title: German cockroach proteases regulate matrix metalloproteinase-9 in human bronchial epithelial cells.
abstract: BACKGROUND
  Matrix metalloproteinases (MMPs) digest extracellular matrix proteins and may play a role in the pathogenesis of bronchial asthma. MMP-9 levels are increased in the bronchoalveolar lavage fluid and sputum of asthmatics compared with that of controls. As exposure to cockroaches is an environmental risk factor for asthma, we sought to investigate the role of German cockroach fecal remnants (frass) on MMP-9 expression.
  
  
  METHODS
  Human bronchial epithelial cells (16HBE14o-) and primary normal human bronchial epithelial cells were treated with cockroach frass in the absence or presence of tumor necrosis factor (TNF)alpha. MMP-9 mRNA, protein levels and pro-MMP-9 activity were determined using real-time polymerase chain reaction (PCR), enzyme-linked immunosorbent assay (ELISA) and zymogram assays. Pretreatment of frass with aprotinin abolished protease activity. PD98059, a chemical inhibitor of extracellular signal regulated kinase (ERK), and SLIGKV, an activator of protease-activated receptor (PAR)-2 were also used. AP-1DNA binding was determined by electrophoretic mobility shift assay (EMSA) and ERK phosphorylation by Western blot analysis.
  
  
  RESULTS
  Cockroach frass augmented TNFalpha-mediated MMP-9 mRNA and protein expression by a mechanism dependent on active serine proteases within frass and not on endogenous endotoxin. Frass increased ERK phosphorylation, and chemical inhibition of ERK attenuated cockroaches' effects on MMP-9. Serine proteases are known to activate the PAR-2 receptor. We found that selective activation of PAR-2 using the peptide SLIGKV augmented TNFalpha-induced MMP-9 protein levels and increased ERK phosphorylation. Frass and SLIGKV each increased AP-1 translocation and DNA binding.
  
  
  CONCLUSIONS
  These data suggest that German cockroach frass contains active serine proteases which augment TNFalpha-induced MMP-9 expression by a mechanism involving PAR-2, ERK and AP-1.
authors.affiliation: Division of Critical Care Medicine, Cincinnati Children's Hospital Medical Center and Cincinnati Children's Research Foundation, Cincinnati, OH 45229, USA.
authors.forename: K
authors.initials: K
authors.lastname: Page
authors.forename: V S
authors.initials: VS
authors.lastname: Hughes
authors.forename: G W
authors.initials: GW
authors.lastname: Bennett
authors.forename: H R
authors.initials: HR
authors.lastname: Wong
mesh_terms: D000818:Animals; D001249:Asthma / Q000201:enzymology / Q000276:immunology; D020048:Blattellidae / Q000737:chemistry / Q000276:immunology*; D001980:Bronchi / Q000201:enzymology / Q000276:immunology* / Q000473:pathology; D002461:Cell Line, Transformed; D004847:Epithelial Cells / Q000201:enzymology / Q000276:immunology* / Q000473:pathology; D048049:Extracellular Signal-Regulated MAP Kinases / Q000037:antagonists & inhibitors / Q000276:immunology / Q000378:metabolism; D005419:Flavonoids / Q000494:pharmacology; D006801:Humans; D019476:Insect Proteins / Q000737:chemistry / Q000276:immunology* / Q000494:pharmacology; D020780:Matrix Metalloproteinase 9 / Q000096:biosynthesis / Q000276:immunology*; D009842:Oligopeptides / Q000494:pharmacology; D012333:RNA, Messenger / Q000096:biosynthesis / Q000276:immunology; D044464:Receptor, PAR-2 / Q000276:immunology / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects / Q000276:immunology*; D018808:Transcription Factor AP-1 / Q000276:immunology / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
chemical_list: D005419:Flavonoids; D019476:Insect Proteins; D009842:Oligopeptides; D012333:RNA, Messenger; D044464:Receptor, PAR-2; D018808:Transcription Factor AP-1; D014409:Tumor Necrosis Factor-alpha; C109968:seryl-leucyl-isoleucyl--glycyl-lysyl-valine; D048049:Extracellular Signal-Regulated MAP Kinases; D020780:Matrix Metalloproteinase 9; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one