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Upregulated miR-224-5p suppresses osteoblast differentiation by increasing the expression of Pai-1 in the lumbar spine of a rat model of congenital kyphoscoliosis

Abstract

Congenital scoliosis is defined by the presence of structural anatomical malformations that arise from failures of vertebral formation or segmentation before and after birth. The understanding of genetic background and key genes for congenital scoliosis is still poor. We herein report that the excess expression of plasminogen activator inhibitor-1 (Pai-1) induced by the upregulation of miR-224-5p is involved in the pathogenesis of congenital kyphoscoliosis through impaired osteoblast differentiation. We first investigated the variety and progression of abnormalities of the lumbar spines in Ishibashi (IS) rats, a rat model of congenital kyphoscoliosis. The rats had already shown fusion and division of the primary ossification center at postnatal day 4. Over time, the rats showed various abnormalities of the lumbar spine, including the fusion of the annular epiphyseal nucleus. At postnatal day 42, spinal curvature was clearly observed due to the fusion of the vertebral bodies. Using a microRNA array, we found that the expression of miR-224-5p was increased in the lumbar spine of the rats at postnatal day 4. The expression of Pai-1, which is involved in osteoblast differentiation regulated by miR-224-5p, was also increased, while the levels of type I collagen, a marker of osteoblast differentiation, were decreased in the lumbar spine. These results indicate that the aberrant expression of miRNA-224-5p and its target genes is involved in the impaired osteoblast differentiation and may provide a partial molecular explanation for the pathogenesis of congenital scoliosis.

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References

  1. 1.

    Goldstein LA, Waugh TR (1973) Classification and terminology of scoliosis. Clin Orthop Relat Res 93:10–22

    Article  Google Scholar 

  2. 2.

    Kose N, Campbell RM (2004) Congenital scoliosis. Med Sci Monit 10:RA104–RA110

    Google Scholar 

  3. 3.

    Chang DG, Suk SI, Kim JH, Ha KY, Na KH, Lee JH (2015) Surgical outcomes by age at the time of surgery in the treatment of congenital scoliosis in children under age 10 years. Spine J 15:1783–1795

    Article  Google Scholar 

  4. 4.

    Morriss GM (1972) Morphogenesis of the malformations induced in rat embryos by maternal hypervitaminosis A. J Anat 113:241–250

    CAS  Google Scholar 

  5. 5.

    Zhao JJ, Sun DG, Wang J, Liu SR, Zhang CY, Zhu MX, Ma X (2008) Retinoic acid downregulates microRNAs to induce abnormal development of spinal cord in spina bifida rat model. Childs Nerv Syst 24:485–492

    Article  Google Scholar 

  6. 6.

    Ishibashi M (1979) Congenital vertebral malformation (Ishibashi rats). In: Kawamata J, Matsushita H (eds) Handbook on animal models of human diseases. Ishiyaku Shuppan, Tokyo, pp 430–434

    Google Scholar 

  7. 7.

    Yamada J, Nikaido H, Moritake S, Maekawa R (1982) Genetic analyses of the vertebral anomalies of the IS strain of rat and the development of a BN congenic line with the anomalies. Lab Anim 16:40–47

    CAS  Article  Google Scholar 

  8. 8.

    Seki T, Shimokawa N, Iizuka H, Takagishi K, Koibuchi N (2008) Abnormalities of vertebral formation and Hox expression in congenital kyphoscoliotic rat. Mol Cell Biochem 312:193–199

    CAS  Article  Google Scholar 

  9. 9.

    Tsunoda D, Iizuka H, Ichinose T, Iizuka Y, Mieda T, Shimokawa N, Takagishi K, Koibuchi N (2016) The Trk family of neurotrophin receptors is downregulated in the lumbar spines of rats with congenital kyphoscoliosis. Mol Cell Biochem 412:11–18

    CAS  Article  Google Scholar 

  10. 10.

    Sonoda H, Iizuka H, Ishiwata S, Tsunoda D, Abe M, Takagishi K, Chikuda H, Koibuchi N, Shimokawa N (2019) The retinol-retinoic acid metabolic pathway is impaired in the lumbar spine of a rat model of congenital kyphoscoliosis. J Cell Biochem 120:15007–15017

    CAS  Article  Google Scholar 

  11. 11.

    Garmire LX, Subramaniam S (2012) Evaluation of normalization methods in mammalian microRNA-Seq data. RNA 18:1279–1288

    CAS  Article  Google Scholar 

  12. 12.

    Moore BT, Xiao P (2013) MiRNAs in bone diseases MicroRNA 2:20–31

    CAS  Google Scholar 

  13. 13.

    Yang N, Wang G, Hu C, Shi Y, Liao L, Shi S, Cai Y, Cheng S, Wang X, Liu Y, Tang L, Ding Y, Jin Y (2013) Tumor necrosis factor α suppresses the mesenchymal stem cell osteogenesis promoter miR-21 in estrogen deficiency-induced osteoporosis. J Bone Miner Res 28:559–573

    CAS  Article  Google Scholar 

  14. 14.

    Sugatani T, Vacher J, Hruska KA (2012) A microRNA expression signature of osteoclastogenesis. Blood 117:3648–3657

    Article  Google Scholar 

  15. 15.

    Li H, Xie H, Liu W, Hu R, Huang B, Tan YF, Xu K, Sheng ZF, Zhou HD, Wu XP, Luo XH (2009) A novel microRNA targeting HDAC5 regulates osteoblast differentiation in mice and contributes to primary osteoporosis in humans. J Clin Invest 119:3666–3677

    CAS  Article  Google Scholar 

  16. 16.

    Zhang Y, Xie RL, Croce CM, Stein JL, Lian JB, van Wijnen AJ, Stein GS (2011) A program of microRNAs controls osteogenic lineage progression by targeting transcription factor Runx2. Proc Natl Acad Sci USA 108:9863–9868

    CAS  Article  Google Scholar 

  17. 17.

    Kim KM, Park SJ, Jung SH, Kim EJ, Jogeswar G, Ajita J, Rhee Y, Kim CH, Lim SK (2012) miR-182 is a negative regulator of osteoblast proliferation, differentiation, and skeletogenesis through targeting FoxO1. J Bone Miner Res 27:1669–1679

    Article  Google Scholar 

  18. 18.

    Luo Y, Cao X, Chen J, Gu J, Zhao J, Sun J (2018) MicroRNA-224 suppresses osteoblast differentiation by inhibiting SMAD4. J Cell Physiol 233:6929–6937

    CAS  Article  Google Scholar 

  19. 19.

    Karner CM, Lee SY, Long F (2017) Bmp induces osteoblast differentiation through both smad4 and mTORC1 signaling. Mol Cell Biol. https://doi.org/10.1128/MCB.00253-16

    Article  Google Scholar 

  20. 20.

    Xu HM, Sui FH, Sun MH, Guo GL (2019) Downregulated microRNA-224 aggravates vulnerable atherosclerotic plaques and vascular remodeling in acute coronary syndrome through activation of the TGF-β/Smad pathway. J Cell Physiol 234:2537–2551

    CAS  Article  Google Scholar 

  21. 21.

    Takafuji Y, Tatsumi K, Ishida M, Kawao N, Okada K, Matsuo O, Kaji H (2019) Plasminogen activator inhibitor-1 deficiency suppresses osteoblastic differentiation of mesenchymal stem cells in mice. J Cell Physiol 234:9687–9697

    CAS  Article  Google Scholar 

  22. 22.

    Jin G, Aobulikasimu A, Piao J, Aibibula Z, Koga D, Sato S, Ochi H, Tsuji K, Nakabayashi T, Miyata T, Okawa A, Asou Y (2018) A small-molecule PAI-1 inhibitor prevents bone loss by stimulating bone formation in a murine estrogen deficiency-induced osteoporosis model. FEBS Open Bio 8:523–532

    CAS  Article  Google Scholar 

  23. 23.

    Tamura Y, Kawao N, Okada K, Yano M, Okumoto K, Matsuo O, Kaji H (2013) Plasminogen activator inhibitor-1 is involved in streptozotocin-induced bone loss in female mice. Diabetes 62:3170–3179

    CAS  Article  Google Scholar 

  24. 24.

    Ghosh AK, Bradham WS, Gleaves LA, De Taeye B, Murphy SB, Covington JW, Vaughan DE (2010) Genetic deficiency of plasminogen activator inhibitor-1 promotes cardiac fibrosis in aged mice: involvement of constitutive transforming growth factor-beta signaling and endothelial-to-mesenchymal transition. Circulation 122:1200–1209

    CAS  Article  Google Scholar 

  25. 25.

    Mao L, Kawao N, Tamura Y, Okumoto K, Okada K, Yano M, Matsuo O, Kaji H (2014) Plasminogen activator inhibitor-1 is involved in impaired bone repair associated with diabetes in female mice. PLoS ONE 9(3):e92686

    Article  Google Scholar 

  26. 26.

    Dawson SJ, Wiman B, Hamsten A, Green F, Humphries S, Henney AM (1993) The two allele sequences of a common polymorphism in the promoter of the plasminogen activator inhibitor-1 (PAI-1) gene respond differently to interleukin-1 in HepG2 cells. J Biol Chem 268:10739–10745

    CAS  Google Scholar 

  27. 27.

    Heaton JH, Dlakic WM, Dlakic M, Gelehrter TD (2001) Identification and cDNA cloning of a novel RNA-binding protein that interacts with the cyclic nucleotide-responsive sequence in the Type-1 plasminogen activator inhibitor mRNA. J Biol Chem 276:3341–3347

    CAS  Article  Google Scholar 

Download references

Acknowledgements

This work was supported by a Grant-in-Aid for Scientific Research (C) (16K10809) from the JSPS (to H.I.) and the Mishima Kaiun Memorial Foundation (2015-3), Japan (to N.S.).

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SI, HS, and NS reproduced the congenital kyphoscoliosis model rats. SI, HS, DT, and NS conducted the experiments of gene and protein expression of miRNAs, Smads, Pai-1, and Col1A1. SI, HS, and YT performed the skeletal preparations and staining of rat lumber spines. SI, HI, HS, DT, HC, NK, and NS contributed to the supervision of the experiments and interpretation of the results. SI, HI, and NS designed the experiments and wrote the manuscript with input from all authors.

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Correspondence to Noriaki Shimokawa.

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The authors declare that they have no conflicts of interest.

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Ishiwata, S., Iizuka, H., Sonoda, H. et al. Upregulated miR-224-5p suppresses osteoblast differentiation by increasing the expression of Pai-1 in the lumbar spine of a rat model of congenital kyphoscoliosis. Mol Cell Biochem 475, 53–62 (2020). https://doi.org/10.1007/s11010-020-03859-8

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Keywords

  • Congenital scoliosis
  • Kyphoscoliosis model rats
  • microRNA array
  • miR-224-5p
  • Plasminogen activator inhibitor-1
  • Osteoblast differentiation