione level seemed to be essential for HSF1 activation by chemical stressors.     

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.     

70-Kilodalton heat shock polypeptides from rainbow trout: characterization of cDNA sequences.

RTG-2 cells, a line of fibroblasts from rainbow trout (Salmo gairdnerii), are induced to synthesize a distinct set of heat-shock polypeptides after exposure to elevated temperature or to low concentrations of sodium arsenite. We isolated and characterized two cDNA sequences, THS70.7 and THS70.14, encoding partial information for two distinct species of 70-kilodalton heat shock polypeptide (hsp70) from these cells. These sequences are identical at 73.3% of the nucleotide positions in their regions of overlap, and their degree of sequence conservation at the polypeptide level is 88.1%. The two derived trout hsp70 polypeptide sequences show extensive homology with derived amino acid sequences for hsp70 polypeptides from Drosophila melanogaster and Saccharomyces cerevisiae. Northern blot analysis of RNA from arsenite-induced RTG-2 cells, with the trout hsp70 cDNAs as probes, revealed the presence of three hsp70 mRNA species. Southern blot analysis of trout testis DNA cleaved with various restriction endonucleases revealed a small number of bands hybridizing to the hsp70 cDNAs, suggesting the existence of a small family of hsp70 genes in this species. Finally, trout hsp70 cDNA sequences cross-hybridized with restriction fragments in genomic DNA from HeLa cells, bovine liver, Caenorhabditis elegans, and D. melanogaster.     

Examination of heat shock protein mRNA accumulation in early Xenopus laevis embryos

Elevation of the incubation temperature of Xenopus laevis neurulae from 22 to 33-35 degrees C induced the accumulation of heat shock protein (hsp) 70 mRNA (2.7 kilobases (kb)) and a putative hsp 87 mRNA (3.2 kb). While constitutive levels of both hsp mRNAs were detectable in unfertilized eggs and cleavage-stage embryos, heat-induced accumulation was not observed until after the mid-blastula stage. Exposure of Xenopus laevis embryos to other stressors, such as sodium arsenite or ethanol, also induced a developmental stage-dependent accumulation of hsp 70 mRNA. To characterize the effect of temperature on hsp 70 mRNA induction, neurulae were exposed to a range of temperatures (27-37 degrees C) for 1 h. Heat-induced hsp 70 mRNA accumulation was first detectable at 27 degrees C, with relatively greater levels at 30-35 degrees C and lower levels at 37 degrees C. A more complex effect of temperature on hsp 70 mRNA accumulation was observed in a series of time course experiments. While continuous exposure of neurulae to heat shock (27-35 degrees C) induced a transient accumulation of hsp 70 mRNA, the temporal pattern of hsp 70 mRNA accumulation was temperature dependent. Exposure of embryos to 33-35 degrees C induced maximum relative levels of hsp 70 mRNA within 1-1.5 h, while at 30 and 27 degrees C peak hsp 70 mRNA accumulation occurred at 3 and 12 h, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Analysis of stress-induced gene expression in fish cell lines exposed to heavy meals and heat shock

We have examined the effect of heavy metals on the expression of two major groups of stress-induced proteins in fish cell lines: the 70 kDa heat-shock proteins (hsp70) and metallothioneins (MTs). The rainbow trout hepatoma (RTH) cell line synthesized the hsp70 protein in response to zinc and heat shock, while chinook salmon embryonic (CHSE) cells synthesized this protein in response to these inducers, as well as cadmium. The synthesis of this 70 kDa protein was correlated with the accumulation of hsp70 mRNA as measured by hybridization to a trout hsp70 gene probe. Heavy metals also induced the synthesis of MT in RTH cells. However, heat shock did not result in induction of MT and its mRNA. Unlike RTH cells, CHSE cells did not synthesize MT following exposure to cadmium or zinc. When these cells were treated with 5-azacytidine prior to heavy metal treatment, accumulation of MT mRNA was observed. Northern blot analysis of total RNA from 5-azacytidine treated CHSE cells, using a trout MT (tMT-B) cDNA probe, indicated that the time-course of induction and the maximal level of MT mRNA accumulation in response to cadmium and zinc paralleled that observed in RTH cells. Copper and dexamethasone were ineffective in inducing MT mRNA in 5-azacytidine-treated CHSE cells. These results indicate that MT is specifically induced in response to heavy metal treatment, whereas the synthesis of hsp70 appears to be a general stress response. Furthermore, MT is differentially regulated by heavy metals and dexamethasone in these cell lines and the expression of MT is cell-type-specific.     

Heat Shock RNA Levels in Brain and Other Tissues After Hyperthermia and Transient Ischemia

A number of studies have demonstrated increased synthesis of heat shock proteins in brain following hyperthermia or transient ischemia. In the present experiments we have characterized the time course of heat shock RNA induction in gerbil brain after ischemia, and in several mouse tissues after hyperthermia, using probes for RNAs of the 70-kilodalton heat shock protein (hsp70) family, as well as ubiquitin. A synthetic oligonucleotide selective for inducible hsp70 sequences proved to be the most sensitive indicator of the stress response whereas a related rat cDNA detected both induced RNAs and constitutively expressed sequences that were not strongly inducible in brain. Considerable polymorphism of ubiquitin sequences was evident in the outbred mouse and gerbil strains used in these studies when probed with a chicken ubiquitin cDNA. Brief hyperthermic exposure resulted in striking induction of hsp70 and several-fold increases in ubiquitin RNAs in mouse liver and kidney peaking 3 h after return to room temperature. The oligonucleotide selective for hsp70 showed equivalent induction in brain that was more rapid and transient than observed in liver, whereas minimal induction was seen with the ubiquitin and hsp70-related cDNA probes. Transient ischemia resulted in 5- to 10-fold increases in hsp70 sequences in gerbil brain which peaked at 6 h recirculation and remained above control levels at 24 h, whereas a modest 70% increase in ubiquitin sequences was noted at 6 h. These results demonstrate significant temporal and quantitative differences in heat shock RNA expression between brain and other tissues following hyperthermia in vivo, and indicate that hsp70 provides a more sensitive index of the stress response in brain than does ubiquitin after both hyperthermia and ischemia.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

T lymphocyte stress response. II. Protection of translation and DNA replication against some forms of stress by prior hyperthermic stress

We have compared the effects of a mild heat shock and febrile temperatures on heat-shock protein (hsp) synthesis and development of stress tolerance in T lymphocytes. Our previous studies demonstrated that febrile temperatures (less than or equal to 41 degrees C) induced the synthesis of hsp110, hsp90, and the constitutive or cognate form of hsp70 (hscp70; a weak induction of the strongly stress-induced hsp70 was also observed. In the studies reported herein, we demonstrate that a mild heat shock (42.5 degrees C) reverses this ratio; that is, hsp70 and not hscp70 is the predominate member of this family synthesized at this temperature. Modest heat shock also enhanced the synthesis of hsp110 and hsp90. In order to assess the relationship between hsp synthesis and the acquisition of thermotolerance, purified T cells were first incubated at 42.5 degrees C (induction temperature) and then subsequently subjected to a severe heat-shock challenge (45 degrees C, 30 min). T cells first incubated at a mild heat-shock temperature were capable of total protein synthesis at a more rapid rate following a severe heat shock than control cells (induction temperature 37 degrees C). This phenomenon, which has been previously termed translational tolerance, did not develop in cells incubated at the febrile temperature (induction temperature 41 degrees C). Protection of translation also extended to immunologically relevant proteins such as interleukin-2 and the interleukin-2 receptor. Because clonal expansion is a critical event during an immune response, the effects of hyperthermic stress on DNA replication (mitogen-induced T cell proliferation) was also evaluated in thermotolerant T cells. DNA synthesis in control cells (induction temperature 37 degrees C) was severely inhibited following heat-shock challenge at 44 degrees C or 45 degrees C; in contrast, T cells preincubated at 42.5 degrees C rapidly recovered their DNA synthetic capacity. T cells preincubated at a febrile temperature were moderately protected against hyperthermic stress. The acquisition of thermotolerance was also associated with enhanced resistance to chemical (ethanol)-induced stress but not to heavy metal toxicity (cadmium) or dexamethasone-induced immunosuppression. These studies suggest that prior hsp synthesis may protect immune function against some forms of stress (e.g., febrile episode) but would be ineffective against others such as elevated glucocorticoid levels which normally occur during an immune response.     

Constitutive heat shock protein 70 (HSC70) expression in rainbow trout hepatocytes: effect of heat shock and heavy metal exposure

The 70-kDa family of heat shock proteins plays an important role as molecular chaperones in unstressed and stressed cells. The constitutive member of the 70 family (hsc70) is crucial for the chaperoning function of unstressed cells, whereas the inducible form (hsp70) is important for allowing cells to cope with acute stressor insult, especially those affecting the protein machinery. In fish, the role of hsc70 in the cellular stress response process is less clear primarily because of the lack of a fish-specific antibody for hsc70 detection. In this study, we purified hsc70 to homogeneity from trout liver using a three-step purification protocol with differential centrifugation, ATP-agarose affinity chromatography and electroelution. Polyclonal antibodies to trout hsc70 generated in rabbits cross-reacted strongly with both purified trout hsc70 protein and also purified recombinant bovine hsc70. Two-dimensional electrophoresis followed by Western blotting confirmed that the isoelectric point of rainbow trout hsc70 was more acidic than hsp70. Using this antibody, we detected hsc70 content in the liver, heart, gill and skeletal muscle of unstressed rainbow trout. Primary cultures of trout hepatocytes subjected to a heat shock (+15 degrees C for 1 h) or exposed to either CuSO(4) (200 microM for 24 h), CdCl(2) (10 microM for 24 h) or NaAsO(2) (50 microM for 1 h) resulted in higher hsp70 accumulation over a 24-h period. However, hsc70 content showed no change with either heat shock or heavy metal exposure suggesting that hsc70 is not modulated by sublethal acute stressors in trout hepatocytes. Taken together, we have for the first time generated polyclonal antibodies specific to rainbow trout hsc70 and this antibody will allow for the characterization of the role of hsc70 in the cellular stress response process in fish.     

The effects of heat shock and acclimation temperature on hsp70 and hsp30 mRNA expression in rainbow trout: in vivo and in vitro comparisons

Heat shock (25° C) of 10° C-acclimated rainbow trout Oncorhynchus mykiss led to increases in heat shock protein 70 (hsp70) mRNA in blood, brain, heart, liver, red and white muscle, with levels in blood being amongst the highest. Hsp30 mRNA also increased with heat shock in all tissues with the exception of blood. When rainbow trout blood was heat shocked in vitro , both hsp70 and hsp30 mRNA increased significantly. In addition, these in vitro experiments demonstrated that blood from fish acclimated to 17° C water had a lower hsp70 mRNA heat shock induction temperature than did 5° C acclimated fish (20 v. 25° C). The hsp30 mRNA induction temperature (25° C), however, was unaffected by thermal acclimation. While increases in hsp70 mRNA levels in blood may serve as an early indicator of temperature stress in fish, tissue type, thermal history and the particular family of hsp must be considered when evaluating stress by these molecular means.     

Heat shock gene expression in Xenopus laevis A6 cells in response to heat shock and sodium arsenite treatments

Continuous exposure of a Xenopus laevis kidney epithelial cell line, A6, to either heat shock (33 degrees C) or sodium arsenite (50 microM) resulted in transient but markedly different temporal patterns of heat-shock protein (HSP) synthesis and HSP 70 and 30 mRNA accumulation. Heat-shock-induced synthesis of HSPs was detectable within 1 h and reached maximum levels by 2-3 h. While sodium arsenite induced the synthesis of some HSPs within 1 h, maximal HSP synthesis did not occur until 12 h. The pattern of HSP 70 and 30 mRNA accumulation was similar to the response observed at the protein level. During recovery from heat shock, a coordinate decline in HSPs and HSP 70 and 30 mRNA was observed. During recovery from sodium arsenite, a similar phenomenon occurred during the initial stages. However, after 6 h of recovery, HSP 70 mRNA levels persisted in contrast to the declining HSP 30 mRNA levels. Two-dimensional polyacrylamide gel electrophoresis revealed the presence of 5 HSPs in the HSP 70 family, of which two were constitutive, and 16 different stress-inducible proteins in the HSP 30 family. In conclusion, heat shock and sodium arsenite induce a similar set of HSPs but maximum synthesis of the HSP is temporally separated by 12-24 h.     

Polyunsaturated fatty acids enhance the heat induced stress response in rainbow trout (Oncorhynchus mykiss) leukocytes

The heat shock response has been studied extensively, yet the molecular signals that trigger the response remain elusive. The dogma of the heat shock response contends that denatured proteins initiate the response, but evidence is accumulating to point to a more complex system in which at least more than one signal is involved in this process. Thermal stress initiates changes in cellular phospholipid membrane physical state, which when acted upon by phospholipases may release lipid mediators that could serve as triggering signals during the heat shock response. We have examined the heat shock response in freshly isolated leukocytes from the pronephros of rainbow trout (Oncorhynchus mykiss). In this study, we show that leukocytes isolated from rainbow trout acclimated to 5 or 19°C express elevated levels of heat shock protein 70 (hsp70) mRNA when heat shocked at 5°C above their respective acclimation temperature and supplementation with exogenous docosahexaenoic acid or arachidonic acid followed by heat shock enhanced levels of hsp70 mRNA. The time course for docosahexaenoic acid induced enhancement of hsp70 mRNA was accelerated compared with heat shock alone, and staurosporine inhibited the docosahexaenoic acid induced increase of hsp70 mRNA. We also provide evidence that phospholipase A₂ is involved in the heat shock response.     

hsp47 and hsp70 gene expression is differentially regulated in a stress- and tissue-specific manner in zebrafish embryos

We have examined differences in the spatial and temporal regulation of stress-induced hsp47 and hsp70 gene expression following exposure of zebrafish embryos to heat shock or ethanol. Using Northern blot analysis, we found that levels of hsp47 and hsp70 mRNA were dramatically elevated during heat shock in 2-day-old embryos. In contrast, ethanol exposure resulted in strong upregulation of the hsp47 gene whereas hsp70 mRNA levels increased only slightly following the same treatment. Whole-mount in situ hybridization analysis revealed that hsp47 mRNA was expressed predominantly in precartilagenous cells, as well as several other connective tissue cell populations within the embryo following exposure to either stress. hsp70 mRNA displayed a very different cell-specific distribution. For example, neither stress induced hsp70 mRNA accumulation in precartilagenous cells. However, high levels of hsp70 mRNA were detectable in epithelial cells of the developing epidermis following exposure to heat shock, but not to ethanol. These cells did not express the hsp47 gene following exposure to either of these stresses. The results suggest the presence of different inducible regulatory mechanisms for these genes which operate in a cell- and stress-specific manner in zebrafish embryos. Dev. Genet. 21:123–133, 1997. © 1997 Wiley-Liss, Inc.     

Identification and characterisation of a regulatory region in the Toxoplasma gondii hsp70 genomic locus

Toxoplasma gondii is an important human and veterinary pathogen. The induction of bradyzoite development in vitro has been linked to temperature, pH, mitochondrial inhibitors, sodium arsenite and many of the other stressors associated with heat shock protein induction. Heat shock or stress induced activation of a set of heat shock protein genes, is characteristic of almost all eukaryotic and prokaryotic cells. Studies in other organisms indicate that heat shock proteins are developmentally regulated. We have established that increases in the expression of bag1/hsp30 and hsp70 are associated with bradyzoite development. The T. gondii hsp70 gene locus was cloned and sequenced. The regulatory regions of this gene were analysed by deletion analysis using beta-galactosidase expression vectors transiently transfected into RH strain T. gondii. Expression was measured at pH 7.1 and 8.1 (i.e. pH shock) and compared to the expression obtained with similar constructs using BAG1 and SAG1 promoters. A pH-regulated region of the Tg-hsp70 gene locus was identified which has some similarities to heat shock elements described in other eukaryotic systems. Green fluorescent protein expression vectors driven by the Tg-hsp70 regulatory region were constructed and stably transfected into T. gondii. Expression of green fluorescent protein in these parasites was induced by pH shock in those lines carrying the Tg-hsp70 regulatory constructs. Gel shift analysis was carried out using oligomers corresponding to the pH-regulated region and a putative DNA binding protein was identified. These data support the identification of a pH responsive cis-regulatory element in the T. gondii hsp70 gene locus. A model of the interaction of hsp70 and small heat shock proteins (e.g. BAG1) in development is presented.     

Loss of a calcium requirement for protein synthesis in pituitary cells following thermal or chemical stress

Ca2+ is required for the maintenance of high rates of translational initiation in GH3 pituitary cells (Chin, K.-V., Cade, C., Brostrom, C.O., Galuska, E.M., and Brostrom, M.A. (1987) J. Biol. Chem. 262, 16509-16514). Following thermal stress at 46 degrees C or chemical stress from exposure to sodium arsenite or 8-hydroxyquinoline, rates of amino acid incorporation in Ca2+-restored GH3 cells were reduced acutely to those of unstressed, Ca2+-depleted control preparations. Sodium arsenite treatment resulted in loss of ability to accumulate polysomes in response to Ca2+. Stressed cells allowed to recover for 2-8 h either with or without Ca2+ in the medium exhibited comparable, increasing rates of amino acid incorporation and the induction of heat shock proteins (hsp). Abolition of the Ca2+-dependent component of translation was proportional to the intensity of the stress. Mild thermal stress (41 degrees C) resulted in the induction of hsp 68 and the retention of Ca2+-dependent protein synthesis; hsp 68 was synthesized in a Ca2+-dependent manner. After arsenite stress, restoration of the Ca2+ requirement for protein synthesis occurred by 24 h, and was preceded by a transitional period during which polysomes accumulated in response to Ca2+ without concomitant increased rates of incorporation. Responses to stress are proposed to include an acute inhibition of normal protein synthesis involving the destruction of Ca2+-stimulated initiation and a protracted period of recovery involving synthesis of the hsp accompanied by Ca2+-independent amino acid incorporation and slowed peptide chain elongation.