title: A protease-resistant form of insulin-like growth factor (IGF) binding protein 4 inhibits IGF-1 actions.
abstract: Smooth muscle cells (SMC) secrete a serine protease that cleaves insulin-like growth factor (IGF) binding protein (IGFBP)-4 into fragments that have low affinity for IGF-1. When IGFBP-4 is added to monolayer cultures of cell types that do not secrete this protease, IGF-1 stimulation of DNA synthesis is significantly inhibited. In contrast, if cell types that secrete this protease are used, IGFBP-4 is a much less potent inhibitor. These studies were conducted to determine whether proteolysis of IGFBP-4 accounted for its reduced capacity to inhibit IGF-1-stimulated DNA synthesis. The cleavage site in IGFBP-4 that the SMC protease uses was determined to be lysine120, histidine121. A protease-resistant mutant form of IGFBP-4 was prepared, expressed, purified, and tested for biologic activity using porcine SMC cultures. Addition of the protease-resistant mutant resulted in inhibition of DNA and cell migration responses to IGF-1. The inhibition was concentration dependent and was maximal when 500 ng/ml (20 nM) of the mutant was added with 20 ng/ml (2.8 nM) of IGF-1. When the mutant was added in the absence of IGF-1, it had no activity. The results show that cleavage of IGFBP-4 at lysine120, histidine121 results in inactivation of the ability of IGFBP-4 to bind to IGF-1. Creation of a mutant form of IGFBP-4 that was not cleaved by the protease resulted in inhibition of IGF-1-stimulated actions. The results suggest that IGFBP-4 can act as a potent inhibitor of the anabolic effects of IGF-1 and that the variables that regulate protease activity may indirectly regulate IGF-1 actions.
authors.affiliation: Department of Medicine, University of North Carolina School of Medicine, Chapel Hill 27599-7170, USA.
authors.forename: C
authors.initials: C
authors.lastname: Rees
authors.forename: D R
authors.initials: DR
authors.lastname: Clemmons
authors.forename: G D
authors.initials: GD
authors.lastname: Horvitz
authors.forename: J B
authors.initials: JB
authors.lastname: Clarke
authors.forename: W H
authors.initials: WH
authors.lastname: Busby
mesh_terms: D000818:Animals; D016466:CHO Cells; D002478:Cells, Cultured; D006224:Cricetinae; D004247:DNA / Q000096:biosynthesis; D018974:Insulin-Like Growth Factor Binding Protein 4 / Q000378:metabolism / Q000494:pharmacology*; D007334:Insulin-Like Growth Factor I / Q000037:antagonists & inhibitors* / Q000378:metabolism; D008666:Metalloendopeptidases / Q000494:pharmacology*; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000378:metabolism; D011266:Pregnancy-Associated Plasma Protein-A; D013552:Swine
chemical_list: D018974:Insulin-Like Growth Factor Binding Protein 4; D007334:Insulin-Like Growth Factor I; D004247:DNA; D008666:Metalloendopeptidases; D011266:Pregnancy-Associated Plasma Protein-A