Inhibition of JAK-STAT signaling stimulates adult satellite cell function

Journal name:
Nature Medicine
Volume:
20,
Pages:
1174–1181
Year published:
DOI:
doi:10.1038/nm.3655
Received
Accepted
Published online
Corrected online

Abstract

Diminished regenerative capacity of skeletal muscle occurs during adulthood. We identified a reduction in the intrinsic capacity of mouse adult satellite cells to contribute to muscle regeneration and repopulation of the niche. Gene expression analysis identified higher expression of JAK-STAT signaling targets in 18-month-old relative to 3-week-old mice. Knockdown of Jak2 or Stat3 significantly stimulated symmetric satellite stem cell divisions on cultured myofibers. Genetic knockdown of Jak2 or Stat3 expression in prospectively isolated satellite cells markedly enhanced their ability to repopulate the satellite cell niche after transplantation into regenerating tibialis anterior muscle. Pharmacological inhibition of Jak2 and Stat3 activity similarly stimulated symmetric expansion of satellite cells in vitro and their engraftment in vivo. Intramuscular injection of these drugs resulted in a marked enhancement of muscle repair and force generation after cardiotoxin injury. Together these results reveal age-related intrinsic properties that functionally distinguish satellite cells and suggest a promising therapeutic avenue for the treatment of muscle-wasting diseases.

At a glance

Figures

  1. Increasing age negatively affects the engraftment capacity of satellite cells.
    Figure 1: Increasing age negatively affects the engraftment capacity of satellite cells.

    (a) Experimental schematic outlining the FACS isolation and immediate transplantation into regenerating TA muscle of immunosuppressed mdx mice between the ages of 6 and 8 weeks. (b) The intrinsic contribution of satellite cells to muscle regeneration in 2- to 4-month-old mdx mice (n = 9 for each time point) with respect to increasing age. Images show dystrophin (green), laminin (red) and DAPI (blue) staining in TA muscle from mdx mice 3 weeks after transplantation. Arrowheads designate dystrophin-positive fibers. Data on adolescent mice are shown in Supplementary Figure 2. Scale bars, 100 μm (larger images); 20 μm (inset). (c) Quantification of the percentage of dystrophin-positive fibers after transplantation of age-related satellite cell populations. Values are relative to cell transplants from 3-month-old mice. *P < 0.05, **P < 0.01. (d) Intrinsic repopulating capacity of transplanted cells into the satellite cell niche. Images show immunofluorescence of Pax7 (red) and ZsGreen (green) 3 weeks after transplantation of age-specific satellite cells into CTX-injured TA muscles of mdx mice. Arrows designate satellite cells positive for both ZsGreen and Pax7, and the arrowhead designates a host Pax7+ satellite cell. Scale bars, 100 μm (larger images); 20 μm (inset). High-magnification images of Pax7 and ZsGreen along with images from adolescent mice are shown in Supplementary Figure 2. (e) Quantification of the repopulating capacity associated with transplanted satellite cells from the muscle of young adult and older adult mice as evidenced by the number of double-positive Pax7+ZsGreen+ cells in relation to the total number of Pax7+ satellite cells. *P < 0.05, **P < 0.01. The data in c and e are shown as the mean ± s.e.m. All statistical significance was calculated by Student's t test.

  2. The transcriptional profile of adult satellite cells is enriched for JAK-STAT signaling pathway members and interactors.
    Figure 2: The transcriptional profile of adult satellite cells is enriched for JAK-STAT signaling pathway members and interactors.

    (a) Comparison between genes enriched (more than twofold) in satellite cells from adolescent compared to older adult mice based on KEGG signaling pathway enrichment. Signaling pathways are ranked on the basis of their NESs. Signaling pathways connected with skeletal muscle regeneration are highlighted in red. (b) Fold changes based on log2-transformed RMA values from satellite cells of adolescent, young adult and older adult mice used to generate global ratio heat maps. Red indicates genes that increased in expression, and blue indicates a decrease in expression. *P < 0.05, **P < 0.01 for adolescent compared to young adult; P < 0.05, P < 0.01 for young adult compared to older adult. The genes highlighted in red are validated by qPCR in c. The data represent pooled replicates of adolescent (n = 5), young adult (n = 8) and older adult (n = 8) mice conducted in biological triplicate. (c) qPCR validation of the microarray data supports the transcriptional changes observed between satellite cells from adolescent, young adult and older adult mice. Data are presented as the mean ± s.e.m. of three biological replicates of freshly sorted satellite cells normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH). **P < 0.01, ***P < 0.001. (d) Quantification of pStat3 protein expression from age-related satellite cell samples (Supplementary Fig. 3f) normalized to Hsp70 protein expression to account for loading. Data represent the mean ± s.e.m. of three biological replicates (n = 3). *P < 0.05, **P < 0.01. All statistical significance was calculated by Student's t test.

  3. Inhibition of JAK-STAT signaling rescues the proliferation defect of satellite cells from the muscle of older adult mice.
    Figure 3: Inhibition of JAK-STAT signaling rescues the proliferation defect of satellite cells from the muscle of older adult mice.

    (a) Effect of siRNA knockdown of Jak2 or Stat3 on the symmetric divisions of satellite stem cells (Pax7+Myf5) on myofibers from Myf5-cre R26R-YFP cells cultured for 42 h in vitro. n = 4. (b) Quantification of the total number of dividing satellite cells on single fibers cultured for 42 h in vitro after siRNA knockdown of Jak2 or Stat3. n = 4. (c) Quantification of the average number of Pax7+ satellite cells per fiber for each age stage after siRNA knockdown. n = 4. (d) Quantification of the percentage of committed satellite cells (Pax7MyoD+) present on single myofibers after 72 h of culture and treatment with siRNAs against Stat3 or Jak2. n = 4. (e) Quantification of symmetric satellite stem cell divisions after JAK-STAT inhibitor treatment for 42 h on myofibers from young adult mice. n = 3. (f) Quantification of the total number of dividing satellite cells on single fibers cultured for 42 h in vitro after pharmacological inhibition of Jak2 or Stat3. n = 3. (g) Quantification of the average number of Pax7+ satellite cells per fiber for each age stage after treatment with small-molecule inhibitors. n = 3. (h) Quantification of the percentage of committed satellite cells (Pax7MyoD+) present on single fibers after 72 h culture in the presence of JAK-STAT inhibitors. n = 3. All data represent the mean ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001. NS, not significant. All statistical significance was calculated by Student's t test.

  4. Knockdown of JAK-STAT pathway members ameliorates the engraftment potential of adult satellite cells.
    Figure 4: Knockdown of JAK-STAT pathway members ameliorates the engraftment potential of adult satellite cells.

    (a) Experimental schematic outlining the FACS isolation, treatment ex vivo with scrambled siRNA, siStat3, siJak2 or a combination of siStat3 and siJak2 followed by transplantation into CTX-treated TA muscle of C57BL/6 mice. (b) Representative images (~200 images total per muscle section) from ex vivo siRNA-treated satellite cells. Images show Pax7 (red), ZsGreen (green) and DAPI (blue) staining in TA muscle from C57BL/6 mice 12 d after transplantation. Arrows designate host satellite cells, and the arrowhead designates a donor-derived satellite cell. Scale bar, 50 μm. (c) Quantification of the percentage of ZsGreen+ satellite cells after siRNA knockdown and transplantation. n = 4. (d) Quantification of the host satellite cells per area after siRNA knockdown and transplantation. n = 4. **P < 0.01, ***P < 0.001. NS, not significant. Statistical significance was calculated by Student's t test. The data in c and d are shown as the mean ± s.e.m.

  5. Administration of small-molecule JAK-STAT inhibitors ex vivo before transplantation into young adult mice enhances engraftment.
    Figure 5: Administration of small-molecule JAK-STAT inhibitors ex vivo before transplantation into young adult mice enhances engraftment.

    (a) Schematic for ex vivo treatment of freshly sorted satellite cells with JAK-STAT inhibitors before transplantation. (b) Quantification of donor satellite cells from young adult and older adult mice after ex vivo treatment with JAK-STAT inhibitors and transplantation into C57BL/6 mice. (c) Quantification of donor satellite cells from young adult and older adult mice after ex vivo treatment with JAK-STAT inhibitors and transplantation into mdx mice. The data in b and c represent the mean ± s.e.m. n = 3. **P < 0.01, ***P < 0.001. Statistical significance was calculated by Student's t test.

  6. Direct injection of JAK-STAT inhibitors in vivo improves muscle regeneration, increases satellite cell number and functionally improves skeletal muscle.
    Figure 6: Direct injection of JAK-STAT inhibitors in vivo improves muscle regeneration, increases satellite cell number and functionally improves skeletal muscle.

    (a) Schematic of in vivo treatment of regenerating TA muscle with JAK-STAT inhibitors. (b) Quantification of the minimal fiber Feret after direct i.m. injection of 5,15 DPP, Tyr AG 490 or a combination of both inhibitors into regenerating TA muscle of young adult and older adult mice. Data represent the mean ± s.e.m., n = 4. **P < 0.01, ***P < 0.001. (c) Quantification of total satellite cells in regenerating TA muscle of young adult or older adult mice after treatment with JAK-STAT signaling inhibitors. Data represent the mean ± s.e.m., n = 4. *P < 0.05, **P < 0.01. (d) Schematic of in vivo treatment of regenerating EDL muscle of young adult mice with JAK-STAT inhibitors and subsequent isolation for functional analysis. (e) Force versus time plots of EDL muscle treated with either vehicle, 5,15 DPP, Tyr AG 490 or a combination of both inhibitors. (f) Quantification of the maximal force determined from EDL muscle treated with JAK-STAT inhibitors. Data represent the mean ± s.e.m. n = 6. *P < 0.05. (g) Fatigue curves indicating the change in percentage of initial muscle force after treatment with JAK-STAT inhibitors. Vehicle (n = 7), 5,15 DPP (n = 6), Tyr AG 490 (n = 6) or a combination of both inhibitors (n = 6). *P < 0.05, **P < 0.01. All statistical significance was calculated by Student's t test.

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Referenced accessions

Gene Expression Omnibus

Change history

Corrected online 22 September 2014

In the version of this article initially published online, the third sentence of the Abstract read “Gene expression analysis identified higher expression of JAK-STAT signaling targets in 3-week-old relative to 18-month-old mice,” when it should have read “Gene expression analysis identified higher expression of JAK-STAT signaling targets in 18-month-old relative to 3-week-old mice.” The error has been corrected for the print, PDF and HTML versions of this article.

References

  1. Mauro, A. Satellite cell of skeletal muscle fibers. J. Biophys. Biochem. Cytol. 9, 493495 (1961).
  2. Schultz, E., Gibson, M.C. & Champion, T. Satellite cells are mitotically quiescent in mature mouse muscle: an EM and radioautographic study. J. Exp. Zool. 206, 451456 (1978).
  3. Montarras, D. et al. Direct isolation of satellite cells for skeletal muscle regeneration. Science 309, 20642067 (2005).
  4. Collins, C.A. et al. Stem cell function, self-renewal, and behavioral heterogeneity of cells from the adult muscle satellite cell niche. Cell 122, 289301 (2005).
  5. Kuang, S., Kuroda, K., Le Grand, F. & Rudnicki, M.A. Asymmetric self-renewal and commitment of satellite stem cells in muscle. Cell 129, 9991010 (2007).
  6. Zammit, P.S. et al. Muscle satellite cells adopt divergent fates: a mechanism for self-renewal? J. Cell Biol. 166, 347357 (2004).
  7. Carlson, M.E. & Conboy, I.M. Loss of stem cell regenerative capacity within aged niches. Aging Cell 6, 371382 (2007).
  8. Augustin, H. & Partridge, L. Invertebrate models of age-related muscle degeneration. Biochim. Biophys. Acta 1790, 10841094 (2009).
  9. Grounds, M.D. Age-associated changes in the response of skeletal muscle cells to exercise and regeneration. Ann. NY Acad. Sci. 854, 7891 (1998).
  10. Allbrook, D.B., Han, M.F. & Hellmuth, A.E. Population of muscle satellite cells in relation to age and mitotic activity. Pathology 3, 223243 (1971).
  11. Shefer, G., Van de Mark, D.P., Richardson, J.B. & Yablonka-Reuveni, Z. Satellite-cell pool size does matter: defining the myogenic potency of aging skeletal muscle. Dev. Biol. 294, 5066 (2006).
  12. Conboy, I.M. & Rando, T.A. The regulation of Notch signaling controls satellite cell activation and cell fate determination in postnatal myogenesis. Dev. Cell 3, 397409 (2002).
  13. Carlson, M.E., Hsu, M. & Conboy, I.M. Imbalance between pSmad3 and Notch induces CDK inhibitors in old muscle stem cells. Nature 454, 528532 (2008).
  14. Conboy, I.M., Conboy, M.J., Smythe, G.M. & Rando, T.A. Notch-mediated restoration of regenerative potential to aged muscle. Science 302, 15751577 (2003).
  15. Brack, A.S. et al. Increased Wnt signaling during aging alters muscle stem cell fate and increases fibrosis. Science 317, 807810 (2007).
  16. Brack, A.S. & Rando, T.A. Intrinsic changes and extrinsic influences of myogenic stem cell function during aging. Stem Cell Rev. 3, 226237 (2007).
  17. Sousa-Victor, P. et al. Geriatric muscle stem cells switch reversible quiescence into senescence. Nature 506, 316321 (2014).
  18. Cosgrove, B.D. et al. Rejuvenation of the muscle stem cell population restores strength to injured aged muscles. Nat. Med. 20, 255264 (2014).
  19. Bernet, J.D. et al. p38 MAPK signaling underlies a cell-autonomous loss of stem cell self-renewal in skeletal muscle of aged mice. Nat. Med. 20, 265271 (2014).
  20. Bentzinger, C.F. & Rudnicki, M.A. Rejuvenating aged muscle stem cells. Nat. Med. 20, 234235 (2014).
  21. Yablonka-Reuveni, Z., Seger, R. & Rivera, A.J. Fibroblast growth factor promotes recruitment of skeletal muscle satellite cells in young and old rats. J. Histochem. Cytochem. 47, 2342 (1999).
  22. Neal, A., Boldrin, L. & Morgan, J.E. The satellite cell in male and female, developing and adult mouse muscle: distinct stem cells for growth and regeneration. PLoS ONE 7, e37950 (2012).
  23. Bosnakovski, D. et al. Prospective isolation of skeletal muscle stem cells with a Pax7 reporter. Stem Cells 26, 31943204 (2008).
  24. Subramanian, A. et al. Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. Proc. Natl. Acad. Sci. USA 102, 1554515550 (2005).
  25. Conboy, I.M. et al. Rejuvenation of aged progenitor cells by exposure to a young systemic environment. Nature 433, 760764 (2005).
  26. Toth, K.G. et al. IL-6 induced STAT3 signalling is associated with the proliferation of human muscle satellite cells following acute muscle damage. PLoS ONE 6, e17392 (2011).
  27. Troy, A. et al. Coordination of satellite cell activation and self-renewal by Par-complex–dependent asymmetric activation of p38α/β MAPK. Cell Stem Cell 11, 541553 (2012).
  28. Pasut, A., Jones, A.E. & Rudnicki, M.A. Isolation and culture of individual myofibers and their satellite cells from adult skeletal muscle. J. Vis. Exp. e50074 (2013).
  29. Cooper, R.N. et al. In vivo satellite cell activation via Myf5 and MyoD in regenerating mouse skeletal muscle. J. Cell Sci. 112, 28952901 (1999).
  30. Uehara, Y., Mochizuki, M., Matsuno, K., Haino, T. & Asai, A. Novel high-throughput screening system for identifying STAT3–SH2 antagonists. Biochem. Biophys. Res. Commun. 380, 627631 (2009).
  31. Eriksen, K.W. et al. Constitutive STAT3-activation in Sezary syndrome: tyrphostin AG490 inhibits STAT3-activation, interleukin-2 receptor expression and growth of leukemic Sezary cells. Leukemia 15, 787793 (2001).
  32. Launay, T., Noirez, P., Butler-Browne, G. & Agbulut, O. Expression of slow myosin heavy chain during muscle regeneration is not always dependent on muscle innervation and calcineurin phosphatase activity. Am. J. Physiol. Regul. Integr. Comp. Physiol. 290, R1508R1514 (2006).
  33. Shuai, K. & Liu, B. Regulation of JAK-STAT signalling in the immune system. Nat. Rev. Immunol. 3, 900911 (2003).
  34. Megeney, L.A., Perry, R.L., LeCouter, J.E. & Rudnicki, M.A. bFGF and LIF signaling activates STAT3 in proliferating myoblasts. Dev. Genet. 19, 139145 (1996).
  35. Yang, Y. et al. STAT3 induces muscle stem cell differentiation by interaction with myoD. Cytokine 46, 137141 (2009).
  36. McKay, B.R. et al. Elevated SOCS3 and altered IL-6 signaling is associated with age-related human muscle stem cell dysfunction. Am. J. Physiol. Cell Physiol. 304, C717C728 (2013).
  37. Chakkalakal, J.V., Jones, K.M., Basson, M.A. & Brack, A.S. The aged niche disrupts muscle stem cell quiescence. Nature 490, 355360 (2012).
  38. Jang, Y.C., Sinha, M., Cerletti, M., Dall'Osso, C. & Wagers, A.J. Skeletal muscle stem cells: effects of aging and metabolism on muscle regenerative function. Cold Spring Harb. Symp. Quant. Biol. 76, 101111 (2011).
  39. Harrison, D.A. The Jak/STAT pathway. Cold Spring Harb. Perspect. Biol. 4, a011205 (2012).
  40. Le Grand, F., Jones, A.E., Seale, V., Scime, A. & Rudnicki, M.A. Wnt7a activates the planar cell polarity pathway to drive the symmetric expansion of satellite stem cells. Cell Stem Cell 4, 535547 (2009).
  41. von Maltzahn, J., Bentzinger, C.F. & Rudnicki, M.A. Wnt7a-Fzd7 signalling directly activates the Akt/mTOR anabolic growth pathway in skeletal muscle. Nat. Cell Biol. 14, 186191 (2012).
  42. Bentzinger, C.F. et al. Fibronectin regulates Wnt7a signaling and satellite cell expansion. Cell Stem Cell 12, 7587 (2013).
  43. Tepass, U. The apical polarity protein network in Drosophila epithelial cells: regulation of polarity, junctions, morphogenesis, cell growth, and survival. Annu. Rev. Cell Dev. Biol. 28, 655685 (2012).
  44. McCaffrey, L.M., Montalbano, J., Mihai, C. & Macara, I.G. Loss of the Par3 polarity protein promotes breast tumorigenesis and metastasis. Cancer Cell 22, 601614 (2012).
  45. Price, F.D. et al. Canonical Wnt signaling induces a primitive endoderm metastable state in mouse embryonic stem cells. Stem Cells 31, 752764 (2013).
  46. Briguet, A., Courdier-Fruh, I., Foster, M., Meier, T. & Magyar, J.P. Histological parameters for the quantitative assessment of muscular dystrophy in the mdx-mouse. Neuromuscul. Disord. 14, 675682 (2004).
  47. Huang, W., Sherman, B.T. & Lempicki, R.A. Bioinformatics enrichment tools: paths toward the comprehensive functional analysis of large gene lists. Nucleic Acids Res. 37, 113 (2009).
  48. Huang, W., Sherman, B.T. & Lempicki, R.A. Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nat. Protoc. 4, 4457 (2009).
  49. Mootha, V.K. et al. PGC-1α–responsive genes involved in oxidative phosphorylation are coordinately downregulated in human diabetes. Nat. Genet. 34, 267273 (2003).

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Author information

  1. These authors contributed equally to this work.

    • Feodor D Price &
    • Julia von Maltzahn

Affiliations

  1. Sprott Center For Stem Cell Research, Ottawa Hospital Research Institute, Regenerative Medicine Program, Ottawa, Ontario, Canada.

    • Feodor D Price,
    • Julia von Maltzahn,
    • C Florian Bentzinger,
    • Nicolas A Dumont,
    • Hang Yin,
    • Natasha C Chang,
    • David H Wilson &
    • Michael A Rudnicki
  2. Department of Cellular and Molecular Medicine, University of Ottawa, Faculty of Medicine, Ottawa, Ontario, Canada.

    • Feodor D Price,
    • Julia von Maltzahn,
    • C Florian Bentzinger,
    • Nicolas A Dumont,
    • Hang Yin,
    • Natasha C Chang &
    • Michael A Rudnicki
  3. Programme de Physiothérapie, Département de Réadaptation, Université Laval, Québec, Canada.

    • Jérôme Frenette
  4. Present address: Fritz-Lipmann Institute for Age Research, Jena, Germany.

    • Julia von Maltzahn

Contributions

F.D.P. designed and carried out experiments, analyzed results and wrote the manuscript. J.v.M. designed and conducted experiments and analyzed results. C.F.B., N.A.D., H.Y. and N.C.C. conducted experiments and analyzed and interpreted data. D.H.W. conducted experiments. J.F. provided expertise in physiological analysis. M.A.R. designed experiments, analyzed results, wrote the manuscript and provided financial support.

Competing financial interests

The authors declare no competing financial interests.

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