Regulation of H2O2 generation in thyroid cells does not involve Rac1 activation

Objectives: The H2O2 generating system of the thyrocyte and the O2− generating system of macrophages and leukocytes present numerous functional analogies. The main constituent enzymes belong to the NADPH oxidase (NOX) family (Duox/ThOX for the thyroid and NOX2/gp91phox for the leukocytes and macrophages), and in both cell types, H2O2 generation is activated by the intra-cellular generation of Ca2+ and diacylglycerol signals. Nevertheless, although the controls involved in these two systems are similar, their mechanisms are different. The main factors controlling O2− production by NOX2 are the cytosolic proteins p67phox and p47phox, and Rac, a small GTP-binding protein. We have previously reported that there is no expression of p67phox and p47phox in thyrocytes. Here, we investigated whether Rac1 is an actor in the thyroid H2O2-generating system. Design and methods: Ionomycin- and carbamylcholine-stimulated H2O2 generation was measured in dog thyroid cells pretreated with the Clostridium difficile toxin B, which inhibits Rac proteins. Activation of Rac1 was measured in response to agents stimulating H2O2 production, using the CRIB domain of PAK1 as a probe in a glutathione S-transferase (GST) pull-down assay. Results: Among the various agents inducing H2O2 generation in dog thyrocytes, carbamylcholine is the only one which activates Rac1, whereas phorbol ester and calcium increase alone have no effect, and cAMP inactivates it. Moreover, whereas toxin B inhibits the stimulation of O2 generation by phorbol ester in leukocytes, it does not inhibit H2O2 generation induced by carbamylcholine and ionomycin in dog thyrocytes. Conclusions: Unlike in leukocytes, Rac proteins do not play a role in H2O2 generation in thyroid cells. A different regulatory cascade for the control of H2O2 generation remains to be defined.