An LGR4 agonist activates the GSK‑3β pathway to inhibit RANK‑RANKL signaling during osteoclastogenesis in bone marrow‑derived macrophages

Jang,Yuria; Lee,Hyeonjoon; Cho,Yongjin; Choi,Eunseo; Jo,Suenghwan; Sohn,Hong Moon; Kim,Beom Chang; Ko,Young Jong; Lim,Wonbong

doi:10.3892/ijmm.2023.5334

An LGR4 agonist activates the GSK‑3β pathway to inhibit RANK‑RANKL signaling during osteoclastogenesis in bone marrow‑derived macrophages

Authors:
- Yuria Jang
- Hyeonjoon Lee
- Yongjin Cho
- Eunseo Choi
- Suenghwan Jo
- Hong Moon Sohn
- Beom Chang Kim
- Young Jong Ko
- Wonbong Lim
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Affiliations: Laboratory of Orthopedic Research, Chosun University Hospital, Gwangju 61452, Republic of Korea, Department of Physics, Chosun University, Gwangju 61452, Republic of Korea
Published online on: December 5, 2023 https://doi.org/10.3892/ijmm.2023.5334
Article Number: 10
Copyright: © Jang et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

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Abstract

The binding between receptor‑activated nuclear factor‑κB (RANK) and the RANK ligand (RANKL) during osteoclast development is an important target for drugs that treat osteoporosis. The leucine‑rich repeat‑containing G‑protein‑coupled receptor 4 (LGR4) acts as a negative regulator of RANK‑RANKL that suppresses canonical RANK signaling during osteoclast differentiation. Therefore, LGR4 agonists may be useful in inhibiting osteoclastogenesis and effectively treating osteoporosis. In the present study, bone marrow‑derived macrophages and a mouse model of RANKL‑induced bone loss were used to investigate the effect of mutant RANKL (MT RANKL), which was previously developed based on the crystal structure of the RANKL complex. In the present study, the binding affinity of wild‑type (WT) RANKL and MT RANKL to RANK and LGR4 was determined using microscale thermophoresis analysis, and the effect of the ligands on the AKT‑glycogen synthase kinase‑3β (GSK‑3β)‑nuclear factor of activated T cells, cytoplasmic, calcineurin‑dependent 1 (NFATc1) signaling cascade was investigated using western blotting and confocal microscopy. In addition, the expression of LGR4 and the colocalization of LGR4 with MT RANKL were analyzed in a mouse model of RANKL‑induced bone loss. The results showed that in osteoclast precursor cells, MT RANKL bound with high affinity to LGR4 and increased GSK‑3β phosphorylation independently of AKT, resulting in the inhibition of NFATc1 nuclear translocation. In the mouse model, MT RANKL colocalized with LGR4 and inhibited bone resorption. These results indicated that MT RANKL may inhibit RANKL‑induced osteoclastogenesis through an LGR4‑dependent pathway and this could be exploited to develop new therapies for osteoporosis.

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1	Hofbauer L, Kuhne C and Viereck V: The OPG/RANKL/RANK system in metabolic bone diseases. J Musculoskel Neuro Inter. 4:268–275. 2004.
2	Wang L, You X, Zhang L, Zhang C and Zou W: Mechanical regulation of bone remodeling. Bone Res. 10:162022.
3	Hanley DA, Adachi JD, Bell A and Brown V: Denosumab: Mechanism of action and clinical outcomes. Int J Clin Pract. 66:1139–1146. 2012.
4	Gowen M, Stroup GB, Dodds RA, James IE, Votta BJ, Smith BR, Bhatnagar PK, Lago AM, Callahan JF, DelMar EG, et al: Antagonizing the parathyroid calcium receptor stimulates parathyroid hormone secretion and bone formation in osteopenic rats. J Clin Invest. 105:1595–1604. 2000.
5	Martin TJ: Bone biology and anabolic therapies for bone: Current status and future prospects. J Bone Metab. 21:8–20. 2014.
6	Sozen T, Ozisik L and Basaran NC: An overview and management of osteoporosis. Eur J Rheumatol. 4:46–56. 2017.
7	Miller PD: Denosumab: Anti-RANKL antibody. Curr Osteoporos Rep. 7:18–22. 2009.
8	Cadieux B, Coleman R, Jafarinasabian P, Lipton A, Orlowski RZ, Saad F, Scagliotti GV, Shimizu K and Stopeck A: Experience with denosumab (XGEVA(R)) for prevention of skeletal-related events in the 10 years after approval. J Bone Oncol. 33:1004162022.
9	Lei MM, Tavares E, Buzgo E, Lou U, Raje N and Yee AJ: Denosumab versus intravenous bisphosphonate use for hypercalcemia in multiple myeloma. Leuk Lymphoma. 63:1–4. 2022.
10	Terpos E, Jamotte A, Christodoulopoulou A, Campioni M, Bhowmik D, Kennedy L and Willenbacher W: A cost-effectiveness analysis of denosumab for the prevention of skeletal-related events in patients with multiple myeloma in four European countries: Austria, Belgium, Greece, and Italy. J Med Econ. 22:766–776. 2019.
11	Benlidayi IC: Denosumab in the treatment of glucocorticoid-induced osteoporosis. Rheumatol Int. 38:1975–1984. 2018.
12	Pittman K, Antill YC, Goldrick A, Goh J and de Boer RH: Denosumab: Prevention and management of hypocalcemia, osteonecrosis of the jaw and atypical fractures. Asia Pac J Clin Oncol. 13:266–276. 2017.
13	Gkoufa A, Angelousi A, Neonaki A, Athanasouli F and Cholongitas E: Severe symptomatic hypocalcemia associated with denosumab administration in a patient with decompensated cirrhosis and renal dysfunction. Ann Pharmacother. 56:853–855. 2022.
14	Anastasilakis AD, Makras P, Yavropoulou MP, Tabacco G, Naciu AM and Palermo A: Denosumab discontinuation and the rebound phenomenon: A narrative review. J Clin Med. 10:1522021.
15	Luo J, Yang Z, Ma Y, Yue Z, Lin H, Qu G, Huang J, Dai W, Li C, Zheng C, et al: LGR4 is a receptor for RANKL and negatively regulates osteoclast differentiation and bone resorption. Nat Med. 22:539–546. 2016.
16	Takegahara N, Kim H and Choi Y: RANKL biology. Bone. 159:1163532022.
17	Yue Z, Niu X, Yuan Z, Qin Q, Jiang W, He L, Gao J, Ding Y, Liu Y, Xu Z, et al: RSPO2 and RANKL signal through LGR4 to regulate osteoclastic premetastatic niche formation and bone metastasis. J Clin Invest. 132:e1445792022.
18	Jin Y and Yang Y: LGR4: A new receptor for a stronger bone. Sci China Life Sci. 59:735–736. 2016.
19	Luo W, Tan P, Rodriguez M, He L, Tan K, Zeng L, Siwko S and Liu M: Leucine-rich repeat-containing G protein-coupled receptor 4 (Lgr4) is necessary for prostate cancer metastasis via epithelial-mesenchymal transition. J Biol Chem. 292:15525–15537. 2017.
20	Elango J, Bao B and Wu W: The hidden secrets of soluble RANKL in bone biology. Cytokine. 144:1555592021.
21	Ko Y, Lee G, Kim B, Park M, Jang Y and Lim W: Modification of the RANKL-RANK-binding site for the immunotherapeutic treatment of osteoporosis. Osteoporos Int. 31:983–993. 2020.
22	Ko YJ, Sohn HM, Jang Y, Park M, Kim B, Kim B, Park JI, Hyun H, Jeong B, Hong C and Lim W: A novel modified RANKL variant can prevent osteoporosis by acting as a vaccine and an inhibitor. Clin Transl Med. 11:e3682021.
23	Jang Y, Sohn HM, Ko YJ, Hyun H and Lim W: Inhibition of RANKL-induced osteoclastogenesis by novel mutant RANKL. Int J Mol Sci. 22:4342021.
24	Romain M, Thiroux B, Tardy M, Quesnel B and Thuru X: Measurement of protein-protein interactions through microscale thermophoresis (MST). Bio Protoc. 10:e35742020.
25	Bartell SM, Kim HN, Ambrogini E, Han L, Iyer S, Ucer SS, Rabinovitch P, Jilka RL, Weinstein RS, Zhao H, et al: FoxO proteins restrain osteoclastogenesis and bone resorption by attenuating H2O2 accumulation. Nat Commun. 5:37732014.
26	Livak KJ and Schmittgen TD: Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods. 25:402–408. 2001.
27	Law SM and Zheng KK: Premise and peril of Wnt signaling activation through GSK-3β inhibition. iScience. 25:1041592022.
28	Fan X, Xiong H, Wei J, Gao X, Feng Y, Liu X, Zhang G, He QY, Xu J and Liu L: Cytoplasmic hnRNPK interacts with GSK3β and is essential for the osteoclast differentiation. Sci Rep. 5:177322015.
29	Cao X: RANKL-RANK signaling regulates osteoblast differentiation and bone formation. Bone Res. 6:352018.
30	Tokuyama N and Tanaka S: Updates of denosumab, anti-RANKL antibody for osteoporosis. Clin Calcium. 24:85–91. 2014.In Japanese.
31	Cipriani C, Piemonte S, Colangelo L, De Martino V, Diacinti D, Ferrone F, Piazzolla V, Fassino V, Nieddu L, Minisola S and Pepe J: Inhibition of the RANKL with denosumab has no effect on circulating markers of atherosclerosis in women with postmenopausal osteoporosis: A pilot study. Endocrine. 71:199–207. 2021.
32	Lasco A, Morabito N, Basile G, Atteritano M, Gaudio A, Giorgianni GM, Morini E, Faraci B, Bellone F and Catalano A: Denosumab inhibition of RANKL and insulin resistance in postmenopausal women with osteoporosis. Calcif Tissue Int. 98:123–128. 2016.
33	Kim AS, Girgis CM and McDonald MM: Osteoclast recycling and the rebound phenomenon following denosumab discontinuation. Curr Osteoporos Rep. 20:505–515. 2022.
34	Anastasilakis AD, Evangelatos G, Makras P and Iliopoulos A: Rebound-associated vertebral fractures may occur in sequential time points following denosumab discontinuation: Need for prompt treatment re-initiation. Bone Rep. 12:1002672020.
35	Anastasilakis AD, Trovas G, Balanika A, Polyzos SA, Makras P and Tournis S: Progression of rebound-associated vertebral fractures following denosumab discontinuation despite reinstitution of treatment: Suppressing increased bone turnover may not be enough. J Clin Densitom. 24:338–340. 2021.
36	Luo J, Zhou W, Zhou X, Li D, Weng J, Yi Z, Cho SG, Li C, Yi T, Wu X, et al: Regulation of bone formation and remodeling by G-protein-coupled receptor 48. Development. 136:2747–2756. 2009.
37	Shi GX, Zheng XF, Zhu C, Li B, Wang YR, Jiang SD and Jiang LS: Evidence of the role of R-spondin 1 and its receptor lgr4 in the transmission of mechanical stimuli to biological signals for bone formation. Int J Mol Sci. 18:5642017.
38	Lee Y, Kim HJ, Park CK, Kim YG, Lee HJ, Kim JY and Kim HH: MicroRNA-124 regulates osteoclast differentiation. Bone. 56:383–389. 2013.
39	Cong F, Wu N, Tian X, Fan J, Liu J, Song T and Fu H: MicroRNA-34c promotes osteoclast differentiation through targeting LGR4. Gene. 610:1–8. 2017.
40	Moon JB, Kim JH, Kim K, Youn BU, Ko A, Lee SY and Kim N: Akt induces osteoclast differentiation through regulating the GSK3beta/NFATc1 signaling cascade. J Immunol. 188:163–169. 2012.
41	Wang M, Liu J, Zhu G and Chen X: Low levels of cadmium exposure affect bone by inhibiting Lgr4 expression in osteoblasts and osteoclasts. J Trace Elem Med Biol. 73:1270252022.
42	Manolagas SC and Parfitt AM: What old means to bone. Trends Endocrinol Metab. 21:369–374. 2010.