Proteolytic degradation of insulin-like growth factor (IGF)-binding protein-3 by porcine ovarian granulosa cells in culture: regulation by IGF-I.
Porcine ovarian granulosa cells in culture secrete glycosylated insulin-like growth factor (IGF)-binding protein-3 (IGFBP-3), which inhibits gonadotropin and IGF action in the ovary. Synthesis of IGFBP-3 is stimulated by IGF-I and attenuated by gonadotropin. The purpose of the present study was to determine whether IGFBP-3 levels were also regulated via proteolysis. Exogenously added nonglycosylated recombinant human IGFBP-3 (rhIGFBP-3) was significantly degraded over time by a soluble serine-specific protease, similar to plasmin, in control cultures and those treated with FSH, insulin, or several other classes of hormones. In contrast, degradation was greatly attenuated by the IGFs. Degraded rhIGFBP-3 exhibited much reduced affinity for [125I]IGF-II, suggesting that degradation could make available IGFs for cellular interaction. The mechanism of IGFBP-3 protease inhibition by IGFs is unclear. Mediation by IGF receptors is unlikely, as insulin at a dose that activated both insulin and type I IGF receptors did not alter intrinsic degradation of IGFBP-3 (as does IGF). Additionally, IGF-I attenuation of IGFBP-3 degradation was not inhibited by antagonism of receptor action with a tyrosine kinase inhibitor. Further, IGF-I inhibited degradation in cell-free conditioned medium. Direct stabilization of IGFBP-3 via binding of IGFs was suggested from these results. However, long R3 IGF-I attenuated IGFBP-3 degradation even though it has low affinity for IGFBPs. Inhibition of the protease by IGFs is also possible. We conclude that IGFs inhibit the degradation of exogenous nonglycosylated rhIGFBP-3. If active in vivo, this may serve to increase endogenous IGFBP-3 levels in follicular fluid.
Grimes
R W
RW
Department of Medicine, Milton S. Hershey Medical Center, Pennsylvania State University, Hershey 17033.
Hammond
J M
JM
D000818:Animals; D015153:Blotting, Western; D002352:Carrier Proteins / Q000378:metabolism*; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D005260:Female; D005640:Follicle Stimulating Hormone / Q000494:pharmacology; D006107:Granulosa Cells / Q000378:metabolism*; D006801:Humans; D007328:Insulin / Q000494:pharmacology; D018969:Insulin-Like Growth Factor Binding Proteins; D007334:Insulin-Like Growth Factor I / Q000494:pharmacology*; D010447:Peptide Hydrolases / Q000378:metabolism*; D011480:Protease Inhibitors / Q000494:pharmacology; D011994:Recombinant Proteins; D013552:Swine
D002352:Carrier Proteins; D007328:Insulin; D018969:Insulin-Like Growth Factor Binding Proteins; D011480:Protease Inhibitors; D011994:Recombinant Proteins; D007334:Insulin-Like Growth Factor I; D005640:Follicle Stimulating Hormone; D010447:Peptide Hydrolases