Dual NADPH oxidases DUOX1 and DUOX2 synthesize NAADP and are necessary for Ca ²⁺ signaling during T cell activation

Abstract

The formation of Ca²⁺ microdomains during T cell activation is initiated by the production of nicotinic acid adenine dinucleotide phosphate (NAADP) from its reduced form NAADPH. The reverse reaction—NAADP to NAADPH—is catalyzed by glucose 6-phosphate dehydrogenase (G6PD). Here, we identified NADPH oxidases NOX and DUOX as NAADP-forming enzymes that convert NAADPH to NAADP under physiological conditions in vitro. T cells express NOX1, NOX2, and, to a minor extent, DUOX1 and DUOX2. Local and global Ca²⁺ signaling were decreased in mouse T cells with double knockout of <i>Duoxa1</i> and <i>Duoxa2</i> but not with knockout of <i>Nox1</i> or <i>Nox2.</i> Ca²⁺ microdomains in the first 15 s upon T cell activation were significantly decreased in <i>Duox2^−/−</i> but not in <i>Duox1^−/−</i> T cells, whereas both DUOX1 and DUOX2 were required for global Ca²⁺ signaling between 4 and 12 min after stimulation. Our findings suggest that a DUOX2- and G6PD-catalyzed redox cycle rapidly produces and degrades NAADP through NAADPH as an inactive intermediate.