Macrophage proliferation is regulated through CSF-1 receptor tyrosines 544, 559, and 807

Colony-stimulating factor-1 (CSF-1)-stimulated CSF-1 receptor (CSF-1R) tyrosine phosphorylation initiates survival, proliferation, and differentiation signaling pathways in macrophages. Either activation loop Y807F or juxtamembrane domain (JMD) Y559F mutations severely compromise CSF-1-regulated proliferation and differentiation. YEF, a CSF-1R in which all eight tyrosines phosphorylated in the activated receptor were mutated to phenylalanine, lacks in vitro kinase activity and in vivo CSF-1-regulated tyrosine phosphorylation. The addition of Tyr-807 alone to the YEF backbone (Y807AB) led to CSF-1-independent but receptor kinase-dependent proliferation, without detectable activation loop Tyr-807 phosphorylation. The addition of Tyr-559 alone (Y559AB) supported a low level of CSF-1-independent proliferation that was slightly enhanced by CSF-1, indicating that Tyr-559 has a positive Tyr-807-independent effect. Consistent with the postulated autoinhibitory role of the JMD Tyr-559 and its relief by ligand-induced Tyr-559 phosphorylation, the addition of Tyr-559 to the Y807AB background suppressed proliferation in the absence of CSF-1, but restored most of the CSF-1-stimulated proliferation. Full restoration of kinase activation and proliferation required the additional add back of JMD Tyr-544. Inhibitor experiments indicate that the constitutive proliferation of Y807AB macrophages is mediated by the phosphatidylinositol 3-kinase (PI3K) and ERK1/2 pathways, whereas proliferation of WT and Y559,807AB macrophages is, in addition, contributed to by Src family kinase (SFK)-dependent pathways. Thus Tyr-807 confers sufficient kinase activity for strong CSF-1-independent proliferation, whereas Tyr-559 maintains the receptor in an inactive state. Tyr-559 phosphorylation releases this restraint and may also contribute to the CSF-1-regulated proliferative response by activating Src family kinase.     

Fibronectin induces macrophage migration through a SFK-FAK/CSF-1R pathway

Integrins, following binding to proteins of the extracellular matrix (ECM) including collagen, laminin and fibronectin (FN), are able to transduce molecular signals inside the cells and to regulate several biological functions such as migration, proliferation and differentiation. Besides activation of adaptor molecules and kinases, integrins transactivate Receptor Tyrosine Kinases (RTK). In particular, adhesion to the ECM may promote RTK activation in the absence of growth factors. The Colony-Stimulating Factor-1 Receptor (CSF-1R) is a RTK that supports the survival, proliferation, and motility of monocytes/macrophages, which are essential components of innate immunity and cancer development. Macrophage interaction with FN is recognized as an important aspect of host defense and wound repair. The aim of the present study was to investigate on a possible cross-talk between FN-elicited signals and CSF-1R in macrophages. FN induced migration in BAC1.2F5 and J774 murine macrophage cell lines and in human primary macrophages. Adhesion to FN determined phosphorylation of the Focal Adhesion Kinase (FAK) and Src Family Kinases (SFK) and activation of the SFK/FAK complex, as witnessed by paxillin phosphorylation. SFK activity was necessary for FAK activation and macrophage migration. Moreover, FN-induced migration was dependent on FAK in either murine macrophage cell lines or human primary macrophages. FN also induced FAK-dependent/ligand-independent CSF-1R phosphorylation, as well as the interaction between CSF-1R and β1. CSF-1R activity was necessary for FN-induced macrophage migration. Indeed, genetic or pharmacological inhibition of CSF-1R prevented FN-induced macrophage migration. Our results identified a new SFK-FAK/CSF-1R signaling pathway that mediates FN-induced migration of macrophages.     

The Cbl protooncoprotein stimulates CSF-1 receptor multiubiquitination and endocytosis, and attenuates macrophage proliferation.

Colony-stimulating factor-1 (CSF-1) activation of the CSF-1 receptor (CSF-1R) causes Cbl protooncoprotein tyrosine phosphorylation, Cbl-CSF-1R association and their simultaneous multiubiquitination at the plasma membrane. The CSF-1R is then rapidly internalized and degraded, whereas Cbl is deubiquitinated in the cytoplasm without being degraded. We have used primary macrophages from gene-targeted mice to study the role of Cbl. Cbl-/- macrophages form denser colonies and, at limiting CSF-1 concentrations, proliferate faster than Cbl+/+ macrophages. Their CSF-1Rs fail to exhibit multiubiquitination and a second wave of tyrosine phosphorylation previously suggested to be involved in preparation of the CSF-1-CSF-1R complex for endocytosis. Consistent with this result, Cbl-/- macrophage cell surface CSF-1-CSF-1R complexes are internalized more slowly, yet are still lysosomally degraded, and the CSF-1 utilization by Cbl-/- macrophages is reduced approximately 2-fold. Thus, attenuation of proliferation by Cbl is associated with its positive regulation of the coordinated multiubiquitination and endocytosis of the activated CSF-1R, and a reduction in the time that the CSF-1R signals from the cell surface. The results provide a paradigm for studies of the mechanisms underlying Cbl attenuation of proliferative responses induced by ligation of receptor tyrosine kinases.     

ERK5/BMK1 is indispensable for optimal colony-stimulating factor 1 (CSF-1)-induced proliferation in macrophages in a Src-dependent fashion

CSF-1, by binding to its high-affinity receptor CSF-1R, sustains the survival and proliferation of monocyte/macrophages, which are central cells of innate immunity and inflammation. The MAPK ERK5 (also known as big MAPK-1, BMK1, or MAPK7) is a 98-kDa molecule sharing high homology with ERK1/2. ERK5 is activated by oxidative stress or growth factor stimulation. This study was undertaken to characterize ERK5 involvement in macrophage signaling that is elicited by CSF-1. Exposure to the CSF-1 of primary human macrophages or murine macrophage cell lines, as well as murine fibroblasts expressing ectopic CSF-1R, resulted in a rapid and sustained increase of ERK5 phosphorylation on activation-specific residues. In the BAC1.2F5 macrophage cell line, ERK5 was also activated by another mitogen, GM-CSF, while macrophage activators such as LPS or IFN-gamma and a number of nonproliferative cytokines failed. Src family kinases were found to link the activation of CSF-1R to that of ERK5, whereas protein kinase C or the serine phosphatases PP1 and PP2A seem not to be involved in the process. Treatment of macrophages with ERK5-specific small interfering RNA markedly reduced CSF-1-induced DNA synthesis and total c-Jun phosphorylation and expression, while increasing the expression of the cyclin-dependent kinase inhibitor p27. Following CSF-1 treatment, the active form of ERK5 rapidly translocated from cytosol to nucleus. Taken together, the results reported in this study show that ERK5 is indispensable for optimal CSF-1-induced proliferation and indicate a novel target for its control.     

The role of atypical protein kinase C in CSF-1-dependent Erk activation and proliferation in myeloid progenitors and macrophages

Colony stimulating factor-1 (CSF-1 or M-CSF) is the major physiological regulator of the proliferation, differentiation and survival of cells of the mononuclear phagocyte lineage. CSF-1 binds to a receptor tyrosine kinase, the CSF-1 receptor (CSF-1R). Multiple pathways are activated downstream of the CSF-1R; however, it is not clear which pathways regulate proliferation and survival. Here, we investigated the role of atypical protein kinase Cs (PKCζ) in a myeloid progenitor cell line that expressed CSF-1R (32D.R) and in primary murine bone marrow derived macrophages (BMMs). In 32D.R cells, CSF-1 induced the phosphorylation of PKCζ and increased its kinase activity. PKC inhibitors and transfections with mutant PKCs showed that optimal CSF-1-dependent Erk activation and proliferation depended on the activity of PKCζ. We previously reported that CSF-1 activated the Erk pathway through an A-Raf-dependent and an A-Raf independent pathway (Lee and States, Mol. Cell. Biol.18, 6779). PKC inhibitors did not affect CSF-1 induced Ras and A-Raf activity but markedly reduced MEK and Erk activity, implying that PKCζ regulated the CSF-1-Erk pathway at the level of MEK. PKCζ has been implicated in activating the NF-κB pathway. However, CSF-1 promoted proliferation in an NF-κB independent manner. We established stable 32D.R cell lines that overexpressed PKCζ. Overexpression of PKCζ increased the intensity and duration of CSF-1 induced Erk activity and rendered cells more responsive to CSF-1 mediated proliferation. In contrast to 32D.R cells, PKCζ inhibition in BMMs had only a modest effect on proliferation. Moreover, PKCζ -specific and pan-PKC inhibitors induced a paradoxical increase in MEK-Erk phosphorylation suggesting that PKCs targeted a common negative regulatory step upstream of MEK. Our results demonstrated that CSF-1 dependent Erk activation and proliferation are regulated differentially in progenitors and differentiated cells.     

IFN-gamma/lipopolysaccharide activation of macrophages is associated with protein kinase C-dependent down-modulation of the colony-stimulating factor-1 receptor.

IFN gamma/LPS treatment increases macrophage tumoricidal and microbicidal activity and inhibits CSF-1-induced macrophage proliferation. The mechanism underlying the latter effect was investigated in the CSF-1-dependent mouse macrophage cell line, BAC-1.2F5. IFN-gamma and LPS together dramatically reduced the total number of CSF-1 receptors (CSF-1R) via selective degradation of the cell surface form. Processing and transport of intracellular CSF-1R to the cell surface were unaffected. IFN-gamma alone had no effect but significantly enhanced LPS-induced CSF-1R down-regulation. The reduction in CSF-1R number was protein kinase C-dependent and involved changes in serine phosphorylation of the receptor at different sites. CSF-1R down-modulation by this mechanism may be important in switching off the energy-consuming processes of CSF-1R-mediated proliferation and chemotaxis in activated macrophages.     

Growth factors of lower vertebrates: characterization of goldfish (Carassius auratus L.) macrophage colony-stimulating factor-1

Colony-stimulating factor-1 (CSF-1) regulates mononuclear cell proliferation, differentiation, and survival. The functions of CSF-1 are well documented in mammals; however, little is known about CSF-1 biology in lower vertebrates. This is the first report on the identification and functional characterization of a fish CSF-1 molecule expressed highly in the spleen and in phorbol 12-myristate 13-acetate-stimulated monocytes. Goldfish CSF-1 is a 199-amino acid protein that possesses the required cysteine residues to form important intra-chain and inter-chain disulfide bonds that allow CSF-1 to form a functional homodimer and to interact with its high affinity receptor, CSF-1R. Recombinant goldfish CSF-1 formed a homodimer and bound to the soluble goldfish CSF-1R. The addition of the recombinant CSF-1 to sorted goldfish progenitor cells, monocytes, and macrophages induced the differentiation of monocytes into macrophages and the proliferation of monocyte-like cells. The proliferation of these cells was abrogated by addition of an anti-CSF-1R antibody as well as the soluble CSF-1R. The ability of the soluble CSF-1R to inhibit CSF-1-induced proliferation represents a novel mechanism for the regulation of CSF-1 function.     

A CSF-1 receptor phosphotyrosine 559 signaling pathway regulates receptor ubiquitination and tyrosine phosphorylation

Receptor tyrosine kinase (RTK) activation involves ligand-induced receptor dimerization and transphosphorylation on tyrosine residues. Colony-stimulating factor-1 (CSF-1)-induced CSF-1 receptor (CSF-1R) tyrosine phosphorylation and ubiquitination were studied in mouse macrophages. Phosphorylation of CSF-1R Tyr-559, required for the binding of Src family kinases (SFKs), was both necessary and sufficient for these responses and for c-Cbl tyrosine phosphorylation and all three responses were inhibited by SFK inhibitors. In c-Cbl-deficient macrophages, CSF-1R ubiquitination and tyrosine phosphorylation were substantially inhibited. Reconstitution with wild-type, but not ubiquitin ligase-defective C381A c-Cbl rescued these responses, while expression of C381A c-Cbl in wild-type macrophages suppressed them. Analysis of site-directed mutations in the CSF-1R further suggests that activated c-Cbl-mediated CSF-1R ubiquitination is required for a conformational change in the major kinase domain that allows amplification of receptor tyrosine phosphorylation and full receptor activation. Thus the results indicate that CSF-1-mediated receptor dimerization leads to a Tyr-559/SFK/c-Cbl pathway resulting in receptor ubiquitination that permits full receptor tyrosine phosphorylation of this class III RTK in macrophages.     

Biology and action of colony-stimulating factor-1

Colony-stimulating factor-1 (CSF-1), also known as macrophage colony-stimulating factor, controls the survival, proliferation, and differentiation of mono-nuclear phagocytes and regulates cells of the female reproductive tract. It appears to play an autocrine and/or paracrine role in cancers of the ovary, endometrium, breast, and myeloid and lymphoid tissues. Through alternative mRNA splicing and differential post-translational proteolytic processing, CSF-1 can either be secreted into the circulation as a glycoprotein or chondroitin sulfate-containing proteoglycan or be expressed as a membrane-spanning glycoprotein on the surface of CSF-1-producing cells. Studies with the op/op mouse, which possesses an inactivating mutation in the CSF-1 gene, have established the central role of CSF-1 in directly regulating osteoclastogenesis and macrophage production. CSF-1 appears to preferentially regulate the development of macrophages found in tissues undergoing active morphogenesis and/or tissue remodeling. These CSF-1 dependent macrophages may, via putative trophic and/or scavenger functions, regulate characteristics such as dermal thickness, male fertility, and neural processing. Apart from its expression on mononuclear phagocytes and their precursors, CSF-1 receptor (CSF-1R) expression on certain nonmononuclear phagocytic cells in the female reproductive tract and studies in the op/op mouse indicate that CSF-1 plays important roles in female reproduction. Restoration of circulating CSF-1 to op/op mice has preliminarily defined target cell populations that are regulated either humorally or locally by the synthesis of cell-surface CSF-1 or by sequestration of the CSF-1 proteoglycan. The CSF-1R is a tyrosine kinase encoded by the c-fms proto-oncogene product. Studies by several groups have used cells expressing either the murine or human CSF-1R in fibroblasts to pinpoint the requirement of kinase activity and the importance of various receptor tyrosine phosphorylation sites for signaling pathways stimulated by CSF-1. To investigate post-CSF-1R signaling in the macrophage, proteins that are rapidly phosphorylated on tyrosine in response to CSF-1 have been identified, together with proteins associated with them. Studies on several of these proteins, including protein tyrosine phosphatase 1C, the c-cbl proto-oncogene product, and protein tyrosine phosphatase-phi are discussed. Mol Reprod Dev 46:4–10, 1997. © 1997 Wiley-Liss, Inc.     

Development and characterization of monoclonal antibodies to murine macrophage colony-stimulating factor

The macrophage-specific CSF (CSF-1), purified from murine L cell-conditioned medium, supports the in vitro proliferation and survival of various murine mononuclear phagocyte colony-forming cells. In this report we describe the production and functional characterization of two monoclonal antibodies (mAb) to CSF-1 obtained from rat X rat hybridomas. These two mAb are functionally distinct and recognize different epitopes on CSF-1. The mAb 5A1 binds to and inhibits the biologic function of CSF-1, and the second mAb (D24) binds CSF-1 but does not neutralize its biologic activity. The mAb 5A1 inhibits colony formation of tissue mononuclear phagocyte colony-forming cells as well as the committed bone marrow stem cells for both granulocytes and monocytes. The extent of colony inhibition by mAb 5A1 is dependent on the tissue origin of colony-forming cells. CSF-1 complexed with mAb 5A1 does not bind to its cell surface receptor of peritoneal exudate macrophages, and mAb 5A1 does not complex with cell-bound CSF-1. Although both bone marrow cell-derived macrophages and J774.1 macrophages bind CSF-1, mAb 5A1 inhibits the proliferation of only bone marrow cell-derived macrophages. The non-neutralizing mAb D24 does not block binding of CSF-1 to its cellular receptor, and it recognizes cell-bound CSF-1.     

The colony-stimulating factor-1 (CSF-1) receptor sustains ERK1/2 activation and proliferation in breast cancer cell lines

Breast cancer is the second leading cause of cancer-related deaths in western countries. Colony-Stimulating Factor-1 (CSF-1) and its receptor (CSF-1R) regulate macrophage and osteoclast production, trophoblast implantation and mammary gland development. The expression of CSF-1R and/or CSF-1 strongly correlates with poor prognosis in several human epithelial tumors, including breast carcinomas. We demonstrate that CSF-1 and CSF-1R are expressed, although at different levels, in 16/17 breast cancer cell lines tested with no differences among molecular subtypes. The role of CSF-1/CSF-1R in the proliferation of breast cancer cells was then studied in MDAMB468 and SKBR3 cells belonging to different subtypes. CSF-1 administration induced ERK1/2 phosphorylation and enhanced cell proliferation in both cell lines. Furthermore, the inhibition of CSF-1/CSF-1R signaling, by CSF-1R siRNA or imatinib treatment, impaired CSF-1 induced ERK1/2 activation and cell proliferation. We also demonstrate that c-Jun, cyclin D1 and c-Myc, known for their involvement in cell proliferation, are downstream CSF-1R in breast cancer cells. The presence of a proliferative CSF-1/CSF-1R autocrine loop involving ERK1/2 was also found. The wide expression of the CSF-1/CSF-1R pair across breast cancer cell subtypes supports CSF-1/CSF-1R targeting in breast cancer therapy.     

Gab2 promotes colony-stimulating factor 1-regulated macrophage expansion via alternate effectors at different stages of development

Colony-stimulating factor 1 (CSF-1) receptor (CSF-1R, or macrophage CSF receptor [M-CSFR]) is the primary regulator of the proliferation, survival, and differentiation of mononuclear phagocytes (MNPs), but the critical CSF-1 signals for these functions are unclear. The scaffold protein Gab2 is a major tyrosyl phosphoprotein in the CSF-1R signaling network. Here we demonstrate that Gab2 deficiency results in profoundly defective expansion of CSF-1R-dependent MNP progenitors in the bone marrow, through decreased proliferation and survival. Reconstitution and phospho-flow studies show that downstream of CSF-1R, Gab2 uses phosphatidylinositol 3-kinase (PI3K)-Akt and extracellular signal-regulated kinase (Erk) to regulate MNP progenitor expansion. Unexpectedly, Gab2 ablation enhances Jun N-terminal protein kinase 1 (JNK1) phosphorylation in differentiated MNPs but reduces their proliferation; inhibition of JNK signaling or reduction of JNK1 levels restores proliferation. MNP recruitment to inflammatory sites and the corresponding bone marrow response is strongly impaired in Gab2-deficient mice. Our data provide genetic and biochemical evidence that CSF-1R, through Gab2, utilizes different effectors at different stages of MNP development to promote their expansion.     

cAMP inhibits CSF-1-stimulated tyrosine phosphorylation but augments CSF-1R-mediated macrophage differentiation and ERK activation

Macrophage colony stimulating factor (M-CSF) or CSF-1 controls the development of the macrophage lineage through its receptor tyrosine kinase, c-Fms. cAMP has been shown to influence proliferation and differentiation in many cell types, including macrophages. In addition, modulation of cellular ERK activity often occurs when cAMP levels are raised. We have shown previously that agents that increase cellular cAMP inhibited CSF-1-dependent proliferation in murine bone marrow-derived macrophages (BMM) which was associated with an enhanced extracellular signal-regulated kinase (ERK) activity. We report here that increasing cAMP levels, by addition of either 8-bromo cAMP (8BrcAMP) or prostaglandin E(1) (PGE1), can induce macrophage differentiation in M1 myeloid cells engineered to express the CSF-1 receptor (M1/WT cells) and can potentiate CSF-1-induced differentiation in the same cells. The enhanced CSF-1-dependent differentiation induced by raising cAMP levels correlated with enhanced ERK activity. Thus, elevated cAMP can promote either CSF-1-induced differentiation or inhibit CSF-1-induced proliferation depending on the cellular context. The mitogen-activated protein kinase/extracellular signal-related protein kinase kinase (MEK) inhibitor, PD98059, inhibited both the cAMP- and the CSF-1R-dependent macrophage differentiation of M1/WT cells suggesting that ERK activity might be important for differentiation in the M1/WT cells. Surprisingly, addition of 8BrcAMP or PGE1 to either CSF-1-treated M1/WT or BMM cells suppressed the CSF-1R-dependent tyrosine phosphorylation of cellular substrates, including that of the CSF-1R itself. It appears that there are at least two CSF-1-dependent pathway(s), one MEK/ERK dependent pathway and another controlling the bulk of the tyrosine phosphorylation, and that cAMP can modulate signalling through both of these pathways.     

Identification of tyrosine-phosphorylated colony-stimulating factor 1 (CSF-1) receptor and a 56-kilodalton protein phosphorylated in intact human cells in response to CSF-1

Colony-stimulating factor 1 (CSF-1) selectively supports the survival, proliferation, and maturation of hemopoietic cells of the monocyte/macrophage lineage. Although the cellular receptor for CSF-1, (the c-fms protein) is a protein-tyrosine kinase activated by the binding of CFS-1, the role of phosphorylation of cellular proteins in CSF-1 signal transduction is poorly understood. Therefore, we examined the CSF-1-stimulated phosphorylation of cellular proteins in human BeWo choriocarcinoma cell line (known to express the c-fms protein). BeWo cells were metabolically labeled with 32Pi, stimulated with recombinant human CSF-1, and extracted with detergent. Phosphotyrosyl proteins were isolated from detergent extracts by affinity chromatography on a highly specific antibody to phosphotyrosine. Rapid phosphorylation of 170-kd protein, followed closely by the phosphorylation of a 56-kd protein, was observed in response to CSF-1. The 170-kd phosphotyrosyl protein bound to wheat germ agglutinin and was secondarily immunoprecipitated with a specific anti-fms serum, consistent with its identity as the CSF-1 receptor. Although purified human macrophages that proliferate in culture in response to CSF-1 are not generally accessible, CSF-1 did stimulate the phosphorylation of a 56-kd protein in intact mononuclear leukocytes from human peripheral blood. Thus, the BeWo cell line may represent a good model for the study of CSF-1-stimulated cellular protein phosphorylation.     

Colony-stimulating factor-1 receptor protein expression is a specific marker for goldfish (Carassius auratus L.) macrophage progenitors and their differentiated cell types

Signaling through the colony-stimulating factor-1 receptor (CSF-1R) mediates the proliferation, differentiation, and activation of macrophages and their progenitors. In this study we report on the use of an anti-goldfish CSF-1R antibody to specifically recognize a population of CSF-1R positive cells from goldfish tissues. Furthermore, using our previously characterized primary kidney macrophage culture system, we show that CSF-1R positive cells include monocytes, macrophages, and their progenitor cells. Freshly isolated progenitor cells had a higher median florescent intensity ratio than those progenitor cells cultured for up to four days. The decrease in CSF-1R expression on the progenitor cells coincides with the appearance and development of monocytes and macrophages. Monocytes were consistently CSF-1R⁺ and maintained the high level of CSF-1R expression as they developed into macrophages. Like that of mammalian systems, CSF-1R is expressed on all macrophage sub-populations (progenitors, monocytes, macrophages), and CSF-1R expression increases with macrophage development in teleosts.     

SHP-1 regulation of p62(DOK) tyrosine phosphorylation in macrophages

SHP-1 plays key roles in the modulation of hematopoietic cell signaling. To ascertain the impact of SHP-1 on colony-stimulating factor-1 (CSF-1)-mediated survival and proliferative signaling, we compared the CSF-1 responses of primary bone marrow macrophages (BMM) from wild-type and SHP-1-deficient motheaten (me/me) mice. CSF-1-induced protein tyrosine phosphorylation levels were similar in wild-type and me/me BMM, except for the constitutive hyperphosphorylation of a 62-kDa phosphoprotein (pp62) in me/me macrophages. pp62 was identified as the RASGAP-associated p62(DOK) and was shown to be the major CSF-1R-associated tyrosine-phosphorylated protein in CSF-1-treated BMM. p62(DOK) was found to be constitutively associated with SHP-1 in BMM and in 293T cells, co-expressing p62(dok) and either wild-type or catalytically inert SHP-1 (SHP-1 C453S). In both cell types, the interaction of SHP-1 with p62(DOK) occurred independently of p62(DOK) tyrosine phosphorylation, but only the tyrosine-phosphorylated p62(DOK) was bound by SHP-1 C453S in a far Western analysis. These findings suggest a constitutive association of SHP-1 and p62(DOK) that is either conformation-dependent or indirect as well as a direct, inducible association of the SHP-1 catalytic domain with tyrosine-phosphorylated p62(DOK). p62(DOK) hyperphosphorylation is not associated with altered CSF-1-induced RAS signaling or proliferation. However, whereas wild-type macrophages undergo cell death following CSF-1 removal, me/me macrophages exhibit prolonged survival in the absence of growth factor. Thus, p62(DOK) is a major SHP-1 substrate whose tyrosine phosphorylation correlates with growth factor-independent survival in macrophages.     

Both granulocyte-macrophage CSF and macrophage CSF control the proliferation and survival of the same subset of alveolar macrophages

The effect of granulocyte-macrophage (GM)-CSF on the proliferation of murine pulmonary alveolar macrophages in vitro was investigated. About 20% of freshly isolated alveolar macrophages formed colonies in both liquid and soft agar cultures in the presence of GM-CSF. GM-CSF was also found to be capable of maintaining the survival of these colony-forming cells in vitro. Moreover, GM-CSF could substitute for CSF-1 in maintaining the survival of CSF-1-responding pulmonary alveolar macrophage colony-forming cells in the absence of CSF-1. The concentration of GM-CSF required for maintaining the survival of colony-forming cells without proliferation was much lower than that required for the proliferation of these cells in vitro. It also enhanced the CSF-1-dependent clonal growth of alveolar macrophages. These data suggest that the colony-forming cells that respond to GM-CSF are the same subset of macrophages that form colonies in the presence of CSF-1. GM-CSF did not inhibit the binding of 125I-CSF-1 to alveolar macrophages at 0 degrees C. However, the preincubation of macrophages with GM-CSF at 37 degrees C resulted in a transient down-regulation of CSF-1 binding activity.     

Functional specificity of cytoplasmic and transmembrane tyrosine kinases: identification of 130- and 75-kilodalton substrates of c-fps/fes tyrosine kinase in macrophages.

c-fps/fes encodes a 92-kDa protein-tyrosine kinase (NCP92) that is expressed at the highest levels in macrophages. To determine if c-fps/fes can mediate the action of the colony-stimulating factor 1 (CSF-1) receptor (CSF-1R) and to identify potential targets of c-fps/fes in macrophages, we have overexpressed c-fps/fes in a CSF-1-dependent macrophage cell line. A 30- to 50-fold overexpression of c-fps/fes partially released these cells from their factor dependence by a nonautocrine mechanism, and this correlated with the tyrosine phosphorylation of two proteins of 130 and 75 kDa (P130 and P75). c-fps/fes did not cause tyrosine phosphorylation or activation of CSF-1 dependent targets, including CSF-1R, Shc, and phosphatidylinositol 3-kinase, and conversely, CSF-1 did not induce tyrosine phosphorylation of P130 and P75. P75 appears to be a novel phosphotyrosyl protein, whereas P130 cross-reacts with a known substrate of v-src. P130 and P75 may be direct substrates of c-fps/fes: P130 was tightly associated with NCP92, and the src homology 2 domain of NCP92 specifically bound phosphorylated P130 and P75 but not the CSF-1-induced phosphotyrosyl proteins, consistent with the possibility that P130 and P75 are physiological targets of c-fps/fes. We conclude that although c-fps/fes can functionally substitute for CSF-1R to a certain extent, these tyrosine kinases act largely independently of each other and that P130 and P75 are novel targets whose mechanisms of action may be unrelated to the signalling pathways utilized by receptor tyrosine kinases.