In the prior competitive renewal of this grant we proposed an experimental plan of three specific aims, to understand the structure-function relationships of thrombopoietin (TPO), identify genes responsible for MK development and to understand the molecular mechanisms that underlie endomitosis (EnM). We have made much progress in pursuit of these aims, and plan to build upon these advances in the continuing studies proposed herein. Specifically, we plan to A) study the mechanisms that regulate thrombopoiesis and the blood levels of TPO in inflammatory conditions, testing two hypotheses positing that inflammatory stimuli either 1) directly affect TPO gene transcription, mRNA stability or its efficiency of translation or 2) affect TPO blood levels indirectly by altering expression of the TPO receptor, c-mpl; B) determine the intramolecular pathways employed by TPO that transmit signals for MK survival, proliferation and differentiation, focusing on the targets of phosphoinositol-3 kinase, the mechanisms by which cell adhesion play a role in megakaryopoiesis and potential signaling roles for molecules recently determined to bind to the cytoplasmic domain of c-Mpl; C) study both the initiation and maintenance events responsible for EnM, testing the role of survivin, INCENP and Aurora kinases, and the integrity of the late anaphase checkpoint in polyploid MKs. Finally, as we recently participated in the cloning and initial characterization of a new cytokine, a fourth specific aim has been added to D) study the intracellular signaling pathways employed by interleukin (IL)-21 and its corresponding receptor. A major reason to study the signaling of this cytokine is that it provides an opportunity to explore the mechanisms by which related cytokines (IL-2, IL-15 and IL-21) employ seemingly identical initial signal transduction pathways but generate specific downstream events to induce distinct physiologic effects.
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