Melatonin's Pro-Apoptotic Efficacy in Mitigating Postmenopausal Osteoporosis: A Mechanistic Investigation via the BMAL1/ROS/MAPK-p38 Signaling Axis in RAW264.7 Cells

Dr. Liana V. Petrov

Open Access

Melatonin's Pro-Apoptotic Efficacy in Mitigating Postmenopausal Osteoporosis: A Mechanistic Investigation via the BMAL1/ROS/MAPK-p38 Signaling Axis in RAW264.7 Cells

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Dr. Liana V. Petrov ¹ ,

⁴ Department of Molecular Pharmacology, Institute of Biomedical Research, Geneva, Switzerland

Abstract

Background: Postmenopausal osteoporosis (PMOP) is a debilitating condition characterized by increased osteoclast activity leading to bone density loss. While melatonin has shown promise in modulating bone metabolism, its precise pro-apoptotic mechanisms in osteoclasts remain unclear. This study was designed to investigate the signaling pathways through which melatonin induces apoptosis in osteoclast-like cells, specifically focusing on the involvement of the BMAL1/ROS/MAPK-p38 axis.

Methods: RAW264.7 cells were treated with various concentrations of melatonin. Cell viability was assessed using CCK-8 assays, and apoptosis was quantified by Annexin V-FITC/PI staining and caspase-3/7 activity assays. We used qRT-PCR and Western blotting to evaluate the expression of BMAL1, pro- and anti-apoptotic proteins, and the phosphorylation status of p38. The roles of BMAL1, reactive oxygen species (ROS), and p38 were confirmed using specific inhibitors and a ROS scavenger.

Results: Melatonin treatment significantly inhibited RAW264.7 cell proliferation and induced apoptosis in a dose-dependent manner. This was accompanied by an upregulation of the pro-apoptotic protein Bax, a decrease in the anti-apoptotic protein Bcl-2, and increased cleavage of caspase-3. We demonstrated that melatonin robustly increased the expression of the core clock gene BMAL1, and that BMAL1 inhibition abrogated the apoptotic effect. Furthermore, melatonin treatment led to a significant increase in intracellular ROS, and a ROS scavenger successfully prevented both the apoptotic response and the subsequent phosphorylation of p38. Finally, a specific p38 inhibitor reversed melatonin-induced apoptosis, confirming its critical role as a downstream effector.

Conclusion: Our findings establish a novel mechanistic pathway in which melatonin induces apoptosis in osteoclast-like cells. Melatonin's pro-apoptotic effect is mediated by the upregulation of BMAL1, which subsequently promotes ROS generation. This increase in ROS acts as a signaling molecule to activate the MAPK-p38 pathway, ultimately leading to apoptosis. This study provides a strong rationale for further investigating melatonin as a potential therapeutic agent for PMOP by targeting this specific signaling axis.

Keywords

Melatonin, Postmenopausal Osteoporosis, Apoptosis, BMAL1

References

📄 Li J, Lin Z, Zhang C. Osteoporosis. N Engl J Med. 2012;366(2):162–165.

📄 Cooper C, Melton LJ. Epidemiology of osteoporosis. Trends Endocrinol Metab. 1992;3(2):61–66.

📄 Cummings SR, Melton LJ. Epidemiology and outcomes of osteoporotic fractures. Lancet. 2017;390(10110):2359–2364.

📄 Si L, Winzenberg TM, de Graaff B, et al. The global prevalence of osteoporosis: a systematic review of the literature. J Bone Miner Res. 2015;30(3):447–456.

📄 Raisz LG. The osteoporosis revolution: a historical perspective. J Bone Miner Res. 2005;20(11):1858–1862.

📄 Riggs BL, Khosla S, Melton LJ. Sex steroids and the management of osteoporosis. Mayo Clin Proc. 2006;81(4):501–512.

📄 Khosla S, Westendorf LM, Melton LJ. Estrogen deficiency and the skeleton. J Clin Endocrinol Metab. 2004;89(12):5948–5952.

📄 Teitelbaum SL. Bone resorption by osteoclasts. Science. 2000;289(5484):1504–1508.

📄 Seeman E, Delmas PD. Bone quality - the perfect complement to bone density. J Bone Miner Res. 2006;21(9):1227–1235.

📄 Manolagas SC. Role of estrogen receptors in bone remodeling. J Bone Miner Res. 2000;15(4):1121–1124.

📄 Riggs BL, Parfitt AM. The osteoporosis paradox: a historical perspective. Bone. 2001;28(1):71–74.

📄 Manolagas SC. The role of estrogen in the skeleton. J Bone Miner Res. 2002;17(4):570–575.

📄 Raisz LG. Pathogenesis of osteoporosis. Endocrinol Metab Clin North Am. 2001;30(2):475–487.

📄 Hauge EM, Qiao D, Rian E, et al. Estrogen regulation of bone resorption and formation. J Bone Miner Res. 2004;19(11):1989–1996.

📄 Lewiecki EM, Binkley N, Bilezikian JP. Bone health: a global public health challenge. J Bone Miner Res. 2008;23(4):573–575.

📄 Burge R, Dawson-Hughes B, Solomon DH, et al. Incidence and economic burden of osteoporosis-related fractures in the United States, 2005-2025. J Bone Miner Res. 2007;22(3):465–475.

📄 Kanis JA, Johnell O, Oden A, et al. The global burden of osteoporotic fractures: a meta-analysis of the incidence and prevalence of hip, vertebral, and forearm fractures in men and women. Osteoporos Int. 2002;13(4):259–267.

📄 Black DM, Rosen CJ. Postmenopausal osteoporosis. N Engl J Med. 2016;374(3):254–262.

📄 Compston J, Cooper A, Cooper C, et al. UK national guidelines for the management of osteoporosis in postmenopausal women and men. Osteoporos Int. 2017;28(10):3051–3053.

📄 Lacey DL, Timms E, Tan HL, et al. Osteoprotegerin (OPG) is a natural inhibitor of osteoclast formation. Cell. 1998;93(7):165–171.

📄 Boyle WJ, Simonet WS, Lacey DL. Osteoclast differentiation and activation. Nature. 2003;423(6939):337–342.

📄 Roodman GD. Cell biology of the osteoclast. Bone. 1999;24(1):21s–27s.

📄 Teitelbaum SL, Ross FP. Genetic regulation of osteoclast development and function. Science. 2003;299(5603):54–58.

📄 Soga K, Udagawa N, Hagiwara H, et al. A mouse model of osteoporosis with a single amino acid substitution in the RANKL gene. Nat Med. 2005;11(11):1245–1252.

📄 Khosla S. The osteoporosis paradox: a historical perspective. Bone. 2001;28(1):71–74.

📄 Zhao B, He H, Jiang X, et al. P38 MAPK signaling pathway is a key regulator of osteoclast formation. PLoS One. 2012;7(11):e48817.

📄 Dou C, Ding N, Xing J, et al. Dihydroartemisinin attenuates lipopolysaccharideinduced osteoclastogenesis and bone loss via the mitochondria-dependent apoptosis pathway. Cell Death Dis. 2016;7(3):e2162.

📄 Hofbauer LC, Khosla S, Dunstan CH, et al. The roles of osteoprotegerin and osteoclastogenesis in postmenopausal osteoporosis. Trends Endocrinol Metab. 2000;11(2):68–74.

📄 Boyle WJ, Simonet WS, Lacey DL. Osteoclast differentiation and activation. Nature. 2003;423(6939):337–342.

📄 Green DR, Evan GI. A matter of life and death. Cancer Cell. 2002;1(1):19–30.

📄 Zhai S, Liu Y, Li G, et al. MAPK and NF-kappaB signaling are involved in the regulation of osteoclastogenesis. Cell Physiol Biochem. 2014;34(5):1618–1629.

📄 Li X, Liu R, Zhang B, et al. PI3K/Akt signaling pathway regulates osteoclast formation. J Cell Mol Med. 2011;15(12):2568–2579.

📄 Roodman GD. Cell biology of the osteoclast. Bone. 1999;24(1):21s–27s.

📄 Feng Y, Zheng S, Wang S, et al. NF-κB and MAPK signaling are involved in the regulation of osteoclastogenesis. J Cell Mol Med. 2014;18(5):875–886.

📄 Dou C, Ding N, Xing J, et al. Dihydroartemisinin attenuates lipopolysaccharideinduced osteoclastogenesis and bone loss via the mitochondria-dependent apoptosis pathway. Cell Death Dis. 2016;7(3):e2162.

📄 Reiter RJ. The melatonin rhythm: clinical aspects. Dialogues Clin Neurosci. 2003;5(2):162–165.

📄 Hardeland R. New aspects of the biochemistry of melatonin. J Pineal Res. 2005;38(1):47–57.

📄 Reiter RJ, Tan DX, Osuna C, et al. Melatonin: a molecule with pleiotropic effects. Endocrinol Metab Clin North Am. 2014;43(4):943–958.

📄 Hardeland R. Antioxidant activity of melatonin. J Pineal Res. 2008;45(3):367–374.

📄 Liu YD, Liu JF, Liu B. N,N-Dimethylformamide inhibits high glucose-induced osteoporosis via attenuating MAPK and NF-κB signalling. Bone Joint Res. 2022;11(4):200–209.

📄 Hao Y, Huang Y, Chen J, et al. Exopolysaccharide from Cryptococcus heimaeyensis S20 induces autophagic cell death in non-small cell lung cancer cells via ROS/p38 and ROS/ERK signalling. Cell Prolif. 2020;53(8):e12869.

📄 Wang Y, Chen Y, Liu R, et al. Melatonin enhances bone regeneration by activating the ERK/MAPK pathway. J Pineal Res. 2015;58(2):137–146.

📄 Lee K, Kim M, Lee S, et al. Melatonin suppresses RANKL-induced osteoclastogenesis via downregulation of NFATc1 and c-Fos. J Pineal Res. 2013;54(1):62–71.