The m6A methyltransferase METTL3 cooperates with demethylase ALKBH5 to regulate osteogenic differentiation through NF-κB signaling

  • Jinjin Yu
  • Lujun Shen
  • Yanli Liu
  • Hong Ming
  • Xinxing ZhuEmail author
  • Maoping ChuEmail author
  • Juntang LinEmail author


As a m6A methylation modifier, METTL3 is functionally involved in various biological processes. Nevertheless, the role of METTL3 in osteogenesis is not determined up to date. In the current study, METTL3 is identified as a crucial regulator in the progression of osteogenic differentiation. Loss of METTL3 significantly augments calcium deposition and enhances alkaline phosphatase activity of mesenchymal stem cells, uncovering an inhibitory role of METTL3 in osteogenesis. More importantly, the underlying molecular basis by which METTL3 regulates osteogenesis is illustrated. We find that METTL3 positively regulates expression of MYD88, a critical upstream regulator of NF-κB signaling, by facilitating m6A methylation modification to MYD88-RNA, subsequently inducing the activation of NF-κB which is widely regarded as a repressor of osteogenesis and therefore suppressing osteogenic progression. Moreover, the METTL3-mediated m6A methylation is found to be dynamically reversed by the demethylase ALKBH5. In summary, this study highlights the functional importance of METTL3 in osteogenic differentiation and METTL3 may serve as a promising molecular target in regenerative medicine, as well as in the field of bone tissue engineering.


METTL3 M6A methylation Osteogenesis NF-κB signaling ALKBH5 



Mesenchymal stem cells




Menstrual blood-derived mesenchymal stem cells


Alkaline phosphatase



This work was supported by grants from the National Natural Science Foundation of China (31502045) and Xinxiang Medical University Foundation (300-505307).

Author contributions

JL, MC, and XZ are responsible for designing the project. JY and LS performed most of the experiments. YL contributed to data analysis. XZ wrote the draft of this manuscript. All authors take part in discussions.

Compliance with ethical standards

Conflict of interest

The authors declare that there is no conflict of interest.

Ethical approval

The MenSCs used in this study were obtained with the informed consent of the donors. All experiments in this manuscript meet the “Declaration of Helsinki” and were approved by the Ethics Committee of Xinxiang Medical University.


  1. 1.
    Barba M, Di Taranto G, Lattanzi W (2017) Adipose-derived stem cell therapies for bone regeneration. Expert Opin Biol Ther 17:677–689. CrossRefPubMedGoogle Scholar
  2. 2.
    Chen M, Xu Y, Zhang T, Ma Y, Liu J, Yuan B, Chen X, Zhou P, Zhao X, Pang F, Liang W (2019) Mesenchymal stem cell sheets: a new cell-based strategy for bone repair and regeneration. Biotechnol Lett 41:305–318. CrossRefPubMedGoogle Scholar
  3. 3.
    Fujii Y, Kawase-Koga Y, Hojo H, Yano F, Sato M, Chung UI, Ohba S, Chikazu D (2018) Bone regeneration by human dental pulp stem cells using a helioxanthin derivative and cell-sheet technology. Stem Cell Res Ther 9:24. CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Shah AR, Cornejo A, Guda T, Sahar DE, Stephenson SM, Chang S, Krishnegowda NK, Sharma R, Wang HT (2014) Differentiated adipose-derived stem cell cocultures for bone regeneration in polymer scaffolds in vivo. J Craniofac Surg 25:1504–1509. CrossRefPubMedGoogle Scholar
  5. 5.
    Zigdon H, Levin L (2012) Stem cell therapy for bone regeneration: present and future strategies. Alpha Omegan 105:35–38PubMedGoogle Scholar
  6. 6.
    Lin TH, Gibon E, Loi F, Pajarinen J, Cordova LA, Nabeshima A, Lu L, Yao Z, Goodman SB (2017) Decreased osteogenesis in mesenchymal stem cells derived from the aged mouse is associated with enhanced NF-kappaB activity. J Orthop Res 35:281–288. CrossRefPubMedGoogle Scholar
  7. 7.
    Liu C, Zhang H, Tang X, Feng R, Yao G, Chen W, Li W, Liang J, Feng X, Sun L (2018) Mesenchymal stem cells promote the osteogenesis in collagen-induced arthritic mice through the inhibition of TNF-alpha. Stem Cells Int 2018:4069032. CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Wang N, Zhou Z, Wu T, Liu W, Yin P, Pan C, Yu X (2016) TNF-alpha-induced NF-kappaB activation upregulates microRNA-150-3p and inhibits osteogenesis of mesenchymal stem cells by targeting beta-catenin. Open Biol. CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Batista PJ, Molinie B, Wang J, Qu K, Zhang J, Li L, Bouley DM, Lujan E, Haddad B, Daneshvar K, Carter AC, Flynn RA, Zhou C, Lim KS, Dedon P, Wernig M, Mullen AC, Xing Y, Giallourakis CC, Chang HY (2014) m(6)A RNA modification controls cell fate transition in mammalian embryonic stem cells. Cell Stem Cell 15:707–719. CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Geula S, Moshitch-Moshkovitz S, Dominissini D, Mansour AA, Kol N, Salmon-Divon M, Hershkovitz V, Peer E, Mor N, Manor YS, Ben-Haim MS, Eyal E, Yunger S, Pinto Y, Jaitin DA, Viukov S, Rais Y, Krupalnik V, Chomsky E, Zerbib M, Maza I, Rechavi Y, Massarwa R, Hanna S, Amit I, Levanon EY, Amariglio N, Stern-Ginossar N, Novershtern N, Rechavi G, Hanna JH (2015) Stem cells. m6A mRNA methylation facilitates resolution of naive pluripotency toward differentiation. Science 347:1002–1006. CrossRefPubMedGoogle Scholar
  11. 11.
    Lin S, Choe J, Du P, Triboulet R, Gregory RI (2016) The m(6)A methyltransferase METTL3 promotes translation in human cancer cells. Mol Cell 62:335–345. CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Zhang C, Chen Y, Sun B, Wang L, Yang Y, Ma D, Lv J, Heng J, Ding Y, Xue Y, Lu X, Xiao W, Yang YG, Liu F (2017) m(6)A modulates haematopoietic stem and progenitor cell specification. Nature 549:273–276. CrossRefPubMedGoogle Scholar
  13. 13.
    Cheng M, Sheng L, Gao Q, Xiong Q, Zhang H, Wu M, Liang Y, Zhu F, Zhang Y, Zhang X, Yuan Q, Li Y (2019) The m(6)A methyltransferase METTL3 promotes bladder cancer progression via AFF4/NF-kappaB/MYC signaling network. Oncogene. CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Maity A, Das B (2016) N6-methyladenosine modification in mRNA: machinery, function and implications for health and diseases. FEBS J 283:1607–1630. CrossRefPubMedGoogle Scholar
  15. 15.
    Vu LP, Pickering BF, Cheng Y, Zaccara S, Nguyen D, Minuesa G, Chou T, Chow A, Saletore Y, MacKay M, Schulman J, Famulare C, Patel M, Klimek VM, Garrett-Bakelman FE, Melnick A, Carroll M, Mason CE, Jaffrey SR, Kharas MG (2017) The N(6)-methyladenosine (m(6)A)-forming enzyme METTL3 controls myeloid differentiation of normal hematopoietic and leukemia cells. Nat Med 23:1369–1376. CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Yue Y, Liu J, He C (2015) RNA N6-methyladenosine methylation in post-transcriptional gene expression regulation. Genes Dev 29:1343–1355. CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Lin Z, Hsu PJ, Xing X, Fang J, Lu Z, Zou Q, Zhang KJ, Zhang X, Zhou Y, Zhang T, Zhang Y, Song W, Jia G, Yang X, He C, Tong MH (2017) Mettl3-/Mettl14-mediated mRNA N(6)-methyladenosine modulates murine spermatogenesis. Cell Res 27:1216–1230. CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Liu J, Yue Y, Han D, Wang X, Fu Y, Zhang L, Jia G, Yu M, Lu Z, Deng X, Dai Q, Chen W, He C (2014) A METTL3-METTL14 complex mediates mammalian nuclear RNA N6-adenosine methylation. Nat Chem Biol 10:93–95. CrossRefPubMedGoogle Scholar
  19. 19.
    Yao QJ, Sang L, Lin M, Yin X, Dong W, Gong Y, Zhou BO (2018) Mettl3-Mettl14 methyltransferase complex regulates the quiescence of adult hematopoietic stem cells. Cell Res 28:952–954. CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Kobayashi M, Ohsugi M, Sasako T, Awazawa M, Umehara T, Iwane A, Kobayashi N, Okazaki Y, Kubota N, Suzuki R, Waki H, Horiuchi K, Hamakubo T, Kodama T, Aoe S, Tobe K, Kadowaki T, Ueki K (2018) The RNA methyltransferase complex of WTAP, METTL3, and METTL14 regulates mitotic clonal expansion in adipogenesis. Mol Cell Biol. CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Selberg S, Blokhina D, Aatonen M, Koivisto P, Siltanen A, Mervaala E, Kankuri E, Karelson M (2019) Discovery of small molecules that activate RNA methylation through cooperative binding to the METTL3-14-WTAP complex active site. Cell Rep 26(3762–3771):e5. CrossRefGoogle Scholar
  22. 22.
    Piette ER, Moore JH (2018) Identification of epistatic interactions between the human RNA demethylases FTO and ALKBH5 with gene set enrichment analysis informed by differential methylation. BMC Proc 12:59. CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Shen F, Huang W, Huang JT, Xiong J, Yang Y, Wu K, Jia GF, Chen J, Feng YQ, Yuan BF, Liu SM (2015) Decreased N(6)-methyladenosine in peripheral blood RNA from diabetic patients is associated with FTO expression rather than ALKBH5. J Clin Endocrinol Metab 100:E148–E154. CrossRefPubMedGoogle Scholar
  24. 24.
    Song H, Feng X, Zhang H, Luo Y, Huang J, Lin M, Jin J, Ding X, Wu S, Huang H, Yu T, Zhang M, Hong H, Yao S, Zhao Y, Zhang Z (2019) METTL3 and ALKBH5 oppositely regulate m(6)A modification of TFEB mRNA, which dictates the fate of hypoxia/reoxygenation-treated cardiomyocytes. Autophagy. CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Zhu X, Yu J, Du J, Zhong G, Qiao L, Lin J (2019) LncRNA HOXA-AS2 positively regulates osteogenesis of mesenchymal stem cells through inactivating NF-kappaB signalling. J Cell Mol Med 23:1325–1332. CrossRefPubMedGoogle Scholar
  26. 26.
    Zou Z, Huang B, Wu X, Zhang H, Qi J, Bradner J, Nair S, Chen LF (2014) Brd4 maintains constitutively active NF-kappaB in cancer cells by binding to acetylated RelA. Oncogene 33:2395–2404. CrossRefPubMedGoogle Scholar
  27. 27.
    Wang H, Hu X, Huang M, Liu J, Gu Y, Ma L, Zhou Q, Cao X (2019) Mettl3-mediated mRNA m(6)A methylation promotes dendritic cell activation. Nat Commun 10:1898. CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.School of PsychologyXinxiang Medical UniversityXinxiangChina
  2. 2.Henan Joint International Research Laboratory of Stem Cell Medicine, College of Biomedical EngineeringXinxiang Medical UniversityXinxiangChina
  3. 3.Children’s Heart Center, the Second Affiliated Hospital and Yuying Children’s Hospital, Institute of Cardiovascular Development and Translational MedicineWenzhou Medical UniversityWenzhouChina
  4. 4.Stem Cell and Biotherapy Engineering Research Center of Henan, College of Life Science and TechnologyXinxiang Medical UniversityXinxiangChina
  5. 5.Synthetic Biology Engineering Lab of Henan Province, College of Life Sciences and TechnologyXinxiang Medical UniversityXinxiangPeople’s Republic of China

Personalised recommendations