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lncRNA HOTAIR Inhibits Mineralization in Osteoblastic Osteosarcoma Cells by Epigenetically Repressing ALPL

  • Aya Misawa
  • Hideo Orimo
Original Research

Abstract

HOTAIR is a lncRNA that plays critical role in gene regulation and chromatin dynamics through epigenetic mechanisms. In this work we studied the physiological role of HOTAIR during the process of mineralization using osteoblastic osteosarcoma cells focusing in ALPL (Tissue Non-Specific Alkaline Phosphatase), a pivotal gene that controls bone formation. HOTAIR knockdown resulted in upregulation of ALPL, increase of alkaline phosphatase (ALP) activity, and enhanced mineralization in osteoblastic SaOS-2 cells cultured in mineralizing medium. Luciferase assays using reporter vectors containing ALPL promoter showed that HOTAIR repression increases ALPL promoter activity. Furthermore, HOTAIR knockdown increased histone H3K4 methylation levels at ALPL promoter region, suggesting that ALPL repression by HOTAIR is regulated by epigenetic mechanisms. This work supports that physiological bone formation is epigenetically regulated by a lncRNA.

Keywords

ncRNA lncRNA Epigenetics Alkaline phosphatase Mineralization Osteoblast 

Notes

Acknowledgements

We are grateful to T. Takizawa and T. Kosuge from the Department of Molecular Medicine and Anatomy, Nippon Medical School, for technical support for Luciferase and ChIP assays. This work was supported by Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (to A.M.) KAKENHI Grant Number: 16K18411.

Compliance with ethical standards

Conflict of interest

The authors have nothing to disclose.

Human and Animal Rights and Informed Consent

This article does not contain any studies involving animals nor human participants.

References

  1. 1.
    Millán JL (2006) Alkaline phosphatases: structure, substrate specificity and functional relatedness to other members of a large superfamily of enzymes. Purinergic Signal 2:335–341.  https://doi.org/10.1007/s11302-005-5435-6 CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Smith M, Weiss MJ, Griffin CA, Murray JC, Buetow KH, Emanuel BS, Henthorn PS, Harris H (1988) Regional assignment of the gene for human liver/bone/kidney alkaline phosphatase to chromosome 1p36.1-p34. Genomics 2:139–143CrossRefPubMedGoogle Scholar
  3. 3.
    Weiss MJ, Ray K, Henthorn PS, Lamb B, Kadesch T, Harris H (1988) Structure of the human liver/bone/kidney alkaline phosphatase gene. J Biol Chem 263:12002–12010PubMedGoogle Scholar
  4. 4.
    Matsuura S, Kishi F, Kajii T (1990) Characterization of a 5′-flanking region of the human liver/bone/kidney alkaline phosphatase gene: two kinds of mRNA from a single gene. Biochem Biophys Res Commun 168:993–1000CrossRefPubMedGoogle Scholar
  5. 5.
    Orimo H (2010) The mechanism of mineralization and the role of alkaline phosphatase in health and disease. J Nippon Med Sch 77:4–12CrossRefPubMedGoogle Scholar
  6. 6.
    Rathbun JC (1948) Hypophosphatasia; a new developmental anomaly. Am J Dis Child 75:822–831CrossRefPubMedGoogle Scholar
  7. 7.
    Bianchi ML (2015) Hypophosphatasia: an overview of the disease and its treatment. Osteoporos Int 26:2743–2757.  https://doi.org/10.1007/s00198-015-3272-1 CrossRefPubMedGoogle Scholar
  8. 8.
    St Hilaire C, Ziegler SG, Markello TC, Brusco A, Groden C, Gill F, Carlson-Donohoe H, Lederman RJ, Chen MY, Yang D, Siegenthaler MP, Arduino C, Mancini C, Freudenthal B, Stanescu HC, Zdebik AA, Chaganti RK, Nussbaum RL, Kleta R, Gahl WA, Boehm M (2011) NT5E mutations and arterial calcifications. N Engl J Med 364:432–442.  https://doi.org/10.1056/NEJMoa0912923 CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Michigami T, Uchihashi T, Suzuki A, Tachikwa K, Nakajima S, Ozono K (2005) Common mutation F310L and T1559del in the tissue-nonspecific alkaline phosphatase gene are related to distinct phenotypes in Japanese patients with hypophosphatasia. Eur J Pediatr 164:277–282.  https://doi.org/10.1007/s00431-004-1612-9 CrossRefPubMedGoogle Scholar
  10. 10.
    Orimo H (2016) Pathophysiology of hypophosphatasia and the potential role of asfotase alfa. Ther Clin Risk Manag 12:777–786.  https://doi.org/10.2147/TCRM.S87956 CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Whyte MP (2017) Hypophosphatasia: an overview for 2017. Bone 102:15–25.  https://doi.org/10.1016/j.bone.2017.02.011 CrossRefPubMedGoogle Scholar
  12. 12.
    Bonewald LF (2011) The amazing osteocyte. J Bone Miner Res 26:229–238.  https://doi.org/10.1002/jbmr.320 CrossRefPubMedGoogle Scholar
  13. 13.
    Lizio M, Harshbarger J, Abugessaisa I, Noguchi S, Kondo A, Severin J, Mungall C, Arenillas D, Mathelier A, Medvedeva YA, Lennartsson A, Drabløs F, Ramilowski JA, Rackham O, Gough J, Andersson R, Sandelin A, Ienasescu H, Ono H, Bono H, Hayashizaki Y, Carninci P, Forrest AR, Kasukawa T, Kawaji H (2017) Update of the FANTOM web resource: high resolution transcriptome of diverse cell types in mammals. Nucleic Acids Res 45:D737-D743.  https://doi.org/10.1093/nar/gkw995 CrossRefPubMedGoogle Scholar
  14. 14.
    Hon CC, Ramilowski JA, Harshbarger J, Bertin N, Rackham OJ, Gough J, Denisenko E, Schmeier S, Poulsen TM, Severin J, Lizio M, Kawaji H, Kasukawa T, Itoh M, Burroughs AM, Noma S, Djebali S, Alam T, Medvedeva YA, Testa AC, Lipovich L, Yip CW, Abugessaisa I, Mendez M, Hasegawa A, Tang D, Lassmann T, Heutink P, Babina M, Wells CA, Kojima S, Nakamura Y, Suzuki H, Daub CO, de Hoon MJ, Arner E, Hayashizaki Y, Carninci P, Forrest AR (2017) An atlas of human long non-coding RNAs with accurate 5′ ends. Nature 543:199–204.  https://doi.org/10.1038/nature21374 CrossRefPubMedGoogle Scholar
  15. 15.
    Iyer MK, Niknafs YS, Malik R, Singhal U, Sahu A, Hosono Y, Barrette TR, Prensner JR, Evans JR, Zhao S, Poliakov A, Cao X, Dhanasekaran SM, Wu YM, Robinson DR, Beer DG, Feng FY, Iyer HK, Chinnaiyan AM (2015) The landscape of long noncoding RNAs in the human transcriptome. Nat Genet 47:199–208.  https://doi.org/10.1038/ng.3192 CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Quek XC, Thomson DW, Maag JL, Bartonicek N, Signal B, Clark MB, Gloss BS, Dinger ME (2015) lncRNAdb v2.0: expanding the reference database for functional long noncoding RNAs. Nucleic Acids Res 43:D168-173.  https://doi.org/10.1093/nar/gku988 CrossRefGoogle Scholar
  17. 17.
    Schmidt LH, Spieker T, Koschmieder S, Schäffers S, Humberg J, Jungen D, Bulk E, Hascher A, Wittmer D, Marra A, Hillejan L, Wiebe K, Berdel WE, Wiewrodt R, Muller-Tidow C (2011) The long noncoding MALAT-1 RNA indicates a poor prognosis in non-small cell lung cancer and induces migration and tumor growth. J Thorac Oncol 6:1984–1992.  https://doi.org/10.1097/JTO.0b013e3182307eac CrossRefPubMedGoogle Scholar
  18. 18.
    Gupta RA, Shah N, Wang KC, Kim J, Horlings HM, Wong DJ, Tsai MC, Hung T, Argani P, Rinn JL, Wang Y, Brzoska P, Kong B, Li R, West RB, van de Vijver MJ, Sukumar S, Chang HY (2010) Long non-coding RNA HOTAIR reprograms chromatin state to promote cancer metastasis. Nature 464:1071–1076.  https://doi.org/10.1038/nature08975 CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Tsai MC, Manor O, Wan Y, Mosammaparast N, Wang JK, Lan F, Shi Y, Segal E, Chang HY (2010) Long noncoding RNA as modular scaffold of histone modification complexes. Science 329:689–693.  https://doi.org/10.1126/science.1192002 CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Bhan A, Mandal SS (2015) LncRNA HOTAIR: a master regulator of chromatin dynamics and cancer. Biochim Biophys Acta 1856:151–164.  https://doi.org/10.1016/j.bbcan.2015.07.001 PubMedPubMedCentralGoogle Scholar
  21. 21.
    Carrion K, Dyo J, Patel V, Sasik R, Mohamed SA, Hardiman G, Nigam V (2014) The long non-coding HOTAIR is modulated by cyclic stretch and WNT/β-CATENIN in human aortic valve cells and is a novel repressor of calcification genes. PLoS ONE 9:e96577.  https://doi.org/10.1371/journal.pone.0096577 CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Orimo H, Shimada T (2006) Effects of phosphates on the expression of tissue-nonspecific alkaline phosphatase gene and phosphate-regulating genes in short-term cultures of human osteosarcoma cell lines. Mol Cell Biochem 282:101–108.  https://doi.org/10.1007/s11010-006-1520-6 CrossRefPubMedGoogle Scholar
  23. 23.
    Orimo H, Shimada T (2008) The role of tissue-nonspecific alkaline phosphatase in the phosphate-induced activation of alkaline phosphatase and mineralization in SaOS-2 human osteoblast-like cells. Mol Cell Biochem 315:51–60.  https://doi.org/10.1007/s11010-008-9788-3 CrossRefPubMedGoogle Scholar
  24. 24.
    Orimo H, Shimada T (2005) Regulation of the human tissue-nonspecific alkaline phosphatase gene expression by all-trans-retinoic acid in SaOS-2 osteosarcoma cell line. Bone 36:866–876.  https://doi.org/10.1016/j.bone.2005.02.010 CrossRefPubMedGoogle Scholar
  25. 25.
    Rodan SB, Imai Y, Thiede MA, Wesolowski G, Thompson D, Bar-Shavit Z, Shull S, Mann K, Rodan GA (1987) Characterization of a human osteosarcoma cell line (Saos-2) with osteoblastic properties. Cancer Res 47:4961–4966PubMedGoogle Scholar
  26. 26.
    Fedde KN (1992) Human osteosarcoma cells spontaneously release matrix-vesicle-like structures with the capacity to mineralize. Bone Miner 17:145–151CrossRefPubMedGoogle Scholar
  27. 27.
    Takayama K, Kaneshiro K, Tsutsumi S, Horie-Inoue K, Ikeda K, Urano T, Ijichi N, Ouchi Y, Shirahige K, Aburatani H, Inoue S (2007) Identification of novel androgen response genes in prostate cancer cells by coupling chromatin immunoprecipitation and genomic microarray analysis. Oncogene 26:4453–4463.  https://doi.org/10.1038/sj.onc.1210229 CrossRefPubMedGoogle Scholar
  28. 28.
    Weiss et al (1988) Structure of the human liver/bone/kidney alkaline phosphatase gene. J Biol Chem 263(24):12002–12010PubMedGoogle Scholar
  29. 29.
    Kiledjian et al (1990) Analysis of the human liver/bone/kidney alkaline phosphatase promoter in vivo and in vitro. Nucleic Acids Res 18(4):957–961CrossRefPubMedPubMedCentralGoogle Scholar
  30. 30.
    Yusa N et al (2000) Transcription factor Sp3 activates the liver/bone/kidney-type alkaline phosphatase promoter in hematopoietic cells. J Leukoc Biol 68(5):772–777.  https://doi.org/10.1189/jlb.68.5.772 PubMedGoogle Scholar
  31. 31.
    Tylzanowski et al (2001) Smad-interacting protein 1 is a repressor of liver/bone/kidney alkaline phosphatase transcription in bone morphogenetic protein-induced osteogenic differentiation of C2C12 cells. J Biol Chem 276(43):40001–40007.  https://doi.org/10.1074/jbc.M104112200 CrossRefPubMedGoogle Scholar
  32. 32.
    Kim HN, Lee JH, Bae SC, Ryoo HM, Kim HH, Ha H, Lee ZH (2011) Histone deacetylase inhibitor MS-275 stimulates bone formation in part by enhancing Dhx36-mediated TNAP transcription. J Bone Miner Res 26:2161–2173.  https://doi.org/10.1002/jbmr.426 CrossRefPubMedGoogle Scholar
  33. 33.
    Delgado-Calle J, Sañudo C, Sánchez-Verde L, García-Renedo RJ, Arozamena J, Riancho JA (2011) Epigenetic regulation of alkaline phosphatase in human cells of the osteoblastic lineage. Bone 49:830–838.  https://doi.org/10.1016/j.bone.2011.06.006 CrossRefPubMedGoogle Scholar
  34. 34.
    Saldana L et al (2011) In search of representative models of human bone-forming cells for cytocompatibility studies. Acta Biomater 7(12):4210–4221.  https://doi.org/10.1016/j.actbio.2011.07.019 CrossRefPubMedGoogle Scholar
  35. 35.
    Czekanska et al (2012) In search of an osteoblast cell model for in vitro research. Eur Cells Mater 24:1–17.  https://doi.org/10.22203/eCM.v024a01 CrossRefGoogle Scholar
  36. 36.
    Vandrovcova et al (2014) Interaction of human osteoblast-like Saos-2 and MG-63 cells with thermally oxidized surfaces of a titanium-niobium alloy. PLoS ONE 9(6):e100475. doi. https://doi.org/10.1371/journal.pone.0100475 CrossRefPubMedPubMedCentralGoogle Scholar
  37. 37.
    Gupta et al (2010) Long non-coding RNA HOTAIR reprograms chromatin state to promote cancer metastasis. Nature 464(7291):1071–1076.  https://doi.org/10.1038/nature08975 CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Division of Metabolism and Nutrition, Department of Biochemistry and Molecular BiologyNippon Medical SchoolTokyoJapan

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