Activation of Hedgehog signaling by loss of GNAS causes heterotopic ossification

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Abstract

Heterotopic ossification, the pathologic formation of extraskeletal bone, occurs as a common complication of trauma or in genetic disorders and can be disabling and lethal. However, the underlying molecular mechanisms are largely unknown. Here we demonstrate that Gαs restricts bone formation to the skeleton by inhibiting Hedgehog signaling in mesenchymal progenitor cells. In progressive osseous heteroplasia, a human disease caused by null mutations in GNAS, which encodes Gαs, Hedgehog signaling is upregulated in ectopic osteoblasts and progenitor cells. In animal models, we show that genetically-mediated ectopic Hedgehog signaling is sufficient to induce heterotopic ossification, whereas inhibition of this signaling pathway by genetic or pharmacological means strongly reduces the severity of this condition. As our previous work has shown that GNAS gain-of-function mutations upregulate WNT–β-catenin signaling in osteoblast progenitor cells, resulting in their defective differentiation and fibrous dysplasia, we identify Gαs as a key regulator of proper osteoblast differentiation through its maintenance of a balance between the Wnt–β-catenin and Hedgehog pathways. Also, given the results here of the pharmacological studies in our mouse model, we propose that Hedgehog inhibitors currently used in the clinic for other conditions, such as cancer, may possibly be repurposed for treating heterotopic ossification and other diseases caused by GNAS inactivation.

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Figure 1: Loss of Gnas in limb mesenchyme leads to heterotopic ossification.
Figure 2: Loss of Gnas in adult subcutaneous tissue leads to heterotopic ossification.
Figure 3: Removal of Gnas from mesenchymal progenitor cells upregulates Hedgehog signaling in vitro and in vivo.
Figure 4: Gαs acts through cAMP and PKA to suppress Hedgehog signaling.
Figure 5: Reducing Hedgehog signaling inhibits heterotopic ossification in vivo and in vitro.
Figure 6: Hedgehog signaling is activated in ectopic bone from individuals with POH, and activation of Hedgehog signaling is sufficient to cause heterotopic ossification.

Change history

  • 18 October 2013

     In the version of this article initially published online, the gene symbol for Prrx1 was misidentified as Prdx1 in four instances. The error has been corrected for the print, PDF and HTML versions of this article.

References

  1. 1

    Shore, E.M. & Kaplan, F.S. Inherited human diseases of heterotopic bone formation. Nat. Rev. Rheumatol. 6, 518–527 (2010).

  2. 2

    Shore, E.M. et al. A recurrent mutation in the BMP type I receptor ACVR1 causes inherited and sporadic fibrodysplasia ossificans progressiva. Nat. Genet. 38, 525–527 (2006).

  3. 3

    Wozney, J.M. et al. Novel regulators of bone formation: molecular clones and activities. Science 242, 1528–1534 (1988).

  4. 4

    Kaplan, F.S., Hahn, G.V. & Zasloff, M.A. Heterotopic ossification: two rare forms and what they can teach us. J. Am. Acad. Orthop. Surg. 2, 288–296 (1994).

  5. 5

    Eddy, M.C. et al. Deficiency of the α-subunit of the stimulatory G protein and severe extraskeletal ossification. J. Bone Miner. Res. 15, 2074–2083 (2000).

  6. 6

    Trüeb, R.M., Panizzon, R.G. & Burg, G. Cutaneous ossification in Albright's hereditary osteodystrophy. Dermatology 186, 205–209 (1993).

  7. 7

    Shore, E.M. et al. Paternally inherited inactivating mutations of the GNAS1 gene in progressive osseous heteroplasia. N. Engl. J. Med. 346, 99–106 (2002).

  8. 8

    Plagge, A., Kelsey, G. & Germain-Lee, E.L. Physiological functions of the imprinted Gnas locus and its protein variants Gαs and XLαs in human and mouse. J. Endocrinol. 196, 193–214 (2008).

  9. 9

    Riminucci, M., Robey, P.G., Saggio, I. & Bianco, P. Skeletal progenitors and the GNAS gene: fibrous dysplasia of bone read through stem cells. J. Mol. Endocrinol. 45, 355–364 (2010).

  10. 10

    Regard, J.B. et al. Wnt/β-catenin signaling is differentially regulated by Gα proteins and contributes to fibrous dysplasia. Proc. Natl. Acad. Sci. USA 108, 20101–20106 (2011).

  11. 11

    Wu, J.Y. et al. Gsα enhances commitment of mesenchymal progenitors to the osteoblast lineage but restrains osteoblast differentiation in mice. J. Clin. Invest. 121, 3492–3504 (2011).

  12. 12

    Jiang, J. & Struhl, G. Protein kinase A and hedgehog signaling in Drosophila limb development. Cell 80, 563–572 (1995).

  13. 13

    Tuson, M., He, M. & Anderson, K.V. Protein kinase A acts at the basal body of the primary cilium to prevent Gli2 activation and ventralization of the mouse neural tube. Development 138, 4921–4930 (2011).

  14. 14

    Jiang, J. & Hui, C.C. Hedgehog signaling in development and cancer. Dev. Cell 15, 801–812 (2008).

  15. 15

    St-Jacques, B., Hammerschmidt, M. & McMahon, A.P. Indian hedgehog signaling regulates proliferation and differentiation of chondrocytes and is essential for bone formation. Genes Dev. 13, 2072–2086 (1999).

  16. 16

    Bastepe, M. et al. Stimulatory G protein directly regulates hypertrophic differentiation of growth plate cartilage in vivo. Proc. Natl. Acad. Sci. USA 101, 14794–14799 (2004).

  17. 17

    Sakamoto, A., Chen, M., Kobayashi, T., Kronenberg, H.M. & Weinstein, L.S. Chondrocyte-specific knockout of the G protein Gsα leads to epiphyseal and growth plate abnormalities and ectopic chondrocyte formation. J. Bone Miner. Res. 20, 663–671 (2005).

  18. 18

    Sakamoto, H. et al. A kinetic study of the mechanism of conversion of α-hydroxyheme to verdoheme while bound to heme oxygenase. Biochem. Biophys. Res. Commun. 338, 578–583 (2005).

  19. 19

    Ogden, S.K. et al. G protein Gαi functions immediately downstream of Smoothened in Hedgehog signalling. Nature 456, 967–970 (2008).

  20. 20

    Riobo, N.A., Saucy, B., Dilizio, C. & Manning, D.R. Activation of heterotrimeric G proteins by Smoothened. Proc. Natl. Acad. Sci. USA 103, 12607–12612 (2006).

  21. 21

    Low, W.C. et al. The decoupling of Smoothened from Gαi proteins has little effect on Gli3 protein processing and Hedgehog-regulated chick neural tube patterning. Dev. Biol. 321, 188–196 (2008).

  22. 22

    Vanden Bossche, L. & Vanderstraeten, G. Heterotopic ossification: a review. J. Rehabil. Med. 37, 129–136 (2005).

  23. 23

    Pignolo, R.J. et al. Heterozygous inactivation of Gnas in adipose-derived mesenchymal progenitor cells enhances osteoblast differentiation and promotes heterotopic ossification. J. Bone Miner. Res. 26, 2647–2655 (2011).

  24. 24

    Huso, D.L. et al. Heterotopic ossifications in a mouse model of Albright hereditary osteodystrophy. PLoS ONE 6, e21755 (2011).

  25. 25

    Nakashima, K. et al. The novel zinc finger-containing transcription factor osterix is required for osteoblast differentiation and bone formation. Cell 108, 17–29 (2002).

  26. 26

    Hill, T.P., Spater, D., Taketo, M.M., Birchmeier, W. & Hartmann, C. Canonical Wnt/β-catenin signaling prevents osteoblasts from differentiating into chondrocytes. Dev. Cell 8, 727–738 (2005).

  27. 27

    Day, T.F., Guo, X., Garrett-Beal, L. & Yang, Y. Wnt/β-catenin signaling in mesenchymal progenitors controls osteoblast and chondrocyte differentiation during vertebrate skeletogenesis. Dev. Cell 8, 739–750 (2005).

  28. 28

    Hu, H. et al. Sequential roles of Hedgehog and Wnt signaling in osteoblast development. Development 132, 49–60 (2005).

  29. 29

    Holmen, S.L. et al. Essential role of β-catenin in postnatal bone acquisition. J. Biol. Chem. 280, 21162–21168 (2005).

  30. 30

    Logan, C.Y. & Nusse, R. The Wnt signaling pathway in development and disease. Annu. Rev. Cell Dev. Biol. 20, 781–810 (2004).

  31. 31

    Keely, S.L. Jr., Corbin, J.D. & Park, C.R. On the question of translocation of heart cAMP-dependent protein kinase. Proc. Natl. Acad. Sci. USA 72, 1501–1504 (1975).

  32. 32

    Seamon, K. & Daly, J.W. Activation of adenylate cyclase by the diterpene forskolin does not require the guanine nucleotide regulatory protein. J. Biol. Chem. 256, 9799–9801 (1981).

  33. 33

    Goodrich, L.V., Milenkovic, L., Higgins, K.M. & Scott, M.P. Altered neural cell fates and medulloblastoma in mouse patched mutants. Science 277, 1109–1113 (1997).

  34. 34

    Ribes, V. & Briscoe, J. Establishing and interpreting graded Sonic Hedgehog signaling during vertebrate neural tube patterning: the role of negative feedback. Cold Spring Harb. Perspect. Biol. 1, a002014 (2009).

  35. 35

    Svärd, J. et al. Genetic elimination of Suppressor of fused reveals an essential repressor function in the mammalian Hedgehog signaling pathway. Dev. Cell 10, 187–197 (2006).

  36. 36

    Ingham, P.W. & McMahon, A.P. Hedgehog signaling in animal development: paradigms and principles. Genes Dev. 15, 3059–3087 (2001).

  37. 37

    Wang, B., Fallon, J.F. & Beachy, P.A. Hedgehog-regulated processing of Gli3 produces an anterior/posterior repressor gradient in the developing vertebrate limb. Cell 100, 423–434 (2000).

  38. 38

    Zhang, Q. et al. Multiple Ser/Thr-rich degrons mediate the degradation of Ci/Gli by the Cul3-HIB/SPOP E3 ubiquitin ligase. Proc. Natl. Acad. Sci. USA 106, 21191–21196 (2009).

  39. 39

    Pan, Y., Wang, C. & Wang, B. Phosphorylation of Gli2 by protein kinase A is required for Gli2 processing and degradation and the Sonic Hedgehog-regulated mouse development. Dev. Biol. 326, 177–189 (2009).

  40. 40

    te Welscher, P. et al. Progression of vertebrate limb development through SHH-mediated counteraction of GLI3. Science 298, 827–830 (2002).

  41. 41

    Matise, M.P., Epstein, D.J., Park, H.L., Platt, K.A. & Joyner, A.L. Gli2 is required for induction of floor plate and adjacent cells, but not most ventral neurons in the mouse central nervous system. Development 125, 2759–2770 (1998).

  42. 42

    Taipale, J. et al. Effects of oncogenic mutations in Smoothened and Patched can be reversed by cyclopamine. Nature 406, 1005–1009 (2000).

  43. 43

    Kim, J., Lee, J.J., Gardner, D. & Beachy, P.A. Arsenic antagonizes the Hedgehog pathway by preventing ciliary accumulation and reducing stability of the Gli2 transcriptional effector. Proc. Natl. Acad. Sci. USA 107, 13432–13437 (2010).

  44. 44

    Lauth, M., Bergstrom, A., Shimokawa, T. & Toftgard, R. Inhibition of GLI-mediated transcription and tumor cell growth by small-molecule antagonists. Proc. Natl. Acad. Sci. USA 104, 8455–8460 (2007).

  45. 45

    Bai, C.B. & Joyner, A.L. Gli1 can rescue the in vivo function of Gli2. Development 128, 5161–5172 (2001).

  46. 46

    Joeng, K.S. & Long, F. The Gli2 transcriptional activator is a crucial effector for Ihh signaling in osteoblast development and cartilage vascularization. Development 136, 4177–4185 (2009).

  47. 47

    Mao, J. et al. A novel somatic mouse model to survey tumorigenic potential applied to the Hedgehog pathway. Cancer Res. 66, 10171–10178 (2006).

  48. 48

    Mukhopadhyay, S. et al. The ciliary G-protein–coupled receptor Gpr161 negatively regulates the Sonic Hedgehog pathway via cAMP signaling. Cell 152, 210–223 (2013).

  49. 49

    Day, T.F. & Yang, Y. Wnt and hedgehog signaling pathways in bone development. J. Bone Joint Surg. Am. 90 (suppl. 1), 19–24 (2008).

  50. 50

    Joeng, K.S., Schumacher, C.A., Zylstra-Diegel, C.R., Long, F. & Williams, B.O. Lrp5 and Lrp6 redundantly control skeletal development in the mouse embryo. Dev. Biol. 359, 222–229 (2011).

  51. 51

    Holmen, S.L. et al. Decreased BMD and limb deformities in mice carrying mutations in both Lrp5 and Lrp6. J. Bone Miner. Res. 19, 2033–2040 (2004).

  52. 52

    de Boer, J. et al. Wnt signaling inhibits osteogenic differentiation of human mesenchymal stem cells. Bone 34, 818–826 (2004).

  53. 53

    Cho, H.H. et al. Endogenous Wnt signaling promotes proliferation and suppresses osteogenic differentiation in human adipose derived stromal cells. Tissue Eng. 12, 111–121 (2006).

  54. 54

    Boland, G.M., Perkins, G., Hall, D.J. & Tuan, R.S. Wnt 3a promotes proliferation and suppresses osteogenic differentiation of adult human mesenchymal stem cells. J. Cell. Biochem. 93, 1210–1230 (2004).

  55. 55

    Chen, Y. β-catenin signaling plays a disparate role in different phases of fracture repair: implications for therapy to improve bone healing. PLoS Med. 4, e249 (2007).

  56. 56

    Kim, J.B. et al. Bone regeneration is regulated by Wnt signaling. J. Bone Miner. Res. 22, 1913–1923 (2007).

  57. 57

    Cooper, K.L. et al. Initiation of proximal-distal patterning in the vertebrate limb by signals and growth. Science 332, 1083–1086 (2011).

  58. 58

    Roselló-Diez, A., Ros, M.A. & Torres, M. Diffusible signals, not autonomous mechanisms, determine the main proximodistal limb subdivision. Science 332, 1086–1088 (2011).

  59. 59

    Zhang, D. et al. ALK2 functions as a BMP type I receptor and induces Indian hedgehog in chondrocytes during skeletal development. J. Bone Miner. Res. 18, 1593–1604 (2003).

  60. 60

    Shimono, K. et al. Potent inhibition of heterotopic ossification by nuclear retinoic acid receptor-γ agonists. Nat. Med. 17, 454–460 (2011).

  61. 61

    Chen, M. et al. Increased glucose tolerance and reduced adiposity in the absence of fasting hypoglycemia in mice with liver-specific Gsα deficiency. J. Clin. Invest. 115, 3217–3227 (2005).

  62. 62

    Logan, M. et al. Expression of Cre Recombinase in the developing mouse limb bud driven by a Prrx1 enhancer. Genesis 33, 77–80 (2002).

  63. 63

    Yu, K. et al. Conditional inactivation of FGF receptor 2 reveals an essential role for FGF signaling in the regulation of osteoblast function and bone growth. Development 130, 3063–3074 (2003).

  64. 64

    Nelson, D.K. & Williams, T. Frontonasal process-specific disruption of AP-2α results in postnatal midfacial hypoplasia, vascular anomalies, and nasal cavity defects. Dev. Biol. 267, 72–92 (2004).

  65. 65

    Mak, K.K., Chen, M.H., Day, T.F., Chuang, P.T. & Yang, Y. Wnt–β-catenin signaling interacts differentially with Ihh signaling in controlling endochondral bone and synovial joint formation. Development 133, 3695–3707 (2006).

  66. 66

    Bai, C.B. & Joyner, A.L. Gli1 can rescue the in vivo function of Gli2. Development 128, 5161–5172 (2001).

  67. 67

    Corrales, J.D., Blaess, S., Mahoney, E.M. & Joyner, A.L. The level of sonic hedgehog signaling regulates the complexity of cerebellar foliation. Development 133, 1811–1821 (2006).

  68. 68

    Long, F., Zhang, X.M., Karp, S., Yang, Y. & McMahon, A.P. Genetic manipulation of hedgehog signaling in the endochondral skeleton reveals a direct role in the regulation of chondrocyte proliferation. Development 128, 5099–5108 (2001).

  69. 69

    Mao, J. et al. A novel somatic mouse model to survey tumorigenic potential applied to the Hedgehog pathway. Cancer Res. 66, 10171–10178 (2006).

  70. 70

    Regard, J.B. et al. Probing cell type-specific functions of Gi in vivo identifies GPCR regulators of insulin secretion. J. Clin. Invest. 117, 4034–4043 (2007).

  71. 71

    Yang, J. et al. Loss of signaling through the G protein, Gz, results in abnormal platelet activation and altered responses to psychoactive drugs. Proc. Natl. Acad. Sci. USA 97, 9984–9989 (2000).

  72. 72

    Livak, K.J. & Schmittgen, T.D. Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCT method. Methods 25, 402–408 (2001).

  73. 73

    Yang, Y., Topol, L., Lee, H. & Wu, J. Wnt5a and Wnt5b exhibit distinct activities in coordinating chondrocyte proliferation and differentiation. Development 130, 1003–1015 (2003).

  74. 74

    Storm, E.E. & Kingsley, D.M. GDF5 coordinates bone and joint formation during digit development. Dev. Biol. 209, 11–27 (1999).

  75. 75

    Bai, C.B., Stephen, D. & Joyner, A.L. All mouse ventral spinal cord patterning by hedgehog is Gli dependent and involves an activator function of Gli3. Dev. Cell 6, 103–115 (2004).

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Acknowledgements

We thank the entire Yang lab for stimulating discussions. We thank J. Fekecs and P. Andre for helping with the data illustration. The work in the Yang and Weinstein labs was supported by the intramural research programs of NHGRI and NIDDK at the US National Institutes of Health (NIH), respectively. We thank R. Caron for his work on the POH lesion histological analyses. The rabbit anti-Gli3 antibody was provided by S. Mackem (NIH NCI), and the dnPKA adenovirus was a gift from C.-M. Fan (Carnegie Institution for Science). The work in the Kaplan and Shore lab was supported by the Progressive Osseous Heteroplasia Association, the University of Pennsylvania Center for Research in FOP and Related Disorders, the Penn Center for Musculoskeletal Disorders (NIH NIAMS P30-AR050950), the Isaac and Rose Nassau Professorship of Orthopaedic Molecular Medicine and NIH NIAMS (R01-AR046831 and R01-AR41916).

Author information

J.B.R., D.M. and Y.Y. designed the experiments and analyzed the data. J.B.R., D.M., F.S.K., E.M.S. and Y.Y. wrote the manuscript. J.B.R., D.M., J.G.-J., M.J. and J.L. carried out the actual experiments. F.S.K. saw the patients with POH. F.S.K. and E.M.S. provided the POH samples and carried out the GLI immunohistochemistry on the human samples. M.C. and L.S.W. generated and provided the Gnasfl/fl mice.

Correspondence to Jean B Regard or Yingzi Yang.

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Regard, J., Malhotra, D., Gvozdenovic-Jeremic, J. et al. Activation of Hedgehog signaling by loss of GNAS causes heterotopic ossification. Nat Med 19, 1505–1512 (2013). https://doi.org/10.1038/nm.3314

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