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Departments of Medicine and Immunology, Baylor College of Medicine, Houston, Texas; and Department of Anatomy, University of California, San Francisco, California
ABSTRACT I. INTRODUCTION II. EVOLUTION OF MATRIX METALLOPROTEINASES A. Development B. Tissue Repair C. Immune Response III. REGULATION OF MATRIX METALLOPROTEINASES IN LUNG: TRANSCRIPTION, TRANSLATION, AND POSTTRANSLATIONAL REGULATION A. Transcriptional and Translational Regulation 1. Transcription factors 2. Growth factors 3. Cytokines/chemokines 4. Endogenous inflammatory intermediates 5. Environmental factors 6. Pathogen-derived mediators 7. Mechanical stress B. Posttranslational Regulation 1. Activation of MMPs 2. Localization of MMPs IV. SPATIOTEMPORAL EXPRESSION OF MATRIX METALLOPROTEINASES IN LUNG: BRANCHING MORPHOGENESIS AND ALVEOLAR DEVELOPMENT A. Branching Morphogenesis B. Angiogenesis C. Alveolarization V. RESURGENCE OF MATRIX METALLOPROTEINASES IN LUNG DISEASES: ACUTE AND CHRONIC VI. MATRIX METALLOPROTEINASES: REDUNDANT VERSUS COMPENSATORY MECHANISM OF ACTION VII. MATRIX METALLOPROTEINASES IN INNATE AND ADAPTIVE IMMUNE FUNCTION A. Evidence for Host Defense B. MMPs as Acute Inflammatory Mediators VIII. PROTEOLYTIC EFFECTOR FUNCTION: MECHANISM OF ACTION IN INFLAMMATION IX. HUMAN GENETIC VARIATION OF MATRIX METALLOPROTEINASES A. MMP Deficiency B. MMP Polymorphisms X. MATRIX METALLOPROTEINASES IN HUMAN INFLAMMATORY LUNG DISEASES A. Bronchopulmonary Dysplasia and Chronic Lung Disease of Prematurity B. Interstitial Lung Disease and Idiopathic Pulmonary Fibrosis C. Asthma D. COPD and Emphysema E. Cystic Fibrosis F. Bronchiolitis Obliterans G. Pulmonary Alveolar Proteinosis XI. MATRIX METALLOPROTEINASES: TO INHIBIT OR NOT INHIBIT? A. Natural Inhibitors B. Synthetic Inhibitors XII. SUMMARY GRANTS ACKNOWLEDGMENTS REFERENCES
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ABSTRACT |
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I. INTRODUCTION |
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Many members of the MMP family are upregulated in the lungs of humans with inflammatory diseases. To understand the biology of MMPs in a variety of relevant human pathological conditions such as allergic inflammation, tissue injury and repair, remodeling, and host defense against pathogens (9, 52, 133), the genetic approach, which uses single or complex deletion of MMP gene(s), has been particularly powerful. However, despite the use of some of these well-established models of lung diseases, no unifying theme on the advisability of inactivating these molecules in all inflammatory conditions has emerged. For instance, deletion of MMP2 is protective in allotransplant models because it significantly reduces cellular infiltration and fibrosis. But, in sharp contrast, deficiency in MMP2 increases susceptibility of mice to lethal asphyxiation in an asthma model (36, 49). Interestingly, lack of MMP2 in humans results in many abnormalities, including an osteolytic and arthritic disorder and other bone deformities (4). Thus, despite the fact that large efforts have been directed towards the development of chemical inhibitors of MMPs, it is not clear whether MMP inhibition is beneficial or harmful and in which situations it would be of clinical help. Among the critical issues that require resolution are the mechanisms of action and function of MMPs under diverse pathological conditions.
The lung has a rich vascular network and is in continuous and direct communication with the outside environment. Thus it is not surprising that besides serving as the conduit for gas exchange, one of the main functions of the lung is its role in host defense, protecting against such affronts as viral and bacterial pathogens and/or other organic/inorganic environmental insults. Although small amounts of MMP2 and MMP14 are present in the lining fluid of the lung under normal conditions, other MMPs such as MMP7, MMP8, MMP9, and MMP12 are upregulated under many pathological conditions. There is considerable evidence that MMPs in the lung play a role in host defense (9, 212, 223). In this review we present evidence from the literature that supports a direct or an indirect role for several of the MMP family members in various lung diseases. The mechanistic insight into the function of proteinases requires the use of carefully crafted models of lung injury and repair. Using examples of work done in animal models of human lung diseases, as well as in translational studies, we explore the expression of MMPs, their mechanisms of action, and their involvement in lung pathology and repair.
In section II, we compare MMPs from lower phylogenetic ranks of species, e.g., invertebrates, with those of higher orders, e.g., mammals, a comparison that may allow lessons from a simple system to provide insights into the most complex biological functions of this family of enzymes. Then, we review the molecular regulation of MMPs and their activation in the lung (see sect. III). Although not formally tested, based on their predicted cleavage sites, collectively, activated forms of MMPs can degrade most of the secreted or cell surface bound proteins in the body; thus many levels of regulation are necessary to prevent proteolytic damage. Following in section IV, we use lung development as a model system in which to explore the spatiotemporal expression of MMPs. In smoking-related human lung diseases such as chronic obstructive pulmonary disease (COPD) and emphysema, where the loss of alveolar space is the main hallmark, understanding the role of MMPs in alveolar generation/regeneration may hold the key to future treatment of these diseases. The upregulation of MMPs in the lung under various pathological conditions is explored in section V. The main issue that we address is whether there is enough evidence to support the notion that resurgence of MMPs in general is harmful in the active or chronic phases of lung diseases. We review and compare in section VI whether the actions of members of the MMP family are redundant or compensatory in nature. The role of MMPs in modulating the immune system perhaps through mediating the cross-talk between the innate and adaptive immune systems is addressed in section VII. In section VIII, we review evidence that MMP cleavage of various proteins alters their effector functions, providing a new paradigm for MMPs' mechanism of action in inflammatory conditions in human lung. Next, we review human genetic variation in MMPs (see sect. IX). In section X, we discuss the role of MMPs in some human lung diseases. Finally, in section XI, we examine whether there is support for inhibition of MMPs in non-cancer-related lung diseases. This is a critical issue, since currently most inhibitors in the market inhibit the broad class of MMPs, which can affect multiple systems. However, there is a great interest in developing designer MMP inhibitors to target the action of individual members.
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II. EVOLUTION OF MATRIX METALLOPROTEINASES |
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Multiple sequence analyses of the different domains indicate that modern, multidomain MMPs evolved early on, before the divergence of vertebrates from invertebrates, from an enzyme with a simpler structure (164). One putative candidate, enterotoxin from Bacteroides fragilis (BFT; GenBank accession no. U90931) has a zinc-binding consensus motif, characteristic of the metalloproteinase family (75), and shares 59% sequence identity with the 27 amino acid stretch in human MMP-1, which includes the catalytic zinc-binding domain and the "Met turn" (75, 307). In addition, purified BFT can cleave monomeric G-actin, gelatin, and azocoll in vitro. Because bacteria first appeared more than 3.5 billion years ago, and eukaryotes around 1.8 billion years ago, it is possible that the metalloproteinases evolved at that time and apparent that MMPs are ancient (164).
Because most of the literature on MMPs is focused on mammals, many comparative studies focus first on identifying the presence of a proteinase and then classifying the proteinase as a matrix-degrading enzyme. This classification is done in several ways: analysis of protein sequence and putative tertiary structures, determination of the substrate specificity, and inhibition of degradatory capacity by known MMP inhibitors. These necessary studies have led to hypotheses that MMPs are critical for regeneration of tissues and development. Now that the presence of MMPs has been unequivocally established in lower phyla, it is time to focus on the structure-function relationship of these MMPs and utilize new model systems for investigating mechanisms for MMP action.
To better understand the roles that MMPs play today in development and disease, it is helpful to understand their historical functions and their function in other organisms. MMPs are widely distributed across phylogenies, but their structures are fairly conserved. Members of the MMP family have been found in soybean (167) and mustard (155) plants and algae (125). In invertebrates, MMP-like activity has been noted in Balanus amphitrite barnacle larvae (158), Strongylocentrotus purpuratus sea urchin embryos (228), Scylla serrata crabs (250), Crassostrea virginica (321) and C. gigas (268) oysters, and Mytilus galloprovincialis mussels (159). The nematodes, Caenorhabditis elegans (288) and Gnathostoma spinigerum (280), have MMPs. Drosophila has two MMPs (149, 150, 206). What remains to be fully answered is whether MMP functions in complex biological processes such as development, tissue repair, and immune response are also conserved.
In vitro, as a group MMPs are able to degrade every type of ECM protein yet tested (256). Conceivably based on this observation, MMPs could be viewed as critical players in development and repair processes. It is clear that Drosophila melanogaster1-MMP (DM1-MMP) is required for proper development of the larval tracheal respiratory system and for pupal head eversion, while DM2-MMP is important for metamorphosis and neural development. Mutant Drosophila, lacking functional DM1-MMP, are smaller in size, grow tracheae that appear to be overstretched and broken in spots, and wander earlier than their wild-type counterparts (206). These researchers also note that at ecdysis from larval/larval molting, flies appear to have difficulty shedding their exoskeleton, which appears to be stuck at the ends of the tracheae. Because new tracheae are formed inside the old tracheae, separated by tracheal fluid, this could imply that there are cuticular defects or possibly a defect in the tracheal lining fluid.
In contrast, MMP2 has been found in developing zebrafish throughout embryonic development and is required for normal embryogenesis (316). In addition, using a novel technique that would best be described as in vivo zymography in the zebrafish model, MMP activity has been visualized throughout embryogenesis in these fish; however, its role in the developing respiratory system remains unclear (54).
XMMP7, the Xenopus homolog to human MMP7, is expressed in macrophages during embryonic development (98) along with XMMP9 (38) and is hypothesized to play a role in macrophage migration.
In organisms from hydra to fish, MMPs are involved with tissue repair. For instance, if MMPs are blocked using the MMP-specific, zinc-chelating inhibitor GM6001, limb regeneration stops in newts (285). In hydra, foot regeneration inhibited by GM6001 is reversible, and upon removal of the inhibitor, foot regeneration resumes (143). Zebrafish also cannot regenerate fins if MMPs are blocked with GM6001 (10). Whether MMPs are involved in gill tissue repair in the fish is unclear. However, in oysters, one study demonstrated increased MMP mRNA expression in gill tissue 18–24 h after exposure to hypoxia. This could represent tissue degradation after oxidative destruction; however, MMPs are often upregulated during angiogenesis. Given their role in tissue regeneration, it seems more likely that they are working to increase gill area or to replace damaged tissue (268). This response is similar to that seen in rats, where systemic treatment with GM6001 delayed epithelial wound healing (175).
The function of MMPs in the innate immune response is a role that evolved before the divergence of vertebrates and invertebrates. In the oyster Perkinsus marinus, MMP-like activity increases after infection with a protozoan parasite (183). In Crassostrea gigas, bacterial challenge results in increased mRNA expression of cg-MMP (89). In response to shell damage and bacterial challenge, cg-TIMP (tissue inhibitor of metalloproteinases) is upregulated in hemocytes in gill (178), suggesting not only that the complexity of MMP regulation seen in mammals evolved early on, but also that the role of MMPs in respiratory system function has long been important. In support of this, adult hydra injected with the MMP inhibitor GM6001 exhibit decreased mucus production, as evidenced by a decrease in cell adhesion (hydra did not stick to a glass rod). The researchers also found decreased peroxidase activity (143). This is suggestive of immune system activity; however, more studies of MMP function in plants and invertebrates will be necessary to answer this question.
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III. REGULATION OF MATRIX METALLOPROTEINASES IN LUNG: TRANSCRIPTION, TRANSLATION, AND POSTTRANSLATIONAL REGULATION |
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A. Transcriptional and Translational Regulation
As emphasized in this review, MMPs are made in different cell types under unique conditions, and thus distinctive inductive and suppressive factors regulate the expression of each of the members of the MMP family. These are detailed comprehensively in several recent reviews; therefore, we will briefly review the main types of regulating factors and provide recent examples (61, 256). Interestingly, most of the same factors that regulate the expression of MMPs in the lung can also act as their substrates, providing a built-in and, most likely, essential component of the regulatory cascades. This coordination between MMP and substrate ensures efficient activation and control of MMPs in vivo (173, 283).
The first step in regulating MMP production occurs at the transcriptional level. Several proteins, including early-growth response gene product 1 (EGR1), nuclear factor kappa B (NF
B), globin transcription factor 1 (GATA1), activator protein 1 family members (AP-1), and signal transducer and activator of transcription 3 (STAT3C), have been shown to affect several members of the MMP gene family. For example, lung fibroblasts from EGR1 null mice fail to upregulate expression of MMP2 in response to stimulation with a mixture of tumor necrosis factor (TNF)-
, interleukin (IL)-1
, and interferon (IFN)-
(189). Interestingly, in endothelial cells EGR1 binds to the MMP14 promoter, also regulating its expression (94). Because MMP14 activates MMP2, this provides a positive-feedback loop for upregulation of MMP2. With the use of a luciferase promoter/reporter assay in cultured fibroblasts, MMP1 transcription increases during cell shape changes and depends on NFB-mediated IL-1 secretion (121, 295). Similarly, GATA-1 in endothelial cells and members of the AP-1 family, c-Jun, JunD, c-Fos, FosB, and Fra-1 proteins in lung cancer cells activate the MMP2 promoter (95. 41). In a mouse model of hyperoxia-induced lung injury, the development of lung damage, including capillary leakage and neutrophil infiltration into alveoli, is mediated by the synthesis and release of MMP9 and MMP12 from neutrophils and alveolar resident cells. This deleterious effect is prevented by overexpression of STAT3C in alveolar epithelial cells, which decreases both transcription and activity of MMP9 and MMP12 (146).
Epidermal growth factor receptor (EGFR) activation upregulates MMP14 in lung fibroblasts (122). MMP2 and MMP9 are upregulated in response to treatment of mice with hepatocyte growth factor (HGF), a treatment that also improves lung fibrosis through a mechanism involving increased myofibroblast apoptosis (176). Deletion of the 1,6-fucosyltransferase (Fut8) gene in mice is associated with abnormal lung parenchyma development consistent with emphysema-like lesions and overexpression of MMP12 and MMP13 (290). Although most likely abnormal lung development plays a significant role in lung lesions seen in mice deficient in Fut8, the unexpected dysregulation of MMPs in the absence of Fut8 is mediated through aberrant transforming growth factor (TGF)-1 receptor signaling, which is known to downregulate MMP1, MMP12, and MMP13 through negative transcriptional regulatory mechanisms (290).
Chemokines have been shown to regulate several members of the MMP family. Indirect evidence shows that activation of CCR1, the receptor for RANTES/CCL5, MCP-1/CCL2, and SDF-1/CXCL12, induces secretion of MMP9 in primary human monocytes (126). In fibroblasts, CCL2 increases expression of MMP1 and MMP2. These increases are mediated by the proinflammatory cytokine IL-1 (308).
In cultured cells, the proinflammatory cytokines IL-1, IL-6, and TNF- upregulate MMPs (132, 147, 308, 311). More importantly, this is also observed in vivo. IL-1 is produced in a biologically inactive form and can be activated by cleavage with MMP9 (237). When IL-1 production is ectopically expressed in the lung epithelium using a Clara cell secretory protein promoter (CCSP) as driver, it causes pulmonary inflammation, spontaneous overexpression of MMP9 and MMP12, disruption of elastin fibers in alveolar septa, and fibrosis in airway walls, a pathology resembling that of emphysema (137).
When IL-13, another proinflammatory cytokine, is administered intranasally, it induces airway hyperresponsiveness (AHR) and increases MMP2 mRNA in lung and both pro- and active MMP2 in bronchoalveolor lavage (49). IL-13 works through STAT6 to induce expression of MMP2 and MMP9 in the lung, and it requires the IL-4/IL-13 receptor-specific signaling chain IL-4R (46, 284; F. Kheradmand and D. Corry, unpublished data). Overexpression of IL-13 using a doxicycline-induced CCSP driver in the adult murine lung increases transcription and translation of MMP2, MMP9, MMP12, MMP13, and MMP14, in a MMP12-dependent manner (135). These mice also exhibit respiratory failure due to increased lung volumes and compliance, mucus metaplasia, and spontaneous inflammation. Some of the IL-13-dependent lung damage can be prevented by administration of the MMP inhibitor GM6001 (318).
IL-17, a T-cell-specific cytokine, has been shown to promote degradation of collagen in vitro through a process that is dependent on upregulation of MMP1, MMP3, and MMP13 (129). Furthermore, when recombinant IL-17 is administered in the airways of mice, it results in an increase in the concentration and activity of MMP9 in the BAL (220). These findings point to the link between adaptive immune system and activation of MMPs in diseases that feature tissue destruction such as arthritis of the joints and emphysema in the lung.
4. Endogenous inflammatory intermediates
Reactive oxygen species (ROS), including singlet O2, hydroxyl radical, superoxide anion, and hydrogen peroxide, are created by the partial reduction of molecular O2 (71). ROS are found in high levels in cigarette smoke and are generated by mitochondria, peroxisomes, and the plasma membranes and vacuoles of activated leukocytes (72, 110). Imbalances in ROS have been implicated in a variety of lung pathologies: too little may impair host defense and too much may result in tissue damage, disease, or cell death (72).
ROS can affect MMP gene expression through activation of several transcription factors. H2O2 production by mitochondria can activate MMP1 gene expression through activation of AP-1 and Ets-1 transcription factors (185). In addition, ROS indirectly activate MMP expression via JNK and ERK signaling pathways (185).
Reactive nitrogen species (RNS) are also involved in inflammatory reactions. In a rat model of pulmonary embolism, MMP2 and MMP9 are upregulated, perhaps indirectly as a result of neutrophil and macrophage influx to the pulmonary artery or directly from oxidative stress associated with pulmonary embolism. L-Arginine, a known oxygen radical scavenger, is also a substrate for nitric oxide synthase (138). Treatment with L-arginine diminishes MMP expression, while inhibition of nitric oxide synthase abrogates the effect of L-arginine (253), suggesting that MMP production is driven by NO or one of its partially reduced forms, such as peroxynitrite. In support of this hypothesis, endogenous NO has been shown to increase MMP1 promoter activity in cultured cells (113).
Ample studies have linked mRNA expression of several proteinases in lungs to cigarette smoke exposure (42, 43, 305). Short (few hours) and prolonged (6 mo) exposure to smoke significantly increases expression of matrix metalloproteinase MMP9 and MMP12 mRNA in lung and antigen presenting cell populations (27). Lungs from mice exposed to cigarette smoke show increased volume fractions of alveolar septa and airspaces by morphometrical analysis and have increased MMP12 in lung macrophages compared with sham-treated mice, pathological changes consistent with those seen in human emphysema (281). These effects may be mediated by ROS, since as mentioned above, cigarette smoke contains high concentrations of ROS (110).
Air pollution may also contain high levels of ROS, but also particulate matter composed of heavy metals. Urban air pollutants have been shown to increase production of MMP2, MMP7, and MMP9 (261, 262). However, since air particulates are composite mixtures, the actual causative agent remains unclear. Interestingly, the MMP activity profile varies depending on the type or fraction of urban pollution administered (2), indicating that the response to pollution is complex and may be driven by several factors.
Destruction of lung by cavity-forming bacterial pathogens has been well documented. Most recently, conditioned media from Mycobacterium tuberculosis (MTb)-infected monocytes, but not direct infection with the organism, was observed to upregulate epithelial cell MMP1 promoter activity, gene expression, and secretion. These effects are dependent on p38 mitogen-activated protein kinase (MAPK) phosphorylation. These data reveal that MTb regulates the expression of MMP1, which can drive lung tissue destruction (64). In contrast in cultured human monocytic cell line (THP-1), infection with both MTb and its cell wall glycolipid stimulate expression and activity of MMP9 (227). The cellular mechanism involved in this process is dependent on signaling through mannose receptors, the activation of protein kinases and transcriptional activation by AP-1 (226). In addition to MTb, other bacteria, including Staphylococcus, can cause similar types of lung damage. Perhaps this example of pathogen-mediated increase in MMP expression is a general response of the host innate immune system to the invading organism (more detail in sect. VII).
In addition to all of the known factors mentioned above, expression of MMPs in the lung is also modulated by the mechanical stimuli that continuously change airway pressure as part of the dynamic breathing cycle. During pathological conditions that cause bronchial constriction, or during mechanical ventilation, lung pressure fluctuates greatly (99, 270, 276, 303). Under experimental conditions, exposure of rats to 4 h of high-volume ventilation results in upregulation of MMP2, MMP9, and MMP14 in the lung, by a mechanism that depends on the upregulation CD147 (74, 100). In cultured cells and in isolated mouse lungs that are stimulated with methacholine, a potent bronchial smooth muscle stimulant, mechanical stress is communicated from stressed (bronchoalveolar) to unstressed (fibroblasts) cells. This elicits increased production of MMP9 and TIMP1, through shedding of cell surface receptors and activation of EGFR (266, 274, 275).
The role of shear stress in upregulation of MMPs is not unique to the lung, since stimulation of cultured synovial cells by shear stress also elevates the mRNA level of MMP1, MMP3, and MMP13 and the three transcription factors c-Fos, Ets-1, and Ets-2, suggesting a new mechanism for tissue degradation in rheumatic joints (263). These effects of mechanical stress may ultimately be caused by production of ROS, since mechanical stress increases ROS in vascular smooth muscle cells. In that case, the ROS generated are responsible for increased gene expression of MMP2 and increased release of pro-MMP2 (86).
B. Posttranslational Regulation
Posttranslational modification of MMPs in the lung is a crucial step in regulating MMP action in vivo and is mediated through activation of latent MMPs. Several MMPs have soluble and cell surface forms, providing another level of regulation through compartmentalization/localization (201, 203). In addition, two endogenous families of inhibitory proteins, TIMP1–4 and 2-macroglobulin, are known to regulate MMPs at the posttranslational level and are briefly discussed in section XI.
Secreted pro-MMPs remain inactive due to the interaction of the unpaired cysteine sulfhydryl group (cysteine switch) in the propeptide domain with the active site containing the zinc ion (256). Of the diverse means of activating MMPs, one common thread is thought to be thiol (sulfhydryl) reactivity, which disrupts the binding of the cysteine switch and exposes the active site of the enzyme (185). Removal of the propeptide containing the cysteine group by enzyme cleavage or disruption of the zinc-cysteine interaction can result in activation of the latent enzyme (256). Several MMPs are exceptions to this rule, including MMP11, MMP28, and all of the transmembrane MMPs (MT1-MT6-MMP), which are activated in the secretory pathway via subtilisin-like furin family serine proteinases (216). Organic mercury compounds, SDS, heat, acidic pH, and ROS have all been shown to activate MMPs in vitro (304). For instance, Pro-MMP1 is activated by H2O2, HOCl, and OH radicals (230). Pro-MMP1 becomes partially activated when ROS oxidate the sulfhydryl group, making an unstable pro-MMP1. At that point, the unstable pro-MMP1 is susceptible to autocatalytic cleavage of the propeptide resulting in active enzyme (185).
Activation of MMP2 on the cell surface critically depends on the binding of pro-MMP2 to MMP14 and TIMP2. This complex, which tethers the zymogen in place, allows a second active MMP14 to cleave the prodomain and release the activated MMP2 (260). This type of multistep, pericellular activation is now also shown to play a role in the activation of pro-MMP-7 where this soluble zymogen is captured and activated on the cell membrane through interaction with CD151, a member of the transmembrane 4 superfamily (248). Furthermore, MMP7 activity is blocked by antibodies to CD151 (248).
Several other proteinases regulate activation of MMPs, including trypsin and cathepsin G (304). In vivo, pro-MMP2 and MMP9 are activated by a chymase proteinase. Mice lacking the mast cell chymase proteinase 4 (mMCP-4) fail to process pro-MMP9 and pro-MMP2 to their active forms, resulting in increased collagen, ear thickness, and a higher content of hydroxyproline in the ear tissue compared with wild-type mice (271).
Plasmin and its activator urokinase plasminogen activator (uPA) can activate an array of proteinases, including many MMPs (304). This system is intricately bound to the MMP system, as the zymogen form of plasmin, plasminogen, is a known substrate for several MMPs (256). Adding further complexity to this system, plasmin not only activates MMPs, it can inactivate the tissue inhibitors of metalloproteinases (TIMPs) (131).
Activation of MMPs in vivo under physiological or pathological conditions, such as sepsis, requires multiple steps, but the exact mechanisms remain unknown. In experimental endotoxemia, lipopolysaccharide (LPS), TNF-, granulocyte colony stimulating factor (G-CSF), and IL-8 result in release of pro-MMP9 in the blood, while activated MMP9 and MMP2 are not detected (224). However, whole blood analysis of MMPs in patients with gram-negative sepsis shows activated forms of MMP2 and MMP9, the levels of which correlate with the severity of sepsis. This indicates that while proinflammatory mediators such as LPS can acutely induce the inactive form of MMPs, the activation process in response to bacterial infection is more complex and regulated differently in vivo (224).
Activity of MMPs can be altered in several ways. The relative fluxes of nitric oxide and superoxide at sites of inflammation are shown to differentially modulate MMP2 activity, because superoxide-derived metabolites increase activity of MMP2 in vitro, while peroxynitrite, which is formed from the interaction between superoxide and nitric oxide, inhibits MMP2 activity (204). MMP activity may also be altered by environmental pollutants. Rats exposed to a combination of particulate matter and ozone experience increased MMP2 activity in bronchoalveolar lavage (BAL) (272).
Compartmentalization of MMPs in inflammatory cells is the most obvious example of this type of regulation. For example, pro-MMP8, pro-MMP9, and pro-MMP25 are packaged into peroxidase-negative granules within neutrophils to be released upon leukocyte activation (67). Peroxidase-positive azurophil granules contain both activators of MMPs, such as serine proteinases and ROS-generating enzymes, and inactivators of MMPs, such as thrombospondin (67). MMP9 and MMP12 can be released from macrophages in response to activation.
In addition, chemokines have been shown to regulate MMP activation by altering their localization. MMP14 is highly expressed on lung endothelial cells, and in its absence, there is decreased angiogenesis induced by the chemokines CCL2 and CXCL8 (6, 78, 320). In human endothelial cells, CCL2 and CXCL8 increase cell surface expression and clustering of MMP14 (78). The cell surface expression pattern of MMP14 is one potential regulator of MMP2 activity, since MMP2 activation would then be dependent on colocalization with MMP14 (93).
Another level of regulation is that MMPs can be either free in the cytosol or bound to the surface of a cell. The cell surface-bound forms of at least two MMPs are thought to enhance inflammatory cell functions. For example, MMP9 is secreted but also has a cell surface-bound form. This active, cell surface form of MMP9 is expressed on neutrophils in response to proinflammatory mediators and can cleave gelatin, type IV collagen, elastin, and 1-proteinase inhibitor in vitro (202). However, in contrast to soluble MMP9, membrane-bound MMP9 is substantially resistant to inhibition by TIMPs and may be responsible for the increase in pericellular proteolytic activity necessary for the proper action of neutrophils in vivo, although this hypothesis has not been formally tested (202). Similarly, neutrophils exposed to proinflammatory cytokines increase cell surface expression of active MMP8. Pericellular collagenase activity from activated neutrophils is attributed to membrane-bound MMP8, and its localization on the cell surface confers resistance to TIMP inhibition (203). MMP8 may be anti-inflammatory during acute lung injury, because MMP8 null mice given intratracheal LPS have significantly greater accumulation of neutrophils in the alveolar space than wild-type mice (203). Together with the cell surface activation of MMP2 and MMP7, these experiments suggest that localization of MMPs to the cell surface is a general means of regulating MMP activity. Additionally, MMP1, MMP2, and MMP9 have been found to bind to specific cell surface proteins such as v3-integrin, 21-integrin, and CD44, respectively (30, 259, 314; for a recent review, see Ref. 213).
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IV. SPATIOTEMPORAL EXPRESSION OF MATRIX METALLOPROTEINASES IN LUNG: BRANCHING MORPHOGENESIS AND ALVEOLAR DEVELOPMENT |
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Very little is known about regeneration of lung after injury/destruction, but it is likely that repair events mimic those during development (317). Thus elucidating the mechanisms involved in alveologenesis will yield information that may facilitate understanding the repair process. In this regard, MMPs represent excellent targets, since these enzymes are critical for alveolar development and often, in lung injury, many of the same MMPs, such as MMP7, MMP8, and MMP9, reappear (303). In many instances, it is unclear whether the presence of MMPs is destructive or beneficial. Knowing how these enzymes function during development will help us to further our understanding of lung repair, possibly providing insight on how to repair emphysematous lungs and lungs damaged from chronic inflammation or injury.
Various MMPs participate in the development of lung from the very beginnings of lung bud formation throughout alveolarization, where some MMPs are more prominent players than others. For instance, in the developing mouse lung, there is no detectable MMP3, MMP9, MMP10, or TIMP1 at any stage (122). Expression of mRNA for MMP15 and MMP20 in fetal and adult lungs shows no significant changes with developmental stage, whereas MMP2 and MMP14 decrease with age (229). MMP21 is expressed transiently during embryonic development with expression peaking around days 11–13.5 as detected by PCR and may be associated with neuronal cells (3, 160). Amidst all the variation, two key MMPs stand out as being of major importance during development: MMP2 and MMP14 (MT1-MMP; Fig. 2).
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Branching morphogenesis of bronchiole structures takes place during the pseuodoglandular stage, which is characterized by dichotomous branching of epithelial tubes (229). This stage occurs from 7–16 wk gestational age in humans and embryonic days 9.5 -16.5 in the mouse. In the mouse, MMP2, MMP14, and TIMP2 are expressed on days 11.5 and 13.5 (122). In humans, MMP1, MMP9, TIMP1, TIMP2, and TIMP3 are detected by immunohistochemistry in fetal epithelium, while MMP2, MMP1, TIMP2, and TIMP3 are expressed only in pulmonary vascular endothelium and media (165).
Branching morphogenesis can be inhibited with high concentrations of the broad spectrum metalloproteinase inhibitor GM6001 (81, 122). Yet, a lower concentration of GM6001 enhances branching (81). One model of branching morphogenesis suggests that cleft formation is driven by accumulation of TGF-, which stimulates ECM deposition and directs branching to either side of the built-up ECM (37, 103). This process is facilitated by TGF--mediated inhibition of MMPs. In support of this hypothesis, TGF-1 and TGF-3 inhibit expression of MMP1 and upregulate expression of TIMP1 in fibroblasts (62). However, TGF- family members do not regulate MMP2, which is highly upregulated during lung development. This relationship points to other, as yet unexplored, and complex interactions between MMP-ECM and growth factors.
Another model for branching morphogenesis suggests that low tissue PO2 could inhibit MMP degradation of tenascin C (TN-C), allowing for deposition of this important ECM component. In support of this model, low tissue PO2 (3%) increases branching in lung, increases expression of TN-C, and also decreases activity of MMP2 (but not its mRNA synthesis), possibly through decreased availability of ROS (79). Inhibition of MMPs with GM6001 results in decreased deposition of TN-C (79), confirming the role of MMPs in lung branching in explants. Potentially, TGF- inhibition of MMPs may promote TN-C deposition, by working in concert with low PO2 to promote branching, since TN-C stimulates cell proliferation in human lung fibroblasts, and this response is amplified in the presence of TGF- (236).
MMPs are shown to stimulate cellular migration, which is another plausible mechanism for their role in branching morphogenesis. MMP14 is expressed in lung tissue early in embryogenesis. MMP14 null embryos after embryonic day 16.5 have a subtle defect in peripheral bronchiolization. However, the role of MMP14 in branching is minimal and redundant (193). Interestingly, lung endothelial cells from the MMP14 null mice show reduced migration (193), possibly due to altered processing of laminin 2-chain. MMP14 and MMP2 cleavage of laminin 2-chain generates chemotactic fragments containing EGF-like motifs that are present in tissues undergoing remodeling (80) and can attract epithelial cells (128). This process suggests another mechanism for MMP14's role in the developing lung. MMP-mediated release of cryptic, bioactive fragments and neoepitopes from ECM macromolecules is detailed in a recent review (181).
The canalicular stage is characterized by extensive angiogenesis and the linking of bronchiolar structures with their capillary interface. In humans this stage occurs from 16 to 24 wk and in mice from embryonic days 16.5 to 17.5. In the human lung, MMP1 and MMP9 are detected throughout all stages. The role of MMPs in angiogenesis is nicely addressed in a recent review by Brauer (28), and a comprehensive review on the role of MMPs in intimal thickening was recently published by Newby (188); thus we will only provide highlights relating to lung development.
Fourteen of the 25 vertebrate MMPs are expressed in vascular smooth muscle cells and endothelial cells, indicating the importance of MMPs in vascular biology (188). During angiogenesis, fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF), and their receptors are important for proper formation of blood vessels; these bioactive molecules can be released from ECM by MMP cleavage (28). Conversely, FGFs and VEGFs can increase expression of MMPs (5, 134). Mice lacking MMP9 have delayed angiogenesis that is likely corrected through compensation by other MMPs, resulting in normal adult mice (286). The dysregulation of angiogenesis is hypothesized to be lack of a critical growth factor in the absence of MMP9. Alternatively, MMP activity can abrogate angiogenesis. Degradation of several ECM components, including plasminogen, thrombospondin, and collagens IV, XV, and XVIII, can generate fragments that are inhibitors of angiogenesis, through MMP-dependent mechanisms, giving MMPs regulatory power over angiogenesis (217).
The final stage, alveolarization, is long lasting (from 36 wk to 3 yr of age in humans) and is characterized by alveolar development and maturation of the capillary network. In mice this stage lasts from postnatal day 5 to day 30 and peaks at day 14 (229). This process requires coordination of ECM remodeling with epithelial morphogenesis and capillary growth. MMP14 (MT1-MMP) is required for alveolarization, because mice lacking this enzyme have decreased alveolar surface area and enlarged airspaces, attributes which could be caused by failure of capillary network formation and defective distal airspace branching (193).
However, the bronchioles of these mice branch properly, although defects in alveoli are apparent from the late saccular stage (8, 193). This led to the hypothesis that the abnormal alveolization results from problems with saccular development and septation (193). Interestingly, these are exactly the same stages impacted by altering TGF- and angiogenesis (163). In addition, treatment with doxicycline, which had MMP inhibitory activity, during postnatal alveolar development (day 4 to day 13) in rats results in enlarged alveolar spaces compared with control rats (107), confirming the role of MMPs in alveolar development.
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V. RESURGENCE OF MATRIX METALLOPROTEINASES IN LUNG DISEASES: ACUTE AND CHRONIC |
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In a mouse model of hyperoxia-induced lung disease, direct O2 toxicity can result in alveolar type I cell injury and death. Lung damage is mediated by the synthesis and release of MMP9 and MMP12 by neutrophils and alveolar macrophages. Overexpression of STAT3C in alveolar epithelial cells results in increased survival, decreased capillary leakage, and suppressed neutrophil infiltration. These deleterious effects were prevented through STAT3C-mediated inhibition of MMP9 and MMP12 (146).
Other indirect evidence for the role of chronic exposure to MMPs in structural lung damage comes from transgenic overexpression models of IFN-, the canonical Th1 cytokine, and IL-13, a key Th2 cytokine. Inducible and targeted expression of IFN- in the lung results in increased alveolar space, lung volume, and pulmonary compliance and is associated with excessive expression of MMP12 and MMP9 (244, 291). The role of IFN- in modulation of MMPs in vivo is most likely an indirect one, because prior studies show that IFN- downregulates MMPs but CXCL10 (IFN-inducible protein of 10 kDa, IP10) upregulated MMP12 in alveolar macrophages (87, 108). Similarly, inducible and targeted expression of IL-13 in mice is associated with increased MMP2, MMP9, and MMP12 expression in the lung, and these mice also develop alveolar and lung enlargement, compliance variations, and respiratory failure, ultimately leading to death, which is prevented when they are crossed with MMP9- or MMP12-deficient mice. This result strongly suggests that the alveolar damage is mediated by MMPs (135).
MMP12 null mice exposed to long-term cigarette smoke fail to recruit macrophages into their lung and do not develop emphysema compared with wild-type littermates (102). These findings are one of the most definitive studies to show that chronic exposure to cigarette smoke induces MMP12 expression in macrophages, which is required for the development of emphysema in mice.
MMP7, an epithelial specific MMP, is constitutively expressed at low levels in the airway epithelia, but its expression is upregulated under a variety of pathological conditions in the lung (60, 211). Short exposure of lung epithelial cells to Pseudomonas aeruginosa or its flagellin results in a strong upregulation of MMP7 that is essential in eradicating this organism, because mice deficient in MMP7 fail to clear the pathogen efficiently (302). However, under chronic conditions, the resurgence of MMP7 may create a more ominous effect. For instance, in cystic fibrosis, MMP7 expression is increased in airway epithelial cells and induced in alveolar type II cells (60). Furthermore, osteopontin, an ECM molecule that distinguishes fibrotic from normal lungs in microarray analysis, upregulates MMP7 expression in lung epithelial cells and may exert a profibrotic effect in idiopathic pulmonary fibrosis (208). These results highlight the potentially different roles of MMPs from acute to chronic conditions.
Pulmonary fibrosis is a chronic condition that results in loss of elastic recoil of the lung. Although evidence for the role of MMPs in the pathogenic processes associated with pulmonary fibrosis and injury to basement membranes is indirect, nonetheless a few studies highlight the potential function of MMPs in this condition. For example, MMP9 expression is increased in the lung in response to intratracheal instillation of bleomycin; however, initiation or evolution of the inflammatory and fibrotic changes that occur are independent of MMP9, because MMP9 null mice have inflammatory cell infiltration and fibrosis equivalent to wild type (19). In addition, MMP9 null mice have disrupted bronchiolization after bleomycin-induced injury, suggesting that MMP9 may be important for repair and may play a more serious role in the long-term recovery from lung injury (19). In addition, in the chronic phase of bleomycin-induced pulmonary fibrosis, attenuation of MMP2 and MMP9 expression and activity in macrophages from mice lacking the C-C chemokine receptor 2 (CCR2–/–) is accompanied by reduced lung remodeling and hydroxyproline content of lung tissues (198), suggesting a role for MCP1 and macrophage infiltration in fibrotic disease.
Another instance of MMP resurgence in chronic inflammation is via CD147. As we discussed earlier in section IV, CD147 is present in the lung during development, but its expression drops to low levels in normal adult lung (39). CD147 has been shown to increase the expression of MMP1, MMP2, MMP3, MMP9, and MMP14 in the lung either directly or through stimulation of the cells adjacent to the tumor mass (90, 264, 265). Not surprisingly, early increases of CD147 have been found in several models of lung damage, such as ventilator-associated acute lung injury (74), LPS (7), and bleomycin-induced lung injury (20).
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VI. MATRIX METALLOPROTEINASES: REDUNDANT VERSUS COMPENSATORY MECHANISM OF ACTION |
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3 wk of age resulting in normal adult mice (286). It is unclear which MMP may be providing the compensatory action. Despite the superficial appearance of redundancy, the differential tissue distribution of these enzymes suggests that they have distinct and nonoverlapping functions in vivo (3, 48, 122, 165). Even more importantly, the apparent compensatory and/or redundant action by other members of the MMP family during normal conditions falls apart when the same null mutations are tested under pathological conditions (48, 49, 144, 166, 170, 302).
Several disease models support the hypothesis that MMPs do not have redundant functions, at least in immune response. First, in an asthma model (Fig. 3), decreased BAL inflammatory cells are observed in MMP2–/– mice; however, the decrease is entirely accounted for by fewer eosinophils (48). Thus observed decreases in neutrophil and eosinophil counts in the BAL of mice deficient in MMP9 reveal similar but nonoverlapping specificity of MMP9 and MMP2 (48).
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VII. MATRIX METALLOPROTEINASES IN INNATE AND ADAPTIVE IMMUNE FUNCTION |
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MMPs are now being recognized as one of the critical mediators for host defense. In recent years, mammalian defensins have been shown to function as effectors of antimicrobial innate immunity. Defensins are small peptides expressed in the leukocytes, mucosal surfaces, and epithelia as prepropeptides (241) from the Paneth cells and require activation by a convertase. In the case of murine -defensin, the convertase is MMP7, an epithelial-specific MMP (301, 302). Mice deficient in MMP7 die more rapidly when exposed to gram-negative bacterial pathogens, and the mechanism for this increased susceptibility is lack of the active form of -defensin in the gut (302). Whether other members of the MMP family act as convertases for other defensins is not known, but based on emerging evidence from animal models of bacterial infection, MMP7 is strongly upregulated as part of mucosal immunity in response to infection (153, 213). Studies in mice have shown that MMP2 and MMP9 are upregulated in response to MTb infection and may be critical in clearance of this organism in the lung (65, 227). Mice infected with MTb and treated with a BB-94, a general inhibitor of MMPs, develop an aberrant Th2 immune response and also develop increased IL-4. This treatment resulted in rapid progression of the disease (105).
Other evidence for the role of MMPs in lung innate immune system originates from the report that pulmonary surfactant proteins regulate the expression of several MMPs in alveolar macrophages. Lung surfactant, which is primarily composed of 90% lipids and 10% proteins, coats the distal airspaces to reduce the alveolar surface tension, a process that is essential in prevention of atelectasis, and in host defense against environmental pathogens (92). Of the four surfactant-associated proteins that have been cloned in mammals, two with hydrophilic properties, surfactant protein (SP)-A and SP-D, play critical roles in innate host defense and adaptive immunity (156, 306). Recombinant SP-D upregulates expression of MMP1, MMP3, and MMP12 in human alveolar macrophages (273). SP-D-mediated upregulation of MMPs is independent of the production of TNF- or IL-1, two potent inducers of MMPs (273). One hypothesis is that specific upregulation of MMPs in alveolar macrophages by SP-D enhances their antimicrobial function in vivo by directly activating macrophages and stimulating the production of more MMPs, which could result in formation of bioactive proteins (Fig. 4).
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SP-D-induced upregulation of MMPs in vitro appears to be in sharp contrast to multiple reports that indicate the absence of SP-D in mice results in accumulation of activated macrophages and proinflammatory enzymes including MMP2, MMP9, and MMP12 in the lung (194, 296). The phenotype of SP-D null (SP-D –/–) mice is quite complex, but these mice show marked inflammation and histological changes in the lung that have similarities with human emphysema (296). However, it is not clear if the absence of SP-D results in inefficient turnover of surfactant proteins or increases susceptibility to infection, which could contribute to the inflammatory changes seen in the lung of SP-D –/– mice. Consistent with this possibility, SP-D –/– mice fare worse during bacterial infection due to decreased cell-surface expression of CD14, a pattern recognition receptor present on many innate immune cells (242). Macrophages without CD14 presumably
