{"paper_id":"dca57d2b-c5d0-40f9-84a7-4aaae713725f","body_text":"Abstract\nPostmenopausal osteoporosis induced by estrogen deficiency causes inadequate new bone formation and affects millions of women worldwide. Melatonin can improve bone mineral density at the femoral neck in postmenopausal women with osteopenia. This study aimed to investigate the mechanism of melatonin in estrogen deficiency-induced osteoporosis by focusing on osteoblast differentiation. 12-week-old female C57BL/6J mice were ovariectomized (OVX) and intraperitoneally injected with 10 or 50 mg/kg of melatonin for 8 weeks. Micro-computerized tomography scanning demonstrated that melatonin alleviated OVX-induced bone loss in a dose-dependent manner. Serum levels of ALP and osteocalcin (OCN) were further increased, whereas tartrate-resistant acid phosphatase level was decreased by melatonin in OVX-treated mice. Melatonin promoted osteoblast differentiation in primary bone marrow mesenchymal stem cells from OVX mice. It also inhibited activation of NLRP3 inflammasome in femoral bone protein and in induced osteoblasts stimulated by OVX. Knockdown of NLRP3 attenuated OVX-induced repression of osteogenic differentiation. The NLRP3 inflammasome activator monosodium urate partly abrogated the effect of melatonin on the expression of osteoblastogenic markers, including Runx2 and OCN. Additionally, the results showed that melatonin suppressed NLRP3 inflammasome activation by regulating Wnt/β-catenin signaling, which was confirmed by the Wnt/β-catenin inhibitor recombinant DKK1. These results indicated that melatonin ameliorates estrogen deficiency-induced osteoporosis and impaired osteogenic differentiation potential by suppressing activation of the NLRP3 inflammasome via mediating the Wnt/β-catenin pathway.\nSimilar content being viewed by others\nReferences\nRachner TD, Khosla S, Hofbauer LC (2011) Osteoporosis: now and the future. Lancet 377(9773):1276–1287. https://doi.org/10.1016/S0140-6736(10)62349-5\nKrassas GE, Papadopoulou P (2001) Oestrogen action on bone cells. J Musculoskelet Neuronal Interact 2(2):143–151\nLiu W, Zhou L, Zhou C, Zhang S, Jing J, Xie L, Sun N, Duan X, Jing W, Liang X, Zhao H, Ye L, Chen Q, Yuan Q (2016) GDF11 decreases bone mass by stimulating osteoclastogenesis and inhibiting osteoblast differentiation. Nat Commun 7:12794. https://doi.org/10.1038/ncomms12794\nRandell KM, Honkanen RJ, Kroger H, Saarikoski S (2002) Does hormone-replacement therapy prevent fractures in early postmenopausal women? J Bone Miner Res 17(3):528–533. https://doi.org/10.1359/jbmr.2002.17.3.528\nde Villiers TJ (2015) The role of menopausal hormone therapy in the management of osteoporosis. Climacteric 18(Suppl 2):19–21. https://doi.org/10.3109/13697137.2015.1099806\nRadogna F, Diederich M, Ghibelli L (2010) Melatonin: a pleiotropic molecule regulating inflammation. Biochem Pharmacol 80(12):1844–1852. https://doi.org/10.1016/j.bcp.2010.07.041\nFavero G, Franceschetti L, Bonomini F, Rodella LF, Rezzani R (2017) Melatonin as an anti-inflammatory agent modulating inflammasome activation. Int J Endocrinol 2017:1835195. https://doi.org/10.1155/2017/1835195\nZhang WL, Meng HZ, Yang RF, Yang MW, Sun GH, Liu JH, Shi PX, Liu F, Yang B (2016) Melatonin suppresses autophagy in type 2 diabetic osteoporosis. Oncotarget 7(32):52179–52194. https://doi.org/10.18632/oncotarget.10538\nAmstrup AK, Sikjaer T, Heickendorff L, Mosekilde L, Rejnmark L (2015) Melatonin improves bone mineral density at the femoral neck in postmenopausal women with osteopenia: a randomized controlled trial. J Pineal Res 59(2):221–229. https://doi.org/10.1111/jpi.12252\nHan Y, Kim YM, Kim HS, Lee KY (2017) Melatonin promotes osteoblast differentiation by regulating osterix protein stability and expression. Sci Rep 7(1):5716. https://doi.org/10.1038/s41598-017-06304-x\nKoyama H, Nakade O, Takada Y, Kaku T, Lau KH (2002) Melatonin at pharmacologic doses increases bone mass by suppressing resorption through down-regulation of the RANKL-mediated osteoclast formation and activation. J Bone Miner Res 17(7):1219–1229. https://doi.org/10.1359/jbmr.2002.17.7.1219\nMundy GR (2007) Osteoporosis and inflammation. Nutr Rev 65(12 Pt 2):S147–S151\nGilbert L, He X, Farmer P, Boden S, Kozlowski M, Rubin J, Nanes MS (2000) Inhibition of osteoblast differentiation by tumor necrosis factor-alpha. Endocrinology 141(11):3956–3964. https://doi.org/10.1210/endo.141.11.7739\nSun X, Hao H, Han Q, Song X, Liu J, Dong L, Han W, Mu Y (2017) Human umbilical cord-derived mesenchymal stem cells ameliorate insulin resistance by suppressing NLRP3 inflammasome-mediated inflammation in type 2 diabetes rats. Stem Cell Res Ther 8(1):241. https://doi.org/10.1186/s13287-017-0668-1\nXu Y, Sheng H, Bao Q, Wang Y, Lu J, Ni X (2016) NLRP3 inflammasome activation mediates estrogen deficiency-induced depression- and anxiety-like behavior and hippocampal inflammation in mice. Brain Behav Immun 56:175–186. https://doi.org/10.1016/j.bbi.2016.02.022\nWang L, Chen K, Wan X, Wang F, Guo Z, Mo Z (2017) NLRP3 inflammasome activation in mesenchymal stem cells inhibits osteogenic differentiation and enhances adipogenic differentiation. Biochem Biophys Res Commun 484(4):871–877. https://doi.org/10.1016/j.bbrc.2017.02.007\nMansoori MN, Shukla P, Kakaji M, Tyagi AM, Srivastava K, Shukla M, Dixit M, Kureel J, Gupta S, Singh D (2016) IL-18BP is decreased in osteoporotic women: prevents Inflammasome mediated IL-18 activation and reduces Th17 differentiation. Sci Rep 6:33680. https://doi.org/10.1038/srep33680\nCao S, Shrestha S, Li J, Yu X, Chen J, Yan F, Ying G, Gu C, Wang L, Chen G (2017) Melatonin-mediated mitophagy protects against early brain injury after subarachnoid hemorrhage through inhibition of NLRP3 inflammasome activation. Sci Rep 7(1):2417. https://doi.org/10.1038/s41598-017-02679-z\nCao Z, Fang Y, Lu Y, Tan D, Du C, Li Y, Ma Q, Yu J, Chen M, Zhou C, Pei L, Zhang L, Ran H, He M, Yu Z, Zhou Z (2017) Melatonin alleviates cadmium-induced liver injury by inhibiting the TXNIP-NLRP3 inflammasome. J Pineal Res. https://doi.org/10.1111/jpi.12389\nYang 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 alpha suppresses the mesenchymal stem cell osteogenesis promoter miR-21 in estrogen deficiency-induced osteoporosis. J Bone Miner Res 28(3):559–573. https://doi.org/10.1002/jbmr.1798\nPark KH, Kang JW, Lee EM, Kim JS, Rhee YH, Kim M, Jeong SJ, Park YG, Kim SH (2011) Melatonin promotes osteoblastic differentiation through the BMP/ERK/Wnt signaling pathways. J Pineal Res 51(2):187–194. https://doi.org/10.1111/j.1600-079X.2011.00875.x\nChen EEM, Zhang W, Ye CCY, Gao X, Jiang LLJ, Zhao TTF, Pan ZZJ, Xue DDT (2017) Knockdown of SIRT7 enhances the osteogenic differentiation of human bone marrow mesenchymal stem cells partly via activation of the Wnt/beta-catenin signaling pathway. Cell Death Dis 8(9):e3042. https://doi.org/10.1038/cddis.2017.429\nYue S, Zhu J, Zhang M, Li C, Zhou X, Zhou M, Ke M, Busuttil RW, Ying QL, Kupiec-Weglinski JW, Xia Q, Ke B (2016) The myeloid heat shock transcription factor 1/beta-catenin axis regulates NLR family, pyrin domain-containing 3 inflammasome activation in mouse liver ischemia/reperfusion injury. Hepatology 64(5):1683–1698. https://doi.org/10.1002/hep.28739\nFeng J, Sun Q, Liu L, Xing D (2015) Photoactivation of TAZ via Akt/GSK3beta signaling pathway promotes osteogenic differentiation. Int J Biochem Cell Biol 66:59–68. https://doi.org/10.1016/j.biocel.2015.07.002\nRubin R, Arzumanyan A, Soliera AR, Ross B, Peruzzi F, Prisco M (2007) Insulin receptor substrate (IRS)-1 regulates murine embryonic stem (mES) cells self-renewal. J Cell Physiol 213(2):445–453. https://doi.org/10.1002/jcp.21185\nFeng X, Zhang Y, Xu R, Xie X, Tao L, Gao H, Gao Y, He Z, Wang H (2010) Lipopolysaccharide up-regulates the expression of Fcalpha/mu receptor and promotes the binding of oxidized low-density lipoprotein and its IgM antibody complex to activated human macrophages. Atherosclerosis 208(2):396–405. https://doi.org/10.1016/j.atherosclerosis.2009.07.035\nDalle Carbonare L, Valenti MT, Zanatta M, Donatelli L, Lo Cascio V (2009) Circulating mesenchymal stem cells with abnormal osteogenic differentiation in patients with osteoporosis. Arthritis Rheum 60(11):3356–3365. https://doi.org/10.1002/art.24884\nWu XB, Li Y, Schneider A, Yu W, Rajendren G, Iqbal J, Yamamoto M, Alam M, Brunet LJ, Blair HC, Zaidi M, Abe E (2003) Impaired osteoblastic differentiation, reduced bone formation, and severe osteoporosis in noggin-overexpressing mice. J Clin Invest 112(6):924–934. https://doi.org/10.1172/JCI15543\nCharles-Harris M, Koch MA, Navarro M, Lacroix D, Engel E, Planell JA (2008) A PLA/calcium phosphate degradable composite material for bone tissue engineering: an in vitro study. J Mater Sci Mater Med 19(4):1503–1513. https://doi.org/10.1007/s10856-008-3390-9\nZhang P, Liu Y, Wang Y, Zhang M, Lv L, Zhang X, Zhou Y (2017) SIRT6 promotes osteogenic differentiation of mesenchymal stem cells through BMP signaling. Sci Rep 7(1):10229. https://doi.org/10.1038/s41598-017-10323-z\nOrtiz F, Acuna-Castroviejo D, Doerrier C, Dayoub JC, Lopez LC, Venegas C, Garcia JA, Lopez A, Volt H, Luna-Sanchez M, Escames G (2015) Melatonin blunts the mitochondrial/NLRP3 connection and protects against radiation-induced oral mucositis. J Pineal Res 58(1):34–49. https://doi.org/10.1111/jpi.12191\nDong Y, Fan C, Hu W, Jiang S, Ma Z, Yan X, Deng C, Di S, Xin Z, Wu G, Yang Y, Reiter RJ, Liang G (2016) Melatonin attenuated early brain injury induced by subarachnoid hemorrhage via regulating NLRP3 inflammasome and apoptosis signaling. J Pineal Res 60(3):253–262. https://doi.org/10.1111/jpi.12300\nZhang Y, Li X, Grailer JJ, Wang N, Wang M, Yao J, Zhong R, Gao GF, Ward PA, Tan DX, Li X (2016) Melatonin alleviates acute lung injury through inhibiting the NLRP3 inflammasome. J Pineal Res 60(4):405–414. https://doi.org/10.1111/jpi.12322\nSon JH, Cho YC, Sung IY, Kim IR, Park BS, Kim YD (2014) Melatonin promotes osteoblast differentiation and mineralization of MC3T3-E1 cells under hypoxic conditions through activation of PKD/p38 pathways. J Pineal Res 57(4):385–392. https://doi.org/10.1111/jpi.12177\nFoo C, Frey S, Yang HH, Zellweger R, Filgueira L (2007) Downregulation of beta-catenin and transdifferentiation of human osteoblasts to adipocytes under estrogen deficiency. Gynecol Endocrinol 23(9):535–540. https://doi.org/10.1080/09513590701556483\nPing Z, Hu X, Wang L, Shi J, Tao Y, Wu X, Hou Z, Guo X, Zhang W, Yang H, Xu Y, Wang Z, Geng D (2017) Melatonin attenuates titanium particle-induced osteolysis via activation of Wnt/beta-catenin signaling pathway. Acta Biomater 51:513–525. https://doi.org/10.1016/j.actbio.2017.01.034\nTakahashi-Yanaga F, Sasaguri T (2009) Drug development targeting the glycogen synthase kinase-3beta (GSK-3beta)-mediated signal transduction pathway: inhibitors of the Wnt/beta-catenin signaling pathway as novel anticancer drugs. J Pharmacol Sci 109(2):179–183\nLadizesky MG, Boggio V, Cutrera RA, Mondelo N, Mastaglia S, Somoza J, Cardinali DP (2006) Melatonin effect on bone metabolism in rats treated with methylprednisolone. J Pineal Res 40(4):297–304. https://doi.org/10.1111/j.1600-079X.2006.00316.x\nBonar SL, Brydges SD, Mueller JL, McGeough MD, Pena C, Chen D, Grimston SK, Hickman-Brecks CL, Ravindran S, McAlinden A, Novack DV, Kastner DL, Civitelli R, Hoffman HM, Mbalaviele G (2012) Constitutively activated NLRP3 inflammasome causes inflammation and abnormal skeletal development in mice. PLoS ONE 7(4):e35979. https://doi.org/10.1371/journal.pone.0035979\nQi M, Zhang L, Ma Y, Shuai Y, Li L, Luo K, Liu W, Jin Y (2017) Autophagy maintains the function of bone marrow mesenchymal stem cells to prevent Estrogen deficiency-induced osteoporosis. Theranostics 7(18):4498–4516. https://doi.org/10.7150/thno.17949\nRhee YH, Ahn JC (2016) Melatonin attenuated adipogenesis through reduction of the CCAAT/enhancer binding protein beta by regulating the glycogen synthase 3 beta in human mesenchymal stem cells. J Physiol Biochem 72(2):145–155. https://doi.org/10.1007/s13105-015-0463-3\nAuthor information\nAuthors and Affiliations\nCorresponding author\nEthics declarations\nConflict of interest\nLijun Xu, Lixia Zhang, Zhifang Wang, Chong Li, Shan Li, Li Li, Qianying Fan, and Lili Zheng declares that they have no conflict of interest.\nEthical Approval\nAll applicable international, national, and/or institutional guidelines for the care and use of animals were followed.\nHuman and Animal Rights and Informed Consent\nThis study was ethically approved by the Institutional Animal Care and Use Committee of The First Affiliated Hospital of Zhengzhou University and performed in accordance with the criteria defined by the rules of the committee.\nRights and permissions\nAbout this article\nCite this article\nXu, L., Zhang, L., Wang, Z. et al. Melatonin Suppresses Estrogen Deficiency-Induced Osteoporosis and Promotes Osteoblastogenesis by Inactivating the NLRP3 Inflammasome. Calcif Tissue Int 103, 400–410 (2018). https://doi.org/10.1007/s00223-018-0428-y\nReceived:\nAccepted:\nPublished:\nVersion of record:\nIssue date:\nDOI: https://doi.org/10.1007/s00223-018-0428-y","source_license":"CC0","license_restricted":false}