Dual Oxidase 2 (Duox2) Regulates Pannexin 1-mediated ATP Release in Primary Human Airway Epithelial Cells via Changes in Intracellular pH and Not H2O2 Production

Abstract

ATP is a key regulator of the innate pulmonary host defense by activating purinergic P2Y2 receptors, which promote chloride secretion by calcium-activated Cl Ϫ channels, inhibit Na ϩ absorption by epithelial Na ϩ channels, increase ciliary beat frequency and airway surface liquid volume, and induce mucin release, thereby activating mucociliary clearance (1-4).Although the effects of ATP on airway epithelial cells have been studied widely, Panx1 channels have only been recognized recently to be involved in ATP release in these tissues (5, 6). Mechanical stress has been shown to be one of the prime stimuli to increase the ATP concentration on the airway surface to a concentration sufficient to activate P2Y2 receptors (7). This ATP release was neither dependent on the intracellular calcium concentration, excluding an exocytotic release mechanism, nor caused by the cystic fibrosis transmembrane conductance regulator. In previous studies, we were the first to show that Panx1, an ortholog of the invertebrate innexin, is expressed at the apical membrane of airway epithelia, contributing to ATP release (5).Pannexin proteins form pannexons, which are channels that open at resting membrane potential because of mechanical stress and in response to extracellular ATP when co-expressed with P2Y2 receptors (8, 9). In addition to stimulated ATP release, resting ATP levels on the apical surface reflect a steady state that, depending on its range, can help autoregulate the release or metabolism of ATP.Currently little is known about the physiological regulation of pannexin channels in the airway epithelium. Most studies have been performed on oocytes or transfected HEK cells. These studies demonstrated that ATP itself is a potent regulator by increasing the permeability of Panx1 initially when coexpressed with P2Y2 receptors but, consequently, causing inhibition as a feedback loop (10, 11).The cytokine IFN-␥ is produced in inflammatory airway diseases such as severe asthma (12, 13) and chronic bronchitis with and without airflow obstruction (14). We have shown that IFN-␥ can increase H 2 O 2 production via increased expression of Duox2, a member of the NADPH oxidase gene family, in ALI 2 cultures (15, 16). In addition to H 2 O 2 , Duox also releases cytosolic H ϩ (17), which could contribute to intracellular acidification. Consequently, a decrease in intracellular pH could decrease mucociliary function by direct inhibition of the ciliary beat frequency (18). In fact, Duox is up-regulated in chronic bronchitis and patients with smoke exposure (15,19,20). Preliminary data also suggested that intracellular pH is essential in

Oxidative stress serves as a key checkpoint for IL‐33 release by airway epithelium

“…For example, hBE cells express connexin 43 (Cx43), which serves as a hemichannel to mediate ATP release in the plasma membrane of various cell types . Exposure of hBE cells to oxidants, such as H 2 O 2 , induces PI3K signaling leading to the activation of AKT, which directly phosphorylates Cx43 on Ser‐369 and Ser‐373 residues, thus inducing Cx43 opening . Therefore, ROS induced by allergen exposure could activate AKT‐dependent phosphorylation of Cx43 and trigger initial ATP secretion.…”

Klotho Inhibits Interleukin-8 Secretion from Cystic Fibrosis Airway Epithelia

“…Human bronchial epithelial cells from cystic fibrosis patients (CF-HBEC) were isolated and cultured using the ALI model as described previously 38 , 50 and with IRB approved protocols. Murine tracheal epithelial cells (MTEC) were obtained from wild type mice and mice (SV129 background), heterozygous and null for the klotho gene 20 .…”

Dual oxidase: a novel therapeutic target in allergic disease

“…Because of the considerable cross‐talk and communication between individual cell types involved in these immune responses (Rivera et al ., ), it is likely that DUOX1 and DUOX2 are both engaged in most cases and their relative contributions may differ depending on the type of pathogen or injury. In some cases, such DUOX‐specific events may be antagonistic, as illustrated by the potential suppression of DUOX1 by viral infection or IFN (Grandvaux et al ., ), but may also be cooperative as highlighted by a recent study indicating that DUOX2 can mediate pannexin‐mediated ATP release in response to hypotonic stress (Krick et al ., ), thereby also resulting in activation of DUOX1.…”