ABSTRACT
The effects of Notch signals on the erythroid/megakaryocytic differentiation of hematopoietic cells were examined. Activation of Notch signals by the intracellular Notch1 or an estradiol-inducible form of Notch1/ER suppressed the expression of the erythroid marker glycophorin A in an erythroid/megakaryocytic cell line K562. Although Mock-transfected K562 cells underwent megakaryocytic differentiation in response to 12-O-tetradecanoylphorbol-13-acetate (TPA), estradiol-activated Notch1/ER induced apoptosis during TPA treatment in the transfectant, which was accompanied by the reduced expression of an antiapoptotic molecule Bcl-XL. Even when apoptosis was prevented by the overexpression of Bcl-XL, activated Notch signals still inhibited TPA-induced megakaryocytic differentiation. As for this mechanism, Notch1/recombination signal binding protein J-kappa-induced HES1 but not HES5 was found to inhibit the function of an erythroid/megakaryocytic lineage-specific transcription factor GATA-1. Although HES1 did not affect the DNA binding activity of GATA-1 in gel shift and chromatin immunoprecipitation assays, it directly bound to GATA-1 and dissociated a critical transcriptional cofactor, p300, from GATA-1. Furthermore, overexpressed HES1 inhibited the development of erythroid and megakaryocytic cells in colony assays. Also, the Notch ligand Jagged1 expressed on NIH3T3 cells suppressed the development of erythroid and megakaryocytic cells from cocultured Lin-Sca-1+ hematopoietic stem/progenitor cells. These results suggest that Notch1 inhibits the development of erythroid/megakaryocytic cells by suppressing GATA-1 activity through HES1.
Subject(s)
DNA-Binding Proteins/metabolism , Erythrocytes/metabolism , Homeodomain Proteins/metabolism , Megakaryocytes/metabolism , Membrane Proteins/metabolism , Transcription Factors/metabolism , Animals , Apoptosis , Basic Helix-Loop-Helix Transcription Factors , Blotting, Northern , Cell Differentiation , Cell Line , Cell Lineage , Cell Nucleus/metabolism , Chromatin Immunoprecipitation , Coculture Techniques , DNA/metabolism , E1A-Associated p300 Protein , Erythroid-Specific DNA-Binding Factors , Estradiol/metabolism , Flow Cytometry , GATA1 Transcription Factor , Glycophorins/metabolism , Hematopoietic Stem Cells/cytology , Humans , Immunoblotting , Immunoprecipitation , K562 Cells , Luciferases/metabolism , Mice , Mice, Inbred BALB C , Microscopy, Fluorescence , Models, Genetic , NIH 3T3 Cells , Nuclear Proteins/metabolism , Phenotype , Proto-Oncogene Proteins c-bcl-2/metabolism , Receptors, Estrogen/metabolism , Receptors, Notch , Retroviridae/genetics , Reverse Transcriptase Polymerase Chain Reaction , Stem Cells/cytology , Tetradecanoylphorbol Acetate/chemistry , Trans-Activators/metabolism , Transcription Factor HES-1 , Transcription, Genetic , Transfection , bcl-X ProteinABSTRACT
Notch and HOXB4 have been reported to expand hematopoietic stem cells (HSCs) in vitro. However, their critical effector molecules remain undetermined. We found that the expression of c-myc, cyclin D2, cyclin D3, cyclin E, and E2F1 was induced or enhanced during Notch1- or HOXB4-induced self-renewal of murine HSCs. Since c-Myc can act as a primary regulator of G(1)/S transition, we examined whether c-Myc alone can induce self-renewal of HSCs. In culture with stem cell factor, FLT3 ligand, and IL-6, a 4-hydroxytamoxifen-inducible form of c-Myc (Myc/ERT) enabled murine Lin(-)Sca-1(+) HSCs to proliferate with the surface phenotype compatible with HSCs for more than 28 days. c-Myc activated by 4-hydroxytamoxifen augmented telomerase activities and increased the number of CFU-Mix about 2-fold in colony assays. Also, in reconstitution assays, HSCs expanded by c-Myc could reconstitute hematopoiesis for more than 6 months. As for the mechanism of c-myc induction by Notch1, we found that activated forms of Notch1 (NotchIC) and its downstream effector recombination signal-binding protein-J kappa (RBP-VP16) can activate the c-myc promoter through the element between -195 bp and -161 bp by inducing the DNA-binding complex. Together, these results suggest that c-Myc can support self-renewal of HSCs as a downstream mediator of Notch and HOXB4.