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

Suresh Acharya, Alexander M. Peters, Ann S. Norton, Gordon K. Murdoch, Rodney A. Hill

Research output: Contribution to journalMeeting Abstractpeer-review


Background NADPH 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.
Approach Using 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.
Results Modulation 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.
Conclusion Nox4 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.
Original languageEnglish
Article number919.1
Pages (from-to)1-1
JournalFASEB Journal
Issue numberS1
Publication statusPublished - Apr 2013


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