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Basic Fibroblast Growth Factor Induces Proteolysis of Secreted and Cell Membrane-Associated Insulin-Like Growth Factor Binding Protein-2 in Human Neuroblastoma Cells¹

Centre for Hormone Research (V.C.R., G.R., N.L.B., G.A.W.), Royal Children’s Hospital Research Institute, Parkville 3052, Victoria, Australia; University of Melbourne, Department of Paediatrics (V.C.R., G.R., N.L.B., G.A.W.), Royal Children’s Hospital, Melbourne 3052, Victoria, Australia; and Department of Medicine (L.A.B.), Austin and Repatriation Medical Centre, Heidelberg 3084, Victoria, Australia

Address all correspondence and requests for reprints to: Associate Professor George A. Werther, Centre for Hormone Research, Royal Children’s Hospital, Flemington Road, Parkville, Victoria 3052, Australia, E-mail: werther{at}cryptic.rch.unimelb.edu.au

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 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.

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