The human granulocyte-macrophage colony-stimulating factor receptor is capable of initiating signal transduction in NIH3T3 cells. Matthias Eder, James D.Griffin ...
The EMBO Journal vol. 1 2 no.4 pp. 1 647 - 1656, 1993
The human granulocyte-macrophage colony-stimulating factor receptor is capable of initiating signal transduction in NIH3T3 cells Matthias Eder, James D.Griffin and Timothy J.Ernst Division of Tumor Immunology, Dana-Farber Cancer Institute 44, Binney St and Harvard Medical School, Boston, MA, USA
Communicated by Leo Sachs
The ability of the receptor for the hematopoietic cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF) to function in non-hematopoietic cells is unknown. NIH3T3 fibroblasts were transfected with cDNAs encoding the a and / subunit of the human GM-CSF receptor and a series of stable transformants were isolated that bound GM-CSF with either low (KD= 860- > 1000 pM) or high affinity (KD= 20-80 pM). Low affinity receptors were not functional. However, the reconstituted high affinity receptors were found to be capable of activating a number of signal transduction pathways, including tyrosine kinase activity, phosphorylation of Raf-1, and the transient induction of c-fos and c-myc mRNAs. The activation of protein tyrosine phosphorylation by GM-CSF in NIH3T3 cells was rapid (< 1 min) and transient (peaking at 5-20 min) and resulted in the phosphorylation of proteins of estimated molecular weights of 42, 44, 52/53 and 58-60 kDa. Some of these proteins co-migrated with proteins from myeloid cells that were phosphorylated on tyrosine residues in response to GM-CSF. In particular, p42 and p44 were identified as mitogen-activated protein kinases (MAP kinases), and the phosphorylation on tyrosine residues of p42 and p44 MAP kinases occurred at the same time as the phosphorylation of Raf-1. However, despite evidence for activation of many mitogenic signal transduction molecules, GM-CSF did not induce significant proliferation of transfected NIH3T3 cells. These results suggest that murine fibroblasts contain signal transducing molecules that can effectively interact with the human GM-CSF receptor, and that are sufficient to activate at least some of the same signal transduction pathways this receptor activates in myeloid cells, including activation of one or more tyrosine kinase(s). However, the level of activation of signal transduction is either below a threshold of necessary activity or at least one mitogenic signal necessary for proliferation is missing. Key words: GM-CSF receptor/MAP kinase/Raf-1/signal transduction/tyrosine kinase
Introduction Granulocyte-macrophage colony-stimulating factor (GMCSF) is a cytokine that stimulates the production of myeloid cells in vitro and in vivo, and also enhances certain functions of mature neutrophils and monocytes such as phagocytosis Oxford University Press
and intracellular killing (Metcalf, 1985; Sachs, 1987; Sieff, 1987; Cannistra and Griffin, 1988). The human receptor for GM-CSF is only expressed by a limited number of cell types (Park et al., 1989) and is a heterodimer consisting of a unique ae chain (Gearing et al., 1989) and a ( chain (Hayashida et al., 1990), which is shared with the IL-3 and IL-5 receptors (Kitamura et al., 1991b; Tavernier et al., 1991). The a chains of these receptors are cytokine-specific binding proteins with low affinity for their respective ligands whereas the ( chain fails to bind any ligand by itself but converts the low affinity binding proteins to high affinity receptors that are biologically active (Kitamura et al., 1991a). Both the a and j3 chains of the GM-CSF receptor belong to the cytokine receptor family (Bazan, 1990). The signal transduction pathways activated by GM-CSF are not well understood. Although the receptor subunits themselves do not have any structural features consistent with a tyrosine kinase (Kaczmarski and Mufti, 1991), one of the first known biochemical activities of the activated receptor is activation of a tyrosine kinase, a feature of virtually all receptors of the cytokine receptor family (Isfort et al., 1988; Kanakura et al., 1990). For the GM-CSF or IL-3 receptors, virtually all of the subsequent biological activities can be blocked by inhibitors of tyrosine kinases (McColl et al., 1991; Duronio et al., 1992). The tyrosine kinase or kinases activated by the GM-CSF receptor have not yet been identified. It has been speculated that members of the cytokine receptor family may interact and activate specific members of the src family of tyrosine kinases. Support for this hypothesis comes from studies showing that IL-3 can rapidly activate p53/56 lyn in myeloid cell lines (Torigoe et al., 1992a) and that the IL-2 receptor can associate with p56 lck in T cells (Hatakeyama et al., 1991). Thus, the lineage specificity of this class of receptors may come from both regulation of receptor expression and from regulation of expression of its 'associated' kinase(s). Functional high affinity GM-CSF receptors have been ectopically reconstituted by transfection in a pro-B cell line (BAF-3) and both GM-CSF and IL-3 receptors have been reconstituted in a T cell line (CTLL-2) (Kitamura et al., 199 la; Hara and Miyajima, 1992). This suggests that BAF-3 and CTLL cells contain cellular signaling molecules sufficient for a mitogenic response after ligand-induced activation of these two receptors. In contrast, we failed to detect any GM-CSF-inducible signal transduction after transfection of the GM-CSF receptor in the Burkitt's lymphoma cell line Raji despite high affinity binding, suggesting some specificity in the expression of signal transducing molecules even in hematopoietic cells (M.Eder,
unpublished data). Human GM-CSF receptors have also been reconstituted by transfection in fibroblasts, although their function has not been tested (Hayashida et al., 1990). The ectopic expression of receptor molecules in fibroblasts can induce proliferation of transfected cells after stimulation with the respective 1647
M.Eder, J.D.Griffin and T.J.Ernst
ligand. For example, the expression of the colony-stimulating factor- I (CSF- 1) receptor, c-fms, in NIH3T3 cells makes these cells responsive to CSF-1 and induces CSF-1-dependent proliferation in agar (Roussel et al., 1987). However, c-fms contains intrinsic tyrosine kinase activity and is known to share many signal transduction components with endogenous fibroblast growth factor receptors such as the epidermal and platelet-derived growth factor (EGF, PDGF) receptors (for review see Cantley et al., 1991). In contrast, it is not clear whether fibroblasts contain signal transduction components such as tyrosine kinases, which could interact with a transfected GM-CSF receptor. In order to examine the requirement for hematopoieticspecific signaling molecules, we have reconstructed human GM-CSF receptors in NIH3T3 cells. Here we report that the high-affinity GM-CSF receptor, when reconstructed in NIH3T3 cells, is functional and can transmit signals which result in activation of tyrosine kinase activity, phosphory-
lation of Raf-1, p42 and p44 MAP kinases and induction of c-fos and c-myc mRNA expression.
Results Transfection of human GMR a//,8 cDNA into NIH3T3 fibroblasts and binding characteristics of transfected cells We transfected NIH3T3 fibroblasts with cDNAs encoding both the a and the j chain of human GMR (pREP8-GMRa and pREP4-GMRO) in order to obtain stable transformants. Transfectants were selected by growth in histidine-free medium supplemented with L-histidinol and hygromycin B and 17 independent clones were isolated. After an initial screening for evidence of signal transduction induced by human GM-CSF in the isolated transformants (see below), binding studies were performed on five independent clones and untransfected NIH3T3 cells to characterize expression of the human GMR. Cells were incubated with human ['25I]GM-CSF and the binding data analyzed by least squares regression analysis (Table I). No specific binding of human GM-CSF was detectable for untransfected NIH3T3 cells or for clone no. 12, suggesting that this clone failed to express a functional GMR a. In contrast, both clone nos 9 and 13 expressed low affinity receptors with KD = 860 and 21000 pM and 7700 and 450 receptors per cell respectively. Both clones presumably fail to express a functional GMR ,B. However, clone a/fl 1 expressed 1865 high affinity receptors/cell (KD = 20 pM, Figure 1) and clone a/$ 14 1325 receptors (KD = 80 pM). Interestingly, low affinity binding sites were not detectable in a/fl 1 or 14 clones, suggesting that the GMR a chain was not overexpressed on the cell surface relative to GMR f.
Table I. Binding characteristics of NIH3T3 cells transfected with cDNAs encoding the a and ,B subunit of the human GM-CSF receptor KD (pmol)
Cells/clones
Bmax
NIH3T3 Ctrl No. 12 No. 13 No. 9 a/IS 14 a/IS 1
0 0 450 7700 1325 1865
0 0 >
loooa 860 81 19
aBecause of the low number of receptors per cell and the highest concentration of 1.3 nM of radiolabeled ligand, the measured value for the KD is necessarily estimated in this particular experiment.
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Bound(pMoI) Fig. 1. Binding of [1251]GM-CSF by NIH3T3 a/,8 1 cells. Duplicate suspensions of a/,S 1 (2.5 x 105 cells) were incubated with [1251]GM-CSF either in the presence or absence of an excess of unlabeled GM-CSF at a final concentration of -425 nM as described in Materials and methods. The specific binding data were fitted by least square regression analysis and Scatchard plots are shown. The insert represents equilibrium binding non-specific binding a// 1. profiles. 0, specific binding a/,3 1; ],
1648
Reconstructed human GM-CSF receptor
Screening for evidence of functional receptors in NIH3T3 fibroblasts transfected with human GMR a//,8 cDNA In order to screen for evidence of functional human GM-CSF receptors in transfected NIH3T3 cells individual isolates of all 17 transfected clones were studied for human GM-CSFinduced signal transduction. Cells were starved overnight in Dulbecco's modified Eagle's medium (DMEM) plus 1 % bovine serum albumin (BSA), stimulated with 20 ng/ml human GM-CSF for 10 min and the induction of protein tyrosine phosphorylation was studied by Western blotting using an anti-phosphotyrosine monoclonal antibody. Three out of 17 clones showed GM-CSF-dependent induction of protein tyrosine phosphorylation and the results from four representative clones are shown in Figure 2A. Whereas no change in protein tyrosine phosphorylation was observed for clones nos 9 (low affinity) and 12 (no binding), GM-CSF induced tyrosine phosphorylation of identical proteins in a/8l 1 and 14 and also in a third responsive clone (Figure 2A and data not shown). Stimulation of clone no. 9 with up to 5 ,tg/ml GM-CSF (- 190 nM) failed to activate protein tyrosine phosphorylation suggesting that only the high affinity receptor containing GMR a and ,B was capable of inducing tyrosine kinase activity in fibroblasts (data not shown). The major proteins phosphorylated on tyrosine residues in response to GM-CSF were of estimated molecular weights 42, 44, a doublet of 52/53 and 58-60 kDa (Figure 2A). Minor proteins of 90 and 120 kDa were detectable in some,
but not all blots. As anticipated, there was no change in the pattern of tyrosine phosphorylated proteins after GM-CSF stimulation in the untransfected NIH3T3 cells (Figure 2B). Furthermore, the observed induction of tyrosine phosphorylation in the responsive clone cd/( 1 was completely abolished by adding an excess of a neutralizing anti-GM-CSF monoclonal antibody (mAb no. 1089, Figure 2B) (Kanakura etal., 1991a). Transient tyrosine phosphorylation induced by GMCSF in GMR a//, transfected NIH3T3 cells The kinetics of GM-CSF-induced tyrosine phosphorylation were investigated in transfected NIH3T3 cells. As shown for clone a/0 1 in Figure 3, tyrosine phosphorylation of p52/53 and p58 -60 was detectable after 30 s and 1 h with no major change in intensity. However, p42 was first clearly detectable after 2 min and p44 was first detected at 5 min. Tyrosine phosphorylation of both proteins peaked in parallel between 5 and 20 min and decreased in intensity after 1 h. Similar results were found for the third GM-CSF-responsive clone (a/0 10, data not shown). Since both the molecular weight and the observed time course of induction suggested that p42 and p44 might be MAP kinases (Okuda et al., 1992), we immunoprecipitated tyrosine phosphorylated proteins from GM-CSF-treated and -untreated a/0 1 and control NIH3T3 cells and immunoblotted the precipitates using a specific anti-MAP kinase antiserum. As shown in Figure 4, GM-CSF induced tyrosine phosphorylation of p42 S:I f
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Fig. 2. (A) Induction of tyrosine phosphorylation in individual NIH3T3 clones transfected with cDNAs encoding GMR a and ,B after stimulation with GM-CSF. Individual clones of NIH3T3 cells transfected with pREP8-GMRa and pREP4-GMR,B (nos 9, 12, cx/, I and cx/f 14) were screened for induction of tyrosine phosphorylation after stimulation with GM-CSF. Cells were starved overnight in DMEM plus 1% BSA and lysed either unstimulated (-) or after stimulation with 20 ng/ml GM-CSF for 10 min (+). Lysates were separated by SDS-PAGE and proteins containing phosphotyrosine were detected by inumunoblotting with anti-phosphotyrosine monoclonal antibody 4G10 as described in Materials and methods. Proteins phosphorylated on tyrosine residues in response to GM-CSF are marked by arrows. Molecular weight markers are on the left in kDa. (B) Inhibition of GM-CSF-induced tyrosine phosphorylation in csIl 1 cells by neutralizing anti-GM-CSF antibody. Untransfected NIH3T3 (NIH3T3 Ctrl.) or ca/l 1 cells were starved overnight in DMEM plus 1% BSA and lysed either unstimulated (0) or after incubation for 10 min with either neutralizing anti-GM-CSF antibody 1089 (Kanakura et al., 1991a, 25 ptg/ml), GM-CSF (5 ng/ml) or both. Immunoblotting was performed as described for (A). Proteins phosphorylated on tyrosine residues in response to GM-CSF are marked by arrows. Tyrosine phosphorylation of p44 MAP kinase in ca/j 1 cells stimulated with GM-CSF is clearly detectable on the original blot, but did not reproduce well in the figure. Molecular weight markers are on the left in kDa.
1649
M.Eder, J.D.Griffin and T.J.Ernst
stimulation with either 20% fetal calf serum (FCS) or PDGF in both oa/3 1 and untransfected NIH3T3 cells with almost identical time courses (Figure 4B and data not shown). FCS also induced tyrosine phosphorylation of proteins of estimated molecular weights of 52/53, 58-60 and 140 kDa (Figure 4B). Interestingly, most of these proteins co-migrated with those phosphorylated in response to GM-CSF, at least at the level of one-dimensional gel electrophoresis. However, the tyrosine phosphorylation of the 140 kDa protein shown
and p44 proteins immunoreactive with anti-MAP kinase antiserum in a/fl 1 cells, but not in untransfected NIH3T3 cells (Figure 4A). The proteins identified by anti-MAP kinase antiserum co-migrated with the p42 and p44 proteins seen in anti-phosphotyrosine blots of whole cell lysates from o/fl 1 cells after stimulation with GM-CSF (Figure 4B). Whereas GM-CSF-induced tyrosine phosphorylation is unique to GMR a/f-expressing clones, p42 and p44 MAP kinases were phosphorylated on tyrosine residues after
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Fig. 3. Time course of tyrosine phosphorylation induced by GM-CSF in a/3 1 cells. a/fl 1 cells were starved overnight and stimulated with 20 ng/ml GM-CSF for 0 to 60 min prior to lysis as described in Materials and methods. Lysates were immunoblotted with anti-phosphotyrosine antibody and proteins phosphorylated on tyrosine residues in response to GM-CSF are marked by arrows. This figure is representative of several experiments on two different cell lines.
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Fig. 4. Tyrosine phosphorylation of MAP kinases induced by GM-CSF in a/c3 I cells. Untransfected NIH3T3 (Ctrl) and ce/B 1 cells were starved overnight and lysed either unstimulated (-) or after stimulation with either 20 ng/ml GM-CSF (GM) or FCS at a final concentration of 20% (FCS,
a/0l 1) for 10 min. Lysates were either immunoprecipitated with anti-phosphotyrosine antibody before immunoblotting with anti-MAP kinase antiserum (A) or whole cell lysates were immunoblotted with anti-phosphotyrosine antibody (B). The migrations of p42 and p44 are indicated by arrows. The arrowhead marks a protein which is only phosphorylated on tyrosine in response to FCS as compared to GM-CSF in oa/f 1 cells. The asterisk indicates a protein either dephosphorylated or with altered electrophoretic mobility in response to both GM-CSF and FCS in a/fl 1 cells. The prestained molecular weight markers where the membrane was cut before incubation with the respective antibodies are shown on the left-hand side.
1650
Reconstructed human GM-CSF receptor
in Figure 4B seems to be unique to stimulation with serum. In the experiment shown in Figure 4B, both GM-CSF and FCS induced either tyrosine dephosphorylation or a mobility shift of protein(s) 72-75 kDa in a/fl 1 cells. In untransfected NIH3T3 cells, only FCS induced this dephosphorylation/mobility shift. Kanakura et al. (199 lb) previously showed that GM-CSF induced the phosphorylation of Raf-I in myeloid cells resulting in an electrophoretic mobility shift of the phosphorylated Raf-I protein. Because of the recently observed link between activation of Raf-1 and MAP kinases (Dent et al., 1992; Howe et al., 1992; Kyriakis et al., 1992), we examined the phosphorylation status of Raf-1 in NIH3T3 cells. Whole cell lysates of unstimulated and stimulated cells were separated by SDS - PAGE and transferred to a nitrocellulose membrane. The upper part of the membrane (molecular weights > 49.5 kDa) was blotted with anti-raf antiserum (Figure SA), and the lower part (molecular weights 1.5-fold increase in DNA synthesis in response to GM-CSF in either a/03 1 or 14 cells. This minimal effect on DNA synthesis which, however, was not observed in untransfected NIH3T3 cells, was true whether confluent or subconfluent cultures were starved in 0, 0.5 or 1.0% FCS, or whether [3H]TdR was added simultaneously with GM-CSF or after an 8 h delay (data not shown). In contrast, FCS (20% final concentration) induced up to a 15-fold increase in DNA synthesis over controls after all starvation conditions. Furthermore, no additional stimulation was induced by GM-CSF when added to cells growing in 10% FCS or when given in combination with PDGF or platelet poor plasma. The thymidine uptake experiments were confirmed by cell cycle analysis after staining of cycling, starved and restimulated cells with propidium iodide (Figure 7). Whereas serum induced synchronized re-entry into the cell cycle of starved cells (NIH3T3 cells, Figure 7 and a/,B 1 cells, data not shown), GM-CSF failed to do so. Similarly, the batch of NIH3T3 cells used in these experiments also proliferated only modestly in response to PDGF after serum starvation, and it is possible that other fibroblast cell lines would have #9 M -CSF
60
0
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Fig. 5. Induction of phosphorylation of Raf-l by GM-CSF in a/cl 1 cells. Untransfected NIH3T3 cells (NIH3T3 Ctrl.), a/(3 1 cells and cells from clone no. 9 (low affinity binding) were starved overnight and stimulated for the indicated periods of time with either FCS (NIH3T3 Ctrl.) or GM-CSF (cx/l( 1, no. 9) respectively. Cells were lysed thereafter and proteins separated by SDS-PAGE and transferred to a nitrocellulose membrane. The membrane was cut along the 49.5 kDa marker and the upper part (A) was immunoblotted with affinty-purified anti-Raf-l antiserum and the lower part (B) with anti-phosphotyrosine monoclonal antibody 4G10 as described in Materials and methods. The bands representing underphosphorylated (p RAF-1) and hyperphosphorylated Raf-l (pp RAF-l) are marked by arrows. Induction of tyrosine phosphorylation of p42 MAP kinase is indicated by the arrow in (B). Tyrosine phosphorylation of p44 MAP kinase is clearly detectable on the original blot, but hard to see on the photograph.
1651
M.Eder, J.D.Griffin and T.J.Ernst B - actin
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Fig. 6. Expression of immediate early response genes in a/,8 1 cells induced by GM-CSF. NIH3T3 cells and a/0 1 cells were starved for 24 h and stimulated for 0 to 24 h with either FCS (NIH3T3), GM-CSF (a/0 1) or PDGF (NIH3T3). Cells were lysed with guanidinium isothiocyanate and RNA was isolated as described in Materials and methods. Total RNA (14 ,ug) per lane was electrophoresed and transferred to nitrocellulose membranes. The membranes were hybridized with 32P-labeled cDNA probes specific for either murine c-fos (2.2 kb transcript), c-nyc (2.4 kb transcript) or B-actin (2.2 kb transcript) respectively. Autoradiographic exposure times were 5 days for c-myc, 6 days for c-fos and 1 day (serum) and 2 days (GM-CSF and PDGF) for fi-actin respectively. Time points are given in h.
different responses to both GM-CSF and PDGF. These experiments suggest that the GM-CSF-induced signal is not sufficient to make the GM-CSF receptor-positive clones a/I 1 and 14 pass the GI/S boundary. We confirmed that the vectors used to express GM-CSF receptors in fibroblasts encoded fully functional and : subunits by introducing the same vectors into the IL-3dependent murine pro-B cell line BAF-3. Stable lines which expressed high affinity human GM-CSF receptors and which could proliferate in response to human GM-CSF were isolated and grown in 10 ng/ml human GM-CSF (data not shown). Production of similar murine hematopoietic cell lines has been reported (Kitamura et al., 1991a). a
Discussion The GM-CSF receptor is a member of the cytokine receptor family which also includes the receptors for IL-3, 2, 4, 5, 6, 7, Epo, G-CSF, leukemia inhibitory factor (LIF), oncostatin M, ciliary neurotrophic factor, growth hormone and prolactin (D'Andrea et al., 1989; Bazan, 1990; Gearing et al., 1991, 1992; Tavernier et al., 1991; Ip et al., 1992). This large receptor family is defined by the presence of several structural motifs including a W-S-X-W-S sequence near the transmembrane domain, conservation of cysteines in the external domains and the canonical composition of two discrete extracellullar folding domains (Bazan, 1990). Moreover, for at least some of the receptors, such as the Epo and IL-2 receptors, limited, but significant, homology in amino acid sequence in the cytoplasmic domains has been observed (Hatakeyama et al., 1989; D'Andrea et al., 1991). At least two subfamilies have been identified that share a common subunit. The IL-3, GM-CSF and IL-5 receptors share a common a subunit, gpl20 (Hayashida et al., 1990; Kitamura et al., 1991b; Tavernier et al., 1991); and the 1652
IL-6, LIF, oncostatin M and ciliary neurotrophic factor receptors also share
a common
subunit,
gpl30
(Gearing
al., 1992; Ip et al., 1992). The human GM-CSF, IL-3 and IL-5 subunits bind their respective ligands with low affinity, and probably do not transduce any proliferative signals (Kitamura et al., 1991a). The common chain of this family, gp 120, does not bind any of the factors by itself, but converts each a subunit to high affinity binding, and is also likely to be responsible for signal transduction (Hayashida et al., 1990; Kitamura et al., 1991a). Signal transduction pathways activated by the receptors of the cytokine receptor family are not well characterized. Although none of these receptors have any intrinsic tyrosine kinase domains, many of them rapidly induce protein tyrosine phosphorylation. In the case of the GM-CSF receptor, activation of a tyrosine kinase occurs within 30 s of ligand binding, even at 4°C, and tyrosine kinase inhibitors block the biological effects of GM-CSF, suggesting that activation of a tyrosine kinase is a very proximal and critical signal transducing event in hematopoietic cells (Kanakura et al., 1990; Okuda et al., 1991). However, in preliminary studies we failed to find evidence for a tyrosine kinase in GM-CSF receptor complexes immunoprecipitated from activated hematopoietic cells. Similar studies have been done with the murine IL-3 receptor protein, AIC2A, again showing that no tyrosine kinase activity was co-precipitated (Schreurs et al., 1991). Nevertheless, it is likely that activation of one or more tyrosine kinase(s) is a critical event in GM-CSF-mediated signal transduction in hematopoietic cells. Although GM-CSF receptors have been found on umbilical vein endothelial cells (Bussolino et al., 1989) and on some carcinoma cell lines (Baldwin et al., 1989), expression of the GM-CSF receptor is largely limited to et
a
Reconstructed human GM-CSF receptor alp 1 / GM-CSF
NIH 3T3 Ctrl. / serum
cycling
starved 3
8 hours i
12 hours I
24 hours
36 hours
48 hours L-
Fig. 7. Propidium iodide staining of starved and restimulated NIH3T3 and a/(3 1 cells. NIH3T3 and a/c( 1 cells were assayed for the re-entry of starved cells into the cell cycle when stimulated with either serum or GM-CSF. Isolated nuclei from either cycling cells or cells starved for 24 h and restimulated with either FCS (NIH3T3) or GM-CSF (c/fl 1) for the indicated periods of time were stained with propidium iodide and analyzed by flow cytometry. Histograms are shown with the X axis representing the amount of DNA per nucleus. The two evident peaks represent 2N and 4N DNA respectively.
hematopoietic cells. It was therefore not clear whether nonhematopoietic cells such as fibroblasts would contain the necessary signal transduction components for function of these receptors. For example, some cytoplasmic signal transducing molecules that have been implicated in GM-CSF and IL-3 signal transduction e.g. p95vav, are expressed only in hematopoietic cells (Katzav et al., 1989; Mui et al., 1992). Similarly, non-myeloid cells may not express the same types of cytoplasmic tyrosine kinases that are presumably necessary to interact with the GM-CSF receptor. It could be anticipated that receptors with intrinsic tyrosine kinase activity might be less lineage-dependent for function. In fact, expression of c-fms, a transmembrane tyrosine kinase receptor for CSF-1, in NIH3T3 cells readily makes these cells CSF-1 responsive (Roussel et al., 1987). Similarly, expression of the EGF receptor in the 32D murine myeloid cell line converts these cells to EGF dependency (Pierce et al., 1988). However, even in the case of these potent tyrosine kinase receptors, there are cell lineage restrictions. For example, transfer of the EGF receptor into the pro-B cell line, BAF-3, is not sufficient to replace IL-2 unless c-myc is also constitutively overexpresssed (Shibuya et al., 1992). In an effort to find whether the GM-CSF receptor could function in a non-lineage-specific manner, we selected murine NIH3T3 fibroblasts for reconstitution studies. When cDNAs encoding both the cy and the j3 subunits were transfected, cell lines were obtained that had either high affinity, low affinity or no binding, consistent with successful
expression of both a and a subunits, ax subunits alone or lack of expression of a subunits respectively. In NIH3T3 cells low affinity GM-CSF receptors were not functional in terms of induction of tyrosine kinase activity, phosphorylation of Raf-1 or induction of c-fos and c-nyc mRNAs. This supports recent data demonstrating that a functional GMR ,B chain is essential for signal transduction in cell lines lacking endogenous expression of AIC2B (Kitamura et al., 1991a). However, GM-CSF was found to induce protein tyrosine phosphorylation of several proteins rapidly over a period of 2-5 min in independent NIH3T3 clones expressing high affinity receptors. Tyrosine phosphorylation of proteins of estimated molecular weights of 42, 44, 52/53 and 58-60 kDa in response to GM-CSF were found. Additional proteins of 90 and 120 kDa were detected in some, but not all experiments. Peak phosphorylation typically occurred by 20 min, followed thereafter by slowly decreasing levels of phosphotyrosine. These effects are kinetically very similar to the effects of GM-CSF in myeloid cell lines such as M07 (Kanakura et al., 1990). However, several of the proteins that are characteristically tyrosine phosphorylated in hematopoietic cells, such as p93 and p70 (Kanakura et al., 1990), were not observed in NIH3T3 cells, although we cannot exclude phosphorylation of these proteins at a low level. The induction of tyrosine phosphorylation in NIH3T3 cells was shown to be due to GM-CSF and not a contaminant by demonstrating that GM-CSF had no effect on control, untransfected cells. Also, the induction of tyrosine kinase activity by GM-CSF could be completely blocked by the
1653
M.Eder, J.D.Griffin and T.J.Ernst
addition of a neutralizing anti-GM-CSF monoclonal antibody. These results do not address the question of whether the GM-CSF receptor in NIH3T3 cells activates the same tyrosine kinase(s) that is activated in myeloid cells. The proteins that are phosphorylated in NIH3T3 cells are substantially different from those phosphorylated in hematopoietic cells, but this may only reflect a difference in expression of the substrates and not the kinase(s). It is apparent that the pattern of tyrosine phosphorylation observed in response to GM-CSF can differ even in different hematopoietic cell lineages. It is also possible that the GM-CSF receptor can activate different kinases in different cells. Support for this possibility comes from the observation that the IL-2 receptor has been reported to activate the lck tyrosine kinase in one cell type (Hatakeyama et al., 1991) and fyn in another (Torigoe et al., 1992b). We compared the patterns of tyrosine phosphorylation in NIH3T3 cells observed after stimulation with either serum, PDGF or GM-CSF, and found p42, p44, p52/53 and p58 -60 phosphorylated on tyrosine in response to all three stimuli. However, tyrosine phosphorylation of a 140- 150 kDa protein seemed to be unique to serum and PDGF as opposed to GM-CSF. Dephosphorylation or a shift in electrophoretic mobility of one phosphotyrosine-containing protein or proteins (p72 -75) was observed in response to either serum, GM-CSF or PDGF in some but not all experiments. We have not previously noted dephosphorylation of any proteins in hematopoietic cells in response to GM-CSF. Two of the proteins that were phosphorylated in response to GM-CSF in NIH3T3 cells have been identified as p42 and p44 MAP kinases. These two kinases are members of a family of serine/threonine kinases which are highly conserved through evolution and which have been implicated in mitogenic signal transduction from many different growth factor receptors, including the EGF and PDGF receptors (for review see Pelech et al., 1990; Sturgill and Wu, 1991), and Okuda et al. (1992) have previously shown that GM-CSF and IL-3 induce tyrosine phosphorylation and activation of p42 and p44 MAP kinases in M07 cells. Recent studies suggest the existence of a signaling cascade involving activation of Raf-1 which then activates MAP kinase-kinase, which in turn phosphorylates and activates MAP kinases (Dent et al., 1992; Howe et al., 1992; Kyriakis et al., 1992). Activated MAP kinases may induce c-fos transcription by phosphorylation of p62TCF and thereby generate a functional transcriptional complex at the serum-responsive element of the c-fos promoter (Gille et al., 1992). MAP kinases have also been reported to phosphorylate c-jun directly (Pulverer et al., 1991). Dephosphorylation of p42 MAP kinase by CD45 eliminates kinase activity, demonstrating the critical role of tyrosine phosphorylation in regulating the activity of MAP kinases (Anderson et al., 1990; Gomez and Cohen, 1991). The activation of the p42 and p44 MAP kinases has been observed so far in all cells which respond to GM-CSF, even in non-proliferative neutrophils (Okuda et al., 1992). The observation that phosphorylation of Raf-1 in human GMR a/3-transfected NIH3T3 cells in response to GM-CSF occurred at the same time as tyrosine phosphorylation of p42 and p44 MAP kinases is in accordance with the proposed model. GM-CSF was also found to induce transient accumulation of c-myc and c-fos mRNA in NIH3T3 cells expressing high 1654
affinity GM-CSF receptors, but not in control NIH3T3 cells. Induction of c-jun mRNA was also observed, but generally to a lesser extent that c-fos and c-myc. Both serum and PDGF also induced c-fos and c-myc mRNA with similar kinetics, further suggesting that there is considerable downstream overlap between the signal transduction pathways of serum, PDGF and GM-CSF in NIH3T3 cells. c-myc and c-fos are known to be induced in hematopoietic cells by GM-CSF (McColl et al., 1991; Schwartz et al., 1991) and c-fos induction may be mediated by activated MAP kinases (Gille et al., 1992). In contrast to c-fos, expression of c-myc, which might be independent of any tyrosine kinase activity (Shibuya et al., 1992), has been closely linked to cell cycle progression and proliferation (Goodrich and Lee, 1992), and it was of some interest that GM-CSF had minimal ability to induce DNA synthesis or proliferation of these cells. However, the NIH3T3 cells used in these experiments were quite sensitive to serum deprivation, and no suitable culture conditions were identified in which either GM-CSF or PDGF could function as an independent growth factor. In the presence of sufficient serum to maintain cell viability, GM-CSF did not augment growth. It may be useful, however, to evaluate the biological effects of GM-CSF in other NIH3T3 cell lines which are less sensitive to serum deprivation. Similarly, GM-CSF could not support the formation of colonies of NIH3T3 cells in agar (data not shown). Thus, in the NIH3T3 cells we studied here, despite the induction of early response genes such as c-myc and c-fos, the ligand-induced activation of the GM-CSF receptor was not sufficient to induce the cells to enter S phase at any significant rate in low serum medium. This was not due to a structural incompatibility between the human receptor and potential murine signal transducing molecules, as demonstrated by the reconstruction of a fully mitogenic human receptor in murine BAF-3 cells. These data suggest that either an additional activation pathway is missing in these NIH3T3 cells, or that the level of activation of the c-fos and c-myc pathways is below a threshold of necessary activity to induce DNA synthesis. Overexpression of c-myc may be useful in further defming the signaling defects of the GM-CSF receptor in NIH3T3 cells. The ability to reconstruct a high affinity, but incompletely functional, receptor in fibroblasts was also observed in the IL-2 receptor system. Expression of the a and f chains of the IL-2 receptor in L929 cells resulted in high affinity binding of IL-2, but no IL-2-dependent proliferation (Minamoto et al., 1990). This led to the conclusion that additional components restricted to hematopoietic cells are missing from fibroblasts. It is possible that the recently identified oy chain is one such component (Takeshita et al., 1992). Similarly, the GM-CSF receptor system we describe here in NIH3T3 cells might lend itself to further characterization of additional subunits of the GM-CSF receptor or components of the signal transduction system that must also be supplied from hematopoietic cells to rescue a fully functional receptor.
Materials and methods Genes and plasmids A cDNA encoding the human GM-CSF receptor (huGMR) a chain was cloned by reverse transcriptase PCR from peripheral blood monocyte mRNA. The cDNA was sequenced and shown to be identical to that previously published (Gearing et al., 1989). pKH97 containing the coding sequence for huGMR ,B (Hayashida et al., 1990) was a gift from Dr A.Miyajima (DNAX Research Institute, Palo Alto, CA). The coding sequence of GMR
Reconstructed human GM-CSF receptor cx was cloned into the XhoI site of pREP8 and the XhoI-XbaI fragment of pKH97, containing the coding sequence of GMR (, was blunt-end cloned into pREP4 (pREP4 and pREP8 were from Invitrogen Corporation, San Diego, CA). The vectors pREP4 and pREP8 each utilize the Rous sarcoma virus LTR for eukaryotic gene expression and, under control of the early SV40 promoter, the non-cross-reactive drug resistance genes, hygromycin B phosphotransferase and histidinol dehydrogenase respectively. The correct orientation of the cDNAs in the resultant plasmids, pREP8-GMRci and pREP4-GMR,B was confirmed by mapping with multiple restriction endonucleases.
Generation of murine fibroblast cell lines expressing huGMR a/,6 chains Plasmids were transfected into NIH3T3 fibroblasts (a gift from Dr R.Weinberg, MIT, Cambridge, MA) by the calcium phosphate precipitation method (Van der Eb and Graham, 1980). Briefly, the cells were plated at S x 105/60 mm tissue culture (TC) dish the day before transfection in 5 ml of DMEM (Gibco, Grand Island, NY) containing 10% FCS (Gibco). Supercoiled DNA, 20 itg of pREP8-GMRct and 200 jig of pREP4-GMR,3, was diluted in 1.5 ml of HEPES-buffered saline (HBS). CaCl2 (0.15 ml of 1.25 M) was added and the DNA was allowed to precipitate at 25°C for 30 min. The precipitant (0.5 ml) was then added to each plate. The plates were incubated for 5 h at 37°C, washed once with Hank's balanced salt solution (HBSS, without Ca or Mg) and 1 ml of 15 % glycerol in HBS was added for 4 min at 25°C. Plates were then washed once with HBSS and cultured in DMEM plus 10% FCS for 48 h. Thereafter, cells were trypsinized and replated into 100 mm TC dishes in L-histidine-free DMEM (Gibco) supplemented with 0.5 mM L-histidinol (Sigma, St Louis, MO) plus 10% FCS. Individual clones were isolated after 1 week and hygromycin B (150 ,ug/ml, Calbiochem, La Jolla, CA) was added for another 7 days. Stable cell lines were maintained in the same medium with 70 Itg/ml hygromycin B.
Binding of 1251-labeled GM-CSF
Control NIH3T3 cells and cells from clones nos 9, 12, 13, a/$ 1 and a/(3 14 were detached from TC flasks by incubation in sterile phosphate-buffered saline (PBS) plus 0.5 mM EDTA and 0.02% sodium azide at 37°C for 15-20 min. 106 cells (control, aI/f 14) and 2.5 x 105 (nos 9, 12, 13 and cr/3 1) were suspended in 100 A1 of binding buffer [RPMI 1640 (Gibco), 25 mM HEPES pH 7.5, 5% BSA] and 50 1l of binding buffer with or without 44.4 jig/ml unlabeled GM-CSF. After the addition of 50 A1 12511 labeled GM-CSF (NEN, Boston, MA) at different concentrations, duplicate samples were incubated for 60 min at 25°C. The samples were then centrifuged (14 000 g, 30 s) and the supernatant discarded. The cell pellet was resuspended in cold binding buffer, layered over a cushion of 0.7 ml of newborn calf serum, and spun in a microfuge at 14 000 g for 5 min at 25°C. After aspirating the supernatant, the cell pellets were cut into borosilicate counting tubes, and radioactivity was counted with an efficiency of 70% (LKB Gammamaster 1277, Uppsala, Sweden). Untransformed specific binding data were iteratively fitted by least square regression analysis using the LIGAND program (Biosoft, Cambridge, UK) (Munson and Rodbard, 1980). KD and Bmax represent the equilibrium dissociation constant and the density of the binding sites per cell in the saturation
experiments respectively.
Stimulation with GM-CSF and cell lysis Transfected NIH3T3 cells were grown in 100
mm dishes to
50-80%
confluence, washed twice with HBSS and starved overnight in DMEM plus either 1% BSA (Sigma) or 0.5 % FCS. Both starvation conditions were shown to give identical results. Cultures were stimulated by adding 20 ng/ml of sterile recombinant human GM-CSF (Schering Corporation, Kenilworth, NJ), incubated at 37°C and washed twice at the given time points in cold HBSS before being lysed with 100-150 pl lysis buffer (20 mM Tris pH 8.0, 137 mM NaCl, 10% glycerol, 1% Nonidet P40, 10 mM EDTA, 100 mM sodium fluoride) containing 1 mM PMSF, 0.15 U/ml aprotinin (Sigma), 2 mM sodium orthovanadate and 20 jg/ml leupeptin (Sigma). The cells and lysates were collected by scraping the dishes with a rubber policeman
and were then incubated for 25 min on ice. Insoluble material was removed by centrifugation at 10 000 g at 4°C for 15 min. To test the effect of the neutralizing anti-GM-CSF antibody 1089 (Kanakura et al., 1991a), GM-CSF (5 ng/ml), 1089 (25 ig/ml) or both of them previously preincubated for 45 min were added to the cultures for 10 min at 37°C before lysis was performed as described.
Gel electrophoresis and immunoblotting Lysates were boiled for 5 min in equal volume of 2 x sample buffer (125 mM Tris pH 6.8, 2% SDS, 288 mM ,B-mercaptoethanol, 20% glycerol, 10 /Ag/ml bromophenol blue) and 180 Ag lysate/lane were electrophoresed on 7.5%
SDS-polyacrylamide gels. After electrophoresis, proteins were electrophoretically transferred to Immobilon P membranes (0.45 Am, Millipore Corporation, Bedford, MA) either at 0.4 A for 4 h or at 0.1 A overnight. The membranes were then blocked with Tris-buffered saline (TBS) plus 5 % BSA for 30 min and incubated with the anti-phosphotyrosine antibody 4G10 (1:5000 in TBS plus 5% BSA) for 4 h (Kanakura et al., 1990). After washing in TBS plus 0.1% BSA, the blots were incubated with an alkaline phosphatase-conjugated goat anti-mouse antiserum (Promega Biotec, Madison, WI, 1:7500 in TBS plus 5% BSA) for 1-2 h, washed, and developed in 100 mM Tris pH 9.5, 100 mM NaCl, 5 mM MgCl2, 330 jg/ml of Nitro blue tetrazolium (Promega) and 165 Ag/ml of 5-bromo-4-chloro-3-indolyl phosphate (Promega). To study tyrosine phosphorylation of MAP kinases, the lysates of cells from two 100 mm plates, either with or without GM-CSF stimulation, were combined and immunoprecipitated for 5 h at 4°C with 40 ll/sample antiphosphotyrosine antibody 4G10 crosslinked to protein-A-sepharose beads (2 mg/ml). The beads were collected and washed three times with lysis buffer. The precipitated proteins were separated by SDS-PAGE, transferred to Immobilon P membranes and blotted with affinity-purified polyclonal MAP kinase antiserum (MAP 2 R2, a gift from Dr S.Pelech, University of British Columbia, Vancouver, BC, 1:40000 in TBS plus 5% BSA) for 3 h (Okuda et al., 1992). The incubation with an aLkaline phosphataseconjugated goat anti-rabbit antiserum (Promega) and the development of the blots were performed as described. Phosphorylation of Raf-1 was studied by SDS-PAGE of whole cell lysates before and after stimulation with subsequent transfer to a nitrocellulose membrane (0.2 1zm, Schleicher and Schuell, Keene, NH). The membrane was blocked with 2% gelatin (Biorad) in TBS for 1 h, washed with TBS + 0.2% Tween 20 (TBST, Biorad) and cut along the 49.5 kDa marker. The upper part was incubated with affinity-purified anti-Raf antiserum (1:3000 in TBS + 5% BSA, a gift from K.Wood) and the lower part with anti-phosphotyrosine antibody 4G10 for 4 h. After washing with TBST, the membranes were incubated with the second antibodies, washed and developed as described.
RNA extraction and Northern blot analysis After starvation for 24 h in DMEM plus 0.5% FCS, samples were stimulated with 20 ng/ml of GM-CSF (see above) for the indicated periods of time. Cells were washed twice with 4°C cold HBSS and lysed in situ with guanidium isothiocyanate. The lysates were extracted with acid phenol and the RNA was precipitated with isopropanol (Chomczynski and Sacchi, 1987). Total RNA (14 jg) was size-fractionated by electrophoresis through a 1% MOPS/formaldehyde agarose gel and transferred to NitroPlus nitrocellulose membranes (0.45 ym, MSI, Westboro, MA). cDNA probes for murine c-fos (- 1.6 kb PstI fragment), murine c-myc (- 1.4 kb XhoI fragment) and murine (3-actin ( -600 bp PstI fragment) respectively, were labeled with [32P]dCTP using the random hexamer priming method (Feinberg and Vogelstein, 1984). After overnight hybridization at 420C in a 50% formamide solution, filters were washed in l x SSC, 0.1 % SDS, at 65 OC and autoradiographed with fluorographic screens (DuPont, Boston, MA) at -700C.
Cell cycle analysis and propidium iodide staining NIH3T3 and a/,8 1 cells were plated in 60 mm TC dishes and grown to - 50% confluence. Cells were detached from the plates by incubation in PBS plus 0.5 mM EDTA either without any starvation or after starvation for 24 h in 0.5% FCS and restimulation with either FCS (20% fial concentration) or GM-CSF (20 ng/ml) for different periods of time. Cells were washed once in PBS and lysed in a buffer containing 0.1% NP-40, 0.1% Na citrate and 50 jig/ml propidium iodide (Sigma). The DNA content per nucleus was analyzed by flow cytometry using an EPICS 750 flow cytometer.
Acknowledgements The cDNAs of murine c-fos and c-myc were gifts from Dr Michael Greenberg and Julia Alberta (both Harvard Medical School, Boston, MA) respectively. This work was supported in part by a grant of the Deutsche Forschungsgemeinschaft (Ed 34/1-1) to M.E. and by PHS grants CA36167 and CA34183.
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