Journal of Bone and Mineral Metabolism

, Volume 31, Issue 1, pp 53–63 | Cite as

Retinoic acid regulates commitment of undifferentiated mesenchymal stem cells into osteoblasts and adipocytes

  • Kunihiro Hisada
  • Kenji Hata
  • Fumitaka Ichida
  • Takuma Matsubara
  • Hideo Orimo
  • Tamaki Nakano
  • Hirohumi Yatani
  • Riko NishimuraEmail author
  • Toshiyuki Yoneda
Original article


Evidence indicates that the balance between osteoblastogenesis and adipogenesis of mesenchymal stem cells (MSCs) is regulated by several hormones, growth factors, and their downstream signaling cascades. Previous studies suggest that retinoic acid (RA) plays a role in osteoblastogenesis and adipogenesis. However, it is unknown whether RA regulates commitment of MSCs into osteoblasts and adipocytes. In this study, we investigated the role of RA in differentiation of MSCs using the C3H10T1/2 cell line. RA stimulated activity and expression of alkaline phosphatase (ALP) and upregulated activity of the ALP gene promoter. The effects of RA were further enhanced by bone morphogenetic protein 2 (BMP2) and resultant Smad signaling. Furthermore, overexpression of Runx2 and Msx2, critical transcription factors for bone formation and BMP2-dependent osteoblastogenesis, enhanced RA-dependent ALP activity. In view of these findings, RA likely stimulates osteoblast differentiation through the BMP2–Smad–Runx2/Msx2 pathway. In contrast, RA markedly inhibited BMP2-induced adipocyte differentiation, suppressing expression of peroxisome proliferator-activated receptor-γ (PPARγ), CCAAT/enhancer-binding protein (C/EBP)α and C/EBPδ, and inhibiting adipogenic function of C/EBPβ, C/EBPδ, and PPARγ. In conclusion, our data suggest that RA regulates commitment of MSCs into osteoblasts and adipocytes by controlling transcriptional regulators.


Retinoic acid Osteoblast Adipocyte Bone morphogenetic protein 



We thank Dr. Minoru Morikawa, Dr. Yoshiaki Ito, Dr. Shizuo Akira, Dr. Bruce M. Spiegelman. and Dr. Takeshi Imamura for providing 3T3-F442A cell line cells, Runx2 cDNA, C/EBPβ and C/EBPδ cDNA, aP2 gene promoter construct, and SBE luciferase construct, respectively. This work was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan, and by the Uehara Memorial Foundation and the Astellas Foundation for Research on Metabolic Disorders.

Conflict of interest

All authors have no conflicts of interest.


  1. 1.
    Owen M (1988) Marrow stromal stem cells. J Cell Sci Suppl 10:63–76PubMedGoogle Scholar
  2. 2.
    Pittenger MF, Mackay AM, Beck SC, Jaiswal RK, Douglas R, Mosca JD, Moorman MA, Simonetti DW, Craig S, Marshak DR (1999) Multilineage potential of adult human mesenchymal stem cells. Science 284:143–147PubMedCrossRefGoogle Scholar
  3. 3.
    Nishimura R, Hata K, Ikeda F, Ichida F, Shimoyama A, Matsubara T, Wada M, Amano K, Yoneda T (2008) Signal transduction and transcriptional regulation during mesenchymal cell differentiation. J Bone Miner Metab 26:203–212PubMedCrossRefGoogle Scholar
  4. 4.
    Burkhardt R, Kettner G, Böhm W, Schmidmeier M, Schlag R, Frisch B, Mallmann B, Eisenmenger W, Gilg T (1987) Changes in trabecular bone, hematopoiesis and bone marrow vessels in aplastic anemia, primary osteoporosis, and old age: a comparative histomorphometric study. Bone (NY) 8:157–164Google Scholar
  5. 5.
    Beresford JN, Bennett JH, Devlin C, Leboy PS, Owen ME (1992) Evidence for an inverse relationship between the differentiation of adipocytic and osteogenic cells in rat marrow stromal cell cultures. J Cell Sci 102:341–351PubMedGoogle Scholar
  6. 6.
    Raisz LG, Kream BE (1983) Regulation of bone formation. N Engl J Med 309:29–35PubMedCrossRefGoogle Scholar
  7. 7.
    Raisz LG, Kream BE (1983) Regulation of bone formation (second of two parts). N Engl J Med 309:83–89PubMedCrossRefGoogle Scholar
  8. 8.
    Yamaguchi A, Komori T, Suda T (2000) Regulation of osteoblast differentiation mediated by bone morphogenetic proteins, hedgehogs, and Cbfa1. Endocr Rev 21:393–411PubMedCrossRefGoogle Scholar
  9. 9.
    Wozney JM, Rosen V, Celeste AJ, Mitsock LM, Whitters MJ, Kriz RW, Hewick RM, Wang EA (1988) Novel regulators of bone formation: molecular clones and activities. Science 242:1528–1534PubMedCrossRefGoogle Scholar
  10. 10.
    Nishimura R, Kato Y, Chen D, Harris SE, Mundy GR, Yoneda T (1998) Smad5 and DPC4 are key molecules in mediating BMP-2-induced osteoblastic differentiation of the pluripotent mesenchymal precursor cell line C2C12. J Biol Chem 273:1872–1879PubMedCrossRefGoogle Scholar
  11. 11.
    Lee KS, Hong SH, Bae SC (2002) Both the Smad and p38 MAPK pathways play a crucial role in Runx2 expression following induction by transforming growth factor-beta and bone morphogenetic protein. Oncogene 21:7156–7163PubMedCrossRefGoogle Scholar
  12. 12.
    Lee KS, Kim HJ, Li QL, Chi XZ, Ueta C, Komori T, Wozney JM, Kim EG, Choi JY, Ryoo HM, Bae SC (2000) Runx2 is a common target of transforming growth factor beta1 and bone morphogenetic protein 2, and cooperation between Runx2 and Smad5 induces osteoblast-specific gene expression in the pluripotent mesenchymal precursor cell line C2C12. Mol Cell Biol 20:8783–8792PubMedCrossRefGoogle Scholar
  13. 13.
    Yagi K, Tsuji K, Nifuji A, Shinomiya K, Nakashima K, DeCrombrugghe B, Noda M (2003) Bone morphogenetic protein-2 enhances osterix gene expression in chondrocytes. J Cell Biochem 88:1077–1083PubMedCrossRefGoogle Scholar
  14. 14.
    Harada S, Rodan GA (2003) Control of osteoblast function and regulation of bone mass. Nature (Lond) 423:349–355CrossRefGoogle Scholar
  15. 15.
    Nishimura R, Hata K, Ikeda F, Matsubara T, Yamashita K, Ichida F, Yoneda T (2003) The role of Smads in BMP signaling. Front Biosci 8:s275–s284PubMedCrossRefGoogle Scholar
  16. 16.
    Tontonoz P, Hu E, Spiegelman BM (1994) Stimulation of adipogenesis in fibroblasts by PPAR gamma 2, a lipid-activated transcription factor. Cell 79:1147–1156PubMedCrossRefGoogle Scholar
  17. 17.
    Wu Z, Xie Y, Bucher NL, Farmer SR (1995) Conditional ectopic expression of C/EBP beta in NIH-3T3 cells induces PPAR gamma and stimulates adipogenesis. Genes Dev 9:2350–2363PubMedCrossRefGoogle Scholar
  18. 18.
    Ahrens M, Ankenbauer T, Schröder D, Hollnagel A, Mayer H, Gross G (1993) Expression of human bone morphogenetic proteins-2 or -4 in murine mesenchymal progenitor C3H10T1/2 cells induces differentiation into distinct mesenchymal cell lineages. DNA Cell Biol 12:871–880PubMedGoogle Scholar
  19. 19.
    Kang S, Bennett CN, Gerin I, Rapp LA, Hankenson KD, MacDougald OA (2007) Wnt signaling stimulates osteoblastogenesis of mesenchymal precursors by suppressing CCAAT/enhancer-binding protein alpha and peroxisome proliferator-activated receptor gamma. J Biol Chem 282:14515–14524PubMedCrossRefGoogle Scholar
  20. 20.
    Ross SE, Hemati N, Longo KA, Bennett CN, Lucas PC, Erickson RL, MacDougald OA (2000) Inhibition of adipogenesis by Wnt signaling. Science 289:950–953PubMedCrossRefGoogle Scholar
  21. 21.
    Moon RT, Bowerman B, Boutros M, Perrimon N (2002) The promise and perils of Wnt signaling through beta-catenin. Science 296:1644–1646PubMedCrossRefGoogle Scholar
  22. 22.
    Takada I, Mihara M, Suzawa M, Ohtake F, Kobayashi S, Igarashi M, Youn MY, Takeyama K, Nakamura T, Mezaki Y, Takezawa S, Yogiashi Y, Kitagawa H, Yamada G, Takada S, Minami Y, Shibuya H, Matsumoto K, Kato S (2007) A histone lysine methyltransferase activated by non-canonical Wnt signalling suppresses PPAR-gamma transactivation. Nat Cell Biol 9:1273–1285PubMedCrossRefGoogle Scholar
  23. 23.
    Tracey KJ, Cerami A (1990) Metabolic responses to cachectin/TNF. A brief review. Ann N Y Acad Sci 587:325–331PubMedGoogle Scholar
  24. 24.
    Suzawa M, Takada I, Yanagisawa J, Ohtake F, Ogawa S, Yamauchi T, Kadowaki T, Takeuchi Y, Shibuya H, Gotoh Y, Matsumoto K, Kato S (2003) Cytokines suppress adipogenesis and PPAR-gamma function through the TAK1/TAB 1/NIK cascade. Nat Cell Biol 5:224–230PubMedCrossRefGoogle Scholar
  25. 25.
    Kaneki H, Guo R, Chen D, Yao Z, Schwarz EM, Zhang YE, Boyce BF, Xing L (2006) Tumor necrosis factor promotes Runx2 degradation through up-regulation of Smurf1 and Smurf2 in osteoblasts. J Biol Chem 281:4326–4333PubMedCrossRefGoogle Scholar
  26. 26.
    Wilkie AO, Tang Z, Elanko N, Walsh S, Twigg SR, Hurst JA, Wall SA, Chrzanowska KH, Jr Maxson RE (2000) Functional haploinsufficiency of the human homeobox gene MSX2 causes defects in skull ossification. Nat Genet 24:387–390PubMedCrossRefGoogle Scholar
  27. 27.
    Satokata I, Ma L, Ohshima H, Bei M, Woo I, Nishizawa K, Maeda T, Takano Y, Uchiyama M, Heaney S, Peters H, Tang Z, Maxson R, Maas R (2000) Msx2 deficiency in mice causes pleiotropic defects in bone growth and ectodermal organ formation. Nat Genet 24:391–395PubMedCrossRefGoogle Scholar
  28. 28.
    Cheng SL, Shao JS, Charlton-Kachigian N, Loewy AP, Towler DA (2003) MSX2 promotes osteogenesis and suppresses adipogenic differentiation of multipotent mesenchymal progenitors. J Biol Chem 278:45969–45977PubMedCrossRefGoogle Scholar
  29. 29.
    Ichida F, Nishimura R, Hata K, Matsubara T, Ikeda F, Hisada K, Yatani H, Cao X, Komori T, Yamaguchi A, Yoneda T (2004) Reciprocal roles of MSX2 in regulation of osteoblast and adipocyte differentiation. J Biol Chem 279:34015–34022PubMedCrossRefGoogle Scholar
  30. 30.
    Hata K, Nishimura R, Ueda M, Ikeda F, Matsubara T, Ichida F, Hisada K, Nokubi T, Yamaguchi A, Yoneda T (2005) A CCAAT/enhancer binding protein beta isoform, liver-enriched inhibitory protein, regulates commitment of osteoblasts and adipocytes. Mol Cell Biol 25:1971–1979PubMedCrossRefGoogle Scholar
  31. 31.
    Hong JH, Hwang ES, McManus MT, Amsterdam A, Tian Y, Kalmukova R, Mueller E, Benjamin T, Spiegelman BM, Sharp PA, Hopkins N, Yaffe MB (2005) TAZ, a transcriptional modulator of mesenchymal stem cell differentiation. Science 309:1074–1078PubMedCrossRefGoogle Scholar
  32. 32.
    Katagiri T, Yamaguchi A, Ikeda T, Yoshiki S, Wozney JM, Rosen V, Wang EA, Tanaka H, Omura S, Suda T (1990) The non-osteogenic mouse pluripotent cell line, C3H10T1/2, is induced to differentiate into osteoblastic cells by recombinant human bone morphogenetic protein-2. Biochem Biophys Res Commun 172:295–299PubMedCrossRefGoogle Scholar
  33. 33.
    Skillington J, Choy L, Derynck R (2002) Bone morphogenetic protein and retinoic acid signaling cooperate to induce osteoblast differentiation of preadipocytes. J Cell Biol 159:135–146PubMedCrossRefGoogle Scholar
  34. 34.
    Choong PF, Martin TJ, Ng KW (1993) Effects of ascorbic acid, calcitriol, and retinoic acid on the differentiation of preosteoblasts. J Orthop Res 11:638–647PubMedCrossRefGoogle Scholar
  35. 35.
    Ng KW, Manji SS, Young MF, Findlay DM (1989) Opposing influences of glucocorticoid and retinoic acid on transcriptional control in preosteoblasts. Mol Endocrinol 3:2079–2085PubMedCrossRefGoogle Scholar
  36. 36.
    Ogston N, Harrison AJ, Cheung HF, Ashton BA, Hampson G (2002) Dexamethasone and retinoic acid differentially regulate growth and differentiation in an immortalised human clonal bone marrow stromal cell line with osteoblastic characteristics. Steroids 67:895–906PubMedCrossRefGoogle Scholar
  37. 37.
    Schwarz EJ, Reginato MJ, Shao D, Krakow SL, Lazar MA (1997) Retinoic acid blocks adipogenesis by inhibiting C/EBPβ-mediated transcription. Mol Cell Biol 17:1552–1561PubMedGoogle Scholar
  38. 38.
    Spiegelman BM, Flier JS (2001) Obesity and the regulation of energy balance. Cell 104:531–543PubMedCrossRefGoogle Scholar
  39. 39.
    Orimo H, Shimada T (2005) Regulation of the human tissue-nonspecific alkaline phosphatase gene expression by all-trans-retinoic acid in SaOS-2 osteosarcoma cell line. Bone (NY) 36:866–876Google Scholar
  40. 40.
    Nishimura R, Hata K, Harris SE, Ikeda F, Yoneda T (2002) Core-binding factor alpha 1 (Cbfa1) induces osteoblastic differentiation of C2C12 cells without interactions with Smad1 and Smad5. Bone (NY) 31:303–312Google Scholar
  41. 41.
    Matsubara T, Kida K, Yamaguchi A, Hata K, Ichida F, Meguro H, Aburatani H, Nishimura R, Yoneda T (2008) BMP2 regulates Osterix through Msx2 and Runx2 during osteoblast differentiation. J Biol Chem 283:29119–29125PubMedCrossRefGoogle Scholar
  42. 42.
    Wang EA, Israel DI, Kelly S, Luxenberg DP (1993) Bone morphogenetic protein-2 causes commitment and differentiation in C3H10T1/2 and 3T3 cells. Growth Factors 9:57–71PubMedCrossRefGoogle Scholar
  43. 43.
    Komori T, Yagi H, Nomura S, Yamaguchi A, Sasaki K, Deguchi K, Shimizu Y, Bronson RT, Gao YH, Inada M, Sato M, Okamoto R, Kitamura Y, Yoshiki S, Kishimoto T (1997) Targeted disruption of Cbfa1 results in a complete lack of bone formation owing to maturational arrest of osteoblasts. Cell 89:755–764PubMedCrossRefGoogle Scholar
  44. 44.
    Mundlos S, Otto F, Mundlos C, Mulliken JB, Aylsworth AS, Albright S, Lindhout D, Cole WG, Henn W, Knoll JH, Owen MJ, Mertelsmann R, Zabel BU, Olsen BR (1997) Mutations involving the transcription factor CBFA1 cause cleidocranial dysplasia. Cell 89:773–779PubMedCrossRefGoogle Scholar
  45. 45.
    Otto F, Thornell AP, Crompton T, Denzel A, Gilmour KC, Rosewell IR, Stamp GW, Beddington RS, Mundlos S, Olsen BR, Selby PB, Owen MJ (1997) Cbfa1, a candidate gene for cleidocranial dysplasia syndrome, is essential for osteoblast differentiation and bone development. Cell 89:765–771PubMedCrossRefGoogle Scholar
  46. 46.
    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
  47. 47.
    Williams JA, Kondo N, Okabe T, Takeshita N, Pilchak DM, Koyama E, Ochiai T, Jensen D, Chu ML, Kane MA, Napoli JL, Enomoto-Iwamoto M, Ghyselinck N, Chambon P, Pacifici M, Iwamoto M (2009) Retinoic acid receptors are required for skeletal growth, matrix homeostasis and growth plate function in postnatal mouse. Dev Biol 328:315–327PubMedCrossRefGoogle Scholar
  48. 48.
    Jackson HA, Sheehan AH (2005) Effect of vitamin A on fracture risk. Ann Pharmacother 39:2086–2090PubMedCrossRefGoogle Scholar
  49. 49.
    Lim LS, Harnack LJ, Lazovich D, Folsom AR (2004) Vitamin A intake and the risk of hip fracture in postmenopausal women: the Iowa Women’s Health Study. Osteoporos Int 15:552–559PubMedCrossRefGoogle Scholar
  50. 50.
    Balkan W, Rodríguez-Gonzalez M, Pang M, Fernandez I, Troen BR (2011) Retinoic acid inhibits NFATc1 expression and osteoclast differentiation. J Bone Miner Metab 29:652–661PubMedCrossRefGoogle Scholar

Copyright information

© The Japanese Society for Bone and Mineral Research and Springer 2012

Authors and Affiliations

  • Kunihiro Hisada
    • 1
    • 2
  • Kenji Hata
    • 1
  • Fumitaka Ichida
    • 1
  • Takuma Matsubara
    • 1
  • Hideo Orimo
    • 3
  • Tamaki Nakano
    • 2
  • Hirohumi Yatani
    • 2
  • Riko Nishimura
    • 1
    Email author
  • Toshiyuki Yoneda
    • 1
  1. 1.Department of Molecular and Cellular BiochemistryOsaka University Graduate School of DentistrySuitaJapan
  2. 2.Department of Fixed ProsthodonticsOsaka University Graduate School of DentistrySuitaJapan
  3. 3.Division of Metabolism and Nutrition, Department of Biochemistry and Molecular BiologyNippon Medical SchoolTokyoJapan

Personalised recommendations