In diabetics, methylglyoxal (MG), a glucose-derived metabolite, plays a noxious role by inducing oxidative stress, which causes and exacerbates a series of complications including low-turnover osteoporosis. In the present study, while MG treatment of mouse bone marrow stroma-derived ST2 cells rapidly suppressed the expression of osteotrophic Wnt-targeted genes, including that of osteoprotegerin (OPG, a decoy receptor of the receptor activator of NF-kappaB ligand (RANKL)), it significantly enhanced that of secreted Frizzled-related protein 4 (sFRP-4, a soluble inhibitor of Wnts). On the assumption that upregulated sFRP-4 is a trigger that downregulates Wnt-related genes, we sought out the molecular mechanism whereby oxidative stress enhanced the sFRP-4 gene. Sodium bisulfite sequencing revealed that the sFRP-4 gene was highly methylated around the sFRP-4 gene basic promoter region, but was not altered by MG treatment. Electrophoretic gel motility shift assay showed that two continuous CpG loci located five bases upstream of the TATA-box were, when methylated, a target of methyl CpG binding protein 2 (MeCP2) that was sequestered upon induction of 8-hydroxy-2-deoxyguanosine, a biomarker of oxidative damage to DNA. These in vitro data suggest that MG-derived oxidative stress (not CpG demethylation) epigenetically and rapidly derepress sFRP-4 gene expression. We speculate that under persistent oxidative stress, as in diabetes and during aging, osteopenia and ultimately low-turnover osteoporosis become evident partly due to osteoblastic inactivation by suppressed Wnt signaling of mainly canonical pathways through the derepression of sFRP-4 gene expression.
References
[1]
Brownlee M (2001) Biochemistry and molecular cell biology of diabetic complications. Nature 414: 813–820. doi: 10.1038/414813a
[2]
Schmidt AM, Yan SD, Wautier JL, Stern D (1999) Activation of receptor for advanced glycation end products: a mechanism for chronic vascular dysfunction in diabetic vasculopathy and atherosclerosis. Circ Res 84: 489–497. doi: 10.1161/01.res.84.5.489
[3]
Shinohara M, Thornalley PJ, Giardino I, Beisswenger P, Thorpe SR, et al. (1998) Overexpression of glyoxalase-I in bovine endothelial cells inhibits intracellular advanced glycation endproduct formation and prevents hyperglycemia-induced increases in macromolecular endocytosis. J Clin Invest 101: 1142–1147. doi: 10.1172/jci119885
[4]
Han Y, Randell E, Vasdev S, Gill V, Gadag V, et al. (2007) Plasma methylglyoxal and glyoxal are elevated and related to early membrane alteration in young, complication-free patients with Type 1 diabetes. Mol Cell Biochem 305: 123–131. doi: 10.1007/s11010-007-9535-1
[5]
Agalou S, Ahmed N, Dawnay A, Thornalley PJ (2003) Removal of advanced glycation end products in clinical renal failure by peritoneal dialysis and haemodialysis. Biochem Soc Trans 31: 1394–1396. doi: 10.1042/bst0311394
[6]
Akhand AA, Hossain K, Kato M, Miyata T, Du J, et al. (2001) Glyoxal and methylglyoxal induce lyoxal and methyglyoxal induce aggregation and inactivation of ERK in human endothelial cells. Free Radic Biol Med 31: 1228–1235. doi: 10.1016/s0891-5849(01)00702-x
[7]
Hamada Y, Fujii H, Kitazawa R, Yodoi J, Kitazawa S, et al. (2009) Thioredoxin-1 overexpression in transgenic mice attenuates streptozotocin-induced diabetic osteopenia: a novel role of oxidative stress and therapeutic implications. Bone 44: 936–941. doi: 10.1016/j.bone.2008.12.011
[8]
Almeida M, Han L, Martin-Millan M, O’Brien CA, Manolagas SC (2007) Oxidative stress antagonizes Wnt signaling in osteoblast precursors by diverting beta-catenin from T cell factor- to forkhead box O-mediated transcription. J Biol Chem 282: 27298–27305. doi: 10.1074/jbc.m702811200
[9]
Hamada Y, Kitazawa S, Kitazawa R, Fujii H, Kasuga M, et al. (2007) Histomorphometric analysis of diabetic osteopenia in streptozotocin-induced diabetic mice: a possible role of oxidative stress. Bone 40: 1408–1414. doi: 10.1016/j.bone.2006.12.057
[10]
Chen JR, Lazarenko OP, Shankar K, Blackburn ML, Badger TM, et al. (2010) A role for ethanol-induced oxidative stress in controlling lineage commitment of mesenchymal stromal cells through inhibition of Wnt/beta-catenin signaling. J Bone Miner Res 25: 1117–1127. doi: 10.1002/jbmr.7
[11]
Gordon MD, Nusse R (2006) Wnt signaling: multiple pathways, multiple receptors, and multiple transcription factors. J Biol Chem 281: 22429–22433. doi: 10.1074/jbc.r600015200
[12]
Kitazawa S, Kitazawa R, Maeda S (1999) Transcriptional regulation of rat cyclin D1 gene by CpG methylation status in promoter region. J Biol Chem 274: 28787–28793. doi: 10.1074/jbc.274.40.28787
[13]
Kani S, Oishi I, Yamamoto H, Yoda A, Suzuki H, et al. (2004) The receptor tyrosine kinase Ror2 associates with and is activated by casein kinase Iepsilon. J Biol Chem 279: 50102–50109. doi: 10.1074/jbc.m409039200
[14]
Matsuda T, Nomi M, Ikeya M, Kani S, Oishi I, et al. (2001) Expression of the receptor tyrosine kinase genes, Ror1 and Ror2, during mouse development. Mech Dev 105: 153–156. doi: 10.1016/s0925-4773(01)00383-5
[15]
Klose RJ, Sarraf SA, Schmiedeberg L, McDermott SM, Stancheva I, et al. (2005) DNA binding selectivity of MeCP2 due to a requirement for A/T sequences adjacent to methyl-CpG. Mol Cell 19: 667–678. doi: 10.1016/j.molcel.2005.07.021
[16]
Ducy P, Schinke T, Karsenty G (2000) The osteoblast: a sophisticated fibroblast under central surveillance. Science 289: 1501–1504. doi: 10.1126/science.289.5484.1501
[17]
Teitelbaum SL (2000) Bone resorption by osteoclasts. Science 289: 1504–1508. doi: 10.1126/science.289.5484.1504
[18]
Yamaguchi A, Komori T, Suda T (2000) Regulation of osteoblast differentiation mediated by bone morphogenetic proteins, hedgehogs, and Cbfa1. Endocr Rev 21: 393–411. doi: 10.1210/edrv.21.4.0403
[19]
Lian JB, Stein GS, Javed A, van Wijnen AJ, Stein JL, et al. (2006) Networks and hubs for the transcriptional control of osteoblastogenesis. Rev Endocr Metab Disord 7: 1–16. doi: 10.1007/s11154-006-9001-5
[20]
Nishimura R, Hata K, Ikeda F, Ichida F, Shimoyama A, et al. (2008) Signal transduction and transcriptional regulation during mesenchymal cell differentiation. J Bone Miner Metab 26: 203–212. doi: 10.1007/s00774-007-0824-2
[21]
Gong Y, Slee RB, Fukai N, Rawadi G, Roman-Roman S, et al. (2001) LDL receptor-related protein 5 (LRP5) affects bone accrual and eye development. Cell 107: 513–523. doi: 10.1016/s0092-8674(01)00571-2
[22]
Van Wesenbeeck L, Cleiren E, Gram J, Beals RK, Benichou O, et al. (2003) Six novel missense mutations in the LDL receptor-related protein 5 (LRP5) gene in different conditions with an increased bone density. Am J Hum Genet 72: 763–771. doi: 10.1086/368277
[23]
Little RD, Carulli JP, Del Mastro RG, Dupuis J, Osborne M, et al. (2002) A mutation in the LDL receptor-related protein 5 gene results in the autosomal dominant high-bone-mass trait. Am J Hum Genet 70: 11–19. doi: 10.1086/338450
[24]
Boyden LM, Mao J, Belsky J, Mitzner L, Farhi A, et al. (2002) High bone density due to a mutation in LDL-receptor-related protein 5. N Engl J Med 346: 1513–1521. doi: 10.1056/nejmoa013444
[25]
Babij P, Zhao W, Small C, Kharode Y, Yaworsky PJ, et al. (2003) High bone mass in mice expressing a mutant LRP5 gene. J Bone Miner Res 18: 960–974. doi: 10.1359/jbmr.2003.18.6.960
[26]
Ambrogini E, Almeida M, Martin-Millan M, Paik JH, Depinho RA, et al. (2010) FoxO-mediated defense against oxidative stress in osteoblasts is indispensable for skeletal homeostasis in mice. Cell Metab 11: 136–146. doi: 10.1016/j.cmet.2009.12.009
[27]
Fatokun AA, Stone TW, Smith RA (2006) Hydrogen peroxide-induced oxidative stress in MC3T3-E1 cells: The effects of glutamate and protection by purines. Bone 39: 542–551. doi: 10.1016/j.bone.2006.02.062
[28]
Fujii H, Hamada Y, Fukagawa M (2008) Bone formation in spontaneously diabetic Torii-newly established model of non-obese type 2 diabetes rats. Bone 42: 372–379. doi: 10.1016/j.bone.2007.10.007
[29]
Grassi F, Tell G, Robbie-Ryan M, Gao Y, Terauchi M, et al. (2007) Oxidative stress causes bone loss in estrogen-deficient mice through enhanced bone marrow dendritic cell activation. Proc Natl Acad Sci U S A 104: 15087–15092. doi: 10.1073/pnas.0703610104
[30]
Lean JM, Jagger CJ, Kirstein B, Fuller K, Chambers TJ (2005) Hydrogen peroxide is essential for estrogen-deficiency bone loss and osteoclast formation. Endocrinology 146: 728–735. doi: 10.1210/en.2004-1021
[31]
Kawano Y, Kypta R (2003) Secreted antagonists of the Wnt signalling pathway. J Cell Sci 116: 2627–2634. doi: 10.1242/jcs.00623
[32]
Qi J, Zhu YQ, Luo J, Tao WH (2006) Hypermethylation and expression regulation of secreted frizzled-related protein genes in colorectal tumor. World J Gastroenterol 12: 7113–7117.
[33]
Marsit CJ, McClean MD, Furniss CS, Kelsey KT (2006) Epigenetic inactivation of the SFRP genes is associated with drinking, smoking and HPV in head and neck squamous cell carcinoma. Int J Cancer 119: 1761–1766. doi: 10.1002/ijc.22051
[34]
Takagi H, Sasaki S, Suzuki H, Toyota M, Maruyama R, et al. (2008) Frequent epigenetic inactivation of SFRP genes in hepatocellular carcinoma. J Gastroenterol 43: 378–389. doi: 10.1007/s00535-008-2170-0
[35]
Suzuki H, Gabrielson E, Chen W, Anbazhagan R, van Engeland M, et al. (2002) A genomic screen for genes upregulated by demethylation and histone deacetylase inhibition in human colorectal cancer. Nat Genet 31: 141–149. doi: 10.1038/ng892
[36]
Kitazawa R, Kitazawa S (2007) Methylation status of a single CpG locus 3 bases upstream of TATA-box of receptor activator of nuclear factor-kappaB ligand (RANKL) gene promoter modulates cell- and tissue-specific RANKL expression and osteoclastogenesis. Mol Endocrinol 21: 148–158. doi: 10.1210/me.2006-0205
[37]
Bocker MT, Tuorto F, Raddatz G, Musch T, Yang FC, et al. (2012) Hydroxylation of 5-methylcytosine by TET2 maintains the active state of the mammalian HOXA cluster. Nat Commun 3: 818. doi: 10.1038/ncomms1826
[38]
Valinluck V, Tsai HH, Rogstad DK, Burdzy A, Bird A, et al. (2004) Oxidative damage to methyl-CpG sequences inhibits the binding of the methyl-CpG binding domain (MBD) of methyl-CpG binding protein 2 (MeCP2). Nucleic Acids Res 32: 4100–4108. doi: 10.1093/nar/gkh739
[39]
David SS, O’Shea VL, Kundu S (2007) Base-excision repair of oxidative DNA damage. Nature 447: 941–950. doi: 10.1038/nature05978
[40]
Hazra TK, Muller JG, Manuel RC, Burrows CJ, Lloyd RS, et al. (2001) Repair of hydantoins, one electron oxidation product of 8-oxoguanine, by DNA glycosylases of Escherichia coli. Nucleic Acids Res 29: 1967–1974. doi: 10.1093/nar/29.9.1967
[41]
Wong VK, Yam JW, Hsiao WL (2003) Cloning and characterization of the promoter region of the mouse frizzled-related protein 4 gene. Biol Chem 384: 1147–1154. doi: 10.1515/bc.2003.127
