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The role of erythropoietin and erythropoietin receptor in regulation of hemopoiesis

University of British Columbia

To analyze the molecular mechanisms by which erythropoietin (Epo) can stimulate proliferation and differentiation of hemopoietic cells, I studied the effects of quantitative and qualitative alterations in the expression of erythropoietin receptors (EpoRs) in hemopoietic cell lines and in the primary bone marrow cells using retrovirus mediated gene transfer to engineer high level expresson of normal and mutant EpoRs in these cells, lnterleukin-3 (IL-3)- dependent murine bone marrow derived Ba/F3 cells engineered to express normal EpoR increased their levels of β-globin mRNA in response to Epo, and this partial differentiation correlated with a marked Epo-induced growth delay, indicating that the transduced EpoR was capable of inducing a distinct set of intracellular events. The tyrosine kinase inhibitor genistein blocked both Epoinduced β-globin mRNA accumulation and proliferation in this model system. In contrast, inhibition of protein kinase C by Compound 3 suppressed only Epo-induced differentiation without affecting proliferation, indicating that the proliferative and differentiation functions of the EpoR can be uncoupled. Mutant EpoRs lacking all intracellular tyrosines were compromised only in proliferative signaling, implying that tyrosine phosphorylation of the EpoR itself is not required for its differentiation function. With IL-3 and Epo costimulation, IL-3 signaling appeared to be dominant, since no increase in β-globin mRNA occurred. Chimeric EpoRs comprising the extracellular domain of the EpoR and the transmembrane and cytoplasmic region of IL-3-R-βIL-3 were capable of inducing β-globin mRNA accumulation, suggesting the existence of a second EpoR subunit responsible for differentiation or that the a subunit of the IL-3 R prevents it. Arguing against the former, a truncated EpoR lacking an intracellular domain possessed no biological activity. Chimeric EpoRs comprising the extracellular domain of the EpoR and the transmembrane and intracellular domains of the IL-3R a subunit were, however, capable of transmitting the Epo-induced mitogenic signal but failed to stimulate accumulation of β-globin mRNA. Moreover, coexpression of EpoR/IL-3Ra with EpoR/IL-3R β-IL-3 suppressed β-globin mRNA accumulation, which implicated an active role for the IL-3-Ra subunit in inhibiting EpoR-specific differentiating signals. Epo also exhibited a marked effect on proliferation of EpoR-transduced primary mouse bone marrow cells. Epo alone supported proliferation of EpoRtransduced CFU-GM and CFU-GEMM in semi-solid and suspension cultures, indicating that Epo was capable of replacing other cytokines normally required for the in vitro proliferation of non-erythroid and multipotent clonogenic progenitors. No Epo-induced proliferation of control cells could be detected in cultures containing high numbers of irradiated EpoR-transduced cells, indicating that Epo stimulated proliferation directly, through activation of the transduced EpoR, and arguing against the possibility of Epo-induced secretion of growth factor(s) within the population of the EpoR-transduced cells. To study effects of ectopic EpoR expression on proliferation of stem cells in vivo, EpoR- and neo-transduced bone marrow cells were transplanted into lethally irradiated mice. Recipients of the EpoR-transduced bone marrow developed within 6-14 weeks severe anemia, leukocytosis characterized by accumulation of undifferentiated blasts, and had significantly increased numbers of all clonogenic progenitor classes, consistent with development of myeloproliferative disease. Bone marrow and spleen cells recovered from the affected mice expressed high levels of surface EpoRs and proliferated in response to Epo, but not in the absence of growth factors, supporting a link between the Epo-induced deregulation in proliferation of the EpoR transduced stem cells and development of neoplasia. Together, the data presented in this thesis provide evidence that EpoRs may influence both proliferative and differentiative decisions of hemopoietic cells subject to their ability to interact with different signalling intermediates.

Thesis/Dissertation

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2009-04-01

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http://hdl.handle.net/2429/6706

Genetics

University of British Columbia

UBCV

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