The Role of Truncated c-kit Receptor (tr-kit) in Maintenance and Differentiation of Hematopoietic Stem Cells and Multipotent Hematopoietic Progenitors.

Abstract

Intrinsic mechanisms that regulate self-renewal of mammalian stem cells are slowly being elucidated. Self-renewal of stem cells in Drosophila and C. elegans is regulated by members of the conserved Pumilio family of RNA-binding proteins. Previously, we have cloned and characterized two mouse and human Pumilio genes (Pum1 and Pum2), which are abundantly transcribed in hematopoietic stem cells (HSC). To study the function of mammalian Pum proteins in HSC and multipotent progenitors, the RNA-binding domain of Pum2 was over-expressed in a stem cell factor (SCF)-dependent HSC-like cell line EML. In the presence of SCF EML cells undergo SCF-dependent self-renewal and remain undifferentiated. In the presence of various cytokines (IL-3, GM-CSF, G-CSF, Epo, Tpo, IL-7, Flt3L) EML cells differentiate into erythroid, granulocytic, megakaryocytic and lymphoid cell lineages in vitro. The over-expression of Pum2-RBD leads to SCF-independent maintenance of EML cells, and suppresses their mutilineage differentiation in the absence of SCF. This uncoupling of the maintenance and differentiation signals in EML cells is accompanied by (a) an increased expression of the full-length c-kit and a novel truncated c-kit receptor called tr-kit, (b) cell intrinsic, SCF-independent activation of c-kit, and (c) constitutive activation of MAPK, PI3K and PLCγ signaling pathways in the absence of SCF. These results indicate that Pum2 could be supporting maintenance of multipotent hematopoietic cells through regulation of SCF/c-kit signaling pathway. An in depth analysis of the pattern of tr-kit expression in murine fetal liver and bone marrow-derived HSC, multipotent progenitors, lineage-committed progenitors and immature blood cells has shown that tr-kit expression is restricted to cell populations highly enriched for HSC and multipotent progenitors. This observation and the finding that an increased expression of tr-kit protein correlates with SCF-independent maintenance of EML cells, suggest that tr-kit could play an important role in SCF-independent activation of full-length c-kit receptor, and participate in the regulation of the balance between maintenance (self-renewal) and differentiation of HSC and multipotent progenitors. The fact that Pum2 and tr-kit are co-expressed in bone marrow cells enriched for HSC and early multipotent progenitors (e.g. Lin-Sca-1+c-kit+ cells), but not in later progenitors (e.g. Lin-Sca-1−c-kit− cells), suggests an exciting possibility that HSC and early multipotent progenitors utilize distinct SCF-dependent and SCF-independent c-kit signaling pathways. In contrast, more differentiated progenitors that lack self-renewal ability and do not express tr-kit, utilize only the canonical SCF-induced c-kit signaling. In this hypothetical model, the survival and maintenance of HSC and multipotent hematopoietic progenitors is mediated through SCF-independent c-kit signaling, whereas their differentiation depends on the canonical SCF-induced c-kit signaling. We are currently studying the effects of Pum2 and tr-kit over-expression and attenuation on (a) HSC and progenitor cell maintenance and differentiation, (b) HoxB4 and Notch1 pathways, involved in HSC maintenance and expansion, and (c) maintenance and differentiation of HSC expressing SLAM receptor CD150. Further study of Pum2 and tr-kit function could provide important new insights into the molecular regulation of two critical elements of self-renewal, inhibition of differentiation and induction of proliferation.