Mapping RANKL- and OPG-expressing cells in bone tissue: the bone surface cells as activators of osteoclastogenesis and promoters of the denosumab rebound effect

Abstract

Denosumab is a monoclonal anti-RANKL antibody that inhibits bone resorption, increases bone mass, and reduces fracture risk. Denosumab discontinuation causes an extensive wave of rebound resorption, but the cellular mechanisms remain poorly characterized. We utilized in situ hybridization (ISH) as a direct approach to identify the cells that activate osteoclastogenesis through the RANKL/OPG pathway. ISH was performed across species, skeletal sites, and following recombinant OPG (OPG:Fc) and parathyroid hormone 1–34 (PTH) treatment of mice. OPG:Fc treatment in mice induced an increased expression of RANKL mRNA mainly in trabecular, but not endocortical bone surface cells. Additionally, a decreased expression of OPG mRNA was detected in bone surface cells and osteocytes of both compartments. A similar but more pronounced effect on RANKL and OPG expression was seen one hour after PTH treatment. These findings suggest that bone surface cells and osteocytes conjointly regulate the activation of osteoclastogenesis, and that OPG:Fc treatment induces a local accumulation of osteoclastogenic activation sites, ready to recruit and activate osteoclasts upon treatment discontinuation. Analysis of publicly available single-cell RNA sequencing (scRNAseq) data from murine bone marrow stromal cells revealed that Tnfsf11⁺ cells expressed high levels of Mmp13, Limch1, and Wif1, confirming their osteoprogenitor status. ISH confirmed co-expression of Mmp13 and Tnfsf11 in bone surface cells of both vehicle- and OPG:Fc-treated mice. Under physiological conditions of human/mouse bone, RANKL is expressed mainly by osteoprogenitors proximate to the osteoclasts, while OPG is expressed mainly by osteocytes and bone-forming osteoblasts.

References

[1.] Delaisse, J. M. et al. Re-thinking the bone remodeling cycle mechanism and the origin of bone loss. Bone141, 115628 (2020).

[2.] FROST, H. M. Dynamics of bone remodeling. Bone Biodyn. 315-334 (1964).

[3.] LassenNE, et al. . Coupling of bone resorption and formation in real time: New knowledge gained from human Haversian BMUs. J. Bone Min. Res., 2017, 32: 1395-1405

[4.] ParfittAM. The coupling of bone-formation to bone-resorption - a critical analysis of the concept and of its relevance to the pathogenesis of osteoporosis. Metab. Bone Dis. Relat., 1982, 4: 1-6

[5.] AndersenTL, et al. . Understanding coupling between bone resorption and formation: Are reversal cells the missing link?. Am. J. Pathol., 2013, 183: 235-246

[6.] ReginsterJY. Antifracture efficacy of currently available therapies for postmenopausal osteoporosis. Drugs, 2011, 71: 65-78

[7.] CompstonJE, McClungMR, LeslieWD. Osteoporosis. Lancet, 2019, 393: 364-376

[8.] TsourdiE, et al. . Fracture risk and management of discontinuation of denosumab therapy: A systematic review and position statement by ECTS. J. Clin. Endocr. Metab., 2021, 106: 264-281

[9.] TsourdiE, et al. . Discontinuation of denosumab therapy for osteoporosis: A systematic review and position statement by ECTS. Bone, 2017, 105: 11-17

[10.] FerrariS, et al. . Further nonvertebral fracture reduction beyond 3 years for up to 10 years of denosumab treatment. J. Clin. Endocr. Metab., 2019, 104: 3450-3461

[11.] BrownJP, et al. . Bone remodeling in postmenopausal women who discontinued denosumab treatment: Off-treatment biopsy study. J. Bone Miner. Res., 2011, 26: 2737-2744

[12.] CosmanF, HuangS, McDermottM, CummingsSR. Multiple vertebral fractures after denosumab discontinuation: FREEDOM and FREEDOM extension trials additional post hoc analyses. J. Bone Min. Res, 2022, 37: 2112-2120

[13.] AnastasilakisAD, et al. . Clinical features of 24 patients with rebound-associated vertebral fractures after denosumab discontinuation: Systematic review and additional cases. J. Bone Min. Res, 2017, 32: 1291-1296

[14.] CummingsSR, et al. . Vertebral fractures after discontinuation of denosumab: A post hoc analysis of the randomized placebo-controlled FREEDOM trial and its extension. J. Bone Min. Res., 2018, 33: 190-198

[15.] FerrariS, LangdahlB. Mechanisms underlying the long-term and withdrawal effects of denosumab therapy on bone. Nat. Rev. Rheumatol., 2023, 19: 307-317

[16.] FassioA, et al. . Changes in Dkk-1, sclerostin, and RANKL serum levels following discontinuation of long-term denosumab treatment in postmenopausal women. Bone, 2019, 123: 191-195

[17.] FontalisA, GossielF, SchiniM, WalshJ, EastellR. The effect of denosumab treatment on osteoclast precursor cells in postmenopausal osteoporosis. Bone Rep., 2020, 13: 100457

[18.] Jähn-Rickert, K. et al. Elevated bone hardness under denosumab treatment, with persisting lower osteocyte viability during discontinuation. Front. Endocrinol. 11, 250 (2020).

[19.] McDonaldMM, et al. . Osteoclasts recycle via osteomorphs during RANKL-stimulated bone resorption. Cell, 2021, 184: 1330

[20.] Fu, Q. et al. Reduced osteoprotegerin expression by osteocytes may contribute to rebound resorption after denosumab discontinuation. JCI Insight8, e167790 (2023).

[21.] KartsogiannisV, et al. . Localization of RANKL (receptor activator of NF kappa B ligand) mRNA and protein in skeletal and extraskeletal tissues. Bone, 1999, 25: 525-534

[22.] MaticI, et al. . Quiescent bone lining cells are a major source of osteoblasts during adulthood. Stem Cells, 2016, 34: 2930-2942

[23.] SilvestriniG, et al. . Detection of osteoprotegerin (OPG) and its ligand (RANKL) mRNA and protein in femur and tibia of the rat. J. Mol. Histol., 2005, 36: 59-67

[24.] GoriF, et al. . The expression of osteoprotegerin and RANK ligand and the support of osteoclast formation by stromal-osteoblast lineage cells is developmentally regulated. Endocrinology, 2000, 141: 4768-4776

[25.] ThomasGP, BakerSU, EismanJA, GardinerEM. Changing RANKL/OPG mRNA expression in differentiating murine primary osteoblasts. J. Endocrinol., 2001, 170: 451-460

[26.] Cawley, K. M. et al. Local production of osteoprotegerin by osteoblasts suppresses bone resorption. Cell Rep.32, 108052 (2020).

[27.] Xiong, J. H. et al. Osteocytes, not osteoblasts or lining cells, are the main source of the RANKL required for osteoclast formation in remodeling bone. Plos One10, e0138189 (2015).

[28.] RootSH, et al. . Perivascular osteoprogenitors are associated with transcortical channels of long bones. Stem Cells, 2020, 38: 769-781

[29.] LimJ, BurclaffJ, HeG, MillsJC, LongF. Unintended targeting of Dmp1-Cre reveals a critical role for Bmpr1a signaling in the gastrointestinal mesenchyme of adult mice. Bone Res., 2017, 5: 16049

[30.] WalkerEC, et al. . Sustained RANKL response to parathyroid hormone in oncostatin M receptor-deficient osteoblasts converts anabolic treatment to a catabolic effect in vivo. J. Bone Miner. Res., 2012, 27: 902-912

[31.] HuangJC, et al. . PTH differentially regulates expression of RANKL and OPG. J. Bone Min. Res., 2004, 19: 235-244

[32.] BaryawnoN, et al. . A cellular taxonomy of the bone marrow stroma in homeostasis and leukemia. Cell, 2019, 177: 1915-1932.e1916

[33.] AbdelgawadME, et al. . Early reversal cells in adult human bone remodeling: osteoblastic nature, catabolic functions and interactions with osteoclasts. Histochemistry Cell Biol., 2016, 145: 603-615

[34.] AndersenTL, et al. . A critical role of the bone marrow envelope in human bone remodeling. J. Bone Min. Res., 2023, 38: 918-928

[35.] FrischT, SorensenMS, OvergaardS, BretlauP. Estimation of volume referent bone turnover in the otic capsule after sequential point labeling. Ann. Otol. Rhinol. Laryngol., 2000, 109: 33-39

[36.] SorensenMS, JorgensenMB, BretlauP. Drift barriers in the postcartilaginous development of the mammalian otic capsule. Eur. Arch. Otorhinolaryngol., 1992, 249: 56-61

[37.] ZehnderAF, et al. . Osteoprotegrin knockout mice demonstrate abnormal remodeling of the otic capsule and progressive hearing loss. Laryngoscope, 2006, 116: 201-206

[38.] ZehnderAF, KristiansenAG, AdamsJC, MerchantSN, McKennaMJ. Osteoprotegerin in the inner ear may inhibit bone remodeling in the otic capsule. Laryngoscope, 2005, 115: 172-177

[39.] EvertsV, et al. . The bone lining cell:: Its role in cleaning Howship’s lacunae and initiating bone formation. J. Bone Miner. Res., 2002, 17: 77-90

[40.] van Dijk ChristiansenP, et al. . Osteoprogenitor recruitment and differentiation during intracortical bone remodeling of adolescent humans. Bone, 2023, 177

[41.] EriksenEF, Eghbali-FatourechiGZ, KhoslaS. Remodeling and vascular spaces in bone. J. Bone Min. Res, 2007, 22: 1-6

[42.] BoyceBF, XingL. Functions of RANKL/RANK/OPG in bone modeling and remodeling. Arch. Biochem Biophys., 2008, 473: 139-146

[43.] YasudaH, et al. . Osteoclast differentiation factor is a ligand for osteoprotegerin/osteoclastogenesis-inhibitory factor and is identical to TRANCE/RANKL. Proc. Natl Acad. Sci. USA, 1998, 95: 3597-3602

[44.] Kim, H. N., et al. Osteocyte RANKL is required for cortical bone loss with age and is induced by senescence. Jci Insight5, e138815 (2020).

[45.] LaceyDL, et al. . Osteoprotegerin ligand is a cytokine that regulates osteoclast differentiation and activation. Cell, 1998, 93: 165-176

[46.] TatsumiS, et al. . Targeted ablation of osteocytes induces osteoporosis with defective mechanotransduction. Cell Metab., 2007, 5: 464-475

[47.] ClarkeB. Normal bone anatomy and physiology. Clin. J. Am. Soc. Nephrol., 2008, 3: S131-S139

[48.] SollingAS, HarslofT, LangdahlB. Treatment with zoledronate subsequent to denosumab in osteoporosis: A 2-year randomized study. J. Bone Miner. Res., 2021, 36: 1245-1254

[49.] SollingAS, HarslofT, JorgensenNR, LangdahlB. Changes in RANKL and TRAcP 5b after discontinuation of denosumab suggest RANKL mediated formation of osteoclasts results in the increased bone resorption. Osteoporos. Int, 2023, 34: 599-605

[50.] TsaiJ, et al. . Denosumab reduces the number of circulating osteoclast precursor cells in postmenopausal women with osteoporosis. J. Bone Miner. Res., 2023, 38: 188

[51.] Drejer, L. A. et al. Trabecular bone deterioration in a postmenopausal female suffering multiple spontaneous vertebral fractures due to a delayed denosumab injection - A post-treatment re-initiation bone biopsy-based case study. Bone Rep.19, 101703 (2023).

[52.] Kim, A. S. et al. Temporal patterns of osteoclast formation and activity following withdrawal of RANKL inhibition. J. Bone Miner. Res.39, 484–497 (2024).

[53.] ReidIR, et al. . Effects of denosumab on bone histomorphometry: the FREEDOM and STAND studies. J. Bone Min. Res., 2010, 25: 2256-2265

[54.] EastellR, et al. . Effects of denosumab on bone turnover markers in postmenopausal osteoporosis. J. Bone Miner. Res., 2011, 26: 530-537

[55.] KristensenHB, AndersenTL, MarcussenN, RolighedL, DelaisseJM. Osteoblast recruitment routes in human cancellous bone remodeling. Am. J. Pathol., 2014, 184: 778-789

[56.] MillerSC, JeeWSS. The bone lining cell: a distinct phenotype?. Calcif. Tissue Int., 1987, 41: 1-5

[57.] ChambersTJ, ThomsonBM, FullerK. Effect of substrate composition on bone resorption by rabbit osteoclasts. J. Cell Sci., 1984, 70: 61-71

[58.] KarsdalMA, FjordingMS, FogedNT, DelaisséJM, LochterA. Transforming growth factor-β-induced osteoblast elongation regulates osteoclastic bone resorption through a p38 mitogen-activated protein kinase- and matrix metalloproteinase-dependent pathway. J. Biol. Chem., 2001, 276: 39350-39358

[59.] Perez-AmodioS, BeertsenW, EvertsV. (Pre-)Osteoclasts induceretraction of osteoblasts before their fusion to osteoclasts. J. Bone Min. Res., 2004, 19: 1722-1731

[60.] FerrierJ, XiaSL, LaganE, AubinJE, HeerscheJN. Displacement and translocation of osteoblast-like cells by osteoclasts. J. Bone Min. Res., 1994, 9: 1397-1405

[61.] NakashimaT, et al. . Evidence for osteocyte regulation of bone homeostasis through RANKL expression. Nat. Med., 2011, 17: 1231-1234

[62.] AndersenTL, et al. . A physical mechanism for coupling bone resorption and formation in adult human bone. Am. J. Pathol., 2009, 174: 239-247

[63.] BorggaardXG, NielsenMH, DelaisseJM, AndreasenCM, AndersenTL. Spatial organization of osteoclastic coupling factors and their receptors at human bone remodeling sites. Front Mol. Biosci., 2022, 9: 896841

[64.] NookaewI, et al. . Refining the identity of mesenchymal cell types associated with murine periosteal and endosteal bone. J. Biol. Chem., 2024, 300

[65.] Weivoda, M. M. et al. Identification of osteoclast-osteoblast coupling factors in humans reveals links between bone and energy metabolism. Nat. Commun. 11, 87 (2020).

[66.] IkedaT, UtsuyamaM, HirokawaK. Expression profiles of receptor activator of nuclear factor kappaB ligand, receptor activator of nuclear factor kappaB, and osteoprotegerin messenger RNA in aged and ovariectomized rat bones. J. Bone Min. Res., 2001, 16: 1416-1425

[67.] MizunoA, et al. . Severe osteoporosis in mice lacking osteoclastogenesis inhibitory factor osteoprotegerin. Biochem. Biophys. Res. Commun., 1998, 247: 610-615

[68.] OnyiaJE, et al. . In vivo demonstration that human parathyroid hormone 1-38 inhibits the expression of osteoprotegerin in bone with the kinetics of an immediate early gene. J. Bone Min. Res., 2000, 15: 863-871

[69.] StreicherC, et al. . Estrogen regulates bone turnover by targeting RANKL expression in bone lining cells. Sci. Rep., 2017, 7

[70.] PereiraRC, et al. . Bone canopies in pediatric renal osteodystrophy. PLoS One, 2016, 11: e0152871

[71.] HaoY, et al. . Dictionary learning for integrative, multimodal and scalable single-cell analysis. Nat. Biotechnol., 2024, 42: 293-304

Funding

Velux Fonden (Velux Foundation)(VELUX25723); Odense University Hospital’s Fund for Free Research (A5111), the Region of Southern Denmark’s Fund for Free Research (A955)