The Effect of Doxycycline on PMA-Induced MUC5B Expression via MMP-9 and p38 in NCI-H292 Cells

Abstract

Objectives: Doxycycline is commonly used in medicine for its bacteriostatic antimicrobial properties. Recent studies have reported that doxycycline also has anti-inflammatory effects. Matrix metalloproteinase (MMP)-9 has been found to be involved in the physiological and pathological process of inflammatory airway disease. Phorbol 12-myristate 13-acetate (PMA), a protein kinase C activator, is known to stimulate the expression of MMP and mucin genes in the airway and intestinal epithelial cells. Therefore, the effects and signal pathways of doxycycline on PMA-induced MUC5B expression dependent MMP-9 in human airway epithelial cells were investigated.

Methods: In human NCI-H292 airway epithelial cells, MUC5B and MMP-9 mRNA expression, MUC5B protein expression, and MMP-9 protein activity after the treatment with PMA, MMP-9 or doxycycline were determined by reverse transcriptase-polymerase chain reaction, enzyme immunoassay, gelatin zymography, and Western blot analysis.

Results: PMA increased MMP-9 and MUC5B expression. MMP-9 increased MUC5B expression. Doxycycline inhibited PMA-induced MUC5B expression, and PMA-induced MMP-9 mRNA expression and protein activity. Doxycycline inhibited phosphorylation of p38 induced by PMA and MMP-9.

Conclusion: The results of this study suggest that doxycycline inhibited PMA-induced MUC5B mRNA expression and protein production through the MMP-9 and p38 pathways in human NCI-H292 airway epithelial cells.

Keywords: Doxycycline; Epithelial cells; Inflammation; MUC5B; Matrix metalloproteinase-9; Mucins; NCI-H292 cell; Phorbol myristate acetate; p38.

Fig. 1

The effect of phorbol 12-myristate 13-acetate (PMA) on MUC5B and matrix metalloproteinase (MMP)-9 mRNA expression. Human NCI-H292 airway epithelial cells were stimulated with PMA. MUC5B and MMP-9 mRNA levels were analyzed by RT-PCR. MUC5B mRNA express ion was significantly increased at all doses of PMA and peaked at 10 nM of PMA. MMP-9 mRNA expression was significantly increased about 17 fold at 5, 10, 25, and 50 nM of PMA. ^*P<0.05 compared with zero value.

Fig. 2

The effect of matrix metalloproteinase (MMP)-9 on MUC5B mRNA expression. Human NCI-H292 airway epithelial cells were stimulated with MMP-9. MUC5B mRNA levels were analyzed by RT-PCR. MUC5B mRNA expression was significantly increased in a dose-dependent manner. ^*P<0.05 compared with zero value.

Fig. 3

The effect of SB203580 and p38 MAPK siRNA on the phosphorylation of p38 MAPK in phorbol 12-myristate 13-acetate (PMA)-induced MUC5B mRNA expression. Human NCI-H292 airway epithelial cells were stimulated with SB203580 before exposure to PMA, and also were transfected with predesigned siRNA targeting p38 MAPK and a negative control siRNA for p38 MAPK before exposure to PMA. MUC5B mRNA expression was analyzed by RT-PCR. (A) SB203580 inhibited PMA-induced MUC5B expression. (B) The knockdown of p38 MAPK by p38 MAPK siRNA significantly blocked PMA-induced MUC5B mRNA expression. ^*P<0.05 compared with PMA alone. ^**P<0.05 compared with control.

Fig. 4

The effect of doxycycline on phorbol 12-myristate 13-acetate (PMA)-induced MUC5B expression. Human NCI-H292 airway epithelial cells were stimulated with doxycycline after the treatment of PMA. MUC5B RNA levels were analyzed by RT-PCR, and MUC5B protein levels of cell lysates and supernatants were analyzed by enzyme-linked immunosorbent assay (ELISA). Doxycycline significantly inhibited PMA-induced MUC5B mRNA expression and protein production in a dose-dependent manner. ^*P<0.05 compared with PMA alone.

Fig. 5

The effect of doxycycline on phorbol 12-myristate 13-acetate (PMA)-induced matrix metalloproteinase (MMP)-9 mRNA expression and protein activity. Human NCI-H292 airway epithelial cells were stimulated with doxycycline after treatment with PMA. MMP-9 mRNA levels were analyzed by RT-PCR, and MMP-9 protein activity was measured by gelatin zymography. (A) Doxycycline significantly inhibited PMA-induced MMP-9 mRNA expression. (B) Doxycycline significantly inhibited PMA-induced MMP-9 protein activity. ^*P<0.05 compared with PMA alone.

Fig. 6

The effects of doxycycline on the phosphorylation of ERK1/2 and p38 after treatment with phorbol 12-myristate 13-acetate (PMA) or matrix metalloproteinase (MMP)-9. Human NCI-H292 airway epithelial cells were stimulated with doxycycline after treatment with PMA or MMP-9. The phosphorylation of ERK1/2 and p38 were detected by Western blot analysis. (A) Doxycycline inhibited PMA induced phosphorylation of p38 in a dose dependent manner, but it did not change the phosphorylation of ERK1/2 significantly. (B) Doxycycline significantly inhibited MMP-9 induced phosphorylation of p38. ^*P<0.05 compared with PMA alone.