ABSTRACT
BACKGROUND/AIMS: The complicated differentiation processes of cells in skeletal muscle against inflammation that induce muscle atrophy are not fully elucidated. Given that skeletal muscle is a secretory organ, we evaluated the effects of inflammation on myogenic signals and myokine expression, and the roles of inflammatory exosomes released by myotubes in myogenic differentiation. METHODS: Inflammation was induced by treatment of fully differentiated C2C12 myotubes with a cytokine mixture of TNF-α and INF-γ. Exosome-like vesicles (ELVs) were isolated from conditioned media of control or inflamed myotubes and incubated with myoblasts. The expression of molecular switches that contribute to myogenic differentiation, including several kinases, their downstream targets, and myokines, were evaluated using immunoblot analysis in inflamed myotubes and in myoblasts treated with ELVs. RESULTS: Inflammation activated molecular mechanisms contributing to muscle atrophy, including AMPK, p-38 MAPK and JNK, while inhibiting Akt-mediated myogenic signals. In addition, inflammation induced myostatin expression with suppression of a myostatin-counteracting myokine, decorin. Well-characterized ELVs released from inflamed myotubes induced myoblast inflammation and inhibited myogenic mechanisms while stimulating atrophic signals. CONCLUSION: Inflammation of skeletal muscle induces muscle atrophy via multiple mechanisms, including the regulation of myokines and kinases. Inflammatory ELVs are likely to contribute to inflammation-induced muscle atrophy.
Subject(s)
Cell Differentiation , Cell-Derived Microparticles/metabolism , MyoD Protein/metabolism , Myostatin/metabolism , AMP-Activated Protein Kinases/metabolism , Animals , Autophagy-Related Proteins/metabolism , Cell Line , Cytokines/pharmacology , Decorin/metabolism , Gene Expression Regulation , JNK Mitogen-Activated Protein Kinases/metabolism , Mice , Muscle Fibers, Skeletal/cytology , Muscle Fibers, Skeletal/metabolism , Myoblasts/cytology , Myoblasts/metabolism , Myogenin/metabolism , Phosphorylation/drug effects , Proto-Oncogene Proteins c-akt/metabolism , Signal Transduction/drug effects , p38 Mitogen-Activated Protein Kinases/metabolismABSTRACT
A potassium channel Kir2.1-associated membrane hyperpolarization is required for myogenic differentiation. However the molecular regulatory mechanisms modulating Kir2.1 channel activities in early stage of myogenesis are largely unknown. A cell surface protein, Cdo functions as a component of multiprotein cell surface complexes to promote myogenesis. In this study, we report that Cdo forms a complex with Kir2.1 during myogenic differentiation, and is required for the channel activity by enhancing the surface expression of Kir2.1 in the early stage of differentiation. The expression of a constitutively active form of the upstream kinase for p38MAPK, MKK6(EE) can restore Kir2.1 activities in Cdo-depleted C2C12 cells, while the treatment with a p38MAPK inhibitor, SB203580 exhibits a similar effect of Cdo depletion on Kir2.1 surface expression. Furthermore, Cdo-/- primary myoblasts, which display a defective differentiation program, exhibit a defective Kir2.1 activity. Taken together, our results suggest that a promyogenic Cdo signaling is critical for Kir2.1 activities in the induction of myogenic differentiation.
Subject(s)
Cell Adhesion Molecules/metabolism , Cell Differentiation , Myoblasts/metabolism , Potassium Channels, Inwardly Rectifying/metabolism , Animals , Blotting, Western , Cell Adhesion Molecules/genetics , Cell Line , Cell Membrane/metabolism , Cells, Cultured , Enzyme Inhibitors/pharmacology , Imidazoles/pharmacology , MAP Kinase Kinase 6/metabolism , Mice, Knockout , Microscopy, Confocal , Myoblasts/cytology , Potassium Channels, Inwardly Rectifying/genetics , Protein Binding , Pyridines/pharmacology , RNA Interference , Signal Transduction , p38 Mitogen-Activated Protein Kinases/antagonists & inhibitors , p38 Mitogen-Activated Protein Kinases/metabolismABSTRACT
KCNQ channels are critical determinants of neuronal excitability, thus emerging as a novel target of anti-epileptic drugs. To date, the mechanisms of KCNQ channel modulation have been mostly characterized to be inhibitory via Gq-coupled receptors, Ca(2+)/CaM, and protein kinase C. Here we demonstrate that methylation of KCNQ by protein arginine methyltransferase 1 (Prmt1) positively regulates KCNQ channel activity, thereby preventing neuronal hyperexcitability. Prmt1+/- mice exhibit epileptic seizures. Methylation of KCNQ2 channels at 4 arginine residues by Prmt1 enhances PIP2 binding, and Prmt1 depletion lowers PIP2 affinity of KCNQ2 channels and thereby the channel activities. Consistently, exogenous PIP2 addition to Prmt1+/- neurons restores KCNQ currents and neuronal excitability to the WT level. Collectively, we propose that Prmt1-dependent facilitation of KCNQ-PIP2 interaction underlies the positive regulation of KCNQ activity by arginine methylation, which may serve as a key target for prevention of neuronal hyperexcitability and seizures.
