0013-7227/91/1292-G939$()3.00/0 Endocrinology Copyright ^ 1991 by The Endocrine Society

Vol. 129, No. 2 Printed in U.S.A.

Insulin-Like Growth Factor (IGF) Binding to Cell Monolayers Is Directly Modulated by the Addition of IGF-Binding Proteins* ROBERT H. McCUSKER, WALKER H. BUSBY, MARLIN H. DEHOFF, CECILIA CAMACHO-HUBNER, AND DAVID R. CLEMMONS Department of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599

ABSTRACT. Insulin-like growth factor-I (IGF-I) binds to specific receptors and IGF-binding proteins (IGFBPs) that are present on cell surfaces. The analysis of [125I]IGF-I binding to human fibroblasts is complicated by IGFBPs on the cell surface and their release into the medium during the binding assay. This release alters the distribution of [125I]IGF-I between type I IGF receptors and both soluble as well as cell surface-associated IGFBPs. In the present study we have determined the effects of three different forms of IGFBPs on [l25I]IGF-I binding to cell surface binding sites of human fetal fibroblasts (GM10 cells) and porcine smooth muscle cells. Human 29,000 mol wt (Mr; IGFBP-1), bovine 34,000 Mr (IGFBP-2), and bovine 46,000 Mr (IGFBP-3) forms of IGFBP were compared. Each of the three IGFBPs inhibited [125I]IGF-I binding to the cell surface of both cell types. This effect was due to increased binding of [''2r>I]IGF-I by the IGFBPs in the assay buffer. At equimolar concentrations, IGFBP-3 was more effective than either IGFBP-1 or IGFBP-2 in blocking cell surface binding.

The addition of increasing concentrations of unlabeled IGF-I in the presence of each IGFBP showed that either IGFBP-1 or IGFBP-3, but not IGFBP-2, resulted in a paradoxical increase in [125I]IGF-I binding to the cell surface. The paradoxical increase occurred in the presence of excess insulin, indicating that unsaturated type I IGF receptors are not required to demonstrate this phenomenon. In a physiological salt solution, the order of affinity of the IGFBPs for IGF-I was IGFBP-3 > IGFBP-1 > IGFBP-2. These differences in affinity appear to account for the differences in IGF-I competition for binding that are seen when each of the three proteins is added. Thus, IGFBPs have the potential to alter the partitioning of IGF-I between cell surfaceassociated IGFBPs, membrane receptors, and the IGFBPs in extracellular fluids. The various forms of IGFBP affect IGF cell surface binding differently, and therefore, each may have distinct effects on IGF target cell actions. (Endocrinology 129: 939-949, 1991)

I

Using molecular cloning techniques, investigators have determined the structures of four forms of IGFBP. Forms with estimated mol wt (Mr) of 39,000-53,000 [defined by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis] are believed to represent alternative glycosylation products of the same core protein, IGFBP-3 (10). Plasma concentrations of this protein are GH dependent (11). Likewise, 34,000, 29,000, and 24,000 Mr proteins (termed IGFBP-2, IGFBP-1, and IGFBP-4, respectively) have been structurally characterized (12-14). Cultured human fibroblasts have been shown to secrete IGFBP-3 (15, 16) and IGFBP-1 (17), porcine smooth muscle cells secrete IGFBP-2 (15), and both cell types secrete a 24,000 Mr form, which is probably IGFBP-4. An additional form with an estimated Mr of 31,000, which is secreted by fibroblasts, appears to be structurally distinct (15). IGFBPs have the potential to modulate IGF binding to two forms of receptors on cell surfaces and thereby modulate the cellular response to IGF-I. Both IGFBP-1 and IGFBP-3 have been shown to potentiate the fibro-

NSULIN-like growth factors (IGF)-I and -II are mitogenic for a variety of cell types (1, 2) and stimulate somatic growth when infused into normal or hypophysectomized animals (3, 4). Within minutes after infusion, IGF-I or IGF-II becomes associated with ligand-specific high affinity IGFBPs1 that are present in the circulation (5, 6). The IGFBPs are also present in extracellular fluids, such as lymph (7), amniotic fluid (8), and cerebral spinal fluid (9) as well as in cell culture supernants. Therefore, these extracellular fluid IGFBPs have the potential to modulate IGF activity by controlling the amount of IGF that is available to bind to cell surface binding sites.

Received February 6,1991. Address all correspondence and requests for reprints to: Robert H. McGusker, Ph.D., 348 MacNider Building, CB 7170, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599. * This work was supported by NIH Grant (AG-02331, to D.R.C.) and a Research and Development Award from the American Diabetes Association (to R.H.M.). 1 We have accepted the nomenclature for the IGFBPs suggested at a Workshop on IGFBPs held in Vancouver, Canada, June 17-19,1989 (Endocrinology 125:2359,1989).

939

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COMPARISON OF IGFBPS

940

blast DNA synthesis response to IGF-I (18, 19), but it is not clear how IGFBP modulation of IGF binding alters the cellular growth response. In previous studies the presence of IGFBPs in the incubation buffer has been shown to directly alter IGF1 binding to type I IGF receptors and cell surfaceassociated IGFBPs (20, 21).. Affinity labeling studies have shown that IGFBPs are associated with fibroblast surfaces, and these cell surface-associated IGFBPs are only partially removed by routine washing procedures before the binding assay (22). These extracellular IGFBPs are released from cell surfaces into the binding assay buffer, and their release results in partitioning of the [125I]IGF-I, such that addition of unlabeled IGF-I causes a paradoxical increase in the amount of [125I]IGFI that is associated with the cell surface (23). Partitioning occurs because the released IGFBPs have a 10-fold greater affinity for IGF-I than those remaining on the cell surface. Since cell surface-associated IGFBPs account for nearly 90% of the IGF-I-binding sites (22), their presence and release markedly affect the availability of IGF-I to bind to the cell surface receptors. The current studies were undertaken to compare the abilities of pure forms of IGFBP to alter [125I]IGF-I partitioning between assay buffer IGFBPs and cell surface-associated binding sites and to determine the molecular basis of any differences that were observed.

Materials and Methods Materials Human IGF-I used for iodination had been purified by a previously described procedure (24) and was iodinated (250350 juCi/Mg) using a chloramine-T method (25). Unlabeled recombinant human IGF-I and IGF-II were purchased from Bachem (Torrance, CA), and insulin was purchased from E. R. Squibb and Sons, Inc. (Princeton, NJ). BSA-linoleic acid (BSA-LA), polyethylene glycol (PEG) 8000, HEPES, and other chemicals were purchased form Sigma Chemical Co. (St. Louis, MO). Dulbecco's Modified Essential Medium (DMEM) and Eagle's Minimum Essential Medium (EMEM) were purchased from Hazelton Systems, Inc. (Denver, PA). Fetal bovine serum and calf serum were purchased form Hyclone Laboratories, Sterile Systems, Inc. (Logan, UT) and Colorado Serum Co. (Denver, CO), respectively. Human immunoglobulins were purchased as an 18% solution (Cutter Biological, Berkeley, CA). Rainbow high mol wt protein standard was purchased from Amersham Corp. (Arlington Heights, IL). The 29,000 Mr (25,272-dalton) IGFBP-1 was purified from human amniotic fluid (8). Bovine 34,000 Mr IGFBP-2 was purified from 19 liters of Madin-Darby bovine kidney (MDBK) cell-conditioned medium. The conditioned medium was first applied to a phenyl-Sepharose (CL-4B, Pharmacia-LRB, Uppsala, Sweden) column and eluted by changing the column buffer to 0.02 M Tris, pH 9.0. Fractions with IGF-binding activity were then applied to an IGF-I affinity column, and activity was

Endo«1991 Voll29«No2

eluted with 0.5 M acetic acid. Final purification was obtained by C4 reverse phase (Vydak, Hesperia, CA) HPLC, as described previously for IGFBP-1 (8). The purified protein eluted as a single peak (our unpublished data). Bovine 46,000 Mr IGFBP3 was purified from bovine placental membranes, using salt extraction of homogenized membranes (0.2 M NaCl and 0.03 M HEPES, pH 7.4), followed by separation through phenyl-Sepharose. The sample was loaded onto phenyl-Sepharose after adjustment to 1.5 M NaCl, 10% (wt/vol) (NH4)2SO4, and 50 mM Tris, pH 7.4. The active fraction eluted in 0.02 M Tris, pH 9.0, and with water. These two active eluates were pooled and concentrated by precipitation with 70% (wt/vol) (NH4)2SO4. The concentrated sample was loaded onto an G-150 Sephdex column in 0.02 M Tris and 0.025 M NaCl, pH 7.2. Active fractions were pooled, then further purified by chromatography through a propylaspartamide column (The Nest Group, Southboro, MA). The sample was loaded in 15% (wt/vol) (NH4)2SO4 and 0.1 M KH2PO4, pH 6.5. The active fractions were eluted with a linear gradient, starting with the loading buffer and changing to 0.01 M KH2PO4, pH 6.5. The activity was pooled, then final purification was obtained with a C3 reverse phase (Alltex, Berkeley, CA) HPLC column, using the same buffers and elution conditions as those described for IGFBP-1 purification with the C4 column. Overall recovery of bovine IGFBP3 was 16%. All three IGFBPs were judged to be greater than 95% pure by SDS-polyacrylamide gel electrophoresis, followed by silver staining. Protein content was determined by amino acid composition analysis. The molarity of the IGFBP solutions was calculated using estimated Mr values (39,000, 34,000, and 29,000 for IGFBP-3, -2, and -1, respectively), since at this time we do not know the size of the amino acid cores for bovine IGFBP-2 and -3. These estimates are subject to some error, since IGFBP-3 may have variable degrees of glycosylation and IGFBP-1 and -2 may migrate anomalously on SDS gels. GM0010A (GM10), fetal human fibroblasts, were purchased from the NIGMS Human Genetic Mutant Cell Repository (Camden, NJ). Porcine vascular smooth muscle (pSM) cells were propagated from aortic explants, using a previously described method (26). These two cell types were compared, since GM10 cells secrete IGFBP-3, and over 90% of added [r25I]IGFI binds to cell surface-associated IGFBPs (22, 23). In contrast, pSM cells secrete IGFBP-2 (15) and have little cell surfaceassociated IGFBPs, as determined by affinity labeling (22). Added [125I] IGF-I binds primarily to type I IGF receptors on pSM cells. Thus, the added IGFBPs may affect binding to the two cell types differentially. Procedures Both fibroblasts and pSM cells were grown in stock cultures and passaged as previously described (27). The pSM cell cultures were maintained in DMEM plus 10% fetal bovine serum, 4 mM glutamine, 100 U/ml penicillin, and 100 Mg/ml streptomycin until used for the binding assays. GM10 fibroblasts were grown in EMEM plus 10% calf serum supplemented with 110 jug/ml pyruvate, 30 fig/m\ asparagine, 21 /ng/ml serine, 100 U/ ml penicillin, and 100 Mg/ml streptomycin. GM10 cell cultures were switched to DMEM plus 100 Mg/ml BSA-LA, 25 /ig/ml cycloheximide, and antibiotics 18 h before performing the binding assay. For binding assays, cells were plated in 2-cm2 mul-

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COMPARISON OF IGFBPS tiwell plates (Falcon 3047, Oxnard, CA) at 1 x 104 cells/cm2 and fed on day 4. Three days later, when the cultures had reached confluency (6-10 X 104 cells/cm2), they were used for binding assays. Binding of [12r>I]IGF-I to cell surfaces was performed as previously described (28, 29). In brief, cells were rinsed twice with PBS, then incubated for 2-3 h (the time needed to reach equilibrium) at 8 C with 0.25 ml assay buffer (EMEM, 20 mM HEPES, and 1 mg/ml BSA, pH 6.4 or 7.4) containing 70,00090,000 cpm [iafiI]IGF-I plus unlabeled IGF-I, IGF-II, insulin, or the IGFBPs at the indicated concentrations. At the end of the incubation period the assay buffer was removed, and the cells were rinsed twice with PBS, then solubilized with 0.3 M NaOH before cell surface-associated [125I] IGF-I was determined by counting with a 7-spectrometer. To assess the [12'r'I]IGF-I-binding activity of the proteins in the assay buffer at the end of the 2- to 3-h incubation, it was collected and added to 12 x 75-mm polystyrene tubes. A 0.25ml aliquot of 1% human immunoglobulin, prepared in assay buffer, and 0.5 ml 25% PEG-8000, prepared in water, was added. The mixture was vortexed, incubated for 20 min at 8 C, and centrifuged; the supernatant was removed; and the pellet was resuspended with 6.25% PEG-8000 and centrifuged at 9000 x g (30). Assay buffer binding protein activity was measured by counting the counts per min of [125I]IGF-I in the final pellet. All results are presented as counts per min of [125I]IGF-I bound to the cell surface or to IGFBPs in the assay buffer. Nonspecific [iar'I]IGF-I precipitated in the assay buffer was 8,000-15,000 cpm and is due to [12BI]IGF-I being associated with the BSA, sticking to the sides of the assay tubes, or trapped in the PEG-protein pellet. This was confirmed to be nonspecific binding by affinity labeling in the presence of 100 ng/ml IGF-I (23). To determine the binding affinity constants (Ka) of the three forms of IGFBP, competition for [12flI]IGF-I binding with unlabeled IGF-I was performed, and the results were analyzed by Scatchard analysis. The indicated amount of each IGFBP was incubated at 22 C with 20,000 cpm [12BI]IGF-I with or without unlabeled IGF-I. After a 2-h incubation, bound and unbound labeled IGF-I were separated by precipitation of the IGFBPbound IGF-I with PEG-8000 as described for the IGFBP binding assays. The incubation (0.25 ml final volume) was performed in the binding assay buffer (EMEM, 20 mM HEPES, and 1 mg/ml BSA, pH 6.4 or 7.4). Nonspecific binding was the amount of labeled peptide precipitated in the absence of added IGFBP. Ligand blot analysis was performed as previously described (30). Purified IGFBPs were electrophoresed through a 12.5% SDS-polyacrylamide gel, transferred to a nitrocellulose filter, and probed for IGF-binding activity with 400,000 cpm [125I] IGF-I. Blocking and probing buffers were as previously described (31). An autoradiogram of the nitrocellulose filter was made using Kodak X-Omat AR film (Eastman Kodak, Rochester, NJ).

Results The three purified IGFBPs that were compared for their ability to modulate [125I]IGF-I binding to the cell surfaces were

941

analyzed for homogeniety by ligand blotting. The relative mobilities of the 46,000 Mr IGFBP-3 (left), the 34,000 Mr IGFBP2 (middle), and the 29,000 Mr IGFBP-1 (right) are shown in Fig. 1. The results demonstrate that the relative sizes are as predicted and that each purified preparation contains only one form of IGFBP. Scatchard analysis of competition by unlabeled IGF-I for binding to each form of IGFBP was determined using physiological salt concentrations (i.e. the binding assay buffer that contains 0.15 M NaCl) at pH 7.4. Both IGFBP-3 and IGFBP1 had curvilinear Scatchard plots, resulting in two affinity estimates. The results show that IGFBP-3 had the highest affinity binding site for IGF-I [Ka = 5.08 X 109 liters/M (Fig. 2]. IGFBP-1 had the next highest affinity component. In contrast, IGFBP-2 had a single affinity estimate. Both IGFBP-1 and IGFBP-3 had a low affinity binding component, with Ka constants similar to that of IGFBP-2 (Kfl = 0.65 x 109 liters/ M). Scatchard analysis of [125I]IGF-I binding to IGFBP-1 was performed at pH 6.4 (Fig. 2, inset). Despite having the same amount of added protein, IGFBP-1 specifically bound 217% more [125I]IGF-I at pH 6.4 vs. pH 7.4. This increased binding was not due to a marked change in affinity for IGF-I (Ka = 2.04 X 109 vs. 2.48 x 109 liters/M for the high affinity site and 0.87 X 109 vs. 0.32 x 109 liters/M for the low affinity site at pH 6.4 and 7.4, respectively), but was due to a 3.0-fold increase in the number of high affinity sites present at pH 6.4 (2.74 x 10~10 M/liter) compared to pH 7.4 (0.92 X 10~10 M/liter). There was no apparent change in the number of low affinity binding sites (4.48 x lO"10 vs. 3.16 x 10"10 M/liter at pH 6.4 and 7.4, respectively). The effect of each of the purified IGFBPs on [125I)IGF-I binding to GM10 fibroblasts that had been exposed to cycloheximide was determined (Fig. 3, right). Cycloheximide exposure has been shown to inhibit the release of endogenous IGFBPs (22, 23) and was added to eliminate their effects on IGF-I binding. Competition for [125I]IGF-I binding to the cell surface was determined in the presence of increasing concentrations of each of the three IGFBPs, unlabeled IGF-I, or insulin. IGFBP-3 was the most effective in reducing [125I]IGF-

-3

MrxlO

— 46

I

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FIG. 1. Ligand blot analysis of the three preparations of IGFBPs. Purified IGFBP-3 (left lane), IGFBP-2 (center lane), and IGFBP-1 (right lane) were electrophoresed through a 12.5% SDS-polyacrylamide gel, electroblotted to nitrocellulose paper, and probed with [125I]IGF-I. The estimated Mr of each form was derived by comparison to defined Mr standards and is shown on the vertical axis.

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COMPARISON OF IGFBPS

942 0.25

hlGFBP-1 0 - 60

Endo • 1991 Vol 129 • No 2

blGFBP-2

blGFBP-3

2.04x109

FIG. 2. Scatchard analysis of IGF-I competition for binding to purified IGFBPs. Scatchard analysis of IGF-I binding to IGFBP-1 (0.5 nM), IGFBP-2 (0.5 nM), and IGFBP-3 (0.1 nM), as described in Materials and Methods. Competition for [125I]IGF-I binding was determined using 0-2.6 nM unlabeled IGF-I. Ligand affinities shown (liters per M) are Ka values, h, Human; b, bovine.

0

1

2

3

K fl =0.32x10 9 .

I ' ^ * K n =0.65x10 9

K«=0.52x109

2 0 10

2 0

Bound (M/LX io ) 16

GM10

pSMC 6-

FIG. 3. Binding of [125I]IGF-I to cell surface binding sites and IGFBPs in the assay buffer using GM10 fibroblast and porcine smooth muscle cell cultures. The binding at pH 7.4 of [125I]IGF-I to the cell surface of cycloheximide-treated GM10 fibroblast monolayers (right) and pSM cells (left) was assessed in the presence of increasing concentrations of IGF-I (O), IGFBP-1 (•), IGFBP-2 (A), IGFBP-3 (A), or insulin (•; top). In addition, the amount of [125I]IGF-I bound in the assay buffer (bottom) was measured. Assay buffer binding was determined by collecting the buffer from each well at the end of the binding assay and separating bound from free ligand by precipitation with PEG, as described in Materials and Methods. All points are the mean ± SEM of duplicate determinations. SEM bars are shown for all points where they are larger than the symbols.

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Peptide (nM) I binding to GM10 cell surfaces compared to IGFBP-1, IGFBP2, insulin, or IGF-I. Half-maximal competition occurred at 0.2, 1.3, 3.2, and 4.2 nM for IGFBP-3, IGFBP-2, IGFBP-1, and IGF-I, respectively. Insulin competed poorly for [125I]IGF-I binding to this cell type, indicating that most of the cell surface binding was not to type I IGF receptors. To determine if the IGFBPs were affecting cell surface [125I] IGF-I association by direct competition, [125I]IGF-I binding to

each of the IGFBPs that had been added was measured in the assay buffer that was collected from each culture (Fig. 3, lower right). Approximately 10,000 cpm [125I]IGF-I was precipitable in the assay buffer of cultures without added IGFBPs. Addition of IGF-I did not change this value, indicating that it was due to nonspecific binding. Addition of increasing concentrations of each of the three IGFBPs increased precipitable [125I]IGF-I. IGFBP-1 and IGFBP-3 bound similar amounts of [I26I]IGF-I

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COMPARISON OF IGFBPS at the highest concentrations tested. In contrast, IGFBP-2 bound less [11!fiI] IGF-I at the highest concentration compared to the amount bound by either IGFBP-1 or IGFBP-3. The lower binding of IGFBP-2 in solution was repeated four times, and this finding was consistent. To determine if these IGFBPs had similar effects on noncycloheximide-exposed cultures, binding was determined using pSM cells (a cell type that does not release IGFBP-3 into the assay buffer). All three IGFBPs decreased [125I]IGF-I binding to the cell surface of pSM cells (Fig. 3, top left). Again, IGFBP3 was more potent in decreasing cell surface [125I]IGF-I binding than IGFBP-1 and IGFBP-2. Analysis of [125I]IGF-I binding to assay buffer showed that added IGFBP-1 and IGFBP-3 bound IGF-I in proportion to their capacity to compete for cell surface binding. In contrast, IGFBP-2 bound similar amounts of [125I] IGF-I in solution compared to IGFBP-1, but reduced cell surface binding to a greater extent. No decrease in the amount of assay buffer-precipitable [12flI]IGF-I was found when unlabeled IGF-I was added to cultures that had no added IGFBPs. To determine if the IGFBPs that were added could associate with cell surfaces, pSM cell cultures were preincubated with the three IGFBPs, and the cultures were washed. [125I]IGF-I cell surface binding was then assessed. Addition of the IGFBPs increased [12rTJIGF-I binding compared to that in control cultures (Table 1). IGFBP-3 appeared to increase cell surface [125I] IGF-I better than did the other two forms. Exposure to IGFBP1 and IGFBP-2 caused equivalent increases in cell surfaceassociated [iaBI]IGF-I. In previous studies the addition of unlabeled IGF-I to monolayer cultures of human fibroblasts was shown to result in a paradoxical increase in cell surface-associated [125I]IGF-I (22, 32, 33). This change is associated with the release of at least two forms of IGFBP into the assay buffer during the binding assay (23). To determine if defined concentrations of one or more of the purified forms of IGFBP could induce similar kinetic changes, competition between [125I]IGF-I and unlabeled IGF-I for binding to cycloheximide-treated GM10 fibroblast TABLE 1. Changes in IGF binding to pSM cell surfaces after exposure to IGFBPs

[125I]IGF-I bound (cpm) Pretreatment

IGF-II Insulin

Control IGFBP-1 (1.2 nM) IGFBP-2 (1.2 nM) IGFBP-3 (0.3 nM)

+ + + +

3233 ± 301 2165 ± 165 2695 ± 107" 2610 ± 346 2931 ± 33°

ND 1249 ± 45 1109 ± 164 1080 ± 126 1090 ± 8

Confluent monolayer cultures of pSM cells were preincubated for 2.5 h at 8 C with assay buffer with or without insulin (5 Mg/m0 or the indicated IGFBP. The cultures were then used to determine [125I]IGFI binding, as described in Materials and Methods. Cell cultures that contained insulin during the pretreatment also had insulin added to the assay buffers during the binding assay. The binding assay was performed with or without the addition of unlabeled IGF-II (100 ng/ ml). Data represent the mean ± SD counts per min bound/well for duplicate determinations. ND, Not determined. " P < 0.05 compared to control plus insulin, by t test.

943

and noncycloheximide-treated pSM cell cultures was determined in the presence of each of the three forms of IGFBP. In the absence of added IGFBPs (Fig. 4, upper right), unlabeled IGF-I blocked [I25I]IGF-I binding to GM10 cells in a concentration-dependent manner, and no paradoxical increase in binding was observed. In the presence of IGFBP-3 (68 pM), the competition curve with unlabeled IGF-I was flat when IGF-I concentrations between 0.3-0.7 nM were used. The presence of IGFBP-3 (172 pM) decreased [125I]IGF-I basal binding, and the addition of unlabeled IGF-I resulted in a paradoxical increase in [125I]IGF-I binding. The maximal increase in [125I]IGF-I binding was 28%. The amount of [125I]IGF-I that bound to the added IGFBP-3 was measured (Fig. 4, lower right). Addition of increasing amounts of IGFBP-3 increased basal binding of [125I] IGF-I in the assay buffer. This increase was due to specific binding, since IGF-I, but not insulin, competed for assay bufferassociated [125I]IGF-I binding. Thus, the paradoxical increase in cell surface [125I]IGF-I binding occurred in parallel with the competitive displacement by unlabeled IGF-I of [125I]IGF-I that was specifically bound to IGFBP-3 in the assay buffer. Increasing competition for cell surface [125I]IGF-I binding also occurred when increasing concentrations of unlabeled IGFI were added to pSM cultures, and no paradoxical increase occurred (Fig. 4, upper left) in the absence of added IGFBP-3. Addition of 68 or 172 pM IGFBP-3 decreased basal [125I]IGF-I binding, and a paradoxical increase in binding became evident with the addition of unlabeled IGF-I. In the presence of IGFBP3 (172 pM), the maximal paradoxical increase in binding with added IGF-I was 36%. Addition of IGFBP-3 caused an increase in the amount of [125I]IGF-I bound in the assay buffer (Fig. 4, lower left). The increased [125I]IGF-I binding that was detected in the assay buffer was specific. In attempting to compare the effects of IGFBP-1 to those of IGFBP-3 on IGF-I binding, competition curves were generated with the addition of IGFBP-1. IGFBP-1 decreased basal [125I] IGF-I cell surface binding to both GM10 and pSM cells. A small paradoxical increase (4.9 ± 1.4; n = 2) in cell surface [125I]IGF-I binding was found in the presence of 3.2 nM IGFBP1 at pH 7.4 (data not shown). Since we had noted that IGFBP1 had a greater affinity for IGF-I at pH 6.4, binding to GM10 fibroblasts and pSM cell cultures was determined in the presence of IGFBP-1 at that pH (Fig. 5). IGFBP-1 (1.2 and 3.2 nM) decreased basal cell surface [125I] IGF-I binding to both GM10 cells (Fig. 5, upper right) and pSM cells {upper left). The presence of IGFBP-1 altered the competition curve. When 3.2 nM IGFBP-1 was used, addition of IGF-I resulted in a paradoxical increase in [125I]IGF-I binding to GM10 fibroblasts. Using cycloheximide-treated GM10 cells, the average maximal paradoxical increase in binding in the presence of 3.2 nM IGFBP-1 was 11.3 ± 4.0% (P < 0.05; n = 3 experiments); using pSM cells, the average maximal increase was 19.9 ±4.1% (P < 0.05; n = 5 experiments). At pH 6.4, the paradoxical increase in cell surface [125I] IGF-I binding was, therefore, considerably greater than that at pH 7.4. Inclusion of insulin (7000 nM) in the assay buffer resulted in greater paradoxical increases. For GM-10 cells, the paradoxical increase was 8% in the absence of insulin and 13% with insulin present (Fig. 5, upper right). Thus, unsaturated type I receptors are not required to generate the

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COMPARISON OF IGFBPS

944

FlG. 4. Alterations in IGF-I binding to GM10 and pSM cells that are induced by incubation with IGFBP-3. The competition between [125I]IGF-I and unlabeled IGF-I (0.3-6.7 nM) or insulin (7000 nM) for binding at pH 7.4 to the cell surface of cycloheximide-treated GM10 fibroblast {right) and pSM cell (left) monolayers was determined in the presence of 0 (O), 68 (•), or 172 pM (A) IGFBP-3. [125I]IGF-I binding to the cell surface (top) and in the assay buffer (bottom) were both determined. All points are the mean ± SEM of duplicate determinations. SEM bars are shown for all points where they are larger than the symbols.

Kudo • 1991 Vol 129 • No 2

I O

Q. O ^^ C O CO

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12--

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0.3 0.7 1.3

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6.7 7000

Peptide (nM) paradoxical increase in cell surface binding. The addition of IGFBP-1 resulted in more [125I] IGF-I being bound in the assay buffer when either GM10 or pSM cells were used. The IGFBP1-associated [125I]IGF-I could be competed by the addition of IGF-I, but not insulin. Despite several attempts we were unable to generate a paradoxical increase in [125I] IGF-I cell surface binding by the addition of increasing concentrations of IGF-I in the presence of IGFBP-2 (Fig. 6). Addition of IGFBP-2 to GM10 (Fig. 6, upper right) and pSM cultures (upper left) decreased [125I]IGFI binding to the cell surface. However, addition of unlabeled IGF-I with IGFBP-2 did not result in a paradoxical increase, even in the presence of insulin. The average maximal paradoxical increases in binding using 2.2 nM IGFBP-2 were 1.9 ± 1.2% (P > 0.05; n = 3) and 0.7 ± 0.7% (P > 0.05; n = 3) for GM10 and pSM cell cultures, respectively. Addition of IGFBP-2 to GM10 (Fig. 6, lower right) and pSM (lower left) cultures resulted in an increased amount of [125I]IGF-I bound in the assay buffer. The bound [125I] IGF-I was competed by thp addition of IGF-I, but not insulin. Since IGFBP-2 and IGFBP-3 have higher affinities for IGFII compared to IGF-I, binding studies were conducted using IGF-II to determine if the paradoxical increase in binding could be detected. IGF-II competed for cell surface [125I]IGF-I binding to both GM10 (Fig. 7, upper left) and pSM (upper right) cells in a dose-dependent manner. With the addition of increasing

concentrations of IGF-II, IGFBP-3 and IGFBP-1 were the most effective in causing the paradoxical increase in [125I]IGF-I binding, followed by IGFBP-2, with the maximal degree of paradoxical increase being 18%, 9%, and 6% for GM10 cells, respectively, and 16%, 21%, and 13% for pSM cells. [12BI]IGFI binding to the IGFBPs in the assay buffer was also measured for both GM10 (Fig. 7, lower left) and pSM (lower right) cultures. The addition of the IGFBPs increased [125I]IGF-I binding, and this increase in bound [125I]IGF-I was competed by the addition of IGF-II. Although the IGFBPs bound similar amounts of [125I]IGF-I, IGF-II was more effective in competing for binding to IGFBP-3 and least effective for IGFBP-2. This order of potency agrees with the relative degree of paradoxical increase in cell surface [125I]IGF-I binding caused by each of the three IGFBPs.

Discussion Three forms of IGFBP were tested to compare their effects on [125I]IGF-I binding to fibroblast and smooth muscle cell surfaces. Direct addition of each of the three forms to the binding assay buffer resulted in decreased binding of [125I] IGF-I to cell surfaces. IGFBP-3 was the most effective of the three IGFBPs, whereas IGFBP-1 was the least effective. Decreased cell surface association of [125I] IGF-I was directly related to the concentration

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COMPARISON OF IGFBPS 10

945 GM10

pSMC 20-

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16-

Fio. 5. Alterations in IGF-I binding to GM10 fibroblasts and pSM cells that are induced by incubation with IGFBP-1. The binding of [iaiI]IGF-I to the cell surface (top) of cycloheximide-treated GM10 fibroblasts (right) or untreated pSM (left) cell monolayers was assessed at pH 6.4 in the absence (O) or presence of 1.2 nM (•) or 3.2 nM (A) IGFBP-1 or 7000 nM insulin plus 3.2 nM IGFBP-1 (V). In addition, [iasI]IGF-I binding to the assay buffer (bottom) was measured. Competition for [12SI]IGF-I binding was assessed by the addition of 0.3-6.7 nM IGF-I or 7000 nM insulin. All points are the mean ± SEM of duplicate determinations, SEM bars are shown for all points where they are larger than the symbols.

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Peptide (nM) of IGFBP added and the subsequent increase in [125I] IGF-I binding by each of the three forms of IGFBP. Thus, the IGFBPs appear to decrease cell surface association of [125I]IGF-I by preventing its association with cell surface binding sites. IGFBP-3, the form that blocks binding most effectively, has the highest affinity for IGFI (23,34-36), suggesting that there is a direct relationship between IGFBP affinity and the decrease in cell surface binding. However, we noted that IGFBP-1 has a high affinity binding site for IGF-I that is greater than the affinity of IGFBP-2, yet IGFBP-2 caused a greater decrease in cell surface-associated binding. This discrepancy could be due to the finding that IGFBP-1 has both low and high affinity IGF-I-binding sites, whereas IGFBP-2 does not. Since low affinity sites of IGFBP-1 represent the majority (~80%) of the binding sites, the net affinity of IGFBP-1 is lower than that of IGFBP-2. In addition, we have previously shown by cross-linking experiments that IGFBP-1 and -3 adhere to cell surfaces (8, 22). Therefore, it is possible that this discrepancy could be explained by more IGFBP-1-[125I-IGF-I] complex becoming associated with cell surfaces during the binding assay compared to IGFBP-2-[125I-IGF-I]. However, our data in Table 1 do not allow us to draw a

definitive conclusion regarding this possibility. Our conclusions are subject to some limitations, since the differences in the affinities of the different forms of IGFBP might have been due to their different sources (bovine IGFBP-2 and -3; human IGFBP-1). However, the purified bovine IGFBP-3 had an affinity constant that was nearly identical to that of IGFBP-3 released from human fibroblasts (23). We have no information at this time concerning possible affinity differences between human and bovine IGFBP-2. A second problem, which limits our ability to extrapolate our findings to an in vivo system, is that the exact pericellular concentrations of the IGFBPs have not been determined. However, the IGFBPs were tested using concentrations that are within the ranges that have been determined to be present in extracellular fluids. Other studies have shown that purified IGFBPs compete for [125I]IGF-I binding to cell membrane preparations or cell surfaces (20, 21, 37), but none has directly compared all three pure proteins. This competition is one of the proposed mechanisms by which the IGFBPs have been postulated to act as inhibitors of the metabolic (21, 38-40) and mitogenic (19, 37, 41) actions of IGF-I. These findings suggest that in some target cell assay

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COMPARISON OF IGFBPS

946

Endo • 1991 Vol 1 2 9 ' N o 2

6-

FIG. 6. Effect of IGFBP-2 on [125I]IGFI binding to GM10 fibroblasts and pSM cells. The binding at pH 7.4 of [125I]IGF1 to the cell surface (top) of cycloheximide-treated GM10 fibroblasts (right) or untreated pSM (left) cell monolayers was assessed in the absence (O) or presence of 0.9 nM (•) or 2.2 nM (A) IGFBP2 or insulin (7000 nM) plus 2.2 nM IGFBP-2 (V). In addition, [125I]IGF-I binding to the assay buffer (bottom) was measured. Competition for [125I]IGF-I binding was assessed by adding IGF-I (0.3-6.7 nM) or insulin (7000 nM). All points are the mean ± SEM of duplicate determinations. SEM bars are shown for all points where they are larger than the symbols.

Cell Surface I 0 I 1

1

0.3 0.7 1.3

6.7 7000

0.3 0.7 1.3

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Peptide (nM) systems the IGFBPs may act directly to limit the access of IGFs to receptors and thereby inhibit cell growth. This conclusion is further supported by studies that have used IGF mutants and truncated forms of IGF-I that bind poorly to IGFBPs. These studies have shown that such variants are more potent than nonmutated IGF-I in stimulating [3H]thymidine incorporation into muscle cells and BALB/C 3T3 fibroblasts, presumably because they do not bind to endogenously secreted IGFBPs (42, 43). The inclusion of a constant amount of each of the three forms of IGFBP in the binding assay buffer, while adding increasing concentrations of unlabeled IGF-I, revealed another difference between each of the three forms of IGFBP. Addition of IGFBP-3 (172 pM) with low concentrations of either unlabeled IGF-I or IGF-II (0.3-1.3 nM) resulted in a paradoxical increase in [125I] IGF-I cell surface association. This form of IGFBP was most effective in causing this phenomenon. IGFBP-1 also caused significant changes, although the increases were less than those found with IGFBP-3. By altering the pH of the assay buffer, we could increase the apparent number of the higher affinity binding sites of IGFBP-1 for IGF-I. This change increased the paradoxical increase

in cell surface [125I] IGF-I binding found in the presence of IGFBP-1. In contrast, despite numerous attempts, we did not note this paradoxical increase with the addition of increasing amounts of IGF-I in the presence of IGFBP-2. However, this effect was detected with IGFII. Since IGFBP-2 has a higher affinity for IGF-II than for IGF-I, this supports the conclusion that induction of the paradoxical increase is related to the affinity of the IGFBP for IGF-I or -II. This conclusion is also reinforced by the finding that IGFBP-3 has the greatest affinity for IGF-I and is associated with the greatest paradoxical increase, whereas IGFBP-2 has the lowest affinity and is associated with the lowest increase. Likewise, we noted that the affinity profiles of IGFBP-3 and IGFBP-1 were curvilinear, whereas that of IGFBP-2 was linear. Therefore, the absolute affinity of each form of IGFBP, the amount of IGFBP that adheres to cell surfaces, as well as the changes in their affinities that occur with cell surface association may play an important role in the extent to which IGF-I is distributed between assay buffer and cell surface binding sites. In previous studies we have reported that IGFBPs in solution had higher affinity for IGF-I than IGFBPs associated with cell surfaces and the type I IGF receptor (44), and this difference in affinity

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COMPARISON OF IGFBPS

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6-

Fio.7. Effect of IGF-II on [1MI]IGF-I binding to GM10 and pSM cells. The binding at pH 7.4 of [125I]IGF-I to the cell surface (top) of cycloheximidetreated GMK) fibroblasts (right) or untreated pSM (left) cell monolayers was assessed in the absence (•) or presence of 3.2 nM 1GFBP-1 (A), 2.2 nM IGFBP2 (A), or 172 pM IGFBP-3 (D). In addition, [iar'I]IGF-I binding to the assay buffer (bottom) was measured. Competition for [l!:sI]IGF-I binding was assessed by adding IGF-II (0.3-6.7 nM). All points are the mean ± SEM of duplicate determinations. SEM bars are shown for all points where they are larger than the symbols.

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10

0.3 0.7

1.3 2.6

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Peptide (nM) was necessary to demonstrate the paradoxical increase in [125I]IGF-I binding (22, 23, 33). Our findings in this study show that IGFBP-2 does not have an affinity for IGF-I that is sufficiently greater than the cell surface binding sites (type I IGF receptor and cell surface-associated IGFBP-3 and -2) to result in a paradoxical increase in [125I]IGF-I cell surface binding. The key to understanding the mechanism by which this paradoxical increase in binding is mediated rests upon two observations. First, we have previously shown that over 90% of fibroblast surface-associated IGF-Ibinding sites are due to cell surface-associated IGFBPs and not to type I IGF receptors (22). More importantly, the affinities of cell surface-associated IGFBP-3 for IGFI (4.9 x 108 and 3.7 x 107 liters/M for high and low affinity sites, respectively) are lower than those in solution (5.0 X 109 and 1.1 x 109 liters/M for higher and lower affinity sites, respectively) (23). Therefore, under equilibrium conditions, added unlabeled IGF-I or IGF-II will preferentially associate with the IGFBPs in the binding assay buffer (rather than those that are associated with cell surfaces), thereby displacing the [125I]IGFI bound to higher affinity soluble sites and thus making it available to bind to the lower affinity cell surface

binding sites. An alternative explanation would be that the addition of low concentrations of IGF-I to medium containing IGFBP-3 or IGFBP-1 causes a major shift in the amount of IGFBP that is cell surface associated. Although we have not directly tested this possibility, our findings are more consistent with the former hypothesis. In addition to the affinity of the IGFBPs in solution, the amount of IGFBP that is present determines the degree of the paradoxical increase in [125I]IGF-I binding (23). In the current studies we have shown that if cell types such as pSM cells [which do not release IGFBPs during the binding assays (22)] are tested, the phenomenon can be replicated by the addition of either IGFBP3 or IGFBP-1 to the binding assay buffer. These findings suggest that constitutive synthesis of particular forms of IGFBP that have either high affinity for IGF-I in solution or become cell surface associated will alter the binding of IGF-I to receptors differently from those that are of lower affinity or do not associate with cell surfaces. The biological significance of the changes in IGF binding induced by IGFBPs has not been defined. It is notable, however, that the addition of either IGFBP-1 (18) or IGFBP-3 (41, 45) to cell cultures does not invariably result in growth inhibition. The studies of Blum et

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COMPARISON OF IGFBPS

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al. (45) and De Mellow and Baxter (19) have shown that addition of IGFBP-3 can potentiate the mitogenic response of human fibroblast cultures to IGF-I above the level seen with IGF-I alone. IGFBP-3 could function by prolonging IGF-I's half-life and thereby enhance the levels that are available to stimulate growth. Addition of IGFBP-1 can also potentiate the mitogenic effect of IGF I on fibroblasts and pSM cells (18). Since IGFBP-1 has an RGD sequence and may act by binding to cell surfaces via a specific (possibly integrin-like) receptor (8), the mechanisms by which it potentiates mitogenic effects may be distinct from those of IGFBP-3. Various normal cell types synthesize and secrete different amounts and forms of IGFBP. For example, muscle cells secrete only the non-GH-dependent forms, as do decidual and endometrial cells (15,46,47). In contrast, human skin fibroblasts secrete predominantly IGFBP-3 (15-17). The current studies demonstrate that the type of IGFBP as well as its concentration in the assay buffer (and presumably in interstitial fluids) will result in different effects on the ability of IGF-I to associate with cell surface binding sites. Also, the IGFBP concentration relative to that of IGF-I will determine the availability of IGF-I for binding to cell surface sites. Therefore, the capacity of a specific cell type to secrete a particular form of IGFBP will greatly influence the expected biological responses. Likewise, the capacity of a particular form of IGFBP to bind to cell surfaces and resultant changes in its affinity for IGF-I that are associated with cell surface association may play a key role in the relative destribution of IGF-I between the soluble IGFBPs and those on the cell surface. The interaction of various forms of IGFBPs with each other in solution has not been investigated. Clearly, studies that test the effect of combinations of IGFBPs in the ratios that are actually found in interstitial fluids will be needed to delineate the relative roles of the IGFBPs in controlling IGF target cell actions.

Acknowledgments The authors wish to thank Dr. Louis E. Underwood for the [125I]IGF-I. We are grateful to Anne Myers and Jennifer O'Lear for preparing this manuscript.

References 1. Zapf J, Schmid CH, Froesch ER1984 Biological and immunological properties of insulin-like growth factors (IGF) I and II. Clin Endocrinol Metab 13:3-30 2. Van Wyk JJ 1985 The somatomedins: biological actions and physiological control mechanisms. In: Li CH (ed) Hormonal Proteins and Peptides. Academic Press, Orlando, pp 81-253 3. Schoenle E, Zapf J, Humbel RE, Froesch ER 1982 Insulin-like growth factor I stimulates growth in hypophysectomized rats. Nature 296:252-253 4. Hizuka N, Takano K, Shizume K, Asakawa K, Miyakawa M, Tanaka I, Horikawa R 1986 Insulin-like growth factor I stimulates

Endo • 1991 Voll29-No2

growth in normal growing rats. Eur J Pharmacol 125:143-146 5. Hodgkinson SC, Davis SR, Burleigh BD, Henderson HV, Gluckman PD 1987 Metabolic clearance rate of insulin-like growth factor-I in fed and starved sheep. J Endocrinol 115:233-240 6. Zapf J, Hauri C, Waldvogel M, Froesch ER 1986 Acute metabolic effects and half-lives of intravenously administered insulin-like growth factors I and II in normal and hypophysectomized rats. J Clin Invest 77:1768-1775 7. Binoux M, Hossenlopp P 1988 Insulin-like growth factor (IGF) and IGF-binding proteins: comparison of human serum and lymph. J Clin Endocrinol Metab 67:509-514 8. Busby WH, Klapper DG, Clemmons DR 1988 Purification of a 31000 dalton insulin like growth factor binding protein from human amniotic fluid. J Biol Chem 263:14203-14210 9. Hossenlopp P, Seurin D, Segovia-Quinson B, Binoux M 1986 Identification of an insulin-like growth factor binding proteins in human spinal fluid with selective affinity for IGF-II. Fed Eur Biochem Soc Lett 208:439-444 10. Wood WI, Cachianes G, Henzel WJ, Winslow GA, Spencer SA, Hellmiss R, Martin JL, Baxter RC 1988 Cloning and expression of the GH dependent insulin like growth factor binding protein. Mol Endocrinol 2:1176-1185 11. Baxter RC, Martin JL 1986 Radioimmunoassay of growth hormone dependent insulin-like growth factor binding protein in human plasma. J Clin Invest 78:1504-1512 12. Brewer MT, Stetler GL, Squires CH, Thompson RC, Busby WT, Clemmons DR 1988 Cloning characterization and expression of a human insulin like growth factor binding protein. Biochem Biophys Res Commun 152:1289-1297 13. Binkert C, Landwehr J, Mary J-L, Schwander J, Heinrich G 1989 Cloning sequence analysis and expression of a cDNA encoding a novel insulin-like growth factor binding protein (IGFBP-2). EMBO J 8:2497-2502 14. Shimasaki S, Uchiyama F, Shimonaka M, Ling N 1990 Molecular cloning of the cDNAs encoding a novel insulin-like growth factorbinding protein from rat and human. Mol Endocrinol 4:1451-1458 15. McCusker RH, Camacho-Hubner C, Clemmons DR 1989 Identification of the types of insulin like growth factor binding proteins that are secreted by muscle cells in vitro. J Biol Chem 264:77957800 16. Martin JL, Baxter RC 1988 Insulin like growth factor binding proteins IGFBP's produced by human skin fibroblasts immunological relationship to other human IGFBP's. Endocrinology 123:1907-1915 17. Hill DJ, Camacho-Hubner C, Rashid P, Strain AJ, Clemmons DR 1989 Insulin like growth factor binding protein secretion by human fibroblasts: dependence on cell density and IGF peptides. J Endocrinol 122:87-98 18. Elgin RG, Busby WH, Clemmons DR 1987 An insulin-like growth factor binding protein enhances the biogic response to IGF-I. Proc Natl Acad Sci USA 84:3254-3258 19. De Mellow JSM, Baxter RC 1988 Growth hormone dependent insulin-like growth factor binding protein both inhibits and potentiates IGF-I stimulated DNA synthesis in skin fibroblasts. Biochem Biophys Res Commun 156:199-204 20. Gopinath R, Walton PE, Etherton TD 1989 An acid stable insulinlike growth factor (IGF)-binding protein from pig serum inhibits binding of IGF-I and IGF-II to vascular endothelial cells. J Endocrinol 120:231-236 21. Ritvos O, Ranta T, Jalkanen J, Suikkari AM, Voutilainen R, Bohn H, Rutanen EM 1988 Insulin-like growth factor (IGF) binding protein from human decidua inhibits the binding and biological action of IGF-I in cultured choriocarcinoma cells. Endocrinology 122:2150-2157 22. Clemmons DR, Elgin RG, Han VKM, Casella SJ, D'Ercole AJ, Van Wyk JJ 1986 Cultured fibroblast monolayers secrete a protein that alters the cellular binding of somatomedin-C/insulin-like growth factor I. J Clin Invest 77:1548-1556 23. McCusker RH, Camacho-Hubner C, Bayne ML, Cascieri MA, Clemmons DR 1990 Insulin-like growth factor (IGF) binding to human fibroblast and glioblastoma cells: the modulating effect of cell released IGF binding proteins (IGFBPs). J Cell Physiol

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COMPARISON OF IGFBPS 144:244-253 24. Svoboda ME, Van Wyk JJ, Klapper DG, Fellows RE, Grissom FE, Schlueter RJ 1980 Purification of somatomedin-C from human plasma: chemical and biological properties, partial sequence analysis and relationship to other somatomedins. Biochemistry 19:790797 25. D'Ercole AJ, Underwood LE, Van Wyk JJ, Decedue CJ, Foushee DB 1976 Specificity, topography and ontogeny of the somatomedin C receptor in mammalian tissues. In: Pecile A, Muller R (eds) Growth Hormone and Related Peptides. Excepta Medica, Amsterdam, pp 190-205 26. Ross R 1971 The smooth muscle cell: growth of smooth muscle in cultures and formation of elastic fibers. J Cell Biol 50:172-186 27. McCusker RH, Camacho-Hubner C, Clemmons DR 1989 Identification of the types of insulin like growth factor binding proteins that are secreted by muscle cells in vitro. J Biol Chem 264:77957800 28. Clemmons DR, Elgin RG 1986 Somatomedin-C binding to cultured fibroblasts is dependent upon donor age and culture density. J Clin Endocrinol Metab 63:996-1001 29. Clemmons DR, Van Wyk JJ, Pledger WJ 1980 Sequential addition of platelet factor and plasma to BALB/c 3T3 fibroblast cultures stimulates somatomedin-C binding early in cell cycle. Proc Natl Acad Sci USA 77:6644-6648 30. McCusker RH, Clemmons DR 1988 Insulin-like growth factorbinding proteins (IGFBPs) secretion by muscle cells: effect of cellular differentiation and proliferation. J Cell Physiol 137:505512 31. Hossenlopp P, Seurin D, Segovia-Quinson B, Hardouin S, Binoux M 1986 Analysis of serum insulin-like growth factor binding proteins using Western blotting: use of the method for titration of the binding proteins and competitive binding studies. Anal Biochem 154:138-143 32. DeVroede MA, Tseng LYH, Katsoyannis PG, Nissley SP, Rechler MM 1986 Modulation of insulinlike growth factor I binding to human fibroblast monolayer cultures by insulin-like growth factor carrier proteins released to the incubation media. J Clin Invest 77:602-613 33. Clemmons DR, Han VKM, Elgin RG, D'Ercole AJ 1987 Alterations in the synthesis of a fibroblast surface associated 35 K protein modulates the binding of somatomedin-C/insulin-like growth factor I. Mol Endocrinol 1:339-347 34. Forbes B, Szabo L, Baxter RC, Ballard FJ, Wallace JC 1988 Classification of the insulin-like growth factor binding proteins into three distinct categories according to their binding specificities. Biochem Biophys Res Commun 157:196-202

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35. Martin JL, Baxter RC 1986 Insulin like growth factor binding protein from human plasma: purification and characterization. J Biol Chem 261:8754-8760 36. Baxter RC, Martin JL, Wood MH 1987 Two immunoreactive binding proteins for insulin-like growth factors in human amniotic fluid: relationship to fetal maturity. J Clin Endocrinol Metab 65:423-431 37. Frauman AG, Tsuzaki S, Moses AC 1989 The binding characteristics and biological effects in FRTL5 cells of placental protein-12, and insulin-like growth factor-binding protein purified from human amniotic fluid. Endocrinology 124:2289-2296 38. Ross M, Francis GL, Szabo L, Wallace JC, Ballard FJ 1989 Insulinlike growth factor (IGF)-binding proteins inhibit the biological activities of IGF-I and IGF-2 but not des-(l-3)-IGF-I. Biochem J 258:267-272 39. Knaur DJ, Smith GL 1980 Inhibition of biologic activity of multiplication stimulating activity by binding to its carrier protein. Proc Natl Acad Sci USA 77:7252-7254 40. Ooi GT, Herington AC 1986 Covalent cross-linking of insulin-like growth factor-1 to a specific inhibitor from human serum. Biochem Biophys Res Commun 137:411-417 41. Blat C, Delbe J, Villaudy J, Chatelain G, Golde A, Harel L 1989 Inhibitory diffusible factor 45 bifunctional activity as a cell growth inhibitor and as an insulin-like growth factor I-binding protein. J Biol Chem 264:12449-12454 42. Cascieri MA, Hayes NS, Bayne ML 1989 Characterization of the increased biological potency in BALB/C 3T3 cells of two analogs of human insulin like growth factor I which have reduced affinity for the 28 K cell-derived binding protein. J Cell Physiol 139:181188 43. Francis GL, Upton FM, Ballard FJ, McNeil KA, Wallace JC 1988 Insulin-like growth factors 1 and 2 in bovine colostrum: sequences and biological activities compared with those of a potent truncated form. Biochem J 251:95-103 44. Jonas HA, Harrison LC 1986 Disulphide reduction alters the immunoreactivity and increases the affinity of insulin-like growth factor-I receptors in human placenta. Biochem J 236:417-423 45. Blum WF, Jenne RW, Reppin F, Kietzmann K, Ranke MB, Bierich JR 1989 Insulin like growth factor I (IGF-I)-binding protein complex is a better mitogen than free IGF-I. Endocrinology 125:766772 46. Rutanen EM, Koistinen R, Sjoberg J 1986 Synthesis of placental protein 12 by human endometrium. Endocrinology 118:1067-1071 47. Rutanen EM, Koistinen R, Wahlstrom T, Bohn H, Ranta T, Seppala M 1985 Synthesis of placental protein 12 by human decidua. Endocrinology 116:1304-1309

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