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First published online 30 January 2007
doi: 10.1242/jcs.03371

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IL-13 mediates the recruitment of reserve cells for fusion during IGF-1-induced hypertrophy of human myotubes

Virginie Jacquemin, Gillian Sandra Butler-Browne, Denis Furling^* and Vincent Mouly^*,

UMR S 787, Inserm/UPMC-Paris 6, Institut de Myologie, Paris, France

View larger version (32K): [in this window] [in a new window]   Fig. 1. IGF-1 induces a new wave of myogenic differentiation. (A) Desmin, Pax7, MyoD, myogenin, p57 and subunit of IGF-1 receptor in myotubes and reserve cells were analyzed separately by western blot, after differential trypsinization at day 3 of differentiation. (B) Cultures were treated with IGF-1 at day 3 of differentiation. The content of MyoD, myogenin and p57 in total cultures (myotubes + reserve cells) was evaluated after 24 hours treatment with IGF-1, by western blot analysis. The amount of each factor relative to that of emerin is shown below. *P<0.05, ***P<0.001.

View larger version (29K): [in this window] [in a new window]   Fig. 2. IGF-1 exclusively signals to myotubes but not to reserve cells. (A) The ability of specific inhibitors of the p38 MAPK (SB203580; SB), p42 MAPK (PD098059; PD), and calcineurin (FK506; FK) to prevent the increase in cell fusion induced by IGF-1 was tested, as well as the ability of LiCl, inhibitor of GSK-3, to mimic IGF-1-induced increase in fusion index. Above each column the significance of the sample versus the control (upper) and versus the IGF-treated sample (lower) is given; NS, non significant; **P<0.01 and ***P<0.001. (B) Calcineurin activity was measured in total cultures at day 3 of differentiation after 15 minutes treatment with IGF-1. (C) Activation of p38 MAPK, p42 MAPK and Akt after treatment with IGF-1 was investigated in total cultures at day 3 of differentiation by western blot analysis using antibodies specific for the phosphorylated forms of the molecules. (D) The phosphorylation of Akt, normalized by total Akt, and p42 MAPK was analyzed in myotubes and reserve cells separately, at day 3 of differentiation, after differential trypsinization, by western blot.

View larger version (19K): [in this window] [in a new window]   Fig. 3. A soluble factor is secreted by myotubes in response to IGF-1 treatment and recruits reserve cells for fusion. (A) The ability of conditioned medium from total cultures (myotubes + reserve cells) or reserve cell cultures (obtained after myotube removal by brief trypsinization) treated with IGF-1 to induce an increase in the fusion index was measured. (B) The ability of neutralizing antibodies directed against IL-13, IL-4, IL-15 to prevent the increase in cell fusion induced by IGF-1 was tested. Mouse immunoglobulins were used as a control. Above each column the significance of the sample versus the control (upper) and versus the IGF-treated sample (lower) is given; NS, non significant; **P<0.01 and ***P<0.001. (C) The effects of IGF-1 on the expression of IL-13, IL-4 and IL-15 was investigated in total cultures, by RT-PCR analysis, and normalized with GAPDH.

View larger version (32K): [in this window] [in a new window]   Fig. 4. Treatment with exogenous IL-13 mimics the increase in fusion index and myotube size observed during IGF-1 induced human myotube hypertrophy. (A) The effects of exogenous treatment with IGF-1, IL-4 or IL-13 on the fusion index were determined. (B) The effects of exogenous treatment with IGF-1 and IL-13 at 5 ng/ml on the mean number of nuclei per myotube was measured. NS, non significant; *P<0.05; **P<0.01. (C) Immunostaining using desmin antibody was performed on cultures treated with IGF-1, IL-13 at 10 ng/ml or neutralizing antibody against IL-13. Nuclei were counterstained with Hoechst. Bar, 100 µm.

View larger version (61K): [in this window] [in a new window]   Fig. 5. IGF-1 induces an increase in the DNA binding activity of the NFAT transcription factors and a nuclear translocation of NFATc2. (A) The NFAT DNA binding activity of the cultures treated by IGF-1 was investigated by EMSA using the consensus NFAT sequence from the IL-13 promoter. A competition with the unlabelled competitor oligo, a mutated probe and supershift using panNFAT antibody or antibodies directed against NFATc1, NFATc2, NFATc3 or NFATc4 were performed. (B) Immunostaining using sera directed against NFATc1, NFATc2, NFATc3 or NFATc4 isoforms was performed after IGF-1 treatment. Nuclei were counterstained with Hoechst. *P<0.05.

View larger version (24K): [in this window] [in a new window]   Fig. 6. IGF-1 stimulates protein metabolism via Akt (A) The ability of specific inhibitors of the p38 MAPK (SB=SB203580), p42 MAPK (PD=PD098059), the calcineurin (FK=FK506) and GSK-3 (LiCl) to prevent the increase in myosin content induced by IGF-1 was tested. Above each column the significance of the sample versus the control (upper) and versus the IGF-treated sample (lower) is given; NS, non significant; *P<0.05; **P<0.01. (B) The phosphorylation of Foxo1, p70S6K, GSK-3 and Akt was investigated by western blot analysis after treatment by IGF-1. (C) The effect of IGF-1 on the Foxo DNA binding activity of the cultures was investigated by EMSA. A competition with the unlabelled competitor oligo and a mutated probe were used to assess signal specificity. (D). The effect of IGF-1 on the expression of atrogin-1 was tested by northern blot analysis after a 3-hour treatment. Two mRNA forms of 2.4 kb and 6.5 kb were detected for atrogin-1, as described previously (Li et al., 2005).

View larger version (18K): [in this window] [in a new window]   Fig. 7. Mechanisms of IGF-1-induced human myotube hypertrophy. (1) IGF-1 induces an increase in the protein content of myotubes via Akt. Within Akt targets, IGF-1 acts on the mTOR/p70S6K and GSK-3 pathways, both of which are involved in the control of protein translocation. IGF-1 also downregulates the Foxo/atrogin-1 protein degradation pathway. (2) IGF-1 induces an increase in the recruitment of reserve cells for fusion via the secretion of IL-13 by myotubes under the control of NFATc2.