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Macrophage Colony-stimulating Factor Promotes Cell Survival through Akt/Protein Kinase B

1999; Elsevier BV; Volume: 274; Issue: 37 Linguagem: Inglês

10.1074/jbc.274.37.26393

ISSN

1083-351X

Autores

Todd W. Kelley, Mandy M. Graham, Andrea I. Doseff, Richard W. Pomerantz, Sey M. Lau, Michael C. Ostrowski, Thomas Franke, Clay B. Marsh,

Tópico(s)

Cytokine Signaling Pathways and Interactions

Resumo

The signaling pathways activated by the macrophage colony-stimulating factor (M-CSF) to promote survival of monocyte and macrophage lineage cells are not well established. In an effort to elucidate these pathways, we have used two cell types responsive to M-CSF: NIH 3T3 fibroblasts genetically engineered to express human M-CSF receptors (3T3-FMS cells) and human monocytes. M-CSF treatment induced M-CSF receptor tyrosine phosphorylation and recruitment of the p85 subunit of phosphatidylinositol 3-kinase (PI3K) to these receptors. These M-CSF receptor events correlated with activation of the serine/threonine kinase Akt. To clarify that PI3K products activate Akt in response to M-CSF, NIH 3T3 fibroblasts expressing mutant human M-CSF receptors (3T3-FMS(Y809F)) that fail to activate Ras in response to M-CSF also exhibit increased Akt kinase activity in response to M-CSF challenge. Furthermore, Akt appears to be the primary regulator of survival in 3T3-FMS cells, as transfection of genes encoding dominant-negative Akt isoforms into these fibroblasts blocked M-CSF-induced survival. In normal human monocytes, M-CSF increased the levels of tyrosine-phosphorylated proteins and induced Akt activation in a PI3K-dependent manner. The PI3K inhibitor LY294002 blocked M-CSF-mediated monocyte survival, an effect that was partially restored by caspase-9 inhibitors. These data suggest that M-CSF may induce cell survival through Akt-induced suppression of caspase-9 activation. The signaling pathways activated by the macrophage colony-stimulating factor (M-CSF) to promote survival of monocyte and macrophage lineage cells are not well established. In an effort to elucidate these pathways, we have used two cell types responsive to M-CSF: NIH 3T3 fibroblasts genetically engineered to express human M-CSF receptors (3T3-FMS cells) and human monocytes. M-CSF treatment induced M-CSF receptor tyrosine phosphorylation and recruitment of the p85 subunit of phosphatidylinositol 3-kinase (PI3K) to these receptors. These M-CSF receptor events correlated with activation of the serine/threonine kinase Akt. To clarify that PI3K products activate Akt in response to M-CSF, NIH 3T3 fibroblasts expressing mutant human M-CSF receptors (3T3-FMS(Y809F)) that fail to activate Ras in response to M-CSF also exhibit increased Akt kinase activity in response to M-CSF challenge. Furthermore, Akt appears to be the primary regulator of survival in 3T3-FMS cells, as transfection of genes encoding dominant-negative Akt isoforms into these fibroblasts blocked M-CSF-induced survival. In normal human monocytes, M-CSF increased the levels of tyrosine-phosphorylated proteins and induced Akt activation in a PI3K-dependent manner. The PI3K inhibitor LY294002 blocked M-CSF-mediated monocyte survival, an effect that was partially restored by caspase-9 inhibitors. These data suggest that M-CSF may induce cell survival through Akt-induced suppression of caspase-9 activation. macrophage colony-stimulating factor phosphatidylinositol 3-kinase Dulbecco's modified Eagle's medium fluoromethyl ketone 7-amino-trifluoromethyl coumarin polyacrylamide gel electrophoresis 4-morpholinepropanesulfonic acid 1,4-piperazinediethanesulfonic acid Monocytes are produced in bone marrow and normally circulate in the bloodstream for 24–48 h (1Cline M.J. Lehrer R.I. Territo C. Golde D.W. Ann. Intern. Med. 1978; 88: 78-88Crossref PubMed Scopus (83) Google Scholar). In the absence of growth factors, circulating monocytes die of apoptosis (2Mangan D.F. Wahl S.M. J. Immunol. 1991; 147: 3408-3412PubMed Google Scholar, 3Mangan D.F. Welch G.R. Wahl S.M. J. Immunol. 1991; 146: 1541-1546PubMed Google Scholar, 4Marsh C.B. Pomerantz R.J. Parker J.M. Winnard A.V. Mazzaferri E.L.J. Moldovan N. Kelley T.W. Beck E. Wewers M.D. J. Immunol. 1999; 162: 6217-6225PubMed Google Scholar). The growth factor M-CSF1 appears to be important in monocyte survival (5Praloran V. Nouv. Rev. Fr. Hematol. 1991; 33: 323-333PubMed Google Scholar), as mice lacking M-CSF suffer deficiencies in circulating monocytes and macrophages (6Wiktor-Jedrzejczak W. Urbanowska E. Aukerman S.L. Pollard J.W. Stanley E.R. Ralph P. Ansari A.A. Sell K.W. Szperl M. Exp. Hematol. (N. Y.). 1991; 19: 1049-1054PubMed Google Scholar), which are restored by injecting exogenous M-CSF (6Wiktor-Jedrzejczak W. Urbanowska E. Aukerman S.L. Pollard J.W. Stanley E.R. Ralph P. Ansari A.A. Sell K.W. Szperl M. Exp. Hematol. (N. Y.). 1991; 19: 1049-1054PubMed Google Scholar). The phenotype of M-CSF-deficient animals illustrates the role of this growth factor in a variety of human diseases, including osteopetrosis (6Wiktor-Jedrzejczak W. Urbanowska E. Aukerman S.L. Pollard J.W. Stanley E.R. Ralph P. Ansari A.A. Sell K.W. Szperl M. Exp. Hematol. (N. Y.). 1991; 19: 1049-1054PubMed Google Scholar), coronary artery disease (7Smith J.D. Trogan E. Ginsberg M. Grigaux C. Tian J. Miyata M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8264-8268Crossref PubMed Scopus (570) Google Scholar), transplant vascular sclerosis (8Shi C.W. Lee W.S. He Q. Zhang D. Fletcher Jr., D.L. Newell J.B. Haber E. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 4051-4056Crossref PubMed Scopus (188) Google Scholar), and cancer (9Nowicki A. Szenajch J. Ostrowska G. Wojtowicz A. Wojtowicz K. Kruszewski A.A. Maruszynski M. Aukerman S.L. Wiktor-Jedrzejczak W. Int. J. Cancer. 1996; 65: 112-119Crossref PubMed Scopus (109) Google Scholar). M-CSF also appears to be important in the survival of human monocytes (4Marsh C.B. Pomerantz R.J. Parker J.M. Winnard A.V. Mazzaferri E.L.J. Moldovan N. Kelley T.W. Beck E. Wewers M.D. J. Immunol. 1999; 162: 6217-6225PubMed Google Scholar, 5Praloran V. Nouv. Rev. Fr. Hematol. 1991; 33: 323-333PubMed Google Scholar,10Stanley E.R. Berg K.L. Einstein D.B. Lee P.S. Pixley F.J. Wang Y. Yeung Y.G. Mol. Reprod. Dev. 1997; 46: 4-10Crossref PubMed Scopus (344) Google Scholar), although the specific intracellular mediators induced by M-CSF to promote cell survival are not well understood.M-CSF is a growth factor that has its highest biological activity as a disulfide-linked dimer (10Stanley E.R. Berg K.L. Einstein D.B. Lee P.S. Pixley F.J. Wang Y. Yeung Y.G. Mol. Reprod. Dev. 1997; 46: 4-10Crossref PubMed Scopus (344) Google Scholar, 11Hamilton J.A. J. Leukocyte Biol. 1997; 62: 145-155Crossref PubMed Scopus (165) Google Scholar). M-CSF dimers cross-link two tyrosine kinase M-CSF receptors (also known as FMS or colony-stimulating factor-1 receptors), inducing auto- and transphosphorylation of tyrosine residues in the cytoplasmic domains of these receptors (11Hamilton J.A. J. Leukocyte Biol. 1997; 62: 145-155Crossref PubMed Scopus (165) Google Scholar). Phosphotyrosine residues in the cytoplasmic domains of M-CSF receptors induce translocation of intracellular signaling molecules via SH2 interactions and activate signaling cascades, resulting in the activation of both PI3K-dependent and Ras/mitogen-activated protein kinase-dependent pathways (12Yeung Y.G. Wang Y. Einstein D.B. Lee P.S. Stanley E.R. J. Biol. Chem. 1998; 273: 17128-17137Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). Similar to normal human monocytes, NIH 3T3 fibroblasts transfected with the gene encoding the human M-CSF receptor (FMS gene) also activate these intracellular signaling pathways in response to human M-CSF (13Roussel M.F. Sherr C.J. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 7924-7927Crossref PubMed Scopus (45) Google Scholar). In contrast, NIH 3T3 fibroblasts expressing mutant forms of human M-CSF receptors containing a phenylalanine residue in place of the tyrosine residue at position 809 (3T3-FMS(Y809F)) do not activate Ras or undergo mitogenesis in response to human M-CSF (14Roussel M.F. J. Cell Sci. Suppl. 1994; 18: 105-108Crossref PubMed Google Scholar, 15Roussel M.F. Downing J.R. Rettenmier C.W. Sherr C.J. Cell. 1988; 55: 979-988Abstract Full Text PDF PubMed Scopus (172) Google Scholar).M-CSF receptors are structurally related to platelet-derived growth factor receptors (16Heldin C.H. Cell. 1995; 80: 213-223Abstract Full Text PDF PubMed Scopus (1427) Google Scholar, 17Roberts W.M. Look A.T. Roussel M.F. Sherr C.J. Cell. 1988; 55: 655-661Abstract Full Text PDF PubMed Scopus (116) Google Scholar). To promote cellular survival, platelet-derived growth factor has been shown to recruit PI3K to the platelet-derived growth factor receptor and to activate the serine/threonine kinase Akt (also known as protein kinase B) (18Franke T.F. Yang S.I. Chan T.O. Datta K. Kazlauskas A. Morrison D.K. Kaplan D.R. Tsichlis P.N. Cell. 1995; 81: 727-736Abstract Full Text PDF PubMed Scopus (1820) Google Scholar, 19Simm A. Hoppe V. Gazit A. Hoppe J. J. Cell. Physiol. 1994; 160: 295-302Crossref PubMed Scopus (14) Google Scholar). Similarly, PI3K and Akt are activated in the insulin receptor activation pathway (20Alessi D.R. Downes C.P. Biochim. Biophys. Acta. 1998; 1436: 151-164Crossref PubMed Scopus (191) Google Scholar), and chimeric receptors constructed of the extracellular domains of M-CSF receptors and the cytoplasmic domains of insulin receptors activate Akt in response to M-CSF (21Dandekar T. Snel B. Huynen M. Bork P. Trends Biochem. Sci. 1998; 23: 324-328Abstract Full Text Full Text PDF PubMed Scopus (817) Google Scholar). PI3K appears to activate Akt by generating 3-phosphorylated second messengers, including phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate (22Franke T.F. Kaplan D.R. Cantley L.C. Toker A. Science. 1997; 275: 665-668Crossref PubMed Scopus (1293) Google Scholar, 23Stephens L. Anderson K. Stokoe D. Erdjument-Bromage H. Painter G.F. Holmes A.B. Gaffney P.R. Reese C.B. McCormick F. Tempst P. Coadwell J. Hawkins P.T. Science. 1998; 279: 710-714Crossref PubMed Scopus (910) Google Scholar, 24Stokoe D. Stephens L.R. Copeland T. Gaffney P.R. Reese C.B. Painter G.F. Holmes A.B. McCormick F. Hawkins P.T. Science. 1997; 277: 567-570Crossref PubMed Scopus (1045) Google Scholar). Recent data suggest that phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate are likely important in Akt activation (21Dandekar T. Snel B. Huynen M. Bork P. Trends Biochem. Sci. 1998; 23: 324-328Abstract Full Text Full Text PDF PubMed Scopus (817) Google Scholar, 22Franke T.F. Kaplan D.R. Cantley L.C. Toker A. Science. 1997; 275: 665-668Crossref PubMed Scopus (1293) Google Scholar), and the inositol 5-phosphatase SHIP and the inositol 3-phosphatase PTEN both suppress Akt activity (25Aman M.J. Lamkin T.D. Okada H. Kurosaki T. Ravichandran K.S. J. Biol. Chem. 1998; 273: 33922-33928Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar, 26Dahia P.L. Aguiar R.C. Alberta J. Kum J.B. Caron S. Sill H. Marsh D.J. Ritz J. Freedman A. Stiles C. Eng C. Hum. Mol. Genet. 1999; 8: 185-193Crossref PubMed Scopus (266) Google Scholar, 27Zundel W. Giaccia A. Genes Dev. 1998; 12: 1941-1946Crossref PubMed Scopus (199) Google Scholar). Membrane-bound phosphatidylinositol 3,4,5-trisphosphate and phosphatidylinositol 3,4-bisphosphate target Akt via interactions with the pleckstrin homology domain in Akt, leading to the stimulation of its serine and threonine kinase activity (28Bellacosa A. Chan T.O. Ahmed N.N. Datta K. Malstrom S. Stokoe D. McCormick F. Feng J. Tsichlis P. Oncogene. 1998; 17: 313-325Crossref PubMed Scopus (455) Google Scholar, 29Testa J.R. Bellacosa A. Leuk. Res. 1997; 21: 1027-1031Crossref PubMed Scopus (25) Google Scholar, 30Andjelkovic M. Alessi D.R. Meier R. Fernandez A. Lamb N.J. Frech M. Cron P. Cohen P. Lucocq J.M. Hemmings B.A. J. Biol. Chem. 1997; 272: 31515-31524Abstract Full Text Full Text PDF PubMed Scopus (895) Google Scholar). Activated Akt can then disengage from the membrane and interact with intracellular signaling elements to promote cellular survival (31Datta S.R. Dudek H. Tao X. Masters S. Fu H. Gotoh Y. Greenberg M.E. Cell. 1997; 91: 231-241Abstract Full Text Full Text PDF PubMed Scopus (4915) Google Scholar, 32Cardone M.H. Roy N. Stennicke H.R. Salvesen G.S. Franke T.F. Stanbridge E. Frisch S. Reed J.C. Science. 1998; 282: 1318-1321Crossref PubMed Scopus (2719) Google Scholar). Akt promotes cellular survival by suppressing the activity of pro-apoptotic proteins and by suppressing the activity of the Forkhead DNA transcription factors. In monocytes, activation of the executioner caspase-3 appears to regulate survival (33Fahy R.J. Doseff A.I. Wewers M.D. J. Immunol. 1999; (in press)PubMed Google Scholar), and recently, Akt has been shown to phosphorylate and inactivate caspase-9, thereby blocking caspase-3 activation (30Andjelkovic M. Alessi D.R. Meier R. Fernandez A. Lamb N.J. Frech M. Cron P. Cohen P. Lucocq J.M. Hemmings B.A. J. Biol. Chem. 1997; 272: 31515-31524Abstract Full Text Full Text PDF PubMed Scopus (895) Google Scholar).There are data to suggest that Akt may be important in monocyte survival, as mice deficient in either SHIP or PTEN suffer expansion of tissue macrophage populations (34Helgason C.D. Damen J.E. Rosten P. Grewal R. Sorensen P. Chappel S.M. Borowski A. Jirik F. Krystal G. Humphries R.K. Genes Dev. 1998; 12: 1610-1620Crossref PubMed Scopus (484) Google Scholar). As previously outlined, both of these phosphatases normally function to suppress Akt activation (25Aman M.J. Lamkin T.D. Okada H. Kurosaki T. Ravichandran K.S. J. Biol. Chem. 1998; 273: 33922-33928Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar,35Wu X.Y. Senechal K. Neshat M.S. Whang Y.E. Sawyers C.L. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 15587-15591Crossref PubMed Scopus (593) Google Scholar). Because circulating monocytes do not proliferate and are precursors to tissue macrophages, these data suggest that Akt may also be important in monocyte survival. In light of these findings, this study was directed at identifying the signaling pathway activated by M-CSF to promote cell survival and at determining the role of PI3K and Akt in this signaling pathway.We find that M-CSF induces tyrosine phosphorylation of human M-CSF receptors and recruitment of the p85 subunit of PI3K to these receptors in 3T3-FMS fibroblasts. Moreover, in these cells, M-CSF induces Akt kinase activity in a PI3K-dependent manner. Akt also appears to be central to M-CSF-induced survival in these fibroblasts, as dominant-negative Akt constructs block M-CSF-induced fibroblast survival. We found a similar M-CSF-induced signaling pathway in normal human monocytes, as M-CSF leads to Akt activation in these cells in a PI3K-dependent manner. In contrast, 3T3-FMS(Y809F) fibroblasts that do not activate Ras in response to M-CSF (14Roussel M.F. J. Cell Sci. Suppl. 1994; 18: 105-108Crossref PubMed Google Scholar) display Akt kinase activity when stimulated with M-CSF. Moreover, PI3K inhibitors block M-CSF-induced Akt activation and survival in normal human monocytes. We found that monocytes express caspase-9, and consistent with an important role for Akt in monocyte survival, caspase-9 inhibitors reverse apoptosis induced by PI3K inhibitors in M-CSF-treated monocytes. These data suggest that PI3K and Akt likely play an important role in M-CSF-induced monocyte survival.DISCUSSIONThis study begins to identify the signaling pathway activated by M-CSF to induce cell survival. Initially using 3T3 fibroblasts that express human M-CSF receptors (3T3-FMS), we have shown that human M-CSF induces tyrosine phosphorylation in the M-CSF receptor, recruiting the p85 subunit of PI3K. This M-CSF-activated pathway results in transient but significant increases in Akt kinase activity, which are not dependent on Ras activation. Akt activation appears to be critical for cell survival in response to M-CSF, as transient transfection of 3T3-FMS cells with the gene encoding a dominant-negative isoform of Akt significantly reduces cell survival as compared with cells transfected with vector alone or wild-type Akt genes when M-CSF is used as the only growth factor. We extended these observations to M-CSF-induced survival in normal human monocytes, a more clinically relevant model. We found that treatment of monocytes with M-CSF results in similarly transient but significant increases in Akt activity that are blocked by pretreatment of cells with the PI3K inhibitor LY294002. Consistent with an important role for PI3K and Akt kinase activity in monocyte survival, the PI3K inhibitor LY294002 also inhibits the ability of M-CSF to protect cells from undergoing apoptosis in a dose-dependent manner. We found that monocytes express caspase-9 and that caspase-9 activity correlates with monocyte apoptosis in response to growth factor deprivation or to PI3K inhibitors in M-CSF-treated cells. These data are consistent with the hypothesis that M-CSF activates monocyte Akt kinase activity, which, in turn, suppresses caspase-9 cleavage and apoptosis. More targeted studies of the effect of dominant-negative or overexpressed wild-type forms of Akt on monocyte survival were, unfortunately, not possible due to the difficulty of transfecting monocytes.Akt is a recognized mediator of cell survival in response to growth factor signaling (38Downward J. Curr. Opin. Cell Biol. 1998; 10: 262-267Crossref PubMed Scopus (1180) Google Scholar, 39Kennedy S.G. Wagner A.J. Conzen S.D. Jordan J. Bellacosa A. Tsichlis P.N. Hay N. Genes Dev. 1997; 11: 701-713Crossref PubMed Scopus (977) Google Scholar). Although the specific mechanism by which Akt promotes cell survival is still unclear, several important mechanisms have been described. First, Akt phosphorylates the serine residue at position 136 in the pro-apoptotic protein BAD (31Datta S.R. Dudek H. Tao X. Masters S. Fu H. Gotoh Y. Greenberg M.E. Cell. 1997; 91: 231-241Abstract Full Text Full Text PDF PubMed Scopus (4915) Google Scholar). This, in turn, causes BAD to be scavenged and neutralized by the cytosolic protein 14-3-3 (31Datta S.R. Dudek H. Tao X. Masters S. Fu H. Gotoh Y. Greenberg M.E. Cell. 1997; 91: 231-241Abstract Full Text Full Text PDF PubMed Scopus (4915) Google Scholar). In the unphosphorylated state, BAD heterodimerizes with the anti-apoptotic protein Bcl-2, leading to cytochrome c release from the mitochondria, which promotes apoptosis (40Gil-Gómez G. Berns A. Brady H.J.M. EMBO J. 1998; 17: 7209-7218Crossref PubMed Scopus (173) Google Scholar, 41Reed J.C. Oncogene. 1998; 17: 3225-3236Crossref PubMed Scopus (936) Google Scholar). This mechanism appears to be functional in modulating cell survival in c-Kit-expressing cells (42Blume-Jensen P. Janknecht R. Hunter T. Curr. Biol. 1998; 8: 779-782Abstract Full Text Full Text PDF PubMed Google Scholar). The second proposed mechanism by which Akt promotes cell survival is through its ability to phosphorylate caspase-9, part of the executioner caspase cascade. Phosphorylation renders caspase-9 unable to be cleaved by the apoptosis-promoting activating factor-1 complex (32Cardone M.H. Roy N. Stennicke H.R. Salvesen G.S. Franke T.F. Stanbridge E. Frisch S. Reed J.C. Science. 1998; 282: 1318-1321Crossref PubMed Scopus (2719) Google Scholar). It was initially thought that maintenance of caspase-9 in its precursor form suppressed the ability of caspase-9 to activate caspase-3, a protease important in the genesis of the apoptotic program (43Faleiro L. Kobayashi R. Fearnhead H. Lazebnik Y. EMBO J. 1997; 16: 2271-2281Crossref PubMed Scopus (339) Google Scholar, 44Thornberry N.A. Lazebnik Y. Science. 1998; 281: 1312-1316Crossref PubMed Scopus (6133) Google Scholar). Recent studies demonstrate that caspase-9 may maintain biological activity in the precursor form (45Stennicke H.R. Deveraux Q.L. Humke E.W. Reed J.C. Dixit V.M. Salvesen G.S. J. Biol. Chem. 1999; 274: 8359-8362Abstract Full Text Full Text PDF PubMed Scopus (415) Google Scholar), although phosphorylation of caspase-9 appears to suppress its activation in vitro (32Cardone M.H. Roy N. Stennicke H.R. Salvesen G.S. Franke T.F. Stanbridge E. Frisch S. Reed J.C. Science. 1998; 282: 1318-1321Crossref PubMed Scopus (2719) Google Scholar). Our data suggest that activated Akt likely phosphorylates caspase-9, suppressing its activity to promote monocyte survival.In addition to suppressing these pro-apoptotic proteins, Akt has also been recently shown to phosphorylate and suppress the activity of FKHRL1, a member of the Forkhead family of transcription factors (46Brunet A. Bonni A. Zigmond M.J. Lin M.Z. Juo P. Hu L.S. Anderson M.J. Arden K.C. Blenis J. Greenberg M.E. Cell. 1999; 96: 857-868Abstract Full Text Full Text PDF PubMed Scopus (5367) Google Scholar). Once dephosphorylated, FKHRL1 can translocate to the nucleus and activate the production of pro-apoptotic proteins to help trigger programmed cell death (46Brunet A. Bonni A. Zigmond M.J. Lin M.Z. Juo P. Hu L.S. Anderson M.J. Arden K.C. Blenis J. Greenberg M.E. Cell. 1999; 96: 857-868Abstract Full Text Full Text PDF PubMed Scopus (5367) Google Scholar). These data suggest that Akt may promote cellular survival through mechanisms in addition to its effects on BAD and caspase-9. Our study adds to these observations to show for the first time that Akt is activated by M-CSF and that the Akt kinase pathway is potentially important in normal human monocytes, presumably by suppressing caspase-9 activation. We also demonstrate that PI3K and Akt are important mediators of survival induced by M-CSF.This study suggests that PI3K and Akt mediate monocyte survival in response to M-CSF. Findings in mutant and transgenic animals support an important role for PI3K and Akt in the monocyte survival pathway. Transgenic animals deficient in the inositol 3-phosphatase PTEN or the inositol 5-phosphatase SHIP suffer macrophage accumulation and organ dysfunction (34Helgason C.D. Damen J.E. Rosten P. Grewal R. Sorensen P. Chappel S.M. Borowski A. Jirik F. Krystal G. Humphries R.K. Genes Dev. 1998; 12: 1610-1620Crossref PubMed Scopus (484) Google Scholar, 47Podsypanina K. Ellenson L.H. Nemes A. Gu J. Tamura M. Yamada K.M. Cordon-Cardo C. Catoretti G. Fisher P.E. Parsons R. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 1563-1568Crossref PubMed Scopus (828) Google Scholar). Interestingly, both PTEN and SHIP can suppress Akt kinase activity (23Stephens L. Anderson K. Stokoe D. Erdjument-Bromage H. Painter G.F. Holmes A.B. Gaffney P.R. Reese C.B. McCormick F. Tempst P. Coadwell J. Hawkins P.T. Science. 1998; 279: 710-714Crossref PubMed Scopus (910) Google Scholar, 24Stokoe D. Stephens L.R. Copeland T. Gaffney P.R. Reese C.B. Painter G.F. Holmes A.B. McCormick F. Hawkins P.T. Science. 1997; 277: 567-570Crossref PubMed Scopus (1045) Google Scholar, 25Aman M.J. Lamkin T.D. Okada H. Kurosaki T. Ravichandran K.S. J. Biol. Chem. 1998; 273: 33922-33928Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar, 48Jacob A. Cooney D. Trindandapani S. Kelley T. Coggeshall K.M. J. Biol. Chem. 1999; 274: 13704-13710Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). Based on our findings that M-CSF induces Akt kinase activity and that Akt is required for 3T3-FMS fibroblast survival in response to M-CSF as the lone growth factor, we speculate that Akt may also play an important role in monocyte survival. Consistent with this, we found evidence for an important role for Akt in monocyte survival via M-CSF. PI3K inhibitors block M-CSF-induced monocyte survival and Akt activation, which ultimately results in caspase-9-dependent apoptosis.In summary, this study shows that M-CSF induces Akt kinase activity in a PI3K-dependent manner in NIH 3T3 fibroblasts expressing human M-CSF receptors and in normal human monocytes. M-CSF-induced recruitment of PI3K to the M-CSF receptor and subsequent PI3K activity are critical in mediating the protective effects of M-CSF on monocyte survival. Transfection studies also demonstrated that Akt is a critical determinant of survival in response to M-CSF in these M-CSF receptor-bearing fibroblasts. Together, these data suggest that PI3K and Akt are important regulators of survival in cell types responsive to M-CSF, at least in part, by suppressing caspase-9 activation. Given these data, these enzymes may represent potential targets for therapies aimed at limiting inappropriate monocyte survival in inflammatory human diseases. Monocytes are produced in bone marrow and normally circulate in the bloodstream for 24–48 h (1Cline M.J. Lehrer R.I. Territo C. Golde D.W. Ann. Intern. Med. 1978; 88: 78-88Crossref PubMed Scopus (83) Google Scholar). In the absence of growth factors, circulating monocytes die of apoptosis (2Mangan D.F. Wahl S.M. J. Immunol. 1991; 147: 3408-3412PubMed Google Scholar, 3Mangan D.F. Welch G.R. Wahl S.M. J. Immunol. 1991; 146: 1541-1546PubMed Google Scholar, 4Marsh C.B. Pomerantz R.J. Parker J.M. Winnard A.V. Mazzaferri E.L.J. Moldovan N. Kelley T.W. Beck E. Wewers M.D. J. Immunol. 1999; 162: 6217-6225PubMed Google Scholar). The growth factor M-CSF1 appears to be important in monocyte survival (5Praloran V. Nouv. Rev. Fr. Hematol. 1991; 33: 323-333PubMed Google Scholar), as mice lacking M-CSF suffer deficiencies in circulating monocytes and macrophages (6Wiktor-Jedrzejczak W. Urbanowska E. Aukerman S.L. Pollard J.W. Stanley E.R. Ralph P. Ansari A.A. Sell K.W. Szperl M. Exp. Hematol. (N. Y.). 1991; 19: 1049-1054PubMed Google Scholar), which are restored by injecting exogenous M-CSF (6Wiktor-Jedrzejczak W. Urbanowska E. Aukerman S.L. Pollard J.W. Stanley E.R. Ralph P. Ansari A.A. Sell K.W. Szperl M. Exp. Hematol. (N. Y.). 1991; 19: 1049-1054PubMed Google Scholar). The phenotype of M-CSF-deficient animals illustrates the role of this growth factor in a variety of human diseases, including osteopetrosis (6Wiktor-Jedrzejczak W. Urbanowska E. Aukerman S.L. Pollard J.W. Stanley E.R. Ralph P. Ansari A.A. Sell K.W. Szperl M. Exp. Hematol. (N. Y.). 1991; 19: 1049-1054PubMed Google Scholar), coronary artery disease (7Smith J.D. Trogan E. Ginsberg M. Grigaux C. Tian J. Miyata M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8264-8268Crossref PubMed Scopus (570) Google Scholar), transplant vascular sclerosis (8Shi C.W. Lee W.S. He Q. Zhang D. Fletcher Jr., D.L. Newell J.B. Haber E. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 4051-4056Crossref PubMed Scopus (188) Google Scholar), and cancer (9Nowicki A. Szenajch J. Ostrowska G. Wojtowicz A. Wojtowicz K. Kruszewski A.A. Maruszynski M. Aukerman S.L. Wiktor-Jedrzejczak W. Int. J. Cancer. 1996; 65: 112-119Crossref PubMed Scopus (109) Google Scholar). M-CSF also appears to be important in the survival of human monocytes (4Marsh C.B. Pomerantz R.J. Parker J.M. Winnard A.V. Mazzaferri E.L.J. Moldovan N. Kelley T.W. Beck E. Wewers M.D. J. Immunol. 1999; 162: 6217-6225PubMed Google Scholar, 5Praloran V. Nouv. Rev. Fr. Hematol. 1991; 33: 323-333PubMed Google Scholar,10Stanley E.R. Berg K.L. Einstein D.B. Lee P.S. Pixley F.J. Wang Y. Yeung Y.G. Mol. Reprod. Dev. 1997; 46: 4-10Crossref PubMed Scopus (344) Google Scholar), although the specific intracellular mediators induced by M-CSF to promote cell survival are not well understood. M-CSF is a growth factor that has its highest biological activity as a disulfide-linked dimer (10Stanley E.R. Berg K.L. Einstein D.B. Lee P.S. Pixley F.J. Wang Y. Yeung Y.G. Mol. Reprod. Dev. 1997; 46: 4-10Crossref PubMed Scopus (344) Google Scholar, 11Hamilton J.A. J. Leukocyte Biol. 1997; 62: 145-155Crossref PubMed Scopus (165) Google Scholar). M-CSF dimers cross-link two tyrosine kinase M-CSF receptors (also known as FMS or colony-stimulating factor-1 receptors), inducing auto- and transphosphorylation of tyrosine residues in the cytoplasmic domains of these receptors (11Hamilton J.A. J. Leukocyte Biol. 1997; 62: 145-155Crossref PubMed Scopus (165) Google Scholar). 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