Protein O-Fucosyltransferase 1 Expression Impacts Myogenic C2C12 Cell Commitment via the Notch Signaling Pathway

Abstract

The Notch signaling pathway plays a crucial role in skeletal muscle regeneration in mammals by controlling the transition of satellite cells from quiescence to an activated state, their proliferation, and their commitment toward myotubes or self-renewal. O-fucosylation on Notch receptor epidermal growth factor (EGF)-like repeats is catalyzed by the protein O-fucosyltransferase 1 (Pofut1) and primarily controls Notch interaction with its ligands. To approach the role of O-fucosylation in myogenesis, we analyzed a murine myoblastic C2C12 cell line downregulated for Pofut1 expression by short hairpin RNA (shRNA) inhibition during the time course of differentiation. Knockdown of Pofut1 affected the signaling pathway activation by a reduction of the amount of cleaved Notch intracellular domain and a decrease in downstream Notch target gene expression. Depletion in Pax7⁺/MyoD⁻ cells and earlier myogenic program entrance were observed, leading to an increase in myotube quantity with a small number of nuclei, reflecting fusion defects. The rescue of Pofut1 expression in knockdown cells restored Notch signaling activation and a normal course in C2C12 differentiation. Our results establish the critical role of Pofut1 on Notch pathway activation during myogenic differentiation.

ACKNOWLEDGMENTS

This work was supported by a French Ministry of Higher Education and Research doctoral fellowship to A.D.V.

We thank Claire Carrion for immunofluorescence advice and excellent colocalization technical assistance, Mélanie Malhouroux for her help with complementation constructs, Nathalie Duprat for DAPT tests, and Rodney O'Connor for his careful assistance in the English drafting of the manuscript.