Expression of Nox4 NADPH Oxidase Splice Variants Generate Hydrogen Peroxide and Modify the Cell Cycle

Abstract

The NADPH oxidase (Nox) family of proteins generate reactive oxygen species (ROS), which regulate redox‐mediated cell signaling and may lead to oxidative stress. Unlike the other catalytic subunits, Nox4 is mostly constitutively active and regulated by its expression levels. It is reported that Nox4 has splice isoforms, including variant D which retains all FADH and NADPH binding sites of the full length Nox4A, but lacks transmembrane domains. The goal of this study was to characterize expression and function of splice variants Nox4A and Nox4D. We first validated the specificity of qPCR primers used for detection of the splice variants by overexpression of Nox4A and Nox4D in Cos22 cells. Next, we examined expression of these isoforms in cancer cell lines. The relative expression of Nox4A and Nox4D was not significantly different in human epithelial colorectal adenocarcinoma cells (CaCo), human Asian endometrial adenocarcinoma (Ishikawa) glioblastoma (LN229), adenocarcinomic human alveolar basal epithelial (A549) and human umbilical vein endothelial (EA.hy926) cells. We next examined the function of Nox4 splice variants and found that expression of Nox4A and Nox4D both increased ROS levels as compared to control transfected cells. It has been suggested that the subcellular targeting of Nox4A and Nox4D may be different. To test this, we measured hydrogen peroxide levels in the cytosol, mitochondria, and nucleus using the genetically targeted HyPer probes. Whereas Nox4A expression increased ROS in each subcellular compartment of Cos22 cells, Nox4D primarily increased the HyPer signal in the nucleus. We next examined the effect of Nox4 on protein thiol oxidation and found that expression of Nox4A caused oxidation of several protein thiols. Finally, Cos22 cells were transfected with Nox4A, Nox4D, or an empty vector and treated with cytokines. The expression of Nox4 isoforms had a differential effect on the percentage of cells in the G0/G1, S, and G2/M cell cycle phase. In summary, Nox4A and Nox4D splice isoforms are detectable in different cell lines, overexpression of Nox4A and Nox4D increase ROS levels in subcellular organelles and the oxidation of multiple protein thiols, and expression of Nox4A and Nox4D alters the cell cycle. The expression of Nox4 splice isoforms may provide specificity for regulation of subcellular redox signaling.Support or Funding InformationDAS was supported by the American Society for Pharmacology and Experimental Therapeutics (ASPET) sponsored Duke SURPH program. FJM is supported by the Office of Research and Development, Department of Veterans Affairs [2I01BX001729] and the National Institutes of Health [HL130039].This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.