Abstract: Matrilysin (matrix metalloproteinase-7) is highly expressed in lungs of patients with pulmonary fibrosis and other conditions associated with airway and alveolar injury. Although matrilysin is required for closure of epithelial wounds ex vivo, the mechanism of its action in repair is unknown. We demonstrate that matrilysin mediates shedding of E-cadherin ectodomain from injured lung epithelium both in vitro and in vivo. In alveolar-like epithelial cells, transfection of activated matrilysin resulted in shedding of E-cadherin and accelerated cell migration. In vivo, matrilysin co-localized with E-cadherin at the basolateral surfaces of migrating tracheal epithelium, and the reorganization of cell-cell junctions seen in wild-type injured tissue was absent in matrilysin-null samples. E-cadherin ectodomain was shed into the bronchoalveolar lavage fluid of bleomycin-injured wild-type mice, but was not shed in matrilysin-null mice. These findings identify E-cadherin as a novel substrate for matrilysin and indicate that shedding of E-cadherin ectodomain is required for epithelial repair. Matrilysin (matrix metalloproteinase-7) is highly expressed in lungs of patients with pulmonary fibrosis and other conditions associated with airway and alveolar injury. Although matrilysin is required for closure of epithelial wounds ex vivo, the mechanism of its action in repair is unknown. We demonstrate that matrilysin mediates shedding of E-cadherin ectodomain from injured lung epithelium both in vitro and in vivo. In alveolar-like epithelial cells, transfection of activated matrilysin resulted in shedding of E-cadherin and accelerated cell migration. In vivo, matrilysin co-localized with E-cadherin at the basolateral surfaces of migrating tracheal epithelium, and the reorganization of cell-cell junctions seen in wild-type injured tissue was absent in matrilysin-null samples. E-cadherin ectodomain was shed into the bronchoalveolar lavage fluid of bleomycin-injured wild-type mice, but was not shed in matrilysin-null mice. These findings identify E-cadherin as a novel substrate for matrilysin and indicate that shedding of E-cadherin ectodomain is required for epithelial repair. Epithelial damage is a prominent feature of several pulmonary diseases. For example, in conducting airways, exfoliated epithelial cells are found in sputum and bronchoalveolar lavage fluid from patients with asthma, and the epithelium is severely damaged in cystic fibrosis and bronchiolitis obliterans.1Dunnill MS Massarella GA Anderson JA A comparison of the quantitative anatomy of the bronchi in normal subjects, in status asthmaticus, in chronic bronchitis, and in emphysema.Thorax. 1969; 24: 176-179Crossref PubMed Scopus (500) Google Scholar, 2Erjefalt JS Persson CGA Airway epithelial repair: breathtakingly quick and multipotentially pathogenic.Thorax. 1997; 52: 1010-1012Crossref PubMed Scopus (69) Google Scholar, 3Laitinen LA Heino M Laitinin A Kava T Haahtela T Damage of the airway epithelium and bronchial reactivity in patients with asthma.Am Rev Respir Dis. 1985; 131: 599-606Crossref PubMed Scopus (967) Google Scholar, 4Jeffery PK Wardlaw AJ Nelson FC Collins JV Kay AB Bronchial biopsies in asthma. An ultrastructural, quantitative study and correlation with hyperreactivity.Am Rev Respir Dis. 1989; 140: 1745-1753Crossref PubMed Scopus (810) Google Scholar, 5Myers JL Katzenstein AL Ultrastructural evidence of alveolar epithelial injury in idiopathic bronchiolitis obliterans-organizing pneumonia.Am J Pathol. 1988; 132: 102-109PubMed Google Scholar Alveolar epithelium is conspicuously damaged in acute lung injury/acute respiratory distress syndrome (ALI/ARDS), desquamative interstitial pneumonitis, and cystic fibrosis, among many other acute and chronic lung diseases.6Bachofen M Weibel ER Structural alterations of lung parenchyma in the adult respiratory distress syndrome.Clin Chest Med. 1982; 3: 35-56Abstract Full Text PDF PubMed Google Scholar, 7Fehrenbach H Alveolar epithelial type II cell: defender of the alveolus revisited.Respir Res. 2001; 2: 33-46Crossref PubMed Scopus (531) Google Scholar An immediate response to epithelial loss is the spreading and migration of intact cells to cover the denuded basement membrane.2Erjefalt JS Persson CGA Airway epithelial repair: breathtakingly quick and multipotentially pathogenic.Thorax. 1997; 52: 1010-1012Crossref PubMed Scopus (69) Google Scholar, 8Adamson IY Hedgecock C Bowden DH Epithelial cell-fibroblast interactions in lung injury and repair.Am J Pathol. 1990; 137: 385-392PubMed Google Scholar, 9Lesur O Arsalane K Lane D Lung alveolar epithelial cell migration in vitro: modulators and regulation processes.Am J Physiol. 1996; 270: L311-L319PubMed Google Scholar Numerous proteins and peptides, including cell adhesion molecules and proteolytic enzymes, regulate the repair process, and failure to restore a functional epithelial barrier contributes to progressive injury, inflammation, and fibrosis. The matrix metalloproteinases (MMPs) are a family of zinc-containing enzymes with proteolytic activity against a wide range of extracellular proteins.10Brinckerhoff CE Matrisian LM Matrix metalloproteinases: a tail of a frog that became a prince.Nature Rev Mol Cell Biol. 2002; 3: 207-214Crossref PubMed Scopus (953) Google Scholar MMP expression is typically limited to tissue remodeling associated with normal and abnormal biological processes, such as development, involution, inflammation, tumor growth, and repair. Matrilysin (MMP-7), unlike many MMPs, is expressed by noninjured, noninflamed mucosal epithelia in most adult human tissues.11Wilson CL Heppner KJ Rudolph LA Matrisian LM The metalloproteinase matrilysin is preferentially expressed by epithelial cells in a tissue-restricted pattern in the mouse.Mol Biol Cell. 1995; 6: 851-869Crossref PubMed Scopus (132) Google Scholar, 12Saarialho-Kere UK Crouch EC Parks WC Matrix metalloproteinase matrilysin is preferentially expressed in human exocrine epithelium.J Invest Dermatol. 1995; 105: 190-196Crossref PubMed Scopus (142) Google Scholar In the human lung, matrilysin is constitutively expressed in tracheal glands and in tracheobronchial epithelium, and expression is acutely up-regulated by injury or exposure to bacteria.13Dunsmore SE Saarialho-Kere UK Roby JD Wilson CL Matrisian LM Welgus HG Parks WC Matrilysin expression and function in airway epithelium.J Clin Invest. 1998; 102: 1321-1331Crossref PubMed Scopus (229) Google Scholar, 14Lopez-Boado YS Wilson CL Parks WC Regulation of matrilysin expression in airway epithelial cells by Pseudomonas aeruginosa flagellin.J Biol Chem. 2001; 276: 41417-41423Crossref PubMed Scopus (75) Google Scholar Work in our laboratory demonstrated that matrilysin is prominently expressed in injured airways and is necessary for airway mucosal repair,13Dunsmore SE Saarialho-Kere UK Roby JD Wilson CL Matrisian LM Welgus HG Parks WC Matrilysin expression and function in airway epithelium.J Clin Invest. 1998; 102: 1321-1331Crossref PubMed Scopus (229) Google Scholar indicating that this a critical extracellular proteinase in repair processes. Matrilysin is also prominently expressed by alveolar epithelium in cystic fibrosis and pulmonary fibrosis.15Cosgrove GP Schwartz MI Geraci MW Brown KK Worthen GS Overexpression of matrix metalloproteinase-7 in pulmonary fibrosis.Chest. 2002; 121: 25S-26SAbstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar, 16Zuo F Kaminski N Eugui E Allard J Yakhini Z Ben-Dor A Lollini L Morris D Kim Y DeLustro B Sheppard D Pardo A Selman M Heller RA Gene expression analyses reveal matrilysin as a key regulator of pulmonary fibrosis in mice and humans.Proc Natl Acad Sci. 2002; 99: 6292-6297Crossref PubMed Scopus (510) Google Scholar Work in our laboratory found that matrilysin is induced in alveolar epithelium in mice after lung injury with bleomycin and expression increases and continues as fibrosis progresses.17Li QL Park PY Parks WC Matrilysin-mediated shedding of syndecan-1 regulates chemokine mobilization and transepithelial efflux of neutrophils in acute lung injury.Cell. 2002; 111: 635-646Abstract Full Text Full Text PDF PubMed Scopus (633) Google Scholar In this model, matrilysin was shown to regulate neutrophil influx by controlling chemokine compartmentalization during the first days after injury. However, the extended production of matrilysin indicates that this MMP serves multiple, distinct roles in repair of lung epithelium. In other words, matrilysin acts on different protein substrates involved in different processes at different stages of repair. Epithelial (E)-cadherin, a transmembrane glycoprotein localized at adherens junctions, mediates calcium-dependent cell-cell adhesion in most epithelia. In cooperation with cytoskeletal structures, E-cadherin is thought to regulate cell differentiation and morphogenesis.18Yap AS Brieher WM Gumbiner BM Molecular and functional analysis of cadherin-based adherens junctions.Annu Rev Cell Dev Biol. 1997; 13: 119-146Crossref PubMed Scopus (681) Google Scholar Reduction or loss of E-cadherin correlates with increased malignancy in tumors and invasiveness in carcinoma cell lines in vitro.19Birchmeier W Behrens J Cadherin expression in carcinomas: role in the formation of cell junctions and the prevention of invasiveness.Biochim Biophys Acta. 1994; 1198: 11-26Crossref PubMed Scopus (924) Google Scholar, 20Chen H Paradies NE Fedor-Chaiken M Brackenbury R E-cadherin mediates adhesion and suppress cell motility via distinct mechanisms.J Cell Science. 1997; 110: 345-356PubMed Google Scholar Although the regulation of cadherin adhesive activity is not completely understood, the level of cadherin gene expression correlates with the strength of adhesion, and the type of cadherin expressed affects the specificity of cell interaction.21Gumbiner BM Regulation of cadherin adhesive activity.J Cell Biol. 2000; 148: 399-404Crossref PubMed Scopus (686) Google Scholar Posttranscriptional mechanisms regulating cadherin adhesion include modulation of cadherin clustering at the cell surface, changes in cadherin interaction with other proteins, and proteolysis of cadherin ectodomains.21Gumbiner BM Regulation of cadherin adhesive activity.J Cell Biol. 2000; 148: 399-404Crossref PubMed Scopus (686) Google Scholar, 22Ito K Okamoto I Araki N Kawano Y Nakao M Fujiyama S Tomita K Mimori T Saya H Calcium influx triggers the sequential proteolysis of extracellular and cytoplasmic domains of E-cadherin, leading to loss of b-catenin from cell-cell contacts.Oncogene. 1999; 18: 7080-7090Crossref PubMed Scopus (115) Google Scholar, 23Marambaud P Shioi J Serban G Georgakopoulos A Sarner S Nagy V Baki L Wen P Efthimiopoulos S Shao Z Wisniewski T Robakis NK A presenilin-1/g-secretase cleavage releases the E-cadherin intracellular domain and regulates disassembly of adherens junctions.EMBO J. 2002; 21: 1948-1956Crossref PubMed Scopus (613) Google Scholar In addition to cleaving extracellular matrix proteins, MMPs mediate shedding of transmembrane and membrane-associated proteins, including E-cadherin.24Sbarba PD Rovida E Transmodulation of cell surface regulatory molecules via ectodomain shedding.Biol Chem. 2002; 383: 69-83Crossref PubMed Scopus (84) Google Scholar For example, exogenous matrilysin and stromelysin-1 (MMP-3) can release an 80-kd E-cadherin fragment from various cancer cell lines25Noe V Fingleton B Jacobs K Crawford HC Vermeulen S Steelant W Bruyneel E Matrisian LM Mareel M Release of an invasion promoter E-cadherin fragment by matrilysin and stromelysin-1.J Cell Sci. 2001; 114: 111-118Crossref PubMed Google Scholar, 26Davies G Jiang WG Mason MD Matrilysin mediates extracellular cleavage of E-cadherin from prostate cancer cells: a key mechanism in hepatocyte growth factor/scatter factor-induced cell-cell dissociation and in vitro invasion.Clin Cancer Res. 2001; 7: 3289-3297PubMed Google Scholar and ectopic expression of stromelysin-1 leads to cleavage of E-cadherin in mammary epithelial cells.27Lochter A Galosy S Muschler J Freedman N Werb Z Bissell MJ Matrix metalloproteinase stromelysin-1 triggers a cascade of molecular alterations that leads to stable epithelial-to mesenchymal conversion and a premalignant phenotype in mammary epithelial cells.J Cell Biol. 1997; 139: 1861-1872Crossref PubMed Scopus (523) Google Scholar Although proteolytic cleavage of E-cadherin ectodomains has been suggested to enhance epithelial repair, the proteinase responsible for this process has not been identified. Here, we provide evidence that matrilysin mediates shedding of E-cadherin from airway epithelium during repair and from alveolar epithelium during progression of bleomycin-induced pulmonary fibrosis. Archival blocks of formalin-fixed, paraffin-embedded normal and diseased human lung tissue were obtained from the Department of Pathology, Washington University School of Medicine. We analyzed specimens from ARDS with diffuse alveolar damage (n = 4), emphysema (n = 5), and desquamative interstitial pneumonitis (n = 3). Specimens from the tumor-free margins from lung adenocarcinoma resections were used as normal controls (n = 5). Sections were stained for matrilysin as described.13Dunsmore SE Saarialho-Kere UK Roby JD Wilson CL Matrisian LM Welgus HG Parks WC Matrilysin expression and function in airway epithelium.J Clin Invest. 1998; 102: 1321-1331Crossref PubMed Scopus (229) Google Scholar For mouse lung and tracheal tissues, 5-μm sections were cut from blocks of frozen lung tissue, air-dried for 5 minutes, and fixed in acetone for 10 minutes at −20°C and methanol for 6 minutes at −20°C. Sections were blocked for 1 hour at room temperature with TBST (25 mmol/L Tris-HCl, pH 7.5, 150 mmol/L NaCl, 0.1% Tween 20) containing 1% bovine serum albumin and 2% goat serum and then incubated with primary antibodies at 37°C. For E-cadherin immunofluorescence on mouse tissues, sections were fixed as above and permeabilized with 1% Triton X-100 in Ca/Mg-phosphate-buffered saline (PBS) (0.01 mol/L phosphate, 0.138 mol/L NaCl, 0.27 mmol/L KCl, 1 mmol/L CaCl2, 0.5 mmol/L MgC12) for 15 minutes before adding primary antibody. Cells were grown on Nunc Lab-Tek II glass chamber slides (Fisher Scientific, Pittsburgh, PA), washed with Ca/Mg-PBS, fixed with 3.7% formaldehyde in Ca/Mg-PBS for 10 minutes, and permeabilized with 1% Triton X-100 in Ca/Mg-PBS for 15 minutes. Fixed cells were blocked with 1% bovine serum albumin in Ca/Mg-PBS for 1 hour at room temperature, incubated with antibodies at 37°C. Immunofluorescence of tissues and cells was imaged by indirect immunofluorescence microscopy and confocal laser-scanning microscopy. Polyclonal antibodies against the catalytic domain of human matrilysin12Saarialho-Kere UK Crouch EC Parks WC Matrix metalloproteinase matrilysin is preferentially expressed in human exocrine epithelium.J Invest Dermatol. 1995; 105: 190-196Crossref PubMed Scopus (142) Google Scholar and against full-length mouse matrilysin28Wilson CL Ouellette AJ Satchell DP Ayabe TL Lopez-Boado YS Stratman JL Hultgren SJ Matrisian LM Parks WC Regulation of intestinal a-defensin activation by the metalloproteinase matrilysin in innate host defense.Science. 1999; 286: 113-117Crossref PubMed Scopus (894) Google Scholar were previously generated in our laboratory. Antibodies against the extracellular domain of human (HECD-1) and mouse (ECCD-2) E-cadherin (Zymed, South San Francisco, CA), and against the cytoplasmic domain of human E-cadherin (Transduction Laboratories, San Diego, CA) were used at the manufacturers' recommended concentrations. Mouse anti-human actin antibody was obtained from Sigma Chemical Co., St. Louis, MO. Fluorescent Alexa 488- and Alexa 568-conjugated antibodies (Molecular Probes, Eugene, OR) were used at the manufacturer's recommended concentrations for indirect immunofluorescence and confocal laser-scanning microscopy. The human lung adenocarcinoma cell lines A549 and Calu3, the human colon carcinoma cell line HT29, and the canine kidney cell line MDCK were obtained from American Type Culture Collection (Manassas, VA). Cultures were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and 50 μg/ml of gentamicin. A construct coding for a full-length human matrilysin cDNA carrying an autoactivating mutation (a gift from Lynn Matrisian, Ph.D., Vanderbilt University, Nashville, TN) was stably expressed in A549 cells under the control of a constitutive CMV promoter. The construct was cloned into pCIneo (Promega, Madison, WI), which contains a neomycin resistance gene, and was transfected into cells using Superfect reagent (Qiagen, Valencia, CA). Neomycin-resistant clones were selected with G418 at 800 mg/ml and were analyzed for matrilysin mRNA expression by Northern blot analysis. Selected clones were screened for matrilysin protein secretion by [35S] labeling. Briefly, cells were cultured to 60 to 70% confluence in basal culture medium then switched to labeling medium (l-methionine and l-cysteine free-Dulbecco's modified Eagle's medium, 5% fetal bovine serum dialyzed against 0.05 mol/L Tris-HCl, pH 7.5, 0.15 mol/L NaCl). After 12 hours, depleted cells were switched to labeling medium supplemented with 50 μCi/ml of [35S]-methionine-cysteine TRAN35S-LABEL (ICN, Costa Mesa, CA). Cells were pulsed overnight, and conditioned medium was collected and centrifuged at 5000 × g to remove cellular debris. Aliquots (600 μl) of conditioned medium were mixed with an equal volume of immunoprecipitation buffer (0.01 mol/L Na3PO4, 0.138 mol/L NaCl, 0.27 mmol/L KCl, 0.8% Triton X-100, 20 mmol/L ethylenediaminetetraacetic acid, 100 mg/ml bovine serum albumin) and precleared with 25 μl of protein A-Sepharose (Zymed) for 1 hour at 4°C. Protein A-Sepharose was removed by centrifugation, and supernatants were incubated with 5 μl per sample of rabbit anti-human matrilysin antiserum overnight at 4°C with gentle agitation. Antibody-antigen complexes were precipitated with protein A-Sepharose, washed three times with immunoprecipitation buffer, and eluted by boiling in 40 μl of 2× sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) buffer. The precipitating reagents were removed by centrifugation, and supernatants were resolved by SDS-PAGE. Gels were dried, and [35S]-labeled matrilysin was visualized by autoradiography. For inhibitor experiments, the hydroxamate MMP inhibitor SC68180A (Pharmacia, St. Louis, MO) was used at 25 μmol/L concentration in culture medium, and the matrilysin selective inhibitor RS-101625 (Roche Bioscience, Palo Alto, CA) was used at 40 nmol/L concentration. All cell culture medium components were purchased from Bio-Whittaker (Walkersville, MD). Uniform 1-mm wounds were made in confluent A549 epithelial monolayers grown on plastic six-well plates. Cultures were rinsed three times with warm PBS, and cells were allowed to heal the wounds throughout 48 hours in regular culture medium. Cultures were photographed at time of wounding and 24 and 48 hours later. Wound area was calculated by measuring average width of the wound and multiplying by wound length. Wound closure was expressed as a percentage of initial wound area. For some experiments, hydroxyurea (Sigma), an inhibitor of cell proliferation, was dissolved in culture medium at 250 μmol/L final concentration, and added at time of wounding. Tracheas from wild-type and matrilysin-null mice were explanted, placed in sterile medium (Dulbecco's modified Eagle's medium) with 5% fetal bovine serum and 50 μg/ml gentamicin), cut, and splayed open along the long axis. Tracheas were wounded by cutting the splayed tracheas into uniform 3-mm pieces, rinsed with sterile PBS to remove cellular debris, and immediately placed into tissue culture wells (four tracheas/well) with just enough medium added to cover the tracheal tissue. Cultures were maintained for 24 hours. We tracked tracheal epithelial migration, or epiboly, along the cut edges of the tissue pieces. At 24 hours, conditioned medium was collected, cleared of cellular debris by centrifugation at 600 × g for 5 minutes, snap-frozen on dry ice, and stored for future analysis. Tracheal tissue was rinsed in PBS and frozen in OCT tissue-freezing medium for immunohistochemical analysis. Tracheal tissues were fixed for 24 hours at 4°C in 2.5% gluturaldehyde/0.1 mol/L Na cacodylate, rinsed for 20 minutes three times in 0.1 mol/L Na cacodylate, and secondarily fixed in 1.25% osmium tetroxide/0.1 mol/L Na cacodylate for 1 hour at room temperature. Fixed tissues were rinsed twice in 0.1 mol/L Na cacodylate and twice in 15% ethanol for 20 minutes each, followed by dehydration in graded ethanols. Tissues were washed twice for 20 minutes in propylene oxide, incubated for 48 hours in 1:1 propylene oxide: Polybed 812 on a rotating shaker, 4 hours in pure Polybed 812 with rotation, and embedded in Polybed 812 overnight in the dessicator, followed by baking at 60°C for 48 hours. Blocks were thin-sectioned and stained with alkaline toluidine blue and observed with a light microscope. Appropriate areas were trimmed for ultra thin sections, which were poststained with 4% uranyl acetate and lead citrate and examined with a Zeiss 902 transmission electron microscope and recorded on electron microscopy film. C57BL6 wild-type and C57BL6 mice carrying a targeted deletion of the matrilysin gene29Wilson CL Heppner KJ Labosky PA Hogan BLM Matrisian LM Intestinal tumorigenesis is suppressed in mice lacking the metalloproteinase matrilysin.Proc Natl Acad Sci USA. 1997; 94: 1402-1407Crossref PubMed Scopus (542) Google Scholar were anesthetized by intraperitoneal injection with 0.1 ml/20 g body weight of mouse cocktail (86.98 mg/kg ketamine/13.4 mg/kg xylazine in 0.9% sterile saline). The MMP-7−/− mice were initially derived on a 129 background and backcrossed >10 generations onto the C57BL6 background. Wild-type mice were age- and strain-matched littermate controls. A small neck incision was made, and mice were administered 0.08 U of bleomycin by direct intratracheal instillation in 50 μl of sterile saline with a 25-gauge needle. Control animals received an equal volume of saline. Incisions were closed with silk suture, and animals were allowed to recover from anesthesia and take food and water ad libitum. Animals were sacrificed 10 days after injury by intraperitoneal injection of pentobarbital (100 mg/kg). Tracheas were cannulated with a 22-gauge 1-inch angiocatheter (Becton-Dickinson, San Diego, CA), and bronchoalveolar lavage (BAL) fluid was collected with 1 ml of sterile 0.9% saline solution. BAL fluid was concentrated 10-fold with Microcon centrifugation columns (Millipore, Bedford, MA), and 20 μl of each sample was boiled in an equal volume of 2× SDS-loading buffer, resolved by SDS-PAGE, and analyzed by Western blot as described below. Similar experiments were performed in C57BL6 mice with a targeted deletion of the macrophage elastase (MMP-12) gene (obtained from Dr. Steven Shapiro, Harvard University, Boston, MA).30Hautamaki RD Kobayashi DK Senior RM Shapiro SD Requirement for macrophage elastase for cigarette smoke-induced emphysema in mice.Science. 1997; 277: 2002-2004Crossref PubMed Scopus (1215) Google Scholar Bronchoalveolar lavage samples from bleomycin-treated C57BL6 gelatinase B (MMP-9)-null mice were obtained from Dr. Robert Senior, Washington University, St. Louis, MO.31Betsuyaku T Fukuda Y Parks WC Shipley JM Senior RM Gelatinase B is required for alveolar bronchiolization after intratracheal bleomycin.Am J Pathol. 2000; 157: 525-535Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar Lungs were removed and either homogenized in RNA STAT-60 reagent from Tel-Test, Inc. (Friendswood, TX) for RNA isolation or snap-frozen in OCT tissue-freezing medium for immunohistochemical analysis. All experiments were performed with the approval of the Washington University School of Medicine animal studies committee. Cultured cells were lysed in 30 mmol/L NaCl, 50 mmol/L Tris-HCl, pH 7.4, 5 mmol/L ethylenediaminetetraacetic acid, 1% Igepal CA630, 1% deoxycholic acid, 0.1% SDS, 1 mmol/L phenylmethyl sulfonyl fluoride, and 20 μg/ml aprotinin; and protein content in lysates quantified by the Bradford assay. Equal quantities (40 μg) of protein/sample were resolved by SDS-PAGE, transferred to Hybond nitrocellulose membranes (Amersham Pharmacia Biotech, Buckinghamshire, UK), and blocked overnight with 5% milk in TBST. Blots were probed with above antibodies at the manufacturers' recommended concentrations and visualized by enhanced chemiluminescence system (Amersham Pharmacia Biotech). For conditioned medium experiments, cells were cultured in serum-free medium for 24 hours. Medium was collected, cellular debris removed by centrifugation at 600 × g for 5 minutes, and 500 μl of medium was concentrated 10-fold as for BAL fluid and analyzed as above. For Northern blotting studies, cultured cells were washed twice with PBS, and total RNA was extracted with RNA STAT-60 reagent according to the manufacturer's protocol. Total RNA (10 μg) was denatured in loading buffer containing ethidium bromide and resolved by electrophoresis through 1.5% agarose gels containing formaldehyde. RNA was transferred to Hybond+ nylon membranes (Amersham Pharmacia Biotech) following the manufacturer's specifications and UV cross-linked. Equivalent loading and integrity of RNA was confirmed by comparing ethidium bromide staining of 18S and 28S rRNA bands. Gel-purified cDNA probes for human matrilysin were labeled by random priming, hybridized to membranes overnight, and visualized by autoradiography. Matrilysin is prominently expressed in cystic fibrosis,13Dunsmore SE Saarialho-Kere UK Roby JD Wilson CL Matrisian LM Welgus HG Parks WC Matrilysin expression and function in airway epithelium.J Clin Invest. 1998; 102: 1321-1331Crossref PubMed Scopus (229) Google Scholar a disease characterized by both fibrosis and chronic infection, and in idiopathic pulmonary fibrosis.16Zuo F Kaminski N Eugui E Allard J Yakhini Z Ben-Dor A Lollini L Morris D Kim Y DeLustro B Sheppard D Pardo A Selman M Heller RA Gene expression analyses reveal matrilysin as a key regulator of pulmonary fibrosis in mice and humans.Proc Natl Acad Sci. 2002; 99: 6292-6297Crossref PubMed Scopus (510) Google Scholar To assess if matrilysin expression is a general feature of alveolar epithelial injury, regardless of the underlying condition, we examined a variety of human lung diseases. Consistent with our previous observations, matrilysin was not expressed in the alveolar epithelium of uninjured human lungs (Figure 1A). However, in specimens of emphysema, desquamative interstitial pneumonitis, and ARDS, strong immunoreactive signal for matrilysin protein was seen in epithelial cells lining damaged alveoli, particularly in cells bordering denuded epithelium (Figure 1; B, C, and E). Based on their morphology and location, matrilysin-positive cells were identified as alveolar type II cells. As in other tissues, matrilysin immunostaining was restricted to epithelial cells and was not seen in interstitial cells and only rarely in inflammatory cells. No immunoreactivity was seen in specimens processed with preimmune serum (Figure 1D). Because it is prominently expressed by injured alveolar epithelium and promotes airway epithelial migration, we evaluated if matrilysin promotes alveolar cell migration in vitro. A549 human lung adenocarcinoma cells have features of alveolar type II cells and have been used in models of alveolar repair in vitro.32Lieber M Smith B Szakal A Nelson-Rees W Todaro G A continuous tumor-cell line form a human lung carcinoma with properties of type II alveolar epithelial cells.Int J Cancer. 1976; 17: 62-70Crossref PubMed Scopus (1003) Google Scholar, 33Planus E Galiacy S Matthay M Laurent V Gavrilovic J Murphy G Clerici C Isabey D Lafuma C d'Ortho M-P Role of collagenase in mediating in vitro alveolar epithelial wound repair.J Cell Sci. 1998; 112: 243-252Google Scholar This cell line does not express matrilysin under basal conditions or in response to stimuli that induce matrilysin expression in other mucosal epithelial cell lines (data not shown). We stably expressed an autoactivating mutant of human promatrilysin (aMat) in A549 cells. This construct has a Val-Gly mutation in the prodomain sequence PRCGVPD that destabilizes the interaction of the cysteine residue with the active site zinc.34Rudolph-Owen LA Cannon P Matrisian LM Overexpression of the matrix metalloproteinase matrilysin results in premature mammary gland differentiation and male infertility.Mol Biol Cell. 1998; 9: 421-435Crossref PubMed Scopus (49) Google Scholar Two clones, aMat8 and aMat16, were identified that expressed high levels of the matrilysin mRNA and secreted activated matrilysin (Figure 2, A and B). Because the cDNA codes for full-length prepromatrilysin, both the 29-kd promatrilysin and 19-kd active forms were detected in the culture medium. Compared to vector-transfected cells, cells expressing activated-matrilysin appeared more spindle-shaped and were more detached from one another (Figure 2C), morphological changes characteristic of a migratory phenotype.35Solic N Davies DE Differential effects of EGF and amphiregulin on adhesion molecule expression and migration of colon carcinoma cells.Exp Cell Res. 1997; 234: 465-476Crossref PubMed Scopus (53) Google Scholar Expression of native full-length human promatrilysin cDNA in A549 cells showed no conversion to the active form of the enzyme and did not induce changes in cell shape or migration (data not shown). Because exogenous matrilysin can cleave E-cadherin from the surface of cultured cells25Noe V Fingleton B Jacobs K Crawford HC Vermeulen S Steelant W Bruyneel E Matrisian LM Mareel M Release of an invasion promoter E-cadherin fragment by matrilysin and stromelysin-1.J Cell Sci.