Proteolysis of insulin-like growth factor-binding protein-1, -2 and -4 : function of fragments

Sammanfattning: The insulin-like growth factors (IGF-I and IGF-II) stimulate cell growth, survival and differentiation, and have insulin-like activity. A family of six IGF-binding proteins (IGFBP-1 through -6) bind to IGFs with high affinity and modulate their activity in the circulation and locally at cell-surface receptors. IGFBPs can potentiate or inhibit IGF-activity. Proteolysis is recognised as the predominant mechanism for IGF release from IGFBPs. IGFBPs have also IGF-independent effects, through the interaction with cell surface structures and extracellular molecules. IGFBP-1 and IGFBP-2 have a C-terminal Arg-Gly-Asp (RGD) sequence, and IGFBP-1 has been shown to increase migration independently of IGF-I by binding to extracellular α5β1-integrins. An IGFBP-1- protease activity has previously been isolated from the urine of a patient with multiple myeloma and an inflammatory skin disease. Although it was not isolated in its active form, the protease-activity was identified as azurocidin. The aim of this thesis was to further characterize this protease-activity and to study the biological effects of the proteolytic IGFBP fragments on migration and on IGF-stimulated proliferation in human dermal fibroblasts. Intact IGFBP-1 and IGFBP-1 fragments, obtained by incubating with the patient-derived protease-activity, were characterized in four RIAs and with SDS–PAGE. Immunoreactivity and size of fragments were compared to in vitro produced fragments. Serum from the patient inhibited IGFBP-1 protease activity; however, immunoreactive IGFBP-1 in patient serum was present at molecular masses consistent with IGFBP-1 fragments, in addition to intact IGFBP-1. We also studied a neutrophil-derived preparation of azurocidin and found that it cleaved IGFBP-1, IGFBP-2 and IGFBP-4. IGFBP-1 bound to IGF-I was also degraded whereas IGF-II was shown to have an inhibitory effect on proteolysis of IGFBP-1. The proteolytically active preparation of neutrophil-derived azurocidin was found to be glycosylated and determined to be 31 kDa by SDS-PAGE. The same cleavage pattern of IGFBP-1 was obtained by both azurocidin-preparations derived from either urine or neutrophils. IGFBP-1, IGFBP-2 and their proteolytic fragments stimulated migration of fibroblasts and the stimulatory effect was abolished by pre-treating cells with an α5β1 integrin antibody. High glucose impaired migration. However, the addition of IGFBP-1, IGFBP-2 or fragments increased migration to normal levels again. IGFBP-2 inhibited IGF-II induced proliferation, while IGFBP-2 fragments had reduced inhibitory effect. Intact phosphorylated IGFBP-1 showed either potentiating or inhibitory effects on IGF-I induced proliferation depending on the confluence of cells, and proteolysis of IGFBP-1 did not change these effects. In conclusion, a novel IGFBP-protease which is associated to inflammation regulates IGF-activity in tissue through cleavage of IGFBPs, resulting in proteolytic IGFBP-fragments with biological effects important for tissue repair.