RESUMEN
Immature double-positive (DP) thymocytes mature into CD4(+)CD8(-) cells in response to coengagement of TCR with any of a variety of cell surface "coinducer" receptors, including CD2. In contrast, DP thymocytes are signaled to undergo apoptosis by coengagement of TCR with CD28 costimulatory receptors, but the molecular basis for DP thymocyte apoptosis by TCR plus CD28 coengagement is not known. In the present study, we report that TCR plus CD28 coengagement does not invariably induce DP thymocyte apoptosis but, depending on the intensity of CD28 costimulation, can induce DP thymocyte maturation. We demonstrate that distinct but interacting signal transduction pathways mediate DP thymocyte maturation signals and DP thymocyte apoptotic signals. Specifically, DP maturation signals are transduced by the extracellular signal-related kinase (ERK)/mitogen-activated protein kinase (MAPK) pathway and up-regulate expression of the antiapoptotic protein Bcl-2. In contrast, the apoptotic response stimulated by CD28 costimulatory signals is mediated by ERK/MAPK-independent pathways. Importantly, when TCR-activated thymocytes are simultaneously coengaged by both CD28 and CD2 receptors, CD28 signals can inhibit ERK/MAPK-dependent Bcl-2 protein up-regulation. Thus, there is cross-talk between the signal transduction pathways that transduce apoptotic and maturation responses, enabling CD28-initiated signal transduction pathways to both stimulate DP thymocyte apoptosis and also negatively regulate maturation responses initiated by TCR plus CD2 coengagement.
Asunto(s)
Apoptosis/inmunología , Antígenos CD28/fisiología , Proteínas Proto-Oncogénicas c-bcl-2/biosíntesis , Transducción de Señal/inmunología , Subgrupos de Linfocitos T/citología , Subgrupos de Linfocitos T/inmunología , Timo/citología , Timo/inmunología , Animales , Antígenos CD28/inmunología , Antígenos CD28/metabolismo , Antígenos CD4/biosíntesis , Antígenos CD8/biosíntesis , Diferenciación Celular/inmunología , Células Cultivadas , Relación Dosis-Respuesta Inmunológica , Femenino , Humanos , Inmunofenotipificación , Activación de Linfocitos , Sistema de Señalización de MAP Quinasas/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Receptor Cross-Talk/inmunología , Receptores de Antígenos de Linfocitos T/inmunología , Receptores de Antígenos de Linfocitos T/metabolismo , Receptores de Antígenos de Linfocitos T/fisiología , Subgrupos de Linfocitos T/enzimología , Subgrupos de Linfocitos T/metabolismo , Timo/enzimología , Timo/metabolismoRESUMEN
Receptors coupled to pertussis toxin (PTX)-sensitive Gi proteins regulate T lymphocyte cytokine secretion, proliferation, and chemotaxis, yet little is known about the molecular mechanisms of Gi protein signaling in mammalian lymphocytes. Using the Jurkat T lymphocyte cell line, we found that a stably expressed Gi protein-coupled receptor (the delta-opioid receptor (DOR1)) stimulates MEK-1 and extracellular signal-regulated kinases 1 and 2 (ERK1 and ERK2) and transcriptional activity by an ERK target, Elk-1, via a mechanism requiring a PTX-sensitive Gi protein. Levels of beta-adrenergic receptor kinase-1 C-terminal fragment that inhibited signaling by Gi protein beta gamma subunits in these cells had no effect on DOR1 stimulation of either MEK-1- or Elk-1-dependent transcription, indicating that this pathway is independent of beta gamma. Analysis of this betagamma-independent pathway indicates a role for a herbimycin A-sensitive tyrosine kinase. Unlike beta gamma-mediated pathways, the beta gamma-independent pathway was insensitive to RasN17, inhibitors of phosphatidylinositol 3-kinase (PI 3-kinase), and constitutive PI 3-kinase activity. The beta gamma-independent pathway regulates downstream events, since blocking it abrogated both Elk-1-dependent transcription and mobilization of the mitogenic transcription factor, AP-1, in response to DOR1 signaling. These results characterize a novel, Ras- and PI 3kinase-independent pathway for ERK activation by Gi protein signaling that is distinct from ERK activation by beta gamma and may therefore be mediated by the alphai subunit.
Asunto(s)
Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Proteínas de Unión al ADN , Proteínas de Unión al GTP/metabolismo , Quinasas de Proteína Quinasa Activadas por Mitógenos , Factor de Transcripción AP-1/metabolismo , Factores de Transcripción , Proteínas ras/metabolismo , Benzoquinonas , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Activación Enzimática , Regulación de la Expresión Génica , Humanos , Células Jurkat , Lactamas Macrocíclicas , MAP Quinasa Quinasa 1 , Toxina del Pertussis , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-fos/metabolismo , Quinonas/farmacología , ARN Mensajero/metabolismo , Receptores Opioides delta/metabolismo , Rifabutina/análogos & derivados , Transducción de Señal , Factores de Virulencia de Bordetella/farmacología , Quinasas de Receptores Adrenérgicos beta , Proteína Elk-1 con Dominio etsRESUMEN
Recent molecular evidence points to transient and/or stage-specific expression of delta- and kappa-opioid receptors by thymic and peripheral T lymphocytes. Since medical treatments or stress commonly increase opioid levels, it is important to understand the mechanisms by which opioids affect T lymphocyte functions. We therefore created and studied a T cell line expressing the cloned delta-opioid receptor (DOR1). DOR1 ligation by a specific DOR1 agonist, deltorphin, augmented IL-2 secretion by synergizing with signals from TCR-CD3 and CD28. Reporter gene constructs were used to map this effect of deltorphin to the AP-1- and NF-AT/AP-1-binding sites of the IL-2 promoter. Although DOR1 signaling increased [Ca2+]i, deltorphin enhanced transcriptional activity of the NF-AT/AP-1-binding site via a mechanism independent of calcineurin and distinct from the effects of elevated [Ca2+]i. Deltorphin also increased accumulation of AP-1 transcription factor complexes, suggesting that DOR1 augments IL-2 secretion by increasing the AP-1 component of the NF-AT/AP-1 transcription factor. These results advance the molecular understanding of opioid effects on lymphocytes, and in addition, demonstrate regulation of IL-2 synthesis and secretion by the novel mechanism of receptor-mediated AP-1 induction.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Interleucina-2/metabolismo , Proteínas Nucleares , Regiones Promotoras Genéticas , Receptores Opioides delta/metabolismo , Factor de Transcripción AP-1/metabolismo , Factores de Transcripción/metabolismo , Secuencia de Bases , Antígenos CD28/inmunología , Complejo CD3/inmunología , Membrana Celular/metabolismo , Humanos , Células Jurkat , Datos de Secuencia Molecular , Factores de Transcripción NFATC , Oligopéptidos/farmacología , Linfocitos T Citotóxicos/inmunologíaRESUMEN
Xenopus oocytes injected with GIRK1 mRNA express inwardly rectifying K+ channels resembling IKACh. Yet IKACh, the atrial G protein-regulated ion channel, is a heteromultimer of GIRK1 and CIR. Reasoning that an oocyte protein might be substituting for CIR, we cloned XIR, a CIR homolog endogenously expressed by Xenopus oocytes. Coinjecting XIR and GIRK1 mRNAs produced large, inwardly rectifying K+ currents responsive to m2-muscarinic receptor stimulation. The m2-stimulated currents of oocytes expressing GIRK1 alone decreased 80% after injecting antisense oligonucleotides specific to the 5' untranslated region of XIR, but GIRK1/CIR currents were unaffected. Thus, GIRK1 without XIR or CIR only ineffectively produces currents in oocytes. This result suggests that GIRK1 does not form native homomultimeric channels.
Asunto(s)
Clonación Molecular , Oocitos/metabolismo , Canales de Potasio de Rectificación Interna , Canales de Potasio/genética , Canales de Potasio/metabolismo , Canales de Potasio/fisiología , Xenopus laevis/metabolismo , Acetilcolina/fisiología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Conductividad Eléctrica , Femenino , Canales de Potasio Rectificados Internamente Asociados a la Proteína G , Datos de Secuencia Molecular , Oligonucleótidos Antisentido/farmacología , Canales de Potasio/efectos de los fármacos , Homología de SecuenciaRESUMEN
The aim of this study was to ascertain whether different types of T-cell receptor (TCR)-mediated [Ca2+]i signals could begin to explain the different cellular responses of mature and immature T cells to ligation of the TCR-CD3 complex. Using a digital fluorescence imaging system, we measured and compared [Ca2+]i of individual cells from immature and mature murine T-cell populations following application of CD3-epsilon monoclonal antibody (mAb). Our approach revealed distinctions among developmental subsets which were not seen by previous measurements of [Ca2+]i in bulk cell populations. The CD3-mediated [Ca2+]i responses of individual thymocytes were very complex. Latencies to peak [Ca2+]i varied greatly among thymocytes, but the responses of splenic T cells were synchronized, novel evidence that the timing of [Ca2+]i responses may be an important informative parameter for TCR-CD3 signalling. In addition, among cells responding to CD3 mAb, higher peak [Ca2+]i responses correlated with maturity (CD4+ CD8+ thymocytes < single-positive thymocytes < splenic T cells). Examination of cells from pp59fyn-deficient mice showed that pp59fyn deficiency affects the amplitude and probability, but not the latency or synchrony, of CD3-mediated [Ca2+]i responses of CD4+ CD8+ and CD4+ CD8- thymocytes. All subsets showed equivalent receptor-independent mobilization of [Ca2+]i. These developmentally distinct [Ca2+]i features most probably reflect meaningful developmental changes in how the TCR-CD3 complex couples to intracellular signalling machinery including pp59fyn. By clearly showing how [Ca2+]i responses change during development, these results support the hypothesis that distinctive types of [Ca2+]i responses drive thymocyte differentiation.