RESUMEN

Candida albicans is an increasingly important pulmonary fungal pathogen. Resident alveolar macrophages are important in host defense against opportunistic fungal infections. Activation of Group IVA cytosolic phospholipase A(2)alpha (cPLA(2)alpha) in macrophages initiates arachidonic acid (AA) release for production of eicosanoids, which regulate inflammation and immune responses. We investigated the ability of C. albicans to activate cPLA(2)alpha in unprimed alveolar macrophages and after priming with granulocyte macrophage colony-stimulating factor (GM-CSF), which regulates alveolar macrophage maturation. AA was released within minutes by GM-CSF-primed but not unprimed alveolar macrophages in response to C. albicans, and was blocked by soluble glucan phosphate (S-GP). The expression of the beta-glucan receptor dectin-1 was increased in GM-CSF-primed macrophages, and AA release from GM-CSF-primed dectin-1(-/-) alveolar macrophages was reduced to basal levels. The enhanced activation of extracellular signal-regulated kinases and phosphorylation of cPLA(2)alpha on Ser-505 that occurred in GM-CSF-primed macrophages were reduced by MEK1 and Syk inhibitors, which also suppressed AA release. At later times after C. albicans infection (6 h), unprimed and GM-CSF-primed macrophages released similar levels of AA. The expression of cyclooxygenase 2 and prostanoid production at 6 hours was higher in GM-CSF-primed macrophages, but the responses were not dependent on dectin-1. However, dectin-1 contributed to the C. albicans-stimulated increase in TNF-alpha production that occurred in GM-CSF-primed macrophages. The results demonstrate that dectin-1 mediates the acute activation of cPLA(2)alpha in GM-CSF-primed alveolar macrophages, but not in the more delayed phase of AA release and GM-CSF-dependent prostanoid production.

Asunto(s)

Ácido Araquidónico/inmunología , Candida albicans/inmunología , Candidiasis/inmunología , Fosfolipasas A2 Grupo IV/inmunología , Macrófagos Alveolares/inmunología , Proteínas de la Membrana/inmunología , Proteínas del Tejido Nervioso/inmunología , Animales , Ácido Araquidónico/metabolismo , Candidiasis/enzimología , Candidiasis/genética , Activación Enzimática/efectos de los fármacos , Activación Enzimática/genética , Activación Enzimática/inmunología , Factor Estimulante de Colonias de Granulocitos y Macrófagos/inmunología , Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Fosfolipasas A2 Grupo IV/metabolismo , Péptidos y Proteínas de Señalización Intracelular/inmunología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Lectinas Tipo C , MAP Quinasa Quinasa 1/inmunología , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Sistema de Señalización de MAP Quinasas/genética , Sistema de Señalización de MAP Quinasas/inmunología , Macrófagos Alveolares/enzimología , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos ICR , Ratones Noqueados , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Fosforilación/efectos de los fármacos , Fosforilación/genética , Fosforilación/inmunología , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Tirosina Quinasas/inmunología , Proteínas Tirosina Quinasas/metabolismo , Quinasa Syk , Factores de Tiempo , Factor de Necrosis Tumoral alfa/inmunología , Factor de Necrosis Tumoral alfa/metabolismo

RESUMEN

Group IVA cytosolic phospholipase A(2) (cPLA(2)alpha) is regulated by phosphorylation and calcium-induced translocation to membranes. Immortalized mouse lung fibroblasts lacking endogenous cPLA(2)alpha (IMLF(-/-)) were reconstituted with wild type and cPLA(2)alpha mutants to investigate how calcium, phosphorylation, and the putative phosphatidylinositol 4,5-bisphosphate (PIP(2)) binding site regulate translocation and arachidonic acid (AA) release. Agonists that elicit distinct modes of calcium mobilization were used. Serum induced cPLA(2)alpha translocation to Golgi within seconds that temporally paralleled the initial calcium transient. However, the subsequent influx of extracellular calcium was essential for stable binding of cPLA(2)alpha to Golgi and AA release. In contrast, phorbol 12-myristate 13-acetate induced low amplitude calcium oscillations, slower translocation of cPLA(2)alpha to Golgi, and much less AA release, which were blocked by chelating extracellular calcium. AA release from IMLF(-/-) expressing phosphorylation site (S505A) and PIP(2) binding site (K488N/K543N/K544N) mutants was partially reduced compared with cells expressing wild type cPLA(2)alpha, but calcium-induced translocation was not impaired. Consistent with these results, Ser-505 phosphorylation did not change the calcium requirement for interfacial binding and catalysis in vitro but increased activity by 2-fold. Mutations in basic residues in the catalytic domain of cPLA(2)alpha reduced activation by PIP(2) but did not affect the concentration of calcium required for interfacial binding or phospholipid hydrolysis. The results demonstrate that Ser-505 phosphorylation and basic residues in the catalytic domain principally act to regulate cPLA(2)alpha hydrolytic activity.

Asunto(s)

Dominio Catalítico , Fosfolipasas A2 Grupo IV/química , Fosfolipasas A2 Grupo IV/metabolismo , Animales , Calcio/metabolismo , Células Cultivadas , Medio de Cultivo Libre de Suero , Activación Enzimática/efectos de los fármacos , Regulación de la Expresión Génica , Fosfolipasas A2 Grupo IV/genética , Humanos , Cinética , Ratones , Ratones Noqueados , Mutación/efectos de los fármacos , Fosforilación , Unión Proteica , Transporte de Proteínas , Acetato de Tetradecanoilforbol/análogos & derivados , Acetato de Tetradecanoilforbol/farmacología

RESUMEN

Group IVA cytosolic phospholipase A(2) (cPLA(2)alpha) initiates eicosanoid production; however, this pathway is not completely ablated in cPLA(2)alpha(-/-) lung fibroblasts stimulated with A23187 or serum. cPLA(2)alpha(+/+) fibroblasts preferentially released arachidonic acid, but A23187-stimulated cPLA(2)alpha(-/-) fibroblasts nonspecifically released multiple fatty acids. Arachidonic acid release from cPLA(2) alpha(-/-) fibroblasts was inhibited by the cPLA(2)alpha inhibitors pyrrolidine-2 (IC(50), 0.03 microM) and Wyeth-1 (IC(50), 0.1 microM), implicating another C2 domain-containing group IV PLA(2). cPLA(2) alpha(-/-) fibroblasts contain cPLA(2)beta and cPLA(2)zeta but not cPLA(2)epsilon or cPLA(2)delta. Purified cPLA(2)zeta exhibited much higher lysophospholipase and PLA(2) activity than cPLA(2)beta and was potently inhibited by pyrrolidine-2 and Wyeth-1, which did not inhibit cPLA(2)beta. In contrast to cPLA(2)beta, cPLA(2)zeta expressed in Sf9 cells mediated A23187-induced arachidonic acid release, which was inhibited by pyrrolidine-2 and Wyeth-1. cPLA(2)zeta exhibits specific activity, inhibitor sensitivity, and low micromolar calcium dependence similar to cPLA(2)alpha and has been identified as the PLA(2) responsible for calcium-induced fatty acid release and prostaglandin E(2) production from cPLA(2) alpha(-/-) lung fibroblasts. In response to ionomycin, EGFP-cPLA(2)zeta translocated to ruffles and dynamic vesicular structures, whereas EGFP-cPLA(2)alpha translocated to the Golgi and endoplasmic reticulum, suggesting distinct mechanisms of regulation for the two enzymes.

Asunto(s)

Ácido Araquidónico/metabolismo , Fibroblastos/metabolismo , Fosfolipasas A/fisiología , Fosfolipasas de Tipo C/fisiología , Secuencia de Aminoácidos , Animales , Citosol/metabolismo , Retículo Endoplásmico/metabolismo , Aparato de Golgi/metabolismo , Fosfolipasas A2 Grupo IV , Insectos , Pulmón/metabolismo , Ratones , Datos de Secuencia Molecular , Fosfoinositido Fosfolipasa C , Fosfolipasas A/genética , Fosfolipasas A/metabolismo , Transporte de Proteínas , Homología de Secuencia de Aminoácido , Fosfolipasas de Tipo C/metabolismo

RESUMEN

In this study, we identify the principal splice variant of human cytosolic phospholipase A(2)beta (cPLA(2)beta) (also known as Group IVB cPLA(2)) present in cells. In human lung, spleen, and ovary and in a lung epithelial cell line (BEAS-2B), cPLA(2)beta is expressed as a 100-kDa protein, not the 114-kDa form originally predicted. Using RNA interference, the 100-kDa protein in BEAS-2B cells was confirmed to be cPLA(2)beta. BEAS-2B cells contain three different RNA splice variants of cPLA(2)beta (beta1, beta2, and beta3). cPLA(2)beta1 is identical to the previously cloned cPLA(2)beta, predicted to encode a 114-kDa protein. However, cPLA(2)beta2 and cPLA(2)beta3 splice variants are smaller and contain internal deletions in the catalytic domain. The 100-kDa cPLA(2)beta in BEAS-2B cells is the translated product of cPLA(2)beta3. cPLA(2)beta3 exhibits calcium-dependent PLA(2) activity against palmitoyl-arachidonyl-phosphatidylethanolamine and low level lysophospholipase activity but no activity against phosphatidylcholine. Unlike Group IVA cPLA(2)alpha, cPLA(2)beta3 is constitutively bound to membrane in unstimulated cells, localizing to mitochondria and early endosomes. cPLA(2)beta3 is widely expressed in tissues, suggesting that it has a generalized function at these unique sites.

Asunto(s)

Endosomas/metabolismo , Mitocondrias/metabolismo , Fosfolipasas A/química , Empalme Alternativo , Secuencia de Aminoácidos , Fosfolipasas A2 Grupo IV , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Fosfatidilcolinas/química , Estructura Terciaria de Proteína , ARN/química , Proteínas Recombinantes/química , Homología de Secuencia de Aminoácido , Distribución Tisular

RESUMEN

Phagocytosis of non-opsonized microorganisms by macrophages initiates innate immune responses for host defense against infection. Cytosolic phospholipase A(2) is activated during phagocytosis, releasing arachidonic acid for production of eicosanoids, which initiate acute inflammation. Our objective was to identify pattern recognition receptors that stimulate arachidonic acid release and cyclooxygenase 2 (COX2) expression in macrophages by pathogenic yeast and yeast cell walls. Zymosan- and Candida albicans-stimulated arachidonic acid release from resident mouse peritoneal macrophages was blocked by soluble glucan phosphate. In RAW264.7 cells arachidonic acid release, COX2 expression, and prostaglandin production were enhanced by overexpressing the beta-glucan receptor, dectin-1, but not dectin-1 lacking the cytoplasmic tail. Pure particulate (1, 3)-beta-D-glucan stimulated arachidonic acid release and COX2 expression, which were augmented in a Toll-like receptor 2 (TLR2)-dependent manner by macrophage-activating lipopeptide-2. However, arachidonic acid release and leukotriene C(4) production stimulated by zymosan and C. albicans were TLR2-independent, whereas COX2 expression and prostaglandin production were partially blunted in TLR2(-/-) macrophages. Inhibition of Syk tyrosine kinase blocked arachidonic acid release and COX2 expression in response to zymosan, C. albicans, and particulate (1, 3)-beta-D-glucan. The results suggest that cytosolic phospholipase A(2) activation triggered by the beta-glucan component of yeast is dependent on the immunoreceptor tyrosine-based activation motif-like domain of dectin-1 and activation of Syk kinase, whereas both TLR2 and Syk kinase regulate COX2 expression.

Asunto(s)

Ciclooxigenasa 2/metabolismo , Macrófagos Peritoneales/metabolismo , Fosfolipasas A/metabolismo , Receptores Inmunológicos/metabolismo , Animales , Ácido Araquidónico/metabolismo , Candida albicans/metabolismo , Células Cultivadas , Eicosanoides/metabolismo , Activación Enzimática , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Lectinas Tipo C , Macrófagos Peritoneales/citología , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas del Tejido Nervioso/metabolismo , Fosfolipasas A2 , Proteínas Tirosina Quinasas/metabolismo , Quinasa Syk , Receptor Toll-Like 2/genética , Receptor Toll-Like 2/metabolismo , Zimosan/metabolismo , Familia-src Quinasas/metabolismo

RESUMEN

PURPOSE: Vascular endothelial growth factor (VEGF) plays an important role in initiation of the angiogenesis that leads to proliferative retinopathy. Several environmental conditions and chemical agents that influence the expression of VEGF can also cause endoplasmic reticulum (ER) stress. The hypothesis for the current study was that expression of VEGF is responsive to conditions that cause ER stress, including amino acid deprivation. METHODS: Confluent cultures of a human retinal pigmented epithelial cell line (ARPE-19) were deprived of amino acids or treated with chemical inducers of ER stress. Treatment with cobalt was used to mimic hypoxia-induced expression of VEGF. Northern blot analysis was used to measure intracellular VEGF mRNA, and ELISA was used to measure secreted VEGF protein. Glucose-regulated protein 78 (GRP78) mRNA levels were compared with those of VEGF. Glyceraldehyde-phosphate dehydrogenase (GAPDH) mRNA was used as a control. RESULTS: Conditions and chemical agents known to activate ER stress response (ERSR) pathways also induced the expression of VEGF. Deprivation of amino acids in the culture medium increased VEGF mRNA expression by 1.3- to 6-fold. Glucose deprivation or treatment of ARPE-19 cells with tunicamycin, brefeldin A, the calcium ionophore A23187, or thapsigargin increased the expression of VEGF mRNA in these cells by 8- to 10-fold. Expression of GRP78 mRNA was well correlated with that of VEGF mRNA under all conditions. These treatments also increased the secretion of VEGF protein by up to twofold. The increase in VEGF mRNA level in response to glutamine deprivation was rapid (greater than 10-fold) and was observed in a physiologically relevant range of glutamine concentrations. The half-life of VEGF mRNA was increased 2.5-fold by glutamine starvation. CONCLUSIONS: These results indicate that VEGF is an ER stress-responsive gene and suggest that cells can respond to nutrient deprivation by increasing VEGF expression through both transcriptional and posttranscriptional mechanisms.

Asunto(s)

Aminoácidos/deficiencia , Hipoxia de la Célula/fisiología , Retículo Endoplásmico/fisiología , Factores de Crecimiento Endotelial/metabolismo , Proteínas de Choque Térmico , Linfocinas/metabolismo , Epitelio Pigmentado Ocular/metabolismo , Estrés Fisiológico/metabolismo , Northern Blotting , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Células Cultivadas , Chaperón BiP del Retículo Endoplásmico , Factores de Crecimiento Endotelial/genética , Ensayo de Inmunoadsorción Enzimática , Gliceraldehído-3-Fosfato Deshidrogenasas/genética , Gliceraldehído-3-Fosfato Deshidrogenasas/metabolismo , Humanos , Linfocinas/genética , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , ARN Mensajero/metabolismo , Factor A de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial Vascular