IL-1β induction of MUC5AC gene expression is mediated by CREB and NF-κB and repressed by dexamethasone

Abstract

Chronic airway diseases are characterized by inflammation and mucus overproduction. The MUC5AC mucin gene is upregulated by the proinflammatory cytokine interleukin-1 β (IL-1β) via activation of cAMP response element-binding protein (CREB) in the NCI-H292 cancer cell line and nuclear factor-κB (NF-κB) in the HBE1 transformed cell line, with each transcription factor binding to a cognate cis site in the proximal or distal region, respectively, of the MUC5AC promoter. We utilized primary differentiated human bronchial epithelial (HBE) and A549 lung adenocarcinoma cells to further investigate the contributions of CREB and NF-κB subunits to the IL-1β-induced upregulation of MUC5AC. Data show that ligand binding of IL-1β to the IL-1β receptor is required to increase MUC5AC mRNA abundance. Chromatin immunoprecipitation analyses show direct binding of CREB to the previously identified cAMP response element site and binding of p65 and p50 subunits to a novel NF-κB site in a mucin-regulatory domain in the proximal promoter and to a previously identified NF-κB site in the distal promoter. P50 binds to both NF-κB sites at 1 h following IL-1β exposure, but is replaced at 2 h by p65 in A549 cells and by a p50/p65 heterodimer in HBE cells. Thus IL-1β activates multiple domains in the MUC5AC promoter but exhibits some cell-specific responses, highlighting the complexity of MUC5AC transcriptional regulation. Data show that dexamethasone, a glucocorticoid that transcriptionally represses MUC5AC gene expression under constitutive conditions, also represses IL-1β-mediated upregulation of MUC5AC gene expression. A further understanding of mechanisms mediating MUC5AC regulation should lead to a honing of therapeutic approaches for the treatment of mucus overproduction in inflammatory lung diseases.

Keywords: IL-1β; MUC5AC mucin; chronic inflammatory lung disease; dexamethasone; gene regulation; inflammatory transcription factors.

Figures

Fig. 1.

Interleukin-1β (IL-1β) increases MUC5AC mRNA abundance in lung epithelial cells in a concentration- and time-dependent manner. Differentiated human bronchial epithelial (HBE) (A) or A549 (B) cells were exposed to IL-1β (1, 10, or 100 ng/ml) or vehicle for 24 h. Differentiated HBE (C) or A549 (D) cells were incubated with 10 ng/ml of IL-1β or vehicle for 0, 1, 2, 4, 6, or 24 h. MUC5AC mRNA levels were quantified by qPCR and normalized to actin mRNA levels. Fold changes were determined by comparing levels of IL-1β-exposed cells to control levels. Experiments were carried out in triplicate using HBE cells from two individuals. Data are expressed as means ± SE. Statistically significant differences were analyzed by one-way ANOVA (Bonferroni method). *P < 0.05; ***P < 0.001.

Fig. 2.

The IL-1β-induced upregulation of the MUC5AC gene utilizes the IL-1β receptor. A549 cells were preexposed to increasing concentrations of IL-1 receptor antagonist (IL-1Ra) (0.25–250 ng/ml) or PBS (carrier control) for 10 min, prior to IL-1β (10 ng/ml) exposure for 6 h. MUC5AC mRNA levels were quantified by qPCR and normalized to actin mRNA levels. Each experiment was performed in duplicate in three independent experiments. Statistical analysis was performed by one-way ANOVA (Bonferroni method). Data are expressed as means ± SE. *P < 0.05 indicates statistically significant differences between IL-1β-exposed and control cells or between IL-1β-exposed cells compared in the presence or absence of IL-1Ra.

Fig. 3.

IL-1β transcriptionally regulates MUC5AC gene expression in A549 cells. Chromatin immunoprecipitation (ChIP) analyses were performed on nuclear lysates isolated from A549 cells following stimulation with IL-1β (10 ng/ml) for 0, 1, 2, 4, 6, or 18 h. Immunoprecipitation (IP) was performed with the antibody of interest and PCR was carried using primers that flank specific regions of the MUC5AC promoter, as shown in (A). B: IP with RNA polymerase II (RNA Pol II) antibody and PCR with primers that span the TATA box and transcriptional start site. C: IP with RNA Pol II antibody and PCR with primers that span the mucin regulatory domain (MRD). D: cells were exposed to IL-1β for 18 h. IP was performed with H4 antibody and PCR with primers that flank the proximal promoter region that includes the TATA box and transcriptional start site. Statistical analysis was performed by the Student's t-test. *P < 0.05; indicates statistically significant differences.

Fig. 4.

ChIP and re-ChIP analysis of CREB and p65 binding to the MUC5AC promoter in A549 cells exposed to IL-1β. A: ChIP analysis following exposure to IL-1β (10 ng/ml) for 0, 1, 2, 4, or 18 h. Nuclear lysates were isolated at the times indicated; DNA-protein complexes were cross-linked, and IP was performed using antibodies to cAMP response element-binding protein (CREB) or to the p65 nuclear factor-κB (NF-κB) subunit. Primers that span the MRD domain were used for PCR analyses of the immune complex. B: ChIP and re-ChIP analysis at 2 h following IL-1β (10 ng/ml) exposure. Nuclear lysates were immunoprecipitated with antibodies to CREB (lanes 1 and 3) or p65 (lanes 2 and 4) following exposure to PBS or IL-1β. For re-ChIP analysis, the eluent of the immune complex after CREB IP was immunoprecipitated with the p65 antibody and analyzed by PCR (lane 5).

Fig. 5.

ChIP and re-ChIP analysis of CREB and p65 binding to the MUC5AC promoter in HBE cells exposed to IL-1β for 2 h. A: ChIP analysis following exposure to PBS or IL-1β (10 ng/ml) for 2 h. DNA-protein complexes isolated from nuclear lysates were cross-linked; IP was performed using antibodies to CREB or the p65 NF-κB subunit. Primers that span the MRD domain were used for qPCR analyses of the immune complex. B: ChIP and re-ChIP analysis at 2 h following PBS or IL-1β (10 ng/ml) exposure. For re-ChIP analysis, the eluent of the immune-complex after CREB IP was immunoprecipitated with the p65 antibody and analyzed by qPCR. Duplicate experiments were carried out using differentiated HBE cells from two individuals. Statistical analysis was performed by the Student's t-test. *P < 0.05; statistically significant differences.

Fig. 6.

Exposure of IL-1β to lung epithelial cells activates binding of p50 and p65 NF-κB subunits to the NF-κB sites in the MRD and distal region of the MUC5AC promoter. A549 (A–D) or primary differentiated HBE (E–H) cells were exposed to IL-1β (10 ng/ml) for 1 h (A, B, E, F) or 2 h (C, D, G, H). Binding of NF-κB subunits was determined by ChIP analysis using anti-p50 and anti-p65 or control IgG (10 μg, Santa Cruz Biotechnology) for IP. The associated DNA was identified by real-time qPCR using primer pairs that encompass the MRD and the NF-κB cis sites in the distal region of the MUC5AC promoter. Values are normalized by input DNA. Data from three independent experiments are expressed as means ± SE. Statistical analysis was performed by Student's t-test. *P < 0.05; statistically significant differences.

Fig. 7.

Dexamethasone (Dex) reduces IL-1β-upregulated MUC5AC expression in HBE cells. A: differentiated HBE cells were preincubated with IL-1β (10 ng/ml) for 18 h, then exposed to Dex (10, 100, or 1,000 nM) or vehicle for 6 h. B: alternatively, cells were preincubated with Dex (10, 100, or 1,000 nM) or vehicle for 6 h, then exposed to IL-1β (10 ng/ml) for 18 h. MUC5AC and actin mRNA levels were quantified by real-time PCR. Results are expressed as means ± SE of three independent experiments in HBE cells from two different individuals. Statistical analysis was performed by one-way ANOVA (Bonferroni method). *P < 0.05; **P < 0.01.

Fig. 8.

Dex and IL-1Ra additively inhibit MUC5AC gene expression. A549 cells were exposed to IL-1Ra (250 ng/ml) or vehicle for 10 min before the addition of IL-1β (10 ng/ml) and Dex (100 nM) for 6 h. MUC5AC and actin mRNA levels were quantified by qRT-PCR and normalized to actin. Each experiment was performed in duplicate from three independent experiments. Statistical analysis was performed by one-way ANOVA (Bonferroni method). *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 9.

Schema of the MUC5AC promoter. Rectangles show putative cAMP response element (CRE) (hatched) and NF-κB (dark gray) cis sites. Triangles above the promoter indicate functional CRE (hatched triangle) (54), NF-κB (dark gray triangle) (16), and glucocorticoid responsive elements (GRE) (white triangle) (6, 8) cis sites. The expanded domain containing two known functional (CRE, GRE3) sites and a newly identified functional NF-κB site is termed a mucin-regulatory domain (MRD). TSS, transcriptional start site.