Phosphorylation-dependent cellular localization and thermoprotective role of heat shock protein 25 in hippocampal progenitor cells

The present study examined phosphorylation-dependent cellular localization and the thermoprotective role of heat shock protein (HSP) 25 in hippocampal HiB5 cells. HSP25 was induced and phosphorylated by heat shock (at 43 degrees C for 3 h). HSP25, which was located in the cytoplasm in the normal condition, translocated into the nucleus after the heat shock. Transfection experiments with hsp27 mutants in which specific serine phosphorylation residues (Ser(78) and Ser(82)) were substituted with alanines or aspartic acids showed that phosphorylation of HSP27 is accompanied by its nuclear translocation. Phosphorylation of mitogen-activated protein kinases (MAPKs) such as p38 MAPK and ERK was markedly increased by the heat shock, and SB203580 (a p38 MAPK kinase inhibitor) and/or PD098059 (a MEK inhibitor) inhibited the phosphorylation of HSP25, indicating that p38 MAPK and ERK are upstream regulators of HSP25 phosphorylation in the heat shock condition. In the absence of heat shock, actin filament stability was not affected by SB203580 and/or PD098059. Heat shock caused disruption of the actin filament and cell death when phosphorylation of HSP25 was inhibited by SB203580 and/or PD098059. In addition, actin filament was more stable in Asp(78,82)-hsp27 (mimics the phosphorylated form) transfected HiB5 cells than in the normal and Ala(78,82)-hsp27 (nonphosphorylative form) transfected cells. In accordance with actin filament stability, the survival rate against the heat shock increased markedly in Asp(15,78,82)-hsp27 expressing HiB5 cells but decreased in Ala(15,78,82)-hsp27 expressing cells. These results support the idea that phosphorylation of HSP25 is critical for the maintenance of actin filament and enhancement of thermoresistance. Interestingly, HSP25 was dephosphorylated and returned to cytoplasm in a recovery time-dependent manner. This phenomenon was accompanied by an increment of apoptotic cell death as determined by nuclear and DNA fragmentation and fluorescence-activated cell sorter analysis. These results suggest that nuclear-translocated HSP25 might function to protect nuclear structure, thereby preventing apoptotic cell death.     

Copine1 C2 domains have a critical calcium-independent role in the neuronal differentiation of hippocampal progenitor HiB5 cells

Copine1 (CPNE1) has tandem C2 domains and an A domain and is known as a calcium-dependent membrane-binding protein that regulates signal transduction and membrane trafficking. We previously demonstrated that CPNE1 directly induces neuronal differentiation via Akt phosphorylation in the hippocampal progenitor cell line, HiB5. To determine which region of CPNE1 is related to HiB5 cell neurite outgrowth, we constructed several mutants. Our results show that over-expression of each C2 domain of CPNE1 increased neurite outgrowth and expression of the neuronal marker protein neurofilament (NF). Even though protein localization of the calcium binding-deficient mutant of CPNE1 was not affected by ionomycin, this mutant increased neurite outgrowth and NF expression in HiB5 cells. Furthermore, Akt phosphorylation was increased by over-expression of the calcium binding-deficient CPNE1 mutant. These results suggest that neither cellular calcium levels nor the localization of CPNE1 affect its function in neuronal differentiation. Collectively, our findings indicating that the C2 domains of CPNE1 play a calcium-independent role in regulating the neuronal differentiation of HiB5 cells.     

The neuroprotective effects of heat shock protein 27 overexpression in transgenic animals against kainate-induced seizures and hippocampal cell death

The 27-kDa heat shock protein (HSP27) has a potent ability to increase cell survival in response to a wide range of cellular challenges. In order to investigate the mode of action of HSP27 in vivo, we have developed transgenic lines, which express human HSP27 at high levels throughout the brain, spinal cord, and other tissues. In view of the particular property of HSP27 compared with other HSPs to protect neurons against apoptosis, we have tested these transgenic lines in a well established in vivo model of neurotoxicity produced by kainic acid, where apoptotic cell death occurs. Our results demonstrate for the first time the marked protective effects of HSP27 overexpression in vivo, which significantly reduces kainate-induced seizure severity and mortality rate (>50%) in two independent lines and markedly reduces neuronal cell death in the CA3 region of hippocampus. This reduced seizure severity in HSP27 transgenic animals was associated with a marked attenuation of caspase 3 induction and apoptotic features. These studies clearly demonstrate that HSP27 has a major neuroprotective effect in the central nervous system in keeping with its properties demonstrated in culture and highlight an early stage in the cell death pathway that is affected by HSP27.     

Cyclin D3 and c-MYC control glucocorticoid-induced cell cycle arrest but not apoptosis in lymphoblastic leukemia cells

Glucocorticoids (GC) induce cell cycle arrest and apoptosis in lymphoblastic leukemia cells. To investigate cell cycle effects of GC in the absence of obscuring apoptotic events, we used human CCRF-CEM leukemia cells protected from cell death by transgenic bcl-2. GC treatment arrested these cells in the G1 phase of the cell cycle due to repression of cyclin D3 and c-myc. Cyclin E and Cdk2 protein levels remained high, but the kinase complex was inactive due to increased levels of bound p27(Kip1). Conditional expression of cyclin D3 and/or c-myc was sufficient to prevent GC-induced G1 arrest and p27(Kip1) accumulation but, importantly, did not interfere with the induction of apoptosis. The combined data suggest that repression of both, c-myc and cyclin D3, is necessary to arrest human leukemia cells in the G1 phase of the cell division cycle, but that neither one is required for GC-induced apoptosis.     

The protective role of HSP90 against 3-hydroxykynurenine-induced neuronal apoptosis

3-hydroxykynurenine (3HK), an endogenous metabolite of tryptophan in the kynurenine pathway, is a potential neurotoxin in several neurodegenerative disorders. Stabilizing protein structure, heat shock proteins (HSPs) have diverse roles as molecular chaperones to mediate stress tolerance. In the present study, we investigated the possible protective role of HSPs against 3HK induced neuronal cell death. Here we report that 3HK induced in a dose- and time-dependent manner neuronal cell death in neuroblastoma SK-N-SH cells. The cell death showed characteristic apoptotic features such as cell shrinkage, plasma membrane blebbing, chromatin condensation, and nuclear condensation and fragmentation. Furthermore, SK-N-SN cells were protected from 3HK induced cytotoxicity by prior elevation of HSPs expression. Our results show that the protective effect was abolished by HSP90 anti-sense oligonucleotides while not by HSP27 and HSP70 anti-sense oligonucleotides. Also, our result shows that HSP90 effectively inhibits caspases activities leading to the apoptosis. These results suggest that 3HK induces apoptosis in neuroblastoma SK-N-SN cells and that HSP90 is major contributing protein component of protection against 3HK induced apoptosis.     

Differential activation of phospholipases by mitogenic EGF and neurogenic PDGF in immortalized hippocampal stem cell lines

In several neuronal systems, nerve growth factor (NGF) and platelet-derived growth factor (PDGF) act as neurogenic agents, whereas epidermal growth factor (EGF) acts as a mitogenic agent. Hippocampal stem cell lines (HiB5) immortalized by the expression of a temperature-sensitive SV40 large T antigen also respond differentially to EGF and PDGF. While EGF treatment at the permissive temperature induces proliferation, the addition of PDGF induces differentiation at the non-permissive temperature. However, the mechanism responsible for these different cellular fates has not been clearly elucidated. In order to clarify possible critical signaling events leading to these distinct cellular outcomes, we examined whether either EGF or PDGF differentially induces the activation of phospholipases, such as phospholipase A(2) (PLA(2)), C (PLC), or D (PLD). Although EGF stimulation did not induce phospholipases, PDGF caused a rapid and transient activation of PLC and PLD, but not PLA(2). When the activation of PLC or PLD was blocked, the neurite outgrowth induced by PDGF was significantly inhibited. Although the activation of PLD occurred faster than PLC, blocking of PLD activity by transient expression of lipase-inactive mutants did not inhibit the induction of PLC activity by PDGF. These results suggest that the differential activation of phospholipases may play an important role in signal transduction by mitogenic EGF and neurotrophic PDGF in HiB5 neuronal hippocampal stem cells. In particular, the activation of phospholipase C and D may contribute to neuronal differentiation by neurogenic PDGF in the HiB5 cells.     

The heat shock protein 70 inhibitor VER155008 suppresses the expression of HSP27, HOP and HSP90β and the androgen receptor,induces apoptosis,and attenuates prostate cancer cell growth

Heat shock proteins (HSPs) are molecular chaperones that play a pivotal role in correct folding, stabilization and intracellular transport of many client proteins including those involved in oncogenesis. HSP70, which is frequently overexpressed in prostate cancer (PCa), has been shown to critically contribute to tumor cell survival, and might therefore represent a potential therapeutic target. We treated both the androgen receptor (AR)-positive LNCaP and the AR-negative PC-3 cell lines with the pharmacologic HSP70 inhibitor VER155008. Although we observed antiproliferative effects and induction of apoptosis upon HSP70 inhibition, the apoptotic effect was more pronounced in AR-positive LNCaP cells. In addition, VER155008 treatment induced G1 cell cycle arrest in LNCaP cells and decreased AR expression. Further analysis of the HSP system by Western blot analysis revealed that expression of HSP27, HOP and HSP90β was significantly inhibited by VER155008 treatment, whereas the HSP40, HSP60, and HSP90α expression remained unchanged. Taken together, VER155008 might serve as a novel therapeutic option in PCa patients independent of the AR expression status.     

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.     

The Role of heat shock protein 27 in extravillous trophoblast differentiation

Trophoblast cells from placental explants differentiate in culture to extravillous trophoblast cells (EVT cells). During trophoblast differentiation heat-shock-protein-27 (HSP27) mRNA and multidrug-resistance-protein-5 (MRP5, transporter of cyclic nucleotides) expression are increased. HSP27 is a regulator of actin filaments structure and dynamic, has a role in cell differentiation and may affect NF-kB activity. In this study we aimed to assess HSP27 level in trophoblast cells and its correlation with motility and differentiation related processes [MMPs activity, nitric oxide (NO), inducible nitric oxide synthase (iNOS), proliferation and MRP5 levels]. We evaluated HSP27 expression in a first trimester human trophoblast explants model designed to assess EVT cells differentiation/migration with/without 6-mercaptopurine (6MP, an EVT inhibitor of migration). We found that HSP27 level is expressed in the nucleous and cytoplasm of non-proliferting villous-trophoblast cells (negative for Ki67) and in the cell periphery and cytoplasm of motile EVT cells. Moreover, 6MP decreased HSP27 nucleous expression that was associated with inhibited MMP2 activity and NO production. Also decreased iNOS expression and increased MRP5 mRNA levels were observed. In conclusion, HSP27 expression is modulated in concordance with migration dependent parameters in trophoblast cells.     

TRAIL/MEKK4/p38/HSP27/Akt survival network is biphasically modulated by the Src/CIN85/c-Cbl complex

Previously, we showed that mitogen-activated protein kinase/extracellular signal-related kinase 4 (MEKK4) is responsible for p38 activation and that its activation during tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) treatment also increases the catalytic activity of Akt. Here, we further investigated how the TRAIL-induced MEKK4/p38/heat shock protein (HSP27)/Akt survival network is modulated by the Src/c-Cbl interacting protein of 85 kDa (CIN85)/c-Cbl complex. TRAIL-induced activation of Akt catalytic activity and phosphorylation were highly correlated with p38/HSP27 phosphorylation, whereas the phosphorylation of p38/HSP27 increased further during incubation with curcumin and TRAIL, which caused significant apoptotic cell death. CIN85, a c-Cbl-binding protein, plays an essential role in connecting cell survival to cell death. The interaction of CIN85 with MEKK4 was increased during the late phase of TRAIL incubation, suggesting that sustained p38 and HSP27 phosphorylation protects cells by preventing further cell death. However, further increases in p38/HSP27 phosphorylation induced by cotreatment with curcumin and TRAIL converted cell fate to death. Taken together, these data demonstrate that phosphorylated p38/HSP27 as biphasic modulators act in conjunction with CIN85 to determine whether cells survive or die in response to apoptotic stress.     

CD44 co-stimulates apoptosis in thymic lymphomas and T cell hybridomas

Thymic lymphomas and hybridomas vary in their sensitivity to dexamethasone (DEX). Identical variance has been demonstrated in our laboratory for apoptosis of such cells by primary thymic epithelial cells or a cell line (TEC). We have also shown that apoptosis induced by TEC was partially mediated by TEC-derived glucocorticoids (GC). We studied the responses of various thymic lymphomas and hybridomas to TEC and DEX. Of these cells, PD1.6 and 2B4 were sensitive whereas B10 were relatively resistant to either inducer. In the present study we found that TEC and DEX synergize in inducing B10 cell apoptosis. B10 cells could also undergo apoptosis by TEC, conditional upon the presence of a TEC-sensitive cell (PD1.6 or 2B4). Contact between TEC and B10 was essential for apoptosis to occur. Thus, TEC may provide two signals, one mediated by GC and the other requiring cell to cell contact. We then analyzed the involvement of co-stimulatory or adhesion molecules in the TEC-induced apoptosis of thymic lymphoma cells. Soluble anti-CD44 antibodies but not anti-CD18, CD2 or CD28, inhibited TEC-induced apoptosis of PD1.6. Dimerization of CD44 by immobilized antibodies augmented DEX-induced apoptosis of all the lymphomas tested. CD44 cross-linkage up-regulated expression of the pro-apoptotic protein Bax, and down-regulated the anti-apoptotic protein, Bclx(L), in the presence of DEX. Taken together, the data suggest that CD44 enhances the apoptotic response of T lymphoma cells to DEX, and that CD44 modulates TEC-induced apoptosis of thymic lymphomas.     

Reduction of HSP70 and HSC70 Heat Shock mRNA Induction by Pentobarbital After Transient Global Ischemia in Gerbil Brain

Abstract: The effect of pentobarbital on the induction of heat shock protein (HSP) 70 and heat shock cognate protein (HSC) 70 mRNAs after transient global ischemia in gerbil brains was investigated by in situ hybridization using cloned cDNA probes selective for each mRNA species. In sham control brains, HSP70 mRNA was scarcely present, whereas HSC70 mRNA was present in most cell populations. After a 5-min occlusion of bilateral common carotid arteries, HSP70 and HSC70 mRNAs were induced together in several cells and were especially dense in hippocampal dentate granule cells at 3 h, but the strong hybridization of the mRNAs continued only in hippocampal CA1 cells by 2 days. At 7 days after the ischemia, CA1 neuronal cell death was apparent, and the HSP70 mRNA disappeared and HSC70 mRNA content returned to the sham level, except for in the CA1 cells. Pretreatment with pentobarbital (40 mg/kg, i.p.) greatly reduced or inhibited the induction of HSP70 and HSC70 mRNAs at both early (3-h) and late (2-day) phases after ischemia. The drug also prevented CA1 cell death at 7 days along with the maintenance of expression of HSC70 mRNA at the sham control level. Hypothermic effects of pentobarbital were noted at 30 and 60 min after the reperfusion, whereas at 2 h there was no statistical significance between the control and drug-treated groups. The great reduction of HSP70 and HSC70 mRNA induction at both early and late phases after ischemia suggests that pentobarbital reduces intra- and/or postischemic stress and may protect CA1 cells from ischemic damage. These effects of the drug may be mainly due to its specific action rather than its hypothermic effects.     

Spontaneous apoptosis and expression of cell surface heat-shock proteins in cultured EL-4 lymphoma cells

Abstract. The expression of heat-shock proteins (HSPs) is enhanced in stressed cells and can protect cells from stress-induced injury. However, existing data about the relationship between apoptosis and HSP expression is contradictory. In this paper, a mouse lymphoma cell death model system is used to detect simultaneously both the process of apoptosis and the level of HSP expression. The model was established after discovering that spontaneous apoptosis and spontaneous cell surface HSP expression occurs in EL-4 mouse lymphoma cells during normal optimal culture conditions. The data show that apoptotic EL-4 cells had higher levels of hsp25, hsp60, hsp70 and hsp90 exposed on the plasma membrane surface than viable cells. The level of surface HSPs was found to increase through several stages of early and late apoptotic death as measured by flow cytometry, with the highest levels observed during the loss of cell membrane phospholipid asymmetry. Heat shock and actinomycin D significantly increased the proportion of apoptotic cells in culture. However, hyperthermia only stimulated a weak and temporary increase in surface HSP expression, whereas actinomycin D strongly elevated the level of surface and intracellular HSPs, particularly in live cells. These results show an associative relationship between apoptosis and HSP expression. The relationship between the progression of cell death and HSP expression suggests a role for membrane HSP expression in programmed cell death.     

Cytochemical identification of HSP110 during early mouse facial development.

Apoptotic cell death constitutes a common phenomenon observed during development. This process plays an important role in the regulation of cell populations and in early differentiation of embryonic organs. Several teratologic situations are considered as resulting in a dramatic increase of the apoptotic process. In mammalian cells, heat shock proteins (HSPs), expressed or increased in response to various stresses, act as molecular chaperones in physiological conditions. In order to determine specific histochemical markers of apoptotic cells in normal craniofacial development, we observed the expression of stress proteins (HSPs) 70, 86, and 110. The apoptotic pattern of mesectodermal cell death areas was confirmed using both nuclear staining (Feulgen) and specific labeling of DNA fragmentation (TUNEL). These areas are localized in the proximal parts of the first and second visceral arches. They are located in mesectodermal and ganglionic cells. Apoptotic mesectodermal populations strongly express HSP110, as shown by the cytochemical identification of HSP110 and by double staining HSP110-TUNEL, suggesting that this protein could be considered as a new marker for apoptotic embryonic cells, and could be used in further teratologic studies to better quantify induced cell death.     

Oral administration of Antioxidant Biofactor (AOBtrade mark) ameliorates ischemia/reperfusion- induced neuronal death in the gerbil

Oxidative damage due to ischemia/reperfusion has been implicated as one of the leading causes for delayed neuronal cell death in a number of neurodegenerative diseases, including stroke. The purpose of this research was to investigate whether oral administration of a fermented grain food mixture (AOB(R)) might offer protective effects against ischemia/reperfusion-induced neuronal damage in Mongolian gerbils, a model known for delayed neuronal death in the hippocampal CA1 region. Histological analysis revealed that AOB administration ad libitum for 3 weeks (preoperative administration) and 1 week (postoperative administration) dose-dependently suppressed the induction of transient ischemia/reperfusion-induced neuronal cell death. TUNEL assay also revealed that AOB suppressed it by inhibiting the induction of apoptosis. A significant increase of superoxide dismutase-like (SOD-like) activity was observed in the hippocampal CA1 region of the AOB-treated gerbil. Furthermore, immunoblot analysis showed that AOB administration down-regulated the expression of heat shock proteins HSP27 and HSP70 in the same region. These results indicated that oral administration of AOB protected against ischemia/reperfusion-induced brain injury by minimizing oxidative damage via its SOD-like activity and inhibiting apoptosis.     

Expression of programmed cell death 5 gene involves in regulation of apoptosis in gastric tumor cells

The protein of programmed cell death 5 (PDCD5) is believed to participate in regulation of apoptosis. Although PDCD5 is reducibly expressed in various human tumors, it is not clear which expression level of PDCD5 is in gastric cancer (GC). In this study, we have systematically employed the approaches of RT-PCR, Real- time PCR, Immunohistochemistry (IHC), Immunofluorescence staining (IFS) and Western blot to determine the PDCD5 expression in GC cells and primary tumors, at mRNA and protein level, respectively. Our data revealed that the positive rate of PDCD5 expression in the gastric tumor tissues was significantly less than that of the normal tissues (14 out of 102 vs 36 out of 51), whereas, the decreased expression of PDCD5 protein was well correlated with the up-regulated expression of Bcl-2 in these tissues, and the up-regulated expression and nuclear translocation of PDCD5 protein were verified in the apoptotic GC cells induced by Diallyl trisulfide (DATS). Furthermore, the survival curve has suggested that the more PDCD5 expressions were found in the patients, the longer the survival periods were. Therefore, our observations lay down a reasonable postulation that PDCD5 may play a key role to regulate the apoptotic processes in the GC cells and gastric tumors.