Effect of pro-inflammatory stimuli on mucin expression and inhibition by secretory leucoprotease inhibitor Siobhan Griffin, Tomas P. Carroll, Catherine M. Greene, Shane J. O’Neill, Clifford C. Taggart* and Noel G. McElvaney Respiratory Research Division, Department of Medicine, Royal College of Surgeons in Ireland, Education and Research Centre, Beaumont Hospital, Dublin 9, Ireland. Summary Stimuli-induced expression of certain mucin genes has been demonstrated to occur as a result of ligand- dependent activation of the epidermal growth factor receptor (EGFR). In particular, MUC5AC expression can be induced by cigarette-smoke, neutrophil elastase and lipopolysaccharide (LPS) following acti- vation of tumour necrosis factor a-converting enzyme. We now show that a large of number of stimuli relevant to the cystic fibrosis lung – neutrophil elastase, LPS, Pam 3Cys-Ser-(Lys)4 Hydrochloride (a lipopeptide analogue), CpG DNA (which mimics bac- terial DNA) and cystic fibrosis bronchoalveolar lavage fluid – can activate MUC1 and 2 expression as well as MUC5AC expression in lung epithelial cells via an EGFR-dependent mechanism. In addition, we demon- strate that the immunomodulatory anti-protease, secretory leucoprotease inhibitor, can inhibit stimuli- induced MUC1, 2 and 5AC expression via a mecha- nism that is primarily dependent on the inhibition of transforming growth factor type alpha release. There- fore, mucin gene expression, induced by cystic fibro- sis respiratory stimuli, can be inhibited by secretory leucoprotease inhibitor indicating its potential impor- tance as an anti-mucin agent in cystic fibrosis and other chronic lung diseases characterized by mucus hypersecretion. Introduction Hypersecretion of mucus is a common pathophysiological feature of cystic fibrosis (CF) and other inflammatory lung diseases. Mucus obstruction of the airways in CF, asthma and chronic bronchitis contributes significantly to mortality and morbidity in these conditions. Mucus, which is com- posed mostly of water and ions with a small percentage of protein (5%), plays an important role in host defence where it binds bacteria and removes it via the mucociliary ladder to the upper airways and oesophagus where it is either expectorated or ingested. To date nine MUC genes have been described in the lung – MUC1, 2, 4, 5AC, 5B, 7, 8, 13 – and very recently, MUC19 (Voynow 2002; Chen et al., 2004). MUC2, 5AC and 5B are secreted, gel- forming mucins and MUC1, 4 and 13 are membrane asso- ciated (Voynow 2002). Although MUC7 is secreted it does not possess the cysteine-rich D domain (a feature of MUC2, 5AC and 5B) and therefore does not oligomerize or contribute to the gel phase of airway secretion (Bobek et al., 1993). MUC5AC is the most prominent mucin of normal airway secretions and its expression is increased in nasal epithelium of individuals with CF and allergic rhinitis (Voynow et al., 1998). In addition, MUC2 expres- sion is elevated in CF nasal epithelium compared with healthy controls (Li et al., 1997). MUC1 has recently been shown to be an adhesion site for Pseudomonas aerugi- nosa and binding of Pseudomonas to MUC1 results in activation of an intracellular mitogen-activated protein kinase pathway (Lillehoj et al., 2001; 2004). In the context of airway inflammation, mucin gene expression can be activated by a number of stimuli. Inter- leukin (IL)-9 can regulate mucin expression via the human calcium-activated chloride channel, hCLCA1 (Hauber et al., 2003). Oxidative stress and activated neutrophils can also increase MUC5AC expression via the epidermal growth factor receptor (EGFR). Neutrophil elastase (NE) has been shown to regulate MUC5AC expression both through an oxidant mechanism and via a tumour necrosis factor a-converting enzyme (TACE-EGFR pathway; Fischer and Voynow, 2002; Kohri et al., 2002): other stimuli have also been shown to regulate MUC5AC expression via the TACE-EGFR pathway including ciga- rette smoke, lipopolysaccharide (LPS) and supernatant from Gram-negative bacteria (Dohrman et al., 1998; Shao et al., 2003; 2004). Gram-positive bacteria-derived lipote- ichoic acid (LTA) can activate MUC2 expression through the platelet-activating factor receptor and a disintegrin and metalloproteinase (ADAM)-10 resulting in the libera- tion of heparin-binding epidermal growth factor (HB-EGF) and activation of the EGFR (Lemjabbar and Basbaum, 2002). Although a number of these stimuli – LPS and LTA – are ligands for the Toll-like receptor family of proteins, Received 26 June, 2006; revised 23 August, 2006; accepted 28 August, 2006. *For correspondence. E-mail ctaggart@rcsi.ie; Tel. (+353) 1 8093712; Fax (+353) 1 8093808. Cellular Microbiology (2007) 9(3), 670–679 doi:10.1111/j.1462-5822.2006.00819.x First published online 4 October 2006 © 2006 The Authors Journal compilation © 2006 Blackwell Publishing Ltd