Both src-dependent and -independent mechanisms mediate phosphatidylinositol 3-kinase regulation of colony-stimulating factor 1-activated mitogen-activated protein kinases in myeloid progenitors.

Colony-stimulating factor-1 (CSF-1) is a homodimeric growth factor secreted by numerous cell types including fibroblasts and bone marrow stroma. It promotes the proliferation, survival, and differentiation of cells of the monocyte/macrophage lineage and their bone marrow progenitors (reviewed in reference 72). The cell surface receptor for CSF-1, the CSF-1R, is normally expressed in monocytes/macrophages, osteoclasts, and trophoblasts and abnormally in a significant number of human breast cancers and other cancers of the female reproductive system (38). The CSF-1R is a receptor tyrosine kinase (RTK) of the platelet-derived growth factor (PDGF) receptor family that also includes c-Kit and the Flt3/Flk2 receptor (reviewed in references 34, 45, and 73). The importance of CSF-1 in vivo is revealed by the functional defects of the naturally occurring osteopetrotic (op/op) mouse, which makes a truncated CSF-1 protein devoid of biological activity (93). These mice exhibit many developmental and functional abnormalities, implying potentially essential roles for CSF-1 in human diseases such as osteopetrosis (94), atherogenesis (77), and tumorigenesis (63). Such abnormalities are the consequence of the severe deficiency observed in certain macrophage populations, including reductions in hematopoietic stem cells and progenitors (94). The latter finding indicates that CSF-1 acts on early precursors as well as on the more mature monocytes and macrophages. CSF-1 binding activates the kinase function of the CSF-1R, leading to receptor autophosphorylation and rapid stimulation of tyrosine phosphorylation on a variety of intracellular signaling molecules. Several tyrosine autophosphorylation sites have been mapped in the CSF-1R, including Tyr 697, Tyr 706, and Tyr 721 in the so-called kinase insert (KI) region that divides the catalytic domain, Tyr 807 in the activation loop of the catalytic domain, and Tyr 559 in the juxtamembrane region. Autophosphorylation converts some of these tyrosines to binding sites for proteins containing Src homology 2 (SH2) domains. Tyr 697 is the binding site for the adapter molecule Grb2 (86), which is constitutively associated with the Ras guanine nucleotide exchange factor (Sos). Tyr 721 binds the p85 regulatory subunit of phosphatidylinositol 3-kinase (PI3-kinase) (68), thus bringing PI3-kinase into proximity with its substrates in the plasma membrane. Tyr 706 is required for STAT1 activation (62), while phosphorylated Tyr 559 allows Src family kinases (SFKs) to bind (2). In addition to molecules that associate directly with the activated CSF-1R, several other proteins also become tyrosine phosphorylated upon CSF-1 binding, including the adapter Shc (55), c-Cbl, which is likely to be involved in the multiubiquitination and downregulation of CSF-1R (50), Ship, a 5′-phosphatase for phosphatidylinositol 3,4,5-trisphosphate (PI-3,4,5-P3) and inositol 1,3,4,5-tetrakisphosphate (56), Gab2, a multidomain docking/scaffolding protein (29), and the tyrosine phosphatases SHP-1 in macrophages (10) and SHP-2 in myeloid progenitors (8). The role of these molecules in CSF-1-mediated signaling has not been determined. Although it has never been directly demonstrated, Grb2 binding to autophosphorylated Tyr 697 is presumed to be the initiating event in the CSF-1-mediated activation of the Ras/ERK (extracellular signal-regulated kinase) pathway (34). However, CSF-1 also induces tyrosine-phosphorylated Shc to associate with Grb2 (55), SHP-2 can serve as an adapter linking activated PDGF and epidermal growth factor (EGF) receptors to the Grb2/Sos complex (4, 54), and overexpression of Gab2 enhances cytokine-stimulated ERK activation (29, 61, 99). There are clearly other potential mechanisms for coupling activated CSF-1R to the Ras/ERK pathway in addition to that mediated by direct docking of Grb2 to the receptor. The well-established paradigm linking Grb2/Sos to MEK/ERK is primarily based on work in fibroblasts and COS cells. In this model, Grb2/Sos is recruited to the plasma membrane, where Sos stimulates the exchange of GTP for GDP on membrane-bound Ras, leading to sequential activation of Raf-1, MEK, and ERK. Depending on cell type and stimulus, variations on this theme include activation of MEK/ERK by Ras-independent mechanisms (7) and utilization of B-Raf instead of Raf-1 (91). Although growth factors can activate A-Raf (79), a physiological role for A-Raf in mediating ERK activation remains to be clarified. Activity of the ERK pathway is also modulated by cross-talk with other intracellular signaling pathways. Many studies have documented the involvement of PI3-kinase in regulating stimulus-induced ERK activation (13, 31, 43, 79), but reports to the contrary also abound (9, 64, 74). Such apparently contradictory findings are likely to reflect, in large part, the different cell types and stimuli used in the various studies. Moreover, the point at which PI3-kinase feeds into the Ras/mitogen-activated protein kinase (MAPK) cascade also varies with different reports, and PI3-kinase has been placed both upstream and downstream of Ras. Two studies have highlighted the importance of signal strength as a determinant of PI3-kinase involvement (21, 92). Thus, under conditions of low receptor occupancy by PDGF in fibroblasts or EGF in COS-7 cells, inhibition of PI3-kinase activity leads to almost complete inhibition of growth factor-induced Ras and ERK activation. As PI3-kinase is not activated by growth factors under these conditions, the suggestion has been made that basal, not growth factor-stimulated, PI3-kinase activity is required for Ras activation (92). At intermediate growth factor concentrations, ERK but not Ras activation is sensitive to PI3-kinase inhibition; at high growth factor concentrations, PI3-kinase activity is not required at all, implying the utilization of redundant pathways for activating Ras/MAPK. These studies have helped to resolve some of the confusion in the literature; however, the generality of these findings and precisely how PI3-kinase modulates growth factor-stimulated ERK activity are still not entirely clear. In some systems, novel (75, 83) and atypical (5, 75) protein kinase C (PKC) isoforms have been implicated as the link between PI3-kinase and the ERK pathway. We have previously shown that CSF-1-stimulated ERK activity is required for optimal protection of CSF-1R-expressing myeloid progenitor cells from apoptosis induced by mitogen deprivation (46). Similarly, expression of a constitutively activated ERK kinase (MEK) in 3T3 fibroblasts was found to restore mitogenicity to a mitogenically defective CSF-1R mutant (11). These results implicate the ERK pathway in positive signaling by the CSF-1R. In contrast, using estrogen-dependent breast cancer cells transfected with the CSF-1R as a model system for studying CSF-1's role in human breast cancer, we found that CSF-1 treatment led to growth arrest as a consequence of ERK-dependent induction of the cyclin-dependent kinase inhibitor p21Waf1 (49). Such disparate behavior illustrates the now well-documented concept that cellular context plays an important role in determining outcome of ERK activation (reviewed in reference 53). A clue to the underlying basis for such differential biological responses might come from a detailed understanding of the pathways emanating from the activated receptor that converge on MEK/ERK in different cell systems. In this report, we have analyzed the contribution of PI3-kinase and Src signaling pathways to CSF-1-mediated ERK activation in the 32D myeloid progenitor cell line transfected with wild-type (WT) or mutant CSF-1R. This cell line lacks endogenous CSF-1R but expresses many of the signaling molecules known to play an important role in cytokine-mediated signaling in hematopoietic cells, such as c-Cbl (50), Ship (56), and Gab2 (29). PI3-kinase activity is found to be required for maximal CSF-1-induced ERK activation; unexpectedly, CSF-1 can stimulate PI3-kinase independent of direct receptor binding. The PI3-kinase requirement occurred at concentrations that optimally activate Akt/PKB, a downstream target of PI3-kinase, indicating that in myeloid cells, CSF-1R activation of PI3-kinase constitutes an integral component of the ERK pathway. We demonstrate that while Src kinase activity played a minimal role in CSF-1-dependent PI3-kinase and ERK activation in cells expressing a significant number of WT CSF-1R molecules, contribution from the Src pathway was more prominent in cells expressing low receptor numbers and was indispensable in cells expressing a mutant CSF-1R lacking the KI. These results imply that CSF-1 can recruit redundant pathways to induce PI3-kinase and ERK activity.