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Soluble c-kit receptor blocks stem cell factor bioactivity in vitro
Leukemia Research 25 (2001) 413– 421 www.elsevier.com/locate/leukres
Soluble c-kit receptor blocks stem cell factor bioactivity in vitro Debra D. Dahlen a, Nancy L. Lin a, Yun-Cai Liu b, Virginia C. Broudy a,* a
Department of Medicine, Di6ision of Hematology, Uni6ersity of Washington, Harbor6iew Medical Center, 325 9th A6enue, Seattle, WA 98104 -2499, USA b La Jolla Institute for Allergy and Immunology, San Diego, CA, USA Received 23 May 2000; accepted 11 September 2000
Abstract Stem cell factor (SCF) is a growth factor that promotes the survival, proliferation, and differentiation of hematopoietic cells. SCF and its receptor, Kit, are normally present in both cell surface and soluble forms. Both forms of Kit can bind SCF. However, the function of soluble Kit is unknown. In order to determine if soluble Kit can modulate SCF activity, we produced a fusion protein, Kit-Fc, comprised of the extracellular domain of murine Kit and the Fc portion of human IgG1 and investigated its ability to bind 125I-SCF and to inhibit SCF-stimulated hematopoietic colony growth in vitro. Stable cell lines expressing Kit-Fc were generated and Kit-Fc was purified to greater than 95% purity. Scatchard analysis demonstrated that Kit-Fc binds iodinated SCF with high affinity (Kd 570 pM). Kit-Fc also bound to transmembrane SCF displayed on the surface of fibroblasts. The murine mast cell line IC2 was engineered to express murine Kit on the cell surface and was demonstrated to proliferate in the presence of SCF. Kit-Fc completely blocked SCF-stimulated proliferation of IC2-Kit cells, but not IL-3-stimulated growth of IC2-Kit cells, demonstrating the specificity of Kit-Fc. We investigated the ability of Kit-Fc to block SCF-stimulated murine hematopoietic colony growth. Kit-Fc blocked SCF-stimulated erythroid colony growth as effectively as a neutralizing anti-Kit monoclonal antibody, ACK2, but did not block erythropoietin-stimulated erythroid colony growth. Likewise, Kit-Fc blocked SCF-stimulated myeloid colony growth as effectively as ACK2 antibody, but did not block IL-3- or GM-CSF-stimulated myeloid colony growth. These results indicate that a form of soluble Kit binds SCF with high affinity, and can specifically block the ability of SCF to stimulate hematopoietic colony growth, suggesting that one function of soluble Kit may be to modulate SCF bioactivity. © 2001 Elsevier Science Ltd. All rights reserved. Keywords: Stem cell factor; Kit; Hematopoiesis; Soluble Receptor
1. Introduction Normal hematopoiesis involves interactions among multiple cytokines, hematopoietic cells and the marrow microenvironment. Stem cell factor (SCF) is a hematopoietic growth factor that promotes the survival, proliferation, and differentiation of hematopoietic cells [1]. SCF exists normally in a transmembrane form dis-
Abbre6iations: SCF, Stem Cell Factor; BFU-E, Burst-Forming Unit-Erythroid; CFU-GM, Colony-Forming Unit GranulocyteMacrophage; FBS; Fetal Bovine Serum; BSA, Bovine Serum Albumin; PSF, Penicillin/Streptomycin/Fungizone; SDS-PAGE, Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis. * Corresponding author. Tel.: + 1-206-3415313; fax: +1-2063415312. E-mail address: [email protected] (V.C. Broudy).
played by marrow stromal cells [2,3] and in a soluble form in the serum [4]. Both forms of SCF have biological activity [5]. SCF triggers its biological effects by binding to a tyrosine kinase receptor, Kit. The phenotype of mice carrying mutations at either the Sl locus, which encodes SCF, or the W locus, which encodes Kit, provides information about the function of this ligand/receptor pair in vivo [6]. Mice lacking either SCF or cell surface Kit are nonviable and die in utero or in the perinatal period with severe macrocytic anemia. Mutations that alter the production of SCF or diminish the tyrosine kinase activity of Kit are associated with a myriad of phenotypic abnormalities including macrocytic anemia, decreased number of tissue mast cells, decreased fertility, and hypopigmentation, demonstrating an essential role for SCF and Kit.
0145-2126/01/$ - see front matter © 2001 Elsevier Science Ltd. All rights reserved. PII: S 0 1 4 5 - 2 1 2 6 ( 0 0 ) 0 0 1 2 2 - 3
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Kit is characterized by an extracellular binding region containing five immunoglobulin-like domains, a hydrophobic transmembrane region and an intracellular region containing a tyrosine kinase domain [7,8]. Binding of SCF induces Kit homodimerization and tyrosine phosphorylation of the receptor, initiating a cascade of signaling events [9]. Kit is displayed on normal hematopoietic cells and hematopoietic cell lines, and on some other cell types [1]. Most human hematopoietic cell lines studied manifest a single class of high-affinity SCF binding sites (Kd 50 – 200 pM), as do normal human hematopoietic cells [10 – 12]. Naturally occurring soluble extracellular domains have been detected for many cytokine receptors, and are generated by alternative mRNA splicing or by proteolytic cleavage of the transmembrane form of the receptor [13,14]. Soluble Kit is proteolytically cleaved from the surface of hematopoietic cells, mast cells, and endothelial cells [10,15,16], and circulates in normal human plasma [17]. Native soluble human Kit and cell surface Kit bind SCF with comparable affinity [10]. A recombinant soluble form of Kit, consisting of only the extracellular domain, can bind SCF, undergo ligand-induced dimerization, and antagonize SCF-induced tyrosine phosphorylation of fibroblast cell surface Kit in vitro [10,18,19]. However, the ability of soluble Kit to modulate hematopoietic colony growth has not been previously investigated. For these reasons we examined the ability of a soluble form of Kit to modulate SCF activity in vitro. We produced a fusion protein (Kit-Fc) consisting of the extracellular portion of murine Kit and the Fc portion of human IgG1. We describe the biochemical and functional properties of Kit-Fc, and examine the ability of Kit-Fc to block the effects of SCF in murine hematopoietic colony assays.
2.2. Cell lines The BHK cell line was maintained in Iscove’s modified Dulbecco’s medium (IMDM; GIBCO, Grand Island, NY) supplemented with 2% heat-inactivated fetal bovine serum (FBS; Summit, Ft. Collins, CO) and 1% penicillin/streptomycin/fungizone (PSF; GIBCO). The murine mast cell line IC2, which lacks endogenous Kit, was transduced with wild type murine c-kit cDNA (obtained from Dr Alan Bernstein, Mt. Sinai Hospital, Toronto, Ontario, Canada) using the MSCV retroviral vector [23]. Transduced cells were selected using G418 (GIBCO) and cloned by limiting dilution to obtain the IC2-Kit cell line. The IC2 and IC2-Kit cell lines were maintained in IMDM supplemented with 10% FBS, 1% PSF, and IL-3 (100 U/ml). The NIH3T3 murine fibroblast cell line was maintained in IMDM supplemented with 10% FBS.
2.3. Expression and purification of Kit-Fc The cDNA for the fusion protein Kit-Fc (Fig. 1) was provided by Dr Yun-Cai Liu [24]. This cDNA contains the extracellular ligand-binding domain of murine c-kit (1581 bp; residues 1–518) and a cDNA encoding the Fc portion of human IgG1 in a pEFneo plasmid vector. BHK cells were transfected with the pEFneo vector encoding Kit-Fc using the calcium phosphate precipitation method (Clontech Laboratories, Inc., Palo Alto, CA) according to the manufacturer’s instructions. Stable cell lines expressing Kit-Fc (BHK-Kit-Fc) were selected in G418. BHK-Kit-Fc cells were grown to 70% confluence, and then were switched to serum-
2. Materials and methods
2.1. Cytokines Purified recombinant rat SCF1-164 was provided by Amgen, Inc. (Thousand Oaks, CA). Recombinant murine granulocyte-macrophage colony-stimulating factor (GM-CSF) and recombinant murine interleukin3 (IL-3) were expressed in baby hamster kidney (BHK) cells and were provided by Dr Kenneth Kaushansky (University of Washington, Seattle, WA). Recombinant human erythropoietin (Epo) was expressed in BHK cells [20]. The ACK2 monoclonal antibody recognizes murine Kit and neutralizes the biological effects of SCF in vitro and in vivo [21,22], and was a generous gift from Dr Shin-Ichi Nishikawa (Kyoto University, Kyoto, Japan). The polyclonal antibody c951 – 960 that recognizes the extracellular domain of Kit was a generous gift from Amgen, Inc.
Fig. 1. Models of the structures of Kit (left panel) and the fusion protein Kit-Fc (right panel). Kit is characterized by an extracellular ligand-binding region containing five immunoglobulin-like domains, a hydrophobic transmembrane region, and an intracellular region with tyrosine kinase activity. Kit-Fc is a chimeric construct comprised of the extracellular domain of murine Kit linked to the Fc portion of human IgG1.
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freee medium (IMDM alone) and incubated for another 48 h. Protein A sepharose beads (Pharmacia LKB, Uppsala, Sweden) were used to purify Kit-Fc from serum-free BHK-Kit-Fc supernatant. Approximately 2 ml of a 50% slurry of protein A beads was mixed with each milliliter of BHK-Kit-Fc supernatant, rocked at 4°C overnight, and the beads were collected into a Pasteur pipette plugged with glass wool. Kit-Fc was eluted off the beads by shift to pH 2.8 using ImmunoPure G Elution Buffer (Pierce, Rockford, IL) as described by the manufacturer. The protein concentration in the column fractions was determined using a modified Bradford protein assay (Bio-Rad Laboratories, Hercules, CA). Aliquots with the highest protein concentration were pooled, dialyzed with phosphate-buffered saline, sterilized using a 0.22 mm filter (Millipore, Bedford, MA) and stored at −20°C. After boiling in sodium dodecyl sulfate (SDS) sample buffer with or without 50 mM dithiothreitol, the purified Kit-Fc protein was analyzed by SDSPAGE [10]. The proteins were transferred to nitrocellulose membranes (Bio-Rad, Richmond, CA), and the identity of Kit-Fc was confirmed by Western blotting with the c951 –960 anti-Kit antibody. The purity of Kit-Fc was analyzed by SDS-PAGE and staining with Coomassie Blue R-250 (Sigma).
2.4. Affinity binding studies Purified recombinant rat SCF1-164 was iodinated by the chloramine-T method [10,16]. The radiologic specific activity of 125I-SCF was determined by self-displacement analysis [25]. Purified Kit-Fc (2 nM) was incubated for 1 h at 37°C with varying concentrations of 125I-SCF (50 pM – 7 nM) with or without a 75-fold excess of unlabeled SCF as previously described [10]. The 125I-SCF-Kit-Fc complexes were immunoprecipitated by the addition of protein A sepharose beads. After a 2 h incubation at room temperature, the beads were separated from the media containing free 125I-SCF by sedimentation through phthalate oil (dibutyl phthalate:dinonyl phthalate 5:3). The dibutyl phthalate oil and the dinonyl phthalate oil were obtained from Sigma and from Fluka (Milwaukee, WI), respectively. Immunoprecipitated and free 125I-SCF were quantitated using a Packard 5330 gamma counter (Packard Instrument Co., Downers Grove, IL). Measurements were performed in duplicate and the binding data was analyzed using the LIGAND program [26] to determine the binding affinity (Kd) of Kit-Fc for SCF. Binding studies were performed in parallel to determine the Kd of murine cell surface Kit. IC2-Kit cells were used for this purpose because five separate binding assays with normal murine marrow cells (up to 4×106 cells/100 ml) failed to yield adequate reproducible data for Scatchard analysis. IC2-Kit cells (5× 105) were
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incubated for 1 h at 37°C with varying amounts of 125 I-SCF (10 pM –5 nM) with or without a 75-fold excess of unlabeled SCF in binding buffer consisting of RPMI 1640 (M.A Bioproducts, Walkersville, MD) supplemented with 1% bovine serum albumin (BSA; Sigma), 50 mM HEPES (pH 7.4), 0.1% sodium azide, and 10 mg/ml cytochalasin B (Sigma). At the end of incubation, the cells were separated from the media containing free 125I-SCF by sedimentation through phthalate oil (dibutyl phthalate:dinonyl phthalate 3.5:2.0). Cell-associated and free 125I-SCF were quantitated and the data were analyzed as described above.
2.5. Cell proliferation assays The function of Kit-Fc was tested in cell proliferation assays using IC2-Kit cells in suspension culture. IC2Kit cells were washed to remove IL-3, then suspended at a concentration of 1.5×105/ml in IMDM supplemented with 10% FBS, 1% PSF, and varying concentrations of SCF (10 –1000 ng/ml) in 24-well plates (Becton Dickinson, Lincoln Park, NJ) to determine the optimal concentration of SCF for cell growth. IC2-Kit cells incubated with IL-3 (100 U/ml) were used as positive controls. IC2-Kit cells were incubated in parallel in IMDM, 10% FBS, 1% PSF, and SCF plus varying concentrations of Kit-Fc (1–10 mg/ml) to examine the effect of Kit-Fc on SCF-dependent cell proliferation. IC2-Kit cells incubated in the presence of the neutralizing anti-Kit receptor monoclonal antibody, ACK2 (25 mg/ml), were used as positive controls for blockade of SCF-dependent cell proliferation. All determinations of cell proliferation were performed in triplicate, and viable cells were quantitated on day 4 on the basis of Trypan blue exclusion using a hemocytometer.
2.6. Hematopoietic colony assays These studies were approved by the Animal Care Committee at the University of Washington. To determine whether purified Kit-Fc could block SCF-stimulated hematopoietic colony growth in vitro, normal murine marrow cells were obtained from the femurs of BDF1 mice (Jackson Laboratory, Bar Harbor, ME) and cultured in erythroid and myeloid hematopoietic colony assays as described previously [22]. For quantitation of burst-forming unit-erythroid (BFU-E)-derived colonies, the marrow cells (0.75×105/ml) were plated in 1.4% methylcellulose in IMDM supplemented with 30% FBS, 1% BSA, 5×10 − 5 M b-mercaptoethanol (Sigma), 1% PSF, and 2 U/ml Epo in triplicate plates with SCF (100 ng/ml) alone or SCF plus either Kit-Fc (10 mg/ml) or ACK2 (25 mg/ml) as previously described. Colony assays performed in the presence of IL-3 (100 U/ml) with or without Kit-Fc or ACK2 served as controls for non-specific toxicity. Hemoglobinized
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Fig. 2. Purification of Kit-Fc (left panel) and analysis of the purity of Kit-Fc (right panel). Serum-free culture media from a BHK cell line expressing the fusion protein Kit-Fc was incubated with protein A sepharose beads. The beads were suspended in SDS-PAGE buffer containing dithiothreitol, size-fractionated on a 7% polyacrylamide gel, transferred to nitrocellulose, and probed with polyclonal anti-Kit antibody c951– 960. Molecular weight markers are indicated. The single prominent band at approximately 110 kDa represents monomeric Kit-Fc (left panel). Purified Kit-Fc was size-fractionated on a 7% polyacrylamide gel under reducing conditions. Coomassie blue staining revealed a single band at approximately 110 kDa (right panel).
colonies were counted after incubation for 8 days at 37°C in a humidified atmosphere containing 5% CO2. For quantitation of colony-forming unit granulocytemacrophage (CFU-GM)-derived colonies, the marrow cells (0.75× 105/ml) were plated in triplicate in 1.4% methylcellulose (Dow Chemical Co., Midland, MI) in IMDM supplemented with 10% FBS, 1% BSA, and SCF (100 ng/ml) plus IL-3 (100 U/ml) with varying concentrations of Kit-Fc (1, 3, or 10 mg/ml). Purified human Fc (Jackson ImmunoResearch, West Grove, PA) was used as a control. Plates containing purified ACK2 (25 mg/ml) served as positive controls for the effects of blockade of SCF-Kit interaction. Colony assays performed in the presence of GM-CSF (10 ng/ ml) with or without Kit-Fc or ACK2 served as controls for non-specific toxicity. After incubation for 5 days, the number of myeloid colonies was counted.
2.7. Binding of Kit-Fc to fibroblasts Murine microenvironmental cells were obtained by culturing marrow cells in IMDM supplemented with 10% FBS. The non-adherent cells were removed, and the adherent cells were trypsinized and replated twice. After a 2 week period in culture, the adherent fibroblast-like cells were detached with versene (Bio-Whittaker, Walkersville, MD), and incubated with Kit-Fc (5 mg/ml) in the absence or presence of SCF (5 mg/ml) for 60 min at room temperature. NIH3T3 cells were incubated with Kit-Fc without or with SCF in parallel. The cells were washed with phosphate-buffered saline containing 0.5% bovine serum albumin, then incubated with donkey anti-human IgG-FITC (Jackson Im-
munoResearch) for 45 min at 4°C, washed, fixed in 0.5% paraformaldehyde, and analyzed by flow cytometry.
3. Results
3.1. Expression of Kit-Fc in BHK cells and purification of Kit-Fc A total of 18 BHK-Kit-Fc subclones were initially screened for production of Kit-Fc by Western blotting using the polyclonal antibody c 951 –960 that recognizes the extracellular domain of Kit. Under reducing conditions, the molecular weight of Kit-Fc was estimated to be approximately 110 kDa (Fig. 2). Under non-reducing conditions, the molecular weight of KitFc was estimated to be approximately 210 kDa (data not shown). This difference in molecular weight found under reducing versus non-reducing conditions is consistent with the predicted dimeric structure of Kit-Fc. Four of the subclones were selected for isolation and purification of Kit-Fc. Repeat analysis of the supernatant after 4 months of culture demonstrated stable production of Kit-Fc by the BHK cells. The modified Bradford protein assay revealed that the concentration of Kit-Fc in the BHK serum-free supernatant was approximately 3 mg/ml. Kit-Fc was purified and concentrated from the serum-free supernatant using protein A Sepharose beads. Analysis of the protein A-purified Kit-Fc by SDS-PAGE and Coomassie Blue R-250 staining demonstrated that the purity of Kit-Fc was greater than 95% (Fig. 2).
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3.3. SCF dose-response cur6e of IC2 -Kit cells Parental IC2 cells proliferate in the presence of IL-3 but do not proliferate in the absence of growth factor or in SCF (data not shown). Expression of Kit in IC2 cells confers the ability to proliferate in the presence of SCF (Fig. 5). Dose-response experiments showed that maximal proliferation of IC2-Kit cells was observed at an SCF concentration of 100 ng/ml.
3.4. Effect of Kit-Fc on SCF-dependent cell proliferation Fig. 3. Scatchard analysis of 125I-SCF binding to Kit-Fc. Purified Kit-Fc (2 nM) was incubated for 1 h at 37°C with varying concentrations of 125I-SCF (50 pM–7 nM) with or without a 75-fold excess of unlabeled SCF. The SCF-Kit-Fc complexes were immunoprecipitated by the addition of protein A sepharose beads. Measurements were performed in duplicate and the data were analyzed using the ligand program. The results of one of 4 similar experiments are shown.
3.2. SCF binding affinity of Kit-Fc and of transmembrane murine Kit The SCF binding affinity of Kit-Fc was determined by equilibrium binding analysis. The soluble obligate dimer Kit-Fc displays high-affinity 125I-SCF binding (Kd 570940 pM, mean9 SEM of four experiments) (Fig. 3). Transmembrane murine Kit expressed on IC2 cells displays somewhat lower affinity 125I-SCF binding (Kd 2 90.2 nM, mean9SEM of 10 experiments) (Fig. 4). Both Kit-Fc and the transmembrane murine Kit exhibit a single class of SCF binding sites. The IC2-Kit cells express approximately 3000 receptors/cell (mean of 10 experiments).
Fig. 4. Scatchard analysis of 125I-SCF binding to cell surface Kit. IC2-Kit cells (5 ×105) were incubated for 1 h at 37°C with varying concentrations of 125I-SCF (10 pM–5 nM), with or without a 75-fold excess of unlabeled SCF. The cells were separated from the free 125 I-SCF by sedimentation through phthalate oil. Cell-associated and free 125I were quantitated using a Packard gamma counter and analyzed using the ligand program. Measurements were performed in duplicate. The results of one of 10 similar experiments are shown.
Kit-Fc was tested for the ability to block SCF-dependent cell proliferation of IC2-Kit cells in suspension culture. A dose-dependent abrogation of the stimulatory effects of SCF on IC2-Kit cell proliferation was observed (Fig. 6). At a concentration of 10 mg/ml, Kit-Fc completely blocked SCF-dependent IC2-Kit cell proliferation. At the lowest dose tested (1 mg/ml) the addition of Kit-Fc to IC2-Kit cells grown in the presence of SCF had a statistically significant effect on SCF-induced IC2-Kit proliferation (PB 0.02, Student’s t-test). The addition of Kit-Fc at a concentration of 10 mg/ml did not inhibit the ability of IC2-Kit cells to proliferate in the presence of IL-3 or GM-CSF (data not shown), indicating that Kit-Fc specifically inhibits SCF-stimulated proliferation.
3.5. Effect of Kit-Fc on erythroid and myeloid colony growth Kit-Fc was tested for the ability to block SCF-stimulated hematopoietic colony growth from normal murine marrow cells in vitro. The addition of Kit-Fc (10 mg/ml) blocked the synergistic effect of SCF plus Epo on BFU-E growth (Fig. 7). The ability of Kit-Fc to block SCF-stimulated erythroid colony growth was comparable to that of the neutralizing anti-Kit monoclonal antibody ACK2. The blockade of SCF-stimulated growth of BFU-E by Kit-Fc was observed to be specific, as the presence or absence of Kit-Fc did not alter the number of BFU-E found in cultures containing Epo plus IL-3 (Fig. 8). Kit-Fc also blocked SCF-stimulated growth of CFUGM (Fig. 9). Moreover, Kit-Fc demonstrated a dosedependent abrogation of the contribution of SCF to myeloid colony growth (Fig. 10). At the lowest dose tested (1 mg/ml) the number of CFU-GM colonies observed in the presence of SCF, IL-3, plus Kit-Fc was lower than the number of CFU-GM colonies observed in the presence of SCF plus IL-3 (PB 0.02, Student’s t-test). At a dose of 10 mg/ml, Kit-Fc was as effective as ACK2 in blocking the effects of SCF on myeloid
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Fig. 5. Dose response of SCF-stimulated IC2-Kit proliferation. IC2-Kit cells (1.5 ×105/ml) were cultured in IMDM supplemented with 10% FBS plus varying concentrations of SCF (0–1000 ng/ml). The data represent the mean 9SEM of triplicate values from one of 3 similar experiments.
colony growth. The addition of Kit-Fc (10 mg/ml) did not decrease the number of myeloid colonies found in the presence of GM-CSF. The Fc portion of Kit-Fc did not suppress myeloid colony growth in the presence of SCF or IL-3 (Table 1).
ability of SCF to induce tyrosine phosphorylation of Kit in a fibroblast cell line [18], but did not explore the effect of soluble Kit on normal hematopoietic colony growth. The concentration of soluble Kit in the circulation in normal adults is approximately 325 ng/ml, while the
3.6. Kit-Fc binding to fibroblasts Kit-Fc binds to the transmembrane form of SCF displayed by microenvironmental cells isolated from murine marrow (Fig. 11), and to SCF displayed by the NIH3T3 fibroblast cell line (Table 2). In the presence of SCF, binding of Kit-Fc to the fibroblasts decreases (Table 2), suggesting that the soluble and transmembrane forms of SCF can compete for Kit-Fc binding.
4. Discussion The present report demonstrates that a soluble form of Kit can bind SCF with high affinity (Kd 570 pM), and can modulate the ability of SCF to stimulate erythroid and myeloid hematopoietic colony growth in vitro. The effects of Kit-Fc are specific for SCF and are dose-dependent. Both the proliferative effects of SCF alone, and the synergistic effects of SCF plus Epo or SCF plus IL-3, are blocked by Kit-Fc. Prior studies have shown that soluble forms of Kit can inhibit the
Fig. 6. Kit-Fc blocks SCF-stimulated IC2-Kit proliferation. IC2-Kit cells were cultured in IMDM supplemented with 10% FBS plus no cytokine, SCF (100 ng/ml), SCF plus the anti-Kit antibody ACK2 (25 mg/ml), or SCF plus varying concentrations of Kit-Fc (1, 3, or 10 mg/ml). The data represent mean 9SEM of triplicate values from one of 2 separate experiments. The lowest concentration of Kit-Fc tested (1 mg/ml) blocked SCF-stimulated growth of IC2-Kit cells by greater than 80% (P B0.02, SCF vs. SCF plus Kit-Fc, Student’s t-test).
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Fig. 7. Effect of Kit-Fc on BFU-E growth. Murine marrow cells (7.5×105/ml) were cultured in semi-solid media in the presence of Epo (2 U/ml), Epo plus SCF (100 ng/ml), Epo plus SCF plus ACK2 (25 mg/ml), or Epo plus SCF plus Kit-Fc (10 mg/ml), and the number of BFU-E was counted on day 8. The data represent the mean 9 SEM of triplicate plates from one of 2 experiments (PB 0.02, Epo plus SCF vs. Epo plus SCF plus Kit-Fc, Student’s t-test).
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Fig. 9. Effect of Kit-Fc on CFU-GM growth. Murine marrow cells (1 × 105/ml) were cultured in semi-solid media in the presence of no added growth factor, SCF (100 ng/ml), SCF plus ACK2 (25 mg/ml), or SCF plus Kit-Fc (10 mg/ml). The number of CFU-GM was counted on day 5. The data represent the mean 9 SEM of triplicate plates from one of 5 similar experiments (PB 0.02, SCF vs. SCF plus Kit-Fc, Student’s t-test).
concentration of SCF is approximately 3 ng/ml [4,17]. These concentrations represent a 30-fold molar excess of soluble Kit over soluble SCF. The present experiments with Kit-Fc employed a range of concentrations of Kit-Fc, and showed that Kit-Fc at a concentration of 10 mg/ml is sufficient to completely ablate the proliferative effects of SCF 100 ng/ml (Figs. 6, 7, 9 and 10). The relative concentrations of Kit-Fc and SCF used in these studies reflect the physiologic differences found in vivo. Soluble Kit purified from human serum is of molecular weight 98 kDa in the absence of SCF [10,17], consistent with predominance of a monomeric form of soluble Kit. However, when native soluble Kit derived
from human serum is incubated with SCF, both monomeric and dimeric forms of Kit are identified [10]. Native soluble Kit binds 125I-SCF with high affinity, approximately 320 pM [10], similar to the affinity of the Kit receptor expressed on the surface of human hematopoietic cells [12]. Additionally, native soluble Kit can compete with cell surface Kit for binding 125I-SCF [10,17]. Taken together, these data support the concept that native soluble Kit is capable of binding SCF, and that one role of soluble Kit may be to downmodulate SCF bioactivity in vivo. Soluble Kit is generated by proteolytic cleavage of cell surface Kit [10,15,16]. Hematopoietic cells, mast cells, and endothelial cells shed Kit from the cell surface. The present results suggest that shedding of Kit
Fig. 8. Specificity of the effect of Kit-Fc on BFU-E growth. Murine marrow cells were cultured as described in Fig. 7 in the presence of Epo (2 U/ml), Epo plus IL-3 (100 U/ml), Epo plus IL-3 plus ACK2 (25 mg/ml), or Epo plus IL-3 plus Kit-Fc (10 mg/ml), and the number of BFU-E was counted. The data represent the mean 9 SEM of triplicate plates from one of 2 similar experiments.
Fig. 10. Dose-dependent effect of Kit-Fc on CFU-GM growth. Murine marrow cells were cultured as described in Fig. 9 in the presence of IL-3 (100 U/ml), SCF (100 ng/ml) plus IL-3, SCF plus IL-3 plus ACK2 (25 mg/ml), or SCF plus IL-3 plus Kit-Fc (1, 3, or 10 mg/ml), and the number of CFU-GM were counted. The data represent the mean 9 SEM of triplicate plates from one of 4 similar experiments. (PB0.02, SCF plus IL-3 vs SCF plus IL-3 plus Kit-Fc 1 mg/ml, Student’s t-test).
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Table 1 Effect of Kit-Fc and Fc on myeloid colony growtha
Table 2 Binding of Kit-Fc to NIH3T3 cellsa
Additions
cCFU-GM/105 Cells
Additions
Mean fluorescence intensity
SCF/Epo/IL-3 (10 U/ml)+0 SCF/Epo/IL-3 (10 U/ml)+ACK2 SCF/Epo/IL-3 (10 U/ml)+Kit-Fc SCF/Epo/IL-3 (10 U/ml)+Fc IL-3 (100 U/ml)+0 IL-3 (100 U/ml)+Kit-Fc IL-3 (100 U/ml)+Fc
47 7 5 73 84 86 85
0 Kit-Fc Kit-Fc+SCF
0.727 0.957 0.768
a Murine marrow cells were cultured in semi-solid media in the presence of SCF (100 ng/ml) plus Epo (1 U/ml) plus a low concentration of IL-3 (10 U/ml), or in a high concentration of IL-3 (100 U/ml) alone, in the absence or presence of ACK2 (25 mg/ml), Kit-Fc (7.5 mg/ml), or Fc (7.5 mg/ml). The data represents the number of myeloid colonies per 105 cells plated (average of duplicate values).
may modulate SCF bioactivity in two ways: by diminishing the density of Kit displayed on the cell surface, and by converting cell surface Kit to soluble Kit, which may serve as an antagonist of SCF bioactivity. The serum levels of soluble Kit increase when the population of cells that release Kit is pathologically expanded, such as in acute myelogenous leukemia [27,28]. Although much is known about intracellular Kit signaling pathways [9], and how these pathways can be biochemically inhibited [29], little is known about how proteolytic cleavage of Kit from the cell surface is regulated. Activation of protein kinase C enhances shedding of Kit [15]. Members of the disintegrin family of metalloproteinases cleave other cytokines and cytokine receptors from the cell surface [30,31], and are candidates for cleavage of Kit. The Kit-Fc fusion protein [24] was selected for study based on the demonstration that a soluble TNF receptor-Fc fusion protein could antagonize TNF activity in clinical studies [32]. The present report demonstrates that Kit-Fc can bind soluble SCF with high affinity, and can also bind to transmembrane SCF on the surface of fibroblasts, in accord with prior results using
a NIH3T3 cells were incubated 9Kit-Fc (1 mg/ml)9SCF (1 mg/ml) for 1 h at 22°C, then with donkey anti-human IgG-FITC for 45 min at 4°C, then analyzed by flow cytometry. The data represent the mean fluorescence intensity of 4500 cells analyzed.
an alkaline phosphatase-tagged form of soluble Kit [33]. Soluble and transmembrane SCF compete for binding of Kit-Fc. Kit-Fc can antagonize the biological effects of soluble SCF in hematopoietic colony assays; whether Kit-Fc can block the biological effects of transmembrane SCF remains to be determined. The availability of Kit-Fc offers the opportunity for in vivo studies to investigate whether soluble forms of Kit can modulate SCF bioactivity in vivo. Soluble and transmembrane SCF have distinct but overlapping roles [1,5,34 –36]. The present report suggests that soluble and transmembrane Kit may also have distinct roles, and that cleavage of Kit from the cell surface may serve to downregulate SCF signaling, both by decreasing the density of cell surface Kit, and by generating a soluble antagonist of SCF that competes with cell surface Kit for SCF binding.
Acknowledgements Supported by NIH grants DK 43719, CA 31615, and 5 T32 HL07093. DD Dahlen provided th econcept, design, assembled and anlayzed the data, drafted the paper and gave final apporval. NL Lin contributed to the study design, provided technical support, collecting and analyzing the data and gave final approval. Y-C Liu provided study materials, gave critical revision to the article and gave final approval. VC Broudy provided the necessary administrative support and funding for the project as well as contributing to the concept, deesign, data interpretation, assistance in tmanuscript preparationk, critical revision and gave final approval.
References
Fig. 11. Binding of Kit-Fc to murine marrow microenvironmental cells. Fibroblasts derived from primary culture of murine marrow cells were incubated without (pale line) or with (black line) Kit-Fc (5 mg/ml), then with donkey anti-human IgG-FITC, and analyzed by flow cytometry.
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D.D. Dahlen et al. / Leukemia Research 25 (2001) 413–421 [4] Langley KE, Bennett LG, Wypych J, Yancik SA, Liu X-D, Westcott KR, Chang DG, Smith KA, Zsebo KM. Soluble stem cell factor in human serum. Blood 1993;81:656. [5] Toksoz D, Zsebo KM, Smith KA, Hu S, Brankow D, Suggs SV, Martin FH, Williams DE. Support of human hematopoiesis in long-term bone marrow cultures by murine stromal cells selectively expressing the membrane-bound and secreted forms of the human homolog of the steel gene product, stem cell factor. Proc Natl Acad Sci USA 1992;89:7350. [6] Besmer P, Manova K, Duttlinger R, Huang EJ, Packer A, Gyssler C, Bachvarova RF. The kit-ligand (steel factor) and its receptor c-kit/W: pleiotropic roles in gametogenesis and melanogenesis, Development 1993; (suppl) 125. [7] Yarden Y, Kuang W-J, Yang-Feng T, Coussens L, Munemitsu S, Dull TJ, Chen E, Schlessinger J, Francke U, Ullrich A. Human proto-oncogene c-kit: A new cell surface receptor tyrosine kinase for an unidentified ligand. EMBO J 1987;6:3341. [8] Qiu F, Ray P, Brown K, Barker PE, Jhanwar S, Ruddle FH, Besmer P. Primary structure of c-kit: Relationship with the CSF-1/PDGF receptor kinase family-oncogenic activation of c-kit involves deletion of extracellular domain and C terminus. EMBO J 1988;7:1003. [9] Linnekin D. Early signaling pathways activated by c-Kit in hematopoietic cells. Intl J Biochem Cell Biol 1999;31:1053. [10] Turner AM, Bennett LG, Lin NL, Wypych J, Bartley TD, Hunt RW, Atkins HL, Langley KE, Parker V, Martin F, Broudy VC. Identification and characterization of a soluble c-kit receptor produced by human hematopoietic cell lines. Blood 1995;85:2052. [11] Abkowitz JL, Broudy VC, Bennett LG, Zsebo KM, Martin FH. Absence of abnormalities of c-kit or its ligand in two patients with Diamond-Blackfan anemia. Blood 1992;79:25. [12] Taniguchi S, Dai C-H, Price JO, Krantz SB. Interferon k downregulates stem cell factor and erythropoietin receptors but not insulin-like growth factor-I receptors in human erythroid colonyforming cells. Blood 1997;90:2244. [13] Rose-John S, Heinrich PC. Soluble receptors for cytokines and growth factors: Generation and biological function. Biochem J 1994;300:281. [14] Heaney ML, Golde DW. Soluble cytokine receptors. Blood 1996;87:847. [15] Yee NS, Langen H, Besmer P. Mechanism of kit ligand, phorbol ester, and calcium-induced down-regulation of c-kit receptors in mast cells. J Biol Chem 1993;268:14189. [16] Broudy VC, Kovach NL, Bennett LG, Lin N, Jacobsen FW, Kidd PG. Human umbilical vein endothelial cells display highaffinity c-kit receptors and produce a soluble form of the c-kit receptor. Blood 1994;83:2145. [17] Wypych J, Bennett LG, Schwartz MG, Clogston CL, Lu HS, Broudy VC, Bartley TD, Parker VP, Langley KE. Soluble kit receptor in human serum. Blood 1995;85:66. [18] Lev S, Yarden Y, Givol D. A recombinant ectodomain of the receptor for the stem cell factor (SCF) retains ligand-induced receptor dimerization and antagonizes SCF-stimulated cellular responses. J Biol Chem 1992;267:10866. [19] Philo JS, Wen J, Wypych J, Schwartz MG, Mendiaz EA, Langley KE. Human stem cell factor dimer forms a complex with two molecules of the extracellular domain of its receptor, Kit. J Biol Chem 1996;271:6895. [20] Broudy VC, Tait JF, Powell JS. Recombinant human erythropoietin: purification and analysis of carbohydrate linkage. Arch Biochem Biophys 1988;265:329.
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[21] Ogawa M, Matsuzaki Y, Nishikawa S, Hayashi S-I, Kunisada T, Sudo T, Kina T, Nakauchi H, Nishikawa S-I. Expression and function of c-kit in hemopoietic progenitor cells. J Exp Med 1991;174:63. [22] Broudy VC, Lin NL, Priestley GV, Nocka K, Wolf NS. Interaction of stem cell factor and its receptor c-kit mediates lodgment and acute expansion of hematopoietic cells in the murine spleen. Blood 1996;88:75. [23] Piao X, Bernstein A. A point mutation in the catalytic domain of c-kit induces growth factor independence, tumorigenicity, and differentiation of mast cells. Blood 1996;87:3117. [24] Liu Y-C, Kawagishi M, Kameda R, Ohashi H. Characterization of a fusion protein composed of the extracellular domain of c-kit and the Fc region of human IgG expressed in a baculovirus system. Biochem Biophys Res Commun 1993;197:1094. [25] Calvo JC, Radicella JP, Charreau EH. Measurement of specific radioactivities in labeled hormones by self-displacement analysis. Biochem J 1983;212:259. [26] Munson PJ, Rodbard D. LIGAND: a versatile computerized approach for characterization of ligand-binding systems. Anal Biochem 1988;107:220. [27] Kawakita M, Yonemura Y, Miyake Y, Ohkubo T, Asou N, Hayakawa K, Nakamura M, Kitoh T, Osawa H, Takatsuki K, Suda T. Soluble c-kit molecule in serum from healthy individuals and patients with haemopoietic disorders. British J Haematol 1995;91:23. [28] Tajima F, Kawatani T, Ishiga K, Nanba E, Kawasaki H. Serum soluble c-kit receptor and expression of c-kit protein and mRNA in acute myeloid leukemia. Eur J Haematol 1998;60:289. [29] Heinrich M, Griffith DJ, Druker BJ, Wait CL, Ott KA, Zigler AJ. Inhibition of c-kit receptor tyrosine kinase activity by STI571, a selective tyrosine kinase inhibitor. Blood 2000;96:925. [30] Peschon JJ, Slack JL, Reddy P, Stocking KL, Sunnarborg SW, Lee DC, Russell WE, Castner BJ, Johnson RS, Fitzner JN, Boyce RW, Nelson N, Kozlosky CJ, Wolfson MF, Rauch CT, Cerretti DP, Paxton RJ, March CJ, Black RA. An essential role for ectodomain shedding in mammalian development. Science 1998;282:1281. [31] Werb Z, Yan Y. A cellular striptease act. Science 1998;282:1279. [32] Moreland LW, Baumgartner SW, Schiff MH, Tindall EA, Fleischmann RM, Weaver AL, Ettlinger RE, Cohen S, Koopman WJ, Mohler K, Widmer MB, Blosch CM. Treatment of rheumatoid arthritis with a recombinant human tumor necrosis factor receptor (p75)-Fc fusion protein. New Engl J Med 1997;337:141. [33] Flanagan JG, Leder P. The kit ligand: a cell surface molecule altered in Steel mutant fibroblasts. Cell 1990;63:185. [34] Tajima Y, Moore MAS, Soares V, Ono M, Kissel H, Besmer P. Consequences of exclusive expression in vivo of kit-ligand lacking the major proteolytic cleavage site. Proc Natl Acad Sci USA 1998;95:11903. [35] Kapur R, Majumdar MK, Xiangli X, McAndrews-Hill M, Schindler K, Williams DA. Signaling through the interaction of membrane-restricted stem cell factor and c-kit receptor tyrosine kinase: genetic evidence for a differential role in erythropoiesis. Blood 1998;91:879. [36] Miyazawa K, Williams DA, Gotoh A, Nishimaki J, Broxmeyer HE, Toyama K. Membrane-bound steel factor induces more persistent tyrosine kinase activation and longer life span of c-kit gene-encoded protein than its soluble form. Blood 1995;85:641– 9.
Soluble c-kit receptor blocks stem cell factor bioactivity in vitro Debra D. Dahlen a, Nancy L. Lin a, Yun-Cai Liu b, Virginia C. Broudy a,* a
Department of Medicine, Di6ision of Hematology, Uni6ersity of Washington, Harbor6iew Medical Center, 325 9th A6enue, Seattle, WA 98104 -2499, USA b La Jolla Institute for Allergy and Immunology, San Diego, CA, USA Received 23 May 2000; accepted 11 September 2000
Abstract Stem cell factor (SCF) is a growth factor that promotes the survival, proliferation, and differentiation of hematopoietic cells. SCF and its receptor, Kit, are normally present in both cell surface and soluble forms. Both forms of Kit can bind SCF. However, the function of soluble Kit is unknown. In order to determine if soluble Kit can modulate SCF activity, we produced a fusion protein, Kit-Fc, comprised of the extracellular domain of murine Kit and the Fc portion of human IgG1 and investigated its ability to bind 125I-SCF and to inhibit SCF-stimulated hematopoietic colony growth in vitro. Stable cell lines expressing Kit-Fc were generated and Kit-Fc was purified to greater than 95% purity. Scatchard analysis demonstrated that Kit-Fc binds iodinated SCF with high affinity (Kd 570 pM). Kit-Fc also bound to transmembrane SCF displayed on the surface of fibroblasts. The murine mast cell line IC2 was engineered to express murine Kit on the cell surface and was demonstrated to proliferate in the presence of SCF. Kit-Fc completely blocked SCF-stimulated proliferation of IC2-Kit cells, but not IL-3-stimulated growth of IC2-Kit cells, demonstrating the specificity of Kit-Fc. We investigated the ability of Kit-Fc to block SCF-stimulated murine hematopoietic colony growth. Kit-Fc blocked SCF-stimulated erythroid colony growth as effectively as a neutralizing anti-Kit monoclonal antibody, ACK2, but did not block erythropoietin-stimulated erythroid colony growth. Likewise, Kit-Fc blocked SCF-stimulated myeloid colony growth as effectively as ACK2 antibody, but did not block IL-3- or GM-CSF-stimulated myeloid colony growth. These results indicate that a form of soluble Kit binds SCF with high affinity, and can specifically block the ability of SCF to stimulate hematopoietic colony growth, suggesting that one function of soluble Kit may be to modulate SCF bioactivity. © 2001 Elsevier Science Ltd. All rights reserved. Keywords: Stem cell factor; Kit; Hematopoiesis; Soluble Receptor
1. Introduction Normal hematopoiesis involves interactions among multiple cytokines, hematopoietic cells and the marrow microenvironment. Stem cell factor (SCF) is a hematopoietic growth factor that promotes the survival, proliferation, and differentiation of hematopoietic cells [1]. SCF exists normally in a transmembrane form dis-
Abbre6iations: SCF, Stem Cell Factor; BFU-E, Burst-Forming Unit-Erythroid; CFU-GM, Colony-Forming Unit GranulocyteMacrophage; FBS; Fetal Bovine Serum; BSA, Bovine Serum Albumin; PSF, Penicillin/Streptomycin/Fungizone; SDS-PAGE, Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis. * Corresponding author. Tel.: + 1-206-3415313; fax: +1-2063415312. E-mail address: [email protected] (V.C. Broudy).
played by marrow stromal cells [2,3] and in a soluble form in the serum [4]. Both forms of SCF have biological activity [5]. SCF triggers its biological effects by binding to a tyrosine kinase receptor, Kit. The phenotype of mice carrying mutations at either the Sl locus, which encodes SCF, or the W locus, which encodes Kit, provides information about the function of this ligand/receptor pair in vivo [6]. Mice lacking either SCF or cell surface Kit are nonviable and die in utero or in the perinatal period with severe macrocytic anemia. Mutations that alter the production of SCF or diminish the tyrosine kinase activity of Kit are associated with a myriad of phenotypic abnormalities including macrocytic anemia, decreased number of tissue mast cells, decreased fertility, and hypopigmentation, demonstrating an essential role for SCF and Kit.
0145-2126/01/$ - see front matter © 2001 Elsevier Science Ltd. All rights reserved. PII: S 0 1 4 5 - 2 1 2 6 ( 0 0 ) 0 0 1 2 2 - 3
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Kit is characterized by an extracellular binding region containing five immunoglobulin-like domains, a hydrophobic transmembrane region and an intracellular region containing a tyrosine kinase domain [7,8]. Binding of SCF induces Kit homodimerization and tyrosine phosphorylation of the receptor, initiating a cascade of signaling events [9]. Kit is displayed on normal hematopoietic cells and hematopoietic cell lines, and on some other cell types [1]. Most human hematopoietic cell lines studied manifest a single class of high-affinity SCF binding sites (Kd 50 – 200 pM), as do normal human hematopoietic cells [10 – 12]. Naturally occurring soluble extracellular domains have been detected for many cytokine receptors, and are generated by alternative mRNA splicing or by proteolytic cleavage of the transmembrane form of the receptor [13,14]. Soluble Kit is proteolytically cleaved from the surface of hematopoietic cells, mast cells, and endothelial cells [10,15,16], and circulates in normal human plasma [17]. Native soluble human Kit and cell surface Kit bind SCF with comparable affinity [10]. A recombinant soluble form of Kit, consisting of only the extracellular domain, can bind SCF, undergo ligand-induced dimerization, and antagonize SCF-induced tyrosine phosphorylation of fibroblast cell surface Kit in vitro [10,18,19]. However, the ability of soluble Kit to modulate hematopoietic colony growth has not been previously investigated. For these reasons we examined the ability of a soluble form of Kit to modulate SCF activity in vitro. We produced a fusion protein (Kit-Fc) consisting of the extracellular portion of murine Kit and the Fc portion of human IgG1. We describe the biochemical and functional properties of Kit-Fc, and examine the ability of Kit-Fc to block the effects of SCF in murine hematopoietic colony assays.
2.2. Cell lines The BHK cell line was maintained in Iscove’s modified Dulbecco’s medium (IMDM; GIBCO, Grand Island, NY) supplemented with 2% heat-inactivated fetal bovine serum (FBS; Summit, Ft. Collins, CO) and 1% penicillin/streptomycin/fungizone (PSF; GIBCO). The murine mast cell line IC2, which lacks endogenous Kit, was transduced with wild type murine c-kit cDNA (obtained from Dr Alan Bernstein, Mt. Sinai Hospital, Toronto, Ontario, Canada) using the MSCV retroviral vector [23]. Transduced cells were selected using G418 (GIBCO) and cloned by limiting dilution to obtain the IC2-Kit cell line. The IC2 and IC2-Kit cell lines were maintained in IMDM supplemented with 10% FBS, 1% PSF, and IL-3 (100 U/ml). The NIH3T3 murine fibroblast cell line was maintained in IMDM supplemented with 10% FBS.
2.3. Expression and purification of Kit-Fc The cDNA for the fusion protein Kit-Fc (Fig. 1) was provided by Dr Yun-Cai Liu [24]. This cDNA contains the extracellular ligand-binding domain of murine c-kit (1581 bp; residues 1–518) and a cDNA encoding the Fc portion of human IgG1 in a pEFneo plasmid vector. BHK cells were transfected with the pEFneo vector encoding Kit-Fc using the calcium phosphate precipitation method (Clontech Laboratories, Inc., Palo Alto, CA) according to the manufacturer’s instructions. Stable cell lines expressing Kit-Fc (BHK-Kit-Fc) were selected in G418. BHK-Kit-Fc cells were grown to 70% confluence, and then were switched to serum-
2. Materials and methods
2.1. Cytokines Purified recombinant rat SCF1-164 was provided by Amgen, Inc. (Thousand Oaks, CA). Recombinant murine granulocyte-macrophage colony-stimulating factor (GM-CSF) and recombinant murine interleukin3 (IL-3) were expressed in baby hamster kidney (BHK) cells and were provided by Dr Kenneth Kaushansky (University of Washington, Seattle, WA). Recombinant human erythropoietin (Epo) was expressed in BHK cells [20]. The ACK2 monoclonal antibody recognizes murine Kit and neutralizes the biological effects of SCF in vitro and in vivo [21,22], and was a generous gift from Dr Shin-Ichi Nishikawa (Kyoto University, Kyoto, Japan). The polyclonal antibody c951 – 960 that recognizes the extracellular domain of Kit was a generous gift from Amgen, Inc.
Fig. 1. Models of the structures of Kit (left panel) and the fusion protein Kit-Fc (right panel). Kit is characterized by an extracellular ligand-binding region containing five immunoglobulin-like domains, a hydrophobic transmembrane region, and an intracellular region with tyrosine kinase activity. Kit-Fc is a chimeric construct comprised of the extracellular domain of murine Kit linked to the Fc portion of human IgG1.
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freee medium (IMDM alone) and incubated for another 48 h. Protein A sepharose beads (Pharmacia LKB, Uppsala, Sweden) were used to purify Kit-Fc from serum-free BHK-Kit-Fc supernatant. Approximately 2 ml of a 50% slurry of protein A beads was mixed with each milliliter of BHK-Kit-Fc supernatant, rocked at 4°C overnight, and the beads were collected into a Pasteur pipette plugged with glass wool. Kit-Fc was eluted off the beads by shift to pH 2.8 using ImmunoPure G Elution Buffer (Pierce, Rockford, IL) as described by the manufacturer. The protein concentration in the column fractions was determined using a modified Bradford protein assay (Bio-Rad Laboratories, Hercules, CA). Aliquots with the highest protein concentration were pooled, dialyzed with phosphate-buffered saline, sterilized using a 0.22 mm filter (Millipore, Bedford, MA) and stored at −20°C. After boiling in sodium dodecyl sulfate (SDS) sample buffer with or without 50 mM dithiothreitol, the purified Kit-Fc protein was analyzed by SDSPAGE [10]. The proteins were transferred to nitrocellulose membranes (Bio-Rad, Richmond, CA), and the identity of Kit-Fc was confirmed by Western blotting with the c951 –960 anti-Kit antibody. The purity of Kit-Fc was analyzed by SDS-PAGE and staining with Coomassie Blue R-250 (Sigma).
2.4. Affinity binding studies Purified recombinant rat SCF1-164 was iodinated by the chloramine-T method [10,16]. The radiologic specific activity of 125I-SCF was determined by self-displacement analysis [25]. Purified Kit-Fc (2 nM) was incubated for 1 h at 37°C with varying concentrations of 125I-SCF (50 pM – 7 nM) with or without a 75-fold excess of unlabeled SCF as previously described [10]. The 125I-SCF-Kit-Fc complexes were immunoprecipitated by the addition of protein A sepharose beads. After a 2 h incubation at room temperature, the beads were separated from the media containing free 125I-SCF by sedimentation through phthalate oil (dibutyl phthalate:dinonyl phthalate 5:3). The dibutyl phthalate oil and the dinonyl phthalate oil were obtained from Sigma and from Fluka (Milwaukee, WI), respectively. Immunoprecipitated and free 125I-SCF were quantitated using a Packard 5330 gamma counter (Packard Instrument Co., Downers Grove, IL). Measurements were performed in duplicate and the binding data was analyzed using the LIGAND program [26] to determine the binding affinity (Kd) of Kit-Fc for SCF. Binding studies were performed in parallel to determine the Kd of murine cell surface Kit. IC2-Kit cells were used for this purpose because five separate binding assays with normal murine marrow cells (up to 4×106 cells/100 ml) failed to yield adequate reproducible data for Scatchard analysis. IC2-Kit cells (5× 105) were
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incubated for 1 h at 37°C with varying amounts of 125 I-SCF (10 pM –5 nM) with or without a 75-fold excess of unlabeled SCF in binding buffer consisting of RPMI 1640 (M.A Bioproducts, Walkersville, MD) supplemented with 1% bovine serum albumin (BSA; Sigma), 50 mM HEPES (pH 7.4), 0.1% sodium azide, and 10 mg/ml cytochalasin B (Sigma). At the end of incubation, the cells were separated from the media containing free 125I-SCF by sedimentation through phthalate oil (dibutyl phthalate:dinonyl phthalate 3.5:2.0). Cell-associated and free 125I-SCF were quantitated and the data were analyzed as described above.
2.5. Cell proliferation assays The function of Kit-Fc was tested in cell proliferation assays using IC2-Kit cells in suspension culture. IC2Kit cells were washed to remove IL-3, then suspended at a concentration of 1.5×105/ml in IMDM supplemented with 10% FBS, 1% PSF, and varying concentrations of SCF (10 –1000 ng/ml) in 24-well plates (Becton Dickinson, Lincoln Park, NJ) to determine the optimal concentration of SCF for cell growth. IC2-Kit cells incubated with IL-3 (100 U/ml) were used as positive controls. IC2-Kit cells were incubated in parallel in IMDM, 10% FBS, 1% PSF, and SCF plus varying concentrations of Kit-Fc (1–10 mg/ml) to examine the effect of Kit-Fc on SCF-dependent cell proliferation. IC2-Kit cells incubated in the presence of the neutralizing anti-Kit receptor monoclonal antibody, ACK2 (25 mg/ml), were used as positive controls for blockade of SCF-dependent cell proliferation. All determinations of cell proliferation were performed in triplicate, and viable cells were quantitated on day 4 on the basis of Trypan blue exclusion using a hemocytometer.
2.6. Hematopoietic colony assays These studies were approved by the Animal Care Committee at the University of Washington. To determine whether purified Kit-Fc could block SCF-stimulated hematopoietic colony growth in vitro, normal murine marrow cells were obtained from the femurs of BDF1 mice (Jackson Laboratory, Bar Harbor, ME) and cultured in erythroid and myeloid hematopoietic colony assays as described previously [22]. For quantitation of burst-forming unit-erythroid (BFU-E)-derived colonies, the marrow cells (0.75×105/ml) were plated in 1.4% methylcellulose in IMDM supplemented with 30% FBS, 1% BSA, 5×10 − 5 M b-mercaptoethanol (Sigma), 1% PSF, and 2 U/ml Epo in triplicate plates with SCF (100 ng/ml) alone or SCF plus either Kit-Fc (10 mg/ml) or ACK2 (25 mg/ml) as previously described. Colony assays performed in the presence of IL-3 (100 U/ml) with or without Kit-Fc or ACK2 served as controls for non-specific toxicity. Hemoglobinized
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Fig. 2. Purification of Kit-Fc (left panel) and analysis of the purity of Kit-Fc (right panel). Serum-free culture media from a BHK cell line expressing the fusion protein Kit-Fc was incubated with protein A sepharose beads. The beads were suspended in SDS-PAGE buffer containing dithiothreitol, size-fractionated on a 7% polyacrylamide gel, transferred to nitrocellulose, and probed with polyclonal anti-Kit antibody c951– 960. Molecular weight markers are indicated. The single prominent band at approximately 110 kDa represents monomeric Kit-Fc (left panel). Purified Kit-Fc was size-fractionated on a 7% polyacrylamide gel under reducing conditions. Coomassie blue staining revealed a single band at approximately 110 kDa (right panel).
colonies were counted after incubation for 8 days at 37°C in a humidified atmosphere containing 5% CO2. For quantitation of colony-forming unit granulocytemacrophage (CFU-GM)-derived colonies, the marrow cells (0.75× 105/ml) were plated in triplicate in 1.4% methylcellulose (Dow Chemical Co., Midland, MI) in IMDM supplemented with 10% FBS, 1% BSA, and SCF (100 ng/ml) plus IL-3 (100 U/ml) with varying concentrations of Kit-Fc (1, 3, or 10 mg/ml). Purified human Fc (Jackson ImmunoResearch, West Grove, PA) was used as a control. Plates containing purified ACK2 (25 mg/ml) served as positive controls for the effects of blockade of SCF-Kit interaction. Colony assays performed in the presence of GM-CSF (10 ng/ ml) with or without Kit-Fc or ACK2 served as controls for non-specific toxicity. After incubation for 5 days, the number of myeloid colonies was counted.
2.7. Binding of Kit-Fc to fibroblasts Murine microenvironmental cells were obtained by culturing marrow cells in IMDM supplemented with 10% FBS. The non-adherent cells were removed, and the adherent cells were trypsinized and replated twice. After a 2 week period in culture, the adherent fibroblast-like cells were detached with versene (Bio-Whittaker, Walkersville, MD), and incubated with Kit-Fc (5 mg/ml) in the absence or presence of SCF (5 mg/ml) for 60 min at room temperature. NIH3T3 cells were incubated with Kit-Fc without or with SCF in parallel. The cells were washed with phosphate-buffered saline containing 0.5% bovine serum albumin, then incubated with donkey anti-human IgG-FITC (Jackson Im-
munoResearch) for 45 min at 4°C, washed, fixed in 0.5% paraformaldehyde, and analyzed by flow cytometry.
3. Results
3.1. Expression of Kit-Fc in BHK cells and purification of Kit-Fc A total of 18 BHK-Kit-Fc subclones were initially screened for production of Kit-Fc by Western blotting using the polyclonal antibody c 951 –960 that recognizes the extracellular domain of Kit. Under reducing conditions, the molecular weight of Kit-Fc was estimated to be approximately 110 kDa (Fig. 2). Under non-reducing conditions, the molecular weight of KitFc was estimated to be approximately 210 kDa (data not shown). This difference in molecular weight found under reducing versus non-reducing conditions is consistent with the predicted dimeric structure of Kit-Fc. Four of the subclones were selected for isolation and purification of Kit-Fc. Repeat analysis of the supernatant after 4 months of culture demonstrated stable production of Kit-Fc by the BHK cells. The modified Bradford protein assay revealed that the concentration of Kit-Fc in the BHK serum-free supernatant was approximately 3 mg/ml. Kit-Fc was purified and concentrated from the serum-free supernatant using protein A Sepharose beads. Analysis of the protein A-purified Kit-Fc by SDS-PAGE and Coomassie Blue R-250 staining demonstrated that the purity of Kit-Fc was greater than 95% (Fig. 2).
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3.3. SCF dose-response cur6e of IC2 -Kit cells Parental IC2 cells proliferate in the presence of IL-3 but do not proliferate in the absence of growth factor or in SCF (data not shown). Expression of Kit in IC2 cells confers the ability to proliferate in the presence of SCF (Fig. 5). Dose-response experiments showed that maximal proliferation of IC2-Kit cells was observed at an SCF concentration of 100 ng/ml.
3.4. Effect of Kit-Fc on SCF-dependent cell proliferation Fig. 3. Scatchard analysis of 125I-SCF binding to Kit-Fc. Purified Kit-Fc (2 nM) was incubated for 1 h at 37°C with varying concentrations of 125I-SCF (50 pM–7 nM) with or without a 75-fold excess of unlabeled SCF. The SCF-Kit-Fc complexes were immunoprecipitated by the addition of protein A sepharose beads. Measurements were performed in duplicate and the data were analyzed using the ligand program. The results of one of 4 similar experiments are shown.
3.2. SCF binding affinity of Kit-Fc and of transmembrane murine Kit The SCF binding affinity of Kit-Fc was determined by equilibrium binding analysis. The soluble obligate dimer Kit-Fc displays high-affinity 125I-SCF binding (Kd 570940 pM, mean9 SEM of four experiments) (Fig. 3). Transmembrane murine Kit expressed on IC2 cells displays somewhat lower affinity 125I-SCF binding (Kd 2 90.2 nM, mean9SEM of 10 experiments) (Fig. 4). Both Kit-Fc and the transmembrane murine Kit exhibit a single class of SCF binding sites. The IC2-Kit cells express approximately 3000 receptors/cell (mean of 10 experiments).
Fig. 4. Scatchard analysis of 125I-SCF binding to cell surface Kit. IC2-Kit cells (5 ×105) were incubated for 1 h at 37°C with varying concentrations of 125I-SCF (10 pM–5 nM), with or without a 75-fold excess of unlabeled SCF. The cells were separated from the free 125 I-SCF by sedimentation through phthalate oil. Cell-associated and free 125I were quantitated using a Packard gamma counter and analyzed using the ligand program. Measurements were performed in duplicate. The results of one of 10 similar experiments are shown.
Kit-Fc was tested for the ability to block SCF-dependent cell proliferation of IC2-Kit cells in suspension culture. A dose-dependent abrogation of the stimulatory effects of SCF on IC2-Kit cell proliferation was observed (Fig. 6). At a concentration of 10 mg/ml, Kit-Fc completely blocked SCF-dependent IC2-Kit cell proliferation. At the lowest dose tested (1 mg/ml) the addition of Kit-Fc to IC2-Kit cells grown in the presence of SCF had a statistically significant effect on SCF-induced IC2-Kit proliferation (PB 0.02, Student’s t-test). The addition of Kit-Fc at a concentration of 10 mg/ml did not inhibit the ability of IC2-Kit cells to proliferate in the presence of IL-3 or GM-CSF (data not shown), indicating that Kit-Fc specifically inhibits SCF-stimulated proliferation.
3.5. Effect of Kit-Fc on erythroid and myeloid colony growth Kit-Fc was tested for the ability to block SCF-stimulated hematopoietic colony growth from normal murine marrow cells in vitro. The addition of Kit-Fc (10 mg/ml) blocked the synergistic effect of SCF plus Epo on BFU-E growth (Fig. 7). The ability of Kit-Fc to block SCF-stimulated erythroid colony growth was comparable to that of the neutralizing anti-Kit monoclonal antibody ACK2. The blockade of SCF-stimulated growth of BFU-E by Kit-Fc was observed to be specific, as the presence or absence of Kit-Fc did not alter the number of BFU-E found in cultures containing Epo plus IL-3 (Fig. 8). Kit-Fc also blocked SCF-stimulated growth of CFUGM (Fig. 9). Moreover, Kit-Fc demonstrated a dosedependent abrogation of the contribution of SCF to myeloid colony growth (Fig. 10). At the lowest dose tested (1 mg/ml) the number of CFU-GM colonies observed in the presence of SCF, IL-3, plus Kit-Fc was lower than the number of CFU-GM colonies observed in the presence of SCF plus IL-3 (PB 0.02, Student’s t-test). At a dose of 10 mg/ml, Kit-Fc was as effective as ACK2 in blocking the effects of SCF on myeloid
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Fig. 5. Dose response of SCF-stimulated IC2-Kit proliferation. IC2-Kit cells (1.5 ×105/ml) were cultured in IMDM supplemented with 10% FBS plus varying concentrations of SCF (0–1000 ng/ml). The data represent the mean 9SEM of triplicate values from one of 3 similar experiments.
colony growth. The addition of Kit-Fc (10 mg/ml) did not decrease the number of myeloid colonies found in the presence of GM-CSF. The Fc portion of Kit-Fc did not suppress myeloid colony growth in the presence of SCF or IL-3 (Table 1).
ability of SCF to induce tyrosine phosphorylation of Kit in a fibroblast cell line [18], but did not explore the effect of soluble Kit on normal hematopoietic colony growth. The concentration of soluble Kit in the circulation in normal adults is approximately 325 ng/ml, while the
3.6. Kit-Fc binding to fibroblasts Kit-Fc binds to the transmembrane form of SCF displayed by microenvironmental cells isolated from murine marrow (Fig. 11), and to SCF displayed by the NIH3T3 fibroblast cell line (Table 2). In the presence of SCF, binding of Kit-Fc to the fibroblasts decreases (Table 2), suggesting that the soluble and transmembrane forms of SCF can compete for Kit-Fc binding.
4. Discussion The present report demonstrates that a soluble form of Kit can bind SCF with high affinity (Kd 570 pM), and can modulate the ability of SCF to stimulate erythroid and myeloid hematopoietic colony growth in vitro. The effects of Kit-Fc are specific for SCF and are dose-dependent. Both the proliferative effects of SCF alone, and the synergistic effects of SCF plus Epo or SCF plus IL-3, are blocked by Kit-Fc. Prior studies have shown that soluble forms of Kit can inhibit the
Fig. 6. Kit-Fc blocks SCF-stimulated IC2-Kit proliferation. IC2-Kit cells were cultured in IMDM supplemented with 10% FBS plus no cytokine, SCF (100 ng/ml), SCF plus the anti-Kit antibody ACK2 (25 mg/ml), or SCF plus varying concentrations of Kit-Fc (1, 3, or 10 mg/ml). The data represent mean 9SEM of triplicate values from one of 2 separate experiments. The lowest concentration of Kit-Fc tested (1 mg/ml) blocked SCF-stimulated growth of IC2-Kit cells by greater than 80% (P B0.02, SCF vs. SCF plus Kit-Fc, Student’s t-test).
D.D. Dahlen et al. / Leukemia Research 25 (2001) 413–421
Fig. 7. Effect of Kit-Fc on BFU-E growth. Murine marrow cells (7.5×105/ml) were cultured in semi-solid media in the presence of Epo (2 U/ml), Epo plus SCF (100 ng/ml), Epo plus SCF plus ACK2 (25 mg/ml), or Epo plus SCF plus Kit-Fc (10 mg/ml), and the number of BFU-E was counted on day 8. The data represent the mean 9 SEM of triplicate plates from one of 2 experiments (PB 0.02, Epo plus SCF vs. Epo plus SCF plus Kit-Fc, Student’s t-test).
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Fig. 9. Effect of Kit-Fc on CFU-GM growth. Murine marrow cells (1 × 105/ml) were cultured in semi-solid media in the presence of no added growth factor, SCF (100 ng/ml), SCF plus ACK2 (25 mg/ml), or SCF plus Kit-Fc (10 mg/ml). The number of CFU-GM was counted on day 5. The data represent the mean 9 SEM of triplicate plates from one of 5 similar experiments (PB 0.02, SCF vs. SCF plus Kit-Fc, Student’s t-test).
concentration of SCF is approximately 3 ng/ml [4,17]. These concentrations represent a 30-fold molar excess of soluble Kit over soluble SCF. The present experiments with Kit-Fc employed a range of concentrations of Kit-Fc, and showed that Kit-Fc at a concentration of 10 mg/ml is sufficient to completely ablate the proliferative effects of SCF 100 ng/ml (Figs. 6, 7, 9 and 10). The relative concentrations of Kit-Fc and SCF used in these studies reflect the physiologic differences found in vivo. Soluble Kit purified from human serum is of molecular weight 98 kDa in the absence of SCF [10,17], consistent with predominance of a monomeric form of soluble Kit. However, when native soluble Kit derived
from human serum is incubated with SCF, both monomeric and dimeric forms of Kit are identified [10]. Native soluble Kit binds 125I-SCF with high affinity, approximately 320 pM [10], similar to the affinity of the Kit receptor expressed on the surface of human hematopoietic cells [12]. Additionally, native soluble Kit can compete with cell surface Kit for binding 125I-SCF [10,17]. Taken together, these data support the concept that native soluble Kit is capable of binding SCF, and that one role of soluble Kit may be to downmodulate SCF bioactivity in vivo. Soluble Kit is generated by proteolytic cleavage of cell surface Kit [10,15,16]. Hematopoietic cells, mast cells, and endothelial cells shed Kit from the cell surface. The present results suggest that shedding of Kit
Fig. 8. Specificity of the effect of Kit-Fc on BFU-E growth. Murine marrow cells were cultured as described in Fig. 7 in the presence of Epo (2 U/ml), Epo plus IL-3 (100 U/ml), Epo plus IL-3 plus ACK2 (25 mg/ml), or Epo plus IL-3 plus Kit-Fc (10 mg/ml), and the number of BFU-E was counted. The data represent the mean 9 SEM of triplicate plates from one of 2 similar experiments.
Fig. 10. Dose-dependent effect of Kit-Fc on CFU-GM growth. Murine marrow cells were cultured as described in Fig. 9 in the presence of IL-3 (100 U/ml), SCF (100 ng/ml) plus IL-3, SCF plus IL-3 plus ACK2 (25 mg/ml), or SCF plus IL-3 plus Kit-Fc (1, 3, or 10 mg/ml), and the number of CFU-GM were counted. The data represent the mean 9 SEM of triplicate plates from one of 4 similar experiments. (PB0.02, SCF plus IL-3 vs SCF plus IL-3 plus Kit-Fc 1 mg/ml, Student’s t-test).
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D.D. Dahlen et al. / Leukemia Research 25 (2001) 413–421
Table 1 Effect of Kit-Fc and Fc on myeloid colony growtha
Table 2 Binding of Kit-Fc to NIH3T3 cellsa
Additions
cCFU-GM/105 Cells
Additions
Mean fluorescence intensity
SCF/Epo/IL-3 (10 U/ml)+0 SCF/Epo/IL-3 (10 U/ml)+ACK2 SCF/Epo/IL-3 (10 U/ml)+Kit-Fc SCF/Epo/IL-3 (10 U/ml)+Fc IL-3 (100 U/ml)+0 IL-3 (100 U/ml)+Kit-Fc IL-3 (100 U/ml)+Fc
47 7 5 73 84 86 85
0 Kit-Fc Kit-Fc+SCF
0.727 0.957 0.768
a Murine marrow cells were cultured in semi-solid media in the presence of SCF (100 ng/ml) plus Epo (1 U/ml) plus a low concentration of IL-3 (10 U/ml), or in a high concentration of IL-3 (100 U/ml) alone, in the absence or presence of ACK2 (25 mg/ml), Kit-Fc (7.5 mg/ml), or Fc (7.5 mg/ml). The data represents the number of myeloid colonies per 105 cells plated (average of duplicate values).
may modulate SCF bioactivity in two ways: by diminishing the density of Kit displayed on the cell surface, and by converting cell surface Kit to soluble Kit, which may serve as an antagonist of SCF bioactivity. The serum levels of soluble Kit increase when the population of cells that release Kit is pathologically expanded, such as in acute myelogenous leukemia [27,28]. Although much is known about intracellular Kit signaling pathways [9], and how these pathways can be biochemically inhibited [29], little is known about how proteolytic cleavage of Kit from the cell surface is regulated. Activation of protein kinase C enhances shedding of Kit [15]. Members of the disintegrin family of metalloproteinases cleave other cytokines and cytokine receptors from the cell surface [30,31], and are candidates for cleavage of Kit. The Kit-Fc fusion protein [24] was selected for study based on the demonstration that a soluble TNF receptor-Fc fusion protein could antagonize TNF activity in clinical studies [32]. The present report demonstrates that Kit-Fc can bind soluble SCF with high affinity, and can also bind to transmembrane SCF on the surface of fibroblasts, in accord with prior results using
a NIH3T3 cells were incubated 9Kit-Fc (1 mg/ml)9SCF (1 mg/ml) for 1 h at 22°C, then with donkey anti-human IgG-FITC for 45 min at 4°C, then analyzed by flow cytometry. The data represent the mean fluorescence intensity of 4500 cells analyzed.
an alkaline phosphatase-tagged form of soluble Kit [33]. Soluble and transmembrane SCF compete for binding of Kit-Fc. Kit-Fc can antagonize the biological effects of soluble SCF in hematopoietic colony assays; whether Kit-Fc can block the biological effects of transmembrane SCF remains to be determined. The availability of Kit-Fc offers the opportunity for in vivo studies to investigate whether soluble forms of Kit can modulate SCF bioactivity in vivo. Soluble and transmembrane SCF have distinct but overlapping roles [1,5,34 –36]. The present report suggests that soluble and transmembrane Kit may also have distinct roles, and that cleavage of Kit from the cell surface may serve to downregulate SCF signaling, both by decreasing the density of cell surface Kit, and by generating a soluble antagonist of SCF that competes with cell surface Kit for SCF binding.
Acknowledgements Supported by NIH grants DK 43719, CA 31615, and 5 T32 HL07093. DD Dahlen provided th econcept, design, assembled and anlayzed the data, drafted the paper and gave final apporval. NL Lin contributed to the study design, provided technical support, collecting and analyzing the data and gave final approval. Y-C Liu provided study materials, gave critical revision to the article and gave final approval. VC Broudy provided the necessary administrative support and funding for the project as well as contributing to the concept, deesign, data interpretation, assistance in tmanuscript preparationk, critical revision and gave final approval.
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