insulin-like growth factor binding protein (igfbp) inhibits igf action on human osteosarcoma cells

JOURNAL OF CELLULAR PHYSIOLOGY 149293-300 (1991)

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 37°C 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, extracellular fluids, and culture media (Nissley and Rechler, 1984). IGFBPs can be divided into three principal groups based on amino acid and gene sequence, immunocross-reactivity, and IGF binding specificities. 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 experimental 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 neoplastic 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 produced 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 osteosarcoma cells.

MATERIALS AND METHODS Materials

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.

294 CAMPBELL AND NOVAK

Bachem (Torrance, CAI. Des3-IGF-I was kindly provided 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 37°C. 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 (approximately 5 days), the monolayers were washed twice with PBS and preincubated for 2 h with serum-free medium at 37°C. 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 37°C. 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 37°C. 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 4°C. 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 transferrin (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 (4°C) 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 competed 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 37°C (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 4°C for 24 h or 37°C 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 incorporation 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-thymidine 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

IGFBP BLOCKS IGF ACTION IN OSTEOSARCOMA CELLS 295

1.0-

0.8--

0.6

0.4

0.2

0.0

0 m 2

-- --

-- -

A

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 prepared for binding experiments as described in Materials and Methods. Either lZ5I-IGF-I (A) or 1251-IGF-II (B) was incubated a t 4°C 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

--cI

50

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 concentrations of either IGF-I or IGF-11. Incubations were terminated after 48 h. Individual points represent mean ? SEM of triplicate determinations 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 maintained 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.

CAMPBELL AND NOVAK 296

0.8

0.6

0.4

0.2

0.0

1.OT

--

--

--

--

T

.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 4°C or 1 h at 37°C. 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 binding studies as described in Materials and Methods. Either 12sI-IGF-I or '251-IGF-II was incubated with increasing concentrations of hIGFBP-1 for 24 h at 4°C. Incubations were terminated and bound lZ5I-IGF determined. Individual points represent mean i SEM of triplicate determinations from one of four and three similar experiments for IGF-I and IGF-11, respectively.

0.1 1 1 0 100

hlGFBP-1, nM

Fig. 5. Failure of hIGFBP-1 to inhibit the binding of '25des31-IGF-I to MG-63 cells. Monolayers were prepared for binding studies as described in Materials and Methods. '251-IGF-I was incubated with increasing concentrations of hIGFBP-1 for 24 h at 4 C (V ) or 1 h at 37"C(*). '251-des3-IGF-I was incubated with increasing concentrations of hIGFBP-1 for 24 h at 4" ( v ). Incubations were terminated and bound '2sI-IGF determined. Individual points represent mean -t SEM of triplicate determinations.

hIGFBP-1 inhibition of IGF binding to MG-63 cells resulted in a loss of IGF-I stimulation of 3H-thymidine incorporation. When increasing concentrations of IGF-I were co-incubated with 5.9 nM hIGFBP-1, the half- maximal concentration of IGF-I shifted from 0.38 mM for IGF-I alone to 1.03 nM in the presence of hIGFBP-1 (Fig. 7). hIGFBP-1 inhibition of IGF-I stimulated 3H- thymidine incorporation into cells was dose dependent (Fig. 8). Whereas hIGFBP-1 completely inhibited the binding of '"I-IGF-I to its receptor i t did not completely suppress IGF-I activity. The effect of hIGFBP-1 was not altered by method of introduction, in contrast to

TABLE 2. Dissociation of '"I-IGF bound to MG-63 cells by IGF and hIGFBP-1'

24 h 96 h % % %I %I

dissociation initial TCA dissociation initial TCA l25IGF.I

68.7 f 1.0 84.3 + 0.7 Control - IGF-I 63.7 k 5.4 84.0 k 4.2 72.6 k 2.6 67.3 5 1.0 hIGFBP-1 55.3 i 0.6 90.0 + 4.7 67.5 5 1.1 83.0 k 1.5

61.7 f 6.5 85.9 k 0.4 Control - IGF-I1 56.0 ?c 2.5 90.3 f 5.1 68.9 * 5.0 62.2 k 3.9 hIGFBP-1 59.2 k 0.6 94.6 + 0.2 62.8 2.4 87.6 k 0.7

-

l z s ~ ~ ~ - ~ ~ -

'Either '2511-IGF-I or '"I-IGF-I1 at 0.15 ng/ml was equilibrated with MG-63 cells at 4°C. After 24 h IGF-I (51 nM), IGF-II(51 nM) or hIGFBP-l(25 nM) was added and incubations were continued at 4°C for 24 or 96 h. Incubations were terminated and dissociation of specifically hound L'511-IGF was determined. Percent dissociation represents control binding for a specific time periodminus hindingin the presenceof either IGF or IGFBP. Control binding was adjustedto 100%. Incubation medium was precipitated with 10%) TCA to determine degradation of labeled IGF. Percent initial TCA represents the TCA-insoluble fraction for each treatment divided by the initial TCA-insoluble fraction of labeled IGF. Values represent the mean k SEM for triplicate determinations.

IGFBP-3 (De Mellow and Baxter, 1988; Blum et al., 1989). When hIGFBP-1 was added as an IGF-BP complex (1.3 nM IGF-I:5.9 nM hIGFBP-l), a 53% reduction in IGF-I-stimulated 3H-thymidine incorporation was observed (mean of two experiments; data not shown). This was similar to concomitant addition of IGF-I and hIGFBP-1 (Fig. 6). Preincubation with 5.9 nM of hIGFBP-1 did not alter the subsequent rate of IGF-I- stimulated 3H-thymidine incorporation (Fig. 91, sub- stantiating hIGFBP-1 failure to bind to cells. Addition- ally, increasing concentrations of hIGFBP-1 alone were without effect (Fig. 71, suggesting that any endogenous production of IGFs by cells, under these experimental conditions, did not result in autocrine regulation. Pos- sibly, any secreted IGFs are immediately sequestered by endogenously produced IGFBPs.

Des3-IGF-I stimulation of DNA synthesis was unaffected by hIGFBP-1 (Table 31, confirming that hIGFBP-1 inhibition of IGF-I activity was by binding competition. The higher level of DNA synthesis observed a t an equal concentration to IGF-I would suggest that endogenous IGFBP may inhibit IGF function.

DISCUSSION From these data we suggest that IGFBPs can regu-

late the biological activity of IGFs in human osteosarcoma cells. Inhibition of IGF bioactivity by IGFBP is

IGFBP BLOCKS IGF ACTION IN OSTEOSARCOMA CELLS

1007

80

60

40

20

297

--

--

-_

--

0 15 30 45 60 75

5 -

4

3

2

1 T

Control

IGF-II

IGFBPl

--

--

--

5 -

4

3

2

1 7

04 * : : : : : : : I .3 1 10

lGFBPl, nM

--

--

I -.

Hours Fig. 8. Inhibition of IGF-I stimulated ”H-thymidine incorporation by increasing concentrations of hIGFBP-1. Cells were seeded in 24-well plates and described in Materials and Methods. Cells were incubated in BT-medium containing 1.3 nM IGF-I and increasing concentrations ofhIGFBP-1. After 48 h incubations the cultures were terminated and thymidine incorporation determined. Individual points represent mean ? SEM of triplicate determinations from one of two similar experiments. Maximal stimulation represents thymidine incorporation due to addition of IGF-I alone.

F ~ ~ . 6, D~~~~~~~~~~~ of I ~ s ~ ~ ~ ~ ~ - ~ I bound to cells by either 25 nM of IGF-II or 25 nM of hIGFBP-l. 12sII-IGF-II was incubated with cells for 24 h, at which time IGF-II o1 hIGFBP-l was added. At indicated times incubations were terminated and bound 1 2 5 ~ - ~ ~ ~ - ~ ~ determined, Indi- vidual points represent mean SEM of triplicate determinations,

t I z X

f d .- U .- E, .E I

r ) I

,::::: : : : : : : : : : : : : : : : : : I : : : I

.05 .1 1 10 50

Peptide, nM

Fig. 7. Blockage of IGF-I dose stimulation of 3H-thymidine incorporation by hIGFBP-1. Wells were seeded in 24-well plates as described in Materials and Methods. Incubations included increasing concentrations of either IGF-I ( A) or hIGFBP-1 (=I, or IGF-I + 5.9 nM hIGFBP-1 (0). Incubations were terminated after 48 h. Individual points represent mean ? SEM of triplicate determinations from one to two similar experiments. Control well contained only BT-medium.

apparently due to sequestration of IGFs which blocks the binding of IGFs to their specific plasma membrane receptors. This hypothesis is supported by the observations that 1) the 251-IGF-BP complex did not bind to cells, 2) lZ5I-hIGFBP-1 alone did not bind to cells, 3) des3-IGF-I was unaffected by hIGFBP-1, and 4) lZ5I- IGFs were dissociated from cells by hIGFBP-1. These results are in agreement with other reports that dem- onstrated the inhibitory activity of hIGFBP-1 in other cell systems (Ritvos et al., 1988; Ross et al., 1989; Rutanen et al., 1988). A 31 kDa IGFBP-1 from amniotic fluid binds to the cell surface and enhances IGF-I binding and stimulation of proliferation in several cell types (Busby et al., 1988). These authors also reported another binding protein of similar molecular weight and identical N-terminal amino acid sequence to the enhancing IGFBP-1, which inhibited the biological

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

IGF-I, nM

Fig. 9. Effect of pre-incubation of IGFBP on 3H-thymidine incorporation into cells. Either BT-medium alone (0 ) or BT-medium + 5.9 nM hIGFBP-1 (A) was added to wells during the 24 h BT-medium incubation. The cells were then washed with BT-medium and increasing concentrations of IGF-I were present for the remaining 48 h. Individual points represent mean 5 SEM of triplicate determinations from one of two similar experiments. Means within a particular IGF-I concentration were not different by Student’s T test (P > 0.05).

action of IGFs. The difference in IGF-I regulation between the two IGFBPs was suggested to be due to differences in cell binding ability. However, possible species and/or cell type differences may influence regulation of IGF-I function by a specific IGFBP. The potentiative ability of IGFBP-1 could not be confirmed by Ross et al., (1989), who attributed their differences to the exclusion of platelet-poor plasma in the culture medium, which was later confirmed by Clemmons and Gardner (1990). Burch et al., (1990) found this same IGFBP-1 to inhibit IGF-I action in cartilage whether added without or with platelet-poor plasma, a condition required for IGFBP-1 potentiative function of IGF-I in smooth muscle cells (Clemmons and Gardner, 1990).

IGFBPs of different molecular weight species, some


TABLE 3. Stimulation of 'H-thymidine incorporation by IGF-I and des3-IGF-I: effect of hIGFBP-I'

Tncnhat,ion W IGF-I activitv

IGF-I des3-IGF-I IGF-I + hIGFBP-I des3-IGF-I + hIGFBP-I

100 147 i 14 59 * 10

180 + 36

"H-thymidme incorporation assays using MG-63 cells were performed as described in Materials and Methods. Treatments were added after a 24 h preincubation period with BT-medium a t the following concentrations: IGF-I (0.64 nM), des3-IGF-I (0.64 nM), and hIGFBP-1 (5.9 nM). Incorporation of 3H-thymidine into DNA was determined after 48 h. Values represent the mean + SEM of four separate experiments. Treatment means differ from IGF-I control ( P < 0.05).

with molecular weights approximating IGFBP-1, are secreted by MG-63 cells (Campbell et al., 1991) and numerous other bone cell cultures (Schmid et al., 1989a,b; Mohan et al., 1989; Ernst and Rodan, 1990; Andress and Birnbaum, 1991; Chen et al., 1991; Tor- ring et al., 1991). It is unclear if osteoblasts or other cell types in bone produce IGFBP-1, but unlike the 150 kDa IGFBP-3 complex, smaller IGFBP's can cross the capillary barrier (Binoux and Hossenlopp, 1988). Trans- capillary permeability of IGFBP-1 has been demon- strated by Bar et al., (1990). I t is possible that IGFBP-1 is available to affect IGF function in the extracellular environment of bone, whether from local or systemic sources.

Other IGFBPs, most produced by bond cells, have been found to differentially affect bone cells; suggesting that various IGFBPs may play opposing regulatory roles in IGF function. IGFBP-4 isolated from a human osteosarcoma cell line is a potent inhibitor of IGF action in bone cells (Mohan et al., 1989). Contrastingly, two IGFBPs isolated from another human osteosarcoma cell line appear to enhance IGF action in osteoblasts (Andress and Birnbaum, 1991). The N-terminal amino acid sequence of the 23 kDa form is essentially homol- ogous to the N-terminal amino acid sequence deduced from the cDNA sequence of IGFBP-6, isolated from a human osteosarcoma cDNA library (Kiefer et al., 1991). Additionally, Ernst and Rodan (1990) suggest that a growth hormone-dependent IGFBP in CM from rat osteoblast-like cells potentiates IGF-I activity in bone. The augmentation of IGF-I response by IGFBP-3 may be explained by slow release of IGF-I over time elimi- nating possible down regulation of the IGF-I receptor by large acute doses of IGF-I (Blum et al., 1989). An IGFBP-3 is also present in CM from MG-63 cells upon stimulation with growth hormone or IGF-I (Campbell, unpublished data). The growth hormone-dependent IGFBP may have a growth-stimulatory role in both normal and neoplastic human bone cells. The stimulatory role for IGFBP-3 is not clear since in porcine systems IGFBP-3 inhibits both binding and bioactivity of IGFs in non-bone cells (Gopinath et al., 1989; Walton et al., 1989). In human fibroblasts, IGFBP-3 has both inhibitory and potentiative effects depending on co- or pre-incubation with IGF-I (De Mellow and Baxter, 1989). The increased sensitivity of an equal molar concentration of des3-IGF-I over IGF-I in stimulating DNA synthesis in MG-63 cells implies that the various endogenous IGFBPs have a net inhibitory effect upon the maximal IGF-1 response.

Association constants for IGFBP-I (Baxter et al., 1987) and IGF receptors in osteoblasts, including MG-63 cells (Centrella et al., 1990; Pollack et al., 19901, are similar a t lo9 L/M. Therefore, IGFBP competition for IGF binding to IGF receptors can occur. The failure of hIGFBP-I to inhibit des3-IGF-I binding or stimulation of MG-63 cells reflects the low binding specificity of des3-IGF-I for IGFBP-1 (Forbes et al., 1988) and confirms that IGFBP-1 effects on MG-63 cells are not due to any toxic effect. Whereas hIGFBP-1 completely inhibited binding of IGF-I to MG-63 cells. IGF-I-stimulated DNA synthesis could not be completely sup- pressed. The reason is not clear but we can suggest two possible explanations: 1) Extracellular proteolytic activity over the 48 h incubation period may have resulted in a loss of IGFBP-1 activity, and 2) other IGFBPs, secreted in response to IGF-1, competed for the binding of IGF-I and having potentiative effects, acted to partially reverse the inhibitory action of hIGFBP-1.

Neoplastic states may influence circulating concentrations of IGFBPs. Plasma of patients with various tumors contains higher levels of IGFBPs (Iino et al., 1986; Rutanen et al., 1984). Serum IGFBP-2 is elevated during extrapancreatic tumor hypoglycemia (Zapf et al., 1990). Indirect in uiuo evidence implies that IGFBP changes during neoplasia may influence circulating IGFs. Plasma levels of IGFs in various neo- plasms are similar to or below concentrations found in the plasma of healthy subjects (Zapf et al., 1981). Furthermore, transplantation of human tumors into a nude mouse system results in lower plasma levels of IGF-I1 peptide (Wilson et al., 1987). However, regulation of IGFs by IGFBPs in cancer remains unclear. Whatever the role of a particular IGFBP may be in bone, regulation of IGFBP production in normal or neoplastic osteoblasts suggests regulation of IGFBP activity. Hormones and growth factors known to effect bone and IGF secretion by bone cells, i.e., parathyroid hormone, 1,25 (OH),D,, glucocorticoid, IGF-I, estradiol, and growth hormone, also influence IGFBP secretion in normal and neoplastic osteoblasts (Schmid et al., 1989a,b; Chen et al., 1991; Torring et al., 1991).

The present study provides evidence for pericellular regulation of IGF activity in bone by hIGFBP-1 by the sequestration of IGFs into bioinactive complexes.

ACKNOWLEDGMENTS We wish to thank Teresa Hentosz for her technical

assistance in cell culture and Kathleen Pater for her help in the preparation of this manuscript. This work was supported in part by Allegheny-Singer Research Institute, The Robert and Mary Weisbrod Foundation, and the Pardee Foundation.

LITERATURE CITED Andress, D.L., and Birnbaum, R.S. (1991) A novel human insulin-like

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insulin-like growth factor binding protein (igfbp) inhibits igf action on human osteosarcoma cells

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