©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Insulin-like Growth Factor (IGF)-binding Protein-3 (IGFBP-3) Functions as an IGF-reversible Inhibitor of IGFBP-4 Proteolysis (*)

(Received for publication, May 26, 1995; and in revised form, July 25, 1995)

John L. Fowlkes Delila M. Serra Carlyn K. Rosenberg Kathryn M. Thrailkill (§)

From the Department of Pediatrics, Duke University Medical Center, Durham, North Carolina 27710

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Previous studies have shown that insulin-like growth factor (IGF)-binding protein-4 (IGFBP-4) is degraded only in the presence of exogenous IGFs; however, we found that cation-dependent proteinase activity present in conditioned medium of MC3T3-E1 osteoblasts degrades I-recombinant human (rh)IGFBP-4 in the absence of IGFs. Addition of IGF-I, IGF-II, or insulin to conditioned medium had little affect on I-rhIGFBP-4 proteolysis, while extraction of IGFs resulted in only a 10% reduction in proteinase activity. Since factors other than IGFs appeared to be involved in regulating IGFBP-4 proteolysis, we hypothesized that IGFBP-3, an IGFBP produced by many cell lines, but not MC3T3-E1 cells, might function as an inhibitor of IGFBP-4 proteolysis. Addition of rhIGFBP-3 to conditioned media inhibited I-rhIGFBP-4 proteolysis by 90%, while IGF-I and IGF-II reversed the inhibitory effects of rhIGFBP-3 in a dose-dependent manner. I-rhIGFBP-4 proteolysis was not inhibited by N-terminal rhIGFBP-3 fragments that bind IGFs, but was inhibited by two synthetic peptides corresponding to sequences contained in the mid-region or C-terminal region of IGFBP-3. Both inhibitory peptides contain highly basic, putative heparin-binding domains and heparin partially reversed the inhibitory effects of rhIGFBP-3 on I-rhIGFBP-4 proteolysis. These data demonstrate that rhIGFBP-3 inhibits IGFBP-4-degrading proteinase activity and binding of IGFs or glycosaminoglycans to IGFBP-3 may induce conformational changes in the binding protein, causing disinhibition of the proteinase.


INTRODUCTION

Insulin-like growth factor (IGF)(^1)-binding proteins (IGFBPs) are a group of six homologous, yet distinct proteins (IGFBPs 1-6) which bind both IGF-I and IGF-II with high affinity (for recent reviews, see (1, 2, 3, 4) ). Among these IGFBPs, IGFBP-4 has been shown to function as an inhibitor of IGF bioactivity(1, 2, 3, 4) . For instance, IGFBP-4 was the only inhibitory IGFBP isolated from conditioned media from a colon adenocarcinoma cell line, although other IGFBPs were present in the conditioned medium(5) . Furthermore, Malpe et al.(6) have demonstrated that treatment of human bone cells with antisense oligonucleotides directed against IGFBP-4 mRNA results in decreased production of IGFBP-4 and a striking increase in cellular proliferation, suggesting that IGFBP-4 plays a major role in regulating cellular proliferation. Since IGFBP-4 has been purified from a number of sources (1, 2, 3, 4) and since mRNA for IGFBP-4 has been detected in all tissues studied by Shimasaki et al.(7) , it is likely that IGFBP-4 may serve to restrain IGF activity in many tissues.

Although IGFBP-4 functions as a potent inhibitor of IGF action, the factors controlling production, secretion, and turnover of IGFBP-4 have only recently been addressed. IGFs have been shown to decrease concentrations of IGFBP-4 in the conditioned media of human fibroblasts (8, 9, 10) , human breast cell carcinoma(11) , and human decidual cells (12, 13) . However, IGF-I or IGF-II have little or no effect on IGFBP-4 mRNA in several cell lines (10, 14) and the effect of IGF-I on IGFBP-4 levels in conditioned media of human fibroblasts (8, 9) and human decidual cells (15) is not blocked by a monoclonal antibody to the type-1 IGF receptor. These observations suggest that IGFs might decrease IGFBP-4 concentrations via posttranslational mechanisms.

Consistent with this hypothesis, recent studies from our laboratory have demonstrated that in human and sheep fibroblasts the addition of IGFs induces IGFBP-4 proteolysis by a proteinase(s) not yet identified (16) . Since this original report, IGF-dependent IGFBP-4 proteolysis has been confirmed in human fibroblasts(17) , and similar IGF-dependent IGFBP-4 proteinase activity has been reported in human decidual cell cultures(15) , vascular smooth muscle cells (18) , and human bone cells(19) . The action of the IGF-dependent IGFBP-4 proteinase(s) provides a novel mechanism through which IGFs can increase their own bioavailability and bioactivity.

In the current study, we use the murine MC3T3-E1 osteoblast cell line to explore the role of IGFs in the regulation and induction of IGFBP-4 proteolysis. Herein, we demonstrate that IGFBP-3 can function in a unique role as an inhibitor of IGFBP-4 proteolysis; however, its inhibitory effects can be reversed by the presence of IGFs.


EXPERIMENTAL PROCEDURES

Materials

Recombinant human (rh)IGFBP-3 produced in Escherichia coli (rhIGFBP-3) was kindly provided by Dr. Christopher Maack, Celtrix Pharmaceuticals, Santa Clara, CA(20) . rhIGFBP-4 was purchased from Austral Biologicals, San Ramon, CA. rhIGF-I was kindly provided by Genentech Inc., South San Francisco, CA, and rhIGF-II was generously supplied by Lilly Research Laboratories, Indianapolis, IN. Recombinant human insulin was from Novo Nordisk Pharmaceuticals Inc., Princeton, NJ. Polyclonal antisera to IGF-I was a gift from Dr. Lewis Underwood, University of North Carolina, Chapel Hill, NC, and a monoclonal antibody to rat IGF-II was purchased from Amano Pharmaceutical Co., Ltd., Nagoya, Japan. Reagents used for SDS-polyacrylamide gel electrophoresis (SDS-PAGE) were purchased from Bio-Rad. Low molecular mass heparin (6 kDa) from porcine intestinal mucosa and all proteinase inhibitors were purchased from Sigma, with the exception of the proteinase inhibitors 3,4-dichloroisocoumarin (3,4-DCI) and L-trans-epoxysuccinyl-leucylamide-(4-guanidino)butane (E-64), which were purchased from Boehringer Mannheim. NaI and Hyperfilm-ECL were obtained from Amersham Corp. Tissue culture plasticware was obtained from Corning Glass Works, Corning, NY. Growth media and cell culture reagents were obtained from Life Technologies, Inc.

MC3T3-E1 Cell Culture and Conditioned Media

Stock cultures of MC3T3-E1 osteoblasts were maintained in minimal essential medium containing 10% (v/v) fetal bovine serum, penicillin (100 units/ml), and streptomycin (100 units/ml), as described previously(21) . Stock cultures were subcultured every 3 days.

To induce cellular differentiation, cells were plated at an initial density of 5 times 10^4 cells/well in 35-mm diameter multiwell plastic culture dishes; cells were then grown in alpha-minimal essential medium/10% fetal bovine serum supplemented with 10 mM beta-glycerol phosphate and 25 µg/ml ascorbic acid. Differentiating cultures were refed every 3 days throughout a 30-day culture period. Under these conditions, these cells display an orderly developmental pattern in culture; replication of preosteoblasts (days 1-10) is followed by growth arrest and the sequential expression of mature osteoblastic characteristics, including increased alkaline phosphatase activity (>day 10), matrix accumulation (days 14-21), osteocalcin expression (>day 21), and eventual mineralization (>day 25)(22) . Every 2-3 days during the 30-day culture period, selected plates were washed and incubated for 48 h in serum-free medium (Dulbecco's modified Eagle's medium/F-12 + 0.1% bovine serum albumin). Cell-free conditioned media collected during these 48-h incubations were used in the subsequent experiments.

Preparation of IGF-extracted MC3T3-E1 Conditioned Media

Endogenous IGF-I and IGF-II present in MC3T3-E1 conditioned media were removed by immunoabsorption using anti-IGF-I and anti-IGF-II protein G-Sepharose. To prepare the immunoaffinity matrix, 18 µl of anti-IGF-I polyclonal antisera and 600 ng of an anti-IGF-II monoclonal antibody were incubated with 150 µl of protein G-Sepharose overnight at room temperature. The matrix was washed extensively with 20 mM Hepes, 1 M NaCl, pH 7.4, to remove excess serum. Immunoabsorption was performed by incubating 400-µl samples of MC3T3-E1 conditioned media with 75 µl of the immunoaffinity matrix overnight at 4 °C in a microcentrifuge tube. The mixtures were centrifuged at 2,000 rpm for 5 min at 4 °C, and the supernatant was removed and used for I-rhIGFBP-4 proteinase assays as described below.

Degradation of I-IGFBP-4 by MC3T3-E1 CM

I-rhIGFBP-4 proteinase assays using cell-free conditioned media were performed as described previously, with minor modifications(16) . rhIGFBP-4 was labeled with NaI using the chloramine-T method to a specific activity of 50 µCi/µg of protein. To detect IGFBP-4 protease activity present in conditioned media, samples of cell-free conditioned medium (50 µl unless indicated otherwise) were incubated with I-IGFBP-4 (25,000 cpm; 1 ng of rhIGFBP-4) at 37 °C for 0-72 h. Proteolytic degradation of I-IGFBP-4 was terminated by the addition of an equal volume of 2 times non-reducing sample buffer(23) , followed by heating at 100 °C for 3 min. Samples and prestained molecular weight markers were then electrophoresed through 15% SDS-polyacrylamide gels, dried under vacuum, and exposed to x-ray film to visualize intact and degraded I-IGFBP-4 fragments. In certain experiments, IGFBP-4 proteinase activity was characterized further in the presence of metal-dependent proteinase inhibitors: EDTA (10 mM) and 1,10-phenanthroline (1 mM); serine proteinase inhibitors: aprotinin (2.5 µg/ml), phenylmethylsulfonylfluoride (10 mM), and 3,4-DCI (100 µM); or the cysteine proteinase inhibitor: E-64 (10 µM). In addition, various concentrations of rhIGF-I, rhIGF-II, intact rhIGFBP-3, rhIGFBP-3 fragments produced by matrix metalloproteinase-3 (MMP-3) (as described below), N-terminal IGFBP-3 fragments that bind IGFs, synthetic peptides corresponding to sequences present in hIGFBP-3, and/or heparin were preincubated with MC3T3-E1 conditioned media for 3 h at 37 °C prior to in vitroI-rhIGFBP-4 protease assay.

Preparation of IGFBP-3 Fragments Produced by MMP-3

Sixty µg of rhIGFBP-3 was digested by 200 ng of MMP-3 (kindly provided by Dr. Hideaki Nagase, University of Kansas Medical Center, Kansas City, KS) in a total volume of 60 µl of 50 mM Tris, pH 7.5, 0.15 M NaCl, 10 mM CaCl(2), 0.02% NaN(3), 0.05% Brij 35 (TNC buffer) for 8 h at 37 °C(24) . The digestion was stopped by the addition of EDTA (final concentration: 10 mM), and the digestion products were analyzed on 15% SDS gels under reducing conditions. Seven rhIGFBP-3 fragments, designated ``a-f,'' were identified as described elsewhere (24) (see Fig. 5). To separate rhIGFBP-3 fragments produced by MMP-3 digestion, the digestion mixture was incubated with 1 ml of heparin-Sepharose (Sigma) overnight at 4 °C. The matrix was washed with 50 mM Tris-HCl, pH 7.4, and heparin-bound fragments were eluted in 50 mM Tris-HCl, pH 7.4, containing 1 M NaCl. All fractions were analyzed by SDS-PAGE and stained with Coomassie Blue. The wash fractions (i.e. 50 mM Tris-HCl, pH 7.4) contained only the smallest IGFBP-3 fragments (fragments e and f) produced by MMP-3, which correspond to the first 100-110 N-terminal amino acids of IGFBP-3. Fragments a-d bound to the heparin column, and all four fragments were eluted with 50 mM Tris-HCl, pH 7.4, containing 1 M NaCl. The inability of fragments e and f to bind heparin-Sepharose was anticipated, since their sequences do not contain either of the two putative heparin-binding sites present in IGFBP-3(2, 25) . In covalent cross-linking studies, fragments e and f bound specifically I-rhIGF-I and I-rhIGF-II with IC values of 8.9 and 3.0 nmol/liter, respectively. (^2)These findings are consistent with a previous report demonstrating that an 88-amino acid N-terminal mutant of IGFBP-3 bound I-IGF-I (20) . Fragments a-f together or fragments e and f together were tested for their abilities to alter I-rhIGFBP-4 degradation by MC3T3-E1 conditioned media as described above.


Figure 5: Inhibition of I-rhIGFBP-4 proteolysis by IGFBP-3 synthetic peptides. I-rhIGFBP-4 was incubated with unconditioned media (lane 1) or MC3T3-E1 conditioned media (50 µl; lanes 2-7) in the absence (lane 2) or presence (lanes 3-7) of increasing concentrations of peptide II (panel A and up triangle), peptide IV (panel B and box), or both peptides II and IV (panel C and circle) as described under ``Experimental Procedures.'' The final concentration for each peptide is as follows: lane 3, 10 µM; lane 4, 20 µM; lane 5, 50 µM; lane 6, 100 µM; lane 7, 200 µM. The percentage of proteolysis was calculated from densitometric data from two to four separate experiments as described under ``Experimental Procedures.''



Preparation of Synthetic hIGFBP-3 Peptides

Peptides based on amino acid sequences contained in both the non-homologous mid-region of hIGFBP-3 (peptides I, II, and III) and in the highly conserved C terminus (peptide IV) (see Fig. 5below) were produced by solid phase peptide synthesis using 9-fluorenylmethoxycarbonyl chemistry. The sequences are as follows: SRLRAYLLPAPPAP (peptide I), KKGHAKDSQRYKVDYESQS (peptide II), TDTQNFSSESKRETEY (peptide III), and DKKGFYKKKQLRPSKGR (peptide IV). All peptides were purified on a Vydac C-8 HPLC column using a Gilson automated HPLC system, and were shown to be >98% pure. Sequence verification was performed by electrospray mass spectrometry. All peptides were synthesized with an additional N-terminal cysteine for use in thiol-coupling reactions. The internal cysteine in peptide IV was acetylmethylated because it is normally involved in a disulfide bond. All synthetic peptides were tested for their abilities to alter I-rhIGFBP-4 degradation by MC3T3-E1 conditioned media as described above.

Statistical Analysis

All experiments were repeated using conditioned media from two to five different experiments. Relative concentrations of intact I-rhIGFBP-4 and fragments of I-rhIGFBP-4 were determined by scanning densitometry (Beckman). Graphic data were normalized to the proteolysis of I-rhIGFBP-4 observed in unconditioned media (i.e. 100% inhibition) and the proteolysis of I-rhIGFBP-4 observed in cell-free conditioned media (i.e. 100% proteolysis). All data are expressed as the mean ± S.E. Statistical significance between groups was determined by paired Student's t test. Curve-fitting and IC values were calculated using InPlot Software (GraphPad Software, San Diego, CA).


RESULTS

Characterization of IGFBP-4 Proteases in MC3T3-E1 Cultures

To determine if MC3T3-E1 osteoblasts produce IGFBP-4-degrading proteinases, conditioned media from different time points during differentiation were examined for their abilities to degrade I-rhIGFBP-4. When incubated with conditioned media from MC3T3-E1 osteoblasts, intact I-IGFBP-4 (28 kDa) was degraded into 20- and 14-kDa fragments (Fig. 1). By 24 h, >50% of the binding protein was degraded (p < 0.0001, n = 4). IGFBP-4 protease activity present in MC3T3-E1 conditioned media increased progressively as osteoblasts matured in culture; maximal proteinase activity was detected in conditioned media from osteoblasts displaying a differentiated phenotype. Therefore, all subsequent studies were performed with conditioned media from >20 day cultures. To characterize further the IGFBP-4-degrading proteinase(s) present in MC3T3-E1 conditioned medium, samples of cell-free conditioned media were analyzed for their ability to degrade I-rhIGFBP-4 in the presence or absence of various protease inhibitors. As shown in Table 1, only the cation-dependent proteinase inhibitors (EDTA and 1,10-phenanthroline) significantly inhibited IGFBP-4-degrading proteinase activity. Serine (phenylmethylsulfonylfluoride, aprotinin, and 3,4-DCI) and cysteine (E-64) proteinase inhibitors had little or no effect on degradation of I-rhIGFBP-4. These data suggested that the IGFBP-4-degrading proteinase in MC3T3-E1 conditioned medium is a cation-dependent proteinase.


Figure 1: Degradation of I-rhIGFBP-4 by MC3T3-E1 conditioned media. MC3T3-E1 media from mature osteoblasts were incubated with I-rhIGFBP-4 for the indicated times as described under ``Experimental Procedures.'' Intact I-rhIGFBP-4 (28 kDa; denoted by &cjs0800;) and proteolytic fragments of I-rhIGFBP-4 (20 and 14 kDa; denoted by ) were separated by SDS-PAGE and detected by autoradiography. Molecular size markers are indicated on the left.





Effects of IGFs on I-rhIGFBP-4 Proteolysis

When IGF-I, IGF-II or insulin were added to MC3T3-E1 conditioned media, only high dose IGF-II (500 ng/ml) induced any significant, although modest, increase in I-rhIGFBP-4 proteolysis (Table 2). Because exogenous IGFs were not required for I-rhIGFBP-4 degradation and exerted only marginal effects on I-rhIGFBP-4 degradation by MC3T3-E1 conditioned medium, we postulated that endogenously produced IGFs by MC3T3-E1 osteoblasts might account for the constitutive degradation of I-rhIGFBP-4. To test this hypothesis, IGF-I and IGF-II were first immunoabsorbed from cell-free MC3T3-E1 conditioned media and IGF-extracted conditioned media were then analyzed for their ability to degrade I-rhIGFBP-4. Using this method, only a 10% (p = 0.03) decrease in the degradation of I-rhIGFBP-4 was observed. In other experiments, direct addition of anti-IGF-II monoclonal antibodies to conditioned media resulted in no change in I-rhIGFBP-4 proteolysis (data not shown). These data suggested that endogenous IGFs contributed only minimally, if at all, to the constitutive degradation of I-rhIGFBP-4 by MC3T3-E1 cells.



Effect of rhIGFBP-3 on I-rhIGFBP-4 Proteolysis

In previous studies using other cell lines, we and others have demonstrated that the addition of IGFs to cells or conditioned media is essential for the induction of IGFBP-4 protease activity (reviewed in (1) -4); in contrast, our current studies failed to demonstrate an absolute requirement for IGFs in inducing IGFBP-4 proteolysis in MC3T3-E1 osteoblasts. To clarify this discrepancy, we searched for differences in the IGF/IGFBP systems between MC3T3-E1 osteoblasts and cell lines previously reported to display only IGF-induced IGFBP-4 proteolysis. Since MC3T3-E1 cells do not produce IGFBP-3(26) , while other cell lines exhibiting IGF-induced IGFBP-4 proteolysis do produce IGFBP-3(15, 16, 17, 18, 19) , we speculated that IGFBP-3 might function as an inhibitor of IGFBP-4 proteolysis. To determine whether exogenous IGFBP-3 inhibits the degradation of I-rhIGFBP-4 by MC3T3-E1 conditioned media, we added rhIGFBP-3 to MC3T3-E1 conditioned media and monitored I-rhIGFBP-4 proteolysis. IGFBP-3 inhibited the degradation of I-rhIGFBP-4 in a dose-dependent manner (Fig. 2). At a concentration of 2 µg/ml, rhIGFBP-3 inhibited the degradation of I-rhIGFBP-4 by 90 ± 0.2% (p = 0.0001, n = 4). Thus, IGFBP-3, at maximal concentrations, was as effective an inhibitor of I-rhIGFBP-4 proteolysis as was either of the metal-dependent proteinase inhibitors (see Table 1).


Figure 2: Inhibition of I-rhIGFBP-3 proteolysis by rhIGFBP-3. I-rhIGFBP-4 was incubated with Dulbecco's modified Eagle's medium containing 0.1% bovine serum albumin (lane 1) or MC3T3-E1 conditioned media in the absence (lane 2) or presence (lanes 3-8) of intact rhIGFBP-3. rhIGFBP-3 was added in the following amounts: lane 3, 5 ng of rhIGFBP-3; lane 4, 10 ng of rhIGFBP-3; lane 5, 25 ng of rhIGFBP-3; lane 6, 50 ng of rhIGFBP-3; lane 7, 100 ng of rhIGFBP-3; lane 8, 500 ng of rhIGFBP-3. Intact I-rhIGFBP-4 and proteolytic fragments of I-rhIGFBP-4 were separated by SDS-PAGE and detected by autoradiography. Molecular size markers are indicated on the left.



Determination of the Epitope(s) in IGFBP-3 Involved in Inhibition of IGFBP-4 Proteolysis

To investigate the mechanism through which rhIGFBP-3 inhibits IGFBP-4-degrading proteinase activity, the effects of (a) intact rhIGFBP-3, (b) rhIGFBP-3 fragments produced by MMP-3, and (c) IGF-binding, N-terminal rhIGFBP-3 fragments were compared for their inhibitory effects on I-rhIGFBP-4 proteolysis by MC3T3-E1 conditioned media. Fig. 3demonstrates that the addition of intact rhIGFBP-3 to MC3T3-E1 conditioned media readily inhibited the degradation of I-rhIGFBP-4 (IC = 0.24 µg/ml). When a mixture of all rhIGFBP-3 fragments produced by MMP-3 (fragments a-f) were added to MC3T3-E1 conditioned media, I-rhIGFBP-4 degradation was also inhibited in a dose-dependent fashion, with 2-fold less potency than intact rhIGFBP-3. In contrast, the addition of IGF-binding, N-terminal fragments of IGFBP-3 (fragments e and f) had little or no effect on I-rhIGFBP-4 proteolysis (IC > 40.0 µg/ml), suggesting that inhibitory activity does not reside in the first 110 amino acids present in IGFBP-3 (see Fig. 4). Since N-terminal IGFBP-3 fragments bind IGF-I and IGF-II, sequestration of endogenous IGFs could not explain the inhibitory effect of IGFBP-3 on I-rhIGFBP-4 proteolysis. These observations support earlier conclusions that endogenous IGFs contribute little to the constitutive IGFBP-4-degrading proteinase activity in MC3T3-E1 conditioned media. Together, these data suggested that the inhibitory domain(s) in IGFBP-3 resides in the non-homologous, mid-region of the molecule and/or in the C-terminal domain (Fig. 4) and that degradation of rhIGFBP-3 fails to destroy the inhibitory effects of rhIGFBP-3 on I-rhIGFBP-4 proteolysis.


Figure 3: Inhibition of I-rhIGFBP-4 proteolysis by rhIGFBP-3 or rhIGFBP-3 fragments. MC3T3-E1 conditioned media (50 µl) were incubated with I-rhIGFBP-4 in the absence or presence of increasing concentrations of rhIGFBP-3 (), rhIGFBP-3 fragments a-f (bullet), or IGF-binding, N-terminal rhIGFBP-3 fragments e and f () as described under ``Experimental Procedures.'' Data were obtained from densitometric analysis of autoradiograms, and the percentage of inhibition was calculated as described under ``Experimental Procedures.''




Figure 4: Schematic representation of hIGFBP-3 demonstrating the sequence of origin of rhIGFBP-3 fragments and IGFBP-3 peptides. Human IGFBP-3 is divided into three distinct domains: the N-terminal domain 1 (amino acids 1-87; white), the non-homologous mid-region domain 2 (amino acids 88-183, shaded), and the C-terminal domain 3 (amino acids 184-264, cross-hatched). The putative heparin-binding domains are represented as black boxes. IGFBP-3 fragments produced by digestion with MMP-3 are designated a-f. Synthetic IGFBP-3 peptides are designated I-IV.



To identify the epitope(s) in the last 150 amino acids of IGFBP-3 that inhibit IGFBP-4 degradation, four synthetic peptides were prepared. Three of these peptides correspond to epitopes present in the mid-region of the IGFBP-3 molecule (peptides I, II, and III), a region that has little or no homology with the other five IGFBPs(1) ; the fourth peptide (peptide IV), corresponds to a region in the C-terminal portion of the binding protein (Fig. 4). Table 3demonstrates that both peptide II and peptide IV, when added to MC3T3-E1 conditioned media at 200 µM/liter, inhibited significantly the degradation of I-rhIGFBP-4. In contrast, peptides I and III, used at the same concentrations, had no discernible inhibitory activities. Fig. 5demonstrates that both peptides II and IV inhibited I-rhIGFBP-4 degradation in a dose-dependent manner (Fig. 5, panels A and B, respectively), and each produced a displacement curve parallel to the other peptide (Fig. 5). However, peptide IV (IC = 25 µm) was approximately 3-fold more potent than peptide II (IC = 74 µM) in inhibiting IGFBP-4-degrading proteinase activity. When used together, the peptides demonstrated no additive effect on inhibiting I-rhIGFBP-4 proteolysis (Fig. 5, panel C).



Effects of IGFs on the Inhibitory Activity of IGFBP-3 and IGFBP-3 Peptides on I-rhIGFBP-4 Proteolysis

Although exogenous IGFs had little or no effect on I-rhIGFBP-4 degradation, IGFs effectively reversed the inhibitory effects of rhIGFBP-3 (Fig. 6). When increasing concentrations IGF-I (Fig. 6, panel A) or IGF-II (Fig. 6, panel B) were added to MC3T3-E1 conditioned media containing I-rhIGFBP-4 and a maximal inhibitory dose of rhIGFBP-3 (2 µg/ml), both ligands effectively reversed the inhibitory effect of rhIGFBP-3 on I-rhIGFBP-4 proteolysis (Fig. 6). A 1:1 IGF-II:rhIGFBP-3 molar ratio produced a 50% reversal of IGFBP-3's inhibitory effect, while molar ratios of greater than 3:1 produced almost 100% reversal. IGF-I (IGF-I:IGFBP-3 IC = 2.9:1) was approximately 3 times less potent than IGF-II in reversing the inhibitory effects of rhIGFBP-3 on I-rhIGFBP-4 proteolysis (Fig. 6).


Figure 6: Reversal of the inhibitory effect of rhIGFBP-3 on I-rhIGFBP-4 proteolysis by IGFs. I-rhIGFBP-4 was incubated with unconditioned media (lane 1) or MC3T3-E1 conditioned media (lanes 2-8) in the absence (lane 2) or presence (lanes 3-8) of rhIGFBP-3 (2 µg/ml) and increasing concentrations of IGF-I (panel A and box) or IGF-II (panel B and ) as described under ``Experimental Procedures.'' The amount of IGF-I or IGF-II added per lane is as follows: lane 4, 10 ng; lane 5, 25 ng; lane 6, 50 ng; lane 7, 100 ng; lane 8, 500 ng. The percentage of proteolysis was calculated from densitometric data obtained from three separate experiments as described under ``Experimental Procedures'' and is expressed as a ratio of IGF:IGFBP-3 added to the sample.



Because IGFs reversed the inhibitory effects of rhIGFBP-3 on I-rhIGFBP-4 proteolysis, IGF-I was examined for its ability to reverse the inhibitory effects of peptides II and IV on I-rhIGFBP-4 proteolysis by MC3T3-E1 conditioned media. As shown in Fig. 7, IGF-I had little or no effect on reversing the inhibitory effects of peptides II and IV on I-rhIGFBP-4 degradation. In covalent cross-linking studies, it was demonstrated that while I-IGF-I and I-IGF-II both bound to intact rhIGFBP-3 or IGFBP-3 fragments e and f, neither radioligand bound to peptides II and IV (data not shown). These data suggest that IGFBP-3 inhibits IGFBP-4 proteolysis via epitopes present within the amino acid sequences contained in peptides II and IV and that IGFs must be able to bind to IGFBP-3 in order to reverse the inhibitory effects of IGFBP-3 on IGFBP-4 proteolysis.


Figure 7: Effect of IGF-I on the inhibition of I-rhIGFBP-4 proteolysis by IGFBP-3 synthetic peptides. I-rhIGFBP-4 was incubated with MC3T3-E1 conditioned media in the absence (lane 1) or presence (lanes 2-9) of various IGFBP-3 synthetic peptides (200 µM): peptide I, lanes 2 and 3; peptide II, lanes 4 and 5; peptide III, lanes 6 and 7; peptide IV, lanes 8 and 9. IGF-I (20 µg/ml) was added to lanes 3, 5, 7, and 9. Intact I-rhIGFBP-4 and proteolytic fragments of I-rhIGFBP-4 were separated by SDS-PAGE and detected by autoradiography. Molecular size markers are indicated on the left.



Effect of Heparin on rhIGFBP-3 Inhibition of I-rhIGFBP-4 Proteolysis

As indicated in Fig. 5, peptides II and IV both contain putative heparin-binding domains(2, 25) . Peptide II contains the sequence KKGHA which resembles a short heparin-binding domain (BBXBX; B = basic amino acid, and X = non-basic amino acid) and peptide IV contains the sequence YKKKQCRP, which resembles a long heparin-binding motif (XBBBXXBX)(25) . This suggested that both of these highly basic, putative heparin-binding domains present in IGFBP-3 could inhibit IGFBP-4-degrading activity in MC3T3-E1 conditioned media; therefore, we next examined the effect of heparin on modulating the inhibitory effect of rhIGFBP-3 on I-rhIGFBP-4 proteolysis. For this purpose, a submaximal dose of rhIGFBP-3 (200 ng/ml) or of peptide IV (20 µM) was added to MC3T3-E1 conditioned medium with or without heparin (100 µg/ml). All solutions were incubated with I-rhIGFBP-4 and processed in parallel with conditioned media containing no additives or heparin alone. As Fig. 8demonstrates, heparin alone (bar 4) had no significant effect on I-rhIGFBP-4 proteolysis. In contrast, when heparin was added to conditioned media containing rhIGFBP-3, heparin reversed the inhibitory effects of rhIGFBP-3 on I-rhIGFBP-4 proteolysis by 33% (p < 0.01) (compare bars 2 and 3). Similarly, heparin almost entirely reversed the inhibitory effects of peptide IV on I-rhIGFBP-4 proteolysis (data not shown).


Figure 8: Effect of heparin on the inhibition of I-rhIGFBP-4 degradation by rhIGFBP-3. MC3T3-E1 conditioned media was incubated with I-rhIGFBP-4 in the absence (bar 1), or the presence of rhIGFBP-3 (200 ng/ml; bars 2 and 3) and/or heparin (100 µg/ml; bars3 and 4) as described under ``Experimental Procedures.'' The percentage of proteolysis was calculated from densitometric data. *, p < 0.01 for comparisons of 1 versus 2 or 3 and 2 versus 3.




DISCUSSION

IGFBP-4 was first isolated from conditioned media from an osteosarcoma cell line and has now been identified in a variety of biological fluids and cellular conditioned media, where it functions as a potent inhibitor of IGF action (for recent reviews, see (1, 2, 3, 4) ). Previously, we demonstrated that IGFs can induce the proteolytic degradation of IGFBP-4 into fragments that display little or no affinity for IGFs, providing a mechanism by which IGFs can directly increase their own bioavailability and/or bioactivity(16) . Subsequently, Conover et al.(17) demonstrated that the degradation of IGFBP-4 increased IGF activity in human fibroblast cultures. How IGFs induce IGFBP-4 proteolysis remains unclear; we (16) and others (15) have hypothesized that IGFs induce IGFBP-4 degradation by binding to IGFBP-4, thus making it more susceptible to proteolysis, while others have proposed that IGFs directly activate an IGFBP-4 proteinase(17) . Insights from the current studies would suggest that neither of these hypotheses is entirely correct, and that a more complex mechanism is involved in IGF-induced IGFBP-4 degradation.

Since other studies have now demonstrated IGF-inducible IGFBP-4 degrading proteinase activity in a number of cell lines(1, 2, 3, 4) , including bone cells(19) , we chose to explore IGFBP-4 proteolysis in a murine bone cell line, MC3T3-E1 osteoblasts. Initial studies demonstrated that the IGFBP-4-degrading proteinase(s) present in MC3T3-E1 conditioned media cleaved the protein into two major fragments and that the proteinase was cation-dependent, making it similar to IGFBP-4-degrading proteinase activity reported in other cell lines(15, 16, 17) . Nevertheless, one major difference existed between the IGFBP-4-degrading proteinase activity observed in MC3T3-E1 conditioned media compared with that produced by other cell lines; no exogenous IGFs were required to induce IGFBP-4 proteolysis(1, 2, 3, 4, 15, 16, 17, 18, 19) . In fact, addition of IGF-I to MC3T3-E1 conditioned media had no effect on I-rhIGFBP-4 degradation, while a maximal dose of IGF-II (500 ng/ml) only modestly increased the degradation of the binding protein. This is in marked contrast to our previous report using human and sheep fibroblast condition media, where we demonstrated that little or no proteolysis of IGFBP-4 occurred during a prolonged incubation (i.e. 72 h), yet with the addition of IGF-I or IGF-II, almost complete proteolysis of IGFBP-4 was achieved(16) . Since MC3T3-E1 osteoblasts secrete both IGF-I and IGF-II(26, 27) , it was possible that endogenous IGFs induced IGFBP-4 proteolysis in this cell line. In this regard, Durham et al.(28) have very recently demonstrated that in certain primary human bone cell lines, IGFBP-4 can be degraded without the addition of IGFs, while in other bone cell lines the addition of IGFs is necessary for IGFBP-4 proteolysis. They suggest that cell lines not requiring exogenous IGFs for IGFBP-4 proteolysis produced more IGFs than those cell lines requiring exogenous IGFs for IGFBP-4 proteolysis. Since immunoabsorption of IGFs from conditioned media or the addition of IGF-binding, N-terminal fragments of IGFBP-3 to conditioned media had little or no effect on inhibiting I-rhIGFBP-4 degradation, endogenous IGFs do not appear to contribute significantly to the constitutive degradation of I-rhIGFBP-4 observed in MC3T3-E1 cells. Furthermore, since many cell lines that display IGF-induced IGFBP-4 proteolysis also produce some IGF-I and/or IGF-II, it seemed unlikely that the presence of IGFs in MC3T3-E1 conditioned media could account for the constitutive nature of the IGFBP-4-degrading proteinase activity.

We have demonstrated that MC3T3-E1 cells secrete immunoreactive IGFBP-2, -4, and -5(26) , while IGFBP-3 is not produced by these cells (26) and the cells produce no IGFBP-3 mRNA(29) . Based on the observation that MC3T3-E1 conditioned media contains little or no IGFBP-3, while other cell lines displaying IGF-dependent IGFBP-4 proteolysis do produce IGFBP-3(15, 16, 17, 18, 19) , we postulated that IGFBP-3 might function as an inhibitor of IGFBP-4 proteolysis. Indeed, the addition of rhIGFBP-3 to conditioned media significantly inhibited the constitutive degradation of I-rhIGFBP-4 by conditioned media from MC3T3-E1 cells in a dose-dependent manner. This demonstrated that exogenous IGFBP-3 is an IGFBP-4 proteinase inhibitor and also suggests that endogenous IGFBP-3 could function as a physiological inhibitor of IGFBP-4 proteolysis since it is present in a variety of biological fluids at concentrations 5-20 times greater (30) than the IC needed to inhibit IGFBP-4 degradation. IGFBP-3 may also be induced in certain cell systems and function as an IGFBP-4 proteinase inhibitor. For instance, Conover and colleagues (31) have demonstrated that treatment of human fibroblasts with phorbol esters induces an inhibitor(s) of IGFBP-4 proteolysis, while Albiston et al.(32) have shown that IGFBP-3 promotor activity is increased when transfected cells are treated with a phorbol ester. Therefore, it is possible that the phorbol ester-induced inhibitor of IGFBP-4 proteolysis is IGFBP-3.

Similar to the effects of intact IGFBP-3 and inhibitory IGFBP-3 fragments, two peptides inhibited IGFBP-4 proteolysis, while two other peptides did not. The feature common to both inhibitory peptides was that each contained one of the two highly basic, putative heparin-binding domains present in IGFBP-3(2, 25) . Further analysis revealed that peptide IV, which contains a long heparin-binding motif, was 3 times more potent in inhibiting IGFBP-4 proteolysis than was peptide II, which contains a short heparin-binding site, and there was no demonstrable synergy when both peptides were used together. This suggests that both peptides work through a similar inhibitory mechanism, although the specifics of the mechanism are currently unknown and are under investigation. Preliminary data from our laboratory demonstrate that other proteins, such as fibronectin and vitronectin, which are commonly found in cell cultures and which contain both long and short forms of heparin-binding motifs, do not inhibit IGFBP-4 proteolysis by MC3T3-E1 conditioned media.^3 However, IGFBP-5, which contains almost the same stretch of basic residues that is present in IGFBP-3 and peptide IV(1) , also inhibits IGFBP-4 proteolysis,^3 suggesting that these basic domains are specific in their ability to inhibit IGFBP-4 proteolysis. These highly basic regions may interact with IGFBP-4 itself, protecting it from proteolysis; however, this mechanism seems doubtful since heterodimerization of IGFBPs has not been reported to date. These regions may function as competitive substrates for the IGFBP-4 proteinase; however, this seems unlikely since IGFBP-4 itself contains no heparin-binding motifs (2) and since a recent study demonstrated that the cleavage site in human IGFBP-4 produced by the IGF-dependent IGFBP-4 proteinase is at Met-Lys(33) , a bond that is not present in either of the inhibitory peptides. Interestingly, Nam et al.(34) have demonstrated recently that an IGFBP-5 synthetic peptide that is 80% homologous with peptide IV significantly inhibited the proteolysis of IGFBP-5 by a partially purified IGFBP-5-degrading proteinase. Thus, together these data suggest that IGFBPs containing heparin-binding domains (i.e. all IGFBPs with the notable exception of IGFBP-4; (2) ) may function as direct inhibitors of the IGFBP-4 proteinase(s). Furthermore, the finding that IGF-I was unable to reverse the inhibitory effects of peptides containing heparin-binding domains suggests that fragments of IGFBP-3 that contain one or more of these domains, but do not contain IGF-binding sites, might function as inhibitors whose effects are not reversible by IGFs.

The mechanism by which IGFs reverse the inhibitory effects of IGFBP-3 on IGFBP-4 degradation is currently unknown; however, one explanation is that binding of IGFs to IGFBP-3 results in a conformational change in the binding protein, making the putative heparin-binding domains less available to inhibit IGFBP-4 proteinase activity. Several lines of investigation support this hypothesis. For instance, in vitro IGFBP-3 binds to cell surfaces and/or cell matrix via glycosaminoglycans and possibly through specific IGFBP-3 cell-surface receptors (see (4) for review). How IGFBP-3 binds to cell monolayers is currently unclear; however, Bar et al.(35) have shown that binding of IGFBP-3 to endothelial cells involves the putative heparin-binding motif contained in the C terminus of IGFBP-3 and in peptide IV used in our study. Despite its association with cell monolayers, IGFBP-3 can be released from cell surfaces when bound to IGFs(4) . Together, these data suggest that unsaturated IGFBP-3 may present certain epitopes (i.e. the putative heparin-binding domains) on its surface, which interact with a variety of molecules such as glycosaminoglycans and cell-surface receptors; however, once bound to IGFs, IGFBP-3 may undergo a conformational change and lose its affinity for these molecules. In the same context, binding of IGFs to IGFBP-3 may alter its conformation in such a way that inhibitory epitopes present in the molecule are no longer available to inhibit the IGFBP-4 proteinase.

In contrast to recent reports by Gockerman and Clemmons (36) and Arai et al.(37) demonstrating that heparin inhibits IGFBP-2 proteinase activity produced by porcine aortic smooth muscle cells and IGFBP-5 proteinase activity produced by human fibroblasts, respectively, our data suggest that heparin has little or no effect on IGFBP-4 degradation by MC3T3-E1 conditioned media. Nevertheless, heparin significantly reversed the inhibitory effects of IGFBP-3 and peptide IV on I-rhIGFBP-4 degradation by MC3T3-E1 conditioned media. The divergent effects of heparin on degradation of these various IGFBPs may simply reflect the differences in the proteinases that are involved. However, it is also possible that heparin alters the inhibition of these proteinases in divergent ways. For example, heparin has been shown to enhance inhibition of thrombin and factor Xa by antithrombin III(38) , but decrease the rate of inhibition of neutrophil elastase by alpha(1)-proteinase inhibitor(39) , although both proteinase inhibitors are members of the serpin family. The mechanism by which heparin interferes with IGFBP-3's ability to inhibit IGFBP-4 proteolysis is unclear; however, association of heparin with heparin-binding sites present in IGFBP-3 may make them less available to inhibit the proteinase. Because heparin has been shown to interfere with binding of IGFs to IGFBP-5 and IGFBP-3(25) , an alternative hypothesis would be that heparin causes dissociation of IGFs from endogenous IGFBPs, making IGFs available for binding to exogenous rhIGFBP-3, thereby partially mitigating the inhibitory effect of IGFBP-3 on IGFBP-4 proteolysis. Regardless of the mechanism, heparin, and possibly other glycosaminoglycans, may have a role similar to the IGFs in inducing IGFBP-4 proteolysis.

IGFBP-3 has been shown to both enhance and inhibit IGF activity in vitro (reviewed in (1, 2, 3, 4) ). Our data would suggest that this dichotomy may be explained partially by the finding that IGFBP-3 inhibits IGFBP-4 proteolysis, thus decreasing IGF activity. However, when bound to IGFs, and possibly glycosaminoglycans, IGFBP-3 loses its capability to inhibit IGFBP-4 proteolysis, thus enhancing IGF activity by facilitating the degradation of IGFBP-4. Fig. 9presents a summary of these studies and a hypothetical scheme of how IGFBP-3 might function in vivo to regulate IGFBP-4 proteolysis. In the absence of IGFBP-3 (and possibly other IGFBPs containing heparin-binding motifs), IGFBP-4 is degraded constitutively (Fig. 9, A) into non-IGF binding fragments. In the presence of IGFBP-3, IGFBP-4 degradation is markedly attenuated (Fig. 9, B). However, the inhibitory effects of IGFBP-3 can be reversed via binding of IGFs to IGFBP-3 (Fig. 9, C). Similarly, if IGFBP-3 is bound to glycosaminoglycans or cell surfaces, heparin-binding domains present in the molecule may be less available for interaction with the proteinase (Fig. 9, D). This scenario may be altered in the event that IGFBP-3 is degraded by proteinases such as MMPs, since these proteinases can cleave the N-terminal IGF-binding domain from the heparin-binding domains present in the mid-region and C-terminal tail of the molecule(24) . In this instance, fragments containing the heparin-binding motif(s) could inhibit proteinase activity; yet since they bind little or no IGFs, their inhibitory effects may not be reversible by IGFs, making them IGF-resistant IGFBP-4 proteinase inhibitors (Fig. 9, E). Together these data suggest a new role for IGFBP-3 as an IGFBP-4 proteinase inhibitor and they exemplify the intricate complexities involved in the IGF/IGFBP/IGFBP-proteinase system.


Figure 9: Hypothetical scheme demonstrating how IGFBP-3 may regulate IGFBP-4 proteolysis and IGF bioavailability in vivo. For details, see text.




FOOTNOTES

*
This work was supported by National Institutes of Health Grant DK02276 and March of Dimes Basil O'Connor Starter Scholar Research Award 5-FY93-0953 (to J. L. F.) and by Duke Children's Miracle Network grant and a grant from the Genentech Foundation for Growth and Development (to K. M. T.). 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 all correspondence should be addressed: Dept. of Pediatrics, Div. of Endocrinology, Box 3080, Duke University Medical Center, Durham, NC 27710. Tel.: 919-684-3772; Fax: 919-684-8613.

(^1)
The abbreviations used are: IGF, insulin-like growth factor; IGFBP, insulin-like growth factor binding protein; rh, recombinant human; MMP-3, matrix metalloproteinase-3; PAGE, polyacrylamide gel electrophoresis; E-64, L-trans-epoxysuccinyl-leucylamide-(4-guanidino)butane; 3,4-DCI, 3,4-dichloroisocoumarin; HPLC, high performance liquid chromatography.

(^2)
J. L. Fowlkes, D. M. Serra, K. Suzuki, and H. Nagase, unpublished data.

(^3)
J. L. Fowlkes, D. M. Serra, and K. M. Thrailkill, unpublished data.


ACKNOWLEDGEMENTS

We thank Dr. Darryl Quarles for providing the MC3T3-E1 osteoblasts for these studies and Drs. Michael Freemark and Jan Enghild for reviewing the manuscript and making helpful suggestions. We also acknowledge the generous gift of rhIGFBP-3 from Dr. Christopher Maack (Celtrix Pharmaceuticals, Santa Clara, CA).


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