Tumor necrosis factor alpha-converting enzyme mediates MUC5AC mucin expression in cultured human airway epithelial cells

Abstract

Ectodomain shedding of epidermal growth factor receptor (EGFR) ligands [e.g., transforming growth factor type alpha (TGF-alpha)] and EGFR phosphorylation are implicated in mucin production in airway epithelial cells. Tumor necrosis factor alpha-converting enzyme (TACE) is reported to cleave precursor of TGF-alpha, with release of soluble mature TGF-alpha in various epithelial tissues. We hypothesized that TACE increases the shedding of TGF-alpha, resulting in EGFR phosphorylation and inducing mucin production in human airway epithelial (NCI-H292) cells. To examine this hypothesis, we stimulated NCI-H292 cells with phorbol 12-myristate 13-acetate (PMA, an activator of TACE) and pathophysiologic stimuli [lipopolysaccharide (LPS) and supernatant from the Gram-negative bacterium Pseudomonas aeruginosa (PA sup)]. PMA, PA sup, and LPS increased MUC5AC gene expression and mucin protein production, effects that were prevented by pretreatment with AG1478, a selective inhibitor of EGFR phosphorylation and by preincubation with an EGFR-neutralizing Ab or with a TGF-alpha-neutralizing Ab, implicating ligand (TGF-alpha)-dependent EGFR phosphorylation in mucin production. These stimuli induced release of soluble TGF-alpha, EGFR phosphorylation, and MUC5AC expression, which were blocked by the metalloprotease inhibitors tumor necrosis factor-alpha 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-alpha 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 ligand-EGFR cascade in response to various stimuli.

Fig. 1.

Effect of PMA on MUC5AC expression. NCI-H292 cells were treated with vehicle or PMA at various concentrations and times. Total RNA was prepared and subjected to RT-PCR; Rig/S15 was used as an internal marker. (A) Effect of increasing concentrations of PMA given for 6 h on RT-PCR-amplified MUC5AC mRNA expression. (B) Effect of PMA (10 ng/ml) given for various times on RT-PCR-amplified MUC5AC mRNA expression. (C) Effect of increasing concentrations of PMA on total MUC5AC protein production during 24 h by ELISA. (D) Effect of PMA (10 ng/ml) given for various times on total MUC5AC protein production by ELISA. Mucins were measured in the cell lysate (shaded areas) and in the supernatant (unshaded areas). Data in C and D are expressed as mean ± SD. *, P < 0.01, compared with vehicle alone.

Fig. 2.

Ligand (TGF-α)-dependent EGFR activation mediates MUC5AC expression by PMA. (A and B) Cells were treated with vehicle alone, PMA alone, PMA after 30-min pretreatment with AG1478 (10 μM), or PMA plus an EGFR-neutralizing Ab (4 ng/ml) for 6 h (A) or 24 h (B). MUC5AC mRNA expression was examined by RT-PCR (A), and mucin production was measured by ELISA (B). *, P < 0.01, compared with PMA alone. (C) Cells were pretreated with an anti-EGFR-neutralizing Ab for 30 min to block EGFR-ligand-binding sites, and then the cells were stimulated with vehicle or PMA at different concentrations for 2 h. Soluble TGF-α in the supernatant was measured by ELISA. *, P < 0.01, compared with vehicle alone. (D) Cells were treated with vehicle alone, PMA alone, or PMA plus a TGF-α-neutralizing Ab (4 ng/ml) for 6 h. MUC5AC mRNA expression was examined by RT-PCR. Results in three separate experiments were similar. (E) Cells were treated with vehicle alone (left), PMA alone (center), or PMA plus a TGF-α-neutralizing Ab (4 ng/ml) (right) for 24 h in the absence or presence of a TGF-α-neutralizing Ab. MUC5AC mucin protein production was examined by ELISA. Mucins were measured in the cell lysate (shaded areas) and in the supernatant (unshaded areas). Data in B, C, and E are expressed as mean ± SD. *, P < 0.01, compared with PMA alone.

Fig. 3.

Effect of inhibitors of metalloprotease and PKC on TGF-α shedding, EGFR phosphorylation, and mucin expression. (A) Cells were pretreated with an anti-EGFR neutralizing Ab for 30 min to block EGFR-ligand-binding sites and then with TAPI-1 (30 μM) or TIMP-3 (10 μg/ml), or with PKC inhibitors [CC and Bisindolylmaleimide I (200 nM), respectively] for 30 min, and then stimulated with PMA (10 ng/ml) for 2 h. Supernatants were collected for measurement of soluble TGF-α by ELISA. *, P < 0.01, compared with PMA alone. (B) Cells were cultured to confluence. After pretreatment with or without inhibitors as shown [TAPI-1 (30 μM), TIMP-3 (10 μg/ml)], cells were stimulated with PMA (10 ng/ml) for various times or with PMA (10 ng/ml) and TGF-α (20 ng/ml) for 5 min. After lysis, EGFR was immunoprecipitated with anti-EGFR mAb and then immunoblotted with anti-phosphotyrosine Ab (PY99, Upper) and reprobed with polyclonal anti-EGFR Ab (Lower). (C) Cells were pretreated with TAPI-1 (10 or 30 μM) or TIMP-3 (10 μg/ml) for 30 min and then treated with PMA (10 ng/ml) for 6 h. MUC5AC gene expression was measured by RT-PCR. (D) Cells were pretreated with inhibitors for 30 min and then stimulated with PMA (10 ng/ml) for 24 h in the presence of the respective inhibitors. MUC5AC mucin production in the cell lysate (shaded areas) and in the supernatant (unshaded areas) was analyzed by ELISA. *, P < 0.01, compared with PMA alone.

Fig. 4.

Effect of knockdown of TACE on PMA-induced TGF-α release, EGFR phosphorylation, and MUC5AC expression. (A) Western blot of NCI-H292 cells treated with siPORT Lipid (transfection agent for siRNA; Ambion) alone (left) or transfected with TACE siRNA (10 nM, center) or luc siRNA (10 nM, right, as negative control) by using anti-TACE mAb. (B) Cells were treated with siPORT Lipid, or transfected with TACE siRNA (10 nM) or luc siRNA (10 nM). Later (48 h), cells were stimulated with PMA (10 ng/ml) for 2 h as described for Fig. 2C. Soluble TGF-α in the supernatant was measured by ELISA. *, P < 0.01, compared with cells treated with siPORT Lipid and transfected with luc siRNA. (C–E) Cells were treated with siPORT Lipid or transfected with TACE siRNA or luc siRNA. Forty-eight hours later, cells were treated with vehicle or PMA (10 ng/ml) for 5 min to examine the effect of knockdown of TACE on EGFR phosphorylation (C) (as described for Fig. 3B), or treated with vehicle or PMA (10 ng/ml) for 6 h to examine MUC5AC mRNA expression (D) or for 24 h to measure mucin protein production by ELISA (E). MUC5AC mucin production in the cell lysate (shaded areas) and in the supernatant (unshaded areas) was analyzed by ELISA. Data in B and E are expressed as mean ± SD. *, P < 0.01, compared with corresponding controls.

Fig. 5.

Role of TACE in PA sup- and LPS-induced EGFR phosphorylation and MUC5AC production. (A) Cells were pretreated with or without EGFR inhibitor, AG1478 (10 μM), or an EGFR-neutralizing Ab (4 ng/ml), or a TGF-α-neutralizing Ab (4 ng/ml) for 30 min, and then were treated with PA sup (1:8 dilution) or LPS (10 μg/ml) for 24 h in the presence or absence of the individual inhibitor or Abs. Mucin protein production was examined by ELISA. *, P < 0.01, compared with PA sup alone or LPS alone. (B) Cells were treated with siPORT Lipid or transfected with TACE siRNA (10 nM) or luc siRNA (10 nM) and incubated for 48 h, or cells were pretreated with TAPI-1 (30 μM) for 30 min. Then the cells were stimulated with PMA (10 ng/ml) for 2 h (as described for Fig. 2C). Soluble TGF-α in the supernatant was measured by ELISA. *, P < 0.01, compared with cells treated with siPORT Lipid and transfected with luc siRNA. (C–F) Cells were pretreated with vehicle or siPORT Lipid, or were transfected with TACE siRNA (10 nM) or luc siRNA (10 nM) and incubated for 48 h, or cells were pretreated with TAPI-1 (30 μM) for 30 min. Then the cells were treated with PA sup (1:8 dilution), PA sup (1:8 dilution) plus TGF-α (20 ng/ml), or with LPS (10 μg/ml) for 30 min to examine EGFR phosphorylation (C) (as described for Fig. 3B). The cells were treated with PA sup (1:8 dilution) or with PA sup plus TGF-2 (20 ng/ml) for 10 h to analyze MUC5AC mRNA expression by RT-PCR (D). The cells were treated with PA sup (1:8 dilution) or with LPS (10 μg/ml) for 24 h to measure mucin protein production by ELISA (E and F). Data in A, B, E, and F are expressed as mean ± SD. *, P < 0.01, compared with corresponding controls.