Positive and negative regulation of basal expression of a yeast HSP70 gene.

The SSA1 gene, one of the heat-inducible HSP70 genes in the yeast Saccharomyces cerevisiae, also displays a basal level of expression during logarithmic growth. Multiple sites related to the heat shock element (HSE) consensus sequence are present in the SSA1 promoter region (Slater and Craig, Mol. Cell. Biol. 7:1906-1916, 1987). One of the HSEs, HSE2, is important in the basal expression of SSA1 as well as in heat-inducible expression. A promoter containing a mutant HSE2 showed a fivefold-lower level of basal expression and altered kinetics of expression after heat shock. A series of deletion and point mutations led to identification of an upstream repression sequence (URS) which overlapped HSE2. A promoter containing a mutation in the URS showed an increased level of basal expression. A URS-binding activity was detected in yeast whole-cell extracts by a gel electrophoresis DNA-binding assay. The results reported in this paper indicate that basal expression of the SSA1 promoter is determined by both positive and negative elements and imply that the positively acting yeast heat shock factor HSF is responsible, at least in part, for the basal level of expression of SSA1.     

Differential gene expression of HSC70/HSP70 in yellowtail cells in response to chaperone-mediated autophagy

A cell line derived from the tailfin of the marine teleost yellowtail fish Seriola quinqueradiata was established to examine cellular temperature regulation in an ectothermic animal. Three cytosolic members of the HSP70 family, heat-shock cognate proteins HSC70-1, HSC70-2 and heat-shock protein HSP70, were isolated from cultured yellowtail cells as stress-responsive biomarkers. Expression of hsp70 was heat-inducible, in contrast to the hsc70-1 gene product, which was expressed constitutively. In addition, expression of hsc70-2 was only induced under severe heat-shock conditions. Subcellular fractionation and immunocytochemistry showed localization of HSC70/HSP70 in the lysosomes, indicating that chaperone-mediated autophagy is induced by heat shock. Thus, chaperone-mediated autophagy is assisted by HSC70/HSP70, and heat-inducible expression of the genes encoding these proteins may be responsible for survival and adaptation under heat-shock conditions in fish cells.     

HSP70 expression and its role in preeclamptic stress

Preeclampsia, a hypertensive pregnancy-specific disorder, has long been analyzed for its association with cellular stress. It still remains one of the most serious complications of pregnancy. It is a multi-system disorder that affects maternal vascular function and fetal growth. The physiopathology of preeclampsia is still unclear, but an imbalance between reactive oxygen species (ROS) and antioxidants, appears to be an important contributing factor. Oxidative stress has been increasingly postulated as a major contributor to endothelial dysfunction in preeclampsia (PE). The ROS promotes lipid oxidation and are known to induce stress proteins, such as hemeoxygenase 1 (HO-1) and heat-shock protein 70 (HSP70). Embryonic and placental cells are highly sensitive to oxidative stress due to their proliferate nature. Endothelial cell dysfunction is suggested to be a part of wider maternal inflammatory reaction responsible for the clinical syndrome of preeclampsia. Part of the dysfunction in endothelial cell and trophoblast is attributed to oxidative stress developed during pregnancy. The disequilibrium in compensatory antioxidant control is proposed as a causative mechanism in the pathophysiology of preeclampsia. HSP70 acts as the secondary line of defense in systems with compromised antioxidant function. This article reviews the differential expression of HSP70 and the effect of mint-tea therapy to modulate preeclamptic oxidative damage.     

Regulation of HSP70 and HSP28 gene expression: absence of compensatory interactions

We have previously reported the lack of HSP28 gene expression during acute and chronic thermotolerance development in L929 cells (J Cell Physiol 152: 118–125, 1992; Cancer Res 52: 5787, 1992). In contrast to HSP28, an extremely high level of inducible HSP70 synthesis was observed. These results led us to investigate the possibility of compensatory interactions between HSP70 and HSP28. To test the hypothesis, L929 cells were transfected with the human HSP28 gene contained in plasmid pCMV27. Data from Western blot and two-dimensional gel electrophoresis of [³H] leucine and [³²P] orthophosphate-labeled proteins showed the synthesis and phosphorylation of HSP28 in transfected cells after heating at 45°C for 10 min. However, the expression of constitutive and inducible HSP70 genes, along with the synthesis of their proteins, was not decreased after heat shock. These results suggest an independent regulation of HSP28 and HSP70 gene expression.     

Seasonal variation in expression pattern of genes under HSP70

Heat shock protein 70 (HSP70) is one of the most abundant and best characterized heat shock protein family that consists of highly conserved stress proteins, expressed in response to stress, and plays crucial roles in environmental stress tolerance and adaptation. The present study was conducted to identify major types of genes under the HSP70 family and to quantify their expression pattern in heat- and cold-adapted Indian goats (Capra hircus) with respect to different seasons. Five HSP70 gene homologues to HSPA8, HSPA6, HSPA1A, HSPA1L, and HSPA2 were identified by gene-specific primers. The cDNA sequences showed high similarity to other mammals, and proteins have an estimated molecular weight of around 70 kDa. The expression of HSP70 genes was observed during summer and winter. During summer, the higher expression of HSPA8, HSPA6, and HSPA1A was observed, whereas the expression levels of HSPA1L and HSPA2 were found to be lower. It was also observed that the expression of HSPA1A and HSPA8 was higher during winter in both heat- and cold-adapted goats but downregulates in case of other HSPs. Therefore, both heat and cold stress induced the overexpression of HSP70 genes. An interesting finding that emerged from the study is the higher expression of HSP70 genes in cold-adapted goats during summer and in heat-adapted goats during winter. Altogether, the results indicate that the expression pattern of HSP70 genes is species- and breed-specific, most likely due to variations in thermal tolerance and adaptation to different climatic conditions.     

Structure and expression of the three MHC-linked HSP70 genes

A duplicated locus encoding the major heat shock-induced protein HSP70 is located in the major histocompatibility complex (MHC) class III region 92 kilobases (kb) telomeric to the C2 gene. Nucleotide sequence analysis of the two intronless genes, HSP70-1 and HSP70-2, has shown that they encode an identical protein product of 641 amino acids. A third intronless gene, HSP70-Hom, has also been identified 4 kb telomeric to the HSP70-1 gene. This encodes a more basic protein of 641 amino acids which has 90% sequence similarity with HSP70-1. In order to investigate the expression of the three (MHC)-linked HSP70 genes individually by northern blot analysis, we have isolated locus-specific probes from the 3 untranslated regions of the genes. The HSP70-1 and HSP70-2 genes have been shown to be expressed at high levels as a 2.4 kb mRNA in cells heat-shocked at 42°C. HSP70-1 is also expressed constitutively at very low levels. The HSP70-Hom gene, which has no heat shock consensus sequence in its 5 flanking sequence, is expressed as a 3 kb mRNA at low levels both constitutively and following heat shock.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession numbers M34267-9.Address correspondence and offprint requests to: R. D. Campbell.     

Specific activation of the human HSP70 promoter by copper sulfate in mosaic transgenic zebrafish

Heat shock proteins (HSPs) play a central role in cell protection and repair upon stresses, such as that caused by heat and heavy metals. Copper sulfate inducibility of a pHhsp70 construct expressing the enhanced green fluorescent protein (EGFP) gene under the control of the exogenous human hsp70 promoter was tested in transfected CHSE 214 cells and transgenic zebrafish (Danio rerio). We developed a transient expression system, using mosaically transgenic zebrafish, which allows rapid analysis of transgenic expression. Transfected CHSE 214 cells which had been exposed to 250 nM and 2.5 microM copper sulfate for up to 24h showed increased EGFP expression in a dose-dependent manner. The 1.5 microM copper sulfate caused stronger EGFP fluorescence than the 1.0 microM copper sulfate in transgenic zebrafish. Most of the expression was spotty and was detected in the gills, dorsal and ventral retina, myotubes of the trunk, and skin epithelium. Transgenic zebrafish exposed to copper sulfate exhibited gross dysmorphogenesis, edema and trunk abnormalities, such as spinal lordosis, in vertebral development 5 days after fertilization. This transgenic zebrafish system was sensitive enough to detect copper sulfate at doses below the median lethal concentration (the LC50 was calculated to be 1.2 microM (95% confidence interval of 0.6-1.9 microM)). These results indicate that zebrafish could be useful transgenic biosensor systems for the detection of xenobiotic toxicants in the environment.     

Correlation of HSP70 expression and cell viability following thermal stimulation of bovine aortic endothelial cells

Thermal preconditioning protocols for cardiac cells were identified which produce elevated HSP70 levels while maintaining high cell viability. Bovine aortic endothelial cells were heated with a water bath at temperatures ranging from 44 to 50 degrees C for periods of 1-30 min. Thermal stimulation protocols were determined which induce HSP70 expression levels ranging from 2.3 to 3.6 times the control while maintaining cell viabilities greater than 90%. An Arrhenius injury model fit to the cell damage data yielded values of A = 1.4 X 10(66) s(-1) and Ea = 4.1 X 10(5) J/mol. Knowledge of the injury parameters and HSP70 kinetics will enhance dosimetry guideline development for thermal stimulation of heat shock proteins expression in cardiac tissue.     

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.     

Cloning and expression of HSP70 gene of sipuncula Phascolosoma esculenta

In this study the gene encoding HSP70 was isolated from Phascoloma esculenta by homologous cloning and rapid amplification of cDNA ends (RACE). The full-length of cDNA (2520 bp) consists of a 5′-terminal untranslated region (UTR) (125 bp), a 3′-terminal UTR (421 bp) with a canonical polyadenylation signal sequence (AATAAA), a poly (A) tail, and an open reading frame (ORF) (1974 bp). The predicted molecular mass and isoelectric point for HSP70 is 71.6 kDa and 5.15, respectively. BLAST analysis showed that P. esculenta HSP70 gene shared high similarity. Classical HSP signature motifs, ATP/GTP-Binding Site Motif A, Bipartite Nuclear Targeting Sequence, the cytosolic HSP70 could be expressed in Escherichia coli BL21. After purification, the recombinant pET-HSP70 protein was used to produce the polyclonal antibody in mice and the specificity of the antibody was confirmed by Western blot analysis. Fluorescent real-time quantitative PCR analysis showed that expression of Hsp70 in sipuncula was increased significantly after exposure to 10 mM Zn for12 h, Cd for 24 h, Cu for 48 h, and was exposure to 37 °C for 24 h sea water.     

Genome-wide expression analysis of HSP70 family genes in rice and identification of a cytosolic HSP70 gene highly induced under heat stress

The heat shock protein 70 (HSP70) gene family plays a key role in protecting plant cells or tissues from thermal or oxidative stress. Although many studies have elucidated the molecular functions of individual family members, genome-wide analysis of this family is still limited, especially for crop species. Our objective was to integrate various meta-profiling data into the context of a phylogenetic tree, which would enable us to perform fine evaluation of functional dominancy or redundancy within this family. Our data indicated that a loss-of-function mutant of a rice cytosolic HSP70 gene (OsctHSP70-1) did not show a clear defective phenotype in response to high temperature because of the existence of another gene family member that was closely clustered with OsctHSP70-1 and had similar expression patterns. Moreover, the second gene showed much stronger anatomical expression. We indirectly analyzed the function of OsctHSP70-1 by studying GUS activity under the control of the endogenous promoter. We also designed a probable interaction network mediated by OsctHSP70-1 and used co-expression analysis among its components to refine the network, suggesting more probable model to explain the function of OsctHSP70-1.     

HSP86 and HSP84 exhibit cellular specificity of expression and co-precipitate with an HSP70 family member in the murine testis

This study extends to the protein level our previous observations, which had established the stage and cellular specificity of expression of hsp86 and hsp84 in the murine testis in the absence of exogenous stress. Immunoblot analysis was used to demonstrate that HSP86 protein was present throughout testicular development and that its levels increased with the appearance of differentiating germ cells. HSP86 was most abundant in the germ cell population and was present at significantly lower levels in the somatic cells. By contrast, the HSP84 protein was detected in the somatic cells of the testis rather than in germ cells. The steady-state levels of HSP86 and HSP84 paralleled the pattern of the expression of their respective mRNAs, suggesting that regulation at the level of translation was not a major mechanism controlling hsp90 gene expression in testicular cells. Immunoprecipitation analysis revealed that a 70-kDa protein coprecipitated with the HSP86/HSP84 proteins in testicular homogenates. This protein was identified as an HSP70 family member by immunoblot analysis, suggesting that HSP70 and HSP90 family members interact in testicular cells. © 1993Wiley-Liss, Inc.     

Effect of chronic hypoxia on proliferation, apoptosis, and HSP70 expression in mouse bronchiolar epithelial cells

Heat shock proteins (HSPs) can be induced by various stresses and play an important role in cell cycle progression. HSP70 has been shown to act as an inhibitor of apoptosis. We studied HSP70 expression in bronchial epithelial cells of C57BL/6 mice and homozygous HPS70 knockout mice (hsp70.1-/-) exposed to chronic hypoxic stress. We also investigated changes in cellular proliferation and apoptosis in relation to HSP70. Lungs were removed from mice after a three-week period of exposure to 10 % O(2). Immunoblots for HSP70 and immunohistochemical staining for HSP70 and Ki-67 were performed. Apoptosis was assessed using the TUNEL assay. The three-week period of hypoxic stress did not change HSP70 levels in total lung tissue, but a significant reduction in HSP70 expression was observed in bronchiolar epithelial cells. In wild type mice, both HSP70 and Ki-67 expression were significantly reduced in bronchiolar epithelial cells. In homozygous HPS70 knockout mice (hsp70.1-/-), apoptosis of bronchiolar epithelial cells was significantly increased. Our results suggest that HSP70 may exert anti-apoptotic effects in mouse bronchiolar epithelial cells.     

Coelomocyte numbers and expression of HSP70 in wounded sea stars during hypoxia

Hypoxia, mainly caused by eutrophication, is a common and growing problem on marine soft bottoms. Echinoderms are known for their ability to regenerate tissue after wounding but hypoxia has a negative influence on regeneration and also on reproduction in echinoderms. We have investigated the cellular and molecular responses to wounding stress and hypoxia in the sea star Asterias rubens by using the total coelomocyte count (TCC) and the expression of heat shock proteins (HSPs). As early as 1 h after wounding, sea stars under hypoxic conditions show significantly increased TCC and, after 6 h, cell numbers increase approximately two-fold. After a 3-h hypoxia exposure of wounded animals, Western blot analysis reveals highly elevated coelomocyte cytoplasmic HSP70 expression. Non-wounded sea stars exposed to hypoxia and wounded animals kept in normoxia show enhanced HSP70 expression only after 24 h. Immunocytochemical analysis has not demonstrated any translocation of HSP70 from the cytoplasm to the nucleus. We conclude that both wounding and hypoxia elicit a stress response in sea stars and that the combined stress produces synergistic effects that may inhibit the initial processes of wound healing and regeneration.