Matrix metalloproteinases in lung repair.
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Animals↗
Biomedical subjects
W C Parks
Publications and source records attributed to W C Parks.
Hypochlorous acid oxygenates the cysteine switch domain of pro-matrilysin (MMP-7). A mechanism for matrix metalloproteinase activation and atherosclerotic plaque rupture by myeloperoxidase.
Myeloperoxidase uses hydrogen peroxide (H2O2) to generate hypochlorous acid (HOCl), a potent cytotoxic oxidant. We demonstrate that HOCl regulates the activity of matrix metalloproteinase-7 (MMP-7, matrilysin) in vitro, suggesting that this oxidant activates MMPs in the artery wall. Indeed, both MMP-7 and myeloperoxidase were colocalized to lipid-laden macrophages in human atherosclerotic lesions. A highly conserved domain called the cysteine switch has been proposed to regulate MMP activity. When we exposed a synthetic peptide that mimicked the cysteine switch to HOCl, HPLC analysis showed that the thiol residue reacted rapidly, generating a near-quantitative yield of products. Tandem mass spectrometric analysis identified the products as sulfinic acid, sulfonic acid, and a dimer containing a disulfide bridge. In contrast, the peptide reacted slowly with H2O2, and the only product was the disulfide. Moreover, HOCl markedly activated pro-MMP-7, an MMP expressed at high levels in lipid-laden macrophages in vivo. Tandem mass spectrometric analysis of trypsin digests revealed that the thiol residue of the enzyme's cysteine switch domain had been converted to sulfinic acid. Thiol oxidation was associated with autolytic cleavage of pro-MMP-7, strongly suggesting that oxygenation activates the latent enzyme. In contrast, H2O2 failed to oxidize the thiol residue of the protein or activate the enzyme. Thus, HOCl activates pro-MMP-7 by converting the thiol residue of the cysteine switch to sulfinic acid. This activation mechanism is distinct from the well-studied proteolytic cleavage of MMP pro-enzymes. Our observations raise the possibility that HOCl generated by myeloperoxidase contributes to MMP activation, and therefore to plaque rupture, in the artery wall. HOCl and other oxidants might regulate MMP activity by the same mechanism in a variety of inflammatory conditions.
Amino Acid Sequence↗
Regulation of matrilysin expression in airway epithelial cells by Pseudomonas aeruginosa flagellin.
Matrilysin (matrix metalloproteinase-7) is expressed by mucosal epithelia throughout the body and functions in host defense by activating murine intestinal alpha-defensins. In normal adult human lung, matrilysin is expressed at low levels in the airway epithelium, but is markedly up-regulated in cystic fibrosis (CF). Because CF lungs support a heavy bacterial load, we assessed if relevant CF pathogens regulate matrilysin expression in human lung epithelial cells. Indeed, acute infection with Pseudomonas aeruginosa (but not Staphylococcus aureus, Haemophilus influenzae, or Klebsiella pneumoniae) induced the expression of matrilysin in Calu-3 lung epithelial cells. Increased matrilysin mRNA levels were detectable at 3 h post-infection and peaked at a 25-fold induction between 6 and 8 h. Both P. aeruginosa CF isolates and laboratory strains induced matrilysin expression to similar levels. Flagellin, the monomeric precursor of bacterial flagella, was identified as the inductive factor released by P. aeruginosa that regulated matrilysin expression. In addition, flagellin-null mutants failed to stimulate matrilysin expression in cultured cells or in lungs infected in vivo. These data show that P. aeruginosa (and specifically flagellin) potently stimulates matrilysin expression in lung epithelial cells and may mediate the overexpression of this proteinase in CF lungs.
Animals↗
Structural analysis of the alpha(2) integrin I domain/procollagenase-1 (matrix metalloproteinase-1) interaction.
Previous studies have established that ligation of keratinocyte alpha(2)beta(1) integrin by type I collagen induces expression of matrix metalloproteinase-1 (MMP-1) and that MMP-1 activity is required for the alpha(2)beta(1) integrin-dependent migration of primary keratinocytes across collagenous matrices. We now present evidence that MMP-1 binds the alpha(2)beta(1) integrin via the I domain of the alpha(2) integrin subunit. Using an enzyme-linked immunosorbent assay with purified human MMP-1 and recombinant alpha(2) integrin I domain, we showed that the alpha(2) integrin I domain specifically bound in a divalent cation-dependent manner to both the pro and active forms of MMP-1, but not to MMP-3 or MMP-13. Although both the I domain and MMP-1 bind divalent cations, MMP-1 bound, in a divalent cation-dependent manner, to alpha(2) integrin I domains containing metal ion-dependent adhesion sites motif mutations that prevent divalent cation binding to the I domain, demonstrating that the metal ion dependence is a function of MMP-1. Using a series of MMP-1-MMP-3 and MMP-1-MMP-13 chimeras, we determined that both the linker domain and the hemopexin-like domain of MMP-1 were required for optimal binding to the I domain. The alpha(2) integrin/MMP-1 interaction described here extends an emerging paradigm in matrix biology involving anchoring of proteinases to the cell surface to regulate their biological activities.
Antigens, CD↗
Pro-collagenase-1 (matrix metalloproteinase-1) binds the alpha(2)beta(1) integrin upon release from keratinocytes migrating on type I collagen.
In injured skin, collagenase-1 (matrix metalloproteinase-1 (MMP-1)) is induced in migrating keratinocytes. This site-specific expression is regulated by binding of the alpha(2)beta(1) integrin with dermal type I collagen, and the catalytic activity of MMP-1 is required for keratinocyte migration. Because of this functional association among substrate/ligand, receptor, and proteinase, we assessed whether the integrin also directs the compartmentalization of MMP-1 to its matrix target. Indeed, pro-MMP-1 co-localized to sites of alpha(2)beta(1) contacts in migrating keratinocytes. Furthermore, pro-MMP-1 co-immunoprecipitated with alpha(2)beta(1) from keratinocytes, and alpha(2)beta(1) co-immunoprecipitated with pro-MMP-1. No other MMPs bound alpha(2)beta(1), and no other integrins interacted with MMP-1. Pro-MMP-1 also provided a substrate for alpha(2)beta(1)-dependent adhesion of platelets. Complex formation on keratinocytes was most efficient on native type I collagen and reduced or ablated on denatured or cleaved collagen. Competition studies suggested that the alpha(2) I domain interacts with the linker and hemopexin domains of pro-MMP-1, not with the pro-domain. These data indicate that the interaction of pro-MMP-1 with alpha(2)beta(1) confines this proteinase to points of cell contact with collagen and that the ternary complex of integrin, enzyme, and substrate function together to drive and regulate keratinocyte migration.
Adult↗
p38 mitogen-activated kinase is a bidirectional regulator of human fibroblast collagenase-1 induction by three-dimensional collagen lattices.
When fibroblasts are cultured in contracting collagen matrices, matrix metalloproteinase-1 (MMP-1, collagenase-1) is induced. In the present study we demonstrate that p38alpha mitogen-activated protein kinase (p38alpha MAPK) plays a bi-directional role in the MMP-1 response to contracting floating collagen lattices (fl-coll). fl-coll, but not attached collagen lattices (att-coll), co-ordinately increased expression of MMP-1 and activities of p38alpha and MKK3/6 (MAPK kinase 3/6). However, treatment of primary fibroblasts cultured in fl-coll with increasing doses of SB203580, an inhibitor of p38alpha and p38beta, caused a bipolar pattern of MMP-1 expression. Partial inhibition of p38 MAPK activity resulted in the lowest level of MMP-1 expression, whereas total inhibition of p38 activity led to MMP-1 levels as high as in the absence of inhibitor. The activation/inhibition of p38alpha was apparently responsible for the observed phenomena, as supported by three lines of evidence. (1) p38alpha was the predominant isoform sensitive to SB203580 in primary fibroblasts. (2) Fibroblasts transfected with increasing dose of a dominant negative p38alpha (p38DN) similarly demonstrated the bipolar pattern of MMP-1 expression induced by fl-coll. (3) The bipolar MMP-1 expression occurred during the gradual, linear inhibition of p38alpha kinase activity by both inhibitors, SB203580 and p38DN. Nuclear factor-kappaB (NF-kappaB), a previously identified positive regulator of MMP-1 expression induced by fl-coll [Xu, Zutter, Santoro and Clark (1998) J. Cell Biol. 140, 709-719] was mediated by fl-coll-activated p38alpha. However, the fl-coll-induced expression of MMP-1 facilitated by p38alpha suppression was maintained independent of NF-kappaB activity, suggesting the existence of a p38alpha-dependent antagonistic pathway. We conclude that fl-coll-induced MMP-1 expression is the net outcome of opposing effects mediated by p38alpha. Therefore, the level of p38alpha kinase activity may provide a fine-tuned control of MMP-1 gene expression in response to biomechanical signals.
Adult↗
Bromination of deoxycytidine by eosinophil peroxidase: a mechanism for mutagenesis by oxidative damage of nucleotide precursors.
Oxidants generated by eosinophils during chronic inflammation may lead to mutagenesis in adjacent epithelial cells. Eosinophil peroxidase, a heme enzyme released by eosinophils, generates hypobromous acid that damages tissue in inflammatory conditions. We show that human eosinophils use eosinophil peroxidase to produce 5-bromodeoxycytidine. Flow cytometric, immunohistochemical, and mass spectrometric analyses all demonstrated that 5-bromodeoxycytidine generated by eosinophil peroxidase was taken up by cultured cells and incorporated into genomic DNA as 5-bromodeoxyuridine. Although previous studies have focused on oxidation of chromosomal DNA, our observations suggest another mechanism for oxidative damage of DNA. In this scenario, peroxidase-catalyzed halogenation of nucleotide precursors yields products that subsequently can be incorporated into DNA. Because the thymine analog 5-BrUra mispairs with guanine in DNA, generation of brominated pyrimidines by eosinophils might constitute a mechanism for cytotoxicity and mutagenesis at sites of inflammation.
Animals↗
Pathogenesis of abdominal aortic aneurysms: a multidisciplinary research program supported by the National Heart, Lung, and Blood Institute.
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Aortic Aneurysm, Abdominal↗
Matrilysin in epithelial repair and defense.
Repair involves an orderly progression of events to reestablish the integrity of the injured tissue. During each stage in this process, secreted proteinases are needed to remodel extracellular matrix, facilitate cell migration, and process latent proteins, among other functions. In lung epithelium, several of these processes are mediated by matrilysin, a matrix metalloproteinase (MMP). Unlike most MMPs, matrilysin is produced by intact, noninjured airway and peribronchial epithelial cells. In other intact epithelial tissues, namely the small intestine, matrilysin functions in host defense by activating the latent form of defensins, a family of antimicrobial peptides. This metalloproteinase may serve a similar function in the lung. Furthermore, in models of airway injury, matrilysin expression is upregulated in migrating epithelial cells, and the activity of this proteinase is required for repair of airway wounds. These observations indicate that matrilysin serves key functions in both epithelial defense and repair.
Animals↗
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.
Animals↗
Epilysin, a novel human matrix metalloproteinase (MMP-28) expressed in testis and keratinocytes and in response to injury.
We have cloned a new human matrix metalloproteinase (MMP-28, epilysin) from human keratinocyte and testis cDNA libraries. Like most MMPs, epilysin contains a signal sequence, a prodomain with a PRCGVTD sequence, a zinc-binding catalytic domain with an HEIGHTLGLTH sequence, and a hemopexin-like domain. In addition, epilysin has a furin activation sequence (RRKKR) but has no transmembrane sequence. The exon-intron organization and splicing pattern of epilysin differ from that of other MMP genes. It has only 8 exons, and 5 exons are spliced at sites not used by other MMPs. Another novel feature of epilysin is that exon 4 is alternatively spliced to a transcript that does not encode the N-terminal half of the catalytic domain. Northern hybridization of tissue RNA indicated that epilysin is expressed at high levels in testis and at lower levels in lungs, heart, colon, intestine, and brain. RNase protection assay with various cell lines indicated that epilysin was selectively expressed in keratinocytes. Recombinant epilysin degraded casein in a zymography assay, and its proteolytic activity was inhibited by EDTA and by batimastat, a selective MMP inhibitor. Immunohistochemical staining showed expression of epilysin protein in the basal and suprabasal epidermis of intact skin. In injured skin, prominent staining for epilysin was seen in basal keratinocytes both at and some distance from the wound edge, a pattern that is quite distinct from that of other MMPs expressed during tissue repair. These findings suggest that this new MMP functions in several tissues both in tissue homeostasis and in repair.
Adult↗
Bacterial exposure induces and activates matrilysin in mucosal epithelial cells.
Matrilysin, a matrix metalloproteinase, is expressed and secreted lumenally by intact mucosal and glandular epithelia throughout the body, suggesting that its regulation and function are shared among tissues. Because matrilysin is produced in Paneth cells of the murine small intestine, where it participates in innate host defense by activation of prodefensins, we speculated that its expression would be influenced by bacterial exposure. Indeed, acute infection (10-90 min) of human colon, bladder, and lung carcinoma cells, primary human tracheal epithelial cells, and human tracheal explants with type 1-piliated Escherichia coli mediated a marked (25-50-fold) and sustained (>24 h) induction of matrilysin production. In addition, bacterial infection resulted in activation of the zymogen form of the enzyme, which was selectively released at the apical surface. Induction of matrilysin was mediated by a soluble, non-LPS bacterial factor and correlated with the release of defensin-like bacteriocidal activity. Bacteria did not induce matrilysin in other cell types, and expression of other metalloproteinases by epithelial cells was not affected by bacteria. Matrilysin was not detected in germ-free mice, but the enzyme was induced after colonization with Bacteroides thetaiotaomicron. These findings indicate that bacterial exposure is a potent and physiologically relevant signal regulating matrilysin expression in epithelial cells.
Adenocarcinoma↗
Structural analysis and promoter characterization of the human membrane-type matrix metalloproteinase-1 (MT1-MMP) gene.
Membrane type-1 matrix metalloproteinase (MT1-MMP) degrades extracellular matrix components directly and indirectly by activation of other matrix metalloproteinases (MMPs). In the present study, we have isolated and characterized the human MT1-MMP gene and its promoter. The gene consists of 10 exons and nine introns spanning more than 10 kilobases (kb). The locations of two exon-intron splicing sites are distinct from the preserved positions among other known MMP genes. Primer extension and RNAse and S1 nuclease protection analyses indicated that there are four major and several minor transcription start sites. The 5'-flanking sequence of the gene contains putative regulatory elements, including one Sp-1 site and four CCAAT-boxes, whereas there is no TATA-box. The Sp-1 binding site was functional, as shown by gel shift and supershift analyses. Transfection studies with promoter constructs containing 0.1 to 7.2 kb of 5'-flanking sequence coupled to a luciferase reporter gene indicated that the promoter contains additional positive and negative regulatory sequences. Deletion of the Sp-1 binding site by site-directed mutagenesis reduced luciferase activity by about 90%, demonstrating the crucial role of this element in maintaining MT1-MMP transcription. Our findings indicate that the human MT1-MMP promoter has distinctive structural and functional features compared with other MMP genes, which may lead to a unique expression pattern and regulation during physiological and pathological processes.
Base Sequence↗
Gelatinase B is required for alveolar bronchiolization after intratracheal bleomycin.
Increased expression of matrix metalloproteinases, particularly gelatinase B (MMP-9), has been described in the lungs in pulmonary fibrosis. Intratracheal bleomycin is often used experimentally to produce lesions resembling human fibrosing alveolitis. To assess the role of gelatinase B in bleomycin-induced fibrosing alveolitis, we instilled bleomycin intratracheally into gelatinase B-deficient mice and gelatinase B+/+ littermates. Twenty-one days after bleomycin the two groups of mice were indistinguishable in terms of pulmonary histology and total lung collagen and elastin. However, the lungs of gelatinase B-deficient mice showed minimal alveolar bronchiolization, whereas bronchiolization was prominent in the lungs of gelatinase B+/+ mice. Gelatinase B was identified immunohistochemically in terminal bronchiolar cells and bronchiolized cells 7 and 14 days after bleomycin in gelatinase B+/+ mice, and whole lung gelatinase B mRNA was increased at the same times. Many bronchiolized cells displayed Clara cell features by electron microscopy. Some bronchiolized cells stained with antibody to helix transcription factor 4, a factor associated with the ciliated cell phenotype. Thus, fibrosing alveolitis develops after intratracheal bleomycin irrespective of gelatinase B. However, gelatinase B is required for alveolar bronchiolization, perhaps by facilitating migration of Clara cells and other bronchiolar cells into the regions of alveolar injury.
Animals↗
Secretion of microbicidal alpha-defensins by intestinal Paneth cells in response to bacteria.
Paneth cells in mouse small intestinal crypts secrete granules rich in microbicidal peptides when exposed to bacteria or bacterial antigens. The dose-dependent secretion occurs within minutes and alpha-defensins, or cryptdins, account for 70% of the released bactericidal peptide activity. Gram-negative bacteria, Gram-positive bacteria, lipopolysaccharide, lipoteichoic acid, lipid A and muramyl dipeptide elicit cryptdin secretion. Live fungi and protozoa, however, do not stimulate degranulation. Thus intestinal Paneth cells contribute to innate immunity by sensing bacteria and bacterial antigens, and discharge microbicidal peptides at effective concentrations accordingly.
Animals↗
Recent advances in molecular biological techniques and their relevance to pulmonary research.
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Animals↗
Regulation of intestinal alpha-defensin activation by the metalloproteinase matrilysin in innate host defense.
Precursors of alpha-defensin peptides require activation for bactericidal activity. In mouse small intestine, matrilysin colocalized with alpha-defensins (cryptdins) in Paneth cell granules, and in vitro it cleaved the pro segment from cryptdin precursors. Matrilysin-deficient (MAT-/-) mice lacked mature cryptdins and accumulated precursor molecules. Intestinal peptide preparations from MAT-/- mice had decreased antimicrobial activity. Orally administered bacteria survived in greater numbers and were more virulent in MAT-/- mice than in MAT+/+ mice. Thus, matrilysin functions in intestinal mucosal defense by regulating the activity of defensins, which may be a common role for this metalloproteinase in its numerous epithelial sites of expression.
Amino Acid Sequence↗
Role of matrix metalloproteinases and their inhibition in cutaneous wound healing and allergic contact hypersensitivity.
Normal wounds can heal by secondary intention (epidermal migration to cover a denuded surface) or by approximation of the wound edges (e.g., suturing). In healing by secondary intention, epidermis-derived MMPs are important. Keratinocyte migration begins within 3-6 hr post injury, as basal cells detach from underlying basal lamina and encounter a dermal substratum rich in type I collagen. Cell contact with type I collagen in vitro stimulates collagenase-1 expression, which is mediated by the alpha 2 beta 1 integrin, the major keratinocyte collagen-binding receptor. Collagenase-1 activity alone is necessary and sufficient for keratinocyte migration over a collagen subsurface. Stromelysins-1 and -2 are also found in the epidermis of normal acute wounds. Stromelysin-2 co-localizes with collagenase-1 and may facilitate cell migration over non-collagenous matrices of the dermis. In contrast, stromelysin-1 is expressed by keratinocytes behind the migrating front and which remain on basal lamina, i.e., the proliferative cell population. Studies with stromelysin-1-deficient mice that suggest this MMP plays a role in keratinocyte detachment from underlying basement membrane to initiate cell migration. In chronic ulcers, MMP levels are markedly elevated, in contrast to their precise temporal and spatial expression in acute wounds. Both collagenase-1 and stromelysin-1 are found in fibroblasts underlying the nonhealing epithelium, and stromelysin-1 expression is especially prominent. Two key questions underlie the use of MMP inhibitors and wound healing: (1) will these agents impair normal reepithelialization in wounds that heal by secondary intention; and (2) can MMP inhibitors be effective therapy for chronic ulcers? The answer to neither is known. Batimastat and marimastat appear not to interfere with normal wound healing, but only in sutured surgical wounds, a situation in which MMP expression has practically no role. We also show the first example of an in vivo immune response, contact hypersensitivity, which is dependent upon MMP activity. Using gene-deficient mice, we demonstrate that stromylysin-1 (MMP-3) is required for sensitization, whereas gelatinase B (MMP-9) is required for timely resolution of the reaction to antigenic challenge.
Animals↗