RESUMEN
CD28 is the major costimulatory molecule on T cells. CD28 activation, in conjunction with T-cell receptor engagement, up-regulates transcription of several cytokines, including interleukin-2 (IL-2), through transcriptional activation of the RE/AP composite element. Although CD28 is not normally expressed on B cells or plasma cells, more than 90% of extramedullary myelomas (a late stage B-cell neoplasm) express CD28. The functional significance of this is unknown. The results of this study demonstrate that CD28 stimulates transcriptional activation of RE/AP-based reporters in B cells and myeloma cells. However, CD28 stimulation does not up-regulate IL-2 production in myeloma cell lines, demonstrating that the IL-2 promoter may not be a relevant RE/AP-containing target of CD28 in myelomas. Instead, an RE/AP composite element has been identified within the promoter of the IL-8 gene, a chemokine that promotes angiogenesis. Furthermore, stimulation of endogenous CD28 expressed by 3 myeloma cell lines increased IL-8 production. Therefore, the study demonstrates that CD28 is functional in myelomas to up-regulate transcription of endogenous genes, including IL-8. The proposal is made that aberrant expression of CD28 may play a role in the progression of multiple myeloma.
Asunto(s)
Antígenos CD28/metabolismo , Antígenos CD28/farmacología , Interleucina-8/biosíntesis , Mieloma Múltiple/patología , Regulación hacia Arriba/efectos de los fármacos , Animales , Linfocitos B/efectos de los fármacos , Linfocitos B/metabolismo , Progresión de la Enfermedad , Humanos , Interleucina-2/genética , Interleucina-8/genética , Ratones , Mieloma Múltiple/metabolismo , Regiones Promotoras Genéticas/efectos de los fármacos , Transducción de Señal , Activación Transcripcional/efectos de los fármacos , Transfección , Células Tumorales CultivadasRESUMEN
The serine/threonine kinase Akt (also known as protein kinase B, PKB) is activated by numerous growth-factor and immune receptors through lipid products of phosphatidylinositol (PI) 3-kinase. Akt can couple to pathways that regulate glucose metabolism or cell survival [1]. Akt can also regulate several transcription factors, including E2F, CREB, and the Forkhead family member Daf-16 [2] [3] [4]. Here, we show that Akt can regulate signaling pathways that lead to induction of the NF-kappaB family of transcription factors in the Jurkat T-cell line. This induction occurs, at least in part, at the level of degradation of the NF-kappaB inhibitor IkappaB, and is specific for NF-kappaB, as other inducible transcription factors are not affected by Akt overexpression. Furthermore, the effect requires the kinase activity and pleckstrin homology (PH) domain of Akt. Also, Akt does not act alone to induce cytokine promoters and NF-kappaB reporters, because signals from other pathways are required to observe the effect. These studies uncover a previously unappreciated connection between Akt and NF-kappaB induction that could have implications for the control of T-cell growth and survival.
Asunto(s)
FN-kappa B/metabolismo , Proteínas Nucleares , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas de Unión al ADN/metabolismo , Regulación de la Expresión Génica , Humanos , Quinasa I-kappa B , Interleucina-2/genética , Células Jurkat , Activación de Linfocitos , Factores de Transcripción NFATC , Fosfatidilinositol 3-Quinasas/metabolismo , Regiones Promotoras Genéticas , Proteínas Serina-Treonina Quinasas/genética , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas c-akt , Factor de Transcripción AP-1/metabolismo , Factores de Transcripción/metabolismoRESUMEN
IL-2 gene transcription in T cells requires both TCR and costimulatory signals. IL-2 promoter activation in Jurkat T cells stimulated with superantigen presented by Raji B cells requires CD28 activation. The addition of rCTLA4Ig, which blocks CD28 binding to its ligand, to the cultures decreased IL-2 promoter activation by >80%. Interestingly, CTLA4Ig did not significantly inhibit the activation of either NF of activated T cells (NFAT) or AP-1 reporters. Therefore, activation of NFAT and AP-1 is insufficient for IL-2 promoter activation. In contrast, an RE/AP reporter was blocked by CTLA4Ig by >90%. Thus, the requirement for CD28 in IL-2 promoter activation appears to be due to RE/AP and not the NFAT or AP-1 sites. In addition, these data suggest that transcriptional activation of RE/AP is not mediated by NFAT, because activation of a NFAT reporter is not affected by the addition of CTLA4Ig.
Asunto(s)
Antígenos CD28/fisiología , Proteínas de Unión al ADN/fisiología , Regulación de la Expresión Génica , Inmunoconjugados , Interleucina-2/biosíntesis , Activación de Linfocitos , Cooperación Linfocítica , Proteínas Nucleares , Regiones Promotoras Genéticas , Secuencias Reguladoras de Ácidos Nucleicos , Factor de Transcripción AP-1/fisiología , Factores de Transcripción/fisiología , Transcripción Genética , Abatacept , Antígenos Bacterianos/inmunología , Antígenos CD , Antígenos de Diferenciación/farmacología , Linfocitos B/efectos de los fármacos , Linfocitos B/metabolismo , Linfoma de Burkitt/patología , Antígeno CTLA-4 , Células Cultivadas , Enterotoxinas/inmunología , Genes Reporteros , Humanos , Interleucina-2/genética , Células Jurkat , Luciferasas/biosíntesis , Luciferasas/genética , Linfocitos , Factores de Transcripción NFATC , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/genética , Superantígenos/inmunología , Linfocitos T/efectos de los fármacos , Linfocitos T/metabolismo , Tacrolimus/farmacología , Transcripción Genética/efectos de los fármacos , Células Tumorales CultivadasRESUMEN
Binding of a T cell to an appropriate antigen-presenting cell (APC) induces the rapid reorientation of the T cell cytoskeleton and secretory apparatus towards the cell-cell contact site in a T cell antigen receptor (TCR) and peptide/major histocompatibility complex-dependent process. Such T cell polarization directs the delivery of cytokines and cytotoxic mediators towards the APC and contributes to the highly selective and specific action of effector T cells. To study the signaling pathways that regulate cytoskeletal rearrangements in T lymphocytes, we set up a conjugate formation assay using Jurkat T cells as effectors and cell-sized latex beads coated with various antibodies as artificial APCs. Here, we report that beads coated with antibodies specific for the TCR-CD3 complex were sufficient to induce T cell polarization towards the bead attachment site, as judged by reorientation of the microtubule-organizing center (MTOC) and localized actin polymerization. Thus, these cytoskeletal changes did not depend on activation of additional coreceptors. Moreover, single subunits of the TCR complex, namely TCR-zeta and CD3epsilon, were equally effective in inducing cytoskeletal polarization. However, mutagenesis of the immunoreceptor tyrosine-based activation motifs (ITAMs), present three times in TCR-zeta and once in CD3epsilon, revealed that the induction of cytoskeletal rearrangements required the presence of at least one intact ITAM. In agreement with this result, lack of functional Lck, the protein tyrosine kinase responsible for ITAM phosphorylation, abolished both MTOC reorientation and polarized actin polymerization. Both inhibitor and transient overexpression studies demonstrated that MTOC reorientation could occur in the absence of Ras activation. Our results suggest that APC-induced T cell polarization is a TCR-mediated event that is coupled to the TCR by the same signaling motif as TCR-induced gene activation, but diverges in its distal signaling requirements.
Asunto(s)
Citoesqueleto/fisiología , Receptores Inmunológicos/fisiología , Linfocitos T/metabolismo , Actinas/química , Actinas/metabolismo , Anticuerpos/fisiología , Sitios de Unión/genética , Complejo CD3/genética , Centrosoma/fisiología , Citoesqueleto/química , Inmunoensayo de Polarización Fluorescente , Expresión Génica/genética , Expresión Génica/fisiología , Regulación de la Expresión Génica/genética , Regulación de la Expresión Génica/fisiología , Genes Dominantes/genética , Genes Dominantes/fisiología , Genes ras/genética , Humanos , Células Jurkat , Proteínas de la Membrana/genética , Microesferas , Mutación/genética , Mutación/fisiología , Fosforilación , Proteínas Tirosina Quinasas/genética , Receptores de Antígenos de Linfocitos T/genética , Receptores de Antígenos de Linfocitos T/inmunología , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/fisiología , Transducción de Señal , Linfocitos T/química , Linfocitos T/inmunología , Activación Transcripcional , Tirosina/fisiología , Proteína Tirosina Quinasa ZAP-70RESUMEN
Mutagenesis studies have demonstrated the requirement for the CD28-responsive element (CD28RE) within the interleukin-2 (IL-2) promoter for transcriptional upregulation by CD28. Here, we demonstrate that CD28 responsiveness is conferred by a composite element containing both the CD28RE and the NF-IL-2B AP-1 sites (RE/AP). Mutations at either site within the RE/AP composite element abolish activity. The RE/AP composite element is a site for signal integration within the IL-2 promoter, since its activation is dependent on at least two separate signalling pathways being activated, through the T-cell receptor, CD28, and/or phorbol myristate acetate. Activation is maximal when all three signals occur simultaneously. By using a panel of CD28 cytoplasmic domain mutants, it was found that the transcriptional activation of the RE/AP composite element correlates exactly with the pattern of IL-2 secretion induced by these mutants upon stimulation. Similar to the upregulation of IL-2 secretion, the transcriptional upregulation of the RE/AP composite element by CD28 is FK506 insensitive. The pattern of activation of the RE/AP composite element is different from that observed for either an NFAT or consensus AP-1 site, implying that RE/AP represents a unique element. Using gel shift analysis, we demonstrate that stimulation by CD28 induces the association of the NF-kappaB family member c-Rel to the CD28RE within the RE/AP composite element. The transcriptional upregulation of IL-2 by CD28 appears, therefore, to be mediated through the RE/AP composite element, involving the association of c-Rel with the CD28RE.
Asunto(s)
Antígenos CD28/fisiología , Interleucina-2/genética , Factor de Transcripción AP-1/fisiología , Transcripción Genética , Regulación hacia Arriba , Secuencia de Aminoácidos , Secuencia de Bases , Sitios de Unión , Línea Celular , Núcleo Celular/metabolismo , Proteínas de Unión al ADN/fisiología , Humanos , Sustancias Macromoleculares , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas c-relRESUMEN
T cell activation by antigen/MHC induces the expression of several genes critical to the immune response, including interleukin-2. T cells from mice deficient for the NF-kappa B family member c-rel cannot activate IL-2 gene expression. However, mutating the NF-kappa B site in the IL-2 promoter has only moderate effects. To investigate additional ways c-Rel could regulate IL-2 gene expression, we determined whether c-rel overexpression could increase the activity of other transcription factors involved in IL-2 promoter regulation: NF-AT, Oct/OAP (ARRE-1), and AP-1. In Jurkat TAg cells, overexpression of c-Rel increased AP-1 activation approximately 17-fold. Moreover, AP-1 activity stimulated by anti-TCR Abs or PMA/ionomycin was further increased by c-Rel overexpression. c-Rel overexpression did not affect NF-AT or ARRE-1 activity. Additionally, overexpression of c-Rel activated the nonconsensus AP-1 site from the IL-2 promoter (NF-IL-2B), although to a lesser extent, approximately sixfold. AP-1 activation required both the DNA binding and transactivation domains of c-Rel. Our results may provide an explanation for the effect on IL-2 gene activation in c-rel-deficient mice.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Interleucina-2/biosíntesis , Proteínas Nucleares , Proteínas Proto-Oncogénicas/metabolismo , Linfocitos T/inmunología , Factor de Transcripción AP-1/metabolismo , Regulación hacia Arriba , Animales , Sitios de Unión , ADN/metabolismo , Genes Reporteros , Humanos , Interleucina-2/genética , Células Jurkat , Ratones , Factores de Transcripción NFATC , Regiones Promotoras Genéticas , Unión Proteica , Conformación Proteica , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas c-rel , Factores de Transcripción/metabolismoRESUMEN
The transcription factor GATA-3 contributes to the expression of several genes critical for T-cell function and development, including the T-cell receptor alpha and beta chains. We report here the isolation of a human cDNA clone which encodes a 3' segment of a novel protein with three zinc fingers. This protein, which we termed G3B, can interact with GATA-3 in vitro. Its mRNA is expressed in T and B cells. Thus, G3B may represent a potential regulator of GATA-3 function.