RESUMEN
Lung endothelial dysfunction is a key feature of acute lung injury (ALI) and clinical acute respiratory distress syndrome (ARDS). Previous studies have identified the lipid-generating enzyme, group V phospholipase A2 (gVPLA2), as a mediator of lung endothelial barrier disruption and inflammation. The current study aimed to determine the role of gVPLA2 in mediating lung endothelial responses to methicillin-resistant Staphylococcus aureus (MRSA, USA300 strain), a major cause of ALI/ARDS. In vitro studies assessed the effects of gVPLA2 inhibition on lung endothelial cell (EC) permeability after exposure to heat-killed (HK) MRSA. In vivo studies assessed the effects of intratracheal live or HK-MRSA on multiple indices of ALI in wild-type (WT) and gVPLA2-deficient (KO) mice. In vitro, HK-MRSA increased gVPLA2 expression and permeability in human lung EC. Inhibition of gVPLA2 with either the PLA2 inhibitor, LY311727, or with a specific monoclonal antibody, attenuated the barrier disruption caused by HK-MRSA. LY311727 also reduced HK-MRSA-induced permeability in mouse lung EC isolated from WT but not gVPLA2-KO mice. In vivo, live MRSA caused significantly less ALI in gVPLA2 KO mice compared to WT, findings confirmed by intravital microscopy assessment in HK-MRSA-treated mice. After targeted delivery of gVPLA2 plasmid to lung endothelium using ACE antibody-conjugated liposomes, MRSA-induced ALI was significantly increased in gVPLA2-KO mice, indicating that lung endothelial expression of gVPLA2 is critical in vivo. In summary, these results demonstrate an important role for gVPLA2 in mediating MRSA-induced lung EC permeability and ALI. Thus, gVPLA2 may represent a novel therapeutic target in ALI/ARDS caused by bacterial infection.
Asunto(s)
Lesión Pulmonar Aguda/enzimología , Lesión Pulmonar Aguda/microbiología , Células Endoteliales/microbiología , Células Endoteliales/patología , Staphylococcus aureus Resistente a Meticilina/fisiología , Fosfolipasas A2/metabolismo , Lesión Pulmonar Aguda/patología , Animales , Permeabilidad de la Membrana Celular/efectos de los fármacos , Células Endoteliales/efectos de los fármacos , Indoles/farmacología , Pulmón/diagnóstico por imagen , Pulmón/microbiología , Pulmón/patología , Staphylococcus aureus Resistente a Meticilina/efectos de los fármacos , Ratones Noqueados , Modelos Biológicos , Fosfolipasas A2/deficienciaRESUMEN
AIMS: Phospholipase A2 (PLA2 ) is a diverse superfamily that hydrolyzes fatty acyl ester bonds at the sn-2 position of phospholipids. The correlation between phospholipid metabolism and the anabolism of neutral lipids remains unclear in yeasts. This study aims to explore the effects of PLA2 on lipid accumulation in the oleaginous yeast Yarrowia lipolytica. METHODS AND RESULTS: This study identified an actively expressed phospholipase A2 gene (PLA2-3, YAIL0_E16060g) in Y. lipolytica by quantitative PCR analysis. The gene PLA2-3 was disrupted in the strain po1gΔKu70 by homologous recombination and in the strain po1g-G3 by a CRISPR-Cas9 system, which caused an increase in stress sensitivity while the cell growth was not altered under fermentative conditions. Lipid production was performed in both flasks and bioreactors. The results showed that the lipid titre and lipid content were improved over 25% and 8-30%, respectively, in PLA2-3 disrupted strains compared to the controls. CONCLUSIONS: Disruption of the phospholipase PLA2-3 gene could effectively improve lipid production in Y. lipolytica. SIGNIFICANCE AND IMPACT OF THE STUDY: This study presented a strategy on improving the lipid production of oleaginous yeasts and a similar strategy might be used in other oleaginous microbes.
Asunto(s)
Proteínas Fúngicas/genética , Metabolismo de los Lípidos , Fosfolipasas A2/genética , Yarrowia/metabolismo , Biocombustibles/microbiología , Reactores Biológicos , Fermentación , Metabolismo de los Lípidos/genética , Lípidos/biosíntesis , Ingeniería Metabólica , Mutación , Fosfolipasas A2/deficiencia , Yarrowia/enzimología , Yarrowia/genéticaRESUMEN
To examine the role of group VIA phospholipase A2 (iPLA2ß) in specific cell lineages in insulin secretion and insulin action, we prepared mice with a selective iPLA2ß deficiency in cells of myelomonocytic lineage, including macrophages (MØ-iPLA2ß-KO), or in insulin-secreting ß-cells (ß-Cell-iPLA2ß-KO), respectively. MØ-iPLA2ß-KO mice exhibited normal glucose tolerance when fed standard chow and better glucose tolerance than floxed-iPLA2ß control mice after consuming a high-fat diet (HFD). MØ-iPLA2ß-KO mice exhibited normal glucose-stimulated insulin secretion (GSIS) in vivo and from isolated islets ex vivo compared to controls. Male MØ-iPLA2ß-KO mice exhibited enhanced insulin responsivity vs. controls after a prolonged HFD. In contrast, ß-cell-iPLA2ß-KO mice exhibited impaired glucose tolerance when fed standard chow, and glucose tolerance deteriorated further when introduced to a HFD. ß-Cell-iPLA2ß-KO mice exhibited impaired GSIS in vivo and from isolated islets ex vivo vs. controls. ß-Cell-iPLA2ß-KO mice also exhibited an enhanced insulin responsivity compared to controls. These findings suggest that MØ iPLA2ß participates in HFD-induced deterioration in glucose tolerance and that this mainly reflects an effect on insulin responsivity rather than on insulin secretion. In contrast, ß-cell iPLA2ß plays a role in GSIS and also appears to confer some protection against deterioration in ß-cell functions induced by a HFD.
Asunto(s)
Fosfolipasas A2 Grupo VI/genética , Células Secretoras de Insulina/metabolismo , Fosfolipasas A2/genética , Animales , Glucemia/genética , Dieta Alta en Grasa/efectos adversos , Glucosa/genética , Intolerancia a la Glucosa/tratamiento farmacológico , Intolerancia a la Glucosa/genética , Prueba de Tolerancia a la Glucosa , Humanos , Insulina/genética , Insulina/metabolismo , Secreción de Insulina/genética , Islotes Pancreáticos/metabolismo , Islotes Pancreáticos/patología , Macrófagos/efectos de los fármacos , Ratones , Ratones Noqueados , Fosfolipasas A2/deficienciaRESUMEN
Biofuel derived from microalgae have several advantages over other oleaginous crops, however, still needs to be improved with its cost aspect and can be achieved by developing of a strain with improved lipid productivity. In this study, the CRISPR-Cas9 system was incorporated to carry out a target-specific knockout of the phospholipase A2 gene in Chlamydomonas reinhardtii. The targeted gene encodes a key enzyme in the Lands cycle. As a result, the mutants showed a characteristic of increased diacylglycerol pool, followed by a higher accumulation of triacylglycerol without being significantly compensated with the cell growth. As a result, the overall lipid productivities of phospholipase A2 knockout mutants have increased by up to 64.25% (to 80.92â¯gâ¯L-1â¯d-1). This study can provide crucial information for the biodiesel industry.
Asunto(s)
Biocombustibles/microbiología , Chlamydomonas reinhardtii/metabolismo , Lípidos/biosíntesis , Fosfolipasas A2/metabolismo , Chlamydomonas reinhardtii/genética , Fosfolipasas A2/deficiencia , Triglicéridos/biosíntesisRESUMEN
Lipid mediators play pivotal roles in colorectal cancer and colitis, but only a limited member of the phospholipase A2 (PLA2) subtypes, which lie upstream of various lipid mediators, have been implicated in the positive or negative regulation of these diseases. Clinical and biochemical evidence suggests that secreted PLA2 group III (sPLA2-III) is associated with colorectal cancer, although its precise role remains obscure. Here we have found that sPLA2-III-null (Pla2g3 -/-) mice are highly resistant to colon carcinogenesis. Furthermore, Pla2g3 -/- mice are less susceptible to dextran sulfate-induced colitis, implying that the amelioration of colonic inflammation by sPLA2-III ablation may underlie the protective effect against colon cancer. Lipidomics analysis of the colon revealed significant reduction of pro-inflammatory/pro-tumorigenic lysophosholipids as well as unusual steady-state elevation of colon-protective fatty acids and their oxygenated metabolites in Pla2g3 -/- mice. Overall, our results establish a role of sPLA2-III in the promotion of colorectal inflammation and cancer, expand our understanding of the divergent roles of multiple PLA2 enzymes in the gastrointestinal tract, and point to sPLA2-III as a novel druggable target for colorectal diseases.
Asunto(s)
Colitis/fisiopatología , Neoplasias Colorrectales/fisiopatología , Susceptibilidad a Enfermedades , Fosfolipasas A2/metabolismo , Animales , Colitis/patología , Colon/patología , Neoplasias Colorrectales/patología , Ratones , Ratones Noqueados , Fosfolipasas A2/deficienciaRESUMEN
PURPOSE: The goal of present study was to elucidate the pathophysiological roles of lysosomal phospholipase A2 (LPLA2) in intraocular pressure (IOP) levels and ocular inflammation. METHODS: C57BL/6 (wild-type) and LPLA2-deficient mice with C57BL/6 background were employed. The IOPs were compared between wild-type and LPLA2-deficient mice during their aging, after topical administration of antiglaucoma medications such as travoprost, dorzolamide, or timolol maleate, or after induction of endotoxin-induced uveitis (EIU) using lipopolysaccharide (LPS). Concerning the EIU, ocular inflammation was also evaluated by immunohistochemical analysis by the anti-glial fibrillary acidic protein (GFAP) antibody. RESULTS: The LPLA2-deficient mice showed higher IOP levels than the wild-type mice until 2 months of age (P = 1.60E-06); in older mice there was no difference between the two groups. Significant differences in the IOP changes between groups in young mice were seen after administration of 0.5% timolol (P < 0.05). Upon induction of EIU by LPS, compared with wild-type mice (P < 0.05), IOPs were significantly elevated in LPLA2-deficient mice at maximum levels of the ocular inflammation (48 h). Immunohistochemical analysis indicated that LPLA2-deficient mice showed more prolonged expression of GFAP at the inner plexiform layer and inner nuclear layer by EIU than that found in the wild-type mice (P < 0.05). CONCLUSIONS: These results confirm that LPLA2 plays a significant role in the control of IOP during mouse ocular development or with ocular inflammation by facilitating the digestion of intraocular insoluble materials.
Asunto(s)
Modelos Animales de Enfermedad , Presión Intraocular/fisiología , Lisosomas/enzimología , Fosfolipasas A2/fisiología , Uveítis/enzimología , Animales , Antihipertensivos/farmacología , Ensayo de Inmunoadsorción Enzimática , Proteína Ácida Fibrilar de la Glía/metabolismo , Inflamación/inducido químicamente , Inflamación/enzimología , Presión Intraocular/efectos de los fármacos , Lipopolisacáridos/toxicidad , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Soluciones Oftálmicas , Fosfolipasas A2/deficiencia , Retina/metabolismo , Uveítis/inducido químicamenteRESUMEN
The mechanisms of poultry particulate matter (PM)-induced agricultural respiratory disorders are not thoroughly understood. Hence, it is hypothesized in this article that poultry PM induces the release of interleukin-8 (IL-8) by lung epithelial cells that is regulated upstream by the concerted action of cytosolic phospholipase A2 (cPLA2) and extracellular signal-regulated kinase (ERK). To test this hypothesis, the widely used cultured human lung epithelial cells (A549) were chosen as the model system. Poultry PM caused a significant activation of PLA2 in A549 cells, which was attenuated by AACOCF3 (cPLA2 inhibitor) and PD98059 (ERK-1/2 upstream inhibitor). Poultry PM induced upstream ERK-1/2 phosphorylation and downstream cPLA2 serine phosphorylation, in a concerted fashion, in cells with enhanced association of ERK-1/2 and cPLA2. The poultry PM-induced cPLA2 serine phosphorylation and IL-8 release were attenuated by AACOCF3, PD98059, and by transfection with dominant-negative ERK-1/2 DNA in cells. The poultry PM-induced IL-8 release by the bone marrow-derived macrophages of cPLA2 knockout mice was significantly lower. For the first time, this study demonstrated that the poultry PM-induced IL-8 secretion by human lung epithelial cells was regulated by cPLA2 activation through ERK-mediated serine phosphorylation, suggesting a mechanism of airway inflammation among poultry farm workers.
Asunto(s)
Células Epiteliales/efectos de los fármacos , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Interleucina-8/metabolismo , Pulmón/citología , Material Particulado/farmacología , Fosfolipasas A2/metabolismo , Aves de Corral , Animales , Ácido Araquidónico/metabolismo , Proteínas Sanguíneas/farmacología , Células de la Médula Ósea/citología , Línea Celular , Relación Dosis-Respuesta a Droga , Activación Enzimática/efectos de los fármacos , Células Epiteliales/citología , Células Epiteliales/metabolismo , Técnicas de Inactivación de Genes , Humanos , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Ratones , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Fosfolipasas A2/química , Fosfolipasas A2/deficiencia , Fosfolipasas A2/genética , Fosforilación/efectos de los fármacos , Transporte de Proteínas/efectos de los fármacos , Serina/metabolismo , Factores de TiempoRESUMEN
Spinal cord injury (SCI) results in permanent loss of motor functions. A significant aspect of the tissue damage and functional loss may be preventable as it occurs, secondary to the trauma. We show that the phospholipase A(2) (PLA(2)) superfamily plays important roles in SCI. PLA(2) enzymes hydrolyze membrane glycerophospholipids to yield a free fatty acid and lysophospholipid. Some free fatty acids (arachidonic acid) give rise to eicosanoids that promote inflammation, while some lysophospholipids (lysophosphatidylcholine) cause demyelination. We show in a mouse model of SCI that two cytosolic forms [calcium-dependent PLA(2) group IVA (cPLA(2) GIVA) and calcium-independent PLA(2) group VIA (iPLA(2) GVIA)], and a secreted form [secreted PLA(2) group IIA (sPLA(2) GIIA)] are up-regulated. Using selective inhibitors and null mice, we show that these PLA(2)s play differing roles. cPLA(2) GIVA mediates protection, whereas sPLA(2) GIIA and, to a lesser extent, iPLA(2) GVIA are detrimental. Furthermore, completely blocking all three PLA(2)s worsens outcome, while the most beneficial effects are seen by partial inhibition of all three. The partial inhibitor enhances expression of cPLA(2) and mediates its beneficial effects via the prostaglandin EP1 receptor. These findings indicate that drugs that inhibit detrimental forms of PLA(2) (sPLA(2) and iPLA2) and up-regulate the protective form (cPLA2) may be useful for the treatment of SCI.
Asunto(s)
Fosfolipasas A2/metabolismo , Traumatismos de la Médula Espinal/enzimología , Animales , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/uso terapéutico , Femenino , Fosfolipasas A2 Grupo II/antagonistas & inhibidores , Fosfolipasas A2 Grupo II/deficiencia , Fosfolipasas A2 Grupo II/metabolismo , Fosfolipasas A2 Grupo IV/antagonistas & inhibidores , Fosfolipasas A2 Grupo IV/deficiencia , Fosfolipasas A2 Grupo IV/genética , Fosfolipasas A2 Grupo IV/metabolismo , Fosfolipasas A2 Grupo VI/antagonistas & inhibidores , Fosfolipasas A2 Grupo VI/deficiencia , Fosfolipasas A2 Grupo VI/metabolismo , Locomoción/efectos de los fármacos , Locomoción/fisiología , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Inhibidores de Fosfolipasa A2 , Fosfolipasas A2/clasificación , Fosfolipasas A2/deficiencia , Receptor Cross-Talk , Subtipo EP1 de Receptores de Prostaglandina E/metabolismo , Traumatismos de la Médula Espinal/tratamiento farmacológico , Traumatismos de la Médula Espinal/patología , Traumatismos de la Médula Espinal/fisiopatologíaRESUMEN
A deficiency of lysosomal phospholipase A2 (LPLA2) causes macrophage-associated phospholipidosis, suggesting that the enzyme is important in the lipid catabolism. Because LPLA2 is secreted by macrophages, extracellular LPLA2 activity may potentially reflect a change in macrophage activation. In this report, the detection of LPLA2 activity in plasma was established by the measurement of the transacylase activity of LPLA2 under acidic conditions. No transacylase activity of LPLA2 was detected in normal human plasma when the plasma was incubated with liposomes consisting of 1,2-dioleoylphosphatidylcholine/sulfatide/N-acetylsphingosine (NAS) at pH 4.5. However, the transacylase activity in the plasma was detected when liposomes consisting of 1,2-dioleoylphosphatidylglycerol/NAS were used as a substrate. To establish the specificity of the assay, ceramide transacylase activity was detected in the plasma of wild-type mice. By contrast, the plasma obtained from LPLA2-knockout mice had no measurable transacylase activity under the same conditions. The enzymatic activity of recombinant LPLA2 was inhibited by treatment with methylarachidonylfluorophosphonate. The inhibitor also suppressed the transacylase activity observed in both normal human and wild-type mouse plasma, establishing that the transacylase activity observed in plasma is due to LPLA2. Plasma LPLA2 activity may be a useful bioassay marker for the identification of LPLA2-related disorders.
Asunto(s)
Pruebas de Enzimas/métodos , Lisosomas/enzimología , Fosfolipasas A2/sangre , Acilación , Aciltransferasas/metabolismo , Animales , Ácidos Araquidónicos/farmacología , Inhibidores Enzimáticos/farmacología , Líquido Extracelular/enzimología , Técnicas de Inactivación de Genes , Humanos , Ratones , Organofosfonatos/farmacología , Inhibidores de Fosfolipasa A2 , Fosfolipasas A2/deficiencia , Fosfolipasas A2/genética , Especificidad por SustratoRESUMEN
Decrease in fat catabolic rate on consuming a high-fat diet contributes to diet-induced obesity. This study used group 1B phospholipase A(2) (Pla2g1b)-deficient mice, which are resistant to hyperglycemia, to test the hypothesis that Pla2g1b and its lipolytic product lysophospholipid suppress hepatic fat utilization and energy metabolism in promoting diet-induced obesity. The metabolic consequences of hypercaloric diet, including body weight gain, energy expenditure, and fatty acid oxidation, were compared between Pla2g1b(+/+) and Pla2g1b(-/-) mice. The Pla2g1b(-/-) mice displayed normal energy balance when fed chow, but were resistant to obesity when challenged with a hypercaloric diet. Obesity resistance in Pla2g1b(-/-) mice is due to their ability to maintain elevated energy expenditure and core body temperature when subjected to hypercaloric diet, which was not observed in Pla2g1b(+/+) mice. The Pla2g1b(-/-) mice also displayed increased postprandial hepatic fat utilization due to increased expression of peroxisome proliferator-activated receptor (PPAR)-alpha, PPAR-delta, PPAR-gamma, cd36/Fat, and Ucp2, which coincided with reduced postprandial plasma lysophospholipid levels. Lysophospholipids produced by Pla2g1b hydrolysis suppress hepatic fat utilization and down-regulate energy expenditure, thereby preventing metabolically beneficial adaptation to a high-fat diet exposure in promoting diet-induced obesity and type 2 diabetes.
Asunto(s)
Ácidos Grasos/metabolismo , Hígado/metabolismo , Lisofosfolípidos/metabolismo , Obesidad/etiología , Fosfolipasas A2/metabolismo , Animales , Grasas de la Dieta/farmacología , Metabolismo Energético , Metabolismo de los Lípidos , Ratones , Obesidad/metabolismo , Oxidación-Reducción , Fosfolipasas A2/deficiencia , Periodo PosprandialRESUMEN
A white adipose-tissue-specific intracellular phospholipase, which releases arachidonic acid from position sn-2 of phospholipids, was recently discovered and named AdPLA. When AdPLA was induced by feeding or by insulin treatment, the arachidonic acid released from phospholipids acted as a precursor for the formation of prostaglandin E(2) (PGE(2)). Subsequent activation of the prostaglandin receptor EP3 caused decreased levels of cAMP that led to decreased lipolysis and increased adiposity. Ablation of AdPLA in knockout mice resulted in the reverse sequence of events, with a decline in arachidonic acid release and prostaglandin synthesis and an increase in levels of cAMP, leading to increased lipolysis and a decline in adiposity, even though food intake was not affected. The newly discovered AdPLA enzyme in white adipose tissue functions as a regulator of lipolysis by acting as an antilipolytic agent mediated by increased PGE(2) formation and decreased intracellular cAMP.
Asunto(s)
Tejido Adiposo Blanco/enzimología , Adiposidad/fisiología , Fosfolipasas A2/metabolismo , Animales , Grasas de la Dieta/metabolismo , Dinoprostona/metabolismo , Metabolismo Energético/fisiología , Humanos , Resistencia a la Insulina/fisiología , Especificidad de Órganos , Fosfolipasas A2/deficiencia , Fosfolipasas A2/fisiología , Triglicéridos/metabolismoRESUMEN
OBJECTIVE: The earliest response of esophageal mucosa to gastric reflux is the development of oxidative damage and inflammation. These processes contribute to the development of metaplasia known as Barrett's esophagus, as well as the progression to malignancy. Secretory phospholipase A(2) is a mediator of inflammation with levels that are increased in Barrett's metaplasia and carcinoma when compared with levels in normal samples. Our goal is to determine the role of secretory phospholipase A(2) in the development of reflux-associated changes in the esophageal mucosa. METHODS: Secretory phospholipase A(2)-deficient mice (C57BL/6, n = 5) and mice known to express high levels of secretory phospholipase A(2) (BALB/c, n = 5) underwent side-to-side surgical anastomosis of the first portion of the duodenum and gastroesophageal junction, allowing exposure of esophageal mucosa to duodenal and gastric contents duodeno-gastroesophageal anastomosis. Control animals (n = 5) of each strain underwent laparotomy with esophagotomy and repair. Tissue was frozen in embedding medium. Hematoxylin and eosin staining and Ki67 and secretory phospholipase A(2) immunohistochemistry were used to evaluate esophageal tissue and its response to duodeno-gastroesophageal anastomosis. RESULTS: Immunofluorescent staining confirmed the absence of secretory phospholipase A(2) in C57BL/6 mice and its presence in BALB/c mice. Hematoxylin and eosin staining demonstrated significant thickening of the esophageal mucosa in response to gastroesophageal reflux in the presence of secretory phospholipase A(2). Mice known to express high levels of secretory phospholipase A(2) also demonstrated increased numbers of proliferating cells. Secretory phospholipase A(2)-deficient mice were immune to the early changes induced by mixed reflux. CONCLUSIONS: The presence of secretory phospholipase A(2) appears necessary for early histologic changes produced by exposure of the esophagus to gastroduodenal contents. This enzyme is identified as a promising target for evaluation of mechanisms of carcinogenesis and chemoprevention of esophageal carcinoma.