Susan J. Atkinson

Cellular Mechanisms for Human Procollagenase-3 (MMP-13) Activation EVIDENCE THAT MT1-MMP (MMP-14) AND GELATINASE A (MMP-2) ARE ABLE TO GENERATE ACTIVE ENZYME

Gelatinase A and membrane-type metalloproteinase (MT1-MMP) were able to process human procollagenase-3 (Mr 60,000) to the fully active enzyme (Tyr85 N terminus; Mr 48,000). MT1-MMP activated procollagenase-3 via a Mr 56,000 intermediate (Ile36 N terminus) to 48,000 which was the result of the cleavage of the Glu84-Tyr85 peptide bond. We have established that the activation rate of procollagenase-3 by MT1-MMP was enhanced in the presence of progelatinase A, thereby demonstrating a unique new activation cascade consisting of three members of the matrix metalloproteinase family. In addition, procollagenase-3 can be activated by plasmin, which cleaved the Lys38-Glu39 and Arg76-Cys77 peptide bonds in the propeptide domain. Autoproteolysis then resulted in the release of the rest of the propeptide domain generating Tyr85 N-terminal active collagenase-3. However, plasmin cleaved the C-terminal domain of collagenase-3 which results in the loss of its collagenolytic activity. Concanavalin A-stimulated fibroblasts expressing MT1-MMP and fibroblast-derived plasma membranes were able to process human procollagenase-3 via a Mr 56,000 intermediate form to the final Mr 48,000 active enzyme which, by analogy with progelatinase A activation, may represent a model system for in vivo activation. Inhibition experiments using tissue inhibitor of metalloproteinases, plasminogen activator inhibitor-2, or aprotinin demonstrated that activation in the cellular model system was due to MT1-MMP/gelatinase A and excluded the participation of serine proteinases such as plasmin during procollagenase-3 activation. We have established that progelatinase A can considerably potentiate the activation rate of procollagenase-3 by crude plasma membrane preparations from concanavalin A-stimulated fibroblasts, thus confirming our results using purified progelatinase A and MT1-MMP. This new activation cascade may be significant in human breast cancer pathology, where all three enzymes have been implicated as playing important roles.

The TIMP2 Membrane Type 1 Metalloproteinase “Receptor” Regulates the Concentration and Efficient Activation of Progelatinase A A KINETIC STUDY

We have used C-terminal domain mutants to further define the role of interactions of progelatinase A and membrane type 1 matrix metalloproteinase (MT1 MMP) in the binding of TIMP2 and in the cell-associated activation of progelatinase A. Soluble constructs of MT1 MMP were used to demonstrate that binding with TIMP2 occurs primarily through N-terminal domain interactions, leaving the C-terminal domain free for interactions with progelatinase A. The rate of autolytic activation of progelatinase A initiated by MT1 MMP cleavage could be potentiated by concentration of the proenzyme by binding to heparin. Residues 568–631 of the progelatinase A C-terminal domain are important in formation of the heparin binding site, since replacement of this region with the corresponding stromelysin-1 sequence abolished binding to heparin and the potentiation of activation. The same region of gelatinase A was required for binding of latent and active enzyme to TIMP2, but residues 418–474 were not important. A similar pattern was seen using cell membrane-associated MT1 MMP; residues 568–631 were required for binding and activation of progelatinase A, whereas residues 418–474 were not. Neither region was required for activation in solution. The addition of TIMP2 to HT1080 membrane preparations expressing MT1 MMP, but depleted of endogenous TIMP2, resulted in potentiation of progelatinase A activation. This effect was dependent upon TIMP2 binding to MT1 MMP rather than at an independent membrane site. Together, the data suggest that TIMP2 forms a receptor with MT1 MMP that regulates the concentration and efficient generation of functionally active gelatinase A.

The Soluble Catalytic Domain of Membrane Type 1 Matrix Metalloproteinase Cleaves the Propeptide of Progelatinase A and Initiates Autoproteolytic Activation REGULATION BY TIMP-2 AND TIMP-3

It has been proposed that the cell-mediated activation of progelatinase A requires binding of the C-terminal domain of the proenzyme to a membrane-associated complex of the membrane type matrix metalloproteinase MT1-MMP and TIMP-2. Subsequent sequential proteolysis of the propeptide by MT1-MMP and gelatinase A is thought to generate the active form of gelatinase A. We have prepared the proform of the catalytic domain of the MT1-MMP and demonstrated that this may be activated in vitro by trypsin proteolysis to yield a functional proteinase capable of cleaving typical metalloproteinase peptide substrates, gelatin and casein. The active catalytic domain of MT1-MMP was also shown to activate progelatinase A to a fully active form. Using the inactive mutant pro-E375A gelatinase A, we dissected the propeptide processing events that occur. MT1-MMP cleaves the propeptide at the sequence Asn37-Leu38 only. Further cleavage of the mutant enzyme propeptide at Asn80-Tyr81, equivalent to that of the active wild type gelatinase A, could only be effected by addition of gelatinase A to the system. TIMP-1 was essentially unable to prevent MT1-MMP processing of wild type or E375A progelatinase A, whereas TIMP-2 and TIMP-3 were good inhibitors of these events. Analysis of the rate of binding of TIMPs to the catalytic domain of MT1-MMP using kinetic methods showed that TIMP-1 is an extremely poor inhibitor of MT1-MMP. In comparison, TIMP-2 and TIMP-3 are excellent inhibitors, binding more rapidly to the catalytic domain of MT1-MMP than to the catalytic domain of gelatinase A. These data demonstrate the basic mechanism of MT1-MMP action on progelatinase A and the reason for the lack of inhibition by TIMP-1 previously demonstrated in cell-based activation studies.

Mechanisms for pro matrix metalloproteinase activation

The activation of pro matrix metalloproteinases (MMPs) by sequential proteolysis of the propeptide blocking the active site cleft is regarded as one of the key levels of regulation of these proteinases. Potential physiological mechanisms including cell‐associated plasmin generation by urokinase‐like plasminogen activator, or the action of cell surface MT1‐MMPs appear to be involved in the initiation of cascades of pro MMP activation. Gelatinase A, collagenase 3 and gelatinase B may be activated by MT‐MMP based mechanisms, as evidenced by both biochemical and cell based studies. Hence the regulation of MT‐MMPs themselves becomes critical to the determination of MMP activity. This includes activation, assembly at the cell surfaces as TIMP‐2 complexes and subsequent inactivation by proteolysis or TIMP inhibition.

The Role of Plasmhogen Activators in the Regulation of Connective Tissue Metalloproteinasesa

The conversion of plasminogen to plasmin is a key event in many physiological and pathologal processes requiring regulated extracellular proteolysis. Direct focal degradation of proteins involved in cell-cell and cell-matrix interactions by plasmin has been described,js as well as activation of other degradative enzymes, notably the matrix rnetall~proteinases~ (MMPs). Complex control of the plasminogen activator cascade has been shown to be required for the movement and re-organization of cells and matrix in events such as ovulation, trophoblast implantation, embryogenesis, and angiogenesis, as well as in the invasion and metastasis of tumors. A further level of regulation is thought to be effected by the release by plasmin of matrix-sequestered growth factors, such as basic fibroblast growth factor and trdnsbrming growth factor /3,677 which are known to regulate the expression of both the plasminogen activatorinhibitor and the MMP-inhibitor systems2

Matrix metalloproteinases in arthritic disease

Chapter summary The role of matrix metalloproteinases in the degradative events invoked in the cartilage and bone of arthritic joints has long been appreciated and attempts at the development of proteinase inhibitors as potential therapeutic agents have been made. However, the spectrum of these enzymes orchestrating connective tissue turnover and general biology is much larger than anticipated. Biochemical studies of the individual members of the matrix metalloproteinase family are now underway, ultimately leading to a more detailed understanding of the function of their domain structures and to defining their specific role in cellular systems and the way that they are regulated. Coupled with a more comprehensive and detailed study of proteinase expression in different cells of joint tissues during the progress of arthritic diseases, it will be possible for the future development and application of highly specific proteinase inhibitors to be directed at specific key cellular events.

Regulation of Matrix Metalloproteinase Activitya

Matrix metalloproteinases (MMPs) are thought to initiate the degradation of the extracellular matrix during the remodeling of connective tissues. A clear understanding of the mechanisms governing the regulation of their activity during normal physiological processes should give further insights into the uncontrolled remodeling occurring in degradative pathologies. Regulation of the MMPs occurs at the level of gene expression, with precise spatial and temporal compartmentalization of both synthesis and secretion by resident cells as well as by those cells invading the tissue. Extracellularly, MMPs are further regulated by the extent of processing of the proform to an active enzyme and by the relative production of the specific inhibitors of MMPs, the tissue inhibitors of metalloproteinases (TIMPs). Furthermore, the potential for association of MMPs with the cell surface or extracellular matrix components further constrains their relationship with substrates, activators and inhibitors, acting as a further regulator of MMP activity. We initiated a program of study of the MMPs and TIMPs to ascertain the relation between their structure and their function, with particular emphasis on the mechanisms of biological regulation. Recombinant wild-type proteins and specific mutants, including deletion and site mutations, have been prepared using a mammalian expression system.’ Aspects of our findings, which illustrate the fundamental importance of the domain structure of MMPs and TIMPs in their biology, are presented here.

Tissue inhibitor of metalloproteinases-2 inhibits the activation of 72 kDa progelatinase by fibroblast membranes

We report that monolayers of human fibroblasts stimulated with concanavalin A were able to activate 72 kDa progelatinase but not 95 kDa progelatinase. The activating capacity of fibroblasts appeared approx. 6 h after concanavalin A stimulation and was blocked by cycloheximide. The activation of 72 kDa progelatinase was readily inhibited by TIMP-2 but only poorly by TIMP-1. Plasma membranes isolated from the fibroblasts were capable of activating 72 kDa progelatinase. The cleavage products of the plasma membrane-mediated activation of 72 kDa progelatinase corresponded to those of organomercurial-induced self-cleavage. Only inhibitors of metalloproteinase self-cleavage inhibited the activating capacity of plasma membrane preparations, although the activating capacity was destroyed by trypsin and heat. As with the fibroblast monolayers, TIMP-2 was a potent inhibitor of the membrane-mediated activation whereas TIMP-1 was less so.

Specific collagenolysis by gelatinase A, MMP-2, is determined by the hemopexin domain and not the fibronectin-like domain

In view of the essential role of the hemopexin domain of the traditional interstitial collagenases, MMP‐1, ‐8, ‐13 and MT1‐MMP (MMP‐14), in determining specific collagen cleavage we have studied the function of this domain in MMP‐2, relative to that of the fibronectin‐like domain that promotes gelatinolysis. Although the fibronectin‐like domain promotes avid binding to collagen, our data demonstrate that the catalytic and hemopexin domains of MMP‐2 are sufficient to effect the critical step in cleavage of rat type I collagen into 3/4 and 1/4 fragments. The mechanism of MMP‐2 cleavage of collagen proceeds in two phases, the first resembling that of the interstitial collagenases, followed by gelatinolysis, promoted by the fibronectin‐like domain.

Activation of pro-(matrix metalloproteinase-2) (pro-MMP-2) by thrombin is membrane-type-MMP-dependent in human umbilical vein endothelial cells and generates a distinct 63 kDa active species.

Thrombin, a critical enzyme in the coagulation cascade, has also been associated with angiogenesis and activation of the zymogen form of matrix metalloproteinase-2 (MMP-2 or gelatinase-A). We show that thrombin activated pro-MMP-2 in a dose- and time-dependent manner in cultured human umbilical-vein endothelial cells (HUVECs) to generate a catalytically active 63 kDa protein that accumulated as the predominant form in the conditioned medium. This 63 kDa thrombin-activated MMP-2 is distinct from the 62 kDa species found following concanavalin A or PMA stimulated pro-MMP-2 activation. Hirudin and leupeptin blocked thrombin-induced pro-MMP-2 activation, demonstrating that the proteolytic activity of thrombin is essential. However, activation was also dependent upon membrane-type-MMP (MT-MMP) action, since it was blocked by EDTA, o-phenanthroline, hydroxamate metalloproteinase inhibitors, tissue inhibitor of metalloproteinase-2 (TIMP-2) and TIMP-4, but not TIMP-1. Thrombin inefficiently cleaved recombinant 72 kDa pro-MMP-2, but efficiently cleaved the 64 kDa MT-MMP-processed intermediate form in the presence of cells. Thrombin also rapidly (within 1 h) increased cellular MT-MMP activity, and at longer time points (>6 h) it increased expression of MT1-MMP mRNA and protein. Thus signalling via proteinase-activated receptors (PARs) may play a role in thrombin-induced MMP-2 activation, though this does not appear to involve PAR1, PAR2, or PAR4 in HUVECs. These results indicate that in HUVECs the activation of pro-MMP-2 by thrombin involves increased MT-MMP activity and preferential cleavage of the MT-MMP-processed 64 kDa MMP-2 form in the presence of cells. The integration of these proteinase systems in the vascular endothelium may be important during thrombogenesis and tissue remodelling associated with neovascularization.