RESUMO
The serine hydrolase inhibitors pyrrophenone and KT195 inhibit cell death induced by A23187 and H2O2 by blocking the release of calcium from the endoplasmic reticulum and mitochondrial calcium uptake. The effect of pyrrophenone and KT195 on these processes is not due to inhibition of their known targets, cytosolic phospholipase A2 and α/ß-hydrolase domain-containing (ABHD) 6, respectively, but represent off-target effects. To identify targets of KT195, fibroblasts were treated with KT195-alkyne to covalently label protein targets followed by click chemistry with biotin azide, enrichment on streptavidin beads and tryptic peptide analysis by mass spectrometry. Although several serine hydrolases were identified, α/ß-hydrolase domain-containing 2 (ABHD2) was the only target in which both KT195 and pyrrophenone competed for binding to KT195-alkyne. ABHD2 is a serine hydrolase with a predicted transmembrane domain consistent with its pull-down from the membrane proteome. Subcellular fractionation showed localization of ABHD2 to the endoplasmic reticulum but not to mitochondria or mitochondrial-associated membranes. Knockdown of ABHD2 with shRNA attenuated calcium release from the endoplasmic reticulum, mitochondrial calcium uptake and cell death in fibroblasts stimulated with A23187. The results describe a novel mechanism for regulating calcium transfer from the endoplasmic reticulum to mitochondria that involves the serine hydrolase ABHD2.
Assuntos
Cálcio/metabolismo , Retículo Endoplasmático/metabolismo , Hidrolases/metabolismo , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular , Relação Dose-Resposta a Droga , Fosfolipases A2 do Grupo IV/antagonistas & inibidores , Fosfolipases A2 do Grupo IV/deficiência , Fosfolipases A2 do Grupo IV/metabolismo , Peróxido de Hidrogênio/antagonistas & inibidores , Peróxido de Hidrogênio/farmacologia , Hidrolases/antagonistas & inibidores , Camundongos , Camundongos Knockout , Inibidores de Fosfolipase A2/farmacologia , Pirrolidinas/farmacologia , Relação Estrutura-AtividadeRESUMO
Cytosolic phospholipase A2α (cPLA2α) mediates agonist-induced release of arachidonic acid from membrane phospholipid for production of eicosanoids. The activation of cPLA2α involves increases in intracellular calcium, which binds to the C2 domain and promotes cPLA2α translocation from the cytosol to membrane to access substrate. The cell permeable pyrrolidine-containing cPLA2α inhibitors including pyrrophenone have been useful to understand cPLA2α function. Although this serine hydrolase inhibitor does not inhibit other PLA2s or downstream enzymes that metabolize arachidonic acid, we reported that it blocks increases in mitochondrial calcium and cell death in lung fibroblasts. In this study we used the calcium indicators G-CEPIA1er and CEPIA2mt to compare the effect of pyrrophenone in regulating calcium levels in the endoplasmic reticulum (ER) and mitochondria in response to A23187 and receptor stimulation. Pyrrophenone blocked calcium release from the ER and concomitant increases in mitochondrial calcium in response to stimulation by ATP, serum and A23187. In contrast, ER calcium release induced by the sarco/endoplasmic reticulum Ca(2+)-ATPase inhibitor thapsigargin was not blocked by pyrrophenone suggesting specificity for the calcium release pathway. As a consequence of blocking calcium mobilization, pyrrophenone inhibited serum-stimulated translocation of the cPLA2α C2 domain to Golgi. The ability of pyrrophenone to block ER calcium release is an off-target effect since it occurs in fibroblasts lacking cPLA2α. The results implicate a serine hydrolase in regulating ER calcium release and highlight the importance of careful dose-response studies with pyrrophenone to study cPLA2α function.
Assuntos
Cálcio/metabolismo , Retículo Endoplasmático/efeitos dos fármacos , Fosfolipases A2 do Grupo IV/antagonistas & inibidores , Pirrolidinas/farmacologia , Trifosfato de Adenosina/farmacologia , Animais , Calcimicina/farmacologia , Ionóforos de Cálcio/farmacologia , Células Cultivadas , Meios de Cultura/química , Meios de Cultura/farmacologia , Retículo Endoplasmático/metabolismo , Inibidores Enzimáticos/farmacologia , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Complexo de Golgi/efeitos dos fármacos , Complexo de Golgi/metabolismo , Fosfolipases A2 do Grupo IV/genética , Fosfolipases A2 do Grupo IV/metabolismo , Pulmão/citologia , Camundongos Knockout , Microscopia de Fluorescência , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Transporte Proteico/efeitos dos fármacos , Soro/química , Tapsigargina/farmacologia , Imagem com Lapso de Tempo/métodosRESUMO
BACKGROUND: The lung is exposed to airborne fungal spores, and fungi that colonize the oral cavity such as Candida albicans, but does not develop disease to opportunistic fungal pathogens unless the immune system is compromised. The Group IVA cytosolic phospholipase A2 (cPLA2α) is activated in response to Candida albicans infection resulting in the release of arachidonic acid for eicosanoid production. Although eicosanoids such as prostaglandins and leukotrienes modulate inflammation and immune responses, the role of cPLA2α and eicosanoids in regulating C. albicans lung infection is not understood. METHODS: The responses of cPLA2α(+/+) and cPLA2α(-/-) Balb/c mice to intratracheal instillation of C. albicans were compared. After challenge, we evaluated weight loss, organ fungal burden, and the recruitment of cells and the levels of cytokines and eicosanoids in bronchoalveolar lavage fluid. The ability of macrophages and neutrophils from cPLA2α(+/+) and cPLA2α(-/-) mice to recognize and kill C. albicans was also compared. RESULTS: After C. albicans instillation, cPLA2α(+/+) mice recovered a modest weight loss by 48 h and completely cleared fungi from the lung by 12 h with no dissemination to the kidneys. In cPLA2α(-/-) mice, weight loss continued for 72 h, C. albicans was not completely cleared from the lung and disseminated to the kidneys. cPLA2α(-/-) mice exhibited greater signs of inflammation including higher neutrophil influx, and elevated levels of albumin and pro-inflammatory cytokines/chemokines (IL1α, IL1ß, TNFα, IL6, CSF2, CXCL1, CCL20) in bronchoalveolar lavage fluid. The amounts of cysteinyl leukotrienes, thromboxane B2 and prostaglandin E2 were significantly lower in bronchoalveolar lavage fluid from C. albicans-infected cPLA2α(-/-) mice compared to cPLA2α(+/+) mice. Alveolar macrophages and neutrophils from uninfected cPLA2α(-/-) mice exhibited less killing of C. albicans in vitro than cells from cPLA2α(+/+) mice. In addition alveolar macrophages from cPLA2α(-/-) mice isolated 6 h after instillation of GFP-C. albicans contained fewer internalized fungi than cPLA2α(+/+) macrophages. CONCLUSIONS: The results demonstrate that cPLA2α contributes to immune surveillance and host defense in the lung to prevent infection by the commensal fungus C. albicans and to dampen inflammation.
Assuntos
Candida albicans/imunologia , Candidíase/imunologia , Pneumopatias Fúngicas/imunologia , Pulmão/imunologia , Macrófagos Alveolares/fisiologia , Neutrófilos/fisiologia , Fosfolipases A2/metabolismo , Animais , Ácido Araquidônico/metabolismo , Movimento Celular , Células Cultivadas , Citocinas/metabolismo , Eicosanoides/metabolismo , Imunidade Inata , Mediadores da Inflamação/metabolismo , Pulmão/microbiologia , Macrófagos Alveolares/microbiologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Neutrófilos/microbiologia , Fosfolipases A2/genética , Fosfolipases A2/imunologiaRESUMO
The pulmonary surfactant phospholipid, 1-palmitoyl-2-oleoylphosphatidylglycerol (POPG), potently inhibits toll-like receptor (TLR)2 and TLR4 signaling from the cell surface of macrophages. Analogs of POPG that vary in polar head group length, hydroxylation, and alkyl branching were synthesized using a phospholipase D-catalyzed transphosphatidylation reaction and a 1-palmitoyl-2-oleoyl phosphatidylcholine substrate. Lipid analogs with C3 and C4 alkyl head group length (POP-propanol and POP-butanol) are less effective than POPG as TLR2 and TLR4 antagonists. However, adding a hydroxyl group at the alkyl chain 3- or 4-position (POP-propanediols or POP-butanediols) greatly increased their inhibitory effects against TLR2 and TLR4. POP-2',2'-dimethylpropanediol is a weak inhibitor of TLR2 and TLR4 activation that results in arachidonic acid release, but an effective inhibitor of TLR4 activation that results in TNF-α production. Addition of an amino group at the alkyl-2 position (POP-2'-aminopropanediol) completely abolished the antagonism of TLRs 2 and 4. Multiple analogs strongly bind to the TLR4 coreceptors, cluster of differentiation 14 (CD14) and myeloid differentiation 2, but competition for di[3-deoxy-D-manno-octulosonyl]-lipid A binding to CD14 is the best predictor of biological activity at the cellular level. Collectively, these findings identify new compounds for antagonizing TLR2 and TLR4 activation and define structural properties of POPG analogs for discriminating between two TLR systems.
Assuntos
Inflamação/tratamento farmacológico , Fosfatidilgliceróis/administração & dosagem , Receptor 2 Toll-Like/genética , Receptor 4 Toll-Like/genética , Animais , Membrana Celular/efeitos dos fármacos , Eicosanoides/administração & dosagem , Eicosanoides/química , Endotoxinas/administração & dosagem , Endotoxinas/química , Humanos , Inflamação/genética , Inflamação/patologia , Receptores de Lipopolissacarídeos/metabolismo , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Fosfatidilgliceróis/química , Surfactantes Pulmonares/administração & dosagem , Surfactantes Pulmonares/química , Células RAW 264.7 , Transdução de Sinais/efeitos dos fármacos , Receptor 2 Toll-Like/antagonistas & inibidores , Receptor 4 Toll-Like/antagonistas & inibidoresRESUMO
In Candida albicans-infected resident peritoneal macrophages, activation of group IVA cytosolic phospholipase A2(cPLA2α) by calcium- and mitogen-activated protein kinases triggers the rapid production of prostaglandins I2 and E2 through cyclooxygenase (COX)-1 and regulates gene expression by increasing cAMP. InC. albicans-infected cPLA2α(-/-)or COX-1(-/-)macrophages, expression ofI l10,Nr4a2, and Ptgs2 was lower, and expression ofTnfα was higher, than in wild type macrophages. Expression was reconstituted with 8-bromo-cAMP, the PKA activator 6-benzoyl-cAMP, and agonists for prostaglandin receptors IP, EP2, and EP4 in infected but not uninfected cPLA2α(-/-)or COX-1(-/-)macrophages. InC. albicans-infected cPLA2α(+/+)macrophages, COX-2 expression was blocked by IP, EP2, and EP4 receptor antagonists, indicating a role for both prostaglandin I2 and E2 Activation of ERKs and p38, but not JNKs, by C. albicansacted synergistically with prostaglandins to induce expression of Il10,Nr4a2, and Ptgs2. Tnfα expression required activation of ERKs and p38 but was suppressed by cAMP. Results using cAMP analogues that activate PKA or Epacs suggested that cAMP regulates gene expression through PKA. However, phosphorylation of cAMP-response element-binding protein (CREB), the cAMP-regulated transcription factor involved inIl10,Nr4a2,Ptgs2, andTnfα expression, was not mediated by cAMP/PKA because it was similar inC. albicans-infected wild type and cPLA2α(-/-)or COX-1(-/-)macrophages. CREB phosphorylation was blocked by p38 inhibitors and induced by the p38 activator anisomycin but not by the PKA activator 6-benzoyl-cAMP. Therefore, MAPK activation inC. albicans-infected macrophages plays a dual role by promoting the cPLA2α/prostaglandin/cAMP/PKA pathway and CREB phosphorylation that coordinately regulate immediate early gene expression.
Assuntos
Candida albicans/fisiologia , Ciclo-Oxigenase 1/imunologia , Regulação da Expressão Gênica , Fosfolipases A2 do Grupo IV/imunologia , Interações Hospedeiro-Patógeno , Macrófagos Peritoneais/imunologia , Proteínas de Membrana/imunologia , 8-Bromo Monofosfato de Adenosina Cíclica/farmacologia , Animais , AMP Cíclico/análogos & derivados , AMP Cíclico/metabolismo , AMP Cíclico/farmacologia , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/imunologia , Ciclo-Oxigenase 1/deficiência , Ciclo-Oxigenase 1/genética , Ciclo-Oxigenase 2/genética , Ciclo-Oxigenase 2/imunologia , Dinoprostona/biossíntese , Epoprostenol/biossíntese , Fosfolipases A2 do Grupo IV/deficiência , Fosfolipases A2 do Grupo IV/genética , Interleucina-10/genética , Interleucina-10/imunologia , Macrófagos Peritoneais/efeitos dos fármacos , Macrófagos Peritoneais/microbiologia , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 1 Ativada por Mitógeno/imunologia , Proteína Quinase 3 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/imunologia , Membro 2 do Grupo A da Subfamília 4 de Receptores Nucleares/genética , Membro 2 do Grupo A da Subfamília 4 de Receptores Nucleares/imunologia , Cultura Primária de Células , Inibidores de Proteínas Quinases/farmacologia , Receptores de Prostaglandina/agonistas , Receptores de Prostaglandina/antagonistas & inibidores , Receptores de Prostaglandina/genética , Receptores de Prostaglandina/imunologia , Transdução de Sinais , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologia , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Proteínas Quinases p38 Ativadas por Mitógeno/imunologiaRESUMO
The most severe complication of influenza is viral pneumonia, which can lead to the acute respiratory distress syndrome. Alveolar epithelial cells (AECs) are the first cells that influenza virus encounters upon entering the alveolus. Infected epithelial cells produce cytokines that attract and activate neutrophils and macrophages, which in turn induce damage to the epithelial-endothelial barrier. Hepatocyte growth factor (HGF)/c-Met and transforming growth factor-α (TGF-α)/epidermal growth factor receptor (EGFR) are well known to regulate repair of damaged alveolar epithelium by stimulating cell migration and proliferation. Recently, TGF-α/EGFR signaling has also been shown to regulate innate immune responses in bronchial epithelial cells. However, little is known about whether HGF/c-Met signaling alters the innate immune responses and whether the innate immune responses in AECs are regulated by HGF/c-Met and TGF-α/EGFR. We hypothesized that HGF/c-Met and TGF-α/EGFR would regulate innate immune responses to influenza A virus infection in human AECs. We found that recombinant human HGF (rhHGF) and rhTGF-α stimulated primary human AECs to secrete IL-8 and granulocyte macrophage colony-stimulating factor (GM-CSF) strongly and IL-6 and monocyte chemotactic protein 1 moderately. Influenza infection stimulated the secretion of IL-8 and GM-CSF by AECs plated on rat-tail collagen through EGFR activation likely by TGF-α released from AECs and through c-Met activated by HGF secreted from lung fibroblasts. HGF secretion by fibroblasts was stimulated by AEC production of prostaglandin E2 during influenza infection. We conclude that HGF/c-Met and TGF-α/EGFR signaling enhances the innate immune responses by human AECs during influenza infections.
Assuntos
Células Epiteliais Alveolares/metabolismo , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Vírus da Influenza A/imunologia , Influenza Humana/imunologia , Interleucina-8/metabolismo , Células Epiteliais Alveolares/virologia , Células Cultivadas , Técnicas de Cocultura , Dinoprostona/metabolismo , Receptores ErbB/metabolismo , Fator de Crescimento de Hepatócito/metabolismo , Humanos , Proteínas Proto-Oncogênicas c-met/metabolismo , Alvéolos Pulmonares/patologia , Transdução de Sinais , Fator de Crescimento Transformador alfa/metabolismoRESUMO
The group IV phospholipase A2 (PLA2) family is comprised of six intracellular enzymes (GIVA, -B, -C, -D, -E, and -F) commonly referred to as cytosolic PLA2 (cPLA2)α, -ß, -γ, -δ, -ε, and -ζ. They contain a Ser-Asp catalytic dyad and all except cPLA2γ have a C2 domain, but differences in their catalytic activities and subcellular localization suggest unique regulation and function. With the exception of cPLA2α, the focus of this review, little is known about the in vivo function of group IV enzymes. cPLA2α catalyzes the hydrolysis of phospholipids to arachidonic acid and lysophospholipids that are precursors of numerous bioactive lipids. The regulation of cPLA2α is complex, involving transcriptional and posttranslational processes, particularly increases in calcium and phosphorylation. cPLA2α is a highly conserved widely expressed enzyme that promotes lipid mediator production in human and rodent cells from a variety of tissues. The diverse bioactive lipids produced as a result of cPLA2α activation regulate normal physiological processes and disease pathogenesis in many organ systems, as shown using cPLA2α KO mice. However, humans recently identified with cPLA2α deficiency exhibit more pronounced effects on health than observed in mice lacking cPLA2α, indicating that much remains to be learned about this interesting enzyme.
Assuntos
Doença , Fosfolipases A2 Citosólicas/metabolismo , Animais , Humanos , Fosfolipases A2 Citosólicas/químicaRESUMO
An immunoassay for IVA phospholipase A2 in human red blood cells is described. The assay is a non-competitive sandwich assay in which increasing amounts of the measured protein produce increased luminescence. The antibodies used in the assay are directed against two unique epitopes of the molecule, which sequentially trap and detect the protein. The standard curve covers the range 0.7ng to 23ng/mL (0.07 to 2.3ng/well). The intra-assay and inter-assay coefficients of variation were 9% and 12%, respectively. Evidence is presented that the assay is specific for the alpha paralog of IV PLA2. The assay allows simple and rapid quantification of IVAPLA2 in red blood cell lysates and other biological fluids.
Assuntos
Epitopos/metabolismo , Eritrócitos/enzimologia , Fosfolipases A2 do Grupo IV/química , Fosfolipases A2 do Grupo IV/metabolismo , Adulto , Animais , Linhagem Celular , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Células Sf9 , Spodoptera , Adulto JovemRESUMO
Perturbation of calcium signaling that occurs during cell injury and disease, promotes cell death. In mouse lung fibroblasts A23187 triggered mitochondrial permeability transition pore (MPTP) formation, lactate dehydrogenase (LDH) release, and necrotic cell death that were blocked by cyclosporin A (CsA) and EGTA. LDH release temporally correlated with arachidonic acid release but did not involve cytosolic phospholipase A2α (cPLA2α) or calcium-independent PLA2. Surprisingly, release of arachidonic acid and LDH from cPLA2α-deficient fibroblasts was inhibited by the cPLA2α inhibitor pyrrophenone, and another serine hydrolase inhibitor KT195, by preventing mitochondrial calcium uptake. Inhibitors of calcium/calmodulin-dependent protein kinase II, a mitochondrial Ca(2+) uniporter (MCU) regulator, also prevented MPTP formation and arachidonic acid release induced by A23187 and H2O2. Pyrrophenone blocked MCU-mediated mitochondrial calcium uptake in permeabilized fibroblasts but not in isolated mitochondria. Unlike pyrrophenone, the diacylglycerol analog 1-oleoyl-2-acetyl-sn-glycerol and CsA blocked cell death and arachidonic acid release not by preventing mitochondrial calcium uptake but by inhibiting MPTP formation. In fibroblasts stimulated with thapsigargin, which induces MPTP formation by a direct effect on mitochondria, LDH and arachidonic acid release were blocked by CsA and 1-oleoyl-2-acetyl-sn-glycerol but not by pyrrophenone or EGTA. Therefore serine hydrolase inhibitors prevent necrotic cell death by blocking mitochondrial calcium uptake but not the enzyme releasing fatty acids that occurs by a novel pathway during MPTP formation. This work reveals the potential for development of small molecule cell-permeable serine hydrolase inhibitors that block MCU-mediated mitochondrial calcium overload, MPTP formation, and necrotic cell death.
Assuntos
Cálcio/metabolismo , Ciclosporina/farmacocinética , Fibroblastos/metabolismo , Mitocôndrias/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Inibidores de Fosfolipase A2/farmacologia , Pirrolidinas/farmacologia , Tapsigargina/farmacologia , Animais , Ácido Araquidônico/genética , Ácido Araquidônico/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/antagonistas & inibidores , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/genética , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Linhagem Celular Transformada , Quelantes/farmacologia , Ciclosporina/farmacologia , Diglicerídeos/farmacologia , Ácido Egtázico/farmacologia , Fibroblastos/patologia , Fosfolipases A2 do Grupo IV/antagonistas & inibidores , Fosfolipases A2 do Grupo IV/farmacologia , Isoenzimas/genética , Isoenzimas/metabolismo , L-Lactato Desidrogenase/genética , L-Lactato Desidrogenase/metabolismo , Camundongos , Camundongos Knockout , Mitocôndrias/genética , Proteínas de Transporte da Membrana Mitocondrial/genética , Poro de Transição de Permeabilidade Mitocondrial , Necrose/genética , Necrose/metabolismo , Necrose/patologiaRESUMO
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
Resolution of neutrophilia characteristic of acute inflammation requires cessation of neutrophil recruitment and removal of tissue neutrophils. Based on in vitro studies, a role in these events was hypothesized for oxidant-generated lysophosphatidylserine (lyso-PS) on recruited neutrophils signaling via the G2A receptor on macrophages. Peritoneal exudate neutrophils harvested from wild type (WT) mice had 5-fold more lyso-PS (lyso-PS(high)) than those of gp91(phox)(-/-) (lyso-PS(low)) mice. Ex vivo engulfment of lyso-PS(high) neutrophils (95% viable) by WT peritoneal macrophages was quantitatively similar to UV-irradiated apoptotic blood neutrophils, although the signaling pathway for the former was uniquely dependent on macrophage G2A. In contrast, lyso-PS(low) neutrophils were poorly engulfed unless presented with exogenous lyso-PS. Enhanced clearance of lyso-PS(high) neutrophils was also seen in vivo following their adoptive transfer into inflamed peritonea of WT but not G2A(-/-) mice, further supporting a requirement for signaling via G2A. To investigate downstream effects of lyso-PS/G2A signaling, antibody blockade of G2A in WT mice reduced macrophage CD206 expression and efferocytosis during peritonitis. Conversely, adoptive transfer of lyso-PS(high) neutrophils early in inflammation in gp91(phox)(-/-) mice led to accelerated development of efferocytic(high) and CD206(high) macrophages. This macrophage reprogramming was associated with suppressed production of pro-inflammatory mediators and reduced neutrophilia. These effects were not seen if G2A was blocked or lyso-PS(low) neutrophils were transferred. Taken together, the results demonstrate that oxidant-generated lyso-PS made by viable tissue neutrophils is an endogenous anti-inflammatory mediator working in vivo to orchestrate the "early" and rapid clearance of recruited neutrophils as well as the reprogramming of "resolving" macrophages.
Assuntos
Transtornos Leucocíticos/congênito , Lipídeos/química , Lisofosfolipídeos/química , Neutrófilos/citologia , Oxidantes/química , Animais , Anti-Inflamatórios/farmacologia , Apoptose , Citocinas/metabolismo , Modelos Animais de Doenças , Feminino , Inflamação , Transtornos Leucocíticos/metabolismo , Macrófagos/citologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , NADPH Oxidases/metabolismo , Peritonite/metabolismo , Transdução de SinaisRESUMO
Group IVα phospholipase A(2) (PLA(2)IVα) is a lipolytic enzyme that catalyzes the hydrolysis of membrane phospholipids to generate precursors of potent inflammatory lipid mediators. Here, the role of PLA(2)IVα in Fc receptor (FcR)-mediated phagocytosis was investigated, demonstrating that PLA(2)IVα is selectively activated upon FcR-mediated phagocytosis in macrophages and that it rapidly translocates to the site of the nascent phagosome. Moreover, pharmacological inhibition of PLA(2)IVα by pyrrophenone reduces particle internalization by up to 50%. In parallel, fibroblasts from PLA(2)IVα knock-out mice overexpressing FcγRIIA and able to internalize IgG-opsonized beads show 50% lower phagocytosis, compared with wild-type cells, and transfection of PLA(2)IVα fully recovers this impaired function. Interestingly, transfection of the catalytically inactive deleted PLA(2)IVα mutant (PLA(2)IVα(1-525)) and point mutant (PLA(2)IVα-S228C) also promotes recovery of this impaired function. Finally, transfection of the PLA(2)IVα C2 domain (which is directly involved in PLA(2)IVα membrane binding), but not of PLA(2)IVα-D43N (which cannot bind to membranes), rescues FcR-mediated phagocytosis. These data unveil a new mechanism of action for PLA(2)IVα, which demonstrates that the membrane binding, and not the enzymatic activity, is required for PLA(2)IVα modulation of FcR-mediated phagocytosis.
Assuntos
Membrana Celular/enzimologia , Fosfolipases A2 do Grupo IV/metabolismo , Macrófagos/enzimologia , Fagocitose/fisiologia , Fagossomos/metabolismo , Substituição de Aminoácidos , Animais , Linhagem Celular Transformada , Membrana Celular/genética , Fibroblastos/citologia , Fibroblastos/enzimologia , Fosfolipases A2 do Grupo IV/genética , Humanos , Macrófagos/citologia , Camundongos , Camundongos Knockout , Mutação de Sentido Incorreto , Fagocitose/efeitos dos fármacos , Fagossomos/genética , Estrutura Terciária de Proteína , Transporte Proteico/efeitos dos fármacos , Transporte Proteico/fisiologia , Pirrolidinas/farmacologia , Receptores de IgG/genética , Receptores de IgG/metabolismoRESUMO
The repellent semaphorin 3A (Sema3A) causes growth cone turning or collapse by triggering cytoskeletal rearrangements and detachment of adhesion sites. Growth cone detachment is dependent on eicosanoid activation of protein kinase C epsilon (PKCε), but the characterization of the phospholipase A(2) (PLA(2) ) that releases arachidonic acid (AA) for eicosanoid synthesis has remained elusive. Here, we show, in rat dorsal root ganglion (DRG) neurons, that Sema3A stimulates PLA(2) activity, that Sema3A-induced growth cone turning and collapse are dependent on the release of AA, and that the primary PLA(2) involved is the group IV α isoform (GIVA). Silencing GIVA expression renders growth cones resistant to Sema3A-induced collapse, and GIVA inhibition reverses Sema3A-induced repulsion into attraction. These studies identify a novel, early step in Sema3A-signaling and a PLA(2) necessary for growth cone repulsion and collapse.
Assuntos
Fosfolipases A2 do Grupo IV/metabolismo , Cones de Crescimento/metabolismo , Neurônios/citologia , Animais , Movimento Celular/fisiologia , Células Cultivadas , Embrião de Mamíferos , Inibidores Enzimáticos/farmacologia , Gânglios Espinais/citologia , Expressão Gênica/efeitos dos fármacos , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Fosfolipases A2 do Grupo IV/genética , Cones de Crescimento/efeitos dos fármacos , Humanos , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Proteína Quinase C/metabolismo , RNA Interferente Pequeno/farmacologia , Ratos , Semaforina-3A/metabolismo , Semaforina-3A/farmacologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Fatores de Tempo , TransfecçãoRESUMO
Intracellular transport occurs through two general types of carrier, either vesicles or tubules. Coat proteins act as the core machinery that initiates vesicle formation, but the counterpart that initiates tubule formation has been unclear. Here, we find that the coat protein I (COPI) complex initially drives the formation of Golgi buds. Subsequently, a set of opposing lipid enzymatic activities determines whether these buds become vesicles or tubules. Lysophosphatidic acid acyltransferase-γ (LPAATγ) promotes COPI vesicle fission for retrograde vesicular transport. In contrast, cytosolic phospholipase A2-α (cPLA2α) inhibits this fission event to induce COPI tubules, which act in anterograde intra-Golgi transport and Golgi ribbon formation. These findings not only advance a molecular understanding of how COPI vesicle fission is achieved, but also provide insight into how COPI acts in intra-Golgi transport and reveal an unexpected mechanistic relationship between vesicular and tubular transport.
Assuntos
Vesículas Revestidas pelo Complexo de Proteína do Envoltório/metabolismo , Complexo I de Proteína do Envoltório/metabolismo , Aciltransferases/antagonistas & inibidores , Aciltransferases/genética , Aciltransferases/metabolismo , Transporte Biológico Ativo , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/ultraestrutura , Complexo de Golgi/metabolismo , Complexo de Golgi/ultraestrutura , Fosfolipases A2 do Grupo IV/metabolismo , Células HeLa , Humanos , Metabolismo dos Lipídeos , Microscopia Eletrônica de Transmissão , Modelos Biológicos , RNA Interferente Pequeno/genéticaRESUMO
Phosphatidylserine (PS) and oxidized PS species have been identified as key ligands on apoptotic cells important for their recognition and removal (efferocytosis) by phagocytes, a requisite step for resolution of inflammation. We have recently demonstrated that lysophosphatidylserine (lyso-PS) generated and retained on neutrophils following short term activation of the NADPH oxidase in vitro and in vivo enhanced their clearance via signaling through the macrophage G-protein-coupled receptor G2A. Here, we investigated the signaling pathway downstream of G2A. Lyso-PS, either made endogenously in apoptosing neutrophils or supplied exogenously in liposomes along with lyso-PS(neg) apoptotic cells, signaled to macrophages in a G2A-dependent manner for their enhanced production of prostaglandin E2 (PGE2) via a calcium-dependent cytosolic phospholipase A2/cyclooxygenase-mediated mechanism. Subsequent signaling by PGE2 via EP2 receptors activated macrophage adenylyl cyclase and protein kinase A. These events, in turn, culminated in enhanced activity of Rac1, resulting in an increase in both the numbers of macrophages efferocytosing apoptotic cells and the numbers of cells ingested per macrophage. These data were surprising in light of previous reports demonstrating that signaling by PGE2 and adenylyl cyclase activation are associated with macrophage deactivation and inhibition of apoptotic cell uptake. Further investigation revealed that the impact of this pathway, either the enhancement or inhibition of efferocytosis, was exquisitely sensitive to concentration effects of these intermediaries. Together, these data support the hypothesis that lyso-PS presented on the surface of activated and dying neutrophils provides a tightly controlled, proresolution signal for high capacity clearance of neutrophils in acute inflammation.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Macrófagos Peritoneais/metabolismo , Neutrófilos/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Adenilil Ciclases/metabolismo , Animais , Apoptose , Western Blotting , Proteínas de Ciclo Celular/genética , Células Cultivadas , AMP Cíclico/metabolismo , Dinoprostona/metabolismo , Feminino , Lisofosfolipídeos/metabolismo , Macrófagos Peritoneais/citologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fagocitose , Fosfolipases A2 Independentes de Cálcio/metabolismo , Prostaglandina-Endoperóxido Sintases/metabolismo , Receptores Acoplados a Proteínas G/genética , Transdução de Sinais , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas em TandemRESUMO
Group IVA cytosolic phospholipase A(2) (cPLA(2)α) catalyzes the first step in the arachidonic acid cascade leading to the synthesis of important lipid mediators, the prostaglandins and leukotrienes. We previously described a patient deficient in cPLA(2)α activity, which was associated with mutations in both alleles encoding the enzyme. In this paper, we describe the biochemical characterization of each of these mutations. Using saturating concentrations of calcium, we showed that the R485H mutant was nearly devoid of any catalytic activity, that the S111P mutation did not affect the enzyme activity, and that the known K651R polymorphism was associated with activity slightly higher than that of the wild type. Using MDCK cells, we showed that translocation to the Golgi in response to serum activation was impaired for the S111P mutant but not for the other mutants. Using immortalized mouse lung fibroblasts lacking endogenous cPLA(2)α activity, we showed that both mutations S111P and R485H/K651R caused a profound defect in the enzyme catalytic activity in response to cell stimulation with serum. Taken together, our results show that the S111P mutation hampers calcium binding and membrane translocation without affecting the catalytic activity, and that the mutation R485H does not affect membrane translocation but blocks catalytic activity that leads to inactivation of the enzyme. Interestingly, our results show that the common K651R polymorphism confers slightly higher activity to the enzyme, suggesting a role of this residue in favoring a catalytically active conformation of cPLA(2)α. Our results define how the mutations negatively influence cPLA(2)α function and explain the inability of the proband to release arachidonic acid for eicosanoid production.
Assuntos
Fosfolipases A2 do Grupo IV/metabolismo , Mutação , Animais , Biocatálise , Linhagem Celular , Cães , Fosfolipases A2 do Grupo IV/deficiência , Fosfolipases A2 do Grupo IV/genética , Humanos , Camundongos , Camundongos Knockout , Transporte ProteicoRESUMO
Mycoplasma pneumoniae is a human pathogen causing respiratory infections that are also associated with serious exacerbations of chronic lung diseases. Membranes and lipoproteins from M. pneumoniae induced a 4-fold increase in arachidonic acid (AA) release from RAW264.7 and a 2-fold increase in AA release from primary human alveolar macrophages. The bacterial lipoprotein mimic and TLR2/1 agonist Pam3Cys and the TLR2/6 agonist MALP-2 produced effects similar to those elicited by M. pneumoniae in macrophages by inducing the phosphorylation of p38(MAPK) and p44/42(ERK1/2) MAP kinases and cyclooxygenase-2 (COX-2) expression. M. pneumoniae induced the generation of prostaglandins PGD(2) and PGE(2) from RAW264.7 cells and thromboxane B(2) (TXB(2)) from human alveolar macrophages. Anti-TLR2 antibody completely abolished M. pneumoniae-induced AA release and TNFα secretion from RAW264.7 cells and human alveolar macrophages. Disruption of the phosphorylation of p44/42(ERK1/2) or inactivation of cytosolic phospholipase A(2)α (cPLA(2)α) completely inhibited M. pneumoniae-induced AA release from macrophages. The minor pulmonary surfactant phospholipid, palmitoyl-oleoyl-phosphatidylglycerol (POPG), antagonized the proinflammatory actions of M. pneumoniae, Pam3Cys, and MALP-2 by reducing the production of AA metabolites from macrophages. The effect of POPG was specific, insofar as saturated PG, and saturated and unsaturated phosphatidylcholines did not have significant effect on M. pneumoniae-induced AA release. Collectively, these data demonstrate that M. pneumoniae stimulates the production of eicosanoids from macrophages through TLR2, and POPG suppresses this pathogen-induced response.
Assuntos
Proteínas de Bactérias/metabolismo , Eicosanoides/metabolismo , Macrófagos Alveolares/metabolismo , Mycoplasma pneumoniae/metabolismo , Fosfatidilgliceróis/metabolismo , Pneumonia por Mycoplasma/metabolismo , Surfactantes Pulmonares/metabolismo , Animais , Proteínas de Bactérias/imunologia , Proteínas de Bactérias/farmacologia , Linhagem Celular , Ciclo-Oxigenase 2/imunologia , Ciclo-Oxigenase 2/metabolismo , Cisteína/análogos & derivados , Cisteína/imunologia , Cisteína/metabolismo , Cisteína/farmacologia , Eicosanoides/imunologia , Fosfolipases A2 do Grupo IV/imunologia , Fosfolipases A2 do Grupo IV/metabolismo , Humanos , Lipopeptídeos/farmacologia , Lipoproteínas/imunologia , Lipoproteínas/metabolismo , Lipoproteínas/farmacologia , Macrófagos Alveolares/imunologia , Camundongos , Quinases de Proteína Quinase Ativadas por Mitógeno/imunologia , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Mycoplasma pneumoniae/imunologia , Fosfatidilgliceróis/imunologia , Fosfatidilgliceróis/farmacologia , Pneumonia por Mycoplasma/imunologia , Surfactantes Pulmonares/imunologia , Surfactantes Pulmonares/farmacologia , Receptores Toll-Like/antagonistas & inibidores , Receptores Toll-Like/imunologia , Receptores Toll-Like/metabolismo , Fator de Necrose Tumoral alfa/imunologia , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Exposure to environmental pollutants, such as polycyclic aromatic hydrocarbons (PAHs) found in coal tar mixtures and tobacco sources, is considered a significant risk factor for the development of heart disease in humans. The goal of this study was to determine the influence of PAHs present at a Superfund site on human coronary artery endothelial cell (HCAEC) phospholipase A(2) (PLA(2)) activity and apoptosis. Extremely high levels of 12 out of 15 EPA high-priority PAHs were present in both the streambed and floodplain sediments at a site where an urban creek and its adjacent floodplain were extensively contaminated by PAHs and other coal tar compounds. Nine of the 12 compounds and a coal tar mixture (SRM 1597A) activated group IVC PLA(2) in HCAECs, and activation of this enzyme was associated with histone fragmentation and poly (ADP) ribose polymerase (PARP) cleavage. Genetic silencing of group IVC PLA(2) inhibited both (3)H-fatty acid release and histone fragmentation by PAHs and SRM 1597A, indicating that individual PAHs and a coal tar mixture induce apoptosis of HCAECs via a mechanism that involves group IVC PLA(2). Western blot analysis of aortas isolated from feral mice (Peromyscus leucopus) inhabiting the Superfund site showed increased PARP and caspase-3 cleavage when compared to reference mice. These data suggest that PAHs induce apoptosis of HCAECs via activation of group IVC PLA(2).
Assuntos
Apoptose/efeitos dos fármacos , Vasos Coronários/efeitos dos fármacos , Endotélio Vascular/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Fosfolipases A2 do Grupo IV/metabolismo , Hidrocarbonetos Policíclicos Aromáticos/toxicidade , Poluentes do Solo/toxicidade , Animais , Animais Selvagens , Caspase 3/química , Caspase 3/metabolismo , Células Cultivadas , Vasos Coronários/enzimologia , Vasos Coronários/metabolismo , Endotélio Vascular/enzimologia , Endotélio Vascular/metabolismo , Exposição Ambiental , Inativação Gênica , Fosfolipases A2 do Grupo IV/antagonistas & inibidores , Fosfolipases A2 do Grupo IV/genética , Histonas/química , Histonas/metabolismo , Humanos , Isoenzimas/antagonistas & inibidores , Isoenzimas/genética , Isoenzimas/metabolismo , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Peromyscus , Poli(ADP-Ribose) Polimerases/química , Poli(ADP-Ribose) Polimerases/metabolismo , Hidrocarbonetos Policíclicos Aromáticos/isolamento & purificação , RNA Mensageiro/metabolismo , Rios , Solo/química , Poluentes do Solo/isolamento & purificação , TennesseeRESUMO
The cytosolic (group IV) phospholipase A(2) (cPLA(2)s) family contains six members. We have prepared recombinant proteins for human α, mouse ß, human γ, human δ, human ε, and mouse ζ cPLA(2)s and have studied their interfacial kinetic and binding properties in vitro. Mouse cPLA(2)ß action on phosphatidylcholine vesicles is activated by anionic phosphoinositides and cardiolipin but displays a requirement for Ca(2+) only in the presence of cardiolipin. This activation pattern is explained by the effects of anionic phospholipids and Ca(2+) on the interfacial binding of mouse cPLA(2)ß and its C2 domain to vesicles. Ca(2+)-dependent binding of mouse cPLA(2)ß to cardiolipin-containing vesicles requires a patch of basic residues near the Ca(2+)-binding surface loops of the C2 domain, but binding to phosphoinositide-containing vesicles does not depend on any specific cluster of basic residues. Human cPLA(2)δ also displays Ca(2+)- and cardiolipin-enhanced interfacial binding and activity. The lysophospholipase, phospholipase A(1), and phospholipase A(2) activities of the full set of mammalian cPLA(2)s were quantified. The relative level of these activities is very different among the isoforms, and human cPLA(2)δ stands out as having relatively high phospholipase A(1) activity. We also tested the susceptibility of all cPLA(2) family members to a panel of previously reported inhibitors of human cPLA(2)α and analogs of these compounds. This led to the discovery of a potent and selective inhibitor of mouse cPLA(2)ß. These in vitro studies help determine the regulation and function of the cPLA(2) family members.
Assuntos
Fosfolipases A2 do Grupo IV/metabolismo , Fosfolipases A2 Citosólicas/metabolismo , Fosfolipídeos/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Sítios de Ligação/genética , Biocatálise/efeitos dos fármacos , Cálcio/metabolismo , Cálcio/farmacologia , Linhagem Celular , Eletroforese em Gel de Poliacrilamida , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Fosfolipases A2 do Grupo IV/química , Fosfolipases A2 do Grupo IV/genética , Humanos , Hidrólise/efeitos dos fármacos , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Cinética , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Molecular , Mutação , Fosfolipases A2 Citosólicas/química , Fosfolipases A2 Citosólicas/genética , Fosfolipídeos/química , Ligação Proteica/efeitos dos fármacos , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , SpodopteraRESUMO
Resident tissue macrophages are activated by the fungal pathogen Candida albicans to release eicosanoids, which are important modulators of inflammation and immune responses. Our objective was to identify the macrophage receptors engaged by C. albicans that mediate activation of group IVA cytosolic phospholipase A(2) (cPLA(2)α), a regulatory enzyme that releases arachidonic acid (AA) for production of prostaglandins and leukotrienes. A comparison of peritoneal macrophages from wild type and knock-out mice demonstrates that the ß-glucan receptor Dectin-1 and MyD88 regulate early release of AA and eicosanoids in response to C. albicans. However, cyclooxygenase 2 (COX2) expression and later phase eicosanoid production are defective in MyD88(-/-) but not Dectin-1(-/-) macrophages. Furthermore, C. albicans-stimulated activation of MAPK and phosphorylation of cPLA(2)α on Ser-505 are regulated by MyD88 and not Dectin-1. In contrast, Dectin-1 mediates MAPK activation, cPLA(2)α phosphorylation, and COX2 expression in response to particulate ß-glucan suggesting that other receptors engaged by C. albicans preferentially mediate these responses. Results also implicate the mannan-binding receptor Dectin-2 in regulating cPLA(2)α. C. albicans-stimulated MAPK activation and AA release are blocked by d-mannose and Dectin-2-specific antibody, and overexpression of Dectin-2 in RAW264.7 macrophages enhances C. albicans-stimulated MAPK activation, AA release, and COX2 expression. In addition, calcium mobilization is enhanced in RAW264.7 macrophages overexpressing Dectin-1 or -2. The results demonstrate that C. albicans engages both ß-glucan and mannan-binding receptors on macrophages that act with MyD88 to regulate the activation of cPLA(2)α and eicosanoid production.