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NELL-1-dependent mineralisation of Saos-2 human osteosarcoma cells is mediated via c-Jun N-terminal kinase pathway activation

International Orthopaedics Aims and scope Submit manuscript

Abstract

Purpose

NELL-1 is a novel osteoinductive growth factor that has shown promising results for the regeneration of bone. Moreover, NELL-1 has been used successfully in bone regeneration in the axial, appendicular and calvarial skeleton of both small and large animal models. Despite increasing evidence of NELL-1 efficacy and future usefulness as an alternative to traditional bone graft substitutes, much has yet to be understood regarding the mechanisms of action of this novel protein. The activation of the mitogen-activated protein kinase (MAPK) pathway has been well studied in the setting of growth factor-mediated changes in osteogenic differentiation.

Methods

In this study, we provide evidence of the involvement of MAPK signalling pathways in NELL-1-induced terminal osteogenic differentiation of Saos-2 human osteosarcoma cells. Activation of extracellular signal-regulated kinase (ERK1/2), P38 and c-Jun N-terminal kinase (JNK) pathways were screened with MAPK signalling protein array after recombinant human (rh)NELL-1 treatment. Next, the mineralisation and intracellular phosphate levels after rhNELL-1 stimulation were assessed in the presence or absence of specific MAPK inhibitors.

Results

Results showed that rhNELL-1 predominantly increased JNK pathway activation. Moreover, the specific JNK inhibitor SP600125 blocked rhNELL-1-induced mineralisation and intracellular phosphate accumulation, whereas ERK1/2 and P38 inhibitors showed no effect.

Conclusions

Thus, activation of the JNK pathway is necessary to mediate terminal osteogenic differentiation of Saos-2 osteosarcoma cells by rhNELL-1. Future studies will extend these in vitro mechanisms to the in vivo effects of NELL-1 in dealing with orthopaedic defects caused by skeletal malignancies or other aetiologies.

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References

  1. Ting K, Vastardis H, Mulliken JB et al (1999) Human NELL-1 expressed in unilateral coronal synostosis. J Bone Miner Res 14:80–89

    Article  PubMed  CAS  Google Scholar 

  2. Zhang X, Carpenter D, Bokui N et al (2003) Overexpression of Nell-1, a craniosynostosis-associated gene, induces apoptosis in osteoblasts during craniofacial development. J Bone Miner Res 18:2126–2134

    Article  PubMed  CAS  Google Scholar 

  3. Zhang X, Kuroda S, Carpenter D et al (2002) Craniosynostosis in transgenic mice overexpressing Nell-1. J Clin Invest 110:861–870

    PubMed  CAS  Google Scholar 

  4. Aghaloo T, Cowan CM, Chou YF et al (2006) Nell-1-induced bone regeneration in calvarial defects. Am J Pathol 169:903–915

    Article  PubMed  CAS  Google Scholar 

  5. Cowan CM, Cheng S, Ting K et al (2006) Nell-1 induced bone formation within the distracted intermaxillary suture. Bone 38:48–58

    Article  PubMed  CAS  Google Scholar 

  6. Li W, Lee M, Whang J et al (2010) Delivery of lyophilized Nell-1 in a rat spinal fusion model. Tissue Eng Part A 16:2861–2870

    Article  PubMed  CAS  Google Scholar 

  7. Li W, Zara JN, Siu RK et al (2011) Nell-1 enhances bone regeneration in a rat critical-sized femoral segmental defect model. Plast Reconstr Surg 127:580–587

    Article  PubMed  CAS  Google Scholar 

  8. Lu SS, Zhang X, Soo C et al (2007) The osteoinductive properties of Nell-1 in a rat spinal fusion model. Spine J 7:50–60

    Article  PubMed  Google Scholar 

  9. Siu RK, Lu SS, Li W et al (2011) Nell-1 protein promotes bone formation in a sheep spinal fusion model. Tissue Eng Part A 17:1123–1135

    Article  PubMed  CAS  Google Scholar 

  10. Zhang X, Qiu X, Lu R, Zhou H, Xue P, Liu X et al (2010) Phenothiazine-based oligomers as novel fluorescence probes for detecting vapor-phase nitro compounds. Talanta 82:1943–1949

    Article  PubMed  CAS  Google Scholar 

  11. Zou X, Shen J, Chen F et al (2011) NELL-1 binds to APR3 affecting human osteoblast proliferation and differentiation. FEBS Lett 585:2410–2418

    Article  PubMed  CAS  Google Scholar 

  12. Sowa H, Kaji H, Yamaguchi T et al (2002) Activations of ERK1/2 and JNK by transforming growth factor beta negatively regulate Smad3-induced alkaline phosphatase activity and mineralization in mouse osteoblastic cells. J Biol Chem 277:36024–36031

    Article  PubMed  CAS  Google Scholar 

  13. Suzuki A, Ghayor C, Guicheux J et al (2006) Enhanced expression of the inorganic phosphate transporter Pit-1 is involved in BMP-2-induced matrix mineralization in osteoblast-like cells. J Bone Miner Res 21:674–683

    Article  PubMed  CAS  Google Scholar 

  14. Shalhoub V, Ward SC, Sun B et al (2011) Fibroblast growth factor 23 (FGF23) and alpha-Klotho stimulate osteoblastic MC3T3.E1 cell proliferation and inhibit mineralization. Calcif Tissue Int 89:140–150

    Article  PubMed  CAS  Google Scholar 

  15. Suzuki A, Palmer G, Bonjour J et al (2000) Stimulation of sodium-dependent phosphate transport and signaling mechanisms induced by basic fibroblast growth factor in MC3T3-E1 osteoblast-like cells. J Bone Miner Res 15:95–102

    Article  PubMed  CAS  Google Scholar 

  16. De Luca A, Gallo M, Aldinucci D et al (2011) Role of the EGFR ligand/receptor system in the secretion of angiogenic factors in mesenchymal stem cells. J Cell Physiol 226:2131–2138

    Article  PubMed  Google Scholar 

  17. Cowan CM, Jiang X, Hsu T et al (2007) Synergistic effects of Nell-1 and BMP-2 on the osteogenic differentiation of myoblasts. J Bone Miner Res 22:918–930

    Article  PubMed  CAS  Google Scholar 

  18. Zhang X, Ting K, Bessette CM et al (2011) Nell-1, a key functional mediator of Runx2, partially rescues calvarial defects in Runx2(+/-) mice. J Bone Miner Res 26:777–791

    Article  PubMed  CAS  Google Scholar 

  19. Jin Z, Mori Y, Yang J et al (2007) Hypermethylation of the nel-like 1 gene is a common and early event and is associated with poor prognosis in early-stage esophageal adenocarcinoma. Oncogene 26:6332–6340

    Article  PubMed  CAS  Google Scholar 

  20. Mori Y, Cai K, Cheng Y et al (2006) A genome-wide search identifies epigenetic silencing of somatostatin, tachykinin-1, and 5 other genes in colon cancer. Gastroenterology 131:797–808

    Article  PubMed  CAS  Google Scholar 

  21. Zhang X, Péault B, Chen W et al (2011) The Nell-1 growth factor stimulates bone formation by purified human perivascular cells. Tissue Eng Part A 17:2497–2509

    Article  PubMed  CAS  Google Scholar 

  22. Chen W, Zhang X, Siu R et al (2011) Nfatc2 is a primary response gene of Nell-1 regulating chondrogenesis in ATDC5 cells. J Bone Miner Res 26:1230–1241

    Article  PubMed  CAS  Google Scholar 

  23. Chen F, Zhang A, Sun S et al (2011) NELL-1, an osteoinductive factor, is a direct transcriptional target of Osterix. PLoS One 6:e24638

    Article  PubMed  CAS  Google Scholar 

  24. Maeda K, Matsuhashi S, Tabuchi K et al (2001) Brain specific human genes, NELL1 and NELL2, are predominantly expressed in neuroblastoma and other embryonal neuroepithelial tumors. Neurol Med Chir (Tokyo) 41:582–588, discussion 589

    Article  CAS  Google Scholar 

  25. Gallea S, Lallemand F, Atfi A et al (2001) Activation of mitogen-activated protein kinase cascades is involved in regulation of bone morphogenetic protein-2-induced osteoblast differentiation in pluripotent C2C12 cells. Bone 28:491–498

    Article  PubMed  CAS  Google Scholar 

  26. Schmitt JM, Hwang K, Winn SR et al (1999) Bone morphogenetic proteins: an update on basic biology and clinical relevance. J Orthop Res 17:269–278

    Article  PubMed  CAS  Google Scholar 

  27. Zhang H, Schulz TJ, Espinoza DO et al (2010) Cross talk between insulin and bone morphogenetic protein signaling systems in brown adipogenesis. Mol Cell Biol 30:4224–4233

    Article  PubMed  CAS  Google Scholar 

  28. Slattery ML, Lundgreen A, Herrick JS et al (2011) Genetic variation in bone morphogenetic protein and colon and rectal cancer. Int J Cancer 130:653–664

    Article  PubMed  Google Scholar 

  29. Slattery ML, Lundgreen A, Herrick JS et al (2011) Genetic variation in the transforming growth factor-beta signaling pathway and survival after diagnosis with colon and rectal cancer. Cancer 117:4175–4183

    Article  PubMed  CAS  Google Scholar 

  30. Hausser HJ, Brenner RE (2005) Phenotypic instability of Saos-2 cells in long-term culture. Biochem Biophys Res Commun 333:216–222

    Article  PubMed  CAS  Google Scholar 

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Acknowledgments

This work was supported by the NIH/NIDCR (grants R21 DE0177711 and RO1 DE01607), UC Discovery Grant 07-10677, T32 training fellowship to A.W.J. (5T32DE007296-14), Eli & Edythe Broad Center of Regenerative Medicine and Stem Cell Research at UCLA Innovation Award, and the Thomas R. Bales Endowed Chair.

Conflict of interest

The authors declare that they have no conflict of interest.

Disclosure

Drs. X.Z, K.T, and C.S. are inventors of NELL-1 related patents. Drs. X.Z, K.T, and C.S are founders of Bone Biologics Inc. which sublicenses NELL-1 patents from the UC Regents.

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Correspondence to Kang Ting or Xinli Zhang.

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Chen, F., Walder, B., James, A.W. et al. NELL-1-dependent mineralisation of Saos-2 human osteosarcoma cells is mediated via c-Jun N-terminal kinase pathway activation. International Orthopaedics (SICOT) 36, 2181–2187 (2012). https://doi.org/10.1007/s00264-012-1590-x

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  • DOI: https://doi.org/10.1007/s00264-012-1590-x

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