Abstract 15121: Nox4D Is the Major Splice Variant of Nox4 NADPH Oxidase Expressed in Vascular Smooth Muscle Cells
Reactive oxygen species (ROS) are increasingly appreciated as key signalling moities in several pathophysiological conditions. NADPH oxidases (Nox) are enzymes that are involved in regulated production of ROS within defined subcellular compartments and hence are critically involved in cell functions such as proliferation, migration, gene expression and apoptosis. Four members of the Nox enzyme family are expressed in the vasculature: Nox1, Nox2, Nox4, and Nox5. Of these, the 65 kDa isoform of Nox4 has been shown to be constitutively active and to produce H2O2. Splice variants of Nox4 have been reported but their tissue specific expression and functional roles are still unclear. We identified a 28kDa Nox4 splice variant, Nox4D, as the major Nox4 variant expressed in A7r5 vascular smooth muscle cells (VSMC) as well as mouse aortae by RT-PCR and western blot. Using confocal miscroscopy, we localized Nox4D to the nucleus in several cell types including VSMC. The cellular localization of Nox4D was further confirmed by subcellular fractionation and identification of Nox4D in the nuclear fraction by western blot. The specificity of Nox4D protein expression and localization were verified using Nox4 specific siRNA and by competitive inhibition using antigenic peptide. Over-expression of Nox4D in VSMC resulted in increased levels of extracellular H2O2 (7.8μmol/mg protein p< .001) compared to mock transfected cells (2.1μmol/mg protein) and this was comparable to H2O2 levels in cells transfected with full length Nox4 (8.6μmol/mg protein, p<.001 compared to mock). Since full-length Nox4 requires association with a p22phox subunit to form a functional complex, we assessed whether Nox4D has a similar requirement. Using co-immunoprecipitation, we confirmed that Nox4D associates with the p22phox subunit and this is critical for its function. p22phox was also localized to the nuclear compartment by immunofluorescence imaging and western blot of cell fractions.
Conclusion: we have identified Nox4D, a 28 kDa splice variant of Nox4, as the major Nox4 expressed in A7r5 VSMC. Nox4D has a nuclear localization and associates with p22phox to form a functional enzyme complex that produces H2O2 at comparable levels to full length Nox4
- © 2010 by American Heart Association, Inc.