Journal of Molecular Histology

, Volume 48, Issue 5–6, pp 389–401 | Cite as

Blockade of LGR4 inhibits proliferation and odonto/osteogenic differentiation of stem cells from apical papillae

  • Meng Zhou
  • Shuyu Guo
  • Lichan Yuan
  • Yuxin Zhang
  • Mengnan Zhang
  • Huimin Chen
  • Mengting Lu
  • Jianrong YangEmail author
  • Junqing MaEmail author
Original Paper


During tooth root development, stem cells from apical papillae (SCAPs) are indispensable, and their abilities of proliferation, migration and odontoblast differentiation are linked to root formation. Leucine-rich repeat-containing GPCR 4 (LGR4) modulates the biological processes of proliferation and differentiation in multiple stem cells. In this study, we showed that LGR4 is expressed in all odontoblast cell lineage cells and Hertwig’s epithelial root sheath (HERS) during the mouse root formation in vivo. In vitro we determined that LGR4 is involved in the Wnt/β-catenin signaling pathway regulating proliferation and odonto/osteogenic differentiation of SCAPs. Quantitative reverse-transcription PCR (qRT-PCR) confirmed that LGR4 is expressed during odontogenic differentiation of SCAPs. CCK8 assays and in vitro scratch tests, together with cell cycle flow cytometric analysis, demonstrated that downregulation of LGR4 inhibited SCAPs proliferation, delayed migration and arrested cell cycle progression at the S and G2/M phases. ALP staining revealed that blockade of LGR4 decreased ALP activity. QRT-PCR and Western blot analysis demonstrated that LGR4 silencing reduced the expression of odonto/osteogenic markers (RUNX2, OSX, OPN, OCN and DSPP). Further Western blot and immunofluorescence studies clarified that inhibition of LGR4 disrupted β-catenin stabilization. Taken together, downregulation of LGR4 gene expression inhibited SCAPs proliferation, migration and odonto/osteogenic differentiation by blocking the Wnt/β-catenin signaling pathway. These results indicate that LGR4 might play a vital role in SCAPs proliferation and odontoblastic differentiation.


LGR4 Stem cells from apical papillae Wnt/β-catenin pathway Odonto/osteogenic differentiation 



This work was supported by the National Natural Science Foundation of China (81371179 and 81771029), Natural Science Foundation of Jiangsu Province (BK20150048), and Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (2014-037).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest with regard to the content of this article.


  1. Bae CH, Kim TH, Ko SO, Lee JC, Yang X, Cho ES (2015) Wntless regulates dentin apposition and root elongation in the mandibular molar. J Dent Res 94:439–445. doi: 10.1177/0022034514567198 CrossRefPubMedPubMedCentralGoogle Scholar
  2. Carmon KS, Gong X, Yi J, Thomas A, Liu Q (2014) RSPO-LGR4 functions via IQGAP1 to potentiate Wnt signaling. Proc Natl Acad Sci USA 111:E1221-1229. doi: 10.1073/pnas.1323106111 CrossRefGoogle Scholar
  3. Chen S et al (2009) Runx2, osx, and dspp in tooth development. J Dent Res 88:904–909. doi: 10.1177/0022034509342873 CrossRefPubMedPubMedCentralGoogle Scholar
  4. Diao S, Lin X, Wang L, Dong R, Du J, Yang D, Fan Z (2017) Analysis of gene expression profiles between apical papillae tissues, stem cells from apical papillae and cell sheet to identify the key modulators in MSCs niche. Cell Prolif. doi: 10.1111/cpr.12337 PubMedGoogle Scholar
  5. Du J, Wang Q, Wang L, Wang X, Yang P (2012) The expression pattern of FHL2 during mouse molar development. J Mol Histol 43:289–295. doi: 10.1007/s10735-012-9409-z CrossRefPubMedGoogle Scholar
  6. Du B et al (2013) Lgr4/Gpr48 negatively regulates TLR2/4-associated pattern recognition and innate immunity by targeting CD14 expression. J Biol Chem 288:15131–15141. doi: 10.1074/jbc.M113.455535 CrossRefPubMedPubMedCentralGoogle Scholar
  7. Du J, Wang Q, Yang P, Wang X (2016) FHL2 mediates tooth development and human dental pulp cell differentiation into odontoblasts, partially by interacting with Runx2. J Mol Histol 47(2):195–202. doi: 10.1007/s10735-016-9655-6 CrossRefPubMedGoogle Scholar
  8. Friedlander LT, Cullinan MP, Love RM (2009) Dental stem cells and their potential role in apexogenesis and apexification. Int Endod J 42:955–962. doi: 10.1111/j.1365-2591.2009.01622.x CrossRefPubMedGoogle Scholar
  9. Glinka A et al (2011) LGR4 and LGR5 are R-spondin receptors mediating Wnt/beta-catenin and Wnt/PCP signalling. EMBO Rep 12:1055–1061. doi: 10.1038/embor.2011.175 CrossRefPubMedPubMedCentralGoogle Scholar
  10. Guo S, Zhang Y, Zhou T, Wang D, Weng Y, Wang L, Ma J (2017) Role of GATA binding protein 4 (GATA4) in the regulation of tooth development via GNAI3. Sci Rep 7:1534. doi: 10.1038/s41598-017-01689-1 CrossRefPubMedPubMedCentralGoogle Scholar
  11. Hou Q et al (2016) LGR4 Is a direct target of MicroRNA-34a and modulates the proliferation and migration of retinal pigment epithelial ARPE-19 cells. PLoS ONE 11:e0168320. doi: 10.1371/journal.pone.0168320 CrossRefPubMedPubMedCentralGoogle Scholar
  12. Huang XF, Chai Y (2012) Molecular regulatory mechanism of tooth root development. Int J Oral Sci 4:177–181. doi: 10.1038/ijos.2012.61 CrossRefPubMedPubMedCentralGoogle Scholar
  13. Huang GT, Sonoyama W, Chen J, Park SH (2006) In vitro characterization of human dental pulp cells: various isolation methods and culturing environments. Cell Tissue Res 324:225–236. doi: 10.1007/s00441-005-0117-9 CrossRefPubMedGoogle Scholar
  14. Huang GT, Sonoyama W, Liu Y, Liu H, Wang S, Shi S (2008) The hidden treasure in apical papillae: the potential role in pulp/dentin regeneration and bioroot engineering. J Endod 34:645–651. doi: 10.1016/j.joen.2008.03.001 CrossRefPubMedPubMedCentralGoogle Scholar
  15. Kawasaki M et al (2014) R-spondins/Lgrs expression in tooth development. Dev Dyn 243:844–851. doi: 10.1002/dvdy.24124 CrossRefPubMedGoogle Scholar
  16. Kim TH, Bae CH, Lee JC, Ko SO, Yang X, Jiang R, Cho ES (2013) beta-catenin is required in odontoblasts for tooth root formation. J Dent Res 92:215–221. doi: 10.1177/0022034512470137 CrossRefPubMedGoogle Scholar
  17. Kim TH, Bae CH, Lee JC, Kim JE, Yang X, de Crombrugghe B, Cho ES (2015) Osterix regulates tooth root formation in a site-specific manner. J Dent Res 94:430–438. doi: 10.1177/0022034514565647 CrossRefPubMedPubMedCentralGoogle Scholar
  18. Lee HK, Park JW, Seo YM, Kim HH, Lee G, Bae HS, Park JC (2016) Odontoblastic inductive potential of epithelial cells derived from human deciduous dental pulp. J Mol Histol 47:345–351. doi: 10.1007/s10735-016-9676-1 CrossRefPubMedGoogle Scholar
  19. Li Z, Zhang W, Mulholland MW (2015) LGR4 and its role in intestinal protection and energy metabolism. Front Endocrinol 6:131. doi: 10.3389/fendo.2015.00131 Google Scholar
  20. Li F, Gu C, Tian F, Jia Z, Meng Z, Ding Y, Yang J (2016) MiR-218 impedes IL-6-induced prostate cancer cell proliferation and invasion via suppression of LGR4 expression. Oncol Rep 35:2859–2865. doi: 10.3892/or.2016.4663 CrossRefPubMedGoogle Scholar
  21. Lian M et al (2016) JAB1 accelerates odontogenic differentiation of dental pulp stem cells. J Mol Histol 47:317–324. doi: 10.1007/s10735-016-9672-5 CrossRefPubMedGoogle Scholar
  22. Liu S et al (2013) Lgr4 gene deficiency increases susceptibility and severity of dextran sodium sulfate-induced inflammatory bowel disease in mice. J Biol Chem 288:8794–8803. doi: 10.1074/jbc.M112.436204 (discussion 8804)CrossRefPubMedPubMedCentralGoogle Scholar
  23. Liu L, Chen W, Li L, Xu F, Jiang B (2017) Inhibition of chondroitin sulfate glycosaminoglycans incorporation affected odontoblast differentiation in cultured embryonic mouse molars. J Mol Histol. doi: 10.1007/s10735-017-9732-5 Google Scholar
  24. Luo J et al (2009) Regulation of bone formation and remodeling by G-protein-coupled receptor 48. Development 136:2747–2756. doi: 10.1242/dev.033571 CrossRefPubMedPubMedCentralGoogle Scholar
  25. Luo W et al (2013) Lgr4 is a key regulator of prostate development and prostate stem cell differentiation. Stem Cells 31:2492–2505. doi: 10.1002/stem.1484 CrossRefPubMedPubMedCentralGoogle Scholar
  26. Luo J et al (2016) LGR4 is a receptor for RANKL and negatively regulates osteoclast differentiation and bone resorption. Nat Med 22:539–546. doi: 10.1038/nm.4076 CrossRefPubMedGoogle Scholar
  27. Mu C et al (2014) Mechanical stress stimulates the osteo/odontoblastic differentiation of human stem cells from apical papillae via erk 1/2 and JNK MAPK pathways. BioMed Res Int 2014:494378. doi: 10.1155/2014/494378 CrossRefPubMedPubMedCentralGoogle Scholar
  28. Nagy MM, Tawfik HE, Hashem AA, Abu-Seida AM (2014) Regenerative potential of immature permanent teeth with necrotic pulps after different regenerative protocols. J Endod 40:192–198. doi: 10.1016/j.joen.2013.10.027 CrossRefPubMedGoogle Scholar
  29. Nakata S, Phillips E, Goidts V (2014) Emerging role for leucine-rich repeat-containing G-protein-coupled receptors LGR5 and LGR4 in cancer stem cells. Cancer Manag Res 6:171–180. doi: 10.2147/CMAR.S57846 PubMedPubMedCentralGoogle Scholar
  30. Park SJ, Bae HS, Park JC (2015) Osteogenic differentiation and gene expression profile of human dental follicle cells induced by human dental pulp cells. J Mol Histol 46:93–106. doi: 10.1007/s10735-014-9604-1 CrossRefPubMedGoogle Scholar
  31. Pawaputanon Na Mahasarakham C et al (2016) BMP-2 enhances Lgr4 gene expression in osteoblastic cells. J Cell Physiol 231:887–895. doi: 10.1002/jcp.25180 CrossRefPubMedGoogle Scholar
  32. Qin Z, Fang Z, Zhao L, Chen J, Li Y, Liu G (2015) High dose of TNF-alpha suppressed osteogenic differentiation of human dental pulp stem cells by activating the Wnt/beta-catenin signaling. J Mol Histol 46:409–420. doi: 10.1007/s10735-015-9630-7 CrossRefPubMedGoogle Scholar
  33. Sohn WJ et al (2014) Contribution of mesenchymal proliferation in tooth root morphogenesis. J Dent Res 93:78–83. doi: 10.1177/0022034513511247 CrossRefPubMedPubMedCentralGoogle Scholar
  34. Sonoyama W, Liu Y, Yamaza T, Tuan RS, Wang S, Shi S, Huang GT (2008) Characterization of the apical papillae and its residing stem cells from human immature permanent teeth: a pilot study. J Endod 34:166–171. doi: 10.1016/j.joen.2007.11.021 CrossRefPubMedPubMedCentralGoogle Scholar
  35. Suzuki S, Haruyama N, Nishimura F, Kulkarni AB (2012) Dentin sialophosphoprotein and dentin matrix protein-1: two highly phosphorylated proteins in mineralized tissues. Arch Oral Biol 57:1165–1175. doi: 10.1016/j.archoralbio.2012.03.005 CrossRefPubMedPubMedCentralGoogle Scholar
  36. Wang J, Liu B, Gu S, Liang J (2012) Effects of Wnt/beta-catenin signalling on proliferation and differentiation of apical papillae stem cells. Cell Prolif 45:121–131. doi: 10.1111/j.1365-2184.2012.00806.x CrossRefPubMedGoogle Scholar
  37. Wang Y, Dong J, Li D, Lai L, Siwko S, Li Y, Liu M (2013) Lgr4 regulates mammary gland development and stem cell activity through the pluripotency transcription factor Sox2. Stem Cells 31:1921–1931. doi: 10.1002/stem.1438 CrossRefPubMedPubMedCentralGoogle Scholar
  38. Wang W, Dang M, Zhang Z, Hu J, Eyster TW, Ni L, Ma PX (2016) Dentin regeneration by stem cells of apical papillae on injectable nanofibrous microspheres and stimulated by controlled BMP-2 release. Acta Biomater 36:63–72. doi: 10.1016/j.actbio.2016.03.015 CrossRefPubMedPubMedCentralGoogle Scholar
  39. Wu J et al (2012) Basic fibroblast growth factor enhances stemness of human stem cells from the apical papillae. J Endod 38:614–622. doi: 10.1016/j.joen.2012.01.014 CrossRefPubMedPubMedCentralGoogle Scholar
  40. Yuan C et al (2016) EphrinB2 stabilizes vascularlike structures generated by endothelial cells and stem cells from apical papillae. J Endod 42:1362–1370. doi: 10.1016/j.joen.2016.05.012 CrossRefPubMedGoogle Scholar
  41. Zhang R, Yang G, Wu X, Xie J, Yang X, Li T (2013) Disruption of Wnt/beta-catenin signaling in odontoblasts and cementoblasts arrests tooth root development in postnatal mouse teeth. Int J Biol Sci 9:228–236. doi: 10.7150/ijbs.5476 CrossRefPubMedPubMedCentralGoogle Scholar
  42. Zhao H, Feng J, Seidel K, Shi S, Klein O, Sharpe P, Chai Y (2014) Secretion of shh by a neurovascular bundle niche supports mesenchymal stem cell homeostasis in the adult mouse incisor. Cell Stem Cell 14:160–173. doi: 10.1016/j.stem.2013.12.013 CrossRefPubMedPubMedCentralGoogle Scholar
  43. Zhou T, Guo S, Zhang Y, Weng Y, Wang L, Ma J (2017) GATA4 regulates osteoblastic differentiation and bone remodeling via p38-mediated signaling. J Mol Histol. doi: 10.1007/s10735-017-9719-2 Google Scholar

Copyright information

© Springer Science+Business Media B.V. 2017

Authors and Affiliations

  1. 1.Jiangsu Key Laboratory of Oral DiseasesNanjing Medical UniversityNanjingChina
  2. 2.Department of Oral and Maxillofacial SurgeryXuzhou Stomatological HospitalXuzhouChina

Personalised recommendations