NOX4 activity is determined by mRNA levels and reveals a unique pattern of ROS generation

Lena Serrander; Laetitia Cartier; Karen Bedard; Botond Banfi; Bernard Lardy; Olivier Plastre; Andrzej Sienkiewicz; Lászlo Fórró; Werner Schlegel; Karl-Heinz Krause

doi:10.1042/BJ20061903

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NOX4 activity is determined by mRNA levels and reveals a unique pattern of ROS generation

Journal article

Open access

Peer reviewed

NOX4 activity is determined by mRNA levels and reveals a unique pattern of ROS generation

Lena Serrander, Laetitia Cartier, Karen Bedard, Botond Banfi, Bernard Lardy, Olivier Plastre, Andrzej Sienkiewicz, Lászlo Fórró, Werner Schlegel and Karl-Heinz Krause

Biochemical journal, Vol.406(1), pp.105-114

08/15/2007

DOI: 10.1042/BJ20061903

PMCID: PMC1948990

PMID: 17501721

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Abstract

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 (IC(50)>100 muM). 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 H(2)O(2), whereas superoxide (O(2)(-)) was almost undetectable. Probes that allow detection of intracellular O(2)(-) 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 O(2)(-) 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.

Animals

Cell Line

Cell Membrane - drug effects

Cell Membrane - enzymology

Cytosol - drug effects

Cytosol - enzymology

Electron Spin Resonance Spectroscopy

Enzyme Induction - drug effects

Enzyme Inhibitors - pharmacology

Ethidium - analogs & derivatives

Ethidium - pharmacology

Gene Expression Regulation, Enzymologic - drug effects

Humans

Hydrogen Peroxide - pharmacology

Mice

Mitochondria - drug effects

NAD - metabolism

NADP - metabolism

NADPH Oxidase 4

NADPH Oxidases - genetics

Nitroblue Tetrazolium - pharmacology

Reactive Oxygen Species - metabolism

RNA, Messenger - genetics

RNA, Messenger - metabolism

Superoxides - metabolism

Tetracycline - pharmacology

Time Factors

Details

Title: Subtitle: NOX4 activity is determined by mRNA levels and reveals a unique pattern of ROS generation
Creators: Lena Serrander - University of Geneva
Laetitia Cartier - University of Geneva
Karen Bedard - University of Geneva
Botond Banfi - Roy J. and Lucille A. Carver College of Medicine
Bernard Lardy - Hôpital Albert Michallon
Olivier Plastre - University of Geneva
Andrzej Sienkiewicz - École Polytechnique Fédérale de Lausanne
Lászlo Fórró - École Polytechnique Fédérale de Lausanne
Werner Schlegel - University of Geneva
Karl-Heinz Krause - University of Geneva
Resource Type: Journal article
Publication Details: Biochemical journal, Vol.406(1), pp.105-114
DOI: 10.1042/BJ20061903
PMID: 17501721
PMCID: PMC1948990
NLM abbreviation: Biochem J
ISSN: 0264-6021
eISSN: 1470-8728
Language: English
Date published: 08/15/2007
Academic Unit: Anatomy and Cell Biology; Otolaryngology; Internal Medicine
Record Identifier: 9984284325702771

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