Examination of KNK437- and quercetin-mediated inhibition of heat shock-induced heat shock protein gene expression in Xenopus laevis cultured cells

We examined the effect of quercetin (3,3',4',5,7-pentahydroxyflavon) and KNK437 (N-formyl-3,4-methylenedioxy-benzylidene-gamma-butyrolactam), a benzylidene lactam compound, on heat-induced heat shock protein (hsp) gene expression in Xenopus laevis A6 kidney epithelial cells. In previous studies, both quercetin and KNK437 inhibited heat shock factor activity resulting in a repression of hsp mRNA and protein accumulation in human cultured cells. In this first study of the effect of these hsp gene expression inhibitors in a non-mammalian cell line, we report that both quercetin and KNK437 reduced the heat shock-induced accumulation of hsp30, hsp47 and hsp70 mRNA in X. laevis cultured cells. However, these inhibitors had no effect on the relative level of a non-heat shock protein mRNA, ef1alpha, in either control or heat shocked cells. Western blot and immunocytochemical analyses revealed that quercetin partially inhibited HSP30 protein accumulation. In contrast, HSP30 protein was not detectable in KNK437-treated cells. Finally, treatment of A6 cells with KNK437 inhibited the heat shock-induced acquisition of thermotolerance, as determined by preservation of actin filaments and cellular morphology using immunocytochemistry and laser scanning confocal microscopy.     

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.     

Spatial pattern of constitutive and heat shock-induced expression of the small heat shock protein gene family, Hsp30, in Xenopus laevis tailbud embryos

We employed whole-mount in situ hybridization and immunohistochemistry to study the spatial pattern of hsp30 gene expression in normal and heatshocked embryos during Xenopus laevis development. Our findings revealed that hsp30 mRNA accumulation was present constitutively only in the cement gland of early and midtailbud embryos, while hsp30 protein was detected until at least the early tadpole stage. Heat shock-induced accumulation of hsp30 mRNA and protein was first observed in early and midtailbud embryos with preferential enrichment in the cement gland, somitic region, lens placode, and proctodeum. In contrast, cytoskeletal actin mRNA displayed a more generalized pattern of accumulation which did not change following heat shock. In heat shocked midtailbud embryos the enrichment of hsp30 mRNA in lens placode and somitic region was first detectable after 15 min of a 33 degrees C heatshock. The lowest temperature capable of inducing this pattern was 30 degrees C. Placement of embryos at 22 degrees C following a 1-h 33 degrees C heat shock resulted in decreased hsp30 mRNA in all regions with time, although enhanced hsp30 mRNA accumulation still persisted in the cement gland after 11 h compared to control. In late tailbud embryos the basic midtailbud pattern of hsp30 mRNA accumulation was enhanced with additional localization to the spinal cord as well as enrichment across the embryo surface. These studies demonstrate that hsp30 gene expression can be detected constitutively in the cement gland of tailbud embryos and that heat shock results in a preferential accumulation of hsp30 mRNA and protein in certain tissues.     

Spatial pattern of constitutive and heat shock‐induced expression of the small heat shock protein gene family,hsp30, in Xenopus laevis tailbud embryos

We employed whole‐mount in situ hybridization and immunohistochemistry to study the spatial pattern of hsp30 gene expression in normal and heatshocked embryos during Xenopus laevis development. Our findings revealed that hsp30 mRNA accumulation was present constitutively only in the cement gland of early and midtailbud embryos, while hsp30 protein was detected until at least the early tadpole stage. Heat shock‐induced accumulation of hsp30 mRNA and protein was first observed in early and midtailbud embryos with preferential enrichment in the cement gland, somitic region, lens placode, and proctodeum. In contrast, cytoskeletal actin mRNA displayed a more generalized pattern of accumulation which did not change following heat shock. In heat shocked midtailbud embryos the enrichment of hsp30 mRNA in lens placode and somitic region was first detectable after 15 min of a 33°C heatshock. The lowest temperature capable of inducing this pattern was 30°C. Placement of embryos at 22°C following a 1‐h 33°C heat shock resulted in decreased hsp30 mRNA in all regions with time, although enhanced hsp30 mRNA accumulation still persisted in the cement gland after 11 h compared to control. In late tailbud embryos the basic midtailbud pattern of hsp30 mRNA accumulation was enhanced with additional localization to the spinal cord as well as enrichment across the embryo surface. These studies demonstrate that hsp30 gene expression can be detected constitutively in the cement gland of tailbud embryos and that heat shock results in a preferential accumulation of hsp30 mRNA and protein in certain tissues. Dev. Genet. 25:365–374, 1999. © 1999 Wiley‐Liss, Inc.     

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.     

Examination of the stress-induced expression of the collagen binding heat shock protein, hsp47, in Xenopus laevis cultured cells and embryos

HSP47 is an endoplasmic reticulum (ER)-resident molecular chaperone involved in collagen production. This study examined the stress-induced pattern of hsp47 gene expression in Xenopus cultured cells and embryos. Sequence analysis revealed that protein encoded by the hsp47 cDNA exhibited 70-77% identity with fish, avian and mammalian HSP47. In A6 kidney epithelial cells hsp47 mRNA and HSP47 were present constitutively and inducible by heat shock but not ER stressors including tunicamycin and A23187, both of which enhanced BiP mRNA. Furthermore A23187 treatment inhibited constitutive accumulation of hsp47 mRNA and retarded heat-induced accumulation of hsp47 and hsp70 mRNA. Interestingly, hsp47 gene expression but not hsp70 or BiP mRNA accumulation was enhanced by treatment with a procollagen-specific stressor, beta-aminopropionitrile. In Xenopus embryos hsp47 mRNA was present constitutively throughout development. In tailbud embryos hsp47 mRNA was enriched in tissues associated with collagen production including notochord, somites and head region. Heat shock-induced accumulation of hsp47 mRNA was enhanced primarily in embryonic tissues already exhibiting hsp47 mRNA accumulation. These studies suggest that the pattern of Xenopus hsp47 gene expression is similar to hsp70 in response to heat shock but also displays unique features including a response to a procollagen-specific stressor and preferential expression in collagen-containing tissues.     

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.     

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.     

Phosphorylation-dependent cellular localization and thermoprotective role of heat shock protein 25 in hippocampal progenitor cells

The present study examined phosphorylation-dependent cellular localization and the thermoprotective role of heat shock protein (HSP) 25 in hippocampal HiB5 cells. HSP25 was induced and phosphorylated by heat shock (at 43 degrees C for 3 h). HSP25, which was located in the cytoplasm in the normal condition, translocated into the nucleus after the heat shock. Transfection experiments with hsp27 mutants in which specific serine phosphorylation residues (Ser(78) and Ser(82)) were substituted with alanines or aspartic acids showed that phosphorylation of HSP27 is accompanied by its nuclear translocation. Phosphorylation of mitogen-activated protein kinases (MAPKs) such as p38 MAPK and ERK was markedly increased by the heat shock, and SB203580 (a p38 MAPK kinase inhibitor) and/or PD098059 (a MEK inhibitor) inhibited the phosphorylation of HSP25, indicating that p38 MAPK and ERK are upstream regulators of HSP25 phosphorylation in the heat shock condition. In the absence of heat shock, actin filament stability was not affected by SB203580 and/or PD098059. Heat shock caused disruption of the actin filament and cell death when phosphorylation of HSP25 was inhibited by SB203580 and/or PD098059. In addition, actin filament was more stable in Asp(78,82)-hsp27 (mimics the phosphorylated form) transfected HiB5 cells than in the normal and Ala(78,82)-hsp27 (nonphosphorylative form) transfected cells. In accordance with actin filament stability, the survival rate against the heat shock increased markedly in Asp(15,78,82)-hsp27 expressing HiB5 cells but decreased in Ala(15,78,82)-hsp27 expressing cells. These results support the idea that phosphorylation of HSP25 is critical for the maintenance of actin filament and enhancement of thermoresistance. Interestingly, HSP25 was dephosphorylated and returned to cytoplasm in a recovery time-dependent manner. This phenomenon was accompanied by an increment of apoptotic cell death as determined by nuclear and DNA fragmentation and fluorescence-activated cell sorter analysis. These results suggest that nuclear-translocated HSP25 might function to protect nuclear structure, thereby preventing apoptotic cell death.     

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.     

Analysis of the expression and function of the small heat shock protein gene, hsp27, in Xenopus laevis embryos

In previous studies, the only small HSPs that have been studied in Xenopus laevis are members of the HSP30 family. We now report the analysis of Xenopus HSP27, a homolog of the human small HSP, HSP27. To date the presence of both hsp30 and hsp27 genes has been demonstrated only in minnow and chicken. Xenopus HSP27 cDNA encodes a 213 aa protein that contains an alpha-crystallin domain as well as a polar C-terminal extension. Xenopus HSP27 shares 71% identity with chicken HSP24 but only 19% identity with Xenopus HSP30C. Northern blot analysis revealed that Xenopus HSP27 gene expression was developmentally regulated. Constitutive and heat shock-induced hsp27 mRNA accumulation was first detectable at the early tailbud stage while HSP27 protein was detected at the tadpole stage. Furthermore, hsp27 mRNA was enriched in selected tissues under both control and heat shock conditions. Whole mount in situ hybridization analysis detected the presence of this message in the lens vesicle, heart, head, somites, and tail region. Purified recombinant HSP27 protein displayed molecular chaperone properties since it had the ability to inhibit heat-induced aggregation of target proteins including citrate synthase, malate dehydrogenase and luciferase. Thus, Xenopus HSP27, like HSP30, is a developmentally-regulated heat-inducible molecular chaperone.     

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.     

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.     

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.