The Role and Content of Endogenous Insulin-Like Growth Factor-Binding Proteins in Bovine Articular Cartilage

Previous work identified insulin-like growth factor (IGF)-binding proteins (IGF-BPs) in chondrocyte culture fluids, but the relationship of these proteins to the composition of intact cartilage was not established. The aim of this work was to analyze the IGF-BP system resident in bovine articular cartilage and to examine its role in IGF-1-regulated proteoglycan (PG) metabolism. Protein extracts of freshly dissected or cultured cartilage slices were analyzed by125I-IGF-2 ligand blotting. Fresh tissue and basal cultured samples (serum-free) from nine animals, aged fetal to adult, contained two major IGF-BPs of ∼31,000 and 24,000–21,500Mr, with the latter doublet being dominant. The 31,000Mrprotein was identified as IGF-BP-2 by specific immunoreactivity with two polyclonal antibodies, and the 24,000–21,500Mrdoublet was identified as IGF-BP-6 by reactivity with a specific polyclonal antibody and by marked preferential affinity for IGF-2 over IGF-1 by ligand blotting. Treatment of the cartilage cultures with 10 ng/ml transforming growth factor-β (TGF-β1) for 1 week led to an accumulation of IGF-BP-2, while IGF-BP-6 was unchanged (ligand blots,n= 6 animals). IGF-1 had a similar but much less pronounced effect. The IGF-BP increase following TGF-β treatment, quantified by charcoal assay, was on average 6-fold, while total protein increased only 1.2-fold (n= 4). By contrast, IGF-1 (10 ng/ml) increased IGF-BP by <2-fold (n= 4), and retinoic acid, at 1 × 10−8M was not effective (n= 3). As before, 10 ng/ml TGF-β or IGF-1 increased proteoglycan synthesis and maintained its homeostasis. IGF-1 analogs with reduced affinity for the IGF-BPs were tested. The effect of an A-chain analog (Thr49, Ser50, Ile51) on PG synthesis was comparable to that of IGF-1, even though the analog had one-half of the IGF-1 affinity for the type I IGF receptor. A B-chain analog, with one-third the receptor affinity of IGF-1, promoted an average 2-fold higher PG synthesis in the linear response range to concentration. Thus, in relation to their respective affinities for the IGF-type I receptor, both IGF analogs were more effective than native IGF-1. These results suggest that an overall effect of the endogenous IGF-BP activity in articular cartilage under the test conditions is the inhibition of IGF-1-stimulated proteoglycan synthesis.