Skip to main content
SpringerLink
Log in

Wnt/β-Catenin Promotes the Osteoblastic Potential of BMP9 Through Down-Regulating Cyp26b1 in Mesenchymal Stem Cells

  • Original Article
  • Published:
Tissue Engineering and Regenerative Medicine Aims and scope

Abstract

BACKGROUND:

All-trans retinoic acid (ATRA) promotes the osteogenic differentiation induced by bone morphogenetic protein 9 (BMP9), but the intrinsic relationship between BMP9 and ATRA keeps unknown. Herein, we investigated the effect of Cyp26b1, a critical enzyme of ATRA degradation, on the BMP9-induced osteogenic differentiation in mesenchymal stem cells (MSCs), and unveiled possible mechanism through which BMP9 regulates the expression of Cyp26b1.

METHODS:

ATRA content was detected with ELISA and HPLC–MS/MS. PCR, Western blot, and histochemical staining were used to assay the osteogenic markers. Fetal limbs culture, cranial defect repair model, and micro–computed tomographic were used to evaluate the quality of bone formation. IP and ChIP assay were used to explore possible mechanism.

RESULTS:

We found that the protein level of Cyp26b1 was increased with age, whereas the ATRA content decreased. The osteogenic markers induced by BMP9 were increased by inhibiting or silencing Cyp26b1 but reduced by exogenous Cyp26b1. The BMP9-induced bone formation was enhanced by inhibiting Cyp26b1. The cranial defect repair was promoted by BMP9, which was strengthened by silencing Cyp26b1 and reduced by exogenous Cyp26b1. Mechanically, Cyp26b1 was reduced by BMP9, which was enhanced by activating Wnt/β-catenin, and reduced by inhibiting this pathway. β-catenin interacts with Smad1/5/9, and both were recruited at the promoter of Cyp26b1.

CONCLUSIONS:

Our findings suggested the BMP9-induced osteoblastic differentiation was mediated by activating retinoic acid signalling, viadown-regulating Cyp26b1. Meanwhile, Cyp26b1 may be a novel potential therapeutic target for the treatment of bone-related diseases or accelerating bone-tissue engineering.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8
Fig. 9

Similar content being viewed by others

References

  1. Ambrosi TH, Scialdone A, Graja A, Gohlke S, Jank AM, Bocian C, et al. Adipocyte accumulation in the bone marrow during obesity and aging impairs stem cell-based hematopoietic and bone regeneration. Cell Stem Cell. 2017;20:771–84.e6.

    Google Scholar 

  2. Nehlin JO, Jafari A, Tencerova M, Kassem M. Aging and lineage allocation changes of bone marrow skeletal (stromal) stem cells. Bone. 2019;123:265–73.

    PubMed  Google Scholar 

  3. Deshpande SS, Gallagher KK, Donneys A, Tchanque-Fossuo CN, Sarhaddi D, Sun H, et al. Stem cell therapy remediates reconstruction of the craniofacial skeleton after radiation therapy. Stem Cells Dev. 2013;22:1625–32.

    PubMed  PubMed Central  Google Scholar 

  4. Bone HG, Wagman RB, Brandi ML, Brown JP, Chapurlat R, Cummings SR, et al. 10 years of denosumab treatment in postmenopausal women with osteoporosis: results from the phase 3 randomised FREEDOM trial and open-label extension. Lancet Diabetes Endocrinol. 2017;5:513–23.

    CAS  Google Scholar 

  5. Negri S, Wang Y, Sono T, Lee S, Hsu GC, Xu J, et al. Human perivascular stem cells prevent bone graft resorption in osteoporotic contexts by inhibiting osteoclast formation. Stem Cells Transl Med. 2020;9:1617–30.

    CAS  PubMed  PubMed Central  Google Scholar 

  6. Long H, Zhu Y, Lin Z, Wan J, Cheng L, Zeng M, et al. miR-381 modulates human bone mesenchymal stromal cells (BMSCs) osteogenesis via suppressing Wnt signaling pathway during atrophic nonunion development. Cell Death Dis. 2019;10:470.

    PubMed  PubMed Central  Google Scholar 

  7. Wang T, Zhang X, Bikle DD. Osteogenic differentiation of periosteal cells during fracture healing. J Cell Physiol. 2017;232:913–21.

    CAS  PubMed  Google Scholar 

  8. Laird NZ, Acri TM, Tingle K, Salem AK. Gene- and RNAi-activated scaffolds for bone tissue engineering: Current progress and future directions. Adv Drug Deliv Rev. 2021;174:613–27.

    CAS  PubMed  PubMed Central  Google Scholar 

  9. Baron R, Kneissel M. WNT signaling in bone homeostasis and disease: from human mutations to treatments. Nat Med. 2013;19:179–92.

    CAS  PubMed  Google Scholar 

  10. Zhuang X, Zhang H, Li X, Li X, Cong M, Peng F, et al. Differential effects on lung and bone metastasis of breast cancer by Wnt signalling inhibitor DKK1. Nat Cell Biol. 2017;19:1274–85.

    CAS  PubMed  Google Scholar 

  11. Matsuoka K, Bakiri L, Wolff LI, Linder M, Mikels-Vigdal A, Patiño-García A, et al. Wnt signaling and Loxl2 promote aggressive osteosarcoma. Cell Res. 2020;30:885–901.

    CAS  PubMed  PubMed Central  Google Scholar 

  12. Kang Q, Song WX, Luo Q, Tang N, Luo J, Luo X, et al. A comprehensive analysis of the dual roles of BMPs in regulating adipogenic and osteogenic differentiation of mesenchymal progenitor cells. Stem Cells Dev. 2009;18:545–59.

    CAS  PubMed  Google Scholar 

  13. Mostafa S, Pakvasa M, Coalson E, Zhu A, Alverdy A, Castillo H, et al. The wonders of BMP9: from mesenchymal stem cell differentiation, angiogenesis, neurogenesis, tumorigenesis, and metabolism to regenerative medicine. Genes Dis. 2019;6:201–23.

    CAS  PubMed  PubMed Central  Google Scholar 

  14. Kawai S, Yoshitomi H, Sunaga J, Alev C, Nagata S, Nishio M, et al. In vitro bone-like nodules generated from patient-derived iPSCs recapitulate pathological bone phenotypes. Nat Biomed Eng. 2019;3:558–70.

    PubMed  Google Scholar 

  15. Wang H, Hu Y, He F, Li L, Li PP, Deng Y, et al. All-trans retinoic acid and COX-2 cross-talk to regulate BMP9-induced osteogenic differentiation via Wnt/β-catenin in mesenchymal stem cells. Biomed Pharmacother. 2019;118:109279.

    CAS  PubMed  Google Scholar 

  16. Roberts C. Regulating retinoic acid availability during development and regeneration: the role of the CYP26 enzymes. J Dev Biol. 2020;8:6.

    CAS  PubMed  PubMed Central  Google Scholar 

  17. Isoherranen N, Zhong G. Biochemical and physiological importance of the CYP26 retinoic acid hydroxylases. Pharmacol Ther. 2019;204:107400.

    CAS  PubMed  PubMed Central  Google Scholar 

  18. Duester G. Retinoic acid synthesis and signaling during early organogenesis. Cell. 2008;134:921–31.

    CAS  PubMed  PubMed Central  Google Scholar 

  19. Richards EJ, McGirr JA, Wang JR, St John ME, Poelstra JW, Solano MJ, et al. A vertebrate adaptive radiation is assembled from an ancient and disjunct spatiotemporal landscape. Proc Natl Acad Sci U S A. 2021;118:e2011811118.

  20. Wang X, Liang T, Zhu Y, Qiu J, Qiu X, Lian C, et al. Melatonin prevents bone destruction in mice with retinoic acid-induced osteoporosis. Mol Med. 2019;25:43.

    PubMed  PubMed Central  Google Scholar 

  21. Stevison F, Hogarth C, Tripathy S, Kent T, Isoherranen N. Inhibition of the all-trans retinoic acid (atRA) hydroxylases CYP26A1 and CYP26B1 results in dynamic, tissue-specific changes in endogenous atRA signaling. Drug Metab Dispos. 2017;45:846–54.

    PubMed  PubMed Central  Google Scholar 

  22. White JA, Ramshaw H, Taimi M, Stangle W, Zhang A, Everingham S, et al. Identification of the human cytochrome P450, P450RAI-2, which is predominantly expressed in the adult cerebellum and is responsible for all-trans-retinoic acid metabolism. Proc Natl Acad Sci U S A. 2000;97:6403–8.

    CAS  PubMed  PubMed Central  Google Scholar 

  23. Shibata M, Pattabiraman K, Lorente-Galdos B, Andrijevic D, Kim SK, Kaur N, et al. Regulation of prefrontal patterning and connectivity by retinoic acid. Nature. 2021;598:483–8.

    CAS  PubMed  PubMed Central  Google Scholar 

  24. Daniel E, Barlow HR, Sutton GI, Gu X, Htike Y, Cowdin MA, et al. Cyp26b1 is an essential regulator of distal airway epithelial differentiation during lung development. Development. 2020;147:181560.

    Google Scholar 

  25. Chang J, Zhong R, Tian J, Li J, Zhai K, Ke J, et al. Exome-wide analyses identify low-frequency variant in CYP26B1 and additional coding variants associated with esophageal squamous cell carcinoma. Nat Genet. 2018;50:338–43.

    CAS  PubMed  Google Scholar 

  26. Jiang S, Zhou F, Zhang Y, Zhou W, Zhu L, Zhang M, et al. Identification of tumorigenicity-associated genes in osteosarcoma cell lines based on bioinformatic analysis and experimental validation. J Cancer. 2020;11:3623–33.

    CAS  PubMed  PubMed Central  Google Scholar 

  27. Shield WP 3rd, Cellini A, Tian H, Wilson K, Dan Y, Abzug JM, et al. Selective agonists of nuclear retinoic acid receptor gamma inhibit growth of HCS-2/8 chondrosarcoma cells. J Orthop Res. 2020;38:1045–51.

    CAS  PubMed  Google Scholar 

  28. Tang Q-Q, Otto T-C, Lane MD. Commitment of C3H10T1/2 pluripotent stem cells to the adipocyte lineage. Proc Natl Acad Sci U S A. 2003;101:9607–11.

    Article  Google Scholar 

  29. Wang Y, Chang WY, Prins GS, van Breemen RB. Simultaneous determination of all-trans, 9-cis, 13-cis retinoic acid and retinol in rat prostate using liquid chromatography-mass spectrometry. J Mass Spectrom. 2001;36:882–8.

    CAS  PubMed  Google Scholar 

  30. Li FS, Li PP, Li L, Deng Y, Hu Y, He BC. PTEN reduces BMP9-induced osteogenic differentiation through inhibiting Wnt10b in mesenchymal stem cells. Front Cell Dev Biol. 2020;8:608544.

    PubMed  Google Scholar 

  31. Chen L, Jiang W, Huang J, He BC, Zuo GW, Zhang W, et al. Insulin-like growth factor 2 (IGF-2) potentiates BMP-9-induced osteogenic differentiation and bone formation. J Bone Min Res. 2010;25:2447–59.

    CAS  Google Scholar 

  32. Niederreither K, Dollé P. Retinoic acid in development: towards an integrated view. Nat Rev Genet. 2008;9:541–53.

    CAS  PubMed  Google Scholar 

  33. Zhang Y, Zolfaghari R, Ross AC. Multiple retinoic acid response elements cooperate to enhance the inducibility of CYP26A1 gene expression in liver. Gene. 2010;464:32–43.

    CAS  PubMed  PubMed Central  Google Scholar 

  34. Foti RS, Diaz P, Douguet D. Comparison of the ligand binding site of CYP2C8 with CYP26A1 and CYP26B1: a structural basis for the identification of new inhibitors of the retinoic acid hydroxylases. J Enzyme Inhib Med Chem. 2016;31:148–61.

    CAS  PubMed  Google Scholar 

  35. Salhotra A, Shah HN, Levi B, Longaker MT. Mechanisms of bone development and repair. Nat Rev Mol Cell Biol. 2020;21:696–711.

    CAS  PubMed  PubMed Central  Google Scholar 

  36. Snyder JM, Zhong G, Hogarth C, Huang W, Topping T, LaFrance J, et al. Knockout of Cyp26a1 and Cyp26b1 during postnatal life causes reduced lifespan, dermatitis, splenomegaly, and systemic inflammation in mice. FASEB J. 2020;34:15788–804.

    CAS  PubMed  Google Scholar 

  37. Laue K, Jänicke M, Plaster N, Sonntag C, Hammerschmidt M. Restriction of retinoic acid activity by Cyp26b1 is required for proper timing and patterning of osteogenesis during zebrafish development. Development. 2008;135:3775–87.

    CAS  PubMed  Google Scholar 

  38. Vogiatzi A, Baltsavia I, Dialynas E, Theodorou V, Zhou Y, Deligianni E, et al. Erf affects commitment and differentiation of Osteoprogenitor cells in cranial sutures via the retinoic acid pathway. Mol Cell Biol. 2021;41:e0014921.

    PubMed  Google Scholar 

  39. Li J, Xie H, Jiang Y. Mucopolysaccharidosis IIIB and mild skeletal anomalies: coexistence of NAGLU and CYP26B1 missense variations in the same patient in a Chinese family. BMC Med Genet. 2018;19:51.

    PubMed  PubMed Central  Google Scholar 

  40. Lind T, Lugano R, Gustafson AM, Norgård M, van Haeringen A, Dimberg A, et al. Bones in human CYP26B1 deficiency and rats with hypervitaminosis a phenocopy Vegfa overexpression. Bone Rep. 2018;9:27–36.

    PubMed  PubMed Central  Google Scholar 

  41. Laue K, Pogoda HM, Daniel PB, van Haeringen A, Alanay Y, von Ameln S, et al. Craniosynostosis and multiple skeletal anomalies in humans and zebrafish result from a defect in the localized degradation of retinoic acid. Am J Hum Genet. 2011;89:595–606.

    CAS  PubMed  PubMed Central  Google Scholar 

  42. Buckley L, Humphrey MB. Glucocorticoid-Induced Osteoporosis. N Engl J Med. 2018;379:2547–56.

    PubMed  Google Scholar 

  43. Rachner TD, Khosla S, Hofbauer LC. Osteoporosis: now and the future. Lancet. 2011;377:1276–87.

    CAS  PubMed  PubMed Central  Google Scholar 

  44. Akiyama M, Ishigaki K, Sakaue S, Momozawa Y, Horikoshi M, Hirata M, et al. Characterizing rare and low-frequency height-associated variants in the Japanese population. Nat Commun. 2019;10:4393.

    CAS  PubMed  PubMed Central  Google Scholar 

  45. Pogoda HM, Riedl-Quinkertz I, Löhr H, Waxman JS, Dale RM, Topczewski J, et al. Direct activation of chordoblasts by retinoic acid is required for segmented centra mineralization during zebrafish spine development. Development. 2018;145:dev159418.

    Google Scholar 

  46. Blum N, Begemann G. Osteoblast de- and redifferentiation are controlled by a dynamic response to retinoic acid during zebrafish fin regeneration. Development. 2015;142:2894–903.

    CAS  PubMed  Google Scholar 

  47. Wang Y, Li YP, Paulson C, Shao JZ, Zhang X, Wu M, et al. Wnt and the Wnt signaling pathway in bone development and disease. Front Biosci (Landmark Ed). 2014;19:379–407.

    Google Scholar 

  48. Huybrechts Y, Mortier G, Boudin E, Van Hul W. WNT Signaling and bone: lessons from skeletal dysplasias and disorders. Front Endocrinol (Lausanne). 2020;11:165.

    Google Scholar 

  49. Colditz J, Thiele S, Baschant U, Garbe AI, Niehrs C, Hofbauer LC, et al. Osteogenic Dkk1 mediates glucocorticoid-induced but not arthritis-induced bone loss. J Bone Miner Res. 2019;34:1314–23.

    CAS  Google Scholar 

  50. Zhang H, Wang J, Deng F, Huang E, Yan Z, Wang Z, et al. Canonical Wnt signaling acts synergistically on BMP9-induced osteo/odontoblastic differentiation of stem cells of dental apical papilla (SCAPs). Biomaterials. 2015;39:145–54.

    CAS  PubMed  Google Scholar 

  51. Lin L, Qiu Q, Zhou N, Dong W, Shen J, Jiang W, et al. Dickkopf-1 is involved in BMP9-induced osteoblast differentiation of C3H10T1/2 mesenchymal stem cells. BMB Rep. 2016;49:179–84.

    CAS  PubMed  PubMed Central  Google Scholar 

  52. Xia MY, Zhao XY, Huang QL, Sun HY, Sun C, Yuan J, et al. Activation of Wnt/β-catenin signaling by lithium chloride attenuates d-galactose-induced neurodegeneration in the auditory cortex of a rat model of aging. FEBS Open Bio. 2017;7:759–76.

    CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

Thanks to Prof. Tongchuan He (the medical center of the University of Chicago) for his generous provision of the cells, recombinant adenoviruses, and pAdtrace361 plasmid of the recombinant adenoviruses. This work was supported by the National Natural Science Foundation of China (NSFC, 81572226), and the National Key Research and Development Program of China. This work was supported by the National Natural Science Foundation of China (NSFC, 81572226), and the National Key Research and Development Program of China (2016YFC1000803).

Author information

Author notes

  1. Xin-Tong Yao and Pei-pei Li have contributed equally to this work.

Authors and Affiliations

  1. Department of Pharmacology, College of Pharmacy, Chongqing Medical University, No. 1 Yixueyuan Road, Yuzhong District, Chongqing, 400016, People’s Republic of China

    Xin-Tong Yao, Pei-pei Li, Yuan-Yuan Yang, Hong-Hong Luo, Yi-Xuan Deng & Bai-Cheng He

  2. Chongqing Key Laboratory for Biochemistry and Molecular Pharmacology, Chongqing, 400016, People’s Republic of China

    Xin-Tong Yao, Pei-pei Li, Yuan-Yuan Yang, Hong-Hong Luo, Yi-Xuan Deng & Bai-Cheng He

  3. Dalian Medical University, Dalian, 116044, Liaoning, People’s Republic of China

    Jiang Liu

  4. Department of Orthopedics, The 960th Hospital of the PLA Joint Logistics Support Force, Ji’nan, 250013, Shandong, People’s Republic of China

    Jiang Liu

  5. Taizhou Food Inspection Centre, Taizhou, 318000, Zhejiang, People’s Republic of China

    Zhen-Ling Luo

  6. Department of Orthopaedics, The Second Affiliated Hospital of Chongqing Medical University, Chongqing, 400010, People’s Republic of China

    Hai-Tao Jiang & Wen-Ge He

Authors
  1. Xin-Tong Yao
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Pei-pei Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Jiang Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Yuan-Yuan Yang
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Zhen-Ling Luo
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Hai-Tao Jiang
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Wen-Ge He
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Hong-Hong Luo
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Yi-Xuan Deng
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Bai-Cheng He
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

XTY designed and performed the experiments, analyzed data, interpreted results and wrote the manuscript. PPL performed the experiments, analyzed data, interpreted results. JL was involved in the collection of clinical specimens. YYY preformed the molecular cloning experiment. ZLL preformed the HPLC–MS/MS experiment and the data analysis. HTJ preformed the molecular mechanism analysis, WGH preformed the RNA sequencing and analysis, HHL and YXD performed part of WB assay; BCH designed the experiments, secured funding and supervised this study and revised the manuscript.

Corresponding author

Correspondence to Bai-Cheng He.

Ethics declarations

Conflict of interest

The authors have declared that no conflicts of interest exist in this work.

Ethical statement

The study protocol was approved by the institutional review board of Chongqing Medical University and the 960th Hospital of the PLA Joint Logistics Support Force (2021–102). Informed consent was comfirmed by the 960th Hospital of the PLA Joint Logistics Support Force.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary Information

Rights and permissions

Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Yao, XT., Li, Pp., Liu, J. et al. Wnt/β-Catenin Promotes the Osteoblastic Potential of BMP9 Through Down-Regulating Cyp26b1 in Mesenchymal Stem Cells. Tissue Eng Regen Med 20, 705–723 (2023). https://doi.org/10.1007/s13770-023-00526-z

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s13770-023-00526-z

Keywords

Search

Navigation