TRAF6 regulates satellite stem cell self-renewal and function during regenerative myogenesis
Sajedah M. Hindi, Ashok Kumar
Sajedah M. Hindi, Ashok Kumar
Published November 30, 2015
Citation Information: J Clin Invest. 2016;126(1):151-168. https://doi.org/10.1172/JCI81655.
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Research Article Muscle biology

TRAF6 regulates satellite stem cell self-renewal and function during regenerative myogenesis

Abstract

Satellite cells are a stem cell population within adult muscle and are responsible for myofiber regeneration upon injury. Satellite cell dysfunction has been shown to underlie the loss of skeletal muscle mass in many acquired and genetic muscle disorders. The transcription factor paired box-protein-7 (PAX7) is indispensable for supplementing the reservoir of satellite cells and driving regeneration in normal and diseased muscle. TNF receptor–associated factor 6 (TRAF6) is an adaptor protein and an E3 ubiquitin ligase that mediates the activation of multiple cell signaling pathways in a context-dependent manner. Here, we demonstrated that TRAF6-mediated signaling is critical for homeostasis of satellite cells and their function during regenerative myogenesis. Selective deletion of Traf6 in satellite cells of adult mice led to profound muscle regeneration defects and dramatically reduced levels of PAX7 and late myogenesis markers. TRAF6 was required for the activation of MAPKs ERK1/2 and JNK1/2, which in turn activated the transcription factor c-JUN, which binds the Pax7 promoter and augments Pax7 expression. Moreover, TRAF6/c-JUN signaling repressed the levels of the microRNAs miR-1 and miR-206, which promote differentiation, to maintain PAX7 levels in satellite cells. We also determined that satellite cell–specific deletion of Traf6 exaggerates the dystrophic phenotype in the mdx (a mouse model of Duchenne muscular dystrophy) mouse by blunting the regeneration of injured myofibers. Collectively, our study reveals an essential role for TRAF6 in satellite stem cell function.

Authors

Sajedah M. Hindi, Ashok Kumar

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Figure 5

Deletion of Traf6 in satellite cells leads to premature differentiation.

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Deletion of Traf6 in satellite cells leads to premature differentiation....
Single myofibers were isolated from EDL muscle of Traf6fl/fl and TRAF6scko mice. Immediately after isolation or after 72h of culturing, myofibers were collected and stained for PAX7 and MyoD. Nuclei were counterstained with DAPI. (A) Representative individual and merged images of freshly isolated and 72h cultured myofibers from Traf6fl/fl and TRAF6scko mice stained with PAX7, MyoD, and DAPI. Scale bars: 20 μm. (B and C) Quantification of number of PAX7+ (B) and MyoD+ (C) cells per myofiber immediately after isolation. Quantification of (D) number of cells per cluster on each myofiber, and (E) percentage of self-renewing (i.e., PAX7+/MyoD–) cells per myofiber following 72h of culturing. Analysis was done using 20–25 myofibers for each mouse at each time point. n = 3 mice in each group for A–E. (F) Traf6+/+ and Traf6–/– myogenic cultures were transfected with vector alone (pcDNA3) or plasmids expressing TRAF6-WT or TRAF6C70A cDNA. After 72h, the cells were pulse-labeled with EdU for 90 minutes and analyzed for the expression of Pax7 protein and EdU incorporation. Nuclei were identified by staining with DAPI. Scale bar: 20 μm. (G–H) Quantification of percentage of PAX7+ cells (G), and PAX7+/EdU+ cells (H) in cultures transfected with vector alone, TRAF6-WT, or TRAF6C70A cDNA. n = 4 in each group. Error bars represent SD. *P < 0.05 (vs. Traf6fl/fl myofiber cultures or Traf6+/+ cultures transfected with vector alone) by unpaired t test (for B–E) and paired t test (for G and H). #P < 0.05 (vs. corresponding Traf6–/– cells transfected with vector alone) by paired t test.