Zoledronic acid in osteoclastogenesis: roles and mechanisms

doi:10.3969/j.issn.2095-4344.1720

Abstract

Abstract:

BACKGROUND: Zoledronic acid has been applied to treat bone resorption, but the exact mechanism is unclear.
OBJECTIVE: To investigate the effects and mechanisms of zoledronic acid on RANKL-induced osteoclastogenesis.
METHODS: The viability of mouse monocyte/macrophage cell line RAW264.7 was measured using cell counting kit-8 assay and then we analyzed the half maximal inhibitory concentration (IC₅₀). RAW264.7 cells were induced by RANKL to differentiate into osteoclasts. The induced cells were identified by rhodamine-conjugated phalloidin and 4,6-diamino-2-phenyl indole. The stage-specific effects of zoledronic acid during osteoclastogenesis were further assessed using tartrate resistant acid phosphatase staining. Bone resorption was examined using pit formation assay. Western blot was finally used to detect the expression of IκBα, P38, JNK, and ERK at protein levels.
RESULTS AND CONCLUSION: The IC₅₀ of zoledronic acid in Raw264.7 cells was 33.9 μmol/L at 48 hours, and zoledronic acid at a concentration of less than IC₅₀ suppressed the formation of osteoclasts. In addition, the number of bone resorption pits and bone resorption area were also decreased after treatment with zoledronic acid as compared with the control group. Zoledronic acid inhibited RANKL-induced IκBα phosphorylation at the protein level. To conclude, zoledronic acid can suppress osteoclastogenesis via the regulation of the nuclear factor-κB signaling pathway.

Key words: osteoclasts, zoledronic acid, bone resorption of osteoclasts, osteoclast differentiation, nuclear factor-κB signaling pathway, receptor activator of nuclear factor kappa B ligand, National Natural Science Foundation of China

CLC Number:

Huang Xiaolin, Liao Jian, Hong Wei, Li Fang, Cheng Yuting, Zhou Qian, Dong Qiang. Zoledronic acid in osteoclastogenesis: roles and mechanisms[J]. Chinese Journal of Tissue Engineering Research, 2019, 23(17): 2716-2721.

Figures/Tables 3

2.2 破骨细胞形成过程 RAW264.7细胞呈聚集生长趋势，体积小，未见多核细胞。在RANKL刺激下，细胞体积增大，成圆形、椭圆形或不规则形状，内含许多空泡样结构，并伸出许多细长的伪足样突起，图2A。罗丹明标记的鬼笔环肽为F-肌动蛋白探针，可选择性标记由F-肌动蛋白组成的肌动蛋白环而呈环形结构。诱导第3天已明显出现多个单核细胞开始融合，肌动蛋白的环形结构清晰可见，第5天达到高峰，环结构变圆变大，单个破骨细胞内可见七八十个细胞核，第7天细胞核开始碎裂，肌动蛋白的环结构也崩解，破骨细胞开始出现凋亡和裂解，见图2B。 2.3 唑来膦酸抑制RANKL诱导的破骨细胞形成观察低于IC50浓度的唑来膦酸对RANKL诱导破骨细胞形成的影响。抗酒石酸酸性磷酸酶染色显示，RANKL诱导组可见较多体积大的抗酒石酸酸性磷酸酶阳性细胞，无论从开始诱导培养至破骨细胞形成前阶段(阶段1)，还是从开始诱导培养至成熟破骨细胞完全形成阶段(阶段1+2)，唑来膦酸处理都可使破骨细胞分化功能几乎被终止。阶段1中破骨细胞数目和面积分别为(23.3±2.1)个/孔、(12.7±0.9)%，阶段1+2中破骨细胞数目和面积分别为(14.3±1.2)个/孔、(9.3± 1.3)%，明显少于对照组[(122.0±2.9)个/孔、(70.8±1.8)%]。破骨细胞开始形成后(阶段2)给予唑来膦酸仍然可见破骨细胞形成，但破骨细胞数目和面积分别为(71.3±2.5)个/孔、(42.0±2.2)%，较对照组分别下降了41.6%，40.7%，见图3。综上所述，唑来膦酸可以显著抑制破骨细胞的形成。 2.4 唑来膦酸对破骨细胞骨吸收的影响上述结果显示唑来膦酸可以明显影响破骨细胞分化，随后通过骨吸收陷窝实验探索破骨细胞骨吸收功能是否也被抑制。结果显示在RANKL组中大量骨吸收陷窝出现在骨磨片表面，吸收陷窝面积较大，陷窝数目较多，形态上表现为不规则形、大小不一；1 μmol/L唑来膦酸处理后，骨吸收陷窝数目和面积都明显减少，见图4。以上结果表明唑来膦酸可显著抑制破骨细胞骨吸收功能。"

References

[1] Tai TW, Chen CY, Su FC, et al. Reactive oxygen species are required for zoledronic acid-induced apoptosis in osteoclast precursors and mature osteoclast-like cells. Sci Rep. 2017;7:44245.

[2] Kanis JA. Diagnosis of osteoporosis and assessment of fracture risk. Lancet. 2002;359(9321):1929-1936.

[3] Frenkel B, White W, Tuckermann J. Glucocorticoid-Induced Osteoporosis. Adv Exp Med Biol. 2015;872:179-215.

[4] Nilsson AG. Bone research: an issue of maturity. J Intern Med. 2015; 277(6):626-629.

[5] Ha H, Shim KS, Ma JY. Water extract of Uncaria sinensis suppresses RANKL-induced bone loss by attenuating osteoclast differentiation and bone resorption. Integr Med Res. 2017;6(4): 434-442.

[6] Ishida M, Kitaura H, Kimura K, et al. Muramyl dipeptide enhances lipopolysaccharide-induced osteoclast formation and bone resorption through increased RANKL expression in stromal cells. J Immunol Res. 2015;2015:132765.

[7] Duan L, de Vos P, Fan M, et al. Notch is activated in RANKL-induced osteoclast differentiation and resorption. Front Biosci. 2008;13:7064-7071.

[8] Liou YM, Chan CL, Huang R, et al. Effect of l-caldesmon on osteoclastogenesis in RANKL-induced RAW264.7 cells. J Cell Physiol. 2018;233(9):6888-6901.

[9] Mundy GR, Yoneda T, Hiraga T. Preclinical studies with zoledronic acid and other bisphosphonates: impact on the bone microenvironment. Semin Oncol. 2001;28(2 Suppl 6):35-44.

[10] 黄晓林,姚超,程余婷,等. 唑来膦酸在骨质疏松状态下影响口腔种植体骨的结合[J].中国组织工程研究, 2018,22(18):2933-2938.

[11] 郑振雨,朱明明,李春娟,等. 唑来膦酸对破骨细胞的分化及骨吸收功能的影响[J].中外健康文摘, 2013,10(36):55-56.

[12] Jia X, Cheng J, Shen Z, et al. Zoledronic acid sensitizes breast cancer cells to fulvestrant via ERK/HIF-1 pathway inhibition in vivo. Mol Med Rep. 2018;17(4):5470-5476.

[13] Zhu M, Zhu Y, Ni B, et al. Mesoporous silica nanoparticles/ hydroxyapatite composite coated implants to locally inhibit osteoclastic activity. ACS Appl Mater Interfaces. 2014;6(8): 5456-5466.

[14] Benford HL, McGowan NW, Helfrich MH, et al. Visualization of bisphosphonate-induced caspase-3 activity in apoptotic osteoclasts in vitro. Bone. 2001;28(5):465-473.

[15] Coxon FP, Helfrich MH, Van't Hof R, et al. Protein geranylgeranylation is required for osteoclast formation, function, and survival: inhibition by bisphosphonates and GGTI-298. J Bone Miner Res. 2000;15(8):1467-1476.

[16] 吴骁伟,李少晗,曹文娟.唑来膦酸影响单个核细胞向破骨细胞分化及RANK表达的研究[J].中华全科医学, 2016, 14(10): 1644-1646.

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

[18] Tanaka S. Emerging anti-osteoclast therapy for rheumatoid arthritis. J Orthop Sci. 2018;23(5):717-721.

[19] Wen H, Liu Y, Li J, et al. Inhibitory effect and mechanism of 1,25-dihydroxy vitamin D3 on RANKL expression in fibroblast-like synoviocytes and osteoclast-like cell formation induced by IL-22 in rheumatoid arthritis. Clin Exp Rheumatol. 2018;36(5):798-805.

[20] Chen PC, Cheng HC, Tang CH. CCN3 promotes prostate cancer bone metastasis by modulating the tumor-bone microenvironment through RANKL-dependent pathway. Carcinogenesis. 2013;34(7): 1669-1679.

[21] 曾勇.蛋白质组学和网络分析鉴定与骨质疏松相关的基因和通路[D]. 北京:北京交通大学,2017.

[22] 周龙,陈曦,罗宗平,等. C57BL/6小鼠骨髓单核细胞分离、培养、纯化及向破骨细胞的分化[J].中国组织工程研究, 2015, 19(6): 940-944.

[23] 高军,由国平,刘陆, 等. 唑来膦酸治疗肺癌骨转移疼痛的评价[J]. 中国肿瘤临床与康复, 2015, 22(3): 374-375.

[24] 王增,卢红阳,翁琳.唑来膦酸抗肿瘤作用的临床研究进展[J]. 中国临床药理学与治疗学, 2011, 16(8): 945-950.

[25] Siddiqi MH, Siddiqi MZ, Kang S, et al. Inhibition of Osteoclast Differentiation by Ginsenoside Rg3 in RAW264.7 Cells via RANKL, JNK and p38 MAPK Pathways Through a Modulation of Cathepsin K: An In Silico and In Vitro Study. Phytother Res. 2015;29(9): 1286-1294.

[26] Kim K, Kim JH, Kim I, et al. TRIM38 regulates NF-κB activation through TAB2 degradation in osteoclast and osteoblast differentiation. Bone. 2018;113:17-28.

[27] Wang X, Chen B, Sun J, et al. Iron-induced oxidative stress stimulates osteoclast differentiation via NF-κB signaling pathway in mouse model. Metabolism. 2018;83:167-176.

[28] Yang JH, Li B, Wu Q, et al. Echinocystic acid inhibits RANKL- induced osteoclastogenesis by regulating NF-κB and ERK signaling pathways. Biochem Biophys Res Commun. 2016;477(4): 673-677.

[29] Lin TH, Tamaki Y, Pajarinen J, et al. Chronic inflammation in biomaterial-induced periprosthetic osteolysis: NF-κB as a therapeutic target. Acta Biomater. 2014;10(1):1-10.