NF-κB mediates IL-1β- and IL-17A-induced MUC5B expression in airway epithelial cells

Abstract

A major pathological feature of chronic airway diseases is the elevated expression of gel-forming mucins. NF-κB activation in airway epithelial cells has been shown to play a proinflammatory role in chronic airway diseases; however, the specific role of NF-κB in mucin gene expression has not been characterized. In this study, we show that the proinflammatory cytokines, IL-1β and IL-17A, both of which use the NF-κB pathway, are potent inducers of MUC5B mRNA expression in both well differentiated primary normal human bronchial epithelial cells and the human bronchial epithelial cell line, HBE1. MUC5B induction by these cytokines was both time- and dose-dependent, and was attenuated by the small molecule inhibitor, NF-κB inhibitor III, as well as p65 small interfering RNA, suggesting that the regulation of MUC5B expression by these cytokines is via an NF-κB-based transcriptional mechanism. Deletion analysis of the MUC5B promoter demonstrated that IL-1β- and IL-17A-induced promoter activity resides within the -4.17-kb to -2.56-kb region relative to the transcriptional start site. This region contains three putative κB-binding sites (NF-κB-1, -3,786/-3,774; NF-κB-2, -3,173/-3,161; and NF-κB-3, -2,921/-2,909). Chromatin immunoprecipitation analysis confirmed enhanced binding of the p50 NF-κB subunit to the NF-κB-3 site after cytokine stimulation. We conclude that an NF-κB-based transcriptional mechanism is involved in MUC5B regulation by IL-1β and IL-17A in airway epithelium. This is the first demonstration of the participation of NF-κB and its specific binding site in cytokine-mediated airway MUC5B expression.

Figures

Figure 1.

Stimulation of MUC5B mRNA expression by IL-1β and IL-17A. (A and C) Dose–response effects of IL-1β and IL-17A on MUC5B mRNA expression. Normal human bronchial epithelial (NHBE) cells grown in air–liquid interface (ALI) conditions for 1 week were starved for 16 hours (without growth factors) before being treated with various concentrations of IL-1β (0–20 ng/ml) and IL-17A (0–50 ng/ml). At 24 hours after cytokine treatment, total RNA was harvested, and MUC5B mRNA was analyzed using SYBR Green quantitative real-time PCR and normalized to a housekeeping gene, GAPDH, as described in Materials and Methods. (B and D) Time course effects of IL-1β and IL-17A treatment on MUC5B mRNA expression. NHBE cells were treated with 10 ng/ml IL-1β (B) or 20 ng/ml IL-17A (D). RNA samples were harvested from these cultures at different time points (0, 12, 24, 48 h) after treatment. MUC5B and GAPDH mRNA levels were quantified using real-time PCR. Triplicate dishes were used for each experiment, and experiments were repeated with cultures derived from two different human donors. Statistically significant: *P < 0.05, **P < 0.01, compared with unstimulated control.

Figure 2.

Effects of cytokines on mucin production and MUC5B glycoprotein expression in NHBE cells. NHBE cells were treated with 10 ng/ml IL-1β and 20 ng/ml IL-17A/F. (A) Medium was collected 24 hours after stimulation for mucin ELISA quantification, as described in the text and Ref. 39. (B) Cells were harvested 24 hours after stimulation and lysed for Western blot analysis with monoclonal antibody (Mab) 5B19-2E specific for MUC5B N-terminal peptide (40) and anti–β-actin antibody, which was used to monitor the protein input. *P < 0.05. CTL, unstimulated control.

Figure 3.

Effects of NF-κB inhibitor III on cytokine-induced MUC5B expression. NF-κB inhibitor III (20 μM) or an equal amount of vehicle (DMSO) was added 1 hour before 10 ng/ml IL-1β (A) or 20 ng/ml IL17A (B) treatment on primary NHBE cells, as described in Materials and Methods. At 12 and 24 hours after treatment, RNA samples were collected from these cultures. SYBR Green quantitative RT-PCR was used to quantify the message levels of MUC5B and GAPDH (or β-actin) in these RNA samples. Triplicate dishes were used for each time point, and experiments were repeated three times for different cultures derived from different donors. Statistically significant: *P < 0.05, **P < 0.01.

Figure 4.

Effects of p65 small interfering RNA (siRNA) on cytokine-induced MUC5B expression. HBE1 cells were treated with p65 siRNA or random oligomer (RO) siRNA as the control treatment (CTL), as described in the text. Two days after siRNA treatment, cells were starved for 6 hours, then treated with IL-1β (B) or IL-17A (C) for 16 hours. The efficiency of siRNA-p65 in reducing endogenous p65 mRNA was confirmed by performing real-time PCR (A). Relative MUC5AC message levels were averaged from triplicate dishes, and the experiment was repeated three times with HBE1 cells from different passage numbers. Statistically significant: **P < 0.01.

Figure 5.

Deletion analysis of MUC5B promoter–reporter gene activities in response to cytokines. HBE1 cells were cotransfected with various MUC5B promoter luciferase reporter constructs and the control pRL-TK plasmid, as described in Materials and Methods. At 2 days after transfection, cells were left unstimulated (CTL) or simulated with IL-1β (A) or IL-17A (B) at the 10-ng/ml level for 16 hours. Luciferase activities in these cells were measured and normalized as described in Materials and Methods. Data from triplicate dishes were averaged and experiments performed in three independent cultures. Statistically significant: **P < 0.01 compared with the CTL case without cytokine treatment.

Figure 6.

Chromatin immunoprecipitation (ChIP) assays on p50 protein binding on MUC5B promoter region in cells after IL-1β treatment. (A) Identification of putative κB-binding sites in the 5′-flanking region of MUC5B promoter. (B) Quantitative PCR analysis of anti-p50 antibody–precipitated and control IgG antibody–precipitated chromatin from HBE1 cells treated with 10 ng/ml of IL-1β for 1 hour. Coprecipitated DNA and the corresponding input DNA before precipitation were quantified by real-time PCR analysis using primers to amplify the MUC5B promoter DNA, which contain different κB-binding sites (top, NF-κB-1; middle, NF-κB-2; bottom, NF-κB-3). Results from the quantitative real-time PCR after normalization with input control DNA template were averaged from three independent experiments. **P < 0.01 compared with non–cytokine-treated control.