title: M-CSF stimulated differentiation requires persistent MEK activity and MAPK phosphorylation independent of Grb2-Sos association and phosphatidylinositol 3-kinase activity.
abstract: Macrophage colony-stimulating factor (M-CSF) is a physiological regulator of monocyte-macrophage lineage. Ectopic expression of the M-CSF receptor (M-CSFR, or Fms) in murine myeloid cell line FDC-P1 (FD/Fms cells) results in M-CSF-dependent macrophage differentiation. Previously, we observed that M-CSF induces two temporally distinct phases of mitogen-activated protein kinase (MAPK) phosphorylation. Here we show that levels of phosphorylated MAPK kinase MEK1 follow the same kinetics as MAPK phosphorylation, characterized by an early and transient phase (the first 30 min of M-CSF stimulation) and a late and persistent phase from 4 h of stimulation. The MEK inhibitor U0126 strongly inhibited both phases of MAPK phosphorylation as well as FD/Fms cell differentiation, indicating that MAPK may relay M-CSF differentiation signaling downstream of M-CSFR. Treatment of FD/Fms cells with U0126 during the first hour of M-CSF stimulation reversibly blocked the early phase of MAPK phosphorylation but did not affect differentiation. In contrast, U0126 still inhibited FD/Fms cell differentiation when its addition was delayed by 24 h. This demonstrated that late and persistent MEK activity is specifically required for macrophage differentiation to occur. Furthermore, disrupting Grb2-Sos complexes with a specific blocking peptide did not prevent FD/Fms cells differentiation in response to M-CSF, nor did it abolish MAPK phosphorylation. The role of phosphatidylinositol 3-kinase (PI 3-kinase), another potential regulator of the MAPK pathway, was examined using the specific inhibitor LY294002. This compound could not impede FD/Fms cell commitment to macrophage differentiation and did not significantly affect MAPK phosphorylation in response to M-CSF. Therefore, M-CSF differentiation signaling in myeloid progenitor cells is mediated through persistent MEK activity but it is not strictly dependent upon Grb2-Sos interaction or PI 3-kinase activity.
authors.affiliation: Centre de Génétique Moléculaire et Cellulaire, UMR CNRS 5534, Bâtiment Gregor Mendel, Université Lyon1, 69622 Villeurbanne , France.
authors.forename: Stéphanie
authors.initials: S
authors.lastname: Gobert Gosse
authors.forename: Caroline
authors.initials: C
authors.lastname: Bourgin
authors.forename: Wang Qing
authors.initials: WQ
authors.lastname: Liu
authors.forename: Christiane
authors.initials: C
authors.lastname: Garbay
authors.forename: Guy
authors.initials: G
authors.lastname: Mouchiroud
mesh_terms: D000818:Animals; D002070:Butadienes / Q000494:pharmacology; D002460:Cell Line; D002867:Chromones / Q000494:pharmacology; D051380:GRB2 Adaptor Protein / Q000378:metabolism*; D048369:MAP Kinase Kinase 1 / Q000037:antagonists & inhibitors / Q000378:metabolism*; D020935:MAP Kinase Signaling System; D016173:Macrophage Colony-Stimulating Factor / Q000502:physiology*; D008264:Macrophages / Q000166:cytology / Q000378:metabolism; D051379:Mice; D009025:Morpholines / Q000494:pharmacology; D023461:Myeloid Progenitor Cells / Q000166:cytology / Q000378:metabolism; D009570:Nitriles / Q000494:pharmacology; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010766:Phosphorylation; D011485:Protein Binding; D016186:Receptor, Macrophage Colony-Stimulating Factor / Q000378:metabolism; D020839:Son of Sevenless Protein, Drosophila / Q000378:metabolism*
chemical_list: D002070:Butadienes; D002867:Chromones; D051380:GRB2 Adaptor Protein; D048369:MAP Kinase Kinase 1; D016173:Macrophage Colony-Stimulating Factor; D009025:Morpholines; D009570:Nitriles; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D016186:Receptor, Macrophage Colony-Stimulating Factor; D020839:Son of Sevenless Protein, Drosophila; C495949:Grb2 protein, mouse; C113580:U 0126; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; C482127:Map2k1 protein, mouse