Abstract
BackgroundDifferentiation and fusion of skeletal muscle myoblasts into multi-nucleated myotubes is required for neonatal development and regeneration in adult skeletal muscle. Herein, we report novel findings that protein kinase C theta (PKCθ) regulates myoblast differentiation via phosphorylation of insulin receptor substrate-1 and ERK1/2.ResultsIn this study, PKCθ knockdown (PKCθshRNA) myotubes had reduced inhibitory insulin receptor substrate-1 ser1095 phosphorylation, enhanced myoblast differentiation and cell fusion, and increased rates of protein synthesis as determined by [3H] phenylalanine incorporation. Phosphorylation of insulin receptor substrate-1 ser632/635 and extracellular signal-regulated kinase1/2 (ERK1/2) was increased in PKCθshRNA cells, with no change in ERK5 phosphorylation, highlighting a PKCθ-regulated myogenic pathway. Inhibition of PI3-kinase prevented cell differentiation and fusion in control cells, which was attenuated in PKCθshRNA cells. Thus, with reduced PKCθ, differentiation and fusion occur in the absence of PI3-kinase activity. Inhibition of the ERK kinase, MEK1/2, impaired differentiation and cell fusion in control cells. Differentiation was preserved in PKCθshRNA cells treated with a MEK1/2 inhibitor, although cell fusion was blunted, indicating PKCθ regulates differentiation via IRS1 and ERK1/2, and this occurs independently of MEK1/2 activation.ConclusionCellular signaling regulating the myogenic program and protein synthesis are complex and intertwined. These studies suggest that PKCθ regulates myogenic and protein synthetic signaling via the modulation of IRS1and ERK1/2 phosphorylation. Myotubes lacking PKCθ had increased rates of protein synthesis and enhanced myotube development despite reduced activation of the canonical anabolic-signaling pathway. Further investigation of PKCθ regulated signaling may reveal important interactions regulating skeletal muscle health in an insulin resistant state.
Highlights
Differentiation and fusion of skeletal muscle myoblasts into multi-nucleated myotubes is required for neonatal development and regeneration in adult skeletal muscle
Protein kinase C theta (PKCθ) is a negative regulator of myogenesis in C2C12 muscle cells To determine how the loss of PKCθ affects differentiation and fusion of myoblasts, PKCθshRNA and scramble cells were exposed to differentiation media for 4 days
Our data supports a model in which PKCθ regulates insulin receptor substrate-1 (IRS1) and extracellular signal-regulated kinase1/2 (ERK1/2) signaling that controls myoblast differentiation and protein synthesis
Summary
Differentiation and fusion of skeletal muscle myoblasts into multi-nucleated myotubes is required for neonatal development and regeneration in adult skeletal muscle. We report novel findings that protein kinase C theta (PKCθ) regulates myoblast differentiation via phosphorylation of insulin receptor substrate-1 and ERK1/2. Skeletal muscle development and the regeneration of adult muscle tissue requires the completion of myogenesis: activation, proliferation, differentiation, and fusion of muscle specific stem cells, known as satellite cells [1]. PI3-kinase is a primary regulator of anabolic and catabolic responses that contribute to the maintenance of skeletal muscle mass, and is activated by IRS1 [10,11]. The theta isoform of the protein kinase C family (PKCθ) phospho-inhibits insulin receptor substrate-1 (IRS1) on ser1101 (homologous to ser1095 mouse numbering), suppressing downstream activation of AKT [12], a target of PI3-kinase and mediator of anabolic and catabolic signaling [10,11]. Further investigation into the cellular signaling dynamics regulated by PKCθ will advance our understanding of the cellular and molecular regulation of the myogenic program
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