Nrf2 epigenetic derepression induced by running exercise protects against osteoporosis

Abstract

Osteoporosis (OP) is a common skeletal disease involving low bone mineral density (BMD) that often leads to fragility fracture, and its development is affected by multiple cellular pathologies and associated with marked epigenetic alterations of osteogenic genes. Proper physical exercise is beneficial for bone health and OP and reportedly possesses epigenetic modulating capacities; however, whether the protective effects of exercise on OP involve epigenetic mechanisms is unclear. Here, we report that epigenetic derepression of nuclear factor erythroid derived 2-related factor-2 (Nrf2), a master regulator of oxidative stress critically involved in the pathogenesis of OP, mediates the significant osteoprotective effects of running exercise (RE) in a mouse model of OP induced by ovariectomy. We showed that Nrf2 gene knockout (Nfe2l2^−/−) ovariectomized mice displayed a worse BMD reduction than the controls, identifying Nrf2 as a critical antiosteoporotic factor. Further, femoral Nrf2 was markedly repressed with concomitant DNA methyltransferase (Dnmt) 1/Dnmt3a/Dnmt3b elevations and Nrf2 promoter hypermethylation in both patients with OP and ovariectomized mice. However, daily 1-h treadmill RE significantly corrected epigenetic alterations, recovered Nrf2 loss and improved the femur bone mass and trabecular microstructure. Consistently, RE also normalized the adverse expression of major osteogenic factors, including osteoblast/osteoclast markers, Nrf2 downstream antioxidant enzymes and proinflammatory cytokines. More importantly, the RE-conferred osteoprotective effects observed in the wild-type control mice were largely abolished in the Nfe2l2^{−/ −} mice. Thus, Nrf2 repression due to aberrant Dnmt elevation and subsequent Nrf2 promoter hypermethylation is likely an important epigenetic feature of the pathogenesis of OP, and Nrf2 derepression is essential for the antiosteoporotic effects of RE.

References

[1] Marcu FL, Bogdan FL, Muţiu G, Lazăr L. The histopathological study of osteoporosis. Rom. J. Morphol. Embryol., 2011, 52:321-325

[2] Rouf RSB, Qureshi NK, Afsana F, Ferdous HS. Osteoporosis: a review. BIRDEM Med. J., 2016, 5:30-36

[3] Peter Vrtačnik JM, Ostan Barbara. Epigenetic mechanisms in bone. Clin. Chem. Lab. Med., 2014, 52:589-608

[4] Francesca M, Cianferotti L, Brandi ML. Epigenetic mechanisms in bone biology and osteoporosis: can they drive therapeutic choices? Int J. Mol. Sci., 2016, 17:1329

[5] Letarouilly JG, Broux O, Clabaut A. New insights into the epigenetics of osteoporosis. Genomics, 2019, 111:793-798

[6] Reppe S et al. Distinct DNA methylation profiles in bone and blood of osteoporotic and healthy postmenopausal women. Epigenetics, 2017, 12:674-687

[7] Moore LD, Le T, Fan G. DNA methylation and its basic function. Neuropsychopharmacology, 2012, 38:23-38

[8] Agrawal K, Das V, Vyas P, Hajdúch M. Nucleosidic DNA demethylating epigenetic drugs – A comprehensive review from discovery to clinic. Pharmacol. Therapeutics., 2018, 188:45-79

[9] Guan H et al. Decitabine represses osteoclastogenesis through inhibition of RANK and NF-κB. Cell. Signal., 2015, 27:969-977

[10] Martyn-St James M, Carroll S. A meta-analysis of impact exercise on postmenopausal bone loss: the case for mixed loading exercise programmes. Br. J. Sports Med., 2008, 43:898-908

[11] Kashimoto RK et al. Physical exercise affects the epigenetic programming of rat brain and modulates the adaptive response evoked by repeated restraint stress. Behav. Brain Res., 2016, 296:286-289

[12] Abel JL, Rissman EF. Running-induced epigenetic and gene expression changes in the adolescent brain. Int. J. Dev. Neurosci., 2013, 31:382-390

[13] Barrès R et al. Acute exercise remodels promoter methylation in human skeletal muscle. Cell Metab., 2012, 15:405-411

[14] Lindholm ME et al. An integrative analysis reveals coordinated reprogramming of the epigenome and the transcriptome in human skeletal muscle after training. Epigenetics, 2014, 9:1557-1569

[15] Greally JM et al. A six months exercise intervention influences the genome-wide DNA methylation pattern in human adipose tissue. PLoS Genet., 2013, 9

[16] Ferioli M et al. Role of physical exercise in the regulation of epigenetic mechanisms in inflammation, cancer, neurodegenerative diseases, and aging process. J. Cell. Physiol., 2019, 234:14852-14864

[17] Benedetti MG, Furlini G, Zati A, Mauro GL. The effectiveness of physical exercise on bone density in osteoporotic patients. Biomed. Res. Int., 2018, 2018:4840531

[18] Howe TE et al. Exercise for preventing and treating osteoporosis in postmenopausal women. Cochrane Database Syst. Rev., 2011, 7:CD000333

[19] Bai XC et al. Oxidative stress inhibits osteoblastic differentiation of bone cells by ERK and NF-κB. Biochem. Biophys. Res. Commun., 2004, 314:197-207

[20] Nojiri H et al. Cytoplasmic superoxide causes bone fragility owing to low-turnover osteoporosis and impaired collagen cross-linking. J. Bone Miner. Res., 2011, 26:2682-2694

[21] Park CK et al. Nrf2 is a novel regulator of bone acquisition. Bone, 2014, 63:36-46

[22] Kansanen E, Kuosmanen SM, Leinonen H, Levonen AL. The Keap1-Nrf2 pathway: mechanisms of activation and dysregulation in cancer. Redox Biol., 2013, 1:45-49

[23] Ibáñez L et al. Effects of Nrf2 deficiency on bone microarchitecture in an experimental model of osteoporosis. Oxid. Med. Cell. Longev., 2014, 2014:1-9

[24] Menale C et al. Absence of dipeptidyl peptidase 3 increases oxidative stress and causes bone loss. J. Bone Miner. Res., 2019, 34:2133-2148

[25] Sun X et al. Octyl itaconate inhibits osteoclastogenesis by suppressing Hrd1 and activating Nrf2 signaling. FASEB J., 2019, 33:12929-12940

[26] Sun X et al. The Nrf2 activator RTA-408 attenuates osteoclastogenesis by inhibiting STING dependent NF-κb signaling. Redox Biol., 2020, 28:101309

[27] Cao H., et al. DNA Demethylation upregulated Nrf2 expression in Alzheimer’s disease cellular model. Front. Aging Neurosci. 7, 244 (2016).

[28] Guo Y, Yu S, Zhang C, Kong AN. Epigenetic regulation of Keap1-Nrf2 signaling. Free Radic. Biol. Med., 2015, 88:337-349

[29] Yu B et al. PGC-1α controls skeletal stem cell fate and bone-fat balance in osteoporosis and skeletal aging by inducing TAZ. Cell Stem Cell, 2018, 23:193-209.e5

[30] Aspera-Werz RH et al. Nicotine and cotinine inhibit catalase and glutathione reductase activity contributing to the impaired osteogenesis of SCP-1 cells exposed to cigarette smoke. Oxid. Med. Cell. Longev., 2018, 2018:1-13

[31] Lee YJ et al. The association between oxidative stress and bone mineral density according to menopausal status of Korean women. Obstet. Gynecol. Sci., 2015, 58:46-52

[32] Benedetti MG, Furlini G, Zati A, Letizia Mauro G. The effectiveness of physical exercise on bone density in osteoporotic patients. BioMed. Res. Int., 2018, 2018:1-10

[33] Santos L, Elliott-Sale KJ, Sale C. Exercise and bone health across the lifespan. Biogerontology, 2017, 18:931-946

[34] Delgado-Calle J et al. Epigenetic regulation of alkaline phosphatase in human cells of the osteoblastic lineage. Bone, 2011, 49:830-838

[35] Reppe S et al. Methylation of BoneSOST, its mRNA, and serum sclerostin levels correlate strongly with fracture risk in postmenopausal women. J. Bone Miner. Res., 2015, 30:249-256

[36] Delgado-Calle J et al. Role of DNA methylation in the regulation of the RANKL-OPG system in human bone. Epigenetics, 2014, 7:83-91

[37] Kitazawa R, Kitazawa S. Methylation status of a single CpG locus 3 bases upstream of TATA-Box of receptor activator of nuclear factor-κB ligand (RANKL) gene promoter modulates cell- and tissue-specific RANKL expression and osteoclastogenesis. Mol. Endocrinol., 2007, 21:148-158

[38] García-Ibarbia C et al. Contribution of genetic and epigenetic mechanisms to Wnt pathway activity in prevalent skeletal disorders. Gene, 2013, 532:165-172

[39] Sun Y. X., Xu A. H., Yang Y., Li J. Role of Nrf2 in bone metabolism. J. Biomed. Sci. 22, 101 (2015).

[40] Hyeon S, Lee H, Yang Y, Jeong W. Nrf2 deficiency induces oxidative stress and promotes RANKL-induced osteoclast differentiation. Free Radic. Biol. Med., 2013, 65:789-799

[41] Manolagas SC. From estrogen-centric to aging and oxidative stress: a revised perspective of the pathogenesis of osteoporosis. Endocr. Rev., 2010, 31:266-300

[42] Bellezza I, Giambanco I, Minelli A, Donato R. Nrf2-Keap1 signaling in oxidative and reductive stress. Biochim. Biophys. Acta. Mol. Cell Res, 2018, 1865:721-733

[43] Fonseca H et al. Voluntary exercise has long-term in vivo protective effects on osteocyte viability and bone strength following ovariectomy. Calcif. Tissue Int., 2011, 88:443-454

[44] Mann V, Huber C, Kogianni G, Jones D, Noble B. The influence of mechanical stimulation on osteocyte apoptosis and bone viability in human trabecular bone. J. Musculoskelet. Neuronal Interact., 2006, 6:408-417

[45] Pellegrini G, Morales C, Wallace T, Plotkin L, Bellido T. Avenanthramides prevent osteoblast and osteocyte apoptosis and induce osteoclast apoptosis in vitro in an Nrf2-independent manner. Nutrients, 2016, 8:423

[46] Tao J et al. Downregulation of Nrf2 promotes autophagy-dependent osteoblastic differentiation of adipose-derived mesenchymal stem cells. Exp. Cell Res., 2016, 349:221-229

[47] Li B et al. Overexpression of DNMT1 leads to hypermethylation of H19 promoter and inhibition of Erk signaling pathway in disuse osteoporosis. Bone, 2018, 111:82-91

[48] Wang C et al. Mechanical stimulation promote the osteogenic differentiation of bone marrow stromal cells through epigenetic regulation of Sonic Hedgehog. Exp. Cell Res., 2017, 352:346-356

[49] Nishikawa K et al. DNA methyltransferase 3a regulates osteoclast differentiation by coupling to an S-adenosylmethionine–producing metabolic pathway. Nat. Med., 2015, 21:281-287

[50] Cai D, Yin S, Yang J, Jiang Q, Cao W. Histone deacetylase inhibition activates Nrf2 and protects against osteoarthritis. Arthritis Res. Ther., 2015, 17:269

[51] Cosman F et al. Clinician’s guide to prevention and treatment of osteoporosis. Osteoporos. Int., 2014, 25:2359-2381

[52] Zhang Q., Yin S., Liu L., Liu Z., Cao W. Rhein reversal of DNA hypermethylation-associated Klotho suppression ameliorates renal fibrosis in mice. Sci. Rep. 6, 34597 (2016).

[53] Li LC, Dahiya R. MethPrimer designing primers for methylation PCRs. Bioinformatics, 2002, 18:1427-1431

[54] Zhang Q et al. Rhein reverses Klotho repression via promoter demethylation and protects against kidney and bone injuries in mice with chronic kidney disease. Kidney Int., 2017, 91:144-156

[55] Zhu X, Chen F, Lu K, Wei A, Jiang Q, Cao W. PPARγ preservation via promoter demethylation alleviates osteoarthritis in mice. Ann. Rheum. Dis., 2019, 78:1420-1429

Funding

National Nature and Science Foundation of China (81670762 and 81970577)

National Nature and Science Foundation of China (81730067 and 81991514)