Advertisement

Cell and Tissue Research

, Volume 357, Issue 1, pp 133–143 | Cite as

Distal-less homeobox 2 promotes the osteogenic differentiation potential of stem cells from apical papilla

  • Binbin Qu
  • Ousheng Liu
  • Xiaodan Fang
  • Haixia Zhang
  • Yuehong Wang
  • Hongzhi Quan
  • Jie Zhang
  • Jing Zhou
  • Jun Zuo
  • Jianxia Tang
  • Zhangui TangEmail author
Regular Article

Abstract

Dental tissue-derived mesenchymal stem cells (MSCs) are a reliable cell source for dental tissue regeneration. However, the molecular mechanisms underlying the directed differentiation of MSCs remain unclear; thus, their use is limited. The histone demethylase, lysine (K)-specific demethylase 4B (KDM4B), plays critical roles in the osteogenic commitment of MSCs by up-regulating distal-less homeobox 2 (DLX2) expression. The DLX2 gene is highly expressed in dental tissue-derived MSCs but the roles of DLX2 in osteogenesis are unclear. Here, we investigate DLX2 function in stem cells from apical papilla (SCAPs). We found that, in vitro, DLX2 expression was up-regulated in SCAPs by adding BMP4 and by inducing osteogenesis. The knock-down of DLX2 in SCAPs decreased alkaline phosphatase (ALP) activity and mineralization. DLX2 depletion affected the mRNA expression of ALP, bone sialoprotein (BSP) and osteocalcin (OCN) and inhibited SCAP osteogenic differentiation in vitro. Over-expression of DLX2 enhanced ALP activity, mineralization and the expression of ALP, BSP and OCN in vitro. In addition, transplant experiments in nude mice confirmed that SCAP osteogenesis was triggered when DLX2 was activated. Furthermore, DLX2 expression led to the expression of the key transcription factor, osterix (OSX) but not to the expression of runt-related transcription factor 2 (RUNX2). Taken together, these results indicate that DLX2 is stimulated by BMP signaling and enhances SCAP osteogenic differentiation by up-regulating OSX. Thus, the activation of DLX2 signaling might improve tissue regeneration mediated by MSCs of dental origin. These results provide insight into the mechanism underlying the directed differentiation of MSCs of dental origin.

Keywords

Distal-less homeobox 2 (DLX2Osteogenic Differentiation Stem cells from apical papilla (SCAPs) Osterix (OSX

Notes

Acknowledgments

This work was supported by the National Sciences Foundation of China (Grant No. 30872895 and 81300841); by a Key Technology Project Grant for Science and Technology, Department of Hunan Province (Grant No. 2008FJ2011); by the Nature Sciences Foundation of Hunan Province (Grant No. S2013J504B); and by a Project Grant for Science and Technology, Department of Hunan Province (Grant No. 2013SK5075).

Disclosure of potential conflicts of interest

The authors declare no potential conflicts of interest.

Supplementary material

441_2014_1833_Fig6_ESM.jpg (17 kb)
Supplementary Fig. 1

DLX2 expression was not changed at 1, 7 and 21 days after culturing SCAPs in osteogenic-inducing medium compared to expression in SCAPs cultured in normal medium. Real-time RT-PCR results show DLX2 mRNA expression levels. GAPDH was used as an internal control. Student’s t-test was performed to determine statistical significance. All error bars represent SD (n = 3). NS no significant difference. Normal normal culture medium; Induced osteogenic-inducing medium. (JPEG 16 kb)

441_2014_1833_MOESM1_ESM.tif (916 kb)
High Resolution Image (TIFF 916 kb)

References

  1. Acampora D, Merlo GR, Paleari L, Zerega B, Postiglione MP, Mantero S, Bober E, Barbieri O, Simeone A, Levi G (1999) Craniofacial, vestibular and bone defects in mice lacking the Distal-less-related gene Dlx5. Development 126:3795–3809PubMedGoogle Scholar
  2. Arthur A, Rychkov G, Shi S, Koblar SA, Gronthos S (2008) Adult human dental pulp stem cells differentiate towards functionally active neurons under appropriate environmental cues. Stem Cells 26:1787–1795PubMedCrossRefGoogle Scholar
  3. Baek WY, Lee MA, Jung JW, Kim SY, Akiyama H, de Crombrugghe B, Kim JE (2009) Positive regulation of adult bone formation by osteoblast-specific transcription factor osterix. J Bone Miner Res 24:1055–1065PubMedCentralPubMedCrossRefGoogle Scholar
  4. Caplan AI (2007) Adult mesenchymal stem cells for tissue engineering versus regenerative medicine. J Cell Physiol 213:341–347PubMedCrossRefGoogle Scholar
  5. Celil AB, Campbell PG (2005) BMP-2 and insulin-like growth factor-I mediate Osterix (Osx) expression in human mesenchymal stem cells via the MAPK and protein kinase D signaling pathways. J Biol Chem 280:31353–31359PubMedCrossRefGoogle Scholar
  6. Chang J, Sonoyama W, Wang Z, Jin Q, Zhang C, Krebsbach PH, Giannobile W, Shi S, Wang CY (2007) Noncanonical Wnt-4 signaling enhances bone regeneration of mesenchymal stem cells in craniofacial defects through activation of p38 MAPK. J Biol Chem 282:30938–30948PubMedCrossRefGoogle Scholar
  7. Chen D, Zhao M, Mundy GR (2004) Bone morphogenetic proteins. Growth Factors 22:233–241PubMedCrossRefGoogle Scholar
  8. Chen S, Gluhak-Heinrich J, Wang YH, Wu YM, Chuang HH, Chen L, Yuan GH, Dong J, Gay I, MacDougall M (2009) Runx2, osx, and dspp in tooth development. J Dent Res 88:904–909PubMedCentralPubMedCrossRefGoogle Scholar
  9. da Cunha JM, da Costa-Neves A, Kerkis I, da Silva MC (2013) Pluripotent stem cell transcription factors during human odontogenesis. Cell Tissue Res 353:435–441PubMedCrossRefGoogle Scholar
  10. Dai J, Kuang Y, Fang B, Gong H, Lu S, Mou Z, Sun H, Dong Y, Lu J, Zhang W, Zhang J, Wang Z, Wang X, Shen G (2013) The effect of overexpression of Dlx2 on the migration, proliferation and osteogenic differentiation of cranial neural crest stem cells. Biomaterials 34:1898–1910PubMedCrossRefGoogle Scholar
  11. Du J, Ma Y, Ma P, Wang S, Fan Z (2013) Demethylation of epiregulin gene by histone demethylase FBXL11 and BCL6 corepressor inhibits osteo/dentinogenic differentiation. Stem Cells 31:126–136PubMedCrossRefGoogle Scholar
  12. Fan Z, Yamaza T, Lee JS, Yu J, Wang SL, Fan G, Shi S, Wang CY (2009) BCOR regulates mesenchymal stem cell function by epigenetic mechanisms. Nat Cell Biol 11:1002–1009PubMedCentralPubMedCrossRefGoogle Scholar
  13. Gronthos S, Mankani M, Brahim J, Robey PG, Shi S (2000) Postnatal human dental pulp stem cells (DPSCs) in vitro and in vivo. Proc Natl Acad Sci USA 97:13625–13630PubMedCentralPubMedCrossRefGoogle Scholar
  14. Harris SE, Guo D, Harris MA, Krishnaswamy A, Lichtler A (2003) Transcriptional regulation of BMP-2 activated genes in osteoblasts using gene expression microarray analysis: role of Dlx2 and Dlx5 transcription factors. Front Biosci 8:s1249–s1265PubMedCrossRefGoogle Scholar
  15. Hassan MQ, Javed A, Morasso MI, Karlin J, Montecino M, van Wijnen AJ, Stein GS, Stein JL, Lian JB (2004) Dlx3 transcriptional regulation of osteoblast differentiation: temporal recruitment of Msx2, Dlx3, and Dlx5 homeodomain proteins to chromatin of the osteocalcin gene. Mol Cell Biol 24:9248–9261PubMedCentralPubMedCrossRefGoogle Scholar
  16. Hassan MQ, Tare RS, Lee SH, Mandeville M, Morasso MI, Javed A, van Wijnen AJ, Stein JL, Stein GS, Lian JB (2006) BMP2 commitment to the osteogenic lineage involves activation of Runx2 by DLX3 and a homeodomain transcriptional network. J Biol Chem 281:40515–40526PubMedCrossRefGoogle Scholar
  17. Holleville N, Matéos S, Bontoux M, Bollerot K, Monsoro-Burq AH (2007) Dlx5 drives Runx2 expression and osteogenic differentiation in developing cranial suture mesenchyme. Dev Biol 304:860–874PubMedCrossRefGoogle Scholar
  18. Jahagirdar BN, Verfaillie CM (2005) Multipotent adult progenitor cell and stem cell plasticity. Stem Cell Rev 1:53–59PubMedCrossRefGoogle Scholar
  19. Jang WG, Kim EJ, Lee KN, Son HJ, Koh JT (2011) AMP-activated protein kinase (AMPK) positively regulates osteoblast differentiation via induction of Dlx5-dependent Runx2 expression in MC3T3E1 cells. Biochem Biophys Res Commun 404:1004–1009PubMedCrossRefGoogle Scholar
  20. Jiang Q, Du J, Yin X, Shan Z, Ma Y, Ma P, Du J, Fan Z (2013) Shh signaling, negatively regulated by BMP signaling, inhibits the osteo/dentinogenic differentiation potentials of mesenchymal stem cells from apical papilla. Mol Cell Biochem 383:85–93PubMedCrossRefGoogle Scholar
  21. Kaback LA, Soung do Y, Naik A, Smith N, Schwarz EM, O’Keefe RJ, Drissi H (2008) Osterix/Sp7 regulates mesenchymal stem cell mediated endochondral ossification. J Cell Physiol 214:173–182PubMedCrossRefGoogle Scholar
  22. Karsenty G, Wagner EF (2002) Reaching a genetic and molecular understanding of skeletal development. Dev Cell 2:389–406PubMedCrossRefGoogle Scholar
  23. Komori T (2006) Regulation of osteoblast differentiation by transcription factors. J Cell Biochem 99:1233–1239PubMedCrossRefGoogle Scholar
  24. Kraus P, Lufkin T (2006) Dlx homeobox gene control of mammalian limb and craniofacial development. Am J Med Genet A 140:1366–1374PubMedCrossRefGoogle Scholar
  25. Lee MH, Kim YJ, Kim HJ, Park HD, Kang AR, Kyung HM, Sung JH, Wozney JM, Kim HJ, Ryoo HM (2003a) BMP-2-induced Runx2 expression is mediated by Dlx5, and TGF-beta 1 opposes the BMP-2-induced osteoblast differentiation by suppression of Dlx5 expression. J Biol Chem 278:34387–34394PubMedCrossRefGoogle Scholar
  26. Lee MH, Kwon TG, Park HS, Wozney JM, Ryoo HM (2003b) BMP-2-induced Osterix expression is mediated by Dlx5 but is independent of Runx2. Biochem Biophys Res Commun 309:689–694PubMedCrossRefGoogle Scholar
  27. Li H, Marijanovic I, Kronenberg MS, Erceg I, Stover ML, Velonis D, Mina M, Heinrich JG, Harris SE, Upholt WB, Kalajzic I, Lichtler AC (2008) Expression and function of Dlx genes in the osteoblast lineage. Dev Biol 316:458–470PubMedCentralPubMedCrossRefGoogle Scholar
  28. Li X, Yang G, Fan M (2012) Effects of homeobox gene distal-less 3 on proliferation and odontoblastic differentiation of human dental pulp cells. J Endod 38:1504–1510PubMedCrossRefGoogle Scholar
  29. Lian JB, Stein GS, Javed A, van Wijnen AJ, Stein JL, Montecino M, Hassan MQ, Gaur T, Lengner CJ, Young DW (2006) Networks and hubs for the transcriptional control of osteoblastogenesis. Rev Endocr Metab Disord 7:1–16PubMedCrossRefGoogle Scholar
  30. Liu Y, Zheng Y, Ding G, Fang D, Zhang C, Bartold PM, Gronthos S, Shi S, Wang S (2008) Periodontal ligament stem cell-mediated treatment for periodontitis in miniature swine. Stem Cells 26:1065–1073PubMedCentralPubMedCrossRefGoogle Scholar
  31. Miura M, Gronthos S, Zhao M, Lu B, Fisher LW, Robey PG, Shi S (2003) SHED: stem cells from human exfoliated deciduous teeth. Proc Natl Acad Sci USA 100:5807–5812PubMedCentralPubMedCrossRefGoogle Scholar
  32. Miyama K, Yamada G, Yamamoto TS, Takagi C, Miyado K, Sakai M, Ueno N, Shibuya H (1999) A BMP-inducible gene, dlx5, regulates osteoblast differentiation and mesoderm induction. Dev Biol 208:123–133PubMedCrossRefGoogle Scholar
  33. Nakashima K, Zhou X, Kunkel G, Zhang Z, Deng JM, Behringer RR, de Crombrugghe B (2002) The novel zinc finger-containing transcription factor osterix is required for osteoblast differentiation and bone formation. Cell 108:17–29PubMedCrossRefGoogle Scholar
  34. Phinney DG, Prockop DJ (2007) Concise review: mesenchymal stem/multipotent stromal cells: the state of transdifferentiation and modes of tissue repair—current views. Stem Cells 25:2896–2902PubMedCrossRefGoogle Scholar
  35. Seo BM, Miura M, Gronthos S, Bartold PM, Batouli S, Brahim J, Young M, Robey PG, Wang CY, Shi S (2004) Investigation of multipotent postnatal stem cells from human periodontal ligament. Lancet 364:149–155PubMedCrossRefGoogle Scholar
  36. Shi S, Wang CY (2004) Bone marrow stromal stem cells for repairing the skeleton. Biotechnol Genet Eng Rev 21:133–143PubMedCrossRefGoogle Scholar
  37. Shi S, Gronthos S, Chen S, Reddi A, Counter CM, Robey PG, Wang CY (2002) Bone formation by human postnatal bone marrow stromal stem cells is enhanced by telomerase expression. Nat Biotechnol 20:587–591PubMedCrossRefGoogle Scholar
  38. Sonoyama W, Liu Y, Fang D, Yamaza T, Seo BM, Zhang C, Liu H, Gronthos S, Wang CY, Wang S, Shi S (2006) Mesenchymal stem cell-mediated functional tooth regeneration in swine. PLoS ONE 1:e79PubMedCentralPubMedCrossRefGoogle Scholar
  39. Takeda S, Bonnamy JP, Owen MJ, Ducy P, Karsenty G (2001) Continuous expression of Cbfa1 in nonhypertrophic chondrocytes uncovers its ability to induce hypertrophic chondrocyte differentiation and partially rescues Cbfa1-deficient mice. Genes Dev 15:467–481PubMedCentralPubMedCrossRefGoogle Scholar
  40. Ulsamer A, Ortuno MJ, Ruiz S, Susperregui AR, Osses N, Rosa JL, Ventura F (2008) BMP-2 induces Osterix expression through up-regulation of Dlx5 and its phosphorylation by p38. J Biol Chem 283:3816–3826PubMedCrossRefGoogle Scholar
  41. Viale-Bouroncle S, Felthaus O, Schmalz G, Brockhoff G, Reichert TE, Morsczeck C (2012) The transcription factor DLX3 regulates the osteogenic differentiation of human dental follicle precursor cells. Stem Cells Dev 21:1936–1947PubMedCentralPubMedCrossRefGoogle Scholar
  42. Ye L, Fan Z, Yu B, Chang J, Al Hezaimi K, Zhou X, Park NH, Wang CY (2012) Histone Demethylases KDM4B and KDM6B Promote Osteogenic Differentiation of Human MSCs. Cell Stem Cell 11:50–61PubMedCentralPubMedCrossRefGoogle Scholar
  43. Yu S, Long J, Yu J, Du J, Ma P, Ma Y, Yang D, Fan Z (2013) Analysis of differentiation potentials and gene expression profiles of mesenchymal stem cells derived from periodontal ligament and Wharton’s jelly of the umbilical cord. Cells Tissues Organs 197:209–223PubMedCrossRefGoogle Scholar
  44. Zhang J, Zhu J, Valverde P, Li L, Pageau S, Tu Q, Nishimura R, Yoneda T, Yang P, Zheng W, Ma W, Chen J (2008) Phenotypic analysis of Dlx5 overexpression in post-natal bone. J Dent Res 87:45–50PubMedCentralPubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2014

Authors and Affiliations

  • Binbin Qu
    • 1
    • 2
    • 3
  • Ousheng Liu
    • 1
    • 2
  • Xiaodan Fang
    • 1
    • 2
  • Haixia Zhang
    • 1
    • 2
    • 4
  • Yuehong Wang
    • 1
    • 2
  • Hongzhi Quan
    • 1
    • 2
  • Jie Zhang
    • 1
    • 2
    • 3
  • Jing Zhou
    • 1
    • 2
    • 3
  • Jun Zuo
    • 1
    • 2
  • Jianxia Tang
    • 1
    • 2
  • Zhangui Tang
    • 1
    • 2
    Email author
  1. 1.Xiangya Stomatology HospitalCentral South UniversityChangshaChina
  2. 2.School of StomatologyCentral South UniversityChangshaChina
  3. 3.Department of Oral and Maxilofacial SurgeryChangsha Stomatological HospitalChangshaChina
  4. 4.Department of Oncology, The Second Xiangya HospitalCentral South UniversityChangshaChina

Personalised recommendations