Airborne particulate matter increases MUC5AC expression by downregulating Claudin-1 expression in human airway cells

Abstract

CLB2.0, a constituent of PM, induces secretion of multiple cytokines and chemokines that regulate airway inflammation. Specifically, IL-6 upregulates CLB2.0-induced MUC5AC and MUC1 expression. Interestingly, of the tight junction proteins examined, claudin-1 expression was inhibited by CLB2.0. While the overexpression of claudin-1 decreased CLB2.0-induced MUC5AC expression, it increased the expression of the anti-inflammatory mucin, MUC1. CLB2.0-induced IL-6 secretion was mediated by ROS. The ROS scavenger N-acetylcysteine inhibited CLB2.0-induced IL-6 secretion, thereby decreasing the CLB2.0-induced MUC5AC expression, whereas CLB2.0-induced MUC1 expression increased. CLB2.0 activated the ERK1/2 MAPK via a ROS-dependent pathway. ERK1/2 downregulated the claudin-1 and MUC1 expressions, whereas it dramatically increased CLB2.0-induced MUC5AC expression. These findings suggest that CLB2.0-induced ERK1/2 activation acts as a switch for regulating inflammatory conditions though a ROS-dependent pathway. Our data also suggest that secreted IL-6 regulates CLB2.0-induced MUC5AC and MUC1 expression via ROS-mediated downregulation of claudin-1 expression to maintain mucus homeostasis in the airway. [BMB Reports 2017; 50(10): 516-521].

Fig. 1

CLB_2.0 induces IL-6 secretion and overexpression in NHBE cells. (A) Cells were treated with CLB_2.0 for 4 h, and a cytokine assay was performed in a dose- and time-dependent manner. (B) After the treatment of CLB_2.0 for 4 h, the total cell lysates were analyzed by Western blot analysis with specific anti-IL-6 antibody. (C) The cells were then treated with CLB_2.0 for 4 h, and their supernatants were collected. The levels of IL-6 in the cell supernatants were measured by ELISA. *P < 0.05 compared with control. Values shown represent the means ± SDs of three technical replicates from a single experiment. Cells were treated with CLB_2.0 (10 mg/ml), IL-6 (30 ng/ml) and both CLB_2.0 (10 mg/ml) and IL-6 (30 ng/ml) for 24 h and their total RNA were collected, and then qRT-PCR for MUC5AC (D) and MUC1 (E) transcript were performed. *P < 0.05 compared to the control, **P < 0.05 compared to CLB_2.0 only.

Fig. 2

Effect of Claudin-1 on CLB_2.0-induced MUC5AC and MUC1 gene expression. (A) After the cells were treated with CLB_2.0 (10 mg/ml) or CLB_2.0 and IL-6 (30 ng/ml) for 24 h, Western blot analysis was performed with specific Claudin-1, ZO-1, and E-cadherin antibodies. Tubulin was used as the loading control. (B) Cells were transfected with either a construct driving the expression of wild-type IL-6 or siRNA specific for IL-6. Cells were then treated with CLB_2.0 (10 mg/ml) for 4 h prior to the generation of total cell lysates, and then Western blot analysis for Claudin-1 was performed. (C) Cells were transfected with either a construct expressing of wild-type Claudin-1 or siRNA specific for Claudin-1, followed by treating with CLB_2.0 (10 mg/ml) for 24 h prior to the generation of total cell lysates, and then qRT-PCR for MUC5AC and MUC1 mucin genes were performed. *P < 0.05 compared to control, **P < 0.05 compared to CLB_2.0-treated cells, and ***P < 0.05 compared to CLB_2.0/wild-type Claudin-1-treated cells.

Fig. 3

Effect of ROS produced by CLB_2.0 on Claudin-1 and mucin genes expression. (A) The cells were pre-incubated in the presence of 50 μM DCFH-DA for 30 min, and then exposed to CLB_2.0 for the indicated times. Cell-associated DCF fluorescence levels were analyzed by flow cytometry. *P < 0.05 compared to the control. (B) The cells were pre-incubated in the presence of N-acetyl-cysteine (NAC) for 1 h in a dose-dependent manner, and then treated CLB_2.0 for 1 h. Cell proliferation assay was performed with CCK-8 (Dojindo; Rockville, MD). (C) After the cells were treated with CLB_2.0 (10 mg/ml) or CLB_2.0 and IL-6 (30 ng/ml) for 5 min, flow cytometry was analyzed to measure the ROS production. *P < 0.05 compared to the control. (D) After the cells were treated with CLB_2.0 (10 mg/ml) or CLB_2.0 and NAC (1 mM; pretreatment for 1 h) for 24 h, Western blot anlaysis (upper panel) and qRT-PCR for MUC5AC and MUC1 (lower panel) mucin genes were performed. *P < 0.05 compared to the control, **P < 0.05 compared to the CLB_2.0-treated cells.

Fig. 4

ERK1/2 plays as a switch molecule to regulate the inflammatory condition. (A) After the cells were treated with CLB_2.0 in a time-dependent manner, Western blots were performed to detect MAPK activation. Tubulin was used as the loading control. (B) After the cells were treated with CLB_2.0 (10 mg/ml) or CLB_2.0 and NAC (1 mM; pretreatment for one hour) for 15 mins, Western blot for ERK1/2 phosphorylation was performed. (C) Cells were transfected with either a construct expressing of wild-type ERK1/2 or siRNA specific for ERK1/2. The cells were then treated with CLB_2.0 (10 mg/ml) for 24 h prior to the generation of total cell lysates, and then qRT-PCR for MUC5AC and MUC1 mucin genes were performed. *P < 0.05 compared to the control, **P < 0.05 compared to the CLB_2.0-treated cells, and ***P < 0.05 compared to the CLB_2.0/wild-type ERK1/2-treated cells. (D) A schematic diagram is presented to show the potential mechanisms for secretion of IL-6, and their physiological roles during the inflammatory responses.