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.     

HSP22, a new member of the small heat shock protein superfamily, interacts with mimic of phosphorylated HSP27 ((3D)HSP27)

Most of the members of the superfamily of mammalian small heat shock or stress proteins are abundant in muscles where they play a role in muscle function and maintenance of muscle integrity. One member of this protein superfamily, human HSP27, is rapidly phosphorylated on three serine residues (Ser(15), Ser(78), and Ser(82)) during cellular response to a number of extracellular factors. To understand better the role of HSP27, we performed a yeast two-hybrid screen of a human heart cDNA library for HSP27-interacting proteins. By using the triple aspartate mutant, a mimic of phosphorylated HSP27, as "bait" construct, a protein with a molecular mass of 21.6 kDa was identified as an HSP27-binding protein. Sequence analysis revealed that this new protein shares an overall sequence identity of 33% with human HSP27. This protein also contains the alpha-crystallin domain in its C-terminal half, a hallmark of the superfamily of small stress proteins. Thus, the new protein itself is a member of this protein superfamily, and consequently we designated it HSP22. According to the two-hybrid data, HSP22 interacts preferentially with the triple aspartate form of HSP27 as compared with wild-type HSP27. HSP22 is expressed predominantly in muscles. In vitro, HSP22 is phosphorylated by protein kinase C (at residues Ser(14) and Thr(63)) and by p44 mitogen-activated protein kinase (at residues Ser(27) and Thr(87)) but not by MAPKAPK-2.     

Recruitment of phosphorylated small heat shock protein Hsp27 to nuclear speckles without stress

During stress, the mammalian small heat shock protein Hsp27 enters cell nuclei. The present study examines the requirements for entry of Hsp27 into nuclei of normal rat kidney (NRK) renal epithelial cells, and for its interactions with specific nuclear structures. We find that phosphorylation of Hsp27 is necessary for the efficient entry into nuclei during heat shock but not sufficient for efficient nuclear entry under control conditions. We further report that Hsp27 is recruited to an RNAse sensitive fraction of SC35 positive nuclear speckles, but not other intranuclear structures, in response to heat shock. Intriguingly, Hsp27 phosphorylation, in the absence of stress, is sufficient for recruitment to speckles found in post-anaphase stage mitotic cells. Additionally, pseudophosphorylated Hsp27 fused to a nuclear localization peptide (NLS) is recruited to nuclear speckles in unstressed interphase cells, but wildtype and nonphosphorylatable Hsp27 NLS fusion proteins are not. The expression of NLS-Hsp27 mutants does not enhance colony forming abilities of cells subjected to severe heat shock, but does regulate nuclear speckle morphology. These data demonstrate that phosphorylation, but not stress, mediates Hsp27 recruitment to an RNAse soluble fraction of nuclear speckles and support a site-specific role for Hsp27 within the nucleus.     

Transfection of human HSP27 in rodent cells: Absence of compensatory regulation between small heat shock proteins

1. 1. We examined rodent cells transfected with an expression plasmid encoding a human small heat shock protein for possible compensatory expression of endogenous heat shock genes. For these investigations, human hsp27 was transfected into CHO cells which express endogenous HSP25.

2. 2. Both endogenous HSP25 and transfected HSP27 were expressed and multiple phosphorylated isoforms were detected upon exposure to thermal stress.

3. 3. Levels of endogenous HSP70 and HSP25 did not appear to be altered by expression of the heterologous heat shock protein.

4. 4. These results suggest that compensatory interactions are not exhibited in the expression of the heat shock genes examined, and that independent regulation may exist not only between the large and small heat shock proteins, but also between individual small heat shock proteins as well.

     

Accumulation of small heat shock proteins, including mitochondrial HSP22, induced by oxidative stress and adaptive response in tomato cells

Changes in gene expression, by application of H₂O₂, O₂°^–generating agents (methyl viologen, digitonin) and gamma irradiation to tomato suspension cultures, were investigated and compared to the well-described heat shock response. Two-dimensional gel protein mapping analyses gave the first indication that at least small heat shock proteins (smHSP) accumulated in response to application of H₂O₂ and gamma irradiation, but not to O₂°^– generating agents. While some proteins seemed to be induced specifically by each treatment, only part of the heat shock response was observed. On the basis of Northern hybridization experiments performed with four heterologous cDNA, corresponding to classes I–IV of pea smHSP, it could be concluded that significant amounts of class I and II smHSP mRNA are induced by H₂O₂ and by irradiation. Taken together, these results demonstrate that in plants some HSP genes are inducible by oxidative stresses, as in micro-organisms and other eukaryotic cells. HSP22, the main stress protein that accumulates following H₂O₂ action or gamma irradiation, was also purified. Sequence homology of amino terminal and internal sequences, and immunoreactivity with Chenopodium rubrum mitochondrial smHSP antibody, indicated that the protein belongs to the recently discovered class of plant mitochondrial smHSP. Heat shock or a mild H₂O₂ pretreatment was also shown to lead to plant cell protection against oxidative injury. Therefore, the synthesis of these stress proteins can be considered as an adaptive mechanism in which mitochondrial protection could be essential.     

Exogenous heat shock protein HSP70 reduces response of human neuroblastoma cells to lipopolysaccharide

The effect of exogenous heat shock protein HSP70 and lipopolysaccharide (LPS) on the production of reactive oxygen species (ROS), TNFα secretion, and mRNA expression by human neuroblastoma SK-N-SH cells. It was shown that exogenous HSP70 protects neuroblastoma cells from the action of LPS. The protection mechanism of HSP70 includes a reduction in the production of ROS and TNFα and a decrease in the expression of TLR4 and IL-1β mRNA in SK-N-SH cells induced by LPS.     

Small heat shock protein 27 (HSP27) associates with tubulin/microtubules in HeLa cells

One of the monoclonal antibodies raised against mitotic HeLa cells (termed as mH3) recognized a 27-kDa protein and stained microtubules in the mitotic spindles of HeLa cells. Immunoscreening of a HeLa cDNA library revealed that mH3 antigen is a small heat shock protein, HSP27. Immunoprecipitation analysis using mH3 suggested that both alpha- and beta-tubulin are associated with HSP27. Further, sucrose-cushioned ultra centrifugation revealed that HSP27 is co-sedimented with taxol-stabilized microtubules. These results indicate that HSP27 associates with tubulin/microtubules in HeLa cells.     

Induction of heat shock protein 27 in rat embryos exposed to hyperthermia

Previously we reported that eight proteins were reproducibly induced in postimplantation rat embryos exposed to a brief heat shock (43°C, 15 min). The major heat-inducible rat embryo protein has now been identified as heat shock protein 72 (Hsp 72). In addition, the induction of Hsp 72 is temporally correlated with induction of thermotolerance. One of the other rat embryo proteins previously shown to be induced by elevated temperature is a heat shock protein of approximately 27 kilodaltons (Hsp 27). In this report we show that this protein is recognized by an antibody directed against a conserved peptide sequence of Hsp 27. Unlike Hsp 72, Hsp 27 is constitutively expressed in the rat embryo in the absence of any thermal stress; however, the level of Hsp 27 is increased approximately 2–3-fold after thermal stress (43°C, 10 min). Immunohistochemical analysis revealed that the constitutively expressed Hsp 27 is localized primarily to cells of the heart, cells that are uniquely resistant to the cytotoxic effects of hyperthermia. After thermal stress, Hsp 27 is expressed in all tissues of the embryo. Finally, our data show that Hsp 27 exists in the rat embryo as three major isoforms indicative of different phosphorylation states. Furthermore, most Hsp 27 in the heart is phosphorylated, whereas in the rest of the embryo, nonphosphorylated Hsp 27 predominates. After thermal stress, levels of phosphorylated isoforms increase dramatically in nonheart tissues of the embryo. Together, these results suggest that Hsp 27 may play a role in the development of thermotolerance in the postimplantation mammalian embryo. © 1996 Wiley-Liss, Inc.     

Effects of prolonged stanozolol treatment on antioxidant enzyme activities, oxidative stress markers, and heat shock protein HSP72 levels in rat liver

The abuse of anabolic-androgenic steroids (AAS) to enhance physical performance is widespread in sport communities despite their reported side effects. Since the biochemical bases for the hepatotoxic effects of these compounds are largely unknown, this investigation was aimed at testing whether prolonged (8 weeks) treatment with high doses (2 mg kg⁻¹ body weight; 5 d wk⁻¹) of stanozolol (ST), either alone or in conjunction with treadmill-exercise training, induced changes in oxidative stress biomarker levels and antioxidant defence systems in rat liver. After ST oral administration, the mean values of serum parameters related to hepatic function were within normal ranges. No changes in protein carbonyl content and in the reduced to oxidized glutathione (GSH/GSSG) ratio were detected in liver homogenates of ST-treated rats, whereas thiobarbituric acid-reactive substances (TBARS) levels resulted increased (P<0.05). Total superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPX) activities were higher (P<0.05) in the liver of treated rats but mitochondrial SOD and glutathione reductase (GR) activities, and the 72 kDa heat shock protein (HSP72) level were not modified. Chronic exercise alone did not change any of the above parameters except for a remarkable enhancement of HSP72 expression; in no case training modified the effects of ST treatment. The present data show that 8 wk ingestion of ST, either with or without concurrent exercise training, can induce oxidative stress in rat liver despite the up-regulation of enzymatic antioxidant activities.     

Overexpression of heat shock protein 27 (HSP27) increases gemcitabine sensitivity in pancreatic cancer cells through S‐phase arrest and apoptosis

We previously established a role for HSP27 as a predictive marker for therapeutic response towards gemcitabine in pancreatic cancer. Here, we investigate the underlying mechanisms of HSP27‐mediated gemcitabine sensitivity. Utilizing a pancreatic cancer cell model with stable HSP27 overexpression, cell cycle arrest and apoptosis induction were analysed by flow cytometry, nuclear staining, immunoblotting and mitochondrial staining. Drug sensitivity studies were performed by proliferation assays. Hyperthermia was simulated using mild heat shock at 41.8°C. Upon gemcitabine treatment, HSP27‐overexpressing cells displayed an early S‐phase arrest subsequently followed by a strongly increased sub‐G1 fraction. Apoptosis was characterized by PARP‐, CASPASE 3‐, CASPASE 8‐, CASPASE 9‐ and BIM‐ activation along with a mitochondrial membrane potential loss. It was reversible through chemical caspase inhibition. Importantly, gemcitabine sensitivity and PARP cleavage were also elicited by heat shock‐induced HSP27 overexpression, although to a smaller extent, in a panel of pancreatic cancer cell lines. Finally, HSP27‐overexpressing pancreatic cancer cells displayed an increased sensitivity also towards death receptor‐targeting agents, suggesting another pro‐apoptotic role of HSP27 along the extrinsic apoptosis pathway. Taken together, in contrast to the well‐established anti‐apoptotic properties of HSP27 in cancer, our study reveals novel pro‐apoptotic functions of HSP27—mediated through both the intrinsic and the extrinsic apoptotic pathways—at least in pancreatic cancer cells. HSP27 could represent a predictive marker of therapeutic response towards specific drug classes in pancreatic cancer and provides a novel molecular rationale for current clinical trials applying the combination of gemcitabine with regional hyperthermia in pancreatic cancer patients.     

Two-dimensional crystallization of a small heat shock protein HSP16.3 on lipid layer

As a member of small heat shock proteins, HSP16.3 was identified as the major membrane-bound protein of Mycobacterium tuberculosis during stationary phase. Previous studies revealed that HSP16.3 was in a nonameric form in solution. Here, two-dimensional crystal of HSP16.3 molecules on lipid monolayer was obtained for the first time. The crystal exhibited p422 symmetry with lattice parameters a=b=90A, gamma=90 degrees. The projection map of untilted crystals showed that the basic unit of the crystal was a rod-like structure with two high-density regions. The three-dimensional map at 2.2 nm resolution revealed a rod-like structure with a dimension of 56A x 32A x 25A, similar to the dimeric forms of M. jannaschii HSP16.5 and wheat HSP16.9. Cross-linking experiments confirmed that HSP16.3 nonamers dissociated into dimers upon interaction with the positively charged lipid layer. Surface plasmon resonance measurements revealed that both electrostatic and hydrophobic forces involved in the formation of the 2D crystal on the lipid monolayer. These results provide a basis for further investigation on the unique dimeric structure of HSP16.3 and its functions in vivo.     

In vivo modifications of the maize mitochondrial small heat stress protein, HSP22

A maize (Zea mays L.) small heat shock protein (HSP), HSP22, was previously shown to accumulate to high levels in mitochondria during heat stress. Here we have purified native HSP22 and resolved the protein into three peaks using reverse phase high performance liquid chromatography. Mass spectrometry (MS) of the first two peaks revealed the presence of two HSP22 forms in each peak which differed in mass by 80 daltons (Da), indicative of a monophosphorylation. Phosphorylation of HSP22 by [gamma-(32)P]ATP was also observed in mitochondria labeled in vitro, but not when purified native HSP22 was similarly used, demonstrating that HSP22 does not autophosphorylate, implicating a kinase involvement in vivo. Collisionally induced dissociation tandem MS (CID MS/MS) identified Ser(59) as the phosphorylated residue. We have also observed forms of HSP22 that result from alternative intron splicing. The two HSP22 proteins in the first peak were approximately 57 Da larger than the two HSP22 proteins in the second peak. MS analysis revealed that the +57-Da forms have an additional Gly residue directly N-terminal of the expected Asp(84), which had been converted to an Asn residue. These results are the first demonstrations of phosphorylation and alternative intron splicing of a plant small HSP.     

Role of small heat shock protein HSP25 in radioresistance and glutathione-redox cycle

Expression of heat shock proteins (HSPs) has been shown to protect mammalian cells exposed to a variety of stress stimuli. Among various HSPs, small HSPs from diverse species were shown to protect cells against oxidative stress. Here, we show that the overexpression of the mouse small hsp gene, hsp25, provides protection against ionizing radiation. Our results demonstrate that the radiation survival of the L929 cells stably transfected with hsp25 was enhanced compared with that of the parental or vector transfected control, L25#1 cells. Our results also demonstrate that the radiation-induced apoptosis was reduced in HSP25 overexpressors. A detailed analysis of glutathione composition of those clones that overexpressed HSP25 revealed the increases of the glutathione pool, which primarily resulted from the increase of reduced glutathione. Our data suggest that higher content of GSH in HSP25 overexpressors was because of a faster reduction of oxidized glutathione (GSSG) to GSH rather than an increased de novo synthesis of GSH. The activities of glutathione reductase (GRd) and glutathione peroxidase (GPx) were greater in HSP25 overexpressors but the activity of gamma-glutamylcysteine synthetase was similar between the transfectants and the control cells. Consistent with our view, a steady state ratio of the GSH/GSSG was greater in the transfectants in comparison with the control L25#1 cells. A difference in the relative ratio became more significant after exposure to the ionizing radiation. To our knowledge, this study provides the first experimental evidence in support of the hypothesis that small HSP plays a key role in radioresistance by modulating the metabolism of glutathione. Based on the results obtained from the current investigation, we propose that HSP25 helps facilitate the glutathione-redox cycle and therefore, enhances glutathione utilization and maintains the cellular glutathione pool in favor of the reduced states.     

Stress-mediated signaling in PC12 cells - the role of the small heat shock protein,Hsp27, and Akt in protecting cells from heat stress and nerve growth factor withdrawal

We have investigated the role of stress-activated signaling pathways and the small heat shock protein, Hsp27, in protecting PC12 cells from heat shock and nerve growth factor (NGF) withdrawal-induced apoptosis. PC12 cells and a stable cell line overexpressing Hsp27 (HSPC cells) were subjected to heat shock. This resulted in the rapid activation of Akt followed by p38 mitogen-activated protein kinase (MAPK) signaling, with phosphorylation and intracellular translocation of Hsp27 also detectable. Hsp27 was found to form an immunoprecipitable complex with Akt and p38 MAPK in both non-stimulated and heat shocked cells, although after heat shock there was a gradual dissociation of Akt and p38 from the Hsp27. Cells were differentiated with NGF and then subjected to NGF withdrawal, a treatment which results in substantial cell death over 24-72 h. Hsp27 was shown to be protective against this treatment, since HSPC cells which overexpress Hsp27 showed significantly less cell death than the parental PC12 cells. In addition, we observed that phosphorylation of Akt was maintained in HSPC cells subjected to heat shock and NGF withdrawal compared with the parental cells. Taken together, our results suggest that Hsp27 may protect Akt from dephosphorylation and may also act in stabilizing Akt.