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Originally published In Press as doi:10.1074/jbc.M005088200 on November 10, 2000

J. Biol. Chem., Vol. 276, Issue 8, 5533-5540, February 23, 2001
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Antiproliferative Effects of Insulin-like Growth Factor-binding Protein-3 in Mesenchymal Chondrogenic Cell Line RCJ3.1C5.18
RELATIONSHIP TO DIFFERENTIATION STAGE*

Anna SpagnoliDagger §, Vivian HwaDagger , William A. Horton, Gregory P. Lunstrum, Charles T. Roberts Jr.Dagger , Francesco Chiarelli||, Monica TorelloDagger , and Ron G. RosenfeldDagger

From the Dagger  Department of Pediatrics, Oregon Health Sciences University, Portland, Oregon 97201, the  Research Department, Shriners Hospital for Children, Portland, Oregon 97201, and the || Department of Pediatrics, University of Chieti, 66013 Chieti, Italy

Chondrogenesis results from a complex equilibrium between chondrocyte proliferation and differentiation. Insulin-like growth factors (IGFs) have a crucial role in chondrogenesis, but their mechanisms of action are not well defined. IGF-binding protein-3 (IGFBP-3) is the major carrier for circulating IGFs in postnatal life, and has been shown to have IGF-independent effects on proliferation of several cancer cell lines. In this study, we have evaluated the IGF-independent and -dependent effects of IGFBP-3 on chondrocyte proliferation and the relationship of these effects with chondrocyte differentiation stage. We used the RCJ3.1C5.18 nontransformed mesenchymal chondrogenic cell line, which, over 2 weeks of culture, progresses through the differentiation pathway exhibited by chondrocytes in the growth plate. We demonstrated that IGFBP-3 inhibited, in a dose-dependent manner (1-30 nM), the proliferation of chondroprogenitors and early differentiated chondrocytes, stimulated by des-(1-3)-IGF-I and longR3-IGF-I (IGF-I analogs with reduced affinity for IGFBP-3), and by insulin and IGF-I. In terminally differentiated chondrocytes, IGFBP-3 retained the ability to inhibit cell proliferation stimulated by IGF-I, but had no effect on cell growth stimulated by insulin, or des-(1-3)-IGF-I or longR3IGF-I. By monolayer affinity cross-linking, we demonstrated a specific IGFBP-3-associated cell-membrane protein of ~20 kDa. We determined that IGFBP-3 has an antiproliferative effect on chondrocytes and, that this effect is related to the differentiation process. In chondroprogenitors and early differentiated chondrocytes, antiproliferative effect of IGFBP-3 is mainly IGF-independent, whereas, following terminal differentiation this effect is IGF-dependent.

* This work was supported in part by a grant from the Medical Research Foundation of Oregon (to A. S.), by a grant from Pharmacia & Upjohn (to A. S.), and by National Institutes of Health Grant CA58110 (to R. G. R.). This work was presented in part at the 81st Annual Meeting of the Endocrine Society, June 12-15, 1999, San Diego, CA.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ To whom correspondence should be addressed: Dept. of Pediatrics (NRC-5), Oregon Health Sciences University, 3181 S.W. Sam Jackson Park Rd., Portland, OR 97201. Tel.: 503-494-0880; Fax: 503-494-0428; E-mail: spagnoli@ohsu.edu.

Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.
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