Functional analysis of Nox4 reveals unique characteristics compared to other NADPH oxidases
Introduction
In recent years it has become evident that the generation of reactive oxygen species (ROS) such as superoxide or hydrogen peroxide is an important cellular mechanism for signal transduction. ROS has been implicated in growth factor receptor signaling, the regulation of various transcription factors involved in proliferation, differentiation and apoptosis, and in GTPase-dependent cytoskeletal rearrangements [1], [2], [3], [4], [5]. In fact, the modification or disruption of signaling pathways involving ROS potentially contributes to human diseases, including vascular disease [6], [7] and cancer [8]. Therefore, it is of great importance to identify the molecular sources for the production of ROS, and understand their regulation.
Currently one of the best characterized sources for ROS production is the oxidative burst generated by phagocytic cells. Upon exposure to invading organisms, phagocytic cells stimulate a NADPH oxidase that catalyzes the one electron reduction of oxygen to superoxide using NADPH as an electron donor [9]. Superoxide can be dismutated to form hydrogen peroxide and other secondary metabolites which, along with the superoxide, are used as microbicidal agents during phagocytosis of pathogens. The multimeric oxidase responsible for host defense is cytochrome b558, which is comprised of the membrane-bound subunits Nox2 (gp91phox) and p22phox, the cytosolic components p47phox, p67phox, p40phox and the small GTPase Rac. Upon stimulation activation of Rac occurs and the cytosolic components undergo conformational changes mediated by phosphorylation. After translocation to the plasma membrane these proteins associate with the heterodimer Nox2/p22phox to facilitate electron transfer and the reduction of O2 to O2− [9], [10], [11]. Most of our knowledge about the molecular components comprising this ROS source, and how these components work together to generate superoxide, is derived from studying genetic mutations in patients suffering from X-linked chronic granulomatous disease (CGD) and from cell-free recombination studies. CGD is a life threatening disease caused by the lack of oxidant generation and the subsequent failure to mount an effective host defense against invading bacteria and fungi [12].
Recent efforts to identify oxidases responsible for ROS generation in non-phagocytic cells led to the discovery of a family of proteins with homology to specific structural regions of Nox2 [13], [14], [15], [16], [17]. These proteins have been termed Nox (NADPH oxidases) and their regulation and biological significance are just beginning to be unraveled. At the DNA level Nox1 is 58% identical to Nox2 and has been implicated in cell transformation [18], [19], MAP-kinase activation [20], and vascular angiogenesis [21]. Nox5 shows overall 27% identity to Nox2 and may be involved in growth and apoptosis of prostate cancer cells [22]. Nox4 has 35% identity to Nox2 and was initially characterized as a kidney NADPH oxidase that might be involved in oxygen sensing or the induction of cellular senescence [23], [24]. Nox4 has since been implicated in a number of vascular pathologies including restenosis [25], diabetic nephropathy [26], and mesangial cell hypertrophy leading to fibrosis of the glomerular microvascular bed [27].
Although knowledge of Nox4 gene expression and its association with biological systems and pathologies is rapidly growing, almost nothing is known about the regulation of Nox4 function at the molecular level. The structural similarities that exist between Nox4 and Nox2 suggest certain common mechanisms of regulation. Nox4 and Nox2 have similar hydrophobicity profiles, suggesting similar arrangement of the transmembrane spanning domains. Nox isoforms have conserved cytoplasmic NADPH and FAD binding domains, as well as four conserved histidines, located in two putative transmembrane domains, which in Nox2 are thought to be required for heme binding and stabilization of p22phox [10], [11], [12]. Numerous unresolved questions remain including whether ROS generation by Nox4 requires assembly with cytosolic factors, as shown for Nox2 and recently for Nox1 [28], [29], [30], or whether Nox4 utilizes a membrane-bound p22phox subunit, also a required element for Nox2 function, or whether Nox4 is regulated by the GTPase Rac. To address these questions, we studied Nox4 function using stably and transiently expressed Nox4 and Nox4 mutants in several epithelial cell lines. We focused on comparing Nox4 to Nox2, and used Nox4 mutant proteins to uncover structural features that may contribute to certain Nox4 functions.
We report here that expression of Nox4 in epithelial cells leads to constitutive ROS production which is most likely generated on internal membranes. ROS production by Nox4 is independent of characterized cytoplasmic oxidase components and seems not to require the Rac1 GTPase. However, ROS production by Nox4 is dependent on p22phox protein. Furthermore, our data show that the presence of Nox4 has a considerable effect on the localization and expression of endogenous p22phox protein.
Section snippets
Construction of Nox4 expression plasmids
Human Nox4 in pcDNA3.0 was modified by adding a Myc epitope to the N-terminus. To generate a Nox4 plasmid for stable expression in HEK293 cells, Nox4 cDNA was transferred into pcDNA3.1(+)hygromycin. N-terminal (EGFP-Nox4) and C-terminal (Nox4-EGFP) EGFP tagged Nox4 were obtained by subcloning the Nox4 cDNA into pEGFPC1 and pEGFPN1 (Clontech), respectively. The Nox4 mutant Nox4-GT was generated by inserting six nucleotides into the Nox4 sequence at position 912 directly after the last predicted
Constitutive ROS generation in HEK293 cell lines stably expressing Nox4
To analyze the Nox4 protein and its function we generated clones of HEK293 cells stably expressing Myc-tagged human Nox4 protein or empty vector control. We chose a parental HEK293 cell line that was consistently negative for Nox4 mRNA. Several hygromycin resistant colonies were selected and tested for the production of H2O2 using the oxidation of homovanillic acid (HVA) as a readout. Analysis of two Nox4-expressing clones, Nox4-2 and Nox4-11, along with two vector controls (EV-1 and EV-6) is
Discussion
Novel NADPH oxidases have been recently identified in epithelial and endothelial cells. Except for their homology to the Nox2 subunit of cytochome b558 of the phagocytic NADPH oxidase and their potential role in intracellular signaling and certain diseases, very little is known about their regulation and their structure-function relationship. To gain mechanistic insights into the regulation of Nox homologs and to uncover structural determinants that affect oxidase function, we investigated Nox4
Acknowledgements
We thank the Lambeth laboratory for kindly providing Nox4 antibody and plasmid, Tom Leto for human NOXO1 and NOXA1 constructs, Mark Quinn, Dirk Roos, Al Jesaitis and John Curnutte for reagents, Greg Hannon for the pGEM-U6 plasmid and advice, and Marc Symons for Rac1 RNAi suggestions. We also acknowledge Katrina Schreiber for excellent administrative assistance and Dorian McGavern, Malcolm Woods, and Becky Diebold for advice and discussions. This work was supported by NIH grants GM37696,
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These authors contributed equally to this work.