Overexpression of cytosolic group IVA phospholipase A2 protects cells from Ca2+ -dependent death

El pdf del artículo es la versión post-print.-- et al.The calcium ionophore ionomycin induces apoptosis-like events in the human embryonic kidney cell line at early times. Plasma membrane blebbing, mitochondrial depolarization, externalization of phosphatidylserine, and nuclear permeability changes can all be observed within 15 min of treatment. However, there is no activation of caspases or chromatin condensation. Expression of a fusion protein containing the enhanced green fluorescent protein (EGFP) and human cytosolic Group IVA phospholipase A2α (EGFP-cPLA 2α) in these cells prevents ionomycin-induced phosphatidylserine externalization and death. Cells expressing the cPLA 2α mutant D43N, which does not bind calcium, retain their susceptibility to ionomycin-induced cell death. Both nonexpressing and EGFP-D43N-cPLA2α-expressing human embryonic kidney cells can be spared from ionomycin-induced cell death by pretreating them with exogenous arachidonic acid. Moreover, during calcium overload, mitochondrial depolarization is significantly lower in the EGFP-cPLA2α- expressing cells than in cells expressing normal amounts of cPLA 2α. These results suggest that early cell death events promoted by an overload of calcium can be prevented by the presence of high levels of arachidonic acid. © 2006 by The American Society for Biochemistry and Molecular Biology, Inc.This work was supported in part by Grant BMC2001- 2244 from the Spanish Ministry of Science and Technology, Grants BFU2004-01886/BMC, SAF2004-04676, and SAF2004-01232 from the Spanish Ministry of Education and Science, and Red Brucella, Red Respira, and Red Temática de Investigación Cardiovascular, from the Instituto de Salud Carlos III.Peer Reviewe

Differential regulation of phospholipase D and phospholipase A2 by protein kinase C in P388D1 macrophages

Activation of P388D1 macrophages by phorbol myristate acetate (PMA) resulted in the translocation of the protein kinase C (PKC) isoforms alpha, delta, and epsilon from the cytosol to membranes. Furthermore, PMA activated phospholipase D (PLD) in these cells, and potentiated the effect of the inflammatory lipid mediator platelet-activating factor (PAF) on PLD activation. PAF also activated phospholipase A2 (PLA2) and enhanced arachidonic acid (AA) release in P388D1 macrophages, and bacterial lipopolysaccharide (LPS) increased the responsiveness of these cells to PAF. In contrast with PLD, PLA2 activation in P388D1 macrophages was found to take place independently of PKC. This was supported by the following evidence: (i) PMA neither induced AA release nor enhanced the PAF response; (ii) inclusion of PMA along with LPS during priming did not have any effect on PAF-stimulated AA release; (iii) down-regulation of PMA-activatable PKC isoforms by chronic treatment with the phorbol ester had no effect on the PAF response; and (iv) the PKC inhibitor staurosporine did not alter the PAF-induced AA release. The present study provides an example of cells in which the direct activation of PKC by phorbol esters does not lead to a primed and/or enhanced AA release. As a unique example in which PKC activation is neither necessary nor sufficient for AA release to occur, this now allows study of the separate and distinct roles for PLD and PLA2 in signal-transduction processes. This has hitherto been difficult to achieve because of the lack of specific inhibitors of these two phospholipases

Lipin-1 integrates lipid synthesis with proinflammatory responses during TLR activation in macrophages

Lipin-1 is a Mg2+-dependent phosphatidic acid phosphatase involved in the de novo synthesis of phospholipids and triglycerides. Using macrophages from lipin-1-deficient animals and human macrophages deficient in the enzyme, we show in this work that this phosphatase acts as a proinflammatory mediator during TLR signaling and during the development of in vivo inflammatory processes. After TLR4 stimulation lipin-1-deficient macrophages showed a decreased production of diacylglycerol and activation of MAPKs and AP-1. Consequently, the generation of proinflammatory cytokines like IL-6, IL-12, IL-23, or enzymes like inducible NO synthase and cyclooxygenase 2, was reduced. In addition, animals lacking lipin-1 had a faster recovery from endotoxin administration concomitant with a reduced production of harmful molecules in spleen and liver. These findings demonstrate an unanticipated role for lipin-1 as a mediator of macrophage proinflammatory activation and support a critical link between lipid biosynthesis and systemic inflammatory responses.This work was supported by the Spanish Ministry of Science and Innovation (Grants SAF2007-60055, SAF2010-18831, and BFU2010-18826) and the Regional Government of Castile and Leon (Grants BIO39/VA04/10 and CSI168A12-1). L.P. and G.L. were supported by predoctoral fellowships from the Spanish Ministry of Science and Innovation (Plan de Formación de Personal Investigador and Plan de Formación de Profesorado Universitario programs). M.V. was supported by a predoctoral fellowship from the Regional Government of Castile and Leon. E.E. was supported by a predoctoral fellowship from the Spanish National Research Council (Junta de Ampliación de Estudios Program). C.G. was supported by a predoctoral fellowship from the University of Valladolid.Peer Reviewe

The hypoxic microenvironment induces stearoyl-coa desaturase-1 overexpression and lipidomic profile changes in clear cell renal cell carcinoma

Clear cell renal cell carcinoma (ccRCC) is the most common histological subtype of renal cell carcinoma (RCC). It is characterized by a high cell proliferation and the ability to store lipids. Previous studies have demonstrated the overexpression of enzymes associated with lipid metabolism, including stearoyl-CoA desaturase-1 (SCD-1), which increases the concentration of unsaturated fatty acids in tumor cells. In this work, we studied the expression of SCD-1 in primary ccRCC tumors, as well as in cell lines, to determine its influence on the tumor lipid composition and its role in cell proliferation. The lipidomic analyses of patient tumors showed that oleic acid (18:1n-9) is one of the major fatty acids, and it is particularly abundant in the neutral lipid fraction of the tumor core. Using a ccRCC cell line model and in vitro-generated chemical hypoxia, we show that SCD-1 is highly upregulated (up to 200-fold), and this causes an increase in the cellular level of 18:1n-9, which, in turn, accumulates in the neutral lipid fraction. The pharmacological inhibition of SCD-1 blocks 18:1n-9 synthesis and compromises the proliferation. The addition of exogenous 18:1n-9 to the cells reverses the effects of SCD-1 inhibition on cell proliferation. These data reinforce the role of SCD-1 as a possible therapeutic target.Fil: Melana Colavita, Juan Pablo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste. Instituto de Química Básica y Aplicada del Nordeste Argentino. Universidad Nacional del Nordeste. Facultad de Ciencias Exactas Naturales y Agrimensura. Instituto de Química Básica y Aplicada del Nordeste Argentino; ArgentinaFil: Mignolli, Francesco. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste. Instituto de Botánica del Nordeste. Universidad Nacional del Nordeste. Facultad de Ciencias Agrarias. Instituto de Botánica del Nordeste; ArgentinaFil: Stoyanoff, Tania Romina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste. Instituto de Química Básica y Aplicada del Nordeste Argentino. Universidad Nacional del Nordeste. Facultad de Ciencias Exactas Naturales y Agrimensura. Instituto de Química Básica y Aplicada del Nordeste Argentino; ArgentinaFil: Aguirre, María Victoria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste. Instituto de Química Básica y Aplicada del Nordeste Argentino. Universidad Nacional del Nordeste. Facultad de Ciencias Exactas Naturales y Agrimensura. Instituto de Química Básica y Aplicada del Nordeste Argentino; ArgentinaFil: Balboa, María A.. Consejo Superior de Investigaciones Científicas; España. Universidad de Valladolid; EspañaFil: Balsinde Rodríguez, Jesús. Consejo Superior de Investigaciones Científicas; España. Universidad de Valladolid; EspañaFil: Rodríguez, Juan Pablo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste. Instituto de Química Básica y Aplicada del Nordeste Argentino. Universidad Nacional del Nordeste. Facultad de Ciencias Exactas Naturales y Agrimensura. Instituto de Química Básica y Aplicada del Nordeste Argentino; Argentin

ISG15 Is a Novel Regulator of Lipid Metabolism during Vaccinia Virus Infection.

Interferon-stimulated gene 15 (ISG15) is a 15-kDa ubiquitin-like modifier that binds to target proteins in a process termed ISGylation. ISG15, first described as an antiviral molecule against many viruses, participates in numerous cellular processes, from immune modulation to the regulation of genome stability. Interestingly, the role of ISG15 as a regulator of cell metabolism has recently gained strength. We previously described ISG15 as a regulator of mitochondrial functions in bone marrow-derived macrophages (BMDMs) in the context of Vaccinia virus (VACV) infection. Here, we demonstrate that ISG15 regulates lipid metabolism in BMDMs and that ISG15 is necessary to modulate the impact of VACV infection on lipid metabolism. We show that Isg15-/- BMDMs demonstrate alterations in the levels of several key proteins of lipid metabolism that result in differences in the lipid profile compared with Isg15+/+ (wild-type [WT]) BMDMs. Specifically, Isg15-/- BMDMs present reduced levels of neutral lipids, reflected by decreased lipid droplet number. These alterations are linked to increased levels of lipases and are independent of enhanced fatty acid oxidation (FAO). Moreover, we demonstrate that VACV causes a dysregulation in the proteomes of BMDMs and alterations in the lipid content of these cells, which appear exacerbated in Isg15-/- BMDMs. Such metabolic changes are likely caused by increased expression of the metabolic regulators peroxisome proliferator-activated receptor-γ (PPARγ) and PPARγ coactivator-1α (PGC-1α). In summary, our results highlight that ISG15 controls BMDM lipid metabolism during viral infections, suggesting that ISG15 is an important host factor to restrain VACV impact on cell metabolism. IMPORTANCE The functions of ISG15 are continuously expanding, and growing evidence supports its role as a relevant modulator of cell metabolism. In this work, we highlight how the absence of ISG15 impacts macrophage lipid metabolism in the context of viral infections and how poxviruses modulate metabolism to ensure successful replication. Our results open the door to new advances in the comprehension of macrophage immunometabolism and the interaction between VACV and the host.We thank the expert technical assistance of Sara Sandoval. We are grateful to Miguel Sánchez-Álvarez who has kindly provided several commercial reagents. We would like to thank the Spanish National Plan for Scientific and Technical Research and Innovation (Plan Estatal de Investigación Científica y Técnica y de Innovación), (Ministry of Health of Spain, State Secretary of R1D and FEDER/FSE).S

Lipin-2 reduces proinflammatory signaling induced by saturated fatty acids in macrophages

Lipin-2 is a member of the lipin family of enzymes, which are key effectors in the biosynthesis of lipids. Mutations in the humanlipin-2 gene are associated with inflammatory-based disorders; however, the role of lipin-2 in cells of the immune system remains obscure. In this study, we have investigated the role of lipin-2 in the proinflammatory action of saturated fatty acids in murine and human macrophages. Depletion of lipin-2 promotes the increased expression of the proinflammatory genes Il6, Ccl2, and Tnfα, which depends on the overstimulation of the JNK1/c-Jun pathway by saturated fatty acids. In contrast, overexpression of lipin-2 reduces the release of proinflammatory factors. Metabolically, the absence of lipin-2 reduces the cellular content of triacylglycerol in saturated fatty acid-overloaded macrophages. Collectively, these studies demonstrate a protective role for lipin-2 in proinflammatory signaling mediated by saturated fatty acids that occurs concomitant with an enhanced cellular capacity for triacylglycerol synthesis. The data provide new insights into the role of lipin-2 in human and murine macrophage biology and may open new avenues for controlling the fatty acid-related low grade inflammation that constitutes the sine qua non of obesity and associated metabolic disorders. © 2012 by The American Society for Biochemistry and Molecular Biology, Inc.This work was supported in part by Spanish Ministry of Science and Innovation Grants SAF2010-18831 and BFU2010-18826 and Regional Government of Castile and Leon Grant BIO39/VA04/10. Supported by a predoctoral fellowship from the Regional Government of Castile and Leon, Spain. Supported by a predoctoral fellowship from the Spanish National Research Council (Junta de Ampliación de Estudios Program). Supported by a predoctoral fellowship from the Spanish Ministry of Science and Innovation.Peer Reviewe

Mycobacterium tuberculosis multi-drug-resistant strain M induces IL-17+ IFNγ- CD4+ T cell expansion through an IL-23 and TGF-β-dependent mechanism in patients with MDR-TB tuberculosis

We have previously reported that T cells from patients with multidrug-resistant tuberculosis (MDR-TB) express high levels of IL-17 in response to the MDR strain M(Haarlem family) of Mycobacterium tuberculosis (M.tuberculosis). Herein, we explore the pathways involved in the induction of h17 cells in MDR-TB patients and healthy tuberculin reactors (PPD+HD) by the M strain and the laboratory strain H37Rv. Our results show that IL-1β and IL-6 are crucial for the H37Rv and M-induced expansion of IL-17+IFNγ¯ and IL-17+IFNγ+ in CD4+ T cells from MDR-TB and PPD+HD. IL-23 plays an ambiguous role in Th1 and Th17 profiles: alone, IL-23 is responsible for M.tuberculosis induced IL-17 and IFNγ expression in CD4+ T cells from PPD+HD whereas, together with TGF-β, it promotes IL-17+IFNγ¯ expansion in MDR-TB. In fact, spontaneous and M.tuberculosis-induced TGF-β secretion is increased in cells from MDR-TB being theM strain the highest inducer. Interestingly, TLR-2 signaling mediates the expansion of IL-17+IFNγ¯ cells and the enhancement of latency-associated protein (LAP) expression in CD14+ and CD4+ T cells from MDR-TB, which suggests that M strain promotes IL-17+IFNγ¯ T cells through a strong TLR-2-dependent TGF-β production by antigenpresenting cells and CD4+ T cells. Finally, CD4+ T cells from MDR-TB patients infected with MDR Haarlem strains show higher IL-17+IFNγ¯ and lower IL-17+IFNγ+ levels than LAM-infected patients. The present findings deepen our understanding on the role of IL-17 in MDR-TB and highlight the influence of the genetic background of the infecting M.tuberculosis strain on the ex vivo Th17 response.Fil: Basile, Juan Ignacio. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Kviatcovsky, Denise. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Romero, María Mercedes. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Balboa, Luciana. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Monteserin, Johana. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud “Dr. C. G. Malbrán”; ArgentinaFil: Ritacco, Gloria Viviana. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud “Dr. C. G. Malbrán”; ArgentinaFil: López, Beatriz. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud “Dr. C. G. Malbrán”; ArgentinaFil: Sabio y García, Carmen Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: García, A.. Gobierno de la Ciudad de Buenos Aires. Hospital de Infecciosas “Dr. Francisco Javier Muñiz”; ArgentinaFil: Vescovo, M.. Gobierno de la Ciudad de Buenos Aires. Hospital de Infecciosas “Dr. Francisco Javier Muñiz”; ArgentinaFil: Gonzalez Montaner, Pablo. Gobierno de la Ciudad de Buenos Aires. Hospital de Infecciosas “Dr. Francisco Javier Muñiz”; ArgentinaFil: Palmero, Domingo. Gobierno de la Ciudad de Buenos Aires. Hospital de Infecciosas “Dr. Francisco Javier Muñiz”; ArgentinaFil: Sasiain, María del Carmen. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: de la Barrera, Silvia Susana. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentin

Essential Role for Ethanolamine Plasmalogen Hydrolysis in Bacterial Lipopolysaccharide Priming of Macrophages for Enhanced Arachidonic Acid Release

Due to their high content in esterified arachidonic acid (AA), macrophages provide large amounts of eicosanoids during innate immune reactions. Bacterial lipopolysaccharide (LPS) is a poor trigger of AA mobilization in macrophages but does have the capacity to prime these cells for greatly increased AA release upon subsequent stimulation. In this work, we have studied molecular mechanisms underlying this phenomenon. By using mass spectrometry-based lipidomic analyses, we show in this work that LPS-primed zymosan-stimulated macrophages exhibit an elevated consumption of a particular phospholipid species, i.e., the ethanolamine plasmalogens, which results from reduced remodeling of phospholipids via coenzyme A-independent transacylation reactions. Importantly however, LPS-primed macrophages show no changes in their capacity to directly incorporate AA into phospholipids via CoA-dependent acylation reactions. The essential role for ethanolamine plasmalogen hydrolysis in LPS priming is further demonstrated by the use of plasmalogen-deficient cells. These cells, while responding normally to zymosan by releasing quantities of AA similar to those released by cells expressing normal plasmalogen levels under the same conditions, fail to show an LPS-primed response to the same stimulus, thus unambiguously demonstrating a cause–effect relationship between LPS priming and plasmalogen hydrolysis. Collectively, these results suggest a hitherto unrecognized role for ethanolamine plasmalogen hydrolysis and CoA-independent transacylation reactions in modulating the eicosanoid biosynthetic response

Regulation of Phagocytosis in Macrophages by Membrane Ethanolamine Plasmalogens

Macrophages, as professional phagocytes of the immune system, possess the ability to detect and clear invading pathogens and apoptotic cells through phagocytosis. Phagocytosis involves membrane reorganization and remodeling events on the cell surface, which play an essential role in innate immunity and tissue homeostasis and the control of inflammation. In this work, we report that cells deficient in membrane ethanolamine plasmalogen demonstrate a reduced capacity to phagocytize opsonized zymosan particles. Amelioration of plasmalogen deficiency in these cells by incubation with lysoplasmalogen results in a significant augmentation of the phagocytic capacity of the cells. In parallel with these increases, restoration of plasmalogen levels in the cells also increases the number and size of lipid rafts in the membrane, reduces membrane fluidity down to levels found in cells containing normal plasmalogen levels, and improves receptor-mediated signaling. Collectively, these results suggest that membrane plasmalogen level determines characteristics of the plasma membrane such as fluidity and the formation of microdomains that are necessary for efficient signal transduction leading to optimal phagocytosis by macrophages

Lipid droplet biogenesis induced by stress involves triacylglycerol synthesis that depends on group VIA phospholipase A2

This work investigates the metabolic origin of triacylglycerol (TAG) formed during lipid droplet (LD) biogenesis induced by stress. Cytotoxic inhibitors of fatty acid synthase induced TAG synthesis and LD biogenesis in CHO-K1 cells, in the absence of external sources of fatty acids. TAG synthesis was required for LD biogenesis and was sensitive to inhibition and down-regulation of the expression of group VIA phospholipase A2 (iPLA2-VIA). Induction of stress with acidic pH, C2-ceramide, tunicamycin, or deprivation of glucose also stimulated TAG synthesis and LD formation in a manner dependent on iPLA2-VIA. Overexpression of the enzyme enhanced TAG synthesis from endogenous fatty acids and LD occurrence. During stress, LD biogenesis but not TAG synthesis required phosphorylation and activation of group IVA PLA2 (cPLA2α). The results demonstrate that iPLA2-VIA provides fatty acids for TAG synthesis while cPLA2α allows LD biogenesis. LD biogenesis during stress may be a survival strategy, recycling structural phospholipids into energy-generating substrates