Abstract
BackgroundMyogenesis is a highly regulated process ending with the formation of myotubes, the precursors of skeletal muscle fibers. Differentiation of myoblasts into myotubes is controlled by myogenic regulatory factors (MRFs) that act as terminal effectors of signaling cascades involved in the temporal and spatial regulation of muscle development. Such signaling cascades converge and are controlled at the level of intracellular trafficking, but the mechanisms by which myogenesis is regulated by the endosomal machinery and trafficking is largely unexplored. The Endosomal Sorting Complex Required for Transport (ESCRT) machinery composed of four complexes ESCRT-0 to ESCRT-III regulates the biogenesis and trafficking of endosomes as well as the associated signaling and degradation pathways. Here, we investigate its role in regulating myogenesis.ResultsWe uncovered a new function of the ESCRT-0 hepatocyte growth factor-regulated tyrosine kinase substrate Hrs/Hgs component in the regulation of myogenesis. Hrs depletion strongly impairs the differentiation of murine and human myoblasts. In the C2C12 murine myogenic cell line, inhibition of differentiation was attributed to impaired MRF in the early steps of differentiation. This alteration is associated with an upregulation of the MEK/ERK signaling pathway and a downregulation of the Akt2 signaling both leading to the inhibition of differentiation. The myogenic repressors FOXO1 as well as GSK3β were also found to be both activated when Hrs was absent. Inhibition of the MEK/ERK pathway or of GSK3β by the U0126 or azakenpaullone compounds respectively significantly restores the impaired differentiation observed in Hrs-depleted cells. In addition, functional autophagy that is required for myogenesis was also found to be strongly inhibited.ConclusionsWe show for the first time that Hrs/Hgs is a master regulator that modulates myogenesis at different levels through the control of trafficking, signaling, and degradation pathways.
Highlights
Myogenesis is a highly regulated process ending with the formation of myotubes, the precursors of skeletal muscle fibers
Hrs expression and distribution are modulated during myoblast differentiation Hrs levels and distribution were evaluated in C2C12 myoblasts cultured in proliferation medium (Pro) and switched to differentiation medium (Diff) to initiate their differentiation into myotubes
Treatment of C2C12 cells with the MG132 proteasome inhibitor significantly restored Hrs protein level at 16 h of differentiation compared to DMSO-treated cells (Fig. 1d). These results indicate that transient decrease of Hrs level at the onset of differentiation is due to proteasomal degradation
Summary
Myogenesis is a highly regulated process ending with the formation of myotubes, the precursors of skeletal muscle fibers. Differentiation of myoblasts into myotubes is controlled by myogenic regulatory factors (MRFs) that act as terminal effectors of signaling cascades involved in the temporal and spatial regulation of muscle development. The tight control of cellular responsiveness to extracellular ligands involves the endocytosis of ligandactivated receptors and their redirection to endosomes or endo-lysosomal compartments In this context, early endosomes and multivesicular bodies (MVBs) are crucial sorting stations that play a key role in the control of the duration of the cellular responsiveness to given ligands by orienting endocytosed complexes to various pathways such as degradation, recycling, scaffolding, sequestration, or catalysis (reviewed in [6]). Different studies revealed that HRS can modulate the autophagic flux [28,29,30]
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