Arylhydrocarbon receptor (AhR) activation in airway epithelial cells induces MUC5AC via reactive oxygen species (ROS) production

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Abstract

The dioxins and dioxin-like compounds in cigarette smoke regulate various immunological responses via the arylhydrocarbon receptor (AhR). These environmental toxicants are known to cause bronchitis, asthma, chronic obstructive pulmonary disease (COPD), and lung cancer. Recent studies have demonstrated that AhR activation upregulates the expression of mucin 5AC, oligomeric mucus/gel-forming (MUC5AC) in the airway epithelial cell line. However, the mechanism for the production of mucin has not been clarified. In this study, we investigated the role and pathway of AhR in airway epithelial cells by using selective agonists and antagonists. After stimulation with or without benzopyrene (B[a]P), an AhR agonist, MUC5AC expression was measured by real-time RT-PCR. The mechanism of AhR-induced MUC5AC expression in airway epithelial cells was studied in terms of the production of cytokine and reactive oxygen species (ROS). Treatment with B[a]P increased ROS generation in NCI-H292 cells. Furthermore, B[a]P-induced MUC5AC upregulation and mucin production were inhibited by AhR siRNA or the use of an antioxidative agent. These results suggest that the AhR-induced increase of mucin production is partially mediated by ROS generation. An antioxidant therapy approach may help to cure AhR-induced mucus hypersecretory diseases.

Introduction

Many noxious agents such as benzopyrene (B[a]P) in cigarette smoke, environmental pollutants, and Yusho oil disease caused by the ingestion of rice bran oil contaminated with dioxins, polychlorinated biphenyls (PCBs), and other related organochlorine compounds not only have harmful effects but also regulate various immunological responses [1], [2], [3], [4]. One of the most toxic dioxins, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), induces the production of tumor necrotizing factor-α (TNF-α) in THP-1, a human promonocytic leukemia cell line, and increases the expression of transforming growth factor-α (TGF-α) mRNA in MCF-10A, a human mammary epithelial cell line [5], [6]. Furthermore, noxious agents such as TCDD and hexachlorobenzene mimic the epithelial growth factor receptor (EGFR) signaling pathway, leading to tyrosine kinase phosphorylation and its physical association with EGFR [7], [8]. On the other hand, TCDD blocks cell differentiation through EGFR signaling in human epidermal keratinocytes [9]. In the respiratory system, these environmental toxicants are known to cause bronchitis, asthma, chronic obstructive pulmonary disease (COPD), and lung cancer [10]. Indeed, the symptoms in patients with chronic bronchitis induced by cigarette smoke and Yusho are defined as cough and excessive mucus production [11], [12].

At present, the biological effects of these dioxins, dioxin-like compounds, and B[a]P are mainly mediated through binding to the arylhydrocarbon receptor (AhR). The AhR is a ligand-activated transcription factor ubiquitously distributed in the body, and after ligation of dioxins to the AhR, the receptor translocates from the cytosol to the nucleus, heterodimerizes with the AhR nuclear translocator (ARNT), and binds to an enhancer sequence, called a dioxin response element (DRE), of several drug metabolizing enzymes, such as cytochrome P450 1A1 (CYP1A1) [13]. It has also been shown that Clara cells in the lung are the most sensitive to AhR stimulation [14], [15]. Wong et al. recently demonstrated that TCDD via AhR-induced mucin production from a Clara-cell-derived cell line [16]. Although IL-1β induction and COX-2 pathway activation might have been responsible for AhR-induced mucin production in the bronchial epithelial cells, the mechanism remains to be determined.

The present study, therefore, investigated the pathway of AhR activation in mucin production using B[a]P. Our data showed that the AhR-induced increase of mucin production was partially mediated by reactive oxygen species (ROS) generation. Thus, the use of antioxidant therapy might be a practical way to treat patients with respiratory symptoms related to dioxin exposure.

Section snippets

Reagents

The human pulmonary mucoepidermoid carcinoma cell line (NCI-H292) and normal human bronchial epithelial (NHBE) cells were obtained from the American Type Culture Collection (Rockville, MD, USA). HaCaT cells, a spontaneously immortalized human keratinocyte cell line, were kindly provided by Dr N.E. Fusenig (DKFZ Heidelberg). B[a]P (benzopyrene), resveratrol (an AhR inhibitor), tumor necrotizing factor-α (TNF-α) and N-acetyl-l-cysteine (NAC) were purchased from Sigma (Poole, UK).

Cell cultures

NCI-H292 and NHBE

AhR activation expressed in airway epithelial cells induces MUC5AC mRNA and mucin production

We primarily investigated the expression of AhR at the mRNA level. HaCaT cells expressing AhR were used as a positive control [17]. As shown in Fig. 1A, AhR mRNA was detected by RT-PCR in NCI-H292 and NHBE cells. Similarly, the protein expression of AhR in airway epithelial cells was examined. NCI-H292, NHBE and HaCaT cells expressed AhR protein, as detected by Western blot (Fig. 1A). Next, we measured MUC5AC from NCI-H292 cells incubated with B[a]P (AhR agonist) or vehicle for 12 h to confirm

Discussion

Dioxins, dioxin-like compounds, and B[a]P act as modulators of immunological response through AhR. Studies using AhR agonists and inhibitors have demonstrated that AhR activation induces cytokine production such as TGF-α, TNF-α, and matrix metalloprotease (MMP), tyrosine kinase phosphorylation, and cell cycle progression through its receptor in human hematocytes and epithelial cells [5], [6], [7], [8], [9], [19], [20]. Recently, Wong et al. reported that TCDD via AhR-induced mucin or MMP

Acknowledgments

This work was supported by the Environment Technology Development Fund of the Ministry of the Environment and in part by the Ministry of Health, Labour, and Welfare of Japan.

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