Pergamon Progress in Growth Factor Research, Vol. 6. Nos. 2--4, pp. 337-344, 1995

Published by Elsevier Science Ltd Printed in Great Britain.

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COMPARISON STUDIES OF IGFBP-5 BINDING TO OSTEOBLASTS AND OSTEOBLAST-DERIVED

EXTRACELLULAR MATRIX

Dennis L. Andress

Department of Medicine at the VA Medical Center and University of Washington, Seattle, WA 98108, U.S.A.

Recent studies have identified a specific membrane protein in osteoblast-like cells which binds intact and carboxy-truncated IGFBP-5 with high affinity. The purpose o f the present study was to evaluate the 1GFBP-5 binding properties o f osteoblast-derived extracellular matrix (ECM), with special interest in determining whether ECM proteo- glycans were necessary for IGFBP-5 binding. Neonatal mouse osteoblasts and the ECM of these cells both bound intact [125I]IGFBP-5 and [1:sI]IGFBP-51-169, though the ECM bound both forms with lower affinity when compared to their cellular binding. Treatment o f the ECM with heparinase or chondroitinase, to remove glycosaminoglycan (GAG) side-chains of proteoglycans, resulted in 20-34% enhanced binding of intact [J2sI]IGFBP-5 and a 92-100% enhancement o f [125I]IGFBP-51-169 binding. Similar enzymatic treatment o f osteoblast monolayers had no effect on the binding of either form of [125I]IGFBP-5. These results indicate that GAGs within ECM secreted by neonatal mouse osteoblasts do not mediate the binding of lGFBP-5. This study also shows that intact and carboxy-truncated IGFBP-5 preferentially bind to the osteoblast surface, but that removal o f GA Gs from osteoblast-derived ECM can increase IGFBP-5 localization to this pericellular space, particularly the carboxy-trun- cated form of IGFBP-5.

Keywords: Insulin-like growth factor binding protein-5 (IGFBP-5), osteoblast, extracellular matrix.

~ T R O D U C T I O N

IGFBP-5 binds to a variety of cultured cells, including osteoblasts [1--4] and endothelial cells [5]. Its position on the cell surface enhances IGF-I action and stim- ulates mitogenesis independent of IGF-I stimulation [1, 2, 4]. While the binding sites for IGFBP-5 have yet to be identified, characterization of its binding to endothelial cells has revealed that glycosaminoglycan (GAG)-containing molecules, such as heparin and heparan sulfate, can compete for IGFBP-5 binding to the cell

Correspondence to: VA Medical Center (111A), 1660 South Columbian Way, Seattle, WA 98108, U.S.A.

337

338 D. L. Andress

surface, suggesting that proteoglycans may somehow be involved [5]. However, heparinase treatment to remove GAG-side chains did not abolish IGFBP-5 binding in those cells [5], leaving open the likelihood of non-proteoglycan binding sites.

In addition to its cell-binding capability, IGFBP-5 binds to extracellular matrix (ECM) derived from endothelial cells [5] and fibroblasts [6]. IGFBP-5 also binds to such ECM proteins as Type II and Type IV collagen, fibronectin and laminin [6]. While IGFBP-5 binds modestly to endothelial ECM [5], its presence in fibroblast- derived ECM is associated with an enhanced growth response of these cells [6]. The additional findings that GAGs are capable of decreasing the binding of IGF-I to IGFBP-5 [7] and can inhibit the degradation of IGFBP-5 [8], adds further strength to the notion that ECM which contains GAGs likely modulates cellular behavior.

While recent studies in cultured osteoblasts have revealed that truncated [1, 2] as well as intact IGFBP-5 [4] bind to the cell surface, only recently have in-depth studies more accurately clarified the mechanism of IGFBP-5 binding to osteoblasts [3]. However, there are no data describing the binding of IGFBP-5 to the ECM derived from osteoblasts. In the current study, osteoblast binding of IGFBP-5 is compared to its binding to ECM derived from neonatal osteoblasts.

METHODS

Materials

Recombinant forms of intact IGFBP-5 and IGFBP-51-169 were expressed and purified as previously described [3]. [125I]IGFBP-5 and [12sI]IGFBP-51-169 were prepared using chloromine-T; specific activities ranged from 100 to 120 /.tCi//~g. Heparin, heparinase and chondroitinase ABC were purchased from Sigma Chemical Co. (St. Louis, MO).

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). The cells were grown for 1 week in 75-cm 2 flasks (Costar) containing DMEM (Gibco) and 10% fetal calf serum (FCS; Hyclone). Nearly confluent cells were released by trypsin-EDTA and plated in 48-well plates in DMEM containing 10% FCS for 24 48 h.

Extracellular Matrix Preparation

Confluent neonatal mouse osteoblasts were removed with trypsin-EDTA, plated onto 48-well plates and grown to confluence over 48 h in DMEM containing 10% FCS. ECM was prepared as described [3]. Briefly, cells were rinsed with ice-cold PBS and the cell membranes removed with 1% Triton X-100 in PBS. Adherent nuclei and cytoplasmic elements were then removed with 25 mM ammonium acetate, pH 9.0. The remaining ECM was rinsed with cold PBS and used immedi- ately for the binding studies.

IGFBP-5 Binding to Osteoblast ECM 339

[125I]IGFBP-5 Binding Studies

Confluent monolayers of neonatal mouse osteoblasts in 48-well plates were incu- bated in serum-free medium for 24 h. The cells were washed three times with PBS and then incubated in 100/11 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 [125I]IGFBP-5 or [125I]IGFBP-51-169 in the absence or presence of varying concentrations of either unlabeled intact IGFBP-5 or IGFBP-51-169. 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 back- ground counts from total c.p.m.

Heparinase and Chondroitinase Treatment

Confluent osteoblast monolayers were grown for 24 h in serum-free DMEM containing 0.1% BSA. Parallel plates were prepared with osteoblast-derived ECM as described above. The cells or ECM were rinsed three times with serum-free DMEM and incubated in binding buffer (DMEM, 25 mM HEPES, 0.1% BSA) without or with 5 U/ml heparinase or 1 U/ml chondroitinase ABC for 1 h at 37°C. At that time the cells or ECM were rinsed three times with cold binding buffer and the [125I]IGFBP-5 binding studies were then performed as described above.

RESULTS

Maximum binding of intact [125I]IGFBP-5 to osteoblast monolayers was 16.1% (of the total c.p.m.) and to osteoblast-derived ECM, 5.2% (Fig. 1A). [I25I]IGFBP- 51-169 binding to osteoblast monolayers was 4% and to ECM, 1.5%. In the compet- itive binding studies (Fig. 1B and 1C), ECM binding of intact [125I]IGFBP-5 and [125I]IGFBP-51-169 occurred with a lower affinity when compared to their binding to osteoblast surfaces. Intact [1251]IGFBP-5 bound to the ECM with a slightly higher affinity when compared to [125I]IGFBP-51-169.

To evaluate whether the presence of GAGs in osteoblast-derived ECM may have a role in IGFBP-5 binding, osteoblast monolayers and osteoblast-derived ECM were treated with either heparinase or chondroitinase before performing the binding studies with intact and carboxy-truncated IGFBP-5. As depicted in Fig. 2, enzy- matic treatment did not alter the binding of either form of [~25I]IGFBP-5 to the cell- surface, as previously shown [3]. In contrast, ECM treatment to remove GAGs resulted in enhanced binding of both forms of IGFBP-5. In particular, the binding of carboxy-truncated IGFBP-5 was enhanced 2-fold after treatment to remove GAGs from proteoglycans presumed to be present in the ECM. These results indi- cate that the binding sites for IGFBP-5 on osteoblast surfaces are different from those within the osteoblast-derived ECM. In both instances, proteoglycans do not appear to function as a binding site for IGFBP-5, since its binding was not reduced by treatment to remove GAG side-chains. In the case of the ECM, proteoglycans may actually interfere with IGFBP-5 binding.

Recent evaluation of the cellular binding site for IGFBP-5 established that neonatal osteoblasts contain a membrane protein that binds intact and carboxy-

340 D. L. Andress

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IGFBP-5 Binding to Osteoblast E C M 341

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FIGURE 1. Binding of intact [ I~I ] IGFBP-5 and [I~I]IGFBP-SJ-169 to mouse osteoblast monolayers and osteoblust-derived extracellular matrix (ECM). Maximal binding to monolayers and ECM (A) and compet- itive binding studies of intact [mlIIGFBP-5 (B) and [l~IIIGFBP-51-1o (C).

truncated IGFBP-5 with relatively high affinity [3]. As summarized in Table 1, the membrane protein is a large molecular-weight structure that is not a proteoglycan and is capable of being down-regulated by prior exposure to either intact or carboxy-truncated IGFBP-5. The latter finding again confirms that the protein is not a proteoglycan and raises the possibility that it functions as a receptor protein.

While GAGs did not appear to mediate IGFBP-5 binding to the osteoblast surface, they are capable of modulating IGFBP-5 binding and internalization [3]. As indicated in Table 2, exposure of osteoblast monolayers to heparin caused a marked reduction in the internalization of intact [~25I]IGFBP-5. In contrast, [125I]IGFBP-51-169 internalization was enhanced by 120%. While the latter finding was due, in part, to enhanced binding of the carboxy-truncated form at low heparin concentrations, this alone would account for only about 25% of the enhanced inter- nalization.

DISCUSSION

The current study shows that intact and carboxy-truncated IGFBP-5 bind to neonatal osteoblast surfaces and to osteoblast-derived ECM with different binding affinities. It does not appear that proteoglycans mediate the binding of IGFBP-5 at either location, since enzymatic treatment to remove GAG-side chains failed to

342 D. L. Andress

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FIGURE 2. Effect of heparinase and chondroitinase treatment on binding of intact [IZSl]IGFBP-5 and 11251]IGFBP-51-1~9 to osteoblast monolayers and osteoblast-derived ECM. (A) Heparinase treatment (B) ~ L .1 J . . . . : . . " . . . . . - - ~ A . - - ^ - - t * D f i f i ~ . . . . . . * . ~ n t . ~ l ~ . t . ~ l ~ . . I t . . . u * * P ~ ' N f l l ~ e I i f l t ~ P q t ~ r n n t r t d ~ l i | t l l t - g

IGFBP-5 Binding to Osteoblast ECM 343

Table 1. Characteristics of the osteoblast membrane protein that binds IGFBP-5*

(l) (2) (3) (4)

Binds and internalizes intact IGFBP-5 and IGFBP-51 169 Lacks GAG-side chains (not a proteoglycan) Absent from osteoblast derived ECM Rapidly down-regulated by exposure to IGFBP-5

*Taken from ref. 3.

Table 2. Effects of heparin on the binding and internalization of intact [I~I]IGFBP-51-169 in mouse osteoblast monolayers*

[I~I]IGFBP-5 and

Bound Internalized

[125I]IGFBP-5 [125I]IGFBP-5H69 [I25I]IGFBP-5 [125I]IGFBP-51-169 (fmol/106 cells)

Control heparin (lO lag/ml)

24.0 4.5 4.8 1.0 4.1 5.6 1.2 2.2

*Adapted from Ref. 3.

decrease the binding of either form. The ECM binding of IGFBP-5 is more in keeping with low-affinity binding that might be expected with abundant binding sites, possibly involving multiple binding proteins. For example, Jones et al. have shown that IGFBP-5 is capable of binding to several ECM proteins typically present in fibroblast-derived ECM [6]. Recent cross-linking studies identified the osteoblast binding site for IGFBP-5 as a membrane protein which was not present in osteoblast-derived ECM [3]. In addition, osteoblast binding of IGFBP-5 was down-regulated by prior exposure to the ligand, suggesting that the membrane protein may function more like a signaling receptor. If true, this would be consis- tent with the known independent effects of carboxy-truncated [3] and intact IGFBP-5 [4] in stimulating the proliferation of osteoblast-like cells.

Surprisingly, the binding of IGFBP-5 to ECM was enhanced after enzymatic treatment to remove GAGs from proteoglycans. This suggests that GAGs may interfere with IGFBP-5 binding to ECM proteins, perhaps through spatial and/or charge effects. Treatment to remove GAGs caused enhancement of IGFBP-51-169 binding to a greater extent than was seen with intact IGFBP-5 binding, suggesting that the N-terminal region of IGFBP-5 may be important in binding to specific proteins in ECM derived from these cells. While the identity of the ECM proteins expressed by the osteoblasts in this study were not identified, it is known that versi- can, a large chondroitin-sulfate proteoglycan, is a component of ECM in the early developmental stages of the osteoblast. Later, versican is replaced by the smaller and less sulfated chondroitin-sulfates, biglycan and decorin [9, 10]. Other ECM proteins that are likely to be present at the early stages (fetal and neonatal) of osteoblast development include fibronectin, osteonectin, Type I collagen and traces of Types III, V and XI collagen [10, 1 1]. Thus, it is possible that in this study the removal of GAGs from versican allowed for increased binding to fibronectin, osteonectin or other proteins in the ECM.

The presence of heparan sulfate-like GAGs in pericellular ECM may influence the cellular binding and internalization of IGFBP-5. As shown in Table 2, the addi-

344 D. L. Andress

tion of heparin during the binding studies resulted in an 83% reduction in the amount of intact [~25I]IGFBP-5 bound to osteoblasts which was similar to the 75% reduction in intact [125I]IGFBP-5 that was internalized. In contrast, heparin caused a 24% increase in [125I]IGFBP-51-169 binding and a 120% increase in its internaliza- tion. While heparin-like molecules may be less important in osteoblast-derived ECM, where chondroitin-sulfates are the predominant GAGs, cell types which secrete heparan-sulfates into the ECM may experience a significant change in the internal concentration of IGFBP-5 in such a way that carboxy-truncated IGFBP-5 becomes the dominant intracellular form.

In summary, IGFBP-5 binds to osteoblast-derived ECM with low affinity compared to its binding to the osteoblast surface. IGFBP-5 binding to the ECM was enhanced following enzymatic treatment to remove GAGs, suggesting that osteoblast-derived proteoglycans are more important in preventing IGFBP-5 binding in this extracellular location. Consequently, the loss of GAGs within osteoblast-derived ECM could result in extracellular sequestration of IGFBP-5, particularly the carboxy-truncated form.

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2. Andress DL, Loop SM, Zapf J, Kiefer MC. Carboxy-truncated insulin-like growth factor binding protein-5 stimulates mitogenesis in osteoblast-like cells. Biochem Biophys Res Commun. 1993; 195: 25-30.

3. Andress DL. Heparin modulates the binding of insulin-like growth factor (IGF-I) binding protein- 5 to a membrane protein in osteoblastic cells. J Biol Chem. 1995; 270: 28,289-28,296.

4. Mohan S, Nakao Y, Honda Y, Landale E, Leser U, Dony C, Lang K, Baylink DJ. Studies on the mechanisms by which insulin-like growth factor (IGF-I) binding protein-4 (IGFBP-4) and IGFBP- 5 modulate IGF actions in bone cells. J Biol Chem. 1995; 270: 20,424-20,431.

5. Booth BA, Boes M, Andress DL, Dake BL, Kiefer MC, Maack C, Linhardt R J, Bar K, Caldwell EEO, Weiler J, Bar RS. IGFBP-3 and IGFBP-5 association with endothelial cells: role of C-termi- nal heparin binding domain. Growth Regul. 1995; 5: 1-17.

6. Jones JI, Gockerman A, Busby Jr. WH, Camacho-Hubner C, Clemmons DR. Extracellular matrix contains insulin-like growth factor binding protein-5: potentiation of the effects of IGF-I. J Cell Biol. 1993; 121: 679-687.

7. Arai T, Parker A J, Busby Jr. WH, Clemmons DR. Heparin, heparan sulfate, and dermatan sulfate regulate formation of the insulin-like growth factor-I and insulin-like growth factor-binding protein complexes. J Biol Chem. 1994; 269: 20,388-20393.

8. Arai T, Arai A, Busby Jr. WH, Clemmons DR. Glycosaminoglycans inhibit degradation of insulin- like growth factor binding protein-5. Endocrinology 1994; 135: 2358-2363.

9. Fisher LW. The nature of proteoglycans of bone. In: Butler WT, ed. The chemistry and biology of mineralized tissues. Birmingham, AL: EBSCO Media; 1985: 188-196.

10. Gehron-Robey P. The biochemistry of bone. Endocrinol Metab Clin N Am. 1989; 18: 859-902. 11. Conn KM and Termine JD. Matrix protein profiles in calf bone development. Bone 1985; 6: 33-36.