Regulated Hydrogen Peroxide Production by Duox in Human Airway Epithelial Cells

Abstract

Hydrogen peroxide (H(2)O(2)) is found in exhaled breath and is produced by airway epithelia. In addition, H(2)O(2) is a necessary substrate for the airway lactoperoxidase (LPO) anti-infection system. To investigate the source of H(2)O(2) produced by airway epithelia, PCR was used to screen nicotinamide adenine dinucleotide phosphate (NADPH) oxidase expression in human airway epithelia redifferentiated at the air-liquid interface (ALI) and demonstrated the presence of Duox1 and 2. Western blots of culture extracts indicated strong expression of Duox, and immunohistochemistry of human tracheal sections localized the protein to the apical portion of epithelial cells. Apical H(2)O(2) production was stimulated by 100 microM ATP or 1 microM thapsigargin, but not 100 microM ADP. Diphenyleneiodonium, an NADPH oxidase inhibitor, and dimethylthiourea, a reactive oxygen species scavenger, both inhibited this stimulation. ATP did not stimulate the basolateral H(2)O(2) production by ALI cultures. ATP and thapsigargin increased intracellular Ca(2+) with kinetics similar to increasing H(2)O(2) production, and thus consistent with the expected Ca(2+) sensitivity of Duox. These data suggest that Duox is the major NADPH oxidase expressed in airway epithelia and therefore a contributor of H(2)O(2) production in the airway lumen. In addition, the data suggest that extracellular H(2)O(2) production may be regulated by stimuli that raise intracellular Ca(2+).

IFNβ/TNFα synergism induces a non-canonical STAT2/IRF9-dependent pathway triggering a novel DUOX2 NADPH Oxidase-mediated airway antiviral response

“…To date, studies based on the use of antioxidant enzymes support the necessity of H 2 O 2 in the LPOdependent antibacterial defense [38]. Further studies suggest that DUOX2 is a source of H 2 O 2 production in the airways [39,40]. However, none of these studies have clearly demonstrated a functional connection between DUOX2-mediated H 2 O 2 production and LPO-dependent airway antibacterial defenses.…”

NADPH oxidases in lung biology and pathology: Host defense enzymes, and more

“…The fact that ROS production by polarized epithelia occurred only apically and could be stimulated by activators of protein kinase C (82), known to activate the phagocyte NADPH oxidase (5), suggested regulated and compartmentalized epithelial ROS production by an NADPH oxidase. The source of epithelial ROS was identified only a few years ago, after initial discovery of prominent expression of the NOX homologs DUOX1 and DUOX2 within the lung (35), and studies on lung tissues using in situ hybridization and immunohistochemistry, which revealed the presence of DUOX1 at the apical surface of tracheobronchial epithelial cells, and DUOX2 within salivary and submucosal glands (19,79,83,84 (88,89), which is due to the presence of DUOX1 (90). Although DUOX expression in the lung is primarily localized to airway or alveolar epithelial cells, recent studies have also suggest the presence of DUOX proteins in lymphocytes (51,91).…”

“…2+ binding domains within DUOX suggests that it can be directly activated by Ca 2+ -mobilizing stimuli. Indeed, Ca 2+ mobilization by various diverse stimuli were been found to promote apical H 2 O 2 production in airway epithelial cells (10,27,84,133). Moreover, in analogy to activation of NOX2 in phagocytes, H 2 O 2 production by airway epithelial cells or thyrocytes was also stimulated in response to activation of protein kinase C (PKC), and Ca 2+ and PKC stimulation appear to act synergistically in promoting H 2 O 2 production (82,105,134,135).…”

“…Since P2 receptor stimulation induces Ca 2+ mobilization, it is not surprising that extracellular ATP is capable of stimulating DUOX-mediated H 2 O 2 production by airway epithelial cells. This appears to be mediated primarily by P2Y receptor stimulation (84,87), which mediates intracellular Ca 2+ mobilization and activates PKC (Fig. 4) (158,159).…”

Dual Oxidase-2 Has an Intrinsic Ca2+-dependent H2O2-generating Activity

“…DUOX genes have been identified in the thyroid gland, where they are strongly expressed (13,14). However, the DUOX are also expressed on the mucosal surfaces of the trachea and the bronchi (15) and in the airway epithelial cells (16,17), where it has been suggested that Duox1 is the isoform responsible for acid production and secretion in airways (16) and plays a critical role in mucin expression (18). DUOX2 was also expressed throughout the digestive tract, where it was found to be functional (19,20), in addition to the salivary gland and rectum (15).…”