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Facultad de Ciencias, Universidad Nacional Autónoma de México; and Instituto Nacional de Enfermedades Respiratorias, Mexico DF, Mexico
Correspondence and requests for reprints should be addressed to Annie Pardo, Ph.D., Universidad Nacional Autónoma de México, Facultad de Ciencias, Ciudad Universitaria, CP 04510, México DF, México. E-mail: aps{at}fciencias.unam.mx
ABSTRACT
Pulmonary fibrosis, the final result of a large variety of interstitial lung diseases, is characterized by an aberrant remodeling of extracellular matrix (ECM) with a profound disturbance of the normal lung architecture. This remodeling includes the exaggerated accumulation of ECM components in the interstitial and alveolar spaces and the disruption of the basement membranes. It has long been accepted that matrix metalloproteases (MMPs) play an important role in the pathogenesis of pulmonary fibrosis, but the exact mechanisms are not well characterized. Several MMPs are strongly up-regulated in human and experimental lung fibrosis, highlighting the dynamic nature of scarring within the lung. MMPs are collectively capable of cleaving all components of the ECM and basement membranes, but importantly, they also process bioactive mediators such as growth factors, cytokines, chemokines, and cell-surface receptors. Moreover, they participate in the initiation of proteinase cascades that impact much broader substrates. Consequently, MMPs may play a central role in several interrelated processes observed in fibrosis such as ECM remodeling, basement-membrane breakdown, epithelial-cell apoptosis, cell migration, and angiogenesis.
Key Words: collagenase • gelatinase • matrilysin • matrix metalloproteinases • pulmonary fibrosis
Extracellular matrix (ECM) is a dynamic structure that plays a key role in lung architecture and homeostasis, providing much of the scaffold necessary for the regulation of cell behavior. ECM in the lung parenchyma comprises numerous molecules, including collagens (mainly type I and III), elastin, proteoglycans, and fibronectin. Specialized structures of the ECM, such as the epithelial and endothelial basement membranes, contribute with other proteins, including laminins, entactin, and collagen IV. The tightly controlled turnover of ECM is critical for maintaining lung morphostasis (1).
Lung fibrosis is a process characterized by an aberrant ECM remodeling with major alterations in both the quantity and composition of ECM. There are at least two important disturbances in the fibrotic lungs: accumulation of ECM in the interstitial and alveolar spaces and disruption of the basement membranes. In advanced stages, the fibrotic lung contains approximately two to three times more ECM than normal, including collagens (I, III, V, VI, and VII), fibronectin, elastin, and proteoglycans. Likewise, breakdown of basement membranes is an important event and it has been postulated that migration of fibroblasts/myofibroblasts into the alveolar spaces occurs through partially interrupted and denuded epithelial basement membranes (2). Thus, lung fibrosis can be minimally conceptualized as resulting from the loss in the regulation of synthesis and degradation of ECM molecules.
In this context, a large number of enzymes from the degradome are involved in ECM remodeling and degradation, primarily the family of matrix metalloproteases (MMPs), whose main characteristics will be illustrated.
MMPs
In humans, MMPs, or matrixins, are represented by 24 genes including duplicated MMP-23 genes. The resulting 23 enzymes of the MMP family are collectively capable of cleaving all components of the ECM and basement membranes (3). MMPs also generate ECM bioactive fragments and neoepitopes from basement-membrane proteins that have further biological effects (4). In addition, MMPs also process bioactive mediators such as growth factors, cytokines, chemokines, and cell-surface receptors, modulating their activity either by direct cleavage or by releasing them from ECM-bound stores. Moreover, they participate in the initiation of proteinase cascades that impact much broader substrates. Consequently, MMPs play a central role in the regulation of multiple cellular functions such as cell proliferation, adhesion, migration, differentiation, angiogenesis, and apoptosis.
The majority of the MMPs are secreted to the extracellular space and some of them are expressed as cell-surface enzymes. However, recent studies show that certain MMPs, such as MMP-1, MMP-2, and MMP-11, can also be found inside the cell and may act on intracellular proteins (5–8).
According to structural and functional characteristics, MMPs have been classified into six different subgroups of closely related members with rather distinctive but often overlapping substrate specificities: collagenases, gelatinases, stromelysins, matrilysins, membrane-type MMPs (MT-MMPs), and other MMPs (9). Recently, a new classification system has been proposed based on MMP structure rather than on their substrate specificity: archetypal MMPs, matrilysins, gelatinases, and furin activable MMPs (10, 11).
The archetypical secreted MMPs are organized around a catalytic structural domain with a conserved zinc-binding site. Incorporated into this site are a signal peptide that destines the protein to the endoplasmic reticulum, an amino-terminal propeptide (pro) domain that preserves the zymogens inactive until they are activated by proteolysis, and a hemopexin carboxy-terminal domain that is important in determining substrate specificity and interactions with endogenous inhibitors (Figure 1).
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REGULATION OF MMPs
MMPs proteolytic activity is regulated at three main levels: transcription, proenzyme activation, and inhibition. Additional mechanisms by which MMPs activity is controlled involve regulation of mRNA stability, translational efficiency, enzyme compartmentalization and secretion, cell-surface recruitment, substrate targeting, shedding, endocytosis, and intracellular degradation.
In general, MMPs levels are usually low in normal adult resting tissues, and with some exceptions, their production and activity are maintained at virtually undetectable levels. By contrast, their expression becomes elevated when there is a challenge to the system, such as wound healing, repair or remodeling processes, in diseased tissues, and even in several cell types grown in culture (13).
MMP expression is cell and tissue specific, temporally variable and complex, and there are several mechanisms by which cells regulate transcription of MMPs. It can be induced by various signals, such as cytokines and growth factors including interleukins and interferons, transforming growth factor ß, epidermal growth factor , keratinocyte growth factor, several fibroblast growth factors, vascular endothelial growth factor, platelet-derived growth factor, tumor necrosis factor 
, and the extracellular MMP inducer (13, 14). Furthermore, the expression of various MMPs can be up- or down-regulated by integrin-derived signals, ECM proteins, physical stress, and changes in cell shape (15).
MMPs are synthesized as inactive proenzymes or zymogens, and their latency is maintained by an unpaired cysteine sulfhydryl group near the C-terminal end of the propeptide domain (16). Although most MMPs are secreted as latent zymogens, there are several exceptions that contain a furinlike motif between their propeptide and catalytic domains and thus can be activated directly inside the cell by members of the proprotein convertase family (12).
The extracellular activation of most MMPs is regulated by a proteolytic cascade and can be initiated by other already activated MMPs or by several serine proteases that can cleave peptide bonds within MMP pro-domains. Activation of MMPs often takes place in the immediate pericellular space, at sites with high affinity for the respective enzyme precursors, such as that seen for pro–MMP-2 activation by the type I transmembrane enzymes (17). Pro–MMP-2 activation by MT1-MMP has been extensively documented and involves formation of a ternary complex consisting of MT1-MMP, TIMP-2, and pro–MMP-2 in a highly orchestrated series of cell-surface events (17, 18).
On the other hand, it has been suggested that although activation of all known MMPs in vitro is accomplished by proteolytic processing of the propeptide, other mechanisms may also participate. For example, in gelatinase B, binding to a ligand or to a substrate may lead to a detachment of the propeptide from the active center of the enzyme, causing its activation (19). In addition, some members of the small integrin-binding ligand N-linked glycoprotein family of proteins have recently been shown to bind and activate specific MMP propeptides (20).
MMP activity either in the extracellular space or membrane bound is blocked by general inhibitors, such as 2-macroglobulin, that are present in the plasma and tissue fluids and by more specific inhibitors, such as the tissue inhibitor of metalloproteases (TIMPs). Four human TIMPs have been identified of which TIMP-1, TIMP-2, and TIMP-4 are secreted extracellularly, whereas TIMP-3 is anchored in the ECM (9).
MMP activity may also be inhibited by some recently described molecules such as the procollagen C-terminal proteinase enhancer, the NC1 domain of type IV collagen, the tissue factor pathway inhibitor-2, and reversion-inducing cysteine-rich protein with kazal motifs (21–24).
MMPs IN FIBROTIC LUNG REMODELING
It has long been accepted that MMPs play an important role in the pathogenesis of pulmonary fibrosis, but the exact mechanisms are not well characterized. There are several interrelated processes—such as ECM remodeling, basement-membrane disruption, epithelial-cell apoptosis, cell migration, and angiogenesis—in which MMPs may play a central role, either by ECM direct cleavage or by generating bioactive mediators. However, the plethora of MMP substrates has significantly broadened and complicated our understanding of the real mechanisms underlying their influence as proteolytic executors and regulators in the pathogenesis of pulmonary fibrosis.
The participation of MMPs in lung fibrosis has been analyzed in several interstitial lung diseases in humans and in experimental models such as those provoked by bleomycin, paraquat plus hyperoxia, and silica (25). Because in the human diseases the detection of the enzymes represents an instantaneous picture, experimental models are used to understand their possible participation in the sequence of processes leading to fibrosis. Also, it is important to have in mind that most (if not all) experimental animal models of fibrosis represent an inflammation-driven lung fibrosis and do not develop the typical progressive and destructive pattern that characterizes idiopathic pulmonary fibrosis (IPF). By contrast, IPF is a chronic and usually fatal lung disorder morphologically characterized by a usual interstitial pneumonia pattern (26, 27). This disease represents primarily, as recently suggested, an epithelial/fibroblastic disorder that is not mimicked by any of the animal models described (28–30).
MMP-7 (MATRILYSIN) IN IPF
Gene-expression signatures of IPF analyzed by oligonucleotide DNA microarray have shown that some MMPs are among the molecules that are most highly increased (30, 31). Among them, MMP-7, also known as matrilysin, is coded for by one of the genes that is most distinctive between fibrotic and normal lungs (30, 31). MMP-7 has a strong affinity for heparin and is able to degrade and process several ECMs as well as a number of bioactive substrates (Table 1). As exemplified in Figure 2, the protein is expressed primarily by the abnormal epithelium in IPF lungs and the active form of the enzyme can be revealed in fibrotic lung extracts (31).
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The generation of mouse models of MMP deficiency has significantly improved our ability to determine the biological function of these enzymes at the molecular and physiological level although apparently very few MMPs are indispensable, as single molecules, for physiologic processes in the mouse (4, 10).
In this context, MMP-7 null mice are normal in appearance and it is of note that they develop attenuated lung fibrosis in response to bleomycin (31, 35). Different studies have suggested that MMP-7 plays a physiologic role in reepithelialization, apoptosis, inflammation, and innate immunity, among others (36).
MMP-1 (INTERSTITIAL COLLAGENASE) IN IPF
MMP-1 is also known as collagenase-1, fibroblast collagenase, and interstitial collagenase. It is the prototype of MMPs capable of degrading fibrillar collagens (types I and III), the most abundant proteins of the ECM (Table 1) (9, 37). MMP-1 expression is low in most normal cells under physiologic conditions, and, like many other MMPs, MMP-1 is undetectable in normal lung in vivo (25, 38–40).
Studies of the transcriptional behavior of lung genes in IPF, and local morphologic expression of the protein have invariably shown a strong up-regulation of MMP-1 (30, 31, 39, 40). This finding in IPF is someway contradictory because the main characteristic of a fibrotic disorder is the exaggerated deposition of fibrillar collagens, which are major substrates of this enzyme. Moreover, MMP-1 has been strongly implicated in diseases that, in contrast to fibrosis, are characterized by exaggerated ECM degradation, such as rheumatoid arthritis and lung emphysema (41, 42). However, this contradiction can be resolved by considering that MMP-1 is primarily localized in reactive alveolar epithelial cells and bronchiolar epithelial cells lining honeycomb cystic spaces and is virtually absent from the interstitial compartment where collagen is being accumulated (Figure 2; References 39, 40). It is interesting to point out that transforming growth factor ß (a strong profibrotic factor) and osteopontin (a recently described fibrogenic mediator) have shown similar effects on fibroblasts, suppressing MMP-1 transcription and up-regulating type I collagen, and TIMP-1 expression (32, 37).
The role of lung epithelial MMP-1 is presently unknown. This enzyme appears to play a pivotal role in epithelial morphogenesis, which depends on specific movement of epithelial cells. Thus, keratinocyte migration on collagen-1 requires specific cleavage of the collagen molecule by collagenase (43). The strong epithelial expression of MMP-1 in IPF lungs suggests that a similar process may occur in vivo in this disease, although reepithelialization seems to be unsuccessful at least partially because it appears to occur when alveoli structures have already disappeared.
As MMP-1 does not have a precise ortholog in the adult mouse or rat, the possible pathological role of this enzyme cannot be explored in experimental models. However, no significant changes in MMP-13, the prevalent collagenase expressed in these rodents, have been found in bleomycin and silica-induced lung fibrosis (44, 45). By contrast, rat lungs with fibrosis induced by the effect of paraquat plus hyperoxia treatment showed a significant reduction in the mRNA expression of both collagenases MMP-8 and MMP-13 (46).
MMP-2 AND MMP-9 IN IPF
Gelatinases (MMP-2 and MMP-9) are the subgroup of MMPs most extensively studied in human interstitial lung diseases and in experimental models of lung fibrosis, probably due to the facility and great sensitivity of revealing gelatinolytic activity through gelatin zymograms (39, 40, 46–51). Both gelatinases contain fibronectin type II–like repeats within their catalytic domain, resulting in a higher binding affinity to gelatin and elastin.
MMP-2 (gelatinase A) is known to degrade a broad range of matrix and nonmatrix substrates (Table 1). It is effective primarily against type IV collagen and other basement-membrane components although a weak ability to degrade stromal collagens has been also reported (52, 53).
It is relevant to indicate that by oligonucleotide microarrays MMP-2 gene expression has been found strongly up-regulated in IPF tissues and the active form is usually increased in bronchoalveolar lavage (BAL) fluids (40). In addition, MMP-2 has been reported to be up-regulated in experimental models of lung fibrosis. The overexpression of MMP-2 and MMP-9 has been mainly associated with their capacity to degrade components of the basement membranes (25, 46, 51–53).
The major physiologic activators of pro–MMP-2 (gelatinase A) are members of the MT-MMP family, and in MT1-MMP this process involves the action of TIMP-2 forming a trimolecular complex of MT1-MMP/TIMP-2/pro–MMP2. TIMP-3, but not TIMP-1, can substitute for TIMP-2 in pro–MMP-2 activation by MT1-MMP (17, 54).
In IPF, MMP-2 is found in alveolar and basal bronchiolar epithelial cells and in fibroblastic foci (39, 40, 47, 55). Interestingly, MT1-, MT2-, MT3-, and MT5-MMPs, the activators of pro–MMP-2, are expressed in similar locations mostly by different types of epithelial cells, a pivotal component in the aberrant remodeling of the lung microenvironment. In a recent study, MT1- and MT2-MMPs were found in alveolar epithelial cells, MT3-MMP in fibroblasts from fibroblastic foci and alveolar epithelial cells, and MT5-MMP in basal bronchiolar epithelial cells and in areas of squamous metaplasia (55). Therefore, the activated form of MMP-2 usually found in BAL fluid of patients with IPF might be attributed to the action of these enzymes.
As mentioned, active MMP-2 (and MMP-9) may provoke the disruption of basement membranes, which seems to be an important event in IPF pathogenesis that enhances the fibroblast invasion into the alveolar spaces (Figure 2). In addition, the disruption of the basement membrane may also contribute to the failure of an orderly repair of the damaged alveolar type I epithelial cells, affecting normal reepithelialization, and moreover, it may have an additional deleterious role by inducing epithelial apoptosis. In fact, the integrity of the basement membrane is required to suppress programmed cell death as has been demonstrated in mammary epithelium and other tissues (56, 57).
MMP-9, also called gelatinase B, contains additionally a type V collagenlike domain that is highly glycosylated, which has been suggested to have an effect on substrate specificity (58).
MMP-9 gene expression and protein have also been shown to be elevated in lungs and BAL fluids from patients with IPF (31, 39, 40, 47, 59). The enzyme has been localized in epithelial cells, neutrophils, and macrophages with some staining in subepithelial fibroblasts. Likewise, in bleomycin-induced pulmonary fibrosis an increase of gelatinase B activity and disruption of the alveolar epithelial basement membrane has been reported (51). Interestingly enough, cigarette-smoke exposure, which induces an increase in BAL fluid neutrophils and MMP-9, potentiates bleomycin-induced lung fibrosis in guinea pigs, suggesting that tobacco smoke creates a profibrotic milieu that may contribute to the increased fibrotic response in this model (60). Likewise, mice deficient in 
-glutamyl transpeptidase, a key enzyme in glutathione and cysteine metabolism, develop less fibrosis and a less pronounced rise in MMP-9 than wild-type mice (61).
In all these models there is a relationship among BAL fluid neutrophils and MMP-9; however, because neutrophils do not synthesize MMP-9 outside the bone marrow, the revealed increased gene expression of MMP-9 in IPF lungs does not reflect the enzyme produced by these cells.
Despite the putative role of MMP-9 in lung fibrosis, the MMP-9 null mice develop fibrosis similar to that of wild-type littermates after bleomycin instillation. However, the lungs of gelatinase B–deficient mice showed minimal alveolar bronchiolization, suggesting that gelatinase B facilitates migration of Clara cells and other bronchiolar cells into the regions of alveolar injury (62). Lung reepithelialization is a complex and not well understood process that should involve a sequence of steps including detachment, migration, reattachment, proliferation, and differentiation of type 2 into type 1 pneumocytes. In this context it has been proposed, based also on tissues other than lung, that MMP-9 plays a role in cellular migration (58).
In summary, MMP-7, MMP-1, MMP-2, and MMP-9 are MMPs whose gene and protein expression is highly elevated in IPF. Additionally, epithelial cells express all these enzymes, suggesting an active role of these cells in matrix remodeling. However, considering the complexity of the biological activities of MMPs, we are far from understanding the biological consequences of the action of these enzymes in the abnormal repair. In this context, this is a story that is still being recounted.
FOOTNOTES
Conflict of Interest Statement: Neither of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript.
(Received in original form January 23, 2006; accepted in final form February 21, 2006)
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