Hsp27 Gene Research Articles

Increased hsp22 RNA levels in Drosophila lines genetically selected for increased longevity.

RNAs for the small heat shock protein (hsp) genes hsp22 and hsp23 are induced during Drosophila aging, suggesting that these genes might have specific functions at late ages. To determine if hsp22 and hsp23 gene expression might correlate with life span, RNA levels for these and additional genes were analyzed throughout the adult life span in a set of five outbred "O" lines, which have been genetically selected for increased longevity, and in five matched control "B" lines. Control ribosomal protein genes rp49 and AP3/RpPO RNA levels were similar in O and B lines. In contrast, hsp22 RNA levels were twofold-tenfold higher in all five O lines relative to all five B lines, while hsp23 exhibited a smaller but significant increase. Thus increased hsp22 and hsp23 RNA levels correlate with the increased life span and increased stress resistance of the genetically selected O lines.

Heat Shock Response of <I>Bactrocera oleae</I> (Diptera: Tephritidae): Genes and Proteins

The heat shock response as revealed by heat inducible puffing and protein patterns, as well as the localization of the major heat shock genes on the salivary gland polytene chromosomes of the olive fruit fly, Bactrocera oleae (Gmelin), a major agricultural pest, have been studied. After in situ hybridization of specific DNA sequences to the polytene chromosomes of B. oleae, the loci of the major heat shock genes hsp70, hsp83, and a putative hsp68 were detected. The two latter genes gave unique signals, and hsp70 hybridized to two adjacent discrete bands. Eight puffs were induced after heat shock, two of which were found to be the loci of the hsp70 and hsp83 genes. The newly synthesized polypeptides from the salivary glands of B. oleae, after gradual temperature elevation from 25 to 42°C, were analyzed in denaturing acrylamide gels. The most heat inducible polypeptides were found to exhibit relative electrophoretic mobilities of 83, 70, 69, 27, 26, and 23 kDa. The thermotolerance of B. oleae assumed by the above results is discussed in comparison with that observed in Drosophila species.

EFFECT OF THE SERINE PROTEASE INHIBITOR N-TOSYL-L-PHENYLALANINE-CHLOROMETHYL KETONE (TPCK) ON MCF-7 MAMMARY TUMOUR CELLS GROWTH AND DIFFERENTIATION

Previous studies from this and other laboratories indicated that the oestrogen-regulated heat shock protein HSP27 is involved in the control of MCF-7 cells growth and differentiation, as it also appears to be in other cell types, including osteoblasts and HL-60 cells. In the latter instance, induction of differentiation is associated with the downregulation of myeloblastin, a serine protease now identified as proteinase 3 (hence its designation as PR3/Mbn), mirrored by an increase in the cellular content of the small heat shock protein HSP27, a substrate to this enzyme. Besides, antisense inhibition of PR3/Mbn production sufficed for inducing HL-60 cells monocytic differentiation. This prompted us to examine the hypothesis that a post-translational control on HSP27 levels (and by this on differentiation) by a serine protease might also be operating in human mammary tumour cells. As part of our attempt to evaluate this hypothesis, the present work consisted of testing the effects of a treatment of MCF-7 cells with the serine protease inhibitor N-tosyl-L-phenylalanine-chloromethyl ketone (TPCK). Our data show that this resulted in a four-fold increase in HSP27 content, associated with a 2.5-fold decrease in growth rate, the formation of cytoplasmic vesicles and increased secretion of 52kDa peptides, identified by Western immunoblot as the isoforms of the oestrogen-regulated protein, cathepsin D. TPCK only affected growth in MDAMB-231 cells (in which HSP27 levels are very low and remained below MCF-7 cells basal levels after treatment) and failed to affect L929 cells, in which the hsp27 gene is silent. This provides circumstantial support for the assumption that effects of TPCK on the MCF-7 cells phenotype are linked to the associated increase in HSP27 content. Our recent demonstration that MCF-7 cells do in fact express PR3/Mbn fits with our concept and opens the way to test it directly, using antisense strategy.

Peroxide accumulation without major mitochondrial alteration in replicative senescence

The aim of this work was to determine how ageing changes were related to each other by studying the establishment of a senescent state in cell culture, rabbit articular chondrocytes. A striking increase of the amount of peroxides appeared at 2/3 of the time of cell growth, which is responsible for an oxidative stress, as shown by the appearance of oxidized proteins, the overexpression of HSP27 gene and the accumulation of HSP27 protein. While no change of the mitochondrial membrane potential was observed all along the cell culture, p21, a protein overproduced in senescent cells, appeared with the peak of peroxides and accumulated.

Transcriptional regulation involving the intronic heat shock element of the rat hsp27 gene

While sequencing the first intron of the rat heat shock protein 27 gene (hsp27), we identified a consensus heat shock regulatory element (HSE). This intronic HSE (i-HSE) is conserved among mammalian hsp27 genes. The aim of this study was to investigate possible effects of this intronic HSE (i-HSE) on transcription from the rat hsp27 promoter. Gel mobility shift assays indicated that the i-HSE bound heat shock transcription factor 1 (HSF1) in a manner equivalent to that of HSE present in hsp27 promoter (p-HSE). The effect of i-HSE on transcription from the hsp27 promoter was evaluated using reporter constructs transiently transfected in the osteosarcoma cell line ROS17/2.8. When inserted 5′ to a 145 bp fragment of the hsp27 promoter not containing p-HSE, a 215 bp fragment of hsp27 intron 1 containing i-HSE enhanced CAT activity and conferred heat shock-inducible activity to the construct. This intronic fragment containing i-HSE also enhanced CAT activity in either normal or heat-shocked culture conditions when inserted 3′ to the CAT open reading frame. However, in chimeric reporter constructs with a 273 bp hsp27 promoter containing p-HSE directly 5′ to CAT reporter, and with a 215 bp fragment containing i-HSE inserted 3′ to the CAT open reading frame, transcription from hsp27 promoter was reduced under normal and heat-stressed culture conditions. Mutation of the i-HSE reversed this effect. Further study is required to define the mechanism by which the i-HSE-containing region of the hsp27 promoter may mediate negative regulation of hsp27 transcription.

Estrogen receptor gene expression and its relation to the estrogen-inducible HSP27 heat shock protein in hormone refractory prostate cancer.

The recent discovery of the classical estrogen receptor alpha (ERalpha) in androgen-insensitive prostate cancer has shed new light on the role of estrogens in endocrine therapy failure. To get more information on downstream events of estrogen signaling in these tumors, we investigated the relation between ERalpha gene expression, and the estrogen-inducible heat shock protein HSP27 in recurrent prostatic adenocarcinomas. Palliative transurethral resection specimens from 50 patients with androgen-insensitive disease were submitted for study. Messenger RNA in situ hybridization for the ERalpha and immunohistochemistry of the HSP27 protein were performed on adjacent sections of an equal number of prostate cancer tissue with and without ERalpha protein expression. Cancerous lesions lacking the nuclear ERalpha at the protein level revealed ERalpha mRNA expression in 15 of 25 cases (60%). A coordinate expression of ERalpha mRNA and HSP27 was observed in 33 of 40 cases (83%), although a significant correlation between ERalpha protein and HSP27 expression was not obtained. Conversely, 90% of neoplastic lesions without detectable levels of ERalpha mRNA and protein also lacked HSP27 immunoreactivity. ERalpha gene expression at the mRNA level significantly correlated with the immunoprofile of the estrogen-inducible HSP27 protein in androgen-insensitive prostatic adenocarcinomas. This may indicate that these tumors harbor functional active estrogen receptors promoting transcriptional activity of the HSP27 gene. Determination of the receptor status by immunohistochemistry is unable to identify neoplastic lesions with established ERalpha mRNA expression in a substantial number of cases. HSP27 may be an additional surrogate biomarker for estrogen-regulated growth in androgen-insensitive prostate cancer.

Polarity of the ecdysone receptor complex interaction with the palindromic response element from the hsp27 gene promoter.

The functional 20-hydroxyecdysone (20E) receptor is a heterodimer of two members of the nuclear hormone receptors superfamily; the product of the EcR (EcR) and of the ultraspiracle (Usp) genes. As most of the natural 20E-response elements are highly degenerated palindromes, we were interested in determining whether or not such asymmetric elements could dictate the defined orientation of the Usp/EcR complex. We have investigated interaction of EcR and Usp DNA-binding domains (EcRDBD and UspDBD, respectively) with the palindromic response element from the hsp27 gene promoter (hsp27pal). The hsp27pal half-sites contribute differently to the binding of the heterodimer components; the 5' half-site exhibits higher affinity for both DBDs than the 3' half-site. This observation, along with data demonstrating that UspDBD exhibits approximate fourfold higher affinity to the 5' half-site than EcRDBD, suggest that UspDBD locates the EcRDBD/UspDBD heterocomplex in the defined orientation (5'-UspDBD-EcRDBD-3') on the hsp27pal sequence. The binding polarity onto hsp27pal is accompanied by different contribution of the UspDBD and EcRDBD C-terminal sequences to the DNA-binding and heterocomplex formation. This is supported by finding that deletion of the C-terminal of EcRDBD region corresponding to the putative A-helix severely decreased binding of the EcRDBD to the hsp27pal. In contrast, UspDBD in which corresponding residues were deleted exhibited the same hsp27pal binding pattern as the wild type UspDBD. Additional truncation comprising the putative T-box, resulted in a reduced binding of the mutated UspDBD. This truncation however, still allowed effective EcRDBD/UspDBD heterodimer formation. Finally we demonstrated that perfect palindromes, composed of two hsp27pal 5' half-sites (or of the related sequence) contain all of the structural information necessary for the anisotropic UspDBD/EcRDBD heterocomplex formation. However, the perfect palindromes bind isolated homomeric DBDs as well as their heterocomplex with higher affinity than imperfect hsp27pal. This is the first report indicating that natural 20E response elements, which with one exception are degenerated palindromes, may act as functionally asymmetric elements in a manner similar to the action of direct repeats in vertebrates.

The Yeast Hsp110 Family Member, Sse1, Is an Hsp90 Cochaperone

In eukaryotes, production of the diverse repertoire of molecular chaperones during normal growth and in response to stress is governed by the heat shock transcription factor HSF. The HSC82 and HSP82 genes, encoding isoforms of the yeast Hsp90 molecular chaperone, were recently identified as targets of the HSF carboxyl-terminal activation domain (CTA), whose expression is required for cell cycle progression during prolonged heat stress conditions. In the present study, we have identified additional target genes of the HSF CTA, which include nearly all of the heat shock-inducible members of the Hsp90 chaperone complex, demonstrating coordinate regulation of these components by HSF. Heat shock induction of SSE1, encoding a member of the Hsp110 family of heat shock proteins, was also dependent on the HSF CTA. Disruption of SSE1 along with STI1, encoding an established subunit of the Hsp90 chaperone complex, resulted in a severe synthetic growth phenotype. Sse1 associated with partially purified Hsp90 complexes and deletion of the SSE1 gene rendered cells susceptible to the Hsp90 inhibitors macbecin and geldanamycin, suggesting functional interaction between Sse1 and Hsp90. Sse1 is required for function of the glucocorticoid receptor, a model substrate of the Hsp90 chaperone machinery, and Hsp90-based repression of HSF under nonstress conditions. Taken together, these data establish Sse1 as an integral new component of the Hsp90 chaperone complex in yeast.

Drosophila melanogaster is polymorphic for a specific repeated (CATA) sequence in the regulatory region of hsp23.

To identify sequence variation associated with a selection response for heat tolerance in Drosophila melanogaster, we sequenced 1400bp of the heat shock protein 23 gene (hsp23) promoter region in four heat-selected and two control lines. The region was found to be variable for a specific (CATA) repeated sequence, and the sequence CTT seems to be a hot spot for mutation. The repeated tetranucleotide sequence was located in several short repeats scattered throughout the entire region. Similar variable repeats are also located downstream the of hsp23 gene in the intergenic region between hsp23 and hsp27. We detected nine different hsp23 alleles. Their frequencies in the selection and control lines seemed to be mainly determined by genetic drift. The function of the CATA repeats is not yet known, though these regions have homology to SAR elements located in the intergenic region between two hsp70 genes, suggesting a similar function.

Expression and chaperone function of hsp30 genes during Xenopus laevis development : Fernando P, Lang L, Miskovic D, and [formula omitted] Dept. of Biology, University of Waterloo, Waterloo, Ontario, Canada, N2L 3G1

Expression and chaperone function of hsp30 genes during Xenopus laevis development : Fernando P, Lang L, Miskovic D, and [formula omitted] Dept. of Biology, University of Waterloo, Waterloo, Ontario, Canada, N2L 3G1

Silencing at Drosophila telomeres: nuclear organization and chromatin structure play critical roles.

Transgenes inserted into the telomeric regions of Drosophila melanogaster chromosomes exhibit position effect variegation (PEV), a mosaic silencing characteristic of euchromatic genes brought into juxtaposition with heterochromatin. Telomeric transgenes on the second and third chromosomes are flanked by telomeric associated sequences (TAS), while fourth chromosome telomeric transgenes are most often associated with repetitious transposable elements. Telomeric PEV on the second and third chromosomes is suppressed by mutations in Su(z)2, but not by mutations in Su(var)2-5 (encoding HP1), while the converse is true for telomeric PEV on the fourth chromosome. This genetic distinction allowed for a spatial and molecular analysis of telomeric PEV. Reciprocal translocations between the fourth chromosome telomeric region containing a transgene and a second chromosome telomeric region result in a change in nuclear location of the transgene. While the variegating phenotype of the white transgene is suppressed, sensitivity to a mutation in HP1 is retained. Corresponding changes in the chromatin structure and inducible activity of an associated hsp26 transgene are observed. The data indicate that both nuclear organization and local chromatin structure play a role in this telomeric PEV.

RNA Polymerase-specific Nucleosome Disruption by Transcription in Vivo

The nucleosomal chromatin structure within genes is disrupted upon transcription by RNA polymerase II. To determine whether this disruption is caused by transcription per se as opposed to the RNA polymerase source, we engineered the yeast chromosomal HSP82 gene to be exclusively transcribed by bacteriophage T7 RNA polymerase in vivo. Interestingly, we found that a fraction of the T7-generated transcripts were 3' end processed and polyadenylated at or near the 3' ends of the hsp82 and the immediately downstream CIN2 genes. Surprisingly, the nucleosomal structure of the T7-transcribed hsp82 gene remained intact, in marked contrast to the disrupted structure generated by much weaker, basal level transcription of the wild type gene by RNA polymerase II under non-heat shock conditions. Therefore, disruption of chromatin structure by transcription is dependent on the RNA polymerase source. We propose that the observed RNA polymerase dependence for transcription-induced nucleosome disruption may be related either to the differential recruitment of chromatin remodeling complexes, the rates of histone octamer translocation and nucleosome reformation during polymerase traversal, and/or the degree of transient torsional stress generated by the elongating polymerase.

Influence of bioenergetic stress on heat shock protein gene expression in nucleated red blood cells of fish.

The physiological and biochemical signals that induce stress protein (HSP) synthesis remain conjectural. In this study, we used the nucleated red blood cells from rainbow trout, Oncorhynchus mykiss, to address the interaction between energy status and HSP gene expression. Heat shock (25 degrees C) did not significantly affect ATP levels but resulted in an increase in HSP70 mRNA. Hypoxia alone did not induce HSP transcription in these cells despite a significant depression in ATP. Inhibition of oxidative phosphorylation with azide, in the absence of thermal stress, decreased ATP by 56% and increased lactate production by 62% but did not induce HSP gene transcription. Inhibition of oxidative phosphorylation and glycolysis with azide and iodoacetic acid respectively, decreased ATP by 79% and prevented lactate production, but did not induce either HSP70 or HSP30 gene transcription in these cells. This study demonstrates that a reduction in the cellular energy status will not induce stress protein gene transcription in rainbow trout red blood cells and may, in fact, limit induction during extreme metabolic inhibition.

Human 27-kDa heat shock protein (hsp27) gene family: chromosomal band assignments and possible involvement in Williams syndrome deletion

The 27-kDa human heat shock protein (hsp27) is expressed in a wide variety of tissues in the absence of stress and is thought to regulate actin filament dynamics. Three related human hsp27 sequences had been previously mapped to chromosomes 3, 9, and X (McGuire et al., 1989). We have used fluorescent in situ hybridization (FISH) to correct and refine the map position of the transcribed hsp27 gene to chromosome 7, band 7q11.23 and its two pseudogenes to chromosome bands 9q21 and Xp11.2. Band 7q11.23 is the site of a deletion associated with Williams syndrome (WS), a congenital developmental disorder involving the vascular, connective tissue, and central nervous systems. We performed FISH mapping of hsp27 DNA to the chromosomes of four WS patients and found that in the majority of cells (77%), a bright signal was found on only one chromosome 7 at band 7q11.23. These results may indicate that the WS deletion includes the gene for the 27-kDa heat shock protein. However, in a small proportion of cells, a second, less intense signal was found on the other chromosome 7. We have interpreted these second weak signals as cross-hybridization due to the repeated DNA sequences (Alu sequences) known to reside within the introns and the flanking regions of this gene. Southern hybridization analysis is currently being performed to substantiate these findings in WS patient DNAs.REFERENCE: McGuire SE, Fuqua SA, Naylor SL, Helin-Davis DA, McGuire WL (1989): Chromosomal assignments of human 27-kDa heat shock protein gene family Somat Cell Mol Genet 15:167-71.

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.

Direct observation of stress response in Caenorhabditis elegans using a reporter transgene.

Transgenic Caenorhabditis elegans expressing jellyfish Green Fluorescent Protein under the control of the promoter for the inducible small heat shock protein gene hsp-16-2 have been constructed. Transgene expression parallels that of the endogenous hsp-16 gene, and, therefore, allows direct visualization, localization, and quantitation of hsp-16 expression in living animals. In addition to the expected upregulation by heat shock, we show that a variety of stresses, including exposure to superoxide-generating redox-cycling quinones and the expression of the human beta amyloid peptide, specifically induce the reporter transgene. The quinone induction is suppressed by coincubation with L-ascorbate. The ability to directly observe the stress response in living animals significantly simplifies the identification of both exogenous treatments and genetic alterations that modulate stress response, and possibly life span, in C. elegans.

Expression of HSP27 results in increased sensitivity to tumor necrosis factor, etoposide, and H2O2 in an oxidative stress-resistant cell line.

The role of HSP27 in cell growth and resistance to stress was investigated using murine fibrosarcoma L929 cells (normally devoid of constitutively expressed small HSPs) and human osteoblast-like SaOS-2 cells stably transfected with a human hsp27 expression vector. Our data showed that our L929 cells were more resistant to oxidative stress than generally observed for this line. Production of HSP27 in these cells led to a marked decrease in growth rate associated with a series of phenotypical changes, including cell spreading, cellular and nuclear hypertrophy, development of an irregular outline, and a tremendous accumulation of actin stress fibers. By contrast, none of these changes was observable in SaOS-2/hsp27 transfectants overexpressing the protein product. Together, these observations are consistent with a cause-to-effect cascade relationship between increased (or induced) HSP27 expression, changes in cytoskeletal organization, and decreased growth. On the other hand, whereas the transfection of the hsp27 gene increased the cell resistance to heat in both cell lines, only in SaOS-2 cells was this associated with protection to the cytotoxic action of tumor necrosis factor-alpha (TNF-alpha) and etoposide. Unexpectedly, L929/hsp27 transfectants exhibited an increased sensitivity to both agents and also to H2O2. These data thus imply that different mechanisms are involved in the cell resistance to heat shock and to the cytotoxic action of TNF-alpha, etoposide, and H2O2. They also plead against the simple view that overexpression of a phosphorylatable HSP27 would necessarily be beneficial in terms of increased cell resistance to any type of stress. Our data further indicate that the role of HSP27 in cellular resistance to stress and in cell proliferation involves different targets and that the ultimate result of its interference with these processes depends on the intracellular context in which the protein is expressed.

Heat shock protein 27 kDa expression and phosphorylation regulates endothelial cell migration.

The effects of enhanced HSP27 expression or expression of a nonphosphorylatable form of HSP27 on the migration of bovine arterial endothelial cells was assessed. Expression of the wild-type protein enhanced migration by twofold compared to control transfectants, whereas expression of the mutant protein retarded migration by 40%. Since homologs of the small heat shock protein inhibit F-actin polymerization in vitro and may alter basolateral F-actin content in vivo, it was postulated that the 27 kDa heat shock protein affects microfilament extension essential for cell motility. Expression of the wild-type protein promoted the generation of long cellular extensions, whereas expression of the dominant negative mutant protein resulted in a marked reduction of lamellipodia and generated aberrant microfilament morphology at the wound edge. Immunofluorescence combined with phalloidin staining demonstrated the colocalization of the HSP27 gene products with lamellipodial microfilament structures. These data suggest that the 27 kDa heat shock protein regulates migration by affecting the generation lamellipodia microfilaments.

Signal transduction pathway leading to Hsp27 and Hsp70 gene expression during myocardial adaptation to stress.

Signal transduction pathway leading to Hsp27 and Hsp70 gene expression during myocardial adaptation to stress.

Histone acetylation facilitates RNA polymerase II transcription of the Drosophila hsp26 gene in chromatin.

A number of activators are known to increase transcription by RNA polymerase (pol) II through protein acetylation. While the physiological substrates for those acetylases are poorly defined, possible targets include general transcription factors, activator proteins and histones. Using a cell-free system to reconstitute chromatin with increased histone acetylation levels, we directly tested for a causal role of histone acetylation in transcription by RNA pol II. Chromatin, containing either control or acetylated histones, was reconstituted to comparable nucleosome densities and characterized by electron microscopy after psoralen cross-linking as well as by in vitro transcription. While H1-containing control chromatin severely repressed transcription of our model hsp26 gene, highly acetylated chromatin was significantly less repressive. Acetylation of histones, and particularly of histone H4, affected transcription at the level of initiation. Monitoring the ability of the transcription machinery to associate with the promoter in chromatin, we found that heat shock factor, a crucial regulator of heat shock gene transcription, profited most from histone acetylation. These experiments demonstrate that histone acetylation can modulate activator access to their target sites in chromatin, and provide a causal link between histone acetylation and enhanced transcription initiation of RNA pol II in chromatin.