Abnormal proteins as the trigger for the induction of stress responses: heat, diamide, and sodium arsenite

Thermotolerance and synthesis of heat shock proteins are induced in cells in response to a variety of environmental stresses. We examined the suggestion of Hightower (1980) that modifications of intracellular proteins may be the triggering event that induces heat shock protein synthesis and thermotolerance. We did so by modifying cellular proteins, using diamide, a sulfhydryl oxidizing agent, and dithio-bis (succinimidyl propionate), an agent that cross-links bifunctional amino groups. Both of these agents induced heat shock proteins and thermotolerance in CHO (HA-1) cells. Furthermore, we observed cross-resistance and self-tolerance with three seemingly unrelated stimuli (diamide, heat, and sodium arsenite). This observation suggests that the induction of protective responses to these stimuli is mediated by a common mechanism. The results support the hypothesis that production of abnormal proteins by various stresses induces the stress responses as well as tolerance.     

Both near ultraviolet radiation and the oxidizing agent hydrogen peroxide induce a 32-kDa stress protein in normal human skin fibroblasts

We have analyzed the pattern of protein synthesis in solar near ultraviolet (334 nm, 365 nm) and near visible (405 nm) irradiated normal human skin fibroblasts. Two hours after irradiation we find that one major stress protein of approximately 32 kDa is induced in irradiated cells. This protein is not induced by ultraviolet radiation at wavelengths shorter than 334 nm and is not inducible by heat shock treatment of these cells. Although sodium arsenite, diamide, and menadione all induced a 32-kDa protein, they also induced the major heat shock proteins. In contrast, the oxidizing agent, hydrogen peroxide, induced the low molecular weight stress protein without causing induction of the major heat shock proteins. A comparison of the 32-kDa proteins induced by sodium arsenite, H2O2, and solar near ultraviolet radiation using chemical peptide mapping shows that they are closely related. These results imply that the pathways for induction of the heat shock response and the 32-kDa protein are not identical and suggest that, at least in the case of radiation and treatment with H2O2, the 32-kDa protein might be induced in response to cellular oxidative stress. This conclusion is supported by the observation that depletion of endogenous cellular glutathione prior to solar near ultraviolet irradiation lowers the fluence threshold for induction of the 32-kDa stress protein.     

Induction of thermotolerance and enhanced heat shock protein synthesis in chinese hamster fibroblasts by sodium arsenite and by ethanol

Synthesis of a family of proteins called “heat shock” proteins is enhanced in cells in response to a wide variety of environmental stresses. This suggests that these proteins may have functions essential to cell survival under stressful conditions. A causative relationship between heat shock protein synthesis and development of thermotolerance would imply that agents known to induce heat shock protein synthesis, such as sodium arsenite, also induce thermotolerance. Conversely, agents known to induce thermotolerance, such as ethanol, would also enhance heat shock protein synthesis. To test this hypothesis, I have examined the effect of sodium arsenite or ethanol treatment on protein synthesis and cell survival in Chinese hamster ovary HA-1 cells. After either sodium arsenite or ethanol treatment, the synthesis of heat shock proteins was greatly enhanced over that of untreated cells. In parallel, cell survival was increased as much as 10⁴-fold when cells exposed to either agent were challenged by a subsequent heat treatment. The synthesis of heat shock proteins correlated well with the development of thermotolerance. A qualitative analysis of individual proteins suggests that the synthesis of 70,000 and 87,000 molecular weight proteins most closely mirrored the development of thermotolerance. The results, therefore, strongly reinforce the hypothesis that a causal relationship exists between the enhanced synthesis of heat shock protein and cell survival under specific stresses.     

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.     

Heat shock response of the rat lens

The sequence relationship between the small heat shock proteins and the eye lens protein alpha-crystallin (Ingolia, T. D., and E. E. Craig, 1982, Proc. Natl. Acad. Sci. USA, 79: 2360-2364) prompted us to subject rat lenses in organ culture to heat shock and other forms of stress. The effects on protein synthesis were followed by labeling with [35S]methionine and analysis by one- and two-dimensional gel electrophoresis and fluorography. Heat shock gave a pronounced induction of a protein that could be characterized as the stress protein SP71. This protein probably corresponds to the major mammalian heat shock protein hsp70. Also two minor proteins of 16 and 85 kD were induced, while the synthesis of a constitutive heat shock-related protein, P73, was considerably increased. The synthesis of SP71 started between 30 and 60 min after heat shock, reached its highest level after 3 h, and had stopped again after 8 h. In rat lenses that were preconditioned by an initial mild heat shock, a subsequent shock did not cause renewed synthesis of SP71. This effect resembles the thermotolerance phenomenon observed in cultured cells. The proline analogue azetidine-2-carboxylic acid, zinc chloride, ethanol, and calcium chloride did not, under the conditions used, induce stress proteins in the rat lens. Sodium arsenite, however, had very much the same effects as heat shock. Calcium ionophore A23187 specifically and effectively induced the synthesis of the glucose-regulated protein GRP78. No special response to stress on crystallin synthesis was noticed.     

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.     

Heat shock induces intestinal-type alkaline phosphatase in rat IEC-18 cells

We demonstrate a previously unknown regulation for intestinal-type alkaline phosphatase (IAP) as a heat shock protein (HSP). Heat shock to rat intestinal epithelial cells (IEC)-18 at 43 degrees C induced the expression of IAP-I and HSP72 mRNAs time dependently (<60 min) but did not induce expression of IAP-II, tissue nonspecific-type alkaline phosphatase (TNAP), or HSP90 as determined by the RT-PCR method. To confirm the identity of the IAP-I gene, we sequenced the amplification product of IAP-I and found the gene to have 99% homology with the sequence of the IAP-I gene in rat intestine. Under the subculture conditions used, no IAP protein was detected in IEC-18 cells, but it became detectable as a 62-kDa band on a Western blot after heat shock. IAP-I was also induced by sodium arsenite, which generates reactive oxygen species and is an inducer of members of the HSP family. Glutathione suppressed activating protein-1 and cAMP response element-binding protein activation caused by heat shock but did not suppress the expression of IAP-I. These results suggest that cellular stress induces the elevation of IAP-I mRNA and protein synthesis. IAP-I may play an important role as a dephosphorylating enzyme under stress conditions.     

Dephosphorylation of S6 and expression of the heat shock response in Drosophila melanogaster.

A basic ribosomal phosphoprotein of 30,000 molecular weight was rapidly dephosphorylated in cultured Drosophila melanogaster cells heat shocked at 37 degrees C. The protein was associated with the 40S ribosomal subunit and had an electrophoretic mobility similar to that of purified rat liver protein S6 on basic two-dimensional polyacrylamide gels as well as a similar partial proteolysis peptide map. In logarithmically growing cultures, this D. melanogaster S6 protein appeared to have a single phosphorylated species consisting of 30 to 40% of the total cellular S6. Thus, the nearly complete dephosphorylation of this protein observed in heat shock involves a large fraction of the cellular S6. The significance of this dephosphorylation in the expression of the heat shock response was investigated by examining the phosphorylation status of S6 in recovery from heat shock and in response to chemical inducers of the heat shock response. During recovery from a 30-min heat shock, the recovery of normal protein synthesis was almost complete in 2 to 4 hr, whereas there was no significant rephosphorylation of S6 for 8 h. Two chemical inducers of the heat shock response, canavanine and sodium arsenite, induced the synthesis of heat shock proteins in D. melanogaster cells. Sodium arsenite also caused an inhibition of normal protein synthesis similar to that observed in heat shock. Neither agent, however, caused significant dephosphorylation of S6. These results suggest that the dephosphorylation of S6, although invariably observed in heat-shocked cells, may in some cases be dissociated from both the induction of heat shock protein synthesis and the turnoff of normal protein synthesis which occur in a heat shock response.     

Heat and sodium arsenite act synergistically on the induction of heat shock gene expression in Xenopus laevis A6 cells

Heat shock protein (HSP) synthesis was studied in the Xenopus epithelial cell line A6 in response to heat and sodium arsenite, either singly or together. Temperatures of 33-35 degrees C consistently brought about the synthesis of HSPs at 87, 73, 70, 54, 31, and 30 kilodaltons (kDa), whereas sodium arsenite at 25-100 microM induced the synthesis of HSPs at 73 and 70 kDa. In cultures exposed to 10 microM sodium arsenite at 30 degrees C, HSP synthesis in the 68- to 73-kDa and 29- to 31-kDa regions was much greater than the HSP synthesis in response to each treatment individually. RNA dot blot analysis using homologous genomic subclones revealed that heat shock induced the accumulation of HSP 70 and 30 mRNAs. The sizes of the HSP 70 and 30 mRNAs determined by Northern hybridization were 2.7 and 1.5 kilobases, respectively. Sodium arsenite (10-100 microM) also induced the accumulation of both HSP 70 and 30 mRNAs. Finally, a mild heat shock (30 degrees C) plus a low concentration of sodium arsenite (10 microM) acted synergistically on HSP 70 and 30 mRNA accumulation in A6 cells. Thus sodium arsenite and heat act synergistically at the level of both HSP synthesis and HSP mRNA accumulation.     

Intracellular localization of Xenopus small heat shock protein, hsp30, in A6 kidney epithelial cells

Small heat shock proteins (shsps) are molecular chaperones that are inducible by environmental stress. In this study, immunocytochemical analysis and laser scanning confocal microscopy revealed that the shsp family, hsp30, was localized primarily in the cytoplasm of Xenopus A6 kidney epithelial cells after heat shock or sodium arsenite treatment. Heat shock-induced hsp30 was enriched in the perinuclear region with some immunostaining in the nucleus but not in the nucleolus. In sodium arsenite-treated cells hsp30 was enriched towards the cytoplasmic periphery as well as showing some immunostaining in the nucleus. At higher heat shock temperatures (35 degrees C) or after 10 microM sodium arsenite treatment, the actin cytoskeleton displayed some disorganization that co-localized with areas of hsp30 enrichment. Treatment of A6 cells with 50 microM sodium arsenite induced a collapse of the cytoskeleton around the nucleus. These results coupled with previous studies suggest that stress-inducible hsp30 acts as a molecular chaperone primarily in the cytoplasm and may interact with cytoskeletal proteins.     

Effect of prostaglandin A1, arsenite and aspirin on stress proteins response in mosquito cells

The stress response of eukaryotic cells is characterized by changes in the metabolism of responding cells, most notably by increased synthesis of a group of proteins known as heat shock (HSP) proteins In this paper the effect of prostaglandin A1 (PGA1), arsenite and aspirin in Aedes albopictus cells was investigated. In cells treated with PGA1 (10 microg/ml) we observed the induction of several polypeptides with molecular masses of 87, 80, 70, 57, 29 and 23 kDa. Immunoblot analysis revealed that arsenite induces a marked synthesis of HSP70, and aspirin administered during the hyperthermic treatment caused a small increase of HSP70 synthesized.     

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.     

Differences in preferential synthesis and redistribution of HSP70 and HSP28 families by heat or sodium arsenite in Chinese hamster ovary cells

Since both heat and sodium arsenite induce thermotolerance, we investigated the differences in synthesis and redistribution of stress proteins induced by these agents in Chinese hamster ovary cells. Five major heat shock proteins (HSPs; Mr 110, 87, 70, 28, and 8.5 kDa) were preferentially synthesized after heat for 10 min at 45.5 degrees C, whereas four major HSPs (Mr 110, 87, 70, and 28 kDa) and one stress protein (33.3 kDa) were preferentially synthesized after treatment with 100 microM sodium arsenite (ARS) for 1 hr. Two HSP families (HSP70a,b,c, and HSP28a,b,c) preferentially relocalized in the nucleus after heat shock. In contrast, only HSP70b redistributed into the nucleus after ARS treatment. Furthermore, the kinetics of synthesis of each member of HSP70 and HSP28 families and their redistribution were different after these treatments. The maximum rates of synthesis of HSP70 and HSP28 families, except HSP28c, were 6-9 hr after heat shock, whereas those of HSP70b and HSP28b,c were 0-2 hr after ARS treatment. In addition, the maximum rates of redistribution of HSP70 and HSP28 families occurred 3-6 hr after heat shock, whereas that of HSP70b occurred immediately after ARS treatment. The degree of redistribution of HSP70b after ARS treatment was significantly less than that after heat treatment. These results suggest that heat treatment but not sodium arsenite treatment stimulates the entry of HSP70 and HSP28 families into the nucleus.     

Arsenite stabilizes IkappaBalpha and prevents NF-kappaB activation in IL-1 beta-stimulated Caco-2 cells independent of the heat shock response

Recent studies suggest that sodium arsenite downregulates NF-kappaB activity by inhibiting phosphorylation and subsequent degradation of IkappaBalpha. Many effects of sodium arsenite are secondary to induction of heat shock proteins. The role of the heat shock response in arsenite-induced inhibition of NF-kappaB, however, is not known. We examined the involvement of the heat shock response in arsenite-induced inhibition of NF-kappaB activity in IL-1beta-stimulated Caco-2 cells, a human colorectal adenocarcinoma cell line with enterocytic properties. Treatment of the cells with IL-1beta resulted in increased IkappaB kinase activity, reduced levels of IkappaBalpha and increased NF-kappaB DNA binding activity. Sodium arsenite blocked all of these responses to IL-1beta without inducing changes in heat shock factor activity or heat shock protein levels. Results from additional experiments showed that the protective effect of sodium arsenite on IkappaBalpha was not influenced by the oxygen radical scavenger catalase or by inhibitors of the MAP-kinase signaling pathway. The present results suggest that sodium arsenite stabilizes IkappaBalpha and prevents NF-kappaB activation in IL-1beta-stimulated Caco-2 cells independent of the heat shock response. In addition, stabilization of IkappaBalpha by sodium arsenite does not require oxygen radical formation or activation of the MAP kinase signaling pathway.     

Involvement of arachidonic acid in chemical stress-induced interleukin-6 synthesis in osteoblast-like cells: comparison with heat shock protein 27 induction

In a previous study, we have demonstrated that sodium arsenite (arsenite) as chemical stress stimulates heat shock protein 27 (HSP27) induction and arachidonic acid release in osteoblast-like MC3T3-E1 cells, and that the response of HSP27 induction is coupled with metabolic activity of the arachidonic acid cascade. In the present study, we examined the effect of exposure to arsenite on the synthesis of interleukin-6 (IL-6) in these cells. Arsenite induced the synthesis of IL-6 after 6 h from the stimulation up to 48 h. The effect of arsenite on IL-6 synthesis was dose-dependent in the range between 10 and 500 microM. The arsenite-induced IL-6 synthesis was enhanced by the pretreatment with indomethacin, an inhibitor of cyclooxygenase. Nordihydroguaiaretic acid, a lipoxygenase inhibitor, significantly amplified the arsenite-induced IL-6 synthesis. Melittin, an activator of phospholipase A2, which by itself hardly affected the levels of IL-6, markedly enhanced the arsenite-induced IL-6 synthesis. These results strongly suggest that chemical stress induces IL-6 synthesis in osteoblasts, and that the IL-6 synthesis is coupled to the arachidonic acid cascade as well as the HSP27 induction by arsenite.     

Heat shock gene expression and cytoskeletal alterations in mouse neuroblastoma cells

The cytoskeleton of neuroblastoma cells, clone Neuro 2A, is altered by two stress conditions: heat shock and arsenite treatment. Microtubules are reorganized, intermediate filaments are aggregated around the nucleus, and the number of stress fibers is reduced. Since both stress modalities induce similar cytoskeletal alterations, no thermic denaturation of one or more cytoskeletal components can be involved in this process. Heat shock proteins are induced both by heat and by arsenite. However, cells treated with arsenite synthesize hsp28 which is not detected in heat-treated cells. Synthesis of all hsps is prevented by addition of actinomycin D or cycloheximide. Under these conditions no alterations are observed in the organization of microtubules and intermediate filaments during heat or arsenite treatment. However, these drugs are not able to prevent the rapid loss of stress fibers. A re-formation of the cytoskeleton during the recovery period proceeds within 3 h and is also found to occur in the presence of a protein synthesis inhibitor. These data suggest that reorganization of microtubules and intermediate filaments during a stress treatment requires the synthesis of a new protein(s), probably hsp(s).     

Reduced activity of the interferon-induced double-stranded RNA-dependent protein kinase during a heat shock stress

Previous studies have shown that the antiviral response induced by interferon in murine cells could be degraded after a heat shock. Here we have confirmed that a similar effect occurs also in interferon-treated human HeLa cells subjected to a heat shock. In addition, we have investigated the fate of the interferon-induced, double-stranded RNA-dependent protein kinase in heat-shocked cells. This protein kinase is a Mr 68,000 protein (p68 kinase) which, when autophosphorylated, catalyzes phosphorylation of the protein synthesis eukaryotic initiation factor-2, thus mediating inhibition of protein synthesis. After heat shock of interferon-treated HeLa cells, the double-stranded RNA-dependent autophosphorylation of p68 kinase in cytoplasmic extracts is greatly reduced whereas the phosphorylation of other cellular proteins is not affected. In vivo, autophosphorylation of p68 kinase is also reduced in heat-shocked cells whereas there is no apparent effect on the phosphorylation state of other proteins. In such cells, the interferon-mediated antiviral response becomes modified according to the virus challenge, i.e. these cells remain resistant to vesicular stomatitis virus but become partially sensitive to encephalomyocarditis virus (EMCV) infection. The reduction in the activity of p68 kinase is due to its reduced nonionic detergent solubility occurring during the heat shock period. The resultant reduced detergent extractibility of p68 kinase is dependent on the intensity of the thermal stress. In contrast to the effect after a heat shock, arsenite treatment of interferon-treated HeLa cells induces heat shock proteins, but neither modifies the antiviral response nor affects the extractibility of p68 kinase. These results indicate that the degradation of the anti-EMCV response and reduced p68 kinase activity occur in response to heat treatment independently of the induction of heat shock proteins. The role of p68 kinase in the mechanism of the antiviral response against EMCV and vesicular stomatitis virus is discussed.     

Cell cycle-specific effects of sodium arsenite and hyperthermic exposure on incorporation of radioactive leucine and phosphate by stress proteins from mouse lymphoma cell nuclei

Cultured mouse lymphoma cells incorporated [3H]leucine and [32P]phosphate into nuclear stress proteins within 3 h after exposure to either elevated temperature (45 degrees C) or sodium arsenite. Radiolabeled proteins were detected by autoradiography after two-dimensional polyacrylamide gel electrophoresis. To determine the cell cycle stage specificity of labeling, nuclei were isolated and sorted into two cell cycle phases using a fluorescent activated cell sorter. After either heat shock or sodium arsenite treatment, the majority of [3H]leucine incorporation into stress proteins occurred during the G0 + G1 phase with minimal labeling in the G2 phase. On the other hand, 32P labeling of stress proteins occurred in both the G0 + G1 and G2 phases after exposure to sodium arsenite, while incorporation of 32P was limited after heat stress. Following sodium arsenite treatment, a distinct set of four stress proteins (80-84 kDa) was detected with [3H]leucine only in G0 + G1 phase, but with [32P]phosphate these stress proteins were labeled in both G0 + G1 and G2. There was differential [32P]phosphate labeling between proteins of the 80-84 kDa set during cell cycling. Individual proteins of this set were isolated from gel plugs after sodium arsenite or heat-shock treatment. Coelectrophoresis of proteins from the two treatment groups showed that they had similar electrophoretic mobilities. All four proteins of the 80-84 kDa set (sodium arsenite induced) possessed similar polypeptide maps after digestion with V8 protease. Cytofluorometric analysis demonstrated a reduction in the number of nuclei in both S and G2 phases of the cell cycle two h after heat shock, but not following sodium arsenite treatment. However, there was a significant depression in the number of nuclei in S and G2 4 h after exposure to sodium arsenite and very modest labeling with 32P of stress proteins was observed at this time.