Regulatory Mechanism of Matrix Metalloprotease-2 Enzymatic Activity by Factor Xa and Thrombin

Abstract

Matrix metalloprotease (MMP)-2 plays a key role in many biological and pathological processes related to cell migration, invasion, and mitogenesis. MMP-2 is synthesized as a zymogen that is activated through either a conformational change or proteolysis of the propeptide. Several activating enzymes for pro-MMP-2 have been proposed, including metalloproteases and serine proteases. The mechanism of pro-MMP-2 activation by metalloproteases is well established, and the most studied activation mechanism involves cleavage of the propeptide by membrane type 1-MMP (MT1-MMP). In contrast, serine protease activation has not been thoroughly studied, although studies suggest that MT1-MMP may be involved in activation by thrombin and plasmin. Here, we demonstrate that factor Xa mediates MT1-MMP-independent processing of pro-MMP-2 in vascular smooth muscle cells and endothelial cells. Factor Xa and thrombin directly cleaved the propeptide on the carboxyl terminal sides of the Arg 98 and Arg 101 residues, whereas plasmin only cleaved the propeptide downstream of Arg 101 . Moreover, processed MMP-2 showed enzymatic activity that was enhanced by intermolecular autoproteolytic processing at the Asn 109 -Tyr peptide bond. In addition to its role in activation, factor Xa rapidly degraded MMP-2, thereby restricting excessive MMP-2 activity. Thrombin also degraded MMP-2, but the degradation was reduced greatly under cell-associated conditions, resulting in an increase in processed MMP-2. Overall, factor Xa and thrombin regulate MMP-2 enzymatic activity through its activation and degradation. Thus, the net enzymatic activity results from a balance between MMP-2 activation and degradation. Matrix metalloprotease (MMP)3 -2 is a member of the zincdependent endopeptidase family, which comprises 24 enzymes (1). MMP-2 plays a key role in many biological and pathological processes, including organ growth, endometrial cycling, wound healing, bone remodeling, tumor invasion, and metastasis (2). This enzyme functions through proteolysis of non-structural extracellular molecules and components of the basement membrane, including type IV collagen, fibronectin, elastin, laminin, aggrecan, and fibrillin (3).Like most MMPs, MMP-2 is synthesized as a zymogen that is activated by conformational change (4) or proteolysis within the propeptide, which may involve membrane type MMPs (MT-MMPs) (5-9). The most studied activation mechanism for pro-MMP-2 is cleavage of the propeptide by MT1-MMP, which requires cooperative activity between MT1-MMP and tissue inhibitor of metalloprotease (TIMP)-2 (5, 10 -12). Serine proteases, such as thrombin, factor Xa, activated protein C, and plasmin as well as the cysteine protease legumain are all known activators of pro-MMP-2 (13-17). (25,26). Both proteases can also elicit endothelial cell and SMC migration through pro-MMP-2 activation and subsequent extracellular matrix degradation (13,27,28). However, despite studies suggesting that MT1-MMP is involved in thrombin-mediated activation of pro-MMP-2, a detailed mechanism...

Thrombin-dependent MMP-2 Activity Is Regulated by Heparan Sulfate

“…In our previous study using metalloprotease inhibitors and pro-MMP-2 mutants incapable of cleavage by MT1-MMP, we showed MT1-MMP-independent activation of pro-MMP-2 by thrombin (26). We further demonstrated that thrombin cleaved the propeptide on the C-terminal side of Arg 98 and Arg 101 followed by intermolecular autoproteolytic cleavage at the Asn 109 -Tyr peptide bond for full enzymatic activity (26). In addition to its role in activation, thrombin also degraded MMP-2, but this degradation was reduced greatly under cell-associated conditions with a concomitant increase in activation (26).…”

Hexane and Chloroform Fractions of Laetiporus sulphrueus var. miniatus Inhibit Thrombin-treated Matrix Metalloproteinase- 2/9 Expression in Human Oral Squamous Carcinoma YD-10B Cells

“…Many reports suggest that thrombin regulates the gelatinolytic activity of MMP-2 and MMP-9 in cancer cell invasion [7][8][9][10]35]. However, the effect of thrombin on human oral squamous carcinoma cells is not fully understood.…”

Dimerization of Matrix Metalloproteinase-2 (MMP-2)

“…Expression Plasmids and Site-directed Mutagenesis-Plasmids for expression of full-length MMP-2 or MMP-2 with C-terminal Myc and His tags were described previously (7). To make an expression plasmid encoding hemopexin-like domaindeleted pro-MMP-2, site-directed mutagenesis was used to convert Pro 466 to a stop codon using a mutagenesis kit (iNtRON Biotechnology, Kyungki-Do, Korea).…”

“…In our previous studies, we demonstrated that thrombin directly cleaved the propeptide on the C-terminal side of Arg 98 and Arg 101 with a preference for Arg 101 ; this was followed by intermolecular autoproteolytic cleavage at the Asn 109 -Tyr peptide bond, resulting in full enzymatic activation (7). We further showed that heparan sulfate was required for thrombin-mediated activation of pro-MMP-2 (25).…”