title: Properties of an insulin-like growth factor-binding protein-4 protease that is secreted by smooth muscle cells.
abstract: Smooth muscle cells (SMC) secrete insulin-like growth factor (IGF)-binding protein-4 (IGFBP-4) and an IGFBP-4 protease. The purpose of this study was to determine the characteristics of this IGFBP-4 protease and to compare its inhibitor profile to those of IGFBP-5 and IGFBP-2 proteases, which are also present in SMC-conditioned medium. Cultured SMC were exposed to serum-free medium for periods of 24-72 h, and the amount of proteolytic activity in the conditioned medium was assessed by its capacity to degrade pure IGFBP-4. Minimal activity (e.g. < 20% of IGFBP-4 degraded in 24 h at 37 C) was present in conditioned medium unless IGF-I or IGF-II was added. This resulted in more than 60% of the intact IGFBP-4 being degraded in 14 h. The activity was a calcium-dependent serine protease and was inhibited by EDTA or 3,4-dicloroisocoumarin. Calcium, but not zinc, could restore proteolytic activity. Heparin alone inhibited IGFBP-4 proteolysis by more than 60%. When heparin cofactor-II and antithrombin-III (AT-III) were added alone, they each had an effect. The combination of heparin plus AT-III was no more active than heparin alone, but the combination of heparin cofactor-II and heparin resulted in near complete inhibition. Peptides that contained the active sites of AT-III or alpha 1-antichymotrypsin were potent inhibitors of the IGFBP-4 protease. The medium also contained proteolytic activities for IGFBP-2 and IGFBP-5. Comparison of the inhibitor profiles for the IGFBP-4 and IGFBP-5 proteolytic activities revealed major differences, but the IGFBP-2 proteolytic activity was very similar to that of the IGFBP-4 protease. IGFBP-4 zymography showed a band with a molecular mass estimate of 48 kilodaltons. In contrast, when IGFBP-2 was used as the substrate, a single band at 36 kilodaltons was visualized. These data taken together with the protease inhibitor results suggest that the IGFBP-2, IGFBP-4, and IGFBP-5 proteases are members of a similar family of calcium-dependent serine proteases, but they are distinct proteases. As IGFBP-4 is a potent inhibitor of IGF action, and the activity of this protease is regulated by IGF exposure, the protease represents a novel system for regulating the actions of IGF-I in this cell type.
authors.affiliation: Department of Medicine, University of North Carolina School of Medicine, Chapel Hill 27599, USA.
authors.forename: A
authors.initials: A
authors.lastname: Parker
authors.forename: A
authors.initials: A
authors.lastname: Gockerman
authors.forename: W H
authors.initials: WH
authors.lastname: Busby
authors.forename: D R
authors.initials: DR
authors.lastname: Clemmons
mesh_terms: D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism; D002478:Cells, Cultured; D004492:Edetic Acid / Q000494:pharmacology; D018974:Insulin-Like Growth Factor Binding Protein 4; D007334:Insulin-Like Growth Factor I / Q000378:metabolism / Q000494:pharmacology; D007335:Insulin-Like Growth Factor II / Q000378:metabolism / Q000494:pharmacology; D008666:Metalloendopeptidases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D009131:Muscle, Smooth, Vascular / Q000201:enzymology*; D011266:Pregnancy-Associated Plasma Protein-A; D011480:Protease Inhibitors / Q000494:pharmacology; D013379:Substrate Specificity; D013552:Swine
chemical_list: D002352:Carrier Proteins; D018974:Insulin-Like Growth Factor Binding Protein 4; D011480:Protease Inhibitors; D007334:Insulin-Like Growth Factor I; D007335:Insulin-Like Growth Factor II; D004492:Edetic Acid; D008666:Metalloendopeptidases; D011266:Pregnancy-Associated Plasma Protein-A