RESUMO
In this study, we examine how infection of murine and human fibroblasts by adenovirus (Ad) serotype 5 (Ad5) affects the expression and activity of cytosolic phospholipase A2 (cPLA2), cyclooxygenase-2 (COX-2), and production of PGs. Our experiments showed that infection with Ad5 is accompanied by the rapid activation of cPLA2 and the cPLA2-dependent release of [3H]arachidonic acid ([3H]AA). Increased expression of COX-2 was also observed after Ad infection, as was production of PGE2 and PGI2. Later, however, as the infection progressed, release of [3H]AA and production of PGs stopped. Late-stage Ad5-infected cells also did not release [3H]AA or PGs following treatment with a panel of biologically diverse agents. Experiments with UV-inactivated virus confirmed that Ad infection is accompanied by the activation of a host-dependent response that is later inhibited by the virus. Investigations of the mechanism of suppression of the PG pathway by Ad5 did not reveal major effects on the expression or activity of cPLA2 or COX-2. We did note a change in the intracellular position of cPLA2 and found that cPLA2 did not translocate normally in infected cells, raising the possibility that Ad5 interferes with the PG pathway by interfering with the intracellular movement of cPLA2. Taken together, these data reveal dynamic interactions between Ad5 and the lipid mediator pathways of the host and highlight a novel mechanism by which Ad5 evades the host immune response. In addition, our results offer insight into the inflammatory response induced by many Ad vectors lacking early region gene products.
Assuntos
Adenovírus Humanos/imunologia , Ciclo-Oxigenase 2/genética , Ciclo-Oxigenase 2/metabolismo , Fosfolipases A/genética , Fosfolipases A/metabolismo , Prostaglandinas/biossíntese , Adenovírus Humanos/genética , Animais , Ácido Araquidônico/antagonistas & inibidores , Ácido Araquidônico/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Ciclo-Oxigenase 2/biossíntese , Dinoprostona/antagonistas & inibidores , Dinoprostona/biossíntese , Ativação Enzimática/imunologia , Epoprostenol/antagonistas & inibidores , Epoprostenol/biossíntese , Fibroblastos/enzimologia , Fibroblastos/metabolismo , Fibroblastos/virologia , Fosfolipases A2 do Grupo IV , Humanos , Líquido Intracelular/enzimologia , Líquido Intracelular/virologia , Camundongos , Fosfolipases A/biossíntese , Fosfolipases A2 , Transporte Proteico/imunologia , TrítioRESUMO
Activation of macrophages and macrophage cell lines by bacterial LPS elicits a delayed phase of PG biosynthesis that appears to be entirely mediated by cyclooxygenase-2 (COX-2). In previous work, we found that a catalytically active group V secreted phospholipase A(2) (sPLA(2)-V) was required for COX-2 induction, but the nature of the sPLA(2)-V metabolite involved was not defined. In this study, we identify lysophosphatidylcholine (lysoPC) as the sPLA(2)-V downstream mediator involved in COX-2 induction by LPS-stimulated macrophages. Inhibition of sPLA(2)-V by RNA interference or by the cell-permeable compound scalaradial blocked LPS-induced COX-2 expression, and this inhibition was overcome by incubating the cells with a nonhydrolyzable lysoPC analog, but not by arachidonic acid or oleic acid. Moreover, inhibition of sPLA(2)-V by scalaradial also prevented the activation of the transcription factor c-Rel, and such an inhibition was also selectively overcome by the lysoPC analog. Collectively, these results support a model whereby sPLA(2)-V hydrolysis of phospholipids upon LPS stimulation results in lysoPC generation, which in turn regulates COX-2 expression by a mechanism involving the transcriptional activity of c-Rel.
Assuntos
Ciclo-Oxigenase 2/biossíntese , Lipopolissacarídeos/farmacologia , Lisofosfatidilcolinas/farmacologia , Macrófagos/enzimologia , Fosfolipases A/fisiologia , Animais , Linhagem Celular Tumoral , Ciclo-Oxigenase 2/metabolismo , Ciclo-Oxigenase 2/fisiologia , Indução Enzimática/imunologia , Inibidores Enzimáticos/farmacologia , Fosfolipases A2 do Grupo V , Homosteroides/farmacologia , Leucemia P388/enzimologia , Leucemia P388/metabolismo , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Macrófagos Peritoneais/enzimologia , Macrófagos Peritoneais/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fosfolipases A/antagonistas & inibidores , Fosfolipases A/biossíntese , Fosfolipases A2 , Proteínas Proto-Oncogênicas c-rel/fisiologia , Sesterterpenos , Terpenos/farmacologiaRESUMO
AIM: To investigate the effects of human beta-defensins on the expression of genes involved in the host immune response of the dental pulp. METHODOLOGY: Human odontoblast-like cells were cultured in Dulbecco's modified Eagle's medium. Cells were stimulated by recombinant human beta-defensins (rhBDs) up to 4 h. RNA was extracted followed by cDNA synthesis (oligo-(dT)-primer). Samples were analysed by real-time polymerase chain reaction (PCR) technology. Genes of interest were: human beta-defensin-1, -2, interleukin (IL)-6, IL-8, tumour necrosis factor-alpha, cyclooxygenase-2, leukotriene-A4-hydrolase, cytosolic phospholipase-A-2 (cPLA(2)), and dentine sialophosphoprotein. Gene expression of beta-actin served as internal standard for normalizing real-time PCR data. Two-way anova and the paired t-test were applied for comparison of the gene expression. RESULTS: In odontoblast-like cells rhBD-2 stimulation led to a down-regulation of the gene expression of hBD-1 (P < 0.05), whilst the mRNA expression of IL-6 (P < 0.05), IL-8 (P < 0.05) and cPLA(2) was increased in response to rhBD-2. CONCLUSION: The results of the present study suggest immune regulatory functions of human beta-defensin-2 in odontoblast-like cells.
Assuntos
Polpa Dentária/imunologia , Imunidade Inata/genética , Odontoblastos/imunologia , beta-Defensinas/imunologia , Adulto , Análise de Variância , Células Cultivadas , DNA Complementar/análise , Polpa Dentária/citologia , Regulação da Expressão Gênica , Fosfolipases A2 do Grupo IV , Humanos , Interleucina-6/biossíntese , Interleucina-6/genética , Interleucina-6/imunologia , Interleucina-8/biossíntese , Interleucina-8/genética , Interleucina-8/imunologia , Fosfolipases A/biossíntese , Fosfolipases A/genética , Fosfolipases A/imunologia , Reação em Cadeia da Polimerase , Proteínas Recombinantes/farmacologia , beta-Defensinas/biossíntese , beta-Defensinas/genéticaRESUMO
Phospholipase A(2)s (PLA(2)s) are key enzymes that catalyze the hydrolysis of membrane phospholipids to release bioactive lipids that play an important role in normal cellular homeostasis. Under certain circumstances, disrupted production of key lipid mediators may adversely impact physiological processes, leading to pathological conditions such as inflammation and cancer. In particular, cytosolic PLA(2)alpha (cPLA(2)alpha) has a high selectivity for liberating arachidonic acid (AA) that is subsequently metabolized by a panel of downstream enzymes for eicosanoid production. Although concentrations of free AA are maintained at low levels in resting cells, alterations in AA production, often resulting from dysregulation of cPLA(2)alpha activity, are observed in transformed cells. In this review, we summarize recent evidence that cPLA(2)alpha plays a role in the pathogenesis of many human cancers. Much of this evidence has been accumulated from functional studies using cPLA(2)alpha-deficient mice, as well as mechanistic studies in cell culture. We also discuss the potential contribution of cPLA(2)alpha and AA to apoptosis, and the regulatory mechanisms leading to aberrant expression of cPLA(2)alpha.
Assuntos
Ácido Araquidônico/biossíntese , Transformação Celular Neoplásica/metabolismo , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Proteínas de Neoplasias/metabolismo , Neoplasias/enzimologia , Fosfolipases A/biossíntese , Animais , Linhagem Celular Tumoral , Citosol/enzimologia , Citosol/patologia , Eicosanoides/biossíntese , Ativação Enzimática , Fosfolipases A2 do Grupo IV , Humanos , Inflamação/enzimologia , Camundongos , Camundongos Mutantes , Fosfolipases A/deficiênciaRESUMO
Secreted phospholipases A(2) (sPLA(2)) form a group of low-molecular weight enzymes that catalyze the hydrolysis of phospholipids. Some sPLA(2)s are likely to play a role in inflammation, cancer, and as antibacterial enzymes in innate immunity. We developed specific and sensitive time-resolved fluroimmunoassays (TR-FIA) for mouse group (G) IB, GIIA, GIID, GIIE, GIIF, GV and GX sPLA(2)s and measured their concentrations in mouse serum and tissues obtained from both Balb/c and C57BL/6J mice. We also analyzed the mRNA expression of the sPLA(2)s by quantitative real-time reverse transcriptase PCR (qPCR). In most tissues, the concentrations of sPLA(2) proteins corresponded to the expression of sPLA(2)s at the mRNA level. With a few exceptions, the sPLA(2) proteins were found in the gastrointestinal tract. The qPCR results showed that GIB sPLA(2) is synthesized widely in the gastrointestinal tract, including esophagus and colon, in addition to stomach and pancreas. Our results also suggest that the loss of GIIA sPLA(2) in the intestine of GIIA sPLA(2)-deficient C57BL/6J mice is not compensated by other sPLA(2)s under normal conditions. Outside the gastrointestinal tract, sPLA(2)s were expressed occasionally in a number of tissues. The TR-FIAs developed in the current study may serve as useful tools to measure the levels of sPLA(2) proteins in mouse serum and tissues in various experimental settings.
Assuntos
Fosfolipases A/biossíntese , Animais , Feminino , Imunoensaio , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Especificidade de Órgãos , Fosfolipases A/metabolismo , Reação em Cadeia da Polimerase , RNA Mensageiro/análise , RNA Mensageiro/genéticaRESUMO
During the course of infection Mycobacterium tuberculosis predominantly resides within macrophages, where it encounters and is often able to resist the antibacterial mechanisms of the host. In this study, we assessed the role of macrophage phospholipases A2 (PLA2s) in defense against M. tuberculosis. Mouse bone marrow-derived macrophages (BMDMs) expressed cPLA2-IVA, cPLA2-IVB, iPLA2-VI, sPLA2-IIE, and sPLA2-XIIA. The expression of cPLA2-IVA was increased in response to M. tuberculosis, gamma interferon, or their combination, and cPLA2-IVA mediated the release of arachidonic acid, which was stimulated by M. tuberculosis in activated, but not unactivated, macrophages. We confirmed that arachidonic acid is highly mycobactericidal in a concentration- and pH-dependent manner in vitro. However, when M. tuberculosis-infected macrophages were treated with PLA2 inhibitors, intracellular survival of M. tuberculosis was not affected, even in inducible nitric oxide synthase-deficient macrophages, in which a major bactericidal mechanism is removed. Moreover, intracellular survival of M. tuberculosis was similar in cPLA2-IVA-deficient and wild-type macrophages. Our results demonstrate that the cytosolic PLA2s are not required by murine BMDMs to kill M. tuberculosis.
Assuntos
Citosol/enzimologia , Macrófagos/microbiologia , Mycobacterium tuberculosis/fisiologia , Fosfolipases A/fisiologia , Animais , Ácido Araquidônico/farmacologia , Citosol/imunologia , Citosol/microbiologia , Macrófagos/citologia , Macrófagos/enzimologia , Macrófagos/imunologia , Camundongos , Fosfolipases A/antagonistas & inibidores , Fosfolipases A/biossíntese , Fosfolipases A2RESUMO
We have previously reported that group V secretory phospholipase A2 (sPLA2) amplifies the action of cytosolic phospholipase A2(cPLA2) alpha in regulating eicosanoid biosynthesis by mouse peritoneal macrophages stimulated with zymosan (Satake, Y., Diaz, B. L., Balestrieri, B., Lam, B. K., Kanaoka, Y., Grusby, M. J., and Arm, J. P. (2004) J. Biol. Chem. 279, 16488-16494). To further understand the role of group V sPLA2, we studied its localization in resting mouse peritoneal macrophages before and after stimulation with zymosan and the effect of deletion of the gene encoding group V sPLA2 on phagocytosis of zymosan. We report that group V sPLA2 is present in the Golgi apparatus and recycling endosome in the juxtanuclear region of resting peritoneal macrophages. Upon ingestion of zymosan by mouse peritoneal macrophages, group V sPLA2 is recruited to the phagosome. There it co-localizes with cPLA2alpha, 5-lipoxygenase, 5-lipoxygenase-activating protein, and leukotriene C4 synthase. Using immunostaining for the cysteinyl leukotrienes in carbodiimide-fixed cells, we show, for the first time, that the phagosome is a site of cysteinyl leukotriene formation. Furthermore, peritoneal macrophages from group V sPLA2-null mice demonstrated a >50% attenuation in phagocytosis of zymosan particles, which was restored by adenoviral expression of group V sPLA2 but IIA not group sPLA2. These data demonstrate that group V sPLA2 contributes to the innate immune response both through regulation of eicosanoid generation in response to a phagocytic stimulus and also as a component of the phagocytic machinery.
Assuntos
Macrófagos Peritoneais/enzimologia , Fagocitose , Fosfolipases A/metabolismo , Zimosan/farmacologia , Animais , Biomarcadores , Células Cultivadas , Eicosanoides/biossíntese , Endossomos/enzimologia , Complexo de Golgi/enzimologia , Fosfolipases A2 do Grupo II , Fosfolipases A2 do Grupo V , Macrófagos Peritoneais/efeitos dos fármacos , Camundongos , Camundongos Knockout , Fagocitose/efeitos dos fármacos , Fagocitose/genética , Fagossomos/enzimologia , Fosfolipases A/biossíntese , Fosfolipases A/deficiência , Fosfolipases A/genética , Fosfolipases A2 , Transporte Proteico/efeitos dos fármacos , Transporte Proteico/genéticaRESUMO
Conjugated linoleic acids (CLAs) were reported to have anti-atherogenic properties in animal feeding experiments. In an attempt to elucidate the molecular mechanisms of these anti-atherogenic effects, the modulatory potential of CLA on cytokine-induced eicosanoid production from smooth muscle cells (SMCs), which contributes to the chronic inflammatory response associated with atherosclerosis, has been investigated in the present study. cis-9, trans-11 CLA and trans-10, cis-12 CLA were shown to reduce proportions of the eicosanoid precursor arachidonic acid in SMC total lipids and to inhibit cytokine-induced NF-kappaB DNA-binding activity, mRNA levels of inducible enzymes involved in eicosanoid formation (cPLA2, COX-2, mPGES), and the production of the prostaglandins PGE2 and PGI2 by TNFalpha-stimulated SMCs in a dose-dependent manner. The effect of 50 micromol/L of either CLA isomer was as effective as 10 micromol/L of the PPARgamma agonist troglitazone in terms of inhibiting the TNFalpha-stimulated eicosanoid production by SMCs. PPARgamma DNA-binding activity was increased by both CLA isomers compared to control cells. Moreover, it was shown that the PPARgamma antagonist T0070907 partially abrogated the inhibitory action of CLA isomers on cytokine-induced eicosanoid production and NF-kappaB DNA-binding activity by vascular SMCs suggesting that PPARgamma signalling is at least partially involved in the action of CLA in human vascular SMCs. With respect to the effects of CLA on experimental atherosclerosis, our findings suggest that the anti-inflammatory effect of CLA is at least partially responsible for the anti-atherogenic effects of CLA observed in vivo.
Assuntos
Dinoprostona/biossíntese , Células Endoteliais/efeitos dos fármacos , Epoprostenol/biossíntese , Ácidos Linoleicos Conjugados/farmacologia , PPAR gama/fisiologia , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Adulto , Benzamidas/farmacologia , Cromanos/farmacologia , Vasos Coronários/citologia , Ciclo-Oxigenase 1/biossíntese , Ciclo-Oxigenase 2/biossíntese , Sistema Enzimático do Citocromo P-450/biossíntese , Citosol/enzimologia , Células Endoteliais/metabolismo , Humanos , Oxirredutases Intramoleculares/biossíntese , Isomerismo , Masculino , NF-kappa B/fisiologia , Fosfolipases A/biossíntese , Piridinas/farmacologia , Tiazolidinedionas/farmacologia , TroglitazonaRESUMO
Gastric cancer is a leading cause of global cancer mortality, but comparatively little is known about the cellular pathways regulating different aspects of the gastric cancer phenotype. To achieve a better understanding of gastric cancer at the levels of systems topology, functional modules, and constituent genes, we assembled and systematically analyzed a consensus gene coexpression meta-network of gastric cancer incorporating >300 tissue samples from four independent patient populations (the "gastrome"). We find that the gastrome exhibits a hierarchical scale-free architecture, with an internal structure comprising multiple deeply embedded modules associated with diverse cellular functions. Individual modules display distinct subtopologies, with some (cellular proliferation) being integrated within the primary network, and others (ribosomal biosynthesis) being relatively isolated. One module associated with intestinal differentiation exhibited a remarkably high degree of autonomy, raising the possibility that its specific topological features may contribute towards the frequent occurrence of intestinal metaplasia in gastric cancer. At the single-gene level, we discovered a novel conserved interaction between the PLA2G2A prognostic marker and the EphB2 receptor, and used tissue microarrays to validate the PLA2G2A/EphB2 association. Finally, because EphB2 is a known target of the Wnt signaling pathway, we tested and provide evidence that the Wnt pathway may also similarly regulate PLA2G2A. Many of these findings were not discernible by studying the single patient populations in isolation. Thus, besides enhancing our knowledge of gastric cancer, our results show the broad utility of applying meta-analytic approaches to genome-wide data for the purposes of biological discovery.
Assuntos
Neoplasias Gástricas/genética , Análise por Conglomerados , Perfilação da Expressão Gênica , Fosfolipases A2 do Grupo II , Humanos , Análise de Sequência com Séries de Oligonucleotídeos , Fosfolipases A/biossíntese , Fosfolipases A/genética , Receptor EphB2/biossíntese , Receptor EphB2/genética , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patologiaRESUMO
Cigarette smoking is a risk factor for colorectal cancer. It is suggested that 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), a tobacco-specific nitrosamine, mediates the carcinogenic action of cigarette smoking by promoting cancer growth. In the present study, the proliferative response of a cultured colon cancer cell line HT-29 to NNK was determined. It was found that NNK dose-dependently stimulated HT-29 cell proliferation. In this regard, the stimulatory action of NNK was abolished by atenolol and ICI 118,551, a beta1- and beta2-selective antagonist, respectively. In addition, cell growth was stimulated by the nonselective adrenergic agonist, noradrenaline, and more effectively by the beta-selective agonist, isoproterenol. The second message cyclic AMP level for beta-adrenoceptor activation was elevated by isoproterenol and NNK treatment. These agents also up-regulated cyclooxygenase-2 expression, cytosolic phospholipase A2 expression, and prostaglandin E2 release. Beta2-adrenoceptor blockade with ICI 118,551, in contrast, significantly decreased cyclooxygenase-2 expression, cytosolic phospholipase A2 expression and prostaglandin E2 release induced by NNK and isoproterenol. To conclude, it is proposed that NNK stimulates HT-29 cell proliferation through beta-adrenoceptors, preferentially beta2 receptors. Activation of the beta-adrenoceptors, and the consequent cyclic AMP elevation coupled with the downstream arachidonic acid pathway, is perhaps an important mechanistic cascade in the promotion of colon cancer growth. These findings partly elucidate the carcinogenic actions of cigarette smoke and shed new light on the novel modulatory role of beta-adrenoceptors in the development of colon cancer.
Assuntos
Agonistas Adrenérgicos beta/farmacologia , Antagonistas Adrenérgicos beta/farmacologia , Carcinógenos/toxicidade , Neoplasias do Colo/patologia , Nitrosaminas/toxicidade , Receptores Adrenérgicos beta/fisiologia , Atenolol/farmacologia , Carcinógenos/antagonistas & inibidores , Processos de Crescimento Celular/efeitos dos fármacos , Processos de Crescimento Celular/fisiologia , Neoplasias do Colo/induzido quimicamente , Neoplasias do Colo/etiologia , AMP Cíclico/metabolismo , Ciclo-Oxigenase 2 , Dinoprostona/metabolismo , Interações Medicamentosas , Células HT29 , Humanos , Isoproterenol/farmacologia , Proteínas de Membrana , Nitrosaminas/antagonistas & inibidores , Norepinefrina/farmacologia , Fosfolipases A/biossíntese , Fosfolipases A/genética , Fosfolipases A2 , Propanolaminas/farmacologia , Prostaglandina-Endoperóxido Sintases/biossíntese , Prostaglandina-Endoperóxido Sintases/genética , Fumaça/efeitos adversos , Nicotiana/efeitos adversos , Nicotiana/química , Regulação para CimaRESUMO
For the past 60 years, dietary intake of essential fatty acids has increased. Moreover, the omega-6 fatty acids have recently been found to play an important role in regulation of gene expression. Proliferation of human prostate cells was significantly increased 48 h after arachidonic acid (AA) addition. We have analyzed initial uptake using nile red fluorescence and we found that the albumin conjugated AA is endocytosed into the cells followed by the induction of RNA within minutes, protein and PGE2 synthesis within hours. Here we describe that AA induces expression of cytosolic phospholipase A2 (cPLA2) in a dose-dependent manner and that this upregulation is dependent upon downstream synthesis of PGE2. The upregulation of cox-2 and cPLA2 was inhibited by flurbiprofen, a cyclooxygenase (COX) inhibitor, making this a second feed-forward enzyme in the eicosanoid pathway. Cox-2 specific inhibitors are known to inhibit colon and prostate cancer growth in humans; however, recent findings show that some of these have cardiovascular complications. Since cPLA2 is upstream in the eicosanoid pathway, it may be a good alternative for a pharmaceutical target for the treatment of cancer.
Assuntos
Ácido Araquidônico/farmacologia , Ácido Araquidônico/farmacocinética , Fosfolipases A/genética , Divisão Celular , Linhagem Celular Tumoral , Ciclo-Oxigenase 2 , Citoplasma/enzimologia , Indução Enzimática/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Proteínas de Membrana , Fosfolipases A/biossíntese , Fosfolipases A2 , Regiões Promotoras Genéticas , Prostaglandina-Endoperóxido Sintases/genética , Neoplasias da Próstata/enzimologia , Proteínas Proto-Oncogênicas c-fos/genética , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
The present study shows that the incubation of human aortic smooth muscle cells (HASMC) and HepG2 cells with atorvastatin and mevastatin as HMG-CoA reductase inhibitors potentiated the interferon-gamma (INF-gamma)-induced group IIA phospholipase A(2) (sPLA(2)-IIA) expression in a dose- and time-dependent manner. The effect of statins on sPLA(2)-IIA expression was reduced by mevalonate, farnesyl pyrophosphate and geranylgeranyl pyrophosphate. Inversely, inhibitors of the farnesyl transferase and geranylgeranyl transferase-I mimicked the effects of statins. Clostridium difficile toxin B (TcdB), Y-27632 and H-1152, functioning as inhibitors of Rho proteins and Rho-associated kinase, also augmented the sPLA(2)-IIA expression in combination with IFN-gamma. The same effects were observed when inhibitors of mitogen-activated/extracellular response protein kinase kinase (MEK), PD98059 or U0126 were used. Further, the Janus kinase-2 (Jak2)-specific inhibitor, AG-490 and inhibitors of nuclear factor-kappaB (NFkappaB) abrogated the sPLA(2)-IIA elevating effects of statins, TcdB and PD98059 in the presence of IFN-gamma. This cytokine alone increased the NFkappaB p65 and CAAT-enhancer-binding protein-beta (C/EBP-beta) activity in HASMC nuclear extract, but only C/EBP-beta was further augmented when the cells were incubated in addition to IFN-gamma with atorvastatin, H-1152, PD98059 or U0126. Moreover, after the incubation of cells with atorvastatin and IFN-gamma the stability of sPLA-(2)IIA mRNA significantly increased in comparison to those after incubation with IFN-gamma alone. In conclusion, the obtained data suggest that (i) the expression of sPLA(2)-IIA is negatively regulated by RhoA/Rho-associated kinase and MEK/ERK signaling pathways and (ii) statins, because of their ability to down-regulate these pathways, can potentiate the IFN-gamma-induced sPLA(2)-II expression at transcriptional and post-transcriptional levels.
Assuntos
Linhagem Celular Tumoral/efeitos dos fármacos , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Interferon gama/farmacologia , Lovastatina/análogos & derivados , Miócitos de Músculo Liso/efeitos dos fármacos , Fosfolipases A/biossíntese , Amidas/farmacologia , Aorta , Atorvastatina , Proteínas de Bactérias/farmacologia , Toxinas Bacterianas/farmacologia , Butadienos/farmacologia , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Carcinoma Hepatocelular , Linhagem Celular Tumoral/metabolismo , Sinergismo Farmacológico , Flavonoides/farmacologia , Fosfolipases A2 do Grupo II , Ácidos Heptanoicos/farmacologia , Humanos , Janus Quinase 2 , Lovastatina/farmacologia , Miócitos de Músculo Liso/metabolismo , NF-kappa B/antagonistas & inibidores , NF-kappa B/metabolismo , Nitrilas/farmacologia , Fosfolipases A2 , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Piridinas/farmacologia , Pirróis/farmacologia , Estabilidade de RNA , Regulação para CimaRESUMO
Cellular pools of free arachidonic acid are tightly controlled through enzymatic release of the fatty acid and subsequent utilization by downstream enzymes including the cyclooxygenases. Arachidonic acid cleavage from membrane phospholipids is accomplished by the actions of phospholipase A(2) (PLA(2)). Upon release, free arachidonic acid provides substrate for the synthesis of eicosanoids. However, under certain conditions, arachidonic acid may participate in ceramide-mediated apoptosis. Disruption of arachidonic acid homeostasis can shift the balance of cell turnover in favor of tumorigenesis, via overproduction of tumor-promoting eicosanoids or alternatively by limiting proapoptotic signals. In the following study, we evaluated the influence of genetic deletion of a key intracellular phospholipase, cytoplasmic PLA(2) (cPLA(2)), on azoxymethane-induced colon tumorigenesis. Heterozygous and null mice, upon treatment with the organotropic colon carcinogen, azoxymethane, developed a significant (P < 0.05) increase in colon tumor multiplicity (7.2-fold and 5.5-fold, respectively) relative to their wild-type littermates. This enhanced tumor sensitivity may be explained, in part, by the attenuated levels of apoptosis observed by terminal deoxynucleotidyl transferase-mediated nick end labeling staining within the colonic epithelium of heterozygous and null mice ( approximately 50% of wild type). The lower frequency of apoptotic cells corresponded with reduced ceramide levels (69% and 46% of wild-type littermates, respectively). Remarkably, increased tumorigenesis resulting from cPLA(2) deletion occurred despite a significant reduction in prostaglandin E(2) production, even in cyclooxygenase-2-overexpressing tumors. These data contribute new information that supports a fundamental role of cPLA(2) in the control of arachidonic acid homeostasis and cell turnover. Our findings indicate that the proapoptotic role of cPLA(2) in the colon may supercede its contribution to eicosanoid production in tumor development.
Assuntos
Neoplasias do Colo/enzimologia , Fosfolipases A/deficiência , Animais , Apoptose/genética , Azoximetano , Neoplasias do Colo/induzido quimicamente , Neoplasias do Colo/genética , Neoplasias do Colo/patologia , Cruzamentos Genéticos , Ciclo-Oxigenase 2 , Citoplasma/enzimologia , Dinoprostona/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fosfolipases A/biossíntese , Fosfolipases A/genética , Fosfolipases A2 , Prostaglandina-Endoperóxido Sintases/metabolismoRESUMO
Although individual mammalian secreted phospholipase A(2) (sPLA(2)) enzymes exhibit unique tissue and cellular distributions, the cell type-specific functions of each enzyme remain largely unknown. In this study, we found by immunohistochemistry that group X sPLA(2) (sPLA(2)-X) is uniquely located in the peripheral neuronal fibers, an observation that was supported by detection of its transcript and protein in the neuronal cell line PC12 and in primary dorsal root ganglia neurons. Adenoviral expression of sPLA(2)-X in PC12 cells facilitated neurite outgrowth, particularly when combined with a suboptimal concentration of nerve growth factor. In neuronally differentiated PC12 cells, sPLA(2)-X was preferentially localized in the Golgi apparatus and growth cones, and proteolytic conversion of the proenzyme to mature enzyme mainly occurred after the secretion process. The neurite-extending ability of sPLA(2)-X depended on the production of its catalytic product, lysophosphatidylcholine. Moreover, nerve growth factor-induced neurite extension of PC12 cells was modestly but significantly attenuated by an anti-sPLA(2)-X antibody or by a small interfering RNA for sPLA(2)-X. These observations suggest the potential contribution of sPLA(2)-X to neuronal differentiation, and possibly repair, under certain conditions.
Assuntos
Neurônios/enzimologia , Fosfolipases A/biossíntese , Fosfolipases A/metabolismo , Adenoviridae/metabolismo , Animais , Northern Blotting , Catálise , Diferenciação Celular , Linhagem Celular , Cromatografia em Camada Fina , DNA Complementar/metabolismo , Gânglios Espinais/metabolismo , Complexo de Golgi/metabolismo , Humanos , Immunoblotting , Imuno-Histoquímica , Lisofosfatidilcolinas/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Confocal , Fator de Crescimento Neural , Neurônios/metabolismo , Células PC12 , Fosfolipases A2 , RNA Interferente Pequeno/metabolismo , Ratos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Espectrometria de Massas por Ionização por Electrospray , Fatores de TempoRESUMO
The present study underlines the importance of phospholipase A2 (PLA2)- and lipoxygenase (LO)-mediated signaling processes in the regulation of inducible nitric oxide synthase (iNOS) gene expression. In glial cells, lipopolysaccharide (LPS) induced the activities of PLA2 (calcium-independent PLA2; iPLA2 and cytosolic PLA2; cPLA2) as well as gene expression of iNOS. The inhibition of cPLA2 by methyl arachidonyl fluorophosphates (MAFP) or antisense oligomer against cPLA2 and inhibition of iPLA2 by bromoenol lactone reduced the LPS-induced iNOS gene expression and NFkappaB activation. In addition, the inhibition of LO by nordihydroguaiaretic acid (NDGA; general LO inhibitor) or MK886 (5-LO inhibitor), but not baicalein (12-LO inhibitor), completely abrogated the LPS-induced iNOS expression. Because NDGA could abrogate the LPS-induced activation of NFkappaB, while MK886 had no effect on it, LO-mediated inhibition of iNOS gene induction by LPS may involve an NFkappaB-dependent or -independent (by 5-LO) pathway. In contrast to LO, however, the cyclooxygenase (COX) may not be involved in the regulation of LPS-mediated induction of iNOS gene because COX inhibition by indomethacin (general COX inhibitor), SC560 (COX-1 inhibitor), and NS398 (COX-2 inhibitor) affected neither the LPS-induced iNOS expression nor activation of NFkappaB. These results indicate a role for cPLA2 and iPLA2 in LPS-mediated iNOS gene induction in glial cells and the involvement of LO in these reactions.
Assuntos
Lipopolissacarídeos/farmacologia , Lipoxigenase/metabolismo , Neuroglia/enzimologia , Óxido Nítrico Sintase/biossíntese , Fosfolipases A/biossíntese , Animais , Araquidonato 5-Lipoxigenase/metabolismo , Ácido Araquidônico/metabolismo , Northern Blotting , Linhagem Celular , Linhagem Celular Tumoral , Núcleo Celular/genética , Ensaio de Desvio de Mobilidade Eletroforética , Indução Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Genes Reporter/genética , Isoenzimas/antagonistas & inibidores , Isoenzimas/biossíntese , Isoenzimas/genética , NF-kappa B/metabolismo , Neuroglia/efeitos dos fármacos , Neuroglia/metabolismo , Óxido Nítrico Sintase/genética , Óxido Nítrico Sintase Tipo II , Fosfolipases A/antagonistas & inibidores , Fosfolipases A/genética , Fosfolipases A2 , RNA/análise , RNA/biossíntese , Ratos , Ativação Transcricional , TransfecçãoRESUMO
The objective of this project was to identify some possible mechanisms by which two common phytochemicals, resveratrol and beta-sitosterol, inhibit the growth of human prostate cancer PC-3 cells. These mechanisms include the effect of the phytochemicals on apoptosis, cell cycle progression, prostaglandin synthesis and the production of reactive oxygen species (ROS). Prostaglandins have been known to play a role in regulating cell growth and apoptosis. PC-3 cells were supplemented with 50 microM resveratrol or 16 microM beta-sitosterol alone or in combination for up to 5 days. Phytochemical supplementation resulted in inhibition in cell growth. beta-Sitosterol was more potent than resveratrol and the combination of the two resulted in greater inhibition than supplementation with either alone. Long-term supplementation with resveratrol or beta-sitosterol elevated basal prostaglandin release but beta-sitosterol was much more potent than resveratrol in this regard. beta-Sitosterol was more effective than resveratrol in inducing apoptosis and the combination had an intermediate effect after 1 day of supplementation. Cells supplemented with resveratrol were arrested at the G1 phase and at the G2/M phase in the case of beta-sitosterol while the combination resulted in cell arrest at the two phases of the cell cycle. beta-Sitosterol increased ROS production while resveratrol decreased ROS production. The combination of the two phytochemicals resulted in an intermediate level of ROS. The observed changes in prostaglandin levels and ROS production by these two phytochemicals may suggest their mediation in the growth inhibition. The reduction in ROS level and increase by resveratrol supplementation in PC-3 cells reflects the antioxidant properties of resveratrol. It was concluded that these phytochemicals may induce the inhibition of tumor growth by stimulating apoptosis and arresting cells at different locations in the cell cycle and the mechanism may involve alterations in ROS and prostaglandin production.
Assuntos
Prostaglandinas/biossíntese , Espécies Reativas de Oxigênio/metabolismo , Sitosteroides/farmacologia , Estilbenos/farmacologia , 6-Cetoprostaglandina F1 alfa/biossíntese , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Ciclo-Oxigenase 1/biossíntese , Ciclo-Oxigenase 2/biossíntese , Dinoprostona/biossíntese , Sinergismo Farmacológico , Humanos , Masculino , Fosfolipases A/biossíntese , Resveratrol , Sitosteroides/administração & dosagem , Estilbenos/administração & dosagemRESUMO
Atherosclerosis is a chronic inflammatory disease of the vessel wall characterized by the accumulation of lipid-laden macrophages and fibrotic material. The initiation of the disease is accompanied by the accumulation of modified lipoproteins in the vessel wall. Group IIa secretory phospholipase A2 (sPLA2 IIa) is a key candidate player in the enzymatic modification of low density lipoproteins. To study the role of sPLA2 IIa in macrophages during atherogenesis, transgenic mice were generated using the human sPLA2 IIa gene and the CD11b promoter. Bone marrow transplantation to LDL receptor-deficient mice was performed to study sPLA2 IIa in atherosclerosis. After 10 weeks of high-fat diet, mice overexpressing sPLA2 IIa in macrophages showed 2.3-fold larger lesions compared with control mice. Pathological examination revealed that sPLA2 IIa-expressing mice had increased collagen in their lesions, independent of lesion size. However, smooth muscle cells or fibroblasts in the lesions were not affected. Other parameters studied, including T-cells and cell turnover, were not significantly affected by overexpression of sPLA2 IIa in macrophages. These data clearly show that macrophage sPLA2 IIa is a proatherogenic factor and suggest that the enzyme regulates collagen production in the plaque and thus fibrotic cap development.
Assuntos
Arteriosclerose/metabolismo , Colágeno/metabolismo , Macrófagos/metabolismo , Fosfolipases A/biossíntese , Animais , Northern Blotting , Transplante de Medula Óssea , Antígeno CD11b/genética , Colesterol/metabolismo , Fibroblastos/metabolismo , Células Espumosas/metabolismo , Fosfolipases A2 do Grupo II , Humanos , Marcação In Situ das Extremidades Cortadas , Lipoproteínas LDL/metabolismo , Camundongos , Camundongos Transgênicos , Modelos Genéticos , Miócitos de Músculo Liso/metabolismo , Necrose , Fenótipo , Fosfolipases/química , Fosfolipases A2 , Regiões Promotoras Genéticas , Receptores de LDL/genética , Receptores de LDL/fisiologia , Fatores de TempoRESUMO
Phospholipase A(2) (PLA(2)) is a superfamily of enzymes that may play a major role in airways inflammation. We investigated the effect of interferon-gamma (IFN-gamma) on the gene expression of 19 different PLA(2) types in human monocyte-derived macrophages and nasal epithelial cells (RPMI 2650). The cells were stimulated with IFN-gamma for different lengths of time (up to 48 h), and the mRNA levels of the different PLA(2) types were determined by reverse transcriptase-PCR (RT-PCR) and normalized to those of the house-keeping gene, GAPDH. It appeared that IFN-gamma clearly increased the expression of secretory PLA(2) IID (but not IIA) in macrophages, while both PLA(2) IID and IIA were upregulated in RPMI 2650 cells. Moreover, after 18 h, the mRNA levels of cytosolic PLA(2) IVA were 2-3 times higher in IFN-gamma-stimulated macrophages than controls, while there was no such effect of IFN-gamma in RPMI 2650 cells. Lipopolysaccharide (LPS) augmented the increased gene expression of PLA(2) IVA but decreased both the basal and the IFN-gamma-induced PLA(2) IID mRNA expression in macrophages (but not in RPMI 2650 cells). The NF-kappaB inhibitor Pyrrolidine dithiocarbamate (PDTC) and the phoshatidylinositol 3-kinase (PI3K) inhibitor wortmannin were employed to get an insight into the mechanism behind these observations. Incubation of macrophages with PDTC had no effect on the LPS impairment of PLA(2) IID gene expression, but inhibited the LPS mediated activation of PLA(2) IVA. No significant effect was noted of PDTC on IFN-gamma stimulation, while PI3K had no effect at all on any of the stimuli used. Furthermore, LPS (but not IFN-gamma) increased the mRNA levels of the nuclear factor (NF)-kappaB inhibitors alpha and xi in macrophages, but not in RPMI 2650 cells. These findings indicate that (a) the gene expression of secretory types PLA(2) IID and IIA in response to IFN-gamma is much dependent on cell type, and (b) the regulation of PLA(2) type IID in human macrophages is clearly different from that of PLA(2) type IVA. (c) PLA(2) IVA is probably under control of both NF-kappaB and IFN-gamma-responsive elements (GRE) or IFN-gamma-activating sites (GAS). The possibility that PLA(2) IID is involved in cytokine-mediated inflammation in the nasal mucosa is inferred, as is the potential role of PLA(2) IID in the host defense against LPS-containing bacteria.
Assuntos
Regulação Enzimológica da Expressão Gênica , Interferon gama/farmacologia , Lipopolissacarídeos/metabolismo , Macrófagos/enzimologia , Monócitos/enzimologia , Fosfolipases A/biossíntese , Linhagem Celular , Citocinas/metabolismo , Primers do DNA/química , Fosfolipases A2 do Grupo II , Humanos , Interferon gama/metabolismo , Ativação de Macrófagos , Macrófagos/metabolismo , Monócitos/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Fosfolipases A2 , Proteínas Recombinantes , Fatores de TempoRESUMO
In colorectal cancer, cyclooxygenase-2 (COX-2) overexpression in stromal cells induces angiogenesis through EP2 prostaglandin E2 receptor signaling. Cytoplasmic phospholipase A2 (PLA2) alpha preferentially hydrolyses arachidonic acid, which is the limiting substrate for prostaglandin production, from membrane phospholipids. We therefore investigated a possible relationship between cytoplasmic PLA2 and COX-2 overexpression in stromal cells, angiogenesis and microsatellite instability in 48 human colorectal adenocarcinomas. Cytoplasmic PLA2 and COX-2 expression in stromal cells and vascular endothelial growth factor (VEGF) expression in tumor cells were evaluated by immunohistochemistry. Microvessel density was assessed in 10 x 400 fields after CD31 staining. Microsatellite instability was evaluated by PCR and immunohistochemistry. A total of 16 tumors had microsatellite instability. We found an overexpression of cytoplasmic PLA2 in superficial stromal cells. These cells corresponded to fibroblasts and myofibroblasts. There was an association between the number of cytoplasmic PLA2 and COX-2-expressing cells (P=0.006). Cytoplasmic PLA2-positive stromal cells usually also expressed COX-2. A high number of cytoplasmic PLA2-positive stromal cells was correlated with a high microvessel density (P=0.002), a strong VEGF (P=0.01) and the absence of microsatellite instability (P=0.001). The coordinate overexpression of cytoplasmic PLA2 and COX-2 in stromal cells could lead to an important prostaglandin production. These results suggest that cytoplasmic PLA2 overexpression in these cells regulates COX-induced angiogenesis probably by providing arachidonic acid, which is the limiting factor for prostaglandin production. The lower number of cytoplasmic PLA2-positive stromal cells in carcinomas with microsatellite instability could be related to their lower microvessel density and VEGF expression.
Assuntos
Neoplasias Colorretais/irrigação sanguínea , Neovascularização Patológica/patologia , Fosfolipases A/biossíntese , Células Estromais/enzimologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Neoplasias Colorretais/enzimologia , Neoplasias Colorretais/genética , Ciclo-Oxigenase 2 , Citoplasma/enzimologia , Fosfolipases A2 do Grupo IV , Humanos , Imuno-Histoquímica , Proteínas de Membrana , Repetições de Microssatélites/genética , Microscopia de Fluorescência , Pessoa de Meia-Idade , Fosfolipases A2 , Prostaglandina-Endoperóxido Sintases/biossíntese , Células Estromais/patologia , Fator A de Crescimento do Endotélio Vascular/biossínteseRESUMO
Although a number of sPLA2 (secretory phospholipase A2) enzymes have been identified in mammals, the localization and functions of individual enzymes in human pathologic tissues still remain obscure. In the present study, we have examined the expression and function of sPLA2s in human lung-derived cells and in human lungs with pneumonia. Group IID, V and X sPLA2s were expressed in cultured human bronchial epithelial cells (BEAS-2B) and normal human pulmonary fibroblasts with distinct requirement for cytokines (interleukin-1b, tumour necrosis factor a and interferon-g). Lentivirus- or adenovirus-mediated transfection of various sPLA2s into BEAS-2B or normal human pulmonary fibroblast cells revealed that group V and X sPLA2s increased arachidonate release and prostaglandin production in both cell types, whereas group IIA and IID sPLA2s failed to do so. Immunohistochemistry of human lungs with pneumonia demonstrated that group V and X sPLA2s were widely expressed in the airway epithelium, interstitium and alveolar macrophages, in which group IID sPLA2 was also positive, whereas group IIA sPLA2 was restricted to the pulmonary arterial smooth muscle layers and bronchial chondrocytes, and group IIE and IIF sPLA2s were minimally detected. These results suggest that group V and X sPLA2s affect lung pathogenesis by facilitating arachidonate metabolism or possibly through other functions.