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 marked preferential affinity over IGF-I. Recombinant human IGFBP-6 (rhIGFBP-6) was synthesized by COS-7 monkey kidney cells that were transiently transfected with a eukaryotic expression vector into which a complementary DNA for IGFBP-6 modified for optimal translation had been inserted. rhIGFBP-6 was similar to IGFBP-6 purified from human cerebrospinal 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 manner, inhibition was complete when rhIGFBP-6 was present in a slight molar excess. In contrast, rhIGFBP-6 had no effect on IGF-I-induced differentiation, even when coincubated in a 5-fold molar excess. These results are consistent with the preferential affinity of IGFBP-6 for IGF-II. As cell association and proteolysis have been associated with the potentiation, rather than the inhibition, of IGF action by IGFBPs, we investigated whether they occurred in the L6A1 myoblast system. After incubation of L6A1 myoblasts with rhIGFBP-6, IGFBP-6 was recovered from the medium, but not from cell lysates or extracellular matrix. In addition, [125I]IGFBP-6 did not bind to myoblast monolayers, and there was no evidence that proteolysis had occurred. Together, these results indicate that rhIGFBP-6 remains intact and soluble and, hence, inhibits IGF-II-induced differentiation. The fidelity of the IGFBP-6 expression system used for these studies will enable us to use this system to determine how structural modifications of the protein affect the modulation of IGF action by IGFBP-6.
Bach
L A
LA
Growth and Development Section, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892.
Hsieh
S
S
Brown
A L
AL
Rechler
M M
MM
D000818:Animals; D001483:Base Sequence; D002352:Carrier Proteins / Q000378:metabolism / Q000494:pharmacology*; D002454:Cell Differentiation / Q000187:drug effects; D002460:Cell Line; D004247:DNA / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D006031:Glycosylation; D000882:Haplorhini; D018976:Insulin-Like Growth Factor Binding Protein 6; D007335:Insulin-Like Growth Factor II / Q000494:pharmacology*; D007668:Kidney / Q000166:cytology / Q000378:metabolism; D008969:Molecular Sequence Data; D009132:Muscles / Q000166:cytology*; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism / Q000494:pharmacology
D002352:Carrier Proteins; D018976:Insulin-Like Growth Factor Binding Protein 6; D011994:Recombinant Proteins; D007335:Insulin-Like Growth Factor II; D004247:DNA