RESUMO
AIM: Non-alcoholic steatohepatitis (NASH) is characterized by steatosis, inflammatory responses and fibrosis. Our previous studies provided evidence that group IVA phospholipase A2 (IVA-PLA2), a key PLA2 isozyme in the arachidonic acid cascade, is involved in the development of NASH. However, which types of cells are critical for the IVA-PLA2-dependent onset and progression of NASH is unclear. We elucidated the effects of the cell-type-specific deficiency of IVA-PLA2 in mice on the development of NASH. MAIN METHODS: Cell-type-specific IVA-PLA2-conditional knockout (cKO) mice and littermate controls were fed a choline-deficient, L-amino-acid-defined, high-fat diet with 0.1% methionine as a NASH model. The degree of hepatic fibrosis was evaluated by staining with picric acid-Sirius red, and the number of activated hepatic stellate cells was determined by immunoblotting and immunostaining for α-smooth muscle actin. Sinusoidal capillarization was analyzed by scanning electron microscopy. KEY FINDINGS: The deposition of collagen and number of activated hepatic stellate cells were markedly reduced in endothelial cell/liver sinusoidal endothelial cell (EC/LSEC)-specific IVA-PLA2 cKO mice but not in hepatocyte-, monocyte/macrophage-, or hepatic stellate cell-specific IVA-PLA2 cKO mice. In addition, EC/LSEC-specific IVA-PLA2-deficient mice showed more fenestrae than control mice fed a CDAHFD, indicating suppression of sinusoidal capillarization. SIGNIFICANCE: These results suggest that ECs/LSECs contribute to the IVA-PLA2-dependent onset and/or progression of NASH. Endothelial IVA-PLA2 is a promising factor for promoting sinusoidal capillarization and the ensuing HSC activation and fibrosis; thus IVA-PLA2 in ECs/LSECs is a potential therapeutic target for NASH.
Assuntos
Capilares/patologia , Células Endoteliais/patologia , Fosfolipases A2 do Grupo IV/fisiologia , Cirrose Hepática/patologia , Neovascularização Patológica/patologia , Hepatopatia Gordurosa não Alcoólica/complicações , Animais , Capilares/enzimologia , Células Endoteliais/enzimologia , Cirrose Hepática/enzimologia , Cirrose Hepática/etiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neovascularização Patológica/enzimologiaRESUMO
Clinical studies revealed that some aged-individuals accumulate a significant number of histopathological Alzheimer´s disease (AD) lesions in their brain, yet without developing any signs of dementia. Animal models of AD represent suitable tools to identify genes that might promote cognitive resilience and hence, this study first set out to identify cognitively resilient individuals in the aged-Tg2576 mouse model. A transcriptomic analysis of these mice identified PLA2G4E as a gene that might confer resistance to dementia. Indeed, a significant decrease in PLA2G4E is evident in the brain of late-stage AD patients, whereas no such changes are observed in early stage patients with AD neuropathological lesions but no signs of dementia. We demonstrated that adeno-associated viral vector-mediated overexpression of PLA2G4E in hippocampal neurons completely restored cognitive deficits in elderly APP/PS1 mice, without affecting the amyloid or tau pathology. These PLA2G4E overexpressing APP/PS1 mice developed significantly more dendritic spines than sham-injected mice, coinciding with the cognitive improvement observed. Hence, these results support the idea that a loss of PLA2G4E might play a key role in the onset of dementia in AD, highlighting the potential of PLA2G4E overexpression as a novel therapeutic strategy to manage AD and other disorders that course with memory deficits.
Assuntos
Doença de Alzheimer/genética , Doença de Alzheimer/terapia , Espinhas Dendríticas , Terapia Genética , Fosfolipases A2 do Grupo IV/fisiologia , Fosfolipases A2 do Grupo IV/uso terapêutico , Hipocampo , Memória Espacial , Animais , Comportamento Animal/fisiologia , Modelos Animais de Doenças , Feminino , Regulação da Expressão Gênica/genética , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos TransgênicosRESUMO
Nonalcoholic steatohepatitis (NASH) is a lifestyle-related disease characterized by hepatic fibrosis with the accumulation of fat and inflammation and can progress to cirrhosis or hepatocellular carcinoma. However, effective pharmacotherapeutic strategies for hepatic fibrosis in NASH remain to be established. Among the initiators of inflammation, we have been investigating the possible involvement of group IVA phospholipase A2 (IVA-PLA2), which catalyzes the initial step in the generation of lipid mediators, including eicosanoids and lysophospholipids, in the progression of hepatic fibrosis. We have recently demonstrated that a lack of IVA-PLA2 alleviates hepatic fibrosis in NASH model mice fed a high-fat and high-cholesterol diet and in CCl4-treated mice. CCl4-induced hepatic fibrosis was also prevented by the administration of an orally active, specific IVA-PLA2 inhibitor even after hepatic fibrosis had developed. Based on these findings suggesting that IVA-PLA2 mediates the cellular responses contributing to the progression of hepatic fibrosis, we have been exploring which types of cells in the liver are involved in IVA-PLA2-mediated hepatic fibrosis using cell-specific IVA-PLA2 knockout mice. The preliminary experimental results suggest that IVA-PLA2 in endothelial cells, but not monocyte-derived cells, plays a role, in part, in the hepatic stellate cell-mediated progression of hepatic fibrosis. In this paper, we discuss the possibility that IVA-PLA2 and/or its related molecules are candidate pharmacotherapeutic targets for NASH treatment.
Assuntos
Fosfolipases A2 do Grupo IV/antagonistas & inibidores , Indóis/uso terapêutico , Terapia de Alvo Molecular , Hepatopatia Gordurosa não Alcoólica/tratamento farmacológico , Hepatopatia Gordurosa não Alcoólica/etiologia , Propionatos/uso terapêutico , Animais , Modelos Animais de Doenças , Fosfolipases A2 do Grupo IV/fisiologia , Humanos , Indóis/administração & dosagem , Indóis/farmacologia , Camundongos , Camundongos Knockout , Hepatopatia Gordurosa não Alcoólica/enzimologia , Propionatos/administração & dosagem , Propionatos/farmacologiaRESUMO
BACKGROUND: Recently, we reported that angiotensin II (Ang II)-induced hypertension is mediated by group IV cytosolic phospholipase A2α (cPLA2α) via production of prohypertensive eicosanoids. Since Ang II increases blood pressure (BP) via its action in the subfornical organ (SFO), it led us to investigate the expression and possible contribution of cPLA2α to oxidative stress and development of hypertension in this brain area. METHODS: Adenovirus (Ad)-green fluorescence protein (GFP) cPLA2α short hairpin (sh) RNA (Ad-cPLA2α shRNA) and its control Ad-scrambled shRNA (Ad-Scr shRNA) or Ad-enhanced cyan fluorescence protein cPLA2α DNA (Ad-cPLA2α DNA) and its control Ad-GFP DNA were transduced into SFO of cPLA2α+/+ and cPLA2α-/- male mice, respectively. Ang II (700 ng/kg/min) was infused for 14 days in these mice, and BP was measured by tail-cuff and radio telemetry. cPLA2 activity, reactive oxygen species production, and endoplasmic reticulum stress were measured in the SFO. RESULTS: Transduction of SFO with Ad-cPLA2α shRNA, but not Ad-Scr shRNA in cPLA2α+/+ mice, minimized expression of cPLA2α, Ang II-induced cPLA2α activity and oxidative stress in the SFO, BP, and cardiac and renal fibrosis. In contrast, Ad-cPLA2α DNA, but not its control Ad-GFP DNA in cPLA2α-/- mice, restored the expression of cPLA2α, and Ang II-induced increase in cPLA2 activity and oxidative stress in the SFO, BP, cardiac, and renal fibrosis. CONCLUSIONS: These data suggest that cPLA2α in the SFO is crucial in mediating Ang II-induced hypertension and associated pathogenesis. Therefore, development of selective cPLA2α inhibitors could be useful in treating hypertension and its pathogenesis.
Assuntos
Angiotensina II/farmacologia , Encéfalo/enzimologia , Fosfolipases A2 do Grupo IV/fisiologia , Hipertensão/etiologia , Espécies Reativas de Oxigênio/metabolismo , Animais , Colágeno/metabolismo , Estresse do Retículo Endoplasmático , Fosfolipases A2 do Grupo IV/antagonistas & inibidores , Fosfolipases A2 do Grupo IV/genética , Hipertensão/tratamento farmacológico , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BLRESUMO
BACKGROUND: The aberrant expression of CD40, a co-stimulatory receptor found on the antigen-presenting cells, is involved in the pathogenesis of various degenerative diseases. Our previous study demonstrated that the reduction of cytosolic phospholipase A2 alpha (cPLA2α) protein overexpression and activation in the spinal cord of a mouse model of ALS, hmSOD1 G93A, inhibited CD40 upregulation in microglia. The present study was designed to determine whether cPLA2α has a direct, participatory role in the molecular events leading to CD40 induction. METHODS: Cultures of primary mouse microglia or BV-2 microglia cell line exposed to lipopolysaccharide (LPS) or interferon gamma (IFNγ) for different periods of time, in order to study the role of cPLA2α in the events leading to CD40 protein induction. RESULTS: Addition of LPS or IFNγ caused a significant upregulation of cPLA2α and of CD40, while prevention of cPLA2α upregulation by a specific oligonucleotide antisense (AS) prevented the induction of CD40, suggesting a role of cPLA2α in the induction of CD40. Addition of LPS to microglia caused an immediate activation of cPLA2α detected by its phosphorylated form, while addition of IFNγ induced cPLA2α activation at a later time scale (4 h). The activation of cPLA2α is mediated by ERK activity. Suppression of cPLA2α activity inhibited superoxide production by NOX2-NADPH oxidase and activation of NF-κB detected by the phosphorylation of p65 on serine 536 at 15 min by LPS and at 4 h by IFNγ. Inhibition of NOX2 prevented NF-κB activation and CD40 induction but did not affect cPLA2α activation, suggesting cPLA2α is located upstream to NOX2 and NF-κB. The activation of cPLA2 by LPS was mediated by both adaptor proteins downstream to LPS receptor; TRIF and MyD88, while the activation of cPLA2α by IFNγ was mediated by the secreted TNF-α at 4 h. The early activation of STAT1α (detected by phospho-serine727 and phoshpo-tyrosine701) by IFNγ and the late activation of STAT1α by LPS were not affected in the presence of cPLA2α inhibitors, indicating that STAT1α is not under cPLA2α regulation. CONCLUSIONS: Our results show for the first time that cPLA2 upregulates CD40 protein expression induced by either LPS or IFNγ, and this regulatory effect is mediated via the activation of NOX2-NADPH oxidase and NF-κB. Cumulatively, our results indicate that cPLA2α may serve as a pivotal amplifier of the inflammatory response in the CNS.
Assuntos
Antígenos CD40/biossíntese , Fosfolipases A2 do Grupo IV/fisiologia , Microglia/metabolismo , Animais , Animais Recém-Nascidos , Linhagem Celular Transformada , Células Cultivadas , Relação Dose-Resposta a Droga , Camundongos , Camundongos Endogâmicos C57BL , Microglia/efeitos dos fármacos , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
Recent findings on the existence of oncogenic fusion genes in a wide array of solid tumors, including head and neck squamous cell carcinoma (HNSCC), suggests that fusion genes have become attractive targets for cancer diagnosis and treatment. In this study, we showed for the first time that a read-through fusion gene JMJD7-PLA2G4B is presented in HNSCC, splicing neighboring jumonji domain containing 7 (JMJD7) and phospholipase A2, group IVB (PLA2G4B) genes together. Ablation of JMJD7-PLA2G4B significantly inhibited proliferation of HNSCC cells by promoting G1 cell cycle arrest and increased starvation-induced cell death compared to JMJD7-only knockdown HNSCC cells. Mechanistically, we found that JMJD7-PLA2G4B modulates phosphorylation of Protein Kinase B (AKT) to promote HNSCC cell survival. Moreover, JMJD7-PLA2G4B also regulated an E3 ligase S-phase kinase-associated protein 2 (SKP2) to control the cell cycle progression from G1 phase to S phase by inhibiting Cyclin-dependent kinase inhibitor 1 (p21) and 1B (p27) expression. Our study provides novel insights into the oncogenic control of JMJD7-PLA2G4B in HNSCC cell proliferation and survival, and suggests that JMJD7-PLA2G4B may serve as an important therapeutic target and prognostic marker for HNSCC development and progression.
Assuntos
Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patologia , Fosfolipases A2 do Grupo IV/genética , Neoplasias de Cabeça e Pescoço/genética , Neoplasias de Cabeça e Pescoço/patologia , Histona Desmetilases com o Domínio Jumonji/genética , Proteínas de Fusão Oncogênica/fisiologia , Biomarcadores Tumorais/genética , Carcinoma de Células Escamosas/diagnóstico , Linhagem Celular Tumoral , Proliferação de Células/genética , Sobrevivência Celular/genética , Fosfolipases A2 do Grupo IV/fisiologia , Células HCT116 , Células HEK293 , Células HT29 , Neoplasias de Cabeça e Pescoço/diagnóstico , Humanos , Histona Desmetilases com o Domínio Jumonji/fisiologia , Células MCF-7 , Proteína Oncogênica v-akt/metabolismo , Proteínas de Fusão Oncogênica/genética , Fosforilação , Carcinoma de Células Escamosas de Cabeça e PescoçoRESUMO
Calpain activation contributes to the development of infection-induced diaphragm weakness, but the mechanisms by which infections activate calpain are poorly understood. We postulated that skeletal muscle calcium-dependent phospholipase A2 (cPLA2) is activated by cytokines and has downstream effects that induce calpain activation and muscle weakness. We determined whether cPLA2 activation mediates cytokine-induced calpain activation in isolated skeletal muscle (C2C12) cells and infection-induced diaphragm weakness in mice. C2C12 cells were treated with the following: 1) vehicle; 2) cytomix (TNF-α 20 ng/ml, IL-1ß 50 U/ml, IFN-γ 100 U/ml, LPS 10 µg/ml); 3) cytomix + AACOCF3, a cPLA2 inhibitor (10 µM); or 4) AACOCF3 alone. At 24 h, we assessed cell cPLA2 activity, mitochondrial superoxide generation, calpain activity, and calpastatin activity. We also determined if SS31 (10 µg/ml), a mitochondrial superoxide scavenger, reduced cytomix-mediated calpain activation. Finally, we determined if CDIBA (10 µM), a cPLA2 inhibitor, reduced diaphragm dysfunction due to cecal ligation puncture in mice. Cytomix increased C2C12 cell cPLA2 activity (P < 0.001) and superoxide generation; AACOCF3 and SS31 blocked increases in superoxide generation (P < 0.001). Cytomix also activated calpain (P < 0.001) and inactivated calpastatin (P < 0.01); both AACOCF3 and SS31 prevented these changes. Cecal ligation puncture reduced diaphragm force in mice, and CDIBA prevented this reduction (P < 0.001). cPLA2 modulates cytokine-induced calpain activation in cells and infection-induced diaphragm weakness in animals. We speculate that therapies that inhibit cPLA2 may prevent diaphragm weakness in infected, critically ill patients.
Assuntos
Diafragma/fisiopatologia , Fosfolipases A2 do Grupo IV/fisiologia , Sepse/enzimologia , Sepse/fisiopatologia , Animais , Linhagem Celular , Diafragma/enzimologia , Diafragma/microbiologia , Camundongos , Debilidade Muscular , Superóxidos/metabolismoRESUMO
Previous studies have demonstrated that membrane tubule-mediated transport events in biosynthetic and endocytic routes require phospholipase A2 (PLA2) activity. Here, we show that cytosolic phospholipase A2ε (cPLA2ε, also known as PLA2G4E) is targeted to the membrane compartments of the clathrin-independent endocytic route through a C-terminal stretch of positively charged amino acids, which allows the enzyme to interact with phosphoinositide lipids [especially PI(4,5)P2] that are enriched in clathrin-independent endosomes. Ablation of cPLA2ε suppressed the formation of tubular elements that carry internalized clathrin-independent cargoes, such as MHC-I, CD147 and CD55, back to the cell surface and, therefore, caused their intracellular retention. The ability of cPLA2ε to support recycling through tubule formation relies on the catalytic activity of the enzyme, because the inactive cPLA2ε(S420A) mutant was not able to recover either tubule growth or transport from clathrin-independent endosomes. Taken together, our findings indicate that cPLA2ε is a new important regulator of trafficking processes within the clathrin-independent endocytic and recycling route. The affinity of cPLA2ε for this pathway supports a new hypothesis that different PLA2 enzymes use selective targeting mechanisms to regulate tubule formation locally during specific trafficking steps in the secretory and/or endocytic systems.
Assuntos
Clatrina/metabolismo , Endocitose , Fosfolipases A2 do Grupo IV/fisiologia , Sequência de Aminoácidos , Sinalização do Cálcio , Endossomos/metabolismo , Fosfolipases A2 do Grupo IV/química , Células HeLa , Humanos , Hidrólise , Dados de Sequência Molecular , Fosfatidilinositóis/metabolismo , Sinais Direcionadores de Proteínas , Transporte ProteicoRESUMO
Increased cytosolic phospholipase A2α (cPLA2α) immunoreactivity and transcript were observed in Alzheimer's disease (AD) brain associated with amyloid deposits. Thus, the present study examined whether cPLA2α upregulation participate in cortical neuron damage induced by aggregated Aß1-42 and determined its role in the signaling events leading to damage, using an antisense technology. Exposure of primary cortical neurons to 1µM aggregated Aß1-42 for 24h induced up-regulation and activation of cPLA2α and apoptotic cell death of about 30% as detected by: cell count, MTT reduction, caspases-3 and -8 activation, DAPI and TUNEL staining, that were prevented by inhibition of cPLA2α up-regulation and activity in the presence of antisense against cPLA2α (AS). cPLA2α was rapidly activated upon addition of aggregated Aß1-42, as determined by its phosphorylated form on serine 505, and this activity was dependent on NADPH oxidase activity. NOX2- and NOX4-NADPH oxidase upregulation at 24h of aggregated Aß1-42 exposure was not affected by the presence of AS, but superoxide production was reduced, probably due to NOX2 inhibition. cPLA2α upregulation led to activation of neutral sphingomyelinase (N-SMase) as its activity was inhibited in the presence of AS, and could be restored by addition of arachidonic acid. Addition of ceramide analog induced caspase-8 activation leading to caspase-3 activation and apoptotic neuronal death. In conclusion, our results suggest that cPLA2α activity plays a crucial role in the signaling cascade leading to apoptotic neuronal death by aggregated Aß1-42 probably via activation of N-SMase, ceramide production and caspases-3 and -8.
Assuntos
Peptídeos beta-Amiloides/toxicidade , Apoptose/genética , Apoptose/fisiologia , Citosol/enzimologia , Fosfolipases A2 do Grupo IV/genética , Fosfolipases A2 do Grupo IV/fisiologia , Neurônios/fisiologia , Fragmentos de Peptídeos/toxicidade , Animais , Caspases/metabolismo , Contagem de Células , Córtex Cerebral/citologia , DNA Antissenso , Ativação Enzimática/genética , Feminino , Gravidez , Ratos , Ratos Sprague-Dawley , Superóxidos/metabolismo , Regulação para Cima/fisiologiaRESUMO
The role of Group IVA cytosolic phospholipase A2 (cPLA2α) activation in regulating macrophage transcriptional responses to Candida albicans infection was investigated. cPLA2α releases arachidonic acid for the production of eicosanoids. In mouse resident peritoneal macrophages, prostacyclin, prostaglandin E2 and leukotriene C4 were produced within minutes of C. albicans addition before cyclooxygenase 2 expression. The production of TNFα was lower in C. albicans-stimulated cPLA2α(+/+) than cPLA2α(-/-) macrophages due to an autocrine effect of prostaglandins that increased cAMP to a greater extent in cPLA2α(+/+) than cPLA2α(-/-) macrophages. For global insight, differential gene expression in C. albicans-stimulated cPLA2α(+/+) and cPLA2α(-/-) macrophages (3 h) was compared by microarray. cPLA2α(+/+) macrophages expressed 86 genes at lower levels and 181 genes at higher levels than cPLA2α(-/-) macrophages (≥2-fold, p<0.05). Several pro-inflammatory genes were expressed at lower levels (Tnfα, Cx3cl1, Cd40, Ccl5, Csf1, Edn1, CxCr7, Irf1, Irf4, Akna, Ifnγ, several IFNγ-inducible GTPases). Genes that dampen inflammation (Socs3, Il10, Crem, Stat3, Thbd, Thbs1, Abca1) and genes involved in host defense (Gja1, Csf3, Trem1, Hdc) were expressed at higher levels in cPLA2α(+/+) macrophages. Representative genes expressed lower in cPLA2α(+/+) macrophages (Tnfα, Csf1) were increased by treatment with a prostacyclin receptor antagonist and protein kinase A inhibitor, whereas genes expressed at higher levels (Crem, Nr4a2, Il10, Csf3) were suppressed. The results suggest that C. albicans stimulates an autocrine loop in macrophages involving cPLA2α, cyclooxygenase 1-derived prostaglandins and increased cAMP that globally effects expression of genes involved in host defense and inflammation.
Assuntos
Candida albicans/imunologia , Eicosanoides/fisiologia , Regulação da Expressão Gênica/imunologia , Fosfolipases A2 do Grupo IV/fisiologia , Macrófagos Peritoneais/metabolismo , Animais , AMP Cíclico/metabolismo , Ciclo-Oxigenase 2/genética , Ciclo-Oxigenase 2/metabolismo , Dinoprostona/biossíntese , Epoprostenol/biossíntese , Imunidade Celular , Leucotrieno C4/biossíntese , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Fator de Necrose Tumoral alfa/biossínteseRESUMO
In mammalian cells, cermide-1-phosphate (C1P) is produced via the ATP-dependent mechanism of converting ceramide to C1P by the enzyme, ceramide kinase (CERK). CERK was first described as a calcium-stimulated lipid kinase that co-purified with brain synaptic vesicles, and to date, CERK is the only identified mammalian enzyme known to produce C1P in cells. C1P has steadily emerged as a bioactive sphingolipid involved in cell proliferation, macrophage migration, and inflammatory events. The recent generation of the CERK knockout mouse and the development of CERK inhibitors have furthered our current understanding of CERK-derived C1P in regulating biological processes. In this chapter, the history of C1P as well as the biological functions attributed to C1P are reviewed.
Assuntos
Ceramidas/fisiologia , Animais , Proliferação de Células , Sobrevivência Celular , Fosfolipases A2 do Grupo IV/fisiologia , Humanos , Fosfotransferases (Aceptor do Grupo Álcool)/fisiologiaRESUMO
Group IVA cytosolic phospholipase A(2) (cPLA(2)α), which preferentially cleaves arachidonic acid from phospholipids, plays a role in apoptosis and tissue injury. Downstream signals in response to tumor necrosis factor (TNF)-α, a mediator of myocardial ischemia-reperfusion (I/R) injury, involve cPLA(2)α activation. This study examined the potential role of cPLA(2)α and its mechanistic link with TNF-α in myocardial I/R injury using cPLA(2)α knockout (cPLA(2)α(-/-)) mice. Myocardial I/R was created with 10-wk-old male mice by 1 h ligation of the left anterior descending coronary artery, followed by 24 h of reperfusion. As a result, compared with wild-type (cPLA(2)α(+/+)) mice, cPLA(2)α(-/-) mice had a 47% decrease in myocardial infarct size, preservation of echocardiographic left ventricle (LV) function (%fractional shortening: 14 vs. 21%, respectively), and lower content of leukotriene B(4) and thromboxane B(2) (62 and 50% lower, respectively) in the ischemic myocardium after I/R. Treatment with the TNF-α inhibitor (soluble TNF receptor II/IgG1 Fc fusion protein, sTNFR:Fc) decreased myocardial I/R injury and LV dysfunction in cPLA(2)α(+/+) mice but not cPLA(2)α(-/-) mice. sTNFR:Fc also suppressed cPLA(2)α phosphorylation in the ischemic myocardium after I/R of cPLA(2)α(+/+) mice. Similarly, sTNFR:Fc exerted protective effects against hypoxia-reoxygenation (H/R)-induced injury in the cultured cardiomyocytes from cPLA(2)α(+/+) mice but not cPLA(2)α(-/-) cardiomyocytes. H/R and TNF-α induced cPLA(2)α phosphorylation in cPLA(2)α(+/+) cardiomyocytes, which was reversible by sTNFR:Fc. In cPLA(2)α(-/-) cardiomyocytes, TNF-α induced apoptosis and release of arachidonic acid to a lesser extent than in cPLA(2)α(+/+) cardiomyocytes. In conclusion, disruption of cPLA(2)α attenuates myocardial I/R injury partly through inhibition of TNF-α-mediated pathways.
Assuntos
Fosfolipases A2 do Grupo IV/deficiência , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Transdução de Sinais/fisiologia , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Fator de Necrose Tumoral alfa/fisiologia , Animais , Células Cultivadas , Etanercepte , Fosfolipases A2 do Grupo IV/genética , Fosfolipases A2 do Grupo IV/fisiologia , Imunoglobulina G/farmacologia , Leucotrieno B4/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Animais , Infarto do Miocárdio/patologia , Infarto do Miocárdio/prevenção & controle , Traumatismo por Reperfusão Miocárdica/patologia , Traumatismo por Reperfusão Miocárdica/fisiopatologia , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Receptores do Fator de Necrose Tumoral , Tromboxano B2/metabolismo , Disfunção Ventricular Esquerda/fisiopatologia , Disfunção Ventricular Esquerda/prevenção & controleRESUMO
The contractile and relaxation characteristics of trabecular meshwork (TM) are presumed to influence aqueous humor (AH) drainage and intraocular pressure. The mechanisms underlying regulation of TM cell contractile properties, however, are not well understood. This study investigates the role of calcium-independent phospholipase A(2) (iPLA(2)), which controls eicosanoid synthesis, in regulation of TM cell contraction and AH outflow using mechanism-based isoform specific inhibitors (R)-bromoenol lactone (R-BEL, iPLA(2)γ specific) and (S)-bromoenol lactone (S-BEL, iPLA(2)ß specific). Immunohistochemical analysis revealed intense staining for both iPLA(2)ß and γ isoforms throughout the TM, juxtacanalicular tissue, and Schlemm's canal of human eye. Inhibition of iPLA(2)γ by R-BEL or small interfering RNA-mediated silencing of iPLA(2)γ expression induced dramatic changes in TM cell morphology, and decreased actin stress fibers, focal adhesions, and myosin light-chain (MLC) phosphorylation. AH outflow facility increased progressively and significantly in enucleated porcine eyes perfused with R-BEL. This response was associated with a significant decrease in TM tissue MLC phosphorylation and alterations in the morphology of aqueous plexi in R-BEL-perfused eyes. In contrast, S-BEL did not affect either of these parameters. Additionally, R-BEL-induced cellular relaxation of the TM was associated with a significant decrease in the levels of active Rho GTPase, phospho-MLC phosphatase, phospho-CPI-17, and arachidonic acid. Taken together, these observations demonstrate that iPLA(2)γ plays a significant and isoform-specific role in regulation of AH outflow facility by altering the contractile characteristics of the TM. The effects of iPLA(2)γ on TM contractile status appear to involve arachidonic acid and Rho GTPase signaling pathways.
Assuntos
Humor Aquoso/fisiologia , Cálcio/metabolismo , Fosfolipases A2 do Grupo IV/fisiologia , Fosfolipases A2 do Grupo VI/genética , Fosfolipases A2 do Grupo VI/metabolismo , Malha Trabecular/fisiologia , Actinas/genética , Actinas/metabolismo , Actomiosina/genética , Actomiosina/metabolismo , Animais , Humor Aquoso/efeitos dos fármacos , Humor Aquoso/enzimologia , Ácido Araquidônico/genética , Ácido Araquidônico/metabolismo , Células Cultivadas , Eicosanoides/genética , Eicosanoides/metabolismo , Adesões Focais/efeitos dos fármacos , Adesões Focais/genética , Adesões Focais/metabolismo , Inativação Gênica/efeitos dos fármacos , Inativação Gênica/fisiologia , Fosfolipases A2 do Grupo IV/genética , Fosfolipases A2 do Grupo IV/metabolismo , Humanos , Imuno-Histoquímica/métodos , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas Musculares , Cadeias Leves de Miosina/genética , Cadeias Leves de Miosina/metabolismo , Naftalenos/farmacologia , Fosfoproteínas Fosfatases/genética , Fosfoproteínas Fosfatases/metabolismo , Monoéster Fosfórico Hidrolases/genética , Monoéster Fosfórico Hidrolases/metabolismo , Fosforilação/efeitos dos fármacos , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Pironas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Fibras de Estresse/efeitos dos fármacos , Fibras de Estresse/genética , Fibras de Estresse/metabolismo , Suínos , Malha Trabecular/efeitos dos fármacos , Malha Trabecular/enzimologia , Proteínas rho de Ligação ao GTP/genética , Proteínas rho de Ligação ao GTP/metabolismoRESUMO
Type 2 diabetes mellitus (T2DM) is the most common human endocrine disease and is characterized by peripheral insulin resistance and pancreatic islet ß-cell failure. Accumulating evidence indicates that mitochondrial dysfunction is a central contributor to ß-cell failure in the evolution of T2DM. As reviewed elsewhere, reactive oxygen species (ROS) produced by ß-cell mitochondria as a result of metabolic stress activate several stress-response pathways. This paper focuses on mechanisms whereby ROS affect mitochondrial structure and function and lead to ß-cell failure. ROS activate UCP2, which results in proton leak across the mitochondrial inner membrane, and this leads to reduced ß-cell ATP synthesis and content, which is a critical parameter in regulating glucose-stimulated insulin secretion. In addition, ROS oxidize polyunsaturated fatty acids in mitochondrial cardiolipin and other phospholipids, and this impairs membrane integrity and leads to cytochrome c release into cytosol and apoptosis. Group VIA phospholipase A2 (iPLA2ß) appears to be a component of a mechanism for repairing mitochondrial phospholipids that contain oxidized fatty acid substituents, and genetic or acquired iPLA2ß-deficiency increases ß-cell mitochondrial susceptibility to injury from ROS and predisposes to developing T2DM. Interventions that attenuate ROS effects on ß-cell mitochondrial phospholipids might prevent or retard development of T2DM.
Assuntos
Diabetes Mellitus Tipo 2/fisiopatologia , Células Secretoras de Insulina/fisiologia , Mitocôndrias/fisiologia , Trifosfato de Adenosina/biossíntese , Apoptose , Ácidos Graxos Insaturados/química , Fosfolipases A2 do Grupo IV/deficiência , Fosfolipases A2 do Grupo IV/fisiologia , Humanos , Resistência à Insulina , Canais Iônicos/fisiologia , Peroxidação de Lipídeos , Membranas Mitocondriais/fisiologia , Proteínas Mitocondriais/fisiologia , Oxirredução , Fosfolipídeos/química , Espécies Reativas de Oxigênio/metabolismo , Proteína Desacopladora 2RESUMO
In the present paper, we report that PLA2G4A (Group IVA phospholipase A2) is important in the development and function of rodent testes. Interstitial cells of rat testes had high PLA2 (phospholipase A2) activity that was very sensitive to the PLA2G4A-preferential inhibitor AACOCF3 (arachidonyl trifluoromethyl ketone). PLA2G4A protein was expressed primarily in the interstitial cells of wild-type mouse testes throughout maturation. Although Pla2g4a knockout (Pla2g4a-/-) male mice are fertile, their sexual maturation was delayed, as indicated by cauda epididymal sperm count and seminal vesicle development. Delayed function of Pla2g4a-/- mice testes was associated with histological abnormalities including disorganized architecture, swollen appearance and fewer interstitial cells. Basal secretion of testosterone was attenuated significantly and steroidogenic response to hCG (human chorionic gonadotropin) treatment was reduced in Pla2g4a-/- mice compared with their Pla2g4a+/+ littermates during the sexual maturation period. Chemical inhibition of PLA2G4A activity by AACOCF3 or pyrrophenone significantly reduced hCG-stimulated testosterone production in cultured rat interstitial cells. AACOCF3 inhibited forskolin- and cAMP analogue-stimulated testosterone production. These results provide the first evidence that PLA2G4A plays a role in male testes physiology and development. These results may have implications for the potential clinical use of PLA2G4A inhibitors.
Assuntos
Fosfolipases A2 do Grupo IV/fisiologia , Células Intersticiais do Testículo/metabolismo , Maturidade Sexual/fisiologia , Testosterona/biossíntese , Fatores Etários , Animais , Células Cultivadas , Fosfolipases A2 do Grupo IB/antagonistas & inibidores , Fosfolipases A2 do Grupo IB/deficiência , Fosfolipases A2 do Grupo IB/fisiologia , Fosfolipases A2 do Grupo IV/antagonistas & inibidores , Humanos , Células Intersticiais do Testículo/enzimologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Ratos , Testosterona/químicaRESUMO
The radioresistant nature of some tumors serves as an obstacle to curative therapy for several poor-prognosis malignancies. The radiosensitivity of a cancer is dependent not only on the intrinsic ability of tumor cells to recover from radiation-induced damage, but also the ability of stromal elements (e.g., vasculature) in the tumor microenvironment to survive and continue proliferating in the face of ionizing radiation. In this regard, it is important to understand the initial events activating radiation-induced signal transduction pathways. Among these events is the activation of cytosolic phospholipase A2 α and the subsequent production of the lipid second messengers. These events occur within minutes following exposure to ionizing radiation, and have been shown to enhance cell viability through a number of prosurvival signaling pathways. Furthermore, inhibition of cytosolic phospholipase A2 α has now been shown to reduce the viability of endothelial cells in culture after exposure to ionizing radiation, as well as slowing the growth of tumors in animal models of cancer.
Assuntos
Fosfolipases A2 do Grupo IV/fisiologia , Neoplasias/radioterapia , Radiossensibilizantes/toxicidade , Terapia Combinada , Fosfolipases A2 do Grupo IV/metabolismo , Humanos , Diester Fosfórico Hidrolases/metabolismo , Radiação IonizanteRESUMO
The regulated expression of ICAM-1 plays an important role in inflammatory processes and immune responses. The present study aimed to determine the in vivo involvement of cytosolic phospholipase A(2)α (cPLA(2)α) in ICAM-1 overexpression during inflammation and to elucidate the cPLA(2)α-specific role in signal events leading to ICAM-1 upregulation in endothelial cells. cPLA(2)α and ICAM-1 upregulation were detected in inflamed paws of mice with collagen-induced arthritis and in periepididymal adipose tissue of mice fed a high-fat diet. Intravenous injection of 2 mg/kg oligonucleotide antisense against cPLA(2)α (AS) that reduced cPLA(2)α upregulation also decreased ICAM-1 overexpression, suggesting a key role of cPLA(2)α in ICAM-1 upregulation during inflammation. Preincubation of endothelial ECV-304 cells that express ICAM-1 and of HUVEC that express ICAM-1 and VCAM-1 with 1 µM AS prevented cPLA(2)α and the adhesion molecule upregulation induced by TNF-α and inhibited their adherence to phagocyte like-PLB cells. Whereas AS did not inhibit NADPH oxidase 4-NADPH oxidase activity, inhibition of oxidase activity attenuated cPLA(2)α activation, suggesting that NADPH oxidase acts upstream to cPLA(2)α. Attenuating cPLA(2)α activation by AS or diphenylene iodonium prevented the induction of cyclooxygenase-2 and the production of PGE(2) that were essential for ICAM-1 upregulation. Inhibition of cPLA(2)α activity by AS inhibited the phosphorylation of both p65 NF-κB on Ser(536) and protein kinase A-dependent CREB. To our knowledge, our results are the first to show that CREB activation is involved in ICAM-1 upregulation and suggest that cPLA(2)α activated by NADPH oxidase is required for sequential phosphorylation of NF-κB by an undefined kinase and CREB activation by PGE(2)-mediated protein kinase A.
Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/fisiologia , Endotélio Vascular/imunologia , Endotélio Vascular/metabolismo , Fosfolipases A2 do Grupo IV/fisiologia , Molécula 1 de Adesão Intercelular/biossíntese , NF-kappa B/fisiologia , Tecido Adiposo/enzimologia , Tecido Adiposo/metabolismo , Tecido Adiposo/patologia , Animais , Artrite Experimental/enzimologia , Artrite Experimental/metabolismo , Artrite Experimental/patologia , Células Cultivadas , Citosol/enzimologia , Citosol/imunologia , Citosol/metabolismo , Gorduras na Dieta/administração & dosagem , Endotélio Vascular/enzimologia , Humanos , Mediadores da Inflamação/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Obesidade/enzimologia , Obesidade/metabolismo , Obesidade/patologia , Transdução de Sinais/imunologia , Transcrição Gênica/imunologia , Regulação para Cima/imunologiaRESUMO
OBJECTIVES: To determine the effect of phospholipase A(2) (PLA(2)) inhibitors on urothelial cell platelet-activating factor (PAF) production in response to tryptase stimulation. METHODS: Urothelial cells isolated from normal human ureters were immortalized with the human papillomavirus type 16E6E7 cell line (TEU-2 cells). PLA(2) activity in TEU-2 cells was measured using (16:0, [(3)H]18:1) plasmenylcholine and phosphatidylcholine substrates in the presence and absence of calcium. [(3)H]PAF production was measured in TEU-2 cells prelabeled with [(3)H] acetic acid. PAF-acetylhydrolase activity was measured by determining the amount of [(3)H] acetate hydrolyzed from [(3)H]PAF incubated with TEU-2 cellular protein. Adherence of human polymorphonuclear leukocyte (PMN) to TEU-2 cells was assessed by measuring myeloperoxidase activity in adherent PMNs after incubation with TEU-2 cells. RESULTS: Most PLA(2) activity measured in TEU-2 cells was determined to be membrane-associated, calcium-independent PLA(2) and selective for plasmenylcholine substrate. Stimulation of TEU-2 cells with tryptase results in increased production of PAF and increased PMN adherence that were inhibited completely by pretreatment with the membrane-associated, calcium-independent PLA(2)γ-selective inhibitor (R)-bromoenol lactone. Pretreatment with the cytosolic PLA(2) inhibitor methyl arachidonyl fluorophosphonate resulted in potentiation of tryptase-stimulated PAF production and PMN adherence to TEU-2 cells that is a result of PAF-acetylhydrolase inhibition. CONCLUSIONS: Tryptase stimulation of TEU-2 cells results in activation of membrane-associated, calcium-independent PLA(2)γ, leading to an increase in PAF production and increased PMN adherence. Inhibition of TEU-2 cell PAF-acetylhydrolase activity with methyl arachidonyl fluorophosphonate potentiated tryptase-stimulated PAF production and PMN adherence.
Assuntos
Fosfolipases A2 do Grupo IV/fisiologia , Fator de Ativação de Plaquetas/biossíntese , Ureter/metabolismo , Urotélio/metabolismo , Cálcio/fisiologia , Células Cultivadas , Humanos , Ureter/citologia , Urotélio/citologiaRESUMO
BACKGROUND: Prostate cancer (PCa) is one of the most common malignancies among men in the United States. Further understanding of the molecular mechanisms underlying PCa tumorigenic development is critical for advancing treatment strategies for PCa. The role of Group VIA phospholipase A2ß (iPLA2ß) in cancers has recently emerged. However, the biological functions of iPLA2ß in PCa development have been minimally investigated and only in vitro studies have been reported. METHODS: We tested the role of iPLA2ß in host cells using an iPLA2ß deficient mouse model and the role of iPLA2ß in tumor cells by comparing the proliferation, migration, and invasion in vitro and tumorigenesis in vivo. CONCLUSIONS: iPLA2ß deficiency did not affect tumor development in C57BL/6 mice injected with syngeneic PCa cell line TRAMP-C1P3 in any of three models (subcutaneous, orthotopic, or intratibia injection) tested, suggesting that host cell iPLA2ß is not required for PCa tumorigenesis and metastasis. In contrast, when iPLA2ß was down-regulated in TRAMP-C1P3 cells, cell proliferation was reduced in vitro and tumor growth was suppressed in vivo compared to control cells. In particular, iPLA2ß was required for lysophosphatidic acid (LPA)-induced migration and invasion in TRAMP-C1P3 cells. We compared human and mouse PCa cells and showed that they shared high similarities in LPA-stimulated effects and signaling pathways. LPA stimulated cell migration and/or invasion via a PI3K-dependent pathway. Together, our results suggest that the tumor cell iPLA2ß-LPA axis may represent a novel target for PCa.