Clin Exp Otorhinolaryngol.  2013 Dec;6(4):237-242.

Effect of Epigallocatechin-3-Gallate on PMA-Induced MUC5B Expression in Human Airway Epithelial Cells

• Song EJ
• Bae CH
• Kim JY
• Kim YW
• Park SY
• Song SY
• Kim YD

Abstract

OBJECTIVES
Among the inflammatory mediators, phorbol 12-myristate 13-acetate (PMA) is associated with the regulation of MUC5B expression in the airway epithelial cells. Epigallocatechin-3-gallate (EGCG) is the major component of green tea extract. The biological activity of EGCG includes reduction of cholesterol and antioxidant activity, as well as anti-inflammatory effect. However, the precise action mechanism of anti-inflammatory effect of EGCG in the airway epithelial cells has not been fully defined. This study investigates the effect and the brief signaling pathway of EGCG on PMA-induced MUC5B expression in the airway epithelial cells.
METHODS
In NCI-H292 airway epithelial cells, the effect and signaling pathway of EGCG on MUC5B expression were investigated using real-time polymerase chain reaction analysis, enzyme immunoassay, immunohistochemical analysis, gelatin zymography assay, and immunoblot analysis.
RESULTS
In NCI-H292 airway epithelial cells, PMA induced MUC5B expression, phosphorylation of p38 mitogen-activated protein kinase (MAPK), and matrix metalloproteinase-9 (MMP-9) expression and protein activity. EGCG significantly decreased PMA-induced MUC5B expression, phosphorylation of p38 MAPK, and MMP-9 expression and protein activity. SB203580 (p38 MAPK inhibitor) significantly decreased PMA-induced MMP-9 expression. In addition, SB203580 and MMP-9 I (MMP-9 inhibitor) significantly decreased PMA-induced MUC5B expression.
CONCLUSION
These results suggest that EGCG down-regulates PMA-induced MUC5B expression through the p38 MAPK dependent MMP-9 signaling pathway in human airway epithelial cells.

Keyword

Epigallocatechin-3-gallate; MUC5B; p38; Matrix metalloproteinase-9; Airway epithelial cell

MeSH Terms

Catechin
Cholesterol
Epithelial Cells*
Gelatin
Humans*
Imidazoles
Immunoenzyme Techniques
Matrix Metalloproteinase 9
p38 Mitogen-Activated Protein Kinases
Phorbols
Phosphorylation
Protein Kinases
Pyridines
Real-Time Polymerase Chain Reaction
Tea
Catechin
Cholesterol
Gelatin
Imidazoles
Matrix Metalloproteinase 9
Phorbols
Protein Kinases
Pyridines
Tea
p38 Mitogen-Activated Protein Kinases

Figure

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  Fig. 1 The effect of EGCG on PMA-induced MUC5B expression. To investigate the effect of EGCG on PMA-induced MUC5B expression, NCI-H292 airway epithelial cells were incubated with PMA in the absence or presence of EGCG. (A) Real-time PCR and (B) ELISA showed that PMA induced MUC5B mRNA expression and protein production, which were significantly decreased by pretreatment with EGCG. EGCG, epigallocatechin-3-gallate; ELISA, enzyme-linked immunosorbent assay; PCR, polymerase chain reaction; PMA, phorbol 12-myristate 13-acetate. *P<0.05 compared with zero value. **P<0.05 compared with PMA alone.

• 

  Fig. 2 The effect of EGCG on PMA-induced phosphorylation of p38 MAPK. To investigate the MAPK signaling pathway of PMA-induced MUC5B expression, NCI-H292 airway epithelial cells were incubated with PMA in the absence or presence of EGCG and phosphorylation of ERK1/2 and p38 MAPK were analyzed by Western blot. (A) As time elapsed, phosphorylation of p38 MAPK was significantly increased: phosphorylation of p38 MAPK peaked at 20 minutes after treatment with PMA, but phosphorylation of ERK1/2 did not increase. (B) EGCG significantly decreased PMA-induced phosphorylation of p38 MAPK. EGCG, epigallocatechin-3-gallate; ERK1/2, extracellular signal related kinase 1/2; MAPK, mitogen-activated protein kinase; PMA, phorbol 12-myristate 13-acetate. *P<0.05 compared with zero value. **P<0.05 compared with PMA alone.

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  Fig. 3 The effect of EGCG on PMA-induced MMP-9 expression. To investigate the signaling pathway associated with MMP-9, NCI-H292 airway epithelial cells were incubated with PMA in the absence or presence of EGCG. (A) Real-time PCR showed that PMA induced MMP-9 mRNA expression and EGCG significantly decreased PMA-induced MMP-9 mRNA expression. (B) Gelatin zymography showed that EGCG inhibited PMA-induced MMP-9 protein activity. To investigate the correlation between MMP-9 and p38 MAPK, NCI-H292 airway epithelial cells were pretreated with SB203580 before being exposed to PMA. (C) Real-time PCR showed that SB203580 significantly decreased PMA-induced MMP-9 mRNA expression. EGCG, epigallocatechin-3-gallate; MAPK, mitogen-activated protein kinase; MMP-9, matrix metalloproteinase-9; PCR, polymerase chain reaction; PMA, phorbol 12-myristate 13-acetate. *P<0.05 compared with zero value. **P<0.05 compared with PMA alone.

• 

  Fig. 4 The signaling pathway of EGCG on PMA-induced MUC5B expression. To investigate the brief signaling pathway of EGCG on PMA-induced MUC5B expression, NCI-H292 airway epithelial cells were pretreated with SB203580 as a p38 MAPK inhibitor or MMP-9 I as a MMP-9 inhibitor before being exposed to PMA. Real-time PCR showed that SB203580 and MMP-9 I significantly decreased PMA-induced MUC5B mRNA expression. EGCG, epigallocatechin-3-gallate; MAPK, mitogen-activated protein kinase; MMP-9 I, matrix metalloproteinase-9 inhibitor; PCR, polymerase chain reaction; PMA, phorbol 12-myristate 13-acetate. *P<0.05 compared with zero value. **P<0.05 compared with PMA alone.

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  Fig. 5 The schematic signaling pathway of EGCG on PMA-induced MUC5B expression. EGCG can down-regulate PMA-induced MUC5B expression, which is associated with p38 MAPK dependent MMP-9 signaling pathway. PMA, phorbol 12-myristate 13-acetate; EGCG, epigallocatechin-3-gallate; MAPK, mitogen-activated protein kinase; MMP-9, matrix metalloproteinase-9.

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