NADPH oxidase‐4 mediated ROS generation modulates C2C12 differentiation via ERK1/2 pathway

Abstract

BackgroundNADPH oxidase 4 (Nox4) enzymatic activity is a major source of intracellular reactive oxygen species (ROS) and plays an important role in balancing oxidation‐reduction within skeletal muscle cells. We hypothesize that changes in Nox4 mediated ROS generation affects the proliferation and differentiation of skeletal muscle cells. Further, ROS plays an important role in cell signaling pathways and this is dependent on the concentration of endogenous ROS in the cells.ApproachUsing mouse myogenic (C2C12) cells, Nox4 concentrations were manipulated via either plasmid mediated over‐expression or siRNA silencing resulting in increased or decreased endogenous ROS concentrations, respectively. Real time RT‐PCR and Western blot demonstrated effective modulation of Nox4 isoform and endogenous ROS production was measured using flow‐cytometry. We further quantified Nox4 protein expression and myogenin using immunofluorescence microscopy. ERK1/2 phosphorylation was determined by Western blot.ResultsModulation of Nox4 and endogenous ROS concentrations decreased the expression of myogenic differentiation markers, Myf5, Myogenin, MyoD1 and Pax7. This is associated with a decrease in cell differentiation percentage in the early stages of myogenesis. These changes are associated with altered MAPK pathway activation; specifically phosphorylation of ERK1/2. In both over‐expression and silencing of Nox4, we observed significant decreases in ERK1/2 phosphorylation.ConclusionNox4 activity determines optimum levels of ROS that are important for cellular processes including proliferation and differentiation of C2C12 myoblasts. Changes in myogenic differentiation pathways in response to Nox4 modulation are mediated via MAPK activation.Funding: USDA/NIFA award number 2010–34479‐20715 “Increasing Shelf Life of Agricultural Commodities, ID”