Thrombospondin Type 1 Repeats Interact with Matrix Metalloproteinase 2

Abstract

Thrombospondins are thought to function as inhibitors of angiogenesis. However, the mechanism(s) of this activity is not well understood. In this study, we have used the yeast two-hybrid system to identify proteins that interact with the thrombospondins 1 (TSP1) and 2 (TSP2) properdin-like type 1 repeats (TSR). One of the proteins identified that interacted with both TSR was matrix metalloproteinase 2 (MMP2). The isolated MMP2 cDNA clone encoded amino acid residues 237-633, which include the fibronectin-like gelatin binding region flanking the catalytic center and the carboxyl hemopexin-like region. Further testing of this clone demonstrated that the TSR interacted with the NH 2 -terminal region of the MMP2 that contains the catalytic domain. The protein interaction observed in yeast was further demonstrated by immunoprecipitation and Western blotting using purified intact TSP1, TSP2, MMP2, and MMP9. Although MMP2 interacted with TSP1 and TSP2 via its gelatin-binding domain or a closely mapping site, neither TSP1 nor TSP2 was degraded by MMP2 in vitro. Tissue culture and in vitro assays demonstrated that the presence of purified TSR and intact TSP1 resulted in inhibition of MMP activity. The ability of TSP1 to inhibit MMP3-dependent activation of pro-MMP9 and thrombin-induced activation of pro-MMP2 suggests that the TSPs may inhibit MMP activity by preventing activation of the MMP2 and MMP9 zymogens.The thrombospondin (TSP) 1 family of proteins includes at least five related extracellular matrix glycoproteins encoded by separate genes (1). Each thrombospondin contains NH 2 -and COOH-terminal globular domains flanking different structural modules ( Ref. 2; Fig. 1A). The modules common to all TSP proteins include epidermal growth factor-like type 2 and Ca 2ϩ -binding type 3 repeats. In addition to these common features, TSP1 and TSP2 share two additional modules: a procollagen homology region and three properdin-like type 1 repeats (TSR).The NH 2 and COOH termini contain regions that are low in cysteine content, do not possess internal repeating motifs, and do not show homology with other proteins (3). The aminoterminal domain is largely responsible for the heparin binding properties of thrombospondins (4, 5). The procollagen region contains a region homologous to a cysteine-rich domain in the NH 2 -terminal propeptide of the ␣ 1 -chain of type 1 procollagen (6). The type 1 repeats are homologous to sequences in a variety of proteins found in protozoans (7-9), invertebrates (10), and mammals (11-16).Thrombospondin 1 has been the most extensively studied TSP protein to date. TSP1 contains a growing number of sites that have been implicated in interactions with more than 30 cell surface and matrix proteins, including structural proteins (e.g. collagen and fibronectin), cell surface receptors (e.g. integrins, syndecans, and CD36), enzymes (e.g. elastase and plasmin), and cytokines (e.g. transforming growth factor-␤1) (17). Because of its ability to interact with such a wide variety of proteins, TSP1 has been implicat...

Identification of molecular markers and signaling pathway in endometrial cancer in Hong Kong Chinese women by genome-wide gene expression profiling

“…Thrombospondin 2 is another potent inhibitor of angiogenesis (Streit et al, 1999). This effect may be due to its ability to inhibit activation of pro-MMP9 protein, as well as downregulate MMP9 expression through a receptor-mediated event (Bein and Simons, 2000;Kamochi et al, 2003). Decreased THBS2 levels may be the result of enhanced MYB expression, which can antagonize transcription of THBS2 (Bein et al, 1998).…”

Gelatinase-mediated migration and invasion of cancer cells

“…Contrasting activities have also been reported, as TSP-1 upregulates MMP-9 expression and stimulates invasion of endothelial cells in vitro (Qian et al, 1997). Yeast two-hybrid assays revealed that the thrombospondin type 1 repeats in TSP-1 and TSP-2 interact with the collagen-binding domain of MMP-2 and MMP-9 indicating the potential inhibition mechanism (Bein and Simons, 2000).…”

“…Other extra domains that are not common to all members of MMPs include the collagenbinding domain (CBD) of gelatinases and the transmembrane domains of MT-MMPs. The CBD domain is composed of three fibronectin type II like repeats and is involved in binding of collagenous substrates and elastin (Steffensen et al, 1995), fatty acids (Berton et al, 2001) and thrombospondins (Bein and Simons, 2000). Although most of the MMPs are secreted proteins, six of them contain a transmembrane domain that is used to anchor these proteins on the cell surface.…”

The effect of thrombospondin-1 on breast cancer metastasis

“…By contrast, TSP-1 is highly expressed by stromal fibroblasts, macrophages and endothelial cells in breast cancer [7]. In the tumor microenvironment, TSP-1 interacts with a wide range of other proteins to affect (1) the level of active transforming growth factor β (TGFβ) [8,9], (2) the level of vascular endothelial cell growth factor (VEGF) [10,11], (3) the composition of the extracellular matrix and the activity of extracellular proteases [10,12], and (4) the survival and migration of endothelial cells [13][14][15]. In addition, TSP-1 may inhibit tumor angiogenesis by down-regulating circulating endothelial cell progenitors [16,17].…”