The Anti-angiogenic Peptide, Loop 6, Binds Insulin-like Growth Factor-1 Receptor

Abstract

Tissue inhibitors of metalloproteinases (TIMPs), the endogenous inhibitors of matrix metalloproteinases, have been shown to possess biological functions that are independent of their ability to inhibit matrix metalloproteinases. We have previously shown that the C-terminal domain of TIMP-2 and, in particular, Loop 6 inhibit capillary endothelial cell proliferation and angiogenesis both in vitro and in vivo. To elucidate the mechanism by which Loop 6 inhibits angiogenesis, we sought to determine whether its biological effects were the result of a known TIMP-2 protein-protein interaction or of a receptor-mediated event. In this study, we identify insulin-like growth factor-1 receptor as a binding partner of Loop 6/TIMP-2 and characterize this interaction on the endothelial cell surface and the consequences of this interaction on downstream receptor signaling. TIMP-2,3 an endogenous regulator of matrix metalloproteinase (MMP) activity, is a multifunctional protein that has been shown to regulate angiogenesis, tumor progression, and metastasis. Studies from our laboratory (1) and others (2) have demonstrated that TIMP-2 can inhibit angiogenesis independent of its ability to inhibit MMP activity. Early studies of the growth modulating effects of TIMP-2 sought to determine whether TIMP-2 could bind to the cell surface of various cell lines (3-7). These studies suggested the existence of at least two potential cell surface receptors: one of higher affinity and at least one of relatively lower affinity. The higher affinity interaction has been shown to involve a trimolecular complex of TIMP-2 with MT1-MMP and pro-MMP-2. Formation of this complex is required for cell surface activation of MMP-2 by MT1-MMP (8 -12). It has been reported that the binding of the N terminus of TIMP-2, T2N, to the cell surface of HT1080 fibrosarcoma cells was an order of magnitude weaker than that of intact TIMP-2 (7, 12). The failure of T2N to completely compete for TIMP-2 binding suggested to us that the C-terminal portion of TIMP-2, T2C, might also be binding to the cell surface.In a structure-function analysis of TIMP-2, we have demonstrated that T2C inhibits angiogenesis in a manner independent of any MMP inhibitory activity. We further demonstrated that Loop 6, a smaller domain of T2C, retained the anti-angiogenic activity (1). We therefore hypothesized that T2C and, more specifically, Loop 6 might bind the endothelial cell surface and that this interaction might mediate the antiangiogenic activity of Loop 6. Until recently, none of the low affinity receptors of TIMP-2 described previously (3-7) had been identified. Seo et al. (2) have now shown that at least one of these interactions involves the integrin ␣3␤1. However, in the course of our studies, we found no evidence of the binding of Loop 6 to ␣3␤1. These results suggested that the anti-angiogenic effects of Loop 6 were mediated by a different mechanism and might be the result of a novel interaction of TIMP-2 at the EC surface.Here, we identify IGF-IR as a novel binding partner of L...

Endogenous Angiogenesis Inhibitor Blocks Tumor Growth via Direct and Indirect Effects on Tumor Microenvironment

“…12,19,21 Indeed, we show that intratumoral angiogenesis is significantly reduced in A549 stables. Because TIMP-2 and AlaϩTIMP-2 are overexpressed in A549 tumors we expect that the antiangiogenic effect might be caused by paracrine mechanisms involving TIMP-2 secretion and interaction with the surface receptors on the ECs.…”

“…12 More recently, TIMP-2 Loop 6, located at the C-terminus of the protein, was shown to inhibit angiogenesis in vivo by direct binding to the insulin-like growth factor receptor I (IGF-IR) on ECs and regulating IGF-IR downstream mitogenic signals. 21 TIMP-2 regulates additional cellular activities including, inhibition of EC migration, myogenesis, and neuronal differentiation, all via an ␣3␤1 integrin-dependent mechanism. [22][23][24][25] We have reported that TIMP-2 inhibits EC migration by inducing the expression of an MMP inhibitor, the reversion-inducing-cystein-rich protein with Kazal motif (RECK), leading to loss of endothelial cell migration.…”

Antagonism of VEGF-A–induced increase in vascular permeability by an integrin α3β1-Shp-1-cAMP/PKA pathway

“…[23][24][25]27,46 TIMP-2 is the only member of the TIMP family that has been shown to selectively inhibit endothelial cell growth and angiogenesis in an MMP-independent fashion. 24,25,[29][30][31] We have shown previously that TIMP-2 reduced phosphorylation of several RTKs after stimulation with their cognate ligands through an increase of Shp-1 with the receptors. 24,30,31,46 This results in a selective alteration in the RTK phosphorylation pattern with subsequent consequences in downstream signaling pathways involving a variety of cellular responses.…”

“…25 These antiangiogenic effects of Loop 6 are mediated by direct binding to insulin-like growth factor receptor I (IGF-IR) and subsequent regulation of IGF-IR signaling pathways. 25,29 TIMP-2 binds to the surface of human microvascular endothelial cells (hMVECs) via interaction with the integrin ␣ 3 ␤ 1 , and this interaction suppresses fibroblast growth factor-2 (FGF-2)-or VEGF-A-induced endothelial cell proliferation in vitro, angiogenesis, and tumorigenesis in vivo. 24,[30][31][32][33] These TIMP-2 antiangiogenic effects are mediated by SH2-containing protein tyrosine phosphatase-1 (Shp-1)-dependent suppression of receptor tyrosine kinase (RTK) signaling pathways and induction of cyclin-dependent kinase (cdk) inhibitor p27 Kip1 , resulting in hypophosphorylation of Rb and cell cycle arrest.…”

Antiangiogenic activity of trabectedin in myxoid liposarcoma: Involvement of host TIMP‐1 and TIMP‐2 and tumor thrombospondin‐1

“…18 TIMP-2 acts by preventing matrix remodeling 23 and by directly affecting endothelial cell response to angiogenic factors through interaction with a3ß1 integrin 24 or insulin-like growth factor-1 receptor. 25 The clinical response of myxoid liposarcomas to trabectedin is often associated with adipogenic differentiation. 6,12 Hence, we hypothesized that trabectedin might impair the ability to tumor cells to induce angiogenesis in connection with its differentiating activity.…”