Advertisement

European Spine Journal

, Volume 27, Issue 8, pp 1757–1766 | Cite as

Combined use of leptin and mechanical stress has osteogenic effects on ossification of the posterior longitudinal ligament

  • Shuai Chen
  • Haifeng Zhu
  • Gangliang Wang
  • Ziang Xie
  • Jiying Wang
  • Jian Chen
Original Article

Abstract

Purpose

To evaluate the effects of leptin/leptin receptor (LepR) combined with mechanical stress on the development of ossification of the posterior longitudinal ligament (OPLL), which is a disease characterized by ectopic bone formation of the posterior longitudinal ligament (PLL) and can lead to radiculopathy and myelopathy.

Methods

Six human samples of the PLL were analyzed for the expression of leptin and LepR by RT-PCR and western blotting. PLL cells were stimulated with leptin and mechanical stress delivered via a Flexcell tension system, and osteogenic differentiation was evaluated by RT-PCR and western blotting analysis of osteogenic marker expression as well as by alkaline phosphatase (ALP) staining and alizarin red S staining. Activation of mitogen-activated protein kinase (MAPK), Janus kinase (JAK) 2-signal transducer, activator of transcription (STAT) 3 and phosphatidylinositol 3-kinase (PI3K)–Akt was evaluated by western blotting.

Results

Samples from the OPLL group had higher LepR mRNA and protein levels and lower leptin levels than those from healthy controls. Exposure to leptin and Flexcell increased the number of ALP-positive cells and calcium nodules in a dose-dependent manner; this effect was accompanied by upregulation of the osteogenic markers osteocalcin, runt-related transcription factor 2 (RUNX2) and osteopontin. Extracellular signal-regulated kinase, P38 MAPK, JAK2, STAT3, PI3K and Akt signaling, was also activated by the combined effects of leptin and mechanical stress.

Conclusions

Leptin and LepR are differentially expressed in OPLL tissues, and the combined use of leptin/LepR and mechanical stress promotes osteogenic differentiation of PLL cells via MAPK, JAK2-STAT3 and PI3K/Akt signaling.

Graphical abstract

These slides can be retrieved under Electronic Supplementary Material.

Keywords

OPLL Leptin Leptin receptor Mechanical stress 

Notes

Funding

This study is supported by the National Natural Science Foundation of China (grant numbers: 81401821, 81601924), Scientific research project of education department of Zhejiang (grant number: Y201330235), Project of Health and Family Planning Commission of Zhejiang Province (grant number: 2016145597), Postdoctor Science Foundation of China (grant number: 2017M612009).

Compliance with ethical standards

Conflict of interest

The authors declare no potential conflict of interest.

Ethical approval

This study was approved by Ethics Committee of Sir Run Run Shaw Hospital.

Supplementary material

586_2018_5663_MOESM1_ESM.pptx (3.4 mb)
Supplementary material 1 (PPTX 3499 kb)

References

  1. 1.
    Tsuyama N (1984) Ossification of the posterior longitudinal ligament of the spine. Clin Orthop Relat Res 184:71–84Google Scholar
  2. 2.
    Maeda S, Ishidou Y, Koga H, Taketomi E, Ikari K, Komiya S, Takeda J, Sakou T, Inoue I (2001) Functional impact of human collagen alpha2(XI) gene polymorphism in pathogenesis of ossification of the posterior longitudinal ligament of the spine. J Bone Miner Res Off J Am Soc Bone Miner Res 16:948–957.  https://doi.org/10.1359/jbmr.2001.16.5.948 CrossRefGoogle Scholar
  3. 3.
    Ohtsuka K, Terayama K, Yanagihara M, Wada K, Kasuga K, Machida T, Matsushima S (1987) A radiological population study on the ossification of the posterior longitudinal ligament in the spine. Arch Orthop Trauma Surg Archiv fur orthopadische und Unfall-Chirurgie 106:89–93CrossRefPubMedGoogle Scholar
  4. 4.
    Terayama K (1989) Genetic studies on ossification of the posterior longitudinal ligament of the spine. Spine 14:1184–1191CrossRefPubMedGoogle Scholar
  5. 5.
    Sakou T, Taketomi E, Matsunaga S, Yamaguchi M, Sonoda S, Yashiki S (1991) Genetic study of ossification of the posterior longitudinal ligament in the cervical spine with human leukocyte antigen haplotype. Spine 16:1249–1252CrossRefPubMedGoogle Scholar
  6. 6.
    Koga H, Sakou T, Taketomi E, Hayashi K, Numasawa T, Harata S, Yone K, Matsunaga S, Otterud B, Inoue I, Leppert M (1998) Genetic mapping of ossification of the posterior longitudinal ligament of the spine. Am J Hum Genet 62:1460–1467.  https://doi.org/10.1086/301868 CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Okawa A, Nakamura I, Goto S, Moriya H, Nakamura Y, Ikegawa S (1998) Mutation in Npps in a mouse model of ossification of the posterior longitudinal ligament of the spine. Nat Genet 19:271–273.  https://doi.org/10.1038/956 CrossRefPubMedGoogle Scholar
  8. 8.
    Kawaguchi Y, Furushima K, Sugimori K, Inoue I, Kimura T (2003) Association between polymorphism of the transforming growth factor-beta1 gene with the radiologic characteristic and ossification of the posterior longitudinal ligament. Spine 28:1424–1426.  https://doi.org/10.1097/01.brs.0000068245.27017.9f PubMedCrossRefGoogle Scholar
  9. 9.
    Furushima K, Shimo-Onoda K, Maeda S, Nobukuni T, Ikari K, Koga H, Komiya S, Nakajima T, Harata S, Inoue I (2002) Large-scale screening for candidate genes of ossification of the posterior longitudinal ligament of the spine. J Bone Miner Res Off J Am Soc Bone Miner Res 17:128–137.  https://doi.org/10.1359/jbmr.2002.17.1.128 CrossRefGoogle Scholar
  10. 10.
    Tanaka T, Ikari K, Furushima K, Okada A, Tanaka H, Furukawa K, Yoshida K, Ikeda T, Ikegawa S, Hunt SC, Takeda J, Toh S, Harata S, Nakajima T, Inoue I (2003) Genomewide linkage and linkage disequilibrium analyses identify COL6A1, on chromosome 21, as the locus for ossification of the posterior longitudinal ligament of the spine. Am J Hum Genet 73:812–822CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Iwasaki K, Furukawa KI, Tanno M, Kusumi T, Ueyama K, Tanaka M, Kudo H, Toh S, Harata S, Motomura S (2004) Uni-axial cyclic stretch induces Cbfa1 expression in spinal ligament cells derived from patients with ossification of the posterior longitudinal ligament. Calcif Tissue Int 74:448–457.  https://doi.org/10.1007/s00223-002-0021-1 CrossRefPubMedGoogle Scholar
  12. 12.
    Okamoto K, Kobashi G, Washio M, Sasaki S, Yokoyama T, Miyake Y, Sakamoto N, Ohta K, Inaba Y, Tanaka H (2004) Dietary habits and risk of ossification of the posterior longitudinal ligaments of the spine (OPLL); findings from a case-control study in Japan. J Bone Miner Metab 22:612–617.  https://doi.org/10.1007/s00774-004-0531-1 CrossRefPubMedGoogle Scholar
  13. 13.
    Kobashi G, Washio M, Okamoto K, Sasaki S, Yokoyama T, Miyake Y, Sakamoto N, Ohta K, Inaba Y, Tanaka H (2004) High body mass index after age 20 and diabetes mellitus are independent risk factors for ossification of the posterior longitudinal ligament of the spine in Japanese subjects: a case-control study in multiple hospitals. Spine 29:1006–1010CrossRefPubMedGoogle Scholar
  14. 14.
    Terayama K, Ohtsuka K, Merlini L, Albisinni U, Gui L (1987) Ossification of the spinal ligament. A radiographic reevaluation in Bologna, Italy. Nihon Seikeigeka Gakkai zasshi 61:1373–1378PubMedGoogle Scholar
  15. 15.
    Chen J, Wang X, Wang C, Yuan W (2011) Rotational stress: role in development of ossification of posterior longitudinal ligament and ligamentum flavum. Med Hypotheses 76:73–76.  https://doi.org/10.1016/j.mehy.2010.08.034 CrossRefPubMedGoogle Scholar
  16. 16.
    Ikeda Y, Nakajima A, Aiba A, Koda M, Okawa A, Takahashi K, Yamazaki M (2011) Association between serum leptin and bone metabolic markers, and the development of heterotopic ossification of the spinal ligament in female patients with ossification of the posterior longitudinal ligament. Eur Spine J Off Publ Eur Spine Soc Eur Spinal Deform Soc Eur Sect Cerv Spine Res Soc 20:1450–1458.  https://doi.org/10.1007/s00586-011-1688-7 CrossRefGoogle Scholar
  17. 17.
    Bartell SM, Rayalam S, Ambati S, Gaddam DR, Hartzell DL, Hamrick M, She JX, Della-Fera MA, Baile CA (2011) Central (ICV) leptin injection increases bone formation, bone mineral density, muscle mass, serum IGF-1, and the expression of osteogenic genes in leptin-deficient ob/ob mice. J Bone Miner Res Off J Am Soc Bone Miner Res 26:1710–1720.  https://doi.org/10.1002/jbmr.406 CrossRefGoogle Scholar
  18. 18.
    Thomas T, Gori F, Khosla S, Jensen MD, Burguera B, Riggs BL (1999) Leptin acts on human marrow stromal cells to enhance differentiation to osteoblasts and to inhibit differentiation to adipocytes. Endocrinology 140:1630–1638.  https://doi.org/10.1210/endo.140.4.6637 CrossRefPubMedGoogle Scholar
  19. 19.
    Turner RT, Kalra SP, Wong CP, Philbrick KA, Lindenmaier LB, Boghossian S, Iwaniec UT (2013) Peripheral leptin regulates bone formation. J Bone Miner Res Off J Am Soc Bone Miner Res 28:22–34.  https://doi.org/10.1002/jbmr.1734 CrossRefGoogle Scholar
  20. 20.
    Philbrick KA, Wong CP, Branscum AJ, Turner RT, Iwaniec UT (2017) Leptin stimulates bone formation in ob/ob mice at doses having minimal impact on energy metabolism. J Endocrinol 232:461–474.  https://doi.org/10.1530/joe-16-0484 CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Hess R, Pino AM, Rios S, Fernandez M, Rodriguez JP (2005) High affinity leptin receptors are present in human mesenchymal stem cells (MSCs) derived from control and osteoporotic donors. J Cell Biochem 94:50–57.  https://doi.org/10.1002/jcb.20330 CrossRefPubMedGoogle Scholar
  22. 22.
    Gordeladze JO, Drevon CA, Syversen U, Reseland JE (2002) Leptin stimulates human osteoblastic cell proliferation, de novo collagen synthesis, and mineralization: impact on differentiation markers, apoptosis, and osteoclastic signaling. J Cell Biochem 85:825–836.  https://doi.org/10.1002/jcb.10156 CrossRefPubMedGoogle Scholar
  23. 23.
    Tenti S, Palmitesta P, Giordano N, Galeazzi M, Fioravanti A (2017) Increased serum leptin and visfatin levels in patients with diffuse idiopathic skeletal hyperostosis: a comparative study. Scand J Rheumatol 46:156–158.  https://doi.org/10.1080/03009742.2016.1188981 CrossRefPubMedGoogle Scholar
  24. 24.
    Considine RV, Sinha MK, Heiman ML, Kriauciunas A, Stephens TW, Nyce MR, Ohannesian JP, Marco CC, McKee LJ, Bauer TL et al (1996) Serum immunoreactive-leptin concentrations in normal-weight and obese humans. N Engl J Med 334:292–295.  https://doi.org/10.1056/nejm199602013340503 CrossRefPubMedGoogle Scholar
  25. 25.
    Kapur S, Amoui M, Kesavan C, Wang X, Mohan S, Baylink DJ, Lau KH (2010) Leptin receptor (Lepr) is a negative modulator of bone mechanosensitivity and genetic variations in Lepr may contribute to the differential osteogenic response to mechanical stimulation in the C57BL/6J and C3H/HeJ pair of mouse strains. J Biol Chem 285:37607–37618.  https://doi.org/10.1074/jbc.M110.169714 CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Hughes-Fulford M (2004) Signal transduction and mechanical stress. Sci STKE Signal Transduct Knowl Environ.  https://doi.org/10.1126/stke.2492004re12 CrossRefGoogle Scholar
  27. 27.
    Boutahar N, Guignandon A, Vico L, Lafage-Proust MH (2004) Mechanical strain on osteoblasts activates autophosphorylation of focal adhesion kinase and proline-rich tyrosine kinase 2 tyrosine sites involved in ERK activation. J Biol Chem 279:30588–30599.  https://doi.org/10.1074/jbc.M313244200 CrossRefPubMedGoogle Scholar
  28. 28.
    Hatton JP, Pooran M, Li CF, Luzzio C, Hughes-Fulford M (2003) A short pulse of mechanical force induces gene expression and growth in MC3T3-E1 osteoblasts via an ERK 1/2 pathway. J Bone Miner Res Off J Am Soc Bone Miner Res 18:58–66.  https://doi.org/10.1359/jbmr.2003.18.1.58 CrossRefGoogle Scholar
  29. 29.
    Matsuda N, Morita N, Matsuda K, Watanabe M (1998) Proliferation and differentiation of human osteoblastic cells associated with differential activation of MAP kinases in response to epidermal growth factor, hypoxia, and mechanical stress in vitro. Biochem Biophys Res Commun 249:350–354.  https://doi.org/10.1006/bbrc.1998.9151 CrossRefPubMedGoogle Scholar
  30. 30.
    Alshaker H, Sacco K, Alfraidi A, Muhammad A, Winkler M, Pchejetski D (2015) Leptin signalling, obesity and prostate cancer: molecular and clinical perspective on the old dilemma. Oncotarget 6:35556–35563.  https://doi.org/10.18632/oncotarget.5574 CrossRefPubMedPubMedCentralGoogle Scholar
  31. 31.
    Kwon O, Kim KW, Kim MS (2016) Leptin signalling pathways in hypothalamic neurons. Cell Mol Life Sci CMLS 73:1457–1477.  https://doi.org/10.1007/s00018-016-2133-1 CrossRefPubMedGoogle Scholar
  32. 32.
    Ren D, Li M, Duan C, Rui L (2005) Identification of SH2-B as a key regulator of leptin sensitivity, energy balance, and body weight in mice. Cell Metab 2:95–104.  https://doi.org/10.1016/j.cmet.2005.07.004 CrossRefPubMedGoogle Scholar
  33. 33.
    Banks AS, Davis SM, Bates SH, Myers MG Jr (2000) Activation of downstream signals by the long form of the leptin receptor. J Biol Chem 275:14563–14572CrossRefPubMedGoogle Scholar
  34. 34.
    Bjorbaek C, Buchholz RM, Davis SM, Bates SH, Pierroz DD, Gu H, Neel BG, Myers MG Jr, Flier JS (2001) Divergent roles of SHP-2 in ERK activation by leptin receptors. J Biol Chem 276:4747–4755.  https://doi.org/10.1074/jbc.M007439200 CrossRefPubMedGoogle Scholar
  35. 35.
    Ohishi H, Furukawa K, Iwasaki K, Ueyama K, Okada A, Motomura S, Harata S, Toh S (2003) Role of prostaglandin I2 in the gene expression induced by mechanical stress in spinal ligament cells derived from patients with ossification of the posterior longitudinal ligament. J Pharm Exp Ther 305:818–824.  https://doi.org/10.1124/jpet.102.047142 CrossRefGoogle Scholar
  36. 36.
    Tahara M, Aiba A, Yamazaki M, Ikeda Y, Goto S, Moriya H, Okawa A (2005) The extent of ossification of posterior longitudinal ligament of the spine associated with nucleotide pyrophosphatase gene and leptin receptor gene polymorphisms. Spine 30:877–880 (discussion 881) CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  • Shuai Chen
    • 1
    • 2
  • Haifeng Zhu
    • 1
    • 2
  • Gangliang Wang
    • 1
    • 2
  • Ziang Xie
    • 1
    • 2
  • Jiying Wang
    • 2
  • Jian Chen
    • 1
    • 2
  1. 1.Department of Orthopaedic Surgery, Sir Run Run Shaw Hospital, School of MedicineZhejiang UniversityHangzhouChina
  2. 2.Key Laboratory of Biotherapy of Zhejiang ProvinceHangzhouChina

Personalised recommendations