RESUMEN
Transcriptional dysregulation as a result of sequestration of essential transcription factors into protein aggregates formed by polyglutamine (polyQ) expansions can lead to late-onset progressive neurodegeneration. DNA microarray analysis of Drosophila expressing polyQ in the compound eye over time revealed large numbers of transcriptional changes at the earliest stages of the disease including repression of the transient receptor potential calcium channels in a polyQ-induced cell death specific manner. While significant differences in expression profiles were found between the Drosophila compound eye and polyQ-sensitive neural cells, a number of possible key overlapping regulators were extracted. Among these, PDK1 was shown to act as a mediator for polyQ-toxicity, suggesting the involvement of the TOR pathway in polyQ-induced neurodegeneration.
Asunto(s)
Proteínas de Drosophila/fisiología , Drosophila melanogaster/genética , Enfermedades Neurodegenerativas/genética , Enfermedades Neurodegenerativas/metabolismo , Péptidos/metabolismo , Fosfatidilinositol 3-Quinasas/fisiología , Proteínas Quinasas Dependientes de 3-Fosfoinosítido , Envejecimiento/genética , Envejecimiento/metabolismo , Animales , Secuencia de Bases , Células Cultivadas , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Regulación de la Expresión Génica/fisiología , Neuronas/metabolismo , Neuronas/fisiología , Análisis de Secuencia por Matrices de Oligonucleótidos , Péptidos/genética , Fosfatidilinositol 3-Quinasas/genética , Plásmidos/genética , Proteínas Quinasas , Proteínas Serina-Treonina Quinasas/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteínas Quinasas S6 Ribosómicas/metabolismo , Transducción de Señal , Serina-Treonina Quinasas TOR , Transcripción Genética/fisiologíaRESUMEN
Apoptosis in corpus luteum (CL) is induced by prolactin (PRL) in female rats. PRL-induced apoptosis in CL is mediated by the Fas/Fas ligand (FasL) system. The CL consists of steroidogenic and non-steroidogenic cells, including immunocytes. Fas mRNA was detected only in the luteal steroidogenic cells, and FasL mRNA was expressed only by the non-steroidogenic CD3-positive luteal immunocytes. Removing the luteal immune cells from the luteal cells inhibited PRL-induced luteal cell apoptosis effectively. Thus, FasL-expressing non-steroidogenic luteal immunocytes are required for PRL-induced luteal cell apoptosis and heterogeneous induction of apoptosis by Fas/FasL in CL.
Asunto(s)
Apoptosis/fisiología , Cuerpo Lúteo/fisiología , Glicoproteínas de Membrana/metabolismo , Receptor fas/metabolismo , Animales , Apoptosis/inmunología , Western Blotting , Complejo CD3/metabolismo , Cuerpo Lúteo/citología , Cuerpo Lúteo/inmunología , Proteína Ligando Fas , Femenino , Células Asesinas Naturales/metabolismo , Ligandos , Prolactina/metabolismo , Ratas , Ratas Wistar , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
In female rats, apoptotic cell death in the corpus luteum is induced by the prolactin (PRL) surge occurring in the proestrous afternoon during the estrous cycle. We have previously shown that this luteolytic action of PRL is mediated by the Fas/Fas ligand (FasL) system. During pregnancy or pseudopregnancy, apoptosis does not occur in the corpus luteum. Progesterone (P4), a steroid hormone secreted from luteal steroidogenic cells, attenuated PRL-induced apoptosis in cultured luteal cells in a dose-dependent manner. P4 significantly decreased the expression of mRNA of Fas, but not FasL, in cultured luteal cells prepared from both proestrous and mid-pseudopregnant rats. These data indicate that P4 suppresses PRL-induced luteal cell apoptosis via reduction of the expression level of Fas mRNA in the corpus luteum, suggesting that P4 acts as an important factor that can change the sensitivity of corpus luteum to PRL.
Asunto(s)
Apoptosis/fisiología , Cuerpo Lúteo/metabolismo , Regulación hacia Abajo , Progesterona/fisiología , Receptor fas/metabolismo , Animales , Estro , Femenino , Masculino , Embarazo , Prolactina/fisiología , Seudoembarazo , ARN Mensajero/genética , Ratas , Ratas Wistar , Receptor fas/genéticaRESUMEN
The Bcl-2/CED-9 family of proteins, which includes both antiapoptotic and proapoptotic members, plays key regulating roles in programmed cell death. We report here the identification and characterization of Drob-1, the first Drosophila member of the Bcl-2/CED-9 family to be isolated. Drob-1 contains four conserved Bcl-2 homology domains (BH1, BH2, BH3, and BH4) and a C-terminal hydrophobic domain. Ectopic expression of Drob-1 in the developing Drosophila eye resulted in a rough-eye phenotype. Furthermore, when overexpressed in Drosophila S2 cells, Drob-1 induced apoptosis accompanied by elevated caspase activity. This Drob-1-induced cell death, however, could not be antagonized by baculovirus p35, a broad-spectrum caspase inhibitor. Drob-1 was localized to the intracytoplasmic membranes, predominantly to the mitochondrial membranes, and a mutant Drob-1 lacking the hydrophobic C terminus lost both its mitochondrial localization and its proapoptotic activity. These results suggest that Drob-1 promotes cell death by inducing both caspase-dependent and -independent pathways at the mitochondria. Our identification of Drob-1 and further genetic analysis should provide increased understanding of the universal mechanisms by which the Bcl-2/CED-9 family members and other related proteins regulate apoptosis.
Asunto(s)
Proteínas de Caenorhabditis elegans , Proteínas de Drosophila , Drosophila/genética , Proteínas de Insectos/genética , Proteínas de la Membrana/genética , Proteínas Proto-Oncogénicas/genética , Secuencia de Aminoácidos , Animales , Animales Modificados Genéticamente , Proteínas Reguladoras de la Apoptosis , Northern Blotting , Células COS , Caspasas/metabolismo , Caspasas/fisiología , Muerte Celular/fisiología , ADN Complementario/química , ADN Complementario/genética , Drosophila/embriología , Drosophila/crecimiento & desarrollo , Activación Enzimática , Ojo/embriología , Ojo/crecimiento & desarrollo , Ojo/metabolismo , Regulación del Desarrollo de la Expresión Génica , Proteínas del Helminto/genética , Hibridación in Situ , Proteínas de Insectos/fisiología , Membranas Intracelulares/química , Proteínas de la Membrana/fisiología , Microscopía Confocal , Mitocondrias/química , Datos de Secuencia Molecular , Fenotipo , Proteínas Proto-Oncogénicas/fisiología , Proteínas Proto-Oncogénicas c-bcl-2/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Alineación de Secuencia , Análisis de Secuencia de ADN , Homología de Secuencia de AminoácidoRESUMEN
The prolactin (PRL) surge in cycling rats during the proestrous afternoon is an inducer of apoptotic cell death in luteal cells. This luteolytic action of PRL is peculiar, because PRL may be categorized as a survival factor, if other known physiological functions of PRL are taken into account. Here we analyzed the underlying molecular/cellular mechanisms of this PRL-induced apoptosis. Corpora lutea (CL) were prepared from the ovary on the proestrous day and cultured with or without PRL (2 microg/ml). An addition of PRL to the culture medium induced DNA breakdown in the nuclei of cells mostly identified as steroidogenic by 3beta-HSD activity staining, and the number of 3beta-HSD-positive cells were significantly decreased, indicating the induction of apoptotic cell death by PRL among luteal cells in culture. Next, the expression of membrane form-Fas ligand (mFasL) in the luteal cell lysate was quantified, because Fas receptor is known to have an exact physiological role in luteolysis. An addition of PRL increased the expression of mFasL. Immunostaining and TUNEL assay on regressing CL revealed that both CD3-positive cells and FasL-positive cells were co-localized in the regions where apoptosis convergently occurred. Moreover, an addition of concanavalin A (ConA), a T-cell specific activator, to the culture mimicked the PRL action by inducing apoptosis in luteal cells and enhancing the expression of mFasL. These data suggest that the CD3-positive T lymphocyte in the CL is at least one of the PRL-effector cell species during the process of luteolysis in rats, and that FasL expression of these cells is upregulated by PRL.
Asunto(s)
Apoptosis , Cuerpo Lúteo/efectos de los fármacos , Cuerpo Lúteo/inmunología , Glicoproteínas de Membrana/fisiología , Neuropéptidos/fisiología , Prolactina/farmacología , Receptores del Factor de Necrosis Tumoral , Animales , Complejo CD3/análisis , Células Cultivadas , Concanavalina A/farmacología , Relación Dosis-Respuesta a Droga , Proteína Ligando Fas , Femenino , Etiquetado Corte-Fin in Situ , Ratas , Ratas Wistar , Factores de Tiempo , Receptor fasRESUMEN
CED-4 protein plays an important role in the induction of programmed cell death in Caenorhabditis elegans through the activation of caspases. However, the precise mechanisms by which it activates caspases remain unknown. To investigate the conservation of CED-4 function in evolution, transgenic Drosophila lines that express CED-4 in the compound eye were generated. Ectopic expression of CED-4 in the eyes induced massive apoptotic cell death through caspase activation. An ATP-binding site (P-loop) mutation in CED-4 (K165R) causes a loss of function in its ability to activate Drosophila caspase, and an ATPase inhibitor blocks the CED-4-dependent caspase activity in Drosophila S2 cells. Immunoprecipitation analysis showed that both CED-4 and CED-4 (K165R) bind directly to Drosophila caspase drICE, and the overexpression of CED-4 (K165R) inhibits CED-4-, ecdysone-, or cycloheximide-dependent caspase activation in S2 cells. Furthermore, CED-4 (K165R) partially prevented cell death induced by CED-4 in Drosophila compound eyes. Thus, CED-4 function is evolutionarily conserved in Drosophila, and the molecular mechanisms by which CED-4 activates caspases might require ATP binding and direct interaction with the caspases.
Asunto(s)
Apoptosis , Proteínas de Caenorhabditis elegans , Proteínas de Unión al Calcio/metabolismo , Caspasas/metabolismo , Proteínas de Drosophila , Proteínas del Helminto/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Animales Modificados Genéticamente , Caenorhabditis elegans , Proteínas de Unión al Calcio/genética , Drosophila/genética , Activación Enzimática , Evolución Molecular , Ojo/anatomía & histología , Ojo/embriología , Genes Reporteros , Proteínas del Helminto/genética , Mutación , Fenotipo , Supresión GenéticaRESUMEN
We identified a Drosophila Apaf-1/CED-4 homolog gene, dapaf-1. Alternative splicing results in two dapaf-1 mRNA species, which encode distinct forms of caspase activator, Dapaf-1L (Apaf-1 type) and Dapaf-1S (CED-4 type). Distinct caspases were activated by these Dapaf-1 isoforms. Loss of Dapaf-1 function resulted in defective cytochrome c-dependent caspase activities and reduced apoptosis in embryo and in larval brain. Dapaf-1 activities were also involved in cell death induced by ectopic expression of reaper in the compound eye. These data suggest that Dapaf-1/cytochrome c-dependent cell death-inducing machinery is present in Drosophila, and the requirement of Dapaf-1/Apaf-1 in neural cell death is conserved through evolution.
Asunto(s)
Apoptosis , Proteínas de Caenorhabditis elegans , Proteínas de Unión al Calcio/química , Caspasas/metabolismo , Proteínas de Drosophila , Drosophila melanogaster/citología , Proteínas del Helminto/química , Proteínas/química , Empalme Alternativo , Secuencia de Aminoácidos , Animales , Factor Apoptótico 1 Activador de Proteasas , Secuencia de Bases , Encéfalo/citología , Encéfalo/embriología , Encéfalo/metabolismo , Proteínas de Unión al Calcio/genética , Caspasas/química , Línea Celular , Clonación Molecular , Grupo Citocromo c/metabolismo , Drosophila melanogaster/embriología , Drosophila melanogaster/enzimología , Drosophila melanogaster/genética , Activación Enzimática , Evolución Molecular , Ojo/citología , Ojo/metabolismo , Proteínas del Helminto/genética , Larva/citología , Larva/metabolismo , Datos de Secuencia Molecular , Mutación/genética , Péptidos/genética , Péptidos/fisiología , Unión Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas/genética , ARN Mensajero/análisis , ARN Mensajero/genética , Homología de Secuencia de AminoácidoRESUMEN
Interleukin (IL)-1 is a major mediator of inflammation and exerts pleiotropic effects on the neuro-immuno-endocrine system. To elucidate pathophysiological roles of IL-1, we have first produced IL-1alpha/beta doubly deficient (KO) mice together with mice deficient in either the IL-1alpha, IL-1beta, or IL-1 receptor antagonist (IL-1ra) genes. These mice were born healthy, and their growth was normal except for IL-1ra KO mice, which showed growth retardation after weaning. Fever development upon injection with turpentine was suppressed in IL-1beta as well as IL-1alpha/beta KO mice, but not in IL-1alpha KO mice, whereas IL-1ra KO mice showed an elevated response. At this time, expression of IL-1beta mRNA in the diencephalon decreased 1.5-fold in IL-1alpha KO mice, whereas expression of IL-1alpha mRNA decreased >30-fold in IL-1beta KO mice, suggesting mutual induction between IL-1alpha and IL-1beta. This mutual induction was also suggested in peritoneal macrophages stimulated with lipopolysaccharide in vitro. In IL-1beta KO mice treated with turpentine, the induction of cyclooxygenase-2 (EC 1.14.99.1) in the diencephalon was suppressed, whereas it was enhanced in IL-1ra KO mice. We also found that glucocorticoid induction 8 h after turpentine treatment was suppressed in IL-1beta but not IL-1alpha KO mice. These observations suggest that IL-1beta but not IL-1alpha is crucial in febrile and neuro-immuno-endocrine responses, and that this is because IL-1alpha expression in the brain is dependent on IL-1beta. The importance of IL-1ra both in normal physiology and under stress is also suggested.
Asunto(s)
Glucocorticoides/metabolismo , Interleucina-1/genética , Receptores de Interleucina-1/antagonistas & inhibidores , Trementina/farmacología , Animales , Peso Corporal/genética , Encéfalo/fisiología , Corticosterona/sangre , Fiebre/inducido químicamente , Fiebre/fisiopatología , Inflamación/fisiopatología , Interleucina-1/fisiología , Lipopolisacáridos/farmacología , Macrófagos Peritoneales/metabolismo , Ratones , Ratones Noqueados , ARN Mensajero/metabolismoRESUMEN
ced-9, a member of the bcl-2 gene family in Caenorhabditis elegans plays a central roles in preventing cell death in worms. Overexpression of human bcl-2 can partially prevent cell death in C. elegans. However, it remains to be elucidated whether ced-9 can regulate cell death when expressed in other organisms. We demonstrated that the CED-9 protein is co-localized with BCL-2 in COS cells and Drosophila Schneider's L2 (SL2) cells, suggesting that the site of CED-9 action is located to specific cytoplasmic compartments. Overexpression of ced-9 only poorly protected cells from the death induced by ced-3 in HeLa cells, but ced-9 significantly reduced the cell death induced by ced-3 in Drosophila SL2 cells. Furthermore, apoptosis of SL2 cells that was induced by a Drosophila cell-death gene, reaper, was shown to be partially prevented by ced-9, bcl-2 and bcl-xL. These results suggest that the signaling pathway that is required for the anti-apoptotic function of bcl-2 family members, including ced-9, is conserved in Drosophila cells. In addition, SL2 cells provide a unique systems for dissecting the main machinery of cell death.
Asunto(s)
Apoptosis/genética , Proteínas de Caenorhabditis elegans , Caspasas , Cisteína Endopeptidasas/genética , Proteínas de Drosophila , Proteínas del Helminto/genética , Proteínas Proto-Oncogénicas/genética , Animales , Proteínas Reguladoras de la Apoptosis , Células COS , Caenorhabditis elegans/genética , Muerte Celular/genética , Línea Celular , Cisteína Endopeptidasas/fisiología , Drosophila melanogaster/citología , Drosophila melanogaster/genética , Regulación de la Expresión Génica , Genes bcl-2/fisiología , Células HeLa , Proteínas del Helminto/metabolismo , Proteínas del Helminto/fisiología , Humanos , Péptidos/fisiología , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas/fisiología , Proteínas Proto-Oncogénicas c-bcl-2RESUMEN
PRL surges in female rats have dual effects of luteal function: either inducing luteolysis during the estrous cycle or rescuing and maintaining luteal function during pseudopregnancy. We analyzed these apparent contradictory effects in relation to apoptosis. The detection of fragmented DNA and in situ 3'-end labeling studies were done on corpora lutea (CL) collected from cycling rats at proestrus 1800 h (P1800 specimen) or pseudopregnant rats on day 6 (psp 6). Distinct DNA ladders were observed in P1800 samples as we previously reported, but only slight ones were found in psp 6 specimen. The effect of PRL on the induction of apoptosis was evaluated in vitro with dispersed luteal tissue. CL from cycling rats were exempted from a PRL surge by pre-treating donors with a dopamine agonist. The extent of apoptotic reaction in P1800 specimen depended on the doses of PRL added to the culture medium. In psp 6 specimen, in contrast, PRL suppressed the apoptotic reaction, increased the cell survival rate (MTT assay), and decreased the cell death rate (LDH assay). Furthermore, PRL enhanced 20 alpha-hydroxysteroid dehydrogenase activity in P1800 specimen but suppressed it in psp 6 specimen. In summary, PRL in rats is either an apoptosis-inducer or -suppressor, depending on the functional state of luteal cells.
Asunto(s)
Apoptosis/fisiología , Cuerpo Lúteo/citología , Prolactina/fisiología , Animales , Apoptosis/efectos de los fármacos , Apoptosis/genética , Muerte Celular , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Cuerpo Lúteo/efectos de los fármacos , Cuerpo Lúteo/enzimología , Cuerpo Lúteo/fisiología , Fragmentación del ADN/efectos de los fármacos , Fragmentación del ADN/genética , Relación Dosis-Respuesta a Droga , Electroforesis en Gel de Agar , Femenino , Hidroxiesteroide Deshidrogenasas/efectos de los fármacos , Hidroxiesteroide Deshidrogenasas/metabolismo , L-Lactato Deshidrogenasa/efectos de los fármacos , L-Lactato Deshidrogenasa/metabolismo , Prolactina/farmacología , Seudoembarazo/patología , Seudoembarazo/fisiopatología , Ratas , Ratas Wistar , Factores de TiempoRESUMEN
We determined whether fragmentation of genomic DNA, one of the hallmarks of apoptosis, occurs during structural luteolysis in cycling rats. Corpora lutea (CL) were collected from rats at each estrous cycle stage (1800 h), and fragmented DNA was extracted. Only CL from rats at the proestrous stage showed distinct DNA fragmentation. To determine the period of occurrence of DNA fragmentation, CL were collected at several points between 1200 h on the day of proestrus and 0600 h on the day of estrus. Distinct DNA fragmentation was observed from 1800 h (proestrus) to 2400 h (proestrus), and the extent was significantly lower at 0600 h (estrus). It is known that prolactin (PRL) induces structural luteolysis in rats. To examine the role of PRL in luteal DNA fragmentation, 2-bromo-alpha-ergocryptine (BE) was used to suppress the PRL surge on the day of proestrus. CL collected at 1800 h from BE-treated rats did not show distinct DNA fragmentation, and PRL injection offset the effect of BE. Histochemical analysis with a 3'-end labeling technique confirmed the occurrence of DNA fragmentation in luteal tissue. These results suggest that apoptotic cell death occurs during PRL-induced structural luteolysis.