RECOMBINANT HUMAN INSULIN-LIKE GROWTH-FACTOR (IGF)-BINDING PROTEIN-6 INHIBITS IGF-II-INDUCED DIFFERENTIATION OF L6A1 MYOBLASTS

Insulin-like growth factor-binding protein-6 (IGFBP-6) is an O-linked glycoprotein that binds insulin-like growth factor-II (IGF-II) with ma rked preferential affinity over IGF-I. Recombinant human IGFBP-6 (rhIG FBP-6) was synthesized by COS-7 monkey kidney cells that were transien tly transfected with a eukaryotic expression vector into which a compl ementary DNA for IGFBP-6 modified for optimal translation had been ins erted. rhIGFBP-6 was similar to IGFBP-6 purified from human cerebrospi nal fluid with respect to IGF binding and O-glycosylation. The effect of rhIGFBP-6 on IGF-induced L6A1 myoblast differentiation was studied using creatine kinase activity as an index of differentiation. rhIGFBP -6 inhibited differentiation initiated by IGF-II in a dose-dependent m anner; inhibition was complete when rhIGFBP-6 was present in a slight molar excess. In contrast, rhIGFBP-6 had no effect on IGF-I-induced di fferentiation, even when coincubated in a 5-fold molar excess. These r esults are consistent with the preferential affinity of IGFBP-6 for IG F-II. As cell association and proteolysis have been associated with th e potentiation, rather than the inhibition, of IGF action by IGFBPs, w e investigated whether they occurred in the L6A1 myoblast system. Afte r incubation of L6A1 myoblasts with rhIGFBP-6, IGFBP-6 was recovered f rom the medium, but not from cell lysates or extracellular matrix. In addition, [I-125]-IGFBP-6 did not bind to myoblast monolayers, and the re was no evidence that proteolysis had occurred. Together, these resu lts indicate that rhIGFBP-6 remains intact and soluble and, hence, inh ibits IGF-II-induced differentiation. The fidelity of the IGFBP-6 expr ession system used for these studies will enable us to use this system to determine how structural modifications of the protein affect the m odulation of IGF action by IGFBP-6.