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New Players in Wnt-mediated Control of Muscle Growth and Repair

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A review of “Barx2 and Pax7 have antagonistic functions in regulation of Wnt signaling and satellite cell differentiation” from Stem Cells by Stuart P. Atkinson.

Satellite cells (SCs) are muscle stem cells [1] which are normally quiescent, activating and forming proliferative myoblasts in response to injury. Previous studies have underlined the importance of several factors in the regulation of SC homeostasis, including Pax7 [2], Barx2 [3] and Wnts [4], although much remains unknown. Now in a study in Stem Cells, physical and functional connections have been delineated between Wnt/βcatenin signaling, Barx2, Pax7, and Myogenic regulatory factors (MRFs) towards the understanding of muscle-specific mechanisms of Wnt signaling [5].

It is known that myoblast differentiation is promoted by Barx2 and inhibited by Pax7, so the regulation of both factors was initially assessed in relation to the influence of Wnt3a. Various Wnts are well understood to be influence either proliferation or differentiation of myoblasts [6-8]. During differentiation, Wnt3a promoted the elongation and fusion of myocytes, with no effect on cell proliferation, and was associated with increased Barx2, but not Pax7, mRNA expression. mRNA comparisons between Barx2+/+ and Barx2‐/‐ primary myoblasts found 22 downregulated and 11 upregulated genes in response to loss of Barx2 and, of these, CyclinD1 and Wif1 were at lower levels while MMP9 was higher and Axin2 was unchanged. Forced expression of Barx2 in a mouse myoblast cell line also induced Axin2 and Cyclin D1 levels.

Promoter-reporter constructs found that Barx2 or β‐catenin upregulated Axin2 in growth and differentiation conditions and synergistically boosted expression in differentiation conditions, but not in growth conditions, which was blocked using a dominant negative (dn) TCF4 which cannot recruit β‐catenin. Similarly, Barx2 or β‐catenin induced the cyclinD1 promoter and dnTCF4 blocked activation, although there was no synergy between Barx2 and β‐catenin. Barx2 also activated a TOPflash reporter gene that contains eight TCF/LEF binding sites in a mouse myoblast cell line, with truncation experiments which showed that loss of the C-terminal or a 17 amino acid basic region reduced TOPflash activation, and loss of the homeodomain prevented activation. Additionally mutation of the TCF/LEF sites on the reporter or co-transfection with a dnTCF4 led to the loss of Barx2-mediated activation, together suggesting a requirement for β‐catenin in Barx2‐mediated activation. Barx2-MyoD and MyoD-β‐catenin interactions have been previously described and, in this study, Barx2/MyoD activated TOPflash to a greater level than Barx2 alone, while MyoD alone had no effect. A similar, but less potent, synergistic effect was also seen for Barx2 and Myf5, MRF4 and myogenin. Co-immunoprecipitation assays with Myc-tagged Barx2 identified endogenous β‐catenin, through the homeodomain and the Barx basic regions, and TCF4 as interacting proteins. Following this, chromatin immunoprecipitation assays found that when the TOPflash promoter was activated by Wnt3a, or by Barx2 and MyoD, there was evidence of β‐catenin or Barx2 recruitment to the promoter, respectively. MyoD recruitment was only observed after Barx2/MyoD co‐transfection, and MyoD transfection alone did not induce β‐catenin recruitment. Interestingly, Pax7 expression repressed basal TOPflash activity and also blocked Barx2- and Barx2/MyoD mediated activation of TOPflash, mediated through the Pax7 homeodomain region. It furthermore antagonised the induction of TOPflash by constitutively active β‐catenin or Wnt3a ligand, and attenuated the activation of the Axin2 and CyclinD1 promoters by β‐catenin. Co-immunoprecipitation assays suggested that Pax7 and β‐catenin interact, and there was also evidence for this reaction in primary myoblasts.

Overall, this elegant study has begun to reveal the integration of intrinsic and extrinsic regulators into a framework with which we can better understand the complex mechanisms which regulate muscle growth and repair (see Figure). It identifies Barx2, MyoD, and Pax7 in the regulation of muscle progenitor differentiation as part of the Wnt effector complex, and suggests that the interplay between these factors may control the myoblast proliferation-differentiation switch.

References

  1. Sambasivan R, Yao R, Kissenpfennig A, et al. Pax7-expressing satellite cells are indispensable for adult skeletal muscle regeneration. Development 2011;138:3647-3656.
  2. Olguin HC and Olwin BB Pax-7 up-regulation inhibits myogenesis and cell cycle progression in satellite cells: a potential mechanism for self-renewal. Dev Biol 2004;275:375-388.
  3. Meech R, Gonzalez KN, Barro M, et al. Barx2 is expressed in satellite cells and is required for normal muscle growth and regeneration. Stem Cells 2012;30:253-265.
  4. Tsivitse S Notch and Wnt signaling, physiological stimuli and postnatal myogenesis. Int J Biol Sci 2010;6:268-281.
  5. Zhuang L, Hulin JA, Gromova A, et al. Barx2 and Pax7 have antagonistic functions in regulation of Wnt signaling and satellite cell differentiation. Stem Cells 2014;
  6. Otto A, Schmidt C, Luke G, et al. Canonical Wnt signalling induces satellite-cell proliferation during adult skeletal muscle regeneration. J Cell Sci 2008;121:2939-2950.
  7. Tanaka S, Terada K, and Nohno T Canonical Wnt signaling is involved in switching from cell proliferation to myogenic differentiation of mouse myoblast cells. J Mol Signal 2011;6:12.
  8. Pansters NA, van der Velden JL, Kelders MC, et al. Segregation of myoblast fusion and muscle-specific gene expression by distinct ligand-dependent inactivation of GSK-3beta. Cell Mol Life Sci 2011;68:523-535.