RESUMEN
The protein p38 mitogen-activated protein kinase (MAPK) delta isoform (p38δ) is a poorly studied member of the MAPK family. Data analysis from The Cancer Genome Atlas database revealed that p38δ is highly expressed in all types of human breast cancers. Using a human breast cancer tissue array, we confirmed elevation in cancer tissue. The breast cancer mouse model, MMTV-PyMT (PyMT), developed breast tumors with lung metastasis; however, mice deleted in p38δ (PyMT/p38δ-/-) exhibited delayed primary tumor formation and highly reduced lung metastatic burden. At the cellular level, we demonstrate that targeting of p38δ in breast cancer cells, MCF-7 and MDA-MB-231 resulted in a reduced rate of cell proliferation. In addition, cells lacking p38δ also displayed an increased cell-matrix adhesion and reduced cell detachment. This effect on cell adhesion was molecularly supported by the regulation of the focal adhesion kinase by p38δ in the human breast cell lines. These studies define a previously unappreciated role for p38δ in breast cancer development and evolution by regulating tumor growth and altering metastatic properties. This study proposes MAPK p38δ protein as a key factor in breast cancer. Lack of p38δ resulted in reduced primary tumor size and blocked the metastatic potential to the lungs.
Asunto(s)
Neoplasias de la Mama/patología , Adhesión Celular , Proliferación Celular , Neoplasias Pulmonares/secundario , Neoplasias Mamarias Experimentales/patología , Proteína Quinasa 13 Activada por Mitógenos/metabolismo , Animales , Mama/patología , Progresión de la Enfermedad , Femenino , Humanos , Células MCF-7 , Neoplasias Mamarias Experimentales/genética , Ratones , Ratones Transgénicos , Proteína Quinasa 13 Activada por Mitógenos/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Análisis de Matrices TisularesRESUMEN
Increasing studies suggest that ceramides differing in acyl chain length and/or degree of unsaturation have distinct roles in mediating biological responses. However, still much remains unclear about regulation and role of distinct ceramide species in the immune response. Here, we demonstrate that alkaline ceramidase 3 (Acer3) mediates the immune response by regulating the levels of C18:1-ceramide in cells of the innate immune system and that Acer3 deficiency aggravates colitis in a murine model by augmenting the expression of pro-inflammatory cytokines in myeloid and colonic epithelial cells (CECs). According to the NCBI Gene Expression Omnibus (GEO) database, ACER3 is downregulated in immune cells in response to lipopolysaccharides (LPS), a potent inducer of the innate immune response. Consistent with these data, we demonstrated that LPS downregulated both Acer3 mRNA levels and its enzymatic activity while elevating C(18:1)-ceramide, a substrate of Acer3, in murine immune cells or CECs. Knocking out Acer3 enhanced the elevation of C(18:1)-ceramide and the expression of pro-inflammatory cytokines in immune cells and CECs in response to LPS challenge. Similar to Acer3 knockout, treatment with C(18:1)-ceramide, but not C18:0-ceramide, potentiated LPS-induced expression of pro-inflammatory cytokines in immune cells. In the mouse model of dextran sulfate sodium-induced colitis, Acer3 deficiency augmented colitis-associated elevation of colonic C(18:1)-ceramide and pro-inflammatory cytokines. Acer3 deficiency aggravated diarrhea, rectal bleeding, weight loss and mortality. Pathological analyses revealed that Acer3 deficiency augmented colonic shortening, immune cell infiltration, colonic epithelial damage and systemic inflammation. Acer3 deficiency also aggravated colonic dysplasia in a mouse model of colitis-associated colorectal cancer. Taken together, these results suggest that Acer3 has an important anti-inflammatory role by suppressing cellular or tissue C(18:1)-ceramide, a potent pro-inflammatory bioactive lipid and that dysregulation of ACER3 and C(18:1)-ceramide may contribute to the pathogenesis of inflammatory diseases including cancer.
Asunto(s)
Ceramidasa Alcalina/genética , Colitis/etiología , Inmunidad Innata/efectos de los fármacos , Lipopolisacáridos/toxicidad , Ceramidasa Alcalina/deficiencia , Animales , Transformación Celular Neoplásica , Ceramidas/análisis , Ceramidas/metabolismo , Colitis/metabolismo , Colitis/patología , Colon/metabolismo , Colon/patología , Citocinas/metabolismo , Sulfato de Dextran/toxicidad , Modelos Animales de Enfermedad , Femenino , Leucocitos Mononucleares/citología , Leucocitos Mononucleares/inmunología , Leucocitos Mononucleares/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Especificidad por Sustrato , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Targeting cell motility, which is required for dissemination and metastasis, has therapeutic potential for ovarian cancer metastasis, and regulatory mechanisms of cell motility need to be uncovered for developing novel therapeutics. Invasive ovarian cancer cells spontaneously formed protrusions, such as lamellipodia, which are required for generating locomotive force in cell motility. Short interfering RNA screening identified class II phosphatidylinositol 3-kinase C2ß (PI3KC2ß) as the predominant isoform of PI3K involved in lamellipodia formation of ovarian cancer cells. The bioactive sphingolipid ceramide has emerged as an antitumorigenic lipid, and treatment with short-chain C6-ceramide decreased the number of ovarian cancer cells with PI3KC2ß-driven lamellipodia. Pharmacological analysis demonstrated that long-chain ceramide regenerated from C6-ceramide through the salvage/recycling pathway, at least in part, mediated the action of C6-ceramide. Mechanistically, ceramide was revealed to interact with the PIK-catalytic domain of PI3KC2ß and affect its compartmentalization, thereby suppressing PI3KC2ß activation and its driven cell motility. Ceramide treatment also suppressed cell motility promoted by epithelial growth factor, which is a prometastatic factor. To examine the role of ceramide in ovarian cancer metastasis, ceramide liposomes were employed and confirmed to suppress cell motility in vitro. Ceramide liposomes had an inhibitory effect on peritoneal metastasis in a murine xenograft model of human ovarian cancer. Metastasis of PI3KC2ß knocked-down cells was insensitive to treatment with ceramide liposomes, suggesting specific involvement of ceramide interaction with PI3KC2ß in metastasis suppression. Our study identified ceramide as a bioactive lipid that limits PI3KC2ß-governed cell motility, and ceramide is proposed to serve as a metastasis-suppressor lipid in ovarian cancer. These findings could be translated into developing ceramide-based therapy for metastatic diseases.
Asunto(s)
Movimiento Celular/efectos de los fármacos , Ceramidas/farmacología , Neoplasias Ováricas/tratamiento farmacológico , Fosfatidilinositol 3-Quinasa/metabolismo , Animales , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Femenino , Humanos , Ratones , Neoplasias Ováricas/enzimología , Neoplasias Ováricas/patologíaRESUMEN
Neutral sphingomyelinase-2 (nSMase2) is a ceramide-generating enzyme that has been implicated in growth arrest, apoptosis and exosome secretion. Although previous studies have reported transcriptional upregulation of nSMase2 in response to daunorubicin, through Sp1 and Sp3 transcription factors, the role of the DNA damage pathway in regulating nSMase2 remains unclear. In this study, we show that doxorubicin induces a dose-dependent induction of nSMase2 mRNA and protein with concomitant increases in nSMase activity and ceramide levels. Upregulation of nSMase2 was dependent on ATR, Chk1 and p53, thus placing it downstream of the DNA damage pathway. Moreover, overexpression of p53 was sufficient to transcriptionally induce nSMase2, without the need for DNA damage. DNA-binding mutants as well as acetylation mutants of p53 were unable to induce nSMase2, suggesting a role of nSMase2 in growth arrest. Moreover, knockdown of nSMase2 prevented doxorubicin-induced growth arrest. Finally, p53-induced nSMase2 upregulation appears to occur via a novel transcription start site upstream of exon 3. These results identify nSMase2 as a novel p53 target gene, regulated by the DNA damage pathway to induce cell growth arrest.
Asunto(s)
Doxorrubicina/farmacología , Esfingomielina Fosfodiesterasa/fisiología , Proteína p53 Supresora de Tumor/fisiología , Regulación hacia Arriba , Daño del ADN , Regulación de la Expresión Génica/efectos de los fármacos , Técnicas de Silenciamiento del Gen , Humanos , Células MCF-7 , ARN Mensajero/efectos de los fármacos , Esfingomielina Fosfodiesterasa/genética , Esfingomielina Fosfodiesterasa/metabolismo , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
p53 is a crucial tumor suppressor that is mutated or deleted in a majority of cancers. Exactly how p53 prevents tumor progression has proved elusive for many years; however, this information is crucial to define targets for chemotherapeutic development that can effectively restore p53 function. Bioactive sphingolipids have recently emerged as important regulators of proliferative, apoptotic and senescent cellular processes. In this study, we demonstrate that the enzyme sphingosine kinase 1 (SK1), a critical enzyme in the regulation of the key bioactive sphingolipids ceramide, sphingosine and sphingosine-1-phosphate (S1P), serves as a key downstream target for p53 action. Our results show that SK1 is proteolysed in response to genotoxic stress in a p53-dependent manner. p53 null mice display elevation of SK1 levels and a tumor-promoting dysregulation of bioactive sphingolipids in which the anti-growth sphingolipid ceramide is decreased and the pro-growth sphingolipid S1P is increased. Importantly, deletion of SK1 in p53 null mice completely abrogated thymic lymphomas in these mice and prolonged their life span by ~30%. Deletion of SK1 also significantly attenuated the formation of other cancers in p53 heterozygote mice. The mechanism of p53 tumor suppression by loss of SK1 is mediated by elevations of sphingosine and ceramide, which in turn were accompanied by increased expression of cell cycle inhibitors and tumor cell senescence. Thus, targeting SK1 may restore sphingolipid homeostasis in p53-dependent tumors and provide insights into novel therapeutic approaches to cancer.
Asunto(s)
Neoplasias/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Animales , Línea Celular , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/metabolismo , Senescencia Celular/genética , Activación Enzimática/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neoplasias/genética , Neoplasias/mortalidad , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Carga Tumoral/genética , Proteína p53 Supresora de Tumor/genéticaRESUMEN
One of the most intriguing enzymes of sphingolipid biology is acid sphingomyelinase (ASMase). In a phospholipase C reaction, ASMase catalyzes the cleavage of the phosphocholine head group of sphingomyelin to generate ceramide. Cumulative efforts of various laboratories over the past 40 years have placed ASMase and its product ceramide at the forefront of lipid research. Activation of the ASMase/ceramide pathway is a shared response to an ever-growing list of receptor and non-receptor mediated forms of cellular stress including: death ligands (TNFalpha, TRAIL, Fas ligand), cytokines (IL-1, IFNgamma), radiation, pathogenic infections, cytotoxic agents and others. The strategic role of ASMase in lipid metabolism and cellular stress response has sparked interest in investigatig the molecular mechanisms underlying ASMase activation. In this article, we review the translational role of the ASMase/ceramide pathway and recent advances on its mechanisms of regulation.
Asunto(s)
Ceramidas/metabolismo , Transducción de Señal , Esfingomielina Fosfodiesterasa/genética , Esfingomielina Fosfodiesterasa/metabolismo , Animales , Muerte Celular , Terapia Genética , Humanos , Metabolismo de los LípidosRESUMEN
The sphingolipid ceramide is intimately involved in the growth, differentiation, senescence, and death of normal and cancerous cells. Mitochondria are increasingly appreciated to play a key role in ceramide-induced cell death. Recent work showed the C16-pyridinium ceramide analogue LCL-30 to induce cell death in vitro by mitochondrial targeting. The aim of the current study was to translate these results to an in vivo model. We found that LCL-30 accumulated in mitochondria in the murine colorectal cancer cell line CT-26 and reduced cellular ATP content, leading to dose- and time-dependent cytotoxicity. Although the mitochondrial levels of sphingosine-1-phosphate (S1P) became elevated, transcription levels of ceramide-metabolising enzymes were not affected. In mice, LCL-30 was rapidly absorbed from the peritoneal cavity and cleared from the circulation within 24 h, but local peritoneal toxicity was dose-limiting. In a model of subcutaneous tumour inoculation, LCL-30 significantly reduced the proliferative activity and the growth rate of established tumours. Sphingolipid profiles in tumour tissue also showed increased levels of S1P. In summary, we present the first in vivo application of a long-chain pyridinium ceramide for the treatment of experimental metastatic colorectal cancer, together with its pharmacokinetic parameters. LCL-30 was an efficacious and safe agent. Future studies should identify an improved application route and effective partners for combination treatment.
Asunto(s)
Apoptosis/efectos de los fármacos , Ceramidas/farmacología , Neoplasias Colorrectales/tratamiento farmacológico , Mitocondrias/efectos de los fármacos , Esfingosina/análogos & derivados , Animales , Caspasas/metabolismo , Proliferación Celular/efectos de los fármacos , Células Cultivadas/efectos de los fármacos , Ceramidas/farmacocinética , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/secundario , Citocromos c/metabolismo , Ensayo de Inmunoadsorción Enzimática , Humanos , Ratones , Ratones Endogámicos BALB C , Estructura Molecular , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Esfingolípidos/metabolismo , Esfingosina/farmacocinética , Esfingosina/farmacología , Tasa de Supervivencia , Células Tumorales CultivadasRESUMEN
The protein kinase C (PKC) family of isoenzymes has been shown to regulate a variety of cellular processes, including receptor desensitization and internalization, and this has sparked interest in further delineation of the roles of specific isoforms of PKC in membrane trafficking and endocytosis. Recent studies have identified a novel translocation of PKC to a juxtanuclear compartment, the pericentrion, which is distinct from the Golgi complex but epicentered on the centrosome. Sustained activation of PKC (longer than 30 min) also results in sequestration of plasma membrane lipids and proteins to the same compartment, demonstrating a global effect on endocytic trafficking. This review summarizes these studies, particularly focusing on the characterization of the pericentrion as a distinct PKC-dependent subset of recycling endosomes. We also discuss emerging insights into a role for PKC as a central hub in regulating vesicular transport pathways throughout the cell, with implications for a wide range of pathobiologic processes, e.g. diabetes and abnormal neurotransmission or receptor desensitization.
Asunto(s)
Membrana Celular/enzimología , Endocitosis , Endosomas/metabolismo , Proteína Quinasa C/metabolismo , Animales , Humanos , Transporte de Proteínas , Transducción de SeñalRESUMEN
BACKGROUND: Sphingolipids are signaling molecules in a range of biological processes. While sphingosine-1-phosphate (S1P) is thought to be abundantly stored in platelets and released upon stimulation, knowledge about the distribution and function of other sphingolipids in blood is lacking. OBJECTIVES: To analyze the sphingolipid content of blood components with special emphasis on dynamic changes in platelets. METHODS: Blood components from mice and humans were prepared by gradient centrifugation and analyzed by liquid chromatography-mass spectrometry. Additionally, murine platelets were activated in vitro and in vivo. RESULTS: Isolated non-activated platelets of mice were devoid of S1P, but instead contained dihydrosphingosine-1-phosphate (dhS1P), along with a high concentration of ceramide. Activation of platelets in vitro led to a loss of dhS1P and an increase in sphingosine, accompanied by a reduction of ceramide content. Platelet activation in vivo led to an immediate and continuous rise of dhS1P in plasma, while S1P remained stable. The sphingolipid distribution of human blood was markedly different from mice. Human platelets contained dhS1P in addition to S1P. CONCLUSIONS: Mouse platelets contain dhS1P instead of S1P. Platelet activation causes loss of dhS1P and breakdown of ceramide, implying ceramidase activation. Release of dhS1P from activated platelets might be a novel signaling pathway. Finally, the sphingolipid composition of mouse and human blood shows large differences, which must be considered when studying sphingolipid biology.
Asunto(s)
Plaquetas/metabolismo , Activación Plaquetaria , Esfingolípidos/sangre , Animales , Anticuerpos , Plaquetas/efectos de los fármacos , Plaquetas/inmunología , Separación Celular , Ceramidas/sangre , Cromatografía Liquida , Eritrocitos/química , Citometría de Flujo , Humanos , Técnicas In Vitro , Leucocitos/química , Lisofosfolípidos/sangre , Ratones , Ratones Endogámicos BALB C , Activación Plaquetaria/efectos de los fármacos , Activación Plaquetaria/inmunología , Glicoproteína IIb de Membrana Plaquetaria/inmunología , Especificidad de la Especie , Espectrometría de Masa por Ionización de Electrospray , Esfingolípidos/metabolismo , Esfingosina/análogos & derivados , Esfingosina/sangre , Trombina/farmacologíaRESUMEN
Despite local and systemic therapies, the National Cancer Institute estimates that prostate cancer will cause over 30,000 deaths in 2006. This suggests that additional therapeutic approaches are needed. The chicken anemia viral protein Apoptin causes tumor-selective apoptosis in human tumor lines independent of p53 and Bcl-2 status. Tet-regulated expression of Apoptin from an adenoviral vector showed cytotoxicity in DU145, PC-3, and LNCaP tumor cells regardless of expression of p53, Bcl-2, Bcl-xL, Bax, survivin, FLIP(S), XIAP, or CIAP. Apoptin expression caused an increase in the tumor suppressor lipid ceramide, which regulates the cellular stress response. Interestingly, 10 of 15 primary prostate cancers examined by Western blotting overexpressed acid ceramidase (AC), suggesting that ceramide deacylation might serve to negate elevated levels of ceramide, creating a more antiapoptotic phenotype. This was confirmed in AC-overexpressing cells in which we observed decreased sensitivity to apoptosis following treatment with Apoptin. Addition of the AC inhibitor LCL204, in combination with Apoptin, augmented cell killing. This effect was also demonstrated in vivo in that Apoptin and LCL204 cotreatment significantly reduced tumor growth in DU145 xenografts (P<0.05). Taken together, our data demonstrated that Apoptin is a promising therapeutic agent for prostate cancer and that its function is improved when combined with acid ceramidase inhibitors.
Asunto(s)
Apoptosis , Proteínas de la Cápside/metabolismo , Ceramidas/metabolismo , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , Adenoviridae/genética , Animales , Apoptosis/efectos de los fármacos , Proteínas de la Cápside/genética , Caspasas/metabolismo , Línea Celular Tumoral , Inhibidores Enzimáticos/farmacología , Galactosilgalactosilglucosilceramidasa/antagonistas & inhibidores , Galactosilgalactosilglucosilceramidasa/metabolismo , Regulación de la Expresión Génica , Genes Reporteros/genética , Terapia Genética , Humanos , Masculino , Ratones , Ratones Desnudos , Fosfoserina/metabolismo , Neoplasias de la Próstata/genética , Esfingolípidos/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Emerging information on sphingolipid metabolism and signaling is leading to a better understanding of cellular processes such as apoptosis, cancer, cell survival and aging. In this review, we discuss the involvement of sphingolipids in these processes and focus on underlying mechanisms based on sphingolipid:protein interactions. Due to the inherent difficulty of studying lipids, we discuss techniques that are useful in the elucidation of these interactions. We classify sphingolipid-binding proteins into four main classes: receptor, effector, enzyme, and transporter. Known structures of sphingolipid-binding proteins are surveyed, and sphingolipid-binding characteristics are described, acknowledging the limitations that there are presently insufficient protein:sphingolipid complexes for more definitive conclusions on this topic. Finally we summarize relevant literature to better inform the reader about sphingolipid:protein interactions.
Asunto(s)
Proteínas Portadoras/química , Metabolismo de los Lípidos , Esfingolípidos/química , Esfingomielina Fosfodiesterasa/química , Animales , Ceramidas/química , Humanos , Modelos Biológicos , Modelos Moleculares , Estructura Molecular , Estructura Terciaria de Proteína , Transducción de Señal , Esfingolípidos/metabolismo , Esfingolípidos/fisiología , Sulfoglicoesfingolípidos/químicaRESUMEN
As of January 2005, there were 1020 gene therapy clinical trials ongoing worldwide with 675 or 66.2% devoted to cancer gene therapy. The majority are occurring in the US and Europe (http://www.wiley.co.uk/genetherapy/clinical/). At the present time, to our knowledge there are no trials that employ gene delivery of Fas Ligand (FasL). As an important note, and in contrast to somatic cell therapy trials, there are no reported deaths due to therapeutic vector administration in any cancer gene therapy trial. That said, from our studies and from the published literature, the issue of gene delivery remains the major obstacle to successfully employing gene therapy for cancer treatment. Numerous laboratories are studying this with many different approaches. My co-workers and I have focused on the delivery issue by using various approaches that address tumor targeting and transgene expression. In addition, we are focusing on enhancing tumor cell killing via the bystander effect and through use of small molecules to enhance bystander activity.
Asunto(s)
Ceramidas/metabolismo , Proteína Ligando Fas/uso terapéutico , Terapia Genética/métodos , Vectores Genéticos/uso terapéutico , Neoplasias de Cabeza y Cuello/terapia , Neoplasias de la Próstata/terapia , Adenoviridae/genética , Animales , Antineoplásicos/uso terapéutico , Efecto Espectador , Ensayos Clínicos como Asunto , Proteína Ligando Fas/genética , Proteína Ligando Fas/metabolismo , Neoplasias de Cabeza y Cuello/genética , Humanos , Masculino , Neoplasias de la Próstata/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/uso terapéutico , Transducción de Señal , TransgenesRESUMEN
In addition to their crucial role in membrane structure in Saccharomyces cerevisiae, sphingolipids serve vital roles in various aspects of yeast biology including endocytosis, intracellular protein transport and stress responses. Although previous studies have unequivocally demonstrated the sphingolipid requirements for these processes, few studies have contributed mechanistic information. We have used a systems approach including microarray, lipidomics and metabolic modelling to better understand (i) biochemical relationships between various branches of sphingolipid metabolism and pathways and contributing pathways such as fatty acid metabolism and phospholipid synthesis, (ii) the changes in cellular sphingolipid composition under various conditions and (iii) the effects of these changes on the transcriptional profiles and subsequently, cell phenotypes. Thus far, these approaches have indicated roles for sphingolipids in major transcriptional changes in response to heat stress, carbon source utilization, sporulation, cell wall integrity and other basic cellular functions. Although the yeast genome is fully sequenced, nearly 50% of all transcribed open reading frames remain uncharacterized with regard to cellular function; therefore, a major advantage of this approach is the ability to identify both biochemical and biological roles for enzymes and their products within broad cellular contexts.
Asunto(s)
Saccharomyces cerevisiae/fisiología , Esfingolípidos/metabolismo , Genómica , Calor , Lípidos , Mutagénesis , Análisis de Secuencia por Matrices de Oligonucleótidos , Saccharomyces cerevisiae/genética , Esfingolípidos/deficiencia , Esfingolípidos/genéticaRESUMEN
Diacylglycerol (DAG) was discovered as a potent lipid second messenger with protein kinase C (PKC) as its major cellular target more than 25 years ago. There is increasing evidence of significant complexity within lipid signaling, and the classical DAG-PKC model no longer stands alone but is part of a larger bioactive lipid universe involving glycerolipids and sphingolipids. Multiple layers of regulation exist among PKC- and DAG-metabolizing enzymes such as phosphatidylcholine (PC)-specific phospholipase D, and cross-talk exists between the glycerolipid and sphingolipid pathways, with PKC at the center. Currently, there is intense interest in the question of whether DAG derived from PC can function as a lipid second messenger and regulate PKC analogous to DAG derived from phosphatidylinositol-4,5-bisphosphate (PIP2). To address these issues and incorporate DAG-PKC and other signaling pathways into an expanded view of cell biology, it will be necessary to go beyond the classical approaches and concepts.
Asunto(s)
Diglicéridos/metabolismo , Fosfolipasa D/metabolismo , Proteína Quinasa C/metabolismo , Transducción de Señal , Animales , Ceramidas/metabolismo , Humanos , Ácidos Fosfatidicos/metabolismoRESUMEN
Sphingolipids, historically described as potential reservoirs for bioactive lipids, presently define a new family of cellular mediators, joining the well-established glycerolipid-derived mediators of signal transduction such as diacylglycerol, phosphatidylinositides, and eicosanoids. Sphingolipid metabolism is clearly involved in the regulation of cell growth, differentiation, and programmed cell death. Indeed, a majority of the greater than four thousand studies conducted on sphingolipids during the past five years were investigations of the role of sphingolipids as cellular bioregulators. Studies spanning more than a decade have shown multiple interactions and intersections of the sphingolipid-mediated pathways and the eicosanoid pathway. This review will discuss the emerging mechanisms by which sphingolipids induce inflammatory responses via the eicosanoid pathway in addition to linking previous literature on sphingolipids and inflammation with newer findings of distinct roles for sphingosine-1-phosphate in regulating cyclooygenase-2 and ceramide-1-phosphate in the regulation of cytosolic phospholipase A2alpha. Finally, the relationship between bioactive sphingolipids and inflammation is discussed.
Asunto(s)
Inflamación/metabolismo , Esfingolípidos/fisiología , Ceramidas/metabolismo , Ciclooxigenasa 2 , Citocinas/metabolismo , Eicosanoides/metabolismo , Isoenzimas/metabolismo , Modelos Moleculares , Fosfolipasas A/metabolismo , Prostaglandina-Endoperóxido Sintasas/metabolismo , Transducción de Señal , Esfingolípidos/química , Esfingolípidos/metabolismo , Esfingosina/análogos & derivados , Esfingosina/metabolismoRESUMEN
Our previous results have indicated that the major cellular pool of sphingomyelin present on the outer leaflet of the plasma membrane is not involved in the ceramide pathway of apoptosis. Thus, in this study we aimed at defining which intracellular pools of sphingomyelin and ceramide are involved in cell death. The bacterial sphingomyelinase (SMase) gene fused with green fluorescent protein was subcloned into mammalian vectors containing sequences that target the fusion proteins to cytoplasm, plasma membrane, mitochondria, Golgi apparatus, endoplasmic reticulum, or nucleus. Transfection of MCF7 breast cancer cells showed for all constructs an increase in SMase activity ranging from 2- to 60-fold, concomitant with an increase in total cellular ceramide levels (10-100%) as compared with vector-transfected cells. Next, the effect of overexpression of the SMase on cell death was examined. Results demonstrate that only when bacterial SMase was targeted to mitochondria did cells undergo apoptosis; its targeting to the other intracellular compartments was ineffective. Further, the results show that apoptosis induced by mitochondrial targeting of bacterial SMase requires SMase catalytic activity, is prevented by the overexpression of Bcl-2, and is mediated by inducing cytochrome c release. These results demonstrate that ceramide induces cell death specifically when generated in mitochondria. The results highlight the significance of compartment-specific lipid-mediated cell regulation, and they offer a novel general approach for these studies.
Asunto(s)
Apoptosis/fisiología , Mitocondrias/metabolismo , Esfingomielinas/metabolismo , Secuencia de Aminoácidos , Apoptosis/efectos de los fármacos , Transporte Biológico , Compartimento Celular/genética , Ceramidas/metabolismo , Grupo Citocromo c/metabolismo , Proteínas Fluorescentes Verdes , Humanos , Hidrólisis , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Mutación , Señales de Clasificación de Proteína/genética , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Transducción de Señal , Esfingomielina Fosfodiesterasa/genética , Esfingomielina Fosfodiesterasa/metabolismo , Esfingomielina Fosfodiesterasa/farmacología , Factores de Tiempo , Transfección , Células Tumorales CultivadasRESUMEN
Protein kinase C (PKC) is known to activate NF-kappaB whereas the lipid mediator ceramide was recently shown to inhibit activation of this transcription factor (1, 2). In this study, the mechanisms by which ceramide interferes with this pathway were examined in Jurkat leukemia and MCF-7 breast cancer cells. Both exogenous and endogenous ceramide inhibited selectively PKC-mediated activation of NF-kappaB by reverting PKC translocation to the membrane. Next, confocal and immunofluorescence studies were performed to evaluate the direct effects of ceramide on PKC. These studies showed that ceramide inhibited translocation of a green fluorescent protein (GFP)-PKCbeta2 fusion protein in response to PMA. A mutant PKC in which autophosphorylation sites were mutated to alanine (PKC-DA) was resistant to ceramide. A kinase-inactive mutant (PKC-KR) was also resistant to ceramide action, and the results were supported using kinase inhibitors of the enzyme. Finally, overexpression of PKC-DA prevented, at least partly, the ability of ceramide to inhibit activation of NF-kappaB. Taken together, these studies show that ceramide has acute effects on translocation of PKC by inducing reverse translocation, and this reversal requires both the kinase activity of PKC and phosphorylation of the autophosphorylation sites.
Asunto(s)
Ceramidas/farmacología , FN-kappa B/efectos de los fármacos , Proteína Quinasa C/metabolismo , Transporte Biológico/efectos de los fármacos , Proteínas Fluorescentes Verdes , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Células Jurkat , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , FN-kappa B/metabolismo , Fosforilación/efectos de los fármacos , Proteína Quinasa C/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Transducción de Señal , Esfingomielina Fosfodiesterasa/farmacología , Acetato de Tetradecanoilforbol/farmacología , Células Tumorales CultivadasRESUMEN
Both glutathione (GSH) depletion and arachidonic acid (AA) generation have been shown to regulate sphingomyelin (SM) hydrolysis and are known components in tumor necrosis factor alpha (TNFalpha)-induced cell death. In addition, both have hypothesized direct roles in activation of N-sphingomyelinase (SMase); however, it is not known whether these are independent pathways of N-SMase regulation or linked components of a single ordered pathway. This study was aimed at differentiating these possibilities using L929 cells. Depletion of GSH with L-buthionin-(S,R)-sulfoximine (BSO) induced 50% hydrolysis of SM at 12 h. In addition, TNF induced a depletion of GSH, and exogenous addition of GSH blocked TNF-induced SM hydrolysis as well as TNF-induced cell death. Together, these results establish GSH upstream of SM hydrolysis and ceramide generation in L929 cells. We next analyzed the L929 variant, C12, which lacks both cytosolic phospholipase A(2) (cPLA(2)) mRNA and protein, in order to determine the relationship of cPLA(2) and GSH. TNF did not induce a significant drop in GSH levels in the C12 line. On the other hand, AA alone was capable of inducing a 60% depletion of GSH in C12 cells, suggesting that these cells remain responsive to AA distal to the site of cPLA(2). Furthermore, depleting GSH with BSO failed to effect AA release, but caused a drop in SM levels, showing that the defect in these cells was upstream of the GSH drop and SMase activation. When cPLA(2) was restored to the C12 line by expression of the cDNA, the resulting CPL4 cells regained sensitivity to TNF. Treatment of the CPL4 cells with TNF resulted in GSH levels dropping to levels near those of the wild-type L929 cells. These results demonstrate that GSH depletion following TNF treatment in L929 cells is dependent on intact cPLA(2) activity, and suggest a pathway in which activation of cPLA(2) is required for the oxidation and reduction of GSH levels followed by activation of SMases.
Asunto(s)
Glutatión/metabolismo , Fosfolipasas A/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Animales , Butionina Sulfoximina/metabolismo , Línea Celular , Supervivencia Celular , Ceramidas/metabolismo , Citosol/enzimología , Hidrólisis , Ratones , Fosfolipasas A/genética , Esfingomielina Fosfodiesterasa/metabolismoRESUMEN
The search for potential targets for ceramide action led to the identification of ceramide-activated protein phosphatases (CAPP). To date, two serine/threonine protein phosphatases, protein phosphatase 2A (PP2A) and protein phosphatase 1 (PP1), have been demonstrated to function as ceramide-activated protein phosphatases. In this study, we show that treatment with either anti-FAS IgM (CH-11) (150 ng/ml) or exogenous d-(e)-C(6-)ceramide (20 microm) induces the dephosphorylation of the PP1 substrates, serine/arginine-rich (SR) proteins, in Jurkat acute leukemia T-cells. The serine/threonine protein phosphatase inhibitor, calyculin A, but not the PP2A-specific inhibitor, okadaic acid, inhibited both FAS- and ceramide-induced dephosphorylation of SR proteins. Anti-FAS IgM treatment of Jurkat cells led to a significant increase in levels of endogenous ceramide beginning at 2 h with a maximal increase of 10-fold after 7 h. A 2-h pretreatment of Jurkat cells with fumonisin B(1) (100 microm), a specific inhibitor of CoA-dependent ceramide synthase, blocked 80% of the ceramide generated and completely inhibited the dephosphorylation of SR proteins in response to anti-FAS IgM. Moreover, pretreatment of Jurkat cells with myriocin, a specific inhibitor of serine-palmitoyl transferase (the first step in de novo synthesis of ceramide), also blocked FAS-induced SR protein dephosphorylation, thus demonstrating a role for de novo ceramide. These results were further supported using A549 lung adenocarcinoma cells treated with d-(e)-C(6-)ceramide. Dephosphorylation of SR proteins was inhibited by fumonisin B(1) and by overexpression of glucosylceramide synthase; again implicating endogenous ceramide generated de novo in regulating the dephosphorylation of SR proteins in response to FAS activation. These results establish a specific intracellular pathway involving both de novo ceramide generation and activation of PP1 to mediate the effects of FAS activation on SR proteins.
Asunto(s)
Ceramidas/biosíntesis , Fumonisinas , Fosfoproteínas Fosfatasas/metabolismo , Retículo Sarcoplasmático/metabolismo , Receptor fas/metabolismo , Western Blotting , Ácidos Carboxílicos/farmacología , Línea Celular , Diacilglicerol Quinasa/metabolismo , Electroforesis en Gel de Poliacrilamida , Activación Enzimática , Inhibidores Enzimáticos/farmacología , Humanos , Células Jurkat , Ácido Palmítico/metabolismo , Monoéster Fosfórico Hidrolasas/metabolismo , Fosforilación , Proteína Fosfatasa 1 , Proteína Fosfatasa 2 , Serina/metabolismo , Esfingolípidos/metabolismo , Treonina/metabolismo , Factores de TiempoRESUMEN
Ceramide accumulation in the cell can occur from either hydrolysis of sphingomyelin or by de novo synthesis. In this study, we found that blocking de novo ceramide synthesis significantly inhibits ceramide accumulation and subsequent cell death in response to tumor necrosis factor alpha. When cells were pre-treated with glutathione, a proposed cellular regulator of neutral sphingomyelinase, inhibition of ceramide accumulation at early time points was achieved with attenuation of cell death. Inhibition of both pathways achieved near-complete inhibition of ceramide accumulation and cell death indicating that both pathways of ceramide generation are stimulated. This illustrates the complexity of ceramide generation in cytokine action.