Loss of Brd4 alleviates pathological bone loss via Slc9b2 suppression in osteoclastogenesis

Abstract

Background: Epigenetic regulation plays a crucial role in skeletal degenerative diseases, including osteoporosis. As an epigenetic reader, bromodomain protein 4 (Brd4) is known as a key driver of gene activation; however, its role in maintaining skeletal homeostasis remains largely unknown.

Methods: We examined Brd4 expression in bone specimens from osteoporotic patients and mouse models, and generated two types of Brd4 conditional knockout mice using Lyz2-Cre and Ctsk-Cre systems. Bone mass, osteoclast differentiation, and metabolic activity were assessed under physiological and pathological conditions, including ovariectomy and lipopolysaccharide (LPS) challenge. Mechanistic analyses were performed using transcriptomic screening, gene overexpression, and pharmacological interventions.

Results: Brd4 expression was markedly elevated in bones from osteoporotic patients and mice compared with normal controls. Deletion of Brd4 increased basal bone mass and prevented bone loss induced by ovariectomy or LPS, primarily by suppressing osteoclastogenesis through inhibition of glycolysis. Unbiased screening identified solute carrier family 9 member B2 (Slc9b2) as a downstream effector of Brd4. Overexpression of Slc9b2 partially rescued the impaired osteoclastogenesis caused by Brd4 depletion. Moreover, phosphatidylserine-containing nanoliposomes loaded with Brd4-targeting PROTACs (e.g., dBET6) effectively suppressed osteoclastogenesis and alleviated pathological bone loss.

Conclusions: Brd4 serves as a crucial regulator of osteoclast metabolism and differentiation. Targeting Brd4 represents a promising therapeutic strategy for the prevention and treatment of osteoporosis and pathological bone loss.

Keywords

Brd4 / osteoclastogenesis / osteoporosis / Slc9b2

References

[1] Zaidi M. Skeletal remodeling in health and disease. Nat Med. 2007; 13(7): 791-801.

[2] Rachner TD, Khosla S, Hofbauer LC. Osteoporosis: now and the future. Lancet (London, England). 2011; 377(9773): 1276-1287.

[3] Nakashima T, Hayashi M, Takayanagi H. New insights into osteoclastogenic signaling mechanisms. Trends Endocrinol Metab. 2012; 23(11): 582-590.

[4] Li B, Lee WC, Song C, Ye L, Abel ED, Long F. Both aerobic glycolysis and mitochondrial respiration are required for osteoclast differentiation. FASEB J. 2020; 34(8): 11058-11067.

[5] Kurotaki D, Yoshida H, Tamura T. Epigenetic and transcriptional regulation of osteoclast differentiation. Bone. 2020; 138: 115471.

[6] Yasui T, Hirose J, Aburatani H, Tanaka S. Epigenetic regulation of osteoclast differentiation. Ann N Y Acad Sci. 2011; 1240: 7-13.

[7] Chiang CM. Brd4 engagement from chromatin targeting to transcriptional regulation: selective contact with acetylated histone H3 and H4. F1000 Biol Rep. 2009; 1: 98.

[8] Sun J, Gui Y, Zhou S, Zheng XL. Unlocking the secrets of aging: epigenetic reader BRD4 as the target to combatting aging-related diseases. J Adv Res. 2023.

[9] Xu YD, Liang XC, Li ZP, et al. Apoptotic body-inspired nanotherapeutics efficiently attenuate osteoarthritis by targeting BRD4-regulated synovial macrophage polarization. Biomaterials. 2024; 306: 122483.

[10] Baud'huin M, Lamoureux F, Jacques C, et al. Inhibition of BET proteins and epigenetic signaling as a potential treatment for osteoporosis. Bone. 2017; 94: 10-21.

[11] Wang K, Zhao Z, Wang X, Zhang Y. BRD4 induces osteogenic differentiation of BMSCs via the Wnt/beta-catenin signaling pathway. Tissue Cell. 2021; 72: 101555.

[12] Najafova Z, Tirado-Magallanes R, Subramaniam M, et al. BRD4 localization to lineage-specific enhancers is associated with a distinct transcription factor repertoire. Nucleic Acids Res. 2017; 45(1): 127-141.

[13] Lamoureux F, Baud'huin M, Rodriguez Calleja L, et al. Selective inhibition of BET bromodomain epigenetic signalling interferes with the bone-associated tumour vicious cycle. Nat Commun. 2014; 5: 3511.

[14] Liu Y, Liu W, Yu Z, et al. A novel BRD4 inhibitor suppresses osteoclastogenesis and ovariectomized osteoporosis by blocking RANKL-mediated MAPK and NF-kappaB pathways. Cell Death Dis. 2021; 12(7): 654.

[15] Caputo VS, Trasanidis N, Xiao X, et al. Brd2/4 and Myc regulate alternative cell lineage programmes during early osteoclast differentiation in vitro. iScience. 2021; 24(1): 101989.

[16] Gregson CL, Armstrong DJ, Bowden J, et al. UK clinical guideline for the prevention and treatment of osteoporosis. Arch Osteoporos. 2022; 17(1): 58.

[17] Zhou X, Cornel EJ, Fan Z, He S, Du J. Bone-targeting polymer vesicles for effective therapy of osteoporosis. Nano Lett. 2021; 21(19): 7998-8007.

[18] Hoque J, Shih YV, Zeng Y, et al. Bone targeting nanocarrier-assisted delivery of adenosine to combat osteoporotic bone loss. Biomaterials. 2021; 273: 120819.

[19] Wen C, Xu X, Zhang Y, Xia J, Liang Y, Xu L. Bone targeting nanoparticles for the treatment of osteoporosis. Int J Nanomedicine. 2024; 19: 1363-1383.

[20] Li M, Zhang X, Ang KS, et al. DISCO: a database of deeply integrated human single-cell omics data. Nucleic Acids Res. 2022; 50(D1): D596-D602.

[21] Stuart T, Butler A, Hoffman P, et al. Comprehensive integration of single-cell data. Cell. 2019; 177(7): 1888-1902. e21.

[22] Korsunsky I, Millard N, Fan J, et al. Fast, sensitive and accurate integration of single-cell data with harmony. Nat Methods. 2019; 16(12): 1289-1296.

[23] Zhong L, Yao L, Tower RJ, et al. Single cell transcriptomics identifies a unique adipose lineage cell population that regulates bone marrow environment. Elife. 2020; 9: e54695.

[24] Yu W, Zhong L, Yao L, et al. Bone marrow adipogenic lineage precursors promote osteoclastogenesis in bone remodeling and pathologic bone loss. J Clin Invest. 2021; 131(2): e140214.

[25] van Dijk D, Sharma R, Nainys J, et al. Recovering gene interactions from single-cell data using data diffusion. Cell. 2018; 174(3): 716-729. e27.

[26] Zhang HT, Gui T, Liu RX, et al. Sequential targeting of YAP1 and p21 enhances the elimination of senescent cells induced by the BET inhibitor JQ1. Cell Death Dis. 2021; 12(1): 121.

[27] Zhang HT, Peng R, Chen S, et al. Versatile nano-PROTAC-induced epigenetic reader degradation for efficient lung cancer therapy. Adv Sci (Weinh). 2022; 9(29): e2202039.

[28] Liu RX, Gu RH, Li ZP, et al. Trim21 depletion alleviates bone loss in osteoporosis via activation of YAP1/beta-catenin signaling. Bone Res. 2023; 11(1): 56.

[29] Yang J, Li S, Li Z, et al. Targeting YAP1-regulated glycolysis in fibroblast-like synoviocytes impairs macrophage infiltration to ameliorate diabetic osteoarthritis progression. Adv Sci (Weinh). 2024; 11(5): e2304617.

[30] Jin ZX, Liao XY, Da WW, Zhao YJ, Li XF, Tang DZ. Osthole enhances the bone mass of senile osteoporosis and stimulates the expression of osteoprotegerin by activating β-catenin signaling. Stem Cell Res Ther. 2021; 12(1): 154.

[31] Garcia-Loza I, Perna-Barrull D, Aguilera E, et al. Targeting macrophages with phosphatidylserine-rich liposomes as a potential antigen-specific immunotherapy for type 1 diabetes. J Autoimmun. 2024; 145: 103196.

[32] Musa RE, Lester KL, Quickstad G, et al. BRD4 binds to active cranial neural crest enhancers to regulate RUNX2 activity during osteoblast differentiation. Development. 2024; 151(2): dev202110.

[33] Dai J, Zhou S, Ge Q, et al. Recruitment of Brd3 and Brd4 to acetylated chromatin is essential for proinflammatory cytokine-induced matrix-degrading enzyme expression. J Orthop Surg Res. 2019; 14(1): 59.

[34] Wang L, Fang B, Fujiwara T, et al. Deletion of ferroportin in murine myeloid cells increases iron accumulation and stimulates osteoclastogenesis in vitro and in vivo. J Biol Chem. 2018; 293(24): 9248-9264.

[35] Battaglino RA, Pham L, Morse LR, et al. NHA-oc/NHA2: a mitochondrial cation-proton antiporter selectively expressed in osteoclasts. Bone. 2008; 42(1): 180-192.

[36] Zhang Z, Ding P, Meng Y, et al. Rational polyelectrolyte nanoparticles endow preosteoclast-targeted siRNA transfection for anabolic therapy of osteoporosis. Sci Adv. 2023; 9(10): eade7379.

[37] Wang ZX, Luo ZW, Li FX, et al. Aged bone matrix-derived extracellular vesicles as a messenger for calcification paradox. Nat Commun. 2022; 13(1): 1453.

[38] Zhang HT, Peng R, Chen S, et al. Versatile nano-PROTAC-induced epigenetic reader degradation for efficient lung cancer therapy. Adv Sci. 2022; 9(29): 162202039.

[39] Yu W, Xie Z, Li J, et al. Super enhancers targeting ZBTB16 in osteogenesis protect against osteoporosis. Bone Res. 2023; 11(1): 30.

[40] Winkeler CL, Kladney RD, Maggi LB, Weber JD. Cathepsin K-Cre causes unexpected germline deletion of genes in mice. PLoS One. 2012; 7(7): e42005.

[41] Devaiah BN, Case-Borden C, Gegonne A, et al. BRD4 is a histone acetyltransferase that evicts nucleosomes from chromatin. Nat Struct Mol Biol. 2016; 23(6): 540-548.

[42] Matsuoka R, Fudim R, Jung S, et al. Structure, mechanism and lipid-mediated remodeling of the mammalian Na(+)/H(+) exchanger NHA2. Nat Struct Mol Biol. 2022; 29(2): 108-120.

[43] Schushan M, Xiang M, Bogomiakov P, Padan E, Rao R, Ben-Tal N. Model-guided mutagenesis drives functional studies of human NHA2, implicated in hypertension. J Mol Biol. 2010; 396(5): 1181-1196.

[44] Prasad H, Dang DK, Kondapalli KC, Natarajan N, Cebotaru V, Rao R. NHA2 promotes cyst development in an in vitro model of polycystic kidney disease. J Physiol. 2019; 597(2): 499-519.

[45] Ha BG, Hong JM, Park JY, et al. Proteomic profile of osteoclast membrane proteins: identification of Na+/H+ exchanger domain containing 2 and its role in osteoclast fusion. Proteomics. 2008; 8(13): 2625-2639.

[46] Hofstetter W, Siegrist M, Simonin A, Bonny O, Fuster DG. Sodium/hydrogen exchanger NHA2 in osteoclasts: subcellular localization and role in vitro and in vivo. Bone. 2010; 47(2): 331-340.

[47] Luhmann T, Germershaus O, Groll J, Meinel L. Bone targeting for the treatment of osteoporosis. J Control Release. 2012; 161(2): 198-213.

[48] Zhou Y, Deng Y, Liu Z, et al. Cytokine-scavenging nanodecoys reconstruct osteoclast/osteoblast balance toward the treatment of postmenopausal osteoporosis. Sci Adv. 2021; 7(48): eabl6432.

[49] Sun DZ, Zhang GL, Xie MY, et al. Softness enhanced macrophage-mediated therapy of inhaled apoptotic-cell-inspired nanosystems for acute lung injury. J Nanobiotechnol. 2023; 21(1): 172.

[50] Ma M, Zeng H, Yang P, Xu J, Zhang X, He W. Drug delivery and therapy strategies for osteoporosis intervention. Molecules. 2023; 28(18): 6652.

[51] Churcher I. Protac-induced protein degradation in drug discovery: breaking the rules or just making new ones?. J Med Chem. 2018; 61(2): 444-452.

[52] Zhang H, Wang L, Cui J, et al. Maintaining hypoxia environment of subchondral bone alleviates osteoarthritis progression. Sci Adv. 2023; 9(14): eabo7868.

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2025 The Author(s). Clinical and Translational Medicine published by John Wiley & Sons Australia, Ltd on behalf of Shanghai Institute of Clinical Bioinformatics.