German cockroach proteases regulate matrix metalloproteinase‐9 in human bronchial epithelial cells

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)α. 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 TNFα‐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 TNFα‐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 TNFα‐induced MMP‐9 expression by a mechanism involving PAR‐2, ERK and AP‐1.