A 28-kDa Splice Variant of NADPH Oxidase-4 Is Nuclear-Localized and Involved in Redox Signaling in Vascular Cells

Abstract

Objective-Reactive oxygen species-generating nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase proteins (Noxs) are involved in cell differentiation, migration, and apoptosis. Nox4 is unique among Noxs in being constitutively active, and its subcellular localization may therefore be particularly important. In this study, we identified and characterized a novel nuclear-localized 28-kDa splice variant of Nox4 in vascular cells. Approach and Results-Nox4 immunoreactivity was noted in the nucleus and nucleolus of vascular smooth muscle cells and multiple other cell types by confocal microscopy. Cell fractionation, sequence analyses, and siRNA studies indicated that the nuclear-localized Nox4 is a 28-kDa splice variant, Nox4D, which lacks putative transmembrane domains. Nox4D overexpression resulted in significant NADPH-dependent reactive oxygen species production as detected by several different methods and caused increased phosphorylation of extracellular-signal-regulated kinase1/2 and the nuclear transcription factor Elk-1. Overexpression of Nox4D could also induce DNA damage as assessed by γ-H2AX phosphorylation. These effects were inhibited by a single amino acid substitution in the Nox4D NADPH-binding region. Conclusions-Nox4D is a nuclear-localized and functionally active splice variant of Nox4 that may have important pathophysiologic effects through modulation of nuclear signaling and DNA damage. Materials and MethodsMaterials and Methods are available in the online-only Supplement. Results Nuclear-Localized Nox4 ImmunoreactivityWe used a well-validated polyclonal antibody directed against the C-terminal region of Nox4 11,20,21 to investigate the localization of endogenous Nox4 by immunofluorescence in several cell types, including VSMC, endothelial cells, fibroblasts, and cardiomyocytes. A diffuse reticular staining pattern through the cell that colocalized with an ER marker, protein disulfide isomerase, was evident in most cell types ( Figures 1A and 2) as reported previously. 15,16 In addition, however, there was strong focal and granular Nox4 immunostaining within the nucleus in all cell types. Electron microscopy indicated that the focal Nox4 staining was in the nucleolus ( Figure 1C). We confirmed that the focal intranuclear staining was nucleolar by costaining with 2 different nucleolar markers, nucleophosmin and fibrillarin ( Figure 1B). Preincubation of the Nox4 antibody with the antigenic peptide against which it was raised completely abolished both the perinuclear and nucleolar staining, verifying the specificity of antigen-antibody interaction ( Figure 1D). Furthermore, treatment with a prevalidated Nox4 siRNA to knock down Nox4 protein expression also abolished staining as compared with a scrambled siRNA ( Figure 3A). Nuclear-Localized Nox4 Is a 28-kDa Nox4 Splice VariantTo confirm nuclear and nucleolar localization, VSMCs were fractionated into membrane and nuclear fractions. Immunoblotting for Nox4 demonstrated an ≈65-kDa band corresponding to the expected molecular size of Nox4 but ...

NADPH Oxidase-generated Hydrogen Peroxide Induces DNA Damage in Mutant FLT3-expressing Leukemia Cells

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“…Up to now, NOX4 have been localized to mitochondria (41,42), nucleus (31)(32)(33), ER (43)(44)(45)(46), and plasma membrane (45). Here for the first time, we localize NOX4 expression in FLT3-ITD expressing cells.…”

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