Requirement for mitogen-activated protein kinase activation in the response of embryonic stem cell-derived hematopoietic cells to thrombopoietin in vitro.
Enforced expression of c-mpl in embryonic stem (ES) cells inactivated for this gene results in protein expression in all the ES cell progeny, producing cells that do not belong to the megakaryocytic lineage and are responsive to PEG-rhuMGDF, a truncated form of human thrombopoietin (TPO) conjugated to polyethylene glycol. These include a primitive cell called BL-CFC, thought to represent the equivalent of the hemangioblast, and all myeloid progenitor cells. In this model, PEG-rhuMGDF was able to potentiate the stimulating effects of other growth factors, including vascular endothelial growth factor, on BL-CFC and a combination of cytokines on the growth of granulocyte macrophage-colony-forming units. The importance of the C-terminal domain of Mpl and of mitogen-activated protein kinase (MAPK) activation in TPO-dependent megakaryocytic differentiation has been well studied in vitro. Here, the role of this domain and the involvement of MAPK in upstream and nonmegakaryocytic cells are examined by using 2 truncated mutants of Mpl (Delta34, deletion of residues 71 to 121 in the C-terminal domain; and Delta3, deletion of residues 71-94) and specific inhibitors of the MAPK pathway. The 2 deleted regions support different functions, mediated by different signals. Residues 71 to 121 were required for PEG-rhuMGDF-dependent growth of BL-CFC, for megakaryocytic and other myeloid progenitors, and for megakaryocyte polyploidization. These responses were mediated by the ERK1-ERK2 MAPK pathway. In contrast, the only function of the sequence comprising residues 71 to 94 was to mediate the synergistic effects of PEG-rhuMGDF with other hematopoietic growth factors. This function is not mediated by MAPK activation.
Filippi
Marie-Dominique
MD
Institut National de la Santé et de la Recherche Médicale U362, Institut Gustave Roussy, Villejuif, France.
Porteu
Françoise
F
Le Pesteur
Françoise
F
Schiavon
Valérie
V
Millot
Gaël A
GA
Vainchenker
William
W
de Sauvage
Frédéric J
FJ
Dubart Kupperschmitt
Anne
A
Sainteny
Francoise
F
D000818:Animals; D001773:Blood Cells / Q000166:cytology / Q000187:drug effects; D002460:Cell Line; D019070:Cell Lineage / Q000187:drug effects; D004357:Drug Synergism; D004622:Embryo, Mammalian / Q000166:cytology; D016228:Endothelial Growth Factors / Q000494:pharmacology; D004789:Enzyme Activation / Q000502:physiology; D006410:Hematopoiesis / Q000187:drug effects; D006412:Hematopoietic Stem Cells / Q000187:drug effects*; D006801:Humans; D008222:Lymphokines / Q000494:pharmacology; D020935:MAP Kinase Signaling System / Q000502:physiology; D051379:Mice; D019950:Mitogen-Activated Protein Kinase 1 / Q000502:physiology; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism / Q000502:physiology*; D011092:Polyethylene Glycols / Q000494:pharmacology; D011994:Recombinant Proteins / Q000494:pharmacology; D013926:Thrombopoietin / Q000494:pharmacology*; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors
D016228:Endothelial Growth Factors; D008222:Lymphokines; D011994:Recombinant Proteins; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors; C103117:polyethylene glycol-recombinant human megakaryocyte growth and development factor; D011092:Polyethylene Glycols; D013926:Thrombopoietin; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases