Insulin-Like growth factor binding protein (IGFBP) inhibits igf action on human osteosarcoma cells

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    Insulin-like Growth Factor Binding Protein (IGFBP) Inhibits IGF Action on Human

    Osteosarcoma Cells PHIL G. CAMPBELL* AND JOSEF F. N O V A K

    Orthopaedic Research laboratory, Allegheny-Singer Research Institute, Pittsburgh, Pennsylvania 1.52 12

    The influence of a human insulin-like growth factor binding protein, hlGFBP-1, on the action of IGFs on human osteosarcoma cells was examined. hlGFBP-1 was found to block binding of IGFs to their receptors on MG-63 cells and subsequent IGF stimulation of DNA synthesis. Concurrent incubation of hlGFBP-1 with either "51-IGF-I or '251-IGF-ll prevented the binding of both '2'1-IGF~ to cells in a dose-dependent manner. hlGFBP-1 inhibition of IGF binding occurred similarly under both 4" and 37C conditions. Additionally, hlGFBP-1 facilitated the dissociation of IGFs bound to cells. The inhibitory effect of hlGFBP-1 on IGF-1 mediated 3H-thymidine incorporation into D N A was dose dependent. hlGFBP-1 did not inhibit binding to or stimulation of growth in MG-63 cells by des3-IGF-I, an IGF-I analog with a 100-fold less affinity for hlGFBP-I. This confirmed that hlGFBP-1 competed for IGF receptor binding sites on MG-63. Since hlGFBP-1 did not bind to cells, inhibition of IGF action was indirect, presumably through the formation of extracellular soluble bioinactive IGF-BP complexes.

    Insulin-like growth factors (IGFs) are associated with specific binding proteins (IGFBPs) in blood, ex- tracellular fluids, and culture media (Nissley and Rechler, 1984). IGFBPs can be divided into three principal groups based on amino acid and gene se- quence, immunocross-reactivity, and IGF binding spec- ificities. IGFBP-1 is growth hormone-independent with a molecular weight ranging from 25 to 34 kDa. IGFBP-1 has been isolated from amniotic fluid (Povoa et al., 1984), human HEP-G2 cells (Povoa et al., 19851, and human decidua (Koistinen et al., 1986). IGFBP-2 is approximately 34 kDa, is exemplified by the IGFBP produced by BRL-3A cell (Mottola et al., 1986) and normal MDBK kidney epithelial cells (Szabo et al., 19881, and is present in cerebrospinal fluid (Romanus et al., 1989). IGFBP-3 is the principal circulating IGFBP, is growth hormone-dependent, and contains an acid-stable 53 kDa component responsible for binding IGFs (Martin and Baxter, 1986). Recently, the number of known IGFBPs have increased to include IGFBP-4 through IGFBP-6 (LaTour et al., 1990; Kiefer et al., 1991).

    It is generally accepted that, in blood, the IGFBPs maintain a reservoir of IGFs by complexing IGFs, thus inhibiting the binding of IGFs to their receptors on endothelial cells (Zapf et al., 1985; Gopinath et al., 1989). In the pericellular environment IGFBPs can both inhibit (Knauer and Smith, 1980; De Mellow and Baxter, 1989; Ritvos et al., 1988; Gopinath et al., 1989; Ross et al., 1989; Walton et al., 1989) and potentiate (De Mellow and Baxter, 1989; klgin et al., 1987; Busby et al., 1988) I,GF activity. The reason for the apparent dichotomy of IGFBP function remains unclear, but Busby et al. (1988) found the association of an IGFBP 0 1991 WILEY-LISS, INC.

    with the cell membrane or matrix necessary for its potentiative action of IGF-I. Differences in experimen- tal conditions resulting in differential IGFBP function have also been suggested (Blum et al., 1989; De Mellow and Baxter, 1988; Ross et al., 1989). Nevertheless, different responses of specific cell types to various IGFBPs cannot be excluded. The exact role of IGFBPs in mechanisms of IGF action remains elusive.

    Like most other cell types, both normal and neoplas- tic bone cells secrete (Blatt et al., 1984; Stracke et al., 1984; Canalis et al., 1988; Ernst and Froesch, 1988; Gray et al., 1989) and respond to IGF-I (Canalis, 1980; Schmid et al., 1984; Canalis et al., 1989; VandePol et al., 1989; Furlanetto, 1988). Additionally, IGFBPs are pro- duced by primary bone cultures (Schmid et al., 1989a,b) and osteosarcoma cells (Mohan et al., 1989; Campbell et al., 1991; Andress and Birnbaum, 1991). These studies prompted us to investigate the effect of IGFBP on IGF binding and biological action in human osteosa- rcoma cells.


    Recombinant human IGF-I1 was purchased from Bachem, Torrance, CA. Recombinant human IGF-I was purchased from IMCERA (Terra Haute, IN) and

    Received July 19, 1990; accepted June 11, 1991. *To whom reprint requestsicorrespondence should be addressed. A preliminary report of this work was presented at the Eleventh Annual Meeting of the American Society for Bone and Mineral Research, Montreal, 1989.


    Bachem (Torrance, CAI. Des3-IGF-I was kindly pro- vided by Dr. P. Walton, GroPrep (Adelaide, Australia). Decidual IGFBP (hIGFBP-l), also referred to as PP,,, was kindly provided by Dr. H. Bohn, Behringwerke (Marburg, Germany). This preparation is immunoaf- finity purified to >99% (Bohn and Kraus, 1980). Molar concentrations of hIGFBP-1 were calculated based on a molecular weight of 34 kDa, estimated under reduced SDS-PAGE conditions. Peptides were iodinated by chloramine T method to a specific activity of 125-300 +Ci/pg and purified by neutral gel chromatography over Sephadex G25 and G75. Bovine insulin, bovine serum albumin (BSA; RIA grade), and transferrin were purchased from Sigma, St. Louis, MO. Most culture reagents were from GIBCO (Grand Island, NY).

    Cell culture Human osteosarcoma cells were obtained from ATCC

    (Rockville, MD). Cells were maintained in aMEM without nucleosidedHam's F-12 Nutrient Mixture (aMEM/F12; 1/1) containing 10% supplemented calf serum (SCS), penicillin (100 Uiml), streptomycin (100 Fgiml), and glutamine (2 mg/ml) in a humidified 5% CO, atmosphere at 37C. All experiments utilized passages 90-100. Cells were released with trypsini EDTA and cell count was determined by hemocytome- ter.

    Binding studies Cells were seeded in 12-well plates (Flow Laborato-

    ries, McLean, VA) a t 40,000 cells/well in 1 ml aMEM/ F12 containing 10% SCS. At confluency (approximate- ly 5 days), the monolayers were washed twice with PBS and preincubated for 2 h with serum-free medium at 37C. The monolayers were washed with ice-cold PBS and incubated with 1 ml binding buffer (0.1 M Hepes, 0.12 M NaC1,5 mM KC1,1.2 mM MgS04, 8 mM glucose, and 1% BSA, pH 7.8) a t 4" and 37C. The binding studies were conducted with labeled IGFs, lZ5I-IGF-I, I2,I-des3-IGF-I, and 1251-IGF-II, added at 19 pM unless otherwise indicated; the binding equilibrium occurred by 24 h a t 4" and 1 h at 37C. Concentrations of competing ligands, incubation time, and temperature are described in figure the table legends. Incubations were terminated by washing the monolayers twice with ice-cold PBS. Cells were solubilized with 1 N NaOH and radioactivity was counted. Non-specific binding (NSB) was determined by using 25 nM IGF-I or 54 nM IGF-11. Specific binding (SB) was determined by subtracting NSB from total binding. ECBOs were analyzed by using LIGAND program as originally developed by P.J. Mun- son and D. Robard and modified for microcomputers by McPherson (1985).

    Complexes of 1251-IGF-I and hIGFBP-1 were formed bz the incubation of 5 pg of hIGFBP-1 with 1 +g

    51-IGF-I in 50 mM phosphate buffer, pH 7.4, over- night a t 4C. Complexes were purified over neutral G75 Sephadex (1 x 50 cm). Equivalent mass of 1251-IGF-I in free or complex form was used in binding experiments.

    Thymidine incorporation experiments Cells were seeded in 24-well plates (Flow Laborato-

    ries, McLean, VA) at 20,000 cells/well in 0.5 ml aMEMi F12, 10% SCS medium and allowed to attach for 24 h.

    Attached cells were washed with serum-free aMEM/ F12 medium and incubated for 48 h in serum-free aMEM/F12 medium containing I00 pg/ml BSA (fatty acid free, Miles, Kankakee, IL) and 10 pg/ml transfer- rin (BT-medium). Preincubations with hIGFBP-1 are described in figure and table legends. For incubations, cells were washed once with serum-free aMEM/F12 medium and peptides were added in 0.5 ml BT-medium. Cultures were terminated after 48 h. During the last 4 h, 0.5 +Ci 3H-thymidine (Moravek Biochemicals, Brea, CA) was present. Wells were washed with 0.5 ml PBS and exposed to 0.5 ml ice cold absolute methanol for 5 min. Methanol was aspirated and 10% TCA (4C) was added. After 15 min, TCA was aspirated and cells were solubilized with 0.5 ml of 0.5 N NaOH and counted for radioactivity. EC,,s were determined by using the LIGAND program as originally developed by P.J. Mun- son and D. Robard and modified for microcomputers by McPherson (1985).

    RESULTS IGF effects on cells

    Human osteosarcoma cells express specific receptors for both IGF-I (type I receptor) and IGF-I1 (type I1 receptor), based on relative binding affinities (Fig. 1). When IGF-I was used as the labeled ligand, competition experiments determined ECSOs of 0.10 nM * 0.01 nM (mean * SEM of 3 experiments) for IGF-I. IGF-I1 exhibited a 16.3-fold lower affinity and insulin a 2,300- fold lower affinity for the IGF-I receptor. When IGF-I1 was the labeled ligand, only IGF-I1 effectively com- peted for 1251-IGF-II binding with an EC,, of 3.06 * 0.76 nM (mean 2 SEM of 3 experiments). Com- petition results were similar whether conducted at 4" or 37C (data not shown). These results correspond with other receptor studies using recombinant IGF prepara- tions (Ewton et al., 1987; Hadsell et al., 1990). Minimal ligand degradation occurred over the incubation period. Both lZ5I-IGF-I and 1251-IGF-II retained greater than 95% of initial TCA-precipitable counts either at 4C for 24 h or 37C for 1 h.

    IGF-I was the principal mitogenic IGF for cells. In 3H-thymidine incorporation experiments, the ECS0 for IGF-I stimulation of DNA synthesis was 0.43 nM, whereas the EC50 for IGF-I1 stimulation was 1.92 nM (Fig. 2). The approximate 5-fold lower ECS0 for IGF-I suggests that stimulation of 3H-thymidine incorpora- tion occurs via the type I receptor. This is supported by experiments using type I receptor antibodies which block both IGF-I and IGF-I1 stimulation of 3H-thymi- dine incorporation in cells (Furlanetto, 1988).

    IGFBP effects on IGF action IGFBP was found to influence the binding of IGFs to

    cells by blocking the interaction of IGFs with their surface receptors. When hIGFBP-1 was co-incubated with 1251-IGF-I, complete inhibition of IGF-I binding resulted (Fig. 3). This occurred when the hIGFBP-1 was added alone at 25 nM or as a 1251-IGF-I/hIGFBP-1 complex. Binding of 1251-IGF-II was similarly affected by hIGFBP-1 (data not shown).

    Increasing concentrations of hIGFBP-1 competed for 1251-IGF-I and 12,1-IGF-II binding to cells in a dose- dependent manner (Fig. 4). hIGFBP-1, competed for








    0 m 2

    -- --

    -- -


    1 10 100 1000 10000

    Competitor, nM

    Fig. 1. Competition for '"I-IGF binding for cells. Confluent culture cells in 12-well plates (approximately 250,000 cellsiwell) were pre- pared for binding experiments as described in Materials and Methods. Either lZ5I-IGF-I (A) or 1251-IGF-II (B) was incubated a t 4C for 24 h in 1 ml with IGF-I, IGF-11, and insulin as competing ligands. Mono- layers were washed with ice-cold PBS and bound '2sII-IGF was determined by counting radioactivity. Individual points represent the mean i SEM of triplicate determinations from one of three similar experiments.

    I U

    8 --

    6 --

    4 --

    2 Ih

    O r 0

    , : : : : I : : : : : : : : I : : : : : : : : : : : .05 .1 1 10

    PeDtide, nM



    Fig. 2. IGF-I and IGF-I1 stimulation of 'H-thymidine incorporation into cells. Wells were seeded in 24-well plates as described in Matrrzals and Methods. Incubations included increasing concentra- tions of either IGF-I or IGF-11. Incubations were terminated after 48 h. Individual points represent mean ? SEM of triplicate determina- tions from one to two similar experiments.

    1251-IGF-I and lZ5I-IGF-II binding with IC,,s of 0.3 ? 0.07 nM (mean of 4 experiments) and 0.18 ? 0.07 nM (mean of 3 experiments), respectively. Incubation of either hIGFBP-1 o r IGF resulted in displacement of bound lZ5I-IGF from MG-63 cells (Table 2). Over the extensive dissociation period of 96 h, hIGFBP-1 main- tained TCA precipitable counts of 1251-IGFs a t 21 and 42% above that of 1251-IGF-I and '251-IGF-II incubated alone, respectively. This would imply that hIGFBP-1 inhibited degradation of labeled IGFs in the incubation medium. However, more extensive experiments to as- sess degradation will be necessary to confirm the present observations. When hIGFBP-1 and IGF were added at equimolar concentrations, hIGFBP-1 dis- placed twice the amount of cell-bound lZ5I-IGF (Fig. 5). This is interesting since the binding affinities of IGFBP-1 and IGF receptors in bone cells are similar (Baxter et al., 1987; Centrella et al., 1990; Pollak et al., 1990) and the IGF binding capacity of IGFBP-1 aver- ages 0.52 mol IGFimol IGFBP (Baxter et al., 1987).

    Inhibition of IGF binding to MG-63 cells was due to the sequestration of IGFs into soluble IGF-BP com-

    Fig. 3. Specific binding of '2sI-IGF-I to cells as influenced by hIGFBP-1. Confluent monolayers of cells were prepared for binding experiments as described in Materials and Methods and incubated with 1251-IGF-I alone (control), 1251-IGF-I + 25 nM hIGFBP-1 added concurrently (hIGFBP-11, or 25 nM hIGFBP-1 complexed to lZ5I-IGF-I (IGF-I hIGFBP-1 complex). After 24 h at 4"C, specifically bound lZ5I-IGF-I was determined. Individual bars represent mean i SEM of triplicate determinations from one of two similar experiments. U.D. represents undetectable.

    plexes, since hIGFBP-1 did not bind to MG-63 cells, either alone (Table 1) or as an IGF-BP complex (Fig. 3). hIGFBP-1 inhibition of IGF binding was unaffected by temperature (Fig. 6). When des3-IGF-I, a variant form of IGF-I which has a much lower binding affinity for IGFBP-1 (Forbes et al., 19881, was used as the labeled ligand only minimal hIGFBP-1 inhibition could be observed (Fig. 6). This confirms that hIGFBP-1 is inhibiting IGF-I binding by complexing IGFs.













    .01 .1 1 10 50

    IGFBPl, nM

    TABLE 1. Failure of IGFBP-1 to bind to MG-63 cells'

    Incubation Unlabeled hIGFBP-1 pM 'Z51-hIGFBP-1 temperature ("C) added bound

    4 None 0.197 f 0.055 11.8 nM 0.185 f 0.032

    0.153 k 0.035 0.150 * 0.049 11.8 nM

    'Confluent monolayer cultures of MG-63 cells in 12-well plates were incubated with 12 pM of '"I-hIGFBP-1 with and without 12 nM unlabeled hIGFBP-1 for 24 h at 4C or 1 h at 37C. Radioactivity hound to monolayers was determined as described in Materials and Methods. Values are the mean * SEM of two separate experiments. No difference between incubations containing or not containing unlabeled hIGFBP-1 could be detected by Student's T test (P > 0.05).

    37 None

    Fig. 4. Inhibition of '251-IGF-I and -11 binding by hIGFBP-1. Mono- layers were prepared for bindi...


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