Direct Comparison of G-CSF Receptor and M-CSF Receptor Signaling Leading to AP-1 and STAT Induction and Identification of Novel Signaling Pathways.

External signals often influence developmental decisions. Whether hematopoietic cytokines contribute to lineage specification or serve to amplify decisions made by an intrinsic transcription factor program remains unresolved. Although much is known about signaling events emanating from the G-CSF Receptor (GR) and the M-CSF Receptor (MR), these signals have not been directly compared in a uniform cellular environment. Reasoning that differences seen could contribute to myeloid lineage choice in bipotent cells such as the granulocyte monocyte progenitor (GMP) or the CFU-GM, we have compared GR and MR signaling via endogenous receptors present in the U937 human leukemia cell line and using a subclone derived from the IL-3 dependent Ba/F3 stem cell line expressing both exogenous GR and MR, introduced by retroviral transduction. FACS analysis confirmed that the large majority of cells in our Ba/F3 line express both MR and GR at similarly high levels. Ba/F3 cells were IL-3 starved for 1hr (which caused no change in signaling for the pathways tested) prior to G-CSF, M-CSF stimulation, or mock addition. Both receptors reproducibly activated JNK and induced c-Jun phosphorylation, and both GR and MR signals activated ERK increasing total c-Fos levels, likely via c-Fos serine phosphorylation. Although only the cytoplasmic domain of MR has been demonstrated to interact with PLCγ, two different PLC-inhibitors (U-73122, a PLCγ-specific inhibitor, and edelfosine, a general PLC inhibitor) reduced ERK phosphorylation and c-Fos expression at 30 min downstream of both G-CSF and M-CSF stimulation. A connection between PLCγ and the ERK pathway had not been previously described within these signaling pathways. Two different ERK inhibitors (U0126 and PD9899) reduced c-Fos expression, confirming a link between ERK and c-Fos in hematopoietic cells. PLC and ERK inhibition not only reduced Fos induction, but also increased JNK activation and c-Jun phosphorylation at 30 min, identifying a novel connection between the ERK and JNK branches of the MAPK pathways. Conversely, JNK inhibition with SP600125 reduced c-Jun phosphorylation without altering ERK activation or c-Fos expression. The balance between c-Jun and c-Fos activation via the signaling pathways we have characterized may contribute to granulocyte versus monocyte lineage specification, as exogenous c-Jun directs monocyte development in cooperation with PU.1 and C/EBPα. For example, perhaps increased c-Fos diverts c-Jun to facilitate granulopoiesis. We did not detect differences in the effects of GR or MR on C/EBPα or PU.1 levels, DNA-binding, or C/EBPα serine 21 phosphorylation in U937 or Ba/F3 cells. On the other hand, GR but not MR signals potently induced STAT1, STAT3, and STAT5 tyrosine phosphorylation and DNA-binding at 30 min in Ba/F3 cells, of potential relevance to both proliferation and granulocytic differentiation induced by G-CSF. We now intend to further delineate G-CSF versus M-CSF activation of the JNK/c-Jun, PLCγ/ERK/c-Fos, and STAT pathways and their effects on granulocyte versus monocyte differentiation, using lineage-negative murine myeloid progenitors.