Regulation of dual oxidase hydrogen peroxide synthesis results in an epithelial respiratory burst

Abstract

Redox status is a central determinant of cellular activities and redox imbalance is correlated with numerous diseases. NADPH oxidase activity results in formation of H₂O₂, that, in turn, sets cellular redox status, a key regulator of cellular homeostasis and responses to external stimuli. Hydrogen peroxide metabolism regulates cell redox status by driving changes in protein cysteine oxidation often via cycling of thioredoxin/peroxiredoxin and glutathione; however, regulation of enzymes controlling synthesis and utilization of H₂O₂ is not understood beyond broad outlines. The data presented here show that calcium-stimulated epithelial Duox H₂O₂ synthesis is transient, independent of intracellular calcium renormalization, H₂O₂ scavenging by antioxidant enzymes, or substrate depletion. The data support existence of a separate mechanism that restricts epithelial H₂O₂ synthesis to a burst and prevents harmful changes in redox tone following continuous stimulation. Elucidation of this H₂O₂ synthesis tempering mechanism is key to understanding cellular redox regulation and control of downstream effectors, and this observation provides a starting point for investigation of the mechanism that controls H₂O₂-mediated increases in redox tone.

Keywords: Cell redox status; Duox; Hydrogen peroxide; NADPH oxidase.

Graphical abstract

Fig. 1

Duox activity is terminated without renormalization of intracellular calcium. Panel A, Heat map of NADPH oxidase mRNA expression in human tissues shows DUOX1 and DUOX2 are the major non-phagocytic NOX forms in most epithelial tissues and DUOX1 is the major form in brain. This highlights the potential importance of Duox in these tissues. The data used for these analyses were obtained from the GTEx Portal on 07/27/2020. The Genotype-Tissue Expression (GTEx) Project was supported by the Common Fund of the Office of the Director of the National Institutes of Health, and by NCI, NHGRI, NHLBI, NIDA, NIMH, and NINDS.Panels B-F, NHBE cultures were loaded with Fura-2 and then simultaneously assessed for H₂O₂ synthesis and [Ca²⁺]_i. Panel B, H₂O₂ synthesis progress curve following stimulation with either ATP (100 μM, red circles) or ionomycin/thapsigargin (10 μM/0.1 μM, black squares). Panels C and D, re-expression of data in Panel B as H₂O₂ synthesis rate (black squares) and [Ca²⁺]_i (red circles) determined by Fura-2. Baseline Fura-2 fluorescence was recorded for 30 min before addition of HRP/Amplex red. Ionomycin treatment prevented renormalization of [Ca²⁺]_i but Duox activity returned toward baseline despite elevated [Ca²⁺]_i (Panels B-D, mean ± s.e.m., n = 6, 2 lung donors). Panel E, H₂O₂ synthesis progress curve after sequential stimulation with ATP (100 μM) followed by ionomycin/thapsigargin (10 μM/0.1 μM) stimulation to same culture. Panel F, replot of synthesis rate (black squares) from Panel E and [Ca²⁺]_i (red circles) (n = 3 cultures, 1 lung donor, H₂O₂ synthesis rate after ionomycin n = 1). The ionomycin-stimulated activity demonstrated that neither NADPH availability nor HRP/Amplex Red depletion could account for the observed termination of Duox activity after ATP stimulation.

Fig. 2

Duox activity is regulated by a shift to an inactive form. H₂O₂ synthesis was assayed in intact cells, homogenates, and a membrane fraction from HEK293T cells expressing either DUOX1/DUOXA1α or DUOX2/DUOXA2. Panel A, H₂O₂ synthesis progress curve following addition of ionomycin/thapsigargin (10 μM/0.1 μM) to intact DUOX1 expressing cells loaded with Fura-2. Panel B, replot of panel A data as rate of H₂O₂ synthesis and [Ca²⁺]_i showing a decrease in H₂O₂ synthesis in elevated [Ca²⁺]_i. Panels C and D progress curves and rate plots of DUOX1 cell-free homogenates with and without auranofin (2 μM) using 12 μM Ca²⁺. Panel E and F, progress curves and rate plots of DUOX2 expressing cells±auranofin (2 μM) using 12 μM Ca²⁺. Auranofin inhibition of GPx and TrxR did not prevent apparent loss of enzyme activity. Panel G, rate plots of DUOX2 homogenates and membranes from post-mitochondrial supernatants using 12 μM Ca²⁺. Panel H, progress curves of DUOX2 homogenates assayed in different [Ca²⁺], fit to the equation shown [20]. 95% confidence levels are plotted and are within the symbols and s.e.m. of the data. All data are means ± s.e.m. n = 3.