Matrix Metalloproteinases (MMPs)

First printed in R&D Systems' 1999 Catalog.

Overview

The MMP family of enzymes contributes to both normal and pathological tissue remodeling. MMPs play a key role in the migration of normal and malignant cells through the body. They also act as regulatory molecules, both by functioning in enzyme cascades and by processing matrix proteins, cytokines, growth factors and adhesion molecules to generate fragments with enhanced or reduced biological effects.

Domain Structure & Function

Figure 1. MMPs can facilitate tumor cell metastasis and angiogenesis. Adapted from Opdenakker, G. & J. Van Damme (1992) Cytokine 4:251.

The matrix metalloproteinases (MMPs) are members of a family of at least 15 Zn-dependent endopeptidases that function extracellularly (Table 1).¹ The MMPs each contain a protease domain that has a conserved HExGHxxGxxHS/T sequence in which the three Histidine residues form a complex with a catalytic Zn atom. In addition, all MMPs contain a regulatory domain (pro-piece) with a conserved PRCGxPD motif that is responsible for maintaining latency in MMPs via binding of the cysteine residue to the active site Zn. The simplest MMP is MMP-7 (matrilysin), which consists of a pro-piece and catalytic domain only. The other MMPs maintain this basic unit but have a variable number of structural domains added. Although most MMPs are secreted proteins, the recently described membrane-type MMPs (MT-MMPs) are anchored to the cell membrane by a transmembrane and intracytoplasmic domain. X-ray crystallography has shown that the catalytic domains of the different MMPs have similar structure, but the topology of the active site clefts differs, accounting for some of the differences in substrate specificities. Differences in the other domains confers further substrate specificity, regulates binding to matrix proteins, and determines interactions with the Tissue Inhibitors of Metalloproteinases (TIMPs), the natural inhibitors of MMP activity.²

Substrates & Nomenclature

Much of the early literature suggested that each MMP had its own particular substrate.¹ This concept led to the use of substrate-focused nomenclature for MMPs such that the collagenases broke down intact fibrillar collagens, gelatinases degraded denatured collagen, and metalloelastase attacked elastin. It is now recognized that MMPs usually degrade multiple substrates, with considerable substrate overlap between individual MMPs. For example, interstitial collagenase (MMP-1) is capable of degrading casein, gelatin, a-1 antitrypsin, myelin basic protein, L-Selectin, pro-TNF and IL-1 beta and pro-MMP-2 and -9. 72-kDa gelatinase (MMP-2) can degrade fibrillar collagen, elastin, IGF-binding proteins, FGF receptor and can activate MMP-1, -9 and -13. MMP-12 is highly active against type IV collagen, gelatin, fibronectin, vitronectin and plasminogen, but it is not very effective at degrading elastin. See Table 1 for a list of substrates that can be cleaved by purified MMPs in vitro.

In an attempt to break the link between name and function, all MMPs are now given an MMP number, such that Interstitial collagenase is MMP-1, etc. (Table 1). There are holes in this system. There is no MMP-4, -5 or -6, as the activities could not be ascribed to a specific gene product, and MMP-18 is known only as a Xenopus enzyme. As with all other enzymes, MMPs have an EC classification, although this lags well behind the MMP designation.

Role in Physiology and Pathology

Although the link between single MMPs and individual substrates is not as direct as once thought, it is clear that as a family, the MMPs are capable of breaking down any extracellular matrix component (see Table 1). In normal physiology, MMPs produced by connective tissue are thought to contribute to tissue remodeling in development, in the menstrual cycle, and as part of repair processes following tissue damage. The obvious destructive capability of MMPs initially focused most research onto diseases that involve breakdown of the connective tissues (e.g., rheumatoid arthritis, cancer and periodontal disease). Leukocytes, particularly macrophages, are major sources of MMP production. MMPs released by leukocytes play vital roles in allowing leukocytes to extravasate and penetrate tissues, a key event in inflammatory disease. Opdenakker proposed that MMP action not only permits leukocyte emigration into tissues and causes tissue damage, it also generates immunogenic fragments of normal proteins that may escalate autoimmune disease. In an analogous way, metastatic cancer cells also use MMPs to get in and out of tissues and to establish a blood supply (Figure 1).⁴ Drug companies have synthesized low molecular weight MMP inhibitors that have shown efficacy in models of these diseases, reinforcing their central role in pathology.⁵

The MMP axis is highly regulated to avoid excessive tissue damage. Most MMPs, with the exception of 72 kDa gelatinase and the MT-MMPs, are not constitutively expressed in normal tissues. Inflammatory cytokines such as IL-1 and TNF, growth factors such as TGF-beta and noxious stimuli are required to initiate transcription. MMPs are also expressed as inactive zymogens (the pro-piece must be dissociated from the catalytic domain before the enzyme is activated). This dissociation can be achieved by autocatalysis or by the action of enzymes such as furin, plasmin or even other MMPs. For example, the activation of pro-MMP-2 occurs at the surface of many cells and is mediated by MT-MMPs. Once activated, MMPs are subject to inactivation by TIMPs and by binding to plasma proteins such as alpha-2 macroglobulin. It is thought that the local balance of MMP expression and activation versus the level of TIMP governs the level of destruction mediated by MMPs. This is of great significance when studying MMP involvement in disease processes.

In order to implicate a particular MMP in a disease, several overlapping approaches have been taken. Each has its advantages and disadvantages (Table 2). Development of a comprehensive picture of MMP involvement in any tissue culture system or in vivo disease model likely would require several of these methods.

The MMP/Cytokine Connection

Figure 2. The MMP/Cytokine Connection

The MMP axis has several areas of overlap with the cytokine network. As described above, inflammatory cytokines or growth factors can regulate the expression of MMPs. Cytokine activation of cells can also lead to increased processing of MMPs from the inactive zymogens to the active enzymes. Cytokines and their receptors can also be substrates for MMP action (Figure 2). Pro-inflammatory cytokine IL-1 beta can be cleaved and inactivated by MMP-1, -2, -3, and -9.⁹ In addition, the degradation of matrix proteins such as decorin can liberate growth factors such as TGF-beta that are sequestered on the matrix.¹⁰ Many membrane-bound cytokines, receptors and adhesion molecules can be released from the cell surface by the action of metalloproteinases, referred to as sheddases or convertases.^{11, 12} This may down regulate cell surface signaling by removal of a receptor or extend the sphere of influence of a molecule by release of a soluble active form. The consequences of this will depend on the molecule. For example, soluble TNF cleaved from the cell surface is pro-inflammatory, whereas TNF receptors cleaved from a cell act as soluble TNF inhibitors. In contrast, the cleaved soluble IL-6 receptor acts to stabilize IL-6 and the complex acts as an IL-6 agonist.

Although classical MMPs can process many of these cell surface molecules, members of the reprolysin or adamalysin clan of metalloenzymes may contribute much of the sheddase or convertase activity at the cell membrane. These enzymes have a catalytic site similar to that of the MMPs, but they have a different domain structure. The best characterized enzyme is the TNF-alpha converting enzyme (TACE or ADAM-17).^13-14 This enzyme was initially isolated from cell membranes using a TNF substrate assay to follow purification. Combined inhibitors of MMP and sheddase activity have been produced. They are active in models of inflammatory diseases, such as multiple sclerosis, where a cytokine drive has been implicated.¹⁵

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