Journal of dental hygiene science (치위생과학회지)

The Korean Society of Dental Hygiene Science (한국치위생과학회)
권호 표지

Effects of Relative Lysyl Oxidase and Hydrogen Peroxide on Odontoblastic Differentiation

인간치수세포 분화과정에서 과산화수소에 대한 Lysyl Oxidase의 역할

  • Lee, Hwa-Jeong (Department of Maxillofacial Tissue Regeneration, School of Dentistry, Kyung Hee University)
  • 이화정 (경희대학교 치의학전문대학원 악안면재생학교실)
  • Published : 2013.09.30
⟨ Previous Next ⟩

Abstract

Although it has been reported that lysyl oxidase (LOX) is involved in odontoblastic differentiation, the role of LOX on odontoblastic differentiation by hydrogen peroxide ($H_2O_2$) have not been clarified. In the present study, we investigated whether $H_2O_2$, reactive oxygen species (ROS), is modulated the messenger RNA (mRNA) expression and activity of LOX during odontoblastic differentiation of human dental pulp (HDP) cells. The mRNA expression was quantified by reverse transcriptase polymerase chain reaction (RT-PCR) analysis, and LOX enzyme activity was measured by high sensitive fluorescent assay. Expression of the odontoblastic differentiation marker genes were assessed in the presence and absence of specific small interfering RNAs (siRNAs) of the LOX and LOXL. The $H_2O_2$-induced mRNA expression of LOX family was significant reduction of LOX, LOXL, and LOXL3 mRNA levels in HDP cells. LOX enzyme activity was increased at $H_2O_2$ 0.3 mM for 24 hours. The mRNA expression of alkaline phosphatase (ALP), osteopontin (OPN), and osteocalcin (OCN) was inhibited by LOX- and LOXL-specific siRNAs whereas the mRNA expression of dentin matrix protein1 (DMP1), and dentin sialophosphoprotein (DSPP) was inhibited by LOX-specific siRNA. In LOX enzyme activity, siRNA-induced knockdown of both LOX and LOXL inhibited the total amine oxidase activity in HDP cells, as in the case of mRNA expression. In conclusion, the essential role of $H_2O_2$ on odontoblastic differentiation suggests that its regulation by LOX may have pharmacologic importance in HDP cells.

과산화수소는 치아미백에 널리 사용되는 물질로 과다 사용시 치수세포에 손상을 일으킬 수 있다. 본 연구의 목적은 활성산소인 과산화수소에 의해 유도되는 상아모세포의 단계별 분화와 LOX isoforms과의 관계를 밝히고자 하였다. 치수세포에 분화유도 배지와 과산화수소를 시간과 농도별로 처리한 후 LOX 유전자 발현은 RT-PCR로 측정하였고, LOX enzyme activity는 고감도 형광분석으로 확인하였다. 또한 가장 많은 발현억제를 보인 LOX와 LOXL을 선택하여 siRNA 처리 후 분화표지자의 발현변화와 LOX enzyme activity를 확인하였다. 1. 과산화수소 처리에 따라 LOX, LOXL, LOXL3 mRNA 발현은 농도와 시간 의존적으로 감소하였으나 LOXL2와 LOXL4 mRNA는 변화가 없었다. 2. 과산화수소 처리된 LOX enzyme activity는 0.3 mM과 24시간에서 가장 많은 증가를 보였다. 3. ALP, OPN, OCN의 mRNA 발현은 LOX와 LOXL siRNAs 모두에서 억제되었고, DMP1과 DSPP는 LOX siRNA에서 더 많은 억제 효과를 보였다. 하지만, 분화단계별(초기, 중기, 말기) 차이는 보이지 않았다. 4. LOX와 LOXL siRNAs를 처리하여 LOX enzyme activity를 측정한 결과 LOX siRNA를 처리한 실험군에서 더 많은 억제효과를 보였다. 이러한 결과는 상아모세포 성장과 분화과정에 낮은 농도의 과산화수소가 분화를 유도하고 여기에 LOX가 관련됨을 알 수 있었다. 결론적으로, 과산화수소는 LOX 유전자 발현조절을 통해 치수세포의 성장과 분화에서 중추적인 역할을 할 것이라고 생각된다.

Keywords

References

  1. Luk K, Tam L, Hubert M: Effect of light energy on peroxide tooth bleaching. J Am Dent Assoc 135: 194-201, 2004. https://doi.org/10.14219/jada.archive.2004.0151
  2. Benetti AR, Valera MC, Mancini MN, Miranda CB, Balducci I: In vitro penetration of bleaching agents into the pulp chamber. Int Endod J 37: 120-124, 2004. https://doi.org/10.1111/j.0143-2885.2004.00761.x
  3. Gokay O, Mujdeci A, Algn E: Peroxide penetration into the pulp from whitening strips. J Endod 30: 887-889, 2004. https://doi.org/10.1097/01.DON.0000128750.29889.06
  4. Sulieman M, Addy M, Macdonald E, Rees JS: The bleaching depth of a 35% hydrogen peroxide based in-office product: a study in vitro. J Dent 33: 33-40, 2005. https://doi.org/10.1016/j.jdent.2004.07.008
  5. Lee DH, Lim BS, Lee YK, Yang HC: Effects of hydrogen peroxide (H2O2) on alkaline phosphatase activity and matrix mineralization of odontoblast and osteoblast cell lines. Cell Biol Toxicol 22: 39-46, 2006. https://doi.org/10.1007/s10565-006-0018-z
  6. Min KS, Lee HJ, Kim SH, et al.: Hydrogen peroxide induces heme oxygenase-1 and dentin sialophosphoprotein mRNA in human pulp cells. J Endod 34: 983-989, 2008. https://doi.org/10.1016/j.joen.2008.05.012
  7. Alliot-Licht B, Bluteau G, Magne D, et al.: Dexamethasone stimulates differentiation of odontoblast-like cells in human dental pulp cultures. Cell Tissue Res 321: 391-400, 2005. https://doi.org/10.1007/s00441-005-1115-7
  8. Butler WT, Ritchie H: The nature and functional significance of dentin extracellular matrix proteins. Int J Dev Biol 39: 169-179, 1995.
  9. Couble ML, Farges JC, Bleicher F, Perrat-Mabillon B, Boudeulle M, Magloire H: Odontoblast differentiation of human dental pulp cells in explant cultures. Calcif Tissue Int 66: 129-138, 2000. https://doi.org/10.1007/PL00005833
  10. Ruch JV, Lesot H, Bègue-Kirn C: Odontoblast differentiation. Int J Dev Biol 39: 51-68, 1995.
  11. Ibaraki K, Termine JD, Whitson SW, Young MF: Bone matrix mRNA expression in differentiating fetal bovine osteoblasts. J Bone Miner Res 7: 743-754, 1992.
  12. Rodan GA, Heath JK, Yoon K, Noda M, Rodan SB: Diversity of the osteoblastic phenotype. Ciba Found Symp 136: 78-91, 1988.
  13. Weinreb M, Shinar D, Rodan GA: Different pattern of alkaline phosphatase, osteopontin, and osteocalcin expression in developing rat bone visualized by in situ hybridization. J Bone Miner Res 5: 831-842, 1990.
  14. Beck GR Jr, Zerler B, Moran E: Phosphate is a specific signal for induction of osteopontin gene expression. Proc Natl Acad Sci U S A 97: 8352-8357, 2000. https://doi.org/10.1073/pnas.140021997
  15. Nagaoka I, Igarashi M, Sakamoto K: Biological activities of glucosamine and its related substances. Adv Food Nutr Res 65: 337-352, 2012. https://doi.org/10.1016/B978-0-12-416003-3.00022-6
  16. Kagan HM, Li W: Lysyl oxidase: properties, specificity, and biological roles inside and outside of the cell. J Cell Biochem 88: 660-672, 2003. https://doi.org/10.1002/jcb.10413
  17. Kagan HM, Trackman PC: Properties and function of lysyl oxidase. Am J Respir Cell Mol Biol 5: 206-210, 1991. https://doi.org/10.1165/ajrcmb/5.3.206
  18. Payne SL, Fogelgren B, Hess AR, et al.: Lysyl oxidase regulates breast cancer cell migration and adhesion through a hydrogen peroxide-mediated mechanism. Cancer Res 65: 11429-11436, 2005. https://doi.org/10.1158/0008-5472.CAN-05-1274
  19. Saito H, Papaconstantinou J, Sato H, Goldstein S: Regulation of a novel gene encoding a lysyl oxidase-related protein in cellular adhesion and senescence. J Biol Chem 272: 8157-8160, 1997. https://doi.org/10.1074/jbc.272.13.8157
  20. Kim EC, Lee HJ, Kim Y: Lysyl oxidase and the lysyl oxidase-like protein modulate odontoblastic differentiation of human dental pulp cells. J Endod 38: 769-773, 2012. https://doi.org/10.1016/j.joen.2012.03.014
  21. Min KS, Kwon YY, Lee HJ, et al.: Effects of proinflammatory cytokines on the expression of mineralization markers and heme oxygenase-1 in human pulp cells. J Endod 32: 39-43, 2006. https://doi.org/10.1016/j.joen.2005.10.012
  22. Chang SW, Lee SI, Bae WJ, et al.: Heat stress activates interleukin-8 and the antioxidant system via Nrf2 pathways in human dental pulp cells. J Endod 35: 1222-1228, 2009. https://doi.org/10.1016/j.joen.2009.06.005
  23. Wei X, Ling J, Wu L, Liu L, Xiao Y: Expression of mineralization markers in dental pulp cells. J Endod 33: 703-708, 2007. https://doi.org/10.1016/j.joen.2007.02.009
  24. Min KS, Lee YM, Hong SO, Kim EC: Simvastatin promotes odontoblastic differentiation and expression of angiogenic factors via heme oxygenase-1 in primary cultured human dental pulp cells. J Endod 36: 447-452, 2010. https://doi.org/10.1016/j.joen.2009.11.021
  25. Palamakumbura AH, Trackman PC: A fluorometric assay for detection of lysyl oxidase enzyme activity in biological samples. Anal Biochem 300: 245-251, 2002. https://doi.org/10.1006/abio.2001.5464
  26. Tang SS, Trackman PC, Kagan HM: Reaction of aortic lysyl oxidase with beta-aminopropionitrile. J Biol Chem 258: 4331-4338, 1983.
  27. Hannon GJ, Rossi JJ: Unlocking the potential of the human genome with RNA interference. Nature 431: 371-378, 2004. https://doi.org/10.1038/nature02870
  28. Ames BN, Shigenaga MK: Oxidants are a major contributor to aging. Ann N Y Acad Sci 663: 85-96, 1992. https://doi.org/10.1111/j.1749-6632.1992.tb38652.x
  29. Allen RG, Tresini M: Oxidative stress and gene regulation. Free Radic Biol Med 28: 463-499, 2000. https://doi.org/10.1016/S0891-5849(99)00242-7
  30. Shackelford RE, Kaufmann WK, Paules RS: Oxidative stress and cell cycle checkpoint function. Free Radic Biol Med 28: 1387-1404, 2000. https://doi.org/10.1016/S0891-5849(00)00224-0
  31. Pischon N, Darbois LM, Palamakumbura AH, Kessler E, Trackman PC: Regulation of collagen deposition and lysyl oxidase by tumor necrosis factor-alpha in osteoblasts. J Biol Chem 279: 30060-30065, 2004. https://doi.org/10.1074/jbc.M404208200
  32. Rodriguez C, Alcudia JF, Martanez-Gonzalez J, Raposo B, Navarro MA, Badimon L: Lysyl oxidase (LOX) downregulation by TNFalpha: a new mechanism underlying TNFalpha-induced endothelial dysfunction. Atherosclerosis 196: 558-564, 2008.
  33. Atsawasuwan P, Mochida Y, Parisuthiman D, Yamauchi M: Expression of lysyl oxidase isoforms in MC3T3-E1 osteoblastic cells. Biochem Biophys Res Commun 327: 1042-1046, 2005. https://doi.org/10.1016/j.bbrc.2004.12.119