Phosphoinositide 3-kinase accelerates necrotic cell death during hypoxia

2001 ◽  
Vol 358 (2) ◽  
pp. 481-487 ◽  
Author(s):  
Toshihiko AKI ◽  
Yoichi MIZUKAMI ◽  
Yoshitomo OKA ◽  
Kazuhito YAMAGUCHI ◽  
Koichi UEMURA ◽  
...  
Keyword(s):  
Cell Death ◽  
Metabolic Acidosis ◽  
Glucose Metabolism ◽  
Dominant Negative ◽  
Hypoxic Cell ◽  
Dependent Manner ◽  
Wild Type ◽  
Promoting Effect ◽  
Rat Cardiomyocytes ◽  
Phosphoinositide 3 Kinase

Using H9c2 cells derived from rat cardiomyocytes, we investigated the mechanism of cell death during hypoxia in the presence of serum and glucose. Hypoxic cell death is by necrosis and is accompanied by metabolic acidosis. Moreover, hypoxic cell death is inhibited by Hepes buffer as well as by 2-deoxyglucose, an inhibitor of glycolysis, indicating that metabolic acidosis should play an essential role in hypoxic injury. The involvement of phosphoinositide 3-kinase (PI 3-kinase), which is known to activate glucose metabolism, was examined using its inhibitor, LY290042, or adenovirus-mediated gene transfer. Hypoxic cell death was inhibited by LY294002 in a dose-dependent manner. Overexpression of dominant negative PI 3-kinase was found to reduce cell death, whereas wild-type PI 3-kinase enhanced it. Dominant negative PI 3-kinase also reduced glucose consumption and acidosis, but this was stimulated by wild-type PI 3-kinase. The data indicate that PI 3-kinase stimulates cell death by enhancing metabolic acidosis. LY294002 significantly reduced glucose uptake, showing that PI 3-kinase regulates glycolysis at the step of glucose transport. These findings indicate the pivotal role of glucose metabolism in hypoxic cell death, and reveal a novel death-promoting effect of PI 3-kinase during hypoxia, despite this enzyme being considered to be a survival-promoting factor.

scholarly journals Active elimination of intestinal cells drives oncogenic growth in organoids

2020 ◽  
Author(s):  
Ana Krotenberg Garcia ◽  
Arianna Fumagalli ◽  
Huy Quang Le ◽  
Owen J. Sansom ◽  
Jacco van Rheenen ◽  
...  
Keyword(s):  
Quality Control ◽  
Cell Death ◽  
Cancer Cells ◽  
Cell Fate ◽  
Crucial Role ◽  
Intestinal Cells ◽  
Dependent Manner ◽  
Wild Type ◽  
Cell Competition ◽  
Type Population

AbstractCompetitive cell-interactions play a crucial role in quality control during development and homeostasis. Here we show that cancer cells use such interactions to actively eliminate wild-type intestine cells in enteroid monolayers and organoids. This apoptosis-dependent process boosts proliferation of intestinal cancer cells. The remaining wild-type population activates markers of primitive epithelia and transits to a fetal-like state. Prevention of this cell fate transition avoids elimination of wild-type cells and, importantly, limits the proliferation of cancer cells. JNK signalling is activated in competing cells and is required for cell fate change and elimination of wild-type cells. Thus, cell competition drives growth of cancer cells by active out-competition of wild-type cells through forced cell death and cell fate change in a JNK dependent manner.

Abstract 165: The 18 kDa FGF-2 Prevents the Doxorubicin-induced Cardiac Damage and Amp-activated Kinase Activation, in vitro and in vivo

2015 ◽  
Vol 117 (suppl_1) ◽  
Author(s):  
Navid Koleini ◽  
Jon Jon Santiago ◽  
Barbara E Nickel ◽  
Robert Fandrich ◽  
Davinder S Jassal ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Cell Death ◽  
Left Ventricular ◽  
Left Ventricular Ejection ◽  
Wild Type ◽  
Rat Cardiomyocytes ◽  
Ventricular Ejection Fraction ◽  
Compound C

Introduction: Protection of the heart from chemotherapeutic (Doxorubicin, DOX) drug-induced toxicity is a desirable goal, to limit side effects of cancer treatments. DOX toxicity has been linked to the activation (phosphorylation) of the AMP-activated kinase, AMPK. The 18 kDa low molecular weight isoform of fibroblast growth factor 2 (Lo-FGF-2) is a known cardioprotective and cytoprotective agent. In this study we have tested the ability of Lo-FGF-2 to protect from DOX-induced damage in rat cardiomyocytes in vitro, and in transgenic mouse models in vivo, in relation to AMPK activation. Methods: Rat neonatal cardiomyocytes in culture were exposed to DOX (0.5 μM) in the presence or absence of pre-treatment Lo-FGF-2 (10 ng/ml). Compound C was used to block phosphorylation (activity) of AMPK. Levels of cell viability/death (using Calcein-AM/Propidium iodide assay), phospho -and total AMPK, and apoptotic markers such as active caspase 3 were analyzed. In addition, transgenic mice expressing only Lo-FGF2, and wild type mice, expressing both high molecular weight (Hi-FGF2) as well as Lo-FGF2 were subjected to DOX injection (20 mg/kg, intraperitoneal); echocardiography was used to examine cardiac function at baseline and at 10 days post-DOX. Results: DOX-induced cell death of cardiomyocytes in culture was maximal at 24 hours post-DOX coinciding with significantly increased in activated (phosphorylated) AMPK. Compound C attenuated DOX-induced cardiomyocyte loss. Pre-incubation with Lo-FGF-2 decreased DOX induced cell death, and also attenuated the phosphorylation of AMPK post-DOX. Relative levels of phospho-AMPK were lower in the hearts of Lo-FGF2-expressing male mice compared to wild type. DOX-induced loss of contractile function (left ventricular ejection fraction and endocardial velocity) was negligible in Lo-FGF2-expressing mice but significant in wild type mice. Conclusion: Lo-FGF-2 protects the heart from DOX-induced damage in vitro and in vivo, by a mechanism likely involving an attenuation of AMPK activity.

scholarly journals RacG Regulates Morphology, Phagocytosis, and Chemotaxis

2006 ◽  
Vol 5 (10) ◽  
pp. 1648-1663 ◽  
Author(s):  
Baggavalli P. Somesh ◽  
Georgia Vlahou ◽  
Miho Iijima ◽  
Robert H. Insall ◽  
Peter Devreotes ◽  
...  
Keyword(s):  
Rho Gtpases ◽  
Actin Polymerization ◽  
Kinase Inhibitor ◽  
Dominant Negative ◽  
Dependent Manner ◽  
Free System ◽  
Mild Phenotype ◽  
Particle Uptake ◽  
Phosphoinositide 3 Kinase

ABSTRACTRacG is an unusual member of the complex family of Rho GTPases inDictyostelium. We have generated a knockout (KO) strain, as well as strains that overexpress wild-type (WT), constitutively active (V12), or dominant negative (N17) RacG. The protein is targeted to the plasma membrane, apparently in a nucleotide-dependent manner, and induces the formation of abundant actin-driven filopods. RacG is enriched at the rim of the progressing phagocytic cup, and overexpression of RacG-WT or RacG-V12 induced an increased rate of particle uptake. The positive effect of RacG on phagocytosis was abolished in the presence of 50 μM LY294002, a phosphoinositide 3-kinase inhibitor, indicating that generation of phosphatidylinositol 3,4,5-trisphosphate is required for activation of RacG. RacG-KO cells showed a moderate chemotaxis defect that was stronger in the RacG-V12 and RacG-N17 mutants, in part because of interference with signaling through Rac1. The in vivo effects of RacG-V12 could not be reproduced by a mutant lacking the Rho insert region, indicating that this region is essential for interaction with downstream components. Processes like growth, pinocytosis, exocytosis, cytokinesis, and development were unaffected in Rac-KO cells and in the overexpressor mutants. In a cell-free system, RacG induced actin polymerization upon GTPγS stimulation, and this response could be blocked by an Arp3 antibody. While the mild phenotype of RacG-KO cells indicates some overlap with one or moreDictyosteliumRho GTPases, like Rac1 and RacB, the significant changes found in overexpressors show that RacG plays important roles. We hypothesize that RacG interacts with a subset of effectors, in particular those concerned with shape, motility, and phagocytosis.

scholarly journals c-Cbl Mediates Ubiquitination, Degradation, and Down-regulation of Human Protease-activated Receptor 2

2005 ◽  
Vol 280 (16) ◽  
pp. 16076-16087 ◽  
Author(s):  
Claire Jacob ◽  
Graeme S. Cottrell ◽  
Daphne Gehringer ◽  
Fabien Schmidlin ◽  
Eileen F. Grady ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Ring Finger ◽  
Dominant Negative ◽  
Dependent Manner ◽  
Wild Type ◽  
Lysosomal Degradation ◽  
Proteolytic Activation ◽  
Gpcr Signaling ◽  
Early Endosomes ◽  
Protease Activated Receptor 2

Mechanisms that arrest G-protein-coupled receptor (GPCR) signaling prevent uncontrolled stimulation that could cause disease. Although uncoupling from heterotrimeric G-proteins, which transiently arrests signaling, is well described, little is known about the mechanisms that permanently arrest signaling. Here we reported on the mechanisms that terminate signaling by protease-activated receptor 2 (PAR2), which mediated the proinflammatory and nociceptive actions of proteases. Given its irreversible mechanism of proteolytic activation, PAR2is a model to study the permanent arrest of GPCR signaling. By immunoprecipitation and immunoblotting, we observed that activated PAR2was mono-ubiquitinated. Immunofluorescence indicated that activated PAR2translocated from the plasma membrane to early endosomes and lysosomes where it was degraded, as determined by immunoblotting. Mutant PAR2lacking intracellular lysine residues (PAR2Δ14K/R) was expressed at the plasma membrane and signaled normally but was not ubiquitinated. Activated PAR2Δ14K/R internalized but was retained in early endosomes and avoided lysosomal degradation. Activation of wild type PAR2stimulated tyrosine phosphorylation of the ubiquitin-protein isopeptide ligase c-Cbl and promoted its interaction with PAR2at the plasma membrane and in endosomes in an Src-dependent manner. Dominant negative c-Cbl lacking the ring finger domain inhibited PAR2ubiquitination and induced retention in early endosomes, thereby impeding lysosomal degradation. Although wild type PAR2was degraded, and recovery of agonist responses required synthesis of new receptors, lysine mutation and dominant negative c-Cbl impeded receptor ubiquitination and degradation and allowed PAR2to recycle and continue to signal. Thus, c-Cbl mediated ubiquitination and lysosomal degradation of PAR2to irrevocably terminate signaling by this and perhaps other GPCRs.

scholarly journals α-Synuclein binds and sequesters PIKE-L into Lewy bodies, triggering dopaminergic cell death via AMPK hyperactivation

2017 ◽  
Vol 114 (5) ◽  
pp. 1183-1188 ◽  
Author(s):  
Seong Su Kang ◽  
Zhentao Zhang ◽  
Xia Liu ◽  
Fredric P. Manfredsson ◽  
Li He ◽  
...  
Keyword(s):  
Cell Death ◽  
Molecular Mechanisms ◽  
Critical Role ◽  
Lewy Bodies ◽  
Cell Loss ◽  
Dominant Negative ◽  
Dependent Manner ◽  
Ph Domain ◽  
Dopaminergic Cell ◽  
Neuroprotective Actions

The abnormal aggregation of fibrillar α-synuclein in Lewy bodies plays a critical role in the pathogenesis of Parkinson’s disease. However, the molecular mechanisms regulating α-synuclein pathological effects are incompletely understood. Here we show that α-synuclein binds phosphoinositide-3 kinase enhancer L (PIKE-L) in a phosphorylation-dependent manner and sequesters it in Lewy bodies, leading to dopaminergic cell death via AMP-activated protein kinase (AMPK) hyperactivation. α-Synuclein interacts with PIKE-L, an AMPK inhibitory binding partner, and this action is increased by S129 phosphorylation through AMPK and is decreased by Y125 phosphorylation via Src family kinase Fyn. A pleckstrin homology (PH) domain in PIKE-L directly binds α-synuclein and antagonizes its aggregation. Accordingly, PIKE-L overexpression decreases dopaminergic cell death elicited by 1-methyl-4-phenylpyridinium (MPP+), whereas PIKE-L knockdown elevates α-synuclein oligomerization and cell death. The overexpression of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) or α-synuclein induces greater dopaminergic cell loss and more severe motor defects in PIKE-KO and Fyn-KO mice than in wild-type mice, and these effects are attenuated by the expression of dominant-negative AMPK. Hence, our findings demonstrate that α-synuclein neutralizes PIKE-L’s neuroprotective actions in synucleinopathies, triggering dopaminergic neuronal death by hyperactivating AMPK.

scholarly journals Torsin ATPases influence chromatin interaction of the Torsin regulator LAP1

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Naemi Luithle ◽  
Jelmi uit de Bos ◽  
Ruud Hovius ◽  
Daria Maslennikova ◽  
Renard TM Lewis ◽  
...  
Keyword(s):  
Chromosome Segregation ◽  
Dominant Negative ◽  
Chromatin Interaction ◽  
Dependent Manner ◽  
Chromatin Binding ◽  
Wild Type ◽  
Nuclear Lamins ◽  
Perinuclear Space ◽  
Aaa Atpases ◽  
Mitotic Chromatin

The inner nuclear membrane is functionalized by diverse transmembrane proteins that associate with nuclear lamins and/or chromatin. When cells enter mitosis, membrane-chromatin contacts must be broken to allow for proper chromosome segregation; yet how this occurs remains ill-understood. Unexpectedly, we observed that an imbalance in the levels of the lamina-associated polypeptide 1 (LAP1), an activator of ER-resident Torsin AAA+-ATPases, causes a failure in membrane removal from mitotic chromatin, accompanied by chromosome segregation errors and changes in post-mitotic nuclear morphology. These defects are dependent on a hitherto unknown chromatin-binding region of LAP1 that we have delineated. LAP1-induced NE abnormalities are efficiently suppressed by expression of wild-type but not ATPase-deficient Torsins. Furthermore, a dominant-negative Torsin induces chromosome segregation defects in a LAP1-dependent manner. These results indicate that association of LAP1 with chromatin in the nucleus can be modulated by Torsins in the perinuclear space, shedding new light on the LAP1-Torsin interplay.

Abi-1-bridged tyrosine phosphorylation of VASP by Abelson kinase impairs association of VASP to focal adhesions and regulates leukaemic cell adhesion

2012 ◽  
Vol 441 (3) ◽  
pp. 889-901 ◽  
Author(s):  
Masahiro Maruoka ◽  
Mizuho Sato ◽  
Yunfeng Yuan ◽  
Masayoshi Ichiba ◽  
Ryosuke Fujii ◽  
...  
Keyword(s):  
Focal Adhesions ◽  
K562 Cells ◽  
Dependent Manner ◽  
Wild Type ◽  
Abelson Kinase ◽  
Leukaemic Cells ◽  
Phosphoinositide 3 Kinase ◽  
Proline Rich Region

Mena [mammalian Ena (Enabled)]/VASP (vasodilator-stimulated phosphoprotein) proteins are the homologues of Drosophila Ena. In Drosophila, Ena is a substrate of the tyrosine kinase DAbl (Drosophila Abl). However, the link between Abl and the Mena/VASP family is not fully understood in mammals. We previously reported that Abi-1 (Abl interactor 1) promotes phosphorylation of Mena and BCAP (B-cell adaptor for phosphoinositide 3-kinase) by bridging the interaction between c-Abl and the substrate. In the present study we have identified VASP, another member of the Mena/VASP family, as an Abi-1-bridged substrate of Abl. VASP is phosphorylated by Abl when Abi-1 is co-expressed. We also found that VASP interacted with Abi-1 both in vitro and in vivo. VASP was tyrosine-phosphorylated in Bcr-Abl-positive leukaemic cells in an Abi-1-dependent manner. Co-expression of c-Abl and Abi-1 or the phosphomimetic Y39D mutation in VASP resulted in less accumulation of VASP at focal adhesions. VASP Y39D had a reduced affinity to the proline-rich region of zyxin. Interestingly, overexpression of both phosphomimetic and unphosphorylated forms of VASP, but not wild-type VASP, impaired adhesion of K562 cells to fibronectin. These results suggest that the phosphorylation and dephosphorylation cycle of VASP by the Abi-1-bridged mechanism regulates association of VASP with focal adhesions, which may regulate adhesion of Bcr-Abl-transformed leukaemic cells.

scholarly journals PKCα regulates the hypertrophic growth of cardiomyocytes through extracellular signal–regulated kinase1/2 (ERK1/2)

2002 ◽  
Vol 156 (5) ◽  
pp. 905-919 ◽  
Author(s):  
Julian C. Braz ◽  
Orlando F. Bueno ◽  
Leon J. De Windt ◽  
Jeffery D. Molkentin
Keyword(s):  
Marker Gene ◽  
Dominant Negative ◽  
Neonatal Rat ◽  
Cardiomyocyte Hypertrophy ◽  
Leucine Incorporation ◽  
Wild Type ◽  
Rat Cardiomyocytes ◽  
Signal Transducers ◽  
Hypertrophic Growth ◽  
Extracellular Signal

Members of the protein kinase C (PKC) isozyme family are important signal transducers in virtually every mammalian cell type. Within the heart, PKC isozymes are thought to participate in a signaling network that programs developmental and pathological cardiomyocyte hypertrophic growth. To investigate the function of PKC signaling in regulating cardiomyocyte growth, adenoviral-mediated gene transfer of wild-type and dominant negative mutants of PKCα, βII, δ, and ε (only wild-type ζ) was performed in cultured neonatal rat cardiomyocytes. Overexpression of wild-type PKCα, βII, δ, and ε revealed distinct subcellular localizations upon activation suggesting unique functions of each isozyme in cardiomyocytes. Indeed, overexpression of wild-type PKCα, but not βII, δ, ε, or ζ induced hypertrophic growth of cardiomyocytes characterized by increased cell surface area, increased [3H]-leucine incorporation, and increased expression of the hypertrophic marker gene atrial natriuretic factor. In contrast, expression of dominant negative PKCα, βII, δ, and ε revealed a necessary role for PKCα as a mediator of agonist-induced cardiomyocyte hypertrophy, whereas dominant negative PKCε reduced cellular viability. A mechanism whereby PKCα might regulate hypertrophy was suggested by the observations that wild-type PKCα induced extracellular signal–regulated kinase1/2 (ERK1/2), that dominant negative PKCα inhibited PMA-induced ERK1/2 activation, and that dominant negative MEK1 (up-stream of ERK1/2) inhibited wild-type PKCα–induced hypertrophic growth. These results implicate PKCα as a necessary mediator of cardiomyocyte hypertrophic growth, in part, through a ERK1/2-dependent signaling pathway.

scholarly journals Glucose Phosphorylation and Mitochondrial Binding Are Required for the Protective Effects of Hexokinases I and II

2007 ◽  
Vol 28 (3) ◽  
pp. 1007-1017 ◽  
Author(s):  
Lin Sun ◽  
Shetha Shukair ◽  
Tejaswitha Jairaj Naik ◽  
Farzad Moazed ◽  
Hossein Ardehali
Keyword(s):  
Tissue Culture ◽  
Cell Death ◽  
Glucose Metabolism ◽  
Mitochondrial Permeability Transition ◽  
Full Length ◽  
Neonatal Rat ◽  
Protective Effects ◽  
Voltage Dependent Anion Channel ◽  
Rat Cardiomyocytes ◽  
Glucose Phosphorylation

ABSTRACT Alterations in glucose metabolism have been demonstrated for diverse disorders ranging from heart disease to cancer. The first step in glucose metabolism is carried out by the hexokinase (HK) family of enzymes. HKI and II can bind to mitochondria through their N-terminal hydrophobic regions, and their overexpression in tissue culture protects against cell death. In order to determine the relative contributions of mitochondrial binding and glucose-phosphorylating activities of HKs to their overall protective effects, we expressed full-length HKI and HKII, their truncated proteins lacking the mitochondrial binding domains, and catalytically inactive proteins in tissue culture. The overexpression of full-length proteins resulted in protection against cell death, decreased levels of reactive oxygen species, and possibly inhibited mitochondrial permeability transition in response to H2O2. However, the truncated and mutant proteins exerted only partial effects. Similar results were obtained with primary neonatal rat cardiomyocytes. The HK proteins also resulted in an increase in the phosphorylation of voltage-dependent anion channel (VDAC) through a protein kinase Cε (PKCε)-dependent pathway. These results suggest that both glucose phosphorylation and mitochondrial binding contribute to the protective effects of HKI and HKII, possibly through VDAC phosphorylation by PKCε.

scholarly journals Myc supercompetitor cells exploit the NMDA receptor to subdue their wild-type neighbours via cell competition

2020 ◽  
Author(s):  
Agnes R. Banreti ◽  
Pascal Meier
Keyword(s):  
Cell Death ◽  
Nmda Receptor ◽  
Cell Growth ◽  
Epithelial Cells ◽  
Fitness Landscape ◽  
Dependent Manner ◽  
Wild Type ◽  
Direct Inhibition ◽  
Cell Competition ◽  
Competitive Behaviour

SUMMARYMyc is a major driver of cell growth in many cancers, but direct inhibition of Myc’s oncogenic activity has been challenging. Interactions between wild-type and Myc-expressing cells cause Myc cells to acquire ‘supercompetitor’ behaviour that increases their fitness and enables them to overtake the tissue by killing their wild-type neighbours through TNF-induced cell death during a process called cell competition. Here we report that the competitive behaviour of Myc, RasV12 cells, and normal epithelial cells, critically depends on the NMDA receptor. Myc cells upregulate NMDAR2 (NR2) to gain supercompetitor status and subdue their wild-type neighbours. Pharmacological inhibition or genetic depletion of NR2 changes the supercompetitor status of oncogenic Myc or RasV12 clones into ‘superlosers’, resulting in their elimination via cell competition by wild-type neighbours in a TNF-dependent manner. Our data demonstrate that that the NMDA receptor (NMDAR) determines cellular fitness during cell competition, and can be targeted to change the fitness landscape of supercompetitive Myc and RasV12 clones, converting them into superlosers.

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