Clin Exp Otorhinolaryngol.  2012 Sep;5(3):161-169. 10.3342/ceo.2012.5.3.161.

Phorbol 12-Myristate 13-Acetate Induces MUC16 Expression via PKCdelta and p38 in Human Airway Epithelial Cells

• Bae CH
• Kim HS
• Song SY
• Kim YD

• KMID: 2134600
• DOI: http://doi.org/10.3342/ceo.2012.5.3.161

Abstract

OBJECTIVES
Phorbol 12-myristate 13-acetate (PMA) is widely used as a protein kinase C (PKC) activator, PKC is involved in the secretion of mucins. MUC16, one of the membrane-bound mucins, is produced in human airway epithelial cells. However, the effect and signaling pathway of PMA on MUC16 expression in human airway epithelial cells has not been reported. Therefore, the effect and brief signaling pathway of PMA on MUC16 expression were investigated in human airway epithelial cells in this study.
METHODS
In the mucin-producing human NCI-H292 airway epithelial cells and the primary cultures of normal nasal epithelial cells, the effect and signaling pathway of PMA on MUC16 expression were investigated using reverse transcriptase-polymerase chain reaction (RT-PCR), real-time PCR, enzyme immunoassay, and immunoblot analysis with several specific inhibitors and small interfering RNA (siRNA) for p38 mitogen-activated protein kinase (MAPK).
RESULTS
PMA increased MUC16 expression, and activated phosphorylation of p38 MAPK. However, it did not activate phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2). SB203580 (p38 MAPK inhibitor) inhibited PMA-induced MUC16 expression, while U0126 (ERK1/2 inhibitor) did not. In addition, the knockdown of p38 MAPK by p38 MAPK siRNA significantly blocked PMA-induced MUC16 mRNA expression. Rottlerin (PKCdelta inhibitor) inhibited PMA-induced MUC16 expression, and also inhibited the phosphorylation of activated p38 MAPK by PMA.
CONCLUSION
These results show for the first time that PMA-induced MUC16 expression is regulated by activation of the PKCdelta and p38 MAPK signaling pathway in human airway epithelial cells.

Keyword

Phorbol 12-myristate 13-acetate; Mucin; MUC16; PKCdelta; p38 MAPK; Airway epithelial cell

MeSH Terms

Acetophenones
Benzopyrans
Butadienes
Epithelial Cells
Humans
Imidazoles
Immunoenzyme Techniques
Mucins
Nitriles
p38 Mitogen-Activated Protein Kinases
Phorbols
Phosphorylation
Phosphotransferases
Protein Kinase C
Protein Kinases
Pyridines
Real-Time Polymerase Chain Reaction
RNA, Messenger
RNA, Small Interfering
Acetophenones
Benzopyrans
Butadienes
Imidazoles
Mucins
Nitriles
Phorbols
Phosphotransferases
Protein Kinase C
Protein Kinases
Pyridines
RNA, Messenger
RNA, Small Interfering
p38 Mitogen-Activated Protein Kinases

Figure

• 

  Fig. 1 The effect of PMA on MUC16 expression in the NCI-H292 airway epithelial cells. MUC16 expression was analyzed using reverse transcriptase-polymerase chain reaction (RT-PCR), real-time PCR. (A) MUC16 expression was significantly increased in a dose-dependent manner. (B) PMA did not induce MUC16 expression in a time-dependent manner. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PMA, phorbol 12-myristate 13-acetate; C, control; T, treatment with PMA. *P<0.05 compared with zero value.

• 

  Fig. 2 The effect of PMA on the phosphorylations of ERK1/2 and p38 MAPK in the NCI-H292 airway epithelial cells. The phosphorylation of ERK1/2 and p38 were detected using Western blot analysis. PMA activated phosphorylation of p38 MAPK, while it did not activate phosphorylation of ERK1/2. ERK, extracellular signal-regulated kinase; PMA, phorbol 12-myristate 13-acetate; MAPK, mitogen-activated protein kinase.

• 

  Fig. 3 The effect of MAPKs inhibitors on PMA-induced MUC16 expression in the NCI-H292 airway epithelial cells. MUC16 expression was analyzed using reverse transcriptase-polymerase chain reaction (RT-PCR), real-time PCR, and ELISA. (A) SB203580 inhibited PMA-induced MUC16 expression. (B) U0126 did not inhibit PMA-induced MUC16 expression. *P<0.05 compared with PMA alone. PMA, phorbol 12-myristate 13-acetate; ERK, extracellular signal-regulated kinase; ELISA, enzyme-linked immunosorbent assay; MAPK, mitogen-activated protein kinase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

• 

  Fig. 4 The effect of p38 MAPK siRNA on the phosphorylation of p38 MAPK in PMA-induced MUC16 mRNA expression in the NCI-H292 airway epithelial cells. MUC16 mRNA expression was analyzed using reverse transcriptase-polymerase chain reaction (RT-PCR), real-time PCR. The knockdown of p38 MAPK by p38 MAPK siRNA significantly blocked PMA-induced MUC16 mRNA expression. MAPK, mitogen-activated protein kinase; PMA, phorbol 12-myristate 13-acetate; siRNA, small interfering RNA; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. *P<0.05 compared with zero value; †P<0.05 compared with negative control.

• 

  Fig. 5 The effect of PKCδ inhibitor on PMA-induced MUC16 expression in the NCI-H292 airway epithelial cells. MUC16 expression was analyzed using reverse transcriptase-polymerase chain reaction (RT-PCR), real-time PCR, and ELISA. Calphostin C (A) and Rottlerin (B) inhibited PMA-induced MUC16 expression. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PMA, phorbol 12-myristate 13-acetate; ELISA, enzyme-linked immunosorbent assay; PKC, protein kinase C. *P<0.05 compared with PMA alone.

• 

  Fig. 6 The effect of PKCδ inhibitor on the phosphorylations of ERK1/2 and p38 MAPK in the NCI-H292 airway epithelial cells. The phosphorylation of ERK1/2 and p38 MAPK were detected using Western blot analysis. (A) The phosphorylation of ERK1/2 was not inhibited by Calphostin C and Rottlerin after treatment with PMA. (B) The phosphorylation of p38 MAPK was inhibited by Calphostin C and Rottlerin after treatment with PMA; the inhibiting effect of Rottlerin was significantly stronger than Calphostin C. PKC, protein kinase C; ERK, extracellular signal-regulated kinase; MAPK, mitogen-activated protein kinase; PMA, phorbol 12-myristate 13-acetate. *P<0.05 compared with PMA alone.

• 

  Fig. 7 PKCδ and p38 MAPK in associated with PMA-induced MUC16 expression in the primary cultures of normal nasal epithelial cells. MUC16 mRNA expression was analyzed using reverse transcriptase-polymerase chain reaction (RT-PCR), real-time PCR. Calphostin C, Rottlerin and SB203580 inhibited PMA-induced MUC16 expression; the inhibiting effect of Rottlerin was significantly stronger than Calphostin C. PKC, protein kinase C; MAPK, mitogen-activated protein kinase; PMA, phorbol 12-myristate 13-acetate; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. *P<0.05 compared with PMA alone.

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