Role of IL-3 receptor alpha isoforms and microRNAs in IL-3-driven myeloid differentiation

Abstract

Blood cell formation involves proliferation of hematopoietic stem cells coupled with progressive maturation and lineage commitment. There is a critical requirement to maintain stem cell numbers by self-renewal. The mechanisms which control both differentiation and the balance between self-renewal and differentiation are poorly understood. The focus of this thesis is on myeloid differentiation. Four myeloid differentiation pathways have been recognized: (1) steady state myeloid differentiation, which maintains the normal levels of mature myeloid cells, (2) cytokine-driven or inducible myelopoiesis which generates additional myeloid effector cells during infections or allergic responses, (3) All-trans retinoic acid (ATRA)-induced differentiation of promyelocytic leukemia cells and (4) CD44-driven differentiation of AML blasts. The aim of the thesis work was to gain a better understanding of IL-3-driven myeloid differentiation. In Chapter 3, investigations of the hIL-3R system in the promyelocytic leukemia line NB4 are presented. NB4 is not only blocked in steady-state granulocytic differentiation but also in cytokine-induced differentiation driven by IL-3 or GM-CSF. The IL-3R system was present at low levels in NB4 but was otherwise normal. Elevation of hIL-3R levels by ectopic expression restored IL-3-induced differentiation, but differentiation was only partial indicating that the PML-RAR{u03B1} protein also interferes with the induction of IL-3-driven differentiation. SiRNA knockdown of hp{u03B2} did not affect ATRA-driven differentiation. In Chapter 4, studies relating to the CD44-induced differentiation pathway are presented. Although it is known that CD44 is a potent inducer of differentiation of promyelocytic leukemia cells, siRNA knockdown of CD44 did not affect ATRA-induced differentiation of NB4 cells and also did not affect IL-3-induced differentiation. In Chapter 5, the development of a new in vitro model for studying the mechanisms of mIL-3-induced myeloid differentiation is presented which utilizes the conditionally immortalized GM progenitor SCF ER-Hoxb8. The data indicate that IL-3-induced differentiation results in significant upregulation of miR-223 and the transcription factors c/EBP{u03B5} and C/EBP{u03B2}. Additionally, the data show an important role for signaling via the IL-3R{u03B1} SP2 isoform in the differentiation of this immortalized GM progenitor. Collectively, the thesis results indicate that the mechanisms involved in the induction of differentiation via the IL-3-driven, steady state, ATRA-induced and CD44-induced pathways are separate but they likely invoke a common granulocytic differentiation pathway which involves upregulation of C/EBP{u03B5}, C/EBP{u03B2} and miR-223. The new IL-3 differentiation model provides the foundation for future studies which could lead to miRNA directed strategies for controlling allergic inflammation and a better understanding of the pathogenesis of AML.