The GM-CSF receptor utilizes ß-catenin and Tcf4 to specify macrophage lineage differentiation.

Granulocyte-macrophage colony stimulating factor (GM-CSF) promotes the growth, survival, differentiation and activation of normal myeloid cells and is essential for fully functional macrophage differentiation in vivo. To better understand the mechanisms by which growth factors control the balance between proliferation and self-renewal versus growth-suppression and differentiation we have used the bi-potent FDB1 myeloid cell line, which proliferates in IL-3 and differentiates to granulocytes and macrophages in response to GM-CSF. This provides a manipulable model in which to dissect the switch between growth and differentiation. We show that, in the context of signaling from an activating mutant of the GM-CSF receptor ß subunit, a single intracellular tyrosine residue (Y577) mediates the granulocyte fate decision. Loss of granulocyte differentiation in a Y577F second-site mutant is accompanied by enhanced macrophage differentiation and accumulation of ß-catenin together with activation of Tcf4 and other Wnt target genes. These include the known macrophage lineage inducer, Egr1. We show that forced expression of Tcf4 or a stabilised ß-catenin mutant is sufficient to promote macrophage differentiation in response to GM-CSF and that GM-CSF can regulate ß-catenin stability, most likely via GSK3ß. Consistent with this pathway being active in primary cells we show that inhibition of GSK3ß activity promotes the formation of macrophage colonies at the expense of granulocyte colonies in response to GM-CSF. This study therefore identifies a novel pathway through which growth factor receptor signaling can interact with transcriptional regulators to influence lineage choice during myeloid differentiation.

Alternative modes of GM-CSF receptor activation revealed using activated mutants of the common beta-subunit.

Granulocyte/macrophage colony-stimulating factor promotes growth, survival, differentiation, and activation of normal myeloid cells and plays an important role in myeloid leukemias. The GM-CSF receptor (GMR) shares a signaling subunit, beta(c), with interleukin-3 and interleukin-5 receptors and has recently been shown to induce activation of Janus kinase 2 (JAK2) and downstream signaling via formation of a unique dodecameric receptor complex. In this study we use 2 activated beta(c) mutants that display distinct signaling capacity and have differential requirements for the GMR alpha-subunit (GMR-alpha) to dissect the signaling pathways associated with the GM-CSF response. The V449E transmembrane mutant selectively activates JAK2/signal transducer and activator of transcription 5 and extracellular signal-regulated kinase (ERK) pathways, resulting in a high level of sensitivity to JAK and ERK inhibitors, whereas the extracellular mutant (FIDelta) selectively activates the phosphoinositide 3-kinase/Akt and IkappaKbeta/nuclear factorkappaB pathways. We also demonstrate a novel and direct interaction between the SH3 domains of Lyn and Src with a conserved proline-rich motif in GMR-alpha and show a selective requirement for Src family kinases by the FIDelta mutant. We relate the nonoverlapping nature of signaling by the activated mutants to the structure of the unique GMR complex and propose alternative modes of receptor activation acting synergistically in the mature liganded receptor complex.

Genetic regulators of myelopoiesis and leukemic signaling identified by gene profiling and linear modeling.

Mechanisms controlling the balance between proliferation and self-renewal versus growth suppression and differentiation during normal and leukemic myelopoiesis are not understood. We have used the bi-potent FDB1 myeloid cell line model, which is responsive to myelopoietic cytokines and activated mutants of the granulocyte macrophage-colony stimulating factor (GM-CSF) receptor, having differential signaling and leukemogenic activity. This model is suited to large-scale gene-profiling, and we have used a factorial time-course design to generate a substantial and powerful data set. Linear modeling was used to identify gene-expression changes associated with continued proliferation, differentiation, or leukemic receptor signaling. We focused on the changing transcription factor profile, defined a set of novel genes with potential to regulate myeloid growth and differentiation, and demonstrated that the FDB1 cell line model is responsive to forced expression of oncogenes identified in this study. We also identified gene-expression changes associated specifically with the leukemic GM-CSF receptor mutant, V449E. Signaling from this receptor mutant down-regulates CCAAT/enhancer-binding protein alpha (C/EBPalpha) target genes and generates changes characteristic of a specific acute myeloid leukemia signature, defined previously by gene-expression profiling and associated with C/EBPalpha mutations.