Effect of inhibitor of Wnt production 2 on osteoclast differentiation

doi:10.12307/2023.448

Abstract

Abstract: BACKGROUND: Abnormal activation of osteoclast-mediated bone resorption leads to the loss of bone mass, damage and deterioration of bone microarchitecture, which is an important cause of osteoporosis. Wnt/β-catenin signaling pathway may be a potential target and tool for the prevention and treatment of osteoporosis. Overactivation of Wnt/β-catenin signaling pathway inhibits osteoclast differentiation. However, the physiological role of Wnt/β-catenin signaling pathway in osteoclast formation remains to be clarified.
OBJECTIVE: To investigate the effect of inhibitor of Wnt production 2 (IWP-2) on proliferation and differentiation of bone marrow-derived macrophages through inhibiting the Wnt/β-catenin signaling pathway.
METHODS: (1) Bone marrow-derived macrophages were extracted. Cell counting kit-8 assay was performed to assess the viability of bone marrow-derived macrophages after being treated with gradient concentrations from 0 to 40 μmol/L IWP-2 for 24, 72, or 120 hours. (2) Bone marrow-derived macrophages induced by 100 μg/L receptor activator of nuclear factor kappa B ligand (RANKL) were treated with 5 or 10 μmol/L IWP-2 and equivalent solvent, and precipitated proteins were collected at 0, 1, 3 and 5 days. The activity of Wnt/β-catenin signaling pathway and the inhibitory effect of IWP-2 on it during osteoclast differentiation were detected by western blot assay. (3) Bone marrow-derived macrophages induced by 100 μg/L RANKL were treated with non-toxic IWP-2 (0, 5, or 10 μmol/L) for 6 days. The effects of IWP-2 on osteoclast differentiation were evaluated by tartrate-resistant acid phosphatase staining. The mRNA expression of osteoclast-related genes including NFATc1, Oscar, CTSK, Acp5, Mmp9, and Dcstamp were detected by real-time fluorescence quantitative PCR.
RESULTS AND CONCLUSION: (1) After treatment with different concentrations of IWP-2 for 24 hours, 10 μmol/L IWP-2 promoted the proliferation of bone marrow-derived macrophages. After treatment with different concentrations of IWP-2 for 72 and 120 hours, the viability of bone marrow-derived macrophages was not significantly affected by IWP-2 at a concentration of 10 μmol/L or less, while high concentrations of IWP-2 (20, 30, and 40 μmol/L) inhibited the proliferation of bone marrow-derived macrophages. (2) After 1, 3, and 5 days of RANKL stimulation, the protein expression of active β-catenin in the control group increased gradually, while that in the 5 and 10 μmol/L IWP-2 treatment group was significantly inhibited. (3) Many large and irregular TRAP-positive multinucleated mature osteoclasts were observed in the control group, while the number and size of the cells decreased in a dose-dependent manner in the IWP-2 treatment groups, suggesting that IWP-2 could inhibit osteoclast differentiation. (4) The expression levels of osteoclast-related genes including NFATc1, Oscar, CTSK, Acp5, Mmp9, and Dcstamp were all down-regulated in a dose-dependent manner after IWP-2 treatment. (5) To conclude, IWP-2 can inhibit osteoclast differentiation in vitro and may play a role in the prevention and treatment of osteoporosis by directly or indirectly inhibiting the expression of osteoclast-elated genes.

Key words: osteoporosis, Wnt/β-catenin signaling pathway, IWP-2, bone marrow-derived macrophage, proliferation, differentiation

CLC Number:

Liao Zirui, Chen Jianquan. Effect of inhibitor of Wnt production 2 on osteoclast differentiation[J]. Chinese Journal of Tissue Engineering Research, 2023, 27(31): 4990-4995.

Figures/Tables 4

References

[1] BUCKWALTER JA, GLIMCHER MJ, COOPER RR, et al. Bone biology. I: Structure, blood supply, cells, matrix, and mineralization. Instr Course Lect. 1996;45:371-386.
[2] COHEN MM JR. The new bone biology: pathologic, molecular, and clinical correlates. Am J Med Genet A. 2006;140(23):2646-2706.
[3] KULAR J, TICKNER J, CHIM SM, et al. An overview of the regulation of bone remodelling at the cellular level. Clin Biochem. 2012;45(12):863-873.
[4] Zhu S, Yao F, Qiu H, et al. Coupling factors and exosomal packaging microRNAs involved in the regulation of bone remodelling. Biol Rev Camb Philos Soc. 2018; 93(1):469-480.
[5] KRISHNAN V, BRYANT HU, MACDOUGALD OA. Regulation of bone mass by Wnt signaling. J Clin Invest. 2006;116(5):1202-1209.
[6] LONG F. Building strong bones: molecular regulation of the osteoblast lineage. Nat Rev Mol Cell Biol. 2011;13(1):27-38.
[7] CHARLES JF, ALIPRANTIS AO. Osteoclasts: more than ‘bone eaters’. Trends Mol Med. 2014;20(8):449-459.
[8] NAKAGAWA N, KINOSAKI M, YAMAGUCHI K, et al. RANK is the essential signaling receptor for osteoclast differentiation factor in osteoclastogenesis. Biochem Biophys Res Commun. 1998;253(2):395-400.
[9] YASUDA H, SHIMA N, NAKAGAWA N, et al. Osteoclast differentiation factor is a ligand for osteoprotegerin/osteoclastogenesis-inhibitory factor and is identical to TRANCE/RANKL. Proc Natl Acad Sci U S A. 1998;95(7):3597-3602.
[10] Consensus development conference: diagnosis, prophylaxis, and treatment of osteoporosis. Am J Med. 1993;94(6):646-650.
[11] DOWNEY PA, SIEGEL MI. Bone biology and the clinical implications for osteoporosis. Phys Ther. 2006;86(1):77-91.
[12] FONSECA H, MOREIRA-GONÇALVES D, CORIOLANO HJ, et al. Bone quality: the determinants of bone strength and fragility. Sports Med. 2014;44(1):37-53.
[13] GONG Y, SLEE RB, FUKAI N, et al. LDL receptor-related protein 5 (LRP5) affects bone accrual and eye development. Cell. 2001;107(4):513-523.
[14] BOYDEN LM, MAO J, BELSKY J, et al. High bone density due to a mutation in LDL-receptor-related protein 5. N Engl J Med. 2002;346(20):1513-1521.
[15] LITTLE RD, CARULLI JP, DEL MASTRO RG, et al. A mutation in the LDL receptor-related protein 5 gene results in the autosomal dominant high-bone-mass trait. Am J Hum Genet. 2002;70(1):11-19.
[16] LIU J, XIAO Q, XIAO J, et al. Wnt/β-catenin signalling: function, biological mechanisms, and therapeutic opportunities. Signal Transduct Target Ther. 2022;7(1):3.
[17] MACDONALD BT, TAMAI K, HE X. Wnt/beta-catenin signaling: components, mechanisms, and diseases. Dev Cell. 2009;17(1):9-26.
[18] NUSSE R, CLEVERS H. Wnt/β-Catenin Signaling, Disease, and Emerging Therapeutic Modalities. Cell. 2017;169(6):985-999.
[19] WEIVODA MM, RUAN M, HACHFELD CM, et al. Wnt Signaling Inhibits Osteoclast Differentiation by Activating Canonical and Noncanonical cAMP/PKA Pathways. J Bone Miner Res. 2019;34(8):1546-1548.
[20] WEI W, ZEVE D, SUH JM, et al. Biphasic and dosage-dependent regulation of osteoclastogenesis by β-catenin. Mol Cell Biol. 2011;31(23):4706-4719.
[21] RIOS-ESTEVES J, RESH MD. Stearoyl CoA desaturase is required to produce active, lipid-modified Wnt proteins. Cell Rep. 2013;4(6):1072-1081.
[22] SHINDOU H, ETO M, MORIMOTO R, et al. Identification of membrane O-acyltransferase family motifs. Biochem Biophys Res Commun. 2009;383(3): 320-325.
[23] LI Y, WU S, LI X, et al. Wnt signaling associated small molecules improve the viability of pPSCs in a PI3K/Akt pathway dependent way. J Cell Physiol. 2020; 235(7-8):5811-5822.
[24] ANASTASILAKIS AD, POLYZOS SA, MAKRAS P. THERAPY OF ENDOCRINE DISEASE: Denosumab vs bisphosphonates for the treatment of postmenopausal osteoporosis. Eur J Endocrinol. 2018;179(1):R31-R45.
[25] COSMAN F. Long-term treatment strategies for postmenopausal osteoporosis. Curr Opin Rheumatol. 2018;30(4):420-426.
[26] DEEKS ED. Denosumab: A Review in Postmenopausal Osteoporosis. Drugs Aging. 2018;35(2):163-173.
[27] YAVROPOULOU MP, MAKRAS P, ANASTASILAKIS AD. Bazedoxifene for the treatment of osteoporosis. Expert Opin Pharmacother. 2019;20(10):1201-1210.
[28] BLACK DM, BAUER DC, SCHWARTZ AV, et al. Continuing bisphosphonate treatment for osteoporosis--for whom and for how long? N Engl J Med. 2012; 366(22):2051-2053.
[29] RACHNER TD, KHOSLA S, HOFBAUER LC. Osteoporosis: now and the future. Lancet. 2011;377(9773):1276-1287.
[30] BONE HG, WAGMAN RB, BRANDI ML, et al. 10 years of denosumab treatment in postmenopausal women with osteoporosis: results from the phase 3 randomised FREEDOM trial and open-label extension. Lancet Diabetes Endocrinol. 2017;5(7): 513-523.
[31] ADLER RA. MANAGEMENT OF ENDOCRINE DISEASE: Atypical femoral fractures: risks and benefits of long-term treatment of osteoporosis with anti-resorptive therapy. Eur J Endocrinol. 2018;178(3):R81-R87.
[32] FUJITA K, JANZ S. Attenuation of WNT signaling by DKK-1 and -2 regulates BMP2-induced osteoblast differentiation and expression of OPG, RANKL and M-CSF. Mol Cancer. 2007;6:71.
[33] LACEY DL, TIMMS E, TAN HL, et al. Osteoprotegerin ligand is a cytokine that regulates osteoclast differentiation and activation. Cell. 1998;93(2):165-176.
[34] BARON R, KNEISSEL M. WNT signaling in bone homeostasis and disease: from human mutations to treatments. Nat Med. 2013;19(2):179-192.
[35] BENNETT CN, LONGO KA, WRIGHT WS, et al. Regulation of osteoblastogenesis and bone mass by Wnt10b. Proc Natl Acad Sci U S A. 2005;102(9):3324-3329.
[36] NAKANISHI R, AKIYAMA H, KIMURA H, et al. Osteoblast-targeted expression of Sfrp4 in mice results in low bone mass. J Bone Miner Res. 2008;23(2):271-277.
[37] BURGERS TA, WILLIAMS BO. Regulation of Wnt/β-catenin signaling within and from osteocytes. Bone. 2013;54(2):244-249.
[38] ZHANG L, FU X, NI L, et al. Hedgehog Signaling Controls Bone Homeostasis by Regulating Osteogenic/Adipogenic Fate of Skeletal Stem/Progenitor Cells in Mice. J Bone Miner Res. 2022;37(3):559-576.