Peptidoglycan from Staphylococcus aureus increases MUC5AC gene expression via RSK1-CREB pathway in human airway epithelial cells

Abstract

Respiratory tract exposure to viruses, air pollutants, or bacterial pathogens can lead to pulmonary diseases. The molecular mechanism of mucous overproduction increased by these pathogens provides the knowledge for developing new therapeutic strategies. There is established in vitro data demonstrating that the overexpression of MUC5AC is induced by peptidoglycan (PGN) derived from Staphylococcus aureus. However, the mechanisms by which PGN activates MUC5AC gene expression in the airway remain unclear. The aim of this study was to identify the mechanism of PGN-induced MUC5AC gene expression. We found that PGN could induce MUC5AC gene expressions in a time- and dose-dependent manner. Moreover, activations of ERK1/2 and JNK increased after treatment of cells with PGN, whereas phosporylation of p38 was undetected. Of these MAPKs, pharmacologic inhibition of ERK1/2 decreased PGN-induced MUC5AC gene expression. In addition, we checked the activation of p90 ribosomal S6 kinase 1 (RSK1) as a downstream signaling target of ERK1/2 in PGN signaling. The activation of RSK1 was prevented by pretreatment with PD98059. We also found that RSK1 mediated the PGN-induced phosphorylation of cAMP response element-binding protein (CREB) and the transcription of MUC5AC. Furthermore, the cAMP-response element (CRE) in the MUC5AC promoter appears to be important for PGN-induced MUC5AC gene expression in NCI-H292 cells.

Fig. 1.. Effect of exotoxins on mucin gene expression. Confluent cells were rendered quiescent in RPMI-1640 with 0.2% FBS for 24 h prior to treatment with PGN (10 μg/ml), exotoxin A (500 ng/ml), or LPS (10 μg/ml) respectively for 24 h, and cell lysates were harvested for RTPCR. β-2-microglobulin (β₂M) was employed as an internal control. The figures shown are representative of three independent experiments. Densitometry results are presented as the means ± SD (Fig. 1A, right panel). NCI-H292 Cells were treated for 24 h with exogenous PGN at the indicated concentrations (B) and times (C). Cell lysates were harvested for real-time quantitative PCR. *p < 0.05 compared to control (vehicle). Data are presented as mean ± SD values of three independent experiments. The figures are representative of three independent experiments.

Fig. 2.. Effect of ERK1/2 on PGN-induced MUC5AC gene expression. (A) Confluent cells were treated with PGN for different times (min), and cell lysates were harvested for Western blot analysis using phospho-specific antibodies. Total ERK1/2 was used as a loading control. (B) Cells were treated with different dose of PD98059, U0126, or SP600125 for 10 min, and cell lystes were performed Western blot analysis. (C) Cells were treated with PD98059 (20 μM) or SP600125 (20 μM) for 2 h, and then stimulated for 24 h with PGN prior to collection of total RNA for real-time RT-PCR analysis of MUC5AC mRNA expression. *p < 0.05 compared to control (vehicle); **p < 0.05 compared to PGN treatment only. The figures shown are representative of three independent experiments.

Fig. 3.. Effect of RSK1 on PGN-induced MUC5AC gene expression. (A) Cells were stimulated for the indicated times with PGN, and then total proteins were collected for Western blot analysis using phospho-RSK1 or MSK1 antibody. Total β-actin was used as a loading control. (B) The cells were pretreated for 2 h with 20 μM PD98059 and then stimulated with PGN for 10 min prior to Western blot analysis. (C) The cells were transiently transfected with RSK1 wild-type construct or siRNA-RSK1. The cells were treated for 24 h prior to real-time RT-PCR. *p < 0.05 compared to control (vehicle); **p < 0.05 compared to PGN treatment only. The figures shown are representative of three independent experiments.

Fig. 4.. Involvement of CREB on PGN-induced MUC5AC transcription. (A) Cells were treated for the different times with PGN, and then total proteins were collected for Western blot. Total β-actin was used as a loading control. (B) The cells were pretreated for 2 h with 20 μM PD98059 or were transiently transfected with either siRNA-RSK1 or siRNA-CREB (C). The cells were harvested for Western blot analysis (upper panel) and the cells were treated for 24 h prior to real-time RT-PCR (lower panel). *p < 0.05 compared to control (vehicle); **p < 0.05 compared to PGN treatment only. (D) Cells were transiently transfected with several MUC5AC promoter luciferase reporter constructs which did or did not contain the CRE motif and were stimulated with PGN for 24 h. Cell lysates were harvested, and reporter assays were performed according to the manufacturer’s instructions (see “Materials and Methods”). *p < 0.05 compared to -776/-1 reporter construct. (E) Site-directed selective mutagenesis was performed to construct CRE-binding site mutants as indicated. Luciferase activities are shown after correction for transfection efficiency using the β-galactosidase activity of the cell lysates. Values shown are means ± S.D. of experiments performed three or more times. *p < 0.05 compared to wild-type reporter construct. The figures shown are representative of three independent experiments.