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Volume 270, Number 47, Issue of November 24, 1995 pp. 28289-28296
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Heparin Modulates the Binding of Insulin-like Growth Factor (IGF) Binding Protein-5 to a Membrane Protein in Osteoblastic Cells (*)

(Received for publication, July 12, 1995; and in revised form, August 30, 1995)

Dennis L. Andress (§)

From the Medical and Research Services, Veterans Administration Medical Center, Seattle, Washington 98108 and the Department of Medicine, University of Washington, Seattle, Washington 98493

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Osteoblast-like cells secrete insulin-like growth factor (IGF) binding protein-5 (IGFBP-5), which may act to enhance IGF-stimulated osteoblast function. We recently demonstrated that carboxyl-truncated IGFBP-5 (IGFBP-5) binds to the osteoblast surface and stimulates mitogenesis by a pathway that is independent of IGF action. The present study was conducted to determine the mechanism of osteoblast binding of IGFBP-5, beginning with the assumption that cell surface glycosaminoglycans may mediate the binding of this heparin binding protein. Intact I-IGFBP-5 and I-IGFBP-5 exhibited one-site binding to mouse osteoblast monolayers with dissociation constants of 28 and 6 nM for intact I-IGFBP-5 and I-IGFBP-5, respectively. Osteoblast binding of intact I-IGFBP-5 was inhibited by low heparin concentrations, while I-IGFBP-5 binding was stimulated by heparin. Treatment of cells with heparinase or chlorate to decrease surface glycosaminoglycan density failed to reduce the binding of either form of IGFBP-5. In contrast, pretreatment of cells with IGFBP-5 caused down-regulation of I-IGFBP-5 binding. Cross-linking studies revealed that both intact I-IGFBP-5 and I-IGFBP-5 bind to proteins in Triton extracts of osteoblast membranes, which were absent in osteoblast-derived matrix. Purification of membrane extracts by IGFBP-5 affinity chromatography revealed a 420-kDa band on reduced SDS-polyacrylamide gels. While the membrane protein internalized both forms of IGFBP-5, heparin treatment inhibited the internalization of intact I-IGFBP-5 but stimulated I-IGFBP-5 internalization. These data indicate that IGFBP-5 binds to and is internalized by an osteoblast membrane protein, which does not appear to be a proteoglycan. Glycosaminoglycans, however, modulate the binding and internalization of IGFBP-5 in a way that may preferentially favor the intracellular accumulation of the carboxyl-truncated form.

INTRODUCTION

Recently, we demonstrated that a carboxyl-truncated form of IGFBP-5, (^1)derived from human osteoblast-like cells, enhanced the mitogenic action of IGF-I in cultured mouse osteoblasts(1, 2) . In addition to its IGF-enhancing action, we also found that human recombinant carboxyl-truncated IGFBP-5 could bind to osteoblast monolayers and stimulate mitogenesis without exogenous IGF-I(3) , similar to the effects of native carboxyl-truncated IGFBP-5(2) . While it was proposed that this intrinsic mitogenic activity was mediated by cell surface binding, the mechanism of IGFBP-5 binding to osteoblasts was not determined.

IGFBP-5 is one of several IGFBPs that bind heparin(4) , most likely due to the presence of one or more putative heparin binding domains(5, 6) . Heparin and other sulfated glycosaminoglycans (GAGs) decrease the binding of IGF-I to IGFBP-5 (7) and inhibit the degradation of intact IGFBP-5(8) . The latter effect, reminiscent of the protection afforded by heparin on fibroblast growth factor degradation(9) , may result from a heparin-induced altered conformation that prevents enzymatic proteolysis. Heparin also modifies the binding and internalization of fibroblast growth factor in cultured cells(10, 11) , acting in competition with the GAG moieties located on cell surface proteoglycans.

Because IGFBP-5 binds to heparin and possibly to GAG-containing proteoglycans located on osteoblast surfaces or within osteoblast-derived extracellular matrix (ECM), we examined whether heparin modifies the binding of IGFBP-5 in primary cultures of normal mouse osteoblasts. This report characterizes the binding properties of two forms of IGFBP-5 and establishes the importance of a high molecular weight osteoblast membrane protein in osteoblastic cells that binds IGFBP-5.

EXPERIMENTAL PROCEDURES

Materials

Recombinant forms of IGFBP-5 were expressed in baculovirus using cDNA that was modified by polymerase chain reaction to encode either amino acids 1-252 (intact) or 1-169 (carboxyl-truncated). The sequences were introduced into the Autographa californica baculovirus using the transfer vector, pAcC13. Approximately 2 µg of the plasmids were cotransfected with 0.5 µg of linearized, wild-type viral DNA into SF-9 cells. Recombinant baculovirus was isolated by plaque purification, and recombinant protein was produced by infecting suspension cultures of SF-9 cells with virus in serum-free medium (Dr. Patricia Olson, Chiron Corp.). Supernatants were harvested 48 h after infection, and both forms of IGFBP-5 were purified by IGF-I affinity chromatography and reversed phase HPLC as described(2) . NaI and S were purchased from Amersham Corp. and [^3H]heparin was obtained from DuPont NEN. I-IGFBP-5 and I-IGFBP-5 were prepared using chloromine-T as described(2) ; specific activities ranged from 100 to 120 µCi/µg. The IGFBP-5 peptides, IGFBP-5 (AVKKDRRKKLT) and IGFBP-5 (RKGFYKRKQCKPSRGRKR), were synthesized and purified by reversed phase HPLC (Fred Hutchinson Cancer Center, Seattle, WA). Heparin, heparan sulfate, dermatan sulfate, chondroitin sulfate A, heparinase and heparin-agarose, type II, and chondroitinase ABC lyase were purchased from Sigma. Disuccinimidyl suberate (DSS) was purchased from Pierce, and collagenase was purchased from Worthington. Heparan sulfate proteoglycan was obtained from Collaborative Biomedical Products. Sodium chlorate was purchased from Aldrich.

Cell Culture

Neonatal (2-3-day-old) mouse osteoblasts were released from calvariae after a 120-min exposure to collagenase (following a 30-min exposure that was discarded) and grown for 1 week in 75-cm^2 flasks (Costar) containing DMEM (Life Technologies, Inc.) and 10% FCS (Hyclone). Nearly confluent cells were then released by trypsin and plated in either 48-well or 6-well plates (Costar) in DMEM containing 10% FCS for 24 or 48 h.

I-IGFBP-5 Binding to Osteoblasts

Confluent monolayers of neonatal mouse osteoblasts in 48-well plates were incubated in serum-free medium for 24 h. The cells were washed three times with phosphate-buffered saline (PBS) and then incubated in 100 µl of assay buffer (20 mM HEPES, 0.1 mg/ml BSA, pH 7.0) for 2 h at 4 °C with 0.2 nM intact I-IGFBP-5 or I-IGFBP-5 in the absence or presence of varying concentrations of unlabeled IGFBP-5, IGFBP-5, heparin, or other glycosaminoglycans shown in Fig. 3. At the end of the incubation period, the buffer was removed, and the cells were rinsed with PBS and solubilized with 1 N NaOH. Radioactivity of the cell lysates was determined, and specific binding was computed by subtracting background counts from total cpm. Binding parameters were determined by the curve-fitting program LIGAND(12) .

Figure 3: Competitive binding of glycosaminoglycans with intact I-IGFBP-5 to mouse osteoblasts. Osteoblast monolayers maintained in serum-free medium were exposed to intact I-IGFBP-5 with increasing concentrations of heparan sulfate, dermatan sulfate, chondrointin sulfate-A, or HSP. Cell-associated radioactivity was determined as described in Fig. 1.


Figure 1: Binding of intact I-IGFBP-5 and I-IGFBP-5 to mouse osteoblasts. Confluent monolayers maintained in serum-free medium were exposed to intact I-IGFBP-5 (A) or I-IGFBP-5 (B) with increasing concentrations of unlabeled IGFBP-5 for 2 h at 4 °C. The cells were washed and solubilized, and the cell-associated radioactivity was determined. Insets depict the Scatchard analysis for each.


[H]Heparin Binding to IGFBP-5

Heparin binding to intact IGFBP-5, IGFBP-5, and IGFBP-5 peptides was determined by the method of Baird et al.(13) . Varying amounts of solubilized IGFBP-5 were applied to nitrocellulose discs and dried for 1 h in a vacuum oven at 90 °C. The nitrocellulose was then wet with 50 mM Tris-buffered saline, pH 7.4, and rinsed three times. Each disk was incubated with 0.1 µCi of [^3H]heparin in 50 mM Tris-buffered saline containing 4% BSA for 16 h at 4 °C. The filters were then rinsed with Tris-buffered saline and transferred to counting vials containing scintillation fluid and counted in a beta counter. Background counts, determined on control filters not containing peptides, were <200 cpm and were subtracted from the total counts to determine specific binding.

Heparin-agarose Chromatography

Intact I-IGFBP-5 and I-IGFBP-5 were applied to a 2-ml column of heparin-agarose equilibrated in 50 mM HEPES, 50 mM NaCl, 0.1 mM PMSF, 0.02% Tween 20, pH 7.5, for 4 h at 4 °C. The column was then washed with 20 ml of equilibration buffer followed by sequential 10-ml volumes of buffer containing increasing concentrations of NaCl. 1-ml fractions were collected and counted in a counter.

Heparinase Treatment of Cells

Mouse osteoblasts were cultured as described above and then grown for 24 h in serum-free DMEM containing 0.1% BSA. The cells were rinsed three times with serum-free DMEM and incubated in binding buffer (DMEM, 25 mM HEPES, 0.1% BSA) without or with 2 units/ml heparinase for 1 h at 37 °C. At that time, the cells were rinsed three times with binding buffer, cooled to 4 °C, and incubated with binding buffer containing intact I-IGFBP-5 or I-IGFBP-5 in the absence or presence of 10 µg/ml unlabeled IGFBP-5 for 2 h at 4 °C. The cells were rinsed three times with PBS and solubilized in 1 N NaOH, and the radioactivity of the cell lysates was determined. The efficiency of the heparinase treatment was assessed by the method of Vassiliou and Stanley(14) . Briefly, cells were incubated with DMEM containing 5% FCS and 20 µCi/ml [S]sulfate for 36 h at 37 °C, rinsed three times with PBS, and incubated with DMEM containing 25 mM HEPES, 0.1% BSA without or with 2 units/ml heparinase for 1 h at 37 °C. The medium was removed for scintillation counting, and the cells were then washed three times with PBS; the remaining cell surface S-labeled material was then released by digestion with 0.25% trypsin for 20 min at 37 °C. Trypsin digestion releases heparinase-resistant S-labeled material from the cell surface in addition to releasing the cells from the culture dishes. The cells were pelleted in a microcentrifuge tube at 1,000 times g for 5 min, and the supernatant (trypsin released) and pellet (heparinase and trypsin resistant, cell associated) radioactivity were quantitated separately. The results are presented as the mean ± S.E. of two experiments performed in duplicate.

Chlorate Treatment of Cells

Subconfluent cultures of mouse osteoblasts were grown in DMEM containing 10% FCS and 5 mM sodium chlorate for 4 days. The medium was then changed to sulfate-free medium containing 5 mM sodium chlorate, 0.1% BSA, and the cells were incubated for 18 h at 37 °C. The cells were then rinsed with PBS and labeled in sulfate-free medium containing either intact I-IGFBP-5 in serum-free medium for 2 h at 4 °C or with SO(4) in medium containing 10% FCS for 18 h at 37 °C. The cells were then rinsed three times with PBS and solubilized, and the cell lysate radioactivity was determined.

Affinity Cross-linking of IGFBP-5 to Membrane Proteins

Affinity labeling of mouse osteoblasts was performed using the method of Massague(15) . Osteoblasts released from primary cultures were plated into 6-well plates (Costar) at 400,000 cells per plate and grown for 24 h in DMEM containing 10% FCS. The medium was then replaced with serum-free DMEM for 18 h, at which time the medium was removed and the cells were rinsed three times with ice-cold binding buffer (128 mM NaCl, 5 mM KCl, 1.2 mM magnesium sulfate, 1.2 mM calcium chloride, 50 mM HEPES, pH 7.7, 2 mg/ml BSA) and incubated for 30 min in cold binding buffer. Fresh binding buffer was then replaced containing I-IGFBP-5 (2 nM) without and with a 50-fold excess of unlabeled IGFBP-5 for 2 h at 4 °C. The buffer was then aspirated, and the cells were rinsed three times with cold binding buffer. Binding buffer without BSA was added to the cells followed by the bifunctional cross-linking agent DSS (0.135 mM, final concentration) for 15 min at 4 °C. The cells were then quickly rinsed with ice-cold detachment buffer (0.25 M sucrose, 10 mM Tris, 1 mM EDTA, pH 7.4, 0.3 mM PMSF), and the cells were removed by scraping in 1 ml of detachment buffer. The cells were centrifuged at 12,000 times g for 2 min, and the pellet was solubilized in 125 mM NaCl, 10 mM Tris, 1 mM EDTA, pH 7.0, 1% Triton X-100, 50 µg/ml leupeptin, 50 µg/ml antipain, 250 µg/ml aprotinin, 500 µg/ml soybean trypsin inhibitor, 500 µg/ml benzamidine hydrochloride, 50 µg/ml pepstatin, 1.5 mM PMSF for 1 h at 4 °C with constant mixing. The insoluble material was removed by centrifugation at 12,000 times g for 15 min, and the supernatant was mixed with electrophoresis sample buffer containing 2-mercaptoethanol and separated by SDS-polyacrylamide gel electrophoresis.

Preparation of Extracellular Matrix

Confluent neonatal mouse osteoblasts were removed with trypsin-EDTA, plated onto 6-well plates (Costar), and grown to confluence over 48 h in DMEM containing 10% FCS. ECM was prepared by two different methods. The first was a modification of the method used by Knudsen et al.(16) in which the cells were rinsed three times with ice-cold PBS and the cell membranes were extracted in 1% Triton X-100/PBS for 10 min on ice followed by removal of nuclei and cytoplasm with 25 mM ammonium acetate, pH 9.0, for 10 min. The remaining ECM was then gently rinsed three times with PBS and used immediately for cross-linking studies. The second method involved detaching the cell monolayer with 5 mM EDTA in PBS at 37 °C for 20 min. Following cell removal the underlying ECM was rinsed three times with PBS and used immediately for cross-linking studies.

IGFBP-5 Affinity Purification of a 420-kDa Membrane Protein

Primary cultures of neonatal mouse osteoblasts were grown to confluence and detached with 1 mM EDTA in PBS. The cells were centrifuged at 500 times g for 10 min at 4 °C, and the cell pellet was resuspended in 10 mM sodium phosphate, pH 7.4, 1 mM EDTA, 0.25 M sucrose, 0.15 M NaCl, 1 mM PMSF, 2 mM iodoacetic acid. The cells were sonicated for 5 s on ice, and the cell lysates were centrifuged at 12,000 times g for 30 min at 4 °C. The supernatant was then centrifuged at 40,000 times g for 1 h at 4 °C, and the pellet was suspended in 50 mM HEPES, pH 7.4, 0.15 M NaCl, 1 mM PMSF, 2 mM magnesium sulfate and centrifuged at 40,000 times g for 1 h at 4 °C. Membrane proteins were extracted in 50 mM HEPES, pH 7.4, 1% Triton X-100, 0.15 mM NaCl, 1 mM PMSF, 2 mM magnesium sulfate overnight at 4 °C with constant agitation and recovered in the supernatant after centrifugation at 12,000 times g for 20 min at 4 °C(17) . The membrane preparation was then applied overnight to an IGFBP-5 affinity column (1 mg of human recombinant intact IGFBP-5 bound to Affi-Gel-15) equilibrated in 50 mM HEPES, pH 7.4, 1% Triton X-100. The column was washed with 200 ml of equilibration buffer at 2 ml/min, followed by 50 ml of 50 mM HEPES, pH 7.4, 0.2 M NaCl, 0.5% Triton X-100, 1 mM PMSF at 1 ml/min. Bound protein was eluted with 4 ml of 10 mM sodium acetate, pH 5.0, 1.5 M NaCl, 0.2% Triton X-100, 1 mM PMSF at 2.5 ml/min and concentrated with a Centricon-30 filtration device in 10 mM sodium acetate, pH 5.0, 0.5 M NaCl, 0.04% Triton X-100, 1 mM PMSF. The eluted protein was separated on a 5% SDS-polyacrylamide gel under reducing conditions and stained with silver.

Down-regulation of Intact I-IGFBP-5 and I-IGFBP-5 Binding to Mouse Osteoblasts

Confluent cultures of mouse osteoblasts were incubated in serum-free DMEM containing 0.1% BSA without or with 3 µg/ml of either intact IGFBP-5 or IGFBP-5 for 1, 2, or 18 h at 37 °C. Surface-bound IGFBP-5 was then removed by rinsing the cells twice with cold 2 M NaCl in 20 mM sodium acetate, pH 4.0, twice with cold 2 M NaCl in 20 mM HEPES, pH 7.5, and twice with cold PBS to remove excess salt. The cells were then incubated in binding buffer (20 mM HEPES, 0.1% BSA, pH 7.0) containing either intact I-IGFBP-5 or I-IGFBP-5 for 2 h at 4 °C. The cells were then rinsed three times with PBS and solubilized, and the radioactivity was then quantitated.

Internalization of I-IGFBP-5

Confluent cultures of mouse osteoblasts on 48-well plates were incubated in serum-free DMEM containing 0.1% BSA for 18 h and rinsed three times with cold serum-free medium. Intact I-IGFBP-5 or I-IGFBP-5 was then added to the cells in DMEM containing 0.1% BSA for 2 h at 37 °C. After incubation, the cells were rinsed twice with PBS, twice with 2 M NaCl in 20 mM HEPES, pH 7.5, and twice with 2 M NaCl in 20 mM sodium acetate, pH 4.0, to remove cell-associated radioactivity(10) . The cells were then extracted with 0.5% Triton X-100 in 0.1 M sodium phosphate, pH 8.1, to release internalized radioactivity. Nonspecific radioactivity was determined in parallel cultures incubated with I-IGFBP-5-containing medium. Nonspecific radioactivity in the Triton extracts was subtracted from the experimental values.

RESULTS

Competition binding assays reveal that both intact I-IGFBP-5 and I-IGFBP-5 specifically bind to osteoblast monolayers (Fig. 1). Maximum binding averaged 24 fmol/10^6 cells for intact I-IGFBP-5 and 4.5 fmol/10^6 cells for I-IGFBP-5; half-maximal displacements occurred with 120 and 30 nM of unlabeled binding protein for intact and carboxyl-truncated IGFBP-5 binding, respectively. Scatchard analysis revealed a best fit for one-site binding for both forms of IGFBP-5; the K(d) for intact IGFBP-5 binding was 28 nM (Fig. 1A), and the K(d) for IGFBP-5 binding was 6 nM (Fig. 1B). It is assumed that IGFBP-5 binding is specific for osteoblast-like cells since these studies were performed with primary cultures that were used within 7 days of plating to minimize growth of potential contaminating cell types, such as fibroblasts.

Because IGFBP-5 is a known heparin binding protein, heparin-like molecules on the cell surface or within the ECM could modify osteoblast binding of IGFBP-5 similar to their action with other heparin binding proteins, such as fibroblast growth factor(10) . To evaluate this possibility, heparin was used as a competitor for IGFBP-5 binding to osteoblast monolayers (Fig. 2). Heparin at low concentrations inhibited the binding of intact I-IGFBP-5 with half-maximal inhibition occurring at 0.3 µg/ml (Fig. 2A). In contrast, osteoblast binding of I-IGFBP-5 was not inhibited until heparin concentrations exceeded 30 µg/ml. Notably, heparin concentrations in the range of 0.3-3 µg/ml enhanced IGFBP-5 binding up to a maximum of 26% above control values (Fig. 2B). Competitive binding studies of intact I-IGFBP-5 with heparan sulfate, dermatan sulfate, chondroitin sulfate-A, and mouse-derived soluble heparan sulfate proteoglycan (HSP) were compared to heparin's inhibition of IGFBP-5 binding to determine whether differences in sulfation of the GAG moieties affected the interaction of GAG side-chains with intact IGFBP-5 (Fig. 3). The relative inhibition of I-IGFBP-5 binding correlated with the degree of GAG sulfation: heparin > heparan sulfate > dermatan sulfate. Neither chondroitin sulfate-A nor HSP inhibited I-IGFBP-5 binding at concentrations of 0.1-3 µg/ml. While the results with the soluble HSP suggest that this particular proteoglycan and its protein backbone does not compete for IGFBP-5 binding, it is possible that the protein structure of other proteoglycans may be competitive.

Figure 2: Competitive binding of heparin with intact I-IGFBP-5 and I-IGFBP-5 to mouse osteoblasts. Osteoblast monolayers maintained in serum-free medium were exposed to intact I-IGFBP-5 (A) or I-IGFBP-5 (B) with increasing concentrations of heparin for 2 h at 4 °C. Cell-associated radioactivity was determined as described in Fig. 1.


To determine whether the contrasting effects of heparin on intact and carboxyl-truncated IGFBP-5 binding were related to possible differences in heparin binding affinity, both forms of IGFBP-5 were immobilized onto nitrocellulose and tested for their ability to bind [^3H]heparin(13) . As shown in Fig. 4A, intact IGFBP-5 bound 4-5-fold more [^3H]heparin than IGFBP-5. The finding that intact IGFBP-5 has a higher affinity for heparin by this method was verified by studies with heparin-agarose chromatography. Intact I-IGFBP-5 eluted from the heparin-agarose column with 0.8 M NaCl while I-IGFBP-5 eluted from the column with 0.24 M NaCl (data not shown). This difference in heparin affinity may be explained on the basis of intact IGFBP-5 having a more effective heparin binding domain compared to the carboxyl-truncated form. For example, a putative heparin binding domain within intact IGFBP-5 is predicted to exist within 201-218 amino acid residues, in which a cluster of basic amino acids with the known heparin binding motif, XBBBXXBX, is found, where B is a basic amino acid(5) . In contrast, IGFBP-5 does not contain a known heparin binding motif, although there is a cluster of basic amino acids in the 133-143 region. To evaluate the relative heparin affinities of these basic regions of IGFBP-5, the synthetic peptides IGFBP-5 and IGFBP-5 were immobilized onto nitrocellulose, and [^3H]heparin binding was quantitated as described by Baird et al.(13) (Fig. 4B). Under these conditions IGFBP-5 consistently bound more heparin than IGFBP-5, although the latter consistently displayed low level heparin binding (background < 200 cpm). While it is unknown whether there were differences in peptide affinity for nitrocellulose, the findings are consistent with the notion that the 201-218 region of IGFBP-5 contains a bona fide heparin binding domain, and its presence may help explain the higher heparin affinity of the intact form of IGFBP-5.

Figure 4: [^3H]Heparin binding to IGFBP-5 and IGFBP-5 peptides. [^3H]Heparin binding to intact IGFBP-5 and IGFBP-5 (A) and IGFBP-5 and IGFBP-5 (B) was determined following the adsorption of the binding proteins to nitrocellulose filters as described under ``Experimental Procedures.'' After washing the filters to remove unbound [^3H]heparin, specific binding of [^3H]heparin onto the filters was determined by subtracting background from total cpm.


While these data suggest that the major differences in heparin inhibition of intact and carboxyl-truncated I-IGFBP-5 binding to osteoblasts are related to their different heparin binding affinities, they do not rule out the possibility that cell surface GAGs are also involved in binding IGFBP-5. To evaluate whether GAGs mediate the binding of IGFBP-5 to the osteoblast surface, confluent monolayers were pretreated with heparinase to remove GAG moieties from osteoblast-surface heparan sulfate proteoglycans before performing binding studies with I-IGFBP-5. As shown in Table 1, pretreatment with heparinase did not alter osteoblast binding of either intact or carboxyl-truncated I-IGFBP-5, suggesting that cell surface GAGs are not the mediators of this interaction. To verify that the heparinase treatment removed cell surface GAGs, heparinase treatments were performed on cells prelabeled with [S]sulfate. Approximately 20% (22 ± 3%) of the cell-associated S-labeled material was released into the medium. An additional 52 ± 4% of the cell-associated S-labeled material was released by trypsin digestion, presumably from chondroitin sulfate and/or dermatan sulfate. In addition, 26 ± 4% of the cell-associated S-labeled material was resistant to both heparinase and trypsin digestion. Thus, even though heparan sulfate proteoglycans may account for less than one-fourth of the total cell surface GAG content, this should be enough to alter binding of IGFBP-5 if GAGs were the principal binding site.

To examine whether sulfation of cell surface proteoglycans are important in mediating IGFBP-5 binding, I-IGFBP-5 binding studies were performed in osteoblasts that were grown in the presence of sodium chlorate to prevent the sulfation of GAG side-chains(10, 28) . As shown in Table 2, chlorate treatment resulted in a 70% reduction in sulfate incorporation, without altering the cellular binding of intact I-IGFBP-5. Similar results were obtained with I-IGFBP-5 binding (data not shown). While these data strongly suggest that proteoglycans are not the primary binding site, they do not completely exclude this possibility owing to the variable resistance of heparan sulfate to chlorate treatment.

To further evaluate the mechanism of IGFBP-5 binding to osteoblasts, affinity labeling of both forms of I-IGFBP-5 to osteoblast monolayers was performed using the bifunctional cross-linking agent DSS. As shown in Fig. 5, both intact and carboxyl-truncated I-IGFBP-5 bound to a 420-kDa Triton-extractable membrane protein (450-kDa band minus 30-kDa IGFBP-5). Affinity labeling was greater for intact than for carboxyl-truncated IGFBP-5, and neither form cross-linked to ECM proteins deposited by osteoblasts (lane 3). When the cells were pretreated with either heparinase or chondroitinase, no decrease in affinity labeling of the 420-kDa protein was evident when compared to untreated cells (Fig. 6). While these results suggest that both forms of IGFBP-5 may bind to the same membrane protein, it is possible that the molecular weight assignments for each are not truly identical and that more than one membrane protein may be present for each form of IGFBP-5.

Figure 5: Affinity labeling of intact I-IGFBP-5 and I-IGFBP-5 to mouse osteoblast monolayers and ECM. Confluent monolayer cultures of mouse osteoblasts maintained in serum-free medium were exposed to binding buffer containing intact I-IGFBP-5 or I-IGFBP-5 in the absence or presence of unlabeled IGFBP-5 for 2 h at 4 °C. After rinsing with binding buffer, the cells were incubated in BSA-free binding buffer containing DSS (final concentration, 0.135 mM) for 15 min at 4 °C. The cells were rinsed, detached, and solubilized with buffer containing 1% Triton X-100 and protease inhibitors as described under ``Experimental Procedures.'' Following centrifugation, the supernatant was mixed with electrophoresis sample buffer containing 2-mercaptoethanol and separated on a 4-10% SDS-polyacrylamide gel. Affinity labeling of intact I-IGFBP-5 to osteoblast-derived ECM (lane 3), prepared as described under ``Experimental Procedures,'' was performed under the same conditions as for the monolayers. Intact I-IGFBP-5 (lanes 1-3) or I-IGFBP-5 (lanes 4 and 5) cross-linked to osteoblast monolayers without (lanes 1 and 4) or with (lanes 2 and 5) unlabeled intact IGFBP-5 (lane 2) or IGFBP-5 (lane 5). Molecular mass standards in kDa are on the left.


Figure 6: Effect of heparinase and chondroitinase on affinity labeling of intact I-IGFBP-5 to osteoblast cells. Confluent monolayers of mouse osteoblasts maintained in serum-free medium containing 0.1% BSA were pretreated with heparinase (10 units/ml) or chondroitinase (1 units/ml) for 1 h at 37 °C. The cells were rinsed and then incubated in binding buffer containing intact I-IGFBP-5 for 2 h at 4 °C before being cross-linked with DSS and analyzed by SDS-polyacrylamide gel electrophoresis as described in Fig. 5.


To determine whether a similar sized membrane protein could be isolated from mouse osteoblasts, Triton X-100 extracts of mouse osteoblast membranes were applied to an IGFBP-5 affinity column. Following extensive washing of the column, protein eluting from the column was identified as a single 420-kDa band on silver stain of a reduced 5% SDS-polyacrylamide gel (Fig. 7). Because of its position at the top of the gel, the molecular weight assignment may be an underestimate.

Figure 7: IGFBP-5 affinity purification of a 420-kDa membrane binding protein from mouse osteoblasts. Triton X-100 extracts of osteoblast membranes were applied to an IGFBP-5 affinity column as described under ``Experimental Procedures.'' After extensive washing of the column, the membrane protein was eluted with 10 mM sodium acetate, pH 5.0, 1.5 M NaCl, 0.2% Triton X-100, 1 mM PMSF, concentrated with a Centricon-30 filter, and separated through a 5% SDS-polyacrylamide gel and silver stained. Numbers on the left represent the molecular mass markers in kDa.


To assess whether the osteoblast binding site for IGFBP-5 was capable of being down-regulated, cells were incubated with IGFBP-5 for various times, and cell-associated IGFBP-5 was removed with 2 M NaCl and 20 mM sodium acetate washes before quantifying surface binding of I-IGFBP-5. Maximal specific binding was 22 fmol/10^6 cells, indicating that the washing conditions did not alter normal binding. As shown in Fig. 8, binding of intact I-IGFBP-5 and I-IGFBP-5 to osteoblast monolayers was reduced to 40 and 35% of maximal binding after 1 and 2 h of preincubation with IGFBP-5, respectively. The inhibitory effect was also seen with a more prolonged exposure time (18 h). This is felt to represent down-regulation of the membrane binding site rather than competition with residual IGFBP-5, since the cells had been extensively washed with 2 M NaCl and 20 mM sodium acetate to remove cell surface IGFBP-5 before performing the I-IGFBP-5 binding studies. Additional cultures labeled with I-IGFBP-5 confirmed that the NaCl-acetate washes remove 89% of I-IGFBP-5 bound at 4 °C (data not shown). Because proteoglycans have not been shown to down-regulate in response to a ligand, these data further support the notion that the membrane protein is not a proteoglycan.

Figure 8: Down-regulation of intact I-IGFBP-5 and I-IGFBP-5 binding to mouse osteoblasts. Confluent cultures of mouse osteoblasts were incubated in serum-free DMEM containing 0.1% BSA without or with 3 µg/ml of either intact IGFBP-5 or IGFBP-5 for 1, 2, or 18 h at 37 °C. To remove surface-bound IGFBP-5, the cells were rinsed twice with cold 2 M NaCl in 20 mM sodium acetate, pH 4.0, twice with 2 M NaCl in 20 mM HEPES, pH 7.5, and twice with cold PBS to remove excess salt. The monolayers were labeled and quantitated for surface binding of I-IGFBP-5 and I-IGFBP-5 as described in Fig. 1.


Because earlier experiments demonstrated that low concentrations of heparin enhanced the binding of I-IGFBP-5, internalization experiments were performed to determine whether the cellular uptake of IGFBP-5 was also increased by heparin. In these experiments, cell-associated radioactivity was removed with 2 M NaCl and 20 mM sodium acetate. As shown in Fig. 9, internalization of I-IGFBP-5 was stimulated by 26% with low heparin concentrations (0.3-1.0 µg/ml) and enhanced by 80 and 120% with heparin concentrations of 3 and 10 µg/ml, respectively. In contrast, internalization of intact I-IGFBP-5 was decreased at all heparin concentrations in a dose-dependent manner, consistent with the inhibitory effect of heparin on the cellular binding of intact IGFBP-5 (Fig. 2).

Figure 9: Effect of heparin on the internalization of I-IGFBP-5 and intact I-IGFBP-5. Confluent osteoblast monolayers maintained in serum-free medium were incubated with I-IGFBP5 (A) or intact I-IGFBP-5 (B) without or with increasing concentrations of heparin for 2 h at 37 °C. The cells were sequentially washed with PBS, 2 M NaCl in 20 mM HEPES, pH 7.5, and 2 M NaCl in 20 mM sodium acetate, pH 4.0, to remove cell-associated I-IGFBP-5. The cells were then extracted with 0.5% Triton X-100 in 0.1 M sodium phosphate, pH 8.1, to release internalized radioactivity. Values represent the mean of triplicate cultures from a representative experiment.


DISCUSSION

These data demonstrate that IGFBP-5 binds to a membrane protein isolated from primary cultures of mouse osteoblast-like cells. Although the exact cell types were not identified, it is presumed that IGFBP-5 binding was specific for osteogenic cells since they were primary cultures enriched for cells of the osteoblast lineage. While the identity of the cell surface protein was not revealed by these studies, its apparent absence from osteoblast-drived ECM suggested that the membrane protein was not a secreted proteoglycan. This notion is further supported by the following findings: solubilized mouse heparan sulfate proteoglycan does not compete for IGFBP-5 binding; heparinase treatment, to remove cell surface GAGs, and chlorate treatment, to prevent sulfation of proteoglycans, do not alter IGFBP-5 binding; IGFBP-5 binding is down-regulated by prior exposure to the ligand; the protein's appearance on affinity cross-linking gels and silver stain is not typical of the diffuse gel mobility, which characterizes large molecular weight proteoglycans. Finally, the membrane protein may be involved in receptor-mediated signal transduction, since earlier studies demonstrated that IGFBP-5 directly stimulates mitogenesis in mouse osteoblasts(3) .

Even though osteoblast-surface GAGs do not mediate binding of IGFBP-5, GAG moieties are important in mediating the effect of heparin on osteoblast binding and internalization of IGFBP-5. Low concentrations of soluble heparin inhibit the cellular binding of intact but not IGFBP-5 apparently because intact IGFBP-5 binds more heparin than IGFBP-5. This may be due, in part, to the heparin binding domain within amino acids 201-218 having a higher affinity for heparin than the 133-143 region. Conformational differences may also play a role if, for example, carboxyl truncation results in a tertiary structure that binds heparin with lower affinity. Since heparin-like molecules are important in many biochemical and cellular functions(18) , it is noteworthy that specific GAG-induced alterations in the function of IGFBP-5 have recently been identified(7, 8) . One particularly relevant finding is that GAG molecules having O-sulfated iduronic regions prevent proteolysis of intact IGFBP-5, the effect of which directly correlates with the extent of GAG sulfation: heparin > heparan sulfate > dermatan sulfate(8) . In the present study, the degree of GAG sulfation was similarly correlated with the inhibition of intact I-IGFBP-5 binding to osteoblasts where heparin was the most inhibitory and dermatan sulfate the least. Chondroitin sulfate-A, which lacks O-sulfated iduronic residues, did not affect IGFBP-5 binding in this study and failed to inhibit IGFBP-5 degradation in the study by Arai et al.(8) . Thus, one potential effect of selected sulfated GAGs that are localized within osteoblast-derived ECM would be to sequester intact IGFBP-5 and allow for cellular binding of IGFBP-5. This may be one explanation for the preferential localization of intact but not truncated IGFBP-5 to fibroblast-derived ECM(19) .

The mechanism for the enhanced internalization of IGFBP-5 by heparin is unknown. While heparin increased osteoblast binding of IGFBP-5, this did not exceed a 26% stimulation (Fig. 2), which corresponds to less than 1 fmol/10^6 cells of additional IGFBP-5 internalized. Thus, the enhanced internalization (an additional 80-120%) induced by higher heparin concentrations (3 and 10 µg/ml) must result from a mechanism other than enhanced binding. For example, heparin may stimulate the internalization of the membrane proteinbulletIGFBP-5 complex by making the complex more resistant to proteolysis. Irrespective of the mechanism, heparin causes a shift in the ratio of IGFBP-5 to intact IGFBP-5 being internalized, resulting in the preferential intracellular accumulation of the carboxyl-truncated fragment. This change may induce specific intracellular events that might otherwise be absent if GAG-associated molecules were not present near the cell surface.

Proteolytic cleavage of IGFBP-5 may be important in determining the extent of IGFBP-5 binding to osteoblasts, especially as it relates to the formation of carboxyl-truncated fragments. Medium conditioned by osteoblast-like cells contains carboxyl-truncated forms of IGFBP-5(2) , and cultured osteoblasts secrete proteases that cleave IGFBPs(20, 21) , including IGFBP-5. While some of the IGFBP-5 proteases may not be unique to osteoblasts(22, 23, 24) , their production of truncated forms of IGFBP-5 having reduced affinity for ECM (19) suggests a mechanism for enhanced cellular binding of IGFBP-5 fragments. Regulation of protease activity would be an additional mechanism for control of IGFBP-5 binding to osteoblasts, either through direct stimulation of enzyme activity (25) or through ECM-associated inhibition of protease action (8) . The ECM constituency would then be important in the cell-specific response to degradation of intact IGFBP-5, depending, in part, on the type and amount of GAG that is present in the pericellular environment.

Binding of IGFBPs to cell surfaces (26, 27, 28, 29, 30) and modulation of IGFBP binding by heparin (28) have been previously reported. While the mechanism for binding was not revealed in those studies, Oh et al. have recently demonstrated that IGFBP-3 binds to specific membrane proteins (20, 26, and 50 kDa) located on human Hs578T breast cancer cells(30) , which may be responsible for mediating the growth inhibitory effect of IGFBP-3(29) . More recently, Booth et al.(28) have shown that intact IGFBP-5 binds to microvascular bovine endothelial cells and that its cellular binding is inhibited by sulfated GAGs but not by heparinase or chlorate treatment of the cells, similar to the results of the present study with mouse osteoblasts. Taken together, these data indicate that IGFBP-3 and IGFBP-5 have specific binding sites on cell surfaces that are not proteoglycans and suggest that their cellular binding may be cell-type dependent. Identifying these membrane binding proteins will be important in determining the mechanisms of the independent actions of IGFBPs in directly stimulating (2) and inhibiting (29) cell growth and differentiated cell functions.

It is likely that IGFBP-5 has a role in normal bone cell physiology through its osteoblast stimulatory effect. IGFBP-5 has been extracted from mineralized bone matrix where it appears to sequester IGF-I and -II for potential use in bone formation(31) . IGFBP-5 production in cultured osteoblasts is stimulated by parathyroid hormone(32, 33) , which may help explain its anabolic action on bone in osteoporosis (34, 35, 36) . The bone loss induced by glucocorticoids (37) may result, in part, from the inhibition of osteoblast-derived IGFBP-5 (38) to cause decreased bone formation. While intact (31) and carboxyl-truncated IGFBP-5 (2) have been shown to enhance IGF-stimulated osteoblast mitogenesis in vitro, their effect on differentiated osteoblast functions, such as collagen synthesis, has not been reported. Nevertheless, endogenous IGFBP-5 may have a role in the anabolic action of IGF-I in experimental osteoporosis(39) , particularly if IGF-I stimulates IGFBP-5 production as well in vivo as it does in vitro(40) . Whether bioactive truncated forms of IGFBP-5 directly promote bone formation in vivo remains to be determined.

FOOTNOTES

*
This work was supported by funds provided by the Research Service of the Department of Veterans Affairs. 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: VA Medical Center (111A), 1660 South Columbian Way, Seattle, WA 98108. Tel.: 206-764-2002; Fax: 206-764-2153.

(^1)
The abbreviations used are: IGFBP, insulin-like growth factor binding protein; IGF, insulin-like growth factor; GAG, glycosaminoglycan; ECM, extracellular matrix; HPLC, high performance liquid chromatography; DSS, disuccinimidyl suberate; DMEM, Dulbecco's modified Eagle's medium; FCS, fetal calf serum; BSA, bovine serum albumin; PBS, phosphate-buffered saline; PMSF, phenylmethylsulfonyl fluoride; HSP, heparan sulfate proteoglycan.

ACKNOWLEDGEMENTS

The technical assistance of Dawn Moran is gratefully acknowledged.

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