title: Primary megakaryocytes reveal a role for transcription factor NF-E2 in integrin alpha IIb beta 3 signaling.
abstract: Platelet integrin alphaIIbbeta3 responds to intracellular signals by binding fibrinogen and triggering cytoskeletal reorganization, but the mechanisms of alphaIIbbeta3 signaling remain poorly understood. To better understand this process, we established conditions to study alphaIIbbeta3 signaling in primary murine megakaryocytes. Unlike platelets, these platelet precursors are amenable to genetic manipulation. Cytokine-stimulated bone marrow cultures produced three arbitrary populations of alphaIIbbeta3-expressing cells with increasing size and DNA ploidy: small progenitors, intermediate-size young megakaryocytes, and large mature megakaryocytes. A majority of the large megakaryocytes bound fibrinogen in response to agonists, while almost none of the smaller cells did. Fibrinogen binding to large megakaryocytes was inhibited by Sindbis virus-mediated expression of isolated beta3 integrin cytoplasmic tails. Strikingly, large megakaryocytes from mice deficient in the transcription factor NF-E2 failed to bind fibrinogen in response to agonists, despite normal surface expression of alphaIIbbeta3. Furthermore, while megakaryocytes from wild-type mice spread on immobilized fibrinogen and exhibited filopodia, lamellipodia and Rho-dependent focal adhesions and stress fibers, NF-E2-deficient megakaryocytes adhered poorly. These studies establish that agonist-induced activation of alphaIIbbeta3 is controlled by NF-E2-regulated signaling pathways that mature late in megakaryocyte development and converge at the beta3 cytoplasmic tail. Megakaryocytes provide a physiologically relevant and tractable system for analysis of bidirectional alphaIIbbeta3 signaling.
authors.affiliation: Department of Vascular Biology, The Scripps Research Institute, La Jolla, California 92037, USA.
authors.forename: M
authors.initials: M
authors.lastname: Shiraga
authors.forename: A
authors.initials: A
authors.lastname: Ritchie
authors.forename: S
authors.initials: S
authors.lastname: Aidoudi
authors.forename: V
authors.initials: V
authors.lastname: Baron
authors.forename: D
authors.initials: D
authors.lastname: Wilcox
authors.forename: G
authors.initials: G
authors.lastname: White
authors.forename: B
authors.initials: B
authors.lastname: Ybarrondo
authors.forename: G
authors.initials: G
authors.lastname: Murphy
authors.forename: A
authors.initials: A
authors.lastname: Leavitt
authors.forename: S
authors.initials: S
authors.lastname: Shattil
mesh_terms: D000818:Animals; D001854:Bone Marrow Cells / Q000166:cytology; D002454:Cell Differentiation / Q000502:physiology; D002478:Cells, Cultured; D004268:DNA-Binding Proteins / Q000502:physiology*; D050981:Erythroid-Specific DNA-Binding Factors; D008533:Megakaryocytes / Q000166:cytology / Q000502:physiology*; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D050983:NF-E2 Transcription Factor; D050984:NF-E2 Transcription Factor, p45 Subunit; D009687:Nuclear Proteins / Q000502:physiology; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000502:physiology*; D015398:Signal Transduction / Q000502:physiology*; D014157:Transcription Factors / Q000502:physiology*
chemical_list: D004268:DNA-Binding Proteins; D050981:Erythroid-Specific DNA-Binding Factors; D050983:NF-E2 Transcription Factor; D050984:NF-E2 Transcription Factor, p45 Subunit; C494693:Nfe2 protein, mouse; D009687:Nuclear Proteins; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D014157:Transcription Factors