Matrix metalloproteinases in lung biology

Despite much information on their catalytic properties and gene regulation, we actually know very little of what matrix metalloproteinases (MMPs) do in tissues. The catalytic activity of these enzymes has been implicated to function in normal lung biology by participating in branching morphogenesis, homeostasis, and repair, among other events. Overexpression of MMPs, however, has also been blamed for much of the tissue destruction associated with lung inflammation and disease. Beyond their role in the turnover and degradation of extracellular matrix proteins, MMPs also process, activate, and deactivate a variety of soluble factors, and seldom is it readily apparent by presence alone if a specific proteinase in an inflammatory setting is contributing to a reparative or disease process. An important goal of MMP research will be to identify the actual substrates upon which specific enzymes act. This information, in turn, will lead to a clearer understanding of how these extracellular proteinases function in lung development, repair, and disease.


Introduction
MMPs comprise a large family of extracellular enzymes that share common structural features, particularly those regions involved in the regulation of proteolytic activity. MMPs, or matrixins, are a subgroup of the much larger metalloproteinase superfamily, which also includes astacin, ADAM, and ADAMTS proteinases, among others [1,2]. Twenty-three different vertebrate MMPs have been cloned to date (Table 1), and additional members continue to be identified.
Numerous studies have assessed the mechanisms controlling MMP expression in lung tissue and lung-derived cells, and to localize the sites of enzyme expression in various diseases. We will not summarize everything that is known about MMPs in the lung, as there are simply too many papers to summarize in a succinct review. We shall instead discuss general and emerging concepts of MMP biology. We will also summarize work on two enzymes, matrilysin and macrophage metalloelastase, as examples of how MMPs can function either beneficially in lung repair or destructively in lung disease.
binds three conserved histidines in the sequence HEXXHXXGXXHS/TXXXXXXM, which also contains a conserved methionine to the carboxy side of the zincbinding site. In the inactive state, the conserved cysteine residue in the pro-domain provides the fourth coordination site for the catalytic zinc ion, and disruption of this bond is necessary for enzyme activity.
In addition to the two conserved regions, MMPs have a variety of specialized domains that contribute to substrate specificity and recognition or interaction with other proteins or molecules. With the exception of matrilysin, CA-MMP, and endometase/matrilysin-2 [3][4][5][6][7], MMPs have a hinge region, which is often proline rich, and a hemopexin-like C-terminal domain. Four of the six membrane-type MMPs (MT1, MT2, MT3, and MT5) have transmembrane and cytosolic domains, whereas MT4-MMP and leukolysin (MT6-MMP) also have C-terminal hydrophobic extensions that act as a glycosylphosphatidylinositol anchoring signal [3,8,9]. The two gelatinases (MMP-2 and MMP-9) have gelatin-binding domains, and CA-MMP has a novel cysteine array motif and an immunoglobulin-like C2-type fold domain [3,10]. MMPs also share a similar gene arrangement, suggesting that they were generated by duplications of an ancestor gene. At least eight of the known human MMP genes (MMP-1, MMP-3, MMP-7, MMP-8, MMP-10, MMP-12, MMP-13, and MMP-20) are clustered on chromosome 11 at 11q21-23 [11 • ]. Other known MMP genes are scattered along chromosomes 1, 8, 12, 14, 16, 20, and 22. Many of the secreted MMPs, such as collagenase-1 and -3, stromelysins 1, 2, and 3, and gelatinase-B, are not expressed in normal, healthy, resting tissues or their production and activity are maintained at nearly undetectable levels. In contrast, some level of MMP expression is seen in any repair or remodeling process, in any diseased or inflamed tissue, and in essentially any cell type grown in culture. Although the qualitative pattern and quantitative levels of MMPs vary among tissues, diseases, tumors, inflammatory conditions, and cell lines, a reasonably safe generalization is that activated cells express MMPs. This is neither a controversial nor a surprising conclusion. After all, in inflammation, cancer, repair, and other remodeling processes, the matrix is turned over, cells are migrating, and secreted factors are processed, and these, among other events, are the functions attributed to MMPs. Some MMPs, including matrilysin, endometase/matrilysin-2, leukolysin, MT5-MMP, and MMP-19, are expressed in healthy tissues [7,[12][13][14], implying roles in tissue homeostasis.

Functions and substrates
As their name suggests, MMPs are thought to be responsible for the turnover and degradation of connective tissue proteins, a function that is clearly performed by several family members. Indeed, cancer patients (including some with lung carcinomas) enrolled in clinical studies to assess the chemotherapeutic effects of a broad-acting synthetic metalloproteinase inhibitor developed reversible skin thickening and joint contractures [15,16]. These sclerotic side effects have been interpreted to indicate that some MMPs, directly or indirectly, are required for the normal catalysis of connective tissue. Although numerous biochemical studies have demonstrated that almost all MMPs can cleave or degrade some protein component(s) of the extracellular matrix, an ability to act on connective tissue protein is not a requirement for membership into the matrix metalloproteinase family. For example, human stromelysin-3 and CA-MMP have no known extracellular matrix substrates [3,17]. As for most protein families, structural The use of genetically defined animal models has allowed investigators to uncover specific and, at times, unexpected functions of MMPs ( Table 2). All of the MMPs targeted to date (Table 2), except MT1-MMP, show no or only a minor phenotype in unchallenged mice, indicating that these enzymes do not serve vital functions in development or homeostasis. The responses of MMP knockout mice to a variety of challenges, in contrast, indicate that these enzymes do serve specific roles in tissue repair, immunity, angiogenesis, host defense, inflammation, and tumor progression, among others (Table 2). It thus seems that several MMPs, at least those that have been knocked out, have evolved to respond to the many insults we experience in our extra-uterine existence. Table 2 Phenotype of matrix metalloproteinase (MMP) knockout mice [56] • Reduction in angiogenesis and tumor growth [57] Stromelysin-1 (MMP-3) No • None [58] • Impaired wound contraction [59] • Reduced contact hypersensitivity response [60] • Accelerated arthritis [58] Matrilysin As for all secreted proteinases, the catalytic activity of MMPs is regulated at four points -gene expression, compartmentalization, enzyme activation, and enzyme inactivation -and is further controlled by substrate availability and affinities. Although each MMP is often described as having a limited substrate specificity, individual enzymes can act on many different proteins, and the spectrum of substrates among many enzymes is actually quite similar. For example, gelatinase-A, gelatinase-B, stromelysin-1, stromelysin-2, matrilysin, macrophage metalloelastase, and collagenase-3 can each degrade non-fibrillar collagens, basement membrane components, fibrillins, fibronectin, and more. Distinct from other MMPs, collagenase-1 and collagenase-2 seem to have a very defined substrate spectrum, being limited to the fibrillar collagens, types I, II, and III. Aside from the collagenases, the set of substrates that overlap among MMPs is more impressive than that of selective substrates.
In a setting such as inflammation, in which essentially all MMPs are present, the shared substrate potential among enzymes would seemingly permit biochemical redundancy. Two processes, however, can hone substrate selectivity: enzyme affinity and compartmentalization. Kinetic studies have demonstrated that specific enzymes degrade some substrates more efficiently than others. For example, both gelatinase-A and gelatinase-B act on cleaved collagen better than other MMPs [30], matrilysin is a more potent proteoglycanse than stromelysin-1 or gelatinase-B [31], macrophage metalloelastase is the most elastolytic enzyme of the MMP family [32], and only the collagenases can cleave native fibrillar collagens. In a complex tissue environment, which contains many types of matrix proteins and many other potential substrates, selectivity of MMP catalysis may thus be directed, in part, by the concentration of a preferred substrate relative to that of other potential substrates within range of a secreted MMP.

Compartmentalization
Where in the tissue environment and by which cells a MMP is expressed and released are equally, if not more important, considerations in regulating the specificity of proteolysis than the affinity of the enzyme-substrate interaction. For example, in a test tube, matrilysin inactivates α 1 -antiproteinase inhibitor much more efficiently than does gelatinase-B. However, in tissue, at least in inflamed dermis, this serpin is selectively cleaved by neutrophilderived gelatinase-B [22 •• ]. Matrilysin, an epithelial cell product, tends to be released lumenally away from the matrix (see later). An important concept is that cells do not indiscriminately release proteases. Rather, proteinases, such as MMPs, are secreted and anchored to the cell membrane, thereby targeting their catalytic activity to specific substrates within the pericellular space. Specific cell-MMP interactions have been reported in recent years, such as the binding of gelatinase-A to the integrin α v β 3 [33], binding of gelatinase-B to CD44 [28], and binding of matrilysin to surface proteoglycans [34]. Pro-gelatinase-A also interacts with tissue inhibitor of metalloproteinases (TIMP)-2 and MT1-MMP on the cell surface, and this trimeric complex is essential for activation of this gelatinase [35,36]. It is likely that other MMPs are also attached to cells via specific interaction to membrane proteins, and determining these anchors will lead to identifying activation mechanisms and pericellular substrates.
Cells also rely on surface receptors to 'sniff out' the presence and location of specific substrates. For matrix substrates, integrin-ligand contacts provide an unambiguous signal informing the cell of which protein it has encountered and, hence, which proteinase is needed and to where the enzyme should be delivered and released. A clear example of this type of spatial regulation is seen with collagenase-1 in human cutaneous wounds. Collagenase-1 is induced in basal epidermal cells (keratinocytes), in response to injury, as the cells move off the basement membrane and contact type I collagen in the underlying dermis [37]. Only basal keratinocytes in contact with dermal type I collagen express collagenase-1, and this inductive response is specifically controlled by the collagen-binding integrin α 2 β 1 , which also directs secretion of the enzyme to the points of cell-matrix contact [38]. This example demonstrates that expression and activity of a specific MMP can be confined to a specific location in an activated tissue (the superficial plane of a denuded epithelium) and to a specific stage of repair (re-epithelialization).

Matrilysin
As already stated, matrilysin, the smallest (28 kDa to be an important component of innate immunity. The αand β-defensins comprise a family of cationic peptides that kill bacteria by membrane disruption [41]. The pro-segment of α-defensin precursors maintains them in an inactive state, and proteolysis at some point in the secretion pathway is needed to remove the pro-domain. In the mouse small intestine, matrilysin is co-expressed and co-sorted with pro-α-defensins in Paneth cells, which are specialized epithelial cells that secrete defensins and other antimicrobial molecules, and this intimate compartmentalization of proteinase and potential substrate suggests that matrilysin activates these peptides in the secretion pathway. Indeed, in matrilysin knockout mice, pro-α-defensins are not processed to their active forms by matrilysin, and deficiency of this enzyme results in impaired bacteriocidal activity in vitro and in vivo [19 •• ]. Because of a lack of defensin activation, matrilysin null mice cannot effectively kill pathogenic Escherichia coli and are themselves killed by doses of Salmonella typhimurium that are not lethal to wild-type mice. Matrilysin thus functions in mucosal immunity by regulating the activity antimicrobial peptides, a function that this MMP may also serve in the lung. Because of the role of matrilysin in innate defense, it became reasonable to hypothesize that the interaction of bacteria with host cells regulates enzyme expression. Bacteria are, after all, an indirect 'substrate' of matrilysin, and the presence of substrates often regulates MMP production. Indeed, matrilysin is strongly induced (> 50-fold) in human and murine mucosal epithelial tissues, including intact human and mouse airway, by bacterial exposure [40 • ]. This induction is remarkably potent and sensitive, requiring relatively short exposure and less than 10 bacteria per epithelial cell in the initial inoculum, and is not mediated by lipopolysaccharide. Large amounts of matrilysin protein, in both its zymogen and activated forms, are released from infected airway epithelial cells and from normal human trachea (Fig. 1). In addition, bacteria-mediated stimulation of matrilysin is restricted to mucosal epithelial cells. Bacterial exposure does not affect the expression of other MMPs examined and does not influence matrilysin expression in other cell types, namely macrophages, fibroblasts, and keratinocytes.
The widespread production of matrilysin in conducting airway epithelium may be initially induced and subsequently sustained by continual, low-level bacterial exposure. Consistent with this idea, matrilysin is seen in adult tissues but is not detected in developed glandular epithelium in utero [42] or in fetal or perinatal mouse tissues [43]. Furthermore, matrilysin is not produced at detectable levels in adult germ-free mice but is expressed in mice with a conventional microflora and in exgerm-free mice colonized with just one species of commensal bacteria [40 • ]. Bacterial exposure thus seems to be the physiologic signal that regulates matrilysin expression in intact epithelium.
Although matrilysin is clearly regulated by bacterial exposure, it is possible that production of this enzyme is also induced in response to injury. Because injury provides an opportunity for infection and infection can lead to injury, some of the epithelial responses associated with either of these events may actually be a key component of both responses. Expression of matrilysin is indeed seen in airway and alveolar epithelial cells at sites of overt damage in emphysema, in fibrotic lungs with diffuse alveolar damage, in inflamed lungs of patients with idiopathic pneumonia syndrome following bone marrow transplantation, and most prominently in lungs from patients with CF [39 •• ]. Because bacterial colonization may be associated with sites of airway injury, especially in the CF samples, it was not clear if injury alone can induce matrilysin expression. A prominent signal for matrilysin protein is, however, seen in migrating epithelial cells in isolated human tracheas that are injured under aseptic conditions (Fig. 2).
These observations demonstrate that matrilysin is induced by injury and is prominently expressed by migrating epithelial cells. Although we do not yet know what process or factor induces matrilysin in epithelial cells at the wound edge, candidates would include a loss of specific cell-cell  In tracheas from wild-type mice, re-epithelialization progresses rapidly; however, wounds in tracheas from matrilysin null mice show no evidence of epithelial migration. Matrilysindeficient mice have in fact shown the most severe woundrepair defect among the MMP knockout mice generated to date (Table 2). However, the mechanism of how matrilysin facilities repair is not known. It may be required to loosen cell-matrix and cell-cell contacts, as has been suggested for other MMPs in other epithelial repair/migration models [38,44 • ]. Furthermore, matrilysin may not be the only MMP involved in repair of airway epithelial wounds. Gelatinase-B is also expressed by injured epithelial cells in distal airways, in response to bleomycin instillation, and deficiency of this MMP leads to excessive bronchiolization [45 • ], suggesting a role in either cell migration or differentiation. In addition, Legrand et al have demonstrated that the activity of gelatinase-B is required for the migration of isolated airway epithelial cells over a matrix substratum [46]. Several MMPs may thus act concurrently on different substrates to facilitate repair. These observations also demonstrate important reparative roles for MMPs but, as is discussed later, overexpression or chronic expression of proteinases may lead to tissue damage.

Macrophage metalloelastase
The progressive structural damage associated with emphysema and other forms of chronic obstructive pulmonary disease (COPD) is due to degradation of selective extracellular matrix components. To determine which proteinases are involved in the development of COPD, one must focus on proteinases that can degrade elastin, a highly proteinase-resistant polymer that normally lasts the human lifespan [47]. The elastin content of the lung parenchyma is decreased in emphysema, while the collagen content is increased [48]. Some studies have proposed that collagenases, such as collagenase-1, contribute to airway enlargement [49], but the accumulation of collagen at sites of emphysematous damage strongly indicates that lung collagenases are acting elsewhere. Elastic fibers in the emphysematous lung are ultrastructurally disorganized and fragmented. Instillation of elastases, but not collagenases, into the lungs of experimental animals causes emphysema, further implicating elastolytic enzymes in human disease.
Several MMPs are produced in human emphysematous lung [50,51]. Furthermore, several studies using transgenic and gene-targeted mice lend support to the role of MMPs in emphysema. For example, induced overexpression of IL-13 results in production of several MMPs, leading to emphysema [52]. Among these MMPs, metalloelastase was a reasonably guilty culprit in the breakdown of alveolar elastin. As already mentioned, this enzyme is a potent elastase, and macrophages from metalloelastase null mice cannot degrade elastin or many other matrix substrates [53].
Exposing proteinase null mice to cigarette smoke provides a highly controlled model to assess the role of specific MMPs in emphysema. Long-term exposure of wild-type mice to cigarette smoke leads to inflammatory cell recruitment followed by alveolar space enlargement that is quite similar to the lesions that develop in humans. Mice deficient in metalloelastase, however, are markedly protected from development of emphysema due to long-term smoke exposure [ [55]. Gene targeting is merely reinforcing this as a major in vivo mechanism of macrophage accumulation in a chronic inflammatory condition.
There are probably several proteinases and inflammatory cells involved in the development of emphysema in humans. The major lesson to be gained from murine models of cigarette smoke-induced emphysema is not that metalloelastase is the sole proteinase responsible for human disease, but that macrophages have the capacity to cause emphysema upon recruitment and activation by cigarette smoke. The predominant macrophage proteinase in mice is metalloelastase; human macrophages probably have a broader spectrum of MMPs (including metalloelastase). Collagenase-1, -2, and -3 also undoubtedly contribute to loss of the airspace; yet, in the small airspace, collagen deposition predominates, leading to airway obstruction complicating simple categorization of collagenases as 'bad guys'.
Moreover, multiple enzymes from different families may cooperate in tissue remodeling at the same time and place. For example, metalloelastase, as well as other MMPs, degrades α 1 -antiproteinase inhibitor, and neutrophil elastase, a serine proteinase, degrades TIMPs. These enzymes, by neutralizing each other's natural inhibitors, can thus amplify overall proteolytic activity. This concept was shown in vivo in studies by Liu et al [22 •• ]. They demonstrated, using a murine model of bullous pemphigoid, that skin blisters form as a result of neutrophil MMP-9 degrading α 1antiproteinase inhibitor, thus allowing unopposed neutrophil elastase activity, which degrades hemidesmosomal proteins. We have not been able to determine a role for MMP-9 in emphysema; however, metalloelastase degrades α 1 -antiproteinase inhibitor, in part explaining why metalloelastase mice are totally protected from smoke-induced emphysema, whereas neutrophil elastase mice are only partially protected.

Conclusion
Several studies have shown that excessive levels of many MMPs are present in chronically inflamed tissues throughout the body. These observations have led to the often held concept that excessive proteolysis damages tissues and impairs healing. Unregulated, excessive proteolysis is indeed probably responsible for the destruction of matrix proteins in emphysema, as well as in arthritis, aneurysms, and other conditions of structural tissues. It seems presumptuous, however, to conclude that all proteinases expressed in diseased tissue contribute to disease pathogenesis. Based on the mechanisms uncovered in matrilysin and MMP-9 null mice, expression of these MMPs in injured airway cells may reflect beneficial roles in re-epithelialization and mucosal defense against bacteria. Because MMPs can both breakdown and activate proteins, we cannot conclude by their presence alone whether a specific proteinase in an inflammatory or injury setting is contributing to a reparative or disease process.  Invest 1998, 102:1321-1331. The observations reported in this paper demonstrate that the matrilysin is induced in response to airway injury and is markedly upregulated in CF, and that its catalytic activity is essential for the repair of airway epithelial wounds.