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. 2008 Dec 5;283(49):34029-36.
doi: 10.1074/jbc.M803012200. Epub 2008 Oct 2.

JAK2/STAT2/STAT3 are required for myogenic differentiation

Kepeng Wang  1 , Chihao Wang, Fang Xiao, Haixia Wang, Zhenguo Wu
Affiliations

JAK2/STAT2/STAT3 are required for myogenic differentiation

Kepeng Wang et al. J Biol Chem. .

Abstract

Skeletal muscle satellite cell-derived myoblasts are mainly responsible for postnatal muscle growth and injury-induced regeneration. However, the cellular signaling pathways that control proliferation and differentiation of myoblasts remain poorly defined. Recently, we found that JAK1/STAT1/STAT3 not only participate in myoblast proliferation but also actively prevent them from premature differentiation. Unexpectedly, we found that a related pathway consisting of JAK2, STAT2, and STAT3 is required for early myogenic differentiation. Interference of this pathway by either a small molecule inhibitor or small interfering RNA inhibits myogenic differentiation. Consistently, all three molecules are activated upon differentiation. The pro-differentiation effect of JAK2/STAT2/STAT3 is partially mediated by MyoD and MEF2. Interestingly, the expression of the IGF2 gene and the HGF gene is also regulated by JAK2/STAT2/STAT3, suggesting that this pathway could also promote differentiation by regulating signaling molecules known to be involved in myogenic differentiation. In summary, our current study reveals a novel role for the JAK2/STAT2/STAT3 pathway in myogenic differentiation.

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Figures

FIGURE 1.
FIGURE 1.
AG490 inhibits myogenic differentiation in both C2C12 and primary myoblasts. Nearly confluent C2C12 cells (A and B) or primary myoblasts (C) were induced to differentiate in DM with either dimethyl sulfoxide (DMSO, vehicle) or different doses of AG490. A, after growing in DM for 24 h (left three panels) or 48 h (right three panels), the cells were fixed and subjected to immunostaining using antibodies against either myogenin or MHC. B, equal amount of WCEs were subjected to immunoblotting to reveal the expression levels of myogenin, MHC, and β-actin (loading control). Con, dimethyl sulfoxide control. C, after growing in DM for 30 h, the primary myoblasts were fixed and subjected to immunostaining for MHC. In A and C, 4′,6′-diamino-2-phenylindole (DAPI) was added to counterstain the nuclei.
FIGURE 2.
FIGURE 2.
Knockdown of JAK2 by siRNA inhibits myogenic differentiation. 50% confluent C2C12 cells (A–D) or primary myoblasts (E) were transfected with either an EGFP-siRNA (control) or JAK2-siRNAs as indicated followed by growth in GM for 24 h. Nearly confluent cells were induced to differentiate in DM for various times as indicated. A, WCEs were subjected to immunoblotting by various antibodies. n.s., nonspecific. B, after cell harvest, total RNA was extracted. The mRNA levels of myogenin were determined by semi-quantitative RT-PCR. GAPDH served as a loading control. C, C2C12 cells were co-transfected with siRNAs and G133-luc. After growth in GM for 24 h and DM for 24 h, the cells were harvested, and WCE were subjected to luciferase assays. Fold change was calculated as the ratio of the luciferase activity of cells transfected with the JAK2-siRNA over that with the EGFP-siRNA. D, C2C12 cells were fixed and subjected to immunostaining for either myogenin (left two columns, DM36 h) or MHC (right two columns, DM48 h). E, primary myoblasts were fixed and subjected to immunostaining for MHC. In D and E, 4′,6′-diamino-2-phenylindole (DAPI) was added to counterstain the nuclei. Among MHC-positive myotubes in three randomly chosen fields (10× magnification), those with 2–3, 4–7, and >8 nuclei were separately counted. The data are presented as the means ± S.D. *, p < 0.05. **, p < 0.01.
FIGURE 3.
FIGURE 3.
Inhibition of JAK2 reduces both the expression levels of MyoD and MEF2 and their transactivating activities. A, C2C12 cells were induced to differentiate for 24 and 48 h in the presence or absence of different doses of AG490. B and C, C2C12 cells were transfected with either the EGFP- or JAK2-siRNA followed by growth in GM for 24 h. The cells were then induced to differentiate for various times as indicated. For A and B, WCEs were subjected to immunoblotting by various antibodies. n.s., nonspecific. For D, mRNA levels of MyoD, MEF2A, and MEF2C were determined by RT-PCR and normalized to that of GAPDH. C and E, C2C12 cells were transfected with various reporter constructs together with either the EGFP- or JAK2-siRNAs followed by growth in GM for 24 h. For C, the cells were induced to differentiate for 18 h before harvest. WCEs were subjected to luciferase assays, and the fold change was determined the same way as that described in the legend for Fig. 2C. For E, after luciferase assays, the remaining WCEs were subjected to immunoblotting to reveal the levels of Gal4 fusion proteins in cells transfected with either the EGFP- or JAK2-siRNA.
FIGURE 4.
FIGURE 4.
Knockdown of either STAT2 or STAT3 inhibits myogenic differentiation. C2C12 cells were transfected with either siRNAs alone (A and B) or siRNAs plus reporter constructs (C) followed by growth in GM for 24 h. The cells were then induced to differentiate for various times as indicated. A, WCEs were subjected to immunoblotting by various antibodies. #1 and #2 denote different sets of siRNA. B, the mRNA levels of myogenin, MEF2A and MEF2C were determined by RT-PCR and normalized to that of GAPDH. C, cells were induced to differentiate for 24 h before harvest followed by luciferase assays. The fold change was determined the same way as that described in the legend for Fig. 2C.
FIGURE 5.
FIGURE 5.
JAK2, STAT2 and STAT3 are activated during myogenic differentiation. Near confluent C2C12 cells were induced to differentiate for various times as indicated. WCEs were subjected to immunoblotting by various antibodies as indicated. To determine the levels of P-JAK2 and P-STAT2, immunoprecipitation (IP) was first performed using our homemade JAK2 and STAT2 antibodies followed by immunoblotting with an antibody against phospho-tyrosine (P-Y).
FIGURE 6.
FIGURE 6.
JAK2/STAT2/STAT3 regulate the expression of IGF2 and HGF at distinct phases of differentiation. C2C12 cells were transfected with different siRNAs as indicated. The cells were induced to differentiate for various times. For F, after growth in GM for 24 h, the cells were starved in serum-free DMEM for 1 h and then treated with or without 50 ng/ml IGF1 for 15 min. For A–C and F, WCEs were subjected to immunoblotting with various antibodies as indicated. For D and E, the mRNA levels of myogenin, HGF, and IGF2 were determined by RT-PCR and normalized to that of GAPDH.
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References

    1. Molkentin, J. D., and Olson, E. N. (1996) Curr. Opin. Genet. Dev. 6 445-453 - PubMed
    1. Sabourin, L. A., and Rudnicki, M. A. (2000) Clin. Genet. 57 16-25 - PubMed
    1. Tapscott, S. J. (2005) Development 132 2685-2695 - PubMed
    1. Black, B. L., and Olson, E. N. (1998) Annu. Rev. Cell Dev. Biol. 14 167-196 - PubMed
    1. McKinsey, T. A., Zhang, C. L., and Olson, E. N. (2002) Trends Biochem. Sci. 27 40-47 - PubMed

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