APC is required for muscle stem cell proliferation and skeletal muscle tissue repair.
Bottom Line: Here we show that conditional genetic disruption of APC in adult muscle stem cells results in the abrogation of adult muscle regenerative potential.By using double knockout strategies, we further prove that this phenotype is attributable to overactivation of β-catenin signaling.Our results demonstrate that in muscle stem cells, APC dampens canonical Wnt signaling to allow cell cycle progression and radically diverge from previous observations concerning stem cells in actively self-renewing tissues.
Affiliation: Institut Cochin, Université Paris-Descartes, Centre National de la Recherche Scientifique (CNRS), UMR 8104, 75014 Paris, France Institut National de la Santé et de la Recherché Médicale (INSERM) U1016, 75014 Paris, France.Show MeSH
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Mentions: To test whether the phenotype of APC mutant cells was a direct consequence of canonical Wnt signaling overactivation, we simultaneously silenced APC and inhibited the Wnt/β-catenin pathway by combining APC and β-catenin siRNAs in vitro (Fig. 4 A). Using this strategy, we were able to completely abrogate transcriptional activation of the Wnt target gene Axin2 transcription by concomitant APC and β-catenin silencing (Fig. 4 B). We further observed the rescue of adequate G1-S checkpoint gene expression in APC;β-catenin double-silenced cells compared with cells only transfected with APC-targeting siRNA (Fig. 4 C). Strikingly, while simple APC silencing blocked BrdU incorporation and decreased the percentage of S-phase cells in proliferating primary myoblasts, silencing of both APC and β-catenin restored normal cell cycle progression (Fig. 4, D and E). In this experimental setup, we observed that the induction of programmed cell death in primary myoblasts after APC silencing requires β-catenin, as quantified by TUNEL assay (Fig. 4 F). To determine whether β-catenin inactivation could compensate for APC loss in vivo, we conditionally deleted both genes in adult satellite cells. We used the Pax7CreERT2 allele to recombine both APC floxed alleles along with one (SC APC-KO; βcat-HET) or two (SC APC-KO; βcat-KO) β-catenin floxed alleles (Brault et al., 2001) in adult mice. Strikingly, cardiotoxin-induced muscle regeneration was completely restored in TA muscles with APC;β-catenin double inactivated satellite cells (Fig. 4 G), as assessed by quantification of satellite cell numbers (Fig. 4 H) and regenerated fiber size (Fig. 4 I) 2 wk after injury. Importantly, recombination of only one β-catenin allele resulted in a partial rescue of APC genetic disruption. In this context, muscle regeneration occurred but satellite cells could not repopulate their niche, and the regenerated myofibers were smaller as compared with single or double inactivated TAs. These data demonstrate that cell cycle arrest and apoptosis after APC inactivation are triggered by β-catenin overactivation. Collectively, these experiments demonstrate that specific levels of canonical Wnt signaling are the major determinant of the observed phenotype in APC-SC-KO mice.
Affiliation: Institut Cochin, Université Paris-Descartes, Centre National de la Recherche Scientifique (CNRS), UMR 8104, 75014 Paris, France Institut National de la Santé et de la Recherché Médicale (INSERM) U1016, 75014 Paris, France.