Pum2 Protein Supports Maintenance and Suppresses Differentiation of Multipotent Hematopoietic Progenitors by Regulating the Function and Activation of c-kit Receptor.

Abstract Intrinsic mechanisms that regulate self-renewal of mammalian stem cells remain largely unknown. Stem cell maintenance and self-renewal in Drosophila and C. elegans are regulated by members of the conserved Pumilio family of RNA-binding proteins. We have previously described cloning and characterization of two mouse and human Pumilio genes (Pum1 and Pum2), which are abundantly transcribed in hematopoietic stem cells (HSC). To study the role of mammalian Pum proteins in HSC, Pum2 was over-expressed in a SCF-dependent multipotent progenitor cell line EML, which has the capacity for multilineage (erythroid, myeloid, B and T lymphoid) differentiation in vitro. In the presence of SCF EML cells undergo SCF-dependent self-renewal, thus remaining undifferentiated and retaining an immature phenotype. When cultured with hematopoietic cytokines (IL-3, GM-CSF, Epo, Tpo) EML cells differentiate into lineage-committed hematopoietic progenitors (e.g. granulocyte/macrophage (CFU-GM), burst-forming unit erythroid (BFU-E) and megakaryocytic (CFU-Meg) progenitors). Pum2 over-expression leads to uncoupling of the survival and differentiation signals in EML cells, and their SCF-independent maintenance. EML cells over-expressing Pum2 (Pum2-EML cells) also exhibit almost complete block of differentiation into multiple lineages in the absence of SCF. Moreover, although the culture with cytokine cocktail (IL-3, Epo, Tpo and GM-CSF) and retinoic acid enhances differentiation capacity of wild type EML cells, it was not sufficient to overcome the differentiation block in Pum2-EML cells. However, the repression of Pum2-EML cell differentiation is a reversible phenomenon, since the addition of SCF to Pum2-EML cell cultures, for at least 48 hours, restores their capacity to undergo multilineage differentiation and generate hematopoietic colonies. The SCF-independent maintenance of Pum2-EML cells seems to be caused by upregulated expression and constitutive activation of the SCF receptor c-kit, and is accompanied by constitutive activation of MAPK, PI3K and PLCγ signaling pathways in the absence of SCF. More importantly, Pum2-EML cells also exhibit upregulated expression and constitutive activation of a novel truncated form of c-kit receptor called tr-kit, which was found previously to be expressed preferentially in HSC. Tr-kit could play a critical role in the SCF-independent activation of the full-length c-kit receptor, leading to SCF-independent maintenance of Pum2-EML cells, and inhibition of their multilineage differentiation. The observation that Pum2-EML cells, maintained with or without SCF, are resistant to treatment with blocking anti-c-kit antibody (ACK2) and c-kit inhibitor STI-571, supports the notion that maintenance and survival of Pum2-EML cells in the presence of SCF is not due to an external activation of c-kit receptor through ligand binding. Taken together, these findings suggest a model in which survival and maintenance of multipotent hematopoietic progenitors are mediated through SCF-independent c-kit signaling, whereas their differentiation depends on the canonical SCF-induced c-kit signaling. In summary, mouse Pum2 protein could play an important role in supporting maintenance of HSC and multipotent progenitors through regulation of the SCF/c-kit signaling pathway, and could represent a part of the mechanism through which HSCs balance their self-renewal and commitment to differentiation.