INTERACTIONS BETWEEN INSULIN-LIKE GROWTH-FACTOR-I (IGF-I) AND THE SYSTEM OF PLASMINOGEN ACTIVATORS AND THEIR INHIBITORS IN THE CONTROL OF IGF-BINDING PROTEIN-3 PRODUCTION AND PROTEOLYSIS IN HUMAN OSTEOSARCOMA CELLS

Limited proteolysis in vivo of insulin-like growth factor-binding prot ein-3 (IGFBP-3) by as yet unidentified serine proteases plays a key ro le in controlling the bioavailability of IGFBP-3-associated insulin-li ke growth factors (IGFs). Both the IGF system and the system of plasmi nogen activators (PAs) and their inhibitors (PAIs) are involved in bon e remodeling, and plasmin has been shown to provoke dissociation of IG FBP-IGF complexes in cultured MG-63 human osteoblasts. The aim of this work was to investigate interactions between IGF-I and the PA/PAI sys tem and their influence on IGFBP-3 production and proteolysis in this cell model. At confluency, MG-63 cells maintained for 3 days in serum- free medium constitutively secreted IGFBP-2 and small amounts of IGFBP -3 and IGFBP-4. As shown by Western ligand and immunoblot analyses of the culture medium and Northern blot analysis of IGFBP-3 messenger RNA , production of these IGFBPs, and of IGFBP-3 in particular, was dose d ependently stimulated by the addition of 12.5-100 ng/ml recombinant hu man (rh) IGF-I. Increasing concentrations of plasminogen (0.05-3.5 mu g/ml) added during the final 12 h of culture reduced the amounts of IG FBP detectable by Western ligand blotting, especially IGFBP-3. This re duction reflected proteolysis, as shown by immunoblotting, which revea led 30-, 20-, and 16-kilodalton fragments of IGFBP-3. In the presence of 25 ng/ml IGF-I, which stimulated IGFBP-3 production, proteolysis wa s reduced by approximately half. Incubation of glycosylated [I-125]rh- IGFBP-3 as substrate in cell-free conditioned medium gave the same res ults. Addition of 50 ng/ml rhIGF-I to conditioned medium (to promote I GFBP-3-rhIGF-I complex formation) failed to diminish plasmin-induced p roteolysis of IGFBP-3. Urokinase PA activity in the conditioned medium decreased significantly when the cells were cultured with rhIGF-I, in dicating a direct action of IGF-I on urokinase PA and/or PAI productio n. Our results support the notion of a regulation loop whereby IGF-I c ontrols its own bioavailability via its action on both IGFBP-3 product ion and the PA/PAI system, which regulates IGFBP-3 proteolysis. The pr oteolytic cleavages of IGFBP-3 caused by plasmin were the same as thos e caused in vivo by serine proteases acting on this IGFBP.