Characterization of proteins associated with heat shock protein hsp27 in the squamous cell carcinoma cell line A431.

Heat shock protein hsp27 is a molecular chaperone and identification of hsp27-binding proteins might help to elucidate its functional role in keratinocyte biology. In the present investigation we used a human epidermal cell carcinoma cell line (A431) transfected with hsp27 (A431/16) to study interference between hsp27 protein and other proteins. Immunoprecipitation experiments with anti-hsp27 antibody revealed a multicomponent complex when analysed by silver staining. By immunoblotting analysis we could demonstrate that hsp27 associates with actin, the mutant form of p53, hsp70 and hsp90. Immunofluorescence analysis showed a co-localization between hsp27 and p53, hsp70 and hsp90. To control for the specificity of the observed interactions, immuno-precipitations with antibodies to actin, p53, hsp70 and hsp90 respectively, were performed. All of the tested proteins demonstrated a coimmunoprecipitation with hsp27. We conclude that hsp27, like the other heat shock proteins, is part of a complex system of molecular chaperones in epidermal keratinocytes.     

Development and tissue-specific distribution of mouse small heat shock protein hsp25

We have investigated the developmental and tissue-specific distribution of the mouse small hsp25 by immunohistology using an antibody that specifically identifies hsp25. Our analysis shows that the relative amount of hsp25 increases during embryogenesis. Through days 13–20 of embryogenesis, hsp25 accumulation is predominant in the various muscle tissues, including the heart, the bladder, and the back muscles. hsp25 is detectable also in neurons of the spinal cord and the purkinje cells. Furthermore analysis of the closely related α, B-crystallin shows that in several tissues, including the bladder, the notochordal sheath and the eye lens both proteins are coexpressed. Our studies demonstrate that mammalian hsp25 accumulation is developmentally regulated during mouse embryogenesis and support the view of an important functional role of small heat shock proteins in normal cell metabolism. © 1993Wiley-Liss, Inc.     

Selective postmortem degradation of inducible heat shock protein 70 (hsp70) mRNAs in rat Brain

Summary 1. Altered mRNA levels in postmortem brain tissue from persons with Alzheimer's disease (AD) or other neurological diseases are usually presumed to be characteristic of the disease state, even though both agonal state (the physiological state immediately premortem) and postmortem interval (PMI) (the time between death and harvesting the tissue) have the potential to affect levels of mRNAs measured in postmortem tissue. Although the possible effect of postmortem interval on mRNA levels has been more carefully evaluated than that of agonal state, many studies assume that all mRNAs have similar rates of degradation postmortem.2. To determine the postmortem stability of inducible heat shock protein 70 (hsp70) mRNAs, themselves unstablein vivo at normal body temperature, rats were heat shocked in order to induce synthesis of the hsp70 mRNAs. hsp70 mRNA levels in cerebellum and cortex were then compared to those of their heat shock cognate 70 (hsc70) mRNAs, as well as to levels of 18S rRNAs, at 0 and at 24 hr postmortem.3. Quantiation of northern blots after hybridization with an hsp70 mRNA-specific oligo probe indicated a massive loss of hsp70 mRNA signal in RNAs isolated from 24-hr postmortem brains; quantitation by slot-blot hybridization was 5- to 15-fold more efficient. Even using the latter technique, hsp70 mRNA levels were reduced by 59% in 24-hr-postmortem cerebellum and by 78% in cortex compared to mRNA levels in the same region of 0-hr-postmortem brain. There was little reduction postmortem in levels of the hsp70 mRNAs or of 18S rRNAs in either brain region.4.In situ hybridization analysis indicated that hsp70 mRNAs were less abundant in all major classes of cerebellar cells after 24 hr postmortem and mRNAs had degraded severalfold more rapidly in neurons than in glia. There was no corresponding loss of intracellular 18S rRNA in any cell type.5. We conclude from these results that the effect of postmortem interval on mRNA degradation must be carefully evaluated when analyzing levels of inducible hsp70 mRNAs, and perhaps other short-lived mRNAs, in human brain.     

Endoplasmic reticulum stress induces the phosphorylation of small heat shock protein, Hsp27

There are several reports describing participation of small heat shock proteins (sHsps) in cellular protein quality control. In this study, we estimated the endoplasmic reticulum (ER) stress-induced response of Hsp27 and alphaB-crystallin in mammalian cells. Treatment targeting the ER with tunicamycin or thapsigargin induced the phosphorylation of Hsp27 but not of alphaB-crystallin in U373 MG cells, increase being observed after 2-10 h and decline at 24 h. Similar phosphorylation of Hsp27 by ER stress was also observed with U251 MG and HeLa but not in COS cells and could be blocked using SB203580, an inhibitor of p38 MAP kinase. Other protein kinase inhibitors, like G?6983, PD98059, and SP600125, inhibitors of protein kinase C (PKC), p44/42 MAP kinase, and JNK, respectively, were without major influence. Prolonged treatment with tunicamycin but not thapsigargin for 48 h caused the second induction of the phosphorylation of Hsp27 in U251 MG cells. Under these conditions, the intense perinuclear staining of Hsp27, with some features of aggresomes, was observed in 10%-20% of the cells.     

Insights into function and regulation of small heat shock protein 25 (HSPB1) in a mouse model with targeted gene disruption

The mammalian small heat shock protein (sHSPs) family is comprised of 10 members and includes HSPB1, which is proposed to play an essential role in cellular physiology, acting as a molecular chaperone to regulate diverse cellular processes. Whilst differential roles for sHSPs are suggested for specific tissues, the relative contribution of individual sHSP family members in cellular and organ physiology remains unclear. To address the function of HSPB1 in vivo and determine its tissue-specific expression during development and in the adult, we generated knock-in mice where the coding sequence of hspb1 is replaced by a lacZ reporter gene. Hspb1 expression marks myogenic differentiation with specific expression first confined to developing cardiac muscles and the vascular system, and later in skeletal muscles with specific expression at advanced stages of myoblast differentiation. In the adult, hspb1 expression was observed in other tissues, such as stratified squamous epithelium of skin, oronasal cavity, tongue, esophagus, and uterine cervix but its expression was most prominent in the musculature. Interestingly, in cardiac muscle hsbp1 expression was down-regulated during the neonatal period and maintained to a relatively low steady-level throughout adulthood. Despite this widespread expression, hspb1-/- mice were viable and fertile with no apparent morphological abnormalities in tissues under physiological conditions. However, at the cellular level and under stress conditions (heat challenge), HSPB1 act synergistically with the stress-induced HSPA1 (HSP70) in thermotolerance development, protecting cells from apoptosis. Our data thus indicate a nonessential role for HSPB1 in embryonic development and for maintenance of tissues under physiological conditions, but also shows that it plays an important role by acting synergistically with other HSPs during stress conditions to exert cytoprotection and anti-apoptotic effects.     

Enhanced stability of alpha B-crystallin in the presence of small heat shock protein Hsp27

Lens alpha-crystallin, alpha A- and alpha B-crystallin, and Hsp27 are members of the small heat shock protein family. Both alpha A- and alpha B-crystallin are expressed in the lens and serve as structural proteins and as chaperones, but alpha B-crystallin is also expressed in nonlenticular organs where Hsp27, rather than alpha A-crystallin, is expressed along with alpha B-crystallin. It is not known what additional function Hsp27 has besides as a heat shock protein, but it may serve, as alpha A-crystallin does in the lens, to stabilize alpha B-crystallin. In this study, we investigate aspects on conformation and thermal stability for the mixture of Hsp27 and alpha B-crystallin. Size exclusion chromatography, circular dichroism (CD), and light scattering measurements indicated that Hsp27 prevented alpha B-crystallin from heat-induced structural changes and high molecular weight (HMW) aggregation. The results indicate that Hsp27 indeed promotes stability of alpha B-crystallin.     

Cloning and nucleotide sequencing of three heat shock protein genes (hsp90, hsc70, and hsp19.5) from the diamondback moth, Plutella xylostella (L.) and their expression in relation to developmental stage and temperature

Heat shock protein genes, hsp90, hsc70, and hsp19.5, were cloned and sequenced from the diamondback moth, Plutella xylostella (L.) by RT-PCR and RACE method. The cDNA sequence analysis of hsp90 and hsp19.5 revealed open reading frames (ORFs) of 2,151 and 522 bp in length, which encode proteins with calculated molecular weights of 82.4 and 19.5 kDa, respectively. Analysis of cDNA from hsc70 revealed an ORF of 1,878 bp coding a protein with a calculated molecular weight of 69.3 kDa. Furthermore, the analysis of genomic DNA from hsc70 confirmed the presence of introns while no introns were apparent in hsp90 and hsp19.5. Southern blot analysis suggested the presence of multiple copies of each gene family in the DBM genome. Detectable expression of hsp19.5 was observed at the pupal stage while expression of hsp90 and hsc70 was detected at both pupal and adult stages. At adult stage, females showed a higher expression of hsp90 and hsc70 than males. An increased expression was observed in all three genes after exposure to a high temperature in both sexes. These results suggest that in addition to a heat shock response, these HSP genes might be involved in other functions during the course of development in DBM.     

The heat shock response in hydra: immunological relationship of hsp60, the major heat shock protein of Hydra vulgaris,to the ubiquitous hsp70 family

The major heat shock protein (hsp) of Hydra vulgaris has recently been found to be a 60 kDa protein. Since in all organisms studied so far, the major heat shock protein is a 70 kDa protein, we have analyzed the relationship of hydra hsp60 to the highly conserved 70 kDa heat shock protein family. Genes and proteins related to the 70 kDa class of stress proteins are present in hydra. However, antibodies known to cross-react with hsp70 proteins in several different organisms do not cross-react with hydra hsp60 suggesting that hsp60 is not related to the conserved hsp70 proteins.     

Attenuated phosphorylation of heat shock protein 27 correlates with tumor progression in patients with hepatocellular carcinoma

Heat shock protein 27 (HSP27) is expressed at high levels in human hepatocellular carcinoma (HCC). We examined correlations of total HSP27 and serine phosphorylated (Ser-15, Ser-78, and Ser-82) HSP27 levels in HCC tissues with clinical and pathologic characteristics in 48 resected HCC specimens. The levels of total and Ser-phosphorylated HSP27 were evaluated by Western blot analysis. Immunohistochemical analysis of HSP27 expression was also performed on some samples. Phosphorylation of HSP27 was detected in all 48 HCC tissues. Levels of phosphorylated HSP27 were correlated inversely with tumor size, microvascular invasion of HCC, and tumor stage by TNM classification. In contrast, only microvascular invasion showed an inverse correlation with total HSP27 levels. The decrease in phosphorylated HSP27 in progressed HCC was also observed by immunohistochemistry. Levels of phosphorylated HSP27 gradually decreased in parallel with HCC progression. Our findings suggest that phosphorylated HSP27 may have a suppressive role in progression of human HCC.     

Divergent evolution of the chloroplast small heat shock protein gene in the genera Rhododendron (Ericaceae) and Machilus (Lauraceae)

BACKGROUND AND AIMS: Evolutionary and ecological roles of the chloroplast small heat shock protein (CPsHSP) have been emphasized based on variations in protein contents; however, DNA sequence variations related to the evolutionary and ecological roles of this gene have not been investigated. In the present study, a basal angiosperm, Machilus, together with the eudicot Rhododendron were used to illustrate the evolutionary dynamics of gene divergence in CPsHSPs. METHODS: Degenerate primers were used to amplify CPsHSP-related sequences from 16 Rhododendron and eight Machilus species that occur in Taiwan. Manual DNA sequence alignment was carried out according to the deduced amino acid sequence alignment performed by CLUSTAL X. A neighbour-joining tree was generated in MEGA using conceptual translated amino acid sequences from consensus sequences of cloned CPsHSP genes from eight Machilus and 16 Rhododendron species as well as amino acid sequences of CPsHSPs from five monocots and seven other eudicots acquired from GenBank. CPsHSP amino acid sequences of Funaria hygrometrica were used as the outgroups. The aligned DNA and amino acid sequences were used to estimate several parameters of sequence divergence using the MEGA program. Separate Bayesian inference of DNA sequences of Rhododendron and Machilus species was analysed and the resulting gene trees were used for detection of putative positively selected amino acid sites by the Codeml program implemented in the PAML package. Mean hydrophobicity profile analysis was performed with representative amino acid sequences for both Rhododendron and Machilus species by the Bioedit program. The computer program SplitTester was used to examine whether CPsHSPs of Rhododendron lineages and duplicate copies of the Machilus CPsHSPs have evolved functional divergence based on the hydrophobicity distance matrix. KEY RESULTS: Only one copy of the CPsHSP was found in Rhododendron. However, a higher evolutionary rate of amino acid substitutions in the Hymenanthes lineage of Rhododendron was inferred. Two positively selected amino acid sites may have resulted in higher hydrophobicity in the region of the alpha-crystallin domain (ACD) of the CPsHSP. By contrast, the basal angiosperm, Machilus, possessed duplicate copies of the CPsHSP, which also differed in their evolutionary rates of amino acid substitutions. However, no apparent relationship of ecological relevance toward the positively selected amino acid sites was found in Machilus. CONCLUSIONS: Divergent evolution was found for both Rhododendron lineages and the paralogues of CPsHSP in Machilus that were directed to the shift in hydrophobicity in the ACD and/or methionine-rich region, which might have played important roles in molecular chaperone activity.     

Chaperone-like activity of the N-terminal region of a human small heat shock protein and chaperone-functionalized nanoparticles

Small heat shock proteins (sHsps) are molecular chaperones employed to interact with a diverse range of substrates as the first line of defense against cellular protein aggregation. The N-terminal region (NTR) is implicated in defining features of sHsps; notably in their ability to form dynamic and polydisperse oligomers, and chaperone activity. The physiological relevance of oligomerization and chemical-scale mode(s) of chaperone function remain undefined. We present novel chemical tools to investigate chaperone activity and substrate specificity of human HspB1 (B1NTR), through isolation of B1NTR and development of peptide-conjugated gold nanoparticles (AuNPs). We demonstrate that B1NTR exhibits chaperone capacity for some substrates, determined by anti-aggregation assays and size-exclusion chromatography. The importance of protein dynamics and multivalency on chaperone capacity was investigated using B1NTR-conjugated AuNPs, which exhibit concentration-dependent chaperone activity for some substrates. Our results implicate sHsp NTRs in chaperone activity, and demonstrate the therapeutic potential of sHsp-AuNPs in rescuing aberrant protein aggregation.     

Effect of methylglyoxal modification and phosphorylation on the chaperone and anti-apoptotic properties of heat shock protein 27

Heat shock protein 27 (Hsp27) is a stress-inducible protein in cells that functions as a molecular chaperone and also as an anti-apoptotic protein. Methylglyoxal (MGO) is a reactive dicarbonyl compound produced from cellular glycolytic intermediates that reacts non-enzymatically with proteins to form products such as argpyrimidine. We found considerable amount of Hsp27 in phosphorylated form (pHsp27) in human cataractous lenses. pHsp27 was the major argpyrimidine-modified protein in brunescent cataractous lenses. Modification by MGO enhanced the chaperone function of both pHsp27 and native Hsp27, but the effect on Hsp27 was at least three-times greater than on pHsp27. Phosphorylation of Hsp27 abolished its chaperone function. Transfer of Hsp27 using a cationic lipid inhibited staurosporine (SP)-induced apoptotic cell death by 53% in a human lens epithelial cell line (HLE B-3). MGO-modified Hsp27 had an even greater effect (62% inhibition). SP-induced reactive oxygen species in HLE-B3 cells was significantly lower in cells transferred with MGO-modified Hsp27 when compared to native Hsp27. In vitro incubation experiments showed that MGO-modified Hsp27 reduced the activity of caspase-9, and MGO-modified pHsp27 reduced activities of both caspase-9 and caspase-3. Based on these results, we propose that Hsp27 becomes a better anti-apoptotic protein after modification by MGO, which may be due to multiple mechanisms that include enhancement of chaperone function, reduction in oxidative stress, and inhibition of activity of caspases. Our results suggest that MGO modification and phosphorylation of Hsp27 may have important consequences for lens transparency and cataract development.     

Activation of protein kinase B (Akt/RAC-protein kinase) by cellular stress and its association with heat shock protein Hsp27

Protein kinase B (PKB, also named as Akt or RAC-protein kinase), that is activated by cellular stress such as heat shock and hyperosmotic treatment, was revealed to be activated by oxidative stress and by chemical stressors of CdCl₂ and NaAsO₂ by measuring the activity of the enzyme immunoprecipitated from the transfected COS-7 cells. Upon stress treatment, a 30-kDa phosphoprotein was co-immunoprecipitated with PKB from the cells metabolic labeled with [³²P]orthophosphate. The phosphoprotein was identified as Hsp27, a small heat shock protein, by immunoblot analysis and co-immunoprecipitation. The association of Hsp27 was specific to PKB as the heat shock protein was not co-immunoprecipitated with other protein kinases such as protein kinase C and PKN. When the cells were treated with H₂O₂, PKB was activated gradually and the association of Hsp27 with PKB increased concurrently with the enhancement of PKB activity. In heat-shocked cells, activation of PKB and the association of Hsp27 were detected immediately after the treatment, and the association of the heat shock protein decreased while PKB kept stimulated activity when the cells were further incubated at 37°C. These results suggest that Hsp27 is involved in the activation process of PKB in the signal transduction pathway of various forms of stress.     

Effects of heat shock on ovary development and hsp83 expression in Tribolium castaneum (Coleoptera: Tenebrionidae)

Heat shock affects reproductive performance in insects including Tribolium castaneum. In this study, the effects of heat shock on ovary development and hsp83 expression in T. castaneum were investigated. Two lines of T. castaneum, H line and C line, from the same base population were established and maintained for five successive generations. In each generation, the newly hatched beetles (within 3 h after eclosion) in the H line were treated with a heat shock at 40°C for 1 h, and those in the C line were raised at normal temperature (30°C) as control treatment. Four traits related to ovary development were measured for the beetles of the fifth generation: days from eclosion to laying the first eggs (T_o), days from eclosion to laying the first hatchable eggs (T_h), ovariole size on the third day after eclosion, and pupal mass of their offspring. The results showed that the beetles of the H line had a significantly longer pre‐oviposition period (0.6 more days) and smaller ovariole size than those of the C line. No significant difference in pupal mass was observed. Applying heat shock to the offspring of the fifth generation of both lines led to significantly higher hsp83 expression in offspring of the C line than in offspring of the H line. Within each line, the hsp83 expression level in offspring with heat shock was significantly higher than that of offspring without heat shock, but the difference in the C line was much larger than that in the H line. We infer from these results that a tradeoff between heat resistance, registered as hsp83 expression, and ovarian development operates under heat stress in T. castaneum. 2009 Wiley Periodicals, Inc.     

Protein kinase C activation inhibits stress-induced synthesis of heat shock protein 27 in osteoblast-like cells: Function of arachidonic acid

Exposure of osteoblast-like MC3T3-E1 cells to sodium arsenite (arsenite) increased the level of heat shock protein 27 (hsp27). The effect of arsenite was dose-dependent in the range of 50 to 200 μM. Arsenite also stimulated arachidonic acid release dose-dependently in the range between 50 and 200 μM in these cells. Both indomethacin, an inhibitor of cyclooxygenase, and nordihydroguaiaretic acid, a lipoxygenase inhibitor, significantly enhanced the arsenite-induced accumulation of hsp27. Melittin, an activator of phospholipase A₂, significantly enhanced the arsenite-induced accumulation of hsp27. 12-O-Tetradecanoylphorbol-13-acetate (TPA), a protein kinase C (PKC)-activating phorbol ester, inhibited the arsenite-induced accumulation of hsp27. In contrast, 4α-phorbol 12, 13-didecanoate (4α-PDD), a PKC-nonactivating phorbol ester, had little effect. TPA suppressed the arsenite-induced arachidonic acid release, but 4α-PDD had little effect. Arsenite no longer affected cAMP accumulation, inositol phosphates formation nor the formation of choline and phosphocholine in these cells. These results suggest that the response to stress of hsp27 is coupled with the metabolic activity of the arachidonic acid cascade, and the activation of PKC inhibits the induction of hsp27 through the suppression of arachidonic acid release in osteoblast-like cells. © 1996 Wiley-Liss, Inc.     

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.     

Induction of a heat shock gene (hsp 70) in rabbit retinal ganglion cells detected by in situ hybridization with plastic-embedded tissue

Elevation of body temperature by 2–3°C induces a 2.7 kilobase hsp70 mRNA species in the rabbit retina within 1 hr. In situ hybridization with thin sections derived from plastic-embedded tissue permitted a higher level of resolution of retinal cell types compared to procedures which involved the use of frozen tissue sections. A prominent induction of hsp70 mRNA in retinal ganglion cells was observed when an hsp70 riboprobe was utilized for in situ hybridization. These results indicate that this neuronal cell type responds rapidly to fever-like body temperatures by inducing one of the major heat shock genes.     

Heat shock protein 27 is associated with irinotecan resistance in human colorectal cancer cells

Heat shock protein (Hsp) in tumor cells has been proposed to enhance their resistance to chemotherapeutic agents. In the present study, we investigated the influence of Hsp expression on the irinotecan resistance of human colorectal cancer cells. Among eight Hsp genes tested in this study, we confirmed that the expression of Hsp27 correlated with irinotecan resistance in colorectal cancer cells. Specific inhibition of Hsp27 expression using an antisense oliogodeoxynucleotide increased the irinotecan sensitivity. On the contrary, an overexpression of Hsp27 decreased the irinotecan sensitivity in colorectal cancer cells. Elevated expression of Hsp27 decreased caspase-3 activity in colorectal cancer cells. The expression level of Hsp27 determined by immunohistochemical analysis correlated with the clinical response to irinotecan in colorectal cancer patients. Hsp27 expression levels of irinotecan-non-responder (mean staining score, 6.28; proportion of high staining score, 64.2%) were significantly higher compared to those of irinotecan-responder (mean staining score, 3.16; proportion of high staining score, 33.3%) (P for t-test=0.045). These findings suggest that Hsp27 is involved in the irinotecan resistance of colorectal cancer cells possibly by reducing caspase-3 activity.     

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.