title: Thrombopoietin stimulates colony-forming unit-megakaryocyte proliferation and megakaryocyte maturation independently of cytokines that signal through the gp130 receptor subunit.
abstract: Thrombopoietin (Tpo), the ligand for the c-Mpl receptor, is a major regulator of megakaryopoiesis. Treatment of mice with Tpo raises the platelet count fourfold within a few days. Conversely, c-mpl knock-out mice have platelet counts that are 15% that of normal. The subunit structure of the c-Mpl receptor is not fully understood. Some cytokines that stimulate megakaryopoiesis (IL-6, IL-11, leukemia inhibitory factor, and oncostatin M) bind to receptors that use gp130 as a signal transduction subunit. For these reasons, we determined whether gp130 function was required for Tpo-induced signal transduction. Murine marrow cells were cultured in semi-solid media in the presence of Tpo or IL-3, with or without a neutralizing anti-gp130 monoclonal antibody (RX187) or a soluble form of c-Mpl receptor (soluble Mpl) that blocks Tpo bioactivity, and the numbers of colony-forming unit-megakaryocyte (CFU-Meg) colonies were counted on day 5. Murine marrow cells were also cultured in suspension under serum-free conditions for 5 days, and megakaryocyte DNA content was measured by flow cytometry, as an index of nuclear maturation. The addition of RX187 did not block Tpo-induced CFU-Meg colony growth nor CFU-Meg nuclear maturation in suspension culture. However, IL-3-induced CFU-Meg colony growth and megakaryocyte nuclear maturation decreased in the presence of RX187. Soluble Mpl completely ablated Tpo-induced CFU-Meg growth, and partially blocked IL-3-stimulated CFU-Meg growth. Thus the effects of Tpo on megakaryopoiesis in vitro do not depend on cytokines that signal through gp130. Furthermore, it is unlikely that gp 130 serves as a beta chain for the c-Mpl receptor, as Tpo signalling is unimpaired in the presence of RX187. In contrast, the effects of IL-3 on CFU-Meg growth are mediated in part through Tpo and through gp130-signalling cytokines.
authors.affiliation: University of Washington, Division of Hematology, Seattle 98195-7710, USA.
authors.forename: V C
authors.initials: VC
authors.lastname: Broudy
authors.forename: N L
authors.initials: NL
authors.lastname: Lin
authors.forename: N
authors.initials: N
authors.lastname: Fox
authors.forename: T
authors.initials: T
authors.lastname: Taga
authors.forename: M
authors.initials: M
authors.lastname: Saito
authors.forename: K
authors.initials: K
authors.lastname: Kaushansky
mesh_terms: D000818:Animals; D015703:Antigens, CD / Q000502:physiology*; D002454:Cell Differentiation / Q000187:drug effects; D003114:Colony-Forming Units Assay; D050822:Cytokine Receptor gp130; D004247:DNA / Q000378:metabolism; D006410:Hematopoiesis / Q000187:drug effects*; D006579:Heterozygote; D007158:Immunologic Techniques; D007377:Interleukin-3 / Q000494:pharmacology; D008533:Megakaryocytes / Q000166:cytology*; D008562:Membrane Glycoproteins / Q000502:physiology*; D051379:Mice; D008810:Mice, Inbred C57BL; D008811:Mice, Inbred DBA; D009363:Neoplasm Proteins; D011518:Proto-Oncogene Proteins / Q000502:physiology*; D018121:Receptors, Cytokine; D053628:Receptors, Thrombopoietin; D015398:Signal Transduction; D013926:Thrombopoietin / Q000494:pharmacology*
chemical_list: D015703:Antigens, CD; C494200:Il6st protein, mouse; D007377:Interleukin-3; D008562:Membrane Glycoproteins; D009363:Neoplasm Proteins; D011518:Proto-Oncogene Proteins; D018121:Receptors, Cytokine; D053628:Receptors, Thrombopoietin; D050822:Cytokine Receptor gp130; C082218:MPL protein, human; D004247:DNA; D013926:Thrombopoietin