RESUMEN
The protein dlk, encoded by the Dlk1 gene, belongs to the Notch epidermal growth factor (EGF)-like family of receptors and ligands, which participate in cell fate decisions during development. The molecular mechanisms by which dlk regulates cell differentiation remain unknown. By using the yeast two-hybrid system, we found that dlk interacts with Notch1 in a specific manner. Moreover, by using luciferase as a reporter gene under the control of a CSL/RBP-Jk/CBF-1-dependent promoter in the dlk-negative, Notch1-positive Balb/c 14 cell line, we found that addition of synthetic dlk EGF-like peptides to the culture medium or forced expression of dlk decreases endogenous Notch activity. Furthermore, the expression of the gene Hes-1, a target for Notch1 activation, diminishes in confluent Balb/c14 cells transfected with an expression construct encoding for the extracellular EGF-like region of dlk. The expression of Dlk1 and Notch1 increases in 3T3-L1 cells maintained in a confluent state for several days, which is associated with a concomitant decrease in Hes-1 expression. On the other hand, the decrease of Dlk1 expression in 3T3-L1 cells by antisense cDNA transfection is associated with an increase in Hes-1 expression. These results suggest that dlk functionally interacts in vivo with Notch1, which may lead to the regulation of differentiation processes modulated by Notch1 activation and signaling, including adipogenesis.
Asunto(s)
Proteínas de la Membrana/metabolismo , Receptores de Superficie Celular/metabolismo , Factores de Transcripción/metabolismo , Células 3T3-L1 , Animales , Secuencia de Bases , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Línea Celular , ADN sin Sentido/genética , Factor de Crecimiento Epidérmico/química , Factor de Crecimiento Epidérmico/genética , Factor de Crecimiento Epidérmico/metabolismo , Regulación de la Expresión Génica , Genes Reporteros , Proteínas de Homeodominio/química , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Técnicas In Vitro , Péptidos y Proteínas de Señalización Intracelular , Luciferasas/genética , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Ratones , Receptor Notch1 , Receptores de Superficie Celular/química , Receptores de Superficie Celular/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Secuencias Repetitivas de Aminoácido , Factor de Transcripción HES-1 , Factores de Transcripción/química , Factores de Transcripción/genética , Transfección , Técnicas del Sistema de Dos HíbridosRESUMEN
The major histocompatibility complex class I (MHC1) molecule plays a crucial role in cytotoxic lymphocyte function. beta 2-Microglobulin (beta 2m) has been demonstrated to be both a structural component of the MHC1 complex and a chaperone-like molecule for MHC1 folding. beta 2m binding to an isolated alpha 3 domain of MHC1 heavy chain at micromolar concentrations has been shown to accurately model the biochemistry and thermodynamics of beta 2m-driven MHC1 folding. These results suggested a model in which the chaperone-like role of beta 2m is dependent on initial binding to the alpha 3 domain interface of MHC1 with beta 2m. Such a model predicts that a mutant beta 2m molecule with an intact MHC1 alpha 3 domain interaction but a defective MHC1 alpha 1 alpha 2 domain interaction would block beta2m-driven folding of MHC1. In this study we generated such a beta 2m mutant and demonstrated that it blocks MHC1 folding by normal beta 2m at the expected micromolar concentrations. Our data support an initial interaction of beta 2m with the MHC1 alpha 3 domain in MHC1 folding. In addition, the dominant negative mutant beta 2m can block T-cell functional responses to antigenic peptide and MHC1.
Asunto(s)
Genes Dominantes , Antígenos de Histocompatibilidad Clase I/genética , Microglobulina beta-2/genética , Sustitución de Aminoácidos , Animales , Secuencia de Bases , Línea Celular , Clonación Molecular , Cartilla de ADN , Genes MHC Clase I , Antígenos de Histocompatibilidad Clase I/metabolismo , Humanos , Linfoma de Células T , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Reacción en Cadena de la Polimerasa , Pliegue de Proteína , Estructura Secundaria de Proteína , Proteínas Recombinantes/metabolismo , Linfocitos T Citotóxicos/inmunología , Transfección , Células Tumorales Cultivadas , Microglobulina beta-2/química , Microglobulina beta-2/metabolismoRESUMEN
There are two distinct phenotypes of T cell cytokine responses that lead to different effector functions and different outcomes in disease processes. Although evidence suggests a possible role of the local microenvironment in the differentiation or localization of T cells with these phenotypes, there are no examples of divergent T cell cytokine phenotypes with the same Ag specificity concurrently existing in different tissue compartments. Using a CD8(+) T cell adoptive transfer model for graft-vs-host disease, we demonstrate that a potent type 2 cytokine response develops in the spleen while a potent type 1 cytokine response simultaneously develops in the testis. These experiments demonstrate for the first time that cytokine production can be oppositely polarized in different organs of the same individual. This may have important implications for organ-specific pathology in infection or autoimmunity: infections or autoimmune diseases that affect multiple organs may have heterogeneity in tissue cytokine responses that is not revealed in systemic lymphocyte cytokine responses. Therefore, attempts to modulate the immune response phenotype may ameliorate pathology in one organ while exacerbating pathology in another.
Asunto(s)
Traslado Adoptivo/métodos , Citocinas/biosíntesis , Subgrupos de Linfocitos T/trasplante , Animales , Linfocitos T CD8-positivos/citología , Linfocitos T CD8-positivos/inmunología , Movimiento Celular/genética , Movimiento Celular/inmunología , Quimiocina CCL2/biosíntesis , Relación Dosis-Respuesta Inmunológica , Inmunofenotipificación , Ganglios Linfáticos/citología , Ganglios Linfáticos/inmunología , Ganglios Linfáticos/metabolismo , Activación de Linfocitos/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Especificidad de Órganos/genética , Especificidad de Órganos/inmunología , Receptores de Quimiocina/biosíntesis , Bazo/citología , Bazo/inmunología , Bazo/metabolismo , Subgrupos de Linfocitos T/citología , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo , Testículo/citología , Testículo/inmunología , Testículo/metabolismo , Células TH1/inmunología , Células TH1/metabolismo , Células Th2/inmunología , Células Th2/metabolismoRESUMEN
Movement of T-lymphocyte cell surface CD43 is associated with both antigen activation of T-cell clones and chemokine induction of T-lymphocyte motility. Here, we demonstrate that CD43 movement away from the site of T-cell receptor ligation occurs in unprimed CD4(+) T cells as well as T-cell clones. The T-cell receptor (TCR)-dependent movement of CD43 in unprimed T cells is associated with a polarized morphology and CD43 accumulation at the uropods of the cells, unlike that reported for primed T cells. The polarization of CD43 has a requirement for Src kinases and occurs in conjunction with lipid raft coalescence. Thymocytes and T-cell hybridomas, cells that have altered responses to TCR activation and lack lipid raft coalescence, do not polarize CD43 as readily as unprimed T cells. The movement of CD43 depends on the cholesterol biosynthetic pathway enzyme 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase. Blockade of this enzyme can specifically prevent CD43 redistribution without affecting cell shape polarization. The likely mechanism of this alteration in CD43 redistribution is through decreased protein prenylation because the cholesterol-dependent lipid rafts still coalesce on activation. These findings suggest that the polarization of cell shape, lipid raft coalescence, and CD43 redistribution on T-cell activation have signaling pathway distinctions. Dissecting out the relationships between various stages of molecular redistribution and lymphocyte activation may facilitate fine-tuning of immunologic responses.
Asunto(s)
Antígenos CD , Inhibidores de Hidroximetilglutaril-CoA Reductasas/farmacología , Lovastatina/análogos & derivados , Lovastatina/farmacología , Microdominios de Membrana/fisiología , Ácido Mevalónico/análogos & derivados , Ácido Mevalónico/farmacología , Pirimidinas/farmacología , Sialoglicoproteínas/inmunología , Linfocitos T/inmunología , Animales , Polaridad Celular/inmunología , Femenino , Leucosialina , Ratones , Ratones Endogámicos BALB C , Microscopía Fluorescente , Bazo/inmunología , Linfocitos T/citología , Linfocitos T/efectos de los fármacos , Familia-src Quinasas/antagonistas & inhibidoresRESUMEN
The EGF-like membrane protein dlk plays a crucial role in the control of cell differentiation. Overexpression of the protein prevents, whereas inhibition of its expression increases, adipocyte differentiation of 3T3-L1 cells in response to Insulin-like Growth Factor I (IGF-1) or insulin. We have investigated whether dlk modulates the signaling pathways known to control this process. We found that the levels of dlk expression modulated signaling through the IGF-1 receptor, causing changes in the activation levels and kinetics of Extracellular-Regulated Kinase/Mitogen-Activated Protein Kinase (ERK/MAPK) that correlated with differentiation outcome. These changes occurred in response to IGF-1 or insulin but not in response to Epidermal Growth Factor. However, the levels of expression of IGF-1 receptor, or the activation of Insulin Receptor Substrate-1 in response to IGF-1, were not affected by the levels of dlk expression. Therefore, dlk appears to modulate ERK/MAPK signaling in response to specific differentiation signals.
Asunto(s)
Diferenciación Celular/fisiología , Membrana Celular/metabolismo , Factor I del Crecimiento Similar a la Insulina/metabolismo , Sistema de Señalización de MAP Quinasas/fisiología , Proteínas de la Membrana/deficiencia , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Receptor IGF Tipo 1/metabolismo , Células 3T3 , Tejido Adiposo/efectos de los fármacos , Tejido Adiposo/metabolismo , Animales , Diferenciación Celular/efectos de los fármacos , Membrana Celular/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/fisiología , Insulina/farmacología , Factor I del Crecimiento Similar a la Insulina/farmacología , Péptidos y Proteínas de Señalización Intracelular , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Proteínas de la Membrana/genética , Ratones , Proteínas Quinasas Activadas por Mitógenos/efectos de los fármacos , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3 , Receptor IGF Tipo 1/efectos de los fármacosRESUMEN
Levels of dlk, an EGF-like homeotic protein, are critical for several differentiation processes. Because growth and differentiation are, in general, exclusive of each other, and increasing evidence indicates that Dlk1 expression changes in tumorigenic processes, we studied whether dlk could also affect cell growth. We found that, in response to glucocorticoids, Balb/c 3T3 cells with diminished levels of dlk expression develop foci-like cells that have lost contact inhibition, display altered morphology, and grow faster than control cell lines. Balb/c 3T3 cells spontaneously growing more rapidly are also dlk-negative cells. Moreover, screening by the yeast two-hybrid system, using Dlk1 constructs as baits, resulted in the isolation of GAS1 and acrogranin cDNAs. Interestingly, these proteins are cysteine-rich molecules involved in the control of cell growth. Taken together, these observations suggest that dlk may participate in a network of interactions controlling how the cells respond to growth or differentiation signals.