RESUMEN
Type 1 diabetes (T1D) development, in part, is due to ER stress-induced ß-cell apoptosis. Activation of the Ca2+-independent phospholipase A2 beta (iPLA2ß) leads to the generation of pro-inflammatory eicosanoids, which contribute to ß-cell death and T1D. ER stress induces iPLA2ß-mediated generation of pro-apoptotic ceramides via neutral sphingomyelinase (NSMase). To gain a better understanding of the impact of iPLA2ß on sphingolipids (SLs), we characterized their profile in ß-cells undergoing ER stress. ESI/MS/MS analyses followed by ANOVA/Student's t-test were used to assess differences in sphingolipids molecular species in Vector (V) control and iPLA2ß-overexpressing (OE) INS-1 and Akita (AK, spontaneous model of ER stress) and WT-littermate (AK-WT) ß-cells. As expected, iPLA2ß induction was greater in the OE and AK cells in comparison with V and WT cells. We report here that ER stress led to elevations in pro-apoptotic and decreases in pro-survival sphingolipids and that the inactivation of iPLA2ß restores the sphingolipid species toward those that promote cell survival. In view of our recent finding that the SL profile in macrophages-the initiators of autoimmune responses leading to T1D-is not significantly altered during T1D development, we posit that the iPLA2ß-mediated shift in the ß-cell sphingolipid profile is an important contributor to ß-cell death associated with T1D.
Asunto(s)
Apoptosis , Estrés del Retículo Endoplásmico , Células Secretoras de Insulina/metabolismo , Lipasa/metabolismo , Proteínas de la Membrana/metabolismo , Esfingolípidos/metabolismo , Apoptosis/genética , Línea Celular , Cromatografía Liquida , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/metabolismo , Estrés del Retículo Endoplásmico/genética , Expresión Génica , Humanos , Lipasa/genética , Lipidómica/métodos , Proteínas de la Membrana/genética , Modelos Biológicos , Espectrometría de Masas en TándemRESUMEN
Phospholipases A2 (PLA2s) catalyze hydrolysis of the sn-2 substituent from glycerophospholipids to yield a free fatty acid (i.e., arachidonic acid), which can be metabolized to pro- or anti-inflammatory eicosanoids. Macrophages modulate inflammatory responses and are affected by Ca2+-independent phospholipase A2 (PLA2)ß (iPLA2ß). Here, we assessed the link between iPLA2ß-derived lipids (iDLs) and macrophage polarization. Macrophages from WT and KO (iPLA2ß-/-) mice were classically M1 pro-inflammatory phenotype activated or alternatively M2 anti-inflammatory phenotype activated, and eicosanoid production was determined by ultra-performance LC ESI-MS/MS. As a genotypic control, we performed similar analyses on macrophages from RIP.iPLA2ß.Tg mice with selective iPLA2ß overexpression in ß-cells. Compared with WT, generation of select pro-inflammatory prostaglandins (PGs) was lower in iPLA2ß-/- , and that of a specialized pro-resolving lipid mediator (SPM), resolvin D2, was higher; both changes are consistent with the M2 phenotype. Conversely, macrophages from RIP.iPLA2ß.Tg mice exhibited an opposite landscape, one associated with the M1 phenotype: namely, increased production of pro-inflammatory eicosanoids (6-keto PGF1α, PGE2, leukotriene B4) and decreased ability to generate resolvin D2. These changes were not linked with secretory PLA2 or cytosolic PLA2α or with leakage of the transgene. Thus, we report previously unidentified links between select iPLA2ß-derived eicosanoids, an SPM, and macrophage polarization. Importantly, our findings reveal for the first time that ß-cell iPLA2ß-derived signaling can predispose macrophage responses. These findings suggest that iDLs play critical roles in macrophage polarization, and we posit that they could be targeted therapeutically to counter inflammation-based disorders.
Asunto(s)
Calcio/metabolismo , Eicosanoides/metabolismo , Fosfolipasas A2 Grupo IV/metabolismo , Macrófagos/metabolismo , Transducción de Señal , Animales , Fosfolipasas A2 Grupo IV/deficiencia , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones TransgénicosRESUMEN
Diabetes is a consequence of reduced ß-cell function and mass, due to ß-cell apoptosis. Endoplasmic reticulum (ER) stress is induced during ß-cell apoptosis due to various stimuli, and our work indicates that group VIA phospholipase A2ß (iPLA2ß) participates in this process. Delineation of underlying mechanism(s) reveals that ER stress reduces the anti-apoptotic Bcl-x(L) protein in INS-1 cells. The Bcl-x pre-mRNA undergoes alternative pre-mRNA splicing to generate Bcl-x(L) or Bcl-x(S) mature mRNA. We show that both thapsigargin-induced and spontaneous ER stress are associated with reductions in the ratio of Bcl-x(L)/Bcl-x(S) mRNA in INS-1 and islet ß-cells. However, chemical inactivation or knockdown of iPLA2ß augments the Bcl-x(L)/Bcl-x(S) ratio. Furthermore, the ratio is lower in islets from islet-specific RIP-iPLA2ß transgenic mice, whereas islets from global iPLA2ß(-/-) mice exhibit the opposite phenotype. In view of our earlier reports that iPLA2ß induces ceramide accumulation through neutral sphingomyelinase 2 and that ceramides shift the Bcl-x 5'-splice site (5'SS) selection in favor of Bcl-x(S), we investigated the potential link between Bcl-x splicing and the iPLA2ß/ceramide axis. Exogenous C6-ceramide did not alter Bcl-x 5'SS selection in INS-1 cells, and neutral sphingomyelinase 2 inactivation only partially prevented the ER stress-induced shift in Bcl-x splicing. In contrast, 5(S)-hydroxytetraenoic acid augmented the ratio of Bcl-x(L)/Bcl-x(S) by 15.5-fold. Taken together, these data indicate that ß-cell apoptosis is, in part, attributable to the modulation of 5'SS selection in the Bcl-x pre-mRNA by bioactive lipids modulated by iPLA2ß.
Asunto(s)
Apoptosis/fisiología , Ceramidas/metabolismo , Fosfolipasas A2 Grupo VI/metabolismo , Células Secretoras de Insulina/metabolismo , Sitios de Empalme de ARN , Empalme del ARN/fisiología , Proteína bcl-X/metabolismo , Animales , Ceramidas/genética , Estrés del Retículo Endoplásmico/fisiología , Fosfolipasas A2 Grupo VI/genética , Humanos , Células Secretoras de Insulina/citología , Ratones , Ratones Noqueados , Ratas , Esfingomielina Fosfodiesterasa/genética , Esfingomielina Fosfodiesterasa/metabolismo , Proteína bcl-X/genéticaRESUMEN
Anaphylaxis is a rapid, life-threatening hypersensitivity reaction. Until recently, it was mainly attributed to histamine released by mast cells activated by allergen crosslinking (XL) of FcεRI-bound allergen-specific IgE. However, recent reports established that anaphylaxis could also be triggered by basophil, macrophage, and neutrophil secretion of platelet-activating factor subsequent to FcγR stimulation by IgG/Ag complexes. We have investigated the contribution of Fyn and Lyn tyrosine kinases to FcγRIIb and FcγRIII signaling in the context of IgG-mediated passive systemic anaphylaxis (PSA). We found that mast cell IgG XL induced Fyn, Lyn, Akt, Erk, p38, and JNK phosphorylation. Additionally, IgG XL of mast cells, basophils, and macrophages resulted in Fyn- and Lyn-regulated mediator release in vitro. FcγR-mediated activation was enhanced in Lyn-deficient (knockout [KO]) cells, but decreased in Fyn KO cells, compared with wild-type cells. More importantly, Lyn KO mice displayed significantly exacerbated PSA features whereas no change was observed for Fyn KO mice, compared with wild-type littermates. Intriguingly, we establish that mast cells account for most serum histamine in IgG-induced PSA. Taken together, our findings establish pivotal roles for Fyn and Lyn in the regulation of PSA and highlight their unsuspected functions in IgG-mediated pathologies.
Asunto(s)
Anafilaxia/inmunología , Inmunoglobulina G/inmunología , Mastocitos/inmunología , Proteínas Proto-Oncogénicas c-fyn/inmunología , Familia-src Quinasas/inmunología , Alérgenos/genética , Alérgenos/inmunología , Anafilaxia/genética , Anafilaxia/patología , Animales , Quinasas MAP Reguladas por Señal Extracelular/genética , Quinasas MAP Reguladas por Señal Extracelular/inmunología , Inmunoglobulina G/genética , MAP Quinasa Quinasa 4/genética , MAP Quinasa Quinasa 4/inmunología , Mastocitos/patología , Ratones , Ratones Noqueados , Fosforilación/genética , Fosforilación/inmunología , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/inmunología , Proteínas Proto-Oncogénicas c-fyn/genética , Receptores de IgG/genética , Receptores de IgG/inmunología , Transducción de Señal/genética , Transducción de Señal/inmunología , Familia-src Quinasas/genéticaRESUMEN
One of the most common molecular changes in cancer is the increased endogenous lipid synthesis, mediated primarily by overexpression and/or hyperactivity of fatty acid synthase (FAS) and acetyl-CoA carboxylase (ACC). The changes in these key lipogenic enzymes are critical for the development and maintenance of the malignant phenotype. Previous efforts to control oncogenic lipogenesis have been focused on pharmacological inhibitors of FAS and ACC. Although they show anti-tumor effects in culture and in mouse models, these inhibitors are nonselective blockers of lipid synthesis in both normal and cancer cells. To target lipid anabolism in tumor cells specifically, it is important to identify the mechanism governing hyperactive lipogenesis in malignant cells. In this study, we demonstrate that lysophosphatidic acid (LPA), a growth factor-like mediator present at high levels in ascites of ovarian cancer patients, regulates the sterol regulatory element binding protein-FAS and AMP-activated protein kinase-ACC pathways in ovarian cancer cells but not in normal or immortalized ovarian epithelial cells. Activation of these lipogenic pathways is linked to increased de novo lipid synthesis. The pro-lipogenic action of LPA is mediated through LPA(2), an LPA receptor subtype overexpressed in ovarian cancer and other malignancies. Downstream of LPA(2), the G(12/13) and G(q) signaling cascades mediate LPA-dependent sterol regulatory element-binding protein activation and AMP-activated protein kinase inhibition, respectively. Moreover, inhibition of de novo lipid synthesis dramatically attenuated LPA-induced cell proliferation. These results demonstrate that LPA signaling is causally linked to the hyperactive lipogenesis in ovarian cancer cells, which can be exploited for development of new anti-cancer therapies.
Asunto(s)
Lipogénesis/efectos de los fármacos , Lisofosfolípidos/farmacología , Neoplasias Ováricas/metabolismo , Transducción de Señal/efectos de los fármacos , Animales , Línea Celular Tumoral , Ácido Graso Sintasas/biosíntesis , Femenino , Subunidades alfa de la Proteína de Unión al GTP G12-G13/metabolismo , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Lisofosfolípidos/metabolismo , Ratones , Proteínas de Neoplasias/metabolismo , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/patología , Receptores del Ácido Lisofosfatídico/biosíntesis , Elementos Reguladores de la TranscripciónRESUMEN
BACKGROUND: Fullerenes are molecules being investigated for a wide range of therapeutic applications. We have shown previously that certain fullerene derivatives (FDs) inhibit mast cell (MC) function in vitro, and here we examine their in vivo therapeutic effect on asthma, a disease in which MCs play a predominant role. OBJECTIVE: We sought to determine whether an efficient MC-stabilizing FD (C(70)-tetraglycolate [TGA]) can inhibit asthma pathogenesis in vivo and to examine its in vivo mechanism of action. METHODS: Asthma was induced in mice, and animals were treated intranasally with TGA either simultaneously with treatment or after induction of pathogenesis. The efficacy of TGA was determined through the measurement of airway inflammation, bronchoconstriction, serum IgE levels, and bronchoalveolar lavage fluid cytokine and eicosanoid levels. RESULTS: We found that TGA-treated mice have significantly reduced airway inflammation, eosinophilia, and bronchoconstriction. The TGA treatments are effective, even when given after disease is established. Moreover, we report a novel inhibitory mechanism because TGA stimulates the production of an anti-inflammatory P-450 eicosanoid metabolites (cis-epoxyeicosatrienoic acids [EETs]) in the lung. Inhibitors of these anti-inflammatory EETs reversed TGA inhibition. In human lung MCs incubated with TGA, there was a significant upregulation of CYP1B gene expression, and TGA also reduced IgE production from B cells. Lastly, MCs incubated with EET and challenged through FcεRI had a significant blunting of mediator release compared with nontreated cells. CONCLUSION: The inhibitory capabilities of TGA reported here suggest that FDs might be used a platform for developing treatments for asthma.
Asunto(s)
Ácido 8,11,14-Eicosatrienoico/análogos & derivados , Asma/tratamiento farmacológico , Fulerenos/farmacología , Ácido 8,11,14-Eicosatrienoico/análisis , Ácido 8,11,14-Eicosatrienoico/metabolismo , Animales , Asma/metabolismo , Broncoconstricción/efectos de los fármacos , Eosinofilia/tratamiento farmacológico , Femenino , Fulerenos/uso terapéutico , Inmunoglobulina E/biosíntesis , Inmunoglobulina E/sangre , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BLRESUMEN
Death of ß-cells due to apoptosis is an important contributor to ß-cell dysfunction in both type 1 and type 2 diabetes mellitus. Previously, we described participation of the Group VIA Ca(2+)-independent phospholipase A(2) (iPLA(2)ß) in apoptosis of insulinoma cells due to ER stress. To examine whether islet ß-cells are similarly susceptible to ER stress and undergo iPLA(2)ß-mediated apoptosis, we assessed the ER stress response in human pancreatic islets. Here, we report that the iPLA(2)ß protein is expressed predominantly in the ß-cells of human islets and that thapsigargin-induced ER stress promotes ß-cell apoptosis, as reflected by increases in activated caspase-3 in the ß-cells. Furthermore, we demonstrate that ER stress is associated with increases in islet iPLA(2)ß message, protein, and activity, iPLA(2)ß-dependent induction of neutral sphingomyelinase and ceramide accumulation, and subsequent loss of mitochondrial membrane potential. We also observe that basal activated caspase-3 increases with age, raising the possibility that ß-cells in older human subjects have a greater susceptibility to undergo apoptotic cell death. These findings reveal for the first time expression of iPLA(2)ß protein in human islet ß-cells and that induction of iPLA(2)ß during ER stress contributes to human islet ß-cell apoptosis. We hypothesize that modulation of iPLA(2)ß activity might reduce ß-cell apoptosis and this would be beneficial in delaying or preventing ß-cell dysfunction associated with diabetes.
Asunto(s)
Apoptosis/fisiología , Retículo Endoplásmico/metabolismo , Células Secretoras de Insulina/enzimología , Islotes Pancreáticos/enzimología , Fosfolipasas A2 Calcio-Independiente/metabolismo , Adulto , Células Cultivadas , Retículo Endoplásmico/efectos de los fármacos , Retículo Endoplásmico/patología , Inhibidores Enzimáticos/farmacología , Femenino , Humanos , Técnicas In Vitro , Células Secretoras de Insulina/efectos de los fármacos , Islotes Pancreáticos/citología , Masculino , Fosfolipasas A2 Calcio-Independiente/efectos de los fármacos , Tapsigargina/farmacologíaRESUMEN
Our recent studies indicate that endoplasmic reticulum (ER) stress causes INS-1 cell apoptosis by a Ca(2+)-independent phospholipase A(2) (iPLA(2)beta)-mediated mechanism that promotes ceramide generation via sphingomyelin hydrolysis and subsequent activation of the intrinsic pathway. To elucidate the association between iPLA(2)beta and ER stress, we compared beta-cell lines generated from wild type (WT) and Akita mice. The Akita mouse is a spontaneous model of ER stress that develops hyperglycemia/diabetes due to ER stress-induced beta-cell apoptosis. Consistent with a predisposition to developing ER stress, basal phosphorylated PERK and activated caspase-3 are higher in the Akita cells than WT cells. Interestingly, basal iPLA(2)beta, mature SREBP-1 (mSREBP-1), phosphorylated Akt, and neutral sphingomyelinase (NSMase) are higher, relative abundances of sphingomyelins are lower, and mitochondrial membrane potential (DeltaPsi) is compromised in Akita cells, in comparison with WT cells. Exposure to thapsigargin accelerates DeltaPsi loss and apoptosis of Akita cells and is associated with increases in iPLA(2)beta, mSREBP-1, and NSMase in both WT and Akita cells. Transfection of Akita cells with iPLA(2)beta small interfering RNA, however, suppresses NSMase message, DeltaPsi loss, and apoptosis. The iPLA(2)beta gene contains a sterol-regulatory element, and transfection with a dominant negative SREBP-1 reduces basal mSREBP-1 and iPLA(2)beta in the Akita cells and suppresses increases in mSREBP-1 and iPLA(2)beta due to thapsigargin. These findings suggest that ER stress leads to generation of mSREBP-1, which can bind to the sterol-regulatory element in the iPLA(2)beta gene to promote its transcription. Consistent with this, SREBP-1, iPLA(2)beta, and NSMase messages in Akita mouse islets are higher than in WT islets.
Asunto(s)
Diabetes Mellitus/etiología , Retículo Endoplásmico/patología , Fosfolipasas A2 Calcio-Independiente/genética , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/fisiología , Transcripción Genética , Animales , Apoptosis , Sitios de Unión , Humanos , Células Secretoras de Insulina , Ratones , Ratones Transgénicos , Unión Proteica , Estrés FisiológicoRESUMEN
Sphingosine-1-phosphate (S1P) is a bioactive lipid that regulates myriad important cellular processes, including growth, survival, cytoskeleton rearrangements, motility, and immunity. Here we report that treatment of Jurkat and U937 leukemia cells with the pan-sphingosine kinase (SphK) inhibitor N,N-dimethylsphingosine to block S1P formation surprisingly caused a large increase in expression of SphK1 concomitant with induction of apoptosis. Another SphK inhibitor, D,L-threo-dihydrosphingosine, also induced apoptosis and produced dramatic increases in SphK1 expression. However, up-regulation of SphK1 was not a specific effect of its inhibition but rather was a consequence of apoptotic stress. The chemotherapeutic drug doxorubicin, a potent inducer of apoptosis in these cells, also stimulated SphK1 expression and activity and promoted S1P secretion. The caspase inhibitor ZVAD reduced not only doxorubicin-induced lethality but also the increased expression of SphK1 and secretion of S1P. Apoptotic cells secrete chemotactic factors to attract phagocytic cells, and we found that S1P potently stimulated chemotaxis of monocytic THP-1 and U937 cells and primary monocytes and macrophages. Collectively, our data suggest that apoptotic cells may up-regulate SphK1 to produce and secrete S1P that serves as a "come-and-get-me" signal for scavenger cells to engulf them in order to prevent necrosis.
Asunto(s)
Apoptosis/fisiología , Lisofosfolípidos/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Esfingosina/análogos & derivados , Antibióticos Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Quimiotaxis de Leucocito/efectos de los fármacos , Quimiotaxis de Leucocito/fisiología , Doxorrubicina/farmacología , Inhibidores Enzimáticos/farmacología , Humanos , Técnicas In Vitro , Células Jurkat , Monocitos/efectos de los fármacos , Monocitos/fisiología , Fosfotransferasas (Aceptor de Grupo Alcohol)/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacos , Esfingosina/metabolismo , Esfingosina/farmacología , Células U937 , Regulación hacia Arriba/efectos de los fármacosRESUMEN
PLA2 (phospholipase A2) enzymes play critical roles in membrane phospholipid homoeostasis and in generation of lysophospholipid growth factors. In the present study, we show that the activity of the cytosolic iPLA2 (calcium-independent PLA2), but not that of the calcium-dependent cPLA2 (cytosolic PLA2), is required for growth-factor-independent, autonomous replication of ovarian carcinoma cells. Blocking iPLA2 activity with the pharmacological inhibitor BEL (bromoenol lactone) induces cell cycle arrest in S- and G2/M-phases independently of the status of the p53 tumour suppressor. Inhibition of iPLA2 activity also leads to modest increases in apoptosis of ovarian cancer cells. The S- and G2/M-phase accumulation is accompanied by increased levels of the cell cycle regulators cyclins B and E. Interestingly, the S-phase arrest is released by supplementing the growth factors LPA (lysophosphatidic acid) or EGF (epidermal growth factor). However, inhibition of iPLA2 activity with BEL remains effective in repressing growth-factor- or serum-stimulated proliferation of ovarian cancer cells through G2/M-phase arrest. Down-regulation of iPLA2b expression with lentivirus-mediated RNA interference inhibited cell proliferation in culture and tumorigenicity of ovarian cancer cell lines in nude mice. These results indicate an essential role for iPLA2 in cell cycle progression and tumorigenesis of ovarian carcinoma cells.
Asunto(s)
Proliferación Celular , Neoplasias Ováricas/prevención & control , Fosfolipasas A/metabolismo , Animales , Apoptosis , Western Blotting , Calcio , División Celular/efectos de los fármacos , División Celular/fisiología , Ciclinas/metabolismo , Citosol/metabolismo , Factor de Crecimiento Epidérmico/farmacología , Femenino , Fase G2/efectos de los fármacos , Fase G2/fisiología , Fosfolipasas A2 Grupo VI , Humanos , Lisofosfolípidos/farmacología , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Naftalenos/farmacología , Neoplasias Ováricas/genética , Neoplasias Ováricas/metabolismo , Fosfolipasas A/antagonistas & inhibidores , Fosfolipasas A/genética , Fosfolipasas A2 , Pironas/farmacología , ARN Interferente Pequeño/farmacología , Fase S/efectos de los fármacos , Fase S/fisiología , Células Tumorales CultivadasRESUMEN
Several lines of evidence suggest that glycerophospholipid mass is maintained through the coordinate regulation of CTP:phosphocholine cytidylyltransferase-alpha (CTalpha) and the group VIA calcium-independent phospholipase A2 (iPLA2). CTalpha expression is modulated by sterol and this is mediated in part through sterol regulatory element binding proteins (SREBP). In this report, we investigate the possibility that iPLA2 expression is controlled in a similar manner. When Chinese hamster ovary (CHO) cells were cultured under sterol-depleted conditions, iPLA2 catalytic activity, mRNA, and protein were induced by between two- and threefold. These inductions were suppressed when the cells were supplemented with exogenous sterols. Luciferase reporter assays indicated that sterol depletion induced transcription of iPLA2, an analysis of the 5' flanking region suggested that the iPLA2 gene contained a putative sterol regulatory element (SRE), and electrophoretic mobility shift assay (EMSA) analysis indicated that this element can bind SREBP-2. Notably, a mutant CHO cell line (SRD4) that constitutively generates mature SREBP proteins exhibited increased iPLA2 activity and expression compared to wild-type cells. These data suggest that iPLA2 expression is regulated in a manner consistent with other important genes in sterol and glycerophospholipid metabolism. Such coordinate regulation may be essential for maintaining the lipid composition of cell membranes.
Asunto(s)
Regulación Enzimológica de la Expresión Génica , Fosfolipasas A/metabolismo , Esteroles/metabolismo , Animales , Secuencia de Bases , Proteínas Potenciadoras de Unión a CCAAT/genética , Proteínas Potenciadoras de Unión a CCAAT/metabolismo , Células CHO , Cricetinae , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Genes Reporteros , Fosfolipasas A2 Grupo VI , Humanos , Datos de Secuencia Molecular , Fosfolipasas A/genética , Fosfolipasas A2 , Regiones Promotoras Genéticas , Proteína 1 de Unión a los Elementos Reguladores de Esteroles , Proteína 2 de Unión a Elementos Reguladores de Esteroles , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
The potent immunosuppressive drug FTY720, a sphingosine analog, induces redistribution of lymphocytes from circulation to secondary lymphoid tissues. FTY720 is phosphorylated in vivo and functions as an agonist for four G-protein-coupled sphingosine-1-phosphate receptors. The identity of the kinase that phosphorylates FTY720 is still not known. Here we report that although both sphingosine kinase type 1 (SphK1) and type 2 (SphK2) can phosphorylate FTY720 with low efficiency, SphK2 is much more effective than SphK1. FTY720 inhibited phosphorylation of sphingosine catalyzed by SphK2 to a greater extent than it inhibits SphK1. Thus, SphK2 may be the relevant enzyme that is responsible for in vivo phosphorylation of FTY720.
Asunto(s)
Inmunosupresores/farmacocinética , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Glicoles de Propileno/farmacocinética , Animales , Biotransformación , Línea Celular , Relación Dosis-Respuesta a Droga , Clorhidrato de Fingolimod , Humanos , Inmunosupresores/farmacología , Ratones , Octoxinol/farmacología , Fosforilación , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Glicoles de Propileno/farmacología , Esfingosina/metabolismo , TransfecciónRESUMEN
BACKGROUND: Patients with localized aggressive periodontitis (LAgP) produce elevated levels of IgG2 both in vivo and in vitro. Responses to the periodontitis-associated pathogens Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis are dominated by IgG2, and these IgG2 responses are associated with reduced extent and severity of disease. Little is known about regulation of the IgG2 subclass, although prostaglandin E2 (PGE2) (a mediator known to polarize responses toward Th-2) and interferon (IFN)-gamma (a Th-1 mediator) both promote IgG2 production. This unusual relationship prompted the hypothesis that, in certain circumstances, PGE2 enhances rather than inhibits IFN-gamma production. METHODS: To test this hypothesis, indomethacin (IND)-treated peripheral blood mononuclear cell (PBL) cultures from healthy volunteers were stimulated with pokeweed mitogen (PWM), and the cultures were manipulated by adding PGE2, rIFN-gamma, rIL-Ialpha, rIL-1beta, rIL-6, or rIL-12. Production of IgG1, IgG2, IFN-gamma, and PGE2 was monitored by enzyme-linked immunosorbent assay. RESULTS: Indomethacin treatment inhibited IgG1 and IgG2 production, and PGE2 restored both immunoglobulins in PWM-stimulated cultures. Remarkably, addition of IFN-gamma also restored IND-suppressed IgG2 but not IgG1. In contrast, addition of rIL (interleukin)-1alpha, rIL-1beta, rIL-6, or rIL-12 did not restore IgG2 responses. Furthermore, IND suppressed IFN-gamma production and PGE2 increased IFN-gamma levels. Kinetic studies indicate that PGE2 production occurred on the first day of culture, followed by IFN-gamma two days later. CONCLUSIONS: These findings support the concept that in certain circumstances, PGE2 production can lead to increased IFN-gamma and that this IFN-gamma selectively promotes IgG2 responses. These data suggest that the elevated PGE2 observed in LAgP patients may contribute to increased IFN-gamma production and help explain elevated IgG2 responses.
Asunto(s)
Dinoprostona/farmacología , Inmunoglobulina G/efectos de los fármacos , Interferón gamma/efectos de los fármacos , Adulto , Análisis de Varianza , Células Cultivadas , Inhibidores de la Ciclooxigenasa/farmacología , Humanos , Indometacina/farmacología , Interleucina-1/farmacología , Interleucina-12/farmacología , Interleucina-6/farmacología , Activación de Linfocitos/efectos de los fármacos , Linfocitos/efectos de los fármacos , Mitógenos de Phytolacca americana/farmacología , Proteínas Recombinantes , Factores de TiempoRESUMEN
A growing body of evidence indicates that the incidence of atherosclerosis is increased in subjects with periodontitis - a chronic infection of the oral cavity. This article summarizes the evidence that suggests periodontitis shifts the lipoprotein profile to be more proatherogenic. LDL-C is elevated in periodontitis and most studies indicate that triglyceride levels are also increased. By contrast, antiatherogenic HDL tends to be low in periodontitis. Periodontal therapy tends to shift lipoprotein levels to a healthier profile and also reduces subclinical indices of atherosclerosis. In summary, periodontal disease alters lipoprotein metabolism in ways that could promote atherosclerosis and cardiovascular disease.
RESUMEN
Activation of phospholipases A(2) (PLA(2)s) leads to the generation of biologically active lipid mediators that can affect numerous cellular events. The Group VIA Ca(2+)-independent PLA(2), designated iPLA(2)beta, is active in the absence of Ca(2+), activated by ATP, and inhibited by the bromoenol lactone suicide inhibitor (BEL). Over the past 10-15 years, studies using BEL have demonstrated that iPLA(2)beta participates in various biological processes and the recent availability of mice in which iPLA(2)beta expression levels have been genetically-modified are extending these findings. Work in our laboratory suggests that iPLA(2)beta activates a unique signaling cascade that promotes beta-cell apoptosis. This pathway involves iPLA(2)beta dependent induction of neutral sphingomyelinase, production of ceramide, and activation of the intrinsic pathway of apoptosis. There is a growing body of literature supporting beta-cell apoptosis as a major contributor to the loss of beta-cell mass associated with the onset and progression of Type 1 and Type 2 diabetes mellitus. This underscores a need to gain a better understanding of the molecular mechanisms underlying beta-cell apoptosis so that improved treatments can be developed to prevent or delay the onset and progression of diabetes mellitus. Herein, we offer a general review of Group VIA Ca(2+)-independent PLA(2) (iPLA(2)beta) followed by a more focused discussion of its participation in beta-cell apoptosis. We suggest that iPLA(2)beta-derived products trigger pathways which can lead to beta-cell apoptosis during the development of diabetes.
Asunto(s)
Apoptosis , Fosfolipasas A2 Grupo VI/metabolismo , Células Secretoras de Insulina/enzimología , Animales , Calcio/metabolismo , Muerte Celular , Proliferación Celular , Diabetes Mellitus/genética , Diabetes Mellitus/metabolismo , Fosfolipasas A2 Grupo VI/antagonistas & inhibidores , Fosfolipasas A2 Grupo VI/genética , Humanos , Ratones , Modelos Biológicos , Transducción de SeñalRESUMEN
In vascular smooth muscle, KCl not only elevates intracellular free Ca(2+) ([Ca(2+)](i)), myosin light chain kinase activity and tension (T), but also can inhibit myosin light chain phosphatase activity by activation of rhoA kinase (ROCK), resulting in Ca(2+) sensitization (increased T/[Ca(2+)](i) ratio). Precisely how KCl causes ROCK-dependent Ca(2+) sensitization remains to be determined. Using Fura-2-loaded isometric rings of rabbit artery, we found that the Ca(2+)-independent phospholipase A(2) (iPLA(2)) inhibitor, bromoenol lactone (BEL), reduced the KCl-induced tonic but not early phasic phase of T and potentiated [Ca(2+)](i), reducing Ca(2+) sensitization. The PKC inhibitor, GF-109203X (> or =3 microM) and the pseudo-substrate inhibitor of PKCzeta produced a response similar to BEL. BEL reduced basal and KCl-stimulated myosin phosphatase phosphorylation. Whereas BEL and H-1152 produced strong inhibition of KCl-induced tonic T (approximately 50%), H-1152 did not induce additional inhibition of tissues already inhibited by BEL, suggesting that iPLA(2) links KCl stimulation with ROCK activation. The cPLA(2) inhibitor, pyrrolidine-1, inhibited KCl-induced tonic increases in [Ca(2+)](i) but not T, whereas the inhibitor of 20-HETE production, HET0016, acted like the ROCK inhibitor H-1152 by causing Ca(2+) desensitization. These data support a model in which iPLA(2) activity regulates Ca(2+) sensitivity.
Asunto(s)
Calcio/metabolismo , Músculo Liso Vascular/enzimología , Fosfolipasas A2 Calcio-Independiente/metabolismo , Cloruro de Potasio/farmacología , Ácido 5,8,11,14-Eicosatetrainoico/farmacología , Animales , Ácido Araquidónico/antagonistas & inhibidores , Cinética , Contracción Muscular/efectos de los fármacos , Tono Muscular/efectos de los fármacos , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/metabolismo , Quinasa de Cadena Ligera de Miosina/metabolismo , Naftalenos/farmacología , Fenilefrina/farmacología , Proteína Quinasa C/antagonistas & inhibidores , Pironas/farmacología , Pirrolidinas/farmacología , Conejos , Quinasas Asociadas a rho/antagonistas & inhibidoresRESUMEN
FTY720 is a potent immunomodulator drug that inhibits the egress of lymphocytes from secondary lymphoid tissues and thymus. FTY720 is phosphorylated in vivo by sphingosine kinase 2 to FTY720-phosphate, which acts as a potent sphingosine-1-phosphate (S1P) receptor agonist. However, in contrast to S1P, FTY720 has no effect on mast-cell degranulation, yet significantly reduces antigen-induced secretion of PGD2 and cysteinyl-leukotriene. Unexpectedly, this effect of FTY720 was independent of its phosphorylation and S1P receptor functions. The rate-limiting step in the biosynthesis of all eicosanoids is the phospholipase A2 (PLA2)-mediated release of arachidonic acid from glycerol phospholipids. Although FTY720 also reduced arachidonic acid release in response to antigen, it had no effect on translocation of cPLA2 or ERK1/2 activation, suggesting that it does not interfere with FcepsilonRI-mediated events leading to cPLA2 activation. Remarkably, however, FTY720 drastically inhibited recombinant cPLA2alpha activity, whereas FTY720-phosphate, sphingosine, or S1P had no effect. This study has uncovered a unique action of FTY720 as an inhibitor of cPLA2alpha and hence on production of all eicosanoids. Our results have important implications for the potential therapeutic mechanism of action of FTY720 in eicosanoid-driven inflammatory disorders such as asthma and multiple sclerosis.
Asunto(s)
Inmunosupresores/farmacología , Fosfolipasas A/antagonistas & inhibidores , Glicoles de Propileno/farmacología , Receptores de Lisoesfingolípidos , Esfingosina/análogos & derivados , Animales , Degranulación de la Célula/efectos de los fármacos , Línea Celular , Cisteína/metabolismo , Citosol , Eicosanoides/antagonistas & inhibidores , Clorhidrato de Fingolimod , Humanos , Leucotrienos/metabolismo , Mastocitos , Fosfolipasas A2 , Prostaglandina D2/metabolismo , Ratas , Receptores de Lisoesfingolípidos/agonistas , Esfingosina/farmacologíaRESUMEN
In this study, we examined whether the documented increase of plasma triglycerides in patients with generalized aggressive periodontitis (GAgP) is associated with changes in lipoprotein subclass distribution and/or LDL-associated platelet-activating factor acetylhydrolase (PAF-AH) activity. Lipoprotein subclasses were analyzed in whole plasma samples using nuclear magnetic resonance methods. Compared with subjects without periodontitis (NP subjects; n = 12), GAgP subjects (n = 12) had higher plasma levels of large, medium, and small VLDL (35.0 +/- 6.7 vs. 63.1 +/- 9.6 nmol/l; P = 0.025), higher levels of intermediate density lipoprotein (24.8 +/- 11.6 vs. 87.2 +/- 16.6 nmol/l; P = 0.006), lower levels of large LDL (448.3 +/- 48.5 vs. 315.8 +/- 59.4 nmol/l; P = 0.098), and higher levels of small LDL (488.2 +/- 104.2 vs. 946.7 +/- 151.6 nmol/l; P = 0.021). The average size of LDL from NP and GAgP subjects was 21.4 +/- 0.2 and 20.6 +/- 0.3 nm, respectively (P = 0.031). Compared with NP subjects, GAgP subjects had a greater number of circulating LDL particles (961.3 +/- 105.3 vs. 1,349.0 +/- 133.2 nmol/l; P = 0.032). Differences in the plasma levels of large, medium, and small HDL were not statistically significant. NP and GAgP subjects had similar plasma levels of total LDL-associated PAF-AH activity; however, LDL of GAgP subjects contained less PAF-AH activity per microgram of LDL protein (1,458.0 +/- 171.0 and 865.2 +/- 134 pmol/min/microg; P = 0.014). These results indicate that, in general, GAgP subjects have a more atherogenic lipoprotein profile and lower LDL-associated PAF-AH activity than NP subjects. These differences may help explain the increased risk of GAgP subjects for cardiovascular disease.
Asunto(s)
1-Alquil-2-acetilglicerofosfocolina Esterasa/metabolismo , Lipoproteínas/clasificación , Lipoproteínas/metabolismo , Periodontitis/metabolismo , Periodontitis/patología , Adulto , Colesterol/sangre , Femenino , Humanos , Masculino , Periodontitis/enzimología , Triglicéridos/sangreRESUMEN
Homeostasis of phosphatidylcholine (PC) is regulated by the opposing actions between CTP:phosphocholine cytidylyltransferase (CT) and the group VIA Ca(2+)-independent phospholipase A(2) (iPLA(2)). We investigated this process during the cell cycle. PC mass doubles during late G(1) and early S phase when its rate of catabolism is lowest. We show that iPLA(2) activity is cell cycle-dependent with peak activity during G(2)/M and late S phase. iPLA(2) activity declines during G(1) and is lowest at the G(1)/S transition and early S phase. The accumulation of PC correlates with decreased iPLA(2) activity, suggesting that regulation of this enzyme contributes to phospholipid accumulation. The levels of 80 kDa iPLA(2) protein do not change and thus cannot account for changes in enzyme activity. Reverse transcriptase and real-time PCR experiments show that splice variant iPLA(2) mRNAs are preferentially expressed during G(2)/M. Immunoblot analyses with an antibody directed against the N terminus of iPLA(2) revealed a approximately 50 kDa protein that is of appropriate size to be the truncated protein encoded by the ankyrin-iPLA(2)-1 splice variant mRNA. The levels of truncated iPLA(2) protein were high in cells in late G(1) and S phase cells that had low iPLA(2) activity and low in G(2)/M cells that had high iPLA(2) activity. The truncated protein co-immunoprecipitated with full-length iPLA(2), indicating a physical interaction between the two proteins. Together, these data suggest that truncated iPLA(2) proteins associate with active iPLA(2) and down-regulate its activity during G(1). This down-regulation may contribute to phospholipid accumulation during the cell cycle.
Asunto(s)
Calcio/química , Fosfolipasas A/química , Empalme Alternativo , Animales , Ancirinas/química , Células CHO , Calcio/metabolismo , Ciclo Celular , División Celular , Separación Celular , Citidililtransferasa de Colina-Fosfato/química , Cricetinae , Regulación hacia Abajo , Citometría de Flujo , Fase G1 , Fase G2 , Humanos , Immunoblotting , Inmunoprecipitación , Células Jurkat , Espectrometría de Masas , Modelos Biológicos , Modelos Genéticos , Fosfolipasa D/metabolismo , Fosfolipasas A2 , Fosfolípidos/química , Estructura Terciaria de Proteína , ARN/metabolismo , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Fase S , Factores de TiempoRESUMEN
Phosphatidylcholine transfer protein (PC-TP) is a cytosolic protein of unknown function that catalyzes intermembrane transfer of phosphatidylcholines in vitro. Using stably transfected CHO cells, we explored the influence of PC-TP on apolipoprotein A-I- and high density lipoprotein 3 (HDL(3))-mediated lipid efflux. In proportion to its cellular level of expression, PC-TP accelerated apolipoprotein A-I-mediated phospholipid and cholesterol efflux as pre-beta-HDL particles. PC-TP increased rates of efflux of both lipids by >2-fold but did not affect mRNA levels or the activity of ATP-binding cassette A1, a plasma membrane protein that regulates apolipoprotein A-I-mediated lipid efflux. Overexpression of PC-TP was associated with only slight increases in HDL(3)-mediated phospholipid efflux and no changes in cholesterol efflux. In scavenger receptor BI-overexpressing cells, PC-TP expression minimally influenced apolipoprotein A-I- or HDL(3)-mediated lipid efflux. PC-TP did not affect cellular phospholipid compositions, phosphatidylcholine contents, or phosphatidylcholine synthetic rates. These findings suggest that a physiological function of PC-TP is to replenish the plasma membrane with phosphatidylcholines that are removed during pre-beta-HDL particle formation due to the activity of ATP-binding cassette A1.