RESUMEN
Cancer cells are known to survive in a hypoxic microenvironment by altering their lipid metabolism as well as their energy metabolism. In this study, Caco-2 cells derived from human colon cancer, were found to have elevated intracellular levels of phosphatidic acid and its lysoform, lysophosphatidic acid (LPA), under hypoxic conditions. Our results suggested that the elevation of LPA in Caco-2 cells was mainly due to the combined increases in cellular levels of lysophosphatidylcholine and lysophosphatidylethanolamine by phospholipase A2 and subsequent hydrolysis to LPA by lysophospholipase D. We detected the Ca2+ -stimulated choline-producing activities toward exogenous lysophosphatidylcholines in whole Caco-2 cell homogenates, indicating their involvement in the LPA production in intact Caco-2 cells.
Asunto(s)
Lisofosfolípidos , Ácidos Fosfatidicos , Humanos , Células CACO-2 , Lisofosfatidilcolinas/metabolismoRESUMEN
Plasmalogen localized in the raft of mammalian cell membranes plays a role in the storage of polyunsaturated fatty acid (PUFA), and exists to a higher extent in malignant cells that survive, and even grow in hypoxic conditions. The biosynthesis of plasmalogen in mammalian cells has been reported to depend on aerobic conditions. Using liquid chromatography-tandem mass spectrometry, we found that the intracellular concentration of plasmalogen species containing a PUFA at the sn-2-position did not change for two days from the start of hypoxic culture in human colorectal cancer-derived Caco2 cells. At the third day of hypoxia, Caco2 cells showed the average increase rate of 2.6 times in ethanolamine plasmalogen and 2.9 times in choline plasmalogen depending on the molecular species compared with those in the second day of hypoxia. In normoxic culture, there was little quantitative change in any species of both ethanolamine and choline plasmalogens for three days. The up-regulations of mRNA of Ca2+-independent phospholipase A2ß and cytoplasmic phospholipase A2γ as well as the down-regulation of lysoplasmalogenase observed in hypoxia were suggested to be responsible for the increase of plasmalogen in Caco2 cells under hypoxia.
Asunto(s)
Neoplasias Colorrectales , Plasmalógenos , Células CACO-2 , Ácidos Grasos Insaturados/metabolismo , Humanos , Hipoxia , FosfolipasasRESUMEN
Sphingolipids constitute a class of bio-reactive molecules that transmit signals and exhibit a variety of physical properties in various cell types, though their functions in cancer pathogenesis have yet to be elucidated. Analyses of gene expression profiles of clinical specimens and a panel of cell lines revealed that the ceramide synthase gene CERS6 was overexpressed in non-small-cell lung cancer (NSCLC) tissues, while elevated expression was shown to be associated with poor prognosis and lymph node metastasis. NSCLC profile and in vitro luciferase analysis results suggested that CERS6 overexpression is promoted, at least in part, by reduced miR-101 expression. Under a reduced CERS6 expression condition, the ceramide profile became altered, which was determined to be associated with decreased cell migration and invasion activities in vitro. Furthermore, CERS6 knockdown suppressed RAC1-positive lamellipodia/ruffling formation and attenuated lung metastasis efficiency in mice, while forced expression of CERS6 resulted in an opposite phenotype in examined cell lines. Based on these findings, we consider that ceramide synthesis by CERS6 has important roles in lung cancer migration and metastasis.
Asunto(s)
Movimiento Celular , Neoplasias Pulmonares/enzimología , Neoplasias Pulmonares/patología , Proteínas de la Membrana/metabolismo , Esfingosina N-Aciltransferasa/metabolismo , Animales , Secuencia de Bases , Línea Celular Tumoral , Ceramidas/metabolismo , Humanos , Masculino , Ratones Desnudos , MicroARNs/genética , MicroARNs/metabolismo , Modelos Biológicos , Metástasis de la Neoplasia , Seudópodos/metabolismo , Resultado del TratamientoRESUMEN
Resveratrol (RSV) is a plant-derived phytoalexin present in plants, whose pleiotropic effects for health benefits have been previously reported. Its anti-cancer activity is among the current topics for novel cancer treatment. Here, effects of RSV on cell proliferation and the sphingolipid metabolism of K562, a human leukemia cell line, were analyzed. Some experiments were also performed in HCT116, a human colon cancer cell line. RSV inhibited cell proliferation of both cell lines. Increased cellular ceramide and decreased sphingomyelin and S1P by RSV were observed in RSV-treated K562 cells. Further analysis revealed that acid sphingomyelinase mRNA and enzyme activity levels were increased by RSV. Desipramine, a functional ASMase inhibitor, prevented RSV-induced ceramide increase. RSV increased ATF3, EGR1, EGR3 proteins and phosphorylated c-Jun and FOXO3. However, co-transfection using these transcription factor expression vectors and ASMase promoter reporter vector revealed positive effects of EGR1 and EGR3 but not others. Electrophoresis mobility shift assay (EMSA) and Chromatin immunoprecipitation (ChIP) assay demonstrated the direct binding of EGR1/3 transcription factors with ASMase 5'-promoter. These results indicate that increased EGR1/3 and ASMase expression play an important role in cellular ceramide increase by RSV treatment.
Asunto(s)
Neoplasias Experimentales/tratamiento farmacológico , Neoplasias Experimentales/metabolismo , Esfingomielina Fosfodiesterasa/metabolismo , Estilbenos/administración & dosificación , Activación Transcripcional/efectos de los fármacos , Anticarcinógenos/administración & dosificación , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Células K562 , Neoplasias Experimentales/patología , Resveratrol , Esfingomielina Fosfodiesterasa/genética , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/genéticaRESUMEN
Sphingolipids make up a family of molecules associated with an array of biological functions, including cell death and migration. Sphingolipids are often altered in cancer, though how these alterations lead to tumor formation and progression is largely unknown. Here, we analyzed non-small-cell lung cancer (NSCLC) specimens and cell lines and determined that ceramide synthase 6 (CERS6) is markedly overexpressed compared with controls. Elevated CERS6 expression was due in part to reduction of microRNA-101 (miR-101) and was associated with increased invasion and poor prognosis. CERS6 knockdown in NSCLC cells altered the ceramide profile, resulting in decreased cell migration and invasion in vitro, and decreased the frequency of RAC1-positive lamellipodia formation while CERS6 overexpression promoted it. In murine models, CERS6 knockdown in transplanted NSCLC cells attenuated lung metastasis. Furthermore, combined treatment with l-α-dimyristoylphosphatidylcholine liposome and the glucosylceramide synthase inhibitor D-PDMP induced cell death in association with ceramide accumulation and promoted cancer cell apoptosis and tumor regression in murine models. Together, these results indicate that CERS6-dependent ceramide synthesis and maintenance of ceramide in the cellular membrane are essential for lamellipodia formation and metastasis. Moreover, these results suggest that targeting this homeostasis has potential as a therapeutic strategy for CERS6-overexpressing NSCLC.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas/patología , Neoplasias Pulmonares/patología , Proteínas de la Membrana/fisiología , Esfingosina N-Aciltransferasa/fisiología , Animales , Apoptosis/efectos de los fármacos , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Línea Celular Tumoral , Ceramidas/metabolismo , Dimiristoilfosfatidilcolina/farmacología , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Masculino , Proteínas de la Membrana/antagonistas & inhibidores , Proteínas de la Membrana/genética , Ratones , MicroARNs/fisiología , Metástasis de la Neoplasia , Fenotipo , Esfingosina N-Aciltransferasa/antagonistas & inhibidores , Esfingosina N-Aciltransferasa/genéticaRESUMEN
Oxygen-requiring enzymes, such as Δ4-desaturase (dihydroceramide desaturase), sphingolipid Δ4-desaturase/C-4-hydroxylase, and fatty acid 2-hydroxylase are involved in ceramide synthesis. We prepared free ceramides, sphingomyelins and glycosphingolipids (GSLs) from cancer cells cultivated under conditions of normoxia and hypoxia, and analyzed these compounds using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Human colon cancer LS174T cells were employed because these cells highly express hydroxyl fatty acids and phytosphingosine (t18:0) which are expected to be greatly influenced by changes in oxygen levels. As expected, the populations of dihydro-species of free ceramide and sphingomyelin with C16:0 non-hydroxy fatty acid were elevated, and the populations of HexCers and Hex2Cers, composed of C16:0 or C16:0 hydroxy fatty acid (C16:0h), and sphingosine (d18:1) or t18:0, were decreased under hypoxia. However, appreciable populations of HexCer and Hex2Cer species of C24:0 or C24:0h and t18:0 remained. These results suggest that the individual species of GSLs with fatty acids possessing different alkyl chain lengths, either non-hydroxy fatty acids or hydroxyl fatty acids, may be metabolized individually.
Asunto(s)
Ceramidas/química , Neoplasias del Colon/metabolismo , Oxígeno/metabolismo , Esfingosina/análogos & derivados , Células CACO-2 , Hipoxia de la Célula , Línea Celular Tumoral , Cromatografía en Capa Delgada , Ácidos Grasos/análisis , Ácidos Grasos/química , Glicoesfingolípidos/química , Humanos , Modelos Lineales , Espectrometría de Masas , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Esfingomielinas/metabolismo , Esfingosina/química , Espectrometría de Masas en TándemRESUMEN
Acid ceramidase (ACDase) metabolizes ceramide to sphingosine, leading to sphingosine 1-phosphate production. Reportedly, ACDase has been upregulated in prostate cancer. However, its regulatory mechanism remains unclear. LNCaP (androgen-sensitive prostate cancer cell line) but not PC3 and DU-145, (androgen-unresponsive cell lines) exhibited the highest ACDase protein. Among three cell lines, ASAH1 mRNA level was not correlated with ACDase protein expression, and the 5'-promoter activity did not show androgen dependency, suggesting the post-transcriptional regulation of ACDase in LNCaP cells. Based on these results, LNCaP was analysed further. Casodex, androgen receptor antagonist, and charcoal-stripped FCS (CS-FCS) decreased ACDase protein and activity, whereas dihydrotestosterone in CS-FCS culture increased ACDase protein and enzyme activity. MG132, a proteasome inhibitor, prevented the decrease of ACDase protein when cultured in CS-FCS, suggesting the involvement of ubiquitin/proteasome system. Reportedly, USP2, a deubiquitinase, plays an important role in LNCaP cells. USP2 siRNA decreased ACDase protein, whereas USP2 overexpression increased ACDase protein of LNCaP cells. However, SKP2, an ubiquitin E3 ligase known to be active in prostate cancer, did not affect androgen-dependent ACDase expression in LNCaP cells. Thus, ACDase regulation by androgen in androgen-sensitive LNCaP cells is mainly due to its prolonged protein half-life by androgen-stimulated USP2 expression.
Asunto(s)
Ceramidasa Ácida/metabolismo , Endopeptidasas/metabolismo , Regulación Neoplásica de la Expresión Génica , Proteínas de Neoplasias/metabolismo , Neoplasias de la Próstata/enzimología , Ceramidasa Ácida/antagonistas & inhibidores , Ceramidasa Ácida/genética , Antagonistas de Andrógenos/farmacología , Andrógenos/farmacología , Antineoplásicos/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/enzimología , Neoplasias de la Mama/metabolismo , Línea Celular Tumoral , Endopeptidasas/química , Endopeptidasas/genética , Inducción Enzimática/efectos de los fármacos , Estabilidad de Enzimas/efectos de los fármacos , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/enzimología , Neoplasias Pulmonares/metabolismo , Masculino , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/genética , Regiones Promotoras Genéticas/efectos de los fármacos , Neoplasias de la Próstata/tratamiento farmacológico , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata Resistentes a la Castración/tratamiento farmacológico , Neoplasias de la Próstata Resistentes a la Castración/enzimología , Neoplasias de la Próstata Resistentes a la Castración/metabolismo , Complejo de la Endopetidasa Proteasomal/efectos de los fármacos , Complejo de la Endopetidasa Proteasomal/metabolismo , Inhibidores de Proteasoma/farmacología , Interferencia de ARN , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Ubiquitina TiolesterasaRESUMEN
Sphingosine kinases (SPHK) are important to determine cells' fate by producing sphingosine 1-phosphate. Reportedly, exogenous SPHK2 overexpression induces cell cycle arrest or cell death. However, the regulatory mechanism of SPHK2 expression has not been fully elucidated. Here, we analyzed this issue using human colon cancer cell lines under various stress conditions. Serum depletion (FCS(-)) but not hypoxia and glucose depletion increased mRNA, protein and enzyme activity of SPHK2 but not SPHK1. In HCT116 cells mostly used, SPHK2 activity was predominant over SPHK1, and serum depletion increased both nuclear and cytoplasmic SPHK2 activity. Based on previous reports analyzing cellular response after serum depletion, the temporal changes of intracellular signaling molecules and candidate transcription factors for SPHK2 were examined using serum-depleted HCT116 cells, and performed transfection experiments with siRNA or cDNA of candidate transcription factors. Results showed that the rapid and transient JNK activation followed by CREB activation was the major regulator of increased SPHK2 transcription in FCS(-) culture. EMSA and ChIP assay confirmed the direct binding of activated CREB to the CREB binding site of 5' SPHK2 promoter region. Colon cancer cells examined continued to grow in FCS(-) culture, although mildly, while hypoxia and glucose depletion suppressed cell proliferation or induced cell death, suggesting the different role of SPHK2 in different stress conditions. Because of the unique relationship observed after serum depletion, we examined effects of siRNA for SPHK2, and found the role of SPHK2 as a growth or survival factor but not a cell proliferation inhibitor in FCS(-) culture.
Asunto(s)
Proliferación Celular/genética , Neoplasias del Colon/genética , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/biosíntesis , Diferenciación Celular , Neoplasias del Colon/patología , Medio de Cultivo Libre de Suero , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/genética , Regulación Neoplásica de la Expresión Génica , Células HCT116 , Humanos , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Unión Proteica , ARN Mensajero/biosíntesisRESUMEN
Multidrug resistance (MDR) is a serious problem faced in the treatment of malignant tumors. In this study, we characterized the expression of non-homologous DNA end joining (NHEJ) components, a major DNA double strand break (DSB) repair mechanism in mammals, in K562 cell and its daunorubicin (DNR)-resistant subclone (K562/DNR). K562/DNR overexpressed major enzymes of NHEJ, DNA-PKcs and DNA ligase IV, and K562/DNR repaired DSB more rapidly than K562 after DNA damage by neocarzinostatin (MDR1-independent radiation-mimetic). Overexpressed DNA-PKcs and DNA ligase IV were also observed in DNR-resistant HL60 (HL60/DNR) cells as compared with parental HL60 cells. Expression level of DNA-PKcs mRNA paralleled its protein level, and the promoter activity of DNA-PKcs of K562/DNR was higher than that of K562, and the 5'-region between -49bp and the first exon was important for its activity. Because this region is GC-rich, we tried to suppress Sp1 family transcription factor using mithramycin A (MMA), a specific Sp1 family inhibitor, and siRNAs for Sp1 and Sp3. Both MMA and siRNAs suppressed DNA-PKcs expression. Higher serine-phosphorylated Sp1 but not total Sp1 of both K562/DNR and HL60/DNR was observed compared with their parental K562 and HL60 cells. DNA ligase IV expression of K562/DNR was also suppressed significantly with Sp1 family protein inhibition. EMSA and ChIP assay confirmed higher binding of Sp1 and Sp3 with DNA-PKcs 5'-promoter region of DNA-PKcs of K562/DNR than that of K562. Thus, the Sp1 family transcription factor affects important NHEJ component expressions in anti-cancer drug-resistant malignant cells, leading to the more aggressive MDR phenotype.
Asunto(s)
ADN Ligasas/genética , Proteína Quinasa Activada por ADN/genética , Resistencia a Antineoplásicos/genética , Inmunoglobulinas/metabolismo , Leucemia/tratamiento farmacológico , Proteínas Nucleares/genética , Daño del ADN/genética , Reparación del ADN por Unión de Extremidades/genética , ADN Ligasa (ATP) , Reparación del ADN/genética , Proteína Quinasa Activada por ADN/metabolismo , Daunorrubicina/administración & dosificación , Regulación Leucémica de la Expresión Génica/efectos de los fármacos , Células HL-60 , Humanos , Inmunoglobulinas/genética , Células K562 , Leucemia/genética , Leucemia/patología , Proteínas Nucleares/metabolismo , Fosforilación , ARN Mensajero/biosíntesisRESUMEN
We previously performed a systematic analysis of free ceramide (Cers) species, the constituent ceramide species of sphingomyelins and neutral glycosphingolipids (NGSLs) using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry with high-energy collision-induced dissociation. As a result, distinct species differences were found among Cers, sphingomyelins and NGSLs in the kidneys. Using this method, we investigated various sphingolipid species from human colon cancer Caco-2 cells as well as the influence of environmental oxygen on these species in detail. Unexpectedly, even in normoxia, all Cers species were composed of dihydrosphingosine (d18:0) and non-hydroxy fatty acid (NFA), and 34% of sphingomyelins were composed of dihydrosphingomyelins with NFA. In contrast, major constituent ceramide species of NGSLs were composed of the usual long-chain base of sphingosine (d18:1) and hydroxy fatty acid (HFA). When the cells were cultured under hypoxic condition for 3 days, all the Cers and nearly 80% of the sphingomyelins were dihydrosphingolipids composed of d18:0-NFAs, but a significant proportion of d18:1-HFAs still remained in the NGSLs. When the cells were transferred from conditions of hypoxia to normoxia again (reoxygenation), Cer species composed of d18:1-NFAs, which were not found in Cers under the original normoxic conditions, appeared. Such Cers were probably synthesized as precursors for the constituent ceramides of sphingomyelins and NGSLs.
Asunto(s)
Ceramidas/análisis , Glicoesfingolípidos Neutros/química , Oxígeno/metabolismo , Esfingomielinas/química , Células CACO-2 , Hipoxia de la Célula , Ceramidas/química , Ceramidas/metabolismo , Ácidos Grasos/análisis , Ácidos Grasos/química , Humanos , Glicoesfingolípidos Neutros/análisis , Glicoesfingolípidos Neutros/metabolismo , Esfingomielinas/análisis , Esfingomielinas/metabolismo , Esfingosina/análogos & derivados , Esfingosina/análisis , Espectrometría de Masas en TándemRESUMEN
Effects of all-trans retinoic acid (ATRA) on sphingomyelinase expression were examined using MCF-7 (ATRA-sensitive) and MDA-MB-231 (ATRA-resistant) breast cancer cells. Increased NSMase activity, NSMase2 mRNA and protein were observed in ATRA-treated MCF-7 but not in ATRA-treated MDA-MB-231. Increased NSMase2 mRNA of ATRA-treated MCF-7 was mostly due to enhanced transcription. Promoter analysis revealed the important 5'-promoter region of NSMase2 between -148 and -42 bp containing three Sp1 sites but no retinoic acid responsive elements. Experiments using mutated Sp1 sites of the NSMase2 promoter, Mithramycin A (a Sp inhibitor) and Sp family over-expression demonstrated the importance of Sp family protein and the three Sp1 sites for ATRA-induced NSMase2 transcription of MCF-7 cells. Although no quantitative change of bound Sp1 on NSMase2 promoter region after ATRA treatment was detected, Sp1 phosphorylation (activation) by ATRA was observed. Interestingly, PKCδ was involved in ATRA-induced increased NSMase2 transcription. ATRA-induced PKCδ phosphorylation and then activated PKCδ phosphorylated Sp1. Chromatin immunoprecipitation (ChIP) assay showed Sp1, RARα and RXRα complex formation in MCF-7 cells regardless of ATRA treatment and ATRA-induced acetylated histone H3 of the 5'-promoter. Thus, NSMase2 mRNA expression enhanced by ATRA was due to increased transcription via phosphorylated Sp1 caused by PKCδ activation, followed by chromatin remodelling with histone H3 acetylation.
Asunto(s)
Esfingomielina Fosfodiesterasa/genética , Transcripción Genética , Tretinoina/farmacología , Supervivencia Celular/efectos de los fármacos , Perfilación de la Expresión Génica , Humanos , Células MCF-7 , ARN Mensajero/genética , ARN Mensajero/metabolismo , Esfingomielina Fosfodiesterasa/metabolismo , Relación Estructura-Actividad , Transcripción Genética/efectos de los fármacos , Tretinoina/uso terapéuticoRESUMEN
Neutral ceramidase (NCDase) is considered to be a critical enzyme for controlling the turnover of ceramide, an important bioactive lipid, which determines cell's fate. All-trans retinoic acid (ATRA) has been reported to induce neuronal differentiation and cell-cycle arrest [Lopez-Carballo, Moreno, Masia, Perez, and Barettino (Activation of the phosphatidylinositol 3-kinase/Akt signalling pathway by retinoic acid is required for neural differentiation of SH-SY5Y human neuroblastoma cells. J Biol Chem 2002:277:25297-304.)]. In this study, we observed that ATRA-induced cellular ceramide accumulation, cell-growth arrest and differentiation accompanied with down-regulation of NCDase in SH-SY5Y cells, without a decrease in sphingosine or sphingosine 1-phosphate. We examined whether the down-regulation of NCDase was involved in the increase in ceramide and cell differentiation. ATRA was found to down-regulate mRNA, protein and the enzyme activity of NCDase. Interestingly, GATA-2 was also decreased with ATRA treatment, and experiments using its expression vector and siRNA and chromatin immunoprecipitation assay demonstrated GATA-2 acted as transcription-factor of NCDase gene expression. By establishing stable transfectants with decreased NCDase expression and activity, we clarified the significance of NCDase down-regulation for ATRA-induced neuronal differentiation. Those sub-clones showed both increased cellular ceramide and reduced cell growth as well as neuronal differentiation phenotypes. These results demonstrate that down-regulation of NCDase through ATRA-induced GATA-2 decrease plays an important role in induction of ceramide accumulation and neuronal differentiation in SH-SY5Y cells.
Asunto(s)
Diferenciación Celular/efectos de los fármacos , Regulación hacia Abajo , Neuroblastoma/patología , Neuronas/efectos de los fármacos , Ceramidasa Neutra/metabolismo , Tretinoina/farmacología , Proliferación Celular/efectos de los fármacos , Regulación hacia Abajo/efectos de los fármacos , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Neuronas/patología , Ceramidasa Neutra/biosíntesis , Ceramidasa Neutra/genética , Relación Estructura-ActividadRESUMEN
We examined alterations of lipid constituents induced by hybrid liposomes (HLs) in cancer cells. As early as 1h after HL treatment, amounts of the raft/caveolae lipids sphingomyelin, ceramide, and ether-type PC were altered. In addition, the structures of caveolae on the cytoplasmic surface of the cell membrane were significantly changed. Our results suggest that alterations of lipid composition in caveolae mediate HL signaling for apoptosis.
Asunto(s)
Caveolas/química , Lípidos/química , Liposomas/química , Neoplasias/química , Apoptosis , Línea Celular Tumoral , Supervivencia Celular , Humanos , Modelos Biológicos , Transducción de Señal , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
Glial cell line-derived neurotrophic factor (GDNF) is important for the development and maintenance of dopamine neurons (Lin et al. [1993] Science 260: 1130-1132). GDNF is neuroprotective in animal models of Parkinson disease, where dopamine neurons show selective degeneration. We previously reported GDNF-induced SPHK1 gene expression in a neuroblastoma cell line, TGW (Murakami et al. [2007] J Neurochem 102: 1585-1594). In the present study, we focused on the regulatory mechanism of GAP43 (GDNF-induced neuronal phenotype) transcription to further elucidate physiological roles of GDNF-induced SPHK1 expression and activity. Stable wild-type (SPHK1-WT) but not dominant-negative SPHK1 (SPHK1-DN) overexpression increased both control- and GDNF-induced GAP43 expression. SPHK1-WT cells showed enhanced GDNF-induced sphingosine 1-phosphate (S1P) secretion compared with mock- and SPHK1-DN cells. Exogenous S1P also increased GAP43 expression. In TGW cells, PD98059, a MEK inhibitor, but not SB203580 (a p38 MAPK inhibitor) and LY294002 (a PI3K inhibitor) inhibited GDNF-induced GAP43 expression, suggesting the MEK/ERK pathway has a major role in GDNF-induced GAP43 transcription. A G-protein-coupled receptor inhibitor, pertussis toxin, and S1P(1) and S1P(3) receptor antagonists (VPC23019 and CAY10444) also inhibited ERK activation. Moreover, both S1P1 and S1P3 were serine-phosphorylated by GDNF, suggesting their activated states. C/EBPß transcription factor was induced by GDNF, and DNA pull-down and chromatin immunoprecipitation assays revealed the C/EBP binding site between -131 bp and -98 bp from the first exon of GAP43. Taken together, our results showed that in TGW cells, GDNF increased SPHK1 transcription, leading to the production and secretion of S1P. Through MEK/ERK pathway, S1P stimulates GAP43 transcription with increased binding of C/EBPß to the 5'-promoter.
Asunto(s)
Proteína GAP-43/genética , Factor Neurotrófico Derivado de la Línea Celular Glial/metabolismo , Lisofosfolípidos/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Esfingosina/análogos & derivados , Transcripción Genética , Secuencia de Bases , Western Blotting , Línea Celular Tumoral , Inmunoprecipitación de Cromatina , Clonación Molecular , Cartilla de ADN , Humanos , Reacción en Cadena de la Polimerasa , Regiones Promotoras Genéticas , Transducción de Señal , Esfingosina/metabolismoRESUMEN
Free ceramides and glycosphingolipids (GSLs) are important components of the membrane microdomain and play significant roles in cell survival. Recent studies have revealed that both fatty acids and long-chain bases (LCBs) are more diverse than expected, in terms of i) alkyl chain length, ii) hydroxylation and iii) the presence or absence of double bonds. Electrospray ionization mass spectrometry and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) have been well utilized to characterize sphingolipids with high throughput, but reports to date have not fully characterized various types of ceramide species such as hydroxyl fatty acids and/or trihydroxy-LCBs of both free ceramides and the constituent ceramides in neutral GSLs. We performed a systematic analysis of both ceramide species, including LCBs with nona-octadeca lengths using MALDI-TOF MS with high-energy collision-induced dissociation (CID) at 20 keV. Using both protonated and sodiated ions, this technique enabled us to propose general rules to discriminate between isomeric and isobaric ceramide species, unrelated to the presence or absence of sugar chains. In addition, this high-energy CID generated (3,5)A ions, indicating Hex 1-4 Hex linkage in the sugar chains. Using this method, we demonstrated distinct differences among ceramide species, including free ceramides, sphingomyelins, and neutral GSLs of glucosylceramides, galactosylceramides, lactosylceramides, globotriaosylceramides and Forssman glycolipids in the equine kidneys.
Asunto(s)
Ceramidas/química , Glicoesfingolípidos Neutros/química , Espectrometría de Masa por Ionización de Electrospray/métodos , Animales , Galactosilceramidas/química , Glucosilceramidas/química , Caballos , Riñón/metabolismo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
The role of sphingolipid metabolic pathway has been recognized in determining cellular fate. Although sphingolipid degradation has been extensively studied, gene expression of human sphingosine 1-phosphate lyase (SPL) catalyzing sphingosine 1-phosphate (S1P) remains to be determined. Among 5 human lung cancer cell lines examined, SPL protein levels paralleled the respective mRNA and enzyme activities. Between H1155 and H1299 cells used for further experiments, higher cellular S1P was observed in H1155 with higher SPL activity compared with H1299 with low SPL activity. GATA-4 has been reported to affect SPL transcription in Dictyostelium discoideum. GATA-4 was observed in H1155 but not in other cell lines. Overexpression of GATA-4 in H1299 increased SPL expression. However, promoter analysis of human SPL revealed that the most important region was located between -136bp and -88bp from the first exon, where 2 Sp1 sites exist but no GATA site. DNA pull-down assay of H1155 showed increased DNA binding of Sp1 and GATA-4 within this promoter region compared with H1299. Electrophoresis mobility shift assay (EMSA), chromatin immunoprecipitation (ChIP) assay, reporter assay using mutated binding motif, and mithramycin A, a specific Sp1 inhibitor, suggest the major role of Sp1 in SPL transcription and no direct binding of GATA-4 with this 5' promoter region. The collaborative role of GATA-4 was proved by showing coimmunoprecipitation of Sp1 and GATA-4 using GST-Sp1 and overexpressed GATA-4. Thus, high SPL transcription of H1155 cells was regulated by Sp1 and GATA-4/Sp1 complex formation, both of which bind to Sp1 sites of the 5'-SPL promoter.
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Aldehído-Liasas/biosíntesis , Regulación Enzimológica de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Neoplasias Pulmonares/enzimología , Proteínas de Neoplasias/biosíntesis , Transcripción Genética , Aldehído-Liasas/genética , Línea Celular Tumoral , Dictyostelium/genética , Factor de Transcripción GATA4/genética , Factor de Transcripción GATA4/metabolismo , Expresión Génica , Humanos , Neoplasias Pulmonares/genética , Proteínas de Neoplasias/genética , Proteínas Protozoarias/biosíntesis , Proteínas Protozoarias/genética , Elementos de Respuesta , Factor de Transcripción Sp1/genética , Factor de Transcripción Sp1/metabolismoRESUMEN
Ceramide is the central lipid in the sphingolipid metabolism. Ceramide kinase (CERK) and its product, ceramide 1-phosphate, have been implicated in various cellular functions. However, the regulatory mechanism of CERK gene expression remains to be determined. Here, we examined CERK mRNA level during all-trans retinoic acid (ATRA)-induced differentiation of a human neuroblastoma cell line, SH-SY5Y. ATRA reduced CERK mRNA and protein levels. Over-expression and small interfering RNA (siRNA) of CERK revealed that CERK is inhibitory against ATRA-induced neuronal differentiation and cell growth arrest. ATRA inhibited the transcriptional activity of 5'-promoter of CERK. Truncation and mutation study suggests that ATRA-responsible region was mainly located in the tandem retinoic acid responsive elements (RARE) between -40 bp and the first exon. The electrophoresis mobility shift assay revealed that ATRA produced two retarded bands, which were erased by antibody against chicken ovalbumin upstream promoter transcription factor I (COUP-TFI), RARalpha, and RXRalpha, respectively. DNA pull-down assay confirmed increased binding of these transcription factors to RARE. Transient expression of RAR, RXR, and COUP-TFI and siRNA transfection of these genes revealed that COUP-TFI inhibited CERK mRNA. Furthermore, chromatin immunoprecipitation assay showed the recruitment of co-repressors as well as three transcription factors. These results suggest that COUP-TFI was the ATRA-responsive suppressive transcription factor of CERK gene transcription.
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Factor de Transcripción COUP I/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Activación Transcripcional/efectos de los fármacos , Tretinoina/farmacología , Sitios de Unión/efectos de los fármacos , Factor de Transcripción COUP I/genética , Muerte Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Ensayo de Cambio de Movilidad Electroforética/métodos , Exones/efectos de los fármacos , Exones/fisiología , Humanos , Inmunoprecipitación/métodos , Datos de Secuencia Molecular , Neuritas/efectos de los fármacos , Neuroblastoma , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Regiones Promotoras Genéticas/efectos de los fármacos , ARN Interferente Pequeño/farmacología , Receptores de Ácido Retinoico/metabolismo , Receptor alfa de Ácido Retinoico , Receptores X Retinoide/metabolismo , Transfección/métodosRESUMEN
Mg(2+)-dependent neutral SMases (NSMases) have emerged as prime candidates for stress-induced ceramide production. Among isoforms identified, previous reports have suggested the importance of NSMase2. However, its activation mechanism has not been precisely reported. Here, we analyzed the mechanism of NSMase2 gene expression by the anti-cancer drug, daunorubicin (DA). DA increased cellular ceramides (C16, C18 and C24) and NSMase activity of a human breast cancer cell line, MCF-7. DA remarkably increased the NSMase2 message and protein, whereas little change in NSMase1 and NSMase3 mRNAs and only a mild increase in acid SMase mRNA were observed. Overexpression and a knock down of NSMase2 indicated that NSMase2 played a role in DA-induced cell death. NSMase2 promoter analysis revealed that three Sp1 motifs located between -148 and -42bp upstream of the first exon were important in basic as well as in DA-induced promoter activity. Consistently, luciferase vectors containing three consensus Sp1-motifs but not its mutated form showed DA-induced transcriptional activation. DA-treated MCF-7 showed increased Sp3 protein. In SL2 cells lacking Sp family proteins, both Sp1 and Sp3 overexpression increased NSMase promoter activity. Increased binding of Sp family proteins by DA to three Sp1 motifs was shown by electrophoresis mobility shift and ChIP assays.