The Cleavage of Pro-Urokinase Type Plasminogen Activator by Stromelysin-1

Abstract

Membrane binding of urokinase type plasminogen activator (u-PA) is thought to play a pivotal role in connective tissue remodeling and invasive processes. We compare the ability of different matrix-metalloproteinases involved in connective tissue turnover to cleave pro-urokinase type plasminogen activator between the catalytic domain and the receptor binding part to investigate a potential role for matrix-metalloproteinases in the regulation of membrane-associated proteolytic activity. We employed several forms of human stromelysin-1 (full length, C-truncated, and recombinant catalytic domain), rabbit C-truncated stromelysin-1, the human gelatinases A and B and the human catalytic domain of neutrophil collagenase. The gelatinases and the collagenase did not separate the receptor binding domain of pro-urokinase type plasminogen activator from the catalytic domain, whereas all stromelysin-1 forms cleaved the glutamic acid 143-leucine 144 bond of pro-urokinase type plasminogen activator. This reaction could be inhibited by specific inhibitors of matrix metalloproteinases and was not affected by inhibitors of serine proteinases. The M(r) 31000 cleavage product with leucine 144 as N-terminus displayed no proteolytic activity towards the pro-urokinase type plasminogen activator substrate pyroGlu-Gly-Arg-pNA-HCI (S2444), but it could be activated by an additional treatment with plasmin. Comparison between full length stromelysin-1 and its C-truncated forms, showed that both exhibited the same cleavage properties towards pro-urokinase type plasminogen activator. Thus, the cleavage of pro-urokinase type plasminogen activator by stromelysin-1 is not influenced by the presence or absence of the C-terminal domain. The recombinant catalytic domain of MMP-3 generated pro-urokinase type plasminogen activator, whereas incubation of pro-urokinase type plasminogen activator with the native forms of human or rabbit stromelysin-1 led to a moderate activation of pro-uPA due to an additional cleavage that is catalyzed by a serine proteinase.

Inhibition of human MDA‐MB‐231 breast cancer cell invasion by matrix metalloproteinase 3 involves degradation of plasminogen

“…This adds to previous reports [31,33,34,45] and provides additional details for a mechanism involving reduced cell-mediated plasminogen activation, through which MMP-3 may inhibit MDA-MB-231 invasion.…”

“…This has followed observations that patterns of MMP over-expression do not always predict aggressive tumour behaviour in either human [19,26,27] or animal tumours [28], that enhanced expression of the MMP inhibitors TIMP-1 and TIMP-2 frequently associates with poor outcome or tumour recurrence [29,30] and that MMPs can degrade components of the plasmin generating system and produce angiostatin, suggesting potential roles for MMPs in the regulation of cellular fibrinolytic activity and in the down-regulation of tumour-associated angiogenesis [31][32][33][34][35][36][37][38].…”

Matrix metalloproteinases: effectors of development and normal physiology

“…They may proteolytically activate latent proteases or inactivate their inhibitors. MMP-3 can cleave urokinase-type plasminogen activator (u-PA), separating the receptor-binding domain from its catalytic domain, without affecting catalytic activity (Orgel et al 1998;Ugwu et al 1998). The biological significance of this finding is unclear, but it is possible that removing receptor binding would result in decreased localization of u-PA to the cell surface.…”

Vascular endothelial growth factor (VEGF) induces rapid prourokinase (pro-uPA) activation on the surface of endothelial cells

“…4,5 It is, however, unclear where the first active uPA originates from: low-rate constant generation of active urokinase by plasmin is one working model; alternatively pro-uPA can become activated by additional enzymes including trypsin, kallikrein, factor XIIa, different cathepsins, and matrix metalloproteinases (MMPs). [6][7][8] VEGF transcriptionally up-regulates urokinase in its proenzyme form and VEGF induces invasion of endothelial cells that are dependent on active uPA, 1 indicating an initial requirement for VEGF-induced pro-uPA activation. How this initial activation of pro-uPA is achieved is unknown.…”