Skip to main content
SpringerLink
Log in

Activating transcription factor 4, an ER stress mediator, is required for, but excessive ER stress suppresses osteoblastogenesis by bortezomib

  • Original Article
  • Published:
International Journal of Hematology Aims and scope Submit manuscript

Abstract

Endoplasmic reticulum (ER) stress is induced in matrix-producing osteoblasts and plays an essential role in osteoblastogenesis. Although the bone anabolic activity of proteasome inhibitors has been demonstrated, the roles of ER stress induced by proteasome inhibition in osteoblastogenesis remain largely unknown. Here we show that bortezomib translationally increases protein levels of activating transcription factor 4 (ATF4), a downstream mediator of ER stress, in bone marrow stromal cells and MC3T3-E1 preosteoblastic cells. The suppression of ATF4 expression by siRNA abrogated osteocalcin expression and mineralized nodule formation by MC3T3-E1 cells induced by bortezomib, indicating a critical role for ATF4 in bortezomib-mediated osteoblastogenesis. However, bortezomib at 20 nM or higher abolished the mineralized nodule formation along with reductions in the expression of osteoblastogenesis mediators β-catenin and Osterix. Furthermore, at 50 nM, bortezomib induced the expression of C/EBP homologous protein (CHOP), suggesting activation of the ATF4–CHOP pro-apoptotic pathway. These results suggest that a low dose of bortezomib induces osteogenic activity, but that, in contrast, excessive ER stress caused by bortezomib at higher doses hampers osteoblastogenesis. Therefore, dosing schedules for proteasome inhibitors warrant further study to maximize anabolic actions without compromising anti-MM activity in patients with multiple myeloma (MM).

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

Similar content being viewed by others

References

  1. Abe M. Targeting the interplay between myeloma cells and the bone marrow microenvironment in myeloma. Int J Hematol. 2011;94:334–43.

    Article  PubMed  Google Scholar 

  2. Giuliani N, Morandi F, Tagliaferri S, Lazzaretti M, Bonomini S, Crugnola M, et al. The proteasome inhibitor bortezomib affects osteoblast differentiation in vitro and in vivo in multiple myeloma patients. Blood. 2007;110:334–8.

    Article  PubMed  CAS  Google Scholar 

  3. Zangari M, Esseltine D, Lee CK, Barlogie B, Elice F, Burns MJ, et al. Response to bortezomib is associated to osteoblastic activation in patients with multiple myeloma. Br J Haematol. 2005;131:71–3.

    Article  PubMed  CAS  Google Scholar 

  4. Ozaki S, Tanaka O, Fujii S, Shigekiyo Y, Miki H, Choraku M, et al. Therapy with bortezomib plus dexamethasone induces osteoblast activation in responsive patients with multiple myeloma. Int J Hematol. 2007;86:180–5.

    Article  PubMed  CAS  Google Scholar 

  5. Zangari M, Aujay M, Zhan F, Hetherington KL, Berno T, Vij R, et al. Alkaline phosphatase variation during carfilzomib treatment is associated with best response in multiple myeloma patients. Eur J Haematol. 2011;86:484–7.

    Article  PubMed  CAS  Google Scholar 

  6. Garrett IR, Chen D, Gutierrez G, Zhao M, Escobedo A, Rossini G, et al. Selective inhibitors of the osteoblast proteasome stimulate bone formation in vivo and in vitro. J Clin Invest. 2003;111:1771–82.

    PubMed  CAS  Google Scholar 

  7. Mukherjee S, Raje N, Schoonmaker JA, Liu JC, Hideshima T, Wein MN, et al. Pharmacologic targeting of a stem/progenitor population in vivo is associated with enhanced bone regeneration in mice. J Clin Invest. 2008;118:491–504.

    PubMed  CAS  Google Scholar 

  8. Pennisi A, Li X, Ling W, Khan S, Zangari M, Yaccoby S. The proteasome inhibitor, bortezomib suppresses primary myeloma and stimulates bone formation in myelomatous and nonmyelomatous bones in vivo. Am J Hematol. 2009;84:6–14.

    Article  PubMed  CAS  Google Scholar 

  9. Qiang YW, Hu B, Chen Y, Zhong Y, Shi B, Barlogie B, et al. Bortezomib induces osteoblast differentiation via Wnt-independent activation of {beta}-catenin/TCF signaling. Blood. 2009;113(18):4319–30.

    Article  PubMed  CAS  Google Scholar 

  10. Murakami T, Saito A, Hino S, Kondo S, Kanemoto S, Chihara K, et al. Signalling mediated by the endoplasmic reticulum stress transducer OASIS is involved in bone formation. Nat Cell Biol. 2009;11:1205–11.

    Article  PubMed  CAS  Google Scholar 

  11. Tohmonda T, Miyauchi Y, Ghosh R, Yoda M, Uchikawa S, Takito J, et al. The IRE1alpha-XBP1 pathway is essential for osteoblast differentiation through promoting transcription of Osterix. EMBO Rep. 2011;12:451–7.

    Article  PubMed  CAS  Google Scholar 

  12. Saito A, Ochiai K, Kondo S, Tsumagari K, Murakami T, Cavener DR, et al. Endoplasmic reticulum stress response mediated by the PERK–eIF2(alpha)–ATF4 pathway is involved in osteoblast differentiation induced by BMP2. J Biol Chem. 2011;286:4809–18.

    Article  PubMed  CAS  Google Scholar 

  13. Hamamura K, Yokota H. Stress to endoplasmic reticulum of mouse osteoblasts induces apoptosis and transcriptional activation for bone remodeling. FEBS Lett. 2007;581:1769–74.

    Article  PubMed  CAS  Google Scholar 

  14. Zhang K, Kaufman RJ. From endoplasmic-reticulum stress to the inflammatory response. Nature. 2008;454:455–62.

    Article  PubMed  CAS  Google Scholar 

  15. Yang X, Matsuda K, Bialek P, Jacquot S, Masuoka HC, Schinke T, et al. ATF4 is a substrate of RSK2 and an essential regulator of osteoblast biology; implication for Coffin-Lowry syndrome. Cell. 2004;117:387–98.

    Article  PubMed  CAS  Google Scholar 

  16. Marie PJ. Transcription factors controlling osteoblastogenesis. Arch Biochem Biophys. 2008;473:98–105.

    Article  PubMed  CAS  Google Scholar 

  17. Yu S, Franceschi RT, Luo M, Fan J, Jiang D, Cao H, et al. Critical role of activating transcription factor 4 in the anabolic actions of parathyroid hormone in bone. PLoS One. 2009;4:e7583.

    Article  PubMed  Google Scholar 

  18. Tominaga H, Maeda S, Hayashi M, Takeda S, Akira S, Komiya S, et al. CCAAT/enhancer-binding protein beta promotes osteoblast differentiation by enhancing Runx2 activity with ATF4. Mol Biol Cell. 2008;19:5373–86.

    Article  PubMed  CAS  Google Scholar 

  19. Obeng EA, Carlson LM, Gutman DM, Harrington WJ Jr, Lee KP, Boise LH. Proteasome inhibitors induce a terminal unfolded protein response in multiple myeloma cells. Blood. 2006;107:4907–16.

    Article  PubMed  CAS  Google Scholar 

  20. Chauhan D, Singh A, Brahmandam M, Podar K, Hideshima T, Richardson P, et al. Combination of proteasome inhibitors bortezomib and NPI-0052 trigger in vivo synergistic cytotoxicity in multiple myeloma. Blood. 2008;111:1654–64.

    Article  PubMed  CAS  Google Scholar 

  21. Rzymski T, Milani M, Singleton DC, Harris AL. Role of ATF4 in regulation of autophagy and resistance to drugs and hypoxia. Cell Cycle. 2009;8(23):3838–47.

    Article  PubMed  CAS  Google Scholar 

  22. Rzymski T, Milani M, Singleton DC, Harris AL. Role of ATF4 in regulation of autophagy and resistance to drugs and hypoxia. Cell Cycle. 2009;8:3838–47.

    Article  PubMed  CAS  Google Scholar 

  23. Matsumoto T, Igarashi C, Takeuchi Y, Harada S, Kikuchi T, Yamato H, et al. Stimulation by 1,25-dihydroxyvitamin D3 of in vitro mineralization induced by osteoblast-like MC3T3-E1 cells. Bone. 1991;12:27–32.

    Article  PubMed  CAS  Google Scholar 

  24. Oshima T, Abe M, Asano J, Hara T, Kitazoe K, Sekimoto E, et al. Myeloma cells suppress bone formation by secreting a soluble Wnt inhibitor, sFRP-2. Blood. 2005;106:3160–5.

    Article  PubMed  CAS  Google Scholar 

  25. Hiasa M, Abe M, Nakano A, Oda A, Amou H, Kido S, et al. GM-CSF and IL-4 induce dendritic cell differentiation and disrupt osteoclastogenesis through M-CSF receptor shedding by up-regulation of TNF-alpha converting enzyme (TACE). Blood. 2009;114:4517–26.

    Article  PubMed  CAS  Google Scholar 

  26. Yang X, Karsenty G. ATF4, the osteoblast accumulation of which is determined post-translationally, can induce osteoblast-specific gene expression in non-osteoblastic cells. J Biol Chem. 2004;279:47109–14.

    Article  PubMed  CAS  Google Scholar 

  27. Xiao G, Jiang D, Ge C, Zhao Z, Lai Y, Boules H, et al. Cooperative interactions between activating transcription factor 4 and Runx2/Cbfa1 stimulate osteoblast-specific osteocalcin gene expression. J Biol Chem. 2005;280:30689–96.

    Article  PubMed  CAS  Google Scholar 

  28. Shirakawa K, Maeda S, Gotoh T, Hayashi M, Shinomiya K, Ehata S, et al. CCAAT/enhancer-binding protein homologous protein (CHOP) regulates osteoblast differentiation. Mol Cell Biol. 2006;26:6105–16.

    Article  PubMed  CAS  Google Scholar 

  29. Jiang HY, Wek RC. Phosphorylation of the alpha-subunit of the eukaryotic initiation factor-2 (eIF2alpha) reduces protein synthesis and enhances apoptosis in response to proteasome inhibition. J Biol Chem. 2005;280:14189–202.

    Article  PubMed  CAS  Google Scholar 

  30. Moreau P, Pylypenko H, Grosicki S, Karamanesht I, Leleu X, Grishunina M, et al. Subcutaneous versus intravenous administration of bortezomib in patients with relapsed multiple myeloma: a randomised, phase 3, non-inferiority study. Lancet Oncol. 2011;12:431–40.

    Article  PubMed  Google Scholar 

  31. Moreau P, Coiteux V, Hulin C, Leleu X, van de Velde H, Acharya M, et al. Prospective comparison of subcutaneous versus intravenous administration of bortezomib in patients with multiple myeloma. Haematologica. 2008;93:1908–11.

    Article  PubMed  CAS  Google Scholar 

  32. Chauhan D, Tian Z, Zhou B, Kuhn D, Orlowski R, Raje N, et al. In vitro and in vivo selective antitumor activity of a novel orally bioavailable proteasome inhibitor MLN9708 against multiple myeloma cells. Clin Cancer Res. 2011;17:5311–21.

    Article  PubMed  CAS  Google Scholar 

  33. Kupperman E, Lee EC, Cao Y, Bannerman B, Fitzgerald M, Berger A, et al. Evaluation of the proteasome inhibitor MLN9708 in preclinical models of human cancer. Cancer Res. 2010;70:1970–80.

    Article  PubMed  CAS  Google Scholar 

  34. Lee EC, Fitzgerald M, Bannerman B, Donelan J, Bano K, Terkelsen J, et al. Antitumor activity of the investigational proteasome inhibitor MLN9708 in mouse models of B-cell and plasma cell malignancies. Clin Cancer Res. 2011;17:7313–23.

    Article  PubMed  CAS  Google Scholar 

  35. Fulciniti M, Tassone P, Hideshima T, Vallet S, Nanjappa P, Ettenberg SA, et al. Anti-DKK1 mAb (BHQ880) as a potential therapeutic agent for multiple myeloma. Blood. 2009;114:371–9.

    Article  PubMed  CAS  Google Scholar 

  36. Raje N, Vallet S. Sotatercept, a soluble activin receptor type 2A IgG-Fc fusion protein for the treatment of anemia and bone loss. Curr Opin Mol Ther. 2010;12:586–97.

    PubMed  CAS  Google Scholar 

  37. Heath DJ, Chantry AD, Buckle CH, Coulton L, Shaughnessy JD Jr, Evans HR, et al. Inhibiting Dickkopf-1 (Dkk1) removes suppression of bone formation and prevents the development of osteolytic bone disease in multiple myeloma. J Bone Miner Res. 2009;24:425–36.

    Article  PubMed  CAS  Google Scholar 

  38. Vallet S, Mukherjee S, Vaghela N, Hideshima T, Fulciniti M, Pozzi S, et al. Activin A promotes multiple myeloma-induced osteolysis and is a promising target for myeloma bone disease. Proc Natl Acad Sci USA. 2010;107:5124–9.

    Article  PubMed  CAS  Google Scholar 

  39. Chantry AD, Heath D, Mulivor AW, Pearsall S, Baud’huin M, Coulton L, et al. Inhibiting activin-A signaling stimulates bone formation and prevents cancer-induced bone destruction in vivo. J Bone Miner Res. 2010;25:2633–46.

    Article  PubMed  Google Scholar 

  40. Takeuchi K, Abe M, Hiasa M, Oda A, Amou H, Kido S, et al. Tgf-Beta inhibition restores terminal osteoblast differentiation to suppress myeloma growth. PLoS One. 2010;5:e9870.

    Article  PubMed  Google Scholar 

  41. Yaccoby S. Osteoblastogenesis and tumor growth in myeloma. Leuk Lymphoma. 2010;51:213–20.

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

This work was supported in part by Grants-in-aid for Scientific Research (C) to M.A., and a National Cancer Center Research and Development Fund (21-8-5) to M.A. from the Ministry of Health, Labor and Welfare of Japan. The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.

Conflict of interest

The authors declare no competing financial interests related to this work.

Author information

Authors and Affiliations

  1. Department of Medicine and Bioregulatory Sciences, University of Tokushima Graduate School of Medicine, 3-18-15 Kuramoto-cho, Tokushima, 770-8503, Japan

    Shingen Nakamura, Ayako Nakano, Asuka Oda, Hiroe Amou, Keiichiro Watanabe, Takeshi Harada, Shiro Fujii, Kyoko Takeuchi, Kumiko Kagawa, Toshio Matsumoto & Masahiro Abe

  2. Department of Transfusion Medicine, Tokushima University Hospital, Tokushima, Japan

    Hirokazu Miki

  3. Department of Molecular Nutrition, Institute of Health Biosciences, University of Tokushima Graduate School, Tokushima, Japan

    Shinsuke Kido

  4. Department of Biomaterials and Bioengineerings, University of Tokushima Graduate School of Oral Sciences, Tokushima, Japan

    Masahiro Hiasa

  5. Division of Internal Medicine, Tokushima Prefectural Central Hospital, Tokushima, Japan

    Shuji Ozaki

Authors
  1. Shingen Nakamura
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Hirokazu Miki
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Shinsuke Kido
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Ayako Nakano
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Masahiro Hiasa
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Asuka Oda
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Hiroe Amou
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Keiichiro Watanabe
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Takeshi Harada
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Shiro Fujii
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Kyoko Takeuchi
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Kumiko Kagawa
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. Shuji Ozaki
    View author publications

    You can also search for this author in PubMed Google Scholar

  14. Toshio Matsumoto
    View author publications

    You can also search for this author in PubMed Google Scholar

  15. Masahiro Abe
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Masahiro Abe.

About this article

Cite this article

Nakamura, S., Miki, H., Kido, S. et al. Activating transcription factor 4, an ER stress mediator, is required for, but excessive ER stress suppresses osteoblastogenesis by bortezomib. Int J Hematol 98, 66–73 (2013). https://doi.org/10.1007/s12185-013-1367-z

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12185-013-1367-z

Keywords

Search

Navigation