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Intronic polyadenylation of PDGFRα in resident stem cells attenuates muscle fibrosis

Abstract

Platelet-derived growth factor receptor α (PDGFRα) exhibits divergent effects in skeletal muscle. At physiological levels, signalling through this receptor promotes muscle development in growing embryos and angiogenesis in regenerating adult muscle1,2. However, both increased PDGF ligand abundance and enhanced PDGFRα pathway activity cause pathological fibrosis3,4. This excessive collagen deposition, which is seen in aged and diseased muscle5,6,7, interferes with muscle function and limits the effectiveness of gene- and cell-based therapies for muscle disorders8,9. Although compelling evidence exists for the role of PDGFRα in fibrosis, little is known about the cells through which this pathway acts. Here we show in mice that PDGFRα signalling regulates a population of muscle-resident fibro/adipogenic progenitors (FAPs) that play a supportive role in muscle regeneration but may also cause fibrosis when aberrantly regulated10,11,12,13. We found that FAPs produce multiple transcriptional variants of Pdgfra with different polyadenylation sites, including an intronic variant that codes for a protein isoform containing a truncated kinase domain. This variant, upregulated during regeneration, acts as a decoy to inhibit PDGF signalling and to prevent FAP over-activation. Moreover, increasing the expression of this isoform limits fibrosis in vivo in mice, suggesting both biological relevance and therapeutic potential of modulating polyadenylation patterns in stem-cell populations.

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Figure 1: Pdgfra undergoes intronic polyadenylation in FAPs, resulting in a truncated protein that increases in abundance during regeneration.
Figure 2: Intronic polyadenylation of Pdgfra inhibits signalling through the receptor, whereas decreasing In-PDGFRα abundance augments signalling.
Figure 3: Intronic polyadenylation of Pdgfra limits activation of FAPs in vitro, and reducing levels of the intronic variant promotes FAP activation.
Figure 4: Downregulation of In-PDGFRα enhances FAP activation and fibrosis, whereas enhancing intronic polyadenylation of PDGFRα attenuates FAP activation and fibrosis.

References

  1. Orr-Urtreger, A., Bedford, M. T., Do, M. S., Eisenbach, L. & Lonai, P. Developmental expression of the α receptor for platelet-derived growth factor, which is deleted in the embryonic lethal Patch mutation. Development 115, 289–303 (1992)

    CAS  PubMed  Google Scholar 

  2. Soriano, P. The PDGFRα receptor is required for neural crest cell development and for normal patterning of the somites. Development 124, 2691–2700 (1997)

    CAS  PubMed  Google Scholar 

  3. Andrae, J., Gallini, R. & Betsholtz, C. Role of platelet-derived growth factors in physiology and medicine. Genes Dev. 22, 1276–1312 (2008)

    Article  CAS  Google Scholar 

  4. Olson, L. E. & Soriano, P. Increased PDGFRα activation disrupts connective tissue development and drives systemic fibrosis. Dev. Cell 16, 303–313 (2009)

    Article  CAS  Google Scholar 

  5. Brack, A. S. et al. Increased Wnt signaling during aging alters muscle stem cell fate and increases fibrosis. Science 317, 807–810 (2007)

    Article  ADS  CAS  Google Scholar 

  6. Goldspink, G., Fernandes, K., Williams, P. E. & Wells, D. J. Age-related changes in collagen gene expression in the muscles of mdx dystrophic and normal mice. Neuromuscul. Disord. 4, 183–191 (1994)

    Article  CAS  Google Scholar 

  7. Conboy, I. M. & Rando, T. A. Aging, stem cells and tissue regeneration: lessons from muscle. Cell Cycle 4, 407–410 (2005)

    Article  ADS  CAS  Google Scholar 

  8. Mann, C. J. et al. Aberrant repair and fibrosis development in skeletal muscle. Skelet. Muscle 1, 21 (2011)

    Article  Google Scholar 

  9. Serrano, A. L. et al. Cellular and molecular mechanisms regulating fibrosis in skeletal muscle repair and disease. Curr. Top. Dev. Biol. 96, 167–201 (2011)

    Article  CAS  Google Scholar 

  10. Uezumi, A., Fukada, S., Yamamoto, N., Takeda, S. & Tsuchida, K. Mesenchymal progenitors distinct from satellite cells contribute to ectopic fat cell formation in skeletal muscle. Nat. Cell Biol. 12, 143–152 (2010)

    Article  CAS  Google Scholar 

  11. Joe, A. W. B. et al. Muscle injury activates resident fibro/adipogenic progenitors that facilitate myogenesis. Nat. Cell Biol. 12, 153–163 (2010)

    Article  CAS  Google Scholar 

  12. Uezumi, A. et al. Fibrosis and adipogenesis originate from a common mesenchymal progenitor in skeletal muscle. J. Cell Sci. 124, 3654–3664 (2011)

    Article  CAS  Google Scholar 

  13. Fiore, D. et al. Pharmacological blockage of fibro/adipogenic progenitor expansion and suppression of regenerative fibrogenesis is associated with impaired skeletal muscle regeneration. Stem Cell Res. 17, 161–169 (2016)

    Article  CAS  Google Scholar 

  14. Tidball, J. G., Spencer, M. J. & St Pierre, B. A. PDGF-receptor concentration is elevated in regenerative muscle fibers in dystrophin-deficient muscle. Exp. Cell Res. 203, 141–149 (1992)

    Article  CAS  Google Scholar 

  15. Zhao, Y. et al. Platelet-derived growth factor and its receptors are related to the progression of human muscular dystrophy: an immunohistochemical study. J. Pathol. 201, 149–159 (2003)

    Article  CAS  Google Scholar 

  16. Ito, T. et al. Imatinib attenuates severe mouse dystrophy and inhibits proliferation and fibrosis-marker expression in muscle mesenchymal progenitors. Neuromuscul. Disord. 23, 349–356 (2013)

    Article  Google Scholar 

  17. Huang, P., Zhao, X. S., Fields, M., Ransohoff, R. M. & Zhou, L. Imatinib attenuates skeletal muscle dystrophy in mdx mice. FASEB J. 23, 2539–2548 (2009)

    Article  CAS  Google Scholar 

  18. Ozsolak, F. et al. Direct RNA sequencing. Nature 461, 814–818 (2009)

    Article  ADS  CAS  Google Scholar 

  19. Boutet, S. C. et al. Alternative polyadenylation mediates microRNA regulation of muscle stem cell function. Cell Stem Cell 10, 327–336 (2012)

    Article  CAS  Google Scholar 

  20. Mueller, A. A., Cheung, T. H. & Rando, T. A. All’s well that ends well: alternative polyadenylation and its implications for stem cell biology. Curr. Opin. Cell Biol. 25, 222–232 (2013)

    Article  CAS  Google Scholar 

  21. Vorlová, S. et al. Induction of antagonistic soluble decoy receptor tyrosine kinases by intronic polyA activation. Mol. Cell 43, 927–939 (2011)

    Article  Google Scholar 

  22. Kawai, T. et al. PPARγ agonist attenuates renal interstitial fibrosis and inflammation through reduction of TGFβ. Lab. Invest. 89, 47–58 (2009)

    Article  CAS  Google Scholar 

  23. Morcos, P. A., Li, Y. & Jiang, S. Vivo-Morpholinos: a non-peptide transporter delivers Morpholinos into a wide array of mouse tissues. Biotechniques 45, 613–614, 616, 618 passim (2008)

    Article  CAS  Google Scholar 

  24. Bansal, R. et al. Novel engineered targeted interferonγ blocks hepatic fibrogenesis in mice. Hepatology 54, 586–596 (2011)

    Article  CAS  Google Scholar 

  25. Du, L. & Gatti, R. A. Potential therapeutic applications of antisense morpholino oligonucleotides in modulation of splicing in primary immunodeficiency diseases. J. Immunol. Methods 365, 1–7 (2011)

    Article  CAS  Google Scholar 

  26. Heinrich, M. C. et al. PDGFRA activating mutations in gastrointestinal stromal tumors. Science 299, 708–710 (2003)

    Article  ADS  CAS  Google Scholar 

  27. Pfaffl, M. W., A new mathematical model for relative quantification in real-time RT–PCR. Nucleic Acids Res. 29, e45 (2001)

    Article  CAS  Google Scholar 

  28. Subramanian, A. et al. Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. Proc. Natl Acad. Sci. USA 102, 15545–15550 (2005)

    Article  ADS  CAS  Google Scholar 

  29. Mehlem, A., Hagberg, C. E., Muhl, L., Eriksson, U. & Falkevall, A. Imaging of neutral lipids by oil red O for analyzing the metabolic status in health and disease. Nat. Protoc. 8, 1149–1154 (2013)

    Article  Google Scholar 

  30. Milacic, M. et al. Annotating cancer variants and anti-cancer therapeutics in reactome. Cancers 4, 1180–1211 (2012)

    Article  CAS  Google Scholar 

  31. Croft, D. et al. The reactome pathway knowledgebase. Nucleic Acids Res. 42, D472–D477 (2014)

    Article  ADS  CAS  Google Scholar 

  32. Robinson, J. T. et al. Integrative genomics viewer. Nat. Biotechnol. 29, 24–26 (2011)

    Article  CAS  Google Scholar 

  33. Thorvaldsdóttir, H., Robinson, J. T. & Mesirov, J. P. Integrative Genomics Viewer (IGV): high-performance genomics data visualization and exploration. Brief. Bioinform. 14, 178–192 (2013)

    Article  Google Scholar 

Download references

Acknowledgements

We would like to thank members of the Rando laboratory, especially A. de Morrée, M. Quarta, J. Shih, B. Yoo, V. Garcia and I. Akimenko for discussions and experimental assistance as well as L. Rott for guidance with FACS. This work was supported by the Glenn Foundation for Medical Research, by grants from the National Institutes of Health (F30 AG043235) and the California Institute for Regenerative Medicine (TG2-01159) to A.A.M., and by grants from the Department of Veterans Affairs and the NIH (P01 AG036695, R01 AG23806, R01 AR062185, and TR01 AG47820) to T.A.R.

Author information

Authors and Affiliations

Authors

Contributions

A.A.M. and T.A.R. conceived the study and were involved in the overall design of experiments. A.A.M. and T.H.C. designed, conducted and analysed the microarray and sequencing experiments as well as the FACS analyses. A.A.M. designed the morpholinos and siRNAs and also performed and analysed the associated experiments to test for their transcriptional effects. A.A.M. and C.T.V.V. designed the viral construct while C.T.V.V. created the construct and performed experiments to analyse expression. A.A.M. and C.T.V.V. designed and performed experiments and data analysis for PDGFRα protein expression and downstream signalling in response to morpholino treatment in vitro and in vivo as well as for muscle fibrosis responses. A.A.M. conducted and analysed the proliferation assays whereas C.T.V.V. performed and evaluated the scratch assays. K.D.F. carried out pilot experiments to assess for PDGFRα signalling in regenerating skeletal muscle and in initial FACS characterization of the FAP population. A.A.M. and T.A.R. wrote the manuscript with input from C.T.V.V. and T.H.C.

Corresponding author

Correspondence to Thomas A. Rando.

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Competing interests

The authors declare no competing financial interests.

Additional information

Reviewer Information Nature thanks F. Rossi, Y. Shi and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Extended data figures and tables

Extended Data Figure 1 PDGFRα is activated specifically in FAPs during muscle regeneration.

a, Tibialis anterior muscles were dissected on the indicated days after injury. The muscle lysates were subjected to western blot to probe for PDGFRα and pPDGFRα using different antibodies, which shows increased expression of pPDGFRα during muscle regeneration. b, Quantification of a (n = 3 individual tibialis anterior muscles per time point). pPDGFRα level refers to expression levels for pPDGFRα normalized to total PDGFRα. Error bars represent s.e.m. c, Immunofluorescence of an uninjured tibialis anterior muscle and a muscle 4.5 days after injury shows an increase in the abundance of pPDGFRα-expressing cells during the regenerative process. (DAPI, blue; laminin, red; pPDGFRα, green). d, Immunofluorescence of an uninjured tibialis anterior muscle from a PDGFRα–eGFP reporter mouse. e, FACS histogram analysis of the GFP signal in a PDGFRα–eGFP reporter heterozygote (green line) and in a wild-type littermate (grey, solid) in cells from uninjured hindlimb muscles. f, FACS plot of cells isolated from uninjured hindlimb muscles of the PDGFRα–eGFP reporter. GFP-positive cells (shown in green) are overlaid upon all cells (shown in grey). Dashed lines represent the gate for the FAP population. g, Histograms showing GFP signal in cell populations isolated from uninjured PDGFRα–eGFP reporter mice, as detected by FACS analysis. hj, The patterns of expression of h, i, and j, as assessed by FACS analysis of BaCl2-injured muscle were similar to the patterns in e, f, and g, respectively, from uninjured muscle. km, Similarly, the patterns of expression of k, l, and m, as evaluated by FACS analysis of glycerol-injured muscle were comparable to the patterns in e, f, and g, respectively, from uninjured muscle.

Source data

Extended Data Figure 2 In-PDGFRα transcript and protein structure.

a, DNA sequencing of 3′ RACE products illustrated in Fig. 1b confirms polyadenylation sites of highly expressed variants. b, Top, the fully-spliced FL-PDGFRα transcript contains 23 exons that encode the corresponding protein domains (bottom). CT, C-terminal region; JMR, juxtramembrane region; KD1, kinase domain 1; KD2, kinase domain 2; KI, interkinase domain; TM, transmembrane region. c, The fully spliced In-PDGFRα transcript contains 16 exons (top) that encode the protein domains (bottom). Red, portions of the transcript and protein that are unique to In-PDGFRα. d, Enlarged view of the genomic sequence that codes for the unique region of the In-PDGFRα protein. In FL-PDGFRα, this region is spliced out of the transcript. e, Map of the locations of amplicons used to assess levels of In-PDGFRα and FL-PDGFRα. Primers amplifying regions of exons 7–8, 11–12, and 15–16 are common to In-PDGFRα and FL-PDGFRα. Primers designated with 16i target the region canonically referred to as intron 16. In FL-PDGFRα, this region is spliced out. In In-PDGFRα, this region becomes the 3′ UTR. Therefore 16–16i and 16i–16i are specific for In-PDGFRα. Primers amplifying regions of exons 18–19, 21–22, exon 23 (23–23) and the 3′ UTR (UTR–UTR) are specific to FL-PDGFRα. f, Levels of In-PDGFRα transcript relative to FL-PDGFRα transcript increase during regeneration. FAPs were collected from uninjured muscles (day 0) or at 3 days after injury and RNA levels were assessed via qPCR. Expression level is plotted as a ratio of In-PDGFRα to FL-PDGFRα normalized to day 0. For In-PDGFRα primers 16–16i and 16i–16i were averaged, whereas for FL-PDGFRα, primers 23–23 and UTR–UTR were averaged. For each time point, n = 3 biological replicates of pooled FAPs. Significance was calculated using an unpaired Student’s t-test, error bars represent s.e.m. g, Western blot using the C-terminal PDGFRα antibody shows knockdown of FL-PDGFRα in response to the 5′ss-AMO.

Source data

Extended Data Figure 3 Phosphorylation of PDGFRα and downstream signalling components in FAPs in response to PDGF-AA stimulation is altered by changes in In-PDGFRα expression.

a, Western blot showing phosphorylation of PDGFRα, PLCγ, Akt and ERK1/2 in response to PDGF-AA stimulation. b, Quantification of data in a. c, Viral overexpression of In-PDGFRα in FAPs results in decreased signalling through Akt and ERK/2 whereas FL-PDGFRα levels remain constant. b, c, n = 3 biological replicates of pooled FAPs per condition except for the ERK1/2 condition in b, where n = 4. Error bars represent s.e.m. *P < 0.05; **P < 0.01; unpaired Student t-tests.

Source data

Extended Data Figure 4 FAPs proliferate in response to PDGF stimulation.

a, EdU incorporation in FAPs stimulated with indicated amounts of PDGF for 20 h and incubated in EdU for the final 4 h. b, EdU incorporation in FAPs incubated with indicated amounts of PDGF and EdU for 24 h. cf, Knockdown of In-PDGFRα by pre-treatment of FAPs with pA-AMOs (c) or In-PDGFRα-specific siRNA (In-siRNA) (d) increases FAP proliferation in response to PDGF-AA, whereas In-PDGFRα upregulation by 5′ss-AMO pre-treatment (e) or viral overexpression (f) decreases proliferation. For a, b, d, f, n = 3 biological replicates of pooled FAPs per condition. Error bars represent s.e.m. *P < 0.05; ****P < 0.0001; using unpaired Student t-tests.

Source data

Extended Data Figure 5 Knockdown of In-PDGFRα and FL-PDGFRα with siRNAs.

a, Schematic of the FL-PDGFRα transcript showing the location of siRNAs designed to knockdown In-PDGFRα (In-siRNA) and FL-PDGFRα (FL-siRNA). Arrows designate relevant polyadenylation sites. bd, FL-siRNA induces transcriptional knockdown of FL-PDGFRα but not In-PDGFRα (b) and protein knockdown of FL-PDGFRα (c). At the same time, In-siRNA induces knockdown of In-PDGFRα (d). In b and d, n = 3 biological replicates of pooled FAPs per condition. The samples in b, and d, were all processed together so that the control condition in b is identical to the control condition in d. Error bars represent s.e.m. *P < 0.05, **P < 0.01; unpaired Student t-tests.

Source data

Extended Data Figure 6 Assessment of molecular pathway changes in response to modulation of In-PDGFRα levels.

a, Ingenuity pathway analysis of control-AMO-treated FAPs stimulated with PDGF-AA (50 ng ml−1) show enrichment for genes associated with fibrosis compared with control cells. b, Western blot showing phosphorylation of SMAD2/3 in response to PDGF-AA stimulation (50 ng ml−1). c, d, Gene set enrichment analysis (GSEA) of FAPs treated with the pA-AMOs compared to control-treated samples were analysed for enrichment of pathways in the Reactome (c) and BioCarta (d) databases30,31. Top sets with a false discovery rate of less than 5% are shown. e, The heat map displays a subset of genes that are upregulated with a false discovery rate of less than 0.1% in the pA-AMO- treated FAPs. The colours indicate the Z-score for expression normalized for each gene. Red, high expression; blue, low expression. f, g, Similarly, enrichment analyses of FAPs treated with the 5′ss-VMO compared to controls were performed using the Reactome (f) and BioCarta (g) databases30,31. h, A heat map displaying top upregulated genes in 5′ss-VMO-treated FAPs with parameters as specified in e is shown.

Extended Data Figure 7 Alteration of In-PDGFRα levels in vivo modulates FAP activity.

a, b, FAPs treated in vitro with pA-VMOs showed a downregulation of In-PDGFRα (n = 3) (a), whereas those treated with the 5′ss-VMO exhibited an upregulation of In-PDGFRα (n = 3) (b). c, d, In vivo treatment with 5′ss-VMO decreases FAP proliferation (n = 16) (c) with a corresponding decrease in cell count (n = 24) (d). Treatment with pA-VMOs does not lead to a significant change in proliferation (P = 0.42). e, f, GSEA of FAPs collected from tibialis anterior muscles treated with pA-VMOs (e) or the 5′ss-VMO (f), compared to control-treated samples, were analysed for enrichment of pathways in the Reactome database30,31. Top sets with a false discovery rate of less than 5% are shown. g, h, Ingenuity pathway analysis of top regulators of gene expression in FAPs treated with pA-AMOs (g) or the 5′ss-VMO (h) compared to control treatment. The top hits are shown, excluding those with the ‘molecular type’ designated as a chemical or drug. For ad, n represents biological replicates of pooled FAPs. *P < 0.05; **P < 0.01, ***P < 0.001, ****P < 0.0001; unpaired Student t-tests. In g, h, two pooled FAP samples per condition were used with the overlap P-value calculated using the Fisher’s exact test. Error bars represent s.e.m.

Source data

Extended Data Figure 8 Alteration of In-PDGFRα levels in vivo modulates muscle fibrosis.

a, b, Fibro–adipose index of muscles treated with pA-VMOs (n = 9) (a) or the 5′ss VMO (control: n = 9, 5′ss-VMO: n = 10) (b). c, Representative images of Gomori-trichrome staining and quantification of fibrosis of BaCl2-treated muscles from aged mice treated with the control VMO (n = 10), pA-VMOs (n = 9) or the 5′ss-VMO (n = 10). For ac, n represents biological replicates consisting of tibialis anterior muscles isolated from individual mice. Scale bars, 100 μm. Error bars represent s.e.m. *P < 0.05; **P < 0.01; unpaired Student’s t test.

Source data

Extended Data Table 1 Pdgfra gene-specific primers used in 3′ RACE
Extended Data Table 2 Primers for Pdgfra variant quantification

Supplementary information

Supplementary Figure 1

This file contains gel source data for the Main and Extended Data Figures. (PDF 1149 kb)

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Mueller, A., van Velthoven, C., Fukumoto, K. et al. Intronic polyadenylation of PDGFRα in resident stem cells attenuates muscle fibrosis. Nature 540, 276–279 (2016). https://doi.org/10.1038/nature20160

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