Identification of Vitronectin as a Novel Insulin-Like Growth Factor-II Binding Protein

Abstract

We have previously reported the presence of a 70 kDa insulin-like growth factor (IGF)-II-specific binding protein in chicken serum using Western ligand blotting approaches. In order to ascertain the identity of this 70 kDa IGF-II binding species, the protein has been purified from chicken serum using a combination of ion-exchange and gel-permeation chromatography. Interestingly, amino acid sequencing of the purified protein revealed that it has the same N-terminal sequence as chicken vitronectin (VN). The protein has the ability to specifically bind IGF-II and not IGF-I as determined by ligand blotting, cross-linking and competitive binding assay approaches. In addition, the protein binds 125I-des(1-6)-IGF-II, suggesting that the interaction with IGF-II is different to those with other characterized IGF-binding proteins. Importantly, we have ascertained that both human and bovine VN also specifically bind IGF-II. These results are particularly relevant in the light of the recent report that the urokinase-type plasminogen activator receptor, a protein that also binds VN, has been shown to associate with the cation-independent mannose-6-phosphate/IGF-II receptor and suggest a possible role for IGF-II in cell adhesion and invasion.

Surface Modification by Complexes of Vitronectin and Growth Factors for Serum-Free Culture of Human Osteoblasts

“…Previously published binding studies have confirmed that VN, IGFBPs, and IGF-I form complexes on tissue culture plastic, that IGFBP-5 modulates binding of 125 I-labeled IGF-I to VN, and that the regulation of cellular activity is due to the proteins bound to the substrate. 12,15 VN is a multifunctional glycoprotein present in blood and in the extracellular matrix. VN contains an RGD sequence, through which it binds to the integrin receptor ␣ v ␤ 3 (␣ v ␤ 3 ), and is involved in cell attachment, spreading, and migration.…”

“…10,11 Furthermore, both systemic and local administration of IGF-I have shown effects on bone cell proliferation and bone formation 11 Investigations have demonstrated that VN can specifically bind proteins of the IGF system and that the insulinlike growth factor-binding proteins (IGFBPs) play an important role in modulating IGF-I binding to VN. 12,13 In addition, it has been demonstrated by our laboratory that MCF-7 breast cancer cells and HaCAT human skin keratinocytes exhibit enhanced functional responses to VN, IGFBP, and IGF-I complexes. 14-16 Therefore, we tested osteoblast attachment, proliferation, and migration on tissue culture plastic and polymer scaffolds treated with complexes containing VN, IGFBP-5, and IGF-I to confirm our hypotheses that IGFs bound to surface-adsorbed VN can stimulate and enhance cell attachment, proliferation, and migration in the absence of serum in two-dimensional monolayer culture and three-dimensional culture on porous biomaterials in vitro.…”

Responses of keratinocytes to substrate-bound vitronectin:growth factor complexes

“…As early as 1981, when EGF was co-purified with somatomedin-B [33], a protein which has subsequently been proposed to be a proteolyticaly-cleaved fragment of VN, there has been evidence that VN plays a critical role in cellular processes through its interaction with growth factors in the ECM. Moreover, the recent discovery that VN binds to IGF-II with high affinity adds further support to this notion [12]. Following this initial finding, our laboratory has found that IGF-I can also bind to VN, albeit indirectly through IGFBP-2, -3, -4, or -5 [10,11].…”

“…Following the initial discovery that the ECM protein vitronectin (VN) binds to insulin-like growth factor-II (IGF-II) [12], novel links between members of the IGF family, IGF-binding proteins (IGFBPs), and VN have also been identified. Specifically, it has been shown that while IGF-II can bind directly to VN, IGF-I associates with VN indirectly through IGFBP-2, -3, -4, or -5 [10].…”

Development of Defined Media for the Serum-Free Expansion of Primary Keratinocytes and Human Embryonic Stem Cells

“…12 This new technology is based on the finding that a synergistic effect occurs between GFs and a specific ECM protein called vitronectin (VN). [13][14][15][16] This has led to the development of novel dimeric, trimeric, and multimeric growth-promoting complexes incorporating GFs, such as insulin-like growth factors (IGFs) and insulin-like growth factor binding proteins (IGFBPs), in conjunction with VN (VN:GF). Further, the addition of these complexes to defined media has been demonstrated to stimulate short-term migration and proliferation in a range of cells, including adult skin-and corneal-derived epithelial cells.…”