In vivo evaluation of hsp27 as an inhibitor of actin polymerization: hsp27 limits actin stress fiber and focal adhesion formation after heat shock

The role of hsp27 as an inhibitor of actin polymerization was considered in the context of the actin cytoskeleton and its relationship with focal adhesion formation. The aim of this study was to evaluate the potential effects of hsp27 on focal adhesion formation as a relevant biological consequence of actin stress fiber formation. When hsp27 was overexpressed in stably transfected cells, cell attachment was delayed and recovery of disrupted stress fibers and focal adhesions was limited. In ROS 17/2.8 cells, heat shock caused the reversible disruption of stress fibers and focal adhesions. The loss of stress fibers and focal adhesions was associated with reduced phosphotyrosine on the focal adhesion kinase (FAK). Microinjection of recombinant 6-His hsp27 and phosphorylated 6-His hsp27 was used to demonstrate that nonphosphorylated hsp27 prevented the recovery of stress fibers and focal adhesions. These results provide in vivo evidence that hsp27 acts as an inhibitor of actin polymerization that can alter cellular interactions with extracellular environments by perturbation of stress fibers, and subsequently focal adhesions.     

Modulation of the Stress-Induced Synthesis of hsp27 and αB-Crystallin by Cyclic AMP in C6 Rat Glioma Cells

Abstract: The possible participation of cyclic AMP in the stress-induced synthesis of two small stress proteins, hsp27 and αB-crystallin, in C6 rat glioma cells was examined by specific immunoassays, western blot analysis, and northern blot analysis. When C6 cells were exposed to arsenite (50–100 µM for 1 h) or heat (42°C for 30 min), expression of hsp27 and αB-crystallin was stimulated, with levels of the two proteins reaching a maximum after 10–16 h of culture. Induction of hsp27 was markedly enhanced when cells were exposed to arsenite in the presence of isoproterenol (20 µM) or epinephrine (20 µM) but not in the presence of phenylephrine. The stimulatory effects of isoproterenol and epinephrine were blocked completely by propranolol, an antagonist of β-adrenergic receptors. Cholera toxin (2 µg/ml), forskolin (20 µM), and dibutyryl cyclic AMP (2.5 mM), all of which are known to increase intracellular levels of cyclic AMP, also stimulated the arsenite- or heat-induced accumulation of hsp27. Treatment of cells with each of these modulators alone did not result in the induction of hsp27. The level of hsp70 in C6 cells, as estimated by western blot analysis, was also enhanced by arsenite or heat stress. However, induction of hsp70 by stress was barely stimulated by isoproterenol. By contrast, induction of αB-crystallin by heat or arsenite stress was suppressed when isoproterenol, cholera toxin, forskolin, or dibutyryl cyclic AMP was present during the stress period. Northern blot analysis of the expression of mRNAs for hsp70, hsp27, and αB-crystallin showed that the modulation of the stress-induced accumulation of the three hsps by the various agents was regulated at the level of the corresponding mRNA. These results indicate that stress responses of hsp70, hsp27, and αB-crystallin in C6 rat glioma cells are regulated differently and, moreover, that when the level of cyclic AMP increases in cells, the response to stress of hsp27 is stimulated but that of αB-crystallin is suppressed.     

Coordinate and independent regulation of αB-crystallin and HSP27 expression in response to physiological stress

α-Crystallins share structural and functional properties with the stress protein hsp27. These polypeptides are expressed at low constitutive levels in many tissues including brain, and αB-crystallin and hsp27 can accumulate in central nervous system glia in a variety of neurological conditions. We report here that heat shock and exposure to transition metals result in an increase in the steady state mRNA level of αB-crystallin and hsp27 in primary cultures of rat forebrain astrocytes. Both exposure to tumour necrosis factor-α and hypertonic conditions result in αB-crystallin mRNA accumulation but no change in the hsp27 mRNA level. Under some of these conditions increased synthesis and accumulation of αB-crystallin and hsp27 protein are also evident. We are unable to detect αA-crystallin mRNA in resting or stressed astrocytes. A novel phenomenon involving a transitory change in stress protein mRNA mobility in Northern blots during induction is reported, which is stress type and cell type independent. The results demonstrate multiple stress regulation of αB-crystallin and hsp27 in cultured astrocytes, suggesting that they can legitimately be regarded as stress proteins in the central nervous system. © 1994 wiley-Liss, Inc.     

Expression of Drosophila's 27 kDa heat shock protein into rodent cells confers thermal resistance.

The role of hsp27, one of Drosophila melanogaster's small heat shock proteins, in the process of thermotolerance was investigated. The coding sequence of hsp27 was subcloned downstream of the human hsp27 promoter which has been shown to be constitutively expressed in Chinese hamster O23 cells. Cellular resistance to a thermal stress was measured two days after transfection by a survival assay following a 3.5 h heat treatment at 44 degrees C. Expression of Drosophila hsp27 was shown to confer thermal resistance to O23 cells in a manner which was dependent on the level of expression of this hsp. Immunoblot analysis confirmed that the thermal resistance was related to the expression of Drosophila hsp27 as none of the endogeneous hsps showed an increased level under these conditions.     

Tumor necrosis factor-α induces changes in the phosphorylation,cellular localization,and oligomerization of human hsp27, a stress protein that confers cellular resistance to this cytokine

The stress protein hsp27 is constitutively expressed in several human cells and shows a rapid phosphorylation following treatment with tumor necrosis factor-α (TNF-α). hsp27 usually displays native molecular mass ranging from 100 to 700 kDa. Here, we have analyzed the TNF-α-mediated changes in the phosphorylation, cellular localization, and structural organization of hsp27 in HeLa cells. We report that the TNF-α-mediated hsp27 phosphorylation is a long-lasting phenomenon that correlates with the cytostatic effect of this cytokine. Following TNF-α treatment, the rapid phosphorylation of hsp27 occurred concomitantly with complex changes in the intracellular distribution and structural organization of this protein. This resulted in the quantitative redistribution of hsp27 toward the soluble phase of the cytoplasm. In addition, during the first 2 h of TNF-α treatment, a transient increase in the native molecular mass of most hsp27 molecules (≤ 700 kDa) occurred. Then, by 4 h of TNF-α treatment, the native size of this stress protein drastically regressed (< 200 kDa). During this phenomenon, the phosphorylated isoforms of hsp27 remained concentrated in the small or medium-sized oligomers (< 300 kDa) of this protein. We also analyzed the properties of human hsp27 in transfected murine L929 cell lines that constitutively express this protein. In these cells, TNF-α induced modifications in the phosphorylation, intracellular distribution, and oligomerization of human hsp27 similar to those observed in HeLa cells. Moreover, the expression of hsp27 in L929 cells was found to correlate with a reduced cytotoxicity of this cytokine. Hence, the complex changes in the phosphorylation, intracellular locale and structural organization of human hsp27 may be related to the protective activity of this protein against the deleterious effects induced by TNF-α.     

Identification and characterization of hic-5/ARA55 as an hsp27 binding protein

hsp27 has been reported to participate in a wide variety of activities, including resistance to thermal and metabolic stress, regulation of growth and differentiation, and acting as a molecular chaperone or a regulator of actin polymerization. We hypothesized that these diverse functions are regulated in a cell- or tissue-specific manner via interaction with various binding proteins. To investigate this hypothesis, we used hsp27 as a "bait" to screen a yeast two-hybrid cDNA library from rat kidney glomeruli and identified a novel hsp27 binding protein, hic-5 (also known as ARA55), a focal adhesion protein and steroid receptor co-activator. Biochemical interaction between hsp27 and hic-5 was confirmed by co-immunoprecipitation, and critical protein.protein interaction regions were mapped to the hic-5 LIM domains and the hsp27 C-terminal domain. Initial analysis of the functional role of hsp27.hic-5 interaction revealed that hic-5 significantly inhibited the protection against heat-induced cell death conferred by hsp27 overexpression in co-transfected 293T cells. In contrast, when a non-hsp27-interacting hic-5 truncation mutant (hic-5/DeltaLIM4) was co-expressed with hsp27, the hic-5 inhibition of hsp27 protection was absent. We conclude that hic-5 is a true hsp27 binding protein and inhibits the ability of hsp27 to provide protection against heat shock in an interaction-dependent manner.     

Identification and characterization of a novel protein from Sertoli cells, PASS1, that associates with mammalian small stress protein hsp27

hsp27 is involved in development of tolerance to stress, possibly by its involvement in molecular chaperoning, maintenance of glutathione status, and/or modulation of microfilament structure and function. We hypothesize that hsp27 function depends on specific association with other proteins. To discover proteins that associate with hsp27, we made a differentiated rat Sertoli cell cDNA expression library and screened it using the yeast two-hybrid system. We obtained a cDNA coding for a novel protein of 428 amino acids that we have named PASS1 (protein associated with small stress proteins 1). BLAST searches did not reveal major similarity of PASS1 to any known protein, but the cDNA sequence matched several mouse EST clones and shares 34% homology with a Caenorhabditis elegans genomic sequence. In vitro, bacterially expressed glutathione S-transferase-PASS1 fusion protein bound to hsp27, and hsp27 was co-immunoprecipitated with c-Myc-tagged PASS1 overexpressed in several cell lines. The region of PASS1 responsible for association with hsp27 was identified as existing predominantly between amino acids 108 and 208 of PASS1. Northern hybridization and Western blot analysis demonstrated that PASS1 is expressed in several tissues, with the highest expression occurring in testis, primarily in Sertoli cells. The presence of a 1.4-kilobase PASS1 mRNA in kidney as well as the 1. 8-kilobase mRNA seen in other tissues suggests that alternate splicing may occur in this organ. Ectopic expression of PASS1 in two cultured cell lines was observed to inhibit the ability of hsp27 to protect cells against heat shock, indicating that PASS1 does interact with hsp27 in the live cell.     

Expression of the small heat shock protein (hsp) 27 in human astrocytomas correlates with histologic grades and tumor growth fractions

Summary 1. Cellular expression and distribution of the stress response small heat shock protein 27 (hsp27) in 39 high-grade astrocytomas (27 glioblastoma multiformes, 12 anaplastic astrocytomas) and in 27 low-grade astrocytomas (grade I–II) were analyzed immunohistochemically.2. The correlation between hsp27 expression and tumor growth fractions of the astrocytomas was examined following Ki-67 immunostaining.3. The hsp27 staining was cell cytoplasmic. The hsp27 immunopositive rate was significantly higher in high-grade astrocytomas; the rates were 74% for glioblastomas, 58% for anaplastic astrocytomas, and 37% for low-grade astrocytomas. The small and large tumor cells, especially in glioblastomas, multinucleated tumor giant cells, tumor cells in the pseudopalisading and necrotic areas, cells of the microvascular endothelial proliferations, and tumor vascular smooth muscles were usually hsp27 positive. The mean percentage of hsp27-positive cells was significantly higher in the glioblastomas alone and in the combined high-grade astrocytomas, compared to the low-grade, and in recurrent rather than in primary high-grade astrocytomas.4. The high-grade astrocytomas had a highly statistical significant Ki-67 labeling index. The Ki-67 labeling indices were significantly higher in the hsp27-positive than the hsp27-negative astrocytomas, irrespective of the histological grade. In the high-grade astrocytomas with a Ki-67 labeling index of five and above, 81% of those tumors were hsp27 positive.5. Thus, a large number of human astrocytomas express hsp27, and hsp27 expression correlates with histological grades of astrocytoma and with tumor growth fractions. This being the case, hsp27 is likely to have a role in the growth of human astrocytomas.     

The effect of neuronal expression of heat shock proteins 26 and 27 on lifespan, neurodegeneration, and apoptosis in Drosophila

Heat shock proteins (Hsps) are chaperones thought to increase lifespan, enhance stress resistance, and prevent apoptosis and neurodegenerative diseases. Our previous study reported that ubiquitous expression of hsp26 or hsp27 extended Drosophila lifespan. The effect of neuronal expression of hsp26 and hsp27 in Drosophila on the above-mentioned functions has not yet been investigated. Here, we show that neuronal expression of hsp26 or hsp27 improved lifespan and increased resistance to oxidative stress. However, only neuronal expression of hsp27 ameliorated Parkinsonism climbing disorder and attenuated mild polyglutamine-induced toxicity. Additionally, neuronal expression of hsp27 specifically partially rescued hid-induced lethality, but was not able to rescue reaper/grim-induced lethality. However, unlike hsp27, neuronal expression of hsp26 did not rescue hid-induced or reaper/grim-induced lethality. In summary, we demonstrate the functional similarities and differences of neuronal expression of hsp26 and hsp27 in adult Drosophila.     

Heat shock protein 27 downregulates the transferrin receptor 1-mediated iron uptake

It has been reported that over-expression of human heat shock protein 27 (hsp27) in murine cells decreased the intracellular iron level [Arrigo, A. P., Virot, S., Chaufour, S., Firdaus, W., Kretz-Remy, C., & Diaz-Latoud, C. (2005). Hsp27 consolidates intracellular redox homeostasis by upholding glutathione in its reduced form and by decreasing iron intracellular levels. Antioxidants & Redox Signalling, 7, 412-422]. However, the mechanism involved is unknown. In this study, the regulation of transferrin receptor 1 (TfR1)-mediated iron uptake by human hsp27 was investigated in CCL39 cells by overexpression of human hsp27 and its dominant-negative mutant (hsp27-3G). The results showed that overexpression of hsp27 diminished intracellular labile iron pool, increased the binding activity of iron regulatory protein (IRP) to iron responsive element (IRE) and the cell surface-expressed TfR1s. However, the increased surface-expressed TfR1s resulted in decrease rather than increase of iron uptake. Further study revealed that overexpression of hsp27 decelerated transferrin endocytosis and recycling, while overexpressed hsp27-3G had a reversal effect. Moreover, flowcytometric analysis showed an enhanced actin polymerization in the cells overexpressing hsp27. In particular, fluorescence imaging of cytoskeleton displayed highly stabilized microfilaments and preferential localization of hsp27 in cortical area of the actin cytoskeleton. In contrast, disruption of actin cytoskeleton by cytochalasin B resulted in acceleration of the endocytosis and recycling of Tf, as well as increase of iron uptake. Meanwhile, the possible involvement of ferroportin 1 in down-regulation of intracellular iron level by overexpression of hsp27 was checked. However, the outcome was negative. Our findings indicate that hsp27 down-regulates TfR1-mediated iron uptake via stabilization of the cortical actin cytoskeleton rather than the classical IRP/IRE mode. The study may also imply that hsp27 protects cells from oxidative stress by reducing cellular iron uptake.     

Non-thermal activation of the hsp27/p38MAPK stress pathway by mobile phone radiation in human endothelial cells: molecular mechanism for cancer- and blood-brain barrier-related effects

Abstract We have examined whether non-thermal exposures of cultures of the human endothelial cell line EA.hy926 to 900 MHz GSM mobile phone microwave radiation could activate stress response. Results obtained demonstrate that 1-hour non-thermal exposure of EA.hy926 cells changes the phosphorylation status of numerous, yet largely unidentified, proteins. One of the affected proteins was identified as heat shock protein-27 (hsp27). Mobile phone exposure caused a transient increase in phosphorylation of hsp27, an effect which was prevented by SB203580, a specific inhibitor of p38 mitogen-activated protein kinase (p38MAPK). Also, mobile phone exposure caused transient changes in the protein expression levels of hsp27 and p38MAPK. All these changes were non-thermal effects because, as determined using temperature probes, irradiation did not alter the temperature of cell cultures, which remained throughout the irradiation period at 37 ± 0.3 °C. Changes in the overall pattern of protein phosphorylation suggest that mobile phone radiation activates a variety of cellular signal transduction pathways, among them the hsp27/p38MAPK stress response pathway. Based on the known functions of hsp27, we put forward the hypothesis that mobile phone radiation-induced activation of hsp27 may (i) facilitate the development of brain cancer by inhibiting the cytochrome c/caspase-3 apoptotic pathway and (ii) cause an increase in blood-brain barrier permeability through stabilization of endothelial cell stress fibers. We postulate that these events, when occurring repeatedly over a long period of time, might become a health hazard because of the possible accumulation of brain tissue damage. Furthermore, our hypothesis suggests that other brain damaging factors may co-participate in mobile phone radiation-induced effects.     

DNA sequence variation and latitudinal associations in hsp23, hsp26 and hsp27 from natural populations of Drosophila melanogaster

Heat shock genes are considered to be likely candidate genes for environmental stress resistance. Nucleotide variation in the coding sequence of the small heat shock genes (hsps) hsp26 and hsp27 from Drosophila melanogaster was studied in flies originating from the Netherlands and eastern Australia. The hsp26 gene was polymorphic for an insertion/deletion of three extra amino acids and two nonsynonymous changes in all populations. The hsp27 gene exhibited two nonsynonymous changes and three synonymous mutations. The hsp26 polymorphism showed a latitudinal cline along the east coast of Australia. This pattern was not confounded by the fact that the shsps are located in the inversion In(3 L)P which also shows a latitudinal cline in eastern Australia. A similar latitudinal cline was found for the previously described variation in hsp23, while frequencies of hsp27 alleles did not change with latitude. These findings suggest that variation at two of the shsps or closely linked loci are under selection in natural populations of D. melanogaster.     

Antioxidant-mediated inhibition of the heat shock response leads to apoptosis

We examined the hypothesis that reactive oxygen species (ROS) contribute to the induction of heat shock proteins (hsps) during stress response. Exposure of HL-60 human myelocytic cells to 42 degrees C induced both hsp72 and hsp27. In the presence of the antioxidant molecules pyrrolidine dithiocarbamate or 1,10-phenanthroline induction of hsp72 and 27 was significantly decreased, while N-acetyl-L-cysteine caused a slight reduction. Prevention of hsp induction was associated with heat sensitization and increased caspase activity, indicating that the cells were undergoing apoptosis. These data suggest that ROS contribute to the induction of hsps and furthermore, that hsp induction and apoptosis are mutually exclusive events within the same cell.     

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.     

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.     

Human hsp27, Drosophila hsp27 and human alphaB-crystallin expression-mediated increase in glutathione is essential for the protective activity of these proteins against TNFalpha-induced cell death.

Expression of small stress proteins (shsp) enhances the survival of mammalian cells exposed to heat or oxidative injuries. Recently, we have shown that the expression of shsp from different species, such as human hsp27, Drosophila hsp27 or human alphaB-crystallin protected murine L929 cells against cell death induced by tumor necrosis factor (TNFalpha), hydrogen peroxide or menadione. Here, we report that, in growing L929 cell lines, the presence of these shsp decreased the intracellular level of reactive oxygen species (ROS). shsp expression also abolished the burst of intracellular ROS induced by TNFalpha. Several downstream effects resulting from the TNFalpha-mediated ROS increment, such as NF-kappaB activation, lipid peroxidation and protein oxidation, were inhibited by shsp expression. We also report that the expression of these different shsp raised the total glutathione level in both L929 cell lines and transiently transfected NIH 3T3-ras cells. This phenomenon was essential for the shsp-mediated decrease in ROS and resistance against TNFalpha. Our results therefore suggest that the protective activity shared by human hsp27, Drosophila hsp27 and human alphaB-crystallin against TNFalpha-mediated cell death and probably other types of oxidative stress results from their conserved ability to raise the intracellular concentration of glutathione.