Doxycycline attenuates acrolein-induced mucin production, in part by inhibiting MMP-9

Abstract

Matrix metalloproteinases (MMPs), especially MMP-9, have been found to increase the expression of epidermal growth factor (EGF) receptor, a possible regulator of acrolein-induced mucin expression in the airway epithelium. The aim of this study was to investigate whether doxycycline, a tetracycline antibiotic that inhibits MMPs, attenuates mucus production and synthesis of mucin MUC5AC in acrolein-exposed rats. Sprague–Dawley rats were exposed to acrolein aerosol [3.0 parts/million (ppm), 6 h/day, 12 days] and they received 20 mg/kg doxycycline daily by gavage, beginning two days before exposure to acrolein until the end of the experiment. The production of mucin glycoproteins and expression of the MMP-9 and MUC5AC genes were measured in rat trachea. The increase in levels of MMP-9 mRNA and protein in airway epithelium after acrolein exposure was accompanied by an increase in MUC5AC mRNA expression. Doxycycline significantly prevented these increases in acrolein-induced expression of MMP-9 and MUC5AC and attenuated mucus production in tracheal epithelium. These results indicate that doxycycline attenuated acrolein-induced mucin synthesis, in part by inhibiting expression of MMP-9. Thus doxycycline may have a prophylactic effect in the treatment of smoking-induced mucus hypersecretion.

Introduction

Mucus is an important protective factor against environmental agents and pathogens, but excessive airway mucus production, termed mucus hypersecretion, may impair mucociliary clearance (Nadel, 2000). Many chronic airway diseases, including chronic obstructive pulmonary disease (COPD), cystic fibrosis, bronchiectasis, and asthma are associated with airway mucus hypersecretion (Perez-Vilar et al., 2003).

Airway mucus is mostly water (95% by weight), and the balance comprises mainly mucous glycoprotein (mucins), other secretory proteins, serum proteins, lipids, and salts. Mucin, the major macromolecular constituent of mucus, is secreted by goblet cells located in the epithelium and by mucus cells in the submucosal glands (Leikauf et al., 2002). In airway tissues from healthy individuals, goblet cells typically express MUC5AC mRNA, while mucus cells express MUC5B mRNA. MUC5AC is a highly inducible gene, whereas MUC5B is expressed constitutively (Leikauf et al., 1989, Williams et al., 2001, Rose and Voynow, 2006, Young et al., 2007, Henke et al., 2007).

Up-regulation of MUC genes and goblet cell hyperplasia (GCH) contribute to and sustain mucin overproduction in the airways of patients with chronic lung diseases. Cigarette smoking, air pollutants and bacterial infection are believed to be common causes of lung inflammation and are all associated with mucus overproduction. Acrolein (CH2═CHCHO) can be found in tobacco smoke at concentrations up to 50 parts per million (ppm), as well as in wood smoke (Altshuler and McPherson, 1963, Ayer and Yeager, 1982). Animals repeatedly exposed to acrolein develop histological changes, including epithelial damage, mucus hypersecretion, and bronchiolitis (Costa et al., 1986, Borchers et al., 1998, Wang et al., 2009a).

Matrix metalloproteinases (MMPs), a family of many structurally related enzymes, degrade components of the extracellular matrix in both physiological and pathophysiological states, and they may play an important role in chronic airway diseases (Murray et al., 1998, Greenlee et al., 2007). MMPs also process, activate, and deactivate a variety of non-matrix soluble factors, including several growth factors and chemokines. Thus, they are considered both proteinases of matrix catalysis and extracellular processing enzymes (Parks and Shapiro, 2001).

Epidermal growth factor (EGF) and its receptor may be involved in mucin secretion, and MMPs may mediate their involvement. Previous studies showed that activation of EGF receptor was responsible for airway mucus production induced by cigarette smoke (Nadel, 2000, Takeyama, 2001). Deshmukh et al. (2008) demonstrated that the EGF receptor and MAPK signaling pathway play an important role in the accumulation of MMP-9 transcripts and protein after acrolein exposure. Indeed, studies have shown that MMP-9, regulated by epidermal growth factor (EGF) receptor, plays a major role in the pathogenesis of inflammatory airway diseases such as COPD, asthma and cystic fibrosis (Wallace and Sandford, 2002, Deshmukh et al., 2008).

The tetracyclines are potent inhibitors of the MMP enzyme family and have been used to reduce tissue degradation in periodontal disease and arthritis (Bokor, 1995, Tamimi et al., 2008, Smieja et al., 2001). Doxycycline is one of the classic tetracycline derivatives that have been studied extensively in various diseases. Doxycycline exerts diverse inhibitory effects on MMP production and activity (Hidalgo and Eckhardt, 2001). It was reported to decrease serum MMP-9 levels by 50% after 6 months of oral administration following myocardial infarction (Brown et al., 2004). Doxycycline has also been reported to decrease the activity of MMP-9 in vitro (Kim et al., 2005), but the effects and mechanisms of doxycycline on acrolein-induced MMP-9 production are not known.

In the present study, levels of MMP-9 mRNA and protein, levels of MUC5AC mRNA, and mucus production were measured in rats exposed to acrolein. Rats were treated with doxycycline before and during the exposure, in order to determine whether the MMP inhibitor can affect acrolein-induced mucin overproduction.