RESUMEN
Developing pre-B cells in the bone marrow alternate between proliferation and differentiation phases. We found that protein arginine methyl transferase 1 (PRMT1) and B cell translocation gene 2 (BTG2) are critical components of the pre-B cell differentiation program. The BTG2-PRMT1 module induced a cell-cycle arrest of pre-B cells that was accompanied by re-expression of Rag1 and Rag2 and the onset of immunoglobulin light chain gene rearrangements. We found that PRMT1 methylated cyclin-dependent kinase 4 (CDK4), thereby preventing the formation of a CDK4-Cyclin-D3 complex and cell cycle progression. Moreover, BTG2 in concert with PRMT1 efficiently blocked the proliferation of BCR-ABL1-transformed pre-B cells in vitro and in vivo. Our results identify a key molecular mechanism by which the BTG2-PRMT1 module regulates pre-B cell differentiation and inhibits pre-B cell leukemogenesis.
Asunto(s)
Proliferación Celular/genética , Ciclina D3/metabolismo , Quinasa 4 Dependiente de la Ciclina/metabolismo , Proteínas Inmediatas-Precoces/genética , Linfopoyesis/genética , Células Precursoras de Linfocitos B/metabolismo , Proteína-Arginina N-Metiltransferasas/genética , Proteínas Supresoras de Tumor/genética , Animales , Puntos de Control del Ciclo Celular , Diferenciación Celular/genética , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Citometría de Flujo , Técnicas de Silenciamiento del Gen , Reordenamiento Génico de Linfocito B/genética , Genes abl/genética , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Proteínas Inmediatas-Precoces/metabolismo , Cadenas Ligeras de Inmunoglobulina/genética , Espectrometría de Masas , Ratones , Células Precursoras de Linfocitos B/citología , Proteína-Arginina N-Metiltransferasas/metabolismo , ARN Interferente Pequeño , Reacción en Cadena en Tiempo Real de la Polimerasa , Proteínas Supresoras de Tumor/metabolismoRESUMEN
Activation-induced deaminase (AID) initiates secondary antibody diversification in germinal center B cells, giving rise to higher affinity antibodies through somatic hypermutation (SHM) or to isotype-switched antibodies through class switch recombination (CSR). SHM and CSR are triggered by AID-mediated deamination of cytosines in immunoglobulin genes. Importantly, AID activity in B cells is not restricted to Ig loci and can promote mutations and pro-lymphomagenic translocations, establishing a direct oncogenic mechanism for germinal center-derived neoplasias. AID is also expressed in response to inflammatory cues in epithelial cells, raising the possibility that AID mutagenic activity might drive carcinoma development. We directly tested this hypothesis by generating conditional knock-in mouse models for AID overexpression in colon and pancreas epithelium. AID overexpression alone was not sufficient to promote epithelial cell neoplasia in these tissues, in spite of displaying mutagenic and genotoxic activity. Instead, we found that heterologous AID expression in pancreas promotes the expression of NKG2D ligands, the recruitment of CD8(+) T cells, and the induction of epithelial cell death. Our results indicate that AID oncogenic potential in epithelial cells can be neutralized by immunosurveillance protective mechanisms.
Asunto(s)
Citidina Desaminasa/biosíntesis , Subfamilia K de Receptores Similares a Lectina de Células NK/biosíntesis , Animales , Linfocitos T CD8-positivos/inmunología , Muerte Celular , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/inmunología , Colon/patología , Citidina Desaminasa/genética , Citidina Desaminasa/inmunología , Pruebas Inmunológicas de Citotoxicidad , Epitelio/metabolismo , Epitelio/patología , Ratones , Ratones Transgénicos , Subfamilia K de Receptores Similares a Lectina de Células NK/inmunología , Páncreas/patologíaRESUMEN
Mature B cells express immunoglobulin M (IgM)- and IgD-isotype B cell antigen receptors, but the importance of IgD for B cell function has been unclear. By using a cellular in vitro system and corresponding mouse models, we found that antigens with low valence activated IgM receptors but failed to trigger IgD signaling, whereas polyvalent antigens activated both receptor types. Investigations of the molecular mechanism showed that deletion of the IgD-specific hinge region rendered IgD responsive to monovalent antigen, whereas transferring the hinge to IgM resulted in responsiveness only to polyvalent antigen. Our data suggest that the increased IgD/IgM ratio on conventional B-2 cells is important for preferential immune responses to antigens in immune complexes, and that the increased IgM expression on B-1 cells is essential for B-1 cell homeostasis and function.
Asunto(s)
Linfocitos B/inmunología , Inmunoglobulina D/inmunología , Inmunoglobulina M/inmunología , Animales , Complejo Antígeno-Anticuerpo/inmunología , Antígenos/inmunología , Sitios de Unión de Anticuerpos/inmunología , Señalización del Calcio/genética , Diferenciación Celular , Línea Celular , Exones de la Región Bisagra/genética , Homeostasis/genética , Inmunidad Humoral/genética , Inmunoglobulina D/genética , Inmunoglobulina M/genética , Ratones , Ratones Noqueados , Ingeniería de Proteínas , Eliminación de Secuencia/genéticaRESUMEN
B-cell antigen receptor (BCR) expression is an important feature of chronic lymphocytic leukaemia (CLL), one of the most prevalent B-cell neoplasias in Western countries. The presence of stereotyped and quasi-identical BCRs in different CLL patients suggests that recognition of specific antigens might drive CLL pathogenesis. Here we show that, in contrast to other B-cell neoplasias, CLL-derived BCRs induce antigen-independent cell-autonomous signalling, which is dependent on the heavy-chain complementarity-determining region (HCDR3) and an internal epitope of the BCR. Indeed, transferring the HCDR3 of a CLL-derived BCR provides autonomous signalling capacity to a non-autonomously active BCR, whereas mutations in the internal epitope abolish this capacity. Because BCR expression was required for the binding of secreted CLL-derived BCRs to target cells, and mutations in the internal epitope reduced this binding, our results indicate a new model for CLL pathogenesis, with cell-autonomous antigen-independent signalling as a crucial pathogenic mechanism.
Asunto(s)
Leucemia Linfocítica Crónica de Células B/metabolismo , Leucemia Linfocítica Crónica de Células B/patología , Receptores de Antígenos de Linfocitos B/metabolismo , Transducción de Señal , Secuencias de Aminoácidos , Autoantígenos/inmunología , Autoantígenos/metabolismo , Señalización del Calcio , Regiones Determinantes de Complementariedad/inmunología , Regiones Determinantes de Complementariedad/metabolismo , Epítopos de Linfocito B/inmunología , Epítopos de Linfocito B/metabolismo , Humanos , Leucemia Linfocítica Crónica de Células B/inmunología , Receptores de Antígenos de Linfocitos B/inmunologíaRESUMEN
Somatic rearrangement of immunoglobulin (Ig) genes is a key step during B cell development. Using pro-B cells lacking the phosphatase Pten (phosphatase and tensin homolog), which negatively regulates phosphoinositide-3-kinase (PI3K) signaling, we show that PI3K signaling inhibits Ig gene rearrangement by suppressing the expression of the transcription factor Ikaros. Further analysis revealed that the transcription factor FoxO1 is crucial for Ikaros expression and that PI3K-mediated down-regulation of FoxO1 suppresses Ikaros expression. Interestingly, FoxO1 did not influence Ikaros transcription; instead, FoxO1 is essential for proper Ikaros mRNA splicing, as FoxO1-deficient cells contain aberrantly processed Ikaros transcripts. Moreover, FoxO1-induced Ikaros expression was sufficient only for proximal V(H) to DJ(H) gene rearrangement. Simultaneous expression of the transcription factor Pax5 was needed for the activation of distal V(H) genes; however, Pax5 did not induce any Ig gene rearrangement in the absence of Ikaros. Together, our results suggest that ordered Ig gene rearrangement is regulated by distinct activities of Ikaros, which mediates proximal V(H) to DJ(H) gene rearrangement downstream of FoxO1 and cooperates with Pax5 to activate the rearrangement of distal V(H) genes.
Asunto(s)
Factores de Transcripción Forkhead/metabolismo , Regulación de la Expresión Génica/fisiología , Genes de Inmunoglobulinas/genética , Factor de Transcripción Ikaros/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Empalme del ARN/fisiología , Recombinación V(D)J/fisiología , Animales , Cartilla de ADN/genética , Citometría de Flujo , Proteína Forkhead Box O1 , Regulación de la Expresión Génica/genética , Factor de Transcripción Ikaros/genética , Immunoblotting , Ratones , Factor de Transcripción PAX5/metabolismo , Fosfohidrolasa PTEN/metabolismo , Plásmidos/genética , Reacción en Cadena de la Polimerasa , Empalme del ARN/genética , Transducción GenéticaRESUMEN
Developing B cells express distinct classes of B cell antigen receptors (BCRs) that differ in their heavy chain (HC). Although only muHC is expressed in early stages, deltaHC-containing BCRs dominate on the surface of mature B cells. The reason for the tightly regulated expression of these receptors is poorly understood. Here we show that muHC was specifically required for precursor BCR (pre-BCR) function and that deltaHC was unable to form a functional pre-BCR. A conserved asparagine (N)-linked glycosylation site at position 46 (N46) in the first conserved domain of muHC was absolutely required for pre-BCR function, and swapping that domain with deltaHC resulted in a functional deltaHC-containing pre-BCR. When tested in the context of the BCR, muHC with a mutant N46 showed normal function, which indicated that N46-glycosylation is specifically required for pre-BCR function. Our results suggest an unexpected mode of pre-BCR function, in which binding of the surrogate light chain to N46 mediates autonomous crosslinking and, concomitantly, receptor formation.
Asunto(s)
Linfocitos B/inmunología , Cadenas Pesadas de Inmunoglobulina/inmunología , Receptores de Células Precursoras de Linfocitos B/inmunología , Receptores de Antígenos de Linfocitos B/inmunología , Animales , Asparagina/inmunología , Linfocitos B/citología , Glicosilación , Ratones , Ratones NoqueadosRESUMEN
Signals from the B cell antigen receptor (BCR), consisting of mu heavy chain (muHC) and conventional light chain (LC), and its precursor the pre-BCR, consisting of muHC and surrogate light chain (SLC), via the adaptor protein SLP-65 regulate the development and function of B cells. Here, we compare the effect of SLC and conventional LC expression on receptor-induced Ca(2+) flux in B cells expressing an inducible form of SLP-65. We found that SLC expression strongly enhanced an autonomous ability of muHC to induce Ca(2+) flux irrespective of additional receptor crosslinking. In contrast, LC expression reduced this autonomous muHC ability and resulted in antigen-dependent Ca(2+) flux. These data indicate that autonomous ligand-independent signaling can be induced by receptor forms other than the pre-BCR. In addition, our data suggest that conventional LCs play an important role in the inhibition of autonomous receptor signaling, thereby allowing further B cell differentiation.
Asunto(s)
Linfocitos B/inmunología , Cadenas Ligeras de Inmunoglobulina/metabolismo , Receptores de Antígenos de Linfocitos B/antagonistas & inhibidores , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Calcio/metabolismo , Línea Celular Tumoral , Humanos , Inmunoglobulina de Cadenas Ligeras Subrogadas , Ligandos , Glicoproteínas de Membrana/metabolismo , Ratones , Fosforilación , Transducción de SeñalRESUMEN
The nonreceptor protein spleen tyrosine kinase (Syk) is a key mediator of signal transduction in a variety of cell types, including B lymphocytes. We show that deregulated Syk activity allows growth factor-independent proliferation and transforms bone marrow-derived pre-B cells that are then able to induce leukemia in mice. Syk-transformed pre-B cells show a characteristic pattern of tyrosine phosphorylation, increased c-Myc expression, and defective differentiation. Treatment of Syk-transformed pre-B cells with a novel Syk-specific inhibitor (R406) reduces tyrosine phosphorylation and c-Myc expression. In addition, R406 treatment removes the developmental block and allows the differentiation of the Syk-transformed pre-B cells into immature B cells. Because R406 treatment also prevents the proliferation of c-Myc-transformed pre-B cells, our data indicate that endogenous Syk kinase activity may be required for the survival of pre-B cells transformed by other oncogenes. Collectively, our data suggest that Syk is a protooncogene involved in the transformation of lymphocytes, thus making Syk a potential target for the treatment of leukemia.
Asunto(s)
Linfocitos B/metabolismo , Diferenciación Celular/fisiología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Traslado Adoptivo , Animales , Linfocitos B/efectos de los fármacos , Linfocitos B/trasplante , Benzamidas , Diferenciación Celular/efectos de los fármacos , Línea Celular , Proliferación Celular/efectos de los fármacos , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Proteínas de Fusión bcr-abl/antagonistas & inhibidores , Proteínas de Fusión bcr-abl/genética , Proteínas de Fusión bcr-abl/metabolismo , Humanos , Mesilato de Imatinib , Péptidos y Proteínas de Señalización Intercelular/farmacología , Péptidos y Proteínas de Señalización Intercelular/fisiología , Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , Péptidos y Proteínas de Señalización Intracelular/genética , Leucemia/genética , Leucemia/patología , Leucemia/terapia , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Oxazinas/farmacología , Fosfolipasa C gamma/genética , Fosfolipasa C gamma/metabolismo , Fosforilación/efectos de los fármacos , Piperazinas/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Proteínas Tirosina Quinasas/genética , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , Piridinas/farmacología , Pirimidinas/farmacología , Receptores de Antígenos de Linfocitos B/genética , Bazo/efectos de los fármacos , Bazo/metabolismo , Bazo/patología , Quinasa Syk , TransfecciónRESUMEN
Signal transduction from the B cell antigen receptor (BCR) involves a multitude of signaling molecules often organized in dynamic protein complexes. The molecular mechanisms operating during signaling are difficult to study solely by loss-of-function analysis. For a better understanding of the transient interaction of signaling molecules and their regulation by feedback loops, as well as their dynamic behavior in living cells, new techniques are required. We have developed a method allowing the reconstitution of the BCR complex and several of its key signaling elements in the evolutionary distant environment of the Drosophila S2 Schneider cell line. With this gain-of-function approach, we study here the assembly of the BCR complex and the control of its transport to the cell surface of S2 cells. We find that without binding to a light chain, the membrane-bound microm heavy chain (micromHC) homodimer, together with the Ig-alpha/Ig-beta heterodimer, can come to the cell surface where it is signaling competent. This finding could have implications for potential signaling functions of such a receptor molecule during pro-/pre-B cell development. We also studied the activation of the BCR-proximal kinase Syk. We found that a truncated Syk mutant lacking the first (N-terminal) SH2 domain and the linker regions, is still regulated by autoinhibition and can only become activated in the presence of the BCR. This indicates that the C-terminal SH2 domain of Syk is the dominant regulatory subunit of this kinase.
Asunto(s)
Precursores Enzimáticos/fisiología , Proteínas Tirosina Quinasas/fisiología , Receptores de Antígenos de Linfocitos B/biosíntesis , Animales , Drosophila melanogaster/inmunología , Drosophila melanogaster/fisiología , Precursores Enzimáticos/inmunología , Inmunoglobulina M/inmunología , Péptidos y Proteínas de Señalización Intracelular , Proteínas Tirosina Quinasas/inmunología , Receptores de Antígenos de Linfocitos B/genética , Receptores de Antígenos de Linfocitos B/inmunología , Quinasa Syk , TransfecciónRESUMEN
SLP-65-/- pre-B cells show a high proliferation rate in vitro. We have shown previously that lambda5 expression and consequently a conventional pre-B cell receptor (pre-BCR) are essential for this proliferation. Here, we show that pre-B cells express a novel receptor complex that contains a micro heavy chain (microHC) but lacks any surrogate (SL) or conventional light chain (LC). This SL-deficient pre-BCR (SL-pre-BCR) requires Ig-alpha for expression on the cell surface. Anti-micro treatment of pre-B cells expressing the SL-pre-BCR induces tyrosine phosphorylation of substrate proteins and a strong calcium (Ca2+) release. Further, the expression of the SL-pre-BCR is associated with a high differentiation rate toward kappaLC-positive cells. Given that B cell development is only partially blocked and allelic exclusion is unaffected in SL-deficient mice, we propose that the SL-pre-BCR is involved in these processes and therefore shares important functions with the conventional pre-BCR.
Asunto(s)
Linfocitos B/metabolismo , Receptores de Antígenos de Linfocitos B/química , Animales , Linfocitos B/citología , Calcio/metabolismo , Diferenciación Celular , Inmunoglobulina A/metabolismo , Ratones , Fosforilación , Tirosina/metabolismoRESUMEN
We have established a protocol allowing transient and inducible coexpression of many foreign genes in Drosophila S2 Schneider cells. With this powerful approach of reverse genetics, we studied the interaction of the protein tyrosine kinases Syk and Lyn with the B cell antigen receptor (BCR). We find that Lyn phosphorylates only the first tyrosine whereas Syk phosphorylates both tyrosines of the BCR immunoreceptor tyrosine-based activation motif (ITAM). Furthermore, we show that Syk is a positive allosteric enzyme, which is strongly activated by the binding to the phosphorylated ITAM tyrosines, thus initiating a positive feedback loop at the receptor. The BCR-dependent Syk activation and signal amplification is efficiently counterbalanced by protein tyrosine phosphatases, the activity of which is regulated by H(2)O(2) and the redox equilibrium inside the cell.