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

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.

List of scientific papers

I. Brandt K, Wang J, Lundell K, Ståhlberg M, von Horn H, Ehrenborg E, Hall K, Jörnvall H, Lewitt M. IGFBP-1 protease activity and IGFBP-1 fragments in a patient with multiple myeloma. Growth Horm IGF Res. 2009 Dec; 19(6):507-12.
https://doi.org/10.1016/j.ghir.2009.05.002

II. Brandt K, Lundell K, Brismar K. Neutrophil-derived azurocidin cleaves insulin-like growth factor-binding protein-1, -2 and -4. Growth Horm IGF Res. 2011 Jun; 21(3):167-73.
https://doi.org/10.1016/j.ghir.2011.04.003

III. Brandt K, Grünler J, Brismar K. Effects of IGFBP-1 and IGFBP-2 fragments on migration and IGF-induced proliferation of human dermal fibroblasts. [Submitted]