Blood cell production is critically dependent on the survival, proliferation and differentiation of hematopoietic stem and progenitor cells, a process regulated by the availability of endocrine growth factors and bone marrow stroma-derived signals. The cloning and availability of thrombopoietin (TPO), the primary regulator of thrombopoiesis and a critical contributor to stem cell physiology, has provided an opportunity to gain important new insights into hematopoietic homeostasis. Our first specific aim will explore the molecular mechanisms by which TPO production is regulated, concentrating on novel modes of hormone production and the alterations in marrow TPO mRNA levels induced by changes in blood platelet count. The relative contribution of marrow TPO production to hematopoiesis will also be established by evaluating a novel animal model of hormone production, a mouse in which the only source of TPO is marrow stroma. The second specific aim will identify the mechanisms by which the carboxyl terminal half of TPO alters its secretory efficiency. Our preliminary studies strongly suggest that TPO contains a prosequence, which functions to enhance folding of the receptor binding domain of the molecule. The proposed studies will identify the cellular site of action, kinetics, and specific sequence required for this function. The results have implications not only for TPO production, but for the entire field of mammalian protein processing. The third specific aim is designed to extend previous studies in which we identified key signaling mediators activated upon TPO receptor engagement. We also determined that the chemokine SDF-1 contributes to megakaryocyte (MK) proliferation. Although several other hematopoietic cytokines act in synergy with TPO to stimulate MK development, the most compelling reason for studying the TPO/SDF-1 interaction is that the two systems are members of distinct receptor classes, potentially shedding new insights on many complex signaling systems. The proposed work is designed to understand how the signals that emanate from these two ligand-receptor systems functionally interact and induce cellular proliferation and/or block cell death. Finally, having shown that TPO exerts favorable effects on hematopoietic stem cells, we will determine whether these effects are mediated by protection against programmed cell death or by stimulation of cellular proliferation. Like all of the aims, these studies will utilize primary cells for in vitro investation and determine their physiologic significance by confirmation in animal models. Together, the proposed work should advance our understanding of hematopoietic regulation, potentially providing new opportunities to rationally intervene to stimulate normal or suppress neoplastic blood cell production.
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