©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Studies on the Mechanisms by Which Insulin-like Growth Factor (IGF) Binding Protein-4 (IGFBP-4) and IGFBP-5 Modulate IGF Actions in Bone Cells (*)

(Received for publication, March 29, 1995)

Subburaman Mohan (1) (2) (3) (4)(§) Yoshihide Nakao (1) (4) Yoko Honda (1) (4) Edwin Landale (1) (4) Ulrike Leser (5) Carola Dony (5) Kurt Lang (5) David J. Baylink (1) (2) (4)

From the  (1)Departments of Medicine, (2)Biochemistry, and (3)Physiology, Loma Linda University, Loma Linda, California 92357, the (4)Mineral Metabolism Laboratory, Pettis Veteran's Administration Medical Center, Loma Linda, California 92357, and (5)Boehringer Mannheim GmbH, Biochemical Research Center, Penzberg, Nonnenwald 2, 82377 Penzberg, Germany

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The growth potentiating effects of the insulin-like growth factor (IGF)-I and IGF-II are modulated by a family of six insulin-like growth factor binding proteins (IGFBPs). Despite the similarity in amino acid sequences of the IGFBPs, their effects on the growth of bone cells differ. Studies on the molecular mechanisms for IGFBP-4 actions revealed that coincubation of bone cells with IGFBP-4 and I-IGF-I or I-IGF-II decreased the binding of both of these ligands in a dose-dependent manner. In addition, IGFBP-4 decreased the binding of IGF-I tracer to purified type I IGF receptor. These data in conjunction with data showing that IGFBP-4 had no effect on cell proliferation induced by analogs of IGF-I or IGF-II, which exhibited >100-fold reduced affinity for binding to IGFBP-4 suggest that IGFBP-4 may inhibit IGF action by preventing the binding of ligand to its membrane receptor. In contrast to IGFBP-4, IGFBP-5 treatment increased the binding of IGF tracer to bone cells but did not increase the binding of I-IGF-I to type I IGF receptor. Studies on the mechanism by which IGFBP-5 increased the binding of I-IGF tracer to bone cells suggest that IGFBP-5 could facilitate IGF binding by a mechanism in which IGFBP-5 has cell surface binding sites independent of IGF receptors. These data in conjunction with the findings that IGFBP-5 potentiated cell proliferation even in the presence of those same IGF analogs that exhibited >200-fold reduced affinity for binding to IGFBP-5, suggest that IGFBP-5 may in part stimulate bone cell proliferation by an IGF-independent mechanism involving IGFBP-5-specific cell surface binding sites.


INTRODUCTION

Studies in our and other laboratories provide strong support to the concept that insulin-like growth factors (IGFs) (^1)function in an autocrine/paracrine manner to regulate the proliferative and differentiative functions of bone cells(1, 2, 3) . The findings that support this concept are as follows. First, IGF-I and IGF-II have been found in bone extracts of several different species, and IGFs are the most abundant growth factors stored in bone(4, 5) . Second, IGFs have been shown to be one of the most abundant mitogens produced by bone cells derived from both fetal and adult bone, and the bone cell production of IGFs is regulated by both local and systemic effectors of bone metabolism(1, 2, 3) . Third, the exogenous addition of IGFs have been shown to stimulate both proliferative and differentiative functions in bone cells(1, 2, 3) . Fourth, there is recent evidence that demonstrates that IGFs increase bone formation parameters in vivo in both rats and humans (6, 7, 8) .

The local activity of IGFs in bone and other tissues is dependent not only on the amount of IGFs produced but also by a family of structurally related proteins that specifically bind to IGFs. Six IGF binding proteins (designated IGFBP-1 through IGFBP-6), different from the IGF receptors, have been purified from human plasma or tissues including bone(9, 10, 11, 12, 13, 14) . In previous studies, we have shown that human bone cells in culture secrete a number of IGFBPs of which the 25-kDa IGFBP-4 is one of the major IGFBPs produced by bone cells. Studies on the biological actions of IGFBP-4 revealed that IGFBP-4 inhibits both IGF-I- and IGF-II-induced cell proliferation in embryonic chick calvaria cells and MC3T3-E1 mouse osteoblasts under all culture conditions tested(15) . In addition, IGFBP-4 has been shown to inhibit the growth of embryonic chick pelvic cartilage in vitro(16) . The inhibitory effect of IGFBP-4 on IGF-induced cell proliferation does not appear to be specific to bone cells since Cheung et al.(17) have shown that IGFBP-4 inhibits IGF-induced cell proliferation in neuroblastoma cell line, and Culouscou and Shyoab et al.(18) have shown that the colon cancer cell growth inhibitor purified from conditioned medium of the HT29 human adenocarcinoma cell line is identical to IGFBP-4.

In contrast to IGFBP-4, studies on the biological actions of IGFBP-5, the major IGFBP stored in human bone, revealed that IGFBP-5 is a stimulator of IGF-induced mouse bone cell proliferation(19) . Similar potentiating effects of IGFBP-5 purified from U2 human osteosarcoma cell conditioned medium have been reported by Andress and Birnbaum(20, 21) . In addition, Jones et al.(22) have demonstrated that when IGFBP-5 was present in cell culture substrata, it potentiated the growth stimulatory effects of IGF-I on fibroblasts. Although Kiefer et al.(23) have demonstrated that recombinant human IGFBP-5 produced in yeast inhibited DNA synthesis in SaOS-2/B10 human osteosarcoma cells, subsequent studies from the same laboratory have shown that recombinant human IGFBP-5 stimulated DNA synthesis in rat osteoblasts(24) . Together, the findings that IGFBPs modulate IGF actions either positively or negatively and that the production of IGFBPs is regulated by various physiological agents(9, 10, 11, 12, 13, 14) support the conclusion that the balance between the stimulatory and inhibitory classes of IGFBPs will determine the degree and extent of IGF-induced cellular responses in target tissues such as bone.

Although IGFBP-4 and IGFBP-5, which share 35% sequence identity with each other, exhibit opposite effects on bone cell proliferation, very little is known about the molecular mechanisms by which these binding proteins exert their effects on bone cell proliferation. There is limited data available in other cell types for IGFBP-1 and IGFBP-3, which have been shown to both stimulate and inhibit IGF actions (25, 26, 27, 28) . Blat et al.(25) have shown that exogenous addition of IGFBP-3 caused a 100% inhibition of DNA synthesis induced by IGF-I in chick embryo fibroblasts. In contrast, preincubation of fibroblasts with IGFBP-3 prior to the addition of IGF-I resulted in the potentiation of IGF-I action(26) . While cell surface attachment of IGFBP-3, which also binds to heparin, has been suggested to be an important mechanism for the potentiation of IGF-I action by IGFBP-3, a number of other mechanisms have also been suggested(27, 28) . The current studies were undertaken to extend the mechanistic data available for the IGFBPs by characterizing the biological effects of IGFBP-4 and IGFBP-5 on MC3T3-E1 mouse osteoblasts and on normal human bone cells under identical culture conditions and by evaluating whether IGFBP-4 and IGFBP-5 modulate their actions on bone cell proliferation by different mechanisms. These data indicate that the molecular mechanisms by which IGFBP-4 and IGFBP-5 affect bone cells are different and that IGFBP-5 may bind bone cells by an IGF-independent mechanism.


EXPERIMENTAL PROCEDURES

Materials

The clonal osteoblastic cell line, MC3T3-E1, was obtained originally from newborn mouse calvarial cells (29) and had been kindly provided by M. Kumegawa (Josai Dental University, Saitama, Japan). Low alkaline phosphatase containing SaOS-2 human osteosarcoma cell line subpopulation was isolated as described previously (30) and had been kindly provided by J. Farley (Loma Linda University, Loma Linda, CA). Untransformed normal human bone cells were derived from bone biopsies as described previously (31) and had been kindly provided by T. A. Linkhart (Loma Linda University, Loma Linda, CA).

Recombinant human (rh) IGFBP-5 and carboxyl-terminal truncated 19- and 10-kDa forms of IGFBP-5 were produced in Escherichia coli and purified by established procedures(32) . IGFBP-4 was purified from human bone cell conditioned medium as described previously, and the homogeneity of the preparation was established by SDS-polyacrylamide gel electrophoresis and amino-terminal amino acid sequence analysis (15, 33, 34) . No detectable levels of IGF-I or IGF-II were found in the purified preparations of IGFBP-4 and IGFBP-5. Recombinant human IGF-I and IGF-II were purchased from Austral Biologicals (Torrance, CA). Recombinant human des-1-3-IGF-I and des-1-6-IGF-II were kindly provided by G. L. Francis (Co-operative Research Center for Tissue Growth and Repair, Adelaide, Australia). Purified human IGF-I receptor was a gift from Y. Fujita-Yamaguchi (City of Hope, Duarte, CA). Dulbecco's modified Eagle's medium (DMEM) was purchased from Life Technologies, Inc. Calf serum was purchased from Hyclone (Logan, UT). Bovine serum albumin (BSA) was purchased from Fluka. [^3H] Methylthymidine and I were purchased from International and Chemical Nuclear (Irvine, CA) and DuPont NEN. All other chemicals used were at least reagent grade and were purchased from Sigma.

Iodination

Iodination of proteins was performed using chloraimine T method as described previously(35) . The specific activity of radiolabeled IGFs varied between 200 and 400 µCi/µg of protein, while that of IGFBPs varied between 100 and 200 µCi/µg of protein. Aliquots of radiolabeled IGFs were stored at -70 °C and used for IGF radioligand assays.

Purification of hBDIGFBP-5

Purification was as described previously (19) with modifications to reduce proteolysis: procedures were executed rapidly and, where possible, at 4 °C; all buffers included 1 mM EDTA (Sigma), 1 mM leupeptin (Boehringer Mannheim), and 1 mM pepstatin (Boehringer Mannheim). After hydroxyapatite and IGF-II affinity chromatography steps, the concentrated sample was applied to a reverse phase HPLC column, 250 mm 7 mm C8 Aquapore column (Applied Biosystems, Foster City CA), and after washing, the bound material was eluted with a gradient of 25% acetonitrile to 35% acetonitrile run over 100 min at a flow rate of 3 ml/min. 2-min fractions were collected and concentrated in a speed-vac apparatus (Savant Instruments Inc., Farmingdale NY), and aliquots of the redissolved material were used for ligand blots and protein sequencing. Ligand blots employed precast 10-20% gradient gels (Jule Inc., New Haven CT), and molecular size was estimated using prestained low molecular weight markers (Bio-Rad). Protein sequencing of both nonreduced and reduced and pyridyl ethylated samples employed an Applied Biosystems model 470A vapor phase sequencer (Biotechnology Instrumentation Facility, University of California, Riverside CA) as described previously(15, 33, 34) .

Cell Culture

The MC3T3-E1 cells were maintained in DMEM supplemented with 5% calf serum and were passaged twice/week by dispersing the cells with trypsin. Osteoblast-like human osteosarcoma cell lines (SaOS-2, MG63) were maintained in DMEM supplemented with 10% calf serum, and the cells were passaged upon reaching confluence by dispersing the cells with trypsin. Untransformed normal human bone cells were cultured in DMEM supplemented with 10% calf serum and were maintained as described previously(35) .

Cell Proliferation Studies

Cell proliferation was determined by using the incorporation of [^3H]thymidine into DNA. For [^3H]thymidine incorporation studies, cells were plated (2,000/well) in 0.05 ml of DMEM containing 0.1% calf serum in 96-well culture plates and incubated for 24 h. 0.05 ml of DMEM containing 0.1% BSA (DMEM/BSA) or 0.05 ml of DMEM/BSA containing the effector was added, and the cells were incubated for an additional 18 h. The cells were then pulse labeled for 4 h with 0.25 µCi of [^3H]methylthymidine. The radioactive medium was removed, and the cells were rinsed twice with phosphate-buffered saline. The cells were then extracted with 0.05 ml of 0.2 N sodium hydroxide, and the extract was transferred to scintillation vial and then counted after mixing with 2 ml of scintillation fluid. Eight replicates were used for each treatment.

Effect of IGFBP on IGF-I or IGF-II Binding to Cells in Monolayer Culture

Cells were plated in DMEM containing 0.1% calf serum in 96-well culture plates as described for [^3H]thymidine incorporation studies. After overnight incubation, the medium was removed, the cultures were rinsed with DMEM/BSA, and 0.03 ml of DMEM/BSA was added. 0.01 ml of DMEM/BSA containing various concentrations of IGFBP was then added, and the cultures were incubated with I-labeled IGF-I or IGF-II (40,000 cpm) for 3 h at room temperature. The labeled medium was discarded, and the cultures were rinsed 5 times with DMEM containing 0.1% BSA. I-Labeled IGF-I or IGF-II bound to cells was extracted with lysis buffer and counted as described previously(34, 35) .

IGFBP Binding to Bone Cells in Monolayer Culture

Cells were plated in DMEM containing 0.1% calf serum in 96-well culture plates as described for [^3H]thymidine incorporation studies. After overnight incubation, the medium was removed, rinsed with DMEM containing 0.1% BSA, and 0.03 ml of DMEM containing 0.1% BSA was added. 0.01 ml of unlabeled IGFBP or vehicle was added to each well, and the cells were incubated at 37 °C for 1 h with 0.01 ml of I-IGFBP (100,000 cpm). At the end of incubation, the labeled medium was discarded, and the cultures were rinsed 5 times with DMEM containing 0.1% BSA. I-IGFBP bound to cells was extracted with lysis buffer and counted as described previously(34, 35) .

IGF-I Binding to IGF-I Receptors in the Presence of IGFBP

Competition of IGF binding to IGF-I receptor by IGFBPs was carried out using human placental type I IGF receptor that was purified as described previously(36) . Briefly, duplicate aliquots of approximately 50 fmol of receptor were incubated in 0.1 ml of 50 mM Tris/HCl buffer (pH 7.4) containing 0.1% Triton X-100, 0.1% BSA, 40,000 cpm of I-IGF-I and various concentrations of IGFBP-4 or IGFBP-5. The reactions were carried out in the absence or presence of unlabeled IGF-I to determine the specificity of the binding. After 24 h of incubation, affinity cross-linking of the receptor bound ligand was performed by adding 1 µl of 10 mM disuccinimidyl suberate prepared freshly in dimethyl sulfoxide. After 15 min of incubation at room temperature, the reaction was quenched by the addition of 10 µl of 1 M Tris, 0.2 M sodium EDTA. 25 µl of 5 electrophoresis sample buffer was then added; the samples were boiled for 3 min and subjected to SDS-polyacrylamide gel electrophoresis using 3-17% gradient gel. At the end of electrophoresis, the gel was fixed and subjected to autoradiography. Cross-linking of I-IGF-I to receptor or IGFBP was quantitated by densitometric scanning of the autoradiograph with Bio-Med Instruments (Fullerton, CA).

IGFBP Assay

0.05 ml of sample containing 2 ng of IGFBP-4 or IGFBP-5 was incubated with 50,000 cpm of I-labeled IGF-I or IGF-II for 60 min at room temperature in 0.1 M Hepes (pH 6.0) containing 0.1% BSA, 0.1% Triton X-100, 0.044 M sodium carbonate, 0.01% sodium azide (0.25 ml volume). Bound and free I-IGF were separated by adding 0.1 ml of 2% bovine serum immunoglobulin and 0.5 ml of polyethylene glycol 8000 and centrifuging(15) . Specific binding was determined by subtracting bound counts in the presence of excess cold IGF from bound counts determined at various concentrations of IGFs or their analogs.

Statistical Analysis

Statistical analysis was performed using Student's t test.


RESULTS

Amino-terminal Amino Acid Sequence of hBDIGFBP

The previously determined amino-terminal amino acid sequence for the hBDIGFBP was similar to but not identical to the contemporaneously determined IGFBP sequences from human osteosarcoma (37) and human placental (38) cDNA library, now named IGFBP-5. To determine whether the hBDIGFBP was identical to or a variant of IGFBP-5, human bone extract, prepared so as to minimize proteolysis, was subjected to IGFBP purification by two successive affinity chromatography steps, first on hydroxyapatite and then on IGF-II, followed by a reverse phase HPLC separation. Purification of hBDIGFBP under these conditions yielded one predominant protein peak with an apparent molecular mass of 29,000 Da that exhibited IGF binding activity on western ligand blot (data not shown). Amino acid sequencing of the amino-terminal residues of nonreduced and of reduced and pyridyl ethylated preparations of the hBDIGFBP and comparison of these sequences with previously described IGFBP sequences, the nomenclature of which has been recently revised(39) , showed that the amino-terminal sequence of the hBDIGFBP is identical to IGFBP-5 (Fig. 1). The purification of hBDIGFBP also yielded a 19-kDa form of IGFBP, which had identical amino-terminal residues to IGFBP-5 (Fig. 1).


Figure 1: Amino terminal protein sequence comparisons of IGFBP-5. IGFBP-5 sequence deduced from cDNA clones derived from human placental (37) and human osteosarcoma library (38) (A); previously described sequence for human bone derived 29-kDa IGFBP (19) (B); nonreduced human bone derived 29-kDa IGFBP (this study) (C); nonreduced human bone-derived 19-kDa IGFBP fragment (this study) (D); reduced and pyridyl ethylated human bone derived 29-kDa IGFBP (this study) (E). Sequences are shown in single-letter amino acid code.



Effects of IGFBP-4 and IGFBP-5 on Basal and IGF-induced Bone Cell Proliferation

Proliferation of the mouse osteoblastic cell line MC3T3-E1 was decreased when it was exposed to IGFBP-4 (Fig. 2). Basal cell proliferation in the absence of exogenous growth factors was inhibited by 50% when MC3T3-E1 cells were exposed to 100 ng/ml IGFBP-4. The stimulation of proliferation associated with exposure to 3 ng/ml IGF-I was also decreased by IGFBP-4. However, IGFBP-4 had negligible effect on the proliferation associated with exposure to 3 ng/ml des-1-3-IGF-I (Fig. 2A), which exhibited >100-fold reduced affinity for IGFBP-4 compared with wild-type IGF-I (Fig. 3A). IGFBP-4 causes a dose-dependent decrease in the stimulation of proliferation associated with exposure to IGF-II but has much less effect on the stimulation of proliferation associated with exposure to des-1-6-IGF-II (Fig. 2B). This analog of IGF-II, like des-1-3-IGF-I, also exhibited >100-fold reduced affinity for IGFBP-4 compared with wild-type IGF-II (Fig. 3B).


Figure 2: Effect of IGFBP-4 and IGFBP-5 on MC3T3-E1 cell proliferation as measured by the incorporation of [^3H]thymidine. Values are expressed as a percent of the BSA-treated control (errorbars give the standard deviation of the measured cpm divided by the control cpm). Panel A shows the effect of IGFBP-4 on basal, IGF-I-, or des-1-3-IGF-I-induced MC3T3-E1 cell proliferation in the absence of exogenous IGFBP-4 (openbars) and in the presence of 100 ng/ml IGFBP-4 (cross-hatchedbars). Panel B shows the effect of IGFBP-4 concentration on cells stimulated with 3 ng/ml IGF-II (shortdash and opensquares) or des-1-6-IGF-II (dots and opendiamonds). PanelC shows the effect of the 29-kDa full-length and the 10-kDa rhIGFBP-5 forms on basal, IGF-I-, or IGF-II-stimulated MC3T3-E1 cell proliferation; 29-kDa IGFBP-5 on basal MC3T3-E1 cell proliferation (longdash and filledsquares); 10-kDa IGFBP-5 on basal cell proliferation (longdash and filledtriangles); 29-kDa IGFBP-5 on 100 ng/ml IGF-I-induced MC3T3-E1 cell proliferation (solidline and filledcircles); 10-kDa IGFBP-5 on 100 mg/ml IGF-I-induced MC3T3-E1 cell proliferation (solidline and filledtriangles); 29-kDa IGFBP- 5 on 100 ng/ml IGF-II-induced MC3T3-E1 cell proliferation (shortdash and filledsquares); 10-kDa IGFBP-5 on 100 ng/ml IGF-II-induced MC3T3-E1 cell proliferation (shortdash and filledtriangles). [^3H]thymidine incorporation in BSA-treated control cultures ranged from 450 to 1200 cpm with standard deviation typically less than 15%.




Figure 3: Competition for binding of I-IGF-I or I-IGF-II to human IGFBP-4 and IGFBP-5 by the IGFs and their analogs. PanelA shows competition for binding of I-IGF-I to IGFBP-4 (opensymbols) and to IGFBP-5 (filledsymbols) by unlabeled IGF-I (solidline and circles), des-1-3-IGF-I (mediumdash and triangles), IGF-II (shortdash and squares) and des-1-6-IGF-II (dottedline and diamonds). PanelBshows the competition for binding of I-IGF-II to IGFBP-4 (opensymbols) and to IGFBP-5 (filledsymbols) by unlabeled IGF-I (solidline and circles), des-1-3-IGF-I (mediumdash and triangles), IGF-II (shortdash and squares) and des-1-6-IGF-II (dottedline and diamonds). Each point represents the mean of four replicate determinations from two experiments.



In contrast to IGFBP-4, exposure of MC3T3-E1 mouse osteoblasts under identical culture conditions to IGFBP-5 results in an increased proliferation of cells both in the absence and in the presence of exogenously added IGFs. Fig. 2C shows that basal cell proliferation was increased by 29-kDa rhIGFBP-5 in a dose-dependent manner. At 100 ng/ml, cell proliferation was increased to 435% of vehicle-treated control (p < 0.001). In addition to stimulating basal cell proliferation, rhIGFBP-5 also stimulated MC3T3-E1 cell proliferation in the presence of 100 ng/ml IGF-I or IGF-II in a dose-dependent manner. The 19- and 10-kDa size variants of IGFBP-5 were also mitogenic when assayed on unstimulated MC3T3-E1 cells or on cells exposed to 100 ng/ml IGF-I or IGF-II (Fig. 2C). However, the 19-kDa variant was less potent than the 29-kDa protein (data not shown) while the 10-kDa variant required 1-2 orders of magnitude higher concentrations to achieve equivalent proliferation (Fig. 2C). Both rhIGFBP-5 and hBDIGFBP-5 exhibited similar potency in stimulating IGF-I-induced cell proliferation. At 10 ng/ml, rhIGFBP-5 and hBDIGFBP-5 increased IGF-I-induced cell proliferation by 92 ± 36% (p < 0.001) and 74 ± 48% (p < 0.001), respectively.

In contrast to IGFBP-4, which did not inhibit cell proliferation induced by either des-1-3-IGF-I or des-1-6-IGF-II, IGFBP-5 potentiated MC3T3-E1 cell proliferation even in the presence of those IGF analogs that exhibited >200-fold reduced affinity for binding to IGFBP-5. Des-1-3-IGF-I and IGF-I at 100 ng/ml stimulated cell proliferation to 304 ± 70 and 225 ± 35% of vehicle treated control, respectively, which were increased further by 27 ± 22 and 20 ± 18%, respectively, upon exposure with 3 ng/ml IGFBP-5 and by 48 ± 15 (p < 0.001) and 56 ± 22% (p < 0.001), respectively, upon exposure with 30 ng/ml IGFBP-5. IGFBP-5 treatment also caused similar increases in cell proliferation induced by des-1-6-IGF-II (data not shown).

The effect of IGFBP-4 and IGFBP-5 on IGF-induced cell proliferation was also tested using untransformed normal human bone cells in serum-free culture. Table 1shows that basal cell proliferation was inhibited by 43 and 41%, respectively, in human bone cells derived from rib and skull by 100 ng/ml IGFBP-4. In addition, 10 ng/ml IGF-II-induced cell proliferation was completely inhibited by the exogenous addition of 100 ng/ml IGFBP-4 in human bone cells derived from both rib and skull. In contrast to IGFBP-4 effects, exogenous addition of 100 ng/ml IGFBP-5-stimulated basal cell proliferation by 50-80% in human bone cells derived from rib and skull in different experiments (data not shown). This increase in cell proliferation does not appear to be an artifact of [^3H]thymidine uptake in the presence of IGFBP-5 since we obtained similar data using the incorporation of bromodeoxy uridine (data not shown). In addition, both IGF-I- and IGF-II-induced cell proliferation was increased by 53 ± 34 and 75 ± 11%, respectively, in the presence of 100 ng/ml rhIGFBP-5 (Table 2).





IGFBP-4 and IGFBP-5 Effects on the Binding of IGFs to Bone Cells

In our studies on the mechanism of IGFBP-4 and IGFBP-5 action, we considered the hypothesis that IGFBP-4 and IGFBP-5 regulate IGF actions in bone cells by modulating the binding of IGFs to their receptors. To examine this hypothesis, the effect of IGFBP-4 and IGFBP-5 on IGF binding to MC3T3-E1 cells was investigated by cell binding assays employing I-labeled IGF-I or IGF-II. These data revealed that IGFBP-4 produced a dose-dependent reduction in the binding of both IGF-I and IGF-II tracer, while IGFBP-5 produced, in different experiments, a dose-dependent increase in the binding of both IGF-I, maximal at 3-4-fold of control binding, and IGF-II, maximal at 1.5-2-fold of control binding (Fig. 4). Similar changes in IGF-I and IGF-II binding were obtained upon treatment of SaOS-2 and MG63 human osteosarcoma cells with IGFBP-4 and IGFBP-5 (data not shown).


Figure 4: Effect of added IGFBP on the binding of I-IGF-I or I-IGF-II to MC3T3-E1 cells. Data are the mean of four replicate determinations from two experiments expressed as the percent of the control determined in the absence of IGFBP for the effect of IGFBP-4 on the binding of I-IGF-I (opencircles) and I-IGF-II (opensquares) and for the effect of IGFBP-5 on the binding of I-IGF-I (filledcircles) and I-IGF-II (filledsquares).



The ability of IGFBP-4 and IGFBP-5 to influence the interaction between IGF-I and the type I receptor was investigated employing IGF-I iodinated with I and a gel shift assay in which the 350-kDa type I receptor migrated much more slowly than 25-kDa IGFBP-4 or 29-kDa IGFBP-5. Upon affinity cross-linking, gel electrophoresis, and autoradiography, I label was found in the 350-kDa IGF-I receptor, which was displaced by unlabeled IGF-I (Fig. 5). Increasing the concentration of IGFBP-4 from 2 to 20 ng/ml resulted in a decrease in the amount of I signal bound to type I receptor and a corresponding increase in the amount of I signal bound to IGFBP-4. These results are consistent with the possibility that IGFBP-4 upon binding to its ligand may modify the affinity of the ligand such that IGFs no longer bind to the receptors. In contrast to IGFBP-4, IGFBP-5 at 2 or 20 ng/ml had no significant effect on the amount of I- signal bound to the type I receptor. In other experiments, IGFBP-5 at concentrations greater than 100 ng/ml resulted in a decrease in the amount of I-labeled tracer bound to the type I receptor and a corresponding increase in the amount of I-labeled signal bound to IGFBP-5 (data not shown). The IGFBP-5-induced increase in I-IGF-I binding in MC3T3-E1 cells was inhibited completely by unlabeled IGF-I but not by insulin or des-1-3-IGF-I, which bind to IGF-I receptor but not to IGFBP-5 (data not shown).


Figure 5: Effect of added IGFBP-4 and IGFBP-5 on the binding of I-IGF-I to type I IGF receptor. Purified type I IGF receptor was incubated with I-IGF-I in the presence or absence of excess of unlabeled IGF-I. Various concentrations of purified IGFBP-4 or IGFBP-5 were added to the reaction mixture as indicated in the figure. The samples were subjected to electrophoresis in a 3-17% of polyacrylamide-gradient gel as described under ``Experimental Procedures.'' and autoradiographed.



IGFBP-5 and IGFBP-4 Binding to Bone Cells

Assaying the ability of IGFBP-4 and IGFBP-5 to bind to MC3T3-E1 cells in serum-free monolayer culture revealed that IGFBP-4 does not bind to cells (2.4 ± 0.2 versus 2.3 ± 0.06% I-labeled IGFBP-4 binding, respectively, in the absence or presence of 4 µg/ml unlabeled IGFBP-4) whereas IGFBP-5 does bind to cells, and this binding is not affected by added IGFBP-4, but it is affected in a dose-dependent manner by added IGFBP-5 (Fig. 6). I-IGFBP-5 binding to MC3T3-E1 cells was decreased by the addition of 4 µg/ml unlabeled IGFBP-5 but not IGFBP-4. Similar evidence for the presence of IGFBP-5 but not IGFBP-4 binding sites have been obtained using SaOS-2 and MG63 human osteosarcoma cells in serum-free culture (data not shown).


Figure 6: Effect of added IGFBP on the binding of I-IGFBP-5 to MC3T3-E1 cells. Monolayer cultures were exposed to I-IGFBP-5 with increasing concentrations of IGFBP-5 (filledcircles) and IGFBP-4 (opencircles) as described under ``Experimental Procedures.'' Data are the mean (± S.D.) of the binding observed in four experiments, expressed as the percent of the binding observed without added IGFBP. Control cpm determined in the absence of IGFBP were 12,156 (S.D.: 604; n = 4).



Similar cell binding assays were employed to investigate the effect of heparin on the binding of I-IGFBP-5 to MC3T3-E1 cells based on the previous evidence that IGFBP-3 binds to heparin(28) . These data revealed that treatment of MC3T3-E1 cells with heparin decreased the binding of I-IGFBP-5 in a dose-dependent manner. At 2 µg/ml, the binding to MC3T3-E1 cells was decreased to 68 ± 2% of control (p < 0.001). In contrast to heparin, treatment of MC3T3-E1 cells with fibronectin, an RGD-containing protein resulted in an increase in the binding of I-IGFBP-5 to MC3T3-E1 cells (163 ± 12% of control at 5 µg/ml fibronectin, p < 0.001). This increase in binding of I-IGFBP-5 to MC3T3-E1 cells was abolished by the addition of 4 µg/ml unlabeled IGFBP-5 but not IGFBP-4. In addition, the increased binding of I-IGFBP-5 to MC3T3-E1 cells in the presence of fibronectin was decreased to 103 ± 6% of control by adding heparin along with fibronectin.

The effect of IGF-I and IGF-II on IGFBP-5 binding to MC3T3-E1 cells was investigated by conducting cell binding assays, carried out in quadruplicate, in which I-IGFBP-5 was exposed to MC3T3-E1 cells. In these studies neither 2 µg/ml IGF-I nor 2 µg/ml IGF-II had any significant effect on the binding of I-IGFBP-5 to MC3T3-E1 cells (94 ± 3 and 103 ± 2% binding in the presence of IGF-I and IGF-II, respectively, compared with control). These data are consistent with the IGF binding results with purified IGF-I receptor and suggest that the increased binding of I-IGF-I to MC3T3-E1 cells in the presence of IGFBP-5 is not to IGF receptors.


DISCUSSION

The findings of this study demonstrate that purified IGFBP-4 and IGFBP-5 exhibited opposite effects on bone cell proliferation under identical culture conditions. IGFBP-4 inhibited while IGFBP-5 stimulated both basal and IGF-induced bone cell proliferation. The potentiation of cell proliferation by IGFBP-5 cannot be explained by contamination of IGFBP-5 preparation with IGFs or other proteins since both bone-derived and recombinant IGFBP-5 exhibited similar potency and since no detectable level of IGF-I or IGF-II were found in either of these preparations. The effects of IGFBP-4 and IGFBP-5 are not peculiar to MC3T3-E1 mouse osteoblasts since similar effects were seen using untransformed normal human bone cells. These two binding proteins, IGFBP-4 and IGFBP-5, also exhibited opposite effects on the binding of radiolabeled IGF ligand to bone cells in monolayer culture. Although IGFBP-4 decreased the binding of both IGF-I and IGF-II in a similar manner, IGFBP-5 treatment increased IGF-I binding much more than that of IGF-II binding to cells in monolayer culture. The reason for greater changes in IGF-I binding compared with IGF-II in the presence of IGFBP-5 is unknown. Additionally, purified IGFBP-4 decreased the binding of IGF-I to the type I receptor. In contrast, IGFBP-5 did not cause an increase in the binding of radiolabeled IGF-I to type I receptor. Although the findings that at 2 and 20 ng/ml, IGFBP-4 but not IGFBP-5 decreased the binding of IGF-I ligand to type I receptor are consistent with the possibility that the complex of IGFBP-5 and IGF-I may bind to the type I receptor, further studies are needed to test this possibility.

The findings of this study demonstrate that the IGFBP-5-induced increase in I-IGF-I binding to bone cells cannot be explained on the basis of increased IGF-I binding to its receptors. In addition, IGFBP-5-induced increase in I-IGF-I binding in MC3T3-E1 cells was inhibited completely by unlabeled IGF-I but not by insulin or des-1-3-IGF-I, which bind to IGF-I receptor but not to IGFBP-5. These data suggest that IGFBP-5 binds to bone cells independent of IGF receptors and thus increases the binding of IGF-I to bone cells. Our results on IGFBP-5 binding to bone cells are similar to studies with cultured mouse osteoblasts using 23-kDa IGFBP-5 by Andress and Birnbaum (21) and studies with cultured fibroblasts using intact IGFBP-3(27) . In regard to the identification of potential binding sites for IGFBP-5, the findings that IGFBP-5 binding to bone cells is not affected by excess of either IGF-I or IGF-II suggest that IGFBP-5 binds to sites independent of IGF receptors. In addition, Jones et al.(22) have shown that IGFBP-5 binds to various extracellular matrix proteins including types III and IV collagen, laminin, and fibronectin. Because IGFBP-5 does not contain an RGD sequence, which has been shown to be involved in the binding of extracelluar matrix proteins to integrin receptors(40) , the findings of this study that fibronectin, an RGD containing protein, increases IGFBP-5 binding to bone cells together with the previous finding that IGFBP-5 binds to fibronectin is consistent with the possibility that IGFBP-5 may bind to integrin receptors via binding to RGD-containing proteins such as fibronectin.

Based on the findings of this study and other previous studies, we have developed models as shown in Fig. 7to explain the potential mechanisms by which IGFBP-4 and IGFBP-5 may modulate IGF actions in bone cells. According to these models, IGFBP-4 inhibits IGF binding to the receptor by binding to IGFs near or at the receptor binding site. The findings that IGFBP-4 inhibited the binding of radiolabeled IGF-I to purified IGF-I receptors and radiolabeled IGF-II to the IGF-II/mannose-6-phosphate receptors in H4-II-E rat hepatoma cells (35) support the conclusion that IGFBP-4 inhibits IGF-induced bone cell proliferation by preventing IGF binding to its receptors. Consistent with this model of IGFBP-4 action are the findings that IGFBP-4 had no significant effect on the mitogenic activity of des-1-3-IGF-I or des-1-6-IGF-II analog, which has >100-fold reduced affinity for IGFBP-4 than wild-type IGF-I or IGF-II. Thus, IGFBP-4 may act like soluble interleukin-1 receptor, which has been shown to inhibit interleukin-1 action by inhibiting the binding of interleukin-1 to its receptors(41) .


Figure 7: Proposed models for IGFBP-4 and IGFBP-5 actions in bone cells (see text for explanation). IGFBP-4 inhibits IGF binding to IGF receptors by binding IGFs near or at the receptor binding site. With regard to IGFBP-5, three alternate models are proposed. In model 1, the complex of IGFBP-5+IGF binds to IGF receptors. In model 2, IGFBP-5 binds to bone cell surface independent of IGF receptors and thus may stimulate cell proliferation via IGF independent mechanism. In model 3, IGFBP-5 binding sites in bone cell surface may recruit IGFs at the surface of IGF responsive cells, enabling the ligand to be easily captured by IGF receptors.



The molecular mechanisms by which IGFBP-5 stimulates IGF-induced bone cell proliferation appear to be more complex than those of IGFBP-4. It is possible that IGFBP-5 binds to IGF at a site distinct from that of IGFBP-4 and that the complex of IGFBP-5 and IGF binds to the receptor with higher affinity than IGFs alone. This would be analogous to a mechanism whereby the affinity of basic FGF to its receptor is increased upon binding to heparin sulfate(42) . Alternately, IGFBP-5 action may be mediated by IGFBP-5 binding to bone cells independent of IGF receptors. In this regard, IGFBP-5 may bind to extracellular matrix proteins such as fibronectin and may recruit and concentrate IGFs near IGF receptors. Based on the findings that the extracellular matrix-bound IGFBP-5 has lower affinity for the ligand than soluble IGFBP-5, Jones et al.(22) have proposed that extracellular matrix bound IGFBP-5 could facilitate delivery of IGF to IGF-I receptors. A similar mechanism of action has been proposed for IGFBP-3 by Conover and Powell(43) . In these studies, it has been proposed that soluble IGFBP-3 and IGFBP-5 may inhibit IGF actions when in high affinity states while extracellular matrix-bound IGFBPs stimulate IGF actions when their affinity is lower and approximates the affinity of the IGF-I receptor. If IGFBP-5 stimulates IGF actions primarily via increasing the delivery of IGFs to their receptors, we would then anticipate IGFBP-5 to be more active in the presence of IGF-I than in the presence of des-1-3-IGF-I, which has >200-fold reduced affinity than IGF-I for IGFBP-5. However, this was not the case. These findings together with the findings that the mitogenic activities of the maximally active concentrations of IGF-I or IGF-II were increased by treatment with IGFBP-5 are consistent with the model that IGFBP-5 may mediate some of its actions via binding to its own receptors.

The idea that IGFBPs can have intrinsic (IGF-independent) biological activity is also evident from other studies. For example, Cohen et al.(44) have shown that the growth rates of the IGFBP-3 transfected fibroblasts, when grown in insulin-containing media, were not restored to those observed in plasmid-transfected control cells, suggesting that the expression of endogenous IGFBP-3 has an inhibitory effect on cell growth, which is IGF independent. Oh et al.(45) have also recently shown evidence for inhibition of cell growth by exogenous IGFBP-3 in Hs578T human breast cancer cells via an IGF-independent mechanism. In addition, Villaudy et al.(46) have found differences in the biological effects between IGFBP-3 and IGFBP-1 and postulated that IGFBP-3 has multifunctional properties and has a function different from its known function to bind IGFs. Although these findings are consistent with the notion that IGFBPs may modulate some of their effects independent of IGFs via binding to their own binding sites/receptors, further studies involving the identification of these signal-transducing receptors and establishment of the cause and effect relationship between IGFBP binding to its potential receptor and IGFBP actions are needed to verify this mode of action for IGFBPs. The isolation of any such high affinity IGFBP receptors has been made more complicated by bone cell production of a number of abundant extracellular matrix proteins to which IGFBP-5 also binds.

In summary, we have demonstrated that 1) IGFBP-4 inhibits while IGFBP-5 stimulates IGF-induced bone cell proliferation under identical culture conditions, 2) IGFBP-4 inhibits the binding of IGF-I tracer to the type I IGF receptor and IGFBP-4 may modulate its effects on bone cell proliferation by acting like soluble interleukin-1 receptor, and 3) IGFBP-5 binds to bone cells and IGFBP-5 may modulate its effects on bone cell proliferation via an IGF-independent mechanism.


FOOTNOTES

*
This work was supported by the National Institutes of Health (AR 31062 and AR 07543), Boehringer Mannheim, Germany, the Veterans Administration, and the Department of Medicine, Loma Linda University. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Research Service (151), Pettis VA Medical Center, 11201 Benton St., Loma Linda, CA 92357. Tel.: 909-422-3101; Fax: 909-796-1680.

(^1)
The abbreviations used are: IGF, insulin-like growth factor; IGFBP, insulin-like growth factor binding protein; rh, recombinant human; BSA, bovine serum albumin; DMEM, Dulbecco's modified Eagle's medium; HPLC, high pressure liquid chromatography; hBDIGFBP, human bone-derived IGFBP.


ACKNOWLEDGEMENTS

We thank Jacquelyn Douglas and Joe Rung-Aroon for excellent technical support and the Jerry L. Pettis Veteran's Administration Hospital Medical Media Production Service for assistance in the preparation of the figures.


REFERENCES

  1. Mohan, S., and Baylink, D. J. (1991) Clin. Ortho. 263,30-48
  2. Baylink, D. J., Finkelman, R. D., and Mohan, S. (1993) J. Bone Miner. Res. 8,S565-S572
  3. Canalis, E., Pash, J., and Varghese, S. (1993) Crit. Rev. Eukaryotic Gene Expression 3,155-166 [Medline] [Order article via Infotrieve]
  4. Bautista, C., Mohan, S., and Baylink, D. J. (1990) Metabolism 39,96-100 [Medline] [Order article via Infotrieve]
  5. Canalis, E., McCarthy, T., and Centrella, M. (1988) Calcif. Tissue Int. 43,346-351 [Medline] [Order article via Infotrieve]
  6. Mueller, K., Cortesi, R. Modrowski, D., and Marie, P. J. (1994) Am. J. Physiol. 267,E1-E6
  7. Johansson, A. G., Lindh, E., and Ljunghall, S. (1992) Lancet 339,1619
  8. Ebeling, P. R., Jones J. D., O'Fallon, W. M., Janes, C. H., and Riggs, B. L. (1993) J. Clin. Endocrinol. & Metab. 77,1384-1387 [Abstract]
  9. Spencer, E. M. (1991) Modern Concepts of Insulin-like Growth Factors , New York, Elsevier
  10. Baxter, R. C., and Martin, J. L. (1989) Prog. Growth Factor Res. 1,49-68 [Medline] [Order article via Infotrieve]
  11. Shimasaki, S., and Ling, N. (1991) Prog. Growth Factor Res. 1,243-266
  12. Cohick, W. S., and Clemmons, D. R. (1993) Annu. Rev. Physiol. 155,131-153
  13. Mohan, S. (1993) Growth Regul. 3,65-78 [Medline] [Order article via Infotrieve]
  14. Rechler, M. M. (1993) Vitam. Horm. 47,1-114 [Medline] [Order article via Infotrieve]
  15. Mohan, S., Bautista, C., Wergedal, J. E., and Baylink, D. J. (1989) Proc. Natl. Acad. Sci. U. S. A. 86,8338-8342 [Abstract]
  16. Schiltz, P. M., Mohan, S., and Baylink, D. J. (1993) J. Bone Miner. Res. 8,391-396 [Medline] [Order article via Infotrieve]
  17. Cheung, P. T., Smith, E. P., Shimasaki, S., Ling, N., and Chernausek, S. D. (1991) Endocrinology 129,1006-1015 [Abstract]
  18. Culouscou, J. M., and Shyoab, M. (1991) Cancer Res. 51,2813-2819 [Abstract]
  19. Bautista, C. M., Baylink, D. J., and Mohan, S. (1991) Biochem. Biophys. Res. Commun. 176,756-763 [Medline] [Order article via Infotrieve]
  20. Andress, D. L., and Birnbaum, R. S. (1991) Biochem. Biophys. Res. Commun. 176,213-218 [Medline] [Order article via Infotrieve]
  21. Andress, D. L., and Brinbaum, R. S. (1992) J. Biol. Chem. 267,22467-22472 [Abstract/Free Full Text]
  22. Jones, J. I., Gockerman, A., Busby, W. H., Camacho-Hubner, C., and Clemmons, D. R. (1993) J. Cell Biol. 121,679-687 [Abstract]
  23. Kiefer, M. C., Schmid, C., Waldvogel, M., Schlapfer, I., Futo, E., Masiarz, F. R., Green, K., Barr, P. J., and Zapf, J. (1992) J. Biol. Chem. 267,12692-12699 [Abstract/Free Full Text]
  24. Schmid, Ch., Schlapfer, I., Froesch, E. R., Kiefer, M., and Zapf, J. (1993) J. Bone Miner. Res. 8,Suppl. 1, S293
  25. Blat, C., Delbe, J., Villaudy, J., Chatelain, G., Golde, A., and Harel, L. (1989) J. Biol. Chem. 264,12449-12454 [Abstract/Free Full Text]
  26. DeMellow, J. S. M., and Baxter, R. C. (1988) Biochem. Biophys. Res. Commun. 156,199-204 [Medline] [Order article via Infotrieve]
  27. Conover, C. A. (1992) Endocrinology 130,3191-3199 [Abstract]
  28. Clemmons, D. R. (1989) in Molecular and Cellular Biology of Insulin-like Growth Factors and Their Receptors (LeRoith, D., and Raizada, M. K., eds) pp. 381-394, Plenum Publishing Corp., New York
  29. Sudo, H., Kodama, H., Amagai, Y., Yamamoto, S., and Kasai, S. (1983) J. Cell Biol. 96,191-196 [Abstract]
  30. Farley, J. R., Hall, S. L., Herring, S., Tarbaux, N. M., Matsuyama, T., and Wergedal, J. E. (1991) Metabolism 40,664-671 [Medline] [Order article via Infotrieve]
  31. Wergedal, J. E., and Baylink, D. J. (1984) Proc. Soc. Exp. Biol. Med. 176,60-69 [Abstract]
  32. Rudolph. R. (1990) in Modern Methods in Protein and Nucleic Acid Analysis (Burdon, R. H., and Knippenberg, P. H., eds) Elsevier, New York
  33. Mohan, S., and Baylink, D. J. (1991) Growth Regul. 1,110-118 [Medline] [Order article via Infotrieve]
  34. Mohan, S., Jennings, J. C., Linkhart, T. A., and Baylink, D. J. (1986) Biochim. Biophys. Acta 884,234-242 [Medline] [Order article via Infotrieve]
  35. Mohan, S., Bautista, C., Herring, S., Linkhart, T. A., and Baylink, D. J. (1990) Endocrinology 126,2534-2542 [Abstract]
  36. Fujita-Yamaguchi, Y., Choi, S., Sakamoto, Y., and Itakura, K. (1983) J. Biol. Chem. 258,5045-5049 [Abstract/Free Full Text]
  37. Shimasaki, S., Shimonaka, M., Zhang, H.-P., and Ling, N. (1991) J. Biol. Chem. 266,10646-10653 [Abstract/Free Full Text]
  38. Kiefer, M. C., Ioh, R. S., Bauer, D. M., and Zapf, J. (1991) Biochem. Biophys. Res. Commun. 176,219-225 [Medline] [Order article via Infotrieve]
  39. Ballard, F. J. (1992) Endocrinology 130,1736-1737 [Medline] [Order article via Infotrieve]
  40. Dzamba, B. J., Bultmann, H., Akiyama, S. K., and Peters, D. M. (1994) J. Biol. Chem. 269,19646-19652 [Abstract/Free Full Text]
  41. Taga, T., and Kishimoto, T. (1992) FASEB J. 7,3387-3396
  42. Givol, D., and Yayon, A. (1992) FASEB J. 6,3362-3369 [Abstract/Free Full Text]
  43. Conover, C. A., and Powell, D. R. (1991) Endocrinology 129,710-716 [Abstract]
  44. Cohen, P., Lamson, G., Okajima, T., and Rosenfeld, R. G. (1993) Mol. Endocrinol. 7,380-386 [Abstract]
  45. Oh, Y., Muller, H. L., Pham, H., Lamson, G., and Rosenfeld, R. G. (1992) Endocrinology 181,3123-3125
  46. Villaudy, J., Blat, C., Drop, S. L. S., Golde, A., and Harel, L. (1994) Growth Factors 10,107-114 [Medline] [Order article via Infotrieve]

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