RESUMO
The generation of B-cell precursors (BCP) from lymphohematopoietic progenitors (LHP) in bone marrow is dependent on signals provided by the receptor tyrosine kinase Flt3 and its ligand, Flt3-ligand (FL). Mice deficient in FL exhibit striking reductions in LHP and BCP. Currently, the mechanism by which Flt3 regulates lymphoid lineage/B-cell development is unknown. Here, we show that haploinsufficiency of FL (FL(+/) (-) ) reduced the numbers of LHP, common lymphoid progenitors, and pro-B cells, suggesting that FL levels set a threshold for B lymphopoiesis. Limiting dilution analysis confirmed reduced BCP frequency in FL(+/) (-) mice. Real-time PCR of LHP from FL(+/) (-) animals showed increased transcripts of the B lineage inhibitor id1. However, targeted deletion of id1 did not restore the lymphoid/B lineage deficiencies in FL(-/-) mice, supporting Id1-independent mechanisms. BrdU incorporation studies established that FL is not essential for the proliferation of Flt3(+) multipotential progenitors. Analysis of FL(-/-) progenitors expressing low levels of Flt3 revealed decreased levels of the pro-survival factor Mcl1. Consequently, the Flt3(+) LHP progeny of Flt3(low) LSK(+) cells exhibited increased Annexin V staining. Together, these data suggest that Flt3 signaling initiates a cascade of events in Flt3(low) precursors that promote the survival of LHP from which BCP are derived.
Assuntos
Apoptose/imunologia , Células Progenitoras Linfoides/citologia , Linfopoese/fisiologia , Proteínas de Membrana/metabolismo , Células Precursoras de Linfócitos B/citologia , Transdução de Sinais/imunologia , Animais , Apoptose/genética , Células da Medula Óssea/citologia , Células da Medula Óssea/imunologia , Contagem de Células , Proliferação de Células , Sobrevivência Celular/genética , Sobrevivência Celular/imunologia , Expressão Gênica/genética , Haploinsuficiência/imunologia , Heterozigoto , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Proteína 1 Inibidora de Diferenciação/genética , Células Progenitoras Linfoides/metabolismo , Linfopoese/efeitos dos fármacos , Proteínas de Membrana/genética , Proteínas de Membrana/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Células-Tronco Multipotentes/citologia , Células-Tronco Multipotentes/efeitos dos fármacos , Células-Tronco Multipotentes/metabolismo , Proteína de Sequência 1 de Leucemia de Células Mieloides , Células Precursoras de Linfócitos B/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Transdução de Sinais/genética , Tirosina Quinase 3 Semelhante a fms/genética , Tirosina Quinase 3 Semelhante a fms/metabolismoRESUMO
Early B cell factor (EBF) is a transcription factor essential for specification and commitment to the B cell fate. In this study, we show downregulation of a developmentally regulated cluster of hoxa genes, notably hoxa9, coincides with induction of EBF at the Pro-B cell stage of B cell differentiation. Analysis of the hematopoietic progenitor compartment in Hoxa9(-/-) mice revealed significantly reduced frequencies and expression levels of Flt3, a cytokine receptor important for lymphoid priming and the generation of B cell precursors (BCPs). We show that Hoxa9 directly regulates the flt3 gene. Chromatin immunoprecipitation analysis revealed binding of Hoxa9 to the flt3 promoter in a lymphoid progenitor cell line. Knockdown of Hoxa9 significantly reduced Flt3 transcription and expression. Conversely, forced expression of Hoxa9 increased Flt3 transcription and expression in a Pro-B cell line that expressed low levels of Flt3. Hoxa9 inversely correlated with ebf1 in ex vivo-isolated bone marrow progenitors and BCPs, suggesting that EBF might function to silence a Hoxa9 transcriptional program. Restoration of EBF function in an EBF(-/-) cell line induced B lineage gene expression but did not directly suppress hoxa9 transcription, revealing alternate mechanisms of Hoxa9 regulation in BCPs. These data provide new insight into Hoxa9 function and regulation during lymphoid and B cell development. Furthermore, they suggest that failure to upregulate Flt3 provides a molecular basis for the lymphoid/early B cell deficiencies in Hoxa9(-/-) mice.
Assuntos
Subpopulações de Linfócitos B/citologia , Subpopulações de Linfócitos B/imunologia , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/imunologia , Proteínas de Homeodomínio/fisiologia , Linfopoese/imunologia , Tirosina Quinase 3 Semelhante a fms/metabolismo , Animais , Subpopulações de Linfócitos B/metabolismo , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Linhagem Celular , Linhagem da Célula/genética , Linhagem da Célula/imunologia , Inativação Gênica/imunologia , Células-Tronco Hematopoéticas/metabolismo , Proteínas de Homeodomínio/genética , Linfopoese/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Transativadores/deficiência , Transativadores/genética , Transativadores/fisiologia , Tirosina Quinase 3 Semelhante a fms/deficiência , Tirosina Quinase 3 Semelhante a fms/genéticaRESUMO
Immediate early gene X-1 (IEX-1) modulates apoptosis, cellular growth, mechanical strain-induced cardiac hypertrophy, and vascular intimal hyperplasia. To determine how IEX-1 alters apoptosis, we performed yeast two-hybrid studies using IEX-1 as the "bait" protein, and examined interactions between IEX-1 and proteins expressed by a human kidney cDNA expression library. We found that IEX-1 interacts with several proteins of which at least four are known to play a role in the regulation of apoptosis: (1) calcium-modulating cyclophilin ligand; (2) tumor necrosis factor-related apoptosis-inducing ligand (tumor necrosis factor superfamily, member 10); (3) ML-1 myeloid cell leukemia gene encoded protein; and (4) BAT3, a gene present in the major histo-compatibility complex. Our data suggest that IEX-1 may regulate apoptosis by directly interacting with various proteins involved in the control of apoptotic pathways.