THEJOURNAL OF BIOLOGICAL CHEMISTRY Vol. 269, No. 32, Issue of August 12, pp. 20388-20393, 1994 0 1994 by The American Societyfor Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Heparin, Heparan Sulfate, and Dermatan Sulfate Regulate Formation of the Insulin-like Growth Factor-I and Insulin-like Growth Factor-binding Protein Complexes* (Received for publication, April 14, 1994, and in revised form, June 1, 1994) Takami Arai, Alex Parker, Walker Busby, Jr., and David R. Clemmons$ From the Department of Medicine, University of North Carolina, School of Medicine, Chapel Hill, North Carolina 27599 The mechanismsby which insulin-like growth factor-I binding toits receptor, thereby inhibiting IGF-I mediated cel- (IGF-I)is released from insulin-like growth factor bind- lular actions (4, 5). On the other hand,the binding of IGF-I to ing proteins (IGFBPs) and then binds to its receptor cell surface or extracellular matrix (ECM) associated IGFBPs have not been defined. This study was designed to de- has been associatedwith potentiation of IGF-I’s cellular actions termine the role of glycosaminoglycans in altering the (3, 5-7). In both situations it remains uncertain how IGF-I is formation of the IGF-I*IGFBP complexes.Heparin inhib- released from IGFBPs in extracellular fluidsor on cell surfaces ited formation of the IGF-1-IGFBP-5 complexand also in order to be able to bind to its receptor, an event that is separated preformed IGF-IsIGFBP-5 complexes.Heparin usually required for optimalbiologic responses. also inhibited formation of the IGF-I-IGFBP-3 complex; Recentlyglycosaminoglycans (GAGS) andproteoglycans however, it did not inhibit formation of complexes be- have been shown to modulate growth factor-stimulated cellular tween IGF-Iand IGFBP-l, -2, or -4. Heparin exposure was associated with a 17-fold decrease in the affinity of IG- actions (8, 9). Basic fibroblast growth factor (b-FGF) binds to FBP-5 for IGF-I. A synthetic peptide that contains resi- bovinecapillary endothelial cell surface heparin-like mol- ecules, which act as a reservoir of b-FGF and modulate the dues from Ar8’l t0Ar8~’of IGFBP-5, and a heparin bind- ing domain prevented the inhibitory effects of heparin metabolism of b-FGF by cells (10). The biological half-life of on formation of the IGF-I*IGFBP-5 complex.It did not acidic FGFis prolonged by heparin, thereby enhancing itscel- directly compete with IGF-Ifor binding to IGFBP-5, sug- lular actions(11). Transforming growth factorp stimulates syn- gesting that heparin binding to this region of IGFBP-5 thesis of decorin (9) and when transforming growth factorp is resulted in a conformational change in IGFBP-5 which bound to decorin itsbiological activity is decreased (12). lowered its affinity for IGF-I. Other glycosaminoglycans Heparin binding sites in many proteins have been charac- that contained 0-linked sulfates in the 2 or 3 carbon po- terized and shown to contain multiple basic amino acids.Two sitions of iduonic acid, e.g. heparan sulfate and derma- motifs X-B-B-X-B-X and X-B-B-B-X-X-B-X (B: basic amino acid, tan sulfate, also inhibited the IGF-1-IGFBP-5 complex X. nonbasic amino acid) have been proposed as consensus se- formation, whereas those that did not, such as keratan quences for heparin binding.All of the forms of IGFBPs except sulfate or hyaluronic acid, had minimal effects. Anionic for IGFBP-4 haveat lease one of these putative heparin bind- polysaccharides that contained 0-sulfate groups in the2 ing sequences (13,141, suggestingthat they havethe potential or 3 positions, such as dextran sulfate, pentosan polysul- to bind to heparin. Previousstudies have shown that heparin fate, and fucoidan, alsohad inhibitory activity. Thefind- can interfere withthe formation of the IGF-I.IGFBP-3 complex ings suggest a role for these compounds in inhibiting (15, 16); therefore, we wondered whether heparin might affect IGF-1-IGFBPinteractions, thus making IGF-I available the binding of IGF-I to other formsof IGFBPs. We report here to bind to its receptor. that heparin inhibited the IGF-I.IGFBP-5 complex formation but had no effect on binding to IGFBP-1, -2, or -4. We have determined some of the structural requirements of heparin Insulin-like growth factor I (IGF-I)’ cellular actions have that are necessary for its inhibitory activity and have defineda been shown to be exerted through the binding of IGF-I to its region of IGFBP-5 that mediates heparin-induced inhibitionof receptor (1); however, most of IGF-I in extracellular fluids is IGF-I binding. bound to insulin-like growth factor binding proteins (IGFBPs). The IGFBPs have been shown tostimulate or to inhibit IGF- EXPERIMENTAL PROCEDURES I-mediated cellular actions (2-4). Binding of IGF-I to high af- Materials-Recombinant human IGFBP-3, IGFBP-4, and IGFBP-5 finity formsof IGFBPs in conditioned medium preventsit from were synthesized in transfected Chinese hamster ovary cells and puri- fied as described previously(17). Recombinant human IGFBP-1(5) and bovine IGFBP-2 (18) were prepared as published previously. Recombi- * This work was supported by Grants HL-26309 and AGO2331 from nant IGF-I was obtained from Bachem, Inc. (Torrance, CA). 1251-IGF-I the National Institutes of Health. The costs of publication of this article was a gift from Dr. Louis E. Underwood (University of North Carolina, were defrayed in part by the payment of page charges. This article must Chapel Hill). 1251-IGFBP-5 wasprepared by a modified chloramine-T therefore be hereby marked ‘‘uduertisernent” in accordance with 18 method (19). Three peptides that contain sequences of IGFBP-5 were U.S.C. Section 1734 solely to indicate this fact. synthesized and purified as described (20). They were designated pep- $ To whom correspondence should be addressed: Dept. of Medicine, CB 7170,University of North Carolina, School of Medicine, Chapel Hill, tide A (residues RzZ1-K-G-F-Y-K-R-K-Q-C-K-P-S-R-G-R-K-Rz3’),peptide NC 27599-7170. Tel.: 919-966-4735; Tel: 919-966-6025. B (residues A151-V-K-K-D-R-R-K-K-L-T’61),and peptide D (residues 134P- The abbreviations used are: IGF, insulin like-growth factor; K-I-F-R-P-K-H-T-R-I-S-E-L-K-A-E160),respectively. Heparin (187 USP IGFBPs, insulin-like growth factor binding proteins; GAG, glycosami- unitdmg) was purchased from Sigma. Heparin, heparan sulfate, chon- noglycan; FGF, fibroblast growth factor; DSS, disuccinimidyl suberate; droitin sulfate A, chondroitin sulfate C, dermatan sulfate, hyaluronic EMEM, Eagle’s minimumessential medium; PAGE, polyacrylamide gel acid, and keratan sulfate were purchased from Seikagaku Kogyo Co. electrophoresis; ECM, extracellular matrix; NDSNAc, N-desulfated N- (Tokyo, Japan). Chemicallymodified heparins, including completely acetylated heparin; CDSNAc, completely desulfated N-acetylated hep- desulfated, N-acetylated heparin (CDSNAc-heparin), completelydesul- arin; CDSNS, completely desulfated N-resulfated heparin. fated, N-resulfated heparin (CDSNS-heparin), and N-desulfated, N- 20388 This is an Open Access article under the CC BY license. Glycosaminoglycans Inhibit IGF-IeIGFBP Complex Formation 20389 acetylated heparin (NDSNAc-heparin) were purchased from Seikagaku KogyoCo. Dextran sulfate (My:<10,000), pentosan polysulfate, and Mrx10-3 fucoidan were purchased from Sigma. Dithiothreitol was purchased - 60 from Sigma. Disuccinimidyl suberate (DSS) was purchased from Pierce - 46 Chemical Co. Dimethyl sulfoxide was purchased from Mallinckrodt Chemical Co. (Paris, KY). Eagle's minimum essential medium (EMEM) - 30 was purchased from Hazelton (Denver, PA). - 21 Affinity Cross-linking Studies-Cross-linking was performed accord- ing to amodification methods described previously (21,22). '2sII-IGF-Ior - 14.5 '2sI-IGFBP-5 (30,000 cpdtube) was added to 100 p1 of EMEM supple- mented with 20 mM HEPES, pH 7.3, and was incubated at room tem- perature with IGFBP-1, -2, -3, -4, or -5 (100 ng/ml) or unlabeled IGF-I B. (100ng/ml). Additional incubations were conducted using '2sI~IGF-I and - 68 heparan sulfate, chondroitin sulfate A or C, dermatan sulfate, keratan sulfate, or hyaluronic acid using 200 pg/ml. Chemically modified hepa- I - 46 I rins, dextran sulfate,pentosan polysulfate, and fucoidan were also - 30 tested at 100 or 200 pg/ml. The synthetic peptides containing IGFBP-5 sequences termed peptides A, B, or D were tested at concentrations - 21 between 0.27 and 27 p~.After a 1-h incubationthe samples were cross- linked by the addition of 10 pl of 5 mM DSS, which had been dissolved in dimethyl sulfoxide, to give a finalconcentration of 0.5 mM DSS for 20 FIG.1. A, inhibitory effects of heparin on forming the Iz5I-IGF- min. The reaction was stopped by adding 10 p1 of 0.5 M Tris-HC1, pH 7.4. 1.IGFBP-5 complex. '2sI-IGF-I (30,000 cpdtube) was added to 100 pl of Samples for SDS-PAGE were exposed to dithiothreitol in a final con- EMEM supplemented with 20 mM HEPES, pH 7.3. The mixture was centration of 0.1 M and Laemmli sample buffer (23). They were electro- incubated with 100 ng/ml of IGFBP-5 and the indicated concentrations phoresed through a 12.5% gel.The gels were fixed with 25% isopropanol of heparin at room temperature. After a 1-h incubation, samples were containing 10% acetic acid and 2.5% glycerol for 30 min then dried and cross-linked by adding DSS in a final concentration of 0.5 mM for 20min autoradiographed using Kodak X-Omat film. In some experiments the and then thereaction was stopped by addition of 10 pl of 0.5 M Tris-HC1, autoradiographic intensities of radiolabeled bands were determined by pH 7.4.