RESUMEN
Rexinoids are compounds that bind to the rexinoid X receptor (RXR) to modulate gene expression and have been proposed as a new class of therapeutics to treat Alzheimer's disease. Different rexinoids will initiate downstream effects that can be quite marked even though such compounds can be structurally similar and have comparable RXR binding affinities. RXR can both homo- and heterodimerize, and these protein-protein interactions and subsequent transactivating potential lead to differential gene expression, depending on the RXR dimeric partner, additional cofactors recruited, and downstream transcription factors that are up- or downregulated. Expression analysis was performed in the U87 human glioblastoma cell line treated with a panel of rexinoids, and our analysis demonstrated that rexinoids with similar RXR EC50 values can have pronounced differences in differential gene expression. Rexinoid binding likely leads to distinctive RXR conformations that cause major downstream gene expression alterations via modulation of RXR interacting proteins. Yeast two-hybrid analysis of RXR bait with two RXR interacting partners demonstrates that rexinoids drive differential binding of RXR to distinctive protein partners. Physiochemical analysis of the rexinoids reveals that the molecules cluster similarly to their gene expression patterns. Thus, rexinoids with similar RXR binding affinities drive differential gene expression by stimulating additional binding patterns in RXR and its homo- and heteropartners, driven by the physicochemical characteristics of these molecules.
Asunto(s)
Glioblastoma , Receptores X Retinoide , Técnicas del Sistema de Dos Híbridos , Humanos , Glioblastoma/metabolismo , Glioblastoma/genética , Línea Celular Tumoral , Receptores X Retinoide/metabolismo , Receptores X Retinoide/genética , Unión Proteica/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacosRESUMEN
In insects, localized tissue injury often leads to global (organism-wide) delays in development and retarded metamorphosis. In Drosophila, for example, injuries to the larval imaginal discs can retard pupariation and prolong metamorphosis. Injuries induced by treatments such as radiation, mechanical damage and induction of localized cell death can trigger similar delays. In most cases, the duration of the developmental delay appears to be correlated with the extent of damage, but the effect is also sensitive to the developmental stage of the treated animal. The proximate cause of the delays is likely a disruption of the ecdysone signaling pathway, but the intermediate steps leading from tissue injury and/or regeneration to that disruption remain unknown. Here, we review the evidence for injury-induced developmental delays, and for a checkpoint or checkpoints associated with the temporal progression of development and the on-going efforts to define the mechanisms involved.
Asunto(s)
Puntos de Control del Ciclo Celular/fisiología , Regulación del Desarrollo de la Expresión Génica/fisiología , Proteínas de Insectos/metabolismo , Insectos/crecimiento & desarrollo , Insectos/fisiología , Animales , Proteínas de Insectos/genética , LarvaRESUMEN
In humans, chronic inflammation, severe injury, infection and disease can result in changes in steroid hormone titers and delayed onset of puberty; however the pathway by which this occurs remains largely unknown. Similarly, in insects injury to specific tissues can result in a global developmental delay (e.g. prolonged larval/pupal stages) often associated with decreased levels of ecdysone - a steroid hormone that regulates developmental transitions in insects. We use Drosophila melanogaster as a model to examine the pathway by which tissue injury disrupts developmental progression. Imaginal disc damage inflicted early in larval development triggers developmental delays while the effects are minimized in older larvae. We find that the switch in injury response (e.g. delay/no delay) is coincident with the mid-3rd instar transition - a developmental time-point that is characterized by widespread changes in gene expression and marks the initial steps of metamorphosis. Finally, we show that developmental delays induced by tissue damage are associated with decreased expression of genes involved in ecdysteroid synthesis and signaling.
Asunto(s)
Drosophila melanogaster/crecimiento & desarrollo , Ecdisteroides/biosíntesis , Discos Imaginales/lesiones , Transducción de Señal/genética , Animales , Cartilla de ADN/genética , Drosophila melanogaster/metabolismo , Regulación del Desarrollo de la Expresión Génica/fisiología , Larva/crecimiento & desarrollo , Larva/metabolismo , Metamorfosis Biológica/fisiología , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
Stage 10 of Drosophila oogenesis can be subdivided into stages 10A and 10B based on a change in the morphology of the centripetal follicle cells (FC) from a columnar to an apically constricted shape. This coordinated cell shape change drives epithelial cell sheet involution between the oocyte and nurse cell complex which patterns the operculum structure of the mature eggshell. We have shown previously that proper centripetal FC migration requires transient expression of the C/EBP encoded by slow border cells (slbo) at 10A, due in part to Notch activation followed by slbo autorepression (Levine et al., 2007). Here we show that decreased slbo expression in the centripetal FC coincides with increased expression of the transcription factor Cut, a Cut/Cux/CDP family member, at 10B. The 10A/10B temporal switch from Slbo to Cut expression is refined by both cross repression between Slbo and Cut, Slbo auto repression and Cut auto activation. High Cut levels are necessary and sufficient to direct polarized, supracellular accumulation of Actin, DE-cadherin and Armadillo associated with apical constriction of the centripetal FC. Separately, Slbo in the border cell rosette and Cut in the pole cells have antagonistic interactions to restrict Fas2 accumulation to the pole cells, which is important for proper border cell migration. The opposing effects of Cut and Slbo in these two tissues reflect the opposing interactions between their respective mammalian homologs CAAT Displacement Protein (CDP; now CUX1) and CAAT Enhancer Binding Protein (C/EBP) in tissue culture.
Asunto(s)
Proteínas Potenciadoras de Unión a CCAAT/fisiología , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/fisiología , Drosophila melanogaster/genética , Regulación del Desarrollo de la Expresión Génica , Proteínas de Homeodominio/metabolismo , Proteínas Nucleares/metabolismo , Factores de Transcripción/metabolismo , Animales , Proteínas Potenciadoras de Unión a CCAAT/genética , Movimiento Celular , Biología Evolutiva/métodos , Proteínas de Drosophila/genética , Células Epiteliales/citología , Femenino , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Genotipo , Mitosis , Modelos Biológicos , Modelos GenéticosRESUMEN
Hemopexin (HPX) binds heme tightly, thus protecting cells from heme toxicity during hemolysis, trauma and ischemia-reperfusion injury. Heme uptake via endocytosis of heme-HPX followed by heme catabolism by heme oxygenase-1 (HMOX1) raises regulatory iron pools, thus linking heme metabolism with that of iron. Normal iron homeostasis requires copper-replete cells. When heme-HPX induces HMOX1, the copper-storing metallothioneins (MTs) are also induced whereas the copper-responsive copper chaperone that delivers copper to Cu, Zn superoxide dismutase, CCS1, is decreased; both are known responses when cellular copper levels rise. Endocytosis of heme-HPX is needed to regulate CCS1 since the signaling ligand cobalt-protoporphyrin (CoPP)-HPX, which does not induce HMOX1 but does co-localize with heme-HPX in endosomes, also decreased CCS1. These observations support that heme-HPX mobilizes copper in cells. The regulation of both hmox1 and mt1 is prevented by the copper-chelator, bathocuproinedisulfonate (BCDS), but not uptake of heme-AlexaFluor-labeled HPX into endosomes. Supporting a role for copper in HMOX1 regulation by heme-HPX, nutritional copper deficiency generated by tetraethylene pentamine or 232 tetraamine prevented HMOX1 induction. Using conditions that mimic maturing endosomes, we found that copper prevents rebinding of heme to apo-HPX. A model is presented in which copper endocytosis together with that of heme-HPX provides a means to facilitate heme export from HPX in the maturing endosomes: heme is needed for hmox1 transcription, while cytosolic copper and CCS1 provide a link for the known simultaneous regulation of hmox1 and mt1 by heme-HPX.
Asunto(s)
Cobre/fisiología , Hemo-Oxigenasa 1/metabolismo , Hemo/farmacología , Hemopexina/farmacología , Proteínas de la Membrana/metabolismo , Animales , Línea Celular Tumoral , Cobre/química , Cobre/deficiencia , Endocitosis/efectos de los fármacos , Endosomas/metabolismo , Activación Enzimática/efectos de los fármacos , Etilenodiaminas/química , Hemo/química , Hemopexina/química , Concentración de Iones de Hidrógeno , Metalotioneína/metabolismo , Ratones , Chaperonas Moleculares/metabolismoRESUMEN
The bunched (bun) gene encodes the Drosophila member of the TSC-22/GILZ family of leucine zipper transcriptional regulators. The bun locus encodes multiple BUN protein isoforms and has diverse roles during patterning of the eye, wing margin, dorsal notum and eggshell. Here we report the construction and activity of a dominant negative allele (BunDN) of the BUN-B isoform. In the ovary, BunDN expression in the follicle cells (FC) resulted in epithelial defects including aberrant accumulation of DE-cadherin and failure to rearrange into columnar FC cell shapes. BunDN expression in the posterior FC led to loss of epithelial integrity associated with extensive apoptosis. BunDN FC phenotypes collectively resemble loss-of-function bun mutant phenotypes. BunDN expression using tissue-specific imaginal disk drivers resulted in characteristic cuticular patterning defects that were enhanced by bun mutations and suppressed by co-expression of the BUN-B protein isoform. These data indicate that BunDN has dominant negative activity useful to identify bun functions and genetic interactions that occur during tissue patterning.
Asunto(s)
Proteínas de Drosophila/metabolismo , Drosophila/fisiología , Animales , Tipificación del Cuerpo , Cadherinas/metabolismo , Forma de la Célula , Drosophila/embriología , Drosophila/metabolismo , Proteínas de Drosophila/genética , Epitelio/anomalías , Epitelio/embriología , Epitelio/fisiología , Femenino , Mutación , Folículo Ovárico/citología , Folículo Ovárico/metabolismoRESUMEN
Ecdysone Receptor (EcR) mediates effects of the hormone ecdysone during larval molts, pupal metamorphosis, and adult female oogenesis. In the ovary, egg chamber formation requires interactions between the somatic follicle cell (FC) epithelium and the germ line nurse cell/oocyte cyst. Previous work has shown EcR is required in the germ line for egg chamber maturation, and here we examine EcR requirements in the FC at late stages of oogenesis. EcR protein is ubiquitous in the FC but its activity is restricted, visualized by activity of the "ligand sensor" hs-GAL4-EcR ligand binding domain fusion and EcRE-lacZ reporter gene expression. GAL4-EcR is activated in the FC by an ecdysone agonist and repressed by tissue-specific Ras GTPase signals. To determine the significance of restricted sites of EcR activity in the FC, we used targeted misexpression of the dominant negative EcR (EcR-DN) molecules EcR(F645A) and EcR(W650A). EcR-DN expression at stage 10 reduced EcRE-lacZ expression in the nurse cell FC and resulted in abnormal FC migrations, including aberrant centripetal migration and dorsal appendage tube formation, leading to the formation of cup-shaped eggs with shortened, branched dorsal appendages at stage 14. Clones of FC expressing EcR-DN displayed cell-autonomous increases in DE-cadherin expression and abnormal epithelial junction formation. EcR-DN expression caused thin eggshell phenotypes that correlated with both reduced levels of chorion gene expression and reduction in chorion gene amplification. Our results indicate that tissue-specific modulation of EcR activity by the Ras signaling pathway refines temporal ecdysone signals that regulate FC differentiation and cadherin-mediated epithelial cell shape changes.