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

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.

Quinone compounds regulate the level of ROS production by the NADPH oxidase Nox4

“…The specificity of AA-861, duroquinone, and tBuBQ for Nox4 over Nox2, suggests either a direct effect of quinone derivatives in the molecular dynamics of electron transfer mediated by Nox4, or an indirect role on a quinone sensitive functional partner of Nox4. In this work, we suggest a new aspect of Nox4 oxidase activity regulation that takes place not only at a transcriptional level as usually described [13,27,45] but also at a post-translational level as described above with quinone molecules. Quinones are bioreactive molecules that are sensitive to redox mechanism.…”

“…To confirm that the observed effect of AA-861 and tBuBHQ was specifically related to Nox4 proteins, we used two other characterized cell lines that over-expressed Nox4: human chondrocyte C-20/A4 [18] and Nox4 T-REx TM in which Nox4 expression could be temporally induced by the addition of tetracycline [27]. Consistently, the constitutive ROS production in Nox4 C-20/A4 cells or in 4 h-induced Nox4 T-REx cells was enhanced after the addition of AA-861 or tBuBHQ (Fig.…”

“…Nox4 is unique among other Nox isoenzymes in that its activity is constitutive and may depend on a specific conformation of the dehydrogenase DH domain that should allow a spontaneous transfer of electrons from NADPH to FAD and to the hemes [24,25]. Data from various studies indicated clearly that Nox4 activity is regulated at the mRNA level, implying that an increase or decrease of ROS production by Nox4 is correlated to an up regulation [9,16,[26][27][28][29][30][31][32][33] or to a decline [34] of Nox4 transcripts. Although it has not been reported yet, post-translational regulation of Nox4 oxidase activity cannot be excluded.…”

Inhibition of Nox4‐dependent ROS signaling attenuates prostate fibroblast activation and abrogates stromal‐mediated protumorigenic interactions

“…Reliable detection of Nox proteins via immunohistochemistry (IHC) is problematic in many tissues,28 including the prostate (not shown). As Nox4 is constitutively active and primarily regulated at the mRNA level,15, 16 we utilized in situ hybridization (ISH) on prostate tissue sections and a PCa tissue microarray (TMA) to identify the cellular origin and semi‐quantify prostatic Nox4 mRNA levels using a highly specific assay with single mRNA transcript sensitivity (Supporting Information, Fig. S1) 29.…”

“…The Nox family comprises seven members (Nox1–5 and Duox1–2) that catalyze the transfer of electrons from molecular oxygen across biological membranes using NADPH as electron donor thereby generating superoxide. Nox4 is unique amongst Nox enzymes as it is constitutively active with hydrogen peroxide (H 2 O 2 ) being the primary ROS detectable 15, 16. ROS play a central role in fibroblast activation 17.…”