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APC is required for muscle stem cell proliferation and skeletal muscle tissue repair.

Parisi A, Lacour F, Giordani L, Colnot S, Maire P, Le Grand F - J. Cell Biol. (2015)

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.

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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.

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APC controls satellite cell proliferation and survival by dampening Wnt/β-catenin signaling. (A–C) RT-qPCR analysis of gene expression in proliferating primary myoblasts after siRNA treatment. Apc and β-catenin levels (A) show efficient reduction of the transcription of genes targeted by siRNA, whereas Axin2 levels (B) reveal that Wnt/β-catenin overactivation is blunted in APC;β-catenin double silenced cells. Cdkn1a induction and concomitant decrease of cdc6 and chek1 expression after APC knockdown reflect a G1/S arrest (C). (D) Quantification of BrdU+ primary myoblasts 48 h after siRNA transfection. (E) Distribution of primary myogenic cells in each cell cycle phase assessed by quantification of propidium iodide (PI) incorporation 48 h after siRNA transfection. P-value refers to S phase. (F) Quantification of apoptotic index by TUNEL staining on primary myoblasts 48 h after siRNA transfection. (G) TA cryosections 14 d after injury immunostained for Pax7 and Laminin. Nuclei were stained with Hoechst. Bars, 50 µm. Inset panels show 2× enlargements. (H) Quantification of sublaminar Pax7+ cells 14 d after injury (normalized by Pax7+ cells of noninjured TA). (I) Quantification of TA muscle fibers cross-sectional area (CSA) 14 d after injury. N.D., not determined. Error bars indicate SEM.
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fig4: APC controls satellite cell proliferation and survival by dampening Wnt/β-catenin signaling. (A–C) RT-qPCR analysis of gene expression in proliferating primary myoblasts after siRNA treatment. Apc and β-catenin levels (A) show efficient reduction of the transcription of genes targeted by siRNA, whereas Axin2 levels (B) reveal that Wnt/β-catenin overactivation is blunted in APC;β-catenin double silenced cells. Cdkn1a induction and concomitant decrease of cdc6 and chek1 expression after APC knockdown reflect a G1/S arrest (C). (D) Quantification of BrdU+ primary myoblasts 48 h after siRNA transfection. (E) Distribution of primary myogenic cells in each cell cycle phase assessed by quantification of propidium iodide (PI) incorporation 48 h after siRNA transfection. P-value refers to S phase. (F) Quantification of apoptotic index by TUNEL staining on primary myoblasts 48 h after siRNA transfection. (G) TA cryosections 14 d after injury immunostained for Pax7 and Laminin. Nuclei were stained with Hoechst. Bars, 50 µm. Inset panels show 2× enlargements. (H) Quantification of sublaminar Pax7+ cells 14 d after injury (normalized by Pax7+ cells of noninjured TA). (I) Quantification of TA muscle fibers cross-sectional area (CSA) 14 d after injury. N.D., not determined. Error bars indicate SEM.

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.


APC is required for muscle stem cell proliferation and skeletal muscle tissue repair.

Parisi A, Lacour F, Giordani L, Colnot S, Maire P, Le Grand F - J. Cell Biol. (2015)

APC controls satellite cell proliferation and survival by dampening Wnt/β-catenin signaling. (A–C) RT-qPCR analysis of gene expression in proliferating primary myoblasts after siRNA treatment. Apc and β-catenin levels (A) show efficient reduction of the transcription of genes targeted by siRNA, whereas Axin2 levels (B) reveal that Wnt/β-catenin overactivation is blunted in APC;β-catenin double silenced cells. Cdkn1a induction and concomitant decrease of cdc6 and chek1 expression after APC knockdown reflect a G1/S arrest (C). (D) Quantification of BrdU+ primary myoblasts 48 h after siRNA transfection. (E) Distribution of primary myogenic cells in each cell cycle phase assessed by quantification of propidium iodide (PI) incorporation 48 h after siRNA transfection. P-value refers to S phase. (F) Quantification of apoptotic index by TUNEL staining on primary myoblasts 48 h after siRNA transfection. (G) TA cryosections 14 d after injury immunostained for Pax7 and Laminin. Nuclei were stained with Hoechst. Bars, 50 µm. Inset panels show 2× enlargements. (H) Quantification of sublaminar Pax7+ cells 14 d after injury (normalized by Pax7+ cells of noninjured TA). (I) Quantification of TA muscle fibers cross-sectional area (CSA) 14 d after injury. N.D., not determined. Error bars indicate SEM.
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fig4: APC controls satellite cell proliferation and survival by dampening Wnt/β-catenin signaling. (A–C) RT-qPCR analysis of gene expression in proliferating primary myoblasts after siRNA treatment. Apc and β-catenin levels (A) show efficient reduction of the transcription of genes targeted by siRNA, whereas Axin2 levels (B) reveal that Wnt/β-catenin overactivation is blunted in APC;β-catenin double silenced cells. Cdkn1a induction and concomitant decrease of cdc6 and chek1 expression after APC knockdown reflect a G1/S arrest (C). (D) Quantification of BrdU+ primary myoblasts 48 h after siRNA transfection. (E) Distribution of primary myogenic cells in each cell cycle phase assessed by quantification of propidium iodide (PI) incorporation 48 h after siRNA transfection. P-value refers to S phase. (F) Quantification of apoptotic index by TUNEL staining on primary myoblasts 48 h after siRNA transfection. (G) TA cryosections 14 d after injury immunostained for Pax7 and Laminin. Nuclei were stained with Hoechst. Bars, 50 µm. Inset panels show 2× enlargements. (H) Quantification of sublaminar Pax7+ cells 14 d after injury (normalized by Pax7+ cells of noninjured TA). (I) Quantification of TA muscle fibers cross-sectional area (CSA) 14 d after injury. N.D., not determined. Error bars indicate SEM.
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.

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.

View Article: PubMed Central - HTML - PubMed

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.

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Related in: MedlinePlus