NOX4 activity is determined by mRNA levels and reveals a unique pattern of ROS generation
Résumé
NOX4 is an enigmatic member of the NOX family of ROS-generating NADPH oxidases. NOX4 has a wide tissue distribution, but the physiological function and activation mechanisms are largely unknown, and its pharmacology poorly understood. We have generated cell lines expressing NOX4 upon tetracycline induction. Tetracycline induced a rapid increase in NOX4 mRNA (1h) followed closely (2h) by a release of reactive oxygen species (ROS). Upon tetracycline withdrawal, NOX4 mRNA levels and ROS release decreased rapidly (<24h). In membrane preparations, NOX4 activity was selective for NADPH over NADH and did not require the addition of cytosol. The pharmacological profile of NOX4 was distinct from other NOX isoforms: diphenyl iodonium (DPI) and thioridazine efficiently inhibited the enzyme, while apocynin and gliotoxin, did not (IC50 > 100 µM). The pattern of NOX4-dependent ROS generation was unique: i) ROS release upon NOX4 induction was spontaneous without need for a stimulus, and ii) the type of ROS released from NOX4 expressing cells was hydrogen peroxide, while superoxide was almost undetectable. Probes that allow detection of intracellular superoxide generation yielded differential results: DHE fluorescence and ACP ESR measurements did not detect any NOX4 signal, while a robust signal was observed with NBT. Thus, most likely NOX4 generates superoxide within an intracellular compartment that is accessible to NBT, but not to DHE and ACP. In conclusion, NOX4 has a distinct pharmacology and pattern of ROS generation. The close correlation between NOX4 mRNA and ROS generation might hint towards a function as inducible NOX isoform.
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