MUC5AC mucin is a major component of mucus in airway epithelial cells (2) and is regulated by means of an epidermal growth factor receptor (EGFR)-signaling pathway (3); ligand-dependent EGFR phosphorylation induces MUC5AC mucin. Airway epithelial cells produce EGFR and its ligands (4, 5). TGF-, EGFR phosphorylation, and MUC5AC expression, which were blocked by the metalloprotease inhibitors tumor necrosis factor- protease inhibitor-1 and tissue inhibitor of metalloprotease-3. We specifically knocked down the expression of metalloprotease TACE by using small interfering RNA for TACE. Knockdown of TACE inhibited PMA-, PA sup-, and LPS-induced TGF- shedding, EGFR phosphorylation, and mucin production. From these results, we conclude that TACE plays a critical role in mucin production by airway epithelial cells by means of a TACE ligandCEGFR cascade in response to various stimuli. Mucus hypersecretion is an important feature of chronic inflammatory airway diseases (1) and contributes to their morbidity and mortality. MUC5AC mucin is a major component of mucus in airway epithelial cells (2) and is regulated by means of an epidermal growth factor receptor (EGFR)-signaling pathway (3); ligand-dependent EGFR phosphorylation induces MUC5AC mucin. Airway epithelial cells produce EGFR and its ligands (4, 5). Transforming growth factor type (TGF-) plays a critical role in EGFR phosphorylation, leading to MUC5AC production in airways. TGF- is synthesized as transmembrane pro-TGF-, which is processed and released (ectodomain shedding) from the cell surface by metalloproteases (6, 7). Tumor necrosis factor -converting enzyme (TACE) has been reported to cleave pro-TGF- into mature soluble TGF- in various epithelial tissues (8). TACE is a member of a disintegrin and metalloprotease (ADAM) family, a group of zinc-dependent transmembrane metalloproteases (9, 10). TACE is produced in a latent form (11, 12) and is activated by agents such as phorbol 12-myristate 13-acetate (PMA) (13) and reactive oxygen species (12, 14), resulting in substrate cleavage. We hypothesized that TACE activation induces MUC5AC mucin production. The hypothesis is that airway epithelial cells can cleave EGFR pro-ligand on their surfaces into mature soluble ligand, which can then bind to and activate EGFR, resulting in mucin production. We used two stimuli to induce mucin production. First, we studied the effect of PMA because PMA is known to activate TACE (13), but it is not known whether it induces mucin expression. Second, we studied the effect of (PA), one of the most common pathogens in hypersecretory diseases. PA is known to stimulate mucin production by means of EGFR phosphorylation (15), but the mechanism by which PA causes EGFR phosphorylation is unknown. First, we examined whether activation of TACE by PMA causes MUC5AC expression in airway epithelial (NCI-H292) cells and, if so, whether increased shedding of soluble TGF- and EGFR phosphorylation are involved in this process. Next, we examined whether inhibition of TACE activity by application of metalloprotease inhibitors and more importantly by specific knockdown of TACE by using small interfering RNA (siRNA) to silence TACE expression could block PMA-induced TGF- release, EGFR phosphorylation, and MUC5AC expression. Finally, we examined whether TACE is also involved in Lobeline hydrochloride PA- and lipopolysaccharide (LPS)-induced TGF- release, EGFR phosphorylation, and mucin production. Here we show that activation of TACE by these stimuli causes ligand (TGF-)-dependent EGFR phosphorylation and up-regulates MUC5AC expression. Inhibition of TACE or knockdown of TACE prevented ligand (TGF-)-dependent EGFR phosphorylation and MUC5AC expression. Materials and Methods Materials. PMA was obtained from Sigma. AG1478, calphostin C (CC), Bisindolylmaleimide I, tissue inhibitor of metalloprotease 3 (TIMP-3), tumor necrosis factor proteinase inhibitor-1 (TAPI-1), EGFR-neutralizing Ab (Ab 3), EGF-neutralizing Ab, and TGF–neutralizing Ab were purchased from Calbiochem. Anti-human TACE Ab, anti-phosphotyrosine (PY99) Ab, and anti-human EGFR Ab were obtained from Santa Cruz Biotechnology. Cell Culture. NCI-H292 cells, a human pulmonary mucoepidermoid carcinoma cell line, were plated at 5C6 105 cells in 2 ml in each well of a 6-well plate or at 1C2 105 cells in 1 ml in each well of a 24-well plate (both 6- and 24-well plates were purchased from BD Biosciences) and were grown in RPMI medium 1640 containing 10% FCS, penicillin (100 units/ml), streptomycin (100 g/ml), and Hepes (25 mM) at 37C in a humidified, 5% Lobeline hydrochloride CO2/95% air, water-jacketed incubator. After the cells reached confluence, they were serum-starved for 24 h to maintain low basal levels of MUC5AC expression. Treatment of Cells with PMA, PA Supernatant (PA Sup), or LPS. After 24 h of serum starvation, cells were treated with stimuli as indicated in each experiment. For inhibitor studies, serum-starved cells were pretreated with inhibitors for 30 min before exposure to stimuli. In studies of PMA, cells were treated with PMA (10 ng/ml) for 1 h. Then the cells were washed three times with serum-free medium and cultured for another 24 h in serum-free medium with the.5and 0.01, compared with PA sup alone or LPS alone. EGFR phosphorylation and by preincubation with an EGFR-neutralizing Ab or with a TGF–neutralizing Ab, implicating ligand (TGF-)-dependent EGFR phosphorylation in mucin production. These stimuli induced release of soluble TGF-, EGFR phosphorylation, and MUC5AC expression, which were blocked by the metalloprotease inhibitors tumor necrosis factor- protease inhibitor-1 and tissue inhibitor of metalloprotease-3. We specifically knocked down the expression of metalloprotease TACE by using small interfering RNA for TACE. Knockdown of TACE inhibited PMA-, PA sup-, and LPS-induced TGF- shedding, EGFR phosphorylation, and mucin production. From these results, we conclude that TACE plays a critical role in mucin VBCH production by airway epithelial cells by means of a TACE ligandCEGFR cascade in response to various stimuli. Mucus hypersecretion is an important feature of chronic inflammatory airway diseases (1) and contributes to their morbidity and mortality. MUC5AC mucin is a major component of mucus in airway epithelial cells (2) and is regulated by means of an epidermal growth factor receptor (EGFR)-signaling pathway (3); ligand-dependent EGFR phosphorylation induces MUC5AC mucin. Airway epithelial cells produce EGFR and its ligands (4, 5). Transforming growth factor type (TGF-) plays a critical role in EGFR phosphorylation, leading to MUC5AC Lobeline hydrochloride production in airways. TGF- is synthesized as transmembrane pro-TGF-, which is processed and released (ectodomain shedding) from the cell surface by metalloproteases (6, 7). Tumor necrosis factor -converting enzyme (TACE) has been reported to cleave pro-TGF- into mature soluble TGF- in various epithelial tissues (8). TACE is a member of a disintegrin and metalloprotease (ADAM) family, a group of zinc-dependent transmembrane metalloproteases (9, 10). TACE is produced in a latent form (11, 12) and is activated by agents such as phorbol 12-myristate 13-acetate (PMA) (13) and reactive oxygen species (12, 14), resulting in substrate cleavage. We hypothesized that TACE activation induces MUC5AC mucin production. The hypothesis is that airway epithelial cells can cleave EGFR pro-ligand on their surfaces into mature soluble ligand, which can then bind to and activate EGFR, resulting in mucin production. We used two stimuli to induce mucin production. First, we studied the effect of PMA because PMA is known to activate TACE (13), but it is not known whether it induces mucin expression. Second, we studied the effect of (PA), one of the most common pathogens in hypersecretory diseases. PA is known to stimulate mucin production by means of EGFR phosphorylation (15), but the mechanism by which PA causes EGFR phosphorylation is unknown. First, we examined whether activation of TACE by PMA causes MUC5AC expression in airway epithelial (NCI-H292) cells and, if so, whether increased shedding of soluble TGF- and EGFR phosphorylation are involved in this process. Next, we examined whether inhibition of TACE activity by application of metalloprotease inhibitors and more importantly by specific knockdown of TACE by using small interfering RNA (siRNA) to silence TACE expression could block PMA-induced TGF- release, EGFR phosphorylation, and MUC5AC expression. Finally, we examined whether TACE is also involved in PA- and lipopolysaccharide (LPS)-induced TGF- release, EGFR phosphorylation, and mucin production. Here we show that activation of TACE by these stimuli causes ligand (TGF-)-dependent EGFR phosphorylation and up-regulates MUC5AC expression. Inhibition of TACE or knockdown of TACE prevented ligand (TGF-)-dependent EGFR phosphorylation and MUC5AC expression. Materials and Methods Materials. PMA was obtained from Sigma. AG1478, calphostin C (CC), Bisindolylmaleimide I, tissue inhibitor of metalloprotease 3 (TIMP-3), tumor necrosis factor proteinase inhibitor-1 (TAPI-1), EGFR-neutralizing Ab (Ab 3), EGF-neutralizing Ab, and TGF–neutralizing Ab were purchased from Calbiochem. Anti-human TACE Ab, anti-phosphotyrosine (PY99) Ab, and anti-human EGFR Ab were obtained from Santa Cruz Biotechnology. Cell Culture. NCI-H292 cells, a human pulmonary mucoepidermoid carcinoma cell line, were plated at 5C6 105 cells in 2 ml in each well of a 6-well plate or at 1C2 105 cells in 1 ml in each well of a 24-well plate (both 6- and 24-well plates were purchased from BD Biosciences) and were grown in RPMI medium 1640 containing 10% FCS, penicillin (100 units/ml), streptomycin (100 g/ml), and Hepes (25 mM) at 37C in a humidified, 5% CO2/95% air, water-jacketed incubator. After the cells reached confluence, they were serum-starved for 24 h to maintain low basal levels of MUC5AC expression. Treatment of Cells with PMA, PA Supernatant (PA Sup), or LPS. After 24 h of serum starvation, cells had been treated with.