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The mpl ligand: molecular and cellular biology of the critical regulator of megakaryocyte development
1Division of Hematology, University of Washington School of Medicine, Seattle.
Abstract:
The development of blood cells requires the interplay of hematopoietic stem and progenitor cells, marrow stroma and polypeptide growth factors. Although many proteins are thought to support the expansion of megakaryocytic precursor cells (e.g., interleukin [IL]-3, c-kit ligand [KL]), identification of the late-acting, lineage-specific growth factor for platelet production, termed Thrombopoietin (Tpo), has remained elusive. Recently, characterization of the proto-oncogene c-mpl revealed structural homology with the hematopoietic cytokine receptor family. Based on the cell of origin of its cDNA, we hypothesized that the ligand for c-mpl might be identical with Tpo. Using BaF3 cells engineered to express c-mpl, we employed a functional expression strategy to clone its cDNA. At low concentrations, the recombinant protein supports the growth of megakaryocytic colonies, alone and together with either IL-3 or KL. For IL-3 this appears to be additive, for KL, true synergy was detected. At higher concentrations, the mpl ligand (ML) alone supported a near maximal number of very large megakaryocytic colonies. Using suspension cultures and human megakaryocytic cell lines, we have also shown that ML induces the terminal differentiation of megakaryocytes by enhancing polyploidization and surface membrane expression of GPIb and IIb/IIIa. Moreover, the development of megakaryocytes in vitro appears to be absolutely dependent on the presence of ML. Following receptor engagement, ML induces tyrosine phosphorylation of a number of membrane associated kinases and adaptor molecules, including SHC, JAK2, PLC-gamma and the mpl receptor itself.(ABSTRACT TRUNCATED AT 250 WORDS)
Insights
The mpl ligand (ML) is identified as Thrombopoietin (Tpo), a crucial factor for platelet production. ML drives megakaryocyte differentiation and proliferation, essential for blood cell development.
Area of Science:
- Hematology
- Molecular Biology
- Cell Signaling
Background:
- Hematopoiesis involves stem cells, marrow stroma, and growth factors.
- Thrombopoietin (Tpo) is the elusive lineage-specific growth factor for platelet production.
- The proto-oncogene c-mpl shares homology with hematopoietic cytokine receptors.
Purpose of the Study:
- To identify the ligand for c-mpl, hypothesized to be Tpo.
- To characterize the function of the mpl ligand (ML) in megakaryopoiesis.
- To investigate the signaling pathways activated by ML.
Main Methods:
- Functional expression cloning using BaF3 cells engineered to express c-mpl.
- Assessing recombinant protein activity on megakaryocytic colony growth.
- Utilizing suspension cultures and human megakaryocytic cell lines.
Main Results:
- Recombinant ML supports megakaryocytic colony growth, showing additive effects with IL-3 and synergy with KL.
- ML alone supports a maximal number of large megakaryocytic colonies at higher concentrations.
- ML induces terminal megakaryocyte differentiation, enhancing polyploidization and GPIb/IIb/IIIa expression.
Conclusions:
- The mpl ligand (ML) is identified as Thrombopoietin (Tpo), essential for megakaryocyte development.
- ML is indispensable for in vitro megakaryocyte development, inducing terminal differentiation.
- ML engagement activates intracellular signaling, including tyrosine phosphorylation of kinases and the mpl receptor.