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.     

Comparison of the effect of heat shock factor inhibitor, KNK437, on heat shock- and chemical stress-induced hsp30 gene expression in Xenopus laevis A6 cells

In this study, we compared the effect of KNK437 (N-formyl-3, 4-methylenedioxy-benzylidene-gamma-butyrolactam), a benzylidene lactam compound, on heat shock and chemical stressor-induced hsp30 gene expression in Xenopus laevis A6 kidney epithelial cells. Previously, KNK437 was shown to inhibit HSE-HSF1 binding activity and heat-induced hsp gene expression. In the present study, Northern and Western blot analysis revealed that pretreatment of A6 cells with KNK437 inhibited hsp30 mRNA and HSP30 and HSP70 protein accumulation induced by chemical stressors including sodium arsenite, cadmium chloride and herbimycin A. In A6 cells subjected to sodium arsenite, cadmium chloride, herbimycin A or a 33 degrees C heat shock treatment, immunocytochemistry and confocal microscopy revealed that HSP30 accumulated primarily in the cytoplasm. However, incubation of A6 cells at 35 degrees C resulted in enhanced HSP30 accumulation in the nucleus. Pre-treatment with 100 microM KNK437 completely inhibited HSP30 accumulation in A6 cells heat shocked at 33 or 35 degrees C as well as cells treated with 10 microM sodium arsenite, 100 microM cadmium chloride or 1 microg/mL herbimycin A. These results show that KNK437 is effective at inhibiting both heat shock- and chemical stress-induced hsp gene expression in amphibian cells.     

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.     

Examination of cadmium-induced expression of the small heat shock protein gene, hsp30, in Xenopus laevis A6 kidney epithelial cells

Cadmium is a highly toxic environmental pollutant that has been classified as a human carcinogen. Toxicological responses to cadmium exposure include respiratory diseases, neurological disorders and kidney damage. In the present study, we have characterized the effect of cadmium on the accumulation of the small heat shock protein (HSP), HSP30, in Xenopus laevis A6 kidney epithelial cells. Incubation of A6 cells with cadmium chloride induced the accumulation of HSP30 protein and hsp30 mRNA. While HSP70 protein and hsp70 mRNA accumulation were also induced, the relative levels of actin remained relatively unaffected. Elevated levels of HSP30 were detected in cells undergoing prolonged exposure of cells to cadmium chloride or in cells recovering from cadmium chloride treatment. Immunocytochemical analysis of cadmium chloride-treated A6 cells revealed HSP30 accumulation primarily in the cytoplasm in a punctate pattern supplemented with larger HSP30 staining structures. Also, HSP30 co-localized with the F-actin cytoskeleton at higher cadmium chloride concentrations. The combination of mild heat shock temperatures plus cadmium chloride concentrations employed in this study resulted in a synergistic accumulation of HSP30 protein and hsp30 mRNA. Finally, in contrast to heat shock, prior exposure of Xenopus A6 cells to cadmium chloride treatment, sufficient to induce the accumulation of HSPs, did not protect the cells against a subsequent thermal challenge.     

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.     

Intracellular localization of the heat shock protein, HSP110, in Xenopus laevis A6 kidney epithelial cells

Heat shock protein 110 (HSP110) is a large molecular mass chaperone that is part of the HSP70/DnaK superfamily. In the present study, we examined the accumulation of HSP110 in Xenopus laevis A6 kidney epithelial cells. Immunoblot analysis, using a homologous antibody, detected the presence of HSP110 in A6 cells maintained at 22 degrees C. The relative levels of HSP110 accumulation increased after heat shock or sodium arsenite treatment. Immunocytochemical analysis revealed that constitutively expressed HSP110 was localized in the cytoplasm in a diffuse granular pattern with enrichment in the nucleus. In A6 cells heat shocked at 33 degrees C or 35 degrees C for 2 to 4 h, HSP110 accumulation was enhanced and detected primarily in the cytoplasm as thread- or spindle-like structures. In contrast, HSP30 was not detected constitutively and heat shock treatment of A6 cells induced a relatively uniform punctate pattern primarily in the cytoplasm. Also, treatment of A6 cells at 35 degrees C for 6 h resulted in the presence of HSP110 and HSP30 enriched in the nucleus of most cells. Finally, A6 cells treated with 25 microM sodium arsenite produced very dense HSP110 structures primarily in the cytoplasm while HSP30 was enriched in the cytoplasm in a granular pattern.     

Examination of the expression of the heat shock protein gene, hsp110, in Xenopus laevis cultured cells and embryos

Eukaryotic organisms respond to various stresses with the synthesis of heat shock proteins (HSPs). HSP110 is a large molecular mass HSP that is part of the HSP70/DnaK superfamily. In this study, we have examined, for the first time, the expression of the hsp110 gene in Xenopus laevis cultured cells and embryos. Sequence analysis revealed that the protein encoded by the hsp110 cDNA exhibited 74% identity with its counterparts in mammals and only 27-29% with members of the Xenopus HSP70 family. Hsp110 mRNA and/or protein was detected constitutively in A6 kidney epithelial cells and was inducible by heat shock, sodium arsenite, and cadmium chloride. However, treatment with ethanol or copper sulfate had no detectable effect on hsp110 mRNA levels. Similar results were obtained for hsp70 mRNA except that it was inducible with ethanol. In Xenopus embryos, hsp110 mRNA was present constitutively during development. Heat shock-inducible accumulation of hsp110 mRNA occurred only after the midblastula stage. Whole mount in situ hybridization analysis revealed that hsp110 mRNA accumulation in control and heat shocked embryos was enriched in selected tissues. These studies demonstrate that Xenopus hsp110 gene expression is constitutive and stress inducible in cultured cells and developmentally- and tissue specifically-regulated during early embryogenesis.     

Sodium arsenite and cadmium chloride induction of proteasomal inhibition and HSP accumulation in Xenopus laevis A6 kidney epithelial cells

Sodium arsenite (NA) and cadmium chloride (CdCl(2)) are relatively abundant environmental toxicants that have multiple toxic effects including carcinogenesis, dysfunction of gene regulation and DNA and protein damage. In the present study, treatment of Xenopus laevis A6 kidney epithelial cells with concentrations of NA (20-30 μM) or CdCl(2) (100-200 μM) that induced HSP30 and HSP70 accumulation also produced an increase in the relative levels of ubiquitinated protein. Actin protein levels were unchanged in these experiments. In time course experiments, the levels of ubiquitinated protein and HSPs increased over a 24h exposure to NA or CdCl(2). Furthermore, treatment of cells with NA or CdCl(2) reduced the relative levels of proteasome chymotrypsin (CT)-like activity compared to control. Interestingly, pretreatment of cells with the HSP accumulation inhibitor, KNK437, prior to NA or CdCl(2) exposure decreased the relative levels of ubiquitinated protein as well as HSP30 and HSP70. A similar finding was made with ubiquitinated protein induced by proteasomal inhibitors, MG132 and celastrol, known to induce HSP accumulation in A6 cells. However, the NA- or CdCl(2)-induced decrease in proteasome CT-like activity was not altered by KNK437 pretreatment. This study has shown for the first time in poikilothermic vertebrates that NA and CdCl(2) can inhibit proteasomal activity and that there is a possible association between HSP accumulation and the mechanism of protein ubiquitination.     

Simultaneous exposure of Xenopus A6 kidney epithelial cells to concurrent mild sodium arsenite and heat stress results in enhanced hsp30 and hsp70 gene expression and the acquisition of thermotolerance

In this study, we examined the effect of concurrent low concentrations of sodium arsenite and mild heat shock temperatures on hsp30 and hsp70 gene expression in Xenopus A6 kidney epithelial cells. RNA blot hybridization and immunoblot analysis revealed that exposure of A6 cells to 1–10 µM sodium arsenite at a mild heat shock temperature of 30 °C enhanced hsp30 and hsp70 gene expression to a much greater extent than found with either stress individually. In cells treated simultaneously with 10 µM sodium arsenite and different heat shock temperatures, enhanced accumulation of HSP30 and HSP70 protein was first detected at 26 °C with larger responses at 28 and 30 °C. HSF1 activity was involved in combined stress-induced hsp gene expression since the HSF1 activation inhibitor, KNK437, inhibited HSP30 and HSP70 accumulation. Immunocytochemical analysis revealed that HSP30 was present in a granular pattern primarily in the cytoplasm in cells treated simultaneously with both stresses. Finally, prior exposure of A6 cells to concurrent sodium arsenite (10 µM) and heat shock (30 °C) treatment conferred thermotolerance since it protected them against a subsequent thermal challenge (37 °C). Acquired thermotolerance was not observed with cells treated with the two mild stresses individually.     

Withaferin A Induces Proteasome Inhibition,Endoplasmic Reticulum Stress,the Heat Shock Response and Acquisition of Thermotolerance

In the present study, withaferin A (WA), a steroidal lactone with anti-inflammatory and anti-tumor properties, inhibited proteasome activity and induced endoplasmic reticulum (ER) and cytoplasmic HSP accumulation in Xenopus laevis A6 kidney epithelial cells. Proteasomal inhibition by WA was indicated by an accumulation of ubiquitinated protein and a decrease in chymotrypsin-like activity. Additionally, immunoblot analysis revealed that treatment of cells with WA induced the accumulation of HSPs including ER chaperones, BiP and GRP94, as well as cytoplasmic/nuclear HSPs, HSP70 and HSP30. Furthermore, WA-induced an increase in the relative levels of the protein kinase, Akt, while the levels of actin were unchanged compared to control. Northern blot experiments determined that WA induced an accumulation in bip, hsp70 and hsp30 mRNA but not eIF-1α mRNA. Interestingly, WA acted synergistically with mild heat shock to enhance HSP70 and HSP30 accumulation to a greater extent than the sum of both stressors individually. This latter phenomenon was not observed with BiP or GRP94. Immunocytochemical analysis indicated that WA-induced BiP accumulation occurred mainly in the perinuclear region in a punctate pattern, while HSP30 accumulation occurred primarily in a granular pattern in the cytoplasm with some staining in the nucleus. Prolonged exposure to WA resulted in disorganization of the F-actin cytoskeleton as well as the production of relatively large HSP30 staining structures that co-localized with F-actin. Finally, prior exposure of cells to WA treatment, which induced the accumulation of HSPs conferred a state of thermal protection since it protected the F-actin cytoskeleton against a subsequent cytotoxic thermal challenge.     

Expression of the small heat shock protein gene, hsp30, in Rana catesbeiana fibroblasts

In the present study, we examined the expression of the Rana catesbeiana small heat shock protein gene, hsp30, in an FT fibroblast cell line. Northern and western blot analyses revealed that hsp30 mRNA or HSP30 protein was not present constitutively but was strongly induced at a heat shock temperature of 35 degrees C. However, treatment of FT cells with sodium arsenite at concentrations that induced hsp gene expression in other amphibian systems caused cell death. Non-lethal concentrations of sodium arsenite (10 microM) induced only minimal accumulation of hsp30 mRNA or protein after 12 h. Immunocytochemical analyses employing laser scanning confocal microscopy detected the presence of heat-inducible HSP30, in a granular or punctate pattern. HSP30 was enriched in the nucleus with more diffuse localization in the cytoplasm. The nuclear localization of HSP30 was more prominent with continuous heat shock. These heat treatments did not alter FT cell shape or disrupt actin cytoskeletal organization. Also, HSP30 did not co-localize with the actin cytoskeleton.     

Sodium arsenite induces heat shock protein 70 expression and protects against secretagogue-induced trypsinogen and NF-kappaB activation

Heat shock proteins (HSPs), induced by a variety of stresses, are known to protect against cellular injury. Recent studies have demonstrated that prior beta-adrenergic stimulation as well as thermal or culture stress induces HSP70 expression and protects against cerulein-induced pancreatitis. The goal of our current studies was to determine whether or not a non-thermal, chemical stressor like sodium arsenite also upregulates HSP70 expression in the pancreas and prevents secretagogue-induced trypsinogen and NF-kappaB activation. We examined the effects of sodium arsenite preadministration on the parameters of cerulein-induced pancreatitis in rats and then monitored the effects of preincubating pancreatic acini with sodium arsenite in vitro. Our results showed that sodium arsenite pretreatment induced HSP70 expression both in vitro and in vivo and significantly ameliorated the severity of cerulein-induced pancreatitis, as evidenced by the markedly reduced degree of hyperamylasemia, pancreatic edema, and acinar cell necrosis. Sodium arsenite pretreatment not only inhibited trypsinogen activation and the subcellular redistribution of cathepsin B, but also prevented NF-kappaB translocation to the nucleus by inhibiting the IkappaBalpha degradation both in vivo and in vitro. We also examined the effect of sodium arsenite pretreatment in a more severe model of pancreatitis induced by L-arginine and found a similarly protective effect. Based on our observations we conclude that, like thermal stress, chemical stressors such as sodium arsenite also induce HSP70 expression in the pancreas and protect against acute pancreatitis. Thus, non-thermal pharmacologically induced stress can help prevent or treat pancreatitis.     

Developmental regulation of heat shock protein synthesis and HSP 70 RNA accumulation during postimplantation rat embryogenesis

Exposure of postimplantation rat embryos on days 9, 10, 11, and 12 of gestation to an in vitro heat shock of 43 degrees C for 30 min results in the induction of heat shock proteins (HSPs) in day 9 and 10 embryos, a severely attenuated response in day 11 embryos, and no detectable response in day 12 embryos. The heat shock response in day 9 embryos (presomite stage) is characterized by the synthesis of HSPs with molecular weights of 28-78 kDa. In heat shocked day 10 embryos, two additional HSPs are induced (34 and 82 kDa). In addition, two HSPs present on day 9 are absent on day 10. In day 11 heat shocked embryos, only three HSPs (31, 39, and 69 kDa) are induced, while in day 12 embryos no detectable HSPs are induced. Northern blot analysis of HSP 70 RNA levels indicates that the accumulation of this RNA, but not actin RNA, varies depending on developmental stage at the time of exposure to heat as well as the duration of the heat shock. Day 9 embryos exhibit the most pronounced accumulation of HSP 70 RNA while embryos on days 10-12 exhibit an increasingly attenuated accumulation of HSP 70 RNA, particularly after the more acute exposures (43 degrees C for 30 or 60 min). Thus, the ability to synthesize HSP 70 and to accumulate HSP 70 RNA changes dramatically as rat embryos develop from day 9 to day 12 (presomite to 31-35 somite stages).     

The cytoplasmic pH, ATP content and total protein synthesis rate during heat-shock protein inducing treatments in yeast

In S. cerevisiae the induction of heat-shock protein (HSP) synthesis is accompanied by a decrease in the cytoplasmic and vacuolar pH as determined by means of [31P]NMR spectroscopy. The relationship of HSP synthesis and acidification of the cytoplasmic pH is dose-dependent under a variety of treatments (temperature increases (23-32 degrees C), addition of 2,4-dinitrophenol (greater than 1 mM), sodium arsenite (greater than 3.75 X 10(-5) M) or sodium cyanide (greater than 10 mM]. Changes in the intracellular pH occur within 5 min after treatment, attain a maximum within 30 min and are subsequently stable. HSPs 98, 85 and 70 show maximum synthesis rates 1-2 h after a 40 degrees C heat shock. The synthesis rates then decline. HSPs 56, 44 and 33 reveal a smaller and slower increase and almost no decrease in the synthesis rate within 4 h at 40 degrees C. The similar dose dependencies of HSP synthesis and cytoplasmic pH. as well as the immediate response of the pH, can also be demonstrated in the mitochondrial mutant of S. cerevisiae (Q0). This result indicates that the heat-shock response is mainly independent of intact oxidative phosphorylation. No correlation was observed between HSP synthesis rate and total intracellular ATP content.     

Food-deprivation induces HSP70 and HSP90 protein expression in larval gilthead sea bream and rainbow trout

Heat shock proteins (HSPs) expression is commonly used as indicators of cellular stress in animals. However, very little is known about either the expression patterns of HSPs or their role in the stress-tolerance phenomenon in early life stages of fish. To this end, we examined the impact of food-deprivation (12 h), reduced oxygen levels (3.5 mg/L for 1 h) and heat shock (HS: + 5 °C for 1 h) on HSP70 and HSP90 protein expression in early life stages of the gilthead sea bream (Sparus aurata), a warm-water aquaculture species. Also, we investigated HSP70 and HSP90 response to food-deprivation (7 days) in early life stages of rainbow trout (Oncorhynchus mykiss), a cool-water aquaculture species, and the tolerance of this larvae to heat shock (either + 5 or + 10 °C for 1 h). Our results clearly demonstrate that food-deprivation enhances HSP70 and HSP90 protein expression in larvae of both species. In gilthead sea bream larvae, the stressors-induced HSP70 and HSP90 (only in the reduced oxygen group) protein expression returned to unstressed levels after 24 h recovery. In fed trout larvae, a + 5 °C heat shock did not elevate HSP70 and HSP90 expression, whereas 100% mortality was evident with a + 10 °C HS. However, food-deprived trout larvae, which had higher HSP70 and HSP90 protein content, survived HS and showed HS-dependent increases in HSP70, but not HSP90 expression. Overall, HSP70 and HSP90 protein expression in early life stages of fish have the potential to be used as markers of nutritional stress, while elevation of the tissue HSPs content may be used as a means to increase stress tolerance during larval rearing.     

HSP_(70)基因表达对高粱育性的影响(英文)

高粱细胞质雄性不育系３１９７Ａ（３Ａ）在常温条件下是不育的（Ｆｉｇｓ．１１＆２）,经热激（４５℃）诱导不同程度地恢复了育性（Ｆｉｇｓ．１３＆４）,为研究其不育机理提供了线索。热激２ｈ后,３Ａ中即可产生一类线粒体热激蛋白（ＨＳＰｓ）。其中,分子量为７０ｋＤ的ＨＳＰ７０含量最高,也最为稳定。不过,３Ａ中ＨＳＰｓ的稳定性弱于保持系３１９７Ｂ（３Ｂ）（Ｆｉｇ．２,Ｐａｎｅｌｓ１～４）。放线菌素Ｄ抑制ＨＳＰｓ的合成,而氯霉素无此作用（Ｆｉｇ．２,Ｐａｎｅｌｓ５＆６）,表明：ＨＳＰｓ是由核基因编码、在细胞质中合成、再跨膜转运到线粒体中的。３Ａ幼穗经热激后,线粒体的总蛋白量猛增了２．７倍（Ｆｉｇ．３）,达到３Ｂ的水平,育性亦变为可育的。Ｆｉｇ．４表明：ＨＳＰ７０反义链ｃＤＮＡ（Ｒ１）能进入到３Ｂ花药细胞中,并与靶ＲＮＡ（ＨＳＣ７０ｍＲＮＡ）结合,而对照、正义链ｃＤＮＡ（Ｄ）链无此反应。由此、再增加一个通用保守序列的反义链ｃＤＮＡ（Ｒ２）、共两个探针（Ｒ１、Ｒ２）,可以检测到：３Ａ在常温下没有能力合成ＨＳＣ７０ｍＲＮＡ（Ｆｉｇ．５）,而在热激条件下,转变为有能力（Ｆｉｇ．６）。启示：３Ａ在热激条件下由不育转变为可育