DESCRIPTION (provided by applicant): Pumilio proteins are evolutionary highly conserved family of RNA-binding proteins that function as translational repressers during embryo development and cell fate specification. Based on their known functions in invertebrates and lower vertebrates it was proposed that a primordial function of Pumilio (Pum) proteins is to sustain proliferation and maintenance of stem cells. We have shown previously that mouse Pum1 and Pum2 genes are transcribed preferentially in hematopoietic stem cells (HSC). To start analyzing their role during blood cell development we have over-expressed the RNA-binding domain of Pum2 in a stem cell factor (SCF)-dependent multipotent hematopoietic progenitor cell line EML, which can differentiate into erythroid, myeloid and lymphoid cell lineages in vitro. The over-expression of Pum2-RBD leads to SCF-independent maintenance of EML cells, and is suppressing their mutilineage differentiation in the absence of SCF. This uncoupling of the survival 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, and (b) cell intrinsic, SCF-independent activation of the c-kit, and its downstream MAPK, PI3K and PLCy signaling pathways. The observations that the expression of tr-kit is restricted to HSC and multipotent progenitors, and that an increased expression of tr-kit protein is associated with SCF- independent maintenance of EML cells, suggest a potentially important role for tr-kit in the regulation of the balance between maintenance (self-renewal) and differentiation of HSC and multipotent progenitors. For example, an interaction of tr-kit with the full-length c-kit receptor could lead to SCF-independent c-kit activation, and could play an important role in the SCF-independent maintenance of EML cells, and suppression of their multilineage differentiation. Since the Pum2 and tr-kit proteins are expressed in bone marrow cells enriched for HSC and early multipotent progenitors, but not in later committed progenitors, we hypothesize that HSC and multipotent progenitors utilize distinct SCF-dependent and SCF-independent c-kit signaling pathways that could regulate their maintenance and differentiation. 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 progenitors is mediated through SCF-independent c-kit signaling, whereas their differentiation depends on the canonical SCF-induced c- kit signaling. In this project we want to test the hypothesis that tr-kit mediates SCF-independent c-kit signaling, and that the regulation of maintenance and differentiation of HSC and multipotent progenitors involves distinct SCF-dependent and SCF-independent c-kit signaling pathways. These studies could provide important insights into the molecular regulation of two critical elements of stem cell self-renewal, inhibition of differentiation and induction of proliferation, and could be relevant for the study of aging HSC and multipotent progenitors.
|Effective start/end date||7/1/06 → 6/30/09|
- National Institutes of Health: $182,732.00
- National Institutes of Health: $156,355.00