Colony-stimulating factor-1(CSF-1) and its receptor, CSF-1R, regulate myelopoiesis, bone remodeling and maturation of the reproductive system, through control of cell growth and apoptosis. CSF-1 transduced signals are targets for therapeutic intervention in bone marrow recovery from chemotherapy and osteoporosis. For these reasons it is important to have a clear understanding of CSF-1-mediated signaling pathways. The experiments described here aim to elucidate the mechanisms CSF-1 uses to activate the phosphatidyl-inositol 3-kinase (PI3K)/Akt survival pathway and characterize their downstream their downstream targets in myelomonocytic cells. They will use both the 32D myeloid cell line and primary murine bone marrow macrophages (BMMs). They have identified two mechanisms the CSF-1R uses to activate PI3K/Akt. The first has been described previously and depends on direct CSF-1R:PI3K association. The second is novel, revealed by their studies of a 32D cell line expressing a CSF-1R deletion mutant (KI) that cannot bind PI3K yet able to stimulate Akt and p70s6 kinase. Evidence is provided implicating both Src kinases and Gab2. The two mechanisms are not redundant in that both are necessary for optimal PI3K activation by CSF-1. Moreover, cells expressing KI-CSF-1R apoptose in the presence of CSF-1 and the survival defect is rescued by expression of a constitutively active PI3K (p110*).
In Aim 1, the will utilize CSF-1R Src binding mutants and Csk-overexpressing cell lines to test the hypothesis that Src kinase mediate the alternate PI3K activation pathway.
In Aim 2, they will utilize Gab2 PI3K binding mutants to test the hypothesize that Gab2-mediated interactions enhance CSF-1 dependent PI3K activation.
In Aim 3, they will use their panel of cell lines expressing CSF-1R, KI-CSF-1R or KI with p100* to define the PI3K/Akt apoptotic targets. They will establish KI cell lines overexpressing constitutively active Akt to define the activation thresholds for different downstream effectors. Primary BMMs will be used to validate key findings from Aims 1-3.
