Abstract
Cigarette smoke (CS) exposure and intrinsic factors such as the NADPH oxidases produce high levels of reactive oxygen species (ROS), ensuing inflammatory tissue injury. We previously demonstrated that CS-generated ROS, particularly hydrogen peroxide (H2O2), impaired adenosine stimulated wound repair. We hypothesized that CS exposure modulates expression of Dual oxidase 1 (Duox-1), a NADPH oxidases known to generate H2O2. To test this hypothesis, we used human bronchial epithelial cell line Nuli-1 and C57BL/6 mice. Cells were treated with 5% CS extract (CSE) for various periods of time, and mice were exposed to whole body CS for six weeks. Both CSE and CS treatment induced increased expression of Duox-1, and silencing of Doux-1 improved the rate of cell wound repair induced by CSE treatment. Nuli-1 cells pretreated with thapsigargin but not calcium ionophore exhibited increased Duox-1 mRNA expression. CSE treatment stimulated PKCα activation, which was effectively blocked by pretreatment with diphenylene iodonium, a NADPH oxidase inhibitor. Compared to control, lungs from CS-exposed mice showed a significant increase in PKCα activity and Duox-1 expression. Collectively, the data demonstrated that CS exposure upregulates expression of Duox-1 protein. This further leads to H2O2 production and PKCα activation, inhibiting A2AAR-stimulated wound repair.
Highlights
We previously demonstrated that Cigarette smoke (CS)-generated reactive oxygen species (ROS), H2O2, is implicated in blunting ADO-mediated wound repair of airway epithelial cells[1,2]
We recently demonstrated that CS exposure blunts ADO-mediated activation of cAMP-dependent Protein Kinase A (PKA) and this was facilitated by the robust activation of protein kinase C (PKC) signaling[2]
Pre-exposure of A2B Adenosine Receptor (A2BAR) knock-out Nuli-1 cells stimulated with CGS21680 to CS extract (CSE) or H2O2, decreased transepithelial resistance (TEER) compared to non-pre-exposed cells implying that A2AAR but not A2BAR is the target of inhibitory effect
Summary
We previously demonstrated that CS-generated ROS, H2O2, is implicated in blunting ADO-mediated wound repair of airway epithelial cells[1,2]. The objective of this study was to determine whether CS exposure of airway epithelial cells activates signaling pathways associated the Duox-1 and 2 generation of H2O2. We recently demonstrated that CS exposure blunts ADO-mediated activation of cAMP-dependent Protein Kinase A (PKA) and this was facilitated by the robust activation of PKC signaling[2]. Duox-1 and/or 2, and the subsequent generation of H2O2 activates PKC, which modulates ADO’s airway epithelial cell repair and recovery
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