Differential effects of IGF-binding proteins, IGFBP-3 and IGFBP-5, on IGF-I action and binding to cell membranes of immortalized human chondrocytes T Matsumoto, T Tsurumoto, M B Goldring 1 and H Shindo Department of Orthopedic Surgery, School of Medicine, Nagasaki University, Nagasaki 852–8501, Japan 1 New England Baptist Bone and Joint Institute and Rheumatology Division, Beth Israel Deaconess Medical Center, Harvard Institutes of Medicine, Boston, Massachusetts 02115, USA (Requests for offprints should be addressed to T Matsumoto, Department of Orthopedic Surgery, School of Medicine, Nagasaki University, Nagasaki, Sakamoto 1–7–1, 852–8501, Japan) Abstract Insulin-like growth factor-I (IGF-I) is an important ana- bolic factor for cartilage tissue and its action is, in part, regulated by IGF-binding proteins (IGFBPs). The object of this study was to investigate the effects of IGFBPs on IGF-I action and on binding of IGF-I to cells using a reproducible immortalized human chondrocyte culture model. Treatment of the C-28/I2 cells with IGF-I or des(1–3)IGF-I in serum-free medium stimulated cell pro- liferation in a dose-dependent manner. However, the effect of des(1–3)IGF-I was more potent, thereby suggest- ing that endogenously produced IGFBPs inhibited IGF action. The stimulatory effect of IGF-I was inhibited significantly by addition of IGFBP-3 but enhanced slightly by IGFBP-5. However, neither IGFBP-3 nor IGFBP- 5 had an effect on basal cell growth. Binding of 125 I- labeled IGF-I to the cells was displaced by both IGFBP-3 and IGFBP-5, although higher concentrations of un- labeled IGFBP-5 were required to displace IGF-I to the same extent as IGFBP-3. Treatment of the cells with IGF-I increased the levels of IGFBP-5 protein measured by Western ligand blotting, and stimulated a correspond- ing increase in IGFBP-5 mRNA while increasing type II collagen mRNA. Our findings indicate that the balance between IGFBP-3 and IGFBP-5 influences IGF receptor binding and its action on chondrocyte proliferation, and may thereby modulate cartilage metabolism. Journal of Endocrinology (2000) 166, 29–37 Introduction The insulin-like growth factor (IGF)-I is a major growth and differentiation factor for cartilage tissue (McQuillan et al. 1986, Ohlsson et al. 1992, Seong et al. 1994) and its influence on chondrocyte functions is regulated, in part, by IGF-binding proteins (IGFBPs) (Lamson et al. 1991, Jones & Clemmons 1995). At present, six IGFBPs (IGFBPs-1 to -6) have been isolated and their cDNAs have been cloned (Shimasaki & Ling 1991). IGFBPs have been detected in culture media of cultured chondrocytes from various species (Froger-Gaillard et al. 1989, Olney et al. 1993, Sunic et al. 1995) and in intact bovine cartilage (Morales 1997). We reported previously that the predominant IGFBPs produced by rat articular chondrocytes were IGFBPs-2, -3, -4 and -5, and that the concentration of IGFBP-5 in conditioned medium was increased by IGF-I in a dose- dependent manner by a transcriptional mechanism via the type 1 IGF receptor (Matsumoto et al. 1996a). We also detected the presence of an IGFBP-5 protease (Matsumoto et al. 1996b), suggesting post-translational modification as an additional mechanism of IGFBP regu- lation. Although the physiological roles of these IGFBPs in cartilage metabolism remain to be clarified, various studies have begun to address how IGFBPs may exert differential effects on the systemic and local activities of IGF-I. In osteoarthritic cartilage, the normal anabolic function of IGF-I may be disrupted. Chondrocytes from animals with experimental arthritis and from patients with osteoarthritis are nonresponsive to IGF-I, although they express increased levels of IGF-I, IGF-I receptor, and IGFBPs-2, -3 and -5 (Middleton & Tyler 1992, Dore et al. 1994, Olney et al. 1996). Since IGFBP-3 and IGFBP-5 have been shown to have both similar and opposing effects in different experimental models, we investigated the inter- action between IGF-I and these IGFBPs in human chondrocytes. 29 Journal of Endocrinology (2000) 166, 29–37 0022–0795/00/0166–0029  2000 Society for Endocrinology Printed in Great Britain Online version via http://www.endocrinology.org