Expression of c-Myc in Response to Colony-stimulating Factor-1 Requires Mitogen-activated Protein Kinase Kinase-1*

Mangeng ChengDagger , Demin Wang§, and Martine F. RousselDagger

From the Departments of Dagger  Tumor Cell Biology and § Biochemistry, St. Jude Children's Research Hospital, Memphis, Tennessee 38105-2794

    ABSTRACT
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Abstract
Introduction
References

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.

    INTRODUCTION
Top
Abstract
Introduction
References

The immediate-early response gene c-myc encodes one of the common effectors of mitogen-regulated proliferation and transformation (1, 2). Myc is a transcription factor that associates in dimeric complexes with its partner Max to activate a series of Myc-responsive genes, at least some of which are necessary for entry into the DNA synthetic (S) phase of the cell division cycle. The critical events underlying Myc control of S phase entry are still unresolved. Myc antagonizes the function of the cyclin-dependent kinase inhibitor p27Kip1 by promoting its degradation and/or sequestration into inactive complexes with other proteins, and this leads in turn to the activation of cyclin E-CDK2 prior to the G1/S transition (3-5). Several direct transcriptional targets of c-Myc have now been identified, which include the dual specificity phosphatase cdc25A (6), ornithine decarboxylase (7), cyclins E and A (8), dihydrofolate reductase (9), alpha -prothymosin (10), RNA helicase MrDb (11), and nucleolin. The combined effects of these and other as yet unrecognized target genes are required to drive quiescent cells into S phase and to maintain proliferating cells in cycle (2, 12).

Mitogen-induced signaling pathway(s) that mediate the induction of c-Myc expression have also largely remained an enigma. The ability of c-Myc to override cell cycle arrest imposed by dominant negative forms of Src kinases suggests that Src-mediated signaling may be responsible for c-Myc expression (13). One of the best characterized growth factor-induced signaling pathways is the Ras/Raf/MEK1/MAPK pathway, and several lines of evidence indicate that it can regulate c-Myc expression. First, the c-myc promoter contains conserved binding sites for Ets family transcription factors, which can be activated by Ras/Raf/MEK/ERK signaling (14-17). Second, inhibition of cell growth caused by overexpression of the carboxyl-terminal catalytic domain of the Ras GTPase-activating protein can be rescued by enforced c-Myc expression (18). Third, induction of c-Myc expression by the oncoprotein v-Abl requires both functional Ras and Raf-1 (19). Finally, overexpression of a dominant negative and growth-suppressive form of Raf-1 (Raf-1-C4B) significantly reduces serum-dependent induction of c-Myc, whereas conditional expression of an inducible Raf-1 fusion protein is in itself sufficient to induce c-Myc expression (20). The downstream effectors of activated Raf-1 are MEK1 and MEK2, but to date neither of these kinases has been linked to regulation of c-Myc expression.

The colony-stimulating factor-1 receptor (CSF-1R), a growth factor receptor with intrinsic tyrosine kinase activity, is expressed specifically in macrophages and trophoblasts (21), and bone marrow-derived macrophages require CSF-1 for cell proliferation, survival, and differentiation (22). Ectopic expression of human CSF-1R in murine fibroblasts endows them with the ability to proliferate in chemically defined medium containing CSF-1 as the sole growth factor (23). One of the major autophosphorylation sites within ligand-stimulated CSF-1R is tyrosine 809, located in the activation loop of the kinase domain (24). Substitution of this residue with phenylalanine (Y809F) leads to reduction but not abolition of receptor protein kinase activity (25). NIH-3T3 cells expressing CSF-1R (Y809F) fail to proliferate in response to CSF-1 and instead arrest in the early G1 phase of the cell cycle (24). These CSF-1-stimulated cells fail to activate MAP kinases, ERK1 and 2, and to induce c-Myc and cyclin D1, both of which are required for entry into S phase (26-28). Enforced expression of c-Myc or D-type cyclins rescues ligand-induced mitogenesis by CSF-1R (Y809F), enabling the cells to proliferate continuously and to form colonies in semisolid medium (26, 27).

We have used macrophages as well as NIH-3T3 fibroblasts expressing different forms of CSF-1R to assess the role of the MEK/ERK pathway in the induction of c-Myc expression. We report that MEK1 activity is necessary for c-Myc expression, although maximal c-Myc induction and S phase entry require the cooperation of additional signaling molecules.

    EXPERIMENTAL PROCEDURES

Cells and Culture Conditions-- A murine macrophage cell line, Bac1.2F5 (29), was grown in Dulbecco's modified Eagle medium supplemented with 15% fetal bovine serum (FBS), 2 mM L-glutamine, 100 units each of penicillin and streptomycin per ml, and 25% L cell conditioned medium (LCM) as a source of murine CSF-1. For CSF-1 stimulation, Bac1.2F5 cells were growth arrested in medium lacking LCM for 18 h, and quiescent cells were restimulated with medium containing LCM for the indicated times. For treatment with the MEK inhibitor PD98059 (kindly provided by Stuart J. Decker, Warner-Lambert Co., Ann Arbor, MI), quiescent cells were pretreated with 60 µM PD98059 or the vehicle control dimethyl sulfoxide (Me2SO) (0.3%) for 30 min before the addition of medium containing LCM with PD98059 or Me2SO.

NIH-3T3 cells stably expressing human CSF-1R or mutant CSF-1R (Y809F) were maintained in Dulbecco's modified Eagle's medium supplemented with 10% FBS, glutamine, and antibiotics (23). For CSF-1 stimulation, cells were growth arrested in serum-free medium (Dulbecco's modified Eagle's medium with 0.4 mg/ml bovine serum albumin, 0.1% FBS, glutamine, penicillin, and streptomycin) for 36 h. Quiescent cells were restimulated to enter the cell cycle with serum-free medium containing purified recombinant human CSF-1 at 10 ng/ml (kindly provided by Steven C. Clark, Genetics Institute, Cambridge, MA).

S phase entry was monitored by incorporation of bromodeoxyuridine (BrdUrd) into newly synthesized DNA (30). To measure cell growth in NIH-3T3 fibroblasts coexpressing CSF-1R (Y809F) and zinc-inducible activated MEK1, cells were growth arrested in serum-free medium, and quiescent cells were switched to medium containing either 25 µM ZnSO4, CSF-1, or both, harvested daily, and counted.

Expression Vectors-- The plasmid pMTCB6-MEK1* contains a mutant, constitutively active murine MEK1 gene under the control of a zinc-inducible metallothionein promoter (31). The plasmid pSRalpha MSV-CSF-1R-tkneo was constructed by inserting human CSF-1R cDNA downstream of the MSV promoter into the EcoRI site of the pSRalpha MSV(EcoRI)-tkneo vector, provided by Dr. Charles Sawyers, UCLA. The plasmid pRK5-Ras N17 was constructed by inserting the murine Ha-Ras N17 cDNA into the NheI and XhoI sites of pRK5 (32). The plasmids pCEP4-HA and pCEP4-HA-ERK1 (K71R), which encode a dominant negative form of murine ERK1 (K71R) (33), were provided by Dr. Melanie H. Cobb (University of Texas Southwestern Medical Center, Dallas).

Transfection and Selection of Cell Lines-- The calcium phosphate precipitation method (34) was used for transfection. The plasmid pMTCB6-MEK1* (20 µg) was cotransfected with pJ6Omega -puro (5 µg), encoding puromycin resistance (35), into NIH-3T3 cells expressing the mutant CSF-1R (Y809F). After selection for 3 weeks in complete medium containing 7.5 µg/ml puromycin, MEK1*-positive subclones were isolated by limiting dilution in 96-well microtiter plates.

For transient transfection, NIH-3T3 cells were cotransfected with pSRalpha MSV-CSF-1R-tkneo together with either a control vector (pCEP4-HA), a vector encoding dominant negative Ras N17 (pRK5-Ras N17), or a vector encoding dominant negative ERK1 (pCEP4-ERK1 (K71R)). 30 h after transfection, cells were growth arrested for 18 h in serum-free medium, and quiescent cells were restimulated with serum-free medium containing 10 ng/ml CSF-1 for 8 h or 14 h as indicated. The transfection efficiency of NIH-3T3 cells was about 10% as determined by transfecting the cells with beta -galactosidase expression plasmid and subsequent cellular staining of beta -galactosidase (data not shown).

ERK Kinase Assay-- The ERK immune complex kinase assay was performed as described previously (36) with minor modifications. Cells were lysed in ERK lysis buffer (50 mM HEPES, pH 7.4, 1% Triton X-100, 150 mM NaCl, 1 mM phenylmethylsulfonyl fluoride, 1 mM dithiothreitol, 4 µg/ml aprotinin, 4 µg/ml pepstatin, 10 mM p-nitrophenyl phosphate, 0.5 mM sodium orthovanadate, 1 mM sodium fluoride, and 10 mM beta -glycerophosphate). Lysates were clarified by centrifugation in an Eppendorf microcentrifuge at 10,000 × g for 10 min, and the protein concentration of the supernatant was measured with a BCA protein assay kit (Pierce Chemical Co.). Cell lysates (400 µg of protein) were mixed with 2 µg of either ERK1 (K-23) or ERK2 (C-14) antibody (Santa Cruz Biotechnology, Santa Cruz, CA) and 20 µl of protein A-Sepharose beads 1:1 in lysis buffer (v/v) and incubated at 4 °C for 2 h. Beads were pelleted by centrifugation, washed four times with ERK lysis buffer, two times with kinase reaction buffer (50 mM HEPES, pH 7.4, containing 10 mM MgSO4), and resuspended in 30 µl of kinase reaction buffer. For the kinase reaction, the beads were incubated with 5 µg of myelin basic protein (Sigma) and 50 µM ATP containing 10 µCi of [32P]ATP (Amersham Pharmacia Biotech) in 30 µl of kinase reaction buffer at 30 °C for 30 min. Reactions were stopped by adding 1/3 volume of sample buffer (50 mM Tris, pH 6.8, 2% SDS, 10% glycerol, 100 mM dithiothreitol, 0.1% bromphenol blue) and heating at 85 °C for 5 min. Labeled proteins were resolved on denaturing polyacrylamide gels, which were dried and subjected to autoradiography.

Northern Blotting-- Total cellular RNA (20 µg/sample) was separated by formaldehyde-agarose gel electrophoresis and transferred to nitrocellulose membranes as described previously (26). Membranes were baked at 85 °C, blocked with Denhardt's solution (37), hybridized with a 32P-labeled mouse c-myc cDNA probe prepared by random priming (Boehringer Mannheim), and washed membranes were subjected to autoradiography (26).

Immunoblotting-- To detect the c-Myc protein, 2 × 106 cells were lysed in 100 µl of gel sample buffer, and cell lysates were boiled for 10 min. After brief sonication, the proteins were resolved by SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membrane (Micron Separations, Westborough, MA). Nonspecific binding sites were blocked by incubation for 1 h at room temperature with Tris-buffered saline (25 mM Tris, pH 8.0, 150 mM NaCl, 2 mM KCl) containing 3% nonfat dry milk. Filters were incubated with the c-Myc antibody (06-340, Upstate Biotechnology, Lake Placid, NY) in Tris-buffered saline and milk for 2 h at room temperature, and sites of antibody binding were identified using horseradish peroxidase-conjugated donkey anti-rabbit IgG followed by enhanced chemiluminescence detection (ECL kit; Amersham Pharmacia Biotech). Detection of cyclin D1 (72-13G-11), CDK4 (RY), human CSF-1R (RA), Myc-tagged active MEK1 (MEK1*) (9E10), and actin (C-11, Santa Cruz Biotechnology) with the indicated antibodies was described previously (31, 38-40).

    RESULTS

Induction of c-Myc by CSF-1 Requires Ras/MEK/ERK Activation-- To address the role of MEK1/2 in CSF-1R-induced c-Myc expression, Bac1.2F5, a macrophage-derived cell line that requires CSF-1 for proliferation, was treated with the specific MEK1/2 inhibitor PD98059 (41, 42). Cells made quiescent by deprivation of CSF-1 for 16 h were pretreated with PD98059 or with Me2SO solvent for 30 min and then restimulated with medium containing CSF-1 together with PD98059 or Me2SO. Treatment with 60 µM PD98059 blocked CSF-1R-induced activation of ERK1 and ERK2 as measured by in vitro kinase assays (Fig. 1A). Analysis of total RNA by Northern blotting demonstrated that the addition of 60 µM PD98059 significantly impaired CSF-1-mediated induction of c-myc expression, reducing c-myc mRNA by 80% by 6 h of treatment (Fig. 1B, lane 5 versus lane 3). In contrast, c-myc mRNA levels were not significantly altered by treatment with Me2SO (Fig. 1B, lanes 6 and 7). PD98059 also inhibited the induction of c-Myc protein in response to CSF-1, whereas Me2SO had no significant effect (Fig. 1C). In Bac1.2F5 macrophages, the expression of CDK4 is dependent on the addition of CSF-1 to the culture medium (39). CDK4 expression was unaffected by PD98059 (Fig. 1C), indicating that this inhibitor did not indiscriminately block CSF-1 signaling.


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Fig. 1.   MEK1/2 activity is required for CSF-1R-mediated c-Myc expression and S phase entry. Panel A, quiescent Bac1.2F5 macrophages were left untreated (-) or were pretreated with 20, 40, or 60 µM PD98059 (PD) or 0.3% Me2SO (DMSO) for 30 min. Cells were restimulated with medium containing CSF-1 with or without the inhibitor at the same concentration. ERK1 kinase activity was measured with myelin basic protein as a substrate. 32P-Labeled proteins were resolved on denaturing polyacrylamide gels, which were dried and subjected to autoradiography. Panel B, quiescent Bac1.2F5 macrophages were left untreated (-) or were pretreated with 60 µM PD98059 or 0.3% Me2SO for 30 min and were restimulated with medium containing CSF-1 with or without the inhibitor. Total RNA extracted at the indicated times after stimulation was hybridized sequentially with 32P-labeled probes specific for c-Myc and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) used for a loading control. Panel C, c-Myc, CDK4, and actin protein levels were measured by immunoblotting with c-Myc antibody (06-340), CDK4 antiserum (RY), or actin antibody (C-11), and the sites of binding were visualized with ECL reagent. Panel D, DNA synthesis was measured in cells pretreated with or without the inhibitor and restimulated with CSF-1 for 18 h. BrdUrd was added to the culture medium between 12 and 18 h of CSF-1 treatment. The cells were fixed and immunostained with BrdUrd antibody, and the percentage of BrdUrd-positive cells was determined by fluorescence microscopy.

Because c-Myc is essential for CSF-1-mediated S phase entry (26, 43), the effect of these inhibitors on entry into the cell cycle was determined. As expected, PD98059-treated quiescent macrophages failed to enter S phase after CSF-1 stimulation, whereas Me2SO-treated cells were not inhibited (Fig. 1D). Similar results were obtained after PD98059 treatment of NIH-3T3 fibroblasts engineered to express CSF-1R (data not shown).

Although PD98059 is a specific inhibitor of MEK1/2, it remained possible that some of its inhibitory effects might be caused by inhibition of other as yet unidentified MEKs. Given that Ras signals via MEKs to activate ERKs, we assessed the effects of dominant negative forms of Ras (N17) and ERK1 (K71R) on c-Myc induction in response to CSF-1. NIH-3T3 cells were transiently transfected with expression plasmids encoding wild-type human CSF-1R alone or together with plasmids encoding Ras (N17) or ERK1 (K71R). Transfected cells growth arrested by serum depletion were stimulated with serum-free medium containing human CSF-1 as the only mitogen (Fig. 2A). Those transfected with CSF-1R alone expressed c-Myc (Fig. 2A, lane 2) and began to enter S phase by 14 h after CSF-1 stimulation (Fig. 2B, lane 2). In contrast, coexpression of CSF-1R with the dominant negative Ras (N17) or ERK1 (K71R) significantly impaired CSF-1R-mediated induction of c-Myc (Fig. 2A, lanes 3-6), and most cells failed to enter S phase (Fig. 2B, lanes 3-6), even though comparable CSF-1R levels were observed in all transfectants (Fig. 2A). Together, these results support the view that signaling through the Ras/MEK/ERK pathway is required for CSF-1-dependent induction of c-Myc and S phase entry.


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Fig. 2.   Dominant negative Ras (N17) or ERK1 (K71R) inhibits CSF-1R-mediated c-Myc expression. Panel A, NIH-3T3 cells were transfected with 30 µg of empty vector pSRalpha MSV (-), with 10 µg of pSRalpha MSV-CSF-1R plus 20 µg of empty vector (lane 2), with 10 µg of pSRalpha MSV-CSF-1R plus 10 µg (lane 3) or 20 µg (lane 4) of pRK5-Ras (N17), or 10 µg (lane 5) or 20 µg (lane 6) of pCEP4-HA-ERK1 (K71R). 30 h post-transfection, cells were serum starved and then restimulated with CSF-1 for 8 h. Cell lysates were assayed for c-Myc, CDK4, and CSF-1R protein levels by immunoblotting with c-Myc antibody (06-340), CDK4 antiserum (RY), or CSF-1R antiserum (RA), respectively. Panel B, DNA synthesis was measured in serum-starved cells restimulated with CSF-1 for 14 h with BrdUrd. Cells were fixed and immunostained with BrdUrd antibody, and the percentage of BrdUrd-positive cells was determined by fluorescence microscopy.

MEK1 Complements Defective Mitogenic Signaling by Mutant CSF-1R (Y809F)-- NIH-3T3 cells that stably express a mutant CSF-1R (Y809F) that is partially defective in its kinase activity fail to induce c-Myc in response to CSF-1 stimulation (26). After stimulation with CSF-1, activation of the wild-type receptor induced a rapid transient peak of ERK1 activation followed by a lower level of activation which persisted in the presence of CSF-1 (Fig. 3, A and B). By contrast, much weaker activation of ERK1 was seen in cells expressing equivalent levels of mutant CSF-1R (Y809F) (Fig. 3, A and B).


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Fig. 3.   ERK1 activation is impaired in CSF-1R (Y809F)-mediated signaling. Quiescent NIH-3T3 cells expressing wild-type (WT) or mutant (Y809F) CSF-1R were stimulated with serum-free medium containing 10 ng/ml human CSF-1 for the indicated times, and ERK1 kinase activity was measured with myelin basic protein (MBP) as a substrate. 32P-Labeled proteins were resolved on denaturing polyacrylamide gels, which were dried and subjected to autoradiography.

To assess whether MEK1 might restore signaling by CSF-1R (Y809F), an expression plasmid encoding a constitutively active, Myc-tagged form of MEK1 (designated MEK1*) expressed under the control of a zinc-inducible metallothionein promoter was introduced into NIH-3T3 cells that stably express mutant CSF-1R (Y809F). Several individual clones were selected by limiting dilution (31). In quiescent serum-starved cells, MEK1* protein was not detected using an antibody to the Myc-epitope tag (Fig. 4A, lane 1). Zinc treatment induced high levels of MEK1* protein expression, which were readily detectable by 6 h of zinc treatment (Fig. 4A, lane 3). In response to MEK1* expression, ERK1 activity and cyclin D1 expression were induced as reported previously (Fig. 4A) (see also Ref. 31). In contrast, MEK1* activation was insufficient to induce c-Myc expression (Fig. 4A, lanes 1-5). As expected, high levels of c-Myc were induced by FBS stimulation (Fig. 4A, lanes 6-10), indicating that the Myc response was specifically refractory to signaling via CSF-1R (Y809F).


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Fig. 4.   Ectopic expression of MEK1* restores c-Myc expression mediated by mutant CSF-1R (Y809F). Panel A, serum-starved NIH-3T3 cells harboring mutant CSF-1R (Y809F) and inducible MEK1* were stimulated with ZnSO4 or FBS and assayed at the indicated times thereafter for MEK1*, c-Myc, cyclin D1, and CDK4 protein levels by immunoblotting with the respective antibodies. Panel B, serum-starved NIH-3T3 cells harboring mutant CSF-1R (Y809F) and inducible MEK1* were treated either with CSF-1 alone or with both ZnSO4 and CSF-1 for the indicated times. c-Myc and CDK4 protein levels were determined by immunoblotting with c-Myc antibody (06-340) and CDK4 antiserum (RY).

Therefore, whereas CSF-1R-mediated c-Myc expression is dependent on MEK1/2 activity (Figs. 1 and 2), induction of c-Myc appears to require CSF-1R-mediated signals in addition to those mediated by the MEK/ERK pathway. To explore this issue further, NIH-3T3 cells coexpressing CSF-1R (Y809F) and inducible MEK1* were serum starved and restimulated with medium containing CSF-1 without or with zinc. Treatment with both zinc and CSF-1 induced c-Myc expression (Fig. 4B, lanes 6-10), whereas, as expected, CSF-1 treatment alone did not (Fig. 4B, lanes 1-5) (24). High levels of c-Myc protein were detected 6 h after stimulation with zinc plus CSF-1 (Fig. 4B, lanes 6-10), correlating with the onset of MEK1* expression in response to zinc treatment alone (Fig. 4A, lanes 1-5). However, in the presence of both CSF-1 and zinc, the levels of Myc achieved were still lower than those detected in response to FBS stimulation, again implying that multiple CSF-1R signaling pathways cooperate in Myc induction.

Ectopic expression of either c-Myc or D-type cyclins in NIH-3T3 cells expressing mutant CSF-1R (Y809F) resensitizes them to the mitogenic effects of CSF-1 and enables them to proliferate continuously (26, 27). MEK1/2 activation was shown to be required for mitogen-dependent c-Myc and cyclin D1 expression (44-48). Although sufficient to induce cyclin D1 expression (see Fig. 4A), zinc-regulated expression of MEK1* alone does not induce S phase entry (31). To address whether zinc-inducible MEK1* could rescue S phase entry and proliferation of cells expressing mutant CSF-1R (Y809F) in response to CSF-1 stimulation, NIH-3T3 cells stably transfected with CSF-1R (Y809F) and zinc-inducible MEK1* were serum starved and restimulated with CSF-1, zinc, or both. As expected, NIH-3T3 cells expressing the wild-type receptor entered S phase in response to CSF-1 stimulation, whereas CSF-1-stimulated cells expressing the mutant CSF-1R (Y809F) failed to enter S phase (Fig. 5A). However, approximately 65% of cells harboring both CSF-1R (Y809F) and inducible MEK1* were able to enter S phase by 18 h (Fig. 5A) and proliferate (Fig. 5B) in medium containing both zinc and CSF-1. Consistent with the observations above that FBS was more efficient than CSF-1 plus zinc in inducing Myc in these cells, their growth rate (doubling time ~60 h) (Fig. 5B) was significantly slower than cells grown in FBS (doubling time, ~24 h). NIH-3T3 cells harboring the wild-type receptor show no such impairment in response to CSF-1 (23). Therefore, even though MEK1*- and CSF-1R (Y809F)-dependent signals can cooperate to rescue cell proliferation in response to CSF-1, other receptor-mediated signals are required for maximal CSF-1-induced cell proliferation.


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Fig. 5.   Ectopic expression of MEK1* rescues S phase entry and proliferation of cells expressing mutant CSF-1R (Y809F). Panel A, serum-starved NIH-3T3 fibroblasts expressing wild-type (WT) and mutant (Y809F) CSF-1R were either left untreated (-) or were stimulated with CSF-1 (+) for 18 h. NIH-3T3 fibroblasts coexpressing CSF-1R (Y809F) and inducible MEK1* were either untreated (-) or treated for 18 h with CSF-1 alone, ZnSO4 alone, or both. Between 12 and 18 h of treatment, BrdUrd was added to the culture medium. Cells were fixed and immunostained with BrdUrd antibody, and the percentage of BrdUrd-positive cells was determined by fluorescence microscopy. Panel B, NIH-3T3 fibroblasts coexpressing CSF-1R (Y809F) and inducible MEK1* were seeded on culture plates for 16 h, growth arrested in serum-free medium, and then treated with serum-free medium containing ZnSO4, CSF-1, or both. Cells were harvested daily and counted.


    DISCUSSION

Our results strongly support the concept that Ras/Raf/MEK/ERK signaling is an obligate pathway for growth factor receptor-mediated c-Myc induction (20). Experiments utilizing the MEK1/2 inhibitor PD98059 as well as dominant negative forms of Ras (N17) and ERK1 (K71R) indicate that activation of Ras and MEK/ERK is necessary for c-Myc expression in response to CSF-1. However, signaling through this pathway is insufficient to guarantee c-Myc induction because the ectopic expression of enzymatically active MEK1* in quiescent cells did not induce c-Myc expression in the absence of other cooperating signals. When cells coexpressing both CSF-1R (Y809F) and MEK1* were stimulated with CSF-1, c-Myc was induced, and the cells entered S phase. Although active MEK1 rescued CSF-1R (Y809F)-mediated c-Myc expression and mitogenesis, the level of c-Myc induction and rate of cell proliferation remained below those obtained using cells expressing wild-type CSF-1R alone. Together, the results imply that several CSF-1R-mediated signaling pathways cooperate with MEK/ERK to induce c-Myc maximally and drive entry into S phase.

Our findings are generally consistent with a previous report that overexpression of a dominant negative form of Raf-1 (Raf-1-C4B) can reduce serum-mediated c-Myc expression (20). Moreover, c-Myc induction by v-Abl, an oncogenic tyrosine kinase, requires functional Ras and Raf-1 (19). However, there are also discrepancies between our results and those of others. Although regulated expression by tamoxifen of a fusion protein between the estrogen receptor and Raf-1 (Raf-ER) was sufficient to induce c-Myc expression and S phase entry in NIH-3T3 fibroblasts (20), our results would suggest that collateral signals in addition to MEK/ERK activation are required for c-Myc expression. One possibility is that Raf-ER might activate pathways in addition to MEK/ERK signaling. For example, cdc25A, a tyrosine phosphatase involved in the regulation of cell proliferation, has been implicated in Raf-1- but not MEK1/2-mediated signaling (49). Constitutive expression of active MEK1 in NIH-3T3 fibroblasts can lead to serum-independent growth (50), and ectopic expression of active MEK1 under the control of the estrogen receptor (MEK1-ER) in these same cells resulted in accelerated S phase entry (51). In contrast, our data indicated that inducible expression of active MEK1 by zinc in the absence of any other growth factors in the medium was not sufficient to promote S phase entry (31). The different levels of active MEK1 achieved in the transfected cell lines might explain the discrepancies between the latter sets of experiments.

Although phosphoinositol 3-kinase is activated quickly in response to CSF-1, its overall activity is not altered in response to CSF-1R (Y809F)-mediated signaling (24). Therefore, it is unlikely to be a candidate for the second signal. Moreover, inhibition of its activation with wortmannin had no effect on CSF-1R-mediated c-Myc expression (data not shown). Microinjection of dominant negative Src constructs or neutralizing antibodies to Src family kinases has shown that they are required for DNA synthesis in response to CSF-1 or platelet-derived growth factor (52). Src family kinases associate with and are activated by growth factor receptors via their Src homology domain-2 and are substrates for the receptor kinases (53, 54). Src family kinases can also activate a pathway that leads to the transcription of c-myc (13). Specifically, constitutively expressed c-Myc rescues a G1 block elicited by a dominant negative form of Src, whereas Fos and Jun rescue a G1 block induced by dominant negative Ras. This has led to the suggestion that Src family kinases induce c-Myc in a Ras-independent fashion (13).

Even though the downstream targets of Src family kinases have not been well characterized, the importance of the Ras/Raf/MEK/ERK signal pathway in transmitting Src-mediated signals has been demonstrated (55, 56). For example, transformation of fibroblasts by oncogenic Src is blocked by inhibiting Ras function with anti-Ras antibody or by overexpression of GTPase-activating protein (57, 58). The Ras/Raf/MEK/ERK pathway is also required for the transcriptional induction of several Src targets including the mitogen-responsive transcription factor Egr-1 (59). Therefore, it is likely that the induction of c-Myc by the Src family kinases is at least in part dependent on Ras function.

    ACKNOWLEDGEMENTS

We thank Carol Bockhold, Zhen Lu, and Rose Mathew for excellent technical assistance. We are indebted to Drs. John Cleveland, Charles Sherr, and Frederique Zindy for criticisms on the manuscript, to Drs. Alan Diehl and Jason Weber for helpful discussions, and to Dr. Julie Cay Jones for scientific editing.

    FOOTNOTES

* This work was supported in part by National Institutes of Health Grant CA-56819, Training Grant T32-CA-70089 (M. C.) Cancer Center Support (Core) Grant CA-21705, and by the American Lebanese Syrian Associated Charities (ALSAC) of St. Jude Children's Research Hospital.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Dept. of Tumor Cell Biology, St. Jude Children's Research Hospital, 332 N. Lauderdale St., Memphis, TN 38105-2794. Tel.: 901-495-3481; Fax: 901-495-2381; E-mail: martine.roussel{at}stjude.org.

    ABBREVIATIONS

The abbreviations used are: MEK, mitogen-activated protein kinase kinase; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; CSF-1R, colony-stimulating factor-1 receptor; FBS, fetal bovine serum; LCM, L cell conditioned medium; Me2SO, dimethyl sulfoxide; BrdUrd, bromodeoxyuridine; MSV, murine sarcoma virus; tk, thymidine kinase; HA, hemagglutinin; ER, estrogen receptor.

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Abstract
Introduction
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