title: Basic fibroblast growth factor induces proteolysis of secreted and cell membrane-associated insulin-like growth factor binding protein-2 in human neuroblastoma cells.
abstract: Insulin-like growth factor (IGF) action in the brain is modulated by IGF-binding proteins (IGFBPs) whose abundance can be altered by other locally expressed growth factors. However, the mechanisms involved are unclear. We here employed the neuroblastoma cell line SK-N-MC as a model to define the mechanisms involved in modulation of IGFBPs in neuronal cells. Western ligand blotting analysis and immunoprecipitation of conditioned media (CM) from SK-N-MC cells showed that in these cells, as in the brain, the most abundantly expressed IGFBP was IGFBP-2. However, IGFBP-2 was barely detectable in CM from cells treated with basic fibroblast growth factor (bFGF) without a change in IGFBP-2 messenger RNA (mRNA) abundance. These CM contained specific IGFBP-2 proteolytic activity, resulting in two IGFBP-2 fragments of 14 and 22 kDa. The activity was inhibited by EDTA/phenylmethylsulfonyl fluoride or aprotinin. Competitive binding studies indicated that IGFBP-2 fragments had reduced binding affinity for IGF-I. bFGF induced IGFBP-3 mRNA and protein. Affinity cross-linking of [125I]IGF-I to neuroblastoma cell membranes followed by immunoprecipitation revealed a approximately 38 kDa [125I]IGF-I/IGFBP-2 complex. Cell surface-associated IGFBP-2 was also susceptible to bFGF-induced proteolysis, with the appearance of a single cross-linked 21-kDa complex with low affinity for IGF-I. These findings indicate that intact IGFBP-2 and the 14-kDa, but not the 22-kDa fragment, bind to the cell surface. Our data suggest that induction of IGFBP-2 proteolysis on neuronal cell surface is a novel mechanism whereby IGF availability is modulated by the local growth factor bFGF.
authors.affiliation: Centre for Hormone Research, Royal Children's Hospital Research Institute, Parkville, Victoria, Australia.
authors.forename: V C
authors.initials: VC
authors.lastname: Russo
authors.forename: G
authors.initials: G
authors.lastname: Rekaris
authors.forename: N L
authors.initials: NL
authors.lastname: Baker
authors.forename: L A
authors.initials: LA
authors.lastname: Bach
authors.forename: G A
authors.initials: GA
authors.lastname: Werther
mesh_terms: D001665:Binding Sites / Q000502:physiology; D002462:Cell Membrane / Q000378:metabolism; D003470:Culture Media / Q000378:metabolism; D004790:Enzyme Induction / Q000502:physiology; D016222:Fibroblast Growth Factor 2 / Q000494:pharmacology*; D006801:Humans; D018971:Insulin-Like Growth Factor Binding Protein 2 / Q000378:metabolism*; D007334:Insulin-Like Growth Factor I / Q000378:metabolism; D009447:Neuroblastoma / Q000378:metabolism* / Q000473:pathology; D010446:Peptide Fragments / Q000378:metabolism; D010447:Peptide Hydrolases / Q000378:metabolism*; D011499:Protein Processing, Post-Translational / Q000502:physiology; D011994:Recombinant Proteins; D014407:Tumor Cells, Cultured
chemical_list: D003470:Culture Media; D018971:Insulin-Like Growth Factor Binding Protein 2; D010446:Peptide Fragments; D011994:Recombinant Proteins; D016222:Fibroblast Growth Factor 2; D007334:Insulin-Like Growth Factor I; D010447:Peptide Hydrolases