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 . Myogenesis is highly regulated by cellular, molecular, and particularly hormonal signals that orchestrate cell mobility, cell contact, hormone sensitivity and the expression of muscle regulatory factors (i.e. MyoD, Myf5, myogenin, and MRF5) [2, 3].
Hormone signaling is critical in the regulation of skeletal muscle mass. Mitogenic signals from insulin and insulin-like growth factor (IGF-1) converge on the insulin receptor substrate (IRS) to regulate cell metabolism, protein synthesis, cell survival, and cell growth by activating phosphoinositide 3-kinase (PI3-kinase)/protein kinase B (PKB or AKT) and extracellular signal-regulated kinase (ERK) signaling pathways [4–9]. However, the kinases and the mechanisms that regulate signal transduction through these cascades, as well as the result on myogenesis, are not completely characterized. Specifically, 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]. Importantly, 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 , a target of PI3-kinase and mediator of anabolic and catabolic signaling [10, 11]. PKCθ also regulates skeletal muscle regeneration in vivo  and myogenesis in vitro [14–16], albeit through mechanisms that are not completely understood. Therefore, further investigation into the cellular signaling dynamics regulated by PKCθ will advance our understanding of the cellular and molecular regulation of the myogenic program.
PKC molecules are intracellular serine/threonine kinases expressed by a variety of cell types involved in diverse functions depending on their structure. PKC molecules are classified as either 1) conventional, containing Ca2+ and diacylglycerol/phorbol binding domains, 2) novel, missing the Ca2+ binding domain and 3) atypical, lacking the Ca2+ and diacylglycerol binding domains . PKCθ is a member of the novel family of PKC molecules and is predominantly expressed in hematopoietic  and skeletal muscle cells .
In skeletal muscle, PKCθ regulates, insulin sensitivity [20–22], muscle cell proliferation and differentiation [14, 16, 23], skeletal muscle regeneration , and expression of acetylcholine receptors in the neuromuscular junction [24–26]. Nonetheless, the contribution of PKCθ to myogenesis is controversial. Studies using human  and chick  primary muscle cells showed that PKCθ expression decreases throughout differentiation, a time associated with increased muscle creatine kinase  and desmin  protein levels, both of which support differentiation and myotube formation. PKCθ was not detected in mouse embryonic myoblasts, which were resistant to the inhibitory effects of phorbol esters and transforming growth factor beta (TGF-β) [27, 28] on myotube formation . Genetic forced expression of PKCθ in mouse embryonic myoblasts prevented myotube formation in the presence of TGFβ and phorbol ester . Moreover, mice with dystrophic muscle have improved skeletal muscle regeneration when PKCθ is globally absent . Taken together, these studies support that PKCθ is a negative regulator of myogenesis and skeletal muscle regeneration. Alternatively, primary muscle cell cultures derived from global PKCθ knockout mice and muscle-specific PKCθ kinase-dead mice have demonstrated a requirement for PKCθ in myogenesis and regeneration . Lastly, in C2C12 muscle cells, PKCθ expression remained constant and overexpression of PKCθ did not impair differentiation .
The overall objective of this study was to investigate how PKCθ regulates cell signaling events that contribute to the advancement of the myogenic program. We hypothesized that PKCθ negatively regulates the myogenic program via IRS1. To test this hypothesis we used a short hairpin-RNA (shRNA) to specifically knockdown PKCθ expression in C2C12 cells (PKCθshRNA), an established cell line for investigating the myogenic program [8, 30–32]. We then investigated how reduced PKCθ affected signaling through the classical insulin signaling pathway in addition to the affect on differentiation and fusion of muscle myoblasts. Our data reveal a PKCθ-regulated myogenic pathway involving serine phosphorylation of IRS1 and phosphorylation of ERK1/2 in the control of myoblast differentiation that enhances our understanding of how PKCθ contributes to myogenic signaling.