Localization of γ-glutamylcysteine synthetase messenger rna expression in lungs of smokers and patients with chronic obstructive pulmonary disease

Abstract

Cigarette smoking results in an oxidant/antioxidant imbalance in the lungs and inflammation, which are considered to be key factors in the pathogenesis of chronic obstructive pulmonary disease (COPD). Glutathione (GSH) is an important protective antioxidant in lung epithelial cells and epithelial lining fluid. De novo GSH synthesis in cells occurs by a two-enzyme process. The rate-limiting enzyme is γ-glutamylcysteine synthetase (γ-GCS), in which the heavy subunit (HS) constitutes most of its catalytic activity. The localization and expression of γ-GCS-HS in specific lung cells as well as possible differences in its expression between smokers with and without COPD have not yet been studied. The purpose of this study was to investigate γ-GCS-HS expression using messenger RNA in situ hybridization in peripheral lung tissue. We studied 23 current or ex-smokers with similar smoking histories with (n = 11; forced expiratory volume in 1 s [FEV₁] < 75% predicted) or without COPD (n = 12; FEV₁ < 84% predicted). We assessed the relations between pulmonary γ-GCS-HS expression, FEV₁ and transforming growth factor-β1 (TGFβ₁), because TGFβ₁ can modulate γ-GCS-HS expression in lung epithelial cells. Gamma-GCS-HS is predominantly expressed by airway and alveolar epithelial cells, alveolar CD68+ cells (macrophages), and endothelial cells of both arteries and veins. In subjects with COPD, semiquantitative analysis revealed higher levels of γ-GCS-HS messenger RNA in alveolar epithelium (1.5 times, p < .04) and a trend for a higher expression in bronchiolar epithelium (1.3 times, p = .075) compared with subjects without COPD. We did not observe a significant correlation between airway and alveolar epithelial γ-GCS-HS expression and TGFβ₁ expression (r = .20), FEV₁ percentage predicted (r = .18), or FEV₁/forced vital capacity ratio (r = .14; p > .05). Our results show that γ-GCS-HS is localized, particularly in lung epithelium, and shows higher expression in smokers with COPD. This suggests a specific role for enhanced GSH synthesis as a mechanism to provide an adaptive response against oxidative stress in patients with COPD.

Introduction

The tripeptide, l-γ-glutamyl-l-cysteinylglycine, or glutathione (GSH), is an ubiquitous nonprotein sulfhydryl compound that has an important role in maintaining intracellular redox balance and cellular defenses against oxidative stress [1], [2]. Depletion of cellular GSH is associated with lung damage produced by a variety of oxidants [1], [3]. GSH is present in high concentrations in the lung epithelial lining fluid (ELF) [4]. It is also important in maintaining the integrity of the airspace epithelium in type II alveolar cells in vitro and in lungs in vivo [3], [5].

GSH is synthesized by two enzymes, γ-glutamylcysteine synthetase (γ-GCS), which is the rate-limiting enzyme, and glutathione synthetase [6]. The γ-GCS holoenzyme exists as a dimer composed of a heavy subunit (γ-GCS-HS) and a light subunit (γ-GCS-LS) [7]. The heavy subunit possesses all the catalytic activity [8]. We [11], [12], [13], [14], [15] as well as other investigators [9], [10] have recently shown that the γ-GCS-HS gene is induced in response to variety of agents such as oxidants, phenolic antioxidants, and tumor necrosis factor-α (TNF-α) in alveolar epithelial cells.

A physiological role for GSH as an antioxidant has been described in numerous inflammatory disorders [16]. Chronic obstructive pulmonary disease (COPD) is a condition characterized by airspace inflammation and progressive and largely irreversible airway obstruction [17]. One of the important events in the pathogenesis of COPD is considered to be the creation of an imbalance between oxidants and antioxidants in the lungs [17]. Cigarette smoke, which contains an estimated 10¹⁴ free radicals per puff, is the major etiological factor in the pathogenesis of COPD [18]. The response of antioxidants such as GSH in response to smoking may be a factor in susceptibility to the development of COPD. We have recently shown that cigarette smoke condensate, oxidants, and TNF-α impose oxidative stress, resulting in transient depletion of intracellular GSH followed by rapid induction of glutathione synthesis in alveolar epithelial cells (A549) in vitro [11], [12], [13], [14], [15]. These data are supported by studies showing elevated levels of GSH in ELF in chronic smokers compared with nonsmokers [19], [20]. Preliminary data showed increased expression of γ-GCS-HS messenger RNA (mRNA) in bronchial biopsies in chronic smokers in contrast to low levels of GSH in ELF and decreased γ-GCS-HS mRNA expression in bronchial biopsies after acute smoking [20], [21]. The site of GSH synthesis in the different cell types in the lungs is unknown, however, its regulation in the lungs of smokers and in patients with COPD has not been studied.

In situ hybridization allows the identification of cells expressing γ-GCS-HS mRNA sequences directly on tissue sections and, hence, the precise localization of γ-GCS-HS mRNA expression. In this study, we used in situ hybridization to investigate the expression and localization of γ-GCS-HS mRNA in lung cells and to study differences in the expression of γ-GCS-HS mRNA in lung tissue of smokers with and without COPD. Previously, we have shown an increase in transforming growth factor-β1 (TGF-β₁) expression in bronchiolar and alveolar epithelium in subjects with COPD [22]. TGF-β₁ has been shown to modulate GSH synthesis in lung cells in vitro [23]. We hypothesized that the increased expression of TGF-β₁ in lungs of COPD patients may be one factor that alters GSH synthesis by its regulatory effect on γ-GCS-HS mRNA. We therefore examined the relation between γ-GCS-HS mRNA expression and TGF-β₁ as well as the relation with measurements of airflow obstruction in patients with COPD.