N-acetyl cysteine inhibits H2O2-mediated reduction in the mineralization of MC3T3-E1 cells by down-regulating Nrf2/HO-1 pathway

Lee, Daewoo;Kook, Sung-Ho;Ji, Hyeok;Lee, Seung-Ah;Choi, Ki-Choon;Lee, Kyung-Yeol;Lee, Jeong-Chae

  • 투고 : 2015.06.11
  • 심사 : 2015.08.20
  • 발행 : 2015.11.30


There are controversial findings regarding the roles of nuclear factor (erythroid-derived 2)-like 2 (Nrf2)/heme oxygenase-1 (HO-1) pathway on bone metabolism under oxidative stress. We investigated how Nrf2/HO-1 pathway affects osteoblast differentiation of MC3T3-E1 cells in response to hydrogen peroxide (H2O2), N-acetyl cysteine (NAC), or both. Exposing the cells to H2O2 decreased the alkaline phosphatase activity, calcium accumulation, and expression of osteoblast markers, such as osteocalcin and runt-related transcription factor-2. In contrast, H2O2 treatment increased the expression of Nrf2 and HO-1 in the cells. Treatment with hemin, a chemical HO-1 inducer, mimicked the inhibitory effect of H2O2 on osteoblast differentiation by increasing the HO-1 expression and decreasing the osteogenic marker genes. Pretreatment with NAC restored all changes induced by H2O2 to near normal levels in the cells. Collectively, our findings suggest that H2O2-mediated activation of Nrf2/HO-1 pathway negatively regulates the osteoblast differentiation, which is inhibited by NAC.


H2O2;N-acetyl cysteine;Nrf2;Osteoblast differentiation;Oxidative stress


  1. Kondo H, Yumoto K, Alwood JS et al (2010) Oxidative stress and gamma radiation-induced cancellous bone loss with musculoskeletal disuse. J Appl Physiol (1985) 108, 152-161
  2. Mody N, Parhami F, Sarafian TA and Demer LL (2001) Oxidative stress modulates osteoblastic differentiation of vascular and bone cells. Free Radic Biol Med 31, 509-519
  3. Bai XC, Lu D, Bai J et al (2004) Oxidative stress inhibits osteoblastic differentiation of bone cells by ERK and NF-kappaB. Biochem Biophys Res Commun 314, 197-207
  4. Banfi G, Iorio EL and Corsi MM (2008) Oxidative stress, free radicals and bone remodeling. Clin Chem Lab Med 46, 1550-1555
  5. Jia P, Xu YJ, Zhang ZL et al (2012) Ferric ion could facilitate osteoclast differentiation and bone resorption through the production of reactive oxygen species. J Orthop Res 30, 1843-1852
  6. Ueno T, Yamada M, Igarashi Y and Ogawa T (2011) N-acetyl cysteine protects osteoblastic function from oxidative stress. J Biomed Mater Res A 99, 523-531
  7. Singh PK, Wise SY, Ducey EJ, Fatanmi OO, Elliott TB and Singh VK (2012) α-Tocopherol succinate protects mice against radiation-induced gastrointestinal injury. Radiat Res 177, 133-145
  8. Zhang Q, Pi J, Woods CG and Andersen ME (2010) A systems biology perspective on Nrf2-mediated antioxidant response. Toxicol Appl Pharmacol 244, 84-97
  9. Shin SM, Yang JH and Ki SH (2013) Role of the Nrf2-ARE pathway in liver diseases. Oxid Med Cell Longev 2013, 763257
  10. Rana T, Schultz MA, Freeman ML and Biswas S (2012) Loss of Nrf2 accelerates ionizing radiation-induced bone loss by upregulating RANKL. Free Radic Biol Med 53, 2298-2307
  11. Maines MD and Panahian N (2001) The heme oxygenase system and cellular defense mechanisms. Do HO-1 and HO-2 have different functions? Adv Exp Med Biol 502, 249-272
  12. Zwerina J, Tzima S, Hayer S et al (2005) Heme oxygenase 1 (HO-1) regulates osteoclastogenesis and bone resorption. FASEB J 19, 2011-2013
  13. Chen JS, Huang PH, Wang CH et al (2011) Nrf-2 mediated heme oxygenase-1 expression, an antioxidant-independent mechanism, contributes to anti-atherogenesis and vascular protective effects of Ginkgo biloba extract. Atherosclerosis 214, 301-309
  14. Yu J, Zhu X, Qi X, Che J and Cao B (2013) Paeoniflorin protects human EA.hy926 endothelial cells against gamma- radiation induced oxidative injury by activating the NF-E2-related factor 2/heme oxygenase-1 pathway. Toxicol Lett 218, 224-234
  15. Hinoi E, Fujimori S, Wang L, Hojo H, Uno K and Yoneda Y (2006) Nrf2 negatively regulates osteoblast differentiation via interfering with Runx2-dependent transcriptional activation. J Biol Chem 281, 18015-18024
  16. Lin TH, Tang CH, Hung SY et al (2010) Upregulation of heme oxygenase-1 inhibits the maturation and mineralization of osteoblasts. J Cell Physiol 222, 757-768
  17. Lee JH and Cho JY (2014) Proteomics approaches for the studies of bone metabolism. BMB Rep 47, 141-148
  18. Neve A, Corrado A and Cantatore FP (2013) Osteocalcin: skeletal and extra-skeletal effects. J Cell Physiol 228, 1149-1153
  19. Jun JH, Lee SH, Kwak HB et al (2008) N-acetyl cysteine stimulates osteoblastic differentiation of mouse calvarial cells. J Cell Biochem 103, 1246-1255
  20. Kim KA, Kook SH, Song JH and Lee JC (2014) A phenolic acid phenethyl urea derivative protects against irradiation- induced osteoblast damage by modulating intracellular redox state. J Cell Biochem 115, 1877-1887
  21. Arai M, Shibata Y, Pugdee K, Abiko Y and Ogata Y (2007) Effects of reactive oxygen species (ROS) on antioxidant system and osteoblastic differentiation in MC3T3-E1 cells. IUBMB Life 59, 27-33
  22. Ahanger AA, Prawez S, Leo MD et al (2010) Pro-healing potential of hemin: an inducer of heme oxygenase-1. Eur J Pharmacol 645, 165-170
  23. Kook YA, Lee SK, Son DH et al (2009) Effects of substance P on osteoblastic differentiation and heme oxygenase-1 in human periodontal ligament cells. Cell Biol Int 33, 424-428
  24. Kozakowska M, Szade K, Dulak J and Jozkowicz A (2013) Role of heme oxygenase-1 in postnatal differentiation of stem cells: a possible cross-talk with microRNAs. Antioxid Redox Signal 20, 1827-1850
  25. Kim GY, Park SY, Jo A et al (2015) Gecko proteins induce the apoptosis of bladder cancer 5637 cell by inhibiting Akt and activating intrinsic caspase cascade. BMB Rep 48, 531-536
  26. Park SS, Kim KA, Lee SY, Lim SS, Jeon YM and Lee JC (2012) X-ray radiation at low doses stimulates differentiation and mineralization of mouse calvarial osteoblasts. BMB Rep 45, 571-576
  27. Heo JS, Lee SY and Lee JC (2010) Wnt/β-catenin signaling enhances osteoblastogenic differentiation from human periodontal ligament fibroblasts. Mol Cells 30, 449-454
  28. Jeon YM, Kook SH, Rho SJ et al (2013) Fibroblast growth factor-7 facilitates osteogenic differentiation of embryonic stem cells through the activation of ERK/Runx2 signaling. Mol Cell Biochem 382, 37-45
  29. Yu JY, Zheng ZH, Son YO, Shi X, Jang YO and Lee JC (2011) Mycotoxin zearalenone induces AIF- and ROSmediated cell death through p53- and MAPK-dependent signaling pathways in RAW264.7 macrophages. Toxicol In Vitro 25, 1654-1663

피인용 문헌

  1. Anti-inflammatory effects and corresponding mechanisms of cirsimaritin extracted from Cirsium japonicum var. maackii Maxim vol.27, pp.14, 2017,
  2. Dalbergioidin (DAL) protects MC3T3-E1 osteoblastic cells against H2O2-induced cell damage through activation of the PI3K/AKT/SMAD1 pathway vol.390, pp.7, 2017,
  3. Omeprazole induces heme oxygenase-1 in fetal human pulmonary microvascular endothelial cells via hydrogen peroxide-independent Nrf2 signaling pathway vol.311, 2016,
  4. Dexmedetomidine attenuates H2O2-induced cell death in human osteoblasts vol.16, pp.4, 2016,
  5. Protective effect of 3-(naphthalen-2-yl(propoxy)methyl)azetidine hydrochloride on hypoxia-induced toxicity by suppressing microglial activation in BV-2 cells vol.49, pp.12, 2016,