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. Author manuscript; available in PMC: 2024 Dec 25.
Published in final edited form as: Biochem Biophys Res Commun. 2023 Oct 24;688:149147. doi: 10.1016/j.bbrc.2023.149147

Enhanced BMP signaling in Cathepsin K-positive tendon progenitors induces heterotopic ossification

Hiroyuki Yamaguchi 1,#, Margaret Li 1,2,#, Megumi Kitami 3,4, Sowmya Swaminathan 1,5, Yuji Mishina 6, Yoshihiro Komatsu 1,7,*
PMCID: PMC10952113  NIHMSID: NIHMS1972849  PMID: 37948912

Abstract

Heterotopic ossification (HO) is abnormal bone growth in soft tissues that results from injury, trauma, and rare genetic disorders. Bone morphogenetic proteins (BMPs) are critical osteogenic regulators which are involved in HO. However, it remains unclear how BMP signaling interacts with other extracellular stimuli to form HO. To address this question, using the Cre-loxP recombination system in mice, we conditionally expressed the constitutively activated BMP type I receptor ALK2 with a Q207D mutation (Ca-ALK2) in Cathepsin K-Cre labeled tendon progenitors (hereafter “Ca-Alk2:Ctsk-Cre”). Ca-Alk2:Ctsk-Cre mice were viable but they formed spontaneous HO in the Achilles tendon. Histological and molecular marker analysis revealed that HO is formed via endochondral ossification. Ectopic chondrogenesis coincided with enhanced GLI1 production, suggesting that elevated Hedgehog (Hh) signaling is involved in the pathogenesis of HO. Interestingly, focal adhesion kinase, a critical mediator for the mechanotransduction pathway, was also activated in Ca-Alk2:Ctsk-Cre mice. Our findings suggest that enhanced BMP signaling may elevate Hh and mechanotransduction pathways, thereby causing HO in the regions of the Achilles tendon.

Keywords: BMP signaling, Hedgehog signaling, Heterotopic ossification, Mechanotransduction, Mouse

Introduction

Heterotopic ossification (HO) is characterized by extraskeletal bone formation in soft tissues, where the bone tissues normally do not develop1,2. HO frequently results in patients with reduced mobility and severe pain, and thus severely affects their quality of life.

Following trauma and/or injuries, the destruction of connective tissue structure occurs and osteogenic growth factors including bone morphogenetic proteins (BMPs) are released and initiate the induction process of ectopic bone formation3. After the discovery of BMPs, accumulated studies have revealed their significant roles in skeletal development4,5. Upon the binding of BMP ligands, BMP type II receptors phosphorylate and activate associated BMP type I receptors, which in turn transduce intracellular signaling by phosphorylating Smad1/5/9, a canonical downstream of BMP signaling6,7. Previous studies have demonstrated that a point mutation in one of the BMP type I receptors ALK2 (R206H) leads to the alteration of ligand selectivity and causes fibrodysplasia ossificans progressiva (FOP), a severe form of HO8,9. Recent work has led to clinical trials to develop therapies for curing FOP10-12. While it is now clear that multiple growth factor signaling pathways such as Hedgehog (Hh) signaling are responsible for HO13,14, recent studies highlight the importance of mechanical cues from the microenvironment during skeletal development15,16. For example, suppression of the mechanotransduction pathway by genetic inactivation and/or a chemical blocker approach inhibits burn and injury-induced HO in mice17,18. These studies demonstrate that dysregulated mechanotransduction pathway also plays a role in the etiology of HO.

In this study, we examined the etiology of HO in the Achilles tendon. Interestingly, Hh and mechanotransduction pathways were elevated during ectopic osteochondrogenesis, suggesting dysregulated molecular and mechanical stimuli may be associated with HO in Ca-Alk2:Ctsk-Cre mice.

Materials and Methods

Animals

To generate Ca-Alk2:Ctsk-Cre mice, we crossed Ca-Alk2 transgenic mice with Ctsk-Cre knock-in mice19,20. To examine Ctsk-Cre activity, we crossed Ctsk-Cre mice with Rosa26-GFP reporter mice21. These mice were maintained in the animal facility of The University of Texas McGovern Medical School in Houston. The experimental protocol (AWC-21-0127) was reviewed and approved by the Animal Welfare Committee and the Institutional Animal Care and Use Committee of The University of Texas McGovern Medical School at Houston.

The sex of animals

In this study, we focused on analyzing male mice to avoid the confounding variable of the hormone cycle in females, which may indirectly affect the bone-related phenotypes in Ca-Alk2:Ctsk-Cre mice.

Micro-computed tomography analysis

Micro-computed tomography (μCT) images were scanned with a CT system at 90 kV energy and 88 μA intensity (CosmoScanGXIII; Rigaku Corporation, Tokyo, Japan). The slices were reconstructed to produce 2D and 3D images, and bone volume was measured using Analyze12.0 (AnalyzeDirect Inc., Overland Park, KS).

Histological Analysis

Hematoxylin and eosin, Safranin O, Picrosirius red staining, and immunostaining analysis were performed as described previously22-25. Primary antibodies used in immunostaining were as follows: p-SMAD1/5/9 (Cell Signaling; 9511, 1:50,), Ki-67 (BD Biosciences; 550609, 1:250), SOX9 (Santa Cruz; sc-20095, 1:50), RUNX2 (Cell Signaling; 12556, 1:250), GLI1 (R&D System; AF3455, 1:100), and p-FAK (Invitrogen; 44-626G, 1:200). Stained slides were examined with an Olympus FluoView FV1000 laser scanning confocal microscope using the software FV10-ASW Viewer (version 4.2). At least three images from each slide were obtained and quantified for histological analysis.

Statistical analysis

A two-tailed Student’s t-test was used for comparisons between the two groups (GraphPad Prism 9 Software). A p-value of less than 0.05 was considered statistically significant.

Results and Discussion

Enhanced BMP signaling in Cathepsin K-labeled tendon progenitors leads to HO in mice.

The previous report demonstrates that Scleraxis (Scx) lineage cells in muscle and tendon contribute to form HO26. A recent study demonstrates that Cathepsin K (Ctsk)-Cre labels a subpopulation of tendon-derived progenitors marked by Scx in mice27. To investigate whether enhanced BMP signaling in Ctsk-Cre labeled tendon progenitors effectively induces HO, we crossed Ca-Alk2 mice with Ctsk-Cre mice (hereafter “Ca-Alk2:Ctsk-Cre”). At 5 months, the Ca-Alk2:Ctsk-Cre mice began to show mobility problems with loss of body weight (Fig. 1A). By crossing with Ctsk-Cre mice and Rosa26-GFP reporter mice, we confirmed that the Achilles tendon was robustly labeled by Ctsk-Cre (Fig. 1B). Micro-computed tomography (μCT) analysis showed spontaneous and progressive tendon ossification in the Achilles tendon with a 100% phenotypic penetrance in Ca-Alk2:Ctsk-Cre mice (Fig. 1C, D). These data suggest that Ctsk-Cre labeled tendon-derived progenitors are critical to forming HO in Ca-Alk2:Ctsk-Cre mice.

Fig. 1. Enhanced BMP signaling causes HO in the Achilles tendons.

Fig. 1.

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(A) Quantification of the body weight of 5-month-old mice (5M). Data are as mean ± SD, n=5-6, Student’s Ttest, *p<0.05. (B) The Ctsk-Cre activity was examined by crossing Ctsk-Cre mice and Rosa26-GFP reporter mice using 5-month-old mice (5M). After obtaining cryosection, tissues were counterstained with DAPI. (C) μCT images of the ankle. Yellow lines indicate the levels of the sectional images. (D) Quantification of the HO volume at 5M. Data are as mean ± SD, n=5-6, Student’s T-test, *p<0.05.

Enhanced BMP signaling stimulates ectopic osteochondrogenesis in the Achilles tendons.

To reveal the mechanisms of how enhanced BMP signaling via ALK2 in tendon-derived progenitors induces HO, we first examined the levels of phosphorylated SMAD1/5/9 using 2-month-old mice when ectopic ossification is still not evident in Ca-Alk2:Ctsk-Cre mice. Achilles tendons of Ca-Alk2:Ctsk-Cre mice exhibited higher levels of phosphorylated SMAD1/5/9 compared with controls (Fig. 2A). Next, we examined the cell proliferation activity in Ca-Alk2:Ctsk-Cre mice. Immunohistological analysis using the Ki-67 antibody showed that the cell proliferation activity is intact in Ca-Alk2:Ctsk-Cre mice (Fig. 2B). However, expression analysis using SOX9 (chondrogenic marker), and RUNX2 (osteogenic marker) antibodies revealed that osteochondrogenic differentiation is ectopically enhanced in the Achilles tendons of Ca-Alk2:Ctsk-Cre mice (Fig. 2C). Histological analysis by Hematoxylin and eosin staining (revealing cell/tissue morphology) (Fig. 2D), Safranin O staining (detecting cartilage) (Fig. 2E) and Picrosirius red staining (detecting collagen fibers) (Fig. 2F, G) further confirmed that HO is formed via endochondral ossification. These data suggest that the formation of HO was not caused by an increase in the proliferation of tendon-derived progenitors but by enhanced osteochondrogenic differentiation in the Achilles tendons.

Fig. 2. Enhanced BMP signaling stimulates ectopic osteochondrogenesis in the Achilles tendons in Ca-Alk2:Ctsk-Cre mice.

Fig. 2.

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(A, B, C) Immunohistochemistry of phospho (P)-SMAD1/5/9, Ki-67, SOX9, and RUNX2, and corresponding quantification of the Achilles tendons in 2-month-old mice (2M). Data are as mean ± SD, n = 4 individual samples in each group. Arrows indicate the nuclear localization of P-SMAD1/5/9 or RUNX2. **p<0.01, ***p<0.001, ****p<0.0001, ns, not significant. (D) Hematoxylin and eosin staining, (E) Safranin O staining, and (F) Picrosirius red staining at 5-month-old mice (5M). Boxes show high-magnification images of each staining. (G) In Picrosirius red staining, the signal intensity of type I collagen was measured and quantified. Data are as mean ± SD, n = 4 individual samples in each group. **p<0.01, ****p<0.0001, ns, not significant.

Altered Hh and mechanotransduction pathways are associated with HO in Ca-Alk2:Ctsk-Cre mice.

Previous studies have shown that connective tissue abnormalities due to either developmental defects or injury can cause HO accompanied by the elevation of Hh signaling28,29. In addition, activation of Hh signaling in the tendon induces ectopic osteochondrogenesis27. Therefore, to reveal whether Hh signaling is dysregulated in Ca-Alk2:Ctsk-Cre mice, we examined GLI1 production. Compared with control mice, high levels of GLI1 were detected in the Ca-Alk2:Ctsk-Cre’s tendon (Fig. 3A). Because the pattern of GLI1 production overlapped with the ectopic SOX9/RUNX2 production in Ca-Alk2:Ctsk-Cre mice (Fig. 2C), these results suggest that enhanced BMP signaling may lead to the dysregulation of Hh signaling.

Fig. 3. Dysregulated Hh and mechanotransduction pathways are associated with HO in Ca-Alk2:Ctsk-Cre mice.

Fig. 3.

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(A) Immunohistochemistry of GLI1 in the Achilles tendons at 2-month-old mice (2M). Data are as mean ± SD, n = 4 individual samples in each group. **p<0.01. (B) Immunohistochemistry of phospho (P)-FAK in the Achilles tendons at 2M. Data are as mean ± SD, n = 4 individual samples in each group. *p<0.05. (C) A schematic model of this study.

Because the Achilles tendon is the largest tendinous structure which bears the highest mechanical stress in the body30, and active mechanotransduction in mesenchymal progenitors impacts the osteochondrogenic cell fate in HO17,18, we hypothesized that in addition to the elevation of Hh signaling, significant amount of mechanical stress in the Achilles tendon may participate in triggering ectopic osteochondrogenesis. To test our hypothesis, levels of phosphorylated focal adhesion kinase (p-FAK), a critical mediator for the mechanotransduction pathway, were examined by immunohistochemistry. Interestingly, higher levels of p-FAK were detected in the Achilles tendon of Ca-Alk2:Ctsk-Cre mice compared with controls (Fig. 3B), suggesting that an altered mechanotransduction pathway may also be involved in the pathogenesis of HO in the Achilles tendons.

To date, numerous studies demonstrate that activation of BMP signaling drives the cell fate of mesenchymal progenitors toward osteochondrogenesis3,5. However, it remains unclear how BMP signaling regulates skeletogenic cell fate in the context of HO. Recently, it has been reported that a self-amplifying loop of Shh and Yap drives the formation of HO31. In addition, mechanotransductive signaling through Yap/Taz/FAK is increased during the formation of trauma-induced HO17. These studies highlight the importance of Hh and mechanotransduction pathways in HO. However, the precise mechanism by which Hh and mechanotransduction pathways in Ca-Alk2:Ctsk-Cre mice remains unclear. For example, what are the Hh ligand(s) to stimulate Hh signaling? Why tendon progenitors in Ca-Alk2:Ctsk-Cre mice are sensitive to mechanical stimuli (Fig. 3C)? Further studies will be required to clarify the mechanisms of how aberrant BMP signaling is associated with dysregulated Hh and mechanotransduction pathways to induce ectopic bone in multiple forms of HO such as injured and traumatic HO, and genetic HO.

Highlight.

  • Cathepsin K-Cre-driven enhanced BMP signaling induces heterotopic ossification (HO) in mice.

  • Ectopic endochondral ossification is associated with the pathogenesis of HO in the tendon.

  • Elevated hedgehog and mechanotransduction pathways are associated with HO in mice.

Acknowledgments

We thank Dr. Brendan Lee for acquiring the Ctsk-Cre mice. This study was supported by NIDCR/NIH R01DE025897 (Y.K.), and the Bone Disease Program of Texas Rolanette and Berdon Lawrence Research Award (H.Y.).

Footnotes

Declaration of Conflict of Interests

The authors declared no potential conflicts of interest for the research, authorship, and/or publication of this article.

References

  • 1.Kaplan FS, Pignolo RJ & Shore EM Heterotopic Ossification: The Keys to the Kingdom. Bone 109, 1–2, doi: 10.1016/j.bone.2018.03.001 (2018). [DOI] [PubMed] [Google Scholar]
  • 2.Agarwal S, Sorkin M & Levi B Heterotopic Ossification and Hypertrophic Scars. Clin Plast Surg 44, 749–755, doi: 10.1016/j.cps.2017.05.006 (2017). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Salazar VS, Gamer LW & Rosen V BMP signalling in skeletal development, disease and repair. Nature reviews. Endocrinology 12, 203–221, doi: 10.1038/nrendo.2016.12 (2016). [DOI] [PubMed] [Google Scholar]
  • 4.Urist MR Bone: formation by autoinduction. Science 150, 893–899, doi: 10.1126/science.150.3698.893 (1965). [DOI] [PubMed] [Google Scholar]
  • 5.Lyons KM & Rosen V BMPs, TGFbeta, and border security at the interzone. Current topics in developmental biology 133, 153–170, doi: 10.1016/bs.ctdb.2019.02.001 (2019). [DOI] [PubMed] [Google Scholar]
  • 6.Sanchez-Duffhues G, Williams E, Goumans MJ, Heldin CH & Ten Dijke P Bone morphogenetic protein receptors: Structure, function and targeting by selective small molecule kinase inhibitors. Bone 138, 115472, doi: 10.1016/j.bone.2020.115472 (2020). [DOI] [PubMed] [Google Scholar]
  • 7.Miyazawa K & Miyazono K Regulation of TGF-beta Family Signaling by Inhibitory Smads. Cold Spring Harbor perspectives in biology 9, doi: 10.1101/cshperspect.a022095 (2017). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Kaplan FS, Al Mukaddam M, Stanley A, Towler OW & Shore EM Fibrodysplasia ossificans progressiva (FOP): A disorder of osteochondrogenesis. Bone 140, 115539, doi: 10.1016/j.bone.2020.115539 (2020). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Shore EM et al. A recurrent mutation in the BMP type I receptor ACVR1 causes inherited and sporadic fibrodysplasia ossificans progressiva. Nature genetics 38, 525–527, doi: 10.1038/ng1783 (2006). [DOI] [PubMed] [Google Scholar]
  • 10.Hatsell SJ et al. ACVR1R206H receptor mutation causes fibrodysplasia ossificans progressiva by imparting responsiveness to activin A. Sci Transl Med 7, 303ra137, doi: 10.1126/scitranslmed.aac4358 (2015). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Shimono K. et al. Potent inhibition of heterotopic ossification by nuclear retinoic acid receptor-gamma agonists. Nature medicine 17, 454–460, doi: 10.1038/nm.2334 (2011). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Lees-Shepard JB et al. Activin-dependent signaling in fibro/adipogenic progenitors causes fibrodysplasia ossificans progressiva. Nature communications 9, 471, doi: 10.1038/s41467-018-02872-2 (2018). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Xu R, Hu J, Zhou X & Yang Y Heterotopic ossification: Mechanistic insights and clinical challenges. Bone 109, 134–142, doi: 10.1016/j.bone.2017.08.025 (2018). [DOI] [PubMed] [Google Scholar]
  • 14.Hwang CD et al. Contemporary perspectives on heterotopic ossification. JCI Insight 7, doi: 10.1172/jci.insight.158996 (2022). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Zhou T. et al. Piezo1/2 mediate mechanotransduction essential for bone formation through concerted activation of NFAT-YAP1-ss-catenin. eLife 9, doi: 10.7554/eLife.52779 (2020). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Wang L. et al. Mechanical sensing protein PIEZO1 regulates bone homeostasis via osteoblast-osteoclast crosstalk. Nature communications 11, 282, doi: 10.1038/s41467-019-14146-6 (2020). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Huber AK et al. Immobilization after injury alters extracellular matrix and stem cell fate. The Journal of clinical investigation 130, 5444–5460, doi: 10.1172/JCI136142 (2020). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Pagani CA et al. Discoidin domain receptor 2 regulates aberrant mesenchymal lineage cell fate and matrix organization. Sci Adv 8, eabq6152, doi: 10.1126/sciadv.abq6152 (2022). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Fukuda T. et al. Generation of a mouse with conditionally activated signaling through the BMP receptor, ALK2. Genesis 44, 159–167, doi: 10.1002/dvg.20201 (2006). [DOI] [PubMed] [Google Scholar]
  • 20.Nakamura T. et al. Estrogen prevents bone loss via estrogen receptor alpha and induction of Fas ligand in osteoclasts. Cell 130, 811–823, doi: 10.1016/j.cell.2007.07.025 (2007). [DOI] [PubMed] [Google Scholar]
  • 21.Madisen L. et al. A robust and high-throughput Cre reporting and characterization system for the whole mouse brain. Nat Neurosci 13, 133–140, doi: 10.1038/nn.2467 (2010). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Noda K, Kitami M, Kitami K, Kaku M & Komatsu Y Canonical and noncanonical intraflagellar transport regulates craniofacial skeletal development. Proceedings of the National Academy of Sciences of the United States of America 113, E2589–2597, doi: 10.1073/pnas.1519458113 (2016). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Yamaguchi H. et al. Temporospatial regulation of intraflagellar transport is required for the endochondral ossification in mice. Developmental biology 482, 91–100, doi: 10.1016/j.ydbio.2021.12.004 (2022). [DOI] [PubMed] [Google Scholar]
  • 24.Yamaguchi H, Meyer MD, He L & Komatsu Y Disruption of Trip11 in cranial neural crest cells is associated with increased ER and Golgi stress contributing to skull defects in mice. Developmental dynamics : an official publication of the American Association of Anatomists 251, 1209–1222, doi: 10.1002/dvdy.461 (2022). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Yamaguchi H. et al. The molecular complex of ciliary and golgin protein is crucial for skull development. Development 148, doi: 10.1242/dev.199559 (2021). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Agarwal S. et al. Scleraxis-Lineage Cells Contribute to Ectopic Bone Formation in Muscle and Tendon. Stem Cells 35, 705–710, doi: 10.1002/stem.2515 (2017). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Feng H. et al. Tendon-derived cathepsin K-expressing progenitor cells activate Hedgehog signaling to drive heterotopic ossification. The Journal of clinical investigation 130, 6354–6365, doi: 10.1172/JCI132518 (2020). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Regard JB et al. Activation of Hedgehog signaling by loss of GNAS causes heterotopic ossification. Nature medicine 19, 1505–1512, doi: 10.1038/nm.3314 (2013). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Liu H, Xu J & Jiang R Mkx-Deficient Mice Exhibit Hedgehog Signaling-Dependent Ectopic Ossification in the Achilles Tendons. Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research 34, 557–569, doi: 10.1002/jbmr.3630 (2019). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Freedman BR, Gordon JA & Soslowsky LJ The Achilles tendon: fundamental properties and mechanisms governing healing. Muscles Ligaments Tendons J 4, 245–255 (2014). [PMC free article] [PubMed] [Google Scholar]
  • 31.Cong Q. et al. A self-amplifying loop of YAP and SHH drives formation and expansion of heterotopic ossification. Sci Transl Med 13, doi: 10.1126/scitranslmed.abb2233 (2021). [DOI] [PMC free article] [PubMed] [Google Scholar]

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