Activation of Dual Oxidases Duox1 and Duox2

Sabrina Rigutto,Candice Hoste,Helmut Grasberger,Milutin Milenkovic,David Communi,Jacques E Dumont,Bernard Corvilain,Françoise Miot,Xavier De Deken

doi:10.1074/jbc.m806893200

Abstract

Dual oxidases were initially identified as NADPH oxidases producing H(2)O(2) necessary for thyroid hormone biosynthesis. The crucial role of Duox2 has been demonstrated in patients suffering from partial iodide organification defect caused by bi-allelic mutations in the DUOX2 gene. However, the Duox1 function in thyroid remains elusive. We optimized a functional assay by co-expressing Duox1 or Duox2 with their respective maturation factors, DuoxA1 and DuoxA2, to compare their intrinsic enzymatic activities under stimulation of the major signaling pathways active in the thyroid in relation to their membrane expression. We showed that basal activity of both Duox isoenzymes depends on calcium and functional EF-hand motifs. However, the two oxidases are differentially regulated by activation of intracellular signaling cascades. Duox1 but not Duox2 activity is stimulated by forskolin (EC(50) = 0.1 microm) via protein kinase A-mediated Duox1 phosphorylation on serine 955. In contrast, phorbol esters induce Duox2 phosphorylation via protein kinase C activation associated with high H(2)O(2) generation (phorbol 12-myristate 13-acetate EC(50) = 0.8 nm). These results were confirmed in human thyroid cells, suggesting that Duox1 is also involved in thyroid hormonogenesis. Our data provide, for the first time, detailed insights into the mechanisms controlling the activation of Duox1-2 proteins and reveal additional phosphorylation-mediated regulation.

Highlights

Dual oxidases (Duox1 and Duox2) belong to the family of NADPH oxidases (Nox), which is composed of five additional enzymes: Nox1–5 [1,2,3]
Additional mechanisms governing their intrinsic activity are different; Duox1 is positively regulated by the cAMP-dependent protein kinase A (PKA)6 cascade, whereas Duox2 is highly induced by activation of protein kinase C (PKC) with very low concentrations of PMA
We first validated our heterologous system: 1) Measurement of H2O2 produced after 1 ␮M ionomycin stimulation of cells transfected with Duox/DuoxA was proportional to the quantity of transfected Duox plasmids (25–500 ng) with a plateau reached at 250 –500 ng of Duox DNA probably caused by a limited amount of DuoxA (Fig. 1A). 2) We observed a linear relationship between Duox-mediated H2O2 generation and the membrane expression of Duox as measured by FACS (Fig. 1B)

Summary

Introduction

Dual oxidases (Duox1 and Duox2) belong to the family of NADPH oxidases (Nox), which is composed of five additional enzymes: Nox1–5 [1,2,3]. The originality of our study is to analyze the specific activity of Duox1 and Duox2 isoenzymes by normalizing the hydrogen peroxide production to the cell surface expression of the respective proteins. In contrast to Duox1, raising calcium concentration was not sufficient to activate Duox2 by Fsk. In all of the conditions, 1 ␮M diphenyleneiodonium, a flavoprotein inhibitor, reduced the H2O2 level produced by the two enzymes, indicating that it derives from an NADPH oxidase (data not shown).

Results

Conclusion