150-kDa oxygen-regulated protein (ORP150) functions as a novel molecular chaperone in MDCK cells

To assess the participationof the 150-kDa oxygen-regulated protein (ORP150) in protein transport,its function in Madin-Darby canine kidney (MDCK) cells was studied.Exposure of MDCK cells to hypoxia resulted in an increase of ORP150antigen and increased binding of ORP150 to GP80/clusterin (80-kDaglycoprotein), a natural secretory protein in this cell line. In ORP150antisense transformant MDCK cells, GP80 was retained within theendoplasmic reticulum after exposure to hypoxia. Metabolic labelingshowed the delay of GP80 maturation in antisense transformants inhypoxia, whereas its matured form was detected in wild-type cells,indicating a role of ORP150 in protein transport, especially inhypoxia. The affinity chromatographic analysis of ORP150 suggested itsability to bind to ATP-agarose. Furthermore, the ATP hydrolysisanalysis showed that ORP150 can release GP80 at a lower ATPconcentration. These data indicate that ORP150 may function as a uniquemolecular chaperone in renal epithelial cells by facilitating proteintransport/maturation in an environment where less ATP is accessible.

     

Expression of 150-kDa oxygen-regulated protein (ORP150) stimulates bleomycin-induced pulmonary fibrosis and dysfunction in mice

Idiopathic pulmonary fibrosis (IPF) involves pulmonary injury associated with inflammatory responses, fibrosis and dysfunction. Myofibroblasts and transforming growth factor (TGF)-β1 play major roles in the pathogenesis of this disease. Endoplasmic reticulum (ER) stress response is induced in the lungs of IPF patients. One of ER chaperones, the 150-kDa oxygen-regulated protein (ORP150), is essential for the maintenance of cellular viability under stress conditions. In this study, we used heterozygous ORP150-deficient mice (ORP150(+/-) mice) to examine the role of ORP150 in bleomycin-induced pulmonary fibrosis. Treatment of mice with bleomycin induced the expression of ORP150 in the lung. Bleomycin-induced inflammatory responses were slightly exacerbated in ORP150(+/-) mice compared to wild-type mice. On the other hand, bleomycin-induced pulmonary fibrosis, alteration of lung mechanics and respiratory dysfunction was clearly ameliorated in the ORP150(+/-) mice. Bleomycin-induced increases in pulmonary levels of both active TGF-β1 and myofibroblasts were suppressed in ORP150(+/-) mice. These results suggest that although ORP150 is protective against bleomycin-induced lung injury, this protein could stimulate bleomycin-induced pulmonary fibrosis by increasing pulmonary levels of TGF-β1 and myofibroblasts.     

The effect of hypoxia on the expression of 150 kDa oxygen-regulated protein (ORP 150) in HeLa cells.

Correct protein folding is an important factor, for the translocation of newly synthesised proteins to specific subcellular compartments, extracellular matrix or to biological fluids. This process is regulated by a group of specific proteins, referred to as chaperones. Many stress conditions, such as oxygen or glucose deprivation, slow down the folding process and cause accumulation of unfolded/misfolded proteins in the cell. Molecular chaperones are induced in these conditions; with some named as oxygen-regulated proteins (ORPs). These bind to unfolded / misfolded proteins to facilitate correct assembly. ORP 150 is the subject of this study. Hypoxia results in an enhancement of ORP 150 expression in several tumour cell lines cultured in vitro. HeLa cells grown in hypoxic conditions (despite an intensive expression of ORP 150) demonstrate higher rates of apoptosis in comparison to those cultured in normoxic conditions. Furthermore, the inhibition of ORP 150 synthesis by transfection of these cells with a specific siRNA resulted in an intensification of apoptosis, as indicated by specific markers of this process; the enhancement of poly ADP-ribose protein cleavage and the increase in Bim protein expression. We conclude from our study that the increase in ORP 150 synthesis protects the cells against the proapoptotic effect of hypoxia.     

Abundant expression of 150-kDa oxygen-regulated protein in mouse pancreatic beta cells is correlated with insulin secretion

The 150-kDa oxygen-regulated protein (ORP150) is a member of glucose-regulated proteins (GRPs), which are induced by stressful conditions such as oxygen or glucose deprivation. Here we investigated the highly abundant expression of ORP150 in mouse pancreas and its relationship with insulin secretion. Immunohistochemical analysis revealed that ORP150 expression was restricted to islets, especially to beta cells. The beta cell-specific expression was also observed in a mouse insulinoma cell line, MIN6, which secretes insulin in response to increased glucose concentration. Furthermore, ORP150 in islets dramatically diminished by fasting, concomitant with reduction of the serum insulin level. These results strongly suggest the role for ORP150 in insulin secretion.     

ORP150 protects against hypoxia/ischemia-induced neuronal death

Oxygen-regulated protein 150 kD (ORP150) is a novel endoplasmic-reticulum-associated chaperone induced by hypoxia/ischemia. Although ORP150 was sparingly upregulated in neurons from human brain undergoing ischemic stress, there was robust induction in astrocytes. Cultured neurons overexpressing ORP150 were resistant to hypoxemic stress, whereas astrocytes with inhibited ORP150 expression were more vulnerable. Mice with targeted neuronal overexpression of ORP150 had smaller strokes compared with controls. Neurons with increased ORP150 demonstrated suppressed caspase-3-like activity and enhanced brain-derived neurotrophic factor (BDNF) under hypoxia signaling. These data indicate that ORP150 is an integral participant in ischemic cytoprotective pathways.     

p53 directly suppresses BNIP3 expression to protect against hypoxia-induced cell death

Hypoxia stabilizes the tumour suppressor p53, allowing it to function primarily as a transrepressor; however, the function of p53 during hypoxia remains unclear. In this study, we showed that p53 suppressed BNIP3 expression by directly binding to the p53-response element motif and recruiting corepressor mSin3a to the BNIP3 promoter. The DNA-binding site of p53 must remain intact for the protein to suppress the BNIP3 promoter. In addition, taking advantage of zebrafish as an in vivo model, we confirmed that zebrafish nip3a, a homologous gene of mammalian BNIP3, was indeed induced by hypoxia and p53 mutation/knockdown enhanced nip3a expression under hypoxia resulted in cell death enhancement in p53 mutant embryos. Furthermore, p53 protected against hypoxia-induced cell death mediated by p53 suppression of BNIP3 as illustrated by p53 knockdown/loss assays in both human cell lines and zebrafish model, which is in contrast to the traditional pro-apoptotic role of p53. Our results suggest a novel function of p53 in hypoxia-induced cell death, leading to the development of new treatments for ischaemic heart disease and cerebral stroke.     

A protective role of 27-kDa heat shock protein in glucocorticoid-evoked apoptotic cell death of hippocampal progenitor cells

Hippocampus is one of the most vulnerable tissues to glucocorticoid (GC). In the present study, we demonstrate that dexamethasone (DEX), a synthetic GC, induces apoptotic cell death in hippocampal progenitor HiB5 cells without any additional insult. Interestingly, expression of 27-kDa heat shock protein (HSP27) was markedly induced by DEX in time- and dose-dependent manners. This induction was dependent on the production of reactive oxygen species (ROS), suggesting that DEX-evoked oxidative damage to HiB5 cells is responsible for the HSP27 induction. To evaluate a possible role of HSP27, we generated two mutant HiB5 cell lines, in which expression of HSP27 was inhibited or enhanced by the over-expression of HSP27 cDNA with antisense or sense orientation (AS-HSP27 and S-HSP27, respectively). DEX-induced apoptotic cell population was significantly increased in AS-HSP27 HiB5 cells and evidently decreased in S-HSP27 cells. These results indicate that HSP27 protects hippocampal progenitor cells from GC-induced apoptotic cell death.     

Protection of apoptotic cell death by protein A

The word Apoptosis or pragrammed cell death is described as the ultimate end of multiple cellular events converging from numerous initiating events to the ultimate death of a cell or organism. Several processes, such as initiation of death signals at the plasma membrane, expression of pro-apoptotic oncoproteins, activation of death proteases, endonucleases etc., that ultimately coalesce to a common irreversible execution phase, lead to cell demise. Counteracting the death signals are cell survival factors. A balance between the cell death and cell survival factors plays a major role in the decision making process as to whether a cell should die or must live. It is, therefore, hypothesized that if the balance can be shifted in favor of cell survival, one might be able to arrest the aging process, save the injured cells or else if the balance is shifted toward cell-kill it might help destroy tumors and other undesirable cells.Protein A (PA) of Staphylococcus aureus has been found to have multifarious biological response modifying properties. It has been shown to possess anti-tumor, anti-toxic, anti-parasitic and antifungal activities. It also acts as a potent immunostimulator. PA can protect bone marrow progenitor cells from zidovudin(AZT)-induced apoptosis and can stimulate immunocyte proliferation, thereby helping to replenish/restore the depleted hematopoietic cell pool. Such ability to replenish hematopoietic cells is a common property of PA observed against a number of toxic drugs/chemicals, such as cyclophosphamide, benzene, aflatoxin, salmonella endotoxin, etc.Interestingly, it was further demonstrated in our laboratory that PA can selectively kill tumor cells without affecting normal cells of the host. A search for the mechanisms of PA action revealed that this bacterial protein could shift the balance between pro- and anti-apoptotic proteins in favor of survival in normal cells, but in favor of cell death in tumor cells at a particular dose level. This unique property of PA suggests that controlled use of such type of Biological Response Modifier might help in controlling both cell growth and death phenomena.     

Neurotoxic 43-kDa TAR DNA-binding protein (TDP-43) triggers mitochondrion-dependent programmed cell death in yeast

Pathological neuronal inclusions of the 43-kDa TAR DNA-binding protein (TDP-43) are implicated in dementia and motor neuron disorders; however, the molecular mechanisms of the underlying cell loss remain poorly understood. Here we used a yeast model to elucidate cell death mechanisms upon expression of human TDP-43. TDP-43-expressing cells displayed markedly increased markers of oxidative stress, apoptosis, and necrosis. Cytotoxicity was dose- and age-dependent and was potentiated upon expression of disease-associated variants. TDP-43 was localized in perimitochondrial aggregate-like foci, which correlated with cytotoxicity. Although the deleterious effects of TDP-43 were significantly decreased in cells lacking functional mitochondria, cell death depended neither on the mitochondrial cell death proteins apoptosis-inducing factor, endonuclease G, and cytochrome c nor on the activity of cell death proteases like the yeast caspase 1. In contrast, impairment of the respiratory chain attenuated the lethality upon TDP-43 expression with a stringent correlation between cytotoxicity and the degree of respiratory capacity or mitochondrial DNA stability. Consistently, an increase in the respiratory capacity of yeast resulted in enhanced TDP-43-triggered cytotoxicity, oxidative stress, and cell death markers. These data demonstrate that mitochondria and oxidative stress are important to TDP-43-triggered cell death in yeast and may suggest a similar role in human TDP-43 pathologies.     

Mammalian prion protein suppresses Bax-induced cell death in yeast

Several lines of evidence suggest that PrP(C), the non-infectious form of the prion protein, may function to protect neurons and other cells from stress or toxicity. In this paper, we report on the use of the yeast Saccharomyces cerevisiae as a model system to assay the cytoprotective activity of PrP(C). The mammalian pro-apoptotic protein, Bax, confers a lethal phenotype when expressed in yeast. Since overexpression of PrP(C) has been found to prevent Bax-mediated cell death in cultured human neurons, we explored whether PrP could also suppress Bax-induced cell death in yeast. We utilized a form of mouse PrP containing a modified signal peptide that we had previously shown is efficiently targeted to the secretory pathway in yeast. We found that this PrP potently suppressed the death of yeast cells expressing mammalian Bax under control of a galactose-inducible promoter. In contrast, cytosolic PrP-(23-231) failed to rescue growth of Bax-expressing yeast, indicating that protective activity requires targeting of PrP to the secretory pathway. Deletion of the octapeptide repeat region did not affect the rescuing activity of PrP, but deletion of a charged region encompassing residues 23-31 partially eliminated activity. We also tested several PrP mutants associated with human familial prion diseases and found that only a mutant containing nine extra octapeptide repeats failed to suppress Bax-induced cell death. These findings establish a simple and genetically tractable system for assaying a putative biological activity of PrP(C).     

Glucose effect on the expression of 150 kDa oxygen-regulated protein in HeLa cells

Chaperones assist in the correct folding of newly synthesised proteins in the endoplasmic reticulum (ER) of cells, this being essential for the translocation of protein molecules to specific subcellular compartments, extracellular matrix or to biological fluids. The biosynthesis of some ER chaperones is regulated by glucose. They are named "glucose-regulated proteins" (GRPs). The function of some GRPs depends on oxygen, a subgroup named "oxygen-regulated proteins" (ORPs). The biosynthesis of ORPs is induced by deprivation of glucose or oxygen. Exposure of HeLa cells to glucose starvation induces the biosynthesis of various GRPs including ORP 150. The expression of ORP 150 is regulated by the concentration of glucose in the culture medium, being induced by a shortage and repressed by a presence of glucose. We have shown that both glucose starvation and transfection of cells with siRNA (specific to ORP 150 mRNA) evoke similar, but quantitatively different, effects. The cells grown for 72 h in a 4.5 mg/ml glucose-containing medium demonstrated low apoptosis (3.7%) whereas in a 0.5 mg/ml glucose-containing medium the apoptosis was increased to 10%. The effect of transfection on apoptosis was distinctly higher with almost 22% of apoptotic cells detected in 72 h cultures. One may conclude that ORP 150 reduces the pro-apoptotic effects of glucose starvation. Such a hypothesis is supported by the observation that the transfection procedure makes HeLa cells resistant to the regulatory effect of glucose on ORP 150 production. The transfected cells do not respond to glucose starvation with an overexpression of ORP 150. It is apparent from our experiments that ORP 150 plays an important role in adaptation of cells to the shortage of glucose and reduces the pro-apoptotic effect of glucose starvation.     

Caspase-2 promotes cytoskeleton protein degradation during apoptotic cell death

The caspase family of proteases cleaves large number of proteins resulting in major morphological and biochemical changes during apoptosis. Yet, only a few of these proteins have been reported to selectively cleaved by caspase-2. Numerous observations link caspase-2 to the disruption of the cytoskeleton, although it remains elusive whether any of the cytoskeleton proteins serve as bona fide substrates for caspase-2. Here, we undertook an unbiased proteomic approach to address this question. By differential proteome analysis using two-dimensional gel electrophoresis, we identified four cytoskeleton proteins that were degraded upon treatment with active recombinant caspase-2 in vitro. These proteins were degraded in a caspase-2-dependent manner during apoptosis induced by DNA damage, cytoskeleton disruption or endoplasmic reticulum stress. Hence, degradation of these cytoskeleton proteins was blunted by siRNA targeting of caspase-2 and when caspase-2 activity was pharmacologically inhibited. However, none of these proteins was cleaved directly by caspase-2. Instead, we provide evidence that in cells exposed to apoptotic stimuli, caspase-2 probed these proteins for proteasomal degradation. Taken together, our results depict a new role for caspase-2 in the regulation of the level of cytoskeleton proteins during apoptosis.     

Differential signaling to apoptotic and necrotic cell death by Fas-associated death domain protein FADD

Two general pathways for cell death have been defined, apoptosis and necrosis. Previous studies in Jurkat cells have demonstrated that the Fas-associated death domain (FADD) is required for Fas-mediated signaling to apoptosis and necrosis. Here we developed L929rTA cell lines that allow Tet-on inducible expression and FK506-binding protein (FKBP)-mediated dimerization of FADD, FADD-death effector domain (FADD-DED), or FADD-death domain (FADD-DD). We show that expression and dimerization of FADD leads to necrosis. However, pretreatment of the cells with the Hsp90 inhibitor geldanamycin, which leads to proteasome-mediated degradation of receptor interacting protein 1 (RIP1), reverts FKBP-FADD-induced necrosis to apoptosis. Expression and dimerization of FADD-DD mediates necrotic cell death. We found that FADD-DD is able to bind RIP1, another protein necessary for Fas-mediated necrosis. Expression and dimerization of FADD-DED initiates apoptosis. Remarkably, in the presence of caspase inhibitors, FADD-DED mediates necrotic cell death. Coimmunoprecipitation studies revealed that FADD-DED in the absence procaspase-8 C/A is also capable of recruiting RIP1. However, when procaspase-8 C/A and RIP1 are expressed simultaneously, FADD-DED preferentially recruits procaspase-8 C/A.     

Preventing apoptotic cell death by a novel small heat shock protein

NCBI database analysis indicated that the human C1orf41 protein (small heat shock-like protein-Hsp16.2) has sequence similarity with small heat shock proteins (sHsps). Since sHsps have chaperone function, and so prevent aggregation of denatured proteins, we determined whether Hsp16.2 could prevent the heat-induced aggregation of denatured proteins. Under our experimental conditions, recombinant Hsp16.2 prevented aggregation of aldolase and glyceraldehyde-3-phosphate dehydrogenase, and protected Escherichia coli cells from heat stress indicating its chaperone function. Hsp16.2 also formed oligomeric complexes in aqueous solution. Hsp16.2 was found to be expressed at different levels in cell lines and tissues, and was mainly localized to the nucleus and the cytosol, but to a smaller extent, it could be also found in mitochondria. Hsp16.2 could be modified covalently by poly(ADP ribosylation) and acetylation. Hsp16.2 over-expression prevented etoposide-induced cell death as well as the release of mitochondrial cytochrome c and caspase activation. These data suggest that Hsp16.2 can prevent the destabilization of mitochondrial membrane systems and could represent a suitable target for modulating cell death pathways.     

Fucoxanthin induced apoptotic cell death in oral squamous carcinoma (KB) cells

Fucoxanthin (Fx) is an active compound commonly found in the many types of seaweed with numerous biological activities. The main goal of this investigation is to explore the effect of Fx against the cell proliferation, apoptotic induction and oxidative stress in the oral squamous (KB) cell line. Cytotoxicity of Fx was determined by MTT assay. The intracellular ROS production, mitochondrial membrane potential (MMP) and apoptosis induction in KB cells were examined through DCFH-DA, Rhodamine-123 and DAPI, and dual staining techniques. Effect of Fx on the antioxidant enzymes and lipid peroxidation in the KB cells was studied through the standard procedures. Fx treated KB cells showed morphological changes and reduced cell survival, which is exhibited by the cytotoxic activity of 50 µM/ml (IC50) Fx against the KB cells. The Fx treatment considerably induced the apoptotosis cells (EB/AO) and decreased the MMP (Rh-123) in KB cells. Further, it was pointed out that there was an increased lipid peroxidation (LPO) with decreased antioxidants (CAT, SOD and GSH). These results concluded that Fx has the cytotoxic effect against KB cells and has the potential to induce the apoptosis via increased oxidative stress. Hence, the Fx can be a promising agent for the treatment of oral cancer and it may lead to the development of cancer therapeutics.