ABSTRACT
Background: Neuron specific enolase (NSE) is a specific biomarker for SCLC. However, the biological roles and aberrant expression of NSE in SCLC have not been well illustrated. Methods: The expression of NSE, miR-93-5p and LINC00657 in SCLC tissues and cell lines were detected using real time quantitative PCR (qRT-PCR) or immunohistochemistry. CCK8 assay was performed to detect cell proliferation. Cell migration and invasion capabilities were investigated by transwell assay. Epithelial-mesenchymal transition (EMT) process was verified by detecting epithelial marker E-cadherin and mesenchymal marker N-cadherin. The direct interactions between miR-93-5p and NSE or LINC00657 were predicted by bioinformatics tools and verified using dual luciferase reporter assay. Results: Upregulated expression of NSE in SCLC tumor tissues were positively associated with advanced tumor stage, distant metastasis and poor overall survival. Overexpression of NSE promoted cell proliferation, migration, invasion and EMT in SCLC cells, while silence of NSE inhibited these effects. Mechanically, NSE expression was positively correlated with LINC00657, and negatively correlated with miR-93-5p. Moreover, NSE was positively regulated by LINC00657 through sponging of miR-93-5p. LINC00657 and miR-93-5p promoted SCLC cell migration, invasion and EMT by NSE-mediated manner. Conclusion: Overall, our study revealed a novel role of NSE in SCLC. NSE was positively regulated by LINC00657 through competitively interacting with miR-93-5p, which may be potential targets for SCLC patients.
Subject(s)
Lung Neoplasms/pathology , MicroRNAs/genetics , Phosphopyruvate Hydratase/physiology , RNA, Long Noncoding/genetics , Small Cell Lung Carcinoma/pathology , Adult , Aged , Aged, 80 and over , Cell Movement/genetics , Cell Proliferation/genetics , Cells, Cultured , China/epidemiology , Epithelial-Mesenchymal Transition/genetics , Female , Gene Expression Regulation, Neoplastic , Humans , Lung Neoplasms/genetics , Lung Neoplasms/mortality , Male , Middle Aged , Neoplasm Invasiveness/genetics , Signal Transduction/genetics , Small Cell Lung Carcinoma/genetics , Small Cell Lung Carcinoma/mortality , Survival AnalysisABSTRACT
Enolase is a crucial enzyme involved in the glycolytic pathway and gluconeogenesis in parasites. It also has been reported to function as a plasminogen receptor and may be involved in tissue invasion. In this study, the biochemical properties of the enolase of Spirometra mansoni (Smenolase) were investigated. The Smenolase gene was found to cluster closely with the enolase genes of Clonorchis sinensis and Echinococcus granulosus, and some functional motifs were identified as conserved. Smenolase was confirmed to be a component of the secretory/excretory products (ESPs) and a circulating antigen of spargana. Recombinant Smenolase expressed in vitro was able to bind to human plasminogen. Smenolase was detected in the eggs, testicles, and vitellaria of adult worms and the tegument of spargana. The transcription level of Smenolase was highest at the gravid proglottid stage. When spargana were cultured with glucose of different concentration in vitro, it was observed that the expression levels of Smenolase in the low-glucose groups were consistent with that of Smenolase in vivo. These results indicate that Smenolase is a critical enzyme involved in supplying energy to support the development and reproduction of the parasite, and it may also play a role in sparganum invasion.
Subject(s)
Helminth Proteins/physiology , Phosphopyruvate Hydratase/physiology , Spirometra/enzymology , Animals , Antigens, Helminth/genetics , Antigens, Helminth/metabolism , Energy Metabolism , Gene Expression Regulation, Developmental , Helminth Proteins/genetics , Helminth Proteins/metabolism , Humans , Phosphopyruvate Hydratase/genetics , Phosphopyruvate Hydratase/metabolism , Plasminogen/metabolism , Sparganum/enzymology , Sparganum/genetics , Spirometra/geneticsABSTRACT
Suppression of ubiquitin proteasome pathway (UPP) and stimulation of caspase-3 are involved in neurodegeneration. Can UPP activators and caspase-3 inhibitors ameliorate neurodegeneration? Here, we found a novel neuronal cell death accompanied with UPP activation and caspase-3 inhibition. Recently, plasmalemmal neuron-specific enolase (NSE) has been identified as one of membrane targets of 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2). 15d-PGJ2 induces neuronal apoptosis via activating caspase-3 and inactivating UPP, whereas the anti-NSE antibody inactivated caspase-3, activated UPP, and caused neuronal cell death. The anti-NSE antibody activated caspase-1 (pyroptosis marker), but not condense chromatin (apoptosis marker). The anti-NSE antibody declined intracellular level of ATP, which is not altered in pyroptosis. The intracellular level of calcium is elevated in necrosis and pyroptosis, but its chelator did not ameliorate the neurotoxicity of anti-NSE. Thiol antioxidants such as N-acetyl cysteine and glutathione reduced the neurotoxicity of 15d-PGJ2 but enhanced that of the anti-NSE antibody. The anti-NSE antibody incorporated propidium iodide into neurons through the disrupted plasma membrane, which are not observed in ferroptosis and autophagic cell death. Thus, the anti-NSE antibody induced neuronal cell death in a novel fashion distinguished from necrosis, necroptosis, apoptosis, pyroptosis, ferroptosis, and autophagic cell death.
Subject(s)
Caspase 3/drug effects , Cell Death/drug effects , Immunoglobulin G/pharmacology , Neurons/drug effects , Phosphopyruvate Hydratase/immunology , Proteasome Endopeptidase Complex/metabolism , Ubiquitin/metabolism , Acetylcysteine/pharmacology , Adenosine Triphosphate/metabolism , Animals , Antioxidants/pharmacology , Calcium Signaling , Caspase 1/metabolism , Cerebral Cortex/cytology , Chromatin/ultrastructure , Enzyme Activation/drug effects , Female , Glutathione/pharmacology , Goats/immunology , HSP70 Heat-Shock Proteins/metabolism , Immunoglobulin G/immunology , MAP Kinase Signaling System/drug effects , Neurites/drug effects , Neurons/cytology , Phosphopyruvate Hydratase/physiology , Pregnancy , Prostaglandin D2/analogs & derivatives , Prostaglandin D2/physiology , Protein Processing, Post-Translational/drug effects , Rabbits/immunology , Rats , Rats, Wistar , Species Specificity , Ubiquitination/drug effectsABSTRACT
BACKGROUND An extensive body of research reveals the clinical value of serum tumor markers in lung cancer patients, including carcinoembryonic antigen (CEA), squamous cell carcinoma antigen (SCCA), cytokeratin-19 fragments (Cyfra21-1), and neuron-specific enolase (NSE), but little is known about the clinical properties of these serum tumor markers in anaplastic lymphoma kinase (ALK)-positive lung cancer patients. MATERIAL AND METHODS We retrospectively analyzed 54 patients harboring ALK rearrangements and 520 patients without ALK rearrangements, and all these patients were treated exclusively by surgery between 2011 and 2016. RESULTS NSE level (P=0.007 for OS) was identified as an independent prognostic factor among patients with resected ALK-positive adenocarcinoma of the lung. CONCLUSIONS A high level of NSE is associated with worse outcome among resected lung adenocarcinoma patients harboring ALK rearrangements.
Subject(s)
Adenocarcinoma of Lung/metabolism , Anaplastic Lymphoma Kinase/genetics , Phosphopyruvate Hydratase/metabolism , Adenocarcinoma/pathology , Adenocarcinoma of Lung/genetics , Adenocarcinoma of Lung/physiopathology , Adult , Aged , Aged, 80 and over , Anaplastic Lymphoma Kinase/metabolism , Antigens, Neoplasm , Biomarkers, Tumor/blood , Carcinoma, Non-Small-Cell Lung/pathology , Carcinoma, Squamous Cell/pathology , China , Female , Humans , Keratin-19 , Lung Neoplasms/pathology , Male , Middle Aged , Phosphopyruvate Hydratase/physiology , Prognosis , Retrospective StudiesABSTRACT
Increased aerobic glycolysis is considered as a hallmark of cancer and targeting key glycolytic enzymes will be a promising therapeutic approach in cancer treatment. Alpha-enolase (ENO1), as a prominent glycolytic enzyme, is upregulated in multiple cancers and its overexpression is involved in tumor cell proliferation and metastasis. In the present study, we aimed to investigate the potential role of ENO1 in the development and progression of gastric cancer (GC). Here, we found that ENO1 expression was upregulated in human GC and was associated with Lauren type, lymph node metastasis (LNM) and TNM stage. Knockdown of ENO1 attenuated GC cell proliferation and metastasis and reversed epithelial-mesenchymal transition (EMT) progress in vitro while ENO1 overexpression did the opposite. ENO1 could modulate AKT signaling pathway in GC cells and the enhanced proliferation and migration ability induced by ENO1 overexpression was impaired after incubation with PI3K inhibitor Ly294002 in SGC7901 cells. Our data demonstrated that ENO1 enhances GC cell proliferation and metastasis through the protein kinase B (AKT) signaling pathway, indicating that ENO1/AKT signaling axis may serve as a potential target for treatment of GC.
Subject(s)
Biomarkers, Tumor/physiology , DNA-Binding Proteins/physiology , Phosphopyruvate Hydratase/physiology , Proto-Oncogene Proteins c-akt/metabolism , Stomach Neoplasms/pathology , Tumor Suppressor Proteins/physiology , Biomarkers, Tumor/genetics , Cell Line, Tumor , Cell Proliferation , DNA-Binding Proteins/genetics , Gene Expression Regulation, Neoplastic , Glycolysis , Humans , Neoplasm Invasiveness , Neoplasm Metastasis , Phosphopyruvate Hydratase/genetics , Signal Transduction , Stomach Neoplasms/genetics , Tumor Suppressor Proteins/geneticsABSTRACT
In the present study, we describe the molecular and biochemical characterization of sunflower (Helianthus annuus L.) enolase (ENO, EC 4.2.1.11) proteins, which catalyze the formation of phosphoenolpyruvate, the penultimate intermediate in the glycolytic pathway. We cloned and characterized three cDNAs encoding different ENO isoforms from developing sunflower seeds. Studies using fluorescently tagged ENOs confirmed the predicted subcellular localization of ENO isoforms: HaENO1 in the plastid while HaENO2 and HaENO3 were found in the cytosol. The cDNAs were used to express the corresponding 6(His)-tagged proteins in Escherichia coli. The proteins were purified to electrophoretic homogeneity, using immobilized metal ion affinity chromatography, and biochemically characterized. Recombinant HaENO1 and HaENO2, but not HaENO3 were shown to have enolase activity, in agreement with data obtained with the Arabidopsis homolog proteins. Site directed mutagenesis of several critical amino acids was used to attempt to recover enolase activity in recombinant HaENO3, resulting in very small increases that were not additive. A kinetic characterization of the two active isoforms showed that pH had similar effect on their velocity, that they had similar affinity for 2-phosphoglycerate, but that the kcat/Km of the plastidial enzyme was higher than that of the cytosolic isoform. Even though HaENO2 was always the most highly expressed transcript, the levels of expression of the three ENO genes were remarkably distinct in all the vegetative and reproductive tissues studied. This indicates that in seeds the conversion of 2-phosphoglycerate to phosphoenolpyruvate takes place through the cytosolic and the plastidial pathways therefore both routes could contribute to the supply of carbon for lipid synthesis. The identity of the main source of carbon during the period of stored products synthesis is discussed.
Subject(s)
Helianthus/enzymology , Phosphopyruvate Hydratase/metabolism , Seeds/growth & development , Cytosol/enzymology , Glucose-6-Phosphate/metabolism , Helianthus/genetics , Helianthus/growth & development , Helianthus/metabolism , Lipid Metabolism , Phosphoenolpyruvate/metabolism , Phosphopyruvate Hydratase/genetics , Phosphopyruvate Hydratase/physiology , Phylogeny , Plastids/enzymology , Protein Conformation , Real-Time Polymerase Chain Reaction , Seeds/enzymology , Seeds/metabolism , Sequence Alignment , Sequence Analysis, DNA , TranscriptomeABSTRACT
In yeast, the import of tRNALys with CUU anticodon (tRK1) relies on a complex mechanism where interaction with enolase 2 (Eno2p) dictates a deep conformational change of the tRNA. This event is believed to mask the tRNA from the cytosolic translational machinery to re-direct it towards the mitochondria. Once near the mitochondrial outer membrane, the precursor of the mitochondrial lysyl-tRNA synthetase (preMsk1p) takes over enolase to carry the tRNA within the mitochondrial matrix, where it is supposed to participate in translation following correct refolding. Biochemical data presented in this report focus on the role of enolase. They show that despite the inability of Eno2p alone to form a complex with tRK1, mitochondrial import can be recapitulated in vitro using fractions of yeast extracts sharing either recombinant or endogenous yeast Eno2p as one of the main components. Taken together, our data suggest the existence of a protein complex containing Eno2p that is involved in RNA mitochondrial import.
Subject(s)
Lysine-tRNA Ligase/physiology , Mitochondria/metabolism , Phosphopyruvate Hydratase/physiology , RNA, Transfer, Lys/metabolism , Saccharomyces cerevisiae/enzymology , Saccharomyces cerevisiae/ultrastructure , Biological Transport , Cation Transport Proteins/metabolism , Mitochondria/enzymology , Multiprotein Complexes/chemistry , Multiprotein Complexes/physiology , Phosphopyruvate Hydratase/metabolism , RNA, Transfer/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae Proteins/physiologyABSTRACT
Adhesion is the first step for Candida species to form biofilms on medical devices implanted in the human host. Both the physicochemical nature of the biomaterial and cell wall proteins (CWP) of the pathogen play a determinant role in the process. While it is true that some CWP have been identified in vitro, little is known about the CWP of pathogenic species of Candida involved in adhesion. On this background, we considered it important to investigate the potential role of CWP of C. albicans, C. glabrata, C. krusei and C. parapsilosis in adhesion to different medical devices. Our results indicate that the four species strongly adher to polyvinyl chloride (PVC) devices, followed by polyurethane and finally by silicone. It was interesting to identify fructose-bisphosphate aldolase (Fba1) and enolase 1 (Eno1) as the CWP involved in adhesion of C. albicans, C. glabrata and C. krusei to PVC devices whereas phosphoglycerate kinase (Pgk) and Eno1 allow C. parapsilosis to adher to silicone-made implants. Results presented here suggest that these CWP participate in the initial event of adhesion and are probably followed by other proteins that covalently bind to the biomaterial thus providing conditions for biofilm formation and eventually the onset of infection.
Subject(s)
Candida/physiology , Cell Adhesion , Cell Wall/chemistry , Equipment and Supplies/microbiology , Membrane Proteins/isolation & purification , Membrane Proteins/physiology , Antifungal Agents/pharmacology , Biocompatible Materials/chemistry , Biofilms/growth & development , Candida/drug effects , Candida/enzymology , Candida/metabolism , Cell Adhesion/drug effects , Cell Wall/enzymology , Cell Wall/metabolism , Fructose-Bisphosphate Aldolase/isolation & purification , Fructose-Bisphosphate Aldolase/physiology , Fungal Proteins/physiology , Humans , Hydrogen Peroxide/pharmacology , Phosphoglycerate Kinase , Phosphopyruvate Hydratase/isolation & purification , Phosphopyruvate Hydratase/physiology , Polyurethanes/chemistry , Polyvinyl Chloride/chemistry , Silicones/chemistryABSTRACT
BACKGROUND: We have previously shown that in pancreatic ductal adenocarcinoma (PDA) cells, the glycolytic enzyme alpha-enolase (ENO1) also acts as a plasminogen receptor and promotes invasion and metastasis formation. Moreover, ENO1 silencing in PDA cells induces oxidative stress, senescence and profoundly modifies PDA cell metabolism. Although anti-ENO1 antibody inhibits PDA cell migration and invasion, little is known about the role of ENO1 in regulating cell-cell and cell-matrix contacts. We therefore investigated the effect of ENO1 silencing on the modulation of cell morphology, adhesion to matrix substrates, cell invasiveness, and metastatic ability. METHODS: The membrane and cytoskeleton modifications that occurred in ENO1-silenced (shENO1) PDA cells were investigated by a combination of confocal microscopy and atomic force microscopy (AFM). The effect of ENO1 silencing was then evaluated by phenotypic and functional experiments to identify the role of ENO1 in adhesion, migration, and invasion, as well as in senescence and apoptosis. The experimental results were then validated in a mouse model. RESULTS: We observed a significant increase in the roughness of the cell membrane due to ENO1 silencing, a feature associated with an impaired ability to migrate and invade, along with a significant downregulation of proteins involved in cell-cell and cell-matrix adhesion, including alpha v/beta 3 integrin in shENO1 PDA cells. These changes impaired the ability of shENO1 cells to adhere to Collagen I and IV and Fibronectin and caused an increase in RGD-independent adhesion to vitronectin (VN) via urokinase plasminogen activator receptor (uPAR). Binding of uPAR to VN triggers integrin-mediated signals, which result in ERK1-2 and RAC activation, accumulation of ROS, and senescence. In shENO1 cancer cells, the use of an anti-uPAR antibody caused significant reduction of ROS production and senescence. Overall, a decrease of in vitro and in vivo cell migration and invasion of shENO1 PDA cells was observed. CONCLUSION: These data demonstrate that ENO1 promotes PDA survival, migration, and metastasis through cooperation with integrins and uPAR.
Subject(s)
Biomarkers, Tumor/physiology , Cell Adhesion , DNA-Binding Proteins/physiology , Integrin alphaVbeta3/genetics , Neoplasm Invasiveness/pathology , Neoplasm Metastasis/pathology , Pancreatic Neoplasms/pathology , Phosphopyruvate Hydratase/physiology , Tumor Suppressor Proteins/physiology , Animals , Apoptosis , Biomarkers, Tumor/genetics , Cell Line, Tumor , Cell Shape , Cellular Senescence , DNA-Binding Proteins/genetics , Gene Expression , Gene Silencing , Humans , Integrin alphaVbeta3/metabolism , Integrins/metabolism , Integrins/physiology , Mice , Pancreatic Neoplasms/enzymology , Pancreatic Neoplasms/metabolism , Phosphopyruvate Hydratase/genetics , Receptors, Urokinase Plasminogen Activator/metabolism , Receptors, Urokinase Plasminogen Activator/physiology , Tumor Suppressor Proteins/geneticsABSTRACT
Rheumatoid arthritis (RA) is the most common form of chronic inflammatory rheumatism. Identifying auto-antigens targeted by RA auto-antibodies is of major interest. Alpha-enolase (ENO1) is considered to be a pivotal auto-antigen in early RA but its pathophysiologic role remains unknown. The main objective of this study was to investigate the in vitro effects of soluble ENO1 on peripheral blood mononuclear cells (PBMC) from healthy donors and RA patients in order to determine the potential pathogenic role of ENO1. ELISA, transcriptomic analysis, experiments of receptor inhibition and flow cytometry analysis were performed to determine the effect, the target cell population and the receptor of ENO1. We showed that ENO1 has the ability to induce early production of pro-inflammatory cytokines and chemokines with delayed production of IL-10 and to activate the innate immune system. We demonstrated that ENO1 binds mainly to monocytes and activates the CD14-dependent TLR4 pathway both in healthy subjects and in RA patients. Our results establish for the first time that ENO1 is able to activate in vitro the CD14-dependent TLR4 pathway on monocytes involving a dual mechanism firstly pro-inflammatory and secondly anti-inflammatory. These results contribute to elucidating the role of this auto-antigen in the pathophysiologic mechanisms of RA.
Subject(s)
Biomarkers, Tumor/physiology , DNA-Binding Proteins/physiology , Leukocytes, Mononuclear/enzymology , Lipopolysaccharide Receptors/metabolism , Phosphopyruvate Hydratase/physiology , Toll-Like Receptor 4/metabolism , Tumor Suppressor Proteins/physiology , Animals , Cattle , Cells, Cultured , Humans , Interleukin-10/biosynthesis , Lipopolysaccharides/pharmacology , Signal Transduction , Tumor Necrosis Factor-alpha/biosynthesisABSTRACT
Enolases are glycolytic enzymes responsible for the ATP-generated conversion of 2-phosphoglycerate to phosphoenolpyruvate. In addition to the glycolytic function, Enolase 1 (ENO1) has been reported up-regulation in several tumor tissues. In this study, we investigated the expression and biologic function of ENO1 in Non-Hodgkin's Lymphomas (NHLs). Clinically, by western blot analysis we observed that ENO1 expression was apparently higher in diffuse large B-cell lymphoma than in the reactive lymphoid tissues. Subsequently, immunohistochemical staining of 144 NHLs suggested that the expression of ENO1 was significantly lower in the indolent lymphomas compared with the progressive lymphomas. Further, we identified ENO1 as an independent prognostic factor, and it was significantly correlated with overall survival of NHL patients. In addition, we found that ENO1 could promote cell proliferation, regulate cell cycle associated gene and PI3K/AKT signaling pathway in NHLs. Finally, we verified that ENO1 participated in the process of lymphoma cell adhesion mediated drug resistance (CAM-DR). Adhesion to FN or HS5 cells significantly protected OCI-Ly8 and Daudi cells from cytotoxicity compared with those cultured in suspension, and these effects were attenuated when transfected with ENO1-siRNA. Based on the study, we propose that inhibition of ENO1 expression may be a novel strategy for therapy for NHLs patients, and it may be a target for drug resistance.
Subject(s)
Biomarkers, Tumor/physiology , Cell Adhesion , Cell Proliferation , DNA-Binding Proteins/physiology , Lymphoma, Non-Hodgkin/enzymology , Phosphopyruvate Hydratase/physiology , Tumor Suppressor Proteins/physiology , Cell Line, Tumor , Coculture Techniques , Drug Resistance, Neoplasm , Female , Humans , Kaplan-Meier Estimate , Lymphoma, Non-Hodgkin/drug therapy , Lymphoma, Non-Hodgkin/mortality , Lymphoma, Non-Hodgkin/pathology , Male , Middle Aged , Proportional Hazards Models , Signal TransductionABSTRACT
Mycoplasma suis belongs to the haemotrophic mycoplasmas, which colonise the red blood cells of a wide range of vertebrates. Adhesion to red blood cells is the crucial step in the unique lifecycle of M. suis. In addition to MSG1 protein, α-enolase is the second adhesion protein of M. suis, and may be involved in the adhesion of M. suis to porcine red blood cells (RBC). To simulate the environment of the RBC, we established the cDNA library of swine peripheral blood mononuclear cells (PBMC). The yeast two-hybrid (Y2H) system was adopted to screen α-enolase interactive proteins in the PBMC line. Alignment with the NCBI database revealed four interactive proteins: beta-actin, 60S ribosomal protein L11, clusterin precursor and endonuclease/reverse transcriptase. However, the M. suis α-enolase interactive proteins in the PBMC cDNA library obtained in the current study provide valuable information about the host cell interactions of the M. suis α-enolase protein.
Subject(s)
Bacterial Adhesion/physiology , Leukocytes, Mononuclear/microbiology , Mycoplasma/physiology , Phosphopyruvate Hydratase/genetics , Phosphopyruvate Hydratase/physiology , Two-Hybrid System Techniques , Actins/genetics , Animals , Clusterin/genetics , DNA, Bacterial/genetics , Gene Library , Leukocytes, Mononuclear/pathology , Leukocytes, Mononuclear/physiology , Mycoplasma/genetics , Mycoplasma Infections/microbiology , Mycoplasma Infections/pathology , Mycoplasma Infections/physiopathology , Swine , Swine Diseases/microbiology , Swine Diseases/pathology , Swine Diseases/physiopathologyABSTRACT
Enolases are glycolytic enzymes in the glycolytic pathway. In order to evaluate the effect of ENO1 on follicle-stimulating hormone receptor (FSHR) mRNA and luteinizing hormone receptor (LHR) mRNA of primary granular cell from goose F1 follicles, the recombinant plasmid adenovirus carrying ENO1 were constructed and infected the primary culture granular cells. The granular cells were randomly divided into three groups: recombinant adenovirus infected (pAd-CMV-ENO1), empty vector infected (pAd-CMV-Null) and no virus (mock control). The expression levels of FSHR mRNA and LHR mRNA of granular cells were examined by qRT-PCR. The results showed the group pAd-CMV-ENO1 had significantly higher FSHR mRNA expression levels than the other two groups (P < 0.05), but had significantly lower LHR mRNA expression levels than the other two groups (P < 0.05). The results suggested that ENO1 could improve the combination rate between FSH and FSHR to accelerate the proliferation and differentiation and steroidogenesis in poultry gonadal tissues.
Subject(s)
Geese/physiology , Granulosa Cells/physiology , Ovarian Follicle/physiology , Phosphopyruvate Hydratase/physiology , Receptors, FSH/physiology , Receptors, LH/physiology , Adenoviridae/genetics , Animals , Blotting, Western/veterinary , Female , Genetic Vectors/genetics , Granulosa Cells/cytology , Granulosa Cells/enzymology , Ovarian Follicle/cytology , Ovarian Follicle/enzymology , Phosphopyruvate Hydratase/genetics , RNA, Messenger/chemistry , RNA, Messenger/genetics , Random Allocation , Receptors, FSH/genetics , Receptors, LH/genetics , Reverse Transcriptase Polymerase Chain Reaction/veterinary , Up-Regulation/genetics , Up-Regulation/physiologyABSTRACT
Adipose tissue-derived stem cells (ADSCs) isolated from adult tissue have pluripotent differentiation and self-renewal capability. The tissue source of ADSCs can be obtained in large quantities and with low risks, thus highlighting the advantages of ADSCs in clinical applications. Valproic acid (VPA) is a widely used antiepileptic drug, which has recently been reported to affect ADSC differentiation in mice and rats; however, few studies have been performed on dogs. We aimed to examine the in vitro effect of VPA on canine ADSCs. Three days of pretreatment with VPA decreased the proliferation of ADSCs in a dose-dependent manner; VPA concentrations of 4 mM and above inhibited the proliferation of ADSCs. In parallel, VPA increased p16 and p21 mRNA expression, suggesting that VPA attenuated the proliferative activity of ADSCs by activating p16 and p21. Furthermore, the effects of VPA on adipogenic, osteogenic or neurogenic differentiation were investigated morphologically. VPA pretreatment markedly promoted neurogenic differentiation, but suppressed the accumulation of lipid droplets and calcium depositions. These modifications of ADSCs by VPA were associated with a particular gene expression profile, viz., an increase in neuronal markers, that is, NSE, TUBB3 and MAP2, a decrease in the adipogenic marker, LPL, but no changes in osteogenic markers, as estimated by reverse transcription-PCR analysis. These results suggested that VPA is a specific inducer of neurogenic differentiation of canine ADSCs and is a useful tool for studying the interaction between chromatin structure and cell fate determination.
Subject(s)
Adipose Tissue/cytology , Cell Proliferation/drug effects , Dogs/physiology , Histone Deacetylase Inhibitors/pharmacology , Stem Cells/physiology , Valproic Acid/pharmacology , Animals , Cyclin-Dependent Kinase Inhibitor p16/genetics , Cyclin-Dependent Kinase Inhibitor p16/physiology , Dose-Response Relationship, Drug , Microtubule-Associated Proteins/genetics , Microtubule-Associated Proteins/physiology , Phosphopyruvate Hydratase/genetics , Phosphopyruvate Hydratase/physiology , RNA/chemistry , RNA/genetics , Real-Time Polymerase Chain Reaction/veterinary , Stem Cells/cytology , Tubulin/genetics , Tubulin/physiology , p21-Activated Kinases/genetics , p21-Activated Kinases/physiologyABSTRACT
The LOS2 gene in Arabidopsis encodes an enolase with 72% amino acid sequence identity with human ENO1. In mammalian cells, the α-enolase (ENO1) gene encodes both a 48 kDa glycolytic enzyme and a 37 kDa transcriptional suppressor protein that are targeted to different cellular compartments. The tumor suppressor c-myc binding protein (MBP-1), which is alternatively translated from the second start codon of ENO1 transcripts, is preferentially localized in nuclei while α-enolase is found in the cytoplasm. We report here that an Arabidopsis MBP-1-like protein (AtMBP-1) is alternatively translated from full-length LOS2 transcripts using a second start codon. Like mammalian MBP-1, this truncated form of LOS2 has little, if any, enolase activity, indicating that an intact N-terminal region of LOS2 is critical for catalysis. AtMBP-1 has a short half-life in vivo and is stabilized by the proteasome inhibitor MG132, indicating that it is degraded via the ubiquitin-dependent proteasome pathway. Arabidopsis plants that over-express AtMBP-1 are hypersensitive to abscisic acid (ABA) during seed germination and show defects in vegetative growth and lateral stem development. AtMBP-1 interacts directly with the E3 ubiquitin ligase AtSAP5 and co-expression of these proteins resulted in destabilization of AtMBP-1 in vivo and abolished the developmental defects associated with AtMBP-1 over-expression. Thus, AtMBP-1 is as a bona fide alternative translation product of LOS2. Accumulation of this factor is limited by ubiquitin-dependent destabilization, apparently mediated by AtSAP5.
Subject(s)
Abscisic Acid/metabolism , Arabidopsis Proteins/physiology , Arabidopsis/enzymology , Phosphopyruvate Hydratase/physiology , Ubiquitin-Protein Ligases/physiology , Alternative Splicing , Arabidopsis/growth & development , Gene Expression Regulation, Plant , Proteasome Endopeptidase Complex/metabolism , Proteolysis , RNA, Messenger/metabolism , Signal Transduction , Stress, Physiological , Ubiquitin/metabolismABSTRACT
γ-Enolase acts as a neurotrophic-like factor promoting growth, differentiation, survival and regeneration of neurons. It is shown in this study to exert a protective effect against amyloid-ß-peptide (Aß)-induced neurotoxicity in rat pheochromocytoma PC12 cells. Aß-induced toxicity was abolished in the presence of the active C-terminal peptide of γ-enolase (γ-Eno) as measured by cell viability, lactate dehydrogenase release, sub-G1 cell population, intracellular reactive oxygen species, mitochondrial functions and apoptotic morphology. γ-Eno caused downregulation of the pro-apoptotic protein Bax and upregulation of the anti-apoptotic protein Bcl-2, as well as reduced caspase-3 activation. Exposure to Aß increased surface expression of p75 neurotrophin receptor (p75(NTR)), and the increase was abolished in the presence of γ-Eno peptide. Further, pretreatment with γ-Eno suppressed the activation of mitogen-activated protein kinases p38 and Jun-N-terminal kinase, which are p75(NTR) downstream effectors in apoptotic signaling. Moreover, Aß triggered γ-enolase co-immunoprecipitation with p75(NTR) as well as their strong association in the perimembrane region as shown by confocal microscopy, which further supports the interaction between these two proteins in cells insulted by Aß peptide. Our results indicate the possible use of γ-enolase C-terminal peptide for treating or preventing Alzheimer's disease.
Subject(s)
Amyloid beta-Peptides/toxicity , Neuroprotective Agents/pharmacology , Peptide Fragments/toxicity , Phosphopyruvate Hydratase/pharmacology , Receptors, Nerve Growth Factor/drug effects , Signal Transduction/drug effects , Amino Acid Sequence , Amyloid beta-Peptides/antagonists & inhibitors , Animals , Apoptosis/drug effects , Caspase 3/metabolism , Enzyme Activation/drug effects , Gene Expression Regulation/drug effects , Humans , Membrane Potential, Mitochondrial/drug effects , Models, Neurological , Molecular Sequence Data , Nerve Tissue Proteins , PC12 Cells , Peptide Fragments/antagonists & inhibitors , Peptide Fragments/chemical synthesis , Peptide Fragments/pharmacology , Phosphopyruvate Hydratase/chemistry , Phosphopyruvate Hydratase/physiology , Proto-Oncogene Proteins c-bcl-2/biosynthesis , Proto-Oncogene Proteins c-bcl-2/genetics , Rats , Receptors, Growth Factor , Receptors, Nerve Growth Factor/physiology , Up-Regulation/drug effects , bcl-2-Associated X Protein/biosynthesis , bcl-2-Associated X Protein/geneticsABSTRACT
Increased glucose catabolism and resistance to cell death are hallmarks of cancers, but the link between them remains elusive. Remarkably, under conditions where caspases are inhibited, the process of cell death is delayed but rarely blocked, leading to the occurrence of caspase-independent cell death (CICD). Escape from CICD is particularly relevant in the context of cancer as apoptosis inhibition only is often not sufficient to allow oncogenic transformation. While most glycolytic enzymes are overexpressed in tumors, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is of particular interest as it can allow cells to recover from CICD. Here, we show that GAPDH, but no other glycolytic enzymes tested, when overexpressed could bind to active Akt and limit its dephosphorylation. Active Akt prevents FoxO nuclear localization, which precludes Bcl-6 expression and leads to Bcl-xL overexpression. The GAPDH-dependent Bcl-xL overexpression is able to protect a subset of mitochondria from permeabilization that are required for cellular survival from CICD. Thus, our work suggests that GAPDH overexpression could induce Bcl-xL overexpression and protect cells from CICD-induced chemotherapy through preservation of intact mitochondria that may facilitate tumor survival and chemotherapeutic resistance.
Subject(s)
Apoptosis/physiology , Caspases/physiology , Glyceraldehyde 3-Phosphate Dehydrogenase (NADP+)/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Up-Regulation/physiology , bcl-X Protein/metabolism , Cell Death/physiology , Cell Line, Tumor , Cell Survival/physiology , HEK293 Cells , HeLa Cells , Humans , Mitochondria/physiology , Phosphoglycerate Kinase/physiology , Phosphopyruvate Hydratase/physiology , Protein Binding/physiologyABSTRACT
Sperm utilize glycolysis to generate ATP required for motility, and several spermatogenic cell-specific glycolytic isozymes are associated with the fibrous sheath (FS) in the principal piece of the sperm flagellum. We used proteomics and molecular biology approaches to confirm earlier reports that a novel enolase is present in mouse sperm. We then found that a pan-enolase antibody, but not antibodies to ENO2 and ENO3, recognized a protein in the principal piece of the mouse sperm flagellum. Database analyses identified two previously uncharacterized enolase family-like candidate genes, 64306537H0Rik and Gm5506. Northern analysis indicated that 64306537H0Rik (renamed Eno4) was transcribed in testes of mice by Postnatal Day 12. To determine the role of ENO4, we generated mice using embryonic stem cells in which an Eno4 allele was disrupted by a gene trap containing a beta galactosidase (beta-gal) reporter (Eno4(+/Gt)). Expression of beta-gal occurred in the testis, and male mice homozygous for the gene trap allele (Eno4(Gt/Gt)) were infertile. Epididymal sperm numbers were 2-fold lower and sperm motility was reduced substantially in Eno4(Gt/Gt) mice compared to wild-type mice. Sperm from Eno4(Gt/Gt) mice had a coiled flagellum and a disorganized FS. The Gm5506 gene encodes a protein identical to ENO1 and also is transcribed at a low level in testis. We conclude that ENO4 is required for normal assembly of the FS and provides most of the enolase activity in sperm and that Eno1 and/or Gm5506 may encode a minor portion of the enolase activity in sperm.
Subject(s)
Infertility, Male/genetics , Phosphopyruvate Hydratase/genetics , Spermatogenesis/genetics , Spermatozoa/abnormalities , Animals , Female , Gene Expression , Male , Mice , Mice, Inbred C57BL , Mice, Transgenic , Mutagenesis/physiology , Organ Specificity/genetics , Phosphopyruvate Hydratase/physiology , Spermatozoa/enzymology , Spermatozoa/ultrastructureABSTRACT
α-Enolase (ENO1) is a multifunctional glycolytic enzyme expressed abundantly in the cytosol. It has been implicated in autoimmune and inflammatory diseases. Serum Abs against ENO1 were reported in rheumatoid arthritis (RA). Cell-surface expression of ENO1 has been found to be increased rapidly in response to inflammatory stimuli, but its expression and function has not been reported in RA. In this study, we show that cell-surface expression of ENO1 is increased on monocytes and macrophages isolated from RA patients but not on those from osteoarthritis patients, and Ab against ENO1 can stimulate these cells to produce higher amounts of proinflammatory mediators, such as TNF-α, IL-1 α/ß, IFN-γ, and PGE(2) via p38 MAPK and NF-κB pathway. The frequency of ENO1-positive cells in synovial fluid mononuclear cells was higher than PBMCs. ENO1-positive cells were also found in the inflamed synovium from RA patients and arthritic ankle tissues of mice with collagen-induced arthritis. Taken together, these findings suggest that Abs against ENO1 present in RA sera may stimulate monocytes and macrophages expressing cell-surface ENO1 and contribute to production of proinflammatory mediators during the effector phase of synovial inflammation.
Subject(s)
Arthritis, Rheumatoid/enzymology , Biomarkers, Tumor/biosynthesis , DNA-Binding Proteins/biosynthesis , Macrophages/enzymology , Monocytes/enzymology , Phosphopyruvate Hydratase/biosynthesis , Synovial Membrane/enzymology , Tumor Suppressor Proteins/biosynthesis , Amino Acid Sequence , Animals , Arthritis, Experimental/enzymology , Arthritis, Experimental/immunology , Arthritis, Experimental/pathology , Arthritis, Rheumatoid/immunology , Arthritis, Rheumatoid/pathology , Biomarkers, Tumor/physiology , Cells, Cultured , Collagen/administration & dosage , DNA-Binding Proteins/physiology , Humans , Inflammation/enzymology , Inflammation/immunology , Inflammation/pathology , Macrophages/immunology , Macrophages/pathology , Male , Mice , Mice, Inbred DBA , Molecular Sequence Data , Monocytes/immunology , Monocytes/pathology , Phosphopyruvate Hydratase/physiology , Synovial Membrane/immunology , Synovial Membrane/pathology , Tumor Suppressor Proteins/physiologyABSTRACT
In order to successfully utilize stem cells for therapeutic applications in regenerative medicine, efficient differentiation into a specific cell lineage and guidance of axons in a desired direction is crucial. Here, we used aligned multi-walled carbon nanotube (MWCNT) sheets to differentiate human mesenchymal stem cells (hMSCs) into neural cells. Human MSCs present a preferential adhesion to aligned CNT sheets with longitudinal stretch parallel to the CNT orientation direction. Cell elongation was 2-fold higher than the control and most of the cells were aligned on CNT sheets within 5° from the CNT orientation direction. Furthermore, a significant, synergistic enhancement of neural differentiation was observed in hMSCs cultured on the CNT sheets. Axon outgrowth was also controlled using nanoscale patterning of CNTs. This CNT sheet provides a new cellular scaffold platform that can regulate morphogenesis and differentiation of stem cells, which could open up a new approach for tissue and stem cell regeneration.