Abstract
Past work has shown that the protein O-fucosyltransferase 1 (POFUT1) is involved in mammal myogenic differentiation program. Pofut1 knockdown (Po –) in murine C2C12 cells leads to numerous elongated and thin myotubes, suggesting significant defects in secondary fusion. Among the few pathways involved in this process, NFATc2/IL-4 is described as the major one. To unravel the impact of POFUT1 on secondary fusion, we used wild-type (WT) C2C12 and Po – cell lines to follow Myf6, Nfatc2, Il-4 and Il-4rα expressions during a 120 h myogenic differentiation time course. Secreted IL-4 was quantified by ELISA. IL-4Rα expression and its labeling on myogenic cell types were investigated by Western blot and immunofluorescence, respectively. Phenotypic observations of cells treated with IL-4Rα blocking antibody were performed. In Po –, we found a decrease in nuclei number per myotube and a downexpression of Myf6. The observed downregulation of Nfatc2 is correlated to a diminution of secreted IL-4 and to the low level of IL-4Rα for reserve cells. Neutralization of IL-4Rα on WT C2C12 promotes myonuclear accretion defects, similarly to those identified in Po –. Thus, POFUT1 could be a new controller of myotube growth during myogenesis, especially through NFATc2/IL-4 signaling pathway.
Highlights
During postnatal skeletal myogenesis, the muscle stem cells called satellite cells play a crucial role in muscle growth, homeostasis and regeneration
To evaluate myonuclear accretion process in C2C12 cells, nuclei number was determined during 120 h of differentiation time course in wild-type (WT) and Pofut1 knockdown (Po –) cell lines; the latter having been created for a previous study [22]
We revealed that the impaired expression of Pofut1 gene encoding enzyme responsible for O-fucosylation of proteins, leads to defects of the secondary fusion process during myogenesis, in particular through NFATc2/interleukin 4 (IL-4) signaling pathway
Summary
The muscle stem cells called satellite cells play a crucial role in muscle growth, homeostasis and regeneration Under activation, these cells commit to a myoblast cell fate and allow cell fusion and fiber formation. The NFATc2/IL-4 (nuclear factor of activated T-cells, cytoplasmic, calcineurin dependent 2/Interleukin-4) pathway is involved in the secondary fusion process [10,11]. It acts on myoblast fusion after the initial formation of MT and is necessary for their growth. An aberrant upregulation of NOTCH signaling pathway [17] and Pax3/7 expression [18] are found in the rhabdomyosarcoma cells to be responsible for the tumor growth. NFATc2 overexpression suppress canonical NOTCH transactivation and expression of HEY (Hairy/Enhancer of Split-related with YRPW motif) genes
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