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Over-expression of Duox1 in thyroid cell lines using the lentivirus technology

par De Deken, Xavier ;Rigutto, Sabrina ;Hoste, Candice ;Milenkovic, Milutin ;Miot, Françoise
Référence Gordon Research Conferences:NOX family NADPH oxidases (2006-10-15: Les Diablerets, Suisse)
Publication Non publié, 2006

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Résumé :

Thyroid cells generate hydrogen peroxide (H2O2) for metabolic purposes. H2O2 is the essential cofactor for iodide oxidation used for thyroid hormone synthesis. In the thyroid, this H2O2 generating system is constituted of Duox1 and Duox2 enzymes which are particular members of the NADPH oxidase family. Duox proteins possess, as NOX5, calcium binding sites regulating their functional activity. It as been demonstrated that physiological H2O2 production in the thyroid is regulated by calcium but also through the cAMP cascade and the IP3-DAG pathway. Using a heterologous system composed of Cos-7 cells expressing Duox1 or Duox2 in combination with their respective maturation factors (DuoxA1 and DuoxA2), we have shown that agents stimulating the cAMP pathway (forskolin or 6-MB-cAMP) positively regulate Duox1 but not Duox2 activity. Duox1 protein is phosphorylated in resting cells and treatment with cAMP analogues increases its phosphorylation status. In vitro phosphorylation experiments demonstrate direct phosphorylation of Duox1 enzyme by purified protein kinase A (PKA). Analysis of Duox1 primary structure reveals three potential phosphorylation sites in Duox1: S955, T1007 and S1217. Substitution of S955 by alanine abolishes Duox1 activity and decreases its degree of phosphorylation. Mutant S1217A diminished also the amount of phosho-Duox1 but leads to hyperactive enzyme which produces 2 times more H2O2 than the wild type. The double Duox1 mutant (S995A/S1217A) produces comparable quantity of H2O2 as wild type and response to calcium and PMA but not to forskolin. These data clearly demonstrate for the first time that Duox1 but not Duox2 activity is positively regulated by the cAMP cascade through direct PKA phosphorylation on the Serine 955. This specific regulation of Duox1 function could allow the thyroid to control precisely the amount of H2O2 produced depending on physiological needs by acting on one of the two Duox isoenzymes expressed in the thyrocyte.