Abstract
BackgroundDual oxidase maturation factor 1 (DUOXA1) has been associated with the maturation of the reactive oxygen species (ROS) producing enzyme, dual oxidase 1 (DUOX1) in the adult thyroid. However, ROS have also been implicated in the development of several tissues. We found that activated muscle satellite cells and primary myoblasts isolated from mice express robust levels of DUOXA1 and that its levels are altered as cells differentiate.ResultsTo determine whether DUOXA1 levels affect muscle differentiation, we used an adenoviral construct (pCMV5-DUOXA1-GFP) to drive constitutive overexpression of this protein in primary myoblasts. High levels of DUOXA1 throughout myogenesis resulted in enhanced H2O2 production, fusion defects, reduced expression of early (myogenin) and late (myosin heavy chain) markers of differentiation, and elevated levels of apoptosis compared to control cells infected with an empty adenoviral vector (pCMV5-GFP). DUOXA1 knockdown (using a DUOXA1 shRNA construct) resulted in enhanced differentiation compared to cells subjected to a control shRNA, and subjecting DUOXA1 overexpressing cells to siRNAs targeting DUOX1 or apoptosis signal-regulating kinase 1 (ASK1) rescued the phenotype.ConclusionsThis study represents the first to demonstrate the importance of DUOXA1 in skeletal muscle myoblasts and that DUOXA1 overexpression in muscle stem cells induces apoptosis and inhibits differentiation through DUOX1 and ASK1.
Highlights
Dual oxidase maturation factor 1 (DUOXA1) has been associated with the maturation of the reactive oxygen species (ROS) producing enzyme, dual oxidase 1 (DUOX1) in the adult thyroid
We further show that a common regulator of apoptosis, apoptosis signal-regulating kinase 1 (ASK1), is a downstream target of DUOXA1-mediated H2O2 production, and that knockdown of either DUOX1 or ASK1 rescues the DUOXA1 overexpression phenotype
Activated satellite cells and primary myoblasts express DUOXA1 To determine whether muscle satellite cells express DUOXA1, myofibre cultures derived from mouse extensor digitorum muscle were examined by immunofluorescent microscopy
Summary
Dual oxidase maturation factor 1 (DUOXA1) has been associated with the maturation of the reactive oxygen species (ROS) producing enzyme, dual oxidase 1 (DUOX1) in the adult thyroid. The process of myogenesis is often studied using activated satellite cells These muscle stem cells, located between the plasma membrane and the basal lamina, form the basis for effective muscle regeneration [1]. Under appropriate stimuli, these normally quiescent cells enter back into the cell cycle, and undergo several rounds of proliferation. The process of myogenesis is characterized by a small percentage of cells that escape differentiation, maintain Pax expression, downregulate MyoD, and return to quiescence [4] These Pax7+/MyoD- cells are thought to maintain a small pool of muscle stem cells, from which future proliferative myoblasts may be derived. Apoptosis is normally regarded as a natural part of differentiation, and identifying factors involved in cell cycle control and survival undoubtedly play an important role in our general understanding of myogenesis and in the etiology of many muscle degenerative diseases
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