Skip to main content

Advertisement

SpringerLink
Log in

Novel method for gain-of-function analyses in primary osteoclasts using a non-viral gene delivery system

  • Original Article
  • Published:
Journal of Bone and Mineral Metabolism Aims and scope Submit manuscript

Abstract

Introduction

Overexpression studies have been commonly used to yield significant advances in cell biology. In vitro osteoclast culturing involves the differentiation of bone marrow-derived monocyte macrophage precursors (BMMs) in medium supplemented with macrophage colony-stimulating factor and receptor activator of nuclear factor-kB ligand (RANKL) into mature osteoclasts. Retroviral vectors are the gold standards for efficient gene delivery into BMMs. While this strategy is effective in BMMs that are in the early stages of differentiation, it is ineffective in RANKL-treated BMMs such as mono- and multinucleated osteoclasts. This study attempted to enhance gene delivery into differentiated BMMs using liposome-mediated RNA transfection.

Material and methods

BMMs were transfected with an EYFP overexpression plasmid or EYFP RNA by lipofection, or transduced with a retroviral vector expressing EYFP. EYFP expression was assessed by flow cytometry.

Results

We performed overexpression analyses using enhanced yellow fluorescent protein (EYFP). Although EYFP expression was observed 24 h after infection of BMMs with a recombinant retrovirus containing EYFP, expression of EYFP was observed within 3 h of transfection with EYFP RNA. Moreover, the efficiency of EYFP RNA for gene delivery into BMMs was comparable to that of retroviral transduction of EYFP. In contrast, while very few BMMs stimulated by RANKL for two days expressed EYFP after retroviral infection, more than half of the cells expressed EYFP after transfection with EYFP RNA.

Conclusion

RNA-mediated gene delivery is quick and easy method for performing gain-of-function analyses in primary osteoclast precursors and mature osteoclasts.

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
Fig. 5

Similar content being viewed by others

References

  1. Takayanagi H (2007) Osteoimmunology: shared mechanisms and crosstalk between the immune and bone systems. Nat Rev Immunol 7:292–304

    Article  CAS  Google Scholar 

  2. Lorenzo J, Horowitz M, Choi Y (2008) Osteoimmunology: interactions of the bone and immune system. Endocr Rev 29:403–440

    Article  CAS  Google Scholar 

  3. Okamoto K, Takayanagi H (2019) Osteoimmunology. Cold Spring Harbor Perspect Med 9:a03145

    Article  Google Scholar 

  4. Theill LE, Boyle WJ, Penninger JM (2002) RANK-L and RANK: T cells, bone loss, and mammalian evolution. Annu Rev Immunol 20:795–823

    Article  CAS  Google Scholar 

  5. Xing L, Schwarz EM, Boyce BF (2005) Osteoclast precursors, RANKL/RANK, and immunology. Immunol Rev 208:19–29

    Article  CAS  Google Scholar 

  6. Ross FP, Teitelbaum SL (2005) alphavbeta3 and macrophage colony-stimulating factor: partners in osteoclast biology. Immunol Rev 208:88–105

    Article  CAS  Google Scholar 

  7. Jimi E, Shuto T, Koga T (1995) Macrophage colony-stimulating factor and interleukin-1 alpha maintain the survival of osteoclast-like cells. Endocrinology 136:808–811

    Article  CAS  Google Scholar 

  8. Otero K, Turnbull IR, Poliani PL, Vermi W, Cerutti E, Aoshi T, Tassi I, Takai T, Stanley SL, Miller M, Shaw AS, Colonna M (2009) Macrophage colony-stimulating factor induces the proliferation and survival of macrophages via a pathway involving DAP12 and beta-catenin. Nat Immunol 10:734–743

    Article  CAS  Google Scholar 

  9. Novack DV, Faccio R (2011) Osteoclast motility: putting the brakes on bone resorption. Ageing Res Rev 10:54–61

    Article  CAS  Google Scholar 

  10. Miyazaki T, Sanjay A, Neff L, Tanaka S, Horne WC, Baron R (2004) Src kinase activity is essential for osteoclast function. J Biol Chem 279:17660–17666

    Article  CAS  Google Scholar 

  11. Ikeda F, Nishimura R, Matsubara T, Tanaka S, Inoue J, Reddy SV, Hata K, Yamashita K, Hiraga T, Watanabe T, Kukita T, Yoshioka K, Rao A, Yoneda T (2004) Critical roles of c-Jun signaling in regulation of NFAT family and RANKL-regulated osteoclast differentiation. J Clin Investig 114:475–484

    Article  CAS  Google Scholar 

  12. Gohda J, Akiyama T, Koga T, Takayanagi H, Tanaka S, Inoue J (2005) RANK-mediated amplification of TRAF6 signaling leads to NFATc1 induction during osteoclastogenesis. EMBO J 24:790–799

    Article  CAS  Google Scholar 

  13. Kim JH, Kim K, Jin HM, Song I, Youn BU, Lee SH, Choi Y, Kim N (2010) Negative feedback control of osteoclast formation through ubiquitin-mediated down-regulation of NFATc1. J Biol Chem 285:5224–5231

    Article  CAS  Google Scholar 

  14. Akiyama T, Bouillet P, Miyazaki T, Kadono Y, Chikuda H, Chung UI, Fukuda A, Hikita A, Seto H, Okada T, Inaba T, Sanjay A, Baron R, Kawaguchi H, Oda H, Nakamura K, Strasser A, Tanaka S (2003) Regulation of osteoclast apoptosis by ubiquitylation of proapoptotic BH3-only Bcl-2 family member Bim. EMBO J 22:6653–6664

    Article  CAS  Google Scholar 

  15. Tanaka S, Miyazaki T, Fukuda A, Akiyama T, Kadono Y, Wakeyama H, Kono S, Hoshikawa S, Nakamura M, Ohshima Y, Hikita A, Nakamura I, Nakamura K (2006) Molecular mechanism of the life and death of the osteoclast. Ann N Y Acad Sci 1068:180–186

    Article  CAS  Google Scholar 

  16. Ikeda F, Matsubara T, Tsurukai T, Hata K, Nishimura R, Yoneda T (2008) JNK/c-Jun signaling mediates an anti-apoptotic effect of RANKL in osteoclasts. J Bone Mineral Res 23:907–914

    Article  CAS  Google Scholar 

  17. Iwasawa M, Miyazaki T, Nagase Y, Akiyama T, Kadono Y, Nakamura M, Oshima Y, Yasui T, Matsumoto T, Nakamura T, Kato S, Hennighausen L, Nakamura K, Tanaka S (2009) The antiapoptotic protein Bcl-xL negatively regulates the bone-resorbing activity of osteoclasts in mice. J Clin Investig 119:3149–3159

    CAS  PubMed  Google Scholar 

  18. Takayanagi H, Kim S, Koga T, Nishina H, Isshiki M, Yoshida H, Saiura A, Isobe M, Yokochi T, Inoue J, Wagner EF, Mak TW, Kodama T, Taniguchi T (2002) Induction and activation of the transcription factor NFATc1 (NFAT2) integrate RANKL signaling in terminal differentiation of osteoclasts. Dev Cell 3:889–901

    Article  CAS  Google Scholar 

  19. Zhao B, Takami M, Yamada A, Wang X, Koga T, Hu X, Tamura T, Ozato K, Choi Y, Ivashkiv LB, Takayanagi H, Kamijo R (2009) Interferon regulatory factor-8 regulates bone metabolism by suppressing osteoclastogenesis. Nat Med 15:1066–1071

    Article  CAS  Google Scholar 

  20. Nishikawa K, Nakashima T, Hayashi M, Fukunaga T, Kato S, Kodama T, Takahashi S, Calame K, Takayanagi H (2010) Blimp1-mediated repression of negative regulators is required for osteoclast differentiation. Proc Natl Acad Sci USA 107:3117–3122

    Article  CAS  Google Scholar 

  21. Nishikawa K, Iwamoto Y, Kobayashi Y, Katsuoka F, Kawaguchi SI, Tsujita T, Nakamura T, Kato S, Yamamoto M, Takayanagi H, Ishii M (2015) DNA methyltransferase 3a regulates osteoclast differentiation by coupling to an S-adenosylmethionine-producing metabolic pathway. Nat Med 21:281

    Article  CAS  Google Scholar 

  22. Nishikawa K, Iwamoto Y, Ishii M (2013) Development of an in vitro culture method for stepwise differentiation of mouse embryonic stem cells and induced pluripotent stem cells into mature osteoclasts. J Bone Miner Metab 32:331

    Article  Google Scholar 

  23. Li X, Zhao X, Fang Y, Jiang X, Duong T, Fan C, Huang CC, Kain SR (1998) Generation of destabilized green fluorescent protein as a transcription reporter. J Biol Chem 273:34970–34975

    Article  CAS  Google Scholar 

  24. Banaszynski LA, Chen LC, Maynard-Smith LA, Ooi AG, Wandless TJ (2006) A rapid, reversible, and tunable method to regulate protein function in living cells using synthetic small molecules. Cell 126:995–1004

    Article  CAS  Google Scholar 

  25. Lagnado CA, Brown CY, Goodall GJ (1994) AUUUA is not sufficient to promote poly(A) shortening and degradation of an mRNA: the functional sequence within AU-rich elements may be UUAUUUA(U/A)(U/A). Mol Cell Biol 14:7984–7995

    Article  CAS  Google Scholar 

  26. Morita S, Kojima T, Kitamura T (2000) Plat-E: an efficient and stable system for transient packaging of retroviruses. Gene Ther 7:1063–1066

    Article  CAS  Google Scholar 

  27. Tanaka S, Takahashi T, Takayanagi H, Miyazaki T, Oda H, Nakamura K, Hirai H, Kurokawa T (1998) Modulation of osteoclast function by adenovirus vector-induced epidermal growth factor receptor. J Bone Mineral Res 13:1714–1720

    Article  CAS  Google Scholar 

  28. Kobayashi Y, Take I, Yamashita T, Mizoguchi T, Ninomiya T, Hattori T, Kurihara S, Ozawa H, Udagawa N, Takahashi N (2005) Prostaglandin E2 receptors EP2 and EP4 are down-regulated during differentiation of mouse osteoclasts from their precursors. J Biol Chem 280:24035–24042

    Article  CAS  Google Scholar 

  29. Matsubara T, Ikeda F, Hata K, Nakanishi M, Okada M, Yasuda H, Nishimura R, Yoneda T (2010) Cbp recruitment of Csk into lipid rafts is critical to c-Src kinase activity and bone resorption in osteoclasts. J Bone Mineral Res 25:1068–1076

    CAS  Google Scholar 

  30. Hsu PD, Lander ES, Zhang F (2014) Development and applications of CRISPR-Cas9 for genome engineering. Cell 157:1262–1278

    Article  CAS  Google Scholar 

  31. Komor AC, Badran AH, Liu DR (2017) CRISPR-based technologies for the manipulation of eukaryotic genomes. Cell 168:20–36

    Article  CAS  Google Scholar 

  32. Mondal P, Krishnamurthy VV, Sharum SR, Haack N, Zhou H, Cheng J, Yang J, Zhang K (2019) Repurposing protein degradation for optogenetic modulation of protein activities. ACS Synth Biol 8:2585–2592

    Article  CAS  Google Scholar 

  33. Bonger KM, Rakhit R, Payumo AY, Chen JK, Wandless TJ (2014) General method for regulating protein stability with light. ACS Chem Biol 9:111–115

    Article  CAS  Google Scholar 

Download references

Acknowledgments

This work was supported by Grants-in-Aid for Scientific Research (B) from the Japan Society for the Promotion of Science (JSPS) (18H02614 to K. N.); and grants from the Takeda Science Foundation (to K.N.); and the Food Science Institute Foundation (Ryoushoku-kenkyukai to K. N.); and Suzuken Memorial Foundation (to K. N.); and Terumo Life Science Foundation (to K. N.); and Fuji Foundation For Protein Research (to K. N.); and The Tojuro Iijima Foundation for Food Science and Technology (to K. N.); and Nakatani Foundation (to K. N.).

Author information

Authors and Affiliations

  1. Laboratory of Cell Biology and Metabolic Biochemistry, Department of Medical Life Systems, Graduate School of Life and Medical Sciences, Doshisha University, Tatara Miyakodani 1-3, Kyotanabe, Kyoto, 610-0394, Japan

    Keizo Nishikawa

  2. Department of Immunology and Cell Biology, Graduate School of Medicine/Frontier Biosciences, Osaka University, Yamada-oka 2-2, Suita, Osaka, 565-0871, Japan

    Keizo Nishikawa & Masaru Ishii

  3. WPI-Immunology Frontier Research Center, Osaka University, Yamada-oka 2-2, Suita, Osaka, 565-0871, Japan

    Keizo Nishikawa & Masaru Ishii

Authors
  1. Keizo Nishikawa
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Masaru Ishii
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Keizo Nishikawa.

Ethics declarations

Conflict of interest

All authors state that they have no conflicts of interest.

Ethical approval

Animal study was approved by the Institutional Animal Care and Use Committee of both Doshisha University and Osaka University.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Nishikawa, K., Ishii, M. Novel method for gain-of-function analyses in primary osteoclasts using a non-viral gene delivery system. J Bone Miner Metab 39, 353–359 (2021). https://doi.org/10.1007/s00774-020-01161-7

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00774-020-01161-7

keywords

Search

Navigation