Dual oxidase 1-dependent MUC5AC mucin expression in cultured human airway epithelial cells

Abstract

Mucus hypersecretion is a prominent manifestation in patients with chronic inflammatory airway diseases. MUC5AC mucin is a major component of airway mucus, and its expression is modulated by a TNF-␣-converting enzyme (TACE)-EGF receptor pathway that can be activated by reactive oxygen species (ROS). Dual oxidase 1 (Duox1), a homologue of glycoprotein p91 phox , is expressed in airway epithelium and generates ROS. We hypothesize that Duox1 activates TACE, cleaving pro-TGF-␣ into soluble TGF-␣, resulting in mucin expression. To examine this hypothesis, we stimulated both normal human bronchial epithelial cells and NCI-H292 airway epithelial cells with phorbol 12-myristate 13-acetate and with human neutrophil elastase. These stimuli induced TACE activation, TGF-␣ release, and mucin expression, effects that were inhibited by ROS scavengers, implicating ROS in TACE activation. Inhibition of epithelial NADPH oxidase or knockdown of Duox1 expression with small interfering RNA prevented ROS generation, TGF-␣ release, and mucin expression by these stimuli, implicating Duox1 in TACE activation and mucin expression. Furthermore, the PKC␦͞PKC inhibitor rottlerin prevented the effects induced by phorbol 12-myristate 13-acetate and human neutrophil elastase, suggesting that PKC␦ and PKC are involved in Duox1 activation. From these results, we conclude that Duox1 plays a critical role in mucin expression by airway epithelial cells through PKC␦͞PKC-Duox1-ROS-TACE-pro-ligand-EGF receptor cascade.EGF receptor ͉ mucus hypersecretion ͉ reactive oxygen species ͉ TNF-␣-converting enzyme ͉ type ␣ TGF

Redox-based regulation of signal transduction: Principles, pitfalls, and promises

“…Various studies have suggested that MMPs and ADAMs can be activated by oxidants or by NO/SNO, which is suggested to involve oxidation and/or S-nitrosylation of the prodomain cysteine residue [137][138][139], although this is generally supported by indirect evidence. Biochemical analysis of oxidative activation of purified MMPs has shown that NO or H 2 O 2 are ineffective at directly activating these proteases, but they can be activated by more potent oxidants such as HOCl or ONOO − [140,141].…”

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

“…Moreover, the airway epithelium also generates airway mucins that facilitate mucociliary clearance of inhaled particulates or microorganisms (103,104). Recent studies suggest that DUOX may participate in these responses as well, as illustrated by DUOX1-mediated production of epithelial mucins, such as MUC1 and MUC5AC, in response to inflammatory mediators such as neutrophil elastase or tumor necrosis factor (TNF)-α (105,106). Similarly, DUOX1 also mediates epithelial production of the neutrophil chemokine IL-8 in response to stimulation by bacterial lipopolysaccharide (LPS) (107).…”

“…4. Critical to the activation of these signaling pathways is the proteolytic cleavage of membrane-associated EGFR ligands by cell surface proteases, such as TNFα-converting enzyme (TACE; ADAM17), a member of the a disintegrin and metalloproteinase (ADAM) family of metzincin metalloproteinases (105,108,121), and matrix metalloproteinases (MMPs) such as gelatinase B (MMP-9), which play critical roles in epithelial host defense and in epithelial repair in response to injury (104,122,123). An important common structural and functional feature shared by the ADAM/MMP proteases is the presence of an invariant cysteine-containing pro-domain, which maintains latency by ligating the active site Zn 2+ , and is susceptible to oxidative disruption (124)(125)(126)(127).…”

“…An important common structural and functional feature shared by the ADAM/MMP proteases is the presence of an invariant cysteine-containing pro-domain, which maintains latency by ligating the active site Zn 2+ , and is susceptible to oxidative disruption (124)(125)(126)(127). Hence, the attractive hypothesis has been put forth that DUOX-derived H 2 O 2 causes direct activation of cell surface MMP/ ADAMs and thereby initiates EGFR-mediated cell signaling (87,105,108) (Fig. 4).…”

“…4) (158,159). Indeed, a number of downstream effects of P2 receptor activation, such as expression of MUC5AC, MMP-9 or IL-8 and stimulated cell migration, have been linked to activation of DUOX1 (87,105,107,108), suggesting a common association of DUOX with epithelial purinergic signaling. Intriguingly, NOX activation has similarly been associated with ATP-mediated purinergic signaling in several other mammalian cell types (160)(161)(162)(163)(164), and in NADPH oxidase-mediated wound responses in plants (165,166), which suggests that the general signaling pathway illustrated in Fig.…”