CSF-1 and TPA stimulate independent pathways leading to lysosomal degradation or regulated intramembrane proteolysis of the CSF-1 receptor

The CSF-1 receptor is a protein-tyrosine kinase that has been shown to undergo regulated intramembrane proteolysis, or RIPping. Here, we have compared receptor downregulation and RIPping in response to CSF-1 and TPA. Our studies show that CSF-1 is a relatively poor inducer of RIPping and that CSF-1-induced receptor downregulation is largely independent of RIPping. TPA is a strong inducer of RIPping and TPA-induced receptor downregulation is mediated by RIPping. We further found that RIPping is dependent on TACE or a TACE-like protease, that CSF-1 and TPA use independent pathways to initiate RIPping, and that the intracellular domain is targeted for degradation through ubiquitination.     

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.     

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.     

Inhibition of the signaling pathways for macrophage proliferation by cyclic AMP. Lack of effect on early responses to colony stimulating factor-1.

Colony stimulating factor-1 (CSF-1) stimulates DNA synthesis in quiescent murine bone marrow-derived macrophages (BMM). CSF-1 action has been shown to involve activation of the CSF-1 receptor kinase. The protein kinase C activator, 12-O-tetradecanoylphorbol 13-acetate (PMA), is itself weakly mitogenic and synergises with CSF-1 for stimulation of BMM DNA synthesis suggesting a possible role for protein kinase C in the stimulation of BMM DNA synthesis. In this report we show that several agents which raise intracellular cAMP (8-bromoadenosine 3':5'-cyclic monophosphate, 3-isobutyl-1-methylxanthine, cholera toxin, and prostaglandin E2) reversibly inhibit DNA synthesis in BMM induced by CSF-1, granulocyte macrophage-colony stimulating factor, interleukin-3, and PMA. The suppressive action of cAMP elevation on the proliferative response to CSF-1 can be manifested even late in the G1 phase of the cell cycle. Several CSF-1-stimulated earlier responses, viz. protein synthesis, Na+/H+ exchange, Na+,K(+)-ATPase and c-myc-mRNA expression, were not inhibited thus showing a striking difference from some other cellular systems involving growth factor-mediated responses. c-fos-mRNA levels were raised and stabilized by the cAMP-elevating agents, and this modulation was not altered by CSF-1. Thus, the signaling pathways in the macrophages involving tyrosine kinase and protein kinase C activation are associated with increased proliferation while those involving elevation of cAMP (and presumably activation of cAMP-dependent protein kinases) appear to have an inhibitory effect.     

Development of a quantitative, high-throughput cell-based enzyme-linked immunosorbent assay for detection of colony-stimulating factor-1 receptor tyrosine kinase inhibitors

Inhibitors of receptor tyrosine kinases are implicated as therapeutic agents for the treatment of many human diseases including cancer, inflammation and diabetes. Cell-based assays to examine inhibition of receptor tyrosine kinase mediated intracellular signaling are often laborious and not amenable to high-throughput cell-based screening of compound libraries. Here we describe the development of a nonradioactive, sandwich enzyme-linked immunosorbent assay (ELISA) to quantify the activation and inhibition of ligand-induced phosphorylation of the colony-stimulating factor-1 receptor (CSF-1R) in 96-well microtiter plate format. The assay involves the capture of the Triton X-100 solubilized human CSF-1R, from HEK293E cells overexpressing histidine epitope-tagged CSF-1R (CSF-1R/HEK293E), with immobilized CSF-1R antibody and detection of phosphosphorylation of the activated receptor with a phosphotyrosine specific antibody. The assay exhibited a 5-fold increase in phosphorylated CSF-1R signal from CSF-1R/HEK293E cells treated with colony-stimulating factor (CSF-1) relative to treated vector control cells. Additionally, using a histidine epitope-specific capture antibody, this method can also be adapted to quantify the phosphorylation state of any recombinantly expressed, histidine-tagged receptor tyrosine kinase. This method is a substantial improvement in throughput and quantitation of CSF-1R phosphorylation over conventional immunoblotting techniques.     

Mutation of Tyr697, a GRB2-binding site, and Tyr721, a PI 3-kinase binding site, abrogates signal transduction by the murine CSF-1 receptor expressed in Rat-2 fibroblasts.

The receptor for the myeloid cell growth factor colony stimulating factor 1 (CSF-1) is a protein tyrosine kinase that is closely related to the PDGF receptor. Ligand binding results in kinase activation and autophosphorylation. Three autophosphorylation sites, Tyr697, Tyr706 and Tyr721, have been mapped to the kinase insert domain. Deletion of the entire kinase insert domain completely abrogates signal transduction by the CSF-1 receptor expressed in Rat-2 fibroblasts. To investigate the function of individual phosphorylation sites present in the CSF-1 receptor kinase insert domain, a number of phosphorylation site mutants were expressed in Rat-2 fibroblasts. Mutation of either Tyr697 or Tyr721 compromised signal transduction by the CSF-1 receptor. A mutant receptor, in which both Tyr697 and Tyr721 were replaced by phenylalanine, has lost all ability to induce changes in morphology or to increase cell growth rate in response to CSF-1. Tyr721 has been identified recently as the binding site for PI 3-kinase. Here we report that GRB2 associates with the CSF-1 receptor upon ligand binding. The phosphorylation on tyrosine of SHC and several other GRB2-associated proteins increased upon stimulation with CSF-1. Tyr697 was identified as a binding site for GRB2. We suggest that PI 3-kinase, GRB2 and some of the GRB2-associated proteins could play an important role in signal transduction by the CSF-1 receptor.     

Activation of Src family kinases by colony stimulating factor-1, and their association with its receptor.

The receptor for the macrophage colony stimulating factor-1 (CSF-1R) is a transmembrane glycoprotein with intrinsic tyrosine kinase activity. CSF-1 stimulation promotes the growth of cells of the macrophage lineage and of fibroblasts engineered to express CSF-1R. We show that CSF-1 stimulation resulted in activation of three Src family kinases, Src, Fyn and Yes. Concomitant with their activation, all three Src family kinases were found to associate with the ligand-activated CSF-1 receptor. These interactions were also demonstrated in SF9 insect cells co-infected with viruses encoding the CSF-1 receptor and Fyn, and the isolated SH2 domain of Fyn was capable of binding the CSF-1R in vitro. Analysis of mutant CSF-1Rs revealed that the 'kinase insert' (KI) domain of CSF-1R was not required for interactions with Src family kinases, but that mutation of one of the receptor autophosphorylation sites, Tyr809, reduced both their binding and enzymatic activation. Because fibroblasts expressing this receptor mutant are unable to form colonies in semi-solid medium or to grow in chemically defined medium in the presence of CSF-1, the Src family kinases may play a physiological role in the mitogenic response to CSF-1.     

Separation and Characterization of the Activated Pool of Colony-Stimulating Factor 1 Receptor Forming Distinct Multimeric Complexes with Signalling Molecules in Macrophages

Colony-stimulating factor 1 (CSF-1) triggers the activation of intracellular proteins in macrophages through selective assembly of signalling complexes. The separation of multimeric complexes of the CSF-1 receptor (CSF-1R) by anion-exchange chromatography enabled the enrichment of low-stoichiometry complexes. A significant proportion of the receptor in CSF-1-stimulated cells that neither possessed detectable tyrosine kinase activity nor formed complexes was separated from the receptor pool displaying autokinase activity that formed chromatographically distinct multimeric complexes. A small pool of CSF-1R formed a multimeric complex with phosphatidylinositol-3 kinase (PI-3 kinase), SHP-1, Grb2, Shc, c-Src, Cbl, and a significant number of tyrosine-phosphorylated proteins in CSF-1-stimulated cells. The complex showed a considerable amount of CSF-1R complex-associated kinase activity. A detectable level of the complex was also present in untreated cells. PI-3 kinase in the multimeric complex displayed low lipid kinase activity despite the association with several proteins. The major pool of activated CSF-1R formed transient multimeric complexes with distinctly different tyrosine-phosphorylated proteins, which included STAT3 but also PI-3 kinase, Shc, SHP-1, and Grb2. A significant level of lipid kinase activity was detected in PI-3 kinase in the latter complexes. The different specific enzyme activities of PI-3 kinase in these complexes support the notion that the activity of PI-3 kinase is modulated by its association with CSF-1R and other associated cellular proteins. Specific structural proteins associated with the separate CSF-1R multimeric complexes upon CSF-1 stimulation and the presence of the distinct pools of the CSF-1R were dependent on the integrity of the microtubular network.     

Colony-stimulating factor 1 activates protein kinase C in human monocytes.

Colony-stimulating factor 1 (CSF-1) is required for the survival, proliferation and differentiation of monocytes. We previously demonstrated that the CSF-1 receptor is linked to a pertussis toxin-sensitive G protein and that the induction of Na+ influx by CSF-1 is a pertussis toxin-sensitive event. The present studies have examined activation of protein kinase C as a potential intracellular signaling event induced by the activated CSF-1 receptor. The results demonstrate that CSF-1 stimulates translocation of protein kinase C activity from the cytosol to membrane fractions. This activation of protein kinase C was sensitive to pretreatment of the monocytes with pertussis toxin. Lipid distribution studies demonstrated that phosphatidylcholine (PC) is the major phospholipid in human monocytes. Moreover, the results indicate that CSF-1 stimulation is associated with decreases in PC, but not in phosphatidylinositol (PI), levels. The absence of an effect of CSF-1 on PI turnover was confirmed by the lack of changes in inositol phosphate production. In contrast, CSF-1 stimulation was associated with increased hydrolysis of PC to phosphorylcholine and diacylglycerol (DAG) in both intact monocytes and cell-free assays. Furthermore, the increase in PC turnover induced by CSF-1 was sensitive to pertussis toxin. The results also demonstrate that the induction of Na+ influx by CSF-1 is inhibited by the protein kinase C inhibitors staurosporine and the isoquinoline derivative H7, but not by HA1004.(ABSTRACT TRUNCATED AT 250 WORDS)     

Colony stimulating factor-1 (CSF-1) stimulates temperature dependent phosphorylation and activation of the RAF-1 proto-oncogene product.

The serine/threonine kinase RAF-1 is phosphorylated in intact macrophages in response to CSF-1 at 37 degrees C The augmented phosphorylation of RAF-1 and a concomitant increase in RAF-1 associated serine/threonine kinase activity are kinetically later events than CSF-1 induced protein tyrosine phosphorylation. Furthermore, phosphoamino acid analysis of RAF-1 reveals the presence of phosphoserine, trace amounts of phosphothreonine but no phosphotyrosine and the phosphorylated RAF-1 does not react with anti-phosphotyrosine antibodies. In contrast to CSF-1 induced protein tyrosine phosphorylation, RAF-1 phosphorylation and activation are temperature dependent and do not occur at 4 degrees C. Furthermore, coprecipitation experiments failed to reveal any noncovalent association of RAF-1 with the CSF-1 receptor. Thus, while RAF-1 is not a direct substrate for the CSF-1 receptor tyrosine kinase in vivo, its temperature dependent phosphorylation and activation represent an intriguing aspect of the CSF-1 response.     

Interactions of phosphatidylinositol kinase, GTPase-activating protein (GAP), and GAP-associated proteins with the colony-stimulating factor 1 receptor.

The interactions of the macrophage colony-stimulating factor 1 (CSF-1) receptor with potential targets were investigated after ligand stimulation either of mouse macrophages or of fibroblasts that ectopically express mouse CSF-1 receptors. In Rat-2 cells expressing the mouse CSF-1 receptor, full activation of the receptor and cellular transformation require exogenous CSF-1, whereas NIH 3T3 cells expressing mouse c-fms are transformed by autocrine stimulation. Activated CSF-1 receptors physically associate with a phosphatidylinositol (PI) 3'-kinase. A mutant CSF-1 receptor with a deletion of the kinase insert region was deficient in its ability to bind functional PI 3'-kinase and to induce PI 3'-kinase activity precipitable with antiphosphotyrosine antibodies. In fibroblasts, CSF-1 stimulation also induced the phosphorylation of the GTPase-activating protein (GAP)-associated protein p62 on tyrosine, although GAP itself was a relatively poor substrate. In contrast to PI 3'-kinase association, phosphorylation of p62 and GAP was not markedly affected by deletion of the kinase insert region. These results indicate that the kinase insert region selectively enhances the CSF-1-dependent association of the CSF-1 receptor with active PI 3'-kinase. The insert deletion mutant retains considerable transforming activity in NIH 3T3 cells (G. Taylor, M. Reedijk, V. Rothwell, L. Rohrschneider, and T. Pawson, EMBO J. 8:2029-2037, 1989). This mutant was more seriously impaired in Rat-2 cell transformation, although mutant-expressing Rat-2 cells still formed small colonies in soft agar in the presence of CSF-1. Therefore, phosphorylation of GAP and p62 through activation of the CSF-1 receptor does not result in full fibroblast transformation. The interaction between the CSF-1 receptor and PI 3'-kinase may contribute to c-fms fibroblast transformation and play a role in CSF-1-stimulated macrophages.     

Characterization of kinase inhibitors using different phosphorylation states of colony stimulating factor-1 receptor tyrosine kinase

It is known that some kinase inhibitors are sensitive to the phosphorylation state of the kinase, and therefore those compounds can discriminate between a phosphorylated and unphosphorylated protein. In this study, we prepared two colony stimulating factor-1 receptor (CSF-1R) tyrosine kinase proteins: one highly phosphorylated by autophosphorylation and the other dephosphorylated by phosphatase treatment. These kinases were subjected to an activity-based assay to investigate the effect of their phosphorylation state on the potency of several kinase inhibitors. Dasatinib, sorafenib, PD173074 and staurosporine showed similar inhibition against different phosphorylation states of CSF-1R, but pazopanib, sunitinib, GW2580 and imatinib showed more potent inhibition against dephosphorylated CSF-1R. Binding analysis of the inhibitors to the two different phosphorylation forms of CSF-1R, using surface plasmon resonance spectrometry, revealed that staurosporine bound to both forms with similar affinity, but sunitinib bound to the dephosphorylated form with higher affinity. Thus, these observations suggest that sunitinib binds preferentially to the inactive form, preventing the activation of CSF-1R. Screening against different activation states of kinases should be an important approach for prioritizing compounds and should facilitate inhibitor design.     

Antibody-induced mitogenicity mediated by a chimeric CD2-c-fms receptor.

A chimeric receptor composed of the extracellular domain of the human T-cell antigen CD2 (T11) joined to the membrane-spanning segment and the intracellular tyrosine kinase domain of the human colony-stimulating factor 1 receptor (CSF-1R) was expressed in murine NIH 3T3 fibroblasts. Stimulation of these cells with monoclonal antibodies to CD2 induced phosphorylation of the chimeric glycoprotein on tyrosine, receptor downmodulation, and mitogenesis. In contrast, neither human CSF-1R nor the chimeric receptor was able to function in interleukin-2-dependent murine T cells. In fibroblasts, then, CSF-1 per se is not required for activation of the receptor kinase or for a biological response, whereas in T cells, CSF-1R may be unable to engage the downstream signal transduction machinery.     

Tyrosine 706 and 807 phosphorylation site mutants in the murine colony-stimulating factor-1 receptor are unaffected in their ability to bind or phosphorylate phosphatidylinositol-3 kinase but show differential defects in their ability to induce early response gene transcription.

The receptor for colony-stimulating factor-1 (CSF-1) is a receptor protein-tyrosine kinase. To study the possible function of CSF-1 receptor autophosphorylation, two autophosphorylation sites, Tyr-706, located in the kinase insert, and Tyr-807, a residue conserved in all protein-tyrosine kinases, were changed independently to either phenylalanine or glycine. Wild-type and mutant receptors were stably expressed in Rat-2 cells. In response to CSF-1, cells expressing Phe- or Gly-706 mutant receptors showed increased growth rate and altered cell morphology. Both the Phe- and Gly-706 mutant receptors associated with and phosphorylated phosphatidylinositol-3 kinase at levels comparable with those of wild-type receptors. However, these mutant receptors differed subtly from each other and from the wild-type receptor in their ability to induce different aspects of the response to CSF-1. The Phe-706 mutant receptor was most strongly affected in its ability to increase growth rate or elevate the levels of c-fos and NGF1A mRNAs, whereas the Gly-706 mutant receptor was most markedly affected in its ability to induce a change in cell morphology or increase the levels of c-jun and NGF1A mRNAs. These findings indicate that Tyr-706 itself, or this region of the receptor, may be important for interaction of the CSF-1 receptor with different signalling pathways. Gly-807 mutant receptors lacked protein-tyrosine kinase activity, failed to respond to CSF-1, and were defective in biosynthetic processing. Phe-807 mutant receptors had 40 to 60% reduced protein-tyrosine kinase activity in vitro. Although cells expressing Phe-807 receptors were able to respond to CSF-1, the changes in growth rate and cell morphology were significantly less than seen with wild-type receptors, and the induction of early response genes was also slightly lower than for the wild-type receptor. In contrast, Phe-807 receptors were equivalent to wild-type receptors when tested for their ability to interact with phosphatidylinositol-3 kinase. These findings indicate that phosphorylation of Tyr-807 may be important for full activation of the receptor.     

Identification of tyrosine 706 in the kinase insert as the major colony-stimulating factor 1 (CSF-1)-stimulated autophosphorylation site in the CSF-1 receptor in a murine macrophage cell line.

The receptor for colony-stimulating factor 1 (CSF-1) is a ligand-activated protein-tyrosine kinase. It has been shown previously that the CSF-1 receptor is phosphorylated on serine in vivo and that phosphorylation on tyrosine can be induced by stimulation with CSF-1. We studied the phosphorylation of the CSF-1 receptor by using the BAC1.2F5 murine macrophage cell line, which naturally expresses CSF-1 receptors. Two-dimensional tryptic phosphopeptide mapping showed that the CSF-1 receptor is phosphorylated on several different serine residues in vivo. Stimulation with CSF-1 at 37 degrees C resulted in rapid phosphorylation on tyrosine at one major site and one or two minor sites. We identified the major site as Tyr-706. The identity of Tyr-706 was confirmed by mutagenesis. This residue is located within the kinase insert domain. There was no evidence that Tyr-973 (equivalent to Tyr-969 in the human CSF-1 receptor) was phosphorylated following CSF-1 stimulation. When cells were stimulated with CSF-1 at 4 degrees C, additional phosphotyrosine-containing phosphopeptides were detected and the level of phosphorylation of the individual phosphotyrosine-containing phosphopeptides was substantially increased. In addition, we show that CSF-1 receptors are capable of autophosphorylation at six to eight major sites in vitro.     

Role of dimerization and modification of the CSF-1 receptor in its activation and internalization during the CSF-1 response.

We have used kinetic and cross-linking approaches to study CSF-1-induced changes in the structure and function of the CSF-1R. Addition of CSF-1 to cells stimulates or stabilizes non-covalent CSF-1R dimerization resulting in activation of the CSF-1R kinase and the tyrosine phosphorylation of the receptor and certain cytoplasmic proteins. The non-covalent dimers become covalently linked via disulfide bonds and/or are subsequently further modified. These modified forms are selectively internalized. Pre-treatment of cells with the alkylating agent, iodoacetic acid (IAA), selectively inhibits covalent dimerization, modification and internalization but enhances protein tyrosine phosphorylation. It is proposed that ligand-induced non-covalent dimerization activates the CSF-1R kinase, whereas the covalent dimerization and subsequent modification lead to kinase inactivation, phosphotyrosine dephosphorylation and internalization of the receptor--ligand complex.     

Expression of c-Myc in response to colony-stimulating factor-1 requires mitogen-activated protein kinase kinase-1

The mitogen-inducible gene c-myc is a key regulator of cell proliferation and transformation. Yet, the signaling pathway(s) that regulate its expression have remained largely unresolved. Using the mitogen-activated protein kinase kinase (MEK1/2) inhibitor PD98059 and dominant negative forms of Ras (N17) and ERK1 (K71R), we found that activation of Ras and extracellular signal-regulated kinase (ERK) is necessary for colony-stimulating factor-1 (CSF-1)-mediated c-Myc expression and DNA synthetic (S) phase entry. Quiescent NIH-3T3 cells expressing a partially defective CSF-1 receptor, CSF-1R (Y809F), exhibited impaired ERK1 activation and c-Myc expression and failed to enter the S phase of the cell division cycle in response to CSF-1 stimulation. Ectopic expression of a constitutively active form of MEK1 in cells expressing CSF-1R (Y809F) rescued c-Myc expression and S phase entry, but only in the presence of CSF-1-induced cooperating signals. Therefore, MEK1 participates in an obligate signaling pathway linking CSF-1R to c-Myc expression, but other signals from CSF-1R must cooperate with the MEK/ERK pathway to induce c-Myc expression and S phase entry in response to CSF-1 stimulation.     

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.     

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.