title: Protease-resistant form of insulin-like growth factor-binding protein 5 is an inhibitor of insulin-like growth factor-I actions on porcine smooth muscle cells in culture.
abstract: IGFs are pleiotrophic mitogens for porcine smooth muscle cells (pSMC) in culture. The effects of IGFs on cells are modulated by various insulin-like growth factor-binding proteins (IGFBP). IGFBP-5 is synthesized by pSMC and binds to the extracellular matrix. However, IGFBP-5 is also secreted into conditioned medium of cultured cells and is cleaved into fragments by a concomitantly produced protease. These fragments have reduced affinity for the IGFs and cleavage makes it difficult to assess the role of intact IGFBP-5. To study the consequence of accumulation of intact IGFBP-5 in medium, we determined the cleavage site in IGFBP-5 and prepared a protease resistant mutant. Amino acid sequencing of purified IGFBP-5 fragments suggested Arg138-Arg139 as the primary cleavage site. Arg138-Arg139-->Asn138-Asn139 mutations were introduced to create protease-resistant IGFBP-5, which has the same affinity for IGF-I as the native protein. This mutant IGFBP-5 remained intact even after 24 h of incubation and it inhibited several IGF-I actions when added to pSMC culture medium. The mutant IGFBP-5 (500 ng/ml) decreased IGF-I stimulated cellular DNA synthesis by 84%, protein synthesis by 77%, and it inhibited IGF-I stimulated migration of pSMC by 77%. It also inhibited IGF-I stimulation of IRS-1 phosphorylation. In contrast, the same amount of native IGFBP-5 did not inhibit IGF-I actions. The significance of inhibitory effects of the protease resistant IGFBP-5 was further demonstrated in pSMC transfected with mutant or native IGFBP-5 cDNAs. The mutant IGFBP-5 accumulated in culture medium of transfected cells, while native IGFBP-5 was degraded into fragments, PSMC overexpressing the mutant IGFBP-5 also responded poorly to IGF-I compared with mock transfected cells. IGF-I (5 ng/ml) increased [35S]methionine incorporation into control cells by 36% above the basal level, but it did not significantly change (4%) in pSMC cultures that were producing the mutant IGFBP-5. In conclusion, the accumulation of protease-resistant IGFBP-5 in the medium was inhibitory to IGF-I actions on pSMC. This suggests that proteolysis can prevent IGFBP-5 from acting as an inhibitor of IGF-I-stimulated effects and that it serves as an important mechanism for regulating cellular responsiveness to IGF-I.
authors.affiliation: Department of Medicine, University of North Carolina School of Medicine, Chapel Hill 27599-7170, USA.
authors.forename: Y
authors.initials: Y
authors.lastname: Imai
authors.forename: W H
authors.initials: WH
authors.lastname: Busby
authors.forename: C E
authors.initials: CE
authors.lastname: Smith
authors.forename: J B
authors.initials: JB
authors.lastname: Clarke
authors.forename: A J
authors.initials: AJ
authors.lastname: Garmong
authors.forename: G D
authors.initials: GD
authors.lastname: Horwitz
authors.forename: C
authors.initials: C
authors.lastname: Rees
authors.forename: D R
authors.initials: DR
authors.lastname: Clemmons
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001011:Aorta; D001120:Arginine; D001216:Asparagine; D002455:Cell Division / Q000187:drug effects; D002478:Cells, Cultured; D017077:Culture Media, Conditioned; D006801:Humans; D018970:Insulin-Like Growth Factor Binding Protein 1 / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D018975:Insulin-Like Growth Factor Binding Protein 5 / Q000096:biosynthesis* / Q000737:chemistry / Q000494:pharmacology*; D007334:Insulin-Like Growth Factor I / Q000494:pharmacology*; D008666:Metalloendopeptidases / Q000378:metabolism*; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry / Q000494:pharmacology; D017354:Point Mutation; D011994:Recombinant Proteins / Q000737:chemistry / Q000494:pharmacology; D013552:Swine; D013936:Thymidine / Q000378:metabolism
chemical_list: D017077:Culture Media, Conditioned; D018970:Insulin-Like Growth Factor Binding Protein 1; D018975:Insulin-Like Growth Factor Binding Protein 5; D010446:Peptide Fragments; D011994:Recombinant Proteins; D007334:Insulin-Like Growth Factor I; D001216:Asparagine; D001120:Arginine; D008666:Metalloendopeptidases; D013936:Thymidine