Skip to main content

Neutralizing antibody against osteopontin attenuates non-alcoholic steatohepatitis in mice


Previously, we reported that an extracellular matrix protein, osteopontin (OPN), is involved in various autoimmune diseases using a neutralizing polyclonal antibody against OPN generated in rabbits. However, the antibody cannot be used for long-term mouse models of chronic inflammatory disease because of the induction of antibodies against anti-OPN rabbit IgG. In this study, we generated a new antibody, anti-mouse OPN mouse IgG (35B6). 35B6 inhibited the cell adhesion of mouse and human OPN to Chinese Hamster Ovary (CHO) cells or CHO cells expressing α4 or α9 integrin. It was reported that OPN is highly expressed and has an important role in a chronic liver disease, non-alcoholic steatohepatitis (NASH). 35B6 injection twice a week for 8 weeks attenuated liver inflammation and fibrosis in a NASH mouse model, suggesting 35B6 is beneficial for the treatment of NASH. 35B6 was preferable to the rabbit anti-OPN antibody for investigating the in vivo function of OPN in mouse models of long-term disease.

This is a preview of subscription content, access via your institution.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7



0.1% methionine on a high fat diet background


type I collagen α1 chain


Chinese Hamster Ovary


extracellular matrix




hepatic stellate cells




non-alcoholic fatty liver disease


non-alcoholic steatohepatitis in mice




smooth muscle actin


  1. Barry ST, Ludbrook SB, Murrison E, Horgan CM (2000a) Analysis of the alpha4beta1 integrin-osteopontin interaction. Exp Cell Res 258:342–351

    CAS  Article  Google Scholar 

  2. Barry ST, Ludbrook SB, Murrison E, Horgan CM (2000b) A regulated interaction between alpha5beta1 integrin and osteopontin. Biochem Biophys Res Commun 267:764–769

    CAS  Article  Google Scholar 

  3. Bayless KJ, Davis GE (2001) Identification of dual alpha 4beta1 integrin binding sites within a 38 amino acid domain in the N-terminal thrombin fragment of human osteopontin. J Biol Chem 276:13483–13489

    CAS  Article  Google Scholar 

  4. Blom T, Franzen A, Heinegard D, Holmdahl R (2003) Comment on "the influence of the proinflammatory cytokine, osteopontin, on autoimmune demyelinating disease". Science 299:1845 author reply 1845

    CAS  Article  Google Scholar 

  5. Brunt EM, Janney CG, Di Bisceglie AM, Neuschwander-Tetri BA, Bacon BR (1999) Nonalcoholic steatohepatitis: a proposal for grading and staging the histological lesions. Am J Gastroenterol 94:2467–2474

    CAS  Article  Google Scholar 

  6. Casini A, Ceni E, Salzano R, Biondi P, Parola M, Galli A, Foschi M, Caligiuri A, Pinzani M, Surrenti C (1997) Neutrophil-derived superoxide anion induces lipid peroxidation and stimulates collagen synthesis in human hepatic stellate cells: role of nitric oxide. Hepatology 25:361–367

    CAS  Article  Google Scholar 

  7. Chabas D, Baranzini SE, Mitchell D, Bernard CC, Rittling SR, Denhardt DT, Sobel RA, Lock C, Karpuj M, Pedotti R, Heller R, Oksenberg JR, Steinman L (2001) The influence of the proinflammatory cytokine, osteopontin, on autoimmune demyelinating disease. Science 294:1731–1735

    CAS  Article  Google Scholar 

  8. Chen W, Zhang J, Fan HN, Zhu JS (2018) Function and therapeutic advances of chemokine and its receptor in nonalcoholic fatty liver disease. Ther Adv Gastroenterol 11:1756284818815184

    Article  Google Scholar 

  9. Coombes JD, Choi SS, Swiderska-Syn M, Manka P, Reid DT, Palma E, Briones-Orta MA, Xie G, Younis R, Kitamura N, Della Peruta M, Bitencourt S, Dolle L, Oo YH, Mi Z, Kuo PC, Williams R, Chokshi S, Canbay A, Claridge LC, Eksteen B, Diehl AM, Syn WK (2016) Osteopontin is a proximal effector of leptin-mediated non-alcoholic steatohepatitis (NASH) fibrosis. Biochim Biophys Acta 1862:135–144

    CAS  Article  Google Scholar 

  10. Denhardt DT, Noda M, O'Regan AW, Pavlin D, Berman JS (2001) Osteopontin as a means to cope with environmental insults: regulation of inflammation, tissue remodeling, and cell survival. J Clin Invest 107:1055–1061

    CAS  Article  Google Scholar 

  11. Diao H, Kon S, Iwabuchi K, Kimura C, Morimoto J, Ito D, Segawa T, Maeda M, Hamuro J, Nakayama T, Taniguchi M, Yagita H, Van Kaer L, Onoe K, Denhardt D, Rittling S, Uede T (2004) Osteopontin as a mediator of NKT cell function in T cell-mediated liver diseases. Immunity 21:539–550

    CAS  Article  Google Scholar 

  12. Fujii M, Shibazaki Y, Wakamatsu K, Honda Y, Kawauchi Y, Suzuki K, Arumugam S, Watanabe K, Ichida T, Asakura H, Yoneyama H (2013) A murine model for non-alcoholic steatohepatitis showing evidence of association between diabetes and hepatocellular carcinoma. Med Mol Morphol 46:141–152

    CAS  Article  Google Scholar 

  13. Helluin O, Chan C, Vilaire G, Mousa S, DeGrado WF, Bennett JS (2000) The activation state of alphavbeta 3 regulates platelet and lymphocyte adhesion to intact and thrombin-cleaved osteopontin. J Biol Chem 275:18337–18343

    CAS  Article  Google Scholar 

  14. Ito K, Kon S, Nakayama Y, Kurotaki D, Saito Y, Kanayama M, Kimura C, Diao H, Morimoto J, Matsui Y, Uede T (2009) The differential amino acid requirement within osteopontin in alpha4 and alpha9 integrin-mediated cell binding and migration. Matrix Biol 28:11–19

    CAS  Article  Google Scholar 

  15. Kahles F, Findeisen HM, Bruemmer D (2014) Osteopontin: a novel regulator at the cross roads of inflammation, obesity and diabetes. Molecular metabolism 3:384–393

    CAS  Article  Google Scholar 

  16. Kanayama M, Kurotaki D, Morimoto J, Asano T, Matsui Y, Nakayama Y, Saito Y, Ito K, Kimura C, Iwasaki N, Suzuki K, Harada T, Li HM, Uehara J, Miyazaki T, Minami A, Kon S, UedeT (2009) Alpha9 integrin and its ligands constitute critical joint microenvironments for development of autoimmune arthritis. J Immunol 182:8015–8025

    CAS  Article  Google Scholar 

  17. Kon S, Ikesue M, Kimura C, Aoki M, Nakayama Y, Saito Y, Kurotaki D, Diao H, Matsui Y, Segawa T, Maeda M, Kojima T, Uede T (2008) Syndecan-4 protects against osteopontin-mediated acute hepatic injury by masking functional domains of osteopontin. J Exp Med 205:25–33

    CAS  Article  Google Scholar 

  18. Kon S, Yokosaki Y, Maeda M, Segawa T, Horikoshi Y, Tsukagoshi H, Rashid MM, Morimoto J, Inobe M, Shijubo N, Chambers AF, Uede T (2002) Mapping of functional epitopes of osteopontin by monoclonal antibodies raised against defined internal sequences. J Cell Biochem 84:420–432

    Article  Google Scholar 

  19. Konno S, Hoshi T, Taira T, Plunkett B, Huang SK (2005) Endotoxin contamination contributes to the in vitro cytokine-inducing activity of osteopontin preparations. J Interferon Cytokine Res 25:277–282

    CAS  Article  Google Scholar 

  20. Kouro H, Kon S, Matsumoto N, Miyashita T, Kakuchi A, Ashitomi D, Saitoh K, Nakatsuru T, Togi S, Muromoto R, Matsuda T (2014) The novel alpha4B murine alpha4 integrin protein splicing variant inhibits alpha4 protein-dependent cell adhesion. J Biol Chem 289:16389–16398

    CAS  Article  Google Scholar 

  21. Li D, He L, Guo H, Chen H, Shan H (2015) Targeting activated hepatic stellate cells (aHSCs) for liver fibrosis imaging. EJNMMI Res 5:71

    Article  Google Scholar 

  22. Li H, Toth E, Cherrington NJ (2018) Asking the right questions with animal models: methionine- and choline-deficient model in predicting adverse drug reactions in human NASH. Toxicol Sci 161:23–33

    CAS  Article  Google Scholar 

  23. Matsumoto M, Hada N, Sakamaki Y, Uno A, Shiga T, Tanaka C, Ito T, Katsume A, Sudoh M (2013) An improved mouse model that rapidly develops fibrosis in non-alcoholic steatohepatitis. Int J Exp Pathol 94:93–103

    CAS  Article  Google Scholar 

  24. Mazzali M, Kipari T, Ophascharoensuk V, Wesson JA, Johnson R, Hughes J (2002) Osteopontin--a molecule for all seasons. Qjm 95:3–13

    CAS  Article  Google Scholar 

  25. Murugaiyan G, Mittal A, Weiner HL (2008) Increased osteopontin expression in dendritic cells amplifies IL-17 production by CD4+ T cells in experimental autoimmune encephalomyelitis and in multiple sclerosis. J Immunol 181:7480–7488

    CAS  Article  Google Scholar 

  26. Papatheodoridi AM, Chrysavgis L, Koutsilieris M, Chatzigeorgiou A (2019) The role of senescence in the development of non-alcoholic fatty liver disease and progression to non-alcoholic steatohepatitis. Hepatology (Jun 23).

  27. Rangaswami H, Bulbule A, Kundu GC (2006) Osteopontin: role in cell signaling and cancer progression. Trends Cell Biol 16:79–87

    CAS  Article  Google Scholar 

  28. Sahai A, Malladi P, Melin-Aldana H, Green RM, Whitington PF (2004) Upregulation of osteopontin expression is involved in the development of nonalcoholic steatohepatitis in a dietary murine model. Am J Physiol Gastrointest Liver Physiol 287:G264–G273

    CAS  Article  Google Scholar 

  29. Shinohara ML, Kim JH, Garcia VA, Cantor H (2008) Engagement of the type I interferon receptor on dendritic cells inhibits T helper 17 cell development: role of intracellular osteopontin. Immunity 29:68–78

    CAS  Article  Google Scholar 

  30. Syn WK, Choi SS, Liaskou E, Karaca GF, Agboola KM, Oo YH, Mi Z, Pereira TA, Zdanowicz M, Malladi P, Chen Y, Moylan C, Jung Y, Bhattacharya SD, Teaberry V, Omenetti A, Abdelmalek MF, Guy CD, Adams DH, Kuo PC, Michelotti GA, Whitington PF, Diehl AM (2011) Osteopontin is induced by hedgehog pathway activation and promotes fibrosis progression in nonalcoholic steatohepatitis. Hepatology 53:106–115

    CAS  Article  Google Scholar 

  31. Syn WK, Oo YH, Pereira TA, Karaca GF, Jung Y, Omenetti A, Witek RP, Choi SS, Guy CD, Fearing CM, Teaberry V, Pereira FE, Adams DH, Diehl AM (2010) Accumulation of natural killer T cells in progressive nonalcoholic fatty liver disease. Hepatology 51:1998–2007

    CAS  Article  Google Scholar 

  32. Takahashi A, Kurokawa M, Konno S, Ito K, Kon S, Ashino S, Nishimura T, Uede T, Hizawa N, Huang SK, Nishimura M (2009) Osteopontin is involved in migration of eosinophils in asthma. Clin Exp Allergy 39:1152–1159

    CAS  Article  Google Scholar 

  33. Uede T (2011) Osteopontin, intrinsic tissue regulator of intractable inflammatory diseases. Pathol Int 61:265–280

    CAS  Article  Google Scholar 

  34. Uede T, Katagiri Y, Iizuka J, Murakami M (1997) Osteopontin, a coordinator of host defense system: a cytokine or an extracellular adhesive protein? Microbiol Immunol 41:641–648

    CAS  Article  Google Scholar 

  35. Weber GF (2001) The metastasis gene osteopontin: a candidate target for cancer therapy. Biochim Biophys Acta 1552:61–85

    CAS  Article  Google Scholar 

  36. Xanthou G, Alissafi T, Semitekolou M, Simoes DC, Economidou E, Gaga M, Lambrecht BN, Lloyd CM, Panoutsakopoulou V (2007) Osteopontin has a crucial role in allergic airway disease through regulation of dendritic cell subsets. Nat Med 13:570–578

    CAS  Article  Google Scholar 

  37. Yamamoto N, Sakai F, Kon S, Morimoto J, Kimura C, Yamazaki H, Okazaki I, Seki N, Fujii T, Uede T (2003) Essential role of the cryptic epitope SLAYGLR within osteopontin in a murine model of rheumatoid arthritis. J Clin Invest 112:181–188

    CAS  Article  Google Scholar 

  38. Yamamoto N, Nakashima T, Torikai M, Naruse T, Morimoto J, Kon S, Sakai F, Uede T (2007) Successful treatment of collagen-induced arthritis in non-human primates by chimeric anti-osteopontin antibody. Int Immunopharmacol 7:1460–1470

    CAS  Article  Google Scholar 

  39. Yokosaki Y, Matsuura N, Sasaki T, Murakami I, Schneider H, Higashiyama S, Saitoh Y, Yamakido M, Taooka Y, Sheppard D (1999) The integrin alpha(9)beta(1) binds to a novel recognition sequence (SVVYGLR) in the thrombin-cleaved amino-terminal fragment of osteopontin. J Biol Chem 274:36328–36334

    CAS  Article  Google Scholar 

Download references


We would like to thank to Junko Miyazaki (Fukuyama University), Kazutaka Matsunami (Immuno-Biological Laboratories Co., Ltd.) and Masaya Takeuchi (Sapporo General Pathology Laboratory Co., Ltd.) for excellent support in the in vivo studies. This study was supported by research grants from JSPS KAKENHI (Grant Numbers 16 K08221 and 19 K07492), the Japan Rheumatism Foundation, the SUHARA Memorial Foundation, the FUGAKU TRUST for Medical Research, and academic support from Pfizer, Chugai, Astellas and Eli Lilly to SK. We thank Edanz Group ( for editing a draft of this manuscript.

Author information




TU and SK designed the study. MH, CK, and SK performed the experiments. SK wrote the manuscript. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Shigeyuki Kon.

Ethics declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Additional information

Publisher’s note

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

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Honda, M., Kimura, C., Uede, T. et al. Neutralizing antibody against osteopontin attenuates non-alcoholic steatohepatitis in mice. J. Cell Commun. Signal. 14, 223–232 (2020).

Download citation


  • Osteopontin
  • Antibody
  • Non-alcoholic steatohepatitis in mice
  • Inflammation
  • Fibrosis