Differential regulation of IGF-binding proteins in rabbit costal chondrocytes by IGF-I and dexamethasone

Cartilage is a primary target tissue for the IGFs. The mitogenic activity o f these peptides is regulated by a family of high-affinity IGF-binding prot eins (IGFBP-1 to -6). We characterized the IGFBPs produced by cultured chon drocytes derived from rib cartilage of prepubertal rabbits. Culture medium, which had been conditioned by these cells for 48 h showed bands of 22 kDa, 24 kDa and a 31/32 kDa doublet by Western ligand blotting with [I-125]IGF- II. When the cells were grown in the presence of increasing amounts of IGF- I or IGF-II, the 31/32 kDa doublet increased in intensity (reaching a plate au of about 11-fold stimulation between 2 and 10 nM IGF-I). The 22 kDa and 24 kDa bands increased only slightly while a 26 kDa band became faintly vis ible. By Western immunoblotting the 31/32 kDa doublet was identified as IGF BP-5. An IGF-I analog with reduced affinity for IGFBPs, Long-R3 IGF-I, also induced IGFBP-5, while insulin was less effective (22-fold stimulation at 10 nM). IGF-I protected IGFBP-5 against proteolytic degradation by conditio ned medium. IGF-I also enhanced the level of IGFBP-5 mRNA. LY294002, a spec ific inhibitor of the intracellular signaling molecule phosphatidylinositol 3-kinase, inhibited stimulation of IGFBP-5 by IGF-I. Dexamethasone suppressed IGFBP-5 (dy 70% at 20 nM) but, at the same time, a 39/41 kDa doublet (presumably IGFBP-3) was induced. IGFBP-5 has been shown in several cell types to enhance the mitogenic activity of IGF-I. IGFBP-3 generally acts as a growth inhibitor. Therefore, the differential effects o f dexamethasone on these regulatory proteins could account, at least in par t, for the growth-arresting effect of this glucocorticoid.