TY - JOUR
T1 - NOX4 activity is determined by mRNA levels and reveals a unique pattern of ROS generation
AU - Serrander, Lena
AU - Cartier, Laetitia
AU - Bedard, Karen
AU - Banfi, Botond
AU - Lardy, Bernard
AU - Plastre, Olivier
AU - Sienkiewicz, Andrzej
AU - Fórró, Lászlo
AU - Schlegel, Werner
AU - Krause, Karl Heinz
PY - 2007/8/15
Y1 - 2007/8/15
N2 - NOX4 is an enigmatic member of the NOX (NADPH oxidase) family of ROS (reactive oxygen species)-generating NADPH oxidases. NOX4 has a wide tissue distribution, but the physiological function and activation mechanisms are largely unknown, and its pharmacology is poorly understood. We have generated cell lines expressing NOX4 upon tetracycline induction. Tetracycline induced a rapid increase in NOX4 mRNA (1 h) followed closely (2 h) by a release of ROS. Upon tetracycline withdrawal, NOX4 mRNA levels and ROS release decreased rapidly (< 24 h). 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: DPI (diphenyleneiodonium chloride) and thioridazine inhibited the enzyme efficiently, whereas 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 H2O2, whereas superoxide (O 2-) was almost undetectable. Probes that allow detection of intracellular O2- generation yielded differential results: DHE (dihydroethidium) fluorescence and ACP (1-acetoxy-3-carboxy-2,2,5, 5-tetramethylpyrrolidine) ESR measurements did not detect any NOX4 signal, whereas a robust signal was observed with NBT. Thus NOX4 probably generates O2- within an intracellular compartment that is accessible to NBT (Nitro Blue Tetrazolium), but not to DHE or 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 an inducible NOX isoform.
AB - NOX4 is an enigmatic member of the NOX (NADPH oxidase) family of ROS (reactive oxygen species)-generating NADPH oxidases. NOX4 has a wide tissue distribution, but the physiological function and activation mechanisms are largely unknown, and its pharmacology is poorly understood. We have generated cell lines expressing NOX4 upon tetracycline induction. Tetracycline induced a rapid increase in NOX4 mRNA (1 h) followed closely (2 h) by a release of ROS. Upon tetracycline withdrawal, NOX4 mRNA levels and ROS release decreased rapidly (< 24 h). 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: DPI (diphenyleneiodonium chloride) and thioridazine inhibited the enzyme efficiently, whereas 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 H2O2, whereas superoxide (O 2-) was almost undetectable. Probes that allow detection of intracellular O2- generation yielded differential results: DHE (dihydroethidium) fluorescence and ACP (1-acetoxy-3-carboxy-2,2,5, 5-tetramethylpyrrolidine) ESR measurements did not detect any NOX4 signal, whereas a robust signal was observed with NBT. Thus NOX4 probably generates O2- within an intracellular compartment that is accessible to NBT (Nitro Blue Tetrazolium), but not to DHE or 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 an inducible NOX isoform.
UR - http://www.scopus.com/inward/record.url?scp=34547850670&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=34547850670&partnerID=8YFLogxK
U2 - 10.1042/BJ20061903
DO - 10.1042/BJ20061903
M3 - Article
C2 - 17501721
AN - SCOPUS:34547850670
SN - 0264-6021
VL - 406
SP - 105
EP - 114
JO - Biochemical Journal
JF - Biochemical Journal
IS - 1
ER -